HPLC Methods For Pharmaceutical. T3

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Doxofylline
Molecular formula: C11H14N4O4
Molecular weight: 266.26
CAS Registry No.: 69975-86-6
Merck Index: 3494
LednicerNo.: 5 144
SAMPLE
Matrix: blood
Sample preparation: Centrifuge, filter (0.45 ixm), inject an aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm internal surface reversed phase Pinkerton, silica derivatized with
glycine-phenylalanine-phenylalanine (Regis) (periodically reverse the column)
Mobile phase: 100 mM pH 6.8 phosphate buffer
Flow rate: 0.3
Injection volume: 10
Detector: UV 275
CHROMATOGRAM
Retention time: 13.94
Limit of detection: <1000 ng/mL
OTHER SUBSTANCES
Extracted: dyphylline, theophylline, caffeine
Noninterfering: acetaminophen, amitriptyline, amphetamine, atropine, benzoylecgonine,
benztropine, caffeine, carbamazepine, carisoprodol, chlorpheniramine, chlorpromazine,
chlorprothixene, cimetidine, cocaine, codeine, dextromethorphan, diazepam, diphenhydra-
mine, diphenoxilate, disopyramide, doxepin, doxylamine, emetine, erythromycin, fluraze-
pam, glutethimide, hydrocortisone, hydromorphone, hydroxyzine, imipramine, Udocaine, lox-
apine, meperidine, meprobamate, methadone, methamphetamine, methapyrilene,
methaqualone, methocarbamol, methylphenidate, nicotine, nordiazepam, nortriptyline,
orphenadrine, papaverine, pentazocine, phenacetin, phencyclidine, phenmetrazine, phe-
nolphthalein, phentermine, phenylpropanolamine, phenytoin, prazepam, procainamide,
procaine, propoxyphene, propranolol, protriptyline, pseudoephedrine, pyrilamine, quinine,
salicylamide, spironolactone, strychnine, terpin hydrate, thioridazine, thiothixene, triam-
terene, trifluoperazine, triflupromazine, trihexyphenidyl, trimeprazine, trimethobenza-
mide, trimethoprim, tripelennamine
KEYWORDS
plasma; serum; direct injection
REFERENCE
Tagliaro,E; Dorizzi,R-J Frigerio,A.; Marigo,M. Non-extraction HPLC method for simultaneous measure-
ment of dyphylline and doxofylline in serum, Clin.Chem., 1990, 36, 113-115.
SAMPLE
Matrix: blood, formulations, tissue
Sample preparation: Elixir. Make up 1 mL elixir to 50 mL with mobile phase. Remove a
5 mL aliquot and add it to 2 mL 500 jxg/mL theophylline in mobile phase, make up to
100 mL with mobile phase, inject a 20 |xL aliquot. Tablets. Finely powder a tablet in a
mortar, weigh out 310 mg and add it to 100 mL mobile phase, let stand for some time,
filter. Add 1 mL filtrate to 2 mL 500 |xg/mL theophylline in mobile phase, make up to 100
mL with mobile phase, inject a 20 |JLL aliquot. Tissue. Homogenize rat brain, extract on
an Extrelut 3 SPE cartridge at pH 8, elute with chloroform, take up the residue in MeOH,
inject an aliquot (from Drug Metab.Dispos. 1989, 17, 437). Serum. Extract on an Extrelut
3 SPE cartridge at neutral pH, elute with chloroform, take up the residue in MeOH, inject
an aliquot (from Drug Metab.Dispos. 1989, 17, 437).
HPLC VARIABLES
Column: 250 mm long 10 |xm LiChrosorb RP-18
Mobile phase: MeCN:water 30:70
Flow rate: 1.5
Detector: UV 273
CHROMATOGRAM
Retention time: 2.6
Internal standard: theophylline (2)
KEYWORDS
serum; rat; brain; elixir; tablets; SPE
REFERENCE
Badini,C; Masera,R; Franzone,J.S. Dosaggio del 2-(7'-teofillinmetil)-l,3-diossolano mediante HPLC [As-
say of 2-(7'-theophyllinmethyl)-l,3-dioxolane using HPLC], Farmaco.[Prat]., 1982, 37, 320-324.
Doxorubicin
Molecular formula: C27H29NO11
CAS Registry No.: 23214-92-8, 25316-40-9 (HCI)
Merck Index: 3495
SAMPLE
Matrix: bile, blood, feces, tissue, urine
Sample preparation: Homogenize tissue in 4% BSA in water to a final concentration of
0.05-2 g/mL. Homogenize feces in 4% BSA in water to a final concentration of 0.03-1 g/
mL. Dilute feces homogenate 20-fold, urine 100-fold, and bile 20-fold with blank human
plasma. 200 |xL Sample + 200 jxL 6% (w/v) pH 9.5 borate buffer + 100 \xL 4 mg/mL IS in
pH 2.05 water, vortex. Mix with 1 mL chloroform: 1-propanol 20:80 for 5 min. (Caution!
Chloroform is a carcinogen!) Centrifuge at 3000 g at 4 for 10 min. Remove the organic
layer and evaporate it under reduced pressure at 43. Reconstitute the residue in 100 |xL
MeCN-.THF 40:1, vortex for 20 s, sonicate for 5 min. Add 300 uL water acidified to pH
2.05, vortex, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 2 pellicular RP material
Column: 100 X 3 7jxm Lichrosorb RP-8
Mobile phase: MeCN:THF:water adjusted to pH 2.05 with perchloric acid 30:1:80
Flow rate: 0.4
Detector: F ex 460 em 550
CHROMATOGRAM
Retention time: 6
Internal standard: daunorubicin (12)
Limit of quantitation: 1.8-2.4 nM
OTHER SUBSTANCES
Extracted: metabolites
KEYWORDS
mouse; plasma; brain; muscle; colon; cecum; small intestine; stomach; liver; gall bladder;
kidney; lung; spleen; heart; ovary; uterus; breast; testis; epididymis; eye; pharma-
cokinetics
REFERENCE
van Asperen,J.; van Tellingen,O.; Beijnen,J.H. Determination of doxorubicin and metabolites in murine
specimens by high-performance liquid chromatography, J.Chromatogr.B, 1998, 712, 129-143.
SAMPLE
Matrix: blood
Sample preparation: Condition an Oasis HLB SPE cartridge (Waters) with 1 mL mobile
phase:water 1:3. 200 u,L Plasma + 500 mL mobile phase:water 1:3 + 100 |xL water, vortex,
add to SPE cartridge, wash with 1 mL mobile phase:water 1:3, elute with 600 JJLL MeCN:
mobile phase 1:1, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 200 X 4.6 10 ^m LiChrosorb RP18
Mobile phase: MeCN:water 29:71 containing 50 mM Na2HPO4 and 0.05% (v/v) triethyl-
amine adjusted to pH 4.6 with citric acid
Flow rate: 1.0
Detector: E, ESA Coulochem II, Model 5014 high-performance analytical cell containing
amperometric electrode +400 mV coupled with coulometric electrode -300 mV, palladium
reference electrode
CHROMATOGRAM
Internal standard: epirubicin (9.4)
OTHER SUBSTANCES
KEYWORDS
plasma; SPE; pharmacokinetics
REFERENCE
Ricciarello,R; Pichini,S.; Pacifici,R.; Altieri,L; Pellegrini,M.; Fattorossi,A.; Zuccaro,R. Simultaneous de-
termination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-
performance liquid chromatography and electrochemical detection, J.Chromatogr.B, 1998, 707, 219-
225.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 5 mL chloroform:isopropanol 80:20, extract, cen-
trifuge at 3000 g for 5 min. Remove the organic layer and evaporate it to dryness under
a stream of nitrogen at 30 in the dark, reconstitute the residue in 100 |xL mobile phase,
inject a 25 JULL aliquot.
HPLC VARIABLES
Column: 125 X 4 Nucleosil 100-5 C18
Mobile phase: MeCN: 10 mM pH 4 ammonium formate buffer 30:70
Flow rate: 1.5
CHROMATOGRAM
Retention time: 4.5
Internal standard: doxorubicin
OTHER SUBSTANCES
Extracted: epirubicin
KEYWORDS
plasma; doxorubicin is IS
REFERENCE
Jakobsen,R; Steiness,E.; Bastholt,L.; Dalmark,M.; Lorenzen,A.; Petersen,D.; Gjedde,S.B.; Sandberg,E.;
Rose,C; Nielsen,O.S. Multiple-dose pharmacokinetics of epirubicin at four different dose levels: stud-
ies in patients with metastatic breast cancer, Cancer Chemother.PharmacoL, 1991, 28, 63-68.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma or blood + 3 mL 100 mM pH 9.5 ammonia-ammonium
chloride buffer + 20 ng daunorubicin + 13.5 mL chloroform:MeOH 2:1, shake mechanically
for 30 min, centrifuge at 3000 g for 10 min, repeat the extraction with 9 mL chloroform.
Combine the organic layers and evaporate them to dryness under a stream of nitrogen
at 30, reconstitute the residue in 3 mL chloroform:MeOH 2:1, evaporate this mixture,
reconstitute the residue in 300 |xL mobile phase, centrifuge a 75 |xL aliquot at 10000 g
for 1 min, inject the supernatant.
HPLC VARIABLES
Column: 250 X 4 5 jxm STR ODS-M (Shimadzu)
Mobile phase: MeCN:bufFer 30:70 (Buffer was 200 mM acetic acid-ammonium formate, pH
4.0.)
Column temperature: 22
Flow rate: 0.7
CHROMATOGRAM
Retention time: 7.7
Internal standard: daunorubicin (16.2)
Limit of detection: 0.5 ng/mL
OTHER SUBSTANCES
Extracted: pirarubicin
KEY WORDS
plasma; whole blood; pharmacokinetics
REFERENCE
Nagasawa,K.; Yokoyama,T.; Ohnishi,N.; Iwakawa,S.; Okumura,K.; Kosaka,Y; Sano,K.; Murakami,R-J
Nakamura,H- Pharmacokinetics of pirarubicin in pediatric patients, J.Pharmacobiodyn., 1991, 14,
222-230.
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Plasma + 250 |JLL 100 ng/mL daunorubicin in mobile phase,
extract with 3 mL MeCN for 10 min, add 100 mg NaCl, shake for 5 min, centrifuge at
995 g for 15 min, let stand at -20 for 1 h. Remove the supernatant and evaporate it to
dryness under a stream of nitrogen at 60, reconstitute the residue in 250 JULL mobile
phase, inject a 100 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 10 jjim Spherisorb phenyl
Column: 250 X 4.6 5 jjim Spherisorb phenyl
Mobile phase: MeCN:30 mM citrate buffer adjusted to pH 4 with formic acid 30:70
Flow rate: 1.5
CHROMATOGRAM
Limit of quantitation: 2 ng/mL
OTHER SUBSTANCES
Extracted: pirarubicin, doxorubicinol
KEYWORDS
plasma; pharmacokinetics
REFERENCE
Jacquet,J.M.; Galtier,M.; Bressolle,E; Jourdan,J. A sensitive and reproducible HPLC assay for doxoru-
bicin and pirarubicin, J.Pharm.BiomedAnaL, 1992, 10, 343-348.
SAMPLE
Matrix: blood, cells
Sample preparation: Thaw cell samples, sonicate (Branson B-12) at 50 W for 20 s. 400
jxL Cell sample or plasma + 200 jxL 200 nM daunorubicin in 100 mM pH 9.3 borate buffer,
add 1.8 mL chloroform:MeOH 80:20, extract, inject a 200-500 jxL aliquot of the organic
phase.
HPLC VARIABLES
Column: 250 X 4 Lichrosorb Si-60
Mobile phase: Chloroform:MeOH:glacial acetic acid:0.3 mM magnesium chloride 72:21:2:3
Flow rate: 1.5
Injection volume: 200-500
CHROMATOGRAM
Retention time: 4.3
Limit of detection: 0.5 nM
OTHER SUBSTANCES
Extracted: metabolites, epirubicin
KEY WORDS
plasma; pharmacokinetics; normal phase
REFERENCE
Tidefelt,U.; Sundman-Engberg,B.; Paul,C. Comparison of the intracellular pharmacokinetics of doxo-
rubicin and 4'-epi-doxorubicin in patients with acute leukemia, Cancer Chemother.Pharmacol., 1989,
24, 225-229.
SAMPLE
Matrix: blood, tissue
Sample preparation: Plasma. Mix plasma with four volumes daunorubicin aglycone in
MeOH, centrifuge, inject a 100 |xL aliquot of the supernatant. Tissue. Tissue + 1 volume
MeOH + 2 volumes 1 M pH 8.5 Tris buffer, homogenize, let stand on ice for 15 min, add
7 volumes MeCN containing daunorubicin aglycone, vortex, let stand at room temperature
for 15 min, centrifuge, inject a 100 |xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 10 |xm LiChrosorb RP-18
Column: 250 X 4.6 10 |xm LiChrosorb RP-18
Mobile phase: Gradient. MeCN:buffer from 15:85 to 50:50 over 10 min, re-equilibrate at
initial conditions for 15 min. (Buffer was 25 mM (NH4)H2PO4 and 30 mM phosphoric acid.)
Flow rate: 1.5
CHROMATOGRAM
Retention time: 9
Internal standard: daunorubicin aglycone (13.5)
Limit of quantitation: 1.5 pmole
OTHER SUBSTANCES
Extracted: metabolites, 13-dihydrodoxorubicin
KEYWORDS
plasma; mouse; lung
REFERENCE
Rose,L.M.; Tillery,K.R; el Dareer,S.M.; Hill,D.L. High-performance liquid chromatographic determina-
tion of doxorubicin and its metabolites in plasma and tissue, J.Chromatogr., 1988, 425, 419-423.
SAMPLE
Sample preparation: Serum. Serum + daunorubicin + 8 mL chloroform:isopropanol 50:50,
extract, centrifuge at 3000 rpm. Remove the organic layer and evaporate it to dryness
under a stream of nitrogen, reconstitute the residue in 100 (JLL mobile phase, inject a 5-
20 (JLL aliquot. Tissue. Homogenize tissue in water, add daunorubicin, add 30 |xL silver
nitrate (33%), extract with 8 mL isopropanol. Remove the organic layer and evaporate it
to dryness under a stream of nitrogen, reconstitute the residue in 100 JXL mobile phase,
inject a 5-20 |iL aliquot.
HPLC VARIABLES
Column: 300 mm long 10 jxm jxBondapak C18
Mobile phase: MeCN:water: 100 mM phosphoric acid 37:37:26
Flow rate: 1.5
CHROMATOGRAM
Internal standard: daunorubicin
Limit of detection: 20 ng/g (tissue), 5 ng/mL (serum)
KEY WORDS
serum; rat; pharmacokinetics; heart; liver; kidney; adrenal; brain; intestine; mouse
REFERENCE
Colombo,!1.; Zucchetti,M.; D'Incalci,M. Cyclosporin A markedly changes the distribution of doxorubicin
in mice and rats, J.Pharmacol.Exp.Ther., 1994, 269, 22-27.
SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. 150 |xL Plasma + 150 |xL MeCN, vortex for 10 s. Centrifuge
at 3000 rpm for 5 min. Remove 200 |xL of the organic layer, evaporate under reduced
pressure, reconstitute the residue in 100 fxL 100 mM pH 3 monobasic phosphate buffer.
Inject a 10 |iL aliquot. Urine. Directly inject a 10 (xL aliquot of urine. (Silanize glassware
with 3% dichlorodimethylsilane in toluene, rinse with MeOH before use.)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm TSK gel ODS/TM silica (Tosoh Co., Japan)
Mobile phase: MeCN:buffer 35:65 (Buffer was 100 mM monobasic phosphate (sic) contain-
ing 0.3% heptafluorobutyric acid, adjusted to pH 3 with NaOH. At the end of the analysis,
wash the column with MeOH and MeOH:water 50:50.)
Flow rate: 1
CHROMATOGRAM
Retention time: 5.5
Limit of detection: 25 nM
Limit of quantitation: 2.5 [xM
OTHER SUBSTANCES
Extracted: metabolites, daunorubicin
KEY WORDS
plasma
REFERENCE
Emara,S.; MoritaJ.; Tamura,K; Razee,S.; Masujima,T.; Mohamed,H.A.; El Gizawy,S.M.; El Rabbat,N.A.
Utility of ion-pair chromatography for analysis of some anthracyclines in plasma and urine,
J.Liq.Chromatogr.Rel.Technol, 1998, 22, 681-692.
SAMPLE
Sample preparation: 1 mL Plasma or urine + 3 mL 10 mM pH 9.0 ammonium chloride
buffer, adjust pH to 9.0 with NaOH, add 6 mL chloroform:MeOH 2:1, shake for 5 min,
centrifuge at 12000 g at 4 for 10 min, remove the organic layer, re-adjust the pH of the
aqueous layer, repeat the extraction. Combine the organic layers and evaporate them to
dryness under a stream of nitrogen, reconstitute the residue in 500 |xL mobile phase,
inject an aliquot.
HPLC VARIABLES
Column: Nova-Pak C18 phenyl
Mobile phase: MeCN:35 mM pH 3.0 ammonium formate buffer 35:65
Flow rate: 0.8
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
Extracted: pirarubicin, metabolites
KEYWORDS
plasma; SPE
REFERENCE
Raber,M.N.; Newman,R.A.; Lu,K; Legha,S.; Gorski,C; Benjamin,R.S.; KrakoffJ.H. Phase I clinical trial
and pharmacokinetic evaluation of 4'-O-tetrahydropyranyladriamycin (THP-adriamycin), Cancer
Chemother.PharmacoL, 1989, 23, 311-315.
SAMPLE
Sample preparation: 1 mL Plasma or urine + daunorubicin + daunorubicinone + 5 mL
chloroformrisopropanol 75:25, shake mechanically for 30 min, centrifuge at 4 at 1200 g
for 15 min. Remove the lower organic layer and evaporate it to dryness under a stream
of nitrogen at room temperature. Add the aqueous phase to 2 mL 50 (plasma) or 500
(urine) mM pH 8.4 borate buffer, add 5 mL chloroformdsopropanol 75:25, shake mechan-
ically for 30 min, centrifuge at 4 at 1200 g for 15 min. Remove the lower organic layer
and add it to the residue from the first extraction, evaporate to dryness under a stream
of nitrogen at room temperature, reconstitute the residue in 600 (xL MeOH:500 mM phos-
phoric acid 50:50, vortex for 30 s, add 2 mL N-hexane, vortex for 30 s, centrifuge, inject
an aliquot of the aqueous phase (omit the hexane wash for urine samples).
HPLC VARIABLES
Guard column: 30-38 |xm pellicular ODS (Whatman)
Column: 150 X 3.9 4 jxm Nova-Pak C18
Mobile phase: MeCNrMeOH: 10 mM pH 1.4 phosphate buffer 25:10:65
Flow rate: 0.58
CHROMATOGRAM
Retention time: 9
Internal standard: daunorubicin (25), daunorubicinone (35)
Limit of quantitation: 12.2 ng/mL (urine), 0.31 ng/mL (plasma)
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Fraier,D.; Frigerio,E.; Pianezzola,E.; Strolin Benedetti,M.; Cassidy,J.; Vasey,R A sensitive procedure for the
quantitation of free and N-(2-hydroxypropyl)methacrylamide polymer-bound doxorubicin (PKl) and some
of its metabolites, 13-dihydrodoxorubicin, 13-dihydrooxorubicinone and doxorubicinone, in human plasma
and urine by reversed-phase HPLC with fluorimetric detection, J.Pharm.BiomedAnaL, 1995, 13, 625-
633.
SAMPLE
Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA),
add 3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove
the organic layer and evaporate it to dryness under a stream of nitrogen at 40, recon-
stitute the residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g
for 2 min, inject a 10 (urine) or 30 (blood) jxL aliquot. (The detector wavelength shown is
the wavelength of maximum absorbance. This will not necessarily be the optimal wave-
length for the separation. Multiple wavelengths from 200-350 nm can be scanned using
a diode-array detector. Otherwise, 220 nm may be a reasonable choice for initial work.
Matrix may interfere.)
HPLC VARIABLES
Guard column: 20 mm long Symmetry C18
Column: 250 X 4.6 5 |mm Symmetry C8 (Waters)
Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A:
B 85:15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at
initial conditions for 7 min.
Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at
1.5 mL/min)
Detector: UV 232.2
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detec-
tion for the creation of a 600-compound library. Application to forensic toxicology, J.ChromatogrA,
1997, 763, 149-163.
SAMPLE
Matrix: bulk
Sample preparation: Prepare a 1 mg/mL solution in water and dilute it with mobile phase
to obtain a 50 jxg/mL solution. Inject a 50 jxL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 fim Nucleosil C18 (Shandon HPLC, UK)
Mobile phase: The ratio MeOHraqueous phase was modified. Aqueous phase was pH 2.2
citrate buffer, Tris buffer, pH 5.1 acetate buffer or pH 5.4 phosphate buffer. Counter ion
was 2.54 mM triethylamine, 2-cyano-2-butylhexanoic acid or heptane sulfonic acid. (De-
tails for preparation mobile phase in paper.)
Column temperature: 20 0.3
Flow rate: 1.0
Detector: UV 494
CHROMATOGRAM
Retention time: not specified
KEYWORDS
ion-pair chromatography
REFERENCE
Pepin,X.; Attali,L.; Domrault,C; Gallet,S.; Metreau,J.M.; Reault,Y.; Cardot,P.J.R; Imalalen,M.; Duber-
net,C; Soma,E.; Couvreur,P. On the use of ion-pair chromatography to elucidate doxorubicin release
mechanism from polyalkylcyanoacrylate nanoparticles at the cellular level, J.Chromatogr.B, 1997,
702, 181-191.
SAMPLE
Matrix: bulk
Sample preparation: Free doxorubicin. Dissolve the sample in water, inject an aliquot.
Bound doxorubicin. Mix 500 |xL of an aqueous solution with 500 |xL 2 M HCl, heat at 50
for 1.5 h, cool to room temperature, inject an aliquot within 2 h.
HPLC VARIABLES
Column: 250 X 4.6 Vydac C18
Mobile phase: Gradient. A was MeCN containing 0.1% trifluoroacetic acid. B was 0.1%
trifluoroacetic acid. A:B from 25:75 to 30:70 in 10 min, to 80:20 in 5 min, maintain at 80:
20 for 15 min.
Detector: UV
CHROMATOGRAM
Retention time: 7.5
Limit of detection: 500 ng/mL
Limit of quantitation: 1 jjig/mL
OTHER SUBSTANCES
Simultaneous: adriamycinone
REFERENCE
Configliacchi,E.; Razzano,G.; Rizzo,V.; Vigevani,A. HPLC methods for the determination of bound and
free doxorubicin, and of bound and free galactosamine, in methacrylamide polymer-drug conjugates,
J.Pharm.BiomedAnal., 1996, 15, 123-129.
SAMPLE
Matrix: formulations
Sample preparation: Dilute a 1 mL aliquot to 50 mL with 0.9% sodium chloride, inject a
25 |JLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 jxm Nucleosil 100-5CN
Mobile phase: MeCN:MeOH:20 mM pH 4.5 ammonium dihydrogen phosphate in water 20:
20:60, containing 10 mM sodium heptanesulfonate
Flow rate: 1.0
Detector: UV 297
CHROMATOGRAM
Retention time: 4.1
OTHER SUBSTANCES
Simultaneous: degradation products, methylparaben, propylparaben, vincristine
KEYWORDS
injections; saline; stability-indicating
REFERENCE
Nyhammar,E.K.; Johansson,S.G.; Seiving,B-E. Stability of doxorubicin hydrochloride and vincristine sul-
fate in two portable infusion-pump reservoirs, Am.J.Health-Syst.Pharm., 1996, 53, 1171-1173.
SAMPLE
HPLC VARIABLES
Column: 220 X 4.6 5 |xm silica (Brownlee)
Mobile phase: MeCN:6.25 mM NaH2PO4 adjusted to pH 3.0 with concentrated phosphoric
acid 40:60
Flow rate: 1
Detector: UV 216
CHROMATOGRAM
Retention time: 7.7
OTHER SUBSTANCES
Simultaneous: dacarbazine, ondansetron
Noninterfering: degradation products
KEYWORDS
injections; 5% dextrose
REFERENCE
King,D.T.; Stewart,J.T. HPLC determination of dacarbazine, doxorubicin, and ondansetron mixture in
5% dextrose injection on underivatized silica with an aqueous-organic mobile phase,
J.Liq.Chromatogr., 1993, 16, 2309-2323.
SAMPLE
HPLC VARIABLES
Column: 300 X 3.9 10 |xm u.Bondapak phenyl
Mobile phase: MeCN:buffer 50:50 (Buffer was 20 mM KH2PO4 adjusted to pH 5.4 with 1
MNaOH
Flow rate: 1
Detector: UV 233
CHROMATOGRAM
Retention time: 5.1
OTHER SUBSTANCES
Simultaneous: methyl paraben, ondansetron, vincristine, degradation products
KEYWORDS
injections; saline
REFERENCE
King,D.T.; Venkateshwaran,T.G.; Stewart,J.T. HPLC determination of a vincristine, doxorubicin, and
ondansetron mixture in 0.9% sodium chloride injection, J.Liq.Chromatogr., 1994, 17, 1399-1411.
SAMPLE
Sample preparation: Dilute with mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 300 X 4.6 5 urn C18
Mobile phase: MeCN: 100 mM NaH2PO4 20:80 adjusted to pH 4.2 with phosphoric acid
Flow rate: 2.5
Detector: UV 300
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: granisetron
KEYWORDS
stability-indicating; injections; saline
REFERENCE
Mayron,D.; Gennaro,A.R. Stability and compatibility of granisetron hydrochloride in i.v. solutions and
oral liquids and during simulated Y-site injection with selected drugs, Am.J.Health-Syst.Pharm.,
1996, 53, 294-304.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 3.9 5 |xm Symmetry C8 (Waters)
Mobile phase: MeCN:20 mM sodium dihydrogen phosphate 20:80 (A) or MeCN:30 mM
sodium dihydrogen phosphate 23:77 (B)
Flow rate: 1
Detector: UV 307 (A), UV 195 (B)
CHROMATOGRAM
Retention time: 11.2 (A), 5.4 (B)
OTHER SUBSTANCES
Simultaneous: granisetron (A), cyclophosphamide (B)
REFERENCE
Zhang,H.; Ye,L.; Stewart,J.T. HPLC determinations of doxorubicin with selected medications in 0.9%
sodium chloride injection USP, J.Liq.Chromatogr.Rel.TechnoL, 1998, 21, 2375-2385.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 jxm IB-SIL C8 (Phenomenex)
Mobile phase: MeCN:20 mM sodium dihydrogen phosphate 40:60
Flow rate: 1
Detector: UV 285
CHROMATOGRAM
Retention time: 7.3
OTHER SUBSTANCES
Simultaneous: morphine
REFERENCE
Zhang,H.; Ye5L.; Stewart,J.T. HPLC determinations of doxorubicin with selected medications in 0.9%
sodium chloride injection USP, J.Liq.Chromatogr.Rel.TechnoL, 1998, 21, 2375-2385.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 X 3.9 10 |xm LiChrosorb Si-60
Mobile phase: MeOH:water 60:40 containing 4 mM disodium citrate and 4 mM tetrabu-
tylammonium bromide, pH 5.9
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Simultaneous: atropine, codeine, dansylamide, dansylcadaverine, methylatropine, napha-
zoline, noscapine, xylometazoline
REFERENCE
Lingeman,H.; van Munster,H.A.; Beynen,J.H.; Underberg,W.J.; Hulshoff,A. High-performance liquid
chromatographic analysis of basic compounds on non-modified silica gel and aluminium oxide with
aqueous solvent mixtures, J.Chromatogr., 1986, 352, 261-274.
SAMPLE
Matrix: tissue
Sample preparation: Flush femurs with MeCNrwater 50:50. Homogenize 3 spleens with
5 mL MeCN:water 50:50. Centrifuge extracts immediately at 1500 rpm for 5 min, inject
an aliquot.
HPLC VARIABLES
Column: 300 X 16 Nucleosil C18 Bondasorb
Mobile phase: MeOH: 10 mM sodium acetate:acetic acid 65:35:1.3
Flow rate: 1
CHROMATOGRAM
Retention time: 9
KEY WORDS
mouse; bone marrow; spleen
REFERENCE
Gibaud,S.; Demoy,M.; Andreux,J.R; Weingarten,C; Gouritin,B.; Couvreur,P. Cells involved in the cap-
ture of nanoparticles in hematopoietic organs, J.Pharm.ScL, 1996, 85, 944-950.
Doxycycline
CAS Registry No.: 564-25-0,17086-28-1 (monohydrate),
24390-14-5 (HCI monohydrate), 83038-87- 3 (fosfatex), 24390-14-5 (hyclate)
Merck Index: 3496
SAMPLE
Matrix: blood
Sample preparation: Add 100 |xL MeCN:phosphoric acidrwater 20:2:78 to 100 jxL plasma,
vortex for 10 s, centrifuge at 14000 g for 30 min. Inject a 50 |xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 12.5 X 4 Zorbax RX-C18
Column: 150 X 4.6 5 |xm Zorbax SB-C8
Mobile phase: MeCN:MeOH:water 14:10:76 containing 100 mM oxalic acid
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Retention time: 6-6.5
KEYWORDS
cat; plasma
REFERENCE
Kordick,D.L.; Papich,M.G.; Breitschwerdt,E.B. Efficacy of enrofloxacin or doxycycline for treatment of
Bartonella henselae or Bartonella clarridgeiae infection in cats, Antimicrob.Agents Chemother., 1997,
41, 2448-2455.
SAMPLE
Matrix: blood
Sample preparation: Add 50 |xL 5 jxg/mL demeclocycline in 1 mM HCl to 250 |xL plasma,
add 50 |xL 6% aqueous ascorbic acid solution, 1 mL phosphate-sulfite buffer, and 6 mL
ethyl acetate, vortex for 2 min, centrifuge at 2600 g for 10 min. Transfer 5 mL of the
organic phase to 100 \LL 0.2% ascorbic acid in MeOH, evaporate to dryness under nitro-
gen, redissolve the residue in 300 jxL mobile phase, add 4 mL n-hexane, vortex, centrifuge
at 2600 g for 5 min, remove the hexane layer by aspiration, inject a 100 |JLL aliquot of the
aqueous phase. (The phosphate-sulfite buffer (pH 6) was 25.2 g sodium sulfite and 36.3
g NaH2PO4 dihydrate in 100 mL .)
HPLCVARIABLES
Guard column: 4 x 4 5 |xm LiChrospher RP-18
Column: 125 X 4 5 juum LiChrospher RP-18
Mobile phase: MeCN:70% perchloric acid:water 29.85:0.25:69.9 containing 0.6 mM diso-
dium EDTA and 5 mM oxalic acid, pH adjusted to 2.5 with 1 M NaOH
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Internal standard: demeclocycline (4.28)
OTHER SUBSTANCES
Extracted: degradation products
KEY WORDS
plasma; turkey
REFERENCE
Santos,M.D.; Vermeersch,H.; Remon,J.R; Schelkens,M.; De Backer,R; Ducatelle,R.; Haesebrouck,F. Val-
idation of a high-performance liquid chromatographic method for the determination of doxycycline
in turkey plasma, J.Chromatogr.B, 1996, 682, 301-308.
SAMPLE
Matrix: eggs, tissue
Sample preparation: Prepare a metal chelate affinity chromatography (MCAC) column by
adding 1.5 mL of thoroughly mixed Chelating Sepharose Fast-Flow suspension in EtOH:
water 20:80 (Pharmacia) to a 150 X 10 glass column, allow to drain, wash with three 2
mL portions of water, add 2 mL 10 mM copper(II) sulfate in water, wash with two 2 mL
portions of water. Condition an SBD-RPS extraction membrane (3M Company, St. Paul,
MN) with 2 mL MeOH and 2 mL 100 mM HCl. Add 20 mL 100 mM pH 4.0 sodium
succinate buffer to 3 g pig kidney, pig muscle, cow liver, or whole chicken egg, vortex for
1 min and shake for 10 min on a horizontal shaker. Add 20 mL MeOH, sonicate for 5 min
and centrifuge at 2666 g for 10 min at 4. Filter the supernatant through a Whatman
541 filter paper. Add the clear supernatant to the MCAC column. Wash sequentially with
2 mL 100 mM sodium succinate buffer, 2 mL water, 2 mL MeOH, 2 mL water, and with
500 fxL Mcllvaine-EDTA-NaCI buffer. Elute with 3 mL Mcllvaine-EDTA-NaCI buffer and
adjust the eluate to pH 1.3 with 400 jxL 4 M HCl. Add the eluate directly to the extraction
membrane to prevent crystallization of EDTA. Wash the membrane with 1 mL 100 mM
HCl and elute with four 250 jxL portions of MeOH:25% ammonia 97:3, evaporate the
eluate to dryness under the nitrogen at 40. Reconstitute the dry residue with 250 JJLL 10
mM oxalic acid in water, vortex, sonicate. Inject a 100 jxL aliquot. (The sodium succinate
buffer was 100 mM succinic acid, pH adjusted to 4.0 with 10 M NaOH. Prepare the
Mcllvaine buffer by dissolving 12.9 g citric acid monohydrate and 10.9 g Na 2HPO4 in 1
L water. The Mcllvaine-EDTA-NaCI buffer was 100 mM EDTA and 500 mM NaCl in
Mcllvaine buffer. Protect all solutions from light.)
HPLC VARIABLES
Guard column: 5 X 3.0 PLRP-S (Polymer Laboratories)
Column: 250 X 4.6 8 |x PLRP-S (Polymer Laboratories)
Mobile phase: Gradient. A was 10 mM oxalic acid in water adjusted to pH 2.0 with 4 M
HCl. B was MeCN. A:B from 85:15 to 60:40 over 16 min.
Flow rate: 1
Detector: F ex 406 em 515 following post-column reaction. The column effluent mixed with
reagent pumped at 1 mL/min and the mixture flowed through a 600 |xL reaction coil to
the detector. (Reagent was 5% zirconyl chloride octahydrate in water stored at 4.)
CHROMATOGRAM
Retention time: 20
Limit of detection: 3.00 ng/g (pig kidney), 1.38 ng/g (pig muscle), 2.35 ng/g (cow liver),
0.80 ng/g chicken egg)
Limit of quantitation: 5 ng/g (pig kidney)
OTHER SUBSTANCES
Extracted: chlortetracycline, oxytetracycline, tetracycline
Also analyzed: demeclocycline
KEYWORDS
cow; liver; pig; kidney; muscle; chicken; metal chelate affinity chromatography; MCAC; SPE
REFERENCE
Croubels,S.M.; Vanoosthuyze,K.E.L; Van Peteghem,C.H. Use of metal chelate affinity chromatography
and membrane-based ion-exchange as clean-up procedure for trace residue analysis of tetracyclines
in animal tissues and egg, J.Chromatogr.B, 1997, 690, 173-179.
SAMPLE
Matrix: milk
Sample preparation: Fill a disposable polypropylene column (Bio-Rad Econo-Pac column)
with Chelating Sepharose Fast Flow (Pharmacia) and condition it with 10 mL water, 1.5
mL 100 mM copper sulfate, and 100 mL water. Condition a 6 mL SupelClean EFfVT-
Chrom P SPE cartridge with 2 mL MeOH and 5 mL water. Homogenize 10 g tissue with
20-30 mL 100 mM pH 4 succinic acid buffer. Centrifuge the homogenate at 2000 g at 10
for 15-20 min. Add the supernatant to the metal chelate affinity column, wash sequen-
tially with 5 mL 500 mM NaCl, 10 mL water, 10 mL MeOH, 10 mL water, and 3 mL
Mcllvaine buffer, discard the clear effluent. Elute with 8 mL Mcllvaine-EDTA-NaCI buf-
fer. Add the eluate to the SPE cartridge under gravity, rinse the column with 2.5 mL
water, add the rinse to the SPE cartridge. Wash the SPE cartridge with 2.5 mL water.
Dry the SPE cartridge by drawing air through it for 2-3 min. Elute with 5 mL MeOH.
Evaporate the eluate to dryness under nitrogen at 40-50, dissolve the residue in 1 mL
water. Inject a 100 |xL aliquot. (Mcllvaine buffer was 500 mM NaCl and 100 mM EDTA
(Carson, M.C. J. AOAC Int. 1993, 76, 329).)
HPLC VARIABLES
Column: 150 X 3.9 5 |xm PLRP-S (Polymer Labs, USA)
Mobile phase: MeOH:5 mM oxalic acid 58:42
Flow rate: 0.5
Detector: MS, HP 5989, NICI, high energy dynode, HP 59980B particle beam interface 60,
helium sheath 40-45 p.s.i., source 250, quadrupole 100, source pressure 1 Torr with
methane reagent gas, m/z 378-483
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: chlortetracycline, demeclocycline, minocycline, oxytetracycline, tetracycline
KEY WORDS
metal chelate affinity chromatography; cow; SPE
REFERENCE
Carson,M.C; Ngoh,M.A.; Hadley,S.W. Confirmation of multiple tetracycline residues in milk and oxy-
tetracycline in shrimp by liquid chromatography-particle beam mass spectrometry, J.Chromatogr.B,
1998, 712, 113-128.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 3 mL 500 mg Bond Elut C18 SPE cartridge with satu-
rated aqueous disodium EDTA. Blend 5 g tissue with two 20 mL portions and one 10 mL
portion of 100 mM pH 4.0 disodium EDTA-Mcllvaine buffer at high speed, centrifuge at
850 g for 5 min each time. Combine the supernatants, centrifuge at 850 g for 15 min,
filter. Add the filtrate to the SPE cartridge, wash with 20 mL water, air-dry by aspiration
for 5 min, elute with 10 mL ethyl acetate followed by 20 mL MeOH:ethyl acetate 5:95,
evaporate the eluate to dryness under reduced pressure at 30, dissolve the residue in
100 |JLL water, inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 2 juum TSK Gel Super Octyl (Tosoh)
Mobile phase: MeCN:0.05% aqueous trifluoroacetic acid 20:80
Flow rate: 0.5
Detector: MS, Finnigan MAT TSQ 7000 Triple-Stage Quadrupole, electrospray voltage 4.5
kV, gas sheath flow 483 kPa nitrogen, collision gas argon, collision offset -25 V, m/z 445
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: chlortetracycline, oxytetracycline, tetracycline
KEY WORDS
cow; SPE; kidney; liver; muscle
REFERENCE
Oka,H.; Ikai,Y; Ito,Y.; Hayakawa,J.; Harada,K.-.; Suzuki,M.; Odani,H.; Maeda,K. Improvement of chem-
ical analysis of antibiotics. XXIII. Identification of residual tetracyclines in bovine tissues by elec-
trospray high-performance liquid chromatography-tandem mass spectrometry, J.Chromatogr.B,
1997, 693, 337-344.
Doxylamine
Molecular formula: C17H22N2O
CAS Registry No.: 469-21-6, 562-10-7 (succinate)
Merck Index: 3497
LednicerNo.: 1 44
SAMPLE
Matrix: blood
Sample preparation: 3 mL Plasma + 10 mL 300 mM NaOH + 5 mL dichloromethane,
agitate in a rocking shaker at a slow speed for 15 min, centrifuge, remove the organic
layer, repeat the extraction. Combine the organic layers and evaporate them to dryness
under a stream of nitrogen at 40, reconstitute the residue in 200 |JLL IS solution, vortex,
centrifuge, inject an 80 |xL aliquot. (Prepare the IS solution as follows. 20 mg Dextro-
amphetamine sulfate + 10 mL 300 mM NaOH + 20 mL dichloromethane, shake for 5
min, repeat the extraction. Combine the organic layers and make up to 50 mL with dich-
loromethane, dilute a 5 mL aliquot to 50 mL with dichloromethane, use this solution.)
HPLC VARIABLES
Column: 300 X 3.9 10 jim jxPorasil
Mobile phase: Chloroform:MeCN:buffer 80:10:10 (Buffer was MeOH:ammonium hydroxide:
ammonium chloride 57:2:1.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Internal standard: dextroamphetamine (12.1)
KEY WORDS
REFERENCE
Kohlhof,K.J.; Stump,D.; Zizzamia,J.A. Analysis of doxylamine in plasma by high-performance liquid
chromatography, J.Pharm.ScL, 1983, 72, 961-962.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform:
isopropanohn-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800
g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum
at 45, reconstitute the residue in 100 JLJLL mobile phase, centrifuge at 2800 g for 5 min,
inject a 50 |ULL aliquot of the supernatant. (Buffer was saturated ammonium chloride
solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)
HPLC VARIABLES
Column: 300 X 3.9 4 jim NovaPack C18
Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 ad-
justed to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session
wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.)
Flow rate: 0.8
Detector: UV 261
CHROMATOGRAM
KEYWORDS
whole blood; plasma; interferences may occurcompounds(all of which are extracted) elute
in this order tenoxicam; iproniazid; methocarbamol; methotrexate; caffeine; nialamide;
colchicine; cytarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfin-
pyrazole; flumazenil; sulpride; morphine; atenolol; toloxatone; terbutaline; albuterol;
phenobarbital; ranitidine; tiapride; phenol; chlormezanone; aspirin; metformin; ritodrine;
codeine; sultopride; amisulpride; naltrexone; lisinopril; benzocaine; nizatidine; nalor-
phine; mephenesin; naloxone; sotalol; carteolol; procainamide; carbamazepine; bromaze-
pam; nalbuphine; nadolol; procarbazine; dihydralazine; omeprazole; strychnine; acebuto-
lol; glutethimide; chlorpropamide; glipizide; triazolam; prazosin; flunitrazepam;
clonazepam; metoclopramide; melphalan; estazolam; tolbutamide; ephedrine; clonidine;
pindolol; clobazam; minoxidil; disopyramide; nitrazepam; dextromethorphan; tofisopam;
zopiclone; debrisoquine; sulindac; alprazolam; cycloguanil; lorazepam; methaqualone; ke-
tamine; piroxicam; metoprolol; nifedipine; quinine; mephentermine; prilocaine; pentazo-
cine; oxazepam; tiaprofenic acid; quinidine; celiprolol; ajmaline; yohimbine; lidocaine;
secobarbital; viloxazine; mepivacaine; meperidine; doxylamine; labetalol; temazepam;
amodiaquine; benperidol; droperidol; hydroxychloroquine; zolpidem; ketoprofen; almino-
profen; cicletanine; moclobemide; chloroquine; cocaine; timolol; nomifensine; ticlopidine;
acenocoumarol; vindesine; mexiletine; dipyridamole; trazodone; pipamperone; pyrimeth-
amine; benazepril; vincristine; metapramine; chlordiazepoxide; oxprenolol; warfarin; clor-
azepate; flecainide; phencyclidine; thiopental; fenfluramine; metipranolol; triprolidine; na-
proxen; buprenorphine; verapamil; buspirone; tianeptine; midazolam; bupivacaine;
carbinoxamine; loprazolam; cetirizine; chlorpheniramine; moperone; cibenzoline; medifox-
amine; astemizole; vinblastine; nicardipine; bisoprolol; diltiazem; glibornuride; reserpine;
aconitine; nitrendipine; diazepam; mianserin; ramipril; haloperidol; tetracaine; alprenolol;
aceprometazine; glibenclamide; chlorophenacinone; doxepin; nimodipine; diphenhydra-
mine; cyclizine; histapyrrodine; phenylbutazone; demexiptiline; clozapine; proguanil; tri-
fluperidol; medazepam; cyamemazine; bumadizone; suriclone; propranolol; acepromazine;
dothiepin; dextromor amide; fenoprofen; dextropropoxyphene; loxapine; betaxolol;
propafenone; promethazine; thioproperazine; methadone; amoxapine; quinupramine; op-
ipramol; cyproheptadine; brompheniramine; mefenidramine; protriptyline; flurbiprofen;
tetrazepam; zorubicin; prazepam; alimemazine; loperamide; imipramine; desipramine;
levomepromazine; hydroxyzine; niflumic acid; penbutolol; fluvoxamine; pimozide; dauno-
rubicin; indomethacin; maprotiline; tropatenine; etodolac; fluoxetine; amitriptyline; nor-
triptyline; tioclomarol; diclofenac; mefloquine; trimipramine; chlorambucil; lidoflazine;
ibuprofen; floctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpipramine;
thioridazine; fentiazac; clemastine; mefenamic acid; fluphenazine; prochlorperazine; pen-
fluridol; bepridil; terfenadine; trifluoperazine
REFERENCE
Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL,
1995, 40, 254-262.
SAMPLE
for 2 min, inject a 10 (urine) or 30 (blood) fxL aliquot. (The detector wavelength shown is
HPLCVARIABLES
1.5 mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
tion for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A,
1997, 763, 149-163.
SAMPLE
Sample preparation: Tablets. Powder tablets, weigh out amount equivalent to about 10
mg, add 75 mL mobile phase, sonicate for 20 min, dilute to 100 mL with mobile phase,
mix, filter (0.45 jxm) (discard first 10 mL of filtrate), inject a 20 |JLL aliquot of the filtrate.
Syrups, elixirs, injectables. Measure out amount equivalent to about 10 mg, add 75 mL
mobile phase, sonicate for 20 min, dilute to 100 mL with mobile phase, mix, inject a 20
IxL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 jjim ixBondapak CN
Mobile phase: MeOH:3 mM ammonium acetate 90:10
Flow rate: 1.3
Detector: UV 254
CHROMATOGRAM
Retention time: 4.9
OTHER SUBSTANCES
Also analyzed: chlorpheniramine, cyclizine, mesoridazine, pentazocine, promethazine, pro-
triptyline, pyrilamine, pyrimethamine, tripelennamine
KEYWORDS
tablets; syrups; elixirs; injections
REFERENCE
Walker,S.T. Liquid chromatographic determination of organic nitrogenous bases in dosage forms: a pro-
gress report, JAssoc.Off.Anal.Chem., 1985, 68, 539-542.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4 ODS (Hitachi)
Mobile phase: MeCN:50 mM phosphoric acid 35:65 containing 300 mM KCl and 300 mM
ammonium chloride, adjusted to pH 3.0 with NaOH
Flow rate: 0.6
Detector: UV 261
REFERENCE
Sugawara,M.; Takekuma,Y; Yamada,H.; Kobayashi,M.; Iseki,K.; Miyazaki,K. A general approach for the
prediction of the intestinal absorption of drugs: regression analysis using the physicochemical prop-
erties and drug-membrane electrostatic interactions, J.Pharm.ScL, 1998, 87, 960-966.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 125 X 4.9 Spherisorb S5W silica
Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL/L 100 mM
NaOH in MeOH, pH 6.7
Flow rate: 2
Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V
CHROMATOGRAM
Retention time: 5.0
OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albu-
terol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacy-
clonal, bamethan, benactyzine, benperidol, benzethidine, benzocaine, benzoctamine,
benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol,
brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine,
butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorpren-
aline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine,
clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine,
deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcar-
bamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, di-
methothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridam-
ole, disopyramide, dothiepin, doxapram, doxepin, droperidol, ephedrine, ergocornine,
ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine,
ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, fla-
voxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine,
hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketan-
serin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclo-
phenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyra-
mine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene,
methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxyproma-
zine, methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine,
metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine,
nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, ox-
ymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, peri-
cyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate,
phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetra-
zine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine,
phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipa-
mazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pi-
zotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen,
procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, pro-
mazine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl,
protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ran-
itidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine,
theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene,
thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, tri-
fluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimetho-
prim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE
Jane,L; McRinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs
on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electro-
chemical oxidation detection, J.Chromatogr., 1985, 323, 191-225.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 |xg/mL, inject
a 10 |xL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 \xm iuiBondapak C18
Mobile phase: MeOH:acetic acid:triethylamine:water 30:1.5:0.5:68
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
Retention time: k' 2.03
REFERENCE
Roos,R.W.; Lau-Cam,C.A. General reversed-phase high-performance liquid chromatographic method for
the separation of drugs using triethylamine as a competing base, J.Chromatogr., 1986, 370, 403-
418.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 cellulose tris(3,5-dimethylphenylcarbamate)
Mobile phase: Hexaneiisopropanol 98:2
Flow rate: 0.5
Detector: UV
CHROMATOGRAM
Retention time: k' 1.80 (of first (+) enantiomer)
KEY WORDS
chiral; a 1.27
REFERENCE
Okamoto,Y.; Aburatani,R.; Hatano,K.; Hatada,K. Optical resolution of racemic drugs by chiral HPLC on
cellulose and amylose tris(phenylcarbamate) derivatives, J.Liq.Chromatogr., 1988, 11, 2147-2163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 Supelcosil LC-ABZ
Mobile phase: MeCN:25 mM pH 6.9 potassium phosphate buffer 35:65
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 2.215, 2.376
OTHER SUBSTANCES
Also analyzed: 6-acetylmorphine, amiloride, amphetamine, benzocaine, benzoyle9gonine,
caffeine, cocaine, codeine, fluoxetine, glutethimide, hexobarbital, hypoxanthine, levor-
phanol, LSD, meperidine, mephobarbital, methadone, methylphenidate, methyprylon, N-
norcodeine, oxazepam, oxycodone, phenylpropanolamine, prilocaine, procaine, terfenadine
REFERENCE
Ascah,T.L. Improved separations of alkaloid drugs and other substances of abuse using Supelcosil LC-
ABZ column, Supelco Reporter, 1993,12(3), 18-21.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 30 X 3.2 7 |xm SI 100 ODS (not commercially available)
Column: 150 X 3.2 7 |xm SI 100 ODS (not commercially available)
Mobile phase: MeCNrbuffer 31.2:68.8 (Buffer was 6.66 g KH2PO4 and 4.8 g 85% phosphoric
acid in 1 L water, pH 2.3.)
Flow rate: 0.5-1
Detector: UV 209, 269
CHROMATOGRAM
Retention time: 1.8
Internal standard: 5-(4-methylphenyl)-5-phenylhydantoin (7.3)
OTHER SUBSTANCES
Also analyzed: aspirin, caffeine, carbamazepine, chlordiazepoxide, chlorprothixene, clona-
zepam, diazepam, ethosuximide, furosemide, haloperidol, hydrochlorothiazide, methocar-
bamol, methotrimeprazine, nicotine, oxazepam, procaine, promazine, propafenone, pro-
pranolol, salicylamide, temazepam, tetracaine, thiopental, triamterene, verapamil,
zolpidem, zopiclone
REFERENCE
Below,E.; Burrmann,M. Application of HPLC equipment with rapid scan detection to the identification
of drugs in toxicological analysis, J.Liq.Chromatogr., 1994, 17, 4131-4144.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Chirex 3020 (Phenomenex)
Mobile phase: Hexane:l,2-dichloroethane:EtOH/trifluoroacetic acid 60:35:5 (EtOH/trifluo-
roacetic acid was premixed 20:1.)
Flow rate: 0.7-1
Next Page
Detector: UV 262
KEYWORDS
chiral; a = 1.07 for enantiomers
REFERENCE
Cleveland,1!. Pirkle-concept chiral stationary phases for the HPLC separation of pharmaceutical race-
mates, J.Liq.Chromatogr., 1995, 18, 649-671.
SAMPLE
Matrix: urine
Sample preparation: Adjust pH of 2 mL urine to 6.0, extract three times with 10 mL
aliquots of dichloromethane, evaporate to dryness under reduced pressure, reconstitute
in MeOH, inject a 100 |xL aliquot.
HPLC VARIABLES
Guard column: 50 x 4.6 50 ^m Supelco LC-CN
Column: 250 X 4.6 5 jxm Supelco LC-CN
Mobile phase: Gradient. A was MeOH: 10 mM KH2PO4 5:95 containing 20 mM triethylam-
ine, pH 7.3. B was MeOH: 10 mM KH2PO4 95:5 containing 20 mM triethylamine, pH 7.3.
A:B 100:0 for 10 min, to 0:100 over 2 min, maintain at 0:100 for 18 min, return to initial
conditions over 2 min, re-equilibrate for 8 min.
Flow rate: 1
Detector: UV 254 or radioactivity
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: metabolites, degradation products
KEY WORDS
monkey
REFERENCE
Holder,C.L.; Korfmacher,W.A.; Rushing,L.G.; Thompson,H.C.,Jr.; Slikker,W.,Jr.; Gosnell,A.B. Formation
of artifactual metabolites of doxylamine following acid hydrolysis, J.Chromatogr., 1987, 419, 113-
122.
Previous Page
Drofenine
Merck Index: 3501
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 4 X 4 5 fxm LiChrospherlOORP-18
Column: 250 X 4 5 jxm Spherisorb ODS 2
Mobile phase: MeCNrbuffer 65:35 (Buffer was 20 mM sodium acetate containing 0.59% di-
n-butylamine, adjusted to pH 4.5 with acetic acid.)
Flow rate: 1.5
Detector: UV 260
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: cyclohexanephenylacetic acid
REFERENCE
Yang,H.; Thyrion,F.C. Determination of six pharmaceuticals and their degradation products in reversed-phase
high performance liquid chromatography by using amine additives, J.Liq.Chromatogr.Rel.TkchnoL, 1998,
21, 1347-1357.
Droloxifene
Merck Index: 3502
SAMPLE
Matrix: blood
Sample preparation: Mix serum with an equal volume of MeCN, mix, centrifuge, inject a
50 |xL aliquot of the supernatant.
HPLCVARIABLES
Column: 150 X 4.6 3 |xm ODS Hypersil
Mobile phase: MeCN:water 66:34 containing 3 mM acetic acid and 2 mM diethylamine
Flow rate: 1
Detector: F ex 260 em 360 following post-column reaction. The column effluent flowed
through a 10 m X 0.3 mm ID knitted PTFE coil irradiated with UV light at 254 nm to
the detector.
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: acetaminophen, atenolol, captopril, clodronate, dexamethasone, dextro-
propoxyphene, diazepam, doxycycline, econazole, enoxaparin, felodipine, flunitrazepam,
furosemide, glibenclamide, indomethacin, insulin, isosorbide mononitrate, megestrol ac-
etate, metoclopramide, mianserin, morphine, nitroglycerin, oxazepam, perphenazine,
phenytoin, pivmecillinam, prochlorperazine, promethazine, ranitidine, tamoxifen
KEYWORDS
post-column reaction; post-column photochemical derivatization; serum; pharmacokinetics
REFERENCE
Lien,E.A.; Anker,G.; Lonning,P.E.; Ueland,P.M. Determination of droloxifene and two metabolites in
serum by high-performance liquid chromatography, Ther.Drug Monit., 1995,17, 259-265.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond Elut benzenesulfonic acid (SCX) SPE car-
tridge with 1 mL MeOH:30% ammonium hydroxide 96.5:3.5, 1 mL MeOH, and 1 mL 1%
acetic acid at 1 mL/min. Add 500 |xL 1% acetic acid, 200 |xL 2 ng/mL IS in mobile phase,
and 200 fiL plasma or serum sequentially to the SPE cartridge, wash with 1 mL water,
wash with 1 mL MeOH, elute with 1 mL MeOH:30% ammonium hydroxide 96.5:3.5.
Evaporate the eluate to dryness under a stream of nitrogen at 50 or reduced pressure
at 75, reconstitute with 100 |xL mobile phase, vortex, centrifuge at 1000 g for 30 s, inject
an 80 |xL aliquot.
HPLC VARIABLES
Column: 100 x 4.6 3 jutm C18 (Rainin)
Mobile phase: MeCN:50 mM sodium phosphate buffer 45:55, adjusted to pH 3.5 with phos-
phoric acid
Flow rate: 2
Detector: F ex 260 em 375 following post-column photolysis. The column effluent passed
through a 3.1 m X 0.25 mm i.d. PTFE coil, where it was irradiated by a Beam Boost 254
nm photochemical reaction lamp, to the detector.
CHROMATOGRAM
Retention time: 3.1
Internal standard: (E)-a-[p-[2-(diethylamino)ethoxy]phenyl]-a'-ethyl-3-stilbenol (K 21.089
E, Klinge Pharma, Munich) (4.8)
Limit of detection: 10 pg/mL
Limit of quantitation: 25 pg/mL
OTHER SUBSTANCES
KEYWORDS
rat; monkey; human; plasma; serum; post-column reaction; pharmacokinetics; SPE; post-
column photochemical derivatization
REFERENCE
Tess,D.A.; Cole,R.O.; Tbler,S.M. Sensitive method for the quantitation of droloxifene in plasma and se-
rum by high-performance liquid chromatography employing fluorimetric detection, J.Chromatogr.B,
1995, 674, 253-260.
Dronabinol
Molecular formula: C2IH30O2
Merck Index: 9349
LednicerNo.: 1 394,4 209
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 200 mg Bond-Elut C18 SPE cartridge with 2 mL
MeOH and 2 mL water. 1 mL Plasma + 2 mL 8 M urea, vortex for 5 s, add 2 mL MeOH,
vortex for 5 s, add to SPE cartridge, pass through within 2-3 min, rinse sample tube with
2 mL water:MeOH:8 M urea 1:2:2 and add rinse to SPE cartridge, wash with 2 mL MeOH:
water 1:1, wash with 1 mL 200 mM HCl, wash with 1 mL MeOH:water 1:1, wash with
1 mL 10 mM NaOH, wash with 3 mL MeOH:water 1:1, centrifuge for 10 min to re-
move remaining fluid, elute with 0.5 mL diethyl ether. Evaporate eluate to dryness
at 40, dissolve residue in 100 fxL MeOH, vortex for 5 s, inject a 10 |xL aliquot. (Purify
8 M urea by passing 50 mL through an activated 3 mL 500 mg Bond-Elut C18 SPE
cartridge.)
HPLC VARIABLES
Guard column: 10 X 2.1 40 fim Chromsep pellicular C18 (Chrompack)
Column: 100 X 4.6 3 uin Chromsep C18 (Chrompack)
Mobile phase: THF:MeOH:5 mM pH 7.0 citrate buffer 7.5:68:24.5
Flow rate: 1
Detector: E, Metrohm glassy carbon working electrode 3 mm diameter (polished daily for
1 min with 0.3 |xm aluminum oxide powder), Ag/AgCl (3 M KCl) reference electrode,
stainless steel auxiliary electrode, polarize working electrode for 20 min at + 960 mV then
decrease to working potential of + 760 mV.
CHROMATOGRAM
Retention time: 14
KEYWORDS
plasma; SPE
REFERENCE
Zweipfenning,P.G.M.; Lisman,J.A.; van Haren,A.Y.N.; Dijkstra,G.R.; Holthuis,J.J.M. Determination of
delta 9-tetrahydrocannabinol in plasma using solid-phase extraction and high-performance liquid
chromatography with electrochemical detection, J.Chromatogr., 1988, 456, 83-91.
SAMPLE
Matrix: blood, saliva
Sample preparation: 1 mL Plasma, serum, or saliva + 50 |xL 1.05 |xg/mL 4-dodecylresor-
cinol in MeOH + 2 mL MeOH + 200 |xL 70% perchloric acid, vortex for 30 s, centrifuge
at 2000 g for 2 min. Remove the supernatant and add it to 1 mL saturated NaCl and 150
IxL toluene, vortex for 30 s, centrifuge at 2000 g for 2 min, inject an aliquot of the organic
phase.
HPLC VARIABLES
Column: 150 X 3.9 jxBondapak C18
Mobile phase: MeOHrwater 22.5:77.5 containing 100 mM sodium monochloroacetate and
25 mM monochloroacetic acid (After each 12 analyses wash the column with MeOH at 3
mL/min for 15 min then re-equilibrate with mobile phase at 3 mL/min for 15 min.)
Flow rate: 3
Detector: E, BAS Model LC-4B, glassy carbon working electrode at + 0.90 V, Ag/AgCl ref-
erence electrode
CHROMATOGRAM
Internal standard: 4-dodecylresorcinol (7.05)
OTHER SUBSTANCES
Simultaneous: metabolites, impurities
KEYWORDS
plasma; serum
REFERENCE
Thompson,L.K.; Cone,E.J. Determination of delta 9-tetrahydrocannabinol in human blood and saliva by
high-performance liquid chromatography with amperometric detection, J.Chromatogr., 1987, 421,
91-97.
SAMPLE
Sample preparation: Plasma. 1-2 mL Plasma + 10-20 |xL (?) 55 (xg/mL cannabinol + 5 mL
MeOH, mix vigorously at 0, centrifuge at 0 at 1000 g for 5 min, suspend the white gel-
like precipitate in 3 mL ice-cold MeOH, centrifuge. Combine the supernatants and evap-
orate them to 2 mL under a stream of air at 65, cool in ice-water for 10-20 min, centrifuge
at 0 at 1000 g for 5 min. Combine the supernatants and add 2 mL dichloromethane,
vortex, add 5 mL hexanes, vortex, centrifuge at 500 g for 5 min, remove the upper organic
layer, extract the lower MeOH/water layer with 2 mL dichloromethane and 5 mL hexanes.
Combine the upper organic layers and evaporate them to dryness under a stream of air
at 65, reconstitute the residue in 1 mL hexanes, evaporate to dryness under reduced
pressure, reconstitute the residue in 200 |xL hexanes, evaporate to dryness under reduced
pressure, reconstitute with 20 |xL MeOH (warm slightly if necessary), add 100 jxL acetone,
mix vigorously, cool in ice-water for 10 min, centrifuge at 0 at 1000 g for 5 min. Remove
the supernatant and add it to 75 |xL EtOH, dry under reduced pressure, reconstitute with
100 uL MeOH, inject a 75 u,L aliquot. Tissue. Homogenize (tissue grinder mortar) 500
mg mouse brain with 3 mL ice-cold MeOH and 10-20 |xL 55 jxg/mL cannabinol at 0,
centrifuge at 0 at 1000 g for 5 min, suspend the pellet in 3 mL ice cold MeOH, centrifuge
at 0 at 1000 g for 5 min. Combine the supernatants and evaporate them to 1.5 mL under
a stream of air at 65, cool in ice-water for 10-20 min, centrifuge at 0 at 1000 g for 5
min, suspend the pellet in 500 |xL ice-cold MeOH:water 80:20, centrifuge. Combine the
supernatants and add 2 mL dichloromethane, vortex, add 1 mL water, vortex, add 5 mL
hexanes, vortex, centrifuge at 500 g for 5 min, remove the upper organic layer, extract
the lower MeOH/water layer with 2 mL dichloromethane and 5 mL hexanes. Combine
the upper organic layers and evaporate them to dryness under a stream of air at 65,
reconstitute the residue in 1 mL hexanes, evaporate to dryness under reduced pressure,
reconstitute the residue in 200 u,L hexanes, evaporate to dryness under reduced pressure,
reconstitute with 20 JAL MeOH (warm slightly if necessary), add 100 jiL acetone, mix
vigorously, cool in ice-water for 10 min, centrifuge at 0 at 1000 g for 5 min. Remove the
supernatant and add it to 75 u,L EtOH, dry under reduced pressure, reconstitute with
100 |xL MeOH, inject a 75 |xL aliquot.
HPLC VARIABLES
Guard column: 50 X 3.9 37-50 |xm ixBondapak C18/Corasil
Column: 300 X 3.9 ixBondapak C18
Mobile phase: MeOH:water 85:15
Flow rate: 1.5
Detector: UV 490 following post-column reaction. The column effluent mixed with the re-
agent pumped at 0.5 mL/min and the mixture flowed through a 4 m X 0.8 mm PTFE coil
to the detector. (Prepare the reagent by dissolving 1 g Fast Blue Salt B in 160 mL water,
adding 200 mL MeOH, and adding 40 mL 10% sodium nitrite in water. Stir for 30 min,
filter (Whatman GF/C), filter (0.45 fxm Gelman Metricel GA-6), discard after 5 h. Protect
from light. At the end of each day flush the post-column reaction system with meOH:
water 50:50, acetone, and MeOH:water 50:50.)
CHROMATOGRAM
Retention time: 7
Internal standard: cannabinol (6)
Limit of detection: 50 ng
OTHER SUBSTANCES
Extracted: 11-hydroxytetrahydrocannabinol
KEYWORDS
post-column reaction; treat glassware with Dri-Film SC-87 (Pierce); mouse; plasma; brain
REFERENCE
Borys,H.K.; Karler,R. Post-column derivatization procedure for the colorimetric analysis of tissue can-
nabinoids separated by high-performance liquid chromatography, J.Chromatogr., 1981,205,303-323.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 1 mmole compound, 4 mmole imidazole, and 2 mmole t-
butyldimethylsilyl chloride in 500 uL DMF, heat on a steam bath for 1 h, add 2 mL 10%
NaOH, extract 3 times with 2 mL portions of petroleum ether bp (30-60). Combine the
extracts and dry them over anhydrous magnesium sulfate, evaporate to dryness.
HPLCVARIABLES
Column: 305 mm X 6.4 mm (o.d.) 10 am ixBondapak C18
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: cannabidiol, cannabinol
Interfering: delta8-tetrahydrocannabinol
KEYWORDS
derivatization
REFERENCE
Knaus,E.E.; Coutts,R.T.; Kazakoff,C.W. The separation, identification, and quantitation of cannabinoids
and their t-butyldimethylsilyl, trimethylsilylacetate, and diethylphosphate derivatives using
high-pressure liquid chromatography, gas-liquid chromatography, and mass spectrometry,
J.Chromatogr.ScL, 1976, 14, 525-530.
SAMPLE
Sample preparation: Dilute with EtOH to a dronabinol concentration of 5-10 mg/mL, add
100 |xL of this solution to 600 |xL 150 |xg/mL tetraphenylethylene in EtOH, inject a 10
IxL aliquot of this solution. (Dilute samples in sesame oil with n-butanol.)
HPLC VARIABLES
Column: 250 X 4.6 10 p,m LiChrosorb RP-8
Mobile phase: MeOH.water 78:22
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 20
Internal standard: tetraphenylethylene (24)
OTHER SUBSTANCES
Simultaneous: impurities, degradation products
REFERENCE
Flora,K.R; Cradock,J.C; Davignon,J.P. Determination of delta 9-tetrahydrocannabinol in pharmaceu-
tical vehicles by high-performance liquid chromatography, J.Chromatogr., 1981, 206, 117-123.
SAMPLE
Sample preparation: Cut capsules with a scalpel, dissolve contents in absolute EtOH so
that the concentration of dronabinol is about 150 |xg/mL, add dodecanophenone, inject a
30 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere ODS + 150 X 4.6 3 |xm Spherisorb ODS II
Mobile phase: MeCN: 1% acetic acid 85:15
Flow rate: 0.5
Detector: UV 220 and UV 280
CHROMATOGRAM
Retention time: 35
Internal standard: dodecanophenone
OTHER SUBSTANCES
Simultaneous: impurities
KEYWORDS
capsules
REFERENCE
Ray,G.; Crook,M.; West,N.; Kwoka,M.; Rehagen,G.; Cox,J.; Murrill,E.; Flora,K. Comparison of the anal-
ysis of delta 9-tetrahydrocannabinol capsules by high-performance liquid chromatography and cap-
illary gas chromatography, J.Chromatogr., 1984, 317, 455-462.
SAMPLE
Matrix: plants
Sample preparation: 100 mg Leaves or 50 mg resin or oil + 1 mL MeOHxhloroform 90:
10, sonicate for 15 min, , filter. Dilute 100 (JLL filtrate with 300 |xL MeOH, inject a 1 jxL
aliquot.
HPLCVARIABLES
Guard column: 20 X 2 3 jxm Spherisorb ODS-I
Column: 200 X 2 3 fxm Spherisorb ODS-I
Mobile phase: Gradient. A was 8.64 g/L 85% orthophosphoric acid in water. B was MeCN.
A:B from 53:47 to 40:60 over 38 min, to 30:70 over 10 min, to 53:47 over 2 min, re-
equilibrate for 10 min. (Wash column with MeCN after use.)
Flow rate: 0.2
Injection volume: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 40
Limit of quantitation: 25 ng
OTHER SUBSTANCES
Simultaneous: related compounds
KEY WORDS
leaves; resin; oil
REFERENCE
Lehmann,T.; Brenneisen,R. High performance liquid chromatographic profiling of cannabis products,
J.Liq.Chromatogr., 1995, 18, 689-700.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 305 mm X 6.4 mm (o.d.) 10 |xm uBondapak C18
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: cannabidiol, cannabinol, delta8-tetrahydrocannabinol
REFERENCE
Knaus,E.E.; Coutts,R.T.; Kazakoff,C.W. The separation, identification, and quantitation of cannabinoids
and their t-butyldimethylsilyl, trimethylsilylacetate, and diethylphosphate derivatives using high-
pressure liquid chromatography, gas-liquid chromatography, and mass spectrometry,
J.Chromatogr.ScL, 1976,14, 525-530.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 x 4.6 Zorbax RX
Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL tri-
ethylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL
triethylamine in 200 mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over
30 min, maintain at 0:100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline,
amitriptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobar-
bital, aspirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotro-
pine, benzphetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenor-
phine, buspirone, butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal,
chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphe-
nesin, chlorpheniramine, chlorpromazine, chlorpropamide, chlortetracycline, cimetidine,
cinchonidine, cinchonine, clenbuterol, clonazepam, clonixin, clorazepate, cocaine, codeine,
colchicine, cortisone, coumarin, cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide,
cymarin, danazol, danthron, dapsone, debrisoquine, desipramine, dexamethasone, dex-
tromethorphan, dextropropoxyphene, diamorphine, diazepam, diclofenac, diethylpropion,
diethylstilbestrol, diflunisal, digitoxin, digoxin, diltiazem, diphenhydramine, dipfyenoxyl-
ate, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, ephedrine, epinephrine,
epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide, etonitazene, etorphine,
eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenproporex, fentanyl, flu-
bendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone, fluphenazine,
furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclamide,
guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone, hy-
dromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indo-
methacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin,
ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam,
loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic
acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, me-
phobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine,
methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsux-
imide, methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylpredni-
solone, methyltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, na-
lorphine, naloxone, naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine,
niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine, nortriptyline, noscapine,
nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, oxytetracycline, papav-
erine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phenacetin, phenazo-
cine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, pheniramine, phen-
obarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenylephrine,
phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid, pro-
gesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, py-
rilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recin-
namine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid,
scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadime-
thoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide,
sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine,
tetracycline, tetramisole, thebaine, theobromine, theophylline, thiabendazole, thiamine,
thiamylal, thiobarbituric acid, thioridazine, thiosalicylic acid, thiothixene, thymol, tola-
zamide, tolazoline, tobutamide, tolmetin, tranylcypromine, triamcinolone, tribenzylamine,
trichloromethiazide, trifluoperazine, trihexyphenidyl, trimethoprim, tripelennamine, tri-
prolidine, tropacocaine, t3nramine, verapamil, vincamine, warfarin, yohimbine,
zoxazolamine
REFERENCE
Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.AnaLToxicoL,
1994, 18, 233-242.
SAMPLE
Matrix: tissue
Sample preparation: Mix 50 mg tissue with 2.5 mL 280 ng/mL 4-dodecylresorcinol in
MeOH using a Teflon pestle, sonicate the homogenates using a Soniprep 150 ultrasonic
disintegrator (MSE Scientific Instruments, Sussex), fitted with a probe assembly with a
3.0 mm diameter tip operated at a 23 jim amplitude. Maintain the homogenates in a ice-
bath and subject to three 2 min sonication intervals. Centrifuge the homogenate at 15000
g at 4 for 30 min, evaporate the supernatant to dryness and reconstitute the residue in
5 mL hexanerethyl acetate 70:30, wash with three 2.5 mL aliquots of 50 mM sulfuric acid,
centrifuge at 3000 g at 4. Evaporate a 4.0 mL aliquot of the organic phase to dryness
using a centrifugal evaporator. Reconstitute the residue in 350 |xL mobile phase:MeOH
25:10, inject a 50 jxL aliquot.
HPLCVARIABLES
Guard column: Newguard RP-18 (Brownlee, Santa Clara, CA)
Column: 300 X 4 10|xm ixBondapak C18
Mobile phase: MeOH:MeCN:10 mM sulfuric acid 21:24:55
Flow rate: 3
Detector: E, Bioanalytical Systems LC-4B dual-electrode, TL-5A glassy carbon working
electrode (1.2 V), Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 5.5
Internal standard: 4-dodecylresorcinol (8.5)
Limit of detection: 1.5 ng
KEYWORDS
brain; rat; pharmacokinetics
REFERENCE
Nyoni,E.C; Sitaram,B.R.; Taylor,D.A. Determination of delta9-tetrahydrocannabinol levels in brain tis-
sue using high-performance liquid chromatography with electrochemical detection, J.Chromatogr.B,
1996, 679, 79-84.
Droperidol
Molecular formula: C22H22FN3O2
Merck Index: 3505
Led nicer No.: 1 308
SAMPLE
Matrix: blood
Sample preparation: Basify plasma containing flurazepam with 500 mM KOH, extract
with diethyl etherrheptane 90:10. Remove the organic layer and evaporate it to dryness,
reconstitute the residue in mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 mm long 5 jxm Spherisorb CN
Mobile phase: MeCN:EtOH:50 mM pH 2.5 phosphate buffer 20:15:65
Detector: UV 200
CHROMATOGRAM
Internal standard: flurazepam (4.86)
KEYWORDS
plasma
REFERENCE
James,J.; Lowe,D.; Karnes,H.T. Determination of histamine from plasma using derivatization with
naphthalene-2,3-dicarboxaldehyde and HPLC with fluorescence detection, Pharm.Res., 1992, 9, S21.
SAMPLE
Matrix: blood
Sample preparation: Condition a Supelclean LC-18 SPE cartridge (Supelco) with two 2
mL aliquots of MeOH, 2 mL water, and 2 mL 50 mM pH 6.9 ammonium acetate, add
1 mL plasma, wash with three 1 mL portions of 150 mM NaCl, wash with 50 |xL
MeOH, elute with 2 mL MeCN. Evaporate the eluate to dryness under a stream of nitro-
gen at 60, reconstitute the residue in 100 |xL MeCN, filter (0.45 |xm), inject a 10 |xL
aliquot.
HPLC VARIABLES
Guard column: ixBondapak C18
Column: 150 X 3.9 5 jjim Novapak C18
Mobile phase: MeCN: 10 mM pH 6.7 ammonium acetate 45:55
Flow rate: 1
Detector: UV 250
CHROMATOGRAM
Retention time: 4.5
Internal standard: nitrazepam (2.5)
OTHER SUBSTANCES
Extracted: flunitrazepam
Noninterfering: atropine, phenoperidine, pancuronium
KEYWORDS
REFERENCE
Guichard,J.; Panteix,G.; Dubost,J.; Baltassat,P.; Roche,C. Simultaneous high-performance liquid chro-
matographic assay of droperidol andflunitrazepamin human plasma. Application to haemodilution
blood samples collected during clinical anaesthesia, J.Chromatogr., 1993, 612, 269-275.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL 200 ng/mL IS in MeOH + 1 mL 50 mM pH
10 borate buffer, vortex briefly, add to an Extrelut 3 SPE cartridge, let stand for 5 min,
elute with 15 mL hexaneidichloromethane 50:50. Add the eluate to 3 mL 50 mM sulfuric
acid, mix for 10 min, centrifuge at 3000 g for 10 min. Remove the aqueous layer and add
it to 6 mL hexane:dichloromethane 50:50, wash for 5 min, centrifuge. Make the aqueous
layer basic with 150 |xL 28% ammonia, extract twice with 3 mL hexane:dichloromethane
50:50. Combine the organic layers and evaporate them to dryness under a stream of
nitrogen at 60, reconstitute the residue in 100 |iL mobile phase, inject a 20 |xL aliquot.
HPLCVARIABLES
Guard column: 30 X 4.6 5 |xm Spherisorb cyano
Column: 250 X 4.6 5 jxm Ultrasphere cyano
Mobile phase: MeCN:buffer 60:40 (Buffer was 50 mM KH2PO4 adjusted to pH 6.5 with
28% ammonia.)
Flow rate: 1
Detector: E, 5100 A Coulochem, 5020 guard cell 1.00 V, 5011 analytical cell, detector 1 0.55
V, detector 2 0.80 V, output of detector 2 is monitored
CHROMATOGRAM
Internal standard: methylrisperidone (R68808) (14.3)
OTHER SUBSTANCES
Extracted: chlorpromazine, clomipramine, cyamemazine, desipramine, flunitrazepam, hal-
operidol, imipramine, pipamperone, risperidone, trihexyphenidyl
Noninterfering: alprazolam, bromazepam, carbamazepine, chlorazepate, diazepam, di-
phenylhydantoin, estazolam, ethylbenzatropine, oxazepam, phenobarbital, triazolam, val-
proic acid
KEYWORDS
plasma; SPE
REFERENCE
Le Moing,J.P.; Edouard,S.; Levron,J.C. Determination of risperidone and 9-hydroxyrisperidone in hu-
man plasma by high-performance liquid chromatography with electrochemical detection,
J.Chromatogr., 1993, 614, 333-339.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform:
isopropanol:n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800
g for 10 min. Remove the lower organic layer and evaporate it to dryness under vacuum
at 45, reconstitute the residue in 100 jxL mobile phase, centrifuge at 2800 g for 5 min,
inject a 50 (xL aliquot of the supernatant. (Buffer was saturated ammonium chloride
solution 25% diluted with water, adjusted to pH 9.5 with 25% ammonia solution.)
HPLC VARIABLES
Column: 300 X 3.9 4 jxm NovaPack C18
Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 ad-
justed to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session
wash the column with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.)
Flow rate: 0.8
Detector: UV 246
CHROMATOGRAM
KEYWORDS
whole blood; plasma; interferences may occurcompounds(all of which are extracted) elute
in this order tenoxicam; iproniazid; methocarbamol; methotrexate; caffeine; nialamide;
colchicine; cytarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfin-
pyrazole; flumazenil; sulpride; morphine; atenolol; toloxatone; terbutaline; albuterol;
phenobarbital; ranitidine; tiapride; phenol; chlormezanone; aspirin; metformin; ritodrine;
codeine; sultopride; amisulpride; naltrexone; lisinopril; benzocaine; nizatidine; nalor-
phine; mephenesin; naloxone; sotalol; carteolol; procainamide; carbamazepine; bromaze-
pam; nalbuphine; nadolol; procarbazine; dihydralazine; omeprazole; strychnine; acebuto-
lol; glutethimide; chlorpropamide; glipizide; triazolam; prazosin; flunitrazepam;
clonazepam; metoclopramide; melphalan; estazolam; tolbutamide; ephedrine; clonidine;
pindolol; clobazam; minoxidil; disopyramide; nitrazepam; dextromethorphan; tofisopam;
zopiclone; debrisoquine; sulindac; alprazolam; cycloguanil; lorazepam; methaqualone; ke-
tamine; piroxicam; metoprolol; nifedipine; quinine; mephentermine; prilocaine; pentazo-
cine; oxazepam; tiaprofenic acid; quinidine; celiprolol; ajmaline; yohimbine; lidocaine;
secobarbital; viloxazine; mepivacaine; meperidine; doxylamine; labetalol; temazepam;
amodiaquine; benperidol; droperidol; hydroxychloroquine; zolpidem; ketoprofen; almino-
profen; cicletanine; moclobemide; chloroquine; cocaine; timolol; nomifensine; ticlopidine;
acenocoumarol; vindesine; mexiletine; dipyridamole; trazodone; pipamperone; pyrimeth-
amine; benazepril; vincristine; metapramine; chlordiazepoxide; oxprenolol; warfarin; clor-
azepate; flecainide; phencyclidine; thiopental; fenfluramine; metipranolol; triprolidine; na-
proxen; buprenorphine; verapamil; buspirone; tianeptine; midazolam; bupivacaine;
carbinoxamine; loprazolam; cetirizine; chlorpheniramine; moperone; cibenzoline; medifox-
amine; astemizole; vinblastine; nicardipine; bisoprolol; diltiazem; glibornuride; reserpine;
aconitine; nitrendipine; diazepam; mianserin; ramipril; haloperidol; tetracaine; alprenolol;
aceprometazine; glibenclamide; chlorophenacinone; doxepin; nimodipine; diphenhydra-
mine; cyclizine; histapyrrodine; phenylbutazone; demexiptiline; clozapine; proguanil; tri-
fluperidol; medazepam; cyamemazine; bumadizone; suriclone; propranolol; acepromazine;
dothiepin; dextromor amide; fenoprofen; dextropropoxyphene; loxapine; betaxolol;
propafenone; promethazine; thioproperazine; methadone; amoxapine; quinupramine; op-
ipramol; cyproheptadine; brompheniramine; mefenidramine; protriptyline; flurbiprofen;
tetrazepam; zorubicin; prazepam; alimemazine; loperamide; imipramine; desipramine;
levomepromazine; hydroxyzine; niflumic acid; penbutolol; fluvoxamine; pimozide; dauno-
rubicin; indomethacin; maprotiline; tropatenine; etodolac; fluoxetine; amitriptyline; nor-
triptyline; tioclomarol; diclofenac; mefioquine; trimipramine; chlorambucil; lidoflazine;
ibuprofen; floctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpipramine;
thioridazine; fentiazac; clemastine; mefenamic acid; fluphenazine; prochlorperazine; pen-
fluridol; bepridil; terfenadine; trifluoperazine
REFERENCE
Tracqui,A-; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL,
1995, 40, 254-262.
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL Bond Elut Certify SPE cartridge with 2 mL MeOH
and 2 mL 100 mM pH 6.0 phosphate buffer, do not allow to dry. 1 mL Blood + 6 mL 100
mM pH 6.0 phosphate buffer, vortex, sonicate, centrifuge, add the supernatant to the SPE
cartridge, wash with water, wash with 1 mM pH 3.3 acetic acid, dry by suction, wash
with 2 mL acetone:chloroform 50:50, elute with 3 mL ethyl acetate:ammonia 98:2. Evap-
orate the eluate under a stream of nitrogen at 40, reconstitute the residue in 50 |xL
MeOH, inject a 10 jxL aliquot.
HPLCVARIABLES
Column: 150 X 3.9 4 jjim Nova-Pack C18
Mobile phase: MeOH:50 mM ammonium acetate 75:25 (Mix column effluent with 50 mM
ammonium acetate pumped at 0.5 mL/min.)
Flow rate: 0.6
Detector: MS, Finnigan MAT TSQ 700 tandem quadrupole, MAT TSP-2 interface, ther-
mospray, selective reaction monitoring m/z 380-165, collision offset -25 V, repeller 100 V,
vaporizer 130, source 200, filament on 200 |xA, argon 2.5 mTorr, multiplier 1500 V,
dynode 15 kV, scan time 1.20 s, MSMSC factor 10
CHROMATOGRAM
Limit of detection: 10 pg
OTHER SUBSTANCES
Extracted: benperidol, dextromoramide, haloperidol, methadone, penfluridol, pimozide, pi-
pamperidone, propoxyphene (dextropropoxyphene)
KEYWORDS
SPE; LC/MS
REFERENCE
Verweij,A.M.; Hordijk,M.L.; Lipman,P.J. Quantitative liquid chromatographic thermospray-tandem
mass spectrometric analysis of some analgesics and tranquilizers of the methadone, butyrophenone,
or diphenylbutylpiperidine groups in whole blood, J.Anal.Toxicol., 1995,19, 65-68.
SAMPLE
Matrix: blood, CSF
Sample preparation: 200 \LL Serum, plasma, or CSF + 300 |xL reagent. Flush column A
to waste with 500 u.L 500 mM ammonium sulfate, inject sample onto column A, flush
column A to waste with 500 |xL 500 mM ammonium sulfate, elute the contents of column
A onto column B with mobile phase, monitor the effluent from column B. (Reagent was
8.05 M guanidine hydrochloride and 1.02 M ammonium sulfate in water.)
HPLCVARIABLES
Column: A 30 X 2.1 40 |xm preparative grade C18 (Analytichem); B 250 X 4.6 10 |xm
Partisil C8
Mobile phase: Gradient. A was 50 mM pH 4.5 KH2PO4. B was MeCN:isopropanol 80:20. A:
B 90:10 for 1 min, to 30:70 over 15 min, maintain at 30:70 for 4 min.
Flow rate: 1.5
Detector: UV 280 for 5 min then UV 254
CHROMATOGRAM
Internal standard: heptanophenone (19.2)
OTHER SUBSTANCES
Extracted: acetazolamide, ampicillin, bromazepam, caffeine, carbamazepine, chloramphen-
icol, chlorothiazide, diazepam, ethionamide, furosemide, isoniazid, methadone, penicillin
G, phenobarbital, phenytoin, prazepam, propoxyphene, pyrazinamide, rifampin, trime-
prazine, trimethoprim
KEYWORDS
plasma; serum; column-switching
REFERENCE
Seifart,H-!L; Kruger,P.B.; Parkin,D.R; van Jaarsveld,P.R; Donald,P.R. Therapeutic monitoring of anti-
tuberculosis drugs by direct in-line extraction on a high-performance liquid chromatography system,
J.Chromatogr., 1993, 619, 285-290.
SAMPLE
for 2 min, inject a 10 (urine) or 30 (blood) jxL aliquot. (The detector wavelength shown is
HPLCVARIABLES
Column: 250 X 4.6 5 jjim Symmetry C8 (Waters)
1.5 mL/min)
Detector: UV 202.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detec-
tion for the creation of a 600-compound library. Application to forensic toxicology, J ChromatognA,
1997, 763, 149-163.
SAMPLE
Sample preparation: Dilute 1:1, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Ultrasphere ODS
Mobile phase: MeOH:20 mM pH 6.8 phosphate buffer 65:35
Flow rate: 1.5
Injection volume: 5
Detector: UV 230
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Simultaneous: degradation products, methylparaben, propylparaben
KEYWORDS
stability-indicating; injections
REFERENCE
Dolezalova,M. Separation and determination of droperidol, methyl- and propylparaben and their deg-
radation products by high-performance liquid chromatography, J.Chromatogr., 1984, 286, 323-r330.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 [ig/mL solution in MeOH, inject a 20 uL aliquot.
HPLC VARIABLES
Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL/L 100 mM
NaOH in MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.4
OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albu-
terol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacy-
clonal, bamethan, benactyzine, benperidol, benzethidine, benzocaine, benzoctamine,
benzphetamine, benzquinamide, bromhexine, bromodiphenhydramine, bromperidol,
brompheniramine, brompromazine, buclizine, bufotenine, bupivacaine, buprenorphine,
butacaine, butethamate, chlorcyclizine, chlorpheniramine, chlorphenoxamine, chlorpren-
aline, chlorpromazine, chlorprothixene, cimetidine, cinchonidine, cinnarizine, clemastine,
clomipramine, clonidine, cocaine, cyclazocine, cyclizine, cyclopentamine, cyproheptadine,
deserpidine, desipramine, dextromoramide, dextropropoxyphene, dicyclomine, diethylcar-
bamazine, diethylpropion, diethylthiambutene, dihydroergotamine, dimethindene, di-
methothiazine, diphenhydramine, diphenoxylate, dipipanone, diprenorphine, dipyridam-
ole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, ephedrine, ergocornine,
ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine, ergotamine,
ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol, fentanyl, fla-
voxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydroxyzine,
hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ketan-
serin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclo-
phenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyra-
mine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene,
methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxyproma-
zine, methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine,
metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine,
nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, ox-
ymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, penthienate, peri-
cyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phenbutrazate,
phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine, phenmetra-
zine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phentolamine,
phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pindolol, pipa-
mazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piritramide, pi-
zotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine, proadifen,
procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolintane, prom-
azine, promethazine, pronethalol, properidine, propiomazine, propranolol, prothipendyl,
protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, quinine, ran-
itidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldiamine,
theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothixene,
thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, tri-
fluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimetho-
prim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE
Jane,L; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs
on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electro-
chemical oxidation detection, J.Chromatogr., 1985, 323, 191-225.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B)
Mobile phase: MeCN:0.025% phosphoric acid:buffer 25:10:5 (A) or 60:25:15 (B) (Buffer was
9 mL concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust pH
to 3.4 with dilute phosphoric acid, make up to 1 L.)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine,
albuterol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atro-
pine, azatadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam,
bupivacaine, buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine,
chlorcyclizine, chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlor-
promazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine,
cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cy-
clizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal, dilti-
azem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, dobu-
tamine, doxapram, doxepin, encainide, ethidium bromide, ethopropazine, fenoprofen,
fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvoxamine, fu-
rosemide* glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hydralazine,
hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydroxyzine,
ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol, Ie-
vorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic
acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone,
methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyl-
dopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, mida-
zolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen,
nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazo-
line, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine,
pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone,
phenyltoloxamine, phenjrtoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, pra-
zosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine, promazine, pro-
methazine, propafenone, propantheline, propiomazine, propofol, propranolol, protriptyl-
ine, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxipride, risperidone,
salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spironolactone, sulfinpyra-
zone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophylline, thiethyl-
perazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tolmetin,
trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine, tri-
methoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone
KEYWORDS
also details of plasma extraction
REFERENCE
Koves,E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology,
J.ChromatogrA, 1995, 692, 103-119.
Dropropizine
CAS Registry No.: 17692-31-8, 99291-24-4 (S-form)
Merck Index: 3507
SAMPLE
Sample preparation: Add 1 mL whole blood or urine to Toxi-Tu.be A (Toxi-Lab, Irvine CA),
stitute the residue with 50 jxL MeCN:water 50:50, vortex for 10 s, centrifuge at17500 g
for 2 min, inject a 10 (urine) or 30 (blood) (xL aliquot. (The detector wavelength shown is
HPLC VARIABLES
Column: 250 X 4.6 5 fxm Symmetry C8 (Waters)
1.5 mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
tion for the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A,
1997, 763, 149-163.
Duloxetine
Molecular formula: C18H19NOS
Merck Index: 3518
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 1 M pH 10 sodium carbonate buffer + 200 |iL
100 ng/mL IS + 6 mL hexanerisopropanol 98:2, shake mechanically for 45 min, centrifuge
at 825 g for 10 min, freeze in MeOH/dry ice. Remove the organic layer and evaporate it
to dryness at 42, reconstitute the residue in 2 mL acetone, add 150 |xL 100 mM pH 10
sodium carbonate buffer, add 75 |xL 500 |xg/mL dansyl chloride, heat at 55 for 30 min.
Evaporate to dryness under a stream of nitrogen at 42, reconstitute the residue in 200
IxL mobile phase, filter, inject a 100 |xL aliquot.
HPLC VARIABLES
Guard column: 12.5 X 4 5 |xm Zorbax ODS
Column: 250 X 4.6 5 u,m Primesphere 5 C18 (Phenomenex)
Mobile phase: MeCN:50 mM ammonium acetate 79:21
Flow rate: 2
CHROMATOGRAM
Retention time: 9.3
Internal standard: N-methyl-3-(l-naphthalenyloxy)-3-phenylpropanamine (Eli Lilly
113821) (10.9)
OTHER SUBSTANCES
Extracted: metabolites, desmethyl duloxetine
KEYWORDS
derivatization; plasma; silanize glassware; pharmacokinetics
REFERENCE
Johnson,J.T.; Oldham,S.W.; Lantz,R.J.; DelongA-F. High performance liquid chromatographic method for the
determination of duloxetine and desmethyl duloxetine in human plasma, J.Liq.Chromatogr.Rel.TkchnoL,
1996,19, 1631-1641.
SAMPLE
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Zorbax SB-CN
Mobile phase: Gradient. A was MeCN:25 mM pH 3.0 potassium dihydrogen phosphate
buffer 20:80. B was MeCN:25 mM pH 3.0 potassium dihydrogen phosphate buffer 75:25.
A:B from 100:0 to 70:30 over 15 min, to 0:100 over 12 min
Flow rate: 1
Detector: UV 215
CHROMATOGRAM
Retention time: 21
OTHERSUBSTANCES
Extracted: impurities
KEYWORDS
pellets; stability-indicating
REFERENCE
Jansen,P.J.; Oren,P.L.; Kemp,C.A.; Maple,S.R.; Baertschi,S.W. Characterization of impurities formed by
interaction of duloxetine HCl with enteric polymers hydroxypropyl methylcellulose acetate succinate
and hydroxypropyl methylcellulose phthalate, J.Pharm.Sci., 1998, 87, 81-85.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Zorbax SB-CN
Mobile phase: Gradient. A was MeCN:water:trifluoroacetic acid 20:80:0.05. B was MeCN:
water:trifluoroacetic acid 75:25:0.05. A:B from 100:0 to 70:30 over 15 min, to 0:100 over
12 min
Flow rate: 1
Detector: MS, Fisons Instruments VG Quattro triple quadrupole, electrospray, positive ion
mode, m/z 297
OTHER SUBSTANCES
Extracted: impurities
KEYWORDS
pellets; stability-indicating
REFERENCE
Jansen,P.J.; Oren,P.L.; Kemp,C.A.; Maple,S.R.; Baertschi,S.W. Characterization of impurities formed by
interaction of duloxetine HCl with enteric polymers hydroxypropyl methylcellulose acetate succinate
and hydroxypropyl methylcellulose phthalate, J.Pharm.Sci., 1998, 87, 81-85.
Dyclonine
CAS Registry No.: 586-60-7, 536-43-6 (HGI)
Merck Index: 3523
SAMPLE
Sample preparation: 1 mL Sample + 18 mL THF:MeCN 30:70, adjust the pH to 6.0 with
1 M ammonium hydroxide, make up to 25 mL with 100 mM pH 6.0 ammonium acetate
buffer, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 |xm fiBondapak phenyl
Mobile phase: MeCN:THF:pH 6.0 ammonium acetate buffer 46:9:45
Flow rate: 2
Detector: UV 282
CHROMATOGRAM
Retention time: 5.8
OTHER SUBSTANCES
Simultaneous: degradation products
KEYWORDS
stability-indicating; oral spray; gel
REFERENCE
Bhagat,H-R; Bhargava,H-N".; Williams,D.A. High-performance liquid chromatographic determination of
dyclonine hydrochloride in the presence of its degradation products, J.Pharm.Biomed.AnaL, 1989, 7,
441-446.
Dyphylline
Merck Index: 3529
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL 0.2 mg/mL -hydroxyethyltheophylline in
buffer + 100 |xL 40% aqueous trichloroacetic acid, vortex for 30 s, let stand for 5 min,
centrifuge at 2000 g for 15 min, inject a 25 (xL aliquot of the supernatant. (Buffer, was 10
mM sodium acetate adjusted to pH 4.0 with glacial acetic acid.)
HPLC VARIABLES
Column: 10 fjum jjiBondapak C18
Mobile phase: MeCN:buffer 6:94 (Buffer was 10 mM sodium acetate adjusted to pH 4.0
with glacial acetic acid.)
Flow rate: 2
Detector: UV 274
CHROMATOGRAM
Retention time: 7.5
Internal standard: p-hydroxyethyltheophylline (9)
OTHER SUBSTANCES
Extracted: theophylline, caffeine, theobromine
I
KEYWORDS
plasma
REFERENCE
Valia,K.H.; Hartman,C.A.; Kucharczyk,N.; Sofia,R.D. Simultaneous determination of dyphylline and the-
ophylline in human plasma by high-performance liquid chromatography, J.Chromatogr., 1980,221,
170-175.
SAMPLE
Matrix: blood
Sample preparation: 50 jxL Serum + 50 JJLL 15 jxg/mL p-hydroxyethyltheophylline in
MeCN + 2 mL chloroform:isopropanol 95:5, mix for 30 s, centrifuge at 3000 g for 3 min.
Remove the organic layer and evaporate it to dryness under a stream of nitrogen, recon-
stitute the residue in 50 |iL MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: ixBondapak C18
Mobile phase: MeCN:buffer 9.75:90.25 (Buffer was 100 mM KH2PO4 adjusted to pH 4.0
with phosphoric acid.) (At the end of each day clean with water for 20 min and MeOH
for 30 min.)
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 4.8
Internal standard: (3-hydroxyethyltheophylline (5.8)
OTHER SUBSTANCES
Extracted: caffeine
Simultaneous: acetaminophen, aspirin, salicylic acid, procainamide, N-acetylprocainamide
Noninterfering: benzoic acid
Interfering: ampicillin, theophylline (separated with MeCN:buffer 8:92)
KEYWORDS
serum
REFERENCE
Ou,C.-N.; Frawley,V.L. Theophylline, dyphylline, caffeine, acetaminophen, salicylate, acetylsalicylate,
procainamide, and N-acetylprocainamide determined in serum with a single liquid-chromatographic
assay, Clin.Chem., 1982, 28, 2157-2160.
SAMPLE
Matrix: blood
Sample preparation: 250 fxL Serum + 50 |xL 30 jig/mL theophylline in water, vortex, add
1.5 g anhydrous sodium sulfite, add 2.5 mL chloroformrMeOH 90:10, shake vigorously for
30 s (work quickly to avoid forming a cake of sodium sulfite), centrifuge at 1000 g for 5
min. Remove the organic layer, filter (Whatman No. 1 paper pre-wetted with chloroform),
rinse filter with 500 JULL chloroform, evaporate the filtrate under a stream of nitrogen at
40. Take up the residue in 100 |xL dichloroethane and add it to 100 |xL 100 mM am-
monium carbonate, vortex for 10 s, centrifuge at 1000 g for 5 min, inject a 10 fxL aliquot
of the aqueous layer.
HPLC VARIABLES
Column: 250 X 4 10 juirn LiChrosorb RP-8
Mobile phase: MeOH.buffer 25:75 (Buffer was 1.5 mL 1M KH2PO4 in 750 mL water, adjust
to pH 3.0 with 900 mM perchloric acid.)
Flow rate: 2
Detector: UV 275
CHROMATOGRAM
Retention time: 3
Internal standard: theophylline (4.5) (alternatively, p-hydroxypropyltheophylline (3.7))
OTHER SUBSTANCES
Simultaneous: theobromine, caffeine
Noninterfering: acetaminophen, acetazolamide, albuterol, amitriptyline, amobarbital, car-
bamazepine, diazoxide, disopyramide, ethosuximide, isoprenaline, nitrazepam, nortrip-
tyline, oxazepam, pentobarbital, phenobarbital, phenytoin, primidone, procainamide, sal-
icylic acid, sulthiame
KEY WORDS
serum; pharmacokinetics
REFERENCE
Paterson,N. High-performance liquid chromatographic method for the determination of diprophylline in
human serum, J.Chromatogr., 1982, 232, 450-455.
SAMPLE
Matrix: blood
Sample preparation: 500 fxL Serum or plasma + 200 JJLL 100 mM pH 7.0 phosphate buffer
+ 3 mL 0.5 |xg/mL 8-chlorotheophylline in isopropanol, stir at 40000 rpm for 5 s using
dental micromotor with a PTFE mixing head, centrifuge at 3500 g for 2 min. Remove the
supernatant and evaporate it to dryness under a stream of nitrogen at 60, reconstitute
the residue in 50 fxL MeOH, inject a 10 jxL aliquot.
HPLC VARIABLES
Guard column: 50 x 3.2 30-38 jim Co:Pell ODS
Mobile phase: MeCN:MeOH:10 mM pH 5.2 sodium acetate buffer 6:3:91
Flow rate: 1.5
Detector: UV 274
CHROMATOGRAM
Retention time: 6.7
Internal standard: 8-chlorotheophylline (8.85)
OTHER SUBSTANCES
Extracted: theophylline, caffeine, proxyphylline, paraxanthine
Simultaneous: cefoxitin
Noninterfering: carbenicillin, cefoperazone, cephacetril, heparin, penicillin G, phenytoin,
phenobarbital
KEYWORDS
plasma; serum; pharmacokinetics
REFERENCE
Wenk,M.; Eggs,B; Follath,F. Simultaneous determination of diprophylline, proxyphylline and theoph-
ylline in serum by reversed-phase high-performance liquid chromatography, J.Chromatogr., 1983,
276, 341-348.
SAMPLE
Matrix: blood
Sample preparation: Centrifuge, filter (0.45 |xm), inject an aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 jxm internal surface reversed phase Pinkerton, silica derivatized with
glycine-phenylalanine-phenylalanine (Regis) (periodically reverse the column)
Mobile phase: 100 mM pH 6.8 phosphate buffer
Flow rate: 0.3
Detector: UV 275
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: doxofylline, theophylline, caffeine
Noninterfering: acetaminophen, amitriptyline, amphetamine, atropine, benzoylecgonine,
benztropine, caffeine, carbamazepine, carisoprodol, chlorpheniramine, chlorpromazine,
chlorprothixene, cimetidine, cocaine, codeine, dextromethorphan, diazepam, diphenhy-
dramine, diphenoxilate, disopyramide, doxepin, doxylamine, emetine, erythromycin,
flurazepam, glutethimide, hydrocortisone, hydromorphone, hydroxyzine, imipramine, Ii-
docaine, loxapine, meperidine, meprobamate, methadone, methamphetamine, methapyr-
ilene, methaqualone, methocarbamol, methylphenidate, nicotine, nordiazepam, nortrip-
tyline, orphenadrine, papaverine, pentazocine, phenacetin, phencyclidine, phenmetrazine,
phenolphthalein, phentermine, phenylpropanolamine, phenytoin, prazepam, procainam-
ide, procaine, propoxyphene, propranolol, protriptyline, pseudoephedrine, pyrilamine, qui-
nine, salicylamide, spironolactone, strychnine, terpin hydrate, thioridazine, thiothixene,
triamterene, trifiuoperazine, triflupromazine, trihexyphenidyl, trimeprazine, trimethob-
enzamide, trimethoprim, tripelennamine
KEYWORDS
plasma; serum; direct injection
REFERENCE
Tagliaro,E; Dorizzi,R.; Frigerio,A.; Marigo,M. Non-extraction HPLC method for simultaneous measure-
ment of dyphylline and doxofylline in serum, Clin.Chem., 1990, 36, 113-115.
Ebastine
Merck Index: 3534
Lednicer No.: 4 48
SAMPLE
Matrix: blood
Sample preparation: SPE (no details)
HPLCVARIABLES
Column: 150 X 4.6 5 |xm Sup RS C18 (Prolabo)
Mobile phase: MeCN:200 mM pH 3.5 acetate buffer:triethylamine 80:20:0.025
Detector: UV 254
CHROMATOGRAM
Retention time: 17
Internal standard: terfenadine (8)
OTHER SUBSTANCES
Extracted: carebastine, metabolites
KEYWORDS
plasma; pharmacokinetics; SPE
REFERENCE
Van Rooij,J.; Schoemaker,H.C; Bruno,R.; Reinhoudt,J.R; Breimer,D.D.; Cohen,A.F. Cimetidine does not influ-
ence the metabolism of the Hi-receptor antagonist ebastine to its active metabolite carebastine,
Br.J.Clin.Pharmacol, 1993, 35, 661-663.
SAMPLE
Matrix: microsomal incubations
Sample preparation: Add 3 mL MeCN to microsomal incubation, centrifuge at 800 g for 10 min,
evaporate an aliquot of the supernatant to dryness in a centrifugal concentrator. Dissolve the
residue in 200 |xL MeOH and inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Inertsil ODS-3 (GL Sciences, Tokyo, Japan)
Mobile phase: Gradient. A was MeCN. B was 12 mM pH 4.5 ammonium acetate. A:B 35:65 for
3 min, to 85:15 over 25 min
Flow rate: 1
Detector: Radioactivity, Radiomatic Flo-One/Beta A-515 detector (Packard Instrumental Co.,
Meriden, CT), column effluent mixed with Ultima FIo-M scintillant pumped at 2 mL/min
OTHER SUBSTANCES
KEYWORDS
human; liver
REFERENCE
Hashizume,T.; Mise,M.; Terauchi,Y.; O,L.; Fujii,T.; Miyazaki,H.; Inaba,T. N-Dealkylation and hydroxylation of
ebastine by human liver cytochrome P450, Drug Metab.Dispos., 1998, 26, 566-571.
1
Ebiratide
Molecular formula: C48H73N11Oi0S
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg C18 SPE cartridge (Varian) with 2 mL MEOH and 2
mL water. 400 JJLL Plasma + 800 |JLL water, mix, add to the SPE cartridge, wash with 1 mL
water, elute with 1 mL 5 mM HCl in MeOH:water 30:70. Evaporate the eluate to dryness in
a freeze dryer, reconstitute with 100 |xL 100 mM pH 9.0 borate buffer, add 100 |JLL 4-(N,N-
dimethylaminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole in MeCN, mix, heat at 50 for 30 min,
inject a 100 |xL aliquot. (Synthesis of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,l,3-benzoxadi-
azole (DBD-F) is as follows. Dissolve 0.5 g magnesium sulfate heptahydrate and 6 g NaOH in
60 mL water, throughout the reaction keep the flask at about 20 with cold water cooling, add
15 mL 30% hydrogen peroxide, add 75 mL MeOH, add 12.1 g powdered benzoyl peroxide in
one go, stir for 10 min, pour into 150 mL 20% sulfuric acid, extract three times with 50 mL
portions of chloroform, determine peroxybenzoic acid concentration by iodometric titration (Tet-
rahedron 1967, 23, 3327). Slowly add 110 mL 1 M peroxybenzoic acid in chloroform to 7 g 2,6-
difluoroaniline dissolved in 100 mL chloroform, stir at room temperature, when reaction is
complete (iodometric titration) wash with 2% sodium thiosulfate, wash with 5% sodium car-
bonate, wash with water, dry over anhydrous sodium sulfate, evaporate to dryness under re-
duced pressure, recrystallize 2,6-difluoronitrosobenzene from EtOH (mp 108.5-109.5). Stir 8.5
g 2,6-difluoronitrosobenzene in 85 mL DMSO at room temperature and add a solution of 3.91
g sodium azide in 85 mL DMSO dropwise, let stand for about 1 h, add to a large volume of
water, extract with ether, dry the extracts over anhydrous sodium sulfate, evaporate to dryness
under reduced pressure and distil to give 4-fluoro-2,l,3-benzoxadiazole as a colorless oil (bp
83712 mm Hg) (J.Chem.Soc.(C) 1970, 1433). Add 11 mL chlorosulfonic acid dropwise to 3 g 4-
fluoro-2,l,3-benzoxadiazole in 10 mL chloroform at 0-10 (use a calcium chloride drying tube),
stir at room temperature for 1 h, reflux for 2 h, cool, slowly pour into ice water, remove the
organic layer, extract the aqueous layer with chloroform, combine the organic layer, wash, dry
over anhydrous magnesium sulfate, evaporate under reduced pressure, take up the residue in
5 mL benzene (Caution! Benzene is a carcinogen!), chromatograph on a 150 X 30 column of
silica gel (100-200 mesh Kanto Chemical) with n-hexane:benzene 50:50, evaporate the appro-
priate fractions to give 4-(chlorosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (CBD-F) as pale yellow
needles (mp 64-66) (Anal. Chem. 1984. 56, 2461). Stir 0.76 g CBD-F in 70 mL MeCN at 0-10
and add 1 g dimethylamine hydrochloride in 10 mL 100 mM pH 10 borax dropwise, adjust pH
to 5 with 1 M HCl, concentrate to about 10 mL under reduced pressure, extract three times
with 200 mL portions of diethyl ether, wash with water, dry over anhydrous magnesium sulfate,
evaporate under reduced pressure, chromatograph on a 500 X 20 column of silica gel with
chloroform, isolate the appropriate fraction and re-chromatograph on the same column with
ethyl acetate:benzene 1:2 to give 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole
(DBD-F) as white needles (mp 124-125) (yield = 1% !). On a Merck no. 5714 60F254 tic plate
eluted with chloroform DBD-F has Rf 0.32 and lies between two other reaction products (An-
alyst 1989, 114, 413). It is also reported that DBD-F can be purchased from Tokyo Kasei.)
HPLCVARIABLES
Column: 150 X 4.6 5C18 (Vydac)
Mobile phase: Gradient. A MeCN:MeOH:50 mM pH 6.0 imidazole nitrate 26.7:13.3:60. B MeCN:
MeOH:50 mM pH 6.0 imidazole nitrate 40:20:40. A:B from 100:0 to 55:45 over 15 min, to 30:
70 over 45 min, re-equilibrate at initial conditions for 15 min.
Flow rate: 1
Detector: Chemiluminescence. The column effluent mixed with the reagent pumped at 1.2 mL/
min and the mixture flowed through a coil at 30 to the detector. (Reagent was 100 mM hy-
drogen peroxide containing 0.5 mM bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate
(Wako, Richmond VA).)
CHROMATOGRAM
Retention time: 33
Limit of detection: 250 fmole
KEY WpRDS
derivatization; rat; plasma; SPE; fluorescence detection is more sensitive
REFERENCE
Hamachi,Y.; Nakashima,K; Akiyama,S. High performance liquid chromatography with peroxyoxalate chemi-
luminescence detection of synthetic peptide, ebiratide, J.Liq.Chromatogr.Rel.Technol, 1997,20,2377-2387.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 100 |xM solution in 100 mM pH 9.0 borate buffer. 200 (xL So-
lution + 200 v, 30 mM DBD-F in MeCN, mix, heat at 50 for 30 min, inject an aliquot. (DBD-
F is 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole. It can be purchased from To-
kyo Kasei Kogyo or synthesized as follows. Dissolve 0.5 g magnesium sulfate heptahydrate and
6 g NaOH in 60 mL water, throughout the reaction keep the flask at about 20 with cold water
cooling, add 15 mL 30% hydrogen peroxide, add 75 mL MeOH, add 12.1 g powdered benzoyl
peroxide in one go, stir for 10 min, pour into 150 mL 20% sulfuric acid, extract three times
with 50 mL portions of chloroform, determine peroxybenzoic acid concentration by iodometric
titration (Tetrahedron 1967,23,3327). Slowly add 110 mL 1M peroxybenzoic acid in chloroform
to 7 g 2,6-difluoroaniline dissolved in 100 mL chloroform, stir at room temperature, when
reaction is complete (iodometric titration) wash with 2% sodium thiosulfate, wash with 5%
sodium carbonate, wash with water, dry over anhydrous sodium sulfate, evaporate to dryness
under reduced pressure, recrystallize 2,6-difluoronitrosobenzene form EtOH (mp 108.5-109.5).
Stir 8.5 g 2,6-difluoronitrosobenzene in 85 mL DMSO at room temperature and add a solution
of 3.91 g sodium azide in 85 mL DMSO dropwise, let stand for about 1 h, add to a large volume
of water, extract with ether, dry the extracts over anhydrous sodium sulfate, evaporate to
dryness under reduced pressure and distil to give 4-fluoro-2,l,3-benzoxadiazole as a colorless
oil (bp 83712 mm Hg) (J.Chem.Soc.(C) 1970, 1433). Add 11 mL chlorosulfonic acid dropwise to
3 g 4-fluoro-2,l,3-benzoxadiazole in 10 mL chloroform at 0-10 (use a calcium chloride drying
tube), stir at room temperature for 1 h, reflux for 2 h, cool, slowly pour into ice water, remove
the organic layer, extract the aqueous layer with chloroform, combine the organic layer, wash,
dry over anhydrous magnesium sulfate, evaporate under reduced pressure, take up the residue
in 5 mL benzene (Caution! Benzene is a carcinogen!), chromatograph on a 150 X 30 column of
silica gel (100-200 mesh Kanto Chemical) with n-hexane:benzene 50:50, evaporate the appro-
priate fractions to give 4-(chlorosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (CBD-F) as pale yellow
needles (mp 64-66) (Anal. Chem. 1984. 56, 2461). Stir 0.76 g CBD-F in 70 mL MeCN at 0-10
and add 1 g dimethylamine hydrochloride in 10 mL 100 mM pH 10 borax dropwise, adjust pH
to 5 with 1 M HCl, concentrate to about 10 mL under reduced pressure, extract three times
with 200 mL portions of diethyl ether, wash with water, dry over anhydrous magnesium sulfate,
evaporate under reduced pressure, chromatograph on a 500 X 20 column of silica gel with
chloroform, isolate the appropriate fraction and re-chromatograph on the same column with
ethyl acetate:benzene 1:2 to give 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole
(DBD-F) as white needles (mp 124-125) (yield = 1% !). On a Merck no. 5714 60F254 tic plate
eluted with chloroform DBD-F has Rf 0.32 and lies between two other reaction products (An-
alyst 1989, 114, 413).)
HPLCVARIABLES
Column: 150 X 4.6 5C18 protein and peptide (Vydac)
Mobile phase: MeCN:50 mM Na2HPO4 40:60 adjusted to pH 7.0 with 20% phosphoric acid
Flow rate: 1
Detector: F ex 440 em 580 or UV 220
CHROMATOGRAM
Retention time: 10
Limit of detection: 0.25 pmole (F)
KEY WpRDS
derivatization
REFERENCE
Hamachi,Y.; Tsujiyama,T.; Nakashima,K; Akiyama,S. High-performance liquid chromatography with fluores-
cence detection of ebiratide using 4-(N,N-dimethylamino-sulphonyl)-7-fluoro-2,l,3-benzoxadiazole as afluor-
genic reagent, Biomed.Chromatogr., 1995, 9, 216-220.
Econazole
Molecular formula: C18H15CI3N2O
CAS Registry No.: 27220-47-9, 68797-31-9 (nitrate)
Merck Index: 3550
Lednicer No.: 2 249
SAMPLE
Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add
3 mL water, mix by gentle inversion for 5 min, centrifuge at 1500 g for 5 min. Remove the
organic layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the
residue with 50 jxL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
a 10 (urine) or 30 (blood) |JLL aliquot. (The detector wavelength shown is the wavelength of
maximum absorbance. This will not necessarily be the optimal wavelength for the separation.
Multiple wavelengths from 200-350 nm can be scanned using a diode-array detector. Otherwise,
220 nm may be a reasonable choice for initial work. Matrix may interfere.)
HPLCVARIABLES
Column: 250 X 4.6 5 \im Symmetry C8 (Waters)
Mobile phase: Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A:B 85:
15 for 6.5 min, 65:35 for 18.5 min, 20:80 for 3 min (step gradient), re-equilibrate at initial
conditions for 7 min.
Flow rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y.; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763,149-
163.
SAMPLE
Sample preparation: Powders. Extract a sample equivalent to about 10 mg econazole twice
with 20 mL portions of MeOH with magnetic stirring, filter extracts, combine, dilute to 50 mL
with MeOH. Remove a 2.5 mL aliquot and add it to 2 mL 200 |xg/mL miconazole in MeOH,
dilute to 10 mL with MeOH, inject a 10 (xL aliquot. Creams. Condition a Baker diol SPE
cartridge with 6 mL dichloromethane. Add a sample equivalent to 10 mg econazole to 30 mL
dichloromethane, sonicate for 2 min, make up to 50 mL with dichloromethane, filter, add 2 mL
of the filtrate to the SPE cartridge. Wash with two 3 mL portions of n-hexane:dichloromethane
4:1, aspirate to dryness, elute with three 1 mL portions of MeOH: 100 mM triethylamine ad-
justed to pH 7.0 with acetic acid 4:1. Combine the eluates and dilute them to 5 mL with MeOH.
Remove a 2.5 mL aliquot and add it to 1 mL 200 |xg/mL miconazole in MeOH, dilute to 5 mL
with MeOH, inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 5 |xm Nova-Pak RP-18
Mobile phase: MeOH:THF:100 mM triethylamine adjusted to pH 7.0 with acetic acid 70:12:18
Flow rate: 0.8
Detector: UV 230
CHROMATOGRAM
Retention time: 2.5
Internal standard: miconazole (3.5)
KEYWORDS
creams; powders; SPE
REFERENCE
Cavrini,V.; Di Pietra,A.M.; Gatti,R. Analysis of miconazole and econazole in pharmaceutical formulations by
derivative UV spectroscopy and liquid chromatography (HPLC), J.Pharm.Biomed.AnaL, 1989, 7, 1535-
1543.
SAMPLE
Sample preparation: Tablets. Powder tablets, weigh out amount equivalent to about 30 mg,
add 100 mL MeOH, sonicate for 5 min, filter. Add a 2 mL aliquot of filtrate to 5 mL of 100 jxg/
mL ketoconazole in MeOH, make up to 25 mL with MeOH, inject 20 |JLL aliquot. Cream. Con-
dition a 500 mg Bond-Elut diol cartridge with 6 mL dichloromethane. Weigh out cream equiv-
alent to about 5 mg of drug, add 30 mL dichloromethane, sonicate for 3 min, make up to 100
mL with dichloromethane, filter. Add a 2 mL aliquot to the cartridge, wash with 2 mL dichlo-
romethane :methanol 4:1, wash with 2 mL dichloromethane, elute with 3 mL MeOH.buffer
85:15. Add eluate to 0.5 mL 100 |xg/mL ketoconazole in MeOH, make up to 5 mL with MeOH,
inject 20 fxL aliquot. (Buffer was 50 mM triethylamine adjusted to pH 7.0 with phosphoric
acid.)
HPLCVARIABLES
Column: 250 X 4.6 5 [xm Spherisorb CN
Mobile phase: THF:buffer 30:70 (Buffer was 50 mM triethylamine adjusted to pH 3.0 with phos-
phoric acid.)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 14
Internal standard: ketoconazole (7)
OTHER SUBSTANCES
Simultaneous: clotrimazole, ketoconazole, bifonazole, tioconazole, isoconazole, miconazole,
fenticonazole
KEYWORDS
tablets; creams
REFERENCE
Di Pietra,A-M.; Cavrini,V.; Andrisano,V.; Gatti,R. HPLC analysis of imidazole antimycotic drugs in pharma-
ceutical formulations, J.Pharm.BiomedAnaL, 1992,10, 873-879.
Edrophonium chloride
Molecular formula: C10H16CINO
Merck Index: 3562
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL 500 ng/mL neostigmine in water + 500 |xL 100 mM
picric acid in 100 mM NaOH (pH adjusted to 7) + 400 |xL 100 mM NaH2PO4 + 12 mL water
saturated dichloromethane, shake vigorously for 5 min, centrifuge at 2000 g for 10 min. Remove
10 mL of the organic phase and add it to 200 |xL 1 mM tetrabutylammonium hydrogen sulfate,
shake vigorously for 30 s, centrifuge at 2000 g for 2 min, discard most of the organic layer,
centrifuge at 2000 g for 1 min, inject a 50 |xL aliquot of the aqueous layer. (Store glassware in
100 mM tetramethylammonium chloride solution and wash 5 times with water before use.)
HPLC VARIABLES
Guard column: 50 X 3.2 30-40 |xm Perisorb RP-2 (Merck)
Column: 150 X 4.6 5 |xm Ultrasphere octyl
Mobile phase: MeCNrwater 20:80 containing 10 mM sodium heptanesulfonate, 10 mM NaH2PO4,
and 2.5 mM tetramethylammonium chloride, pH adjusted to 3 with concentrated sulfuric acid
Flow rate: 2
Detector: UV 214
CHROMATOGRAM
Retention time: 3.5
Internal standard: neostigmine (5)
KEY WORDS
REFERENCE
De Ruyter,M.G.M.; Cronnelly,R.; Castagnoli,N.,Jr. Reversed-phase, ion-pair liquid chromatography of quater-
nary ammonium compounds: determination of pyridostigmine, neostigmine and edrophionium in biological
fluids, J.Chromatogr., 1980, 183, 193-201.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 50 X 4.6 5 |xm Suplex pKb-100 (Supelco)
Mobile phase: MeCN:buffer 30:70 (Buffer was 10 mM pH 7.0 sodium phosphate containing 5
mM sodium dodecyl sulfate.)
Flow rate: 2
Detector: UV 210
CHROMATOGRAM
Retention time: 1.6
OTHER SUBSTANCES
Simultaneous: 4'-methylphenazone, neostigmine bromide
REFERENCE
Supelco Catalog, 1994, p. 771.
EDTA
CAS Registry No.: 60-00-4, 150-38-9 (tri Na salt), 64-02-8 (tetra Na salt), 6381-92-6 (di Na salt di-
hydrate), 139-33-3 (di Na salt), 25102-12-9 (di K salt dihydrate), 2001- 94-7 (di K salt), 58167-76-3 (di
K salt monohydrate), 23411-34-9 (Ca di Na salt hydrate), 62-33-9 (Ca di Na salt)
Merck Index: 3559
SAMPLE
Matrix: cell suspensions
Sample preparation: Centrifuge cell suspension at 14000 rpm for 2 min, dilute if necessary,
inject a 30 pX aliquot.
HPLCVARIABLES
Column: 150 X 4.6 LC18 (Supelco)
Mobile phase: MeCN:buffer 0.5:99.5 (Buffer was >1.5>mL/L acetic acid containing 1 g/L ammo-
nium acetate, 0.5 g/L l,2-diaminopropane-N,N,N ,N -tetraacetic acid, 0.5 mL/L ammonium hy-
droxide, and 0.1 mL/L triethylamine.) (After 2.5 min wash column with MeCN:water 60:40
containing 0.5 g/L l,2-diaminopropane-N,N,N',N'-tetraacetic acid, and 0.1 mL/L triethylamine
for 5.5 min, re-equilibrate for 4 min.)
Flow rate: 1
Detector: UV 360 (as ferric complex)
KEYWORDS
edetic acid measured as ferric complex
REFERENCE
Lauff,J.J.; Steele,D.B.; Coogan,L.A.; Breitfeller,J.M. Degradation of the ferric chelate of EDTA by a pure culture
of an Agrobacterium sp., Appl.Environ.Microbiol., 1990, 56, 3346-3353.
SAMPLE
Matrix: fertilizer
Sample preparation: Prepare an aqueous solution, filter (paper), filter (0.2 |xm), inject an
aliquot.
HPLCVARIABLES
Guard column: Ion Pac AG7 (Dionex)
Column: 10 jxm Ion Pac AS 7 (Dionex)
Mobile phase: 70 mM nitric acid
Flow rate: 0.5
Detector: UV 330 following post-column reaction with 1 g/L Fe(NO3)3.9H2O in 2% perchloric acid
CHROMATOGRAM
Retention time: 5
KEYWORDS
post-column reaction
REFERENCE
Vande Gucht,I. Determination of chelating agents in fertilizers by ion chromatography, J.ChromatogrA, 1994,
671, 359-365.
SAMPLE
Sample preparation: Dilute with water to give an EDTA concentration of 0.01%, mix with an
equal volume of 0.02% cupric nitrate, inject a 25 (xL aliquot.
HPLC VARIABLES
Guard column: 10 \jjm Adsorbosphere C18
Column: 150 X 4.5 5 |xm Resolve C18 (Waters)
Mobile phase: MeCN:buffer:water 20:20:80 (Buffer was 195 mL water and 6 mL 1 M tetrabu-
tylammonium hydroxide in MeOH adjusted to pH 6.5 0.1 with 1 M phosphoric acid.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 8.5
OTHER SUBSTANCES
Simultaneous: sorbic acid
Noninterfering: benzalkonium chloride, hydroxyethyl cellulose, Miranol 2MCA, phenylmercuric
nitrate, polyvinyl alcohol, propylene glycol, thimerosal
KEYWORDS
ophthalmic solutions; derivatization; complexation
REFERENCE
Hall,L.; Takahashi,L. Quantitative determination of disodium edetate in ophthalmic and contact lens care
solutions by reversed-phase high-performance liquid chromatography, J.Pharm.Sci., 1988, 77, 247-250.
SAMPLE
Sample preparation: Inject a 20 (xL aliquot.
HPLC VARIABLES
Column: 250 X 4.1 10 |xm Anion/R (Alltech)
Mobile phase: MeCN:buffer 25:75 (Buffer was 11 mM nitric acid containing 2 mM cupric nitrate,
pH adjusted to 3.0 with 10 M NaOH.)
Flow rate: 1
Detector: UV 250
CHROMATOGRAM
Retention time: 8
KEYWORDS
stability-indicating; rugged; ophthalmic solution; complexation; copper complexes
REFERENCE
Kord,A.S.; Tumanova,L; Matier,W.L. A novel HPLC method for determination of EDTA in a cataract inhibiting
ophthalmic drug, J.Pharm.BiomedAnaL, 1995, 13, 575-580.
SAMPLE
Sample preparation: Dilute ophthalmic cleanser 1:25 with 100 |xM ferric chloride hexahydrate
in water, inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 3 |xm Adsorbosphere HS C18
Mobile phase: 50 mM Tetrabutylammonium hydrogen sulfate in water
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 5.2
OTHER SUBSTANCES
KEYWORDS
derivatization; complexation; ophthalmic cleanser; stability-indicating
REFERENCE
Tran,G.; Chen,C; Miller,R.B. HPLC method for the determination of EDTA in an ophthalmic cleanser,
J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 1499-1508.
SAMPLE
Matrix: solutions
Sample preparation: Mix a 10 juiM solution of the ligand with a 100 jxM solution of lutetium
chloride, pass through a 70 X 20 column of 70-130 |xm AG 50W-X8 cation-exchange resin
(sodium form, Bio-Rad), inject an aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Prodigy 5 ODS-2 (Phenomenex)
Mobile phase: Gradient. A was 1 mM potassium sulfate containing 3 mM tetrapropylammonium
bromide. B was MeCN:3 mM potassium sulfate 4:96. A:B 100:0 for 4 min, to 0:100 over 3 min.
Flow rate: 1
Detector: F ex 360 em 500 following post-column reaction. The column effluent mixed with the
reagent pumped at 0.3 mL/min and the mixture flowed through a 5.1 m X 0.5 mm ID knitted
PTFE coil. The effluent from the coil mixed with 1 M NaOH pumped at 0.3 mL/min and the
mixture flowed to the detector. (Reagent was 1 mM trans-l,2-diaminocyclohexane-N,N,N',N'-
tetraacetic acid (CDTA) in 1 mM 8-hydroxyquinoline-5-sulfonic acid, adjusted to pH 2.8 with
acetic acid.)
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
Simultaneous: trans-l,2-diammocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), diethylenetria-
minepentaacetic acid (DTPA), ethylene glycol-bis(p-aminoethylether) N,N,N',N'-tetraacetic acid
(EGTA), nitrilotriacetic acid (NTA)
KEY WpRDS
derivatization; complexation; post-column reaction; use a metal-free system
REFERENCE
Ye,L.; Lucy,C.A. Ion chromatographic determination of chelating ligands based on the postcolumn formation of
ternary fluorescent complexes, J.Chromatogr.A, 1996, 739, 307-315.
SAMPLE
Matrix: water
Sample preparation: Filter (0.2 |xm cellulose nitrate) river water, pass the filtrate through a
10 X 5 column filled with SPE 7090 sulfonic acid material (Baker), collect 2-6 mL, evaporate
to dryness in an oven at 90, add 1 mL buffer, add 20 |xL iron solution, heat at 90 for 3 h,
cool, add 40 |xL 50 mM tetrabutylammonium bromide in buffer, inject a 200 |xL aliquot. (Buffer
was 15 mM formic acid containing 5 mM sodium formate and 1 mM tetrabutylammonium
bromide. The iron solution was 1 mM ferric nitrate in 10 mM nitric acid.)
HPLC VARIABLES
Guard column: 4 x 4 Lichrocart (Merck)
Column: 250 X 4 Lichrocart RP-18 (Merck)
Mobile phase: MeCN:buffer 8:92 (Buffer was 15 mM formic acid containing 5 mM sodium for-
mate, pH 3.3.)
Flow rate: 1
Detector: UV 258
CHROMATOGRAM
Retention time: 7.6
KEYWORDS
protect from light; river water; derivatization; complexation; SPE
REFERENCE
Nowack,B.; Kari,F.G.; Hilger,S.U.; Sigg,L. Determination of dissolved and adsorbed EDTA species in water and
sediments by HPLC, Anal.Chem., 1996, 68, 561-566.
Eflornithine
Molecular formula: C6H12F2N2O2
CAS Registry No.: 67037-37-0, 68278-23-9 (HCI), 96020-91-6 (HCI monohydrate)
Merck Index: 3564
Lednicer No.: 4 2
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 20 |xL 250 (xg/mL 4-amino-3-hydroxybutyric acid in
water + 400 |xL MeOH, centrifuge at 800 g for 20 min. Remove the supernatant and add it to
200 |xL 20 mM pH 7.5 phosphate buffer, mix an aliquot of this mixture with an equal volume
of reagent, inject. (Reagent was 10 mg o-phthalaldehyde in 1 mL EtOH, add 100 jxL 2-mer-
captoethanol, add 10 mL 100 mM pH 7.5 phosphate buffer, store in the dark, freshly prepare
every 3 days.)
HPLCVARIABLES
Guard column: 37-50 jxm Bondapak C18/corasil
Column: 5 |xm C18 Radial-Pak (Waters)
Mobile phase: Gradient. A was MeOH:isopropanol:100 mM pH 7.5 phosphate buffer 5:3:92. B
was MeOH:MeCN:isopropanol:water 80:5:5:10. A:B 80:20 for 3 min, to 50:50 over 3 min, main-
tain at 50:50 for 5 min, re-equilibrate at initial conditions for 7 min.
Flow rate: 0.2 for 1 min then 1.5
CHROMATOGRAM
Retention time: 19
Internal standard: 4-amino-3-hydroxybutyric acid (13)
KEYWORDS
plasma; derivatization
REFERENCE
Smithers,J. A precolumn derivatization high-performance liquid chromatographic (HPLC) procedure for the
quantitation of difluoromethylornithine in plasma, Pharm.Res., 1988, 5, 684-686.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 u-L 1 mg/mL norvaline in water + 2 mL ice-cold 80%
EtOH, vortex for 30 s, centrifuge at 3000 rpm for 5 min, remove the supernatant, extract the
residue twice more with 2 mL portions of ice-cold 80% EtOH. Combine the supernatants and
dry them under vacuum at 50, dissolve in 2 mL water and 2 mL buffer. 100 |xL Mixture + 100
(JLL 6 mg/mL dansyl chloride in acetone, let stand at room temperature in the dark for 4 h, add
800 |JLL water adjusted to pH 8.5 with NaOH, vortex for 15 s, centrifuge at 3000 rpm, inject a
25-50 |xL aliquot. (Buffer was 500 mM sodium bicarbonate adjusted to pH 8.5 with 1 M NaOH.)
HPLC VARIABLES
Guard column: 15 X 4.6 5 |xm C8 (Rainin)
Column: 150 X 4.6 5 juim C8 (Rainin)
Mobile phase: Gradient. A was THF: 10 mM sodium acetate adjusted to pH 4.18 with 1 M acetic
acid 5:95. B was MeCN:THF 90:10. A:B from 100:0 to 63.3:36.7 over 19 min, to 0:100 over 2
min, re-equilibrate at 100:0 for 7 min.
Flow rate: 1.5 for 21 min, 2.0 for 2 min, 1.75 for 3 min, 1.5 for 2 min
Detector: UV 330
CHROMATOGRAM
Internal standard: norvaline (17.66)
KEYWORDS
serum; derivatization; pharmacokinetics
REFERENCE
Cohen,J.L.; Ko,RJ; Lo,A.T.L.; Shields,M.D.; Gilman,T.M. High-pressure liquid chromatographic analysis of
eflornithine in serum, J.Pharm.ScL, 1989, 78, 114-116.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 jxm Ultrasphere ion pair
Mobile phase: Buffer prepared from 0.92 g L-proline and 1 g CuSO4.5H2O in 1 L water, pH
adjusted to 5.5 with 1 mL 5 M NaOH.
Flow rate: 0.5
Detector: F ex 340 em 455 following post-column derivatization. The column effluent and the
reagent pumped at 0.35 mL/min were mixed and allowed to flow through a 3 m X 0.3 mm i.d.
PTFE reaction coil at 50 to the detector. (Reagent was 800 mg o-phthalaldehyde in 10 mL
EtOH + 500 mM pH 10.5 potassium borate buffer, filter (0.45 |xm), add 3 mL Brij 35, add 2.5
mL 2-mercaptoethanol, store in the dark.)
CHROMATOGRAM
Retention time: 6.4 (+), 13.9 (-)
KEYWORDS
REFERENCE
Wagner,J.; Gaget,C; Heintzelmann,B-; Wolf,E. Resolution of the enantiomers of various a-substituted ornithine
and lysine analogs by high-performance liquid chromatography with chiral eluant and by gas chromatog-
raphy on Chirasil-Val, Anal.Biochem., 1987, 164, 102-116.
Efonidipine
Molecular formula: C34H38N3O7P
CAS Registry No.: 111011 -76-8 (HCI ethanol)
Merck Index: 3566
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL 100 ng/mL IS in MeOH + 40 |xL 10 mM diethyl-
amine, extract twice with 5 mL portions of ether. Combine the organic layers and evaporate
them to dryness under a stream of nitrogen, reconstitute the residue in 100 (xL mobile phase,
inject a 70 |xL aliquot.
HPLC VARIABLES
Column: 50 X 4.6 2.5 |xm Inertsil ODS
Flow rate: 1
Detector: MS, Hewlett-Packard 1090L and 5988A, ion source 276, probe stem 95-106, probe
tip 165-175, m/z 631
CHROMATOGRAM
Retention time: 4.4
Internal standard: heptadeutero-efonidipine (m/z 638)
KEYWORDS
REFERENCE
Nakabeppu,H.; Asada,M.; Oda,T.; Shinozaki,Y; Yajima,T. Plasma and urinary metabolites of efonidipine hy-
drochloride in man, Xenobiotica, 1996, 26, 229-239.
SAMPLE
Matrix: urine
Sample preparation: Condition a 1 mL 100 mg Bond Elut C18 SPE cartridge with 2 mL MeCN
and 2 mL water. 5 mL Urine + 10 jxL 5 |xg/mL IS in MeOH, mix, add to the SPE cartridge,
wash with 3 mL water, wash with 2 mL MeCN:water 30:70, wash with 1 mL MeCN:water 45:
55, elute with 2 mL MeCN:water 70:30. Add 500 |xL 1 M NaOH and 5 mL diethyl ether to the
eluate, shake, centrifuge. Remove the organic layer and evaporate it to dryness, reconstitute
the residue in 50 |xL n-hexane:EtOH 2:1, inject a 15 (xL aliquot.
HPLC VARIABLES
Guard column: 4 x 4 LiChrospher Si60
Column: 250 X 4.6 Nucleosil 50-5
Mobile phase: n-Hexane:MeOH:chloroform 120:15:5
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: ()-2-[benzyl(phenyl)amino]ethyl l,4-dihydro-2,6-dimethyl-4-(3-nitro-
phenyl)-5-(4,4,6,6-tetramethyl-2-oxo-1,3,2-dioxaphosphorinan-2-yl)-3-pyridinecarboxylate hy-
drochloride (P00-851743) (10)
KEYWORDS
normal phase; pharmacokinetics; SPE
REFERENCE
Nakabeppu,H.; Asada,M.; Oda,T.; Shinozaki,Y.; Yajima,T. Plasma and urinary metabolites of efonidipine hy-
drochloride in man, Xenobiotica, 1996, 26, 229-239.
Eliprodil
Molecular formula: C20H23CIFNO
CAS Registry No.: 119431 -25-3, 136634-88-3 (HCI)
SAMPLE
Sample preparation: Plasma. 200 |JLL Plasma + 200 |xL pH 6.5 buffer + 20 |xL 200 U/mL p-
glucuronidase (from E. coli, Sanofi-Pasteur), mix, heat at 37 for 24 h, make up to 1 mL with
water, add 20 |JLL 1.25 |xg/mL IS, add 1 mL pH 12 buffer, vortex, add 6 mL n-hexane, shake at
40 rpm for 10 min, centrifuge at 15 at 900 g for 5 min, centrifuge at -20 at 900 g for 8 min.
Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 70, add
200 |xL 0.1% (S)-(+)-l-(l-naphthyl)ethyl isocyanate in MeCN under a stream of nitrogen, vortex
for 30 s, heat at 70 for 40 min, evaporate to dryness under a stream of nitrogen, add 250
IxL MeCN:pH 4.5 buffer 40:60, vortex, inject a 150 (xL aliquot on to column A and elute to
waste with mobile phase A, after 5 min elute the contents of column A on to column B with
mobile phase B, after 2.5 min remove column A from the circuit, elute column B with mobile
phase B, monitor the effluent from column B. (Backflush column A with MeCN:water 70:30,
MeCN, MeOH, and THF, then re-equilibrate with MeCN:water 50:50 at 2 mL/min.) Urine. 100
IxL Urine + 100 JJLL pH 6.5 buffer + 50 jxL 200 U/mL (3-glucuronidase (from E. coli, Sanofi-
Pasteur), mix, heat at 37 for 24 h, make up to 1 mL with water, add 20 |JLL 20 |xg/mL IS, add
1 mL pH 12 buffer, vortex, add 6 mL n-hexane, shake at 40 rpm for 10 min, centrifuge at 15
at 900 g for 5 min, centrifuge at -20 at 900 g for 8 min. Remove the organic layer and evaporate
it to dryness under a stream of nitrogen at 70, add 200 JULL 0.1% (S)-(+)-l-(l-naphthyl)ethyl
isocyanate in MeCN under a stream of nitrogen, vortex for 30 s, heat at 70 for 40 min, evap-
orate to dryness under a stream of nitrogen, add 250 |xL MeCN.pH 4.5 buffer 40:60, vortex,
inject a 50 |xL aliquot on to column A and elute to waste with mobile phase A, after 5 min elute
the contents of column A on to column B with mobile phase B, after 2.5 min remove column A
from the circuit, elute column B with mobile phase B, monitor the effluent from column B.
(Backflush column A with MeCN.water 70:30, MeCN, MeOH, and THF, then re-equilibrate
with MeCN.water 50:50 at 2 mL/min.) (Prepare pH 6.5 buffer by adding 20 mL 136.08 mg/mL
KH2PO4 to 780 mL water, adjusting pH to 6.5 with 1 M KOH, and making up to 1 L with
water. Prepare pH 12 buffer by dissolving 26.5 g sodium carbonate and 21 g sodium bicarbonate
in 800 mL water, adjust pH to 12 with about 30 mL 30% NaOH, make up to 1 L with water.
Prepare pH 4.5 buffer by dissolving 6.8 g KH2PO4 in 1 L water.)
HPLC VARIABLES
Column: A 20 X 4.6 5 |xm Supelguard LC8; B 20 X 4.6 40 |xm Pelliguard LC8 (Supelco) + 150
X 4.6 5 |xm Hypersil C8 BDS
Mobile phase: A MeCN.water 50:50; B MeCN:MeOH:buffer 56:2:42 (Prepare buffer by dissolving
6.8 g KH2PO4 and 3.4 mL orthophosphoric acid in 4 L water, pH 2.6.)
Flow rate: A 2; B 1.2
CHROMATOGRAM
Retention time: 16 (S-(+)), 17 (R-(-))
Internal standard: (+)-a-(3,4-dichlorophenyl)-4[(4-fluorophenyl)methyl]piperidine-l-ethanol hy-
drochloride (SL83.0601-10, Synthelabo Recherche, Bagneux, France) (19, 21)
Limit of quantitation: 0.75 ng/mL (plasma), 50 ng/mL (urine)
KEY WpRDS
derivatization; plasma; column-switching; pharmacokinetics; chiral
REFERENCE
Malavasi,B.; Ripamonti,M.; Rouchouse,A.; Ascalone,V. Stereoselective determination of unchanged and glu-
curoconjugated eliprodil, a new anti-ischaemic drug, in human plasma and urine by precolumn derivatiza-
tion and column-switching high-performance liquid chromatography with fluorescence detection,
J.ChromatogrA, 1996, 729, 323-333.
Emedastine
CAS Registry No.: 87233-61-2, 87233-62-3 (fumarate)
Merck Index: 3597
SAMPLE
Matrix: perfusate
HPLC VARIABLES
Column: 150 X 5 Inertsil ODS
Mobile phase: MeCN:buffer 50:50 (Buffer was 25 mM pH 2.4 phosphate buffer containing 0.25%
sodium lauryl sulfate.)
Flow rate: 1.4
Detector: UV 280
KEYWORDS
rabbit; pharmacokinetics
REFERENCE
Harada,S.; Takahashi,Y.; Nakagawa,H. Transdermal administration of emedastine, Biol.Pharm.BulL, 1993,16,
884-888.
Emetine
CAS Registry No.: 483-18-1, 316-42-7 (2HCI)
Merck Index: 3600
SAMPLE
residue with 50 |xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
a 10 (urine) or 30 (blood) JJLL aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
mL/min)
Detector: UV 202.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y.; P6pin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
163.
Enalapril
CAS Registry No.: 75847-73-3, 76095-16-4 (maleate)
Merck Index: 3605
LednicerNo.: 4 58,81-84
SAMPLE
Sample preparation: Add 1 mL whole blood or urine to Tbxi-Tube A (Toxi-Lab, Irvine CA), add
a 10 (urine) or 30 (blood) |xL aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
Column: 250 X 4.6 5 jutm Symmetry C8 (Waters)
mL/min)
Detector: UV 211.1
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.ChromatognA, 1997, 763,149-
163.
SAMPLE
Sample preparation: Add MeOH: 100 mM pH 4.5 phosphate buffer 20:80 to powdered capsules
or tablets so as to give an enalapril concentration of ca. 46 |xg/mL, stir for 15 min, inject a 20
fxL aliquot.
HPLC VARIABLES
Column: 250 X 4.5 5 ixm Hypersil ODS
Mobile phase: MeCN:MeOH:20 mM pH 2.5 sodium heptanesulfonate 35.15:1.85:63
Flow rate: 1
Detector: UV 215
CHROMATOGRAM
KEYWORDS
capsules; tablets
REFERENCE
Bonazzi,D.; Gotti,R.; Andrisano,V.; Cavrini,V. Analysis of ACE inhibitors in pharmaceutical dosage forms by
derivative UV spectroscopy and liquid chromatography (HPLC), J.Pharm.Biomed.Anal., 1997,16,431438.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4 10 \um LiChrosorb RP-18
Mobile phase: MeCNrbuffer 30:70 (Buffer was 67 mM KH2PO4 adjusted to pH 2.4 with phos-
phoric acid.)
Flow rate: 1
Detector: UV 211
CHROMATOGRAM
OTHERSUBSTANCES
Simultaneous: benazepril, cilazapril
REFERENCE
Gumieniczek,A.; Przyborowski,L. Determination of benazepril and cilazapril in Pharmaceuticals by high per-
formance liquid chromatography, J.Liq.Chromatogr.Rel.TechnoL, 1997, 20, 2135-2142.
Enalaprilat
Molecular formula: C18H24N2O5.2H2O
CAS Registry No.: 76420-72-9, 84680-54-6 (dihydrate)
Merck Index: 3606
SAMPLE
Matrix: blood
Sample preparation: Condition a 500 mg Bond Elut SCX SPE cartridge with 3 mL MeOH and
12 mL 1% acetic acid. Add 1 mL plasma to the SPE cartridge, wash with 3 mL water, wash
with 3 mL MeCN, dry under vacuum, elute with 2 mL 500 mM HCl in MeCN. Evaporate the
eluate to dryness under a stream of nitrogen, reconstitute with 100 |xL 50 mM triethylamine
in MeCN, vortex for 1 min, add 50 jxL 60 mM ethyl chloroformate in MeCN, vortex for 1 min,
add 200 |xL 3 mM L-leucine-(4-methyl-7-coumarinylamide) in MeOH, vortex for 1 min, let stand
for 4 min, evaporate to dryness under a stream of nitrogen, reconstitute with 200 |xL MeCN:
water 50:50, inject a 5-15 JULL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 (xm Axxiom octyl (Richard Scientific, Novato)
Mobile phase: Gradient. A was 1 L water containing 2 mL 85% phosphoric acid. B was 1 L
MeCN containing 2 mL 85% phosphoric acid. A:B 52:48 for 15 min, to 10:90 over 15 min.
CHROMATOGRAM
Retention time: 38
KEYWORDS
plasma; derivatization; SPE
REFERENCE
Levai,E; Liu,C.-M.; Tse,M.M.; Lin,E.T. Pre-column fluorescence derivatization using leucine-coumarnylamide
for HPLC determination of mono- and dicarboxylic acids in plasma, Ada Physiol.Hung., 1995, 83, 39-46.
SAMPLE
Sample preparation: Formed from the hydrolysis of enalapril with NaOH.
HPLCVARIABLES
Column: 5 jxm Ultrasphere ODS
Mobile phase: MeCN:50 mM phosphate buffer 12:88, pH 3.2
Flow rate: 1
Detector: UV
CHROMATOGRAM
KEYWORDS
perfusate buffer; rat
REFERENCE
Friedman,D.L; Amidon,G.L. Passive and carrier-mediated intestinal absorption components of two angiotensin
converting enzyme (ACE) inhibitor prodrugs in rats: enalapril and fosinopril, Pharm.Res., 1989, 6, 1043-
1047.
SAMPLE
Sample preparation: Dissolve tablets in MeCNrI mM pH 2 KH2PO4 50:50, centrifuge, inject a
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherisorb C8
Mobile phase: MeCN:buffer 35:65 (Buffer was 1 mM KH2PO4 adjusted to pH 2 with phosphoric
acid.)
Flow rate: 2.5
Detector: UV 215
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Simultaneous: degradation products, enalapril, felodipine
KEYWORDS
tablets
REFERENCE
Qin,X.-Z.; DeMarco,J.; Ip,D.P. Simultaneous determination of enalapril, felodipine and their degradation prod-
ucts in the dosage formulation by reversed-phase high-performance liquid chromatography using a Spher-
isorb C8 column, J.ChromatognA, 1995, 707, 245-254.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 300 X 3.9 ixBondapak phenyl
Mobile phase: MeOH:water:85% phosphoric acid 45:55:0.05
Column temperature: 30-40
Detector: UV 215-220
REFERENCE
Ranadive,S.A.; Chen,A.X.; Serajuddin,A.T. Relative lipophilicities and structural-pharmacological considera-
tions of various angiotensin-converting enzyme (ACE) inhibitors, Pharm.Res., 1992, 9, 1480-1486.
SAMPLE
Matrix: urine
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 10 mL MeOH, 10 mL water,
and 20 mL 100 mM HCl. 1 mL Urine + 10 JULL 6 M nitric acid, vortex for 30 s, add to the SPE
cartridge, wash with 20 mL 100 mM HCl, elute with 3 mL MeCN.water 10:90, elute with 6
mL water. Combine the eluates and evaporate the MeCN under a stream of air at 65, add 25
(xL 6 M nitric acid, add this solution to the SPE cartridge, wash with 10 mL chloroform, elute
with 6 mL MeOH. Evaporate the eluate to dryness under a stream of air at 65, wash the
residue with 1 mL MeCN, reconstitute with 500 |xL MeOHxhloroform 10:90, vortex for 30 s.
Put this solution in an another tube, evaporate to dryness, reconstitute with 100 |xL mobile
phase, inject a 10 u,L aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 |xm ixBondapak C18
Mobile phase: MeCN:MeOH:THF:15 mM pH 2.9 KH2PO4 6:1:1:92
Flow rate: 1.5
Detector: UV 206
CHROMATOGRAM
Internal standard: enalaprilat
OTHER SUBSTANCES
Extracted: lisinopril
KEYWORDS
SPE; enalaprilat is IS
REFERENCE
Wong,Y.-c.; Charles,B.G. Determination of the angiotensin-converting enzyme inhibitor lisinopril in urine using
solid-phase extraction and reversed-phase high-performance liquid chromatography, J.Chromatogr.B, 1995,
673, 306-310.
Encainide
CAS Registry No.: 66778-36-7, 37612-13-8, 66794-74-9 (HCI)
Merck Index: 3609
Lednicer No.: 3 56
SAMPLE
Matrix: blood
Sample preparation: 2 mL Serum or plasma + 100 jxL 10 jxg/mL trimethobenzamide in MeOH
+ 1 mL buffer + 10 mL n-butyl chloride:isopropanol 95:5, shake for 10 min, centrifuge at 500
g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of air at
40, reconstitute the residue in 200 jxL chloroform and 200 [xL 25 mM HCl, vortex for 10 s,
centrifuge at 500 g for 3-5 min, inject a 50-60 |xL aliquot of the upper aqueous layer. (Buffer
was 630 mL of a solution containing 1 M boric acid and 1 M KCl + 370 mL 1 M sodium
carbonate, adjust pH to 9.0.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm cyanopropyldimethylsilyl (PCN) (Supelco)
Mobile phase: MeCN:0.06% phosphoric acid 15:85 containing 0.01% octylamine
Flow rate: 2 (After analysis wash out system with MeCN:water 20:80.)
Detector: UV 254
CHROMATOGRAM
Retention time: 7
Internal standard: trimethobenzamide (5.5)
OTHER SUBSTANCES
Simultaneous: dipyridamole, oxazepam
Noninterfering: amiodarone, caffeine, chloral hydrate, chlordiazepoxide, diazepam, ethosuxim-
ide, flecainide, lidocaine, methadone, mexiletine, nicotine, phenobarbital, phenytoin, primidone,
procainamide, propranolol, quinidine, tocainide, tricyclic antidepressants
KEYWORDS
plasma; serum
REFERENCE
Dasgupta,A-l RosenzweigJ.B.; Turgeon,J.; Raisys,V.A. Encainide and metabolites analysis in serum or plasma
using a reversed-phase high-performance liquid chromatographic technique, J.Chromatogr., 1990,526,260-
265.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 fxL 5 jxg/mL 4-methylpropranolol in water + 500 u,L
buffer + 5 mL dichloromethanerdiethyl ether 70:30, shake vigorously for 5 min, centrifuge at
2000 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of
nitrogen at 45, reconstitute the residue in 200 |xL mobile phase, inject a 100 |xL aliquot. (Buffer
was pH 10.5 prepared from 200 mM sodium carbonate and 200 mM sodium bicarbonate.)
HPLC VARIABLES
Guard column: 10 mm long 5 |xm Resolve (Waters)
Column: 150 X 4.6 5 |jim Resolve (Waters)
Mobile phase: MeOH:water:methanesulfonic acid:triethylamine 400:4:0.02:0.02
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 7.0
Internal standard: 4-methylpropranolol (3.6)
OTHER SUBSTANCES
Simultaneous: amiodarone, propranolol, sotalol
Noninterfering: acebutolol, caffeine, disopyramide, hydroquinidine, nadoxolol, quinidine, theo-
bromine, theophylline
KEYWORDS
plasma
REFERENCE
Poirier,J.-M.; Lebot,M.; Cheymol,G. Analysis of encainide and its three major metabolites in plasma by column
liquid chromatography, J.Chromatogr., 1990, 534, 223-227.
SAMPLE
Matrix: blood
Sample preparation: Automated SPE by ASPEC system. Condition a C18 Clean-Up SPE car-
tridge (CEC 18111, Worldwide Monitoring) with 2 mL MeOH then 2 mL water. 1 mL Plasma
+ 1 mL 400 ng/mL protriptyline in water, vortex, add to column, wash with 3 mL water, wash
with 3 mL 750 mL/L methanol. Elute with three aliquots of 300 jxL 0.1 M ammonium acetate
in MeOH. Add 0.5 mL 0.5 M NaOH and 4 mL 50 mL/L isopropanol in heptane to eluate, mix
thoroughly. Allow 5 min for phase separation. Remove upper heptane phase and add it to 300
U.L 0.1 M phosphoric acid (pH 2.5), mix, separate, inject a 100 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Guard column: LC-8-DB (Supelco)
Column: 150 X 4.6 LC-8-DB (Supelco)
Mobile phase: MeCN:buffer 35:65 (Buffer was 10 mL/L triethylamine in water adjusted to pH
5.5 with glacial acetic acid.)
Flow rate: 2
Detector: UV 228
CHROMATOGRAM
Retention time: 1.7
Internal standard: protriptyline (4)
OTHERSUBSTANCES
Extracted: acetazolamide, amitriptyline, chlordiazepoxide, chlorimipramine, chlorpromazine,
desipramine, dextromethorphan, diazepam, diphenhydramine, doxepin, fentanyl, flecainide,
fluoxetine, flurazepam, haloperidol, hydroxyethylflurazepam, ibuprofen, imipramine, maproti-
line, methadone, methaqualone, midazolam, norchlorimipramine, nordiazepam, nordoxepin,
norfluoxetine, nortriptyline, norverapamil, promazine, propafenone, propoxyphene, proprano-
lol, protriptyline, temazepam, trazodone, trimipramine, verapamil
Noninterfering: acetaminophen, acetylmorphine, amiodarone, amobarbital, amphetamine, ben-
droflumethiazide, benzocaine, benzoylecgonine, benzthiazide, butalbital, carbamazepine, chlo-
rothiazide, clonazepam, cocaine, codeine, cotinine, cyclosporine, cyclothiazide, desalkylflura-
zepam, diamorphine, dicumerol, ephedrine, ethacrynic acid, ethanol, ethchlorvynol,
ethosuximide, furosemide, glutethimide, hydrochlorothiazide, hydrocodone, hydroflumethia-
zide, hydromorphone, lorazepam, mephentermine, meprobamate, methamphetamine, methar-
bital, methoxsalen, methoxyphenteramine, methsuximide, methylcyclothiazide, metoprolol,
MHPG, monoacetylmorphine, morphine, normethsuximide, oxazepam, oxycodone, oxymor-
phone, pentobarbital, phencyclidine, phenteramine, phenylephrine, phenytoin, polythiazide,
primidone, prochlorperazine, salicylic acid, sulfanilamide, THC-COOH, theophylline, thiazo-
lam, thiopental, thioridazine, tocainide, trichloromethiazide, trifluoperazine, valproic acid,
warfarin
Interfering: lidocaine, mexiletine, pentazocine, quinidine
KEYWORDS
plasma; SPE
REFERENCE
Nichols,J.H.; Charlson,J.R.; Lawson,G.M. Automated HPLC assay of fluoxetine and norfluoxetine in serum,
Clin.Chem., 1994, 40, 1312-1316.
SAMPLE
Sample preparation: 1 mL Plasma or urine + 200 JJLL 1 (plasma) or 50 (urine) (xL/mL ethaverine
hydrochloride in water + 100 (plasma) or 500 (urine) |xL 500 mM pH 8.5 TRIS-HCl buffer + 2
g NaCl + 5 mL butyl chloridedsopropanol 95:5, vortex for 15 s, centrifuge at 3000 g for 10 min,
repeat the extraction. Combine the organic layers and evaporate them to dryness in a vortex-
evaporator at 45, reconstitute the residue in 50 |xL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Zorbax SiI
Mobile phase: EtOH:water:methanesulfonic acid 500:25:0.5 (plasma) or 500:10:0.25 (urine)
Flow rate: 1.2
Detector: UV 270
CHROMATOGRAM
Retention time: 11.6 (plasma), 13.4 (urine)
Internal standard: ethaverine (6.9 (plasma), 7.6 (urine))
Limit of detection: 1 ng (plasma)
Limit of quantitation: 2.5 ng (plasma)
OTHER SUBSTANCES
Simultaneous: quinidine
Noninterfering: acetaminophen, alprazolam, aspirin, cephalexin, chloral hydrate, diazepam, di-
goxin, dipyridamole, docusate, flurazepam, furosemide, hydrochlorothiazide, ibuprofen, isosor-
bide dinitrate, lidocaine, lorazepam, meclizine, mexiletine, nifedipine, norfloxacin, oxazepam,
ranitidine, tocainide, triamterene, triazolam
KEYWORDS
REFERENCE
Turgeon,J.; Funck-Brentano,C; Gray,H-T.; Roden,D.M. Improved high-performance liquid chromatographic as-
say for encainide and its metabolites in human body fluids, J.Chromatogr., 1989, 490, 165-174.
SAMPLE
Matrix: bulk
Sample preparation: 0.01-5 |xg Encainide + 50 |xL MeCN:N,N-diisopropylethylamine 90:10 +
50 |xL (-)-menthyl chloroformaterMeCN 10:90, heat at 60 for 2 h, evaporate to dryness, recon-
stitute in mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere silica
Mobile phase: Hexane:ethyl acetate:triethylamine 85:15:1
Flow rate: 1
Detector: UV 261
CHROMATOGRAM
Retention time: 20 (+), 21 (-)
KEYWORDS
chiral; derivatization; normal phase
REFERENCE
Prakash,C; Jajoo,H-K.; Blair,I.A.; Mayol,R.F. Resolution of enantiomers of the antiarrhythmic drug encainide
and its major metabolites by chiral derivatization and high-performance liquid chromatography,
J.Chromatogr., 1989, 493, 325-335.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B)
Mobile phase: MeCN:0.025% phosphoric acid:buffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 mL
concentrated phosphoric acid and 10 mL triethylamine in 900 mL water, adjust pH to 3.4 with
dilute phosphoric acid, make up to 1 L.)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetaminophen, acetazolamide, acetophenazine, albu-
terol, alprazolam, amitriptyline, amobarbital, amoxapine, antipyrine, atenolol, atropine, aza-
tadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bupivacaine,
buspirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine, chlordi-
azepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlorpropamide,
chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonaze-
pam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dantrolene,
desipramine, diazepam, diclofenac, diflunisal, diltiazem, diphenhydramine, diphenidol, diphen-
oxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, ethidium
bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam,
flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, hom-
atropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine,
hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labe-
talol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic
acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone, meth-
dilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyldopa, meth-
ylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam, moclobem-
ide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizatidine,
norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, pen-
tazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital, phenol, phe-
nolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenôin, pimozide, pindolol,
piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine, prochlorpera-
zine, procyclidine, promazine, promethazine, propafenone, propantheline, propiomazine, pro-
pofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine,
remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spirono-
lactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophyl-
line, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide, tol-
metin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimeprazine,
trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Enilconazole
Merck Index: 3622
SAMPLE
Matrix: food
Sample preparation: Condition a 3 mL 500 mg Sep-Pak Vac Silica SPE cartridge with 10 mL
MeOH and 10 mL ethyl acetate. Condition a 3 mL 300 mg Bond Elut PRS cartridge with 10
mL water and 10 mL MeOH. Place the Sep Pak Vac Silica cartridge on top of the Bond Elut
PRS cartridge. Citrus fruit. Slice and homogenize citrus fruit with a mixer. 5 g Aliquot of
sample + 2Og anhydrous sodium sulfate + 1.5 g anhydrous sodium hydrogen phosphate + 30
mL ethyl acetate, blend at high-speed, centrifuge at 3100 rpm for 8 min, remove the super-
natant. Re-extract with 20 mL ethyl acetate, combine the supernatants. Add the crude extract
to the double SPE cartridge, allow to pass at ca. 1 mL/min, wash with 5 mL ethyl acetate,
remove the Sep-Pak Vac Silica cartridge. Wash the second SPE cartridge with 10 mL MeOH
and 10 mL 100 mM NaCl, elute with 10 mL mobile phase, inject a 20 (JLL aliquot. Banana. 10
g Homogenized sample + 4Og anhydrous sodium sulfate + 50 mL ethyl acetate, extract as
described above. Re-extract with 30 mL ethyl acetate, combine the supernatants. Add the crude
extract to the double SPE cartridge, allow to pass at ca. 1 mL/min, wash with 5 mL ethyl
acetate, remove the Sep-Pak Vac Silica cartridge. Wash the second SPE cartridge with 10 mL
MeOH and 10 mL 100 mM NaCl, elute with 10 mL mobile phase, inject a 20 JJLL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 jjim TSKgel ODS-80Ts (TOSOH, Japan)
Mobile phase: MeCN:MeOH:water 40:30:30 containing 10 mM sodium 1-tridecanesulfonate, ad-
justed to pH 2.5 with phosphoric acid
Flow rate: 1
Detector: UV 225
CHROMATOGRAM
Retention time: 17
Limit of detection: 100 ng/g (citrus fruit), 50 ng/g (banana)
OTHER SUBSTANCES
Extracted: thiabendazole
KEY WORDS
citrus fruit; banana; SPE
REFERENCE
Ito,Y.; Ikai,Y.; 0ka,H.; Hayakawa,J.; Kagami,T. Application of ion-exchange cartridge clean-up in food analysis.
I. Simultaneous determination of thiabendazole and imazalil in citrus fruit and banana using high-perfor-
mance liquid chromatography with ultraviolet detection, J.ChromatogrA, 1998, 810, 81-87.
Enocitabine
Merck Index: 3624
SAMPLE
Matrix: blood, bone marrow fluid, CSF, urine
Sample preparation: Plasma, CSF. 1 mL Plasma or CSF + 2 mL THF, mix, keep at 0 for 1 h,
centrifuge at 1000 g for 15 min, filter (0.45 |xm) the supernatant, inject a 10 |xL aliquot. Blood,
bone marrow fluid. 1 mL Blood or bone marrow fluid + 1 mL water + 3 mL THF, mix, keep at
0 for 1 h, centrifuge at 1000 g for 15 min, filter (0.45 |xm) the supernatant, inject a 10 (xL
aliquot. Urine. Centrifuge urine at 12000 g for 15 min, filter, inject a 10 |xL aliquot.
HPLCVARIABLES
Column: u-Bondapak C18
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
KEYWORDS
REFERENCE
Ueda,T.; Nakamura,T.; Ando,S.; Kagawa,D.; Sasada,M.; Uchino,H.; Johno,L; Akiyama,Y. Pharmacokinetics of
N4-behenoyl-l-(3-D-arabinofuranosylcytosine in patients with acute leukemia, Cancer Res., 1983,43, 3412-
3416.
Enoxacin
CAS Registry No.: 74011 -58-8
Merck Index: 3625
SAMPLE
Matrix: blood
Sample preparation: Filter 1 mL plasma using a micropartition system (MPS-I, Amicon, MA)
while centrifuging at 2000 g for 20 min at 10, inject an aliquot of the ultrafiltrate.
HPLC VARIABLES
Column: 250 X 4.6 Spherisorb ODS-2 endcapped
Mobile phase: MeCN.buffer 13:87 containing 5 mM tetrabutylammonium sulfate, adjusted to
pH 2.5 with 1 M NaOH (Buffer was 100 mM citric acid containing 200 mM ammonium
perchlorate.)
Flow rate: 1
Detector: UV 271
CHROMATOGRAM
Internal standard: ofloxacin (10.54)
KEYWORDS
plasma; ultrafiltrate
REFERENCE
Zlotos,G.; Biicker,A.; Kinzig-Schippers,M.; Sorgel,F.; Holzgrabe,U. Plasma protein binding of gyrase inhibitors,
J.Pharm.ScL, 1998, 87, 215-220.
SAMPLE
Matrix: blood
Sample preparation: 50 fxL Plasma + I m L 100 mM pH 7.0 K2HPO4 adjusted to pH 7.0 with
85% orthophosphoric acid + 100 |xL 300 |xg/mL nalidixic acid in water + 3 mL dichloromethane:
isoamyl alcohol 9:1, shake vigorously for 10 min, centrifuge at 2270 g for 10 min. Remove 2
mL of the organic phase and evaporate it to dryness under a stream of nitrogen at 40. Recon-
stitute residue in 100 |xL MeOH:50 mM NaOH 2:1, vortex, inject a 10 JULL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |jim Chemcosorb 5-ODS-H
Mobile phase: MeOH:5 mM sodium lauryl sulfate 2:1, adjusted to pH 2.5 with 85% phosphoric
acid (Better separation obtained at pH 2.35, J.Chromatogr. 1990, 530, 186.)
Flow rate: 0.6
Detector: UV 300
CHROMATOGRAM
Retention time: 6.2
Internal standard: nalidixic acid (5.0)
OTHER SUBSTANCES
Extracted: fenbufen, felbinac
Interfering: ofloxacin, norfloxacin
KEYWORDS
plasma; rat
REFERENCE
Katagiri,Y.; Naora,K.; Ichikawa,N.; Hayashibara,M.; Iwamoto,K. Simultaneous determination of ofloxacin, fen-
bufen and felbinac in rat plasma by high-performance liquid chromatography, J.Chromatogr., 1988, 431,
135-142.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 50 |xL 80 [xg/mL ciprofloxacin in water + 500 JULL 7%
perchloric acid, vortex for 10 s, centrifuge at >700 g for 10 min, inject a 20 JJLL aliquot of the
supernatant.
HPLC VARIABLES
Column: 100 X 8 u-Bondapak C18 Radial-PAK
Mobile phase: MeOH: 18 mM KH2PO4 containing 0.13 mM heptanesulfonic acidxoncentrated
phosphoric acid 30:70:0.1
Flow rate: 3
Detector: UV 268
CHROMATOGRAM
Retention time: 4.6
Internal standard: ciprofloxacin
KEYWORDS
serum
REFERENCE
Griggs,D.J.; Wise,R. A simple isocratic high-pressure liquid chromatographic assay of quinolones in serum,
J.Antimicrob.Chemother., 1989, 24, 437-445.
SAMPLE
Matrix: blood
Sample preparation: 100 JJLL Plasma + 100 |xL saturated sodium bicarbonate + 50 jxL 20 jxg/
mL ciprofloxacin in saturated sodium bicarbonate + 5 mL chloroform:isopropanol 90:10, shake
on a rotary mixer for 15 min, centrifuge at 800 g for 5 min. Evaporate organic layer under
nitrogen at 45, sonicate residue with 100 |xL mobile phase, inject 25 |xL aliquot.
HPLCVARIABLES
Guard column: 10 X 4.9 Spherisorb ODS
Column: 250 X 4.9 Spherisorb S5 ODS2
Mobile phase: MeCN:buffer 15:85 adjusted to pH 3.0 with 85% phosphoric acid immediately
before use (Buffer was 4.54 g KH2PO4 + 5.94 g Na2HPO4.2H2O + 1.49 g tetrabutylammonium
hydrogen sulfate per L.)
Flow rate: 1.3
Detector: UV 280
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
Simultaneous: theophylline, norfloxacin
KEYWORDS
plasma; rat
REFERENCE
Davis,J.D.; Aarons,L.; Houston,J.B. Simultaneous assay of fluoroquinolones and theophylline in plasma by
high-performance liquid chromatography, J.Chromatogr., 1993, 621, 105-109.
SAMPLE
Matrix: blood
Sample preparation: 150 |xL Plasma + 75 |xL 10% trichloroacetic acid + 600 |xL chloroform,
vortex for 5 min, centrifuge at 13800 g for 10 min. Remove 500 JJLL of the organic layer and
evaporate it to dryness under reduced pressure, reconstitute the residue in 150 |xL mobile
HPLC VARIABLES
Column: 80 X 4.6 5 |xm Zorbax C8
Mobile phase: MeOH:0.01% trifluoroacetic acid 25:75
Flow rate: 1.2
CHROMATOGRAM
Retention time: 5.8
Internal standard: enoxacin
OTHER SUBSTANCES
Extracted: norfloxacin
KEYWORDS
plasma; rat; enoxacin is IS
REFERENCE
Hussain,M.S.; Chukwumaeze-Obiajunwa,V.; Micetich,R-(j. Sensitive high-performance liquid chromatographic
assay for norfloxacin utilizing fluorescence detection, J.Chromatogr.B, 1995, 663, 379-384.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 3 mL MeCN, shake at 1000 rpm for 30 s, centrifuge at
2600 g for 10 min. Remove 3 mL of the supernatant and evaporate it to dryness under a stream
of nitrogen at 60, reconstitute the residue in 100 JJLL 60 mM KH2PO4 adjusted to pH 2.6 with
orthophosphoric acid, inject a 30 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3 7 |xm Separon SGX C18 (Tessek, Prague)
Mobile phase: THFrbuffer 5.5:94.5 (Buffer was 60 mM KH2PO4 adjusted to pH 2.6 with ortho-
phosphoric acid:triethylamine 97:3 containing 50 |xg/mL sodium azide. (Caution! Sodium azide
is carcinogenic and toxic and must not be discharged to the plumbing system!))
Flow rate: 0.7
CHROMATOGRAM
Retention time: 5.0
Internal standard: enoxacin
OTHER SUBSTANCES
Extracted: ofloxacin
KEY WORDS
plasma; enoxacin is IS
REFERENCE
Macek,J.; Ptcek,P. Determination of ofloxacin in human plasma using high-performance liquid chromatogra-
phy and fluorescence detection, J.Chromatogr.B, 1995, 673, 316-319.
SAMPLE
Matrix: blood, CSF, tissue
Sample preparation: Plasma, CSF. 100 |xL Plasma or CSF, add 1.0 mL 100 mM phosphate
buffer and 1 |xg IS in MeOH. Extract with 5 mL chloroform (Caution! Chloroform is a carcin-
ogen!) containing 1.0% ethyl chloroformate by shaking with a reciprocal shaker for 10 min.
Remove 4 mL organic phase, dry it with rotary evaporator at 40, reconstitute the residue in
100 |xL mobile phase and inject a 20 |xL aliquot. Tissue. Homogenize brain sample in 100 mM
pH 7.0 phosphate buffer 1:4. Mix 1 mL homogenate with IS, extract with 5 mL dichloromethane
by shaking with a reciprocal shaker for 10 min. Remove 4 mL organic phase and back-extract
with 4 mL 1 mM sodium hydroxide, extract the aqueous phase as described above for the
plasma, inject a 20 JJLL aliquot.
HPLCVARIABLES
Column: 250 X 6 Nucleosil 5C18
Mobile phase: MeOH:5 mM sodium lauryl sulfate 60:40, adjusted to pH 2.5 with phosphoric acid
Flow rate: 1.2
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: felbinac
KEYWORDS
plasma; brain; rat; derivatization
REFERENCE
Ohtani,H; Noma,S.; Kawakami,J.; Sawada,Y.; Iga,T. Lack of potentiation with felbinac patch on the convulsive
toxicity of enoxacin in rats, Biol.Pharm.Bull, 1996,19, 995-997.
SAMPLE
Matrix: blood, nasal secretions, saliva, sweat, tears, urine
Sample preparation: Plasma, saliva. 100 |xL Plasma or saliva + 25 |xM MeCN:perchloric acid
80:20, centrifuge, inject a 10 |xL aliquot of the supernatant. Urine. Dilute urine 1:20 or 1:200
with 100 mM pH 4.5 acetate buffer, inject a 10 |xL aliquot. Nasal secretions. Collect nasal
secretions on 25 mg cotton for 20 min, add 300 |JLL isotonic NaCl, after 1 h squeeze, centrifuge
the solution, inject a 50 |xL aliquot of the supernatant. Tears, sweat. Inject tears and sweat
directly.
HPLCVARIABLES
Column: 5 |xm Spherisorb ODS II C18
Mobile phase: MeCN:buffer 12:88 to 14:86 (Buffer was 100 mM citric acid containing 40 mM
ammonium perchlorate and 5 mM tetrabutylammonium hydroxide.)
Flow rate: 1-2
Detector: UV 340
CHROMATOGRAM
Limit of quantitation: 78 ng/mL (nasal secretions), 1.95 jig/mL (urine), 39 ng/mL (plasma, sa-
liva, tears, sweat)
OTHER SUBSTANCES
Simultaneous: metabolites, oxoenoxacin
KEYWORDS
REFERENCE
Jaehde,U.; Sorgel,E; Naber,K.G.; Ziircher,J.; Schunack,W. Distribution kinetics of enoxacin and its metabolite
oxoenoxacin on excretory fluids of healthy volunteers, Antimicrob.Agents Chemother., 1995, 39, 2092-
2097.
SAMPLE
Matrix: blood, saliva
Sample preparation: 500 |xL Plasma or saliva + 50 |xL 50 ng/mL difloxacin, vortex briefly, add
500 (JLL 100 mM pH 7.4 phosphate buffer, add 4 mL dichloromethane, add 1 mL isopropanol,
vortex for 30 s, shake gently for 30 min, centrifuge at 1500 g for 20 min. Remove the lower
residue in 500 |JLL mobile phase, inject a 50-200 |xL aliquot.
HPLC VARIABLES
Guard column: ixBondapak C18 Guard-Pak
Column: 300 X 3.9 10 jxm ixBondapak C18
Mobile phase: MeOH:buffer 35:100 (Buffer was 5.44 g KH2PO4 and 4 mL tetrabutylammonium
hydroxide in 1 L water, adjust pH to 2.5 with 85% phosphoric acid.)
Flow rate: 2
Detector: UV 268
CHROMATOGRAM
Retention time: 5.2
Internal standard: difloxacin (8.8)
OTHER SUBSTANCES
Extracted: ciprofloxacin, theophylline
Simultaneous: 1,7-dimethylxanthine
Noninterfering: 1,3-dimethyluric acid, hypoxanthine, 1-methyluric acid, 1-methylxanthine, 3-
methylxanthine, 7-methylxanthine, theobromine
Interfering: caffeine
KEY WORDS
REFERENCE
Zhai,S.; Korrapati,M.R.; Wei,X.; Muppalla,S.; Vestal,R.E. Simultaneous determination of theophylline, enoxacin
and ciprofloxacin in human plasma and saliva by high-performance liquid chromatography, J. Chromatogr.B,
1995, 669, 372-376.
SAMPLE
Sample preparation: Plasma. Mix 100 (JLL plasma with 900 |xL 100 mM phosphate buffer, 100
(xL 10 fxg/mL IS and 5 mL chloroform:ethyl chloroformate 99:1, shake for 10 min, centrifuge
at 1620 g for 5 min, evaporate the organic phase under reduced pressure, dissolve the residue
in 100 |xL MeOH:50 mM NaOH 2:1, inject a 20 |xL aliquot. Tissue. Homogenate the cerebrum
sample with 4 volumes of 100 mM phosphate buffer. Mix 1 mL homogenate with 100 u.L 10
fjLg/mL IS and 5 mL dichloromethane, shake for 10 min, centrifuge at 1620 g for 5 min. Mix 4
mL 1 mM NaOH with 4 mL organic phase, shake for 10 min, centrifuge it at 1620 g for 5 min,
collect 3 mL aqueous phase and treat in a manner similar to that for the plasma samples,
except for the IS addition. Inject a 20 JJLL aliquot. (Caution! Chloroform is a carcinogen!)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Nucleosil 5 C 18
Mobile phase: MeOH:5 mM sodium dodecylsulfate adjusted to pH 2.5 with phosphoric acid
Flow rate: 0.8
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: foscarnet
KEYWORDS
plasma; brain; mouse; pharmacokinetics; derivatization
REFERENCE
Matsuo,H-; Ryu,M.; Nagata,A.; Uchida,T.; Kawakami,J.-L; Yamamoto,K.; Iga,T.; Sawada,Y. Neurotoxicody-
namics of the interaction between ciprofloxacin and foscarnet in mice, Antimicrob.Agents Chemother., 1998,
42, 691-694.
SAMPLE
Sample preparation: Blood. Briefly vortex 500 |xL plasma, 20 imL 8 jxg/mL pefloxacin, and 500
IxL pH 7.4 phosphate buffer, add 5 mL dichloromethanedsopropanol 80:20, vortex for 30 s,
shake gently for 15 min on an electric shaker, centrifuge at 1500 g for 10 min. Separate the
lower layer using phase-separating filter paper (Whatman 1 PS), evaporate the organic phase
under nitrogen at 25, dissolve the residue in 100 |xL mobile phase, inject a 20|xL aliquot.
Tissue. Pulverize prostatic tissue under liquid nitrogen, weigh a 200 mg aliquot, add 20 JJIL
pefloxacin, vortex briefly, incubate at 4 for 2 h, add 500 jxL pH 7.4 phosphate buffer and 5 mL
dichloromethane:isopropanol 80:20, vortex for 30 s, shake gently for 15 min on an electric
shaker, centrifuge at 1500 g for 10 min. Separate the lower layer using phase-separating filter
paper (Whatman 1 PS), evaporate the organic phase under nitrogen at 25, dissolve the residue
in 100 |xL mobile phase, inject a 20|xL aliquot. (The pH 7.4 phosphate buffer was 28.2 g K2HPO4
and 5.17 g KH2PO4 in 1 L water.)
HPLCVARIABLES
Guard column: 4 X 4 5 fim LiChrospher 100 RP-18
Column: 250 X 4 5 jjun Nucleosil C18
Mobile phase: MeCNrpH 2.1 buffer 20.9:79.1 (Prepare the mobile phase by dissolving 18.1 g
citric acid and 4.1g ammonium perchlorate in about 300 mL distilled water, add 209 mL MeCN,
dilute to 1 L with water, and add 3 mL tetrabutylammonium hydroxide. Filter through a 0.45
ixm HV Millipore filter.)
Flow rate: 0.9
Detector: UV 340
CHROMATOGRAM
Retention time: 5.2
Internal standard: pefloxacin (6.8)
Limit of detection: 10 ng/mL (plasma), 25 ng/mL (tissue)
Limit of quantitation: 20 ng/mL (plasma), 50 ng/mL (tissue)
OTHER SUBSTANCES
Extracted: 4-oxo-enoxacin
Noninterfering: amikacin, ciprofloxacin, fosfomycin, ofloxacin, rifampicin, roxithromycin, tobra-
mycin, vancomycin
KEYWORDS
pharmacokinetics; plasma; prostatic tissue; prostate
REFERENCE
Hamel,B-; Audran,M.; Costa,R; Bressolle,F. Reversed-phase high-performance liquid chromatographic deter-
mination of enoxacin and 4-oxo-enoxacin in human plasma and prostatic tissue. Application to a pharma-
cokinetic study, J.ChromatogrA, 1998, 812, 369-379.
SAMPLE
Sample preparation: Plasma. Condition a Sep-Pak C18 SPE cartridge with 1 mL 4% phosphoric
acid in MeOH and 10 mL water. Add 1 mL plasma to the SPE cartridge, wash with 6 mL
water, elute with 1 mL 4% phosphoric acid in MeOH, wash out with 1 mL water. Combine the
eluates and make up to 2 mL with water, inject a 50 |xL aliquot. Urine. 100 (xL urine + 900
(xL mobile phase, inject a 10 fxL aliquot.
HPLCVARIABLES
Guard column: (xBondapak C18/Corasil
Column: 300 X 3.9 ^Bondapak C18
Mobile phase: MeCN:MeOH:water 5:30:70 containing 1.74 g K2HPO4 and 20 mg sodium hep-
tanesulfonate, pH adjusted to 3 with phosphoric acid
Flow rate: 2
OTHER SUBSTANCES
KEYWORDS
plasma; SPE; this procedure can be used for enoxacin (see Infection 1986; 14 (Suppl. 3); S203)
REFERENCE
Gutzler,R; de Vries,J.X. Bestimmung von Norfloxacin in Plasma und Urin durch Hochdruckflussigkeitschro-
matographie, Fortschr.Antimikr.Antineoplast.Chemother., 1984, 3, 673-677.
SAMPLE
Sample preparation: Plasma. 500 JJLL Plasma + 50 |JLL 20 |jLg/mL difloxacin + 200 jxL MeCN:
60% perchloric acid 80:20, vortex, centrifuge for 5 min, inject a 75 |xL aliquot of the superna-
tant. Urine. 200 |xL Urine + 500 |xL water + 200 jxL 200 jig/mL difloxacin + 200 |xL pH 7.4
phosphate buffer. Pass through an Analytichem C18 SPE cartridge, inject a 10 |JLL aliquot of
the eluate.
HPLC VARIABLES
Column: 100 X 4.6 5 ^m RAC II Partisil ODS 3
Mobile phase: MeCN:buffer 15:85 (Buffer was 100 mM citrate containing 650 |xL/L 20% tetra-
butylammonium hydroxide and 450 mg/L ammonium perchlorate.)
Flow rate: 1
Detector: UV 340
CHROMATOGRAM
Internal standard: difloxacin
Limit of quantitation: 833 ng/mL (urine), 25 ng/mL (plasma)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Grasela,T.H.,Jr.; Schentag,J.J.; Sedman,AJ-; Wilton,J.H.; Thomas,D.J.; Schultz,R.W.; Lebsack,M.E.; Kin-
kel,A.W. Inhibition of enoxacin absorption by antacids or ranitidine, Antimicrob.Agents Chemother., 1989,
33, 615-617.
SAMPLE
residue with 50 (JLL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 268.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y.; Ppin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:50 mM phosphoric acid 25:75 containing 200 mM KCl
Flow rate: 0.6
Detector: UV 265
REFERENCE
Sugawara,M.; Takekuma,Y; Yamada,H.; Kobayashi,M.; Iseki,K; Miyazaki,K. A general approach for the pre-
diction of the intestinal absorption of drugs: regression analysis using the physicochemical properties and
drug-membrane electrostatic interactions, J.Pharm.Sci., 1998, 87, 960-966.
SAMPLE
Matrix: solutions
Sample preparation: Filter (0.45 |xm) a solution in MeCN:water 10:90, inject an aliquot of the
filtrate.
HPLC VARIABLES
Column: 250 X 4 5 |jim LiChrospher 100 RP-18
Mobile phase: MeCN.buffer 7:93 (Buffer was 25 mM phosphoric acid adjusted to pH 3.89 with
100 mM tetrabutylammonium hydroxide.)
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 7.8
OTHER SUBSTANCES
Simultaneous: ciprofloxacin, fleroxacin, norfloxacin, ofloxacin (UV 295), pipemidic acid
REFERENCE
Barbosa,J.; Berges,R.; Sanz-Nebot,V. Solvatochromic parameter values and pH in aqueous-organic mixtures
used in liquid chromatography. Prediction of retention of a series of quinolones, J.Chromatogr.A, 1996, 719,
27-36.
Enoximone
Molecular formula: C12H12N2O2S
CAS Registry No.: 77671 -31 -9
Merck Index: 3627
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 |xL 4.16 |xg/mL IS in MeOH + 3 mL MeCN, vortex,
add 2 mL 100 mM pH 7.5 sodium phosphate buffer, centrifuge at 900 g for 20 min. Remove 6
mL of the supernatant and add it to 9 mL ethyl acetate, shake on a reciprocating shaker for
20 min, centrifuge. Remove 10 mL of the organic layer and evaporate it to dryness under a
stream of nitrogen at 50-55, reconstitute the residue in 200 |xL MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 313
CHROMATOGRAM
Retention time: 6.0
Internal standard: 1,3-diethyl-1,3-dihydro-4-(4-methoxybenzoyl)-5-methyl-2H-imidazol-2-one
(MDL 17,043) (11.5)
KEYWORDS
plasma; dog; pharmacokinetics
REFERENCE
Chan,K.Y.; Lang,J.F.; Okerholm,R.A. Quantitative determination of cardiotonic agent MDL 17,043 in plasma
by reversed-phase high-performance liquid chromatography, J.Chromatogr., 1983, 272, 396400.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 (xL 6 u,g/mL ID in MeOH + 3 mL MeCN, vortex,
centrifuge at 900 g for 20 min. Remove the supernatant and add it 9 mL ethyl acetate, shake
for 20 min, centrifuge for 5 min. Remove 10 mL of the upper organic layer and evaporate it to
dryness under a stream of nitrogen at 50-55, reconstitute the residue in 100 JJLL MeOH, inject
a 20 u,L aliquot.
HPLCVARIABLES
Column: 250 X 4.6 6 urn Zorbax CN
Flow rate: 1
Detector: UV 313
CHROMATOGRAM
Retention time: 6.8
Internal
(1 5) standard: l-ethyl-4-methyl-5-[p-methoxybenzoyl]-4-imidazolin-2-one (MDL 18,763)
. .'
Limit of quantitation: 62.5 ng/mL
OTHER SUBSTANCES
Noninterfering: chlorthalidone, clonidine, digitalis, digoxin, furosemide, hydralazine, hydro-
chlorothiazide, isosorbide dinitrate, levothyroxine (synthroid), methyldopa, nitroglycerin, phen-
azopyridine, sulfamethoxazole (gantanol), tolazamide (tolinase), zomepirac
KEYWORDS
REFERENCE
Chan,K.Y; Ohlweiler,D.R; Lang,J.E; Okerholm,R.A. Simultaneous analysis of a new cardiotonic agent, MDL
17,043, and its major sulfoxide metabolite in plasma by high-performance liquid chromatography,
J.Chromatogr., 1984, 306, 249-256.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL 3 jxg/mL + 1.5 mL MeCN, vortex for 15 s, centrifuge
at 3000 g for 10 min. Add the supernatant to 5 mL ethyl acetate, rotate at 20 rpm for 20 min,
centrifuge at 3000 g for 10 min. Remove the organic layer and evaporate it to dryness under
a stream of nitrogen at 40-45, reconstitute the residue in 120 fxL MeOH:water 20:80, inject a
20 |JLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Ultrasphere ODS
Mobile phase: Gradient. MeCN:water 86:14 for 3 min, to 10:90 (step gradient), maintain at 10:
90 for 6 min, re-equilibrate at initial conditions for 10 min.
Flow rate: 1
Detector: UV 365
CHROMATOGRAM
Internal standard: MDL 82249 (7.75)
OTHER SUBSTANCES
Noninterfering: acenocoumarol, allopurinol, amikacin, amiloride, amiodarone, aspirin, broma-
zepam, captopril, cefoperazone, ceftriaxone, chlorazepate, clonidine, digitoxin, dihydralazine,
diltiazem, dipyridamole, doxycycline, flumequine, furosemide, gentamicin, heparin, hydro-
chlorothiazide, isosorbide dinitrate, lidocaine, lorazepam, meperidine, mezlocillin, minocycline,
nalidixic acid, netilmicin, nifedipine, nitrazepam, nitroglycerin, penicillin G, propafenone, pro-
tamine, sodium nitroprusside, spironolactone, ticarcillin
KEYWORDS
REFERENCE
Tarral,E.; Jehl,R; Gallion,C; Monteil,H. Dosage de Fenoximone et de son principal metabolite dans Ie serum
et l'urine par chromatographie liquide a haute performance [Determination of enoximone and its principle
metabolite in serum and urine using high performance liquid chromatography], Therapie, 1990, 45, 1-6.
SAMPLE
Matrix: blood, dialysate
Sample preparation: The dialyzer had two 300 mm path length blocks in series. The membrane
was Cuprophan C-type (Technicon) with a cut-off of 10000 Daltons. Dialyze 600 (JLL serum
against 10 mM ammonium phosphate buffer, pump the buffer continuously through the dia-
lyzer and through a trace-enrichment column packed with 20 mg 5 |xm Hypersil ODS at 2.5
mL/min, after 3 min flush the contents of the trace-enrichment column onto the analytical
column with the mobile phase, after 3 min remove the trace-enrichment column from the
circuit, monitor the effluent from the analytical column.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Spherisorb ODS II
Mobile phase: Gradient. A was bufferrwater 4:96. B was MeCN.buffer.water 70:4:26. A:B 85:15
for 2.5 min, to 40:60 over 4 min, return to initial conditions over 0.5 min, re-equilibrate for 4
min. (Buffer was 38.5 g ammonium acetate in 800 mL water, adjust the pH to 5.0 with glacial
acetic acid, make up to 1 L with water.)
Flow rate: 2
Detector: UV 335
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
KEYWORDS
serum; column-switching; trace enrichment; pharmacokinetics
REFERENCE
Cooper,J.D.; Turnell,D.C. Automatic preparation of human serum samples for analysis of the drug enoximone
and its sulphoxide metabolite using high-performance liquid chromatography, J.Chromatogr., 1986, 380,
109-116.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 1 mL water + 100 |xL 6 (xg/mL IS in MeOH, add
to a Bond Elut C18 SPE cartridge, wash with 18 mL water, elute with 3 mL MeOH. Evaporate
the eluate to dryness under a stream of nitrogen at 40, reconstitute the residue in 200 |xL
mobile phase, inject a 20 |xL aliquot. Urine. 300 |xL Urine + 700 |xL 1 M pH 9.0 Tris-HCl buffer
+ 1 mL water + 100 |xL 6 |Ag/mL IS in MeOH, add to a Bond Elut C18 SPE cartridge, wash
with 18 mL water, elute with 3 mL MeOH. Evaporate the eluate to dryness under a stream of
nitrogen at 40, reconstitute the residue in 200 JULL mobile phase, inject a 20 JLJLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Zorbax CN
Flow rate: 1
Detector: UV 340
CHROMATOGRAM
Internal standard: MDL 18,763
KEYWORDS
REFERENCE
Morita,S.; Sawai,Y.; Heeg,J.E; Koike,Y. Pharmacokinetics of enoximone after various intravenous administra-
tions to healthy volunteers, J.Pharm.Sci., 1995, 84, 152-157.
Enprostil
Molecular formula: C23H28O6
Merck Index: 3629
Lednicer No.: 4 10
Next Page
SAMPLE
Matrix: blood
Sample preparation: Prepare a SPE cartridge by adding 150 mg 40 [xm Bondesil phenyl (An-
alytichem) to a 5 mL column. Condition SPE cartridge with 2 mL MeOH, 2 mL water, and 1
mL 20 mM pH 3.0 sodium acetate buffer. 2 mL Plasma + IS (500-1000 cpm), filter (20 jim),
add to SPE cartridge, wash with 300 JJLL pH 3 buffer, wash with 1 mL MeOHrwater 40:60,
wash with 300 |xL 0.3% acetic acid, wash with 1 mL water, elute with 1.5 mL MeOHiwater 60:
40. Evaporate the eluate under a stream of nitrogen, reconstitute in 125 u,L 1 mg/mL 2-bro-
moacetyl-6-methoxynaphthalene in MeCN and 100 |xL 34.6 mg/mL 18-crown-6 in MeCN, add
about 2 mg anhydrous sodium sulfate, add about 2 mg potassium carbonate, shake gently for
1 h, evaporate under nitrogen, dissolve the residue in 250 |xL dichloromethane. Inject a 200
|JLL aliquot onto a 100 X 4.6 5 (xm Spheri-5 silica column, elute to waste with dichloromethane:
MeCN 20:70, elute a 1 min fraction containing the analyte onto a 220 X 4.6 5 jxm Spheri-5
silica column, elute this column with the same mobile phase, collect a fraction containing the
analyte. Evaporate the fraction to dryness and reconstitute it in 1 mL dichloromethane, add
it to an AASP silica SPE cartridge (Analytichem), elute the contents of the cartridge onto
column A with mobile phase A for 1 min, elute column A to waste with mobile phase A, elute
a 1 min fraction containing the analyte and mix it with water pumped at 2 mL/min, the
combined effluent flows onto column B. At the end of this time elute column B with mobile
phase B onto column C, elute a 1 min fraction containing the analyte and mix it with water
pumped at 0.15 mL/min, store the diluted column effluent in a 600 jxL sample loop. Flush the
contents of the sample loop onto column D with mobile phase C for 8 min, elute the contents
of column D with mobile phase D onto column E, monitor the effluent from column E.
HPLCVARIABLES
Column: A 250 X 4.6 7 |xm Chemcosorb 7CN (Dychrom); B 250 X 4.6 5 |xm Spheri-5 C18; C 250
X 1 5 jxm Hypersil C18; D 30 X 1 5 fxm Hypersil C18; E 150 X 1 3 (xm Hypersil C18
Mobile phase: A MeOH:water:acetic acid 40:60:0.1; B MeOH:water:acetic acid 50:50:0.1; C wa-
ter; D MeOH:water 40:60
Flow rate: A 1; B 0.05; C 0.1; D 0.05
Detector: F ex 325 em 450 (Corrion S40-450 and LL-400 filters), laser fluorescence, specially
constructed apparatus
CHROMATOGRAM
Retention time: 20 (of the corresponding acid)
Internal standard: tritiated enprostil acid
KEY WORDS
plasma; SPE; derivatization; column-switching; normal phase; reversed phase; heart cut; micro-
bore; laser fluorescence
REFERENCE
Kiang,C.H.; Nolan,T.; Huang,BL.; Lee,C.P. Determination of femtomole/milliliter concentrations of enprostil
acid in human plasma using high-performance liquid chromatography-laser-induced fluorescence detection,
J.Chromatogr., 1991, 567, 195-212.
Enrofloxacin
Merck Index: 3630
Lednicer No.: 5 125
SAMPLE
M a t r i x : blood
Previous Page
SAMPLE
Matrix: blood
Sample preparation: Prepare a SPE cartridge by adding 150 mg 40 [xm Bondesil phenyl (An-
alytichem) to a 5 mL column. Condition SPE cartridge with 2 mL MeOH, 2 mL water, and 1
mL 20 mM pH 3.0 sodium acetate buffer. 2 mL Plasma + IS (500-1000 cpm), filter (20 jim),
add to SPE cartridge, wash with 300 JJLL pH 3 buffer, wash with 1 mL MeOHrwater 40:60,
wash with 300 |xL 0.3% acetic acid, wash with 1 mL water, elute with 1.5 mL MeOHiwater 60:
40. Evaporate the eluate under a stream of nitrogen, reconstitute in 125 u,L 1 mg/mL 2-bro-
moacetyl-6-methoxynaphthalene in MeCN and 100 |xL 34.6 mg/mL 18-crown-6 in MeCN, add
about 2 mg anhydrous sodium sulfate, add about 2 mg potassium carbonate, shake gently for
1 h, evaporate under nitrogen, dissolve the residue in 250 |xL dichloromethane. Inject a 200
|JLL aliquot onto a 100 X 4.6 5 (xm Spheri-5 silica column, elute to waste with dichloromethane:
MeCN 20:70, elute a 1 min fraction containing the analyte onto a 220 X 4.6 5 jxm Spheri-5
silica column, elute this column with the same mobile phase, collect a fraction containing the
analyte. Evaporate the fraction to dryness and reconstitute it in 1 mL dichloromethane, add
it to an AASP silica SPE cartridge (Analytichem), elute the contents of the cartridge onto
column A with mobile phase A for 1 min, elute column A to waste with mobile phase A, elute
a 1 min fraction containing the analyte and mix it with water pumped at 2 mL/min, the
combined effluent flows onto column B. At the end of this time elute column B with mobile
phase B onto column C, elute a 1 min fraction containing the analyte and mix it with water
pumped at 0.15 mL/min, store the diluted column effluent in a 600 jxL sample loop. Flush the
contents of the sample loop onto column D with mobile phase C for 8 min, elute the contents
of column D with mobile phase D onto column E, monitor the effluent from column E.
HPLCVARIABLES
Column: A 250 X 4.6 7 |xm Chemcosorb 7CN (Dychrom); B 250 X 4.6 5 |xm Spheri-5 C18; C 250
X 1 5 jxm Hypersil C18; D 30 X 1 5 fxm Hypersil C18; E 150 X 1 3 (xm Hypersil C18
Mobile phase: A MeOH:water:acetic acid 40:60:0.1; B MeOH:water:acetic acid 50:50:0.1; C wa-
ter; D MeOH:water 40:60
Flow rate: A 1; B 0.05; C 0.1; D 0.05
Detector: F ex 325 em 450 (Corrion S40-450 and LL-400 filters), laser fluorescence, specially
constructed apparatus
CHROMATOGRAM
Retention time: 20 (of the corresponding acid)
Internal standard: tritiated enprostil acid
KEY WORDS
plasma; SPE; derivatization; column-switching; normal phase; reversed phase; heart cut; micro-
bore; laser fluorescence
REFERENCE
Kiang,C.H.; Nolan,T.; Huang,BL.; Lee,C.P. Determination of femtomole/milliliter concentrations of enprostil
acid in human plasma using high-performance liquid chromatography-laser-induced fluorescence detection,
J.Chromatogr., 1991, 567, 195-212.
Enrofloxacin
Merck Index: 3630
Lednicer No.: 5 125
SAMPLE
M a t r i x : blood
Sample preparation: Add 20 |xL 10 |xg/mL IS in MeOH:0.1% trifluoroacetic acid 15:85 and 5
|xL (sic) MeCN to 300 |xL plasma. Centrifuge at 600 g for 10 min. Evaporate the supernatant
under nitrogen at 40 for 30 min. Reconstitute the residue in 200 |xL MeOH:0.1% trifluoroacetic
acid 15:85. Inject a 50 jxL aliquot.
HPLC VARIABLES
Mobile phase: MeCN: water trifluoroacetic acid 19:81:0.02
Flow rate: 1
Detector: UV 279
CHROMATOGRAM
Retention time: 5.6-5.8
Internal standard: norfloxacin (3.8-3.9)
OTHER SUBSTANCES
Extracted: ciprofloxacin
KEY WORDS
cat; plasma
REFERENCE
Kordick,D.L.; Papich,M.G.; Breitschwerdt,E.B. Efficacy of enrofloxacin or doxycycline for treatment of Barto-
nella henselae or Bartonella clarridgeiae infection in cats, Antimicrob.Agents Chemother., 1997, 41, 2448-
2455.
SAMPLE
Matrix: blood
HPLC VARIABLES
Mobile phase: MeCN-.buffer 20:80 containing 5 mM tetrabutylammonium sulfate, adjusted to
perchlorate.)
Flow rate: 1
Detector: UV 279
CHROMATOGRAM
Internal standard: ciprofloxacin (4.82)
KEYWORDS
REFERENCE
J.Pharm.Sci., 1998, 87, 215-220.
SAMPLE
Matrix: blood, milk
Sample preparation: 500 |xL Milk or plasma + 500 (xL MeCN: 100 mM NaOH, vortex for 10-15
s, filter (Centricon-3, 3000 Dalton cut-off) while centrifuging at 4000 g for 30 min, inject a 50-
150 |xL aliquot of the ultrafiltrate.
HPLCVARIABLES
Column: 250 X 4.6 3 jxm Spherisorb phenyl
Mobile phase: MeCN:MeOH:triethylamine:85% phosphoric acid:water 9:9:0.45:0.4:81.15 con-
taining 5 mM dodecanesulfonate
Flow rate: 1
Detector: UV 278
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
plasma; cow; ultrafiltrate
REFERENCE
Tyczkowska,K.L.; Voyksner,R.D.; Anderson,K.L.; Papich,M.G. Simultaneous determination of enrofloxacin and
its primary metabolite ciprofloxacin in bovine milk and plasma by ion-pairing liquid chromatography,
J.Chromatogr.B, 1994, 658, 341-348.
SAMPLE
Sample preparation: Serum. 500 uL Serum + 500 uL MeCN:100 mM NaOH 50:50, vortex for
10-15 s, filter (Amicon Centricon-10, 10000 Daltons) while centrifuging at 2677 g for 30 min,
inject a 30-120 |xL aliquot of the ultrafiltrate. Tissue. Cut up prostate tissue with a scalpel.
Weigh out 100-130 mg tissue, make up to 500 jxL with MeCN:100 mM NaOH 50:50, sonicate
for 30 min, filter (Amicon Centricon-10, 10000 Daltons) while centrifuging at 2677 g for 30
min, inject a 80-120 JJLL aliquot of the ultrafiltrate.
HPLCVARIABLES
Column: 100 X 4.6 3 |xm Spherisorb phenyl
Mobile phase: MeCN:MeOH:water 15:2:83 containing 3 mM dodecanesulfonate, 1.5 mM oc-
tanesulfonate, 0.4% phosphoric acid, and 0.4% triethylamine
Detector: UV 278.6
CHROMATOGRAM
OTHER SUBSTANCES
KEY WORDS
serum; dog; prostate; ultrafiltrate
REFERENCE
Tyczkowska,K; Hedeen,K.M.; Aucoin,D.R; Aronson,A.L. High-performance liquid chromatographic method for
the simultaneous determination of enrofloxacin and its primary metabolite ciprofloxacin in canine serum
and prostatic tissue, J.Chromatogr., 1989, 493, 337-346.
SAMPLE
Matrix: milk
Sample preparation: Condition a 500 mg Bond Elut LRC PRS SPE cartridge with 5 mL MeOH
and 5 mL extracting solution 65:35. Add 25 mL extracting solution to 5 mL milk, shake for 15
s, add 4 g anhydrous sodium sulfate, shake for 15 s, centrifuge at 3000 rpm at 5 for 5 min.
Remove the supernatant and repeat the extraction with 25 mL extracting solution as before
except do not add any more sodium sulfate, mix mechanically, centrifuge, combine the super-
natants, add 25 mL 1% acetic acid, shake for 10-15 s. Freeze for 30 min to facilitate precipi-
tation, centrifuge at 2500 rpm at 5 for 10 min. Add 75 mL to the SPE cartridge, pass the
entire sample through the cartridge, then add 2 mL MeOH, wash with 5 mL water, wash with
2 mL MeOH. Elute with 2.5 mL 25% ammonium hydroxide-MeOH. Evaporate to dryness under
nitrogen at 55, dissolve the residue in 2 mL 1% acetic acid, sonicate for 1 min, vortex for 20
s, filter (0.45 jxm), inject an aliquot. (Extracting solution was 1% aqueous acetic acid:EtOH 1:
99.)
HPLC VARIABLES
Column: 150 X 4.6 5 (xm Inertsil
Mobile phase: MeCN:2% acetic acid 15:85
Flow rate: 1
Detector: F ex 278 em 450, with a 418 nm cut-off filter
CHROMATOGRAM
Retention time: 4.2
Limit of detection: 0.3 ppb
Limit of quantitation: 5 ppb
OTHER SUBSTANCES
Extracted: ciprofloxacin, difloxacin, sarafloxacin
KEYWORDS
SPE
REFERENCE
Roybal,J.E.; Pfenning,A.R; Turnipseed,S.B.; Walker,C.C; Hurlbut,J.A. Determination of four fluoroquinolones
in milk by liquid chromatography, J.AOAC Int., 1997, 80, 982-987.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 4 |xm NovaPak C18
Mobile phase: MeCN:MeOH:buffer:acetic acid 2.5:10:86.5:1 containing 20 mM triethylamine
(The pH 2.7 buffer was 0.4% diammonium hydrogen phosphate in water containing 0.4% (?)
tetrabutylammonium hydrogen sulfate.)
Flow rate: 1
Detector: UV 279
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ciprofloxacin, ofloxacin
REFERENCE
Cester,C.C; Toutain,P.L. A comprehensive model for enrofloxacin to ciprofloxacin transformation and disposi-
tion in dog, J.Pharm.Sci., 1997, 86, 1148-1155.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 10 mL 500 mg Bond Elut LRC PRS SPE cartridge with 2 mL
MeOH and 2 mL equilibrating solution. 2 g Catfish muscle + 18 mL extracting solution, ho-
mogenize for 20 s, centrifuge at 3000 rpm for 5 min, decant the supernatant. Add another 18
mL extracting solution to the pellet and homogenize again, centrifuge at 3000 rpm for 5 min,
combine the supernatants. Add 20 mL 1% glacial acetic acid, freeze for 30 min, centrifuge at
2500 rpm at 4 for 10 min. Add the extracts to the SPE cartridge, wash with 2 mL MeOH, 5
mL water, and 2 mL MeOH. Let the SPE cartridge dry for 30 s. Elute with 2 mL MeOH:30%
ammonium hydroxide 80:20, dry the eluate under nitrogen at 50. Reconstitute the residue in
500 |JLL mobile phase, filter (0.45 ixm), inject an aliquot. (The extracting solution was EtOH:
water-.glacial acetic acid 98:1:1. The equilibrating solution was extracting solution: 1% glacial
acetic acid 35:20.)
HPLC VARIABLES
Column: 150 X 2 5 ^m Inertsil Phenyl
Mobile phase: MeCN:2% formic acid 14:86
Flow rate: 0.35
Detector: MS, Hewlett-Packard 5989, Model 59987A electrospray, nitrogen drying gas 40 mL/
min, 260, nebulizing gas nitrogen, 80 psi, m/z 245
CHROMATOGRAM
Limit of detection: 10 ppb
OTHER SUBSTANCES
KEY WORDS
catfish; muscle; SPE
REFERENCE
Turnipseed,S.B.; Walker,C.C; Roybal,J.E.; Pfenning,A.P.; Hurlbut,J.A. Confirmation offluoroquinolonesin cat-
fish muscle by electrospray liquid chromatography/mass spectrometry, J.AOAC Int., 1998, 81, 554-562.
SAMPLE
Matrix: tissue
Sample preparation: Soxhlet extract 20 g finely chopped (20 mesh or finer) tissue with 130 mL
dichloromethane:MeOH 90:10 for 15 h at 5 exchanges/hour. If no aqueous layer is present filter
through a glass wool plug, wash though with 20 mL and 10 mL dichloromethane. (If an aqueous
layer is present add 1 mL 1 M pH 7 phosphate buffer, filter through glass wool plug, separate
layers, extract aqueous layer with 20 mL and 10 mL dichloromethane, combine the dichloro-
methane layers.) Concentrate the dichloromethane extracts nearly to dryness (3-ball Snyder
column), add 10 mL hexane, concentrate nearly to dryness (^ 1 mL). Add the residue to 10
mL 100 mM pH 2 phosphate buffer using three 10 mL portions of hexane (wash original flask
with two 10 mL portions of warm dichloromethane (A)), shake vigorously for 2 min, discard
hexane layer, wash aqueous layer with 10 mL hexane, wash aqueous layer with dichloro-
methane (A). Extract the dichloromethane layer with a fresh 10 mL portion of 100 mM pH 2
phosphate buffer. Combine the aqueous layers, adjust pH to 12 with 1.7 mL 5 M NaOH, add
20 mL dichloromethane, shake vigorously for 1 min, centrifuge, separate layers. Extract di-
chloromethane layer with 10 mL 100 mM pH 12 buffer. Combine the aqueous layers, adjust
the pH to 7.0 with 600 |JLL 30% phosphoric acid, add 30 mL dichloromethane, shake vigorously
for 1 min, extract aqueous layer again with 15 mL dichloromethane. Combine dichloromethane
layers, filter through a glass wool plug, add 1 mL 5% diethylene glycol in dichloromethane,
evaporate to dryness under vacuum at 50, dissolve the residue in 1 mL mobile phase, inject
a 10 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Supelcosil LC-18 DB
Mobile phase: MeCN:water:triethylamine 20:75:5, adjusted to pH 3.5 with orthophosphoric acid
Flow rate: 1
CHROMATOGRAM
Retention time: 5.6
KEYWORDS
chicken; turkey; liver; muscle; skin
REFERENCE
Waggoner,T.B.; Bowman,M.C. Spectrofluorometric determination of BAY Vp 2674 residues in poultry tissues,
JAssoc.OffAnal.Chem., 1987, 70, 813-818.
SAMPLE
Matrix: tissue
Sample preparation: Wash a 500 mg 2.8 mL Bond-Elut SCX cartridge with 1% HOAc in EtOH.
Homogenize 2 g muscle tissue in 20 mL 1% HOAc in EtOH, sonicate 3 min, centrifuge at 4200
g for 5 min, decant supernatant. Repeat extraction, combine supernatants, centrifuge at 4200
g for 5 min. Pass supernatants through SPE cartridge, wash cartridge with 5 mL MeOH, 10
mL water, 5 mL MeOH, and elute with 25% aqueous ammonia (specific gravity 0.88) in MeOH.
Evaporate eluate to dryness under a stream of nitrogen at 50, evaporation of the final portion
is aided by the addition of 1 mL MeCN. Add 1 mL mobile phase, vortex 15 s, sonicate 3 min,
centrifuge at 1860 g for 5 min, filter (0.45 |xm), inject 20 |xL.
HPLC VARIABLES
Column: 250 X 4.6 Zorbax RXC8
Mobile phase: MeCNrbuffer 20:80 (Buffer was 0.68 mL orthophosphoric acid in 900 mL water,
taken to pH 3.0 with triethylamine, made up to 1 L.)
Flow rate: 0.5
CHROMATOGRAM
Retention time: 12
Limit of quantitation: < 10 ng/g
OTHER SUBSTANCES
Simultaneous: ciprofloxacin
KEYWORDS
muscle; pig; cow; muscle; SPE
REFERENCE
Tarbin,J.A.; Tyler,D.J.; Shearer,G. Analysis of enrofloxacin and its metabolite ciprofloxacin in bovine and por-
cine muscle by high-performance liquid chromatography following cation exchange clean-up, Food Ad-
dit.Contam., 1992, 9, 345-350.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 500 mg Bond Elut C18 cartridge with 5 mL MeOH and 10
mL water. Homogenize a 5 g tissue sample with 100 mL MeCN:0.2% metaphosphoric acid 30:
70 at high speed, filter through ca. 2 mm Hyflo Super-Cel coated on a suction funnel. Evaporate
filtrate under reduced pressure at 50 to about 30 mL. Apply remaining solution to the SPE
cartridge, wash with 20 mL water, elute with 10 mL MeOH. Evaporate eluate to dryness under
reduced pressure, dissolve in 1 mL mobile phase, inject a 10 jxL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Wakosil II 5C18 HG (Wako)
Mobile phase: MeCN:50 mM pH 2.4 phosphate buffer 20:80 containing 2.5 mM 1-heptanesul-
fonic acid
Flow rate: 0.6
CHROMATOGRAM
Retention time: 15
Limit of detection: 10 ng/g
OTHER SUBSTANCES
Simultaneous: benofloxacin, danofloxacin, ofloxacin
KEYWORDS
chicken; SPE
REFERENCE
Horie,M.; Saito,K; Nose,N.; Nakazawa,H. Simultaneous determination of benofloxacin, danofloxacin, enroflox-
acin and ofloxacin in chicken tissues by high-performance liquid chromatography, J.Chromatogr.B, 1994,
653, 69-76.
Ephedrine
Molecular formula: C10H15NO
CAS Registry No.: 90-81-3 (DL), 134-71-4 (DL HCI), 50-98-6 (L HCI),
134-72-5 (L sulfate), 299-42-3 (-), 50906-05-3 ((-) hemihydrate)
Merck Index: 3645
Lednicer No.: 1 66
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-pak C18 SPE cartridge with EtOH, 5% aqueous bovine
serum albumin, and water. 100-500 |xL Plasma + 100 ng (l)-norephedrine + 2 mL 500 mM pH
7.0 phosphate buffer, add to the SPE cartridge, wash with 5 mL water, wash with 3 mL EtOH:
water 20:80, elute with 8 mL EtOH. Evaporate the eluate to dryness, reconstitute the residue
in 100 |xL MeCN, add 100 |JLL 6 mg/mL dansylchloride in MeCN containing 0.03% triethylam-
ine, heat at 50 for 20 min, evaporate to dryness under a stream of nitrogen, reconstitute with
1 mL EtOH:water 90:10, add to an 18 X 6 column containing 80 mg carboxymethyl Sephadex
LH-20 (0.95 meq/g), wash with EtOH:water 90:10, elute with 6 mL 50 mM methylamine in
EtOH:water 90:10 at 0.1 mL/min. Evaporate the eluate to dryness, reconstitute with 50-100
IxL mobile phase, inject an aliquot.
HPLC VARIABLES
Guard column: 10 X 4 5 |xm Shimpack G-ODS guard column (Shimadzu)
Column: 150 X 6 5 |xm Shimpack CLC-ODS (Shimadzu)
Mobile phase: MeOH:0.6% pH 6.5 phosphate buffer 80:30
Flow rate: 1.3
CHROMATOGRAM
Internal standard: (l)-norephedrine (10)
OTHER SUBSTANCES
Extracted: pseudoephedrine
KEYWORDS
plasma; SPE; guinea pig; human; derivatization; pharmacokinetics
REFERENCE
Shao,G.; Wang,D.-S.; Wu,R; Chen,S.-J.; Luo,X. Separation and determination of (l)-ephedrine and (d)-pseudo-
ephedrine in plasma by high-performance liquid chromatography with fluorescence detection,
J.Liq.Chromatogr., 1995, 18, 2133-2145.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroformiisopropanol:
n-heptane 60:14:26, shake gently horizontally for 10 min, centrifuge at 2800 g for 10 min.
Remove the lower organic layer and evaporate it to dryness under vacuum at 45, reconstitute
the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of
the supernatant. (Buffer was saturated ammonium chloride solution 25% diluted with water,
adjusted to pH 9.5 with 25% ammonia solution.)
HPLC VARIABLES
Column: 300 X 3.9 4 |xm NovaPack C18
Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted
to pH 2.6 with concentrated orthophosphoric acid.) (At the end of each session wash the column
with water for 1 h and MeOH for 1 h, re-equilibrate for 30 min.)
Flow rate: 0.8
Detector: UV 259
CHROMATOGRAM
KEYWORDS
whole blood; plasma; interferences may occur-compounds (all of which are extracted) elute in
this order tenoxicam; iproniazid; methocarbamol; methotrexate; caffeine; nialamide; colchicine;
cytarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfinpyrazole; fluma-
zenil; sulpride; morphine; atenolol; toloxatone; terbutaline; albuterol; phenobarbital; ranitidine;
tiapride; phenol; chlormezanone; aspirin; metformin; ritodrine; codeine; sultopride;
amisulpride; naltrexone; lisinopril; benzocaine; nizatidine; nalorphine; mephenesin; naloxone;
sotalol; carteolol; procainamide; carbamazepine; bromazepam; nalbuphine; nadolol; procarba-
zine; dihydralazine; omeprazole; strychnine; acebutolol; glutethimide; chlorpropamide; glipi-
zide; triazolam; prazosin; flunitrazepam; clonazepam; metoclopramide; melphalan; estazolam;
tolbutamide; ephedrine; clonidine; pindolol; clobazam; minoxidil; disopyramide; nitrazepam;
dextromethorphan; tofisopam; zopiclone; debrisoquine; sulindac; alprazolam; cycloguanil; lor-
azepam; methaqualone; ketamine; piroxicam; metoprolol; nifedipine; quinine; mephentermine;
prilocaine; pentazocine; oxazepam; tiaprofenic acid; quinidine; celiprolol; ajmaline; yohimbine;
lidocaine; secobarbital; viloxazine; mepivacaine; meperidine; doxylamine; labetalol; temaze-
pam; amodiaquine; benperidol; droperidol; hydroxychloroquine; zolpidem; ketoprofen; almino-
profen; cicletanine; moclobemide; chloroquine; cocaine; timolol; nomifensine; ticlopidine; acen-
ocoumarol; vindesine; mexiletine; dipyridamole; trazodone; pipamperone; pyrimethamine;
benazepril; vincristine; metapramine; chlordiazepoxide; oxprenolol; warfarin; clorazepate; fle-
cainide; phencyclidine; thiopental; fenfluramine; metipranolol; triprolidine; naproxen; bupren-
orphine; verapamil; buspirone; tianeptine; midazolam; bupivacaine; carbinoxamine; loprazo-
lam; cetirizine; chlorpheniramine; moperone; cibenzoline; medifoxamine; astemizole;
vinblastine; nicardipine; bisoprolol; diltiazem; glibornuride; reserpine; aconitine; nitrendipine;
diazepam; mianserin; ramipril; haloperidol; tetracaine; alprenolol; aceprometazine; glibenclam-
ide; chlorophenacinone; doxepin; nimodipine; diphenhydramine; cyclizine; histapyrrodine;
phenylbutazone; demexiptiline; clozapine; proguanil; trifluperidol; medazepam; cyamemazine;
bumadizone; suriclone; propranolol; acepromazine; dothiepin; dextromoramide; fenoprofen;
dextropropoxyphene; loxapine; betaxolol; propafenone; promethazine; thioproperazine; metha-
done; amoxapine; quinupramine; opipramol; cyproheptadine; brompheniramine; mefenidra-
mine; protriptyline; flurbiprofen; tetrazepam; zorubicin; prazepam; alimemazine; loperamide;
imipramine; desipramine; levomepromazine; hydroxyzine; niflumic acid; penbutolol; fluvox-
amine; pimozide; daunorubicin; indomethacin; maprotiline; tropatenine; etodolac; fluoxetine;
amitriptyline; nortriptyline; tioclomarol; diclofenac; mefloquine; trimipramine; chlorambucil;
lidoflazine; ibuprofen; floctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpi-
pramine; thioridazine; fentiazac; clemastine; mefenamic acid; fluphenazine; prochlorperazine;
penfluridol; bepridil; terfenadine; trifluoperazine
REFERENCE
TracquiA; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
a 10 (urine) or 30 (blood) (JLL aliquot. (The detector wavelength shown is the wavelength of
HPLCVARIABLES
Column: 250 X 4.6 5 |jim Symmetry C8 (Waters)
mL/min)
Detector: UV 206.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
4163.
SAMPLE
Matrix: bulk
Sample preparation: Prepare a 10 mg/mL solution in 500 mM sodium bicarbonate solutions,
extract a 10 mL aliquot twice with 15 mL portions of dichloromethane. Combine the extracts
and add 10 |xL phenylisothiocyanate, evaporate to dryness under a stream of air, reconstitute
with 10 mL MeOH, inject a 10 |xL aliquot.
HPLCVARIABLES
Guard column: 70 X 2.1 COrPELL ODS
Mobile phase: MeOH.water.acetic acid 45:54:1
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
Simultaneous: lidocaine, phenylpropanolamine, pseudoephedrine
KEYWORDS
derivatization
REFERENCE
Noggle,F.T.,Jr.; Clark,C.R. Liquid chromatographic analysis of samples containing cocaine, local anesthetics,
and other amines, J.Assoc.Off.Anal.Chem.y 1983, 66, 151-157.
SAMPLE
Matrix: bulk
Sample preparation: Mix a 1 mg/mL solution in 1 M sodium carbonate with 2 mL 5 mg/mL 8-
quinolinesulfonyl chloride in acetone, heat at 65 for 20 min, cool, extract twice with 30 mL
portions of chloroform. Combine the extracts and dry them over anhydrous magnesium sulfate,
evaporate to dryness under a stream of air, reconstitute, inject an aliquot.
HPLC VARIABLES
Guard column: 70 X 2.1 CoiPell ODS
Mobile phase: MeCN:water:acetic acid 40:59:1
Flow rate: 1.5
Detector: UV 254, UV 280
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Simultaneous: amphetamine, methamphetamine, phenmetrazine, phentermine, phenylpropan-
olamine, pseudoephedrine
KEYWORDS
derivatization
REFERENCE
Noggle,F.T.,Jr.; Clark,C.R. Liquid chromatographic determination of primary and secondary amines as 8-quin-
olinesulfonyl chloride derivatives, J.Assoc.Off.Anal.Chem., 1984, 67, 687-691.
SAMPLE
Matrix: bulk
Sample preparation: Mix 200 jjimole amine with 500 ixmole (lS)-(+)-camphor-10-sulfonyl chlo-
ride, 10 mL diethyl ether, and 10 mL 1 M NaOH, stir vigorously for 1 h, acidify with concen-
trated HCl, extract three times with diethyl ether. Combine the organic layers and wash them
three times with water, evaporate to dryness, reconstitute with 1 mL MeOH, inject a 10 JJLL
aliquot.
HPLC VARIABLES
Column: 200 X 4.6 5 |xm Silica 100-RP 18
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: k' 7.16, 8.26 (enantiomers)
OTHER SUBSTANCES
Also analyzed: amphetamine, bamethan, norpseudoephedrine, 1-phenylethylamine
KEYWORDS
derivatization; chiral
REFERENCE
Vogt,C; Jira,T.; Beyrich,T. HPLC-Trennung racemischer Amine nach Derivatisierung mit (lS)-(+)-Campher-10-
sulfonylchlorid, Pharmazie, 1990, 45, 691.
SAMPLE
Sample preparation: Syrup. Dilute syrup with an equal volume of water. 2 mL Diluted syrup
+ 2 mL 1% dansyl chloride in acetone + 200 |xL 1.5 M sodium carbonate, heat at 45 2 in
the dark for 20 min, cool, add 3 mL water, add 500 |xL benzene (Caution! Benzene is a carcin-
ogen!), shake. Remove the organic layer and evaporate it to dryness, reconstitute the residue
in mobile phase, inject a 10 |xL aliquot. Capsules. Sonicate the contents of a capsule in 20 mL
water for 10 min, centrifuge at 2000 g for 10 min. 2 mL Supernatant + 2 mL 1% dansyl chloride
in acetone + 200 |xL 1.5 M sodium carbonate, heat at 45 2 in the dark for 20 min, cool, add
3 mL water, add 500 JXL benzene (Caution! Benzene is a carcinogen!), shake. Remove the
organic layer and evaporate it to dryness, reconstitute the residue in mobile phase, inject a 10
jxL aliquot.
HPLC VARIABLES
Column: 250 X 2.8 10 |jim silica gel SI 100 (Merck)
Mobile phase: Diisopropyl ether:isopropanol:concentrated ammonia 48:2:0.3 (Caution! Diisopro-
pyl ether readily forms explosive peroxides!)
Detector: UV 254 or F ex 354 em 476
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Simultaneous: cephaeline, emetine, codeine (not derivatized, detect at UV 254 only)
KEY WORDS
derivatization; syrup; capsules; normal phase
REFERENCE
Frei,R-W.; Santi,W.; Thomas,M. Liquid chromatography of dansyl derivatives of some alkaloids and the appli-
cation to the analysis of Pharmaceuticals, J.Chromatogr., 1976, 116, 365-377.
SAMPLE
Sample preparation: Dilute formulation 1:10. Remove a 1 mL aliquot and add it to 1.5 mL
water and 2.5 mL 20 (xg/mL ephedrine, inject an 80 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Nucleosil C18
Mobile phase: MeCN:buffer 35:65 containing 0.05% sodium tetradecyl sulfate (Buffer was 0.83
mM phosphoric acid adjusted to pH 5.0 with triethylamine. Use a 50 X 4.6 5-25 |xm LiChroprep
Si 60 column before the injector. Wash column with MeCN:83 mM phosphoric acid 40:60 after
use.)
Flow rate: 2.5
Detector: UV
CHROMATOGRAM
Retention time: 10
Internal standard: ephedrine
OTHER SUBSTANCES
Simultaneous: ergonovine (ergometrine), oxytocin
KEYWORDS
injections; ephedrine is IS
REFERENCE
Pask-Hughes,R.A.; Corran,P.H.; Calam,D.H. Assay of the combined formulation of ergometrine and oxytocin
by high-performance liquid chromatography, J.Chromatogr., 1981, 214, 307-315.
SAMPLE
Sample preparation: Powder tablet and add 50 mg to 50 mL MeCN:20 mM pH 3.8 phosphate
buffer 3:97, sonicate for 5 min, filter (0.5 |xm), inject a 20 |xL aliquot of the filtrate.
HPLCVARIABLES
Guard column: Supelguard pre-column containing 5 |jim Suplex pKblOO (Supelco)
Column: 150 X 4.6 5 (xm Suplex pKblOO (Supelco)
Mobile phase: Gradient. MeCN:20 mM pH 3.8 phosphate buffer at 3:97 for 3 min, to 15:85 over
5 min, stay at 15:85 for 4 min, re-equilibrate for 8 min.
Flow rate: 1.5
CHROMATOGRAM
Limit of quantitation: 10 jxg/mL
OTHER SUBSTANCES
Simultaneous: methamphetamine, amphetamine, caffeine, 3,4-methylenedioxyamphetamine,
N-methyl-3,4-methylenedioxyamphetamine, N-ethyl-3,4-methylenedioxyamphetamine
KEYWORDS
tablets
REFERENCE
Longo,M.; Martines,C; Rolandi,L.; Cavallaro,A. Simple and fast determination of some phenethylamines in
illicit tablets by base-activated reversed phase HPLC, J.Liq.Chromatogr., 1994, 77, 649-658.
SAMPLE
Sample preparation: Dilute nasal solution 10-fold with water, filter (0.45 |xm), inject a 10 jxL
aliquot of the filtrate.
HPLC VARIABLES
Column: 125 X 4 5 |xm Aluspher RP-select B (Merck)
Mobile phase: Gradient. MeCN: 1 mM NaOH from 10:90 to 80:20 over 25 min.
Flow rate: 1.2
Detector: UV 224 or UV 259
CHROMATOGRAM
Retention time: 3
Limit of detection: 5 ng (UV 224)
OTHER SUBSTANCES
Simultaneous: naphazoline, oxymetazoline, xylometazoline
KEYWORDS
nasal solutions
REFERENCE
De Orsi,D.; Gagliardi,L.; Cavazzutti,G.; Mediati,M.G.; Tonelli,D. Simultaneous determination of ephedrine and
2-imidazolines in pharmaceutical formulations by reversed-phase HPLC, J.Liq.Chromatogr., 1995, 18,
3233-3242.
SAMPLE
Sample preparation: Dilute syrup with mobile phase to a concentration of 5-100 jxg/mL, shake,
filter, inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 jxm 80 A Ultrasphere CN
Mobile phase: MeCN:water:EtOH 60:38:2 containing 1 mM perchloric acid
Flow rate: 1
Detector: Conductivity, zero suppression 2, range 1 or 10
CHROMATOGRAM
Retention time: 9.4
OTHER SUBSTANCES
Simultaneous: bromhexine, chlorpheniramine, codeine, dextromethorphan, diphenhydramine,
papaverine, phenylephrine
KEYWORDS
syrup; indirect conductometric detection; presence of compound causes a decrease in mobile phase
conductivity
REFERENCE
Lau,0.-W.; Mok,C.-S. High-performance liquid chromatographic determination of active ingredients in cough-
cold syrups with indirect conductometric detection, J.Chromatogr.A, 1995, 693, 45-54.
SAMPLE
Matrix: solutions
Sample preparation: Mix 40 mL of a 325-375 mM amine solution in THF with 150 mL 10%
potassium carbonate, add dropwise 40 mL 275-325 mM reagent in THF, heat at 50 while
maintaining at pH 8 or above for 3 h, cool, extract with chloroform. Evaporate the extracts to
dryness, reconstitute, inject an aliquot. (Prepare reagent (l-[(4-nitrophenyl)sulfonyl]prolyl chlo-
ride) as follows. Mix 40-45 mmoles L-(-)-proline, 40 mL THF, and 200 mL 10% potassium
carbonate, add 37-43 mmoles 4-nitrobenzenesulfonyl chloride in 40 mL THF dropwise, heat at
50 and maintain at pH 8 or above for 3 h, cool, acidify to pH 2, extract with chloroform.
Extract the organic layers with potassium carbonate in water. Acidify the aqueous layer and
extract it with chloroform. Dry the chloroform layer and evaporate it to dryness, recrystallize
the resulting l-[(4-nitrophenyl)sulfonyl]proline from petroleum ether and benzene (Caution!
Benzene is a carcinogen!). Stir 15 mmoles l-[(4-nitrophenyl)sulfonyl]proline in 100 mL benzene
and add 75 mmoles thionyl chloride in 50 mL benzene dropwise, heat at 35-40 until the
reaction is complete (about 48 h; monitor by IR), evaporate to dryness, recrystallize from n-
heptane to give l-[(4-nitrophenyl)sulfonyl]prolyl chloride (mp 110-110.5).)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Zorbax ODS
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 5, 6 (enantiomers)
OTHER SUBSTANCES
Simultaneous: pseudoephedrine
KEYWORDS
REFERENCE
Clark,C.R.; Barksdale,J.M. Synthesis and liquid chromatographic evaluation of some chiral derivatizing agents
for resolution of amine enantiomers, Anal. Chem., 1984, 56, 958-962.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH at a concentration of 1 mg/mL, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 5 Spherisorb S5W
Mobile phase: MeOH:buffer 90:10 (Buffer was 94 mL 35% ammonia and 21.5 mL 70% nitric
acid in 884 mL water, adjust the pH to 10.1 with ammonia.)
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHERSUBSTANCES
Simultaneous: methamphetamine, mescaline, mephentermine, buprenorphine, dextromoram-
ide, phenoperidine, fentanyl, etorphine, piritramide, noscapine, papaverine, naloxone, dex-
tropropoxyphene, nalorphine, phenazocine, norpipanone, levallorphan, hydroxypethidine,
normethadone, meperidine, dipipanone, diamorphine, pentazocine, acetylcodeine, monoacetyl-
morphine, thebacon, oxycodone, thebaine, norlevorphanol, methadone, benzylmorphine, ethyl-
morphine, morphine-N-oxide, codeine, codeine-N-oxide, morphine, ethoheptazine, morphine-3-
glucuronide, pholcodine, norpethidine, hydrocodone, dihydrocodeine, dihydromorphine,
levorphanol, norcodeine, normorphine, pemoline, benzphetamine, diethylpropion, mazindol,
tranylcypromine, caffeine, fenethyline, phendimetrazine, methylphenidate, phenelzine, epi-
nephrine, pipradol, phenylpropanolamine, fencamfamin, chlorphentermine, norpseudoephed-
rine, fenfluramine, methylenedioxyamphetamine, amphetamine, normetanephrine, 4-
hydroxyamphetamine, bromo-STP, STP, prolintane, 2-phenethylamine, tyramine,
trimethoxy amphetamine
Noninterfering: dopamine, levodopa, methyldopa, methyldopate, norepinephrine
Interfering: phenylephrine, pseudoephedrine, methylephedrine, dimethylamphetamine
REFERENCE
Law,B.; Gill,R.; Moffat,A.C. High-performance liquid chromatography retention data for 84 basic drugs of fo-
rensic interest on a silica column using an aqueous methanol eluent, J.Chromatogr., 1984, 301, 165-172.
SAMPLE
Matrix: solutions
Sample preparation: Mix 1 mL of an aqueous solution with 1 mL 100 mM nickel sulfate in
water, 1 mL 20% aqueous ammonia, and 5 mL chloroform xarbon disulfide 98:2, shake vigor-
ously for 1 min, wash the organic layer with three 2 mL portions of water, filter (phase-sepa-
ration paper). Evaporate the filtrate to dryness under a stream of nitrogen, reconstitute with
1 mL mobile phase, inject a 10 |xL aliquot. (Copper may also be used with electrochemical
detection or UV detection at 270 nm.)
HPLCVARIABLES
Guard column: 30 X 4 40 |xm LiChrosorb RP-18
Column: 250 X 4 7 |xm LiChrosorb RP-18
Mobile phase: MeOH:20 mM pH 5.8 sodium acetate buffer 80:20 containing 5 mM lithium
perchlorate
Flow rate: 1.5
Detector: UV 325, E, Merck-Clevenot E 230, Model LCC 231 thin-layer electrolytic cell with a
glassy carbon electrode at +0.7 V, standard calomel reference electrode
CHROMATOGRAM
Limit of detection: 1 fmole (E), 1 nmole (UV)
OTHER SUBSTANCES
Simultaneous: methamphetamine
Also analyzed: acebutolol, alprenolol, flecainide, propranolol
KEYWORDS
derivatization; complexation
REFERENCE
Leroy,R; Nicolas,A. Determination of secondary amino drugs as their metal dithiocarbamate complexes by
reversed-phase high-performance liquid chromatography with electrochemical detection, J.Chromatogr.,
1984, 317, 513-521.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mIVL 100 mM NaOH in
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.8
OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albuterol, al-
prenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacyclonal, bamethan,
benactyzine, benperidol, benzethidine, benzocaine, benzoctamine, benzphetamine, benzquin-
amide, bromhexine, bromodiphenhydramine, bromperidol, brompheniramine, brompromazine,
buclizine, bufotenine, bupivacaine, buprenorphine, butacaine, butethamate, chlorcyclizine,
chlorpheniramine, chlorphenoxamine, chlorprenaline, chlorpromazine, chlorprothixene, cimet-
idine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine, cy-
clizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dextro-
propoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene,
dihydroergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipa-
none, diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine,
droperidol, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, er-
gosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, feno-
terol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol,
hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine,
ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclo-
phenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine,
mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene,
methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine,
methylergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, mor-
azone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortrip-
tyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pe-
cazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone,
phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide,
phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine,
phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, pimino-
dine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pi-
renzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, pri-
maquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine,
prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, pro-
thipendyl, protriptyline, prox3nnetacaine, pseudoephedrine, pyrimethamine, quinidine, qui-
nine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyldi-
amine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine,
thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, tra-
zodone, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, tri-
methoprim, trimipramine, tripelennamine, triprolidine, tiyptamine, verapamil, xylometazoline
REFERENCE
Jane,L; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica
columns using non-aqueous ionic eluents. II. Application of UV,fluorescenceand electrochemical oxidation
detection, J.Chromatogr., 1985, 323, 191-225.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 |xg/mL, inject a 10
IxL aliquot.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Simultaneous: phenylpropanolamine, pseudoephedrine
REFERENCE
Roos,R.W.; Lau-Cam,C.A. General reversed-phase high-performance liquid chromatographic method for the
separation of drugs using triethylamine as a competing base, J.Chromatogr., 1986, 370, 403-418.
SAMPLE
Matrix: solutions
Sample preparation: 100 juiL 100 JULM Solution + 300 JJLL buffer + 500 JULL 1 mM dansyl chloride
in acetone, mix, heat at 45 in the dark for 1 h, dilute with MeCN:water 50:50, inject a 20 |xL
aliquot. (Prepare buffer by adjusting 10 mM sodium bicarbonate to pH 9.0 with NaOH.)
HPLC VARIABLES
Guard column: 30 X 4.6 Spheri-5 RP-18
Column: 250 X 4.6 Inertsil ODS-2
Mobile phase: MeCN:water 70:30 containing 1 mM imidazole, pH adjusted to 7.0 with nitric
acid
Flow rate: 1
Detector: Chemiluminescence following post-column reaction. The column effluent mixed with
the reagent pumped at 1 mL/min and the mixture flowed through a 300 mm X 0.25 mm ID
coil to the detector. (Prepare the reagent by dissolving 112 mg bis(2,4,6-trichlorophenyl) oxalate
in 500 mL MeCN, add 8.6 mL 30% hydrogen peroxide, sonicate.), F ex 343 em 530
CHROMATOGRAM
Retention time: 9
Limit of detection: 4 fmole (chemiluminescence), 50 fmole (F)
OTHER SUBSTANCES
Simultaneous: benzylamine, N-isopropylbenzylamine, methamphetamine, N-methylphenethyl-
amine, phenylbutylamine, phenylethylamine, phenylpropanolamine, phenylpropylamine
KEYWORDS
derivatization; post-column reaction; comparison with other derivatization reagents
REFERENCE
Hayakawa,K.; Hasegawa,K; Imaizumi,N.; Wong,O.S.; Miyazaki,M. Determination of amphetamine-related
compounds by high-performance liquid chromatography with chemiluminescence and fluorescence detec-
tions, J.Chromatogr., 1989, 464, 343-352.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 220 X 2.1 Spheri-5 RP-8
Mobile phase: Gradient. A was 0.08% diethylamine and 0.09% phosphoric acid in water, pH 2.3.
B was MeCN:water 90:10 containing 0.08% diethylamine and 0.09% phosphoric acid. A:B 95:
5 for 2 min, to 0:100 over 15 min (?), maintain at 0:100 for 5 min.
Flow rate: 0.5
Detector: UV 200
CHROMATOGRAM
Retention time: 5.5
OTHER SUBSTANCES
Simultaneous: diethylpropion, phenylpropanolamine, amphetamine, methamphetamine, phen-
termine, fenfluramine
Also analyzed: amitriptyline, chlordiazepoxide, chlorpromazine, desalkylflurazepam, desipra-
mine, desmethyldoxepin, diazepam, doxepin, flurazepam, imipramine, mesoridazine, norchlor-
diazepoxide, nordiazepam, nortriptyline, oxazepam, prazepam, promazine, thioridazine, thi-
othixene, trifluoperazine
REFERENCE
Rainin Catalog, Cl-94, 1994, p. 7.24.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Zorbax RX
Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine
in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 200
mL water, make up to 1 L with MeCN. A:B from 100:0 to 0:100 over 30 min, maintain at 0:
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHERSUBSTANCES
Also analyzed: acepromazine, acetaminophen, acetophenazine, albuterol, aminophylline, ami-
triptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspi-
rin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphet-
amine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone,
butabarbital, butacaine, butethal, caffeine, carbamazepine, carbromal, chloramphenicol, chlor-
diazepoxide, chloroquine, chlorothiazide, chloroxylenol, chlorphenesin, chlorpheniramine,
chlorpromazine, chlorpropamide, chlortetracycline, cimetidine, cinchonidine, cinchonine, clen-
buterol, clonazepam, clonixin, clorazepate, cocaine, codeine, colchicine, cortisone, coumarin,
cyclazocine, cyclobenzaprine, cyclothiazide, cyheptamide, cymarin, danazol, danthron, dapsone,
debrisoquine, desipramine, dexamethasone, dextromethorphan, dextropropoxyphene, diamor-
phine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, diflunisal, digitoxin, digoxin, dil-
tiazem, diphenhydramine, diphenoxylate, diprenorphine, dipyrone, disulfiram, dopamine, dox-
apram, doxepin, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide,
etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fenpro-
porex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymesterone,
fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glyben-
clamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone,
hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indometh-
acin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine,
ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazin-
dol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, me-
pacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine,
mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, meth-
azolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, meth-
yldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, meto-
prolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline,
naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, nor-
epinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphen-
butazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, per-
santine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine,
phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenyl-
butazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primi-
done, probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine,
puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopola-
mine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sul-
faethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sul-
fasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole,
thebaine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid,
thioridazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tol-
metin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine,
trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapa-
mil, vincamine, warfarin, yohimbine, zoxazolamine
REFERENCE
Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 |xL aliquot of a 100-500 jxg/mL solution in mobile phase.
HPLC VARIABLES
Column: 100 X 4.6 5 jxm Hypersil C8 MOS 100A coated with phosphatidylcholine (95% pure
soybean lecithin, Epikuron, Lucas Meyer & Co.) (Coat column by recycling a 1 mM solution of
phosphatidylcholine in MeOH:water 80:20 for 24 h.)
Mobile phase: MeCN:35 mM pH 7.4 sodium phosphate buffer 40:60
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: amoxicillin, antipyrine, carbamazepine, chlorpheniramine, chlorpromazine, clo-
nidine, codeine, desipramine, diphenhydramine, dipyridamole, flufenamic acid, haloperidol, hy-
droxyzine, imipramine, indomethacin, lidocaine, megestrol acetate, metoprolol, nabumetone,
nadolol, phenobarbital, phenol, promazine, propranolol, pyrilamine, quinidine, ropinirole, tes-
tosterone, thioridazine, tolfenamic acid, verapamil
Noninterfering: acetaminophen, aspirin, azathioprine, caffeine, carprofen, chlorambucil, cimet-
idine, fenoterol, flurbiprofen, ibuprofen, ketoprofen, ranitidine, salicylic acid, sulfamethoxazole,
theophylline, thioguanine, tiaprofenic acid, trimethoprim, valproic acid
KEYWORDS
comparison with capillary electrophoresis
REFERENCE
Hanna,M.; de Biasi,V.; Bond,B.; Salter,C; Hutt,A.J.; Camilleri,P. Estimation of the partitioning characteristics
of drugs: A comparison of a large and diverse drug series utilizing chromatographic and electrophoretic
methodology, Anal. Chem., 1998, 70, 2092-2099.
SAMPLE
Matrix: urine
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 5 mL MeOH, 3 mL MeCN:
10 mM ammonium acetate 40:60 adjusted to pH 3 with acetic acid, and 5 mL water. 5 mL
Urine + 5 mL 500 mM ammonium acetate, adjusted to pH 9.5 with ammonia, mix, add to the
SPE cartridge, wash with 20 mL 5 mM pH 9.5 ammonium acetate, wash with 0.5 mL water.
Elute with 2 mL MeCN: 10 mM ammonium acetate 40:60 adjusted to pH 3 with acetic acid,
inject a 50 |xL aliquot of the eluate.
HPLC VARIABLES
Column: 150 X 4.6 L-column ODS (Chemical Inspection & Testing Institute, Tokyo)
Mobile phase: Gradient. MeCN: 100 mM ammonium acetate 0:100 for 1 min, to 40:60 over 20
min.
Flow rate: 1
Detector: UV 210; MS Shimadzu model QP-1100EX thermospray, vaporizer temperature from
170 to 150 over 20 min. SIM, m/z 166
CHROMATOGRAM
Retention time: 14
Limit of detection: 2-40 ng/mL
OTHER SUBSTANCES
Extracted: 6-acetylmorphine, amphetamine, benzoylecgonine, cocaine, methamphetamine,
methylephedrine, morphine, morphine-3-glucuronide, morphine-6-glucuronide
KEYWORDS
SPE
REFERENCE
Tatsuno,M.; Nishikawa,M.; Katagi,M.; Tsuchihashi,H- Simultaneous determination of illicit drugs in human
urine by liquid chromatography-mass spectrometry, J.Anal.Toxicol., 1996, 20, 281-286.
SAMPLE
Matrix: urine
Sample preparation: 500 |xL Urine + N-ethylnordiazepam + chlorpheniramine + 100 |xL buffer,
centrifuge at 11000 g for 30 s, inject a 500 |xL aliquot onto column A with mobile phase A, after
0.6 min backflush column A with mobile phase A to waste for 1.6 min, elute column A with 250
IxL mobile phase B, with 200 |xL mobile phase C, and with 1.15 mL mobile phase D. Elute
column A to waste until drugs start to emerge then elute onto column B. Elute column B to
waste until drugs started to emerge, then elute onto column C. When all the drugs have
emerged from column B remove it from the circuit, elute column C with mobile phase D,
monitor the effluent from column C. Flush column A with 7 mL mobile phase E, with mobile
phase D, and mobile phase A. Flush column B with 5 mL mobile phase E then with mobile
phase D. (Buffer was 6 M ammonium acetate adjusted to pH 8.0 with 2 M KOH.)
HPLC VARIABLES
Column: A 10 X 2.1 12-20 |xm PRP-I spherical poly(styrene-divinylbenzene) (Hamilton); B 10 X
3.2 11 |xm Aminex A-28 (Bio-Rad); C 25 X 3.2 5 |xm C8 (Phenomenex) + 150 X 4.6 5 fjim silica
(Macherey-Nagel)
Mobile phase: A 0.1% pH 8.0 potassium borate buffer; B 6 mM KH2PO4 containing 5 mM tetra-
methylammonium hydroxide, and 2 mM dimethyloctylamine, pH adjusted to 6.50 with phos-
phoric acid; C MeCNibuffer 40:60 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylam-
monium hydroxide, and 2 mM dimethyloctylamine, pH adjusted to 6.50 with phosphoric acid.);
D MeCN:buffer 33:67 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylammonium
hydroxide, and 2 mM dimethyloctylamine, pH adjusted to 6.50 with phosphoric acid.); E MeCN:
buffer 70:30 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylammonium hydroxide,
and 2 mM dimethyloctylamine, pH adjusted to 6.50 with phosphoric acid.)
Column temperature: ambient (column A), 40 (columns B and C)
Flow rate: A 5; B-E 1
CHROMATOGRAM
Internal standard: N-ethylnordiazepam (k' 2.1), chlorpheniramine (k' 5.9)
OTHER SUBSTANCES
Extracted: methamphetamine, desipramine, nortriptyline, diphenhydramine, methadone, imip-
ramine, flurazepam, amitriptyline, morphine, codeine, hydromorphone, hydrocodone, caffeine,
cotinine, benzoylecgonine, secobarbital, oxazepam, phenobarbital, nordiazepam, diazepam,
phenylpropanolamine
Interfering: phentermine, amphetamine, phenmetrazine, lidocaine, pentazocine
KEY WORDS
column-switching
REFERENCE
Binder,S.R.; Regalia,M.; Biaggi-McEachern,M.; Mazhar,M. Automated liquid chromatographic analysis of drugs
in urine by on-line sample cleanup and isocratic multi-column separation, J.Chromatogr., 1989, 473, 325-
341.
SAMPLE
Matrix: urine
Sample preparation: 5 mL Urine + 25 JJLL 1 mg/mL phenylpropanolamine in mobile phase +
100 |xL 10 M NaOH + 2 mL diethyl ether + 3 g sodium sulfate, shake for 20 min, centrifuge
at 1200 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of
nitrogen, reconstitute the residue in 100 jxL mobile phase, inject a 20 JULL aliquot.
HPLCVARIABLES
Column: 125 X 4 5 |xm LiChrospher 60 RP Select B
Mobile phase: 200 mM pH 5.5 phosphate buffer containing 150 mM triethylamine (Wash column
with MeOH for 15 min and with water for 15 min at the end of each day.)
Flow rate: 1.3
Detector: UV 215
CHROMATOGRAM
Retention time: 8
Internal standard: phenylpropanolamine (11.5)
OTHER SUBSTANCES
Extracted: norephedrine, norpseudoephedrine, pseudoephedrine, N-methylephedrine, ethyl-
ephedrine
Noninterfering: amfepramone, amphetamine, caffeine, chlorphentermine, cocaine, codeine, cro-
propamide, crotethamide, dimethylamphetamine, etamivan, fencamfamine, heptaminol, lep-
tazol, lidocaine, methoxamine, methylamphetamine, methylphenidate, nicotine, niketamine,
meperidine, phendimetrazine, phenmetrazine, pipradol, procaine, prolintane, strychnine
REFERENCE
Imaz,C; Carreras,D.; Navajas,R.; Rodriguez,C; Rodriguez,A.F.; Maynar,J.; Cortes,R. Determination of ephed-
rines in urine by high-performance liquid chromatography, J.Chromatogr., 1993, 631, 201-205.
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 0.5 mL 1% trichloroacetic acid, centrifuge at 5200 g for 10
min, filter (0.2 (xm), inject 20 |xL aliquot
HPLC VARIABLES
Column: 250 X 4 Lichrospher 5|xm 60 RP-select B
Mobile phase: Gradient. MeCN:50 mM pH 3.2 potassium phosphate buffer from 10:90 to 50:50
over 15 min.
Flow rate: 1.5
CHROMATOGRAM
Retention time: 5
OTHERSUBSTANCES
Extracted: morphine, phenylpropanolamine, lidocaine, diphenhydramine, nortriptyline, cocaine,
benzoylecgonine, norpropoxyphene, nordiazepam
Also analyzed: amitriptyline, amphetamine, meperidine, codeine, (different gradient)
REFERENCE
Li,S.; Gemperline,P.J.; Briley,K.; Kazmierczak,S. Identification and quantitation of drugs of abuse in urine
using the generalized rank annihilation method of curve resolution, J.Chromatogr.B, 1994, 655, 213-223.
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 10 mg p-glucuronidase/arylsulfatase (Helix pomatia,
Sigma), heat at 37 overnight, add an equal volume of buffer, centrifuge at 2000 g for 5 min,
inject an aliquot of the supernatant onto column A with mobile phase A and elute to waste.
After 2.5 min backflush the contents of column A onto column B with mobile phase B, monitor
the effluent from column B. Re-equilibrate both columns for 12.5 min before the next injection.
(Buffer was 200 mM boric acid adjusted to pH 9.5 with 5 M NaOH.)
HPLC VARIABLES
Column: A 10 X 4.6 5 jxm Spherisorb cyanopropyl; B 250 X 4.6 Capcell Pak C18 UG-120
(Shiseido)
Mobile phase: A water; B Gradient. MeCNibuffer from 3:97 to 30:70 over 30 min, to 40:60 over
8 min (Buffer was 3.4 mIVL phosphoric acid adjusted to pH 3.0 with 5 M NaOH.)
Flow rate: A 1.25; B 1
Detector: UV 220
CHROMATOGRAM
Retention time: 8.3
OTHER SUBSTANCES
Extracted: acebutolol, alprenolol, amphetamine, atenolol, bopindolol, codeine, labetalol, meto-
prolol, morphine, nadolol, oxprenolol, pindolol, propranolol, timolol
KEYWORDS
column-switching
REFERENCE
Saarinen,M.T.; Sirn,H.; Riekkola,M.-L. Screening and determination of p-blockers, narcotic analgesics and
stimulants in urine by high-performance liquid chromatography with column switching, J.Chromatogr.B,
1995, 664, 341-346.
SAMPLE
Matrix: urine
Sample preparation: Inject 15 |xL urine, inject a mixture of 5 JJLL 20 mM 9-fluorenylmethyl
chloroformate in MeCN and 45 jxL water, and inject 10 |xL buffer on to column A and elute to
waste with mobile phase A. After 2.8 min backflush the contents of column A on to column B
with mobile phase B and start the gradient, monitor the effluent from column B. At the end
of the run condition column A with 1 mL mobile phase A. (Buffer was 4% sodium bicarbonate
adjusted to pH 10 with 10% NaOH.)
HPLCVARIABLES
Column: A 20 X 2.1 30 jxm Hypersil ODS-C18; B 125 X 4 5 |xm LiChrospher 100 PR-C18
Mobile phase: A water; B Gradient. MeCN:water from 40:60 to 70:30 over 15 min. to 100:0 over
5 min.
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: amphetamine, methamphetamine, norephedrine, 3-phenylpropylamine, pseudoephedrine
KEYWORDS
column-switching; derivatization; on-column derivatization
REFERENCE
Herrez~Hernandez,R.; Campins-Falc6,P.; Sevillano-Cabeza,A. Determination of amphetamine and related
compounds in urine using on-line derivatization in octadecyl silica columns with 9-fluorenylmethyl chlo-
roformate and liquid chromatography, J.Chromatogr.B, 1996, 679, 69-78.
Epicillin
Merck Index: 3651
SAMPLE
Matrix: perfusate
Sample preparation: Vortex perfusate, centrifuge at 11600 g for 5 min, inject an aliquot of the
supernatant.
HPLC VARIABLES
Guard column: 20 X 2 5 |xm Hypersil ODS
Column: 150 X 4.6 5 |xm Hypersil ODS
Mobile phase: MeOH:buffer 35:65 (Buffer was 50 mM KH2PO4 containing 0.1% triethylamine
adjusted to pH 3 with orthophosphoric acid.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 6.0
REFERENCE
Erah,P.O.; Barrett,D.A.; Shaw,P.N. Reversed-phase high-performance liquid chromatographic assay methods
for the analysis of a range of penicillins in in vitro permeation studies, J.Chromatogr.B, 1998, 705, 63-69.
Epidermal growth factor

Molecular formula: indeterminate
Molecular weight: 6201
Merck Index: 3569
SAMPLE
Matrix: blood
Sample preparation: Filter (0.2 jim polysulfone) plasma, inject a 200 |xL aliquot on to column
A and elute to waste with mobile phase A, after 8 min elute column A to waste with mobile
phase B, after 4 min elute the contents of column A on to column B with mobile phase B, after
6 min remove column A from the circuit, elute column B with mobile phase C (start the gra-
dient), monitor the effluent from column B. Re-equilibrate column A with mobile phase A for
17.6 min.
HPLC VARIABLES
Column: A 24 X 4 immunoaffinity (column preparation details in paper); B 20 X 4.6 polysulfo-
ethyl aspartamide (The Nest Group, Southborough, MA)
Mobile phase: A 0.9% NaCl containing 10 mM potassium phosphate and 20 ppm sodium azide,
pH 7.4 (Caution! Sodium azide is carcinogenic and should not be discharged to the plumbing
system!); B MeOH:100 mM pH 2.50 glycine/HCl buffer 50:50; C Gradient. A was MeCN:5 mM
pH 3.0 potassium phosphate buffer 25:75. B was MeCN:600 mM KCl containing 5 mM potas-
sium phosphate, pH 3.0 25:75. A:B 80:20 for 1 min, to 75:25 over 5 min, to 65:35 over 1 min,
to 40:60 over 7.5 min, return to initial conditions over 0.1 min, re-equilibrate for 3 min.
Flow rate: A 0.6; B 0.6; C 1
Detector: UV 220 or immunoassay
CHROMATOGRAM
Retention time: 25 QiEGF 1-47), 28 QiEGF 1-48), 32 QiEGF 1-53)
KEYWORDS
plasma; column-switching
REFERENCE
Kagel,J.R.; Rossi,D.T.; Nordblom,G.D.; Dudeck,R.C; Barksdale,C.M.; Kuo,B.-S.; Wright,D.S. Considerations in
the development of a sensitive HPLC assay for human epidermal growth factors in human plasma,
J.Pharm.Biomed.Anal., 1995, 13, 1205-1213.
SAMPLE
HPLC VARIABLES
Column: 150 X 4.6 5 iim TSKgel C18 (TOSOH)
Mobile phase: MeCNrIO mM pH 6.0 diethylenetriamine phosphate 22:78
Flow rate: 0.5
Detector: UV 214
CHROMATOGRAM
Retention time: 26.72 (a-form)
OTHER SUBSTANCES
Simultaneous: deamidated epidermal growth factor (p-form)
REFERENCE
Son,K.; Kwon,C. Stabilization of human epidermal growth factor (hEGF) in aqueous formulations, Pharm.Res.,
1995, 12, 451-454.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in 200 mM pH 5.6 ammonium acetate, concentrate 10-
fold under vacuum, inject a 200-400 jxL aliquot
HPLC VARIABLES
Guard column: Bio-Sil ODS-IO (Bio-Rad)
Column: two 300 X 3.9 (xBondapak C18 columns in series
Mobile phase: MeCN:buffer 26:74 (Buffer was 50 mM acetic acid adjusted to pH 5.6 with
triethylamine.)
Flow rate: 0.5
Detector: UV 280
CHROMATOGRAM
Retention time: 58 (a-form), 65 (p-form)
KEY WORDS
mouse
REFERENCE
Matrisian,L.M.; Larsen,B.R.; Finch,J.S.; Magun,B.E. Further purification of epidermal growth factor by high-
performance liquid chromatography, Anal.Biochem., 1982, 125, 339-351.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Spherisorb S5 ODS2 fully end-capped
Mobile phase: Gradient. A was 155 mM NaCl containing 0.2% pentadecafluorooctanoic acid. B
was MeCNdsopropanol 50:50 containing 0.2% pentadecafluorooctanoic acid. A:B from 65:35 to
50:50 over 50 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 26 (a-form), 28 (p-form)
KEYWORDS
mouse; full discussion of factors affecting selectivity
REFERENCE
Smith,J.A.; O'Hare,M.J. Reversed-phase high-performance liquid chromatography of mouse epidermal growth
factor and its congeners: mobile phase optimization with ion-pairing additives, J.Chromatogr., 1984, 299,
13-28.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 |xm ODS Hypersil C18
Mobile phase: Gradient. A was 155 mM NaCl adjusted to pH 2.1 with HCl. B was MeCN. A:B
100:0 for 5 min, to 90:10 over 5 min, to 52.5:47.5 over 50 min.
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 35
KEYWORDS
mouse
REFERENCE
Smith,J.A.; Ham,J.; Winslow,D.R; O'Hare,M.J.; Rudland,P.S. The use of high-performance liquid chromatog-
raphy in the isolation and characterisation of mouse and rat epidermal growth factors and examination of
apparent heterogeneity, J.Chromatogr., 1984, 305, 295-308.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: Two 300 X 4.6 10 |xm fiBondapak C18 columns in series
Mobile phase: MeCN: 10 mM pH 7.0 diethylenetriamine phosphate 25:75
Flow rate: 0.5
Detector: UV 280
CHROMATOGRAM
Retention time: 35 (a-form), 42 (p-form), 54 (gamma-form)
KEYWORDS
mouse
REFERENCE
DiAugustine,R.R; Walker,M.R; Klapper,D.G.; Grove,R.L; Willis,W.D.; Harvan,D.J.; Hernandez,O. p-epidermal
growth factor is the des-asparaginyl form of the polypeptide, J.Biol.Chem., 1985, 260, 2807-2811.
SAMPLE
Matrix: solutions
Sample preparation: Filter (Amicon YM-2, 2000 cut-off), inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Vydac 218TPS
Mobile phase: Gradient. MeCN:buffer 18:82 for 20 min, to 66:34 over 20 min, maintain at 66:
34 for 10 min. (Buffer was 10 mM pH 7.2 sodium phosphate buffer.)
Detector: UV 280
CHROMATOGRAM
Retention time: 28
KEYWORDS
human
REFERENCE
Engler,D.A.; Matsunami,R-K.; Campion,S.R.; Stringer,C.D.; Stevens,A.; Niyogi,S.K. Cloning of authentic human
epidermal growth factor as a bacterial secretory protein and its initial structure-function analysis by site-
directed mutagenesis, J.Biol.Chem., 1988, 263, 12384-12390.
Epinastine
Molecular formula: C16H15N3
CAS Registry No.: 80012-43-7 (HCI)
Merck Index: 3655
SAMPLE
Matrix: blood
Sample preparation: Add 100 u,L 600 ng/mL diphenidol in water and 600 |xL 100 mM sodium
carbonate to 100 |xL plasma. Add 5 mL dichloromethane, shake on a reciprocal shaker for 10
min, centrifuge at 1000 g for 8 min, dry 4 mL of the lower organic phase in a rotary evaporator
at 45, dissolve the residue in 50 jxL mobile phase, inject a 20 JLJLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Cosmosil 5C18-MS (Nacalai Tesque, Japan)
Next Page
Mobile phase: MeOHrbuffer 36:64 (Buffer was 0.3% triethylamine adjusted to pH 4.5 with phos-
phoric acid.)
Flow rate: 1.3
Detector: UV 220
CHROMATOGRAM
Retention time: 8.1
Internal standard: diphenidol (14.8)
KEYWORDS
plasma
REFERENCE
Ohtani,H.; Kotaki,H.; Sawada,Y.; Iga,T. Quantitative determination of epinastine in plasma by high-perfor-
mance liquid chromatography, J.Chromatogr.B, 1996, 683, 281-284.
SAMPLE
Sample preparation: Blood. Dilute 1 mL plasma with 100 u,L 1 M pH 9 phosphate buffer and
100 (JLL water, add 8 mL chloroform and extract. (Caution! Chloroform is a carcinogen!) Evap-
orate the organic layer, dissolve the residue in 400 (xL mobile phase, inject a 200 |JLL aliquot.
Tissue. Homogenize the brain with 2-fold the weight of water. Dilute 1.5 mL brain homogenate
with 500 |xL 1 M pH 9 phosphate buffer, add 8 mL chloroform, extract. Evaporate the organic
layer and dissolve the residue in 400-500 uL mobile phase. Inject 200 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Intersil PH
Mobile phase: MeCN:0.018% TFA 15:85
Flow rate: 0.7
Detector: UV 220
CHROMATOGRAM
Limit of quantitation: 5 ng/mL (plasma), 15 ng/mL (brain)
KEYWORDS
brain; cat; mouse; pharmacokinetics; plasma; rat
REFERENCE
Kato,M.; Nishida,A.; Aga,Y.; Rita,J.; Kudo,Y.; Narita,H.; Endo,T. Pharmacokinetic and pharmacodynamic eval-
uation of central effect of the novel antiallergic agent betotastine besilate, Arzneimittelforschung, 1997,47,
1116-1124.
Epinephrine
CAS Registry No.: 51-43-4 (-), 51-42-3 ((-) bitartrate), 329-65-7 (racemic)
Merck Index: 3656
Lednicer No.: 1 95
SAMPLE
M a t r i x : blood
Previous Page
Mobile phase: MeOHrbuffer 36:64 (Buffer was 0.3% triethylamine adjusted to pH 4.5 with phos-
phoric acid.)
Flow rate: 1.3
Detector: UV 220
CHROMATOGRAM
Retention time: 8.1
Internal standard: diphenidol (14.8)
KEYWORDS
plasma
REFERENCE
Ohtani,H.; Kotaki,H.; Sawada,Y.; Iga,T. Quantitative determination of epinastine in plasma by high-perfor-
mance liquid chromatography, J.Chromatogr.B, 1996, 683, 281-284.
SAMPLE
Sample preparation: Blood. Dilute 1 mL plasma with 100 u,L 1 M pH 9 phosphate buffer and
100 (JLL water, add 8 mL chloroform and extract. (Caution! Chloroform is a carcinogen!) Evap-
orate the organic layer, dissolve the residue in 400 (xL mobile phase, inject a 200 |JLL aliquot.
Tissue. Homogenize the brain with 2-fold the weight of water. Dilute 1.5 mL brain homogenate
with 500 |xL 1 M pH 9 phosphate buffer, add 8 mL chloroform, extract. Evaporate the organic
layer and dissolve the residue in 400-500 uL mobile phase. Inject 200 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Intersil PH
Mobile phase: MeCN:0.018% TFA 15:85
Flow rate: 0.7
Detector: UV 220
CHROMATOGRAM
KEYWORDS
REFERENCE
Kato,M.; Nishida,A.; Aga,Y.; Rita,J.; Kudo,Y.; Narita,H.; Endo,T. Pharmacokinetic and pharmacodynamic eval-
1116-1124.
Epinephrine
CAS Registry No.: 51-43-4 (-), 51-42-3 ((-) bitartrate), 329-65-7 (racemic)
Merck Index: 3656
Lednicer No.: 1 95
SAMPLE
M a t r i x : blood
Sample preparation: Plasma. Prepare a SPE column by adding 500 jxL of a 20% suspension of
19-40 jxm Toyopak SP (strong cation-exchange sulfopropyl resin, Na+ (Toyo Soda)) in water to
a 35 X 6 column, wash with two 1 mL portions of 2 M LiOH, wash with two 5 mL portions of
water, wash with two 1 mL portions of EtOH: 12 M HCl 90:10, wash with two 5 mL portions
of water, wash with three 1 mL portions of buffer. 500 |xL Plasma + 25 JJLL 10 nM isoproterenol
+ 500 |xL buffer, mix, add to the SPE column, wash with two 5 mL portions of water, wash
with 1 mL MeCN:water 50:50, elute with 300 jxL 600 |JLM potassium ferricyanide in 600 mM
KCl.MeCN 50:50, add 50 |xL reagent to the eluate, heat at 37 for 40 min, cool in ice-water,
inject a 100 |xL aliquot. Urine. 10 jxL Urine + 1 mL MeCN:500 mM KCl 60:40 + 10 |xL 500 nM
isoproterenol + 10 JJLL 75 mM potassium hexacyanoferrate(III) + 100 |xL reagent, heat at 37
for 40 min, inject a 100 |xL aliquot (J. Chromatogr. 1986, 380, 229). (Prepare buffer by mixing
8 volumes 250 mM LiOH in 200 mM phosphoric acid with 1 volume 200 mM phosphoric acid,
pH 5.8. Prepare reagent by dissolving 212 mg 1,2-diphenylethylenediamine in 10 mL 100 mM
HCl, pH 6.7.)
HPLC VARIABLES
Column: 150 X 4.6 5 ^m TSK-gel ODS-120T (Toyo Soda)
Mobile phase: MeCN:MeC)H:50 mM pH 7.0 Tris-HCl buffer 50:10:40 (Wash with MeCN:MeOH:
water 50:10:40 for 15 min at the end of each day.)
Flow rate: 1
Detector: F ex 345 em 485 (plasma), F ex 350 em 480 (urine)
CHROMATOGRAM
Retention time: 5
Internal standard: isoproterenol (8)
Limit of detection: 7 pM
OTHER SUBSTANCES
Extracted: dopamine, norepinephrine
KEYWORDS
derivatization; plasma; SPE
REFERENCE
Mitsui,A-; Nohta,H.; Ohkura,Y. High-performance liquid chromatography of plasma catecholamines using 1,2-
diphenylethylenediamine as precolumn fluorescence derivatization reagent, J.Chromatogr., 1985, 344, 61
70.
SAMPLE
Matrix: blood
Sample preparation: Prepare a 20 X 5 polypropylene column packed with CM-Sephadex pre-
swollen in water, wash with 5 mL 2 M HCl, wash with 10 mL water, wash with 10 mL 100
mM pH 7 phosphate buffer. 1 mL Plasma + 30 (xL 80 ng/mL N-methyldopamine, apply to
column, wash with 5.5 mL water (A), elute with 3 mL 0.5 M perchloric acid. Collect eluate,
add 2 mL 1.5 M pH 9.3 Tris buffer containing 60 mM EDTA, add 20 mg alumina, vortex for 2
min, discard supernatant, add 2 mL water to alumina, mix, centrifuge at 3000 g for 3 min,
repeat water wash, remove as much water as possible, elute catecholamines from alumina with
100 |xL 100 mM acetic acid with vortexing for 2 min, centrifuge, inject 25 u,L aliquot of super-
natant. (Wash water A contains levodopa, carbidopa, DOPAC, and O-methyldopa.)
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Nucleosil C18
Mobile phase: MeCN:MeOH:25 mM sodium acetate 4:4:92 containing 0.2 mM 1-octanesulfonic
acid and 0.3 mM disodium EDTA, pH was adjusted to pH 3 with acetic acid
Flow rate: 0.9
Detector: E, ESA Coulochem 5100 A, 5010 A analytical cell, first electrode +0.25 V, second elec-
trode -0.30 V
CHROMATOGRAM
Retention time: 9
Internal standard: N-methyldopamine (16)
OTHER SUBSTANCES
Simultaneous: norepinephrine, dopamine
KEYWORDS
plasma
REFERENCE
Betto,R; Ricciarello,G.; Giambenedetti,M.; Lucarelli,C; Ruggeri,S.; Stocchi,F. Improved high-performance liq-
uid chromatographic analysis with double detection system for L-dopa, its metabolites and carbidopa in
plasma of parkinsonian patients under L-dopa therapy, J.Chromatogr., 1988, 459, 341-349.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 250 |xL 1 ng/mL a-methylnorepinephrine + 1 mL buffer
+ 5 mL n-heptane containing 4.6 mM tetraoctylammonium bromide and 10 mL/L 1-octanol,
shake for 2 min, centrifuge at 20 at 1000 g for 5 min, freeze in acetone/dry ice. Remove the
organic phase and add it to 2 mL 1-octanol and 200 ^xL 80 mM acetic acid, shake, centrifuge
at 20 at 1000 g for 5 min, freeze in acetone/dry ice. Discard the organic phase, thaw the
aqueous phase and add it to 1 mL 10 mM HCl, 1 mL buffer, and 5 mL n-heptane containing
4.6 mM tetraoctylammonium bromide and 10 mL/L 1-octanol, shake for 2 min, centrifuge at
20 at 1000 g for 5 min, freeze in acetone/dry ice. Remove the organic phase and add it to 2
mL 2 M pH 8.6 ammonia/ammonium chloride buffer containing 13.4 mM EDTA, shake, freeze
in dry ice/acetone. Remove the organic layer and add it to 2 mL 1-octanol and 150 (xL 80 mM
acetic acid, shake, centrifuge at 20 at 1000 g for 5 min, freeze in dry ice/acetone, discard the
organic layer. Thaw the aqueous layer and add it to 250 |xL MeCN, 50 (xL 1.75 M pH 7.05
bicine, and 100 |ULL 100 mM 1,2-diphenylethylenediamine in 100 mM HCl, add 20 \LL 20 mM
potassium ferricyanide in water, heat at 37 in the dark for 1 h, keep at 20 in the dark, inject
a 100 |xL aliquot. (Buffer was 2 M pH 8.6 ammonia/ammonium chloride buffer containing 8.9
mM diphenylborate-ethanolamine complex and 13.4 mM EDTA. Stir buffer with 45 g/L acti-
vated alumina for 2 h before use. Wash 1-octanol with 80 mM acetic acid. Recrystallize 1,2-
diphenylethylenediamine from toluene:light petroleum (bp 60-80) 10:90, dry overnight at 60.)
HPLC VARIABLES
Column: 100 X 4.6 3 |xm Cp MicroSpher C18 (Chrompack)
Flow rate: 1
CHROMATOGRAM
Retention time: 4
Internal standard: a-methylnorepinephrine (3)
OTHER SUBSTANCES
Extracted: dihydroxybenzylamine, dopamine, isoproterenol, norepinephrine
KEYWORDS
plasma; derivatization; comparison with electrochemical detection
REFERENCE
van der Hoorn,F.A.J.; Boomsma,E; Man in 't Veld,A.J.; Schalekamp,M.A.D.H. Determination of catecholamines
in human plasma by high-performance liquid chromatography: comparison between a new method with
fluorescence detection and an established method with electrochemical detection, J.Chromatogr., 1989,487,
17-28.
SAMPLE
Matrix: blood
Sample preparation: Pack a 65 X 15 SPE column with 50 mg WA-4 alumina (Sigma). Add 500
IxL plasma to the SPE column, add 1 mL buffer, rotate for 15 min, wash three times with water
(aspirating to dryness each time), centrifuge to dryness, add 200 |xL 100 mM pH 1.2 perchloric
acid, mix, let stand for 15 min, centrifuge the SPE column at 1000 g for 3 min, inject an aliquot
of the effluent. (Buffer was 45 g Tris and 5 g EDTA in 200 mL water, pH adjusted to 8.6 with
concentrated HCl.)
HPLC VARIABLES
Guard column: 50 x 4.6 5 |xm reversed-phase
Column: 250 X 4.6 5 |xm ODS Spherisorb
Mobile phase: Buffer contained 1.4% monochloroacetic acid, 0.47% NaOH, and 0.075% EDTA,
finally pH adjusted to 3.0 with NaOH or monochloroacetic acid and 6 mg% sodium octylsulfate
added.
Flow rate: 1
Detector: E, Bioanalytical Systems LC-4B, TL-5 transducer with a glassy carbon electrode, +650
mV, 1 nA, Ag/AgCl reference electrode
OTHER SUBSTANCES
Extracted: norepinephrine, dopamine
KEYWORDS
plasma; rabbit; human; SPE
REFERENCE
Ganhao,M.E; Hattingh,J.; Hurwitz,M.L.; Pitts,N.I. Evaluation of a simple plasma catecholamine extraction
procedure prior to high-performance liquid chromatography and electrochemical detection, J.Chromatogr.,
1991, 564, 55-66.
SAMPLE
Matrix: blood
Sample preparation: Plasma. 1 mL Plasma + 125 |xL 2 ng/mL a-methylnorepinephrine + 1 mL
buffer + 5 mL n-heptane containing 4.6 mM tetraoctylammonium bromide and 10 ml/L 1-
octanol, shake for 2 min, centrifuge at 1000 g for 5 min, freeze in dry ice/acetone. Remove the
organic phase and add it to 2 mL 1-octanol (saturated with 80 mM acetic acid) and 200 JJLL 80
mM acetic acid, shake, centrifuge at 1000 g for 5 min. Freeze the aqueous layer and remove
the organic layer. Add 1 mL 10 mM HCl, 1 mL buffer, and 5 mL n-heptane containing 4.6 mM
tetraoctylammonium bromide and 10 mL/L 1-octanol to the aqueous phase. Shake, centrifuge,
freeze, remove the organic layer and add it to 2 mL 2 M pH 8.6 ammonia-ammonium chloride
buffer containing 13.4 mM EDTA (but no complex). Freeze, remove the organic layer and add
it to 2 mL 1-octanol and 150 JJLL 80 mM acetic acid, shake, centrifuge at 1000 g for 5 min.
Freeze, remove the organic layer and add the aqueous layer to 200 jxL MeCN, 50 |xL 1.75 M
pH 6.95 bicine buffer containing 1% EDTA, 100 |xL 100 mM 1,2-diphenylethylenediamine in
100 mM HCl, and 20 |xL 20 mM potassium ferricyanide in water. Heat at 37 in the dark for
1 h, inject a 75 (JLL aliquot (keep it in the dark in the autosampler). Urine. 100 (xL Urine + 1
mL 10 mM HCl + 125 (JLL 40 ng/mL a-methylnorepinephrine + 1 mL buffer + 5 mL n-heptane
containing 4.6 mM tetraoctylammonium bromide and 10 mL/L 1-octanol, shake for 2 min,
centrifuge at 1000 g for 5 min, freeze in dry ice/acetone. Remove the organic phase and add it
to 2 mL 1-octanol (saturated with 80 mM acetic acid) and 200 |xL 80 mM acetic acid, shake,
centrifuge at 1000 g for 5 min. Freeze the aqueous layer and remove the organic layer. Add 1
mL 10 mM HCl, 1 mL buffer, and 5 mL n-heptane containing 4.6 mM tetraoctylammonium
bromide and 10 mL/L 1-octanol to the aqueous phase. Shake, centrifuge, freeze, remove the
organic layer and add it to 2 mL 1-octanol and 150 JJIL 80 mM acetic acid, shake, centrifuge at
1000 g for 5 min. Freeze, remove the organic layer and add the aqueous layer to 200 |xL MeCN,
50 |xL 1.75 M pH 6.95 bicine buffer containing 1% EDTA, 100 fxL 100 mM 1,2-diphenylethyl-
enediamine in 100 mM HCl, and 20 |xL 20 mM potassium ferricyanide in water. Heat at 37
in the dark for 1 h, inject a 50 |xL aliquot (keep it in the dark in the autosampler). (Buffer was
a 2 M pH 8.6 ammonia-ammonium chloride buffer containing 8.9 mM diphenyl borate-etha-
nolamine complex and 13.4 mM EDTA.)
HPLC VARIABLES
Column: 100 X 4.6 3 jim PhaseSep C18 ODS2
Mobile phase: Gradient. A was MeCN:MeOH:50 mM pH 7.0 sodium acetate buffer 20:4:76. B
was MeCN:MeOH:50 mM pH 7.0 sodium acetate buffer 60:10:30. A:B 40:60 for 3 min, go to 0:
100 over 0.5 min, stay at 0:100 for another 4.5 min. (After the last sample flush column with
60 mL MeCN:MeOH:water 70:10:20.)
Flow rate: 1
CHROMATOGRAM
Retention time: 3.5
Internal standard: a-methylnorepinephrine (2.5)
Limit of detection: 0.3-0.6 pg
OTHERSUBSTANCES
Simultaneous: norepinephrine, dopamine, epinine
Interfering: a-methyldopa
KEYWORDS
plasma
REFERENCE
Boomsma,R; Alberts,G.; van der Hoorn,RA.J.; Man in 't Veld,A.J.; Schalekamp,M.A.D.H. Simultaneous deter-
mination of free catecholamines and epinine and estimation of total epinine and dopamine in plasma and
urine by high-performance liquid chromatography with fluorimetric detection, J.Chromatogr., 1992, 574,
109-117.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 5 mg alumina + 10 jxL 10 (xM IS in 10 mM perchloric
acid + 100 |xL pH 8.7 Tris-HCl buffer, stir for 10 min, centrifuge at 3000 g for 1 min, discard
the supernatant. Wash the alumina with two 500 |xL portions of water, add 100 (xL 100 mM
perchloric acid, mix for 1 min, centrifuge at 3000 g for 1 min, inject a 50 JJLL aliquot of the
supernatant. (Heat 20 g alumina (WA-4, Sigma) with 200 mL 2 M HCl at 100 for 1 h with
gentle mixing, decant the supernatant, wash with twenty 200 mL portions of water, filter (Toyo
Roshi No. 2 paper), dry at 120 overnight.)
HPLC VARIABLES
Column: 150 X 4.6 catecholpak (JASCO)
Mobile phase: MeCN:50 mM pH 3.20 potassium acetate:50 mM pH 3.20 potassium phosphate
buffer 3:92.15:4.85 containing 1 mM sodium hexanesulfonate
Flow rate: 0.5
Detector: Chemiluminescence (Kenko filter Y-46) following post-column reaction. The column
effluent mixed with reagent 1 pumped at 0.25 mL/min and the mixture flowed through a 15
m X 0.5 mm i.d. knitted PTFE coil at 80. The effluent from the coil mixed with reagent 2
pumped at 1.4 mL/min and this mixture flowed to the detector. (Reagent 1 was 105 mM ethyl-
enediamine (semiconductor grade) and 175 mM imidazole in MeCN:EtOH 90:10. Reagent 2
was 0.25 mM bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (Wako), 150 mM hy-
drogen peroxide, and 110 mM trifluoroacetic acid in dioxane.ethyl acetate 50:50 (Caution! Di-
oxane is a carcinogen!).)
CHROMATOGRAM
Retention time: 16
Internal standard: 3,4-dihydroxybenzylamine (17)
OTHER SUBSTANCES
KEYWORDS
human; rat; plasma; SPE; post-column reaction
REFERENCE
Higashidate,S.; Imai,K Determination of femtomole concentrations of catecholamines by high-performance
liquid chromatography with peroxyoxalate chemiluminescence detection, Analyst, 1992, 117, 1863-1868.
SAMPLE
Matrix: blood
Sample preparation: Filter (Ultrafree-MC with 10000 molecular mass cut-off, Millipore) 100
IxL plasma while centrifuging at 15000 g for 15 min. Mix 50 |xL ultrafiltrate and 10 |xL 140
ng/mL 3-methoxytyramine in Ringer solution, inject a 5 |JLL aliquot.
HPLC VARIABLES
Column: 150 X 1 5 |xm Inertsil-2 ODS
Mobile phase: MeCN:THF:water 6:0.8:93.2 containing 0.48 g/L sodium 1-octanesulfonate, 2 g/L
NaH2PO4, 8.82 g/L sodium citrate, 10 mg/L EDTA, and 1 mIVL diethylamine, pH adjusted to
3.2 with concentrated orthophosphoric acid.
Flow rate: 0.06
Injection volume: 5
Detector: E, Bioanalytical Systems BAS-4C, glassy carbon working electrodes, upstream +0.75
V, downstream +0.05 V (measuring electrode), Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 3.5
Internal standard: 3-methoxytyramine (11)
OTHER SUBSTANCES
Extracted: dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, serotonin, 5-hydroxyin-
doleacetic acid, homovanillic acid
KEYWORDS
plasma; microbore; rat; ultrafiltrate
REFERENCE
Cheng,F.-C; Yang,L.-L.; Kuo,J.-S.; Yang,M.C.M.; Yu,P.-C. Rapid assay of the monoamine content in small vol-
umes of rat plasma, J.Chromatogr.B, 1994, 653, 9-16.
SAMPLE
Matrix: blood, food, peptides, plants, tissue
Sample preparation: Hydrolyze peptide with 6 M HCl containing 0.2% 3,3'-thiodipropionic acid
at 110 for 24 h, evaporate to dryness, reconstitute with 50-200 jxL 0.1% HCl containing 0.2%
3,3'-thiodipropionic acid. Homogenize (Ultra-Turrax) 0.1-1 g food, tissue, plant material, Iy-
ophilized plasma, or lyophilized tissue in 10 mL 250 nM IS in 100 mM HCl containing 0.2%
3,3'-thiodipropionic acid at 20000 rpm for 2 min, sonicate for =^30 min, centrifuge at 5000 g for
20 min, discard fat layer, filter (Millipore ultrafiltration insert (MW cutoff 5000) prewashed
with 200 fxL 100 mM HCl containing 0.2% 3,3'-thiodipropionic acid) 3 mL supernatant while
centrifuging at 3500 g for 1 h. Mix 20 |xL deproteinized sample (or 10 |xL peptide hydrolysate)
with 180 |xL buffer, vortex, add 200 |xL reagent, mix, heat at 70 for 15 min with mixing at 1
min and 12 min, cool in an ice bath for 5 min, centrifuge at 10000 g for 10 s, add 400 |xL
diluent, mix thoroughly, centrifuge at 15000 g for 5 min, inject a 10 jxL aliquot of the super-
natant. (Prepare buffer by dissolving 630 mg sodium bicarbonate in 40 mL water, adjusting
pH to 8.6 with NaOH, and making up to 50 mL with water. Prepare reagent by sonicating 40
mg dabsyl chloride in 10 mL acetone for 10 min, then filtering into brown vials and storing at
-20. Prepare diluent by mixing 50 mL MeCN, 25 mL EtOH, and 25 mL mobile phase A.)
HPLC VARIABLES
Guard column: present but not specified
Column: 150 X 3.9 4 |xm Novapak C18
Mobile phase: Gradient. A was DMF:9 mM NaH2PO4 containing 0.16% triethylamine, adjusted
to pH 6.55 with phosphoric acid. B was MeCN:water 80:20. A:B 92:8 for 2 min, to 80:20 over
5 min (Waters convex curve 5), to 65:35 over 28 min (Waters concave curve 7), to 50:50 over
10 min, to 0:100 over 21 min, maintain at 0:100 for 11 min, return to initial conditions over
0.5 min, re-equilibrate for 12.5 min.
Flow rate: 1
Detector: UV 436
CHROMATOGRAM
Internal standard: norleucine (40.90), norvaline (35.06)
OTHER SUBSTANCES
Extracted: amino acids dopamine, histamine, norepinephrine, taurine
KEYWORDS
rinse glass and plasticware with 70% EtOH and water and dry before use; derivatization; cheese;
meat; sausage; fish; plasma
REFERENCE
KrauseJ.; Bockhardt,A.; Neckermann,H.; Henle,T.; Klostermeyer,H. Simultaneous determination of amino
acids and biogenic amines by reversed-phase high-performance liquid chromatography of the dabsyl deriv-
atives, J.ChromatogrA, 1995, 715, 67-79.
SAMPLE
Sample preparation: Serum. Add 1 mL serum to 100 mg activated aluminum oxide suspended
in 1 mL pH 8.7 Tris-HCl buffer, stir, let stand for 10 min. Discard the supernatant and wash
the solid three times with 5 mL portions of water, wash the solid with 3 mL MeOH, dry under
reduced pressure, elute with 3 mL 4 M acetic acid. Evaporate the eluate to dryness under
reduced pressure, reconstitute the residue with 90 |xL water, inject a 10 |xL aliquot. Urine. 5
mL Urine + 5.3 mL 2 M HCl, heat at 100 for 20 min, cool to room temperature, add 1 mL 50
mM disodium EDTA, adjust the pH to 8.5 with dilute ammonia, add 500 mg 200 mesh alu-
minum oxide (Wako), shake for 10 min, filter, wash the solid with 10 mL water, elute with 5
mL 300 mM acetic acid, inject an aliquot of the eluate.
HPLCVARIABLES
Column: 250 X 3.6 10-25 |xm Hitachi 3011 C resin
Mobile phase: 50 mM K2HPO4 containing 0.05% phosphoric acid
Flow rate: 0.6
Detector: F ex 383 em 486 following post-column reaction. The column effluent mixed with 1%
2-cyanoacetamide in water pumped at 0.5 mL/min and with buffer pumped at 1 mL/min and
the mixture flowed through a 5 m X 0.5 mm ID PTFE coil at 100 1 to the detector. (Buffer
was 600 mM boric acid containing 750 mM KOH.)
CHROMATOGRAM
Retention time: 7
Limit of detection: 0.28 pmole
OTHERSUBSTANCES
KEYWORDS
post-column reaction; serum; SPE
REFERENCE
Honda,S.; Takahashi,M.; Araki,Y; Kakehi,K. Postcolumn derivatization of catecholamines with 2-cyanoacetam-
ide for nuorimetric monitoring in high-performance liquid chromatography, J.Chromatogr., 1983,274, 4 5 -
52.
SAMPLE
Sample preparation: Condition a 150 |xL Toyopak IC-SP S (sulfopropyl resin, H+ form) SPE
cartridge (Tosoh) with 10 mL water. Plasma. 700 |xL Plasma + 50 |xL 700 nM 3,4-dihydroxy-
benzylamine + 350 (JLL 2 M perchloric acid, mix, centrifuge at 4 at 1000 g for 15 min. Remove
a 700 |xL aliquot of the supernatant and add it to 30 |JLL 2 M potassium carbonate, centrifuge
at 4 at 1000 g for 5 min. Add a 500 |xL aliquot of the supernatant to the SPE cartridge, wash
with 1 mL water, wash with 500 JJLL EtOH:water 50:50, wash with 5 mL water, elute with 500
(JLL 2 M sodium perchlorate, filter (0.2 |xm), inject a 50 |xL aliquot of the filtrate. Urine. Acidify
urine collected over 24 h with 10 mL 6 M HCl. 500 [xL Urine + 25 |xL 10 |xM 3,4-dihydroxy-
benzylamine + 25 JLJLL 40 |xM ferulic acid + 500 |JLL 1 M perchloric acid, mix, centrifuge at 4 at
1000 g for 15 min. Remove a 700 JJLL aliquot of the supernatant and add it to 30 |JLL 2 M
potassium carbonate, centrifuge at 4 at 1000 g for 5 min, add a 500 JJLL aliquot of the super-
natant to the SPE cartridge, wash with 1.5 mL water, wash with 500 |xL EtOH:water 50:50,
wash with 5 mL water, elute with 500 jxL 2 M sodium perchlorate, filter (0.2 (xm), inject a 50
IxL aliquot of the filtrate.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm TSK-gel ODS-80TM (Tosoh)
Mobile phase: Gradient. A was buffer. B was MeCN:MeOH:buffer 8:12:80, pH 3.1. A:B 100:0 for
4 min, to 60:40 over 8 min, to 0:100 over 2 min, maintain at 0:100 for 16 min, return to initial
conditions (step gradient), re-equilibrate for 20 min. Buffer was 60 mM pH 3.1 citric acid
containing 32 mM Na2HPO4, 1.7 mM sodium hexanesulfonate, and 0.1 mM disodium EDTA(J.
Chromatogr. 1989, 467, 237).
Flow rate: 1
Detector: F ex 345 em 480 following post-column reaction. The column effluent passed through
a Hitachi 655A electrochemical detector with carbon cloth electrodes; working electrode at
+0.68 V versus reference electrode (200 mM equimolar mixture of potassium hexacyanofer-
rate(II) and potassium hexacyanoferrate(III) containing 200 mM potassium nitrate and 200
mM KOH). The effluent from the electrochemical detector mixed with 20 mM meso-l,2-di-
phenylethylenediamine in 50 mM HCl pumped at 0.4 mL/min and with 1 M glycine containing
490 mM KOH and 3 mM potassium hexacyanoferrate(III) pumped at 0.4 mL/min. This mixture
flowed through a 10 m X 0.47 mm ID coil at 80 to the detector (J. Chromatogr. 1989, 467,
237).
CHROMATOGRAM
Internal standard: 3,4-dihydroxybenzylamine (12.5)
OTHER SUBSTANCES
Extracted: dopamine, levodopa, metanephrine, 3-methoxytyramine, norepinephrine
KEYWORDS
post-column reaction; plasma; SPE
REFERENCE
Nohta,EL; Yamaguchi,E.; Ohkura,Y.; Watanabe,H- Measurement of catecholamines, their precursor and metab-
olites in human urine and plasma by solid-phase extraction followed by high-performance liquid chroma-
tography with fluorescence derivatization, J.Chromatogr., 1989, 493, 15-26.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 3 mL ice-cold MeOH:500 mM perchloric acid 98:
2, centrifuge at 4 at 4000 g for 3 min. Remove 200 JJLL of the supernatant and add it to 100
IxL 80 ng/mL N-methyldopamine, evaporate to dryness under vacuum, reconstitute in 200 JJLL
mobile phase, inject a 5-20 |xL aliquot. Urine. 1 mL Urine + 50 mL water, inject a 10 JJLL aliquot.
(To deconjugate adjust pH to 1, flush with nitrogen, heat in a boiling water bath for 1 h, dilute
with 50 mL water, inject a 10 |xL aliquot.)
HPLC VARIABLES
Column: 150 X 4.6 5 (xm Supelcosil LC-18
Mobile phase: MeOH: 13 mM sodium acetate containing 0.5 mM sodium 1-octanesulfonate and
0.5 mM disodium EDTA 14:86, pH 3.10
Flow rate: 1
Detector: E, ESA Model 5100 A Coulochem, Model 5011 A analytical cell, first electrode +0.40
V, second electrode -0.30 V
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Extracted: methyldopa, norepinephrine, dopamine, dihydroxyphenylacetic acid, 3-O-methyl-
methyldopa, homovanilic acid
KEYWORDS
REFERENCE
Lucarelli,C; Betto,R; Ricciarello,G.; Grossi,G. High-performance liquid chromatographic determination of L-3-
(3,4-dihydroxyphenyl)-2-methylalanine (a-methyldopa) in human urine and plasma, J.Chromatogr., 1991,
541, 285-296.
SAMPLE
Sample preparation: Condition a Toyopak IC-SP S sulfopropyl resin, H+ form, SPE cartridge
(Tosoh) with 10 mL water and 2 mL 200 mM pH 5.0 sodium phosphate buffer. Plasma. 700 |JLL
Plasma + 30 (xL 700 nM isoproterenol + 50 (xL 7 (xM 3,4-dihydroxyphenylpropanoic acid + 350
|xL 2 M perchloric acid, mix, centrifuge at 4 at 1000 g for 15 min. Remove a 700 |xL aliquot
of the supernatant and adjust the pH to 1.5-2.0 with about 150 |xL 2 M potassium carbonate,
centrifuge at 4 at 1000 g for 5 min, add the supernatant to the SPE cartridge, wash with 10
mL water, elute with 300 jxL MeOH:2 M sodium perchlorate 7:93, filter (cellulose acetate mem-
brane), inject a 100 |xL aliquot of the filtrate. Urine. Collect human urine for 24 h in the
presence of 10 mL 6 M HCl. 500 jxL, Urine + 10 jxL 15 JJLM isoproterenol + 25 |JLL 800 |xM 3,4-
dihydroxyphenylpropanoic acid + 500 |xL 1 M perchloric acid, mix, centrifuge at 4 at 1000 g
for 15 min. Remove a 700 ^xL aliquot of the supernatant and adjust the pH to 1.5-2.0 with
about 130 |xL 2 M potassium carbonate, centrifuge at 4 at 1000 g for 5 min, add the super-
natant to the SPE cartridge, wash with 1.5 mL water, wash with 500 |xL EtOHiwater 50:50,
wash with 5 mL water, elute with 500 |xL 1.5 M KCl in MeOH:100 mM HCl 7:93, filter (cellulose
acetate membrane), inject a 100 |xL aliquot of the filtrate.
HPLC VARIABLES
Mobile phase: MeOH:buffer 7:93 (Buffer was 30 mM pH 2.5 citrate buffer containing 0.4 mM
sodium octanesulfonate.)
Flow rate: 0.8
Detector: F ex 350 em 480 following post-column reaction. The column effluent mixed with re-
agent A pumped at 0.3 mL/min and the mixture flowed through a 3 m X 0.5 mm ID stainless
steel coil at 90. The effluent from this coil mixed with reagent B pumped at 0.3 mL/min and
the mixture flowed through a 10 m X 0.5 mm ID stainless steel coil at 90 and through a i m
X 0.5 mm ID stainless steel cooling coil to the detector (Anal. Sci. 1991, 7, 257). (Reagent A
was 10 mM sodium periodate containing 3 mM potassium ferricyanide. Reagent B was 30 mM
meso-l,2-diphenylethylenediamine in EtOH:water 70:30 containing 130 mM sodium
methylate.)
CHROMATOGRAM
Retention time: 20
OTHER SUBSTANCES
Extracted: dopamine, levodopa, metanephrine, 3-methoxytyramine, norepinephrine, normeta-
nephrine
KEYWORDS
REFERENCE
Jeon,H--K; Nohta,H; Ohkura,Y. High-performance liquid chromatographic determination of catecholamines
and their precursor and metabolites in human urine and plasma by postcolumn derivatization involving
chemical oxidation followed by fluorescence reaction, Anal.Biochem., 1992, 200, 332-338.
SAMPLE
Sample preparation: 2 mL Plasma or 1 mL urine + dihydroxybenzylamine + 20 mg Sigma WA4
alumina + 200 JLL 1 M pH 8.6 Tris-EDTA buffer, mix for 10 min, discard plasma. Wash the
alumina three times with 3 mL water and dry it. Add 125 jxL 500 mM phosphoric acid, after
1 min inject a 100 jxL aliquot. (Ann. Clin. Biochem. 1985, 22, 194-203)
HPLC VARIABLES
Column: 250 X 4.5 5 |xm Ultratechsphere
Mobile phase: Per liter 75 mmol citric acid, 58.5 mmol NaH2PO4, 0.2 mmol disodium EDTA, and
4.4 mmol heptanesulfonic acid, pH adjusted to 3.4, made up to a final volume of 2 L, add 200
mL MeOH
Flow rate: 1
Detector: E, ESA Coulochem conditioning cell +0.35 V, first electrode +0.05 V, second electrode
-0.35 V
CHROMATOGRAM
Internal standard: dihydroxybenzylamine (10.53)
OTHER SUBSTANCES
Simultaneous: levodopa, metanephrine, norepinephrine, 3-methoxytyrosine, normetanephrine,
dihydroxyphenylacetic acid, dopamine
KEYWORDS
plasma
REFERENCE
Dutton,J.; Copeland,L.G.; Playfer,J.R.; Roberts,N.B. Measuring L-dopa in plasma and urine to monitor therapy
of elderly patients with Parkinson disease treated with L-dopa and a dopa decarboxylase inhibitor,
Clin.Chem., 1993, 39, 629-634.
SAMPLE
residue with 50 (JLL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
a 10 (urine) or 30 (blood) jxL aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 5 mg amino acids in 10 mL MeCN:water:triethylamine 50:50:
0.55. Remove a 50 u-L aliquot and add it to 50 uL 0.66% 2,3,4,6-tetra-O-benzoyl-p-D-glucopy-
ranosyl isothiocyanate (Fluka) in MeCN, shake mechanically for 30 min, add 10 \LL 0.26%
ethanolamine in MeCN, shake for 10 min, make up to 1 mL with MeCN, inject a 10 |JLL aliquot.
HPLC VARIABLES
Column: 25 X 4 (sic) 5 |xm LiChrospher 100 RP-18
Flow rate: 1
Detector: UV 231
CHROMATOGRAM
Retention time: k' 22.61, k' 25.67 (enantiomers)
OTHER SUBSTANCES
Simultaneous: phenylephrine
KEYWORDS
REFERENCE
Lobell,M.; Schneider,M.R 2,3,4,6-Tetra-O-benzoyl-p-D-glucopyranosyl isothiocyanate: an efficient reagent for
the determination of enantiomeric purities of amino acids, p-adrenergic blockers and alkyloxiranes by high-
performance liquid chromatography using standard reversed-phase columns, J.Chromatogr., 1993, 633,
287-294.
SAMPLE
Matrix: cell cultures
Sample preparation: Centrifuge at 20000 g for 5 min, dilute the supernatant 10-50-fold, inject
a 30 |JLL aliquot.
HPLC VARIABLES
Column: 125 X 3 3 |xm Nucleosil 100C18
Mobile phase: MeOH:buffer 5:95, pH adjusted to 3.0 with 5 M NaOH (Buffer was 50 mM citric
acid containing 30 mM phosphoric acid, 0.75 mM octylsulfate, and 0.5 mM EDTA.)
Flow rate: 0.7
Detector: E, Waters 460, glassy carbon electrode
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: norepinephrine
REFERENCE
Ghindilis,A.L.; Michael,N.; Makower,A. A new sensitive and simple method for detection of catecholamines
from adrenal chromaffin cells, Pharmazie, 1995, 50, 599-600.
SAMPLE
Matrix: enzyme incubations
Sample preparation: Prepare a SPE column by adding 500 |xL of a 20% suspension of 19-40
|xm Toyopak SP (strong cation-exchange sulfopropyl resin, Na+ (Toyo Soda)) in water to a 35
X 6 column, wash with two 2 mL portions of 2 M NaOH, wash with 5 mL water, wash with 2
mL 2 M HCl, wash with 10 mL water. 350 |xL Enzyme incubation at pH 8.5 + 50 ^xL 3 M
trichloroacetic acid + 50 jxL 2 jxM isoproterenol, centrifuge at 4 at 1000 g for 10 min, add a
300 jxL aliquot of the supernatant to the SPE column, wash with 10 mL water, wash with 3
mL 200 mM pH 5.5 phosphate buffer, wash with 10 mL water, elute with 2 mL EtOHrI M
NaCl 70:30. Add 100 |xL 100 mM pH 6.7 1,2-diphenylethylenediamine in 100 mM HCl and 100
(JLL 15 mM potassium ferricyanide to the eluate, heat at 37 for 40 min, inject a 100 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 TSK-gel ODS-120T (Toyo Soda)
Mobile phase: MeCN:MeOH:50 mM pH 7.0 Tris-HCl buffer 52:3:45 (Wash with MeCN:MeOH:
Flow rate: 1
CHROMATOGRAM
Retention time: 8
KEY WpRDS
derivatization; SPE
REFERENCE
Lee,M.; Nohta,H-l Ohkura,Y.; Yoo,B- Determination of phenylethanolamine N-methyltransferase by high-per-
formance liquid chromatography with fluorescence detection, J.Chromatogr., 1985, 348, 407-415.
SAMPLE
HPLC VARIABLES
Column: 250 x 4.6 10 |xm Whatman PXS ODS-3 C18
Mobile phase: MeCN:MeOH:water:85% phosphoric acid: sodium octanesulfonate 10:20:70:0.5:
0.108 (v/v/v/v/w)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Simultaneous: milrinone
KEYWORDS
injections; 10% calcium chloride; 7.5% sodium bicarbonate; stability-indicating
REFERENCE
Wilson,T.D.; Forde,M.D. Stability of milrinone and epinephrine, atropine sulfate, lidocaine hydrochloride, or
morphine sulfate injection, Am. J.Hosp.Pharm., 1990, 47, 2504-2507.
SAMPLE
Sample preparation: Tablets. Dissolve powdered tablets in 10 mM HCl, filter if necessary, inject
an aliquot. Injections, solutions. Dilute with 10 mM HCl, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Partisil-5 ODS-3
Mobile phase: MeOH:buffer 30:70 (Buffer was 10 mM sodium 1-octanesulfonate in 0.2% acetic
acid.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: norepinephrine, levonordefrin, isoproterenol, phenylephrine, metaraminol,
impurities
KEYWORDS
tablets; injections; ophthalmic solutions; inhalation solutions
REFERENCE
Smela,M.J.,Jr.; Stromberg,R- Liquid chromatographic determination of six sympathomimetic drugs in dosage
forms, JAssoc.Off.Anal.Chem., 1991, 74, 289-291.
SAMPLE
Sample preparation: Tablets. Grind tablets, weigh out a portion, dissolve in 50 mL mobile
phase, sonicate, filter (No. 4 sintered glass plate), dilute, inject an aliquot. Capsules. Dissolve
10 capsules (without opening) in 100 mL mobile phase, sonicate, inject an aliquot. Injections,
ampules, sprays. Dilute, inject an aliquot.
HPLC VARIABLES
Column: 120 X 4.6 Spherisorb C18 ODS-2
Mobile phase: Isopropanol:buffer 5:95 (Buffer was 100 mM sodium dodecyl sulfate containing
25 mM Na2HPO4, pH adjusted to 3.0 with HCl.)
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: carbidopa, dopamine, hydrochlorothiazide, isoproterenol, levodopa, methyldopa,
norepinephrine, phenylephrine
KEYWORDS
tablets; capsules; injections; ampules; sprays
REFERENCE
Villanueva Camaiias,R.M.; Sanchis Mallols,J.M.; Torres Lapasi6,J.R.; Ramis-Ramos,G. Analysis of pharmaceu-
tical preparations containing catecholamines by micellar liquid chromatography with spectrophotometric
detection, Analyst, 1995, 120, 1767-1772.
SAMPLE
Matrix: solutions
Sample preparation: Dilute with 5% dextrose, inject a 20 jxL aliquot.
HPLC VARIABLES
Column: Waters microparticulate C18
Mobile phase: MeOH:350 mM acetic acid and 5 mM sodium heptanesulfonate 20:80
Flow rate: 1.6-2.0
CHROMATOGRAM
OTHER SUBSTANCES
Interfering: dopamine
REFERENCE
Williams,D.A.; Fung,E.Y.Y.; Newton,D.W. Ion-pair high-performance liquid chromatography of terbutaline and
catecholamines with aminophylline in intravenous solutions, J.Pharm.ScL, 1982, 71, 956-958.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 2.15 (tails)
OTHER SUBSTANCES
Simultaneous: monoacetylmorphine, thebacon, oxycodone, thebaine, norlevorphanol, metha-
done, benzylmorphine, ethylmorphine, morphine-N-oxide, codeine, codeine-N-oxide, morphine,
ethoheptazine, morphine-3-glucuronide, pholcodeine, norpethidine, hydrocodone, dihydroco-
deine, dihydromorphine, levorphanol, norcodeine, normorphine, pemoline, benzphetamine, die-
thylpropion, mazindol, tranylcypromine, caffeine, fenethyline, phendimetrazine, methylpheni-
date, phenelzine, chlorphentermine, norpseudoephedrine, phentermine, fenfluramine,
metnylenedioxyamphetamine, amphetamine, normetanephrine, 4-hydroxyamphetamine,
bromo-STP, STP, prolintane, 2-phenethylamine, tyramine, trimethoxyamphetamine, phenyl-
ephrine, pseudoephedrine, ephedrine, methylephedrine, dimethylamphetamine, methamphet-
amine, mescaline, mephentermine, buprenorphine, dextromoramide, phenoperidine, fentanyl,
etorphine, piritramide, noscapine, papaverine, naloxone, dextropropoxyphene, nalorphine,
phenazocine, norpipanone, levallorphan
Interfering: pipradol, phenylpropanolamine, fencamfamin, hydroxypethidine, normethadone,
meperidine, dipipanone, diamorphine, pentazocine, acetylcodeine
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Inject a 250 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Nucleosil 5-C18
Mobile phase: 50 mM Potassium perchlorate containing 250 (xL/L 3% copper acetate in water
and 10 g/L sodium acetate, pH adjusted to 4.45 with acetic acid
Flow rate: 1
Detector: F ex 400 em 500 following post-column reaction. The column effluent flowed through
the reactor then through a 3.5 m X 0.8 mm ID coil of PTFE tubing at 30. The effluent from
the coil mixed with reducing solution pumped at 1 (?) mL/min and this mixture flowed through
a 2 m X 0.8 mm ID PTFE coil at 30 to the detector. (Prepare the reactor as follows. Dissolve
75.3 g manganese nitrate in 500 mL water, add 50 g 18-35 mesh silica gel (Macherey-Nagel),
stir vigorously, slowly add 31.6 g potassium permanganate in 500 mL water, stir for 30 min,
filter (500 |xm sieve), wash until no permanganate color is left, dry in a desiccator, pack in a
50 X 2.1 stainless steel tube. The reducing solution was 266 g NaOH, 13.4 g anhydrous sodium
sulfite, and 9 mL 2-mercaptoethanol in 1 L water. Note that some Nucleosil 5-C18 column
packings do not give separation at pH 4.45. In this case it is necessary to use 50 mM perchloric
acid as mobile phase and mix the column effluent with pH 4.4 sodium acetate buffer before it
enters the reactor.)
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Riiter,J.; Kurz,U.R; Neidhart,B. Solid phase reactors as an analytical tool in the determination of urinary
noradrenaline and adrenaline, J.Liq.Chromatogr., 1985, 8, 2475-2496.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Partisil ODS-3
Mobile phase: MeOH:buffer 30:70 (Buffer was 10 mM octanesulfonic acid in 0.2% acetic acid.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: isoproterenol, levonordefrin, metaraminol, phenylephrine
REFERENCE
Phenomenex Catalog, 1994, p. 1.077.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
rin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benzphe-
tamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone,
apram, doxepin, dronabinol, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide,
REFERENCE
Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.ToxicoL, 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Hexane:l,2-dichloroethane:EtOH/trifluoroacetic acid 55:35:10 (EtOH/trifluoroac-
etic acid was premixed 20:1.)
Flow rate: 0.7-1
Detector: UV 282
KEYWORDS
REFERENCE
Cleveland,T. Pirkle-concept chiral stationary phases for the HPLC separation of pharmaceutical racemates,
J.Liq.Chromatogr., 1995, 18, 649-671.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (glass potters) with 5 volumes of 100 mM perchloric acid
containing 1.9 mM sodium bisulfite, centrifuge at 10000 g at 4 for 30 min. Filter (0.22 |xm)
the supernatant, add dihydroxybenzylamine, inject a 5-20 |JLL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 Hypersil H5 ODS
Mobile phase: EtOH.buffer 2:98 (Buffer was 13.8 g NaH2PO4, 60 mg disodium EDTA, and 20
mg 1-octanesulfonic acid in 1 L water, pH adjusted to 3.70 with phosphoric acid.)
Flow rate: 1
Detector: E, Unicam PU 4022, 70 mV
CHROMATOGRAM
Retention time: 9
Internal standard: dihydroxybenzylamine
OTHER SUBSTANCES
KEYWORDS
adrenal; fetal
REFERENCE
Garcia,J.C; Blanco,L.; McPherson,M.; Leiva,A.; Maci&s,R. High-performance liquid chromatographic determi-
nation of norepinephrine, epinephrine and dopamine in human foetal adrenal gland, J.Chromatogr.B, 1994,
656, 77-80.
SAMPLE
Matrix: urine
Sample preparation: Acidify urine with 1% (v/v) 6 M HCl. 6-10 mL Swine urine or 1-2.5 mL
rat urine, centrifuge at 4000 g for 30 min, add 200 ng 3,4-dihydroxybenzylamine hydrobromide
and 15 mL lg/L EDTA, adjust to pH 6.45-6.55 with HCl or NaOH. Add the mixture to a cation-
exchange resin SPE cartridge (Bio-Rad), wash twice with 10 mL water and with 5 ml water,
elute with 8 mL 10 g/L boric acid. Dilute boric acid eluate with an equal volume of mobile
phase, inject a 60 |xL aliquot. (Procedure for determining methoxycatecholamines is also
described.)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Kromasil C8
Mobile phase: MeOH:buffer 15:85 (Mobile phase was 300 mL MeOH, 1.5 mL 200 mg/mL 1-
octanesulfonic acid, 100 mL 1 M sodium acetate, and about 1 L water. The pH was adjusted
to pH 3.8 with citric acid and made up to 2 L with water.)
Flow rate: 0.6
Detector: E, Bioanalytical Systems, glassy carbon electrode + 650 mV, Ag/AgCl reference
electrode
CHROMATOGRAM
Internal standard: 3,4-dihydroxybenzylamine hydrobromide (10.31)
OTHER SUBSTANCES
KEYWORDS
pig; rat; SPE; pharmacokinetics
REFERENCE
Hay,M.; Mormede,R Determination of catecholamines and methoxycatecholamines excretion patterns in pig
and rat urine by ion-exchange liquid chromatography with electrochemical detection, J.Chromatogr.B, 1997,
703, 15-23.
SAMPLE
Matrix: urine
Sample preparation: Acidify urine by adding 1% 6 M HCl. 5 mL Acidified urine + 1 mL 7.5%
disodium EDTA, adjust pH to 8.5 with 1 M NaOH, add 250 mg alumina (previously treated
with 2 M HCl), shake for 5 min, decant the supernatant, wash the alumina three times with
5 mL portions of water. Place the alumina in a 4 mm ID glass column, elute with 250 mM
acetic acid in water, collect 2.5 mL eluate, inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 1000 X 2.1 Zipax SCX (DuPont)
Mobile phase: MeCN:50 mM NaH2PO4 5:95
Flow rate: 0.8
reagent pumped at 0.4 mL/min and the mixture flowed through a 10 m X 0.5 mm PTFE coil
at 75 0.1 to the detector. (Reagent was 500 mM borate buffer adjusted to pH 9.7 with NaOH.)
CHROMATOGRAM
Retention time: 9
Limit of quantitation: 0.25 ng
OTHER SUBSTANCES
KEYWORDS
post-column reaction; SPE
REFERENCE
Nimura,N.; Ishida,K.; Kinoshita,T. Novel post-column derivatization method for the fluorimetric determination
of norepinephrine and epinephrine, J.Chromatogr., 1980, 221, 249-255.
SAMPLE
Matrix: urine
Sample preparation: Add disodium EDTA and sodium metabisulfite to urine. 100 JJLL Urine +
2 mL water, vortex, add 1 mL reagent 1, add 5 mL reagent 2, shake vigorously for 2 min,
centrifuge at 2000 g for 2 min, freeze in dry ice/acetone. Remove the organic layer and add it
to 200 JJLL 80 mM acetic acid and 2 mL n-octanol saturated with acetic acid, shake vigorously
for 2 min, centrifuge at 2000 g for 2 min, freeze in dry ice/acetone until the aqueous layer is
just solid, remove the organic layer. Thaw out the aqueous layer and add it to 1 mL reagent 1
and 5 mL reagent 2, shake vigorously for 2 min, centrifuge at 2000 g for 2 min, freeze in dry
ice/acetone. Remove the organic layer and add it to 200 |xL 80 mM acetic acid and 2 mL n-
octanol saturated with acetic acid, shake vigorously for 2 min, centrifuge at 2000 g for 2 min,
freeze in dry ice/acetone until the aqueous layer is just solid, remove the organic layer. Thaw
out the aqueous layer and add 100 |xL Bicine buffer, 250 |xL MeCN, and 100 |xL 100 mM 1,2-
diphenylethylenediamine in 100 mM HCl, vortex, add 20 (xL 20 mM potassium ferricyanide,
vortex, heat at 37 for 40 min, cool to room temperature, inject a 100 (xL aliquot. (Prepare
reagent 1 by dissolving 214 g ammonium chloride and 10 g disodium EDTA in 2 L water, adjust
pH to 8.3-8.5 with concentrated ammonium hydroxide, add 4.0 g diphenylborate-ethanolamine
complex, stir for several hours until a clear solution is obtained. Prepare reagent 2 by dissolving
2.5 g tetraoctylammonium bromide and 10 mL n-octanol (saturated with acetic acid) in 1 L n-
heptane. Prepare Bicine buffer by dissolving 14.3 g Bicine (N,N-bis(2-hydroxyethyl)glycine) and
359 mg anhydrous sodium acetate in 45 mL water, stir overnight until dissolved, adjust pH to
7.30 with concentrated NaOH, make up to 50 mL with water. Note that concentration of 1,2-
diphenylethylenediamine is not given in paper. Other authors have used 100 mM
(J.Chromatogr. 1989, 487, 17; 1992, 574, 109; 1992, 583, 236).)
HPLC VARIABLES
Column: 150 X 4.6 5 jjiin Ultrasphere ODS
was MeCN:MeOH:50 mM pH 7.0 sodium acetate buffer 50:10:40. A:B 75:25 for 1 min, to 10:
90 over 7 min, return to initial conditions over 1 min.
Flow rate: 1
Detector: F ex 365 em 418 (cutoff filter)
CHROMATOGRAM
Retention time: 5
Limit of detection: <0.4 nM
OTHER SUBSTANCES
Simultaneous: isoproterenol
KEYWORDS
derivatization; protect from light
REFERENCE
Moleman,R; van Dijk,J. Determination of urinary norepinephrine and epinephrine by liquid chromatography
with fluorescence detection and pre-column derivatization, Clin.Chem., 1990, 36, 732-736.
SAMPLE
Matrix: urine
Sample preparation: Add 10-15 mL 6 M HCl to a 24 h volume of urine. 2 mL 3 M Tris buffer
containing 30 mM EDTA + 500 |JLL 10 |xM dihydrobenzylamide in 100 mM perchloric acid + 2
mL 3 M tris buffer containing 30 mM EDTA + 100 |xL 5 M NaOH + 4 mL acidified urine, mix,
add to a 1 mL SPE column containing 200 mg alumina (70-230 mesh-ASTM (Touzart et Ma-
tignon) at 3 mL/min, wash with 9 mL water at 12 mL/min, force through 0.5 mL air, elute
with 1 mL 150 mM perchloric acid at 0.75 mL/min, mix eluate, inject a 100 |xL aliquot. (The
pH of the Tris buffer is such that the pH of the mixture applied to the SPE column is 7.75-
8.0.)
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Nucleosil 100/C18
Mobile phase: MeOH.buffer 36:64 (Buffer was 75 mM NaH2PO4, 0.15 mM EDTA, and 6 mM
sodium heptanesulfonate, pH 3.96.)
Flow rate: 1.25
CHROMATOGRAM
Retention time: 7.6
Internal standard: dihydrobenzylamide hydrobromide (9.6)
OTHER SUBSTANCES
Simultaneous: levodopa, methyldopa
KEYWORDS
SPE
REFERENCE
Said,R.; Robinet,D.; Barbier,C; Sartre, J.; Huguet,C. Fully automated high-performance liquid chromatographic
assay for the analysis of free eateeholamines in urine, J.Chromatogr., 1990, 530, 11-18.
SAMPLE
Matrix: urine
Sample preparation: 100 u,L Urine + 125 |xL 218.6 nM a-methylnorepinephrine in 10 mM HCl
+ 1 mL 10 mM HCl + 1 mL reagent + 5 mL 4.6 mM tetraoctylammonium bromide in n-heptane:
1-octanol 99:1, shake for 2 min, centrifuge at 20 at 1000 g for 5 min, freeze in dry ice/acetone.
Remove the organic phase and add it to 2 mL 1-octanol saturated with 80 mM acetic acid and
200 |JLL 80 mM acetic acid, shake, centrifuge at 20 at 1000 g for 5 min, freeze in dry ice/
acetone. Discard the organic layer and add 1 mL 10 mM HCl to the aqueous layer, add 2 mL
1-octanol saturated with 80 mM acetic acid, add 150 uX 80 mM acetic acid, shake, centrifuge
at 20 at 1000 g for 5 min, freeze in dry ice/acetone. Discard the organic layer and add 200 jxL
MeCN and 50 JJLL buffer to the aqueous layer, add 100 IJLL 100 mM 1,2-diphenylethylenediamine
in 100 mM HCl, add 20 |xL 20 mM potassium ferricyanide in water, heat at 37 in the dark
for 1 h, inject a 50 |JLL aliquot. (Reagent was 8.9 mM diphenylborate-ethanolamine complex in
2 M pH 8.6 ammonia/ammonium chloride buffer containing 13.4 mM EDTA. Buffer was 1.75
M pH 7.05 bicine in water containing 1% EDTA. Recrystallize 1,2-diphenylethylenediamine
from tolueneilight petroleum (bp 60-80) 10:90, dry overnight at 60.)
HPLC VARIABLES
Column: 100 X 4.6 3 [xm Cp MicroSpher C18 (Chrompack)
Mobile phase: MeCN:MeOH:50 mM pH 7.0 sodium acetate 40:8:50 (At the end of the day flush
column with 60 mL MeCN.MeOH.water 70:10:20.)
Flow rate: 1
CHROMATOGRAM
Retention time: 4
Internal standard: a-methylnorepinephrine (Janssen, Beerse, Belgium) (3)
Limit of quantitation: 1.6 nM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
van der Hoorn,F.A.J.; Boomsma,R; Man in 't Veld,A.J.; Schalekamp,M.A.D.H. Improved measurement of uri-
nary eateeholamines by liquid-liquid extraction, derivatization and high-performance liquid chromatogra-
phy with fluorimetric detection, J.Chromatogr., 1991, 563, 348-355.
SAMPLE
Matrix: urine
Sample preparation: Condition a 100 mg Bakerbond C-18 SPE cartridge with 2 mL MeOH and
2 mL buffer I. Heat urine at 50, centrifuge, remove a 500 |xL aliquot and add it to 1 mL buffer
II (?), add 20 (xL 860 ng/mL dihydroxybenzylamine, shake for 5 min, add a 1 mL aliquot to the
SPE cartridge, wash with 2 mL buffer I, wash with 1 mL MeOHibuffer I 50:50, wash with 500
JJLL water, elute with 2 mL 1 M acetic acid, inject a 20 |xL aliquot. (Buffer I was 200 mM
ammonium chloride containing 0.05% EDTA and 0.4% tetrabutylammonium iodide, pH ad-
justed to 8.0 0.1. Buffer II was 2 M ammonium chloride containing 0.5% EDTA and 1.2%
tetrabutylammonium iodide, pH adjusted to 8.0 0.1.)
HPLC VARIABLES
Column: 150 X 3 5 urn Separon SGX C-18 (Tessek)
Mobile phase: MeOH.buffer 5:95-7:93 (Buffer was 50 mM pH 3.0 0 . 1 phosphate buffer con-
taining 50 mM EDTA, 1 mM sodium octanesulfonate, and 1 mM NaCl.)
Detector: E, AMOR 400 mV (SunChrom)
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
SPE
REFERENCE
Brandsteterova,E.; Krajnak,K; Skacani,I. HPLC analysis of urinary catecholamines using affinity SPE pro-
cedure, Pharmazie, 1995, 50, 825-826.
SAMPLE
Matrix: urine
Sample preparation: Adjust urine to pH 3.0 with 6 M HCl, centrifuge at 1600 g for 10 min.
900 |xL Supernatant + 100 |xL 2.5 |xM N-methyldopamine, mix, inject a 100 |xL aliquot on to
column A and elute to waste with mobile phase A, after 5 min elute the contents of column A
on to column B with mobile phase B, monitor the effluent from column B.
HPLC VARIABLES
Column: A 35 X 4 TSK-precolumn-CA 2 (Tosoh); B 150 X 4.6 mixed mode (C18/cation exchange)
(AUtech)
Mobile phase: A 15 mM pH 6.0 citric acid/trisodium citrate buffer; B 200 mM pH 6.0 citric acid/
trisodium citrate buffer
Flow rate: 0.7
Detector: E, Mitsubishi 8 channel, 150 mV
CHROMATOGRAM
Internal standard: N-methyldopamine (30 mV) (20)
OTHER SUBSTANCES
Extracted: dopamine (30 mV), metanephrine (380 mV), 3-methoxytyramine (340 mV), norepi-
nephrine (150 mV), normetanephrine (380 mV), serotonin (150 mV)
KEYWORDS
column-switching
REFERENCE
Mashige,R; Matsushima,Y.; Miyata,C; Yamada,R.; Kanazawa,H.; Sakuma,L; Takai,N.; Shinozuka,N.; Ohk-
ubo,A.; Nakahara,K. Simultaneous determination of catecholamines, their basic metabolites and serotonin
in urine by high-performance liquid chromatography using a mixed-mode column and an eight-channel
electrochemical detector, Biomed.Chromatogr., 1995, 9, 221-225.
SAMPLE
Matrix: urine
Sample preparation: Condition a 10 X 2 20 mg 15-25 |xm PLRP-S polymer-based SPE cartridge
(Spark Holland) with 1 mL MeOH, with 0.5 mL water, and with 1.5 mL 200 mM pH 8.5
ammonia/ammonium chloride buffer containing 0.05% EDTA. Collect 24 h urine with 10 mL 6
M HCl, final pH 1-3. Dilute 4-fold with buffer. Inject a 200 ^L aliquot onto the SPE cartridge,
wash with 1 mL 200 mM pH 8.5 ammonia/ammonium chloride buffer containing 0.05% EDTA,
wash with 250 JJLL MeOH:200 mM pH 8.5 ammonia/ammonium chloride buffer 20:80, wash
with water at 1 mL/min for 2.25 min. Elute the contents of the SPE cartridge onto column A
with the mobile phase for 30 s then remove the SPE cartridge from the circuit, elute column
A with mobile phase onto column B, after 1.25 min elute column A to waste with mobile phase
and elute column B with mobile phase, monitor the effluent from column B. (Buffer was 2 M
pH 8.5 ammonia/ammonium chloride containing 0.5% EDTA, 0.1% diphenylborate, and 18 ng/
mL dihydroxybenzylamine.)
HPLC VARIABLES
Column: A 30 X 4.6 C18 (Brownlee); B 250 X 4.6 5 |xm Ultrasphere IP C18
Mobile phase: MeCN:MeOH:buffer 15:8:100, apparent pH adjusted to 3.2 with 1.5 M ortho-
phosphoric acid (Buffer was 50 mM KH2P(X containing 1 mM sodium heptane sulphate and
0.07 mM EDTA.)
Flow rate: 0.8
Detector: E, ESA Model 5100A, Model 5021 conditioning cell, Model 5011 analytical cell, oxidiz-
ing electrode +350 mV, screen electrode +100 mV, quantifying electrode -300 mV
CHROMATOGRAM
Retention time: 7
Internal standard: dihydroxybenzylamine (8)
OTHER SUBSTANCES
Extracted: norepinephrine, dopamine
KEYWORDS
SPE; column-switching
REFERENCE
Pastoris,A.; Cerutti,L.; Sacco,R; De Vecchi,L.; Sbaffi,A. Automated analysis of urinary catecholamines by high-
performance liquid chromatography and on-line sample pretreatment, J.Chromatogr.B, 1995, 664, 287-
293.
SAMPLE
Matrix: urine
Sample preparation: Acidify urine to pH 2.0-3.5 with 5 M HCl, centrifuge at 7000 g for 10 min,
inject a 10-500 |JLL aliquot on to column A and elute to waste with mobile phase A, after 10
min backflush the contents of column A on to column B with mobile phase B, elute with mobile
phase B, monitor the effluent from column B.
HPLC VARIABLES
Column: A nitrophenylboronic acid modified copolymer (U.S. Patent 4 767 529 (Chem.Abs. 1988,
108, 71698t)); B 53 X 4.6 1.5 |xm MICRA, NPS RP-18 (MICRA Scientific, Northbrook)
Mobile phase: A 20 mM (NHJ2HPO4 containing 10 mM EDTA, adjusted to pH 8.7 with 25%
ammonia solution; B 10 mM NaH2PO4 containing 0.1 mM dodecanesulfonic acid, adjusted to
pH 2.5 with orthophosphoric acid
Flow rate: A 0.5; B from 0.2 to 0.5 over 2 min, maintain at 0.5
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
KEYWORDS
column-switching
REFERENCE
Rudolphi,A.; Boos,K.-S.; Seidel,D. Coupled-column HPLC analysis of free urinary catecholamines using re-
stricted access affinity precolumn and micro-particulate nonporous silica analytical column, Chromatogra-
phia, 1995, 41, 645-650.
Epinine
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
apram, doxepin, dronabinol, ephedrine, estradiol, estriol, estrone, ethacrynic acid,
ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, feno-
profen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil,
fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glu-
tethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone,
hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imip-
ramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, iver-
mectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetaze-
pam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic
acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobar-
bital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene,
methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicy-
late, methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone,
methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, na-
phazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitra-
zepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone,
oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbi-
tal, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine,
REFERENCE
18, 233-242.
Epirizole
Merck Index: 3659
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 251
OTHER SUBSTANCES
Also analyzed: albendazole, prochlorperazine
REFERENCE
Epirubicin
Molecular formula: C27H29NO1 ,
Merck Index: 3660
SAMPLE
Matrix: blood
Sample preparation: Slowly add 200 (xL MeCN:100 mM orthophosphoric acid 80:20 to 200 |xL
plasma in a centrifuge tube. Vortex the white precipitate for 30 s and then centrifuge at 1500
g until a compact pellet and clear supernatant is obtained, inject a 20 jxL aliquot of the
supernatant.
HPLC VARIABLES
Guard column: 10 mm long C18
Column: 250 X 4.6 5|xm Spherisorb ODSl
Mobile phase: MeCN:buffer 35:65. (Buffer was 60 mM Na2HPO4 containing 0.05% triethylamine,
adjusted to pH 4.2 with citric acid.)
Flow rate: 1
CHROMATOGRAM
Limit of quantitation: 8.6 pmol/mL
OTHER SUBSTANCES
KEYWORDS
REFERENCE
BarkerJ.K; Crawford,S.M.; Fell,A-F- Determination of plasma concentrations of epirubicin and its metabolites
by high-performance liquid chromatography during a 96-h infusion in cancer chemotherapy,
J.Chromatogr.B, 1996, 681, 323-329.
SAMPLE
Matrix: blood
Sample preparation: Condition an Oasis HLB SPE cartridge (Waters) with 1 mL mobile phase:
water 1:3. 200 |xL Plasma + 500 mL mobile phase:water 1:3 + 100 ^xL 120 ng/mL IS in water,
vortex, add to the SPE cartridge, wash with 1 mL mobile phase:water 1:3, elute with 600 |xL
MeCN:mobile phase 1:1, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 200 X 4.6 10 jmm LiChrosorb RP18
Mobile phase: MeCN:water 29:71 containing 50 mM Na2HPO4 and 0.05% (v/v) triethylamine
adjusted to pH 4.6 with citric acid
Flow rate: 1.0
Detector: E, ESA Coulochem II, Model 5014 high-performance analytical cell containing amper-
ometric electrode +400 mV coupled with coulometric electrode -300 mV, palladium reference
electrode
CHROMATOGRAM
Retention time: 9.4
Internal standard: doxorubicin (12.0)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Ricciarello,R.; Pichini,S.; Pacifici,R.; Altieri,L; Pellegrini,M.; Fattorossi,A.; Zuccaro,R. Simultaneous determi-
nation of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance
liquid chromatography and electrochemical detection, J.Chromatogr.B, 1998, 707, 219-225.
SAMPLE
Matrix: blood
Sample preparation: Plasma + 100 ng daunorubicin hydrochloride + 1 mL pH 8.4 phosphate
buffer + 8 mL chloroform: 1-heptanol 90:10, shake mechanically for 30 min, centrifuge at 3300
rpm for 10 min. Remove the lower organic layer and evaporate it to 2 mL under a stream of
nitrogen. Add the residue to 200 |xL 300 mM phosphoric acid, vortex. Remove the aqueous
phase and add it to 2 mL n-hexane, vortex, centrifuge, inject a 100-150 (JLL aliquot of the
aqueous phase.
HPLCVARIABLES
Column: 250 X 4 7 |xm Lichrosorb RP-18
Mobile phase: MeOH:water 70:30 containing 0.5% acetic acid and 2.5 mM sodium
heptanesulfonate
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
KEYWORDS
REFERENCE
Hu,O.Y.-R; Chang,S.-R; Jame,J.-M.; Chen,K.-Y. Pharmacokinetic and pharmacodynamic studies with 4'-epi-
doxorubicin in nasopharyngeal carcinoma patients, Cancer Chemother.Pharmacol., 1989, 24, 332-337.
SAMPLE
Matrix: blood
Sample preparation: Condition a C2 SPE cartridge with 1 mL MeOH, 500 IJLL water and 500
JJLL buffer. 1 mL Plasma + daunorubicin + 500 JJLL water, mix, add to SPE cartridge, wash with
500 |JLL buffer, elute contents of SPE cartridge with mobile phase onto column for 1 min, remove
SPE cartridge, elute column with mobile phase, monitor the effluent. (Buffer was 19 mM
NaH2PO4 adjusted to pH 4.0 with 100 mM phosphoric acid:MeCN 90:10.)
HPLCVARIABLES
Column: 100 X 5 5 ixm Apex II ODS (Jones Chromatography)
Mobile phase: MeCN:buffer 1:2.25 (Buffer was 19 mM NaH2PO4 adjusted to pH 4.0 with 100
mM phosphoric acid.)
Flow rate: 1
CHROMATOGRAM
Retention time: 7.5
Internal standard: daunorubicin (22)
OTHER SUBSTANCES
KEYWORDS
plasma; SPE
REFERENCE
Dobbs,N.A.; Twelves,C.J. Measurement of epidoxorubicin and its metabolites by high-performance liquid chro-
matography using an advanced automated sample processor, J.Chromatogr., 1991, 572, 211-217.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 50 ng doxorubicin + 5 mL chloroformdsopropanol 80:20,
extract, centrifuge at 3000 g for 5 min. Remove the organic layer and evaporate it to dryness
under a stream of nitrogen at 30 in the dark, reconstitute the residue in 100 |xL mobile phase,
HPLC VARIABLES
Column: 125 X 4 Nucleosil 100-5 C18
Mobile phase: MeCN: 10 mM pH 4 ammonium formate buffer 30:70
Flow rate: 1.5
CHROMATOGRAM
Retention time: 5.6
Internal standard: doxorubicin (4.5)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Jakobsen,R; Steiness,E.; Bastholt,L.; Dalmark,M.; Lorenzen,A.; Petersen,D.; Gjedde,S.B.; Sandberg,E.;Rose,C;
Nielsen,O.S. Multiple-dose pharmacokinetics of epirubicin at four different dose levels: studies in patients
with metastatic breast cancer, Cancer Chemother.Pharmacol, 1991, 28, 63-68.
SAMPLE
Sample preparation: Thaw cell samples, sonicate (Branson B-12) at 50 W for 20 s. 400 |iL Cell
sample or plasma + 200 |xL 200 nM daunorubicin in 100 mM pH 9.3 borate buffer, add 1.8 mL
chloroform:MeOH 80:20, extract, inject a 200-500 |xL aliquot of the organic phase.
HPLCVARIABLES
Column: 250 X 4 Lichrosorb Si-60
Mobile phase: Chloroform:MeOH:glacial acetic acid:0.3 mM magnesium chloride 72:21:2:3
Flow rate: 1.5
CHROMATOGRAM
Retention time: 3.9
OTHER SUBSTANCES
Extracted: metabolites, doxorubicin
KEY WORDS
REFERENCE
Tidefelt,U.; Sundman-Engberg,B.; Paul,C. Comparison of the intracellular pharmacokinetics of doxorubicin and
4'-epi-doxorubicin in patients with acute leukemia, Cancer Chemother.Pharmacol., 1989, 24, 225-229.
SAMPLE
Sample preparation: Plasma. Condition a 100 mg C8 Bond Elut SPE cartridge with 2 mL
MeOH and 2 mL 10 jjig/mL desipramine hydrochloride in 50 mM pH 7 sodium phosphate buffer.
Add 300 jxL plasma to the SPE cartridge, wash with 1 mL water, wash with 1 mL 10 |xg/mL
desipramine hydrochloride in 50 mM pH 7 sodium phosphate buffer, elute with 500 jxL MeOH.
Evaporate the eluate under a stream of nitrogen, reconstitute in 300 fiL mobile phase, inject
a 100 fxL aliquot. Tissue. Tissue + 1 volume MeOH + 2 volumes 1 M pH 8.5 Tris buffer,
homogenize, let stand on ice for 15 min, add 7 volumes MeCN containing daunorubicin, vortex,
let stand at room temperature for 15 min, centrifuge, inject a 100 JULL aliquot of the supernatant.
HPLCVARIABLES
Column: 150 X 4.6 5 jxm Supelcosil LC-8-DB
Mobile phase: MeCN:50 mM pH 1.8 phosphoric acid 27:73
Flow rate: 1.5
CHROMATOGRAM
Retention time: 4.5
Internal standard: daunorubicin (for tissue)
OTHER SUBSTANCES
KEYWORDS
plasma; rat; liver; pharmacokinetics; SPE
REFERENCE
Hall,K.S.; Endresen,L.; Schjerven,L.; Rugstad,H.E. The influence of partial hepatectomy on the pharmacoki-
netics of preoperatively injected 4'-epidoxorubicin in rats, Cancer Chemother.Pharmacol., 1990, 26, 444-
448.
SAMPLE
Sample preparation: Homogenize (UltraTurrax T25) tissue with 5 volumes of MeOHiwater 5:
95 at 13500 rpm for about 2 min, centrifuge at 3200 g for about 2 min, remove the supernatant,
centrifuge at 4000 g for 2 min, dilute 1:10 with MeOHiwater 5:95. Inject a 50 |JLL aliquot of
plasma or tissue homogenate onto column A with mobile phase A, elute column A with mobile
phase A to waste for 10 min, backflush the contents of column A onto column B with mobile
phase B, after 5 min remove column A from the circuit and flush it to waste with mobile phase
A, elute column B with mobile phase B and monitor the effluent.
HPLC VARIABLES
Column: A 20 X 4 25 |xm C4-alkyl-diol silica (Pinkerton type); B 250 X 4 5 jjiin LiChrospher 60
RP-Select B
Mobile phase: A MeOH:water 5:95; B MeCN:buffer 30:70 (Buffer was 0.1% triethylamine in
water adjusted to pH 2.0 with trichloroacetic acid.)
Flow rate: 1
CHROMATOGRAM
Retention time: 9
Limit of quantitation: 50 pg
OTHER SUBSTANCES
KEYWORDS
column-switching; protect from light; tumor; liver; plasma; pharmacokinetics
REFERENCE
Rudolphi,A.; Vielhauer,S.; Boos,K.-S.; Seidel,D.; Bathge,I.-M.; Berger,H- Coupled-column liquid chromato-
graphic analysis of epirubicin and metabolites in biological material and its application to optimization of
liver cancer therapy, J.Pharm.BiomedAnaL, 1995, 13, 615-623.
SAMPLE
Sample preparation: Condition a C18 Sep-Pak SPE cartridge with 3 mL MeOH, 3 mL MeOH:
water 50:50, and 10 mL 50 mM pH 8.9 Na2HPO4. 1 mL Plasma or urine + 200 ng daunorubicin
+ 2 mL 0.9% NaCl, mix, add to the SPE cartridge, wash with 3 mL 50 mM pH 8.9 Na2HPO4,
elute with four 500 JULL aliquots of chloroform:MeOH 2:1. Evaporate the eluates to dryness
under vacuum while centrifuging, dissolve the residue in 200 |xL MeCN:7 mM pH 2.6 Na2HPO4
40:60, vortex gently, centrifuge at 3000 g for 15 min, inject an aliquot of the supernatant.
HPLC VARIABLES
Column: 100 X 8 ixBondapak phenyl Radial-Pak
Mobile phase: MeCN:buffer 30:70 (Buffer was 7 mM Na2HPO4 adjusted to pH 2.6 with formic
acid.)
Flow rate: 3
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Tjuljandin,S.A.; Doig,R.G.; Sobol,M.M.; Watson,D.M.; Sheridan,W.P.; Morstyn,G.; Mihaly,G.; Green,M.D. Phar-
macokinetics and toxicity of two schedules of high dose epirubicin, Cancer Res., 1990, 50, 5095-5101.
SAMPLE
Sample preparation: Emulsion. 500 jxL Emulsion + 10 mL 400 |xg/mL hydroquinone in MeOH
+ 40 mL 0.1% Tween 80, shake until homogeneous, inject a 10 JJLL aliquot. Drug release me-
dium. 1 mL Drug release medium + 200 (xL 100 |jig/mL hydroquinone, mix, inject a 50 |xL
aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 ^m Cosmosil 10 C18 (Nacalai Tesque)
Mobile phase: Gradient. MeCN:10 mM pH 3.0 phosphate buffer 2:98 for 1 min, to 45:55 over
5.5 min, maintain at 45:55 for 2 min, return to initial conditions over 1 min.
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
Internal standard: hydroquinone (4.2)
Limit of detection: 1 |xg/mL
OTHER SUBSTANCES
Simultaneous: carboplatin, mitomycin C, iomeprol
KEYWORDS
emulsions; drug release medium; injections
REFERENCE
Yamazoe,K.; Horiuchi,T.; Sugiyama,T.; Katagiri,Y. Simultaneous high-performance liquid chromatographic de-
termination of carboplatin, epirubicin hydrochloride and mitomycin C in a Lipiodol emulsion,
J.ChromatogrA, 1996, 726, 241-245.
Epoetin
Molecular formula: C8O9H13OiN229O240S5
Molecular weight: 30400 400
CAS Registry No.: 113427-24-0 (alfa), 122312-54-3 (beta)
Merck Index: 3729
SAMPLE
Matrix: dialysate
Sample preparation: Equilibrate 7 mL pre-swollen DEAE-Sephacel gel (Pharmacia) with ace-
tate buffer (40 mM acetic acid + 2.5 mM calcium chloride adjusted to pH 4.5 with 1 M NaOH),
pour the gel into a 1 cm diameter column. Add the dialysate (corresponding to 3.7 mg epoetin)
to the column at a flow rate of 0.5 mL/min. Elute starting with pH 4.5 acetate buffer and
continuing with pH 4.5 acetate buffer containing 15, 30, 60, 150, 350, and 1000 mM NaCl.
Collect seven fractions at elution volumes of about 0-35 mL, 35-70 mL, 70-125 mL, 125-160
mL, 160-185 mL, 185-195 mL, and 195-210 mL, adjust the pH of each fraction to 7.5 with 1
M Tris, concentrate by ultra-filtration on PM 10 Amicon membranes, inject an aliquot of the
filtrate.
HPLC VARIABLES
Column: 600 X 7.5 TSK G 2000 SW (Pharmacia)
Mobile phase: 20 mM pH 7.4 HEPES-NaOH buffer containing 150 mM NaCl
Flow rate: 0.5
Detector: UV 280
REFERENCE
GokanaA; Winchenne,J.J.; Ben-Ghanem,A-; AhadedA-; Cartron,J.R; Lambin,P. Chromatographic separation
of recombinant human erythropoietin isoforms, J.ChromatognA, 1997, 791, 109-118.
Epoprostenol
CAS Registry No.: 35121-78-9, 61849-14-7 (sodium salt)
Merck Index: 8061
LednicerNo.: 3 10
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1 mg/mL solution in 10 mM NaOH, inject a 20-100 jxL aliquot.
HPLC VARIABLES
Column: 150 X 4.1 10 jxm PRP-I styrene-divinylbenzene copolymer (Hamilton)
Mobile phase: MeCN:10 mM NaOH 21:79, measured pH 12.3 (At the end of each day clean the
column with 40 mL water and 20 mL MeCN.)
Flow rate: 1
Detector: UV 206
CHROMATOGRAM
Retention time: 4.5
Limit of quantitation: 5 |xg/mL
OTHER SUBSTANCES
Simultaneous: 6-ketoprostaglandin FIa, 6-ketoprostaglandin El, prostaglandin A2, prostaglan-
din B2, prostaglandin D2, dinoprost, dinoprostone, thromboxane B2
REFERENCE
Skrinska,V.; Thomas,G. High-performance liquid chromatography of prostacyclin, J.Chromatogr., 1983, 277,
287-291.
Eprinomectin
Molecular formula: C 50 H 75 NOi 4 (component B1a), C 49 H 73 NOi 4 (component Bib)
Molecular weight: 914.14 (component Bi a ), 900.12 (component Bib)
CAS Registry No.: 123997-26-2, 133305-88-1 (component Bi a ), 133305-89-2 (component B1b)
Merck Index: 3667
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 2 ng IS, add 3 mL MeCN, vortex for 60 s centrifuge
at 3000 rpm for 12 min. Add 1 mL water, vortex, centrifuge. Condition a 3 mL 200 mg C18
SPE cartridge (Waters) with 4 mL MeCN, 4 mL MeCNxhloroform 1:1 containing 0.1% 1-meth-
ylimidazole, 4 mL MeCN and 4 mL 100 mM sodium phosphate buffer. Add the supernatant to
the SPE cartridge, wash with two 4 mL portions of MeCN:water 1:2, dry under positive nitro-
gen pressure for 3 min. Elute with 5 mL MeCNxhloroform 50:50 containing 0.1% 1-methylim-
idazole, evaporate the eluate to dryness under a stream of nitrogen at 45 for 55 min. Recon-
stitute the dry residue in 100 |xL MeCN:l-methylimidazole 70:30 and vortex. Add 150 |xL
MeCN:trifluoroacetic anhydride 70:30, mix, inject a 100 fjuL aliquot. (Caution! Chloroform is a
carcinogen!)
HPLC VARIABLES
Guard column: Zorbax ODS
Column: 250 X 4.6 Zorbax RX-C18
Mobile phase: Gradient. A was MeCN:MeOH:water:triethylamine:orthophosphoric acid 50:44:6:
0.2:0.2. B was MeCN:MeOH:water:triethylamine:orthophosphoric acid 50:40:10:0.2:0.2. A:B 0:
100 for 12 min, to 100:0 over 2 min, 100:0 for 11 min, re-equilibrate at 0:100 for 3 min.
Flow rate: 1
CHROMATOGRAM
Internal standard: L-648 548 (Merck) (24)
KEYWORDS
cow; plasma; derivatization; SPE
REFERENCE
Antonian,L.; DeMontigny,R; Wislocki,P.G. An automated method for the determination of subnanogram con-
centrations of eprinomectin in bovine plasma, J.Pharm.Biomed.AnaL, 1998, 16, 1363-1371.
Eptastigmine
Molecular formula: C2IH33N3O2
CAS Registry No.: 101246-68-8, 121652-76-4 (tartrate)
Merck Index: 3672
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 40 |xL 50 ng/mL physostigmine + 1 mL 500 mM sodium
bicarbonate + 5 mL n-hexane, shake for 10 min, centrifuge at 1500 g for 5 min. Remove 4 mL
of the organic layer and evaporate it to dryness under a stream of nitrogen at 30, reconstitute
the residue in 100 |xL MeCN:MeOH 50:50, inject a 70 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm silica (Violet)
Mobile phase: MeCN:MeOH:80 mM ammonium nitrate 50:40:10, pH 8.90
Flow rate: 1
Detector: E, glassy carbon electrode +0.75 V
CHROMATOGRAM
Retention time: 5
Internal standard: physostigmine (6)
KEYWORDS
REFERENCE
ImbimbOjB-P-; Licini,M.; Schettino,M.; Mosca,A.; Onelli,E.; Zecca,L.; Giustina,A. Relationship between phar-
macokinetics and pharmacodynamics of eptastigmine in young healthy volunteers, J.Clin.Pharmacol., 1995,
35, 285-290.
Next Page
Equilin
Molecular formula: Ci8H20O2
Merck Index: 3676
SAMPLE
Matrix: blood
Sample preparation: Mix 9.7 mL antiserum (J. Steroid Biochem. 1980, 13, 1291) with 34 mL
0.4% rivanol solution, vortex on ice for 10 min, centrifuge at 3000 rpm at 4 for 10 min. Add
1.1 g activated charcoal to the supernatant, stir for 15 min on ice, centrifuge at 3000 rpm for
10 min, filter (0.45 jxm). Lyophilize, add 3.5 mL pH 7.6 sodium phosphate buffer, determine
the content of protein, add 2.2 mL Affi-gel 10 (Bio-Rad, pre-rinsed with 10 mL water and pH
7.6 sodium phosphate buffer). Stir the suspension at 4 overnight, add 220 |xL mL 1 M pH 8.0
ethanolamine, stir the adsorbent for 1 h, wash with 200 mL water, 200 mL MeCN.water 90:
10, and 100 mL sodium phosphate buffer until the absorbance of the eluate at 280 nm disap-
pears. Pack a 1 mL portion into a 6 mm diameter pipette tip (silanized with trichloromethyl-
silane), wash with 10 mL water and 10 mL pH 7.3 phosphate buffer. Add 1 mL plasma to the
immunoaffinity column, wash with 5 mL water. Elute with 4 mL MeCNrwater 90:10, inject a
50 |xL aliquot.
HPLC VARIABLES
Column: A 150 X 4.6 5 |xm Cosmosil AR (Nacalai Tesque Inc.); B 150 X 1 5 ujn Capcell Pak C18
Mobile phase: A MeCN:MeOH:60 mM pH 5.0 acetate buffer 30:9:60; B MeOH:100 mM pH 5.0
ammonium acetate buffer 10:40
Flow rate: 1 (A),0.005 (B)
Detector: E, ESA Coulochem 510 A, + 100 mV for first electrode, + 800 mV for the second elec-
trode (A); MS, Hitachi M-1000H, ESI, drift voltage -50 V, nebulizer 150, m/z 413 (B)
CHROMATOGRAM
Retention time: 21 (A), 16 (B)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Ikegawa,S.; Itoh,M.; Murao,N.; Kijima,H.; Suzuki,M.; Fujiyama,T.; Goto,J.; Tohma,M. Immunoaffinity extrac-
tion for liquid chromatographic determination of equilin and its metabolites in plasma, Biomed.Chromatogr.,
1996,10, 73-77.
Previous Page
Ergocornine
Merck Index: 3685
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jxg/mL solution in MeOH, inject a 20 JJLL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.2
OTHER SUBSTANCES
droperidol, ephedrine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergo-
sinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol,
fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hy-
droxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine,
thipendyl, protriptyline, proxjmcietacaine, pseudoephedrine, pyrimethamine, quinidine, qui-
nine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyidi-
methoprim, trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE
Ergocristine
Merck Index: 3687
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 u,L aliquot.
HPLC VARIABLES
Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL/L 100 mM NaOH in
MeOH, pH 6.7
Flow rate; 2
CHROMATOGRAM
R e t e n t i o n time: 1.1
OTHER SUBSTANCES
droperidol, ephedrine, ergocornine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosi-
nine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol,
mesoridazine, metaraminol, methadone, methampnetamine, methapyrilene, methdilazene,
methotrimeprazine, methoxamine, methoxyphenamine, methoxjrpromazine, methylephedrine,
thipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, qui-
REFERENCE
columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electrochemical oxidation
Ergocristinine
Merck Index: 3688
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.1
OTHER SUBSTANCES
none, diprenorphine, dipyridamole, disop3rramide, dothiepin, doxapram, doxepin, doxylamine,
droperidol, ephedrine, ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, ergosi-
REFERENCE
Jane,L; McKinnon,A-; Flanagan,R-J- High-performance liquid chromatographic analysis of basic drugs on silica
Ergocryptine
CAS Registry No.: 511-09-1 (a) 20315-46-2 (p)
Merck Index: 3689
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jig/mL solution in MeOH, inject a 20 |xL aliquot.
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.2
OTHER SUBSTANCES
droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergometrine, ergosine, ergosi-
thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcjrpromine, tra-
REFERENCE
Jane,L; McKinnonÂ..; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica
Ergoloid mesylates
Merck Index: 3692
Dihydroergocomine R = CH(CH3)2
Dihydroergocristine R = CH 2 C 6 H 5
Dihydro-a-ergocryptine R = CH2CH(CH3)2
Dihydro-p-ergocryptine R = CH(CH3)CH2CH3
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 jxL 1 |xg/mL dihydroergotamine mesylate in water +
30 fxL 5 M NaOH + 7 mL chloroform, shake on a reciprocal shaker for 10 min, centrifuge at
nitrogen at 40, reconstitute the residue in 100 jxL mobile phase, inject a 10-30 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4 10 n-m RP-8 (Merck)
Mobile phase: MeCN.buffer 60:40 (Buffer was 9 mM NaH2PO4 and 9 mM Na2HPO4, pH 7.2.)
Flow rate: 1
CHROMATOGRAM
Retention time: 7.9 (dihydroergocristine)
Internal standard: dihydroergotamine mesylate (5.2)
Limit of detection: 0.5-0.7 ng/mL (F), 5-10 ng/mL (UV)
OTHER SUBSTANCES
Simultaneous: dihydroergocornine, dihydroergocryptine
KEYWORDS
plasma; rat
REFERENCE
Zecca,L.; Bonini,L.; Bareggi,S.R. Determination of dihydroergocristine and dihydroergotamine in plasma by
high-performance liquid chromatography withfluorescencedetection, J.Chromatogr., 1983, 272, 401-405.
SAMPLE
Matrix: blood, urine, tissue
Sample preparation: Homogenize tissue with 4 volumes of water, centrifuge. 1 mL Plasma,
urine, or tissue homogenization supernatant + 50 (xL 1 (plasma) or 10 (urine, tissue) (xg/mL a-
dihydroergocristine + 100 jxL 1 M HCl, vortex, add 5 mL hexane, extract, centrifuge. Remove
the aqueous phase and add it to 100 |xL 2 M NaOH, extract with 7 mL chloroform, centrifuge.
Remove 5 mL of the organic layer and evaporate it to dryness, reconstitute the residue in 70
|JLL mobile phase, inject a 50 \LL aliquot.
HPLCVARIABLES
Column: 125 X 4 5 jjim LiChrospher 100 RP 18
Mobile phase: MeCNrbuffer 43:57 (Buffer was 10 mM pH 7.2 Na2HPO4ZKH2PO4.)
Flow rate: 1
CHROMATOGRAM
Retention time: 8.9 (dihydroergocriptine)
Internal standard: a-dihydroergocristine (10.2)
Limit of detection: 2 ng/g (tissue), 10 ng/mL (urine), 0.1 ng/mL (plasma)
KEYWORDS
rat; plasma; kidney; heart; lung; spleen; liver; brain; pharmacokinetics
REFERENCE
Coppi,G.; Silingardi,S. Pharmacokinetics of a-dihydroergocriptine in rats after single intravenous and single
and repeated oral administrations, Biopharm.Drug Dispos., 1995, 16, 333-342.
Ergonovine
Merck Index: 3694
SAMPLE
M a t r i x : blood
Sample preparation: 300 |xL Plasma + 300 u,L MeCN, centrifuge at 11000 g for 4 min, inject a
HPLC VARIABLES
Guard column: 75 X 2.1 10 |xm pellicular reversed phase (Chrompack no. 028653)
Column: 250 X 4.6 5 jxm Spherisorb 5-ODS
Mobile phase: MeCN.buffer 35:65 (Buffer was 67 mM KH2PO4 containing 0.5 mL/L (?)
triethylamine.)
Flow rate: 1.2
CHROMATOGRAM
KEYWORDS
REFERENCE
de Groot,A.N.J.A.; Vree,T.B.; Hekster,Y.A.; BaarsAM.; Van den Biggelaar-Martea,M.; vanDongen,RW.J. High-
performance liquid chromatography of ergometrine and preliminary pharmacokinetics in plasma of men,
J.Chromatogr., 1993, 613, 158-161.
SAMPLE
Sample preparation: Dilute formulation 1:10. Remove a 1 mL aliquot and add it to 1.5 mL
water and 2.5 mL 20 jig/mL ephedrine, inject an 80 jxL aliquot.
HPLC VARIABLES
Mobile phase: MeCNibuffer 35:65 containing 0.05% sodium tetradecyl sulfate (Buffer was 0.83
mM phosphoric acid adjusted to pH 5.0 with triethylamine. Use a 50 X 4.6 5-25 |xm LiChroprep
Si 60 column before the injector. Wash column with MeCN:83 mM phosphoric acid 40:60 after
use.)
Flow rate: 2.5
Detector: UV
CHROMATOGRAM
Retention time: 18
Internal standard: ephedrine (10)
OTHER SUBSTANCES
Simultaneous: oxytocin
KEYWORDS
injections
REFERENCE
Pask-Hughes,R.A.; Corran,P.H.; Calam,D.H. Assay of the combined formulation of ergometrine and oxytocin
SAMPLE
Sample preparation: Injections. 1 mL Injection (200 jxg/mL) + 300 mg NaCl + 200 (xL 10%
ammonia + 5 mL dichloromethane, shake vigorously for 10 min, let stand for a few min. Re-
move 4 mL of the organic layer and evaporate it to dryness under a stream of nitrogen, recon-
stitute the residue in 4 mL water, mix an aliquot with an equal volume of 20 jxg/mL 17a-
hydroxyprogesterone in MeOH, inject a 20 |xL aliquot. Tablets. Weigh out amount of powdered
tablets equivalent to about 200 |xg compound, add 1 mL water, sonicate for 2 min, add 300 mg
NaCl, add 200 |xL 10% ammonia, add 5 mL dichloromethane, shake vigorously for 10 min, let
stand for a few min. Remove 4 mL of the organic layer and evaporate it to dryness under a
stream of nitrogen, reconstitute the residue in 4 mL water, mix an aliquot with an equal volume
of 20 jxg/mL 17a-hydroxyprogesterone in MeOH, inject a 20 jxL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:50 mM pH 3.5 acetate buffer 40:60 containing 1.5 mM triethylamine
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 7.5
Internal standard: 17a-hydroxyprogesterone (12)
OTHER SUBSTANCES
Simultaneous: benzyl alcohol, methylergonovine
Noninterfering: ascorbic acid
KEYWORDS
injections; tablets
REFERENCE
Tokunaga,H.; Kimura,T.; Kawamura,J. Determination of ergometrine maleate and methylergometrine maleate
in pharmaceutical preparations by high-performance liquid chromatography, Chem.Pharm.Bull.(Tokyo),
1983, 31, 3988-3993.
SAMPLE
Sample preparation: Grind tablet, add 100 |xL 90% formic acid for each 100 |xg ergotamine
tartrate, swirl to thoroughly wet sample, make up to 100 mL with MeOH, mix, filter (paper),
dilute filtrate with MeOH (if necessary) so that the ergotamine tartrate concentration is 3 mg/
L, inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 C18 (Alltech)
Mobile phase: MeCN:water:triethylamine 70:30:0.05
Flow rate: 1
CHROMATOGRAM
Retention time: 2.5
OTHER SUBSTANCES
Simultaneous: ergotamine, ergotaminine
Noninterfering: caffeine
KEYWORDS
tablets
REFERENCE
Cieri,U.R. Determination of ergotamine tartrate in tablets by liquid chromatography with fluorescence detec-
tion, JAssoc.OffAnaLChem., 1987, 70, 538-540.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.2
OTHER SUBSTANCES
droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergosine, ergosi-
thipendyl, protriptyline, prox3maetacaine, pseudoephedrine, pyrimethamine, quinidine, qui-
REFERENCE
Jane,L; McKinnon,A.; Flanagan,R-J- High-performance liquid chromatographic analysis of basic drugs on silica
SAMPLE
Matrix: solutions
fxL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 |xm |jiBondapak C18
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in mobile phase, inject a 25-150 |xL aliquot.
HPLC VARIABLES
Column: 250 mm long 5 |xm Hypersil C18 ODS
Mobile phase: MeCNtIO mM ammonium carbonate 30:70
Flow rate: 2
CHROMATOGRAM
Retention time: 2.4
OTHER SUBSTANCES
Simultaneous: methylergonovine (methylergometrine), methysergide
REFERENCE
Bredberg,U.; Paalzow,L. Pharmacokinetics of methysergide and its metabolite methylergometrine in the rat,
Drug Metab.Dispos., 1990, 18, 338-343.
SAMPLE
Matrix: wheat
Sample preparation: Grind wheat to pass 2 mm screen. 25 g Ground wheat + 10 mL 4% am-
monia in water + 100 mL ethyl acetate, shake vigorously on a wrist-action shaker for 15 min,
filter (paper). Extract 50 mL filtrate twice with 25 mL 1% sulfuric acid. Combine aqueous
extracts, add 50 mL 4% ammonia in water, extract twice with 25 mL dichloromethane shaking
gently for 30 s each time. Dry extracts over 10-20 g anhydrous sodium sulfate for 10 min in
the dark, filter, wash solid with 25 mL dichloromethane. Evaporate filtrate to near dryness at
40 under reduced pressure, rinse into a tube with two 2 mL portions of dichloromethane,
evaporate to dryness under a stream of nitrogen, reconstitute in 1 mL mobile phase, inject a
20 (xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 10 jxm PRP-I polystyrene-divinylbenzene (Hamilton)
Mobile phase: MeCN:water 45:55 containing 6.6 g/L (NHJ2HPO4
Flow rate: 0.3
Detector: F ex not specified em 418 (cut-off filter)
CHROMATOGRAM
Retention time: 7
Limit of detection: < 18 ng/g
OTHER SUBSTANCES
Extracted: ergonovinine, ergotamine, ergocryptine, ergocristine, ergotaminine, ergocryptinine,
ergocristinine
REFERENCE
Ware,G.M.; Carman,A.S.; Francis,O.J.; Kuan,S.S. Liquid chromatographic determination of ergot alkaloids in
wheat, J.Assoc.Off.Anal.Chem., 1986, 69, 697-699.
Ergosine
Merck Index: 3695
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.1
OTHER SUBSTANCES
droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, er-
gosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, feno-
zodone, trifiuoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, tri-
REFERENCE
Ergotamine
Merck Index: 3703
SAMPLE
Sample preparation: Grind tablet, add 100 u,L 90% formic acid for each 100 u,g ergotamine
tartrate, swirl to thoroughly wet sample, make up to 100 mL with MeOH, mix, filter (paper),
dilute filtrate with MeOH (if necessary) so that the ergotamine tartrate concentration is 3 rag/
L, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 C18 (Alltech)
Flow rate: 1
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
Simultaneous: ergonovine, ergotaminine
Noninterfering: caffeine
KEYWORDS
tablets
REFERENCE
Cieri,U.R. Determination of ergotamine tartrate in tablets by liquid chromatography with fluorescence detec-
tion, JAssocOffAnaLChem., 1987, 70, 538-540.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 (xg/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.3
OTHER SUBSTANCES
gosine, ergosinine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol,
phenindamine, pneniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine,
REFERENCE
SAMPLE
Matrix: solutions
|xL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 u,m ixBondapak C18
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN: 10 mM ammonium carbonate 50:50
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 11
OTHER SUBSTANCES
Simultaneous: impurities, ergocristine, ergosine, 8-hydroxyergotamine
REFERENCE
Gazdag,M.; Szepesi,G. Selection of high-performance liquid chromatographic methods in pharmaceutical anal-
ysis. IV. Selection of most applicable separation system, J.Chromatogr., 1989, 464, 279288.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 X 3.9 10 |xm ixBondapak phenyl
Mobile phase: MeCN:0.1% pH 3.25 acetic acid containing 1.25 mM heptanesulfonic acid 65:35
Flow rate: 1.3
Detector: UV 254
REFERENCE
Fernandez Otero,G.C; Lucangioli,S.E.; Carducci,C.N. Adsorption of drugs in high-performance liquid chro-
matography injector loops, J.Chromatogr.A, 1993, 654, 87-91.
SAMPLE
Matrix: solutions
Sample preparation: Pass 20 mL of a solution in water (?) through an Empore C18 SPE disc.
Wash with 2.5 mL water, add 1 mL MeCN:25 mM pH 3.0 phosphate buffer 65:35, let soak for
3 min, elute, inject an aliquot of the eluate.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Spherisorb S5 ODS2
Mobile phase: MeCN: 10 mM KH2PO4 30:70
Flow rate: 1.3
Detector: UV 254
OTHER SUBSTANCES
Noninterfering: excipients
KEYWpRDS
comparison with capillary electrophoresis; SPE
REFERENCE
Lucangioli,S.E.; Rodriguez,V.G.; Fernandez Otero,G.C; Vizioli,N.M.; Carducci,C.N. Development and validation
of capillary electrophoresis methods for pharmaceutical dissolution assays, J.Capillary Electrophor., 1997,
4, 27-31.
SAMPLE
Matrix: wheat
Sample preparation: Grind wheat to pass 2 mm screen. 25 g Ground wheat + 10 mL 4% am-
monia in water + 100 mL ethyl acetate, shake vigorously on a wrist-action shaker for 15 min,
filter (paper). Extract 50 mL filtrate twice with 25 mL 1% sulfuric acid. Combine aqueous
extracts, add 50 mL 4% ammonia in water, extract twice with 25 mL dichloromethane shaking
gently for 30 s each time. Dry extracts over 10-20 g anhydrous sodium sulfate for 10 min in
the dark, filter, wash solid with 25 mL dichloromethane. Evaporate filtrate to near dryness at
40 under reduced pressure, rinse into a tube with two 2 mL portions of dichloromethane,
evaporate to dryness under a stream of nitrogen, reconstitute in 1 mL mobile phase, inject a
20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 10 fim PRP-I polystyrene-divinylbenzene (Hamilton)
Mobile phase: MeCN:water 45:55 containing 6.6 g/L (NHJ2HPO4
Flow rate: 0.3
Detector: F ex not specified em 418 (cut-off filter)
CHROMATOGRAM
Retention time: 15
Limit of detection: < 95 ng/g
OTHER SUBSTANCES
Extracted: ergonovinine, ergonovine, ergocryptine, ergocristine, ergotaminine, ergocryptinine,
ergocristinine
REFERENCE
Ware,G.M.; Carman,A.S.; Francis,O.J.; Kuan,S.S. Liquid chromatographic determination of ergot alkaloids in
wheat, J.Assoc.Off.Anal.Ckem., 1986, 69, 697-699.
Erythrityl tet ran it rate

Merck Index: 3716
SAMPLE
Sample preparation: Powder tablets, weigh out a portion equivalent to 3 mg erythrityl tetra-
nitrate, add to 10 mL 75 |xg/mL nitroglycerin in MeOH, sonicate for 2 min, shake mechanically
for 30 min, filter, inject an aliquot
HPLC VARIABLES
Guard column: 40 X 4.6 ixBondapak C18/Corasil
Column: 300 X 3.9 10 jjim ixBondapak C18
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 31
Internal standard: nitroglycerin (14)
OTHER SUBSTANCES
Simultaneous: pentaerythritol tetranitrate, isosorbide dinitrate
KEYWORDS
tablets
REFERENCE
Olsen,C.S.; Scroggins,H.S. High-performance liquid chromatographic determination of the nitrate esters iso-
sorbide dinitrate, pentaerythritol tetranitrate, and erythrityl tetranitrate in various tablet forms,
J.Pharm.Scl, 1984, 73, 1303-1304.
Erythromycin
Molecular formula: C 37 H 67 NOi 3
CAS Registry No.: 114-07-8, 41342-53-4 (ethylsuccinate),
96128-89-1 (acistrate), 3521-62-8 (estolate),
304-63-2 (gluheptonate), 23067-13-2 (gluheptonate),
3847-29-8 (lactobionate), 134-36-1 (propionate),
643-22-1 (stearate), 84252-03-9 (stinoprate)
Merck Index: 3720
SAMPLE
Sample preparation: Homogenize (Physcotron) liver with 4 volumes of ice-cold saline. Evapo-
rate 100 |xL 3 jjLg/mL oleandomycin in MeOH to dryness under dry nitrogen. Put 200 |xL plasma
or liver homogenate into the tube. Add 2 mL MTBE and 5 (JLL 1 M NaOH and shake mechan-
ically for 5 min. Centrifuge at 1500 g for 10 min, transfer the upper layer transfer into a glass
tube and evaporate it to dryness under dry nitrogen. Rinse the inner wall of the tube with 200
IxL MeOH and evaporate to dryness. Dissolve the residue in 30 |xL MeOH and inject a 10 jxL
aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 |xm Cosmosil 5-C18 (Nacalai Tesque)
Mobile phase: MeCN: 100 mM pH 6.6 sodium acetate buffer 50:50
Flow rate: 0.6
Detector: E, 1.1 V, Ag/AgCl reference electrode IS oleandomycin (8.5)
CHROMATOGRAM
Limit of detection: 100 jxg/mL (plasma); 0.5 fxg/g (liver)
KEYWORDS
rat; plasma; liver; pharmacokinetics
REFERENCE
Hanada,E.; Ohtani,H.; Kotaki,H.; Sawada,Y.; Iga,T. Determination of erythromycin concentrations in rat
plasma and liver by high-performance liquid chromatography with amperometric detection,
J.Chromatogr.B, 1997, 692, 478-482.
SAMPLE
Matrix: bulk, formulations
Sample preparation: Erythromycin estolate bulk. Dissolve the bulk powder in mobile phase B
for a final concentration of 5-6 mg/mL and sonicate for 30 s. Inject an aliquot. Erythromycin
estolate capsules. Powder the contents of capsules. Prepare a 5 mg/mL suspension in mobile
phase B, sonicate for 2 min, filter (0.45 |jim), discard the first 5 mL of the filtrate. Inject an
aliquot. Erythromycin ethylsuccinate powder. Prepare as described for estolate. Erythromycin
ethylsuccinate tablets. Powder the tablets, prepare a 15 mg/mL suspension in mobile phase B,
sonicate for 2 min, filter (0.45 |xm). Discard the first few mL of filtrate. Inject an aliquot.
Erythromycin ethylsuccinate powder for oral suspension. Prepare a 500 mg/mL suspension in
100 mL mobile phase B, sonicate for 15 min, make up the supernatant to 100 mL with mobile
phase B, filter (0.45 |xm). Inject an aliquot. Erythromycin stearate powder. Prepare a 5-6 mg/
mL solution in MeOH, inject an aliquot. Erythromycin stearate tablets. Powder the tablets and
prepare a 30 mg/mL suspension in MeOH, sonicate for 5 min, filter (0.45 |xm), discard the first
few mL of the filtrate, inject an aliquot.
HPLC VARIABLES
Guard column: 5 |xm Inertsil ODS-2
Column: 150 X 4.6 5 jxm Inertsil ODS-2 (A) or 250 X 4.6 5 |xm Inertsil ODS-2 (B)
Mobile phase: Gradient. A was MeCN:buffer 10:90. B was MeCN:buffer 75:25. A:B from 90:10
to 0:100 over 10 min, maintain at 0:100. (Prepare mobile phase A as follows. Mix 60 mL 200
mM pH 6.5 ammonium phosphate buffer with 60 mL 200 mM pH 6.5 tetrabutylammonium
sulfate buffer and 200 mL water. Add 100 mL MeCN and make up to 1 L with water. Prepare
mobile phase B as described for A except use 750 mL MeCN.)
Flow rate: 1.3
Detector: UV 205
CHROMATOGRAM
Retention time: 9.5 (estolate, column A), 14.5 (ethylsuccinate, column A), ca. 26 (stearate, col-
umn B), ca. 26 (gluceptate, column B), ca. 26 (lactobionate, column B)
OTHER SUBSTANCES
KEYWORDS
powder; capsules; tablets
REFERENCE
Nasr,M.M.; Stanley,C.M High performance liquid chromatographic assay of erythromycin salts and esters in
bulk and pharmaceutical dosage forms, J.Liq.Chromatogr.Rel.TechnoL, 1998, 21, 1147-1160.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 1 mL 100 mg Bond-Elut diol SPE cartridge with 1 mL chlo-
roform (Caution! Chloroform is a carcinogen!). Mix 2 g minced muscle tissue with 800 \LL water.
Stir, vortex for 1 min at maximum speed, let stand for 15 min. Add 2 mL pH 8 buffer, mix
briefly, add 10 mL chloroform. Stir at 100 rpm for 15 min, centrifuge at 4000 g for 10 min,
discard the aqueous layer, filter the chloroform layer through glass wool. Add the filtrate to
the SPE cartridge, wash with 500 jxL chloroform, dry under vacuum, elute with three 200 |xL
portions of MeOH: 100 mM ammonium acetate 50:50, inject a 200 JJLL aliquot of the eluate.
(Buffer was 33.46 g K2HPO4 and 1.046 g KH2PO4 in 1 L water.)
HPLC VARIABLES
G u a r d c o l u m n : 4 x 4 5 fxm C18
Column: 125 X 4 5 |xm Lichrospher RP18
Mobile phase: Gradient. A was MeCN. B was MeOH. C was 0.1% trifluoroacetic acid in water.
A:B:C from 20:20:60 to 25:55:20 in 10 (?) min
Flow rate: 0.5
Detector: MS, HP Model 5989 A, desolvation chamber 60, source 280 and 300 in negative and
positive chemical ionization mode, respectively, with methane as reagent, quadrupole 100,
particle beam nebulizer helium 345 kPa, scan m/z 442.3-619.4 in NCI and 734.4-576.2 in PCI
CHROMATOGRAM
Retention time: 6.4
OTHER SUBSTANCES
Extracted: josamycin, spiramycin, tilmicosin, tylosin
KEYWORDS
muscle; cow; SPE
REFERENCE
Delepine,B.; Hurtaud-Pessel,D.; Sanders,P. Multiresidue method for confirmation of macrolide antibiotics in
bovine muscle by liquid chromatography/mass spectrometry, J.AOAC Int., 1996, 79, 397-404.
Esculin
Molecular formula: Ci5Hi6O9
Merck Index: 3739
SAMPLE
residue with 50 jxL MeCN.water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 202.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Esmolol
CAS Registry No.: 81147-92-4, 103598-03-4, 81161 -17-3 (HCI)
Merck Index: 3741
Lednicer No.: 4 27
SAMPLE
Matrix: blood
Sample preparation: 1 mL Blood + 6 mL dichloromethane + 100 |xL 200 mM NaOH + 5 jxL 50
|xg/mL IS in water, vortex for 10 s, shake on a mechanical shaker for 10 min, centrifuge at
1000 g for 10 min. Remove the organic layer and add it to 100 |xL 2.5 mM sulfuric acid, vortex
for 1 min, centrifuge for 5 min, inject a 50 |xL portion of the aqueous layer.
HPLCVARIABLES
Guard column: 30 X 3.2 37-53 |xm Whatman C18 guard column
Column: 100 X 5 10 |xm Radial-Pak CN (Waters)
Mobile phase: MeOH:60 mM KH2PO4:triethylamine 25:75:0.1 adjusted to pH 3.15 with 85%
phosphoric acid
Flow rate: 1.8
Detector: UV 221
CHROMATOGRAM
Retention time: 2.6
Internal standard: 3-methyloxy-O-demethylencainide (3.9)
OTHER SUBSTANCES
Simultaneous: amiodarone, atropine, caffeine, cimetidine, digoxin, diazepam, disopyramide
(norpace), encainide, flecainide, imipramine, lidocvaine, nimodipine, prazepam, procainamide,
propafenone, propranolol, quinidine, theophylline
Interfering: captopril
KEYWORDS
pig; whole blood
REFERENCE
Fan,C.-D.; Zhao,H.; Chow,M.S.S. Simple and rapid high-performance liquid chromatographic assay for esmolol,
J.Chromatogr., 1991, 570, 217-223.
SAMPLE
Matrix: blood
Sample preparation: 1 mL plasma + 5 mL dichloromethane, shake for 10 min, centrifuge at 4
at 1900 g for 10 min. Remove 4 mL of the organic phase and add it to 600 |xL 100 mM pH 2.8
NaH2PO4, shake, centrifuge, inject a 100 |xL aliquot. (To determine metabolites mix 500 |JLL
aqueous phase (left after initial extraction) with 500 JJLL 7% perchloric acid, centrifuge for 5
min, inject a 100 JJLL aliquot of the supernatant.)
HPLC VARIABLES
Guard column: 5 |xm Lichrocart 4-4 RP18 (Merck)
Column: 150 X 3.9 5 |xm Nova-Pak C18
Mobile phase: MeCNrIO mM pH 2.4 NaH2PO4 40:60 containing 0.2 mM sodium dodecylsulfate
(For metabolites use MeCNrIO mM pH 2.4 NaH2PO4 17.5:82.5 containing 0.2 mM sodium
dodecylsulfate.)
Flow rate: 1
Detector: UV 229
CHROMATOGRAM
Retention time: 5.4
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Jahn,R; Eckrich,B.; Schneidrowski,B.; Volz-Zang,C; Schulte,B.; Mutschler,E.; Palm,D. (Bi-Adrenoceptor subtype
selective antagonism of esmolol and its major metabolite in vitro and in man. Investigations using tricre-
sylphosphate as red blood cell carboxylesterase inhibitor, Arzneimittelforschung, 1995, 45, 536-541.
SAMPLE
Sample preparation: Dilute with water to an expected esmolol hydrochloride concentration of
10 |xg/mL. Remove a 100 |xL aliquot and add it to 100 |xL 15 |xg/mL IS, inject a 15 |xL aliquot.
HPLC VARIABLES
Guard column: jxBondapak C18 Guard-Pak
Column: 100 x 8 jxBondapak C18 Radial-Pak
Mobile phase: MeOH.10 mM pH 2.69 KH2PO4 40:60
Flow rate: 2.8
Detector: UV 229
CHROMATOGRAM
Internal standard: methyl-4-[4-[2-hydroxy-3-[(2-methylethyl)amino]propoxy]phenyl]butyrate
hydrochloride (8.36)
KEYWORDS
stability-indicating; 5% dextrose; injections
REFERENCE
Wiest,D.B.; Garner,S.S.; Childress,L.M. Stability of esmolol hydrochloride in 5% dextrose injection,
Am.J.Health-Syst.Pharm., 1995, 52, 716-718.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeOH.buffer 30:70 (Buffer was pH 4.0 phosphate buffer (ionic strength = 0.1)
containing 2.86 mM N,N-dimethyloctylamine, pH readjusted to 4.00 with 85% phosphoric acid.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: acebutolol, bunitrolol, carazolol, celiprolol, mepindolol, metoprolol, timolol
REFERENCE
Hamoir,T.; Verlinden,Y.; Massart,D.L. Reversed-phase liquid chromatography of 0-adrenergic blocking drugs
in the presence of a tailing suppressor, J.Chromatogr.Sci., 1994, 32, 14[94]20.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 12 |xm l-myristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine
chemically bonded to silica (Regis)
Mobile phase: MeCN: 100 mM pH 7.0 phosphate buffer 20:80
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: acebutolol, alprenolol, antazoline, atenolol, betaxolol, bisoprolol, bopindolol, bu-
pranolol, carteolol, celiprolol, chloropyramine, chlorpheniramine, cicloprolol, cimetidine, cin-
narizine, cirazoline, clonidine, dilevalol, dimethindene, diphenhydramine, doxazosin, famoti-
dine, isothipendyl, ketotifen, metiamide, metoprolol, moxonidine, nadolol, naphazoline,
nifenalol, nizatidine, oxprenolol, pheniramine, phentolamine, pindolol, pizotyline (pizotifen),
practolol, prazosin, promethazine, propranolol, pyrilamine (mepyramine), ranitidine, roxati-
dine, sotalol, tiamenidine, timolol, tramazoline, tripelennamine, triprolidine, tymazoline, UK-
14,304
REFERENCE
Kaliszan,R.; Nasal,A.; Turowski,M. Binding site for basic drugs on oti-acid glycoprotein as revealed by chemo-
metric analysis of biochromatographic data, Biomed.Chromatogr., 1995, 9, 211-215.
Estazolam
Molecular formula: C16H11CIN4
Merck Index: 3744
SAMPLE
Matrix: blood
Sample preparation: 500 fxL Serum + 20 uL 20 (xg/mL IS + 200 |xL 1 M potassium carbonate
+ 3 mL chloroform, mix for 2 min, centrifuge at 1200 g for 5 min, aspirate aqueous phase.
Evaporate the organic phase under a stream of nitrogen at 40. Dissolve the residue in 100 (xL
mobile phase, inject a 20 JULL aliquot. (Caution! Chloroform is a carcinogen!)
HPLC VARIABLES
Column: 100 X 4.6 2 |xm TSK gel Super-ODS (A) or 100 X 4.6 5 |xm Hypersil ODS-C18 (B)
Mobile phase: MeCN:5 mM pH 6 NaH2PO4 45:55
Flow rate: 0.65
Detector: UV 254
CHROMATOGRAM
Internal standard: diazepam (29.8 (A), 77.5 (B))
Limit of quantitation: 5 ng/mL (A)
OTHER SUBSTANCES
Extracted: bromazepam, chlordiazepoxide, clonazepam, etizolam, flutazolam, haloxazolam, lor-
azepam, nitrazepam, oxazolam, triazolam
Simultaneous: alprazolam
Noninterfering: barbital, carbamazepine, cloxazolam, ethosuximide, hexobarbital, mexazolam,
oxazepam, pentobarbital, phenobarbital, phenytoin, primidone, trimethadione
KEYWORDS
serum
REFERENCE
Tanaka,E.; Terada,M.; Misawa,.; Wakasugi,C. Simultaneous determination of twelve benzodiazepines in human
serum using a new reversed-phase chromatographic column on a 2-|xm porous microspherical silica gel,
J.Chromatogr.B, 1996, 682, 173-178.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 2 mL water + 2 mL 100 mM NaOH, mix gently, add 8 mL
diethyl ether, shake for 15 min, centrifuge at 2500 rpm for 5 min. Remove 4 mL of the organic
layer and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue
in 100 jxL mobile phase, vortex for 30 s, inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 50 X 4.6 Shim-pack FLC-C8 (Shimadzu)
Mobile phase: MeOHibuffer 53:47 (Buffer was 5 mM Na2HPO4 adjusted to pH 6.0 with phos-
phoric acid.)
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
Retention time: 4
Internal standard: estazolam
OTHER SUBSTANCES
Extracted: diazepam, nordiazepam, clorazepate, temazepam, oxazepam
Simultaneous: sulpride, bromazepam, nitrazepam, flunitrazepam
Noninterfering: haloperidol, trihexyphenidyl
Interfering: triazolam
KEYWORDS
serum; estazolam is IS
REFERENCE
Tada,K; Moroji,T.; Sekiguchi,R.; Motomura,H.; Noguchi,T. Liquid-chromatographic assay of diazepam and its
major metabolites in serum, and application to pharmacokinetic study of high doses of diazepam in schi-
zophrenics, Clin.Chem., 1985, 31, 1712-1715.
SAMPLE
Matrix: blood
Sample preparation: 500 IJLL Plasma + 20 JJLL 2.5 |xg/mL norprazepam in MeOH + 50 JJLL buffer
+ 6 mL diethyl ether:dichloromethane 2:1, agitate, centrifuge. Remove the organic phase and
evaporate to dryness under vacuum at 45, dissolve the residue in 50 |xL MeOH, inject a 20
JJLL aliquot. (Prepare buffer as follows. Solution A was 6.18 g boric acid + 7.46 g KCl in 100 mL
water. Solution B was 10.6 g sodium carbonate in 100 mL water. Mix 63 mL solution A and 37
mL solution B and adjust pH to 9.5.)
HPLCVARIABLES
Column: 150 X 4.6 5 |xm Nova Pak C18
Mobile phase: MeCNrMeOH.buffer 23:13:64 (Buffer was 94 mL 200 mM NaH2PO4 + 6 mL 200
mM Na2HPO4, adjusted to pH 5.0 with 100 mM HCl.)
Flow rate: 1.3
Detector: UV 242
CHROMATOGRAM
Internal standard: norprazepam (18.6)
OTHER SUBSTANCES
Simultaneous: alprazolam, bromazepam, chlordiazepoxide, clobazam, diazepam, flumazenil,
flunitrazepam, loflazepate, nitrazepam, norflunitrazepam, tofizopam, triazolam
Noninterfering: acepromazine, aceprometazine, amylobarbital, aprobarbital, barbital, brallo-
barbital, butalbital, caffeine, carbamazepine, chlorpromazine, cyclobarbital, ethosuximide, hep-
tabarbital, hexobarbital, loprazolam, medazepam, midazolam, pentobarbital, phenobarbital,
phenytoin, prazepam, secobarbital, theophylline, thiopental, vinylbarbital
Interfering: oxazepam, clonazepam, lorazepam
KEYWORDS
plasma
REFERENCE
Boukhabza,A-; Lugnier,A.A.; Kintz,R; Mangin,P. Simultaneous HPLC analysis of the hypnotic benzodiazepines
nitrazepam, estazolam, flunitrazepam, and triazolam in plasma, J.Anal.ToxicoL, 1991, 15, 319-322.
SAMPLE
Matrix: blood
Sample preparation: Inject 100-200 |xL plasma onto column A with mobile phase A and elute
to waste, after 5 min backflush the contents of column A onto column B with mobile phase B,
after 5 min remove column A from the circuit, elute column B with mobile phase B, monitor
the effluent from column B. Wash column A with MeCN:water 60:40 at 1 mL/min for 6 min
then re-equilibrate with pH 7.5 buffer for 10 min.
HPLC VARIABLES
Column: A 45 X 4 12 |xm TSK-gel G 3 PW (Tosohass); B 75 X 4.6 Ultrasphere ODS C18 3 |xm
Mobile phase: A 50 mM pH 7.5 phosphate buffer; B Gradient. A was MeCN. B was 65 mM
KH2PO4 + 1% diethylamine adjusted to pH 5.4 with phosphoric acid. A:B 22:78 for 5 min, to
25:75 over 10 min, to 60:40 over 15 min.
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: alprazolam, bromazepam, chlordiazepoxide, clobazam, clonazepam, clorazepate, clo-
tiazepam, desmethylclobazam, desmethyldiazepam, diazepam, flunitrazepam, loflazepate, lor-
azepam, medazepam, nitrazepam, prazepam, temazepam, tetrazepam, tofisopam, triazolam
Noninterfering: carbamazepine, phenytoin, ethosuximide, phenobarbital, primidone, valproic
acid
Interfering: oxazepam
KEYWORDS
REFERENCE
Lacroix,C; Wojciechowski,E; Danger,P. Monitoring of benzodiazepines (clobazam, diazepam and their main
active metabolites) in human plasma by column-switching high-performance liquid chromatography,
J.Chromatogr., 1993, 617, 285-290.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroformdsopropanol:
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 223
CHROMATOGRAM
KEYWORDS
whole blood; plasma; interferences may occur-compounds (all of which are extracted) elute in this
order tenoxicam; iproniazid; methocarbamol; methotrexate; caffeine; nialamide; colchicine; cy-
tarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfinpyrazole; flumazenil;
sulpride; morphine; atenolol; toloxatone; terbutaline; albuterol; phenobarbital; ranitidine; tia-
pride; phenol; chlormezanone; aspirin; metformin; ritodrine; codeine; sultopride; amisulpride;
naltrexone; lisinopril; benzocaine; nizatidine; nalorphine; mephenesin; naloxone; sotalol; car-
teolol; procainamide; carbamazepine; bromazepam; nalbuphine; nadolol; procarbazine; dihy-
dralazine; omeprazole; strychnine; acebutolol; glutethimide; chlorpropamide; glipizide; triazo-
lam; prazosin; flunitrazepam; clonazepam; metoclopramide; melphalan; estazolam;
REFERENCE
Tracqui,A-; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
a 10 (urine) or 30 (blood) [xL aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
mL/min)
Detector: UV 221.6
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
Gaillard,Y; Pepin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149
163.
Estradiol
CAS Registry No.: 50-28-2, 113-38-2 (dipropionate),
979-32-8 (valerate), 57-91-0 (a- estradiol), 50-50-0 (benzoate),
313-06-4 (cypionate), 4956-37-0 (enanthate), 3571-53-7 (undecylenate)
Merck Index: 3746
LednicerNo.: 1 162; 2 136
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 1 mL 1 mM tetrapentylammonium bromide in 1 M NaOH,
mix, add 5 mL 1 mM 1-pyrenesulfonyl chloride (Molecular Probes, Eugene OR) in dichloro-
methane, vortex for 10 min, centrifuge at 1800 rpm for 10 min. Remove the organic layer and
evaporate it to dryness under reduced pressure, reconstitute the residue in mobile phase, inject
a 20 [xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 [xm Ultramex C8
Mobile phase: MeCNrwater 75:25
Flow rate: 1.5
Detector: UV 348, F ex 350 em 385, F ex 325 (Ar laser)
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Simultaneous: equilin, estrone
KEYWORDS
derivatization; serum
REFERENCE
DeSilva,K.H.; Vest,F.B.; Karnes,HT- Pyrene sulphonyl chloride as a reagent for quantitation of oestrogens in
human serum using HPLC with conventional and laser-induced fluorescence detection, Bio-
med.Chromatogr., 1996, 10, 318-324.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763,149
163.
SAMPLE
Matrix: solutions
Sample preparation: Add 25 mL receptor fluid to a 100 mg LiChrolut RP 18 SPE cartridge
using vacuum. Elute with 4 mL MeCN, evaporate the eluate, reconstitute the residue in 1 mL
MeCN. Inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 ixm Lichrospher 100 RP C18
Mobile phase: MeCN.water 60:40
Flow rate: 1.0
Detector: F ex 225 no emission filter
KEYWORDS
SPE
REFERENCE
Rohr,U.D.; Altenburger,R.; Kissel,T. Pharmacokinetics of the transdermal reservoir membrane system deliv-
ering p-estradiol: In vitro/in vivo-correlation, Pharm.Res., 1998,15, 877-882.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 226
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: progesterone
REFERENCE
Kim,D.-D.; Kim,J.L.; Chien,Y.W. Mutual hairless rat skin permeation-enhancing effect of ethanol/water system
and oleic acid, J.Pharm.ScL, 1996, 85, 1191-1195.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 X 3.9 jxBondapack C18
Mobile phase: MeCN:50 mM pH 6 potassium phosphate buffer 47:53
Flow rate: 1.5
Detector: UV 208
CHROMATOGRAM
Retention time: 13-14
Internal standard: estradiol-3-acetate
OTHER SUBSTANCES
Simultaneous: mestranol, 17a-ethinyl estradiol
Noninterfering: ketoconazole, fluconazole, itraconazole, miconazole, a-naphthoflavone, quini-
dine, sulfaphenazole, troleandomycin
KEYWORDS
estradiol-3-acetate is IS
REFERENCE
Schmider,J.; Greenblatt,D.J.; von Moltke,L.L.; Karsov,D.; Vena,R.; Friedman,H.L.; Shader,R.I. Biotransforma-
tion of mestranol to ethinyl estradiol in vitro: The role of cytochrome P-450 2C9 and metabolic inhibitors,
J.Clin.PharmacoL, 1997, 37, 193-200.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 jxm Ultrasphere
Mobile phase: MeCN:EtOH:water 54:1:45
Flow rate: 1.5
Detector: UV 270
CHROMATOGRAM
Retention time: 2.4 (17p-estradiol)
REFERENCE
Fridriksdottir,H.; Loftsson,T.; Gudmundsson,J.A.; Bjarnason,G.J.; Kjeld,M.; Thorsteinsson,T. Design and in
vivo testing of 17 p-estradiol-HP p CD sublingual tablets, Pharmazie, 1996, 51, 39-42.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize 2.5 g tissue with 10 mL acetone for 20 s, sonicate for 5 min,
centrifuge at 3200 rpm. Decant the supernatant into a silanized tube. Add 8 mL acetone to the
pellet and repeat the extraction. Combine the supernatants. Add to a 5 mL pipette tip con-
taining 1.5 g alumina (80-200 mesh, Brockman activity 1) followed by an Econo-Column filled
with 1.0 g AGMP-I resin (Bio-Rad), allow to pass through by gravity. Wash with four 1 mL
portions of acetone:water 95:5. Remove the alumina column, wash the ion-exchange column
with 1 mL acetone:water 95:5, elute with four 1 mL portions of 10% acetic acid in acetone.
Evaporate the combined eluates to dryness with nitrogen at 40. Add 500 |xL water to the
residue, extract twice with 2 mL potions of ether. Combine the ether layers and evaporate
them to dryness. Reconstitute the residue in mobile phase B. Inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelco silica
Mobile phase: Gradient. A was hexane. B was MeOH:hexane:2-propanol 45:40:15. A:B from 100:
0 to 60:40 over 15 min.
Flow rate: 2.0
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diethylstilbestrol, zeranol
Simultaneous: estrone, zeralenol, zeralenone, zeralanone
KEYWORDS
chicken; muscle; normal phase; SPE
REFERENCE
Medina,M.B.; Sherman,J.T. High performance liquid chromatographic separation of anabolic oestrogens and
ultraviolet detection of 17p-oestradiol, zeranol, diethylstilboestrol or zearalenone in avian muscle tissue
extracts, Food Addit.Contam., 1986, 3, 263-272.
Estramustine
Molecular formula: C23H3ICI2NO3
CAS Registry No.: 2998-57-4, 52205-73-9
(phosphate sodium)
Merck Index: 3749
Lednicer No.: 3 83
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL EtOH + 2 mL buffer, mix well, add 12 mL hexane,
shake slowly for 15 min on a reciprocating shaker, centrifuge at 1200 g at 5 for 10 min. Remove
10 mL of the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute
the residue in 100 |xL hexane:EtOH 92.5:7.5, inject a 20 |xL aliquot. (Buffer was 630 mL of a
solution containing 61.8 g/L boric acid and 74.6 g/L KCl and 370 mL 106 g/L sodium carbonate
solution, shake well, adjust pH to 9.0 with sodium carbonate solution (if necessary), store at
35-37.)
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Partisil PXS 5/25 silica gel
Mobile phase: Hexane:EtOH 92.5:7.5
Flow rate: 1.5
Detector: F ex 195 em 250 (cut-off filter)
CHROMATOGRAM
Retention time: 7.2
OTHER SUBSTANCES
Simultaneous: estrone, estradiol
KEYWORDS
plasma; normal phase; normal phase more sensitive than reverse phase; pharmacokinetics
REFERENCE
Brooks,M.A.; Dixon,R. Determination of estramustine and its 17-keto metabolite in plasma by high-perfor-
mance liquid chromatography, J.Chromatogr., 1980, 182, 387-394.
SAMPLE
Matrix: blood
Sample preparation: 0.5-1 mL Plasma + 1 mL 500 mM pH 7 phosphate buffer + 12 mL hexane:
ethyl acetate 70:30, extract. Remove a 10 mL aliquot of the organic layer and evaporate it to
dryness under a stream of nitrogen at 50, reconstitute the residue in 100 |xL mobile phase,
inject a 20-50 |xL aliquot. (Hydrolyze 500 JAL plasma by adding 500 |xL 200 mM pH 5 acetate
buffer and 100 |xL beef liver p-glucuronidase (Sigma) or 10 |xL p-glucuronidase/sulfatase (GIu-
sulase), heat at 37 overnight, add 1 mL 500 mM pH 7 phosphate buffer + 12 mL hexane:ethyl
acetate 70:30, extract. Remove a 10 mL aliquot of the organic layer and evaporate it to dryness
under a stream of nitrogen at 50, reconstitute the residue in 100 jxL mobile phase, inject a
20-50 |JLL aliquot.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Partisil 5/25 silica gel
Flow rate: 1.5
CHROMATOGRAM
Retention time: 7.2
OTHER SUBSTANCES
Extracted: estromustine, estrone, estradiol, metabolites
KEYWORDS
plasma; rat; dog; human; pharmacokinetics; normal phase
REFERENCE
Dixon,R.; Brooks,M.; Gill,G. Estramustine phosphate: Plasma concentrations of its metabolites following oral
administration to man, rat and dog, Res.Commun.Chem.Pathol.PharmacoL, 1980, 27, 1729.
Estriol
CAS Registry No.: 50-27-1, 113-22-4
(16,17-bis(sodium hemisuccinate))
Merck Index: 3750
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
apram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estrone, ethacrynic acid,
fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glu-
zepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxjnnorphone,
oxyphenbutazone, ox3rtetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbi-
puromycin, pyrilamine, pjn-ithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
thioridazine, thiosalicylic acid, thiothixene, thjmaol, tolazamide, tolazoline, tobutamide, tol-
REFERENCE
Hill,D.W.; Kind,A-J- Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.Toxicol., 1994,
18, 233-242.
Estrogens, conjugated
Molecular formula: C18H18O2 (equilenin), C18H22O2 (17a-dihydroequilin), C18H20O2 (equilin), C18H22O2

(estrone), C18H24O2 (estradiol)
Molecular weight: 266.34 (equilenin), 272.39 (estradiol), 268.36 (equilin), 270.37 (17a-dihydro-
equilin), 270.39 (estrone)
CAS Registry No.: 474-86-2 (equilin), 50-28-2 (estradiol), 57-91-0 (a-estradiol), 517-09-9 (equilenin),
53-16-7 (estrone), 338-67-5 (estrone sodium sulfate), 481-97-0 (estrone hydrogen sulfate)
Merck Index: 3216 (17a-dihydroequilin), 3675 (equilenin), 3676 (equilin), 3746 (estradiol), 3751
(estrone)
LednicerNo.: 1 156 (estrone); 1 162 (estradiol); 2 136 (estradiol)
SAMPLE
Matrix: blood
Sample preparation: 0.5-1 mL Plasma + 1 mL 500 mM pH 7 phosphate buffer + 12 mL hexane:
ethyl acetate 70:30, extract. Remove a 10 mL aliquot of the organic layer and evaporate it to
inject a 20-50 |xL aliquot. (Hydrolyze 500 jxL plasma by adding 500 |JLL 200 mM pH 5 acetate
buffer and 100 |JLL beef liver p-glucuronidase (Sigma) or 10 JAL p-glucuronidase/sulfatase (GIu-
sulase), heat at 37 overnight, add 1 mL 500 mM pH 7 phosphate buffer + 12 mL hexaneiethyl
acetate 70:30, extract. Remove a 10 mL aliquot of the organic layer and evaporate it to dryness
under a stream of nitrogen at 50, reconstitute the residue in 100 |xL mobile phase, inject a
20-50 |xL aliquot.)
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Partisil 5/25 silica gel
Flow rate: 1.5
CHROMATOGRAM
Retention time: 6.2 (estrone)
OTHER SUBSTANCES
Extracted: estromustine, estradiol, estramustine, metabolites
KEYWORDS
plasma; rat; dog; human; pharmacokinetics; normal phase
REFERENCE
Dixon,R.; Brooks,M.; Gill,G. Estramustine phosphate: Plasma concentrations of its metabolites following oral
administration to man, rat and dog, Res.Commun.Chem.Pathol.PharmacoL, 1980, 27, 17-29.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 5 mL water + 1 mL 2 jxg/mL equilenin in MeOH + 50 (JLL
0.1 M NaOH to adjust pH to 10, vortex briefly after each addition, shake with 10 mL dichlo-
romethane for 10 min, centrifuge at 2000 g for 10 min. Wash organic layer twice with 2 mL
water, centrifuge 5 min, evaporate 8 mL of organic phase to dryness at 40 under a stream of
nitrogen, reconstitute residue in 150 |xL mobile phase, inject 25 |xL aliquot
HPLC VARIABLES
Column: 300 X 4 10 juum ixBondapak C18
Mobile phase: MeOH.buffer 65:35 (Buffer was 10 mL 200 mM acetic acid + 15 mL 200 mM
sodium acetate made up to 1 L, pH 4.8.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 5.3 (equilenin)
Internal standard: equilenin
OTHER SUBSTANCES
Simultaneous: hydrocortisone, deoxycortisol, triamcinolone, prednisone, dexamethasone,
betamethasone
KEYWORDS
plasma; equilenin is IS
REFERENCE
Bouquet,S.; Brisson,A.M.; Gombert,J. Dosage du cortisol et du ll-de"soxycortisol plasmatiques par chromato-
graphie liquide haute performance [Cortisol and 11-desoxycortisol determination in blood by high perfor-
mance liquid chromatography], ATITLBIOZ.Clin.(Paris), 1981, 39, 189-191.
SAMPLE
Matrix: blood
Sample preparation: 100 jxL Serum + 500 |xL water + 100 |xL 10 |xg/mL 3,7-dimethoxynavone
in EtOH + 8 mL diethyl ether, shake, centrifuge at 4 at 1000 g for 5 min, freeze in acetone/
dry ice. Remove the organic layer and dry it over anhydrous sodium sulfate, evaporate to
dryness under a stream of nitrogen, reconstitute the residue in 100 jxL MeOHiwater 40:60,
HPLCVARIABLES
Column: 250 X 4.6 3 |xm NS-GeI C18
Mobile phase: Gradient. MeOH:water from 40:60 to 55:45, maintain at 55:45 for 24 min, to 80:
20 over 25 min
Flow rate: 1
CHROMATOGRAM
Internal standard: 3,7-dimethoxyflavone (47)
OTHER SUBSTANCES
Extracted: aldosterone, androstenedione, dehydroepiandrosterone, deoxycorticosterone, 11-deox-
ycortisol, estradiol, hydrocortisone, 17-hydroxyprogesterone, pregnenolone, progesterone
KEYWORDS
serum
REFERENCE
Ueshiba,H; Segawa,M.; Hayashi,T.; Miyachi,Y.; Irie,M. Serum profiles of steroid hormones in patients with
Cushing's syndrome determined by a new HPLC/RIA method, Clin.Chem., 1991, 37, 1329-1333.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 3 urn Nucleosil C18
Mobile phase: MeOH:dichloromethane:2-propanol:water 45:5:7.5:42.5
Detector: UV 280
CHROMATOGRAM
Retention time: 16.80 (17a-dihydroequilenin), 19.30 (17a-dihydroequilin), 22.52 (17a-estradiol),
25.77 (equilenin), 28.62 (equilin), 32.19 (estrone)
REFERENCE
Novakovic,J.; Pacakova,V.; Sevcik,J.; Cserhati,T. Quantitative structure-chromatographic retention relation-
ship study of six underivatized equine estrogens, J.Chromatogr.B, 1996, 681, 115-123.
SAMPLE
Matrix: solutions
|xL aliquot.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
Retention time: k' 1.43 (estrone)
REFERENCE
Roos,RW*> Lau-Cam,C.A. General reversed-phase high-performance liquid chromatographic method for the
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:THF:water 12.9:22.4:64.7
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: ethinyl estradiol, mestranol, norethindrone, norethindrone acetate, norgestrel
REFERENCE
Gazdag,M.; Szepesi,G.; Szeleczki,E. Selection of high-performance liquid chromatographic methods in phar-
maceutical analysis. I. Optimization for selectivity in reversed-phase chromatography, J.Chromatogr., 1988,
454, 83-94.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm LiChrosorb Si 60
Mobile phase: Hexane:dioxane:isopropanol 95:3:2
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 11 (estrone)
OTHER SUBSTANCES
Simultaneous: ethinyl estradiol, mestranol, norethindrone, norethindrone acetate, norgestrel
KEY WORDS
normal phase
REFERENCE
Gazdag,M.; Szepesi,G.; Fabian-Varga,K. Selection of high-performance liquid chromatographic methods in
pharmaceutical analysis. II. Optimization for selectivity in normal-phase systems, J.Chromatogr., 1988,
454, 95-107.
Next Page
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a solution in MeOH.
HPLC VARIABLES
Column: Radial-PAK jxBondapak C18
Flow rate: 2
Detector: UV 254 or 214
CHROMATOGRAM
Retention time: 2.5 (estriol), 5.5 (testosterone), 5.6 (17p-estradiol), 6.9 (estrone), 16.3
(progesterone)
KEYWORDS
testosterone and estradiol interfere
REFERENCE
Erkoc,F.U.; Ozsar,S.; Giiven,B.; Kalkandelen,G.; Ugrar,E. High-performance liquid chromatographic analysis
of steroid hormones, J.Chromatogr.Sci., 1989, 27, 86-90.
SAMPLE
Matrix: solutions
Sample preparation: Prepare an aqueous solution, inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 3.5 |xm Zorbax SB C18
Mobile phase: MeCN:MeOH:buffer 15:45:40 (Buffer was 10 mM KH2PO4 and 50 mM tetrabu-
tylammonium chloride, pH adjusted to 3.0 with 1 M HCl.)
Flow rate: 0.9
Detector: UV 220
CHROMATOGRAM
Retention time: 11.2 (estrone), 8.4 (estrone-3-phosphate)
OTHER SUBSTANCES
Simultaneous: estriol, 17p-estradiol, 17p-estradiol-3-phosphate
REFERENCE
Miller,R.B.; Chen,C. A stability-indicating HPLC method for the determination of 17[3-estradiol-3-phosphate in
an ophthalmic solution, Chromatographia, 1995, 40, 204-206.
Estrone
CAS Registry No.: 53-16-7, 338-67-5 (sodium sulfate),
481-97-0 (hydrogen sulfate)
Merck Index: 3751
Lednicer No.: 1 156
SAMPLE
Matrix: blood
Previous Page
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a solution in MeOH.
HPLC VARIABLES
Column: Radial-PAK jxBondapak C18
Flow rate: 2
CHROMATOGRAM
Retention time: 2.5 (estriol), 5.5 (testosterone), 5.6 (17p-estradiol), 6.9 (estrone), 16.3
(progesterone)
KEYWORDS
testosterone and estradiol interfere
REFERENCE
Erkoc,F.U.; Ozsar,S.; Giiven,B.; Kalkandelen,G.; Ugrar,E. High-performance liquid chromatographic analysis
of steroid hormones, J.Chromatogr.Sci., 1989, 27, 86-90.
SAMPLE
Matrix: solutions
Sample preparation: Prepare an aqueous solution, inject a 20 JJLL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:buffer 15:45:40 (Buffer was 10 mM KH2PO4 and 50 mM tetrabu-
tylammonium chloride, pH adjusted to 3.0 with 1 M HCl.)
Flow rate: 0.9
Detector: UV 220
CHROMATOGRAM
Retention time: 11.2 (estrone), 8.4 (estrone-3-phosphate)
OTHER SUBSTANCES
Simultaneous: estriol, 17p-estradiol, 17p-estradiol-3-phosphate
REFERENCE
Miller,R.B.; Chen,C. A stability-indicating HPLC method for the determination of 17[3-estradiol-3-phosphate in
an ophthalmic solution, Chromatographia, 1995, 40, 204-206.
Estrone
CAS Registry No.: 53-16-7, 338-67-5 (sodium sulfate),
481-97-0 (hydrogen sulfate)
Merck Index: 3751
Lednicer No.: 1 156
SAMPLE
Matrix: blood
Sample preparation: Hydrolyse serum or plasma with (3-glucuronidase and sulfatase, extract
with diethyl ether. Remove the organic layer and evaporate it to dryness under a stream of
nitrogen at room temperature, reconstitute the residue in 40 jxL buffer, add 100 |xL 1.5 mg/
mL dansyl chloride in acetone, shake vigorously for 30 s, heat at 100 for 5 min, inject a 20
jxL aliquot. (Prepare buffer by adjusting the pH of 4 g/L sodium bicarbonate in water to 10.5
with 5 M NaOH.)
HPLC VARIABLES
Column: 250 X 2.6 PAH-10 C18 (Perkin-Elmer)
Mobile phase: Gradient. MeCN:water from 60:40 to 95:5 over 15 min (Perkin-Elmer curve 1),
maintain at 95:5 for 10 min. (Flush column with MeCN at 0.1 mL/min overnight.)
Flow rate: 1
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Extracted: diethylstilbestrol, estriol, hexestrol, zanone, zenone, zeranol
KEY WpRDS
derivatization; cow; sheep; plasma; serum; LOD is too high for practical detection of compounds
in serum and plasma.
REFERENCE
Rhys Williams,A.T.; Winfield,S.A.; Belloli,R.C. Dns derivatization of anabolic agents with high-performance
liquid chromatographic separation and fluorescence detection, J.Chromatogr., 1982, 240, 224-229.
SAMPLE
Matrix: blood
Sample preparation: 0.5 mL Serum + 0.5 mL MeCN:water 1:1, vortex 15 s, add 3 mL MeCN,
shake 1 min, centrifuge at 1800 rpm for 10 min. Remove supernatant and dry it under a stream
of nitrogen at 55, add 2 mL MeCNrMeOH 1:1, vortex 15 s, centrifuge at 1800 rpm for 10 min.
Remove supernatant and dry it under a stream of nitrogen at 55. Reconstitute in 200 |xL
MeCN:water 1:1.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Beckman ODS
Mobile phase: A 2% tetrabutylammonium hydroxide adjusted to pH 3 with phosphoric acid. B
MeCN:water 33:67 A:B was 6.5:93.5
Flow rate: 0.8
CHROMATOGRAM
Retention time: 41
OTHER SUBSTANCES
Simultaneous: estrone, equilin, estradiol, equilin sulfate, 17-a-dihydroequilin sulfate
KEY WORDS
serum
REFERENCE
Su,S.Y.; Shiu,G.K; Simmons,J.; Viswanathan,C.T.; Skelly,J.P. High performance liquid chromatographic anal-
ysis of six conjugated and unconjugated estrogens in serum, Biomed.Chromatogr., 1992, 6, 265268.
SAMPLE
Matrix: culture media
Sample preparation: Extract culture medium twice with 2 volumes of ether, combine the ex-
tracts and evaporate them to dryness, reconstitute with MeOH, inject an aliquot.
HPLC VARIABLES
Column: 300 X 3.9 Techopak 10 C18 (HPLC Technology)
Mobile phase: MeOH:0.5% pH 3.0 (NH4)H2PO4 62:39
Flow rate: 0.7
Detector: UV 280, radioactivity
CHROMATOGRAM
Retention time: 17
OTHER SUBSTANCES
Extracted: estradiol
KEYWORDS
tritium labeled
REFERENCE
Wild,M.J.; Rudland,P.S.; Back,D.J. Metabolism of the oral contraceptive steroids ethynylestradiol and norges-
timate by normal (Huma 7) and malignant (MCF-7 and ZR-75-1) human breast cells in culture, J.Steroid
Biochem.Mol.BioL, 1991, 39, 535-543.
SAMPLE
Sample preparation: Finely powder tablets. Weigh out an amount equivalent to 3 mg pipera-
zine estrone sulfate, add 10 mL mobile phase containing 100 |xg/mL biphenyl, shake 30 min,
inject 10 |xL aliquot.
HPLC VARIABLES
Column: 250 x 4.8 Brownlee RP-18
Mobile phase: MeCN:20 mM pH 5.0 phosphate buffer 55:45 containing 3 mM cetyltrimethylam-
monium bromide
Flow rate: 2
Detector: UV 225
CHROMATOGRAM
Internal standard: biphenyl (k' 11.04)
OTHER SUBSTANCES
Simultaneous: a-estradiol sulfate, (3-estradiol sulfate, a-estradiol, 0-estradiol, equilin sulfate,
estrone, methylparaben, propylparaben
KEY WORDS
tablets
REFERENCE
Carignan,G.; Lodge,B.A.; Skakum,W. Analysis of piperazine estrone sulfate in tablets by ion-pair high-perfor-
SAMPLE
Sample preparation: Dissolve in mobile phase
HPLC VARIABLES
Mobile phase: MeOH:25 mM KH2PO4 40:60
Flow rate: 0.8
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: 17a-estradiol sulfate, 17(3-estradiol sulfate, equilin sulfate, 17a-dihydroequilin
sulfate, 17p-dihydroequilin sulfate, equilenin sulfate, 17a-dihydroequilenin sulfate
KEYWORDS
intravenous formulations; tablets
REFERENCE
Flann,B-; Lodge,B- Analysis of estrogen sulphate mixtures in pharmaceutical formulations by reversed-phase
chromatography, J.Chromatogr., 1987, 402, 273-282.
SAMPLE
Sample preparation: Dissolve in mobile phase
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere Octyl
Mobile phase: MeCN:MeOH:buffer 32:18:50 (Buffer was 1.7 mM cetyltrimethylammonium phos-
phate and 25 mM KH2PO4.)
Flow rate: 0.9
Detector: UV 200
CHROMATOGRAM
KEYWORDS
intravenous formulations; tablets; estrone sulfate; equilin sulfate; 17a-dihydroequilin sulfate; 17(3-
dihydroequilin sulfate; equilenin sulfate; 17a-dihydroequilenin sulfate; estrone; equilin; 17a-
dihydroequilin; 17p-dihydroequilin; equilenin; 17p-dihydroequilenin; 17a-estradiol sulfate;
17p-estradiol sulfate; 17a-estradiol; 17p-estradiol
REFERENCE
Flann,B.; Lodge,B- Analysis of estrogen sulphate mixtures in pharmaceutical formulations by reversed-phase
SAMPLE
Sample preparation: 1 mL Microsomal incubation + 5 mL ethyl acetate, vortex, centrifuge at
2000 g for 8 min, remove organic phase, repeat extraction. Combine the organic layers and
evaporate them to dryness under a stream of nitrogen, reconstitute the residue in 100 |xL
MeOH:water 50:50, inject a 50 uL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 jxm Ultracarb 30 ODS (Phenomenex)
Mobile phase: Gradient. MeCN:0.1% acetic acid in MeOH:0.1% acetic acid in water 16:12:72 for
3 min, to 20:21:59 over 25 min (Waters no. 3 convex gradient), to 24:23:53 over 10 min (linear),
to 55:24:21 over 10 min (linear), to 92:5:3 over 1 min, maintain at 92:5:3 for 7 min, return to
initial conditions over 15 min.
Flow rate: 1.2
Detector: UV 280
CHROMATOGRAM
Retention time: 50
OTHER SUBSTANCES
Extracted: estradiol, metabolites
KEYWORDS
rat
REFERENCE
Suchar,L.A.; Chang,R.L.; Rosen,R.T.; Lech, J.; Conney,A.H. High-performance liquid chromatography separation
of hydroxylated estradiol metabolites: Formation of estradiol metabolites by liver microsomes from male
and female rats, J.Pharmacol.Exp.Ther., 1995, 272, 197-206.
SAMPLE
Matrix: solutions
Sample preparation: dissolve in ethanol
HPLCVARIABLES
Column: 150 X 4.6 5 |xm Spherisorb S5-0DS
Mobile phase: Gradient. MeOH:20 mM ammonium sulfate from 30:70 to 100:0 over 35 min
Flow rate: 1
Detector: F ex 214 em 340 (cut-off) or UV 280
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Simultaneous: estriol-3-sulfate, 17p-estradiol-3-sulfate, estriol, estrone, 17p-estradiol, estrone-
3-sulfate, estriol, estrone
REFERENCE
Simonian,M.H.; Capp,M.W. Reversed-phase high-performance liquid chromatography of steroid 3-sulfates and
the corresponding unconjugated steroids, J.Chromatogr., 1984, 287, 97-104.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 0.5 mg/mL solution in MeOH, inject a 5 |xL aliquot.
HPLC VARIABLES
Mobile phase: Gradient. A was 150 mM phosphoric acid and 50 mM triethylamine. B was MeCN:
water 80:20 containing 150 mM phosphoric acid and 50 mM triethylamine. A:B 100:0 for 2.2
min then to 0:100 over 30 min.
Flow rate: 2
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: acetaminophen, aprobarbital, butabarbital, chlordiazepoxide, chloroxylenol,
chlorpromazine, clenbuterol, cortisone, danazol, diflunisal, doxapram, fluoxymesterone, mefen-
amic acid, methyltestosterone, nicotine, oxazepam, phentermine, phenylpropanolamine, pro-
gesterone, sulfamethazine, sulfanilamide, testosterone propionate, tranylcypromine,
tripelennamine
Interfering: testosterone
KEYWORDS
details for purification of triethylamine in paper
REFERENCE
Hill,D.W.; Kind,A.J. The effects of type B silica and triethylamine on the retention of drugs in silica based
reverse phase high performance chromatography, J.Liq.Chromatogr., 1993, 16, 3941-3964.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
apram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, ethacrynic acid,
fluoxymesterone, nuphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glu-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Mix 10 |xL 0.5 mM compound in anhydrous benzene containing 100 mM
quinuclidine with 10 |xL 25 mM DBD-COCl in anhydrous benzene (Caution! Benzene is a
carcinogen!), heat at 60 for 15 min, add 980 |xL MeCN:water:acetic acid 50:50:1, inject a 2 |xL
aliquot. (Purify quinuclidine by sublimation. DBD-COCl was 4-(N-chloroformylmethyl-N-
methyl)amino-7-N,N-dimethylaminosulfonyl-2,l,3-benzoxadiazole. Synthesis of DBD-COCl is
as follows. Dissolve 0.5 g magnesium sulfate heptahydrate and 6 g NaOH in 60 mL water,
throughout the reaction keep the flask at about 20 with cold water cooling, add 15 mL 30%
hydrogen peroxide, add 75 mL MeOH, add 12.1 g powdered benzoyl peroxide in one go, stir for
10 min, pour into 150 mL 20% sulfuric acid, extract three times with 50 mL portions of chlo-
roform, determine peroxybenzoic acid concentration by iodometric titration (Tetrahedron 1967,
23, 3327). Slowly add 110 mL 1 M peroxybenzoic acid in chloroform to 7 g 2,6-difluoroaniline
dissolved in 100 mL chloroform, stir at room temperature, when reaction is complete (iodo-
metric titration) wash with 2% sodium thiosulfate, wash with 5% sodium carbonate, wash with
water, dry over anhydrous sodium sulfate, evaporate to dryness under reduced pressure, re-
crystallize 2,6-difluoronitrosobenzene form EtOH (mp 108.5-109.5). Stir 8.5 g 2,6-difluoronitro-
sobenzene in 85 mL DMSO at room temperature and add a solution of 3.91 g sodium azide in
85 mL DMSO dropwise, let stand for about 1 h, add to a large volume of water, extract with
ether, dry the extracts over anhydrous sodium sulfate, evaporate to dryness under reduced
pressure and distil to give 4-fluoro-2,l,3-benzoxadiazole as a colorless oil (bp 83712 mm Hg)
(J.Chem.Soc.(C) 1970, 1433). Add 11 mL chlorosulfonic acid dropwise to 3 g 4-fluoro-2,l,3-
benzoxadiazole in 10 mL chloroform at 0-10 (use a calcium chloride drying tube), stir at room
temperature for 1 h, reflux for 2 h, cool, slowly pour into ice water, remove the organic layer,
extract the aqueous layer with chloroform, combine the organic layer, wash, dry over anhydrous
magnesium sulfate, evaporate under reduced pressure, take up the residue in 5 mL benzene
(Caution! Benzene is a carcinogen!), chromatograph on a 150 X 30 column of silica gel (100-
200 mesh Kanto Chemical) with n-hexane:benzene 50:50, evaporate the appropriate fractions
to give 4-(chlorosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (CBD-F) as pale yellow needles (mp 64-
66) (Anal. Chem. 1984, 56, 2461). Stir 0.76 g CBD-F in 70 mL MeCN at 0-10 and add 1 g
dimethylamine hydrochloride in 10 mL 100 mM pH 10 borax dropwise, adjust pH to 5 with 1
M HCl, concentrate to about 10 mL under reduced pressure, extract three times with 200 mL
portions of diethyl ether, wash with water, dry over anhydrous magnesium sulfate, evaporate
under reduced pressure, chromatograph on a 500 X 20 column of silica gel with chloroform,
isolate the appropriate fraction and re-chromatograph on the same column with ethyl acetate:
benzene 1:2 to give 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (DBD-F) as
white needles (mp 124-125) (yield = 1% !) (Analyst 1989, 114, 413). On a Merck no. 5714 60F254
tic plate eluted with chloroform DBD-F has Rf 0.32 and lies between two other reaction prod-
ucts. It is also reported that DBD-F can be purchased from Tokyo Kasei (TCI America, Portland
OR). Stir N-methylglycine and 2.3 g sodium carbonate in water at room temperature, add 880
mg DBD-F in 40 mL MeCN dropwise, stir for 1 h, evaporate to remove the MeCN, wash twice
with 50 mL portions of ethyl acetate. Acidify the aqueous phase with HCl and extract it twice
with 300 mL portions of ethyl acetate. Wash the organic layer twice with 100 mL portions of
saturated aqueous NaCl, dry over anhydrous magnesium sulfate, evaporate to dryness under
reduced pressure, recrystallize from ethyl acetate to give 4-(N-carboxymethyl-N-methyl)amino-
7-dimethylaminosulfonyl-2,l,3- benzoxadiazole (DBD-COOH) as orange-yellow crystals (mp
209-210). Add 3.5 mL oxalyl chloride and 24 \xL DMF to 1 g DBD-COOH in anhydrous benzene,
stir at room temperature for 30 min, reflux for 1 h, evaporate to dryness, add 20 mL dry
benzene to the residue, filter, evaporate the filtrate to give 4-(N-chloroformylmethyl-N-
methyl)amino-7-N,N-dimethylaminosulfonyl-2,l,3-benzoxadiazole (DBD-COCl) as yellow crys-
tals (mp 102).)
HPLCVARIABLES
Column: 150 X 4.6 5 (xm Cosmosil 5C18
Mobile phase: Gradient. MeCN.water from 50:50 to 100:0 over 20 min, maintain at 100:0 for 1
h.
Flow rate: 1
Injection volume: 2
CHROMATOGRAM
Retention time: 15
KEYWORDS
derivatization
REFERENCE
Imai,K; Fukushima,T.; Yokosu,H. A novel electrophilic reagent, 4-(A^-chloroformylmethyl-N-methyl)amino-7-
iV,iV-dimethylaminosulphonyl-2,l,3-benzoxadiazole (DBD-COCl) for fluorometric detection of alcohols, phe-
nols, amines and thiols, Biomed.Chromatogr., 1994, 8, 107-113.
Ethacrynic acid
Molecular formula: C13H12CI2O4
Merck Index: 3761
Lednicer No.: 1 120
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 20 jxL 750 u,g/mL 4-(2,4-dichlorophenoxy)butyric acid in
MeOH + 20 |xL MeOH, vortex briefly, add 3 mL 1 M HCl, vortex, add 5 mL ethyl acetate, rock
for 5 min, centrifuge at 400 g for 5 min. Remove the upper ethyl acetate layer and evaporate
it to dryness under a stream of nitrogen at 30, reconstitute the residue in 400 |xL MeCN:
buffer 30:70, inject a 200 jxL aliquot. (Buffer was water containing 0.25% triethylamine and
0.25% phosphoric acid.)
HPLC VARIABLES
Guard column: 15 X 3.2 7 fim Aquapore C18 (Brownlee)
Column: 100 X 4.6 5 |xm Hypersil ODS C18
Mobile phase: MeCN:MeOH:buffer 13:32:55 (Buffer was water containing 0.25% triethylamine
and 0.25% phosphoric acid.)
Flow rate: 2
Detector: UV 280
CHROMATOGRAM
Internal standard: 4-(2,4-dichlorophenoxy)butyricacid (10.1)
KEYWORDS
plasma
REFERENCE
LaCreta,F.R; Brennan,J.M.; Tinsley,P.W.; OT)wyer,P.J. High-performance liquid chromatographic determina-
tion of ethacrynic acid in human plasma, J.Chromatogr., 1991, 571, 271-276.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 50 JJLL 500 ng/mL mefenamic acid or indomethacin
+ 1 mL 100 mM HCl + 10 mL dichloromethane, rotate for 10 min, centrifuge at 1500 g for 15
min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 45.
Redissolve the residue in mobile phase, inject a 20 |xL aliquot. Urine. 50 |xL Urine + 1 mL
mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 75 X 4.6 3 |xm Supelcosil LC-8
Mobile phase: MeCN:50 mM phosphoric acid 45:55
Flow rate: 1
Detector: UV 235
CHROMATOGRAM
Retention time: 4.0
Internal standard: mefenamic acid (8) or indomethacin (5)
OTHER SUBSTANCES
Simultaneous: naproxen, flunixin, thiosalicylic acid, phenylbutazone
KEYWORDS
plasma
REFERENCE
Singh,A.K.; Jang,Y.; Mishra,U.; Granley,K. Simultaneous analysis of flunixin, naproxen, ethacrynic acid, in-
domethacin, phenylbutazone, mefenamic acid and thiosalicyclic acid in plasma and urine by high-perfor-
mance liquid chromatography and gas chromatography-mass spectrometry, J.Chromatogr., 1991,568, 351-
361.
SAMPLE
Sample preparation: Condition a 3 mL 200 mg Bond Elut C18 SPE cartridge with two column
volumes of MeOH and one column volume of water. Plasma. Acidify 4 mL plasma with 30 |xL
86% phosphoric acid. 1 mL Acidified plasma + 100 |xL 1 M HCl + 70 |xL 3 |xg/mL diclofenac in
MeOH, add to the SPE cartridge, wash with 1 column volume water, wash with 4 mL EtOH:
water 9:1, elute with 800 |xL EtOH. Evaporate the eluate to dryness, reconstitute the residue
with 100 |xL mobile phase, inject a 70 |xL aliquot. Urine. Acidify 10 mL urine with 50 |JLL 86%
phosphoric acid. 500 |JLL Acidified urine + 100 |xL 1 M HCl + 70 |JLL 7 \xg/mL 4-(2,4-dichloro-
phenoxy)butyric acid, add to the SPE cartridge, wash with 1 column volume water, wash with
4 mL EtOH:water 9:1, elute with 800 |xL EtOH. Evaporate the eluate to dryness, reconstitute
the residue with 100 |xL mobile phase, inject a 70 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 3 jxm Spherisorb ODS II
Mobile phase: MeCN:MeOH:THF:0.2% phosphoric acid 32:13:1.5:50 (plasma) or 32:13:2:55
(urine)
Flow rate: 0.9 (plasma), 0.8 (urine)
Detector: UV 275
CHROMATOGRAM
Retention time: 19 (plasma), 30 (urine)
Internal standard: 4-(2,4-dichlorophenoxy)butyric acid (32), diclofenac (37)
KEYWORDS
SPE; plasma; pharmacokinetics
REFERENCE
Voith,B-l Spahn-Langguth,H-; Mutschler,E. New specific and sensitive HPLC-assays for ethacrynic acid and its
main metabolite -the cysteine conjugate -in biological material, J.Pharm.Biomed.AnaL, 1995, 13, 1373-
1382.
SAMPLE
Sample preparation: Powder tablets, weigh out amount equivalent to 50 mg ethacrynic acid,
add 100 mL EtOHiwater 50:50, stir for 15 min, filter. Dilute a 5 mL aliquot of the filtrate to
20 mL with EtOHiwater 50:50. Add a 3 mL aliquot of this solution to 1.5 mL 6 |xg/mL 4-
acetylbiphenyl in EtOH:water 50:50, make up to 20 mL with mobile phase, inject a 10 |xL
aliquot.
HPLC VARIABLES
Column: 150 X 3.9 5 |xm Hypersil RP-18
Mobile phase: MeCN:50 mM pH 3.0 ammonium phosphate buffer 56:44
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Internal standard: 4-acetylbiphenyl
KEYWORDS
tablets
REFERENCE
Cavrini,V.; Bonazzi,D.; Di Pietra,A.M.; Gatti,R. Determination of ethacrynic acid in pharmaceutical formula-
tions by difference ultraviolet spectrophotometry after derivatisation with N-acetylcysteine, Analyst, 1989,
114, 1307-1310.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
apram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, ethosuximide,
REFERENCE
Hill,D.W.; Rind,AJ- Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeOH:water 80:20, inject a 6 |xL aliquot.
HPLCVARIABLES
Guard column: 5 X 4 10 (xm LiChrosorb RP-8
Column: 100 X 4.6 5 |xm Spheri RP-18 (Brownlee)
Mobile phase: MeOH:water 80:20 containing 2 g/L lithium perchlorate
Flow rate: 0.5
Injection volume: 6
Detector: E, ESA Model 5100A Coulochem, model 5020 guard cell +950 mV, Model 5010 analyt-
ical cell + 400 mV, palladium reference electrode, following post-column photolysis. The effluent
from the column flowed through a 10 m X 0.3 mm coil of PTFE tubing and was irradiated at
254 nm with a Sylvania GTE 8 W low-pressure lamp.
CHROMATOGRAM
Limit of detection: 1.33 jig/mL
OTHER SUBSTANCES
Also analyzed: bendroflumethiazide, butizide, chlorthalidone, furosemide, hydrochlorothiazide
KEYWORDS
post-column reaction; post-column photochemical derivatization
REFERENCE
Macher,M.; Wintersteiger,R. Improved electrochemical detection of diuretics in high-performance liquid chro-
matographic analysis by postcolumn on-line photolysis, J.Chromatogr.A, 1995, 709, 257-264.
SAMPLE
Matrix: urine
Sample preparation: 2 mL Urine + 0.5 g solid buffer I (pH 5-5.5), vortex 15 s, add 4 mL ethyl
acetate, agitate for 10 min, centrifuge at 600 g for 5 min. Remove organic layer and vortex it
with 2 mL 5% aqueous lead acetate for 10 s, centrifuge at 600 g for 5 min, remove and keep
organic phase. 2 mL Urine + 0.5 g solid buffer II (pH 9-9.5), vortex 15 s, add 4 mL ethyl acetate,
agitate for 10 min, centrifuge at 600 g for 5 min. Remove organic layer and combine it with
previous organic layer. Evaporate to dryness at 50 under a stream of nitrogen, reconstitute in
300 (JLL 50 jxg/mL p-hydroxyethyltheophylline in MeOH, inject 5 JJLL aliquot. (Solid buffer I was
KH2PO4:Na2HPO4 99:1, solid buffer II was NaHCO3:K2CO3 3:2.)
HPLCVARIABLES
Column: 250 X 4.6 5 |xm HP Hypersil ODS (A) or HP LiChrosorb RP-18 (B)
Mobile phase: Gradient. MeCN:buffer from 15:85 at 2 min to 80:20 at 20 min (Buffer was 50
mM NaH2PO4 containing 16 mM propylamine hydrochloride, adjusted to pH 3 with concen-
trated phosphoric acid.)
Flow rate: 1
Injection volume: 5
CHROMATOGRAM
Internal standard: p-hydroxyethyltheophylline (3.7 (A), 4.4 (B))
OTHER SUBSTANCES
Extracted: furosemide, metolazone, amiloride, acetazolamide, chlorothiazide, hydrochlorothia-
zide, quinethazone, triamterene, hydroflumethiazide, chlorthalidone, dichlorphenamide, tri-
chloromethiazide, methyclothiazide, benzthiazide, cyclothiazide, polythiazide, bendroflumethi-
azide, probenecid, spironolactone, canrenone, flumethiazide
Noninterfering: acetaminophen, aspirin, caffeine, diflunisal, fenoprofen, ibuprofen, indometha-
cin, methocarbamol, naproxen, phenylbutazone, sulindac, tetracycline, theobromine, theoph-
ylline, tolmetin, trimethoprim, verapamil
Interfering: bumetanide
REFERENCE
Cooper,S.E; Masse",R.; Dugal,R. Comprehensive screening procedure for diuretics in urine by high-performance
SAMPLE
Matrix: urine
Sample preparation: Make 5 mL urine alkaline (pH 9-10), add 2 g NaCl, extract twice with 6
mL ethyl acetate. Combine the organic layers and evaporate them to dryness under a stream
of nitrogen, reconstitute the residue in 200 JULL MeCN/water, inject a 10-20 JLJLL aliquot.
HPLC VARIABLES
Column: 100 X 4 5 |xm SGE 100 GL-4 C18P (Scientific Glass Engineering)
Mobile phase: MeCN.MeOH.water.trifluoroacetic acid 15:15:70:0.5
Flow rate: 0.8 or 1
Detector: MS, ZAB2-SEQ (VG), PSP source coupled to LC, source 250, probe 240-260, scan m/
z 200-550 or UV 270
CHROMATOGRAM
Retention time: 4.8
Limit of detection: 50 ng (by MS)
OTHER SUBSTANCES
Extracted: probenecid, bumetanide, spironolactone
REFERENCE
Ventura,R.; Fraisse,D.; Becchi,M.; Paisse,O.; Segura,J. Approach to the analysis of diuretics and masking
agents by high-performance liquid chromatography-mass spectrometry in doping control, J.Chromatogr.,
1991, 562, 723-736.
SAMPLE
Matrix: urine
Sample preparation: Buffer urine to 4.9 by mixing with an equal volume of pH 4.9 200 mM
sodium phosphate buffer. Inject a 40 |xL aliquot onto column A with mobile phase A, after 3
min backflush the contents of column A onto column B with mobile phase B and start the
gradient. At the end of the run re-equilibrate for 10 min.
HPLC VARIABLES
Column: A 20 X 4 5 |xm Hypersil octadecylsilica ODS; B 200 X 4.6 5 |xm Shiseido SG-120 poly-
mer-based C18
Mobile phase: A water; B Gradient. MeCN:buffer from 7:93 to 15:85 over 3.5 min, to 50:50 over
8.5 min, maintain at 50:50 for 11 min (Buffer was 6.9 g NaH2PO4.H2O in 1 L water, pH adjusted
to 3.1 with phosphoric acid.)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: acetazolamide, amiloride, bendroflumethiazide, benzthiazide, bumetanide, caffeine,
carbamazepine, chlorothiazide, chlorthalidone, clopamide, dichlorfenamide, furosemide, hy-
drochlorothiazide, metyrapone, probenecid, spironolactone, triamterene, trichlorcnethiazide
KEYWORDS
column-switching; optimum detection wavelengths vary for each drug
REFERENCE
Saarinen,M.; Sir6n,H.; Riekkola,M.-L. A column switching technique for the screening of diuretics in urine by
high performance liquid chromatography, J.Liq.Chromatogr., 1993, 16, 4063-4078.
SAMPLE
Matrix: urine
Sample preparation: 5 mL Urine + 50 |xL 100 jig/mL 7-propyltheophylline in MeOH + 200 |xL
ammonium chloride buffer + 2 g NaCl, extract with 6 mL ethyl acetate by rocking at 40 move-
ments/min for 20 min and centrifuging at 800 g for 5 min, repeat extraction, combine organic
layers, evaporate to dryness at 40 under a stream of nitrogen. Reconstitute in 200 |xL MeCN:
water 15:85 and inject 20 (xL aliquots. (Ammonium chloride buffer was 28 g ammonium chlo-
ride in 100 mL water with the pH adjusted to 9.5 with concentrated ammonia solution.)
HPLCVARIABLES
Mobile phase: Gradient. MeCN: 100 mM ammonium acetate adjusted to pH 3 with concentrated
phosphoric acid. From 10:90 to 15:85 over 2 min to 55:45 over 3 min to 60:40 over 3 min. Kept
at 60:40 for 1 min, decreased to 10:90 over 1 min and equilibrated at 10:90 for 2 min.
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 6.9
Internal standard: 7-propyltheophylline (4.5)
OTHER SUBSTANCES
Simultaneous: xipamide, bumetanide, acetazolamide, amiloride, buthiazide, benzthiazide, can-
renone, caffeine, clopamide, chlorthalidone, diclofenamide, cyclothiazide, furosemide, hydro-
chlorothiazide, mesocarb, morazone, piretanide, probenecid, spironolactone, torsemide,
triamterene
Interfering: polythiazide, bendroflumethiazide
REFERENCE
Ventura,R.; Nadal,T.; Alcalde,R; Pascual,J.A.; Segura,J. Fast screening method for diuretics, probenecid and
other compounds of doping interest, J.ChromatogrA, 1993, 655, 233-242.
SAMPLE
Matrix: urine
Sample preparation: Direct injection into column A with mobile phase A for 1 min then back
flush onto column B with mobile phase B.
HPLC VARIABLES
Column: A 20 X 2.1 30 |xm Hypersil ODS-C18; B 250 X 4 5 |xm Hypersil ODS-C18
Mobile phase: A Water; B Gradient. MeCN:buffer 15:85 for 1.5 min then to 80:20 over 8 min.
Keep at 80:20 for 2.5 min then re-equilibrate with 15:85. (Buffer was 50 mM NaH2PO4 + 1.4
mL propylamine hydrochloride per liter adjusted to pH 3 with concentrated phosphoric acid.)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 10
Limit of detection: 200 ng/mL.
OTHER SUBSTANCES
Simultaneous: acetazolamide, amiloride, bendroflumethiazide, chlorthalidone, cyclothiazide, fu-
rosemide, hydrochlorothiazide, probenecid, spironolactone, triamterene
Interfering: bumetanide
REFERENCE
Campins-Falco,R; Herraez-Hern&ndez,R.; Sevillano-Cabeza,A. Column-switching techniques for screening of
diuretics and probenecid in urine samples, Anal.Chem., 1994, 66, 244-248.
Ethambutol
CAS Registry No.: 74-55-5, 1070-11-7 (di HCI)
Merck Index: 3764
Lednicer No.: 1 222
SAMPLE
Matrix: blood
Sample preparation: Add 100 |xL 8 ng/mL octylamine in MeCN and 100 |xL 4 M NaOH to 100
IxL plasma, extract with 4 mL chloroform (Caution! Chloroform is a carcinogen!), agitate for
10 min, centrifuge at 3000 rpm for 5 min, discard the aqueous phase. Add 100 |xL 400 ng/mL
phenylethylisocyanate in MeCN to the organic layer, mix for 1 min, evaporate to dryness under
a stream of nitrogen, reconstitute the residue with 100 |xL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 30 X 4.6 C18 (Interchim, France)
Column: 150 X 4.6 3 |xm Hypersil C18
Flow rate: 1
Detector: UV 200
CHROMATOGRAM
Retention time: 8.9
Internal standard: octylamine (13.7)
OTHERSUBSTANCES
Noninterfering: amphotericin B, dapsone, didanosine, fluconazole, isoniazid, itraconazole, pyr-
azinamide, pyrimethamine, rifampicin, rifabutine, sparfloxacin, zidovudine
KEYWORDS
derivatization; plasma
REFERENCE
Chenevier,R; Massias,L.; Gueylard,D.; Farinotti,R. Determination of ethambutol in plasma by high-perfor-
mance liquid chromatography after pre-column derivatization, J.Chromatogr.B, 1998, 708, 310-315.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 2 mL 4 M NaOH + 100 uL 1 mg/mL IS + 8 mL chloroform,
agitate for 20 min, centrifuge. Remove the organic layer and evaporate it to dryness under a
stream of nitrogen, reconstitute the residue in 100 |xL MeCN, inject a 50 \xL aliquot.
HPLCVARIABLES
Mobile phase: MeCN:water 50:50 containing 0.04 mM copper sulfate and 2 M ammonia (The
mobile phase was saturated with silica using a 250 X 4.6 column packed with 50 |xm high-
porosity silica (Alltech).)
Flow rate: 1.5
Detector: UV 270
CHROMATOGRAM
Retention time: 5.7
Internal standard: (S,S)-N,N'-bis(hydroxymethyl-l-ethyl)ethylenediamine (7) (Reflux 5 mmoles
1,2-dibromoethane with 50 mmole (S)-2-aminopropan-l-ol at 110 for 25 min, cool to 30, add
12 mmoles potassium in 5 mL propanol, cool in an ice bath, filter, concentrate the filtrate under
reduced pressure, take up the residue in acetone:propanol 1:1, cool in an ice bath, filter, con-
centrate the filtrate under reduced pressure, add 1 mL EtOH, filter off the product, dry the
IS.)
OTHER SUBSTANCES
Noninterfering: isoniazid, pyrazinamide, rifampin
KEYWORDS
plasma; derivatization; complexation; copper complexes
REFERENCE
Lacroix,C; Cerutti,R; Nouveau,J.; Menager,S.; Lafont,O. Determination de Tethambutol plasmatique par chro-
matographie liquide et detection spectrophotome'trique ultraviolette [Determination of ethambutol in
plasma by liquid chromatography and ultraviolet spectrophotometric detection], J.Chromatogr., 1987,415,
85-94.
SAMPLE
Sample preparation: Plasma. 200 |xL Plasma + 300 |xL 5 M NaOH + 5 mL diethyl ether, vortex
for 1 min, centrifuge at 1200 g for 3 min, remove the organic layer, repeat the extraction.
Combine the organic layers and add them to 200 |iL 10 mM phosphoric acid, vortex for 1 min,
centrifuge at 1200 g for 3 min. Remove the aqueous phase and add it to 300 (xL 200 mM pH
7.5 borate buffer, vortex for 10 s, add 40 JJLL 4 mg/mL 4-fluoro-7-nitrobenzo-2-oxa-l,3-diazole
in MeCN, heat at 80 for 30 min, add 50 |xL 1 M phosphoric acid, cool in dry ice/acetone for 1
min, add 2 mL ethyl acetate, vortex for 1 min, centrifuge at 1200 g for 3 min. Discard the
upper organic layer and add 50 |AL 5 M NaOH to the lower aqueous phase, add 2 mL ethyl
acetate:MeOH 90:10, mix for 1 min, centrifuge at 1200 g for 3 min. Remove the upper organic
in 250 |xL 10 mM pH 2.5 phosphoric acid, inject a 200 (xL aliquot. Urine. Dilute 100 |JLL urine
to 20 mL with water. 200 |xL Diluted urine + 300 jxL 200 mM pH 7.5 borate buffer, vortex for
10 s, add 40 |xL 4 mg/mL 4-fluoro-7-nitrobenzo-2-oxa-l,3-diazole in MeCN, heat at 80 for 30
min, add 50 jxL 1 M phosphoric acid, cool in dry ice/acetone for 1 min, add 2 mL ethyl acetate,
vortex for 1 min, centrifuge at 1200 g for 3 min. Discard the upper organic layer and add 50
IxL 5 M NaOH to the lower aqueous phase, add 2 mL ethyl acetate:MeOH 90:10, mix for 1
min, centrifuge at 1200 g for 3 min. Remove the upper organic layer and evaporate it to dryness
under a stream of nitrogen at 37, reconstitute the residue in 1 mL 10 mM pH 2.5 phosphoric
acid, inject a 200 |xL aliquot.
HPLC VARIABLES
Guard column: 37-53 |xm Pellicular ODS (Whatman)
Column: 250 X 4.6 5 |xm Spherisorb CN
Mobile phase: MeCN:buffer 30:70 (Buffer was 10 mM phosphoric acid adjusted to pH 2.5 with
10 M KOH.)
Flow rate: 1
CHROMATOGRAM
Retention time: 14
Limit of quantitation: 10 ng/mL (plasma), 10 jxg/mL (urine)
KEYWORDS
derivatization; plasma; 4-chloro-7-nitrobenzo-2-oxa-l,3-diazole is a less reactive derivatizing
reagent
REFERENCE
Breda,M.; Marrari,R; Pianezzola,E.; Strolin Benedetti,M. Determination of ethambutol in human plasma and
urine by high-performance liquid chromatography with fluorescence detection, J.ChromatogrA, 1996, 729,
301-307.
SAMPLE
Matrix: solutions
Sample preparation: Mix a 2 mL aliquot of a 25 nM (?) solution of ethambutol in benzene
(Caution! Benzene is a carcinogen!) with 5 mL 400 mM (R)-(-)-a-methoxyphenylacetyl chloride
in benzene and 300 |xL pyridine, stir vigorously at 40 for 30 min, pour into 5 mL ether and 5
mL water. Remove the ether layer and wash it with 5 mL 5% HCl, dry over anhydrous mag-
nesium sulfate, filter, evaporate to dryness under reduced pressure, reconstitute with 500 jxL
chloroform, inject a 5 (xL aliquot. (Synthesis of (RM-)-a-methoxyphenylacetyl chloride is as
follows. Reflux (R)-(-)-a-methoxyphenylacetic acid with a 5 to 10-fold excess of thionyl chloride
on a steam bath for 5 min, add benzene, evaporate under reduced pressure, repeat this pro-
cedure 3 or 4 times to remove excess thionyl chloride, distil at 70-7570.2 mm Hg to give (R)-
(-)-a-methoxyphenylacetyl chloride (J. Org. Chem. 1968, 33, 1142).)
HPLCVARIABLES
Column: 250 X 4 5 |xm LiChrosorb Si 60
Mobile phase: Chloroform:ethyl acetate 90:10
Flow rate: 0.7
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 9 (-), 9.5 (+), 10.2 (meso)
OTHER SUBSTANCES
Simultaneous: 2-amino-l-butanol
KEYWORDS
derivatization; normal phase; chiral
REFERENCE
Gamberini,G.; Ferioli,V. Determination of optical purity by high performance liquid chromatography of com-
pounds of pharmaceutical interest, Farmaco./TratJ., 1988, 43, 357-363.
SAMPLE
Matrix: solutions
Sample preparation: Mix 1 mL of a 16-320 |xg/mL solution in pH 4.4 KH2PO4 buffer with 100
JJLL 1.32 mg/mL L-glycine in KH2PO4, inject a 20 u,L aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 u.m Chiral-SilOO D-VaICu (Serva)
Mobile phase: 50 mM pH 4.4 KH2PO4 containing 1 mM copper(II) sulfate
Flow rate: 0.3
Detector: UV 260
CHROMATOGRAM
Retention time: 12.7 (+), 14.2 (meso)
Internal standard: L-glycine
Limit of detection: 1 fxg/mL
OTHER SUBSTANCES
Simultaneous: impurities, 2-amino-l-butanol
KEYWORDS
derivatization; complexation; chiral; D-enantiomer is not separated from L-enantiomer but they
are separated from meso-form
REFERENCE
Ferioli,V.; Gamberini,G.; Rustichelli,C; Vezzalini,F. Direct determination of non-UV-absorbing compounds by
high-performance liquid chromatography, Farmaco, 1994, 49, 411-413.
Ethaverine
Merck Index: 3773
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 15 (xL 2 |xg/mL papaverine HCl in MeOH + 200 |xL 4 M
NaOH + 5 mL diethyl ether, vortex for 5 min, centrifuge at 2000 g for 10 min, remove the
organic layer, extract the aqueous layer with 2 mL diethyl ether, centrifuge. Combine the
organic layers and add them to 500 (xL 1 M HCl, vortex for 1 min, centrifuge at 2000 g for 10
min. Remove the aqueous layer and add it to 500 jxL 4 M NaOH, vortex for 1 min, centrifuge
nitrogen at 37, reconstitute the residue in 25 |xL MeOH, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 |jim Partisil 10 ODS
Mobile phase: MeOH:0.1% KH2PO4 65:35
Flow rate: 2
Detector: UV 238
CHROMATOGRAM
Retention time: 8.5
Internal standard: papaverine (3.5)
KEYWORDS
REFERENCE
Brodie,R.R.; Chasseaud,L.R; Walmsley,L.M.; Soegtrop,H.H.; DarraghA; O'Kelly,D.A. Determination of the an-
tispasmodic agent ethaverine in human plasma by high-performance liquid chromatography, J.Chromatogr.,
1980, 182, 379-386.
SAMPLE
Sample preparation: 1 mL Plasma or urine + 200 (xL water + 100 (plasma) or 500 (urine) fiL
500 mM pH 8.5 TRIS-HCl buffer + 2 g NaCl + 5 mL butyl chloride:isopropanol 95:5, vortex for
15 s, centrifuge at 3000 g for 10 min, repeat the extraction. Combine the organic layers and
evaporate them to dryness in a vortex-evaporator at 45, reconstitute the residue in 50 (xL
mobile phase, inject an aliquot.
HPLC VARIABLES
Mobile phase: EtOH:water:methanesulfonic acid 500:25:0.5 (plasma) or 500:10:0.25 (urine)
Flow rate: 1.2
Detector: UV 270
CHROMATOGRAM
Internal standard: ethaverine
OTHER SUBSTANCES
Extracted: encainide
Simultaneous: quinidine
Noninterfering: acetaminophen, alprazolam, aspirin, cephalexin, chloral hydrate, diazepam, di-
goxin, dipyridamole, docusate, flurazepam, furosemide, hydrochlorothiazide, ibuprofen, isosor-
bide dinitrate, lidocaine, lorazepam, meclizine, mexiletine, nifedipine, norfloxacin, oxazepam,
ranitidine, tocainide, triamterene, triazolam
KEYWORDS
plasma; ethaverine is IS
REFERENCE
Turgeon,J.; Funck-Brentano,C; Gray,H-T.; Roden,D.M. Improved high-performance liquid chromatographic as-
say for encainide and its metabolites in human body fluids, J.Chromatogr., 1989, 490, 165-174.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 fxm Supelco C18
Mobile phase: MeCNrbuffer 70:30 (Buffer contained 2.88% sodium lauryl sulfate and 1.248%
NaH2PO4 adjusted to pH 3 with orthophosphoric acid.)
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: moxaverine, drotaverine, papaverine, codeine
REFERENCE
Girgis,E.H. Ion-pair reversed-phase liquid chromatographic identification and quantitation of papaverine con-
geners, J.Pharm.ScL, 1993, 82, 503-505.
Ethchlorvynol
Molecular formula: C7H9CIO
Merck Index: 3774
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Serum + 200 jxL 50 |xg/mL hexobarbital in MeCN + 25 jxL glacial
acetic acid, vortex for 10 s, centrifuge for 1 min, inject a 30-100 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: (xBondapak C18
Mobile phase: Gradient. MeCN:7.5 g/L NaH2PO4 adjusted to pH 3.2 with phosphoric acid 5:95
to 22:78 over 24 min, to 45:55 over 10 min, maintain at 45:55 for 5 min. Re-equilibrate with
5:95 for 5 min.
Flow rate: 3
Detector: UV 210
CHROMATOGRAM
Internal standard: hexobarbital (20.6)
OTHER SUBSTANCES
Extracted: acetaminophen, amobarbital, butabarbital, butalbital, chlordiazepoxide, diazepam,
flurazepam, glutethimide, methaqualone, methyprylon, nitrazepam, pentobarbital, phenobar-
bital, phenytoin, primidone, salicylic acid, secobarbital, theophylline
Simultaneous: amitriptyline, caffeine, clomipramine, codeine, desipramine, ethotoin, imipra-
mine, lidocaine, methsuximide, nirvanol, nortriptyline, oxazepam, procainamide, phenylpro-
panolamine, propranolol, quinidine
Interfering: mesantoin
KEYWORDS
serum
REFERENCE
Kabra,P.M.; Stafford,B-E.; Marton,L.J. Rapid method for screening toxic drugs in serum with liquid chroma-
tography, JAnal.Toxicol., 1981, 5, 177-182.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 6-10 u,L aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 Supelguard LC-I (Supelco)
Column: 250 X 4.6 5 ^m Supelcosil LC-I (Supelco)
Mobile phase: MeOH.MeCN.buffer 17.5:17.5:65 (Buffer was 2.72 g KH2PO4 in 1.9 L water, pH
adjusted to 6.3 with about 2 mL 1 M NaOH, made up to 2 L.)
Flow rate: 2
Detector: UV 204
CHROMATOGRAM
Internal standard: 5-ethyl-5-p-tolybarbituric acid (tolybarb) (4.80)
OTHER SUBSTANCES
Simultaneous: acetaminophen, acetanilide, barbital, butabarbital, butalbital, caffeine, carba-
mazepine, chloramphenicol, cimetidine, cyheptamide, diazoxide, diflunisal, disopyramide, eth-
chlorvynol, ethosuximide, glutethimide, heptabarbital, hexobarbital, ibuprofen, indomethacin,
ketoprofen, mefenamic acid, mephenytoin, methaqualone, methyl salicylate, methyprylon, na-
proxen, nirvanol, oxphenylbutazone, phenacetin, phenobarbital, phensuximide, phenylbuta-
zone, phenytoin, primidone, salicylamide, secobarbital, sulindac, theophylline, thiopental,
tolmetin
Noninterfering: N-acetylcysteine, N-acetylprocainamide, amikacin, ampicillin, aspirin, chlor-
propamide, codeine, diphylline, gentamicin, gentisic acid, meprobamate, morphine, netilmicin,
quinidine, salicylic acid, sulfamethoxazole, tetracycline, tobramycin, trimethoprim, valproic
acid, vancomycin
Interfering: methsuximide, procainamide, mephobarbital, pentobarbital
REFERENCE
Meatherall,R.; Ford,D. Isocratic liquid chromatographic determination of theophylline, acetaminophen, chlo-
ramphenicol, caffeine, anticonvulsants, and barbiturates in serum, Ther.Drug Monit, 1988, 10, 101-115.
Ethinyl estradiol
Merck Index: 3780
Lednicer No.: 1 162
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 500 |xL 10 M NaOH, shake on a slow rotatory mixer for
5 min, add 5 mL diethyl ether, rotomix 10 min, centrifuge at 700 g for 5 min, repeat extraction.
Combine organic layers, evaporate to dryness under a stream of nitrogen at 37, dissolve in
250 |xL mobile phase, inject aliquot.
HPLC VARIABLES
Column: 150 X 3.9 4 jxm Novapack C18
Mobile phase: MeCN:MeOH.buffer 35:15:50 (Buffer was 50 mM KH2PO4 adjusted to pH 3.6 with
phosphoric acid.)
Flow rate: 1.6
Detector: E, Waters Model 464 pulsed electrochemical detector, + 1 V versus Ag/AgCl
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: estriol, estradiol, estrone, heparin
Noninterfering: pentobarbital
KEYWORDS
plasma; rabbit
REFERENCE
Fernandez,N.; Garcia,J.J.; Diez,M.J.; Teran,M.T.; Sierra,M. Rapid high-performance liquid chromatographic
assay of ethynyloestradiol in rabbit plasma, J.Chromatogr., 1993, 619, 143-147.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 10 |xL IS in water, extract twice by shaking for 1 min
with 1.2 mL dichloromethane, evaporate organic layer below 40 under reduced pressure, dis-
solve residue in 100 |xL MeCN. Add 10 JXL reagent 1, add 10 |xL reagent 2, heat at 50 for 15
min, cool to room temperature, add 100 |xL water, add 200 jxL MeOH:water 1:1, add to Sep-
Pak C18 cartridge, wash vial with 2 mL MeOH:water 1:1 and add washings to cartridge, wash
cartridge with 40 mL MeOH:water 1:1, elute with 5 mL MeOH. Concentrate eluent to 500 |xL
by evaporation at 40 under reduced pressure, inject 20 (xL aliquot. (Reagent 1 was 30 mg 2-
(4-carboxvphenyl)-5,6-dimethylbenzimidazole in 3 mL pyridine, add 700 mg 4-piperidinopyri-
dine, dilute to 10 mL with MeCN. Reagent 2 was 700 mg l-isopropyl-3-(3-dimethylaminopro-
pyl)carbodiimide perchlorate in 10 mL MeCN.)
HPLC VARIABLES
Guard column: 50 X 4 5 |xm Wakosil 5C18
Column: 300 X 4 5 |xm Wakosil 5C18
Flow rate: 0.7
CHROMATOGRAM
Internal standard: sec-butyl p-hydroxybenzoate (14.3)
Limit of detection: 1-2 pg/mL
OTHER SUBSTANCES
Simultaneous: estriol, estradiol, equilin, equilenin, estrone, estetrol, 4-hydroxyestradiol, 2-
hydroxyestradiol
KEYWORDS
plasma; equilin and equilenin not resolved
REFERENCE
Katayama,M.; Taniguchi,H. Determination of estrogens in plasma by high-performance liquid chromatography
after pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole, J.Chromatogr., 1993,
616, 317-322.
SAMPLE
Matrix: blood, perfusate
Sample preparation: 200 |xL Plasma or perfusate + 5 mL dichloromethane, vortex, centrifuge
for 10 min. Remove a 4.5 mL aliquot of the lower organic layer and evaporate it to dryness
under a stream of nitrogen, reconstitute the residue in 200 |xL mobile phase, inject a 50 |xL
aliquot.
HPLC VARIABLES
Column: LiChrocart 100 RP-18
Mobile phase: MeOH:isopropanol:dichloromethane:water 40:9:4:47
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Internal standard: ethinyl estradiol (17a-ethynylestradiol)
OTHER SUBSTANCES
Extracted: digoxin
KEYWORDS
plasma; rat; ethinyl estradiol is IS
REFERENCE
Su,S.-R; Huang,J.-D. Inhibition of the intestinal digoxin absorption and exsorption by quinidine, Drug Me-
tab.Dispos., 1996, 24, 142-147.
SAMPLE
Sample preparation: 5 Tablets + 2 glass beads + 25 mL 50 |xg/mL dibutyl phthalate in MeOH,
vortex 15 min or until tablets have completely disintegrated, sonicate 5 min, filter (2 |xm),
HPLC VARIABLES
Column: 50 X 4.5 5|xm IBM C18
Mobile phase: MeOHiTHF:water 10:25:65
Flow rate: 2.1
Detector: UV 230
CHROMATOGRAM
Retention time: 3.5
Internal standard: dibutyl phthalate
OTHER SUBSTANCES
Simultaneous: norgestimate, degradation products
KEYWORDS
tablets; stability-indicating
REFERENCE
Lane,P.A.; Mayberry,D.O.; Young,R.W. Determination of norgestimate and ethinyl estradiol in tablets by high-
performance liquid chromatography, J.Pharm.ScL, 1987, 76, 44-47.
SAMPLE
Matrix: solutions
Sample preparation: Extract 15 mL water with dichloromethane, evaporate organic layer, take
up residue in 3 mL mobile phase, inject 50 |xL aliquot.
HPLC VARIABLES
Column: reverse phase
CHROMATOGRAM
Internal standard: mestranol
REFERENCE
de Leede,L.G.J.; Govers,C.P.M.; de Nijs,H. A multi-compartment vaginal ring system for independently ad-
justable release of contraceptive steroids, Contraception, 1986, 34, 589-602.
Ethionamide
Molecular formula: C8H10N2S
Merck Index: 3783
Lednicer No.: 1 255
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL octadecyl SPE cartridge (J.T. Baker) with one volume
of MeOH and one volume of water. 200 |xL Serum + 4 |xL 200 jxg/mL prothionamide in MeCN:
20 mM Na2HPO4 50:50, vortex for 5 s, add to the SPE cartridge, wash with 400 JJLL water, wash
with 50 |xL MeOH:water 90:10, dry under vacuum for 15 min, elute with 1 mL MeOH. Evap-
orate the eluate under nitrogen at 40, reconstitute in 200 |xL MeCN:20 mM Na2HPO4 25:75,
vortex for 5 s, inject a 20 (JLL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 5 (xm Hypersil ODS
Mobile phase: MeCN:20 mM Na2HPO4 25:75
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Internal standard: prothionamide (7.24)
OTHER SUBSTANCES
Noninterfering: acetaminophen, p-aminosalicylic acid, aspirin, amikacin, capreomycin, etham-
butol, isoniazid, kanamycin, pyrazinamide, streptomycin, rifampin
Interfering: phenobarbital
KEYWORDS
serum; SPE
REFERENCE
Peloquin,C.A.; James,G.T.; McCarthy,E. Improved high-performance liquid chromatographic assay for the de-
termination of ethionamide in serum, J.Chromatogr., 1991, 563, 472-475.
SAMPLE
Matrix: blood, CSF
Sample preparation: 200 |xL Serum, plasma, or CSF + 300 |xL reagent. Flush column A to
waste with 500 |xL 500 mM ammonium sulfate, inject sample onto column A, flush column A
to waste with 500 |xL 500 mM ammonium sulfate, elute the contents of column A onto column
B with mobile phase, monitor the effluent from column B. (Reagent was 8.05 M guanidine
hydrochloride and 1.02 M ammonium sulfate in water.)
HPLC VARIABLES
Column: A 30 X 2.1 40 |xm preparative grade C18 (Analytichem); B 250 X 4.6 10 |xm Partisil
C8
Mobile phase: Gradient. A was 50 mM pH 4.5 KH2PO4. B was MeCNdsopropanol 80:20. A:B 90:
10 for 1 min, to 30:70 over 15 min, maintain at 30:70 for 4 min.
Flow rate: 1.5
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: acetazolamide, ampicillin, bromazepam, caffeine, carbamazepine, chloramphenicol,
chlorothiazide, diazepam, droperidol, furosemide, isoniazid, methadone, penicillin G, pheno-
barbital, phenytoin, prazepam, propoxyphene, pyrazinamide, rifampin, trimeprazine,
trimethoprim
KEYWORDS
serum; plasma; column-switching
REFERENCE
Seifart,H.L; Kruger,P.B.; Parkin,D.P.; van Jaarsveld,P.R; Donald,P.R. Therapeutic monitoring of antitubercu-
losis drugs by direct in-line extraction on a high-performance liquid chromatography system, J.Chromatogr.,
1993, 619, 285-290.
Ethopabate
Merck Index: 3791
SAMPLE
Matrix: meat
Sample preparation: Homogenize (Ultra-Turrax TP 18/10) 3 g tissue with 1 mL water and 4
mL acetone for 6 s, centrifuge at 5000 rpm for 3 min. Transfer 4 mL supernatant, add 5 mL
dichloromethane, mix for 5 s, centrifuge at 3000 rpm for 3 min. Evaporate the organic layer
to dryness under a stream of nitrogen at 60. Dissolve residue in 500 jxL MeOH:buffer 70:30,
place in freezer at -20 for 5 min. Filter (Costar Spin-X with 0.2 |xm nylon membrane) while
centrifuging at 5600 g for 3 min. Inject a 20 |xL aliquot of the filtrate. (Prepare buffer by
dissolving 4.45 g sodium heptanesulfonate and 1.8 g Na2HPO4.2H2O in 750 mL water, adjust
pH to 6.3 with 5 M phosphoric acid, adjust pH to 6.0 with 1 M phosphoric acid, make up to 1
L with water.)
HPLC VARIABLES
Guard column: 20 X 4.6 5 |xm Supelcosil LC-ABZ+Plus
Column: 250 X 4.6 5 jxm Supelcosil LC-ABZ+Plus
Flow rate: 0.8
CHROMATOGRAM
Retention time: ca. 8.3
Limit of quantitation: 1 ng/g
KEYWORDS
chicken meat
REFERENCE
Hormazabal,V.; Yndestad,M. Rapid assay for the determination of residues of amprolium and ethopabate in
chicken meat by HPLC, J.Liq.Chromatogr.Rel.Technol, 1996, 19, 2517-2525.
Ethopropazine
Merck Index: 3793
Lednicer No.: 1 373
SAMPLE
Matrix: blood
Sample preparation: Add IS and 300 jxL MeCN to 100 |xL plasma, vortex, centrifuge for 2 min.
Remove the supernatant and add it to 300 |xL pH 5.9 sodium phosphate buffer and 3 mL hexane
and vortex for 45 s. Centrifuge at 250Og for 3 min. Evaporate the supernatant under a stream
of nitrogen and reconstitute with mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 100 X 4.6 ODS
Mobile phase: MeCN:MeOH:25 mM potassium phosphate 25:25:50 containing 0.75 mIVL 2 M
sulfuric acid and 0.25 mIVL triethylamine
Flow rate: 1.0
Detector: UV 250
CHROMATOGRAM
Retention time: 7.7
Internal standard: orphenadrine (5.7)
Limit of quantitation: 12.5 mg/mL
KEYWORDS
plasma; rat; pharmacokinetics
REFERENCE
Padovani,P.K; Timby,D.M.; Wright,M.R.; Kapil,R.P. Quantitative analysis of DMP 851 in rat and dog plasma
by liquid-liquid extraction and reverse-phase high performance liquid chromatography with ultraviolet de-
tection (Abstract 3318), Pharm.Res., 1997, 14, S568.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 3.1
OTHER SUBSTANCES
idine, cinchonidine, cinnarizine, clemastine, elomipramine, clonidine, cocaine, cyclazocine, cy-
gosine, ergosinine, ergotamine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol,
fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, fiurazepam, haloperidol, hy-
REFERENCE
Jane,I.; McKinnon,A.; Flanagan,R-J- High-performance liquid chromatographic analysis of basic drugs on silica
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1 mg/mL solution in MeOH, inject a 5 JULL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 \im Lichrosphere cyanopropyl
Mobile phase: Carbon dioxide.MeOH.isopropylamine 90:10:0.05
Flow rate: 3
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: triflupromazine, carphenazine, methotrimeprazine, promazine, perphenazine,
chlorprothixene, thiothixene, reserpine, acetophenazine, promethazine, propiomazine,
deserpidine
Interfering: methotrimeprazine
KEYWORDS
SFC; pressure 200 bar
REFERENCE
Berger,T.A.; Wilson,W.H. Separation of drugs by packed column supercritical fluid chromatography. 1. Pheno-
thiazine antipsychotics, J.Pharm.Sci., 1994, 83, 281-286.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 |im Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
oxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, encainide,
ethidium bromide, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flur-
biprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatro-
pine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hy-
droxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac, labetalol,
levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic acid,
meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone, methdila-
zine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyldopa, methyl-
phenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam, moclobemide,
morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizatidine, nor-
epinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, pentaz-
ocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital, phenol, phe-
nolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenytoin, pimozide, pindolol,
lactone, sulfinpjnâzone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophyl-
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: Make up a 500 ng/mL solution in mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm KK-CARNU (a-R-naphthyl)ethylurea (YMC)
Mobile phase: Hexane:l,2-dichloroethane:EtOH:trifluoroaceticacid 400:150:100:1
Flow rate: 1
Detector: F ex 254 em 280 (filter)
CHROMATOGRAM
Retention time: 10.94, 12.37 (enantiomers)
KEY WORDS
chiral
REFERENCE
Ponder,G.W.; Butram,S.L.; Adams,A-(j.; Ramanathan,C.S.; Stewart,J.T. Resolution of promethazine, ethopro-
pazine, trimeprazine and trimipramine enantiomers on selected chiral stationary phases using high-per-
formance liquid chromatography, J.Chromatogr.A, 1995, 692, 173-182.
Ethosuximide
Merck Index: 3794
Lednicer No.: 1 228
SAMPLE
Matrix: blood
Sample preparation: Mix 500 jxL plasma with 500 |JLL MeCN and 2 |xg IS for 30 s, centrifuge
at 2700 g for 5 min, inject an aliquot of the supernatant.
HPLC VARIABLES
Column: 150 X 4.6 5 \im Ultrasphere C18
Mobile phase: MeCN:MeOH:10 mM pH 7.4 phosphate buffer 15:35:50
Flow rate: 1
Detector: UV 219
CHROMATOGRAM
Internal standard: 2-hydroxy-2-ethyl-2-phenylacetamide(17)
OTHER SUBSTANCES
Extracted: carbamazepine, clonazepam, D,L-2-hydroxy-2-ethyl-2-phenylpropionamide (HEPP),
phenobarbital, phenytoin, primidone
KEYWORDS
rat; plasma
REFERENCE
Martinez de Muiioz,D.; Arenas,R; Chavez Gonzalez,O. Liquid chromatographic assay in plasma of one of the
members of a new series of anticonvulsants: D,L-3-hydroxy-3-ethyl-3-phenylpropionamide, J. Chromatogr.B,
1996, 678, 377-383.
SAMPLE
Matrix: blood
Sample preparation: Inject a 5 (xL aliquot of serum directly.
HPLC VARIABLES
Column: 100 X 4.6 5-10 \im Silicalite (by sieving Silicalite, 3M Co.(?))
Mobile phase: MeNC:20 mM pH 6.9 phosphate buffer 10:90
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: methamphetamine, sulfamethoxazole, primidone
KEYWORDS
serum
REFERENCE
Ambrose,D.L.; Fntz,J.S. High-performance liquid chromatographic determination of drugs and metabolites in
human serum and urine using direct injection and a unique molecular sieve, J.Chromatogr.B, 1998, 709,
89-96.
SAMPLE
Matrix: blood
Sample preparation: Add 200 JJLL 2 |xg/mL thymol in MeCN to 200 |xL serum, vortex for 10 s,
centrifuge at 7000 g for 5 min, inject 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 Resolve C18-5 (Waters)
Mobile phase: MeCN:isopropanol:50 mM pH 3.0 phosphate buffer 25:15:60
Flow rate: 0.7
Detector: UV 220
CHROMATOGRAM
Retention time: 2.3
Internal standard: thymol (18.5)
OTHER SUBSTANCES
Extracted: primidone, phenobarbital, phenytoin, carbamazepine, valproic acid
KEYWORDS
human; plasma
REFERENCE
Kondo,K.; Nakamura,M.; Nishioka,R.; Kawai,S. Direct method of determination of valproic acid in serum by
high performance liquid chromatography, Anal.ScL, 1985, 1, 385-387.
SAMPLE
Matrix: blood
Sample preparation: 500 IJLL Plasma + 100 |xL heptabarbital in MeOH + 500 |xL 400 mM pH
7.0 sodium phosphate buffer + 10 mL ethyl acetate, extract. Evaporate the extract to dryness
at 50, reconstitute the residue in 20 |xL MeOH, inject a 3 |xL aliquot.
HPLC VARIABLES
Guard column: 50 X 2.1 Whatman Co:Pell ODS
Column: 125 X 4.5 5 |xm SAS Hypersil
Mobile phase: MeCNrbuffer 20:80 (Buffer was 5 mM tetrabutylammonium hydroxide adjusted
to pH 7.5 with phosphoric acid.)
Flow rate: 1.6
Injection volume: 3
Detector: UV 200
CHROMATOGRAM
Retention time: 3.4
Internal standard: heptabarbital (9.8)
Limit of quantitation: 16 |xM
OTHER SUBSTANCES
Extracted: primidone, phenobarbital, pheneturide, carbamazepine, phenytoin
Simultaneous: phenylethylmalonamide, sulthiame, sulfamethoxazole, ethotoin, butabarbital,
pentobarbital, methsuximide, cyclobarbital, ethylphenacemide, amobarbital, glutethimide,
secobarbital
Interfering: barbital
KEYWORDS
plasma; horse
REFERENCE
Christofides,J.A.; Fry,D.E. Measurement of anticonvulsants in serum by reversed-phase ion-pair liquid chro-
matography, Clin.Chem., 1980, 26, 499-501.
SAMPLE
Matrix: blood
Sample preparation: 200 (xL Serum or plasma + 200 |xL 20 |xg/mL IS in MeOH:water 10:90 +
75 IJLL glacial acetic acid, vortex for 30 s, add 5 mL chloroform, shake for 5 min, centrifuge at
2000 rpm for 5 min. Remove the organic layer and evaporate it to dryness under a stream of
nitrogen, reconstitute the residue in 200 |xL mobile phase, inject a 40 |xL aliquot.
HPLC VARIABLES
Guard column: 30 X 2.1 Permaphase ETH (DuPont)
Column: 250 X 4.6 CLC 1 C8 (DuPont)
Mobile phase: MeCN.buffer 35:65 (Buffer was 20 mM KH2PO4 and 1 mM K2HPO4 adjusted to
pH 5.6.)
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
Retention time: 2.3
Internal standard: alphenal (5-allyl-5-phenylbarbituric acid) (4.4)
OTHER SUBSTANCES
Extracted: phenytoin, carbamazepine, primidone, phenobarbital
Simultaneous: amobarbital, chlordiazepoxide, codeine, cortisol, ethotoin, glutethimide, hexobar-
bital, mephenytoin, mephobarbital, metharbital, methsuximide, nitrazepam, pentobarbital,
phenacetin, phensuximide, secobarbital
Noninterfering: acetaminophen, acetazolamide, amphetamine, bilirubin, caffeine, diazepam, di-
menhydrinate, meperidine, meprobamate, methamphetamine, methaqualone, methylpheni-
date, nicotine, propoxyphene, theophylline, valproate
KEYWORDS
plasma; serum
REFERENCE
Rydzewski,R.S.; Gadsden,R.H.; Phelps,C.A. Simultaneous rapid HPLC determination of anticonvulsant drugs
in plasma and correlation with EMIT, Ann.Clin.Lab.ScL, 1980, 10, 89-94.
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Serum + 50 JJLL 7 |xg/mL IS in water + 1 mL buffer, vortex for 10
s, add 5 mL n-hexane:ether:n-propanol 49:49:2, shake gently for 20 min, centrifuge at 1000 g
for 5 min, repeat the extraction. Combine the organic layers and evaporate them to dryness
under a stream of nitrogen at 50, reconstitute the residue in 300 JJLL mobile phase, inject a
50-100 |xL aliquot. (Buffer was 10 mM sodium acetate: 10 mM acetic acid 88.5:11.5, pH 5.5.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Partisil 5 ODS-3
Mobile phase: MeCN:buffer 28:72 (Buffer was 300 |xL 1 M KH2PO4 and 50 jxL 900 mM phos-
phoric acid in 1.8 L water, pH 4.4.)
Flow rate: 2.8
Detector: UV 195
CHROMATOGRAM
Retention time: 1.8
Internal standard: 5-(4-methylphenyl)-5-phenylhydantoin(11.5)
OTHER SUBSTANCES
Extracted: carbamazepine, phenytoin, secobarbital
Simultaneous: mephobarbital, paramethadione, phenobarbital
Noninterfering: chlorazepate, clonazepam, diazepam, thioridazine, valproic acid
Interfering: primidone
KEY WORDS
serum
REFERENCE
Levine,H.L.; Cohen,M.E.; Duffner,RK; Kustas,K.A.; Shen,D.D. An improved high-pressure liquid chromato-
graphic assay for secobarbital in serum, J.Pharm.Sci., 1982, 71, 1281-1283.
SAMPLE
Matrix: blood
Sample preparation: 400 |xL Serum or plasma + 400 fxL 10 jxg/mL IS in acetone, vortex for 10
s, centrifuge at 4500-5000 g for 1 min, remove the supernatant to another tube, centrifuge for
30 s, inject a 5-7.5 JJLL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:buffer 17:28:55, final pH 6.8-7.0 (Buffer was 400 |xL 1 M KH2PO4
in 1 L water, pH adjusted to 6.0 with 900 mM phosphoric acid.)
Flow rate: 0.7
Injection volume: 5-7.5
Detector: UV 195
CHROMATOGRAM
Retention time: 6.8
Internal standard: tolybarb (5-ethyl-5-(p-methylphenyl)barbituric acid) (13.8)
OTHER SUBSTANCES
Extracted: carbamazepine, N-desmethylmethsuximide, phenobarbital, phenytoin, primidone
Simultaneous: acetaminophen, butalbital, caffeine, hexobarbital, methsuximide, phenacetin,
phenylethylmalonamide, salicylic acid
KEYWORDS
plasma; serum
REFERENCE
Szabo,G.K; Browne,T.R. Improved isocratic liquid-chromatographic simultaneous measurement of phenytoin,
phenobarbital, primidone, carbamazepine, ethosuximide, and N-desmethylmethsuximide in serum,
Clin.Chem., 1982, 28, 100-104.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Serum + 50 fxL 10 jig/mL IS in MeCN, vortex for 10 s, centrifuge
at 3000 g for 1 min, remove the supernatant and place it in another tube, centrifuge for 1 min,
inject a 20 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: 100 X 8 5 jxm Nova Pak C18 Radial pak
Mobile phase: MeCN:MeOH:acetone:buffer 8:21:10:61 adjusted to pH 7.95 0.02 with NaOH
(Buffer was 1.36 g/L KH2PO4.)
Flow rate: 2.8
Detector: UV 200
CHROMATOGRAM
Internal standard: tolybarb (5-ethyl-5-(p-methylphenyl)barbituric acid) (4.89)
OTHER SUBSTANCES
Extracted: primidone, phenobarbital, carbamazepine, phenytoin, metabolites
Simultaneous: acetaminophen, N-acetylprocainamide, aspirin, ampicillin, caffeine, cephapirin,
chloramphenicol, digoxin, disopyramide, hexobarbital, indomethacin, lidocaine, mephobarbital,
methsuximide, nafcillin, pentobarbital, phenylethylmalonamide, procainamide, quinidine, sa-
licylic acid, secobarbital, sulfamerazine, sulfamethazine, terbutaline, tetracycline, theobromine,
theophylline
Noninterfering: acetazolamide, amikacin, cephalosporin C, gentamicin, propranolol, sulfadia-
zine, sulfamethoxazole, sulfisoxazole, tobramycin, valproic acid, verapamil
KEYWORDS
serum
REFERENCE
Ou,C.-N.; Rognerud,C.L. Simultaneous measurement of ethosuximide, primidone, phenobarbital, phenytoin,
carbamazepine, and their bioactive metabolites by liquid chromatography, Clin.Chem., 1984, 30, 1667-
1670.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Serum + 100 |xL buffer + 1.5 mL IS in 5% isopropanol in chloro-
form, vortex for 30 s, centrifuge. Remove the organic layer and evaporate it to dryness under
a stream of air at room temperature, reconstitute the residue in 100 |xL mobile phase, inject
a 6-10 uL aliquot. (Buffer was 13.6 g KH2PO4 in 90 mL water, pH adjusted to 6.8 with about
3 mL 10 M NaOH, made up to 100 mL.)
HPLC VARIABLES
Column: 250 X 4.6 5 pjn Supelcosil LC-I (Supelco)
Mobile phase: MeOH:MeCN:buffer 17.5:17.5:65 (Buffer was 2.72 g KH2PO4 in 1.9 L water, pH
Flow rate: 2
Detector: UV 204
CHROMATOGRAM
Internal standard: methsuximide (5.30)
OTHER SUBSTANCES
Extracted: acetaminophen, amobarbital, caffeine, carbamazepine, chloramphenicol, mephobar-
bital, methsuximide, pentobarbital, phenobarbital, phenytoin, primidone, secobarbital, theoph-
ylline, thiopental
Also analyzed: acetanilide, N-acetylcysteine, N-acetylprocainamide, ampicillin, aspirin, buta-
barbital, butalbital, chlorpropamide, codeine, cyheptamide, diazoxide, diflunisal, diphylline, di-
sopyramide, ethchlorvynol, gentisic acid, glutethimide, heptabarbital, hexobarbital, ibuprofen,
indomethacin, ketoprofen, mefenamic acid, mephenytoin, methaqualone, methyl salicylate,
methyprylon, morphine, naproxen, nirvanol, oxphenylbutazone, phenacetin, phensuximide,
phenylbutazone, procainamide, salicylamide, salicylic acid, sulfamethoxazole, sulindac, tolme-
tin, trimethoprim, vancomycin
Noninterfering: amikacin, gentamicin, meprobamate, netilmicin, quinidine, tetracycline, tobra-
mycin, valproic acid
Interfering: barbital, cimetidine
KEYWORDS
serum
REFERENCE
ramphenicol, caffeine, anticonvulsants, and barbiturates in serum, Ther.Drug Monit, 1988, 10, 101115.
SAMPLE
Matrix: blood
Sample preparation: Prepare an SPE cartridge by plugging the end of a 1 mL disposable pipette
tip with glass wool and adding about 100 mg Chromosorb P/NAW. Add 50 |xL plasma then 50
jxL 10 |xg/mL tolylphenobarbital in 200 mM HCl to the SPE cartridge, let stand for 2 min,
elute with 1 mL chloroform:isopropanol 6:1. Evaporate the eluate to dryness under a stream
of nitrogen at 30, reconstitute the residue in 100 |JLL mobile phase, inject a 15 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 jim Supelcosil-LC-8
Flow rate: 3.3
Detector: UV 208
CHROMATOGRAM
Internal standard: tolylphenobarbital (7.57)
OTHER SUBSTANCES
Extracted: theophylline, caffeine, primidone, carbamazepinediol, phenacemide, methyprylon,
nirvanol, phenobarbital, chloramphenicol, butabarbital, carbamazepine epoxide, mephenytoin,
pentobarbital, amobarbital, carbamazepine, glutethimide, phenytoin, secobarbital, meth-
aqualone
Noninterfering: acetaminophen, amikacin, amitriptyline, clonazepam, cyclospor-
ine,desipramine, diazepam, digoxin, disopyramide, gentamicin, imipramine, lidocaine, metho-
trexate, N-acetylprocainamide, netilmicin, nortriptyline, procainamide, quinidine, salicylic
acid, sulfamethoxazole, tobramycin, trimethoprim, valproic acid, p-hydroxyphenobarbital,
vancomycin
KEY WORDS
plasma; SPE
REFERENCE
Svinarov,D.A.; Dotchev,D.C. Simultaneous liquid-chromatographic determination of some bronchodilators, an-
ticonvulsants, chloramphenicol, and hypnotic agents, with Chromosorb P columns used for sample prepa-
ration, Clin.Chem., 1989, 35, 1615-1618.
SAMPLE
Matrix: blood
Sample preparation: 400 /xL Plasma + 100 jxL water, mix for 10 s, add 1 mL isopropanol, vortex
for 30 s, centrifuge at 1800 g for 5 min. Remove a 1 mL aliquot of the supernatant and add it
to 300 |xL 10 mM NaOH, evaporate to dryness under a stream of nitrogen at 50, add 200 |xL
2.5 mM 4-(bromomethyl)-7-methoxycoumarin in MeCN, add 300 |xL of a solution of 2,2'-dini-
trobiphenyl in MeCN, add 100 mg potassium carbonate, shake at 70 for 1.5 h, inject a 15 jxL
aliquot.
HPLC VARIABLES
Column: 150 X 3.9 4 (xm Nova-Pak C18
Mobile phase: MeCN:MeOH:water 20:20:60
Flow rate: 1.3
Detector: UV 320
CHROMATOGRAM
Retention time: 10
Internal standard: 2,2'-dinitrobiphenyl (14)
Limit of detection: 7 pmole
OTHER SUBSTANCES
Noninterfering: acetazolamide, carbamazepine, phenobarbital, primidone, valproic acid
KEY WpRDS
REFERENCE
Chen,S.-H.; Wu,H.-L.; Wu,J.-K; Kou,H.-S.; Wu,S.-M. Determination of ethosuximide in plasma by derivatiza-
tion and high performance liquid chromatography, J.Liq.Chromatogr.Rel.TechnoL, 1997, 20, 1579-1589.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 ^m Symmetry C8 (Waters)
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Mix 1 mL microsomal incubation with 500 mg/mL ice-cold 200 mg/mL
trichloroacetic acid solution and 500 |xL 200 (xg/mL IS, centrifuge at 9600 g for 10 min. Filter
the supernatant (0.2 (xm nylon syringe filter), inject a 20 |JLL aliquot.
HPLCVARIABLES
Column: 150 X 3.9 C18 NovaPak
Mobile phase: Gradient. MeCN:water from 3:97 to 35:65 over 6.4 min, return to initial condition
over 0.6 min, re-equilibrate for 9 min
Flow rate: 2.0
Detector: UV 195
CHROMATOGRAM
Retention time: 6.7
Internal standard: 3,3-dimethylglutarimide (6.2)
OTHER SUBSTANCES
KEYWORDS
liver; rat
REFERENCE
Sarver,J.G.; Bachmann,K.A.; Zhu,D.; Klis,W.A. Ethosuximide is primarily metabolized by CYP3A when incu-
bated with isolated rat liver microsomes, Drug Metab.Dispos., 1998, 26, 78-82.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 3.2 7 (xm SI 100 ODS (not commercially available)
Mobile phase: MeCN:buffer 31.2:68.8 (Buffer was 6.66 g KH2PO4 and 4.8 g 85% phosphoric acid
in 1 L water, pH 2.3.)
Flow rate: 0.5-1
CHROMATOGRAM
Retention time: 2.0
OTHER SUBSTANCES
Also analyzed: aspirin, caffeine, carbamazepine, chlordiazepoxide, chlorprothixene, clonazepam,
diazepam, doxylamine, furosemide, haloperidol, hydrochlorothiazide, methocarbamol, metho-
trimeprazine, nicotine, oxazepam, procaine, promazine, propafenone, propranolol, salicylamide,
temazepam, tetracaine, thiopental, triamterene, verapamil, zolpidem, zopiclone
REFERENCE
Below,E.; Burrmann,M. Application of HPLC equipment with rapid scan detection to the identification of drugs
in toxicological analysis, J.Liq.Chromatogr., 1994, 17, 4131-4144.
SAMPLE
Matrix: solutions
Next Page
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
apram, doxepin, dronabinol, ephedrine, epinephrine, epinine, estradiol, estriol, estrone,
puromycin, pjnilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
faethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapjrridine, sul-
metin, tranylc^romine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine,
REFERENCE
Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.Toxicol., 1994,
18, 233-242.
Previous Page
Ethotoin
Merck Index: 3795
Lednicer No.: 1 245
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL water + 1 mL phenytoin solution + 5 mL 100 mM
trisodium phosphate + 10 mL ether, extract, centrifuge. Discard the ether layer and adjust the
pH of the aqueous phase to 6.8 with 400 (JLL 2 M HCl, extract with 10 mL ether, centrifuge.
Remove the organic layer and evaporate it to dryness, reconstitute the residue in 250 JXL
MeCN, inject a 10 (xL aliquot.
HPLC VARIABLES
Column: 300 X 4 jjiBondapak C18
Mobile phase: MeCN:10 mM pH 4.4 sodium phosphate 30:70
Flow rate: 2.2
Detector: UV 195
CHROMATOGRAM
Retention time: 2.5
Internal standard: phenytoin (6.3)
KEYWORDS
REFERENCE
Meyer,M.C; Holcombe,BJ; Burckart,G.J.; Raghow,G.; Yau,M.K. Nonlinear ethotoin kinetics,
Clin.Pharmacol.Ther., 1983, 33, 329-334.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Serum + 1 mL saturated NaCl solution + 1 mL chloroform, vortex
for 10 s, centrifuge. Remove the organic layer and evaporate it to dryness under vacuum at
50, reconstitute the residue in 50 jxL EtOH, inject a 25 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Chiralcel CA-I
Mobile phase: EtOHiwater 95:5
Flow rate: 0.5
Detector: UV 254
CHROMATOGRAM
Retention time: 11.9 (d), 14.6 (1)
OTHER SUBSTANCES
Simultaneous: primidone, phenytoin
Interfering: carbamazepine, phenobarbital
KEYWORDS
serum; chiral; pharmacokinetics
REFERENCE
Inotsume,N.; Fujii,J.; Honda,M.; Nakano,M.; Higashi,A.; Matsuda,I. Stereoselective analysis of the enantiomers
of ethotoin in human serum using chiral stationary phase liquid chromatography and gas chromatography-
mass spectrometry, J.Chromatogr., 1988, 428, 402-407.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 3 |xL aliquot of a MeOH solution.
HPLC VARIABLES
Column: 125 X 4.5 5 jjim SAS Hypersil
Mobile phase: MeCN:bufifer 20:80 (Buffer was 5 mM tetrabutylammonium hydroxide adjusted
Flow rate: 1.6
Injection volume: 3
Detector: UV 200
CHROMATOGRAM
Retention time: 6.7
Internal standard: heptabarbital (9.8)
OTHER SUBSTANCES
Simultaneous: amobarbital, barbital, butabarbital, carbamazepine, cyclobarbital, ethotoin,
ethylphenacemide, glutethimide, methsuximide, pentobarbital, pheneturide, phenobarbital,
phenylethylmalonamide, phenytoin, primidone, secobarbital, sulfamethoxazole, sulthiame
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 100 X 4.6 3 |xm 208HS3410 (Vydac)
Mobile phase: Gradient. MeCN:water from 15:85 to 60:40 over 10 min.
Flow rate: 1.5
Detector: UV 210 (?)
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
Simultaneous: barbital, carbamazepine, diazepam, mephenytoin, methsuximide, phenacemide,
phenobarbital, phensuximide
REFERENCE
Vydac HPLC Catalog, 1994, p. 26.
Ethylmorphine
CAS Registry No.: 76-58-4, 6746-59-4 (HCI, dihydrate),
6696-59-9 (methiodide)
Merck Index: 3876
LednicerNo.: 1 287
SAMPLE
Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA)5 add
residue with 50 (xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
a 10 (urine) or 30 (blood) (xL aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
Column: 250 X 4.6 5 fim Symmetry C8 (Waters)
mL/min)
Detector: UV 211.1
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Ethynodiol
CAS Registry No.: 1231-93-2, 297-76-7 (diacetate)
Merck Index: 3905
LednicerNo.: 1 165
SAMPLE
Sample preparation: Powder tablets (60 mesh), weigh out amount equivalent to one tablet, add
2 mL 50 [Lg/mL BHT in MeCNrwater 80:20, shake 30 min, centrifuge
HPLC VARIABLES
Column: 250 X 3.2 Altex RP-2 express series
Flow rate: 1.75
CHROMATOGRAM
Retention time: k' 22.65 (ethynodiol diacetate)
Internal standard: BHT (butylated hydroxytoluene) (k' 16.54)
OTHER SUBSTANCES
Simultaneous: mestranol, ethinyl estradiol, degradation products
KEYWORDS
tablets
REFERENCE
Carignan,G.; Lodge,B-A.; Skakum,W. Quantitative analysis of ethynodiol diacetate and ethinyl estradiol/mes-
tranol in oral contraceptive tablets by high-performance liquid chromatography, J.Pharm.ScL, 1982, 71,
264-266.
Etidocaine
Merck Index: 3907
Lednicer No.: 2 95
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL 1 M NaOH, vortex for 15 s, add 5 mL diethyl
ether, shake on a reciprocating shaker for 10 min, centrifuge. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 100 JJLL mobile
phase, inject an 80 |xL aliquot.
HPLCVARIABLES
Column: 150 X 3.2 10 \xm ixBondapak C18
Mobile phase: MeCN:50 mM pH 5.80 Na2HPO4 25:75
Flow rate: 0.9
Detector: UV 210
CHROMATOGRAM
Internal standard: etidocaine
OTHER SUBSTANCES
Extracted: 2,6-pipecolylxylidine, lidocaine, bupivacaine, mepivacaine
Noninterfering: metabolites, 2,3-chloroprocaine, theophylline, mexiletine, quinidine, disopyr-
amide, verapamil, phenobarbital, phenytoin, carbamazepine, ethosuximide, digoxin, theobro-
mine, caffeine, furosemide, phenprocoumon, aldactone
KEYWORDS
plasma; etidocaine is IS
REFERENCE
Ha,H--R; Funk,B-; Gerber,H.R.; Follath,F. Determination of bupivacaine in plasma by high-performance liquid
chromatography, AnesthAnalg., 1984, 63, 448-450.
SAMPLE
Matrix: blood
Sample preparation: Condition a diol AASP SPE cartridge (Jones Chromatography) with 1 mL
MeOH, 1 mL water, 1 mL mobile phase, 0.5 mL MeOH, 0.5 mL toluene. 200 |xL Plasma + 100
|xL 100 mM K2HPO4, mix, add 1 mL toluene, vortex for 1 min, centrifuge at 12000 g for 1 min.
Add 750 |xL of the toluene layer to the SPE cartridge, wash with 500 |xL MeCN, elute the
contents of the SPE onto the column with mobile phase (MeCN used for purge (6 strokes) and
afterwash (10 strokes), valve reset time 2 min).
HPLC VARIABLES
Guard column: 20 X 2 30-40 |xm Co:Pell ODS
Column: 150 X 4.6 Spherisorb 5 CN
Mobile phase: MeCN:water 40:60 containing 10 mM phosphoric acid
Flow rate: 1
Detector: E, Environmental Science Associates Model 5100A Coulochem, screen mode, electrode
1 +0.7 V, electrode 2 +0.9 V, palladium reference electrode, guard cell +1.2 V (before injection
valve)
CHROMATOGRAM
Internal standard: etidocaine
OTHER SUBSTANCES
Extracted: prilocaine
KEYWORDS
plasma; SPE; etidocaine is IS
REFERENCE
Whelpton,R.; Dudson,P.; Cannell,H.; Webster,K. Determination of prilocaine in human plasma samples using
high-performance liquid chromatography with dual-electrode electrochemical detection, J.Chromatogr.,
1990, 526, 215-222.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 uX. 1 M NaOH + 3 mL heptanerethyl acetate 90:10,
shake for 2 min, centrifuge at 1200 g for 10 min. Remove the organic phase and add it to 50
IxL 50 mM sulfuric acid, shake for 2 min, centrifuge at 1200 g for 5 min. Remove the aqueous
phase and add it to 820 |xg sodium acetate, inject a 40 |xL aliquot. (The sodium acetate was
measured out by adding 50 JJLL 200 mM sodium acetate in MeOH to the tube and evaporating
the MeOH.)
HPLC VARIABLES
Column: 250 X 4 10 |xm ixBondapak C18
Mobile phase: MeCNrIO mM NaH2PO4 20:80, adjusted to pH 2.1
Flow rate: 1
Detector: UV 205
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Extracted: bupivacaine
KEYWORDS
plasma; rabbit
REFERENCE
Le Guevello,R; Le Corre,R; Chevanne,R; Le Verge,R- High-performance liquid chromatographic determination
of bupivacaine in plasma samples for biopharmaceutical studies and application to seven other local an-
aesthetics, J.Chromatogr., 1993, 622, 284-290.
SAMPLE
Sample preparation: 2 mL Whole blood, plasma, or urine + 1 mL saturated sodium carbonate
+ 20 |JLL 100 (xg/mL dibucaine, add to a 3 mL Extrelut SPE cartridge, elute with 15 mL di-
chloromethane. Evaporate eluate to dryness under a stream of nitrogen at 40, reconstitute in
100 |xL 10 mM HCl, add 3 mL diethyl ether, vortex for 20 s, centrifuge at 2800 g for 5 min,
inject a 40 jxL aliquot of the aqueous layer.
HPLC VARIABLES
Guard column: 5 X 6 ixBondapak Guard Pak
Column: 300 X 3.9 10 |xm |jiBondapak C18
Mobile phase: MeCN: 100 mM ammonium acetate 50:50
Flow rate: 1.5
Detector: UV 230
CHROMATOGRAM
Retention time: 14
Internal standard: dibucaine (18)
OTHER SUBSTANCES
Extracted: prilocaine, lidocaine, bupivacaine,
Also analyzed: procaine, butacaine, tetracaine, p-aminobenzoic acid, articaine, o-toluidine, caf-
feine, amphetamine, ephedrine, epinephrine, morphine, monoacetylmorphine, diamorphine,
ethylmorphine, codeine, acetylcodeine
KEYWORDS
whole blood; plasma; SPE
REFERENCE
Rop,P.R; Grimaldi,R; Bresson,M.; Fornaris,M.; Viala,A- Liquid chromatographic analysis of cocaine, benzoyl-
ecgonine, local anaesthetic agents and some of their metabolites in biological fluids, J.Liq. Chromatogr.,
1993, 16, 2797-2811.
Etidronic acid
Molecular formula: C2H8O7P2
CAS Registry No.: 2809-21-4, 7414-83-7 (disodium salt)
Merck Index: 3908
SAMPLE
Sample preparation: Dilute with water to a concentration of 400 jxg/mL, inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 75 X 4.6 6 |xm IC-Pak HR anion-exchange (Waters)
Mobile phase: 7.2 mM nitric acid:7.2 mM potassium nitrate 40:60, pH 2.7
Flow rate: 0.8
Detector: UV 240
CHROMATOGRAM
Retention time: 8.5
KEYWORDS
injections; indirect UV detection; rugged
REFERENCE
Tsai,E.W.; Chamberlin,S.D.; Forsyth,R.J.; BeIl9C; Ip,D.R; Brooks,M.A. Determination of bisphosphonate drugs
in pharmaceutical dosage formulations by ion chromatography with indirect UV determination,
J.Pharm.Biomed.Anal, 1994, 12, 983-991.
SAMPLE
Sample preparation: Dilute injections 100-fold, inject a 20 fxL aliquot. Disintegrate a 5 mg
tablet in 100 mL water, sonicate for 5 min, centrifuge an aliquot at 3600 g for 4 min, inject a
HPLC VARIABLES
Column: 150 X 4.6 10 jxm IC-PAK Anion HC (Waters)
Mobile phase: 1.5 mM Nitric acid containing 0.5 mM copper(II) nitrate (Prepare column by
pumping ILC Regenerant A (Waters) and 100 mM nitric acid for 30 min.)
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Retention time: 5.8
OTHER SUBSTANCES
Simultaneous: alendronate, clodronate, neridronate, olpadronate, pamidronate
KEY WpRDS
derivatization; complexation; injections; tablets
REFERENCE
Sparidans,R.W.; Den Hartigh,J.; Vermeij,P. High-performance ion-exchange chromatography with in-line com-
plexation of bisphosphonates and their quality control in pharmaceutical preparations, J.Pharm.
BiomedAnaL, 1995,13, 1545-1550.
Etizolam
Molecular formula: C17H15CIN4S
Merck Index: 3919
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 20 ^L 20 |xg/mL IS + 200 |xL 1 M potassium carbonate
Evaporate the organic phase under a stream of nitrogen at 40. Dissolve the residue in 100 jxL
mobile phase, inject a 20 |xL aliquot. (Caution! Chloroform is a carcinogen!)
HPLC VARIABLES
Flow rate: 0.65
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: bromazepam, chlordiazepoxide, clonazepam, estazolam, flutazolam, haloxazolam, lor-
KEYWORDS
serum
REFERENCE
serum using a new reversed-phase chromatographic column on a 2-jxm porous microspherical silica gel,
J.Chromatogr.B, 1996, 682, 173-178.
SAMPLE
Matrix: urine
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 5 mL MeOH, 10 mL di-
chloromethane :MeOH 9:1, 5 mL MeOH, and 10 mL water. Condition a Sep-Pak Silica SPE
cartridge with 20 mL dichloromethane, 20 mL dichloromethane :MeOH 9:1, and 20 mL dich-
loromethane, then air dry. 10 mL Urine adjusted to pH 5 with acetic acid, add 2.5 mL 500 mM
pH 5.0 acetate buffer, add 3000 U p-glucuronidase, incubate at 37 for 24 h, make alkaline
with ammonia, centrifuge at 1200 g for 15 min, add the supernatant to the C18 SPE cartridge,
wash with 5 mL water, wash with 5 mL MeOH:water 20:80, wash with 2 mL water, elute with
7 mL dichloromethane:MeOH 9:1. Evaporate the eluate to dryness under vacuum, dissolve the
residue in 5 mL dichloromethane :MeOH 99:1, add to the silica SPE cartridge, wash with 20
mL dichloromethane, wash with 25 mL dichloromethane:MeOH 99:1, elute with 20 mL dich-
loromethane:MeOH 9:1. Evaporate the eluate to dryness under vacuum, dissolve the residue
in 100 |JLL mobile phase, inject a 20 uL aliquot. (MeOH for silica SPE cartridge was distilled
and dried over 3 A molecular sieve, then 0.1% water added just before use.)
HPLCVARIABLES
Column: 100 X 8 10 jjim Radial-Pak C18
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 17
Internal standard: etizolam
OTHER SUBSTANCES
Simultaneous: triazolam
KEYWORDS
SPE; etizolam is IS
REFERENCE
Inoue,T.; Suzuki,S.-I. High-performance liquid chromatographic determination of triazolam and its metabolites
in human urine, J.Chromatogr., 1987, 422, 197-204.
Etodolac
Merck Index: 3920
SAMPLE
residue with 50 fxL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 225.2
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y.; Pe*pin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
163.
SAMPLE
Sample preparation: Centrifuge cell suspension at 2000 g for 4 min. Remove a 2 mL aliquot of
the supernatant and add it to 200 JULL 100 jjug/mL IS in DMF, mix, add 200 JULL 5 M HCl, extract
twice with 3 mL portions of toluene. Combine the organic layers and evaporate them to dryness
under a stream of nitrogen, reconstitute the residue in 1 mL dichloromethane, add 20 JULL 10
mg/mL 1-hydroxybenzotriazole in dichloromethane:pyridine 99:1, add 300 |xL 10 mg/mL l-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in dichloromethane, add 300 jxL 10
mg/mL (-)-(S)-a-methylbenzylamine in dichloromethane, let stand for 30 min, evaporate to dry-
ness, reconstitute with 500 jxL mobile phase, inject a 10 jxL aliquot.
HPLCVARIABLES
Guard column: 10 mm long Techsphere ODS (HPLC Technology, Macclesfield UK)
Column: 250 X 5 5 |xm Techsphere ODS (HPLC Technology, Macclesfield UK)
Mobile phase: MeCN:7.5 mM NaH2PO4 65:35, containing 5 mM sodium pentanesulfonate, pH
adjusted to 2.8 with phosphoric acid
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: (R)-ibuprofen (k' 5.21)
Limit of detection: 1 (xg/mL
KEYWORDS
REFERENCE
Thomason,M.J.; Hung,Y.-F.; Rhys-Williams,W.; Hanlon,G.W.; LloydA-W. Indirect enantiomeric separation of 2-
arylpropionic acids and structurally related compounds by reversed phase HPLC, J.Pharm.Biomed.AnaL,
1997, 25, 1765-1774.
Etonitazene
Merck Index: 3929
Lednicer No.: 1 325
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
ethacrynic acid, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fen-
proporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymester-
one, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, gly-
benclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone,
puromycin, p3rrilamine, p^thyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
18, 233-242.
Etoposide
CAS Registry No.: 33419-42-0, 117091-64-2 (phosphate)
Merck Index: 3931
SAMPLE
Matrix: blood
Sample preparation: Mix 500 |xL serum, pleural effusion, or urine with 100 |xL IS solution and
400 |xL pH 9.0 ammonium acetate. Add to an Extrelut-1 SPE cartridge. After 15 min, elute
with 3 mL and 4 mL portions of dichloromethane. Evaporate the eluate to dryness under a
stream of nitrogen at 40, reconstitute the residue with 100 |xL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 (xm LiChrospher 100 CN
Mobile phase: MeCN: 100 mm pH 3.5 acetate buffer 30:70
Flow rate: 1
CHROMATOGRAM
Retention time: 4.5
Internal standard: BRL 43693A (Smith Kleine Beecham) (9.5)
OTHER SUBSTANCES
Extracted: granisetron
Also analyzed: domperidone, metoclopramide, ondansetron
Noninterfering: cisplatin, carboplatin, dexamethasone
KEYWORDS
serum; SPE; pharmacokinetics
REFERENCE
Wada,L; Satoh,M.; Takeda,T.; Nakabayashi,T.; Honma,T.; Saitoh,H.; Takada,M.; Hirano,K. A rapid assay of
granisetron in biological fluids from cancer patients, Biol.Pharm.Bull., 1998, 21, 535537.
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 500 mg Bond-Elut PH SPE cartridge with 6 mL MeOH
and 3 mL 20 mM pH 5.5 ammonium acetate. Mix 500 |xL serum with 500 jxL 20 mM pH 5.5
ammonium acetate, 50 |xL 760 mM sodium dodecyl sulfate and 50 |xL 800 ng/mL IS in MeOH.
Add to the SPE cartridge, wash with 3 mL 20 mM ammonium acetate and 3 mL MeOH:water
10:90. Elute with 2 mL MeOH, evaporate to dryness at 43 under reduced pressure, reconstitute
in 150 ^L MeOH:water 36:64.
HPLCVARIABLES
Column: 300 X 3.9 Bondclone 10 C18 (Phenomenex, Torrance, CA, USA)
Mobile phase: MeOH:40 mM pH 6.9 KH2PO4:0.14 mM 1-heptanesulfonic acid 40:60:0.6
Flow rate: 2
CHROMATOGRAM
Retention time: 14
Internal standard: podofilox (28)
KEYWORDS
REFERENCE
Manouilov,K.K.; McGuire,T.R.; Gordon,B-Cr.; Gwilt,P.R. Assay for etoposide in human serum using solid-phase
extraction and high-performance liquid chromatography with fluorescence detection, J.Chromatogr.B, 1998,
707, 342-346.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 200 jxL 500 ng/mL 2-acetamidophenol in water.
Add 4 mL 500 mM pH 7.2 NaH2PO4, add with 5 mL ethyl etheridichloromethane 2:1, vortex
for 1 min. Centrifuge at 4500 rpm for 5 min. Evaporate the organic layer to dryness under a
stream of nitrogen at 45, reconstitute with 200 juiL mobile phase, inject a 100 JULL aliquot.
HPLC VARIABLES
Guard column: 40 X 4 35-50 jjim Corasil C18
Mobile phase: MeOH:75 mM pH 3.8 acetate buffer 45:55
Flow rate: 1
Detector: E, Bioanalytical Systems LC-4B, glassy carbon electrode +800 mV, Ag/AgCl reference
electrode
CHROMATOGRAM
Retention time: 5.6
Internal standard: 2-acetomidophenol (3.0)
KEYWORDS
REFERENCE
Pe*rez-Urizar,J.; Picazo,Y.E; Navarro-Gonzalez,B-; Flores-Murrieta,F.J.; Castaneda-Hernandez,G. A new rapid
and economical high performance liquid chromatographic assay with electrochemical detection for the de-
termination of etoposide (VP-16) in human plasma samples, J.Liq.Chromatogr.Rel.TechnoL, 1996,19, 939-
947.
SAMPLE
Matrix: blood
Sample preparation: Filter 1 mL plasma (Centrifree micropartition device, molecular mass cut-
off 30000, Amicon, USA) using a 33 fixed angle centrifuge (Beckman ModelG56R) at 2000 g
and 25 for 30 min. To the ultrafiltrate add 50 jiL 10 |xg/mL teniposide in MeOH and 1 mL
chloroform (Caution! Chloroform is a carcinogen!), agitate slowly for 20 min, centrifuge at 1000
g for 5 min. Evaporate the organic phase to dryness under vacuum at 40, dissolve the dry
extract in 50 u-L MeOH, inject a 25 (xL aliquot. Alternatively, add 10 |xg teniposide and 8 mL
chloroform (Caution! Chloroform is a carcinogen!) to 1 mL plasma, agitate slowly for 20 min,
centrifuge at 1000 g for 5 min, evaporate the organic phase to dryness, dissolve the dry extract
in 100 |xL MeOH, inject a 25 jxL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 fxm 125 A ixBondapak Phenyl (Waters)
Mobile phase: MeCN.water.glacial acid 35:64:1
Flow rate: 1
CHROMATOGRAM
Retention time: 6.5
Internal standard: teniposide (18)
OTHER SUBSTANCES
Noninterfering: alizapride, doxorubicin, furosemide, idarubicin, ranitidine, vinblastine,
vinorelbine
KEYWORDS
REFERENCE
Robieux,L; Aita,R; Sorio,R-; Toffoli,G.; Boiocchi,M. Determination of unbound etoposide concentration in ul-
trafiltered plasma by high-performance liquid chromatography withfluorimetricdetection, J.Chromatogr.B,
1996, 686, 35-41.
SAMPLE
HPLC VARIABLES
Column: 150 X 3.9 5 (xm Symmetry C8
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Simultaneous: idarubicin
KEYWORDS
0.9% NaCl; injections
REFERENCE
Zhang,H.; Ye,L.; Stewart, J.T. HPLC determination of idarubicin-etoposide and idarubicin-ondansetron mixtures
in 0.9% sodium chloride injection USP, J.Liq.Chromatogr.Rel.TechnoL, 1998, 21, 979-988.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 |xm cyano
Mobile phase: MeCN:20 mM sodium acetate 26:74, pH adjusted to 4.0 with acetic acid
F l o w rate: 1
Detector: UV 290
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: granisetron (UV 300)
KEYWORDS
REFERENCE
Mayron,D.; Gennaro,A.R. Stability and compatibility of granisetron hydrochloride in i.v. solutions and oral
liquids and during simulated Y-site injection with selected drugs, Am. J.Health-Syst.Pharm., 1996,53,294-
304.
Etorphine
Merck Index: 3932
Lednicer No.: 2 321
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 (xL aliquot.
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.4
OTHER SUBSTANCES
gosine, ergosinine, ergotamine, ethopropazine, etoxeridine, fenethazine, fenfluramine, feno-
thipendyl, protriptyline, proxjmoLetacaine, pseudoephedrine, pyrimethamine, quinidine, qui-
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
ethacrynic acid, ethosuximide, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen,
fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymes-
terone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide,
glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocor-
tisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, in-
domethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ke-
tamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine,
mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol,
mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine,
thioridazine, thiosalicylic acid, thiothixene, th3nnol, tolazamide, tolazoline, tobutamide, tol-
REFERENCE
18, 233-242.
Etoxeridine
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 [ig/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.3
OTHER SUBSTANCES
gosine, ergosinine, ergotamine, ethopropazine, etorphine, fenethazine, fenfluramine, fenoterol,
methoprim, trimipramine, tripelennamine, triprolidine, trj^ptamine, verapamil, xylometazoline
REFERENCE
Jane,Io*, McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica
Etretinate
Merck Index: 3935
SAMPLE
Matrix: bile, blood, perfusate, tissue
Sample preparation: Homogenize 1 g tissue and 4 mL ice-cold pH 7.4 Krebs-Henseleit buffer.
Dilute bile with an equal volume of 200 mM pH 5 sodium acetate buffer. 100 (xL Plasma,
perfusate, diluted bile, or tissue homogenate + 20 |xL MeCN + 350 JULL MeCN:l-butanol 50:50
+ 20 JiL 37.6 |xg/mL retinyl acetate, vortex for 1 min, add 300 |xL 1 g/mL K2HPO4 in water,
vortex for 30 s, centrifuge at 13600 g for 3 min, inject a 200 |xL aliquot of the organic layer.
(Hydrolyze conjugates in bile as follows. 100 jxL Diluted bile + 8 |xL 100000 U/mL p-glucuron-
idase (Helix pomatia, Sigma), heat at 37 for 5 h.)
HPLC VARIABLES
Guard column: 10 mm long Supelcosil LC-18 guard column
Mobile phase: Gradient. A was MeCN.buffer 20:80. B was MeCN. A:B 65:35 for 10 min, 31:69
for 17 min (step gradient), re-equilibrate for 6 min. (Buffer was 0.8 g ammonium acetate and
10 mL glacial acetic acid in 200 mL water.)
Flow rate: 1.5
Detector: UV 350
CHROMATOGRAM
Internal standard: retinyl acetate (25.0)
OTHERSUBSTANCES
Extracted: acitretin, cis-acitretin, metabolites
KEYWORDS
rat; liver; plasma; protect from light
REFERENCE
Decker,M.A.; Zimmerman,C.L. Simultaneous determination of etretinate, acitretin and their metabolites in
perfusate, perfusate plasma, bile or hepatic tissue with reversed-phase high-performance liquid chroma-
tography, J.Chromatogr.B, 1995, 667, 105-113.
SAMPLE
Matrix: blood
Sample preparation: 0.5-2 mL Plasma + 100 |xL pH 7 phosphate buffer + 2 mL diethyl ether:
ethyl acetate 50:50, vortex gently for 5 min, centrifuge at 2000 g for 10 min. Remove the organic
layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 30-
100 (xL MeOH, inject a 25 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeOH: 1% aqueous acetic acid 85:15
Flow rate: 1.5
Detector: UV 350
CHROMATOGRAM
Retention time: 32
OTHER SUBSTANCES
Extracted: 13-cis-acitretin, tretinoin, 4-oxo-13-cis-retinoic acid, isotretinoin, acitretin
Noninterfering: antidepressants, benzodiazepines, psoralen
KEYWORDS
plasma; handle under yellow light
REFERENCE
Bun,H.; al-Mallah,N.R.; Aubert,C; Cano,J.P. High-performance liquid chromatography of aromatic retinoids
and isotretinoin in biological fluids, Methods Enzymol, 1990, 189, 167-172.
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Plasma + 10 jxL 9.8 jxg/mL retinyl palmitate in MeOH + 1.5 mL
EtOH + 500 |JLL 2 M HCl, vortex for 30 s, add 5 mL water, vortex for 30 s, add 7.5 mL n-
hexane, rotate for 15 min, centrifuge at 1500 g for 6 min. Remove the organic layer and evap-
orate it to dryness under a stream of nitrogen, reconstitute the residue in 150 |xL mobile phase,
inject a 50 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm CP-Spher Si 5 um (Chrompack)
Mobile phase: Dichloromethane:acetic acid 99.8:0.2 (?)
Detector: UV 350
CHROMATOGRAM
Retention time: 6.5
Internal standard: retinyl palmitate (6)
OTHER SUBSTANCES
Extracted: 13-cis acitretin, all-trans-acitretin
KEYWORDS
plasma; normal phase; pharmacokinetics; protect from light
REFERENCE
De Leenheer,A.R; Lambert,W.E.; De Bersaques,J.R; Kint,A.H. High-performance liquid chromatographic de-
termination of etretinate and all-trans- and 13-cis-acitretin in human plasma, J.Chromatogr., 1990, 500,
637-642.
SAMPLE
Matrix: blood
Sample preparation: 200 JLJLL Plasma + 50 JULL 1 juig/mL retinoic acid in methyl acetate + 500 (xL
pH 7.4 phosphate buffer + 200 (JLL methyl acetate + 4 mL diethyl ether, rotate at 20 rpm for
10 min, centrifuge at 2000 g for 5 min. Remove the organic layer and evaporate it to dryness
under a stream of nitrogen, reconstitute the residue in 200 |xL mobile phase. Inject a 150 (xL
aliquot.
HPLC VARIABLES
Column: 250 mm long 5 jxm LiChrosorb Si 60
Mobile phase: Hexane:methyl benzoate:propionic acid 375:25:1
Flow rate: 2
Detector: UV 365
CHROMATOGRAM
Retention time: 3.2
Internal standard: retinoic acid (5.2)
OTHER SUBSTANCES
Extracted: acitretin, isoacitretin
KEYWORDS
plasma; rat; normal phase; pharmacokinetics
REFERENCE
McNamara,P.J.; Blouin,R-A. Pharmacokinetic profile of two aromatic retinoids (etretinate and acitretin) in the
obese Zucker rat, J.Pharm.ScL, 1990, 79, 301-304.
SAMPLE
Matrix: blood
Sample preparation: 500 u,L Plasma + 1 mL 100 ng/mL Ro 12-7554 and 100 ng/mL isotretinoin
in EtOH, vortex, stand at 4 for 15 min, centrifuge at 1800 g for 3 min, inject a 500 |xL aliquot
onto column A with mobile phase A and elute for 7 min, elute column A in backflush mode with
mobile phase A for 3 min, backflush contents of column A onto column B with mobile phase B
and start the gradient for mobile phase B. At the end of the process flush the lines with
component B of mobile phase B, re-equilibrate columns for 4 min. (Keep sample at 20 in the
autosampler.)
HPLC VARIABLES
Column: A 14 X 4.6 37-50 (xm Bondapak C18 Corasil (column fitted with 3 jxm sieves not glass
fiber filters); B 30 X 4 5 jim Spherisorb ODS 1 + 125 X 4 5 |xm Spherisorb ODS 1
Mobile phase: A MeCN: 1% ammonium acetate 10:90; B Gradient. A was MeCN:water: 10% am-
monium acetate:acetic acid 600:400:4:30. B was MeCN:water: 10% ammonium acetate:acetic
acid 850:146:4:10. A:B 100:0 to 0:100 over 8 min, stay at 0:100 for 11 min.
Detector: UV 360
CHROMATOGRAM
Retention time: 21
Internal standard: Ro 12-7554 (ethyl all-trans-9-(2,6-dichloro-4-methoxy-m-tolyl)-3,7-dimethyl-
2,4,6,8-nonatetraenoate) (24) and isotretinoin (17)
Limit of detection: 0.5-1 ng/mL
OTHER SUBSTANCES
Simultaneous: acitretin, 13-cis-acitretin, metabolites
KEYWORDS
REFERENCE
Wyss,R. Determination of retinoids in plasma by high-performance liquid chromatography and automated
column switching, Methods EnzymoL, 1990, 189, 146-155.
SAMPLE
Sample preparation: 100 JXL Culture media + 200 |xL ice-cold EtOH, mix thoroughly, let stand
for 15 min, centrifuge at 12000 g for 15 min, inject an aliquot of the supernatant.
HPLC VARIABLES
Guard column: Whatman CO:PELL ODS guard column
Column: 100 X 8 5 |xm Nova-Pak C18 (radial-packed)
Mobile phase: MeOH: 100 mM pH 7.0 ammonium acetate 90:10
Flow rate: 1
Detector: UV 340
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: isotretin, motretinid, acitretin, all-trans-retinoic acid, retinal, Vitamin A (retinol)
REFERENCE
Kochhar,D.M.; Penner,J.D.; Minutella,L.M. Biotransformation of etretinate and developmental toxicity of etre-
tin and other aromatic retinoids in teratogenesis bioassays, Drug Metab.Dispos., 1989, 17, 618-624.
SAMPLE
Matrix: perfusate
Sample preparation: 500 |xL Perfusate + 1 mL acetone + 20 |xL 26 jjug/mL retinyl acetate, vortex
for 1 min, centrifuge at 4 at 1300 g for 15 min, inject an aliquot of the supernatant.
HPLC VARIABLES
Guard column: LC-18 pellicular (Supelco)
Column: 150 X 4.6 5 |xm Supelcosil C18
Mobile phase: MeCN:water 84:16 containing 0.8 g/L ammonium acetate and 10 mL/L glacial
acetic acid
Flow rate: 1.5
Detector: UV 350
CHROMATOGRAM
Internal standard: retinyl acetate
OTHER SUBSTANCES
Extracted: acitretin
KEYWORDS
do not use PTFE or plastic; rat
REFERENCE
Pithavala,Y.K.; Odishaw,J.L.; Han,S.; Wiedmann,T.S.; Zimmerman,C.L. Retinoid absorption from simple and
mixed micelles in the rat intestine, J.Pharm.ScL, 1995, 84, 1360-1365.
Eugenol
Merck Index: 3944
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
ethacrynic acid, ethosuximide, etonitazene, famotidine, fenbendazole, fencamfamine, fenopro-
fen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluox-
ymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, gluteth-
imide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone,
puromycin, p3ailamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
Hill,D.W.; Kind,A-J- Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnalToxicol, 1994,
18, 233-242.
Exifone
Merck Index: 3958
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 1 mL 100 mM citric acid + 8 mL diethyl ether,
shake for 10 min, centrifuge at 4 at 2400 g for 5 min. Remove the organic layer and evaporate
it to dryness under a stream of nitrogen, reconstitute the residue in 200 |xL mobile phase,
inject a 20 |xL aliquot. Urine. 1 mL Urine + 1 mL pH 7 phosphate buffer (Normadose, Prolabo)
+ 8 mL diethyl ether, shake for 10 min, centrifuge at 4 at 2400 g for 5 min. Remove the organic
layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 250
|xL mobile phase, inject a 20 (xL aliquot.
HPLCVARIABLES
Guard column: Guard-Pack C18 (Waters)
Column: 100 X 5 4 jjim Novapak C18 RCM 8x10
Mobile phase: MeCN:300 mM orthophosphoric acid 15:85, final pH 2.2
Flow rate: 0.9
Detector: E, Environmental Sciences Model 5100 A, ESA 5011 analytical cell, oxidative mode,
cell II +0.30 V, cell I and guard cell not used
CHROMATOGRAM
Retention time: 8
Limit of quantitation: 1 nM
KEYWORDS
plasma; silanize all glassware; pharmacokinetics
REFERENCE
Descombe,J.-J.; Doumont,G.; Picard,M. Determination of exifone in human plasma and urine by high-perfor-
mance liquid chromatography with electrochemical detection, J.Chromatogr., 1989, 496, 345-353.
Factor VIII
Merck Index: 3963
SAMPLE
Matrix: solutions
Sample preparation: Add 3 mL Factor VIII solution in water to a column of Sephadex G-25
(Pharmacia), elute with buffer, monitor at UV 280, all protein elutes in void volume, inject a
10 mL aliquot. (Buffer was 50 mM imidazole HCl, 150 mM NaCl, 0.02% sodium azide, pH 7.0.)
HPLC VARIABLES
Guard column: 75 X 25 37 mL TSK 6000PW (Toyo Soda)
Column: 600 X 25 300 mL bead size 17 |xm TSK 5000PW (Toyo Soda)
Mobile phase: 50 mM imidazole HCl, 150 mM NaCl, 0.02% sodium azide, pH 7.0
Flow rate: 8.5
Detector: UV 254 or bioassay
CHROMATOGRAM
Retention time: 12
KEYWORDS
Preparative; SEC
REFERENCE
Herring,S.W.; Shitanishi,KT.; Moody,K.E.; Enns,R.K. Isolation of human factor VIILC by preparative high-
performance size-exclusion chromatography, J.Chromatogr., 1985, 326, 217-224.
SAMPLE
Matrix: solutions
it to dryness under a stream of nitrogen, reconstitute the residue in 200 |xL mobile phase,
inject a 20 |xL aliquot. Urine. 1 mL Urine + 1 mL pH 7 phosphate buffer (Normadose, Prolabo)
+ 8 mL diethyl ether, shake for 10 min, centrifuge at 4 at 2400 g for 5 min. Remove the organic
layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 250
|xL mobile phase, inject a 20 (xL aliquot.
HPLCVARIABLES
Guard column: Guard-Pack C18 (Waters)
Column: 100 X 5 4 jjim Novapak C18 RCM 8x10
Mobile phase: MeCN:300 mM orthophosphoric acid 15:85, final pH 2.2
Flow rate: 0.9
Detector: E, Environmental Sciences Model 5100 A, ESA 5011 analytical cell, oxidative mode,
cell II +0.30 V, cell I and guard cell not used
CHROMATOGRAM
Retention time: 8
KEYWORDS
plasma; silanize all glassware; pharmacokinetics
REFERENCE
Descombe,J.-J.; Doumont,G.; Picard,M. Determination of exifone in human plasma and urine by high-perfor-
mance liquid chromatography with electrochemical detection, J.Chromatogr., 1989, 496, 345-353.
Factor VIII
Merck Index: 3963
SAMPLE
Matrix: solutions
Sample preparation: Add 3 mL Factor VIII solution in water to a column of Sephadex G-25
(Pharmacia), elute with buffer, monitor at UV 280, all protein elutes in void volume, inject a
10 mL aliquot. (Buffer was 50 mM imidazole HCl, 150 mM NaCl, 0.02% sodium azide, pH 7.0.)
HPLC VARIABLES
Guard column: 75 X 25 37 mL TSK 6000PW (Toyo Soda)
Column: 600 X 25 300 mL bead size 17 |xm TSK 5000PW (Toyo Soda)
Mobile phase: 50 mM imidazole HCl, 150 mM NaCl, 0.02% sodium azide, pH 7.0
Flow rate: 8.5
CHROMATOGRAM
Retention time: 12
KEYWORDS
Preparative; SEC
REFERENCE
Herring,S.W.; Shitanishi,KT.; Moody,K.E.; Enns,R.K. Isolation of human factor VIILC by preparative high-
performance size-exclusion chromatography, J.Chromatogr., 1985, 326, 217-224.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 100 X 1 Mono Q gel HR 10/10 (Pharmacia)
Mobile phase: Gradient. 20 mM Tris HCl containing 50 mM calcium chloride, pH 6.8 with a
linear gradient up to 600 mM NaCl
Flow rate: 2
Detector: UV 280
CHROMATOGRAM
R e t e n t i o n time: 50
REFERENCE
Andersson,L.O.; Forsman,N.; Huang,K; Larsen,K; Lundin,A.; Pavlu,B-J Sandberg,H.; Sewerin,K; Smart, J. Iso-
lation and characterization of human factor VIII: molecular forms in commercial factor VIII concentrate,
cryoprecipitate, and plasma, Proc.Nat.Acad.Sci.USA, 1986, 83, 2979-2983.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in water, dilute with mobile phase, centrifuge at 10000 g for 10
min, filter (Millex 0.22 jim SLGVO25 BS), inject a 50-500 aliquot.
HPLC VARIABLES
Column: HR10/30 Superose 6 (Pharmacia)
Mobile phase: 20 mM Tris and 20 mM citric acid buffer, pH 7.4 containing 400 mM NaCl (A) or
20 mM pH 7.4 triethanolamine buffer containing 150 mM NaCl (B)
Flow rate: 0.5 (A) or 0.3 (B)
REFERENCE
Dawes,J.; Freeman,L.; Dawson,N.J.; Pepper,D.S.; Barrowcliffe,T.W. High molecular weight aggregate content
of heated and unheated factor VIII products determined by fast-protein liquid chromatography, Vox Sang.,
1990, 58, 30-34.
Famciclovir
Merck Index: 3971
SAMPLE
M a t r i x : blood
Sample preparation: 200 |xL Blood + 600 |xL 16% trichloroacetic acid, centrifuge, inject an
aliquot of the supernatant.
HPLC VARIABLES
Guard column: C18 Guard-Pak (Waters)
Column: 100 X 8 Nova-Pak C18 (in a Z module)
Mobile phase: Gradient. A was 50 mM sodium hydrogen phosphate. B was MeOH:water 80:20
containing 5 mM NaH2PO4. A:B 99:1 for 1.5 min, to 5:95 over 18.5 min, re-equilibrate at initial
Flow rate: 1.6
Detector: UV 254
CHROMATOGRAM
Retention time: 13.5 (as penciclovir, the active metabolite)
OTHER SUBSTANCES
Extracted: acyclovir
KEYWORDS
mouse; pharmacokinetics
REFERENCE
Boyd,M.R.; Bacon,T.H.; Sutton,D. Antiherpesvirus activity of 9-(4-hydroxy-3-hydroxymethylbut-l-yl) guanine
(BRL 39123) in animals, Antimicrob.Agents Chemother., 1988, 32, 358-363.
SAMPLE
Sample preparation: Condition a Bond Elut SCX strong cation exchange SPE cartridge with 1
mL MeOH, 1 mL water, and I m L l mM pH 7.0 Na2HPO4 buffer. 1 mL 500 |xL Plasma or 100
jxL urine + 100 |JLL 20-50 |xg/mL IS in water + 100-500 |xL 16% trichloroacetic acid, add the
supernatant to the SPE cartridge, wash with 1 mL MeOH: 1 mM pH 7.0 Na2HPO4 buffer 20:
80, elute with 1 mL MeOH:100 mM pH 11.0 K2HPO4 buffer 25:75, inject an aliquot.
HPLC VARIABLES
Column: 3 |xm Apex 1 ODS
Mobile phase: Gradient. A was MeOH:10 mM pH 7.0 Na2HPO4 buffer 7:93. B was MeOH:10 mM
pH 7.0 Na2HPO4 buffer 35:65. A:B from 100:0 to 0:100 over 4 min, maintain at 0:100 for 1.5
min, return to 0:100 over 1 min.
Flow rate: 2
Detector: UV 305
CHROMATOGRAM
Retention time: 6
Internal standard: 6-deoxy-9-(4-hydroxy-2-hydroxymethylbut-l-yl)guanine(BRL 44056) (2.8)
Limit of detection: 2000 ng/mL (urine), 500 ng/mL (plasma)
OTHER SUBSTANCES
Extracted: penciclovir, metabolites
KEYWORDS
plasma; human; rat; dog; SPE
REFERENCE
Winton,C.R; Fowles,S.E.; Pierce,D.M.; Hodge,A.V. Gradient high-performance liquid chromatographic method
for the analysis of the pro-drug famciclovir and its metabolites, including the active anti-viral agent pen-
ciclovir, in plasma and urine, Anal.Proc, 1990, 27, 181-182.
SAMPLE
Sample preparation: Mix 200 (xL microsomal incubation with 200 |xL MeOH, centrifuge for 3-
5 min in a bench-top microcentrifuge, inject a 20 (xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: ixBondapak C18 Guard-Pack
Column: 250 X 4.6 5 \xm Spherisorb ODS 2
Mobile phase: MeCN:0.5 mM pH 4.6 ammonium acetate buffer 9:91
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Retention time: 20
OTHER SUBSTANCES
Simultaneous: metabolites
KEYWORDS
human; guinea pig; rabbit; rat; liver
REFERENCE
Rashidi,M.R.; Smith,J.A.; Clarke,S.E.; Beedham,C. In vitro oxidation of famciclovir and 6-deoxypenciclovir by
aldehyde oxidase from human, guinea pig, rabbit, and rat liver, Drug Metab.Dispos., 1997, 25, 805-813.
Famotidine
Molecular formula: C8H15N7O2S3
Merck Index: 3972
Lednicer No.: 2 37
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 fim Symmetry C8 (Waters)
mL/min)
Detector: UV 202.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
HPLC VARIABLES
Column: Nova Pak C18
Mobile phase: MeCN:0.1% acetic acid:10 mM pH 7.8 (NH4)H2PO4 10:23:74
Flow rate: 1
Detector: UV 300
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: cefmetazole
KEYWORDS
stability-indicating; injections; 5% dextrose
REFERENCE
Lee,D.K.T.; Wong,C.-Y.; Wang,D.-R; Chang,L.-C; Wu,K.-H. Stability of cefmetazole sodium and famotidine,
Febantel
Merck Index: 3982
Lednicer No.: 4 35
SAMPLE
Matrix: blood
Sample preparation: 4 mL Plasma + 2 mL pH 7.4 phosphate buffer + 20 mL diethyl ether,
shake mechanically for 10 min, place in a freezer for 30 min, repeat the extraction. Combine
the organic layers and evaporate them to dryness under a stream of nitrogen at 45, reconsti-
tute the residue in 200 JJLL DMF, inject a 20 JULL aliquot.
HPLC VARIABLES
Column: 250 X 4 10 fxm RP 8
Mobile phase: Gradient. MeCN: 1% phosphoric acid 20:80 to 60:40 over 10 min
Flow rate: 2
Detector: UV 290
CHROMATOGRAM
OTHER SUBSTANCES
KEY WORDS
plasma; sheep
REFERENCE
Delatour,R; Tiberghien,M.R; Besse,S. An HPLC procedure for the quantification of five metabolites of febantel
in sheep serum, J.Vet.Pharmacol.Ther., 1983, 6, 233-235.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 jxL 10 jxg/mL albendazole in MeOH + 200 JULL 500
mM ammonium hydroxide (to adjust pH to 11) + 200 mg NaCl + 5 mL distilled diethyl ether,
roll for 15 min, remove 4 mL supernatant, repeat extraction, remove 5 mL supernatant. Com-
bine the organic layers and evaporate them to dryness under a stream of nitrogen, reconstitute
the residue in 60 |xL MeOH, sonicate for 2 min, inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:l% acetic acid 43:57
Flow rate: 0.9
Detector: UV 292
CHROMATOGRAM
Retention time: 6.2
Internal standard: albendazole (1.6)
OTHER SUBSTANCES
Extracted: fenbendazole, oxfendazole, oxfendazole sulfone
KEYWORDS
plasma; sheep
REFERENCE
Landuyt,J.; Debackere,M.; Delbeke,R; McKellar,Q. A high performance liquid chromatographic method for the
determination of febantel and its major metabolites in lamb plasma, Biomed.Chromatogr., 1993, 7, 78-81.
Felbamate
Merck Index: 3988
SAMPLE
Matrix: CSF
Sample preparation: Dilute CSF with an equal volume of a 10 jxg/mL solution of IS in MeCN:
30 mM pH 3.0 phosphate buffer 20:80, vortex, centrifuge at about 2000 g at 0 for 10 min,
HPLC VARIABLES
Column: 150 X 4.6 3 jxm Spherisorb ODS2
Mobile phase: MeCN:MeOH:30 mM pH 3.0 KH2PO4 12:6:82
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Internal standard: 2-phenyl-3-carbamoyl-3-oxypropyl allophanate (14.5)
OTHER SUBSTANCES
KEYWORDS
rat
REFERENCE
Romanyshyn,L.A.; Wichmann,J.K.; Kucharczyk,N.; Sofia,RD. Simultaneous determination of felbamate and
four metabolites in rat cerebrospinal fluid by high-performance liquid chromatography, J.Chromatogr., 1993,
622, 223-228.
SAMPLE
Matrix: blood
Sample preparation: 100 uX Plasma or serum + 100 |xL 100 |xg/mL IS in MeCN, vortex, let
stand for 10 min. Centrifuge at 15000 rpm for 5 min. Mix the supernatant with an equal volume
of water and inject an aliquot.
HPLC VARIABLES
Column: 40 X 3.2 3 |jim Brownlee VeloSep RP-18
Mobile phase: Gradient. A was MeCN. B was water. A:B from 0:100 to 22:78 over 11 min, to
100:0 over 1.5 min, maintain at 100:0 for 0.5 min, to 0:100 over 1 min, maintain at 0:100 for
6 min.
Flow rate: 0.8 for 11 min, to 1.5 over 2 min, to 0.8 over 6 min, hold at 0.8 for 1 min
Detector: UV 210
CHROMATOGRAM
Retention time: 9.2
Internal standard: 2-methyl-2-phenyl-l,3-propanediol dicarbamate (10.8)
OTHER SUBSTANCES
Extracted: carbamazepine, carbamazepine-10,ll-epoxide, clonazepam, ethosuximide, hexobar-
bital, ibuprofen, phenytoin, valproic acid
Simultaneous: phenobarbital, primidone
KEYWORDS
plasma; serum
REFERENCE
Behnke,C.E.; Reddy,M.N. Determination of felbamate concentration in pediatric samples by high-performance
liquid chromatography, Ther.Drug Monit., 1997, 19, 301-306.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 500 jxL 1 M pH 5.0 sodium acetate buffer + 50 IJLL 400
jxg/mL W-509 in MeOH, vortex for 15 s, add 4 mL dichloromethane:ethyl acetate 2:1, shake
for 5 min, repeat extraction. Combine the organic layers and evaporate them to dryness under
a stream of nitrogen at 40. Reconstitute the residue in 200 |xL mobile phase, inject a 20 |xL
aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherisorb Octyl C8
Mobile phase: MeOH:MeCN:THF:10 mM pH 6.5 ammonium phosphate buffer 16:11:7:66
Flow rate: 1.5
Detector: UV 215
CHROMATOGRAM
Retention time: 3.7
Internal standard: W-509 (4.7)
OTHERSUBSTANCES
Simultaneous: phenytoin, carbamazepine, 5-(p-hydroxyphenyl)-5-phenylhydantoin, cyhepta-
mide, carbamazepinediol, carbamazepine-10,11-epoxide, metabolites
Also analyzed: ethosuximide, primidone, phenobarbital, ethotoin, lorazepam, phenyl-
ethylmalonamide
KEYWORDS
plasma
REFERENCE
Remmel,R-P-; Miller,S.A.; Graves,N.M. Simultaneous assay of felbamate plus carbamazepine, phenytoin, and
their metabolites by liquid chromatography with mobile phase optimization, Ther.Drug Monit, 1990, 12,
90-96.
SAMPLE
Matrix: blood
Sample preparation: 100 JJLL Plasma + 200 jxL 40 |xg/mL IS in MeCN, vortex, centrifuge at
2500 g at 0 for 15 min, inject a 10 |xL aliquot of the supernatant.
HPLCVARIABLES
Column: 150 X 4.6 3 |xm Spherisorb ODS2
Mobile phase: MeCN:buffer 25:75 (Buffer was 2.2 g K2HPO4 in 740 mL water, adjust pH to 6.50
with phosphoric acid, make up to 750 mL with water.)
Flow rate: 0.7
Detector: UV 210
CHROMATOGRAM
Retention time: 5.4
Internal standard: 2-methyl-2-phenyl-l,3-propanediol dicarbamate (7.3)
KEYWORDS
plasma; dog
REFERENCE
Clark,L.A.; Wichmann,J.K; Kucharczyk,N.; Sofia,R.D. Determination of the anticonvulsant felbamate in beagle
dog plasma by high-performance liquid chromatography, J.Chromatogr., 1992, 573, 113-119.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 100 |xL 40 |xg/mL IS in MeOH:water 4:96 + 1 mL 1 M
NaOH saturated with ammonium sulfate, vortex, add 8 mL MTBExhloroform 75:25, rotate for
45 min, centrifuge at 2000 g at 0 for 15 min. Remove 7-7.5 mL of the organic layer and
evaporate it to dryness under vacuum at 50, reconstitute the residue in 200 JJLL mobile phase,
vortex, inject a 100 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:20 mM pH 6.8 (NH4)2HPO4 15:85
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Internal standard: 2-phenyl-3-carbamoyl-3-oxypropyl allophanate (18.5)
OTHER SUBSTANCES
KEYWORDS
plasma; rat; dog
REFERENCE
Romanyshyn,L.A.; Wichmann,J.K; Kucharczyk,N.; Sofia,R.D. Simultaneous determination of felbamate and
three metabolites in rat and dog plasmas by high-performance liquid chromatography, J.Chromatogr., 1993,
622, 229-234.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Serum + 200 JJLL 12 jxg/mL acetoacetanilide in MeCN, centrifuge
at 14000 g for 15 s, inject a 50 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: 125 X 4 5 |xm C8 (Merck)
Mobile phase: MeCN:20 mM pH 6.1 phosphate buffer 12.5:87.5
Flow rate: 1.8
Detector: UV 205
CHROMATOGRAM
Internal standard: acetoacetanilide (8.4)
OTHER SUBSTANCES
Noninterfering: acetaminophen, N-acetylprocainamide, amikacin, carbamazepine, digoxin, di-
sopyramide, ethosuximide, lidocaine, phenobarbital, phenytoin, primidone, procainamide, quin-
idine, salicylic acid, theophylline, valproic acid, vancomycin
KEYWORDS
serum; comparison with capillary electrophoresis
REFERENCE
Shihabi,Z.K; Oles,K.S. Felbamate measured in serum by two methods: HPLC and capillary electrophoresis,
Clin.Chem., 1994, 40, 1904-1908.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 50 |xL 330 |xg/mL IS in MeOH + 3 mL dichloromethane,
rotate for 5 min, centrifuge for 5 min. Remove the organic layer and evaporate it to dryness
under a stream of air at 40, reconstitute the residue in 200 |xL mobile phase, inject a 35 |xL
aliquot. Alternatively, condition a 6 mL C18 SPE cartridge (J.T.Baker) with 2 mL MeOH, 2 mL
water, I m L l M KOH, and 1 mL water. 500 |xL Serum + 50 JJLL 330 |xg/mL IS in MeOH, add
to the SPE cartridge, wash with 1 mL 100 mM pH 7.4 phosphate buffer, wash with 1 mL water,
elute with 2 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen, reconsti-
tute the residue in 200 |xL mobile phase, inject a 35 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Zorbax C18
Mobile phase: MeCN:MeOH:THF:buffer 5.25:10.25:12.5:72 (Buffer was 17 g KH2PO4 in 250 mL
water, pH 7.4.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Internal standard: 2-methyl-2-phenyl-l,3-propanediol dicarbamate (methyl felbamate) (7.0)
Limit of quantitation: 5 [LgImL
OTHER SUBSTANCES
Simultaneous: carbamazepine, carbamazepine epoxide, phenytoin
Noninterfering: amobarbital, butabarbital, butalbital, caffeine, lidocaine, pentobarbital, phe-
nobarbital, primidone, secobarbital, theophylline
KEYWORDS
serum; SPE; valproic acid; comparison with GC
REFERENCE
Gur,R; Poklis,A.; Saady,J.; Costantino,A. Chromatographic procedures for the determination of felbamate in
serum, JAnal.ToxicoL, 1995, 19, 499-503.
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Plasma or serum + 200 |xL IS in MeCN, mix, let stand, centrifuge.
Remove the supernatant and wash it with hexane, centrifuge, inject a aliquot.
HPLC VARIABLES
Mobile phase: MeCN:phosphate buffer 20:80
Detector: UV 214
CHROMATOGRAM
Retention time: 3.0
Internal standard: W509 (4.5)
OTHER SUBSTANCES
Simultaneous: phenobarbital
KEYWORDS
serum; plasma
REFERENCE
Wong,S.H.Y.; Sasse,E.; Schroeder,J.; Rodgers,J.; Pearson,L.; Neicheril,J.; Radewahn,K; Morris,G.L. Felbamate
monitoring by reversed-phase and automated prestation liquid chromatography (Abstract 201), Ther.Drug
Monit, 1995, 17, 433-433.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Ultra-Turrax Tissuemizer) with four volumes of water for 1
min. 100 |xL Homogenate + 100 (JLL 12.5 |xg/mL IS in mobile phase + 2 mL water, vortex for 1
min, centrifuge at 600 g for 10 min. 2 mL Supernatant + 8 mL ethyl acetate, vortex for 1 min,
vacuum at about 50, reconstitute the residue in 100 (xL mobile phase, inject an 80 |xL aliquot.
HPLC VARIABLES
Guard column: Resolve C18 (Waters)
Mobile phase: MeCN:MeOH:buffer 15:5:80 (Buffer was 2.3 g K2HPO4.3H2O in 950 mL water,
adjust pH to 6.8 with phosphoric acid, make up to 1 L with water.)
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 11
Internal standard: 2-methyl-2-phenyl-l,3-propanediol dicarbamate (18)
OTHER SUBSTANCES
KEYWORDS
rat; brain; heart; pharmacokinetics
REFERENCE
Jacala,A.; Adusumalli,V.E.; Kucharczyk,N.; Sofia,R.D. Determination of the anticonvulsant felbamate and its
three metabolites in brain and heart tissue of rats, J.Chromatogr., 1993, 614, 285-292.
Felbinac
Merck Index: 3989
Lednicer No.: 4 32
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 20 |xL 20 |xg/mL IS in MeOH. Vortex for 1 min
with dichloromethane:diethyl ether 80:20, centrifuge at 1000 g for 10 min, separate the organic
layer. Add 4 mL dichloromethane-diethyl ether 80:20, repeat the same extraction procedure
twice, evaporate the organic phase to dryness under a stream of nitrogen, add 100 uL 10 mM
NaOH to the residue, shake for 5 min, inject a 20 |JLL aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 10 |xm Vydac AXGU
Column: 250 X 4.6 5 |xm Adsorbosphere SAX
Mobile phase: MeCN:100 mM pH 7.0 phosphate buffer 10:90
Flow rate: 1.2
Detector: UV 280
CHROMATOGRAM
Retention time: 6.8
Internal standard: 2-[4-(2-furoyl)phenyl]propionic acid (3.9)
OTHER SUBSTANCES
Extracted: fenbufen, pefloxacin
KEYWORDS
plasma
REFERENCE
Carlucci,G.; Palumbo,G.; Mazzeo,P. Simple and rapid analysis of pefloxacin, fenbufen and felbinac in human
plasma using high-performance liquid chromatography, J.Liq.Chromatogr.Rel.Technol., 1996, 19, 1107-
1115.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL serum with 20 uL 20 |xg/mL IS in MeOH and 100 |xL 20 mM
NaOH. Vortex for 1 min with 4 ml dichloromethane:diethylether 80:20, centrifuge at 2000 g
for 10 min, remove 3 mL organic phase. Repeat the same extraction procedure twice. Combine
organic layers, evaporate to dryness under a stream of nitrogen. Add 100 |xL 20 mM NaOH to
the residue, shake for 5 min, inject a 20 (xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |mm Supelcosil LC-SAX
Mobile phase: MeCN;100 mM pH 7.0 phosphate buffer 10:90
Flow rate: 1.2
Detector: UV 280
CHROMATOGRAM
Retention time: 7.2
Internal standard: 2-[4-(2'-furoyl)phenyl]propionic acid (3.5)
OTHER SUBSTANCES
Extracted: fenbufen, lomefloxacin
KEYWORDS
plasma
REFERENCE
Carlucci,G.; Mazzeo,R; Palumbo,G. Simultaneous determination of lomefloxacin, fenbufen and felbinac in hu-
man plasma using high performance liquid chromatography, Chromatographia, 1996, 43, 261-264.
SAMPLE
Matrix: blood
Sample preparation: 50 fxL Plasma + 1 mL 100 mM pH 7.0 K2HPO4 adjusted to pH 7.0 with
85% orthophosphoric acid + 100 JJLL 200 |xg/mL N-phenylanthranilic acid in water + 3 mL
dichloromethaneiisoamyl alcohol 9:1, shake vigorously for 10 min, centrifuge at 2270 g for 10
min. Remove 2 mL of the organic phase and evaporate it to dryness under a stream of nitrogen
at 40. Reconstitute residue in 100 (xL MeOH:50 mM NaOH 2:1, vortex, inject a 10 uX aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Chemcosorb 5-ODS-H
Flow rate: 0.6
Detector: UV 275
CHROMATOGRAM
Retention time: 9.8
Internal standard: N-phenylanthranilic acid (15.0)
OTHER SUBSTANCES
Extracted: fenbufen, nalidixic acid, norfloxacin, ofloxacin, enoxacin
KEYWORDS
plasma; rat
REFERENCE
Katagiri,Y; Naora,K.; Ichikawa,N.; Hayashibara,M.; Iwamoto,K. Simultaneous determination of ofloxacin, fen-
135-142.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Plasma + 1 mL 100 mM pH 7.0 K2HPO4 adjusted to pH 7.0 with
85% orthophosphoric acid + 100 |xL 300 |xg/mL nalidixic acid in water + 3 mL dichloromethane:
stitute residue in 100 |xL MeOH:50 mM NaOH 2:1, vortex, inject a 10 fxL aliquot.
HPLC VARIABLES
acid
Flow rate: 0.6
Detector: UV 275
CHROMATOGRAM
Retention time: 10
Internal standard: nalidixic acid (5)
OTHER SUBSTANCES
Extracted: fenbufen, ciprofloxacin
KEYWORDS
REFERENCE
Naora,K; Katagiri,Y.; Ichikawa,N.; Hayashibara,M.; Iwamoto,K. Simultaneous high-performance liquid chro-
matographic determination of ciprofloxacin, fenbufe and felbinac in rat plasma, J.Chromatogr., 1990, 530,
186-191.
SAMPLE
Matrix: urine
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 10 mL ethyl acetate and dry
by aspiration of air. Evaporate an aliquot of 100 |xL 20 |xg/mL IS in MeOH to dryness at 37.
Add 1 mL urine, vortex, add 250 |xL 1 M pH 5.0 acetate buffer, vortex. Add 250 |xL of the
mixture to the SPE cartridge, dry by aspiration of air, elute with 3 mL ethyl acetate, evaporate
the eluate to dryness under a stream of nitrogen at 37, reconstitute the residue in 200 u,L
mobile phase, inject a 10-30 (xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Inertsil ODS-2
Flow rate: 0.9
Detector: UV 230
CHROMATOGRAM
Retention time: 8
Internal standard: indomethacin (18.5)
OTHER SUBSTANCES
Extracted: diclofenac, ibuprofen, fenbufen, flurbiprofen, ketoprofen, loxoprofen, mefenamic acid,
naproxen, piroxicam, sulindac
KEYWORDS
SPE
REFERENCE
Hirai,T.; Matsumoto,S.; KishiJ. Simultaneous analysis of several non-steroidal anti-inflammatory drugs in
human urine by high-performance liquid chromatography with normal solid-phase extraction,
J.Chromatogr.B, 1997, 692, 375-388.
Felodipine
Molecular formula: C18H19CI2NO4
Merck Index: 3991
Lednicer No.: 4 106
SAMPLE
Matrix: bile, urine
Sample preparation: Dilute 20-200 |xL bile or urine to 1 mL with water, adjust pH to 2.2 with
1 M phosphate buffer, extract twice with 6 mL ether with gentle inversion for 1 min. Remove
the organic phases and evaporate them under a stream of nitrogen, dissolve the residue in 250
fiL mobile phase A, inject the whole amount.
HPLC VARIABLES
Column: 60-5 Polygosil C18 (Macherey-Nagel)
Mobile phase: Gradient. A was MeOH: 10 mM tetrapropylammonium hydrogen sulfate in 50 mM
pH 5.0 phosphate buffer 5:95. B was MeOH:10 mM tetrapropylammonium hydrogen sulfate in
50 mM pH 5.0 phosphate buffer 2:1. A:B 100:0 for 2 min then to 0:100 over 35 min.
Flow rate: 1.2
Detector: UV 254
OTHER SUBSTANCES
KEYWORDS
rat
REFERENCE
Sutfin,T.A; Gabrielsson,M.; Regardh,C.G. Effects of biliary excretion on the disposition of felodipine and me-
tabolites in the rat, Xenobiotica, 1987, 17, 1203-1214.
SAMPLE
Matrix: blood
Sample preparation: Adjust pH of 200-300 |xL whole blood to 12 with 100 |xL 1 M NaOH, add
150 |xL 10% EtOH, extract with 4 mL diethyl ether for 30 min, freeze in dry ice. Remove the
organic phase and evaporate it to dryness under a stream of nitrogen. Reconstitute the residue
with 250 |xL mobile phase, inject a 200 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.5 5 |xm Nucleosil C-18
Mobile phase: MeOH:water 70:30 containing 20 mM perchloric acid and 80 mM sodium chlorate
Flow rate: 1.1
Detector: UV 254
OTHER SUBSTANCES
KEYWORDS
whole blood
REFERENCE
Sutfin,T.A.; Gabrielsson,M.; Regardh,C.G. Effects of biliary excretion on the disposition of felodipine and me-
tabolites in the rat, Xenobiotica, 1987, 27, 1203-1214.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 |xL 100 ng/mL IS in MeOH, mix, equilibrate for 10
min, add 25 |xL 4 M NaOH, mix, add 4 mL n-pentane:dichloromethane 1:1, mix on a Sarstedt
CM-9 whirl-mixer for 1 h, centrifuge at 2000 g for 5 min. Remove the organic layer and evap-
orate it to dryness under vacuum at 30, dissolve the residue in 40 |iL mobile phase, inject a
20 (JLL aliquot.
HPLC VARIABLES
Guard column: 20 X 2 Perisorb RP-18 (Upchurch)
Column: 250 X 4.6 Chiralcel OJ (Daicel)
Mobile phase: n-Hexane:isopropanol 87.5:12.5
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 15.9 (S), 21.9 (R)
Internal standard: 3-(2-hydroxy-2-methyl)ethyl-5-methyl-2,6-dimethyl-4-(2-nitrophenyl)-3,5-
pyridine dicarboxylate (Bay r 9590)
Limit of detection: 0.1 ng/mL obtainable using off-line GC-ECD detection of HPLC column
effluent, 5 ng/mL (UV)
OTHER SUBSTANCES
Simultaneous: metabolites, nitrendipine, nimodipine, nisoldipine, isradipine, niguldipine, nil-
vadipine, nicardipine
KEYWORDS
plasma; chiral
REFERENCE
Soons,P.A.; Roosemalen,M.C.M.; Breimer,D.D. Enantioselective determination of felodipine and other chiral
dihydropyridine calcium entry blockers in human plasma, J.Chromatogr., 1990, 528, 343-356.
SAMPLE
HPLC VARIABLES
mIVmin)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763,
149[9]4163.
SAMPLE
Sample preparation: Dissolve tablets in MeCN: 1 mM pH 2 KH2PO4 50:50, centrifuge, inject a
50 JJLL aliquot of the supernatant.
HPLC VARIABLES
Mobile phase: MeCN:buffer 35:65 (Buffer was 1 mM KH2PO4 adjusted to pH 2 with phosphoric
acid.)
Flow rate: 2.5
Detector: UV 215
CHROMATOGRAM
Retention time: 31
OTHER SUBSTANCES
Simultaneous: enalapril, enalaprilat
KEYWORDS
tablets
REFERENCE
Qin,X.-Z.; DeMarco,J.; Ip,D.P. Simultaneous determination of enalapril, felodipine and their degradation prod-
ucts in the dosage formulation by reversed-phase high-performance liquid chromatography using a Spher-
isorb C8 column, J.ChromatognA, 1995, 707, 245-254.
SAMPLE
Sample preparation: Adjust pH to 2.2 with 1 M phosphate buffer, extract with ether for 1 h,
centrifuge at 1000 rpm for 15 min, freeze in dry ice/EtOH. Remove the organic phase and
evaporate it under a stream of nitrogen, dissolve the residue in 200 u,L MeOH, inject a 40 |xL
aliquot.
HPLC VARIABLES
Guard column: Brownlee Newguard RP-18 pre-column
Column: 150 X 4.5 5 \\jm Polygosil C18 (Macherey-Nagel)
Mobile phase: Gradient. A was MeOH: 10 mM tetrapropylammonium hydrogen sulfate in 50 mM
pH 5.0 phosphate buffer 5:95. B was MeOH: 10 mM tetrapropylammonium hydrogen sulfate in
50 mM pH 5.0 phosphate buffer 2:1. A:B 65:35 for 2 min then to 0:100 over 35 min, stay at 0:
100 for another 27 min.
Flow rate: 0.8
Detector: UV 280
CHROMATOGRAM
Retention time: 43
OTHER SUBSTANCES
REFERENCE
Baarnhielm,C; Backman,A.; Hoffmann,K.J.; Weidolf,L. Biotransformation of felodipine in liver microsomes
from rat, dog, and man, Drug Metab.Dispos., 1986, 14, 613-618.
SAMPLE
Matrix: microsomal incubations, perfusate
Sample preparation: Extract using a 100 mg 1 mL Bond Elut C2 SPE cartridge, elute with
MeOH, evaporate eluate to dryness under a stream of nitrogen, reconstitute the residue in 100
|xL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 X 5 Hichrom HiRPB deactivated reverse-phase
Mobile phase: MeOH:25 mM pH 5.0 N,N,^N'-tetramethylethylenediamine phosphate buffer
75:25
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Internal standard: felodipine
OTHER SUBSTANCES
Extracted: nitrendipine
KEYWORDS
rat; liver; felodipine is IS; SPE
REFERENCE
Walker,D.K.; Humphrey,M.J.; Smith,D.A. Importance of metabolic stability and hepatic distribution to the
pharmacokinetic profile of amlodipine, Xenobiotica, 1994, 24, 243-250.
SAMPLE
Matrix: perfusate
HPLC VARIABLES
Column: 100 X 8 5 |xm Novapak C18 radial compression
Flow rate: 2
Detector: UV 237
OTHER SUBSTANCES
Also analyzed: nicardipine, nifedipine, nimodipine, nitrendipine
REFERENCE
Diez,L; Colom,H.; Moreno,J.; Obach,R.; Peraire,C; Domenech,J. A comparative in vitro study of transdermal
absorption of a series of calcium channel antagonists, J.Pharm.Sci., 1991, 80, 931-934.
SAMPLE
Matrix: solutions
Sample preparation: Centrifuge at 2000 g at 37 for 15 min.
HPLC VARIABLES
Column: 100 X 8 5 |xm C18 Novapak
Flow rate: 2
Detector: UV 237
OTHER SUBSTANCES
Also analyzed: nicardipine, nifedipine, nimodipine, nitrendipine
KEYWORDS
buffers
REFERENCE
Diez,I.; Colom,H.; Moreno,J.; Obach,R-> Peraire,C; Domenech,J. A comparative in vitro study of transdermal
absorption of a series of calcium channel antagonists, J.Pharm.ScL, 1991, 80, 931-934.
Felypressin
Merck Index: 3992
SAMPLE
Sample preparation: Inject a 20 |xL aliquot of the undiluted formulation onto column A and
elute to waste with mobile phase A, after 14 min elute the contents of column A onto column
B with mobile phase B, monitor the effluent from column B, after 9 min re-equilibrate column
A with mobile phase A for 7 min.
HPLC VARIABLES
Column: A 25 X 4 4 fjim Superspher 60 RP-8; B 50 X 4 4 |xm Superspher 60 RP-8e
Mobile phase: AMeCN:50 mM pH 6.0 phosphate buffer 12:88; B MeCN:50 mM pH 6.0 phosphate
buffer 20:80
Flow rate: 1
reagent pumped at 0.25 mL/min and the mixture flowed through a 5 m X 0.5 mm ID knitted
PTFE coil to the detector. (Reagent was 300 |xg/mL fluorescamine in MeCN containing 0.1%
Brij-35.)
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
Noninterfering: prilocaine
KEYWORDS
post-column reaction; injections; column-switching; rinse all glass and plastic ware with 1M acetic
acid and then water to minimize adsorption.
REFERENCE
Svensson,M.; Groningsson,K. Liquid chromatographic determination of felypressin using a column-switching
technique and post-column derivatization, J.Chromatogr., 1990, 521, 141-147.
Fenbendazole
Merck Index: 4000
Lednicer No.: 3 176
SAMPLE
Matrix: abomasal fluid, blood, duodenal fluid, rumen fluid
Sample preparation: 4 mL Plasma, rumen fluid, abomasal fluid, or duodenal fluid + 4 mL pH
7.4 phosphate buffer + 20 mL ether, shake on a rotary mixer for 10 min, remove 16 mL of the
ether layer, add 20 mL ether, shake on a rotary mixer for 10 min, remove 20 mL of the ether
layer. Combine the ether layers and evaporate them under a stream of nitrogen at 60 to
dryness, reconstitute in 50 ^xL MeOH, sonicate, inject a 5 jxL aliquot.
HPLCVARIABLES
Column: 100 X 8 ODS Hypersil 10
Mobile phase: MeOH:50 mM ammonium carbonate 65:35
Flow rate: 1.5
Injection volume: 5
Detector: UV 292
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Extracted: albendazole, oxfendazole, cambendazole, thiabendazole, mebendazole, oxibendazole,
parbendazole
KEYWORDS
plasma; sheep
REFERENCE
Bogan,J.A.; Marriner,S. Analysis of benzimidazoles in body fluids by high-performance liquid chromatography,
J.Pharm.Scl, 1980, 69, 422-423.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Sep-Pak Vac C18 SPE cartridge with 3 mL MeOH, 1
mL water, and 5 mL 100 mM sodium carbonate. Mix 1.5 mL heparinized plasma with 1.5 mL
100 mM sodium carbonate, add to the SPE cartridge, wash with 5 mL 100 mM sodium car-
bonate, dry the cartridge in air for 30 s, elute with 750 uX. MeCN:0.2% phosphoric acid 50:50,
vortex the eluate (protect from light), inject an aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 5 jxm BST Nucleosil C18
Mobile phase: MeCN:buffer 33:67 (Buffer was 50 mM KH2PO4 adjusted to pH 3.0 with 20%
phosphoric acid.)
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
Extracted: praziquantel
KEYWORDS
dog; pharmacokinetics; plasma; SPE
REFERENCE
Morovj6n,G.; Csokan,R; Makranaszki,L.; Abdellah-Nagy,E.A.; T6th,K. Determination of fenbendazole, prazi-
quantel and pyrantel pamoate in dog plasma by high-performance liquid chromatography, J.ChromatognA,
1998, 797, 237-244.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 40 jxL 10 |xg/mL mebendazole in MeOHiDMF 99:1 + 1 mL
50 mM pH 8 borate buffer + 5 mL ethyl acetate, vortex for 20 s, centrifuge at 2500 g for 5 min.
Remove 4 mL of the organic layer and evaporate it to dryness under a stream of nitrogen at
50 for about 50 min, reconstitute the residue in 200 |xL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 125 X 4.6 5 ujn Hypersil SAS
Mobile phase: MeCN:bufifer 30:70 (Buffer was 5 mM phosphoric acid adjusted to pH 5.9 with
tetraethylammonium hydroxide.)
Flow rate: 2
Detector: UV 300
CHROMATOGRAM
Retention time: 8.6
Internal standard: mebendazole (3.2)
OTHER SUBSTANCES
KEYWORDS
plasma; sheep; pharmacokinetics
REFERENCE
Lehr,K.H.; Damm,P. Simultaneous determination of fenbendazole and its two metabolites and two triclaben-
dazole metabolites in plasma by high-performance liquid chromatography, J.Chromatogr., 1986, 382, 355-
360.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 |xL 10 jjig/mL albendazole in MeOH + 200 |xL 500
the residue in 60 uL MeOH, sonicate for 2 min, inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:l% acetic acid 43:57
Flow rate: 0.9
Detector: UV 292
CHROMATOGRAM
Retention time: 2.6
OTHER SUBSTANCES
Extracted: febantel, oxfendazole, oxfendazole sulfone
KEYWORDS
plasma; sheep
REFERENCE
SAMPLE
Matrix: blood, feces, tissue, urine
Sample preparation: Plasma or liver homogenate. 1 mL Plasma or liver homogenate (S9) + 20
JJLL concentrated ammonium hydroxide, vortex, add to a 1 mL Chem Elut SPE cartridge (An-
alytichem), rinse tube with 100 |xL dichloromethane, add rinse to the SPE cartridge, elute SPE
cartridge with two 4 mL portions of dichloromethane. Evaporate the eluate to dryness under
a stream of nitrogen at 50, reconstitute the residue in 100 fxL mobile phase, inject a 20 |xL
aliquot. Urine. 1 mL Urine + 20 fxL concentrated ammonium hydroxide, vortex, add to a 1 mL
Chem Elut SPE cartridge (Analytichem), rinse tube with 100 jxL dichloromethane, add rinse
to the SPE cartridge, elute SPE cartridge with two 4 mL portions of dichloromethane. Evap-
orate the eluate to dryness under a stream of nitrogen at 50, reconstitute the residue in 500
/xL 2 M HCl and 1 mL benzene (Caution! Benzene is a carcinogen!), vortex, centrifuge at 2000
rpm for 5 min, wash twice more with benzene. Add the aqueous layer to 500 |xL concentrated
ammonium hydroxide, extract twice with 1 mL dichloromethane. Combine the extracts and
wash them with 1 mL water. Evaporate the dichloromethane layer to dryness under a stream
of nitrogen, reconstitute the residue in 100 |xL mobile phase, inject a 10 |xL aliquot. Feces. 2
g Feces + 8 mL water + 1 mL concentrated ammonia, homogenize by stirring, add to a 10 mL
Chem Elut SPE cartridge (Analytichem) with a cotton ball on the top of the column, elute with
two 12 mL portions of ethyl acetate. Evaporate the eluate to dryness under a stream of nitrogen
at 50, reconstitute the residue in 2 mL 2 M HCl and 4 mL benzene (Caution! Benzene is a
carcinogen!), vortex, centrifuge at 2000 rpm for 5 min, wash three more times with benzene.
Add the aqueous layer to 1 mL concentrated ammonium hydroxide, extract twice with 1 mL
dichloromethane. Combine the extracts and wash them with 1 mL water. Evaporate the di-
chloromethane layer to dryness under a stream of nitrogen, reconstitute the residue in 500 |xL
mobile phase, inject a 5 (xL aliquot.
HPLC VARIABLES
Column: 300 X 4 MicroPak MCH 10 octadecylsilane
Mobile phase: MeCN:0.05 N phosphoric acid 78:22
Flow rate: 0.35 for 14 min, increasing to 2 over 2 min, maintain at 2
Detector: UV 290
CHROMATOGRAM
Retention time: 17
Limit of detection: 720 ng/g (goat feces), 200 ng/g (duck feces), 10 ng/mL (urine, plasma, tissue)
OTHER SUBSTANCES
KEY WORDS
plasma; goat; duck; chicken; rat; rabbit; liver; sheep; SPE
REFERENCE
Barker,S.A.; Hsieh,L.C; Short,C.R. Methodology for the analysis of fenbendazole and its metabolites in plasma,
urine, feces, and tissue homogenates, Anal.Biochem., 1986,155, 112-118.
SAMPLE
Sample preparation: Dissolve sample in MeOH containing 10% formic acid, dilute with mobile
phase, inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 jxm Hypersil C18
Mobile phase: MeOH:buffer 19:81, pH 3.9 (Buffer was prepared by dissolving 6.6 g dibasic am-
monium phosphate in 1 L water and adjusting to pH 3.9 with phosphoric acid.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 7.8
OTHER SUBSTANCES
Simultaneous: albendazole, niclosamide, oxyclozanide
KEYWORDS
tablets; powder; liquid formulations
REFERENCE
van Tonder,E.C; de Villiers,M.M.; Handford,J.S.; Malan,C.E.R; Du Preez,J.L. Simple, robust and accurate high-
performance liquid chromatography method for the analysis of several anthelmintics in veterinary formu-
lations, J.Chromatogr.A, 1996, 729, 267-272.
SAMPLE
Matrix: milk
Sample preparation: Condition a 3 mL Bond Elut silica SPE cartridge with 2 mL dichloro-
methane. 10 g Milk + 5 mL 1 M sodium carbonate, mix, add 150 mL ethyl acetate, add 1 mL
10 mg/mL BHT in ethyl acetate, blend (tissuemizer) at high speed for 5 min, add 10 g anhy-
drous sodium sulfate, blend for 1 min, let settle for 2-3 min, filter (No. 41 paper), add another
150 mL ethyl acetate to the sodium sulfate, blend for 2 min, filter. Combine the filtrates and
evaporate them to dryness under vacuum. Rinse out flask with two 10 mL portions of hexane
and two 10 mL portions of 1 M phosphoric acid. Combine rinses, shake vigorously for 2 min,
extract the hexane layer with two 10 mL portions of 1 mL phosphoric acid. Combine all the
aqueous layers and wash them with 5 mL hexane, adjust the pH to 8-9 by slowly adding about
9 mL 10 M KOH (use an ice bath), add 50 mL ethyl acetate, shake vigorously for 2 min, repeat
extraction. Filter the organic layers through 40 g anhydrous sodium sulfate, wash the sodium
sulfate with 25 mL ethyl acetate. Combine the organic layers, add 200 |xL 10 mg/mL BHT in
ethyl acetate, evaporate to dryness under vacuum. Take up the residue in two 3 mL portions
of dichloromethane and add them to the SPE cartridge, wash with 5 mL dichloromethane,
elute with 5 mL dichloromethane:MeOH 75:25. Evaporate the eluate to dryness under nitrogen,
reconstitute in 1 mL mobile phase, vortex, filter (0.2 |xm), inject an aliquot.
HPLC VARIABLES
Guard column: 30 X 4.6 pellicular C18 (Alltech)
Column: 250 X 4.6 5 jxm Hypersil ODS
Mobile phase: MeOH.buffer 70:30 (Buffer was 1.15 g (NH4)H2PO4 in 950 mL water, adjust pH
to 7.0 with dilute ammonia, make up to 1 L with water.)
Flow rate: 1
Detector: UV 298
CHROMATOGRAM
Retention time: 13
KEYWORDS
cow; SPE
REFERENCE
Tai,S.S.; Cargile,N.; Barnes,C.J. Determination of thiabendazole, 5-hydroxythiabendazole, fenbendazole, and
oxfendazole in milk, JAssoc.Off.Anal.Chem., 1990, 73, 368-373.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 120 X 4.6 7 |jim LiChrosorb RP8
Mobile phase: MeOH:33 mM phosphoric acid 50:50
Flow rate: 2
Detector: UV 245 following post-column reaction. The column effluent flowed through a 20 m X
0.5 mm ID knitted PTFE coil irradiated by a 15 w low-pressure mercury lamp (Original Hanau
TNN 15/32) to the detector., F ex 300 em 342 following post-column reaction. The column
effluent flowed through a 20 m X 0.5 mm ID knitted PTFE coil irradiated by a 15 w low-
pressure mercury lamp (Original Hanau TNN 15/32) to the detector.
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Uihlein,M.; Schwab,E. A novel reactor for photochemical post-column derivatization in HPLC, Chromatogra-
phia, 1982, 15, 140-146.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, fencamfamine, fenoprofen, fen-
proporex, fentanyl, flubendazole, flufenamic acid, fiunitrazepam, 5-fluorouracil, fluoxymester-
trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tjrramine, verapa-
REFERENCE
Hill,D.W.; Kind,AJ- Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 1 g silica SPE cartridge with 6 mL ethyl acetate-hexane 20:
80. 5 g tissue + 500 jxL 4 M potassium carbonate, vortex for 10 s, add 10 mL ethyl acetate,
vortex for 10 s, shake at 500 rpm for 10 min, centrifuge at 3400 g for 6 min. Decant the
supernatant. Repeat the extraction with another 10 mL ethyl acetate. Combined extracts + 80
mL hexane + 2 g anhydrous sodium sulfate, shake, let stand until it becomes transparent.
Filter over an S & S 589.1 filter paper circle. Add the filtrate to the SPE cartridge, dry the
column in a stream of nitrogen for 10 min. Elute with 3 mL acetic acid:MeOH 3:97, evaporate
to dryness under a stream of nitrogen at 37. Dissolve the residue in 550 |xL 50 mM ammonium
phosphate, add 280 |xL MeCN and 170 |xL MeOH, sonicate for 5 min, centrifuge at 15800 g for
at least 8 min. Inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 2.1 40 |xm pellicular reversed phase stainless steel
Column: two 100 X 3.0 5 fim Lichrosorb RP8 glass columns in series
Mobile phase: MeCN:MeOH:50 mM pH 5.0 phosphate buffer 28:17:55 (Prepare buffer as follows.
Dissolve 5.75 g ammonium phosphate in 950 mL water, adjust to pH 5.0 with 1 M NaOH.
Make up to 1000 mL with water.)
Flow rate: 0.6
Detector: UV 297
CHROMATOGRAM
Retention time: 15
Limit of detection: 500 pg (on column), 3.0 |xg/kg (in muscle tissue)
OTHER SUBSTANCES
KEYWORDS
muscle; trout; eel; metabolites; SPE
REFERENCE
Iosifidou,E.G.; Haagsma,N. High-performance liquid chromatographic determination of fenbendazole and its
metabolites, sulphoxide and sulphone, in fish muscle tissue, J.Liq.Chromatogr.Rel.TechnoL, 1996,19,1819-
1830.
SAMPLE
Matrix: tissue
Sample preparation: Wash 22 g bulk 40 |xm 18% load end-capped C18 material (Analytichem)
in a syringe barrel with 100 mL hexane, with 100 mL dichloromethane, and with 100 mL
MeOH and dry under vacuum aspiration. Gently blend 2 g C18 material, 0.5 g liver, and 10
(xL 40 jjig/mL mebendazole in DMF in a glass pestle for 1 min until homogeneous in appearance.
Place in a 10 mL syringe barrel plugged with filter paper (Whatman No. 1), cover with filter
paper, compress to 4.5 mL, place a 100 (xL pipette tip on the barrel to restrict flow, wash with
8 mL hexane, elute with 8 mL MeCN. Pass the eluate through 0.5 g activated alumina (EM
Science Type F-20 80-200 mesh) between filter paper in a 10 mL syringe barrel (wash column
with 4 mL MeCN just before use). Evaporate the eluate to dryness under a stream of nitrogen,
reconstitute the residue in 100 |xL MeOH and 400 JJLL 17 mM phosphoric acid, sonicate for 5-
10 min, centrifuge at 17000 g for 5 min, filter the supernatant (0.45 |xm), inject a 20 JJLL aliquot.
HPLCVARIABLES
Column: 300 X 4 10 |xm Micro Pak ODS (Varian)
Mobile phase: MeCN: 17 mM phosphoric acid 40:60
Flow rate: 1
Detector: UV 290
CHROMATOGRAM
Retention time: 14
Internal standard: mebendazole (9)
OTHER SUBSTANCES
Extracted: albendazole, thiafendazole, oxfendazole
KEYWORDS
matrix solid-phase dispersion; liver
REFERENCE
Long,A-R; Malbrough,M.S.; Hsieh,L.C; Short,C.R.; Barker,S.A. Matrix solid phase dispersion isolation and
liquid chromatographic determination of five benzimidazole anthelmintics in fortified beef liver,
JAssoc.Off.Anal.Chem., 1990, 73, 860-863.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 2.8 mL 500 mg 40 |xm 60 A Bond Elut silica SPE cartridge
with 2 mL dichloromethane. 10 g Minced tissue + 5 mL 1 M sodium carbonate + 150 mL ethyl
acetate + 1 mL 10 mg/mL BHT in ethyl acetate, blend (Waring) at high speed for 5 min, add
80 g anhydrous sodium sulfate, blend at low speed for 1 min. Decant the organic layer and
filter it (No. 41 paper), add 150 mL ethyl acetate to material remaining in blender, blend at
low speed for 2-3 min, filter, wash solid with 10 mL EtOH. Combine all the filtrates and
evaporate them to dryness under vacuum at 30-35 (beware of bumping). Rinse out flask with
two 10 mL portions of hexane and two 10 mL portions of 1 M phosphoric acid, combine rinses,
shake vigorously for 2 min, allow to separate for 10 min, extract the hexane layer twice more
with 10 mL portions of 1 M phosphoric acid. Combine all the aqueous layers and wash them
with 10 mL hexane, adjust the pH of the aqueous layer to 8.5 1.0 by slowly adding about 9
mL 10 M KOH while using an ice bath. Extract twice with 50 mL ethyl acetate (2 min shaking),
pass ethyl acetate layers through 40 g anhydrous sodium sulfate, wash the sodium sulfate with
25 mL ethyl acetate. Combine the ethyl acetate layers, add 200 |xL 10 mg/mL BHT in ethyl
acetate, evaporate to dryness under vacuum at 30-35 (beware of bumping). Rinse out flask
with three 3 mL portions of dichloromethane, add rinses to the SPE cartridge, wash with 5
mL dichloromethane, elute with 5 mL dichloromethane:MeOH 75:25. Evaporate the eluate to
dryness under a stream of nitrogen, reconstitute the residue in 1 mL mobile phase, vortex,
filter (0.2 jxm), inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 30 X 4.6 Brownlee RP-18 Spheri-10 MPLC
Column: 250 X 4.6 5 ^m C18 (Alltech)
Mobile phase: MeOH:buffer 70:30 (Buffer was 1.15 g (NH4)H2PO4 in 950 mL water, adjust pH
Flow rate: 1
Detector: UV 298
CHROMATOGRAM
Retention time: 14
Limit of detection: <800 ppb
OTHER SUBSTANCES
Simultaneous: chloramphenicol
Noninterfering: amprolium, chlortetracycline, erythromycin, levamisole, morantel, oxytetracy-
cline, phenothiazine, sulfadimethoxine, sulfamethazine, sulfaquinoxaline
KEYWORDS
cow; liver; SPE
REFERENCE
LeVan,L.W.; Barnes,C.J. Liquid chromatographic method for multiresidue determination of benzimidazoles in
beef liver and muscle: collaborative study, J.Assoc.Off.Anal.Chem., 1991, 74, 487-493.
SAMPLE
Matrix: tissue
Sample preparation: 3 g Pulverized tissue + 7 mL water, homogenize (Silverson) for 1 min, add
20 mL MeOH, sonicate for 15 min, centrifuge at 2000 g at 5 for 10 min. Remove 10 mL of the
supernatant and add it to 8 mL light petroleum (bp 40-60), shake gently for 30 s, centrifuge.
Add the aqueous phase to 14 mL 500 mM NaH2PO4 and 8 mL diethyl etheriethyl acetate 60:
40, shake gently for 30 s, centrifuge, repeat the extraction with 5 mL and 3 mL portions of
diethyl etheriethyl acetate 60:40. Combine the upper organic layers and evaporate them to
dryness under a stream of nitrogen at 60, reconstitute the residue in 200 jxL MeCN: water 50:
50, sonicate for 5 min, inject a 50 \LL aliquot.
HPLCVARIABLES
Column: 125 X 4 LiChrosorb RP18
Mobile phase: MeCN:THF:water 50:10:40 containing 50 mM ammonium acetate
Flow rate: 1
Detector: MS, Vestec Model 20IA, thermospray, positive-ion chemical ionization, electron beam
250 |xA, electron multiplier 2000 V, source block 250, tip heater 250, lens assembly 150,
vaporizer probe 10 below take-off point, m/z 300
CHROMATOGRAM
Retention time: 2.7
KEYWORDS
sheep; muscle; liver; pharmacokinetics; LC-MS
REFERENCE
Blanchflower,W.J.; Cannavan,A.; Kennedy,D.G. Determination of fenbendazole and oxfendazole in liver and
muscle using liquid chromatography-mass spectrometry, Analyst, 1994, 119, 1325-1328.
Fenbufen
Merck Index: 4003
Lednicer No.: 2 126
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL Plasma with 500 (xL 50 mM pH 7.0 phosphate buffer for 1
min, add 2 mL dichloromethane, vortex for 1 min, shake for 5 min. Centrifuge at 100 g for 10
min, separate the organic layer, repeat the extraction procedure twice, evaporate the combined
organic layers to dryness under reduced pressure. Reconstitute the residue with 200 fxL 20
mM NaOH, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 fxm Supelcosil LC-SAX
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Retention time: 3.5
Internal standard: fenbufen
OTHER SUBSTANCES
Extracted: furprofen, rufloxacin
KEYWORDS
plasma; fenbufen is IS
REFERENCE
Carlucci,G.; Mazzeo,P. Simultaneous determination of furprofen and rufloxacin in human plasma by high-
performance liquid chromatography, J.Chromatogr.Sci., 1996, 34, 182184.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 20 |xL 20 u-g/mL IS in MeOH. Vortex for 1 min
with dichloromethane:diethyl ether 80:20, centrifuge at 1000 g for 10 min, separate the organic
twice, evaporate the organic phase to dryness under a stream of nitrogen, add 100 jxL 10 mM
NaOH to the residue, shake for 5 min, inject a 20 u,L aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 10 (xm Vydac AXGU
Flow rate: 1.2
Detector: UV 280
CHROMATOGRAM
Retention time: 5.3
Internal standard: 2-[-(2-furoyl)phenyl]propionic acid (3.9)
OTHER SUBSTANCES
Extracted: felbinac, pefloxacin
KEYWORDS
plasma
REFERENCE
1115.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL serum with 20 u,L 20 |xg/mL IS in MeOH and 100 |JLL 20 mM
NaOH. Vortex for 1 min with 4 ml dichloromethanerdiethylether 80:20, centrifuge at 2000 g
organic layers, evaporate to dryness under a stream of nitrogen. Add 100 u,L 20 mM NaOH to
the residue, shake for 5 min, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 10 jim Vydac AXGU
Column: 250 X 4.6 5 |xm Supelcosil LC-SAX
Flow rate: 1.2
Detector: UV 280
CHROMATOGRAM
Retention time: 5.5
OTHER SUBSTANCES
Extracted: felbinac, lomefloxacin
KEYWORDS
plasma
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroformrisopropanol:
the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 uL aliquot of
HPLCVARIABLES
Column: 300 X 3.9 4 jim NovaPack C18
Flow rate: 0.8
Detector: UV 285
CHROMATOGRAM
KEYWORDS
REFERENCE
Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond-Elut C18 SPE cartridge with 1 mL MeOH. 1 mL
Serum + 6 |xL 1 mg/mL ketoprofen in MeOH + 1 mL water + 20 |xL saturated ammonium
sulfate solution + 60 JJLL concentrated HCl, vortex for 3 min, add to the SPE cartridge, wash
with three 1 mL portions of water, allow to dry for 3 min, elute with five 500 |xL portions of
MeOH. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue
in 1 mL mobile phase, inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 jxm Spheri-5 cyano
Mobile phase: MeCN:MeOH:water:phosphoric acid 21:22:56.5:0.5
Flow rate: 0.5
Detector: F ex 248 em 335 (filter) following post-column reaction. The column effluent flowed
through a knitted 7.9 m X 0.3 mm ID PTFE coil irradiated with an SC3-9 UV lamp (UVP, San
Gabriel CA) and cooled with a fan to the detector.
CHROMATOGRAM
Retention time: 8.5
Internal standard: ketoprofen (6.5)
OTHER SUBSTANCES
KEY WORDS
post-column reaction; post-column photochemical derivatization; serum; SPE
REFERENCE
Siluveru,M.; Stewart,J.T. Determination of fenbufen and its metabolites in serum by reversed-phase high-
performance liquid chromatography using solid-phase extraction and on-line post-column ultraviolet irra-
diation and fluorescence detection, J.Chromatogr.B, 1996, 682, 89-94.
SAMPLE
residue with 50 JJLL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Symmetry C8 (Waters)
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:50 mM phosphoric acid 40:60 adjusted to pH 5.5 with NaOH
Flow rate: 0.6
Detector: UV 283
OTHER SUBSTANCES
Also analyzed: carbamazepine, indomethacin, ketoprofen, a.-naphthoquinone, naproxen,
tolmetin
REFERENCE
SAMPLE
Matrix: urine
Add 1 mL urine, vortex, add 250 JJLL 1 M pH 5.0 acetate buffer, vortex. Add 250 |xL of the
mobile phase, inject a 10-30 (JLL aliquot.
HPLCVARIABLES
Flow rate: 0.9
Detector: UV 230
CHROMATOGRAM
Retention time: 9.5
OTHER SUBSTANCES
Extracted: diclofenac, ibuprofen, felbinac, flurbiprofen, ketoprofen, loxoprofen, mefenamic acid,
KEYWORDS
SPE
REFERENCE
Hirai,T.; Matsumoto,S.; Kishi,I. Simultaneous analysis of several non-steroidal anti-inflammatory drugs in
J.Chromatogr.B, 1997, 692, 375-388.
Fenbutrazate
Merck Index: 4005
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.2
OTHER SUBSTANCES
gosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenflura-
mine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, hal-
operidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl,
isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamyl-
amine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol,
mepyramine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene,
methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine,
methylephedrine, methylergonovine, methysergide, metoclopramide, metopimazine, metopro-
lol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nom-
ifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaver-
ine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine,
phenadoxone, phenampromide, phenazocine, phendimetrazine, phenelzine, phenglutarimide,
REFERENCE
Fencamfamine
Molecular formula: C15H21N
Merck Index: 4006
Lednicer No.: 1 74
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenoprofen, fenpro-
epinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, ox5rphen-
butazone, oxjrtetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, per-
REFERENCE
Hill,D.W.; Kind,A.J- Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994,
18, 233-242.
Fenchone
Molecular formula: C10H16O
Merck Index: 4008
SAMPLE
M a t r i x : solutions
HPLC VARIABLES
Column: 250 X 1 5 jim LiChrosorb RP18
Mobile phase: EtOH:water 35:65 containing 10 mM a-cyclodextrin and 0.5 mM tri-O-methyl-a-
cyclodextrin
Flow rate: 0.04
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: camphor
REFERENCE
Nowakowski,R.; BielejewskaA-l Duszczyk,K; Sybilska,D. Chiral discrimination by high-performance liquid
chromatography with joint use of two cyclodextrin additives, J.Chromatogr.A, 1997, 782, 1-11.
Fenethazine
Merck Index: 4013
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 4.4
Next Page
OTHER SUBSTANCES
gosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenfluramine, fenoterol,
REFERENCE
Fenfluramine
Molecular formula: C12H16F3N
Merck Index: 4015
Lednicer No.: 1 70
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 0.6 M pH 9.8 carbonate buffer + 40 |xL 5 (xg/mL
maprotiline in 10 mM HCl + 5 mL 200 g/L ethyl acetate in n-heptane, mix by rocking for 10
min, centrifuge at 1500 g for 10 min. Remove organic layer and add it to 150 |xL 100 mM HCl,
mix 10 min, centrifuge at 1500 g for 10 min. Discard organic layer and evaporate aqueous
layer at 45 in a vacuum centrifuge for 1 h. Take up residue in 50 JJLL 1 M pH 10.3 carbonate
buffer and 25 |xL 10 mg/mL dansyl chloride in MeCN, vortex, allow to react at room temper-
Previous Page
OTHER SUBSTANCES
gosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenfluramine, fenoterol,
REFERENCE
Fenfluramine
Molecular formula: C12H16F3N
Merck Index: 4015
Lednicer No.: 1 70
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 0.6 M pH 9.8 carbonate buffer + 40 |xL 5 (xg/mL
layer at 45 in a vacuum centrifuge for 1 h. Take up residue in 50 JJLL 1 M pH 10.3 carbonate
ature for 45 min, evaporate at 45 in a vacuum centrifuge for 20 min, reconstitute in 125 JJUL
MeCN:water 75:25, vortex, centrifuge for 3-5 min, inject a 25-40 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:25 mM KH2PO4 75:25 + 500 JULL/L orthophosphoric acid + 600 juiL/L n-
butylamine
Flow rate: 2
Detector: F ex 235 em 470 (cut-off)
CHROMATOQRAM
Internal standard: maprotiline (12.8)
OTHER SUBSTANCES
Simultaneous: fluoxetine, propranolol, clovoxamine, fluvoxamine, amoxapine, desipramine, pro-
triptyline, nortriptyline, sertraline, norfluoxetine
Noninterfering: amitriptyline, imipramine, clomipramine, trimipramine, mianserin, chlordiaz-
epoxide, trazodone, cyclobenzaprine, nomifensine, bupropion, metoprolol, atenolol, pindolol,
tranylcypromine, moclobemide, thioridazine, citalopram, clozapine, carbamazepine, doxepin,
loxapine
KEYWORDS
plasma
REFERENCE
Suckow,R.R; Zhang,M.R; Cooper,T.B. Sensitive and selective liquid-chromatographic assay of fluoxetine and
norfluoxetine in plasma with fluorescence detection after precolumn derivatization, Clin.Chem., 1992, 38,
1756-1761.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL 1.25 jxg/mL IS in water + 200 JULL water, vortex,
add 400 jxL 1 M trichloroacetic acid, vortex, centrifuge at 2500 g for 10 min. Remove 900 |xL
of the supernatant and add it to 250 (xL 1 M NaOH, add 2 mL reagent, vortex for 2 s, let stand
for 90 min. Remove a 1 mL aliquot of the lower organic layer and evaporate it to dryness under
a stream of nitrogen, reconstitute the residue in 200 |xL dichloromethane, inject a 35 JJLL ali-
quot. (Reagent was 10 jxM 3,5-dinitrophenylisocyanate in dichloromethane. Prepare as follows.
Stir 6.40 g 3,5-dinitrobenzoyl chloride in 100 mL glacial acetic acid, add 1.8Og sodium azide
in small increments, stir for 1 h, add 300 mL cold water. Filter off the 3,5-dinitrobenzyl azide
precipitate and wash it with a small portion of water. Dry overnight in a vacuum desiccator.
Reflux 25 mg 3,5-dinitrobenzyl azide dissolved in 5 mL toluene for 10 min, cool to room tem-
perature, make up to 50 mL with dichloromethane, dilute an aliquot 1:200 with dichlorome-
thane to give a 10 uM solution of 3,5-dinitrophenylisocyanate. Prepare fresh daily. CAUTION!
3,5-Dinitrobenzyl azide may be explosive and 3,5-dinitrophenylisocyanate may be toxic!)
HPLCVARIABLES
Column: 250 X 4.6 5 |xm (R)-naphthylurea Chiral (Supelco)
Mobile phase: Hexane:isopropanol:MeCN 89:9:2
Flow rate: 1.2 for 15 min, 3.5 for 13 min, 1.2 for 7 min
Detector: UV 235
CHROMATOGRAM
Retention time: 10 (d), 11.5 (1)
Internal standard: p-methylphenethylamine (20.7, first peak)
OTHER SUBSTANCES
KEYWORDS
plasma; conduct analyses under yellow light; chiral; pharmacokinetics; derivatization
REFERENCE
Zeng,J.-N.; Dou,L.; Duda,M.; Stuting,H.H. New chiral high-performance liquid chromatographic methodology
used for the pharmacokinetic evaluation of dexfenfluramine, J.Chromatogr.B, 1994, 654, 231-248.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chlorofornv.isopropanol:
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 264
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
HPLC VARIABLES
G u a r d column: 20 mm long Symmetry C18
Mobile p h a s e : Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A:B 85:
Column t e m p e r a t u r e : 30
Flow r a t e : 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5
mL/min)
Detector: UV 207.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Dissolve the contents of a 150 mg capsule in 1 L water, stir for 1 h, let
stand for 30 min, centrifuge an aliquot at 1250 g for 5 min. Dilute 1 mL of the supernatant to
50 mL with water. Remove a 1 mL aliquot and add it to 100 |xL 1 |xg/mL IS, add 100 |JLL 100
mM NaOH, add 2 mL reagent, vortex, let stand for 15 min, inject a 30 |xL aliquot of upper
aqueous layer (sic). (Reagent was 20 JXM 3,5-dinitrophenylisocyanate in dichloromethane. Pre-
pare as follows. Stir 6.40 g 3,5-dinitrobenzoyl chloride in 100 mL glacial acetic acid, add 1.80
g sodium azide in small increments, stir for 1 h, add 300 mL cold water. Filter off the 3,5-
dinitrobenzyl azide precipitate and wash it with a small portion of water. Dry overnight in a
vacuum desiccator. Reflux 25 mg 3,5-dinitrobenzyl azide dissolved in 5 mL toluene for 10 min,
cool to room temperature, make up to 50 mL with dichloromethane, dilute an aliquot 1:100
with dichloromethane to give a 20 jxM solution of 3,5-dinitrophenylisocyanate. Prepare fresh
daily. CAUTION! 3,5-Dinitrobenzyl azide may be explosive and 3,5-dinitrophenylisocyanate
may be toxic!)
HPLC VARIABLES
Column: 250 X 4.6 5 fim (R)-naphthylurea Chiral (Supelco)
Mobile phase: Hexanerdichloromethane:MeOH:MeCN 81:17.5:1:0.5 (For 1-isomer use 85:13.5:1:
0.5 and 200 (xM reagent.) (The exact ratios are very important.)
Flow rate: 1.5
Detector: UV 235
CHROMATOGRAM
Retention time: 10.3 (d)
Internal standard: -methylphenethylamine (17.6)
OTHER SUBSTANCES
KEYWORDS
conduct analyses under yellow light; chiral; derivatization; capsules
REFERENCE
Dou,L.; Zeng,J.-N.; Gerochi,D.D.; Duda,M.R; Stuting,H.H. Chiral high-performance liquid chromatography
methodology for quality control monitoring of dexfenfluramine, J.ChromatogrA, 1994, 679, 367-374.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: methadone, benzylmorphine, ethylmorphine, morphine-N-oxide, codeine, co-
deine-N-oxide, morphine, ethoheptazine, morphine-3-glucuronide, pholcodeine, norpethidine,
hydrocodone, dihydrocodeine, dihydromorphine, levorphanol, norcodeine, normorphine, pemo-
line, benzphetamine, diethylpropion, mazindol, tranylcypromine, caffeine, fenethyline, phen-
dimetrazine, methylphenidate, phenelzine, epinephrine, pipradol, phenylpropanolamine, fen-
camfamin, normetanephrine, 4-hydroxy amphetamine, bromo-STP, STP, prolintane,
2-phenethylamine, tyramine, trimethoxyamphetamine, phenylephrine, pseudoephedrine,
ephedrine, methylephedrine, dimethylamphetamine, methamphetamine, mescaline, mephen-
termine, buprenorphine, dextromoramide, phenoperidine, fentanyl, etorphine, piritramide, nos-
capine, papaverine, naloxone, dextropropoxyphene, nalorphine, phenazocine, norpipanone, lev-
allorphan, hydroxypethidine, normethadone, meperidine, dipipanone, diamorphine,
pentazocine
Interfering: chlorphentermine, norpseudoephedrine, methylenedioxyamphetamine, ampheta-
mine, acetylcodeine, monoacetylmorphine, thebacon, oxycodone, thebaine, norlevorphanol
REFERENCE
Law,B; Gill,R.; Moffat,AC. High-performance liquid chromatography retention data for 84 basic drugs of fo-
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 (jug/mL solution in MeOH, inject a 20 |xL aliquot.
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.1
OTHERSUBSTANCES
gosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenoterol,
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
B was MeCNrwater 90:10 containing 0.08% diethylamine and 0.09% phosphoric acid. A:B 95:
Flow rate: 0.5
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: diethylpropion, phenylpropanolamine, ephedrine, amphetamine, methampheta-
mine, phentermine
Also analyzed: amitriptyline, chlordiazepoxide, chlorpromazine, desalkylflurazepam, desipra-
REFERENCE
SAMPLE
Matrix: urine
Sample preparation: Condition a Bond Elut C8 SPE cartridge with MeOH, water, and buffer.
Dilute human and dog urine with an equal volume of buffer and add to the SPE cartridge,
wash with water, elute with 200 jxL MeOH. Evaporate the eluate to dryness under a stream
of nitrogen, reconstitute in MeOH, inject an aliquot. Inject mouse and rat urine directly. (Buffer
was 100 mM pH 9.0 phosphate buffer.)
HPLCVARIABLES
Mobile phase: Gradient. A was MeOH. B was MeOH:50 mM phosphoric acid 5:95. C was MeOH
water 5:95. D was MeOHiwater 70:30. A:B:C:D from 30:70:0:0 to 0:0:0:100 over 20 min, to 70:
30:0:0 over 5 min, maintain at 70:30:0:0 for 35 min, return to initial conditions over 1 min.
Flow rate: 1
Detector: Radioactivity or UV
CHROMATOGRAM
Retention time: 50
OTHER SUBSTANCES
KEYWORDS
mouse; rat; dog; human; pharmacokinetics; SPE
REFERENCE
Marchant,N.C; Breen,M.A.; Wallace,D.; Bass,S.; Taylor,A.R.; Ings,R.M.J.; Campbell,D.B.; Williams,J. Compar-
ative biodisposition and metabolism of 14C-()-fenfluramine in mouse, rat, dog and man, Xenobiotica, 1992,
22, 1251-1266.
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 200 JULL 5 M NaOH + 3 mL diethyl ether, vortex for 2 min,
centrifuge at 2200 g for 5 min, freeze in acetone-dry ice. Remove the organic layer and add it
to 120 |xL 500 mM sulfuric acid, vortex for 2 min, centrifuge at 2200 g for 5 min. Remove the
aqueous layer and evaporate traces of ether with a stream of nitrogen, inject a 100 fxL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 20 jxm jxBondapak C18
Mobile phase: MeCN:50 mM KH2PO4 25:75
Flow rate: 1.3
Detector: UV 210
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
REFERENCE
Gross,A.S.; Phillips,A.C; Boutagy,J.; Shenfield,G.M. Determination of dexfenfluramine and nordexfenfluramine
in urine by high-performance liquid chromatography using ultraviolet detection, J.Chromatogr., 1993, 621,
115-120.
SAMPLE
Matrix: urine
Sample preparation: 2 mL Urine + 200 |JLL 5 jig/mL IS in water + 400 |xL water, vortex for 5
s, add 400 |xL 1 M NaOH, vortex, centrifuge at 2500 g for 10 min. Remove the supernatant
and add it to 5 mL hexane:ethyl acetate 70:30, shake gently by hand for 1 min, let stand for
10 min. Remove 4 mL of the upper organic layer and evaporate it to about 30 JJLL under a
stream of nitrogen, add 1 mL reagent, vortex for 2 s, let stand for 30 min, inject a 35 JJLL
aliquot. (Reagent was 20 |xM 3,5-dinitrophenylisocyanate in dichloromethane. Prepare as fol-
lows. Stir 6.40 g 3,5-dinitrobenzoyl chloride in 100 mL glacial acetic acid, add 1.80 g sodium
azide in small increments, stir for 1 h, add 300 mL cold water. Filter off the 3,5-dinitrobenzyl
azide precipitate and wash it with a small portion of water. Dry overnight in a vacuum des-
iccator. Reflux 25 mg 3,5-dinitrobenzyl azide dissolved in 5 mL toluene for 10 min, cool to room
temperature, make up to 50 mL with dichloromethane, dilute an aliquot 1:100 with dichloro-
methane to give a 20 |xM solution of 3,5-dinitrophenylisocyanate. Prepare fresh daily. CAU-
TION! 3,5-Dinitrobenzyl azide may be explosive and 3,5-dinitrophenylisocyanate maybe toxic!)
HPLC VARIABLES
Column: 250 X 4.6 5 jmm (R)-naphthylurea Chiral (Supelco)
Mobile phase: Hexane:isopropanol:MeCN 90.5:7.5:2 (d isomer) or 89:9:2 (1 isomer)
Column temperature: ambient (1 isomer), 35 (d isomer)
Flow rate: 1.2
Detector: UV 235
CHROMATOGRAM
Retention time: 9.3 (d isomer), 10.7 (1 isomer)
Internal standard: p-methylphenethylamine (19.5, first peak, d-isomer system) (18.8,1-isomer
system, first peak)
OTHER SUBSTANCES
KEYWORDS
conduct analyses under yellow light; derivatization
REFERENCE
Zeng,J.-N.; Dou,L.; Duda,M.; Stuting,H.H. New chiral high-performance liquid chromatographic methodology
used for the pharmacokinetic evaluation of dexfenfluramine, J.Chromatogr.B, 1994, 654, 231248.
Fenofibrate
Molecular formula: C20H21CIO4
Merck Index: 4019
SAMPLE
Sample preparation: Condition a 200 mg Analytichem C8 SPE cartridge with 2 portions of
MeOH and 2 portions of water, do not allow to dry. 1 mL Plasma or urine + 5 |xg/mL naproxen
in MeOH + 500 jxL water + 250 jxL 1 M HCl, mix, add to the SPE cartridge, wash with 2
portions of water, elute with 500 |xL MeCN. Evaporate the eluate to 150 |xL under vacuum,
HPLCVARIABLES
Guard column: Bondapak C18/Corasil
Column: 100 X 8 10 |xm Radial-Pak C8 (Waters)
Mobile phase: MeCNrbuffer 35:65 (Buffer was 2.72 g KH2PO4 in 1 L water, pH adjusted to 3
with phosphoric acid.)
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Retention time: 7 (as fenofibric acid)
Internal standard: naproxen (4.5)
KEYWORDS
plasma; SPE
REFERENCE
Ramusino,A.C; Carozzi,A. Simple and rapid method for determining procetofenic acid, an active metabolite of
procetofen, in biological fluids by solid-phase extraction and high-performance liquid chromatography,
J.Chromatogr., 1986, 383, 419-424.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: feces, urine
Sample preparation: Urine. Add 3-5 mL urine to a C18 Sep-Pak SPE cartridge, wash with 10
mL water, elute with 10 mL MeOH. Evaporate the eluate to dryness under reduced pressure,
take up the residue in 200 |xL MeOH, inject an aliquot. Feces. Vortex 2 mL homogenized feces
with 10 mL MeOH for 90 s, centrifuge, evaporate the supernatant to dryness under reduced
pressure, take up the residue in 5 mL water, add to a C18 Sep-Pak SPE cartridge, wash with
10 mL water, elute with 10 mL MeOH. Evaporate the eluate to dryness under reduced pressure,
take up the residue in 200 JXL MeOH, inject an aliquot.
HPLC VARIABLES
Column: 100 X 8 RadialPAK |j,Bondapak
Mobile phase: MeOH:water 60:40 containing 1% glacial acetic acid
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Retention time: 68
OTHER SUBSTANCES
KEYWORDS
SPE
REFERENCE
Weil,A; Caldwell,J.; Strolin-Benedetti,M. The metabolism and disposition of 14C-fenofibrate in human vol-
unteers, Drug Metab.Dispos., 1990, 18, 115-120.
Fenoldopam
Molecular formula: C16H16CINO3
CAS Registry No.: 67227-56-9, 67227-57-0 (monomethanesulfonate)
Merck Index: 4020
Lednicer No.: 4 147
SAMPLE
Matrix: blood
Sample preparation: Add 4.75 mL plasma to 250 |xL 10% ascorbic acid before storage at -20.
1 mL Plasma + 50 |xL 500 ng/mL IS in 50 mM acetic acid + 5 mL ethyl acetate + 500 |xL 0.5
mM Na2HPO4, shake on a reciprocal shaker at 60 cycles/min for 10 min, centrifuge at 2000 g
for 10 min. Remove 4.5 mL of the organic layer and evaporate it to dryness under a stream of
nitrogen at 40, reconstitute the residue in 100 |xL 200 |xg/mL 2,3,4,6-tetra-O-acetyl-p-D-glu-
copyranosyl isothiocyanate in ethyl acetate (freshly prepared), add 50 |xL 1% triethylamine in
ethyl acetate (freshly prepared), let stand at room temperature for 1 h, evaporate to dryness
under a stream of nitrogen at 40, reconstitute the residue in 200 |xL MeCN:50 mM acetic acid
30:70, inject a 5-50 |xL aliquot.
HPLC VARIABLES
Guard column: 30 X 2.1 butylsilica (Pierce)
Column: 220 X 2.1 7 (Jim Aquapore butylsilica (Pierce)
Mobile phase: MeCN:MeOH:buffer:water 17:15:28:42 (Prepare buffer by dissolving 22 g sodium
acetate trihydrate, 21 g citric acid monohydrate, 9.8 g NaOH, and 0.63 g disodium EDTA in 2
L water, pH 5.6. Recycle mobile phase.)
Flow rate: 0.3
Detector: E, ESA, guard electrode +0.2 V, working electrode 1 -0.20 V, working electrode 2
+0.20 V
CHROMATOGRAM
Retention time: 3 (S), 6 (R)
Internal standard: 2,3,4,5-tetrahydro-l-phenyl-lH-3-benzazepine-7,8-diol (SK&F 38393-A) (25,
30 (enantiomers))
KEYWORDS
derivatization; chiral; plasma
REFERENCE
Boppana,V.K.; Geschwindt,L.; Cyronak,M.J.; Rhodes,G. Determination of the enantiomers of fenoldopam in
human plasma by reversed-phase high-performance liquid chromatography after chiral derivatization,
J.Chromatogr., 1992, 592, 317-322.
Fenoprofen
CAS Registry No.: 31879-05-7, 34507-40-5 (calcium salt), 53746-45-5 (calcium salt dihydrate)
Merck Index: 4021
Lednicer No.: 2 67
SAMPLE
Matrix: blood
Sample preparation: Activate a 1 mL Bond-Elut C8 SPE cartridge with 2 mL MeOH then 1
mL 10 mM HCl, do not allow it to dry completely. Sonicate 1 mL whole blood for 20-30 min
then apply to cartridge. Wash with 100 |JLL water, elute with three 500 |xL portions of MeOH:
MeCN: 1% aqueous ammonium hydroxide 50:20:30, combine eluents and evaporate to dryness
under a stream of nitrogen at 40. Redissolve in 1 mL MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.5 5 |xm Spherisorb ODS
Mobile phase: MeCN:MeOH:buffer 35:13:52 (Buffer was water adjusted to pH 3.2 with ortho-
phosphoric acid
Flow rate: 1
Detector: UV 250
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Simultaneous: ketoprofen, acetaminophen, salicylic acid, naproxen, ibuprofen, indomethacin
KEYWORDS
whole blood; SPE
REFERENCE
Moore,C.M.; Tebbett,I.R. Rapid extraction of anti-inflammatory drugs in whole blood for HPLC analysis, Fo-
rensic Sci.Int, 1987, 34, 155-158.
SAMPLE
Matrix: blood
Sample preparation: 500 |JLL Plasma + 200 jxL 2 M HCl + 6 mL ice-cold hexane:diethyl ether
8:2, extract, centrifuge at 1500 g for 10 min. Remove 5 mL of organic layer and evaporate it
to dryness under a stream of nitrogen. Dissolve in 250 (xL isopropanol:water 2:8, inject a 40
IxL aliquot.
HPLC VARIABLES
Column: 100 X 4.0 AGP (EnantioPac)
Mobile phase: 20 mM pH 6.7 phosphate buffer containing 0.5% isopropanol and 5 mM
dimethyloctylamine
Flow rate: 0.5
Detector: UV 220
CHROMATOGRAM
Retention time: 34 (R), 42 (S)
OTHER SUBSTANCES
Simultaneous: ibuprofen
KEY WORDS
plasma; chiral
REFERENCE
Menzel-Soglowek,S.; Geisslinger,G.; Brune,K. Stereoselective high-performance liquid chromatographic deter-
mination of ketoprofen, ibuprofen and fenoprofen in plasma using a chiral c^-acid glycoprotein column,
J.Chromatogr., 1990, 532, 295-303.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 50 (xL MeOH:water 1:4 + 200 |xL 1 M sulfuric acid + 3
mL isooctane:isopropanol 95:5, vortex 30 s, centrifuge at 1800 g for 5 min. Remove organic
layer and evaporate it to dryness. Add 300 |xL 50 mM triethylamine in MeCN and 50 JJLL 6
mM ethylchloroformate in MeCN, wait 30 s, add 25 |xL 0.1% (S)-naphthylethylamine in MeCN:
triethylamine 98:2, after 3 min add 25 |xL 2.5% ethanolamine in MeCN, inject 2-30 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Partisil ODS 3 RAC
Mobile phase: MeCN:water:acetic acid:triethylamine 60:40:0.1:0.02, final pH 5.0 (After every
third injection flush with MeCN for 6 min at 1.6 mL/min, equilibrate with mobile phase for 9
min.)
Flow rate: 1.2
CHROMATOGRAM
Internal standard: fenoprofen
OTHER SUBSTANCES
Extracted: ibuprofen
KEY WORDS
plasma; chiral; UV 232 (see Clin.Chem. 1988; 34; 493); derivatization; fenoprofen is IS
REFERENCE
Lemko,C.H.; Caille,G.; Foster,R.T. Stereospecific high-performance liquid chromatographic assay of ibuprofen:
improved sensitivity and sample processing efficiency, J.Chromatogr., 1993, 619, 330-335.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 125 |xL 40 mM decanoic acid in MeCN, mix. Dialyze a
100 |xL sample against 20 mM pH 7.0 phosphate buffer using a Gilson Cuprophane membrane
(molecular mass cut-off 15 kDa). Continuously pump the buffer through the dialysis cell and
through column A at 3 mL/min for 9.6 min, backflush the contents of column A onto column B
with the mobile phase, monitor the effluent from column B. (After each injection flush plasma
channel with 1 mL 0.05% Triton X-100, with I m L l mM HCl, and with 2 mL water. After
each injection flush buffer channel with 3 mL 20 mM pH 7.0 phosphate buffer and condition
column A with 1 mL 20 mM pH 7.0 phosphate buffer.)
HPLC VARIABLES
Column: A 10 X 2 40 fun Bondesil C18 (Analytichem); B 250 X 3.1 5 |xm C18 (RoSiI Research
Separation Laboratories)
Flow rate: 1
Detector: UV 272
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: flurbiprofen (UV 247), ibuprofen (UV 264), ketoprofen (UV 261), naproxen (UV 272)
KEYWORDS
plasma; dialysis; column-switching
REFERENCE
Herraez-Hernandez,R.; Van de Merbel,N.C; Brinkman,U.A.T. Determination of the total concentration of
highly protein-bound drugs in plasma by on-line dialysis and column liquid chromatography: application
to non-steroidal anti-inflammatory drugs, J.Chromatogr.B, 1995, 666, 127-137.
SAMPLE
Matrix: blood
the residue in 100 jxL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 jxL aliquot of
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 272
CHROMATOGRAM
KEYWORDS
lam; prazosin; flunitrazepam; clonazepam; metoclopr amide; melphalan; estazolam;
REFERENCE
254-262.
SAMPLE
Matrix: blood
Sample preparation: 500 IJLL Plasma + 50 |xL water + 200 |xL 20% sulfuric acid + 6 mL n-butyl
chloride, vortex for 5 min, centrifuge at 950 g for 5 min, freeze in dry ice/acetone. Remove the
organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue
in 300 fxL 50 mM triethylamine, sonicate for 1 min, vortex for 30 s, add 50 |xL 6 mM ethyl
chloroformate, let stand for 30 s, add 25 jxL 10 mM S-(-)-l-(l-naphthyl)ethylamine, let stand
for 3 min, add 25 |xL MeCN:ethanolamine 40:1. Evaporate to dryness under a stream of nitro-
gen at 40, reconstitute the residue in 250 JULL mobile phase, inject a 25 |ULL aliquot.
HPLC VARIABLES
Guard column: 15 X 3.2 7 |xm Newguard RP-18
Column: 150 X 4.6 5 jxm Inertsil ODS-2
Mobile phase: MeCN:water (pH 3.0) 66.5:33.5
Flow rate: 1.2
CHROMATOGRAM
OTHER SUBSTANCES
KEY WpRDS
derivatization; chiral; plasma; fenoprofen is IS
REFERENCE
Lau,Y.Y. Determination of ibuprofen enantiomers in human plasma by derivatization and high-performance
liquid chromatography with fluorescence detection, J.Liq.Chromatogr.Rel.Technol., 1996, 19, 21432153.
SAMPLE
Sample preparation: Plasma. 500 |xL Plasma + 50 |xL 50 jxg/mL ketoprofen in 10 mM NaOH
+ 100 |xL 600 mM sulfuric acid + 3 mL isooctanerisopropanol 95:5, vortex for 30 s, centrifuge
at 3000 rpm for 5 min. Remove the organic layer and add it to 2.5 mL water, vortex for 15 s,
centrifuge for 3 min. Remove the aqueous layer and add it to 200 |xL 600 mM sulfuric acid,
add 2.5 mL chloroform, vortex for 15 s, centrifuge for 3 min. Remove the organic layer and
evaporate it to dryness under reduced pressure, reconstitute with 100 (xL 50 mM triethylamine
in MeCN, add 50 |xL 60 mM ethyl chloroformate in MeCN, after 30 s add 50 |xL 1 M 1-leuci-
namide in MeOH containing 1 M triethylamine, after 2 min add 50 |xL water, inject a 10-40
[xL aliquot. Urine. 500 JXL Urine + 250 |xL 1 M NaOH, mix, add 300 JXL 600 mM sulfuric acid,
add 50 |xL 50 |xg/mL ketoprofen in 10 mM NaOH, add 3 mL isooctanedsopropanol 95:5, vortex
for 30 s, centrifuge at 3000 rpm for 5 min. Remove the organic layer and add it to 2.5 mL
water, vortex for 15 s, centrifuge for 3 min. Remove the aqueous layer and add it to 200 |xL
600 mM sulfuric acid, add 2.5 mL chloroform, vortex for 15 s, centrifuge for 3 min. Remove
the organic layer and evaporate it to dryness under reduced pressure, reconstitute with 100
IxL 50 mM triethylamine in MeCN, add 50 |xL 60 mM ethyl chloroformate in MeCN, after 30
s add 50 (JLL 1 M 1-leucinamide in MeOH containing 1 M triethylamine, after 2 min add 50 |xL
water, inject a 10-40 |xL aliquot.
HPLC VARIABLES
Guard column: 20 mm long 37-53 u-m reverse phase
Mobile phase: MeCN:70 mM KH2PO4:triethylamine 65:35:0.02, pH 6.0
Flow rate: 1 (plasma), 1.2 (urine)
CHROMATOGRAM
Retention time: 16.3 (R, urine), 19.1 (R, plasma or S, urine), 22.0 (S, plasma)
Internal standard: ketoprofen (9, 10 (enantiomers))
KEYWORDS
plasma; derivatization; pharmacokinetics; chiral
REFERENCE
Mehvar,R.; Jamali,F. Stereospecific high-performance liquid chromatographic (HPLC) assay of fenoprofen en-
antiomers in plasma and urine, Pharm.Res., 1988, 5, 5356.
SAMPLE
Sample preparation: Plasma. Adjust pH of 1 mL of plasma immediately to 2-4 with 50 (xL 85%
phosphoric acid, add 2 mL MeCN, centrifuge, evaporate the MeCN, extract with 3 mL ethyl
acetate. Evaporate the organic solvent to dryness, reconstitute in 250 |xL mobile phase, 10 jxL
0.125 mM ketoprofen, and 20 jxL 0.2 mM flunoxaprofen, inject a 100 jxL aliquot. Urine. 500
IJLL Urine + 500 (xL mobile phase + 50 JJLL 0.25 mM ketoprofen, inject a 100 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jmm Ultrasphere ODS
Mobile phase: Gradient. MeCNrIO mM pH 2.5 tetrabutylammonium hydrogen sulfate 25:75 for
15 min, 32:68 for 10 min, 38:62 for 12 min (step gradient).
Flow rate: 1
Detector: UV 272
CHROMATOGRAM
Internal standard: ketoprofen (29.9), flunoxaprofen (42.2)
OTHER SUBSTANCES
Extracted: metabolites, glucuronides
KEY WORDS
REFERENCE
Volland,C; Sun,H; Benet,L.Z. Stereoselective analysis of fenoprofen and its metabolites, J.Chromatogr., 1990,
534, 127-138.
SAMPLE
Sample preparation: 100 |xL Urine or rat plasma or 500 (JLL human plasma + 50 |xL MeOH: 10
mM NaOH 10:90 + 200 JULL 600 mM sulfuric acid + 3 mL isooctane.isopropanol 95:5, vortex for
30 s, centrifuge at 1800 g for 5 min. Remove the organic layer and evaporate it to dryness,
reconstitute the residue in 200 jxL 50 mM triethylamine in MeCN, add 50 /xL 6 mM ethyl
chloroformate in MeCN, vortex for 30 s, add 50 (xL 500 mM R-(+)-a-phenylethylamine in MeCN:
triethylamine 80:20, vortex briefly, let stand for 2 min, add 1 mL 250 mM HCl, add 3 mL
chloroform, vortex for 30 s, centrifuge at 1800 g for 2 min. Remove the organic layer and
evaporate it to dryness, reconstitute the residue in 200 |JLL mobile phase, inject a 10-150 |xL
aliquot.
HPLCVARIABLES
Guard column: 20 X 4.6 37-53 (xm reversed-phase
Column: 100 X 4.6 5 ^m C18 (Phenomenex)
Mobile phase: MeCN:water:acetic acid:triethylamine 46.5:53.5:0.1:0.03, pH 4.9
Flow rate: 1.6
Detector: UV 225
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
derivatization; human; chiral; rat; fenoprofen is IS; plasma
REFERENCE
Wright,M.R.; Sattari,S.; Brocks,D.R.; Jamali,F. Improved high-performance liquid chromatographic assay
method for the enantiomers of ibuprofen, J.Chromatogr., 1992, 583, 259-265.
SAMPLE
Sample preparation: Plasma. 500 (JLL Plasma + 100 JJIL 600 mM sulfuric acid + 4 mL 2,2,4-
trimethylpentanerisopropanol 95:5, vortex for 10 s, centrifuge at 1800 g for 5 min. Remove the
organic layer and evaporate it to dryness under reduced pressure, reconstitute the residue in
180 fxL mobile phase, vortex for 10 s, inject a 100 |xL aliquot. Urine. 500 |xL Urine + 100 |xL
600 mM sulfuric acid + 4 mL 2,2,4-trimethylpentane:isopropanol 95:5, vortex for 10 s, centri-
fuge at 1800 g for 3 min. Remove the organic layer and add it to 3 mL water, vortex for 10 s,
centrifuge for 3 min. Remove the aqueous phase and add it to 200 |xL 600 mM sulfuric acid
and 3 mL chloroform, vortex for 10 s, centrifuge for 3 min. Remove the organic phase and
evaporate it to dryness, reconstitute the residue in 180 |xL mobile phase, vortex for 10 s, inject
a 100 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Chiralpak AD amylose carbamate (Chiral Technologies)
Mobile phase: Hexane:isopropanol:trifluoroaceticacid 80:19.9:0.1
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ketoprofen
KEYWORDS
plasma; chiral; fenoprofen is IS
REFERENCE
Carr,R.A.; Caille,G.; Ngoc,A.H.; Foster,R.T. Stereospecific high-performance liquid chromatographic assay of
ketoprofen in human plasma and urine, J.Chromatogr.B, 1995, 668, 175-181.
SAMPLE
Sample preparation: Plasma. 2 mL Plasma + 250 JJLL 1 M HCl + 50 jxL prenazone solution,
vortex briefly, extract twice with 5 mL portions of diethyl ether for 15 min each time. Combine
tute the residue in 200 jxL MeCN: 1% aqueous acetic acid 50:50, inject an aliquot. Urine. 500
|xL Urine + 250 |xL 1 M HCl + 50 JJLL prenazone solution, mix, extract with 5 mL diethyl ether
for 15 min. Remove the organic layer and add it to 1 mL 1% sodium bicarbonate solution
(freshly prepared), vortex. Remove the organic layer and evaporate it to dryness under a stream
of nitrogen at 36, reconstitute the residue in 200 jxL MeCN: 1% aqueous acetic acid 50:50,
inject an aliquot. (To hydrolyze conjugates mix 500 |xL urine, 100 |xL 1M pH 5.2 sodium acetate
buffer, and 20 jxL enzyme solution, heat at 56 for 2.5 h, cool, proceed as above. The enzyme
solution was Sue Helix Pomatia containing 100 000 Fishman U/mL of p-glucuronidase and 1
000 000 Roy U/mL of arylsulfatase (IBF).)
HPLC VARIABLES
Column: 100 X 3 5 |xm Nucleosil
Mobile phase: Gradient. MeCN: 1% aqueous acetic acid 50:50 for 7 min, to 80:20 over 0.6 min,
maintain at 80:20 for 3.4 min, re-equilibrate at initial conditions for 8 min. (For urine isocratic
MeCN: 1% aqueous acetic acid 50:50.)
Flow rate: 0.5
Detector: UV 230
CHROMATOGRAM
Internal standard: prenazone
KEYWORDS
horse; plasma; pharmacokinetics
REFERENCE
Delbeke,F.T.; Landuyt,J.; Debackere,M. Disposition of human drug preparations in the horse. IV. Orally ad-
ministered fenoprofen, J.Pharm.BiomedAnaL, 1995, 13, 1041-1047.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: 10 mg Compound + 10 mg l-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride + 2 drops 3,5-dimethylaniline + 1.5 mL dichloromethane, mix, after 30 min add
1 mL 1 M HCl, shake vigorously. Remove the lower organic layer and dry it over anhydrous
magnesium sulfate, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm D N-(3,5-dinitrobenzoyl)phenylglycine (Regis)
Mobile phase: Hexanedsopropanol 80:20
Flow rate: 2
CHROMATOGRAM
Retention time: k' 2.50 (for first enantiomer)
OTHER SUBSTANCES
Also analyzed: carprofen, cicloprofen, etodolac, flurbiprofen, ibuprofen, ketoprofen, naproxen,
pirprofen, tiaprofenic acid
KEYWORDS
derivatization; a = 1.25; chiral
REFERENCE
Pirkle,W.H.; Murray,P.G. The separation of the enantiomers of a variety of non-steroidal anti-inflammatory
drugs (NSAIDS) as their anilide derivatives using a chiral stationary phase, J.Liq.Chromatogr., 1990, 13,
2123-2134.
SAMPLE
the supernatant and add it to 200 |xL 100 jxg/mL IS in DMF, mix, add 200 |xL 5 M HCl, extract
under a stream of nitrogen, reconstitute the residue in 1 mL dichloromethane, add 20 |xL 10
mg/mL 1-hydroxybenzotriazole in dichloromethaneipyridine 99:1, add 300 |xL 10 mg/mL l-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in dichloromethane, add 300 |xL 10
mg/mL (-)-(S)-a-methylbenzylamine in dichloromethane, let stand for 30 min, evaporate to dry-
ness, reconstitute with 500 jxL mobile phase, inject a 10 |xL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: (S)-naproxen (100 jxg/mL)
Limit of detection: 5 jxg/mL
KEYWORDS
REFERENCE
Thomason,M.J.; Hung,Y.-R; Rhys-Williams,W.; Hanlon,G.W.; Lloyd,A.W. Indirect enantiomeric separation of 2-
arylpropionic acids and structurally related compounds by reversed phase HPLC, J.Pharm.Biomed.Anal.,
1997, 15, 1765-1774.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 4 X 4 5 ixm LiChroCart LiChrospher 100 RP-18
Column: 125 X 4 5 |xm LiChroCart LiChrospher 100 RP-18
Mobile phase: MeCN:pH 4.8 sodium acetate 38:62
Flow rate: 1.5
Detector: UV 223
CHROMATOGRAM
OTHER SUBSTANCES
REFERENCE
Dominkus,M.; Nicolakis,M.; Kotz,R.; Wilkinson,F.E.; Kaiser,R.R.; Chlud,K. Comparison of tissue and plasma
levels of ibuprofen after oral and topical administration, Arzneimittelforschung, 1997, 46, 1138-1143.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
phine, diazepam, diclofenac, diethylpropion, diethylstilbestrol, difiunisal, digitoxin, digoxin, dil-
ethacrynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fen-
proporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymester-
puromycin, p5nilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Mix 1 mL 100 |xg/mL compound in dichloromethane with 300 |xL 100 |xg/
mL 1-hydroxybenzotriazole in dichloromethane:pyridine 99:1, 300 |xL 1.1 mg/mL l-ethyl-3-di-
methylaminopropylcarbodiimide hydrochloride in dichloromethane, and 300 |xL 300 |xg/mL
benzylamine in dichloromethane, vortex, let stand at room temperature for 1.5 h, evaporate to
dryness under a stream of nitrogen, reconstitute the residue in 500 |JLL MeOH, inject an aliquot.
HPLCVARIABLES
Column: 150 X 4.6 10 fxm EXP BlOl tris(4-methylbenzoate) cellulose on silica (Bio-Rad)
Mobile phase: MeOH:buffer 70:30 (Prepare buffer solution by dissolving 14.05 g sodium per-
chlorate in water, adjust pH to 2.0, make up to 1 L with water.)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: benoxaprofen (MeOH:buffer 80:20), carprofen, flurbiprofen, ibuprofen, ketopro-
fen, pirprofen, tiaprofenic acid
KEYWORDS
REFERENCE
Van Overbeke,A.; Baeyens,W.; Van den Bossche,W.; Dewaele,C. Separation of 2-arylpropionic acids on a cellu-
lose based chiral stationary phase by RP-HPLC, J.Pharm.Biomed.Anal, 1994, 12, 901-909.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 125 X 3 Ecocart LiChrospher 100 RP-18
Mobile phase: MeCN: 100 mM pH 7.4 KH2PO4 30:70
Flow rate: 0.4
Injection volume: 2.5
CHROMATOGRAM
Retention time: 4.6
OTHER SUBSTANCES
Simultaneous: lonazolac (F ex 282 em 345)
REFERENCE
Baeyens,W.R.G.; Van Der Weken,G.; Lievens,L.; Van Overbeke,A. LC-study of lonazolac, naproxen and related
non-steroidal anti-inflammatory drugs in a classical and a narrow-bore set-up applying UV and fluorescence
detection, Biomed.Chromatogr., 1995, 9, 263-264.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.5 5 fxm Ultrasphere ODS
Mobile phase: MeCN: 10 mM tetrabutylammonium buffer 45:55
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
REFERENCE
Bischer,A.; Iwaki,M.; Zia-Amirhosseini,R; Benet,L.Z. Stereoselective reversible binding properties of the glu-
curonide conjugates of fenoprofen enantiomers to human serum albumin, Drug Metab.Dispos., 1995, 23,
900-903.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 jim LiChrospher 100 RP-8 (B)
Mobile phase: MeCN:0.025% phosphoric acid.buffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 mL
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ethidium bromide, ethopropazine, fentanyl, flavoxate, fiuoxetine, fluphenazine, flurazepam,
zine, procyclidine, promazine, promethazine, prooafenone, propantheline, propiomazine, pro-
lactone, sulfinpâzone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophyl-
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: 1 mL 5 mM fenoprofen in dichloromethane + 300 |xL 1 mg/mL hydroxy-
benzotriazole in dichloromethane.pyridine 99:1 + 300 |xL 11 mg/mL l-ethyl-3-dimethylamino-
propylcarbodiimide in dichloromethane + 300 |xL 3.47 mg/mL 1-naphthylamine (Caution! 1-
Naphthylamine in a carcinogen!) in dichloromethane, vortex, let stand for 1 h, evaporate to
dryness under a stream of nitrogen, reconstitute with 5 mL MeOH, inject an aliquot.
HPLCVARIABLES
Column: 150 X 2.1 Tolycellulose EXP BlOl (tris(4-methylbenzoate)cellulose covalently bonded
to 10 |xm aminopropylsilica)
Mobile phase: MeOH.buffer 85:15 (Buffer was 14.05 g/L sodium perchlorate adjusted to pH 2.0.)
Flow rate: 0.21
Injection volume: 1
CHROMATOGRAM
Retention time: k' 3.21 (first enantiomer)
OTHER SUBSTANCES
Also analyzed: flurbiprofen, ibuprofen, ketoprofen, tiaprofenic acid
KEYWORDS
derivatization; narrow-bore; chiral; a=1.31; (see Biomed. Chromatogr. 1995; 9; 292)
REFERENCE
Van Overbeke,A-; Baeyens,W.; Van Der Weken,G.; Van de Voorde,L; Dewaele,C. Comparative chromatographic
study on the chiral separation of the 1-naphthylamine derivative of ketoprofen on cellulose-based columns
of different sizes, Biomed.Chromatogr., 1995, 9, 289-290.
Fenoterol
CAS Registry No.: 13392-18-2, 1944-12-3 (HBr), 1944-10-1 (HBr)
Merck Index: 4022
Lednicer No.: 2 38
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.6
OTHER SUBSTANCES
mine, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, fiurazepam, haloperidol,
thipendyl, protriptyline, proxymetacaine, pseudoephedrine, p^methamine, quinidine, qui-
REFERENCE
Jane,L; McRinnon,A.; Flanagan,R-J- High-performance liquid chromatographic analysis of basic drugs on silica
Fenoverine
Merck Index: 4023
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y; P6pin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
163.
Fenozolone
Merck Index: 4028
SAMPLE
HPLC VARIABLES
Column temperature; 30
mL/min)
Detector: UV 220.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.ChromatogrA, 1997, 763,149-
163.
Fenproporex
Merck Index: 4036
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
camfamine, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymes-
butazone, oxj^tetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, per-
puromycin, pjîlamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
18, 233-242.
Fenprostalene
Merck Index: 4037
Lednicer No.: 4 9
SAMPLE
Matrix: solutions
Next Page
HPLC VARIABLES
Mobile phase: MeOH:20 mM acetic acid 55:45
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
REFERENCE
Johnson,D.M.; Taylor,W.F.; Thompson,G.F.; Pritchard,R.A. Degradation of fenprostalene in aqueous solution,
J.Pharm.Sci., 1983, 72, 946-948.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN.water 35:65 to 45:55
Detector: UV 219
CHROMATOGRAM
Retention time: 22
REFERENCE
Johnson,D.M.; Taylor,W.F. Degradation of fenprostalene in polyethylene glycol 400 solution, J.Pharm.Sci.,
1984, 73, 1414-1417.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Polytron Model PT-10-ST/PT-35) 60 g liver with 240 mL cold
water, adjust to pH 2 with 2 M HCl, extract three times with an equal volume of ethyl acetate.
Combine the extracts, neutralize with solid sodium bicarbonate, dry over anhydrous sodium
sulfate, evaporate to 50 mL under vacuum, evaporate to dryness, take up the residue in 50
mL MeOH:water 90:10, wash with 40 mL hexane, evaporate the MeOH/water phase to dryness,
dissolve the residue in MeOH, chromatograph on silica gel 60F-254 tic plates (EM Science)
with ethyl acetate:MeOH 80:20, scrape off the fenprostalene layer, desorb with 100 JJLL mobile
HPLC VARIABLES
Column: jiBondapak C18
Mobile phase: MeOH: 10 mM acetic acid 60:40
Flow rate: 1
Detector: UV 225
KEYWORDS
pig; liver
REFERENCE
Spires,H.R.; Bowen,J.L.; Tomlinson,R.V.; Donahue,D.J. Pharmacokinetic and tissue residue characteristics of
fenprostalene, a prostaglandin F2 a analog, in swine, Am. J. Vet.Res., 1990, 51, 386-390.
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Fentanyl
CAS Registry No.: 437-38-7, 990-73-8 (citrate)
Merck Index: 4043
Lednicer No.: 1 299
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 50 |xL 5 M NaOH + 100 |xL MeCN + 600 JXL hexane,
mix, centrifuge at 2000 rpm for 5 min. Evaporate the organic phase under nitrogen at 30 for
about 10 min. Reconstitute the residue in 100 JJLL MeCN:50 mM pH 3.0 phosphate buffer 50:
50, inject a 50 jxL aliquot. (Use silanized glassware.)
HPLCVARIABLES
Column: 100 X 8 4 |xm cyano column (Waters)
Flow rate: 2.5
Detector: UV 210
CHROMATOGRAM
Retention time: 6.2
OTHER SUBSTANCES
Noninterfering: ampicillin, calcium chloride, clafoxan, diazepam, dobutamine, dopamine, furo-
semide, gentamicin, morphine, midazolam, pavulon, phenytoin, vitamin K
KEYWORDS
plasma
REFERENCE
Bansal,R.; Aranda,J.V. High-performance liquid chromatography microassay for the simultaneous determina-
tion of fentanyl and its major metabolites in biological samples, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19,
353-364.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL BondElut C18 SPE cartridge once with 1 M HCl, twice
with MeOH, and once with water, remove the liquid completely with suction each time. Add
250 |xL IS solution and 250 u,L serum to the column at 1 mL/min, wash twice with water and
once with MeCN draining the column completely after each wash, elute with 250 |xL eluting
solution, centrifuge for 20 s to remove last of eluate, inject a 5 JJLL aliquot of the eluate. (Prepare
IS solution by adding 40 |xL 1 mg/mL N-pentyl-2,6-pipecoloxylidide (l-pentyl-N-(2,6-dimethyl-
phenyl)-2-piperidinecarboxamide, pentyl-PPX) in MeOH to 10 mL 100 mM NaH2PO4. Eluting
solution was 2.5 mL 35% perchloric acid in 100 mL MeOH.)
HPLC VARIABLES
Guard column: 15 X 3.2 7 |xm RP-8 (Applied Biosystems)
Column: 150 X 4.6 5 jxm Ultrasphere octyl
Mobile phase: MeCNrIO mM KH2PO4 25:80, pH 5.2
Flow rate: 1.5
Injection volume: 5
Detector: UV 205
CHROMATOGRAM
Internal standard: N-pentyl-2,6-pipecoloxylidide (l-pentyl-N-(2,6-dimethylphenyl)-2-piperidi-
necarboxamide, pentyl-PPX) (14.5)
OTHER SUBSTANCES
Extracted: bupivacaine, mepivacaine, meperidine
Noninterfering: acetaminophen, codeine, epinephrine, morphine, diazepam
KEYWORDS
serum; SPE
REFERENCE
Gupta,R.N.; Dauphin,A. Column liquid chromatographic determination of bupivacaine in human serum using
solid-phase extraction, J.Chromatogr.B, 1994, 658, 113-119.
SAMPLE
Matrix: blood
with 3 mL 750 mIVL methanol. Elute with three aliquots of 300 |xL 0.1 M ammonium acetate
IxL 0.1 M phosphoric acid (pH 2.5), mix, separate, inject a 100 |xL aliquot of the aqueous phase.
HPLCVARIABLES
Mobile phase: MeCNibuffer 35:65 (Buffer was 10 mL/L triethylamine in water adjusted to pH
Flow rate: 2
Detector: UV 228
CHROMATOGRAM
Retention time: 2.8
OTHER SUBSTANCES
Extracted: acetazolamide, amitriptyline, chlordiazepoxide, chlorimipramine, chlorpromazine, de-
sipramine, dextromethorphan, diazepam, doxepin, encainide, fluoxetine, flurazepam, hydroxy-
ethymurazepam, ibuprofen, imipramine, lidocaine, maprotiline, methadone, methaqualone,
mexiletine, midazolam, norchlorimipramine, nordiazepam, norfluoxetine, nortriptyline, nor-
verapamil, pentazocine, promazine, propafenone, propoxyphene, propranolol, protriptyline,
quinidine, temazepam, trimipramine, verapamil
warfarin
Interfering: diphenhydramine, flecainide, nordoxepin, haloperidol (reduced), trazodone
KEY WORDS
plasma; SPE
REFERENCE
Nichols,J.H.; Charlson,J.R.; Lawson,G.M. Automated HPLC assay of fluoxetine and norfiuoxetine in serum,
Clin.Chem., 1994, 40, 1312-1316.
SAMPLE
Sample preparation: 50 jxL Plasma or urine + 50 jxL 4 M NaOH + 100 JJLL MeCN + 500 |JLL n-
hexane, vortex for 30 s, centrifuge at 2000 rpm for 5 min. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen at 30, reconstitute the residue in 100 (xL
mobile phase, inject a 50 ^xL aliquot.
HPLC VARIABLES
Column: 100 X 8 4 |xm Nova pak cyano
Flow rate: 2.5
Detector: UV 214
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Extracted: alfentanil, sufentanil
KEY WORDS
plasma
REFERENCE
Bansal,R; Aranda,J.V. Simultaneous microassay of alfentanil, fentanil, and sufentanil by high performance
liquid chromatography, J.Liq.Chromatogr., 1995, 18, 339-348.
SAMPLE
residue with 50 |xL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 255.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Inject a 20 |xL aliquot
HPLC VARIABLES
Column: 100 X 3.9 4 jjum Radial Pak phenyl (Waters)
Mobile phase: MeOH:buffer 65:35 (Buffer was 5 mM pH 4.8 phosphate buffer containing 1.4
mM tetrabutylammonium hydroxide.)
Flow rate: 3
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: degradation products, bupivacaine
KEYWORDS
REFERENCE
Tu,Y.H.; Stiles,M.L.; Allen,L.V.,Jr. Stability of fentanyl citrate and bupivacaine hydrochloride in portable pump
reservoirs, Am. J.Hosp.Pharm., 1990, 47, 2037-2040.
SAMPLE
Sample preparation: 100 JJLL Injection solution + 400 |xL 2.5 jxg/mL haloperidol in water, inject
a 100 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Brownlee C18
Mobile phase: MeCN:MeOH:10 mM NaH2PO4 24:31:45, pH adjusted to 5.0 with 2 M KOH
Flow rate: 1.7
Detector: UV 210
CHROMATOGRAM
Internal standard: haloperidol (9.74)
OTHER SUBSTANCES
Extracted: sufentanil
KEY WORDS
injections
REFERENCE
Dewell,W.M.,Jr.; Khandaghabadi,M.; D'Souza,M.J.; Solomon,H.M. High-performance liquid chromatographic
determination of fentanyl and sufentanil returned from the operating room, Am. J.Hosp.Pharm., 1993, 50,
2374-2375.
SAMPLE
Sample preparation: Dilute 5-fold with mobile phase, inject a 10 |xL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 fxm Spherisorb phenyl
Mobile phase: MeOH:buffer 65:35 (Buffer was 5 mM pH 4.8 KH2PO4 containing 1.4 mM tetra-
butylammonium hydroxide.)
Flow rate: 1.5
Detector: UV 229
CHROMATOGRAM
Retention time: 5.1
OTHER SUBSTANCES
Noninterfering: midazolam
KEYWORDS
injections; 5% dextrose; stability-indicating
REFERENCE
Bhatt-Mehta,V.; Johnson,C.E.; Leininger,N.; Agarwal,M. Stability of fentanyl citrate and midazolam hydro-
chloride during simulated intravenous coadministration, Am.J.Health-Syst.Pharm., 1995, 52, 511513.
SAMPLE
Sample preparation: 500 |JLL Microsomal incubation + 500 |xL DMSO, vortex, centrifuge, filter,
inject an aliquot of the filtrate.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Spherisorb ODS-2
Mobile phase: Gradient. A was 1 M ammonium acetate. B was MeCN:MeOH:THF:l M ammo-
nium acetate 30:20:40:10. A:B from 100:0 to 35:65 over 40 min.
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 40
OTHER SUBSTANCES
KEYWORDS
human; liver; pharmacokinetics
REFERENCE
Tateishi,T.; Wood,A.J.J.; Guengerich,F.R; Wood,M. Biotransformation of tritiated fentanyl in human liver mi-
crosomes. Monitoring metabolism using phenylacetic acid and 2-phenylethanol, Biochem.Pharmacol., 1995,
50, 1921-1924.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:water adjusted to pH 3 with phosphoric acid 70:30
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Internal standard: codeine
REFERENCE
Vanbever,R.; Le Boulenge\E.; Pr6at,V. Transdermal delivery of fentanyl by electroporation. I. Influence of elec-
trical factors, Pharm.Res., 1996, 13, 559-565.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH at a concentration of 1 mg/mL, inject a 20 |JLL aliquot.
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: tranylcypromine, caffeine, fenethyline, phendimetrazine, methylphenidate, phe-
nelzine, epinephrine, pipradol, phenylpropanolamine, fencamfamin, chlorphentermine, nor-
pseudoephedrine, phentermine, fenfluramine, methylenedioxyamphetamine, amphetamine,
normetanephrine, 4-hydroxyamphetamine, bromo-STP, STP, prolintane, 2-phenethylamine, ty-
ramine, trimethoxyamphetamine, phenylephrine, pseudoephedrine, ephedrine, methylephed-
rine, dimethylamphetamine, methamphetamine, mescaline, mephentermine, nalorphine, phen-
azocine, norpipanone, levallorphan, hydroxypethidine, normethadone, meperidine, dipipanone,
diamorphine, pentazocine, acetylcodeine, monoacetylmorphine, thebacon, oxycodone, thebaine,
norlevorphanol, methadone, benzylmorphine, ethylmorphine, morphine-N-oxide, codeine, co-
deine-N-oxide, morphine, ethoheptazine, morphine-3-glucuronide, pholcodine, norpethidine,
hydrocodone, dihydrocodeine, dihydromorphine, levorphanol, norcodeine, normorphine
Interfering: pemoline, benzphetamine, diethylpropion, mazindol, buprenorphine, dextromoram-
ide, phenoperidine, etorphine, piritramide, noscapine, papaverine, naloxone, dextro-
propoxyphene
REFERENCE
Law,B-; Gill,R-; Moffat,A-C. High-performance liquid chromatography retention data for 84 basic drugs of fo-
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 JJLL aliquot.
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.6
OTHER SUBSTANCES
ine, pargyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine,
phenadoxone, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine,
phenglutarimide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine,
phenothiazine, phenoxybenzamine, phentolamine, phenylephrine, phenyltoloxamine, physo-
stigmine, piminodine, pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperido-
late, pipradol, pirenzepine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine,
prilocaine, primaquine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, pro-
heptazine, prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, pro-
pranolol, prothipendyl, protriptyline, prox5maetacaine, pseudoephedrine, pyrimethamine, quin-
idine, quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine,
thenyldiamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine,
methoprim, trimipramine, tripelennamine, triprolidine, trjrptamine, verapamil, xylometazoline
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in mobile phase.
HPLC VARIABLES
Guard column: 15 X 3.2 7 |xm Applied Biosystems pre-column
Column: 100 X 2 10 |xm ixPorasil
Mobile phase: MeCN:5 mM pH 3.75 sodium acetate 80:20
Flow rate: 1
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: buprenorphine, nalbuphine, ethylmorphine, morphine, codeine, meperidine,
tramadol
Noninterfering: thiopentone, succinylcholine, pancuronium, diazepam, atropine, neostigmine
Interfering: butorphanol
REFERENCE
Ho,S.-T.; Wang,J.-J.; Ho,W.; Hu,O.Y.-P. Determination of buprenorphine by high-performance liquid chroma-
tography with fluorescence detection: application to human and rabbit pharmacokinetic studies,
J.Chromatogr., 1991, 570, 339-350.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
camfamine, fenoprofen, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymes-
butazone, oxj^tetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, per-
santine, phenacetin, phenazocine, phenazopjrridine, phencyclidine, phendimetrazine,
puromycin, pîlamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
trihex5^phenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tjrramine, verapa-
REFERENCE
Hill,D.W.; KindA-J- Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:0.025% phosphoric acidibuffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 mL
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ethidium bromide, ethopropazine, fenoprofen, flavoxate, fluoxetine, fluphenazine, flurazepam,
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Fenthion
Molecular formula: C10H15O3PS2
Merck Index: 4044
SAMPLE
Matrix: blood
Sample preparation: 1.5 mL Serum + 2 mL 200 mM pH 7.0 phosphate buffer, add to an Extrelut
No. 3 SPE column, let stand for 10 min, elute with 15 mL n-hexane:diethyl ether 80:20. Evap-
orate the eluate to dryness under a stream of nitrogen at 40, reconstitute the residue with
150 JJLL MeOH-.water 70:30, inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 fjim fiBondapak C18
Mobile phase: Gradient. MeOH:water from 70:30 to 90:10.
Flow rate: 1
Detector: MS, Hitachi Model M-2000, APCI non-equilibrium interface, vaporizer 250, nebulizer
400, ionization needle electrode current 5 |JLA, drift voltage 230 V, vacuum 0.0001 Pa, ion-
source slit 500 (Jim, collector slit 400 |xm, accelerated electrical potential 4 kV, secondary elec-
tronic step-up tube potential 1.3 kV, positive-ion mode
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diazinon, dichlorvos, dimethoate, dimethylvinphos, ediphenphos, IBP, isoxathon, mal-
athion, phenthoate, propaphos, pyridafenthion
KEYWORDS
serum; SPE; m/z 279
REFERENCE
Kawasaki,S.; Ueda,H.; Itoh,H.; Tadano,J. Screening of organophosphorus pesticides using liquid chromatog-
raphy-atmospheric pressure chemical ionization mass spectrometry, J.Chromatogr., 1992, 595, 193-202.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve in 1 mL 0.5 M NaOH, heat at 75 for 45 min, cool, add 2 drops
methyl isobutyl ketone, add 2 drops 0.1% dansyl chloride in acetone, shake well, heat at 65
for 30 min, cool, acidify with 10% HCl, add 300 |xL benzene (Caution! Benzene is a carcinogen!),
extract, inject a 1-10 |JLL aliquot.
HPLC VARIABLES
Column: 1000 X 2.4 Zipax coated with 0.5% p,p'-oxydipropionitrile
Mobile phase: HexanerMeOH 95:5
Flow rate: 0.78
Detector: F ex Turner filter no. 811 em Turner filter no. 817
CHROMATOGRAM
Retention time: 3
KEYWORDS
derivatization; normal phase
REFERENCE
Frei,R.W.; Lawrence,J.F. Fluorigenic labelling in high-speed liquid chromatography, J.Chromatogr., 1973, 83,
321-330.
SAMPLE
Matrix: solutions
Sample preparation: Condition a 10 X 2 SPE column packed with 40 jjim octadecylsilica (Spark
Holland) with 10 mL MeCN, 10 mL MeOH, and 10 mL water at 2 mL/min. Add nitric acid to
a final concentration of 0.5% to water sample, filter (0.45 |xm), add a 150 mL aliquot to the
SPE column at 3 mL/min, wash with 3 mL distilled water, elute the contents of the SPE column
on to the analytical column with the mobile phase.
HPLC VARIABLES
Column: 250 X 4 4 |xm Superspher 60 RP-8 endcapped C8 (Merck)
Mobile phase: Gradient. A was MeCNiMeOH 80:20. B was water. A:B from 10:90 to 40:60 over
10 min, maintain at 40:60 for 5 min, to 90:10 over 33 min, return to initial conditions over 5
min.
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Limit of detection: 65 ng/L
OTHER SUBSTANCES
Simultaneous: azinphos-ethyl, azinphos-methyl, chlorfenvinphos, dichlorvos, fenitrothion, mal-
athion, mevinphos, parathion-ethyl, parathion-methyl
Interfering: diazinon
KEYWORDS
groundwater; wastewater; SPE
REFERENCE
Lacorte,S.; Barcel6,D. Improvements in the determination of organophosphorus pesticides in ground- and
wastewater samples from interlaboratory studies by automated on-line liquid-solid extraction followed by
liquid chromatography-diode array detection, J.Chromatogr.A, 1996, 725, 85-92.
Fentiazac
Molecular formula: C17H12CINO2S
Merck Index: 4045
Lednicer No.: 4 96
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 247
CHROMATOGRAM
KEYWORDS
REFERENCE
Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL7 1995, 40,
254-262.
Fenticonazole
Molecular formula: C24H20CI2N2OS
Merck Index: 4047
Lednicer No.: 4 93
SAMPLE
add 100 mL MeOH, sonicate for 5 min, filter. Add a 2 mL aliquot of filtrate to 5 mL of 100 |xg/
mL ketoconazole in MeOH, make up to 25 mL with MeOH, inject 20 |xL aliquot. Cream. Con-
mL with dichloromethane, filter. Add a 2 mL aliquot to the cartridge, wash with 2 mL di-
chloromethane :methanol 4:1, wash with 2 mL dichloromethane, elute with 3 mL MeOH:buffer
inject 20 jxL aliquot. (Buffer was 50 mM triethylamine adjusted to pH 7.0 with phosphoric
acid.)
HPLC VARIABLES
Mobile phase: THFibuffer 30:70 (Buffer was 50 mM triethylamine adjusted to pH 3.0 with phos-
phoric acid.)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 32
OTHER SUBSTANCES
Simultaneous: clotrimazole, ketoconazole, bifonazole, tioconazole, isoconazole, econazole,
miconazole
KEYWORDS
tablets; creams
REFERENCE
Di Pietra,A.M.; Cavrini,V.; Andrisano,V.; Gatti,R. HPLC analysis of imidazole antimycotic drugs in pharma-
ceutical formulations, J.Pharm.Biomed.Anal, 1992, 10, 873-879.
Feprazone
Merck Index: 4053
SAMPLE
Sample preparation: Plasma. 2 mL Plasma + 250 |xL 1 M HCl + 5 mL diethyl ether, mix,
centrifuge at 1100 g for 10 min. Remove the organic phase, repeat the extraction, combine the
organic extracts, evaporate under nitrogen at 40, reconstitute the residue in 200 |xL mobile
phase, vortex, inject a 20 |xL aliquot. Urine. 1 mL Urine + 250 jxL 1 M HCl + 5 mL diethyl
ether, rotate for 15 min. Remove the organic layer, add 1 mL 1% sodium hydrogen carbonate,
vortex for 1 min. Remove the organic layer and evaporate it under nitrogen at 40, reconstitute
the residue in 200 |JLL mobile phase, inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 100 X 3 5 jxm Nucleosil
Mobile phase: MeCN:water:acetic acid 40:59.4:0.6
Flow rate: 0.8
Detector: UV 245
CHROMATOGRAM
Retention time: 8.3
Internal standard: feprazone
OTHER SUBSTANCES
Extracted: tiaprofenic acid (UV 310)
Noninterfering: alclofenac, diclofenac, fenoprofen, flunixin, flurbiprofen, ibuprofen, indometha-
cin, naproxen, oxyphenbutazone, phenylbytazone, piroxicam
KEYWORDS
plasma; feprazone is IS
REFERENCE
Delbeke,F.T.; Baert,K; De Backer,P. Disposition of human drug preparations in the horse. Vl. Tiaprofenic acid,
J.Chromatogr.B, 1997, 704, 207-214.
Fibrinolysin
Molecular weight: about 90000
CAS Registry No.: 9004-09-5, 9001-90-5
Merck Index: 7678
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 100 X 6 Asahipak GS-520-AHA-ABA (Prepare by suspending 10 g Asahipak-GS gel
(Asahi Chemical Industry) in water, sonicate for 5 min, wash with 200 mL water, wash with
200 mL dioxane, suspend in 100 mL dioxane, add 3.24 g l,l'-carbonyldiimidazole, stir gently
for 15 min at room temperature, wash with 200 mL dioxane, suspend in 200 mL 1 M sodium
bicarbonate containing 1 M 6-aminohexanoic acid, shake at 4 for 25 h, wash with 200 mL
water, wash with 100 mL 1 M NaCl, wash with 200 mL water. Suspend 2 g of the gel in 15
mL 200 mM pH 4.752-(morpholino)ethanesulfonic acid/NaOH buffer, add 288 mg l-ethyl-3-(3-
dimethylaminopropyl)carbodiimide monohydrochloride, stir gently for 30 min, add 28.3 mg p-
aminobenzamide monohydrochloride, adjust pH three times to 4.75 with 1 M HCl or 1 M NaOH
at 30 min intervals, shake gently at room temperature for 24 h, wash with 150 mL water,
wash with 100 mL 50 mM NaOH containing 1 M NaCl, wash with 100 mL 50 mM HCl con-
taining 1 M NaCl, wash with water until washings are neutral. Caution! Dioxane is a carcin-
ogen. It may be possible to use acetone instead.)
Mobile phase: Gradient. Buffer A, after 10 min buffer B, after 25 min buffer B containing 40
mM 6-hexanoic acid, after 40 min buffer B containing 40 mM 6-hexanoic acid and 1 M urea
(step gradient). Buffer A was 50 mM pH 6.5 sodium phosphate. Buffer B was 50 mM pH 7.4
sodium phosphate containing 100 mM NaCl.
Flow rate: 1.8
Detector: F ex 285 em 340 (?)
CHROMATOGRAM
Retention time: 50
OTHER SUBSTANCES
Simultaneous: plasminogens
REFERENCE
Ito,N.; Noguchi,K; Kazama,M.; Shimura,K; Kasai,K.-I. Separation of human Glu-plasminogen, Lys-plasmin-
ogen and plasmin by high-performance affinity chromatography on Asahipak GS gel coupled with p-ami-
nobenzamide, J.Chromatogr., 1985, 348, 199-204.
Finasteride
Merck Index: 4125
Lednicer No.: 5 49
SAMPLE
Matrix: blood
Sample preparation: Condition a 6 mL 300 mg Carbograph SPE cartridge (Alltech) with 2 mL
water and 4 mL MeOH. Add 100 jxL 10 jxg/mL IS to 1.0 mL plasma, briefly vortex, add the
mixture to the SPE cartridge. Wash with 2 mL water and 4 mL MeOHrwater 20:80. Elute with
2 mL MeOHrchloroform 20:80 (Caution! Chloroform is a carcinogen!). Centrifuge eluate at 1000
g for 10 min, filter through a WTP 500 nm filter, evaporate to dryness with a stream of nitrogen
under vacuum. Reconstitute the residue in 1.0 mL mobile phase, vortex, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 40 |xm Pelliguard (Supelco)
Column: 250 X 4.6 5 |xm reversed-phase Symmetry (Waters)
Mobile phase: MeCN:40 mM pH 4.0 orthophosphoric acid 45:55
Flow rate: 1.2
Detector: UV 215
CHROMATOGRAM
Internal standard: 4-androstene-3,17-dione (9.8)
KEYWORDS
plasma; SPE
REFERENCE
Carlucci,G.; Mazzeo,P. Finasteride in biological fluids: extraction and separation by a graphitized carbon black
cartridge and quantification by high-performance liquid chromatography, J.Chromatogr.B, 1997, 693, 245
248.
SAMPLE
Matrix: blood
Sample preparation: Non-buffered extraction. 1 mL Plasma + 100 fiL 100 ng/mL IS in MeOH.
Add 7 mL MTBE, mix and rotate for 15 min, centrifuge, remove the organic layer and evaporate
it to dryness under a stream of nitrogen at 50. Reconstitute the residue in 300 JJLL mobile
phase, inject an aliquot. Buffered extraction. 1 mL Plasma + 100 |xL 100 ng/mL IS in MeOH.
Add 1 mL 200 mM pH 9.8 carbonate buffer and 7 mL MTBE. Extract as described above. Inject
an aliquot.
HPLCVARIABLES
Column: 30 X 4.6 3 |xm BDS Hypersil C18 (A), 50 X 2 3 |xm BDS Hypersil C18 (B)
Mobile phase: MeCNrwater containing 0.1% formic acid 90:10
Flow rate: l(A), 0.2 (B)
Detector: MS, PE Sciex API IIIplus tandem MS, heated nebulizer 500, corona discharge needle
+4 JJLA, APCI positive ion, nebulizing gas air 550 kPa, auxiliary gas at 2 mL/min, curtain gas
nitrogen at 0.9 mL/min, orifice +50 V, interface heater 60, collision gas argon, m/z 373 (A),
Turbo ion spray, nebulizing gas air 410 kPa, auxiliary gas at 6 mL/min, curtain gas at ImL/
min, orifice +30 V, interface heater 60, m/z 373 (B)
CHROMATOGRAM
Retention time: 0.5 (A), 1 (B)
Internal standard: finasteride analog (1.1)
Limit of detection: 25 pg/mL (B)
KEYWORDS
plasma
REFERENCE
Matuszewski,B-K.; Constanzer,M.L.; Chavez-Eng,C.M. Matrix effect in quantitative LC/MS/MS analyses of
biological fluids: A method for determination of finasteride in human plasma at picogram per milliliter
concentrations, Anal Chem., 1998, 70, 882-889.
Flavoxate
Merck Index: 4135
Lednicer No.: 2 392
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 fjig/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention t i m e : 2.8
OTHER SUBSTANCES
mine, fenoterol, fentanyl, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol,
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 ixm Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B)
Mobile phase: MeCN:0.025% phosphoric acidrbuffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 mL
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
KEYWORDS
also details of plasma extraction; also acebutolol; acepromazine; acetaminophen; acetazolamide;
acetophenazine; albuterol; alprazolam; amitriptyline; amobarbital; amoxapine; antipyrine;
atenolol; atropine; azatadine; baclofen; benzocaine; bromocriptine; brompheniramine; brotizo-
lam; bupivacaine; buspirone; butabarbital; butalbital; caffeine; carbamazepine; cetirizine; chlor-
cyclizine; chlordiazepoxide; chlormezanone; chloroquine; chlorpheniramine; chlorpromazine;
chlorpropamide; chlorprothixene; chlorthalidone; chlorzoxazone; cimetidine; cisapride; clomi-
pramine; clonazepam; clonidine; clozapine; cocaine; codeine; colchicine; cyclizine; cyclobenza-
prine; dantrolene; desipramine; diazepam; diclofenac; diflunisal; diltiazem; diphenhydramine;
diphenidol; diphenoxylate; dipyridamole; disopyramide; dobutamine; doxapram; doxepin; dro-
peridol; encainide; ethidium bromide; ethopropazine; fenoprofen; fentanyl; fluoxetine; fiuphen-
azine; flurazepam; flurbiprofen; fluvoxamine; furosemide; glutethimide; glyburide; guaifenesin;
haloperidol; homatropine; hydralazine; hydrochlorothiazide; hydrocodone; hydromorphone;
hydroxychloroquine; hydroxyzine; ibuprofen; imipramine; indomethacin; ketoconazole; ketopro-
fen; ketorolac; labetalol; levorphanol; lidocaine; loratadine; lorazepam; lovastatin; loxapine; ma-
zindol; mefenamic acid; meperidine; mephenytoin; mepivacaine; mesoridazine; metaproterenol;
methadone; methdilazine; methocarbamol; methotrexate; methotrimeprazine; methoxamine;
methyldopa; methylphenidate; metoclopramide; metolazone; metoprolol; metronidazole; mida-
zolam; moclobemide; morphine; nadolol; nalbuphine; naloxone; naphazoline; naproxen; nifedi-
pine; nizatidine; norepinephrine; nortriptyline; oxazepam; oxycodone; oxymetazoline; paroxe-
tine; pemoline; pentazocine; pentobarbital; pentoxifylline; perphenazine; pheniramine;
phenobarbital; phenol; phenolphthalein; phentolamine; phenylbutazone; phenyltoloxamine;
phenytoin; pimozide; pindolol; piroxicam; pramoxine; prazepam; prazosin; probenecid; procain-
amide; procaine; prochlorperazine; procyclidine; promazine; promethazine; propafenone; pro-
pantheline; propiomazine; propofol; propranolol; protriptyline; quazepam; quinidine; quinine;
racemethorphan; ranitidine; remoxipride; risperidone; salicylic acid; scopolamine; secobarbital;
sertraline; sotalol; spironolactone; sulfinpyrazone; sulindac; temazepam; terbutaline; terfena-
dine; tetracaine; theophylline; thiethylperazine; thiopental; thioridazine; thiothixene; timolol;
tocainide; tolbutamide; tolmetin; trazodone; triamterene; triazolam; trifluoperazine; triflupro-
mazine; trimeprazine; trimethoprim; trimipramine; verapamil; warfarin; xylometazoline; yo-
himbine; zopiclone
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Flecainide
CAS Registry No.: 54143-55-4, 54143-56-5 (acetate)
Merck Index: 4136
Lednicer No.: 3 59
SAMPLE
Matrix: blood
with 3 mL 750 mL/L methanol. Elute with three aliquots of 300 JJLL 0.1 M ammonium acetate
JJLL 0.1 M phosphoric acid (pH 2.5), mix, separate, inject a 100 JULL aliquot of the aqueous phase.
HPLC VARIABLES
Mobile phase: MeCNibuffer 35:65 (Buffer was 10 mL/L triethylamine in water adjusted to pH
Flow rate: 2
Detector: UV 228
CHROMATOGRAM
Retention time: 3.1
OTHER SUBSTANCES
sipramine, dextromethorphan, diazepam, encainide, fluoxetine, flurazepam, hydroxyethylflur-
azepam, ibuprofen, imipramine, lidocaine, maprotiline, methadone, methaqualone, mexiletine,
midazolam, norchlorimipramine, nordiazepam, norfluoxetine, nortriptyline, norverapamil, pen-
tazocine, promazine, propafenone, propoxyphene, propranolol, protriptyline, quinidine, tema-
zepam, trimipramine, verapamil
warfarin
Interfering: diphenhydramine, doxepin, fentanyl, haloperidol, nordoxepin, trazodone
KEYWORDS
plasma; SPE
REFERENCE
Clin.Chem., 1994, 40, 1312-1316.
SAMPLE
Matrix: blood
the residue in 100 (xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 jxL aliquot of
HPLC VARIABLES
Mobile phase: MeOH.THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted
Flow rate: 0.8
Detector: UV 299
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: blood, gastric contents, tissue, urine
Sample preparation: Homogenize 15 g liver with water to a final volume of 90 mL, dilute 1 to
10 with water. Homogenize gastric contents, dilute 1 to 100 with water. Dilute urine 1 to 25.
1 mL Whole blood, diluted liver homogenate, diluted gastric contents, or diluted urine + 50 jxL
100 |xg/mL clomipramine, mix, inject an aliquot.
HPLCVARIABLES
Guard column: 20 mm long Hypersil ODS
Column: 150 mm long Hypersil ODS
Mobile phase: MeCN:10 mM Na2HPO4:n-nonylamine 40:60:0.12, pH 3
Flow rate: 1
Detector: UV 295
CHROMATOGRAM
Retention time: 3
Internal standard: clomipramine (5.5)
KEYWORDS
liver; whole blood
REFERENCE
Sadler,D.W.; Quigley,C. Unsuspected self-poisoning with flecainide and alcohol, J.Forensic ScL, 1995,40, 9 0 3 -
905.
SAMPLE
Sample preparation: Dilute urine 1:100. 1 mL Plasma or diluted urine + 100 |xL 4 (xg/mL IS
in water + 1 mL water + 1 mL 1 M NaOH + 8 mL distilled diethyl ether, shake for 15 min,
centrifuge at 1000 g for 5 min. Remove the organic layer and evaporate it to dryness under a
stream of nitrogen, reconstitute the residue in 1 mL freshly prepared 49 mM l-[(4-nitro-
phenyl)sulfonyl]-L-prolyl chloride in ethyl acetate, add 100 |xL 0.016% triethylamine in ethyl
acetate, vortex for 5 s, heat at 80 for 2 h, cool, wash with 3 mL 600 mM HCl, centrifuge for
5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen,
reconstitute the residue in 200 (xL mobile phase, inject a 40-180 |AL aliquot. (Synthesis of 1-
[(4-nitrophenyl)sulfonyl]-L-prolyl chloride is as follows. Stir 40-45 mmoles L-(-)-proline in 40
mL THF and 200 mL 10% potassium carbonate, add 37-43 mmoles 4-nitrophenylsulfonyl chlo-
ride in 40 mL THF dropwise, heat at 50 for 3 h and maintain at pH 8 or above, cool, acidify
to pH 2 and extract into chloroform. Extract with 10% potassium carbonate, acidify the aqueous
layer, extract into chloroform. Dry the chloroform extract, evaporate, recrystallize from petro-
leum ether and benzene (Caution! Benzene is a carcinogen!). Stir 15 mmoles of the 4-nitro-
phenylsulfonylproline in 100 mL benzene under reflux condenser fitted with a calcium sulfate
drying tube, add dropwise a five-fold molar excess of thionyl chloride in 50 mL benzene, heat
at 35-40 until formation of the acid chloride is complete (about 48 h, monitor by IR spectros-
copy), evaporate, recrystallize product from HPLC-grade heptane (mp 110-110.5).)
HPLC VARIABLES
Guard column: 50 mm long octadecyl Pellicular ODS (Whatman)
Column: 300 X 3.9 Bondapak C18
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 29.8 (R), 31.9 (S)
Internal standard: (R,S)-N-(2-piperidylmethyl)-2,3-bis-(2,2,2-trifiuoroethoxy)benzamide (R,
25.5, S, 27.9)
KEYWORDS
REFERENCE
Alessi-Severini,S.; Jamali,R; Pasutto,F.M.; Coutts,R.T.; Gulamhusein,S. High-performance liquid chromato-
graphic determination of the enantiomers of flecainide in human plasma and urine, J.Pharm.Sci., 1990,
79, 257-260.
SAMPLE
Sample preparation: Extract ground tablets containing 10-50 mg of the compound with 100
mL MeOH, filter. Add 500 |xL 100 jxg/mL IS in MeOH to 1 mL filtrate, make up to 10 mL with
MeOH. Inject a 50 (JLL aliquot.
HPLCVARIABLES
Column: 250 X 4 10 |xm LiChrosorb
Mobile phase: MeCN:MeOH:buffer 30:60:10 (Buffer was 67 mM KH2PO4 adjusted to pH 2.9 with
phosphoric acid.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 3.30 (flecainide acetate)
Internal standard: amiodarone (7.46)
Limit of quantitation: 4 fxg/mL
KEYWORDS
tablets
REFERENCE
Paw,B.; Przyborowski,L.; Slawik,T. Determination of flecainide acetate in tablets by HPLC and UV-spectro-
photometry, Pharmazie, 1998, 53, 97-98.
SAMPLE
Matrix: solutions
water, 1 mL 20% aqueous ammonia, and 5 mL ehloroformxarbon disulfide 98:2, shake vigor-
1 mL mobile phase, inject a 10 |xL aliquot. (Copper may also be used with electrochemical
detection or UV detection at 270 nm.)
HPLC VARIABLES
Column: 250 X 4 7 jxm LiChrosorb RP-18
perchlorate
Flow rate: 1.5
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: acebutolol, alprenolol, ephedrine, methamphetamine, propranolol
KEYWORDS
REFERENCE
Leroy,P.; Nicolas,A. Determination of secondary amino drugs as their metal dithiocarbamate complexes by
1984, 317, 513-521.
SAMPLE
Matrix: solutions
Sample preparation: Mix a 50 (JLL aliquot of a solution in MeOH:triethylamine 99:1 with 20 u.L
0.1% FLOPIC in dry toluene, vortex briefly, let stand at room temperature in the dark for 30
min, add 50 |xL 1% ethanolamine in MeOH, let stand at room temperature for 15 min, evap-
orate to dryness under reduced pressure, reconstitute with 100 JJLL mobile phase, sonicate for
30 s, inject a 20 |xL aliquot. (FLOPIC is (-)-(S)-flunoxaprofen isocyanate; synthesis is as follows.
Dissolve 1 g (+)-(S)-flunoxaprofen in 30 mL acetone, cool to 0, add a solution of 500 JULL tri-
ethylamine in 2 mL acetone dropwise, add a solution of 370 JJLL ethyl chloroformate in 2 mL
acetone dropwise, stir at 0 for 15 min, add a solution of 250 mg sodium azide in 1 mL water
dropwise (Caution! Sodium azide is highly toxic!), stir for 1 h, pour into 60 mL ice water, stir
for 10 min, filter, wash the solid with two 50 mL aliquots of ice-water, dry under reduced
pressure to obtain flunoxaprofen azide. Dissolve 100 mg flunoxaprofen azide in 3 mL dry tol-
uene, reflux for 10-15 min, cool to room temperature, filter. Evaporate the filtrate to dryness
under reduced pressure and dry under reduced pressure to obtain FLOPIC as a crystalline
solid (mp 93-94), store in a desiccator under reduced pressure.)
HPLC VARIABLES
Column: 150 X 3.9 4 |xm Nova Pak C18 (A) or 200 X 4.6 5 jjim Nucleosil cyano (B)
Mobile phase: MeOH:water:THF 62:35:3 (A) or n-hexane:isopropanol:diethylamine 95:5:0.05 (B)
Flow rate: 1
CHROMATOGRAM
Retention time: 29.0 (R (A)), 31.0 (S (A)), 22.2 (R (A)), 26.8 (S (B))
OTHER SUBSTANCES
Simultaneous: mexiletine (system A only)
KEY WpRDS
REFERENCE
Martin,E.; Quinke,K; Spahn,H.; Mutschler,E. (-MS)-Flunoxaprofen and (-MS)-naproxen isocyanate: two new
fluorescent chiral derivatizing agents for an enantiospecific determination of primary and secondary amines,
Chirality, 1989, 2, 223-234.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 Spherisorb S5SCX
Mobile phase: MeOH:MeCN:water 40:40:20 containing 25 mM perchloric acid
Flow rate: 2
CHROMATOGRAM
Retention time: 4.5
Internal standard: benzimidazole (7)
OTHER SUBSTANCES
Simultaneous: propranolol
REFERENCE
Croes,K; McCarthy,P.T.; Flanagan,R.J. HPLC of basic drugs and quaternary ammonium compounds on micro-
p articulate strong cation-exchange materials using methanolic or aqueous methanol eluents containing an
ionic modifier, J.ChromatogrA, 1995, 693, 289-306.
SAMPLE
Matrix: solutions
Sample preparation: Mix 20 jxL of a 1 mM solution in MeOH or water with 50 jn-L pH 8 borate
buffer and 50 JJLL 18 mM 2-(6-methoxy-2-naphthyl)-l-propyl chloroformate in acetone, vortex,
let stand at room temperature for 30 min, add 100 jxL 10 mM trans-4-hydroxy-L-proline in
water, mix, let stand for 2 min, add 2 mL dichloromethane, vortex for 30 s. Remove the organic
layer and evaporate it to dryness under reduced pressure, reconstitute the residue in 100 jxL
mobile phase, inject an aliquot. Prepare 2-(6-methoxy-2-naphthyl)-l-propyl chloroformate as
follows. Stir 1.5 mmoles lithium aluminum hydride in THF, slowly add 2 mmoles (S)-naproxen
in 20 mL anhydrous THF, reflux for 1 h, evaporate most of the solvent, cautiously add water
with stirring, acidify with 6 N HCl, extract three times with diethyl ether. Combine the organic
layers and dry them over anhydrous sodium sulfate, evaporate to dryness, chromatograph on
silica gel with dichloromethane:MeOH 100:2 (flash chromatography), evaporate eluate to dry-
ness, dry under vacuum over KOH to give 2-(6-methoxy-2-naphthyl)propanol as a white solid
(mp 92-3). Stir 0.5 mmoles 2-(6-methoxy-2-naphthyl)propanol and 0.5 mmoles triethylamine
in 10 mL dry toluene at 0, add 1 mL 20% phosgene in toluene (Caution! Phosgene is highly
toxic, perform reaction in a chemical fume hood!) (Fluka), stir for 4 h, filter, evaporate to
dryness under reduced pressure, dry under vacuum to give 2-(6-methoxy-2-naphthyl)-l-propyl
chloroformate (mp 60). Store under vacuum over phosphorus pentoxide at room temperature.)
HPLC VARIABLES
Column: 250 X 4 5 |xm Zorbax-SIL
Mobile phase: n-Hexane:isopropanol 100:1.5
Flow rate: 1.5
Detector: UV 230, F ex 270 em 365
CHROMATOGRAM
Retention time: k' 14.4 (R-(-)), k' 15.2 (S-(+))
OTHER SUBSTANCES
Simultaneous: metoprolol, tocainide
Interfering: propafenone
KEY WORDS
derivatization; chiral; normal phase
REFERENCE
Biischges,R.; Linde,H.; Mutschler,E.; Spahn-Langguth,H. Chloroformates and isothiocyanates derived from 2-
arylpropionic acids as chiral reagents: synthetic routes and chromatographic behaviour of the derivatives,
J.ChromatogrA, 1996, 725, 323-334.
Fleroxacin
Merck Index: 4137
Lednicer No.: 5 125
SAMPLE
Matrix: bile, blood, urine
Sample preparation: Dilute urine 1:20. Dilute bile 1:10. 500 JJLL Serum, diluted urine, or diluted
bile + 3.2 mL dichloromethane, vortex, rotate at 20 rpm for 10 min, centrifuge at 1000 g for
10 min. Remove 3 mL of the lower organic phase and add it to 200 IJLL 100 mM NaOH, rotate
at 20 rpm for 30 min, centrifuge at 1000 g for 10 min, inject a 20 JJLL aliquot of the aqueous
layer.
HPLC VARIABLES
Column: 150 X 4.6 5 ^m Ultrasphere C18
Mobile phase: MeCN:buffer 10:90, pH adjusted to 2 with 14.6 M phosphoric acid (Buffer was 10
mM NaH2PO4 containing 5 mM tetrabutylammonium bromide.)
Flow rate: 2
CHROMATOGRAM
Retention time: 3
Limit of detection: 25 ng/mL (bile), 50 ng/mL (urine), 2.5 ng/mL (serum)
OTHER SUBSTANCES
Noninterfering: amikacin, aztreonam, carbamazepine, cephalosporins, ciprofloxacin, clavulanic
acid, difloxacin, digitoxin, digoxin, fosfomycin, furosemide, gentamycin, imipenem, lidocaine,
netilmicin, norfloxacin, pefloxacin, penicillins, phenobarbital, phenytoin, primidone, procain-
amide, quinidine, rifampin, salicylic acid, teicoplanin, temafloxacin, theophylline, tobramycin,
valproic acid, vancomycin
Interfering: ofloxacin
KEYWORDS
serum; human; rabbit
REFERENCE
Koechlin,C; Jehl,E; Linger,L.; Monteil,H. High-performance liquid chromatography for the determination of
three newfluoroquinolones,fleroxacin,temafloxacin and A-64730, in biological fluids, J.Chromatogr., 1989,
491, 379-387.
SAMPLE
Matrix: blood
HPLC VARIABLES
Mobile phase: MeCN:buffer 22:78 containing 5 mM tetrabutylammonium sulfate, adjusted to
perchlorate.)
Flow rate: 1
Detector: UV 287
CHROMATOGRAM
KEYWORDS
REFERENCE
Zlotos,G.; Biicker,A.; Kinzig-Schippers,M.; Sorgel,R; Holzgrabe,U. Plasma protein binding of gyrase inhibitors,
J.Pharm.ScL, 1998, 87, 215-220.
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Serum + 500 JJLL 7% perchloric acid, vortex for 10 s, centrifuge at
>700 g for 10 min, inject a 20 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: 150 X 3.9 Nova-Pak C18
Flow rate: 0.8
Detector: F ex 288 em 475 bandpass filter
CHROMATOGRAM
Retention time: 5.5
OTHER SUBSTANCES
KEYWORDS
serum
REFERENCE
JAntimicrob.Chemother., 1989, 24, 437-445.
SAMPLE
Matrix: blood
Sample preparation: Filter (0.2 jjim cellulose acetate, Schleicher & Schuell) while centrifuging
at 4 at 2500 g for 5 min, maintain filtrate at 4, inject a 5 |xL aliquot of the filtrate onto column
A and elute to waste with mobile phase A, collect the effluent containing fleroxacin in a sample
loop and backflush the contents of this loop onto column B with mobile phase B, elute column
B with mobile phase B and monitor the effluent.
HPLC VARIABLES
Column: A 40 X 6 12 |xm TSKgel PWxI methacrylate polymer gel (Toso-Haas); B 250 X 4.6 5
|jim Zorbax Rx-C8
Mobile phase: A Isopropanol:30 mM pH 6.2 potassium phosphate buffer 10:90; B MeCN:50 mM
pH 2.7 potassium phosphate buffer 18:82
Injection volume: 5
Detector: UV 287
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Extracted: Ro 23-9424 (a pro-drug)
KEYWORDS
plasma; column-switching; heart-cut; pharmacokinetics
REFERENCE
Szuna,A.J.; Blain,R.W. Determination of a new antibacterial agent (Ro 23-9424) by multidimensional high-
performance liquid chromatography with ultraviolet detection and direct plasma injection, J.Chromatogr.,
1993, 620, 211-216.
SAMPLE
Matrix: blood
Sample preparation: 250 |xL Serum + 20 |xL 30 |xg/mL pipemidic acid + 250 jxL 25% sodium
sulfate, vortex, add 3.5 mL chloroform, shake at low speed for 10 min, centrifuge at 1155 g for
10 min. Remove the lower organic layer and add it to 200 |xL 1 M NaOH, shake fast for 20
min, centrifuge at 1155 g for 10 min, inject a 150 |xL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 10 jxm Nucleosil C18
Mobile phase: MeCN:MeOH:10 mM phosphate buffer containing 5 mM tetrabutylammonium
hydrogen sulfate 13:6:81
Flow rate: 1
Detector: F ex 274
CHROMATOGRAM
Internal standard: pipemidic acid
KEYWORDS
REFERENCE
Bertino,J.S.,Jr.; Nafziger,A.N.; Wong,M.; Stragand,L.; Puleo,C. Effect of a fat- and calcium-rich breakfast on
pharmacokinetics of fleroxacin administered in single and multiple doses, Antimicrob.Agents Chemother.,
1994, 38, 499-503.
SAMPLE
Matrix: blood
Sample preparation: 250 |xL Serum + 20 |JLL (?) 30 (xg/mL pipemidic acid + 250 |xL 25% sodium
sulfate, vortex, add 3.5 mL chloroform, shake at low speed for 10 min, centrifuge at 1155 g for
10 min. Remove the organic layer and add it to 200 |xL 1 M NaOH, shake quickly for 20 min,
centrifuge at 1155 g for 10 min, inject a 150 u,L aliquot of the aqueous layer.
HPLC VARIABLES
Column: 10 (xm Nucleosil C18
Mobile phase: MeCNrMeOH: 10 mM phosphate buffer containing 5 mM tetrabutylammonium
sulfate 13:6:81
Flow rate: 1
Detector: F ex 274 (?)
CHROMATOGRAM
KEYWORDS
pharmacokinetics; serum
REFERENCE
Bertino,J.S.,Jr.; Nafziger,A.N. Pharmacokinetics of oral fleroxacin in male and premenopausal female volun-
teers, Antimicrob.Agents Chemother., 1996, 40, 789-791.
SAMPLE
Sample preparation: Plasma. 100 |xL Plasma + 25 jxL MeCN:70% perchloric acid 80:20 con-
taining IS, mix vigorously, centrifuge. Inject a 5 or 50 |xL aliquot. Dialysate. Dilute 20 |xL
dialysate with 980 |xL 50 mM sodium dihydrogen phosphate buffer containing IS. Mix thor-
oughly. Inject a 5 or 10 (LJLL aliquot.
HPLC VARIABLES
Column: Spherisorb ODS II
Mobile phase: MeCN containing 2 mM tetrabutyl ammonium hydrogen sulfate:100 mM citric
acid buffer containing 5 mM ammonium perchlorate 87:13, adjusted to pH 2.2
Flow rate: 1.2
CHROMATOGRAM
Retention time: 6.6
Internal standard: pipemidic acid (3.2)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Uehlinger,G.E.; Schaedeli,R; Kinzig,M.; Sorgel,E; Frey,F.J. Pharmacokinetics of fleroxacin after multiple oral
dosing in patients receiving regular hemodialysis, Antimicrob.Agents Chemother., 1996, 40, 19031909.
SAMPLE
Matrix: blood, intestinal efflux
Sample preparation: Intestinal efflux. Freeze intestinal efflux at -80, lyophilize, reconstitute
with 1 mL ofloxacin in MeOH: 100 mM phosphoric acid 50:50, centrifuge at 3000 rpm for 10
min, inject a 20 |xL aliquot. Serum. Deproteinize serum with MeOH containing ofloxacin, ex-
tract with dichloromethane at pH 7.5.
HPLCVARIABLES
Column: 150 X 3.9 Novapack C18
Mobile phase: MeCN:buffer 16:84 (Buffer was 10 mM pH 3.0 potassium phosphate buffer con-
taining 25 mM sodium heptanesulfonate (PIC B7) and 20 mM triethylamine.)
Flow rate: 1.5
CHROMATOGRAM
Internal standard: ofloxacin
KEYWORDS
serum; rat
REFERENCE
Rubinstein,E.; Dautrey,S.; Farinoti,R.; St.Julien,L.; Ramon,J.; Carbon,C. Intestinal elimination of sparfloxacin,
fleroxacin, and ciprofloxacin in rats, Antimicrob.Agents Chemother., 1995, 39, 99-102.
SAMPLE
Sample preparation: 500 JULL Plasma + 100 |JLL 1 jjLg/mL ISl in 10 mM HCl + 1 mL pH 7.5
Sorensen buffer + 7 mL dichloromethanerisoprcpanol 70:30, extract by turning head over head
at 70 rotations/min for 10 min, centrifuge at 2000 g for 10 min. Remove 5 mL of the organic
in 500 |xL mobile phase, vortex, inject an aliquot. Urine. 500 juiL Urine + 100 JJLL 1 mg/mL IS2
in 10 mM HCl + 500 JJLL 1 M acetic acid + 500 |iL 25 mM sodium dodecyl sulfate in water + 7
mL dichloromethane:isopropanol 70:30, extract by turning head over head at 70 rotations/min
for 10 min, centrifuge at 2000 g for 10 min. Remove 5 mL of the organic layer and evaporate
it to dryness under a stream of nitrogen at 35, reconstitute the residue in 500 |xL mobile
phase, vortex, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm TSK-GeI ODS 120T (Toyo Soda)
Mobile phase: MeOH:5 mM tetrabutylammonium hydrogen sulfate 21:79 (plasma) or 24.5:74.5
(urine)
Column temperature: ambient (urine), 27 (plasma)
Flow rate: 0.8
CHROMATOGRAM
Retention time: 7.5 (plasma), 9 (urine)
Internal standard: ISl 6-fluoro-l-(2-fluoroethyl)-l,4-dihydro-7-(4-methyl-l-piperazinyl)-4-oxo-3-
quinolinecarboxylic acid (12), IS2 pipemidic acid (7)
Limit of quantitation: 10-20 ng/mL
KEYWORDS
plasma; protect from light; pharmacokinetics
REFERENCE
Dell,D.; Partos,C; Portmann,R. The determination of a new trifluorinated quinolone, fleroxacin, its N-demethyl,
and N-oxide metabolites in plasma and urine by high performance liquid chromatography with fluorescence
detection, J.Liq.Chromatogr., 1988, 11, 1299-1312.
SAMPLE
Sample preparation: Plasma. 200 |xL Plasma + 1 mL 50 ng/mL ISl in MeCN:water 95:5, vortex,
centrifuge at 14 at 1000 g for 10 min. Remove the supernatant and evaporate it to dryness
under a stream of nitrogen at 38, reconstitute the residue in 200-400 |xL mobile phase, inject
a 20-30 JJLL aliquot onto column A and column B in series, after 1.5 min remove column A from
the circuit and backflush it to waste, elute column B with mobile phase and monitor the efflu-
ent. Urine. 100 |xL Urine + 100 |xL 100 |xg/mL IS2 in water + 5 mL mobile phase, inject a 20
|JLL aliquot onto column A and column B in series, after 1.5 min remove column A from the
circuit and backflush it to waste, elute column B with mobile phase and monitor the effluent.
HPLC VARIABLES
Column: A 10 X 4 Nucleosil 5-C18 (replaced every 60-80 samples); B 250 X 4.6 5 |xm TSK ODS-
120T (Toyo Soda)
Mobile phase: MeOH:buffer 28:72, pH adjusted to 2.6 with 40% phosphoric acid (Buffer was 50
mM KH2PO4 containing 10 mM tetrabutylammonium hydrogen sulfate.)
Flow rate: 1
CHROMATOGRAM
Retention time: 7.7
Internal standard: ISl 6-fluoro-l-(2-fluoroethyl)-l,4-dihydro-7-(4-methyl-l-piperazinyl)-4-oxo-3-
quinoline carboxylic acid (AM-735) (13.0), IS2 pipemidic acid (6.0)
Limit of detection: 1 ng/mL (plasma)
OTHER SUBSTANCES
Noninterfering: ciprofloxacin, norfloxacin, pefloxacin
KEYWORDS
plasma; column-switching; protect from light; pharmacokinetics
REFERENCE
Heizmann,R; Dell,D.; Eggers,H.; Gora,R. Determination of the newfluoroquinolonefleroxacinand its N-de-
methyl and N-oxide metabolites in plasma and urine by high-performance liquid chromatography with
fluorescence detection, J.Chromatogr., 1990, 527, 91-101.
SAMPLE
Matrix: cells
Sample preparation: Incubate cells in 2 mL 100 mM pH 3.0 glycine-HCl buffer for 2 h at room
temperature, centrifuge at 5600 g for 5 min, inject an aliquot.
HPLC VARIABLES
Column: Bondapak C18
Mobile phase: MeCN:25 mM phosphoric acid adjusted to pH 3.0 with tetrabutylammonium hy-
droxide 25:75
Flow rate: 1.5
OTHER SUBSTANCES
Also analyzed: ciprofloxacin, lomefloxacin, norfloxacin, ofloxacin, temafloxacin
REFERENCE
Pascual,A.; Garcia,L; Conejo,M.C; Perea,E.J. Fluorometric and high-performance liquid chromatographic mea-
surement of quinolone uptake by human neutrophils, Eur.J.Clin.Microbiol.Infect.Dis., 1991, 10, 969-971.
SAMPLE
Matrix: milk
Sample preparation: Milk + pipemidic acid + phosphate buffer, extract with dichloromethane:
isopropanol 70:30, centrifuge. Remove an aliquot of the organic layer and evaporate it to dry-
ness under a stream of nitrogen, reconstitute the residue in mobile phase, add n-hexane, mix
thoroughly, centrifuge, inject an aliquot of the aqueous phase.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm ODS-120T (Toyo Soda)
Mobile phase: MeOH: 5 mM tetrabutylammonium hydrogen sulfate 28:72
Flow rate: 0.8
CHROMATOGRAM
KEYWORDS
protect from light; pharmacokinetics
REFERENCE
Dan,M.; Weidekamm,E.; Sagiv,R.-; Portmann,R.; Zakut,H. Penetration offleroxacininto breast milk and phar-
macokinetics in lactating women, Antimicrob.Agents Chemother., 1993, 37, 293-296.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 125 X 4.6 3 ^m ODS-Hypersil
Mobile phase: MeOH:THF:670 mM pH 3.0 phosphate buffer 20:0.8:79.2 plus 2 g/L tetrabutylam-
monium hydrogen sulfate and 2 mL/L 85% phosphoric acid
Flow rate: 1
Detector: UV 278
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: photodegradation products, ofloxacin
Interfering: ciprofloxacin
REFERENCE
Tiefenbacher,E.M.; Haen,E.; Przybilla,B.; Kurz,H. Photodegradation of some quinolones used as antimicrobial
therapeutics, J.Pharm.ScL, 1994, 83, 463-467.
SAMPLE
Matrix: solutions
nitrate.
HPLC VARIABLES
Column: 250 X 4 5 |xm LiChrospher 100 RP-18
Mobile phase: MeCN.buffer 7:93 (Buffer was 25 mM phosphoric acid adjusted to pH 3.89 with
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Simultaneous: ciprofloxacin, enoxacin, norfloxacin, ofloxacin (UV 295), pipemidic acid
REFERENCE
Barbosa,J.; Berge"s,R.; Sanz-Nebot,V. Solvatochromic parameter values and pH in aqueous-organic mixtures
27-36.
SAMPLE
Matrix: urine
Sample preparation: 250 JJIL Urine + 20 jxL 30 jxg/mL pipemidic acid + 250 JJLL 500 mM pH 7.5
phosphate buffer, vortex, add 3.5 mL chloroform, shake at low speed for 10 min, centrifuge at
1155 g for 10 min. Remove the lower organic layer and add it to 200 |xL 1/15 M pH 12.5 sodium
phosphate, shake fast for 20 min, centrifuge at 1155 g for 10 min, inject a 150 fxL aliquot of
the aqueous layer.
HPLC VARIABLES
Column: 10 u-m Nucleosil C18
Mobile phase: MeCNiMeOH: 10 mM phosphate buffer containing 5 mM tetrabutylammonium
hydrogen sulfate 13:6:81
Flow rate: 1
Detector: F ex 274
CHROMATOGRAM
KEYWORDS
pharmacokinetics
REFERENCE
Bertino,J.S.,Jr.; Nafziger,A.N.; Wong,M.; Stragand,L.; Puleo,C. Effect of a fat- and calcium-rich breakfast on
pharmacokinetics of fleroxacin administered in single and multiple doses, Antimicrob.Agents Chemother.,
1994, 38, 499-503.
Flezelastine
Molecular formula: C29H30FH3O
SAMPLE
Sample preparation: Adjust pH of 750 (xL microsomal incubation to 10 with 100 mM NaOH,
add 3 mL cyclohexanerethyl acetate 80:20, shake mechanically for 10 min, centrifuge at 2500
g for 10 min, repeat the extraction. Combine the organic layers and evaporate them to dryness
under a stream of nitrogen, reconstitute with a solution of azelastine in MeOH, evaporate to
dryness under a stream of nitrogen, reconstitute with 200 |JLL MeOH, inject a 20 fxL aliquot.
HPLC VARIABLES
Guard column: 4 x 4 5 |xm LiChrospher Si-60
Column: 250 X 4 5 |jim LiChrospher Si-60
Mobile phase: MeOH containing 0.033% perchloric acid
Flow rate: 0.5 for 17 min then 0.9
CHROMATOGRAM
Retention time: 15
Internal standard: azelastine (27)
OTHER SUBSTANCES
KEYWORDS
human; rat; cow; pig; liver
REFERENCE
Paris,S.; Blaschke,G.; Locher,M.; Borbe,H.O.; Engel,J. Investigation of the stereoselective in vitro metabolism
of the chiral antiasthmatic/antiallergenic drug flezelastine by high-performance liquid chromatography and
capillary zone electrophoresis, J.Chromatogr.B, 1997, 691, 463471.
Floctafenine
Molecular formula: C20Hi7F3N2O4
Merck Index: 4140
Lednicer No.: 3 184
SAMPLE
M a t r i x : blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform risopropanol:
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 353
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
HPLCVARIABLES
Column: 250 X 4.6 5 fjum Symmetry C8 (Waters)
mL/min)
Detector: UV 209.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Flosequinan
Molecular formula: C11H10FNO2S
Merck Index: 4146
Lednicer No.: 5 127
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 10 fxL 100 |xg/mL IS in MeCN:MeOH 50:50 + 5 mL chlo-
roform, shake for 10 min, centrifuge at 1800 g for 10 min. Remove 4 mL of the organic layer
and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 100
|JLL MeOH, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 jxm Chiralcel OD
Mobile phase: MeOH:EtOH 78:22
Flow rate: 0.7
Detector: UV 320
CHROMATOGRAM
Retention time: 12.1 (R(+)), 7.9 (S(-))
Internal standard: ()-7-chloro-l-methyl-3-methylsulfinyl-4-quinolone(8.9, 14.6)
OTHER SUBSTANCES
KEYWORDS
plasma; chiral; pharmacokinetics
REFERENCE
Kashiyama,E.; Odomi,M.; Shimizu,T. Stereospecific and simultaneous high-performance liquid chromato-
graphic assay of flosequinan and its metabolites in human plasma, J.Chromatogr.B, 1994, 652, 179185.
SAMPLE
Sample preparation: Condition a 1 mL Baker-10 octadecyl C18 SPE cartridge (J.T. Baker) with
MeOH then water. 100 |xL Plasma or urine + 25 JJLL 2 (plasma) or 10 (urine) |xg/mL IS in water,
add to the SPE cartridge, wash with 2 column volumes of water, elute with 200 (xL MeOH,
inject a 2 (urine) or 5 (plasma) aliquot of the eluate.
HPLC VARIABLES
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Retention time: 3.4
Internal standard: 7-chloro-l-methyl-3-methylsulfinyl-4-quinolone (7.8)
OTHER SUBSTANCES
Noninterfering: acetaminophen, amiodarone, diazepam, digoxin, furosemide, lidocaine, nitro-
glycerin, procainamide, propranolol, quinidine
KEYWORDS
plasma; SPE
REFERENCE
Slegowski,M.B.; Miller,C; Porter,R.S. Simplified high-performance liquid chromatographic determination of
flosequinan and its metabolite in plasma, serum and urine, J.Chromatogr., 1988, 425, 227232.
SAMPLE
Matrix: incubations
Sample preparation: 2 mL Incubation + 10 |xg IS + 4 mL chlorobutane:l,2-dichloroethane 80:
20, shake for 15 min, centrifuge. Remove the upper organic layer and evaporate it to dryness
100 uX aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Hypersil 3ODS
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Internal standard: 7-chloro-l-methyl-3-methylsulfinyl-4-quinolone (BTS 49037)
OTHER SUBSTANCES
Next Page
REFERENCE
Lee,S.C; Renwick,A.G. Sulphoxide reduction by rat intestinal flora and by Escherichia coli in vitro,
Biochem.PharmacoL, 1995, 49, 1567-1576.
Floxacillin
Molecular formula: C19H17CIFN3O5S
CAS Registry No.: 5250-39-5,1847-24-1 (sodium salt),
34214-51-2 (sodium monohydrate)
Merck Index: 4147
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond Elut C18 SPE cartridge with 2 mL MeCN and 1
mL 10 mM pH 2 Na2HPO4. 250 |xL Plasma + 100 |xL 20 |xg/mL dicloxacillin sodium in water,
add 400 |xL MeCN at -15 while vortexing, add 700 |xL 10 mM pH 2 Na2HPO4, centrifuge at
8000 g for 10 min. Add the supernatant to the SPE cartridge, wash with 1 mL water, elute
with two 500 |xL portions of MeCN:water 35:65 containing 10 mM Na2HPO4 (pH adjusted to 6
with phosphoric acid), inject a 20 |xL aliquot of the eluate.
HPLC VARIABLES
Column: 100 x 2 5 jjim ODS Hypersil
Mobile phase: MeCN:water 40:60 containing 10 mM Na2HPO4, pH adjusted to 2 with ortho-
phosphoric acid
Flow rate: 0.5
Detector: UV 220
CHROMATOGRAM
Retention time: 2.5
Internal standard: dicloxacillin (3.5)
OTHER SUBSTANCES
Simultaneous: cloxacillin
KEYWORDS
REFERENCE
Hung,C.T.; Lim,J.K.C; Zoest ,A-R-; Lam,F.C. Optimization of high-performance liquid chromatographic analysis
for isoxazolyl penicillins using factorial design, J.Chromatogr., 1988, 425, 331-341.
SAMPLE
Matrix: blood
Sample preparation: 100 (JLL Plasma + 100 u,L dicloxacillin in water + 25 |xL glacial acetic acid
+ 2 mL ethyl acetate, vortex for 30 s, centrifuge at 2000 g for 5 min. Remove the supernatant
JJLL mobile phase, inject a 10-20 JJLL aliquot.
HPLCVARIABLES
Column: 40 X 3.2 RP18 VeloSep (Brownlee)
Mobile phase: MeCN: 10 mM pH 7 phosphate buffer 18:82
Flow rate: 1.2
Detector: UV 220
Previous Page
REFERENCE
Lee,S.C; Renwick,A.G. Sulphoxide reduction by rat intestinal flora and by Escherichia coli in vitro,
Biochem.PharmacoL, 1995, 49, 1567-1576.
Floxacillin
Molecular formula: C19H17CIFN3O5S
CAS Registry No.: 5250-39-5,1847-24-1 (sodium salt),
34214-51-2 (sodium monohydrate)
Merck Index: 4147
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond Elut C18 SPE cartridge with 2 mL MeCN and 1
mL 10 mM pH 2 Na2HPO4. 250 |xL Plasma + 100 |xL 20 |xg/mL dicloxacillin sodium in water,
add 400 |xL MeCN at -15 while vortexing, add 700 |xL 10 mM pH 2 Na2HPO4, centrifuge at
8000 g for 10 min. Add the supernatant to the SPE cartridge, wash with 1 mL water, elute
with two 500 |xL portions of MeCN:water 35:65 containing 10 mM Na2HPO4 (pH adjusted to 6
with phosphoric acid), inject a 20 |xL aliquot of the eluate.
HPLC VARIABLES
Column: 100 x 2 5 jjim ODS Hypersil
Mobile phase: MeCN:water 40:60 containing 10 mM Na2HPO4, pH adjusted to 2 with ortho-
phosphoric acid
Flow rate: 0.5
Detector: UV 220
CHROMATOGRAM
Retention time: 2.5
OTHER SUBSTANCES
Simultaneous: cloxacillin
KEYWORDS
REFERENCE
Hung,C.T.; Lim,J.K.C; Zoest ,A-R-; Lam,F.C. Optimization of high-performance liquid chromatographic analysis
for isoxazolyl penicillins using factorial design, J.Chromatogr., 1988, 425, 331-341.
SAMPLE
Matrix: blood
Sample preparation: 100 (JLL Plasma + 100 u,L dicloxacillin in water + 25 |xL glacial acetic acid
+ 2 mL ethyl acetate, vortex for 30 s, centrifuge at 2000 g for 5 min. Remove the supernatant
JJLL mobile phase, inject a 10-20 JJLL aliquot.
HPLCVARIABLES
Column: 40 X 3.2 RP18 VeloSep (Brownlee)
Flow rate: 1.2
Detector: UV 220
CHROMATOGRAM
Retention time: 2.8
OTHER SUBSTANCES
Simultaneous: carbamazepine, phenytoin, phenobarbital
Noninterfering: acetaminophen, N-acetylprocainamide, amikacin, amitriptyline, caffeine, chlo-
ramphenicol, cyclosporine, digoxin, ethosuximide, gentamicin, lidocaine, nortriptyline, metho-
trexate, primidone, procainamide, quinidine, salicylic acid, theophylline, tobramycin, valproic
acid, vancomycin, metabolites
KEYWORDS
plasma
REFERENCE
Charles,B.G.; Foo,C.C; Gath,J. Rapid column liquid chromatographic analysis of flucloxacillin in plasma on a
microparticulate pre-column, J.Chromatogr.B, 1994, 660, 186-190.
Floxuridine
Merck Index: 4148
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL water, vortex 10 s, add 200 (xL water containing
30% w/v trichloroacetic acid and 30% w/v perchloric acid, vortex 10 s, place in an ice bath for
2 min, centrifuge at 3000 g for 20 min, inject a 20 (xL aliquot of the supernatant.
HPLC VARIABLES
Column: 100 X 4.6 5(xm Hypersil-ODS
Mobile phase: 20 mM Na2HPO4 adjusted to pH 2.0 with orthophosphoric acid
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 7
Internal standard: floxuridine
OTHER SUBSTANCES
Extracted: allopurinol, oxipurinol
KEYWORDS
plasma; floxuridine is IS
REFERENCE
Hung,C.T.; Zoest,A.R.; Perrier,D.G. Analysis of allopurinol and oxipurinol in plasma by reversed phase HPLC,
J.Liq.Chromatogr., 1986, 9, 2471-2483.
SAMPLE
Matrix: blood
Sample preparation: 100 JULL Serum + 5 JLJLL 42 JULM 5-chlorouracil in acetone, mix, let stand at
room temperature for 3 min, add 500 |xL 100 mM pH 3.5 potassium phosphate buffer, add 2
mL ethyl acetate, vortex for 2 min, centrifuge at 1000 g for 5 min. Remove a 1.4 mL aliquot
of the organic layer and evaporate it to dryness under reduced pressure. Add 20 mg solid
potassium bicarbonate:anhydrous sodium sulfate 1:7 to the residue, add 50 (xL 1.3 mM 3-
bromomethyl-6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone in acetone, add 50 |xL 1.5 mM 18-
crown-6 in acetone, heat at 50 for 20 min, cool, inject a 10 |xL aliquot. (Silanize all glassware.
Synthesize 3-bromomethyl-6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone as follows. Stir 483 g
veratrole in 1.45 L acetic acid at 15 for 1 h, add 683 g concentrated nitric acid (d 1.05) over 1
h (maintain the temperature below 40 by cooling and regulating the rate of addition of the
nitric acid). Continue stirring and add 2.127 L fuming nitric acid (d 1.50) over 1 h while main-
taining the temperature below 30, let stand for 2 h, pour into a large volume of cold water,
filter, wash the solid with water until the washings are neutral, recrystallize from EtOH to
give 4,5-dinitroveratrole (mp 129.5-130.5) (J. Am. Chem. Soc. 1946, 68, 1536). Reflux 5 g 4,5-
dinitroveratrole in 200 mL benzene (Caution! Benzene is a carcinogen!), add 100 g 60 mesh
iron powder and 20 mL concentrated HCl in small portions over 1 h, reflux for 4 h, add 10 mL
water, reflux for 2 h, cool, make alkaline with 2.5 M NaOH, extract several times with 200 mL
portions of benzene. Combine the organic layers and evaporate them to dryness, add 10 mL
concentrated HCl, recrystallize from EtOH to give l,2-diamino-4,5-dimethoxybenzene mono-
hydrochloride as very slightly pink needles (mp 240) (Anal. Chim. Acta 1982, 134, 39). Heat
2.5 mmoles l,2-diamino-4,5-dimethoxybenzene hydrochloride and 2.4 mmoles pyruvic acid in
30 mL 500 mM HCl on a boiling water bath for 2 h, cool with ice-water, filter. Wash the
precipitate with water and dry it under vacuum, recrystallize from MeOH:water 90:10 to give
6,7-dimethoxy-3-methyl-2(lH)-quinoxalinone as yellow needles (mp 255) (Chem. Pharm. Bull.
1985, 33, 3493). Treat 1 g 6,7-dimethoxy-3-methyl-2(lH)-quinoxalinone dissolved in 50 mL
anhydrous MeOH with a solution of diazomethane in ether, evaporate to dryness under reduced
pressure, dissolve the residue in 5 mL ethyl acetate, chromatograph on a 250 X 35 column
filled with 130 g 70-230 mesh silica gel 60 (Merck) using n-hexane:ethyl acetate 25:75 to give
6,7-dimethoxy-l,3-dimethyl-2(lH)-quinoxalinone as yellow needles (mp 170-171). Dissolve 350
mg 6,7-dimethoxy-l,3-dimethyl-2(lH)-quinoxalinone in 3 mL acetic acid, add 350 mg anhy-
drous sodium acetate, add 2 mL 1.5 M bromine in acetic acid, heat at 100 for 15 min, cool,
add 10 mL ether, filter, wash the solid 2 or 3 times with small portions of ether. Combine the
filtrate and washings and evaporate them to dryness, dissolve the residue in 5 mL ethyl ace-
tate, chromatograph on a 250 X 35 column filled with 130 g 70-230 mesh silica gel 60 (Merck)
using ether, evaporate the main fraction to dryness, recrystallize the residue from n-hexane:
ethyl acetate 50:50 to give 3-bromomethyl-6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone as yel-
low needles (mp 161-163) (J. Chromatogr. 1985, 346, 227). 3-Bromomethyl-6,7-dimethoxy-l-
methyl-2(lH)-quinoxalinone is also available from Dojindo Molecular Technologies, Inc., 3
Bethesda Metro Center, Suite 700, Bethesda MD 20814; (301) 664-8448; www.dojindo.co.jp.)
HPLC VARIABLES
Column: 100 X 8 10 |xm Radial Pak C18 (Waters) (Wash with MeOH at 2 mL/min for 20 min at
the end of each day.)
Mobile phase: Gradient. MeOH:water 35:65 for 15 min, 50:50 for 25 min (step gradient), re-
equilibrate at initial conditions for 20 min.
Flow rate: 1.5
CHROMATOGRAM
Internal standard: 5-chlorouracil (32.5)
OTHER SUBSTANCES
Extracted: 5-fluorouracil
KEY WpRDS
derivatization; serum; pharmacokinetics
REFERENCE
Yamaguchi,M.; Nakamura,M.; Kuroda,N.; Ohkura,Y. Determination of 5-fluorouracil and 5-fluoro-2'-deoxyuri-
dine in human serum by high-performance liquid chromatography with fluorescence detection, Anal.Sci.,
1987, 3, 75-79.
SAMPLE
Matrix: blood
Sample preparation: Add 250 |xL serum to a 20 X 7 DEAE-Cellulofine AM anion-exchange
column (Seikagaku Tokyo), elute with 3.5 mL 1 mM HCl, discard the first 0.5 mL eluate, collect
the next 3 mL eluate. Evaporate the eluate to 0.5 mL under reduced pressure, add 15 mL ethyl
acetate, shake, centrifuge. Remove the organic layer and evaporate it to dryness, reconstitute
the residue in 800 JJLL anhydrous acetone, add 100 JJLL 750 |xg/mL 4-bromomethyl-6,7-dime-
thoxycoumarin in acetone, add 100 (xL 250 (xg/mL 18-crown-6 in acetone, add 1.5 mg anhydrous
potassium carbonate, heat at 70 for 15 min (protect from atmospheric moisture with a calcium
chloride drying tube), cool, inject an aliquot
HPLC VARIABLES
Column: 200 X 4 5 (xm Nucleosil 5 C18
Flow rate: 0.8
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Extracted: ftorafur, 5-fluorouracil
KEYWORDS
derivatization; serum; protect from light; SPE
REFERENCE
Yoshida,S.; Adachi,T.; Hirose,S. 4-Bromomethyl-6,7-dimethoxycoumarin as a fluorescence reagent for precol-
umn derivatization of 5-fluorouracil compounds in high-performance liquid chromatography, J. Chromatogr.,
1988, 430, 156-162.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL LC-SCX Supelclean strong cation-exchange SPE car-
tridge (Supelco) with 2 mL MeOH, 1 mL 100 mM copper(II) sulfate solution, and 3 mL 50 mM
pH 7 phosphate buffer, do not allow to dry. 300 \xL Serum + 5-bromouracil, add to the SPE
cartridge, wash with 2 mL 50 mM pH 7 phosphate buffer, wash with 2 mL MeOH, elute with
700 |JLL 1.7 M ammonia solution, add 70 |xL glacial acetic acid to the eluate, mix thoroughly,
inject a 20 \xL aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 5 jmm Supelguard LC-18-S (Supelco)
Column: 250 X 4.6 5 |xm Supelcosil LC-18-S ODS
Mobile phase: Gradient. A was MeOH:50 mM pH 6.5 phosphate buffer 60:40. B was 50 mM pH
6.5 phosphate buffer.
Flow rate: 1
Detector: UV 269
CHROMATOGRAM
Retention time: 13
Internal standard: 5-bromouracil (12)
OTHER SUBSTANCES
Extracted: doxifluridine, 5-fluorouracil, 5-fluorouridine monophosphate, metabolites
KEYWORDS
serum; SPE
REFERENCE
Guerrieri,A.; Palmisano,R; Zambonin,RG.; De Lena,M.; Lorusso,V. Solid-phase extraction of fluoropyrimidine
derivatives on a copper-modified strong cation exchanger: determination of doxifluridine, 5-fluorouracil and
its main metabolites in serum by high-performance liquid chromatography with ultraviolet detection,
J.Chromatogr., 1993, 617, 71-77.
SAMPLE
Matrix: blood, peritoneal fluid
Sample preparation: Plasma. 1 mL Plasma + 10 |xL 100 |xM bromouridine + 70 |xL perchloric
acid, mix thoroughly, let stand at 4 for at least 12 h, centrifuge for 5 min. Remove the super-
natant and adjust the pH to 7 with 5 M KOH, let stand on ice for 2 h, inject a 20 u-L aliquot.
Peritoneal fluid. 1 mL Peritoneal fluid + 10 |xL 100 u-M bromouridine, dilute 1:100 with water,
inject a 20 JJLL aliquot.
HPLC VARIABLES
Guard column: 12.5 X 4.6 5 |xm Zorbax RX
Column: 250 X 4.6 5 |xm Zorbax RX
Mobile phase: Gradient. A was 25 mL pH 2.5 ammonium phosphate. B was MeCN:25 mM pH
7.5 ammonium phosphate 7:93. A:B 100:0 for 5 min, to 0:100 over 10 min, maintain at 0:100
for 10 min, return to initial conditions over 1 min, re-equilibrate for 20 min.
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Internal standard: bromouridine (18)
OTHER SUBSTANCES
Extracted: 5-fluorouracil
KEYWORDS
REFERENCE
Smith-Rogers,J.A.; Tong,W.R; Duafala,M.E.; Markman,M.; Bertino,J.R. High-performance liquid chromato-
graphic method for the simultaneous measurement of floxuridine andfluorouracilin human body fluids,
J.Chromatogr., 1991, 566, 147-154.
SAMPLE
Sample preparation: Add 100 |JLL 10% perchloric acid and 20 |xL 200 ixg/mL IS to 100 |xL serum
or homogenized tissue. Shake for 2 min and centrifuge at 2000 g for 10 min. Filter (45 |xm)
supernatant, inject a 10 jxL aliquot of the filtrate.
HPLCVARIABLES
Guard column: 45 X 4.6 5 |xm ODS Hypersil (VDS Optilab)
Column: 250 X 4.6 5 |xm ODS Hypersil (VDS Optilab)
Mobile phase: MeOH:99% acetic acidiwater 3:0.5:96.95
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: 5-bromouracil (10.34)
Limit of quantitation: 200 ng/mL (serum); 600 ng/mL (tissue)
OTHER SUBSTANCES
Extracted: metabolites, fluorouracil
KEYWORDS
serum; rat; liver; tumor; kidney; spleen; peritoneum; gastric mucosa; lung; heart; pancreas
REFERENCE
Jung,M.; Berger,G.; Pohlen,U.; Pauser,S.; Reszka,R; Buhr,H-J- Simultaneous determination of 5-fluorouracil
and its active metabolites in serum and tissue by high-performance liquid chromatography, J. Chromatogr. B,
1997, 702, 193-202.
SAMPLE
Sample preparation: 0.5 g Tissue or 1 mL plasma + 5 JJLL 1 M sulfuric acid (lung and heart
only) + 2 |xL 1 M sulfuric acid (plasma only) + 500 |JLL 200 mg/mL sodium sulfate (liver and
kidney only) + 50 |xL 1 M pH 6 sodium acetate (liver only) + 50 (xL 1 M pH 5 sodium acetate
(kidney only) + 100 |xL 2% trichloroacetic acid (lung and heart only) + 15 mL n-propanol:ether
(liver 16:84, kidney 20:80, lung 88:12, heart 40:60, plasma 88:12), sonicate for 30 s, shake for
15 min, centrifuge for 15 min. Remove the organic layer and evaporate it to dryness under
reduced pressure, reconstitute the residue in 1 mL 50 mM ammonium phosphate (liver pH 11,
kidney pH 3, lung pH 2.5, heart pH 5, plasma pH 2.5), inject a 20 fxL aliquot. (From J.
Liq.Chromatogr. 1994, 17, 1621.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherisorb 5 ODS 2
Mobile phase: MeCN:50 mM phosphate buffer 0.5:99.5 (Liver pH 3, kidney pH 6, lung pH 5,
heart pH 5, plasma pH 2.5)
Column temperature: 10 (kidney), 35 (lung), 20 (heart), 25 (plasma), 15 (liver)
Flow rate: 1
Detector: UV 200
CHROMATOGRAM
Retention time: 18 (plasma), 28 (lung), 30 (liver), 32 (kidney), 36 (heart)
Internal standard: flucytosine (for plasma) (4), 4-chlorouracil (for tissue) (17 (lung), 18 (liver),
11 (kidney), 19 (heart))
Limit of quantitation: 670 ng/g plasma, 110 ng/g (heart), 90 ng/g (lung), 210 ng/g (kidney), 500
ng/g (liver)
OTHER SUBSTANCES
Extracted: metabolites, 5-fluorouracil
KEYWORDS
plasma; rabbit; liver; kidney; lung; heart; pharmacokinetics
REFERENCE
Del Nozal,M.J.; Bernal,J.L.; Pampliega,A.; Marinero,R; Pozuelo,M. Determination of the concentrations of 5-
fluorouracil and its metabolites in rabbit plasma and tissues by high-performance liquid chromatography,
J.Chromatogr.B, 1994, 656, 397-405.
SAMPLE
Sample preparation: Inject a 10 JJLL aliquot.
HPLC VARIABLES
Column: 30 mm long 3 |xm C18 (Perkin-Elmer)
Mobile phase: MeOHrwater 0.5:95.5 (sic)
Flow rate: 1
Detector: UV 268
CHROMATOGRAM
Retention time: 2
KEYWORDS
stability-indicating; saline; injections
REFERENCE
Smith,J.A.; Morris,A.; Duafala,M.E.; Bertino,J.R.; Markman,M.; Kleinberg,M. Stability of floxuridine and leu-
covorin calcium admixtures for intraperitoneal administration, Am. J.Hosp.Pharm., 1989, 46, 985-989.
SAMPLE
Sample preparation: Dilute formulation 1:100 with water, inject a 50 \LL aliquot.
HPLC VARIABLES
Guard column: 5 X 4 35-60 jxm Perisorb RP18
Column: 250 X 4 10 |xm LiChrosorb RP18
Mobile phase: MeOH:300 mM sodium acetate 2:98
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: fluorouracil
KEYWORDS
injections; water
REFERENCE
Sadjak,A.; Wintersteiger,R. Compatibility of morphine, baclofen, floxuridine and fluorouracil in an implantable
medication pump, Arzneimittelforschung, 1995, 45, 93-98.
SAMPLE
Matrix: solutions
Sample preparation: Prepare 50 mg/mL or 1 mg/mL solutions in 0.9% sodium chloride injection,
dilute 1:1000 or 1:100 with mobile phase, inject an aliquot.
HPLCVARIABLES
Column: Bakerbond C18
Mobile phase: MeOH:water 0.5:99.5
Flow rate: 1
Detector: UV 268
CHROMATOGRAM
Retention time: 4.6
OTHER SUBSTANCES
KEYWORDS
stability-indicating
REFERENCE
Stiles,M.L.; Allen,L.V.,Jr.; Prince,S.J. Stability of deferoxamine mesylate, floxuridine, fluorouracil, hydromor-
phone hydrochloride, lorazepam, and midazolam hydrochloride in polypropylene infusion-pump syringes,
SAMPLE
Matrix: solutions
Sample preparation: Add 10 mg of 4-bromomethyl-7-methoxycoumarin and 10 mg potassium
carbonate to 5 mL of a solution in DMSO, after 5 min at room temperature add 4-nitrobenzoic
acid.
HPLCVARIABLES
Column: 200 X 4 5 |xm Nucleosil 5 C18
Mobile phase: MeCN.MeOH.water 5:29:66
Flow rate: 0.6
CHROMATOGRAM
Internal standard: chlorodeoxyuridine (16.5)
OTHER SUBSTANCES
Simultaneous: inosine, thymine, uracil, uridine
KEYWORDS
derivatization; some details of serum extraction in paper
REFERENCE
Yoshida,S.; Hirose,S.; Iwamoto,M. Use of 4-bromomethyl-7-methoxycoumarin for derivatization of pyrimidine
compounds in serum analysed by high-performance liquid chromatography with fluorimetric detection,
J.Chromatogr., 1986, 383, 61-68.
Flubendazole
Merck Index: 4154
Lednicer No.: 2 354
SAMPLE
Sample preparation: Add 1 mL whole blood or urine to Tbxi-Tube A (Tbxi-Lab, Irvine CA), add
HPLC VARIABLES
mL/min)
Detector: UV 211.1
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
ethaciynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fen-
camfamine, fenoprofen, fenproporex, flufenamic acid, flunitrazepam, 5-fluorouracil, fluoxymes-
butazone, oxêtracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, per-
faethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapjnridine, sul-
REFERENCE
Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.Toxicol., 1994,
18, 233-242.
Fluconazole
Molecular formula: C13H12F2N6O
Merck Index: 4158
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL 700 |xg/mL IS + 1.0 mL 1 M KOH, mix, extract
twice with 3 mL portions of ethyl acetate. Combine the organic layers and extract with 2 mL
1 M HCl. Add 1.75 mL 1 M KOH to the aqueous layer and extract twice with 3 mL portions
of ethyl acetate. Combine the organic layers and evaporate them to dryness under a stream of
dry nitrogen, reconstitute the residue in 100 |xL MeCN: 10 mM pH 9.0 ammonium phosphate
50:50, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 3 |xm Microsorb C18
Mobile phase: Gradient. MeCN: 10 mM pH 9.0 ammonium phosphate 10:90 for 1 min, to 60:40
over 15 min, maintain at 60:40 for 2 min.
Flow rate: 0.5
Detector: UV 260
CHROMATOGRAM
Retention time: 7
Internal standard: a-(2,4-dichlorophenyl)-lH-imidazole-l-ethanol (9)
Limit of quantitation: 12 |jig/mL
KEYWORDS
REFERENCE
Black,D.J.; Kunze,K.L.; Wienkers,L.C; Gidal,B.E.; Seaton,T.L.; McDonnell,N.D.; Evans,J.S.; Bauwens,J.E.;
Trager,W.F. Warfarin-fluconazole II. A metabolically based drug interaction: In vivo studies, Drug Me-
tab.Dispos., 1996, 24, 422-428.
SAMPLE
Matrix: blood
Sample preparation: Add 500 jxL MeCN containing an excess of sodium carbonate to 500 jxL
plasma, vortex for 10 s, centrifuge at 2000 g for 10 min, dilute a 350 |xL aliquot of the super-
natant with 700 |xL distilled water, inject an aliquot.
HPLC VARIABLES
Column: 125 X 4 5 juim Ultrabase C8 (SFCC, Neuilly Plaisance, France)
Flow rate: 1
Detector: UV 260
CHROMATOGRAM
Retention time: 3.6
OTHER SUBSTANCES
Noninterfering: amikacin, amoxicillin, carbamazepine, cilastatin, clavulanic acid, epoxycar-
bamazepine, fiunitrazepam, imipenem, methotrexate, midazolam, ofloxacin, phenobarbital,
phenytoin, piperacillin, teicoplanin, theophylline, thiopental, tobramycin, valproic acid,
vancomycin
KEYWORDS
plasma
REFERENCE
Cociglio,M.; Brandissou,S.; Alric,R.; Bressolle,F. High-performance liquid chromatographic determination of
fluconazole in plasma, J.Chromatogr.B, 1996, 686, 11-17.
SAMPLE
Matrix: blood
Sample preparation: Condition a 6 mL 500 mg Bakerbond C18 SPE cartridge with 2 column
volumes of MeOH and 2 column volumes of buffer. 1 mL Plasma + 100 (xL 100 |xg/mL IS in
buffer + 2 mL buffer, mix, add to the SPE cartridge, wash with 1 mL buffer, wash with 1 mL
MeOH:buffer 15:85, dry under vacuum (8-9 inches Hg) for 1 min, elute with 500 |xL MeOH
without vacuum, elute with 500 |xL MeOH under 3-4 inches Hg vacuum. Combine the eluates
and evaporate them to dryness under a stream of nitrogen at 40, reconstitute with 200 |xL
mobile phase, inject a 15 JJL aliquot. (Buffer was 100 mM pH 6.0 sodium phosphate buffer.)
HPLC VARIABLES
Column: 250 X 4 5 |xm Nucleosil C18
Mobile phase: MeCN:25 mM pH 7.0 Tris buffer 25:75
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 5
Internal standard: 2-(2,4-difluorophenyl)-l-fluoro-l,3-bis(lH-l,2,4-triazol-l-yl)propan-2-ol (Pfizer
UK-54373) (8)
KEYWORDS
plasma; comparison with capillary electrophoresis; pharmacokinetics; SPE
REFERENCE
von Heeren,R; Tanner,R.; Theurillat,R.; Thormann,W. Determination offluconazolein human plasma by mi-
cellar electrokinetic capillary chromatography with detection at 190 nm, J.ChromatognA, 1996, 745, 165-
172.
SAMPLE
Sample preparation: Plasma. Extract 100 |xL plasma with 5 mL dichloromethane, centrifuge
at 1000 g for 15 min. Evaporate the organic to dryness under a stream of nitrogen. Reconstitute
the residue with 100 uL mobile phase, inject a 20 |JLL aliquot. Dialysate. Inject a 10 JJLL aliquot
directly.
HPLCVARIABLES
Column: 200 X 2.1 5 |xm HP ODS
Mobile phase: MeCN-.buffer 13:87 (Buffer was 20 mm ammonium monobasic phosphate, ad-
justed to pH 7 with 5 M NaOH.)
Flow rate: 0.5
Detector: UV 210
CHROMATOGRAM
Internal standard: UK-51,060 (plasma)
KEYWORDS
REFERENCE
Yang,H.; Wang,Q.; Elmquist,W.F. Fluconazole distribution to the brain: a crossover study in freely-moving rats
using in vivo microdialysis, Pharm.Res., 1996, 13, 1570-1575.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y; P6pin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for the
creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-163.
Flucytosine
Molecular formula: C4H4FN3O
Merck Index: 4161
SAMPLE
Matrix: blood
Sample preparation: 500 jxL Plasma + 50 |xL 100 |xg/mL 5-chlorouracil in MeOH + 30 |xL 1 M
pH 4.0 sodium acetate buffer + 300 |xL 20% sodium sulfate in water + 8 mL diethyl ether.n-
propanol 75:25, vortex for 5 min, centrifuge at 3000 rpm for 10 min. Remove the organic layer
and evaporate it to dryness under vacuum using a freeze dryer, reconstitute the residue in 1
mL water, vortex, filter (0.22 |xm), inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 150 X 6 5 jxm YMC A-312 octadecylsilane (Yamamura Chemical)
Flow rate: 1.5
Detector: UV 275
CHROMATOGRAM
Retention time: 8
Internal standard: 5-chlorouracil (3)
KEYWORDS
dog; plasma; pharmacokinetics
REFERENCE
Bonny, J.-D.; Kyowa,M. Use of in vitro release tests for the prediction of the in vivo behavior and the develop-
ment offlucytosinecontrolled-release capsules, J.Pharm.Sci., 1995, 84, 619-623.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Dilute 30 |xL of the injection to 100 mL with buffer, add 50 |xL 10 mg/mL
5-flucytosine in water, inject a 20 |JLL aliquot. (Buffer contained 1 g/L Na2HPO4^H2O and 0.363
g/L KH2PO4, pH 7.4.)
HPLCVARIABLES
Column: 150 X 4.6 5 jim Supelcosil LC 18
Mobile phase: 10 mM KH2PO4 adjusted to pH 6.8 with 25% KOH
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: flucytosine
OTHER SUBSTANCES
Simultaneous: vidarabine phosphate
KEYWORDS
injections; water; flucytosine is IS
REFERENCE
Kwee,M.S.L.; Stolk,L.M.L. Formulation of a stable vidarabine phosphate injection, Pharm.Weekbl.fSci]., 1984,
0, 101-104.
SAMPLE
Sample preparation: Tablets. Powder tablets, weigh out amount equivalent to 5 mg flucytosine,
add 50 mL water, stir for 15 min, filter. 1 mL Filtrate +1.5 mL 27 |xg/mL thymine in buffer,
make up to 10 mL with buffer, inject an aliquot. Injections. Dilute with water to a drug con-
centration of 100 |xg/mL. 1 mL Sample + 1.5 mL 27 |xg/mL thymine in buffer, make up to 10
mL with buffer, inject an aliquot. (Buffer was 70 mM KH2PO4 adjusted to pH 3.0 with HCl.)
HPLC VARIABLES
Mobile phase: 9 mM Sodium heptanesulfonate adjusted to pH 2.8 with phosphoric acid
Flow rate: 0.8
Detector: UV 266
CHROMATOGRAM
Retention time: 6.3
Internal standard: thymine (4.85)
OTHER SUBSTANCES
Simultaneous: 5-fluorouracil
KEYWORDS
tablets; injections
REFERENCE
Cavrini,V.; Bonazzi,D.; Di Pietra,A.M. Analysis of flucytosine dosage forms by derivative UV spectroscopy and
liquid chromatography, J.Pharm.Biomed.Anal., 1991, 9, 401-407.
SAMPLE
Sample preparation: Dilute 60 JJLL to 10 mL with 40 u-g/mL p-aminobenzoic acid in MeCN:
water 25:75, inject a 5 JJLL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 ^m ixBondapak C18
Mobile phase: MeCN:buffer 25:75 (Buffer was 2 mL glacial acetic acid and 700 mg 1-octanesul-
fonic acid in 750 mL water.)
Flow rate: 1
Injection volume: 5
Detector: UV 285
CHROMATOGRAM
Retention time: 3.2
Internal standard: p-aminobenzoic acid (4.7)
OTHER SUBSTANCES
KEY WORDS
stability-indicating; oral liquids
REFERENCE
Wintermeyer,S.M.; Nahata,M.C. Stability of flucytosine in an extemporaneously compounded oral liquid, An-
timicrob.Agents Chemother., 1996, 40, 407-409.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Guard column: 10 (xm precolumn (Beckman Instruments Inc.)
Column: 150 X 4.6 5 uin C18 Altex Ultrasphere ODS
Mobile phase: Isocratic. MeOH:buffer 15:85 containing 2.5 mM sodium pentanesulfonate and
2.5 mM sodium heptanesulfonate. Gradient. A was MeOH containing 2.5 mM sodium pentane-
sulfonate and 2.5 mM sodium heptanesulfonate. B was buffer containing 2.5 mM sodium pen-
tanesulfonate and 2.5 mM sodium heptanesulfonate. A:B 0:100 for 2 min, to 30:70 over 1 min,
maintain at 30:70. (Buffer was 50 mM phosphoric acid containing 50 mM KH2PO4, pH 2.5.)
Flow rate: 1
Detector: UV 254; UV 285
CHROMATOGRAM
Retention time: 3.76 (isocratic), 9.20 (gradient)
Internal standard: 5-methylcytosine (5.66 (isocratic), 12.62 (gradient))
Limit of quantitation: 12.5 (xM
OTHER SUBSTANCES
Simultaneous: barbituric acid, cytosine, fluorouracil, hydroxycytosine, uracil, urea
REFERENCE
Biondi,L.; Nairn,J.G. High performance liquid chromatographic assay for 5-fluorouracil and 5-fluorocytosine,
J.Liq.Chromatogr., 1985, 8, 1881-1892.
SAMPLE
Matrix: solutions
Sample preparation: Centrifuge and filter cell solutions (0.22 |xm), inject an aliquot.
HPLC VARIABLES
Guard column: Guard-PAK C18 (Waters)
Column: 150 X 3.9 5 |xm NOVA PAK C18
Mobile phase: MeOH:50 mM pH 6.0 KH2PO4 3:97
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 2.5
REFERENCE
Koga,H. High-performance liquid chromatography measurement of antimicrobial concentrations in polymor-
phonuclear leukocytes, Antimicrob.Agents Chemother., 1987, 31, 1904-1908.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1
Detector: UV 200
CHROMATOGRAM
Retention t i m e : 4
OTHER SUBSTANCES
Simultaneous: 5-fluorouracil, floxuridine
REFERENCE
J.Chromatogr.B, 1994, 656, 397-405.
Fludarabine
Molecular formula: G10H12FN5O4
Merck Index: 4162
Lednicer No.: 4 167
SAMPLE
Matrix: blood
Sample preparation: Condition a 500 mg Bakerbond SPE cartridge packed with LiChrosorb
RP-18 with 2 mL MeOH, 2 mL water and 1 mL MeOH. Mix 500 |xL plasma with 100 JJIL 30
juLg/mL mercaptopurine in MeOH, make up to 3 mL with MeOH. Centrifuge at 1100 g for 15
min, add 1.5 mL supernatant to the SPE cartridge, elute at 50 |xL/min flow-rate, inject a 20
IxL aliquot.
HPLC VARIABLES
Column: 200 X 4 7 jjim Lichrosorb RP-18
Mobile phase: MeOH: pH 4.15 phosphate buffer 20:80
Flow rate: 1
Detector: UV 262
CHROMATOGRAM
Internal standard: mercaptopurine (3.30)
KEYWORDS
plasma; SPE
REFERENCE
Misztal,G.; Paw,B- Determination of fludarabine phosphate in human plasma using reversed phase high-per-
formance liquid chromatography, Pharmazie, 1996, 51, 733-734.
SAMPLE
Matrix: blood
Sample preparation: Isolate mononuclear cells from 10 mL blood by a standard step-gradient
density centrifugation procedure. Wash once with PBS, resuspend in 500 JJLL water, add 500
|JLL 800 mM perchloric acid, centrifuge at 400 g for 5 min, wash the pellet with 500 |xL 400
mM perchloric acid, centrifuge at 400 g for 5 min. Combine supernatants, neutralize with 10
M KOH, bring to pH 7 with 1 M KOH (Universal indicator paper), cool in ice, centrifuge at
400 g for 5 min, inject a 50-2000 |xL aliquot of the supernatant. (PBS was 8.1g NaCl, 0.22 g
KCl, 1.14 g NaHPO4 (sic), 0.27 g KH2PO4 in 1 L water, pH 7.4.)
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Partisil 10 SAX
Mobile phase: Gradient. A was 5 mM pH 2.8 (NH4)H2PO4. B was 750 mM pH 3.5 (NH4)H2PO4.
A:B from 70:30 to 0:100 over 30 min (concave gradient, Waters no. 9). (At the start of each day
pump through 20 mL 2 M (NH4)H2PO4 then inject 100 |xL 100 mM disodium EDTA into the
initial mobile phase.)
Flow rate: 3
Detector: UV 262
CHROMATOGRAM
Retention time: 29 (as triphosphate, Fara-ATP)
OTHER SUBSTANCES
Extracted: ara-CTP (cytarabine triphosphate), ATP, CTP, UTP, GTP
KEYWORDS
mononuclear cells
REFERENCE
Gandhi,V.; Danhauser,L.; Plunkett,W. Separation of 1-p-D-arabinofuranosylcytosine 5'-triphosphate and 9-p-D-
arabinofuranosyl-2-fluoroadenine 5'-triphosphate in human leukemia cells by high-performance liquid chro-
matography, J.Chromatogr., 1987, 413, 293-299.
SAMPLE
Sample preparation: Plasma. Filter (Amicon CF) while centrifuging at 1000 g for 20 min, filter
(0.45 |xm) the ultrafiltrate, inject an aliquot. Urine. 250 |xL Urine + 500 JJLL 150 mM Ba(OH)2,
vortex, add 500 |xL 5% ZnSO4.7H2O, vortex, centrifuge at 1700 g for 15 min, filter (0.45 |xm),
inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere C18
Mobile phase: MeOH: 10 mM pH 4.15 (NH4)H2PO4 6:94
Flow rate: 1-1.2
Detector: UV 254
CHROMATOGRAM
Retention time: 55
OTHER SUBSTANCES
Extracted: fludarabine phosphate
KEYWORDS
plasma; pharmacokinetics; ultrafiltrate
REFERENCE
Hersh,M.R.; Kuhn,J.G.; Phillips,J.L.; Clark,G.; Ludden,T.M.; von Hoff,D.D. Pharmacokinetic study of fludara-
bine phosphate (NSC 312887), Cancer Chemother.Pharmacol., 1986, 17, 277-280.
SAMPLE
Matrix: dialysate
HPLC VARIABLES
Column: 80 X 4.6 C18 (Perkin-Elmer)
Mobile phase: MeOHrIO mM pH 7 KH2PO4 20:80
Flow rate: 1
Detector: UV 265
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Simultaneous: araA, adenosine, 2-chloro-2'-arabinofluoro-2'-deoxyadenosine, 2-chloro-2'-deoxy-
adenosine, 2-chloroadenosine, 5'-chloro-5'-deoxyadenosine, 2'-deoxyadenosine
REFERENCE
Reichelova,V.; Liliemark,J.; Albertioni,F. Structure-activity relationships of 2-chloro-2'-ara&mo-fluoro-2'-
deoxyadenosine and related analogues: Protein binding, lipophilicity, and retention in reversed-phase LC,
J.Liq.Chromatogr., 1995, 18, 1123-1135.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 80 X 4.6 3 juim (Perkin-Elmer)
Mobile phase: MeOH: 10 mM pH 6.8 potassium phosphate buffer 20:80
Flow rate: 1
Detector: UV 265
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Simultaneous: cladribine, analogs, degradation products
REFERENCE
Reichelova,V.; Liliemark,J.; Albertioni,F. Liquid chromatographic study of acid stability of 2-chloro-2'-ara&mo-
fluoro-2'-deoxyadenosine, 2-chloro-2'-deoxyadenosine and related analogues, J.Pharm.Biomed.Anal., 1995,
13, 711-714.
Fludeoxyglucose F18
Molecular formula: C6H1118FO5
Merck Index: 4163
SAMPLE
Matrix: cell incubations
Sample preparation: Wash cells with PBS, freeze in liquid nitrogen, add 3 mL 400 mM per-
chloric acid, let stand at 4 for 30 min, centrifuge at 12000 g at 4, extract with 3 mL 500 mM
trioctylamine in 1,1,2-trichlorotrifluoroethane, filter (0.22 (xm) the inorganic phase, inject an
aliquot.
HPLC VARIABLES
Guard column: LiChrosorb RP 18-5
Column: 100 X 8 Radial-PAK Partisil 10 SAX (Waters)
Mobile phase: Gradient. A was MeOH:15 mM pH 3.8 (NH4)H2PO4 3:97. B was MeOH:750 mM
pH 4.8 (NH4)H2PO4 3:97. A:B from 100:0 to 0:100 over 35 min (concave gradient), re-equilibrate
for 15 min.
Flow rate: 2
Detector: Radioactivity (Berthold LB 506 C-I) (with Quick Szint Flow 306 pumped at 4 mL/min)
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
KEY WORDS
C14 labeled; chondrocytes
REFERENCE
Fedders,G.; Kock,R.; Van de Leur,E.; Greiling,H. A radiochemical high-performance liquid chromatographic
method for the analysis of 2-fluoro-2-deoxy-D-glucose-derived metabolites in human chondrocytes,
Anal.Biochem., 1993, 211, 81-86.
SAMPLE
Matrix: reaction mixtures
Sample preparation: Evaporate reaction mixture in the presence of active charcoal and pass
through a 100 X 10 column of Merck Kieselgel 60 for dry column chromatography using 70
mL MeCN:water 99.7:0.3, evaporate the eluate, take up the residue in 1 mL water, inject an
aliquot.
HPLC VARIABLES
Guard column: 30 X 3.6 Aminex HPX-87C (Bio-Rad)
Column: 300 X 7.8 Aminex HPX-87P (Bio-Rad) (The column contains lead which is washed off.
Periodically backflush with 100 mM lead nitrate at 0.1 mL/min overnight.)
Mobile phase: Water
Flow rate: 0.1
Detector: RI (at 18)
CHROMATOGRAM
KEYWORDS
F18 labeled
REFERENCE
Oberdorfer,R; Hull,W.E.; Traving,B.C; Maier-Borst,W. Synthesis and purification of 2-deoxy-2-[18F]fluoro-D-
glucose and 2-deoxy-2-[18F]fluoro-D-mannose: characterization of products by IH- and 19F-NMR spectros-
copy, Int.J.Rad.Appl.Instrum.[A]., 1986, 37, 695-701.
SAMPLE
Matrix: tissue hydrolyzate
Sample preparation: Centrifuge at 12000 g for 10 min, inject an aliquot.
HPLC VARIABLES
Guard column: 20 X 4 APS Hypersil
Column: 250 X 4 BioSil Amino 5S (Bio-Rad)
Flow rate: 1
Detector: Radioactivity (with Quick Szint Flow 302)
CHROMATOGRAM
Retention time: 7
KEYWORDS
C14 labeled
REFERENCE
Fedders,G.; Kock,R.; Van de Leur,E.; Greiling,H. The metabolism of 2-fluoro-2-deoxy-D-glucose in human chon-
drocytes and its incorporation into keratan sulfate proteoglycans, Eur.J.Biochem., 1994, 219, 10631071.
Fludrocortisone
Merck Index: 4166
Lednicer No.: 1 192
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherex C18 (Phenomenex USA)
Mobile phase: MeOH:THF:water 3:25:72
Flow rate: 1.0
Detector: UV 254
CHROMATOGRAM
OTHERSUBSTANCES
Simultaneous: 11-deoxycortisol, dexamethasone, hydrocortisone, methylprednisolone, pred-
nisolone
REFERENCE
McWhinney,B.C; Ward,G.; Hickman,P.E. Improved HPLC method for simultaneous analysis of cortisol, 11-
deoxycortisol, prednisolone, methylprednisolone, and dexamethasone in serum and urine, CUn. Chem., 1996,
42, 979-981.
SAMPLE
Matrix: solutions
Sample preparation: Inject 20 |xL aliquot of a MeOH solution.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Hypersil 5-ODS
Mobile phase: THF:water 23:77
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Retention time: k' 7.87 (fludrocortisone), k' 28.04 (fludrocortisone acetate)
Internal standard: methylprednisolone (k' 11.36)
OTHER SUBSTANCES
Simultaneous: metabolites, betamethasone, corticosterone, cortisone, defiazacort, deoxycorticos-
terone, dexamethasone, fluorocortisone, fluorocortisone acetate, hydrocortisone, 21-hydroxy-
deflazacort, lla-hydroxyprogesterone, methylprednisolone, prednisolone, prednisone, triamcin-
olone acetonide, triamcinolone
REFERENCE
Santos-Montes,A.; Gonzalo-Lumbreras,R.; Gasco-Lopez,A.I.; Izquierdo-Hornillos,R. Extraction and high-perfor-
mance liquid chromatographic separation of defiazacort and its metabolite 21-hydroxydeflazacort. Appli-
cation to urine samples, J.Chromatogr.B, 1994, 657, 248-253.
SAMPLE
Matrix: urine
Sample preparation: Equilibrate a Sephadex G-25M column with 100 mM pH 7.0 phosphate
buffer. Condition a Bond-Elut C18 SPE cartridge with 1 mL MeCN, 4 mL acetone:water 20:
80, and 4 mL water. 2 mL Urine + 500 (xL 500 mM pH 5.0 acetate buffer + 50 jxL MeOH +
160 |xL 100000 Fishmann U/mL p-glucuronidase and 800000 Roy U/mL arylsulfatase (from
Helix pomatia, Boehringer Mannheim), heat at 37 for 24 h, filter (0.45 jxm), add to the Se-
phadex column, wash with three 2 mL portions of 100 mM pH 7.0 phosphate buffer, elute with
four 2 mL portions of 100 mM pH 7.0 phosphate buffer. Add the eluate to the SPE cartridge,
wash with 4 mL water, wash with 4 mL acetone:water 20:80, elute with 1 mL MeCN. Evaporate
the eluate to dryness under a stream of nitrogen, reconstitute the residue in 100 JJLL MeOH,
add 20 |xL cupric acetate solution, let stand at room temperature for 1 h, add 100 |xL reagent,
heat at 60 for 40 min, cool, centrifuge briefly at 1000 g, inject a 100 jxL aliquot of the super-
natant. (Cupric acetate solution was 0.7 g cupric acetate in water diluted to 100 mL with
MeOH. Reagent was 7 mM l,2-diamino-4,5-methylenedioxybenzene in water containing 200
mM p-mercaptoethanol and 250 mM sodium hydrosulfite, store in the dark at 4, stable for at
least 2 weeks. Prepare l,2-diamino-4,5-methylenedioxybenzene as follows. Add 5 g l,2-(meth-
ylenedioxy)-4-nitrobenzene to 37.5 mL concentrated nitric acid and 12.5 mL glacial acetic acid,
pour the yellow-colored solution into water, recrystallize the l,2-dinitro-4,5-methylenedioxy-
benzene from EtOH (Rec.Trav.Chim.Pays-Bas 1930, 49, 45). Dissolve 5 g l,2-dinitro-4,5-meth-
ylenedioxybenzene in 200 mL benzene (Caution! Benzene is a carcinogen!), add 100 g 80 mesh
iron powder, add 20 mL concentrated HCl in small portions over 1 h while heating the mixture
under reflux. Reflux for 4 h, add 10 mL water, reflux for 2 h, cool, make alkaline with 2.6 M
NaOH, extract three times with 200 mL portions of benzene. Combine the extracts, evaporate
to dryness to give l,2-diamino-4,5-methylenedioxybenzene, mix with 10 mL concentrated HCl,
recrystallize from EtOH to give l,2-diamino-4,5-methylenedioxybenzene dihydrochloride, mp
176-9 (Chem.Pharm.Bull. 1987, 35, 687).)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm L-Column ODS (Chemicals Inspection and Testing Institute, Tokyo)
Mobile phase: MeOH:MeCN:500 mM ammonium acetate 50:10:40 (After each injection wash
with MeOH:water 80:20 for 20 min for 20 min, re-equilibrate for 20 min.)
Flow rate: 1
CHROMATOGRAM
Internal standard: fludrocortisone
OTHER SUBSTANCES
Extracted: hydrocortisone, tetrahydroaldosterone, aldosterone
Noninterfering: cortisone, corticosterone, hydroxycorticosteroids
KEY WORDS
SPE; derivatization; fludrocortisone is IS
REFERENCE
Yoshitake,T.; Ishida,J.; Sonezaki,S.; Yamaguchi,M. High performance liquid chromatographic determination of
3a, 5p-tetrahydroaldosterone and cortisol in human urine with fluorescence detection, Biomed.
ChromatogK, 1992, 6, 217-221.
Flufenamic acid
Molecular formula: C14H10F3NO2
Merck Index: 4167
Lednicer No.: 1 110
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 40 JULL 100 jxg/mL mefenamic acid in MeOH + 1 mL 1 M
hydrochloric acid + 6 mL dichloromethane, shake mechanically for 20 min. Centrifuge at 2000
g for 5 min, evaporate organic phase to dryness under a gentle stream of nitrogen at 40.
Reconstitute the residue with 50 JJLL MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 5 jxm Nucleosil C18
Mobile phase: MeOHrwater 77:23
Flow rate: 0.8
Detector: UV 280
CHROMATOGRAM
Retention time: 7.2
Internal standard: mefenamic acid (8.6)
KEYWORDS
REFERENCE
Cerretani,D.; Micheli,L.; Fiaschi,L.; Giorgi,G. High-performance liquid chromatography of flufenamic acid in
rat plasma, J.Chromatogr.B, 1996, 678, 365-368.
SAMPLE
Sample preparation: 100-300 mg Gel ointment + 3 mL MeOH, mix vigorously, filter (0.2 (xm),
inject a 10 uL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 |xm jxBondapak C18
Mobile phase: MeOH
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
KEYWORDS
ointment
REFERENCE
Yamamura,K; Yamada,J.-L; Yotsuyanagi,T. High-performance liquid chromatographic assay of antiinflamma-
tory drugs incorporated in gel ointments. Separation and stability testing, J.Chromatogr., 1985, 331, 383-
388.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
camfamine, fenoprofen, fenproporex, fentanyl, flunitrazepam, 5-fluorouracil, fluoxymesterone,
REFERENCE
Hill,D.W.; Kind ,A. J- Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 JJLL aliquot of a 100-500 jjig/mL solution in mobile phase.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Hypersil C8 MOS 100A coated with phosphatidylcholine (95% pure
phosphatidylcholine in MeOHrwater 80:20 for 24 h.)
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: amoxicillin, antipyrine, carbamazepine, chlorpheniramine, chlorpromazine, clon-
idine, codeine, desipramine, diphenhydramine, dipyridamole, ephedrine, haloperidol, hydro-
xyzine, imipramine, indomethacin, lidocaine, megestrol acetate, metoprolol, nabumetone, na-
dolol, phenobarbital, phenol, promazine, propranolol, pyrilamine, quinidine, ropinirole,
testosterone, thioridazine, tolfenamic acid, verapamil
idine, fenoterol, fiurbiprofen, ibuprofen, ketoprofen, ranitidine, salicylic acid, sulfamethoxazole,
KEYWORDS
REFERENCE
methodology, Anal Chem., 1998, 70, 2092-2099.
Fluindione
Merck Index: 4168
SAMPLE
Matrix: blood
Sample preparation: Add 100 jxL plasma to a siliconized tube containing 50 |xL 2 |xg/mL IS in
MeOH and 100 JUIL MeCN. Vortex for 30 s, centrifuge at 3000 g for 10 min. Mix a 150 JULL
aliquot of the supernatant with 150 |xL 0.9% NaCl, inject a 100 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:67 mM Na2HPO4 buffer adjusted to pH 7.2 with orthophosphoric acid 23:
77 (At the end of each chromatographic session rinse the HPLC system with 200 mL MeCN:
water 50:50.)
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Retention time: 4.2
Internal standard: coumarin (8.5)
OTHER SUBSTANCES
Also analyzed: acenocoumarol, brodifacoum, bromadiolone, chlorphacinone, coumatetralyl, di-
fenacoum, diphenadione, ethyl biscoumacetate, phenidione, warfarin
KEYWORDS
plasma
REFERENCE
Aymard,G.; Legrand,M.; Comets,E.; Mentre,R; Diquet,B. Rapid and simple micromethod for the quantification
of fluindione in human plasma using high-performance liquid chromatography, J.Chromatogr.B, 1998, 707,
169-173.
Flumazenil
CAS Registry No.: 78755-81 -4
Merck Index: 4169
LednicerNo.: 4 220
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 25 jxL EtOH + 25 |xL 3 |xg/mL ISl and 7.6 |xg/mL IS2 in
EtOH + 1 mL 100 mM Na2HPO4 adjusted to pH 10.5 with NaOH + 5 mL diethyl ether:di-
chloromethane 60:40, vortex for 30 s, centrifuge at 4 at 2000 g for 10 min. Remove the organic
phase and add it to 1 mL 100 mM Na2HPO4 adjusted to pH 10.5 with NaOH, vortex for 30 s,
centrifuge at 4 at 2000 g for 5 min. Remove the organic layer and evaporate it to dryness
under a stream of nitrogen at 40, reconstitute the residue in 50 JJLL mobile phase, inject a 1-
15 JJLL aliquot.
HPLCVARIABLES
Column: 100 X 4.6 3 |xm CP-Microspher C18 (Chrompack)
Mobile phase: Gradient. A was MeOH:buffer 1:2. B was MeOH:water 80:20. A:b 93.8:6.2 for 5.5
min, to 60:40 over 0.15 min, maintain at 60:40 for 11.3 min, to 2.5:97.5 over 0.5 min, maintain
at 2.5:97.5 for 3.5 min, return to initial conditions over 0.5 min (Buffer was 6 g/L NaH2PO4
and 1 mL/L triethylamine adjusted to pH 7.00 with NaOH.)
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Retention time: 5.0
Internal standard: ISl ethyl 7-chloro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[l,5-a][l,4]
benzodiazepine-3-carboxylate (Ro 15-305) (6.4), IS2 climazolam (17.0)
OTHER SUBSTANCES
Extracted: midazolam
KEYWORDS
REFERENCE
Vletter,A.A.; Burm,A.G.L.; Breimer,L.T.M.; Spierdijk,J. High-performance liquid chromatographic assay to de-
termine midazolam and flumazenil simultaneously in human plasma, J.Chromatogr., 1990, 530, 177-185.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum or plasma + 1 mL saturated Na2HPO4 + 100 |xL 10 fjug/mL
IS + 5 mL diisopropyl ether :isopropanol 95:5 (Caution! Diisopropyl ether readily forms explo-
sive peroxides!), shake for 5 min, centrifuge. Remove the organic layer and evaporate it to
dryness at 35, reconstitute the residue in 50 jxL mobile phase, vortex, inject a 20 JJLL aliquot.
HPLCVARIABLES
Column: 100 X 3 Chrompack CP spher C8
Mobile phase: MeCN:50 mM NaH2PO4 70:30 adjusted to pH 2.2 with 85% orthophosphoric acid
Flow rate: 0.8
Detector: UV 210
CHROMATOGRAM
Retention time: 2.1
Internal standard: N-6-dimethyl-2-(4-methylphenyl)-N-propylimidazo[ 1,2-a]pyridine-3-aceta-
mide methanesulfonate (Synthelabo France) (6.4)
OTHER SUBSTANCES
Extracted: prothipendyl, zolpidem
KEYWORDS
serum; plasma; pharmacokinetics
REFERENCE
Debailleul,G.; Khalil,F.A.; Lheureux,P. HPLC quantification of zolpidem and prothipendyl in a voluntary in-
toxication, JAnal.ToxicoL, 1991,15, 35-37.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with water, MeOH, and 100 mM
ammonium acetate. Add 200 |xL plasma to the SPE cartridge, wash with 100 mM ammonium
acetate, elute with MeOHrIOO mM ammonium acetate 3:1. Evaporate the eluate to dryness
under reduced pressure, dissolve the residue in 200 (xL mobile phase, inject a 20 JJIL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 Hitachi gel 3056 octadecylsilica
Mobile phase: MeOH: 100 mM ammonium acetate 60:40
Flow rate: 1
Detector: MS, Hitachi M1000, APCI, nebulizer 260, vaporizer 399
CHROMATOGRAM
Retention time: 2.7
Limit of detection: 0.5-2.5 ng/mL
OTHER SUBSTANCES
Simultaneous: atipamezole, atropine, butorphanol, ketamine, medetomidine, midazolam,
xylazine
KEYWORDS
plasma; SPE; dog
REFERENCE
Kanazawa,H-; Nagata,Y.; Matsushima,Y.; Takai,N.; Uchiyama,H-; Nishimura,R-; Takeuchi,A- Liquid chroma-
tography-mass spectrometry for the determination of medetomidine and other anaesthetics in plasma,
J.Chromatogr., 1993, 631, 215-220.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform:isopropanol:
the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 jxL aliquot of
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 244
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Sample preparation: 500 |JLL Plasma or urine + 50 |xL 4 |xg/mL flurazepam in MeOH + 1 mL
100 mM pH 9 sodium phosphate buffer + 4 mL dichloromethanerdiethyl ether 60:40, shake at
45 rpm for 15 min, centrifuge at 10 at 1870 g for 10 min. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 80 |xL
MeOH, inject a 30 |xL aliquot. (Deconjugate urine as follows. 250 |xL Urine + 750 |xL pH 5.4
acetate buffer + 500 U p-glucuronidase, heat at 37 for 18 h, add 20 JULL 5 M NaOH, centrifuge,
proceed as above using 5 mL dichloromethaneidiethyl ether.)
HPLC VARIABLES
Guard column: 30 X 4.6 30 |xm C8
Column: 100 X 8 4 |xm Nova Pak C18
Mobile phase: MeCN:40 mM sodium phosphate buffer 32:68 containing 1 mL/L triethylamine,
final pH 7.2
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Retention time: 3.8
OTHER SUBSTANCES
Extracted: midazolam, 4-hydroxymidazolam, 1-hydroxymethylmidazolam
Noninterfering: alfentanil, atropine, bupivacaine, lignocaine, neostigmine
KEYWORDS
plasma
REFERENCE
Chan,K.; Jones,R.D.M. Simultaneous determination of flumazenil, midazolam and metabolites in human bio-
logical fluids by liquid chromatography, J.Chromatogr., 1993, 619, 154-160.
SAMPLE
HPLCVARIABLES
Column: 150 X 4.6 5 jxm Spherisorb C18
Mobile phase: MeOH:THF:isopropanol:water 30:3.5:1.5:65
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: methylparaben, propylparaben, phenol, degradation products
Noninterfering: aminophylline, cimetidine, dobutamine, dopamine, famotidine, lidocaine, pro-
cainamide, ranitidine
KEYWORDS
REFERENCE
Olsen,K.M.; Gurley,BJ.; Davis,G.A.; Christensen,R.; Monaghan,M.S. Stability of flumazenil with selected drugs
in 5% dextrose injection, Am. J.Hosp.Pharm., 1993, 50, 1907-1912.
Flumequine
Molecular formula: C14H12FNO3
Merck Index: 4172
LednicerNo.: 3 186
SAMPLE
Matrix: blood
Sample preparation: Mix 100 |xL plasma with 100 JJLL pH 6.0 phosphate buffer and 1 mL ethyl
acetate, shake, centrifuge at 20000 g for 5 min, collect 800 |xL the organic phase, dry under
nitrogen at 45. Add 300 |xL pH 7.8 phosphate buffer and 300 |xL hexane to the residue, shake,
centrifuge at 20000 g for 5 min, inject a 100 (JLL aliquot of the aqueous phase.
HPLC VARIABLES
Guard column: 4 X 4 C18 (Merck)
Column: 125 X 4 5 |xm Lichrospher RP Select B
Mobile phase: MeCN:DMF:water:orthophosphoricacid 18:28:40.5:13.5
Flow rate: 0.8
Detector: UV 324 (A), F ex 320 em 365 (B)
CHROMATOGRAM
Retention time: 6.4
Limit of quantitation: 5 ng/mL (F), 100 ng/mL (UV)
OTHER SUBSTANCES
Noninterfering: ciprofloxacin, danofloxacin, enrofioxacin, marbofloxacin, nalidixic acid, oxolinic
acid
KEYWORDS
sheep; plasma
REFERENCE
Delmas,J.M.; Chapel,A.M.; Sanders,P. Determination of flumequine and 7-hydroxyflumequine in plasma of
sheep by high-performance liquid chromatography, J.Chromatogr.B, 1998, 712, 263-268.
SAMPLE
Matrix: tissue
Sample preparation: 2 g Minced tissue + 100 |xL 5 |xg/mL IS in water + 4 g anhydrous sodium
sulfate, mix until homogenized. Add 10 ml ethyl acetate, shake mechanically for 10 min, cen-
trifuge at 1500 g for 10 min. Transfer organic layer into another tube and repeat extraction on
the tissue pellet with 10 mL ethyl acetate. Evaporate combined organic phases under a stream
of nitrogen at 50. Dissolve residue in 1 mL MeCN:2.7 mM pH 2.5 oxalic acid 50:50, vortex,
sonicate for 5 min, filter through 0.45 |xm filter (GHP Acrodisc GF, Gelman Sciences, USA).
Inject a 100 |JLL aliquot. (1 mg/mL IS stock solution was prepared in 20 mM NaOH, it was
diluted to working concentration with water immediately before use.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultrabase C18 (Shandon, UK)
Mobile phase: Gradient. A was MeCN. B was 2.7 mM pH 2.5 oxalic acid. A:B from 10:90 to 70:
30 over 20 min, maintain at 70:30 for 5 min, return to initial conditions over 5 min.
Flow rate: 0.8
CHROMATOGRAM
Retention time: 20
Internal standard: ibafloxacin (21.3)
OTHER SUBSTANCES
KEYWORDS
kidney; pig
REFERENCE
Guyonnet,J.; Pacaud,M.; Richard,M.; Doisi,A.; Spavone,E; Hellings,R Routine determination of fiumequine in
kidney tissue of pig using automated liquid chromatography, J.Chromatogr.B, 1996, 679, 177-184.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize 1 g tissue with 5 mL acetone, centrifuge, decant and save
the supernatant, repeat the extraction. Add 2 mL acetone, 3 mL hexane, and 6 mL 3% NaCl
to the combined supernatants, extract, centrifuge, discard the hexane layer. Add the extract to
25 mL chloroform (Caution! Chloroform is a carcinogen!), mix, separate the phases, add 2.5
mL 100 mM pH 9.0 Na3PO4 buffer and one drop 1 M NaOH to the chloroform phase, mix,
separate the phases, discard the chloroform layer. Wash the aqueous layer with 2.5 mL chlo-
roform, centrifuge the aqueous layer. Dialyze a 740 |xL aliquot of the supernatant (in 2 portions)
against 3.9 mL 20 mM pH 5.0 sodium phosphate buffer pumped at 0.6 mL/min using a Cu-
prophan 15000 MW cut-off cellulose acetate membrane. After washing column A with 500 (JLL
MeCNiwater 50:50 and 500 IJLL 20 mM pH 5.0 Na3PO4 buffer the dialysate flowed through
column A to waste. Wash column A with 500 jxL 20 mM pH 5.0 sodium phosphate buffer, elute
the contents of column A onto column B with mobile phase, elute with mobile phase, monitor
the effluent from column B. (Wash the donor channel with 2 mL 0.01% Triton X-100 in 20 mM
pH 5.0 sodium phosphate buffer. Wash the acceptor channel with 3 mL 20 mM pH 5.0 sodium
phosphate buffer. Regenerate the membrane with 2 mL 100 mM pH 9.0 sodium phosphate
buffer (donor channel) and 3 mL 20 mM pH 5.0 sodium phosphate buffer (acceptor channel).)
HPLC VARIABLES
Column: A 5.8 X 4.6 Hypersil ODS; B 150 X 4.6 PLRP-S (Polymer Labs., UK)
Mobile phase: MeCN:THF:20 mM pH 5.0 Na3PO4 buffer 20:15:65
Flow rate: 0.6
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Extracted: oxolinic acid
KEYWORDS
column switching; chicken; liver; dialysis
REFERENCE
Eng,G.Y.; Maxwell,R.J.; Cohen,E.; Piotrowski,E.G.; Fiddler,W. Determination of fiumequine and oxolinic acid
in fortified chicken tissue using on-line dialysis and high-performance liquid chromatography with fluores-
cence detection, J.Chromatogr.A, 1998, 799, 349-354.
Flumethasone
Molecular formula: C22H28F2O5
CAS Registry No.: 2135-17-3,2002-29-1 (pivalate)
Merck Index: 4173
Lednicer No.: 1 200
SAMPLE
Matrix: blood
Sample preparation: Add 1 mL serum to a Sep Pak C18 SPE cartridge, wash with 4 mL water,
elute with 4 mL MeOH, evaporate to dryness under vacuum, reconstitute in 50 |xL MeCN:
water 30:70, inject whole sample.
HPLCVARIABLES
Column: 250 X 4.6 5 |jim Ultrasphere ODS
Flow rate: 1
Detector: enzyme immunoassay of fractions
CHROMATOGRAM
Retention time: 18
Limit of detection: 0.3 pg
OTHER SUBSTANCES
Extracted: dexamethasone, betamethasone, triamcinolone
Noninterfering: endogenous steroids
KEYWORDS
serum; SPE; horse
REFERENCE
Friedrich,A.; Schulz,R.; Meyer,H.H. Use of enzyme immunoassay and reverse-phase high-performance liquid
chromatography to detect and confirm identity of dexamethasone in equine blood, Am. J. Vet Res., 1992, 53,
2213-2220.
SAMPLE
Matrix: blood
Sample preparation: Condition a Tef Elutor C18 cartridge with two 3 mL portions of MeOH
then two 3 mL portions of water. Heat 1 mL plasma at 50 for 10 min, add to cartridge, wash
with 2 mL water, 1 mL MeOH:water 10:90, 4 mL acetone:water 20:80, apply suction to car-
tridge for 10 min to air dry. Elute with 1 mL MeOH, evaporate eluent at 45 under nitrogen,
reconstitute with 50 (JLL mobile phase, inject 25 JJLL aliquot.
HPLCVARIABLES
Column: 100 X 2 3 jxm C18 Hypersil
Mobile phase: MeCN:THF:water 8:10:82, containing 5 mIVL triethylamine, pH adjusted to 6.5
with citric acid
Flow rate: 0.6
Detector: UV 242
CHROMATOGRAM
Internal standard: flumethasone
OTHER SUBSTANCES
Simultaneous: dexamethasone, prednisone, hydrocortisone, adrenosterone, prednisolone, es-
triol, corticosterone, methylprednisolone, cortisone, hydroxyprogesterone, testosterone, deoxy-
corticosterone, fluorometholone, spironolactone, equilenin, estrone, estradiol, progesterone, di-
phenhydramine, propranolol, aspirin, theophylline, imipramine, desipramine, indomethacin,
amitriptyline, nortriptyline, nordiazepam, diazepam, chlordiazepoxide, tripelennamine, car-
bamazepine, probenecid, phenobarbital
Noninterfering: caffeine, nicotine, cotinine, chlorothiazide, acetazolamide, phenytoin, phenira-
mine, cephalothin, primidone, acebutolol, hydrochlorothiazide, quinine, acetophenetidin, furo-
semide, aldosterone, triamcinolone, ephedrine, allopurinol, phenylephrine
KEYWORDS
plasma; SPE; flumethasone is IS
REFERENCE
Hariharan,M.; Naga,S.; VanNoord,T.; Kindt,E.K. Simultaneous assay of corticosterone and cortisol in plasma
by reversed-phase liquid chromatography, Clin.Chem., 1992, 38, 346-352.
SAMPLE
Matrix: blood
Sample preparation: Condition a 2 mL 200 mg Tef Elutor C18 SPE cartridge (Versa Prep) with
3 mL MeOH and two 3 mL portions of water. Heat 1 mL plasma at 50 for 10 min, add to SPE
cartridge, wash with 2 mL water, wash with 1 mL MeOH:water 10:90, wash with 4 mL acetone:
water 20:80, air-dry for 10 min, elute with 1 mL MeOH. Evaporate the eluate to dryness under
a stream of nitrogen at 45, reconstitute the residue in 50 |JLL mobile phase, inject a 25 JJLL
aliquot.
HPLC VARIABLES
Column: 100 X 2 3 |xm Hypersil
Mobile phase: MeCN:THF:water 8:10:82 containing 5 mL/L triethylamine, pH adjusted to 6.5
with citric acid
Flow rate: 0.6
Detector: UV 242
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Extracted: cortisone, corticosterone, hydrocortisone
Simultaneous: acebutolol, acetazolamide, acetophenetidin, adrenosterone, aldosterone, amitrip-
tyline, androsten-3,17-dione, aspirin, carbamazepine, cephalothin, chlordiazepoxide, chloro-
thiazide, dehydrocorticosterone, deoxycorticosterone, deoxycortisol, desipramine, dexametha-
sone, diazepam, diphenhydramine, equilenin, estradiol, estriol, estrone, fluorometholone,
furosemide, hydrochlorothiazide, hydroxycorticosterone, hydroxyprogesterone, hydroxyproges-
terone, imipramine, indomethacin, methylhydroxyprogesterone, methylprednisolone, nandro-
lone, nordiazepam, nortriptyline, pheniramine, phenobarbital, phenytoin, prednisolone, pred-
nisone, primidone, probenecid, progesterone, propranolol, quinine, spironolactone, testosterone,
theophylline, triamcinolone, tripelennamine
Noninterfering: caffeine, nicotine, cotinine, ephedrine, allopurinol, phenylephrine
KEYWORDS
serum; SPE; flumethasone is IS
REFERENCE
Hariharan,M.; Naga,S.; VanNoord,T.; Kindt,E.K. Assay of human plasma cortisone by liquid chromatography:
normal plasma concentrations (between 8 and 10 a.m.) of cortisone and corticosterone, J.Chromatogr., 1993,
613, 195-201.
SAMPLE
Sample preparation: Ointment. Weigh out 1-1.5 g ointment, add 10 mL chloroform:hexane 50:
50, warm until the ointment dissolves, add to a silica Sep-Pak SPE cartridge, rinse container
with 10 mL hexanexhloroform 50:50, add rinse to SPE cartridge, discard all eluates, rinse
container with 5 mL MeOH, add rinse to SPE cartridge, elute with 15 mL MeOH, collect all
MeOH eluates, make up to 25 mL with MeOH, inject a 25 |iL aliquot. Cream. Weigh out 1 g
cream and add it to 10 mL MeOH, shake for 5 min, sonicate for 5 min, inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 C8 (Brownlee)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 8.4 (flumethasone pivalate)
KEYWORDS
ointment; cream; SPE
REFERENCE
Lodge,B-A.; Vincent,A. Analysis of flumethasone pivalate formulations by high-performance liquid chromatog-
raphy, J.Chromatogr., 1984, 301, 477-480.
SAMPLE
Matrix: liposomal preparations
Sample preparation: Dilute 1000-fold with MeOH/water, inject a 20 |L aliquot.
HPLC VARIABLES
Column: C18
Mobile phase: MeOH:l% acetic acid 70:30
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Interfering: dexamethasone
REFERENCE
Devoisselle,J.-M.; Vion-Dury,J.; Confort-Gouny,S.; Coustaut,D.; Cozzone,P.J. Liposomes containing fluorinated
steroids: an analysis based on photon correlation and fluorine-19 nuclear magnetic resonance spectroscopy,
J.Pharm.ScL, 1992, 81, 249-254.
SAMPLE
Matrix: solutions
Sample preparation: Condition a Bond Elut C18 SPE cartridge with 4 mL water then 3 mL
MeOH. Add aqueous steroid solution to cartridge, elute with MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 237
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: betamethasone 17-valerate, dexamethasone
KEYWORDS
for flumethasone 21-acetate; SPE
REFERENCE
Valenta,C; Janout,H- Corticosteroid analysis by HPLC with increased sensitivity by use of precolumn concen-
tration, J.Liq.Chromatogr., 1994, 17, 1141-1146.
SAMPLE
Matrix: synovial fluid
Sample preparation: 100 |xL Synovial fluid + 10 |xL 10 (xg/mL flumethasone in MeOH + 1 mL
100 niM NaOH + 10 mL dichloromethane, shake for 10 min, centrifuge at 8400 g for 10 min.
Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40, re-
constitute the residue in 100 |JLL mobile phase, vortex, inject the whole amount.
HPLC VARIABLES
Column: 100 X 8 10 (xm Radial Pak B silica (Waters)
Mobile phase: Dichloromethane:MeOH:glacial acetic acid 96.8:2.4:0.8
Flow rate: 1.4
Detector: UV 254
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Extracted: methylprednisolone, methylprednisolone acetate
KEYWORDS
normal phase; cow; flumethasone is IS
REFERENCE
Alvinerie,M.; Toutain,P.L. Determination of methylprednisolone and methylprednisolone acetate in synovial
fluid using high-performance liquid chromatography, J.Chromatogr., 1984, 309, 385-390.
Flunarizine
Molecular formula: C26H26F2N2
CAS Registry No.: 52468-60-7, 30484-77-6 (2.HCI)
Merck Index: 4179
LednicerNo.: 2 31
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 |mm Symmetry C8 (Waters)
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.ChromatogrA, 1997, 763, 149-
163.
Flunisolide
CAS Registry No.: 3385-03-3, 77326-96-6 (hemihydrate),
4533-89-5 (acetate)
Merck Index: 4180
Lednicer No.: 2 181
SAMPLE
Matrix: blood
Sample preparation: Add 30 |xL IS to 1 mL serum, add 5 mL diethyl ether/dichloromethane
(ratio not given), mix for 30 min. Centrifuge and freeze at -80 for 15 min. Evaporate the
supernatant under a stream of nitrogen, reconstitute the residue in 200 |JLL mobile phase. Inject
a 50 |ULL aliquot.
HPLC VARIABLES
Column: Lichrospher RP Select B
Mobile phase: MeCN:20 mM ammonium acetate buffer 80:20
Flow rate: 1
Detector: MS, PE-Sciex API 300, negative ion mode
CHROMATOGRAM
Internal standard: budesonide
KEYWORDS
REFERENCE
M6llmann,H.; Derendorf,H.; Barth,J.; Meibohm,B.; Wagner,M.; Krieg,M.; Weisser,H.; Knoller,J.; M6llmann,A.;
Hochhaus,G. Phaimacokinetic/pharmacodynamic evaluation of systemic effects of flunisolide after inhala-
tion, J.Clin.PharmacoL, 1997, 37, 893-903.
Next Page
SAMPLE
Sample preparation: Dissolve in mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 10 fxm Spherisorb ODS
Mobile phase: MeCN: water .-acetic acid 35:64:1
Detector: UV 240
CHROMATOGRAM
Internal standard: nuocinonide
KEYWORDS
nasal spray
REFERENCE
Yu,C.D.; Jones,R-E.; Henesian,M. Cascade impactor method for the droplet size characterization of a metered-
dose nasal spray, J.Pharm.ScL, 1984, 73, 344-348.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
REFERENCE
T6kes,L.; Cho,D.; Maddox,M.L.; Chaplin,M.D.; Chu,N.I. Isolation and identification of an oxidatively defluori-
nated metabolite offlunisolidein man, Drug Metab.Dispos., 1981, 9, 485-486.
Flunitrazepam
Molecular formula: Ci6H12FN3O3
Merck Index: 4181
Lednicer No.: 2 406
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL plasma with 3 mL toluenedsoamyl alcohol 95:5 at 1000 rpm
for 90 s, centrifuge at 2600 g for 10 min. Evaporate a 2.5 mL aliquot of the upper organic layer
to dryness under nitrogen at 40, reconstitute with 100 yiL mobile phase, inject a 25 jxL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 5 pun Nucleosil 120-5 C18
Previous Page
SAMPLE
HPLC VARIABLES
Column: 10 fxm Spherisorb ODS
Mobile phase: MeCN: water .-acetic acid 35:64:1
Detector: UV 240
CHROMATOGRAM
Internal standard: nuocinonide
KEYWORDS
nasal spray
REFERENCE
Yu,C.D.; Jones,R-E.; Henesian,M. Cascade impactor method for the droplet size characterization of a metered-
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
REFERENCE
T6kes,L.; Cho,D.; Maddox,M.L.; Chaplin,M.D.; Chu,N.I. Isolation and identification of an oxidatively defluori-
nated metabolite offlunisolidein man, Drug Metab.Dispos., 1981, 9, 485-486.
Flunitrazepam
Molecular formula: Ci6H12FN3O3
Merck Index: 4181
Lednicer No.: 2 406
SAMPLE
Matrix: blood
Sample preparation: Vortex 1 mL plasma with 3 mL toluenedsoamyl alcohol 95:5 at 1000 rpm
for 90 s, centrifuge at 2600 g for 10 min. Evaporate a 2.5 mL aliquot of the upper organic layer
to dryness under nitrogen at 40, reconstitute with 100 yiL mobile phase, inject a 25 jxL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 5 pun Nucleosil 120-5 C18
Column: 250 X 4 5 |xm Nucleosil 120-5 C 18
Mobile phase: MeOH:buffer 47:53 (Buffer was 100 mM Na2HPO4 adjusted to pH 7.8 with or-
thophosphoric acid.)
Flow rate: 1.2
Detector: UV 302
CHROMATOGRAM
Internal standard: flunitrazepam
OTHER SUBSTANCES
Extracted: omeprazole
KEYWORDS
plasma; flunitrazepam is IS
REFERENCE
Macek,J.; Ptcek,R; Klima,J. Determination of omeprazole in human plasma by high-performance liquid chro-
matography, J.Chromatogr.B, 1997, 689, 239-243.
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 221
CHROMATOGRAM
KEY WORDS
REFERENCE
254-262.
SAMPLE
Sample preparation: Condition a 300 mg Bond Elut Certify LRC SPE cartridge with 2 volumes
of the elution mixture, MeOH, and pH 6 phosphate buffer. Add 30 JULL 10 (jug/mL IS in MeOH
and 5 mL pH 6 phosphate buffer to 1 mL serum, plasma or urine. Vortex, centrifuge at 10000
rpm for 5 min. Add the supernatant to the SPE cartridge and allow to pass through at 1.0 mL/
min. Wash with 5 mL MeOH:water 20:80, 5 mL 1 M orthophosphoric acid, 5 mL MeOH, and
5 mL chloroform. Elute with 2 mL chloroform:isopropanol:ammonia 70:28:2. Add 20 |xL eth-
ylene glycol to the effluent, evaporate at 50 under vacuum. Add 30 |xL mobile phase to the
residue. Inject a 20 jxL aliquot. (Caution! Chloroform is a carcinogen! Prepare the SPE elution
mixture as follows. Add 400 JJLL ammonia to 19.6 mL chloroformdsopropanol 70:30.)
HPLCVARIABLES
Column: 250 X 4.0 LiChrospher 60 RP-Select B
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: methylclonazepam (19.97)
OTHER SUBSTANCES
Simultaneous: alprazolam, bromazepam, brotizolam, clobazam, clonazepam, desmethyldiaze-
pam, diazepam, lorazepam, midazolam, methylclonazepam, nitrazepam, oxazepam, temaze-
pam, triazolam
Noninterfering: amphetamine, atropine, barbital, caffeine, cocaine, codeine, dronabinol, flu-
phenazine, haloperidol, imipramine, morphine, phenobarbital, secobarbital
KEYWORDS
serum; plasma; SPE
REFERENCE
Deinl,L; Mahr,G.; von Meyer,L. Determination of flunitrazepam and its main metabolites in serum and urine
by HPLC after mixed-mode solid-phase extraction, JAnal.ToxicoL, 1998, 22, 197-202.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
camfamine, fenoprofen, fenproporex, fentanyl, flubendazole, 5-fluorouracil, fluoxymesterone,
thioridazine, thiosalicylic acid, thiothixene, th3rmol, tolazamide, tolazoline, tobutamide, tol-
REFERENCE
18, 233-242.
Flunixin
Molecular formula: Ci4H11F3N2O2
CAS Registry No.: 38677-85-9, 42461-84-7 (meglumine salt)
Merck Index: 4182
Lednicer No.: 2 281
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma or serum + 4 mL 250 ng/mL naproxen in MeCN, vortex for
30 s, centrifuge at 1000 g for 15 min. Remove 4 mL of the supernatant and evaporate it to
dryness under a stream of nitrogen at 37, reconstitute the residue in 500 (xL mobile phase,
HPLC VARIABLES
Guard column: 50 mm long 30 |xm pellicular ODS
Column: 250 mm long 5 |xm Spherisorb ODS I
Mobile phase: MeCNrMeOH: 1% pH 3.0 acetate buffer 30:20:50
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Retention time: 9.0
OTHER SUBSTANCES
Extracted: phenylbutazone, oxyphenbutazone
KEYWORDS
plasma; serum; horse; pharmacokinetics; for dogs see Am.J.Vet.Res. 1985; 46; 235
REFERENCE
Hardee,G.E.; Lai,J.-W.; Moore,J.N. Simultaneous determination of flunixin, phenylbutazone, oxyphenbutazone
and 7-hydroxyphenylbutazone in equine plasma by high-performance liquid chromatography: With appli-
cation to pharmacokinetics, J.Liq.Chromatogr., 1982, 5, 1991-2003.
SAMPLE
Matrix: blood, milk
Sample preparation: Plasma. 1 mL Plasma + 10 (xL 6 jxg/mL IS + 1 mL 1 M HCl + 3 mL water,
add to a Clin Elut SPE cartridge, elute with two 8 mL portions of dichloromethane. Evaporate
the eluate to dryness under a stream of nitrogen at 50, reconstitute the residue in 200 JXL
MeOH, vortex for 30 s, inject a 50 |xL aliquot. Milk. 25 mL Milk + 2.5 mL 1 M HCl, mix briefly,
centrifuge at 10000 rpm in a refrigerated centrifuge for 10 min. Remove the supernatant and
wash the pellet with 15 mL 1 M HCl, centrifuge at 10000 rpm in a refrigerated centrifuge for
10 min. Combine the supernatants, add a 5 mL aliquot to 150 JJLL 10 u-g/mL IS and 5 mL
hexane, rotate for 5 min, centrifuge at 1000 g for 5 min, add the aqueous phase to a Clin Elut
SPE cartridge, elute with two 8 mL portions of dichloromethane. Evaporate the eluate to dry-
ness under a stream of nitrogen at 50, reconstitute the residue in 200 \LL MeOH, vortex for
30 s, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 40 |xm COiPELL ODS
Column: Radial Compression C18 (Waters)
Flow rate: 2
Detector: UV 280
CHROMATOGRAM
Retention time: 3.2
Internal standard: Sch 13476 (4.8)
KEYWORDS
plasma; cow; SPE
REFERENCE
Neff-Davis,C.A.; Bosch,K An HPLC method for the determination of flunixin in bovine plasma and milk,
J.Vet.Pharmacol.Ther., 1985, S, 331-334.
SAMPLE
Sample preparation: 5 mL Blood or 10 mL urine + 2.5 (blood) or 5 (urine) jxg flufenamic acid,
acidify with 2 M pH 4.5 acetate buffer, add dichloromethane:EtOH 95:5, agitate for 3 min,
centrifuge at 2000 g for 10 min, repeat extraction. Combine the organic phases, wash with
saturated sodium bicarbonate solution, evaporate to dryness under a stream of air at 45,
reconstitute in MeOH, inject an aliquot.
HPLCVARIABLES
Guard column: 4 X 4 5 |xm Lichrospher 100 RP-18
Column: 125 X 4 5 |xm Lichrospher 100 RP-18
Mobile phase: MeOH:buffer 70:30 (Buffer was 200 mM Na2HPO4 and 100 mM citric acid, pH
3.2.)
Flow rate: 1
Detector: UV 284
CHROMATOGRAM
Internal standard: flufenamic acid
Limit of quantitation: 100 ng/mL (urine), 250 ng/mL (blood)
KEYWORDS
horse; pharmacokinetics
REFERENCE
Araiijo,A.C; Salvadori,M.C; Velletri,M.E.; Camargo,M.M.A. Influence of furosemide on the detection of flunixin
meglumine in horse urine samples, JAnal.ToxicoL, 1990, 14, 146-148.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 50 |xL 500 ng/mL mefenamic acid or indomethacin
+ 1 mL 100 mM HCl + 10 mL dichloromethane, rotate for 10 min, centrifuge at 1500 g for 15
min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 45.
Redissolve the residue in mobile phase, inject a 20 |xL aliquot. Urine. 50 |xL Urine + 1 mL
mobile phase, inject a 20 (xL aliquot.
HPLC VARIABLES
Column: 75 X 4.6 3 fxm Supelcosil LC-8
Flow rate: 1
Detector: UV 235
CHROMATOGRAM
Retention time: 2.8
Internal standard: mefenamic acid (8) or indomethacin (5)
OTHER SUBSTANCES
Simultaneous: naproxen, thiosalicylic acid,ethacrynic acid, phenylbutazone
KEYWORDS
plasma
REFERENCE
Singh,A.K; Jang,Y.; Mishra,U.; Granley,K. Simultaneous analysis of flunixin, naproxen, ethacrynic acid, in-
mance liquid chromatography and gas chromatography-mass spectrometry, J.Chromatogr., 1991,568,351-
361.
SAMPLE
Matrix: milk
Sample preparation: Condition a 3 mL C18 Bakerbond SPE cartridge with 5 mL ethyl acetate
and 5 mL water. 5 mL Milk + 200 fxL 4000 U/mL p-glucuronidase (type IX-A, Sigma) in 100
mM pH 6.8 phosphate buffer, heat at 37 for 2 h, cool, acidify to pH 3.0-3.5 with about 350-
450 IJLL 1 M HCl, while vortexing gently, add 5.4-5.6 g 60-200 mesh silica gel (Baker No. 3405-
1), mix thoroughly for 2 min, place in a 300 X 25 glass column on top off 0.5-1 g untreated
silica gel, place 0.5-1 g untreated silica gel on the top of the column, wash with 50 mL water-
saturated dichloromethane:hexane 30:70, elute with 50 mL ethyl acetate. Wash the eluate with
25 mL pH 3.5 HCl for 15 s, extract ethyl acetate layer twice with NaOH solution by shaking
for 15 s. Combine the aqueous extracts and adjust the pH to 5.0-5.5 with 1 M HCl, add to the
SPE cartridge, elute with 5 mL ethyl acetate. Evaporate the eluate to dryness under a stream
of nitrogen at 50-55, reconstitute the residue in 500 |xL MeOH:buffer 50:50, sonicate briefly,
filter (0.45 |xm), inject a 100 (xL aliquot of the filtrate. (NaOH solution was 15 mL 100 mM
NaOH and 1 mL 1 M NaOH. Buffer was 2.5 mL 1 M tetrabutylammonium dihydrogen phos-
phate and 10 mL 100 mM NaOH made up to 500 mL with water, pH 6.)
HPLC VARIABLES
Guard column: 20 X 4 5 |xm Hypersil C18 ODS
Column: 200 X 4.6 5 |xm Hypersil C18 ODS
Mobile phase: MeOH:buffer 58:42 (Buffer was 2.5 mL 1 M tetrabutylammonium dihydrogen
phosphate and 10 mL 100 mM NaOH made up to 500 mL with water, pH 6.)
Flow rate: 1
Detector: UV 285
CHROMATOGRAM
Retention time: 5.7
KEYWORDS
cow; SPE
REFERENCE
Rupp,H.S.; Holland,D; Munns,R.K; Turnipseed,S.B.; Long,A.R. Determination of flunixin in milk by liquid
chromatography with confirmation by gas-chromatography/mass spectrometry and selected ion monitoring,
JAOAC Int., 1995, 78, 959-967.
SAMPLE
Matrix: urine
Sample preparation: Condition a 3 mL Bond Elut Certify II (C8 plus strong anion exchanger)
SPE cartridge with 3 mL MeOH and 3 mL water. Adjust pH of urine to 7 with 1 M NaOH or
1 M HCl, centrifuge at 500 g for 15 min. Add 3 mL of the supernatant to the SPE cartridge at
0.2 mL/min, wash with two 2.5 mL portions of water, wash with two 2 mL portions of MeOH,
dry for 20 min under full vacuum, wash with two 2 mL portions of hexane, elute with two 2
mL portions of hexaneracetic acid 90:10, add 2 jxg ketoprofen. Evaporate to dryness under a
stream of nitrogen, reconstitute in 100 |xL hexanedsopropanol 50:50, inject an aliquot.
HPLC VARIABLES
Column: 100 X 2 5 jim Hypersil SI
Mobile phase: Gradient. Hexane:isopropanol containing 5% water from 98:2 to 70:30 over 8 min,
to 0:100 over 4 min, maintain at 0:100 for 1 min, re-equilibrate at initial conditions for 5 min.
Flow rate: 0.4
Injection volume: 3
Detector: UV 280 or MS, Hewlett-Packard HP5989A quadrupole, HP 59980B PB interface at 65
with helium pressure of 275 kPa, 10 kV high energy dynode, source 275, particle beam, EI,
CI (methane)
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: alclofenac, aminoantipyrine, bumadizone, clonixin, diclofenac, diflunisal, dipy-
rone, famprofazone, fenbufen, fenclofenac, fenoprofen, floctafenine, flufenamic acid, flurbipro-
fen, ibufenac, ibuprofen, indomethacin, indoprofen, isopyrin, isoxepac, ketorolac, meclofenamic
acid, naproxen, nefopam, niflumic acid, oxaprozin, phenazone, phenazopyridine, phenylbuta-
zone, piroxicam, propyphenazone, salicylamide, sulindac, suprofen, tenoxicam, tiaprofenic acid,
tolmetin, zomepirac
KEYWORDS
horse; SPE; normal phase
REFERENCE
Stanley,S.M.; Owens,N.A.; Rodgers,J.P. Detection of flunixin in equine urine using high-performance liquid
chromatography with particle beam and atmospheric pressure ionization mass spectrometry after solid-
phase extraction, J.Chromatogr.B, 1995, 667, 95-103.
Flunoxaprofen
Molecular formula: Ci 6 H 12 FNO 3
Merck Index: 4183
SAMPLE
Sample preparation: 50 JAL Plasma or urine + 10 |xL 1 M NaOH, heat at 37 for 2 h, add 10
(JiLlM HCl, extract with 1 mL ethyl acetate. Remove the organic layer and evaporate it to
dryness, reconstitute the residue in 100 JJLL 50 mM triethylamine in MeCN, add 50 jxL 60 mM
ethyl chloroformate in MeCN, let stand for 2 min, add 50 |xL 1 M L-leucinamide in 1 M tri-
ethylamine in MeOH. Evaporate, take up the residue in 100 |xL mobile phase, inject a 100 |xL
aliquot. (Hydrolysis of glucuronides may be omitted.)
HPLC VARIABLES
Flow rate: 1
Detector: UV 272
CHROMATOGRAM
Internal standard: flunoxaprofen
OTHER SUBSTANCES
Extracted: ketoprofen, fenoprofen
KEYWORDS
plasma; chiral; flunoxaprofen is IS; derivatization
REFERENCE
Volland,C; Sun,H-; Benet,L.Z. Stereoselective analysis of fenoprofen and its metabolites, J.Chromatogr., 1990,
534, 127-138.
Fluocinolone acetonide
Merck Index: 4185
Lednicer No.: 1 202; 3 94
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 1.2 mL dichloromethane, shake 1 min, repeat extraction.
Combine organic layers and evaporate a 2 mL aliquot under reduced pressure below 40. Dis-
solve the residue in 100 |xL MeCN, add 10 |xL reagent 1, add 10 JJLL reagent 2, heat at 70 for
20 min, cool to room temperature, add 100 \xL water, add 200 |JLL MeOH:water 1:1, apply to a
Sep-Pak C18 cartridge, wash vial into cartridge with 2 mL MeOH:water 1:1, wash cartridge
with 40 mL MeOH:water 1:1, elute with 5 mL MeOH. Concentrate eluate to 500 JJLL by evap-
oration under reduced pressure below 40, inject 20 |JLL aliquot. (Reagent 1 was 30 mg 2-(4-
carboxyphenyl)-5,6-dimethylbenzimidazole in 3 mL pyridine, add 700 mg 4-piperidinopyridine,
dilute to 10 mL with MeCN. Reagent 2 was 700 mg l-isopropyl-3-(3-dimethylaminopro-
pyl)carbodiimide perchlorate in 10 mL MeCN. Prepare 2-(4-carboxyphenyl)-5,6-dimethylben-
zimidazole as follows. Add 13 g 4-carboxybenzaldehyde (terephthalaldehydic acid) in 400 mL
EtOH dropwise to 4,5-dimethyl-l,2-phenylenediamine in 400 mL EtOH in an ice bath, after 1
h reflux for 8 h, cool to room temperature, collect the precipitate, recrystallize three times from
MeOH:water 50:50 to give 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole as a white amor-
phous product (mp >300) (J.Chromatogr. 1991, 585, 219). 4-Piperidinopyridine is not commer-
cially available but 4-dimethylaminopyridine or 4-pyrrolidinopyridine can be used instead al-
though interferences are greater (J. Chromatogr. 1991, 585, 219). Alternatively
4-piperidinopyridine can be synthesized as follows. Add 200 mmoles piperidine dropwise with
stirring to 15 g phosphorus pentoxide and 9.51 g 4-hydroxypyridine, heat at 250 for 7 h,
cautiously pour onto 200 g ice, add 400 mL 1 M NaOH, add 200 mL ether. Remove the ether
layer and extract the aqueous layer three times with 100 mL portions of ether. Combine the
organic layers and dry them over anhydrous potassium carbonate, evaporate, distil the residue,
recrystallize from petroleum ether (bp 80-100) to give 4-piperidinopyridine (bp 167-170711
mm Hg; mp 79-80) (Synthesis 1978, 844). Alternatively, add 1.94 g 4-bromopyridine hydro-
chloride to 5 mL 50% NaOH, add 5 mL piperidine, add 2.72 g benzyltriethylammonium bro-
mide, heat at 100 for 5 h, remove excess piperidine by distillation, add 25 mL water, extract
four times with 25 mL portions of benzene. Combine the organic layers and dry them over
anhydrous sodium sulfate, boil the residue with petroleum ether to give 4-piperidinopyridine
(mp 80) (Syn. Commun. 1979, 9, 251). Prepare l-isopropyl-3-(3-dimethylaminopropyl)
carbodiimide perchlorate as follows. Stir 1.41 moles isopropylisocyanate in 750 mL dichloro-
methane at 5, add 144 g 3-dimethylaminopropylamine (N,N-dimethyl-l,3-propanediamine)in
250 mL dichloromethane at such a rate that the temperature does not exceed 10, add 500 mL
triethylamine, add 300 g p-toluenesulfonyl chloride in 300 mL dichloromethane at such a rate
that the temperature does not exceed 10, reflux for 3 h, add 400 g anhydrous sodium carbonate,
add 3.5 L ice water, stir vigorously for 30 min, remove the organic phase. Extract the aqueous
phase three times with 500 mL portions of dichloromethane. Combine the organic layers and
dry them over anhydrous sodium sulfate, evaporate under reduced pressure, distil the residue
to give l-isopropyl-3-(3-dimethylaminopropyl)carbodiimide (bp 91-92710 mm Hg (Ber. 1941,
74B, 1285)) (cf. Org. Syn. 1973, Coll. Vol. V, 555). Prepare pyridine perchlorate from pyridine
and 20% perchloric acid, crystallize from EtOH (Ber. 1926, 59, 446). Add 18 g pyridine per-
chlorate in portions to 100 mmoles l-isopropyl-3-(3-dimethylaminopropyl)carbodiimide stirred
in 200 mL dichloromethane at 0, let stand for 30 min, filter, add 200 mL anhydrous diethyl
ether to the filtrate. Filter off the precipitate and recrystallize it from dichloromethane/diethyl
ether to give l-isopropyl-3-(3-dimethylaminopropyl)carbodiimide perchlorate (mp 88-90)
(Chem. Pharm. Bull. 1985, 33, 5375).)
HPLC VARIABLES
Guard column: 50 X 4.6 7 jxm Zorbax ODS
Column: 250 X 4.6 7 ^m Zorbax ODS
Mobile phase: MeOH:water 75:25 containing 5 mM tetramethylammonium hydrogen sulfate
Flow rate: 0.4
CHROMATOGRAM
Internal standard: fluocinolone acetonide
Limit of detection: 0.6 pg/mL
OTHER SUBSTANCES
Extracted: triamcinolone, triamcinolone acetonide
Simultaneous: aldosterone, cortisone, hydrocortisone, corticosterone, fluocinolone acetonide, tri-
amcinolone, triamcinolone acetonide, dexamethasone
KEYWORDS
plasma; derivatization; fluocinolone acetonide is IS
REFERENCE
Katayama,M.; Masuda,Y.; Taniguchi,H. Determination of corticosteroids in plasma by high-performance liquid
chromatography after pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole,
J.Chromatogr., 1993, 612, 33-39.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 (xL water containing 5 |xg/mL 2,3-diaminonaphthalene
and 3.5 |xg/mL 18-hydroxy-ll-deoxycorticosterone + 1 mL 250 mM NaOH + 7 mL diethyl ether,
shake on a rotary shaker for 15 min, repeat extraction. Combine the organic layers and evap-
orate them to dryness under a stream of nitrogen at 30-40, reconstitute the residue in 70 |xL
MeOHrIOO mM perchloric acid 50:50, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 4 |jtm Nova-Pak C18
Mobile phase: Gradient. A was 58 mM NaH2PO4 containing 6 mM sodium heptanesulfonate,
adjusted to pH 3.1 with concentrated phosphoric acid. B was MeCN:MeOH 85:15. A:B from
100:0 to 78:22 over 5 min, to 70:30 over 12 min, maintain at 70:30 for 4 min, to 65:35 over 9
min.
Flow rate: 1
Detector: UV 245, 256, 343
CHROMATOGRAM
Retention time: 24.75 (fluocinolone acetonide)
Internal standard: 2,3-diaminonaphthalene (10.71), 18-hydroxy-ll-deoxycorticosterone (15.85)
Limit of detection: 1-10 ng/mL (245 nm)
OTHER SUBSTANCES
Extracted: betamethasone, chloroquine, corticosterone, cortisone, dexamethasone, fluendrenol-
ide, fluorometholone, fluprednisolone, hydrocortisone, hydroxychloroquine, 178-hydroxyproges-
terone, meprednisone, methylprednisolone, methylprednisolone acetate, paramethasone, pred-
nisolone, prednisone, progesterone, triamcinolone
Noninterfering: aspirin, ibuprofen, indomethacin, phenylbutazone, pregnenolone
KEYWORDS
serum
REFERENCE
Volin,P. Simple and specific reversed-phase liquid chromatographic method with diode-array detection for si-
multaneous determination of serum hydroxychloroquine, chloroquine and some corticosteroids,
J.Chromatogr.B, 1995, 666, 347-353.
SAMPLE
Sample preparation: Dissolve 5 g cream containing 0.00925% fluocinonide, 0.00365% procinon-
ide, and 0.0021% ciprocinonide in 2.5 mL THF, add norethindrone, dilute to 25 mL with MeOH,
centrifuge, inject a 25 uL aliquot onto column A with mobile phase A and allow components to
elute from column A to column B for 7 min. After 7 min remove column A from circuit, monitor
effluent from column B. Back-flush column A with mobile phase B for 5 min, equilibrate column
A with mobile phase A for 5 min before next injection.
HPLCVARIABLES
Column: A 30 X 4.6 5 |im Spheri-5 ODS (Brownlee); B 70 X 2.1 Whatman CorPell ODS + 250
X 4.6 5 |xm Ultrasphere C18
Mobile phase: A MeCN:THF:water 43:4:53; B MeOH:THF 75:25
CHROMATOGRAM
Retention time: 8 (fluocinolone acetonide)
Internal standard: norethindrone (12)
OTHER SUBSTANCES
Simultaneous: procinonide, ciprocinonide, fluocinonide
KEY WORDS
creams; column-switching
REFERENCE
Conley,D.L.; Benjamin,E.J. Automated high-performance liquid chromatographic column switching technique
for the on-line clean-up and analysis of drugs in topical cream formulations, J.Chromatogr., 1983,257,337
344.
SAMPLE
Sample preparation: 11.25 g Cream + 100 mL cyclohexane + 50 mL MeOH, shake vigorously
for 3 min, let stand for 15 min. Remove the lower layer and add it to 140 mL water and 100
mL chloroform, shake for 3 min, allow phases to separate. Remove a 3 mL aliquot of the
chloroform layer and add it to 300 |xL 0.028% hydrocortisone in EtOH, add 1 mL 3 mg/mL
acenaphthene-5-sulfonyl hydrazine in EtOHrtoluene 10:90, evaporate to dryness under reduced
pressure at 60, reconstitute with 200 (xL mobile phase, inject an aliquot. (Preparation of ace-
naphthene-5-sulfonyl hydrazine is as follows. Dissolve 20 g acenaphthene in 100 g nitroben-
zene, cool to 0, add 9 mL chlorosulfonic acid dropwise with stirring, maintain the temperature
below 5, when the addition is complete allow the temperature to rise to 20 over 30 min, add
500 mL water. Remove the aqueous layer and neutralize it with solid sodium carbonate, heat
and add NaCl until precipitation occurs, cool in an ice bath for 1 h, filter, heat at 140 to remove
traces of water and nitrobenzene to give acenaphthene-5-sulfonic acid sodium salt as a pale
yellow solid (mp >300). Grind 10 g acenaphthene-5-sulfonic acid sodium salt with 3.5 g phos-
phorus pentachloride in a mortar for 3 min, add ice and water, extract with 100 mL ethyl
acetate. Wash the ethyl acetate layer with 5% sodium bicarbonate and with water until neutral,
dry over anhydrous sodium sulfate, evaporate the ethyl acetate under a stream of nitrogen,
chromatograph on a 300 X 20 column of silica gel H with toluene to give acenaphthene-5-
sulfonyl chloride (mp 98-101) as the first yellow band to elute. Cool a solution of 1 g ace-
naphthene-5-sulfonyl chloride in 3 mL THF to 10 and pass nitrogen through the solution, add
400 jxL 85% hydrazine hydrate dropwise with stirring, maintain the temperature between 10
and 15, stir for a further 15 min. Filter the upper THF layer through Celite, wash the Celite
with 1 mL THF. Stir the filtrate vigorously and add two 10 mL portions of water, cool in a
refrigerator for 1 h, filter the precipitate, wash with water, dry, recrystallize from EtOH to give
acenaphthene-5-sulfonyl hydrazine (mp 132-4).)
HPLC VARIABLES
Column: 500 X 1 10 |xm silica
Mobile phase: Tolueneidioxane 90:10 (Caution! Dioxane is a carcinogen!)
CHROMATOGRAM
Internal standard: hydrocortisone
KEY WORDS
derivatization; cream; normal phase; for fluocinolone acetonide
REFERENCE
Gifford,L.A.; Owusu-Daaku,F.T.K.; Stevens,A-L Acenaphthene fluorescence derivatization reagents for use in
high-performance liquid chromatography, J.Chromatogr.A, 1995, 715, 201-212.
Fluocinonide
Merck Index: 4186
SAMPLE
Sample preparation: Dissolve 5 g cream containing 0.00925% fluocinonide, 0.00365% procinon-
ide, and 0.0021% ciprocinonide in 2.5 mL THF, add norethindrone, dilute to 25 mL with MeOH,
centrifuge, inject a 25 |xL aliquot onto column A with mobile phase A and allow components to
elute from column A to column B for 7 min. After 7 min remove column A from circuit, monitor
effluent from column B. Back-flush column A with mobile phase B for 5 min, equilibrate column
A with mobile phase A for 5 min before next injection.
HPLC VARIABLES
Column: A 30 X 4.6 5 |xm Spheri-5 ODS (Brownlee); B 70 X 2.1 Whatman Co:Pell ODS + 250
X 4.6 5 |xm Ultrasphere C18
Mobile phase: A MeCN:THF:water 43:4:53; B MeOH:THF 75:25
CHROMATOGRAM
Retention time: 18
Internal standard: norethindrone (12)
OTHER SUBSTANCES
Simultaneous: procinonide, ciprocinonide, fluocinolone acetonide
KEYWORDS
creams; column-switching
REFERENCE
Conley,D.L.; Benjamin,E.J. Automated high-performance liquid chromatographic column switching technique
for the on-line clean-up and analysis of drugs in topical cream formulations, J.Chromatogr., 1983,257,337-
344.
SAMPLE
HPLC VARIABLES
Column: 10 u-m Spherisorb ODS
Detector: UV 240
CHROMATOGRAM
Internal standard: fluocinonide
OTHER SUBSTANCES
Simultaneous: flunisolide
KEYWORDS
nasal spray; fluocinonide is IS
REFERENCE
Yu,C.D.; Jones,R.E.; Henesian,M. Cascade impactor method for the droplet size characterization of a metered-
Fluocortolone
CAS Registry No.: 152-97-6, 303-40-2 (21-hexanoate)
Merck Index: 4188
SAMPLE
Matrix: blood
Sample preparation: Add 150 |xL MeOH to 1 mL plasma. Add 500 |xL 100 mM NaOH and 2
mL dichloromethane, shake for 10 min, centrifuge at 2500 g for 10 min, evaporate a 1.9 mL
aliquot of the supernatant under a stream of nitrogen at 45. Reconstitute the residue in 50
|xL MeOH, inject 17 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 4 5 |xm LiChrospher RP 18
Column: 250 X 4 5 jxm Lichrospher RP 18
Mobile phase: MeOH:THF:water 110:2.5:100
Flow rate: 1
Detector: UV 252
CHROMATOGRAM
Internal standard: fluocortolone
OTHER SUBSTANCES
Extracted: hydrocortisone, triamcinolone
KEYWORDS
plasma
REFERENCE
Doppenschmitt,S.A.; Scheidel,B-; Harrison,E; Surmann,J.P. Simultaneous determination of triamcinolone ace-
tonide and hydrocortisone in human plasma by high-performance liquid chromatography, J.Chromatogr.B,
1996, 682, 79-88.
Fluorometholone
Merck Index: 4213
Lednicer No.: 1 203
SAMPLE
M a t r i x : blood
Sample preparation: 2 mL Plasma + 5 mL hexane, shake horizontally at 60 cycles/min for 10
min, centrifuge at 1000 g for 5 min, discard the hexane layer, add 8 mL dichloromethane,
shake horizontally at 60 cycles/min for 10 min, centrifuge at 1000 g for 5 min, repeat extraction
with 8 mL dichloromethane. Combine the dichloromethane layers and add 300 mg anhydrous
sodium sulfate, shake horizontally at 200 cycles/min for 5 min, centrifuge at 1000 g for 5 min.
constitute the residue in 200 |xL mobile phase, add 2 mL hexane, vortex for 1 min, centrifuge
at 1000 g for 5 min, discard the hexane layer, inject a 100 JULL aliquot of the mobile phase layer.
HPLCVARIABLES
Guard column: 30 X 4.6 5 |jim Spheri-5 RP-18
Mobile phase: MeCN:water:glacial acetic acid 33:62:5
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: fluorometholone
OTHERSUBSTANCES
KEYWORDS
plasma; fluorometholone is IS
REFERENCE
Hopkins,N.K; Wagner,C.M.; Brisson,J.; Addison,T.E. Validation of the simultaneous determination of methyl-
prednisolone and methylprednisolone acetate in human plasma by high-performance liquid chromatogra-
phy, J.Chromatogr., 1992, 577, 87-93.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 5 mL hexane, shake horizontally at 60 cycles/min for 10
min, centrifuge at 1000 g for 5 min. Remove the aqueous layer and add it to 8 mL dichloro-
methane, shake horizontally at 60 cycles/min for 10 min, centrifuge at 1000 g for 5 min, repeat
the extraction. Combine the organic layers and add them to 300 mg anhydrous sodium sulfate,
shake horizontally at 200 cycles/min for 5 min, centrifuge at 1000 g for 5 min. Remove the
residue in 200 jxL mobile phase, add 2 mL hexane, vortex for 1 min, centrifuge at 1000 g for
5 min, inject a 100 jxL aliquot of the aqueous layer.
HPLC VARIABLES
Guard column: 30 X 4.6 5 |xm Brownlee guard column
Mobile phase: MeCN.water.glacial acetic acid 33:62:5
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: fluorometholone
OTHER SUBSTANCES
KEYWORDS
plasma; fluorometholone is IS
REFERENCE
Hopkins,N.K.; Wagner,C.M.; Brisson,J.; Addison,T.E. Validation of the simultaneous determination of methyl-
prednisolone and methylprednisolone acetate in human plasma by high-performance liquid chromatogra-
phy, J.Chromatogr., 1992, 577, 87-93.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL water containing 5 (xg/mL 2,3-diaminonaphthalene
and 3.5 (xg/mL 18-hydroxy-ll-deoxycorticosterone + 1 mL 250 mM NaOH + 7 mL diethyl ether,
MeOH: 100 mM perchloric acid 50:50, inject a 20 JJLL aliquot.
HPLCVARIABLES
Mobile phase: Gradient. A was 58 mM NaH2PO4 containing 6 mM sodium heptanesulfonate, ad-
justed to pH 3.1 with concentrated phosphoric acid. B was MeCN:MeOH 85:15. A:B from 100:0
to 78:22 over 5 min, to 70:30 over 12 min, maintain at 70:30 for 4 min, to 65:35 over 9 min.
Flow rate: 1
Detector: UV 245, 256, 343
CHROMATOGRAM
OTHER SUBSTANCES
ide, fluocinolone acetonide, fluprednisolone, hydrocortisone, hydroxychloroquine, 178-hydroxy-
progesterone, meprednisone, methylprednisolone, methylprednisolone acetate, paramethasone,
prednisolone, prednisone, progesterone, triamcinolone
KEYWORDS
serum
REFERENCE
J.Chromatogr.B, 1995, 666, 347-353.
SAMPLE
Matrix: solutions
Sample preparation: Dilute in an appropriate solvent, inject an aliquot.
HPLCVARIABLES
Guard column: RC18 Guardpak (Waters)
Flow rate: 1.5
Detector: UV 246
CHROMATOGRAM
Retention time: 6.8
OTHER SUBSTANCES
Simultaneous: 20-a-dihydrofluorometholone, prednisolone
Interfering: prednisolone acetate
REFERENCE
Richman,J.B.; Tang-Liu,D.D.-S. A corneal perfusion device for estimating ocular bioavailability in vitro,
J.Pharm.ScL, 1990, 79, 153-157.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.5 5 jjum Ultrasphere octyl
Mobile phase: MeCN:triethylamine:1.65% glacial acetic acid 505:0.65:495, pH 4.35
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: bacitracin, cortisone acetate, diazepam, diclofenac, flurbiprofen, hydrocortisone
acetate, imipramine, indomethacin, ketoprofen, ketorolac tromethamine, levobunolol, meclo-
fenamic acid, metipranolol, neomycin, prednisolone acetate, proparacaine, propranolol, salicylic
acid, sulfacetamide, suprofen
Noninterfering: acebutolol, acetaminophen, acetazolamide, alprenolol, apraclonidine, atenolol,
atropine, betamethasone, betaxolol, bupivacaine, caffeine, cyclopentolate, dexamethasone, di-
phenhydramine, erythromycin, haloperidol, lidocaine, phenylephrine, polymyxin B, procaine,
scopolamine, timolol, tropicamide
KEYWORDS
human; rabbit
REFERENCE
Riegel,M.; Ellis,P.P. High-performance liquid chromatography assay for antiinflammatory agents diclofenac and
flurbiprofen in ocular fluids, J.Chromatogr.B, 1994, 654, 140-145.
Fluorouracil
Merck Index: 4219
Lednicer No.: 3 155
SAMPLE
Matrix: blood
Sample preparation: Mix 150 |xL plasma with 150 JXL 10% trichloroacetic acid in water, vortex
for 30 s, centrifuge at 5000 g for 5 min, inject a 50 |xL aliquot of the clear supernatant.
HPLC VARIABLES
Guard column: 9 |xm Aminex HPX-87H
Column: 300 X 7.8 9 |xm Aminex HPX-87H
Mobile phase: 5 mM sulfuric acid
Flow rate: 0.5
Detector: UV 265
CHROMATOGRAM
Retention time: 24
OTHER SUBSTANCES
Simultaneous: cisplatin, 5-fluoro-2'-deoxyuridine-5'-monophosphate, 5-fluoro-2'-deoxyuridine, 5-
fluorouridine, thymidine, uric acid, uridine
Noninterfering: alizapride, cytosine, hypoxanthine, methylprednisolone, methotrexate, metoclo-
pramide, xanthine
Interfering: uracil
KEYWORDS
REFERENCE
Compagnon,R; Thiberville,L.; Moore,N.; Thuillez,C; Lacroix,C. Simple high-performance liquid chromato-
graphic method for the quantitation of 5-fluorouracil in human plasma, J.Chromatogr.B, 1996, 677, 380
383.
SAMPLE
Matrix: blood
Sample preparation: Mix 500 u,L plasma with 50 |xL 5 (xg/mL 5-chlorouracil, add to an unac-
tivated C18 Chem Elut SPE cartridge (Varian). Elute with four 2 mL portions of MeOH:ethyl
acetate 5:95. Flush the SPE cartridge with nitrogen at 1 mL/min. Evaporate collected eluate
to dryness under a stream of nitrogen at 40 for 30 min. Reconstitute the residue in 200 (xL
water, sonicate for 10 min, filter (0.45 u,m HV4 membrane, Millipore) Inject a 20 |xL aliquot;
SPE
HPLC VARIABLES
Column: 150 X 4.6 5 jjum Kromasil C18 (Touzart and Matignon, France)
Flow rate: 0.6
Detector: UV 268
CHROMATOGRAM
Retention time: 5.8
Internal standard: 5-chlorouracil (10.7), 5-iodouracil (22.1)
OTHER SUBSTANCES
Simultaneous: metabolites (UV 275)
KEYWORDS
plasma; SPE
REFERENCE
Joulia,J.M.; Pinguet,R; Grosse,P.Y.; Astre,C; Bressolle,F. Determination of 5-fluorouracil and its main metab-
olites in plasma by high-performance liquid chromatography: Application to a pharmacokinetic study,
J.Chromatogr.B, 1997, 692, 427-435.
SAMPLE
Matrix: blood
Sample preparation: Mix 200 |xL plasma with 50 u,L 500 mM pH 8.0 phosphate buffer, 100 |xL
500 ng/mL 5-chlorouracil in water, and 8 mL ethyl acetate. Shake for 30 min and centrifuge
at 2200 g for 10 min. Remove the ethyl acetate layer and evaporate it. Reconstitute the residue
in 500 [xL mobile phase with sonication for 10 min. Inject a 150 jxL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 Develosil 60-3 (Nomura Chemical, Japan)
Mobile phase: n-Hexane:ethyl acetateiformic acid:water 50:0.5:0.5:0.3
Flow rate: 0.9 for 20 min, 0.4 for 27 min, 0.9 for 8 min
Detector: UV 264
CHROMATOGRAM
Internal standard: 5-chlorouracil
KEYWORDS
plasma; rat
REFERENCE
Fuse,E.; Takai,K; Okuno,K; Kobayashi,S. Hepatic extraction ratio of 5-fluorouracil in rats. Dose dependence
and effect of uracil and interleukin-2, Biochem.Pharmacol., 1996, 52, 561-568.
SAMPLE
Matrix: blood
Sample preparation: Add 50 (JLL 1 M pH 4.8 sodium acetate buffer to 1 mL plasma (to adjust
pH to 6.0), add 250 jxL 200 mg/mL saturated sodium sulfate solution, vortex, add 7 mL water-
saturated ethyl acetate, vortex vigorously for 90 s, centrifuge for 5 min. Add 1 mL 50 mM pH
11 phosphate buffer to a 6 mL aliquot of the organic layer, vortex, centrifuge. Aspirate the
organic layer to waste, flush the residual solvent from the aqueous portion with nitrogen for
about 10 min, add 10 |xL 1 M sulfuric acid (to adjust the pH to neutral), inject a 125 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm YMC ODS-AQ C18
Mobile phase: Gradient. A was MeOH: 10 mM pH 5.5 potassium phosphate buffer 50:50. B was
10 mM pH 5.5 potassium phosphate buffer. A:B 0:100 for 5 min, from 0:100 to 50:50 in 1 min,
maintain at 50:50 for 3 min, from 50:50 to 0:100 in 1 min, maintain at 0:100 for 10 min
Flow rate: 1.2
Detector: UV 266
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: uracil, 5-chlorouracil
KEYWORDS
plasma
REFERENCE
Coe,R.A.; Earl,R.A.; Johnson,E.T.C; Lee,J.W. Determination of 5-fluorouracil in human plasma by a simple
and sensitive reversed-phase HPLC method, J.Pharm.Biomed.Anal., 1996, 14, 1733-1741.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 500 jxL physiological saline + 100 |xL 500 mM NaH2PO4
+ 8 mL ethyl acetate, extract, centrifuge. Remove the organic layer and evaporate it to dryness
under reduced pressure, reconstitute the residue in 1 mL 500 |xg/mL 4-bromomethyl-7-
methoxycoumarin in acetone:MeCN 1:2 containing 100 |xg/mL 18-crown-6 and 1 mg/mL potas-
sium carbonate, reflux in the dark for 45 min, cool, add valeric acid, reflux for 5 min, dilute
with acetone, inject an aliquot.
HPLC VARIABLES
Flow rate: 0.8
CHROMATOGRAM
Retention time: 7
Internal standard: valeric acid (8)
OTHER SUBSTANCES
Extracted: tegafur
KEY WpRDS
REFERENCE
Iwamoto,M.; Yoshida,S.; Hirose,S. Fluorescence determination of 5-fluorouracil and l-(tetrahydro-2-furanyl)-5-
fluorouracil in blood serum by high-performance liquid chromatography, J.Chromatogr., 1984, 310, 151-
157.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Serum + 5 jxL 42 |xM 5-chlorouracil in acetone, mix, let stand at
room temperature for 3 min, add 500 (xL 100 mM pH 3.5 potassium phosphate buffer, add 2
mL ethyl acetate, vortex for 2 min, centrifuge at 1000 g for 5 min. Remove a 1.4 mL aliquot
of the organic layer and evaporate it to dryness under reduced pressure. Add 20 mg solid
potassium bicarbonate:anhydrous sodium sulfate 1:7 to the residue, add 50 u,L 1.3 mM 3-
bromomethyl-6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone in acetone, add 50 |JLL 1.5 mM 18-
crown-6 in acetone, heat at 50 for 20 min, cool, inject a 10 jxL aliquot. (Silanize all glassware.
Synthesize 3-bromomethyl-6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone as follows. Stir 483 g
veratrole in 1.45 L acetic acid at 15 for 1 h, add 683 g concentrated nitric acid (d 1.05) over 1
h (maintain the temperature below 40 by cooling and regulating the rate of addition of the
nitric acid). Continue stirring and add 2.127 L fuming nitric acid (d 1.50) over 1 h while main-
taining the temperature below 30, let stand for 2 h, pour into a large volume of cold water,
filter, wash the solid with water until the washings are neutral, recrystallize from EtOH to
give 4,5-dinitroveratrole (mp 129.5-130.5) (J. Am. Chem. Soc. 1946, 68, 1536). Reflux 5 g 4,5-
dinitroveratrole in 200 mL benzene (Caution! Benzene is a carcinogen!), add 100 g 60 mesh
iron powder and 20 mL concentrated HCl in small portions over 1 h, reflux for 4 h, add 10 mL
water, reflux for 2 h, cool, make alkaline with 2.5 M NaOH, extract several times with 200 mL
portions of benzene. Combine the organic layers and evaporate them to dryness, add 10 mL
concentrated HCl, recrystallize from EtOH to give l,2-diamino-4,5-dimethoxybenzene mono-
hydrochloride as very slightly pink needles (mp 240) (Anal. Chim. Acta 1982, 134, 39). Heat
2.5 mmoles l,2-diamino-4,5-dimethoxybenzene hydrochloride and 2.4 mmoles pyruvic acid in
30 mL 500 mM HCl on a boiling water bath for 2 h, cool with ice-water, filter. Wash the
precipitate with water and dry it under vacuum, recrystallize from MeOH:water 90:10 to give
6,7-dimethoxy-3-methyl-2(lH)-quinoxalinone as yellow needles (mp 255) (Chem. Pharm. Bull.
1985, 33, 3493). Treat 1 g 6,7-dimethoxy-3-methyl-2(lH)-quinoxalinone dissolved in 50 mL
anhydrous MeOH with a solution of diazomethane in ether, evaporate to dryness under reduced
pressure, dissolve the residue in 5 mL ethyl acetate, chromatograph on a 250 X 35 column
filled with 130 g 70-230 mesh silica gel 60 (Merck) using n-hexane:ethyl acetate 25:75 to give
6,7-dimethoxy-l,3-dimethyl-2(lH)-quinoxalinone as yellow needles (mp 170-171). Dissolve 350
mg 6,7-dimethoxy-l,3-dimethyl-2(lH)-quinoxalinone in 3 mL acetic acid, add 350 mg anhy-
drous sodium acetate, add 2 mL 1.5 M bromine in acetic acid, heat at 100 for 15 min, cool,
add 10 mL ether, filter, wash the solid 2 or 3 times with small portions of ether. Combine the
filtrate and washings and evaporate them to dryness, dissolve the residue in 5 mL ethyl ace-
tate, chromatograph on a 250 X 35 column filled with 130 g 70-230 mesh silica gel 60 (Merck)
using ether, evaporate the main fraction to dryness, recrystallize the residue from n-hexane:
ethyl acetate 50:50 to give 3-bromomethyl-6,7-dimetnoxy-l-methyl-2(lH)-quinoxalinone as yel-
low needles (mp 161-163) (J. Chromatogr. 1985, 346, 227). 3-Bromomethyl-6,7-dimethoxy-l-
methyl-2(lH)-quinoxalinone is also available from Dojindo Molecular Technologies, Inc., 3
Bethesda Metro Center, Suite 700, Bethesda MD 20814; (301) 664-8448; www.dojindo.co.jp.)
HPLC VARIABLES
Column: 100 X 8 10 |xm Radial Pak C18 (Waters) (Wash with MeOH at 2 mIVmin for 20 min at
the end of each day.)
Mobile phase: Gradient. MeOH:water 35:65 for 15 min, 50:50 for 25 min (step gradient), re-
equilibrate at initial conditions for 20 min.
Flow rate: 1.5
CHROMATOGRAM
Internal standard: 5-chlorouracil (32.5)
OTHER SUBSTANCES
Extracted: floxuridine
KEY WpRDS
derivatization; serum; pharmacokinetics
REFERENCE
Yamaguchi,M.; Nakamura,M.; Kuroda,N.; Ohkura,Y. Determination of 5-fluorouracil and 5-fluoro-2'-deoxyuri-
dine in human serum by high-performance liquid chromatography with fluorescence detection, Anal.ScL,
1987, 3, 75-79.
SAMPLE
Matrix: blood
Sample preparation: Add 250 |xL serum to a 20 X 7 DEAE-Cellulofine AM anion-exchange
column (Seikagaku Tokyo), elute with 3.5 mL 1 mM HCl, discard the first 0.5 mL eluate, collect
the next 3 mL eluate. Evaporate the eluate to 0.5 mL under reduced pressure, add 15 mL ethyl
acetate, shake, centrifuge. Remove the organic layer and evaporate it to dryness, reconstitute
the residue in 800 |xL anhydrous acetone, add 100 |xL 750 jxg/mL 4-bromomethyl-6,7-dime-
thoxycoumarin in acetone, add 100 |xL 250 (ig/mL 18-crown-6 in acetone, add 1.5 mg anhydrous
potassium carbonate, heat at 70 for 15 min (protect from atmospheric moisture with a calcium
chloride drying tube), cool, inject an aliquot
HPLC VARIABLES
Flow rate: 0.8
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Extracted: floxuridine, ftorafur
KEYWORDS
derivatization; serum; protect from light
REFERENCE
Yoshida,S.; Adachi,T.; Hirose,S. 4-Bromomethyl-6,7-dimethoxycoumarin as a fluorescence reagent for precol-
umn derivatization of 5-fluorouracil compounds in high-performance liquid chromatography, J. Chromatogr.,
1988, 430, 156-162.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL LC-SCX Supelclean strong cation-exchange SPE car-
tridge (Supelco) with 2 mL MeOH, 1 mL 100 mM copper(II) sulfate solution, and 3 mL 50 mM
pH 7 phosphate buffer, do not allow to dry. 300 |xL Serum + 5-bromouracil, add to the SPE
cartridge, wash with 2 mL 50 mM pH 7 phosphate buffer, wash with 2 mL MeOH, elute with
700 (xL 1.7 M ammonia solution, add 70 |xL glacial acetic acid to the eluate, mix thoroughly,
HPLC VARIABLES
Guard column: 20 X 4.6 5 |xm Supelguard LC-18-S (Supelco)
Column: 250 X 4.6 5 |xm Supelcosil LC-18-S ODS
Mobile phase: Gradient. A was MeOH:50 mM pH 6.5 phosphate buffer 60:40. B was 50 mM pH
6.5 phosphate buffer.
Flow rate: 1
Detector: UV 269
CHROMATOGRAM
Retention time: 6.5
Internal standard: 5-bromouracil (12)
OTHER SUBSTANCES
Extracted: doxifluridine, floxuridine, 5-fluorouridine monophosphate, metabolites
KEYWORDS
serum; SPE
REFERENCE
Guerrieri,A.; Palmisano,E; Zambonin,P.G.; De Lena,M.; Lorusso,V. Solid-phase extraction of fluoropyrimidine
derivatives on a copper-modified strong cation exchanger: determination of doxifluridine, 5-fluorouracil and
its main metabolites in serum by high-performance liquid chromatography with ultraviolet detection,
J.Chromatogr., 1993, 617, 71-77.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL 100 (xg/mL niacinamide, add 2 mL MeCN dropwise,
centrifuge at 3000 g for 30 min. Remove the supernatant and evaporate it to dryness, recon-
stitute the residue in 1 mL saturated ammonium sulfate, add 5 mL diethyl etherdsopropanol
80:20, shake for 15 min, centrifuge at 3000 g for 15 min, repeat the extraction. Combine the
organic phases and evaporate them to dryness, reconstitute the residue in 1 mL mobile phase,
inject an aliquot.
HPLC VARIABLES
Guard column: 5 |xm LiChrosorb RP-18
Mobile phase: pH 7.0 phosphate buffer (|x = 0.05)
Flow rate: 1
Detector: UV 265
CHROMATOGRAM
Retention time: 5
Internal standard: niacinamide
KEYWORDS
REFERENCE
Vandenbosch,C; van Belle,S.; de Smet,M.; Taton,G.; Bruynseels,V.; Vandenhoven,G.; Massart,D.L. Determi-
nation of leucovorin and 5-fluorouracil in plasma by high-performance liquid chromatography,
J.Chromatogr., 1993, 612, 77-85.
SAMPLE
Matrix: blood
Sample preparation: 100 JJLL Plasma + 100 |xL 20 |xg/mL 5-bromouracil + 8 mL ethyl acetate,
shake for 20 min, centrifuge at 2500 rpm for 20 min. Remove 7 mL of the organic layer and
evaporate it to dryness under nitrogen or at 60. Dissolve residue in 200 |xL mobile phase,
inject a 20 uL aliquot.
HPLCVARIABLES
Column: 150 X 6 Shimpack CLS-ODS (Shimadzu)
Mobile phase: 0.5 mM phosphoric acid
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Internal standard: 5-bromouracil
KEYWORDS
plasma; rat
REFERENCE
Lee,C.K; Uchida,T.; Kitagawa,K; Yagi,A.; Kim,N.-S.; Goto,S. Skin permeability of various drugs with different
lipophilicity, J.Pharm.ScL, 1994, 83, 562-565.
SAMPLE
Matrix: blood
Sample preparation: 100 JULL Plasma + 50 jxL 15 |xg/mL 5-bromouracil + 1 mL 0.365% HCl + 8
mL ethyl acetate, shake for 10 min, centrifuge at 1006 g for 10 min. Remove 7 mL of the
residue in 200 uL mobile phase, inject a 20 fxL aliquot.
HPLC VARIABLES
Column: 150 X 6 Shimpack CLS-ODS
Mobile phase: Water:phosphoric acid 100:0.1
Flow rate: 1.7
Detector: UV 270
CHROMATOGRAM
Internal standard: 5-bromouracil
KEYWORDS
REFERENCE
Umejima,H.; Kikuchi,A.; Kim,N.-S.; Uchida,T.; Goto,S. Preparation and evaluation of Eudragit gels. VIII. Rectal
absorption of 5-fluorouracil from Eudispert hv gels in rats, J.Pharm.ScL, 1995, 84, 199-202.
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Plasma + 50 |xL 50 |xg/mL stavudine + 1 mL ice-cold MeCN5 mix
vigorously for 30 s, centrifuge at 9000 g for 7 min. Remove the supernatant and add it to excess
crystalline magnesium sulfate, mix for 2 min, centrifuge at 9000 g for 10 min. Remove the
supernatant and evaporate it to dryness under a stream of nitrogen at room temperature,
reconstitute the residue in 200 |JLL mobile phase, inject a 15-150 fxL aliquot.
HPLC VARIABLES
Guard column: 25 X 2.3 PRP-I (Hamilton)
Column: 250 X 4.1 10 |xm PRP-I (Hamilton)
Mobile phase: MeCN:5 mM pH 11.1 tetrabutylammonium hydroxide 16:84
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 4.6
Internal standard: stavudine (5.5)
OTHER SUBSTANCES
Extracted: 4-deoxy-5-fluorouracil (UV 313), tegafur
KEYWORDS
REFERENCE
Jarugula,V.R.; Boudinot,F.D. High-performance liquid chromatographic determination of 5-fluorouracil and its
prodrugs, tegafur and 4-deoxy-5-fluorouracil, in rat plasma, J.Chromatogr.B, 1996, 677, 199-203.
SAMPLE
Matrix: blood, peritoneal fluid
Sample preparation: Plasma. 1 mL Plasma + 10 |xL 100 u-M bromouridine + 70 jxL perchloric
acid, mix thoroughly, let stand at 4 for at least 12 h, centrifuge for 5 min. Remove the super-
natant and adjust the pH to 7 with 5 M KOH, let stand on ice for 2 h, inject a 20 |xL aliquot.
Peritoneal fluid. 1 mL Peritoneal fluid + 10 |xL 100 u,M bromouridine, dilute 1:100 with water,
inject a 20 u-L aliquot.
HPLC VARIABLES
Guard column: 12.5 X 4.6 5 |xm Zorbax RX
Column: 250 X 4.6 5 |xm Zorbax RX
Mobile phase: Gradient. A was 25 mL pH 2.5 ammonium phosphate. B was MeCN:25 mM pH
7.5 ammonium phosphate 7:93. A:B 100:0 for 5 min, to 0:100 over 10 min, maintain at 0:100
for 10 min, return to initial conditions over 1 min, re-equilibrate for 20 min.
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 6-7
Internal standard: bromouridine (18)
OTHER SUBSTANCES
Extracted: floxuridine
KEYWORDS
REFERENCE
Smith-Rogers,J.A.; Tong,W.R; Duafala,M.E.; Markman,M.; Bertino,J.R. High-performance liquid chromato-
graphic method for the simultaneous measurement of floxuridine and fluorouracil in human body fluids,
J.Chromatogr., 1991, 566, 147-154.
SAMPLE
Sample preparation: Add 100 |xL 10% perchloric acid and 20 |xL 200 |xg/mL IS to 100 |xL serum
or homogenized tissue. Shake for 2 min and centrifuge at 2000 g for 10 min. Filter (45 jxm)
supernatant, inject a 10 |xL aliquot of the filtrate.
HPLC VARIABLES
Guard column: 45 X 4.6 5 |xm ODS Hypersil (VDS Optilab)
Column: 250 X 4.6 5 jjum ODS Hypersil (VDS Optilab)
Mobile phase: MeOH:99% acetic acid:water 3:0.5:96.95
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: 5-bromouracil (10.34)
Limit of quantitation: 100 ng/mL (serum); 300-500 ng/mL (tissue)
OTHER SUBSTANCES
Extracted: metabolites, fioxuridine
KEYWORDS
serum; rat; liver; tumor; kidney; spleen; peritoneum; gastric mucosa; lung; heart; pancreas
REFERENCE
Jung,M.; Berger,G.; Pohlen,U.; Pauser,S.; Reszka,R.; Buhr,H.J. Simultaneous determination of 5-fluorouracil
and its active metabolites in serum and tissue by high-performance liquid chromatography, J. Chromatogr. B,
1997, 702, 193-202.
SAMPLE
Sample preparation: 0.5 g Tissue or 1 mL plasma + 5 |xL 1 M sulfuric acid (lung and heart
only) + 2 JJLL 1 M sulfuric acid (plasma only) + 500 |xL 200 mg/mL sodium sulfate (liver and
kidney only) + 50 uX. 1 M pH 6 sodium acetate (liver only) + 50 \xL 1 M pH 5 sodium acetate
(kidney only) + 100 JULL 2% trichloroacetic acid (lung and heart only) + 15 mL n-propanol:ether
(liver 16:84, kidney 20:80, lung 88:12, heart 40:60, plasma 88:12), sonicate for 30 s, shake for
15 min, centrifuge for 15 min. Remove the organic layer and evaporate it to dryness under
reduced pressure, reconstitute the residue in 1 mL 50 mM ammonium phosphate (liver pH 11,
kidney pH 3, lung pH 2.5, heart pH 5, plasma pH 2.5), inject a 20 |xL aliquot. (From J.
Liq.Chromatogr. 1994, 17, 1621.)
HPLC VARIABLES
Mobile phase: MeCN:50 mM phosphate buffer 0.5:99.5 (Liver pH 3, kidney pH 6, lung pH 5,
heart pH 5, plasma pH 2.5)
Column temperature: 10 (kidney), 35 (lung), 20 (heart), 25 (plasma), 15 (liver)
Flow rate: 1
Detector: UV 200
CHROMATOGRAM
Retention time: 5 (plasma), 7 (liver), 7.5 (kidney), 8 (lung), 9 (heart)
Internal standard: flucytosine (for plasma) (4), 4-chlorouracil (for tissue) (18 (liver), 11 (kidney),
17 (lung), 19 (heart))
Limit of quantitation: 300 ng/g plasma, 800 ng/g (heart), 100 ng/g (lung), 240 ng/g (kidney),
570 ng/g (liver)
OTHER SUBSTANCES
Extracted: metabolites, fioxuridine
KEYWORDS
plasma; rabbit; liver; kidney; lung; heart; pharmacokinetics
REFERENCE
J.Chromatogr.B, 1994, 656, 397-405.
SAMPLE
residue with 50 |JLL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 204
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.Ay 1997, 763,149-
163.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 1-10 ng 5-fluorouracil in 100 JJLL DMSO, add 400 |xL 2.5 mg/mL
4-bromomethyl-7-methoxycoumarin in DMSO, add 5 mg potassium carbonate, shake at room
temperature for 15 min, add 500 uL water, centrifuge at 15600 g for 5 min, inject a 100 |xL
aliquot of the supernatant onto column A and elute to waste with mobile phase A, after 9.5
min divert the effluent containing the derivatized 5-fluorouracil onto column B, after another
2 min elute column B with mobile phase B, monitor the effluent from column B.
HPLCVARIABLES
Column: A 150 X 4.6 5 jxm CPS-Hypersil cyanopropyl; B 150 X 4.6 5 |jun ODS-Hypersil
Mobile phase: A MeOHrwater 50:50; B MeOH:water 60:40
Column temperature: 35 (column A only)
Flow rate: 1
CHROMATOGRAM
Retention time: 17
KEYWORDS
derivatization; column-switching; heart-cut
REFERENCE
Kindberg,C.G.; Slavik,M.; Riley,C.M.; Stobaugh,J.F. High-performance liquid chromatography of 5-fluorouracil
after derivatization with 4-bromomethyl-7-methoxycoumarin. Characterization of the derivative and the
use of column switching for the improvement of resolution and the enhancement of sensitivity,
J.Pharm.Biomed.AnaL, 1989, 7, 459-469.
SAMPLE
Sample preparation: Condition a 500 mg SCX Tech-Elut strong cation exchange SPE cartridge
(HPLC Technology) with 6 mL MeOH and five 2 mL portions of buffer. Tablets. Powder tablets,
weigh out amount equivalent to 500 mg flucytosine, add 50 mL water, stir for 15 min, filter. 1
mL Filtrate + 1.5 mL 27 jxg/mL thymine in buffer, make up to 10 mL with buffer, add 2 mL
to the SPE cartridge, wash with 1 mL buffer, collect all the eluates, inject an aliquot. Injections.
Dilute with water to a drug concentration of 10 mg/mL. 1 mL Sample + 1.5 mL 27 |xg/mL
thymine in buffer, make up to 10 mL with buffer, add 2 mL to the SPE cartridge, wash with
1 mL buffer, collect all the eluates, inject an aliquot. (Buffer was 70 mM KH2PO4 adjusted to
pH 3.0 with HCl.) (To measure flucytosine as well as fluorouracil omit the SPE step.)
HPLCVARIABLES
Mobile phase: 9 mM Sodium heptanesulfonate adjusted to pH 2.8 with phosphoric acid
Flow rate: 0.8
Detector: UV 266
CHROMATOGRAM
Retention time: 4.0
Internal standard: thymine (4.85)
OTHER SUBSTANCES
Simultaneous: flucytosine (when SPE is omitted)
KEYWORDS
tablets; injections; SPE
REFERENCE
Cavrini,V.; Bonazzi,D.; Di Pietra,A.M. Analysis of flucytosine dosage forms by derivative UV spectroscopy and
liquid chromatography, J.Pharm.Biomed.AnaL, 1991, 9, 401-407.
SAMPLE
Sample preparation: Dilute formulation 1:500 with water, inject a 50 jxL aliquot.
HPLC VARIABLES
Guard column: 5 x 4 35-60 |xm Perisorb RP18
Column: 250 X 4 10 u,m LiChrosorb RP18
Mobile phase: MeOH:300 mM sodium acetate 2:98
Detector: UV 254
CHROMATOGRAM
Retention time: 4.7
OTHER SUBSTANCES
Simultaneous: floxuridine
KEYWORDS
injections; water
REFERENCE
medication pump, Arzneimittelforschung, 1995, 45, 93-98.
SAMPLE
Sample preparation: Dilute 1 mL formulation to 10 mL with mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 150 X 4.6 jxBondapak phenyl
Mobile phase: MeCN:MeOH:0.01% acetic acid:0.005 N sulfonic acid 20:15:40:25
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 3.5
OTHER SUBSTANCES
Simultaneous: metoclopramide
KEY WORDS
REFERENCE
Wang,D.-R; Chang,L.-C; Lee,D.K.T.; Wong,C.-Y. Stability of fluorouracil-metoclopramide hydrochloride admix-
ture, Am.J.Health-Syst.Pharm., 1995, 52, 98-99.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 fjim C18
Flow rate: 2.5
Detector: UV 300
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: cytarabine, granisetron
KEYWORDS
REFERENCE
304.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 10 |xm precolumn (Beckman Instruments Inc.)
Column: 150 X 4.6 5 |xm C18 Altex Ultrasphere ODS
Mobile phase: Isocratic. MeOH:buffer 15:85 containing 2.5 mM sodium pentanesulfonate and
2.5 mM sodium heptanesulfonate. Gradient. A was MeOH containing 2.5 mM sodium penta-
nesulfonate and 2.5 mM sodium heptanesulfonate. B was buffer containing 2.5 mM sodium
pentanesulfonate and 2.5 mM sodium heptanesulfonate. A:B 0:100 for 2 min, to 30:70 over 1
min, maintain at 30:70. (Buffer was 50 mM phosphoric acid containing 50 mM KH2PO4, pH
2.5.)
Flow rate: 1
Detector: UV 254; UV 285
CHROMATOGRAM
Retention time: 2.52 (isocratic), 4.36 (gradient)
Internal standard: 5-methylcytosine (5.66 (isocratic), 12.62 (gradient))
Limit of quantitation: 12.5 \xM
OTHER SUBSTANCES
Simultaneous: barbituric acid, cytosine, flucytosine, hydroxycytosine, uracil, urea
REFERENCE
Biondi,L.; Nairn,J.G. High performance liquid chromatographic assay for 5-fluorouracil and 5-fluorocytosine,
J.Liq.Chromatogr., 1985, 8, 1881-1892.
SAMPLE
Matrix: solutions
Sample preparation: Dilute a 50 mg/mL sample 1:100 with mobile phase and inject an aliquot.
HPLC VARIABLES
Mobile phase: 5 mM pH 7.8 K2HPO4
Flow rate: 0.5
Detector: UV 214
CHROMATOGRAM
Retention time: 6.6
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Am.J.Health-SystPharm., 1996, 53, 1583-1588.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of an aqueous solution.
HPLC VARIABLES
Column: 100 X 8 Resolve C18
Mobile phase: MeCN:50 mM ammonium dihydrogen phosphate 4:96, pH adjusted to 3.5 with
H3PO4
Flow rate: 1.7
Detector: UV 268
CHROMATOGRAM
Retention time: 2.8
OTHER SUBSTANCES
Simultaneous: fluorouridine
REFERENCE
Dorta,M.J.; Munguia,0.; Farina,J.B.; Martin,V.S.; Llabr6s,M. Stability indicating high performance liquid chro-
matography methods for 5-fluorouridine in aqueous solution, Arzneimittelforschung, 1997, 47, 13881392.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
rin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benz-
phetamine, berberine, bibucaine, bromazepan, brompheniramine, buprenorphine, buspirone,
camfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, fluoxymesterone,
REFERENCE
Hill,D.W.; Kind,A-J- Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 mm long 5 |xm Microsorb-MV C18
Mobile phase: MeCN:pH 4 sodium acetate buffer 2:98
Flow rate: 1.5
Detector: UV 270
REFERENCE
Phillips,C.A.; Michniak,B.B. Transdermal delivery of drugs with differing lipophilicities using azone analogs
as dermal penetration enhancers, J.Pharm.Sci., 1995, 84, 1427-1433.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Ultra-Turrax) 1 g tissue with 0.05-1 (xg IS and 15 mL ice-
cold MeCN, rinse homogenizer with 10 mL ice-cold MeCN, centrifuge at 6000 rpm for 15 min,
remove the supernatant, wash the pellet with 5 mL MeCN, centrifuge for 5 min. Combine the
supernatants, remove a 5 mL aliquot, evaporate to dryness under a stream of nitrogen at room
temperature, reconstitute the residue in 50 |xL n-hexane:ethyl acetate:water 40:80:1, chromat-
ograph on a 125 X 4 5 jxm Spherisorb Si column (A) and a 200 X 4.6 5 jxm Spherisorb Si
column (B) with n-hexane:ethyl acetate:water 40:80:1 at 1 mL/min using column-switching.
Initially elute the columns in series, after 3 min (after 5-fluorouracil has eluted from column
A to column B) elute only column B, monitor the effluent from column B at 266 nm, collect the
appropriate fraction. (Backflush column A to waste for 6 min before the next injection.) Evap-
orate the eluate to dryness under a stream of nitrogen, reconstitute with 10 |xL acetone, add
1 mg freshly-powdered potassium carbonate, add 5 |xL 200 jjig/mL 18-crown-6 in acetone, add
20 |xL 750 |xg/mL 4-bromomethyl-7-methoxycoumarin in acetone, heat at 70 for 25 min, inject
a 1 |xL aliquot.
HPLC VARIABLES
Guard column: 20 X 1.6 3 |xm Nukleosil-120 (MZ Analysentechnik)
Column: 125 X 1.6 3 |xm Nukleosil-120 (MZ Analysentechnik)
Flow rate: 0.06
Injection volume: 1
CHROMATOGRAM
Retention time: 27
Internal standard: 5-chlorouracil (31)
KEY WpRDS
derivatization; microbore; human; pig; liver
REFERENCE
Jochheim,C; Janning,R; Marggraf,U.; Lofner,T.M.; Hasse,F.; Linscheid,M. A procedure for the determination
of 5-fluorouracil in tissue using microbore HPLC andfluorescencedetection, AnaLBiochem., 1994,227,285-
291.
Fluoxetine
Molecular formula: C17H18F3NO
Merck Index: 4222
LednicerNo.: 3 32
SAMPLE
Matrix: blood
Sample preparation: Add 250 |xL 2 M sodium carbonate to 500 uL plasma. Add 100 JJLL 1 |xg/
mL IS in MeOH, extract with 10 mL n-hexane. Shake for 30 min and centrifuge at 3000 g for
10 min. Cool in a dry ice-acetone bath. Add 200 |xL 0.3% phosphoric acid to upper organic layer.
Shake for 10 min and centrifuge at 3000 g for 10 min. Separate the organic layer. Inject a 100
JJLL aliquot of the acidic aqueous layer.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm C18 Symmetry (Waters Millipore, USA)
Mobile phase: MeCN:67 mM potassium phosphate buffer adjusted to pH 3.0 with phosphoric
acid 35:65 (After each chromatographic session wash the column with 200 mL MeCN:water
50:50.)
Flow rate: 1.2
Detector: UV 226, UV 254, UV 400
CHROMATOGRAM
Internal standard: clovoxamine (6.5)
Limit of quantitation: 5 ng/mL (UV 226, UV 400); 7 ng/mL (UV 254)
OTHER SUBSTANCES
Extracted: metabolites, amitriptyline, clomipramine, desipramine, imipramine, maprotiline,
nortriptyline
Simultaneous: amineptine, carbamazepine, chlordiazepoxide, clorazepate, clozapine, cyamema-
zine, desmethylmaproptiline, desmethylvenlafaxine, doxepin, flunitrazepam, fluvoxamine, hal-
operidol, levomepromazine, lorazepam, loxapine, mianserine, sulpiride, trimipramine, venla-
faxine, viloxazine, zolpidem, zopiclone
Noninterfering: diazepam, valproic acid
Interfering: chlorpromazine, clonazepam
KEYWORDS
plasma
REFERENCE
Aymard,G.; Livi,R; Pham,Y.T.; Diquet,B. Sensitive and rapid method for the simultaneous quantification of five
antidepressants with their respective metabolites in plasma using high-performance liquid chromatography
with diode-array detection, J.Chromatogr.B, 1997, 700, 183-189.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 400 jxL 50 |xg/mL IS in MeOH, make up to 2 mL
with MeOH, centrifuge at 1100 g for 10 min. Remove a 1 mL aliquot of the supernatant, add
2 mL 1 M NaOH and 2 mL diethyl ether, shake for 10 min, repeat the extraction. Combine
the ether extracts and dry them over anhydrous sodium sulfate. Evaporate to dryness under
a stream of nitrogen and reconstitute the residue in 1 mL MeOH. Inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCNipH 2.7 phosphate buffer 90:10
Flow rate: 1.0
Detector: UV 254
CHROMATOGRAM
Internal standard: chlorprothixene (5.20)
OTHER SUBSTANCES
Simultaneous: amitriptyline, azathioprine, diazepam, doxepin, fludarabine, imipramine,
mercaptopurine,
Interfering: methadone, pentazocine, piritramide, tramadol
KEYWORDS
plasma
REFERENCE
Misztal,G.; Hopkala,H. Determination of fluoxetine in human plasma using reversed phase HPLC, Pharmazie,
1997, 52, 854-856.
SAMPLE
Matrix: blood
Sample preparation: Add 400 jiL 330 mM NaOH to 2 mL plasma. Mix for 5 min, extract with
14 mL n-hexane:isoamyl alcohol 98.5:1.5 for 20 min. Centrifuge at 2500 g for 5 min and collect
the organic layer. Adjust to pH 2 with 400 |xL HCl, shake for 1 min, centrifuge at 1500 g for
10 min. Inject a 100 |xL aliquot of the aqueous layer.
HPLCVARIABLES
Guard column: 15 X 4.6 5 |xm LiChrospher RP-18
Column: 250 X 4.6 5 jim Spherisorb ODS-2
Mobile phase: MeCN:5 mM n-octylamine in water 40:60, adjusted to pH 6.4 with orthophos-
phoric acid (After use, wash the column with water for 15 min, MeCNrwater 50:50 for 15 min,
and MeCN for 5 min.)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: amisulpride, diazepam, flunitrazepam, fluvoxamine, haloperidol, imipramine,
maprotiline, paroxetine, amitriptyline, clomipramine, mianserin
KEYWORDS
plasma
REFERENCE
Gennaro,M.C; Abrigo,C; Angelino,S.; Albert,U.; Bogetto,E; Maina,G.; Prolo,R; Ravizza,L. Determination of
fluoxetine and norfluoxetine in human plasma by ion-interaction RP-HPLC, J.Liq.Chromatogr.Rel.Technol.,
1997, 20, 3017-3028.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Serum + 20 jxL 5 |xg/mL IS in MeOH + 100 |xL 5 M NaOH, vortex
for 30 s, add 2 mL hexane, vortex for 30 s, centrifuge at 3000 g for 3 min. Remove the organic
layer and evaporate it under a gentle stream of nitrogen at 20, reconstitute the residue in 50
JJLL mobile phase, vortex for 30 s, inject a 20 jxL aliquot.
HPLC VARIABLES
Guard column: RP C18 (Brownlee)
Column: 150 X 4.6 5 |xm Microsorb MV
Mobile phase: MeCN.water 55:45 containing 10 mM triethylamine, adjusted to pH 4.8 with 85%
phosphoric acid
Flow rate: 1
Detector: UV 226
CHROMATOGRAM
Retention time: 4.7
OTHER SUBSTANCES
Extracted: norfluoxetine
KEYWORDS
mouse; serum; pharmacokinetics
REFERENCE
Holladay,J.W.; Dewey,M.J; Yoo,S.D. Quantification of fluoxetine and norfluoxetine serum levels by reversed-
phase high-performance liquid chromatography with ultraviolet detection, J.Chromatogr.B, 1997, 704,259-
263.
SAMPLE
Sample preparation: 100 JJLL Serum or 200 JULL brain homogenate + 20 jxL 5.0 |xg/mL clomipra-
mine in MeOH + 100 uL 5.0 M NaOH + 2 mL hexane, vortex for 30 s, centrifuge at 3000 g for
5 min. Evaporate organic layer under a gentle stream of nitrogen at 20. Reconstitute residue
with 50 u,L mobile phase, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: Microsorb MV C18 (Rainin, Woburn, USA)
Mobile phase: MeCN:water 55:45 containing 10 mM triethylamine, pH adjusted to 4.8 with 85%
phosphoric acid
Flow rate: 1.0
Detector: UV 226
CHROMATOGRAM
Internal standard: clomipramine
OTHER SUBSTANCES
KEYWORDS
serum; brain; mouse; pharmacokinetics
REFERENCE
Holladay,J.W.; Dewey,M.J.; Yoo,S.D. Pharmacokinetics and antidepressant activity of fluoxetine in transgenic
mice with elevated serum a-l-acid glycoprotein levels, Drug Metab.Dispos., 1998, 26, 20-24.
SAMPLE
Sample preparation: 100 |xL Serum or weighed homogenized brain + 1 mL 600 mM pH 9.8
sodium carbonate-sodium bicarbonate buffer containing 100 ng/mL IS. Add 7 mL ethyl acetate:
n-heptane 20:80, mix vigorously for 1.5 min, centrifuge at 3000 g for 10 min. Mix the organic
layer with 200 jxL 25 mM potassium dihydrogen phosphate adjusted to pH 2.3 with 85% phos-
phoric acid. Mix for 1 min, centrifuge at 3000 g for 10 min, discard the organic layer, inject a
50 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Guard column: 4 x 4 5 |xm C8 endcapped (Merck, Germany)
Column: 125 X 4 4 |xm C8 endcapped (Merck, Germany)
Mobile phase: MeCN:buffer 42:58 (Buffer was water containing 100 |xL/L perchloric acid and
1.5 g/L tetramethylammonium perchlorate.)
Flow rate: 1.2
Detector: UV 227
CHROMATOGRAM
Internal standard: protriptyline (8.1)
Next Page
OTHERSUBSTANCES
Simultaneous: acetopromazine, aceprometazine, amitriptyline, bromazepam, chlorpromazine,
ciamemazine, clomipramine, clorazepate, demethyldomipramine, diazepam, flunitrazepam,
imipramine, nitrazepam, nordiazepam, nortriptyline
KEY WORDS
serum; brain; mouse; human; pharmacokinetics
REFERENCE
Alvarez,J.-C; Bothua,D.; Collignon,L; Advenier,C; Spreux-Varoquaux,O. Determination of fluoxetine and its
metabolite norfluoxetine in serum and brain areas using high-performance liquid chromatography with
ultraviolet detection, J.Chromatogr.B, 1998, 707, 175-180.
SAMPLE
residue with 50 uL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Fluoxymesterone
Merck Index: 4223
Lednicer No.: 1 175
Previous Page
OTHERSUBSTANCES
Simultaneous: acetopromazine, aceprometazine, amitriptyline, bromazepam, chlorpromazine,
ciamemazine, clomipramine, clorazepate, demethyldomipramine, diazepam, flunitrazepam,
imipramine, nitrazepam, nordiazepam, nortriptyline
KEY WORDS
serum; brain; mouse; human; pharmacokinetics
REFERENCE
Alvarez,J.-C; Bothua,D.; Collignon,L; Advenier,C; Spreux-Varoquaux,O. Determination of fluoxetine and its
metabolite norfluoxetine in serum and brain areas using high-performance liquid chromatography with
ultraviolet detection, J.Chromatogr.B, 1998, 707, 175-180.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Fluoxymesterone
Merck Index: 4223
Lednicer No.: 1 175
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 10 jxL 20 fig/mL 6a-methylprednisolone in MeOH, mix
thoroughly, add 10 mL dichloromethane, shake for 1 h, centrifuge for 15 min. Discard the
aqueous layer, wash the organic layer with 1 mL 100 mM NaOH and 1 mL water (vortex for
30 s and centrifuge for 15 min each time). Remove the organic layer and evaporate it to dryness
under a stream of nitrogen at 40-45, reconstitute the residue in 200 |xL mobile phase, inject
a 50 |JLL aliquot onto column A in series with column B and elute with mobile phase. After 2.1
min remove column A from the circuit and backflush it with mobile phase at 1 mL/min for 15
min, elute column B with mobile phase and monitor the effluent.
HPLC VARIABLES
Column: A 30 mm long Spherisorb silica; B 250 X 4.6 6 |xm Zorbax silica
Mobile phase: Butyl chloride:THF:MeOH:phosphoric acid 880:100:15:0.5 (Butyl chloride was
50% water-saturated.)
Flow rate: 2
Detector: UV 236
CHROMATOGRAM
Retention time: 14
Internal standard: 6a-methylprednisolone (24)
KEYWORDS
serum; normal phase; column-switching; pharmacokinetics
REFERENCE
Capponi,V.J.; Cox,S.R.; Harrington,E.L.; Wright,C.E.; Antal,E.J.; Albert,K.S. Liquid chromatographic assay for
fluoxymesterone in human serum with application to a preliminary bioavailability study, J.Pharm.Sci.,
1985, 74, 308-311.
SAMPLE
Sample preparation: Oils. 1 mL Sample + 25 mL MeOH:water 90:10, shake vigorously for 5
min, centrifuge, inject a 10 |xL aliquot of the supernatant. Tablets. Grind a tablet to a fine
powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 |xm), discard first 5 mL of filtrate,
inject a 10 |xL aliquot of the remaining filtrate. Suspensions (aqueous). Make up 5 mL to 50
mL with MeOH, filter (0.45 |xm), discard first 5 mL of filtrate, inject a 10 |xL aliquot of the
remaining filtrate.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: 4.4
OTHER SUBSTANCES
Simultaneous: ethisterone, methandrostenolone, nandrolone, norgestrel, testosterone, dehy-
droepiandrosterone (UV 210), mibolerone, methyltestosterone, methandriol (UV 210), noreth-
indrone acetate, calusterone, mesterolone (UV 210), norethandrolone, trenbolone acetate, ben-
zyl benzoate, nandrolone acetate, testosterone acetate, stanozolol, oxymetholone, nandrolone
propionate, methenolone acetate, testosterone propionate, aspirin, caffeine, formebolone, ben-
zyl alcohol, testolactone, cortisone
Interfering: norethindrone, oxandrolone (UV 210), boldenone
KEYWORDS
oils; tablets; suspensions
REFERENCE
Walters,M.J.; Ayers,RL; Brown,D.J. Analysis of illegally distributed anabolic steroid products by liquid chro-
matography with identity confirmation by mass spectrometry or infrared spectrophotometry,
J.Assoc.Off.Anal.Chem., 1990, 73, 904-926.
SAMPLE
Sample preparation: Crush tablets, weigh out amount equivalent to 10 mg steroid, dissolve in
10 mL MeOH, sonicate for 15 min, filter. 1 mL Filtrate + 5 mL MeOH + 4 mL water, inject a
25 JJLL aliquot.
HPLC VARIABLES
Mobile phase: Gradient. MeOH:water from 70:30 to 100:0 over 15 min, maintain at 100:0 for 15
min.
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 7.2
OTHER SUBSTANCES
Simultaneous: boldenone, boldenone acetate, boldenone undecylenate, clostebol acetate, danazol
(UV 280), methandriol, methandriol-3-acetate, methandriol dipropionate, methandrostenolone,
methyltestosterone, nandrolone, nandrolone decanoate, nandrolone phenylpropionate, nandro-
lone propionate, stanolone, stanozolol, testosterone, testosterone acetate, testosterone cypio-
nate, testosterone enanthate, testosterone isobutyrate, testosterone propionate, testosterone
undecanoate
Noninterfering: oxandrolone, oxymetholone, testosterone decanoate, testosterone isocaproate
KEYWORDS
tablets
REFERENCE
Lurie,I.S.rling,A.R.; Meyers,RR The determination of anabolic steroids by MECC, gradient HPLC, and capil-
lary GC, J.Forensic ScL, 1994, 39, 74-85.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: ixBondapak ODS
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 10
Internal standard: fluoxymesterone
OTHER SUBSTANCES
Simultaneous: triamcinolone acetonide
KEYWORDS
fluoxymesterone is IS
REFERENCE
Kirschbaum,J. High-pressure liquid chromatography of triamcinolone acetonide: effect of different octadecyl-
silane columns on mobility, J.Pharm.ScL, 1980, 69, 481-482.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: Phenyl (Waters) or 12% ODS (Whatman)
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 7.7 (phenyl), 12.0 (ODS)
OTHER SUBSTANCES
REFERENCE
Kirschbaum,J.; Clay,R.; Poet,R. HPLC steroid analyses: generic column description and variable selectivity,
Anal.Chem.Symp.Ser. (Adv.Steroid Anal.), 1982, 10, 361-366.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve at 100 jxg/mL in MeOH.
HPLC VARIABLES
Guard column: 70 X 2.1 Whatman CO:Pell ODS
Column: 300 X 3.9 Bondex C18
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Simultaneous: boldenone, nandrolone, methandrostenolone, testosterone, danazol, methyl-
testosterone
REFERENCE
Noggle,F.T.,Jr.; Clark,C.R.; DeRuiter,J. Liquid chromatographic and spectral analysis of the 17-hydroxy ana-
bolic steroids, J.Chromatogr.Sci., 1990, 28, 162-166.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 0.5 mg/mL solution in MeOH, inject a 5 JJLL aliquot.
HPLC VARIABLES
Flow rate: 2
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
chlorpromazine, clenbuterol, cortisone, danazol, diflunisal, doxapram, estrone, mefenamic acid,
methyltestosterone, nicotine, oxazepam, phentermine, phenylpropanolamine, progesterone,
sulfamethazine, sulfanilamide, testosterone, testosterone propionate, tranylcypromine,
tripelennamine
Interfering: 2-naphthoxyacetic acid
KEYWORDS
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
camfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, flu-
phenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glybenclam-
ide, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone,
mescaline, mesoridazine, methadone, methamphetamine, methap3rrilene, methaqualone, meth-
yldopamine, methylphenidate, methylprednisolone, methyltestosterone, meth^rylon, meto-
epinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, ox^hen-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 5 |xL aliquot of a 10 jxg/mL solution in MeOH.
HPLCVARIABLES
Mobile phase: MeCNrIO mM ammonium acetate buffer 45:55
Flow rate: 0.5
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: boldenone, epimethandienone, epitestosterone, 6p-hydroxymethandienone,
methandienone, norethindrone, oxymetholone (UV 280), trenbolone
REFERENCE
Barr6n,D.; Pascual,J.A.; Segura,J.; Barbosa,J. Prediction of LC retention of steroids using solvatochromic pa-
rameters, Chromatographia, 1995, 41, 573-580.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 (xL aliquot of a solution in MeOH:water 50:50.
HPLC VARIABLES
Column: 250 X 4 7 |xm LichroCAKT RP-8 (Merck)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 4.5
OTHERSUBSTANCES
Simultaneous: medrogestone, mestranol, norethindrone, progesterone, testosterone propionate
REFERENCE
Gau,Y.S.; Sun,S.W.; Chem,R-R--L- Optimization of high-performance liquid chromatographic separation for pro-
gestogenic, estrogenic, and androgenic steroids using factorial design, J.Liq.Chromatogn, 1995, 18, 2373-
2382.
Flupentixol
Molecular formula: C23H25F3N2OS
CAS Registry No.: 2709-56-0, 30909-51-4 (decanoate),
2413-38-9(2.HCI)
Merck Index: 4224
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 228.7
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 juig/mL solution in MeOH, inject a 20 jxL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.0
OTHER SUBSTANCES
mine, fenoterol, fentanyl, flavoxate, fluopromazine, fluphenazine, flurazepam, haloperidol, hy-
thipendyl, protriptyline, prox5rmetacaine, pseudoephedrine, pyrimethamine, quinidine, qui-
REFERENCE
Fluphenazine
Molecular formula: C22H26F3N3OS
CAS Registry No.: 69-23-8, 146-56-5 (di HCI),
2746-81-8 (enanthate)
Merck Index: 4226
Lednicer No.: 1 383
SAMPLE
Matrix: blood
Sample preparation: 1-5 mL Plasma + 1 mL 1 M NaOH + hexanes, extract for 30 min, centri-
fuge. Remove a 9 mL aliquot of the organic phase and evaporate it to dryness at 30 under a
stream of nitrogen. Dissolve the residue in 100 |xL mobile phase, inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 10 |xm Micropak CN (Varian)
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Retention time: 8.7
OTHER SUBSTANCES
Simultaneous: acetophenazine, amitriptyline, benztropine, butaperazine, carphenazine, chlor-
promazine, promethazine, haloperidol, imipramine, mesoridazine, nortriptyline, orphenadrine,
piperacetazine, promazine, thioridazine, thiothixene, trifluoperazine, triflupromazine, trihex-
yphenidyl, trimeprazine, metabolites
KEYWORDS
plasma
REFERENCE
Curry,S.H.; Brown,E.A.; Hu,0.Y.-R; Perrin,J.H. Liquid chromatographic assay of phenothiazine, thioxanthene
and butyrophenone neuroleptics and antihistamines in blood and plasma with conventional and radial
compression columns and UV and electrochemical detection, J.Chromatogr., 1982, 231, 361-376.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 jxL 1 fxg/mL loxapine in isopropanolidiethylamine
99.9:0.1 + 250 |xL 25% potassium carbonate containing 0.1% diethylamine + 5 mL hexane:
isoamyl alcohol 97:3, vortex for 30 s, centrifuge at 500 g for 3 min. Remove the organic layer
and add it to 100 |JLL 250 mM HCl, vortex for 30 s, inject a 50 |xL aliquot of the aqueous phase.
HPLCVARIABLES
Guard column: 50 X 4.6 40 yna C8 (Supelco)
Mobile phase: MeCN:water:diethylamine:85% phosphoric acid 53.3:45.1:1:0.4, pH adjusted to 7.2
with NaOH or phosphoric acid
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Internal standard: loxapine (k' 7.18)
OTHER SUBSTANCES
Extracted: amitriptyline, chlorpromazine, desipramine, desmethldiazepam, desmethylchlordi-
azepoxide, diazepam, doxepin, haloperidol, imipramine, nortriptyline, thiothixene
Noninterfering: molindone, perphenazine, trifluoperazine
Interfering: chlordiazepoxide, desmethyldoxepin, oxazepam
KEYWORDS
plasma
REFERENCE
Kiel,J.S.; Abramson,R.K.; Morgan,S.L.; Voris,J.C. A rapid high performance liquid chromatographic method for
the simultaneous measurement of six tricyclic antidepressants, J.Liq.Chromatogr., 1983, 6, 2761-2773.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 40 ng/mL chlorpromazine in water, vortex for a few
s, add 500 |xL 650 mM sodium carbonate, vortex for a few s, add 7 mL pentane:ethyl acetate
75:25, mix for 15 min, let stand for 5 min. Remove the upper organic layer and evaporate it
to dryness under a stream of nitrogen at 65, reconstitute the residue in 40 |xL MeCN, mix for
a few s, inject a 30 jxL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Spherisorb cyano
Mobile phase: MeCN:MeOH:100 mM pH 7 ammonium acetate 90:5:5
Flow rate: 1.5
Detector: E, ESA 5100A, Model 5020 guard cell +1.00 V, model 5011 analytical cell, cell 1 +0.50
V, cell 2 +0.75 V
CHROMATOGRAM
Internal standard: chlorpromazine (10.87)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Cooper, J.K.; Hawes,E.M.; Hubbard,J.W.; McKay,G.; Midha,K.K. An ultrasensitive method for the measurement
of fluphenazine in plasma by high-performance liquid chromatography with coulometric detection,
Ther.Drug Monit, 1989, 11, 354-360.
SAMPLE
Matrix: blood
the residue in 100 u,L mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 260
CHROMATOGRAM
Limit of detection: < 120 ng/mL
KEYWORDS
mine; protriptyline; fiurbiprofen; tetrazepam; zorubicin; prazepam; alimemazine; loperamide;
amitriptyline; nortriptyline; tioclomarol; diclofenac; mefioquine; trimipramine; chlorambucil;
REFERENCE
254-262.
SAMPLE
residue with 50 |xL MeCNiwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLCVARIABLES
Column: 250 X 4.6 5 jjum Symmetry C8 (Waters)
mL/min)
Detector: UV 259.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Extract ground tablets containing 1 mg with 10 mL MeOH, shake for 30
min, centrifuge at 2000 rpm for 5 min. Remove a 5 mL aliquot of the supernatant and add it
to 10 mL 1.25 mg/mL norephedrine hydrochloride in MeOH, make up to 25 mL with MeOH,
HPLC VARIABLES
Column: 150 X 4.6 5 (xm Zorbax CN
Mobile phase: MeOH:MeCN:25 mM pH 4.5 acetate buffer 30:40:30
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Internal standard: norephedrine (2.38)
OTHER SUBSTANCES
Interfering: desipramine, promazine
KEYWORDS
tablets
REFERENCE
Beaulieu,N.; Gagn6,C; Lovering,E.G. Liquid chromatographic determination of identity, content, and content
uniformity of desipramine, fluphenazine, and promazine, J.Assoc.Off.Anal.Chem., 1986, 69, 178-179.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 |AL aliquot.
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.0
OTHER SUBSTANCES
mine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, flurazepam, haloperidol, hy-
REFERENCE
Jane,I.; McKinnon,A-; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica
SAMPLE
Matrix: solutions
jxL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 |xm jjuBondapak C18
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
Retention time: k' 0.53 (fluphenazine), k' 3.49 (fluphenazine decanoate), k' 2.49 (fluphenazine
enanthate)
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Inject 1 mL onto column A. Elute column A onto column B with mobile
phase for 30 s then remove it from the circuit. Elute column B with mobile phase and monitor
the effluent.
HPLC VARIABLES
Column: A 10 X 6 packed with 40 jxm material from a Bond Elut cartridge (cat. no. 620303); B
100 X 4 3 jxm Spherisorb ODS Superpac
Mobile phase: MeCN:85% phosphoric acid:triethylamine:water 49.55:0.225:0.225:50
Flow rate: 0.65
Detector: UV 238
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: alprazolam, amitriptyline, chlorpromazine, chlorprothixene, clomipramine, des-
clomipramine, desmethylimipramine, diazepam, flunitrazepam, imipramine, levomepromazine,
maprotiline, nortriptyline, perphenazine, promethazine, thioridazine sulfoxide, thioridazine,
thioridazine sulfone, trimipramine, zimeldine
Noninterfering: carbamazepine, clonazepam, lorazepam, nitrazepam, oxazepam, phenytoin
Interfering: haloperidol, protriptyline, zuclopenthixol
KEYWORDS
column-switching
REFERENCE
Svensson,C; Nyberg,G.; Martensson,E. High-performance liquid chromatographic quantitation of amitriptyline
and nortriptyline in dialysate from plasma or serum using on-line solid-phase extraction, J.Chromatogr.,
1988, 432, 363-369.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Zorbax KX
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
camfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-
fluorouracil, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin, glutethimide, glyben-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 jxm LiChrospher 100 RP-8 (B)
Mobile phase: MeCN:0.025% phosphoric acid:buffer 25:10:5 (A) or 60:25:15 (B) (Buffer was 9 rnL
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
tadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bupivacaine, bus-
pirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine,
chlordiazepoxide, chlormezanone, chloroquine, chlorpheniramine, chlorpromazine, chlorpro-
pamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine,
clonazepam, clonidine, clozapine, cocaine, codeine, colchicine, cyclizine, cyclobenzaprine, dan-
trolene, desipramine, diazepam, diclofenac, diflunisal, diltiazem, diphenhydramine, diphenidol,
diphenoxylate, dipyridamole, disopyramide, dobutamine, doxapram, doxepin, droperidol, en-
cainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluraze-
pam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol,
homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloro-
quine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac,
labetalol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefen-
amic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone,
methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, methyldopa,
methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam, moclo-
bemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, nizati-
dine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemo-
line, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital,
phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phen3rtoin, pimo-
zide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine,
prochlorperazine, procyclidine, promazine, promethazine, propafenone, propantheline, pro-
piomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemethorphan,
ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, so-
talol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetra-
caine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide,
tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, tri-
meprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine,
zopiclone
KEYWORDS
details of plasma extraction
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Flupirtine
Merck Index: 4227
Lednicer No.: 4 102
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 30 yJL 10 (xg/mL IS in 10 mM HCl + 40 ^L 1 M NaOH +
8 mL diethyl ether, rotate for 15 min, centrifuge. Remove 5 mL of the organic layer and add
it to 10 mL n-hexane and 400 |xL 1 M HCl, vortex for 1 min, centrifuge at 1000 g, inject a 30-
200 (xL aliquot of the acidic aqueous phase.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm ODS-Hypersil
Mobile phase: Gradient. A was 10 mM pH 3 phosphate buffer. B was MeCNiMeOH 50:50. A:B
from 40:60 to 30:70 over 8 min, maintain at 30:70 for 3 min.
Flow rate: 1.4
CHROMATOGRAM
Retention time: 6.0
Internal standard: [2-amino-6-[[(2,4-dimethylphenyl)methyl]amino]-3-pyridinyl]carbamic acid
ethyl ester (9.38)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Niebch,G.; Borbe,H.O.; Hummel,T.; Kobal,G. Dose-proportional plasma levels of the analgesic flupirtine mal-
eate in man. Application of a new HPLC assay, Arzneimittelforschung, 1992, 42, 13431345.
SAMPLE
Sample preparation: Plasma. 500 |xL Plasma + 50 |xL 2 |xg/mL IS in MeOH + 1 mL MeCN,
vortex for 5 s, centrifuge at 2000 g for 5 min. Remove the supernatant and evaporate it to
dryness under a stream of air at 37, reconstitute the residue in 150 |xL mobile phase, mix for
1 min, centrifuge for 2 min at 2000 g, inject a 20 |xL aliquot. Urine. 500 |xL Urine + 50 |xL 2
|xg/mL IS in MeOH + 50 |xL 10% ammonium hydroxide, vortex for 5 s, add 5 mL dichloro-
methane, shake on a reciprocating shaker for 5 min, centrifuge at 2000 g for 5 min. Remove 4
mL of the organic phase and evaporate it to dryness under a stream of air at 37, reconstitute
the residue in 150 |xL mobile phase, mix for 1 min, centrifuge for 2 min at 2000 g, inject a 20
fiL aliquot.
HPLC VARIABLES
Guard column: 70 X 2.3 30-38 |xm Co:Pell ODS
Column: 250 X 4.6 5 (xm Ultrasphere ODS
Mobile phase: MeOH:MeCN:5 mM phosphate buffer 32:32:36, pH adjusted to 6.7
Flow rate: 1.4
CHROMATOGRAM
Internal standard: 2-amino-3-carbethoxyamino-6-(2,4-dimethylbenzylamino)pyridine (6.08)
OTHER SUBSTANCES
Noninterfering: aspirin, carbamazepine, clonazepam, diazepam, methylprednisolone, phenyt-
oin, valproic acid
KEYWORDS
REFERENCE
Narang,RK; Tourville,J.E; Chatterji,D.C; Gallelli,J.F. Quantitation of flupirtine and its active acetylated me-
tabolite by reversed-phase high-performance liquid chromatography using fluorometric detection,
J.Chromatogr., 1984, 305, 135-143.
Flurazepam
Molecular formula: C2IH23GIFN3O
CAS Registry No.: 17617-23-1,1172-18-5 (HCI)
Merck Index: 4233
SAMPLE
Matrix: blood
Sample preparation: 200 JJLL Serum + 200 |xL 50 |xg/mL hexobarbital in MeCN + 25 |xL glacial
acetic acid, vortex for 10 s, centrifuge for 1 min, inject a 30-100 (xL aliquot of the supernatant.
HPLCVARIABLES
Column: jxBondapak C18
5:95 for 5 min.
Flow rate: 3
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
ethchlorvynol, glutethimide, methaqualone, methyprylon, nitrazepam, pentobarbital, pheno-
barbital, phenytoin, primidone, salicylic acid, secobarbital, theophylline
mine, lidocaine, mesantoin, methsuximide, nirvanol, nortriptyline, oxazepam, procainamide,
phenylpropanolamine, propranolol, quinidine
Interfering: desmethyldoxepin
KEYWORDS
serum
REFERENCE
tography, JAnal.Toxicol., 1981, 5, 177-182.
SAMPLE
Matrix: blood
Sample preparation: Basify plasma with 500 mM KOH, extract with diethyl ethenheptane 90:
10. Remove the organic layer and evaporate it to dryness, reconstitute the residue in mobile
HPLC VARIABLES
Column: 250 mm long 5 (xm Spherisorb CN
Mobile phase: MeCN:EtOH:50 mM pH 2.5 phosphate buffer 20:15:65
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: droperidol
KEYWORDS
flurazepam is IS; plasma
REFERENCE
James,J.; Lowe,D.; Karnes,H-T. Determination of histamine from plasma using derivatization with naphtha-
lene-2,3-dicarboxaldehyde and HPLC with fluorescence detection, Pharm.Res., 1992, 9, S21.
SAMPLE
Matrix: blood
Sample preparation: Rock 5 mL whole blood + 10 mL water + 8.5 mL Na2WO4 in a 50 mL
stoppered tube for 1 min, add 6 mL NiCl2, rock for 5 min, add 15 mL l-chlorobutane:isobutyl
alcohol:THF 40:40:20, centrifuge at 2500 g for 15 min. Remove organic phase and repeat the
process. Filter all organic phases through a 40-90 jjim filter and evaporate to dryness in a 100
mL porcelain dish at a moderate temperature in a sand bath. Take up residue in 500 |xL MeCN:
water 80:20, inject a 20 |xL aliquot. (Na2WO4 prepared by mixing 10 g Na2WO4.2H2O in 38 mL
of 2 M NaOH and 2.5 g of NaHCO3 and making up to 100 mL. NiCl2 was 17% w/v NiCl2 in
water.)
HPLCVARIABLES
Mobile phase: A = MeCN; B = 20 mM n-hexylamine adjusted to pH 4 with 85% phosphoric acid.
A:B from 25:75 to 40:60 over 25 min to 50:50 over another 5 min
Detector: UV 230
CHROMATOGRAM
Retention time: 11
Limit of detection: 0.30 ppm
OTHER SUBSTANCES
Extracted: bromazepam, clonazepam, diazepam, flunitrazepam, medazepam, nitrazepam,
oxazepam
Also analyzed: buprenorphine, caffeine, cocaine, codeine, diamorphine, ethylmorphine, lido-
caine, methaqualone, morphine, naloxone, noscapine, papaverine, pentazocine, procaine
KEYWORDS
whole blood
REFERENCE
Bernal,J.L.; Del Nozal,M.J.; Rosas,V.; Villarino,A. Extraction of basic drugs from whole blood and determination
by high performance liquid chromatography, Chromatographia, 1994, 38, 617623.
SAMPLE
Matrix: blood
with 3 mL 750 mL/L methanol. Elute with three aliquots of 300 |xL 0.1 M ammonium acetate
(JLL 0.1 M phosphoric acid (pH 2.5), mix, separate, inject a 100 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Flow rate: 2
Detector: UV 228
CHROMATOGRAM
Retention time: 6.6
OTHER SUBSTANCES
desipramine, dextromethorphan, diazepam, diphenhydramine, doxepin, encainide, fentanyl,
flecainide, haloperidol, ibuprofen, imipramine, lidocaine, maprotiline, methadone, methaqua-
lone, mexiletine, midazolam, nordiazepam, nordoxepin, norfluoxetine, nortriptyline, norvera-
pamil, pentazocine, promazine, propafenone, propoxyphene, propranolol, protriptyline, quini-
dine, temazepam, trazodone, trimipramine, verapamil
warfarin
Interfering: fluoxetine, hydroxyethylflurazepam, norchlorimipramine
KEYWORDS
plasma; SPE
REFERENCE
Nichols, J.H.; Charlson,J.R.; Lawson,G.M. Automated HPLC assay of fluoxetine and norfluoxetine in serum,
Clin.Chem., 1994, 40, 1312-1316.
SAMPLE
Sample preparation: 500 \xL Plasma or urine + 50 |xL MeOH + 1 mL 100 mM pH 9 sodium
phosphate buffer + 4 mL dichloromethane:diethyl ether 60:40, shake at 45 rpm for 15 min,
centrifuge at 10 at 1870 g for 10 min. Remove the organic layer and evaporate it to dryness
under a stream of nitrogen at 40, reconstitute the residue in 80 |xL MeOH, inject a 30 |xL
aliquot. (Deconjugate urine as follows. 250 |xL Urine + 750 jxL pH 5.4 acetate buffer + 500 U
P-glucuronidase, heat at 37 for 18 h, add 20 |xL 5 M NaOH, centrifuge, proceed as above using
5 mL dichloromethane:diethyl ether.)
HPLCVARIABLES
Guard column: 30 X 4.6 30 ^m C8
Column: 100 X 8 4 |xm Nova Pak C18
Mobile phase: MeCN:40 mM sodium phosphate buffer 32:68 containing 1 mL/L triethylamine,
final pH 7.2
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Internal standard: flurazepam
OTHER SUBSTANCES
Extracted: flumazenil, midazolam, 4-hydroxymidazolam, 1-hydroxymethylmidazolam
Noninterfering: alfentanil, atropine, bupivacaine, lignocaine, neostigmine
KEYWORDS
plasma; flurazepam is IS
REFERENCE
Chan,K; Jones,RD-M. Simultaneous determination of flumazenil, midazolam and metabolites in human bio-
logical fluids by liquid chromatography, J.Chromatogr., 1993, 619, 154-160.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jxg/mL solution in MeOH, inject a 20 u,L aliquot.
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.3
OTHER SUBSTANCES
mine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, haloperidol, hy-
methoprim, trimipramine, tripelennamine, triprolidine, trjrptamine, verapamil, xylometazoline
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
% for 2 min, to 0:100 over 15 min, maintain at 0:100 for 5 min.
Flow rate: 0.5
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: chlordiazepoxide, desalkylflurazepam, diazepam, norchlordiazepoxide, nordiaze-
pam, oxazepam, prazepam
REFERENCE
Rainin Catalog 1991-2, p. 3.26.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.5
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: norchlordiazepoxide, chlordiazepoxide, nordiazepam, desalkylflurazepam, oxa-
zepam, diazepam, prazepam
Also analyzed: amitriptyline, amphetamine, chlorpromazine, desipramine, desmethyldoxepin,
diethylpropion, doxepin, ephedrine, fenfluramine, imipramine, mesoridazine, methampheta-
mine, nortriptyline, phentermine, phenylpropanolamine, promazine, thioridazine, thiothixene,
trifluoperazine
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
cainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphen-
azine, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol,
phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenytoin, pimo-
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1-10 |xg/mL solution in water, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Hypersil SCX/C18
Mobile phase: MeCN:25 mM pH 3 Na2HPO4 50:50
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: amitriptyline, barbital, benzoic acid, butabarbital, clomipramine, clonazepam,
desipramine, diazepam, furosemide, imipramine, nitrazepam, phenobarbital, phenol, phenol-
phthalein, pindolol, propranolol, resorcinol, salicylic acid, secobarbital, terbutaline, xylazine
KEYWORDS
effect of mobile phase pH on capacity factor is discussed
REFERENCE
Walshe,M.; Kelly,M.T.; Smyth,M.R.; Ritchie,H. Retention studies on mixed-mode columns in high-performance
liquid chromatography, J.ChromatogrA, 1995, 708, 31-40.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a solution in mobile phase.
HPLC VARIABLES
Column: Nova-Pak C18
Mobile phase: MeOH.buffer 85:15 (Buffer was 90.7 mL 66.7 mM Na2HPO4 and 9.3 mL 66.7 mM
KH2PO4 made up to 1 L with water, pH 7.6.)
Flow rate: 5 (sic)
Detector: UV (wavelength not given)
CHROMATOGRAM
Limit of detection: 10OnM
OTHER SUBSTANCES
Simultaneous: chlordiazepoxide, diazepam, nitrazepam
KEYWORDS
comparison with capillary electrophoresis; capillary GC; and polarography
REFERENCE
McGrath,G.; McClean,S.; O'Kane,E.; Smyth,W.R; Tagliaro,F. Study of the capillary zone electrophoretic behav-
iour of selected drugs, and its comparison with other analytical techniques for their formulation assay,
J.ChromatogrA, 1996, 735, 237-247.
SAMPLE
Matrix: urine
Sample preparation: 2 mL Urine + 3 mL 500 mM NaOH, vortex for 30 s, add 12 mL diethyl
ether, rotate for 5 min, centrifuge at 2500 rpm for 5 min. Remove the ether layer and evaporate
it to dryness under a stream of nitrogen, reconstitute the residue in 2 mL mobile phase, inject
an aliquot.
HPLCVARIABLES
Column: 100 X 5 5 jxm Waters Radial-Pak C18
Mobile phase: MeOH:200 mM NaCl 65:35
Flow rate: 1.2
Detector: E, Bioanalytical Systems LC4B, glassy carbon working electrode operated in parallel
mode, stainless steel auxiliary electrode, electrode potentials +1.0 V and 0.85 V, Ag/AgCl ref-
erence electrode following a 9.144 m X 0.5 mm i.d. figure eight Teflon tubing UV irradiation
unit maintained at 0-5 with an ice bath
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: midazolam, clonazepam, demoxepam, diazepam, benzophenone
REFERENCE
Selavka,C.M.; Krull,I.S.; Lurie,I.S. Photolytic derivatization for improved LCEC determinations of pharmaceu-
ticals in biological fluids, J.Chromatogr.Scl, 1985, 23, 499-508.
SAMPLE
Matrix: urine
Sample preparation: 500 |xL Urine + N-ethylnordiazepam + chlorpheniramine + 100 (xL buffer,
IxL mobile phase B, with 200 |xL mobile phase C, and with 1.15 mL mobile phase D. Elute
HPLC VARIABLES
Column: A 10 X 2.1 12-20 juum PRP-I spherical poly(styrene-divinylbenzene) (Hamilton); B 10 X
3.2 11 |xm Aminex A-28 (Bio-Rad); C 25 X 3.2 5 jjim C8 (Phenomenex) + 150 X 4.6 5 |xm silica
(Macherey-Nagel)
phoric acid; C MeCN:buffer 40:60 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylam-
CHROMATOGRAM
Next Page
OTHER SUBSTANCES
Extracted: morphine, codeine, hydromorphone, hydrocodone, caffeine, cotinine, benzoylecgonine,
secobarbital, oxazepam, phenobarbital, nordiazepam, diazepam, phenylpropanolamine, phen-
termine, amphetamine, phenmetrazine, lidocaine, ephedrine, pentazocine, methamphetamine,
desipramine, nortriptyline, diphenhydramine, methadone
Interfering: imipramine, amitriptyline
KEYWORDS
column-switching
REFERENCE
in urine by on-line sample cleanup and isocratic multi-column separation, J.Chromatogr., 1989,473, 325-
341.
Flurbiprofen
Merck Index: 4234
Lednicer No.: 1 86
SAMPLE
Matrix: aqueous humor
Sample preparation: 100 |xL Aqueous humor + 500 uX. MeCN + 30 jxL 400 ng/mL (+)-naproxen
in MeOH, mix mechanically for 90 s, centrifuge at 3000 g for 20 min. Remove the supernatant
and dry it under nitrogen at room temperature, dissolve the residue in 50 jxL mobile phase by
swirl-mixing for 1 min, centrifuge at 3000 g for 20 s, reduce volume to 20-30 |xL, inject an
aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diclofenac, indomethacin, meclofenamic acid
Simultaneous: bacitracin, cortisone acetate, diazepam, fluorometholone, hydrocortisone acetate,
imipramine, ketoprofen, ketorolac tromethamine, levobunolol, metipranolol, neomycin, pred-
nisolone acetate, proparacaine, propranolol, salicylic acid, sulfacetamide, suprofen
KEYWORDS
human; rabbit
Previous Page
OTHER SUBSTANCES
Interfering: imipramine, amitriptyline
KEYWORDS
column-switching
REFERENCE
in urine by on-line sample cleanup and isocratic multi-column separation, J.Chromatogr., 1989,473, 325-
341.
Flurbiprofen
Merck Index: 4234
Lednicer No.: 1 86
SAMPLE
Sample preparation: 100 |xL Aqueous humor + 500 uX. MeCN + 30 jxL 400 ng/mL (+)-naproxen
and dry it under nitrogen at room temperature, dissolve the residue in 50 jxL mobile phase by
aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diclofenac, indomethacin, meclofenamic acid
KEYWORDS
human; rabbit
REFERENCE
SAMPLE
Sample preparation: 100 |xL Aqueous humor + 500 (JLL MeCN + 30 |xL 400 ng/mL (+)-naproxen,
mix mechanically for 90 s, centrifuge at 3000 g for 20 min. Remove supernatant and dry it
under nitrogen at room temperature. Dissolve residue in 50 |xL mobile phase by swirl mixing
for 1 min, centrifuge at 3000 g for 20 s. For concentrations of < 20 ng/mL, reduce volume to
20-30 |xL under nitrogen.
HPLC VARIABLES
Mobile phase: 505 mL MeCN containing 0.65 mL triethylamine + 495 mL 1.65% glacial acetic
acid, apparent pH 4.35
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diclofenac
Simultaneous: bacitracin, cortisone, diazepam, fluorometholone, hydrocortisone, imipramine, in-
domethacin, ketoprofen, ketorolac, levobunolol, meclofenamic acid, metipranolol, neomycin,
prednisolone, proraracaine, propranolol, salicylic acid, sulfacetamide, suprofen
scopolamine, timolol, tropicamide.
KEYWORDS
rabbit; human
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: Add 125 jxL MeCN to 50 |xL plasma, mix, centrifuge at 900Og for 10 min,
inject a 10 u,L aliquot of the supernatant.
HPLC VARIABLES
Guard column: 5 \xm C18 (Brownlee)
Column: 250 X 4.6 5 |xm RP C18 (Hibar, Merck, Germany)
Mobile phase: MeCN.waterrphosphoric acid 60:40:0.05
Flow rate: 1.5
CHROMATOGRAM
Retention time: 3.4
KEYWORDS
rat; plasma; pharmacokinetics
REFERENCE
Park,K-M.; Gao,Z.-G.; Kim,C.-K. Assay of flurbiprofen in rat plasma using HPLC with fluorescence detection,
SAMPLE
Matrix: blood
Sample preparation: Mix 500 |xL plasma with 50 jxL 200 |xg/mL IS in MeOH. Add 500 |xL 1 M
HCl, extract with 10 mL dichloromethane. Separate the organic layer and evaporate it under
a stream of nitrogen at 30. Reconstitute the residue with 500 JJLL mobile phase. Inject a 50 |xL
aliquot.
HPLC VARIABLES
Column: 150 X 3.9 10 \xm C18 jjuBondapak
Mobile phase: MeCNiMeOH: 1% pH 5.8 glacial acetic acid 19:19:62
Flow rate: 1.8
Detector: UV 260
CHROMATOGRAM
Internal standard: 2-(2'-chloro-4-biphenyl)propionicacid (7.99)
KEYWORDS
plasma
REFERENCE
Pargal,A.; Kelkar,M.G.; Nayak,P.J. The effect of food on the bioavailability of ibuprofen and flurbiprofen from
sustained release formulations, Biopharm.Drug Dispos., 1996, 17, 511519.
SAMPLE
Matrix: blood
Sample preparation: 500 JiL Plasma + 1 mL water + ibuprofen (15 |xg per 100 |xL) + 2 mL 10%
trichloroacetic acid, mix, add 5 mL hexane, mix for 15 min, centrifuge at 800 g for 5-10 min,
repeat extraction. Combine hexane layers and evaporate them under a stream of nitrogen at
37-40. Reconstitute with 300 |xL chloroform, add 200 jxL 65 mg/mL l,l'-carbonyldiimidazole
in chloroform, let stand 5-10 min at room temperature, add 10 |xL glacial acetic acid, vortex
briefly, let stand 5-10 min at room temperature, add 50 jxL S-(a)-methylbenzylamine, mix
briefly, let stand for 30 min at room temperature, add 3 mL 0.5 M ammonium hydroxide, add
5 mL hexane, mix gently for 15 min. Remove hexane and wash it with 3 mL 1 M HCl, 3 mL
0.5 M ammonium hydroxide, and 3 mL 1 M HCl (with 15 min mixing each time). Evaporate
hexane under a stream of nitrogen at 37, dissolve in 150 JJLL mobile phase, inject 25 |JLL aliquot.
HPLC VARIABLES
Guard column: Brownlee RP18
Column: 250 X 4.6 5 ixm Ultrasphere ODS
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Internal standard: ibuprofen (17 S, 19 R)
OTHER SUBSTANCES
KEYWORDS
plasma; derivatization; chiral
REFERENCE
Knadler,M.R; Hall,S.D. High-performance liquid chromatographic analysis of the enantiomers of flurbiprofen
and its metabolites in plasma and urine, J.Chromatogr., 1989, 494, 173-182.
SAMPLE
Matrix: blood
Sample preparation: 500 (xL Plasma + 1.25 |xg S-Naproxen + 200 JXL 2 M HCl + 6 mL hexane:
diethyl ether 8:2 (ice cold), extract, centrifuge at 1500 g for 5 min. Remove 5 mL of organic
layer and evaporate it to dryness under a stream of nitrogen, redissolve in 500 jxL 30 mM pH
7.5 phosphate buffer, inject 50-100 |xL aliquot
HPLC VARIABLES
Column: 100 X 4 5 jxm Grom AGP
Mobile phase: 2-Propanol:20 mM pH 6.5 phosphate buffer 5:95 containing 1 mM
dimethyloctylamine
Flow rate: 0.8
Detector: UV 246
CHROMATOGRAM
Internal standard: S-naproxen (5.2)
KEYWORDS
plasma; chiral
REFERENCE
Geisslinger,G.; Menzel-Soglowek,S.; Schuster,O.; Brune,K. Stereoselective high-performance liquid chromato-
graphic determination of flurbiprofen in human plasma, J.Chromatogr., 1992, 573, 163167.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 150 |xL 1 M phosphoric acid + 5 mL hexane:ether 80:
20, extract. Remove the organic layer and evaporate it to dryness, reconstitute the residue in
200 (xL mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 4 jxm Nova Pak C18
Mobile phase: MeCN:water:acetic acid 59:40.5:0.5
Flow rate: 1.3
Detector: UV 233
CHROMATOGRAM
Internal standard: flurbiprofen
OTHER SUBSTANCES
KEYWORDS
plasma; flurbiprofen is IS
REFERENCE
al-Meshal,M.A.; El-Sayed,Y.M.; al-Balla,S.R.; Gouda,M.W. The effect of colestipol and cholestyramine on ibu-
profen bioavailability in man, Biopharm.Drug Dispos., 1994, 15, 463-471.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL 1 M HCl + 100 JJIL water + 6 mL etherrhexane 20:
80, shake for 10 min, centrifuge at 900 g for 5 min. Remove 4 mL of the organic layer and
10 mM NaOH, sonicate for 3 min, add 50 |xL 100 mM pH 7.0 phosphate buffer containing 0.1%
dimethyloctylamine, sonicate for 3 min, inject a 5 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 3 5 fxm Chiral-AGP (ChromTech)
Column: 100 X 4 5 [xm Chiral-AGP (ChromTech)
Mobile phase: Gradient. A was 10 mM pH 7.0 phosphate buffer containing 1 mM dimethyl-
octylamine. B was isopropanol:10 mM pH 7.0 phosphate buffer 50:50 containing 1 mM dimeth-
yloctylamine. A:B 99.2:0.8 for 5 min, to 59:41 over 10 min, re-equilibrate for 10 min.
Flow rate: 0.9
Injection volume: 5
Detector: UV 220 for 7 min, then UV 245
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
plasma; chiral; flurbiprofen is IS
REFERENCE
de Vries,J.X.; Schmitz-Kummer,E.; Siemon,D. The analysis of ibuprofen enantiomers in human plasma by high-
performance liquid chromatography on an al-acid glycoprotein chiral stationary phase, J.Liq.Chromatogr.,
1994, 17, 2127-2145.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + + 25 jxL 2 M HCl, vortex for 15 s, add 2 mL isooctane:
isopropanol 85:15, rotate for 5 min, centrifuge at 3000 rpm for 10 min. Remove the upper
residue in 25 |xL 5 mg/mL 5-bromoacetyl acenaphthene in MeCN, add 10 (JLL 3% triethylamine
in MeCN, vortex for 30 s, heat at 75 for 5 min, evaporate to dryness under reduced pressure,
reconstitute with 25 (xL MeCN, inject a 20 (xL aliquot. (Prepare 5-bromoacetyl acenaphthene
as follows. Add 43 g bromoacetyl chloride to 43 g acenaphthene dissolved in 200 mL dichloro-
ethane, cool to -5 in an ice/salt bath, stir vigorously and add 38 g aluminum chloride in small
portions over 90 min, do not allow temperature to go above 3, place under reduced pressure
for 30 min, add an excess of crushed ice. Separate the dichloroethane layer and wash it with
two 100 mL portions of dilute HCl, wash with 100 mL 5% sodium carbonate solution. Dry the
organic layer over anhydrous magnesium sulfate, remove the solvent under reduced pressure,
allow the oily residue to solidify, remove liquid by blotting with filter paper. Purify the solid
by chromatography on a 300 X 20 column of 60-120 mesh silica gel, elute with toluene, un-
reacted acenaphthene elutes first followed by 5-bromoacetyl acenaphthene (mp 87-90).)
HPLC VARIABLES
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
rat; plasma; protect from light; derivatization; flurbiprofen is IS
REFERENCE
Gifford,L.A.; Owusu-Daaku,RT.K.; Stevens,A.J. Acenaphthenefluorescencederivatization reagents for use in
high-performance liquid chromatography, J.Chromatogr.A, 1995, 715, 201212.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 125 |xL 40 mM decanoic acid in MeCN, mix. Dialyze a
100 jxL sample against 20 mM pH 7.0 phosphate buffer using a Gilson Cuprophane membrane
(molecular mass cut-off 15 kDa). Continuously pump the buffer through the dialysis cell and
through column A at 3 mL/min for 9.6 min, backflush the contents of column A onto column B
with the mobile phase, monitor the effluent from column B. (After each injection flush plasma
channel with 1 mL 0.05% Triton X-100, with I m L l mM HCl, and with 2 mL water. After
each injection flush buffer channel with 3 mL 20 mM pH 7.0 phosphate buffer and condition
column A with 1 mL 20 mM pH 7.0 phosphate buffer.)
HPLCVARIABLES
Column: A 10 X 2 40 ujn Bondesil C18 (Analytichem); B 250 X 3.1 5 |xm C18 (RoSiI Research
Separation Laboratories)
Flow rate: 1
Detector: UV 247
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: fenoprofen (UV 272), ibuprofen (UV 264), ketoprofen (UV 261), naproxen (UV 272)
KEYWORDS
plasma; dialysis; column-switching
REFERENCE
Herrez-Hernndez,R.; Van de Merbel,N.C; Brinkman,U.A.T. Determination of the total concentration of
highly protein-bound drugs in plasma by on-line dialysis and column liquid chromatography: application
to non-steroidal anti-inflammatory drugs, J.Chromatogr.B, 1995, 666, 127-137.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 100 (xL 25 |xg/mL indomethacin + 500 |xL 600 mM
sulfuric acid + 15 mL dichloromethane, mix for 20 min, centrifuge at 2000 g for 5 min. Remove
the organic layer and evaporate it to dryness under a stream of nitrogen at room temperature,
reconstitute the residue in 100 jxL 50 mM triethylamine in MeCN + 50 u,L 60 mM ethyl chlo-
roformate in MeCN, vortex for 30 s, add 50 (xL 100 mM L-leucinamide in MeOH:triethylamine
100:14, let stand for 2 min, add 50 jiL water, inject a 10-50 |xL aliquot of the reaction mixture.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Ultrabase C18 (Shandon)
Mobile phase: MeCN:60 mM KH2PO4:triethylamine 49:51:0.1
Flow rate: 1.8
Detector: UV 275
CHROMATOGRAM
Retention time: 3.5 (R-(-)), 4.4 (S-(+))
OTHER SUBSTANCES
Extracted: ibuprofen (UV 225), ketoprofen
KEYWORDS
plasma; chiral; derivatization
REFERENCE
Pehourcq,E; Lagrange,R; Labat,L.; Bannwarth,B- Simultaneous measurement of flurbiprofen, ibuprofen, and
ketoprofen enantiomer concentrations in plasma using L-leucinamide as the chiral coupling component,
J.Liq.Chromatogr., 1995, 18, 3969-3979.
SAMPLE
Matrix: blood
the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |JLL aliquot of
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 247
CHROMATOGRAM
KEYWORDS
whole blood; plasma; interferences may occurcompounds (all of which are extracted) elute in this
amine; pimozide; daunorubicin; indomethacin; maprotiline; tropatenine; etodolac; fiuoxetine;
lidoflazine; ibuprofen; fioctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpi-
REFERENCE
Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J,Forensic ScL, 1995, 40,
254-262.
SAMPLE
Matrix: blood, milk
Sample preparation: 200 (JLL Plasma or milk + 20 (xL 30% perchloric acid + 200 |JLL 2 |xg/mL
IS in MeOH, vortex for 2 min, add 20 |xL 5 M NaOH, centrifuge at 5000 g. Remove a 200 JJLL
aliquot of supernatant and add it to 200 |xL mobile phase, mix, centrifuge, inject a 50 JJLL
aliquot.
HPLC VARIABLES
Guard column: Waters C18 Guard Pak
Column: 100 X 8 10 |xm ixBondapak C8 Radial Pak
Mobile phase: MeCNrMeOH: 1% acetic acid 30:30:40
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 6
Internal standard: 2-(2'-chloro-4-biphenylyl)propionic acid (7)
KEYWORDS
plasma
REFERENCE
Johnson,V.A.; Wilson,J.T. Flurbiprofen analysis in plasma and breast milk by high-performance liquid chro-
SAMPLE
Matrix: blood, synovial fluid
Sample preparation: 0.5 mL Plasma or synovial fluid + 200 JJLL 2 M HCl + 5 mL hexane, tumble
10 min on a rotary mixer, centrifuge at 10 000 g for 5 min. Remove organic layer and evaporate
it to dryness under vacuum centrifugation. Reconstitute residue in 150 |JLL MeOH + 100 fxL
water, vortex mix, inject aliquot.
HPLC VARIABLES
Guard column: 20 X 2 Perisorb RP18 30-40 fjim pellicular
Column: 125 X 4.6 5 jutm Spherisorb ODS 1
Mobile phase: MeOH.water 63:37 adjusted to pH 3.3 with phosphoric acid
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
KEYWORDS
plasma; flurbiprofen is IS
REFERENCE
Blagbrough,I.S.; Daykin,M.M.; Doherty,M.; Pattrick,M.; Shaw,P.N. High-performance liquid chromatographic
determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man, J.Chromatogr.,
1992, 578, 251-257.
SAMPLE
Sample preparation: 100 JULL Serum or urine + 50 jxL 1 M NaOH, let stand at room temperature
for 20 min (to hydrolyse conjugates), add 50 jxL 1 M HCl, add 1 mL 1 jxg/mL IS in MeCN, add
2 mL 50 mM pH 2.6 potassium phosphate buffer, centrifuge at 2000 rpm for 10 min, inject 100
jxL aliquot of supernatant.
HPLC VARIABLES
Guard column: 30 X 4.6 5 |xm Brownlee RP-8
Column: 300 X 3.9 jjuBondapak C18
Mobile phase: THF:50 mM pH 2.6 potassium phosphate 45:55
Flow rate: 1.9
CHROMATOGRAM
Retention time: 10
Internal standard: (RS)-2-(2-methoxy-4-biphenyl)propionic acid (8)
KEYWORDS
serum
REFERENCE
Adams,W.J.; Bothwell,B.E.; Bothwell,W.M.; VanGiessen,G.J.; Kaiser,D.G. Simultaneous determination of flur-
biprofen and its major metabolite in physiological fluids using liquid chromatography with fluorescence
detection, Anal Chem., 1987, 59, 1504-1509.
SAMPLE
Sample preparation: 500 |xL Urine + 100 |xL 1 M NaOH (to hydrolyze conjugates), vortex. 500
|xL Plasma or 600 |xL basified urine + 200 |xL 600 mM sulfuric acid, vortex, add 50 |xL 100 |xg/
mL ketoprofen in water + 3 mL isooctanerisopropanol 96:5, mix vigorously for 45 s, centrifuge
at 3000 rpm for 5 min. Remove the top layer and add it to 3 mL water, mix vigorously, cen-
trifuge for 5 min, discard the organic layer. Add the aqueous layer to 350 jxL 600 mM sulfuric
acid, add 3 mL chloroform, mix, centrifuge for 5 min. Remove the organic layer and evaporate
it to dryness under reduced pressure, reconstitute the residue in 100 |xL 50 mM triethylamine
in MeCN, after 30 s add 50 |xL 60 mM ethylchloroformate in MeCN, after 30 s add 50 |xL 1 M
L-leucinamide, after 2 min add 50 |xL water, inject a 10-50 |iL aliquot.
HPLC VARIABLES
Guard column: 50 mm long 10 |xm Partisil 5 ODS-3
Mobile phase: MeCN:67 mM KH2PO4:triethylamine 35:65:0.02
Flow rate: 1
Detector: UV 250
CHROMATOGRAM
Retention time: 17 (-), 21 (+)
Internal standard: ketoprofen (UV 275) (8 (-), 10 (+))
KEYWORDS
REFERENCE
Berry,B.W.; Jamali,F. Stereospecific high-performance liquid chromatographic (HPLC) assay of flurbiprofen in
biological specimens, Pharm.Res., 1988, 5, 123-125.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Serum. Activate an SPE cartridge filled with 100 mg 40-63 |xm silica gel
(Merck) with 1 mL MeOH and dry in a hot air oven at 100 for 1 h, equilibrate with 1 mL
dichloromethane before use. 500 |xL Serum + 100 fxL 1 M HCl, mix, add 1 mL 1 M pH 3.8
sodium phosphate buffer, mix, add 3 mL diethyl ether, rock for 20 min, centrifuge at 1000 g
for 2 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at
40, reconstitute the residue in 100 |xL 1 mg/mL l-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide hydrochloride in dichloromethane, add 100 |xL 1 mg/mL 1-hydroxybenzotriazole in dich-
loromethane, add 100 |JLL 1 mg/mL (R)-(+)-l-(l-naphthyl)ethylamine in dichloromethane, vortex
briefly, let stand at room temperature for 2 h, add to the SPE cartridge, elute with two 1 mL
portions of dichloromethane:MeCN 90:10. Combine all the eluate and evaporate it to dryness
under a stream of nitrogen at 40, reconstitute the residue in 100 JJLL mobile phase, inject a 50
|JLL aliquot. Urine. Activate an SPE cartridge filled with 100 mg 40-63 |xm silica gel (Merck)
with 1 mL MeOH and dry in a hot air oven at 100 for 1 h, equilibrate with 1 mL dichloro-
methane before use. 500 jxL Urine + 100 |xL 1 M HCl, mix, add 1.5 mL 1 M pH 3.8 sodium
phosphate buffer, mix, add 5 mL hexane:isopropanol 90:10, rock for 20 min, centrifuge at 1000
g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen
at 40, reconstitute the residue in 100 jxL 1 mg/mL l-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide hydrochloride in dichloromethane, add 100 (xL 1 mg/mL 1-hydroxybenzotriazole in
dichloromethane, add 100 JJLL 1 mg/mL (R)-(+)-l-(l-naphthyl)ethylamine in dichloromethane,
vortex briefly, let stand at room temperature for 2 h, add to the SPE cartridge, elute with two
1 mL portions of dichloromethaneiMeCN 90:10. Combine all the eluate and evaporate it to
HPLC VARIABLES
Guard column: 10 X 2.1 40-63 |xm pellicular C18 (Alltech)
Column: 150 X 3.9 5 jxm Resolve C18 (Waters)
Flow rate: 1.5
CHROMATOGRAM
OTHER SUBSTANCES
Interfering: ibuprofen (a metabolite of ibuprofen interferes with the R enantiomer)
KEYWORDS
flurbiprofen is IS; derivatization; chiral; serum; SPE
REFERENCE
Tan,S.C; Jackson,S.H.D.; Swift,C.G.; Hutt,A. J. Enantiospecific analysis of ibuprofen by high performance liquid
chromatography: Determination of free and total drug enantiomer concentrations in serum and urine, Chro-
matographia, 1997, 46, 23-32.
SAMPLE
Matrix: bulk
Sample preparation: 10 mg Compound + 10 mg l-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride + 2 drops 3,5-dimethylaniline + 1.5 mL dichloromethane, mix, after 30 min add
1 mL 1 M HCl, shake vigorously. Remove the lower organic layer and dry it over anhydrous
magnesium sulfate, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm D N-(3,5-dinitrobenzoyl)phenylglycine (Regis)
Flow rate: 2
CHROMATOGRAM
Retention time: k' 2.27 (for first enantiomer)
OTHER SUBSTANCES
Also analyzed: carprofen, cicloprofen, etodolac, fenoprofen, ibuprofen, ketoprofen, naproxen, pir-
profen, tiaprofenic acid
KEYWORDS
derivatization; a = 1.26; chiral
REFERENCE
Pirkle,W.H.; Murray,RG. The separation of the enantiomers of a variety of non-steroidal anti-inflammatory
drugs (NSAIDS) as their anilide derivatives using a chiral stationary phase, J.Liq.Chromatogr., 1990, 13,
2123-2134.
SAMPLE
the supernatant and add it to 200 |xL 200 |xg/mL IS in DMF, mix, add 200 |xL 5 M HCl, extract
mg/mL 1-hydroxybenzotriazole in dichloromethane:pyridine 99:1, add 300 |xL 10 mg/mL l-(3-
mg/mL (-MS)-a-methylbenzylamine in dichloromethane, let stand for 30 min, evaporate to dry-
ness, reconstitute with 500 |xL mobile phase, inject a 10 |xL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: (S)-naproxen (k' 3.15)
KEYWORDS
REFERENCE
1997, 15, 1765-1774.
SAMPLE
Matrix: dialysate
Sample preparation: Inject a 10 JJLL aliquot of dialysate (pH 7.4 isotonic phosphate buffer) con-
taining 250 ng/mL naproxen.
HPLCVARIABLES
Guard column: 37-50 jxm Corasil C18
Flow rate: 1.1
CHROMATOGRAM
Retention time: 4
Internal standard: naproxen (F ex 262 em 356) (2.5)
KEYWORDS
mouse; rat; pharmacokinetics
REFERENCE
Evrard,P.A.; Deridder,G.; Verbeeck,R.K. Intravenous microdialysis in the mouse and the rat: Development and
pharmacokinetic application of a new probe, Pharm.Res., 1996, 13, 12-17.
SAMPLE
Sample preparation: 300 JJLL Microsomal incubation + 20 |xL 6 M HCl, centrifuge at 3000 g for
10 min, inject a 100 |xL aliquot of the supernatant.
HPLCVARIABLES
Column: 250 X 4 5 (xm Lichrospher RP-18
Mobile phase: MeCN:water:trifluoroacetic acid 65:145:0.08
Flow rate: 1.5
Detector: UV 273
CHROMATOGRAM
Retention time: 70
Internal standard: 1-naphthol-p-D-glucuronide
OTHER SUBSTANCES
Extracted: metabolites glucuronides
KEYWORDS
rat; human; liver
REFERENCE
Hamdoune,M.; Mounie,J.; Magdalou,J.; Masmoudi,T.; Goudonnet,H.; Escousse,A. Characterization of the in
vitro glucuronidation of flurbiprofen enantiomers, Drug Metab.Dispos., 1995, 23, 343-348.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 Spherisorb S10-ODS2
Mobile phase: MeCN:water:acetic acid 60:35:0.5
Flow rate: 1
Detector: UV 280
OTHER SUBSTANCES
Simultaneous: mefenamic acid
REFERENCE
Galia,E.; Nicolaides,E.; Horter,D.; Lbbenberg,R.; Reppas,C; Dressman,J.B. Evaluation of various dissolution
media for predicting in vivo performance of class I and II drugs, Pharm.Res., 1998, 15, 698-705.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.5 5 |xm Altex C18
Mobile phase: MeOH:water:acetic acid 67:32.5:0.5
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 8
REFERENCE
J.Pharm.ScL, 1990, 79, 153-157.
SAMPLE
Matrix: solutions
Sample preparation: Mix 100 |xL of a 1-200 |xM solution of carboxylic acid in dichloromethane
with 100 |xL 800 |xM (-)-APMB in dichloromethane, 100 |xL 1.6 mM 2,2'-dipyridyl disulfide in
dichloromethane, and 100 \LL 1.6 mM triphenylphosphine in dichloromethane, let stand at
room temperature for 20 min. Evaporate to dryness under a stream of nitrogen, reconstitute
the residue in 400 jxL mobile phase, inject a 10 |xL aliquot. ((-)-APMB is (-)-2-[4-(l-amino-
ethyl)phenyl]-6-methoxybenzoxazole. Synthesis is as follows. Hydrogenate 5-methoxy-2-nitro-
phenol in EtOH over platinum oxide to give 2-amino-5-methoxyphenol (J. Org. Chem. 1957,
22, 220). It should be possible to prepare ethyl 4-acetylbenzimidate hydrochloride
(CH3COCeH4CC = NH)OC2H5.HC1) by passing dry hydrogen chloride into a mixture of 4-acetyl-
benzonitrile and 1.2-1.5 equivalents EtOH in an inert solvent (e.g., benzene, chloroform, di-
oxane, ether, nitrobenzene (Caution! Benzene, chloroform, and dioxane are carcinogens!) at 0-
5, the benzimidate should crystallize from the mixture in 7-10 days (J. Chem. Soc. 1942, 103).
Add a solution of 5.5 g 2-amino-5-methoxyphenol in 200 mL MeOH to 9 g ethyl 4-acetylben-
zimidate hydrochloride, stir at 60-70 for 4 h, evaporate to dryness under reduced pressure,
recrystallize from EtOH to give 4-(6-methoxy-2-benzoxazolyl)acetophenone as fine orange-yel-
low crystals (mp 167) (J. Chromatogr. 1990, 532, 65). Add 7.0 g hydroxylamine hydrochloride
and 8.2 g sodium acetate to 10.1 g 4-(6-methoxy-2-benzoxazolyl)acetophenone in 500 mL EtOH:
water 95:5, reflux for 1 h, pour into ice-water, filter, recrystallize from EtOH:water 90:10 to
give 4-(6-methoxy-2-benzoxazolyl)acetophenone oxime as faint reddish needles (mp 212). Dis-
solve 4.7 g 4-(6-methoxy-2-benzoxazolyl)acetophenone oxime in 300 mL MeOH, add 3 g 10%
palladium on charcoal, add 10.5 g ammonium formate, reflux for 30 min, filter, evaporate the
filtrate to dryness under reduced pressure. Take up the residue in 100 mL 5% HCl and wash
the aqueous phase with 100 mL ethyl acetate. Adjust the pH of the aqueous layer to 13-14
with 10% NaOH and extract with 200 mL ethyl acetate. Wash the organic layer with 100
mL water and dry it over anhydrous sodium sulfate, evaporate to dryness under reduced pres-
sure to give racemic 2-[4-(l-aminoethyl)phenyl]-6-methoxybenzoxazole. Dissolve 3.6 g racemic
2-[4-(l-aminoethyl)phenyl]-6-methoxybenzoxazole in 50 mL EtOH and add 3.5 g (S)-(-)-a-meth-
oxy-a-trifluoromethylphenylacetic acid, allow to stand overnight at 5. Collect the precipitate
and fractionally crystallize it from EtOH 4 times. Take up the final product in 5% NaOH and
extract it with ethyl acetate, wash the organic layer with water, dry over anhydrous sodium
sulfate, evaporate to dryness under reduced pressure, recrystallize from EtOH to give (-)-2-[4-
(l-aminoethyl)phenyl]-6-methoxybenzoxazole as pale yellow crystals (mp 74).)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm TSK gel ODS-80TM (Tosoh)
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: ibuprofen, naproxen
KEYWORDS
REFERENCE
Kondo,J.; Imaoka,T.; Kawasaki,T.; Nakanishi,A.; Kawahara,Y. Fluorescence derivatization reagent for resolu-
tion of carboxylic enantiomers by high-performance liquid chromatography, J.Chromatogr., 1993, 645, 7 5 -
81.
SAMPLE
Matrix: solutions
Sample preparation: Mix 100 JJLL of a solution of the carboxylic acid in MeCN with 100 JJLL 2
mM (S)-l-(4-dansylaminophenyl)ethylamine in MeCN, add 100 |xL 3 mM 2,2'-dipyridyl disul-
fide (Aldrithiol-2) in MeCN, add 100 |xL 3 mM triphenylphosphine in MeCN, vortex, let stand
at room temperature for 3 h, inject a 5 u,L aliquot. (Synthesis of (S)-l-(4-dansylamino-
phenyl)ethylamine is as follows. Add 2.2 g di-tert-butyl dicarbonate dropwise to a stirred so-
lution of 2 g (S)-a-methyl-4-nitrobenzylamine hydrochloride ((S)-l-(4-nitrophenyl)ethylamine
hydrochloride) and 1.1 g triethylamine in 20 mL MeCN at 0, stir at room temperature for 1
h, evaporate to dryness under reduced pressure. Dissolve the residue in 50 mL ethyl acetate
and wash with 10% aqueous citric acid, wash with water, dry over anhydrous sodium sulfate,
evaporate to dryness under reduced pressure to give (S)-N-tert-butoxycarbonyl-l-(4-nitro-
phenyl)ethylamine as white crystals (mp 86-89). Add 200 mg 5% PdC to a solution of 2 g
(S)-N-tert-butoxycarbonyl-l-(4-nitrophenyl)ethylamine in 40 mL MeOH, stir, hydrogenate at
room temperature for 3 h, filter, evaporate the filtrate to dryness to obtain (S)-N-tert-butoxy-
carbonyl-l-(4-aminophenyl)ethylamine. Stir 1.4 g (S)-N-tert-butoxycarbonyl-l-(4-aminophenyl)
ethylamine in 10 mL MeCN and 50 mL 100 mM pH 9.0 sodium bicarbonate, add a solution of
1.9 g dansyl chloride in 30 mL MeCN dropwise while maintaining the pH of the solution at
9.0 with 1 M NaOH, stir at room temperature for 20 min, stir at 45 for 2 h, cool to room
temperature, extract three times with 30 mL portions of ethyl acetate. Combine the organic
layers and evaporate them to dryness under reduced pressure, recrystallize the residue from
benzene/hexane to obtain (S)-N-tert-butoxycarbonyl-l-(4-dansylaminophenyl)ethylamine as
pale-yellow crystals (mp 97-101) (Caution! Benzene is a carcinogen!). Add 2 mL concentrated
HCl to a solution of 1.5 g (S)-N-tert-butoxycarbonyl-l-(4-dansylaminophenyl)ethylamine in 10
mL MeOH, stir at room temperature for 30 min, evaporate to dryness under reduced pressure.
Dissolve the residue in 30 mL water and adjust the pH to 8.0 with sodium bicarbonate, extract
three times with 30 mL portions of ethyl acetate. Combine the organic layers and evaporate
them to dryness under reduced pressure, recrystallize the residue from EtOH to obtain (S)-I-
(4-dansylaminophenyl)ethylamine as pale-yellow crystals (mp 157-160; [a]D28 -11.1 (c = 0.2 in
MeCN)). (The (R)-enantiomer can be prepared in an exactly analogous fashion.))
HPLC VARIABLES
Column: 150 X 4.6 5 |xm ODS-80TM (Tosoh)
Mobile phase: MeCN:50 mM pH 6.5 sodium acetate buffer 65:35
Flow rate: 1
Injection volume: 5
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: ibuprofen, phenoprofen, 2-phenylpropionic acid, pranoprofen, naproxen
KEYWORDS
REFERENCE
Iwaki,K; Bunrin,T.; Kameda,Y.; Yamazaki,M. Resolution and sensitive detection of carboxylic acid enantiomers
using fluorescent chiral derivatization reagents by high-performance liquid chromatography,
J.Chromatogr.A, 1994, 662, 87-93.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 jjim ChiralpakAD (Daicel)
Mobile phase: Carbon dioxide.MeOH 96:4
Flow rate: 2.5
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: fenoprofen, ibuprofen, ketoprofen, naproxen
KEYWORDS
SFC; 250 bar; chiral
REFERENCE
Kot,A.; Sandra,R; Venema,A. Sub- and supercritical fluid chromatography on packed columns: A versatile tool
for the enantioselective separation of basic and acidic drugs, J.Chromatogr.Sci., 1994, 32, 439-448.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: Ultrasphere ODS
Mobile phase: MeOH:water:acetic acid 67:32.5:0.5
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Simultaneous: flurbiprofen amide
KEYWORDS
rabbit; buffer
REFERENCE
Tang-Liu,D.D.-S.; Richman,J.B.; Weinkam,R.J.; Takruri,H. Effects of four penetration enhancers on corneal
permeability of drugs in vitro, J.Pharm.Sci., 1994, 83, 85-90.
SAMPLE
Matrix: solutions
Sample preparation: Mix 1 mL 100 jjig/mL compound in dichloromethane with 300 |xL 100 |xg/
mL 1-hydroxybenzotriazole in dichloromethane:pyridine 99:1, 300 JJLL 1.1 mg/mL l-ethyl-3-di-
methylaminopropylcarbodiimide hydrochloride in dichloromethane, and 300 u,L 300 u,g/mL
benzylamine in dichloromethane, vortex, let stand at room temperature for 1.5 h, evaporate to
dryness under a stream of nitrogen, reconstitute the residue in 500 u,L MeOH, inject an aliquot.
HPLC VARIABLES
Column: 150 X 4.6 10 |xm EXP BlOl tris(4-methylbenzoate) cellulose on silica (Bio-Rad)
Mobile phase: MeOH:buffer 70:30 (Prepare buffer solution by dissolving 14.05 g sodium per-
chlorate in water, adjust pH to 2.0, make up to 1 L with water.)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: benoxaprofen (MeOH:buffer 80:20), carprofen, fenoprofen, ibuprofen, ketoprofen,
pirprofen, tiaprofenic acid
KEYWORDS
REFERENCE
Van Overbeke,A.; Baeyens,W.; Van den Bossche,W.; Dewaele,C. Separation of 2-arylpropionic acids on a cellu-
lose based chiral stationary phase by RP-HPLC, J.Pharm.Biomed.AnaL, 1994, 12, 901-909.
SAMPLE
Matrix: solutions
Sample preparation: Mix 1 mL 100 jxg/mL compound in dichloromethane with 300 |xL 1 mg/
mL 1-hydroxybenzotriazole in dichloromethane:pyridine 99:1, 300 JJIL 1 mg/mL l-ethyl-3-di-
methylaminopropylcarbodiimide hydrochloride in dichloromethane, and 300 |xL 1-1.5 mg/mL
benzylamine in dichloromethane, vortex, let stand at room temperature for 1.5 h, wash with
1 mL 250 mM HCl, wash with 1 mL water. Remove the organic layer and evaporate it to
dryness under a stream of nitrogen, reconstitute the residue in MeOH, inject an aliquot.
HPLC VARIABLES
Column: 10 |xm EXP BlOl 4-methylbenzoate cellulose on silica gel(Bio-Rad)
Mobile phase: MeOH:50 mM pH 1.5 perchlorate buffer 80:20
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 7, 8
OTHER SUBSTANCES
Simultaneous: ketoprofen
KEYWORDS
REFERENCE
Van Overbeke,A.; Baeyens,W.; Van den Bossche,W.; Dewaele,C. Enantiomeric separation of amide derivatives
of some 2-arylpropionic acids by HPLC on a cellulose-based chiral stationary phase, J.Pharm.Biomed.Anal.,
1994, 12, 911-916.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: IsopropanolrlOO mM KH2PO4:formic acid 54:100:0.1
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: acemetacin; diclofenac; indomethacin; lonazolac; ketoprofen; naproxen; piroxi-
cam; sulindac; tenoxicam
REFERENCE
Baeyens,W.R.G.; Van Der Weken,G.; Van Overbeke,A.; Zhang,Z.D. Preliminary results on the LC-separation of
non-steroidal anti-inflammatory agents in conventional and narrow-bore RP set-ups applying columns with
different internal diameters, Biomed.Chromatogr., 1995, 9, 261-262.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ethidium bromide, ethopropazine, fenoprofen, fentanyl, fiavoxate, fluoxetine, fiuphenazine, flur-
azepam, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatro-
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 100 jxM solution in buffer, inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 column containing riboflavin binding proteins (Prepare as follows. Add ri-
boflavin to saturate protein of egg yolk, homogenize with 3 volumes buffer, centrifuge, add the
supernatant to a 500 X 30 column of DEAE-cellulose (Whatman) equilibrated with buffer, wash
extensively with buffer to remove bound protein, elute riboflavin binding proteins (RFBP) with
buffer containing 200 mM NaCl (RFBP has intense yellow color, absorption at 455 nm). Purify
RFBP on a Sephadex G-100 column with 50 mM pH 7.5 Tris-HCl buffer as eluent, remove the
bound riboflavin by extensive dialysis at pH 3.0. Add 4.5 g N,N-disuccinylimidyl carbonate to
3 g Nucleosil 5NH2 slurried in MeCN, filter, wash with MeCN, wash with 50 mM pH 7.5
phosphate buffer. Suspend 300 mg RFBP in 50 mM phosphate buffer, add the activated silica,
mix gently for 2 h using a rotary evaporator, filter, wash with sterile water, wash with isopro-
panol:water 1:2, pack in a 100 X 4.6 column.) (Buffer was 100 mM pH 5.3 sodium acetate.)
Mobile phase: 50 mM pH 5.5 KH2PO4
Flow rate: 0.8
Detector: UV
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: isradipine, ketoprofen, nimodipine, suprofen
KEYWORDS
chiral; a = 1.13
REFERENCE
Massolini,G.; De Lorenzi,E.; Ponci,M.C; Gandini,C; Caccialanza,G.; Monaco,H-L. Egg yolk riboflavin binding
protein as a new chiral stationary phase in high-performance liquid chromatography, J.ChromatognA, 1995,
704, 55-65.
SAMPLE
Matrix: solutions
Sample preparation: 1 mL 5 mM flurbiprofen in dichloromethane + 300 |xL 1 mg/mL hydroxy-
benzotriazole in dichloromethane:pyridine 99:1 + 300 |xL 11 mg/mL l-ethyl-3-dimethylamino-
propylcarbodiimide in dichloromethane + 300 JXL 3.47 mg/mL 1-naphthylamine (Caution! 1-
Naphthylamine in a carcinogen!) in dichloromethane, vortex, let stand for 1 h, evaporate to
dryness under a stream of nitrogen, reconstitute with 5 mL MeOH, inject an aliquot.
HPLC VARIABLES
Column: 150 X 2.1 Tolycellulose EXP BlOl (tris(4-methylbenzoate)cellulose covalently bonded
to 10 |xm aminopropylsilica)
Mobile phase: MeOHrbuffer 85:15 (Buffer was 14.05 g/L sodium perchlorate adjusted to pH 2.0.)
Flow rate: 0.21
Injection volume: 1
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: fenoprofen, ibuprofen, ketoprofen, tiaprofenic acid
KEYWORDS
derivatization; narrow-bore; chiral; a=2.18; (see Biomed. Chromatogr. 1995; 9; 292)
REFERENCE
Van OverbekeA-; Baeyens,W.; Van Der Weken,G.; Van de VoordeJ.; Dewaele,C. Comparative chromatographic
study on the chiral separation of the 1-naphthylamine derivative of ketoprofen on cellulose-based columns
of different sizes, Biomed.Chromatogr., 1995, 9, 289-290.
SAMPLE
Matrix: urine
by aspiration of air. Evaporate an aliquot of 100 jxL 20 jjig/mL IS in MeOH to dryness at 37.
Add 1 mL urine, vortex, add 250 JJLL 1 M pH 5.0 acetate buffer, vortex. Add 250 IJLL of the
the eluate to dryness under a stream of nitrogen at 37, reconstitute the residue in 200 |JLL
HPLC VARIABLES
Column: 150 X 4.6 5 \xm Inertsil ODS-2
Flow rate: 0.9
Detector: UV 230
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Extracted: diclofenac, ibuprofen, felbinac, fenbufen, ketoprofen, loxoprofen, mefenamic acid, na-
proxen, piroxicam, sulindac
KEYWORDS
SPE
REFERENCE
J.Chromatogr.B, 1997, 692, 375-388.
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 1 mL 6 M HCl, heat at 90 for 30 min, cool to room tem-
perature, add ibuprofen (15 jxg per 100 |xL), add 5 mL dichloroetharie, mix 15 min, centrifuge
at 800 g for 5-10 min. Evaporate organic layer under a stream of nitrogen at 37-40. Recon-
stitute with 300 |xL chloroform, add 200 |xL 65 mg/mL l,l'-carbonyldiimidazole in chloroform,
let stand 5-10 min at room temperature, add 10 (xL glacial acetic acid, vortex briefly, let stand
5-10 min at room temperature, add 50 (xL S-(a)-methylbenzylamine, mix briefly, let stand for
30 min at room temperature, add 3 mL 0.25 M ammonium hydroxide, add 5 mL dichloroethane,
mix gently for 15 min. Remove organic layer and wash it with 3 mL 0.25 M ammonium hy-
droxide and twice with 3 mL 1 M HCl (with 15 min mixing each time). Evaporate organic layer
under a stream of nitrogen at 37, dissolve in 150 JJLL MeCN:water 45:55, inject 20-30 |xL
aliquot.
HPLC VARIABLES
Guard column: Brownlee RP18
Mobile phase: MeCN:50 mM acetic acid 55:45
Flow rate: 1
Detector: F ex 200 em 320 (cut-off) (flurbiprofen), UV 232 (ibuprofen)
CHROMATOGRAM
Internal standard: ibuprofen (24 S, 27 R)
OTHER SUBSTANCES
KEY WORDS
REFERENCE
Knadler,M.R; Hall,S.D. High-performance liquid chromatographic analysis of the enantiomers of flurbiprofen
and its metabolites in plasma and urine, J.Chromatogr., 1989, 494, 173-182.
SAMPLE
Matrix: urine
Sample preparation: Dilute urine 20-fold with 100 mM pH 2.0 phosphate buffer, extract twice
with two volumes of ethyl acetate, centrifuge at 5000 g for 5 min. Combine the organic layers
and evaporate them to dryness under a stream of nitrogen below 30. Reconstitute in 0.2-1 mL
HPLC VARIABLES
Guard column: 4 X 4 5 |xm LiChrospher 100 RP-18
Column: 250 X 4 5 jxm LiChrospher CH-18
Mobile phase: MeOH: 10 mM pH 6.0 phosphate buffer 80:20 containing 2.5 mM cethexonium
bromide (Rinse with 100 mL MeOH:EtOH:water 50:25:25 at the end of the day.)
Flow rate: 1
Detector: UV 273
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: glucuronides, pirprofen
REFERENCE
Liu,H.-R; Leroy,R; Nicolas,A.; Magdalou,J.; Siest,G. Evaluation of a versatile reversed-phase high-performance
liquid chromatographic system using cethexonium bromide as ion-pairing reagent for the analysis of glu-
curonic acid conjugates, J.Chromatogr., 1989, 493, 137-147.
Flutamide
Merck Index: 4242
Lednicer No.: 3 57
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Vigorously mix 500 |xL microsomal incubation with 5 mL dichloro-
methane. Evaporate the dichloromethane layer under a stream of nitrogen at 30. Reconstitute
the residue in 100 |xL MeOH. Inject an aliquot.
HPLCVARIABLES
Column: 300 X 3.9 CP-18 ixBondapak
Mobile phase: Gradient. A was MeOH:water 60:40. B was MeOH. A:B from 100:0 to 60:40 over
40 min, flush column with MeOH for 10 min, re-equilibrate at initial conditions
Flow rate: 1
Detector: UV; Radioactivity (Radiometer FIo-I)
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
radiolabeled
REFERENCE
Shet,M.D.; McPhaul,M.; Fisher,C.W.; Stallings,N.R.; Estabrook,R.W. Metabolism of the antiandrogenic drug
(flutamide) by human CYP1A2, Drug Metab.Dispos., 1997, 25, 1298-1303.
Flutazolam
Molecular formula: C19H18CIFN2O3
Merck Index: 4243
SAMPLE
Matrix: blood
Sample preparation: 500 jxL Serum + 20 ^L 20 (xg/mL IS + 200 JJLL 1 M potassium carbonate
Evaporate the organic phase in a water bath under a stream of nitrogen at 40. Dissolve residue
in 100 |xL mobile phase, inject a 20 |xL aliquot. (Caution! Chloroform is a carcinogen!)
HPLC VARIABLES
Flow rate: 0.65
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: bromazepam, chlordiazepoxide, clonazepam, estazolam, etizolam, haloxazolam, lor-
KEYWORDS
serum
REFERENCE
serum using a new reversed-phase chromatographic column on a 2-(xm porous microspherical silica gel,
J.Chromatogr.B, 1996, 682, 173-178.
Flutoprazepam
Molecular formula: C19H16CIFN2O
Merck Index: 4245
SAMPLE
Matrix: blood
Sample preparation: 200 fiL Plasma + 25 |xL 1 |xg/mL o-chlorodiazepam in MeOH, vortex gen-
tly, extract twice with 1 mL portions of benzene (Caution! Benzene is a carcinogen!) with shak-
ing. Combine the extracts evaporate them to dryness under a stream of nitrogen at 30-35,
reconstitute the residue in 100 JJLL mobile phase, inject a 10-90 |xL aliquot.
HPLCVARIABLES
Mobile phase: MeCNrIO mM KH2PO4 50:50 adjusted to pH 4.5 with orthophosphoric acid
Flow rate: 1.2
Detector: UV 229
CHROMATOGRAM
Retention time: 16
Internal standard: o-chlorodiazepam
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Conti,L; Sarati,S.; Caccia,S. Propranolol does not alter flutoprazepam kinetics and metabolism in the rat,
Eur.J.Drug Metab.Pharmacokinet, 1991, 16, 53-58.
Flutrimazole
Molecular formula: C22H16F2N2
Merck Index: 4247
SAMPLE
Matrix: formulations, tissue
Sample preparation: Formulations. 500 mg Cream + 30 mL EtOH, heat mixture to 50 to melt
it, cool, dilute to 50 mL, filter through a 0.45 |xm filter. Inject a 25 JJLL aliquot of the filtrate.
Tissue. Add 3 mL MeOH to cleaned and cut skin, sonicate for 15 min, inject an aliquot.
HPLC VARIABLES
Guard column: 10 |xm jjuBondapak C18
Column: 10 |xm ixBondapak C18
Mobile phase: MeOH:pH 7.0 phosphate buffer 80:20
Flow rate: 1
Detector: UV 225
CHROMATOGRAM
KEY WORDS
cream; skin
REFERENCE
Ramis,J.; Conte,L.; Segado,X.; Forn,J.; Lauroba,J.; Calpena,A.; Escribano,E.; Domenech,J. Influence of for-
mulation on the in vitro transdermal penetration of flutrimazole, Arzneimittelforschung, 1997, 47, 1139-
1144.
Fluvastatin
Merck Index: 4250
Lednicer No.: 5 105
SAMPLE
Matrix: blood
Sample preparation: Mix 500 fxL plasma with 500 |xL MeCN and 500 |xL pH 6.0 phosphate
buffer, extract with 5 mL MTBE by shaking for 30 min, centrifuge at 1200 g for 5 min, evap-
orate the organic phase under nitrogen. Dissolve the residue in 400 |xL MeCNrwater 40:60,
vortex 3 times for 30 s, inject a 20-300 |xL aliquot.
HPLC VARIABLES
Guard column: 15 X 3.2 7 (xm Brownlee Si
Column: 250 X 4.6 10 |xm Chiralcel OD-R (Daicel Chemical Industries, Japan)
Mobile phase: MeCN:buffer 40:60 (Buffer was pH 2.5 phosphate buffer (I = 0.04).)
Flow rate: 0.5
CHROMATOGRAM
Retention time: 28.0 (3S, 5R), 30.0 (3R, 5S)
KEYWORDS
chiral; plasma
REFERENCE
Toreson,H-; Eriksson,B-M. Determination of fluvastatin enantiomers and the racemate in human blood plasma
by liquid chromatography and fluorimetric detection, J.Chromatogr.A, 1996, 729, 13-18.
SAMPLE
Matrix: blood
Sample preparation: Mix 500 |xL plasma with 100 JJLL 500 nM IS, 500 |xL MeCN, and 500 |xL
pH 6.0 phosphate buffer, extract with 5 mL MTBE by shaking for 30 min, centrifuge at 1200
g for 5 min, evaporate the organic phase under nitrogen. Dissolve the residue in 400 |xL mobile
phase, vortex 3 times for 30 s, inject an aliquot.
HPLC VARIABLES
Guard column: 15 X 3.2 7 |xm Brownlee CN
Column: 150 X 4.6 5 |xm Zorbax Rx-C8
Mobile phase: MeOH:pH 6.0 phosphate buffer: 100 mM tetrabutylammonium fluoride 60:25:15
Flow rate: 1
Detector: F ex 305 em 390 following post-column photolysis. The column effluent flowed through
a knitted 1OmX 0.3 mm PTFE coil irradiated at 254 nm to the detector.
CHROMATOGRAM
Internal standard: Sandoz compound 63-267 ([R,S,-(E)-] ()-7-(3-(4-fluorophenyl)-l-(l-methyl-
ethyl)-lH-indol-2yl)-(3,5-dihydroxy-6-methyl)-6-heptenoicacid, monosodium salt) (20.5)
Limit of quantitation: 500 pM
OTHER SUBSTANCES
KEY WORDS
plasma; post-column reaction; post-column photochemical derivatization
REFERENCE
Toreson,H.; Eriksson,B--M. Determination of fluvastatin enantiomers and the racemate in human blood plasma
by liquid chromatography and fluorimetric detection, J.Chromatogr.A, 1996, 729, 13-18.
Fluvoxamine
Merck Index: 4251
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 JJLL 900 ng/mL IS + 4 mL 300 mM trisodium phos-
phate, extract with 400 JJLL diisopropyl ether for 20 min, centrifuge. Evaporate upper organic
layer to dryness under a stream of nitrogen, dissolve residue in 100 |xL MeCN , inject a 50 |xL
aliquot. (Caution! Diisopropyl ether readily forms explosive peroxides!)
HPLC VARIABLES
Column: 150 X 4.6 3 |xm Supelcosil LC-SI
Mobile phase: MeCN:MeOH:concentrated ammonia 87.5:12.0:0.5
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 2.1
Internal standard: 5-(pyrrolidinylpropyliden)-10,1 l-dihydro-5H-dibenzo[a,d]cyclohepten (4.1)
OTHER SUBSTANCES
Noninterfering: amitriptyline, citalopram, clomipramine, desmethylclomipramine, imipramine,
nortriptyline, risperidone
KEYWORDS
REFERENCE
Carrillo,J.A.; Dahl,M.-L.; Svensson,J.-O.; Alm,C; Rodriguez,L; Bertilsson,L. Disposition offluvoxaminein hu-
mans is determined by the polymorphic CYP2D6 and also by the CYP1A2 activity, Clin.Pharmacol.Ther.,
1996, 60, 183-190.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 20 JJLL 10 |xg/mL clovoxamine + 120 |xL 2 M NaOH + 4
mL heptaneiisopropanol 98:2, shake for 30 min, centrifuge at 3000 g for 10 min. Remove the
organic layer and add it to 100 |xL 100 mM HCl, shake for 20 min, centrifuge at 3000 g for 10
min, inject a 20 jxL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 120 X 4.6 5 ^m Nucleosil C8
Mobile phase: MeCN:buffer 36:64 (Buffer was 16 mM KH2PO4 adjusted to pH 2.5 with concen-
Flow rate: 1
Detector: UV 215
CHROMATOGRAM
Retention time: 4.3
OTHER SUBSTANCES
Simultaneous: amitriptyline, chlorimipramine, desipramine, doxepin, imipramine, nortripty-
line, trimipramine
KEYWORDS
plasma
REFERENCE
Foglia,J.R; Birder,L.A.; Perel,J.M. Determination of fluvoxamine in human plasma by high-performance liquid
chromatography with ultraviolet detection, J.Chromatogr., 1989, 495, 295-302.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 200 |xL 1 |xg/mL metapramine in MeOH + 2 mL 1 M pH
10.0 phosphate buffer + 6 mL diethyl ether:hexane 50:50, shake for 15 min, centrifuge at 4000
g for 5 min. Remove the organic layer and add it to 2 mL 62.5 mM sulfuric acid, vortex for 5
min, centrifuge at 4000 g for 5 min. Remove the aqueous phase and add it to 1 mL 500 mM
NaOH, vortex, add 6 mL hexane:diethyl ether 50:50, shake for 10 min, centrifuge at 4000 g
50, reconstitute the residue in 100 mM sodium carbonate, add 10 (JLL 10 mg/mL dansyl chloride
in acetone, vortex for 1 min, heat at 45 for 30 min, evaporate under a stream of nitrogen at
50. Reconstitute the residue in 200 |xL MeCN:water 45:55, inject a 100 jxL aliquot.
HPLC VARIABLES
Mobile phase: Gradient. MeCN:water from 45:55 to 65:35 over 10 min, maintain at 65:35 for 20
min
Flow rate: 1.5
Detector: F (Fluorichrom ex 7.54 and 7.60 filters, em 3.71 and 4.76 filters
CHROMATOGRAM
Retention time: 19
Internal standard: metapramine (28)
OTHER SUBSTANCES
Noninterfering: alimemazine, alprazolam, amineptine, amitriptyline, caffeine, clobazam, clom-
ipramine, clorazepate, cyamemazine, diazepam, demethyldiazepam, flunitrazepam, levome-
promazine, loprazolam, lorazepam, meprobamate, nitrazepam, oxazepam, triazolam, viloxazine
KEYWORDS
plasma; protect from light; derivatization
REFERENCE
Pommery,J.; Lhermitte,M. High performance liquid chromatographic determination of fluvoxamine in human
plasma, Biomed.Chromatogr., 1989, 3, 177-179.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 500 JULL 2 M sodium bicarbonate + 100 yiL 0.1 or 1 |xg/mL
clovoxamine in MeOH + 10 mL hexane, shake horizontally for 20 min, centrifuge at 2000 g for
10 min. Remove the upper organic layer and evaporate it to dryness under a stream of nitrogen
at 35, reconstitute the residue in 200 JJLL mobile phase, inject a 50 (xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 5 \xm Resolve spherical silica (Waters)
Mobile phase: MeOH:MeCN:THF:water:diethylamine 98.59:1:0.2:0.2:0.01
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 4.7
OTHER SUBSTANCES
Simultaneous: alimemazine, amitriptyline, chlorpromazine, clomipramine, desipramine, fluox-
etine, haloperidol, imipramine, levomepromazine, norfluoxetine, nortriptyline, propericiazine,
trimipramine, viloxazine
Noninterfering: metabolites, amineptine, buspirone, clobazam, clonazepam, clorazepate, diaze-
pam, flunitrazepam, lorazepam, nitrazepam, oxazepam
Interfering: imipramine
KEYWORDS
plasma; human; rat; normal phase; pharmacokinetics
REFERENCE
Van Der Meersch-Mougeot,V.; Diquet,B. Sensitive one-step extraction procedure for column liquid chromato-
graphic determination of fluvoxamine in human and rat plasma, J.Chromatogr., 1991, 567, 441-449.
SAMPLE
Matrix: blood
Sample preparation: Add 10 u,L 20 |xg/mL oxaprotiline in MeOH to 990 |xL plasma or serum.
Inject 100 |JLL plasma or serum onto column A with mobile phase A and elute to waste, after
15 min elute column A onto column B with mobile phase B for 2 min. Remove column A from
circuit and re-equilibrate it with mobile phase A for 5 min. Chromatograph on column B with
mobile phase B.
HPLC VARIABLES
Column: A 20 X 4.6 10 |xm Hypersil MOS C8; B 20 X 4.6 5 |xm Hypersil CPS CN + 250 X 4.6
5 |xm Nucleosil 100 CN
Mobile phase: A MeOH:water 5:95; B MeOHrMeCN: 10 mM pH 6.8 potassium phosphate buffer
188:578:235
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 6.2
Internal standard: oxaprotiline (9.5)
OTHER SUBSTANCES
Simultaneous: metoclopramide, doxepin, amitriptyline, clomipramine, fluoxetine, imipramine,
norfluoxetine, nortriptyline, desipramine, maprotiline
Noninterfering: haloperidol, spiroperidol, pimozide, fluspirilene, trifluperidol, perazine, chlor-
diazepoxide, clobazam, diazepam, nordiazepam, flurazepam, lorazepam, nitrazepam, oxaze-
pam, carbamazepine
Interfering: clozapine
KEYWORDS
REFERENCE
Hartter,S.; Wetzel,H.; Hiemke,C. Automated determination offluvoxaminein plasma by column-switching high-
performance liquid chromatography, Clin.Chem., 1992, 38, 2082-2086.
SAMPLE
Matrix: blood
Sample preparation: For each 1 mL plasma or serum add 10 |xL 14 |xg/mL trimipramine in
MeOH. Inject serum or plasma directly onto column A with mobile phase A, elute with mobile
phase A to waste. After 15 min elute column A onto column B (foreflush) with mobile phase B.
After 2 min remove column A from the circuit, elute column B with mobile phase B, monitor
the effluent from column B. Re-equilibrate column A with mobile phase A.
HPLCVARIABLES
Column: A 20 X 4.6 10 ^m Hypersil MOS C8; B 20 X 4.6 5 |xm Hypersil CPS CN + 250 X 4.6
5 fjum Nucleosil 100 CN
Mobile phase: A MeOH:water 5:95; B MeCN:MeOH:buffer 578:188:235 (Buffer was 10 mM
K2HPO4 adjusted to pH 6.8 with 85% phosphoric acid.)
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Internal standard: trimipramine (6.5)
Limit of detection: 1 ng/mL (with three injections onto column A before switching), 5-10 ng/mL
OTHERSUBSTANCES
Extracted: metabolites, amitriptyline, clomipramine, desipramine, doxepin, imipramine, mapro-
tiline, nortriptyline
Noninterfering: chlordiazepoxide, clobazam, clozapine, diazepam, flurazepam, fluspirilene, hal-
operidol, nitrazepam, oxazepam, perazine, pimozide, spiroperidol, trifluperidol
KEYWORDS
REFERENCE
Hartter,S.; Hiemke,C. Column switching and high-performance liquid chromatography in the analysis of am-
itriptyline, nortriptyline and hydroxylated metabolites in human plasma or serum, J.Chromatogr., 1992,
578, 273-282.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 10 |xL 100 |xg/mL nortriptyline in water + 100 uX. 1 M
pH 7.6 K2HPO4, vortex for 5 s, add 6 mL ethyl acetate, vortex for 1 min, centrifuge at 1900 g
45, reconstitute the residue in 250 |xL 40 mM sodium bicarbonate, add 20 uL 10 mg/mL dansyl
chloride in acetone, add 750 |xL acetone, vortex for 30 s, let stand at room temperature at 22
for 15 min. Evaporate under a stream of nitrogen at 45, reconstitute in 1 mL mobile phase,
vortex for 1 min, centrifuge at 1900 g for 10 min, inject a 100 |xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 15 X 3.2 7 fxm NewGuard RP-8 (Brownlee)
Column: 250 X 4.6 5 |xm Supelcosil LC-18-DB
Mobile phase: MeCN: 10 mM pH 7.2 potassium phosphate 85:15
Flow rate: 1.5
Detector: F (wavelengths not given)
CHROMATOGRAM
Retention time: 5.3
Internal standard: nortriptyline (9.7)
OTHER SUBSTANCES
Simultaneous: desipramine
KEYWORDS
REFERENCE
Pullen,RH.; Fatmi,A.A. Determination of fluvoxamine in human plasma by high-performance liquid chroma-
tography with fluorescence detection, J.Chromatogr., 1992, 574, 101-107.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 0.6 M pH 9.8 carbonate buffer + 40 \xh 5 (xg/mL
layer at 45 in a vacuum centrifuge for 1 h. Take up residue in 50 |JLL 1 M pH 10.3 carbonate
ature for 45 min, evaporate at 45 in a vacuum centrifuge for 20 min, reconstitute in 125 |xL
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Supelcosil LC-18
Mobile phase: MeCN:25 mM KH2PO4 75:25 + 500 |xL/L orthophosphoric acid + 600 jxL/L n-
butylamine
Flow rate: 2
Detector: F ex 235 em 470 (cut-ofD
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: fluoxetine, propranolol, clovoxamine, fenfluramine, amoxapine, desipramine, pro-
triptyline, nortriptyline, sertraline, norfluoxetine
loxapine
KEYWORDS
plasma
REFERENCE
Suckow,R-F.; Zhang,M.R; Cooper,T.B. Sensitive and selective liquid-chromatographic assay of fluoxetine and
norfluoxetine in plasma with fluorescence detection after precolumn derivatization, Clin.Chem., 1992, 38,
1756-1761.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL BondElut C18 SPE cartridge with 1 mL 1 M HCl, 1 mL
MeOH, 1 mL water, and 1 mL 1% potassium carbonate. 700 |xL Serum + 50 |xL 5 |xg/mL
trimipramine in 5% potassium bicarbonate + 700 |xL MeCN, vortex, centrifuge at 1500 g for 5
min, add supernatant to SPE cartridge (at ca. 1 mL/min). Wash with 2 mL water and 1 mL
MeCN, elute with 250 (JLL MeOH:35% perchloric acid 20:1 by gravity (10 min) then centrifuge
for 20 s to remove rest of eluant, inject a 50 jxL aliquot of the eluate.
HPLC VARIABLES
Guard column: 15 mm 7 |xm Brownlee RP-8
Mobile phase: MeCNiwater 37.5:62.5 containing 0.5 g/L tetramethylammonium perchlorate and
0.5 mL/L 7% perchloric acid
Flow rate: 1.5
Detector: UV 215
CHROMATOGRAM
Retention time: 6.8
OTHER SUBSTANCES
Extracted: amitriptyline, clomipramine, doxepin, fluoxetine, maprotiline, nortriptyline
Interfering: desmethylmaprotiline, desipramine, imipramine, protriptyline
KEYWORDS
serum; SPE
REFERENCE
Gupta,R-N. An improved solid phase extraction procedure for the determination of antidepressants in serum
by column liquid chromatography, J.Liq.Chromatogr., 1993,16, 2751-2765.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Serum + 5 mL 300 mM sodium phosphate + 400 |xL diisopropyl
ether (Caution! Diisopropyl ether readily forms explosive peroxides!) + imipramine, mix for 20
min, centrifuge for 10 min, inject an aliquot of the organic layer.
HPLCVARIABLES
Column: 150 X 4.6 3 |xm Apex Silica (Jones Chromatography)
Mobile phase: MeCN:MeOH:25% ammonia 345:65:1.7
Flow rate: 1.3
Detector: UV 254
CHROMATOGRAM
Retention time: 3.5
Internal standard: imipramine
KEYWORDS
serum; normal phase; pharmacokinetics
REFERENCE
Spigset,O.; Carleborg,L.; Hedenmalm,K; Dahlqvist,R- Effect of cigarette smoking on fluvoxamine pharmaco-
kinetics in humans, Clin.Pharmacol.Ther., 1995, 58, 399-403.
SAMPLE
Matrix: blood
the residue in 100 jxL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 253
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + oxprotiline, make alkaline with borate buffer, extract with
cyclohexane:dichloromethane 60:40. Remove the organic layer and extract it with acid, inject
an aliquot of the acid layer.
HPLC VARIABLES
Column: C18 DB (Supelco)
Mobile phase: MeCN:pH 2.5 phosphate buffer 37:63
Detector: UV 254
CHROMATOGRAM
Internal standard: oxprotiline
OTHER SUBSTANCES
Extracted: bromazepam, clobazam, diazepam, lorazepam, oxazepam
KEYWORDS
serum
REFERENCE
Vandenberghe,H-; MacDonald,J.C. Analysis of fluvoxamine, clobazam and other benzodiazepines on the same
HPLC system (Abstract 40), Ther.Drug Monit., 1995, 17, 393-393.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Pulverize a tablet, add 100 mL MeOH, shake mechanically for 5 min,
centrifuge an aliquot at 3000 rpm for 5 min. Remove a 50 |xL aliquot of the supernatant and
add it to 50 JJLL 100 |xg/mL propyl paraben in MeOH, make up to 1 mL with MeCN, inject a
20 |xL aliquot.
HPLC VARIABLES
Column: 100 X 8 10 |xm ixBondapack C18
Mobile phase: MeCN:50 mM ammonium acetate 60:40 adjusted to pH 5.2 with glacial acetic
acid
Flow rate: 0.8
Detector: UV 273
CHROMATOGRAM
Retention time: 9
Internal standard: propyl paraben (7)
KEYWORDS
tablets
REFERENCE
Foda,N.H. Quantitative analysis of fluvoxamine maleate in tablet formulations by HPLC, J.Liq.Chromatogr.,
1995, 18, 1591-1601.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flur-
azepam, flurbiprofen, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatro-
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Sample preparation: 8 mL Urine + 2 mL 100 mM pH 9.5 borate buffer, mix, filter (0.2 |xm).
Remove a 500 |xL aliquot of the filtrate and add it to 100 IJLL 10 mM NaCN in 20 mM pH 9.5
borate buffer, add 500 jxL 1 mM naphthalene-2,3-dicarboxaldehyde in MeOH, mix, let stand
at room temperature for 20 min, add 100 |xL 100 mM glycine in 20 mM pH 9.5 borate buffer,
mix, let stand for 10 min, add 500 jxL hexane:toluene 50:50, extract. Remove a 400 |xL aliquot
of the organic layer and add it to 800 |xL dichloromethane, mix, inject a 35 |xL aliquot.
HPLC VARIABLES
Column: 250 X 3.1 5 |xm LiChrosorb Si-60
Mobile phase: Dichloromethane:MeOH 99.8:0.2
Flow rate: 0.5
Next Page
Detector: Chemiluminescence (418 nm cutoff filter) following post-column reaction. The column
effluent mixed with 50 mM hydrogen peroxide in MeCN:dichloromethane 50:50 containing 0.5
mM triethylamine pumped at 0.1 mL/min and with 5 mM bis(2,4,6-trichlorophenyl) oxalate in
dichloromethane pumped at 0.1 mL/min and the mixture flowed into the detector.
CHROMATOGRAM
Retention time: 7
KEYWORDS
REFERENCE
Kwakman,RJ.M.; Koelewijn,H.; Kool,L; Brinkman,U.A.T.; de Jong,G.J. Naphthalene- and anthracene-2,3-di-
aldehyde as precolumn labelling reagents for primary amines using reversed- and normal-phase liquid
chromatography with peroxyoxalate chemiluminescence detection, J.Chromatogr., 1990, 511, 155-166.
Folic acid
Merck Index: 4253
SAMPLE
Matrix: blood, formulations, urine
Sample preparation: Tablets. Powder tablets, dissolve in water, inject a 10 |xL aliquot. Injec-
tions. Dilute with water, inject a 10 jxL aliquot. Plasma, urine. Condition a Lichrolut RP-18
(Merck) SPE cartridge with 3 mL MeOH and 3 mL water. Mix 40 JJLL plasma or 100 |xL urine
with twice the volume of MeCN for 2 min, add 100 |JLL water, centrifuge at 3500 rpm for 15
min, evaporate the supernatant under nitrogen at 45 to remove the organic solvents, add
slowly to the SPE cartridge, collect the eluate. Evaporate to dryness under a stream of nitrogen
at 45. Reconstitute the residue with 500 |xL MeOH containing 4.2 (xg/mL IS. Inject a 10 |xL
aliquot.
HPLC VARIABLES
Column: 250 X 4 5 jxm Lichrosorb RP-18
Mobile phase: Gradient. A was MeOH. B was 50 mM ammonium acetate. A:B from 5:95 to 15:
85 over 6 min, to 30:70 over 7 min, maintain at 30:70 over 7 min
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Internal standard: xanthine (4.65)
OTHER SUBSTANCES
Extracted: ascorbic acid, niacin, niacinamide, riboflavin, vitamin B12
KEYWORDS
plasma; SPE; tablets; injections
REFERENCE
Papadoyannis,I.N.; Tsioni,G.K.; Samanidou,V.E Simultaneous determination of nine water and fat soluble vi-
tamins after SPE separation and RP-HPLC analysis in pharmaceutical preparations and biological fluids,
Previous Page
Detector: Chemiluminescence (418 nm cutoff filter) following post-column reaction. The column
effluent mixed with 50 mM hydrogen peroxide in MeCN:dichloromethane 50:50 containing 0.5
mM triethylamine pumped at 0.1 mL/min and with 5 mM bis(2,4,6-trichlorophenyl) oxalate in
dichloromethane pumped at 0.1 mL/min and the mixture flowed into the detector.
CHROMATOGRAM
Retention time: 7
KEYWORDS
REFERENCE
Kwakman,RJ.M.; Koelewijn,H.; Kool,L; Brinkman,U.A.T.; de Jong,G.J. Naphthalene- and anthracene-2,3-di-
aldehyde as precolumn labelling reagents for primary amines using reversed- and normal-phase liquid
chromatography with peroxyoxalate chemiluminescence detection, J.Chromatogr., 1990, 511, 155-166.
Folic acid
Merck Index: 4253
SAMPLE
tions. Dilute with water, inject a 10 jxL aliquot. Plasma, urine. Condition a Lichrolut RP-18
(Merck) SPE cartridge with 3 mL MeOH and 3 mL water. Mix 40 JJLL plasma or 100 |xL urine
with twice the volume of MeCN for 2 min, add 100 |JLL water, centrifuge at 3500 rpm for 15
at 45. Reconstitute the residue with 500 |xL MeOH containing 4.2 (xg/mL IS. Inject a 10 |xL
aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ascorbic acid, niacin, niacinamide, riboflavin, vitamin B12
KEYWORDS
REFERENCE
Papadoyannis,I.N.; Tsioni,G.K.; Samanidou,V.E Simultaneous determination of nine water and fat soluble vi-
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Bulk. Dilute 32 mg bulk drug in 100 mL IS solution, mix well. Remove a
4 mL aliquot and make it up to 100 mL with IS solution, inject an aliquot. Tablets. Powder
tablets, weigh out amount equivalent to 300 jxg folic acid, add 25 mL IS solution, flush tube
with nitrogen, shake vigorously for 5 min, filter (Millipore type HA, 0.45 (xm), flush filtrate
container with nitrogen, inject an aliquot. Capsules. Weigh out amount of capsule filling equiv-
alent to 300 |xg folic acid, add 30 mL hexane, shake vigorously for 5 min, centrifuge at 1070 g
for 15 min. Remove the hexane and dry the residue at 60. Add 25 mL IS solution to the dry
residue, flush tube with nitrogen, shake vigorously for 5 min, filter (Millipore type HA, 0.45
ixm), flush filtrate container with nitrogen, inject an aliquot. (IS solution was 40 mg methyl
paraben, 240 mL MeOH, 650 mL water, 12 mL 40% tetrabutylammonium hydroxide in water,
2.04 g KH2PO4, and 30 mL 100 mg/mL pentetic acid in 750 mM ammonium hydroxide made
up to 1 L with water.)
HPLC VARIABLES
Mobile phase: MeOH.buffer 24:76 adjusted to pH 7.0 with phosphoric acid and ammonium hy-
droxide (Buffer was 7.5 mL 40% tetrabutylammonium hydroxide in water, 2.04 g KH2PO4, and
7 mL 1 M phosphoric acid in 760 mL water.)
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Retention time: 12
Internal standard: methyl paraben (17)
OTHER SUBSTANCES
Simultaneous: degradation products, p-aminobenzoic acid
Noninterfering: vitamins A, B6, B12, C, D, E, niacin, thiamine, pantothenic acid
KEYWORDS
protect from light; capsules; tablets
REFERENCE
Tafolla,W.H.; Sarapu,A.C.; Dukes,G.R. Rapid and specific high-pressure liquid chromatographic assay for folk
acid in multivitamin-mineral pharmaceutical preparations, J.Pharm.Sci., 1981, 70, 1273-1276.
SAMPLE
Matrix: formula, milk
Sample preparation: Mix 8.0 g powdered infant milk with 10 mL water to it. Mix the diluted
powder or 10.5 g liquid infant milk with 1 g solid trichloroacetic acid, shake thoroughly with
magnetic stirring for 10 min, centrifuge at 1250 g for 10 min, add 3 mL 4% trichloroacetic acid
to the solid residue, mix thoroughly for 10 min, centrifuge, discard the solid phase. Combine
the two acid extracts and make up to 10 mL with 4% trichloroacetic acid, filter (0.45 |xm), inject
an aliquot of the filtrate.
HPLC VARIABLES
Guard column: 5 |xm Tracer Spherisorb ODS 2 C18 (Teknokroma, Spain)
Column: 250 X 4.6 5 jxm Tracer Spherisorb ODS 2 C18 (Teknokroma, Spain)
Mobile phase: MeOH:buffer 15:85 (Buffer was 5 mM octanesulfonic acid and 0.5% triethylamine,
pH 3.6.)
Flow rate: 1
Detector: UV 261 for 6 min, UV 287 for 2 min, UV 290 for 5 min, UV 282 for 3 min, UV 268 for
3.5 min, UV 361 for 20.5 min, UV 246 for 20 min
CHROMATOGRAM
Retention time: 13
Limit of quantitation: ^300 ng/mL
OTHER SUBSTANCES
Extracted: thiamine, riboflavin, pyridoxine, vitamin B12, niacinamide, pyridoxal, pyridoxamine
REFERENCE
Albala-Hurtado,S.; Veciana-Nogues,M.; Izquierdo-Pulido,M.; Marin6-Font,A. Determination of water-soluble vi-
tamins in infant milk by high-performance liquid chromatography, J.Chromatogr.A, 1997, 778, 247-253.
SAMPLE
Sample preparation: Dilute injections with water, inject a 50 JJLL aliquot. Dissolve tablets or
capsule contents in water (warm if necessary), filter (0.5 (xm PTFE), inject a 50 JJLL aliquot of
the filtrate. (Dissolve tablets or other formulations containing proteinaceous material in water
at 60, add 5% trichloroacetic acid (to pH 4.4), filter, inject a 50 jxL aliquot.)
HPLC VARIABLES
Guard column: pellicular Corasil
Column: 10 |xm u-Bondapak C18
Mobile phase: Gradient. A was prepared by dissolving 1 g sodium dioctylsulfosuccinate in 170
mL MeOH, add 10 mL concentrated formic acid, make up to 800 mL with water, adjust pH to
2.5 with 1 M KOH, make up to 1 L. B was prepared by dissolving 1 g sodium dioctylsulfo-
succinate in 450 mL MeOH, add 10 mL concentrated formic acid, make up to 800 mL with
water, adjust pH to 4.6, make up to 1 L. A:B 100:0 for 19 min then 0:100 (step gradient) or A:
B from 100:0 to 0:100 over 25 min (concave curve 9), maintain at 0:100 for 3 min, return to
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Retention time: 7 (step gradient), 8 (curve gradient)
OTHER SUBSTANCES
Simultaneous: niacin (UV 254), niacinamide (UV 254), pyridoxamine, thiamine (UV 254), ribo-
flavin (UV 254), pyridoxine, ascorbic acid
KEY WORDS
injections; capsules; tablets
REFERENCE
Woollard,D.C. New ion-pair reagent for the high-performance liquid chromatographic separation of B-group
vitamins in Pharmaceuticals, J.Chromatogr., 1984, 301, 470-476.
SAMPLE
HPLC VARIABLES
Column: 100 X 4 3 ^m Hypersil BDS-C18
Mobile phase: Gradient. MeCN:water adjusted to pH 2.1 from 0.3:99.7 to 25:75 over 11 min
Flow rate: 0.5
Detector: UV 220
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Simultaneous: biotin, caffeine, citric acid, niacinamide, niacin, pantothenic acid, riboflavin, sac-
charin, thiamine, pyridoxine, vitamin B12, ascorbic acid
KEYWORDS
tablets
REFERENCE
Hewlett Packard Leaflet 12-5091-7351 EUS, 1993.
SAMPLE
Sample preparation: Dilute liquid multivitamin formulations, filter (0.45 fxm), inject an aliquot.
HPLC VARIABLES
Mobile phase: Gradient. A was 10 mM KH2PO4 containing 5 mM sodium hexanesulfonate ad-
justed to pH 2.8 with phosphoric acid. B was MeOH. A:B from 90:10 to 71.8:28.2 over 4 min,
maintain at 71.8:28.2 for 1.5 min, to 50:50 over 6.5 min, maintain at 50:50 for 5 min, return
to initial conditions over 5 min
Flow rate: 1
Injection volume: 5
Detector: UV 272
CHROMATOGRAM
Internal standard: theobromine (8)
OTHER SUBSTANCES
Simultaneous: niacin, niacinamide, thiamine, riboflavin, pyridoxine (UV 290)
KEYWORDS
liquid multivitamins; degas solutions with helium; protect from light
REFERENCE
Blanco,D.; Snchez,L.A.; Guti6rrez,M.D. Determination of water soluble vitamins by liquid chromatography
with ordinary and narrow-bore columns, J.Liq.Chromatogr., 1994, 17, 1525-1539.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 30 X 4.6 10 ixm octadecylsilica (Brownlee)
Mobile phase: Gradient. MeCN:33 mM pH 2.3 sodium phosphate buffer from 7.2:92.8 to 11.3:
88.7 over 15 min. (At the end of each day flush system with water then 50-75 mL MeOH. Place
a column of 37-53 |xm silica (Whatman) between pump and injection valve.)
Flow rate: 1
Detector: F ex 365 em >415 (filter) following post-column reaction. The column effluent mixed
with the reagent pumped at 0.23 mL/min and the mixture flowed through a 5 m X 0.8 mm ID
coil of PTFE tubing at 60 to the detector. (Reagent was 0.005% calcium hypochlorite (HTH
dry chlorine, Olin, Overland KS) in 100 mM K2HPO4 containing 200 mM NaCl.)
CHROMATOGRAM
Retention time: 22
KEYWORDS
REFERENCE
Gregory,J.F.,Ill; Sartain,D.B.; Day,BRF. Fluorometric determination of folacin in biological materials using
high performance liquid chromatography, J.Nutr., 1984, 114, 341-353.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 3.3
OTHER SUBSTANCES
Simultaneous: biotin, niacin, pantothenic acid, riboflavin, niacinamide
REFERENCE
MetaCkem Catalog, 1995, p. 21.
SAMPLE
Matrix: tissue
HPLC VARIABLES
Guard column: 20 X 2 pellicular C18
Column: Econosphere C18
Mobile phase: Gradient. A was 16 mM sodium phosphate and 4 mM tetrabutylammonium phos-
phate, pH 6.0. B was MeCN: 16 mM sodium phosphate and 4 mM tetrabutylammonium phos-
phate, pH 6.0 20:80. A:B 100:0 for 5 min, to 40:60 over 30 min.
Flow rate: 1.5
Detector: UV 290
CHROMATOGRAM
Retention time: 21
OTHER SUBSTANCES
Extracted: other folates
KEYWORDS
rat; liver
REFERENCE
Rebello,T. Trace enrichment of biological folates on solid-phase adsorption cartridges and analysis by high-
pressure liquid chromatography, Anal.Biochem., 1987, 166, 55-64.
Fonazine
CAS Registry No.: 7456-24-8, 7455-39-2 (methanesulfonate)
Merck Index: 4260
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 jxL aliquot.
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.7
OTHER SUBSTANCES
dihydroergotamine, dimethindene, diphenhydramine, diphenoxylate, dipipanone, diprenor-
phine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol,
ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergo-
sinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol,
fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol, hydro-
xyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine, ke-
tanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclo-
REFERENCE
Formoterol
Merck Index: 4272
SAMPLE
Matrix: bulk
Sample preparation: Dissolve in mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Chiralcel OJ
Mobile phase: EtOH:heptane 30:70
Detector: UV 365
CHROMATOGRAM
Retention time: k' 1.38 ((RRM+)), k' 2.00 ((SSM-))
KEYWORDS
chiral
REFERENCE
Francotte,E.R.; Richert,R Applications of simulated moving-bed chromatography to the separation of the en-
antiomers of chiral drugs, J.Chromatogr.A, 1997, 769, 101-107.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 10 (xmole compound (as free base or hydrochloride) in 500 |xL
MeCN, add 250 JJLL 5% sodium carbonate (for hydrochlorides only), add 500 |JLL 100 mM reagent
in MeCN, vortex for 1 min, heat at 60 for 2 h, add 100 ixmole L-proline, heat at 60 for 30
min. Remove a 100 |xL aliquot and dilute it with mobile phase, neutralize with acetic acid,
inject a 10 |xL aliquot. Prepare the reagent ((R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexy-
lisothiocyanate) as follows. Add 0.7 mL carbon disulfide to 6 mL (lR,2R)-(-)-l,2-diaminocycloh-
exane, 12 mL water, and 12 mL EtOH, heat the oil bath to 80, add 2.8 mL carbon disulfide
dropwise (making sure that the product does not start to precipitate), when addition is complete
reflux for 1 h, acidify with 500 |xL 5 M HCl, reflux for 12 h, cool, filter, wash the solid with a
little cold EtOH to give trans-4,5-tetramethyleneimidazolidine-2-thione as a white fluffy solid
(mp 148-150) (Tetrahedron 1993, 49, 4419). Stir 7.97 g 3,5-dinitrobenzoyl chloride in 30 mL
dichloroethane at 50, add a solution of 6 g trans-4,5-tetramethyleneimidazolidine-2-thione in
120 mL dichloroethane containing a catalytic amount of 4-(dimethylamino)pyridine over 15
min, reflux for 2 h, remove the crystals of (R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexyliso-
thiocyanate by filtration, evaporate the filtrate to dryness and dissolve the residue in 60 mL
dichloroethane, reflux for 16 h to obtain more (R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexyl-
isothiocyanate (mp >250, [a]546 = -133 (c = 1) in MeCN).
HPLC VARIABLES
Column: 125 X 4 5 |xm Lichrospher 60 RP Select B
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: acebutolol, alprenolol, atenolol, carazolol, carvedilol, methamphetamine, meti-
pranolol, metoprolol, nifenanol, nitrilo atenolol, oxprenolol, pindolol, propranolol, xamoterol
KEYWORDS
REFERENCE
Kleidernigg,O.R; Posch,K; Lindner,W. Synthesis and application of a new isothiocyanate as a chiral derivatiz-
ing agent for the indirect resolution of chiral amino alcohols and amines, J.ChromatognA, 1996, 729, 3 3 -
42.
SAMPLE
Matrix: urine
Sample preparation: Condition a 100 mg silica SPE cartridge with 3 mL MeOH and 3 mL ethyl
acetate. Briefly vortex 1 mL urine with 100 (xL 2.5 mg/mL IS in MeOH and 100 |xL 250 mM
pH 8.0 phosphate buffer, add 3 mL ethyl acetate, tumble at 25 rpm for 30 min, centrifuge at
4000 rpm for 10 min. Remove the organic layer and add it to the SPE cartridge, wash with 1
mL ethyl acetate, wash with 10 mL 5% isopropanol in water, centrifuge at 4000 rpm for 10
min. Elute with 3 mL MeOH, evaporate the eluate to dryness at 30 under a stream of nitrogen,
reconstitute the residue with 100 juiL mobile phase, inject a 100 fjiL aliquot.
HPLC VARIABLES
Guard column: 10 X 4 Chiral AGP (Baker, Deventer, Netherlands)
Column: 100 X 4 Chiral AGP (Baker, Deventer, Netherlands)
Mobile phase: Isopropanol:50 mM pH 7.0 phosphate buffer 1.5:100, containing 1 mM KCl and
a small quantity of EDTA (sic)
Flow rate: 0.9
Detector: E, Waters EC 460, glassy carbon electrode +0.63 V, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 8.7 (R,R), 11.3 (S,S)
Internal standard: diastereomeric formoterol (Either R,S or S,R, prepared by preparative
HPLC, details in paper) (15.5)
Limit of detection: 60 pmol/L (R,R), 75 pmol/L (S,S)
KEYWORDS
SPE; chiral; comparison with GC/MS; pharmacokinetics
REFERENCE
Butter,J.J.; van den Berg,B.T.J.; Portier,E.J.G.; Kaiser,G.; van Boxtel,C.J. Determination by HPLC with elec-
trochemical detection of formoterol RR and SS enantiomers in urine, J.Liq.Chromatogr.Rel.Technol., 1996,
19, 993-1005.
Foscarnet sodium
Molecular formula: CNa3O5P
Merck Index: 4277
SAMPLE
Matrix: blood, CSF, urine
Sample preparation: Plasma. 100 |xL Plasma + 900 jxL 10 mM pyrophosphoric acid + 25 mg
charcoal, after 30 s filter (Amicon MPS-I with YMT membrane or Centricon 30 Microcencen-
trator) while centrifuging at 1000-2000 g for 15 min, dilute the ultrafiltrate 10-fold with 1 mM
pyrophosphoric acid, inject a 20 (JLL aliquot. (For high concentrations of foscarnet charcoal may
be omitted.) Urine. 100 jxL Urine + 900 |xL 10 mM pyrophosphoric acid + 25 mg charcoal,
vortex for 30 s, filter (Millipore SJHVL04NS), dilute the filtrate 10-fold with 1 mM pH 5.8
pyrophosphoric acid, inject a 20 |xL aliquot. CSF. Dilute CSF 100-fold with 1 mM pH 5.8 py-
rophosphoric acid, inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 3 |xm UltroPac C18 (LKB)
Mobile phase: MeOH:pH 5.8 57 mM phosphate buffer 25:75 containing 1 mM tetrahexylam-
monium hydrogen sulfate and 0.2 mM pyrophosphoric acid
Flow rate: 1
Detector: E, Environmental Sciences 5100 Coulochem, 5020 guard cell +0.75 V (placed after
injector), 5010 analytical cell, cell 1 +0.75 V, cell 2 +0.90 V (monitored)
CHROMATOGRAM
Limit of quantitation: 30 fxM (urine), 500 nM (plasma)
KEYWORDS
REFERENCE
Pettersson,K.-J.; Nordgren,T.; Westerlund,D. Determination of phosphonoformate (foscarnet) in biological fluids
by ion-pair reversed-phase liquid chromatography, J.Chromatogr., 1989, 488, 447-455.
SAMPLE
Sample preparation: Plasma. Filter (Amicon Centricon 30) 1 mL plasma while centrifuging at
1500 g for 20 min, heat 200 |xL of the ultrafiltrate in a boiling water bath for 20 min, cool, add
25-50 IJLL 671.8 |xM hydrochlorothiazide in MeOH:water 50:50, vortex for 10 s, add 900 |xL
EtOH, mix for 20 s. Remove a 100 JJLL aliquot and dilute it to 1 mL with 1 mM pyrophosphoric
acid, mix for 20 s, inject a 20 jxL aliquot. Urine. 1 mL Urine + 9 mL 10 mM pyrophosphoric
acid + 250 mg activated charcoal, vortex for 30 s. Filter (Amicon Centricon-30) 2 mL while
centrifuging at 1500 g for 5 min. 200 |xL Ultrafiltrate + 200 |xL 20 mM NaOH + 400 |xL EtOH,
heat at 56 for 30 min, cool, add 671.8 yM hydrochlorothiazide in MeOHiwater 50:50, mix for
10 s. Remove a 200 JJLL aliquot and dilute it 10-fold with 1 mM pyrophosphoric acid, mix for
20 s, inject a 20-40 JJLL aliquot.
HPLC VARIABLES
Column: 4 |xm Nova-Pak C18
Mobile phase: MeOH:60 mM pH 5.8 buffer 30:70 containing 1 mM tetrahexylammonium hydro-
gen sulfate and 0.2 mM pyrophosphoric acid, pH adjusted to 5.8
Flow rate: 0.7
Detector: E, ESA Coulochem Model 5100A, guard cell +0.99 V, analytical cell 1 +0.50 V, analyt-
ical cell 2 +0.95 V
CHROMATOGRAM
Internal standard: hydrochlorothiazide (18.9)
Limit of detection: 14 JJLM
KEYWORDS
plasma; ultrafiltrate; pharmacokinetics
REFERENCE
Hassanzadeh,M.K; Aweeka,F.T.; Wu,S.; Jacobson,M.A.; Gambertoglio,J.G. Determination of phosphonoformic
acid in human plasma and urine by high-performance liquid chromatography with electrochemical detec-
tion, J.Chromatogr., 1990, 525, 133-140.
SAMPLE
HPLC VARIABLES
Mobile phase: MeOH:5 mM sulfuric acid 5:95 containing 0.904 g/L tetrahexylammonium hydro-
gen sulfate
Flow rate: 1.5
Detector: UV 254
KEYWORDS
injections; saline
REFERENCE
Woods,K; Steinmann,W.; Bruns,L.; Neels,J.T. Stability of foscarnet sodium in 0.9% sodium chloride solution,
Am.J.Hosp.Pharm., 1994, 51, 88-90.
Fosfosal
Molecular formula: C7H7O6P
Merck Index: 4281
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep Pak C18 SPE cartridge with 2 mL MeOH. Add 500 |xL
Plasma to the SPE cartridge, elute with 500 JJIL water. Collect all the eluate and centrifuge at
2500 rpm for 15 min, inject a 50 |xL aliquot of the supernatant.
HPLCVARIABLES
Guard column: 10 |xm ixBondapak C18
Mobile phase: MeOH:5 mM tetrabutylammonium phosphate (PIC A) 30:70
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 5.5
KEYWORDS
plasma; rat; dog; pharmacokinetics; SPE; also for humans (see Int.J.Clin.Pharmacol.ther.Toxicol.
1988; 26; 421)
REFERENCE
Ramis,J.; Mis,R.; Forn,J. Pharmacokinetics of fosfosal in rats and dogs, Arzneimittelforschung, 1989, 39, 7 4 -
77.
Fosinopril
Molecular formula: C30H46NO7P
CAS Registry No.: 98048-97-6, 97825-24-6,
88889-14-9 (sodium salt)
Merck Index: 4282
LednicerNo.: 5 66
SAMPLE
HPLC VARIABLES
Mobile phase: MeCN:MeOH:0.05% phosphate buffer 65:8:27, pH adjusted to 3.5 with phosphoric
acid
Flow rate: 3
Detector: UV
KEYWORDS
perfusate buffer; rat
REFERENCE
Friedman,D.L; Amidon,G.L. Passive and carrier-mediated intestinal absorption components of two angiotensin
converting enzyme (ACE) inhibitor prodrugs in rats: enalapril and fosinopril, Pharm.Res., 1989, 6, 1043-
1047.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in mobile phase at a concentration of 100 |xg/mL.
HPLC VARIABLES
Column: 150 X 3.9 5 |xm RESOLVE (Waters)
Mobile phase: MeCN:water:orthophosphoric acid 4000:15:2
Flow rate: 1
Detector: UV 205
CHROMATOGRAM
OTHER SUBSTANCES
REFERENCE
Kirschbaum,J.; Noroski,J.; Cosey,A.; Mayo,D.; Adamovics,J. High-performance liquid chromatography of the
drug fosenopril, J.Chromatogr., 1990, 507, 165-170.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeOH:water:85% phosphoric acid 72:28:0.2
REFERENCE
Ranadive,S.A.; Chen,A.X.; Serajuddin,A.T. Relative lipophilicities and structural-pharmacological considera-
tions of various angiotensin-converting enzyme (ACE) inhibitors, Pharm.Res., 1992, 9, 1480-1486.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 X 4 10 |jim alkylphenyl (Column Resolution Inc.)
Mobile phase: MeOH:0.2% phosphoric acid 72:28
Flow rate: 2
Detector: UV 215
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: fosinoprilat, degradation products
KEYWORDS
REFERENCE
Lozano,R.; Warren,F.V.,Jr.; Perlman,S.; Joseph,J.M. Quantitative analysis of fosinopril sodium by capillary zone
electrophoresis and liquid chromatography, J.Pharm.Biomed.Anal., 1995, 23, 139-148.
Fosphenytoin
Molecular formula: C16H15N2O6P
SAMPLE
Sample preparation: Dilute injection with MeCNrwater 20:80, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 5 X 6 5 jxm Guard-Pak Resolve C18
Column: 150 x 3.9 5 ^m Resolve C18
Mobile phase: MeCN:water:85% phosphoric acid 25:75:0.09, containing 5 mM tetrabutylammo-
nium sulfate
Flow rate: 2
Detector: UV 210
CHROMATOGRAM
Retention time: 9.1
Limit of quantitation: 15 |xg/ml
OTHER SUBSTANCES
KEYWORDS
injections; stability-indicating
REFERENCE
Fischer,J.H.; Cwik,M.J.; Luer,M.S.; Sibley,C.B.; Deyo,K.L. Stability of fosphenytoin sodium with intravenous
solutions in glass bottles, polyvinyl chloride bags, and polypropylene syringes, Ann.Pharmacother., 1997,
31, 553-559.
Fotemustine
Molecular formula: C9H19CIN3O5P
Merck Index: 4285
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg CBA Bond Elut SPE cartridge with 1 mL MeOH and
1 mL water. Centrifuge blood at 5000 g for 2-3 min, freeze plasma in dry ice/hexane within 1
min. Thaw within 3 min by immersion in a 50 water bath. 1 mL Thawed plasma + 500 |xL
2.5 |xg/mL IS in 100 mM citric acid, vortex for 5 s, centrifuge for 5 min, add a 1 mL aliquot of
the supernatant to the SPE cartridge, wash with 1 mL water, elute with 200 |xL MeOH into a
vial containing 50 |xL 100 mM acetic acid, inject a 25 |xL aliquot.
HPLC VARIABLES
Column: 125 X 5 5 (im Spherisorb ODS
acid
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 9.1
Internal standard: l-methyl-3-isobutyl-8-vmyl-2,6-dioxopurine(S10338) (7.2)
OTHER SUBSTANCES
Extracted: lomustine, carmustine, metabolites
KEYWORDS
plasma; protect from light; pharmacokinetics; SPE; monkey; human
REFERENCE
Gordon,B.H.; Richards,R.R; Hiley,M.R; GrayAJ.; Ings,R.M.; Campbell,D.B. A new method for the measure-
ment of nitrosoureas in plasma: an h.p.l.c. procedure for the measurement of fotemustine kinetics, Xeno-
biotica, 1989, 19, 329-339.
SAMPLE
Matrix: blood
Sample preparation: Add fotemustine in ethanol PBS to plasma. Mix 100 |xL plasma with 600
jxL diethyl ether, rotate, centrifuge. Remove 400 jxL of the organic layer and evaporate it to
dryness, reconstitute the residue in 100 |xL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 mm long 4 |xm Novapack
Mobile phase: EtOH:l% acetic acid (pH 3) 25:75
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 7.6
Internal standard: carmustine (5.6)
KEYWORDS
plasma; rat
REFERENCE
Meulemans,A-; Giroux,B.; Hannoun,R; Henzel,D.; Bizzari,J.R; Mohler,J. Permeability of two nitrosoureas, car-
mustine and fotemustine in rat cortex, Chemotherapy, 1989, 55, 313319.
Fructose
Merck Index: 4295
SAMPLE
Matrix: beverages, plants
Sample preparation: Beverages. Dilute 50-fold, filter (0.22 |xm), inject an aliquot of the filtrate.
Plants. Heat 1 g barley leaves and 10 mL EtOH.water 80:20 at 100 in a sealed tube for 15-
30 min. Evaporate the liquid phase to dryness, reconstitute with water, pass through Analy-
tichem trimethylaminopropyl and cyclohexyl SPE cartridges, inject an aliquot.
HPLC VARIABLES
Column: 300 X 6.5 Sugar-Pak I (Waters)
Mobile phase: water
Flow rate: 0.4
Detector: F ex 360 em 470 following post-column reaction. The effluent from the column passed
through a 75 X 3.8 reactor containing Dowex 50 W X 2 sulfonic-acid type styrene divinylben-
zene copolymer at 100 and mixed with 30 mM benzamidine in 1 M KOH pumped at 1 mL/
min. This mixture flowed through a 530 |xL reaction coil (Varian PCR-I) at 100 to the detector.
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: dextrose, sucrose
KEYWORDS
barley; SPE; post-column reaction
REFERENCE
Coquet,A-; Haerdi,W.; Degli Agosti,R.; Veuthey,J.-L. Determination of sugars by liquid chromatography with
post-column catalytic derivatization andfluorescencedetection, Chromatographia, 1994, 38, 12-16.
Furaltadone
Merck Index: 4315
Lednicer No.: 1 229
SAMPLE
residue with 50 JJLL MeCN.water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
Column: 250 X 4.6 5 jim Symmetry C8 (Waters)
mL/min)
Detector: UV 347.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: milk
Sample preparation: Mix 50 mL cow milk with 25 mL 20% trichloroacetic acid, let stand for
15 min. Filter the samples and wash with water. Adjust the pH to 4.5-5 with NaOH, make up
to 100 mL with water. Take a 25 mL aliquot and add it to a Sep-Pak Plus C18 SPE cartridge,
elute with 2.5 mL mobile phase, pass nitrogen through eluate for at least 2 min (to remove
oxygen), inject an aliquot.
HPLC VARIABLES
Guard column: Symmetry C18
Mobile phase: MeCNiIOOmM aqueous sodium perchloraterglacial acetic acid 28:72:0.5
Flow rate: 1
Detector: E, ESA Coulochem II, Model 5011 analytical cell, porous carbon electrode -600 V5 Model
5021 conditioning cell
CHROMATOGRAM
Retention time: 2.5
OTHER SUBSTANCES
Extracted: furazolidone, nitrofurantoin
KEYWORDS
cow; SPE
REFERENCE
Galeano Diaz,T.; Guiberteau Cabanillas,A.; Acedo Valenzuela,M.L; Correa,C.A.; Salinas,F. Determination of
nitrofurantoin, furazolidone and furaltadone in milk by high-performance liquid chromatography with elec-
trochemical detection, J.ChromatogrA, 1997, 764, 243-248.
Furazolidone
Merck Index: 4320
Lednicer No.: 1 229
SAMPLE
Matrix: blood, eggs
Sample preparation: Dilute 1 mL serum or 0.5 mL egg yolk to 3 mL with water, mix, add to
an Extrelut-3 SPE cartridge, elute with 14 mL ethyl acetate. Evaporate the eluate to dryness
under a stream of nitrogen at 40, reconstitute the residue in 500 JLJLL mobile phase, centrifuge
at 10000 rpm for 6 min, inject a 50 |JLL aliquot of the supernatant.
HPLC VARIABLES
Column: 150 X 4.6 5C18 HG (Wako)
Flow rate: 1
Detector: UV 358
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Noninterfering: chlortetracycline, oxolinic acid, oxytetracycline, sulfonamides, tylosin
KEY WORDS
pig; serum; chicken; SPE
REFERENCE
Yoshida,K.; Kondo,F. Liquid chromatographic determination of furazolidone in swine serum and avian egg,
JAOAC Int., 1995, 78, 1126-1129.
SAMPLE
Matrix: blood, milk
Sample preparation: Extract 1 mL plasma or milk with 5 mL dichloromethane in acidic me-
dium (pH 3), mix, centrifuge. Remove organic phase and evaporate it to dryness at 50 under
a stream of nitrogen. Take up residue in MeCN and inject an aliquot. (Protect from light during
extraction procedure.)
HPLCVARIABLES
Column: 75 X 4.6 Beckman XL 3 |xm ODS
Flow rate: 1
Detector: UV 364
CHROMATOGRAM
Internal standard: furazolidone
OTHER SUBSTANCES
Simultaneous: nitrofurantoin
KEYWORDS
plasma; furazolidone is IS
REFERENCE
Pons,G.; Rey,E.; Richard,M.O.; Vauzelle,R; Francoual,C; Moran,C; d'Athis,R; Badoual,J.; 01ive,G. Nitrofu-
rantoin excretion in human milk, Dev.Pharmacol.Ther., 1990, 14, 148152.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 347.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Add 30 mL MeCN to 10 g homogenized shelled eggs, liver, or muscle,
blend at low speed for 2 min, centrifuge at 1000 g for 5 min, add 10 mL 10% NaCl solution
and 50 mL dichloromethane to the supernatant, shake for a few min. Filter the lower organic
layer through 5 g anhydrous sodium sulfate, evaporate the filtrate to dryness using a rotary
vacuum evaporator at 45, redissolve the residue in 1 mL MeCN:MeOH:20 mM pH 4.6 sodium
acetate 10:50:40, inject an aliquot. (Protect from light. Wash the 1 mL of MeCN:MeOH:20 mM
pH 4.6 sodium acetate 10:50:40 three times with 1 mL n-hexane before use.)
HPLC VARIABLES
Guard column: 10 X 4.6 (xBondapak C18
Column: 150 X 4.6 5 |xm Spherisorb ODS2 S5
Flow rate: 1
Detector: UV 362
CHROMATOGRAM
Retention time: 8.2
Limit of detection: 2.5 ng/g
OTHER SUBSTANCES
Extracted: nitrofurazone, furaltadone
KEYWORDS
chicken; liver; muscle
REFERENCE
Draisci,R; Giannetti,L.; Lucentini,L.; Palleschi,L.; Brambilla,G.; Serpe,L.; Gallo,P. Determination of nitrofuran
residues in avian eggs by liquid chromatography-UV photodiode array detection and confirmation by liquid
chromatography-ionspray mass spectrometry, J.Chromatogr.A, 1997, 777, 201211.
SAMPLE
layer through 5 g anhydrous sodium sulfate, evaporate the nitrate to dryness using a rotary
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil L C18-DB
Mobile phase: MeCN:water 50:50 containing 1 mM ammonium acetate and 0.025% acetic acid
Flow rate: 0.6
Detector: MS, PESCIEX API I, ionspray interface 5500 V, OR 60 V, m/z 226, split the column
effluent so that 0.03 mL/min enters the MS
CHROMATOGRAM
Retention time: 6.3
KEY WORDS
REFERENCE
Draisci,R.; Giannetti,L.; Lucentini,L.; Palleschi,L.; Brambilla,G.; Serpe,L.; Gallo,P. Determination of nitrofuran
chromatography-ionspray mass spectrometry, J. ChromatognA, 1997, 777, 201-211.
SAMPLE
Matrix: egs, milk, tissue
Sample preparation: Centrifuge milk at 2000 g for 10 min, freeze at -20 for 15 min, dilute a
10 mL aliquot with 10 mL saline, add 2 mL 1% sodium azide. Blend (Stomacher) 10 g homog-
enized tissue with 30 mL saline for 3 min, centrifuge at 2000 g, mix 20 mL of the supernatant
with 2 mL 1% sodium azide. Dilute 10 mL homogenized egg with 10 mL saline, add 3 mL 10%
sodium azide solution. Dialyze sample using a Cuprophan membrane (10000-15000 dalton cut-
off) against water pumped at 0.36-1.44 mL/min for 3-9 min, pass the water through column A,
flush the column with pure water for 8 min, backflush the contents of column A onto column
B with mobile phase, after 5 min remove column A from the circuit, elute column B with mobile
phase, monitor the effluent from column B. To increase sensitivity a number of sample batches
can be dialyzed before the contents of column A are analyzed. (Caution! Sodium azide is car-
cinogenic, mutagenic, and highly toxic! Do not discharge to the sink!)
HPLC VARIABLES
Column: A 60 X 4.6 37-50 |xm Bondapak C18/Corasil; B 250 X 4.6 5 \im Hypersil ODS
Mobile phase: MeCNrIOO mM pH 5 acetate buffer 20:80
Flow rate: 1
Detector: UV 365
CHROMATOGRAM
Retention time: 11
Limit of detection: 5 ng/mL (milk), 2 ng/g (tissue), 1 ng/g (eggs)
OTHER SUBSTANCES
Extracted: furaltadone, nitrofurantoin, nitrofurazone
KEYWORDS
protect from light; cow; muscle; dialysis
REFERENCE
Aerts,M.M.; Beek,W.M.; Brinkman,U.A. On-line combination of dialysis and column-switching liquid chroma-
tography as a fully automated sample preparation technique for biological samples. Determination of ni-
trofuran residues in edible products, J.Chromatogr., 1990, 500, 453-468.
SAMPLE
Matrix: feed
Sample preparation: Add 15 mL water to 5 g ground feed sample, let stand for 5 min, add 35
mL MeCN:MeOH 50:50, shake on a mechanical shaker for 30 min, filter through a glass fiber
filter. Pass the filtrate through a column dry-packed with 4 g neutral alumina (Sigma), discard
the first 4 mL of eluate, collect the next 8 mL eluate, inject a 20 (xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN: 10 mM pH 6.0 sodium acetate 20:80
Flow rate: 1.5
Detector: UV 365
CHROMATOGRAM
Retention time: 7.2
Limit of detection: 1 |xg/g
REFERENCE
McCracken,R.J.; Kennedy,D.G. Determination of furazolidone in animal feeds using liquid chromatography
with UV and thermospray mass spectrometric detection, J.ChromatognA, 1997, 771, 349-354.
SAMPLE
Matrix: feed
Sample preparation: Grind feed to pass 20 mesh. 10 g Feed + 5 mL water, swirl, let stand for
5 min, add 50 mL DMF:water 95:5, shake vigorously for 15 s, let stand in the dark at room
temperature overnight, filter (paper). Add 15 mL of the filtrate to 5 g alumina (Alcoa F-20, 80-
200 mesh) in a 300 X 10 glass column, discard first several mL of eluate, collect remaining
eluate, inject an aliquot
HPLCVARIABLES
Guard column: 100 X 2 ixBondapak C18/Corasil
Column: 300 X 4 jxBondapak C18
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Extracted: carbadox, nitrofurazone
KEYWORDS
protect from light
REFERENCE
Thorpe,V.A. Sample preparation of carbadox, furazolidone, nitrofurazone, and ethopabate in medicated feeds
for high pressure liquid chromatography, JAssoc.OffAnal.Chem., 1980, 63, 981-984.
SAMPLE
Matrix: feed, formulations, milk
Sample preparation: Formulations. Dissolve formulation in DMF, filter, inject a 10 |xL aliquot.
Feeds. Stir 10 g finely ground feeds with 40 mL DMF for 30 min, centrifuge, filter, wash
residues with DMF, dilute to 50 mL with DMF, inject a 10 jxL aliquot. Milk. Lyophilize 200
mL milk, wash with 75 mL MeCN during 15 min. Extract residue with 15 mL DMF with
stirring for 30 min, wash residue with a mixture of 25 mL MeCN+ 5 mL DMF. Combine all
organic solutions and evaporate to dryness in vacuum. Treat residue with DMF, filter, dilute
to 25 mL with DMF, filter before analysis, inject a 10 (xL aliquot.
HPLCVARIABLES
Column: 33 X 4.6 Perkin-Elmer Pecosphere 3x3 CR C18
Mobile phase: MeCN: 100 mM pH 3.2 sodium acetate/acetic acid 10:90
Flow rate: 2
Detector: UV 360
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: nitrofurantoin, furaltadone
REFERENCE
Galeano Diaz,T.; Lopez Martinez,L.; Martinez Galera,M.; Salinas,F. Rapid determination of nitrofurantoin,
furazolidone and furaltadone in formulations, feed and milk by high performance liquid chromatography,
J.Liq.Chromatogr., 1994, 17, 457-475.
SAMPLE
Matrix: feeds
Sample preparation: Add 15 mL water to 5 g ground feed sample, let stand for 5 min, add 35
mL MeCNrMeOH 50:50, shake on a mechanical shaker for 30 min, filter through a glass fiber
filter. Pass the filtrate through a column dry-packed with 4 g neutral alumina (Sigma), discard
the first 4 mL of eluate, collect the next 8 mL eluate, inject a 20 JJLL aliquot.
HPLC VARIABLES
Guard column: RP18 4-4 guard column (Merck)
Column: 125 X 4 5 |xm LiChrospher RP18 (end-capped) (Merck)
Mobile phase: MeCNrIOO mM ammonium acetate 35:65
Flow rate: 1
Detector: MS, Hewlett-Packard Model HP5989A, positive ion mode, source 200, thermospray
stem 125, multiplier voltage 2000 V, dynode voltage 8000 V, m/z 243
CHROMATOGRAM
REFERENCE
McCracken,R.J.; Kennedy,D.G. Determination of furazolidone in animal feeds using liquid chromatography
with UV and thermospray mass spectrometric detection, J.Chromatogr.A, 1997, 771, 349-354.
SAMPLE
Matrix: milk
HPLC VARIABLES
Column: 150 X 3.9 4 jjim Nova Pak C18
Mobile phase: MeCN: 10OmM aqueous sodium perchlorate:glacial acetic acid 28:72:0.5
Flow rate: 1
Detector: E, ESA Coulochem II, Model 5011 analytical cell, porous carbon electrode -600 V, Model
CHROMATOGRAM
Retention time: 2.7
Limit of detection: 5-6 ppb
OTHER SUBSTANCES
Extracted: furaltadone, nitrofurantoin
KEYWORDS
cow; SPE
REFERENCE
Galeano Diaz,T.; Guiberteau Cabanillas,A.; Acedo Valenzuela,M.L; Correa,C.A.; Salinas,F. Determination of
SAMPLE
Matrix: shrimp
Sample preparation: Condition a 6 mL Bond Elut C18 SPE cartridge with 5 mL MeOH and 5
mL water. Homogenize (Tissuemizer) 5 g finely-chopped (by hand) shrimp and 20 mL MeCN
at medium speed for 45 s, centrifuge at 3000 rpm for 5 min, decant the supernatant. Add 30
mL hexane saturated with MeCN to the supernatant, shake for 30 s, discard the hexane layer.
Add 10 mL EtOH to the MeCN layer, evaporate under reduced pressure at 45 to 2-5 mL (until
liquid looks milky), add 2 mL EtOH, continue evaporation until there is 2 mL of a thick liquid,
add 2 mL EtOH, evaporate to dryness. Add 2 mL water to the residue, sonicate for 5 min, add
to the SPE cartridge, wash with 4 mL water, elute at s3 mL/min with 5 mL MeCN. Evaporate
the eluate to dryness under a stream of nitrogen at ^45 (remove promptly when dry), recon-
stitute the residue in 1 mL mobile phase, filter (0.45 |xm), inject a 50 |xL aliquot.
HPLCVARIABLES
Guard column: 20 X 4 5 |xm ODS Hypersil C18
Column: 200 X 4.6 5 fjum ODS Hypersil C18
Mobile phase: MeCN: 1% aqueous acetic acid 25:75
Flow rate: 1
Detector: UV 375
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Extracted: nitrofurazone
KEYWORDS
SPE
REFERENCE
Rupp,H.S.; Munns,R.K; Long,A.R. Simultaneous determination of nitrofurazone and furazolidone in shrimp
(Penaeus vannamei) muscle tissue by liquid chromatography with UV detection, J.AOAC Int., 1993, 76,
1235-1239.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 200 X 4.6 5 ixm Hypersil SAS or 150 X 4.6 5 |xm Hypersil SAS
Mobile phase: MeCNrbuffer 30:70 (Mobile phase was 340 mL 100 mM citric acid, 5 mL 100 mM
trisodium citrate, and 5 mL 100 mM Na2EDTA made up to 500 mL with MeCN.)
Flow rate: 2
Detector: UV 370
CHROMATOGRAM
Retention time: 2.1
OTHER SUBSTANCES
Simultaneous: oxytetracycline, tetracycline, chlortetracycline
REFERENCE
Murray,J.; McGill,A.S.; Hardy,R. Development of a method for the determination of oxytetracycline in trout,
Food Addit.Contam., 1987, 5, 77-83.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeOH:water 30:70, inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 5 (xm Resolve spherical C18 (Waters)
Flow rate: 1
Detector: UV 305
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
Simultaneous: carbadox, nitrofurazone
Noninterfering: pyrantel
KEYWORDS
protect from light
REFERENCE
Roybal,J.E.; Munns,R.K.; Shimoda,W. Liquid chromatographic determination of carbadox residues in animal
feed, JAssoc.Off.Anal.Chem., 1985, 68, 653-657.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in chloroform at a concentration of 1 |xg/mL, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4 5 |jim Lichrospher RP-18
Mobile phase: MeCN: 10 mM sodium acetate 20:80, pH 5
Flow rate: 1.6
Detector: UV 365
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: degradation products, carbadox, nitrofurazone, nitrofurantoin, furaltadone
REFERENCE
Kaniou,L; Zachariadis,G.; Kalligas,G.; Tsoukali,H.; Stratis,J. Separation and determination of carbadox, nitro-
furazone, nitrofurantoin, furazolidone, and furaltadone in their mixtures by thin layer and high perfor-
mance liquid chromatography, J.Liq.Chromatogr., 1994, 17, 1385-1398.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Waring blender) 10 g muscle, liver, or kidney in 100 mL
EtOH for 5 min, let stand for 5 min, filter through 10 g Celite 545 on top of a sintered glass
filter, rinse blender with 100 mL EtOH and filter rinse. Add 25 mL 3.6% aqueous metaphos-
phoric acid to the combined filtrates, evaporate to 25 mL under reduced pressure at 45. Re-
move residue, rinse out flask with 5 mL hexane and 3 mL water, combine, centrifuge at 0 at
27000 g for 30 min, discard hexane, rinse surface with 5 mL hexane, discard hexane. Remove
aqueous layer, rinse out tube twice with 3 mL portions of water, combine, add 10 mL 1 M
KH2PO4, make up to 100 mL with water, extract three times for 5 min with 50 mL ethyl acetate.
Combine the extracts and dry them over 15 g anhydrous sodium sulfate, filter through glass
wool, evaporate to dryness under reduced pressure at 45. Take up residue in 3 mL ethyl
acetate and add to alumina column, rinse flask with 2 mL ethyl acetate and add rinse to
column. Elute with 20 mL EtOH:MeOH:ethyl acetate 10:10:80 and combine all the eluate.
Evaporate to dryness under reduced pressure at 45, reconstitute in 500 |xL mobile phase, inject
a 100 (xL aliquot. (Prepare alumina column by slurrying 1 g aluminum oxide (Baker) in 20 mL
ethyl acetate and adding to a 200 X 6 glass chromatographic column.)
HPLC VARIABLES
Guard column: Brownlee 10 |xm RP-GU MPLC C-8
Column: 250 X 4.6 Brownlee RP-10A C-8
Mobile phase: MeCN:EtOH:10 mM ammonium acetate 25:5:70, pH 6.8
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Retention time: 8.7
OTHER SUBSTANCES
Extracted: quinoxaline-2-carboxylic acid, carbadox, nitrofurazone, desoxycarbadox
KEYWORDS
protect from light; pig; muscle; liver; kidney
REFERENCE
MacIntosh,A.L; Neville,G.A. Liquid chromatographic determination of carbadox, desoxycarbadox, and nitro-
furazones in pork tissues, J.Assoc.Off.Anal.Chem., 1984, 67, 958-962.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 500 mg Bond Elut C18 SPE cartridge with 5 mL MeOH and
10 mL water. Homogenize 5 g tissue with 100 mL MeOH:0.2% metaphosphoric acid 40:60 for
2 min, filter through a 1 mm layer of Hyflo Super-Cel. Evaporate the filtrate under reduced
pressure at 40 to 10 mL, add the residue to the SPE cartridge, wash with 10 mL water, elute
with 10 mL MeOH. Evaporate the eluate to dryness under reduced pressure, take up the
residue in 1 mL mobile phase, inject a 10 |xL aliquot.
HPLCVARIABLES
Guard column: 15 X 3.2 Newguard RP-8
Column: 150 X 4.6 5 |xm Inertsil ODS
Mobile phase: MeCN:5 mM oxalic acid 45:55
Flow rate: 0.5
Detector: UV 265
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Extracted: sulfamonomethoxine, sulfadimethoxine, sulfisozole, nalidixic acid, oxolinic acid, pi-
romidic acid, sodium nifurstyrenate
KEYWORDS
fish; SPE
REFERENCE
Horie,M.; Saito,K; Hoshino,Y.; Nose,N.; Nakazawa,H.; Yamane,Y. Simultaneous determination of residual syn-
thetic antibacterials in fish by high-performance liquid chromatography, J.Chromatogr., 1991, 538, 484
491.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Ultra-Turrax TP 18/2) 3 g ground muscle + 200 |xL 1 |xg/mL
nitrofurazone in water + 6.8 mL MeCN for 6 s, centrifuge at 5000 rpm for 5 min. Remove 6.5
mL of the supernatant and add it to 2 mL 5 M NaCl, shake vigorously for 10 s, centrifuge at
3000 rpm for 2 min. Remove the organic layer and evaporate it to dryness under a stream of
nitrogen at 43, reconstitute the residue in 250 |xL MeCN:buffer 20:80, add 1 mL hexane, mix
(Whirlimixer), centrifuge for 4 min, discard the hexane layer, filter (Costar Spin-X 0.22 ^m
cellulose acetate) while centrifuging at 5600 g for 4 min, inject a 20 |xL aliquot of the filtrate.
(Buffer was 20 mM sodium 1-heptanesulfonate and 10 mM Na2HPO4, pH adjusted to 6.0 with
phosphoric acid.)
HPLCVARIABLES
Guard column: 20 X 4.6 5 |xm Supelcosil LC-ABZ
Column: 250 X 4.6 5 |xm Supelcosil LC-ABZ
Mobile phase: MeCN:buffer 25:75 (Buffer was 4.45 g sodium 1-heptanesulfonate and 9.5 g
Na3PO4.12H2O in 750 mL water, adjust pH to 2.5 with 5 M phosphoric acid, make up to 1 L
with water.)
Flow rate: 1
Detector: UV 365
CHROMATOGRAM
Retention time: 7.5
Internal standard: nitrofurazone (5.5)
KEYWORDS
cow; muscle
REFERENCE
Hormazbal,V.; Yndestad,M. Simple and rapid method of analysis for furazolidone in meat tissues by high-
performance liquid chromatography, J.Liq.Chromatogr., 1995, 18, 1871-1877.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 500 mg Bond-Elut NH2 SPE cartridge with 20 mL chloroform:
MeOH 70:30 and 10 mL hexane. Homogenize 2 g pulverized tissue with 40 mL MeOH:buffer
30:70 for 1 min, centrifuge at 2000 rpm for 15 min, evaporate to about 15 mL under reduced
pressure at 40, add 25 mL dichloromethane, shake gently for 1 min, centrifuge at 1500 rpm
for 10 min. Remove the lower organic layer and evaporate it to near dryness under reduced
pressure at 30. Resuspend the residue in 2 mL dichloromethane and 6 mL hexane, add to the
SPE cartridge, wash with 5 mL hexane:dichloromethane 50:50, elute with 5 mL chloroform:
MeOH 70:30. Evaporate the eluate to dryness under a stream of nitrogen at 50, reconstitute
the residue in 100 |xL mobile phase, inject a 50 |xL aliquot. (Buffer was 23 g Na2HPO4 and 14.2
g citric acid in 1 L water, pH 3.6.)
HPLCVARIABLES
Guard column: RP18 4-4 (Merck)
Column: 125 X 4 5 (xm Lichrospher RP18 (end-capped)
Flow rate: 1
Detector: MS, Vestec LC-MS Model 201A, thermospray, positive-ion mode, filament-assisted ion-
ization, electron-beam current 300 |xA, block 250,+ tip 245, lens 140, vaporizer probe 180,
electron multiplier 2000 V, SIM m/z 243 (M + NH4)
CHROMATOGRAM
Retention time: 3.6
KEYWORDS
protect from light; pig; liver; kidney; diaphragm; muscle; pharmacokinetics; SPE
REFERENCE
McCracken,R.J.; Blanchflower,W.J.; Rowan,C; McCoy,M.A.; Kennedy,D.G. Determination of furazolidone in
porcine tissue using thermospray liquid chromatography-mass spectrometry and a study of the pharma-
cokinetics and stability of its residues, Analyst, 1995, 120, 2347-2351.
Furosemide
Molecular formula: C12H11CIN2O5S
Merck Index: 4331
Lednicer No.: 1 134; 2 87
SAMPLE
Matrix: blood
Sample preparation: Filter 900 or 1350 JJLL serum through a Tosoh Plastic filter (Japan) while
centrifuging at 3000 or 5000 g for 15 min. Inject an aliquot.
HPLC VARIABLES
Column: Superspher 100RP-18e
Flow rate: 1
Detector: UV 285
KEYWORDS
serum; pharmacokinetics; ultrafiltrate
REFERENCE
Takamura,N.; Maruyama,T.; Otagiri,M. Effects of uremic toxins and fatty acids on serum protein binding of
furosemide: possible mechanism of the binding defect in uremia, Clin.Chem., 1997, 43, 2274-2280.
SAMPLE
Matrix: blood
Sample preparation: Add 100 (xL 10 |xg/mL nitrazepam in MeOH to 1 mL plasma, vortex, add
500 |xL 200 mM pH 9.0 glycine buffer and 5 mL ethyl acetate, extract. Centrifuge at 700 g for
10 min, evaporate 4 mL of the organic phase to dryness under nitrogen at 60, reconstitute the
residue in 200 fxL mobile phase, inject a 20 uL aliquot.
HPLCVARIABLES
Mobile phase: MeCN:buffer 69:31 (Mobile phase was 690 mL MeCN, 310 mL water, and 9 mL
glacial acetic acid, adjusted to pH 5.0 with 5 M NaOH.)
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Retention time: 5.2
Internal standard: nitrazepam (10.5)
KEYWORDS
pharmacokinetics; plasma
REFERENCE
Jankowski,A.; Skorek-Jankowska,A.; Lamparczyk,H. Determination and pharmacokinetics of a furosemide-
amiloride drug combination, J.Chromatogr.B, 1997, 693, 383-391.
SAMPLE
Matrix: blood
Sample preparation: Filter 170 |xL serum (UFC30HV00 membrane filter, 0.45 |xm, Millipore)
and inject a 100 |xL aliquot of the filtrate onto column A, elute to waste with mobile phase A,
after 4.6 min elute the contents of column A onto column B with mobile phase A:B 82:18, after
7 min elute column B with mobile phase C, monitor the effluent from column B. (After 7 min
remove column A from the circuit and wash it with mobile phase A:B 40:60 for 4 min then
equilibrate it with mobile phase A for 5 min. After 11 min re-equilibrate column B with mobile
phase A for 6 min.)
HPLC VARIABLES
Column: A 10 (xm Guard-pak ixBondapak C18 (Waters); B 150 X 4.6 5 iim YMC Pack ODS A-
type (Yamamura Chemicals, Japan)
Mobile phase: A 20 mM pH 7 phosphate buffer; B MeCN; C MeCN:20 mM pH 7 phosphate
buffer 35:65 containing 15 mM tetra-ra-butylammonium
Column temperature: 40 (column B)
Flow rate: 2
Detector: UV 271
CHROMATOGRAM
KEYWORDS
column-switching; serum
REFERENCE
Okuda,T.; Yamashita,K; Motohashi,M. High-performance liquid chromatography using on-line solid-phase ex-
traction: determination of furosemide in human serum, J.Chromatogr.B, 1996, 682, 343-348.
SAMPLE
Matrix: blood, perilymph, tissue
Sample preparation: Add 5 uL 1100 |xg/mL p-nitrophenol to 50 |xL serum. Homogenize tissue
in MeOH.water 80:20, centrifuge at 4 at 500 g for 5 min, remove 100 |xL of the resulting
supernatant and add internal standard. Pool perilymph to yield a 3 |xL perilymph sample.
Acidify all samples with 5 |xL 2 M phosphoric acid (except 3 (xL for perilymph), extract with
200 JJIL ethyl acetate, dry under nitrogen, reconstitute in 30 fiL mobile phase, inject a 15 JJIL
aliquot. (Perform all extractions in the dark.)
HPLCVARIABLES
Column: 250 X 4.6 5 u-m C18 (Beckmann)
Mobile phase: MeOH:10 mM pH 5.5 KH2PO4 buffer 37:63
Flow rate: 1.5
Detector: UV 235
CHROMATOGRAM
Internal standard: p-nitrophenol (8.30)
Limit of detection: < 100 ng/mL
OTHER SUBSTANCES
Extracted: metabolites, furosemide glucuronide
KEY WORDS
rat; serum; kidney; liver
REFERENCE
Mills,C.D.; Whitworth,C; Ryback,L.R; Henley,C.M. Quantification of furosemide from serum and tissues using
high-performance liquid chromatography, J.Chromatogr.B, 1997, 701, 6570.
SAMPLE
Sample preparation: Plasma. Mix 500 |xL plasma with 30 |JLL 10 mg/mL warfarin in MeOH, 50
JLJLL 6 M hydrochloric acid and 3 mL diethyl ether, vortex for 30 s, centrifuge at 2000 g for 10
min, evaporate ether layer in a 45 water bath under a stream of nitrogen. Reconstitute the
residue in 100 jxL MeOH and inject a 20 JAL aliquot. Urine. Mix 200 |xL urine with 20 jxL 6 M
hydrochloric acid, 40|xL 10 mg/mL warfarin in MeOH and 8 mL diethyl ether, vortex for 30 s,
evaporate to dryness in a 45 water bath under a stream of nitrogen. Reconstitute the residue
in 100 |xL mobile phase and inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 10 |xm ixBondapak C18 (plasma), 150 X 3.9 5 |xm Resolve Spherical C18
(urine)
Mobile phase: MeCN:bufFer 38:62 (Buffer was 10 mM potassium dihydrogen phosphate adjusted
Flow rate: 1.5
CHROMATOGRAM
Retention time: 3
Internal standard: warfarin (9)
Limit of detection: 2-3 ng/ mL
OTHER SUBSTANCES
Simultaneous: quinidine, sulfamethoxazole
Noninterfering: metabolites, carbamazepine, cimetidine, diazepam, disopyramide, fluvoxamine,
meclofenamate, metoclopramide, phenobarbital, phenylbutazone, phenytoin, ranitidine, the-
ophylline, trimethoprim
KEYWORDS
REFERENCE
Abou-Auda,H.S.; Al-Yamani,M.J.; Morad,A.M.; Bawazir,S.A.; Khan,S.Z.; al-Khamis,K.I. High-performance liq-
uid chromatographic determination of furosemide in plasma and urine and its use in bioavailability studies,
J.Chromatogr.B, 1998, 710, 121-128.
SAMPLE
Sample preparation: Plasma. Mix 400 jxL plasma with 700 uX. MeOH:acetone 60:40, centrifuge
at 3000 rpm for 10 min. Add 400 |xL of the supernatant to 50 |xL 50 ng/mL IS, filter (0.5 |xm),
inject an aliquot. Urine. Mix 1 mL urine with 700 |xL pH 4.8 acetate buffer and 300 (xL 100
ng/mL IS, filter (0.5 u,m), inject an aliquot.
HPLC VARIABLES
Guard column: 10 X 4 5 |xm Shim-pack G-ODS
Column: 150 X 6 5 |xm Shim-pack CLC-ODS
Mobile phase: Gradient. A was MeCN:water 20:80 containing 0.3% acetic acid. B was MeCN:
water 80:20 containing 0.3% acetic acid. A:B 88:12 for 5 min, 60:40 for 5 min, 50:50 for 10 min,
88:12 for 10 min (step gradient).
Flow rate: 1
CHROMATOGRAM
Internal standard: bumetanide (17.4)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Yagi,N.; Kiuchi,T.; Satoh,H.; Terashima,Y.; Kenmotsu,H.; Sekikawa,H.; Takada,M. Bioavailability and diuretic
effect of furosemide following administration of tablets and retarded capsules to human subjects,
Biol.Pharm.BulL, 1996, 19, 616-622.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 234.6
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
HPLC VARIABLES
Column: 300 X 4.6 5 ^m C18
Mobile phase: MeCNrIOO mM NaH2PO4 20:80 adjusted to pH 4.2 with phosphoric acid
Flow rate: 1.2
Detector: UV 228
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
304.
SAMPLE
Matrix: formulations, urine
Sample preparation: Tablets. Pulverize tablets, add MeOH, shake for 20 min, filter, wash solid
with MeOH, dilute filtrate with mobile phase, inject a 20 |xL aliquot. Urine. 2 mL Urine + 2
mL 1 M pH 3.25 KH2PO4 + 4 mL ethyl acetate, vortex for 20 min, centrifuge at 734 g for 5
min. Remove the organic layer and. evaporate it to dryness under a stream of nitrogen at 40,
reconstitute the residue in 2 mL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: jxBondapak C18
Mobile phase: MeCN:water 40:60 containing 5 mM KH2PO4ZK2HPO4, pH adjusted to 4.25
Flow rate: 1
Detector: E, EG&G Princeton Applied Research PAR Model 400, glassy carbon working electrode
+1200 mV, Ag/AgCl reference electrode (At the end of each day clean electrode with mobile
phase of MeOH at 1.5 mL/min, -800 mV for 2 min then +1600 mV for 15 min.)
CHROMATOGRAM
Retention time: 7.7
OTHER SUBSTANCES
Extracted: piretanide
KEYWORDS
tablets; pharmacokinetics
REFERENCE
Barroso,M.B.; Jimenez,R.M.; Alonso,R.M.; Ortiz,E. Determination of piretanide and furosemide in pharmaceu-
ticals and human urine by high-performance liquid chromatography with amperometric detection,
J.Chromatogr.B, 1996, 675, 303-312.
SAMPLE
Sample preparation: Tablets. Pulverize tablets, add MeOH, shake for 30 min, sonicate for 5
min, filter (Albet 242 paper), wash solid with MeOH, make up filtrate to 50 mL with MeOH,
inject a 20 JJLL aliquot. Urine. Adjust pH of 2 mL urine to 10.0 with 2 M KOH, add 1.5 mg
NaCl, add 4 mL ethyl acetate, shake for 10 min, centrifuge at 2500 rpm for 5 min. Remove the
residue in 2 mL mobile phase, sonicate, inject a 20 JJIL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 fjum ^Bondapak C18
Mobile phase: MeCN.water 30:70 containing 5 mM KH2PO4ZK2HPO4, pH adjusted to 5.5
Flow rate: 1
Detector: E, EG&G Princeton Applied Research PAR Model 400, glassy carbon working electrode
+1300 mV, Ag/AgCl reference electrode (At the end of each day clean electrode with mobile
phase of MeOH at 1.5 mL/min, -800 mV for 2 min then +1600 mV for 5 min.)
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: triamterene
KEYWORDS
tablets; pharmacokinetics
REFERENCE
Barroso,M.B.; Alonso,R.M.; Jimnez,R.M. Simultaneous determination of the diuretics triamterene and furo-
semide in pharmaceutical formulations and urine by HPLC-EC, J.Liq.Chromatogr.Rel.Technol., 1996, 19,
231-246.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Ultrasphere C18
Mobile phase: THF:glacial acetic acid:water 40:1:59
Flow rate: 1
Detector: UV 276
REFERENCE
Walter,E.; Janich,S.; Roessler,B.J.; Hilfinger,J.M.; Amidon,G.L. HT29-MTX/Caco-2 cocultures as an in vitro
model for the intestinal epithelium: In vitro-in vivo correlation with permeability data from rats and hu-
mans, J.Pharm.Sci., 1996, 85, 1070-1076.
SAMPLE
Matrix: urine
Sample preparation: Inject 5 jxL urine onto column A and elute to waste with mobile phase A,
after 1 min backflush the contents of column A onto column B with mobile phase B. Monitor
the effluent from column B.
HPLC VARIABLES
Column: A 20 X 2.1 30 |xm Hypersil ODS-C18; B 125 X 4 5 |xm LiChrospher 100 RP 18
Mobile phase: A 50 mM pH 3 phosphate buffer; B MeCN:50 mM pH 3 phosphate buffer 60:40
(Prepare buffer as follows. Dissolve 3.45 g NaH2PO4 monohydrate in 500 mL water containing
750 |xL propylamine hydrochloride, adjust to pH 3 with concentrated phosphoric acid.)
Flow rate: 1
Injection volume: 5
CHROMATOGRAM
Retention time: 7.7
OTHER SUBSTANCES
Extracted: amiloride, bumetanide, triamterene
KEYWORDS
column-switching
REFERENCE
Campins-Falc6,R; Herraez-Hernandez,R.; Pastor-Navarro,M.D. Analysis of diuretics in urine by column-switch-
ing chromatography andfluorescencedetection, J.Liq.Chromatogr.Rel.Technol., 1997, 20, 1867-1885.
Furprofen
Molecular formula: Ci 4 H 12 O 4
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 20 |xL 100 |xg/mL IS in 20 mM NaOH. Add 500
IJLL 50 mM pH 7.0 phosphate buffer, vortex for 1 min, add 2 mL dichloromethane, vortex for 1
min, shake for 5 min. Centrifuge at 100 g for 10 min, separate the organic layer, repeat the
extraction procedure twice, evaporate the combined organic lowers to dryness under reduced
pressure. Reconstitute with 200 |xL 20 mM NaOH, inject a 20 |xL aliquot.
HPLCVARIABLES
Guard column: 20 X 4.6 10 jxm Vydac AXGU
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Retention time: 4.9
Internal standard: fenbufen (3.5)
OTHER SUBSTANCES
Extracted: rufloxacin
KEYWORDS
SPE; plasma
REFERENCE
Carlucci,G.; Mazzeo,P. Simultaneous determination of furprofen and rufloxacin in human plasma by high-
performance liquid chromatography, J.Chromatogr.Sci., 1996, 34, 182-184.
Fusidic acid
Merck Index: 4340
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Weigh out ointment containing 2.14 mg sodium fusidate, take up in 100
mL MeOH:water 20:80. Remove a 200 JJIL aliquot and add it to 150 jxL 20 mM tetrahexylam-
monium bromide in 100 mM pH 7.0 phosphate buffer and 100 jxL 4.2 mg/mL 2-bromoacetyl-
6-methoxynaphthalene in acetone, mix, let stand at room temperature for 5 min, add 150 |xL
8.9 |xg/mL IS in MeCN, sonicate at room temperature for 1 min, inject a 50 |xL aliquot. (Prepare
2-bromoacetyl-6-methoxynaphthalene by stirring equimolar amounts of 2-acetyl-6-methoxy-
naphthalene (Janssen Chimica, Belgium) and phenyltrimethylammonium tribromide in THF
at room temperature for 3 h (Phosphorus and Sulfur 1985, 25, 357), purify by column chro-
matography on silica gel with chloroform.petroleum ether 50:50 (mp 109-112) (Chromatogra-
phia 1992, 33, 13).)
HPLC VARIABLES
Mobile phase: MeCN.MeOH.water 51:34:15
Flow rate: 1.6
CHROMATOGRAM
Retention time: 10
Internal standard: nonanoic acid naphthacyl ester (Prepare as follows. Dissolve 2 mmoles non-
anoic acid and 1 mmole 2-bromoacetyl-6-methoxynaphthalene in 10 mL anhydrous MeCN, add
500 |xL triethylamine, heat to 60 for 30 min, cool, add 30 mL water, extract three times with
10 mL portions of ether. Combine the extracts and wash them with 5% sodium bicarbonate
solution and with three 10 mL portions of water, dry over anhydrous sodium sulfate, evaporate
to dryness under reduced pressure, recrystallize from MeOH/water (mp 66-8) (Chromatogra-
phia 1992, 33, 13).) (5.5)
KEYWORDS
derivatization; ointment
REFERENCE
Gatti,R.; Gotti,R.; Bonazzi,D.; Cavrini,V. A comparative evaluation of three detectors in the HPLC analysis of
sodium fusidate, Farmaco, 1996, 51, 115-119.
Gabapentin
Merck Index: 4343
SAMPLE
Matrix: blood
1 mL buffer, do not allow to go dry. Condition an Empore C18 SPE membrane by adding 500
jxL MeOH, force through three drops, discard MeOH remaining in reservoir, add 500 jxL water,
force through three drops, discard water remaining in reservoir. Add 200 |xL 3 jig/mL IS in
buffer to the SPE cartridge, add 200 JJLL serum and force through at 1 drop/s, add 200 |xL
buffer, force all liquid through, elute with 500 JJLL MeOH. Add 100 JJLL saturated sodium tetra-
borate solution and 50 jxL 5% 2,4,6-trinitrobenzenesulfonic acid in water to the eluate, mix,
heat at 50 for 10 min, add 500 jxL 250 mM acetic acid, centrifuge at 12500 g for 2 min, add
the supernatant to the SPE membrane, force through using a syringe or by centrifuging at
100-120 g for 5 min, wash with 500 |AL MeCN:water 20:80, elute with 75 |AL MeCN then 125
[iL water, mix the eluates, inject a 50 |xL aliquot. (Buffer was saturated sodium tetraborate
solution diluted with three volumes of water.)
HPLC VARIABLES
Guard column: 20 X 2 30 |xm Permaphase ETH (DuPont)
Column: 250 X 4.6 Ultrasphere C18
Mobile phase: MeCN:water:acetic acid.n-butylamine 52:48:0.1:0.01 (pH should not exceed 4.5)
(Connect a 150 X 4.6 37-53 |xm silica (Whatman) column between pump and injector.)
Flow rate: 1.2
Detector: UV 340
CHROMATOGRAM
Retention time: 10
Internal standard: l-(aminomethyl)cycloheptaneacetic acid (13)
OTHER SUBSTANCES
Noninterfering: acetaminophen, N-acetylprocainamide, amikacin, caffeine, carbamazepine
epoxide, carbamazepine, chlordiazepoxide, demoxepam, desalkymurazepam, desmethylchlor-
diazepoxide, desmethyldiazepam, diazepam, disopyramide, ethosuximide, flurazepam, genta-
micin, lidocaine, phenobarbital, phenytoin, primidone, procainamide, quinidine, theophylline,
tobramycin, valproic acid, vancomycin
KEYWORDS
SPE; derivatization; pharmacokinetics
REFERENCE
Lensmeyer,G.L.; Kempf/T.; Gidal,BE'> Wiebe,D.A. Optimized method for determination of gabapentin in serum
by, Ther.DrugMonit, 1995, 17, 251-258.
SAMPLE
Matrix: blood
REFERENCE
Gatti,R.; Gotti,R.; Bonazzi,D.; Cavrini,V. A comparative evaluation of three detectors in the HPLC analysis of
sodium fusidate, Farmaco, 1996, 51, 115-119.
Gabapentin
Merck Index: 4343
SAMPLE
Matrix: blood
1 mL buffer, do not allow to go dry. Condition an Empore C18 SPE membrane by adding 500
jxL MeOH, force through three drops, discard MeOH remaining in reservoir, add 500 jxL water,
force through three drops, discard water remaining in reservoir. Add 200 |xL 3 jig/mL IS in
buffer to the SPE cartridge, add 200 JJLL serum and force through at 1 drop/s, add 200 |xL
buffer, force all liquid through, elute with 500 JJLL MeOH. Add 100 JJLL saturated sodium tetra-
borate solution and 50 jxL 5% 2,4,6-trinitrobenzenesulfonic acid in water to the eluate, mix,
heat at 50 for 10 min, add 500 jxL 250 mM acetic acid, centrifuge at 12500 g for 2 min, add
the supernatant to the SPE membrane, force through using a syringe or by centrifuging at
100-120 g for 5 min, wash with 500 |AL MeCN:water 20:80, elute with 75 |AL MeCN then 125
[iL water, mix the eluates, inject a 50 |xL aliquot. (Buffer was saturated sodium tetraborate
solution diluted with three volumes of water.)
HPLC VARIABLES
Mobile phase: MeCN:water:acetic acid.n-butylamine 52:48:0.1:0.01 (pH should not exceed 4.5)
(Connect a 150 X 4.6 37-53 |xm silica (Whatman) column between pump and injector.)
Flow rate: 1.2
Detector: UV 340
CHROMATOGRAM
Retention time: 10
Internal standard: l-(aminomethyl)cycloheptaneacetic acid (13)
OTHER SUBSTANCES
Noninterfering: acetaminophen, N-acetylprocainamide, amikacin, caffeine, carbamazepine
epoxide, carbamazepine, chlordiazepoxide, demoxepam, desalkymurazepam, desmethylchlor-
diazepoxide, desmethyldiazepam, diazepam, disopyramide, ethosuximide, flurazepam, genta-
micin, lidocaine, phenobarbital, phenytoin, primidone, procainamide, quinidine, theophylline,
tobramycin, valproic acid, vancomycin
KEYWORDS
SPE; derivatization; pharmacokinetics
REFERENCE
Lensmeyer,G.L.; Kempf/T.; Gidal,BE'> Wiebe,D.A. Optimized method for determination of gabapentin in serum
by, Ther.DrugMonit, 1995, 17, 251-258.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 100 IJLL 2 M perchloric acid, vortex for 10 s, centrifuge
at 15000 g for 3 min. Remove a 50 |xL aliquot and add it to 200 |xL MeOH, add 200 |xL buffer,
add 50 jxL reagent, mix, let stand at room temperature for 5 min, inject a 20 JJLL aliquot.
(Prepare buffer weekly by adjusting the pH of 500 mM boric acid to 9.5 with 1 M NaOH.
Prepare reagent weekly by dissolving 50 mg o-phthalaldehyde in 4.5 mL MeOH, add 500 |xL
buffer, add 50 |xL 3-mercaptopropionic acid.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere octadecyl
Mobile phase: MeCN:MeOH:buffer 30:30:40 (Prepare the buffer by diluting 7.5 mL glacial acetic
acid to 400 mL with water, adding 40 mg EDTA, and adjusting the pH to 3.7 with 3 M NaOH.)
Flow rate: 1.5
CHROMATOGRAM
Internal standard: l-(aminomethyl)cycloheptaneacetic acid (Parke-Davis) (15)
OTHER SUBSTANCES
Noninterfering: alanine, arginine, aspartic acid, carbamazepine, clobazam, clonazepam, cyste-
ine, felbamate, glutamic acid, glycine, histidine, isoleucine, lamotrigine, leucine, lysine, me-
thionine, oxcarbazepine, phenobarbital, phenylalanine, phenytoin, primidone, proline, rema-
cemide, serine, threonine, tiagabine, tyrosine, valine, valproic acid, vigabatrin
KEY WpRDS
REFERENCE
Forrest,G.; Sills,G.J.; Leach,J.P.; Brodie,M.J. Determination of gabapentin in plasma by high-performance liq-
uid chromatography, J.Chromatogr.B, 1996, 681, 421-425.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 500 |xL IS solution + 1 mL MeCN, vortex for 5 min,
centrifuge for 15 min. Mix 6 |xL buffer, 6 |xL reagent, and 6 |xL supernatant, let stand for 1
min, inject the whole amount. (Prepare IS solution by dissolving 100 mg gamma-phenyl-
gamma-aminobutyric acid and 10 mg l-(aminomethyl)cycloheptane acetic acid in 500 mL
MeCN and 500 mL water. Prepare buffer by dissolving 15.5 mg boric acid in 500 mL water
and adjusting to pH 9.5 with concentrated NaOH. Prepare reagent by mixing 100 mg o-phthal-
dialdehyde, 9 mL MeOH, 1 mL buffer, and 100 JJLL mercaptoethanol.)
HPLCVARIABLES
Column: 250 X 4 5 jxm BANsil C18 (ASMT, Enger, Germany)
Mobile phase: Gradient. A was MeCN:MeOH:0.1% pH 2 phosphoric acid 10:10:80. B was MeCN:
MeOH 50:50. A.B 90:10 for 1 min, to 30:70 over 25 min, maintain at 30:70 for 3 min, return
to initial conditions over 0.1 min, re-equilibrate for 3.9 min.
Flow rate: 1
CHROMATOGRAM
Internal standard: gamma-phenyl-gamma-aminobutyric acid (Marion Merrel Dow) (23.4), 1-
(aminomethyl)cycloheptane acetic acid (Go-3609, Parke Davis) (28.3)
OTHER SUBSTANCES
Extracted: vigabatrin
KEY WpRDS
derivatization; serum; degas mobile phase continuously with helium
REFERENCE
Juergens,U.H.; May,T.W.; Rambeck,B. Simultaneous HPLC determination of vigabatrin and gabapentin in se-
rum with automated pre-injection derivatization, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 1459-1471.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 50 jxL IS solution + 200 |xL 1.2 M perchloric acid, vortex,
centrifuge at 3000 rpm for 5 min. Remove the supernatant and add it to 50 |xL 5% 2,4,6-
trinitrobenzenesulfonic acid in water, add 50 |xL 50% NaOH, vortex, let stand at room tem-
perature for 30 min, add 200 (xL 6 M HCl, add 100 |xL saturated NaCl, add 6 mL cyclohexane,
shake for 10 min, centrifuge at 3000 rpm for 10 min. Remove the organic layer and evaporate
it to dryness under a stream of nitrogen at 55, reconstitute the residue in 200 |xL EtOHrmobile
phase 10:90, inject a 75 JJLL aliquot.
HPLC VARIABLES
Column: 5 (xm Spherisorb ODSIII
Mobile phase: MeCN: 100 mM pH 4 ammonium acetate 54:46
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Retention time: 5.2
Internal standard: l-(aminomethyl)heptaneacetic acid (Parke-Davis) (13.3)
KEYWORDS
derivatization; plasma; dog; pharmacokinetics
REFERENCE
Stevenson,C.M.; Radulovic,L.L.; Bockbrader,H.N.; Fleisher,D. Contrasting nutrient effects on the plasma levels
of an amino acid-like antiepileptic agent from jejeunal administration in dogs, J.Pharm.Sci., 1997,86,953-
957.
SAMPLE
Sample preparation: Add 50 |xL serum or urine (diluted between 1:50 and 1:200) to 1 mL MeOH
containing 3.4 |xg 7-phenyl-7-amino-n-butyric acid, vortex for 15 s, centrifuge at 2000 g for 10
min, mix 6 |xL of the supernatant with 3 |JLL reagent, inject an aliquot. (Reagent was 10 mL
30 mg/mL o-phthaldialdehyde and 200 (xL 2-mercaptoethanol made up to 50 mL with 400 mM
pH 9.5 borate buffer (Ther. Drug Monit. 1991, 13, 251).)
HPLC VARIABLES
Column: 125 X 3 5 |xm Superspher 60 RP-Select B (Merck)
Mobile phase: Gradient. A was MeCN. B was 20 mM KH2PO4 buffer. A:B from 22:78 to 37:63 in
12 min, from 37:63 to 55:45 in 6 min, from 55:45 to 80:20 in 1.5 min, maintain at 80:20 for 2
min
Flow rate: 0.7
CHROMATOGRAM
Internal standard: ^-phenyl-^-amino-n-butyric acid (14.8)
OTHER SUBSTANCES
Extracted: vigabatrin
KEY WpRDS
derivatization; serum; urine
REFERENCE
Wad,N.; Kramer,G. Sensitive high-performance liquid chromatographic method withfluorometricdetection for
the simultaneous determination of gabapentin and vigabatrin in serum and urine, J.Chromatogr.B, 1998,
705, 154-158.
SAMPLE
Sample preparation: Plasma. 500 |xL Plasma + 10 |xL IS in water + 5 drops 2 M perchloric
acid, vortex vigorously for a few s, centrifuge at 15000 g for 2 min. Remove the supernatant
and add it to 500 |xL 1 M sodium bicarbonate, add 50 |xL 2 M 2,4,6-trinitrobenzenesulfonic
acid in water, adjust pH to 8.5 with 100 mM NaOH, let stand for 30 min, add 2 drops of 25%
HCl, add 3 mL toluene, shake for 10 min, centrifuge at 5000 g for 2 min. Remove the upper
organic layer and evaporate it to dryness under reduced pressure at 40, reconstitute the res-
idue in 100 |xL 200 mM pH 8.5 sodium borate buffer, wash with 1 mL cyclohexane:toluene 90:
10 by vortexing for 1 min, inject a 10-50 |xL aliquot of the aqueous phase. Urine. 10-100 |xL
Urine + 10 |xL 200 |xg/mL IS in water, add 500 |xL 1 M sodium bicarbonate, add 50 uX. 2 M
2,4,6-trinitrobenzenesulfonic acid in water, adjust pH to 8.5 with 100 mM NaOH, let stand for
30 min, add 2 drops of 25% HCl, add 3 mL toluene, shake for 10 min, centrifuge at 5000 g for
2 min. Remove the upper organic layer and evaporate it to dryness under reduced pressure at
40, reconstitute the residue in 100 |iL 200 mM pH 8.5 sodium borate buffer, wash with 1 mL
cyclohexanertoluene 90:10 by vortexing for 1 min, inject a 10-50 |xL aliquot of the aqueous
phase.
HPLCVARIABLES
Mobile phase: MeCN:0.5% acetic acid 58:42
Flow rate: 1
Detector: UV
CHROMATOGRAM
Internal standard: l-(aminomethyl)cycloheptaneacetic acid (13.2)
KEYWORDS
plasma; derivatization; pharmacokinetics
REFERENCE
Hengy,H.; Kolle,E.U. Determination of gabapentin in plasma and urine by high-performance liquid chroma-
tography and pre-column labelling for ultraviolet detection, J.Chromatogr., 1985, 341, 473-478.
Gadodiamide
Molecular formula: C16H26GdN5O8
Merck Index: 4345
SAMPLE
Sample preparation: Serum. Filter (Millipore Ultrafree-MC, type PTGC, 10 000 NMWL Filter
unit) serum while centrifuging at 4 at 5000 g for 1 h, inject a 10 |xL aliquot of the ultrafiltrate.
Urine. Centrifuge urine at 4 at 15000 g for 10 min, dilute a 100 |xL aliquot of the supernatant
with 400 jxL water, inject a 10 |xL aliquot.
HPLC VARIABLES
Guard column: 20 X 2.1 5 u.m Supelguard LC-19-DB (Supelco)
Mobile phase: 10 mM Triethylammonium acetate containing 2 mM EDTA, pH adjusted to 6.5-
7.0 with 1 M acetic acid or 1 M NaOH
Flow rate: 0.3
Detector: UV 658 following post-column reaction with the reagent pumped at 0.3 mL/min. (Re-
agent was 100 mM nitric acid containing 0.15 mM Arsenazo III and 10 mM urea, filter (0.45
|jim), sonicate, discard after 2 days.)
CHROMATOGRAM
Retention time: 4
Limit of detection: 1.1 |xM (unne), 0.3 |xM (serum)
Limit of quantitation: 10 |xM (urine), 2 (JLM (serum)
OTHER SUBSTANCES
Extracted: gadopentetate dimeglumine
KEY WORDS
serum; ultrafiltrate; post-column reaction
REFERENCE
Hvattum,E.; Normann,P.T.; Jamieson,G.C; Lai,J.-J.; Skotland,T. Detection and quantitation of gadolinium che-
lates in human serum and urine by high-performance liquid chromatography and post-column derivatiza-
tion of gadolinium with Arsenazo III, J.Pharm.BiomedAnaL, 1995, 13, 927-932.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 6 Asahipak ODP-50
Mobile phase: MeCNipH 6.8 Tris-HCl buffer 1.5:98.5 containing 75 u,M n-octylamine
Flow rate: 1.2
Detector: UV 215
CHROMATOGRAM
Retention time: 2.4
OTHER SUBSTANCES
Simultaneous: caldiamide, similar chelates of other metals
REFERENCE
Okazaki,O.; Kurata,T.; Yoshioka,N.; Hakusui,H. Pharmacokinetics and stability of caldiamide sodium in rats,
Arzneimittelforschung, 1996, 46, 79-83.
Gadopentetic acid
Molecular formula: C14H20GdN3O10
Merck Index: 4347
SAMPLE
Sample preparation: Serum. Filter (Millipore Ultrafree-MC, type PTGC, 10 000 NMWL Filter
unit) serum while centrifuging at 4 at 5000 g for 1 h, inject a 10 |xL aliquot of the ultrafiltrate.
Urine. Centrifuge urine at 4 at 15000 g for 10 min, dilute a 100 /xL aliquot of the supernatant
with 400 |xL water, inject a 10 |xL aliquot.
HPLC VARIABLES
Guard column: 20 X 2.1 5 urn Supelguard LC-19-DB (Supelco)
Mobile phase: 10 mM Triethylammonium acetate containing 2 mM EDTA, pH adjusted to 6.5-
7.0 with 1 M acetic acid or 1 M NaOH
Flow rate: 0.3
Detector: UV 658 following post-column reaction with the reagent pumped at 0.3 mL/min. (Re-
agent was 100 mM nitric acid containing 0.15 mM Arsenazo III and 10 mM urea, filter (0.45
|xm), sonicate, discard after 2 days.)
CHROMATOGRAM
Retention time: 11
OTHER SUBSTANCES
Extracted: gadodiamide
KEYWORDS
serum; ultrafiltrate; post-column reaction
REFERENCE
Hvattum,E.; Normann,P.T.; Jamieson,G.C; Lai,J.-J.; Skotland,T. Detection and quantitation of gadolinium che-
lates in human serum and urine by high-performance liquid chromatography and post-column derivatiza-
tion of gadolinium with Arsenazo III, J.Pharm.Biomed.AnaL, 1995, 13, 927-932.
Gallamine triethiodide
Molecular formula: C30H60IsN3O3
Merck Index: 4361
SAMPLE
M a t r i x : blood
Sample preparation: 500 |xL Plasma + 500 (xL MeCN, vortex for 2 min, centrifuge at 15000 g
at 4 for 30 min. Remove a 500 |JLL aliquot of the supernatant and add it to 500 |xL mobile
phase, mix for 2 min, inject a 5 |xL aliquot.
HPLCVARIABLES
Column: 300 X 3.9 10 |xm (xBondapak octadecylsilane
Mobile phase: MeCN:buffer 2:98 adjusted to pH 6.0 with 1 M phosphoric acid (Buffer was 5 mM
octanesulfonic acid and 5 mM Na2HPO4.)
Flow rate: 0.2
Injection volume: 5
Detector: UV 230
CHROMATOGRAM
KEYWORDS
plasma
REFERENCE
Shao,M.J.; Fallon,K.D.; Khalil,S.N.; Abouleish,E. Quantitation of gallamine (Flaxedil) in human plasma using
SAMPLE
Matrix: blood
Sample preparation: 50 jxL Serum + 10 |JLL 100 |xg/mL d-tubocurarine chloride in water + 50
JJLL 10% sodium tungstate:335 mM sulfuric acid 50:50, vortex for 15 s, centrifuge at 12800 g
for 2 min, inject a 30 JJLL aliquot of the supernatant.
HPLCVARIABLES
Mobile phase: MeOH:7.5 mM tetrabutylammonium hydrogen sulfate 10:90
Flow rate: 1.8
Detector: UV 229
CHROMATOGRAM
Internal standard: d-tubocurarine chloride (10.0)
OTHER SUBSTANCES
Noninterfering: barbiturates, alcuronium, metocurine, neostigmine, edrophonium
KEYWORDS
serum; rat; pharmacokinetics
REFERENCE
Ramzan,I.M. Determination of the neuromuscular blocking drug gallamine in rat serum using high-perfor-
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 |xL aliquot of a 0.5-400 u,g/mL solution in mobile phase.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Nucleosil octadecylsilyl
Mobile phase: MeCNibuffer 31:69 containing 100 mM sodium perchlorate (Buffer was 50 mM
phosphoric acid adjusted to pH 3 with NaOH.)
Flow rate: 1
Detector: UV 200
CHROMATOGRAM
Retention time: 12
REFERENCE
Mourier,P.A. Determination of gallamine and its impurities by reversed-phase ion-pair high-performance liquid
chromatography and comparison with thin-layer chromatography, J.Chromatogr., 1989, 462, 281-292.
Gallopamil
Merck Index: 4369
SAMPLE
Matrix: blood
Sample preparation: Condition column A with two 995 |xL portions of mobile phase at 3 mL/
min and then with 995 |xL solution B. Wash the donor channel of the dialyser (Gilson ASTED
XL fitted with a Cuprophan cellulose acetate membrane with a molecular mass cut-off of 15000)
with 2 mL solution A at 3 mL/min and wash the acceptor channel with 2 mL solution B. Dialyze
370 |xL plasma against 9 mL solution B is pumped through the acceptor channel at 1 mL/min.
Pass the dialysate through column A, backflush the contents of column A onto column B with
mobile phase, elute with mobile phase, monitor the effluent from column B. (Solution A was
10 mM pH 3 acetate buffer containing 0.01% Triton X-100 and 50 |xg/mL sodium azide. Solution
B was 10 mM pH 3 acetate buffer containing 50 jig/mL sodium azide.)
HPLCVARIABLES
Column: A 30 |xm Nucleosil CN; B 5 |xm LiChrospher 100 RP-18 guard column + 4 |xm Super-
spher 100 RP-18
Mobile phase: MeCN:2-aminoheptane:buffer 25:0.5:75 (Buffer was 10 mM sodium acetate ad-
justed to pH 3.0 with acetic acid.)
Flow rate: 0.9
CHROMATOGRAM
Retention time: 15
Internal standard: gallopamil
OTHER SUBSTANCES
Simultaneous: norverapamil, verapamil
KEYWORDS
dialysate; column-switching; plasma; gallopamil is IS
REFERENCE
Ceccato,A.; Chiap,R; Hubert,R; Toussaint,B.; Crommen,J. Automated determination of verapamil and norve-
rapamil in human plasma with on-line coupling of dialysis to high-performance liquid chromatography and
fluorimetric detection, J.Chromatogr A, 1996, 750, 351-360.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 JJLL 60 ng/mL norverapamil in water + 100 |xL 2 M
KOH, vortex gently for 5 s, add 5 mL hexane:MTBE 80:20, rotate at 45 rpm for 30 min,
centrifuge at 1150 g for 5 min, freeze at -80 for 20 min. Remove the organic layer and add it
to 100 (JLL 50 mM pH 3 KHgPO4, shake mechanically at high speed for 5 min, centrifuge for 5
min, freeze at -80 for 20 min, discard the organic layer, inject an aliquot of the aqueous layer.
HPLC VARIABLES
Guard column: 20 mm long 5 (xm Supelcosil LC-18-DB
Mobile phase: MeCNibuffer 38:62 (Buffer was 50 mM KH2PO4 containing 5 mM 1-octanesulfonic
acid and 1 mM triethylamine, pH adjusted to 3.0 with phosphoric acid.)
Flow rate: 2
Detector: F ex 205 em 0-56 filter (Kopp) (Emission maximum is 310 nm.)
CHROMATOGRAM
Internal standard: norverapamil (8.0)
OTHER SUBSTANCES
Simultaneous: hydrochlorothiazide, procainamide, propranolol, quinidine, triamterene
KEYWORDS
REFERENCE
McLean,A.M.; Babcock-Atkinson,E.; Rein,K; Ruggirello,D.A.; Gonzalez,M.A.; Noonan,P.K. High-performance
liquid chromatographic (HPLC) assay using fluorescence detection for the simultaneous determination of
gallopamil and norgallopamil in human plasma, Pharm.Res., 1987, 4, 327-331.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 0.9% NaCl + 120 |JLL 2 M NaOH + 5 mL n-hexane,
vortex for 30 s, mix for 10 min, centrifuge at 2500 g for 20 min, repeat extraction with 4 mL
n-hexane. Combine the organic phases and evaporate them to dryness, rinse walls with 1
mL n-hexane, evaporate to dryness, dissolve residue in 50 |xL isopropanol, inject a 25 |JLL
aliquot.
HPLCVARIABLES
Guard column: 50 X 4.6 Chiralpak AD (Baker)
Column: 250 X 4.6 Chiralpak AD (Baker)
Mobile phase: n-Hexane:isopropanol 90:10 with 0.1% diethylamine
Flow rate: 1
CHROMATOGRAM
Retention time: 11 (S-(-)), 14 (R-(+))
OTHER SUBSTANCES
Simultaneous: norverapamil
Also analyzed: verapamil
KEYWORDS
plasma; chiral
REFERENCE
Fieger,H; Blaschke,G. Direct determination of the enantiomeric ratio of verapamil, its major metabolite nor-
verapamil, and gallopamil in plasma by chiral high-performance liquid chromatography, J.Chromatogn,
1992, 575, 255-260.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve a sample in water, mix a 1 mL aliquot with 100 jxL 13.36 (xg/m
IS, inject an aliquot.
HPLCVARIABLES
Column: 100 X 4.0 5 |xm Chiral-AGP (Baker)
Mobile phase: MeCN:pH 6.8 (I = 0.01) ammonium acetate 11:89 (Buffer was adjusted to pH 6.8
with ammonium hydroxide or acetic acid)
Flow rate: 0.9
Detector: UV 225; MS, Finnigan MAT SSQ 710A, interface particle beam, desolvation chamber
45, nebulizing gas helium, electron impact mode 70 eV, source 250, filament current 200 |JLA,
electron multiplier 1500 V, m/z 45-400
CHROMATOGRAM
Retention time: 9.39 ((2RM+)), 14.26 ((2SM-))
Internal standard: procaine hydrochloride (5.09)
Limit of detection: 154 ng/mL ((2RM+)), 163 ng/mL ((2SM-))
OTHER SUBSTANCES
Also analyzed: verapamil
KEYWORDS
chiral
REFERENCE
Rustichelli,C; Ferioli,V.; Gamberini,G. Resolution of the enantiomers of verapamil and gallopamil by chiral
liquid chromatography-mass spectrometry, Chromatographia, 1997, 44, 477483.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve a sample in water and inject an aliquot.
HPLCVARIABLES
Column: 100 X 4.0 5 |xm Chiral-AGP (Baker)
Mobile phase: Gradient. A was MeCN. B was isopropanol. C was pH 6.8 (I = 0.01) ammonium
acetate adjusted to pH 6.8 with ammonium hydroxide or acetic acid. A:B:C from 11:1:88 to 7:
1:92 over 1.5 min, maintain at 7:1:92 for 35 min, to 11:1:88 over 5 min
Flow rate: 0.9
Detector: UV 225
CHROMATOGRAM
Retention time: 11 ((2RH+)), 22 ((2SH-))
OTHER SUBSTANCES
Simultaneous: verapamil
KEYWORDS
chiral
REFERENCE
Rustichelli,C; Ferioli,V.; Gamberini,G. Resolution of the enantiomers of verapamil and gallopamil by chiral
liquid chromatography-mass spectrometry, Chromatographia, 1997, 44, 477-483.
Ganciclovir
Merck Index: 4374
LednicerNo.: 5 146
SAMPLE
Matrix: blood
Sample preparation: Add 10 (xL 200 |xg/mL acyclovir in MeOH to 500 JJLL plasma, add 1 mL
MeCN, vortex briefly, centrifuge at 2000 g for 3 min, add 2 mL chloroform (Caution! Chloroform
is a carcinogen!) to the supernatant, vortex. Remove the aqueous supernatant layer, remove
traces of the organic solvent under a stream of nitrogen at 80 for 3 min, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4 5 |xm LiChrocart RP8
Mobile phase: MeCN: 10 mM pH 5 ammonium acetate buffer 2:98
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 6
Internal standard: acyclovir (7)
KEYWORDS
plasma
REFERENCE
Cociglio,M.; Peyriere,H.; Hillaire-Buys,D.; Alric,R- Application of a standardized coextractive cleanup procedure
to routine high-performance liquid chromatography assays of teicoplanin and ganciclovir in plasma,
J.Chromatogr.B, 1998, 705, 79-85.
SAMPLE
Matrix: blood
Sample preparation: 500 (xL Plasma + 2 |xg 9-methylxanthine + 50 JJLL 35% perchloric acid,
centrifuge at 4 at 2000 g for 15 min, inject a 20 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: 150 X 4.6 3 fjim Hypersil ODS
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 7
Internal standard: 9-methylxanthine (4.5)
OTHER SUBSTANCES
Noninterfering: acyclovir, allopurinol, azathioprine, caffeine, guanine, guanosine, hypoxanthine,
mercaptopurine, oxypurinol, theophylline, uric acid, xanthine
KEYWORDS
plasma
REFERENCE
Boulieu,R.; Bleyzac,N.; Ferry,S. Modified high-performance liquid chromatographic method for the determi-
nation of ganciclovir in plasma from patients with severe renal impairment, J.Chromatogr., 1991,571, 331-
333.
SAMPLE
Matrix: blood
Sample preparation: 500 IJLL Plasma + 50 |xL 35% perchloric acid, centrifuge at 4 at 2000 g
for 15 min, inject a 20 (xL aliquot of the supernatant.
HPLC VARIABLES
Column: 3 |xm Hypersil ODS
Flow rate: 1.5
Detector: UV 254
KEYWORDS
plasma
REFERENCE
Boulieu,R.; Bleyzac,N. Stability of ganciclovir in blood samples, J.Pharm.Biomed.AnaL, 1994,12, 1205-1207.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 4 mL MeCN: 10 mM pH 3.2 phosphate buffer,
mix, centrifuge at 12000 rpm for 15 min. Remove the supernatant, filter, inject an aliquot.
Tissue. Homogenize tissue in MeCN/PBS, centrifuge at 12000 rpm for 15 min. Remove the
supernatant, filter, inject an aliquot.
HPLC VARIABLES
Mobile phase: MeCNiIO mM KH2PO4 20:80 (A) or MeOHrIO mM KH2PO4 5:95 containing 1 mM
tetramethylammonium perchlorate (B)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 3 (A), 8 (B)
KEYWORDS
plasma; rat; brain; lung; pharmacokinetics
REFERENCE
Brewster,M.E.; Raghavan,K.; Pop,E.; Bodor,N. Enhanced delivery of gangiclovir to the brain through the use
of redox targeting, Antimicrob.Agents Chemother., 1994, 38, 817-823.
SAMPLE
Sample preparation: Dilute withmobile phase, add hypoxanthine (final hypoxanthine concen-
tration 10 |xg/mL), inject a 5 (xL aliquot.
HPLC VARIABLES
Mobile phase: 6 mM (NH4)H2PO4 adjusted to pH 2.5 with 0.33 M phosphoric acid
Flow rate: 2
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Internal standard: hypoxanthine (3.94)
KEYWORDS
injections; 10% dextrose; 25% dextrose; stability-indicating
REFERENCE
Johnson,C.E.; Jacobson,P.A.; Chan,E. Stability of gangiclovir sodium and amino acids in parenteral nutrient
solutions, Am.J.Hosp.Pharm., 1994, 51, 503-508.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Bioblock stirpack homogenizer) 10 mg tissue in 700 (xL ice-
cold 600 mM perchloric acid at 6000 rpm for 2.5 min, centrifuge at 4 at 2000 g for 15 min,
adjust the pH of the supernatant to 7-8 with NaOH. Remove a 200 (xL aliquot and add 10 |xL
alkaline phosphatase (type VII-NT, 10000 glycine U/mg protein, Sigma), heat at 37 for 30 min,
evaporate, reconstitute in 30 |JLL mobile phase, inject a 20 jxL aliquot. (The alkaline phospha-
tase step may be omitted.)
HPLCVARIABLES
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 7
Limit of detection: 0.9 pmole/g
KEYWORDS
heart
REFERENCE
Bleyzac,N.; Boulieu,R- High-performance liquid chromatographic determination of ganciclovir nucleotides in
human myocardial tissue, J.Chromatogr.B, 1994, 658, 173-176.
Gemeprost
Merck Index: 4393
Lednicer No.: 4 11
SAMPLE
Matrix: blood
Sample preparation: Acidify plasma to pH 3.5 with 2 M HCl, pass through a column of Am-
berlite XKD-2 with a bed volume equal to half the sample volume, wash with water until the
eluate is neutral, elute with MeOH (equal to half the original volume), inject an aliquot.
HPLC VARIABLES
Column: 200 X 10 25-40 |xm Polygosil 60 C18 glass column
Detector: Radioactivity
KEY WORDS
REFERENCE
Dimov,V.; Green,K; Bygdeman,M.; Christensen,N.J. Metabolism of 16, 16-dimethyl-ra7is-delta2-prostaglandin
E1 methyl ester (ONO-802) following intravenous and vaginal administration to pregnant women, Drug
Metab.Dispos., 1986, 14, 494-502.
Gentamicin
CAS Registry No.: 1403-66-3, 1405-41-0 (sulfate)
Merck Index: 4398
SAMPLE
Matrix: blood
Sample preparation: Prepare a column of 150 mg dry silicic acid in a Pasteur pipette plugged
with glass wool (10 mm height) and treat with 1 mL water. 500 fxL Serum + 1.5 mL water,
add to the column, rinse the tube with 1 mL water, add this water to the column, discard the
eluate, add 500 jxL reagent, let stand for 30 s, elute, discard the eluate, elute with 1.5 mL
MeOH. Vortex the eluate, centrifuge, protect from light, inject a 20 JJLL aliquot. (Elution was
performed under positive pressure from a rubber bulb. Reagent was prepared by dissolving 1
g of boric acid in 38 mL water, adjust pH to 10.4 with 450 g/L KOH, add 2 mL 100 mg/mL o-
phthalaldehyde in MeOH, add 400 (xL 2-mercaptoethanol. Prepare fresh each week.)
HPLCVARIABLES
Guard column: 23 X 3.9 ixBondapak C18/Porasil B
Column: 300 X 3.9 10 |xm ixBondapak
Mobile phase: MeOHibuffer 21:79 (Buffer was 1% triethylamine adjusted to pH 6.2 0.1 with
phosphoric acid.)
Flow rate: 2
CHROMATOGRAM
Retention time: 4.8 (Cl), 6.8 (CIa), 8.6 (C2)
Limit of detection: 80 ng/mL (CIa, C2), 20 ng/mL (Cl)
KEYWORDS
serum; derivatization; SPE; pharmacokinetics
REFERENCE
Rumble,R-H.; Roberts,M.S. High-performance liquid chromatographic assay of the major components of gen-
tamicin in serum, J.Chromatogr., 1987, 419, 408-413.
SAMPLE
Matrix: blood
Sample preparation: 50 jxL Serum + 20 |xL water + 50 jxL buffer, vortex for 15 s, add 200 |xL
MeCN, vortex for 15 s, centrifuge at 2000 g for 5 min. Filter (0.45 |xm, Millex-HV4) the super-
natant and add 300 fxL of the filtrate to 20 |xL 250 mg/mL l-fluoro-2,4-dinitrobenzene in MeCN.
Heat at 80 for 2 h, cool rapidly to room temperature, filter (0.45 fxm, Millex-HV4), inject a 50
|xL aliquot of the filtrate. (Buffer was prepared by dissolving 3.81 g disodium tetraborate de-
cahydrate in water, adjusting pH to 10 with NaOH, and making up to 100 mL with water.)
HPLCVARIABLES
Guard column: 33 X 4.6 5 (xm C18 (Perkin-Elmer)
Flow rate: 2.2
Detector: UV 365
CHROMATOGRAM
Internal standard: gentamicin CIa
OTHER SUBSTANCES
Extracted: netilmicin
KEYWORDS
serum; guinea pig; human; derivatization; gentamicin CIa is IS
REFERENCE
Dionisotti,S.; Bamonte,E; Gamba,M.; Ongini,E. High-performance liquid chromatographic determination of
netilmicin in guinea-pig and human serum by fluorodinitrobenzene derivatization with spectrophotometric
SAMPLE
Matrix: blood
Sample preparation: 50 JULL Serum + 20 jxL water + 50 JULL buffer, vortex for 15 s, add 200 JULL
MeCN, vortex for 15 s, centrifuge at 2000 g for 5 min. Filter (0.45 |xm, Millex-HV4) the super-
natant and add 300 |xL of the filtrate to 20 (xL 250 mg/mL l-fluoro-2,4-dinitrobenzene in MeCN.
Heat at 80 for 2 h, cool rapidly to room temperature, filter (0.45 |xm, Millex-HV4), inject a 50
fxL aliquot of the filtrate. (Buffer was prepared by dissolving 3.81 g disodium tetraborate de-
cahydrate in water, adjusting pH to 10 with NaOH, and making up to 100 mL with water.)
HPLCVARIABLES
Guard column: 33 X 4.6 5 |xm C18 (Perkin-Elmer)
Flow rate: 2.2
Detector: UV 365
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
serum; guinea pig; human; derivatization; gentamicin CIa is IS
REFERENCE
Riley,C.M. Stability of milrinone and digoxin, furosemide, procainamide hydrochloride, propranolol hydrochlo-
ride, quinidine gluconate, or verapamil hydrochloride in 5% dextrose injection, Am.J.Hosp.Pharm., 1988,
45, 2079-2091.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Plasma +20 JJLL water + 50 |xL buffer, vortex for 15 s, add 200 |JLL
MeCN, vortex for 20 s, centrifuge at 2000 g for 5 min. Filter (Millex-HV4) the supernatant.
Heat 200 |xL filtrate and 20 JJLL 250 mg/mL l-fluoro-2,4-dinitrobenzene in MeCN at 80 for 1
h, cool, inject a 50 JJLL aliquot. (Buffer was 3.81 g disodium tetraborate decahydrate in water,
adjust pH to 10 with NaOH, make up to 100 mL with water.)
HPLC VARIABLES
Guard column: 25 X 4 10 jxm LiChroCART RP 18
Column: 250 X 4 5 jxm LiChrosorb RP 18
Mobile phase: MeCN:water 70:30 containing 1 mL/L acetic acid
Flow rate: 2
Detector: UV 365
CHROMATOGRAM
Retention time: 10.0 (gentamicin CIa)
OTHER SUBSTANCES
Extracted: isepamicin
Noninterfering: ampicillin, aspirin, captopril, cefazolin, cefotaxime, ceftazidime, ceftriaxone,
cephalosporins, chlorpromazine, diazepam, heparin, propranolol, sulfamethoxazole, sulpiride,
trimethoprim, verapamil
KEYWORDS
plasma; guinea pig; human; derivatization; gentamicin CIa is IS
REFERENCE
Dionisotti,S.; Bamonte,R; Scaglione,E; Ongini,E. Simple measurement of isepamicin, a new aminoglycoside
antibiotic, in guinea pig and human plasma, using high-performance liquid chromatography with ultraviolet
detection, Ther.Drug Monit, 1991, 13, 73-78.
SAMPLE
Matrix: blood, broth
Sample preparation: Condition a 3 mL 100 mg Isolute CBA-bonded (carboxypropyl) silica SPE
cartridge (Jones Chromatography) with 1 mL MeOH and 1 mL 20 mM pH 7.4 phosphate buffer.
Add 1 mL plasma or broth to the SPE cartridge, wash with 2 mL 20 mM pH 7.4 phosphate
buffer, wash with 4 mL 200 mM pH 9.0 borate buffer, dry with 30 mL air, elute with 1 mL
MeCN:200 mM pH 10.5 borate buffer 50:50, force out all the liquid with air. 1 mL Eluate +
200 (xL 800 mM boric acid + 200 |xL 2.5 mM 9-fluorenylmethyl chloroformate in MeCN, let
stand at room temperature for 15 min, add 25 |xL 100 mM glycine, let stand for 2 min, inject
a 50 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCNiwater 87:13
Flow rate: 1
CHROMATOGRAM
Retention time: 18 (Cla), 20 (C2), 22 (C2J, 24 (C1)
KEYWORDS
REFERENCE
Stead,D.A.; Richards,R-M.E. Sensitive fluorimetric determination of gentamicin sulfate in biological matrices
using solid-phase extraction, pre-column derivatization with 9-fluorenylmethyl chloroformate and reversed-
phase high-performance liquid chromatography, J.Chromatogr.B, 1996, 675, 295302.
SAMPLE
Matrix: blood, dialysate, urine
Sample preparation: Plasma. Condition a 3 mL Baker cyanopropylsilane CN SPE cartridge
with 2 mL MeOH, 2 mL water, and 2 mL buffer. 1 mL Plasma + 100 |JLL 100 |xg/mL dibekacin
in water, vortex for 15 s, add 1 mL buffer, vortex for 15 s, centrifuge at 3100 g at 4 for 7 min,
add to SPE cartridge, wash with 500 jxL water, wash with 250 |xL mobile phase, elute to
dryness. Elute with 250 |xL mobile phase, inject an aliquot of the eluate. Urine, dialysate.
Dilute 1:100 with water, add 100 |JLL 100 |xg/mL dibekacin per 1 mL of sample, mix well, inject
a 100 |JLL aliquot. (Buffer was 0.94 g sodium hexanesulfonate in 300 mL water, add 500 JXL
glacial acetic acid, dilute to 500 mL with water.)
HPLC VARIABLES
Guard column: 10 X 4.6 5 jxm Hypersil C18
Mobile phase: MeOH:buffer 15:85 (Buffer was 3.48 g sodium hexanesulfonate + 28.4 g sodium
sulfate in 2 L water, acidify to pH 3.4 with 2 mL glacial acetic acid.)
Flow rate: 1.1
Detector: F ex 338 em 418 (bandpass filter) following post-column reaction. The column effluent
mixed with the reagent pumped at 0.4 mL/min and the mixture flowed through a 3 m X 0.05
mm i.d. knitted PTFE reaction coil at 25 to the detector (Derivatizing reagent was 0.4 g o-
phthalaldehyde in 3 mL MeOH added to 390 mL buffer, add 2 mL p-mercaptoethanol, make
up to 500 mL with water, store at 4. Buffer was 1 M pH 10.4 borate from equal volumes of 1
M KOH and boric acid.)
CHROMATOGRAM
Retention time: 11, 17, 17.5, 22
Internal standard: dibekacin
OTHER SUBSTANCES
Simultaneous: kanamycin, isepamicin, tobramycin, netilmicin
KEYWORDS
post-column reaction; SPE; plasma
REFERENCE
Maloney,J.A.; Awni,W.M. High-performance liquid chromatographic determination of isepamicin in plasma,
urine and dialysate, J.Chromatogr., 1990, 526, 487-496.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y.; PSpin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 7631149-
163.
SAMPLE
Matrix: bulk
Sample preparation: Prepare a 2 mg/mL solution in 20 mM pH 9.0 borate buffer, remove a 5
mL aliquot and add it to 15 mL 150 mM 2,4-dinitrofluorobenzene in MeOH (prepare fresh
daily), heat at 100 for 45 min, cool, make up to 250 mL with mobile phase, discard the upper
aqueous phase, inject a 20 |xL aliquot of the lower organic phase.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm LiChrosorb SI-100
Mobile phase: Chloroform:THF:water 65:17.5:0.2
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Retention time: 8, 12, 16
KEYWORDS
normal phase; derivatization
REFERENCE
Tsuji,K; Goetz,J.R; VanMeter,W.; Gusciora,K.A. Normal-phase high-performance liquid chromatographic de-
termination of neomycin sulfate derivatized with l-fluoro-2,4-dinitrobenzene, J.Chromatogr., 1979, 175,
141-152.
SAMPLE
Sample preparation: Bulk. Weigh out amount equivalent to 100 mg gentamicin, dissolve in 100
mL water. Remove a 20 mL aliquot and add it to 10 mL reagent, make up to 50 mL with
MeOH, heat at 90 for 15 min, cool for 5 min, inject a 20 |xL aliquot. Injections. 500 \xL of a
5% injection + 19.5 mL water + 10 mL reagent, heat at 90 for 15 min, cool for 5 min, inject a
20 (JLL aliquot. (Reagent was 400 mg o-phthalaldehyde in 4 mL MeOH, add 38 mL buffer, add
0.8 mL thioglycolic acid, adjust the pH to 10.4 with 45% KOH. Buffer was 6.18 g boric acid in
200 mL water, adjust pH to 10.4 with 45% KOH, make up to 250 mL with water.)
HPLC VARIABLES
Guard column: 45 X 4.6 Vydac reversed phase
Column: 150 X 4.6 Ultrasphere ODS
Mobile phase: Gradient. A was MeOH:water:acetic acid 700:250:50 containing 5 g/L sodium hep-
tanesulfonate. B was MeOH. A:B 100:0 for 2 min, to 75:25 over 3 min, maintain at 75:25.
Flow rate: 1.5
Detector: UV 330
CHROMATOGRAM
Retention time: 5.07 (Cl), 11.06 (CIa), 12.67 (C2a), 13.77 (C2)
KEYWORDS
injections; derivatization
REFERENCE
Albracht,J.H.; de Wit,M.S. Analysis of gentamicin in raw material and in pharmaceutical preparations by high-
performance liquid chromatography, J.Chromatogr., 1987, 389, 306-311.
SAMPLE
Sample preparation: Prepare a 100-200 |xg/mL solution in water, inject a 20 JLJLL aliquot.
HPLCVARIABLES
Guard column: 50 X 4 Carbopac PA-I anion-exchange (Dionex)
Column: 250 X 4 Carbopac PA-I anion-exchange (Dionex)
Mobile phase: Gradient. Water: 10 mM NaOH from 70:30 to 50:50 over 15 min, re-equilibrate
for 5 min.
Flow rate: 1
Detector: E, Dionex PED-I pulsed amperometric detector, gold working electrode, amperometry
mode, El 0.10 V, t l 300 ms, E2 0.60 V t2 120 ms, E3 -0.80 V t3 300 ms following post-column
reaction. The effluent from the column was mixed with 500 mM NaOH pumped at 0.5 mL/min
and flowed through a mixing coil (Dionex RDM) to the detector.
CHROMATOGRAM
Retention time: 5.91 (CIa), 6.66 (C2), 8.04 (C2a), 10.05 (Cl)
OTHER SUBSTANCES
Noninterfering: cefazolin, clindamycin, cloxacillin, kanamycin, neomycin, penicillin G,
tobramycin
KEYWORDS
post-column reaction; injections
REFERENCE
Kaine,L.A.; Wolnik,K.A. Forensic investigation of gentamicin sulfates by anion-exchange ion chromatography
with pulsed electrochemical detection, J.Chromatogr.A, 1994, 674, 255-261.
SAMPLE
Sample preparation: Mix 2 g cream with 3 mL n-butanol, add 5 mL 2% sulfuric acid, mix
thoroughly. Separate lower aqueous layer and re-extract the organic layer with another portion
of sulfuric acid. Combine the aqueous layers and make up to 100 mL with water. Filter a
portion of the extract through a 0.45 |xm Nylon 66 syringe filter. Dilute the filtrate with water,
inject a 10 |xL. aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Metachem Inertsil C8
Mobile phase: 200 mM Sodium sulfate containing0.3 mM sodium 1-heptanesulfonate and 0.1%
acetic acid
Flow rate: 1
agent pumped at 1.0 mL/min and this mixture flowed through a 9 m X 0.25 mm LD. stainless
steel coil to the detector. (Reagent was 800 mg o-phthalaldehyde and 1 mL mercaptoethanol
in 10 mL MeOH diluted to 1 L with 2.5% boric acid and adjusted to pH 10 with 2.5% KOH.)
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: paromomycin
KEYWORDS
cream; post-column reaction
REFERENCE
Pick,J.; Olson,L.L.; Ellis,W.Y.; Lim,R Development and validation of a method to extract and quantitate
paromomycin and gentamicin from an Aquaphilic cream formulation, J.Pharm.Biomed.Anal., 1997, 16,
131-137.
SAMPLE
Sample preparation: Dilute 150 |xL sample to 5 mL, inject an aliquot.
HPLCVARIABLES
Guard column: C18 precolumn filter
Mobile phase: MeCN:200 mM KH2PO4 30:70 adjusted to pH 6.5
Flow rate: 2
Detector: UV 260
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
stability-indicating; ophthalmic solutions
REFERENCE
McBride,H.A.; Martinez,D.R.; Trang,J.M.; Lander,R.D.; Helms,H.A. Stability of gentamicin sulfate and tobra-
mycin sulfate in extemporaneously prepared ophthalmic solutions at 8 degrees C,Am.J.Hosp.Pharm., 1991,
48, 507-509.
Gentian violet
Merck Index: 4401
SAMPLE
Matrix: feed
Sample preparation: Prepare a Sephadex column by slurrying 5 g Sephadex LH-20 with 100
mL MeOHiwater 90:10 in a beaker, let stand for 4 h, add to a 300 X 19 column, wash out
beaker with MeOH:water 90:10 and add this to the column. Grind feed to pass 1 mm sieve
and mix for at least 24 h on a revolving mixer. 10 g Ground feed + 50 mL MeOH: 1 M HCl (99:
1), shake vigorously on a mechanical shaker for 20 min, centrifuge at 2000 rpm for 15 min,
decant, repeat extraction. Combine the extracts, mix well, evaporate an aliquot containing 5
jmg gentian violet to dryness under reduced pressure at 48-50. Reconstitute the residue in 2
mL MeOH:water 90:10, add to the Sephadex column, rinse the flask three times with 2 mL
portions and once with a 6 mL portion of MeOH:water 90:10, add the rinses to the column,
discard the eluates, elute with 6-6.5 mL MeOH:water 90:10, wash the column tip with 1 mL
MeOH:water 90:10, evaporate the eluate to 500 |xL under a stream of nitrogen at 48-50, add
2 mL EtOH, evaporate to dryness under a stream of nitrogen at 48-50, repeat process, recon-
stitute the residue in 1 mL MeOH, inject a 3 |xL aliquot.
HPLC VARIABLES
Guard column: pellicular C18 (Alltech)
Column: 150 X 3.9 4 jxm Nova-Pak RP-C18
Mobile phase: MeOH:buffer 85:15 (Buffer was 10 mM KH2PO4 adjusted to pH 3.0 with phos-
phoric acid.)
Flow rate: 0.75
Injection volume: 3
Detector: UV 588
CHROMATOGRAM
Retention time: 4.4
KEYWORDS
SPE
REFERENCE
Martinez,E.E.; Shimoda,W. Modified liquid chromatographic method for determination of gentian violet in
animal feed, JAssoc.Off.Anal.Chem., 1989, 72, 742-745.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeOH, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm CN (Alltech)
Mobile phase: MeOH: 100 mM sodium acetate 60:40 containing 50 mg/L disodium EDTA, pH
adjusted to 4.5 with aldehyde free glacial acetic acid
Flow rate: 0.8
Detector: E, Bioanalytical Systems LC-4B, glassy carbon working electrode +1.000 V, Ag/AgCl
reference electrode
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: metabolites, leucogentian violet
Interfering: methylene blue
REFERENCE
Roybal,J.E.; Munns,R.K.; Hurlbut,J.A.; Shimoda,W. High-performance liquid chromatography of gentian violet,
its demethylated metabolites, leucogentian violet and methylene blue with electrochemical detection,
J.Chromatogr., 1989, 467, 259-266.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 2.1 5 |xm Ultracarb C18 (Phenomenex)
Flow rate: 0.4
Detector: MS, Hewlett-Packard Model 5989A, source 250, solvation chamber 45, particle beam
nebulizer helium 40-45, scan m/z 80-500
OTHER SUBSTANCES
Simultaneous: brilliant green, leucogentian violet, leucomalachite green, malachite green, pen-
tamethyl gentian violet, tetramethyl gentian violet
REFERENCE
Turnipseed,S.B.; Roybal,J.E.; Rupp,H.S.; Hurlbut,J.A.; Long,A.R. Particle beam liquid chromatography-mass
spectrometry of triphenylmethane dyes: application to confirmation of malachite green in incurred catfish
tissue, J.Chromatogr.B, 1995, 670, 55-62.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 6 mL neutral alumina SPE cartridge (J.T. Baker) and a 2.8
mL 500 mg Bond Elut PRS SPE cartridge with 5 mL MeCN. Place the alumina SPE cartridge
on the top of the PRS SPE cartridge using a Bond Elut adapter. Mix 3 mL 250 mg/mL hy-
droxylamine hydrochloride in water with 5 mL 50 mM p-toluene sulfonic acid, 20 mL 100 mM
ammonium acetate adjusted to pH 4.5 with glacial acetic acid, and 20 g fish tissue. Homogenize
at 20000 rpm for 1 min (Ultra-Turrax T25 Tissuemizer, Tekmar, USA). Add 90 mL MeCN to
the sample and homogenize for 10 s. Shake vigorously by hand for 1 min. Add 20 g basic
alumina (Brockman activity I), shake for 1 min. Centrifuge, decant the supernatant. Add 30
mL MeCN to the residue, extract, combine the supernatants. Add 100 mL water, 50 mL di-
chloromethane, and 20 mL diethylene glycol to the supernatants, shake vigorously, separate
the bottom layer. Again add 50 mL dichloromethane, shake for 1 min, combine the separated
dichloromethane layers. Concentrate to 2-3 mL under reduced pressure at 65. Add 2 mL di-
chloromethane and 5 mL MeCN then add the mixtures to the SPE cartridges. Rinse the flask
twice with 5 mL portions of MeCN and add the rinses to the SPE cartridges. Wash with 5 mL
MeCN to waste. Remove the alumina cartridge. Wash the PRS cartridge with 2 mL water and
with 1 mL MeCN: 100 mM ammonium acetate buffer 50:50 adjusted to pH 4.5 with glacial
acetic acid. Elute with 2 mL MeCN: 100 mM ammonium acetate buffer 50:50 adjusted to pH
4.5 with glacial acetic acid and collect in a tube containing 500 |xL 2.5 mg/mL hydroxylamine
hydrochloride in water. Inject a 100 jxL aliquot.
HPLCVARIABLES
Guard column: 20 X 2.0 pellicular C18
Column: 150 X 4.6 5 urn SynChropak SCD-100 (SynChrom)
Mobile phase: MeCN:buffer 55:45 (Buffer was 400 mg ammonium acetate and 1 mL triethylam-
ine in 400 mL water, adjusted to pH 3.0 with glacial acetic acid, and made up to 450 mL with
water.)
Flow rate: 2.0
Detector: E, ESA Coulochem Model, oxidative electrochemical cell (EC) +900 mV; UV 588; F ex
265 em 360
CHROMATOGRAM
Retention time: 7.9
OTHER SUBSTANCES
Extracted: metabolites, malachite green
KEYWORDS
SPE; catfish; trout
REFERENCE
Rushing,L.G.; Hansen,E.B.,Jr. Confirmation of malachite green, gentian violet and their leuco analogs in catfish
and trout tissue by high-performance liquid chromatography utilizing electrochemistry with ultraviolet-
visible diode array detection and fluorescence detection, J.Chromatogr.B, 1997, 700, 223-231.
SAMPLE
Matrix: tissue
Sample preparation: Prepare an alumina column by adding 5 g alumina (Alcoa type F-20, 80-
200 mesh, activated chromatographic grade) to a 75 X 16 column. Condition a Bond Elut
disposable LRC carboxylic acid SPE cartridge with 10 mL MeCN. 25 g Ground tissue + 100
mL MeCN:buffer 80:20, shake vigorously for 1 min, let stand for 30 min, shake for 1 min,
protect from light, let stand overnight, centrifuge at 2000 rpm for 15 min, filter (paper) the
supernatant, add 100 mL MeCN:buffer 80:20 to the residue, shake vigorously for 15 s, protect
from light, let stand for 1 h, centrifuge, filter (paper). Combine the filtrates, add 25 mL water,
add 2 mL diethylene glycol, extract with 100 mL dichloromethane. Evaporate the dichloro-
methane extract just to dryness, add 5 mL MeCN to the residue, add to the alumina column,
rinse flask three times with 5 mL portions of MeCN, add the rinses to the column, elute with
10 mL MeCN, call all the eluates and evaporate them just to dryness. Reconstitute the residue
in 25 mL dichloromethane, add 15 mL citrate buffer, shake vigorously for 30 s, remove the
organic layer, extract the aqueous layer with two 25 mL portions of dichloromethane. Combine
the organic layers and evaporate them just to dryness. Reconstitute with 10 mL MeCN, add a
5 mL aliquot to the SPE cartridge, wash with 5 mL MeCN, discard all eluates, elute with two
2 mL portions of acidic MeOH, evaporate eluate under a stream of nitrogen at 40 to 1 mL,
inject a 20 |xL aliquot. (Citrate buffer (pH 6-7) was prepared fresh just before use from 1 part
1 M HCl and 2 parts saturated sodium citrate. Acidic MeOH was 95 mL, 5 mL water and 2
mL concentrated HCl, dilute a 1 mL aliquot of this solution to 100 mL with MeOH.)
HPLC VARIABLES
Column: 250 X 4.6 5 jim CN (Alltech)
Mobile phase: MeCN:buffer 50:50 (Buffer was 100 mM sodium acetate adjusted to pH 4.5 with
acetic acid containing 50 mg/L EDTA.)
Flow rate: 1
Detector: E, Bioanalytical Systems LC-4B, glassy carbon electrode 1.000 V, Ag/AgCl reference
electrode
CHROMATOGRAM
Retention time: 16
Limit of detection: <10 PPB
OTHER SUBSTANCES
Extracted: metabolites, leucogentian violet
KEYWORDS
chicken; liver; muscle; SPE; protect from light
REFERENCE
Roybal,J.E.; Munns,R.K.; Hurlbut,J.A.; Shimoda,W. Determination of gentian violet, its demethylated metab-
olites, and leucogentian violet in chicken tissue by liquid chromatography with electrochemical detection,
JAssoc.Off.Anal.Chem., 1990, 73, 940-command.946.
SAMPLE
Matrix: tissue
Sample preparation: Condition at 6 mL 1 g neutral alumina SPE cartridge (J.T. baker) with 5
mL MeCN. Condition a 2.8 mL 0.5 g Bond Elut PRS SPE cartridge with 5 mL MeCN. 10 g
Muscle tissue + 3 mL 250 mg/mL hydroxylamine hydrochloride in water + 5 mL 50 mM p-
toluenesulfonic acid + 10 mL 100 mM pH 4.5 ammonium acetate buffer, homogenize (Tekmar
Ultra-Turrax T25 tissuemizer) at 20000 rpm for 1 min, add 90 mL MeCN, homogenize for 10
s, shake vigorously by hand for 1 min, add 20 g basic alumina (Brockman activity I, Fisher
Scientific), shake vigorously for 1 min, centrifuge, decant, add 30 mL MeCN to the residue,
extract, centrifuge, decant. Combine the supernatants and add 100 mL water, add 50 mL
dichloromethane, add 2 mL diethylene glycol, shake vigorously by hand for 1 min, let stand
for 45 min, remove the lower organic layer, add 50 mL dichloromethane, shake for 1 min, let
stand for 5 min, remove the lower organic layers. Combine the organic layers and evaporate
them to 2-5 mL under reduced pressure at 65, add 2 mL dichloromethane, add 5 mL MeCN,
add the mixture to the alumina SPE cartridge on top of the PRS SPE cartridge, rinse the flask
with two 5 mL portions of MeCN, add the rinses to the SPE cartridges, wash the SPE cartridges
with 5 mL MeCN, wash with 1 mL solvent, elute with 1.5 mL solvent, add 500 |xL water to
the eluate, inject a 100 jxL aliquot. (Solvent was MeCNrIOO mM ammonium acetate 50:50,
adjusted to pH 4.5 with glacial acetic acid.)
HPLC VARIABLES
Guard column: 20 X 2 pellicular CN
Column: 250 X 4.6 5 |xm LC-CN (Supelco)
Mobile phase: MeCNibuffer 60:40 (Prepare buffer by dissolving 3.85 g ammonium acetate in 380
mL water, adjust to pH 4.5 with glacial acetic acid, make up to 400 mL with water.)
Flow rate: 1
Detector: UV 588 following post-column oxidation. The column effluent passed through a 20 X
2 column packed with lead(IV) oxide to the detector (Mallinckrodt)
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: leucogentian violet
Simultaneous: methyl violet
KEYWORDS
fish; muscle; catfish; SPE; post-column reaction
REFERENCE
Rushing,L.G.; Webb,S.R; Thompson,H.C.,Jr. Determination of leucogentian violet and gentian violet in catfish
tissue by high-performance liquid chromatography with visible detection, J.Chromatogr.B, 1995, 674,125-
131.
SAMPLE
Matrix: tissue
Sample preparation: Condition at 6 mL 1 g neutral alumina SPE cartridge (J.T. baker) with 5
mL MeCN. Condition a 2.8 mL 0.5 g Bond Elut PRS SPE cartridge with 5 mL MeCN. 20 g
Muscle tissue + 3 mL 250 mg/mL hydroxylamine hydrochloride in water + 5 mL 50 mM p-
toluenesulfonic acid in water + 20 mL 100 mM pH 4.5 ammonium acetate buffer, homogenize
(Tekmar Ultra-Turrax T25 tissuemizer) at 20000 rpm for 1 min, add 90 mL MeCN, homogenize
for 10 s, shake vigorously by hand for 1 min, add 20 g basic alumina (Brockman activity I,
Fisher Scientific), shake vigorously for 1 min, centrifuge, decant, add 30 mL MeCN to the
residue, extract, centrifuge, decant. Combine the supernatants and add 100 mL water, add 50
mL dichloromethane, add 2 mL diethylene glycol, shake vigorously by hand for 1 min, let stand
for 45 min, remove the lower organic layer, add 50 mL dichloromethane, shake for 1 min, let
stand for 5 min, remove the lower organic layer, repeat the extraction with 50 mL dichloro-
methane. Combine the organic layers and evaporate them to 2-5 mL under reduced pressure
at 65, add 2 mL dichloromethane, add 5 mL MeCN, add the mixture to the alumina SPE
cartridge on top of the PRS SPE cartridge, rinse the flask with two 5 mL portions of MeCN,
add the rinses to the SPE cartridges, wash the SPE cartridges with 5 mL MeCN. Discard the
alumina cartridge, wash the PRS cartridge with 2 mL water, wash with 1 mL solvent, elute
with 2 mL solvent, add 500 jxL 2.5 mg/mL hydroxylamine hydrochloride in water to the eluate,
inject a 100 (JLL aliquot. (Solvent was MeCN: 100 mM ammonium acetate 50:50, adjusted to pH
HPLC VARIABLES
Guard column: 20 X 2 pellicular C18
Column: 150 X 4.6 5 jjum SynChropak SCD-100 (SynChrom)
Mobile phase: MeCN:buffer 55:45 (Prepare buffer by dissolving 400 mg ammonium acetate and
1 mL triethylamine in 400 mL water, adjust to pH 3.6 with glacial acetic acid, make up to 450
mL with water.)
Flow rate: 2
Detector: UV 588 following post-column oxidation. The column effluent passed through a 20 X
2 column packed with lead(IV) oxide (Mallinckrodt) to the detector.
CHROMATOGRAM
Retention time: 8.2
OTHER SUBSTANCES
Extracted: leucogentian violet, leucomalachite green, malachite green, methyl violet
KEYWORDS
fish; muscle; catfish; trout; SPE; post-column reaction
REFERENCE
Rushing,L.G.; Thompson,H.C.,Jr. Simultaneous determination of malachite green, gentian violet and their
leuco metabolites in catfish and trout tissue by high-performance liquid chromatography with visible de-
tection, J.Chromatogr.B, 1997, 688, 325-330.
Gentisic acid
Merck Index: 4404
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
triptyline amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, aspi-
ethacrynic acid, ethosnximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fen-
fluorouracil, fluoxymesterone, fluphenazine, gitoxigenin, glipizide, glunixin, glutethimide, gly-
zepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxjrmorphone,
thioridazine, thiosalicylic acid, thiothixene, th3rmol, tolazamide, tolazoline, tobutamide, tol-
REFERENCE
18, 233-242.
Glafenine
Molecular formula: Ci9H17CIN2O4
Merck Index: 4443
SAMPLE
Matrix: blood, formulations
Sample preparation: Blood. Add 3 mL MeOH.aqueous ammonia solution 99.5:0.5 to 500 |xL
serum, shake vigorously, centrifuge at 300 rpm for 15 min, evaporate the supernatant to dry-
ness under a stream of nitrogen, dissolve the residue in 200 JXL MeOH, filter (0.2 jxm Fluore-
pore), inject an aliquot. Tablets. Dissolve 200 mg (2 tablets) in 500 mL 100 mM HCl, stir at
100 rpm, filter, dilute 2 mL of the filtrate to 100 mL with 100 mM HCl, inject an aliquot.
HPLCVARIABLES
Mobile phase: MeOH:water:H3PO4 40:60:0.25, pH adjusted to 3.5 with 100 mM NaOH
Flow rate: 1.5
Detector: UV 360
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
rat; serum; tablets
REFERENCE
Hassan,S.S.M.; Elnemma,E.M.; Abbas,A.B. Determination of glafenine in dosage forms and serum by thin layer
densitometry and high performance liquid chromatography, J.Pharm.Biomed.Anal., 1997, 16, 215-221.
Glibomuride
Molecular formula: Ci6H26N2O4S
Merck Index: 4447
Lednicer No.: 2 117
SAMPLE
Matrix: blood
the residue in 100 IJLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of
HPLC VARIABLES
Column: 300 X 3.9 4 |jim NovaPack C18
Flow rate: 0.8
Detector: UV 229
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
Gliclazide
Merck Index: 4448
SAMPLE
residue with 50 (xL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Glimepiride
Merck Index: 4449
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 40 JJLL 10 ixg/mL ISl in MeOH containing 10 jxg/mL IS2 +
1 mL 50 mM pH 1 HC1/KC1 buffer + 5 mL diethyl ether, shake for 20 min, centrifuge at 2500
g for 5 min. Remove 4 mL of the organic layer and evaporate it to dryness under a stream of
nitrogen at 30, reconstitute the residue in 100 |xL reagent, heat at 100 for 20 min, evaporate
to dryness under a stream of nitrogen at 60, reconstitute with 200 |xL initial mobile phase,
inject a 100 |xL aliquot. (Prepare reagent by dissolving 30 JJLL 2,4-dinitrofluorobenzene in 10
mL n-butyl acetate.)
HPLC VARIABLES
Mobile phase: Gradient. MeCN:50 mM perchloric acid 40:60 for 6 min, to 58:42 (step gradient),
maintain at 58:42 for 8 min, re-equilibrate at initial conditions for 2 min.
Flow rate: 2
Detector: UV 350
CHROMATOGRAM
Internal standard: ISl (l-[4-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-l-carboxamido)ethyl]phenyl
sulfonyl]-3-(4-ethylcyclohexyl)urea) (13.4), IS2 (144-[2-(5-chloro~2-methoxyphenyl-l-carboxam-
ido)ethyl]phenylsulfonyl]-3-(4-hydroxycyclohexyl)urea) (3.4)
OTHER SUBSTANCES
Simultaneous: glibornuride, glyburide, tolbutamide
KEYWORDS
derivatization; serum; silanize glassware with dichlorodimethylsilane; pharmacokinetics
REFERENCE
Lehr,K.H.; Damm,R Simultaneous determination of the sulphonylurea glimepiride and its metabolites in hu-
man serum and urine by high-performance liquid chromatography after pre-column derivatization,
J.Chromatogr., 1990, 526, 497-505.
Glipizide
Molecular formula: C2IH27N5O4S
Merck Index: 4451
Lednicer No.: 2 117
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Glucagon
CAS Registry No.: 9007-92-5, 16941-32-5
Merck Index: 4455
HisSerGInGIyThrPheThrSerAspTyrSerLysTyrLeuAspSerArg
ThrAsnMetLeuTrpGinVaIPheAspGInAlaArg
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1 mg/mL solution in dilute HCl, inject a 2.5 jxL aliquot.
HPLC VARIABLES
Column: 300 X 4 10 fxm Polygosil C18 (Macherey Nagel) (Condition column as follows. Elute
with a linear gradient from MeOH to toluene, pump 10 mL 0.1 g/mL chlorodimethyloctylsilane
in toluene through at 0.2 mL/min at 48, flush (at 1 mL/min) with a linear gradient of MeOH,
MeOH:water:trifluoroacetic acid 50:50:0.1, MeOH, and MeOHxhloroform 50:50, flush overnight
at 0.2 mL/min with MeOH with a gradient.)
Mobile phase: MeOH:water:trifluoroacetic acid 64:36:0.1
Flow rate: 1.2
Detector: UV 205
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Interfering: secretin
KEYWORDS
Pig
REFERENCE
01ieman,C; Sedlick,E.; Voskamp,D. In situ silylation of an octadecylsilyl-silica stationary phase applied to the
analysis of peptides, such as secretin and glucagon, J.Chromatogr., 1981, 207, 421-424.
Gluconolactone
Merck Index: 4465
SAMPLE
HPLC VARIABLES
Column: 250 X 4.1 PRP-X300 (Hamilton)
Mobile phase: 50 mM pH 4.5 NaH2PO4
Flow rate: 0.5
Detector: UV 200
CHROMATOGRAM
Retention time: 5.3
OTHER SUBSTANCES
Simultaneous: gluconic acid, dextrose
REFERENCE
Baxter Scientific Products Catalog, 1990-1, p. 123.
SAMPLE
Matrix: urine
Sample preparation: Lyophilize a 5 mL aliquot of urine at -50 for 18 h, reconstitute the residue
in 5 mL DMF, centrifuge at 3000 g for 10 min. 1 mL Supernatant + 300 JJLL phenylisocyanate,
heat at 100 for 1 h, cool, add 500 |xL MeOH, inject an aliquot.
HPLC VARIABLES
Column: 220 X 4.6 5 |xm ODS 224 RP18 (Brownlee)
Flow rate: 2
Detector: UV
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Extracted: galactonolactone, galactitol
Simultaneous: dextrose, galactose, allose, myoinositol, sorbitol, mannitol
KEYWORDS
derivatization
REFERENCE
Rakotomanga,S.; Baillet,A.; Pellerin,R; Baylocq-Ferrier,D. Simultaneous determination of gluconolactone, ga-
lactonolactone and galactitol in urine by reversed-phase liquid chromatography: application to galactosemia,
J.Chromatogn, 1991, 570, 277-284.
Glutethimide
Merck Index: 4485
Lednicer No.: 1 257
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Serum + 200 |xL 50 fjug/mL hexobarbital in MeCN + 25 (xL glacial
acetic acid, vortex for 10 s, centrifuge for 1 min, inject a 30-100 (JLL aliquot of the supernatant.
HPLC VARIABLES
Column: |iBondapak C18
5:95 for 5 min.
Flow rate: 3
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
ethchlorvynol, flurazepam, methaqualone, methyprylon, nitrazepam, pentobarbital, phenobar-
bital, phenytoin, primidone, salicylic acid, secobarbital, theophylline
mine, lidocaine, mesantoin, methsuximide, nirvanol, nortriptyline, oxazepam, procainamide,
phenylpropanolamine, propranolol, quinidine
KEYWORDS
serum
REFERENCE
Kabra,P.M.; Stafford,B.E.; Marton,L.J. Rapid method for screening toxic drugs in serum with liquid chroma-
tography, J.AnalToxicol, 1981, 5, 177-182.
SAMPLE
Matrix: blood
IxL 10 jjLg/mL tolylphenobarbital in 200 mM HCl to the SPE cartridge, let stand for 2 min,
elute with 1 mL chloroform:isopropanol 6:1. Evaporate the eluate to dryness under a stream
of nitrogen at 30, reconstitute the residue in 100 |xL mobile phase, inject a 15 |xL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 ^m Supelcosil-LC-8
Flow rate: 3.3
Detector: UV 208
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: theophylline, caffeine, barbital, ethosuximide, primidone, carbamazepinediol, phen-
acemide, methyprylon, nirvanol, phenobarbital, chloramphenicol, butabarbital, carbamazepine
epoxide, mephenytoin, pentobarbital, amobarbital, carbamazepine, phenytoin, secobarbital,
methaqualone
Noninterfering: acetaminophen, amikacin, amitriptyline, clonazepam, cyclosporine, desipra-
mine, diazepam, digoxin, disopyramide, gentamicin, imipramine, lidocaine, methotrexate, N-
acetylprocainamide, netilmicin, nortriptyline, procainamide, quinidine, salicylic acid, sulfa-
methoxazole, tobramycin, trimethoprim, valproic acid, p-hydroxyphenobarbital, vancomycin
KEYWORDS
plasma; SPE
REFERENCE
ration, Clin.Chem., 1989, 35, 1615-1618.
SAMPLE
Matrix: blood
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 259
CHROMATOGRAM
KEYWORDS
amitriptyline; nortriptyline; tioclomarol; dicloienac; mefloquine; trimipramine; chlorambucil;
REFERENCE
254-262.
SAMPLE
Sample preparation: Cool 1 mL microsomal incubation to 0, add 3 mL ethyl acetate, shake in
a reciprocal shaker for 10 min, centrifuge at 2500 g for 10 min. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen, reconstitute with running buffer, inject an
aliquot.
HPLCVARIABLES
Guard column: 4 X 4 10 um RP 8 (Merck)
Column: 250 X 4 5 jxm Superspher RP 8
Mobile phase: MeCN: 10 mM pH 6.5 tetrabutylammonium hydrogen sulfate 30:70
Flow rate: 0.7
Detector: UV 210
CHROMATOGRAM
Retention time: 33
OTHER SUBSTANCES
KEYWORDS
comparison with CE; rat; liver
REFERENCE
Weinz,C; Blaschke,G.; Schiebel,H.-M. Investigation of the stereoselective in vitro biotransformation of gluteth-
imide by high-performance liquid chromatography and capillary electrophoresis, J.Chromatogr.B, 1997,690,
233-242.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 1 5 urn LiChrosorb RP18
cyclodextrin
Flow rate: 0.04
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: mephobarbital
KEYWORDS
chiral
REFERENCE
Nowakowski,R.; Bielejewska,A.; Duszczyk,K; Sybilska,D. Chiral discrimination by high-performance liquid
chromatography with joint use of two cyclodextrin additives, J.Chromatogr.A, 1997, 782, 111.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: MeCN:water:glacial acetic acid 4:84:12 containing 4.84 g/L Trizma, pH 2.3
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: acetaminophen, caffeine, cefazolin, cimetidine, ergotamine, heparin, metham-
phetamine, propranolol, salicylic acid, sulfamethoxazole, theobromine, theophylline, tobutam-
ide, trimethoprim
Noninterfering: amitriptyline, amobarbital, ampicillin, butabarbital, butalbital, celbenine,
chlordiazepoxide, chlorpromazine, clorazepate, desipramine, diazepam, doxepin, ethchlorvynol,
fluphenazine, hydroxyzine, ibuprofen, imipramine, isoniazid, lidocaine, mephobarbital, mesor-
idazine, methaqualone, methyluric acid, naprotyline, nordiazepam, nortriptyline, oxazepam,
pentobarbital, perphenazine, phenelzine, phenmetrazine, phenobarbital, phenylbutazone,
phenytoin, prednisolone, prednisone, procainamide, prochlorperazine, promazine, prometha-
zine, propoxyphene, protriptyline, pyrilamine, secobarbital, thioridazine, thiothixene, timolol,
trazodone, triazolam, trifluoperazine
REFERENCE
Osterloh,J.; Yu,S. Simultaneous ion-pair and partition liquid chromatography of acetaminophen, theophylline
and salicylate with application to 500 toxicologic specimens, Clin.Chim.Acta, 1988, 175, 239-248.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 10 urn Chiralcel OJ
Mobile phase: HexanerEtOH 60:40
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 8.73 (R-(+)), 16.20 (S-(-))
OTHER SUBSTANCES
Simultaneous: 4-hydroxyglutethimide
KEYWORDS
chiral
REFERENCE
Aboul-Enein,H.Y.; Islam,M.R. Isocratic high-performance liquid chromatographic resolution of glutethimide
enantiomers and their 4-hydroxyglutethimide metabolites using cellulose tribenzoate chiral stationary
phase, J.Chromatogr.ScL, 1990, 28, 307-310.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: 6-acetylmorphine, amiloride, amphetamine, benzocaine, benzoylecgonine, caf-
feine, cocaine, codeine, doxylamine, fluoxetine, hexobarbital, hypoxanthine, levorphanol, LSD,
meperidine, mephobarbital, methadone, methylphenidate, methyprylon, N-norcodeine, oxaze-
pam, oxycodone, phenylpropanolamine, prilocaine, procaine, terfenadine
REFERENCE
Ascah,T.L. Improved separations of alkaloid drugs and other substances of abuse using Supelcosil LC-ABZ
column, Supelco Reporter, 1993, 12(3), 18-21.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
fluorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide,
REFERENCE
Hill,D.W.; Kind,A-J- Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.ToxicoL, 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 jjum LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
tadine, baclofen, benzocaine, bromocriptine, brompheniramine, brotizolam, bupivacaine, bu-
spirone, butabarbital, butalbital, caffeine, carbamazepine, cetirizine, chlorcyclizine,
azine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glyburide, guaifenesin, haloperidol,
amic acid, meperidine, mephenjrtoin, mepivacaine, mesoridazine, metaproterenol, methadone,
zopiclone
KEYWORDS
Next Page
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: MeOH
Flow rate: 0.5
Detector: UV 210
CHROMATOGRAM
Retention time: 10 (R-(+)), 16 (S-(-))
OTHER SUBSTANCES
Simultaneous: metabolites, 5-hydroxyglutethimide
KEYWORDS
chiral
REFERENCE
233-242.
G Iy bu ride
Merck Index: 4486
Lednicer No.: 2 139
SAMPLE
Matrix: blood
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 229
Previous Page
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: MeOH
Flow rate: 0.5
Detector: UV 210
CHROMATOGRAM
Retention time: 10 (R-(+)), 16 (S-(-))
OTHER SUBSTANCES
Simultaneous: metabolites, 5-hydroxyglutethimide
KEYWORDS
chiral
REFERENCE
233-242.
G Iy bu ride
Merck Index: 4486
Lednicer No.: 2 139
SAMPLE
Matrix: blood
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 229
CHROMATOGRAM
KEYWORDS
amitriptyline; nortriptyline; tioclomarol; diclofenac; menoquine; trimipramine; chlorambucil;
lidoflazine; ibuprofen; noctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpi-
REFERENCE
TracquiÂ..; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-eompound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763,149-
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
glunixin, guaiacol, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocortisone,
epinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, ox5rphen-
REFERENCE
18, 233-242.
Glycerin
Merck Index: 4493
SAMPLE
Matrix: bulk
Sample preparation: Prepare an aqueous solution, inject an aliquot.
HPLCVARIABLES
Guard column: 10 |xm Guard-pak (Waters)
Column: 250 X 4.6 5 (xm Ultrasphere ODS C18
Flow rate: 1
Detector: RI
REFERENCE
Cannon,J.M.; Brown,R.D.; Murrill,E.M.; Jameson,C.W. Identification of components in iodinated glycerol,
J.Pharm.ScL, 1989, 78, 48-51.
SAMPLE
Matrix: bulk
HPLCVARIABLES
Guard column: 10 |xm Guard-pak (Waters)
Column: 250 X 4.6 5 jxm Ultrasphere ODS C18
Flow rate: 1
Detector: RI
REFERENCE
Vinas,R; Ldpez Erroz,C; Hernandez Canals,A.; Hernandez C6rdoba,M. Liquid chromatographic analysis of
sulfonamides in foods, Chromatographia, 1995, 40, 382-386.
SAMPLE
Sample preparation: Remove the water from 10 |xL syrup under reduced pressure for 10 min,
reconstitute with 2 mL pyridine. Remove a 25 |xL aliquot and add it to 75 |xL reagent, shake
well, let stand at room temperature for 10 min, evaporate to dryness under reduced pressure
at room temperature, flush the tube with a stream of air or nitrogen, add 2 mL 5% sodium
carbonate solution containing 2.5 mg/mL 4-dimethylaminopyridine, shake or sonicate for 5 min,
extract with 2 mL chloroform. Wash the extract with 2 mL 5% sodium bicarbonate solution,
wash twice with 3 mL portions of 50 mM HCl containing 5% NaCl, inject an aliquot. (Prepare
reagent by dissolving 100 mg 4-nitrobenzoyl chloride in pyridine with gentle warming.)
HPLCVARIABLES
Column: 150 X 3 5 |xm LiChrosorb SI 60
Mobile phase: n-Hexane:chloroform:MeCN 10:3:1.9 containing 0.1% water
Flow rate: 1.4
Detector: UV 260
CHROMATOGRAM
Retention time: 2.5
OTHER SUBSTANCES
Simultaneous: dextrose, fructose, propylene glycol, saccharose, sorbitol
KEYWORDS
syrup; derivatization; normal phase
REFERENCE
Nachtmann,R; Budna,K.W. Sensitive determination of derivatized carbohydrates by high-performance liquid
chromatography, J.Chromatogn, 1977, 136, 279-287.
SAMPLE
Sample preparation: Condition a C18 Sep-Pak SPE cartridge with 2 mL MeOH and 20 mL
water. Dilute formulation ten-fold with water, add a 0.5 mL aliquot to the SPE cartridge, elute
with three 1 mL portions of water, inject a 10 |xL aliquot of the eluate.
HPLC VARIABLES
Column: 8 mm i.d. C18 radial compression (Waters)
Mobile phase: Water
Flow rate: 1
Detector: RI
CHROMATOGRAM
Retention time: 3.4
OTHER SUBSTANCES
Simultaneous: dihydroxyacetone, dioxane, ethylene glycol, formic acid, glyceraldehyde, meth-
ylglyoxal, propylene glycol
KEYWORDS
SPE
REFERENCE
Bobin,M.R; Martini,M.C; Gudefin,A.; Cotte,J. Dosage de Ia dihydroxyacetone dans les Emulsions [Assay of
dihydroxyacetone in emulsions], Farmaco.[Prat]., 1983, 38, 403-414.
SAMPLE
Sample preparation: Weigh out 1-2 mL, add 20 mL water, mix thoroughly, make up to 100 mL
with water, mix thoroughly, inject a 25 (xL aliquot.
HPLC VARIABLES
Guard column: Micro-Guard ion exclusion cartridge (Bio-Rad)
Column: 300 X 7.8 Aminex HPX-87H (Bio-Rad)
Mobile phase: 6.5 mM sulfuric acid
Flow rate: 0.8
Detector: RI
CHROMATOGRAM
REFERENCE
Del Grosso,A.V.; May,J.C. Gas chromatographic, liquid chromatographic, and titrimetric procedures for deter-
mination of glycerin in allergenic extracts and diagnostic antigens: comparative study, J.Assoc.Off.
Anal.Chem., 1987, 70, 825-828.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10 |xL aliquot of an aqueous solution.
HPLC VARIABLES
Column: 250 X 4.6 5 ^m Supelcosil LC-NH2
Flow rate: 1
Detector: RI
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Simultaneous: dextrose, fructose, maltose, sucrose
REFERENCE
Johnson,J.M.; Harris,C.H. Selecting the most effective filtration media for HPLC analysis of saccharides,
J.Chromatogr.Sci, 1987, 25, 267-269.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: ION-300
Mobile phase: 2.5 mM sulfuric acid
Flow rate: 0.4
Detector: RI
CHROMATOGRAM
Retention time: 24
OTHER SUBSTANCES
Simultaneous: acetic acid, citric acid, dextrose, EtOH, fructose, lactic acid, malic acid, MeOH,
tartaric acid
REFERENCE
Keystone Scientific Catalog, 1993-4, p. 45.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: Hamilton PRP-X300
Mobile phase: MeCN:0.5 mM sulfuric acid 10:90
Flow rate: 3
Injection volume: 1
Detector: RI
OTHER SUBSTANCES
Simultaneous: i-butanol, n-butanol, s-butanol, t-butanol, EtOH, isopropanol, MeOH, n-propanol
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: Shodex Sugar SP 0810P and SP 0810
Mobile phase: water
Flow rate: 0.5
Detector: RI
CHROMATOGRAM
Retention time: 27
OTHER SUBSTANCES
Simultaneous: arabinose, dextrose, fructose, galactose, lactose, lactulose, mannitol, pullulan P-
10, raffinose, sorbitol, stachyose, sucrose, xylitol
REFERENCE
Majors,R.E. Polymeric liquid chromatography column technology in Japan, LCGC, 1993, 11, 778-788.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 100 X 7.8 Sarasep AL-I (Sarasep, MetaChem)
Mobile phase: 50 mg/L dicalcium EDTA
Flow rate: 0.7
Detector: RI
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Simultaneous: EtOH, isoamyl alcohol, n-amyl alcohol
REFERENCE
MetaChem Catalog, 1994, p. 65.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: RSpak DC-613 (Shodex, Phenomenex)
Flow rate: 1
Detector: RI
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
Simultaneous: meso-erythrite, arabite, xylite, mannite, sorbite
REFERENCE
Glycopyrrolate
Molecular formula: Ci9H28BrNO3
Merck Index: 4511
SAMPLE
HPLCVARIABLES
Mobile phase: MeCN:buffer 45:55 (Buffer was 10 mM KH2PO4 adjusted to pH 4.0 with 1 M
KOH.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Limit of detection: 6.9 jjig/mL
OTHER SUBSTANCES
Simultaneous: ondansetron
KEYWORDS
injections; saline
REFERENCE
Venkateshwaran,T.G.; King,D.T.; Stewart,J.T. HPLC determination of ondansetron-atropine and ondansetron-
glycopyrrolate mixtures in 0.9% sodium chloride injection, J.Liq.Chromatogr., 1995, 18, 2647-2659.
SAMPLE
Matrix: urine
Sample preparation: Condition a 500 mg 14 mL 40 |xm CCX-2 cation-exchange SPE cartridge
(Worldwide Monitoring) with two 2.5 mL aliquots of MeOH, two 2.5 mL aliquots of water, and
two 2.5 mL aliquots of 100 mM pH 7.00 phosphate buffer, do not allow to dry. 5 mL Urine + 3
mL 100 mM pH 7.00 phosphate buffer + 12.5 ng mepenzolate + 5 mL water, centrifuge at 800
g for 5 min, add to the SPE cartridge, wash with 5 mL MeOH, wash with 5 mL water, dry
under vacuum for 5 min, elute with 4 mL MeOH:500 mM pH 3.00 ammonium acetate 95:5 (all
flow rates were 1-2 mL/min). Evaporate the eluate under a stream of nitrogen at 60, recon-
stitute in 100 fxL MeOH, inject a 10 jxL aliquot.
HPLC VARIABLES
Column: 150 X 4.1 10 (xm LiChroma (Chromatographic Specialties)
Mobile phase: MeOH:50 mM pH 3.0 ammonium acetate 80:20
Flow rate: 0.8
Detector: MS, Sciex API III triple quadrupole, ion spray interface, split column effluent 95:5
before entering detector, nebulizing gas air at 550 kPa, collision gas argon, curtain gas nitrogen,
positive-ion mode, m/z 318 and 116
CHROMATOGRAM
Retention time: 2.3
Internal standard: mepenzolate (m/z 340 and 130) (2.1)
KEYWORDS
SPE; horse
REFERENCE
Matassa,L.C; Woodard,D.; Leavitt,R-K.; Firby,P.; Beaumier,P. Solid-phase extraction techniques for the deter-
mination of glycopyrrolate from equine urine by liquid chromatography-tandem mass spectrometry and gas
chromatography-mass spectrometry, J.Chromatogr., 1992, 573, 43-48.
Gonadorelin
CAS Registry No.: 33515-09-2,
51952-41-1 (HCI), 52699-48-6 (sulfate)
Merck Index: 5500
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Analytichem weak cation-exchange (carboxymethylhy-
drogen form, CBA) SPE cartridge with 1 mL 1% trifluoroacetic acid in MeOH, 1 mL MeOH,
and 2 mL water. Add 1 mL plasma to the SPE cartridge, rinse the tube with 1 mL water, add
the rinse to the SPE cartridge, wash with 1 mL 1% trifluoroacetic acid in water, wash with 2
mL water, wash with 2 mL MeOH, elute with 2 mL 1% trifluoroacetic acid in MeOH. Evaporate
the eluate to dryness under a stream of nitrogen, reconstitute the residue in 100 |xL MeOH:
buffer 50:50, inject a 5-75 |xL aliquot. (Buffer was 5.7 g monochloroacetic acid, 2.0 g NaOH,
and 0.2 g disodium EDTA in 1 L water, pH 3.2.) [Procedure was not necessarily validated for
this compound.]; SPE
HPLC VARIABLES
Column: 250 X 2 5 |xm Ultrasphere octyl
Mobile phase: Gradient. A was MeOH containing 10 mM sodium octanesulfonate. B was buffer
containing 10 mM sodium octanesulfonate. A:B from 45:55 to 70:30 over 30 min, maintain at
70:30 for 1 h. (Buffer was 5.7 g monochloroacetic acid, 2.0 g NaOH, and 0.2 g disodium EDTA
Flow rate: 0.3
Detector: F ex 390 em 470 following post-column reaction. The column effluent mixed with 400
mM NaOH pumped at 0.15 mL/min and 0.05% ninhydrin pumped at 0.05 mL/min and the
mixture flowed through a 12 m X 0.33 mm i.d. reaction coil at 70 to the detector.
CHROMATOGRAM
Retention time: 22
OTHER SUBSTANCES
Simultaneous: adrenocorticotropin, angiotensin I, angiotensin II, angiotensin III, atrial natri-
uretic peptide, bombesin, bradykinin, somatoliberin, vasopressin
KEYWORDS
plasma; SPE; post-column reaction
REFERENCE
Rhodes,G.R.; Boppana,V.K. High-performance liquid chromatographic analysis of arginine-containing peptides
in biological fluids by means of a selective post-column reaction with fluorescence detection, J.Chromatogr.,
1988, 444, 123-131.
SAMPLE
HPLC VARIABLES
Column: 100 X 5 Nucleosil 5 C8
Mobile phase: MeOH.buffer 23:77 (Buffer was 100 mM phosphoric acid adjusted to pH 3.0 with
triethylamine.)
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Retention time: 23
OTHER SUBSTANCES
REFERENCE
Storring,P.L.; Corran,P.H.; Gaines Das,R.E.; Calam,D.H. The International Reference Preparation of Gonado-
relin for Bioassay: a comparison with different preparations of synthetic luteinizing hormone releasing
hormone using physicochemical methods of analysis, different bioassays and immunoassay, J.Endocrinol.,
1982, 95, 95-103.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 4.6 Zorbax ODS
Mobile phase: MeCN.water.pH 3.0 triethylamine phosphate buffer 14:43:43
Flow rate: 1.5
Detector: UV 210
CHROMATOGRAM
KEYWORDS
validation
REFERENCE
Bi,M.; Singh, J. Modified HPLC method for quantification of luteinizing hormone-releasing hormone (Abstract
3367), Pharm.Res., 1997,14, S585.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 125 X 4 5 jxin LiChrospher 100 RP-18
Mobile phase: MeCN:0.1% trifluoroacetic acid 16:84
Flow rate: 1
Detector: UV 214
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
REFERENCE
Hoitink,M.A.; Beijnen,J.H.; BuItA; van der Houwen,O.A.G.J.; Nijholt,J.; Underberg,W.J.M. Degradation ki-
netics of gonadorelin in aqueous solution, J.Pharm.Sci., 1996, 85, 1053-1059.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 125 X 4 5 jjim Lichrosphere 100 RP-18
Mobile phase: MeCNiO. 1% trifluoroacetic acid 16:84
Flow rate: 1
Detector: UV 214
OTHER SUBSTANCES
REFERENCE
Hoitink,M.A.; Beijnen,J.H.; Boschma,M.U.S.; Bult,A.; Hop,E.; Nijholt,J.; Versluis,C; Wiese,G.; Under-
berg,W.J.M. Identification of the degradation products of gonadorelin and three analogues in aqueous so-
lution, Anal. Chem., 1997, 69, 4972-4978.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 X 3.9 10 jjum uJBondapak C18
Mobile phase: Gradient. A was 0.1% phosphoric acid. B was MeCN:0.1% phosphoric acid 70:30.
A:B from 95:5 to 30:70 over 20 min.
Flow rate: 1
Detector: UV 206 or RIA
CHROMATOGRAM
Retention time: 17
OTHER SUBSTANCES
Simultaneous: protirelin, somatostatin, substance P
REFERENCE
McDermott,J.R.; Smith,A.I.; Biggins,J.A.; Al-Noaemi,M.C; Edwardson,J.A. Characterization and determina-
tion of neuropeptides by high-performance liquid chromatography and radioimmunoassay, J.Chromatogr.,
1981, 222, 371-379.
SAMPLE
Matrix: solutions
Sample preparation: Cool in ice while mixing 200 fxL solution, 100 |xL 4 mM benzoin in 2-
methoxyethanol, 100 |ULL mercaptoethanol solution, and 200 |xL 2 M KOH, heat on a boiling
water bath for 5 min, cool in ice-water for 2 min, add 200 jxL 4 M HCl:buffer 50:50, inject a
100 |xL aliquot. (Prepare mercaptoethanol solution by dissolving 780 mg p-mercaptoethanol
and 2.52 g sodium sulfite in 80 mL water, make up to 100 mL with water. Prepare buffer by
dissolving 12.11 g Tris in 80 mL water, adjusting pH to 9.2 with concentrated HCl, and making
up to 100 mL with water.)
HPLC VARIABLES
Column: 300 X 3.9 10 |xm jxBondapak phenyl
Mobile phase: Gradient. MeOH:water:500 mM pH 8.5 Tris-HCl buffer 50:35:15 for 2 min, to 80:
5:15 over 24 min, maintain at 80:5:15 for 2 min.
Flow rate: 0.8
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: N-a-acetylarginine, agmatine, angiotensin I, angiotensin II, angiotensin III, ar-
ginine, argininosuccinic acid, bradykinin, canavanine, creatine, creatinine, guanidine, guani-
dinoacetic acid, guanidinobutyric acid, guanidinopropionic acid, guanidinosuccinic acid, ho-
moarginine, methylguanidine, neurotensin, phenylguanidine, taurocyamine, tuftsin
KEY WpRDS
derivatization
REFERENCE
Kai,M.; Miyazaki,T.; Yamaguchi,M.; Ohkura,Y. High-performance liquid chromatography of guanidino com-
pounds using benzoin as a pre-column fluorescent derivatization reagent, J.Chromatogr., 1983, 268, 417-
424.
SAMPLE
Matrix: solutions
Sample preparation: Cool in ice while mixing 100 JXL of an aqueous solution, 50 |JLL 5 mM
benzoin in 2-methoxyethanol, 50 |xL mercaptoethanol solution, and 100 (xL 0.8 M KOH, heat
on a boiling water bath for 1.5 min, add 100 \xL 1.6 M HCLl M pH 8.5 Tris-HCl buffer 50:50,
inject a 100 JJLL aliquot. (Prepare mercaptoethanol solution by dissolving 780 mg p-mercapto-
ethanol and 2.52 g sodium sulfite in 80 mL water, make up to 100 mL with water.)
HPLCVARIABLES
Column: 15 X 4 (sic) 5 |xm LiChrosorb RP-18
Flow rate: 0.8
CHROMATOGRAM
Retention time: 22
OTHER SUBSTANCES
Simultaneous: angiotensin I, angiotensin II, angiotensin III, leupeptin acid, substance P, tuftsin
KEY WpRDS
derivatization
REFERENCE
Kai,M.; Miyazaki,T.; Sakamoto,Y.; Ohkura,Y. Use of benzoin as pre-columnfluorescencederivatization reagent
for the high-performance liquid chromatography of angiotensins, J.Chromatogr., 1985, 322, 473-477.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a solution in saline or Sorensen's buffer.
HPLCVARIABLES
Column: 150 X 4.6 5 jjim Econosphere endcapped C18
Mobile phase: MeCN:water 80:20 containing 0.1% trifluoroacetic acid
Detector: UV 278
CHROMATOGRAM
Retention time: 5.2
OTHER SUBSTANCES
REFERENCE
Miller,L.L.; Kolaskie,C.J.; Smith,G.A.; Rivier,J. Transdermal iontophoresis of gonadotropin releasing hormone
(LHRH) and two analogues, J.Pharm.Sci., 1990, 79, 490-493.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 TSKgel ODS-120T
Mobile phase: Gradient. A was MeOHiwater 20:80 containing 0.05% trifluoroacetic acid. B was
MeOH:water 50:50 containing 0.05% trifluoroacetic acid. A:B from 100:0 to 0:100 over 1 h.
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: angiotensin I, angiotensin II, a-endorphin, p-endorphin, calcitonin (human), pro-
tirelin (TRH), somatostatin
REFERENCE
Varian Catalog, 1993, p. 182.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 200 X 3 Spherisorb S5ODS-2
Mobile phase: Gradient. A was 0.05% phosphoric acid containing 0.5% (NH4)2SO4. B was MeCN.
A:B from 82:18 to 64:36 over 25 min, maintain at 64:36 for 2.5 min, return to initial conditions
over 1 min, re-equilibrate for 6.5 min. or Isocratic MeCN:0.05% phosphoric acid containing
0.5% (NHJ2SO4 24:76
Flow rate: 0.5
Detector: UV 210
CHROMATOGRAM
Retention time: 9 (gradient), 2.5 (isocratic)
OTHER SUBSTANCES
Simultaneous: buserelin, deslorelin, goserelin, leuprolide, nafarelin
KEYWORDS
REFERENCE
Corran,P.H.; Sutcliffe,N. Identification of gonadorelin (LHRH) derivatives: comparison of reversed-phase high-
performance liquid chromatography and micellar electrokinetic chromatography, J.Chromatogr., 1993,636,
87-94.
SAMPLE
Matrix: tissue
Sample preparation: Condition a Bond Elut C18 SPE cartridge with 3 mL water and 3 mL
MeCN (in this order ?). Homogenize 400 mg brain tissue with 2 mL 100 mM HCl, add 20 (JLL
10 |xM IS, add 2 mL acetone, mix, centrifuge at 2450 g for 15 min. Remove the supernatant
and add it to 220 yJu 1 M sodium bicarbonate and 500 /JLL 100 mM disodium EDTA, centrifuge
at 2450 g for 15 min. Remove the supernatant and evaporate it to remove the acetone, dilute
the aqueous residue with 2 mL water, add to the SPE cartridge, wash with 1 mL water, wash
with 3 mL 100 mM HCl, wash with two 3 mL portions of dichloromethane, wash with 1 mL
water, wash with 2 mL 100 mM pH 8.0 phosphate buffer, wash with 2 mL water, elute with 2
mL MeCN: 100 mM pH 2.3 phosphate buffer 70:30. Evaporate the eluate under reduced pres-
sure, make up to 400 |xL with water, inject a 100 |xL aliquot.
HPLCVARIABLES
Column: 200 X 4 5 um TSKgel ODS-120T (Tosoh)
Mobile phase: Gradient. A was MeCN:300 mM pH 2.3 sodium phosphate buffer.water 1:20:79.
B was MeCN:300 mM pH 2.3 sodium phosphate buffer:water 60:20:20. A:B from 90:10 to 55:
45 over 33 min, maintain at 55:45 for 7 min, to 0:100 (step gradient), maintain at 0:100.
Flow rate: 1
mM benzoin in 1.6 M KOH containing 700 mM 2-mercaptoethanol and this mixture flowed
through a 15 m X 0.33 mm ID PTFE coil at 76 1. The effluent from this coil mixed with
500 mM Tris containing 2.1 M HCl pumped at 0.4 mL/min and this mixture flowed to the
detector.
CHROMATOGRAM
Internal standard: [D-Phen]-neurotensin (40.0)
OTHER SUBSTANCES
Extracted: bradykinin, dynorphin 1-8, kallidin, leucine enkephalin-Arg, methionine enkephalin-
Arg-Gly-Leu, methionine enkephalin-Arg-Phe, a-neoendorphin, p-neoendorphin, neurotensin,
substance P, vasopressin
KEYWORDS
post-column reaction; rat; brain; SPE
REFERENCE
Ohno,M.; Kai,M.; Ohkura,Y. High-performance liquid chromatographic determination of substance P-like ar-
ginine-containing peptide in rat brain by on-line post-column fluorescence derivatization with benzoin,
J.Chromatogr., 1989, 490, 301-310.
SAMPLE
Matrix: tissue
Sample preparation: Tissue homogenate + MeCN, vortex for 10 s, centrifuge at 10000 g for 15
min. Remove the supernatant and evaporate it to dryness under a stream of nitrogen, recon-
stitute the residue in 100 jxL mobile phase, inject an aliquot.
HPLCVARIABLES
Mobile phase: Gradient. MeCN:0.6% aqueous ethanolamine, pH 3.0 14:86 for 5 min, to 30:70
over 8 min, maintain at 30:70 for 7 min, re-equilibrate at initial conditions for 5 min.
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
KEY WORDS
rabbit; rectal mucosa; vaginal mucosa; nasal mucosa
REFERENCE
Han,K.; Park,J.S.; Chung,Y.B.; Lee,M.J.; Moon,D.C; Robinson,J.R. Identification of enzymatic degradation
products of luteinizing hormone releasing hormone (LHRH)/[D-Ala6] LHRH in rabbit mucosal homogenates,
Pharm.Res., 1995, 12, 1539-1544.
Gonadotropin
Molecular weight: ca. 39500
Merck Index: 2273
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 300 X 4 10 |xm u-Bondapak alkylphenyl
Mobile phase: Gradient. A was 100 mM pH 7.0 ammonium acetate. B was MeCN:water 60:40
containing 15 mM trifluoroacetic acid, pH 2.0. A:B from 100:0 to 0:100 over 90 min.
Flow rate: 1.2
Detector: UV 278
CHROMATOGRAM
Retention time: 30 (ctl), 32 (a2), 33 (pi), 34 (2) (subunits)
REFERENCE
Grego,B.; Hearn,M.T.W. High-performance liquid chromatography of amino acids, peptides and proteins. LXIII.
Reversed-phase high-performance liquid chromatographic characterisation of several polypeptide and pro-
tein hormones, J.Chromatogr., 1984, 336, 25-40.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Vydac C4 300 A no. 214TP54
Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in water. B was 0.1% trifluoroacetic acid
in MeCN. A:B from 100:0 to 40:60 over 90 min.
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 50 (a-1), 52 (a-2), 50 ((3-1), 54 (p-2) (subunits)
REFERENCE
Pollak,S.; Halpine,S.; Chait,B.T.; Birken,S. High resolution high performance liquid chromatography finger-
printing of purified human chorionic gonadotropin demonstrates that oxidation is a cause of hormone het-
erogeneity, Endocrinology, 1990, 126, 199-208.
Goserelin
Merck Index: 4547
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 125 X 4 5 |xm Lichrosphere 100 RP-18
Mobile phase: MeCNrO. 1% trifluoroacetic acid 22:78
Flow rate: 1
Detector: UV 214
OTHER SUBSTANCES
Also analyzed: triptorelin
REFERENCE
Hoitink,M.A.; Beijnen,J.H.; Boschma,M.U.S.; Bult,A.; Hop,E.; Nijholt,J.; Versluis,C; Wiese,G.; Under-
berg,W. J.M. Identification of the degradation products of gonadorelin and three analogues in aqueous so-
lution, Anal.Chem., 1997, 69, 4972-4978.
Gossypol
Merck Index: 4549
SAMPLE
Matrix: blood
Sample preparation: 400 |xL serum + 500 |xL saturated disodium EDTA, mix, let stand for 10
min, adjust to pH 8.0 with concentrated NaOH solution using a micro-electrode, add 50 \LL 1
g/mL L-phenylalanine methyl ester in MeCN, let stand for 10 min, adjust pH to 7.00 with
concentrated sulfuric acid, add 2 mL ether, vortex, centrifuge, repeat extraction. Combine the
organic layers and evaporate them to dryness under reduced pressure, reconstitute the residue
in 500 |xL MeCN, inject a 50 jxL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:THF:buffer 76:2:22 (Buffer was 10 mM KH2PO4 adjusted to pH 2.35 with
phosphoric acid.)
Flow rate: 2.5
Detector: UV 250
CHROMATOGRAM
KEYWORDS
serum; derivatization; chiral
REFERENCE
Matlin,S.A.; BelengueiyA..; Vince,RM.; Stein,R. Analysis of gossypol enantiomers in human serum,
J.Liq.Chromatogr., 1990, 13, 2261-2268.
Gramicidin
Molecular formula: C60Hg2N12O10
113-73-5 (gramicidin S)
Merck Index: 4552
SAMPLE
Matrix: saliva
Sample preparation: Mix saliva with an equal volume of EtOH, centrifuge.
HPLC VARIABLES
Mobile phase: MeCN:water:phosphoric acid 47.5:52.5:0.01
Flow rate: 2.5
Detector: UV 220
CHROMATOGRAM
Retention time: 13 (valin-gramicidin A)
KEYWORDS
pharmacokinetics
REFERENCE
Kreuzig,E; Nahler,G. Salivary levels of gramicidin after use of a tyrothricin lozenge and a tyrothricin gargle/
mouth-wash, Int.J.Clin.Pharmacol.Res., 1983, 3, 65-70.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 X 7.8 Ultrastyragel 1000 A (Waters)
Mobile phase: THF
Flow rate: 1
Injection volume: 2
CHROMATOGRAM
Retention time: 7.9 (dimers), 8.4 (monomer)
REFERENCE
Bano,M.C; Braco,L.; Abad,C. New high-performance liquid chromatography-based methodology for monitoring
the conformational transitions of self-associating hydrophobic peptides, incorporated into liposomes,
J.Chromatogr., 1988, 458, 105-116.
Granisetron
Merck Index: 4557
Lednicer No.: 5 118
SAMPLE
Matrix: blood
Sample preparation: Filter (Amicon Centricon-10 microconcentrator, 10000 daltons molecular
weight cutoff) 150 JXL plasma while centrifuging at 5 at 5000 g for 100 min, inject a 10 fxL
aliquot of the ultrafiltrate.
HPLCVARIABLES
Column: 100 X 4.6 5 |xm Spheri-5 silica
Flow rate: 1
Detector: UV 305
CHROMATOGRAM
Retention time: 3.4
KEYWORDS
plasma; guinea pig; ultrafiltrate
REFERENCE
Capacio,B-R; Byers,C.E.; Jackson,T.K.; Matthews,R-L. An HPLC method for the determination of granisetron
in guinea pig plasma, JAnaLToxicoL, 1993,17, 151-155.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond Elut C2 SPE cartridge with two 1 mL portions
of MeOH, 1 mL water, and two 1 mL portions of buffer. 1 mL Plasma + 100 |xL 40 ng/mL IS
in water + 500 \LL buffer, add to the SPE cartridge, wash with 1 mL MeCN:water 40:60, remove
all wash solvent, elute with 800 |xL MeOH. Evaporate the eluate to dryness under a stream
of nitrogen at 50. Add 30 |xL 10% trimethylsilyldiazomethane in hexane and 30 |xL 2% N ,N-
diisopropylethylamine in MeOH to the residue, heat at 50 for 20 min, cool to room tempera-
ture, evaporate to dryness under a stream of nitrogen at 50, reconstitute in 300 (xL MeOH:
water 10:90, centrifuge at 1700 g for 5 min, inject a 150 \LL aliquot. (Phosphate buffer was
4.33 g Na2HPO4 and 3.04 g NaH2PO4.2H2O in 50 mL water.)
HPLCVARIABLES
Guard column: 15 X 3.2 NewGuard RP-18
Column: 250 X 4.6 5 fxm Develosil ODS-5 (Nomura Chemical)
Mobile phase: MeOH:buffer 30:70 (Buffer was 15.4 g ammonium acetate and 20 mL tetra-n-
butylammonium hydroxide in 1.7 L water, adjust pH to 4.70 with glacial acetic acid, make up
to 2 L with water.)
Flow rate: 1
CHROMATOGRAM
Retention time: 9.5
Internal standard: 2-methyl-M-(endo-9-methyl-9-azabicyclo[3.3. l]non-3-yl)-2H-indazole-3-car-
boxamide hydrochloride (6)
OTHER SUBSTANCES
KEYWORDS
plasma; SPE; derivatization
REFERENCE
Kudoh,S.; Sato,T.; Okada,H.; Kumakura,H.; Nakamura,H. Simultaneous determination of granisetron and 7-
hydroxygranisetron in human plasma by high-performance liquid chromatography with fluorescence detec-
tion, J.Chromatogr.B, 1994, 66O1 205-210.
SAMPLE
Matrix: blood
Sample preparation: Condition a C2 SPE cartridge (Analytichem) with 1 mL MeOH, 1 mL
water, and 1 mL 1 M pH 7.0 phosphate buffer. 1 mL Plasma + 50 |xL water + 50 JULL 100 ng/
mL IS in water + 500 |xL 1 M pH 7.0 phosphate buffer, add immediately to SPE cartridge,
wash with 1 mL water, wash with 1 mL MeCN:water 40:60, remove wash solvent completely,
elute with 1 mL MeOH, elute with 1 mL MeOH:trifluoroacetic acid 99:1. Combine the eluates
and evaporate them to dryness under a stream of nitrogen at 40, reconstitute the residue in
200 |xL MeOH.water 10:90, vortex for 30 s, centrifuge at 2000 g for 5 min, transfer to a fresh
vial, centrifuge at 2000 g for 5 min, inject a 10-65 |xL aliquot.
HPLC VARIABLES
Guard column: 30 X 2.1 C8 (ABI Instruments)
Column: 150 X 2.1 Zorbax Rx C8
Mobile phase: MeCN:buffer 19:81 (Buffer was 0.95 g sodium hexanesulfonate in 405 mL 100
mM pH 4.7 acetate buffer.)
Flow rate: 0.3
Detector: E, ESA, El 0.15 V, E2 0.35 V (measuring electrode), guard cell 0.4 V (before injector)
followed by F ex 305 em 360
CHROMATOGRAM
Retention time: 17 (F)
Internal standard: 8-methyl-8-azabicyclo[3.2.1]oct-3-yl l-methyl-lH-indazole-3-carboxylate
(BRL 43704) (21) (F)
OTHER SUBSTANCES
KEYWORDS
plasma; SPE; fluorescence detection for granisetron and some metabolites; electrochemical detec-
tion for other metabolites
REFERENCE
Boppana,V.K Simultaneous determination of granisetron and its 7-hydroxy metabolite in human plasma by
reversed-phase high-performance liquid chromatography utilizing fluorescence and electrochemical detec-
tion, J.ChromatogrA, 1995, 692, 195-202.
SAMPLE
Matrix: blood
Sample preparation: 500 u,L Plasma + 7 jxg IS, vortex for 10 s, add 1.5 mL toluene, add 250
JXL buffer, shake for 20 min, centrifuge at 3000 g for 10 min. Remove 1 mL of the organic layer
IxL mobile phase, vortex for 10 s, inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:buffer 15:85 (Buffer was 100 mM NaH2PO4 adjusted to pH 4.5 with ortho-
phosphoric acid.)
Flow rate: 2
CHROMATOGRAM
Internal standard: N-(l-naphthyl)ethylenediamine dihydrochloride (3.60)
OTHER SUBSTANCES
Noninterfering: metabolites, anthracyclines, cimetidine, cisplatin, dexamethasone, etoposide,
fluorouracil, methotrexate, methylprednisolone, ondansetron
KEYWORDS
plasma
REFERENCE
Pinguet,F.; Bressolle,E; Martel,R; Salabert,D.; Astre,C. High-performance liquid chromatographic determina-
tion of granisetron in human plasma, J.Chromatogr.B, 1996, 675, 99-105.
SAMPLE
Matrix: blood, pleural effusion, urine
Sample preparation: Mix 500 JJLL serum, pleural effusion, or urine with 100 jxL IS solution and
400 |xL pH 9.0 ammonium acetate. Add to an Extrelut-1 SPE cartridge. After 15 min, elute
with 3 mL and 4 mL portions of dichloromethane. Evaporate the eluate to dryness under a
stream of nitrogen at 40, reconstitute the residue with 100 |xL mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 10 |xm LiChroCART LiChrospher 100 CN
Mobile phase: MeGN:100 mM pH 3.5 acetate buffer 30:70
Flow rate: 1
CHROMATOGRAM
Internal standard: BRL 43693A (Smith Kline Beecham) (9.5)
OTHER SUBSTANCES
Extracted: etoposide
Simultaneous: domperidone, metoclopramide, ondansetron
Noninterfering: cisplatin, carboplatin, dexamethasone
KEYWORDS
REFERENCE
Wada,L; Satoh,M.; Takeda,T.; Nakabayashi,T.; Honma,T.; Saitoh,H.; Takada,M.; Hirano,K. A rapid assay of
granisetron in biological fluids from cancer patients, Biol.PharmBull., 1998, 21, 535-537.
SAMPLE
Sample preparation: 1 mL Plasma or urine + 100 JJLL IS in water + 500 |xL 100 mM pH 12
phosphate buffer + 3 mL toluene, shake mechanically for 30 min, centrifuge. Remove the or-
ganic layer and evaporate it to dryness under a stream of nitrogen at 60, reconstitute the
residue in 100 |xL mobile phase, inject an 80 |xL aliquot.
HPLC VARIABLES
Guard column: 50 mm long unspecified
Column: 250 X 4.5 10 JJLM Apex CN
Mobile phase: MeOH.buffer 97:3 (Buffer was 50 mM sodium acetate containing 0.25% triethy-
lamine adjusted to pH 6.0.)
Flow rate: 1
CHROMATOGRAM
Internal standard: endo-l-methyl-O-(9-methyl-9-azabicyclo(3,3,l)non-3-yl)-lH-indazole-3-car-
boxylate (BRL 43704) (19.2)
KEYWORDS
plasma; human; rat; dog
REFERENCE
Clarkson,A.; Coates,P.E.; Zussman,B.D. A specific h.p.l.c. method for the determination of BRL 43694 in plasma
and urine, Br.J.Clin.Pharmacol, 1988, 25, 136P.
SAMPLE
Sample preparation: Dilute 1-mg granisetron hydrochloride injection with 0.9% NaCl or 5%
dextrose to a granisetron concentration of 100 |xg/mL, inject a 20 |xL aliquot of the solution.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherisorb Cyano
Mobile phase: MeCNrIOO mM NaH2PO4 15:85
Flow rate: 2
Detector: UV 302
CHROMATOGRAM
Retention time: 9.1
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Quercia,R.A.; Zhang,J.; Fan,C; Chow,M.S.S. Stability of granisetron hydrochloride in polypropylene syringes,
Am.J.Health-SystPharm., 1996, 53, 2744-2746.
SAMPLE
Sample preparation: Filter (0.2 |xm nylon), inject a 50 |xL aliquot of the filtrate.
HPLC VARIABLES
Column: 250 X 4.6 5 fjtm Accubond CN (J & W)
Mobile phase: MeCN:buffer 60:40 (Buffer was 20 mM KH2PO4 containing 5 mM octanesulfonic
acid, adjusted to pH 6.0 with 1 M NaOH.)
Flow rate: 1.5
Detector: UV 305
CHROMATOGRAM
Retention time: 7.5
KEYWORDS
injections; saline; 5% dextrose; stability-indicating
REFERENCE
Chung,K.C; Chin,M.A.; Gill,M.A. Stability of granisetron hydrochloride in a disposable elastomeric infusion
device, Am. J.Health-Syst.Pharm., 1995, 52, 1541-1543.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 fim Spherisorb CN
Mobile phase: MeCN: 100 mM pH 4.5 NaH2PO4 15:85
Detector: UV 300
CHROMATOGRAM
Retention time: 6.8
Internal standard: propylparaben (3.3)
OTHER SUBSTANCES
Simultaneous: dexamethasone, methylprednisolone
KEYWORDS
injections; 5% dextrose; saline; water
REFERENCE
Pinguet,R; Rouanet,R; Martel,R; Fabbro,M.; Salabert,D.; Astre,C. Compatibility and stability of granisetron,
dexamethasone, and methylprednisolone in injectable solutions, J.Pharm.ScL, 1995, 84, 267-268.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 10 ^m cyano
Flow rate: 2
Detector: UV 300
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: dexamethasone (UV 228)
KEYWORDS
stability-indicating; injections; saline; 5% dextrose; apple juice; orange juice; soft drinks
REFERENCE
Mayron,D.; GennaroA-R- Stability and compatibility of granisetron hydrochloride in i.v. solutions and oral
liquids and during simulated Y-site injection with selected drugs, Am. J.Health-Syst.Pharm., 1996,53,294
304.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1
Detector: UV 307
CHROMATOGRAM
Retention time: 4.5
OTHERSUBSTANCES
Simultaneous: doxorubicin (11.2)
REFERENCE
Zhang,H.; Ye,L.; Stewart,J.T. HPLC determinations of doxorubicin with selected medications in 0.9% sodium
chloride injection USP, J.Liq.Chromatogr.Rel.TechnoL, 1998, 21, 2375-2385.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4 Nucleosil C18
Mobile phase: MeOH:THF:buffer 30:5:65 (Buffer was 100 mM triethylamine adjusted to pH 3.0
with nitric acid.)
Flow rate: 0.8
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: ondansetron, tropisetron
REFERENCE
Barbato,R; Immacolata La Rotonda,M.; Quaglia,F. Retention behaviour of anti-emetic serotonin antagonists in
reversed phase high performance liquid chromatography, Farmaco, 1995, 50, 875-880.
Grepafloxacin
CAS Registry No.: 119914-60-2, 146863-02-7 ( ) ,
161967-81-3 (() HCI)
Merck Index: 4567
SAMPLE
Matrix: blood
Sample preparation: Extract 200 |xL plasma with dichloromethanern-butanol 95:5 and evapo-
rate to dryness. Treat the organic layer residue with Mosher's acid chloride (R-(-)- or S-(+)-a-
methoxy-a -trifluoromethylphenylacetic acid chloride) and triethanolamine in dichloromethane
for 1 hr. Reconstitute, inject an aliquot.
HPLC VARIABLES
Column: 150 X 3.2 5|xm ODS
Mobile phase: MeCN:0.2% phosphoric acid 70:30
CHROMATOGRAM
Retention time: 12.7 (S-(-)), 13.5 (R-(+))
KEY WpRDS
REFERENCE
Tata,RN.V.; Bramer,S.L. Enantiomeric assay of grepafloxacin in plasma (Abstract 4162), Pharm.Res., 1997,14,
S684.
Griseofulvin
Molecular formula: C17H17CIO6
Merck Index: 4571
Lednicer No.: 1 314
SAMPLE
M a t r i x : blood
Sample preparation: 100 |xL Plasma + 100 (JLL p-phenylphenol in MeCN, vortex, centrifuge,
inject a 20 JJLL aliquot of the supernatant.
HPLC VARIABLES
Column: 150 mm long 5 |xm Novapack
Mobile phase: MeCN:0.1 M acetic acid, pH 3.5 45:55
Flow rate: 1
Detector: UV 290
CHROMATOGRAM
Internal standard: p-phenylphenol (4.5)
KEYWORDS
REFERENCE
Vudathala,G.K; Rogers,J.A. Oral bioavailability of griseofulvin from aged griseofulvin: lipid coprecipitates: in
vivo studies in rats, J.Pharm.ScL, 1992, 82, 1166-1169.
SAMPLE
Matrix: blood, CSF, gastric contents, urine
Sample preparation: 200 |xL Serum, urine, CSF, or gastric fluid + 300 |xL reagent. Flush column
A to waste with 500 |xL 500 mM ammonium sulfate, inject sample onto column A, flush column
A to waste with 500 (xL 500 mM ammonium sulfate, backflush the contents of column A onto
column B with mobile phase, monitor the effluent from column B. (Reagent was 8.05 M gua-
nidine HCl and 1.02 M ammonium sulfate in water.)
HPLCVARIABLES
Column: A 40 |xm preparative grade C18 (Analytichem); B 75 X 2.1 pellicular C18 (Whatman)
+ 250 X 4.6 5 fun C8 end-capped (Whatman)
Mobile phase: Gradient. A was 50 mM pH 4.5 KH2PO4. B was MeCNiisopropanol 80:20. A:B 90:
10 for 1 min, to 30:70 over 20 min.
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Internal standard: heptanophenone (19)
OTHERSUBSTANCES
Extracted: acetaminophen, allobarbital, azinphos, barbital, brallobarbitone, bromazepam, bu-
tethal, caffeine, carbamazepine, carbaryl, cephaloridine, chloramphenicol, chlordiazepoxide,
chlorothiazide, chlorvinphos, clothiapine, cocaine, coomassie blue, desipramine, diazepam, di-
phenhydramine, dipipanone, ethylbromphos, flufenamic acid, formothion, indomethacin, lido-
caine, lorazepam, malathion, medazepam, midazolam, oxazepam, paraoxon, penicillin G, pen-
tobarbital, prazepam, propoxyphene, prothiophos, quinine, salicylic acid, secobarbital,
strychnine, sulfamethoxazole, theophylline, thiopental, thioridazine, trimethoprim
KEYWORDS
serum; column-switching
REFERENCE
Kruger,P.B.; Albrecht,C.F.De V.; Jaarsveld,P.P. Use of guanidine hydrochloride and ammonium sulfate in com-
prehensive in-line sorption enrichment of xenobiotics in biological fluids by high-performance liquid chro-
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 292.6
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
Sample preparation: Mix an excess of griseofulvin with 8 mL water or with 8 mL 6-7 mM
sodium dodecyl sulfate. Shake in a 25 water bath for 48 h, filter through a 0.45 ^m. filter.
Discard the first a few mL, inject a 150 JJLL aliquot of the rest.
HPLCVARIABLES
Column: 140 x 4.6 5|xm cyanopropyl methyl silane bonded silica (Supelco Inc., PA)
Flow rate: 1
Detector: UV 295
REFERENCE
Rao,V.M.; Lin,M.; Larive,C.K; Southard,M.Z. A mechanistic study of griseofulvin dissolution into surfactant
solutions under laminar flow conditions, J.Pharm.Sci., 1997, 86, 1132-1137.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in mobile phase, inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Spherisorb 5 ODS
Mobile phase: MeCN: 10 mM phosphoric acid 34:66, pH 2.82
Flow rate: 1
Detector: UV 248
CHROMATOGRAM
Internal standard: griseofulvin
OTHER SUBSTANCES
Simultaneous: methoxsalen
KEYWORDS
SPE; griseofulvin is IS
REFERENCE
Kucov,D.; Maryskov&,D.; Davidkov&,R; Gasparic,J. High-performance liquid chromatographic determination
of methoxsalen in plasma after liquid-solid extraction, J.Chromatogr., 1993, 614, 340-344.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 |xm RP 18
Detector: UV 245 and 315
CHROMATOGRAM
REFERENCE
De Smidt,J.H.; Grit,M.; Crommelin,D.J.A. Dissolution kinetics of griseofulvin in mixed micellar solutions,
J.Pharm.Sci., 1994, 83, 1209-1212.
Guaiacol
Merck Index: 4575
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
Gaillard,Y.; Pe"pin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHERSUBSTANCES
glunixin, glutethimide, halazepam, haloperidol, hydrochlorothiazide, hydrocodone, hydrocorti-
sone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, in-
yldopamine, methylphenidate, methylprednisolone, methyltestosterone, methjrprylon, meto-
metin, tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifiuoperazine,
trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tâmine, verapa-
REFERENCE
Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnaLToxicol., 1994,
18, 233-242.
Guaifenesin
Merck Index: 4582
Lednicer No.: 1 118
SAMPLE
Matrix: blood
Sample preparation: 500 jxL Plasma + 100 jxL 2.5 mg/mL O-desmethylnaproxen in MeOH +
400 |xL acetone, homogenize for 10 min, centrifuge at 1000 g for 15 min. Remove supernatant
and evaporate it to dryness under a stream of air at 35. Take up residue in 500 u,L mobile
phase, inject 10 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeOH: 10 mM pH 6.5 citrate buffer 10:90
Flow rate: 2
CHROMATOGRAM
Retention time: 37
Internal standard: O-desmethylnaproxen (26)
OTHER SUBSTANCES
KEYWORDS
plasma; horse
REFERENCE
Ketelaars,H.C; Peters,J.G.; Anzion,R.B.; Van Ginneken,C.A. Isolation, partial identification and quantitative
determination of four guaiphenesin glucuronides in plasma and urine of the horse by high-performance
SAMPLE
a 10 (urine) or 30 (blood) \xh aliquot. (The detector wavelength shown is the wavelength of
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve in mobile phase, inject a 20 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 jxm Chiralcel OD
Mobile phase: EtOH:heptane 20:80
Flow rate: 1
Detector: UV 365
CHROMATOGRAM
Retention time: k' 0.57 ((RH-)), k'1.72 ((SH+))
KEYWORDS
chiral
REFERENCE
Francotte,E.R.; Richert,R Applications of simulated moving-bed chromatography to the separation of the en-
antiomers of chiral drugs, J.Chromatogr.A, 1997, 769, 101-107.
Guanabenz
Molecular formula: C8H8CI2N4
Merck Index: 4585
Lednicer No.: 2 123
SAMPLE
Sample preparation: Finely powder tablets, weigh out amount equivalent to 30 mg guanabenz
acetate, add 190 mL MeCN, sonicate for a few minutes, make up to 200 mL with MeCN, filter.
Remove a 14-59 mL aliquot and add it to 9 mL 1 mg/mL carbamazepine in MeCN, make up
to 100 mL with MeCN, inject a 20 JAL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 10 |xm octadecylsilane (Perkin-Elmer)
Mobile phase: MeCN.buffer 35:65 (Buffer was 4 mM KH2PO4 adjusted to pH 3.25 with phos-
phoric acid.)
Flow rate: 2.5
Detector: UV 265
CHROMATOGRAM
Retention time: 2
Internal standard: carbamazepine (3)
OTHER SUBSTANCES
Simultaneous: mefruside, impurities
KEYWORDS
tablets
REFERENCE
Vio,L.; Mamolo,M.G.; Furlan,G. Quantitative high pressure liquid chromatographic determination of guana-
benz and mephruside in pharmaceutical formulations, Farmaco.fPratJ., 1988, 43, 2736.
SAMPLE
Sample preparation: Dilute a 500 |xL aliquot of syrup with water to give a clonidine concen-
tration of 5 |xg/mL, filter (0.22 u,m), inject a 15 JJLL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Zorbax TMS trimethylsilyl
Mobile phase: MeOH:buffer 65:35 (Buffer was 2.2 mM KH2PO4 and 16 mM Na2HPO4, pH 7.9.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 7
Internal standard: guanabenz
OTHER SUBSTANCES
Simultaneous: clonidine
KEYWORDS
syrup; guanabenz is IS
REFERENCE
Levinson,M.L.; Johnson,C.E. Stability of an extemporaneously compounded clonidine hydrochloride oral liquid,
Am.J.Hosp.Pharm., 1992, 49, 122-125.
Guanethidine
Molecular formula: Ci0H22N4
CAS Registry No.: 55-65-2, 645-43-2 (monosulfate), 60-02-6 (sulfate)
Merck Index: 4589
Lednicer No.: 1 282
SAMPLE
Matrix: blood
Sample preparation: Inject 200 |xL serum onto column A and elute to waste with mobile phase
A, after 2 min elute the contents of column A onto column B with mobile phase B, after another
3 min remove column A from the circuit, elute column B with mobile phase B, monitor the
effluent from column B. Flush column A with mobile phase C for 10 min, re-equilibrate with
mobile phase A for 7 min.
HPLC VARIABLES
Column: A 35 X 4.6 10 ^m TSK precolumn BSA-ODS (Tosoh); B 150 X 4.6 5 ^m TSKgel ODS-
80TM (Tosoh)
Mobile phase: A 50 mM NaH2PO4 adjusted to pH 3.0 with 50 mM phosphoric acid; B MeCN:
buffer 30:70, containing 7 g/L sodium 1-octanesulfonate (Buffer was 50 mM NaH2PO4 adjusted
to pH 3.0 with 50 mM phosphoric acid.); C MeCN:water 50:50.
Flow rate: 1
Detector: F ex 39 em 500 following post-column reaction. The effluent from column B mixed with
1 M NaOH pumped at 0.3 mL/min and with 6 g/L ninhydrin in water pumped at 0.3 mL/min
and the mixture flowed through a 10 m X 0.5 mm ID PTFE coil at 56 to the detector.
CHROMATOGRAM
Retention time: 16
KEYWORDS
serum; post-column reaction; column-switching; rat; human; pharmacokinetics
REFERENCE
Inamoto,Y.; Inamoto,S.; Hanai,T.; Takahashi,Y.; Kadowaki,K.; Kinoshita,T. Development of automated highly
sensitive analytical system for guanethidine sulfate in serum, J.Liq.Chromatogr.Rel.TechnoL, 1997, 20,
2099-2108.
SAMPLE
Sample preparation: Grind tablets, add 3-20 mL MeCN.water 15:85, sonicate for 10 min, filter,
make up to 100 mL with MeCN:water 15:85. Remove a 500 |xL aliquot and add it to 300 |xL
250 |xg/mL procaine hydrochloride in water, make up to 10 mL with mobile phase, inject a 50
(xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm ASI chromosphere 3869 octadecylsilane (Analytical Sciences, Inc.)
Mobile phase: MeCN:50 mM NaH2PO4 30:70 containing sodium pentanesulfonate, pH adjusted
to 2.5 with concentrated phosphoric acid
Flow rate: 1
Detector: E, Metrohm model E-611, Bioanalytical Systems KeI F cell, glassy carbon electrode +
1300 mV, auxiliary platinum electrode, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 3.5
Internal standard: procaine hydrochloride (5.7)
OTHER SUBSTANCES
Simultaneous: hydrochlorothiazide
KEYWORDS
tablets; not stability-indicating
REFERENCE
Stewart,J.T.; Clark,S.S. Liquid chromatographic determination of guanethidine salts and hydrochlorothiazide
using electrochemical detection and ion-pair techniques, J.Pharm.Sci., 1986, 75, 413-415.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: ixBondapak CN
Mobile phase: MeCN:0.1% pH 7.36 ammonium acetate 50:50
Flow rate: 2
Detector: UV 206
CHROMATOGRAM
Retention time: 2
KEYWORDS
rabbit; buffer
REFERENCE
Tang-Liu,D.D.-S.; Richman,J.B.; Weinkam,R.J.; Takruri,H. Effects of four penetration enhancers on corneal
permeability of drugs in vitro, J.Pharm.ScL, 1994, 83, 85-90.
Guanfacine
Merck Index: 4590
Lednicer No.: 3 40
SAMPLE
residue with 50 jmL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Gusperimus
Molecular formula: Ci 7 H 37 N 7 O 3
CAS Registry No.: 104317-84-2, 84937-45-1 ((-)-form tri HCI), 85468-01-5 (tri HCI)
Merck Index: 4610
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 10 |xg IS, mix well, make up to 10 mL with water, add to
a 50 X 6 column of CM-Sephadex C-25, wash with 10 mL 300 mM NaCl, elute with 10 mL
400 mM NaCl, add the eluate to a Sep-Pak C18 SPE cartridge, wash with 5 mL water, elute
with 10 mL MeOH. Evaporate the eluate to dryness under reduced pressure, reconstitute the
residue in 200 |xL mobile phase, vortex, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: Guard Pak C18 (Waters)
Column: 150 X 4.6 Cosmosil 5C18-P (Nacalai Tesque)
Mobile phase: MeCNrbuffer 9:91 (Buffer was 10 mM pH 3 phosphate buffer containing 5 mM
sodium pentanesulfonate.)
Flow rate: 1
Detector: UV 205
CHROMATOGRAM
Retention time: 8.6
Internal standard: l-amino-20-guanidino-ll-hydroxy-4,9,12-triazaeicosane-10,13-dione (Heat
33.7 mmole 8-guanidinooctanamide, 30.7 mmole glyoxylspermidine hydrochloride, 33.7 mmole
glutaric +acid, and 2 g water at 60 for 8 h, dilute with water, chromatograph on CM-Sephadex
C-25(Na ) using gradient elution with water and 1 M NaCl. Evaporate the eluate fraction
containing the product to dryness, extract the residue with MeOH, chromatograph the extract
on Sephadex LH-20 with MeOH, evaporate the eluate to dryness to get the compound.) (18.6)
KEY WORDS
dog; plasma; SPE; pharmacokinetics
REFERENCE
Nakanuma,R.; Watanabe,K; Yamashita,K.; Mizuguchi,S.; Hashimoto,Y.; Nakamura,T.; Umezawa,H- High-per-
formance liquid chromatographic determination of deoxyspergualin in dog plasma with ultraviolet detec-
tion, J.Chromatogr., 1990, 527, 208-213.
SAMPLE
Matrix: blood
Sample preparation: Filter (Amicon MPS-I with 14 mm YM20 membrane) 1 mL plasma while
centrifuging at 800 g for 1 h. Remove a 400 |xL aliquot of the ultrafiltrate and add it to 50 |xL
500 mM pH 6.8 phosphate buffer, add 50 |xL 10 mM NaCN in water, add 100 (xL 2 mM naph-
thalene-2,3-dicarboxaldehyde in MeCN, let stand at room temperature for 15 min, add 50 (xL
500 mM pH 3.0 sodium acetate buffer, inject an aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 jutm ODS Hypersil
Mobile phase: MeCN: 100 mM KH2PO4:phosphoric acid 52:48:0.8 containing 18 mM sodium do-
decyl sulfate
Flow rate: 2
CHROMATOGRAM
Retention time: 14
KEYWORDS
derivatization; plasma; ultrafiltrate
REFERENCE
Sprancmanis,L.A.; Riley,C.M.; Stobaugh,J.F. Determination of the anticancer drug, 15-deoxyspergualin, in
plasma ultrafiltrate by liquid chromatography and precolumn derivatization with naphthalene-2,3-dicar-
boxaldehyde/cyanide, J.Pharm.BiomedAnal., 1990, 8, 165-175.
SAMPLE
Sample preparation: Plasma. 750 |xL Plasma + 30 |xL 70% perchloric acid, vortex, centrifuge
at 15600 g for 5 min, filter (0.22 |xm) the supernatant, inject a 10-200 |xL aliquot of the filtrate.
Urine. Dilute urine 10 to 25-fold with water, centrifuge, filter (0.22 jxm) the supernatant, inject
a 10-200 |xL aliquot of the filtrate.
HPLCVARIABLES
Guard column: 15 X 3.2 7 |xm Newguard RP 18
Column: two 100 X 4.6 5 |xm RP 18 columns in series (Brownlee)
Mobile phase: Gradient. A was MeCN: 100 mM pH 2.55 NaH2PO4 containing 8 mM octanesul-
fonic acid and 0.1 mM EDTA 2:98. B was MeCN:200 mM pH 3.1 NaH2PO4 containing 8 mM
octanesulfonic acid 30:70. A:B from 55:45 to 25:75 over 20 min.
Flow rate: 1
reagent pumped at 0.7 mL/min and flowed through a 2 m long reaction coil at 41 to the
detector. The reagent was 500 mM pH 8.8 potassium borate buffer containing 0.8 g/L o-phthal-
dialdehyde, 1% MeOH, and 0.06% 2-mercaptoethanol.
CHROMATOGRAM
Retention time: 22
OTHER SUBSTANCES
KEYWORDS
use plasticware; plasma; post-column reaction; pharmacokinetics
REFERENCE
Muindi,J.R; Lee,S.-J.; Baltzer,L.; Jakubowski,A.; Scher,H.L; Sprancmanis,L.A.; Riley,C.M.; Vander Velde,D.;
Young,C.W. Clinical pharmacology of deoxyspergualin in patients with advanced cancer, Cancer Res., 1991,
52, 3096-3101.
Halazepam
Molecular formula: C17H12CIF3N2O
Merck Index: 4619
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond-Elut C8 SPE cartridge with 2 mL MeOH and 2 mL
water, do not allow to dry. Add 100 |xL 5 ng/mL diazepam in 1 M pH 10.5 glycine buffer then
1 mL plasma to the SPE cartridge, wash with 2 mL water, wash with 50 jxL MeOH, elute with
three 200 |xL aliquots of MeOH. Combine the eluates and evaporate them to dryness under a
stream of nitrogen at 37, reconstitute the residue in 100 |xL mobile phase, inject a 50-80 JJLL
aliquot.
HPLC VARIABLES
Column: 100 X 4.6 3 jxm Adsorbosphere C8
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Internal standard: diazepam (7.76)
OTHER SUBSTANCES
E x t r a c t e d : nordiazepam
Simultaneous: alprazolam, chlordiazepoxide, clonazepam, desmethyldiazepam, 3-hydroxyhala-
zepam, lorazepam, methylclonazepam, oxazepam, prazepam, quazepam, temazepam, triazolam
KEYWORDS
REFERENCE
Gupta,S.K; Ellinwood,E.H. Liquid chromatographic assay and pharmacokinetics of halazepam and its metab-
olite in humans, J.Pharm.Sci., 1990, 79, 822-825.
SAMPLE
Sample preparation: 2.5 mL Microsomal incubation + 2.5 mL acetone, add 30 jxL diazepam in
MeOH, add 2.5 mL chloroform, centrifuge. Remove the organic layer and evaporate it to dry-
ness under reduced pressure at 40, reconstitute the residue in 100 JJLL mobile phase, inject an
aliquot.
HPLC VARIABLES
Column: 250 X 6.2 7 |xm Zorbax silica
KEYWORDS
use plasticware; plasma; post-column reaction; pharmacokinetics
REFERENCE
Muindi,J.R; Lee,S.-J.; Baltzer,L.; Jakubowski,A.; Scher,H.L; Sprancmanis,L.A.; Riley,C.M.; Vander Velde,D.;
Young,C.W. Clinical pharmacology of deoxyspergualin in patients with advanced cancer, Cancer Res., 1991,
52, 3096-3101.
Halazepam
Merck Index: 4619
SAMPLE
Matrix: blood
water, do not allow to dry. Add 100 |xL 5 ng/mL diazepam in 1 M pH 10.5 glycine buffer then
1 mL plasma to the SPE cartridge, wash with 2 mL water, wash with 50 jxL MeOH, elute with
three 200 |xL aliquots of MeOH. Combine the eluates and evaporate them to dryness under a
stream of nitrogen at 37, reconstitute the residue in 100 |xL mobile phase, inject a 50-80 JJLL
aliquot.
HPLC VARIABLES
Column: 100 X 4.6 3 jxm Adsorbosphere C8
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
OTHER SUBSTANCES
E x t r a c t e d : nordiazepam
Simultaneous: alprazolam, chlordiazepoxide, clonazepam, desmethyldiazepam, 3-hydroxyhala-
zepam, lorazepam, methylclonazepam, oxazepam, prazepam, quazepam, temazepam, triazolam
KEYWORDS
REFERENCE
Gupta,S.K; Ellinwood,E.H. Liquid chromatographic assay and pharmacokinetics of halazepam and its metab-
olite in humans, J.Pharm.Sci., 1990, 79, 822-825.
SAMPLE
ness under reduced pressure at 40, reconstitute the residue in 100 JJLL mobile phase, inject an
aliquot.
HPLC VARIABLES
Column: 250 X 6.2 7 |xm Zorbax silica
Mobile phase: Hexane:dichloromethane:isopropanol 77:20:3
Flow rate: 2
Detector: UV 232
CHROMATOGRAM
Retention time: 10
Internal standard: diazepam (12)
OTHER SUBSTANCES
Extracted: metabolites, oxazepam
KEYWORDS
human; liver; normal phase; pharmacokinetics
REFERENCE
Lu,X.-L.; Guengerich,F.R; Yang,S.K. Stereoselective metabolism of prazepam and halazepam by human liver
microsomes, Drug Metab.Dispos., 1991, 19, 637-642.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
glunixin, glutethimide, glybenclamide, haloperidol, hydrochlorothiazide, hydrocodone, hydro-
cortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine,
indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin,
ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, lox-
apine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, me-
gestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, me-
pivacaine, mescaline, mesoridazine, methadone, methamphetamine, methap5nilene,
oxjrphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbi-
REFERENCE
Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnaLToxicoL, 1994,
18, 233-242.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize brain in 100 mM NaOH (5 mL/g) by sonication. Add 250 jxL
homogenate (ca. 50 mg tissue equivalent) to 250 |xL 100 mM pH 13 NaOH, vortex thoroughly.
Add 2 mL toluene, mix (50 inversions), centrifuge at 15000 rpm at 4 for 20 min. Dry the
organic phase under a stream of nitrogen. Add 2 mL toluene to the aqueous phase and repeat
the extraction. Combine the organic layers and dry under a stream of nitrogen. Reconstitute
the residue in 100 |JLL mobile phase, inject a 40 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 3 |xm Rainin C8 Microsorb
Mobile phase: MeCN:MeOH:25 mM potassium phosphate buffer 18.5:16.5:65
Flow rate: 0.9
Detector: UV 240
CHROMATOGRAM
Retention time: 6.2
Internal standard: halazepam
OTHER SUBSTANCES
Extracted: midazolam
KEYWORDS
brain; rat; halazepam is IS
REFERENCE
Jiang,Q.; Walton,N.Y.; Gunawan,S.; Treiman,D.M. High-performance liquid chromatographic determination of
midazolam in rat brain, J.Chromatogr.B, 1996, 683, 276-280.
Halcinonide
Molecular formula: C24H32CtFO5
Merck Index: 4621
Lednicer No.: 2 187
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10-60 \xL aliquot.
HPLCVARIABLES
Column: 150 X 2 5 fxm Spherisorb ODSl
Flow rate: 0.16-0.30
Detector: UV 240
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
REFERENCE
Gardner,R-S.; Walker,M.; Hollingsbee,D.A. A sensitive high-performance liquid chromatographic method for the
assessment of percutaneous absorption of topical corticosteroids, J.Pharm.Biomed.AnaL, 1990, 8, 1083-
1085.
Halofantrine
Molecular formula: C26H30CI2F3NO
Merck Index: 4626
Lednicer No.: 3 76
SAMPLE
Matrix: blood
Sample preparation: Add 1 ml MeCNiEtOH 99:1 to 500 |xL erythrocyte pellet, vortex for 1 min,
centrifuge at 2000 g for 5 min. Collect the supernatant, evaporate to dryness under nitrogen,
reconstitute with 100 |xL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 jxm AD Chiralpak amylase tris-3,5-dimethyl phenylcarbamate
Mobile phase: Hexane:2-propanol:2-butanol:diethylamine 95:3:2:0.5
Flow rate: 0.3
CHROMATOGRAM
Internal standard: ( S)-dichloro-[2-(dibutylamino)methyl]-6-(trifluoromethyl)-9-phenanthrene-
methanol (SmithKline Beecham) (21)
OTHER SUBSTANCES
KEYWORDS
erythrocytes; chiral; do the extraction procedure in silanized tubes
REFERENCE
Gorichon,E.; Martin,C; Bangchang,K.N.; Karbwang,J.; Thuiller,A.; Farinotti,R.; Gimenez,F. Chiral chromato-
graphic method to determine the enantiomers of halofantrine and its main chiral desbutyl metabolite in
erythrocytes, J.Chromatogr.B, 1998, 712, 259-262.
SAMPLE
Matrix: blood
Sample preparation: Add 200 |xL 2 jxg/mL IS to 500 (xL plasma. Add 950 |xL MeCN, vortex for
1 min, centrifuge at 700 g for 2 min. Add 2-8 mL MTBE, vortex for 2 min, centrifuge at 700 g
for 5 min. Remove the upper organic phase and add it to 100 |xL 5 mM HCl in MeCN, evaporate
under a stream of nitrogen at 35. Reconstitute the residue with 200 (xL MeCN, vortex for 1
min, centrifuge at 700 g for 2 min. Inject a 25 |xL aliquot.
HPLC VARIABLES
Guard column: 15 X 3.2 7 |xm Newguard RP-8 (Perkin Elmer)
Mobile phase: MeCN.water 75:25 containing 0.2% sodium dodecyl sulfate (w/v) and 0.2% glacial
acetic acid (v/v)
Flow rate: 1.5
Detector: UV 257
CHROMATOGRAM
Internal standard: 2,6-dichloro-6-trifluoromethyl-9-(l-[2-(dibutyl-amino)ethyl])phenanthrene-
methanol.HCl (SmithKline Beecham)
KEY WORDS
plasma; dog
REFERENCE
Porter,C.J.H.; Caliph,S.M.; Charman,W.N. Differences in pre- and post-prandial plasma lipid profiles affect the
extraction efficiency of a model highly lipophilic drug from beagle dog plasma, J.Pharm.Biomed.Anal., 1997,
16, 175-180.
SAMPLE
Matrix: blood
Sample preparation: Add 300 |xL MeCN to 100 |xL rat plasma with containing IS, vortex. Cen-
trifuge for 2 min and remove the supernatant. Add 200 |xL ammonium hydroxide and 2 mL
MTBE:hexane 50:50. Vortex for 45 s, centrifuge at 2500 g for 3 min. Evaporate the organic
layer to dryness. Add 250 mM (+)-di-O-acetyl L-tartaric acid anhydride in acetic acididichlo-
romethane 20:80, heat at 45 for 30 min. Inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 C18
Mobile phase: MeCN:25 mM potassium phosphate/sulfuric acid/triethylamine solution 53.5:46.5
containing 1.8 g/L sodium dodecyl sulfate
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Retention time: 19.3 ((+)-enantiomer), 21.7 ((-)-enantiomer)
Internal standard: imipramine (13.8)
KEYWORDS
plasma; rat; derivatization; chiral
REFERENCE
Padovani,P.K; Timby,D.M.; Wright,M.R.; Kapil,R.R Quantitative analysis of DMP 851 in rat and dog plasma
SAMPLE
Matrix: blood
Sample preparation: 500 jxL Serum + 50 jxL 4 jxg/mL IS + 1 mL MeCN, vortex for 20 s, cen-
trifuge at 1200 g for 10 min, add to a conditioned 2.8 mL 500 mg Bond Elut C8 SPE cartridge,
wash with two 1 mL portions of MeCN, elute with 1 mL MeCN: 1 M HCl 90:10. Add the eluate
to 1 mL 28% ammonium hydroxide and 6 mL dichloromethane, rotate at 15 rpm for 25 min,
stream of nitrogen at 40, reconstitute the residue in 150 |JLL mobile phase, inject a 25 (JLL
aliquot.
HPLC VARIABLES
Mobile phase: MeCN:water 75:25 containing 0.2% sodium dodecyl sulfate and 0.2% glacial acetic
acid
Flow rate: 1.5
Detector: UV 257
CHROMATOGRAM
Retention time: 8.3
Internal standard: 2,4-dichloro-9-(2-dibutylamino-l-hydroxy)ethyl-6-trifluoromethylphenan-
threne (BL 22312) (11.5)
OTHER SUBSTANCES
Noninterfering: chloroquine, dapsone, mefloquine, primaquine, proguanil, pyrimethamine, qui-
nine, sulfadoxine, tetracycline
KEYWORDS
serum; treat glassware with 0.2% aquasil; pharmacokinetics; SPE
REFERENCE
Keeratithakul,D.; Teja-Isavadhann,R; Shanks,G.D.; Webster,H.K; Edstein,M.D. An improved high-perfor-
mance liquid chromatographic method for the simultaneous measurement of halofantrine and desbutyl-
halofantrine in human serum, Ther.Drug Monit, 1991, 13, 64-68.
SAMPLE
Matrix: blood
Sample preparation: Dried blood. Allow 500 |xL blood to dry on a strip of filter paper. Store at
room temperature, protect from dust and sunlight. Add 15 |xL 10 |xg/mL IS in water to each
strip and allow to dry at 37 for 30 min. Cut strip into small pieces, add 1 mL 10 mM perchloric
acid, vortex, let stand at room temperature for 5 min, add 2 mL MeCN, vortex at high speed
for 30 s, add 1 mL ammonia, mix thoroughly, add 5 mL hexane, vortex at moderate speed for
1 min, centrifuge at 2000 g for 5 min. Remove the organic layer and evaporate it to dryness
under a stream of nitrogen at 37, reconstitute the residue in 120 |xL mobile phase, inject a 50
|JLL aliquot. Whole blood, plasma. 500 |xL Whole blood or plasma + 15 |xL 10 |xg/mL IS in water
+ 2 mL MeCN, vortex for 30 s, centrifuge at 2000 g for 5 min. Remove the supernatant and
add it to 500 jxL ammonia, vortex, add 5 mL hexaneidiethyl ether 50:50, vortex for 1 min,
stream of nitrogen at 37, reconstitute the residue in 120 |xL mobile phase, inject a 50 |xL
aliquot.
HPLCVARIABLES
Guard column: 10 X 4.6 5 (xm CN precolumn RP-18 endcapped (Merck)
Column: 250 X 4.6 Hypersil 5 ODS
Mobile phase: MeCN:wateritriethylamine 65:35:1 adjusted to pH 4 with orthophosphoric acid
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 8.5
Internal standard: 2,4-dichloro-9-(2-dibutylamino-l-hydroxy)ethyl-6-trifluoromethylphenan-
threne (11)
OTHER SUBSTANCES
Simultaneous: diazepam
Noninterfering: acetaminophen, chlorcycloguanil, chloroquine, chlorproguanil, cycloguanil,
mefloquine, phenobarbital, proguanil, pyrimethamine, quinidine, quinine, sulfadoxine
KEYWORDS
plasma; whole blood; dried blood; pharmacokinetics; diazepam may be used as IS
REFERENCE
Mberu,E.K; Muhia,D.K; Watkins,W.M. Measurement of halofantrine and its major metabolite desbutylhalo-
fantrine in plasma and blood by high-performance liquid chromatography: a new methodology,
J.Chromatogr., 1992, 581, 156-160.
SAMPLE
Matrix: blood
Sample preparation: Condition a 500 mg Bakerbond SPE cartridge with three 3 mL portions
of MeCN and three 3 mL portions of solvent. 1 mL Plasma + 3 mL solvent, add to the SPE
cartridge, elute with 2 mL solvent. Collect all the eluate and add it to 5 fiL 10 |xg/mL desipra-
mine hydrochloride in n-hexane:EtOH:2-butanol 93:4.5:2.5, evaporate to dryness under a
stream of nitrogen at 40, reconstitute the residue in 1 mL mobile phase, inject a 200 (xL
aliquot. (Solvent was MeCN:triethylamine:EtOH 99:1:1 adjusted to pH 4 with 1 M HCl.)
HPLC VARIABLES
Guard column: 50 X 4.6 10 |xm Chiralpak AD amylose tris-3,5-dimethylphenylcarbamate
(Daicel)
Column: 250 X 4.6 10 |xm Chiralpak AD amylose tris-3,5-dimethylphenylcarbamate (Daicel)
Mobile phase: n-Hexane:EtOH:2-butanol:diethylamine 93:4.5:2.5:0.1
Flow rate: 0.3
CHROMATOGRAM
Retention time: 15.5 (+), 18 (-)
Internal standard: desipramine (32)
OTHER SUBSTANCES
KEYWORDS
SPE; plasma; pharmacokinetics; chiral
REFERENCE
Terefe,H.; Blaschke,G. Direct determination of the enantiomers of the antimalarial drug halofantrine and its
active metabolite N-desbutylhalofantrine in human plasma, J.Chromatogr.B, 1994, 657, 238-242.
SAMPLE
Matrix: blood
Sample preparation: 500 (JLL Plasma + 30 |xL 100 |xg/mL IS in MeCN:water 80:20, vortex at
high speed and add 2 mL MeCN, centrifuge at 1800 g for 3 min. Remove the supernatant and
add it to 500 JJLL ammonium hydroxide and 5 mL MTBE:hexane 50:50, vortex at high speed
for 90 s, centrifuge at 1800 g. Remove the upper organic layer and evaporate it to dryness
under a stream of nitrogen at 25, reconstitute the residue in 300 |xL 250 mM (+)-di-O-acetyl-
L-tartaric acid anhydride in acetic acid:dichloromethane 20:80 (freshly prepared), heat at 45
for 30 min, add 300 JJLL MeOH, evaporate to dryness under a stream of nitrogen at 25, recon-
stitute with 170 |xL mobile phase, inject a 30-100 jxL aliquot.
HPLC VARIABLES
Guard column: Guard-Pak ODS (Waters)
Mobile phase: MeCN:buffer 53.5:46.5 containing 0.9 g/L sodium dodecyl sulfate (Buffer was 25
mM KH2PO4 containing 1.5 mIVL 2 M sulfuric acid and 0.5 mL/L triethylamine, pH 5.0.)
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Retention time: 11.5 (+), 13.3 (-)
Internal standard: ( )-2,4-dichloro-a-[2-(dibutylamino)ethyl]-6-(trifluoromethyl)-9-phenanthre-
nemethanol (SK&F 99123) (17.0, 20.7 (enantiomers))
KEYWORDS
REFERENCE
Brocks,D.R.; Dennis,M.J.; Schaefer,W.H. A liquid chromatographic assay for the stereospecific quantitative
analysis of halofantrine in human plasma, J.Pharm.Biomed.AnaL, 1995, 13, 911-918.
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 200 mg Bond Elut C8 SPE cartridge with 2 mL MeOH
and 2 mL buffer. Prepare whole blood by freezing then thawing, dilute with an equal volume
of water, centrifuge at 5000 g for 5 min. 1 mL Plasma or whole blood supernatant + 100 (JLL
10 |jLg/mL IS in MeCN:water 50:50 + 2 mL MeCN, vortex for 15 s, centrifuge at 1500 g for 10
min, add the supernatant to the SPE cartridge, allow to dry under vacuum for 1 min, wash
with two 2 mL portions of buffer, wash with two 2 mL portions of MeOH:water 50:50, elute
with four 750 |xL portions of ethyl acetate:acetic acid 98:2. Evaporate the eluate to dryness
under a stream of nitrogen at 40, reconstitute the residue in 200 (plasma) or 100 (whole blood)
|iL mobile phase, inject a 50 JJLL aliquot. (Buffer was 1 g/L potassium bicarbonate.)
HPLCVARIABLES
Column: 250 X 4 5 |xm Lichrospher 60 RP select B C8
Mobile phase: MeCN:water 35:65 containing 1% triethylamine, adjusted to pH 4 with ortho-
phosphoric acid
Flow rate: 1.1
CHROMATOGRAM
Retention time: 6.8
Internal standard: N-tert-butyl-3-hydroxy-( 1,3-dichloro-6-trimioromethyl-9-phenanthryl)pro-
pionamide hydrate (S76,395-0, Aldrich) (12.0)
Limit of detection: 10.8 ng/mL (whole blood), 6.1 ng/mL (plasma)
Limit of quantitation: 14.8 ng/mL (whole blood), 12.4 ng/mL (plasma)
OTHER SUBSTANCES
Noninterfering: chloroquine, mefloquine, proguanil, pyrimethamine, quinine, sulfadoxine
KEYWORDS
REFERENCE
Gaillard,Y; Pr6vosto,J.-M.; Cheminel,V.; Soares,O.; Chaulet,J.-F. New solid-phase extraction for an improved
high-performance liquid chromatographic procedure for the quantitation of halofantrine and monodesbu-
tylhalofantrine in blood or plasma, J.Chromatogr.B, 1995, 668, 315-321.
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Plasma + 1 mL MeCN + 200 |xL 2 |xg/mL IS in MeCN, vortex for
2 min, centrifuge, add 8 mL MTBE, vortex for 2 min, centrifuge at 700 g for 5 min. Remove 8
mL of the upper organic layer and add it to 100 |xL 5 mM HCl in MeCN, evaporate to dryness
under a stream of nitrogen at 35, reconstitute the residue in 200 |xL MeCN, inject a 25 (JLL
aliquot.
HPLC VARIABLES
Guard column: 15 X 3.2 7 |xm Aquapore (Analytical Biosystems)
Mobile phase: MeCN:water 75:25 containing 0.2% sodium dodecyl sulfate and 0.2% glacial acetic
acid
Flow rate: 1.5
Detector: UV 257
CHROMATOGRAM
Retention time: 7.8
Internal standard: 2,4- dichloro- 6 -trifluoromethyl- 9 - [ 1 - (2 - (dibutylamino)ethyl]pehnanthrene-
methanol hydrochloride (10.4)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Humberstone,A.J.; Currie,G.J.; Porter,C.J.H.; Scanlon,M.J.; Charman,W.N. A simplified liquid chromatography
assay for the quantitation of halofantrine and desbutylhalofantrine in plasma and identification of a deg-
radation product of desbutylhalofantrine formed under alkaline conditions, J.Pharm.BiomedAnal., 1995,
13, 265-272.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 258.2
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Adjust pH of 1.75 mL microsomal incubation to pH 9 with 100 mM NaOH,
add 3.5 mL n-hexane:diethyl ether 70:30, shake mechanically for 15 min, centrifuge at 2500 g
for 15 min, repeat extraction. Combine the organic layers and evaporate them to dryness under
a stream of nitrogen at 40, reconstitute the residue in 1 mL mobile phase, inject a 20 |xL
aliquot.
HPLCVARIABLES
Guard column: 50 X 4.6 10 |xm Chiralcel OD cellulose tris-3,5-dimethylphenylcarbamate
Column: 250 X 4.6 10 jxm Chiralcel OD cellulose tris-3,5-dimethylphenylcarbamate
Mobile phase: n-Hexane:isopropanol:diethylamine 90:10:0.1
Flow rate: 0.3
Detector: UV 260
CHROMATOGRAM
Retention time: 27 (+), 31.5 (-)
OTHER SUBSTANCES
KEYWORDS
rat; liver; chiral
REFERENCE
Terefe,H.; Blaschke,G. Direct determination of the enantiomers of halofantrine and its pharmacologically active
metabolite N-desbutylhalofantrine by high-performance liquid chromatography, J.Chromatogr., 1993, 615,
347-351.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve a sample in MeOH to a concentration of about 1 mg/mL, inject
an aliquot.
HPLCVARIABLES
Column: 100 X 4.6 5 jxm Spherisorb SCX
Mobile phase: MeOHrwater 80:20 containing 20 mM ammonium formate and 2.3 mL/L trifluo-
roacetic acid
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: cimetidine, clomipramine, haloperidol, minoxidil, reserpine, verapamil
REFERENCE
Law,N.; Appleby,J.R.G. Re-evaluation of strong cation-exchange high-performance liquid chromatography for
the analysis of basic drugs, J.Chromatogr.A, 1996, 725, 335-341.
Haloperidol
Molecular formula: C21H23CIFNO2
CAS Registry No.: 52-86-8, 74050-97-8 (decanoate)
Merck Index: 4629
Lednicer No.: 1 306
SAMPLE
Matrix: blood
Sample preparation: Mix 0.5 mL plasma with 25 jxL 1 |xg/mL IS in MeOH . Vortex for 30 s,
add 5 mL ether and 3 mL 100 mM HCl, vortex for 1 min, centrifuge at 3000 rpm for 3 min.
Remove the aqueous phase, add 7 mL chloroform (Caution! Chloroform is a carcinogen!) and
500 |xL 1 M NaOH. Shake the mixture for 2 min, remove the chloroform phase, evaporate it
to dryness under vacuum at 45. Reconstitute the residue with 500 |xL mobile phase, vortex
for 1 min, inject a 20 |L aliquot.
HPLCVARIABLES
Mobile phase: MeOH:buffer 55:45 (Buffer was water containing 200 mM ammonium acetate,
adjusted to pH 7.1-7.3 with acetic acid.)
Flow rate: 1.5
Detector: UV 249
CHROMATOGRAM
Retention time: 6.3
KEYWORDS
REFERENCE
El-Sayed,Y.M.; Khidr,S.H.; Niazy,E.M. High-performance liquid chromatographic assay for the determination
of haloperidol in plasma, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 125-134.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 500 |xL 600 mM pH 10 sodium carbonate/bicarbonate buffer
+ 8 mL heptaneiisoamyl alcohol 98:2, shake at 250 cycles/min for 5 min, centrifuge at 1500 g
for 10 min. Freeze the aqueous layer, evaporate the heptane layer to dryness under a gentle
stream of nitrogen at 60. Dissolve the residue in 75 |iL mobile phase, inject a 65 IJLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 LiChroCart
Mobile phase: MeOH:40 mM pH 7.0 ammonium acetate buffer 90:10
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Internal standard: haloperidol
OTHER SUBSTANCES
Simultaneous: amitriptyline, citalopram, chlorprothixene, clomipramine, clozapine, desipra-
mine, desmethylcitalopram, desmethylclomipramine, desmethylsertraline, diltiazem, fluoxe-
tine, fluphenazine, 10-hydroxyamitriptyline, 8-hydroxyclomipramine, 8-hydroxydesmethyl-
clomipramine, 10-hydroxynortriptyline, hydroxyzine, imipramine, methotrimeprazine
sulfoxide, mianserine, norfluoxetine, nortriptyline, paroxetine, perphenazine, sertraline,
zuclopenthixol
Noninterfering: carbamazepine, clonazepam, flunitrazepam, nitrazepam, oxazepam,
oxcarbazepine
KEYWORDS
serum; haloperidol is IS
REFERENCE
Olesen,O.V.; Linnet,K. Simplified high-performance liquid chromatographic method for determination of ris-
peridone and 9-hydroxyrisperidone in serum from patients comedicated with other psychotropic drugs,
J.Chromatogr.B, 1997, 698, 209-216.
SAMPLE
Matrix: blood
Sample preparation: After each extraction or wash step, centrifuge the sample at 2000 g for 5
min. 2 mL Plasma + 50 JJLL 500 ng/mL IS + 500 fxL 2 M NaOH + 8 mL n-hexane:isoamyl alcohol
99:1, extract gently for 20 min on rotator in a horizontal position, centrifuge at 2000 g for 5
min. Extract the organic layer with 1 mL 200 mM HCl for 15 min by vigorous shaking, discard
the organic layer, wash the aqueous layer with 5 mL n-hexane:isoamyl alcohol 99:1, discard
the organic layer. Add 500 fiL2M NaOH, extract with 7 mL n-hexane:isoamyl alcohol 99:1 for
15 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at
37, reconstitute the residue in 50 jxL mobile phase, inject a 1 jxL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:2 mM ammonium formate 45:55 adjusted to pH 3.0 with formic acid
Flow rate: 50 ^xL/min
Injection volume: 1
Detector: MS, API 100 Sciex, electrospray, nitrogen as the nebulizing and curtain gas, orifice
voltages 20, 60, 120, m/z 376.2
CHROMATOGRAM
Retention time: 6.6
Internal standard: chlorohaloperidol (8.7)
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Hoja,H.; Marquet,P.; Verneuil,B-; Lotfi,H.; Dupuy,J.L.; Penicaut,B-J Lachtre,G. Determination of haloperidol
and its reduced metabolite in human plasma by liquid chromatography-mass spectrometry with electro-
spray ionization, J.Chromatogr.B, 1997, 688, 275-280.
SAMPLE
Matrix: blood
Sample preparation: 10 mL Plasma or whole blood + 1 mL 1 M NaOH, extract twice with 10
mL hexane for 30 min. Remove the organic layers and evaporate them to dryness under a
stream of nitrogen, reconstitute the residue in 1 mL 100 mM HCl, add 5 mL chloroform, vortex
for 1 min, centrifuge. Remove a 4.5 mL aliquot of the organic layer and evaporate it to dryness,
reconstitute the residue in 100 jxL mobile phase, inject a 50 JXL aliquot. (It is implied, but not
explicitly stated in the paper, that this extraction procedure works for this compound.)
HPLC VARIABLES
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Retention time: 5.1
OTHER SUBSTANCES
promazine, imipramine, mesoridazine, nortriptyline, orphenadrine, piperacetazine, promazine,
promethazine, thioridazine, thiothixene, trifluoperazine, triflupromazine, trihexyphenidyl,
trimeprazine
Interfering: fluphenazine
KEYWORDS
plasma; whole blood
REFERENCE
Curry,S.H.; Brown,E.A.; Hu,O.Y.-R; Perrin,J.H. Liquid chromatographic assay of phenothiazine, thioxanthene
compression columns and UV and electrochemical detection, J.Chromatogr., 1982, 231, 361376.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 |xL 1 |xg/mL loxapine in isopropanol:diethylamine
and add it to 100 |xL 250 mM HCl, vortex for 30 s, inject a 50 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Guard column: 50 X 4.6 40 |xm C8 (Supelco)
Column: 250 X 4.6 5 |jim Supelcosil C8
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: amitriptyline, chlordiazepoxide, chlorpromazine, desipramine, desmethldiazepam,
desmethylchlordiazepoxide, desmethyldoxepin, diazepam, doxepin, fluphenazine, imipramine,
oxazepam
Interfering: nortriptyline, thiothixene
KEYWORDS
plasma
REFERENCE
Kiel,J.S.; Abramson,R.K.; Morgan,S.L.; Voris,J.C. A rapid high performance liquid chromatographic method for
the simultaneous measurement of six tricyclic antidepressants, J.Liq.Chromatogr., 1983, 6> 27612773.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut C18 SPE cartridge with 2 mL MeOH, 2 mL water,
2 mL MeOH, and 2 mL water. 1 mL Plasma or serum + 2 mL 100 mM pH 9.0 borate buffer,
vortex for 10 s. Add to the SPE cartridge, wash with 5 mL water, elute with 1.6 mL 200 mM
HCl in MeOH. Evaporate the eluate to dryness at 80, reconstitute in 50 jxL 600 ng/mL tria-
zolam in mobile phase, centrifuge at 3000 rpm for 5 min, inject a 15 JJLL aliquot of the
supernatant.
HPLC VARIABLES
Column: 250 X 4 10 jxm Hitachi ODS-3056
Mobile phase: MeCN:THF:l% acetate:triethylamine 28.2:1.9:69.5:0.4
Flow rate: 1.6
Detector: UV 245
CHROMATOGRAM
Retention time: 8
Internal standard: triazolam (14)
OTHER SUBSTANCES
Simultaneous: metabolites, amitriptyline, carpipramine, chlocapramine, clomipramine, chlor-
promazine, clorazepam, diazepam, estazolam, flunitrazepam, fluphenazine, haloxazolam, imip-
ramine, levomepromazine, maprotiline, moperone, nitrazepam, perphenazine, promethazine,
triazolam, trifluperidol, trimipramine
Interfering: amoxapine
KEYWORDS
plasma; serum; SPE
REFERENCE
Hayakari,M.; Hashimoto,Y.; Kita,T.; Murakami,S. A rapid and simplified extraction of haloperidol from plasma
or serum with Bond Elut C18 cartridge for analysis by high performance liquid chromatography, Forensic
ScLInL, 1987, 35, 73-81.
SAMPLE
Matrix: blood
Sample preparation: 500 \LL Serum + 250 |xL di-iso-propyl ether:n-butyl alcohol 7:3 containing
400 ng/mL minaprine, centrifuge 2 min, shake, centrifuge 5 min, inject 50 (JLL aliquot of top
organic layer.
HPLC VARIABLES
Guard column: 30 X 4.6 5 |xm Brownlee cyano spheri-5
Column: 250 X 4.6 5 |xm Altex ultrasphere cyano
Mobile phase: MeCN:THF:water:2 M ammonium formate (pH 4.0) 700:100:195:5
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: 6
Internal standard: minaprine (5.5)
OTHER SUBSTANCES
Also analyzed: diltiazem, nortriptyline, amitriptyline, haloperidol, desipramine, imipramine,
clomipramine, propafenone, amiodarone, verapamil
KEYWORDS
serum
REFERENCE
Mazzi,G. Simple and practical high-performance liquid chromatographic assay of some tricyclic drugs, halo-
peridol, diltiazem, verapamil, propafenone, and amiodarone, Chromatographia, 1987, 24, 313-316.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 40 |xL 1 |jig/mL chlorohaloperidol in MeOH + 2 mL pH 11
Normex buffer, vortex for 1 min, add to a 3 mL Extrelut SPE cartridge, elute with diethyl
ether. Evaporate the eluate to dryness under a stream of air at 40, reconstitute the residue
in 100 |xL 10 mM HCl, vortex, add 2 mL hexane, shake on a whirlmixer for 20 s, centrifuge at
2800 g for 5 min, inject a 20-40 ILJLL aliquot of the aqueous layer.
HPLC VARIABLES
Mobile phase: MeCN:25 mM KH2PO4:water 45:50:5
Flow rate: 0.8
Detector: UV 220
CHROMATOGRAM
Retention time: 9
Internal standard: chlorohaloperidol (11)
OTHER SUBSTANCES
Simultaneous: alimemazine, amisulpride, amitriptyline, biperiden, caffeine, carbamazepine,
clobazam, clomipramine, clorazepate, cyamemazine, desipramine, diazepam, ethybenztropine,
ethyl loflazepate, flunitrazepam, imipramine, levomepromazine, loflazepate, lorazepam, nor-
diazepam, oxazepam, sulpride, sultopride, tiapride, triazolam, trihexiphenidyl, trimipramine,
tropazepine, viloxazine
Noninterfering: heptaminol, meprobamate
Interfering: nortriptyline
KEYWORDS
SPE; plasma
REFERENCE
Cahard,C; Rop,P.R; Conquy,T.; Viala,A. High-performance liquid chromatographic analysis of haloperidol and
hydroxyhaloperidol in plasma after solid-phase extraction, J.Chromatogr., 1990, 532, 193202.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL 200 ng/mL IS in MeOH + 1 mL 50 mM pH 10
borate buffer, vortex briefly, add to an Extrelut 3 SPE cartridge, let stand for 5 min, elute with
15 mL hexaneidichloromethane 50:50. Add the eluate to 3 mL 50 mM sulfuric acid, mix for 10
min, centrifuge at 3000 g for 10 min. Remove the aqueous layer and add it to 6 mL hexane:
dichloromethane 50:50, wash for 5 min, centrifuge. Make the aqueous layer basic with 150 |xL
28% ammonia, extract twice with 3 mL hexane:dichloromethane 50:50. Combine the organic
layers and evaporate them to dryness under a stream of nitrogen at 60, reconstitute the
residue in 100 |xL mobile phase, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere cyano
ammonia.)
Flow rate: 1
Detector: E, 5100 A Coulochem, 5020 guard cell 1.00 V, 5011 analytical cell, detector 1 0.55 V,
detector 2 0.80 V, output of detector 2 is monitored
CHROMATOGRAM
Retention time: 21.1 (haloperidol), 14.9 (reduced haloperidol)
OTHER SUBSTANCES
Extracted: chlorpromazine, clomipramine, cyamemazine, desipramine, droperidol, flunitraze-
pam, imipramine, pipamperone, risperidone, trihexyphenidyl
Noninterfering: alprazolam, bromazepam, carbamazepine, chlorazepate, diazepam, diphenylhy-
dantoin, estazolam, ethylbenzatropine, oxazepam, phenobarbital, triazolam, valproic acid
KEYWORDS
plasma; SPE
REFERENCE
Le Moing,J.R; Edouard,S.; Levron,J.C. Determination of risperidone and 9-hydroxyrisperidone in human
plasma by high-performance liquid chromatography with electrochemical detection, J.Chromatogr., 1993,
614, 333-339.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 (xL 40 ng/mL chlorohaloperidol in MeCN + 500 |xL
saturated sodium carbonate, mix well, add 7 mL pentane:dichloromethane 90:10, shake in a
Vibrax shaker for 10 min, centrifuge at 18 at 1725 g for 10 min. Remove the organic layer
(xL MeCN, inject an aliquot.
HPLCVARIABLES
Mobile phase: MeCN:MeOH:40 mM ammonium acetate 86:6:8
Flow rate: 0.8
Detector: E, ESA Coulochem Model 5100A, Model 5011 porous graphite analytical cell, electrode
1 0.6 V (screening), electrode 2 0.95 V (detection), Model 5020 guard cell 1 V (between pump
and injector)
CHROMATOGRAM
Retention time: 12
Internal standard: chlorohaloperidol (11)
OTHER SUBSTANCES
Noninterfering: acetaminophen, benztropine, clonazepam, clozapine, fluphenazine, ibuprofen,
lorazepam, pseudoephedrine, trihexiphenidyl
KEYWORDS
REFERENCE
Aravagiri,M.; Marder,S.R.; Van Putten,T.; Marshall,B.D. Simultaneous determination of plasma haloperidol
and its metabolite reduced haloperidol by liquid chromatography with electrochemical detection. Plasma
levels in schizophrenic patients treated with oral or intramuscular depot haloperidol, J.Chromatogr.B, 1994,
656, 373-381.
SAMPLE
Matrix: blood
jxL 0.1 M phosphoric acid (pH 2.5), mix, separate, inject a 100 jxL aliquot of the aqueous phase.
HPLC VARIABLES
Mobile phase: MeCNrbuffer 35:65 (Buffer was 10 mL/L triethylamine in water adjusted to pH
Flow rate: 2
Detector: UV 228
CHROMATOGRAM
Retention time: 3.4 (haloperidol), 2.5 (reduced haloperidol)
OTHER SUBSTANCES
desipramine, diazepam, encainide, fluoxetine, flurazepam, hydroxyethylflurazepam, ibuprofen,
imipramine, lidocaine, maprotiline, methadone, methaqualone, mexiletine, midazolam, nor-
chlorimipramine, nordiazepam, norfluoxetine, nortriptyline, norverapamil, pentazocine, pro-
mazine, propafenone, propoxyphene, propranolol, protriptyline, quinidine, temazepam, trimi-
pramine, verapamil
warfarin
Interfering: dextromethorphan, diphenhydramine, doxepin, fentanyl, flecainide, nordoxepin,
trazodone
KEYWORDS
plasma; SPE
REFERENCE
Clin.Chem., 1994, 40, 1312-1316.
SAMPLE
Matrix: blood
the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 (xL aliquot of
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 221
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL Bond Elut Certify SPE cartridge with 2 mL MeOH and
2 mL 100 mM pH 6.0 phosphate buffer, do not allow to dry. 1 mL Blood + 6 mL 100 mM pH
6.0 phosphate buffer, vortex, sonicate, centrifuge, add the supernatant to the SPE cartridge,
wash with water, wash with 1 mM pH 3.3 acetic acid, dry by suction, wash with 2 mL acetone:
chloroform 50:50, elute with 3 mL ethyl acetate:ammonia 98:2. Evaporate the eluate under a
stream of nitrogen at 40, reconstitute the residue in 50 JXL MeOH, inject a 10 JJLL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 4 |xm Nova-Pack C18
Mobile phase: Gradient. MeOH:50 mM ammonium acetate 65:35 for 1 min, to 75:25 over 4 min,
maintain at 75:25 for 20 min (Mix column effluent with 50 mM ammonium acetate pumped at
0.5 mL/min.)
Flow rate: 0.6
Detector: MS, Finnigan MAT TSQ 700 tandem quadrupole, MAT TSP-2 interface, thermospray,
selective reaction monitoring m/z 376-165, collision offset -27 V, repeller 100 V, vaporizer 130,
source 200, filament on 200 JJLA, argon 2.5 mTorr, multiplier 1500 V, dynode 15 kV, scan time
1.20 s, MSMSC factor 10
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: benperidol, dextromoramide, droperidol, methadone, penfluridol, pimozide, pipam-
peridone, propoxyphene (dextropropoxyphene)
KEYWORDS
SPE; LC/MS
REFERENCE
Verweij,A.M.; Hordijk,M.L.; Lipman,P.J. Quantitative liquid chromatographic thermospray-tandem mass spec-
trometric analysis of some analgesics and tranquilizers of the methadone, butyrophenone, or diphenylbu-
tylpiperidine groups in whole blood, J.Anal.ToxicoL, 1995, 19, 65-68.
SAMPLE
Sample preparation: 1 mL Blood, urine, or gastric contents or 1 g tissue homogenate + 500 |xL
buffer + 8 mL n-hexane:ethyl acetate 70:30, mix on a rotary mixer for 10 min, centrifuge at
nitrogen, reconstitute the residue in 100 |xL 12.5 mM NaOH in MeOH:water 50:50, inject a 50
|xL aliquot. (Buffer was 13.8 g potassium carbonate in 100 mL water, pH adjusted to 9.5 with
concentrated HCl.)
HPLCVARIABLES
Guard column: 4 X 4 30 |xm LiChrocart Aluspher RP-select B (Merck)
Mobile phase: Gradient. A was 12.5 mM NaOH in MeOH. B was 12.5 mM NaOH in water. A:B
10:90 for 5 min, to 90:10 over 15 min, maintain at 90:10 for 5 min, return to initial conditions
over 1 min, re-equilibrate for 5 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 17
OTHER SUBSTANCES
Extracted: alprenolol, amitriptyline, bromazepam, carbamazepine, chlordiazepoxide, chlorpro-
mazine, clonazepam, desipramine, diazepam, flunitrazepam, nitrendipine, nordiazepam, nor-
triptyline, pindolol, zolpidem
Also analyzed: acebutolol, acetaminophen, alprazolam, amphetamine, atenolol, betaxolol, bro-
tizolam, caffeine, camazepam, captopril, chloroquine, clobazam, clomipramine, clothiapine, clo-
tiazepam, cloxazolam, cocaine, codeine, diclofenac, dihydralazine, dihydrocodeine, dihydroer-
gotamine, diphenhydramine, domperidone, doxepin, droperidol, ergotamine, ethyl loflazepate,
fenethylline, fluoxetine, flupentixol, flurazepam, furosemide, gliclazide, hydrochlorothiazide,
hydroxyzine, ibuprofen, imipramine, ketazolam, loprazolam, lorazepam, lormetazepam, ma-
protiline, medazepam, mepyramine, methadone, methaqualone, methyldopa, methylphenidate,
metoclopramide, metoprolol, mexiletine, mianserin, midazolam, minoxidil, morphine, nadolol,
nitrazepam, oxprenolol, papaverine, pentazocine, phenprocoumon, phenylbutazone, pipamper-
one, piritramide, practolol, prazepam, prazosin, promazine, promethazine, propoxyphene, pro-
pranolol, prothipendyl, quinine, sotalol, sulpride, thioridazine, trazodone, triazolam, trimipra-
mine, tripelennamine, tyramine, verapamil, yohimbine
REFERENCE
Lambert,W.E.; Meyer,E.; De Leenheer,A.P. Systematic toxicological analysis of basic drugs by gradient elution
of an alumina-based HPLC packing material under alkaline conditions, JAnaLToxicoL, 1995,19, 73-78.
SAMPLE
Sample preparation: Blood or serum. 1 mL Blood or serum + 1 |xg cianopramine + 1 mL water,
vortex, add 1 mL 200 mM sodium carbonate, vortex, add 6 mL hexane:l-butanol 95:5, gently
agitate for 30 min, centrifuge at 2500 g for 5 min. Remove the organic layer and add it to 100
JJLL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the organic
layer and inject a 30 |xL aliquot of the aqueous layer. Liver homogenate. 0.5 mL Liver ho-
mogenate + 10 |xg cianopramine + 500 jxL 2% sodium tetraborate + 8 mL hexaneil-butanol 95:
5, gently agitate for 30 min, centrifuge at 2500 g for 5 min. Remove the organic layer and add
it to 400 |xL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the
organic layer and inject a 30 |xL aliquot of the aqueous layer.
HPLC VARIABLES
Guard column: 15 X 3.2 7 |xm RP-18 Newguard (Applied Biosystems)
Column: 100 X 4.6 5 |xm Brownlee Spheri-5 RP-18
Mobile phase: MeCN: 100 mM NaH2PO4:diethylamine 40:57.5:2.5
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
Internal standard: cianopramine (8.93)
OTHER SUBSTANCES
Simultaneous: amitriptyline, amoxapine, benztropine, brompheniramine, chlorpheniramine,
chlorpromazine, clomipramine, cyproheptadine, desipramine, diphenhydramine, dothiepin,
doxepin, fluoxetine, imipramine, loxapine, maprotiline, mesoridazine, methadone, metoclo-
pramide, mianserin, moclobemide, nomifensine, nordoxepin, norfluoxetine, nortriptyline, pen-
tobarbital, pheniramine, promethazine, propoxyphene, propranolol, protriptyline, quinidine,
quinine, sulforidazine, thioridazine, thiothixene, tranylcypromine, trazodone, trihexyphenidyl,
trimipramine, triprolidine
Noninterfering: dextromethorphan, norphetidine, phenoxybenzamine, prochlorperazine,
trifluoperazine
Interfering: meperidine, norpropoxyphene, northiaden
KEYWORDS
serum; whole blood; liver
REFERENCE
McIntyre,I.M.; King,C.V.; Skafidis,S.; Drummer,O.H. Dual ultraviolet wavelength high-performance liquid chro-
matographic method for the forensic or clinical analysis of seventeen antidepressants and some selected
metabolites, J.Chromatogr., 1993, 621, 215-223.
SAMPLE
residue with 50 |JLL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr A, 1997, 763,149
163.
SAMPLE
Sample preparation: Weigh out capsule contents equivalent to 400 |xg haloperidol, dissolve in
30 mL MeOH, add 1 mL 100 |xg/mL isothipendyl hydrochloride in MeOH, filter (paper), wash
filter with MeOH, make up filtrate to 50 mL with MeOH. Dilute a 1 mL aliquot to 10 mL,
HPLC VARIABLES
Column: 250 X 4 5 |xm Econosphere C18
Mobile phase: MeOH:water:triethylamine 80:20:0.15, pH adjusted to 7.8 with phosphoric acid
Flow rate: 1.8
Detector: UV 254
CHROMATOGRAM
Retention time: 4
Internal standard: isothipendyl (7)
OTHERSUBSTANCES
Simultaneous: propantheline bromide
KEYWORDS
capsules
REFERENCE
Sane,R.T.; Ghadge,J.K.; Jani,A.B.; Vaidya,A.J.; Kotwal,S.S. Simultaneous high-performance liquid chromato-
graphic determination of haloperidol with propantheline bromide, nalidixic acid with phenazopyridine hy-
drochloride, and dipyridamole with aspirin in combined dosage (forms), Indian Drugs, 1992, 29, 240-244.
SAMPLE
Sample preparation: 100 u-L Injection solution + 400 |xL 2.5 |xg/mL haloperidol in water, inject
a 100 |xL aliquot.
HPLCVARIABLES
Column: 100 X 4.6 5 |xm Brownlee C18
Mobile phase: MeCN:MeOH:10 mM NaH2PO4 24:31:45, pH adjusted to 5.0 with 2 M KOH
Flow rate: 1.7
Detector: UV 210
CHROMATOGRAM
Internal standard: haloperidol
OTHER SUBSTANCES
Simultaneous: fentanyl, sufentanil
KEYWORDS
injections; haloperidol is IS
REFERENCE
Dewell,W.M.,Jr.; Khandaghabadi,M.; D'Souza,M.J.; Solomon,H.M. High-performance liquid chromatographic
determination of fentanyl and sufentanil returned from the operating room, Am.J.Hosp.Pharm., 1993, 50,
2374-2375.
SAMPLE
Matrix: hair
Sample preparation: Wash hair in water, rinse 3 times with MeOH, dry, weigh. 5-25 mg Washed
hair + 1 mL 1 M NaOH, heat at 70 for 30 min, adjust pH to 9.5-10. 1 mL Extract + 1 jxg
protriptyline + 1 mL water + 1 mL 200 mM sodium carbonate buffer, mix, extract with hexane:
butanol 95:5 for 20 min. Remove the organic layer and add it to 100 |xL 0.2% orthophosphoric
acid, mix for 20 min, inject a 30 |xL aliquot of the aqueous layer.
HPLCVARIABLES
Column: 100 X 4.6 Spheri-5 RP-C18
Mobile phase: MeCN:buffer 40:60 (Buffer was 1.2 L 100 mM pH 7.0 NaH2PO4 + 30 mL
diethylamine.)
Flow rate: 2
Detector: UV 214
CHROMATOGRAM
Retention time: 2.5
OTHER SUBSTANCES
Extracted: amitriptyline, clomipramine, desipramine, dothiepin, doxepin, imipramine, mian-
serin, nortriptyline
KEYWORDS
may be interferences
REFERENCE
Couper,F.J.; McIntyre,I.M.; Drummer,O.H. Extraction of psychotropic drugs from human scalp hair, J.Forensic
ScL, 1995,40,83-86.
SAMPLE
Matrix: solutions
an aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Spherisorb SCX
Mobile phase: MeOH:water 80:20 containing 20 mM ammonium formate and 2.3 mL/L trifluo-
roacetic acid
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 5.5
OTHER SUBSTANCES
Simultaneous: cimetidine, clomipramine, halofantrine, minoxidil, reserpine, verapamil
REFERENCE
the analysis of basic drugs, J.ChromatogrA, 1996, 725, 335-341.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jxg/mL solution in MeOH, inject a 20 jxL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.0
OTHER SUBSTANCES
mine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, hy-
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 30 X 2 5 |xm Hypersil CPS
Column: 250 X 4.6 5 |xm Hypersil CPS-5
Flow rate: 1
CHROMATOGRAM
Retention time: 13 (haloperidol), 11.5 (reduced haloperidol)
Internal standard: pirenzepine (8)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Tomlinson,A.J.; Benson,L.M.; Landers,J.P.; Scanlan,G.R; Fang,J.; Gorrod,J.W.; Naylor,S. Investigation of the
metabolism of the neuroleptic drug haloperidol by capillary electrophoresis, J.ChromatogrA, 1993, 652,
417-426.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.5-1
CHROMATOGRAM
Retention time: 3.0
OTHER SUBSTANCES
diazepam, doxylamine, ethosuximide, furosemide, hydrochlorothiazide, methocarbamol, meth-
otrimeprazine, nicotine, oxazepam, procaine, promazine, propafenone, propranolol, salicylam-
ide, temazepam, tetracaine, thiopental, triamterene, verapamil, zolpidem, zopiclone
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
glunixin, glutethimide, glybenclamide, guaiacol, hydrochlorothiazide, hydrocodone, hydrocor-
mescaline, mesoridazine, methadone, methamphetamine, methapjrrilene, methaqualone, meth-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
azine, flurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin,
dine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxyraetazoline, paroxetine, pemo-
talol, spironolactone, sulfinpâzone, sulindac, temazepam, terbutaline, terfenadine, tetra-
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 |xL aliquot of a 100-500 |xg/mL solution in mobile phase.
HPLC VARIABLES
Column: 100 X 4.6 5 jxm Hypersil C8 MOS 100A coated with phosphatidylcholine (95% pure
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
idine, codeine, desipramine, diphenhydramine, dipyridamole, ephedrine, flufenamic acid, hy-
droxyzine, imipramine, indomethacin, lidocaine, megestrol acetate, metoprolol, nabumetone,
KEYWORDS
REFERENCE
SAMPLE
Matrix: tissue
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 5 mL MeOH and 5 mL water.
Homogenize kidney with a kitchen grinder. Weigh out a 5 g sample and add 20 mL MeCN with
continuous gentle mixing, mix vigorously on a vibromixer at 1500 rpm for 30 s, sonicate for 2
min, centrifuge at 4000 g for 5 min. Mix 7.5 mL sample extract and 40 mL 10% NaCl and add
to SPE cartridge, wash with 1 mL 10 mM sulfuric acid, wash with 2 mL air, elute with 2 mL
acidic MeCN. Place eluate in a washed tube and evaporate to 300 |xL at 70 under a stream
of nitrogen, mix gently, add 1 mL n-hexane, mix on a vibromixer for 30 s, centrifuge at 2000
g, inject a 50 |xL aliquot of the aqueous phase. (Acidic MeCN was 1 mL 50 mM sulfuric acid
and 100 mL MeCN. The washed tube was prepared by rinsing with concentrated ammonia,
water, and acetone and drying under a stream of nitrogen.)
HPLCVARIABLES
Guard column: 10 X 2.1 37-50 ^m Bondapak C18
Column: 300 X 3.9 Bondapak C18
Mobile phase: MeCN.water 55:45 containing 2.46 g/L anhydrous sodium acetate, pH adjusted
to 6.5 with acetic acid
Flow rate: 1.2
Detector: UV 240
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Extracted: azaperol, carazolol, acepromazine, xylazine, azaperone, propiomazine, chlorpro-
mazine
KEYWORDS
SPE; pig; kidney
REFERENCE
Keukens,H.J.; Aerts,M.M.L. Determination of residues of carazolol and a number of tranquilizers in swine
kidney by high-performance liquid chromatography with ultraviolet and fluorescence detection,
J.Chromatogr., 1989, 464, 149-161.
SAMPLE
Matrix: tissue
water. Cut pig kidney or liver into small pieces and homogenize. 5 g Homogenate + 10 mL
MeCN, shake, vortex for 30 s, sonicate for 3 min, vortex for 30 s, sonicate for 3 min, centrifuge
at 10000 g for 20 min. Add 7.5 mL supernatant + 40 mL 10% NaCl to the SPE cartridge at
about 1 mL/min, do not allow cartridge to dry out, wash with 850 |xL 10 mM sulfuric acid, dry
with air, elute with 3.5 mL acidic MeCN. Evaporate the eluate to dryness under a stream of
nitrogen at 50, reconstitute the residue in 300 |xL 10 mM sulfuric acid, vortex briefly, add 1
mL hexane, vortex for 30 s, centrifuge at 2000 g for 5 min, inject an aliquot of the aqueous
layer. (Acidic MeCN was 1 mL 50 mM sulfuric acid in 100 mL MeCN.)
HPLC VARIABLES
Guard column: Hypersil 5 |xm SAS Cl
Column: 250 mm long 5 |xm Hypersil SAS Cl
Mobile phase: MeCN:water 50:50 containing 0.77 g/L ammonium acetate
Flow rate: 2
Detector: E, ESA Model 5100A Coulochem, first electrode +0.4 V, second electrode (which was
monitored) +0.7 V, Model 5020 guard cell after pump but before injector at +0.75 V
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: azaperol, acepromazine, carazolol, azaperone, xylazine, propriomazine, chlorpro-
mazine
KEYWORDS
SPE; pig; kidney; liver
REFERENCE
Rose,M.D.; Shearer,G. Determination of tranquilisers and carazolol residues in animal tissue using high-per-
formance liquid chromatography with electrochemical detection, J.Chromatogr., 1992, 624, 471-477.
Halothane
Molecular formula: C2HBrCIF3
Merck Index: 4634
SAMPLE
Matrix: solutions
Sample preparation: Mix 50 jxL phosphate buffer containing isoflurane and 50 jxL 0.05 mM
toluene in MeOH, inject a 20 (xL aliquot.
HPLC VARIABLES
Guard column: RCSS Guard-Pak ixBondapak C18 precolumn cartridge
Column: 100 X 8 4 |xm Nova-Pak C18 Radial Compression Module
Flow rate: 3.5
Detector: UV 203
CHROMATOGRAM
Retention time: 6
Internal standard: toluene (12)
Limit of detection: 0.001 mM
OTHER SUBSTANCES
Simultaneous: enflurane, isoflurane
KEYWORDS
buffer
REFERENCE
Janicki,P.K; Erskine,W.A.R.; James,M.RM. High-performance liquid chromatographic method for the direct
determination of the volatile anaesthetics halothane, isoflurane and enflurane in water and in physiological
buffer solutions, J.Chromatogr., 1990, 518, 250-253.
Haloxazolam
Molecular formula: C17H14BrFN2O2
Merck Index: 4635
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 20 |xL 20 u,g/mL IS + 200 (xL 1 M potassium carbonate
Evaporate the organic phase under a stream of nitrogen at 40. Dissolve the residue in 100 |xL
HPLCVARIABLES
Flow rate: 0.65
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: bromazepam, chlordiazepoxide, clonazepam, estazolam, etizolam, flutazolam, lora-
zepam, nitrazepam, oxazolam, triazolam
KEYWORDS
serum
REFERENCE
serum using a new reversed-phase chromatographic column on a 2-fim porous microspherical silica gel,
J.Chromatogr.B, 1996, 682, 173-178.
Heparin
Molecular weight: 6000-30000
CAS Registry No.: 9005-49-6,9041-08-1 (Na salt)
Merck Index: 4685
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 500 |xL 10 M NaOH, shake on a slow rotatory mixer for
5 min, add 5 mL diethyl ether, rotomix 10 min, centrifuge at 700 g for 5 min, repeat extraction.
Combine organic layers, evaporate to dryness under a stream of nitrogen at 37, dissolve in
250 |xL mobile phase, inject aliquot.
HPLC VARIABLES
Column: 150 X 3.9 4 (xm Novapack C18
Mobile phase: MeCN:MeOH:buffer 35:15:50 (Buffer was 50 mM KH2PO4 adjusted to pH 3.6 with
phosphoric acid.)
Flow rate: 1.6
Detector: E, Waters Model 464 pulsed electrochemical detector, + 1 V versus Ag/AgCl
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: ethinylestradiol, estrone, estriol, estradiol
Noninterfering: pentobarbital
KEYWORDS
plasma; rabbit
REFERENCE
Fernandez,N.; Garcia,J.J.; Diez,M.J.; Tern,M.T.; Sierra,M. Rapid high-performance liquid chromatographic
assay of ethynyloestradiol in rabbit plasma, J.Chromatogr., 1993, 619, 143-147.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 fjim Ultrasphere ODS
Mobile phase: MeCN:water:glacial acetic acid 4:84:12 containing 4.84 g/L Trizma, pH 2.3
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: acetaminophen, caffeine, cefazolin, cimetidine, ergotamine, glutethimide, meth-
amphetamine, propranolol, salicylic acid, sulfamethoxazole, theobromine, theophylline, tobu-
tamide, trimethoprim
Noninterfering: amitriptyline, amobarbital, ampicillin, butabarbital, butalbital, celbenine,
chlordiazepoxide, chlorpromazine, clorazepate, desipramine, diazepam, doxepin, ethchlorvynol,
fluphenazine, hydroxyzine, ibuprofen, imipramine, isoniazid, lidocaine, mephobarbital, mesor-
idazine, methaqualone, methyluric acid, naprotyline, nordiazepam, nortriptyline, oxazepam,
pentobarbital, perphenazine, phenelzine, phenmetrazine, phenobarbital, phenylbutazone,
phenytoin, prednisolone, prednisone, procainamide, prochlorperazine, promazine, prometha-
zine, propoxyphene, protriptyline, pyrilamine, secobarbital, thioridazine, thiothixene, timolol,
trazodone, triazolam, trifluoperazine
REFERENCE
Osterloh,J.; Yu,S. Simultaneous ion-pair and partition liquid chromatography of acetaminophen, theophylline
and salicylate with application to 500 toxicologic specimens, Clin.Chim.Acta, 1988, 175, 239-248.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 X 7.5 Ultropac TSK G 2000 SW (LKB)
Mobile phase: 100 mM NaCl
Flow rate: 0.5
Detector: UV 206
CHROMATOGRAM
Retention time: 15, 21, 29
KEYWORDS
SEC
REFERENCE
de Vries,J.X. Analysis of heparins by size-exclusion and reversed-phase high-performance liquid chromatog-
raphy with photodiode-array detection, J.Chromatogr., 1989, 465, 297-304.
Next Page
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 mg/mL solution in mobile phase, inject a 20 |iL aliquot.
HPLC VARIABLES
Column: TSK G3000SW and TSK G2000SW in series (Tosoh)
Mobile phase: 500 mM sodium sulfate
Flow rate: 0.5
Detector: UV 234 or RI
KEYWORDS
SEC
REFERENCE
Ahsan,A.; Jeske,W.; Hoppensteadt,D.; Lormeau,J.C; Wolf,H.; Fareed,J. Molecular profiling and weight deter-
mination of heparins and depolymerized heparins, J.Pharm.Sci, 1995, 84, 724-727.
SAMPLE
Matrix: urine
Sample preparation: Mix 9 mL urine with 600 |xL 5% hexadecylpyridinium chloride, keep at
0 for 4 h. Centrifuge at 2300 g for 15 min, wash the precipitate twice with 1.5 mL 0.1%
hexadecylpyridinium chloride. Dissolve the precipitate in 1 mL 2.5 M NaCl. Centrifuge at 2300
g for 15 min. Add 11 mL EtOH:water 85:15 to he supernatant, keep overnight at 0. Centrifuge
at 2300 g for 15 min at 4. Dry the precipitate under reduced pressure, re-dissolve in 50 jxL
200 mM pH 8.0 Tris-HCl buffer. Add 10 |xL of an aqueous solution containing 0.1 U chondro-
itinase ABC, incubate at 37. for 3 h. Add 250 |xL EtOH, keep overnight at 4 dg. Centrifuge at
4 at 2300 g for 15 min. Wash the precipitate with three 1 mL portions of EtOH:water 75:25.,
dry under reduced pressure. Redissolve in 50 JJLL 100 mM acetate buffer containing 10 mM pH
7.0 calcium acetate. Mix 10 JJLL sample with 10 |xL 100 mM acetate buffer containing 10 mM
pH 7.0 calcium acetate and 30 JJLL aqueous heparin lyase solution, incubate at 37 for 12 h.
Inject a 2 JJLL aliquot. (The heparin lyase solution contained 4 mU heparin lyase I, 0.4 mU
heparin lyase II, and 0.4 mU heparin lyase III.)
HPLCVARIABLES
Column: 150 X 2.0 5 |xm TSK gel Amide-80 column (TOSOH, Japan)
Mobile phase: MeCN:water:200 mM pH 7.0 sodium phosphate buffer:3.0 M ammonium chloride
32:10:1:1
Flow rate: 0.4
Injection volume: 2
2-cyanoacetamide solution containing 500 mM NaOH pumped at 0.25 mL/min and this mixture
flowed through a 10 m X 0.5 mm reaction coil at 110 and a 2 m X 0.25 mm cooling coil to the
detector.
CHROMATOGRAM
Retention time: 7.1 (S UA-GIcNAc), 8.5 (8 UA-GlcNAc6S), 10.5 (S UA2S-GlcNac6S), 11.0 (8 UA-
^ GIcNS), 8.9 (8 UA2S-GlcNS), 16.1 (8 UA-GlcNS6S), 21.2 (8 UA2s-GlcNS6S)
Limit of detection: 2 pmol
KEYWORDS
REFERENCE
Toyoda,H.; Nagashima,T.; Hirata,R.; Toida,T.; Imanari,T. Sensitive high-performance liquid chromatographic
method withfluorometricdetection for the determination of heparin and heparan sulfate in biological sam-
ples: application to human urinary heparan sulfate, J.Chromatogr.B, 1997, 704, 19-24.
Previous Page
Hesperidin
Molecular formula: C28H34Oi5
Merck Index: 4705
SAMPLE
Sample preparation: Split each plasma and urine sample into two fractions. Mix 3 mL 1 M pH
4.5 sodium acetate buffer with one 3 mL plasma portion or one 10 mL urine portion, incubate
at 37 for 20 h with 10,000 U p-glucuronidase (Helix Pomatia, Sigma). Condition a Sep-Pak
C18 SPE cartridge with 6 mL EtOHrwater 95:5 and 10 mL water. Add the sample to the SPE
cartridge. Wash with 4 mL MeOH:water 10:90, elute the flavanones with 3 mL MeOH, reduce
the plasma eluent volume to 500 jxL under a stream of nitrogen. Inject 25 or 50 jxL aliquots.
HPLCVARIABLES
Mobile phase: MeOH.water.glacial acetic acid 47:50.5:2.5
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Extracted: hesperitin, naringenin, naringin, narirutin
KEYWORDS
plasma; SPE
REFERENCE
Ameer,B.; Weintraub,R.A.; Johnson,J.V.; Yost,R.A.; Rouseff,R.L. Flavanone absorption after naringin, hesperi-
din, and citrus administration, Clin.Pharmacol.Ther., 1996, 60, 34-40.
SAMPLE
Sample preparation: Condition a 2.4 mL 500 mg 40 |xm Bond Elut C2 SPE cartridge with 2
mL MeOH and 2 mL water. Adjust pH of 1-2 mL urine or plasma to 5.5 with buffer, add to the
SPE cartridge, wash with two 2 mL portions of water, elute with 1 mL MeOH. Add the eluate
to 100 |iL 4 M pH 5.5 ammonium acetate, centrifuge at 1000 g for 5 min, inject an aliquot of
the supernatant. (Buffer was 10 mM ammonium acetate adjusted to pH 5.5 with acetic acid.)
HPLCVARIABLES
Column: 300 X 3.8 10 jxm jiBondapak C18
Mobile phase: Isopropanol:buffer 20:80 (Buffer was 10 mM ammonium acetate adjusted to pH
5.5 with acetic acid.)
Flow rate: 1
Detector: UV 303
CHROMATOGRAM
Internal standard: hesperidin
OTHER SUBSTANCES
Extracted: flavone acetic acid
KEYWORDS
SPE; hesperidin is IS; plasma
REFERENCE
Cummings,J.; Kerr,D.J.; Kaye,S.B.; Smyth,J.F. Optimisation of a reversed-phase high-performance liquid chro-
matographic method for the determination of flavone acetic acid and its major human metabolites in plasma
and urine, J.Chromatogr., 1988, 431, 77-85.
SAMPLE
Matrix: fruit
Sample preparation: Grind 1.2 g dried fruit, extract with 50 mL EtOHrwater 80:20 at 90 for
2 h. Filter the solution and evaporate it to dryness at vacuum. Dissolve 120 mg residue in 50
mL MeOH, filter (0.45 (xm nylon Acrodisk), inject a 3 |xL aliquot.
HPLC VARIABLES
Column: 150 X 2.1 5|xm Waters Symmetry C18 (Waters USA)
Mobile phase: Gradient. A was water containing 0.6% acetic acid. B was MeOH. A:B 80:20 to
60:40 over 12 min; maintain at 60:40 for 7 min, to 0:100 over 11 min, maintain at 0:100 for 3
min, to 80:20 over 2 min.
Flow rate: 0.2
Injection volume: 3
Detector: UV 290; MS, HP 5989 B electrospray, quadrupole temperature 150., EM 2173 V, pos-
itive mode, drying gas nitrogen, 360., nebulizing gas nitrogen 0.55 MPa, m/z 611
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: hesperitin, isonaringin, naringenin, naringin, neohesperidin, nobiletin, tangeritin
KEYWORDS
sour orange; orange
REFERENCE
He,X.-g.; Lian,L.-z.; Lin,L.-z.; Bernart,M.W. High-performance liquid chromatography-electrospray mass spec-
trometry in phytochemical analysis of sour orange (Citrus aurantium L.), J.ChromatognA, 1997, 791,127
134.
SAMPLE
Matrix: juice
Sample preparation: Centrifuge 10 mL juice at 2500 g for 10 min, filter 1.2 u-m, inject a 20 |xL
aliquot.
HPLC VARIABLES
Guard column: 50 X 4.6 Spheri-5 C18
Column: 125 X 3.6 3 |xm C18 (Supelco)
Mobile phase: MeCN:water:glacial acetic acid 20:79.5:0.5
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Extracted: narirutin, naringin, neohesperidin
KEYWORDS
orange; grapefruit
REFERENCE
Rouseff,R.L. Liquid chromatographic determination of naringin and neohesperidin as a detector of grapefruit
juice in orange juice, J.Assoc.Off.Anal.Chem., 1988, 71, 798-802.
SAMPLE
Matrix: juice
Sample preparation: Condition a 360 mg Sep-Pak C18 SPE cartridge with 2 mL MeOH and
two 4 mL portions of water. Centrifuge 15 mL orange juice at 8000 rpm for 15 min, add 2 mL
of the supernatant to the SPE cartridge, wash with 2 mL water, wash with 1.5 mL MeOH:
water 25:75, elute with 1.5 mL THF, inject a 20 JJLL aliquot.
HPLC VARIABLES
Guard column: 20 mm long LC-NH2 (Supelco)
Column: 250 X 4.6 Bondesil C18 (Analytichem)
Mobile phase: Gradient. MeCN:THF:2% acetic acid 5:12:83 for 22 min, to 0:35:65 over 6 min,
maintain at 0:35:65 for 12 min, return to initial conditions over 2 min, re-equilibrate for 10
min.
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Extracted: caffeic acid, coumaric acid, ferulic acid, narirurin, sinapic acid
KEYWORDS
orange; SPE
REFERENCE
Rouseff,R.L.; Dettweiler,G.R.; Swaine,R.M.; Naim,M.; Zehavi,U. Solid-phase extraction and HPLC determina-
tion of 4-vinyl guaiacol and its precursor, ferulic acid, in orange juice, J.Chromatogr.Sci., 1992, 30, 383-
387.
SAMPLE
Matrix: juice
Sample preparation: Grapefruit. 1 g Grapefruit juice concentrate + 1 mL 1.8 mg/mL rhoifolin
in MeOH, vortex for 1 min, centrifuge at 25000 g for 15 min, remove the supernatant. Add 1
mL MeOH to the solid and stir with a spatula, vortex for 1 min, centrifuge, repeat extraction.
Combine the supernatants and add 2 mL water, filter (1 |xm glass fiber), filter (0.2 (xm, Anotop),
inject a 20 |xL aliquot of the filtrate. Orange. 1 g Orange juice concentrate + 0.4 mL 1.8 mg/
mL rhoifolin in MeOH + 3 mL MeOH, vortex for 30 s, heat at 55 for 15 min, mix for 30 s,
centrifuge at 25000 g for 15 min, remove the supernatant. Add 2 mL MeOH and 1 mL water
to the solid, vortex for 30 s, heat at 55 for 15 min, vortex for 30 s, centrifuge. Combine the
supernatants and add 4 mL water, vortex, filter (1 fxm glass fiber), filter (0.2 jjim, Anotop),
inject a 20 jutL aliquot of the filtrate.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Alltima C18 (Alltech)
Mobile phase: MeCN:water:isopropanol:formic acid 11.5:79:9.5:0.1
Flow rate: 0.6
Detector: UV 283
CHROMATOGRAM
Retention time: 21
Internal standard: rhoifolin (UV 335) (30)
OTHER SUBSTANCES
Simultaneous: naringin, narirutin
KEYWORDS
concentrate; orange; grapefruit
REFERENCE
Bronner,W.E.; Beecher,G.R. Extraction and measurement of prominent flavonoids in orange and grapefruit
juice concentrates, J.ChromatogrA, 1995, 705, 247-256.
SAMPLE
Matrix: plants
Sample preparation: Grind wood, bark, or leaves and extract with MeOHrwater 80:20 at room
temperature for 24 h, filter, remove the MeOH under reduced pressure, extract the aqueous
layer with diethyl ether. Dry the organic layer and evaporate it to dryness, reconstitute with
MeOH, inject a 20 |xL aliquot.
HPLCVARIABLES
Guard column: Hypersil ODS
Column: 200 X Hypersil ODS
Mobile phase: Gradient. A was MeOH containing 0.1% phosphoric acid. B was water containing
0.1% phosphoric acid. A:B from 20:80 to 100:0 over 40 min, maintain at 100:0 for 5 min.
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
Extracted: flavanoids, phenolic acids
KEYWORDS
wood; bark; leaves
REFERENCE
Conde,E.; Cadahia,E.; Garcia-Vallejo,M.C. HPLC analysis of flavonoids and phenolic acids and aldehydes in
Eucalyptus spp, Chromatographia, 1995, 41, 657-660.
SAMPLE
Matrix: plants
Sample preparation: Extract leaves eight times with 5 mL MeOH at 60, combine the extracts,
filter, evaporate to dryness, reconstitute in 1 mL MeOH, filter (0.22 |xm), inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 10 fjim C18 (Vydac)
Mobile phase: Gradient. MeCN:3% acetic acid from 1:99 to 10:90 over 15 min, to 15:85 over 15
min, to 25:75 over 20 min, to 35:65 over 20 min, to 50:50 over 15 min, maintain at 50:50 for
20 min.
Flow rate: 1
CHROMATOGRAM
Internal standard: isorhamnetin 3-rutinoside (38)
OTHER SUBSTANCES
Simultaneous: caffeic acid, chlorogenic acid, daticoside, dihydrorobinetin, robinin, rutin
REFERENCE
Ficarra,R.; Ficarra,R; Tommasini,S.; Calabro,M.L.; Ragusa,S.; Barbera,R.; Rapisarda,A. Leaf extracts of some
Cordia species: Analgesic and anti-inflammatory activities as well as their chromatographic analysis, Far-
maco, 1995, 50, 245-256.
SAMPLE
Matrix: plants
Sample preparation: 500 mg Plant material + 7 mL MeOHrwater 70:30, stir at room temper-
ature for 30 min, centrifuge at 1500 g for 5 min, repeat extraction 3 times. Combine extracts,
filter (No. 1 paper), add 2.5 mL 1.172 mg/mL p-tert-octylphenol in MeOH, make up to 25 mL
with MeOH:water 70:30, inject a 5 JJLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Cosmosil 5C18 (Nacalai Tesque)
Mobile phase: Gradient. A was MeCN:buffer 10:90. B was MeCN:MeOH:l% acetic acid 45:45:
10. A:B from 90:10 to 75:25 over 14 min, maintain at 75:25 for 8 min, to 70:30 over 8 min, to
20:80 over 5 min, to 0:100 over 10 min, maintain at 0:100 over 10 min, return to initial con-
ditions over 5 min. (Buffer contained 20 mM sodium acetate and 419.7 mM acetic acid.)
Flow rate: 0.8
Injection volume: 5
Detector: UV 280
CHROMATOGRAM
Internal standard: p-tert-octylphenol (54.2)
OTHER SUBSTANCES
Extracted: emodin, gallic acid, honokiol, magnolol, naringin, sennoside A, sennoside B
REFERENCE
Sheu,S.-J.; Lu,C.-F. Determination of eight constituents of Hsiao-cheng-chi-tang by high-performance liquid
chromatography, J.ChromatogrA, 1995, 704, 518-523.
Hetacillin
CAS Registry No.: 3511-16-8, 5321-32-4 (K salt)
Merck Index: 4706
Lednicer No.: 1 414
SAMPLE
Matrix: milk
Sample preparation: 500 |xL Milk + 500 |xL MeCN:MeOH:water 40:20:40, vortex for 10-15 s,
filter (Centricon-10, molecular mass cut-off filter 10000 daltons) with centrifuging at 2677 g
for 30 min, inject a 10-100 |xL aliquot of the ultrafiltrate.
HPLC VARIABLES
Column: 220 X 2.1 5 |xm Spheri-5 phenyl microbore (UV detection) or 220 X 4.6 5 (xm Spheri-5
phenyl microbore (MS detection)
Mobile phase: MeCN:85% phosphoric acid:triethylamine:water 20:0.4:0.4:79.2 containing 5 mM
dodecanesulfonate (UV) or isopropanol:acetic acid in 200 mM ammonium acetate:water 10:2:
88 (MS)
Flow rate: 0.2-0.45 (UV) or 0.8-1.2 (MS)
Detector: UV 220 or MS, Finnigan MAT 4800 quadrupole, thermospray, source 320, vaporizer
120, pulsed positive ion negative ion
CHROMATOGRAM
Retention time: 8.3 (UV), 14.5 (MS) [as ampicillin]
Limit of detection: 200 ng/mL (MS), 75 ng/mL (100)
OTHER SUBSTANCES
Also analyzed: cloxacillin, amoxicillin
KEYWORDS
ultrafiltrate; LC-MS; hetacillin is rapidly converted to ampicillin
REFERENCE
Voyksner,R.D.; Tyczkowska,K.L.; Aronson,A.L. Development of analytical methods for some penicillins in bo-
vine milk by ion-paired chromatography and confirmation by thermospray mass spectrometry,
J.Chromatogr., 1991, 567, 389-404.
SAMPLE
Matrix: solutions
Sample preparation: React the antibiotic, triethylamine, and l-(2,5-dihydroxyphenyl)-2-bro-
moethanone in a 1:2:4 molar ratio in DMF at 45 for 2 h (use 18-crown-6 to make the potassium
salt soluble), inject a 10 |xL aliquot. (Preparation of l-(2,5-dihydroxyphenyl)-2-bromoethanone
is as follows. Stir 27.6 g 1,4-dimethoxybenzene and 28 mL bromoacetyl bromide at 0, add 53.4
g aluminum bromide over 10 min (an exothermic reactions ensues), let stand at room temper-
ature for 12 h, add 100 mL 48% HBr, add 100 g ice, stir for 1 h, extract twice with 200 mL
portions of diethyl ether. Combine the extracts and wash them 3 times with 200 mL portions
of water, dry over 40 g anhydrous magnesium sulfate, evaporate to dryness, recrystallize the
product 3 times from EtOH to yield l-(2,5-dihydroxyphenyl)-2-bromoethanone monobromoace-
tate (mp 105-107). Dissolve H g l-(2,5-dihydroxyphenyl)-2-bromoethanonemonobromoacetate
in 200 mL warm dry MeOH saturated with HBr, stir for 18 h, add 200 mL water, cool to -10.
Collect the yellow solid and dry it under vacuum at 50 for 48 h, recrystallize from toluene:
heptane 50:50 then toluene to obtain l-(2,5-dihydroxyphenyl)-2-bromoethanone as yellow nee-
dles (mp 117-119).)
HPLCVARIABLES
Column: 250 X 4 7 |xm RP-18 LiChrocart (Merck)
Mobile phase: MeOH: 100 mM pH 6.5 sodium acetate 58:42
Flow rate: 1
Detector: E, Bioanalytical Systems Model LC4B, glassy carbon electrode 0.8 V, Ag/AgCl reference
electrode
CHROMATOGRAM
Retention time: 7.8
OTHER SUBSTANCES
Simultaneous: carbenicillin, cephapirin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin,
penicillin G
KEY WpRDS
derivatization
REFERENCE
Munns,R.K; Roybal,J.E.; Shimoda,W.; Hurlbut,J.A. l-(4-Hydroxyphenyl)-, l-(2,4-dihydroxyphenyl)-and l-(2,5-
dihydroxyphenyl)-2-bromoethanones: new labels for determination of carboxylic acids by high-performance
liquid chromatography with electrochemical and ultraviolet detection, J.Chromatogr., 1988, 442, 209-218.
Hexachlorophene
Molecular formula: Ci3H6CI6O2
Merck Index: 4716
SAMPLE
Matrix: solutions
Sample preparation: Dissolve 4-20 |xg in 1 mL 5% NaOH, add 30 |xL p-anisoyl chloride, vortex
for 1 min, let stand at room temperature for 20 min, add 9 mL water, vortex for 2 min, extract
three times with 10 mL portions of hexane. Combine the extracts and evaporate them to dry-
ness under a stream of nitrogen, reconstitute the residue in 1 mL butyl chloride, inject a 10
IxL aliquot.
HPLCVARIABLES
Column: 610 X 2.3 36-40 |xm SiI-X silica (Nester-Faust)
Mobile phase: Hexane :n-butyl chloride 55:45
Flow rate: 0.7
Detector: UV 254
CHROMATOGRAM
KEYWORDS
REFERENCE
Porcaro,P.J.; Shubiak,P. Detection of nanogram quantities of hexachlorophene by ultraviolet liquid chromatog-
raphy, Anal Chem., 1972, 44, 1865-1867.
Hexobarbital
Merck Index: 4742
Lednicer No.: 1 273
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm YMC GEL, ODS-AM coated with poly-(R)-l-(a-naphthyl)ethyl meth-
acrylamide (Prepare (R)-l-(a-naphthyDethyl methacrylamide by reacting methacryl chloride
with (R)-l-(a-naphthyl)ethylamine. Prepare poly-(R)-l-(a-naphthyl)ethyl methacrylamide by
polymerizing this compound in anhydrous benzene/THF with 2,2'-azobis(isobutyronitrile) (Cau-
tion! Benzene is a carcinogen!). Average molecular weight = 2500. Coat 4 g 5 |xm YMC GEL,
ODS-AM with 0.8 g of this polymer using dichloromethane as a solvent.)
Flow rate: 0.7
CHROMATOGRAM
Retention time: k' 6.86 (a = 1.08)
KEYWORDS
chiral
REFERENCE
Oi,N.; Hashimoto,S.; Ishizuka,N.; Ohtake,J. Enantiomer separation with poly-(R)-l (a-naphthyl)-ethyl-meth-
acrylamide coated on ODS silica gel by reversed phase HPLC, Biomed.Chromatogr., 1997,11, 296-297.
Histamine
CAS Registry No.: 51-45-6,51-74-1 (phosphate)
Merck Index: 4756
SAMPLE
Matrix: bacterial culture
Sample preparation: Prepare a column by filling a 1 mL pipette tip with 200 mg Extrelut,
condition with 250 JJLL buffer. Add 10 JULL bacterial culture and 10 jxL IS solution to the column,
elute with 1.3 mL ethyl methyl ketone iisopropanol 90:10 (prepared just prior to use) or with
1.3 mL ethyl methyl ketone. Add 200 |xL reagent to the eluate, vortex for 15 s, let stand for
30 min, dilute 1 volume of the supernatant with 1 volume of MeOH and 2 volumes of water,
inject an aliquot. (Prepare buffer by dissolving 50 mmoles ascorbic acid and 50 mmoles
Na2HPO4 in 80 mL 1 M NaOH with stirring, adjust pH to 12.5 with 10 mM NaOH, make up
to 100 mL with water, store in completely filled vials. Prepare the IS solution by adding 12 |xL
pentylamine to 100 mL 100 mM HCl. Purify ethyl methyl ketone by passing 20 mL through 4
g acidic aluminum oxide (Merck). Prepare reagent each day by dissolving 10 mg o-phthaldi-
aldehyde and 50 (xL ethanethiol in 1 mL MeOH and adding 9 mL 400 mM pH 9.5 sodium
borate buffer.)
HPLC VARIABLES
Column: 125 X 2 Superspher 100 RP-18 (Merck)
Mobile phase: MeOH:buffer 85:15 (Buffer was 7% triethylamine adjusted to pH 7.5 with acetic
acid.)
Flow rate: 0.2
Injection volume: 1
CHROMATOGRAM
Internal standard: pentylamine (7.45)
OTHER SUBSTANCES
Extracted: cadaverine, isobutylamine, phenethylamine, putrescine, tryptamine, tyramine
KEYWORDS
SPE; derivatization
REFERENCE
Bilic,N. Rapid identification of biogenic amine-producing bacterial cultures using isocratic high-performance
liquid chromatography, J.Chromatogr.A, 1996, 719, 321-326.
SAMPLE
Matrix: beverages
Sample preparation: Condition a 500 mg SAX SPE cartridge (Varian) with two 5 mL portions
of MeOH and two 5 mL portions of water. Condition a 1000 mg C18 SPE cartridge with two 5
mL portions of MeOH two 5 mL portions of 200 mM pH 4.5 sodium decanesulfonate. Adjust
pH of 15 mL red wine to 8, pass through the SAX SPE cartridge, adjust the pH of the eluate
to 4.5, add 100 |xL 200 mM sodium decanesulfonate, add this solution to the C18 SPE cartridge,
elute with 3 mL MeOH. Mix 10 |xL reagent and 2 JJLL eluate for 1 min, inject the whole amount.
(Prepare reagent by dissolving 45 mg o-phthalaldehyde and 200 |xL mercaptoethanol in 1 mL
MeOH and making up to 10 mL with Buffer. Prepare buffer by dissolving 3.81 g sodium tetra-
borate in 100 mL water and adjusting pH to 10.5 with 10 M NaOH.)
HPLC VARIABLES
Guard column: ODS Basic (Teknokroma, Barcelona)
Column: 250 X 4.6 5 |xm ODS Basic (Teknokroma, Barcelona)
Mobile phase: Gradient. A was THF:50 mM pH 7 (?) sodium acetate 1:99. B was MeOH. A:B
from 45:55 to 20:80 over 25 min, maintain at 20:80 for 3 min, return to initial conditions over
2 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
Extracted: amylamine, butylamine, cadaverine, ethanolamine, ethylamine, hexylamine, isopro-
pylamine, methylamine, 3-methylbutylamine, phenethylamine, propylamine, putrescine, trypt-
amine, tyramine
KEY WORDS
wine; derivatization; SPE
REFERENCE
Busto,O.; Guasch,J.; Borrull,F. Improvement of a solid-phase extraction method for determining biogenic
amines in wines, J.ChromatogrA, 1995, 718, 309-317.
SAMPLE
Matrix: blood
Sample preparation: Chill 20 mL whole blood in ice water, add 1 mL reagent, invert several
time, centrifuge at 4 at 4000 g for 10 min. Remove the plasma and make it 0.4 M in perchloric
acid by adding concentrated perchloric acid, mix, let stand at 4 for 15 min, centrifuge at 4 at
20000 g for 20 min. Remove a 2 mL aliquot and adjust the pH to 7.0 0.2 with 0.5 M KOH,
add 400 |xL reagent, add 2 g NaCL, add 2 mL ethyl acetate, shake for 1 min, centrifuge at
3400 g, repeat the extraction. Combine the organic layers, add 2 mL 35 mM pH 10.0 0 . 1
Na2HPO4 buffer, shake for 1 min, centrifuge at 3400 g, repeat the wash, evaporate the ethyl
acetate layer to 100 (xL with a stream of nitrogen, inject a 10-50 JJLL aliquot. (Prepare reagent
as follows. Dissolve 500 mg boric acid in 19 mL water, adjust the pH to 10.40 0.02 with 450
g/L KOH, add 17.5 mg o-phthalaldehyde dissolved in 200 JJLL MeOH, add 40 jxL fresh 2-mer-
captoethanol, store under nitrogen at 5, stable for 7 days (J.Chromatogr. 1979, 162, 293).)
HPLC VARIABLES
Guard column: Co:Pell ODS
Column: 300 X 4 10 |jim ixBondapak phenyl
Mobile phase: Gradient. MeCN:25 mM pH 5.10 NaH2PO4 25:75 for 15 min, then MeOH:25 mM
pH 5.10 NaH2PO4 45:55 for 35 min (step gradient).
Flow rate: 1.5
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Extracted: dopamine, norepinephrine, octopamine, serotonin, tyramine
KEYWORDS
pig; whole blood; derivatization
REFERENCE
Davis,T.R; Gehrke,C.W.,Jr.; Williams,C.H.; Gehrke,C.W.; Gerhardt,KO. Pre-column derivatization and high-
performance liquid chromatography of biogenic amines in blood of normal and malignant hyperthermic
pigs, J.Chromatogr., 1982, 228, 113-122.
SAMPLE
Matrix: blood
Sample preparation: Mix plasma with pH 9.7 buffer containing 8.6 mM cyanide and 17.2 mM
naphthalene-2,3-dicarboxaldehyde, let stand for 20 min, add 200 mM taurine, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultramex C8
Detector: F
CHROMATOGRAM
KEYWORDS
REFERENCE
James,J.; Lowe,D.; Karnes,H.T. Determination of histamine from plasma using derivatization with naphtha-
lene-2,3-dicarboxaldehyde and HPLC with fluorescence detection, Pharm.Res., 1992, 9, S21.
SAMPLE
Matrix: blood
Sample preparation: Condition a CBA (carboxylic acid) SPE cartridge (Analytichem) with 1 mL
MeOH and 2 mL 10 mM pH 7 phosphate buffer. 500 juiL Plasma + 100 jxL 1 mg/mL betazole
+ 2 mL ice-cold 10 mM pH 7 phosphate buffer, add to the SPE cartridge at 0.2-0.3 mL/min,
dry for 30 s, wash with 1 mL hexane, elute with 1 mL MeOH: 100 mM HCl 60:40. Evaporate
the eluate to dryness under a stream of nitrogen at 60, reconstitute the residue in 100 |xL 200
mM pH 9 sodium borate buffer, add 50 |xL 20 |xg/mL fluorescamine in MeCN, vortex, store at
4, inject a 50 u,L aliquot.
HPLCVARIABLES
Guard column: Pelliguard LC-8 (Supelco)
Column: 250 X 4.6 5 |xm Ultramex C8
Mobile phase: MeCN:500 mM pH 7 imidazole buffer 20:80
Flow rate: 1
CHROMATOGRAM
Internal standard: betazole (26.9)
KEYWORDS
plasma; SPE; derivatization
REFERENCE
Lowe,D.R.; March,C; James,J.E.; Karnes,H.T. A high-performance liquid chromatographic method for hista-
mine in plasma using solid phase extraction and fluorescamine derivatization, J.Liq.Chromatogr., 1994,17,
3563-3570.
SAMPLE
Matrix: blood
Sample preparation: Condition a CBA SPE cartridge with 1 mL MeOH and 2 mL 10 mM pH
7 phosphate buffer. 500 |xL Plasma + 2 mL chilled 10 mM pH 7 phosphate buffer, mix, add to
the SPE cartridge, dry for 30 min, wash with 1 mL hexane, elute with 1 mL MeOH: 100 mM
HCl 40:60. Evaporate to dryness under a stream of nitrogen at 60, reconstitute the residue in
100 IJLL 200 mM pH 9.7 borate buffer. Remove a 10 JJLL aliquot and add it to 40 |xL 20 |xg/mL
fluorescamine in 200 mM pH 9.7 sodium borate buffer and 50 |JLL 200 mM pH 9.7 sodium
borate buffer, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultramex C-8
Mobile phase: MeCN:500 mM pH 7 imidazole nitrate buffer 20:80
Flow rate: 1
CHROMATOGRAM
KEYWORDS
derivatization; plasma; SPE; details of chemiluminescence detection are also given in paper
REFERENCE
Walters,D.L.; James,J.E.; Vest,F.B.; Karnes,H.T. A comparison of fluorescence versus chemiluminescence de-
tection for analysis of the fluorescamine derivative of histamine by HPLC, Biomed.Chromatogr., 1994, 8,
207-211.
SAMPLE
at 110 for 24 h, evaporate to dryness, reconstitute with 50-200 jxL 0.1% HCl containing 0.2%
3,3'-thiodipropionic acid at 20000 rpm for 2 min, sonicate for ^30 min, centrifuge at 5000 g for
with 200 JJLL 100 mM HCl containing 0.2% 3,3'-thiodipropionic acid) 3 mL supernatant while
centrifuging at 3500 g for 1 h. Mix 20 |JLL deproteinized sample (or 10 |xL peptide hydrolysate)
with 180 |xL buffer, vortex, add 200 |xL reagent, mix, heat at 70 for 15 min with mixing at 1
min and 12 min, cool in an ice bath for 5 min, centrifuge at 10000 g for 10 s, add 400 jxL
diluent, mix thoroughly, centrifuge at 15000 g for 5 min, inject a 10 |xL aliquot of the super-
HPLC VARIABLES
to pH 6.55 with phosphoric acid. B was MeCNrwater 80:20. A:B 92:8 for 2 min, to 80:20 over
Flow rate: 1
Detector: UV 436
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: amino acids dopamine, epinephrine, norepinephrine, taurine
KEYWORDS
REFERENCE
KrauseJ.; Bockhardt,A.| Neckermann,H.; Henle,T.; Klostermeyer,H. Simultaneous determination of amino
SAMPLE
Matrix: bulk
Sample preparation: Evaporate 100 |xL of a solution of histamine in MeOH to dryness under
nitrogen, add 100 |xL 0.3 mM N-(4-aminobutyl)-N-ethylisoluminol isothiocyanate in MeCN:
water:triethylamine 88:10:2 to the residue, vortex for 10 s, heat at 80 for 1 h. Add 600 |xL
mobile phase to the reaction mixture, mix, inject a 100 JJLL aliquot. (Synthesis of N-(4-amino-
butyl)-N-ethylisoluminol isothiocyanate is as follows. Dissolve 110 mg N-(4-aminobutyl)-N-
ethylisoluminol in 500 mL 100 mM sodium carbonate, add 100 |xL thiophosgene, stir at room
temperature for 2.5 h, adjust pH to 1 with 100 mL 1 M HCl, extract twice with 250 mL portions
of ethyl acetate, combine the extracts and evaporate them to dryness under reduced pressure
at 30-35. Dissolve the residue in 4 mL DMF, add 100 mL ice-cold water, store at 4 overnight,
collect the precipitate by filtration (0.45 |xm), dry under vacuum for 16 h to obtain N-(4-ami-
nobutyl)-N-ethylisoluminol isothiocyanate. Store at 4 in the dark.)
HPLC VARIABLES
Guard column: 10 X 2.0 10 |xm PLRP-S (Polymer Labs., UK)
Column: 250 X 4.0 5 |xm Asahipak ODP-50 (Hewlett-Packard)
Mobile phase: Gradient. A was MeCN: 10 mM pH 10.5 sodium carbonate buffer 30:70 containing
5 mM tetraheptylammonium bromide. B was MeCN: 10 mM pH 10.5 sodium carbonate buffer
70:30. A:B 100:0 for 22 min, to 0:100 (step gradient), maintain at 0:100 for 6 min, re-equilibrate
at initial conditions for 30 min
Flow rate: 0.8
Detector: E, ESA Coulochem guard cell, Model 5020, porous graphite electrode, Pd/PdO reference
electrode
CHROMATOGRAM
Retention time: 20
Limit of detection: 1.5 pmol
KEYWORDS
derivatization
REFERENCE
Steijger,O.M.; Kamminga,D.A.; Brummelhuis,A.; Lingeman,H. Liquid chromatography with luminol-based elec-
trochemiluminescence detection. Determination of histamine, J.ChromatognA, 1998, 799, 57-66.
SAMPLE
Matrix: bulk
Sample preparation: Mix 0.5-5 nmole histamine with 50 nmole luminarin 2 in 500 |xL acetone,
add 100 |xL 100 mM dimethylaminopyridine in acetone, evaporate to dryness, let stand in the
dark for 1.5 !!,reconstitute with 200 JULL acetone, inject an aliquot. (Dry acetone over 0.4 nm
molecular sieve. Luminarin 2, N-[6-[(2,5-dioxo-l-pyrrolidinyl)oxy]-6-oxohexyl]-2,3,6,7-tetrahy-
dro-ll-oxo-lH,5H,HH-[l]benzopyrano[6,7,8-ij]quinolizine-9-acetamide, may be obtained from
Eurobio, Les Ulis, France. Synthesis is as follows. Reflux (with protection from moisture and
with stirring) 2.12 g 8-hydroxyjulolidine, 2.22 g diethyl 1,3-acetonedicarboxylate (oxo-3-glutaric
acid ethyl ester, Fluka), 1.71 g anhydrous zinc chloride, and 6 mL EtOH for 24 h, cool, add to
200 mL water, extract with 200 mL ethyl acetate, extract with 100 mL ethyl acetate. Combine
the organic layers and wash them with water, dry over magnesium sulfate, evaporate to dry-
ness, recrystallize from 5 parts ethyl acetate to give ethyl 2,3,6,7-tetrahydro-ll-oxo-
lH,5H,llH-[l]benzopyrano[6,7,8-ij]qiiinolizine-9-acetate. Heat 2 g of this compound with 42
mL 1.2% NaOH in water and 40 mL MeOH at 45 for 1 h, cool, wash with 50 mL chloroform,
wash with 40 mL chloroform. Degas the aqueous phase and acidify it with 16 mL 3 M HCl,
stir for 15 min, adjust pH to 6.5 with 13 mL 2.5 M NaOH, filter. Wash the precipitate with
water and dry it to obtain 2,3,6,7-tetrahydro-ll-oxo-lH,5H,llH-[l]benzopyrano[6,7,8-
ij]quinolizine-9-acetic acid. React 30 g potassium carbonate with 30 g methyl 6-aminohexanoate
hydrochloride (Fluka) for 30 h, filter. Stir 11.26 g of 2,3,6,7-tetrahydro-ll-oxo-lH,5H,llH-
[l]benzopyrano[6,7,8-ij]quinolizine-9-acetic acid, 10.62 g disuccinimidyl oxalate (dihydroxysuc-
cinimide carbonate), 3.81 g anhydrous triethylamine, and 560 mL dry MeCN protected from
moisture at room temperature for 1 h, stir at 35-40 for 1 h, add the methyl 6-aminohexanoate
filtrate, stir for 8 h, add 20 g ethanolamine, stir for 30 min, filter, wash with water, remove
the solvent, chromatograph on a silica gel column with dichloromethane, dichloromethane :THF
85:15, dichloromethane:THF 75:25, recrystallize from ethyl acetate to give methyl N-(2,3,6,7-
tetrahydro-ll-oxo-lH,5H,HH-[l]benzopyrano[6,7,8-ij]quinolizine-9-yl)acetyl-6-aminohexano-
ate (yield 36%). Heat 1.25 g of this compound with 42 mL 1.2% NaOH in water and 40 mL
MeOH at 45 for 1 h, cool, wash with 50 mL chloroform, wash with 40 mL chloroform. Degas
the aqueous phase and acidify it with 16 mL 3 M HCl, stir for 15 min, adjust pH to 6.5 with
13 mL 2.5 M NaOH, filter. Wash the precipitate with water and dry it to obtain N-(2,3,6,7-
tetrahydro-ll-oxo-lH,5H,HH-[l]benzopyrano[6,7,8-ij]quinolizine-9-yl)acetyl-6-aminohexanoic
acid. React 750 mg of this acid with 912 mg triethylamine and 512 mg disuccinimidyl oxalate
(dihydroxysuccinimide carbonate) in 27 mL MeCN for 6 h, filter, evaporate, chromatograph on
a silica gel column with dichloromethane, dichloromethane :THF 85:15, and dichloromethane:
THF 75:25 to obtain Luminarin 2 (yield 19%) (World Pat. 89 12,052; Chem. Abstr. 1990, 113,
23889n).)
HPLC VARIABLES
Column temperature: 40 (chemiluminescence only)
Flow rate: 2 (F) or 1.5 (chemiluminescence)
Detector: F ex 390 em 490, Chemiluminescence. 1.1 mM Bis(2,4,6-trichlorophenyl) oxalate in
methyl acetate pumped at 0.3 mL/min and 400 mM hydrogen peroxide in THF pumped at 0.3
mL/min mixed in a 292 |xL capillary tube and this mixture mixed with the column effluent.
The resulting mixture flowed through a 60 |xL PTFE capillary at 40 to a Kratos FS970 detector
fitted with a 470 nm long-pass filter.
CHROMATOGRAM
Limit of detection: 100 fmole (F), 50 fmole (chemiluminescence)
KEYWORDS
derivatization; comparison with o-phthalaldehyde derivatization
REFERENCE
Tbd,M.; Legendre,J.-Y.; Chalom,J.; Kouwatli,H.; Poulou,M.; Farinotti,R.; Mahuzier,G. Primary and secondary
amine derivatization with luminarins 1 and 2: separation by liquid chromatography with peroxyoxalate
chemiluminescence detection, J.Chromatogr., 1992, 594, 386-391.
SAMPLE
Sample preparation: 500 |xL Cell suspension in buffer A containing 0.1% bovine serum albumin
+ 1.5 mL ice cold buffer A, centrifuge at 4 at 250 g for 10 min, remove the supernatant, add
2 mL buffer A to the pellet, boil this mixture for 3 min, cool on ice, centrifuge at 4 at 500 g
for 10 min, remove the supernatant. Dry a 50-200 JJLL aliquot of each supernatant in a vacuum
centrifuge and reconstitute the residue with 100 |xL buffer B, add 20 jxL reagent, inject an
aliquot. (Buffer A contained 150 mM NaCl, 4 mM KCl, 1 mM calcium chloride, 1.2 mM mag-
nesium sulfate, 2.46 mM Na2HPO4, 0.615 mM ICH2PO4, 5.6 mM glucose, and 10 mM HEPES,
pH 7.4. Buffer B was THF.100 mM pH 9.5 sodium tetraborate buffer 30:70. The reagent was
prepared by mixing equal volumes of 3.8 mM o-phthaldialdehyde in MeOH (freshly prepared)
and 2.5 mL/L mercaptoethanol in MeOH.)
HPLC VARIABLES
Column: 100 X 3.2 3 |xm Phase-II ODS (Bioanalytical Systems)
Mobile phase: MeCN:MeOH:buffer 14:16:70 containing 1 mM disodium EDTA (Buffer was 100
mM sodium phosphate buffer containing 0.4% triethylamine, pH 6.4.)
Flow rate: 0.6
Detector: E, Bioanalytical Systems Model LC4B, LC17A thin-layer electrochemical cell, glassy
carbon working electrode +0.5 V, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 10
Limit of detection: <0.1 pmole
KEYWORDS
rat mast cells; derivatization
REFERENCE
Jensen,T.B.; Marley,P.D. Development of an assay for histamine using automated high-performance liquid
chromatography with electrochemical detection, J.Chromatogr.B, 1995, 670, 199-207.
SAMPLE
Matrix: cheese, tissue
Sample preparation: Add 1,7-diaminopentane as IS. Homogenize (Polytron) 10 g cheese with
two 20 mL portions of 100 mM HCl. Homogenize (Polytron) 10 g tissue with three 15 mL
portions of 5% trichloroacetic acid. Saturate the extracts with NaCl, adjust the pH to 11.5.
Remove a 5 mL aliquot and add it to 5 mL butanol (cheese) or butanolichloroform 50:50 (tissue)
vortex for 5 min, repeat extraction twice more. Combine the organic extracts and remove a 1
mL aliquot, add 2 drops 1 M HCl, evaporate to dryness under reduced pressure, reconstitute
the residue in 1 mL 100 mM HCl, add 0.5 |JLL saturated sodium bicarbonate, add 1 mL 5 mg/
mL dansyl chloride, heat at 40 for 1 h, evaporate to dryness under reduced pressure, recon-
stitute with MeCN, inject an aliquot.
HPLC VARIABLES
Column: 150 X 1.6 3 jxm Spherisorb 3S TG
Mobile phase: Gradient. MeCN:water 65:35 for 1 min, to 80:20 over 4 min, to 90:10 over 1 min.
Flow rate: 0.8
Detector: UV 254
CHROMATOGRAM
Retention time: 3.7
Internal standard: 1,7-diaminopentane (4.2)
OTHER SUBSTANCES
Extracted: cadaverine, 2-phenylethylamine, putrescine, spermidine, spermine, tryptamine,
tyramine
KEYWORDS
derivatization; fish; salmon; tuna; salami
REFERENCE
Moret,S.; Conte,L.S. High-performance liquid chromatographic evaluation of biogenic amines in foods. An anal-
ysis of different methods of sample preparation in relation to food characteristics, J.Chromatogr.A, 1996,
729, 363-369.
SAMPLE
Matrix: fish, food, wine
Sample preparation: Filter (0.45 (xm) wine, fruit juice or vegetable juice, dilute 1:5 or 1:20. 50
|xL Diluted wine, diluted juice, fish extract, or cheese extract (neutralize strongly acidic samples
if necessary) + 200 |xL 200 mM boric acid adjusted to pH 8.5 with 30% KOH + 200 |xL 3 mM
9-fluorenylmethyl chloroformate in acetone, mix for 3 min at room temperature, add 50 |xL
reagent, mix for 3 min. Remove an 80 |xL aliquot and add it to 320 JJLL initial mobile phase,
inject a 20 |xL aliquot. (Reagent was 3 mL heptylamine in 15 mL MeCN, adjusted to pH 7-8
with 175 mL 100 mM HCl. Extract fish as follows. Homogenize 20 g fish and 10 mL 100 mM
HCl, add 40 mL 100 mM HCl, centrifuge, decant, extract residue with two 40 mL and one 20
mL portions of 100 mM HCl, filter, make up the extracts to 100 (?) mL with 100 mM HCl.
Extract cheese as follows. Homogenize 25 g cheese and 18.75 mL 100 mM HCl, suspend with
40 mL 100 mM HCl, centrifuge, extract the residue twice with 20 mL portions of 100 mM HCl.
Combine and filter the extracts, make up to 100 mL with 100 mM HCl.)
HPLC VARIABLES
Column: 250-4 Supersphere 60 RP-8 (Merck)
Mobile phase: Gradient. A was MeCN: 100 mM pH 4.4 sodium acetate buffer 50:50. B was MeCN.
A:B 100:0 for 7 min, to 90:10 over 5 min, to 70:30 over 15 min, maintain at 70:30 for 6 min,
to 10:90 over 7 min, to 0:100 over 3 min, maintain at 0:100 for 9 min, return to 100:0 over 1
min, re-equilibrate for 7 min.
Flow rate: 1.2 (0.05 when not in use)
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: cadaverine, phenylethylamine, putrescine, spermidine, spermine, tyramine
KEYWORDS
wine; fruit juice; vegetable juice; fish; cheese; derivatization
REFERENCE
Kirschbaum,J.; Luckas,B; Beinert,W.-D. Pre-column derivatization of biogenic amines and amino acids with
9-fluorenylmethyl chloroformate and heptylamine, J.ChromatogrA, 1994, 661, 193-199.
SAMPLE
Matrix: food
Sample preparation: Add 10 mL light petroleum to 5 g natural canned tuna or tuna canned in
oil, extract, centrifuge at about 13 g for 15 min, discard the organic layer, extract the remaining
solid with two or more 10 mL portions of light petroleum. Homogenize the defatted sample
with 10 mL 600 mM perchloric acid, filter, add 10 mL 100 mM NaOH, extract with five 25 mL
portions of butanol. Combine the organic layers and extract with 20 mL light petroleum and
five 10 mL portions of 100 mM HCl, dilute sample to 50 mL with water. Add 1 mL 1 M NaOH
to 2 mL aliquot of the diluted sample, let stand for 5 min, add 1 mL derivatizing solution, let
stand 5-10 min until the solution reaches a brown color, add 500 mg NaCl, extract with two 3
mL portions of ethyl acetate, centrifuge, evaporate the organic layer under nitrogen to 100 |xL,
make up to 2 mL with mobile phase, inject a 20 |xL aliquot within 30-40 min. (Prepare the
derivatizing solution as follows: Mix 1 g sodium tetraborate heptahydrate, 50 mg o-phthalal-
dehyde, 50 jxL p-mercaptoethanol, and 1 mL MeOH, dilute to 50 mL with 1 M NaOH (pH 10).)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherisorb ODS2 RP-18
H3PO4.)
Flow rate: 0.8
CHROMATOGRAM
Retention time: 7.5
Limit of detection: 5 |xg/g
OTHER SUBSTANCES
Extracted: histidine
KEY WpRDS
derivatization; tuna; fish
REFERENCE
Frattini,V.; Lionetti,C. Histamine and histidine determination in tuna fish samples using high-performance
liquid chromatography. Derivatization with o-phthalaldehyde and fluorescence detection or UV detection of
"free" species, J.Chromatogr.A, 1998, 809, 241-4J245.
SAMPLE
Matrix: food
Sample preparation: Cheese, chocolate. Homogenize (stomacher) 5 g ground cheese or chocolate
with 45 mL 70 mM trisodium citrate at 45 for 5 min. Remove a 3 mL aliquot and add it to 3
mL 600 mM trichloroacetic acid, mix, centrifuge at 4 at 10000 g for 10 min, suspend the pellet
in 3 mL 300 mM trichloroacetic acid, centrifuge. Combine the supernatants, filter (0.45 jxm),
make up the filtrate to 10 mL with water, inject an aliquot. Wine. 3 mL Wine + 3 mL 600 mM
trichloroacetic acid, centrifuge, filter the supernatant, inject an aliquot of the filtrate. Fish,
sauerkraut. Blend 200 g fish or sauerkraut with 200 mL water for 3 min. Remove a 3 mL
aliquot and add it to 3 mL 600 mM trichloroacetic acid, centrifuge, filter the supernatant, inject
HPLC VARIABLES
Guard column: 30 X 3 Corasil C18
Column: 100 X 8 10 (xm Nucleosil C18 radial-compression
Mobile phase: DMSO:buffer 47:53 (Prepare mobile phase by dissolving 16 g ninhydrin and 1.2
g hydrindantin in 322 mL DMSO with sonication for 10 min, add 350 mL 1.8 M pH 5.00 sodium
acetate buffer, add 2 g sodium dodecyl sulfate dissolved in a mixture of 618 mL DMSO and
710 mL water, flush constantly with nitrogen. Flush column with DMSO:water 50:50 at the
end of each day.)
Flow rate: 1
Detector: UV 546 following post-column reaction. The column effluent flowed through a 10 m X
0.25 mm PTFE tube coiled in a figure 8 at 145 to the detector.
CHROMATOGRAM
Retention time: 16
Limit of detection: 800 ng/g (sauerkraut), 300 ng/g (wine)
OTHER SUBSTANCES
Extracted: cadaverine, phenylethylamine, putrescine, tryptamine, tyramine
KEYWORDS
post-column reaction; cheese; chocolate; wine; fish; sauerkraut; tuna
REFERENCE
Joosten,H.M.L.J.; 01ieman,C. Determination of biogenic amines in cheese and some other food products by
high-performance liquid chromatography in combination with thermo-sensitized reaction detection,
J.Chromatogr., 1986, 356, 311-319.
SAMPLE
Matrix: food
Sample preparation: Dilute vinegar, wine, or juice 10-fold with water, centrifuge at 2000 g for
15 min. Remove a 20 |JLL aliquot and mix it with 150 (xL buffer and 400 jxL 5 mM 2-naphthox-
ycarbonyl chloride in MeCN, let stand for 3 min, add 50 (JLL 20 mM glycine in water, let stand
for 3 min, add 500 u,L MeCN:500 mM pH 4.4 sodium acetate buffer 75:25, mix, inject a 20 uX,
aliquot. (Prepare buffer by adjusting the pH of 33.43 g boric acid in 950 mL water to 9.0 with
20% KOH, make up to 1 L with water. Synthesis of 2-naphthoxycarbonyl chloride is as follows.
Dissolve 30 mmoles 2-naphthol and 30 moles quinoline in 18 g toluene and 5 g dichloro-
methane, cool to 0, add 47 mL 1.93 M phosgene in toluene, warm on a steam bath for 10 min,
filter, evaporate to remove the solvent, distil the residue (bp 150-15279 mm Hg, take up the
distillate in ether, crystallize by adding ligroin to obtain 2-naphthoxycarbonyl chloride (mp 66)
(J. Am. Chem. Soc.1951, 73, 2080).)
HPLC VARIABLES
Column: 250 X 4.6 4 |xm Superspher 60 RP-18e
Mobile phase: Gradient. A was MeCN: 100 mM pH 4.4 sodium acetate 40:60. B was MeCN. A:B
77:23 for 7 min, to 65:35 over 31 min, to 30:70 over 3 min, maintain at 30:70 for 5 min, to 0:
100 over 1 min, maintain at 0:100 for 6 min, return to initial conditions over 1 min, re-equil-
ibrate for 6 min.
Flow rate: 1.1 for 46 min, to 1.5 over 1 min, maintain at 1.5 for 13 min
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: cadaverine, histamine, 2-phenylethylamine, putrescine, spermidine, spermine,
tyramine
KEYWORDS
derivatization; vinegar; wine; juice
REFERENCE
Kirschbaum,J.; Busch,L; Bruckner,H. Determination of biogenic amines in food by automated pre-column de-
rivatization with 2-naphthyloxycarbonyl chloride (NOC-Cl), Chromatographia, 1997, 45, 263-268.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1 mg/mL solution in 10 mM HCl, inject a 1-2 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4 7 |xm 300 A Nucleosil C8
Mobile phase: MeOH:buffer 15:85 (Buffer was 50 mM pH 3.1 NaH2PO4 containing 0.5 mM di-
sodium EDTA and 5 mM sodium 1-pentanesulfonate or sodium 1-octanesulfonate.)
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Simultaneous: metabolites, methylhistamine, histidine, methylimidazole acetic acid
REFERENCE
Hermann,K; Frank,G.; Ring,J. High-performance liquid chromatography for the separation of histamine, its
precursor, and metabolites: Application to biological samples, J.Liq.Chromatogr., 1995, 18, 189-204.
SAMPLE
Matrix: tears
Sample preparation: 10 jxL Tears + 40 fxL 200 mM pH 9.1 sodium borate buffer + 50 JJLL 200
|xg/mL fluorescamine in MeCN, mix vigorously, inject a 20 (xL aliquot.
HPLC VARIABLES
Guard column: 20-40 |xm LiChroprep RP-8
Column: 10 |xm Nucleosil C8 or 10 ^m RP-8 (Merck)
Mobile phase: MeCN:4 mM pH 3.5 KH2PO4 65:35
Flow rate: 0.5
CHROMATOGRAM
Retention time: 4
KEYWORDS
derivatization
REFERENCE
Bettero,A-J Angi,M.R.; Moro,R; Benassi,C.A. Histamine assay in tears byfluorescaminederivatization and high-
SAMPLE
Matrix: tears
Sample preparation: 10 |xL Tears + 40 JJLL 100 mM pH 9.1 borate buffer, mix, add 50 |xL 200
(xg/mL fluorescamine in MeCN with vigorous stirring, inject a 20 |xL aliquot on to column A,
divert the fraction containing the derivatized histamine on to column B then remove column
A from the circuit (details are sketchy).
HPLC VARIABLES
Column: A 20-40 jxm RP-8 (Merck); B 10 jjim RP-8 (Merck)
Flow rate: 0.5
CHROMATOGRAM
Retention time: 4
KEY WpRDS
derivatization; column-switching; heart cut
REFERENCE
Bettero,A.; Galiano,R; Benassi,C.A.; Angi,M.R. A rapid HPLC technique for determining levels of histamine in
tears from normal and inflamed human eyes, Food Chem.Toxicol., 1985, 23, 303-304.
SAMPLE
Matrix: tissue
Sample preparation: Prepare SPE (A) column by washing 100-200 mesh Dowex 50 W with
excess 2 M HCl, with water, with 2 M NaOH, and with water. Equilibrate the resin with 200
mM pH 6.5 sodium phosphate buffer and pack in a 16 X 5 glass column. Prepare SPE (B)
column by washing cellulose-phosphate fibrous cation-exchanger (Sigma) in a 13 X 17 glass
column with excess 100 mM NaOH, with water, with 100 mM HCl, and with water until the
pH reaches 5-6. Homogenize (glass homogenizer) rat brain hypothalamus with 500 (xL ice-cold
3% perchloric acid, rinse homogenizer 3 times with 500 |xL portions of 3% perchloric acid.
Combine the homogenate and rinses, add IS, centrifuge at 4 at 10000 g for 30 min, add the
supernatant to SPE column (A), wash with 5 mL water, wash with 4 mL 2 M HCl, elute with
2.5 mL 3.5 M HCl. Evaporate the eluate to dryness, reconstitute the residue in 800 |JLL water,
add 150 /JLL buffer, add 100 JJLL 20 mM sulfosuccinimidyl-3-(4-hydroxyphenyl)propionate (sulfo
B-H, Pierce) in water, vortex for 30 s, adjust pH to 5.5-6.0 with 100 mM HCl, add to SPE
column (B), wash with 5 mL water, wash with 6 mL 1 mM HCl, elute with 2.5 mL 100 mM
HCl. Discard the first 500 |xL eluate, evaporate the next 200 |xL to dryness, reconstitute with
1 volume water, add 9 volumes water to a total volume of 0.11-1 mL, inject a 100 |xL aliquot.
(Prepare buffer by mixing 100 mM sodium carbonate and 100 mM sodium bicarbonate in a 10:
1 ratio.)
HPLC VARIABLES
Guard column: jmBondapak C18/Corasil
Mobile phase: MeCN: 140 mM sodium acetate 73:17 containing 3.89 M (sic) 1-octanesulfonic acid
and 56 mg/L (?) EDTA, adjusted to pH 3.48 with glacial acetic acid
Flow rate: 1
Detector: E, ESA Coulochem 5100A, Model 5011 analytical cell, cell 1 0.47 V, cell 2 (monitored)
0.56 V, oxidative screen mode
CHROMATOGRAM
Retention time: 8.4
Internal standard: Na-methylhistamine (11.6)
OTHER SUBSTANCES
Extracted: N^-methylhistamine
Simultaneous: histidine, spermidine
KEYWORDS
derivatization; rat; brain; SPE
REFERENCE
Mine,K; Jacobson,KA.; Kirk,K.L.; Kitajima,Y.; Linnoila,M. Simultaneous determination of histamine and N
tau-methylhistamine with high-performance liquid chromatography using electrochemical detection,
AnaLBiochem., 1986, 152, 127-135.
SAMPLE
Matrix: tissue
Sample preparation: Sonicate 10 g tuna with 50 mL 1200 ppm triethylamine in MeOH for 10
min, filter, filter (0.45 |xm) again, add 25 |xL filtrate to 10 mg 3,5-dinitrobenzoyl derivatized
silica, heat at 60 for 10 min, elute with 1 mL MeCN, inject a 20 JULL aliquot of the eluate.
(Prepare 3,5-dinitrobenzoyl derivatized silica as follows. Heat 4.7 g 4-hydroxy-3-nitrobenzoic
acid, 7.5 mL thionyl chloride, 750 |xL pyridine, and 60 mL benzene (Caution! Benzene is a
carcinogen!) at 55 for 4 h, cool to room temperature, filter, evaporate under reduced pressure
to obtain 4-hydroxy-3-nitrobenzoyl chloride. Store 10 jjum LiChrosorb Si-100 silica in a desic-
cator over saturated aqueous LiCl solution for 2 weeks. 5 g Silica + 8.4 g 62% bis(2-hydroxy-
ethyl)-3-aminopropyltriethoxysilane (Petrarch Systems, Bristol PA) in EtOH, add 40 mL EtOH:
pyridine 99.5:0.5 (?), reflux with stirring under nitrogen for 6 h, cool, filter, wash silica with
three 25 mL portions of MeOH and four 25 mL portions of dichloromethane, dry under nitrogen
at 40 overnight. Heat 5 g 4-hydroxy-3-nitrobenzoyl chloride, 300 |JLL pyridine, 5 g silica, and
60 mL benzene (previously dried over anhydrous sodium sulfate) at 70-75 for 2 h, filter, wash
silica with three 75 mL portions of DMF and three 75 mL portions of dichloromethane, dry
under vacuum at 40 for 12 h. Stir 400 mg silica, 600 mg 3,5-dinitrobenzoyl chloride, 25 mL
benzene, and 300 |xL pyridine at room temperature for 24 h, filter, wash with three 25 mL
portions of dichloromethane to give 3,5-dinitrobenzoyl derivatized silica.)
HPLC VARIABLES
Column: 250 X 4 5 |xm LiChrosphere C18
Mobile phase: MeCN:water 50:50 containing 0.05% ammonium hydroxide
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 2
KEYWORDS
derivatization; tuna; fish
REFERENCE
Zhou.F.-X.,; Wahlberg,J.; Krull,I.S. Silica based 3,5-dinitrobenzoyl (DNB) reagent for off-line derivatization of
amine nucleophiles in HPLC, J.Liq.Chromatogr., 1991, 14, 1325-1350.
SAMPLE
Matrix: tissue
Sample preparation: Incubate chick embryo retinas in 4 mL medium at 37 for 4 h, centrifuge
at 500 g for 1 min. For each 20 mg solid add 1 mL 20 |xM 1,7-diaminoheptane in 200 mM
perchloric acid, suspend, sonicate (Soniprep 150) with 10 |xm amplitude in 10 s bursts, centri-
fuge at 20000 g for 15 min, neutralize with KOH, centrifuge. Remove a 1 mL aliquot and add
it to 1 mL 2 M NaOH, add 5 (xL benzoyl chloride, vortex briefly, let stand for 20 min, add 2
mL saturated NaCl, extract with 2 mL diethyl ether, centrifuge. Remove the upper organic
phase and wash it with 2 mL 100 |xM NaOH, dry the organic layer over a few mg anhydrous
sodium sulfate, centrifuge. Remove the organic layer and evaporate it to dryness under a
stream of nitrogen, reconstitute the residue in 2 mL mobile phase, inject a 20 jxL aliquot.
(Medium was pH 7.4 serum- and glutamine-free Eagle's minimum essential medium containing
25 mM 4-(2-hydroxyethyl)-l-piperazineethane sulfonic acid (HEPES), 100 U/mL penicillin, and
100 jxg/mL streptomycin.)
HPLCVARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: 1,7-diaminoheptane (5.4)
Limit of quantitation: 1.25 \JM
OTHER SUBSTANCES
Extracted: N-acetylcadaverine, N-acetylputrescine, Nâcetylspermidine, Nâcetylspermine, ca-
daverine, putrescine, spermidine, spermine
KEYWORDS
derivatization; retina; chicken
REFERENCE
Taibi,G.; Schiavo,M.R. Simple high-performance liquid chromatographic assay for polyamines and their mon-
oacetyl derivatives, J.Chromatogr., 1993, 614, 153-158.
SAMPLE
Matrix: tissue
Sample preparation: Make a slurry of 40 |xm Bakerbond carboxylic acid material in MeOH and
prepare SPE columns by adding an aliquot containing 150 mg material to a Pasteur pipette
plugged with glass fiber prefilter material (Sartorius). Wash column with 2 mL water, 2 mL 1
M HCl, 2 mL water, and 4 mL 200 mM pH 6.4 sodium phosphate buffer. Homogenize (Ultra-
Turrax) rat heart and 500 ng 1-methylhistamine in ice-cold 50 mM pH 8.5 Tris-HCl buffer,
sonicate (MSE sonifier, 12 W, 12 |xm peak-peak) for 2 min with repeated intervals of 5 s, add
perchloric acid to a final concentration of 300 mM, heat at 100 for 5 min, neutralize with KOH,
cool to 0, centrifuge at 4 at 2000 g for 5 min, adjust pH of supernatant to 6.4 with phosphate
buffer, add 1-methylhistamine, add to the SPE column, wash with 4 mL 50 mM pH 6.4 diso-
dium EDTA, wash with 4 mL water, elute with 1 mL 1 M HCl. Evaporate the eluate to dryness
under a stream of nitrogen at 40 over about 15 min, reconstitute with 100 |xL water, add 400
IxL 50 mM pH 9.1 sodium borate, with continuous vigorous stirring add 500 |xL 200 |xg/mL
fluorescamine in MeCN (freshly prepared), stir for 1 min, evaporate to dryness under a stream
of nitrogen at 40, reconstitute with 1 mL mobile phase, inject a 20 (xL aliquot.
HPLCVARIABLES
Column: 200 X 3 5 |xm Inertsil ODS-2 (see J. Chromatogr. B 1994, 657, 261)
Mobile phase: MeCN:MeOH:water:phosphoric acid 15:10:75:0.2 adjusted to pH 6.87 with am-
monium hydroxide (see J. Chromatogr. B 1994, 657, 261)
Flow rate: 0.4
CHROMATOGRAM
Retention time: 15
Internal standard: 1-methylhistamine (25)
OTHER SUBSTANCES
Extracted: 3-methylhistamine
KEYWORDS
SPE; derivatization; rat; heart
REFERENCE
van Haaster,C.M.C.J.; Engels,W.; Lemmens,P.J.M.R.; Hornstra,G.; van der Vusse,G.J. Rapid and highly sen-
sitive high-performance liquid chromatographic method for the determination of histamine and 3-methyl-
histamine in biological samples using fluorescamine as the derivatizing agent, J.Chromatogr., 1993, 617,
233-240.
SAMPLE
Matrix: tissue
Sample preparation: Blend 50 g tissue and 75 mL 5% trichloroacetic acid in a Waring Blendor
at high speed for 2 min, centrifuge for 2 min, extract the solid twice more with 75 mL portions
of 5% trichloroacetic acid, filter the supernatants through a glass wool plug, wash the funnel
with 5% trichloroacetic acid, make up the filtrate to 250 mL with 5% trichloroacetic acid.
Remove a 10 mL aliquot and add it to 4 g NaCl, 1 mL 50% NaOH, and 5 mL chloroform:n-
butanol 50:50, shake vigorously for 2 min, centrifuge for 5 min, remove the upper organic layer,
repeat the extraction twice more with 5 mL portions of chloroform :n-butanol 50:50. Combine
the organic layers and add them to 15 mL n-heptane, extract three times with 1 mL portions
of 200 mM HCl, inject a 20 |xL aliquot (J.Assoc.Off.Anal.Chem. 1978, 61, 139).
HPLCVARIABLES
Column: PRP-X200 Cationic (Hamilton)
Flow rate: 0.5
Detector: UV 210
CHROMATOGRAM
Retention time: 2
KEYWORDS
fish
REFERENCE
Kalligas,G.; KaniouJ.; Zachariadis,G.; Tsoukali,H.; Epivatianos,R Thin layer and high pressure liquid chro-
matographic determination of histamine in fish tissues, J.Liq.Chromatogr., 1994, 17, 2457-2468.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Polytron) 200 mg tissue with 5 volumes of 1 M perchloric
acid, centrifuge at 10000 g for 20 min. Neutralize the supernatant with 10 M KOH, centrifuge
at 10000 g for 20 min, remove a 20 |xL aliquot of the supernatant and add it to 10 jxL reagent,
mix well, add 30 fxL 10% sodium carbonate in EtOH:water 5:95, mix well, inject a 10 \LL aliquot.
(Prepare reagent prior to use by mixing equal volumes of 20 mM sulfanilic acid in 1 M HCl
and 200 mM sodium nitrite solution.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm TSK-ODS (Toso)
Mobile phase: Gradient. A was EtOH:150 mM pH 6.0 sodium acetate buffer 5:95. B was MeCN:
water 60:40. A:B from 100:0 to 55:45 over 30 min.
Flow rate: 1
Detector: UV 420
CHROMATOGRAM
Limit of detection: <7 nmole/g
OTHER SUBSTANCES
Extracted: carnosine, histidine, tyrosine
Noninterfering: anserine, 1-methylhistidine
KEY WORDS
derivatization; fish; muscle; mackerel
REFERENCE
Sato,M.; Nakano,T.; Takeuchi,M.; Kumagai,T.; Kanno,N.; Nagahisa,E.; Sato,Y. Specific determination of his-
tamine in fish by high-performance liquid chromatography after diazo coupling, Biosci.Biotechnol.Biochem.,
1995, 59, 1208-1210.
SAMPLE
Matrix: tissue
Sample preparation: 10 g Homogenized fish + 15 mL 600 mM perchloric acid, stir magnetically
for 10 min, centrifuge at 3000 rpm for 10 min, remove the supernatant, add 10 mL 600 mM
perchloric acid to the residue, stir magnetically for 10 min, centrifuge at 3000 rpm for 10 min.
Combine the supernatants, make up to 25 mL with 600 mM perchloric acid, filter (0.45 |xm),
inject a 20 JAL aliquot of the filtrate.
HPLC VARIABLES
Mobile phase: Gradient. A was 100 mM sodium acetate containing 10 mM sodium octanesulfon-
ate, adjusted to pH 5.20 with acetic acid. B was MeCNrbuffer 66:34 (Buffer was 200 mM sodium
acetate containing 10 mM sodium octanesulfonate, adjusted to pH 4.50 with acetic acid.) A:B
from 80:20 to 20:80 over 50 min, maintain at 20:80 for 2 min, return to initial conditions over
2 min, re-equilibrate for 10 min.
Flow rate: 1
reagent pumped at 0.5 mL/min, the mixture flowed throug;h a 200 cm X 0.25 mm i.d. coil of
stainless steel tubing to the detector. (Prepare reagent by dissolving 15.5 g boric acid and 13.1
g KOH in 500 mL water, adjust pH to 10.5-11 with 30% KOH (if necessary), add 1.5 mL 30%
Brij-35, add 1.5 mL mercaptoethanol, add 2.5 mL 40 jig/mL o-phthalaldehyde in MeOH, mix.
Protect from light, prepare fresh daily.)
CHROMATOGRAM
Retention time: 35
OTHER SUBSTANCES
Extracted: agmatine, cadaverine, creatinine, (S-phenylethylamine, putrescine, serotonin, sper-
midine, spermine, tryptamine, tyramine
KEYWORDS
post-column reaction; fish
REFERENCE
Veciana-Nogues,M.T.; Hernandez-Jover,T.; Marine-Font,A.; Vidal-Carou,M.D.C. Liquid ehromatographic
method for determination of biogenic amines in fish and fish products, J.AOAC Int., 1995, 78, 1045-1050.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Ultraturrax) 5 g fish, 10 mL 200 mM perchloric acid, and
100 |JLL 800 |xg/mL 1,3-diaminopropane dihydrochloride in water at -20 and 20000 rpm and
centrifuge at 2 at 2500 g for 20 min. Remove a 100 |xL aliquot of the supernatant and add it
to 200 JJLL saturated sodium bicarbonate solution, add 400 JJLL 7.5 mg/mL dansyl chloride in
acetone, agitate, heat at 60 in the dark for ?, add 100 |xL 100 mg/mL L-proline in water,
agitate, let stand in the dark at room temperature for 30 min, add 500 JJLL toluene, agitate.
Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute
the residue in 300 |xL MeCN, filter, inject an aliquot.
HPLC VARIABLES
Guard column: 30 X 4.6 5 (xm Brownlee C18
Column: 250 X 4.6 5 fxm Kromasil C18
min, to 95:5 over 5 min, maintain at 95:5 for 7 min, re-equilibrate at initial conditions for 10
min.
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: 1,3-diaminopropane (12.5)
OTHER SUBSTANCES
Extracted: cadaverine, putrescine, spermidine, spermine
KEY WpRDS
derivatization; fish
REFERENCE
Malle,R; Vall6,M.; Bouquelet,S. Assay of biogenic amines involved in fish decomposition, J.AOAC Int., 1996,
79, 43-49.
SAMPLE
Matrix: urine, blood
Sample preparation: Prepare a 500 JJLL SPE cartridge of Amberlite CG-50 resin and wash it
with 2 mL water. Add urine or blood to the SPE cartridge, wash with 2 mL water, wash with
two 2 mL portions of 500 mM pH 6.5 sodium acetate, wash with two 2 mL portions of water,
elute with 400 |xL 2 M perchloric acid, elute with two 1 (urine) or 0.2 (blood) mL portions of
mobile phase, inject an aliquot of the eluate.
HPLC VARIABLES
Mobile phase: MeOH: 100 mM KH2PO4 25:75 containing 56 |xg/mL sodium dodecyl sulfate and
3.2 mL/L 10 M NaOH
Flow rate: 1.2
Detector: E, ESA model 5100A, detector 1 +0.85 V, detector 2 +1.12 V, guard cell +1.15 V
CHROMATOGRAM
Retention time: 11
OTHER SUBSTANCES
Extracted: N-methylhistamine
KEYWORDS
guinea pig; whole blood; SPE
REFERENCE
Houdi,A.A.; Crooks,P.A.; Van Loon,G.R.; Schubert,C.A. A simple and sensitive determination of histamine and
Ntau-methylhistamine in biological fluids by high-performance liquid chromatography with electrochemical
detection, J.Pharm.ScL, 1987, 76, 398-401.
SAMPLE
Matrix: wine
Sample preparation: 20 jxL Wine + 50 |xL buffer, mix, add 100 JJLL 8 mg/mL 9-fluorenylmethyl
chloroformate in MeCN, mix, let stand for 3 min, add 50 |xL 500 mM ammonia, mix, let stand
for 3 min, add 300 |xL MeCN:water:acetic acid 80:12:8, inject a 20 (xL aliquot. (Prepare buffer
by adjusting the pH of 200 mM boric acid to 8.5 with 5 M NaOH.)
HPLC VARIABLES
Mobile phase: Gradient. A was MeCN:2-octanol 99:1. B was MeCN:water:phosphoric acid:di-
methylcyclohexylamine 15:83.12:0.88:1, pH 2.7. A:B 15:85 for 18 min, 38:62 over 0.1 min, to
40:60 over 6.9 min, maintain at 40:60 for 5 min, to 42:58 over 37 min, to 85:15 over 36 min,
maintain at 85:15 for 7 min.
Flow rate: 0.3
Injection volume: 2
CHROMATOGRAM
Retention time: 100
OTHER SUBSTANCES
Extracted: agmatine, arginine, cadaverine, histidine, ornithine, phenylalanine, phenylethylam-
ine, putrescine, spermidine, spermine, tyramine, tyrosine
KEYWORDS
derivatization
REFERENCE
Bauza,T.; BlaiseÂ..; Daumas,E; Cabanis,J.C. Determination of biogenic amines and their precursor amino acids
in wines of the Vall6e du Rhdne by high-performance liquid chromatography with precolumn derivatization
and fluorimetric detection, J.ChromatogrA, 1995, 707, 373-379.
SAMPLE
Matrix: wine
Sample preparation: Wash 5 g Amberlite CG-50 with water, add 5 mL 10 M NaOH, let stand
for 30 min, rinse with water 3 times, add 25 mL 5 M HCl to a pH of 2, wash, mix with 5 mL
10 M NaOH, wash with 1 volume pH 7 buffer. Filter (0.45 |xm) wine, add 10 mL to the resin
in a 40 X 10 column, wash with water, elute with 10 mL 1 M HCl. Evaporate the eluate to 1
mL under reduced pressure, add heptylamine, derivatize with reagent, inject an aliquot. (Re-
agent was 45 mg phthalaldehyde in 1 mL MeOH, add 200 |xL 2-mercaptoethanol, make up to
10 mL with buffer, prepare daily. Buffer was 3.81 g sodium tetraborate in 100 mL water, adjust
to pH 10.5 with 10 M NaOH.)
HPLC VARIABLES
Mobile phase: Gradient. A was THF:water 1:99 containing 0.03% triethanolamine. B was MeOH.
A:B from 40:60 to 20:80 over 25 min, re-equilibrate for 3 min. (Every 10 analyses flush with
MeCN:80.8 mM acetic acid 30:70 for 1 h.)
Flow rate: 1
CHROMATOGRAM
Retention time: 4.5
Internal standard: heptylamine (21.5)
OTHER SUBSTANCES
Simultaneous: biogenic amines
KEYWORDS
derivatization; SPE
REFERENCE
Busto,O.; Mestres,M.; Guasch,J.; Borrull,F. Determination of biogenic amines in wine after clean-up by solid-
phase extraction, Chromatographia, 1995, 40, 404-410.
SAMPLE
Matrix: wine
Sample preparation: Mix 10 |xL wine with 6 (xL pH 8.8 borate buffer (Waters), add 0.5 |xL 10
mM 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate in MeCN (Waters), mix, let stand for
5 min, inject a 10 |xL aliquot.
HPLC VARIABLES
Guard column: Spherisorb ODS-2
Column: 250 X 4.6 5 fxm Spherisorb ODS-2
Mobile phase: Gradient. A was THF:50 mM sodium acetate. B was MeOH. A:B 75:25 for 5 min,
to 20:80 over 20 min, to 0:100 (step gradient), maintain at 0:100 for 3 min, return to initial
conditions, re-equilibrate for 2 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 9
Internal standard: heptylamine
OTHER SUBSTANCES
Extracted: ammonia, amylamine, butylamine, cadaverine, ethanolamine, ethylamine, hexylam-
ine, isopropylamine, methylamine, 3-methylbutylamine, phenethylamine, propylamine, pu-
trescine, pyrrolidine, tyramine
KEYWORDS
derivatization
REFERENCE
Busto,O.; Guasch,J.; Borrull,F. Determination of biogenic amines in wine after precolumn derivatization with
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, J.Chromatogr.A, 1996, 737, 205-213.
Histrelin
Merck Index: 4760
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 \im SP-150 C8 (DuPont)
Mobile phase: Gradient. A was 654 mL triethylamine, 444 mL 85% phosphoric acid, and 29.1 g
sodium butanesulfonate in 8 L water, pH 2.4. B was MeCN containing enough ethyl acetate to
make the absorbance at 210 nm the same as that of mobile phase A. A:B from 85:15 to 78:22
over 25 min (Perkin-Elmer concave curve 4), maintain at 78:22 for 10 min
Flow rate: 1.6
CHROMATOGRAM
Retention time: 26
OTHER SUBSTANCES
REFERENCE
Oyler,A.R; Naldi,R.E.; Lloyd,J.R.; Graden,D.A.; Shaw,C.J.; Cotter,M.L. Characterization of the solution deg-
radation products of histrelin, a gonadotropin releasing hormone (LH/RH) agonist, J.Pharm.ScL, 1991,80,
271-275.
Homatropine
CAS Registry No.: 87-00-3, 51-56-9 (HBr)
Merck Index: 4766
SAMPLE
Matrix: plants
Sample preparation: Dissolve alkaloids in 1 mL MeOH, inject aliquot.
HPLC VARIABLES
Column: 150 X 4.1 5 ^m Hamilton PRP-I
Mobile phase: MeCN: 100 mM pH 10.4 ammonium acetate
Flow rate: 1
Detector: MS thermospray, VG Trio-2, ion source 150, vaporizer tip 170, repeller electrode 150
V, m/z 276
CHROMATOGRAM
Internal standard: homatropine
OTHER SUBSTANCES
Simultaneous: hyoscyamine, scopolamine
KEYWORDS
total run time 6 min; homatropine is IS
REFERENCE
Auriola,S.; Martinsen,A.; Oksman-Caldentey,K.M.; Naaranlahti,T. Analysis of tropane alkaloids with thermo-
spray high-performance liquid chromatography-mass spectrometry, J.Chromatogr., 1991, 562, 737-744.
SAMPLE
Matrix: solutions
JULL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 jim ixBondapak C18
Mobile phase: MeOH.acetic acid:triethylamine:water 15:1.5:0.5:83
Flow rate: 1.5
Detector: UV 230
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Simultaneous: scopolamine, methscopolamine, tropic acid, atropine methyl, atropine
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH or water to 0.1%.
HPLC VARIABLES
Column: two 250 mm p-cyclodextrin bonded phase columns in series (Advanced Separation
Technologies)
Mobile phase: MeOH: 1% pH 4.1 aqueous triethylammonium acetate 4:96
Flow rate: 0.5
Injection volume: 1
Detector: UV
CHROMATOGRAM
Retention time: k' 1.98 (d-isomer)
KEYWORDS
chiral; optical isomers are separated
REFERENCE
Armstrong,D.W.; Han,S.M.; Han,Y.I. Separation of optical isomers of scopolamine, cocaine, homatropine, and
atropine, Anal.Biochem., 1987, 167, 261-264.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Hexane:isopropanol:diethylamine 80:20:0.1
Flow rate: 0.5
Detector: UV
CHROMATOGRAM
KEYWORDS
chiral; a 3.13
REFERENCE
Okamoto,Y.; Aburatani,R.; Hatano,K; Hatada,K. Optical resolution of racemic drugs by chiral HPLC on cel-
lulose and amylose tris(phenylcarbamate) derivatives, J.Liq.Chromatogr., 1988, 11, 2147-2163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 fjum LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
haloperidol, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloro-
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Hyaluronic acid
Merck Index: 4793
SAMPLE
Matrix: bulk
Sample preparation: Mix 100 |xL of a 1-500 jxg/mL solution of hyaluronic acid in water with
40 JULL 100 mM pH 5.2 citrate/phosphate buffer, add 10 500 U/mL hyaluronate 4-glycanohydro-
lase (sheep testis, Type V, Sigma) in water, heat at 37 for 5 h, heat in a boiling water bath
for 3 min, evaporate to dryness. Reconstitute with 20 |xL 300 mM NaOH, add 20 |xL 500 mM
l-(4-methoxy)phenyl-3-methyl-5-pyrazolone in MeOH, heat at 70 for 20 min, add 20 jxL 300
mM HCl, add 200 |xL water, add 200 (xL ethyl acetate saturated with water, shake vigorously,
discard the organic phase, repeat the ethyl acetate wash twice more. Evaporate the aqueous
phase to dryness and reconstitute the residue in 200 jxL MeCN:water 15:85, inject a 20 jxL
aliquot. (Synthesis of l-(4-methoxy)phenyl-3-methyl-5-pyrazolone is as follows. Reflux 5.6 g 4-
methoxyphenylhydrazine hydrochloride, 5.45 g sodium acetate trihydrate, and 4.16 g ethyl
acetoacetate in 40 mL EtOH for 2 h, cool, evaporate to dryness, dissolve the residue in 10 mL
EtOH, filter, evaporate the filtrate to dryness, dissolve the residue in a small volume of ben-
zenerethyl acetate 80:20 (Caution! Benzene is a carcinogen!), chromatograph on a column of
150 g silica gel 60 (Merck) equilibrated with benzene:ethyl acetate 80:20, collect 5 mL fractions
(monitor by TLC using Merck silica gel 60 F254 eluted with benzene:ethyl acetate 80:20, UV
detection, Rf 0.41). Combine the appropriate fractions and evaporate them to dryness, recrys-
tallize the residue from MeOH to give l-(4-methoxy)phenyl-3-methyl-5-pyrazolone (Anal.
Biochem. 1991, 199, 256).)
HPLC VARIABLES
Column: 150 X 6 Cosmosil 5C18-AR
Flow rate: 0.8
Detector: UV 249
CHROMATOGRAM
Retention time: 9 (hexasaccharide), 10 (tetrasaccharide), 16 (disaccharide)
KEY WpRDS
derivatization
REFERENCE
Kakehi,K; Ueda,M.; Suzuki,S.; Honda,S. Determination of hyaluronic acid by high-performance liquid chro-
matography of the oligosaccharides derived therefrom as l-(4-methoxy)phenyl-3-methyl-5-pyrazolone deriv-
atives, J.Chromatogr., 1993, 630, 141-146.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1.5 mg/mL solution of sodium hyaluronate in 0.9% saline, filter
0.22 |xm, inject a 10 jxL aliquot.
HPLC VARIABLES
Guard column: TSK G6000 PW (Toyo Soda)
Column: 300 X 7.5 TSK G6000 PW (Toyo Soda)
Mobile phase: 3 mM pH 7.0 NaH2PO4 containing 150 mM NaCl and 0.02% sodium azide
Flow rate: 1
Detector: RI
CHROMATOGRAM
REFERENCE
Beaty,N.B.; Tbw,W.R; Mello,R.J. Relative molecular weight and concentration determination of sodium hyalu-
ronate solutions by gel-exclusion high-performance liquid chromatography, Anal.Biochem., 1985,147, 387
395.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 X 8 Shodex OHpak KB-803 (Showa Denko)
Mobile phase: 200 mM NaCl
Flow rate: 0.5
Detector: UV 210
CHROMATOGRAM
Retention time: 12
KEYWORDS
GPC
REFERENCE
Nakamura,T.; Majima,M.; Kubo,K.; Takagaki,K.; Tamura,S.; Endo,M. Hyaluronidase assay using fluorogenic
hyaluronate as a substrate, Anal.Biochem., 1990, 191, 21-24.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10-20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 TSKgel NH2-60
Mobile phase: MeCN:buffer 54:46 (Buffer was 40 mM Tris-HCl borate buffer adjusted to pH 7.5
with HCl containing 5 mM sodium sulfate.)
Flow rate: 0.5
Detector: F ex 346 em 410 following post-column reaction. The effluent from the column was
mixed with 300 mM NaOH (pumped at 0.25 mL/min) and 1% 2-cyanoacetamide (pumped at
0.25 mL/min). The mixture passed through a 10 m X 0.5 mm i.d. PTFE coil at 105 and a 2 m
X 0.25 mm i.d. PTFE coil at 25 to the detector.
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: chondroitin sulfate, dermatan sulfate
KEYWORDS
REFERENCE
Akiyama,H.; Saito,M.; Qiu,G.; Toida,T.; Imanari,T. Analytical studies on hyaluronic acid synthesis by normal
human epidermal keratinocytes cultured in a serum-free medium, Biol.Pharm.Bull., 1994, 17, 361-364.
SAMPLE
Matrix: synovial fluid
Sample preparation: Centrifuge synovial fluid, dilute 40 jxL to 500 |xL with initial mobile phase,
inject an aliquot.
HPLC VARIABLES
Guard column: LC 18 (Supelco)
Column: 50 X 4.6 Supelcosil LC 318
Mobile phase: Gradient. A was 20 mM NaH2PO4 containing 150 mM NaCl, pH 6.5. B was MeCN.
A:B from 100:0 to 40:60 over 50 min.
Flow rate: 1
Detector: UV 229
CHROMATOGRAM
REFERENCE
Brun,R; De Galateo,A.; Camporese,A; Cortivo,R.; Abatangelo,G. Analysis of hyaluronic acid in synovial fluid
by reversed-phase liquid chromatography, J.Chromatogr., 1990, 526, 530-534.
Hydralazine
Merck Index: 4800
SAMPLE
Matrix: blood
Sample preparation: Add 8-12 mL blood to 125 IU lithium heparin in ice-cold tubes, centrifuge
at 8000 g for 30 s, add 1 mL plasma to 75 (xL 50% sodium nitrite in a tube kept on ice, add
150 IJLL 16.7 |xM 4-methylhydralazine in 10 mM HCl, add 2 mL 20 mM HCl (perform the
preceding procedure as rapidly as possible), vortex briefly, allow to stand at 20 1 for 10.0
min, add 1 mL 1 M NaOH/0.6 M sodium tetraborate buffer (pH 10), add chloroform, shake at
110 rpm for 5 min, centrifuge at 1100 g for 10 min. Remove the organic layer and evaporate
phase, inject a 50 (JLL aliquot.
HPLCVARIABLES
Column: 10 (xm ixBondapak phenyl
Mobile phase: MeCN: 1.5 mM aqueous phosphoric acid 15:85
Flow rate: 2
CHROMATOGRAM
Retention time: 6.7
Internal standard: 4-methylhydralazine (10)
OTHER SUBSTANCES
Simultaneous: metabolites, propranolol, quinidine
KEYWORDS
REFERENCE
Reece,P.A.; Cozamanis,!; Zacest,R. Selective high-performance liquid chromatographic assays for hydralazine
and its metabolites in plasma of man, J.Chromatogr., 1980,181, 427-440.
SAMPLE
Matrix: blood
Sample preparation: 3 mL Whole blood + 20 |xL p-anisaldehyde + 8 |xL 5 |xg/mL 4-methylhy-
dralazine in 10 mM HCl, vortex for 15 s, let stand at room temperature for 10 min, add 10 mL
hexane, shake horizontally at 180 strokes/min for 10 min, centrifuge at 1000 g for 10 min.
the residue in 100 |xL MeOH, inject the whole amount.
HPLC VARIABLES
Column: 300 X 3.9 ixBondapak CN
Flow rate: 2
Detector: UV 365
CHROMATOGRAM
Retention time: 3.5
Internal standard: 4-methylhydralazine (6)
OTHER SUBSTANCES
Noninterfering: propranolol, furosemide, hydrochlorothiazide, digoxin, nitroglycerin
KEYWORDS
whole blood; derivatization
REFERENCE
Ludden,T.M.; Ludden,L.K.; Wade,K.E.; Allerheiligen,S.R. Determination of hydralazine in human whole blood,
J.Pharm.ScL, 1983, 72, 693-695.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 20 mM disodium EDTA + 1 mL 500 mM HCl + 1
mL 10 mM 2-hydroxy-l-naphthaldehyde, vortex for 15 s, keep at 25 for 90 min, add 50 |xL 1
|xg/mL methyl red, add 7 mL dichloromethane, vortex for 5 min, centrifuge at 4500 rpm for 15
min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 50,
reconstitute the residue in 100 (xL MeCN, inject a 20 (xL aliquot.
HPLC VARIABLES
Column: 250 X 4 3 |xm Spherisorb ODS-2
Mobile phase: MeCN:buffer 80:20, pH 3 (Buffer was 0.75% phosphoric acid and 0.5% triethy-
lamine in water.)
Flow rate: 0.7
Detector: UV 406
CHROMATOGRAM
Retention time: 6.4
Internal standard: methyl red (6)
OTHER SUBSTANCES
Extracted: dihydralazine
KEYWORDS
REFERENCE
Maries,J.; Man,J.; Garcia,R.; Font,G. Liquid chromatographic determination of hydralazine in human plasma
with 2-hydroxy-l-naphthaldehyde pre-column derivatization, J.Pharm.BiomedAnal., 1990, 5, 795-798.
SAMPLE
Sample preparation: Injections. Dilute 1.5 mL of a 20 mg/mL injection to 100 mL with water,
remove a 10 mL aliquot and add it to 3 mL 0.2% hydrochlorothiazide, make up to 100 mL with
water, inject a 20 |JLL aliquot. Tablets. Grind tablets to a fine powder, weigh out amount equiv-
alent to about 10 mg hydralazine, mix thoroughly with 2 mL 500 mM HCl, make up to 100
mL with water, shake for 2-3 min, filter, discard first 15 mL. 15 mL Filtrate + 1.5 mL 0.2%
hydrochlorothiazide, make up to 50 mL with water, inject a 20 jxL aliquot.
HPLC VARIABLES
Mobile phase: MeOH: 15 mM KH2PO4:glacial acetic acid 0.5:99.4:0.1
Flow rate: 3
Detector: UV 256
CHROMATOGRAM
Retention time: 5
Internal standard: hydrochlorothiazide (8)
OTHER SUBSTANCES
Simultaneous: phenylpropanolamine
KEYWORDS
injections; tablets
REFERENCE
Das Gupta,V. Quantitation of hydralazine hydrochloride in pharmaceutical dosage forms using high-perfor-
mance liquid chromatography, J.Liq.Chromatogr., 1985, #, 2497-2509.
SAMPLE
Sample preparation: Powder tablets, sonicate for 10 min with enough 5 mM HCl to give a 30
|xg/mL solution, filter, inject an aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 jjim Spherisorb CN
Mobile phase: MeOHibuffer 20:80 (Buffer was 7 mM sodium heptanesulfonate:50 mM triethyl-
amine adjusted to pH 3.1 with dilute phosphoric acid 80:20.)
Flow rate: 1
Detector: UV 260
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Simultaneous: dihydralazine, phthalazine
KEYWORDS
tablets
REFERENCE
Di Pietra,A.M.; Roveri,R; Gotti,R.; Cavrini,V. Spectrophotometric and chromatographic (HPLC) analysis of
hydralazine, dihydralazine and hydrazine after derivatization with 2-nitrocinnamaldehyde, Farmaco, 1993,
48, 1555-1567.
SAMPLE
Sample preparation: Powder tablets, add MeCN:5 mM sodium octanesulfonate 15:85, sonicate,
inject an aliquot.
HPLC VARIABLES
Mobile phase: MeCN:5 mM sodium octanesulfonaterphosphoric acid 15:85:0.045
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
OTHERSUBSTANCES
KEYWORDS
tablets
REFERENCE
Lessen,T.; Zhao,D.C. Interactions between drug substances and excipients. 1. Fluorescence and HPLC studies
of triazolophthalazine derivatives from hydralazine hydrochloride and starch, J.Pharm.Sci., 1996,85, 326-
329.
SAMPLE
Matrix: perfusate
Sample preparation: 30 |xL Perfusate (artificial CSF) + 10 fxL 200 mM perchloric acid. Mix a
25 JJLL aliquot with 12.5 |xL reagent, let stand for 2 min, inject an aliquot. (Prepare a stock
solution by dissolving 27 mg o-phthalaldehyde in 1 mL MeOH, add 5 JJLL p-mercaptoethanol,
add 9 mL 100 mM pH 9.3 sodium tetraborate containing 10 |xM EDTA. This solution is good
for 5 days in a sealed amber bottle at room temperature. Prepare the working reagent by
diluting 1 mL of the stock solution with 3 mL 100 mM pH 9.3 sodium tetraborate containing
10 JJLM EDTA, allow to stand for 24 h before use.)
HPLC VARIABLES
Column: two columns 150 X 4.6 5 |xm M.S. Gel C18 (ESA)
Mobile phase: MeOHrbuffer 8:92 adjusted to pH 3.0 with phosphoric acid (Buffer was 54 mM
NaH2PO4 containing 1.24 mM sodium heptanesulfonate.)
Flow rate: 1.2
Detector: E, ESA Coulochem Electrode Array System Model 5500, detector temp 33, oxidation
potential 0 V
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: apomorphine, dopamine, isoproterenol, methoxamine, morphine, norepinephrine,
phenylephrine
KEY WORDS
rat; derivatization
REFERENCE
Acworth,I.N.; Yu,J.; Ryan,E.; Gariepy,KC; Gamache,P.; Hull,K; Maher,T. Simultaneous measurement of mon-
oamine, amino acid, and drug levels, using high performance liquid chromatography and coulometric array
technology: application to in vivo microdialysis perfusate analysis, J.Liq.Chromatogr., 1994, 17, 685-705.
SAMPLE
Matrix: solutions
Sample preparation: Make up a solution in 40 mM sodium formate and 62 mM formic acid
buffer (pH 3.5), inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 10 jxm CN (Waters)
Column: 150 X 4.6 5 |xm Ultrasphere CN
Mobile phase: MeOH:buffer 15:85 (Buffer was 40 mM sodium formate and 62 mM formic acid,
pH 3.5.)
Flow rate: 1
Detector: UV 258
CHROMATOGRAM
Retention time: 3.7
Internal standard: phenylephrine (2.7)
OTHER SUBSTANCES
Simultaneous: phthalazine, degradation products
REFERENCE
Halasi,S.; Nairn,J.G. Quantitative determination of hydralazine hydrochloride and phthalazine in aqueous
solutions by high performance liquid chromatography, J.Liq.Chromatogr., 1989, 12, 2397-2403.
SAMPLE
Matrix: solutions
Sample preparation: Add a 1 mL aliquot of a solution in MeOHrwater 70:30 to 1 mL pH 4.5
acetate buffer, add 50 |xL acetic acid, add 1 mL 2.2 mM 2-nitrocinnamaldehyde in EtOH, heat
at 70 for 50 min, cool to room temperature, inject an aliquot.
HPLCVARIABLES
Mobile phase: MeCN:buffer 65:35 (Prepare buffer by adjusting the pH of 50 mM triethylamine
to 3.3 with dilute phosphoric acid.)
Flow rate: 1
Detector: UV 390
CHROMATOGRAM
Retention time: 5.1
OTHER SUBSTANCES
Simultaneous: hydrazine (UV 350)
KEY WpRDS
derivatization
REFERENCE
Di Pietra,A.M.; Roveri,R; Gotti,R.; Cavrini,V. Spectrophotometric and chromatographic (HPLC) analysis of
hydralazine, dihydralazine and hydrazine after derivatization with 2-nitrocinnamaldehyde, Farmaco, 1993,
48, 1555-1567.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 12 |xm Dynamax C18 (Rainin)
Mobile phase: MeCN:50 mM acetic acid 20:80
Flow rate: 2
Detector: UV 241
CHROMATOGRAM
Retention time: 1.6
OTHER SUBSTANCES
Simultaneous: metabolites, 3-methyl-s-triazolo[3,4-a]phthalazine
REFERENCE
Hickman,D.; Palamanda,J.R.; Unadkat,J.D.; Sim,E. Enzyme kinetic properties of human recombinant arylam-
ine N-acetyltransferase 2 allotypic variants expressed in Escherichia coli, Biochem.Pharmacol., 1995, 50,
697-703.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
haloperidol, homatropine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloro-
dine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxjrmetazoline, paroxetine, pemo-
zopiclone
KEYWORDS
Next Page
REFERENCE
J.Chromatogr.A, 1995, 692, 103-119.
Hydrochlorothiazide
Molecular formula: C7H8CIN3O4S2
Merck Index: 4822
Lednicer No.: 1 358
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum +100 |xL 1.25 |xg/mL IS + 5 mL MTBE, vortex for 2 min.
Centrifuge at 2700 g for 5 min and evaporate the organic phase to dryness under a stream of
nitrogen. Dissolve the residue in 200 |xL water, add 3 mL toluene, vortex for 2 min, centrifuge
at 2700 g for 10 min, discard the toluene layer . Add 3 mL toluene, vortex, centrifuge, discard
the toluene layer. Evaporate the aqueous layer to dryness under a stream of nitrogen. Recon-
stitute the residue in 200 (xL mobile phase. Inject a 100 |xL aliquot.
HPLC VARIABLES
Guard column: 4 X 4 5 |xm RP-C18
Column: 250 X 4 5 |xm LiChrospher 100 RP-C18
Mobile phase: MeCN:THF:200mM pH 7.5 phosphate buffer 5:10:85
Flow rate: 1
Detector: UV 273
CHROMATOGRAM
Internal standard: hydroflumethiazide
KEYWORDS
REFERENCE
Vervaet,C; Remon,J.P. Bioavailability of hydrochlorothiazide from pellets, made by extrusion/spheronisation,
containing polyethylene glycol 400 as a dissolution enhancer, Pharm.Res., 1997,14, 1644-1646.
SAMPLE
Matrix: blood
Sample preparation: 200 |JLL Serum + 20 |xL 1 M pH 7.0 phosphate buffer, extract with 5 mL
MTBE, vortex for 20 s, centrifuge at 2500 g for 10 min. Remove the organic layer and add it
to 10 JJLL 20 jjig/mL IS in MeOH, evaporate to dryness at 80 in a vacuum centrifuge, reconsti-
tute the residue with 180 |xL mobile phase, inject a 30 |xL aliquot.
HPLC VARIABLES
Column: 125 X 4 5 |xm endcapped LichroCART RP18
Mobile phase: MeCN:7.5 mM pH 7.3 phosphate buffer 10:90
Flow rate: 0.8
Detector: E, ESA Coulochem II, coulometric cell 5011, first electrode +450 mV, second electrode
+630 mV; UV 254
Previous Page
REFERENCE
J.Chromatogr.A, 1995, 692, 103-119.
Hydrochlorothiazide
Merck Index: 4822
Lednicer No.: 1 358
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum +100 |xL 1.25 |xg/mL IS + 5 mL MTBE, vortex for 2 min.
Centrifuge at 2700 g for 5 min and evaporate the organic phase to dryness under a stream of
nitrogen. Dissolve the residue in 200 |xL water, add 3 mL toluene, vortex for 2 min, centrifuge
at 2700 g for 10 min, discard the toluene layer . Add 3 mL toluene, vortex, centrifuge, discard
the toluene layer. Evaporate the aqueous layer to dryness under a stream of nitrogen. Recon-
stitute the residue in 200 (xL mobile phase. Inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4 5 |xm LiChrospher 100 RP-C18
Mobile phase: MeCN:THF:200mM pH 7.5 phosphate buffer 5:10:85
Flow rate: 1
Detector: UV 273
CHROMATOGRAM
KEYWORDS
REFERENCE
Vervaet,C; Remon,J.P. Bioavailability of hydrochlorothiazide from pellets, made by extrusion/spheronisation,
containing polyethylene glycol 400 as a dissolution enhancer, Pharm.Res., 1997,14, 1644-1646.
SAMPLE
Matrix: blood
Sample preparation: 200 |JLL Serum + 20 |xL 1 M pH 7.0 phosphate buffer, extract with 5 mL
MTBE, vortex for 20 s, centrifuge at 2500 g for 10 min. Remove the organic layer and add it
to 10 JJLL 20 jjig/mL IS in MeOH, evaporate to dryness at 80 in a vacuum centrifuge, reconsti-
tute the residue with 180 |xL mobile phase, inject a 30 |xL aliquot.
HPLC VARIABLES
Column: 125 X 4 5 |xm endcapped LichroCART RP18
Mobile phase: MeCN:7.5 mM pH 7.3 phosphate buffer 10:90
Flow rate: 0.8
Detector: E, ESA Coulochem II, coulometric cell 5011, first electrode +450 mV, second electrode
+630 mV; UV 254
CHROMATOGRAM
Retention time: 5.8
Limit of quantitation: 5 ng/mL (E)
KEYWORDS
serum
REFERENCE
Richter,K; Oertel,R.; Kirch,W. New sensitive method for the determination of hydrochlorothiazide in human
serum by high-performance liquid chromatography with electrochemical detection, J.Chromatogr.A, 1996,
729, 293-296.
SAMPLE
Sample preparation: Serum. Condition a Bakerbond C18 SPE cartridge with 3 mL MeOH and
3 mL water. Mix 100 |xL serum with 200 |xl MeCN, vortex for 2 min, add 100 |xL 4.08 jjug/mL
IS in MeOH, mix, centrifuge at 4000 rpm for 15 min. After the removal of the organic solvent
add the supernatant to the SPE cartridge, dry under vacuum, wash with 3 mL water, elute
with 3 mL MeOH. Evaporate to dryness under a stream of nitrogen at 45, dilute to 100 (xL
with MeOH. Inject a 20 (xL aliquot. Tablets. Powder tablets. Prepare an 1-3 |xg/mL solution of
hydrochlorothiazide in MeOH containing 4.08 |xg/mL IS. Inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 100 X 4.6 5 fxm Nucleosil C18
Flow rate: 1.3
Detector: UV 270
CHROMATOGRAM
Internal standard: hydroflumethiazide (5.28)
OTHER SUBSTANCES
Noninterfering: captopril
KEYWORDS
tablets; plasma; SPE
REFERENCE
Papadoyannis,I.N.; Samanidou,V.R; Georga,K.A.; Georgarakis,E. High performance liquid chromatographic de-
termination of hydrochlorothiazide (HCT) in pharmaceutical preparations and human serum after solid
phase extraction, J.Liq.Chromatogr.Rel.TechnoL, 1998, 21, 1671-1683.
SAMPLE
a 10 (urine) or 30 (blood) u,L aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
Column: 250 X 4.6 5 (xm Symmetry C8 (Waters)
mL/min)
Detector: UV 226.3
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
Sample preparation: Dilute 25 mL 9.6 mg/mL chlorothiazide in MeOH with 1 mL 380 mg/L IS
in 0.1% phosphoric acid, inject an aliquot.
HPLC VARIABLES
Column: A 250 X 2 J sphere ODS-M80; B 150 X 4.6 5 |xm Beckman Ultrasphere C18
Mobile phase: A Gradient. MeCN:0.1% formic acid from 0:100 to 30:70 over 20 min. B Gradient.
MeCNiO. 1% phosphoric acid 0:100 to 30:70 over 12 min.
Detector: A MS, Finnigan Model TSQ-7000 triple-quadrupole, nebulizer nitrogen 260; B UV 270
CHROMATOGRAM
Retention time: 22
Internal standard: ethylparaben
OTHER SUBSTANCES
KEYWORDS
photolysis
REFERENCE
Revelle,L.K; Musser,S.M.; Rowe,B.J.; Feldman,I.C. Identification of chlorothiazide and hydrochlorothiazide
UV-A photolytic decomposition products, J.Pharm.Sci., 1997, 86, 631-634.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Partisil ODSl
Mobile phase: MeOH:50 mM pH 3.0 phosphoric acid 10:90
Flow rate: 1.5
Detector: radioactivity detection
OTHER SUBSTANCES
Also analyzed: atenolol, cimetidine, ranitidine
KEYWORDS
14C labeled
REFERENCE
CollettA-; Sims,E.; Walker,D.; He,Y.-L.; Ayrton,J.; Rowland,M.; Warhurst,G. Comparison of HT29-18-d and
Caco-2 cell lines as models for studying intestinal paracellular drug absorption, Pharm.Res., 1996,13,216-
221.
SAMPLE
Matrix: urine
Sample preparation: Dilute 10 mL urine to 15 mL with water, add to Extrelut-20 cartridge,
elute with 60 mL ethyl acetate:isopropanol 85:15. Evaporate under vacuum at 50, filter, dry
under nitrogen, reconstitute the residue in 100 JJLL acetone. Add 100 |xL 1 mg/mL 3-bromo-
methylpropylphenazone in acetone, mix with 1 mg anhydrous potassium carbonate, make up
to 200 |xL with acetone. Let stand at 105 5 for 60 min. Cool the reaction mixture, dry under
a gentle stream of nitrogen. Reconstitute the residue with 500 |xL MeCN, shake for 2 min.
inject a 10 |xL aliquot. (3-Bromomethylpropylphenazone is produced by the reaction of propyl-
phenazone with bromine and recrystallized from chloroform:diethyl ether 1:2. (Caution! Chlo-
roform is a carcinogen!))
HPLC VARIABLES
Mobile phase: MeCN:MeOH:50 mM sodium acetate 34:8:28, adjusted to pH 6.5 with acetic acid
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 27.7 (derivatized), 3.3 (underivatized)
OTHER SUBSTANCES
Extracted: captopril
KEYWORDS
derivatization; SPE
REFERENCE
Khedr,A.; El-Sherief,H. 3-Bromomethyl-propyphenazone as a new derivatization reagent for high performance
liquid chromatography of captopril and hydrochlorothiazide with UV-detection, Biomed.Chromatogr., 1998,
12, 57-60.
Hydrocodone
CAS Registry No.: 125-29-1,34195-34-1 (bitartrate hydrate),
143-71-5 (bitartrate)
Merck Index: 4826
Lednicer No.: 1 288
SAMPLE
Sample preparation: 500 |xL or 1.0 mL Sample + 1.0 mL water + 500 |JLL 1 M NaOH + 15 mL
dichloromethane, shake at 100 cpm for 20 min. Centrifuge at 2500 rpm for 5 min, evaporate
organic layer to dryness under a gentle stream of nitrogen at 35 to 40. Dissolve residue in 5
mL MeOH, inject a 20 to 80 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Alltech C8
Mobile phase: MeCN.pH 4.5 buffer 18:82 (Buffer was 10 mM KH2PO4 and 50 mM potassium
nitrate.)
Flow rate: 1.4
Detector: UV 280
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
Noninterfering: chorpheniramine
KEYWORDS
suspensions
REFERENCE
Hadzija,B.W.; Shrewsbury,R-P- Determination of hydrocodone in Tussionex extended-release suspension by
high-performance liquid chromatography (HPLC), J.Forensic ScL, 1996, 41, 878-880.
Hydrocortisone
CAS Registry No.: 50-23-7,13609-67-1 (butyrate), 57524-89-7
(valerate), 50-03-3 (acetate), 3863-59-0 (phosphate), 6000-74-4
(sodium phosphate), 125-04-2 (21-sodium succinate), 508-96-3
(tebutate), 74050-20-7 (aceponate), 72590-77-3 (buteprate), 508-99-6 (cypionate), 83784-20-7 (hem-
isuccinate monohydrate), 2203-97-6 (hemisuccinate), 2203-97-6 (succinate), 5752489-7 (valerate)
Merck Index: 4828
LednicerNo.: 1 190
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge. Mix 1 mL plasma with 134.0 ng
hydrocortisone-d5 and 74.56 ng cortisone-d5. Add the sample to the SPE cartridge, wash with
8 mL water, elute with 4 mL ethyl acetate, evaporate the eluate to dryness at 70 under a
stream of nitrogen, dissolve the residue in 30 jxL# mobile phase, filter (0.45 |xm), inject a 20 JJLL
aliquot.
HPLCVARIABLES
Column: 125 X 4.0 4 |xm LiChroCART Superspher 100
Mobile phase: AMeOH:THF:50 mM ammonium formate 17:53:180; B MeCN:50 mM ammonium
formate 35:65
Flow rate: 0.6 (A); 1.3 (B)
Detector: MS, Shimadzu LCMS-QP1000EX Model 750 B, thermospray, vaporizer control 155,
vaporizer tip 195, vapor 274, ion source block 295, tip heater 305, m/z 363
CHROMATOGRAM
Retention time: 13 (A)
Internal standard: hydrocortisone-d5, cortisone-d5
OTHER SUBSTANCES
Extracted: cortisone, prednisolone, prednisone
KEYWORDS
plasma; SPE
REFERENCE
Shibasaki,H.; Furuta,T.; Kasuya,Y. Quantification of corticosteroids in human plasma by liquid chromatogra-
phy-thermospray mass spectrometry using stable isotope dilution, J.Chromatogr.B, 1997, 692, 7-14.
SAMPLE
Matrix: blood
Sample preparation: Add 100 |JLL MeOH and 50 |xL 1 a,g/mL fluocortolone in MeOH to 1 mL
plasma. Add 500 jxL 100 mM NaOH and 2 mL dichloromethane, shake for 10 min, centrifuge
at 2500 g for 10 min, evaporate a 1.9 mL aliquot of the supernatant under a stream of nitrogen
at 45. Reconstitute the residue in 50 |xL MeOH, inject 17 JJLL aliquot.
HPLCVARIABLES
Guard column: 10 X 4 5 |xm LiChrospher RP 18
Column: 250 X 4 5 ^m Lichrospher RP 18
Mobile phase: MeOH:THF:water 110:2.5:100
Flow rate: 1
Detector: UV 252
CHROMATOGRAM
Internal standard: fluocortolone
OTHER SUBSTANCES
Extracted: triamcinolone
KEYWORDS
plasma
REFERENCE
Doppenschmitt,S.A.; Scheidel,B; Harrison,E; Surmann,J.P. Simultaneous determination of triamcinolone ace-
tonide and hydrocortisone in human plasma by high-performance liquid chromatography, J.Chromatogr.B,
1996, 682, 79-88.
SAMPLE
Sample preparation: Condition a 3 mL 500 mg Sep-Pak Vac C18 SPE cartridge with 3 mL
MeOH and 3 mL water. 1 mL Serum or urine + 500 |xL 200 mM pH 3.85 acetate buffer (serum
only) + 400 |xL 2.5 |xM IS in mobile phase, mix, centrifuge. Add the supernatant to the SPE
cartridge, wash with 3 mL acetone:water 20:80, 3 mL water, and 3 mL hexane. Elute with 3
mL diethyl ether into tubes containing 1 mL 200 mM NaOH, vortex, centrifuge. Dry the organic
layer under a stream of nitrogen. Reconstitute the residue in 250 |xL mobile phase, mix for 5
min. Inject a 60 |xL aliquot.
HPLCVARIABLES
Flow rate: 1.0
Detector: UV 254
CHROMATOGRAM
Internal standard: fludrocortisone (15.9)
OTHER SUBSTANCES
Extracted: 11-deoxycortisol, dexamethasone, methylprednisolone, prednisolone
KEYWORDS
serum; SPE
REFERENCE
McWhinney,B.C; Ward,G.; Hickman,P.E. Improved HPLC method for simultaneous analysis of cortisol, 11-
deoxycortisol, prednisolone, methylprednisolone, and dexamethasone in serum and urine, Clin.Chem., 1996,
42, 979-981.
SAMPLE
Sample preparation: Condition a Sep-Pak Plus C18 SPE cartridge with 7 mL MeOH and 14
mL water. Add 40 ng 6p-hydroxycortisone to 400 |xL plasma or urine, add the mixture to the
SPE cartridge, wash with 6 mL water, 3 mL MeOH.water 12:88, and 3 mL petroleum ether,
elute with 5 mL ethyl acetate. Dry the eluate under reduced pressure at 40, add 200 JJLL MeCN:
triethylamine 90:10 and MeCN:0.1% quinuclidine 20:80 to the residue, vortex. Add 200 |JLL
0.02% 9-anthroyl nitrile and a few molecular sieves (4A), let stand for 30 min, evaporate under
reduced pressure at 40, dissolve the residue in 200 jxL acetone, dilute with 2 mL n-hexane.
Add the mixture to a Sep-Pak Plus Silica SPE cartridge, wash with 14 mL 1,2-dichloroethane,
elute with 5 mL ethyl acetate. Evaporate the eluate under reduced pressure at 40, reconstitute
the residue in 200 fxL mobile phase, inject a 30-60 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Cosmosil 5SL (Nacalai Tesque, Japan)
Mobile phase: Dioxane:ethyl acetate.chloroform.n-hexane.pyridine 58.1:11.6:11.6:16.3:2.4 (Cau-
tion! Dioxane and chloroform are carcinogens!)
Flow rate: 1 for 45 min, to 1.2 over 5 min
CHROMATOGRAM
Retention time: 24
Internal standard: 6p-hydroxycortisone (86)
OTHER SUBSTANCES
Extracted: cortisone, 6p-hydroxycortisol, 6p-hydroxyprednisolone, prednisolone, prednisone
KEYWORDS
derivatization; plasma; urine; SPE; normal phase
REFERENCE
Shibata,N.; Hayakawa,T.; Takada,K.; Hoshino,N.; Minouchi,T.; Yamaji,A- Simultaneous determination of glu-
cocorticoids in plasma or urine by high-performance liquid chromatography with precolumn fluorimetric
derivatization by 9-anthroyl nitrile, J.Chromatogr.B, 1998, 706, 191-199.
SAMPLE
a 10 (urine) or 30 (blood) ^xL aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
Column: 250 X 4.6 5 um Symmetry C8 (Waters)
mL/min)
Detector: UV 242.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
Sample preparation: Prepare solutions in MeCN, dilute to an appropriate concentration with
HPLC VARIABLES
Column: 120 X 4.6 5 jxm octadecyl Bakerbond
Mobile phase: MeCN:water 30:70 containing 16 mM p-cyclodextrin
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 0.7
OTHER SUBSTANCES
Simultaneous: testosterone, prednisone, cortisone, 17a-methyltestosterone, 17a-hydroxyproges-
terone
REFERENCE
Zarzycki,P.K; Wierzbowska,M.; Lamparczyk,H. The influence of temperature on the high performance liquid
chromatographic separation of steroids using mobile phases modified with p-cyclodextrin,
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 X 4.1 10 |xm Versapack C18 (Alltech)
Flow rate: 1
Detector: UV 254
OTHER SUBSTANCES
Simultaneous: hydrocortisone, hydrocortisone acetate
REFERENCE
Michniak,B.B.; Player,M.R.; Sowell,J.W. Synthesis and in vitro transdermal penetration enhancing activity of
lactam N-acetic acid esters, J.Pharm.Sci., 1996, 85, 150-154.
SAMPLE
Matrix: urine
Sample preparation: Condition a 10 mL 200 mg MCF Isolute SPE cartridge with two 3 mL
portions of EtOH and two 3 mL portions of water. Centrifuge urine at 4000 g for 30 in, filter
through a 0.22 |xm filter unit. Dilute 0.75-3mL urine to 4 mL with water. Add 30 ng IS. Add
to the SPE cartridge. Wash with three 3 mL portions of water, 3 mL MeOHrIO mM NaOH 30:
70, twice with 3 mL water and with 3 mL MeOHrIO mM HCl 30.70. Elute with 3 mL EtOH.
Evaporate eluate under vacuum and reconstitute the residue with 150 |xL mobile phase. Inject
a 100 (xL aliquot.
HPLCVARIABLES
Column: 100 X 3.2 5 |xm Nucleosil 120-C18
Flow rate: 0.5
Detector: UV 254
CHROMATOGRAM
Internal standard: dexamethasone (22.01)
OTHER SUBSTANCES
Extracted: metabolites, cortisone
KEYWORDS
SPE; human; pig
REFERENCE
Hay,M.; Morm&de,R Improved determination of urinary cortisol and cortisone, or corticosterone and 11-dehy-
drocorticosterone by high-performance liquid chromatography with ultraviolet absorbance detection,
J.Chromatogr.B, 1997, 702, 33-39.
SAMPLE
Matrix: urine
Sample preparation: Activate 3-mL 500 mg Bakerbond C18 cartridge with 2 mL MeOH and 2
mL water. Filter sample. Add 25 |xL 8 (xM IS in MeOH to 2 mL urine, add to the SPE cartridge,
wash with two 2 mL portions of 25 mM borate buffer and with 200 mIVL acetone in water.
Add 1 mL hexane and air-dry under reduced pressure for 4 min. Elute with two 1 mL portions
of ethyl acetate. Dry the eluate under a stream of nitrogen and dissolve in 75 JULL 400 mL/L
MeOH, inject a 25 (JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm LiChrospher 100 C18
Flow rate: 1
Detector: UV 242
CHROMATOGRAM
Internal standard: 6 a-methylprednisolone (39.3-40.1)
OTHER SUBSTANCES
Simultaneous: metabolites, alprazolam, amlodipine, aspirin, carbamazepine, citalopram, corti-
costerone, cortisone, dexamethasone, digoxin, enalapril, ferrous sulfate, fluoxetine, furosemide,
gabapentin, 5-hydroxyindoleacetic acid, lamotrigine, metyrapone, naproxen, oxazepam, oxcar-
bazepine, oxybutynin, phenobarbital, phenytoin, prednisone, spironolactone, valproic acid,
vigabatrin, zopiclone
Noninterfering: octreotide
Interfering: prednisolone
KEYWORDS
SPE; comparison with RIA
REFERENCE
Turpeinen,U.; Markkanen,HL; Valimaki,M.; Stenman,U.-H. Determination of urinary free cortisol by HPLC,
Clin.Chem., 1997, 43, 1386-1391.
SAMPLE
Matrix: urine
Sample preparation: 10 mL Urine + 40 |JLL 25 |xg/mL corticosterone, vortex briefly, add 1 mL
100 mM NaOH, vortex briefly, add 3 mL dichloromethane, rotate at 20 rpm for 45 min, cen-
trifuge at 1000 g for 15 min, discard the aqueous layer, centrifuge at 1000 g for 10 min, discard
the aqueous layer, add 150 mg NaCl, break up emulsion, centrifuge for 10 min. Remove the
residue in 150 jxL MeOH, inject an aliquot.
HPLC VARIABLES
Mobile phase: Gradient. MeOHiwater from 30:70 to 44:56 over 6 min, maintain at 44:56 for 14
min, return to initial conditions over 3 min, re-equilibrate for 5 min.
Flow rate: 1
Detector: UV 246
CHROMATOGRAM
Internal standard: corticosterone (17.8)
OTHER SUBSTANCES
Extracted: cortisone
REFERENCE
Lee,Y.S.; Lorenzo,B-J-5 Koufis,T.; Reidenberg,M.M. Grapefruit juice and itsflavonoidsinhibit lip-hydroxyste-
roid dehydrogenase, Clin.Pharmacol.Ther., 1996, 59, 62-71.
Hydroflumethiazide
Molecular formula: C8H8F3N3O4S2
Merck Index: 4830
Lednicer No.: 1 358
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 5 mL MTBE, vortex for 2 min. Centrifuge at 2700 g for
5 min and evaporate the organic phase to dryness under a stream of nitrogen. Dissolve the
residue in 200 JJLL water, add 3 mL toluene, vortex for 2 min, centrifuge at 2700 g for 10 min,
discard the toluene layer. Add 3 mL toluene, vortex, centrifuge, discard the toluene layer.
Evaporate the aqueous layer to dryness under a stream of nitrogen. Reconstitute the residue
in 200 JJLL mobile phase. Inject a 100 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4 5 |xm LiChrospher RP-C18
Mobile phase: MeCN:THF:200 mM pH 7.5 phosphate buffer 5:10:85
Flow rate: 1
Detector: UV 273
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: hydrochlorothiazide
KEYWORDS
hydroflumethiazide is IS; serum
REFERENCE
Vervaet,C; Remon,J.R Bioavailability of hydrochlorothiazide from pellets, made by extrusion/spheronisation,
containing polyethylene glycol 400 as a dissolution enhancer, Pharm.Res., 1997, 14, 1644-1646.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 25 jxL 10 mg/mL hydroflumethiazide in water + 1 mL 1
M pH 10 sodium carbonate-bicarbonate buffer + 5 mL ethyl acetate, vortex 1 min, centrifuge
at 1250 g for 5 min. Remove the ethyl acetate layer and evaporate at 45 under nitrogen.
Dissolve in 100 (xL mobile phase, inject 50 (xL aliquot.
HPLCVARIABLES
Column: 125 X 4.6 5 jjum Spherisorb ODSII
Mobile phase: MeCN:MeOH:buffer 10:9:100 (Buffer was 15.54 g tetraethylammonium hydroxide
and 2.9 g 89% orthophosphoric acid in 500 mL water, pH was 2.8.)
Flow rate: 1.2
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: amiloride (detection by F), hydrochlorothiazide (detection by UV)
KEYWORDS
plasma; hydroflumethiazide is IS
REFERENCE
Van der Meer,M.J.; Brown,L.W. Simultaneous determination of amiloride and hydrochlorothiazide in plasma
by reversed-phase high-performance liquid chromatography, J.Chromatogr., 1987, 423, 351-357.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + ImL buffer + 200 JULL water + 6 mL ethyl acetate, shake
for 5 min, centrifuge at 900 g for 5 min. Remove 5 mL organic layer and evaporate at 37 under
a stream of nitrogen. Reconstitute with 100 |xL MeOH, sonicate twice at 37 for 1 min, cool at
2-8 for 2 h to obtain a clear solution, inject a 20 |xL aliquot. (Buffer was 0.38 g ammonium
acetate in 500 mL water and acidified to pH 5.0 with glacial acetic acid.)
HPLC VARIABLES
Guard column: 40 X 4 35-50 |xm C18 Corasil
Column: 125 X 4 5 fxm Nucleosil 100-5 C18
Mobile phase: Gradient. A was MeCN:acetic acid:water 25:1:975. B was MeCN:acetic acid:water
500:1:500. A:B 100:0 to 36:64 over 16 min, re-equilibrate at 100:0 for 24 min before next
injection
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: acebutolol, acenocoumarol, acetaminophen, aspirin, allopurinol, ambroxol
amoxicillin, atenolol, bendroflumethiazide, benzbromarone, bezafibrate, biperiden, bisacodyl,
bromazepam, butizide, caffeine, captopril, cimetidine, ciprofioxacin, clobutinol, clonidine, cotin-
ine, diazepam, diclofenac, digitoxin, digoxin, dihydrocodeine, dihydroergotamine, diltiazem,
doxepin, doxycycline, enalapril, erythromycin, fenoterol, furosemide, glibenclamide, heparin,
hypoxanthine, ibuprofen, indomethacin, isosorbide mononitrate, lisinopril, lovastatin, mapro-
tiline, methyldigoxin, methyldopa, metoclopramide, metoprolol, metronidazole, midazolam,
naloxone, nifedipine, nicotine, norfloxacin, ofloxacin, oxazepam, oxipurinol, penicillin V, pen-
toxyfylline, phenacetin, phenazone, propyphenazone, phenprocoumon, ranitidine, salicylic acid,
sotalol, sulfamethoxazole, trimethoprim, terbutaline, theophylline, tilidine, timolol, triamter-
ene, uric acid, verapamil, ascorbic acid, warfarin, xanthine, purine and pyrimidine bases, nu-
cleosides, nucleotides
Interfering: amiloride
KEYWORDS
plasma; hydroflumethiazide is IS
REFERENCE
de Vries,J.X.; Voss,A. Simple determination of hydrochlorothiazide in human plasma and urine by high per-
formance liquid chromatography, BiomedChromatogr., 1993, 7, 12-14.
SAMPLE
Matrix: blood, middle ear fluid
Sample preparation: 75 |xL Plasma or middle ear effusion + 50 u.L water, mix, add 25 jxL 10%
perchloric acid, vortex, add 25 |xL KCl solution. Mix, centrifuge, remove supernatant, add 25
JJLL pH 10.4 800 mM Na2HPO4 to the supernatant, inject a 6 uL aliquot.
HPLC VARIABLES
Guard column: 20 X 3.2 Brownlee C8 precolumn
Column: 150 X 4.6 5 jxm Zorbax C8
Mobile phase: MeOH:MeCN:10 mM NaH2PO4 10:2:88
Flow rate: 1.4
Injection volume: 6
Detector: UV 230
CHROMATOGRAM
Retention time: 6.4
OTHER SUBSTANCES
Extracted: amoxicillin
KEYWORDS
plasma; chinchilla; hydroflumethiazide is IS
REFERENCE
Erdmann,G.R.; Walker,K; Giebink,G.S.; Canafax,D.M. High performance liquid chromatographic analysis of
amoxicillin in microliter volumes of chinchilla middle ear effusion and plasma, J.Liq.Chromatogr., 1990,
13, 3339-3350.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve in solvent, inject an aliquot. (Solvent was 750 mg KCl in 10 mL
1 M HCl, add 400 mL water, add 400 mL MeOH, make up to 1 L with water.)
HPLC VARIABLES
G u a r d c o l u m n : 5 x 4 7 |xm Nucleosil-100 p h e n y l
Column: 300 X 4 7 |xm Nucleosil-100 phenyl
Flow rate: 1.5
Detector: UV 270
CHROMATOGRAM
Retention time: 3.1
OTHER SUBSTANCES
Simultaneous: bendroflumethiazide, degradation products
REFERENCE
Frontini,R.; Mielck,J.B. Determination and quantitation of bendroflumethiazide and its degradation products
using HPLC, J.Liq.Chromatogr., 1992, 15, 2519-2528.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1.3
Injection volume: 20 JJLL
Detector: UV 270
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: hydrochlorothiazide
Noninterfering: captopril
REFERENCE
Papadoyannis,I.N.; Samanidou,V.R; Georga,K.A.; Georgarakis,E. High performance liquid chromatographic de-
termination of hydrochlorothiazide (HCT) in pharmaceutical preparations and human serum after solid
phase extraction, J.Liq.Chromatogr.Rel.TechnoL, 1998, 21, 1671-1683.
SAMPLE
Matrix: solutions
JJLL aliquot.
HPLCVARIABLES
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
REFERENCE
SAMPLE
Matrix: urine
300 |xL 50 |xg/mL p-hydroxyethyltheophylline in MeOH, inject 5 |xL aliquot. (Solid buffer I was
KH2PO4INa2HPO4 99:1, solid buffer II was NaHCO3-K2CO3 3:2.)
HPLC VARIABLES
Flow rate: 1
Injection volume: 5
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: furosemide, metolazone, amiloride, acetazolamide, chlorothiazide, hydrochlorothia-
zide, quinethazone, triamterene, chlorthalidone, dichlorphenamide, trichloromethiazide, meth-
yclothiazide, benzthiazide, cyclothiazide, polythiazide, bendroflumethiazide, ethacrynic acid,
bumetanide, probenecid, spironolactone, canrenone, flumethiazide
REFERENCE
Cooper,S.R; Masse,R.; Dugal,R. Comprehensive screening procedure for diuretics in urine by high-performance
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 1 mL buffer + 200 |xL water + 6 mL ethyl acetate, shake
for 5 min, centrifuge at 900 g for 5 min. Remove 5 mL organic layer and evaporate at 37 under
a stream of nitrogen. Reconstitute with 100 jxL mobile phase, inject a 20 JJLL aliquot. (Buffer
was 0.38 g ammonium acetate in 500 mL water and acidified to pH 5.0 with glacial acetic
acid.)
HPLC VARIABLES
Guard column: 40 X 4 35-50 jxm C18 Corasil
Column: 125 X 4 5 |xm Nucleosil 100-5 C18
Mobile phase: MeCNracetic acid:water 120:1:880
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: amiloride, acebutolol, acenocoumarol, acetaminophen, aspirin, allopurinol, am-
broxol amoxicillin, atenolol, bendroflumethiazide, benzbromarone, bezafibrate, biperiden, bis-
acodyl, bromazepam, butizide, caffeine, captopril, cimetidine, ciprofloxacin, clobutinol, cloni-
dine, cotinine, diazepam, diclofenac, digitoxin, digoxin, dihydrocodeine, dihydroergotamine,
diltiazem, doxepin, doxycycline, enalapril, erythromycin, fenoterol, furosemide, glibenclamide,
heparin, hypoxanthine, ibuprofen, indomethacin, isosorbide mononitrate, lisinopril, lovastatin,
maprotiline, methyldigoxin, methyldopa, metoclopramide, metoprolol, metronidazole, midazo-
lam, naloxone, nifedipine, nicotine, oxazepam, oxipurinol, penicillin V, pentoxyfylline, phenac-
etin, phenazone, propyphenazone, phenprocoumon, ranitidine, salicylic acid, sotalol, sulfame-
thoxazole, trimethoprim, terbutaline, theophylline, tilidine, timolol, triamterene, uric acid,
verapamil, ascorbic acid, warfarin, xanthine, purine and pyrimidine bases, nucleosides,
nucleotides
Interfering: norfloxacin and ofloxacin
KEYWORDS
hydroflumethiazide is IS
REFERENCE
de Vries,J.X.; Voss,A. Simple determination of hydrochlorothiazide in human plasma and urine by high per-
formance liquid chromatography, Biomed.Chromatogr., 1993, 7, 12-14.
Hydrogen peroxide
Molecular formula: H2O2
Merck Index: 4839
SAMPLE
Matrix: beverages
Sample preparation: Mix 3.45 mL 500 mM pH 5.0 potassium phosphate buffer, 250 |xL MeOH,
and 100 JJLL antifoaming reagent, pass nitrogen gas through the mixture for a few min, add 1
mL beverage, add 100 jxL 1000 U/mL catalase (Boehringer Mannheim) in water (purge with
nitrogen before use), add 100 |xL 100 mg/mL 4-amino-3-penten-2-one (Fluoral-P, Eastman) in
MeCN, bubble nitrogen at 200 mL/min through the mixture, heat at 30 for 10 min, add to a
Sep-Pak C18 SPE cartridge, wash with a little water, elute with 5 mL MeCN:water 50:50,
inject a 20 |xL aliquot of the eluate. (Prepare antifoaming reagent by diluting concentrated
silicon polymer (Sigma) with water to give a 0.1% suspension.)
HPLC VARIABLES
Flow rate: 1
CHROMATOGRAM
Retention time: 3.5
OTHER SUBSTANCES
KEY WpRDS
derivatization; SPE
REFERENCE
Hamano,T.; Mitsuhashi,Y; Yamamoto,S. Determination of hydrogen peroxide in beverages by high-performance
liquid chromatography with fluorescence detection, J.Chromatogr., 1987, 411, 423-429.
Hydromorphone
Merck Index: 4847
Lednicer No.: 1 288
SAMPLE
Matrix: bile, blood, tissue
Sample preparation: 250 jxL Bile, 3 mL blood, or 5 mL tissue homogenate + 1 mL 200 |xg/mL
nalorphine in water + 2 mL 200 mM pH 8.9 sodium borate buffer + 5 (bile) or 10 (blood, tissue)
mL chloroform:isopropanol 90:10, rotate gently for 20 min, centrifuge at 2000 rpm for 10 min.
Remove the organic layer and add it to 2 mL 500 mM HCl, rotate for 20 min, centrifuge for 5
min. Remove 1.8 mL of the upper aqueous phase, adjust to pH 8.6 0.2 by carefully adding
powdered ammonium carbonate until the solution was saturated, add 5 mL ethyl acetate:
isopropanol 90:10, rotate for 20 min, centrifuge for 5 min. Remove 4.8 mL of the upper organic
in 50 JJLL MeOH, vortex for 30 s, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 30 X 4.6 5 jxm RP-18 Spheri-5
Mobile phase: MeOH:50 mM pH 7 phosphate buffer 40:60 Place a 70 X 2 30-38 |xm Co-Pell ODS
column before the injection valve.)
Flow rate: 2
Detector: E, Environmental Sciences Associates Model 5100, porous graphite electrode Wl 900
mV W2 400 mV, difference in electrolysis current monitored
CHROMATOGRAM
Retention time: 5
Internal standard: nalorphine (14.72)
OTHER SUBSTANCES
Extracted: codeine, morphine, norcodeine, normorphine
Simultaneous: acetaminophen, atropine, epinephrine, ethylmorphine, hydrocodone, hydroxy-
zine, naloxone, oxycodone, pentazocine, phenylpropanolamine, pseudomorphine, scopolamine,
secobarbital
Noninterfering: brompheniramine, chloroprocaine, dextromethorphan, diazepam, diphenhydra-
mine, fentanyl, flurazepam, meperidine, methadone, neostigmine, propoxyphene
REFERENCE
Hepler,B.R.; Sutheimer,C; SunshineJ.; Sebrosky,G.R Combined enzyme immunoassay-LCEC method for the
identification, confirmation, and quantitation of opiates in biological fluids, J.Anal.Toxicol, 1984,8, 78-90.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 1 M sodium bicarbonate + 15 mL diethyl ether,
rotate for 20 min, centrifuge at 800 g for 15 min. Remove the organic phase and add it to 200
IxL 17 mM phosphoric acid, mix vigorously for 15 s, centrifuge for 5 min. Remove the aqueous
phase and evaporate it to dryness under a stream of air at 45, reconstitute the residue in 200
JXL 17 mM phosphoric acid, inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 25 X 4.6 5 |xm Hi-Chrom reversible octyl (Regis)
Mobile phase: MeCN:30 mM pH 4 KH2PO4:0.3% sodium octanesulfonate 15:75:9
Flow rate: 1.5
Detector: E, BAS LC4B, glassy carbon working electrode 0.9 V, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 8.9
Internal standard: hydromorphone
OTHER SUBSTANCES
Extracted: oxymorphone
KEYWORDS
rat; plasma; hydromorphone is IS
REFERENCE
Lam,G.; Williams,R-M.; Whitney,C.C. Electrochemical determination of oxymorphone in rat plasma by ion-pair
reversed-phase high-performance liquid chromatography, J.Chromatogr., 1987, 413, 309-314.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Baxter C18 SPE cartridge with 3 mL MeOH and 3 mL
water. 1 mL Plasma + 2 mL 500 mM pH 9.3 ammonium sulfate + 30 |xL 1 |jig/mL naltrexone,
add to the SPE cartridge, wash with 3 mL 5 mM pH 9.3 ammonium sulfate, wash with 3
mL water, dry under vacuum, elute with 1 mL MeOH:triethylamine 99.5:0.5. Evaporate the
eluate to dryness under a stream of nitrogen at room temperature, reconstitute the residue in
100 |JLL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: C18 (Upchurch)
Mobile phase: MeOH:50 mM Na2HPO^ 15:85 containing 3 mM 1-heptanesulfonic acid, pH ad-
justed to 3.5 with orthophosphoric acid
Flow rate: 0.8
Detector: E, ESA Coulochem, guard cell + 650 mV, analytical cell +250 mV and +600 mV
(monitored)
CHROMATOGRAM
Retention time: 8.0
Internal standard: naltrexone (16.4)
OTHER SUBSTANCES
Extracted: morphine
KEYWORDS
plasma; SPE
REFERENCE
Bouquillon,A.L; Freeman,D.; Moulin,D.E. Simultaneous solid-phase extraction and chromatographic analysis
of morphine and hydromorphone in plasma by high-performance liquid chromatography with electrochem-
ical detection, J.Chromatogr., 1992, 577, 354-357.
SAMPLE
Sample preparation: Add 1 tablet to 95 mL water, place on a steam bath for 15 min, cool, mix
for 15 min, sonicate, allow to stand, filter, inject 13 (xL aliquot
HPLC VARIABLES
Mobile phase: MeOH:buffer 25:75 (Buffer was 0.01 N KH2PO4 + 50 mM KNO3, adjusted to pH
4.5 with 3 N phosphoric acid.)
Flow rate: 1.1
Detector: UV 283
CHROMATOGRAM
Retention time: 5.2
OTHER SUBSTANCES
Simultaneous: hydrocodone, p-aminophenol, acetaminophen, codeine, p-chloroacetanilide
KEYWORDS
tablets
REFERENCE
Wallo,W.E.; D'Adamo,A. Simultaneous assay of hydrocodone bitartrate and acetaminophen in a tablet formu-
lation, J.Pharm.Scl, 1982, 71, 1115-1118.
SAMPLE
Sample preparation: Dilute 1:5, inject a 20 |xL aliquot.
HPLCVARIABLES
Mobile phase: MeCN:20 mM KH2PO4 50:50, pH adjusted to 5.40 with 1 M NaOH
Flow rate: 1.5
Detector: UV 216
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
Simultaneous: morphine, ondansetron
KEYWORDS
REFERENCE
Trissel,L.A.; Xu,Q.; Martinez, J.F.; Fox,J.L. Compatibility and stability of ondansetron hydrochloride with mor-
phine sulfate and with hydromorphone hydrochloride in 0.9% sodium chloride injection at 4, 22, and 32
pC, Am.J.Hosp.Pharm., 1994, 51, 2138-2142.
SAMPLE
HPLCVARIABLES
Column: 300 X 3.9 10 (xm ixBondapak phenyl
Mobile phase: MeCN:20 mM KH2PO4 adjusted to pH 6.0 with 1 M KOH 50:50
Flow rate: 1
Detector: UV 235
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: morphine, bupivacaine
KEYWpRDS
saline; injections
REFERENCE
Venkateshwaran,T.G.; Stewart,J.T. HPLC determination of morphine-hydromorphone-bupivacaine and mor-
phine-hydromorphone-tetracaine mixtures in 0.9% sodium chloride injection, J.Liq.Chromatogr., 1995,18,
565-578.
SAMPLE
HPLCVARIABLES
Column: 220 X 4.6 5 |xm Brownlee silica (Applied Biosystems)
Mobile phase: MeOH:10 mM KH2PO4 adjusted to pH 4.0 with 10% phosphoric acid 25:75
Flow rate: 1
Detector: UV 235
CHROMATOGRAM
Retention time: 8.7
OTHER SUBSTANCES
Simultaneous: tetracaine, morphine
KEYWpRDS
saline; injections
REFERENCE
565-578.
SAMPLE
HPLCVARIABLES
Flow rate: 1.25
Detector: UV 290
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
304.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 mg/mL solution in 0.9% sodium chloride, dilute 1:100 with
HPLC VARIABLES
Mobile phase: MeOH.buffer 15:85 adjusted to pH 3.5 with o-phosphoric acid (Buffer was 15 mM
sodium dihydrogen phosphate containing 3 mM 1-heptanesulfonic acid.)
Flow rate: 0.8
Detector: UV 230
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
KEYWORDS
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Gradient. A was MeCN containing 1 mg/mL heptanesulfonic acid. B was 50 mM
pH 2.2 phosphoric acid containing 1 mg/mL heptanesulfonic acid. A:B 12.5:87.5 for 2.5 min, to
48.5:51.5 over 13.5 min, maintain at 48.5:51.5 for 4 min
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
Simultaneous: dexamethasone, diphenhydramine, creatinine, methyl paraben, propyl paraben,
degradation products
KEYWORDS
stability-indicating; buffer
REFERENCE
Walker,S.E.; DeAngelis,C; Iazzetta,J.; Eppel,J.G. Compatibility of dexamethasone sodium phosphate with hy-
dromorphone hydrochloride or diphenhydramine hydrochloride, Am. J.Hosp.Pharm., 1991, 48, 2161-2166.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
glunixin, glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide,
hydrocodone, hydroxyquinoline, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin,
isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, ke-
toprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol,
mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepa-
crine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mes-
caline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, metha-
zolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa,
methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, me-
toprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, na-
proxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepi-
nephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone,
faethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanil amide, sulfapyridine, sul-
REFERENCE
Hill,D.W.; KindÂ..J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnaLToxicoL, 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
azine, nurazepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin,
haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydroxychloroquine,
KEYWORDS
REFERENCE
J.Chromatogr.A, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Sample preparation: 500 |xL Urine + N-ethylnordiazepam + chlorpheniramine + 100 |JLL buffer,
centrifuge at 11000 g for 30 s, inject a 500 JJLL aliquot onto column A with mobile phase A, after
JJLL mobile phase B, with 200 |xL mobile phase C, and with 1.15 mL mobile phase D. Elute
HPLCVARIABLES
3.2 11 |xm Aminex A-28 (Bio-Rad); C 25 X 3.2 5 jxm C8 (Phenomenex) + 150 X 4.6 5 |xm silica
(Macherey-Nagel)
phoric acid; C MeCNibuffer 40:60 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylam-
D MeCNrbuffer 33:67 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylammonium
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: caffeine, cotinine, benzoylecgonine, secobarbital, oxazepam, phenobarbital, nordiaze-
pam, diazepam, phenylpropanolamine, phentermine, amphetamine, phenmetrazine, lidocaine,
ephedrine, pentazocine, methamphetamine, desipramine, nortriptyline, diphenhydramine,
methadone, imipramine, flurazepam, amitriptyline, morphine, codeine, hydrocodone
KEYWORDS
column-switching
REFERENCE
341.
Hydroquinone
Merck Index: 4853
SAMPLE
Matrix: air
Sample preparation: Condition a Sep Pak silica SPE cartridge with 10 mL dichloromethane
and dry with helium at 5 L/min. Pull air through a 0.80 fjum cellulose ester membrane filter
and the SPE cartridge at 2 L/min for 1 h, desorb the filter with 5 mL 1% acetic acid with
sonication for 10 min, elute the SPE cartridge with 5 mL 1% acetic acid, inject aliquots of the
eluates.
HPLC VARIABLES
Mobile phase: Gradient. A was 1% acetic acid. B was MeCN:acetic acid 99:1. A:B from 0:100 to
90:10 over 10.5 min, to 78:22 to 24.5 min, to 0:100 (step gradient), maintain at 0:100 for 5 min,
re-equilibrate for 12 min.
Flow rate: 2
Detector: F ex 304 em 338 for 6.3 min, ex 280 em 325 for 7.7 min, ex 257 em 330 for 5.3 min,
ex 342 em 464 for 4.7 min, ex 285 em 310 for 11 min
CHROMATOGRAM
Retention time: 5
Limit of detection: 0.16 jxg/cu.m.
OTHER SUBSTANCES
Simultaneous: catechol, cresol, 3-methylcatechol, phenol, scopoletin
KEYWORDS
SPE
REFERENCE
Risner,C.H. The quantification of hydroquinone, catechol, phenol, 3-methylcatechol, scopoletin, m+p-cresol and
o-cresol in indoor air samples by high-performance liquid chromatography, J.Liq.Chromatogr., 1993, 16,
4117-4140.
SAMPLE
Sample preparation: Emulsion. 500 JJLL Emulsion + 10 mL 400 jig/mL hydroquinone in MeOH
+ 40 mL 0.1% Tween 80, shake until homogeneous, inject a 10 jxL aliquot. Drug release me-
dium. 1 mL Drug release medium + 200 |xL 100 |xg/mL hydroquinone, mix, inject a 50 |xL
aliquot.
HPLCVARIABLES
Column: 250 X 4.6 10 |xm Cosmosil 10 C18 (Nacalai Tesque)
Mobile phase: Gradient. MeCNrIO mM pH 3.0 phosphate buffer 2:98 for 1 min, to 45:55 over
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
Retention time: 4.2
Internal standard: hydroquinone
OTHER SUBSTANCES
Simultaneous: carboplatin, epirubicin, iomeprol, mitomycin C
KEYWORDS
emulsions; drug release medium; injections; hydroquinone is IS
REFERENCE
Yamazoe,K; Horiuchi,T.; Sugiyama,T.; Katagiri,Y. Simultaneous high-performance liquid chromatographic de-
J.ChromatogrA, 1996, 726, 241-245.
SAMPLE
Matrix: solutions
Sample preparation: Aqueous food simulants. Pipette 1.0 mL 200 mg/L IS in MeOH into a 25
mL volumetric flask and dilute to the mark with the food simulant obtained from migration
experiment, shake. Repeat the procedure to obtain a duplicate sample, filter a portion through
a 200 nm membrane filter, inject a 20 |xL aliquot. Olive oil simulants. Weigh 25 g olive oil food
simulant obtained from migration experiment into a beaker, pour oil into a separating funnel,
allow beaker to drain for 30 s. Rinse it with 25 mL hexane and add washes to separating
funnel. Add 1.0 mL 200 mg/L IS in MeOH into funnel and mix. Add 10 mL water, shake
vigorously by hand for 30 s, allow to stand for 5 min. Collect aqueous phase and reextract oil
with a 10 mL water. Combine aqueous extracts, make up to 25 with water, filter the extracts
through a small cotton plug to remove any entrained oil. Repeat the procedure to obtain a
duplicate sample. Inject a 20 |xL aliquot. (Aqueous food simulants were: distilled water, 3%
acetic acid in water; EtOH.water 15:85.)
HPLC VARIABLES
Column: 250 X 4.6 5 |mm Hypersil ODS
Mobile phase: MeCNibuffer 15:85 (Prepare mobile phase as follows. Dissolve 7.5 g sodium di-
hydrogen orthophosphate in 800 mL water, add 150 mL MeCN and adjust to pH 3.6 with glacial
acetic acid. Make up to 1000 mL with water.)
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 4.2
Internal standard: 2-methyl-l,3-dihydroxybenzene (7.4)
Limit of detection: 300-500 ng/g
OTHER SUBSTANCES
Extracted: pyrocatechol, resorcinol
KEYWORDS
aqueous food simulants; olive oil simulants
REFERENCE
Philo,M.R.; Jickells,S.M.; Castle,L. Testing for compliance with migration limits: Determination of 1, 2-, 1,3-,
and 1,4-dihydroxybenzenes in food-simulating solvents by liquid chromatography, J.AOAC Int., 1996, 79,
746-750.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 (xm LiChrosorb RP 18
Mobile phase: MeOH:10 mM pH 5.5 potassium phosphate buffer 3.5:96.5
Flow rate: 2-3
Detector: UV 254
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Simultaneous: catechol, phenol, phenyl glucuronide, phenyl glucoside, phenyl galactopyrano-
side, phenyl sulfate, resorcinol
REFERENCE
Beyer,J.; Frank,G. Hydroxylation and conjugation of phenol by the frog Rana temporaria, Xenobiotica, 1985,
15, 277-280.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: 50 mM pH 3.0 sodium phosphate buffer
Flow rate: 1
Detector: E, ESA, Model 5020 porous graphite analytical cell, Tl 0.60 V, T2 0.82 V (monitored),
guard cell 0.85 V (before injector)
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Simultaneous: oxidized glutathione
REFERENCE
O'Gara,C.Y.; Maddipati,K.R.; Marnett,L.J. A sensitive electrochemical method for quantitative hydroperoxide
determination, Chem.Res.ToxicoL, 1989, 2, 295-300.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 JJIL aliquot of a solution in 1% acetic acid.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere-ODS C18
Mobile phase: Gradient. A was MeCN:acetic acid 99:1. B was 1% acetic acid in water. A:B from
0:100 to 10:90 over 10 min, to 20:80 over 25 min, wash with A for 6 min, re-equilibrate for 14
min.
Flow rate: 2
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Simultaneous: catechol (F ex 280 em 325), phenol (ex 274 em 298), resorcinol (F ex 284 em 313)
REFERENCE
Risner,C.H.; Cash,S.L. A high-performance liquid chromatographic determination of major phenolic compounds
in tobacco smoke, J.Chromatogr.ScL, 1990, 28, 239-244.
SAMPLE
Matrix: urine
Sample preparation: Condition a 500 mg Bond Elut SAX SPE cartridge with 3 mL MeOH and
3 mL water. Dilute 125 |xL urine to 4 mL with water, adjust to pH 4.5 with ascorbic acid, add
12.5 |xL enzyme solution, heat at 37 for 48 h, add to the SPE cartridge, wash with 3 mL 5
mM pH 7 phosphate buffer. Acidify the eluate to pH <3 with concentrated HCl, add 5 mL ether,
vortex, repeat the extraction twice. Combine the organic layers and evaporate them to dryness
under reduced pressure at 30, reconstitute the residue in 1 mL 1% aqueous phosphoric acid,
inject a 20 jxL aliquot. (The enzyme solutions used to deconjugate glucuronides and sulfate
esters were p-glucuronidase/arylsulfatase (Merck, 4114), arylsulfatase (Sigma, S 1629), and p-
glucuronidase diluted 1:6 with water (Boehringer, 127051).)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Nucleosil ODS
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 3.2
OTHER SUBSTANCES
Extracted: catechol, phenol
KEYWORDS
mouse; SPE
REFERENCE
Schad,H.; Schafer,R; Weber,L.; Seidel,H.J. Determination of benzene metabolites in urine of mice by solid-
phase extraction and high-performance liquid chromatography, J.Chromatogr., 1992, 593, 147151.
Next Page
SAMPLE
Matrix: urine
Sample preparation: Filter (0.2 |xm), inject an aliquot directly. Hydrolyze conjugates by heating
with 6 M HCl at 37 for 18 h, inject an aliquot.
HPLC VARIABLES
Column: 150 X 3.9 5 jim Resolve C18 (Waters)
Mobile phase: MeOH: 1.5% trifluoroacetic acid on water 10:90
Flow rate: 0.5
Detector: UV or radioactivity
CHROMATOGRAM
Retention time: 1.8
OTHER SUBSTANCES
Extracted: phenol, phenyl glucuronide, phenyl sulfate
KEYWORDS
rat
REFERENCE
Hughes,M.R; Hall,L.L. Disposition of phenol in rat after oral, dermal, intravenous, and intratracheal admin-
istration, Xenobiotica, 1995, 25, 873-883.
Hydroxychloroquine
Molecular formula: C18H26CIN3O
Merck Index: 4863
Lednicer No.: 1 342
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 500 |JLL 5 M NaOH + 100 |xL 10 (xg/mL chloroquine in
MeOH + 5 mL hexane:diethyl ether 50:50, vortex for 1 min, centrifuge at 1000 g for 10 min,
freeze in dry ice/acetone, remove the organic layer. Thaw out the aqueous layer and repeat the
extraction. Combine the organic layers and evaporate them to dryness under vacuum, recon-
stitute the residue in 100 |xL mobile phase, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 5 |xm cyano (Regis)
Column: 75 X 4.6 3 |xm Ultremex cyano (Phenomenex)
Mobile phase: 20 mM Dimethyloctylamine phosphate:60 mM ammonium acetate 40:60, pH ad-
justed to 4.5 (Dimethyloctylamine phosphate was prepared by adding phosphoric acid to N,N-
dimethyloctylamine to precipitate the salt.)
Flow rate: 0.6
Detector: UV 320
CHROMATOGRAM
Retention time: 15
Internal standard: chloroquine (23)
OTHER SUBSTANCES
Previous Page
SAMPLE
Matrix: urine
Sample preparation: Filter (0.2 |xm), inject an aliquot directly. Hydrolyze conjugates by heating
with 6 M HCl at 37 for 18 h, inject an aliquot.
HPLC VARIABLES
Column: 150 X 3.9 5 jim Resolve C18 (Waters)
Mobile phase: MeOH: 1.5% trifluoroacetic acid on water 10:90
Flow rate: 0.5
CHROMATOGRAM
Retention time: 1.8
OTHER SUBSTANCES
Extracted: phenol, phenyl glucuronide, phenyl sulfate
KEYWORDS
rat
REFERENCE
Hughes,M.R; Hall,L.L. Disposition of phenol in rat after oral, dermal, intravenous, and intratracheal admin-
istration, Xenobiotica, 1995, 25, 873-883.
Hydroxychloroquine
Merck Index: 4863
Lednicer No.: 1 342
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 500 |JLL 5 M NaOH + 100 |xL 10 (xg/mL chloroquine in
MeOH + 5 mL hexane:diethyl ether 50:50, vortex for 1 min, centrifuge at 1000 g for 10 min,
freeze in dry ice/acetone, remove the organic layer. Thaw out the aqueous layer and repeat the
extraction. Combine the organic layers and evaporate them to dryness under vacuum, recon-
stitute the residue in 100 |xL mobile phase, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 5 |xm cyano (Regis)
Column: 75 X 4.6 3 |xm Ultremex cyano (Phenomenex)
Mobile phase: 20 mM Dimethyloctylamine phosphate:60 mM ammonium acetate 40:60, pH ad-
justed to 4.5 (Dimethyloctylamine phosphate was prepared by adding phosphoric acid to N,N-
dimethyloctylamine to precipitate the salt.)
Flow rate: 0.6
Detector: UV 320
CHROMATOGRAM
Retention time: 15
Internal standard: chloroquine (23)
OTHER SUBSTANCES
KEYWORDS
plasma; rabbit; pharmacokinetics
REFERENCE
Iredale,J.; Wainer,I.W. Determination of hydroxychloroquine and its major metabolites in plasma using se-
quential achiral-chiral high-performance liquid chromatography, J.Chromatogr., 1992, 573, 253-258.
SAMPLE
Matrix: blood
Sample preparation: Serum. 200 |xL Serum + 50 |JLL 1 |xg/mL IS in water + 50 |xL 4 M NaOH
+ 200 |xL MTBE, vortex for 30 s, centrifuge at 9950 g for 2 min, inject 100 |xL of the organic
layer. Whole blood. 100 |xL Whole blood + 500 |xL water + 50 jxL 1 |xg/mL IS in water + 50 JAL
4 M NaOH + 200 |JLL MTBE, vortex for 30 s, centrifuge at 9950 g for 2 min, inject 100 JJLL of
the organic layer. Dried blood. Spread 100 (JLL whole blood on a 70 X 30 mm piece of filter
paper, allow to dry, cut paper into 10 X 5 mm strips, add 100 (xL 1 |xg/mL IS in water, add 1.5
mL 0.5 M NaOH, vortex for 30 s, let stand for 30 min at room temperature, add 300 |JLL MTBE,
vortex for 30 s, centrifuge at 2000 g for 5 min, inject a 100 |xL aliquot of the organic layer.
HPLC VARIABLES
Column: 150 X 5 5 |xm Spherisorb S5SCX sulfophenylpropyl-modified silica
Mobile phase: MeOH:water 98.5:1.5 containing 9.41 g/L ammonium perchlorate, adjust appar-
ent pH to 8.0 with 220 mL/L 50 mM NaOH in MeOH
Flow rate: 1.5
Detector: F ex 215 em no filter
CHROMATOGRAM
Retention time: 8
Internal standard: 6,8-dichloro-4-(l-methyl-4-diethylaminobutylamino)quinoline (5)
Limit of quantitation: 5 ng/mL (serum), 10 ng/mL (whole blood, dried blood)
OTHER SUBSTANCES
Extracted: chloroquine, quinine, metabolites
Simultaneous: acebutolol, N-acetylprocainamide, atenolol, butriptyline, chlorpromazine, desi-
pramine, flecainide, fluoxetine, imipramine, labetalol, maprotiline, mepacrine, metoprolol, mex-
iletine, norbutriptyline, normaprotiline, procainamide, propranolol, sotalol
Noninterfering: amitriptyline, amodiaquin, carbamazepine, clomipramine, dapsone, diazepam,
dothiepin, doxepin, fluvoxamine, lorazepam, mefloquine, nitrazepam, norclomipramine, nordi-
azepam, nordothiepin, nordoxepin, nortriptyline, primaquine, proguanil, pyrimethamine
KEYWORDS
serum; whole blood; dried blood
REFERENCE
Croes,K; McCarthy,P.T.; Flanagan,R.J. Simple and rapid HPLC of quinine, hydroxychloroquine, chloroquine,
and desethylchloroquine in serum, whole blood, and filter paper-adsorbed dry blood, J.Anal.Toxicol., 1994,
18, 255-260.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Whole blood + 2 mL 20 ng/mL propranolol hydrochloride in water,
vortex for 10 s, sonicate for 10 min, centrifuge at 3000 rpm for 20 min. Remove 2 mL of the
supernatant and add it to 1 mL 100 mM NaOH and 8 mL ethyl acetaterisopropanol 90:10,
vortex for 30 s, centrifuge at 3000 rpm for 15 min. Remove the organic layer and evaporate it
to dryness under a stream of air at 35-40, reconstitute the residue in 250 (JLL MeOH, vortex
for 30 s, inject a 10-100 |xL aliquot.
HPLCVARIABLES
Guard column: 10 X 3 7 |xm cyano (Applied Biosystems)
Column: 250 X 4.6 5 jutm cyanopropyl (Baxter/Burdick & Jackson)
Mobile phase: MeCN:MeOH:buffer 50:30:20 (Buffer was 25 mM K2HPO4 adjusted to pH 6.0 with
phosphoric acid.)
Flow rate: 2
Detector: F ex 230 em 385 (370 nm cut-off filter)
CHROMATOGRAM
Internal standard: propranolol (4)
OTHER SUBSTANCES
KEYWORDS
whole blood
REFERENCE
Wei,Y.; Nygard,G.A.; Khalil,S.K.W. A HPLC method for the separation and quantification of the enantiomers
of hydroxychloroquine and its three major metabolites, J.Liq.Chromatogr., 1994, 17, 34793490.
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 222
CHROMATOGRAM
KEYWORDS
REFERENCE
TracquijA-; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL water containing 5 |xg/mL 2,3-diaminonaphthalene
orate them to dryness under a stream of nitrogen at 30-40, reconstitute the residue in 70 JJLL
MeOH: 100 mM perchloric acid 50:50, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 4 jjum Nova-Pak C18
Mobile phase: Gradient. A was 58 mM NaH2PO4 containing 6 mM sodium heptanesulfonate, ad-
justed to pH 3.1 with concentrated phosphoric acid. B was MeCN:MeOH 85:15. A:B from 100:0
to 78:22 over 5 min, to 70:30 over 12 min, maintain at 70:30 for 4 min, to 65:35 over 9 min.
Flow rate: 1
Detector: UV 245, 256, 343
CHROMATOGRAM
Limit of detection: 2 ng/mL (343 nm)
OTHER SUBSTANCES
Extracted: betamethasone, chloroquine, corticosterone, cortisone, dexamethasone, fluocinolone
acetonide, fluendrenolide, fluorometholone, fluprednisolone, hydrocortisone, 178-hydroxypro-
gesterone, meprednisone, methylprednisolone, methylprednisolone acetate, paramethasone,
prednisolone, prednisone, progesterone, triamcinolone
KEYWORDS
serum
REFERENCE
J.Chromatogr.B, 1995, 666, 347-353.
SAMPLE
Matrix: blood, erythrocytes, urine
mL buffer. Hemolyze erythrocytes in water 1:3. Dilute urine with water 1:99. Add 1 mL plasma,
hemolyzed erythrocytes, or diluted urine to the SPE cartridge, wash with 4 mL buffer, wash
with 2 mL MeOH:buffer 50:50, elute with 3 mL MeOH:ammonia 99:1. Evaporate the eluate to
dryness under a stream of nitrogen at 30, reconstitute the residue in the initial mobile phase,
vortex, inject a 50 jxL aliquot. (Prepare buffer by mixing equal volumes of 100 mM ammonium
formate and 100 mM ammonia solution, pH 9.2.)
HPLC VARIABLES
Guard column: 10 X 4 Inertsil
Column: 250 X 4 5 |xm Inertsil
Mobile phase: Gradient. A was MeCN. B was MeOH:25% ammonia solution 92.5:7.5. A:B 78:22
for 3 min, then to 65:35 over 2 min (Waters curve no. 3), maintain at 65:35 for 20 min, return
to 78:22 over 5 min (Waters curve no. 3).
Flow rate: 0.85
CHROMATOGRAM
Internal standard: hydroxychloroquine
OTHER SUBSTANCES
Extracted: chloroquine, quinine, monodesethylchloroquine, bidesethylchloroquine
Simultaneous: halofantrine, quinidine
Noninterfering: proguanil, cycloguanil, 4-chlorophenylbiguanide, amodiaquine, mefloquine,
pyrimethamine, sulfadoxine, cinchonine, cinchonidine
KEYWORDS
SPE; hydroxychloroquine is IS; plasma
REFERENCE
Chaulet,J.-R; Robet,Y.; Prevosto,J.-M.; Soares,O.; Brazier,J.-L. Simultaneous determination of chloroquine and
quinine in human biological fluids by high-performance liquid chromatography, J.Chromatogr., 1993, 613,
303-310.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in mobile phase, inject an aliquot.
HPLCVARIABLES
Guard column: Chiral-AGP
Column: 150 x 4.6 Chiral-AGP (Regis)
Mobile phase: MeCN:EtOH:30 mM pH 7.0 sodium phosphate buffer 1:20:79 containing 5 mM
N,N-dimethyloctylamine
Flow rate: 0.9
Detector: UV 320
CHROMATOGRAM
KEYWORDS
chiral
REFERENCE
Iredale,J.; WainerJ.W. Determination of hydroxychloroquine and its major metabolites in plasma using se-
quential achiral-chiral high-performance liquid chromatography, J.Chromatogr., 1992, 573, 253-258.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in mobile phase, inject a 10-100 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4 5 ^m Chiral-AGP (ChromTech)
Mobile phase: MeCN:isopropanol:buffer 1:5:94 (Buffer was 50 mM (NH4)H2PO4 containing 5 mM
dihexylamine, pH adjusted to 7.0 with 3 M NaOH.)
Flow rate: 1
CHROMATOGRAM
Retention time: 10 (S(+)), 13 (R(-))
KEYWORDS
chiral
REFERENCE
Wei,Y.; Nygard,G.A.; Khalil,S.K.W. A HPLC method for the separation and quantification of the enantiomers
of hydroxychloroquine and its three major metabolites, J.Liq.Chromatogr., 1994, 17, 3479-3490.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 5 Spherisorb S5SCX
Mobile phase: MeOH:water 98.5:1.5 containing 80 mM ammonium perchlorate, adjusted to pH
8.0 with 50 mM NaOH in MeOH
Flow rate: 1.5
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Simultaneous: hydroquinine, quinine, chloroquine
REFERENCE
Croes,K; McCarthy,P.T.; Flanagan,R.J. HPLC of basic drugs and quaternary ammonium compounds on micro-
particulate strong cation-exchange materials using methanolic or aqueous methanol eluents containing an
ionic modifier, J.ChromatogrA, 1995, 693, 289-306.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
haloperidol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hy-
epinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pemoline, penta-
zocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital, phenol, phe-
lactone, sulfinpâzone, sulindac, temazepam, terbutaline, terfenadine, tetracaine, theophyl-
KEYWORDS
REFERENCE
J.Chromatogr.A, 1995, 692, 103-119.
Hydroxyprogesterone
Merck Index: 4886
Lednicer No.: 1 176
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL 2 |xg/mL dexamethasone in EtOHiwater 10:90 +
100 |xL 250 mM NaOH + 7 mL etherdichloromethane 60:40, vortex for 30 s, centrifuge at 2000
rpm for 5 min. Remove 6 mL of the organic layer and evaporate it to dryness under a stream
of air at 40, reconstitute the residue in 100 jxL dichloromethane:EtOH:water 95:4:1, inject a
50 |L aliquot.
HPLC VARIABLES
Column: 250 X 4.5 5 |xm Partisil silica
Mobile phase: Dichloromethane:EtOH:water 95:4:1
Flow rate: 1.5
Detector: UV 239
CHROMATOGRAM
Retention time: 2.5 (17-hydroxyprogesterone)
Internal standard: dexamethasone (11.5)
OTHER SUBSTANCES
Extracted: corticosterone, 11-deoxycortisol, hydrocortisone, 6a-methylprednisolone, predniso-
lone, prednisone, progesterone
KEYWORDS
plasma; normal phase
REFERENCE
Scott,N.R.; Chakraborty,J.; Marks,V. Determination of prednisolone, prednisone, and cortisol in human plasma
by high-performance liquid chromatography, Anal.Biochem., 1980, 108, 266268.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond-Elut C18 SPE cartridge with MeOH and water. Add 500
JJLL plasma to the SPE cartridge, wash with 2 mL water, wash with 2 mL MeOH:water 20:80,
elute with two 500 |xL aliquots of MeOH. Evaporate the eluate to dryness under a stream of
nitrogen, reconstitute the residue in 50 JJLL MeOH:water 20:80, inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 100 X 1 5 |xm Hypersil ODS
Flow rate: 0.1
CHROMATOGRAM
Retention time: 8 (17a-hydroxyprogesterone)
OTHER SUBSTANCES
Extracted: androstenedione, 20a-hydroxy-4-pregnen-3-one, norethindrone, progesterone,
testosterone
KEYWORDS
microbore; rat; plasma; SPE
REFERENCE
Taylor,R.B.; Kendle,K.E.; Reid,R.G.; Hung,C.T. Chromatography of progesterone and its major metabolites in
rat plasma using microbore high-performance liquid chromatography columns with conventional injection
and detection systems, J.Chromatogr., 1987, 385, 383-392.
SAMPLE
Matrix: blood
Sample preparation: 100 jxL Serum + 500 jxL water + 100 jxL 10 jxg/mL 3,7-dimethoxyflavone
in EtOH + 8 mL diethyl ether, shake, centrifuge at 4 at 1000 g for 5 min, freeze in acetone/
dry ice. Remove the organic layer and dry it over anhydrous sodium sulfate, evaporate to
dryness under a stream of nitrogen, reconstitute the residue in 100 |xL MeOH:water 40:60,
inject a 50 JXL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 3 |xm NS-GeI C18
Mobile phase: Gradient. MeOH:water from 40:60 to 55:45, maintain at 55:45 for 24 min, to 80:
20 over 25 min
Flow rate: 1
CHROMATOGRAM
Internal standard: 3,7-dimethoxyflavone (47)
OTHER SUBSTANCES
Extracted: aldosterone, androstenedione, dehydroepiandrosterone, deoxycorticosterone, 11-deox-
ycortisol, estradiol, estrone, hydrocortisone, pregnenolone, progesterone
KEYWORDS
serum
REFERENCE
Ueshiba,H.; Segawa,M.; Hayashi,T.; Miyachi,Y.; Irie,M. Serum profiles of steroid hormones in patients with
Cushing's syndrome determined by a new HPLC/RIA method, Clin.Chem., 1991, 37, 1329-1333.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL water containing 5 |xg/mL 2,3-diaminonaphthalene
MeOH: 100 mM perchloric acid 50:50, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 150 X 3.9 4 jjim Nova-Pak C18
min.
Flow rate: 1
Detector: UV 245, 256, 343
CHROMATOGRAM
Retention time: 29.26 (178-hydroxyprogesterone)
OTHER SUBSTANCES
ide, fluocinolone acetonide, fluorometholone, fluprednisolone, hydrocortisone, hydroxychloro-
quine, meprednisone, methylprednisolone, methylprednisolone acetate, paramethasone, pred-
nisolone, prednisone, progesterone, triamcinolone
KEYWORDS
serum
REFERENCE
J.Chromatogr.B, 1995, 666, 347-353.
SAMPLE
Sample preparation: Make up 1 mL injection to 100 mL with EtOH, remove a 1.2 mL aliquot
and add it to 1 mL 1 mg/mL 17-hydroxyprogesterone in EtOH. Dilute this mixture to 50 mL
with EtOH, inject an aliquot.
HPLCVARIABLES
Column: 300 X 4 fjiBondapak CN
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 7 (hydroxyprogesterone caproate)
Internal standard: 17-hydroxyprogesterone (3)
OTHER SUBSTANCES
Simultaneous: benzyl benzoate
Noninterfering: polyethylene glycol 4000, mystristyl-gamma-picolinium chloride, methylcellu-
lose, thimerosal
KEYWORDS
injections
REFERENCE
Das Gupta,V. Quantitation of hydroxyprogesterone caproate, medroxyprogesterone acetate, and progesterone
by reversed-phase high-pressure liquid chromatography, J.Pharm.ScL, 1982, 71, 294-297.
SAMPLE
Sample preparation: Injections. 1 mL Injection (200 fxg/mL) + 300 mg NaCl + 200 |xL 10%
stitute the residue in 4 mL water, mix an aliquot with an equal volume of 30 jjig/mL 17a-
hydroxyprogesterone in MeOH, inject a 20 jxL aliquot. Tablets. Weigh out amount of powdered
of 30 |xg/mL 17a-hydroxyprogesterone in MeOH, inject a 20 |JLL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 12
Internal standard: 17a-hydroxyprogesterone
OTHER SUBSTANCES
Simultaneous: benzyl alcohol, ergonovine, methylergonovine
KEYWORDS
injections; tablets; 17a-hydroxyprogesterone is IS
REFERENCE
1983, 31, 3988-3993.
SAMPLE
Sample preparation: Grind tablets containing about 1 mg levothyroxine, add 4.5 mL 0.5 mg/
mL hydroxyprogesterone caproate in MeOH, add 20.5 mL 10 mM NaOH in MeOH:water 75:
25, shake intermittently for 5 min, filter, discard first 5 mL filtrate, inject a 25 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:0.1% phosphoric acid in water 35:65
Flow rate: 3
Detector: UV 225
CHROMATOGRAM
Retention time: 8 (hydroxyprogesterone caproate)
Internal standard: hydroxyprogesterone caproate
OTHER SUBSTANCES
Simultaneous: levothyroxine
KEYWORDS
tablets; hydroxyprogesterone caproate is IS
REFERENCE
Das Gupta,V.; Odom,C; Bethea,C; Plattenburg,J. Effect of excipients on the stability of levothyroxine sodium
tablets, J.Clin.Pharm.Ther., 1990, 15, 331-336.
SAMPLE
Matrix: ileostomy effluent
Sample preparation: Dilute ileostomy effluent 1:2 by weight with water. Extract 3 g aliquot
three times with 10 mL dichloromethane by shaking for 1 min and centrifuging at 2000 rpm
for 2 min. Wash combined extracts successively with 2 mL 0.1 M NaOH and 4 mL water by
shaking for 30 s and centrifuging for 1 min then dry the organic layer under air at 40. Take
up the extract in 1 mL MeOH, add 1.1 mL water and apply to C18 Bond Elut SPE cartridge.
Wash with 10 mL water, wash with 5 mL MeOH.water 45:55, elute with 2 mL MeOH. Add 50
fjiL 20 |xg/mL progesterone to the eluate, dry at 40, take up in 100 |xL MeOH, inject 10 |xL
aliquot.
HPLC VARIABLES
Guard column: Bondapak C18/Corasil
Mobile phase: MeOH:50 mM pH 3.0 sodium phosphate buffer 55:45
Flow rate: 3
Detector: UV 254 and 238
CHROMATOGRAM
Retention time: 6.0
Internal standard: progesterone (11.6)
OTHER SUBSTANCES
Extracted: beclomethasone alcohol, beclomethasone 17-monopropionate, beclomethasone
dipropionate
KEYWORDS
SPE; 17-hydroxyprogesterone is IS
REFERENCE
Levine,D.S.; Raisys,V.A.; Ainardi,V. Coating of oral beclomethasone dipropionate capsules with cellulose acetate
phthalate enhances delivery of topically active antiinflammatory drug to the terminal ileum, Gastroenter-
ology, 1987, 92, 1037-1044.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm RP-18 C18 (Brownlee)
Detector: UV 254
KEYWORDS
for 17a-hydroxyprogesterone
REFERENCE
Kane,M.R; Tsuji,K. Radiolytic degradation scheme for 60Co-irradiated corticosteroids, J.Pharm.Scl, 1983, 72,
30-35.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 6 5 |jim Shim-pack CLC-ODS
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 14.6 (17a)
OTHER SUBSTANCES
Simultaneous: cortisone, estriol, cortisol, corticosterone, 11-deoxycortisol, androstenedione,
prednisone acetate, 11-deoxycorticosterone, testosterone, dexamethasone acetate, estradiol, es-
trone, progesterone
REFERENCE
Wei,J.Q.; Wei,J.L.; Zhou,X.T. Optimization of an isocratic reversed phase liquid chromatographic system for
the separation of fourteen steroids using factorial design and computer simulation, Biomed.Chromatogr.,
1990, 4, 3-38.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 25 |xg/mL solution in mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Partisil 10 ODS-I
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: androsterone (UV 210), cortexolone (UV 240), cortisone (UV 240), estradiol (UV
280), estrone (UV 280), ethinyl estradiol (UV 280), ethisterone (UV 240), hydrocortisone (UV
240), lynestrenol (UV 210), medroxyprogesterone acetate (UV 240), medroxyprogesterone (UV
240), methandienone (UV 240), methylandrostenediol (UV 210), methylprednisolone acetate
(UV 240), methylprednisolone (UV 240), methyltestosterone (UV 240), nandrolone (UV 240),
norethisterone (UV 240), prednisolone acetate (UV 240), prednisolone (UV 240), prednisone
(UV 240), pregnenolone (UV 210), progesterone (UV 240), testosterone (UV 240)
REFERENCE
Sadlej-Sosnowska,N. Structure retention relationship for steroid hormones. Functional groups as structural
descriptors, J.Liq.Chromatogr., 1994, 17, 2319-2330.
SAMPLE
Matrix: solutions
Sample preparation: Inject 20 |xL aliquot of a MeOH solution.
HPLC VARIABLES
Column: 250 X 4.6 5 jim Hypersil 5-ODS
Mobile phase: THF:water 23:77
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Retention time: k' 11.86 (lla-hydroxyprogesterone)
Internal standard: methylprednisolone (k' 11.36)
OTHER SUBSTANCES
Simultaneous: metabolites, betamethasone, corticosterone, cortisone, deflazacort, deoxycorticos-
terone, dexamethasone, fludrocortisone, fludrocortisone acetate,fluorocortisone,fluorocortisone
acetate, hydrocortisone, 21-hydroxydeflazacort, methylprednisolone, prednisolone, prednisone,
triamcinolone acetonide, triamcinolone
REFERENCE
Santos-Montes,A.; Gonzalo-Lumbreras,R.; Gasco-Lopez,A.L; Izquierdo-Hornillos,R- Extraction and high-perfor-
mance liquid chromatographic separation of deflazacort and its metabolite 21-hydroxydeflazacort. Appli-
cation to urine samples, J.Chromatogr.B, 1994, 657, 248-253.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Nucleosil phenyl
Mobile phase: Gradient. Carbon dioxide:MeOH from 98:2 to 78:22 over 40 min
Flow rate: 2
Detector: UV
CHROMATOGRAM
Retention time: 9.7
OTHER SUBSTANCES
Simultaneous: testosterone, estradiol, norethisterone, hydrocortisone, estriol, other steroids
KEYWORDS
SFC; 200 bar
REFERENCE
Hanson,M. Aspects of retention behaviour of steroids in packed column supercritical fluid chromatography,
Chromatographia, 1995, 40, 58-68.
SAMPLE
Matrix: tissue
Sample preparation: Extract 70-125 mg tissue four times with 5 mL portions of etherxhloro-
form 80:20. Combine the organic layers and evaporate them to dryness under a stream of
nitrogen, reconstitute the residue in 100 (xL MeOH, inject an aliquot.
HPLC VARIABLES
Column: 80 mm long 10 |xm octadecylsilane radial compression (Radial-Pak) (Waters)
Mobile phase: Gradient. A was MeOH:water 50:50. B was MeOH. A:B form 100:0 to 70:30 over
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 25 (17-hydroxyprogesterone)
OTHER SUBSTANCES
Extracted: androstenedione, deoxycortisol, hydrocortisone, testosterone
Simultaneous: estriol, estradiol, pregnenolone, progesterone, testosterone enanthate, testoster-
one propionate
KEYWORDS
tumor
REFERENCE
KessleiyM.J. Analysis of steroids from normal and tumor tissue by HPLC, Clin.Chim.Acta, 1982, 125, 21-30.
SAMPLE
Matrix: urine
Sample preparation: 3 mL Urine + 1.5 jxg betamethasone + 100 mg K2HPO4 + 500 mg anhy-
drous sodium sulfate + 5 mL diethyl ether, shake mechanically for 10 min, centrifuge at 2500
g for 5 min. Remove the organic layer and evaporate it to dryness under vacuum, reconstitute
the residue in 200 |xL MeOH, filter (0.45 |xm), inject a 15 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 jjim Hypersil ODS
Mobile phase: Gradient. MeCN:water from 4:96 to 30:70 over 10 min, to 45:55 over 5 min, to
50:50 over 3 min
Flow rate: 1
Detector: UV 246
CHROMATOGRAM
Internal standard: betamethasone (12.83)
OTHER SUBSTANCES
Extracted: corticosterone, cortisone, deoxycorticosterone, hydrocortisone, prednisolone, predni-
sone, triamcinolone, triamcinolone acetonide
REFERENCE
Park,S.-J.; Kim,Y.-J.; Pyo,H.-S.; Park,J. Analysis of corticosteroids in urine by HPLC and thermospray LC/MS,
JAnal.ToxicoL, 1990, 14, 102-108.
SAMPLE
Matrix: urine
Sample preparation: 3 mL Urine + 0.25 g NaCl, adjust pH to 9.0 with 0.5 g Na2HPO4, add 4
mL dichloromethane, vortex 1 min, centrifuge at 3700 g for 3 min. Remove organic phase and
dry it over anhydrous sodium sulfate. Evaporate a 3 mL aliquot to dryness under vacuum,
reconstitute residue with 200 JULL 5 |xg/mL IS in MeOH, inject a 20 JULL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Hypersil ODS
Flow rate: 1
Detector: UV 245
CKROMATOGRAM
Retention time: 19
Internal standard: methylprednisolone (9)
OTHER SUBSTANCES
Simultaneous: triamcinolone, triamcinolone acetonide, prednisolone, corticosterone, hydroxy-
progesterone, fluorocortisone acetate, cortisone, hydrocortisone, fluorocortisone, betametha-
sone, dexamethasone, prednisone
KEYWORDS
SPE also discussed
REFERENCE
Santos-Montes,A.; Gonzalo-Lumbreras,R.; Gasco-Lopez,A.L; Izquierdo-Hornillos,R. Solvent and solid-phase ex-
traction of natural and synthetic corticoids in human urine, J.Chromatogr.B, 1994, 652, 83-89.
Hydroxypropyl methylcellulose
Merck Index: 4889
SAMPLE
Sample preparation: Dilute with an equal volume of MeOH.water 40:60, inject an 80 JULL
aliquot.
HPLCVARIABLES
Column: 300 X 7.8 Ultrahydrogel 250 250 A cross-linked methacrylate gel (waters)
Mobile phase: MeOH:buffer 20:80 (Buffer was 95 mM boric acid containing 10 mM KCl, 13 |JLM
sodium borate, and 1.5 mM dextrose.) (At the end of each set of analyses rinse instrument and
column with 0.5% sodium azide. (Caution! Sodium azide is carcinogenic, highly toxic, and can
form explosive heavy metal azides. Do not discharge to the plumbing system!)
Flow rate: 1
Detector: RI
CHROMATOGRAM
OTHERSUBSTANCES
Simultaneous: PEG 400
KEYWORDS
ophthalmic solutions; SEC
REFERENCE
Delker,G.; Chen,C; Miller,R.B. Size exclusion chromatographic determination of hydroxypropyl methyl cellu-
lose and polyethylene glycol 400 in an ophthalmic solution, Chromatographia, 1995, 41, 263-266.
Hydroxyquinoline
Merck Index: 4890
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
hydrocodone, hydrocortisone, ibogaine, ibuprofen, iminostilbene, imipramine, indomethacin, is-
ocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine, keto-
profen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine, mazindol,
mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, megestrol, mepa-
crine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mes-
caline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone, metha-
zolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa,
nephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, ox5nnorphone,
puromycin, pyrilamine, pjnithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.Toxicol, 1994,
18, 233-242.
Hydroxyurea
Molecular formula: CH4N2O2
Merck Index: 4896
SAMPLE
Matrix: blood
Sample preparation: Mix 500 |xL serum, 20 |xL 70% perchloric acid, and 20 |xL 15.5 mM meth-
ylurea, centrifuge at 12000 g for 5 min. Mix 200 |JLL supernatant with 700 |JLL BUN acid reagent
(No. 535-3, Sigma) and 600 |xL BUN color reagent (No. 535-5, Sigma), place the mixture in a
boiling water bath to form colored complexes. After 10 min cool the mixture in ice water, inject
a 25-100 |xL aliquot of the colored solution.
HPLC VARIABLES
Column: 300 X 3.9 Bondclone 10 C18 (Phenomenex)
Flow rate: 1.7
Detector: UV 449
CHROMATOGRAM
Retention time: 6.5
Internal standard: methylurea (12.2)
KEYWORDS
serum; derivatization
REFERENCE
Manouilov,K.K.; McGuire,T.R.; Gwilt,P.R. Colorimetric determination of hydroxyurea in human serum using
high-performance liquid chromatography, J.Chromatogr.B, 1998, 708, 321-324.
SAMPLE
Matrix: blood
Sample preparation: Vortex plasma with two volumes of 5% trichloroacetic acid for 30 s, cen-
trifuge at 7000 g for 5 min, inject a 5 jxL aliquot of the supernatant.
HPLC VARIABLES
Column: 100 X 3.2 3 jxm Phase-II ODS (Bioanalytical Systems)
Mobile phase: 50 mM sodium acetate containing 5 mM tetrabutylammonium hydroxide, ad-
justed to pH 6.75 0.02 with 50 mM acetic acid
Flow rate: 0.5
Injection volume: 5
Detector: E, Bioanalytical Systems LC-4B, glassy carbon electrode + 800 mV, Ag/AgCl reference
electrode
CHROMATOGRAM
Retention time: 1.1 (elute for 5 min to remove plasma components)
Limit of detection: 20 |xM
OTHER SUBSTANCES
Noninterfering: acetaminophen, caffeine, carbamazepine, ethosuximide, morphine, phenobar-
bital, phenytoin, salicylic acid, valproic acid
KEYWORDS
plasma
REFERENCE
Havard,J.; Grygiel,J.; Sampson,D. Determination by high-performance liquid chromatography of hydroxyurea
in human plasma, J.Chromatogr., 1992, 584, 270-274.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Serum + 200 |xL trichloroacetic acid, vortex for 30 s, centrifuge at
5000 g for 5 min, pass the supernatant through a 1 mL Bond Elut C18 SPE cartridge, inject
an aliquot of the eluate.
HPLC VARIABLES
Guard column: 20 mm long Supelguard LC 18 (Supelco)
Column: 150 X 4.6 Supelcosil LC 18
Mobile phase: 50 mM Sodium acetate adjusted to pH 6.75 with acetic acid
Flow rate: 0.8
Detector: E, ESA Coulochem II, Model 5020 guard cell 650 mV, Model 5011 analytical cell 600
mV
CHROMATOGRAM
Limit of quantitation: 1.3 |xM
KEYWORDS
pharmacokinetics; serum; SPE
REFERENCE
Villani,R; Maserati,R.; Regazzi,M.B.; Giacchino,R.; Lori,F. Pharmacokinetics of hydroxyurea in patients in-
fected with human immunodeficiency virus type I, J.Clin.Pharmacol., 1996, 36, 117121.
Hydroxyzine
Molecular formula: C21H27CIN2O2
CAS Registry No.: 68-88-2, 2192-20-3 (di HCI),
10246-75-0 (pamoate)
Merck Index: 4897
Lednicer No.: 1 59, 4 118
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL 1 |xg/mL triprolidine + 250 |xL 10% KOH + 5 mL
ether, vortex, centrifuge. Remove ether layer and add it to 100 |xL 0.5% phosphoric acid, vortex,
centrifuge, remove most of ether layer and discard it, remove traces of ether by nitrogen at
room temperature for 2-3 min, inject all of aqueous layer.
HPLCVARIABLES
Column: Waters CN reverse-phase radial compression
Mobile phase: MeCNibuffer 27:73 (Buffer was 75 mM pH 3.0 phosphate buffer containing 20
mM dibutylamine and 50 ng/mL triprolidine.)
Flow rate: 1
Detector: UV 229
CHROMATOGRAM
Retention time: 6.9
Internal standard: triprolidine (3.6)
KEYWORDS
serum
REFERENCE
Simons,F.E.R.; Simons,K.J.; Frith,E.M. The pharmacokinetics and antihistaminic of the H1 receptor antagonist
hydroxyzine, J.Allerg.Clin.Immunol., 1984, 73, 69-75.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 50 |xL 3 ixg/mL IS + 1 mL 1 M pH 5.0 sodium citrate buffer
+ 3 mL ethyl acetate, vortex 1 min, centrifuge at 4000 rpm for 15 min, remove organic layer,
repeat extraction. Combine organic layers and add 200 |xL 1.7% phosphoric acid, vortex 1 min,
centrifuge 5 min, remove and discard ethyl acetate layer, remove traces of ethyl acetate from
aqueous layer using a stream of nitrogen, inject.
HPLC VARIABLES
Column: radial 4 (xm NovoPak C18 radial compression
Mobile phase: MeCNibuffer 46:54 (Buffer was 10 mM pH 2.9 KH2PO4 + 20 mM sodium 1-
decanesulfonate.)
Flow rate: 1.4
Detector: UV 229
CHROMATOGRAM
Retention time: 3.6
Internal standard: P-265, an ethoxy derivative of cetirizine (6.8)
OTHER SUBSTANCES
Simultaneous: cetirizine
KEYWORDS
serum
REFERENCE
Simons,K.J.; Watson,W.T.A.; Chen,X.Y.; Simons,F.E.R. Pharmacokinetic and pharmacodynamic studies of the
Hi-receptor antagonist hydroxyzine in the elderly, Clin.Pharmacol.Ther., 1989, 45, 9-14.
SAMPLE
Matrix: blood
the residue in 100 JULL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 jxL aliquot of
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 230
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: blood, CSF
Sample preparation: Plasma. Centrifuge blood at 7000 rpm, decant 100 (xL plasma. Mix 100
|xL plasma with 200 |xL acetone, centrifuge at 7000 rpm for 5 min. Evaporate the supernatant
under a stream of nitrogen, reconstitute the residue with mobile phase, inject an aliquot. CSF.
Add 25 JJLL water to 25 |xL CSF, mix with 50 JJLL acetone, centrifuge at 7000 rpm for 5 min.
Evaporate the supernatant under a stream of nitrogen, reconstitute the residue with mobile
HPLC VARIABLES
Column: Regis SPS phenyl
Mobile phase: MeCNrpH 3.0 water 21:79 containing 9 mM decanesulfonic acid
KEYWORDS
REFERENCE
Chou,K.-J.; Donovan,M.D. Distribution of antihistamines into the CSF following intranasal delivery, Bio-
pharm.Drug Dispos., 1997, 18, 335-346.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Prepare solutions in mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 120 X 4.0 5 (xm Lichrosorb RP-18
Mobile phase: MeCN:buffer 30:70 (Buffer was 9.8% triethylamine and 0.35% sodium methane-
sulfonate with pH adjusted to 2.85 with sulfuric acid.)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
OTHER SUBSTANCES
REFERENCE
Simpson,D.; Skellern,G.G.; Miller,J.H.MB. Method for the control of known impurities in hydroxyzine hydro-
chloride, J.Pharm.Biomed.AnaL, 1996,14, 1371-1375.
SAMPLE
Sample preparation: 5 mL Formulation + 5 mL IS solution, make up to 50 mL with MeOH,
inject 10 |xL aliquot. (IS solution was 0.2 mg p-nitroacetophenone and 2.5 mg isobutyrophenone
per mL of MeOH.)
HPLC VARIABLES
Mobile phase: MeCN:water:reagent 25:60:15, pH 2.6 (Reagent was MeOH containing 0.06% sul-
furic acid, 0.5% sodium sulfate, and 0.02% sodium heptanesulfonate.)
Flow rate: 2
Detector: UV 257
CHROMATOGRAM
Retention time: 9
Internal standard: p-nitroacetophenone (6) and isobutyrophenone (13)
OTHER SUBSTANCES
Simultaneous: benzyl alcohol, benzoic acid, benzaldehyde, p-chlorobenzoic acid, p-chlorobenzal-
dehyde, p-chlorobenzophenone
KEYWORDS
REFERENCE
Menon,G.N.; Norris,B.J. Simultaneous determination of hydroxyzine hydrochloride and benzyl alcohol in in-
jection solutions by high-performance liquid chromatography, J.Pharm.Sci., 1981, 70, 697-698.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.2
OTHER SUBSTANCES
operidol, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine,
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 jig/mL, inject a 10
JJLL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 (xm ixBondapak C18
Flow rate: 1.5
Detector: UV 230
CHROMATOGRAM
Retention time: 12
REFERENCE
separation of drugs using triethylamine as a competing base, J.Chromatogr., 1986, 370, 403418.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1 mg/mL solution in MeOH, inject a 5 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 u.m Lichrosphere cyanopropyl
Mobile phase: Carbon dioxide.MeOH.isopropylamine 90:10:0.05
Flow rate: 3
Injection volume: 5
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: benactyzine, buclizine, perphenazine, thioridazine, amitriptyline, desipramine,
imipramine, nortriptyline, protriptyline
KEYWORDS
REFERENCE
Berger,T.A.; Wilson,W.H. Separation of drugs by packed column supercritical fluid chromatography. 2. Anti-
depressants, J.Pharm.ScL, 1994, 83, 287-290.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 |im LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
cainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, fiavoxate, fluoxetine, fluphen-
droxychloroquine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac,
methdilazine, methocarbamol, methotrexate, metnotrimeprazine, methoxamine, methyldopa,
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 100 X 4.6 5 jjum Hypersil C8 MOS 100A coated with phosphatidylcholine (95% pure
phosphatidylcholine in MeOH.water 80:20 for 24 h.)
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
idine, codeine, desipramine, diphenhydramine, dipyridamole, ephedrine, flufenamic acid, hal-
operidol, imipramine, indomethacin, lidocaine, megestrol acetate, metoprolol, nabumetone, na-
KEYWORDS
REFERENCE
methodology, Anal.Chem., 1998, 70, 2092-2099.
Ibafloxacin
Merck Index: 4919
SAMPLE
Matrix: tissue
Sample preparation: Mix 2 g minced tissue with 4 g anhydrous sodium sulfate until homoge-
nized. Add 10 ml ethyl acetate, shake mechanically for 10 min, centrifuge at 1500 g for 10 min.
Transfer organic layer into another tube and repeat extraction on the tissue pellet with 10 mL
ethyl acetate. Evaporate combined organic phases under a stream of nitrogen at 50. Dissolve
residue in 1 mL MeCN:2.7 mM pH 2.5 oxalic acid 50:50, vortex, sonicate for 5 min, filter
through 0.45 |xm filter (GHP Acrodisc GF, Gelman Sciences, USA).. Inject a 100 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 jjim Ultrabase C18 (Shandon, UK)
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 100 X 4.6 5 jjum Hypersil C8 MOS 100A coated with phosphatidylcholine (95% pure
phosphatidylcholine in MeOH.water 80:20 for 24 h.)
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
operidol, imipramine, indomethacin, lidocaine, megestrol acetate, metoprolol, nabumetone, na-
KEYWORDS
REFERENCE
Ibafloxacin
Merck Index: 4919
SAMPLE
Matrix: tissue
Sample preparation: Mix 2 g minced tissue with 4 g anhydrous sodium sulfate until homoge-
nized. Add 10 ml ethyl acetate, shake mechanically for 10 min, centrifuge at 1500 g for 10 min.
Transfer organic layer into another tube and repeat extraction on the tissue pellet with 10 mL
ethyl acetate. Evaporate combined organic phases under a stream of nitrogen at 50. Dissolve
residue in 1 mL MeCN:2.7 mM pH 2.5 oxalic acid 50:50, vortex, sonicate for 5 min, filter
through 0.45 |xm filter (GHP Acrodisc GF, Gelman Sciences, USA).. Inject a 100 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 jjim Ultrabase C18 (Shandon, UK)
Mobile phase: Gradient. A was MeCN. B was 2.7 mM pH 2.5 oxalic acid. A:B from 10:90 to 70:
30 over 20 min, maintain at 70:30 for 5 min, return to initial conditions over 5 min.
Flow rate: 0.8
CHROMATOGRAM
Internal standard: ibafloxacin
OTHER SUBSTANCES
Extracted: flumequine
KEYWORDS
kidney; pig; ibafloxacin is IS
REFERENCE
Guyonnet,J.; Pacaud,M.; Richard,M.; Doisi,A.; Spavone,R; Hellings,P. Routine determination of flumequine in
kidney tissue of pig using automated liquid chromatography, J.Chromatogr.B, 1996, 679, 177-184.
lbogaine
Merck Index: 4920
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jxg/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.7
OTHER SUBSTANCES
operidol, hydroxyzine, hyoscine, imipramine, indapamine, iprindole, isothipendyl, isoxsuprine,
REFERENCE
Jane,L; McKmnon,A.; Flanagan,RJ- High-performance liquid chromatographic analysis of basic drugs on silica
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
camfamine, fenoprofen, fenproporex, fentanyl, fiubendazole, flufenamic acid, flunitrazepam, 5-
hydrocodone, hydrocortisone, hydromorphone, ibuprofen, iminostilbene, imipramine, indometh-
REFERENCE
18, 233-242.
lbopamine
Merck Index: 4921
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Plasma + 500 |JLL pH 7.4 phosphate buffer, mix, add 9 mL dioxane
(Caution! Dioxane is a carcinogen!), mix, centrifuge at high speed for 30 s, inject a 20 JJLL aliquot
of the supernatant.
HPLCVARIABLES
G u a r d column: ixBondapak C18 Guard-Pak
Mobile p h a s e : Dioxane:buffer 35:65 (Caution! Dioxane is a carcinogen!) (Buffer was 100 mM
NaH 2 PO 4 containing 0.11 mM 9,10-dimethoxyanthracene-2-sulfonate and 0.11 mM 1-hepta-
nesulfonic acid, pH adjusted to 3 with phosphoric acid. Purify 9,10-dimethoxyanthracene-2-
sulfonate (Fluka) by Soxhlet extraction with dichloroethane before use.)
Flow r a t e : 0.5
Detector: F ex 380 em 452 following post-column extraction. The column effluent mixed with
dichloroethane pumped at 1 mL/min and the mixture flowed through a 1 m X 0.25 mm i.d.
stainless steel coil to a phase separator (Anal. Chim. Acta 1987, 192, 267) then the organic
phase flowed through the detector.
CHROMATOGRAM
R e t e n t i o n time: k' 3.3
OTHER SUBSTANCES
Also analyzed: dibenzoyl dopamine, dibudop, dipivaloyl dopamine
KEYWORDS
plasma; post-column extraction; post-column reaction
REFERENCE
Haas,M.; Moolenaar,E; Kluppel,A.C.; Dijkstra,D.; Meijer,D.K; De Zeeuw,D. Determination of dopaminergic
prodrugs by high-performance liquid chromatography followed by post-column ion-pair extraction,
J.Chromatogr.B, 1997, 693, 484-488.
SAMPLE
Sample preparation: Plasma. 2 mL Plasma + 10 |xL 10 |xg/mL dihydroxybenzylamine in 200
mM acetic acid + 40 mg alumina (Bioanalytical Systems) + 2 mL tris buffer, vortex, shake on
a wrist action shaker for 15 min. Discard the liquid, add 1 mL water to the alumina, vortex
for 2 min, discard the liquid, repeat the wash. Add 500 |xL 200 mM acetic acid to the alumina,
vortex for 15-20 min, inject a 50 |xL aliquot of the liquid. Urine. Dilute urine 1:20 with water.
Remove a 250 uL aliquot and add it to 10 jxL 20 |xg/mL a-ethyldopamine in 200 mM acetic
acid, add 20 mg alumina (Bioanalytical Systems), add 500 |xL tris buffer, vortex, shake on a
wrist action shaker for 15 min. Discard the liquid, add 1 mL water to the alumina, vortex for
2 min, discard the liquid, repeat the wash. Add 500 (xL 200 mM acetic acid to the alumina,
vortex for 15-20 min, inject a 50 jxL aliquot of the liquid. (To deconjugate mix 650 (xL plasma
or urine, 500 |xL acetate buffer, 50 (xL 40 mg/mL penicillamine in water, and 250 |xL sulfatase,
heat at 37 for 16 h. Remove a 250 |xL aliquot and add 10 |xL 10 jxg/mL dihydroxybenzylamine
in 200 mM acetic acid (plasma) or 20 |xg/mL a-ethyldopamine in 200 mM acetic acid (urine),
add 20 mg alumina, add 500 jxL tris buffer, proceed as above. Tris buffer was 60.5 g tris in
250 mL water, pH adjusted to 8.6 with phosphoric acid. Acetate buffer was 27.2 g sodium
acetate trihydrate and 11.6 mL glacial acetic acid in 1 L water, pH 4.7. To prepare sulfatase
solution first condition a Sep-Pak C18 SPE cartridge with 15 mL water. Prepare a solution of
15000 U sulfatase H-5 (Sigma) in 2 mL water, add to the SPE cartridge, elute with 3 mL water,
collect 5 mL effluent and use this solution.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere C18 IP
Mobile phase: MeCN.buffer 1 3:97 (plasma) or MeCN.buffer 2 18:82 (urine) (Buffer 1 was 28.3
g monochloroacetic acid, 9.35 g NaOH, 1.0 g disodium EDTA, and 50 mg sodium octyl sulfate
in 2 L water, adjust pH to 3.0 with monochloroacetic acid. Buffer 2 was 22.0 g sodium acetate,
21.0 g citric acid, 9.8 g NaOH, 0.67 g disodium EDTA, and 75 mL glacial acetic acid in 2 L
water, adjust pH to 5.0 with 5 M NaOH.)
Flow rate: 1
Detector: E, BAS LC-4B, TL-5 thin-layer glassy-carbon electrode +0.65 V, Ag/AgCl reference
electrode
CHROMATOGRAM
Retention time: 23 (plasma), 13 (urine) (of epinine, the active metabolite)
Internal standard: dihydroxybenzylamine (9), a-ethyldopamine (10)
OTHER SUBSTANCES
Extracted: dihydroxyphenylacetic acid
KEYWORDS
REFERENCE
Gifford,R.; Randolph,W.C; Heineman,F.C; Ziemniak,J.A. Analysis of epinine and its metabolites in man after
oral administration of its pro-drug ibopamine using high-performance liquid chromatography with electro-
chemical detection, J.Chromatogr., 1986, 381, 83-93.
lbufenac
Merck Index: 4924
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 33 X 4.6 3 ^m C18 (Perkin Elmer, Norwalk, CT)
Mobile phase: MeCN:buffer 40:60 (Prepare mobile phase as follows. Dissolve 4 mL concentrated
phosphoric acid in 600 mL water and mix with 400 mL MeCN.)
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: dicloxacillin, ibuprofen, ketoprofen
Noninterfering: acetaminophen, N-acetylprocainamide, amikacin, amitriptyline, aspirin, az-
treonam, barbituric acid, brompheniramine, cafazolin, caffeine, carbamazepine, carbamazepine
epoxide, cephalexin, chlorpheniramine, clonazepam, clotrimazole, desipramine, desmethyldox-
epin, digitoxin, digoxin, disopyramide, doxepin, ethosuximide, felbamate, gentamicin, imipe-
nem, imipramine, lidocaine, maprotiline, mephenytoin, mephobarbital, metharbital, methsux-
imide, methylsuccinimide, nortriptyline, paramethadione, phenacemide, phenobarbital,
phensuximide, phenylpropanolamine, phenytoin, primidone, protriptyline, sulfamethoxazole,
theophylline, tobramycin, trimethadione, trimethoprim, vancomycin.
KEY WORDS
plasma; rapid HPLC; ibufenac is IS
REFERENCE
Rifai,N.; Lafi,M.; Sakamoto,M.; Law,T. Measurement of plasma ketoprofen by a rapid high-performance liquid
chromatography assay, Ther.Drug Monit., 1997, 19, 175-178.
Ibuprofen
CAS Registry No.: 15687-27-1, 58560-75-1 ( mixture), 61054-06-6 (Al salt), 112017-99-9 (piconol)
Merck Index: 4925
Lednicer No.: 1 86; 2 218, 356
SAMPLE
Matrix: blood
Sample preparation: Mix 500 |xL plasma with 50 |xL 200 |xg/mL IS in MeOH. Add 500 |xL 1 M
HCl, extract with 10 mL dichloromethane. Separate the organic layer and evaporate it under
a stream of nitrogen at 30. Reconstitute the residue with 500 |xL mobile phase. Inject a 50 |JLL
aliquot.
HPLC VARIABLES
Column: 150 X 3.9 4 fim C18 Novapak
Mobile phase: MeCN:10 mM pH 6.05 dipotassium hydrogen orthophosphate 25:75
Flow rate: 1.4
Detector: UV 225
CHROMATOGRAM
Retention time: 5.2
Internal standard: (4-n-pentylphenyl) acetic acid (7.9)
KEYWORDS
plasma
REFERENCE
Pargal,A.; Kelkar,M.G.; Nayak,P.J. The effect of food on the bioavailability of ibuprofen and fiurbiprofen from
sustained release formulations, Biopharm.Drug Dispos., 1996, 17, 511-519.
SAMPLE
Matrix: blood
Sample preparation: Inject a 5 |xL aliquot of serum directly.
HPLC VARIABLES
Column: 100 X 4.6 5-10 |xm Silicalite (by sieving Silicalite, 3M Co.(?))
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
KEYWORDS
serum
REFERENCE
89-96.
SAMPLE
Matrix: blood
Sample preparation: Centrifuge plasma or serum at 11300 g for 7 min, inject a 200 |JLL aliquot
onto column A, elute to waste with mobile phase A, after 5 min backflush the contents of column
A onto column B with mobile phase B, after 5 min remove column A from the circuit, elute
column B with mobile phase B, monitor the effluent from column B. (Reequilibrate column A
with mobile phase A for 4 min.)
HPLC VARIABLES
Column: A 20 X 4.0 BioTrap 500 C18 (ChromTech); B 100 x 4.6 5 |xm CT-SiI C18
Mobile phase: A 2-Propanol:30 mM pH 7.0 sodium phosphate buffer containing 10 mM octanoic
acid 2:98; B MeCN:30 mM pH 7.0 sodium phosphate buffer 35:65
CHROMATOGRAM
Retention time: 8.0
KEYWORDS
REFERENCE
Hermansson,J.; Grahn,A.; Hermansson,I. Direct injection of large volumes of plasma/serum on a new biocom-
patible extraction column for the determination of atenolol, propranolol and ibuprofen. Mechanisms for the
improvement of chromatographic performance, J.Chromatogr.A, 1998, 797, 251-263.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 50 |xL 50 |xg/mL fenoprofen in water + 200 |xL 20%
sulfuric acid + 6 mL n-butyl chloride, vortex for 5 min, centrifuge at 950 g for 5 min, freeze in
dry ice/acetone. Remove the organic layer and evaporate it to dryness under a stream of nitro-
gen, reconstitute the residue in 300 |xL 50 mM triethylamine, sonicate for 1 min, vortex for 30
s, add 50 |xL 6 mM ethyl chloroformate, let stand for 30 s, add 25 |xL 10 mM S-(-)-l-(l-naph-
thyl)ethylamine, let stand for 3 min, add 25 jxL MeCN:ethanolamine 40:1. Evaporate to dryness
under a stream of nitrogen at 40, reconstitute the residue in 250 JJLL mobile phase, inject a 25
|xL aliquot.
HPLCVARIABLES
Guard column: 15 X 3.2 7 jxm Newguard RP-18
Column: 150 X 4.6 5 jjum Inertsil ODS-2
Mobile phase: MeCN.water (pH 3.0) 66.5:33.5
Flow rate: 1.2
CHROMATOGRAM
Retention time: 11.3 (S-(+)), 12.3 (R-(-))
Internal standard: fenoprofen (7.7 (S), 8.5 (R))
KEYWORDS
REFERENCE
Lau,Y.Y. Determination of ibuprofen enantiomers in human plasma by derivatization and high-performance
liquid chromatography withfluorescencedetection, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 2143-2153.
SAMPLE
Matrix: blood, synovial fluid, tissue
Sample preparation: Plasma, synovial fluid. Acidify 1 mL plasma or synovial fluid with 100 (JLL
1 M HCl, add 100 (JLL 3 |xg/mL IS. Add 10 mL diethyl ether, stir for 10 min, centrifuge at 6000
rpm for 5 min, remove the ether phase, repeat the extraction twice more. Evaporate the organic
phase at 40. Dissolve the residue in 1 mL MeOH, inject a 50 |xL aliquot. Tissue. Weigh out
about 500 mg frozen tissue, add 5 mL water, 5 mL 100 mM HCL, and 100 jxL 3 fxg/mL IS.
Homogenize the mixture using an Ultraturrax at 13000 rpm, add 30 mL diethyl ether and 2
mL each of Carrez solutions I and II. Vortex, centrifuge at 6000 rpm for 5 min, remove the
ether phase. Repeat the same extraction procedure twice with 30 mL portions of diethyl ether.
Evaporate the combined ether phases at 40. Dissolve the residue in 1 mL MeOH, inject a 50
|xL aliquot.
HPLC VARIABLES
Guard column: 4 X 4 5 |xm LiChroCart LiChrospher 100 RP-18
Column: 125 X 4 5 |xm LiChroCart LiChrospher 100 RP-18
Mobile phase: MeCN:pH 4.8 sodium acetate 38:62
Flow rate: 1.5
Detector: UV 223
CHROMATOGRAM
KEYWORDS
plasma; subcutis; musculature; fasciae; pharmacokinetics
REFERENCE
Dominkus,M.; Nicolakis,M.; Kotz,R.; Wilkinson,F.E.; Kaiser,R.R.; Chlud,K. Comparison of tissue and plasma
levels of ibuprofen after oral and topical administration, Arzneimittelforschung, 1997, 46, 11381143.
SAMPLE
Sample preparation: Filter plasma (0.2 jxm membrane) and inject a 100 |xL aliquot onto column
A, elute with mobile phase A onto column B, after 6 min backflush the contents of column B
onto column C with mobile phase B, elute with mobile phase B, monitor the effluent from
column C.
HPLC VARIABLES
Column: A 150 X 4.6 5 |xm CAPCELL PAK MF (Shiseido, Japan); B 35 X 4.6 5 |xm CAPCELL
PAK C18 (Shiseido, Japan); C 250 X 4 5|i,m CAPCELL PAK C18 (Shiseido, Japan)
Mobile phase: A MeCN:50 mM pH 7.0 phosphate buffer 5:95; B MeCN:50 mM pH 7.0 phosphate
buffer 27:73
Flow rate: 1
Detector: UV 223
CHROMATOGRAM
KEYWORDS
comparison with capillary electrophoresis; column-switching; plasma; rat
REFERENCE
Kang,S.H.; Chang,S.-Y.; Do,K.-C; Chi,S.-C; Chung,D.S. High-performance liquid chromatography with a col-
umn-switching system and capillary electrophoresis for the determination of ibuprofen in plasma,
J.Chromatogr.B, 1998, 712, 153-160.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 urn Symmetry C8 (Waters)
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Serum. Activate an SPE cartridge filled with 100 mg 40-63 |xm silica gel
(Merck) with 1 mL MeOH and dry in a hot air oven at 100 for 1 h, equilibrate with 1 mL
dichloromethane before use. 500 (xL Serum + 10 uX 100 jxg/mL flurbiprofen in MeCN + 100
fxL 1 M HCl, mix, add 1 mL 1 M pH 3.8 sodium phosphate buffer, mix, add 3 mL diethyl ether,
rock for 20 min, centrifuge at 1000 g for 2 min. Remove the organic layer and evaporate it to
dryness under a stream of nitrogen at 40, reconstitute the residue in 100 jxL 1 mg/mL l-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in dichloromethane, add 100 JJLL 1
mg/mL 1-hydroxybenzotriazole in dichloromethane, add 100 |xL 1 mg/mL (R)-(+)-l-(l-naph-
thyDethylamine in dichloromethane, vortex briefly, let stand at room temperature for 2 h, add
to the SPE cartridge, elute with two 1 mL portions of dichloromethane:MeCN 90:10. Combine
all the eluate and evaporate it to dryness under a stream of nitrogen at 40, reconstitute the
residue in 100 |xL mobile phase, inject a 50 u,L aliquot. Urine. Activate an SPE cartridge filled
with 100 mg 40-63 |xm silica gel (Merck) with 1 mL MeOH and dry in a hot air oven at 100
for 1 h, equilibrate with 1 mL dichloromethane before use. 500 u,L Urine + 10 (xL 100 jxg/mL
flurbiprofen in MeCN + 100 (JLL 1 M HCl, mix, add 1.5 m L l M p H 3.8 sodium phosphate buffer,
mix, add 5 mL hexane:isopropanol 90:10, rock for 20 min, centrifuge at 1000 g for 5 min.
constitute the residue in 100 (xL 1 mg/mL l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride in dichloromethane, add 100 |xL 1 mg/mL 1-hydroxybenzotriazole in dichlorome-
thane, add 100 |xL 1 mg/mL (R)-(+)-l-(l-naphthyl)ethylamine in dichloromethane, vortex
briefly, let stand at room temperature for 2 h, add to the SPE cartridge, elute with two 1 mL
portions of dichloromethane:MeCN 90:10. Combine all the eluate and evaporate it to dryness
under a stream of nitrogen at 40, reconstitute the residue in 100 u,L mobile phase, inject a 50
|JLL aliquot.
HPLCVARIABLES
Guard column: 10 X 2.1 40-63 |xm pellicular C18 (Alltech)
Flow rate: 1.5
CHROMATOGRAM
Internal standard: flurbiprofen (quantitation uses the peak for the (S)-enantiomer) (14.5 (R),
17.8 (S))
KEYWORDS
derivatization; chiral; serum; SPE
REFERENCE
Tan,S.C; Jackson,S.H.D.; Swift,C.G.; Hutt,A.J. Enantiospecific analysis of ibuprofen by high performance liquid
chromatography: Determination of free and total drug enantiomer concentrations in serum and urine, Chro-
matographia, 1997, 46, 23-32.
SAMPLE
the supernatant and add it to 200 JJLL 1 jig/mL IS in DMF, mix, add 200 |xL 5 M HCl, extract
under a stream of nitrogen, reconstitute the residue in 1 mL dichloromethane, add 20 |JLL 10
mg/mL 1-hydroxybenzotriazole in dichloromethane :pyridine 99:1, add 300 |xL 10 mg/mL l-(3-
ness, reconstitute with 500 (xL mobile phase, inject a 10 [xL aliquot.
HPLC VARIABLES
Column: 250 X 5 5 jim Techsphere ODS (HPLC Technology, Macclesfield UK)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: (S)-naproxen (k' 3.05)
KEY WpRDS
REFERENCE
1997, 15, 1765-1774.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN: water-.glacial acetic acid 40:60:0.1
Flow rate: 2.5
Detector: UV 232
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: naproxen
KEYWORDS
plasma
REFERENCE
Niazi,S.K; Alam,S.M.; Ahmad,S.I. Partial-area method in bioequivalence assessment: Naproxen, Bio-
pharm.Drug Dispos., 1997, 18, 103-116.
SAMPLE
Matrix: solutions
Sample preparation: Mix 50 u,L of a 0.001-5 mM solution in MeCN with 50 |xL 1 mM DNS-
APy in MeCN containing 50 mM 2,2'-dipyridyl disulfide and 50 mM triphenylphosphine, let
stand at room temperature for 30 min. Remove a 10 JJLL aliquot and dilute it to 100 |xL with
MeCN, inject a 2 jxL aliquot. (Synthesis of DNS-APy, l-(5-dimethylamino-l-naphthalenesul-
fonyl)-(S)-3-aminopyrrolidine, is as follows. Cool a solution of 16.4 g (R)-(+)-l-benzyl-3-pyrrol-
idinol in 164 mL pyridine to +5, add 19.35 g p-toluenesulfonyl chloride, stir at +10 for 48 h,
evaporate to dryness, chromatograph using dichloromethane:acetone 95:5 to obtain (3R)-3-[(4-
tolylsulfonyl)oxy]-l-(phenylmethyl)pyrrolidine (mp 68). Heat a solution of (3R)-3-[(4-tolylsul-
fonyl)oxy]-l-(phenylmethyl)pyrrolidine in 200 mL anhydrous DMF to 65, add 33.5 g sodium
azide (Caution! Sodium azide is highly toxic!), stir at 60 for 7 h, filter, evaporate the filtrate
to dryness under reduced pressure, dissolve the residue in ethyl acetate, wash twice with water,
dry over anhydrous magnesium sulfate, evaporate to obtain (3S)-3-azido-l-(phenylmeth-
yl)pyrrolidine as an oil. Add 3.5 g 10% palladium on carbon under nitrogen to a solution of
7.05 g (3S)-3-azido-l-(phenylmethyl)pyrrolidine in 34.8 mL 1 M HCl in water and 245 mL
EtOH, hydrogenate at atmospheric pressure for 30 min, add 3.5 g catalyst, hydrogenate for 2
h, filter, add 34.8 mL 1 M HCl to the filtrate, evaporate to dryness under reduced pressure,
take up the residue in 70 mL EtOH, filter, evaporate the filtrate to dryness under reduced
pressure, repeat this operation twice, crystallize with the minimum amount of EtOH to obtain
(3S)-(+)-3-aminopyrrolidine dihydrochloride (J. Med. Chem. 1992, 35, 4205). (3S)-(+)-Amino-
pyrrolidine dihydrochloride is also reported to be available from Tokyo Kasei. Stir 800 mg (3S)-
(+)-3-aminopyrrolidine dihydrochloride and 2 mL triethylamine in 800 mL MeCN at 0-10, add
a solution of 440 mg dansyl chloride in 80 mL MeCN dropwise, stir in the dark for 30 min,
evaporate to dryness under reduced pressure, dissolve the residue in 200 mL 5% HCl, wash
twice with 40 mL portions of dichloromethane. Adjust the pH of the organic layer to 13-14 with
5% NaOH, extract twice with 10 mL portions of dichloromethane. Combine the organic layers
and wash them with 80 mL water. Dry the organic layer over anhydrous sodium sulfate, evap-
orate to dryness under reduced pressure, dissolve the residue in dichloromethane:MeOH 90:
10, chromatograph on silica gel with dichloromethane:MeOH 90:10. Collect the greenish-yellow
fluorescent band and evaporate it under reduced pressure to obtain DNS-APy as a greenish-
yellow oil.)
HPLC VARIABLES
Column: 150 X 4.6 5 *jim TSK gel ODS-80TM (Tosoh)
Flow rate: 1
Injection volume: 2
CHROMATOGRAM
Retention time: 30 ((SH+)), 32 ((RH-))
KEY WpRDS
REFERENCE
Al-Kindy,S.; Santa,T.; Fukushima,T.; Homma,H.; Imai,K. l-(5-Dimethylamino-l-naphthalenesulphonyl)-(S)-3-
aminopyrrolidine (DNS-Apy) as a fluorescence chiral labelling reagent for carboxylic acid enantiomers,
Biomed.Chromatogr., 1997, 11, 137-142.
SAMPLE
Matrix: urine
by aspiration of air. Evaporate an aliquot of 100 JLJLL 20 jxg/mL IS in MeOH to dryness at 37.
mobile phase, inject a 10-30 |xL aliquot.
HPLC VARIABLES
Flow rate: 0.9
Detector: UV 230
CHROMATOGRAM
Retention time: 24
OTHER SUBSTANCES
Extracted: diclofenac, felbinac, fenbufen, flurbiprofen, ketoprofen, loxoprofen, mefenamic acid,
KEY WORDS
SPE
REFERENCE
J.Chromatogr.B, 1997, 692, 375-388.
SAMPLE
Matrix: urine
Sample preparation: Condition an Isolute KLAX SPE cartridge (International Sorbent Technol-
ogy, UK) with 3 mL MeOH, 3 mL water, and 3 mL 100 mM pH 2.0 acetate buffer. Add 1 mL
100 mM pH 2.0 acetate buffer to 1 mL urine, mix, add to the SPE cartridge. Wash with 2 mL
water, 2 mL MeOH.water 40:60, 2 mL hexane containing 10% triethylamine, and 2 mL hexane,
elute with 1 mL MeOH containing 10 mM HCl. Evaporate the eluate to dryness under a stream
of nitrogen at 50, reconstitute the dry residue with 50 |xL MeOH, add 100 JJLL reagent. Add
10 |JLL diethyl phosphorocyanidate and let stand for 5 min at room temperature. Add 40 jxL
water and inject a 10 JJLL aliquot. (The reagent was 28 mM Nepsilon-dansyl-O-methyl-L-lysine
in MeOH. Preparation is as follows. Dissolve 53.2 mg Nepsilon-dansyl-L-lysine in 2 mL MeOH,
add 8 mL benzene and 400 jxL 10% trimethylsilyldiazomethane in hexane, stir for 30 min at
room temperature, evaporate to dryness under a stream of nitrogen at 40. Reconstitute the
dry residue in 5 mL MeOH to a final concentration of 28 mM. (Caution! Benzene is a carcin-
ogen!) The free amino group of the reagent reacts with the free carboxylic acid group of
ibuprofen.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm L-column ODS (Chemicals Inspection and Testing Institute, Japan)
Flow rate: 1
CHROMATOGRAM
Retention time: 15 (-), 15.9 (+)
Limit of detection: 4 pmol
KEYWORDS
chiral; SPE; derivatization
REFERENCE
Hayamizu,T.; Kudoh,S.; Nakamura,H. Methylated Nepsilon-dansyl-L-lysine as a fluorogenic reagent for the
chiral separation of carboxylic acids, J.Chromatogr.B, 1998, 710> 211-218.
lbutilide
CAS Registry No.: 122647-31 -8,
122647-32-9 (fumarate)
Merck Index: 4927
Lednicer No.: 5 23
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Bond-Elut C18 SPE cartridge with 1 mL MeCN:
acetone:triethylamine 50:50:0.2, with 1 mL 0.1% triethylamine in water, and 1 mL water. 0.1-
1 mL Plasma + 1 mL buffer + 100 (xL 100-250 ng/mL IS in MeCN: 10 mM ammonium acetate
30:70, mix, add to the SPE cartridge, wash with 1 mL water, wash with 2 mL MeCN:MeOH:
water 25:25:50, wash with 1 mL water, dry under vacuum for 10 min, wash with 300 |xL
hexane, dry under vacuum for 5 min, elute with 500 (xL MeCN:acetone:triethylamine 50:50:
0.2. Evaporate the eluate to dryness under a stream of nitrogen at 30, reconstitute the residue
in 10 |xL 0.1% acetic acid in MeCN and 100 jxL 0.1% 1-naphthylisocyanate in MeCN, mix, heat
at 30 for 10 min, add 600 MeOH:water:trifluoroacetic acid 10:90:0.1, inject a 600 JJLL aliquot
onto column A and elute to waste with mobile phase A, after 8 min backflush the contents of
column A onto column B with mobile phase C, after 3.1 min elute column B to waste with
mobile phase D, after 6.1 min divert the fraction containing the drugs onto column C, after 2
min elute the contents of column C onto column D with mobile phase E, elute column E with
mobile phase E, monitor the effluent from column E. Before the next injection flush column A
with mobile phase B for 22.1 min and re-equilibrate with mobile phase A for 2 min. (Buffer
was 50 mM pH 7.0 NaH2PO4 containing 0.1% triethylamine.)
HPLCVARIABLES
Column: A two 15 X 3.2 7 |xm Newguard in series; B 20 X 4 4 jxm Sentry Nova-Pak C18 + 75
X 4.6 3 fxm Ultremex 3 C8; C 30 X 4.6 Spheri-5 RP-18; D 100 X 4.6 3 ^m Pirkle covalent
dinitrophenyl-D-phenylglycine (Regis)
Mobile phase: A MeOH:water 40:60; B MeCN:MeOH:water:trifluoroacetic acid:triethylamine 40:
40:20:0.1:0.1; C MeCN:MeOH:water:trifluoroacetic acid:triethylamine 25:5:70:0.1:0.1; D MeCN:
water:trifluoroacetic acid:triethylamine 52:48:0.1:0.1; E MeOH:trifluoroacetic acid triethylam-
ine 100:0.3:0.3
Flow rate: 1
CHROMATOGRAM
Retention time: 11.7 (+), 12.6 (-)
Internal standard: N-[4-[4-(ethyloctylamino)-l-hydroxybutyl]phenyl]methanesulfonamide (Up-
john U-74747) (IS detected using F ex 224 em 340 (cut-off filter) detector between columns B
# and C) (4)
KEY WpRDS
derivatization; SPE; plasma; column-switching; heart-cut; human; rat; rabbit; dog; chiral
REFERENCE
Hsu,C.-y.L.; Walters,R.R. Assay of the enantiomers of ibutilide and artilide using solid-phase extraction, de-
rivatization, and achiral-chiral column-switching high-performance liquid chromatography,e/. Chromatogr. B,
1995, 667, 115-128.
SAMPLE
Matrix: blood
Sample preparation: Basify 1 mL serum with 500 mM pH 10 carbonate buffer, extract with 1-
chlorobutane. Extract the organic layer with 37.5 mM sulfuric acid. Basify the sulfuric acid
layer (?) and extract it with 1-chlorobutane. Evaporate the organic layer to dryness, add 50 JJLL
130 ng/mL IS, add 50 (xL 0.1% triethylamine in MeCN, evaporate to dryness, add 10 |xL 0.1%
acetic acid in MeCN, add 100 |xL 220 |xL/L naphthylisocyanate in MeCN, heat at 30 for 10
min, evaporate to dryness, reconstitute with 100 |xL MeCN, use a column-switching technique
during the first 6 min.
HPLC VARIABLES
Column: 100 X 4.6 3 |xm 100 A D-phenylglycine Pirkle column
Mobile phase: MeOH:triethylamine:trifluoroacetic acid 100:0.038:0.038
Flow rate: 1
CHROMATOGRAM
Retention time: 10.5 (+), 11.6 (-)
Internal standard: U-71175
KEYWORDS
chiral; derivatization; serum
REFERENCE
Bhandarkar,S.V.; Neau,S.H. Development of a chiral separation assay for suprofen using an al-acid glycopro-
tein column (Abstract APQ 1117), Pharm.Res., 1996, 13, S32.
ldarubicin
Merck Index: 4931
LednicerNa: 5 47
SAMPLE
Matrix: blood
Sample preparation: Condition a 6 mL Bondelut C18 SPE cartridge with 3 mL MeOH and 3
ml MeOH:phosphate buffer 1:2. 1 mL Plasma + 10-100 JJLL 1 ixg/mL daunorubicin hydrochloride
in water + 1 mL 10 mM pH 8 phosphate buffer containing 600 nM tetrabutylammonium bro-
mide + 1 mL MeOH, add to the SPE cartridge, wash with 4 mL MeOH:water 25:75, elute with
3 mL 30 mM phosphoric acid in MeOH. Add the eluate to 100 jxL 100 mM KH2PO4, evaporate
to 100-400 |JLL under vacuum at 25, inject a 10-100 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-CN
Mobile phase: Gradient. A was MeCN: 10 mM KH2PO4 22:78. B was MeCN: 10 mM KH2PO4
containing 6 mM phosphoric acid 70:30. A:B from 90:10 to 80:20 over 9 min.
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
plasma; SPE
REFERENCE
Camaggi,C.M.; Carisi,R; Strocchi,E.; Pannuti,F. High-performance liquid chromatographic analysis of idaru-
bicin and fluorescent metabolites in biological fluids, Cancer Chemother.Pharmacol., 1992, 30, 303-306.
SAMPLE
HPLC VARIABLES
Column: 150 X 3.9 5 u-m Symmetry C8
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 9.6
OTHER SUBSTANCES
Simultaneous: etoposide
KEYWORDS
REFERENCE
Zhang,H.; Ye,L.; Stewart,J.T. HPLC determination of idarubicin-etoposide and idarubicin-ondansetron mixtures
in 0.9% sodium chloride injection USP, J.Liq.Chromatogr.Rel.Technol., 1998, 21, 979-988.
SAMPLE
HPLC VARIABLES
Flow rate: 1.8
Detector: UV 254
CHROMATOGRAM
Retention time: 7.1
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Zhang,H.; Ye,L.; Stewart,J.T. HPLC determination of idarubicin-etoposide and idarubicin-ondansetron mixtures
in 0.9% sodium chloride injection USP, J.Liq.Chromatogr.Rel.TechnoL, 1998, 21, 979-988.
ldebenone
Merck Index: 4932
SAMPLE
Sample preparation: Serum. 100 JJLL serum + 100 |xL 100 ng/mL vitamin K3 in EtOH, shake,
add 4 mL cyclohexane:benzene 20:80 (Caution! Benzene is a carcinogen!), vortex for 2 min,
a stream of nitrogen, reconstitute the residue in 100 JAL EtOH, filter (0.45 |xm), inject a 20 |xL
aliquot. Tissue. 50-100 mg Brain tissue + 500 |xL 100 mM perchloric acid + 20 jxL 100 ng/mL
vitamin K3 in EtOH, homogenize in a glass homogenizer at 4, add 6 mL cyclohexanerbenzene
80:20 (Caution! Benzene is a carcinogen!), vortex for 2 min, centrifuge at 1500 g for 10 min.
the residue in 100 |xL EtOH, filter (0.45 |xm), inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 150 X 6 5 |xm Shim-pack CLC-ODS (Shimadzu)
Mobile phase: MeOH:water 85:15 containing 50 mM sodium perchlorate
Flow rate: 1
Detector: E, EICOM ECD 100, glassy carbon working electrode +0.7 V, Ag/AgCl reference elec-
trode following post-column catalytic reduction. The column effluent flowed through a 10 X 4.6
column packed with 10 jxm 5% platinum on alumina catalyst (Toa Electronics) to the detector.
(Purge catalyst column with water at 10 mL/min for 5 min before use.)
CHROMATOGRAM
Retention time: 7.8
Internal standard: vitamin K3 (4.9)
KEYWORDS
rat; serum; brain; post-column reaction
REFERENCE
Wakabayashi,H.; Nakajima,M.; Yamato,S.; Shimada,K. Determination of idebenone in rat serum and brain by
high-performance liquid chromatography using platinum catalyst reduction and electrochemical detection,
J.Chromatogr., 1992, 573, 154-157.
Idoxuridine
Molecular formula: C9H11IN2O5
Merck Index: 4934
SAMPLE
Sample preparation: Inject a 20 u,L aliquot.
HPLC VARIABLES
Column: Aquapore RP.300 (Brownlee)
Mobile phase: Water.acetic acid 99:1
Flow rate: 1
Detector: UV 290
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: 5-iodo-2'-deoxycytidine
KEYWORDS
rabbit; GPC
REFERENCE
Sincholle,D.; Conduzorgues,J.-R; Mbatchi,B.; Masse,J.-P. Corneal penetration and metabolism of the antiher-
petic 5-iodo-2'-deoxycytidine, Curr.Eye Res., 1985, 4, 627-629.
SAMPLE
Matrix: blood
Sample preparation: 500 (xL Serum + 25 |xL 20 jig/mL 5-iodouridine in mobile phase + 1 mL
saturated ammonium sulfate + 50 |xL pH 6.7 ammonium phosphate + 4 mL ethyl acetate,
vortex for 15 min, centrifuge at 2000 g for 10 min. Remove the organic layer and evaporate it
to about 500 |xL under a stream of air, add 200 |xL 500 mM NaOH, vortex for 15 min, centrifuge
at 2000 g for 10 min, inject a 50 |JLL aliquot of the aqueous phase.
HPLC VARIABLES
Column: 220 X 4.6 5 |xm C18 (Brownlee)
Mobile phase: MeCN: 10 mM potassium phosphate buffer 5:95
Flow rate: 1.5
Detector: UV 290
CHROMATOGRAM
Internal standard: 5-iodouridine (7.74)
OTHER SUBSTANCES
KEYWORDS
serum
REFERENCE
Belotto,N.; Reiner,V.; Verga,R; Canobbio,G.; Lietti,R; Magnani,R; Lucarelli,C. Determination of 2'-deoxy-5-
iodouridine and its metabolite 5-iodouracil by high-performance liquid chromatography with ultraviolet
absorbance detection in human serum, J.Chromatogr., 1991, 572, 327-332.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL 10 |xg/mL bromodeoxyuridine in water + 2 mL
saturated ammonium sulfate solution + 100 u,L pH 6.7 ammonium phosphate buffer + 8 mL
ethyl acetate, shake for 15 min, centrifuge at 1200 g for 10 min. Remove the organic layer and
evaporate it to 1 mL under a stream of air, add 400 jxL 500 mM KOH, shake for 15 min,
centrifuge at 1000 g for 10 min, inject a 3-30 JJLL aliquot of the aqueous phase (J.Chromatogr.
1985, 341, 217).
HPLC VARIABLES
Mobile phase: MeOH:50 mM pH 7.3 ammonium phosphate buffer 12:88
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Internal standard: bromodeoxyuridine
KEYWORDS
dog; plasma
REFERENCE
Smith,D.E.; Brenner,D.E.; Knutsen,C.A.; Deremer,S.J.; Terrio,P.A.; Johnson,N.J.; Stetson,P.L.; Ensminger,W.D.
Mutual kinetic interaction between 5-fluorouracil and bromodeoxyuridine or iododeoxyuridine in dogs, Drug
Metab.Dispos., 1993, 21, 277-283.
SAMPLE
Sample preparation: Dilute with water to an idoxuridine concentration of 0.1%. Remove a 15
mL aliquot and add it to 2 mL sulfathiazole solution, make up to 20 mL with water, inject a
10 |xL aliquot. (Prepare sulfathiazole solution by dissolving 120 mg sulfathiazole in 10 mL
EtOH, make up to 100 mL with water.)
HPLC VARIABLES
Flow rate: 1.7
Detector: UV 254
CHROMATOGRAM
Retention time: 8
Internal standard: sulfathiazole (12.5)
OTHER SUBSTANCES
KEYWORDS
eye drops
REFERENCE
Carr,G.P.R. The development of British Pharmacopeia monographs for idoxuridine eye drops using high-pres-
sure liquid chromatography for assay and for controlling related impurities, J.Chromatogr., 1978,157,171-
184.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 2 jxL aliquot of a 0.1% solution.
HPLC VARIABLES
Flow rate: 1
Injection volume: 2
Detector: UV 262
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
REFERENCE
Szepesi,G.; Gazdag,M.; Mezei,J. Investigation of the hydrolytic decomposition of 5-iodine-2'-desoxyuridine by
high-performance liquid chromatography, Pharmazie, 1980, 35, 602-604.
lfosfamide
Molecular formula: C7H15CI2N2O2P
CAS Registry No/. 3778-73-2
Merck Index: 4937
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg CH Bond Elut SPE cartridge with two 1 mL aliquots
of MeOH and 1 mL water adjusted to pH 4 with phosphoric acid. 1 mL Serum + 1 mL 25 mM
pH 4 phosphate buffer + 200 |xL water adjusted to pH 4 with phosphoric acid, add this mixture
to the SPE cartridge in two 1 mL aliquots, allow to elute through cartridge for 2-3 min then
dry by applying a full vacuum for 5 min. Wash with 1 mL MeCN:water adjusted to pH 4 with
phosphoric acid 10:90, elute with 1 mL MeOH, evaporate to dryness under a stream of nitrogen,
reconstitute in 250 |xL mobile phase, inject a 20 |xL aliquot onto column A, after about 6.5 min
switch effluent containing ifosfamide from column A onto column B for 42 s, elute column B
and monitor the effluent.
HPLC VARIABLES
Column: A 50 X 4.6 5 jmi Spherisorb Cl; B 100 X 4 Chiral-AGP Ct1 acid glycoprotein (Chromtech)
Flow rate: 1
Detector: UV 195
CHROMATOGRAM
KEYWORDS
serum; chiral; SPE; column-switching
REFERENCE
Corlett,S.A.; Chrystyn,H. Enantiomeric separation of R- and S-ifosfamide and their determination in serum
from clinical subjects, J.Chromatogr.B, 1994, 654, 152-158.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with three 1 mL portions of
MeOH and three 1 mL portions of 25 uM cyclohexylamine in 500 mM pH 8.0 sodium phosphate
buffer containing 1 M NaCl. 500 |xL Plasma (stabilized with semicarbazide) + 500 |xL 500 mM
pH 8.0 sodium phosphate buffer containing 1 M NaCl + 100 (JLL 100 mg/mL sodium diethyl-
dithiocarbamate, heat at 70 for 30 min, cool to 0, add to the SPE cartridge, wash three times
with 25 |JLM cyclohexylamine in 500 mM pH 8.0 sodium phosphate buffer containing 1 M NaCl,
elute with 1 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 30,
reconstitute the residue in 100 JJLL MeCN:water 27.5:72.5, inject a 10 |xL aliquot.
HPLCVARIABLES
Column: 125 X 4 5 |xm LichroCART RP8
Mobile phase: MeCN:water 32:68 containing 10 mM sodium phosphate buffer and 20 mM cy-
clohexylamine, pH 7.0
Detector: UV 280
CHROMATOGRAM
Retention time: 4 (as ifosforamide mustard, the active metabolite)
Limit of quantitation: 450 nmoles
KEYWORDS
REFERENCE
Kaijser,G.P.; Beijnen,J.H.; Rozendom,E.; BuItA; Underberg,W.J.M. Analysis of ifosforamide mustard, the ac-
tive metabolite of ifosfamide, in plasma, J.Chromatogr.B, 1996, 686, 249-255.
SAMPLE
HPLC VARIABLES
Column: 100 X 4.6 5 jxm ODS-Hypersil
Flow rate: 1.5
Detector: UV 210
CHROMATOGRAM
Retention time: 1.5
KEYWORDS
injections; saline; water; stability-indicating
REFERENCE
Munoz,M.; Girona,V.; Pujol,M.; Dur&n,S.; Vicente,R; Sole,L.-A. Stability of ifosfamide in 0.9% sodium chloride
solution or water for injection in a portable i.v. pump cassette, Am.J.Hosp.Pharm., 1992, 49, 1137-1139.
SAMPLE
HPLC VARIABLES
Column: 300 X 4.6 5 (xm C18
Flow rate: 1.8
Detector: UV 198
CHROMATOGRAM
OTHERSUBSTANCES
KEYWORDS
REFERENCE
304.
SAMPLE
Sample preparation: Add solid NaCl to a 500 |xL aliquot of the reaction mixture until some
solid remains undissolved, add 250 |xL MeCN, stir for 5 min, inject a 20 jxL aliquot of the upper
layer.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Microsorb C8
Flow rate: 1
Detector: UV 190
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: cyclophosphamide
REFERENCE
Lunn,G.; Sansone,E.B.; Andrews ,A. W.; Hellwig,L.C. Degradation and disposal of some antineoplastic drugs,
J.Pharm.ScL, 1989, 78, 652-659.
lloprost
Merck Index: 4940
SAMPLE
Sample preparation: 100 mL 5% Dextrose injection + 25 mL chloroform + 1 mL 10 |xg/mL 2-
naphthoic acid in MeOH, shake vigorously for 1-2 min. Remove the organic layer and evaporate
it to dryness under a stream of air, reconstitute the residue in 1 mL MeOH, inject a 70 |xL
aliquot.
HPLCVARIABLES
Mobile phase: MeCN:MeOH:20 mM pH 3.0 KH2PO4 40:14.4:45.6
Flow rate: 1.8
Detector: UV 207
CHROMATOGRAM
Retention time: 8.24 (4R), 8.96 (4S)
Internal standard: 2-naphthoic acid (4.5)
KEYWORDS
injections; 5% dextrose; diastereoisomers
REFERENCE
Scypinski,S.; Lanzano,R-K; Soltero,R.A. Determination of iloprost in 5% dextrose in water solution by reversed-
phase high performance liquid chromatography, J.Pharm.ScL, 1990, 79, 934-937.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: Radial Pak C18 (Waters)
Mobile phase: Gradient. MeCN:0.1% acetic acid from 26:74 to 35:65 over 3 min, maintain at 35:
65 for 22 min, to 50:50 over 5 min, to 100:0 over 15 min.
Flow rate: 2
CHROMATOGRAM
Retention time: 18.5 (R), 20 (S)
KEYWORDS
stereoisomers
REFERENCE
Tsai,A.-L; Vijjeswarapu,H.; Wu,K.K. Interaction between platelet receptor and iloprost isomers, Biochim.
BiophysActa, 1988, 942, 220-226.
SAMPLE
Matrix: urine
Sample preparation: Inject an aliquot directly.
HPLC VARIABLES
Guard column: 50 X 4.6 not otherwise specified
Column: 250 X 4.6 Lichrosorb RP-18
Mobile phase: Gradient. A was MeOH containing 1 mL/L acetic acid. B was water containing 1
mL/L acetic acid. A:B from 30:70 to 48:52 over 60 min, to 55:45 over 80 min, to 80:20 over 60
min.
Flow rate: 2
CHROMATOGRAM
Retention time: 170
OTHER SUBSTANCES
KEYWORDS
rat; radiolabelled
REFERENCE
Krause,W.; Hiimpel,M.; Hoyer,G.-A. Biotransformation of the stable prostacyclin analogue, iloprost, in the rat,
DrugMetab.Dispos., 1984, 12, 645-651.
lmidapril
Merck Index: 4947
SAMPLE
Sample preparation: Condition a 100 mg Bond Elut C18 SPE cartridge with 3 mL MeOH, with
3 mL water, and with 1 mL 100 mM HCl. Condition a 100 mg Bond Elut SI silica SPE cartridge
with 1 mL acetone and 3 mL n-hexane. Plasma. 1 mL Plasma + 200 jxL 2 M HCl + 200 |xL
water + 10 mL acetone, shake for 10 min, centrifuge at 1500 g for 10 min. Remove 10 mL of
the supernatant and evaporate it to dryness under reduced pressure at 45, reconstitute the
residue in 1 mL 100 mM HCl, add 10 mL diethyl ether, shake for 5 min, centrifuge at 1500 g
for 5 min. Add the aqueous layer to the C18 SPE cartridge, wash with 2 mL 100 mM HCl,
wash with 1 mL MeOH:100 mM HCl 20:80, elute with 1 mL MeOHiIOO mM HCl 60:40. Evap-
orate the eluate to dryness, reconstitute in 200 (JLL MeOH: 100 mM HCl 60:40, evaporate to
dryness into a vial, reconstitute in 25 jxL water and 25 JLJLL acetone, sonicate for 10 min, add
50 |AL 0.2% ISl in acetone, heat at 40 for 1 h, evaporate to dryness under reduced pressure
at 45, dissolve in 50 |xL acetone and 1 mL hexane, add to the silica SPE cartridge, wash with
1 mL n-hexane.ethyl acetate 70:30, elute with 1 mL n-hexane:ethyl acetate 50:50, evaporate
the eluate to dryness, reconstitute in 300 |xL 20 ng/mL 9-anthrylmethyl myristate in MeCN:
water 80:20, inject a 200 |xL aliquot. Urine. 1 mL Urine + 200 |xL water + 200 |xL IS2 + 15
mL 500 mM Na2HPO4, make up to 25 mL with water, remove a 2 mL aliquot and add it to 10
mL diethyl ether, shake for 5 min, centrifuge at 1500 g for 5 min, discard the organic layer,
add 10 mL chloroform, shake for 5 min, centrifuge at 1500 g for 5 min. Add 1 mL of the aqueous
layer to the C18 SPE cartridge, wash with 2 mL 100 mM HCl, wash with 1 mL MeOH: 100
mM HCl 20:80, elute with 1 mL MeOH: 100 mM HCl 60:40. Evaporate the eluate to dryness,
reconstitute in 200 |xL MeOH: 100 mM HCl 60:40, evaporate to dryness into a vial, reconstitute
in 25 JULL water and 25 jxL acetone, sonicate for 10 min, add 50 jxL 0.2% 9-anthryldiazomethane
in acetone, heat at 40 for 1 h, evaporate to dryness under reduced pressure at 45, dissolve in
50 |JLL acetone and 1 mL hexane, add to the silica SPE cartridge, wash with 1 mL n-hexane:
ethyl acetate 70:30, elute with 1 mL n-hexane:ethyl acetate 50:50, evaporate the eluate to
dryness, reconstitute in 250 |xL MeCN:water 80:20, inject a 25 (JLL aliquot. (Synthesis of 9-
anthryldiazomethane is as follows. Stir 8.8 g 9-anthraldehyde and 8.5 g 80% hydrazine hydrate
in 150 mL EtOH at room temperature for 3 h, filter off the solid 9-anthraldehyde hydrazone
and dry under vacuum (mp 124-6) (Bull. Chem. Soc. Jpn. 1967, 40, 691). Dissolve 220 mg 9-
anthraldehyde hydrazone in 100 mL anhydrous ether, add 800 mg activated manganese di-
oxide, follow the reaction by reverse-phase HPLC using MeCN at 0.4 mL/min and UV 254. At
the end of the reaction filter off the manganese and wash it with 20 mL ether, evaporate the
filtrate to obtain 9-anthryldiazomethane (mp 64-6) (Anal.Biochem. 1980, 107, 116 and 1983,
132 456). Prepare activated manganese dioxide as follows. Stir a solution of 20 g potassium
permanganate in 250 mL water at room temperature, add 10 g activated carbon (Nuchar C-
190 or C-190N), stir for 16 h, filter (Buchner funnel), wash 4 times with 50 mL portions of
water, dry in air, dry in an oven at 105-110 for 8-24 h (J.Org.Chem. 1970, 35, 3971).)
HPLC VARIABLES
Guard column: 4 X 4 5 jxm Lichrosorb Si60
Column: 250 X 4 5 |jim Hypersil MOS-5 dimethyloctyl
Mobile phase: MeCN:0.02% triethylamine 80:20, pH adjusted to 3.0 with 10% phosphoric acid
Flow rate: 1
CHROMATOGRAM
Internal standard: 9-anthryldiazomethane (ISl) (17), (S)-3-(N-[(S)-l-ethoxycarbonylundecyl]-L-
alanyl)-l-methyl-2-oxoimidazoline-4-carboxylic acid (IS2) (42)
Limit of detection: 10 ng/mL (urine), 0.2 ng/mL (plasma)
OTHER SUBSTANCES
KEYWORDS
plasma; protect from light during and after derivatization; derivatization; SPE
REFERENCE
Tagawa,K; Hayashi,K; Mizobe,M.; Noda,K. Highly sensitive determination of imidapril, a new angiotensin I-
converting enzyme inhibitor, and its active metabolite in human plasma and urine using high-performance
liquid chromatography with fluorescent labelling reagent, J.Chromatogr., 1993, 617, 95-103.
SAMPLE
Matrix: bulk
Sample preparation: 5 mg Imidapril hydrochloride + 15 mg L-alanine-p-naphthylamide hy-
drobromide + 100 |JLL chloroform + 50 |xL pyridine + 2 mL 4.5 g/L N,N'-dicyclohexylcarbodiimide
in chloroform, shake vigorously, let stand for 1 h, wash with 2 mL 1 M HCl, wash with two 2
mL portions of water. Remove a 1 mL aliquot and dilute it to 5 mL with chloroform, inject a
20 |xL aliquot.
HPLCVARIABLES
Mobile phase: Chloroform:MeOH:EtOH:diethylamine 600:10:2:0.1
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 5 (SRS), 5.5 (SRR), 7.5 (SSS, SSR), 15 (RSS), 17.5 (RRR), 19 (RRS), 21 (RSS)
Limit of detection: 0.05% of larger isomer
KEY WpRDS
REFERENCE
Nishi,H.; Yamasaki,K.; Kokusenya,Y.; Sato,T. Optical resolution of imidapril hydrochloride by high-performance
liquid chromatography and application to the optical purity testing of drugs, J.Chromatogr.A, 1994, 672,
125-133.
SAMPLE
Sample preparation: Grind tablets, weigh out amount equivalent to 25 mg imidapril, add 40
mL MeOH:water 40:60, shake vigorously for 10 min, make up to 50 mL with MeOH:water 40:
60, filter (0.45 |xm), inject a 20 |xL aliquot of the filtrate.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Nucleosil 5C8
Mobile phase: MeOH: 10 mM pH 2.7 phosphate buffer 40:60
Flow rate: 1
Detector: UV 215
CHROMATOGRAM
Retention time: 4 (SRR, RSS), 6 (SSR, RRS), 7 (SRS, RSR)
Limit of detection: 0.05% for each diastereomer
OTHER SUBSTANCES
Simultaneous: degradation products, imidaprilat
KEYWORDS
tablets
REFERENCE
Nishi,H.; Yamasaki,K; Kokusenya,Y.; Sato,T. Optical resolution of imidapril hydrochloride by high-performance
liquid chromatography and application to the optical purity testing of drugs, J.Chromatogr.A, 1994, 672,
125-133.
lmipenem
CAS Registry No.: 64221-86-9, 74431-23-5 (monohydrate)
Merck Index: 4954
Lednicer No.: 4 181
SAMPLE
Matrix: blood
Next Page
Sample preparation: Filter (Ultrafree MC) plasma at 6000 g for 10 min, inject a 10 JULL aliquot
of the ultrafiltrate.
HPLC VARIABLES
Column: 150 X 3.9 4 |jLin Nova Pak C18
Mobile phase: 200 mM pH 7.2 borate buffer (Prepare the buffer by dissolving 12.4 g boric acid
in 1 L water and adjusting pH to 7.2 with 1 M NaOH.)
Flow rate: 1
Detector: UV 300
CHROMATOGRAM
KEYWORDS
REFERENCE
Garcia-Capdevila,L.; L6pez-Calull,C; Arroyo,C; Moral,M.A.; Mangues,M.A.; Bonal,J. Determination of imipe-
nem in plasma by high-performance liquid chromatography for pharmaceutical studies in patients,
J.Chromatogr.B, 1997, 692, 127-132.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10 |xL aliquot of a 400 |xg/mL solution.
HPLC VARIABLES
Column: 150 X 4.6 Microsorb C8 80-315
Mobile phase: 1 mM KH2PO4 adjusted to pH 6.8 with 500 mM NaOH
Flow rate: 1.5
Detector: UV 300
REFERENCE
Connolly,M.; Debenedetti,P.G.; Tung,H.-H. Freeze crystallization of imipenem, J.Pharm.ScL, 1996, 85, 174-
177.
lmipramine
Merck Index: 4955
LednicerNo.: 1 401; 2 420; 3 32; 4 146 201 203
SAMPLE
Matrix: blood
Sample preparation: Add 250 |xL 2 M sodium carbonate to 500 jxL plasma. Add 100 |xL 1 [Lg/
10 min. Cool in a dry ice-acetone bath. Add 200 |xL 0.3% phosphoric acid to the upper organic
layer. Shake for 10 min and centrifuge at 3000 g for 10 min. Separate the organic layer. Inject
a 100 |xL aliquot of the acidic aqueous layer.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm C18 Symmetry (Waters Millipore, USA)
Previous Page
Sample preparation: Filter (Ultrafree MC) plasma at 6000 g for 10 min, inject a 10 JULL aliquot
of the ultrafiltrate.
HPLC VARIABLES
Column: 150 X 3.9 4 |jLin Nova Pak C18
Mobile phase: 200 mM pH 7.2 borate buffer (Prepare the buffer by dissolving 12.4 g boric acid
in 1 L water and adjusting pH to 7.2 with 1 M NaOH.)
Flow rate: 1
Detector: UV 300
CHROMATOGRAM
KEYWORDS
REFERENCE
Garcia-Capdevila,L.; L6pez-Calull,C; Arroyo,C; Moral,M.A.; Mangues,M.A.; Bonal,J. Determination of imipe-
nem in plasma by high-performance liquid chromatography for pharmaceutical studies in patients,
J.Chromatogr.B, 1997, 692, 127-132.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10 |xL aliquot of a 400 |xg/mL solution.
HPLC VARIABLES
Column: 150 X 4.6 Microsorb C8 80-315
Mobile phase: 1 mM KH2PO4 adjusted to pH 6.8 with 500 mM NaOH
Flow rate: 1.5
Detector: UV 300
REFERENCE
Connolly,M.; Debenedetti,P.G.; Tung,H.-H. Freeze crystallization of imipenem, J.Pharm.ScL, 1996, 85, 174-
177.
lmipramine
Merck Index: 4955
LednicerNo.: 1 401; 2 420; 3 32; 4 146 201 203
SAMPLE
Matrix: blood
Sample preparation: Add 250 |xL 2 M sodium carbonate to 500 jxL plasma. Add 100 |xL 1 [Lg/
10 min. Cool in a dry ice-acetone bath. Add 200 |xL 0.3% phosphoric acid to the upper organic
layer. Shake for 10 min and centrifuge at 3000 g for 10 min. Separate the organic layer. Inject
a 100 |xL aliquot of the acidic aqueous layer.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm C18 Symmetry (Waters Millipore, USA)
50:50.)
Flow rate: 1.2
CHROMATOGRAM
Limit of quantitation: 5 ng/mL (UV 226, UV 400); 7 ng/mL (UV 254)
OTHER SUBSTANCES
Extracted: metabolites, amitriptyline, clomipramine, desipramine, fluoxetine, maprotiline,
nortriptyline
Simultaneous: amineptine, chlordiazepoxide, chlorpromazine, clonazepam, clorazepate, cloza-
pine, cyamemazine, desmethylmaproptiline, desmethylvenlafaxine, doxepin, flunitrazepam,
haloperidol, levomepromazine, lorazepam, loxapine, mianserine, sulpiride, trimipramine, ven-
lafaxine, viloxazine, zolpidem, zopiclone
Interfering: carbamazepine, fluvoxamine
KEYWORDS
plasma
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 1600 ng clomipramine in MeOH + 2 mL 1 M NaOH + 5
mL hexane:isoamyl alcohol 99:1, shake mechanically for 15 min, centrifuge at 1686 g for 5
min. Remove the organic phase and add it to 200 (xL 0.05% orthophosphoric acid, shake for 15
min, centrifuge for 5 min, inject a 50 fxL aliquot of the aqueous phase.
HPLC VARIABLES
Guard column: jxBondapak/Porasil
Mobile phase: MeCN:buffer 40:60 (Buffer was 13.68 g KH2PO4 in 2 L water, adjusted to pH 4.7
with dilute KOH.)
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Extracted: amitriptyline, desipramine, nortriptyline
Simultaneous: chlordiazepoxide, chlorpromazine, cimetidine, clomipramine, diazepam, doxepin,
flurazepam, lorazepam, oxazepam, pentobarbital, perphenazine, phenobarbital, phenytoin,
prochlorperazine, propoxyphene, secobarbital, thioridazine, trifluoperazine
Noninterfering: acetaminophen, codeine, meperidine
KEYWORDS
plasma
REFERENCE
Wong,S.H.Y.; McCauley,T. Reversed phase high-performance liquid chromatographic analysis of tricyclic anti-
depressants in plasma, J.Liq.Chromatogr., 1981, 4, 849-862.
SAMPLE
Matrix: blood
reconstitute the residue in 100 |xL mobile phase, inject a 50 |xL aliquot. (It is implied, but not
HPLCVARIABLES
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
promazine, fluphenazine, haloperidol, mesoridazine, nortriptyline, orphenadrine, piperaceta-
zine, promethazine, thioridazine, trifluoperazine, trinupromazine, trihexyphenidyl,
trimeprazine
Interfering: promazine, thiothixene
KEYWORDS
plasma; whole blood
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 200 |xL 10 jjug/mL protriptyline in water + 200 |xL 80 g/L
NaHCO3 + 5 mL hexane, vortex for 15 s, centrifuge for 5 min. Remove the hexane layer and
evaporate it in a stream of nitrogen at 60. Reconstitute in 100 u,L mobile phase, vortex for 15
s, inject a 50 JLJLL aliquot.
HPLC VARIABLES
Column: 300 X 4 10 |xm ixBondapak CN
Mobile phase: MeCN:MeOH:5 mM phosphate buffer 60:15:25, adjusted to pH 7.0
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Internal standard: protriptyline (12.20)
OTHER SUBSTANCES
Simultaneous: trimipramine, doxepin, amitriptyline, desmethyldoxepin, nortriptyline, desipra-
mine, chlorpromazine, procainamide, thioridazine, propranolol, propoxyphene, disopyramide,
maprotiline
Noninterfering: caffeine, theophylline, salicylic acid, chlordiazepoxide, methaqualone, diaze-
pam, acetaminophen, trifluoperazine
KEYWORDS
serum
REFERENCE
Koteel,R; Mullins,R.E.; Gadsden,R-H. Sample preparation and liquid-chromatographic analysis for tricyclic
antidepressants in serum, Clin.Chem., 1982, 28, 462-466.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond-Elut C18 column with 2 volumes MeOH then 2 volumes
water. Add 1 mL serum then 200 |xL 700 ng/mL promazine in MeOH:0.1 M HCl 13:87 to each
column, wash with 2 volumes water, wash with 2 volumes 0.1 M acetic acid, wash with MeOH/
water, add 200 |xL 10 mM ammonium acetate in MeOH, wait for 30 s, elute with vacuum,
repeat elution process two more times. Combine eluates and evaporate them to dryness at 56-
8 under compressed air. Reconstitute with 200 \xL mobile phase, vortex 10 s, inject 75-100 JJLL
aliquot. (MeOH/water was 500 mL MeOH:water 65:35 plus 25 |xL concentrated HCl.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelco silica
Mobile phase: EtOH:MeCN:t-butylamine 98:2:0.05 (Mix 1 gallon EtOH with 77 mL MeCN and
1.9 mL t-butylamine.)
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 4.0
Internal standard: promazine (5.2)
OTHER SUBSTANCES
Extracted: amitriptyline, desipramine, desmethyldoxepin, doxepin, nortriptyline, protriptyline
Simultaneous: procainamide, zimeldine, morphine, codeine, trifluoperazine, desmethyldisopyr-
amide, 10-hydroxynortriptyline, prochlorperazine, oxaprotiline, 2-hydroxydesipramine, chlor-
pheniramine, maprotiline, norzimeldine, iminostilbene, desmethylchlordiazepoxide, buprion,
diazepam, demoxepam, chlordiazepoxide, propoxyphene, dextropropoxyphene, cocaine, oxa-
pam, trimipramine, mianserin, trimeprazine, loxepin, fluphenazine, methadone, trifluopro-
mazine, phenteramine, chlorimipramine, perphenazine, quinidine, thioridazine, hydroxyamox-
apine, meperidine, chlorpromazine, disopyramide, amphetamine, 2-hydroxyimipramine
Noninterfering: thiopropazine
Interfering: iprindole, pyrilamine, promethazine, prolixin, amoxapine, N-acetylprocainamide
KEYWORDS
serum; normal phase; SPE
REFERENCE
Beierle,F.A.; Hubbard,R.W. Liquid chromatographic separation of antidepressant drugs: I. Tricyclics, Ther.Drug
Monit, 1983, 5, 279-292.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 |xL 1 |xg/mL loxapine in isopropanol:diethylamine
and add it to 100 u,L 250 mM HCl, vortex for 30 s, inject a 50 |xL aliquot of the aqueous phase.
HPLCVARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
desmethylchlordiazepoxide, desmethyldoxepin, doxepin, fluphenazine, haloperidol, nortripty-
line, oxazepam, thiothixene
Interfering: diazepam
KEYWORDS
plasma
REFERENCE
Kiel,J.S.; Abramson,RK.; Morgan,S.L.; Voris,J.C. A rapid high performance liquid chromatographic method for
the simultaneous measurement of six tricyclic antidepressants, J.Liq.Chromatogr., 1983, 6, 27612773.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Serum or plasma + 100 |xL 1 |xg/mL IS in water + 0.5 mL water,
vortex, extract with 10 mL toluene:isoamyl alcohol 99:1 for 10 min on a rotator, centrifuge for
5 min. Remove upper organic layer, evaporate under a stream of nitrogen at 37, take up in
150 fjuL mobile phase, vortex for 2 min, add 0.5 mL hexane, vortex briefly, centrifuge for 5 min,
discard upper hexane layer, inject a 100 |xL aliquot of the lower layer.
HPLC VARIABLES
Column: 250 X 4 Bio-Sil ODS-IO (Bio-Rad)
Mobile phase: MeCN:pH 4.5 50 mM phosphate buffer 30:70 (Buffer was 6.9 g KH2PO4 in 1 L
adjusted to pH 4.5 with orthophosphoric acid.)
Flow rate: 2.5
Detector: UV 202
CHROMATOGRAM
Retention time: 8.4
Internal standard: U-31485 (6.9)
OTHER SUBSTANCES
Extracted: desipramine, protriptyline
Noninterfering: N-acetylprocainamide, amitriptyline, caffeine, chlordiazepoxide, chlorproma-
zine, diazepam, flurazepam, lorazepam, oxazepam, prazepam, procainamide, propranolol,
thioridazine
Interfering: alprazolam, nortriptyline, triazolam
KEYWORDS
plasma; serum
REFERENCE
McCormick,S.R.; Nielsen,J.; Jatlow,R Quantification of alprazolam in serum or plasma by liquid chromatog-
raphy, Clin.Chem., 1984, 30, 1652-1655.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 37 jxL 2 ixg/mL IS in MeOH + 500 |xL pH 10 borate
buffer + 1.5 mL hexanerisoamyl alcohol 95:5, shake for 10 min. Evaporate the organic layer to
dryness under a stream of nitrogen, reconstitute in 100 |xL MeOH, inject a 50 |xL aliquot. (The
borate buffer was prepared as follows. Prepare a solution of 61.8 g boric acid and 74.6 g KCl
in 1 L water. Add 630 mL of this solution to 370 mL 106 g/L sodium carbonate solution. Adjust
pH to 10.0 with 6 M NaOH and store at 35-37.)
HPLCVARIABLES
Column: 250 X 4.6 Zorbax SiI
Mobile phase: MeOHiammonium hydroxide 998:2
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 5
Internal standard: N-desmethylclomipramine hydrochloride (10)
OTHER SUBSTANCES
Extracted: amitriptyline, nortriptyline, desipramine, 2-hydroxyimipramine, 2-hydroxydesipra-
mine, metabolites
Also analyzed: doxepin, desmethyldoxepin, desmethylclomipramine, clomipramine, maprotiline,
protriptyline
Noninterfering: chlordiazepoxide, diazepam, flurazepam, oxazepam, thioridazine
KEYWORDS
plasma
REFERENCE
Sutfin,T.A.; D'Ambrosio,R; Jusko,W.J. Liquid-chromatographic determination of eight tri- and tetracyclic an-
tidepressants and their major active metabolites, Clin.Chem., 1984, 30, 471-474.
SAMPLE
Matrix: blood
Sample preparation: Evaporate 200 |xL 1 jjug/mL clomipramine in MeOH into a tube, add 2 mL
plasma, add 2 mL pH 10 Titrisol buffer (Merck), add 8 mL diethyl ether, shake for 15 min,
centrifuge at 2800 g for 5 min. Remove the organic phase and shake it with 100 |JLL 50 mM
phosphoric acid for 15 min, centrifuge at 2800 g for 10 s. Remove the aqueous layer and vortex
it with 2 mL diethyl ether for 10 s, centrifuge at 2800 g. Discard the organic layer and inject
a 10-50 |xL aliquot of the aqueous layer.
HPLC VARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 8.9
Internal standard: clomipramine (13)
OTHER SUBSTANCES
Simultaneous: desipramine, trimipramine
Noninterfering: alimemazine, alprazolam, amineptine, amitriptyline, caffeine, carbamazepine,
citalopram clobazam, desmethylflunitrazepam, diazepam, dibenzepine, estazolam, ethyl lofla-
zepate, indalpine, loprazolam, lorazepam, meprobamate, nitrazepam, norclobazam, nordiaze-
pam, nortriptyline, oxazepam, triazolam, viloxazine
Interfering: monodesmethyltrimipramine, flunitrazepam, levomepromazine
KEYWORDS
plasma
REFERENCE
Pok Phak,R.; Conquy,T.; Gouezo,E; Viala,A.; Grimaldi,F. Determination of metapramine, imipramine, trimip-
ramine and their major metabolites in plasma by reversed-phase column liquid chromatography,
J.Chromatogr., 1986, 375, 339-347.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Analytichem cyanopropyl SPE cartridge with 1 mL
water and 1 mL MeOH, do not allow to dry. Add 1 mL serum + 250 u,L 50 mM sodium n-
heptanesulfonic acid to the SPE cartridge, wash with 1 mL water, 1 mL MeOH:water 50:50,
air dry cartridge, elute with 1 mL MeOHitriethylamine 99.2:0.8, evaporate eluate to dryness
under a stream of nitrogen at 40, reconstitute residue with 250 jxL mobile phase, inject a 50
(xL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5|xm Spherisorb cyanopropyl
Mobile phase: MeOH:20 mM phosphoric acid containing 0.05% N,N-diethyloctylamine 55:45, pH
was 2.4
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: doxepin
KEY WORDS
serum; imipramine is IS; SPE
REFERENCE
Emm,T.; Lesko,L.J.; Perkal,M.B. Simultaneous determination of doxepin and nordoxepin in serum using high-
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut C-18 SPE cartridge twice with MeOH and twice
with water. 500 |xL Serum + 50 uX 1 |xg/mL N-propionylprocainamide in 2.5 mM HCl, add to
SPE cartridge, wash with 2 volumes water, wash with 2 volumes 0.1 M acetic acid, wash with
1 volume MeOH:2.5 mM HCl 10:90. Add 200 |xL 10 mM acetic acid and 5 mM diethylamine
in MeOH to column, let stand 1 min, elute under vacuum, repeat, evaporate eluents to dryness
under nitrogen at room temperature, reconstitute in 100 |xL mobile phase, inject a 40 |xL
aliquot.
HPLCVARIABLES
Guard column: Pelliguard LC-CN (Supelco)
Column: 150 X 4.6 5 |jim Supelcosil LC-PCN
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Internal standard: N-propionylprocainamide (6)
OTHER SUBSTANCES
Extracted: amitriptyline, desipramine, doxepin, nortriptyline, protriptyline, trimipramine
Simultaneous: atropine, butalbital, chlorpromazine, maprotiline, methadone, norpropoxyphene,
phenylpropanolamine, procainamide, prochlorperazine, promethazine, propranolol, quinidine,
trifluoperazine, trimeprazine
Noninterfering: acetaminophen, allopurinol, amikacin, amoxapine, amytal, bretylium, caffeine,
carbamazepine, carisoprodol, chloramphenicol, chlordiazepoxide, chlorpropamide, clonazepam,
codeine, diazepam, disopyramide, droperidol, ethinamate, ethinamate, ethosuximide, fluphen-
azine, flurazepam, furosemide, gentamicin, haloperidol, hydrochlorothiazide, hydroxyzine, ibu-
profen, kanamycin, lidocaine, loxapine, meperidine, mephobarbital, meprobamate, methaqua-
lone, methotrexate, morphine, nafcillin, naloxone, neomycin, perphenazine, phenacetin,
phenobarbital, phenytoin, prazepam, primidone, procaine, propoxyphene, reserpine, salicylam-
ide, salicylic acid, secobarbital, spironolactone, theophylline, thiopental, thioridazine, tobra-
mycin, valproic acid, verapamil
Interfering: hydroxynortriptyline
KEYWORDS
serum; SPE
REFERENCE
Lin,W.-N.; Frade,P.D. Simultaneous quantitation of eight tricyclic antidepressants in serum by high-perfor-
mance liquid chromatography, Ther.Drug Monit., 1987, 9, 448455.
SAMPLE
Matrix: blood
Sample preparation: 500 |JLL Serum + 250 |iL di-iso-propyl ether:n-butyl alcohol 7:3 containing
800 ng/mL minaprine, centrifuge 2 min, shake, centrifuge 5 min, inject 50 |xL aliquot of top
organic layer.
HPLC VARIABLES
Guard column: 30 X 4.6 5 |xm Brownlee cyano spheri-5
Column: 250 X 4.6 5 |xm Altex ultrasphere cyano
Mobile phase: MeCN:THF:water:2 M ammonium formate (pH 4.0) 700:100:195:5
Flow rate: 1.5
Detector: UV 248
CHROMATOGRAM
Retention time: 7.5
Internal standard: minaprine (5.5)
OTHER SUBSTANCES
Simultaneous: desipramine, clomipramine
Also analyzed: diltiazem, nortriptyline, amitriptyline, haloperidol, propafenone, amiodarone,
verapamil
KEYWORDS
serum
REFERENCE
Mazzi,G. Simple and practical high-performance liquid chromatographic assay of some tricyclic drugs, halo-
peridol, diltiazem, verapamil, propafenone, and amiodarone, Chromatographia, 1987, 24, 313-316.
SAMPLE
Matrix: blood
Sample preparation: Inject 200 |xL serum onto column A and elute with mobile phase A for 10
min then back-flush column A onto column B with mobile phase B for 4 min. Elute column B
with mobile phase B and monitor the effluent. Remove column A from circuit and wash with
MeCN:water 60:40 for 6 min then with mobile phase A for 10 min.
HPLC VARIABLES
Column: A 40 X 4 TSKprecolumn PW (Tosoh); B 150 X 4 TSKgel ODS-80TM (Tosoh)
Mobile phase: A 50 mM pH 7.5 potassium phosphate; B MeCN:100 mM pH 2.7 potassium phos-
phate 32.5:67.5, containing 0.2 g/L sodium 1-heptanesulfonate
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Simultaneous: amitriptyline, amoxapine, clomipramine, doxepin, desipramine, maprotiline,
trimipramine
KEYWORDS
serum; column-switching; use gradient to determine metabolites
REFERENCE
Matsumoto,K.; Kanba,S.; Kubo,H.; Yagi,G.; Iri,H.; Yuki,H. Automated determination of drugs in serum by col-
umn-switching high-performance liquid chromatography. IV. Separation of tricyclic and tetracyclic antide-
pressants and their metabolites, Clin.Chem., 1989, 35, 453-456.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 |xL 10 mM HCl + 200 |xL 10% ammonium carbonate
(final pH 8.7), vortex gently, add 5 mL MTBE, extract (Vibrax VXR2) for 20 min, centrifuge at
4 at 1720 g for 10 min, remove the organic layer. Add 5 mL dichloromethane to the aqueous
layer, shake for 20 min on a reciprocating shaker, centrifuge at 0 at 1720 g for 10 min. Combine
tute the residue in 100 JJLL 10 mM HCl, wash with 2 mL MTBE, wash with 2 mL hexane, inject
HPLCVARIABLES
Mobile phase: MeCN:MeOH:40 mM ammonium acetate 24:40:36 containing 0.04% triethylam-
ine, pH adjusted to 7.3 with glacial acetic acid
Flow rate: 1.2
Detector: UV 237
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diltiazem
Simultaneous: alprazolam, amitriptyline, desipramine, loxapine, nortriptyline
Noninterfering: clomipramine
KEYWORDS
plasma; imipramine is IS
REFERENCE
Yeung,P.K.R; Montague,T.J.; Tsui,B.; McGregor,C. High-performance liquid chromatographic assay of diltiazem
and six of its metabolites in plasma: application to a pharmacokinetic study in healthy volunteers,
J.Pharm.Sci., 1989, 78, 592-597.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 200 |xL saturated potassium carbonate, mix, add 5 mL
hexaneiisopropanol 98:2, shake at 230 rpm for 15 min, centrifuge at 800 g for 10 min. Remove
the organic layer and add it to 100 JJLL 0.5% orthophosphoric acid, shake for 15 min, centrifuge
at 800 g at 5 for 10 min, inject a 50 |xL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Spherisorb ODS-I
Mobile phase: MeCN:water:l M NaH2PO4 55:35:10
Flow rate: 1.8
Detector: UV 205
CHROMATOGRAM
Retention time: 6.4
OTHER SUBSTANCES
Simultaneous: diphenhydramine
KEYWORDS
REFERENCE
Selinger,K; Prevost,J.; Hill,H.M. High-performance liquid chromatography method for the determination of
diphenhydramine in human plasma, J.Chromatogr., 1990, 526, 597-602.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 10 |xL 1 mg/mL 8-hydroxychloroimipramine + 500 |xL 0.6
M pH 10.4 carbonate buffer + 5 mL ethyl acetate:heptane 20:80, shake for 2.5 min, centrifuge
at 3000 g for 10 min. Remove organic layer and add it to 125 |xL pH 2.4 25 mM KH2PO4, shake
for 2.5 min, centrifuge at 3000 g for 10 min. Remove the aqueous layer and put it in a rotary
evaporator for 25 min to remove traces of organic solvent. Inject a 50 |xL aliquot.
HPLCVARIABLES
Column: 120 X 4.6 5 fjum Nucleosil C18
Mobile phase: MeCN:bufTer 30:70 (Buffer was 10 Mm KH2PO4 + 5 mM tetramethylammonium
chloride adjusted to pH 2.4 with concentrated phosphoric acid.)
Flow rate: 1
Detector: E, ESA Coulochem Model 5100A, detector 1 +0.2 V, detector 2, +0.68 V, guard cell 0.70
V, gain 12 X 10, response time 0.4 s; UV 215
CHROMATOGRAM
Internal standard: 8-hydroxychloroimipramine (6.35)
Limit of detection: 0.5 ng/mL (electrochemical)
Limit of quantitation: 15 ng/mL (UV)
OTHER SUBSTANCES
Simultaneous: 2-hydroxydesipramine, 2-hydroxyimipramine, desipramine, chlorodesipramine,
chloroimipramine, mesoridazine
Noninterfering: doxepin, nordoxepin, amitriptyline, fluoxetine, norfluoxetine, triazolam,
alprazolam
KEYWORDS
plasma
REFERENCE
Foglia,J.R; Sorisio,D.; Perel,J.M. Determination of imipramine, desipramine and their hydroxy metabolites by
reversed-phase chromatography with ultraviolet and coulometric detection, J.Chromatogr., 1991,572, 247-
258.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 3 JJLL 20 ng/mL clobazam + 1 mL saturated sodium borate
(adjusted to pH 11 with 6 M NaOH) + 5 mL n-hexane, mix 2 min, centrifuge at 3000 g for 10
min. Remove organic phase and evaporate to dryness under a stream of helium at 30. Recon-
stitute in 20 |xL mobile phase, inject a 10 |xL aliquot.
HPLCVARIABLES
Guard column: 20 mm long Pelliguard LC-8 40 |xm (Supelco)
Column: 150 X 4.6 C8 5 |xm (Supelco)
Mobile phase: MeCN:buffer 50:50 (Buffer was 1.2 mL butylamine in 1 L 10 mM NaH2PO4, pH
adjusted to 3 with phosphoric acid.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: clobazam (k' 1.344)
OTHER SUBSTANCES
Extracted: desipramine, nortriptyline, amitriptyline, clomipramine
Simultaneous: nitrazepam, lorazepam, clonazepam, triazolam, flunitrazepam, alprazolam, di-
azepam, haloperidol, maprotiline
KEYWORDS
serum
REFERENCE
Segatti,M.R; Nisi,G.; Grossi,R; Mangiarotti,M.; Lucarelli,C. Rapid and simple high-performance liquid chro-
matographic determination of tricyclic antidepressants for routine and emergency serum analysis,
J.Chromatogr., 1991, 536, 319-325.
SAMPLE
Matrix: blood
Sample preparation: For each 1 mL plasma or serum add 10 |xL 14 (xg/mL trimipramine in
MeOH. Inject serum or plasma directly onto column A with mobile phase A, elute with mobile
phase A to waste. After 15 min elute column A onto column B (foreflush) with mobile phase B.
After 2 min remove column A from the circuit, elute column B with mobile phase B, monitor
the effluent from column B. Re-equilibrate column A with mobile phase A.
HPLC VARIABLES
Column: A 20 X 4.6 10 urn Hypersil MOS C8; B 20 X 4.6 5 |xm Hypersil CPS CN + 250 X 4.6
5 |xm Nucleosil 100 CN
Mobile phase: A MeOHrwater 5:95; B MeCNrMeOH:buffer 578:188:235 (Buffer was 10 mM
K2HPO4 adjusted to pH 6.8 with 85% phosphoric acid.)
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Limit of detection: 1 ng/mL (with three injections onto column A before switching), 5-10 ng/mL
OTHER SUBSTANCES
Extracted: metabolites, amitriptyline, clomipramine, desipramine, doxepin, fluvoxamine, ma-
protiline, nortriptyline
Noninterfering: chlordiazepoxide, clobazam, clozapine, diazepam, flurazepam, fluspirilene, hal-
operidol, nitrazepam, oxazepam, perazine, pimozide, spiroperidol, trifluperidol
KEYWORDS
REFERENCE
Hartter,S.; Hiemke,C. Column switching and high-performance liquid chromatography in the analysis of am-
itriptyline, nortriptyline and hydroxylated metabolites in human plasma or serum, J.Chromatogr., 1992,
578, 273-282.
SAMPLE
Matrix: blood
Sample preparation: Add 10 |xL 20 fxg/mL oxaprotiline in MeOH to 990 IJLL plasma or serum.
Inject 100 |xL plasma or serum onto column A with mobile phase A and elute to waste, after
mobile phase B.
HPLC VARIABLES
Column: A 20 X 4.6 10 |xm Hypersil MOS C8; B 20 X 4.6 5 |xm Hypersil CPS CN + 250 X 4.6
5 jxm Nucleosil 100 CN
Mobile phase: A MeOH:water 5:95; B MeOHiMeCN: 10 mM pH 6.8 potassium phosphate buffer
188:578:235
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 9.7
OTHER SUBSTANCES
Simultaneous: clozapine, fluvoxamine, metoclopramide, fluoxetine, norfluoxetine, nortriptyline,
desipramine, maprotiline, doxepin, clomipramine, amitriptyline
pam, carbamazepine
Interfering: oxaprotiline
KEYWORDS
REFERENCE
Hartter,S.; Wetzel,H.; Hiemke,C. Automated determination of fluvoxamine in plasma by column-switching high-
SAMPLE
Matrix: blood
MeOH, 1 mL water, and 1 mL 1% potassium carbonate. 700 |xL Serum + 50 )JLL 5 |jig/mL
trimipramine in 5% potassium bicarbonate + 700 (xL MeCN, vortex, centrifuge at 1500 g for 5
MeCN, elute with 250 |xL MeOH:35% perchloric acid 20:1 by gravity (10 min) then centrifuge
for 20 s to remove rest of eluant, inject a 50 jxL aliquot of the eluate.
HPLC VARIABLES
Guard column: 15 mm 7 |xm Brownlee RP-8
Column: 150 X 4.6 5 jxm Ultrasphere Octyl
Mobile phase: MeCN:water 37.5:62.5 containing 0.5 g/L tetramethylammonium perchlorate and
Flow rate: 1.5
Detector: UV 215
CHROMATOGRAM
Retention time: 7.1
OTHER SUBSTANCES
Extracted: amitriptyline, clomipramine, desipramine, doxepin, fluoxetine, maprotiline,
protriptyline
Interfering: desmethylmaprotiline, fluvoxamine, nortriptyline
KEYWORDS
serum; SPE
REFERENCE
Gupta,R.N. An improved solid phase extraction procedure for the determination of antidepressants in serum
by column liquid chromatography, J.Liq.Chromatogr., 1993, 16, 27512765.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL 200 ng/mL IS in MeOH + 1 mL 50 mM pH 10
borate buffer, vortex briefly, add to an Extrelut 3 SPE cartridge, let stand for 5 min, elute with
15 mL hexane:dichloromethane 50:50. Add the eluate to 3 mL 50 mM sulfuric acid, mix for 10
min, centrifuge at 3000 g for 10 min. Remove the aqueous layer and add it to 6 mL hexane:
dichloromethane 50:50, wash for 5 min, centrifuge. Make the aqueous layer basic with 150 JXL
28% ammonia, extract twice with 3 mL hexane:dichloromethane 50:50. Combine the organic
residue in 100 u,L mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 u-m Ultrasphere cyano
Mobile phase: MeCN.buffer 60:40 (Buffer was 50 mM KH2PO4 adjusted to pH 6.5 with 28%
ammonia.)
Flow rate: 1
Detector: E, 5100 A Coulochem, 5020 guard cell 1.00 V, 5011 analytical cell, detector 1 0.55 V,
detector 2 0.80 V, output of detector 2 is monitored
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: chlorpromazine, clomipramine, cyamemazine, desipramine, droperidol, flunitraze-
pam, haloperidol, pipamperone, risperidone
Noninterfering: alprazolam, bromazepam, carbamazepine, chlorazepate, diazepam, diphenylhy-
dantoin, estazolam, ethylbenzatropine, oxazepam, phenobarbital, triazolam, valproic acid
Interfering: trihexyphenidyl
KEYWORDS
plasma; SPE
REFERENCE
Le Moing,J.R; Edouard,S.; Levron,J.C. Determination of risperidone and 9-hydroxyrisperidone in human
614, 333-339.
SAMPLE
Matrix: blood
JJLL 0.1 M phosphoric acid (pH 2.5), mix, separate, inject a 100 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Flow rate: 2
Detector: UV 228
CHROMATOGRAM
Retention time: 4.6
OTHER SUBSTANCES
sipramine, dextromethorphan, diazepam, diphenhydramine, doxepin, encainide, fentanyl, fle-
cainide, fluoxetine, flurazepam, haloperidol, hydroxyethylflurazepam, ibuprofen, lidocaine,
methadone, methaqualone, mexiletine, midazolam, norchlorimipramine, nordiazepam, nordox-
epin, norfluoxetine, norverapamil, pentazocine, promazine, propafenone, propoxyphene, pro-
pranolol, protriptyline, quinidine, temazepam, trazodone, trimipramine
warfarin
Interfering: maprotiline, nortriptyline, verapamil
KEYWORDS
plasma; SPE
REFERENCE
Nichols, J.H.; Charlson,J.R.; Lawson,G.M. Automated HPLC assay of fluoxetine and norfluoxetine in serum,
Clin.Chem., 1994, 40, 1312-1316.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 100 jxL EtOH + 500 |xL 500 mM NaOH, mix, add to an
Extrelut-1 SPE cartridge, let stand for 10 min, elute with 5 mL n-hexane:isoamyl alcohol 98:
2. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue in 100
JJLL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 125 X 4.6 5 |xm Partisphere silica (Whatman)
Mobile phase: Hexane:EtOH:dichloromethane:diethylamine 77:18:5:0.003
Flow rate: 1.3
Detector: UV 214
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Extracted: clomipramine
Simultaneous: amitriptyline, desipramine, doxepin, fluoxetine
KEYWORDS
plasma; SPE; normal phase; imipramine is IS
REFERENCE
Altieri,L; Pichini,S.; Pacifici,R.; Zuccaro,P. Improved clean-up procedure for the high-performance liquid chro-
matographic assay of clomipramine and its demethylated metabolite in human plasma, J.Chromatogr.B,
1995, 669, 416-417.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 500 mM pH 9 phosphate buffer, vortex briefly, add
7 mL n-heptane:isoamyl alcohol 98.5:1.5, shake on a rotating shaker at 32 rpm for 15 min,
centrifuge at 1500 g for 5 min. Remove 6 mL of the organic layer and evaporate it to dryness
under a stream of nitrogen at 60, reconstitute the residue in 200 |xL mobile phase, vortex for
10 s, centrifuge at 1500 g for 3 min, inject a 100 |xL aliquot of the supernatant.
HPLCVARIABLES
Guard column: 20 X 4.6 5 |xm Supelguard LC-8-DB (Supelco)
Mobile phase: MeCN:MeOH:buffer 20:25:55 (Buffer was 50 mM pH 3.0 KH2PO4 containing 0.2%
triethylamine.)
Flow rate: 1
Detector: UV 235
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ticlopidine
KEYWORDS
REFERENCE
Dal Bo,L.; Verga,R; Marzo,A.; Ambrosoli,L.; Poli,A. Determination of ticlopidine in human plasma by high-
performance liquid chromatography and ultraviolet absorbance detection, J.Chromatogr.B, 1995,665,404-
409.
SAMPLE
Matrix: blood
HPLC VARIABLES
Column: 300 X 3.9 4 y,m NovaPack C18
Flow rate: 0.8
Detector: UV 251
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: blood
Sample preparation: 100 (xL Serum + 25 JJLL 5 |xg/mL clomipramine in MeOH, vortex for 30 s,
add 100 |xL 5 M NaOH, add 2 mL hexane, vortex for 30 s, centrifuge at 3000 g for 3 min.
constitute the residue in 50 JJLL mobile phase, vortex for 30 s, inject a 20 (JLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 jim Microsorb MV octadecyl
Mobile phase: MeCN: 10 mM triethylamine 60:40, pH adjusted to 3.0 with 85% phosphoric acid
Flow rate: 1
Detector: UV 260
CHROMATOGRAM
Retention time: 6.0
OTHER SUBSTANCES
Extracted: desipramine
KEYWORDS
REFERENCE
Yoo,S.D.; Holladay,J.W.; Fincher,T.K; Dewey,M.J. Rapid microsample analysis of imipramine and desipramine
by reversed-phase high-performance liquid chromatography with ultraviolet detection, J.Chromatogr.B,
1995, 668, 338-342.
SAMPLE
Matrix: blood, CSF, tissue
Sample preparation: Homogenize brain tissue in 4 mL 50 mM pH 7.4 Tris-HCl. 500 jxL Serum
or 200 |xL CSF or 1 mL homogenate + 100 jxL 2 M NaOH + IS + 5 mL hexane:dichloromethane
60:40, shake for 15 min, centrifuge at 4000 rpm for 10 min. Remove the organic layer and
evaporate it to dryness, reconstitute the residue in 100 |xL mobile phase, inject a 30 |xL aliquot.
HPLC VARIABLES
Column: 125 X 4 5 jjum Lichrospher RP select B
Mobile phase: MeCN:0.1% pH 4 diethylamine in water 40:60
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Internal standard: desmethylclomipramine chlorhydrate (6.85)
OTHER SUBSTANCES
Extracted: metabolites, desipramine
KEYWORDS
rat; serum; brain; pharmacokinetics
REFERENCE
Besret,L.; Debruyne,D.; Rioux,R; Bonvalot,T.; Moulin,M.; Zarifian,E.; Baron,J.-C. A comprehensive investiga-
tion of plasma and brain regional pharmacokinetics of imipramine and its metabolites during and after
chronic administration in the rat, J.Pharm.ScL, 1996, 85, 291-295.
SAMPLE
Sample preparation: Serum. 1 mL Serum + 800 ng nortriptyline + 4 mL MeOH + 5 mL 2.5%
perchloric acid, shake vigorously, centrifuge at 11000 g for 15 min. Add supernatant to 1 mL
4 M KOH, centrifuge. Add supernatant (9 mL) to 10 mL diethyl ethenethyl acetate 85:15,
shake for 15 min. Remove 8 mL of organic phase and evaporate it to dryness under a stream
of nitrogen. Dissolve residue in 200 JJLL mobile phase buffer:MeOH 9:1, inject 100 JXL aliquot.
Tissue. 2 g Brain tissue + 10 mL 2.5% perchloric acid + 8 mL MeOH + 1.6 |xg nortriptyline,
homogenize, centrifuge at 11000 g for 15 min. Add supernatant to 4 mL 4 M KOH, centrifuge.
Add supernatant to 20 mL diethyl ether:ethyl acetate 3:1, shake for 15 min. Remove 8 mL of
organic phase and evaporate it to dryness under a stream of nitrogen. Dissolve residue in 200
(xL mobile phase buffer:MeOH 9:1, inject 100 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Cosmosil 5C18
Mobile phase: MeOH.THF.buffer 45:17:88 (Buffer was 1% triethylamine adjusted to pH 3.0 with
phosphoric acid.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: nortriptyline (14.2)
Limit of detection: 10 ng/g (tissue)
OTHER SUBSTANCES
Simultaneous: desipramine
KEYWORDS
serum; rat
REFERENCE
Sugita,S.; Kobayashi,A.; Suzuki,S.; Yoshida,T.; Nakazawa,K. High-performance liquid chromatographic deter-
mination of imipramine and its metabolites in rat brain, J.Chromatogr., 1987, 421, 412-417.
SAMPLE
Sample preparation: Blood or serum. 1 mL Blood or serum + 1 jxg cianopramine + 1 mL water,
vortex, add 1 mL 200 mM sodium carbonate, vortex, add 6 mL hexanerl-butanol 95:5, gently
IxL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the organic
layer and inject a 30 jxL aliquot of the aqueous layer. Liver homogenate. 0.5 mL Liver ho-
mogenate + 10 (xg cianopramine + 500 |xL 2% sodium tetraborate + 8 mL hexane:l-butanol 95:
it to 400 JXL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the
organic layer and inject a 30 jxL aliquot of the aqueous layer.
HPLC VARIABLES
Guard column: 15 X 3.2 7 \xm RP-18 Newguard (Applied Biosystems)
Column: 100 X 4.6 5 jjum Brownlee Spheri-5 RP-18
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
doxepin, fluoxetine, haloperidol, loxapine, maprotiline, meperidine, mesoridazine, methadone,
metoclopramide, mianserin, moclobemide, nomifensine, nordoxepin, norfluoxetine, norpropox-
yphene, northiaden, nortriptyline, pentobarbital, pheniramine, promethazine, propoxyphene,
propranolol, protriptyline, quinidine, quinine, sulforidazine, thioridazine, tranylcypromine, tra-
zodone, trihexyphenidyl, trimipramine, triprolidine
trifluoperazine
Interfering: thiothixene
KEYWORDS
REFERENCE
SAMPLE
Matrix: blood, tissue, urine
Sample preparation: Serum, urine. 500 JJLL Serum or urine + 100 jxL 2 u-g/mL diazepam + 200
IxL 20% sodium carbonate + 500 |xL water + 3 mL n-hexane:isoamyl alcohol 98.5:1.5, mix for
2 min, centrifuge at 1200 g for 5 min. Remove the organic phase and evaporate it under a
gentle stream of nitrogen at about 40. Dissolve the residue in 100 |JLL mobile phase, inject a
10 |xL aliquot. Tissue. Homogenize 1 g sample with 9 mL 100 mM HCl and 100 |xL 20 |xg/mL
diazepam, centrifuge at 15000 g for 10 min. Add 500 (JLL 20% sodium carbonate and 4 mL n-
hexanerisoamyl alcohol 98.5:1.5 to 1 mL of the supernatant, mix for 5 min. Remove the organic
phase and evaporate it under a gentle stream of nitrogen at about 40. Dissolve the residue in
100 |xL mobile phase, filter by microconcentrator (Microcon-30, Grace). Inject a 10 |JLL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 2 |xm TSK gel Super-Octyl (A) or 100 X 4.6 5 |jim Hypersil MOS-C8 (B),
(Yokogawa, Japan)
Mobile phase: MeOH:20 mM pH 7 KH2PO4 60:40
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
Internal standard: diazepam (4.4, A)
Limit of quantitation: 50 ng/mL (serum, urine) (A), 500 ng/mL (tissue) (A)
OTHER SUBSTANCES
Extracted: amitriptyline, amoxapine, clomipramine, desipramine, dothiepin, doxepin, maproti-
line, melitracen, mianserin, nortriptyline
Noninterfering: barbital, carbamazepine, ethosuximide, hexobarbital, lofepramine, pentobarbi-
tal, phenobarbital, phenytoin, primidone, sulpiride, trimethadione, trimipramine
KEYWORDS
serum; brain; liver
REFERENCE
Tanaka,E.; Terada,M.; Nakamura,T.; Misawa,S.; Wakasugi,C. Forensic analysis of eleven cyclic antidepressants
in human biological samples using a new reversed-phase chromatographic column of 2 |xm porous micro-
spherical silica gel, J.Chromatogr.B, 1997, 692, 405-412.
SAMPLE
Sample preparation: 1 mL Plasma or urine + 20 |xL 10 |xg/mL pericyazine in MeOH, mix, add
0.2 mL 1 M sodium carbonate buffer to adjust pH to 9.6. Add 10 mL distilled diethyl ether,
shake on an automatic shaker for 10 min, centrifuge at 1000 g for 10 min in a refrigerated
centrifuge. Remove the upper organic layer and add 100|xL 100 mM orthophosphoric acid.
Shake for 10 min and centrifuge at 1000 g for 10 min. Discard the top layer and inject a 50
|JLL aliquot of the acid layer.
HPLC VARIABLES
Guard column: 40 X 4.6 10 |xm RP-18
Column: 300 X 3.9 Bondclone 10 C18 (Phenomenex)
Mobile phase: MeCN: 100 mM K2HPO4, adjust pH to 6.0 with orthophosphoric acid
Flow rate: 2
Detector: E, EDT Chromajet, oxidation cell +1.00 V
CHROMATOGRAM
Internal standard: pericyazine (6.2)
OTHER SUBSTANCES
KEYWORDS
plasma; pharmacokinetics; silanize glassware
REFERENCE
Chen,A.G.; Wing,Y.K.; Chiu,H.; Lee,S.; Chen,C.N.; Chan,K. Simultaneous determination of imipramine, desip-
ramine and their 2- and 10-hydroxylated metabolites in human plasma and urine by high-performance
liquid chromatography, J.Chromatogr.B, 1997, 693, 153-158.
SAMPLE
Sample preparation: 1 mL Plasma or urine + 1 mL 600 mM pH 11.3 K2CO3 + 100 |JLL 5 (plasma)
or 20 (urine) [iM 2-hydroxydesmethylclomipramine in EtOH + 5 mL heptane:MTBE 1:1 + 5%
n-butanol, vortex 1 min, centrifuge at 1400 g for 10 min, freeze at -50 (dry ice/ethanol). Remove
organic layer and add it to 1 mL 20 mM HCl, vortex 1 min, centrifuge at 1400 g for 10 min,
freeze at -50 (dry ice/ethanol). Discard organic layer. Thaw out aqueous layer and make al-
kaline (pH 11) by adding 500 |xL 600 mM pH 11.3 K2CO3. Add 3 mL heptane:MTBE 1:1 + 5%
n-butanol, vortex 1 min, centrifuge at 1400 g for 10 min, freeze at -50 (dry ice/ethanol). Remove
organic layer and evaporate it to dryness at 50 under a stream of nitrogen. Dissolve residue
in 100 |JLL mobile phase, vortex for 5 s, centrifuge at 1400 g for 1 min, inject a 20 |JLL aliquot.
HPLC VARIABLES
Guard column: 20 X 4 7 |xm 120 A Nucleosil
Column: 250 X 4 5 |xm 100 A Nucleosil RP-phenyl
Mobile phase: MeCN:buffer 30:70 (Buffer was 14.05 g sodium perchlorate and 1.6 mL 60% per-
chloric acid in 5 L water, pH 2.5.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Internal standard: 2-hydroxydesmethylclomipramine (10.02)
Limit of detection: 10 nM (urine), 5 nM (plasma)
OTHER SUBSTANCES
Simultaneous: desipramine, metabolites
KEYWORDS
plasma
REFERENCE
Nielsen,K.K; Brsen,K. High-performance liquid chromatography of imipramine and six metabolites in human
plasma and urine, J.Chromatogr., 1993, 612, 87-94.
SAMPLE
Sample preparation: 100 |xL Serum or urine + 25 |xL 5 |xg/mL clomipramine in MeOH + 100
|JLL 5 M NaOH + 2 mL hexane, vortex for 30 s, centrifuge at 5000 rpm for 3 min. Remove the
HPLC VARIABLES
Column: 10 X 4.6 5 |xm Microsorb MV C18
Mobile phase: MeCN: 10 mM triethylamine in water 60:40, pH adjusted to 3.0 with 85% phos-
phoric acid
Flow rate: 1
Detector: UV 260
CHROMATOGRAM
Internal standard: clomipramine
OTHER SUBSTANCES
Extracted: desipramine
KEYWORDS
REFERENCE
Yoo,S.D.; Holladay,J.W.; Fincher,T.K; Baumann,H.; Dewey,M.J. Altered disposition and antidepressant activity
of imipramine in transgenic mice with elevated a-l-acid glycoprotein, J.Pharmacol.Exp.Ther., 1996, 276,
918-922.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 |i,m Symmetry C8 (Waters)
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Crush tablet or capsule, to 2 mg amitriptyline add 20 mL MeOH, shake
30 min, centrifuge at 2000 rpm for 5 min, to 5 mL supernatant add 4 mL 1.25 mg/mL nor-
ephedrine.HCl in MeOH, dilute to 10 mL with MeOH.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Zorbax CN
Mobile phase: MeCN:MeOH:25 mM pH 4.8 sodium acetate-acetic acid buffer 35:45:20
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Retention time: 3.8
Internal standard: norephedrine (2.7)
OTHER SUBSTANCES
Also analyzed: chlorpromazine, amitriptyline, thioridazine, trifluoperazine
KEYWORDS
tablets; capsules
REFERENCE
Lovering,E.G.; Beaulieu,N.; Lawrence,R.C; Sears,R-W. Liquid chromatographic method for identity, assay, and
content uniformity of five tricyclic drugs, J.Assoc.Off.Anal.Chem., 1985, 68, 168171.
SAMPLE
Matrix: hair
hair + 1 mL 1 M NaOH, heat at 70 for 30 min, adjust pH to 9.5-10. 1 mL Extract + 1 fig
protriptyline + 1 mL water + 1 mL 200 mM sodium carbonate buffer, mix, extract with hexane:
butanol 95:5 for 20 min. Remove the organic layer and add it to 100 u,L 0.2% orthophosphoric
acid, mix for 20 min, inject a 30 JULL aliquot of the aqueous layer.
HPLC VARIABLES
diethylamine.)
Flow rate: 2
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: amitriptyline, clomipramine, desipramine, dothiepin, doxepin, haloperidol, mian-
serin, nortriptyline
KEYWORDS
REFERENCE
Couper,F.J.; McIntyre,I.M.; Drummer,O.H. Extraction of psychotropic drugs from human scalp hair, J.Forensic
ScL, 1995,40,83-86.
SAMPLE
Sample preparation: Add 200 |xL MeCN to 200 |xL microsomal incubation, centrifuge at 3000
g for 5 min, inject an aliquot of the supernatant.
HPLCVARIABLES
Column: 150 X 4.6 Zorbax Phenyl
Mobile phase: MeCNibuffer 50:50 (Buffer was 20 mM perchloric acid adjusted to pH 2.5 with
NaOH.)
Flow rate: 1
Detector: Radioactivity, Inus p-Ram using Inus Tru-Count scintillation fluid at a flow rate of 5
mL/min
CHROMATOGRAM
Retention time: 4.7
OTHER SUBSTANCES
KEYWORDS
human; liver
REFERENCE
Obach,R.S. Nonspecific binding to microsomes: Impact on scale-up of in vitro intrinsic clearance to hepatic
clearance as assessed through examination of warfarin, imipramine, and propranolol, Drug Metab.Dispos.,
1997, 25, 1359-1369.
SAMPLE
Sample preparation: Condition a C18 SepPak SPE cartridge with 5 mL MeOH and 5 mL buffer.
1 mL Microsomal incubation + 500 (xL buffer, mix, add a 500 |xL aliquot to the SPE cartridge,
wash with 4 mL MeOH:buffer containing 0.01% ascorbic acid 5:95, elute with 5 mL MeOH,
evaporate eluate to dryness, reconstitute in mobile phase, inject an aliquot. (Buffer was 100
mM pH 3.0 potassium acetate buffer containing 5 mM n-heptanesulfonic acid.)
HPLC VARIABLES
Guard column: used but not specified
Column: 100 X 8 4 |xm Nova-Pak C18
Mobile phase: Gradient. MeOH:MeCN:buffer 20:35:45 for 20 min, then 30:50:20 for 25 min, then
35:60:5
Flow rate: 0.4
Detector: UV 254
CHROMATOGRAM
Retention time: 33
OTHER SUBSTANCES
Extracted: metabolites, desipramine, lofepramine
KEY WORDS
rat; human; liver; SPE
REFERENCE
Strandgarden,K; Gunnarsson,RO. Metabolism of lofepramine and imipramine in liver microsomes from rat
and man, Xenobiotica, 1994, 24, 703-711.
SAMPLE
Sample preparation: 1 mL Microsomal incubation + 500 |xL 2 M pH 12 sodium carbonate + 5
mL ether, vortex, centrifuge at 1000 g for 10 min. Remove the organic layer and add it to 1
mL 100 mM HCl, vortex, centrifuge. Remove the aqueous layer and add it to 100 |xL 2 M pH
12 sodium carbonate and 1 mL ether, extract. Remove the organic layer and evaporate it to
dryness under a stream of nitrogen, reconstitute the residue in 100 JJLL mobile phase, inject a
50 JJLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-PCN
Mobile phase: MeCN:MeOH:10 mM pH 7 K2HPO4 40r35r25
Flow rate: 1.4
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
rat; liver; brain
REFERENCE
Sequeira,D.J.; Strobel,H.W. High-performance liquid chromatographic method for the analysis of imipramine
metabolism in vitro by liver and brain microsomes, J.Chromatogr.B, 1995, 673, 251-258.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:50 mM phosphoric acid 50:50 containingl50 mM KCl
Flow rate: 0.6
Detector: UV 251
REFERENCE
Sugawara,M.; Takekuma,Y; Yamada,H.; Kobayashi,M.; Iseki,K.; Miyazaki,K. A general approach for the pre-
drug-membrane electrostatic interactions, J.Pharm.ScL, 1998, 87, 960-966.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jig/mL solution in MeOH, inject a 20 JJLL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
operidol, hydroxyzine, hyoscine, ibogaine, indapamine, iprindole, isothipendyl, isoxsuprine,
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 u-g/mL, inject a 10
JJLL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 fxm ixBondapak C18
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 500 |jig/mL solution in MeOHrwater 50:50, inject a 5 JJLL aliquot.
HPLC VARIABLES
in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL triethylamine in 1 L
MeCN:water 20:80. A:B from 100:0 to 0:100 over 30 min. (Purify triethylamine as follows. Wash
neutral alumina (Merck) 3 times with 2 bed volumes of pentane, 3 times with 2 bed volumes
of dichloromethane, and 3 times with 2 bed volumes of MeOH, allow solvent to evaporate in a
fume hood overnight, heat alumina at 130 for 2 h. Prepare a 14 cm column of the washed
alumina in a 290 X 22 tube, pass through a head volume of MeOH, pass through triethylamine.
When triethylamine starts to elute discard the first 20 mL, use the next 20 mL, discard the
column.)
Flow rate: 2
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: acetophenone, amphetamine, desipramine, ethylmorphine, mefenamic acid,
methamphetamine, morphine, phenylbutazone, salicylic acid
KEYWORDS
also details of isocratic elution
REFERENCE
Hill,D.W. Evaluation of alkyl bonded silica and solvent phase modifiers for the efficient elution of basic drugs
on HPLC, J.Liq.Chromatogr., 1990, 13, 3147-3175.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 u.m Adsorbosphere C18 (PEEK column) (retention times are longer and
peaks broader with stainless steel column)
Mobile phase: MeCN:20 mM pH 3.2 KH2PO4 23.4:76.6 containing 0.05% nonylamine
Flow rate: 1.2
Detector: UV 214
CHROMATOGRAM
Retention time: 11
OTHER SUBSTANCES
Simultaneous: ami trip tyline, desmethyldoxepin, desipramine, doxepin, loxapine, maprotiline,
nortriptyline, trazodone
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Econosil C8
Mobile phase: MeCNrbuffer 30:70 (Buffer was 20 mM KH2PO4 and 14 mM triethylamine ad-
justed to pH 3.0 with phosphoric acid.)
Detector: UV 210
CHROMATOGRAM
Retention time: 8.3
Limit of quantitation: < 1000 ng/mL
OTHER SUBSTANCES
Simultaneous: amitriptyline, amoxapine, carbamazepine, nortriptyline
Also analyzed: doxepin, desipramine, protriptyline, cyclobenzaprine, maprotiline
KEYWORDS
UV spectra given
REFERENCE
Ryan,T.W. Identification and quantification of tricyclic antidepressants by UV-photodiode array detection with
multicomponent analysis, J.Liq.Chromatogr., 1993, 16, 1545-1560.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 0.5
Detector: UV 200
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
Simultaneous: desmethyldoxepin, doxepin, desipramine, nortriptyline, amitriptyline
Also analyzed: amphetamine, chlordiazepoxide, chlorpromazine, desalkylflurazepam, diazepam,
diethylpropion, ephedrine, fenfluramine, flurazepam, mesoridazine, methamphetamine, nor-
chlordiazepoxide, nordiazepam, oxazepam, phentermine, phenylpropanolamine, prazepam, pro-
mazine, thioridazine, thiothixene, trifluoperazine
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Lichrosphere cyanopropyl
Mobile phase: Carbon dioxide:MeOH:isopropylamine 90:10:0.05
Flow rate: 3
Injection volume: 5
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: benactyzine, buclizine, hydroxyzine, perphenazine, thioridazine, amitriptyline,
desipramine, nortriptyline, protriptyline
KEYWORDS
REFERENCE
Berger,T.A.; Wilson,W.H. Separation of drugs by packed column supercritical fluid chromatography. 2. Anti-
depressants, J.Pharm.Scl, 1994, 83, 287-290.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
fiuorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide,
hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, indo-
methacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ke-
mepacrine, meperidine, mephentermine, mephen3rtoin, mephesin, mephobarbital, mepivacaine,
naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, ninumic acid, nitrazepam, nor-
epinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxj^phen-
puromycin, pyrilamine, pjrrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Supelcosil LC-DP (A) or 250 X 4 5 /xm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
droxychloroquine, hydroxyzine, ibuprofen, indomethacin, ketoconazole, ketoprofen, ketorolac,
dine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxjnnetazoline, paroxetine, pemo-
phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenôin, pimo-
tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifiuoperazine, triflupromazine, tri-
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1-10 jxg/mL solution in water, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 *mi Hypersil SCX/C18
Mobile phase: MeCN:25 mM pH 3 Na2HPO4 50:50
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: amitriptyline, barbital, benzoic acid, butabarbital, clomipramine, clonazepam,
desipramine, diazepam, flurazepam, furosemide, nitrazepam, phenobarbital, phenol, phenol-
phthalein, pindolol, propranolol, resorcinol, salicylic acid, secobarbital, terbutaline, xylazine
KEYWORDS
effect of mobile phase pH on capacity factor is discussed
REFERENCE
Walshe,M.; Kelly,M.T.; Smyth,M.R.; Ritchie,H. Retention studies on mixed-mode columns in high-performance
liquid chromatography, J.ChromatogrA, 1995, 708, 31-40.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
operidol, hydroxyzine, indomethacin, lidocaine, megestrol acetate, metoprolol, nabumetone,
KEYWORDS
REFERENCE
SAMPLE
Matrix: urine
Sample preparation: 500 |xL Urine + N-ethylnordiazepam + chlorpheniramine + 100 jxL buffer,
IxL mobile phase B, with 200 JJLL mobile phase C, and with 1.15 mL mobile phase D. Elute
HPLC VARIABLES
3.2 11 fjim Aminex A-28 (Bio-Rad); C 25 X 3.2 5 jxm C8 (Phenomenex) + 150 X 4.6 5 |xm silica
(Macherey-Nagel)
CHROMATOGRAM
OTHER SUBSTANCES
Interfering: flurazepam, amitriptyline
KEYWORDS
column-switching
REFERENCE
341.
SAMPLE
Matrix: vitreous humor
Sample preparation: 600 JULL Vitreous humor + 3 mL 0.1 M NaCl + 50 |xL 4 |xg/mL desmethyl-
clomipramine in water, mix for a few s, add to a C18 SepPak attached to a 5 mL syringe, allow
to flow through (10-15 min). Wash with 1 mL 0.1 M NaCl, wash with 1 mL water, wash 3 mL
reagent by gravity. Elute with 3 mL MeOH and push air through to remove as much as possible.
Evaporate in vacuum at 37, vortex with 50 |xL mobile phase for 1 min, inject 25 |xL aliquot.
(Reagent was isopropanol:n-heptane:l M sulfuric acid 40:320:1.)
HPLC VARIABLES
Guard column: 50 X 4.6 30 jxm Permaphase ETH
Column: 250 X 4.6 5-6 |xm Zorbax cyanopropyl
Mobile phase: MeCN:0.5 M acetic acid:n-butylamine 40:60:0.0022
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Retention time: 22
Internal standard: Desmethylclomipramine
OTHER SUBSTANCES
Simultaneous: amitriptyline, doxepin, nortriptyline, metabolites
Noninterfering: acetaminophen, N-acetylprocainamide, amikacin, caffeine, carbamazepine,
chloramphenicol, clonazepam, cyclosporine, diazepam, digoxin, disopyramide, ethosuximide,
flurazepam, gentamicin, haloperidol, kanamycin, lidocaine, meprobamate, methapyriline,
methaqualone, methotrexate, methyprylon, netilmicin, pentazocine, pentobarbital, phenobar-
bital, phenytoin, prazepam, primidone, procainamide, propranolol, quinidine, salicylic acid, sec-
obarbital, streptomycin, theophylline, tobramycin, tocainide, valproic acid, vancomycin.
REFERENCE
Evenson,M.A.; Engstrand,D.A. A SepPak HPLC method for tricyclic antidepressant drugs in human vitreous
humor, JAnal.ToxicoL, 1989, 13, 322-325.
lndalpine
Merck Index: 4965
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 uL 1 |xg/mL IS in MeOH + 200 |xL 5 M NaOH, vortex
for 1 min, add 4 mL dichloromethane, shake for 15 min, centrifuge at 3000 g for 10 min, repeat
extraction. Combine the organic layers and evaporate them to dryness under a stream of ni-
trogen at 38, reconstitute the residue in 50 |JLL mobile phase, vortex, centrifuge at 3000 g for
5 min, inject a 10 |JLL aliquot.
HPLC VARIABLES
Guard column: 20 X 3 Bondapak C18/Corasil
Column: 300 X 3.9 10 um fxBondapak C18
Mobile phase: MeOH: 10 mM K2HPO4:acetic acid 50:50:1
Flow rate: 1
CHROMATOGRAM
Retention time: 8
Internal standard: 4-[(5-methoxy-3-indolyl)methyl]piperidine (PK 26042) (5)
OTHER SUBSTANCES
KEY WORDS
REFERENCE
Jozefczak,C; Ktorza,N.; Uzan,A. High-performance liquid chromatographic determination of indalpine, a new
non-tricyclic antidepressant, in human plasma: identification and simultaneous measurement of its major
plasma metabolite, J.Chromatogr., 1982, 230, 87-95.
SAMPLE
Sample preparation: 200 JJLL Plasma + 10 IJLL 1 |xg/mL IS in MeOH + 50 |xL 1 M NaOH + 4 mL
dichloromethane, shake on an alternating agitator for 20 min, centrifuge at -4 at 2000 g for
15-20 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen
at 38, reconstitute the residue in 100 |xL mobile phase, inject a 50 |xL aliquot. Tissue. Mouse
brain + 25 |xL 1 jig/mL IS in MeOH + 3 mL 1 M perchloric acid, homogenize in a potter
apparatus (Ultraturax), centrifuge at 2000 g for 15 min. Remove the supernatant and adjust
the pH to 12 with 1 M NaOH, add 4 mL dichloromethane, shake on an alternating agitator
for 20 min, centrifuge at -4 at 2000 g for 15-20 min. Remove the organic layer and evaporate
phase, inject a 50 (xL aliquot.
HPLC VARIABLES
Mobile phase: MeOH:water:5 mM hexanesulfonic acid 52:48:2
Flow rate: 0.8
Detector: E, Metrohm 641 VA, 1000 mV, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 5.8
Internal standard: 5-methoxy-3-(4-piperidylmethyl)indole (3.6) (structure shown is 3-(4-
piperidylmethyDindole)
OTHER SUBSTANCES
KEYWORDS
mouse; plasma; brain; pharmacokinetics
REFERENCE
Joulin,Y.; Doare,L.; Diquet,B. Micromethod for the determination of indalpine in mouse plasma using high-
performance liquid chromatography with electrochemical detection, J.Chromatogr., 1986, 381, 457-463.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:MeOH:buffer 13:35:52 (Buffer was 6.5 g/L (?) KH2PO4 adjusted to pH 3
with orthophosphoric acid.)
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Retention time: 2.9
OTHER SUBSTANCES
Simultaneous: amitriptyline, bromazepam, chlorpromazine, clobazam, clomipramine, desipra-
mine, diazepam, flunitrazepam, haloperidol, imipramine, levomepromazine, lorazepam, ma-
protiline, metapramine, mianserin, oxazepam, prochlorperazine, triazolam
Noninterfering: meprobamate
REFERENCE
Rouquette,C; Hecquet,D.; Pommereau,X.; Gardere,J.J.; Brachet-Liermain,A. Metapramine overdose: report of
two cases and analytical determinations, J.Anal.Toxicol., 1985, 9, 275-277.
lndapamide
Merck Index: 4969
Lednicer No.: 2 349
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.0
OTHER SUBSTANCES
operidol, hydroxyzine, hyoscine, ibogaine, imipramine, iprindole, isothipendyl, isoxsuprine,
Next Page
REFERENCE
Indeloxazine hydrochloride
Merck Index: 4972
Lednicer No.: 4 59
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 300 ng/mL viloxazine in water + 500 |xL 200 mM
ammonium hydroxide + 4 mL ether, extract, centrifuge. Remove the organic layer and evapo-
rate it to dryness at 45, reconstitute the residue in 100 jxL 3 mg/mL sodium bicarbonate and
200 |xL 125 |xg/mL dansyl chloride in acetone, heat at 45 for 20 min, cool, add 4 mL ether.
Wash the ether solution twice with 3 mL water and evaporate it to dryness at 45. Take up
the residue in 100 |JLL n-heptane, inject a 3-5 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4 5 |xm LiChrosorb SI-60
Mobile phase: n-Heptane:ethyl acetate 20:3
Flow rate: 1.5
CHROMATOGRAM
Retention time: 3
Internal standard: viloxazine (4)
KEYWORDS
plasma; normal phase; derivatization
REFERENCE
Kamimura,H.; Sasaki,H-l Yokoi,K; Kawamura,S. Determination of indeloxazine in plasma by liquid chroma-
tography and gas chromatography-mass spectrometry, J.Pharm.ScL, 1985, 74, 559-561.
Indinavir
CAS Registry No.: 150378-17-9, 157810-81 -6 (sulfate)
Merck Index: 4979
SAMPLE
M a t r i x : blood
Sample preparation: 300 (xL Plasma + 300 jxL 50 mM pH 9.0 ammonium dihydrogen phosphate
buffer + 30 (xL 10.5 |xg/mL IS in water, vortex for 10 s, add 3 mL diethyl ether, vortex for 30
Previous Page
REFERENCE
Indeloxazine hydrochloride
Merck Index: 4972
Lednicer No.: 4 59
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 300 ng/mL viloxazine in water + 500 |xL 200 mM
ammonium hydroxide + 4 mL ether, extract, centrifuge. Remove the organic layer and evapo-
rate it to dryness at 45, reconstitute the residue in 100 jxL 3 mg/mL sodium bicarbonate and
200 |xL 125 |xg/mL dansyl chloride in acetone, heat at 45 for 20 min, cool, add 4 mL ether.
Wash the ether solution twice with 3 mL water and evaporate it to dryness at 45. Take up
the residue in 100 |JLL n-heptane, inject a 3-5 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4 5 |xm LiChrosorb SI-60
Mobile phase: n-Heptane:ethyl acetate 20:3
Flow rate: 1.5
CHROMATOGRAM
Retention time: 3
Internal standard: viloxazine (4)
KEYWORDS
plasma; normal phase; derivatization
REFERENCE
Kamimura,H.; Sasaki,H-l Yokoi,K; Kawamura,S. Determination of indeloxazine in plasma by liquid chroma-
tography and gas chromatography-mass spectrometry, J.Pharm.ScL, 1985, 74, 559-561.
Indinavir
CAS Registry No.: 150378-17-9, 157810-81 -6 (sulfate)
Merck Index: 4979
SAMPLE
M a t r i x : blood
Sample preparation: 300 (xL Plasma + 300 jxL 50 mM pH 9.0 ammonium dihydrogen phosphate
buffer + 30 (xL 10.5 |xg/mL IS in water, vortex for 10 s, add 3 mL diethyl ether, vortex for 30
s, keep at -20 for 30 min. Remove the ether layer and evaporate it to dryness under nitrogen.
Reconstitute the residue with 150 |xL 10 mM pH 5.5 ammonium dihydrogen phosphate buffer,
centrifuge at 750 g for 5 min, inject a 35 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 5 |xm Delta-pak C4 (Waters)
Mobile phase: MeCN:buffer 35:65 (Buffer was 10 mM ammonium dihydrogen phosphate and 1
mM 1-heptanesulfonic acid sodium salt, pH adjusted to 4.8 with ammonium hydroxide.)
Flow rate: 0.6
Detector: UV 210
CHROMATOGRAM
Retention time: 13.2 0.5
Internal standard: methylindinavir (methyl at 5 position on pyridine) (14.1 0.7)
OTHER SUBSTANCES
Simultaneous: nelfinavir, ritonavir, saquinavir
Noninterfering: didanosine, lamivudine, stavudine, zalcitabine, zidovudine
KEYWORDS
REFERENCE
Iayewardene,AL-; Zhu,R; Aweeka,F.T.; Gambertoglio,J.G. Simple high-performance liquid chromatographic de-
termination of the protease inhibitor indinavir in human plasma, J.Chromatogr.B, 1998, 707, 203-211.
SAMPLE
Matrix: blood
Sample preparation: Add 50 jxL 1 jxg/mL IS in MeCN:water 50:50 to 1 mL plasma and vortex.
Add 1 mL 100 mM pH 9.5 borate buffer, vortex, add 8 mL isopropanolxhloroform 1:15 (Caution!
Chloroform is a carcinogen!), mix on a flat-bed shaker for 15 min, centrifuge at 1500 g for 5
min. Remove the lower organic layer and evaporate it under a stream of nitrogen at 45.
Reconstitute the residue in 1 mL mobile phase, inject a 50 u,L aliquot.
HPLC VARIABLES
Column: 50 X 3.9 5 |jim Symmetry C8
Mobile phase: MeCN:water 40:60 containing 2 mM ammonium acetate
Flow rate: 1
Detector: MS, PE-SCIEX API IIIplus triple quadrupole, heated nebulizer, corona discharge nee-
dle (+4.5 |iA), nebulizer 500, collision gas argon at 260 X 1012 atoms/cm2, nebulizing gas ni-
trogen at 80 psi and 0.6 mL/min, orifice +75 V, electron multiplier -4.0 kV, dwell time 400 ms
with a 30 ms pause time between scans, Ql at m/z 523, Q2 at m/z 512, Q3 at m/z 273
CHROMATOGRAM
Retention time: 1.5
Internal standard: structure given in paper
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Woolf,E.; Haddix,H-M; Matuszewski,B- Determination of an in vivo metabolite of a human immunodeficiency
virus protease inhibitor in human plasma by high-performance liquid chromatography with tandem mass
spectrometry, J.ChromatogrA, 1997, 762, 311-319.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 |xL MeCNrwater 50:50 + 25 |JLL 1 |xg/mL IS in MeCN,
vortex, add 1 mL 100 mM pH 9.5 borate buffer, vortex, add 8 mL MTBE, mix on a Glas Col
(Terre Haute, IN) RD 350 rotator for 15 min, centrifuge at 1500 g for 5 min, freeze the aqueous
phase in a dry ice-acetone bath. Decant the organic layer and evaporate it under a stream of
nitrogen at 42. Reconstitute the residue in 175 |xL mobile phase, inject a 6 |xL aliquot.
HPLC VARIABLES
Column: 50 X 2 3 |jim BDS Hypersil C8
Mobile phase: MeCN:water 40:60 containing 7 mM pH ammonium acetate, adjusted to pH 4.9
with formic acid
Flow rate: 0.2
Injection volume: 6
Detector: MS, PE-Sciex API IIIplus triple quadrupole, turbo-ion spray, turbo probe at 500, aux-
iliary gas nitrogen, nebulizing gas nitrogen at 80 psi, positive ion mode, interface sprayer at
+4 kV, sampling orifice at +60 V, m/z 614
CHROMATOGRAM
Retention time: 2.6
Internal standard: indinavir analog (4.2)
OTHER SUBSTANCES
Extracted: hexadeuterated indinavir (m/z 620)
KEYWORDS
REFERENCE
Woolf,E.J.; Matuszewski,B.K. Simultaneous determination of unlabeled and deuterium-labeled indinavir in
human plasma by high-performance liquid chromatography ith tandem mass spectrometric detection,
J.Pharm.ScL, 1997, 86, 193-198.
SAMPLE
Sample preparation: Add four volumes of MeCN to microsomal incubation, centrifuge at 1500
g for 10 min, evaporate supernatant to dryness under nitrogen at 40, reconstitute the residue
in 100-120 JJLL mobile phase, inject an 80 jxL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Hypersil 5C18 (Phenomenex)
Mobile phase: Gradient. A was MeCN. B was 0.3% pH 6 triethylamine in water. A:B from 25:
75 to 45:55 over 18 min
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
human; liver
REFERENCE
Koudriakova,T.; Iatsimirskaia,E.; Utkin,I.; Gangl,E.; Vouros,R; Storozhuk,E.; Orza,D.; Marinina,J.; Gerber,N.
Metabolism of the human immunodeficiency virus protease inhibitors indinavir and ritonavir by human
intestinal microsomes and expressed cytochrome P4503A4/3A5: Mechanism-based inactivation of cyto-
chrome P4503Aby ritonavir, Drug Metab.Dispos., 1998, 26, 552-561.
SAMPLE
Sample preparation: Add four volumes of 1-chlorobutane containing 50 ng/mL 5-methoxypsor-
alen to microsomal incubation, mix vigorously, centrifuge at 1500 g for 10 min, evaporate 1-
chlorobutane extract to dryness under nitrogen, reconstitute the residue in 100 |xL mobile
phase, inject an 80 JJLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Hypersil 5C18 (Phenomenex)
Mobile phase: Gradient. A was MeCN. B was 0.3% pH 6 triethylamine in water. A:B from 25:
75 to 45:55 in 18 min
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Internal standard: 5-methohypsoralen (14.6)
OTHER SUBSTANCES
KEYWORDS
human; liver
REFERENCE
Koudriakova,T.; Iatsimirskaia,E.; Utkin,L; Gangl,E.; Vouros,R; Storozhuk,E.; Orza,D.; Marinina,J.; Gerber,N.
Metabolism of the human immunodeficiency virus protease inhibitors indinavir and ritonavir by human
intestinal microsomes and expressed cytochrome P4503A4/3A5: Mechanism-based inactivation of cyto-
chrome P4503A by ritonavir, Drug Metab.Dispos., 1998, 26, 552-561.
lndobufen
Molecular formula: Ci8H17NO3
Merck Index: 4991
SAMPLE
Matrix: blood
Sample preparation: 0.5-1 mL Plasma + 500 jxL water + 100 |xL 600 mM sulfuric acid + 40 mg
NaCl + 4 mL diethyl ether, extract on a rotamixer, centrifuge at 1200 g. Remove the organic
layer and evaporate it to dryness under a stream of air at 30, add 5 drops toluene, evaporate
to dryness under a stream of air at 30, reconstitute the residue in 200 |xL 50 mM triethylamine
in MeCN, add 100 |JLL 60 mM ethyl chloroformate in MeCN, after 30 s add 100 |xL 1 M 1-
leucinamide hydrochloride in MeOH containing 1 M triethylamine, after 2 min add 500 |xL 250
mM HCl, extract with 4 mL ethyl acetate. Evaporate the organic layer to dryness under a
stream of air at 30, reconstitute the residue with 500 |xL MeOH, inject a 10 |xL aliquot.
HPLC VARIABLES
Guard column: 30 X 4 Perisorb RP-18 (Merck)
Column: 250 X 4 7 |xm LiChroCart RP-18 (Merck)
Flow rate: 2
Detector: UV 275
CHROMATOGRAM
Retention time: 6 (-), 8 (+) (assignment tentative)
Internal standard: indobufen
OTHER SUBSTANCES
Extracted: indoprofen
KEYWORDS
plasma; derivatization; chiral; indobufen is IS
REFERENCE
Bjorkman,S. Determination of the enantiomrs of indoprofen in blood plasma by high-performance liquid chro-
matography after rapid derivatization by means of ethyl chloroformate, J.Chromatogr., 1985,339, 339-346.
SAMPLE
Matrix: blood
Sample preparation: 0.5-1 mL Plasma + 100 jxL 600 mM sulfuric acid + 40 mg NaCl + 4 mL
diethyl ether, extract by rotation, centrifuge at 1200 g. Remove the organic layer and evaporate
it to dryness under a stream of air at 30, add 5 drops of toluene, evaporate to dryness under
a stream of air. Reconstitute the residue in 200 |xL 50 mM triethylamine in MeCN, add 100
JXL 60 mM ethyl chloroformate in MeCN, let stand for 30 s, add 100 |xL 1 M leucinamide
hydrochloride in MeOH containing 1 M triethylamine, let stand for 2 min, add 500 |xL 250 mM
HCl, extract with 4 mL ethyl acetate. Remove the organic layer and evaporate it to dryness
under a stream of air at 30, reconstitute in 500 |AL MeOH, inject a 10 |xL aliquot
(J.Chromatogr. 1985, 339, 339).
HPLC VARIABLES
Column: 300 X 3.9 10 \im ixBondapak C18
Flow rate: 1.5
Detector: UV 275
CHROMATOGRAM
KEYWORDS
chiral; derivatization
REFERENCE
Perrone,G.; Farina,M. High-performance liquid chromatographic method for direct resolution of the indobufen
enantiomeric components, J.Chromatogr., 1990, 520, 373-378.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Chiralcel OD
Mobile phase: Hexane:isopropanol:formic acid 80:20:0.5
Flow rate: 1.5
Detector: UV 270
CHROMATOGRAM
KEYWORDS
chiral; a = 1.33
REFERENCE
Perrone,G.; Farina,M. High-performance liquid chromatographic method for direct resolution of the indobufen
enantiomeric components, J.Chromatogr., 1990, 520, 373-378.
SAMPLE
Matrix: urine
Sample preparation: 500 JJLL Urine + 50 |xL 5000 U/mL p-glucuronidase (Sigma) + 2 mL 100
mM pH 5 acetate buffer, heat at 37 for 16 h, add S-indoprofen, add 1 mL 1 M HCl, extract
with diethyl ether. Remove the organic phase and extract it with 500 JJLL 1 M NaOH. Remove
the aqueous phase and add it to 1 mL 1 M HCl, extract with diethyl ether. Remove the organic
layer and evaporate it to dryness under a stream of air at 30, add 5 drops of toluene, evaporate
to dryness under a stream of air. Reconstitute the residue in 200 JJLL 50 mM triethylamine in
MeCN, add 100 |xL 60 mM ethyl chloroformate in MeCN, let stand for 30 s, add 100 |xL 1 M
leucinamide hydrochloride in MeOH containing 1 M triethylamine, let stand for 2 min, add
500 |JLL 250 mM HCl, extract with 4 mL ethyl acetate. Remove the organic layer and evaporate
it to dryness under a stream of air at 30, reconstitute in 500 |xL MeOH, inject a 10 JJIL aliquot
(J.Chromatogr. 1985, 339, 339).
HPLC VARIABLES
Column: 125 x 4 4 |xm Lichrocart Superspher (Merck)
Detector: UV 275
CHROMATOGRAM
Internal standard: indoprofen
KEYWORDS
chiral; derivatization; pharmacokinetics
REFERENCE
Strolin Benedetti,M.; Frigerio,E.; Tamassia,V.; Noseda,G.; Caldwell,J. The dispositional enantioselectivity of
indobufen in man, Biochem.Pharmacol., 1992, 43, 2032-2034.
SAMPLE
Matrix: urine
Sample preparation: Inject an aliquot.
HPLCVARIABLES
Column: 250 X 4 LiChrosorb C18
Mobile phase: Gradient. MeCN:water 25:75 containing 0.1% trifluoroacetic acid for 30 min then
MeCNiwater 40:60 containing 0.1% trifluoroacetic acid for 20 min (step gradient).
Flow rate: 2
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
rat; mouse
REFERENCE
Grubb,N.; Caldwell,J.; Strolin-Benedetti,M. Excretion balance and urinary metabolism of indobufen in rats and
mice, Biochem.Pharmacol., 1993, 46, 759-761.
lndocyanine green
Molecular formula: C43H47N2NaO6S2
Merck Index: 4992
SAMPLE
Matrix: blood
Sample preparation: 0.5 mL Plasma +10 jxL 250 (xg/mL 1-acetamidopyrene in MeOH + 200 |xL
1 M ammonium sulfate + 800 JJLL cold MeCN, vortex for 30 s, store at -20 for at least 30 min,
vortex, centrifuge at 1500 g for 30 min. Remove 400 |xL of the upper organic layer and evap-
orate it under a stream of nitrogen. Reconstitute with 100 |xL mobile phase, vortex for 30 s,
HPLCVARIABLES
Mobile phase: MeCNibuffer 47:53 (Buffer was 6.805 g potassium monophosphate in 1 L water,
adjust pH to 6.00 with 10 M NaOH.)
Flow rate: 1
Detector: UV 214
CHROMATOGRAM
Retention time: 7.2
Internal standard: 1-acetamidopyrene (9.7)
OTHER SUBSTANCES
Simultaneous: lorazepam, antipyrine
Noninterfering: adenosine, albuterol, alphenal, aspirin, caffeine, carbamazepine, cefazolin,
cephalexin, cephalothin, cimetidine, ciprofloxacin, claforan, desipramine, enoxacin, fleroxacin,
furosemide, hydralazine, hydrochlorothiazide, minoxidil, norfloxacin, phenytoin, propafenone,
sulindac, teicoplanin, theophylline, vancomycin
KEYWORDS
plasma
REFERENCE
Awni,W.M.; Bakker,L.J. Antipyrine, indocyanine green, and lorazepam determined in plasma by high-pressure
liquid chromatography, Clin.Chem., 1989, 35, 2124-2126.
SAMPLE
Matrix: blood
Sample preparation: 25 |xL Plasma + 25 (xL cold (-20) 60 jjig/mL IS in acetone, vortex for 30
s, centrifuge at 13000 g for 1 min, inject a 10-20 jxL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm octylsilane (Alltech)
Flow rate: 1.5
CHROMATOGRAM
Retention time: 7.3
Internal standard: l,l>,3,3,3',3>-hexamethyl-4,4',5,57-dibenzo-2,2>-indotricarbocyanine perchlo-
rate (Eastman Kodak) (8.7)
KEYWORDS
REFERENCE
Pollack,G.M.; Brouwer,K.L.R.; Demby,K.B.; Jones,J.A. Determination of hepatic blood flow in the rat using
sequential infusions of indocyanine green or galactose, Drug Metab.Dispos., 1990, 15, 197202.
lndomethacin
CAS Registry No.: 53-86-1, 74252-25-8 (sodium salt trihydrate)
Merck Index: 4998
Lednlcer No.: 1 318; 2 345; 3 165
SAMPLE
Sample preparation: 100 JJLL Aqueous humor + 500 |xL MeCN + 30 |xL 400 ng/mL (+)-naproxen
and dry it under nitrogen at room temperature, dissolve the residue in 50 (xL mobile phase by
aliquot.
HPLCVARIABLES
Column: 150 X 4.5 5 u-m Ultrasphere octyl
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diclofenac, flurbiprofen, meclofenamic acid
KEYWORDS
human; rabbit
REFERENCE
SAMPLE
Sample preparation: 200 JULL Aqueous humor + 200 |xL MeCN, vortex for 15 s, centrifuge at
2000 rpm for 10 min, inject a 20 |xL aliquot of the supernatant.
HPLCVARIABLES
Guard column: 4 X 4 5 u-m Lichrospher 100 C18
Column: 125 X 4 5 pun Lichrospher 100 C18
Mobile phase: MeOH:50 mM pH 3 sodium phosphate buffer 65:35
Flow rate: 1
Detector: E, Shimadzu L-ECD-6A, glassy carbon working electrode 1.20 V, Ag/AgCl reference
electrode or UV 266
CHROMATOGRAM
Retention time: 7.5
Limit of detection: 50 ng/mL (UV), 20 ng/mL (E)
KEYWORDS
rabbit
REFERENCE
Baudrit,O.; Fabre,H. Evaluation of electrochemical and fluorescence detection in liquid chromatography for the
assay of indomethacin in aqueous humor samples, J.Liq.Chromatogr., 1995, 18, 3283-3299.
SAMPLE
Matrix: bile, blood, gastric contents, urine
Sample preparation: Plasma. 300 |xL Plasma + 1 mL MeCN, vortex for 30 s, centrifuge at 3500
rpm. Remove the supernatant and evaporate it to 100 fxL under a stream of nitrogen at 50,
inject a 20 |xL aliquot. Urine, bile, gastric fluid. 300 (xL Urine, bile, or gastric fluid + 100 u-L
5 M NaOH, let stand at room temperature for 15 min, adjust the pH with 100 |xL 28.3%
phosphoric acid, add 1 (urine, bile) or 1.5 (gastric fluid) mL MeCN, vortex for 30 s, centrifuge
at 3500 rpm. Remove the supernatant and evaporate it to 100 |JLL under a stream of nitrogen
at 50, inject a 20 (urine), 35 (bile), or 40 (gastric fluid) |xL aliquot. (Borate buffer was 12.4 g
boric acid and 10 mL 1 M NaOH made up to 1 L with water, pH 7.2.)
HPLCVARIABLES
Guard column: Microguard reverse-phase (Bio-Rad)
Column: 100 X 8 Radial-PAK C18 in a radial compression module
Mobile phase: MeCNibuffer 70:30 (Buffer was 6.8 g/L KH2PO4 adjusted to pH 3.0 with 85%
phosphoric acid.)
Flow rate: 2
Detector: UV 340
CHROMATOGRAM
Retention time: 13
Internal standard: indomethacin
OTHER SUBSTANCES
Extracted: sulindac
KEYWORDS
plasma; indomethacin is IS
REFERENCE
Musson,D.G.; Vincek,W.C; Constanzer,M.L.; Detty,T.E. Analytical methods for the determination of sulindac
and metabolites in plasma, urine, bile, and gastric fluid by liquid chromatography using ultraviolet detec-
tion, J.Pharm.ScL, 1984, 73, 1270-1273.
SAMPLE
Matrix: blood
Sample preparation: Mix 500 JJLL serum with 100 jxL MeOH:50 mM pH 3 sodium phosphate
buffer 50:50, vortex for 5 s. Add 1 mL MeCN, vortex for 1 min. Centrifuge the mixture at 14000
rpm for 5 min, decant the clear upper layer. Add 500 JJLL MeCN to the pellet, mix, centrifuge
at 14000 rpm for 5 min. Combine the upper layers and evaporate at 40. Reconstitute the
residue with 300 |JLL MeOH:50 mM pH 3.0 sodium phosphate buffer 40:60 containing 0.5%
sodium metabisulfate (sic). Vortex for 30 s and inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 x 4.6 5 |xm Techsil C18 (HPLC Technology, Macclesfield)
Mobile phase: MeCN:50 mM phosphate buffer 46:63, adjusted to pH 3.0 with NaOH
Flow rate: 1.2
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: sulindac
KEYWORDS
indomethacin is IS; serum
REFERENCE
Kanfer,L; Brown,C; Koninigs,M.; Swarbrick,J. Absorption of sulindac from a novel (Pro-SorbTM) liquid for-
mulation, Biopharm.Drug Dispos., 1996, 17, 209-221.
SAMPLE
Matrix: blood
Sample preparation: Precipitate 100 (xL serum with 200 |xL 2 |xg/mL IS in MeCN, centrifuge
at 12 000 g for 5 min. Inject a 50 |xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 4 X 4 5 |xm LiChroCART LiChrospher 60 RP Select B
Column: 125 x 4 5 jim LiChroCART LiChrospher 60 RP Select B
Mobile phase: MeCNibuffer 60:40 (Buffer was 25 mM pH 3.0 triethylammonium phosphate con-
taining 2% MeCN.)
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Internal standard: mefenamin (2.94)
KEYWORDS
serum
REFERENCE
Hannak,D.; Scharbert,R; Kattermann,R. Stepwise binary gradient high-performance liquid chromatographic
system for routine drug monitoring, J.ChromatognA, 1996, 728, 307-310.
SAMPLE
Matrix: blood
Sample preparation: 500 u.L Plasma + 250 u,L 1 M sulfuric acid + 5 mL 24 ng/mL p-phenyl-
phenol in dichloromethane, vortex for 10 s, centrifuge at 500 g for 5 min. Remove 4 mL of the
in 100 JJLL MeOH, inject a 20-30 JULL aliquot.
HPLC VARIABLES
Mobile phase: Gradient. MeOH: 100 mM pH 5 acetate buffer 45:55 for 3 min, to 62:38 over 2
min, maintain at 62:38 for 10 min.
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Internal standard: p-phenylphenol (12.43)
OTHER SUBSTANCES
Simultaneous: acetaminophen, caffeine, carbamazepine, ethosuximide, fenoprofen, naproxen,
phenobarbital, phenytoin, primidone, quinidine, salicylic acid, sulindac, theophylline, tolmetin,
valproic acid
Noninterfering: ibuprofen
KEYWORDS
plasma
REFERENCE
Shimek,J.L.; Rao,N.G.S.; Wahba Khalil,S.K High performance liquid chromatographic analysis of tolmetin,
indomethacin and sulindac in plasma, J.Liq.Chromatogr., 1981, 4, 19872013.
SAMPLE
Matrix: blood
Sample preparation: 200 (JLL Plasma + 100 |xL pH 2 dilute sulfuric acid + 1 mL MeCN, vortex
for 30 s, centrifuge at 2500 rpm for 5 min. Remove the supernatant and evaporate it to dryness
under a stream of nitrogen at 40, reconstitute the residue in 200 (JLL mobile phase, inject a 10
|xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 (xm Zorbax CN
Mobile phase: MeCN:4% aqueous acetic acid 45:55
Flow rate: 1
Detector: UV 340
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Extracted: sulindac
KEYWORDS
REFERENCE
Clark,C.R.; McMillian,C.L.; Hoke,J.R; Campagna,K.D.; Ravis,W.R. Liquid chromatographic determination of
sulindac and metabolites in serum, J.Chromatogr.Sci., 1987, 25, 247-251.
SAMPLE
Matrix: blood
Sample preparation: Activate a 1 mL Bond-Elut C8 SPE cartridge with 2 mL MeOH then 1
mL 10 mM HCl, do not allow it to dry completely. Sonicate 1 mL whole blood for 20-30 min
then apply to cartridge. Wash with 100 |xL water, elute with three 500 |xL portions of MeOH:
MeCN: 1% aqueous ammonium hydroxide 50:20:30, combine eluents and evaporate to dryness
under a stream of nitrogen at 40. Redissolve in 1 mL MeOH, inject a 20 |xL aliquot.
HPLCVARIABLES
Mobile phase: MeCN.MeOH.bufFer 35:13:52 (Buffer was water adjusted to pH 3.2 with ortho-
phosphoric acid
Flow rate: 1
Detector: UV 250
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: ketoprofen, acetaminophen, salicylic acid, naproxen, fenoprofen, ibuprofen
KEYWORDS
whole blood; SPE
REFERENCE
Moore,C.M.; TebbettJ.R. Rapid extraction of anti-inflammatory drugs in whole blood for HPLC analysis, Fo-
rensic ScLInL, 1987, 34, 155-158.
SAMPLE
Matrix: blood
Sample preparation: Wash a Sep-Pak C18 cartridge with 2 mL MeOH, 5 mL water, and 1 mL
0.25 mM pH 3.0 ammonium phosphate buffer. 20-200 |xL Plasma + 100 |xL MeOH + 20 |xL 50
|xg/mL indomethacin in MeOH + 100 |xL 0.25 mM pH 3.0 ammonium phosphate buffer + 100
IxL water, vortex for 2 min, centrifuge at 1800 g for 10 min. Add the supernatant to the car-
tridge, wash with 5 mL water, elute twice with 5 mL portions of MeOH. Evaporate eluate to
dryness under vacuum, dissolve the residue in 1 mL mobile phase, inject a 20 (JLL aliquot.
HPLCVARIABLES
Column: 100 X 4.6 5 jjim Brownlee RP18
Mobile phase: MeOHrbuffer 75:25 (Buffer prepared by diluting 0.25 mM ammonium phosphate
buffer adjusted to pH 3.0 with orthophosphoric acid.)
Detector: E ESA Coulochem Model 5100 A, + 0.9 V
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: sulindac, piroxicam, diflunisal
KEYWORDS
plasma
REFERENCE
Kazemifard,A.G.; Moore,D.E. Liquid chromatography with amperometric detection for the determination of
non-steroidal anti-inflammatory drugs in plasma, J.Chromatogr., 1990, 533, 125-132.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 1 M pH 10.0 Tris buffer containing 150 ng/mL
indomethacin + 8 mL diethyl ether, vortex 3 min, freeze for 1 h. Remove organic phase and
evaporate it to dryness at room temperature. Dissolve residue in 200 JJLL MeOH, inject 20 |JLL
aliquot.
HPLCVARIABLES
Column: 70 X 4.6 3 jxm Ultrasphere XL ODS
Mobile phase: MeOH:20 mM ammonium acetate buffer (pH 5.0) 65:35
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: dipyridamole
KEY WORDS
REFERENCE
Barberi,M.; Merlin,J.L.; Weber,B. Sensitive determination of free and plasma protein-bound dipyridamole by
high-performance liquid chromatography, J.Chromatogr., 1991, 565, 511515.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut phenyl SPE cartridge with 5 mL MeOH and 5 mL
water. Adjust pH of 500 jxL plasma to 3.4 with 345 mM citrate buffer, add to SPE cartridge,
wash with water, dry, elute with 5 mL hexanerdiethyl ether 50:50. Evaporate the eluate to
dryness under a stream of nitrogen at 40, reconstitute the residue in 200 |xL MeOH, inject a
25 |JIL aliquot.
HPLC VARIABLES
Guard column: 6 x 4 4 jxm Nova-Pack C18
Column: 150 X 4.6 5 ^m Ultrasphere ODS
Mobile phase: MeCN:20 mM ammonium sulfate 55:45
Flow rate: 1.5
Detector: UV 340
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
Extracted: phenylbutazone, oxyphenbutazone, suxibuzone
KEYWORDS
plasma; SPE; indomethacin is IS
REFERENCE
Caturla,M.C; Cusido,E. Solid-phase extraction for the high-performance liquid chromatographic determination
of indomethacin, suxibuzone, phenylbutazone and oxyphenbutazone in plasma, avoiding degradation of
compounds, J.Chromatogr., 1992, 581, 101-107.
SAMPLE
Matrix: blood
Sample preparation: 20 |xL serum + 20 |xL MeCN, vortex for a few s, centrifuge at 10000 g for
2 min, inject a 20 |xL aliquot of the supernatant.
HPLC VARIABLES
Mobile phase: MeCN:25 mM phosphate buffer 35:65 containing 30 mM hydrogen peroxide, ad-
justed to pH 7.0 with 1 M NaOH
Flow rate: 1
a 15 m X 0.5 mm ID stainless steel coil at 180 then a 3 m X 0.5 mm ID stainless steel coil
at 15 to the detector.
CHROMATOGRAM
Retention time: 10
KEYWORDS
post-column reaction; serum; pharmacokinetics
REFERENCE
Kubo,H.; Umiguchi,Y; Kinoshita,T. Fluorometric determination of indomethacin in serum by high performance
liquid chromatography using in-line oxidation with hydrogen peroxide, J.Liq.Chromatogr., 1993, 16, 465-
474.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Plasma + 250 |xL 0.75 |xg/mL mefenamic acid in MeCN + 50 |xL
MeCN, vortex, centrifuge at 9000 g for 3 min. Remove 250 |xL of the supernatant and evaporate
it to dryness under vacuum, dissolve the residue in 50 |xL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 Alltech 5 |xm C18 bonded-phase silica
Column: 250 X 4.6 Vydac column packed with Merck 5 |xm C18 bonded-phase silica
Mobile phase: MeCN: 10 mM phosphoric acid 60:40, pH 2.6
Flow rate: 0.9
Detector: UV 280
CHROMATOGRAM
Retention time: 6.3
Internal standard: mefenamic acid (9.2)
KEYWORDS
plasma
REFERENCE
Niopas,L; Mamzoridi,K. Determination of indomethacin and mefenamic acid in plasma by high-performance
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 60 jjum Separon SGX C18 SPE cartridge with 5 mL
MeOH, 5 mL water, and 5 mL buffer. 250 |xL Blood + 500 |xL water, shake for 5 min, sonicate
for 5 min, let stand at room temperature for 5 min, add 1 mL buffer, shake for 5 min, centrifuge
at 1930 g for 10 min, add supernatant to cartridge, wash with 5 mL buffer, wash with 10
mL water, dry with vacuum for 5 min, elute with dichloromethane. Evaporate eluate to dryness
under a stream of nitrogen, dissolve in 100 |xL mobile phase, inject 10 |xL aliquot. (Buffer was
66 mM KH2PO4 adjusted to pH 2.0 with phosphoric acid.)
HPLC VARIABLES
Column: 150 X 3.3 5 |xm Separon SGX C18 glass column
Mobile phase: MeOH water 220:100, adjusted to pH 3.0 with 5% perchloric acid
Flow rate: 1.3
Detector: UV 222
CHROMATOGRAM
Retention time: 7.8
OTHER SUBSTANCES
KEYWORDS
SPE; indomethacin is IS; rabbit; human
REFERENCE
Sochor,J.; Klimes,J.; Zahradnicek,M.; Sedlacek,J. High-performance liquid chromatographic assay for ibuprofen
in whole blood using solid-phase extraction, J.Chromatogr.B, 1994, 654, 282-286.
SAMPLE
Matrix: blood
mobile phase. I m L Serum + 1 drop saturated ammonium sulfate solution + 1 drop 1 M HCl,
vortex for 3 min, add to the SPE cartridge, wash with six 500 (JLL portions of wash solvent,
elute with four 250 |xL aliquots of mobile phase, combine the eluates, vortex, inject a 100 |xL
aliquot. (Wash solvent was MeCNrwater adjusted to pH 3.0 with phosphoric acid 20:80.)
HPLC VARIABLES
Column: 150 X 4.6 5 jxm Ultrasphere C8
Flow rate: 0.5
Detector: F ex 232 em 335 (filter) following post-column photolysis. The effluent from the column
flowed through a 7.9 m X 0.3 mm i.d. coil of PTFE irradiated by an SC3-9 UV lamp (UVP)
(cooled with air) to the detector.
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Extracted: sulindac
KEYWORDS
serum; post-column reaction; SPE; indomethacin is IS; post-column photochemical derivatization
REFERENCE
Siluveru,M.; Stewart,J.T. Determination of sulindac and its metabolites in human serum by reversed-phase
high-performance liquid chromatography using on-line post-column ultraviolet irradiation and fluorescence
detection, J.Chromatogr.B, 1995, 673, 91-96.
SAMPLE
Matrix: blood
Sample preparation: Erythrocytes. 500 |xL Erythrocytes + 900 |xL water, shake for 5 min, son-
icate for 5 min, let stand at room temperature for 5 min, add 400 jxL 3 M HCl, shake for 5
min, add 6 mL dichloromethane, shake, centrifuge at 1930 g for 5 min. Remove 5 mL of the
in 100 |xL mobile phase, inject a 10 JJLL aliquot. Plasma. Acidify 500 JJLL plasma gradually with
900 IJLL 1 M HCl, shake, add 200 |xL 3 M HCl, shake for 5 min, add 6 mL dichloromethane,
shake, centrifuge at 1930 g for 5 min. Remove 5 mL of the organic layer and evaporate it to
dryness under a stream of nitrogen, reconstitute the residue in 100 |xL mobile phase, inject a
10 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.3 5 jxm C18 glass column (Tessek)
Mobile phase: MeOH:water 66:30 adjusted to pH 3.0 with 5% perchloric acid
Flow rate: 1.3
Detector: UV 222
CHROMATOGRAM
Retention time: 7.8
OTHER SUBSTANCES
Simultaneous: diazepam, phenylanthranilic acid
KEYWORDS
plasma; erythrocytes; rabbit; indomethacin is IS
REFERENCE
Sochor,J.; Klimes,J.; Sedl&cek,J.; Zahradnicek,M. Determination of ibuprofen in erythrocytes and plasma by
high-performance liquid chromatography, J.Pharm.Biomed.Anal., 1995, 13, 899903.
SAMPLE
Matrix: blood
the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 JJIL aliquot of
HPLC VARIABLES
Column: 300 X 3.9 4 um NovaPack C18
Flow rate: 0.8
Detector: UV 253
CHROMATOGRAM
KEYWORDS
lam; prazosin; nunitrazepam; clonazepam; metoclopr amide; melphalan; estazolam;
REFERENCE
254-262.
SAMPLE
A to waste with 500 (xL 500 mM ammonium sulfate, backflush the contents of column A onto
HPLC VARIABLES
+ 250 X 4.6 5 |xm C8 end-capped (Whatman)
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
phenhydramine, dipipanone, ethylbromphos, flufenamic acid, formothion, griseofulvin, lido-
caine, lorazepam, malathion, medazepam, midazolam, oxazepam, paraoxon, penicillin G, pen-
KEYWORDS
REFERENCE
SAMPLE
Sample preparation: Plasma. 50 |xL Plasma + 150 u-L 8 u,g/mL mefenamic acid in MeOH, mix,
centrifuge at 15000 rpm, filter (0.45 |xm), inject an aliquot. Tissue. Homogenize liver in ice-
cold 10 mM pH 7.4 phosphate buffer, 1 mL homogenate + 2 mL 8 |xg/mL mefenamic acid in
MeOH, mix, centrifuge at 15000 rpm, inject an aliquot.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:water:acetic acid 65:10:25:1
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: mefenamic acid
KEYWORDS
plasma; rat; liver
REFERENCE
Ogiso,T.; Iwaki,M.; Kinoshita,T.; Tanino,T.; Paku,T. Pharmacokinetics of indomethacin octyl ester (prodrug)
and indomethacin produced from the prodrug, J.Pharm.Sci., 1994, 83, 34-37.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 50 jiL 500 ng/mL mefenamic acid + 1 mL 100
mM HCl + 10 mL dichloromethane, rotate for 10 min, centrifuge at 1500 g for 15 min. Remove
the organic layer and evaporate it to dryness under a stream of nitrogen at 45. Redissolve the
residue in mobile phase, inject a 20 uX aliquot. Urine. 50 JJLL Urine + 1 mL mobile phase, inject
a 20 |xL aliquot.
HPLC VARIABLES
Column: 75 X 4.6 3 jim Supelcosil LC-8
Flow rate: 1
Detector: UV 235
CHROMATOGRAM
Retention time: 5
Internal standard: mefenamic acid (8)
OTHER SUBSTANCES
Simultaneous: naproxen, flunixin, thiosalicylic acid, ethacrynic acid, phenylbutazone
KEYWORDS
plasma
REFERENCE
Singh,A.K.; Jang,Y.; Mishra,U.; Granley,K. Simultaneous analysis of flunixin, naproxen, ethacrynic acid, in-
mance liquid chromatography and gas chromatography-mass spectrometry, J.Chromatogr., 1991,568, 3 5 1 -
361.
SAMPLE
Sample preparation: Plasma. 100 |xL Plasma + 400 |xL 200 mM perchloric acid, centrifuge at
3000 g, inject a 20 |xL aliquot of the supernatant. Urine. Dilute 1:1 with water, inject a 20 |xL
aliquot.
HPLCVARIABLES
Guard column: 75 X 2.1 pellicular reverse phase (Chrompack no. 28653)
Column: 250 X 4.6 5 \xm Cp Spherisorb ODS (Chrompack)
Mobile phase: Gradient; MeCN:5 g/L orthophosphoric acid from 80:20 to 60:40 (sic, probably 20:
80 to 40:60) over 30 min then stay there for 5 min, then to initial conditions over 5 min,
equilibrate for 2 min before next injection
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: metabolites, glucuronides
KEYWORDS
plasma; pharmacokinetics; also details of preparative procedure
REFERENCE
Vree,T.B.; Van den Biggelaar-Martea,M.; Verwey-van Wissen,C.P.W.G.M. Determination of indomethacin, its
metabolites and their glucuronides in human plasma and urine by means of direct gradient high-perfor-
mance liquid chromatographic analysis. Preliminary pharmacokinetics and effect of probenecid,
J.Chromatogr., 1993, 616, 271-282.
SAMPLE
a 10 (urine) or 30 (blood) JULL aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
mL/min)
Detector: UV 201.7
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: 500 |xL Plasma + 100 |xL 5 mg/mL indomethacin in MeOH, filter (Sarto-
rius SM 13243 ultrafiltration unit at 4000 g for 30 min). Add filtrate to a dry Chem Elut column
(modified diatomaceous earth), leave 3 to 5 min, elute with 6 mL diethyl ether, evaporate eluant
under a stream of nitrogen at room temperature, sonicate residue with 100 |xL MeOH for 10
min, vortex, inject 20 |xL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 jim pJBondapak C18
Mobile phase: MeOH: 100 mM pH 4.0 sodium acetate buffer 55:45
Flow rate: 1
Detector: E, ESA Coulochem 5100A dual electrode with an ESA guard cell, + 0.65 V
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: dipyridamole
KEYWORDS
REFERENCE
Barberi-Heyob,M.; Merlin,J.L.; Pons,L.; Calco,M.; Weber,B. A sensitive isocratic liquid chromatography assay
for the determination of dipyridamole in plasma with electrochemical detection, J.Liq.Chromatogr., 1994,
17, 1837-1848.
SAMPLE
Sample preparation: Reconstitute 1 mg indomethacin sodium trihydrate injection with 2 mL
water, inject a 5 |iL aliquot.
HPLC VARIABLES
Column: 250 X 4.2 5 jxm Ultrasphere ODS C18 (Beckman)
Mobile phase: MeCN:50 mM H3PO4 60:40
Flow rate: 1
Injection volume: 5
Detector: UV 260
CHROMATOGRAM
KEYWORDS
injections
REFERENCE
Walker,S.E.; Gray,S.; Schmidt,B. Stability of reconstituted indomethacin sodium trihydrate in original vials
and polypropylene syringes, Am. J.Health-Syst.Pharm., 1998, 55, 154-158.
SAMPLE
Sample preparation: 250 |xL Microsomal incubation, 150 |xL ice-cold MeCN and 2.5 ng keto-
profen, centrifuge. Extract the mixture with 4 mL ethyl acetate, centrifuge at 3000 rpm for 10
min, remove the organic fraction and evaporate it under a gentle stream of nitrogen at 40.
Dissolve the residue in 30 JJLL MeOH and dilute to 60 jxL with water. Inject a 30 (JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm RP-18 (Kanto Chemical, Tokyo)
Mobile phase: MeCNiwater 40:60 containing 0.6% acetic acid
Flow rate: 1
Detector: UV 260
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
liver; pharmacokinetics
REFERENCE
Nakajima,M.; Inoue,T.; Shimada,N.; Tokudome,S.; Yamamoto,T.; Kuroiwa,Y. Cytochrome P450 2C9 catalyzes
indomethacin O-demethylation in human liver microsomes, Drug Metab.Dispos., 1998, 26, 261-266.
SAMPLE
Matrix: perfusate
Sample preparation: Mix 100 nL perfusate with 600 nL perfusion fluid containing IS, inject an
aliquot. (Perfusion fluid contained 104 mM NaCl, 25 mM sodium bicarbonate, 2.3 mM sodium
biphosphate, 10 mM sodium acetate, 1.2 mM calcium chloride, 1 mM magnesium sulfate, 5
mM KCl, 5 mM dextrose, and 5 mM alanine.)
HPLCVARIABLES
Flow rate: 0.13
Detector: F ex 295 em 376 following post-column reaction. The column effluent mixed with 4 M
NaOH pumped at 0.0013 mIVmin and the mixture flowed through a 130 |xL PTFE coil at 64
to the detector.
CHROMATOGRAM
Internal standard: phenylbutazone
KEYWORDS
post-column reaction; microbore
REFERENCE
De Zeeuw,D.; Leinfelder,J.L.; Brater,D.C. Highly sensitive measurement of indomethacin using a high perfor-
mance liquid chromatographic technique combined with post column in-line hydrolysis, J.Chromatogr.,
1986, 380, 157-162.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 264
OTHER SUBSTANCES
Also analyzed: carbamazepine, fenbufen, ketoprofen, a.-naphthoquinone, naproxen, tolmetin
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4 OmniPac PAX-500 (Dionex)
Mobile phase: Gradient. A was MeCN: 10 mM sodium carbonate 18:82. B was MeCN:50 mM
sodium carbonate 33:67. A:B from 100:0 to 0:100 over 10 min.
Flow r a t e : 1
Detector: UV 254
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
Simultaneous: tolmetin, aspirin, ibuprofen, fenbufen, naproxen, carprofen, diflunisal
REFERENCE
Slingsby,R.W.; Rey,M. Determination of Pharmaceuticals by multi-phase ehromatography: Combined reversed
phase and ion exchange in one column, J.Liq.Chromatogr., 1990, 23, 107-134.
SAMPLE
Matrix: solutions
Sample preparation: Sample + 400 (JLL 5 mM DBD-PZ + 70 mM diethylphosphorocyanidate in
MeCN, react for 6 h, inject a 1 jxL aliquot. (Synthesis of 4-(N,N-dimethylaminosulfonyl)-7-N-
piperazino-2,l,3-benzoxadiazole (DBD-PZ) is as follows. Dissolve 0.5 g magnesium sulfate hep-
tahydrate and 6 g NaOH in 60 mL water, throughout the reaction keep the flask at about 20
with cold water cooling, add 15 mL 30% hydrogen peroxide, add 75 mL MeOH, add 12.1 g
powdered benzoyl peroxide in one go, stir for 10 min, pour into 150 mL 20% sulfuric acid,
extract three times with 50 mL portions of chloroform, determine peroxybenzoic acid concen-
tration by iodometric titration (Tetrahedron 1967, 23, 3327). Slowly add 110 mL 1 M peroxy-
benzoic acid in chloroform to 7 g 2,6-difluoroaniline dissolved in 100 mL chloroform, stir at
room temperature, when reaction is complete (iodometric titration) wash with 2% sodium thi-
osulfate, wash with 5% sodium carbonate, wash with water, dry over anhydrous sodium sulfate,
evaporate to dryness under reduced pressure, recrystallize 2,6-difluoronitrosobenzene form
EtOH (mp 108.5-109.5). Stir 8.5 g 2,6-difluoronitrosobenzene in 85 mL DMSO at room tem-
perature and add a solution of 3.91 g sodium azide in 85 mL DMSO dropwise, let stand for
about 1 h, add to a large volume of water, extract with ether, dry the extracts over anhydrous
sodium sulfate, evaporate to dryness under reduced pressure and distil to give 4-fluoro-2,l,3-
benzoxadiazole as a colorless oil (bp 83712 mm Hg) (J.Chem.Soc.(C) 1970, 1433). Add 11 mL
chlorosulfonic acid dropwise to 3 g 4-fluoro-2,l,3-benzoxadiazole in 10 mL chloroform at 0-10
(use a calcium chloride drying tube), stir at room temperature for 1 h, reflux for 2 h, cool,
slowly pour into ice water, remove the organic layer, extract the aqueous layer with chloroform,
combine the organic layer, wash, dry over anhydrous magnesium sulfate, evaporate under
reduced pressure, take up the residue in 5 mL benzene (Caution! Benzene is a carcinogen!),
chromatograph on a 150 X 30 column of silica gel (100-200 mesh Kanto Chemical) with n-
hexane:benzene 50:50, evaporate the appropriate fractions to give 4-(chlorosulfonyl)-7-fluoro-
2,1,3-benzoxadiazole (CBD-F) as pale yellow needles (mp 64-66) (Anal. Chem. 1984. 56, 2461).
Stir 0.76 g CBD-F in 70 mL MeCN at 0-10 and add 1 g dimethylamine hydrochloride in 10
mL 100 mM pH 10 borax dropwise, adjust pH to 5 with 1 M HCl, concentrate to about 10 mL
under reduced pressure, extract three times with 200 mL portions of diethyl ether, wash with
water, dry over anhydrous magnesium sulfate, evaporate under reduced pressure, chromato-
graph on a 500 X 20 column of silica gel with chloroform, isolate the appropriate fraction and
re-chromatograph on the same column with ethyl acetateibenzene 1:2 to give 4-(N,N-dimethyl-
aminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (DBD-F) as white needles (mp 124-125) (yield =
1% !). On a Merck no. 5714 60F254 tic plate eluted with chloroform DBD-F has Rf 0.32 and lies
between two other reaction products (Analyst 1989, 114, 413). It is also reported that DBD-F
can be purchased from Tokyo Kasei (TCI America, Portland OR). Add 123 mg 4-(N,N-dimethyl-
aminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole in 20 mL MeCN dropwise to 129 mg piperazine
in 20 mL MeCN at room temperature, stir for 30 min, evaporate under reduced pressure,
dissolve residue in 50 mL 5% HCl, wash three times with 20 mL ethyl acetate, discard ethyl
acetate extracts, adjust pH of aqueous solution to 13-14 with 5% NaOH, extract five times with
50 mL ethyl acetate, combine extracts, wash with 20 mL water, dry over anhydrous sodium
sulfate, evaporate under vacuum to give 4-(N,N-dimethylaminosulfonyl)-7-N-piperazino-2,l,3-
benzoxadiazole as orange crystals (mp 121-2).)
HPLCVARIABLES
Flow rate: 1
Injection volume: 1
CHROMATOGRAM
Retention time: 8
Limit of detection: 3.9 fmol
OTHER SUBSTANCES
REFERENCE
Toyo'oka,T.; Ishibashi,M.; Takeda,Y.; Nakashima,K.; Akiyama,S.; Uzu,S.; Imai,K. Precolumn fluorescence tag-
ging reagent for carboxylic acids in high-performance liquid chromatography: 4-substituted-7-aminoalkyl-
amino-2,l,3-benzoxadiazoles, J.Chromatogr., 1991, 588, 61-71.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, iminos-
tilbene, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketam-
ine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine,
REFERENCE
Hill,D.W.; Kind^-J- Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Isopropanol:100 mM KH2PO4:formic acid 54:100:0.1
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
KEYWORDS
solutions acemetacin; diclofenac; flurbiprofen; lonazolac; ketoprofen; naproxen; piroxicam; sulin-
dac; tenoxicam
REFERENCE
Baeyens,W.R.G.; Van Der Weken,G.; Van Overbeke,A; Zhang,Z.D. Preliminary results on the LC-separation of
non-steroidal anti-inflammatory agents in conventional and narrow-bore RP set-ups applying columns with
different internal diameters, Biomed.Chromatogr., 1995, 9, 261-262.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:water 45:55, pH adjusted to 3.5 with acetic acid
Detector: UV 280
CHROMATOGRAM
Internal standard: clomethacin
OTHER SUBSTANCES
Also analyzed: diclofenac, phenylbutazone
REFERENCE
Guterres,S.S.; Fessi,H.; Barratt,G.; Puisieux,R; Devissaguet,J.-P. Poly(D,L-lactide) nanocapsules containing
non-steroidal anti-inflammatory drugs: Gastrointestinal tolerance following intravenous and oral adminis-
tration, Pharm.Res., 1995, 12, 1545-1547.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
chlordiazepoxide, chlormezanone, chloroquine, ehlorpheniramine, chlorpromazine, chlorpro-
cainide, ethidium bromide, ethopropazine, fenoprofen, fentanyl, fiavoxate, fluoxetine, fluphen-
droxychloroquine, hydroxyzine, ibuprofen, imipramine, ketoconazole, ketoprofen, ketorolac, Ia-
betalol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefen-
tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, trifiupromazine, tri-
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 JJLL aliquot of a 250 ng/mL solution in pH 2.5 buffer.
HPLC VARIABLES
Mobile phase: MeCNrpH 6.0 acetate/citrate buffer 45:55
Detector: UV 254
CHROMATOGRAM
Retention time: 14
KEYWORDS
microcolumn
REFERENCE
Streel,B.; Ceccato,A.; Chiap,R; Hubert,R; Crommen,J. Injection-generated solvent and pH gradients for sample
enrichment on injection of large volumes in microcolumn liquid chromatography, Biomed.Chromatogr., 1995,
9, 254-256.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 10 X 4.6 10 jim Spherisorb silica
Column: 120 X 2 5 |xm Spherisorb ODS
Flow rate: 0.378
Detector: UV 254
REFERENCE
Lough,W.J.; Mills,M.J.; Maltas,J. Analyte adsorption in liquid chromatography valve injectors for samples in
non-eluting solvents, J.ChromatogrA, 1996, 726, 67-75.
SAMPLE
Matrix: solutions
Sample preparation: Filter (0.45 |xm), dilute the filtrate with mobile phase, inject an aliquot.
HPLCVARIABLES
Mobile phase: MeCNrIO mM pH 4 acetate buffer 50:50
Detector: UV 242
REFERENCE
Okimoto,K.; Rajewski,R.A.; Uekama,K.; Jona,J.A.; Stella,V.J. The interaction of charged and uncharged drugs
with neutral (HP-(S-CD) and anionically charged (SBE7-(5-CD) p-cyclodextrins, Pharm.Res., 1996,13, 256-
264.
SAMPLE
Matrix: solutions
HPLC VARIABLES
phosphatidylcholine in MeOHiwater 80:20 for 24 h.)
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
operidol, hydroxyzine, imipramine, lidocaine, megestrol acetate, metoprolol, nabumetone, na-
KEYWORDS
REFERENCE
Hanna,M.; de Biasi,V.; Bond,B-J Salter,C; Hutt,A.J.; Camilleri,P. Estimation of the partitioning characteristics
lndoramin
Merck Index: 5000
Lednicer No.: 2 344
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
lnositol
CAS Registry No.: 87-89-8, 573-35-3 (monophosphate)
Merck Index: 5008
SAMPLE
Matrix: solutions
HPLCVARIABLES
CoUtmn: 300 X 8.7 Aminex HPX-87C Ca++ (Bio-rad)
Mobile phase: Water
Flow rate: 0.6
Detector: E, pulsed amperometric detector (Dionex ?), El 0.1 V, Tl 300 ms, E2 0.6 V, T2 120
ms, E3 -0.8 V, T3 300 ms, following post-column reaction. The column effluent mixed with 100
mM NaOH pumped at 0.2 mL/min and the mixture flowed to the detector.
CHROMATOGRAM
Retention time: 13.64 (myo-inositol), 14.16 (chiro-inositol), 11.16 (scyllo-inositol), 19.58 (neo-
inositol)
OTHER SUBSTANCES
Also analyzed: 2-deoxygalactitol, 2-deoxyribositol, dextrose, fucitol, fucose, galactitol, galactose,
mannitol, mannose, perseitol, sorbitol
KEYWORDS
REFERENCE
Wang,W.T.; Safar,J.; Zopf,D. Analysis of inositol by high-performance liquid chromatography, Anal.Biochem.,
1990, 188, 432-435.
Insulin
Molecular formula: C257H383N65O77S6(human)
Molecular weight: 5807.69 (human)
CAS Registry No.: 9004-10-8 (injection), 8049-62-5 (zinc suspension), 11061-68-0 (human), 12584-
58-6 (pig), 11070-73-8 (cow), 9004-14-2 (neutral insulin), 8049-62-5 (isophane insulin), 9004-17-5
(protamine zinc suspension)
Merck Index: 5011
SAMPLE
HPLC VARIABLES
Column: 250 X 4 5 |xm Nucleosil RP18
Mobile phase: MeCN:buffer 24:76 (w/w) (Prepare buffer by dissolving 9.8 g 85% phosphoric acid
and 28.4 g sodium sulfate in 1 L water.)
Flow rate: 1
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Kunkel,A.; Giinter,S.; Dette,C; Watzig,H. Quantitation of insulin by capillary electrophoresis and high-perfor-
mance liquid chromatography. Method comparison and validation, J.Chromatogr.A, 1997, 781, 445-455.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 Synchropak C4
Mobile phase: Gradient. A was 0.05% trifluoroacetic acid in water. B was 0.05% trifluoroacetic
acid in MeCN. A:B from 74:26 to 38:62 over 15 min.
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Retention time: 4.7
REFERENCE
Ho,H.-O.; Hsiao,C.-C; Sheu,M.-T. Preparation of microemulsions using polyglycerol fatty acid esters as surfac-
tant for the delivery of protein drugs, J.Pharm.ScL, 1996, 85, 138-143.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Zorbax 300 A SB-C3
Mobile phase: Gradient. A was MeCN:water:trifluoroacetic acid 5:95:0.1. B was MeCN:water:
trifluoroacetic acid 5:95:0.085. A:B from 85:15 to 47:53 over 20 min.
Flow rate: 1
Detector: UV 215
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Simultaneous: angiotensin II, carbonic anhydrase, cytochrome C, leucine enkephalin, lysozyme,
myoglobin, RNAase
REFERENCE
Ricker,R.D.; Sandoval,L.A.; Permar,B.J.; Boyes,B.E. Improved reversed-phase high performance liquid chro-
matography columns for biopharmaceutical analysis, J.Pharm.Biomed.Anal., 1996,14, 93-105.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Protein & Peptide C18 (Vydac)
Mobile phase: MeCNrbuffer 26:74 (Buffer was 28.4 g sodium sulfate and 2.7 mL phosphoric acid
in 1 L water, pH adjusted to 2.3 with ethanolamine (if necessary).)
Flow rate: 0.8
Detector: UV 214
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
REFERENCE
Yomota,C; Yoshii,Y.; Takahata,T.; Okada,S. Separation of B-3 monodesamidoinsulin from human insulin by
high-performance liquid chromatography under alkaline conditions, J.ChromatogrA, 1996, 721, 89-96.
lnterferon
CAS Registry No.: 76543-88-9 (aA), 99210-65-8 (a2B), 98059-61-1 (gamma-1B)
Merck Index: 5015
Lednicer No.: 4 1
SAMPLE
Matrix: solutions
Sample preparation: Terminate the enzymatic hydrolysis by adding 10 JJLL 1% trifluoroacetic
acid, 100 |xL 8 M guanidine hydrochloride, inject an aliquot of the enzymatic digests.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Vydac C18
Mobile phase: Gradient. A was 0.1% trifluoroacetic acid. B was MeCN:water 90:10 containing
0.1% trifluoroacetic acid. A:B from 52:48 to 45:55 in 45 min, to 20:80 over 2.5 min.
Flow rate: 0.6
Detector: UV 214
CHROMATOGRAM
Retention time: 20
OTHER SUBSTANCES
REFERENCE
Gitlin,G.; Tsarbopoulos,A.; Patel,S.T.; Sydor,W.; Pramanik,B.N.; Jacobs,S.; Westreich,L.; Mittelman,S.;
Bausch,J.N. Isolation and characterization of a monomethioninesulfoxide variant of interferon a-2b,
Pharm.Res., 1996, 13, 762-769.
lodamide
Molecular formula: C12H11I3N2O4
Merck Index: 5031
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 100 jxL 500 |xg/mL iodopyracet in water + 5 mL
MeOH, centrifuge at 1500 g for 20 min. Remove the supernatant and evaporate it to dryness
under a stream of air at 65, reconstitute the residue in 300 |xL MeOHiwater 90:10, let stand
for 10 min, inject a 10 JJLL aliquot. Urine. 100 |xL Urine + 900 |xL mobile phase + 100 |xL 500
|xg/mL iodopyracet in water, inject a 10 |xL aliquot.
HPLCVARIABLES
Column: 100 X 4.6 5 |m LiChrosorb RP-8
Mobile phase: MeOH:water 85:15 containing 10 mM tetrabutylammonium hydrogen sulfate and
10 mM Tris
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 8
Internal standard: iodopyracet (11)
KEYWORDS
plasma
REFERENCE
Hekman,R; Van Ginneken,C.A. Rapid determination of renal contrast media in biological fluids by means of
SAMPLE
Sample preparation: Plasma. 0.5-1 mL Plasma + 1 mL 1 M HCl + 5 mL ethyl acetate, shake
for 10 min, centrifuge, repeat extraction. Combine the organic layers and evaporate them under
a stream of nitrogen at 60, add 3 mL 100 mM NaOH, shake for 10 min, centrifuge, discard
the organic layer. Remove the aqueous layer and add it to 500 |xL 1 M HCl, add 5 mL ethyl
acetate, shake for 10 min, centrifuge, repeat extraction. Combine the organic layers and evap-
orate them to dryness under a stream of nitrogen at 60, reconstitute the residue in 100 |xL
mobile phase, inject a 10-20 jxL aliquot. Urine. 100 |xL Urine + 1 mL 1 M HCl + add 5 mL
ethyl acetate, shake for 10 min, centrifuge, repeat extraction. Combine the organic layers and
evaporate them to dryness under a stream of nitrogen at 60, reconstitute the residue in 400
|xL mobile phase, inject a 10-20 ^L aliquot.
HPLC VARIABLES
Column: 250 X 2.6 10 |xm ODS-HC Sil-X-1 (Perkin-Elmer)
Mobile phase: MeCN:water:85% phosphoric acid 4:96:0.03
Flow rate: 1
Detector: UV 235
CHROMATOGRAM
Retention time: 2.5
Internal standard: iodamide
OTHER SUBSTANCES
Extracted: o-iodohippurate, iothalamate
KEYWORDS
plasma; iodamide is IS
REFERENCE
Boschi,S.; Marchesini,B. High-performance liquid chromatographic method for the simultaneous determination
of iothalamate and o-iodohippurate, J.Chromatogr., 1981, 224, 139-143.
lodinated glycerol
Molecular formula: C6H11IO3
Merck Index: 5033
SAMPLE
Matrix: bulk
HPLC VARIABLES
Guard column: 10 jjim Guard-pak (Waters)
Column: 250 X 4.6 5 u-m Ultrasphere ODS C18
Flow rate: 1
Detector: RI
KEYWORDS
this procedure determines glycerol, a component of iodinated glycerol
REFERENCE
Cannon,J.M.; Brown,R.D.; Murrill,E.M.; Jameson,C.W. Identification of components in iodinated glycerol,
J.Pharm.Scl, 1989, 78, 48-51.
lodochlorhydroxyquin
Molecular formula: GgH5CIINO
Merck Index: 5052
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL concentrated perchloric acid, vortex for 30 s, cen-
trifuge at 15 at 5000 rpm for 15 min. Remove 500 |xL of the supernatant and add it to 5 mL
ether, vortex for 10 s, centrifuge at 15 for 10 min, repeat extraction. Add 10 mL ether to the
original precipitate, vortex for 1 min, centrifuge at 15 at 5000 rpm for 15 min. Combine the
extracts and dry them over anhydrous sodium sulfate, evaporate to dryness under a stream of
nitrogen at 38, reconstitute the residue in 500 |xL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 40 X 5 RP-18-MPLC (Brownlee)
Column: 250 X 2.6 ODS-HC-SIL-X-I (Perkin-Elmer)
Mobile phase: MeOH:50 mM phosphoric acid 80:20 (Flush column with MeOH at the end of
each day.)
Flow rate: 1
Detector: UV 256
CHROMATOGRAM
Retention time: 5
Limit of quantitation: 1 (xg/mL
OTHER SUBSTANCES
Noninterfering: hydrocortisone
KEYWORDS
plasma
REFERENCE
Ezzedeen,F.W.; Masoud,A-N.; Stohs,S.J.; Lerman,S.J. High-performance liquid chromatographic analysis of
iodochlorhydroxyquin in plasma, J.Pharm.Sci., 1981, 70, 889-891.
SAMPLE
Sample preparation: Weigh out 30 mg of bulk drug or an amount of cream equivalent to 30
mg iodochlorhydroxyquin, add 70 mL THF, shake vigorously until the cream has dissolved,
make up to 100 mL with THF. Remove a 5 mL aliquot and add it to 1 mL pyridine and 1
mL acetic anhydride, heat at 60 for 15 min, cool, add 15 mL 450 |xg/mL testosterone acetate
in 94:6 butyl chloride:THF, mix thoroughly. Remove a 3 mL aliquot and evaporate it to dryness
under a stream of nitrogen at 40, reconstitute the residue in 15 mL mobile phase with gentle
warming and vigorous shaking, inject an aliquot.
HPLC VARIABLES
Mobile phase: Butyl chloride:water-saturated butyl chloride:THF:glacial acetic acid 55:55:3:2
Flow rate: 2-3
Detector: UV 254
CHROMATOGRAM
Retention time: 6
Internal standard: testosterone acetate (8)
OTHER SUBSTANCES
KEYWORDS
cream; normal phase; derivatization; acetylation
REFERENCE
Kubiak,E.J.; Munson,J.W. Analysis of iodochlorhydroxyquin in cream formulations and bulk drugs by high-
performance liquid chromatography, J.Pharm.Sci., 1982, 71, 872-875.
SAMPLE
Sample preparation: Weigh out ointment, cream, or bulk drug containing 30 mg of iodochlor-
hydroxyquin, add 40-50 mL THF, dissolve with heating, make up to 100 mL with THF. Remove
a 5 mL aliquot and add it to 1 mL 10 mg/mL nickel chloride in MeOH, add 5 mL 0.4 mg/mL
diphenylamine in MeOH, make up to 50 mL with MeOH, filter, inject an aliquot.
HPLCVARIABLES
Mobile phase: MeCN:MeOH:water 30:20:50 containing 1 mM NiCl2
Flow rate: 1.2
Detector: UV 273
CHROMATOGRAM
Retention time: 7.5
Internal standard: diphenylamine (11)
OTHER SUBSTANCES
Simultaneous: chloroxine, iodoquinol
KEYWORDS
ointment; creams; separated as Ni chelates
REFERENCE
Wojtowicz,E.J. Reverse-phase high-performance liquid chromatographic determination of halogenated 8-hy-
droxyquinoline compounds in pharmaceuticals and bulk drugs, J.Pharm.ScL, 1984, 73, 14301433.
SAMPLE
Sample preparation: Ointment. 50 mg Ointment + 10 mL ether, vortex until dissolved. Remove
a 200 |xL aliquot and add phenyl salicylate in mobile phase, evaporate to dryness under a
stream of nitrogen at 40, reconstitute the residue in 10 mL mobile phase, warm for 1 min on
a steam bath, vortex for 1 min, cool. Remove an aliquot, dilute with mobile phase, inject an
aliquot. Cream. Suspend 50 mg cream in 10 mL mobile phase by vortexing. Remove an aliquot
and add phenyl salicylate in mobile phase, evaporate to dryness under a stream of nitrogen at
40, suspend the residue in 10 mL mobile phase, warm for 1 min on a steam bath, vortex for
1 min, cool. Remove an aliquot, dilute with mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 250 X 2.6 ODS-HC-SIL-X (Perkin-Elmer)
each day.)
Flow rate: 1
Detector: UV 256
CHROMATOGRAM
Retention time: 7.5
Internal standard: phenyl salicylate (5.25)
OTHER SUBSTANCES
Simultaneous: hydrocortisone
KEYWORDS
ointment; cream
REFERENCE
Ezzedeen,F.W.; Stohs,S.J.; Masoud,A.N. High-performance liquid chromatographic analysis of iodochlorhy-
droxyquin and hydrocortisone in ointments and creams, J.Pharm.ScL, 1983, 72, 1036-1039.
SAMPLE
Matrix: feces, tissue, urine
Sample preparation: Urine. 1-5 mL Urine + 1-5 mL diethyl ether, vortex for 10 s, centrifuge at
15 at 3000 g for 10 min. Remove the organic layer and dry it over anhydrous sodium sulfate,
evaporate to dryness under a stream of nitrogen at 40, reconstitute the residue in mobile
phase, inject a 20 |JLL aliquot. (Hydrolyze conjugates by adjusting pH to 5 with 1 M acetate
buffer, add B-glucuronidase (Sigma) to a final concentration of 200 U/mL, heat at 37 for 2 h,
neutralize with 3 M NaOH, add 1-5 mL dieftiyl e!her, vortexlor 10 s, centrifuge a t l ^ aVSOOO
g for 10 min. Remove the organic layer and dry it over anhydrous sodium sulfate, evaporate
to dryness under a stream of nitrogen at 40, reconstitute in 500 |xL benzene (Caution! Benzene
is a carcinogen!), add to an alumina column, wash with 2 mL benzene:pyridine 7:1, wash with
2 mL acetone, wash with 2 mL 100 mM acetic acid in MeOH, wash with 2 mL MeOH. Remove
the alumina and add it to 1 mL saturated aqueous sodium fluoride, extract twice with 5 mL
diethyl ether. Combine the extracts and evaporate them to dryness, reconstitute the residue
in mobile phase, inject a 20 |xL aliquot.) Tissue. Homogenize (Potter-Elvehjem) 1 g liver and 2
mL mobile phase, place homogenate in another tube, rinse original tube with 1 mL mobile
phase, add rinse to homogenate, add 5 mL diethyl ether, vortex for 1 min, centrifuge at 3000
g for 10 min, repeat extraction twice. Combine the organic layers and evaporate them to dry-
ness, reconstitute in 500 |xL benzene (Caution! Benzene is a carcinogen!), add to an alumina
column, wash with 2 mL benzene:pyridine 7:1, wash with 2 mL acetone, wash with 2 mL 100
mM acetic acid in MeOH, wash with 2 mL MeOH. Remove the alumina and add it to 1 mL
saturated aqueous sodium fluoride, extract twice with 5 mL diethyl ether. Combine the extracts
and evaporate them to dryness, reconstitute the residue in mobile phase, inject a 20 |xL aliquot.
Feces. Homogenize (Potter-Elvehjem) 1 g feces and 5 mL mobile phase, adjust pH to 5 with 1
M acetate buffer, add p-glucuronidase (Sigma) to a final concentration of 200 U/mL, heat at
37 for 2 h, neutralize with 3 M NaOH, add 1-5 mL diethyl ether, vortex for 10 s, centrifuge
at 15 at 3000 g for 10 min. Remove the organic layer and dry it over anhydrous sodium sulfate,
evaporate to dryness under a stream of nitrogen at 40, reconstitute in 500 JJLL benzene (Cau-
tion! Benzene is a carcinogen!), add to an alumina column, wash with 2 mL benzene:pyridine
7:1, wash with 2 mL acetone, wash with 2 mL 100 mM acetic acid in MeOH, wash with 2 mL
MeOH. Remove the alumina and add it to 1 mL saturated aqueous sodium fluoride, extract
twice with 5 mL diethyl ether. Combine the extracts and evaporate them to dryness, reconsti-
tute the residue in mobile phase, inject a 20 |iL aliquot.
HPLC VARIABLES
Column: 250 X 2.6 ODS-HC-SIL-X-I (Perkin-Elmer)
each day.)
Flow rate: 1
Detector: UV 256
CHROMATOGRAM
Retention time: 7.5
Limit of quantitation: 250 ng/g (feces), 500 ng/g (liver), 200 ng/mL (urine)
KEY WORDS
liver; dog; SPE
REFERENCE
Ezzedeen,F.W.; Stohs,S.J.; Stublar,M. Analysis of iodochlorhydroxyquin in biological materials by high-perfor-
lodoquinol
Molecular formula: C9H5I2NO
Merck Index: 5063
SAMPLE
Sample preparation: Cream, bulk. Weigh out cream or bulk drug containing 20 mg of iodo-
quinol, add 40-50 mL THF, dissolve with heating, make up to 100 mL with THF. Remove a 5
mL aliquot and add it to 1 mL 10 mg/mL nickel chloride in MeOH, add 3 mL 0.4 mg/mL
diphenylamine in MeOH, make up to 50 mL with MeOH, filter, inject an aliquot. Tablets. Finely
powder tablets, weigh out amount equivalent to 200 mg iodoquinol, add 150 mL THF, haet on
a steam bath for a few min, shake mechanically for 30 min, make up to 250 mL with THF.
Remove a 25 mL aliquot and make up to 100 mL with THF. Remove a 5 mL aliquot and add
it to 1 mL 10 mg/mL nickel chloride in MeOH, add 3 mL 0.4 mg/mL diphenylamine in MeOH,
make up to 50 mL with MeOH, filter, inject an aliquot.
HPLC VARIABLES
Column: 300 X 3.9 fjiBondapak phenyl
Mobile phase: MeCN:MeOH:water 30:20:50 containing 1 mM NiCl2
Flow rate: 1.2
Detector: UV 273
CHROMATOGRAM
Retention time: 9
Internal standard: diphenylamine (11)
OTHER SUBSTANCES
Simultaneous: chloroxine, iodochlorhydroxyquin
KEYWORDS
tablets; creams; separated as Ni chelates
REFERENCE
Wojtowicz,E.J. Reverse-phase high-performance liquid chromatographic determination of halogenated 8-hy-
droxyquinoline compounds in Pharmaceuticals and bulk drugs, J.Pharm.ScL, 1984, 73, 1430-1433.
lofratol
Molecular formula: C3IH36I6N6O13
SAMPLE
Sample preparation: Plasma. Add 30 fjuL 2 mg/mL IS in water to 100 |xL plasma. Add 30 JULL
35% perchloric acid. Agitate and centrifuge at 3500 g for 10 min. Inject a 10 |JLL aliquot of the
clear supernatant. Urine. Dilute 1 mL urine with 2 mL water, centrifuge at 4500 g for 15 min.
Add 100 |xL 5 mg/mL IS, 100 |xL glacial acetic acid, and an ion-exchange resin mixture (1 g
Duolite A-30B + 900 mg Amberlite IR-120). Dilute the suspension to 5 mL with water. Agitate
for 30 min and centrifuge at 3500 g for 5 min. Inject a 10 fxL aliquot of the supernatant.
HPLC VARIABLES
Mobile phase: MeCN:5 mM pH 4.5 potassium dihydrogen phosphate 5:95
Flow rate: 1
Detector: UV 242
CHROMATOGRAM
Internal standard: iopamidol (4.1)
Limit of detection: 75 ng/mL (plasma), 430 ng/mL (urine)
Next Page
KEYWORDS
plasma; rat; human
REFERENCE
Arbughi,T.; Bertani,R; Celeste,R.; Grotti,A.; Tirone,P. High-performance liquid chromatographic determination
of the X-ray imaging contrast agent, iofratol, in plasma and urine, J.Chromatogr.B, 1997, 701, 103-113.
lohexol
Merck Index: 5068
SAMPLE
Matrix: blood
Sample preparation: Mix serum with an equal volume of MeCN, vortex for 15 s, centrifuge at
14000 g for 30 s, dilute the supernatant 100-fold with mobile phase, inject a 10 u,L aliquot.
HPLC VARIABLES
Column: 40 X 3.2 3 |xm Velosep RP-18 (Applied Biosystems)
Mobile phase: 8 mM pH 2 Phosphoric acid
Flow rate: 1
Detector: UV 254
KEYWORDS
serum
REFERENCE
Shihabi,Z.K.; Constantinescu,M.S. lohexol in serum determined by capillary electrophoresis, Clin.Chem., 1992,
38, 2117-2120.
SAMPLE
Matrix: blood
Sample preparation: 50 JJLL Serum + 50 |xL 250 |xg/mL acetaminophen in 100 mM HCl, add to
SPE cartridge containing 150 mg 80-100 mesh Chromosorb P/NAW, elute with 1 mL ethyl
acetate:MeOH 5:1, add the eluate to 50 |xL 100 mM HCl, vortex for 15 s, centrifuge at 10000
g for 3 min, inject a 20 |xL aliquot of the lower aqueous phase.
HPLC VARIABLES
Column: 5 jxm C8
Mobile phase: MeCN:20 mM pH 3.3 phosphoric acid 2.5:97.5
Detector: UV 254
CHROMATOGRAM
Internal standard: acetaminophen
Limit of detection: <1 jxg/mL
OTHER SUBSTANCES
Extracted: aminohippuric acid (PAH)
KEYWORDS
serum; SPE
Previous Page
KEYWORDS
plasma; rat; human
REFERENCE
lohexol
Merck Index: 5068
SAMPLE
Matrix: blood
Sample preparation: Mix serum with an equal volume of MeCN, vortex for 15 s, centrifuge at
14000 g for 30 s, dilute the supernatant 100-fold with mobile phase, inject a 10 u,L aliquot.
HPLC VARIABLES
Column: 40 X 3.2 3 |xm Velosep RP-18 (Applied Biosystems)
Mobile phase: 8 mM pH 2 Phosphoric acid
Flow rate: 1
Detector: UV 254
KEYWORDS
serum
REFERENCE
Shihabi,Z.K.; Constantinescu,M.S. lohexol in serum determined by capillary electrophoresis, Clin.Chem., 1992,
38, 2117-2120.
SAMPLE
Matrix: blood
Sample preparation: 50 JJLL Serum + 50 |xL 250 |xg/mL acetaminophen in 100 mM HCl, add to
SPE cartridge containing 150 mg 80-100 mesh Chromosorb P/NAW, elute with 1 mL ethyl
acetate:MeOH 5:1, add the eluate to 50 |xL 100 mM HCl, vortex for 15 s, centrifuge at 10000
g for 3 min, inject a 20 |xL aliquot of the lower aqueous phase.
HPLC VARIABLES
Column: 5 jxm C8
Mobile phase: MeCN:20 mM pH 3.3 phosphoric acid 2.5:97.5
Detector: UV 254
CHROMATOGRAM
Internal standard: acetaminophen
Limit of detection: <1 jxg/mL
OTHER SUBSTANCES
Extracted: aminohippuric acid (PAH)
KEYWORDS
serum; SPE
REFERENCE
Andreeva,M.; Rapondjieva,A.; Deskova,D.; TishkovJ.; Svinarov,D. Liquid chromatographic determination of
iohexol and PAH with Chromosorb P column used for sample preparation (Abstract 175), Ther.Drug Monit.,
1995, 17, 427.
SAMPLE
Sample preparation: Plasma. 50 |xL Plasma + 150 |xL buffer, centrifuge at 1000 g at 0 for 10
min, inject a 10 |xL aliquot. Tissue. Weigh 2 testes, add 5 mL buffer, homogenize (Kinematica
type PT 10/35, setting 7.5) for 1 min, vortex, centrifuge at 1000 g at 0 for 10 min. Remove a
50 |xL aliquot of the supernatant and add it to 150 (xL buffer, centrifuge at 1000 g at 0 for 10
min, inject a 10 |xL aliquot. (Buffer was 5 mM metaphosphoric acid and 5 mM disodium EDTA.)
HPLC VARIABLES
Guard column: C18 Bondapak guard column
Column: 300 X 3.9 10 |xm ^Bondapak C18
Mobile phase: MeCN:buffer 2.5:97.5-5:95 (Buffer was 100 mM NaH2PO4 and 0.2 mM Na2EDTA
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 7.5 (two isomers seen)
OTHER SUBSTANCES
Also analyzed: iopamidol, diatrizoate
KEYWORDS
plasma; mouse; testes
REFERENCE
Harapanhalli,R.S.; Yaghmai,V.; Patel,Y.D.; Baker,S.R.; Rao,D.V. Assay of radiographic contrast agents in mice
plasma and testes by high-performance liquid chromatography, Anal.Chem., 1993, 65, 606612.
Iopamidol
Merck Index: 5071
SAMPLE
Sample preparation: Plasma. 50 JJLL Plasma + 150 |xL buffer, centrifuge at 1000 g at 0 for 10
min, inject a 10 |xL aliquot. Tissue. Weigh 2 testes, add 5 mL buffer, homogenize (Kinematica
type PT 10/35, setting 7.5) for 1 min, vortex, centrifuge at 1000 g at 0 for 10 min. Remove a
50 (JLL aliquot of the supernatant and add it to 150 |xL buffer, centrifuge at 1000 g at 0 for 10
min, inject a 10 |xL aliquot. (Buffer was 5 mM metaphosphoric acid and 5 mM disodium EDTA.)
HPLCVARIABLES
Guard column: C18 Bondapak guard column
Column: 300 X 3.9 10 fjim ixBondapak C18
Mobile phase: MeCN:buffer 2.5:97.5-5:95 (Buffer was 100 mM NaH2PO4 and 0.2 mM Na2EDTA
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 9.5
OTHER SUBSTANCES
Also analyzed: iohexaol, diatrizoate
KEYWORDS
plasma; mouse; testes
REFERENCE
Harapanhalli,R.S.; Yaghmai,V.; Patel,Y.D.; Baker,S.R.; Rao,D.V. Assay of radiographic contrast agents in mice
plasma and testes by high-performance liquid chromatography, Azia/. Chem., 1993, 65, 606612.
SAMPLE
Sample preparation: Plasma. Add 30 |xL water to 100 |xL plasma. Add 30 |JLL 35% perchloric
acid. Agitate and centrifuge at 3500 g for 10 min. Inject a 10 (xL aliquot of the clear superna-
tant. Urine. Dilute 1 mL urine with 2 mL water, centrifuge at 4500 g for 15 min. Add 100 |xL
5 mg/mL IS, 100 |xL glacial acetic acid, and an ion-exchange resin mixture ( I g Duolite A-30B
+ 900 mg Amberlite IR-120). Dilute the suspension to 5 mL with water. Agitate for 30 min and
centrifuge at 3500 g for 5 min. Inject a 10 |xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 30 X 4.0 7 |xm LiChrosorb RP-8
Column: 250 X 4.6 5 jxm LiChrosorb RP-8
Mobile phase: MeCN:5 mM pH 4.5 potassium dihydrogen phosphate 5:95
Flow rate: 1.0
Detector: UV 242
CHROMATOGRAM
R e t e n t i o n t i m e : 4.1
OTHER SUBSTANCES
Extracted: iofratol
KEY VVORDS
iopamidol is IS; plasma; rat; human
REFERENCE
lothalamate
Molecular formula: C11H9I3N2O4C7H17NO5 (meglumine),
C11H8I3N2NaO4 (sodium salt)
Molecular weight: 809.13 (meglumine), 635.90 (sodium salt)
CAS Registry No.: 13087-53-1, 2276-90-6 (iothalamic acid), 6284-40-8 (meglumine),
17692-74-9 C25I radioactive agent), 15845-98-4 (131I radioactive agent), 1225-20-3 (sodium salt)
SAMPLE
Sample preparation: Dilute urine 10-fold with water. Add 200 |xL MeCN containing 20 |xg/mL
p-aminobenzoic acid to 100 /xL diluted urine, vortex briefly, centrifuge at 12000 g for 4 min,
HPLCVARIABLES
Guard column: 5 |xm Ultrasphere C18
Column: 250 mm 5 |xm Primesphere C18 (Torrance, USA)
Mobile phase: MeOH:buffer 18:82 (Buffer was 50 mM NaH2PO4 with 0.5 mM tetrabutyl am-
monium hydrogen sulfate with an unadjusted pH of 4.11.)
Flow rate: 0.8
Detector: UV 254
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Extracted: p-aminohippuric acid
KEYWORDS
serum
REFERENCE
Agarwal,R. Chromatographic estimation of iothalamate and p-aminohippuric acid to measure glomerular fil-
tration rate and effective renal plasma flow in humans, J.Chromatogr.B, 1998, 705, 3-9.
SAMPLE
Sample preparation: Mix a dilution of the injectable solution in water with a mixture of 2,4-
dinitrobenzenesulfonyl chloride (DNBS-Cl) and sodium carbonate in acetone at ambient tem-
perature for 1 hr. Dilute the reaction mixture with 25% MeOH and inject a 20 |iL aliquot.
HPLC VARIABLES
Mobile phase: MeOHrbuffer 25:75 (Buffer was 20 mM tetrapropylammonium hydroxide adjusted
to pH 3.4 with orthophosphoric acid.)
Flow rate: 1.3
Detector: UV 262
CHROMATOGRAM
Limit of quantitation: 1 jxg
OTHER SUBSTANCES
Simultaneous: diatrizoate, meglumine
KEYWORDS
injections; only meglumine is derivatized under these conditions
REFERENCE
Lau-Cam,C.A.; Roos,R.W. HPLC method with spectrophotometric detection for the simultaneous assay of meg-
lumine and its counterions iothalamic acid or diatrizoic acid in radiographic solutions for injection (Abstract
3372), Pharm.Res., 1997, 14, S586.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma or urine + 0.5-5 |xg p-aminobenzoic acid + 200 |xL MeCN,
vortex for a few s, centrifuge at 800 g for 5 min, inject a 5 jxL aliquot of the supernatant.
HPLC VARIABLES
Mobile phase: MeCN:0.04% pH 2.5 0.05 phosphoric acid 3.5:96.5
Flow rate: 1.5
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 6
Internal standard: p-aminobenzoic acid (8)
OTHER SUBSTANCES
KEYWORDS
plasma; dog; human; pharmacokinetics
REFERENCE
Prueksaritanont,T.; Chen,M.L.; Chiou,W.L. Simple and micro high-performance liquid chromatographic method
for simultaneous determination of p-aminohippuric acid and iothalamate in biologicalfluids,J.Chromatogr.,
1984, 306, 89-97.
SAMPLE
Matrix: blood
Sample preparation: Add barbital to plasma. 100 fxL Plasma + 500 |xL MeOH, vortex for 15 s,
centrifuge at 2500 rpm for 10 min. Remove the organic layer and evaporate it to dryness under
a stream of nitrogen, reconstitute the residue in buffer, inject a 15-20 u,L aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Octyl C8 (Rainin)
Mobile phase: MeOH:MeCN:buffer 90:10:300 (Buffer was 6.44 g KH2PO4, 7.04 g K2HPO4, and
14 mL 500 mM dodecyltriethylammonium phosphate (Regis) in 4 L water.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: barbital (15.9)
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Jayewardene,A-L-; Seneviratne,A.K.; Gambertoglio,J.G. Paired ion reversed-phase HPLC assay for the simul-
taneous determination of iothalamic acid and para aminohippuric acid in plasma, J.Liq.Chromatogr., 1994,
17, 2395-2412.
SAMPLE
Sample preparation: Plasma. 0.5-1 mL Plasma + 18 |xg iodamide + 2 jjig hippuric acid + 1 mL
1 M HCl + 5 mL ethyl acetate, shake for 10 min, centrifuge, repeat extraction. Combine the
organic layers and evaporate them under a stream of nitrogen at 60, add 3 mL 100 mM NaOH,
shake for 10 min, centrifuge, discard the organic layer. Remove the aqueous layer and add it
to 500 iuiLlM HCl, add 5 mL ethyl acetate, shake for 10 min, centrifuge, repeat extraction.
Combine the organic layers and evaporate them to dryness under a stream of nitrogen at 60,
reconstitute the residue in 100 (xL mobile phase, inject a 10-20 jxL aliquot. Urine. 1 mL Urine
+ 10 (JLL 10% iodamide in water, mix. Remove a 100 |xL aliquot and add it to 1 mL 1 M HCl,
add 5 mL ethyl acetate, shake for 10 min, centrifuge, repeat extraction. Combine the organic
residue in 400 JAL mobile phase, inject a 10-20 [xL aliquot.
HPLC VARIABLES
Column: 250 X 2.6 10 (xm ODS-HC Sil-X-1 (Perkin-Elmer)
Mobile phase: MeCN:water:85% phosphoric acid 4:96:0.03
Flow rate: 1
Detector: UV 235
CHROMATOGRAM
Retention time: 2.0
Internal standard: iodamide (2.5), hippuric acid (5.6)
OTHER SUBSTANCES
Extracted: o-iodohippurate
KEYWORDS
REFERENCE
Boschi,S.; Marchesini,B. High-performance liquid chromatographic method for the simultaneous determination
of iothalamate and o-iodohippurate, J.Chromatogr., 1981, 224, 139-143.
SAMPLE
Sample preparation: 200 |xL Plasma + 200 |xL 100 mM NaH2PO4 + 400 |xL MeCN, mix for 5
s, let stand at 4 for 15 min, centrifuge at 10500 g for 1 min. Remove the supernatant and add
it to 2 mL dichloromethane, mix for 5 min, centrifuge at 4800 g for 10 min, inject a 5-50 |xL
aliquot of the upper aqueous phase. Urine. Centrifuge urine at 4800 g for 10 min, dilute 1:10
with 50 mM NaH2PO4, inject an aliquot.
HPLC VARIABLES
Mobile phase: MeCN:water 7.5:92.5 containing 5.50 g/L NaH2PO4.H20,1.80 g/L Na2HPO4.2H2O,
and 20 mg/L tetrabutylammonium bromide, pH 6.4 (plasma) or MeCN.water 5:95 containing
5.50 g/L NaH2PO4-H20,1.80 g/L Na2HPO4.2H2O, and 22.5 mg/L tetrabutylammonium bromide,
pH 6.4 (urine)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: cefotetan (UV 280)
KEYWORDS
REFERENCE
Kees,R; Grobecker,H.; Naber,K.G. High-performance liquid chromatographic analysis of cefotetan epimers in
human plasma and urine, J.Chromatogr., 1984, 305, 363-371.
SAMPLE
Sample preparation: Plasma. 500 |xL Plasma + 500 |xL 1 M HCl, vortex for 10 s, add 6 mL
ethyl acetate, vortex for 20 s, centrifuge at 4 at 1700 g for 10 min. Remove the organic layer
and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 400 JJLL 25
mM pH 3 KH2PO4, add 500 (xL dichloromethane, shake gently horizontally for 5 min, centrifuge
at 1700 g for 5 min, inject a 20 |xL aliquot of the aqueous phase. Urine. Dilute 1:10 with water.
Remove a 1 mL aliquot and add it to 1 mL 1 M HCl, vortex for 10 s, add 6 mL ethyl acetate,
vortex for 20 s, centrifuge at 4 at 1700 g for 10 min. Remove the organic layer and evaporate
it to dryness under a stream of nitrogen, reconstitute the residue in 400 |xL 25 mM pH 3
KH2PO4, add 500 |xL dichloromethane, shake gently horizontally for 5 min, centrifuge at 1700
g for 5 min, inject a 20 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Guard column: 10 X 3 anion-exchange guard column (Chrompack)
Column: 250 X 4.6 Partisil 10 SAX
Mobile phase: MeCN:25 mM pH 3 phosphate buffer 15:85
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: acipimox, allopurinol, aspirin, atenolol, captopril, chlorthalidone, clonidine, dig-
itoxin, digoxin, diltiazem, dipyridamole, enalapril, furosemide, gemfibrozil, hydralazine, hy-
drochlorothiazide, ibopamine, insulin, inulin, isosorbide dinitrate, ot-methyldopa, nicardipine,
nifedipine, prazosin, propranolol, salicylic acid, simvastatin, trinitrin, verapamil
KEYWORDS
REFERENCE
Gaspari,R; Mainardi,L.; Ruggenenti,P.; Remuzzi,G. High-performance liquid chromatographic determination
of iothalamic acid in human plasma and urine, J.Chromatogr., 1991, 570, 435-440.
SAMPLE
Matrix: urine
Sample preparation: Dilute urine 1:100 or 1:500. 200 |xL Diluted urine + 50 |xL barbital solu-
tion, vortex for 15 s, inject a 20-30 JJLL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:10 mM pH 7.5 potassium phosphate buffer:0.5 M dodecyl triethyl-
ammonium phosphate 6:94:300:0.6 (0.5 M Dodecyl triethylammonium phosphate was Q-12, Ion
pair reagent, Regis Chemical Co.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: barbital (14.5)
OTHER SUBSTANCES
REFERENCE
Seneviratne,A.K.; Jayewardene,A.L.; Gambertoglio,J.G. Paired-ion reversed-phase HPLC assay for the deter-
mination of iothalamic acid and para aminohippuric acid in urine, J.Pharm.Biomed.Anal., 1994,12, 1311-
1316.
Ipecac
Molecular formula: C28H38N2O4, C29H40N2O4,
Molecular weight: 466.62,480.65
CAS Registry No.: 483-17-0, 483-18-1
Merck Index: 5086
SAMPLE
Matrix: blood, emesis, urine
Sample preparation: 2 mL Plasma, whole blood, urine, or emesis (diluted 1:100) + 100 |xL 10
ng/mL N-propylprocainamide in water + 2 mL saturated sodium borate + 7 mL n-butyl chloride,
vortex for 30 s, centrifuge. Remove the organic phase and add it to 200 |xL 10 mM HCl, vortex,
centrifuge, inject a 30-50 (xL aliquot of the aqueous phase.
HPLC VARIABLES
Mobile phase: MeOH:25 mM pH 8.0 Na2HPO4 72:28
Flow rate: 1.7
CHROMATOGRAM
Retention time: 3 (cephaeline), 4.1 (emetine)
Internal standard: N-propylprocainamide (1.7)
KEYWORDS
plasma; whole blood
REFERENCE
Crouch,D.J.; Moran,D.M.; Finkle,B.S.; Peat,M.A. Quantitative analysis of emetine and cephaeline by reversed-
phase high performance liquid chromatography with fluorescence detection, JAnal.ToxicoL, 1984, 8, 6 3 -
65.
SAMPLE
Matrix: blood, vomit
Sample preparation: Mix 250 |xL vomit or 500 ^LL serum with an equal volume of 10% am-
monium hydroxide for 5 min, add 4 mL ether, mix. Remove the organic layer and evaporate it
to dryness under vacuum at 25, reconstitute the residue in 10 (vomit) or 0.2 (serum) mL 0.01%
HCl. Mix 1 mL vomit solution with 50 JJLL 4 mg/mL acrinol or 200 JJLL serum solution with 50
|xL 200 |xg/mL acrinol, inject a 100 \xh aliquot.
HPLC VARIABLES
Column: 150 x 4.6 5 ^m TSK gel ODS-80TM
Mobile phase: MeOH:buffer 54:46 (Buffer was 10 mM sodium 1-heptanesulfonate adjusted to
pH 4 with glacial acetic acid.)
Flow rate: 1
CHROMATOGRAM
Internal standard: acrinol
KEYWORDS
dog; serum; pharmacokinetics; assay determines cephaeline, a constituent of ipecac
REFERENCE
Teshima,D.; Suzuki,A.; Otsubo,K; Higuchi,S.; Aoyama,T.; Shimozono,Y.; Saita,M.; Noda,K. Efficacy of emetic
and United State Pharmacopoeia ipecac syrup in prevention of drug absorption, Chem.Pharm.Bull.(Tokyo),
1990, 38, 2242-2245.
SAMPLE
Sample preparation: Dry 1-5 g cell culture at 60, powder, extract with MeOH in a glass per-
colator for 72 h. Filter (paper) extract, wash solid with MeOH at 45, concentrate filtrate under
reduced pressure, filter (0.5 |xm), dilute filtrate with MeOH, inject a 2.5 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4 10 |xm silica gel (Spectra Physics)
Mobile phase: Chloroform:MeOH:diethylamine 90:10:0.2
Flow rate: 0.5
Detector: UV 280
CHROMATOGRAM
Retention time: 4.5 (emetine), 5 (cephaeline)
KEYWORDS
normal phase
REFERENCE
Jha,S.; Sahu,N.R; Mahato,S.B. Production of the alkaloids emetine and cephaeline in callus cultures of
Cephaelis ipecacuanha, Planta Med., 1988, 54, 504-506.
SAMPLE
Sample preparation: Syrup. Dilute syrup with an equal volume of water. 2 mL Diluted syrup
+ 2 mL 1% dansyl chloride in acetone + 200 |JLL 1.5 M sodium carbonate, heat at 45 2 in
the dark for 20 min, cool, add 3 mL water, add 500 |xL benzene (Caution! Benzene is a carcin-
ogen!), shake. Remove the organic layer and evaporate it to dryness, reconstitute the residue
in mobile phase, inject a 10 |xL aliquot. Capsules. Sonicate the contents of a capsule in 20 mL
water for 10 min, centrifuge at 2000 g for 10 min. 2 mL Supernatant + 2 mL 1% dansyl chloride
in acetone + 200 jxL 1.5 M sodium carbonate, heat at 45 2 in the dark for 20 min, cool, add
3 mL water, add 500 u,L benzene (Caution! Benzene is a carcinogen!), shake. Remove the
organic layer and evaporate it to dryness, reconstitute the residue in mobile phase, inject a 10
(xL aliquot.
HPLC VARIABLES
Column: 250 X 2.8 10 |xm silica gel SI 100 (Merck)
Mobile phase: Diisopropyl ether.isopropanol.concentrated ammonia 48:2:0.3 (Caution! Diisopro-
pyl ether readily forms explosive peroxides!)
Detector: UV 254 or F ex 358 em 492 (cephaeline) or ex 356 em 481 (emetine)
CHROMATOGRAM
Retention time: 2.5 (cephaeline), 3.5 (emetine)
OTHER SUBSTANCES
Simultaneous: ephedrine, codeine (not derivatized, detect at UV 254 only)
KEYWORDS
derivatization; syrup; capsules; normal phase
REFERENCE
Frei,RW.; Santi,W.; Thomas,M. Liquid chromatography of dansyl derivatives of some alkaloids and the appli-
cation to the analysis of Pharmaceuticals, J.Chromatogr., 1976, 116, 365-377.
SAMPLE
Sample preparation: Mix 10 g linctus with 10-20 mL 200 jxg/mL ethyl 4-hydroxybenzoate in
MeCN:mobile phase 10:90, make up to 100 mL with mobile phase, inject a 10-25 |xL aliquot.
Mix 10 pastilles with 50 mL 200 |xg/mL ethyl 4-hydroxybenzoate in MeCN:mobile phase 10:
90, make up to 200 mL with mobile phase, inject a 10-25 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 u.Bondapak C18
Mobile phase: MeOH.water 40:60 containing 1 g/L sodium 1-heptanesulfonate and 1 mL/L or-
thophosphoric acid
Flow rate: 2
CHROMATOGRAM
Retention time: 8 (cephaeline), 15 (emetine)
Internal standard: ethyl 4-hydroxybenzoate (5)
Limit of quantitation: 5 fxg/g
OTHER SUBSTANCES
Simultaneous: dihydroemetine (UV 214), emetamine (UV 214), O-methylpsychotrine (UV 214),
tetrahydroemetine (UV 214)
KEYWORDS
stability-indicating; linctus; pastilles; rugged
REFERENCE
Elvidge,D.A.; Johnson,G.W.; Harrison,J.R. Selective, stability-indicating assay of the major ipecacuanha alka-
loids, emetine and cephaeline, in pharmaceutical preparations by high-performance liquid chromatography
using spectrofluorimetric detection, J.Chromatogr., 1989, 463, 107-118.
SAMPLE
Sample preparation: 2 mL Sample + 1 mL 200 |xg/mL emetine hydrochloride in water, make
up to 10 mL with mobile phase, filter (0.45 |xm), inject a 50-100 (JLL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Technosphere RP C-8 (HPLC Technology)
Mobile phase: MeCN:40 mM tetramethylammonium bromide: 1 M acetic acid 80:15:5 (apparent
pH 4.5)
Flow rate: 1.5
Detector: UV 260
CHROMATOGRAM
Internal standard: emetine
OTHER SUBSTANCES
Simultaneous: benzalkonium (C 12, C14, C16), tetrahydrozoline, naphazoline
KEYWORDS
nasal; ophthalmic; emetine is IS
REFERENCE
Santoni,G.; Medica,A.; Gratteri,R; Furlanetto,S.; Pinzauti,S. High-performance liquid chromatographic deter-
mination of benzalkonium and naphazoline or tetrahydrozoline in nasal and ophthalmic solutions, Farmaco,
1994, 49, 751-754.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 X 4 C18 Micropak SP
Flow rate: 2
CHROMATOGRAM
Retention time: 4.1 (cephaeline), 6.2 (emetine)
REFERENCE
Lachman,M.R; Romeo,R.; McComb,R.B. Emetine identified in urine by HPLC, with fluorescence and ultravi-
olet/diode array detection, in a patient with cardiomyopathy, Clin.Chem., 1989, 35, 499-502.
Ipriflavone
Merck Index: 5090
SAMPLE
Matrix: bulk
Sample preparation: Inject a 10 jxL aliquot of a solution in MeOH.
HPLC VARIABLES
Column: 30 X 4.6 3 |xm ODS C18 (Perkin-Elmer)
Mobile phase: MeCN:buffer 50:50 (Buffer was 10 mM triethylamine adjusted to pH 2.5 with
orthophosphoric acid.)
Flow rate: 1.2
Detector: UV 225
CHROMATOGRAM
Retention time: 3.4
OTHER SUBSTANCES
REFERENCE
Sustacha,K; Chac6n,M.; Lucero,M.L.; Orjales,A. Determination of ipriflavone and its synthetic impurities by
high-performance liquid chromatography using diode-array detection, J.Chromatogr.A, 1996, 719,245-250.
lprindole
Merck Index: 5091
Lednicer No.: 1 318
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
operidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, isothipendyl, isoxsuprine,
methotrimeprazine, methoxamine, methox^henamine, methoxypromazine, methylephedrine,
REFERENCE
Iproniazid
Merck Index: 5094
Lednicer No.: 1 254
SAMPLE
Matrix: blood
the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 JJLL aliquot of
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 264
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
lrbesartan
Merck Index: 5097
SAMPLE
Sample preparation: Condition a 3 mL 100 mg Isolute Cyano SPE cartridge (Jones Chroma-
tography) with 2 mL MeOH and 2 mL 0.85% phosphoric acid. Plasma. Mix 1.0 mL 0.85%
phosphoric acid and 100 |xL 1 |xg/mL IS in 0.85% phosphoric acid with 250 |xL plasma. Pass
the mixture slowly through the SPE cartridge, wash with 3 mL 0.85% phosphoric acid, wash
with 1 mL hexane. Elute with 1 mL of mixture MeOH:0.85% phosphoric acid 50:50. Inject a
20 JXL aliquot of the eluate. Urine. Mix 1.0 mL 0.85% phosphoric acid and 100 |JLL 1 |jig/mL IS
in 0.85% phosphoric acid with 250 |xL urine. Pass the mixture slowly through the SPE car-
tridge, wash with 4 mL 0.85% phosphoric acid. Elute with 1 mL of mixture MeOH:0.85%
phosphoric acid 50:50. Inject a 20 |xL aliquot of the eluate.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm YMC-ODS-AQ
Mobile phase: MeCN:buffer 50:50 (Prepare buffer by adding 1 mL triethylamine to 1 L water,
adjust pH to 3.5 with 85% phosphoric acid.)
Flow rate: 0.8
CHROMATOGRAM
Retention time: 6.6
Internal standard: BMS-190462 (9.4)
KEYWORDS
plasma; urine; SPE; pharmacokinetics
REFERENCE
Chang,S.-Y.; Whigan,D.B.; Vachharajani,N.N.; Patel,R. High-performance liquid chromatographic assay for the
quantitation of irbesartan (SR 47436/BMS-186295) in human plasma and urine, J.Chromatogr.B, 1997,
702, 149-155.
lrinotecan
Merck Index: 5104
SAMPLE
Matrix: bile
Sample preparation: Dilute bile, inject an aliquot.
HPLC VARIABLES
Column: 300 X 7.2 TSKgel-ODS 80Tm (Tosoh)
Mobile phase: Gradient. MeOH: 100 mM pH 4.0 phosphate buffer from 50:50 to 60:40 over 20
min, maintain at 60:40 for 20 min.
Flow rate: 3
Detector: UV 254
CHROMATOGRAM
Retention time: 16
OTHER SUBSTANCES
KEYWORDS
rat; preparative for metabolites
REFERENCE
Atsumi,R.; Suzuki,W.; Hakusui,H. Identification of the metabolites of irinotecan, a new derivative of campto-
thecin, in rat bile and its biliary excretion, Xenobiotica, 1991, 21, 1159-1169.
SAMPLE
Matrix: blood
Sample preparation: Mix 10 u,L plasma with 100 jxM diisopropyl fluorophosphate, 125 ng/mL
camptothecin and 375 |xL MeOH with an automatic mixer (S-100, Taitec, Saitama, Japan) for
30 s, centrifuge at 3000 rpm for 10 min. Evaporate the supernatant on a Speed Vac Plus
SCIlOA (Savant Instruments. Inc., Farmingdale, NY) and dissolve the residue in 200 |xL THF:
50 mM pH 2.0 KH2PO4 containing 5 mM heptanesulfonate 25:75. Centrifuge at 10000 rpm for
3 min, inject 20 or 50 |xL aliquot.
HPLC VARIABLES
Guard column: 15 x 3.5 5 fjim TSK-GEL ODS-80TS (Toyo soda, Tokyo)
Column: 150 X 4.6 5 |xm TSK-GEL ODS-80TS (Toyo soda, Tokyo)
Mobile phase: THF:50mM pH 4.0 KH2PO4 containing 5mM heptanesulfonate 25:75
Flow rate: 0.8
Injection volume: 20 or 50
CHROMATOGRAM
Internal standard: camptothecin
KEYWORDS
plasma; mouse; pharmacokinetics
REFERENCE
Ohdo,S.; Makinosumi,T.; Ishizaki,T.; Yukawa,E.; Higuchi,S.; Nakano,S.; Ogawa,N. Cell cycle-dependent chron-
otoxicity of irinotecan hydrochloride in mice, J.Pharmacol.Exp.Ther., 1997, 283, 1383-1388.
SAMPLE
Matrix: blood
Sample preparation: Determination of lactone form. 1 mL Plasma + 100 |JLL 25 ng/mL IS in
MeOH: 10 mM HCl 40:60 + 800 mg solid NaCl, extract with 7.5 mL MeCN:n-butyl chloride 20:
80 for 5 min, centrifuge at 4000 g for 2 min. Rotate quickly by hand to break the gels, centrifuge
at 4000 g for 5 min. Mix the organic layer with 50 jxL DMSO, dry under a gentle stream of
nitrogen at 60 to approximately 50 JJLL. Reconstitute the residue in 100 JJLL MeOH and 100 |xL
perchloric acid:water 1:500, vortex for 5 s, inject a 100 |xL aliquot. Total determination. Mix
250 JJIL plasma with 500 |xL cold (-20) MeOH:perchloric acid:water 20:1:20, centrifuge at 24000
g for 5 min, inject a 100 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: 100 X 4.6 5 u-m Hypersil ODS
Mobile phase: MeOH:buffer 40:60 adjusted to pH 5.5 with HCl (A, lactone form determination)
or MeOH:buffer 35:65 adjusted to pH 5.5 with HCl (B, total determination). (Buffer was 100
mM ammonium acetate containing 10 mM tetrabutylammonium sulfate.)
Flow rate: 1
CHROMATOGRAM
Internal standard: camptothecin (6.5, A)
Limit of quantitation: 200 pg/mL (lactone form), 2.0 ng/mL (total)
OTHER SUBSTANCES
Extracted: active metabolite
Noninterfering: acetaminophen, alizapride, codeine, dexamethasone, domperidone, metoclo-
pramide, morphine, ranitidine
KEYWORDS
REFERENCE
de Bruijn,R; Verweij,J.; Loos,W.J.; Nooter,K; Stoter,G.; Sparreboom,A. Determination of irinotecan (CPT-Il)
and its active metabolite SN-38 in human plasma by reversed-phase high-performance liquid chromatog-
raphy withfluorescencedetection, J.Chromatogr.B, 1997, 698, 277-285.
SAMPLE
Matrix: blood
Sample preparation: Add 1 mL MeCNiMeOH 50:50 to 500 JJLL plasma at -20, vortex for 10 s.
Centrifuge at 10000 g at 4 for 3 min, mix 70 |xL supernatant with 70 |xL mobile phase at 0,
vortex, inject a 20 JJLL aliquot.
HPLCVARIABLES
Guard column: 10 X 3 C18 (Chrompack, Netherlands)
Mobile phase: MeCNiIOOmM pH 6.4 ammonium acetate:triethylamine 15.6:80:0.1 containing 5
mM tetrabutylammonium phosphate
Flow rate: 1.5
CHROMATOGRAM
Retention time: 5.7 (carboxylate form), 11.1 (lactone form)
OTHER SUBSTANCES
Extracted: metabolites (F ex 385 em 525)
Noninterfering: lurtotecan, topotecan
KEYWORDS
REFERENCE
Herben,V.M.M.; Mazee,D.; van Gortel-van Zomeren,D.M.; Zeedijk,S.; Schellens,J.H.M.; ten Bokkel Huin-
ink,W.W.; Beijnen,J.H. Sensitive determination of the carboxylate and lactone forms of the novel antitumor
drug irinotecan and its active metabolite in plasma by HPLC, J.Liq.Chromatogr.Rel.Technol., 1998, 21,
1541-1558.
SAMPLE
Matrix: blood
1 mL water at 0.5 mL/s. 100 |xL Plasma + 50 |xL 1 |xg/mL camptothecin in 10 mM HCl + 850
(xL 10 mM HCl, mix, add to the SPE cartridge at 2.4 mL/min, wash with 1.5 mL water at 6
mL/min, wash with 1 mL 10 mM HCl at 6 mL/min, elute with 1.5 mL acidic MeOH at 2.4 mL/
min. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue in
250 [ih mobile phase, inject an aliquot. (Prepare IS solution by sonicating 1 mg camptothecin
in 25 mL MeOH at room temperature for 20 min, dilute to 10 |xg/mL with water, dilute to 1
|xg/mL with 10 mM HCl. Prepare acidic MeOH by mixing 100 JJLL concentrated HCl with 100
mL MeOH.)
HPLCVARIABLES
Guard column: 22 X 3.5 10 |xm Nucleosil C18
Column: 300 X 3.9 10 jjim Nucleosil octadecylsilane
Mobile phase: MeCN: 100 mM KH2PO4 34:66 containing 3 mM sodium heptanesulfonate, ad-
justed to pH 4 with 1 M HCl
Flow rate: 1
CHROMATOGRAM
Retention time: 5.5
Internal standard: camptothecin (9)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Barilero,L; Gandia,D.; Armand,J.-R; Mathieu-Bou6,A.; R6,M.; Chabot,G.G. Simultaneous determination of the
camptothecin analogue CPT-Il and its active metabolite SN-38 by high-performance liquid chromatography:
application to plasma pharmacokinetic studies in cancer patients, J.Chromatogr., 1992, 575, 275280.
SAMPLE
Matrix: blood
Sample preparation: Evaporate 50 (JLL 1 |xg/mL camptothecin in acetone into the bottom of a
tube under a stream of nitrogen, add 50 |xL plasma, add 100 |xL ice-cold MeCN:MeOH 50:50,
vortex for 5 s, centrifuge at 8000 g briefly. Remove a 100 |xL aliquot of the supernatant and
add it to 70 |xL 75 mM pH 6.4 ammonium acetate buffer, vortex briefly, inject a 5-20 |xL aliquot.
HPLCVARIABLES
Guard column: Guard-Pak Nova-Pak C18
Column: 100 X 5 4 |xm Nova-Pak Radial-Pak C18
Mobile phase: MeCN:75 mM pH 6.4 ammonium acetate buffer 22:78 containing 5 mM tetra-
butylammonium phosphate (PIC A)
Flow rate: 1.5
CHROMATOGRAM
Retention time: 4.2 (carboxylate form), 8.2 (lactone form)
Internal standard: camptothecin (6.5 (carboxylate form), 10.5 (lactone form))
OTHER SUBSTANCES
KEYWORDS
plasma; pharmacokinetics; carboxylate form is the inactive form
REFERENCE
Rivory,L.R; Robert,J. Reversed-phase high-performance liquid chromatographic method for the simultaneous
quantitation of the carboxylate and lactone forms of the camptothecin derivative irinotecan, CPT-Il and
its metabolite SN-38 in plasma, J.Chromatogr.B, 1994, 661, 133-141.
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Plasma + 50 |xL 1.25 |xg/mL camptothecin in MeOH:0.5 M HCl
97.5:2.5 + 750 |xL MeOH, vortex for 10 s, centrifuge at 20 at 10000 g for 5 min. Remove the
supernatant and evaporate it to dryness under reduced pressure, reconstitute with 400 |xL
mobile phase adjusted to pH 2.0, vortex for 10 s, centrifuge at 20 at 10000 g for 5 min, inject
a 100 |xL aliquot of the supernatant.
HPLCVARIABLES
Guard column: 15 X 3.2 TSK guardgel ODS-120T
Column: 150 X 4.6 5 |xm TSK gel ODS-80Ts
Mobile phase: MeCN:50 mM Na2HPO4 28:72 containing 5 mM sodium 1-heptanesulfonate, ad-
justed to pH 3.0 with orthophosphoric acid (Prepared by dissolving 17.9 g Na2HPO4.12H2O and
1.1 g sodium 1-heptanesulfonate hydrate in 1 L water and adding 388 mL MeCN, adjust pH
to 3.0 with orthophosphoric acid.)
Flow rate: 1
Detector: F ex 380 em 556 or ex 370 em 430
CHROMATOGRAM
Retention time: 5.4
Internal standard: camptothecin (8.8)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Sumiyoshi,H.; Fujiwara,Y.; Ohune,T.; Yamaoka,N.; Tamura,K.; Yamakido,M. High-performance liquid chro-
matographic determination of irinotecan (CPT-Il) and its active metabolite (SN-38) in human plasma,
J.Chromatogr.B, 1995, 670, 309-316.
SAMPLE
Matrix: blood, feces, urine
Sample preparation: Plasma, urine. Thaw frozen plasma or urine sample in a waterbath, vor-
tex, add 250 |xL plasma or 250 |xL plasma:diluted urine 50:50 to 500 u,L MeOH:5% (w/v) aque-
ous perchloric acid 50:50. Mix for 5 min, centrifuge at 24000 g for 5 min, dilute the upper
aqueous layer from plasma and urine extracts 2-fold and 10-fold, respectively, with mobile
phase, inject a 100-200 JJLL aliquot. Feces. Homogenize feces with 5 volumes of 5% (w/v) perch-
loric acid in water using five 1 min bursts from an Ystral X1020 tissue homogenizer at 20500
r.p.m. Centrifuge at 24000 g for 10 min, dilute with one volume of drug-free human plasma,
and process further as described for the urine sample.
HPLC VARIABLES
Guard column: 4 x 4 LiChroCart endcapped RP-18 Merck
Mobile phase: MeOH: 100 mM ammonium acetate containing 10 mM tetrabutylammonium sul-
phate 30:70, pH adjusted to 5.3 with HCl
Flow rate: 1.0
CHROMATOGRAM
Limit of quantitation: 10 ng/mL (plasma), 100 ng/mL (feces, urine)
OTHER SUBSTANCES
KEY WORDS
REFERENCE
Sparreboom,A-; de Bruijn,R; de Jonge,M.J.A.; Loos,W.J.; Stoter,G.; Verweij,J.; Nooter,K. Liquid chromato-
graphic determination of irinotecan and three major metabolites in human plasma, urine and feces,
J.Chromatogr.B, 1998, 712, 225-235.
SAMPLE
Sample preparation: Condition an Analytichem C18 SPE cartridge with 1.5 mL MeOH and 1.5
mL water. Homogenize (PTFE homogenizer) tissue with four volumes ice-cold 150 mM KCl,
centrifuge at 2 at 9000 g for 20 min. Dilute plasma, serum, or tissue homogenate supernatant
10-fold with 100 mM HCl, add to the SPE cartridge, elute the contents of the SPE cartridge
on to the column with the mobile phase.
HPLCVARIABLES
Guard column: RP-18
Column: 250 X 4 LiChrosorb RP-18
Mobile phase: MeCN:EtOH:0.8% ammonium carbonate 50:25:25
Flow rate: 1
CHROMATOGRAM
Retention time: 6.6
KEY WORDS
mouse; plasma; serum; liver; epithelium; pharmacokinetics; SPE
REFERENCE
Kaneda,N.; Nagata,H.; Furuta,T.; Yokokura,T. Metabolism and pharmacokinetics of the camptothecin analogue
CPT-Il in the mouse, Cancer Res., 1990, 50, 1715-1720.
lsepamicin
Molecular formula: C22H43N5Oi2
Merck Index: 5121
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 0.5-5.0 |xg IS and 2 mL EtOH. Add 7 mL dichlo-
romethane and 1 mL water, mix, centrifuge. Inject an aliquot of the aqueous supernatant onto
column A, elute to waste with mobile phase A, after 4 min elute the contents of column A onto
column B with mobile phase B, elute column B with mobile phase B. Mix the effluent from
column B with o-phthalaldehyde at 0.2 mL/min and monitor.
HPLC VARIABLES
Column: A 3.9 X 4.0 10 |xm Guard Pak Cyano (Waters); B 150 X 4.6 5 ujn Shandon Hypersil
C18
Mobile phase: A 17 mM acetic acid containing 10 mM hexanesulfonic acid; B 17 mM acetic acid
containing 10 mM hexanesulfonic acid, 100 mM sodium acetate, and 3.53 M MeOH
Detector: F ex 338 em 450 (cut-off filter) following post-column reaction. The column effluent
mixed with o-phthalaldehyde pumped at 0.2 mL/min and the mixture flowed to the detector.
CHROMATOGRAM
Retention time: 7.4
Internal standard: dibekacin (9.5)
OTHER SUBSTANCES
Simultaneous: aspirin, caffeine, chlorpheniramine, gentamicin, neomycin, netilmicin, sisomicin
KEYWORDS
plasma; pharmacokinetics; comparison with RIA and microbiological assay; post-column reaction;
column-switching
REFERENCE
Lin,C.-.; Veals,J.; Korduba,C; Hilbert,M.J.; Nomeir,A. Analysis of isepamicin in human plasma by radioim-
munoassay, microbiologic assay, and high-performance liquid chromatography, Ther.Drug Monit., 1997,19,
675-681.
SAMPLE
Matrix: blood
Sample preparation: Dry blood on gauze, soak gauze in 500 (xL 500 mM Na2HPO4 at 35 for
30 min, inject a 50 jxL aliquot.
HPLC VARIABLES
Mobile phase: 16 mM Sodium sulfate containing 5 mM 1-heptanesulfonic acid (PIC B-7)
Flow rate: 0.6
Detector: F ex 360 em 440 following post-column reaction. The column effluent was mixed with
reagent pumped at 0.3 mL/min and flowed through a 5 m X 0.25 mm i.d. coil of PTFE tubing
to the detector. (Prepare reagent by dissolving 300 mg o-phthalaldehyde in 500 mL MeOH, add
1.25 mL p-mercaptoethanol, add 500 mL 400 mM pH 10.4 potassium borate buffer.)
CHROMATOGRAM
Retention time: 12
KEYWORDS
post-column reaction; dried blood
REFERENCE
Shoshihara,M.; Kase,K; Yoshizawa,E.; Takao,M.; Fujimoto,T. Column liquid chromatographic determination of
isepamicin in nasal cavity using gauze, J.Chromatogr., 1990, 529, 473-478.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Plasma + 20 |xL 10 mg/mL gentamicin CIa in water + 50 |xL buffer,
vortex for 15 s, add 200 |xL MeCN, vortex for 20 s, centrifuge at 2000 g for 5 min. Filter (Millex-
HV4) the supernatant. Heat 200 |xL filtrate and 20 |xL 250 mg/mL l-fluoro-2,4-dinitrobenzene
in MeCN at 80 for 1 h, cool, inject a 50 ^xL aliquot. (Buffer was 3.81 g disodium tetraborate
decahydrate in water, adjust pH to 10 with NaOH, make up to 100 mL with water.)
HPLCVARIABLES
Guard column: 25 X 4 10 fxm LiChroCART RP 18
Column: 250 X 4 5 jxm LiChrosorb RP 18
Mobile phase: MeCN:water 70:30 containing 1 mL/L acetic acid
Flow rate: 2
Detector: UV 365
CHROMATOGRAM
Internal standard: gentamicin CIa (10.0)
OTHER SUBSTANCES
Noninterfering: ampicillin, aspirin, captopril, cefazolin, cefotaxime, ceftazidime, ceftriaxone,
cephalosporins, chlorpromazine, diazepam, heparin, propranolol, sulfamethoxazole, sulpiride,
trimethoprim, verapamil
KEYWORDS
plasma; guinea pig; human; derivatization
REFERENCE
Dionisotti,S.; Bamonte,R; Scaglione,R; Ongini,E. Simple measurement of isepamicin, a new aminoglycoside
antibiotic, in guinea pig and human plasma, using high-performance liquid chromatography with ultraviolet
detection, Ther.Drug Monit., 1991, 13, 73-78.
SAMPLE
with 2 mL MeOH, 2 mL water, and 2 mL buffer. 1 mL Plasma + 100 |xL 100 |xg/mL dibekacin
add to SPE cartridge, wash with 500 |JLL water, wash with 250 JJLL mobile phase, elute to
dryness. Elute with 250 jxL mobile phase, inject an aliquot of the eluate. Urine, dialysate.
Dilute 1:100 with water, add 100 |xL 100 |xg/mL dibekacin per 1 mL of sample, mix well, inject
a 100 JULL aliquot. (Buffer was 0.94 g sodium hexanesulfonate in 300 mL water, add 500 |xL
HPLCVARIABLES
Guard column: 10 X 4.6 5 fjun Hypersil C18
Mobile phase: MeOHrbuffer 10:90 (Buffer was 3.76 g sodium hexanesulfonate + 28.4 g sodium
Flow rate: 1.1
CHROMATOGRAM
Retention time: 6.7
Internal standard: dibekacin (17)
OTHER SUBSTANCES
Simultaneous: kanamycin, gentamicin, tobramycin, netilmicin
KEYWORDS
REFERENCE
SAMPLE
Sample preparation: 1 mL Plasma or urine + 0.5-5 |xg dibekacin + 2 mL EtOH, mix, add 7 mL
dichloromethane, add 1 mL water, centrifuge, inject an aliquot of the aqueous supernatant on
to column A and elute to waste with mobile phase A, after 4 min elute the contents of column
A on to column B with mobile phase B, monitor the effluent from column B.
HPLC VARIABLES
Column: A 3.9 X 4 10 jxm Guard Pak with Cyano insert; B 150 X 4.6 5 |xm Hypersil C18
Mobile phase: A 17 mM Acetic acid containing 10 mM hexanesulfonate; B MeOH:buffer 14.3:
85.7 (Mobile phase contained 100 mM sodium acetate, 17 mM acetic acid, 10 mM hexanesul-
fonate, and 3.53 M MeOH.)
Detector: F ex 338 (band-pass filter) em 418-700 and 450 cut-off niters following post-column
reaction with o-phthalaldehyde derivatizing reagent pumped at 0.2 mL/min.
CHROMATOGRAM
KEY WORDS
plasma; pharmacokinetics; post-column reaction; column-switching
REFERENCE
Lin,C.-C; Radwanski,E.; Korduba,C; Cayen,M.; Affrime,M. Pharmacokinetics of intravenously administered
isepamicin in men, Antimicrob.Agents Chemother., 1995, 39, 2774-2778.
Isocarboxazid
Merck Index: 5172
Lednicer No.: 1 233
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 200 |xL 1.5 M NaOH + 10 |xL 200 |xg/mL IS in 100 mM
HCl, vortex, add 5 mL hexane:ethyl acetate 80:20, shake mechanically at low speed for 10 min,
centrifuge at 10 at 1100 g for 10 min. Remove the organic layer and add it to 500 |xL 2 M
HCl, shake mechanically for 5 min, centrifuge at 10 at 1100 g for 5 min. Remove the aqueous
phase and add it to 200 jxL saturated K2HPO4, vortex, inject a 50 JJLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm LC-18 (Supelco)
Mobile phase: MeOH:5 mM octanesulfonic acid 50:50
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 6.4
Internal standard: 5-methyl-3-isoxazolecarboxylic acid 2-(2-propyl-l-phenyl)hydrazide (Ro 5-
1226) (15.5)
KEYWORDS
REFERENCE
Powell,M.L.; Town,C; Henderson,L.; Buck,C. Determination of marplan in human plasma using high-perfor-
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
tilbene, imipramine, indomethacin, isoniazid, isoproterenol, isoxsuprine, ivermectin, ketamine,
yldopamine, methylphenidate, methylprednisolone, methyltestosterone, methj^prylon, meto-
naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, nifiumic acid, nitrazepam, nor-
puromycin, pyrilamine, pjnnthyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
18, 233-242.
lsoetharine
CAS Registry No.: 530-08-5, 2576-92-3 (HCI), 7279-75-6 (mesylate)
Merck Index: 5185
Lednicer No.: 2 9
SAMPLE
Matrix: blood
Sample preparation: 25-150 |xL Plasma + 100 |xL 50 ng/mL colterol mesylate in 25 mM sulfuric
acid + 2 mL 2% boric acid + 2 g ammonium sulfate + 10 mL 0.5% di(2-ethylhexyl)phosphoric
acid in benzene (Caution! Benzene is a carcinogen!), shake, centrifuge. Remove the organic
phase and add it to 130 |xL 25 mM sulfuric acid, shake, centrifuge, inject a 100 |xL aliquot of
the aqueous phase.
HPLC VARIABLES
Mobile phase: MeOH:buffer 12.5:87.5 (Buffer was 100 mM sodium sulfate adjusted to pH 2.8
phosphoric acid then adjusted to pH 3.0 with NaOH.)
Flow rate: 1.2
Detector: E, Bioanalytical Systems LC-4, TL-5 glassy carbon electrode +0.60 V, Ag/AgCl refer-
ence electrode
CHROMATOGRAM
Retention time: 9.3
Internal standard: colterol mesylate (7.5)
KEYWORDS
REFERENCE
Park,G.B.; Koss,R.R; Utter,J.; Mayes,B.A.; Edelson,J. Determination of isoetharine in plasma by reversed-
phase chromatography with amperometric detection, J.Pharm.ScL, 1982, 71, 932-934.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.7-1
Detector: UV 282
KEYWORDS
REFERENCE
J.Liq.Chromatogr., 1995, 18, 649-671.
Isofezolac
Merck Index: 5188
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 1 mL 100 mM pH 4.4 citrate buffer + 100 |xL 40
|xg/mL IS in MeOH + 15 mL diethyl ether, agitate, centrifuge at 3000 g for 5 min. Remove the
residue in 0.5-3 mL mobile phase, inject a 20 |xL aliquot. Urine. 100 JJLL Urine + 1 mL 100 mM
pH 4.4 citrate buffer + 100 |xL 40 jxg/mL IS in MeOH + 100 JJLL enzyme solution containing
100000 U/mL p-glucuronidase and 1000000 U/mL arylsulfatase (I.B.F.), heat at 37 for 16 h,
add 15 mL diethyl ether, agitate, centrifuge at 3000 g for 5 min. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen at 40, reconstitute the residue in 1-2 mL
HPLC VARIABLES
Column: 150 X 4.6 5 n,m LiChrosorb RP 8
Mobile phase: MeCN:water:200 mM pH 3 phosphate buffer 65:15:20
Flow rate: 1.5
CHROMATOGRAM
Retention time: 2.7
Internal standard: l-phenyl-3,4-di-p-chlorophenylpyrazole-5-acetic acid (4.5)
Limit of detection: 10 ng/mL (F)
KEYWORDS
REFERENCE
Bannier,A.; Brazier,J.L. Determination of isofezolac in biological fluids by reversed-phase liquid column chro-
lsoflupredone
Molecular formula: C2IH27FO5
Merck Index: 5190
SAMPLE
Sample preparation: Cream. Weigh out cream containing 1 mg diflorasone diacetate, add 30
mL 40 jxg/mL isoflupredone acetate in water-saturated chloroform, shake for 30 min, centrifuge
at 2000 rpm for 15 min, inject a 10 |xL aliquot of the lower chloroform layer. Ointment. Weigh
out ointment containing 0.5 mg diflorasone diacetate, add 15 mL 40 |xg/mL isoflupredone ac-
etate in water-saturated chloroform, shake for 30 min, centrifuge at 2000 rpm for 15 min,
inject a 10 |xL aliquot of the lower chloroform layer. Bulk. Dissolve 1.5 mg bulk drug in 50 mL
40 jig/mL isoflupredone acetate in water-saturated chloroform, inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 3 |xm silica gel (Perkin-Elmer part 0258-1500)
Mobile phase: Butyl chloride:dichloromethane:THF:acetic acid 70:25:2:3 (Butyl chloride and
dichloromethane were saturated with water.)
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Retention time: 24 (isoflupredone acetate)
Internal standard: isoflupredone acetate
OTHER SUBSTANCES
Simultaneous: related compounds, diflorasone diacetate
KEYWORDS
cream; ointment; normal phase; isoflupredone acetate is IS
REFERENCE
Shaw,M.C; Vanderwielen,A.J. Liquid chromatographic assay for diflorasone diacetate in cream and ointment
formulations, J.Pharm.ScL, 1984, 73, 1606-1608.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm SMOO (Brownlee)
Mobile phase: Butyl chloride:THF:MeOH:glacial acetic acid 88:2.5:2.5:2.5 (Butyl chloride was
50% water saturated.)
Detector: UV 254
CHROMATOGRAM
Retention time: 16 (isoflupredone), 10 (isoflupredone acetate
OTHER SUBSTANCES
Simultaneous: bromoprednisolone acetate, prednisolone, fluoroprednisone acetate
KEYWORDS
normal phase
REFERENCE
Kane,M.R; Tsuji,K. Radiolytic degradation scheme for 60Co-irradiated corticosteroids, J.Pharm.ScL, 1983, 72,
30-35.
lsoflurane
Molecular formula: C3H2CIF5O
Merck Index: 5191
SAMPLE
Matrix: solutions
Sample preparation: Mix 50 |xL phosphate buffer containing isoflurane and 50 |xL 0.05 mM
toluene in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: RCSS Guard-Pak (xBondapak C18 precolumn cartridge
Column: 100 X 8 4 |xm Nova-Pak C18 Radial Compression Module
Flow rate: 3.5
Detector: UV 203
CHROMATOGRAM
Retention time: 5
Internal standard: toluene (12)
Limit of detection: 0.2 mM
OTHER SUBSTANCES
Simultaneous: enflurane, halothane
KEYWORDS
buffer
REFERENCE
Janicki,P.K.; Erskine,W.A.R.; James,M.F.M. High-performance liquid chromatographic method for the direct
determination of the volatile anaesthetics halothane, isoflurane and enflurane in water and in physiological
buffer solutions, J.Chromatogr., 1990, 518, 250-253.
Isoniazid
Merck Index: 5203
Lednicer No.: 1 254
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 |xL 30|xg/mL IS in water, mix, add 2 g ammonium
sulfate, add 40 mL water saturated n-butanol:chloroform 30:70, shake for 10 min, centrifuge
at 500 g for 10 min. Transfer the organic layer into a tube, add 1 mL 1 M sulfuric acid, shake
for 10 min, centrifuge at 500 g for 10 min, inject a 250 jxL aliquot of the upper aqueous layer.
(Caution! Chloroform is a carcinogen !)
HPLC VARIABLES
Guard column: lOfxm (xBondapak C18
Column: 115 X 8 5 |xm (xBondapak C18 radial compression
Mobile phase: EtOH: 1 mM dioctyl sulfosuccinate 45:55, adjusted to pH 2.50
Flow rate: 4
Detector: UV 254
CHROMATOGRAM
Internal standard: l-benzoyl-2-isonicotinoylhydrazine (8.9)
OTHER SUBSTANCES
Extracted: acetylisoniazid
KEYWORDS
plasma
REFERENCE
Holdiness,M.R. High pressure liquid chromatographic determination of isoniazid and acetylisoniazid in human
plasma, J.Liq.Chromatogr., 1982, 5, 707-714.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 50 JULL 50 jutg/mL phenelzine + 400 (xL 10% acetic acid +
7 mL diethyl ether:dichloromethane 2:1, shake, centrifuge at 2059 g for 10 min. Remove the
aqueous layer and add it to 600 |xL 10% acetic acid, add 300 jxL 0.1% salicaldehyde in EtOH,
heat at 60 for 30 min, cool to room temperature, add 1 mL 1 M K2PO4 (sic), extract with 7 mL
diethyl ether, centrifuge at 2059 g for 10 min, repeat extraction. Combine the organic layers
and evaporate them to dryness under a stream of nitrogen at 40, reconstitute the residue in
200 |JLL buffer, inject a 20 |xL aliquot. (Buffer was 5 mM heptanesulfonic acid in MeCN.water:
triethylamine 70:30:0.4.)
HPLC VARIABLES
Guard column: 50 X 4.6 30 |xm C8
Column: 250 X 4.6 Spherisorb S5 ODS2 C18
Mobile phase: Gradient. MeCN:buffer:water 0:75:25 for 5 min, 15:85:0 for 12 min (step gradient).
(Buffer was 5 mM heptanesulfonic acid in MeCN:water:triethylamine 70:30:0.4.)
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Internal standard: phenelzine (11.09)
OTHER SUBSTANCES
Extracted: hydrazine, monoacetylhydrazine
KEYWORDS
derivatization
REFERENCE
Walubo,A.; Smith,R; Folb,P.I. Comprehensive assay for pyrazinamide, rifampicin and isoniazid with its hydra-
zine metabolites in human plasma by column liquid chromatography, J.Chromatogr.B, 1994, 658, 391396.
SAMPLE
Matrix: blood
Sample preparation: 250 |JLL Plasma + 150 |xL 10% zinc sulfate in water, mix, centrifuge at
1000 g for 1 min. Remove a 250 |xL aliquot of the supernatant and add it to 100 jxL MeOH,
mix, centrifuge, inject a 5 fxL aliquot of the supernatant.
HPLCVARIABLES
Column: 250 X 4.6 5 urn LC-CN (Supelco)
Mobile phase: Isopropanol:water 5:95 containing 5 g/L ammonium formate
Flow rate: 1
Injection volume: 5
Detector: E, ESA Model 5100A, Model 5011 analytical cell with first detector +0.6 V and second
(monitored) detector +0.8 V, Model 5020 guard cell +1.0 V between pump and injector
CHROMATOGRAM
Retention time: 4.4
KEYWORDS
rat; plasma
REFERENCE
Hansen,E.B.,Jr.; Dooley,KL.; Thompson,H.C.,Jr. High-performance liquid chromatographic analysis of the an-
tituberculosis drugs aconiazide and isoniazid, J.Chromatogr.B, 1995, 670, 259-266.
SAMPLE
Matrix: blood
Sample preparation: 250 |xL Plasma + 50 |xL 1.5% p-hydroxybenzaldehyde in MeOH + 40 |xL
20% trichloroacetic acid in water, vortex thoroughly, centrifuge at 8000 g for 10 min, let stand
on ice for 30 min, centrifuge at 8000 g for 10 min, inject a 30 jxL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 20 X 2 30-40 |xm Perisorb RP18
Column: 100 X 8 4 |xm Radial-Pak Novapak C18
Mobile phase: MeOH:water:20% tetraethylammonium hydroxide:70% perchloric acid 24:76:0.05:
0.05, apparent pH 2.3
Flow rate: 2
Detector: UV 350
CHROMATOGRAM
Retention time: 4.4
KEYWORDS
REFERENCE
Schall,R.; Muller,F.O.; Duursema,L.; Groenewoud,G.; Hundt,H.K.L.; Middle,M.V.; Mogilnicka,E.M.; Swart,K.J.
Relative bioavailability of rifampicin, isoniazid and ethambutol from a combination tablet vs. concomitant
administration of a capsule containing rifampicin and a tablet containing isoniazid and ethambutol, Arz-
neimittelforschung, 1995, 45, 1236-1239.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 100 |xL 30% trichloroacetic acid, mix, centrifuge at 2000
g for 5 min. Remove a 100 JJLL aliquot of the supernatant and add it to 20 |xL 0.1% trans-
cinnamaldehyde in MeOH, let stand for 10 min, add 20 jxL 1 M KOH, inject an aliquot.
HPLCVARIABLES
Column: 125 X 3.9 4 |xm Nova-pak C18
Mobile phase: MeCN:water:triethylamine:acetic acid 40:60:0.2:0.1, pH 5 1
Flow rate: 1.3
Detector: UV 340
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: aceprometazine, adrafinil, allopurinol, alprostadil, altretamine, atenolol (ten-
ormine), baclofen, bendroflumethiazide, benserazide, betamethasone, bisoprolol, bromocriptine,
caffeine, captopril, chlorpromazine, clomipramine, clonazepam, cortisone, cyamemazine, dife-
barbamate, dothiepin (dosulepin), ethosuximide, fenspiride, flumazenil, fluoxetine, fluvoxam-
ine, halofantrine, hydrochlorothiazide, hydroxyzine, ibuprofen, imipramine, levamisol, levo-
dopa, maprotiline, medifoxamine, metopimazine, midazolam, nafronyl (naftidrofuryl),
naftazone, naproxen, nicergoline, nitrazepam, nordazepam, nortriptyline, penfluridol, pheno-
barbital, pimozide, pipamperone, pipotizine, primidone, pyrazinamide, pyridoxine, quinine, ri-
fampin, selegiline (deprenyl), streptomycin, tetrazepam, theophylline, thioproperazide, tia-
pride, triazolam, trihexyphenidyl, trimeprazine (alimemazine), trimipramine, tropatepine,
KEYWORDS
REFERENCE
Sadeg,N.; Pertat,N.; Dutertre,H.; Dumontet,M. Rapid, specific and sensitive method for isoniazid determination
in serum, J.Chromatogr.B, 1996, 675, 113-117.
SAMPLE
Matrix: blood, CSF
Sample preparation: 200-500 |xL Plasma or CSF + 50 |xL 50 jig/mL phenelzine sulfate + 100
IxL 10% (?) aqueous acetic acid + 5 mL n-hexane, shake for 30 min, centrifuge at 1870 g for 10
min. Discard the organic layer. Add 300 (xL 0.1% salicaldehyde in EtOH and 400 |JLL 10%
aqueous acetic acid to the aqueous layer, heat at 60 for 30 min, cool, add 1 mL 1 M pH 6.5
K2HPO4, shake for 10 s, add 5 mL diethyl ether, shake for 10 min, centrifuge at 1870 g for 10
reconstitute the residue in 50 |xL mobile phase, inject a 25 |xL aliquot.
HPLCVARIABLES
Guard column: 30 X 4.6 30 |xm C8 (Waters)
Column: 300 X 3.9 10 |xm jjiBondapak C18
Mobile phase: MeCN:water:triethylamine 70:30:0.4 containing 5 mM heptanesulfonic acid, pH
adjusted to 6.0 with acetic acid
Flow rate: 1
Detector: UV 320
CHROMATOGRAM
Retention time: 1.6
Internal standard: phenelzine sulfate (3)
OTHER SUBSTANCES
Extracted: hydrazine
Noninterfering: p-aminosalicylic acid, pyrazinamide, rifampin
KEYWORDS
plasma; rabbit; derivatization
REFERENCE
Walubo,A.; Chan,K; Wong,C.L. Simultaneous assay for isoniazid and hydrazine metabolite in plasma and
cerebrospinal fluid in the rabbit, J.Chromatogr., 1991, 567, 261-266.
SAMPLE
Matrix: blood, CSF
Sample preparation: 200 jxL Serum, plasma, or CSF + 300 (xL reagent. Flush column A to
waste with 500 JULL 500 mM ammonium sulfate, inject sample onto column A, flush column A
to waste with 500 JJLL 500 mM ammonium sulfate, elute the contents of column A onto column
HPLCVARIABLES
Column: A 30 X 2.1 40 |jim preparative grade C18 (Analytichem); B 250 X 4.6 10 |xm Partisil
C8
Flow rate: 1.5
CHROMATOGRAM
OTHER SUBSTANCES
chlorothiazide, diazepam, droperidol, ethionamide, furosemide, methadone, penicillin G, phe-
nobarbital, phenytoin, prazepam, propoxyphene, pyrazinamide, rifampin, trimeprazine,
trimethoprim
KEY WORDS
REFERENCE
Seifart,H.L; Kruger,P.B.; Parkin,D.R; van Jaarsveld,P.F; Donald,P.R. Therapeutic monitoring of antitubercu-
losis drugs by direct in-line extraction on a high-performance liquid chromatography system, J. Chromatogr.,
1993, 619, 285-290.
SAMPLE
Sample preparation: Centrifuge 1.5 mL whole blood, CSF, or urine at 3000 g for 3 min, add
500 |xL of the supernatant to 500 |xL 10% trichloroacetic acid, centrifuge at 10000 g for 1 min.
Remove a 200 |xL aliquot and add it to 20 (xL water, add 40 |xL 1% trans-cinnamaldehyde in
MeOH, let stand at room temperature for 10 min, inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Partisil 5 C8
Mobile phase: Gradient. A was 50 mM KH2PO4. B was MeCN:isopropanol 80:20. A:B 60:40 for
1 min, to 30:70 over 9 min, maintain at 30:70 for 4.5 min, re-equilibrate at initial conditions
for 4 min.
Flow rate: 1
Detector: UV 340
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Extracted: hydrazine
KEY WORDS
whole blood; derivatization
REFERENCE
Seifart,H.L; Gent,W.L.; Parkin,D.R; van Jaarsveld,P.R; Donald,P.R. High-performance liquid chromatographic
determination of isoniazid, acetylisoniazid and hydrazine in biological fluids, J.Chromatogr.B, 1995, 674,
269-275.
SAMPLE
Sample preparation: Condition a 3 mL CN-bonded maxi-clean cartridge (Alltech) with 5 mL
MeOH and 2 mL 1% aqueous acetic acid. 2 mL Plasma or urine + 1 mL isopropanolrchloroform
50:50 + 1 |xg rifampin, shake for 30 s, add to the SPE cartridge with rinses, collect the eluate,
inject an aliquot of the eluate.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Excalibar C18-CN (Alltech)
Mobile phase: MeOH:5 mM tetra-n-butylammonium hydroxide 80:20 adjusted to pH 3.0 with
phosphoric acid
Flow rate: 1.5
Detector: UV 265
CHROMATOGRAM
Retention time: 2.7
Internal standard: rifampin (4.3)
OTHER SUBSTANCES
Extracted: pyrazinamide
KEYWORDS
plasma; SPE
REFERENCE
Gaitonde,C.D.; Pathak,P.V. Rapid liquid chromatographic method for the estimation of isoniazid and pyrazin-
amide in plasma and urine, J.Chromatogr., 1990, 532, 418-423.
SAMPLE
Matrix: milk
Sample preparation: Condition a 3 mL 500 mg C18 SPE cartridge (J.T. Baker) with 6 mL
MeOH and 6 mL water. 80 mL milk + 20 mL 20% trichloroacetic acid in water, mix, let stand
at room temperature for 5 min, centrifuge at 5500 g for 15 min. Filter (0.45 (xm) 75 mL of the
liquid, add 1 mL 1% cinnamaldehyde in MeOH to the filtrate, mix for a few s, let stand at
room temperature for 15 min, add to the SPE cartridge at 5 mL/min, dry for 10 min, wash
with 3 mL mobile phase at 0.2 mL/min, wash with 5 mL n-hexane at 0.3 mL/min, dry for 10
min, elute with 6 mL MeOH. Evaporate the eluate under reduced pressure at 35-40, recon-
stitute the residue with 200 |xL mobile phase, inject an aliquot.
HPLCVARIABLES
Guard column: 4 X 4 5 |xm LiChrospher 100-CN
Column: 250 X 4 5 |xm LiChrospher 100-CN
Mobile phase: MeOH:water 40:60 containing 0.41 g/L sodium acetate trihydrate and 10 mIVL
glacial acetic acid
Flow rate: 1
Detector: UV 330
CHROMATOGRAM
Retention time: 7.4
KEYWORDS
cow; SPE; derivatization
REFERENCE
Defilippi,A.; Piancone,G.; Costa Laia,R.; Balla,S.; Tibaldi,G.P. High-performance liquid chromatography with
UV detection and diode-array UV confirmation of isonicotinic acid hydrazide in cattle milk, J.Chromatogr.B,
1994, 656, 466-471.
SAMPLE
Matrix: milk
Sample preparation: Condition a 3 mL 500 mg 40 |xm phenyl SPE cartridge (J.T. Baker) with
6 mL MeOH and 6 mL water. 80 mL Milk + 20 mL 20% trichloroacetic acid in water, let stand
at room temperature for 5 min, centrifuge at 5500 g for 15 min, filter (0.45 |xm cellulose acetate)
a 75 mL aliquot. Add the nitrate to 1 mL 1% cinnamaldehyde in MeOH, mix for a few s, let
stand at room temperature for 15 min. Add 70 mL of the sample to the SPE cartridge at 3 mL/
min, wash with 3 mL MeOH:water 40:60 at 3 mL/min, dry under vacuum for 1 min, wash with
3 mL MeOH:water 46:54 at 3 mL/min, dry under vacuum for 1 min, wash with 3 mL n-hexane
at 3 mL/min, dry under vacuum for 1 min, elute with 6 mL MeOH. Evaporate the eluate under
vacuum, reconstitute in 233 |JIL mobile phase, inject an aliquot.
HPLC VARIABLES
Guard column: 4 X 4 5 (xm LiChrosphere 100 RP18
Column: 250 X 4 5 jjum LiChrosphere 100 RP18
Mobile phase: MeCN:MeOH:buffer 41:13:46 (Buffer was 1% ammonium acetate adjusted to pH
5.5 with acetic acid.)
Flow rate: 1
Detector: UV 330
CHROMATOGRAM
KEYWORDS
cow; SPE; derivatization
REFERENCE
Defilippi,A.; Piancone,G.; Costa Laia,R; Tibaldi,G.P. An HPLC screening method for the detection of isonicotinic
acid hydrazide in cattle milk, Chromatographia, 1995, 40, 170-174.
SAMPLE
Sample preparation: Centrifuge, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Microsorb C8
Mobile phase: MeOH:0.4 g/L (NH4)H2PO4 + 0.1% triethylamine (pH 10.0) 10:90
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 5.3
REFERENCE
Lunn,G.; Sansone,E.B. Reductive destruction of dacarbazine, procarbazine hydrochloride, isoniazid, andipron-
iazid, Am.J.Hosp.Pharm., 1987, 44, 2519-2524.
SAMPLE
Matrix: solution
Sample preparation: Mix 1 mL pH 7.4 phosphate buffer or hepatocyte solution with 3 mL ethyl
acetate:n-butanol 2:1, 300 |xL 500 mM tetra-n-butylammonium hydroxide, and 100 |xL 80 |xg/
mL IS in water. Shake the mixture for 20 min, centrifuge at 850 g for 15 min, remove 2.5 mL
of the upper organic layer, mix with 1 mL 0.2% hydrobromic acid, shake for 20 min, centrifuge
at 850 g for 15 min, discard the upper organic layer, inject a 5 |xL aliquot of the aqueous
solution.
HPLCVARIABLES
Column: 250 X 4.6 5 u-m TSK-gel ODS-80Ts
Flow rate: 0.8
Injection volume: 5
Detector: UV 265
CHROMATOGRAM
Internal standard: 6-methylnicotinic acid (8)
OTHER SUBSTANCES
Simultaneous: acetylisoniazid, isonicotinic acid
KEYWORDS
rat; hepatocytes
REFERENCE
Ono,Y.; Noda,A.; Zaima,Y.; Jitsufuchi,N.; Eto,S.; Noda,H. Determination of isonicotinic acid in the presence of
isoniazid and acetylisoniazid. Studies on isonicotinic acid formation from isoniazid in isolated rat hepato-
cytes, J.Chromatogr.B, 1996, 677, 339-343.
SAMPLE
Matrix: solutions
Sample preparation: Centrifuge and filter cell solutions (0.22 (xm), inject an aliquot.
HPLCVARIABLES
Flow rate: 3
Detector: UV 254
CHROMATOGRAM
Retention time: 3.5
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, imino-
Next Page
stilbene, imipramine, indomethacin, isocarbostyril, isoproterenol, isoxsuprine, ivermectin, ke-

thioridazine, thiosalicylic acid, thiothixene, thjnnol, tolazamide, tolazoline, tobutamide, tol-
trihex^henidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapa-
REFERENCE
Hill,D.W.; KindÂ-.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: pH 7.5 sodium phosphate buffer
Flow rate: 2
Detector: UV 266
CHROMATOGRAM
Retention time: 5.4
OTHER SUBSTANCES
Simultaneous: metabolites, acetylisoniazid
REFERENCE
697-703.
Isoproterenol
Molecular formula: CnH17NO3
CAS Registry No.: 7683-59-2, 51-30-9 (HCI), 6700-39-6 (sulfate dihydrate), 299-95-6 (sulfate)
Merck Index: 5236
SAMPLE
Matrix: blood
Previous Page
stilbene, imipramine, indomethacin, isocarbostyril, isoproterenol, isoxsuprine, ivermectin, ke-

trihex^henidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapa-
REFERENCE
Hill,D.W.; KindÂ-.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: pH 7.5 sodium phosphate buffer
Flow rate: 2
Detector: UV 266
CHROMATOGRAM
Retention time: 5.4
OTHER SUBSTANCES
Simultaneous: metabolites, acetylisoniazid
REFERENCE
697-703.
Isoproterenol
CAS Registry No.: 7683-59-2, 51-30-9 (HCI), 6700-39-6 (sulfate dihydrate), 299-95-6 (sulfate)
Merck Index: 5236
SAMPLE
Matrix: blood
Sample preparation: Plasma. Prepare a SPE column by adding 500 JULL of a 20% suspension of
19-40 |xm Toyopak SP (strong cation-exchange sulfopropyl resin, Na+ (Toyo Soda)) in water to
of water, wash with three 1 mL portions of buffer. 500 |iL Plasma + 500 JJLL buffer, mix, add
to the SPE column, wash with two 5 mL portions of water, wash with 1 mL MeCN:water 50:
50, elute with 300 jxL 600 jxM potassium ferricyanide in 600 mM KCl:MeCN 50:50, add 50 |xL
reagent to the eluate, heat at 37 for 40 min, cool in ice-water, inject a 100 JJLL aliquot. Urine.
10 IJLL Urine + 1 mL MeCN:500 mM KCl 60:40 + 10 (xL 75 mM potassium hexacyanoferrate(III)
+ 100 |xL reagent, heat at 37 for 40 min, inject a 100 |xL aliquot (J. Chromatogr. 1986, 380,
229). (Prepare buffer by mixing 8 volumes 250 mM LiOH in 200 mM phosphoric acid with 1
volume 200 mM phosphoric acid, pH 5.8. Prepare reagent by dissolving 212 mg 1,2-diphenyl-
ethylenediamine in 10 mL 100 mM HCl, pH 6.7.)
HPLCVARIABLES
Column: 150 X 4.6 5 |xm TSK-gel ODS-120T (Toyo Soda)
Flow rate: 1
CHROMATOGRAM
Retention time: 8
Internal standard: isoproterenol
OTHER SUBSTANCES
Extracted: dopamine, epinephrine, norepinephrine
KEYWORDS
derivatization; plasma; SPE; isoproterenol is IS
REFERENCE
Mitsui,A; Nohta,H.; Ohkura,Y. High-performance liquid chromatography of plasma catecholamines using 1,2-
diphenylethylenediamine as precolumn fluorescence derivatization reagent, J. Chromatogr., 1985, 344, 61
70.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 250 |xL 1 ng/mL a-methylnorepinephrine + 1 mL buffer
+ 5 mL n-heptane containing 4.6 mM tetraoctylammonium bromide and 10 mIVL 1-octanol,
organic phase and add it to 2 mL 1-octanol and 200 |xL 80 mM acetic acid, shake, centrifuge
in dry ice/acetone. Remove the organic layer and add it to 2 mL 1-octanol and 150 |xL 80 mM
organic layer. Thaw the aqueous layer and add it to 250 JJLL MeCN, 50 |xL 1.75 M pH 7.05
bicine, and 100 |xL 100 mM 1,2-diphenylethylenediamine in 100 mM HCl, add 20 |xL 20 mM
a 100 |xL aliquot. (Buffer was 2 M pH 8.6 ammonia/ammonium chloride buffer containing 8.9
diphenylethylenediamine from toluenerlight petroleum (bp 60-80) 10:90, dry overnight at 60.)
HPLC VARIABLES
Column: 100 X 4.6 3 |xm Cp MicroSpher C18 (Chrompack)
Flow rate: 1
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Extracted: dihydroxybenzylamine, dopamine, epinephrine, norepinephrine
KEYWORDS
REFERENCE
van der Hoorn,F.A.J.; Boomsma,F.; Man in 't Veld,A-J-; Schalekamp,M.A.D.H. Determination of catecholamines
17-28.
SAMPLE
Matrix: blood
Sample preparation: Plasma. 1 mL Plasma + 1 mL buffer + 5 mL n-heptane containing 4.6
mM tetraoctylammonium bromide and 10 mL/L 1-octanol, shake for 2 min, centrifuge at 1000
g for 5 min, freeze in dry ice/acetone. Remove the organic phase and add it to 2 mL 1-octanol
(saturated with 80 mM acetic acid) and 200 |xL 80 mM acetic acid, shake, centrifuge at 1000
g for 5 min. Freeze the aqueous layer and remove the organic layer. Add 1 mL 10 mM HCl, 1
mL buffer, and 5 mL n-heptane containing 4.6 mM tetraoctylammonium bromide and 10 mL/
L 1-octanol to the aqueous phase. Shake, centrifuge, freeze, remove the organic layer and add
it to 2 mL 2 M pH 8.6 ammonia-ammonium chloride buffer containing 13.4 mM EDTA (but no
complex). Freeze, remove the organic layer and add it to 2 mL 1-octanol and 150 JJLL 80 mM
acetic acid, shake, centrifuge at 1000 g for 5 min. Freeze, remove the organic layer and add
the aqueous layer to 200 |xL MeCN, 50 |JLL 1.75 M pH 6.95 bicine buffer containing 1% EDTA,
100 (xL 100 mM 1,2-diphenylethylenediamine in 100 mM HCl, and 20 JJLL 20 mM potassium
ferricyanide in water. Heat at 37 in the dark for 1 h, inject a 50 |xL aliquot (keep it in the
dark in the autosampler). Urine. 100 jxL Urine + 1 mL 10 mM HCl + 1 mL buffer + 5 mL n-
heptane containing 4.6 mM tetraoctylammonium bromide and 10 mL/L 1-octanol, shake for 2
min, centrifuge at 1000 g for 5 min, freeze in dry ice/acetone. Remove the organic phase and
add it to 2 mL 1-octanol (saturated with 80 mM acetic acid) and 200 |xL 80 mM acetic acid,
shake, centrifuge at 1000 g for 5 min. Freeze the aqueous layer and remove the organic layer.
Add 1 mL 10 mM HCl, 1 mL buffer, and 5 mL n-heptane containing 4.6 mM tetraoctylammo-
nium bromide and 10 mL/L 1-octanol to the aqueous phase. Shake, centrifuge, freeze, remove
the organic layer and add it to 2 mL 1-octanol and 150 |xL 80 mM acetic acid, shake, centrifuge
at 1000 g for 5 min. Freeze, remove the organic layer and add the aqueous layer to 200 |xL
MeCN, 50 |xL 1.75 M pH 6.95 bicine buffer containing 1% EDTA, 100 jxL 100 mM 1,2-diphen-
ylethylenediamine in 100 mM HCl, and 20 |xL 20 mM potassium ferricyanide in water. Heat
at 37 in the dark for 1 h, inject a 20 |xL aliquot (keep it in the dark in the autosampler).
(Buffer was a 2 M pH 8.6 ammonia-ammonium chloride buffer containing 8.9 mM diphenyl
borate-ethanolamine complex and 13.4 mM EDTA.)
HPLC VARIABLES
Column: 100 X 4.6 3 (xm Spherisorb ODS2
100 over 0.1 min, stay at 0:100 for another 10 min. Equilibrate at initial conditions for 4 min
before next sample.
Flow rate: 1
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: dobutamine, epinephrine, dopamine, epinine, norepinephrine, metabolites
KEYWORDS
plasma; isoproterenol is IS; derivatization
REFERENCE
Alberts,G.; Boomsma,E; Man in 't Veld,A.J.; Schalekamp,M.A.D.H. Simultaneous determination of catechol-
amines and dobutamine in human plasma and urine by high-performance liquid chromatography with
fluorimetric detection, J.Chromatogr., 1992, 583, 236-240.
SAMPLE
Matrix: blood
Sample preparation: Mix plasma and N-methyldopamine, add to a TOYOPAK SP strong cati-
onic exchange SPE cartridge (Tosoh), elute with MeCN:600 mM KCl 50:50 containing 0.6 mM
potassium hexacyanoferrate (III), derivatize eluate with 1,2-diphenylethylenediamine, inject
an aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Nucleosil 5C18
Mobile phase: MeOH:50 mM Tris-HCl buffer 80:20, adjusted to pH 7.0
Flow rate: 1
Detector: F
KEYWORDS
plasma; guinea pig; SPE; derivatization; pharmacokinetics
REFERENCE
Ohtani,H.; Yamamoto,K.; Sawada,Y.; Iga,T. Antibronchospasmic, tachycardiac, and hypokalaemic effects of L-
isoproterenol in guinea-pigs, Biopharm.Drug Dispos., 1995, 16, 745753.
SAMPLE
(Tosoh) with 10 mL water and 2 mL 200 mM pH 5.0 sodium phosphate buffer. Plasma. 700 JJLL
Plasma + 50 JJLL 7 (xM 3,4-dihydroxyphenylpropanoic acid + 350 JXL 2 M perchloric acid, mix,
centrifuge at 4 at 1000 g for 15 min. Remove a 700 (xL aliquot of the supernatant and adjust
the pH to 1.5-2.0 with about 150 |xL 2 M potassium carbonate, centrifuge at 4 at 1000 g for
5 min, add the supernatant to the SPE cartridge, wash with 10 mL water, elute with 300 |xL
MeOH:2 M sodium perchlorate 7:93, filter (cellulose acetate membrane), inject a 100 |xL aliquot
of the filtrate. Urine. Collect human urine for 24 h in the presence of 10 mL 6 M HCl. 500 |xL
Urine + 25 |xL 800 (xM 3,4-dihydroxyphenylpropanoic acid + 500 |xL 1 M perchloric acid, mix,
centrifuge at 4 at 1000 g for 15 min. Remove a 700 JJLL aliquot of the supernatant and adjust
the pH to 1.5-2.0 with about 130 |JLL 2 M potassium carbonate, centrifuge at 4 at 1000 g for
5 min, add the supernatant to the SPE cartridge, wash with 1.5 mL water, wash with 500 JJLL
EtOH:water 50:50, wash with 5 mL water, elute with 500 ^L 1.5 M KCl in MeOH:100 mM
HCl 7:93, filter (cellulose acetate membrane), inject a 100 |xL aliquot of the filtrate.
HPLC VARIABLES
Column: 250 X 4.6 5 (xm TSK-gel ODS-80TM (Tosoh)
Flow rate: 0.8
x 0.5 mm ID stainless steel cooling coil to the detector (Anal. Sci. 1991, 7, 257). (Reagent A
meso-l,2-diphenylethylenediamine in EtOHiwater 70:30 containing 130 mM sodium
methylate.)
CHROMATOGRAM
Retention time: 60
OTHER SUBSTANCES
Extracted: dopamine, epinephrine, levodopa, metanephrine, 3-methoxytyramine, norepineph-
rine, normetanephrine
KEYWORDS
post-column reaction; plasma; SPE; isoproterenol is IS
REFERENCE
Jeon,H.-K.; Nohta,H.; Ohkura,Y. High-performance liquid chromatographic determination of catecholamines
chemical oxidation followed by fluorescence reaction, Anal.Biochem., 1992, 200, 332-338.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Partisil-5 ODS-3
Mobile phase: MeOHibuffer 30:70 (Buffer was 10 mM sodium 1-octanesulfonate in 0.2% acetic
acid.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: norepinephrine, epinephrine, levonordefrin, phenylephrine, metaraminol,
impurities
KEYWORDS
REFERENCE
Smela,M.J.,Jr.; Stromberg,R. Liquid chromatographic determination of six sympathomimetic drugs in dosage
SAMPLE
HPLCVARIABLES
Mobile phase: Isopropanohbuffer 5:95 (Buffer was 100 mM sodium dodecyl sulfate containing
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Simultaneous: carbidopa, dopamine, epinephrine, hydrochlorothiazide, levodopa, methyldopa,
KEYWORDS
REFERENCE
Villanueva Camanas,R.M.; Sanchis Mallols,J.M.; Torres Lapasi6,J.R.; Ramis-Ramos,G. Analysis of pharmaceu-
SAMPLE
Matrix: perfusate
Sample preparation: 30 jxL Perfusate (artificial CSF) + 10 |xL 200 mM perchloric acid. Mix a
25 |xL aliquot with 12.5 u<L reagent, let stand for 2 min, inject an aliquot. (Prepare a stock
solution by dissolving 27 mg o-phthalaldehyde in 1 mL MeOH, add 5 |xL p-mercaptoethanol,
10 (xM EDTA, allow to stand for 24 h before use.)
HPLC VARIABLES
Column: two columns 150 X 4.6 5 |xm M.S. Gel C18 (ESA)
Mobile phase: MeOHibuffer 8:92 adjusted to pH 3.0 with phosphoric acid (Buffer was 54 mM
Flow rate: 1.2
potential 70 mV
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: apomorphine, dopamine, hydralazine, methoxamine, morphine, norepinephrine,
phenylephrine
KEYWORDS
rat; derivatization
REFERENCE
Acworth,I.N.; Yu,J.; Ryan,E.; Gariepy,KC; Gamache,R; Hull,K; Maher,T. Simultaneous measurement of mon-
oamine, amino acid, and drug levels, using high performance liquid chromatography and coulometric array
technology: application to in vivo microdialysis perfusate analysis, J.Liq.Chromatogr., 1994, 17, 685-705.
SAMPLE
Matrix: solutions
Sample preparation: Dilute with 5% dextrose, inject a 20 JXL aliquot.
HPLC VARIABLES
Flow rate: 1.6-2.0
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: theophylline, terbutaline
Interfering: methyldopate
REFERENCE
catecholamines with aminophylline in intravenous solutions, J.Pharm.ScL, 1982, 71, 956-958.
SAMPLE
Matrix: solutions
fiL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 jjim jxBondapak C18
Mobile phase: Acetic acid:triethylamine:water 1.5:0.5:98
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: Hexane:isopropanol:trichloroacetic acid 80:15:5
Flow rate: 0.5
Detector: UV
CHROMATOGRAM
KEY WORDS
chiral; a 1.28
REFERENCE
Okamoto,Y.; Aburatani,R.; Hatano,K.; Hatada,K. Optical resolution of racemic drugs by chiral HPLC on cel-
SAMPLE
Matrix: solutions
Sample preparation: 1 mL Saline solution + 100 U.L ice-cold 5 M acetic acid + 10 u,L 270 mM
disodium EDTA, stir, inject an aliquot.
HPLCVARIABLES
Guard column: Guard-Pak CN (Waters)
Mobile phase: MeCN:buffer 9:91 adjusted to pH 3.6 with 8.7 M phosphoric acid (Buffer was 70
mM Na2HPO^ containing 5 mM sodium heptanesulfonate and 0.1 mM disodium EDTA.) (Re-
circulate mobile phase.)
Flow rate: 1
Detector: E, Waters Model 410, glassy carbon working electrode + 0.825 V, Ag/AgCl reference
electrode
CHROMATOGRAM
Retention time: 4.2
OTHER SUBSTANCES
Simultaneous: 3-O-methylisoprenaline
KEYWORDS
saline
REFERENCE
Bryan,L.J.; O'Donnell,S.R. Analysis of the O-methylated metabolites of isoprenaline, adrenaline and noradren-
aline in physiological salt solutions by high-performance liquid chromatography with electrochemical de-
tection, J.Chromatogn, 1989, 487, 29-39.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: epinephrine, levonordefrin, metaraminol, phenylephrine
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
fluorouracil, fluoxymesterone, fiuphenazine, furosemide, gentisic acid, gitoxigenin, glipizide,
stilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoxsuprine, ivermectin, ke-
faethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfap3rridine, sul-
thioridazine, thiosalicylic acid, thiothixene, thjnuol, tolazamide, tolazoline, tobutamide, tol-
metin, tranylcj^promine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine,
trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tjnramine, verapa-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.7-1
Detector: UV 282
KEY WORDS
REFERENCE
Cleveland,!1. Pirkle-concept chiral stationary phases for the HPLC separation of pharmaceutical racemates,
J.Liq.Chromatogr., 1995, 18, 649-671.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Sumchiral CSP 10 (Sumika Chemical Analysis Service)
Mobile phase: n-Hexane:l,2-dichloroethane:MeOH:trifiuoroaceticacid 250:140:20:1
Flow rate: 1
CHROMATOGRAM
KEYWORDS
chiral
REFERENCE
Oi,N.; Kitahara,H.; Aoki,F. Direct enantiomer separations by high-performance liquid chromatography with
chiral urea derivatives as stationary phases, J.ChromatogrA, 1995, 694, 129-134.
SAMPLE
Matrix: solutions
Sample preparation: Swab surface with mobile phase, shake swab with mobile phase for 20
min, filter (0.20 |xm PDVF membrane), inject a 50 JJLL aliquot of the filtrate.
HPLC VARIABLES
Mobile phase: MeOH:buffer 10:90 (Buffer was 50 mM KH2PO4 containing 5 mM sodium 1-pen-
tanesulfonate and 100 |xM disodium EDTA, pH adjusted to 3.6 with phosphoric acid.)
Flow rate: 1
Detector: E, Bioanalytical Systems, thin-layer glassy carbon electrode +0.65 V, Ag/AgCl reference
electrode
CHROMATOGRAM
Retention time: 6.9
KEYWORDS
surface contamination
REFERENCE
Elrod,L.,Jr.; Schmit,J.L.; Morley,J.A. Determination of isoproterenol sulfate on surfaces using high-performance
liquid chromatography with electrochemical detection, J.Chromatogr.A, 1996, 723, 235-241.
lsosorbide dinitrate
Merck Index: 5245
SAMPLE
Matrix: blood
Sample preparation: 3 mL Plasma + 30 jxL 1 jxg/mL nitroglycerin in n-hexane + 12 mL di-
chloromethane:ethyl acetate 1:1, shake mechanically at 250 cycles/min for 5 min, centrifuge at
550 g at 4 for 5 min. Remove the organic phase and evaporate it to about 20 jxL under a
stream of nitrogen at room temperature, inject.
HPLC VARIABLES
Column: 250 X 4 10 |xm Zorbax NH2
Mobile phase: n-Hexane:MeOH 95:5
Flow rate: 5
Detector: Thermal energy analyzer, Thermo Electron Corp. Model 502A, furnace temp 575, ar-
gon 15 mL/min, oxygen 25 mL/min, MeOH/dry ice slush bath
CHROMATOGRAM
Retention time: 3.3
Internal standard: nitroglycerin (5.0)
OTHER SUBSTANCES
Simultaneous: isosorbide mononitrate
KEYWORDS
plasma
REFERENCE
Maddock,J.; Lewis,P.A.; Woodward A-1 Massey,P.R.; Kennedy,S. Determination of isosorbide dinitrate and its
mononitrate metabolites in human plasma by high-performance liquid chromatography-thermal energy
analysis, J.Chromatogr., 1983, 272, 129-136.
SAMPLE
Sample preparation: Prepare a 500 |xg/mL aqueous solution, filter, inject a 5 |xL aliquot.
HPLC VARIABLES
Column: 200 X 2.1 5 u-m Hypersil ODS
Flow rate: 0.4 for 3 min, to 0.6 over 0.5 min, maintain at 0.6 for 9.5 min, return to 0.4 over 0.5
min
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: isosorbide mononitrate
Noninterfering: lactose
REFERENCE
Azcona,T.; Martin-Gonzalez,A.; Zamorano,R; Pascual,C; Grau,C; Garcia de Mirasierra,M. New methods for
the assay of 5-isosorbide mononitrate and its validation, J.Pharm.Biomed.Anal., 1991, 9, 725-729.
SAMPLE
Sample preparation: Dissolve in water.
HPLC VARIABLES
Column: 300 X 3.9 ixBondapack phenyl
Mobile phase: MeCN:THF:water 26:10:64
Flow rate: 2
Detector: UV 218
CHROMATOGRAM
Retention time: 6
Internal standard: isosorbide dinitrate
OTHER SUBSTANCES
Simultaneous: nitroglycerin, isosorbide dinitrate is IS
KEYWORDS
injections
REFERENCE
Baaske,D.M.; Carter,J.E.; Amann,A.H. Rapid and accurate stability-indicating assay for nitroglycerin,
J.Pharm.Sci., 1979, 68, 481-483.
SAMPLE
Sample preparation: Powder tablets, weigh out a portion equivalent to 1 mg isosorbide dini-
trate, add to 10 mL 75 |xg/mL nitroglycerin in MeOH, sonicate for 2 min, shake mechanically
for 30 min, filter, inject an aliquot
HPLC VARIABLES
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 9.5
OTHER SUBSTANCES
Simultaneous: pentaerythritol tetranitrate, erythrityl tetranitrate
KEY WORDS
tablets
REFERENCE
J.Pharm.Sci., 1984, 73, 1303-1304.
SAMPLE
Sample preparation: Weigh out an amount of finely powdered tablets or capsules equivalent to
about 25 mg of drug. Add 50 mL buffer, shake for 30 min, add 10 mL 5 mg/mL nitroglycerin
in MeOH, make up to 100 mL with buffer, filter (0.45 |xm), inject a 20 |xL aliquot. If the sample
clumps when the buffer is added, agitate with a stirring rod and sonicate. (Buffer was MeOH:
200 mM ammonium acetate buffer:water 55:10:35.)
HPLC VARIABLES
Guard column: 50 X 6.4 25-37 |xm Whatman Co-Pell ODS
Mobile phase: MeOH:200 mM ammonium acetate buffer:water 55:10:35
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Simultaneous: isosorbide mononitrate, saccharin, pentaerythritol tetranitrate
KEYWORDS
tablets; capsules; stability-indicating
REFERENCE
Carlson,M.; Thompson,R.D.; Snell,R.P. Determination of isosorbide dinitrate in pharmaceutical products by
HPLC, J.Chromatogr.ScL, 1988, 26, 574-578.
SAMPLE
Sample preparation: Inject directly.
HPLCVARIABLES
Column: 250 X 4.6 LiChrosorb 10 RP 8
Flow rate: 2
Detector: UV 214
CHROMATOGRAM
Retention time: 3.8
OTHER SUBSTANCES
Simultaneous: nitroglycerin
KEYWORDS
saline
REFERENCE
Martens,H.J.; de Goede,P.N.; van Loenen,A.C. Sorption of various drugs in polyvinyl chloride, glass, and poly-
ethylene-lined infusion containers, Am. J.Hosp.Pharm., 1990, 47, 369-373.
SAMPLE
HPLCVARIABLES
Column: C18
Flow rate: 1.3
Detector: UV 220
CHROMATOGRAM
Internal standard: isosorbide dinitrate
OTHER SUBSTANCES
Simultaneous: nitroglycerin
KEYWORDS
injections; 5% dextrose; isosorbide dinitrate is IS
REFERENCE
Pramar,Y.; Das Gupta,V.; Gardner,S.N.; Yau,B. Stabilities of dobutamine, dopamine, nitroglycerin and sodium
nitroprusside in disposable plastic syringes, J.Clin.Pharm.Ther., 1991, 16, 203-207.
SAMPLE
Matrix: solutions
Sample preparation: 500 (JLL Buffer solution + 500 |xL 1% trifluoroacetic acid, inject a 5 |xL
aliquot.
HPLC VARIABLES
Column: 150 X 4.6 YMC R-ODS-I 5-ST
Mobile phase: MeCN:water 45:55 containing 0.05% trifluoroacetic acid
Flow rate: 1.1
Injection volume: 5
Detector: UV 230
KEY WORDS
buffer
REFERENCE
Fukuyama,S.; Hirasawa,Y; Cox,D.; Koda,S.; Kita,Y. Acceleration of nitric oxide (NO) release from FK409, a
spontaneous NO releaser, in the presence of sulfhydryl-bearing compounds, Pharm.Res., 1995, 12, 1948-
1952.
lsosorbide mononitrate
Merck Index: 5245
SAMPLE
Matrix: blood
Sample preparation: 3 mL Plasma + 30 jiL 1 jjug/mL nitroglycerin in n-hexane + 12 mL di-
chloromethane:ethyl acetate 1:1, shake mechanically at 250 cycles/min for 5 min, centrifuge at
550 g at 4 for 5 min. Remove the organic phase and evaporate it to about 20 JJLL under a
stream of nitrogen at room temperature, inject.
HPLCVARIABLES
Column: 250 X 4 10 |xm Zorbax NH2
Mobile phase: n-Hexane:MeOH 95:5
Flow rate: 5
Detector: Thermal energy analyzer, Thermo Electron Corp. Model 502A, furnace temp 575, ar-
gon 15 mL/min, oxygen 25 mL/min, MeOH/dry ice slush bath
CHROMATOGRAM
Retention time: 5.8 (2-isomer), 8.4 (5-isomer)
Internal standard: nitroglycerin (5.0)
Limit of detection: 1-1.2 ng/mL (5-isomer), 0.5-0.8 ng/mL (2-isomer)
Limit of quantitation: 1.66 ng/mL (5-isomer), 0.86 ng/mL (2-isomer)
OTHER SUBSTANCES
Simultaneous: isosorbide dinitrate
KEYWORDS
plasma
REFERENCE
Maddock,J.; Lewis,P.A.; Woodward,A.; Massey,RR.; Kennedy,S. Determination of isosorbide dinitrate and its
mononitrate metabolites in human plasma by high-performance liquid chromatography-thermal energy
analysis, J.Chromatogr., 1983, 272, 129-136.
SAMPLE
Sample preparation: Prepare a 500 fig/mL aqueous solution, filter, inject a 5 JJLL aliquot.
HPLC VARIABLES
Column: 200 X 2.1 5 p-m Hypersil ODS
Flow rate: 0.4 for 3 min, to 0.6 over 0.5 min, maintain at 0.6 for 9.5 min, return to 0.4 over 0.5
min
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
Retention time: 3.06 (2 isomer), 3.49 (5 isomer)
OTHER SUBSTANCES
Simultaneous: isosorbide dinitrate
Noninterfering: lactose
REFERENCE
Azcona,T.; Martin-Gonzalez ,A.; Zamorano,R; Pascual,C; Grau,C; Garcia de Mirasierra,M. New methods for
the assay of 5-isosorbide mononitrate and its validation, J.Pharm.BiomedAnal., 1991, 9, 725-729.
SAMPLE
in MeOH, make up to 100 mL with buffer, filter (0.45 jjim), inject a 20 |xL aliquot. If the sample
200 mM ammonium acetate bufferrwater 55:10:35.)
HPLC VARIABLES
Guard column: 50 X 6.4 25-37 fim Whatman Co-Pell ODS
Mobile phase: A MeOH:200 mM ammonium acetate buffer:water 55:10:35; B MeOH:200 mM
ammonium acetate buffer:water 20:10:70
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 3.1 (mobile phase A), 7.20 (2-isomer, mobile phase B), 8.55 (5-isomer, mobile
phase B), 46.2 (dinitrate, mobile phase B)
Internal standard: nitroglycerin (8, mobile phase A)
OTHER SUBSTANCES
Simultaneous: saccharin, isosorbide dinitrate, pentaerythritol tetranitrate
KEYWORDS
tablets; capsules
REFERENCE
Carlson,M.; Thompson,R-D-; Snell,R-P- Determination of isosorbide dinitrate in pharmaceutical products by
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 10 mm long reversed-phase pellicular (Chrompack)
Mobile phase: MeCN:MeOH:buffer 16:8:76 (Buffer was 10 mL triethylamine in 760 mL water
adjusted to pH 6.8 with acetic acid.)
Flow rate: 2
Detector: UV 230
CHROMATOGRAM
Retention time: 3.6 (5 isomer)
OTHER SUBSTANCES
Simultaneous: acenocoumarol, acetaminophen, alizapride, alpiropride, amisulpride, aspirin, caf-
feine, carbamazepine, clonazepam, codeine, metoclopramide, nitrazepam, nitrofurantoin,
theophylline
Noninterfering: amitriptyline, cisplatin, furosemide, indomethacin, isosorbide dinitrate, or-
phenadrine, propranolol
KEY WORDS
plasma; SPE
REFERENCE
de Jong,A.R; Wittebrood,A.J.; du Ch&tinier,W.M.; Bron,J. Liquid chromatographic analysis of alizapride and
metoclopramide in human plasma and urine using solid-phase extraction, J.Chromatogr., 1987, 419, 233-
242.
lsothipendyl
Molecular formula: Ci6H19N3S
Merck Index: 5248
Lednicer No.: 1 430
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 248.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.A, 1997, 763, 149-
163.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jxg/mL solution in MeOH, inject a 20 JJLL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 4.4
OTHER SUBSTANCES
operidol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isoxsuprine,
REFERENCE
columns using non-aqueous ionic eluents. IL Application of UV,fluorescenceand electrochemical oxidation
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 12 ^m l-myristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
narizine, cirazoline, clonidine, dilevalol, dimethindene, diphenhydramine, doxazosin, esmolol,
famotidine, ketotifen, metiamide, metoprolol, moxonidine, nadolol, naphazoline, nifenalol, ni-
zatidine, oxprenolol, pheniramine, phentolamine, pindolol, pizotyline (pizotifen), practolol, pra-
zosin, promethazine, propranolol, pyrilamine (mepyramine), ranitidine, roxatidine, sotalol, tia-
menidine, timolol, tramazoline, tripelennamine, triprolidine, tymazoline, UK-14,304
REFERENCE
Kaliszan,R; Nasal,A.; Turowski,M. Binding site for basic drugs on c^-acid glycoprotein as revealed by chemo-
Isoxicam
Merck Index: 5258
Lednicer No.: 2 394
SAMPLE
Sample preparation: Plasma. 500 |xL Plasma + 250 (xL 1 M HCl + 5 mL diethyl ether, shake
at 240 rpm on an orbital shaker for 6 min, centrifuge at 900 g at 4 for 10 min. Remove the
organic phase and evaporate it to dryness at 37 under nitrogen. Reconstitute the residue in
250 fxL MeCN:water 1:1, vortex for 1 min, inject a 50 |xL aliquot. Urine, bile. 500 |xL Urine or
bile + 250 (xL 1 M HCl + 5 mL diethyl ether, shake at 240 rpm on an orbital shaker for 6 min,
centrifuge at 900 g at 4 for 10 min. Remove the organic phase, wash it with 2 mL pH 4.9 citric
acid-phosphate buffer, and evaporate it to dryness at 37 under nitrogen. Reconstitute the
residue in 250 |xL MeCN:water 1:1, vortex for 1 min, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: Techsil ClO CN guard column
Column: 200 X 3.9 10 jim Techsil ClO CN (HPLC Technology)
Mobile phase: MeCNrwater 22:78 (10:90 for bile analyses) containing 50 mM NaH2PO4, final pH
3.5
Flow rate: 2.5
Detector: UV 365
CHROMATOGRAM
Retention time: 7.6, 17 (for bile)
Internal standard: isoxicam
OTHER SUBSTANCES
Simultaneous: piroxicam, 5-hydroxypiroxicam
KEYWORDS
plasma; isoxicam is IS
REFERENCE
Milligan,P.A. Determination of piroxicam and its major metabolites in the plasma, urine and bile of humans
SAMPLE
Matrix: blood
Sample preparation: 50 fxL Serum + 20 |xL 100 mM pH 4.8 citrate buffer + 5 |xL MeOH + 3
mL dichloromethane, shake for 45 s, centrifuge at 4000 rpm. Remove the organic layer and
mobile phase, mix for 10 s, centrifuge, inject a 20 u,L aliquot.
HPLCVARIABLES
Column: 150 X 4.6 7 ^m Separon SGX CN
Flow rate: 0.4
Detector: UV 350
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: piroxicam
KEYWORDS
serum; isoxicam is IS
REFERENCE
Migulla,H.; Alken,R.G.; Huller,H. Mikromethode zur Bestimmung der Piroxicamkonzentration im Serum [Mi-
cromethod for the determination of piroxicam concentration in serum], Pharmazie, 1988, 43, 866-867.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 (xL MeOH + 200 |xL 1 M HCl, vortex at slow speed
for 30 s, add 10 mL dichloromethane, shake vigorously for 30 s, centrifuge at 2500 rpm for 5
min. Remove the organic phase and add it to 20 mg anhydrous sodium sulfate, filter, evaporate
to dryness under a stream of nitrogen at 35, reconstitute the residue in 200 |JLL MeOH, vortex
for 30 s, centrifuge at 15000 g for 5 min, inject 20 u,L of the supernatant.
HPLC VARIABLES
Column: 250 X 4 5 |xm LiChrospher 60 RP-Select B
Mobile phase: MeOH:water:acetic acid 48:45:7, pH 2.47
Flow rate: 1.1
Detector: UV 340
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: droxicam
KEYWORDS
protect from light; plasma; isoxicam is IS
REFERENCE
Maya,M.T.; Pais,J.R; Morais,J.A. A rapid method for the determination of piroxicam in plasma by high-perfor-
mance liquid chromatography, J.Pharm.Biomed.Anal., 1995, 13, 319-322.
SAMPLE
Sample preparation: 1 mL Plasma or tissue + 700 mg potassium carbonate + 1 mL THF + 500
|xL EtOH, vortex for 30 s, centrifuge at 2000 g for 5 min. Remove the supernatant and evap-
orate it to dryness under a stream of nitrogen at 70. Reconstitute residue in 100 |xL THF,
vortex for 5 s, filter (0.5 |xm), inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 3 jxm Novapak C18
Mobile phase: THF:water 45:55 with 1% acetic acid and 5 mM 1-heptanesulfonic acid (PIC B-
7, Waters)
Flow rate: 0.7
Detector: UV 313
CHROMATOGRAM
Retention time: 8.0
OTHER SUBSTANCES
Simultaneous: piroxicam
KEYWORDS
plasma; rat; skin; muscle; isoxicam is IS
REFERENCE
Cerretani,D.; Micheli,L.; Fiaschi,A.L; Giorgi,G. Rapid and sensitive determination of piroxicam in rat plasma,
muscle and skin by high-performance liquid chromatography, J.Chromatogr., 1993, 614, 103-108.
SAMPLE
Sample preparation: 1 mL Plasma or tissue + 700 mg potassium carbonate + 1 mL THF + 500
JJLL EtOH, vortex for 30 s, centrifuge at 2000 g for 5 min. Remove the supernatant and evap-
orate it to dryness under a stream of nitrogen at 70. Reconstitute residue in 100 (JLL THF,
vortex for 5 s, filter (0.5 |xm), inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 3 ^m Novapak C18
Mobile phase: THF.water 45:55 with 1% acetic acid and 5 mM 1-heptanesulfonic acid (PIC B-
7, Waters)
Flow rate: 0.7
Detector: UV 313
CHROMATOGRAM
Retention time: 8.0
OTHER SUBSTANCES
Simultaneous: piroxicam
KEYWORDS
plasma; rat; skin; muscle; isoxicam is IS
REFERENCE
Jin,L.; Lau,C.E. Determination of alprazolam and its major metabolites in serum microsamples by high-per-
formance liquid chromatography and its application to pharmacokinetics in rats, J.Chromatogr.B, 1994,
654, 77-83.
SAMPLE
Sample preparation: Plasma. 500 |iL Plasma + 500 |xL water + 100 |xL THF + 250 |xL 200 mM
citric acid + 1 mL 2 jjig/mL piroxicam in toluene, shake for 10 min at moderate speed, centrifuge
at 2000 g for 5 min. Remove 600 |xL of the toluene layer and evaporate it to dryness under a
stream of air at 67, reconstitute the residue in 300 |xL THF, inject a 30 JJLL aliquot. Urine. 1
mL Urine + 100 |xL THF + 100 |xL 1 M HCl + 3 mL 600 ng/mL PD 79,703 in toluene, shake
for 10 min, centrifuge at 2000 g for 5 min. Remove 2 mL of the toluene layer and evaporate it
to dryness under a stream of air at 67, reconstitute the residue in 300 |xL THF, inject a 30
|JLL aliquot.
HPLC VARIABLES
Guard column: 40 X 2 Corasil C18
Mobile phase: THF:water:glacial acetic acid 45:54:1 containing 5 mM 1-heptanesulfonic acid
(plasma) or MeCN:water:glacial acetic acid 50:49:1 containing 5 mM 1-heptanesulfonic acid
(urine)
Flow rate: 1.5
Detector: UV 320
CHROMATOGRAM
Retention time: 8.1 (plasma), 6 (urine)
Internal standard: piroxicam (4.1), 4-hydroxy-2-methyl-2H-l,2-benzothiazine-3-carboxanilide
1,1-dioxide (PD 79,703) (14)
KEYWORDS
plasma
REFERENCE
Daftsios,A.C; Johnson,E.L.; Keeley,F.J.; Gryczko,C; Rawski,V. High-performance liquid chromatographic anal-
ysis of isoxicam in human plasma and urine, J.Chromatogr., 1984, 305, 145151.
SAMPLE
Sample preparation: Plasma. 100 |xL Plasma + 50 JJLL 100 u,g/mL piroxicam in MeCN + 300
(xL MeCN, vortex vigorously for 1 min, centrifuge at 2000 g for 2 min, inject a 20 |JLL aliquot
of the supernatant. Urine. 1 mL Urine + 50 |xL 40 |xg/mL diazepam in MeCN + 100 |xL satu-
rated KH2PO4 adjusted to pH 2.4 with orthophosphoric acid + 3 mL dichloromethane, vortex
for 2 min, centrifuge at 2000 g for 2 min. Remove the organic layer and evaporate it to dryness
under a stream of nitrogen at 50, reconstitute the residue in 50 jxL MeCN, inject a 20 |xL
aliquot.
HPLC VARIABLES
Column: 250 X 4 10 |xm LiChrosorb RP18 ODS
Mobile phase: MeCNibuffer 50:50 (plasma) or 45:55 (urine) (Buffer was 50 mM KH2PO4 adjusted
to pH 3.0 with orthophosphoric acid.)
Flow rate: 2
Detector: UV 325
CHROMATOGRAM
Internal standard: piroxicam (2.4), diazepam (5.8)
OTHER SUBSTANCES
Noninterfering: allopurinol, cephalexin, chlorothiazide, digoxin, doxepin, furosemide, hydrala-
zine, hydrochlorothiazide, imipramine, labetalol, mepenzolate, methyldopa, metoprolol, minox-
idil, naproxen, prazosin, propranolol, sulfamethoxazole, sulfinpyrazone, trifluoperazine,
trimethoprim
KEYWORDS
plasma
REFERENCE
Bury,R.W. Liquid chromatographic assay of isoxicam in human plasma and urine, J.Chromatogr., 1985, 337,
156-159.
SAMPLE
Sample preparation: 500 JJLL Plasma or urine + 1.1 mL 100 mM NaOH + 250 JJLL 1 M HCl + 5
mL diethyl ether, shake mechanically for 5 min, centrifuge at 1150 g at 4 for 5 min. Remove
the ether layer and evaporate it to dryness at 35 under a stream of nitrogen. Reconstitute the
residue in 250 |xL 50 mM TRIS, inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 10 jxm ixBondapak CN (plasma) or 300 X 3.9 10 |xm ixBondapak C18 (urine)
Mobile phase: MeCNiwater 25:75 containing 50 mM NaH2PO4, final pH 3.2 (plasma) or THF:5
mM sodium octylsulfonate buffer:glacial acetic acid 45:54:1 (urine)
Flow rate: 1.5
Detector: UV 365
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: 5'-hydroxypiroxicam, piroxicam
KEYWORDS
plasma; isoxicam is IS; (see J.Chromatogr. 1984; 305; 145)
REFERENCE
Richardson,C.J.; Ross,S.G.; Blocka,K.L.; Verbeeck,R.K. High-performance liquid chromatographic analysis of
piroxicam and its major metabolite 5'-hydroxypiroxicam in human plasma and urine, J.Chromatogr., 1986,
382, 382-388.
SAMPLE
Next Page
mL water. Filter 30 mL microsomal incubation, add filtrate to SPE cartridge, elute with MeOH.
Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue, inject an
aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 [im C18 (Alltech)
Mobile phase: Gradient. MeCN:200 mM pH 6.5 ammonium acetate 10:90 for 5 min, to 20:80
over 5 min, maintain at 20:80 for 10 min, to 50:50 over 10 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 35
OTHER SUBSTANCES
KEYWORDS
rabbit; liver; SPE
REFERENCE
Woolf,T.R; Black,A.; Chang,T. In vitro metabolism of isoxicam by horseradish peroxidase, Xenobiotica, 1989,
19, 1369-1377.
Isoxsuprine
Merck Index: 5259
Lednicer No.: 1 69
SAMPLE
Matrix: blood
Sample preparation: Dilute blood with an equal volume of water. 1 mL Plasma or 900 |xL
diluted blood + 0.9-1 mL buffer + 5 mL freshly distilled ethyl acetate, vortex for 1 min, cen-
trifuge at 1750 g for 7 min. Remove the organic layer and evaporate it almost to dryness under
a stream of nitrogen at 57, evaporate the final 500 jxL at room temperature, reconstitute the
residue in 100 JULL MeCN, vortex for 15 s, inject the whole amount. (Buffer was 26.5 g sodium
carbonate and 21 g sodium bicarbonate in 500 mL water, pH 9.48.)
HPLC VARIABLES
Column: 300 X 3.9 10 |xm ixBondapak phenyl (plasma) or 200 X 4.6 5 |jim Spheri-5 RP-18 (blood)
Mobile phase: MeCN:0.05% orthophosphoric acid 17:83 (plasma) or 63:37 (blood)
Flow rate: 2
Detector: F ex 200 no emission filter or UV 254
CHROMATOGRAM
Retention time: 15.1 (plasma), 16.3 (blood)
Internal standard: isoxsuprine hydrochloride
OTHER SUBSTANCES
Extracted: ritodrine
Simultaneous: fenoterol
Previous Page
mL water. Filter 30 mL microsomal incubation, add filtrate to SPE cartridge, elute with MeOH.
Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue, inject an
aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 [im C18 (Alltech)
Mobile phase: Gradient. MeCN:200 mM pH 6.5 ammonium acetate 10:90 for 5 min, to 20:80
over 5 min, maintain at 20:80 for 10 min, to 50:50 over 10 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 35
OTHER SUBSTANCES
KEYWORDS
rabbit; liver; SPE
REFERENCE
Woolf,T.R; Black,A.; Chang,T. In vitro metabolism of isoxicam by horseradish peroxidase, Xenobiotica, 1989,
19, 1369-1377.
Isoxsuprine
Merck Index: 5259
Lednicer No.: 1 69
SAMPLE
Matrix: blood
Sample preparation: Dilute blood with an equal volume of water. 1 mL Plasma or 900 |xL
diluted blood + 0.9-1 mL buffer + 5 mL freshly distilled ethyl acetate, vortex for 1 min, cen-
trifuge at 1750 g for 7 min. Remove the organic layer and evaporate it almost to dryness under
a stream of nitrogen at 57, evaporate the final 500 jxL at room temperature, reconstitute the
residue in 100 JULL MeCN, vortex for 15 s, inject the whole amount. (Buffer was 26.5 g sodium
carbonate and 21 g sodium bicarbonate in 500 mL water, pH 9.48.)
HPLC VARIABLES
Column: 300 X 3.9 10 |xm ixBondapak phenyl (plasma) or 200 X 4.6 5 |jim Spheri-5 RP-18 (blood)
Mobile phase: MeCN:0.05% orthophosphoric acid 17:83 (plasma) or 63:37 (blood)
Flow rate: 2
Detector: F ex 200 no emission filter or UV 254
CHROMATOGRAM
Retention time: 15.1 (plasma), 16.3 (blood)
Internal standard: isoxsuprine hydrochloride
OTHER SUBSTANCES
Extracted: ritodrine
Simultaneous: fenoterol
Noninterfering: acetaminophen, albuterol, betamethasone, bupivacaine, caffeine, chloral hy-
drate, dexamethasone, diazepam, lignocaine, meperidine, metoclopramide, morphine, nitraze-
pam, terbutaline
KEYWORDS
isoxsuprine is IS; plasma; whole blood
REFERENCE
Gross,AS.; Brown,K.E; Baird-Lambert,J.A.; Nation,R.L. Determination of ritodrine in blood and plasma by
high-performance liquid chromatography with fluorescence detection, J.Chromatogr., 1987, 416, 400408.
SAMPLE
Matrix: blood
Sample preparation: 500 fiL Plasma + nylidrin + 500 |xL buffer, mix briefly, add 50-75 mg
resin, mix at 10-25 rpm for 30 min, discard the supernatant, wash twice with 1 mL buffer, add
500 JJLL 50 mg/mL KOH in MeOH:water 50:50, mix for 30 min, inject a 20 jxL aliquot of the
eluate. (Buffer was 100 mM citric acid:200 mM Na2HPO4 29:71, pH 6.5 (Mcllvaine buffer) Wash
20-50 mesh Dowex HCR-S resin twice with water and allow it to equilibrate in buffer).
HPLC VARIABLES
Guard column: 25 X 4.6 5 |xm Spherisorb ODS-I
Mobile phase: MeCN:MeOH:buffer 30:18:52 containing 1.8 mM octanesulfonic acid (Buffer was
30 mM KH2PO4 adjusted to pH 3.0 with concentrated orthophosphoric acid.)
Flow rate: 1
Detector: E, Gynotek M20, glassy carbon working electrode 950 mV, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 8.5
Internal standard: nylidrin (11)
Limit of detection: 1 ng/mL (from 2 mL plasma)
KEYWORDS
horse; plasma; pharmacokinetics; SPE
REFERENCE
Hashem,A.; Lubczyk,B. Determination of isoxsuprine in equine plasma by high-performance liquid chromatog-
raphy with electrochemical detection, J.Chromatogr., 1991, 563, 216-223.
SAMPLE
Matrix: blood
Sample preparation: Condition a Baker 500 mg C18 SPE cartridge with 3 mL MeOH and 3
mL water. 1 mL Serum + 714 units p-glucuronidase (E. coli, Sigma), heat at 37 overnight, add
2 mL water, add to the SPE cartridge at 0.15 mIVs, wash with 3 mL water, elute with 2 mL
MeOHrtriethylamine 99:1. Evaporate the eluate to dryness under a stream of nitrogen, recon-
stitute the residue in 200 IJLL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4 5 |xm LC 8 DB (Supelco)
Mobile phase: Gradient. MeOH:buffer 5:95 for 1 min, to 95:5 over 20 min, maintain at 95:5 for
5 min, re-equilibrate at initial conditions for 10 min. (Buffer was 100 mM sodium acetate
containing 0.1% triethylamine adjusted to pH 3.4 with 85% phosphoric acid.)
Flow rate: 1.2
Detector: UV 276
CHROMATOGRAM
KEYWORDS
serum; horse; SPE; pharmacokinetics
REFERENCE
Pompa,G.; Caloni,R; Montana,M.; Pasqualucci,C. Prolonged presence of isoxsuprine in equine serum after oral
administration, Xenobiotica, 1994, 24, 339-346.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jxg/mL solution in MeOH, inject a 20 \xL aliquot.
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.6
OTHER SUBSTANCES
droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergociyptine, ergometrine, er-
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
tilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, ivermectin, ketam-
ine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, loxapine,
faethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfap5nridine, sul-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 0.7-1
Detector: UV 276
KEYWORDS
REFERENCE
J.Liq.Chromatogr., 1995, 18, 649-671.
lsradipine
Merck Index: 5260
Lednicer No.: 4 107
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 4 ng/mL IS in water + 100 (xL 2 M NaOH + 6 mL
dichloromethane, mix on a rotary mixer for 20 min, centrifuge at 1500 g for 5 min (if emulsions
form stir with a class rod and re-centrifuge). Remove the organic layer and evaporate it under
a stream of nitrogen at 40, add 500 |xL dichloromethane, vortex, evaporate under a stream of
nitrogen at 40, reconstitute in 100 |xL mobile phase, vortex for 30 s, allow to stand for 10 min,
vortex, inject a 75 JXL aliquot.
HPLCVARIABLES
Column: 150 X 3.9 4 (xm Nova-pak C18
Mobile phase: MeOH:10 mM dibutylamine phosphate (Waters D-4) 50:50, pH 2.8-3.0
Flow rate: 1
Detector: UV 325
CHROMATOGRAM
Retention time: 12
Internal standard: PY 108-068 (diethyl ester of isradipine) (13)
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Boutagy,J.; Rumble,R; Dunagan,F. Determination of isradipine and the oxidative pyridine metabolite in human
plasma by high-performance liquid chromatography, J.Chromatogr., 1989, 487, 483-488.
SAMPLE
residue with 50 JJLL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: cells, culture medium
Sample preparation: Cells. To 100 |xL containing 40 X 106 cell suspension add 100 jxL of a
methanolic solution of p-phenoxyphenol and benzoylated spermine so as to give concentrations
of 10.63 |xm and 2815 nM, respectively. Vortex for 1 min, sonicate for 3 min, add 200 |xL MeOH,
vortex for 1 min, centrifuge at 2000 g for 5 min, inject a 50 |xL aliquot of the supernatant.
Culture medium. To 5 mL culture medium add 200 |xL of a methanolic solution of p-phenox-
yphenol and benzoylated spermine so as to give concentrations of 10.63 |xm and 2815 nM,
respectively. Adjust the pH to 8.0 with 1 M NaOH, add 5 mL chloroform (Caution! Chloroform
is a carcinogen!), mix at 30 rpm for 30 min, centrifuge at 1000 g for 5 min, remove the chlo-
roform layer. Adjust the aqueous phase to pH 3.0 with 1 M HCl, add 1 mL 100 mM pH 3 citrate
buffer and 1 mL 37 mM tetrabutylammonium hydrogen sulfate. Extract the aqueous phase
with 5 mL chloroform at 30 rpm for 30 min, centrifuge at 1000 g for 5 min. Combine the
chloroform layers and evaporate them to dryness under a stream of nitrogen, dissolve the
residue in 300 JJLL mobile phase, inject a 50 |xL aliquot.
HPLCVARIABLES
Column: 100 X 4.6 5 |xm Chromspher C8 (Chrompack)
Mobile phase: MeOHiTHF-.buffer 43.6:1:55.4 (Buffer was water containing 160 mM NaH2PO4
and 17 mM tetrabutylammonium hydrogen sulfate, pH 3.0.)
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 37
Internal standard: p-phenoxyphenol (19), benzoylated spermine (33)
Limit of detection: 110 pmol/106 (cells), 210 nM (culture medium)
Limit of quantitation: 350 pmol/106 (cells), 700 nM (culture medium)
OTHER SUBSTANCES
REFERENCE
Bidouil,S.; Dubois,J.; Hanocq,M. Isocratic high-performance liquid chromatographic method for the separation
of isradipine and its main metabolites. Application to in vitro metabolization by h3A4/OR cells,
J.Chromatogr.B, 1997, 693, 359-366.
SAMPLE
Sample preparation: Shake bottle by hand, dilute a 1 mL aliquot with MeOH:95% EtOH 1:1
to an expected isradipine concentration of 100 |xg/mL, filter (0.22 jim), inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 (xm Spheri-5 ODS (Applied Biosystems)
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
KEYWORDS
suspensions; stability-indicating
REFERENCE
MacDonald,J.L.; Johnson,C.E.; Jacobson,P. Stability of isradipine in an extemporaneously compounded oral
liquid, Am. J.Hosp.Pharm., 1994, 51, 2409-2411.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 100 |JLM solution in buffer, inject a 20 |JLL aliquot.
HPLC VARIABLES
panolrwater 1:2, pack in a 100 X 4.6 column.) (Buffer was 100 mM pH 5.3 sodium acetate.)
Flow rate: 0.8
Detector: UV
CHROMATOGRAM
OTHERSUBSTANCES
Simultaneous: flurbiprofen, ketoprofen, nimodipine, suprofen
KEYWORDS
chiral; a = 1.28
REFERENCE
Massolini,G.; De Lorenzi,E.; Ponci,M.C; Gandini,C; Caccialanza,G.; Monaco,H.L. Egg yolk riboflavin binding
protein as a new chiral stationary phase in high-performance liquid chromatography, J.ChromatognA, 1995,
704, 55-65.
Itraconazole
Molecular formula: C35H38CI2N8O4
Merck Index: 5262
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 1 mg/mL IS in MeCN, add 500 mg KCl, vortex,
centrifuge at 1500 g for 5 min, inject an aliquot of the supernatant.
HPLC VARIABLES
Column: 250 X 4 5 /xm LiChrospher RP8
Flow rate: 1.5
Detector: UV 263
CHROMATOGRAM
Internal standard: R51012 (14-15) (Janssen, France)
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Cociglio,M.; Hillaire-Buys,D.; Alric,R. Prevalidation statistical design to assess analytical methods. Example
of a quick liquid chromatographic assay of itraconazole in serum, J.Chromatogr.B, 1997, 698, 225233.
SAMPLE
Matrix: blood
Sample preparation: 250 |xL serum + IS + 50 |xL 0.3 N barium hydroxide + 50 |xL 0.4 N zinc
sulfate + 1 mL MeCN, vortex, centrifuge at 3521 g for 15 min, evaporate the supernatant to
dryness under a stream of nitrogen, reconstitute with 250 JJLL mobile phase, inject an aliquot.
HPLCVARIABLES
Guard column: 7.5 X 4.6 5 |xm Alltech Alltima C18
Column: 250 X 4.6 5 jxm Alltech Alltima C18
Flow rate: 1
Detector: UV 263
CHROMATOGRAM
Internal standard: saperconazole
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Christensen,K.J.; Gubbins,P.O.; Gurley,B.J.; Bowman,J.L.; Buice,R.G. Relative bioavailability of itraconazole
from an extemporaneously prepared suspension and from the marketed capsules, Am.J.Health-Syst.Pharm.,
1998, 55, 261-265.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut SPE cartridge (No. 607101) with 1 mL MeOH and
1 mL water. 1 mL Serum + 100 |xL 1 |xg/mL IS in MeOH, mix, add to the SPE cartridge, wash
with 1-2 mL water, wash with 1-2 mL MeOH:water 50:50, elute with 400 (JLL MeOH:triethyl-
amine.'concentrated orthophosphoric acid 99.7:0.3:0.3, inject a 20 |JLL aliquot of the eluate.
HPLCVARIABLES
Column: 100 X 4.6 MPLC RP-18 Spheri-5
Mobile phase: MeCN:water 60:40 containing 20 mM triethylamine, pH adjusted to 2.3 with
phosphoric acid
Flow rate: 1
CHROMATOGRAM
Retention time: 3
Internal standard: cis-4-[4-[4-[4-[[2-(2,4-dichlorophenyl)-2-(lH-l,2,4-triazol-l-ylmethyl)-l,3-di-
oxolan-4-yl]methoxy]phenyl]-l-piperazinyl]phenyl]-2,4-dihydro-5-methyl-2-(3-methylbutyl)-3H-
l,2,4-triazol-3-one (R 51 012) (4)
OTHER SUBSTANCES
Noninterfering: amphotericin B, cefaclor, imipenem, flucytosine, gentamycin, ketoconazole,
netilmicin, salicylic acid, sulfamethoxazole, tienamycin, tobramycin, trimethoprim
KEYWORDS
serum; SPE
REFERENCE
Allenmark,S.; Edebo,A.; Lindgren,K. Determination of itraconazole in serum with high-performance liquid
chromatography and fluorescence detection [letter], J.Chromatogr., 1990, 532, 203-206.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma or serum + 300 |JLL 250 nM IS in MeOH, vortex for 1 min,
centrifuge at 2000 g for 2 min, inject a 250 |xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 50 X 4.6 20 |xm Ultrasphere ODS
Mobile phase: MeCN:water:diethylamine 60:40:0.05 (At the end of each day flush column with
MeOH:DMSO 90:10.)
Flow rate: 1.5
Detector: UV 261
CHROMATOGRAM
Retention time: 6.1
l,2,4-triazol-3-one (R51012) (8.5)
OTHER SUBSTANCES
Noninterfering: amoxicillin, amphotericin B, ampicillin, cimetidine, diazepam, erythromycin,
gentamycin, griseofulvin, ketoconazole, miconazole, nystatin, penicillin G, prednisolone, sul-
famethoxazole, trimethoprim, zidovudine
KEYWORDS
plasma; serum
REFERENCE
Badcock,N.R. Micro-scale method for itraconazole in plasma by reversed-phase high-performance liquid chro-
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 1 mL 50 mM sodium borate + 100 |xL 10 |xg/mL IS in
MeOH + 200 jxL MeOH, extract twice with 4 mL aliquots of heptane:isoamyl alcohol 95:5 in a
rotary mixer. Combine the organic phases and add them to 2 mL 1 M sulfuric acid, extract.
Discard the organic phase and add 600 |xL concentrated ammonium hydroxide to the aqueous
phase. Extract the aqueous phase twice with 2.5 mL portions of heptane:isoamyl alcohol 95:5.
reconstitute the residue in 100 |xL MeCN:water 60:40, inject a 20 (xL aliquot.
HPLC VARIABLES
Guard column: C18
Column: 100 X 4.5 3 |xm Hypersil octadecylsilane
Mobile phase: MeCN:water 40:60 containing 0.03% diethylamine adjusted to pH 7.8 with dilute
orthophosphoric acid
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
l,2,4-triazol-3-one (R51012) (k' 10.8)
OTHER SUBSTANCES
Extracted: metabolites, hydroxyitraconazole
KEYWORDS
serum
REFERENCE
Law,D.; Moore,C.B.; Denning,D.W. Bioassay for serum itraconazole concentrations using hydroxyitraconazole
standards, Antimicrob.Agents Chemother., 1994, 38, 1561-1566.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 1.5 mL MeOH, shake for 5 min, centrifuge. Remove the
supernatant and evaporate it to dryness, reconstitute the residue in 100 |xL MeOH, inject a
20 uL aliquot. Alternatively, condition a 100 mg Bakerbond C-18 SPE cartridge with 2 mL
MeOH and 2 mL water. 1 mL Plasma + 1 mL water, add to the SPE cartridge, wash with 2
mL water containing 100 u,L MeCN, elute with 2 mL MeOH or MeCN. Evaporate the eluate
to dryness, reconstitute the residue in 100 uX MeOH, inject a 20 JLL aliquot.
HPLC VARIABLES
Column: 150 X 3 5 Jim Separon SGX C-18 (Tessek, Prague)
Mobile phase: MeOH:water:triethylamine 72:28:0.05
Flow rate: 1.1
Detector: UV (wavelength not specified)
CHROMATOGRAM
Retention time: 9
Internal standard: R 51012 (13)
Limit of detection: 10 ng/mL (?)
OTHER SUBSTANCES
KEYWORDS
SPE; plasma; serum
REFERENCE
Brandsteterova,E.; Kubalec,P.; Rady,A; Krcmry,V. Determination of itraconazole and its metabolites in plasma
using SPE-HPLC, Pharmazie, 1995, 50, 597-599.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond-Elut C18 SPE cartridge with 2 mL 95% EtOH
and 2 mL MeCNrwater 15:85. 200 |xL Plasma or whole blood + 50 |xL 100 |xM testosterone
propionate in MeOH + 3 mL MeCN.water 15:85, vortex for 30 s, add to the SPE cartridge,
wash with 9 mL MeCN:water 30:70, dry, elute with 200 (JLL 95% EtOH, inject a 10 jxL aliquot
of the eluate.
HPLC VARIABLES
Column: 50 X 4.6 5 |xm Supelcosil LC-8DB
Mobile phase: MeOH:buffer 72.5:27.5 (Buffer was 25 mM K2HPO4 adjusted to pH 3 with 670
mM phosphoric acid.)
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Internal standard: testosterone propionate (3.60)
OTHER SUBSTANCES
Extracted: doxepin
Noninterfering: acetaminophen, N-acetylprocainamide, amitriptyline, aspirin, barbituric acid,
brompheniramine, caffeine, carbamazepine, chloramphenicol, chlorpheniramine, clonazepam,
desipramine, desmethyldoxepin, digitoxin, digoxin, disopyramide, ethosuximide, felbamate,
gentamicin, ibuprofen, imipramine, lidocaine, maprotiline, mephenytoin, mephobarbital, meth-
arbital, methsuximide, methylsuccinimide, nortriptyline, paramethadione, phenacemide, phe-
nobarbital, phensuximide, phenylpropanolamine, phenytoin, primidone, procainamide, protrip-
tyline, quinidine, theophylline, tobramycin, trimethadione, valproic acid, vancomycin
Interfering: clotrimazole
KEYWORDS
plasma; SPE; whole blood
REFERENCE
Rifai,N.; Sakamoto,M.; Law,T.; Platt,O.; Mikati,M.; Armsby,C.C; Brugnara,C. HPLC measurement, blood dis-
tribution, and pharmacokinetics of oral clotrimazole, potentially useful antisickling agent, Clin.Chem.,
1995, 41, 387-391.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond-Elut C18 SPE cartridge with 2 mL 95% EtOH
and 2 mL MeCN:water 15:85. 100 |xL Plasma + 50 |xL 4 |xg/mL + 3 mL MeCN:water 15:85,
vortex for 30 s, add to the SPE cartridge, wash with 9 mL MeCN:water 40:60, dry the SPE
cartridge, elute with 500 |xL dichloromethane:MeOH 50:50. Evaporate the eluate to dryness,
reconstitute the residue in 100 fxL mobile phase, inject a 10 u,L aliquot.
HPLC VARIABLES
Column: 50 X 4.6 5 |xm Supelcosil LC-I
Mobile phase: MeCN:MeOH:25 mM pH 6.3 K2HPO4 30:30:40
Flow rate: 2
Detector: UV 263
CHROMATOGRAM
Retention time: 2.1
Internal standard: R051012 (Jannsen) (2.8)
OTHER SUBSTANCES
Noninterfering: acetaminophen, N-acetylprocainamide, amikacin, amitriptyline, aspirin, bar-
bituric acid, brompheniramine, caffeine, carbamazepine epoxide, carbamazepine, chloramphen-
icol, chlorpheniramine, clonazepam, clotrimazole, desipramine, desmethyldoxepin, digitoxin,
digoxin, disopyramide, doxepin, ethosuximide, felbamate, gentamicin, ibuprofen, imipramine,
lidocaine, maprotiline, mephenytoin, mephobarbital, metharbital, methsuximide, methylsuc-
cinimide, nortriptyline, paramethadione, phenacemide, phenobarbital, phensuximide, phenyl-
propanolamine, phenytoin, primidone, protriptyline, theophylline, tobramycin, trimethadione,
vancomycin
KEYWORDS
SPE; plasma
REFERENCE
Rifai,N.; Sakamoto,M.; Platt,O.; Brugnara,C. A high-performance liquid chromatographic assay for the deter-
mination of itraconazole concentration using solid-phase extraction and small sample volume, Ther.Drug
Monit, 1995, 17, 522-525.
SAMPLE
Sample preparation: Tissue. Homogenize 25 mg tissue with 1 mL MeOH. Add 50 |xL 2.5 |xg/
mL IS in MeOH, vortex. Centrifuge the sample at 833 g for 15 min. Evaporate the supernatant
to dryness under a stream of nitrogen. Reconstitute the residue in 300 (JLL MeOH, inject an
aliquot. Plasma. Add 50 JULL 12.5 jxg/mL IS to 100 jxL plasma, vortex. Add 300 uL MeOH, vortex
for 1 min. Centrifuge the sample at 833 g for 5 min. Inject a 190 jxL aliquot.
HPLCVARIABLES
Guard column: Novapak Guard-Pak
Column: 100 X 8 4 |xm Novapak C18
Mobile phase: MeCN:diethylamine:water 58:0.05:42, adjusted to pH 2.45 with 85% phosphoric
acid
Flow rate: 1.5
CHROMATOGRAM
Internal standard: R51012 (Janssen Research Diagnostics, USA) (18.5)
OTHER SUBSTANCES
KEYWORDS
plasma; serum; liver; bird
REFERENCE
Cox,S.K.; Orosz,S.; Burnette,J.; Frazier,D. Microassay for determination of itraconazole and hydroxyitracona-
zole in plasma and tissue biopsies, J.Chromatogr.B, 1997, 702, 175-180.
SAMPLE
Sample preparation: Homogenize (Ultra-Turrax) tissue in water 1:4. 1-2 mL Plasma or homo-
genate + 100 |xL 2-10 |xg/mL IS in MeOH + 50 mM pH 7.8 phosphate buffer, extract twice with
4 mL heptanerisoamyl alcohol 98.5:1.5 for 10 min. Combine the organic layers and add them
to 3 mL 50 mM sulfuric acid, extract, centrifuge at 1000 g. Remove the aqueous phase and
adjust the pH to 9 with concentrated ammonia, extract twice with 2 mL heptane.isoamyl al-
cohol 98.5:1.5. Combine the organic layers and evaporate them to dryness under a stream of
nitrogen at 55, reconstitute the residue in 100 |xL mobile phase, inject a 40 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.1 5 fun RSiL C 18HL octadecyl (Alltech)
Mobile phase: MeCN:water 60:40 containing 0.05% diethylamine
Flow rate: 0.5
Detector: UV 263
CHROMATOGRAM
Retention time: 4.3
^ l,2,4-triazol-3-one (R 51 012) (5.8)
KEYWORDS
plasma; human; rat; pharmacokinetics
REFERENCE
Woestenborghs,R.; Lorreyne,W.; Heykants,J. Determination of itraconazole in plasma and animal tissues by
SAMPLE
Sample preparation: Tissue. Homogenize (Baxter Scientific disposable tissue grinder) esopha-
geal tissue with 20 volumes of cold phosphate-buffered saline (pH 7.8). 250 uL Homogenate +
1 mL MeCN + 1 uL 1 mg/mL IS, vortex for 1 min, centrifuge at 1000 g for 5 min. Remove the
supernatant and dry in air for 30 min, reconstitute in 100 |JLL mobile phase, inject an aliquot.
Plasma. 250 |JLL Plasma + 1 mL MeCN + 1 |xL 1 mg/mL IS, vortex for 1 min, centrifuge at
1000 g for 5 min. Remove the supernatant and dry in air for 30 min, reconstitute in 100 |xL
HPLC VARIABLES
Column: 150 X 3.9 Nova-pak C18
Mobile phase: MeCN:10 mM K2HPO4 60:40, final pH adjusted to 7.8 with 85% phosphoric acid
Flow rate: 1.3
Detector: UV 263
CHROMATOGRAM
Internal standard: R51012 (Research Diagnostics Inc.) (6.00) Remarks
Limit of detection: 10 ng/g, 5 ng/mL
OTHER SUBSTANCES
Noninterfering: ampicillin, cefazolin, cefoperazone, ceftriaxone, cefuroxime, clindamycin, flucon-
azole, folic acid, minocycline, nafcillin, norfloxacin, rifampin, tetracycline, vancomycin, zalci-
tabine, zidovudine
KEYWORDS
plasma
REFERENCE
Darouiche,R.O.; Setoodeh,A.; Anaissie,E.J. Potential use of a simplified method for determination of itracona-
zole levels in plasma and esophageal tissue by using high-performance liquid chromatography, Antimi-
crob.Agents Chemother., 1995, 39, 757-759.
SAMPLE
Sample preparation: 250 |xL Syrup + 1.5 mL DMF, make up to 10 mL with mobile phase. 250
|JLL Solution + 500 |xL 1 mg/mL IS, make up to 10 mL with mobile phase, inject a 5 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 juim Spheri-5 ODS
Mobile phase: MeCN:water:diethylamine 60:40:0.05
Flow rate: 1
Injection volume: 5
Detector: UV 263
CHROMATOGRAM
Retention time: 7
Internal standard: R51012 (Janssen) (11)
KEYWORDS
syrup; stability-indicating; suspensions
REFERENCE
Jacobson,P.A.; Johnson,C.E.; Walters,J.R. Stability of itraconazole in an extemporaneously compounded oral
liquid, Am.J.Health-SystPharm., 1995, 52, 189-191.
Ivermectin
Molecular formula: C48H74O14(BIa)
Molecular weight: 875.11 (B1a)
70161-11-4 (B1a), 70209-81-3 (B1b)
Merck Index: 5264
SAMPLE
Matrix: blood
Sample preparation: Mix 5 mL Serum with 5.0 mL MeOH:buffer 20:80. Pass through an im-
munoaffinity column (80 X 7 mm, 1.0 mL bed volume, IgG + CNBr-activated Sepharose 4B,
Pharmacia, Sweden) at 1.2 mL/min. Wash with 20 mL MeOHrbuffer 10:90 and 5 mL MeOH:
water 10:90. Elute with 5 mL MeOH. Evaporate the eluate to dryness at 55. Add 1 mL MeOH
to the residue, vortex for 15 s, inject an aliquot. (Buffer was prepared by dissolving 200 mg
KH2PO4, 2.9 g Na2HPO4.12 H2O, 200 mg KCl, and 18.8 g NaCl in 900 mL water, adjusting to
pH 7.4 with 2 M NaOH, and making up to 1 L with water.)
HPLC VARIABLES
Column: 220 X 4.6 5 fxm Spheri-5 RP-18
Flow rate: 1.0
Detector: UV 245
CHROMATOGRAM
Retention time: 6.5
KEYWORDS
serum; sheep; immunoaffinity; SPE
REFERENCE
Li, J.; Zhang,S. Immunoaffinity column cleanup and liquid chromatographic method for determining ivermectin
in sheep serum, J.AOAC Int., 1996, 79, 1300-1302.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 4 mL MeCN:water 50:50.
Condition a Sep-Pak silica SPE cartridge with 4 mL MeCN and 4 mL dichloromethane. 5 mL
Serum + 5 mL MeCN:water 50:50, add to the C18 SPE cartridge, wash with 4 mL MeCN:water
50:50, blow out excess solvent, elute the C18 SPE cartridge onto the silica SPE cartridge with
4 mL MeCN:dichloromethane 10:90, discard the C18 cartridge and elute the silica cartridge
with 4 mL MeCN. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute
the residue in 1 mL mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 100 X 8 4 |xm Nova-Pak radial PAK
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Retention time: 4
KEYWORDS
cow; serum; SPE
REFERENCE
Oehler,D.D.; Miller,J.A. Liquid chromatographic determination of ivermectin in bovine serum,
SAMPLE
Matrix: blood
Sample preparation: Condition a 500 mg Sep-Pak C18 SPE cartridge with 4 mL MeCN, 5 mL
chloroform, 4 mL MeCN, and 4 mL water. 1 mL Plasma + 500 |xL MeCN + 500 |xL 1 ng/mL
IS in MeCN, mix for 15 s, centrifuge at 2500 g for 10 min, add the supernatant to the SPE
cartridge, dry under vacuum for 15 min, elute with 5 mL chloroform. Evaporate the eluate to
dryness under a stream of nitrogen at <50, reconstitute with 100 |xL N-methylimidazole:MeCN
1:1, add 150 JXL trifluoroacetic anhydride :MeCN 1:2, let stand for <30 s, inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 u-m Zorbax C8
Mobile phase: MeCN:THF:water 40:40:20
Flow rate: 1
CHROMATOGRAM
Retention time: 18 (ivermectin Bla)
Internal standard: avermectin Bla (12.5)
KEY WORDS
plasma; SPE; cow; derivatization
REFERENCE
de Montigny,R; Shim,J.S.K; Pivnichny,J.V. Liquid chromatographic determination of ivermectin in animal
plasma with trifluoroacetic anhydride and iV-methylimidazole as the derivatization reagent,
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL C18 SPE cartridge (J.T. Baker) with 4 mL MeCN, 5 mL
chloroform, 4 mL MeCN, and 4 mL water. 1 mL Plasma + 50 JJLL 86-285 ng/mL IS in MeCN,
vortex for 15 s, add 1 mL MeCN, mix, centrifuge at 1130 g for 15 min, add the supernatant to
the SPE cartridge. Reconstitute the residue in 3.5 mL MeCNrwater 1:2, vortex for 15 s, cen-
trifuge at 1130 g for 15 min, add the supernatant to the SPE cartridge. Wash the SPE cartridge
with 4 mL MeCN:water 1:2, dry under vacuum for 1 h, elute with 5 mL MeCNxhloroform 50:
50. Evaporate the eluate to dryness under a stream of nitrogen at 45, reconstitute with two
100 jxL portions of MeCN, evaporate to dryness under a stream of nitrogen at room tempera-
ture. Reconstitute with 100 |xL N-methylimidazole:MeCN 1:1, add 150 (xL trifluoroacetic an-
hydride:MeCN 1:2, let stand for 1.7 min, inject a 150 (xL aliquot.
HPLC VARIABLES
Column: 100 X 2 3 \xm MOS-Hypersil-2
Flow rate: 0.3
CHROMATOGRAM
Internal standard: ivermectin monosaccharide (20)
KEYWORDS
plasma; SPE; dog; narrow bore; derivatization
REFERENCE
Rabel,S.R.; Stobaugh,J.R; Heinig,R.; Bostick,J.M. Improvements in detection sensitivity for the determination
of ivermectin in plasma using chromatographic techniques and laser-inducedfluorescencedetection with
automated derivatization, J.Chromatogr., 1993, 617, 79-86.
SAMPLE
Sample preparation: Whole blood, serum. Condition a 1 mL Bond Elut C18 SPE cartridge with
2 mL MTBE, 2 mL MeCN, and 2 mL MeCN:water 50:50. 500 JJLL Whole blood or serum + 20
(JLL IS solution + 50 (JLL 200 mM zinc sulfate solution + 500 JJLL MeCN, vortex, centrifuge for 5
min, add to the SPE cartridge, wash with 2 mL MeCN:water 50:50, elute with 2 mL MTBE.
Evaporate the eluate and dissolve the residue in 150 |xL mobile phase, inject a 50 |xL aliquot.
Muscle. Condition a 3 mL Bond Elut C18 SPE cartridge with 4 mL MTBE, 4 mL MeCN, and
4 mL MeCN:water 50:50. Weigh out 1 g muscle, add 50 yJJg IS solution, add 6 mL MeCN:
water 50:50, homogenize (Vertis 45), rinse blades and jar with 2 mL MeCN:water 50:50, add
200 mL (sic) 200 mM zinc sulfate, mix, centrifuge, add the supernatant to the SPE cartridge,
wash with 4 mL MeCN:water 50:50, elute with 4 mL MTBE. Evaporate the eluate and dissolve
the residue in 150 fiL mobile phase, inject a 50 |xL aliquot.
HPLCVARIABLES
Column: 70 X 4.6 3 |xm Ultrasphere XL ODS
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Retention time: 5.6
Internal standard: dehydroivermectin (13.4) (Prepare by evaporating 1 mL 278 fig/mL iver-
mectin in MeCN into a tube. Add 200 jiL 1-methylimidazole, add 300 |xL acetic anhydride, add
900 |xL DMF, mix well, heat at 60 for 15 min, add 4 mL MeCN, pass through a silica SPE
cartridge, Evaporate the eluate to 500 |xL, make up to 20 mL with MeCN, use this solution.)
Limit of detection: 2 ng/g (tissue), 2 ng/mL (whole blood, serum)
KEYWORDS
whole blood; serum; muscle; human; cow; SPE
REFERENCE
Dickinson,C.M. Improved high-performance liquid chromatographic method for quantitation of ivermectin in
whole blood, serum or muscle tissue, J.Chromatogr., 1990, 528, 250-257.
SAMPLE
Matrix: feces
Sample preparation: Stir 5 g feces with 25 mL MeOH for 25 min, centrifuge at 1500 g for 15
min, concentrate the supernatant to 7 mL under reduced pressure at 80, centrifuge at 1500 g
for 15 min, add procaine to a concentration of 50 ppm, make up to 10 mL with MeOH, filter
(0.50 |xm PTFE), inject a 20 |xL aliquot of the filtrate.
HPLC VARIABLES
Column: 300 X 9 Bondclone 10C18 (Phenomenex)
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Internal standard: procaine (7.3)
KEYWORDS
cow
REFERENCE
Bernal,J.L.; Del Nozal,M.J.; Salas,M.; Galante,E.; Lumaret,J.P. HPLC determination of ivermectin in cattle
dung following subcutaneous injection, J.Liq.Chromatogr., 1994, 17, 2429-2444.
SAMPLE
Matrix: milk
Sample preparation: Prepare a SPE cartridge by adding 2 g 40 |xm Bondesil C18 18% load
endcapped (Varian) to a 25 mL syringe barrel fitted with a 20 |xm frit, wash with 5 mL petro-
leum ether, 5 mL acetone, and two 5 mL aliquots of MeOH, aspirate with full vacuum for <5
s (A). Condition a 500 mg Bond Elut LRC silica SPE cartridge with 3 mL hexane:ethyl acetate
60:40 (B). Condition a 500 mg Bond Elut LRC silica SPE cartridge with 4 mL chloroform (C).
25 mL Milk + 200 |xL 500 ng/mL abamectin (avermectins) in MeOH, mix, add 5 mL to the SPE
cartridge (A), mix milk with C18 material, let stand for 2 min, wash spatula with water, wash
with two 5 mL portions of water, elute with 10 mL ethyl acetate, allow eluate to pass through
a 5 cm layer of anhydrous sodium sulfate. Evaporate the eluate to dryness under a stream of
nitrogen below 50, add 2 mL hexaneiethyl acetate 60:40 to the oily residue, vortex, sonicate
for 1 min, add mixture to SPE cartridge (B), rinse in with 1 mL hexane:ethyl acetate 60:40,
wash with 5 mL hexane:ethyl acetate 60:40, elute with 5 mL MeOH:ethyl acetate 50:50. Evap-
orate the eluate to dryness under a stream of nitrogen below 60 (this residue should have no
moisture in it), reconstitute the residue in 100 (xL reagent, vortex gently for a few s, heat at
95 for 1 h, cool, add 1 mL chloroform, vortex, add to SPE cartridge (C), wash in with three 1
mL portions of chloroform, elute with 2 mL chloroform. Collect all the eluate and evaporate it
to dryness under a stream of nitrogen below 60, reconstitute in 500 jxL MeOH, inject a 50 (JLL
aliquot. (Prepare reagent by sequentially mixing 900 |JLL DMF, 300 IJLL acetic anhydride, and
200 |AL N-methylimidazole just before use.)
HPLC VARIABLES
Guard column: Newguard RP-18 (Brownlee)
Column: 250 X 4.6 5 |jim Econosil C18
Flow rate: 1
CHROMATOGRAM
Retention time: 15
Internal standard: abamectin (avermectins) (10.5)
KEYWORDS
cow; SPE; MSPD; derivatization; silylate glassware
REFERENCE
Schenck,F.J. Isolation and quantification of ivermectin in bovine milk by matrix solid phase dispersion (MSPD)
extraction and liquid chromatographic determination, J.Liq.Chromatogr., 1995, 18, 349-362.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
tilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, ke-
REFERENCE
18, 233-242.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 3 mL 500 mg Isolute C8 end-capped SPE cartridge (Inter-
national Sorbent Technologies) with 5 mL MeCN and 5 mL MeCNrwater 1:2 containing 0.1%
triethylamine. Condition a 3 mL 500 mg Isolute silica SPE cartridge (International Sorbent
Technologies) with 5 mL ethyl acetate:hexane 40:60. Homogenize (Tissuemizer) 5 g tissue in
25 mL MeCN, centrifuge at 3000 rpm for 5 min, decant supernatant into 50 mL water and 75
JULL triethylamine. mix, pass through the C8 SPE cartridge at 2 mL/min, discard eluate, dry
column under vacuum for 5 min, elute with 5 mL MeCN. Dry the eluate under a stream of
nitrogen at 50-55, resolubilize dry residues in 5 mL of ethyl acetate:hexane 40:60, vortex
briefly, pass through the silica SPE cartridge at 2 mL/min. Rinse the reservoir with 5 mL ethyl
acetate:hexane 40:60, add the rinse to the SPE cartridge. Dry the SPE cartridge for 5 min,
elute with 5 mL MeOH:ethyl acetate 50:50, dry the eluate under a stream of nitrogen at 50-
55. Reconstitute the extract with 200 jutL of fresh methylimidazole:MeCN 50:50, vortex briefly,
add 300 (xL of fresh trifluoroacetic anhydride:MeCN 1:2, vortex briefly, dry under a stream of
nitrogen at 50-55 for 15 min. Add 500 |JLL MeOH:ammonium acetate:molecular sieves 4:1:1),
vortex briefly, dry under a stream of nitrogen at 50-55. Add 1 mL MeCN, vortex thoroughly,
filter (0.45 fxm), inject a 50 (xL aliquot.
HPLCVARIABLES
Column: 200 X 4.6 5 |xm Hypersil ODS (C 18)
Mobile phase: MeCN-water 90:10
Flow rate: 1.0
CHROMATOGRAM
Retention time: 11 (BIb), 12.5 (BIa)
OTHER SUBSTANCES
Extracted: doramectin
KEYWORDS
SPE; derivatization; salmon; muscle
REFERENCE
Rupp,H.S.; Turnipseed,S.B.; Walker,C.C; Roybal,J.E.; Long,A-R Determination of ivermectin in salmon muscle
tissue by liquid chromatography with fluorescence detection, J.AOAC Int., 1998, 81, 549-553.
SAMPLE
Matrix: tissue
Sample preparation: Condition a C8 Bond-Elut SPE cartridge with 5 mL MeCN and 5 mL
MeCN:water 30:70 containing 0.1% triethylamine. Mix 5 g muscle with 15 mL MeCN in a high
speed blender for 1 min. Centrifuge at 3000 rpm for 5 min. Dilute 15 mL of the supernatant
with 35 mL water, add 50 u,L triethylamine, add to the SPE cartridge, elute with 5 mL MeCN,
evaporate the eluate to dryness under a stream of nitrogen at 60. Dissolve the residue in 1
mL MeOH, filter (0.45 |xm Millipore filter). Transfer a 500 (xL aliquot to a silanized 3 mL
reaction vial, evaporate to dryness. Add 100 uX derivatization reagent, heat at 95 for 1 h. Cool
to room temperature, add 1 mL chloroform, vortex, add quantitatively to a Sep-Pak silica SPE
cartridge with 3-4 mL chloroform. Elute with 9 mL chloroform, evaporate the combined chlo-
roform eluates to dryness. Dissolve the residue in 500 |xL MeOH, filter (0.45 |jim Millipore
filter), inject a 50 \LL aliquot. (Caution! Chloroform is a carcinogen! Derivatization reagent was
1-methylimidazole:acetic anhydride:DMF 2:6:9, freshly prepared.)
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Shandon ODS Hypersil C18
Flow rate: 1
CHROMATOGRAM
Retention time: 14
Limit of detection: 500-1000 ng/kg
KEYWORDS
pig; muscle; SPE; derivatization
REFERENCE
NordlanderJ.; Johnsson,H. Determination of ivermectin residues in swine tissues--an improved clean-up pro-
cedure using solid-phase extraction, Food Addit.Contam., 1990, 7, 79-82.
SAMPLE
Matrix: tissue
Sample preparation: Add 15 mL MeOH to 5 g ' homogenized liver, shake thoroughly by hand
and again using a shaking apparatus at medium speed for 1 h. Adjust the volume to 20 mL
with MeOH, shake, centrifuge at 2000 g for 5 min. Mix 10 mL supernatant with 40 mL phos-
phate buffer A, add to immunoaffinity column (column preparation described in detail in paper)
at 1.2 mL/min, wash with 40 mL MeOHrphosphate buffer B 10:90 and 10 mL MeOHrwater 20:
80. Elute with 3 mL MeOH, evaporate the eluate to less than 1 mL on a rotary evaporator at
55, vortex with 5 mL ethyl acetate for 15 s. Collect the organic layer, evaporate to dryness at
55, redissolve the residue in 1 mL mobile phase by vortexing for 15 s, filter (0.45 jxm filter),
inject a 100 |xL aliquot of the filtrate. (Phosphate buffer A was 200 mg KH2PO4, 2900 mg
Na2HPO4, 200 mg KCl, and 8800 mg NaCl in 900 mL water adjusted to pH 7.4 with 2 M NaOH
and diluted to 1 L with water. Phosphate buffer B was 200 mg KH2PO4, 2900 mg Na2HPO4,
200 mg KCl, and 29.3 g NaCl in 900 mL water adjusted to pH 7.4 with 2 M NaOH and diluted
to 1 L with water.)
HPLC VARIABLES
Column: 220 X 4.6 5 jxm Brownlee C18
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Retention time: 15
Limit of detection: 2 mg/g
Limit of quantitation: 5 mg/g
KEY WORDS
immunoaffinity; liver; pig; SPE
REFERENCE
Li,J.S.; Li,X.W.; Hu,H.B. Immunoaffinity column cleanup procedure for analysis of ivermectin in swine liver,
J.Chromatogr.B, 1997, 696, 166-171.
SAMPLE
Matrix: tissue
Sample preparation: Condition a Bond-Elut C8 SPE cartridge (Analytichem International) with
5 mL MeCN and 5 mL MeCN:water 30:70 containing 0.1% triethylamine. Mix 5 g muscle with
15 mL MeCN in a high speed blender for 1 min, centrifuge at 3000 rpm for 5 min. Dilute 15
mL supernatant with 35 mL water, add 50 u,L triethylamine, add the entire mixture to the
SPE cartridge, elute with 5 mL MeCN, evaporate the eluate to dryness under a stream of
nitrogen at 60. Dissolve the residue in 1 mL MeOH and filter (0.45 |xm). Evaporate a 500 fxL
aliquot of the filtrate to dryness in a silanized vial, add 100 JJLL derivatization reagent (1-
methyl-imidazole:acetic anhydride:dimethylformamide 2:6:9, freshly prepared), heat in an oven
at 95 for 1 h. Cool to room temperature, add 1 mL chloroform and vortex briefly. (Caution!
Chloroform is a carcinogen!) Transfer quantitatively to a Sep-Pak silica SPE cartridge with 3-
4 mL chloroform, elute with 9 mL chloroform, evaporate the combined chloroform eluates to
dryness. Dissolve the residue in 500 u,L MeOH, filter (0.45 |xm), inject a 50 (JLL aliquot of the
filtrate.
HPLC VARIABLES
Column: 250 X 4.6 5 p-m Hypersil ODS C18 (Shandon)
Flow rate: 1
CHROMATOGRAM
Retention time: 14 (dihydroavermectin BlA)
Limit of quantitation: 500-1000 ng/g
KEYWORDS
derivatization; muscle; SPE; pig
REFERENCE
Nordlander,L; Johnsson,H. Determination of ivermectin residues in swine tissuesan improved clean-up pro-
cedure using solid-phase extraction, Food Addit.Contam., 1990, 7, 79-82.
SAMPLE
Matrix: tissue
Sample preparation: Condition a Bond Elut C8 SPE cartridge with 5 mL MeCN and 5 mL
MeCN:water:triethylamine 30:70:0.1. Homogenize (Silverson) 5 g frozen minced tissue with 15
mL MeCN at full speed for 1 min, centrifuge at 4 at 2000 g for 10 min. Remove a 13 mL
aliquot of the supernatant and add it to 35 mL water and 50 (xL triethylamine, elute with 5
mL MeCN. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute with 200
IxL l-methylimidazole:MeCN 1:1, add 300 |xL trifluoroacetic anhydride:MeCN 1:2, mix, store
cold, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Partisil 5 ODS-3
Flow rate: 1.8
CHROMATOGRAM
Retention time: 6 (ivermectin Bi8)
KEYWORDS
SPE; salmon; derivatization; brain; gill; kidney; liver; muscle; skin; spleen
REFERENCE
Kennedy,D.G.; Cannavan,A.; Hewitt,S.A.; Rice,D.A.; Blanchflower,W.J. Determination of ivermectin residues in
the tissues of Atlantic salmon (Salmo salar) using HPLC withfluorescencedetection, Food Addit.Contam.,
1993, 10, 579-584.
SAMPLE
Matrix: tissue
and 5 mL MeCN:water:triethylamine 30:70:0.1. Homogenize (Polytron) 5 g tissue and 15 mL
MeCN for 20 s, rinse probe with 5 mL MeCN, shake mechanically at high speed for 5 min,
centrifuge at 2000 g for 5 min. Re-extract the solid with 10 mL MeCN. Add the supernatants
to the alumina column. Combine the eluates, add 70 mL water, add 100 (JLL triethylamine, mix,
add to the C18 SPE cartridge, pull air through the SPE cartridge for 3 min, elute with 5 mL
MeCN. Evaporate the eluate to dryness under a stream of nitrogen at 60, reconstitute the
residue in 100 u-L freshly prepared reagent, vortex for 15 s, heat at 95-100 for 45 min, cool,
add 1 mL chloroform, vortex, add to a 2.8 mL 500 mg Bond Elut silica SPE cartridge, elute
with three 3 mL aliquots of chloroform. Combine the eluates and evaporate them to dryness
under a stream of nitrogen at 60, reconstitute the residue in 1 mL MeOH, filter, inject a 40
|JLL aliquot. (Prepare alumina column as follows. Shake 94 g Brockman Activity I neutral alu-
mina (Fisher) and 6 mL water for 45 min, add 4.5 g alumina to an 8 mL column with a frit.
Reagent was 200 |xL 1-methylimidazole, 600 (xL acetic anhydride, and 900 |xL DMF.)
HPLC VARIABLES
Guard column: 30 X 4.6 RP-18 (Brownlee)
Column: 250 X 4.6 RP-18 OD-224 (Brownlee)
Flow rate: 1.8
CHROMATOGRAM
Retention time: 9.3
KEYWORDS
SPE; cow; pig; sheep; fish; liver; muscle; derivatization
REFERENCE
Salisbury,C.D.C. Modified method for the determination of ivermectin residues in animal tissues, JAOACInt.,
1993, 76, 1149-1151.
SAMPLE
Matrix: tissue
MeCNrwater 10:90. 4 g Minced meat or liver + 40 mL MeCN + 3.5 mL water, vortex for 2 min,
centrifuge at 2000 rpm for 10 min, remove supernatant, repeat extraction with 20 mL MeCN
and 3.5 mL water. Combine the supernatants and evaporate them to 6 mL under reduced
pressure (all the MeCN should be removed), add 6 mL water to the residue, add to the SPE
cartridge, pull air through the cartridge for 10 min, elute with 5 mL MeCN. Evaporate the
eluate under a stream of nitrogen, add 150 |xL trifluoroacetic anhydride:MeCN 1:2 to the res-
idue, add 100 |JLL N-methylimidazole:MeCN 50:50, shake, store in the dark, inject a 10-50 fxL
aliquot.
HPLC VARIABLES
Mobile phase: MeOH:water 95:5 (At the end of the day flush column with 30 mL MeOH.)
Flow rate: 1.5
CHROMATOGRAM
Retention time: 11
KEYWORDS
meat; liver; cow; pig; muscle; SPE; derivatization
REFERENCE
Degroodt,J.M.; Wyhowski de Bukanski,B-; Srebrnik,S. Determination of ivermectin residues in meat and liver
by HPLC and fluorometric detection, J.Liq.Chromatogr., 1994, 17, 1419-1426.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 3 mL Bakerbond C8 SPE cartridge with 5 mL MeCN and 5
mL MeCN:water:triethylamine 30:70:0.1. Prepare a 55 mm column of 70-230 mesh Kiselgel 60
(Merck) in a Pasteur pipette, condition with 3 mL hexanedsopropanol 60:40. Homogenize
(Polytron) 5 g blended tissue with 12 mL MeCN, rinse homogenizer with 3 mL MeCN, centri-
fuge the mixture at 4000 rpm for 10 min. Remove the supernatant and make up to 50 mL with
water, add 50 |xL triethylamine, shake, add to the SPE cartridge, elute with 5 mL MeCN at 1
drop/s. Evaporate the eluate to 300 |xL under a stream of nitrogen at 40, transfer to a smaller
vial with MeCN, evaporate to dryness under a stream of nitrogen at 60, reconstitute the
residue in 100 |xL l-methylimidazole:MeCN 50:50, cool in an ice bath, add 150 JJLL trifluoro-
acetic anhydride:MeCN 1:2, shake at room temperature for 1 min, add to the column, rinse
the vial with 500 |xL hexanedsopropanol 60:40, add the rinse to the column, elute with 1 mL
hexane:isopropanol 60:40. Evaporate the eluate to dryness under a stream of nitrogen with
heating, reconstitute with 250 |xL MeCN, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 5 (xm LiChrospher 100 RP-18
Column: 125 X 4 5 ixm LiChrospher 60 RP-select B
Flow rate: 1
CHROMATOGRAM
Retention time: 3.5
Limit of quantitation: 2.5 ppb
KEY WORDS
derivatization; muscle; liver; pig; cow; SPE
REFERENCE
Guggisberg,D.; Sievi,M.; Koch,H. Methode zur quantitativen Bestimmung von Ivermectin in Fleisch und Leber
mit HPLC und Vorsaulenderivatisation [Method for the quantitative determination of ivermectin in meat
and liver by HPLC and pre-column derivatization], Mitteilungen aus dem Gebiete der Lebensmittelunter-
suchung und Hygiene, 1994, 85, 395-405.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 6 mL 500 mg Bakerbond C18 SPE cartridge with three 5 mL
portions of MeOH, 5 mL MeCN, and three 5 mL portions of MeCN:water:triethylamine 30:70:
0.1. Condition a Waters silica SPE cartridge with 8 mL chloroform. Homogenize (Ultraturrax)
5 g minced tissue with 15 mL MeCN at high speed for 3 min, rinse blade with 2 mL MeCN,
sonicate for 15 min, centrifuge at 3000 rpm for 5 min, filter (paper), extract the residue again
with 10 mL MeCN, wash the filter with 3 mL MeCN. Combine the organic layers and add 70
mL water and 100 (xL triethylamine, stir thoroughly, add to the C18 SPE cartridge, wash with
two 5 mL portions of MeCN:water 50:50, elute with 7 mL MTBE at 2 mL/min. Store the eluate
overnight at -20, remove the organic layer and evaporate it to dryness under a stream of
nitrogen at 50, reconstitute the residue in 3 mL MeOH, add 100 uL water, add 3 mL hexane,
vortex, remove the hexane layer, repeat the hexane wash. Extract the combined hexane layers
with 1 mL MeOH. Combine the MeOH layers and evaporate them to dryness under a stream
of nitrogen at 50, heat in a vacuum oven at 50 for 30 min, reconstitute the residue in 150 JJLL
1-methylimidazole.acetic anhydride:DMF 2:3:9 (freshly prepared), vortex for 30 s, heat at 100
for 1 h, cool, add 1 mL chloroform, vortex, add to the silica SPE cartridge, elute with three 3
mL portions of chloroform. Evaporate the eluate to dryness under a stream of nitrogen at 50,
reconstitute the residue in 400 |JLL MeOH, vortex, inject a 20 jxL aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 5 |xm Supelcosil LC-18
Flow rate: 1.8
CHROMATOGRAM
Retention time: 7
KEYWORDS
derivatization; SPE; liver; muscle; fat; guinea pig; cow; pig; horse; sheep; pharmacokinetics
REFERENCE
Dusi,G.; Curatolo,M.; Fierro,A.; Faggionato,E. Determination of the antiparasitic drug ivermectin in liver, mus-
cle and fat tissue samples from swine, cattle, horses and sheep using HPLC with fluorescence detection,
Josamycin
51016-68-3 (propionate)
Merck Index: 5280
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 231.1
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr.Ay 1997, 763,149
163.
SAMPLE
Matrix: bulk
HPLC VARIABLES
Column: 250 X 4.6 8 |xm 1000 A PLRP-S (Polymer Labs., UK)
Mobile phase: MeCN:buffer:water 52:20:28 (Prepare buffer by mixing 200 mM K3PO4 with 200
mM K2HPO4 to obtain a pH of 10.0.)
Flow rate: 1
Detector: UV 232
CHROMATOGRAM
Retention time: 20 (josamycin propionate)
OTHER SUBSTANCES
Simultaneous: josamycin, leucomycin A4 propionate, josamycin 2',9-dipropionate, josamycin
3",9-dipropionate, platenomycin Al propionate
REFERENCE
Roets,E.; Lepoudre,X.; Van Rompaey,V.; Velghe,G.; Liu,L.; Hoogmartens,J. Liquid chromatography ofjosamycin
propionate on poly(styrene-divinylbenzene), J.Chromatogr.A, 1998, 812, 303-308.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 1 mL 100 mg Bond-Elut diol SPE cartridge with 1 mL chlo-
roform (Caution! Chloroform is a carcinogen!). Mix 2 g minced muscle tissue with 800 |xL water.
Stir, vortex for 1 min at maximum speed, let stand for 15 min. Add 2 mL pH 8 buffer, mix
briefly, add 10 mL chloroform. Stir at 100 rpm for 15 min, centrifuge at 4000 g for 10 min,
discard the aqueous layer, filter the chloroform layer through glass wool. Add the filtrate to
the SPE cartridge, wash with 500 |xL chloroform, dry under vacuum, elute with three 200 |xL
portions of MeOH: 100 mM ammonium acetate 50:50, inject a 200 |xL aliquot of the eluate.
(Buffer was 33.46 g K2HPO4 and 1.046 g KH2PO4 in 1 L water.)
HPLC VARIABLES
G u a r d c o l u m n : 4 x 4 5 (xm C18
Column: 125 X 4 5 fim Lichrospher RP18
Mobile phase: Gradient. A was MeCN. B was MeOH. C was 0.1% trifluoroacetic acid in water.
A:B:C from 20:20:60 to 25:55:20 in 10 (?) min
Flow rate: 0.5
Detector: MS, HP Model 5989 A, desolvation chamber 60, source 280 and 300 in negative and
positive chemical ionization mode, respectively, with methane as reagent, quadrupole 100,
particle beam nebulizer helium 345 kPa, scan m/z 735.4-828.5 in NCI and 828.5-769.4 in PCI
CHROMATOGRAM
Retention time: 7.2
Limit of detection: 50 |xg/kg
OTHER SUBSTANCES
Extracted: erythromycin, spiramycin, tilmicosin, tylosin
KEYWORDS
muscle; cow; SPE
REFERENCE
Del6pine,B-; Hurtaud-Pessel,D.; Sanders,P. Multiresidue method for confirmation of macrolide antibiotics in
bovine muscle by liquid chromatography/mass spectrometry, J.AOAC Int., 1996, 79, 397-404.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 500 mg Bond Elut SCX SPE cartridge (Varian) with 5 mL
MeOH and 10 mL 100 mM pH 4.4 KH2PO4 buffer. Homogenize 5 g tissue with 100 mL MeOH:
0.3% metaphosphoric acid 30:70 at high speed for 2 min, filter through 2 mm Hyflo Super-Cel
coated on a suction funnel (when filtering liver or kidney add several grams of Hyflo Super-
Cel to the homogenized solution before filtration). Evaporate the filtrate to ca. 20 mL under
reduced pressure at 45, add to the SPE cartridge, wash with 10 mL distilled water and 5 mL
100 mM pH 8.9 K2HPO4 buffer, elute with 10 mL MeOH, evaporate the eluate to dryness under
reduced pressure at 45, dissolve the residue in 1 mL MeCN:50 mM pH 4.5 NaH2PO4 buffer
30:70, inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 \ym Puresil 5C18 (Waters)
Mobile phase: Gradient. A:B from 60:40 to 0:100 over 16 min. A was buffer. B was MeCN:buffer
40:60 (Buffer was 2.5 g KH2PO4 dihydrate and 0.65 mL 85% phosphoric acid dissolved in 1 L
distilled water, pH 2.5.)
Flow rate: 1
Detector: UV 232 for 9 min, UV 287 for 2 min, UV 232 for 4 min
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: leucomycin (kitasamycin), mirosamicin, spiramycin, tylosin
KEYWORDS
meat; SPE
REFERENCE
Horie,M.; Saito,K.; Ishii,R.; Yoshida,T.; Haramaki,Y.; Nakazawa,H. Simultaneous determination of five ma-
crolide antibiotics in meat by high-performance liquid chromatography, J.Chromatogr.A, 1998, 812, 295-
302.
SAMPLE
Matrix: tissue
Sample preparation: Blend (Virtis model 45 with U-shaped blades) 2.5 g tissue with 20 mL
MeCN: 10 mM pH 6.0 phosphate buffer 65:35 for 10 min, centrifuge at 5 at 8500 g for 5 min.
Remove the supernatant and adjust the volume to 25 mL with MeCN: 10 mM pH 6.0 phosphate
buffer 65:35. Remove a 1.5 mL aliquot and add it to 5 mL isooctane, shake for 10 min, centrifuge
at 5 at 3000 g for 5 min, discard the organic layer. Add 500 (xL reagent to the aqueous layer,
mix, heat at 90 for 2 h, cool, inject a 100 |xL aliquot. (Prepare reagent by dissolving 1 g
cyclohexa-l,3-dione and 25 g ammonium acetate in 60 mL water and 8 mL concentrated HCl,
make up to 100 mL with water. Store at 5, discard after 1 month.)
HPLCVARIABLES
Guard column: 4 x 4 5 |xm LiChrospher 100 RP-18 end capped
Column: 125 X 4 5 |xm LiChrospher 100 RP-18 end capped
Mobile phase: MeCN:MeOH:10 mmole pH 6.0 phosphate buffer 45:5:50
Flow rate: 1.5
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: spiramycin, tylosin
Noninterfering: acetaldehyde, benzaldehyde, erythromycin, formaldehyde
KEYWORDS
pig; muscle; liver; kidney; fat; derivatization
REFERENCE
Leroy,P.; Decolin,D.; Nicolas,A.; Archimbault,P. Determination of josamycin residues in porcine tissues using
high-performance liquid chromatography with pre-column derivatization and spectrofluorometric detection,
Analyst, 1994, 119, 2743-2747.
Kanamycin
Molecular formula: C18H36N4O11(A)
Molecular weight: 484.51 (A)
CAS Registry No.: 8063-07-8, 25389-94-0 (A sulfate),
59-01-8(A), 4696-78-8(B)
Merck Index: 5293
SAMPLE
Sample preparation: Plasma. 300 JJIL Plasma + 30 (xL water + 100 (xL2M perchloric acid,
vortex for 2-3 s, centrifuge at 1000 g for 5 min. Remove the supernatant and neutralize it with
1.5 M NaOH, add 300 |xL buffer, add 400 |xL DMSO, add 100 |xL 2% 2,4-dinitrofluorobenzene
in EtOH, vortex, heat at 64 for 30 min, add 3 mL toluene, vortex, centrifuge, discard the upper
toluene layer, add 3 mL MeCN:toluene 50:50, vortex for 5-10 s. Remove the upper organic layer
and evaporate it to dryness under a stream of nitrogen at 30-40, reconstitute the residue in
1 mL MeCN:water 50:50, inject a 20 JJLL aliquot. Urine. Dilute urine 100-fold with water. 300
fxL Diluted urine + 30 |JLL water + 300 |xL buffer + 400 |xL DMSO + 100 (xL 2% 2,4-dinitro-
fluorobenzene in EtOH, vortex, heat at 64 for 30 min, add 3 mL toluene, vortex, centrifuge,
discard the upper toluene layer, add 3 mL MeCN:toluene 50:50, vortex for 5-10 s. Remove the
upper organic layer and evaporate it to dryness under a stream of nitrogen at 30-40, recon-
stitute the residue in 1 mL MeCN:water 50:50, inject a 20 |xL aliquot. (Prepare buffer by mixing
80 mL 100 mM Na2HPO4 and 20 mL 100 mM NaH2PO4, adding 1 g Tris HCl, and adjusting
the pH to 7.8 with 6 M HCl.)
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Zorbax SB-C18
Mobile phase: MeOH:water 64:36, adjusted to pH 3.0 with phosphoric acid
Flow rate: 2
Detector: UV 350
CHROMATOGRAM
Retention time: 24.0 (kanamycin B)
Internal standard: kanamycin B (24.0)
OTHER SUBSTANCES
Extracted: paromomycin
KEYWORDS
derivatization; plasma; pharmacokinetics; kanamycin B is IS
REFERENCE
Lu, J.; Cwik,M.; Kanyok,T. Determination of paromomycin in human plasma and urine by reversed-phase high-
performance liquid chromatography using 2,4-dinitrofluorobenzene derivatization, J.Chromatogr.B, 1997,
695, 329-335.
SAMPLE
Matrix: bulk
aqueous phase, inject a 20 u-L aliquot of the lower organic phase.
HPLCVARIABLES
Column: 250 X 4.6 5 jim LiChrosorb SMOO
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Retention time: 18
KEYWORDS
REFERENCE
Tsuji,K.; Goetz,J.R; VanMeter,W.; Gusciora,KA. Normal-phase high-performance liquid chromatographic de-
141-152.
SAMPLE
Matrix: fermentation solutions
Sample preparation: 5 mL Fermentation broth + 5 mL saturated aqueous solution of Tris + 20
mL MeCN, centrifuge at 3000 rpm for 10 min. Remove a 1 mL aliquot of the supernatant and
add it to 3 mL 150 mM 2,4-dinitrofluorobenzene in MeOH, heat at 100 under a reflux con-
denser for 45 min, make up to 4 mL with mobile phase, inject an aliquot.
HPLC VARIABLES
Flow rate: 1.2
Detector: UV 350
CHROMATOGRAM
Retention time: 11.28 (kanamycin B)
OTHER SUBSTANCES
Extracted: apramycin, tobramycin
KEY WpRDS
derivatization
REFERENCE
Harangi,J.; Dek,M.; N&nasi,R; Bacsa,G. Determination of the major factors of fermentation of the nebramycin
complex by high performance liquid chromatography, J.Liq.Chromatogr., 1984, 7, 8393.
SAMPLE
Sample preparation: Dilute 3 mg/mL ophthalmic suspension in water with 10 mM sulfuric acid
to a tobramycin concentration of 240 |xg/mL. Mix 4 mL diluted suspension with 10 mL 10 mg/
mL 2,4-dinitrofluorobenzene in EtOH and 10 mL 15 mg/mL tris(hydroxymethyl)aminomethane
in water:dimethylsulfoxide 20:80. Heat at 70 2. for 20 min, allow to cool slightly for 2 min
and add 24 mL MeCN. Allow to cool to room temperature, make up to 50 mL with MeCN,
HPLCVARIABLES
Mobile phase: MeCN:buffer 55:45 (Prepare mobile phase as follows. Dissolve 2.0 g tris (hydrox-
ymethyl)aminomethane in 960 mL water, add 20 mL 0.5 M sulfuric acid and 1200 mL MeCN.)
Flow rate: 1.5
Detector: UV 365
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Simultaneous: neamine, nebramine, tobramycin
KEYWORDS
derivatization; ophthalmic suspension
REFERENCE
Russ,H.; McCleary,D.; Katimy,R.; Montana,J.L.; Miller,R.B.; Krishnamoorthy,R.; Davis,C.W. Development and
validation of a stability-indicating HPLC method for the determination of tobramycin and its related sub-
stances in an ophthalmic suspension, J.Liq.Chromatogr.Rel.TechnoL, 1998, 21, 2165-2181.
SAMPLE
Sample preparation: 50 |xL Buffered reaction mixture + 50 |xL isopropanol + 50 uL reagent,
heat at 60 for 10 min, centrifuge at 1000 g for 2 min, immediately inject a 50 |xL aliquot of
the supernatant. (Reagent was 80 mM o-phthalaldehyde and 250 mM thioglycolic acid in 1 M
boric acid, pH adjusted to 10.4 with 40% KOH.)
HPLC VARIABLES
Column: 100 X 5 Hypersil ODS
Mobile phase: A was MeOH:water:acetic acid 50:45:5 containing 5 g/L heptanesulfonic acid. B
was MeOH:water:acetic acid 75:20:5 containing 5 g/L heptanesulfonic acid. A:B 60:40.
Flow rate: 2
Detector: UV 330
CHROMATOGRAM
Retention time: 19 (kanamycin A)
KEYWORDS
derivatization
REFERENCE
Lovering,A.M.; White,L.O.; Reeves,D.S. Identification of aminoglycoside-acetylating enzymes by high-pressure
liquid chromatographic determination of their reaction products, Antimicrob.Agents Chemother., 1984, 26,
10-12.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeCN, inject a 50 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:20 mM buffer 52:48 (Buffer was 2.68 g KH2PO4 in 1 L water adjusted to
pH 3.0 with phosphoric acid.)
Flow rate: 2
Detector: UV 340
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Simultaneous: amikacin
Noninterfering: acetaminophen, acetazolamide, N-acetylprocainamide, amobarbital, ampicillin,
amitriptyline, caffeine, cefamandole, cefoxime, cefoxitin, cephalothin, clindamycin, chloram-
phenicol, chlordiazepoxide, diazepam, erythromycin, ethosuximide, gentamicin, nitrofurantoin,
penicillin G, pentobarbital, phenobarbital, phenytoin, primidone, procainamide, quinidine,
salicylic acid, secobarbital, tetracycline, theophylline, tobramycin, vancomycin
REFERENCE
Kabra,P.M.; Bhatnager,P.K.; Nelson,M.A. Liquid chromatographic determination of amikacin in serum with
spectrophotometric detection, J.Chromatogr., 1984, 307, 224-229.
SAMPLE
Matrix: solutions
Sample preparation: 50 |xL Buffer solution + 25 ^xL 242 mg/mL pH 10.4 Tris buffer + 100 |JLL
MeCNrwater 50:50 + 30 |xL 250 mg/mL 2,4,6-trinitrobenzenesulfonic acid in MeCN:water 80:
20, vortex for 10 s, heat at 70 for 15 min, add 2 mL chloroform, shake horizontally at 180
cycles/min for 5 min, centrifuge at 750 g for 5 min. Remove the lower organic layer and evap-
orate it to dryness under a stream of nitrogen, reconstitute the residue in 200 (xL MeCN, vortex,
HPLCVARIABLES
Column: 250 X 4.6 5 |jim Ultrasphere octyl
Mobile phase: MeCN:50 mM KH2PO4 62:38, pH adjusted to 3.5 with phosphoric acid
Flow rate: 2.5
Detector: UV 340
CHROMATOGRAM
Retention time: 6.4
OTHER SUBSTANCES
Simultaneous: tobramycin
KEY WpRDS
derivatization
REFERENCE
Dash,A.K; Suryanarayanan,R. A liquid-chromatographic method for the determination of tobramycin,
J.Pharm.BiomedAnaL, 1991, 9, 237-245.
Ketamine
Molecular formula: C13H16CINO
Merck Index: 5306
Lednicer No.: 1 57
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 20 |xL IS + 350 |xL 200 mM pH 13 borate buffer, mix.
Extract with 5 mL dichloromethane:ethyl acetate 80:20, vortex at 60 rpm for 10 min, centrifuge
at 150Og at 15 for 3 min, remove organic phase and extract again with 3 mL dichloromethane:
ethyl acetate 80:20. Combine the organic layers and evaporate them to dryness under a stream
of nitrogen. Reconstitute the residue in 500 |xL dichloromethane:ethyl acetate 80:20, extract
with 2 mL 2 M HCl. Remove the aqueous layer and evaporate it to dryness at 45. Reconstitute
the residue in 100 (xL mobile phase, inject a 60 |xL aliquot.
HPLC VARIABLES
Column: 125 X 4 5 jxm Purospher RP-18e (Merck)
Flow rate: 1.5
Detector: UV 210
CHROMATOGRAM
Internal standard: nortilidine (6.29)
Limit of quantitation: 5 |xg/L
OTHER SUBSTANCES
Simultaneous: atropine, buprenorphine, diazepam, dopamide, furosemide, nalbuphine, omepra-
zole, phenobarbital, phytomenadione, propofol
KEYWORDS
REFERENCE
Bolze,S.; Boulieu,R- HPLC determination of ketamine, norketamine, and dehydronorketamine in plasma with
a high-purity reversed-phase sorbent, Clin.Chem., 1998, 44, 560-564.
SAMPLE
Matrix: blood
Sample preparation: Activate two 130 mg Sep-Pak Light C18 SPE cartridges with 1 mL MeOH
and 1 mL water. Add 1 mL plasma onto SPE cartridge, wash with 1 mL water, 2 mL 5 mM
pH 9.6 ammonium sulfate buffer containing 3% MeCN, and 1 mL 5 mM pH 9.6 ammonium
sulfate buffer containing 20% MeCN. Displace washing solution with 200 |xL 20 mM pH 2.1
phosphoric acid buffer containing 25% MeCN and elute with 500 jxL of the same solution. Mix
eluate with 1 mL 40 mM pH 11.5 sodium hydroxide. Add the mixture onto the second SPE
cartridge, wash with 2 mL 5 mM pH 9.6 ammonium sulfate buffer containing 3% MeCN and
with 1 mL 5 mM pH 9.6 ammonium sulfate buffer containing 20% MeCN. Displace washing
solution with 200 (JLL 20 mM pH 2.1 phosphoric acid buffer containing 25% MeCN and elute
with 500 |AL of the same solution. Evaporate the eluate in a vacuum centrifuge to about 150
|AL, add 12 |xL 1 mM sodium hydroxide immediately before injection, inject the whole volume
on the column. (Buffers were adjusted with ammonia. SPE flow-rate at all steps was approx-
imately 1.5 mL/min.)
HPLC VARIABLES
Column: 150 X 4.0 5 |xm Chiral AGP (Chrom Tech, Sweden)
Mobile phase: MeOH:10 mM pH 7.0 KH2PO4 16:84 (Buffer was adjusted with potassium
hydroxide.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Limit of detection: 1.7 ng/mL (S), 2.0 ng/mL (R)
OTHER SUBSTANCES
Extracted: metabolite
KEYWORDS
plasma; SPE; chiral
REFERENCE
Svensson,J.O.; Gustafsson,L.L. Determination of ketamine and norketamine enantiomers in plasma by solid-
phase extraction and high-performance liquid chromatography, J.Chromatogr.B, 1996, 678, 373-376.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL bisnortilidine in mobile phase + 100 jxL 3 M NaOH,
mix, add 5.75 mL ice-cold cyclohexane, agitate at 4 for 15 min, repeat extraction with 3 mL
ice-cold cyclohexane. Combine the organic layers and evaporate them to dryness under a
stream of nitrogen, reconstitute the residue in 500 |xL cyclohexane, evaporate to dryness under
a stream of nitrogen, reconstitute in 0.2-1 mL mobile phase, mix for 30 s, let stand for 2 min,
inject a 50-100 |xL aliquot.
HPLCVARIABLES
Column: 100 X 4 AGP (Grom)
Mobile phase: Isopropanol:20 mM pH 7 phosphate buffer 2.5:97.5
Flow rate: 0.5
Detector: UV 215
CHROMATOGRAM
Internal standard: bisnortilidine (ethyl trans-2-amino-l-phenyl-3-cyclohexene-l-carboxylatehy-
drochloride) (one enantiomer only, separated by HPLC) (15)
OTHER SUBSTANCES
KEY WORDS
chiral; plasma; pharmacokinetics
REFERENCE
Geisslinger,G.; Menzel-Soglowek,S.; Kamp,H.-D.; Brune,K. Stereoselective high-performance liquid chromato-
graphic determination of the enantiomers of ketamine and norketamine in plasma, J.Chromatogr., 1991,
568, 165-176.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with water, MeOH, and 100 mM
ammonium acetate. Add 200 |xL plasma to the SPE cartridge, wash with 100 mM ammonium
acetate, elute with MeOH:100 mM ammonium acetate 3:1. Evaporate the eluate to dryness
under reduced pressure, dissolve the residue in 200 |xL mobile phase, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 Hitachi gel 3056 octadecylsilica
Flow rate: 1
Detector: MS, Hitachi M1000, APCI, nebulizer 260, vaporizer 399
CHROMATOGRAM
Retention time: 6.2
OTHER SUBSTANCES
Simultaneous: atipamezole, atropine, butorphanol, flumazenil, medetomidine, midazolam,
xylazine
KEYWORDS
plasma; SPE; dog
REFERENCE
Kanazawa,H.; Nagata,Y.; Matsushima,Y.; Takai,N.; Uchiyama,H.; Nishimura,R.; Takeuchi,A. Liquid chroma-
tography-mass spectrometry for the determination of medetomidine and other anaesthetics in plasma,
J.Chromatogr., 1993, 631, 215-220.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 500 (xL MeCN:water 85% phosphoric acid 20:78:2, vortex
for 10-15 s, filter (Amicon Centricon-10 microseparation system, 10000 molecular mass cut-off)
while centrifuging at 3000 g for 30 min, inject a 20-100 jxL aliquot of the ultrafiltrate.
HPLC VARIABLES
Column: 100 X 4.6 3 |xm Spherisorb phenyl
Mobile phase: MeCN:MeOH:10 mM NaH4PO4:85% phosphoric acid 10:30:59.8:0.2
Detector: UV 215
CHROMATOGRAM
Retention time: 5.1
OTHER SUBSTANCES
KEYWORDS
horse; serum; ultrafiltrate
REFERENCE
Seay,S.S.; Aucoin,D.R; Tyczkowska,K.L. Rapid high-performance liquid chromatographic method for the deter-
mination of ketamine and its metabolite dehydronorketamine in equine serum, J.Chromatogr., 1993, 620,
281-287.
SAMPLE
Matrix: blood
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 269
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 jjum Symmetry C8 (Waters)
mL/min)
Detector: UV 202.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm YMC GEL, ODS-AM coated with poly-(R)-l-(a-naphthyl)ethyl meth-
acrylamide (Prepare (R)-l-(a-naphthyDethyl methacrylamide by reacting methacryl chloride
with (R)-l-(a-naphthyl)ethylamine. Prepare poly-(R)-l-(a-naphthyl)ethyl methacrylamide by
polymerizing this compound in anhydrous benzene/THF with 2,2'-azobis(isobutyronitrile) (Cau-
tion! Benzene is a carcinogen!). Average molecular weight = 2500. Coat 4 g 5 |xm YMC GEL,
ODS-AM with 0.8 g of this polymer using dichloromethane as a solvent.)
Mobile phase: MeCN:0.5M sodium perchlorate 40:60
Flow rate: 1
CHROMATOGRAM
Retention time: k' 3.18 (a = 1.21)
OTHER SUBSTANCES
Also analyzed: propranolol
KEYWORDS
chiral
REFERENCE
Oi,N.; Hashimoto,S.; Ishizuka,N.; Ohtake,J. Enantiomer separation with poly-(R)-l-(a-naphthyl)-ethyl-meth-
acrylamide coated on ODS silica gel by reversed phase HPLC, Biomed.Chromatogr., 1997, 11, 296-297.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
stilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, iso-
xsuprine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam, lormetazepam, lox-
apine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid,
megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital,
mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, meth-
aqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate,
methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone,
puromycin, pjrrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 Sumchiral CSP 10 (Sumika Chemical Analysis Service)
Mobile phase: n-Hexane:EtOH:trifluoroacetic acid 200:40:0.6
Flow rate: 1
CHROMATOGRAM
KEYWORDS
chiral; a = 1.12
REFERENCE
Oi,N.; Kitahara,H.; Aoki,F. Direct enantiomer separations by high-performance liquid chromatography with
chiral urea derivatives as stationary phases, J.Chromatogr.A, 1995, 694, 129-134.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 CSP-4 (Prepare as follows. Add a solution of 1.07 g L-valyl-L-valyl-L-valine
isopropylester (Bunseki Kagaku 1079, 28, 125) in 30 mL dry dioxane (Caution! Dioxane is a
carcinogen!) dropwise to a mixture of 2.2 g 2,4,6-trichloro-l,3,5-triazine (cyanuric chloride) in
20 mL dry dioxane stirred at 0, add 3 g anhydrous sodium carbonate at room temperature,
stir, filter, evaporate to give a colorless solid. Dissolve 8.3 g of this solid in 30 mL dry dioxane,
add 2 g N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, add 1.5 g anhydrous sodium carbon-
ate, reflux with stirring for 40 h, filter, add 3 g dried 10 |xm LiChrosorb Si 100, reflux with
slow stirring for 10 h, cool, filter. Wash the solid with dioxane, MeOH, and diethyl ether, dry
under reduced pressure (J.Chromatogr. 1984, 292, 427).)
Mobile phase: Hexane:EtOH:trifluoroacetic acid 96:4:0.24
Detector: UV
CHROMATOGRAM
KEYWORDS
chiral; a = 1.09
REFERENCE
Oi,N.; Kitahara,H.; Matsushita,Y.; Kisu,N. Enantiomer separation by gas and high-performance liquid chro-
matography with tripeptide derivatives as chiral stationary phases, J.Chromatogr.A, 1996, 722, 229-232.
Ketanserin
Merck Index: 5307
Lednicer No.: 3 193
SAMPLE
Matrix: blood
Sample preparation: 25 |xL Serum + 50 |xL MeOH, vortex for 30 s, centrifuge at 13600 g for 5
min, inject a 25 JJLL aliquot of the supernatant.
HPLC VARIABLES
Mobile phase: MeCN:buffer:water 31:50:19 (Buffer was 2% acetic acid adjusted to pH 7.0 with
ammonium hydroxide.)
Flow rate: 1
Detector: F ex 225 em no emission filter
CHROMATOGRAM
Retention time: 7.7
OTHER SUBSTANCES
KEYWORDS
rat; serum; pharmacokinetics
REFERENCE
Wong,Y.W.; Skinner,M.H. Rapid method for the determination of ketanserin in rat serum by high-performance
liquid chromatography with fluorimetric detection, J.Chromatogr., 1991, 571, 318-323.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 JXL aliquot.
HPLC VARIABLES
MeOH5 pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.4
OTHER SUBSTANCES
isoxsuprine, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine, meclo-
thipendyl, protriptyline, proxymetacaine, pseudoephedrine, pîmethamine, quinidine, qui-
REFERENCE
Jane,I.; McKinnonA-', Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica
Ketoconazole
Molecular formula: C26H28CI2N4O 4
Merck Index: 5313
Lednicer No.: 3 132
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 2 |xg clotrimazole + hexanerisoamyl alcohol 98.5:1.5,
vortex, centrifuge. Remove the organic layer and evaporate it to dryness, reconstitute the res-
idue in 250 |xL MeCN, inject an aliquot.
HPLC VARIABLES
Column: 150 X 3.9 NovaPak C18
Mobile phase: MeCN:MeOH:50 mM phosphate buffer 40:5:55
Detector: UV 220
CHROMATOGRAM
KEYWORDS
REFERENCE
von Moltke,L.L.; Greenblatt,D.J.; Harmatz,J.S.; Duan,SX; Harrel,L.M.; Cotreau-Bibbo,M.M.; Pritchard,G.A.;
Wright,C.E.; Shader,R.I. Triazolam biotransformation by human liver microsomes in vitro: Effects of
metabolic inhibitors and clinical confirmation of a predicted interaction with ketoconazole,
J.PharmacoLExp.Ther., 1996, 276, 370-379.
SAMPLE
Matrix: blood, microsomal incubations
Sample preparation: Vortex 1 mL plasma or microsomal incubation with 200 |xL 5 |xg/mL di-
azepam and 100 JJLL 5 M NaOH solution for 10 s, add 5 mL butan-l-ol:hexane 2:98, vortex for
1 min, centrifuge at 2000 g and 4 for 5 min, evaporate the organic phase to dryness at 40
using a vacuum vortex evaporator, reconstitute the residue in 200 |xL mobile phase, inject a
50 |xL aliquot.
HPLC VARIABLES
Column: 5 |xm Nova-Pak C18
Mobile phase: MeCN:buffer 35:65 (Buffer was water containing 1% triethylamine, adjusted to
pH 3 with orthophosphoric acid.)
Flow rate: 2
Detector: UV 240
CHROMATOGRAM
Retention time: 6.3
Internal standard: diazepam
OTHER SUBSTANCES
Extracted: amitriptyline, nortriptyline
Noninterfering: furafylline, hydroxyamitriptyline, hydroxynortriptyline, quinidine, mephenyt-
oin, triacetyloleandomycin
KEYWORDS
human; liver; rat; plasma
REFERENCE
Ghahramani,R; Lennard,M.S. Quantitative analysis of amitriptyline and nortriptyline in human plasma and
liver microsomal preparations by high-performance liquid chromatography, J.ChromatognB, 1996, 685,
307-313.
SAMPLE
a 10 (urine) or 30 (blood) |AL aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
mL/min)
Detector: UV 202.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 125 X 4 5 |xm Hibar RP 18 (Merck)
Mobile phase: MeCN:water:diethylamine 48:55:0.02
Flow rate: 1.2
Injection volume: 20, 100
Detector: UV 254
REFERENCE
Galia,E.; Nicolaides,E.; Hbrter,D.; Lobenberg,R.; Reppas,C; Dressman,J.B. Evaluation of various dissolution
Ketoprofen
Merck Index: 5316
Lednicer No.: 2 64
SAMPLE
Matrix: blood
Sample preparation: Mix 50 JJLL plasma with 150 JJLI 25 jxg/mL ibufenac in MeCN:water 95:5,
vortex for 30 s, centrifuge for 1 min using a Beckman Microfuge (Beckman Instruments, Palo
Alto, CA). Inject a 20 gmL aliquot of the supernatant.
HPLC VARIABLES
Column: 33 X 4.6 3 |xm C18 (Perkin Elmer, Norwalk, CT)
Mobile phase: MeCN:buffer 40:60 (Prepare mobile phase as follows. Dissolve 4 mL concentrated
phosphoric acid in 600 mL water and mix with 400 mL MeCN.)
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Retention time: 1.1
Internal standard: ibufenac (2.6)
Limit of quantitation: 5 fig/mL
OTHER SUBSTANCES
Simultaneous: dicloxacillin, ibuprofen
Noninterfering: acetaminophen, N-acetylprocainamide, amikacin, amitriptyline, aspirin, az-
treonam, barbituric acid, brompheniramine, cafazolin, caffeine, carbamazepine, carbamazepine
epoxide, cephalexin, chlorpheniramine, clonazepam, clotrimazole, desipramine, desmethyldox-
epin, digitoxin, digoxin, disopyramide, doxepin, ethosuximide, felbamate, gentamicin, imipe-
nem, imipramine, lidocaine, maprotiline, mephenytoin, mephobarbital, metharbital, methsux-
imide, methylsuccinimide, nortriptyline, paramethadione, phenacemide, phenobarbital,
phensuximide, phenylpropanolamine, phenytoin, primidone, protriptyline, sulfamethoxazole,
theophylline, tobramycin, trimethadione, trimethoprim, vancomycin.
KEYWORDS
plasma
REFERENCE
Rifai,N.; Lafi,M.; Sakamoto,M.; Law,T. Measurement of plasma ketoprofen by a rapid high-performance liquid
chromatography assay, Ther.Drug Monit., 1997, 19, 175-178.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg C 18 SPE cartridge (Varian) with 1 mL MeOH
and 1 mL 100 mM pH 2.5 phosphate buffer. Mix 100 |xL 50 jxg/mL 3,4-dimethoxybenzoic acid
in MeOH with 1 mL plasma. Vortex with 360 mg ammonium sulfate for 30 min to deproteinate
the plasma. Centrifuge at 8000 g at 4. for 30 min, acidify the supernatant with 3 ml 100 mM
sulfuric acid. Add the supernatant to the SPE cartridge, wash twice with 750 JULL MeOH: 100
mM pH 2.5 phosphate buffer 20:80. Elute with two 500 |xL portions of MeOH:50 mM pH 7.4
phosphate buffer 75:25. Inject a 40 (xL aliquot.
HPLC VARIABLES
Guard column: Supelco C18
Column: 250 X 4.6 Chirex 3005 [(R)-l-naphtylglycine and 3,5-dinitrobenzoic acid] (Phenomenex,
Torrance, CA, USA)
Mobile phase: 20 mM ammonium acetate in MeOH
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Internal standard: 3,4-dimethoxybenzoic acid (18.1)
KEYWORDS
SPE; chiral; pharmacokinetics
REFERENCE
Boisvert,J.; Caille",G.; McGilveray,I.J.; Qureshi,S.A. Quantification of ketoprofen enantiomers in human plasma
based on solid-phase extraction and enantioselective column chromatography, J.Chromatogr.B, 1997, 690,
189-193.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 1 mL 1 M pH 2.0 phosphate buffer + IS, extract with 7
mL diethyl ether, evaporate, reconstitute the residue in 250 jxL MeCN:5 mM pH 7.0 phosphate
buffer 10:90, inject 50 (xL aliquot.
HPLC VARIABLES
Guard column: 4 X 4 LiChrocart
Column: 125 X 3 5 |xm Ecocart packed with LiChrospher 100 RP-18
Flow rate: 0.6
Detector: UV 260
CHROMATOGRAM
KEYWORDS
horse; plasma
REFERENCE
Baeyens,W.R.G.; Van Der Weken,G.; Van Overbeke,A.; Corveleyn,S.; Remon,J.R; Deprez,P. Comparative nar-
row-bore high-performance liquid chromatographic determination of ketoprofen in horse plasma, Bio-
med.Chromatogr., 1998, 12, 167-169.
SAMPLE
Matrix: blood
Sample preparation: Acidify plasma with an equal volume of 100 mM pH 3.2 phosphoric acid,
inject a 20 |xL aliquot onto column A and elute to waste with mobile phase A. After 5 min elute
the contents of column A onto column B with mobile phase B, monitor the effluent from column
B. (Before each run wash column A with MeCNrIOO mM pH 3.2 phosphate buffer 20:80 for 3
min, with water for 1 min, with MeOH for 3 min, and with mobile phase A. Equilibrate column
B with mobile phase B.)
HPLCVARIABLES
Column: A 10 X 4 5 |xm Develosil NH2-5 (Nomura Chemical Co., Japan) + 10 X 4 Nucleosil 5CN
(Macherey-Nagel, Dtiren, Germany); B 150 X 4.6 Ultron ES-OVM G bonded silica column
(Shinwa Kako Co., Japan)
Mobile phase: A 100 mM pH 3.2 phosphate buffer; B MeOH: 100 mM pH 3.2 phosphate buffer
20:80
Flow rate: 0.8
Detector: UV 265
CHROMATOGRAM
Retention time: 9.6 (S(+)), 12.0 (R(-))
KEYWORDS
column-switching; chiral; plasma
REFERENCE
Tamai,G.; Edani,M.; Imai,H- Determination of ketoprofen enantiomers in plasma by solid phase extraction and
column switching high performance liquid chromatography, Anal.ScL, 1991, 7, 29-32.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 u,L tolmetin solution + 500 uX pH 1.8 phosphate
buffer, extract with 1-butanol/MTBE. Remove the organic layer and add it to 500 uX pH 6.1
ammonium acetate buffer, mix, inject an aliquot of the aqueous layer.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Cosmosil C18
Mobile phase: MeCN:250 mM pH 5.0 ammonium acetate buffer 20:80
Flow rate: 1.8
Detector: UV 350
CHROMATOGRAM
Internal standard: tolmetin (UV 258)
KEYWORDS
REFERENCE
Shah,A.K; Wei,G.; Lanman,R.C; Bhargava,V.O.; Weir,S.J. Percutaneous absorption of ketoprofen from differ-
ent anatomical sites in man, Pharm.Res., 1996, 13, 168-172.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond-Elut C18 SPE cartridge with 1 mL MeOH. 1 mL
Serum + 1 mL water + 20 jxL saturated ammonium sulfate solution + 60 jxL concentrated HCl,
vortex for 3 min, add to the SPE cartridge, wash with three 1 mL portions of water, allow to
dry for 3 min, elute with hve 500 |xL portions of MeOH. Evaporate the eluate to dryness under
a stream of nitrogen, reconstitute the residue in 1 mL mobile phase, inject a 100 JJLL aliquot.
HPLCVARIABLES
Column: 100 X 4.6 5 |xm Spheri-5 cyano
Mobile phase: MeCN:MeOH:waterrphosphoric acid 21:22:56.5:0.5
Flow rate: 0.5
Detector: F ex 248 em 335 (filter) following post-column reaction. The column effluent flowed
through a knitted 7.9 m X 0.3 mm ID PTFE coil irradiated with an SC3-9 UV lamp (UVP, San
Gabriel CA) and cooled with a fan to the detector.
CHROMATOGRAM
Retention time: 6.5
Internal standard: ketoprofen
OTHER SUBSTANCES
Extracted: fenbufen
KEYWORDS
ketoprofen is IS; post-column reaction; post-column photochemical derivatization; serum; SPE
REFERENCE
Siluveru,M.; Stewart,J.T. Determination of fenbufen and its metabolites in serum by reversed-phase high-
performance liquid chromatography using solid-phase extraction and on-line post-column ultraviolet irra-
diation and fluorescence detection, J.Chromatogr.B, 1996, 682, 89-94.
SAMPLE
Matrix: blood
Sample preparation: 100 u>L Serum + 200 |xL MeCN, vortex for 30 s, centrifuge at 14000 g for
30 s, inject a 20 u,L aliquot of the supernatant.
HPLC VARIABLES
Column: 150 X 4.6 5 jxm Econosphere CN
Mobile phase: MeCN:water:phosphoric acid 4:100:0.02
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 7
KEYWORDS
comparison with capillary electrophoresis; serum
REFERENCE
Friedberg,M.; Shihabi,Z.K Ketoprofen analysis in serum by capillary electrophoresis, J.Chromatogr.B, 1997,
695, 193-198.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
the supernatant and add it to 200 |xL 4 mg/mL IS in DMF, mix, add 200 JJLL 5 M HCl, extract
mg/mL 1-hydroxybenzotriazole in dichloromethanerpyridine 99:1, add 300 |xL 40 mg/mL l-(3-
ness, reconstitute with 500 |xL mobile phase, inject a 10 (xL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: phenylacetic acid (k' 2.50)
KEYWORDS
REFERENCE
1997, 15, 1765-1774.
SAMPLE
Sample preparation: 250 |xL Microsomal incubation, 150 IJLL ice-cold MeCN and 2.5 ng keto-
profen, centrifuge. Extract the mixture with 4 mL ethyl acetate, centrifuge at 3000 rpm for 10
min, remove the organic fraction and evaporate it under a gentle stream of nitrogen at 40.
Dissolve the residue in 30 jxL MeOH and dilute to 60 |xL with water. Inject a 30 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 \xm RP-18 (Kanto Chemical, Tokyo)
Mobile phase: MeCNiwater 40:60 containing 0.6% acetic acid
Flow rate: 1
Detector: UV 260
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: metabolites, indomethacin
KEYWORDS
liver; pharmacokinetics; ketoprofen is IS
REFERENCE
Nakajima,M.; Inoue,T.; Shimada,N.; Tokudome,S.; Yamamoto,T.; Kuroiwa,Y. Cytochrome P450 2C9 catalyzes
indomethacin O-demethylation in human liver microsomes, Drug Metab.Dispos., 1998, 26, 261-266.
SAMPLE
Matrix: permeate
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 260
OTHER SUBSTANCES
Also analyzed: carbamazepine, fenbufen, indomethacin, a.-naphthoquinone, naproxen, tolmetin
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Mix 50 |xL of a 0.001-5 mM solution in MeCN with 50 uL 1 mM DNS-
APy in MeCN containing 50 mM 2,2'-dipyridyl disulfide and 50 mM triphenylphosphine, let
stand at room temperature for 30 min. Remove a 10 jxL aliquot and dilute it to 100 jxL with
MeCN, inject a 2 |xL aliquot. (Synthesis of DNS-APy, l-(5-dimethylamino-l-naphthalenesul-
fonyl)-(S)-3-aminopyrrolidine, is as follows. Cool a solution of 16.4 g (R)-(+)-l-benzyl-3-pyrrol-
idinol in 164 mL pyridine to +5, add 19.35 g p-toluenesulfonyl chloride, stir at +10 for 48 h,
evaporate to dryness, chromatograph using dichloromethane:acetone 95:5 to obtain (3R)-3-[(4-
tolylsulfonyl)oxy]-l-(phenylmethyl)pyrrolidine (mp 68). Heat a solution of (3R)-3-[(4-tolylsul-
fonyl)oxy]-l-(phenylmethyl)pyrrolidine in 200 mL anhydrous DMF to 65, add 33.5 g sodium
azide (Caution! Sodium azide is highly toxic!), stir at 60 for 7 h, filter, evaporate the filtrate
to dryness under reduced pressure, dissolve the residue in ethyl acetate, wash twice with water,
dry over anhydrous magnesium sulfate, evaporate to obtain (3S)-3-azido-l-(phenylmeth-
yDpyrrolidine as an oil. Add 3.5 g 10% palladium on carbon under nitrogen to a solution of
7.05 g (3S)-3-azido-l-(phenylmethyl)pyrrolidine in 34.8 mL 1 M HCl in water and 245 mL
EtOH, hydrogenate at atmospheric pressure for 30 min, add 3.5 g catalyst, hydrogenate for 2
h, filter, add 34.8 mL 1 M HCl to the filtrate, evaporate to dryness under reduced pressure,
take up the residue in 70 mL EtOH, filter, evaporate the filtrate to dryness under reduced
pressure, repeat this operation twice, crystallize with the minimum amount of EtOH to obtain
(3S)-(+)-3-aminopyrrolidine dihydrochloride (J. Med. Chem. 1992, 35, 4205). (3S)-(+)-Amino-
pyrrolidine dihydrochloride is also reported to be available from Tokyo Kasei. Stir 800 mg (3S)-
(+)-3-aminopyrrolidine dihydrochloride and 2 mL triethylamine in 800 mL MeCN at 0-10, add
a solution of 440 mg dansyl chloride in 80 mL MeCN dropwise, stir in the dark for 30 min,
evaporate to dryness under reduced pressure, dissolve the residue in 200 mL 5% HCl, wash
twice with 40 mL portions of dichloromethane. Adjust the pH of the organic layer to 13-14 with
5% NaOH, extract twice with 10 mL portions of dichloromethane. Combine the organic layers
and wash them with 80 mL water. Dry the organic layer over anhydrous sodium sulfate, evap-
orate to dryness under reduced pressure, dissolve the residue in dichloromethane:MeOH 90:
10, chromatograph on silica gel with dichloromethane:MeOH 90:10. Collect the greenish-yellow
fluorescent band and evaporate it under reduced pressure to obtain DNS-APy as a greenish-
yellow oil.)
HPLCVARIABLES
Column: 150 X 4.6 5 fxm TSK gel ODS-80TM (Tosoh)
Flow rate: 1
Injection volume: 2
CHROMATOGRAM
Retention time: 37 ((SH+)), 40 ((RH-))
OTHER SUBSTANCES
Simultaneous: pranoprofen
KEY WpRDS
REFERENCE
Al-Kindy,S.; Santa,T.; Fukushima,T.; Homma,H.; Imai,K. l-(5-Dimethylammo-l-naphthalenesulphonyl)-(S)-3-
aminopyrrolidine (DNS-Apy) as a fluorescence chiral labelling reagent for carboxylic acid enantiomers,
SAMPLE
Matrix: urine
by aspiration of air. Evaporate an aliquot of 100 (JLL 20 (xg/mL IS in MeOH to dryness at 37.
Add 1 mL urine, vortex, add 250 jxL 1 M pH 5.0 acetate buffer, vortex. Add 250 |xL of the
the eluate to dryness under a stream of nitrogen at 37, reconstitute the residue in 200 uL
HPLCVARIABLES
Flow rate: 0.9
Detector: UV 230
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Extracted: diclofenac, ibuprofen, felbinac, fenbufen, flurbiprofen, loxoprofen, mefenamic acid, na-
KEYWORDS
SPE
REFERENCE
J.Chromatogr.B, 1997, 692, 375-388.
Ketorolac
CAS Registry No,: 74103-06-3, 74103-07-4 (tromethamine)
Merck Index: 5318
Lednicer No.: 4 81
SAMPLE
Matrix: blood
Sample preparation: Dilute 0.1-1 mL plasma to 1 mL with water, add 100 (xL 2 |xg/mL IS in
MeOH:water 90:10, add 100 |xL 500 mM pH 3 sodium acetate buffer, add 6 mL hexane:ethyl
acetate 70:30, vortex vigorously for 5 min. Centrifuge at 2000 rpm for 2-5 min, place in dry
ice/isopropanol or dry ice/MeOH bath to freeze the aqueous layer. Decant the organic layer and
evaporate it to dryness under a stream of nitrogen at 38. Add 500 |xL MeOH:water 90:10 and
3 mL hexane, sonicate for 15 s, vortex vigorously for 3 min, let stand for at least 5 min, discard
the hexane layer. Evaporate the remaining MeOH/water to dryness under a stream of nitrogen
at 38. Add 100 |xL MeOH and 100 |xL mobile phase, sonicate for 30 s, vortex for 30 s, inject a
20 JiL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 4 ^m Nova Pak C18
Mobile phase: MeCN:0.05% phosphoric acid 34:66
Flow rate: 1
Detector: UV 317
CHROMATOGRAM
Retention time: 5.5
Internal standard: RS-37414-000, ()-5-p-fluorobenzoyl-l,2-dihydro-3H-pyrrolo[l,2a]-pyrrole-l-
carboxylic acid (7)
KEYWORDS
REFERENCE
Tsina,L; Chu,R; Kaloostian,M.; Pettibone,M.; Wu,A- HPLC method for the determination of ketorolac in human
plasma, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 957-967.
SAMPLE
Matrix: blood
Sample preparation: Condition a 10 X 3 C18 SPE cartridge (Analytichem) with 2 mL MeOH,
2 mL water, and 2 mL 50 mM pH 3.5 sodium acetate at 2 mL/min. 550 JJLL Plasma + 550 |xL
0/9% NaCl, vortex vigorously, add 25 jxL 10 jjug/mL ketoprofen in MeOH:water 10:90, add 1 mL
to the SPE cartridge at 1 mL/min, wash with 1 mL 50 mM pH 3.5 sodium acetate at 1 mL/
min, wash with 1.5 mL MeOHrO. 1% acetic acid 20:80 at 1.5 mL/min, elute the contents of the
cartridge on to the column with mobile phase.
HPLC VARIABLES
Column: 100 X 8 4 fxm Nova-pak C18 radial pak
Mobile phase: Gradient. MeCN:0.1% acetic acid from 30:70 to 60:40 over 10 min, maintain at
60:40 for 2 min, to 100:0 over 3 min.
Flow rate: 2
Detector: UV 313 for 7.2 min then UV 258
CHROMATOGRAM
Retention time: 6.7
KEYWORDS
plasma; SPE
REFERENCE
Sola,J.; Pruiionosa,J.; Colom,H.; Peraire,C; Obach,R. Determination of ketorolac in human plasma by high-
performance liquid chromatography after automated on-line solid-phase extraction, J.Liq.Chroma-
togr.Rel.TechnoL, 1996, 19, 89-99.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 100 |JLL 600 mM sulfuric acid + 3 mL isooctane:isopro-
panol 95:5, vortex for 30 s, centrifuge at 1800 g for 5 min. Remove the organic layer and
evaporate it to dryness, reconstitute the residue in 100 JJLL 2 mg/mL 4-(dimethylamino)pyridine
in MeCN, add 100 (xL 60 mM trichloroethyl chloroformate in MeCN, add 1 M L-leucinamide
in MeCN, let stand for 2 min, add 500 |xL 250 mM HCl, extract with chloroform. Remove the
organic layer and evaporate it to dryness, reconstitute the residue in mobile phase, inject a
10-100 JJLL aliquot. (A 7% conversion of S to R is observed during the derivatization procedure.
No racemization is observed using a direct procedure with a chiral column.)
HPLC VARIABLES
Mobile phase: MeCN:60 mM KH2PO4itriethylamine 30:70:0.02
Flow rate: 1
Detector: UV 310
CHROMATOGRAM
Internal standard: ketorolac
OTHER SUBSTANCES
Extracted: tiaprofenic acid
KEYWORDS
derivatization; chiral; plasma; ketorolac is IS
REFERENCE
Vakily,M.; Jamali,F. Pharmacokinetics of tiaprofenic acid in humans: Lack of stereoselectivity in plasma using
both direct and precolumn derivatization methods, J.Pharm.Sci., 1996, 85, 638-642.
Ketotifen
Merck Index: 5319
Lednicer No.: 3 239
SAMPLE
Sample preparation: Blood. Dilute 1 mL plasma with 100 |xL 1 M pH 9 phosphate buffer and
100 |JLL water, add 8 mL diethyl ether, extract. Evaporate the organic layer and dissolve the
residue in 400 |JLL mobile phase. Inject 200 |xL aliquot. Tissue. Homogenize the brain with 2
fold the weight of water. Dilute 1500 |xL brain homogenate with 500 |xL 1 M pH 9 phosphate
buffer, add 8 mL diethyl ether, extract. Evaporate the organic layer, dissolve the residue in
400-500 |xL mobile phase. Inject a 200 |xL aliquot.
HPLC VARIABLES
Column: 250 X 6 Intersil ODS-2
Mobile phase: MeCN:0.018% TFA 20:80 (plasma), MeCN:0.018% TFA 15:85 (tissue)
Flow rate: 0.7
Detector: UV 300
CHROMATOGRAM
KEYWORDS
REFERENCE
Kato,M.; Nishida,A.; Aga,Y.; Kita,J.; Kudo,Y; Narita,H.; Endo,T. Pharmacokinetic and pharmacodynamic eval-
1116-1124.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 12 (xm l-m3rristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
famotidine, isothipendyl, metiamide, metoprolol, moxonidine, nadolol, naphazoline, nifenalol,
nizatidine, oxprenolol, pheniramine, phentolamine, pindolol, pizotyline (pizotifen), practolol,
prazosin, promethazine, propranolol, pyrilamine (mepyramine), ranitidine, roxatidine, sotalol,
tiamenidine, timolol, tramazoline, tripelennamine, triprolidine, tymazoline, UK-14,304
REFERENCE
Kaliszan,R.; Nasal,A.; Turowski,M. Binding site for basic drugs on cti-acid glycoprotein as revealed by chemo-
Labetalol
Merck Index: 5341
fold the weight of water. Dilute 1500 |xL brain homogenate with 500 |xL 1 M pH 9 phosphate
buffer, add 8 mL diethyl ether, extract. Evaporate the organic layer, dissolve the residue in
400-500 |xL mobile phase. Inject a 200 |xL aliquot.
HPLC VARIABLES
Column: 250 X 6 Intersil ODS-2
Mobile phase: MeCN:0.018% TFA 20:80 (plasma), MeCN:0.018% TFA 15:85 (tissue)
Flow rate: 0.7
Detector: UV 300
CHROMATOGRAM
KEYWORDS
REFERENCE
Kato,M.; Nishida,A.; Aga,Y.; Kita,J.; Kudo,Y; Narita,H.; Endo,T. Pharmacokinetic and pharmacodynamic eval-
1116-1124.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 12 (xm l-m3rristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
famotidine, isothipendyl, metiamide, metoprolol, moxonidine, nadolol, naphazoline, nifenalol,
REFERENCE
Kaliszan,R.; Nasal,A.; Turowski,M. Binding site for basic drugs on cti-acid glycoprotein as revealed by chemo-
Labetalol
Merck Index: 5341
SAMPLE
Matrix: amniotic fluid, blood, fetal tracheal fluid
Sample preparation: 50-500 |xL Plasma, fetal tracheal fluid, or amniotic fluid + water to 750
(xL total volume, add 500 JJLL pH 9.5 carbonate buffer, add 6 mL ethyl acetate, extract. Remove
the organic layer and extract it with 600 jxL 10 mM phosphoric acid, inject a 60 uX aliquot of
the aqueous layer.
HPLC VARIABLES
Guard column: 10 X 3 Chiral AGP (Regis)
Column: 100 X 4 10 u,m Chiral AGP (Regis)
Mobile phase: 20 mM phosphate buffer containing 15 mM tetrabutylammonium phosphate, de-
gas with helium for 30 min, adjust pH to 7.10 with phosphoric acid
Flow rate: 0.5
CHROMATOGRAM
Retention time: 19 (SR), 23 (SS), 28 (RS), 34 (RR)
KEYWORDS
plasma; sheep; diastereomers; chiral
REFERENCE
Doroudian,A.; Yeleswaram,K.; Rurak,D.W.; Abbott,F.S.; Axelson,J.E. Sensitive high-performance liquid chro-
matographic method for direct separation of labetalol stereoisomers in biological fluids using an cti-acid
glycoprotein stationary phase, J.Chromatogr., 1993, 619, 79-86.
SAMPLE
Matrix: bile, perfusate
Sample preparation: 500 jxL Perfusate or 100 JJLL bile + 1 mL 1 M pH 10.3 carbonate buffer +
5 mL acid-washed diethyl ether, vortex, centrifuge. Remove the organic layer and add it to 125
|JLL 0.5% phosphoric acid, extract, inject a 10 JJLL aliquot of the aqueous layer. (Deconjugate 500
(JLL perfusate with 250 JJLL 8000 U/mL p-D-glucuronidase/aryl sulfatase in 200 mM pH 4.5
sodium acetate buffer, heat at 40 for 1 h, proceed as above.)
HPLC VARIABLES
Column: 100 X 8 4 |xm Novapak phenyl radial compression
Mobile phase: MeCN:water:triethylamine 23:77:1 adjusted to pH 3.6 with concentrated phos-
phoric acid
Flow rate: 3
CHROMATOGRAM
Retention time: 6.9
Internal standard: labetalol
OTHER SUBSTANCES
Extracted: propranolol
KEYWORDS
sheep; liver; labetalol is IS
REFERENCE
Ring,J.A.; Ghabrial,H.; Ching,M.S.; Shulkes,A.; Smallwood,R-A.; Morgan,D.J. Fetal hepatic propranolol metab-
olism. Studies in the isolated perfused fetal sheep liver, Drug Metab.Dispos., 1995, 23, 190-196.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL water + 100 |xL 20% sodium metabisulfite (freshly
prepared) + 1 mL 1 M pH 10.2 sodium carbonate + 8 mL ether, shake gently for 10 min on a
reciprocating shaker, centrifuge at 2000 rpm for 10 min. Remove the organic layer and evap-
orate it to dryness under a stream of nitrogen, reconstitute the residue in 100 |xL mobile phase,
centrifuge for 4 min, inject a 50 jxL aliquot.
HPLC VARIABLES
Mobile phase: MeOH:buffer 50:50 (Buffer was 10 mM potassium phosphate adjusted to pH 3.4
with 5 M HCl.)
CHROMATOGRAM
Retention time: 4.8
Internal standard: labetalol
OTHER SUBSTANCES
KEYWORDS
plasma; labetalol is IS
REFERENCE
Drummer,O.H.; McNeil,J.; Pritchard,E.; Louis,W.J. Combined high-performance liquid chromatographic pro-
cedure for measuring 4-hydroxypropranolol and propranolol in plasma: Pharmacokinetic measurements
following conventional and slow-release propranolol administration, J.Pharm.Sci., 1981, 70, 1030-1032.
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Plasma + 1 mL buffer + 10 mL chloroformrisopropanol 100:2 ,
shake on a horizontal reciprocal shaker at 30 strokes/min for 10 min, centrifuge at 1000 g for
10 min. Remove 8 mL of the organic phase and evaporate it to dryness under a stream of
nitrogen, dissolve the residue in 50 |xL MeCN:MeOH:100 mM HCl 30:20:5, inject a 25 |xL
aliquot. (Buffer was 3.80 g/L sodium bicarbonate and 0.504 g/L sodium carbonate, pH 8.8.)
HPLCVARIABLES
Column: 150 X 6 5 |xm Asahipack ODP 50 (octadecyl-bonded polymer gel)
Mobile phase: MeCN:50 mM pH 11.5 diethylamine 84:16 which was 36 mM in NaCl
Flow rate: 0.9
CHROMATOGRAM
Retention time: 9.5 (RR-SS), 10.5 (RS-SR)
KEYWORDS
plasma; rat; pharmacokinetics; diastereomers
REFERENCE
Grellet,J.; Michel-Gueroult,R; Ducint,D.; Saux,M.C. Sensitive high-performance liquid chromatographic
method for the determination of labetalol diastereoisomers in plasma samples without derivatization,
J.Chromatogr.B, 1994, 652, 59-66.
SAMPLE
Matrix: blood
the residue in 100 JJLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 229
CHROMATOGRAM
KEYWORDS
imipramine; desipramine; levomepromazine; hydroxyzine; ninumic acid; penbutolol; fluvox-
REFERENCE
254-262.
SAMPLE
Matrix: bulk
Sample preparation: 1 mg Labetalol + 3 mg reagent + 100 |xL MeCN + 50 |xL water + 3 |xL
triethylamine, vortex, heat at 60 for 1 h. Remove a 150 |xL aliquot and add it to 400 |xL MeCN,
vortex, inject a 5 |JLL aliquot. (Prepare the reagent, (4S-cis)-2,2-dimethyl-5-isothiocyanato-4-
phenyl-l,3-dioxane (PHEDIT), as follows. Add dropwise 5 g (4S,5S)-(+)-5-amino-2,2-dimethyl-
4-phenyl-l,3-dioxane in 150 mL dichloromethane to 5 g l,l'-thiocarbonyldiimidazole stirred in
150 mL dichloromethane, stir at room temperature for 3 h, wash the reaction mixture three
times with 300 mL portions of 5% sodium bicarbonate, wash three times with 300 mL portions
of water. Dry the organic layer over anhydrous sodium sulfate for 20 min, evaporate to dryness
under reduced pressure, recrystallize from EtOH to give (4S-cis)-2,2-dimethyl-5-isothiocyanato-
4-phenyl-l,3-dioxane as a pale yellow solid (mp 105-6).)
HPLC VARIABLES
Column: 150 x 3.9 4 |xm Nova-Pak C18
Mobile phase: MeOH:20 mM pH 4.60 (NH4)H2PO4 63:37
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 19.7 (RS or SR), 21.4 (RS or SR), 23.8 (SS), 30.0 (RR)
KEYWORDS
derivatization; chiral; comparison with other derivatization reagents
REFERENCE
Desai,D.M.; Gal,J. Reversed-phase high-performance liquid chromatographic separation of the stereoisomers
of labetalol via derivatization with chiral and non-chiral isothiocyanate reagents, J.Chromatogr., 1992,579,
165-171.
SAMPLE
Matrix: bulk
Sample preparation: 1 mg Labetalol + 3 mg 1-naphthalenemethyl isothiocyanate (Trans World
Chemicals, Chevy Chase MD) + 100 JJLL MeCN + 50 |xL water + 3 |xL triethylamine, vortex,
heat at 60 for 1 h. Remove a 150 |xL aliquot and add it to 400 |xL MeCN, vortex, inject a 5
JULL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 4 jjim Nova-Pak C18
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 10.42 (RS/SR), 14.73 (SS/RR)
KEY WpRDS
derivatization; comparison with other derivatization reagents
REFERENCE
Desai,D.M.; Gal,J. Reversed-phase high-performance liquid chromatographic separation of the stereoisomers
of labetalol via derivatization with chiral and non-chiral isothiocyanate reagents, J.Chromatogr., 1992,579,
165-171.
SAMPLE
Sample preparation: Urine. Condition a Bond Elut Certify LRC SPE cartridge with 6 mL water.
Mix 3.75 mL urine with 750 JLJLL 1 M pH 9.0 borate buffer, centrifuge at 734 g for 5 min. Add
a 3 mL aliquot to the SPE cartridge. Wash with 2 mL water, 1 mL 100 mM pH 4.0 acetate
buffer, and 2 mL MeOH using vacuum (5 mmHg). Dry the SPE cartridge under vacuum (<150
mmHg) for 5 min. Elute with 2 mL 2% ammonia in chloroform:isopropanol 60:40 using vacuum
(2 mmHg) (Caution! Chloroform is a carcinogen!). Evaporate the eluate to dryness under a
gentle stream of nitrogen at 60. Dissolve the residue in 1 mL mobile phase, inject an aliquot.
Tablets. Crush and mix tablets to a fine powder, weigh, dissolve in water, shake for 20 min,
filter (Whatman No. 41 filter-paper), wash, make up to a fixed volume. Dilute an aliquot with
mobile phase and inject an aliquot.
HPLC VARIABLES
Guard column: jx-Bondapak C18
Column: 250 X 4.6 5 jmm Supelcosil ABZ + Plus
Mobile phase: MeCN:water 30:70 containing 5 mM acetate buffer adjusted to pH 4.5 with acetic
acid or 1 M KOH
Flow rate: 1
Detector: E, PAR Model 400, glassy carbon cell + 1.3 V, Ag/AgCl reference electrode, platinum
auxiliary electrode in the DC mode with 5-s low-pass filter time constant, current range 20-
10OnA
CHROMATOGRAM
Limit of detection: 5 ng/mL (solutions), 10 ng/mL (urine)
Limit of quantitation: 20 /xg/mL (urine)
KEYWORDS
tablets; SPE
REFERENCE
Ceniceros,C; Maguregui,M.L; Jime*nez,R.M.; Alonso,R.M. Quantitative determination of the (3-blocker labetalol
in Pharmaceuticals and human urine by high-performance liquid chromatography with amperometric de-
tection, J.Chromatogr.B, 1998, 705, 97-103.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: MeOH:buffer 40:60 (Buffer was pH 4.0 phosphate buffer (ionic strength = 0.1)
containing 3.33 mM N,N-dimethyloctylamine, pH readjusted to 4.00 with 85% phosphoric acid.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: bisoprolol, carvedilol, metipranolol, oxprenolol, talinolol, toliprolol
REFERENCE
Hamoir,T.; Verlinden,Y.; Massart,D.L. Reversed-phase liquid chromatography of (3-adrenergic blocking drugs
in the presence of a tailing suppressor, J.Chromatogr.Sci., 1994, 32, 14-20.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: MeCN:40 mM pH 4.3 acetate buffer 75:25
Flow rate: 1.5
Detector: UV 278
OTHER SUBSTANCES
Extracted: dextromethorphan
REFERENCE
Abdel-Moety,E.M.; Al-Deeb,O.A.; Khattab,N.A. Determination of dextromethorphan hydrobromide in bulk form
and dosage formulations by high-performance liquid chromatography, J.Liq.Chromatogr., 1995, 18, 4127-
4134.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1
Detector: UV 308
CHROMATOGRAM
Retention time: 23, 25, 28, 33 (diastereomers)
KEYWORDS
chiral
REFERENCE
J.Liq.Chromatogr., 1995, 18, 649-671.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 jjim Supelcosil LC-DP (A) or 250 X 4 5 jxm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
droxychloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketopro-
fen, ketorolac, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol,
mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, meth-
adone, methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine, meth-
yldopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam,
moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedipine, ni-
zatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxetine, pe-
moline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, phenobarbital,
zide, pindolol, piroxicarn, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine,
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: tissue
Sample preparation: Weigh out brain tissue and homogenize in 4 volumes 400 mM perchloric
acid using a Tamson motor-driven PTFE/glass homogenizer at 1400 rpm to give a final tissue
concentration of 25 mg/mL in the perchloric acid. For each 1 mL of homogenate add 40 jxL 1.48
(jug/mL propranolol in 200 mM sulfuric acid, centrifuge at 3000 g for 15 min. Remove 1 mL
supernatant and add it to 10 |xL 10 M NaOH and 350 |xL buffer, vortex for 10 s, add 8 mL
diethyl ether, shake mechanically for 45 min, centrifuge at 2000 g for 8 min. Remove the
organic layer and add it to 200 |xL 200 mM sulfuric acid, shake mechanically for 15 min,
centrifuge at 2000 g for 8 min. Remove the aqueous layer and heat it at 45 for 1 h to remove
traces of ether, inject a 50 JULL aliquot. (Buffer was 90 g sodium carbonate and 32 g potassium
carbonate in 1 L, pH 9.0.)
HPLC VARIABLES
Guard column: 20 X 4.6 5 \LVCL LC-18-DB (Supelchem)
Column: 250 X 4.6 5 |xm LC-18-DB (Supelchem)
Mobile phase: MeCN:50 mM NaH2PO4:triethylamine 35:65:0.1, adjusted to pH 3.0 with ortho-
phosphoric acid
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 5.5
Internal standard: propranolol (7.3)
OTHER SUBSTANCES
Simultaneous: clenbuterol
KEYWORDS
rat; brain
REFERENCE
Botterblom,M.H.A.; Feenstra,M.G.R; Erdtsieck-Ernste,E.B.H.W. Determination of propranolol, labetalol and
clenbuterol in rat brain by high-performance liquid chromatography, J.Chromatogr., 1993, 613, 121-126.
SAMPLE
Matrix: urine
HPLC VARIABLES
Column: A 10 X 4.6 5 |xm Spherisorb cyanopropyl; B 250 X 4.6 Capcell Pak C18 UG-120
(Shiseido)
Mobile phase: A water; B Gradient. MeCNibuffer from 3:97 to 30:70 over 30 min, to 40:60 over
8 min (Buffer was 3.4 mL/L phosphoric acid adjusted to pH 3.0 with 5 M NaOH.)
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: acebutolol, alprenolol, amphetamine, atenolol, bopindolol, codeine, ephedrine, meto-
prolol, morphine, nadolol, oxprenolol, pindolol, propranolol, timolol
KEYWORDS
column-switching
REFERENCE
Saarinen,M.T.; Sir6n,H.; Riekkola,M.-L. Screening and determination of fJ-blockers, narcotic analgesics and
1995, 664, 341-346.
Lacidipine
Merck Index: 5344
Lednicer No.: 5 82
SAMPLE
Matrix: blood
Sample preparation: Condition a 500 mg Bondelut C18 SPE cartridge with 3 mL MeOH and
2 mL MeCN:water 50:50. 3 mL Plasma + 3 mL MeCN, centrifuge, add the supernatant to the
SPE cartridge, wash with 2 mL MeCN:water 50:50, wash with 1.5 mL basic washing solution,
wash with 1.5 mL acid washing solution, wash with 2 mL MeCN:water 50:50, elute with two
1.5 mL portions of MeCN. Evaporate the eluate, reconstitute in 100 |xL mobile phase, store at
4, inject an 82 |JLL aliquot. (Basic washing solution was MeCN:water:33% ammonia 10:88:2.
Acid washing solution was MeCN:water:88% orthophosphoric acid 10:89:1.)
HPLCVARIABLES
Guard column: 30 X 4.6 30-40 |xm RP-8 (Merck)
Flow rate: 1
CHROMATOGRAM
Retention time: 4.5
KEYWORDS
plasma; human; dog; rat; horse; protect from light; pharmacokinetics; SPE; automation of this
procedure reported (see J. Chromatogr. B 1995; 669; 383)
REFERENCE
Pellegatti,M.; Braggio,S.; Sartori,S.; Franceschetti,E; Bolelli,G.F. Validation of a high-performance liquid chro-
matographic-radioimmunoassay method for the determination of lacidipine in plasma, J.Chromatogr., 1992,
573, 105-111.
Lactulose
Merck Index: 5360
SAMPLE
Matrix: beverages, juice, milk
Sample preparation: Orange juice. Dilute orange juice 100-fold with water, filter (Millipore HV,
0.45 (xm), dilute nitrate 10-fold, inject an aliquot. Beverages. Dilute soft drinks 1000-fold with
water, inject an aliquot. Milk. Dilute 5 mL milk to 100 mL with mobile phase, filter (Millipore
HV, 0.45 |xm), dilute nitrate 50-fold, inject an aliquot.
HPLC VARIABLES
Guard column: 30 X 4.6 Cation H (Bio-Rad)
Column: 300 X 3.8 9 jutm HPX 87-H Aminex (Bio-Rad)
Mobile phase: 10 mM Sulfuric acid
Flow rate: 0.5
Detector: E following post-column reaction, Hewlett-Packard 1049A programmable electrochem-
ical detector, Metrohm detector cell, cuprous oxide working electrode +550 mV, glassy carbon
auxiliary electrode, Ag/AgCl (3 M KCl) reference electrode. The column effluent mixed with
200 mM NaOH pumped at 0.4 mL/min, the mixture flowed through a 220 X 0.8 single-bead
string reactor packed with 0.6 mm glass beads to the detector. (Prepare cuprous oxide electrode
as follows. Stir 300 mg conductive carbon cement (Gerhard Neubauer, Miinster), 60 mg cuprous
oxide (Fluka), and 300 |xL acetone until a thick paste forms as the acetone evaporates. Pack
conductive carbon cement into the base of a 3 mm diameter cavity carbon paste electrode base
(Metrohm), allow to dry, polish with dry emery paper (grade 2/0, Oakey), remove surface layer
with an acetone-soaked tissue, pack the paste into the cavity, allow to dry overnight, polish
with dry emery paper (grade 2/0), 3 (xm imperial micro finishing film sheet (3M), 0.3 |xm
imperial micro finishing film sheet (3M), and 0.05 |xm alumina particles on a Buehler pad,
sonicate for 2 min in water (Anal. Chim. Acta 1995, 300, 5).)
CHROMATOGRAM
Limit of detection: 2 \xM
OTHER SUBSTANCES
Also analyzed: arabinose, cellobiose, dextrose, fructose, fucose, galactitol, galactose, galacturonic
acid, lactose, lyxose, maltose, mannitol, mannose, myo-inositol, raffinose, rhamnose, ribose,
sorbose, sucrose, xylose
KEYWORDS
orange juice; soft drinks; post-column reaction; fruit
REFERENCE
Huang,X.; Pot,J.J.; Kok,W.T. Determination of sugars by liquid chromatography and amperometric detection
with a cuprous oxide modified electrode, Chromatographia, 1995, 40, 684-689.
SAMPLE
Sample preparation: Urine. Dilute urine 1:10 to 1:40 with water, add a 1 mL aliquot to 1 mL
250 jxg/mL melibiose, add Amberlite IR-120 H+ to occupy one third of the volume, inject a 25
(JLL aliquot of the supernatant. Plasma. 200 fxL Plasma + 200 |xL 250 |xg/mL melibiose, mix,
add 200 u,L ice cold 35 mg/mL 5-sulfosalicylic +acid, let stand on ice for 20 min, centrifuge at
9000 g for 5 min, mix with Amberlite IR-120 H :Amberlite IRA 400 Cl' 40:60, centrifuge, inject
a 25 JJIL aliquot of the supernatant.
HPLC VARIABLES
Guard column: Carbopac PA-I (Dionex)
Column: 250 X 40 Carbopac PA-I (Dionex)
Mobile phase: 120 mM NaOH containing 0.5 mM zinc acetate (urine) or 160 mM NaOH con-
taining 0.675 mM zinc acetate (plasma) (At the end of each plasma sample wash with 1 M
NaOH for 4 min.)
Flow rate: 1
Detector: E, Dionex pulsed electrochemical detector, detection potential -0.01 V (0-0.5 s), oxi-
dation potential +0.75 V (0.51-0.64 s), reduction potential -0.75 V (0.65-0.75 s), integration
period 0.05-0.5 s
CHROMATOGRAM
Internal standard: melibiose (4.0 (plasma), 4.6 (urine))
OTHER SUBSTANCES
Extracted: mannitol, 3-O-methylglucose, dextrose
KEYWORDS
plasma
REFERENCE
Fleming,S.C; Kynaston,J.A.; Laker,M.R; Pearson,A.D.J.; Kapembwa,M.S.; Griffin,G.E. Analysis of multiple
sugar probes in urine and plasma by high-performance anion-exchange chromatography with pulsed elec-
trochemical detection. Application in the assessment of intestinal permeability in human immunodeficiency
virus infection, J.Chromatogr., 1993, 640, 293-297.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: water
Flow rate: 0.5
Detector: RI
CHROMATOGRAM
Retention time: 19
OTHER SUBSTANCES
Simultaneous: arabinose, dextrose, fructose, galactose, glycerol, lactose, mannitol, pullulan P-
REFERENCE
SAMPLE
Matrix: urine
Sample preparation: Condition a 600 mg Maxi-Clean C18 SPE cartridge with 5 mL MeOH and
5 mL water. Add 2-3 mL urine to the SPE cartridge and pass it through the cartridge. Discard
the first 1 mL, collect the residual volume and dilute it 1:1 with water. Dilute a 200 JJLL aliquot
with 1.8 mL water containing 75 |xm/mL cellobiose, add 400 g/L Amberlite IRA-400 resin Cl"
form (Fluka). Vortex for 10 s, centrifuge at 3000 g for 2 min. Filter (Micro-spin centrifuge
cartridge, Nylon 66, 0.2 |xm, Alltech) a 400 |xL aliquot of the supernatant while centrifuging
at 3000 g for 5 min. Inject a 50 jxL aliquot.
HPLCVARIABLES
Guard column: Benson Carbohydrate BC-100 Ca2+ (Alltech)
Column: 300 X 6.5 10 |xm Alltech 700 CH Carbohydrate (Alltech)
Mobile phase: Water
Flow rate: 0.5
Detector: ELSD, Varex MKIII (Alltech), drift tube temperature 120, carrier gas flow (air) 41.67
cm3/s
CHROMATOGRAM
Internal standard: cellobiose (7.55)
OTHER SUBSTANCES
Extracted: dextrose, mannitol
Simultaneous: fructose, galactose
KEYWORDS
SPE; pharmacokinetics
REFERENCE
Marsilio,R.; D'Antiga,L.; Zancan,L.; Dussini,N.; Zaccello,F. Simultaneous HPLC determination with light-scat-
tering detection of lactulose and mannitol in studies of intestinal permeability in pediatrics, Clin.Chem.,
1998, 44, 1685-1691.
SAMPLE
Matrix: urine
Sample preparation: Dilute urine 2.5-20 fold with water, add a 1 mL aliquot to 1 mL water
containing 250 |xg/mL arabinose and 25 |xg/mL cellobiose, add 0.5 g washed Amberlite IR-120
H:Amberlite IRA400 Cl 40:60, vortex, centrifuge, filter (0.2 jjum), inject a 50 |xL aliquot of the
supernatant.
HPLCVARIABLES
Column: 250 X 40 HPIC-AS6 (Dionex)
Mobile phase: 150 mM NaOH
Flow rate: 1
Detector: E, Dionex pulsed electrochemical detector, gold working electrode, detection potential
-0.05 V, oxidation potential +0.6 V, reduction potential -0.95 V, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 6
Internal standard: arabinose (4), cellobiose (9)
OTHER SUBSTANCES
Extracted: mannitol
REFERENCE
Fleming,S.C; Kapembwa,M.S.; Laker,M.R; Levin,G.E.; Griffin,G.E. Rapid and simultaneous determination of
lactulose and mannitol in urine, by HPLC with pulsed amperometric detection, for use in studies of intes-
tinal permeability, Clin.Chem., 1990, 36, 797-799.
SAMPLE
Matrix: urine
Sample preparation: Centrifuge, dilute 10-20 fold with water, inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 300 X 6.5 Sugar Pak I cation-exchange in calcium form
Mobile phase: Water containing 1 mL/L of a 50 g/L calcium EDTA solution
Flow rate: 0.5
Detector: RI
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Extracted: mannitol
REFERENCE
Willems,D.; Cadranel,S.; Jacobs,W. Measurement of urinary sugars by HPLC in the estimation of intestinal
permeability: evaluation in pediatric clinical practice, Clin.Chem., 1993, 39, 888-890.
SAMPLE
Matrix: urine
Sample preparation: 2 mL Urine + 500 mg washed and mixed Duolite ion-exchange resin
(BDH), vortex for 10 s, centrifuge at 3000 g for 10 min, filter (0.2 um) the supernatant, inject
an aliquot.
HPLC VARIABLES
Guard column: Direct-Connect polymeric guard column (Alltech)
Column: 250 X 4.6 5 |xm Kromasil NH2 (Alltech)
Flow rate: 1
Detector: RI
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Extracted: mannitol, L-rhamnose, urea
REFERENCE
Miki,K.; Butler,R.; Moore,D.; Davidson,G. Rapid and simultaneous quantification of rhamnose, mannitol, and
lactulose in urine by HPLC for estimating intestinal permeability in pediatric practice, Clin.Chem., 1996,
42, 71-75.
SAMPLE
Matrix: urine
Sample preparation: 10 |xL Urine + 200 |xL reagent, heat at 65 for 16 h, cool to room temper-
ature, inject a 5 |xL aliquot of the clear supernatant. (Prepare reagent by dissolving 5 mg Fmoc-
hydrazine in 1 mL MeCN, add 10 JJLL buffer. Buffer was 1.44 M formic acid containing 600 mM
NaOH. Prepare Fmoc-hydrazine as follows. Dissolve 1 g 9-fluorenylmethyl chloroformate in
100 mL EtOH, add this solution dropwise with stirring to 10 mL hydrazine hydrate (Caution!
Hydrazine hydrate is a carcinogen!), stir for 30 min, filter off the precipitate, wash it twice
with 20 mL portions of ice-cold EtOH, dry at room temperature.)
HPLCVARIABLES
Guard column: 10 X 4.6 3 |xm Spherisorb ODS II
Mobile phase: Gradient. Isopropanolrisobutyl alcoholrwater 6:6:88 for 13 min, to 80:0:20 (step
gradient), maintain at 80:0:20 for 6 min, re-equilibrate at initial conditions.
Injection volume: 5
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Extracted: 3-O-methyl-D-glucose, rhamnose, xylose
KEYWORDS
derivatization
REFERENCE
Rooyakkers,D.R.; van Eijk,H.M.H.; Deutz,N.E.R Simple and sensitive multi-sugar-probe gut permeability test
by high-performance liquid chromatography with fluorescence labelling, J.Chromatogr.A, 1996, 730, 99-
105.
Lamivudine
Molecular formula: C8H1, N3O3S
Merck Index: 5365
Lednicer No.: 5 99
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Cyclobond I acetyl
Mobile phase: 0.2% Triethylamine in water, adjusted to pH 7.2 with glacial acetic acid
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 6.3 (-), 6.7 (+)
KEYWORDS
chiral
REFERENCE
Coates,J.A.; Cammack,N.; Jenkinson,H.J.; MuttonJ.M.; Pearson,B.A.; Storer,R.; Cameron,J.M.; Penn,C.R. The
separated enantiomers of 2'-deoxy-3'-thiacytidine (BCH 189) both inhibit human immunodeficiency virus
replication in vitro, Antimicrob.Agents Chemother., 1992, 36, 202-205.
Lamotrigine
Molecular formula: C9H7CI2N5
CAS Registry No.: 84057-84-1 or 84507-84-1 (?)
Merck Index: 5367
Lednicer No.: 4 120
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Serum + 100 JJLL 30 mg/L IS in water + 200 |xL 25% saturated
ammonium acetate, mix. Add the sample to the reservoir of a primed 4 mm/1 mL Empore C8
SPE disk cartridge suspended in a test tube (16 X 100 mm). Force the liquid then 500 |xL
water through the disk by centrifuging at 100-120 g for 5 min. Suspend disk cartridge in a
tube, elute the drug with 100 JJLL MeCN and 300 |xL water. Combine the eluates, inject a 50
IxL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Zorbax Stable-Bond CN
Mobile phase: MeCN:MeOH:acetic acid:triethylamine: water 15:12.5:0.1:0.06:72.5 (Connect a
250 X 4.6 column dry packed with 37-53 |xm silica gel (Whatman) as a mobile-phase saturating
column between the pump and the injector .)
Flow rate: 1.2
Detector: UV 214
CHROMATOGRAM
Retention time: 5.5
Internal standard: cyheptamide (14)
OTHER SUBSTANCES
Extracted: carbamazepine, carbamazepine diol, carbamazepine epoxide, 5-(p-hydroxyphenyl)-5-
phenylhydantoin, phenytoin
Simultaneous: acetaminophen, N-acetylprocainamide, amikacin, caffeine, chlordiazepoxide,
clonazepam, desmethylchlordiazepoxide, desmethyldiazepam, diazepam, digoxin, disopyram-
ide, erythromycin, ethosuximide, felbamate, flurazepam, gabapentin, gentamicin, lidocaine,
methotrexate, nitrazepam, oxazepam, phenylethylmalonamide, phenobarbital, primidone,
quinidine, salicylate, temazepam, theophylline, tobramycin, valproic acid, vancomycin
KEYWORDS
serum; SPE
REFERENCE
Lensmeyer,G.L.; Gidal,B.E.; Wiebe,D.A. Optimized high-performance liquid chromatographic method for de-
termination of lamotrigine in serum with concomitant determination of phenytoin, carbamazepine, and
carbamazepine epoxide, Ther.Drug Monit., 1997, 19, 292-300.
SAMPLE
Matrix: blood
Sample preparation: Mix 550 |xL plasma with 100 |xL 500 mM monochloroacetic acid in 500
mM pH 2.5 ammonium orthophosphate buffer. Dialyze two 300 |xL aliquots against 5 mM pH
7.0 potassium phosphate buffer pumped at 0.25 mL/min for 4 min each using a 15 kD Cupro-
phan membrane. The dialysate passed through column A and was washed through with 500
JJLL 1 mM pH 7.0 potassium phosphate buffer. Elute the glucuronide metabolite from column
A onto column B with mobile phase. After 30 s remove column A from the circuit, elute column
B with mobile phase, elute column A with 200 |xL MeCNiwater 10:90 and 500 (xL 1 mM pH
7.0 potassium phosphate buffer to waste, at the 4 min mark elute lamotrigine and the meth-
ylated metabolite from column A onto column B with mobile phase, elute column B with mobile
phase, monitor the effluent from column B. (At the end of the process flush the donor channel
with 1.5 mL 1 mM pH 7.0 potassium phosphate buffer and flush column A with 500 IJLL 5 mM
pH 7.0 potassium phosphate buffer.)
HPLCVARIABLES
Column: A 70 mg 10 |xm Hypersil ODS (Shandon) in a Prelute cartridge; B 150 X 4.6 5 (xm
Kromasil C8 (Technicol, UK)
Mobile phase: Gradient. A was 50 mM pH 4.15 ammonium phosphate buffer containing 20 mM
diethylamine hydrochloride. B was MeCN:500 mM pH 4.15 ammonium phosphate buffer con-
taining 20OmM mM diethylamine hydrochloride buffer:water 60:10:30. A:B 86:14 for 3 min, to
69:31 over 0.2 min, maintain at 69:31 for 10.2 min, to 0:100 over 0.1 min, maintain at 0:100
for 1.5 min, return to 86:14 over 0.2 min, re-equilibrate at initial conditions for 3.8 min
Flow rate: 1.5
Detector: UV 270 for 12.5 min, then UV 215
CHROMATOGRAM
Retention time: 9.6
OTHER SUBSTANCES
KEYWORDS
plasma; column-switching; dialysis
REFERENCE
Cooper, J.D.H.; Shearsby,N.J.; Taylor,J.E.; Fook Sheung,C.T.C. Simultaneous determination of lamotrigine and
its glucuronide and methylated metabolites in human plasma by automated sequential trace enrichment
of dialysates and gradient high-performance liquid chromatography, J.Chromatogr.B, 1997, 702, 227-233.
SAMPLE
Matrix: blood
Sample preparation: Add 1.5 mL MeCN to 500 |xL serum, centrifuge, evaporate the superna-
tant to dryness, redissolve the residue in 200 u-L water. Inject onto column A, wash with MeCN:
water 10:90 or MeOH:water 20:80 for 20 min, backflush the contents of column A onto column
B with mobile phase, elute with mobile phase, monitor the effluent from column B.
HPLCVARIABLES
Column: A 25 X 4 25 urn pore diameter 6 nm LiChrospher RP-18 ADS (Merck); B 125 X 4 5 |xm
endcapped LiChroCART HPLC-cartridge RP-18 (Merck)
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 3.1
OTHER SUBSTANCES
Extracted: oxprenolol
KEYWORDS
REFERENCE
Oertel,R.; Richter,K; Gramatte",T.; Kirch,W. Determination of drugs in biological fluids by high-performance
liquid chromatography with on-line sample processing, J.Chromatogr.A, 1998, 797, 203-209.
SAMPLE
Matrix: blood
Sample preparation: Add an equal volume of 1 |xg/mL IS in MeCN to 0.2-1 mL plasma, vortex
briefly, add excess sodium carbonate, vortex to form a saturated solution, centrifuge at 6 at
1500 g for 10 min. Remove the MeCN supernatant (top layer) and dilute it by half with water,
HPLC VARIABLES
Column: 250 X 4 5 |xm LiChrospher 100CN
Mobile phase: MeCN: 10 mM pH 3.5 ammonium acetate buffer 55:45
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Retention time: 10
Internal standard: 3,5-diamino-6-(2-methoxyphenyl)-l,2,4-triazine(A725C) (12)
OTHER SUBSTANCES
Noninterfering: carbamazepine, clonazepam, phenobarbital, phenytoin, valproic acid
KEYWORDS
plasma
REFERENCE
Cociglio,M.; Alric,R.; Bouvier,O. Performance analysis of a reversed-phase liquid chromatographc assay of
lamotrigine in plasma using solvent-demixing extraction, J.Chromatogr., 1991, 572, 269-276.
SAMPLE
Matrix: blood
Sample preparation: 300 |xL Plasma + 50 |xL 1.7 M perchloric acid, vortex for 20 s, centrifuge
at 1500 g for 4 min, add 35 (xL 4 M K2HPO4, shake gently, allow to settle, inject a 50 |xL aliquot
of the supernatant.
HPLC VARIABLES
Column: 250 X 4.6 5 (xm Ultrasphere C18
Flow rate: 1.5
Detector: UV 306
CHROMATOGRAM
Retention time: 4.5
Limit of quantitation: 1.2 (xM
OTHER SUBSTANCES
Noninterfering: anticonvulsant drugs
KEYWORDS
plasma
REFERENCE
Boutagy,J.; Dell'Anna,M. Simplified and rapid HPLC procedure for analysis of lamotrigine in plasma (Abstract
107), Ther.DrugMonit, 1995, 27, 410-410.
SAMPLE
Matrix: blood
Sample preparation: 200 |iL Serum + 100 |xL 10 jjug/mL IS in MeOH + 200 |JLL 2 M NaOH + 1
mL ethyl acetate, vortex for 1 min, centrifuge for 5 min. Remove the organic layer and evap-
orate it to dryness under a stream of nitrogen, reconstitute the residue in 100 |xL MeOH, inject
a 25 (xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm RP-8 (Brownlee/Applied Biosystems)
Mobile phase: MeCN:water:buffer 20:79:1 (The buffer was 20.7 g NaH 2 PO 4 -H 2 O and 14.2 g
Na 2 HPO 4 in 500 mL water, pH 6.5.)
Flow rate: 1.6
Detector: UV 306
CHROMATOGRAM
Retention time: 7.2
Internal standard: 6-(2-methoxyphenyl)-l,2,4-triazine-3,5-diamine(BWC430C78) (4)
OTHER SUBSTANCES
Simultaneous: carbamazepine
Noninterfering: N-acetylprocainamide, ethosuximide, phenobarbital, phenytoin, primidone, pro-
cainamide, quinidine, theophylline, valproic acid
KEYWORDS
serum
REFERENCE
Fraser,A.D.; MacNeil,W.; Isner,A.R; Camfield,P.R. Lamotrigine analysis in serum by high-performance liquid
chromatography, Ther.Drug Monit., 1995, 17, 174-178.
SAMPLE
Matrix: blood
Sample preparation: 50 u-L Plasma + 100 jxL MeCN, centrifuge, inject a 5 |xL aliquot of the
supernatant.
HPLCVARIABLES
Column: 100 X 4.6 3.5 |xm Zorbax SB
Flow rate: 1.5
Injection volume: 5
Detector: UV 310
CHROMATOGRAM
Retention time: 1.9
Limit of detection: <1 |xM
OTHER SUBSTANCES
Extracted: carbamazepine (UV 220), carbamazepine epoxide (UV 220), hydroxycarbamazepine
(UV 220), oxcarbazepine (UV 220), phenobarbital (UV 220), phenytoin (UV 220)
Also analyzed: ibuprofen, naproxen, trimethoprim
KEYWORDS
plasma
REFERENCE
Lessing,U.; Vielemeyer,O.; Heilmann,P.; Schoneshofer,M. Routine determination of serum primidone levels with
a fully automated liquid chromatographic method: Comparison with an immuno-assay-technique (Abstract
100), Ther.Drug Monit, 1995, 17, 408.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond-Elut SPE cartridge with two 1 mL portions of
MeOH and with two 1 mL portions of buffer. 100 |xL Serum + 50 |iL 6 u,g/mL acetanilide in
MeOH:water 50:50 + 800 ^L buffer, mix, add to the SPE cartridge, wash with 1 mL buffer,
elute with 250 |xL MeOH, inject a 40 |xL aliquot. (Buffer was 10 mM pH 3.5 phosphate buffer
containing 5 mM sodium octanesulfonate.)
HPLC VARIABLES
Column: 125 X 4 4 |xm ODS LiChroCART C18
Mobile phase: MeCNrbuffer 27:73 (Buffer was 10 mM pH 3.5 phosphate buffer containing 5 mM
Flow rate: 1
Detector: UV 265
CHROMATOGRAM
Internal standard: acetanilide (3.4)
OTHER SUBSTANCES
Simultaneous: carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, primidone,
zonisamide
Noninterfering: valproic acid
KEYWORDS
serum; SPE
REFERENCE
Yamashita,S.; Furuno,K; Kawasaki,H.; Gomita,Y; Yoshinaga,H.; Yamatogi,Y.; Ohtahara,S. Simple and rapid
analysis of lamotrigine, a novel antiepileptic, in human serum by high-performance liquid chromatography
using a solid-phase extraction technique, J.Chromatogr.B, 1995, 670, 354-357.
SAMPLE
Sample preparation: Tablets. Powder tablets, weigh out amount corresponding to 10.9 mg la-
motrigine, add 100 mL MeOH, sonicate for 5 min, centrifuge an aliquot at 3500 g for 15 min.
Remove a 5 mL aliquot of the supernatant and make up to 25 mL with mobile phase. Remove
a 4 mL aliquot of this solution and add it to 5 mL 5.5 (xg/mL in mobile phase, make up to 25
mL with mobile phase, inject a 20 |xL aliquot. Plasma. Condition a 3 mL 200 mg Bond Elut
C8 SPE cartridge with 1 volume of MeOH and 1 volume of water. 40 JJLL Plasma + 200 JJLL 1.1
jxg/mL IS in MeOH + 80 ixL MeCN, centrifuge at 3500 g for 15 min. Remove the supernatant
and evaporate it so as to remove the organic solvents under a stream of nitrogen at 45, add
the residue to the SPE cartridge, wash with 2 volumes of water, elute with 1 volume of 10 mM
HCl in MeCN. Evaporate the eluate to dryness under a stream of nitrogen at 45, reconstitute
the residue in 200 |xL mobile phase, inject a 20 fxL aliquot. Urine. Condition a 3 mL 200 mg
Bond Elut C8 SPE cartridge with 1 volume of MeOH and 1 volume of water. 100 |xL Urine +
200 |xL 1.1 ixg/mL IS in MeOH + 200 |xL MeCN, centrifuge at 3500 g for 15 min. Remove the
supernatant and evaporate it so as to remove the organic solvents under a stream of nitrogen
at 45, add the residue to the SPE cartridge, wash with 2 volumes of water, elute with 1 volume
of 10 mM HCl in MeCN. Evaporate the eluate to dryness under a stream of nitrogen at 45,
reconstitute the residue in 200 (xL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Lichrosorb RP-8
Mobile phase: MeCN:buffer 28:72 (Buffer was 56 mL 1 M acetic acid and 50 mL 1 M NaOH
made up to 1 L with water, pH 5.6.)
Flow rate: 1
Detector: UV 306
CHROMATOGRAM
Retention time: 6.5
Internal standard: 5-diamino-6-(2-methoxyphenyl)-l,2,4-triazine(BW725C78) (3.5)
Limit of detection: 1.2 ng (urine), 1.1 ng (plasma)
Limit of quantitation: 3.0 ng (urine), 2.8 ng (plasma)
KEYWORDS
plasma; tablets; SPE
REFERENCE
Papadoyannis,I.N.; Zotou,A.C; Samanidou,V.F. Solid-phase extraction study and RP-HPLC analysis of lamo-
trigine in human biological fluids and in antiepileptic tablet formulations, J.Liq.Chromatogr., 1995, 18,
2593-2609.
SAMPLE
Sample preparation: Whole blood. Condition a 100 mg C18 SPE cartridge (Burdick and Jack-
son/Baxter) with 2 mL MeOH, 2 mL water, and 1 mL 50 mM pH 1.2 phosphoric acid buffer.
250 |xL Whole blood + 750 (xL pH 1.2 phosphoric acid buffer containing 15 mM sodium dodecyl
sulfate + 50 |xL 30 |xg/mL IS in MeOHiwater 50:50, vortex for 15 s, centrifuge at 13000 g for
7 min, add the supernatant to the SPE cartridge, wash with 300 |xL 50 mM phosphoric acid
buffer, elute with 1 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen
below 40, reconstitute the residue in 250 jxL mobile phase, filter (0.45 jxm), inject a 50 jxL
aliquot. Urine. Acidify urine with 20% acetic acid, filter (0.45 u,m). 1 mL Urine + 1 mL 1 M pH
11.0 NaH2PO4 + 50 fiL 15 |xg/mL IS in MeOH:water 50:50, extract twice with 3 mL ethyl
acetate:MTBE 50:50. Combine the organic layers and evaporate them to dryness under a
stream of nitrogen at 40, reconstitute the residue in 250 fxL mobile phase, inject an aliquot.
(To analyze glucuronide mix 100 jxL urine with 400 JJLL 50 mM pH 1.2 phosphoric acid buffer
containing 15 mM sodium dodecyl sulfate, inject a 10-50 u,L aliquot (MeCN:buffer 30:70).)
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Spherisorb C8
Mobile phase: MeCN.buffer 33:67 (whole blood) or 40:60 (urine) (Buffer was 50 mM pH 2.2
phosphoric acid containing 10 mM sodium dodecyl sulfate.) (After each injection flush with
MeCN:buffer 67:33 for 5 min, re-equilibrate for 5 min.)
Flow rate: 1.5
Detector: UV 277
CHROMATOGRAM
Retention time: 19.9 (whole blood), 9.8 (urine)
Internal standard: 3,5-diamino-6-(2-methoxyphenyl)-l,2,4-triazine (BW A725C) (11.9 (whole
blood), 7.0 (urine))
OTHER SUBSTANCES
Simultaneous: carbamazepine, cyheptamide, desmethyldiazepam, diazepam, ethosuximide, fel-
bamate, lorazepam, oxazepam, phenobarbital, phenytoin, temazepam, tolybarbital
KEYWORDS
guinea pig; whole blood; SPE; pharmacokinetics
REFERENCE
Sinz,M.W.; Remmel,R.P. Analysis of lamotrigine and lamotrigine 2-N-glucuronide in guinea pig blood and urine
by reserved-phase ion-pairing liquid chromatography, J.Chromatogn, 1991, 571, 217-230.
Lansoprazole
Molecular formula: C16H14F3N3O2S
Merck Index: 5373
LednicerNo.: 5 115
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 100 |xL 25 |xg/mL isobutyl p-hydroxybenzoate in dichlo-
romethane + 3 mL diethyl etherrdichloromethane 70:30, extract, centrifuge, repeat extraction.
Add 500 |xL diethyl etherrdichloromethane: propylene glycol 70:30:0.5 to the supernatants,
evaporate to dryness with a stream of nitrogen, reconstitute in 500 |xL mobile phase, inject a
100 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm TSK gel ODS-120T (Tosoh)
Mobile phase: MeCN:water:n-octylamine 38:62:0.1 adjusted to pH 7 with 85% phosphoric acid
Flow rate: 1
Detector: UV 285
CHROMATOGRAM
Internal standard: isobutyl p-hydroxybenzoate (23)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Aoki,L; Okumura,M.; Yashiki,T. High-performance liquid chromatographic determination of lansoprazole and
its metabolites in human serum and urine, J.Chromatogr., 1991, 571, 283-290.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL 20 |xg/mL n-butyl p-hydroxybenzoate + 100 jxL
MeOH, vortex for a few s, add 7 mL MTBE, vortex for 45 s, centrifuge at 4 at 1500 g for 10
rinse the tube with 1 mL MTBE, evaporate to dryness under a stream of nitrogen at 30,
reconstitute the residue in 100 |xL MeOH, vortex for 1 min. Dilute a 100 |xL aliquot 10-fold
with water, inject 1 mL water then the diluted plasma extract onto column A, wash with 1 mL
water, elute the contents of column A onto column B with the mobile phase, elute with the
mobile phase and monitor the effluent from column B.
HPLC VARIABLES
Column: A 10 mm long 10 |xm Nucleosil CN (SFCC); B 10 mm long 5 |xm Nucleosil C18 + 250
X 4.6 5 |xm Nucleosil C18
Mobile phase: MeCN:phosphoric acid:250 mM KH2PO4:acetic acid:triethylamine:water 36:10.5:
4:0.15:0.15:49.2
Flow rate: 2
Detector: UV 285
CHROMATOGRAM
Retention time: 11
Internal standard: n-butyl p-hydroxybenzoate (23)
OTHER SUBSTANCES
Noninterfering: acebutolol, allopurinol, amiloride, atenolol, phenobarbital, pindolol, prazosin,
quinidine, ranitidine, sotalol
KEYWORDS
plasma; column-switching; pharmacokinetics
REFERENCE
Landes,B.D.; Miscoria,G.; Flouvat,B. Determination of lansoprazole and its metabolites in plasma by high-
performance liquid chromatography using a loop column, J.Chromatogr., 1992, 577, 117-122.
SAMPLE
Matrix: blood
Sample preparation: 500 [iL Plasma + 150 JXL 4 (xg/mL omeprazole in water + 5 mL diethyl
etheridichloromethane 70:30, vortex for 5 min, centrifuge at 6 at 300-850 g for 10 min. Remove
4 mL of the organic layer and evaporate to dryness under reduced pressure at room temper-
ature, reconstitute the residue in 500 |xL buffer, refrigerate until injection, inject a 100 |JLL
aliquot. (Buffer was MeCN:water 35:65 containing 1 mL/L n-octylamine and 5 mM N-aceto-
hydroxamic acid, pH adjusted to 7.5 with 85% phosphoric acid.)
HPLC VARIABLES
Column: 150 or 250 X 4.6 5 fjim Hi-Chrom Reversible octadecylsilane (Regis)
Mobile phase: MeCN:water 35:65 containing 1 mL/L n-octylamine and 5 mM N-acetohydroxamic
acid, pH adjusted to 7.0 with 85% phosphoric acid
Flow rate: 1 for 15 min then 2.5
Detector: UV 285
CHROMATOGRAM
Internal standard: omeprazole (7.6)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Karol,M.D.; Granneman,G.R.; Alexander,K. Determination of lansoprazole and five metabolites in plasma by
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in EtOH, inject a 10-20 (xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Chiralpak AD (Daicel)
Mobile phase: HexaneiEtOH 65:35
Flow rate: 1
Detector: UV 302
CHROMATOGRAM
Retention time: k' 2.91, a 1.15
OTHER SUBSTANCES
Also analyzed: timoprazole, omeprazole, pantoprazole
KEYWORDS
chiral
REFERENCE
Balme*r,K.; Persson,B.-A.; Lagerstrom,P.-O. Stereoselective effects in the separation of enantiomers of omepra-
zole and other substituted benzimidazoles on different chiral stationary phases, J.Chromatogr.A, 1994,660,
269-273.
Lasalocid A
Merck Index: 5384
SAMPLE
Matrix: blood
Sample preparation: Cow, dog. 10 mL Whole blood + 1 mL 1 M NaOH, shake vigorously for
10-15 s, let stand at room temperature for 5 (cow) or 20 (dog) min, add 20 mL ethyl acetate,
shake vigorously by hand for 50-60 s, centrifuge at 350 g for 10-15 min. Remove 1-10 mL of
the organic layer and evaporate it to dryness under a stream of nitrogen at 60, reconstitute
the residue in 1 mL mobile phase, vortex vigorously for 1 min, inject an aliquot. Chicken. 10
mL Whole blood + 20 mL ethyl acetate, shake vigorously by hand for 10-15 s, shake on a
reciprocal shaker at high speed for 5 min, centrifuge at 350 g for 10-15 min. Remove 1-10 mL
of the organic layer and evaporate it to dryness under a stream of nitrogen at 60, reconstitute
the residue in 1 mL mobile phase, vortex vigorously for 1 min, inject an aliquot. Rat, mouse.
1 mL Whole blood + 10 mL ethyl acetate, shake vigorously by hand for 30-40 s, shake on a
reciprocal shaker at high speed for 5 min, shake vigorously by hand for 10-15 s, centrifuge at
350 g for 10-15 min. Remove 9 mL of the organic layer and evaporate it to dryness under a
stream of nitrogen at 60, reconstitute the residue in 1 mL mobile phase, vortex vigorously for
1 min, inject an aliquot.
HPLC VARIABLES
Column: 250 mm long 5 |xm Partisil PXS 5/25
Mobile phase: Hexane:THF:MeOH:triethylamine:ammonium hydroxide 81:14:2:2:1 shake for 30-
40 s, let stand for 25-30 min, discard the lower phase, add 10 mL/L THF, do not degas or filter.
(Place a 10 |xm Partisil column before the injector. Flush system with hexane for 30 min at 2
mL/min at the end of each day.)
Flow rate: 0.9
CHROMATOGRAM
Retention time: 6-8
KEYWORDS
whole blood; mouse; rat; cow; dog; chicken; normal phase
REFERENCE
Kaykaty,M.; Weiss,G. Lasalocid determination in animal blood by high-performance liquid chromatography
fluorescence detection, J.Agric.Food Chem., 1983, 31, 81-84.
SAMPLE
Matrix: feed, premix
Sample preparation: Premix. 2 g Premix + 100 mL acidified MeOH, sonicate at 40 for 2 min,
cool, make up to 250 mL with acidified MeOH, mix thoroughly, let stand for 1 h, filter (0.45
|xm) an aliquot, dilute the filtrate with acidified MeOH to produce a 4 |xg/mL solution, inject
a 20 |xL aliquot. Feed. Grind feed to pass a 1 mm sieve, add 100 mL acidified MeOH, sonicate
at 40 for 2 min, cool, make up to 250 mL with acidified MeOH, mix thoroughly, let stand for
1 h, filter (0.45 |xm) an aliquot, inject a 20 |xL aliquot of the filtrate. (Acidified MeOH was
MeOHxoncentrated HCl 99.5:0.5.)
HPLC VARIABLES
Column: 250 or 125 X 4 5 |xm C18
Mobile phase: MeOHibuffer 95:5 (Prepare buffer by dissolving 1.36 g KH2PO4 in 500 mL water,
add 3 mL phosphoric acid, add 10 mL 1,5-dimethylhexylamine, adjust pH to 4.0 with 20%
phosphoric acid, make up to 1 L with water.)
Flow rate: 1.2
CHROMATOGRAM
Retention time: 7
REFERENCE
Analytical Methods Committee, Determination of lasalocid sodium in poultry feeds and premixes, Analyst,
1995, 120, 2175-2180.
SAMPLE
Matrix: solutions
Sample preparation: Condition a Mega Bond Elut silica gel SPE cartridge with benzene (Cau-
tion! Benzene is a carcinogen!). Evaporate a solution in MeOH to dryness, add 5 mL 5.28 mg/
mL 1-bromoacetylpyrene in MeCN, add 5 mL 1.28 mg/mL Kryptofix 222 in MeCN, heat at 50
for 1.5 h, cool. Either inject this solution directly or evaporate it to dryness, dissolve the residue
in 5 mL benzenexhloroform 50:50, rinse out the flask with two 5 mL portions of benzene:
chloroform 50:50, filter, add the filtrate to the SPE cartridge, elute with two 5 mL portions of
benzene:acetone 70:30. Evaporate the eluate to dryness, reconstitute the residue in 10 mL
MeCN, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Develosil 5C18
Flow rate: 1
CHROMATOGRAM
Retention time: 15
Internal standard: 18,19-dihydrosalinomycin (25), 18,19-dihydro-20-ketosalinomycin(16.5)
OTHER SUBSTANCES
Simultaneous: narasin, monensin, salinomycin
KEYWORDS
derivatization; SPE
REFERENCE
Asukabe,H.; Murata,H.; Harada,K.-L; Suzuki,M.; Oka,H.; Ikai,Y. Improvement of chemical analysis of antibi-
otics. XX. Basic study on high-performance liquid chromatographic determination of four polyether anti-
biotics pre-derivatized with 1-bromoacetylpyrene, J.ChromatognA, 1993, 657, 349-356.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Tissuemizer) 10 g tissue and 25 mL solvent for 1 min, wash
blades with 3-4 mL solvent, combine with homogenate, shake for 30 min, centrifuge at 800 g
for 15 min, decant. Add 25 mL solvent to residue, mix thoroughly, shake vigorously for 1 min,
centrifuge at 800 g for 15 min, add supernatants to a 75 X 20 column of 80-200 mesh alumina
(Fisher), rinse container onto column with 25 mL solvent, add 100 mL solvent to the column.
Combine all the eluates and add 100 mL 5% NaCl, shake vigorously, let stand 2-3 min, add 30
mL dichloromethane, shake vigorously for 30 s, repeat extraction twice. Combine the dichlo-
romethane layers and evaporate them to dryness under reduced pressure at 48-50, reconsti-
tute the residue in 1 mL solvent, add to a 75 X 20 column of 25-100 |xm Sephadex LH-20,
rinse flask with two 3.5 mL portions of solvent and add the rinses to the column, add 10 mL
solvent to the column, discard the first 18 mL of eluate, add 10 mL solvent to the column,
collect this fraction, evaporate to dryness under a stream of nitrogen at 48-50, make up to 1
mL with solvent, mix, add 500 JJIL 9-anthryldiazomethane solution, let stand in the dark for
30 min, evaporate to dryness under a stream of nitrogen at 48-50, reconstitute in 1 mL hexane.
Condition a Baker-10 silica SPE cartridge with 5-10 mL hexane, do not allow to dry. Add the
hexane solution to the SPE cartridge, rinse tube with 9 mL hexane, add rinse to the SPE
cartridge. Wash SPE cartridge with 10 mL hexane: dichloromethane 50:50, with 10 mL hexane:
dichloromethane 20:80, with 10 mL dichloromethane, and with 1 mL MeOH. Elute with 1 mL
MeOH, inject a 20 |xL aliquot of the eluate. (Solvent was MeOH:water 80:20. Prepare 9-an-
thryldiazomethane solution as follows. Add 1100 g manganous sulfate tetrahydrate in 1.5 L
water and 1170 mL 40% NaOH over 1 h to a hot stirred solution of 960 g potassium perman-
ganate in 6 L water, stir for 1 h, centrifuge, wash solid with water until washings are colorless,
dry solid at 100-120, grind the activated manganese dioxide to a fine powder. Add 8.5 g 85%
hydrazine hydrate (Caution! Hydrazine hydrate is a carcinogen!) to 8.8 g 9-anthraldehyde dis-
solved in 150 mL EtOH, stir at room temperature for 3 h, filter off solid, dry under vacuum,
recrystallize from EtOH to give 9-anthraldehyde hydrazone as red-yellow crystals, mp 124-6.
Dissolve 220 mg 9-anthraldehyde hydrazone in 100 mL anhydrous ethyl ether, add 800 mg
activated manganese dioxide, add 600 |xL EtOH saturated with KOH, stir vigorously for 30
min, filter (glass fiber), wash solid with 20 mL anhydrous ethyl ether, evaporate to reduce
volume, make up to 100 mL with anhydrous ethyl ether, store in a dark flask in the dark in a
refrigerator. Discard after 30 days (J.Assoc.Off.Anal.Chem. 1985, 68, 1149).)
HPLC VARIABLES
Column: 200 X 4.6 5 ^m RP-C8 (Hewlett-Packard)
Mobile phase: Gradient. A was MeCN. B was MeCN:water 10:90. A:B 20:80 for 9 min, 10:90 for
7 min, 20:80 for 1 min.
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: monensin, salinomycin
KEYWORDS
cow; liver; SPE; protect from light; derivatization
REFERENCE
Martinez,E.E.; Shimoda,W. Liquid chromatographic determination of multiresidue fluorescent derivatives
of ionophore compounds, monensin, salinomycin, narasin, and lasalocid, in beef liver tissue,
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Polytron) 10 g minced liver and 40 mL MeCN for 15-30 s,
scrape down sides and shaft, homogenize for 15-30 s, centrifuge at 5-10 at 2000-2500 rpm for
15-20 min, decant 23 mL supernatant, add 23 mL hexane, shake vigorously for 15-20 s, cen-
trifuge at 1500-2000 rpm. Evaporate 20 mL of the lower MeCN layer to dryness under a stream
of nitrogen at 55-65, add 1 mL water saturated with mobile phase, vortex for 15-20 s, add 2
mL mobile phase, vortex for 15-20 s, centrifuge at 2000-2500 rpm for 15-20 min, recentrifuge
if top layer is not clear, inject an aliquot of the top layer.
HPLC VARIABLES
Column: Two Partisil 10 PXS 10/25 columns in series
Mobile phase: THF:MeOH:hexane:mixture 3.75:0.75:20.5:75, do not filter or degas. (Mixture was
150 mL THF + 30 mL MeOH + 10 mL ammonium hydroxide + 810 mL hexane, mix, let stand
for 1 h, discard lower layer.) (Place a silica column between pump and injector.)
Flow rate: 2
CHROMATOGRAM
Retention time: 7
Limit of detection: 0.24-0.47 ppm
KEYWORDS
normal phase; liver; cow
REFERENCE
Newkirk,D.R.; Barnes,C.J. Liquid chromatographic determination and gas chromatographic-mass spectromet-
ric confirmation of lasalocid sodium in bovine liver: interlaboratory study, J.Assoc.Off.Anal.Chem., 1989,
72, 581-584.
SAMPLE
Matrix: tissue
Sample preparation: 10 g Minced tissue + 50 mL MeCN, homogenize for 2 min, sonicate for 5
min, centrifuge at 1860 g for 5 min, repeat extraction. Combine the supernatants and add 30
mL carbon tetrachloride and 20 mL saturated NaCl, shake for 1 min, filter the organic layer
through anhydrous sodium sulfate and phase separating paper, evaporate to dryness. Transfer
the residue to a Bond-Elut silica SPE cartridge with three 2 mL aliquots of hexane, wash with
5 mL chloroform, elute with 10 mL chlorofornv.MeOH 95:5. Evaporate the eluate to dryness,
transfer to a small vial with three 2 mL portions of hexane, evaporate to dryness with a stream
of nitrogen, reconstitute in 500 |xL mobile phase, inject a 10 |xL aliquot.
HPLC VARIABLES
Guard column: 5 x 3 PLRP-S styrene-divinylbenzene copolymer (Polymer Labs)
Column: 250 X 4.6 5 jim PLRP-S styrene-divinylbenzene copolymer (Polymer Labs)
Mobile phase: MeCNiIO mM pH 10.0 disodium tetraborate (borax) 60:40
Flow rate: 1
CHROMATOGRAM
Retention time: 5.5
KEYWORDS
chicken; muscle; SPE
REFERENCE
Tarbin,J.A.; Shearer,G. Improved high-performance liquid chromatographic procedure for the determination of
lasalocid in chicken tissues and egg using polymeric and porous graphitic carbon columns, J.Chromatogr.,
1992, 579, 177-183.
SAMPLE
Matrix: tissue, eggs
Sample preparation: 2 g Minced tissue or egg + 25 mL MeCN, homogenize for 2 min, sonicate
for 5 min, centrifuge at 1860 g for 5 min, repeat extraction. Combine the supernatants and
add 50 mL carbon tetrachloride and 20 mL saturated NaCl, shake for 1 min, filter the organic
layer through anhydrous sodium sulfate and phase separating paper, evaporate to dryness.
Transfer the residue to a Bond-Elut silica SPE cartridge with three 2 mL aliquots of hexane,
wash with 5 mL chloroform, elute with 10 mL chloroform:MeOH 95:5. Evaporate the eluate to
dryness, transfer to a small vial with three 2 mL portions of hexane, evaporate to dryness with
a stream of nitrogen, reconstitute in 500 jxL mobile phase, inject a 25 uL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 7 |xm Hypercarb porous graphitic carbon (Shandon)
Mobile phase: MeCN containing 5% 1,1,3,3-tetramethylguanidine
Flow rate: 0.5
CHROMATOGRAM
Retention time: 7
Limit of detection: 10 ng/g (eggs), 2 ng/g (tissue)
KEYWORDS
chicken; muscle; SPE
REFERENCE
Tarbin,J.A.; Shearer,G. Improved high-performance liquid chromatographic procedure for the determination of
lasalocid in chicken tissues and egg using polymeric and porous graphitic carbon columns, J.Chromatogr.,
1992, 579, 177-183.
Lazabemide
Merck Index: 5407
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 jxL 400 mM NaOH, vortex, add 10 mL MTBE:l-butanol
80:20, shake at 30 rpm on a rotating shaker for 20 min, centrifuge at 2000 g for 5 min. Remove
9 mL of the organic layer and add it to 500 |xL 0.17% phosphoric acid, extract for 20 min,
centrifuge for 5 min. Remove the aqueous layer and add it to 500 |xL buffer, add 500 jxL 50
jutg/mL fluorescamine in MeCN (prepare fresh each day) with constant vortexing, after 10 min
remove the MeCN by evaporation under reduced pressure for exactly 10 min, vortex, inject a
100 |xL aliquot. (Prepare buffer by dissolving 30 g Na2HPO4 in water, add 24 mL 1 M NaOH,
make up to 1 L with water.)
HPLC VARIABLES
Column: 125 X 4 LiChroCART Superspher 60 RP-8e (Merck)
Mobile phase: MeCN:water 32:68 containing 100 mM NaH2PO4 and 5 mM Na2HPO4, pH 5.9
0.1
Flow rate: 1
CHROMATOGRAM
Retention time: 3.7
OTHER SUBSTANCES
Noninterfering: benserazide, dopamine, levodopa
KEYWORDS
derivatization; plasma; rat; human
REFERENCE
Wyss,R.; Philipp,W. Determination of the monoamine oxidase B inhibitor Ro 19-6327 in plasma by high-per-
formance liquid chromatography using precolumn derivatization with fluorescamine andfluorescencede-
tection, J.Chromatogr., 1990, 507, 187-198.
Lenampicillin
Merck Index: 5460
SAMPLE
Matrix: blood
Sample preparation: 0.5 mL Plasma + 1 mL MeOH, stir for 5 min, centrifuge at 2400 g for 10
min. Remove 1 mL supernatant, add 2 |xg cefazolin, inject an aliquot.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 225
CHROMATOGRAM
Retention time: 9 (measured as ampicillin peak)
Internal standard: cefazolin (14)
KEYWORDS
plasma
REFERENCE
Marzo,A.; Monti,N.; Ripamonti,M.; Arrigoni Martelli,E.; Picari,M. High-performance liquid chromatographic
assay of ampicillin and its prodrug lenampicillin, J.Chromatogr., 1990, 507, 235-239.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 225
CHROMATOGRAM
Retention time: 20
Internal standard: o-tolylpiperazine (8.5)
OTHER SUBSTANCES
Simultaneous: ampicillin
REFERENCE
Marzo,A.; Monti,N.; Ripamonti,M.; Arrigoni Martelli,E.; Picari,M. High-performance liquid chromatographic
assay of ampicillin and its prodrug lenampicillin, J.Chromatogr., 1990, 507, 235-239.
Letrozole
Merck Index: 5474
SAMPLE
Matrix: blood
Sample preparation: Mix plasma with 50 |xL 6.12 |xM IS, adjust to pH 13.0, add to an Extrelut
SPE cartridge. Elute with 6 mL and 5 mL portions of dichloromethanerdiethyl ether 40:60,
evaporate the eluates to dryness, dissolve the residue in 500 |xL pH 7.0 phosphate buffer, wash
with 2 mL hexane, inject a 120 |xL aliquot of the aqueous phase.
HPLCVARIABLES
Column: 5 |xm LiChrospher RP 8
Mobile phase: MeCNrpH 7.0 phosphate buffer 42:58
Flow rate: 1
Detector: UV 234
CHROMATOGRAM
Internal standard: CGS 18320 B (4,44 IH-l,3-diazol-l-ylmethylene]bis-benzonitrile)
KEYWORDS
REFERENCE
SioufiA; Sandrenan,N.; Godbillon,J.; Trunet,P.; Czendlik,C; Howald,H.; Pfister,C; Ezzet,F. Comparative bio-
availability of letrozole under fed and fasting conditions in 1 healthy subjects after a 2.5 mg single oral
administration, Biopharm.Drug Dispos., 1997,18, 489-497.
Leucovorin
CAS Registry No.: 58-05-9, 1492-18-8 (Ca salt),
6035-45-6 (Ca salt pentahydrate)
Merck Index: 4254
SAMPLE
M a t r i x : blood
Sample preparation: Mix 20 jxL serum with 20 |xL 2 M perchloric acid, vortex for a few seconds,
HPLC VARIABLES
Column: 150 X 4.6 5 \im Inertsil ODS-2 (GL Sciences, Japan)
Mobile phase: MeCN:25 mM pH 7.0 sodium phosphate buffer 8:92 containing 25 mM hydrogen
peroxide
Flow rate: 1
a 5 m X 0.5 mm i.d. stainless-steel reaction coil at 160 and a 3 m X 0.5 mm i.d. stainless-
steel coil at 15 to the detector.
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Extracted: methotrexate
KEYWORDS
serum; post-column reaction
REFERENCE
Kubo,H-; Umiguchi,Y; Fukumoto,M.; Kinoshita,T. Fluorometric determination of methotrexate in serum by
high-performance liquid chromatography using in-line oxidation with hydrogen peroxide, Anal.ScL, 1992,
5, 789-792.
SAMPLE
Matrix: blood
Sample preparation: Add 2 mg ascorbic acid to each 1 mL blood, centrifuge at 800 g in the
cold. 1 mL Plasma + 10 (xL 30 (xg/mL methotrexate in 1 mg/mL ascorbic acid +1.5 mL MeOH,
vortex, centrifuge at 800 g. Remove the supernatant and evaporate it to dryness under a stream
of nitrogen, reconstitute the residue in 200 jxL water, inject the whole amount.
HPLCVARIABLES
Mobile phase: Gradient. A was 250 mM pH 5.0 phosphate buffer. B was MeOH:250 mM pH 5.0
phosphate buffer 1:1. A:B 100:0 for 15 min, to 50:50 over 15 min, to 0:100 over 5 min, maintain
at 0:100 for 5 min.
Flow rate: 2
Detector: UV 310
CHROMATOGRAM
Retention time: 21
Internal standard: methotrexate (29)
KEYWORDS
plasma
REFERENCE
Wainer,l.W.; Stiffin,R-M. Direct resolution of the stereoisomers of leucovorin and 5-methyltetrahydrofolate us-
ing a bovine serum albumin high-performance liquid chromatographic chiral stationary phase coupled to
an achiral phenyl column, J.Chromatogr., 1988, 424, 158-162.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut RP-18 SPE cartridge with four 2 mL portions of
MeOH and three 2 mL portions of 100 mM pH 4.7 TrisP buffer. Add 1 mg/mL ascorbic acid to
plasma. Dilute 500 JLJLL plasma six fold with 9 mg/mL NaCl containing 1 mg/mL ascorbic acid,
add 200 |xL 8.5 mg/mL phosphoric acid, add to the SPE cartridge, wash with 500 |xL 10 mM
pH 4.7 TrisP buffer, elute with 1.5 mL eluant. Evaporate the eluate to dryness under a stream
of nitrogen at 37 for 30 min, reconstitute the residue in 250 JJLL 9 mg/mL NaCl containing 1
mg/mL ascorbic acid, inject a 40 |xL aliquot onto column A and elute with mobile phase, collect
600 |xL effluent (monitor with detector A) in a sample loop and inject it onto column B, elute
with mobile phase and monitor the effluent from column B with detector B. (TrisP was
tris(hydroxyethyl)methylaminomethane phosphate. Eluant was MeOH: 10 mM TrisP buffer 75:
25, pH 7 containing 150 fxg/mL ascorbic acid.)
HPLC VARIABLES
Column: A 119 X 2 4 |xm Superspher RP-8 (Merck) (Condition column with MeOH:water 30:70
every 100-200 injections.); B 150 X 4.5 7 |xm chiral protein 2 HSA-BP human serum albumin
(Societe Francaise Chromato Colonne)
Mobile phase: 1-Propanol:200 mM Na2HPO4 2:98 adjusted to pH 6.2 with 8.5 g/L phosphoric
acid
Column temperature: 35 (column B)
Detector: A UV 313; B E, ESA Coulochem 5100 A, Model 5020 guard cell, Model 5010 analytical
cell, El 0.30 V, E2 0.55 V (monitored), guard cell is placed before second injection valve
CHROMATOGRAM
Retention time: 6 (1), 10 (d) (after injection onto column B)
KEYWORDS
plasma; narrow bore; column-switching; heart cut; chiral; SPE
REFERENCE
Etienne,M.-C; Speziale,N.; Milano,G. HPLC of folinic acid diastereoisomers and 5-methyltetrahydrofolate in
plasma, Clin.Chem., 1993, 39, 82-86.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bakerbond C18 SPE cartridge with 1 mL MeOH and 1 mL
1% acetic acid. 1 mL Plasma + 1 mL 5% acetic acid, add to the SPE cartridge, elute with
MeCN:pH 7.0 phosphate buffer (ionic strength 0.1) 20:80, inject a 100 |xL aliquot of the eluate
onto column A with mobile phase A. Collect the fraction containing leucovorin (about 8 min)
in a 250 (xL sample loop and inject onto column B with mobile phase B, monitor the effluent
from column B. (For achiral determination use only column A and mobile phase A, LOQ 50 ng/
mL.)
HPLC VARIABLES
Column: A 5 |xm Novapack C18 pre-column + 300 X 4 5 jxm Novapack RP-18; B 7 |xm Resolvosil
BSA chiral pre-column + 150 X 4 7 |xm Resolvosil BSA chiral (Macherey-Nagel)
Mobile phase: A Gradient. MeCN:pH 7.0 phosphate buffer (ionic strength 0.1) from 0:100 to 30:
70 over 10 min, to 100:0 over 8 min.; B pH 7.0 phosphate buffer (ionic strength 0.1)
Detector: UV 290
CHROMATOGRAM
KEYWORDS
plasma; SPE; chiral; column-switching; heart-cut
REFERENCE
Vandenbosch,C; van Belle,S.; de Smet,M.; Taton,G.; Bruynseels,V.; Vandenhoven,G.; Massart,D.L. Determi-
nation of leucovorin and 5-fluorouracil in plasma by high-performance liquid chromatography,
J.Chromatogr., 1993, 612, 77-85.
SAMPLE
Matrix: blood, CSF
Sample preparation: 250 |xL Plasma or CSF + 25 |xL 10 mg/mL ascorbic acid + 250 |xL ice-cold
1.5 M perchloric acid, vortex, let stand in ice-water for 5 min, centrifuge at 4 at 3000 g for 5
min. Remove 350 JJLL of the supernatant with 50 fxL 8 M potassium acetate, keep in ice-water
for 2 min, centrifuge at 4 at 3000 g for 2 min, inject a 100 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: 200 X 3 5 |xm Hypersil ODS glass column
Mobile phase: Gradient. A was 10 mM ammonium formate adjusted to pH 3.5 with HCl. B was
MeCN: 10 mM ammonium formate 25:75 adjusted to pH 3.5 with HCl. A:B from 85:15 to 5:95
Flow rate: 0.4
Detector: UV 305
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Extracted: methotrexate, N5-methyltetrahydrofolate
KEYWORDS
plasma
REFERENCE
van Tellingen,O.; van der Woude,H-R; Beijnen,J.H.; van Beers,C.J.T.; Nooyen,W.J. Stable and sensitive method
for the simultaneous determination of N5-methyltetrahydrofolate, leucovorin, methotrexate and 7-hydroxy-
methotrexate in biological fluids, J.Chromatogr., 1989, 488, 379-388.
SAMPLE
Sample preparation: Serum. 500 |xL Serum + 250 |xg ascorbic acid + 750 |xL MeCN, vortex,
centrifuge at 400 g for 1 min, add 7 mL chloroform, mix, centrifuge at 400 g for 1 min. 400 JJLL
Aqueous phase + 2 mL mobile phase A, inject the whole amount through a 125 X 4 40 juim
silica (Supelco) column on to column A and elute to waste with mobile phase A, backflush the
contents of column A on to column B and elute to waste with mobile phase B, collect the fraction
containing leucovorin in a 1 mL sample loop, inject this fraction on to column C and elute with
mobile phase C, monitor the effluent from column C. Urine. Condition a 1 mL C18 SPE car-
tridge (Supelco) with 2 mL water, 2 mL MeOH, and 2 mL mobile phase A. Dilute urine with
4 volumes of mobile phase A, add to the SPE cartridge, wash with 2 mL mobile phase A, elute
with 2 mL MeCN:water 50:50. Wash the eluate with chloroform, filter (Waters ultrafiltration
cartridge) the aqueous layer while centrifuging, dilute 400 |xL of the ultrafiltrate with 2 mL
mobile phase A, inject the whole amount through a 125 X 4 40 jxm silica (Supelco) column on
to column A and elute to waste with mobile phase A, backflush the contents of column A on to
column B and elute to waste with mobile phase B, collect the fraction containing leucovorin in
a 1 mL sample loop, inject this fraction on to column C and elute with mobile phase C, monitor
the effluent from column C.
HPLC VARIABLES
Column: A 30 X 4 5 ^m C18; B 250 X 2 3 |jim C18 (Macherey-Nagel); C 150 X 4.6 7 ^m Resolvosil
BSA-7
Mobile phase: A 5 mM tetrabutylammonium phosphate (low-UV Pic A) adjusted to pH 6.5 with
phosphoric acid; B Isopropanolibuffer 7.5:92.5 adjusted to pH 5 with phosphoric acid (Buffer
was 1.5 mM sodium phosphate containing 0.75 mM tetrabutylammonium phosphate (low-UV
PIC A).); C 28 mM Phosphate buffer containing 0.6 mM sodium azide (Caution! Sodium azide
is toxic! Do not discharge to plumbing system!)
Column temperature: 40 (B and C only, A is ambient)
Flow rate: A 2; B 0.15; C 0.4
CHROMATOGRAM
Retention time: 26 (6S), 30 (6R)
OTHER SUBSTANCES
Extracted: metabolites, 5-methyltetrahydrofolate
KEYWORDS
serum; chiral; column-switching; heart-cut; SPE; pharmacokinetics
Next Page
REFERENCE
Schleyer,E.; Reinhardt,J.; Unterhalt,M.; Hiddemann,W. Highly sensitive coupled-column high-performance liq-
uid chromatographic method for the separation and quantitation of the diastereomers of leucovorin and 5-
methyltetrahydrofolate in serum and urine, J.Chromatogr.B, 1995, 669, 319-330.
SAMPLE
HPLC VARIABLES
Mobile phase: MeOH:buffer 21:79 (Buffer was 15 mL 1 M tetrabutylammonium hydroxide in
MeOH + 850 mL water, pH adjusted to 7.5 0.1 with 2 M NaH2PO4, make up to 875 mL with
water.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 4
KEYWORDS
stability-indicating; saline; injections
REFERENCE
Smith,J.A.; Morris,A.; Duafala,M.E.; Bertino,J.R.; Markman,M.; Kleinberg,M. Stability offloxuridineand leu-
covorin calcium admixtures for intraperitoneal administration, Am. J.Hosp.Pharm., 1989, 46, 985-989.
SAMPLE
Matrix: solutions
Sample preparation: Prepare aqueous solutions, stabilize them with ascorbic acid (1 mg/mL
water), flush with nitrogen, refrigerate, keep in brown glass vials. Inject a 10 (xL aliquot.
HPLC VARIABLES
Guard column: 5 |xm Lichrospher 60 RP-select B
Column: 125 X 3 3 |xm Hypersil BDS
Mobile phase: Gradient. A was MeCN. B was 5 mM monobasic potassium phosphate adjusted
to pH 2.3 with phosphoric acid. A:B 7:93 for 5 min, to 13:87 over 15 min, to 21:79 over 6 min,
maintain at 21:79 for 1 min, back to 7:93 over 2 min, re-equilibrate at initial conditions for 5
min.
Flow rate: 0.5
Detector: F ex 295 em 355 following post-column photochemical derivatization. The column ef-
fluent flowed through a 10 m X 0.3 mm ID Teflon tube irradiated with a 254 nm mercury lamp
to the detector.
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
Simultaneous: metabolites, methotrexate
KEYWORDS
post-column photochemical derivatization; post-column reaction
REFERENCE
Mandl,A.; Lindner,W. Improved detection of leucovorin in mixed folates and antifolates by reversed-phase liquid
chromatography and on-line post-column UV irradiation, Chromatographia, 1996, 43, 327-330.
Previous Page
Levallorphan
Merck Index: 5485
SAMPLE
Sample preparation: Dilute urine 3:1 or more with water. Mix 1 mL CSF, plasma, or diluted
urine with 500 |xL saturated sodium carbonate, add 5 mL hexane containing 0.1% n-octylamine.
Vortex for 60 s, centrifuge at 2000 g for 10 min. Re-extract aqueous phase with 5 mL hexane
containing 0.1% n-octylamine, evaporate the combined hexane extracts to dryness under a
stream of nitrogen in a 50 water bath. Reconstitute residue with 150 (xL 100 mM hydrochloric
acid, inject a 100 |xL aliquot.
HPLCVARIABLES
Guard column: 10 X 4.6 5 |xm CN
Column: 220 X 4.6 5 |xm Brownlee Spheri-5CN (Applied Biosystems, USA)
Mobile phase: MeCN:n-octylamine:water 19:0.05:80.95 adjusted to pH 2.8 with phosphoric acid
Flow rate: 1
CHROMATOGRAM
Retention time: 7.4
Internal standard: levallorphan
OTHER SUBSTANCES
KEYWORDS
plasma; levallorphan is IS
REFERENCE
Kimiskidis,V.K.; Kazis,A.D.; NiopasJ. Simultaneous determination of dextromethorphan and dextrorphan in
human plasma, urine and cerebrospinal fluid by HPLC with fluorescence detection, JJAq.
Chromatogr.Rel.TechnoL, 1996, 19, 1267-1275.
SAMPLE
Matrix: blood, saliva, urine
Sample preparation: Add 100 (xL 28% ammonium hydroxide and 5 mL n-butanol:hexane 10:90
to 5 mL urine, 1 mL plasma or 3 mL saliva. Rotate for 30 min and centrifuge at 4500 rpm for
10 min. Remove the upper organic layer and extract it with 300 uX 100 mM HCl by vortexing
for 20 min, centrifuge for 5 min. Inject a 40 |xL (urine) or 200 JJLL (plasma, saliva) aliquot of
the acidic layer.
HPLC VARIABLES
Column: Zorbax RP-phenyl
Mobile phase: MeCN: 10 mM potassium phosphate 50:50 adjusted to pH 4.0 with 8.5% phos-
phoric acid
Flow rate: 1.0
CHROMATOGRAM
Retention time: 8.6
OTHER SUBSTANCES
KEYWORDS
levallorphan is IS; plasma
REFERENCE
Hu,O.Y.-R; Tang,H.-S.; Lane,H.-Y.; Chang,W.-H.; Hu,T.-M. Novel single-point plasma or saliva dextromethor-
phan method for determining CYP2D6 activity, J.Pharmacol.Exp.Ther., 1998, 285, 955-960.
SAMPLE
Sample preparation: 500 |xL Microsomal incubation + 500 |xL MeOH:water:zinc sulfate 50:45:
5, centrifuge at 10000 g for 3 min, extract the supernatant twice with 3 mL dichloromethane.
Combine dichloromethane extracts, dry under a stream of nitrogen, dissolve the residue in 200
JXL MeOH. Inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm polymer C18 (Astec, Whippany, NJ)
Mobile phase: MeCN:50 nM (sic) pH 9.0 ammonium carbonate buffer 60:40 (Buffer was adjusted
to pH 9.0 with ammonium hydroxide.)
Flow rate: 0.7
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: dextromethorphan, dextrorphan
KEYWORDS
liver; levallorphan is IS
REFERENCE
Vielnascher,E.; Spatzenegger,M.; Mayrhofer,A.; Klinger,P.; Jager,W. Metabolism of dextromethorphan in human
liver microsomes: a rapid HPLC assay to monitor cytochrome P450 2D6 activity, Pharmazie, 1996,52, 586
588.
Levamisole
CAS Registry No.: 14769-73-4, 16595-80-5 (HCI), 5036-02-2 (racemic), 5086-74-8 (racemic HCI)
Merck Index: 5486
Lednicer No.: 4 217
SAMPLE
Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Tbxi-Lab, Irvine CA), add
residue with 50 jxL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLCVARIABLES
mL/min)
Detector: UV 213.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 10 mL MeOH and 20 mL
water. Add 2 mL cell culture to the SPE cartridge, wash with 20 mL water, elute with 10 mL
MeOHrwater 80:20, inject a 10 JULL portion of the eluate.
HPLC VARIABLES
Column: 100 X 8 4 jxm RCM Nova-Pak C18
Flow rate: 1.3
Detector: UV 245
CHROMATOGRAM
Retention time: 2.2
OTHER SUBSTANCES
KEYWORDS
SPE
REFERENCE
Shu,Y.-Z.; Kingston,D.G.L; Van Tassell,R.L.; Wilkins,T.D. Metabolism of levamisole, an anti-colon cancer drug,
by human intestinal bacteria, Xenobiotica, 1991, 21, 737-750.
SAMPLE
Matrix: milk
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 10 mL ethyl acetate, with
10 mL MeOH, and with 10 mL water. Acidify 50 mL milk to pH 4.6 with 6 M HCl, centrifuge
at 10 at 4200 g for 15 min, remove the supernatant from the solid and the fat layer. Adjust
the pH of the supernatant to 11.0-11.2 with a few drops of 40% NaOH, add a 25 mL aliquot
to the SPE cartridge, elute with 10 mL water-saturated ethyl acetate. Evaporate the eluate to
dryness under reduced pressure at 35, reconstitute the residue in 200 |iL mobile phase.
HPLC VARIABLES
Guard column: normal phase universal guard cartridge kit (Whatman)
Column: 250 X 2.1 Spherisorb S5W
Mobile phase: DichloromethaneiMeOH 95:5
Flow rate: 0.2
Detector: UV 254
CHROMATOGRAM
Retention time: 12
KEYWORDS
normal phase; SPE; detection with GC for higher sensitivity
REFERENCE
Chappell,C.G.; Creaser,C.S.; Shepherd,M.J. Modified on-column interface for coupled high-performance liquid
chromatography-gas chromatography and its application to the determination of levamisole in milk,
J.Chromatogr., 1992, 626, 223-230.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
stilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol,
isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, loraze-
pam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medaze-
pam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, me-
phesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine,
methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide,
methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, meth-
yltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone,
naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic
acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, ox-
ymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine,
pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendi-
metrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phenter-
mine, phenylbutazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, predni-
solone, primidone, probenecid, progesterone, propiomazine, propranolol, propylparaben,
pseudoephedrine, puromycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine,
quinine, ranitidine, recinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, sal-
icylic acid, scopolamine, scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sul-
fadimethoxine, sulfaethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide,
sulfapyridine, sulfasoxizole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, theba-
ine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thior-
idazine, thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin,
tranylcypromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexy-
phenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vin-
camine, warfarin, yohimbine, zoxazolamine
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Inject 10 |JLL of a solution in RPMI-1640.
HPLC VARIABLES
Column: 250 X 4.6 5 |jim Hypersil MS C8
Mobile p h a s e : MeCN:water 75:25 containing 0.02% dimethyloctylamine and 0.02% trifluoroac-
etic acid
Detector: radioactivity
CHROMATOGRAM
KEYWORDS
tritium labelled
REFERENCE
Ho,N.RH.; Sims,S.M.; Vidmar,T.J.; Day,J.S.; Barsuhn,C.L.; Thomas,E.M.; Geary,T.G.; Thompson,D.R Theoret-
ical perspectives on anthelmintic drug discovery: Interplay of transport kinetics, physicochemical properties,
and in vitro activity of anthelmintic drugs, J.Pharm.ScL, 1994, 83, 1052-1059.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 3 mL Bakerbond Cyano SPE cartridge with 5 mL ethyl ace-
tate:hexane 50:50, do not allow to dry out. Pulverize frozen tissue samples to a fine powder. 3
g Powdered tissue + 2 g anhydrous sodium sulfate + 9 mL ethyl acetate + 500 JJLL 50% KOH
(w/v), homogenize (Silverson) for 1 min, centrifuge at 2000 rpm for 10 min. Remove 6 mL of
the ethyl acetate extract and add it to 6 mL hexane, mix, add to the SPE cartridge, wash with
5 mL chloroform:hexane 50:50, dry under vacuum for 10 min, elute with two 2 mL portions of
MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 70, reconstitute the
residue in 200 JULL, sonicate for 5 min, inject a 50 JULL aliquot.
HPLC VARIABLES
Guard column: 5 jxm LiChrospher 60 RP-select B guard column
Column: 125 X 4 5 |jim LiChrospher 60 RP-select B
Mobile phase: MeCN:THF:triethylamine:water 35:5:0.2:59.8 containing 100 mM ammonium
acetate
Flow rate: 1
Detector: MS, Hewlett-Packard 5989A, thermospray, vaporizer 15 below take-off temperature,
ion source 250, quadrupole 100, electron multiplier +200 V with respect to autotune voltage,
SIM, m/z 205
CHROMATOGRAM
Retention time: 3.5
Limit of detection: <3 ng/g
KEYWORDS
sheep; liver; kidney; muscle; SPE; LC-MS; thermospray
REFERENCE
Cannavan,A.; Blanchflower,W.J.; Kennedy,D.G. Determination of levamisole in animal tissues using liquid chro-
matography-thermospray mass spectrometry, Analyst, 1995, 120, 331-333.
Levobunolol
Merck Index: 5488
Lednicer No.: 2 110,215,280
SAMPLE
Sample preparation: Blood. Add MeCN to blood so that the ratio is 1:5. Remove a 1 mL aliquot
and add 10 |xL 2 |xg/mL metoprolol in water, add 1 mL water, adjust pH to 9.8-10.2 with 7-10
drops 100 mM NaOH, add 2 mL benzene (CAUTION! Benzene is a carcinogen!), shake on an
automatic shaker for 30 min, centrifuge, remove the organic phase and extract the aqueous
layer again with 2 mL benzene for 20 min. Combine the organic layers and evaporate them
under reduced pressure at 30, dissolve the residue in 50 jxL mobile phase, inject a 10-50 |xL
aliquot and determine amount of dihydrolevobunolol present (DHL-A). Add MeCN to blood so
that the ratio was 1:5. Remove a 1 mL aliquot and add 10 [LL 2 |xg/mL metoprolol in water,
add 1 mL water, adjust pH to 9.8-10.2 with 7-10 drops 100 mM NaOH, add 2 mL benzene,
shake on an automatic shaker for 30 min, centrifuge, remove the organic phase and extract
the aqueous layer again with 2 mL benzene for 20 min. Combine the organic layers and evap-
orate them under reduced pressure at 30, dissolve the residue in 200 |xL MeOH, add 5 mg
sodium borohydride, let stand at room temperature in a closed tube for 30 min, add 1 mL
water, add 300 mg NaCl, extract with 3 mL benzene for 20 min, centrifuge. Remove the organic
layer and evaporate it under reduced pressure, dissolve the residue in 50 JJLL mobile phase,
inject a 10-50 |xL aliquot and determine the amount of dihydrolevobunolol (DHL-B) that is now
present which represents the total amount of levobunolol and dihydrolevobunolol originally
present. Determine amount of levobunolol originally present by subtracting DHL-A from DHL-
B. Urine. 20-1000 jxL Urine + 200 ng metoprolol + 1 mL 200 mM pH 10.2 sodium borate buffer
(Sorensen), adjust pH to 9.8-10.2 with 100 mM NaOH (if necessary), add 500 mg NaCl, extract
with 4 mL benzene for 30 min, proceed as described above for blood samples. (Levobunolol
itself is not fluorescent so it must be reduced to the fluorescent derivative dihydrolevobunolol.
Addition of MeCN to freshly drawn blood stopped enzymatic conversion of levobunolol to
dihydrolevobunolol.)
HPLC VARIABLES
Column: 250 X 4.6 10 |xm |iondapak C18
Mobile phase: MeOH:water 48:52 containing 0.4% phosphoric acid and 0.2% heptanesulfonic
acid
Flow rate: 2
CHROMATOGRAM
Retention time: 3.8 (dihydrolevobunolol)
Internal standard: metoprolol (4.6)
Limit of detection: 0.5-1 ng/mL
KEYWORDS
human; dog
REFERENCE
Hengy,H-; K6lle,E.-U. Determination of levobunolol and dihydrolevobunolol in blood and urine by high-perfor-
mance liquid chromatography usingfluorescencedetection, J.Chromatogr., 1985, 338, 444-449.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 4.5 5 |xm Altex C18
Mobile phase: MeOH:water:phosphoric acid 48:52:0.1, containing 2 g/L sodium heptanesulfo-
nate, pH adjusted to 3.5 with 3 M NaOH
Flow rate: 1.5
Detector: UV 255
CHROMATOGRAM
REFERENCE
J.Pharm.ScL, 1990, 79, 153-157.
Levodopa
Merck Index: 5490
SAMPLE
Matrix: amniotic fluid, blood, CSF, urine
Sample preparation: Plasma. Condition a 100 mg Bond Elut SCX (propylbenzenesulfonic acid,
H+ form) SPE cartridge with 1 mL 50 mM HCl, 1 mL MeOH, 2 mL water, and 1 mL 50 rnM
HCl. 100 |xL Plasma + 100 |xL 250 JJLM norleucine in 100 mM HCl + 10 mg solid sulfosalicylic
acid + 800 |xL acetone or MeOH, mix, centrifuge, add a 50 jxL aliquot to the SPE cartridge,
wash with 2 mL water, elute with two 500 |xL portions of MeOH:water:triethylamine 40:40:20,
dry the eluate under vacuum, add 10 [LL MeOH: 1 M sodium acetate:triethylamine 40:40:20,
dry under vacuum at 70 mTorr, reconstitute with 20 (JLL MeOH:triethylamine:water:phenyli-
sothiocyanate 70:10:10:10, let stand at room temperature for 20 min, evaporate to dryness
under vacuum, reconstitute with 100 jxL MeCN:5 mM pH 7.4 sodium phosphate buffer 5:95,
inject a 20 JJLL aliquot. Dried blood. Add 25 fxL 250 |xM norleucine in 100 mM HCl to a 6 mm
filter paper disc containing dried blood, add 100 \LL MeCN, let stand for 30 min, centrifuge,
remove a 75 |xL aliquot of the supernatant, evaporate to dryness under reduced pressure, add
10 |xL MeOH: 1 M sodium acetate:triethylamine 40:40:20, dry under vacuum at 70 mTorr, re-
constitute with 20 JJLL MeOH:triethylamine:water:phenylisothiocyanate 70:10:10:10, let stand
at room temperature for 2 min, evaporate to dryness under vacuum, reconstitute with 50 |xL
MeCN:5 mM pH 7.4 sodium phosphate buffer 5:95, inject a 20 (xL aliquot. Amniotic fluid, CSF.
Mix amniotic fluid or CSF with an equal volume of 250 |xM norleucine in 100 mM HCl, filter
(Centrifree 10000 MW cutoff) while centrifuging at 2200 g. Evaporate a 50 |xL aliquot of the
ultrafiltrate to dryness under vacuum, add 10 |xL MeOH: 1 M sodium acetate:triethylamine 40:
40:20, dry under vacuum at 70 mTorr, reconstitute with 20 JJLL MeOH:triethylamine:water:
phenylisothiocyanate 70:10:10:10, let stand at room temperature for 20 min, evaporate to dry-
ness under vacuum, reconstitute with 50 (CSF) or 100 (amniotic fluid) |xL MeCN:5 mM pH 7.4
sodium phosphate buffer 5:95, inject a 20 JJLL aliquot. Urine. Dilute urine with water to a
creatinine concentration of 1 mM, mix an aliquot with an equal volume of 250 |xM norleucine
in 100 mM HCl, filter (Centrifree 10000 MW cutoff) while centrifuging at 2200 g. Evaporate a
50 |xL aliquot of the ultrafiltrate to dryness under vacuum, add 10 JJLL MeOH: 1 M sodium
acetate:triethylamine 40:40:20, dry under vacuum at 70 mTorr, reconstitute with 20 |xL MeOH:
triethylamine:water:phenylisothiocyanate 70:10:10:10, let stand at room temperature for 20
min, evaporate to dryness under vacuum, reconstitute with 100 |xL MeCN:5 mM pH 7.4 sodium
phosphate buffer 5:95, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 Pico-Tag amino acid column (Waters)
Mobile phase: Gradient. A was MeCN:70 mM pH 6.55 sodium acetate 2.5:97.5. B was MeCN:
MeOH:water 45:15:40
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: norleucine (55.07)
OTHER SUBSTANCES
Extracted: p-alanine, alanine, alloisoleucine, a-aminoadipic acid, 4-aminobenzoic acid, gamma-
aminobutyric acid, p-amino-n-butyric acid, gamma-amino-n-butyric acid, 4-aminohippuric acid,
P-aminoisobutyric acid, 4-aminophenylacetic acid, a-aminophenylacetic acid, 3-amino-3-phen-
ylpropionic acid, 8-amino-n-valeric acid, ammonia, anserine, arginine, asparagine, aspartic
acid, aspartylglucosamine, carnosine, citrulline, cystathionine, cysteic acid, cysteine, eysteine-
homocysteine (mixed disulfide), cystine, ethanolamine, ethionine, ethylamine, galactosamine,
glucosamine, glutamic acid, glutamine, glutathionine (oxidized), glycine, glycylglycine, gly
cylhistidine, glycylleucine, glycylphenylalanine, glycyltyrosine, histidine, homoarginine, hom-
ocitrulline, homoserine, homocystine, 3-hydroxyanthranilic acid, 3-hydroxykynurenine, hy-
droxyproline, isoleucine, kynurenine, leucine, lysine, methionine sulfone, methionine, 3-meth-
ylhistidine, 1-methylhistidine, ornithine, phenylalanine, phosphoethanolamine, phosphoserine,
proline, sarcosine, serine, serotonin, taurine, threonine, tromethamine, tryptophan, tyrosine,
valine
Noninterfering: cadaverine, 2-phenylethylamine
KEYWORDS
derivatization; SPE; ultrafiltrate; plasma; dried blood
REFERENCE
Davey,J.R; Ersser,R.S. Amino acid analysis of physiological fluids by high-performance liquid chromatography
with phenylisothiocyanate derivatization and comparison with ion-exchange chromatography,
J.Chromatogr., 1990, 528, 9-23.
SAMPLE
Matrix: blood
Sample preparation: Mix 450 |xL plasma with 50 JJLL 310 |x g/L iso-homovanilic acid in anti-
oxidant solution. Dialyze this solution using a Carnegie Medicin (Stockholm) microdialysis
system. Perfuse 10 mm microdialysis probes with Ringer solution at 2 |x L/min. Collect dialy-
sate for each plasma sample over 20 min in a vial containing 80 jxL antioxidant solution, inject
a 100 jxL aliquot. (Antioxidant solution was 10 mM HCl containing 1 g/L sodium metabisulfite
and 0.1 g/L Na2EDTA.)
HPLC VARIABLES
Guard column: 30 X 4 Bondapak C18/Corasil 37-50 |x m
Column: 250 X 4.8 5 jjim ODS (Beckman, CA)
Mobile phase: MeOH:buffer 20:80 (Buffer was 70 mM pH 2.55 NaH2PO4 containing 2.08 mM
sodium octanesulfonate and 80 |x M EDTA.)
Flow rate: 1
Detector: E , Waters 460 containing an electrochemical cell fitted with a glassy carbon working
electrode and an Ag/AgCl reference electrode; the detector potential is + 0.8 V vs the reference
electrode
CHROMATOGRAM
Retention time: 3.5
Internal standard: iso-homovanilic acid (10)
Limit of detection: 1.1 nM/L
OTHERSUBSTANCES
Extracted: dihydroxyphenylacetic acid, dopamine, homovanilic acid
KEYWORDS
plasma; pharmacokinetics; dialysate
REFERENCE
Dethy,S.; Laute,M.A.; Van Blercom,N.; Damhaut,R; Goldman,S.; Hildebrand,J. Microdialysis-HPLC for plasma
levodopa and metabolites monitoring in parkinsonian patients, Clin.Chem., 1997, 43, 740-744.
SAMPLE
Matrix: blood
Sample preparation: Total levodopa. Add 300 fxL 60 mM trichloroacetic acid to 1 mL plasma,
place the mixture in an ice bath for 10 min, centrifuge at 5000 g for 10 min, inject a 20 jxL
aliquot of the supernatant. Unbound levodopa. Add 250 |xL plasma to 5 |JLL 4 M orthophosphoric
acid, centrifuge through a membrane Minicent 10 (Bio-Rad) at 8000 g at 25 for 30 min, inject
a 20 |xL aliquot of the ultrafiltrate.
HPLCVARIABLES
Column: 45 X 4.6 5 (xm C18 (Bio-Rad)
Mobile phase: MeCN: 1.4 mM sodium dodecylsulphate:50 mM KH2PO4 16:42:42, pH 2.8
Flow rate: 1
Detector: E, ESA Coulochem 5100, analytical cell +50 mV, -300 mV, conditioning cell +300 mV
CHROMATOGRAM
Retention time: 3.7
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Rizzo,V.; Memmi,M.; Moratti,R.; Melzi,E.; Perucca,E. Concentrations of L-dopa in plasma and plasma ultra-
filtrates, J.Pharm.BiomedAnaL, 1996, 14, 1043-1046.
SAMPLE
Matrix: blood
Sample preparation: 0.5 mL Plasma + 50 |xL 4 M perchloric acid + 50 |JLL 1 |xg/mL dihydroxy-
benzylamine in 0.1 M perchloric acid, centrifuge at 1500 g for 10 min. Remove 300 |xL super-
natant and centrifuge it at 1600 g through a 0.2 fim regenerated cellulose filter, inject a 20 |xL
aliquot.
HPLCVARIABLES
Guard column: 20 X 4.6 5 |xm Biophase ODS + 5Ox 4.6 Pelliguard LC-18
Column: 250 X 4.6 5 fjun Biophase ODS or 250 X 4.6 Phase II ODS (both from Bioanalytical
Systems)
Mobile phase: MeOHrbuffer 5:95 (Buffer was 20 mM sodium citrate, 100 mM NaH2PO4, 0.15
mM, and 1.25 mM heptanesulfonic acid, pH 3.2.)
Flow rate: 1-1.5
Detector: E, Bioanalytical Systems LC-150 in dual-parallel mode, channel 1 700 mV 200 nA f.s.
for levodopa and 3-O-methyldopa, channel 2 560 mV 10 nA f.s. for dopamine, carbidopa, and
dihydroxyphenylacetic acid, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 5.1
OTHER SUBSTANCES
Simultaneous: 3-O-methyldopa, dopamine, carbidopa, dihydroxyphenylacetic acid
KEYWORDS
plasma
REFERENCE
Cedarbaum,J.M.; Williamson,R-; Kutt,H. Simultaneous determination of levodopa, its metabolites and carbi-
dopa in clinical samples, J.Chromatogr., 1987, 415, 393-399.
SAMPLE
Matrix: blood
mM pH 7 phosphate buffer. Add 1 mL plasma to column, elute with 5.5 mL water, discard first
1 mL. Add next 4.5 mL to 0.5 mL 0.5 M perchloric acid, centrifuge, inject 10 |xL aliquot of
supernatant.
HPLC VARIABLES
Column: 250 x 4.6 5 |xm Nucleosil C18
Flow rate: 0.9
trode -0.30 V
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Simultaneous: O-methyldopa, carbidopa, dihydroxyphenylacetic acid (DOPAC)
KEYWORDS
plasma
REFERENCE
plasma of parkinsonian patients under L-dopa therapy, J.Chromatogr., 1988, 459, 341349.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 JULL 1.25 jutg/mL a-ethyldopa in 0.1 M HCl + 100 jxL 4
M perchloric acid, vortex, centrifuge at 2000 g for 10 min, inject a 60 (JLL aliquot of the super-
natant (keep sample tray at 6 1).
HPLC VARIABLES
Guard column: 45 X 4.6 37-40 |xm Whatman pellicular-ODS followed by 45 X 4.6 5 |xm Ultra
sphere-IP C18
Column: 250 X 4.6 5 |xm Ultrasphere IP C18
Mobile phase: MeOH:20 mM orthophosphoric acid and 4 mM sodium octanesulfonate 25:75 ad-
justed to pH 2.8 0.05 with 50% NaOH
Flow rate: 1
Detector: E, BAS LC-4B, 0.75 V vs Ag/AgCl, 5 nA full scale for carbidopa, 20 nA full scale for 3-
O-methyldopa and levodopa
CHROMATOGRAM
Retention time: 5.9
Internal standard: a-ethyldopa (15.4)
OTHER SUBSTANCES
Extracted: carbidopa, 3-O-methyldopa
KEY WORDS
plasma; stabilize plasma sample immediately with EDTA and 2 mg/mL sodium metabisulfite
REFERENCE
Titus,D.C; August,T.R; Yeh,K.C; Eisenhandler,R.; Bayne,W.R; Musson,D.G. Simultaneous high-performance
liquid chromatographic analysis of carbidopa, levodopa and 3-O-methyldopa in plasma and carbidopa,
levodopa and dopamine in urine using electrochemical detection, J.Chromatogr., 1990, 534, 87-100.
SAMPLE
Matrix: blood
Sample preparation: 4 mL Plasma + 500 u,L 20 mg/mL ascorbic acid solution, vortex for 30 s.
1 mL Aliquot + 75 mg acid washed alumina + 100 /uuL 1 jutg/mL 3,4-dihydroxybenzylamine
hydrobromide in buffer, vortex, add 1 mL 1.5 M pH 8.6 TRIS buffer, shake at 230 oscillations/
min for 15 min. Allow to settle and discard plasma, wash the alumina twice by shaking with
5 mL water for 10 min. To the washed alumina add 900 (xL buffer, vortex for 20 s, allow to
settle, inject a 50 |xL aliquot of the supernatant. (Buffer was 200 mM phosphoric acid contain-
ing 3.3 |xm EDTA and 6.7 jxm potassium metabisulfite.)
HPLCVARIABLES
Mobile phase: MeCN:buffer 8:92 (Buffer was pH 2.6 550 mM NaH2PO4 containing 1 mM sodium
octyl sulfate and 0.7 mM EDTA.)
Flow rate: 1.5
Detector: E, Bioanalytical Systems LC-4B, glassy carbon electrode, Ag/AgCl reference electrode,
0.75 V.
CHROMATOGRAM
Retention time: 3.3
OTHER SUBSTANCES
Extracted: carbidopa
Noninterfering: caffeine, ibuprofen, aspirin, nicotine, acetaminophen, theophylline
KEYWORDS
plasma; SPE
REFERENCE
Miller,R.B.; Dehelean,L.; B61anger,L. Determination of carbidopa and levodopa in human plasma by high-
performance liquid chromatography with electrochemical detection, Chromatographia, 1993, 35, 607-612.
SAMPLE
Matrix: blood
Sample preparation: 0.5 mL Plasma + 25 |xL 2 jxg/mL 3,4-dihydroxybenzylamine in 0.4 M
perchloric acid + 25 jxL 70% perchloric acid, vortex 1 min, keep on ice for 1 min, store at -80.
Allow to thaw at +4, vortex 1 min, centrifuge at 1200 g at +4 for 15 min. Remove 300 |xL
supernatant and add it to 200 |xL 2 M pH 4.5 potassium citrate buffer, centrifuge at 1200 g at
+4 for 10 min, inject 50 fxL of the supernatant.
HPLC VARIABLES
Column: 250 X 4.6 5 |m Spherisorb C18
Mobile phase: MeOH:MeCN:buffer 8:4:88 (Buffer was 50 mM pH 3.2 phosphate containing 3.5
mM heptanesulfonic acid and 0.05 mM EDTA.)
Flow rate: 1
Detector: E, ESA Model 5100, Model 5020 guard cell +0.6 V, Model 5010 analytical cell, DET 1
+0.35 V, DET 2 -0.35 V, both DET 1 and DET 2 monitored
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: 3-O-methyldopa, dopamine, L-DOPA methyl ester
KEYWORDS
plasma; rat; human
REFERENCE
Rondelli,L; Acerbi,D.; Mariotti,E; Ventura,P. Simultaneous determination of levodopa methyl ester, levodopa,
3-O-methyldopa and dopamine in plasma by high-performance liquid chromatography with electrochemical
SAMPLE
Sample preparation: Plasma. 20 JULL Plasma + 180 |xL isoproterenol in 100 mM perchloric acid
containing 10 |xM disodium EDTA, centrifuge at 4 at 4000 rpm for 10 min. Filter (0.45 |xm
cellulose acetate) the supernatant and inject a 10 |xL aliquot of the supernatant. Tissue. Ho-
mogenize (Polytron PT 10-35) 50 mg brain tissue + 500 uL isoproterenol in 100 mM perchloric
acid containing 10 \xM disodium EDTA at 15000 rpm for 10 s, centrifuge at 4 at 4000 rpm for
10 min. Filter (0.45 |xm cellulose acetate) the supernatant and inject a 10 uL aliquot of the
supernatant.
HPLCVARIABLES
Guard column: Supelcosil LC-18-DB
Column: Supelcosil LC-18-DB
Mobile phase: MeOH: 10 mM pH 4.4 citrate buffer 10:90 containing 10 |xM disodium EDTA and
0.5 mM sodium 1-octanesulfonate
Flow rate: 1
Detector: E, EICOM Co. ECD-100, +0.7 V, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 4
KEYWORDS
plasma; rat; brain; pharmacokinetics
REFERENCE
Sato,S.; Koitabashi,T.; Koshiro,A- Pharmacokinetic and pharmacodynamic studies of L-dopa in rats. I. Phar-
macokinetic analysis of L-dopa in rat plasma and striatum, Biol.Pharm.Bull., 1994, 17, 1616-1621.
SAMPLE
cartridge (Tosoh) with 10 mL water. Plasma. 700 |xL Plasma + 50 |xL 700 nM 3,4-dihydroxy-
benzylamine + 350 |xL 2 M perchloric acid, mix, centrifuge at 4 at 1000 g for 15 min. Remove
a 700 |xL aliquot of the supernatant and add it to 30 |xL 2 M potassium carbonate, centrifuge
at 4 at 1000 g for 5 min. Add a 500 |xL aliquot of the supernatant to the SPE cartridge, wash
with 1 mL water, wash with 500 jxL EtOH.water 50:50, wash with 5 mL water, elute with 500
|xL 2 M sodium perchlorate, filter (0.2 |xm), inject a 50 |JLL aliquot of the filtrate. Urine. Acidify
urine collected over 24 h with 10 mL 6 M HCl. 500 pX. Urine + 25 |xL 10 jxM 3,4-dihydroxy-
benzylamine + 25 |JLL 40 jxM ferulic acid + 500 |xL 1 M perchloric acid, mix, centrifuge at 4 at
1000 g for 15 min. Remove a 700 |xL aliquot of the supernatant and add it to 30 u,L 2 M
potassium carbonate, centrifuge at 4 at 1000 g for 5 min, add a 500 |JLL aliquot of the super-
natant to the SPE cartridge, wash with 1.5 mL water, wash with 500 jxL EtOH:water 50:50,
wash with 5 mL water, elute with 500 jxL 2 M sodium perchlorate, filter (0.2 jxm), inject a 50
(xL aliquot of the filtrate.
HPLC VARIABLES
Column: 150 X 4.6 5 ^m TSK-gel ODS-80TM (Tosoh)
Mobile phase: Gradient. A was buffer. B was MeCN:MeOH:bufifer 8:12:80, pH 3.1. A:B 100:0 for
Chromatogr. 1989, 467, 237).
Flow rate: 1
237).
CHROMATOGRAM
Retention time: 9.5
Limit of detection: 12 nM (urine), 10 nM (plasma)
OTHER SUBSTANCES
Extracted: dopamine, epinephrine, metanephrine, 3-methoxytyramine, norepinephrine
KEYWORDS
REFERENCE
Nohta,H-; Yamaguchi,E.; Ohkura,Y.; Watanabe,H- Measurement of catecholamines, their precursor and metab-
SAMPLE
(Tosoh) with 10 mL water and 2 mL 200 mM pH 5.0 sodium phosphate buffer. Plasma. 700 jxL
Plasma + 30 |xL 700 nM isoproterenol + 50 jxL 7 |iM 3,4-dihydroxyphenylpropanoic acid + 350
|JLL 2 M perchloric acid, mix, centrifuge at 4 at 1000 g for 15 min. Remove a 700 |xL aliquot
of the supernatant and adjust the pH to 1.5-2.0 with about 150 jxL 2 M potassium carbonate,
mL water, elute with 300 |xL MeOH:2 M sodium perchlorate 7:93, filter (cellulose acetate mem-
brane), inject a 100 |xL aliquot of the filtrate. Urine. Collect human urine for 24 h in the
presence of 10 mL 6 M HCl. 500 |xL Urine + 10 |xL 15 |xM isoproterenol + 25 |xL 800 |xM 3,4-
for 15 min. Remove a 700 jxL aliquot of the supernatant and adjust the pH to 1.5-2.0 with
about 130 JJLL 2 M potassium carbonate, centrifuge at 4 at 1000 g for 5 min, add the super-
wash with 5 mL water, elute with 500 (xL 1.5 M KCl in MeOH:100 mM HCl 7:93, filter (cellulose
acetate membrane), inject a 100 |xL aliquot of the filtrate.
HPLC VARIABLES
Column: 250 X 4.6 5 ^m TSK-gel ODS-80TM (Tosoh)
Flow rate: 0.8
meso-l,2-diphenylethylenediamine in EtOH.water 70:30 containing 130 mM sodium
methylate.)
CHROMATOGRAM
Retention time: 23
Limit of detection: 7-9 nM
OTHER SUBSTANCES
Extracted: dopamine, epinephrine, metanephrine, 3-methoxytyramine, norepinephrine, normet-
anephrine
KEYWORDS
REFERENCE
Jeon,H.-K; Nohta,H.; Ohkura,Y. High-performance liquid chromatographic determination of catecholamines
chemical oxidation followed byfluorescencereaction, Anal.Biochem., 1992, 200, 332-338.
SAMPLE
alumina + 200 \xL 1 M pH 8.6 Tris-EDTA buffer, mix for 10 min, discard plasma. Wash the
alumina three times with 3 mL water and dry it. Add 125 |JLL 500 mM phosphoric acid, after
1 min inject a 100 jxL aliquot. (Ann. Clin. Biochem. 1985, 22, 194-203)
HPLC VARIABLES
Column: 250 X 4.5 5 ^m Ultratechsphere
mL MeOH
Flow rate: 1
-0.35 V
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: norepinephrine, metanephrine, epinephrine, 3-methoxytyrosine, normetaneph-
rine, dihydroxyphenylacetic acid, dopamine
KEYWORDS
plasma
REFERENCE
Clin.Chem., 1993, 39, 629-634.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Powder tablets or contents of capsules, weigh out an amount equivalent
to about 100 mg levodopa, add 30 mL 0.1 M HCl, sonicate, make up to 50 mL with 0.1 M HCl,
mix, filter (0.45 |xm), discard first 5 mL filtrate. 5 mL Filtrate + 10 mL 2 mg/mL methyldopa
in 0.1 M HCl, make up to 100 mL with mobile phase, mix, inject a 20 jxL aliquot.
HPLCVARIABLES
Mobile phase: 3% aqueous acetic acid
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Retention time: 3
Internal standard: methyldopa (4.5)
KEYWORDS
capsules; tablets
REFERENCE
Ting,S. Liquid chromatographic determination of levodopa and levodopa-carbidopa in solid dosage forms: col-
laborative study, JAssoc.OffAnaLChem., 1987, 70, 987-990.
SAMPLE
Sample preparation: Dissolve in mobile phase, filter, inject an aliquot.
HPLC VARIABLES
Mobile phase: MeOH:50 mM ammonium acetate adjusted to pH 4.1 with 0.6 M acetic acid 1:99
Flow rate: 0.9
Detector: E, Coulochem model 5100A, screen electrode +0.3 V, sample electrode +0.6 V and UV
280
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: hydroxydopa, carbidopa, methyldopa, methoxytyrosine, methylcarbidopa,
impurities
KEYWORDS
stability-indicating; tablets
REFERENCE
Kafil,J.B.; Dhingra,B.S. Stability-indicating method for the determination of levodopa, levodopa-carbidopa and
related impurities, J.ChromatogrA, 1994, 667, 175-181.
SAMPLE
HPLC VARIABLES
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Simultaneous: carbidopa, dopamine, epinephrine, hydrochlorothiazide, isoproterenol, methyl-
dopa, norepinephrine, phenylephrine
KEYWORDS
REFERENCE
Villanueva Camanas,R-M.; Sanchis Mallols,J.M.; Torres Lapasi6,J.R; Ramis-Ramos,G. Analysis of pharmaceu-
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 100 (xg/mL solution in mobile phase.
HPLC VARIABLES
Column: 150 X 4 5 ixm Crownpak CR(+) immobilized crown ether
Mobile phase: MeOH:0.1% pH 1.9 perchloric acid 15:85
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: baclofen, norephedrine, primaquine
KEYWORDS
chiral; comparison with capillary electrophoresis
REFERENCE
Nishi,H.; Nakamura,K; Nakai,H.; Sato,T. Separation of enantiomers and isomers of amino compounds by
capillary electrophoresis and high-performance liquid chromatography utilizing crown ethers,
J.ChromatogrA, 1997, 757, 225-235.
SAMPLE
Matrix: tissue
Sample preparation: Prepare a 70 X 5 SPE column of Sephadex G 10 in a Pasteur pipette,
wash with 3 mL 20 mM ammonia and 3 mL 10 mM formic acid, let stand for 10 days. Ho-
mogenize up to 150 mg rat brain in 1 mL 100 mM perchloric acid, centrifuge at 4000 g at 4
for 15 min, add 500 |xL of the supernatant to the SPE column, wash with 2.5 mL 10 mM formic
acid, elute with 1 mL 10 mM formic acid followed by 1.5 mL 5 mM Na2HPO4, inject an aliquot
of the eluate.
HPLC VARIABLES
Column: Nucleosil 5 C18
Mobile phase: pH 5.5 Buffer prepared from 200 mM Na2HPO4 and 100 mM citric acid
Flow rate: 0.8
Detector: E, rotating disc electrode, 500 mV
CHROMATOGRAM
Retention time: 4.5
Limit of detection: 0.015 nmole/g
OTHER SUBSTANCES
Extracted: dopamine, uric acid
KEYWORDS
rat; brain; SPE
REFERENCE
Westerink,B.H.C; Mulder,T.B A. Determination of picomole amounts of dopamine, noradrenaline, 3,4-dihy-
droxyphenylacetic acid, homovanillic acid, and 5-hydroxyindolacetic acid in nervous tissue after one-step
purification on Sephadex G-IO, using high-performance liquid chromatography with a novel type of elec-
trochemical detection, J.Neurochem., 1981, 36, 1449-1462.
SAMPLE
Matrix: urine
Sample preparation: 100 JJLL Urine + 100 JJLL solution containing 55 mM ascorbic acid and 55
mM disodium EDTA + 25 JJLL 1.25 |xg/mL a-ethyldopa in 0.1 M HCl + 25 mg alumina + 1 mL
2 M pH 8.6 Tris-HCl buffer in a microfilter tube (Centrex, Schleicher & Schuell), vortex 5 min,
allow to stand for 10 min, filter off water, wash with 5 mL water, add 5 mL water, centrifuge
at 3000 g, vortex with 400 JJLL 0.2 M perchloric acid containing 11 mM disodium EDTA and 0.4
M sodium metabisulfite, centrifuge at 9000 g for 5 min, inject 50 |xL of filtrate. (Stabilize each
10 mL urine sample immediately with 0.5 mL 0.1 M HCl and 1 mL solution containing 55 mM
ascorbic acid and 55 mM disodium EDTA.)
HPLC VARIABLES
Guard column: 40 X 4.6 Bio-Sil ODS-10 (Bio-Rad)
Mobile phase: MeOH:water 22.5:77.5 containing 20 mM citric acid, 20 mM Na2HPO4, 4 mM
sodium octanesulfonate, and 0.05 mM disodium EDTA, pH adjusted to 2.74 0.01 with 2 M
citric acid
Detector: E, BAS LC-4B, 0.54 V vs Ag/AgCl, 50 nA full scale
CHROMATOGRAM
Retention time: 6
Internal standard: a-ethyldopa (14)
OTHER SUBSTANCES
Extracted: carbidopa, dopamine
KEYWORDS
SPE
REFERENCE
Titus,D.C; August,T.R; Yeh,KC; Eisenhandler,R.; Bayne,W.E; Musson,D.G. Simultaneous high-performance
liquid chromatographic analysis of carbidopa, levodopa and 3-O-methyldopa in plasma and carbidopa,
levodopa and dopamine in urine using electrochemical detection, J.Chromatogr., 1990, 534, 87100.
Levonordefrin
CAS Registry No.: 829-74-3, 138-61-4 (racemic HCI)
Merck Index: 6785
Lednicer No.: 1 68
SAMPLE
HPLC VARIABLES
Mobile phase: MeOHibuffer 30:70 (Buffer was 10 mM sodium 1-octanesulfonate in 0.2% acetic
acid.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 12
OTHERSUBSTANCES
Simultaneous: norepinephrine, epinephrine, isoproterenol, phenylephrine, metaraminol,
impurities
KEYWORDS
REFERENCE
forms, J.Assoc.Off.Anal.Chem., 1991, 74, 289-291.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 u.m Partisil ODS-3
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Simultaneous: epinephrine, isoproterenol, metaraminol, phenylephrine
REFERENCE
Levorphanol
CAS Registry No.: 77-07-6, 5985-38-6 (tartrate dihydrate), 125-72-4 (tartrate)
Merck Index: 5496
Lednicer No.: 1 293
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 20 ng levallorphan, mix, add 1 mL 1M pH 9 borate buffer,
add 10 mL hexanerethyl acetate 90:10, shake for 10 min, centrifuge at 2500 rpm for 10 min.
Remove 9 mL of the organic layer and evaporate it to dryness under a stream of nitrogen at
50, reconstitute the residue in 200 |xL mobile phase, inject a 100 |xL aliquot.
HPLCVARIABLES
Column: 300 X 4 10 |xm jjiBondapak C18
Mobile phase: MeCN:buffer 30:70 containing 0.1 mM EDTA (Buffer was 10 mM NaCl adjusted
to pH 4.8 with 1 M HCl.)
Flow rate: 1
Detector: E, Bioanalytical Systems LC-4B, glassy carbon electrode +1.00 V, Ag/AgCl reference
electrode
CHROMATOGRAM
Retention time: 6.5
Internal standard: levallorphan (9.5)
OTHER SUBSTANCES
Simultaneous: 6-acetylmorphine, diamorphine, oxymorphine, pentazocine
Noninterfering: acetaminophen, 1-a-acetylmethodol, caffeine, codeine, hydrocodone, hydromor-
phine, meperidine, morphine, oxycodone, propoxyphene
KEYWORDS
REFERENCE
Lucek,R.; Dixon,R. Quantitation of levorphanol in plasma using high-performance liquid chromatography with
electrochemical detection, J.Chromatogr., 1985, 341, 239-243.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 (JLL 2.58 jxg/mL laudanosine in MeCN + 500 (xL sat-
urated sodium carbonate solution, vortex for 10 s, add 5 mL chloroform, vortex for 10 s, mix
on a rocking mixer for 40 min, centrifuge at 2000 g for 25 min. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 400 jxL mobile
phase, inject a 300 |xL aliquot. (Hydrolyze conjugates by heating 1 mL plasma with 1 mL 3000
U/mL p-glucuronidase (Helix pomatia type H-I (Sigma)) in 100 mM pH 5.0 sodium citrate at
37 for 2 h, proceed as above.)
HPLC VARIABLES
Column: 150 X 4.6 Spherisorb 5-CN
Mobile phase: MeCN:water:triethylamine 10:89:1 adjusted to pH 6 with orthophosphoric acid
Flow rate: 1
CHROMATOGRAM
Internal standard: laudanosine (8.603)
OTHER SUBSTANCES
Extracted: guaifenesin
KEYWORDS
plasma
REFERENCE
Stavchansky,S.; Demirbas,S.; Reyderman,L.; Chai,C.-K. Simultaneous determination of dextrorphan and guai-
fenesin in human plasma by liquid chromatography with fluorescence detection, J.Pharm.Biomed.Anal.,
1995, 13, 919-925.
SAMPLE
Sample preparation: Dilute urine 3:1 or more with water. Vortex 1 mL CSF, plasma, or diluted
urine with 100 |xL 100 ng/mL IS, add 500 uL saturated sodium carbonate, mix, add 5 mL
hexane containing 0.1% n-octylamine. Vortex for 60 s, centrifuge at 2000 g for 10 min. Re-
extract aqueous phase with 5 mL hexane containing 0.1% n-octylamine, evaporate the com-
bined hexane extracts to dryness under a stream of nitrogen in a 50 water bath. Reconstitute
residue with 150 u,L 100 mM HCl, inject a 100 u,L aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 5 |xm CN
Column: 220 X 4.6 5 |xm Brownlee Spheri-5CN (Applied Biosystems, USA)
Mobile phase: MeCN:n-octylamine:water 19:0.05:80.95 adjusted to pH 2.8 with phosphoric acid
Flow rate: 1
CHROMATOGRAM
Retention time: 5.3
Limit of quantitation: 1 ng/mL (CSF, plasma), 5 ng/mL (urine)
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Kimiskidis,V.K.; Kazis,A.D.; Niopas,I. Simultaneous determination of dextromethorphan and dextrorphan in
human plasma, urine and cerebrospinal fluid by HPLC with fluorescence detection, J.Liq.
Chromatogr.Rel.TechnoL, 1996, 19, 1267-1275.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 100 JJLL 1 |xg/mL pholcodine in water + 500 uX.
saturated sodium carbonate, mix, add 4 mL diethyl ether:chloroform:isopropanol 20:9:1, mix
on a rotary mixer for 10 min, centrifuge at 2000 g for 10 min. Remove the organic layer and
add it to 100 uL 100 mM HCl, mix on a rotary mixer for 10 min, centrifuge at 2000 g for 5
min, inject a 10-50 |xL aliquot of the aqueous layer. Urine. 500 (xL Urine + 50 |xL 50 (xg/mL
pholcodine in water + 500 u,L saturated sodium carbonate, mix, add 4 mL diethyl etherxhlo-
roform:isopropanol 20:9:1, mix on a rotary mixer for 10 min, centrifuge at 2000 g for 10 min.
Remove the organic layer and add it to 100 |xL 100 mM HCl, mix on a rotary mixer for 10
min, centrifuge at 2000 g for 5 min, inject a 10-50 |xL aliquot of the aqueous layer. (If desired,
hydrolyse 500 |xL plasma or urine with 500 |xL 8000 U/mL p-glucuronidase (Helix pomatia,
type H-I, Sigma) in 200 mM pH 5 acetate buffer at 37 for 16 h, proceed as above.)
HPLC VARIABLES
Column: 150 X 4.6 5 jxm Spherisorb cyano
Mobile phase: MeCN:water:triethylamine 17:82.94:0.06, adjusted to pH 3.0 with orthophos-
phoric acid
Flow rate: 1
CHROMATOGRAM
Retention time: 5.1
Internal standard: pholcodine (6.3)
OTHER SUBSTANCES
Extracted: metabolites, dextromethorphan
Noninterfering: acetaminophen, clofibrate, codeine, cyclophosphamide, diclofenac, digoxin, dox-
epin, doxorubicin, estrogens, flucloxacillin, folic acid, furosemide, metformin, metoclopramide,
miconazole, minoxidil, morphine, nifedipine, nitroglycerin, norcodeine, norethisterone, oxaze-
pam, oxethazaine, prednisolone, pseudoephedrine, quinine, spironolactone, temazepam, tol-
butamide, warfarin
KEY WORDS
plasma
REFERENCE
Chen,Z.R.; Somogyi,A.A.; Bochner,F. Simultaneous determination of dextromethorphan and three metabolites
in plasma and urine using high-performance liquid chromatography with application to their disposition in
man, Ther.Drug Monit., 1990, 12, 97-104.
SAMPLE
Sample preparation: 600 |xL Microsomal incubation + 600 |xL saturated sodium carbonate,
place on ice, add 150 |xL 12.5 |xg/mL betaxolol, extract with 5 mL ethyl acetate. Remove the
organic layer and add it to 300 |xL 0.5% orthophosphoric acid, extract, inject an aliquot of the
aqueous layer.
HPLC VARIABLES
Column: 5 mm i.d. 4 |xm Nova-Pak phenyl radial-Pak
Mobile phase: MeCN:MeOH:0.05% orthophosphoric acid 24:10:66
Flow rate: 1.6
CHROMATOGRAM
Retention time: 5.5
Internal standard: betaxolol
OTHER SUBSTANCES
KEYWORDS
rat; liver
REFERENCE
Laslett,T.J.; Alvarez,R; Nation,R.L.; Evans,A.M.; Scott,S.D.; Stupans,I. Effect of cyclophosphamide administra-
tion on the activity and relative content of hepatic P4502D1 in rat, Xenobiotica, 1995, 25, 1031-1039.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: buprenorphine, dextromoramide, phenoperidine, fentanyl, etorphine, piritram-
ide, noscapine, papaverine, naloxone, dextropropoxyphene, nalorphine, phenazocine, norpipa-
none, levallorphan, hydroxypethidine, normethadone, meperidine, dipipanone, diamorphine,
pentazocine, acetylcodeine, monoacetylmorphine, thebacon, oxycodone, thebaine, norlevor-
phanol, methadone, benzylmorphine, ethylmorphine, morphine-N-oxide, codeine, codeine-N-ox-
ide, morphine, ethoheptazine, morphine-3-glucuronide, pholcodeine, norpethidine, hydroco-
done, dihydrocodeine, dihydromorphine, norcodeine, normorphine, pemoline, benzphetamine,
diethylpropion, mazindol, tranylcypromine, caffeine, fenethyline, phendimetrazine, methyl-
phenidate, phenelzine, epinephrine, pipradol, phenylpropanolamine, fencamfamin, chlorphen-
termine, norpseudoephedrine, phentermine, fenfluramine, methylenedioxyamphetamine, am-
phetamine, normetanephrine, 4-hydroxyamphetamine, bromo-STP, STP, prolintane,
ephedrine, methylephedrine, dimethylamphetamine, methamphetamine, mescaline, mephen-
termine
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
feine, cocaine, codeine, doxylamine, fluoxetine, glutethimide, hexobarbital, hypoxanthine, LSD,
meperidine, mephobarbital, methadone, methylphenidate, methyprylon, N-norcodeine, oxaze-
pam, oxycodone, phenylpropanolamine, prilocaine, procaine, terfenadine
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
isoxsuprine, ivermectin, ketamine, ketoprofen, lidocaine, lorazepam, lormetazepam, loxapine,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: MeCN:0.025% phosphoric acid:buffer 60:25:15 (Buffer was 9 mL concentrated
phosphoric acid and 10 mL triethylamine in 900 mL water, adjust pH to 3.4 with dilute phos-
phoric acid, make up to 1 L.)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
azepam, flurbiprofen, fluvoxamine, furosemide, glutethimide, glyburide, guaifenesin, haloper-
idol, homatropine, hydralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxy-
chloroquine, hydroxyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen,
ketorolac, labetalol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefenamic
pofol, propranolol, protriptyline, quazepam, quinidine, quinine, racemetnorphan, ranitidine,
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 1 mL 100 mM pH 5 acetate buffer + 20 |xL p-glucuronidase/
P-arylsulfatase (Helix pomatia, Boehringer Mannheim) + 50 |xL 600 mM sodium azide in water,
heat at 37 for 12 h. Inject a 100 |xL aliquot onto column A and elute to waste at 0.5 mL/min,
after 3 min elute contents of column A onto column B at 1.4 mL/min, monitor the effluent from
column B.
HPLCVARIABLES
Column: A 30 X 4 10 |xm LiChrosorb CN; B 250 X 4.6 5 |xm Spherisorb phenyl
Mobile phase: MeCN: 10 mM KH2PO4 50:50, adjusted to pH 4 with phosphoric acid
Flow rate: A 0.5; B 1.4
CHROMATOGRAM
Retention time: 9.5
Limit of detection: <50 ng/mL (UV)
OTHER SUBSTANCES
KEYWORDS
column-switching
REFERENCE
Motassim,N.; Decolin,D.; Le Dinh,T.; Nicolas,A-; Siest,G. Direct determination of dextromethorphan and its
three metabolites in urine by high-performance liquid chromatography using a precolumn switching system
for sample clean-up, J.Chromatogr., 1987, 422, 340-345.
SAMPLE
Matrix: urine
Sample preparation: Condition a Bond Elut silica modified with carboxylic acid ion-exchange
groups SPE cartridge with 1 mL MeCN: 100 mM HCl 40:60 and 1 mL water. Adjust 1 mL urine
to pH 5.0-5.5, add p-glucuronidase/arylsulfatase (Helix pomatia (Boehringer Mannheim), heat
at 37 for 18 h, add 1 mL to the SPE cartridge, wash with 1 mL water, wash with 500 |xL 100
mM HCl, elute with 1 mL MeCN: 100 mM HCl 40:60, inject a 20 |xL aliquot of the eluate.
HPLCVARIABLES
Column: 250 mm long 5 jxm Zorbax phenyl
Mobile phase: MeCN:100 mM KH2PO4 45:55, adjusted to pH 4
Flow rate: 1.5
CHROMATOGRAM
Retention time: 4.8
Limit of detection: 20 ng/mL (F)
OTHER SUBSTANCES
KEYWORDS
SPE
REFERENCE
Jacqz-Aigrain,E.; Menard,Y.; Popon,M.; Mathieu,H. Dextromethorphan phenotypes determined by high-perfor-
mance liquid chromatography and fluorescence detection, J.Chromatogr., 1989, 495, 361-363.
SAMPLE
Matrix: urine
Sample preparation: Condition a 3 mL 200 mg Bond Elut C18 SPE cartridge with 6 mL MeOH,
6 mL water, and 4 mL 100 mM pH 9.2 sodium carbonate buffer. 750 u,L Urine + 750 |xL 100
mM pH 5.0 sodium acetate buffer containing 20 |xL p-glucuronidase-arylsulfatase (Helix po-
matia, 100000 Fisherman units/mL, Boehringer Mannheim) + 50 |xL 600 mM sodium azide,
heat at 37 for 18 h. 250 (xL Hydrolysed urine + 100 |xL 10 |xg/mL levallorphan tartrate in
water + 2 mL 100 mM pH 9.2 sodium carbonate, add to SPE cartridge, wash with 2 mL water,
wash with 1 mL MeCN, elute with 3 mL MeOH:MeCN:2% phosphoric acid 50:30:20. Evaporate
the eluate to dryness under a stream of nitrogen at 70, reconstitute the residue in 500 JJLL
mobile phase, inject a 20 |xL aliquot. (For low concentrations of dextromethorphan: 500 |xL
Hydrolysed urine + 100 (xL 1 (xg/mL levallorphan tartrate in water + 2 mL 100 mM pH 9.2
sodium carbonate, add to SPE cartridge, wash with 2 mL water, wash with 1 mL MeCN, elute
with 3 mL MeOH:MeCN:2% phosphoric acid 50:30:20. Evaporate the eluate to dryness under
a stream of nitrogen at 70, reconstitute the residue in 250 JJLL mobile phase, inject a 120 JJLL
aliquot.)
HPLC VARIABLES
Guard column: 15 X 3.2 RP-2 (Brownlee)
Column: 250 X 4.6 5 |xm Zorbax phenyl
Mobile phase: MeCN:MeOH:10 mM pH 2.5 phosphate buffer containing 2.5 mM 1-octanesulfonic
acid 27:13:60
Flow rate: 1
CHROMATOGRAM
Retention time: 7.0
Internal standard: levallorphan tartrate (9.5)
OTHER SUBSTANCES
KEYWORDS
SPE
REFERENCE
Wenk,M.; Todesco,L.; Keller,B-; Follath,F. Determination of dextromethorphan and dextrorphan in urine by
high-performance liquid chromatography after solid-phase extraction, J.Pharm.Biomed.AnaL, 1991,9,341-
344.
SAMPLE
Matrix: urine
Sample preparation: 250 JJIL Urine + 1.25 jjig levallorphan + 250 |xL 140 mM pH 5 sodium,
acetate buffer, mix, add 25 jxL p-glucuronidase (glucurase, from bovine liver, 5000 U/mL,
Sigma), heat at 37 overnight, add 1.5 mL pH 11.3 glycine buffer, add 6 mL hexanerbutanol
90:10, shake vigorously for 10 min, centrifuge at 1500 g for 10 min. Remove the organic layer
IxL mobile phase, inject a 10 jxL aliquot.
HPLC VARIABLES
Column: Nucleosil 7 C6H5
Mobile phase: MeCN:MeOH:buffer 20:10:70 (Buffer was 10 mM phosphate buffer containing 2.5
mM sodium 1-octanesulfonate, adjusted to pH 2.5 with concentrated phosphoric acid.)
Flow rate: 1.3
CHROMATOGRAM
Retention time: 6
Internal standard: levallorphan (9)
OTHER SUBSTANCES
REFERENCE
Caslavska,J.; Hufschmid,E.; Theurillat,R.; Desiderio,C; Wolfisberg,H.; Thormann,W. Screening for hydroxyla-
tion and acetylation polymorphisms in man via simultaneous analysis of urinary metabolites of mephe-
nytoin, dextromethorphan and caffeine by capillary electrophoretic procedures, J.Chromatogr.B, 1994, 656,
219-231.
SAMPLE
Matrix: urine
Sample preparation: 250 jxL Urine + 5000 U p-glucuronidase in 1 M pH 5.0 sodium acetate
buffer, heat at 37 for 18 h, add 500 |xL saturated sodium carbonate, add 10 mL hexane:
triethylamine 99.9:0.1. Remove the organic layer, dry, reconstitute the residue in 250 |xL mobile
phase, inject a 5-50 JJLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Selectosil (Phenomenex)
Mobile phase: MeCN: 10 mM pH 3.0 potassium phosphate buffer 30:70
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
REFERENCE
Marinac,J.S.; Foxworth,J.W.; Willsie,S.K. Dextromethorphan polymorphic hepatic oxidation (CYP2D6) in
healthy black american adult subjects, Ther.Drug Monit., 1995, 17, 120-124.
Levothyroxine sodium
Molecular formula: C15H10I4NNaO4
CAS Registry No.: 55-03-8, 25416-65-3
(hydrate), 51-48-9 (free acid)
Merck Index: 5497
Lednicer No.: 1 97
SAMPLE
Matrix: blood
Sample preparation: Equilibrate a Sep-Pak silica cartridge with 5 mL ethyl acetate. 1 mL
Serum + 3 mL 5% trichloroacetic acid + 4 mL ethyl acetate, vortex vigorously, centrifuge at
1500 g for 5 min. Remove organic layer and repeat extraction twice with 3 mL portions of ethyl
acetate. Combine extracts, evaporate to about 1.5 mL, add to Sep-Pak cartridge. Wash with 8
mL ethyl acetate, elute with 4 mL MeOH.ammonium hydroxide (90:10). Evaporate the eluent
to dryness under nitrogen, reconstitute in 100 (xL MeOH, inject.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere LP.
Mobile phase: MeCN:buffer 35:65 (Buffer was 13.6 g sodium acetate, 1.0 g cupric sulfate pen-
tahydrate, 0.92 g L-proline, and 0.34 g silver nitrate.)
Flow rate: 1.5
Detector: E, Bioanalytical Systems Inc. TL-5 KeI-F glassy carbon thin-layer cell, LC-4 electronic
controller, +0.78 V, 2-5 nA/V
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Extracted: dextrothyroxine, triiodothyronine
KEYWORDS
serum; SPE
REFERENCE
Hay,I.D.; Annesley,T.M.; Jiang,N.S.; Gorman,C.A. Simultaneous determination of D- and L-thyroxine in human
serum by liquid chromatography with electrochemical detection, J.Chromatogr., 1981, 226, 383-390.
SAMPLE
Sample preparation: Weigh out powder equivalent to about 65 mg thyroid, add 5 mL enzyme
solution, mix well, incubate at 37 for 28 h, agitate after 4-8 h and after 20-24 h, add 2 mL
deactivating solution, mix well, centrifuge at 2000 rpm for 5-10 min, if necessary filter (0.45
imm). (The enzyme solution was about 150 protease units/mL of bacterial protease from Strep-
tomyces griseus in 110 mM NaCL + 40 mM Tris buffer + 50 mM methimazole (pH adjusted to
8.4 0.05 with 6 M HCl) reducing buffer. Deactivating solution was 1:100 phosphoric acid:
MeCN.)
HPLC VARIABLES
Column: 300 X 4 u-Bondapak C18
Flow rate: 1.5
Detector: UV 225
CHROMATOGRAM
Retention time: 22
OTHER SUBSTANCES
Simultaneous: liothyronine, L-3,3',5'-triiodothyronine
KEYWORDS
tablets
REFERENCE
Richheimer,S.L.; Jensen,C.B. Determination of liothyronine and levothyroxine in thyroid preparations by liquid
SAMPLE
Sample preparation: Grind a tablet, add 50 |xg 3,3',5'-triiodothyronine, add 20 mL solvent A,
stir for 10 min, add 40 mL solvent B, stir for 30 min, filter. Remove the upper layer and wash
it six times with 15 mL portions of water saturated with butanol, evaporate under vacuum at
40-42, reconstitute in 2.5 mL 3% ammonium hydroxide in MeOH, inject an aliquot (Anal.Lett.
1979, 12, 1201). (Prepare the solvents by mixing 1.8 L 1-butanol, 1.35 L water and 450 mL
concentrated HCl, shake vigorously for 20 min, allow to separate. The lower layer was solvent
A and the upper layer was solvent B.)
HPLC VARIABLES
Guard column: 25 X 2.5 Co:Pel ODS
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Internal standard: 3,3',5'-triiodothyronine
OTHER SUBSTANCES
Simultaneous: liothyronine
KEYWORDS
protect from light; tablets
REFERENCE
Rapaka,R.S.; Knight,P.W.; Prasad,V.K. Reversed-phase high-performance liquid chromatographic analysis of
liothyronine sodium and levothyroxine sodium in tablet formulations: preliminary studies on dissolution
and content uniformity, J.Pharm.Sci., 1981, 70, 131-134.
SAMPLE
Sample preparation: Powder tablets, weigh out amount equivalent to about 200 |xg sodium
levothyroxine, add 10 mL mobile phase, sonicate for 5 min, centrifuge. Filter (0.45 |xm, 25 mm
Acrodisc CR, Gelman) the supernatant, inject a 200 uX aliquot.
HPLC VARIABLES
Guard column: 40 X 4 40 |xm RP 201SC pellicular (Vydac)
Column: 300 X 4 ixBondapak C18
Mobile phase: MeCNrbuffer 60:40 (Buffer was pH 3.0 containing 5 mM 1-octanesulfonic acid and
5 mM tetramethylammonium chloride.)
Flow rate: 2
Detector: UV 230
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
Simultaneous: liothyronine, 3,5-diiodo-L-thyronine
KEYWORDS
REFERENCE
Richheimer,S.L.; Amer,T.M. Stability-indicating assay, dissolution, and content uniformity of sodium levothy-
roxine in tablets, J.Pharm.Sci., 1983, 72, 1349-1351.
SAMPLE
Sample preparation: Grind tablets containing about 1 mg levothyroxine, add 4.5 mL 0.5 mg/
mL hydroxyprogesterone caproate in MeOH, add 20.5 mL 10 mM NaOH in MeOH:water 75:
25, shake intermittently for 5 min, filter, discard first 5 mL filtrate, inject a 25 jxL aliquot.
HPLC VARIABLES
Mobile phase: MeCNrO. 1% phosphoric acid in water 35:65
Flow rate: 3
Detector: UV 225
CHROMATOGRAM
Retention time: 5
Internal standard: hydroxyprogesterone caproate (8)
KEYWORDS
tablets
REFERENCE
Das Gupta,V.; Odom,C; Bethea,C; Plattenburg,J. Effect of excipients on the stability of levothyroxine sodium
tablets, J.Clin.Pharm.Ther., 1990, 15, 331-336.
SAMPLE
Sample preparation: Condition a 13 mm Empore C18 SPE disk (Baker) with 2.5 mL MeOH
and 2.5 mL water at 1.5 mL/min. Dissolve tablet in dissolution medium (?). Pass 40 mL through
the SPE disk, wash with 2.5 mL water, dry, add 1 mL MeOH and let it soak in for 3 min, elute
at 0.5 mL/min, dilute the eluate to 2 mL with 50 mM pH 2.5 phosphate buffer, inject a 20 |JLL
aliquot.
HPLCVARIABLES
Column: 125 X 4 5 jxm LiChrospher cyano
Mobile phase: MeCN:water:phosphoric acid 45:55:0.05
Flow rate: 1.6
Detector: UV 225
KEYWORDS
tablets; SPE; comparison with capillary electrophoresis
REFERENCE
Carducci,C.N.; Lucangioli,S.E.; Rodriguez,V.G.; Fernandez Otero,G.C. Application of extraction disks in disso-
lution tests of clenbuterol and levothyroxine tablets by capillary electrophoresis, J.ChromatogrA, 1996,
730, 313-319.
SAMPLE
Matrix: solutions
Sample preparation: Take up 1.5 mg levothyroxine in 200 |xL 100 mM sodium bicarbonate and
400 |JLL reagent, stir in an ice bath for 30 min, evaporate to dryness below 30, add 100 (xL
trifluoroacetic acid to the dry residue, let stand for 30 min at room temperature, add 2 mL 1
M sodium bicarbonate, centrifuge. Remove the precipitate and dissolve it in 600 JJLL MeOH:20
mM NaOH 50:50, inject a 15 jxL aliquot. Reagent was 7 mg/mL BOC-L-Leu-SU (tert-butyloxy-
L-leucine-N-hydroxysuccinimide ester) in MeOH, prepared immediately before use.)
HPLCVARIABLES
Column: 150 X 3.2 7 |xm LiChrosorb KP-18
Mobile phase: MeOH:water 60:40 containing 0.05% methanesulfonic acid
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 9
Limit of detection: 0.05% of the D form
OTHER SUBSTANCES
Simultaneous: dextrothyroxine, impurities
KEYWORDS
REFERENCE
Lankmayr,E.R; Budna,K.W.; Nachtmann,F. Separation of enantiomeric iodinated thyronines by liquid chro-
matography of diastereomers, J.Chromatogr., 1980, 198, 471-479.
SAMPLE
Matrix: solutions
Sample preparation: 20 |xL Solution + 40 |xL 50 mM pH 8.5 borate buffer + 40 jxL 4 mM dabsyl
chloride in MeCN, mix, heat at 70 for 15 min, add 100 |xL 25 mM pH 6.5 sodium acetate
buffer, inject an aliquot.
HPLC VARIABLES
Guard column: 10 X 3 30 |jim Chromspher C18 (Chrompaek)
Column: 100 X 3 5 |xm Chromspher C18 (Chrompaek)
Mobile phase: Gradient. A was MeOH:25 mM pH 6.5 sodium acetate buffer 70:30. B was MeOH:
25 mM pH 6.5 sodium acetate buffer 90:10. A:B from 100:0 to 0:100 over 15 min, maintain at
0:100 for 5 min.
Flow rate: 0.7
Detector: UV 436
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: 3,5-diiodothyronine, 3,5-diiodotyrosine, liothyronine, 3-monoiodotyrosine, thy-
ronine, 3,3',5-triiodothyronine, tyrosine
KEYWORDS
derivatization; comparison with other derivatization procedures
REFERENCE
Doorn,L.; Jansen,E.H.; Van Leeuwen,F.X. Comparison of high-performance liquid chromatographic detection
methods for thyronine and tyrosine residues in toxicological studies of the thyroid, J.Chromatogr., 1991,
553, 135-142.
SAMPLE
Matrix: solutions
Sample preparation: 100 |xL Solution + 100 |xL 500 mM pH 7.7 borate buffer + 100 jxL 2.5 mM
9-fluorenylmethyl chloroformate in dry acetone, mix, let stand at room temperature for 45 s,
add 200 |xL 12 mM 1-adamantamine in MeCN, inject an aliquot.
HPLC VARIABLES
Guard column: 10 X 3 30 |xm Chromspher C18 (Chrompack)
Column: 100 X 3 5 fxm Chromspher C18 (Chrompack)
Mobile phase: Gradient. A was MeOH:50 mM pH 4.2 sodium acetate buffer 40:60. B was MeCN:
MeOH:50 mM pH 4.2 sodium acetate buffer 20:60:20. A:B from 100:0 to 0:100 over 40 min.
Flow rate: 0.7
Detector: UV 260
CHROMATOGRAM
Retention time: 33
OTHER SUBSTANCES
Simultaneous: 3,5-diiodothyronine, 3,5-diiodotyrosine, liothyronine, 3-monoiodotyrosine, thy-
ronine, 3,3',5-triiodothyronine
KEYWORDS
REFERENCE
553, 135-142.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Phenomenex cyano-bonded silica
Mobile phase: MeCN:water:phosphoric acid 400:600:1
Next Page
Flow rate: 1.5

Detector: UV 225
CHROMATOGRAM
Retention time: 8.6
OTHER SUBSTANCES
Simultaneous: degradation products, liothyronine
REFERENCE
Won,C.M. Kinetics of degradation of levothyroxine in aqueous solution and in solid state, Pharm.Res., 1992,
9, 131-137.
SAMPLE
Matrix: tissue
Sample preparation: 100 |xL Thyroid tissue + 200 jxL MeCN, mix, centrifuge. Remove a 100
IxL aliquot of the supernatant and add it to 100 |xL 4 nM dabsyl chloride in MeCN, heat at 70
for 10 min, add 400 |xL MeOH:50 mM pH 7.0 phosphate buffer 50:50, inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: Gradient. A was MeOH:25 mM pH 6.5 sodium acetate 56:44. B was MeOH. A:B
from 80:20 to 35:65 over 15 min, maintain at 35:65 for 3 min, to 0:100 over 1 min, maintain
at 0:100 for 2 min.
Flow rate: 1
Detector: UV 436
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diiodothyronine (T2), liothyronine (T3)
KEYWORDS
derivatization; thyroid
REFERENCE
Jansen,E.H.J.M.; van den Berg,R.H.; Both-Miedema,R.; Doorn,L. Advantages and limitations of pre-column
derivatization of amino acids with dabsyl chloride, J.Chromatogr., 1991, 553, 123-133.
Lidocaine
CAS Registry No.: 137-58-6, 6108-05-0 (HCI monohydrate), 73-78-9 (HCI)
Merck Index: 5505
Lednicer No.: 1 16
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 200 |xL 2 jjig/mL IS in MeOH, add 2 mL water
and 2 mL MeCN, vortex gently, set aside for 3 min, centrifuge at 2200 g for 20 min. Separate
the clear supernatant, add 500 (xL 200 mM NaOH and extract with 6 mL n-hexane by vortexing
for 2 min. Centrifuge at 2200 g for 15 min. Evaporate 5 mL of the organic phase to dryness
under reduced pressure. Reconstitute the residue in 120 |xL mobile phase. Inject a 100 |xL
aliquot.
Previous Page
Flow rate: 1.5

Detector: UV 225
CHROMATOGRAM
Retention time: 8.6
OTHER SUBSTANCES
Simultaneous: degradation products, liothyronine
REFERENCE
9, 131-137.
SAMPLE
Matrix: tissue
Sample preparation: 100 |xL Thyroid tissue + 200 jxL MeCN, mix, centrifuge. Remove a 100
IxL aliquot of the supernatant and add it to 100 |xL 4 nM dabsyl chloride in MeCN, heat at 70
for 10 min, add 400 |xL MeOH:50 mM pH 7.0 phosphate buffer 50:50, inject a 20 |xL aliquot.
HPLC VARIABLES
at 0:100 for 2 min.
Flow rate: 1
Detector: UV 436
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diiodothyronine (T2), liothyronine (T3)
KEYWORDS
REFERENCE
Lidocaine
CAS Registry No.: 137-58-6, 6108-05-0 (HCI monohydrate), 73-78-9 (HCI)
Merck Index: 5505
Lednicer No.: 1 16
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 200 |xL 2 jjig/mL IS in MeOH, add 2 mL water
and 2 mL MeCN, vortex gently, set aside for 3 min, centrifuge at 2200 g for 20 min. Separate
the clear supernatant, add 500 (xL 200 mM NaOH and extract with 6 mL n-hexane by vortexing
for 2 min. Centrifuge at 2200 g for 15 min. Evaporate 5 mL of the organic phase to dryness
under reduced pressure. Reconstitute the residue in 120 |xL mobile phase. Inject a 100 |xL
aliquot.
HPLC VARIABLES
Guard column: 10 X 3 5 jjim AGP bonded silica (ChromTech, Hagersten, Sweden)
Column: 150 X 4 5 micro.m AGP bonded silica (ChromTech, Hagersten, Sweden)
Mobile phase: Isopropanol:buffer 4:96 (Prepare mobile phase by adding 4% isopropanol and 0.6%
diethylamine to 8 mM sodium dihydrogen phosphate containing 100 mM NaCl, adjust to pH
7.05 with 50% phosphoric acid.)
Flow rate: 0.9
Detector: UV 214
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
KEY WORDS
REFERENCE
Abraham,I.; Fawcett,J.R; Kennedy,J.; Kumar,A.; Ledger,R. Simultaneous analysis of lignocaine and bupiva-
caine enantiomers in plasma by high-performance liquid chromatography, J.Chromatogr.B, 1997, 703, 203-
208.
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL Bond-elut C2 SPE cartridge with 2 mL MeOH and 2
mL water. Apply 1 mL plasma to the cartridge, wash with 1 mL water, wash with 1 mL MeOH:
water 50:50, wash with 1 mL MeCN, elute with 2 mL 1 M NaChMeOH 5:95. Dry the eluate
under vacuum, resuspend in 200 |JLL MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 10 mm long C18 (Waters)
Column: 250 X 4.6 Supelcosil LC-8-DB
Mobile phase: MeCN:20 mM phosphoric acid containing 200 |JLL/L triethylamine 10:90
Flow rate: 1.7
Detector: UV 263
CHROMATOGRAM
Internal standard: tocainide (9.7)
Limit of quantitation: 200 ng/L
OTHER SUBSTANCES
Noninterfering: acetaminophen, N-acetylprocainamide, amitriptyline, bupivicaine, caffeine, car-
bamazepine, chloramphenicol, cyclosporin A, desipramine, diazepam, disopyramide, doxepin,
ethosuximide, flecainide, fluoxetine, ibuprofen, imipramine, naproxen, norchlordiazepoxide,
nordiazepam, nordoxepine, nortriptyline, phenobarbital, phenytoin, primidone, procainamide,
quinidine, salicylic acid, theophylline, valproic acid
KEYWORDS
serum; comparison with fluorescence polarization immunoassay; SPE
REFERENCE
O'Neal,C.L.; Poklis,A. Sensitive HPLC for simultaneous quantification of lidocaine and its metabolites
monoethylglycinexylidide and glycinexylidide in serum, Clin.Chem., 1996, 42, 330331.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 |L 40 |xg/mL etidocaine hydrochloride in water + 100
IxL 1 M NaOH, vortex for 15 s, add 5 mL diethyl ether, shake on a reciprocating shaker for 10
min, centrifuge. Remove the organic layer and evaporate it to dryness under a stream of ni-
trogen, reconstitute the residue in 100 |xL mobile phase, inject a 80 (xL aliquot.
HPLC VARIABLES
Flow rate: 0.9
Detector: UV 210
CHROMATOGRAM
Retention time: 4.5
Internal standard: etidocaine (12.0)
OTHER SUBSTANCES
Extracted: 2,6-pipecolylxylidine, bupivacaine, mepivacaine
Noninterfering: metabolites, 2,3-ehloroprocaine, theophylline, mexiletine, quinidine, disopyr-
KEYWORDS
plasma
REFERENCE
Ha,H.-R.; Funk,B.; Gerber,H.R.; Follath,F. Determination of bupivacaine in plasma by high-performance liquid
SAMPLE
Matrix: blood
Sample preparation: Filter plasma (0.22 |xm), inject a 10 u,L aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 u,m GFF-S5-80 internal-surface reversed phase "Pinkerton" (Regis)
Mobile phase: THF: 100 mM potassium phosphate 10:90, pH 7.2
Flow rate: 0.8
Detector: UV 220
CHROMATOGRAM
KEYWORDS
plasma; direct injection
REFERENCE
Nakagawa,T.; Shibukawa,A-; Shimono,N.; Kawashima,T.; Tanaka,H.; Haginaka,J. Retention properties of in-
ternal-surface reversed-phase silica packing and recovery of drugs from human plasma, J.Chromatogr.,
1987, 420, 297-311.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Serum + 100 |xL 1 M pH 9.0 borate buffer + 1 mL chlorofornr.EtOH
82.5:17.5, mix, centrifuge. Remove the organic layer and evaporate it to dryness, reconstitute
the residue in 50 |xL mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 300 X 2 10 |xm (xBondapak C18
Mobile phase: MeOH:MeCN:buffer 12:16:72 (Buffer was 31 mM sodium acetate adjusted to pH
5.1 with 40% phosphoric acid containing 0.15 mM tetrabutylammonium phosphate.)
Flow rate: 0.3
Detector: UV 230
CHROMATOGRAM
Retention time: 10
Internal standard: lidocaine
OTHER SUBSTANCES
Extracted: caffeine, cocaine, metabolites
Simultaneous: barbital, phenobarbital, flumazepil, mazindol, hexobarbital, nicotine, procaine,
cotinine
Noninterfering: amphetamine, desipramine, tetracaine, methadone, reserpine, buspirone, di-
azepam, haloperidol, chlordiazepoxide, oxazepam, midazolam, clonazepam, chlorpromazine,
pentobarbital
KEYWORDS
serum; rat; lidocaine is IS
REFERENCE
Lau,C.E.; Ma,R; Falk,J.L. Simultaneous determination of cocaine and its metabolites with caffeine in rat serum
microsamples by high-performance liquid chromatography, J.Chromatogr., 1990, 532, 95-103.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 JXL 1 M NaOH + 3 mL heptanerethyl acetate 90:10,
JJLL 50 mM sulfuric acid, shake for 2 min, centrifuge at 1200 g for 5 min. Remove the aqueous
phase and add it to 820 |xg sodium acetate, inject a 40 uX aliquot. (The sodium acetate was
measured out by adding 50 |xL 200 mM sodium acetate in MeOH to the tube and evaporating
the MeOH.)
HPLC VARIABLES
Column: 250 X 4 10 |xm fjiBondapak C18
Mobile phase: MeCN:10 mM NaH2PO4 5:95, adjusted to pH 2.1
Flow rate: 1
Detector: UV 205
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Extracted: procaine
KEYWORDS
plasma; rabbit
REFERENCE
Le Guevello,R; Le Corre,P.; Chevanne,R; Le Verge,R. High-performance liquid chromatographic determination
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL 2 M NaOH, mix, add 3 mL n-hexane, shake for 1
min, centrifuge at 3500 rpm for 10 min. Remove the organic layer and evaporate it to dryness
with nitrogen under vacuum, reconstitute the residue in 200 |JLL mobile phase, vortex, inject a
100 |xL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 3.8
OTHER SUBSTANCES
KEYWORDS
plasma; lidocaine is IS
REFERENCE
Murillo,L; Costa,J.; Salva,P. Determination of bupivacaine in human plasma by HPLC, J.Liq.Chromatogr.,
1993, 16, 3509-3514.
SAMPLE
Matrix: blood
stoppered tube for 1 min, add 6 mL NiCl2, rock for 5 min, add 15 mL dichloromethanedsobutyl
process. Filter all organic phases through a 40-90 |xm filter and evaporate to dryness in a 100
water.)
HPLC VARIABLES
Mobile phase: A = MeCN; B = 20 mM n-propylamine adjusted to pH 5 with 85% phosphoric
acid. A:B from 15:85 to 20:80 over 5 min to 45:55 over another 15 min to 65:35 over another 5
min
Detector: UV 230
CHROMATOGRAM
Retention time: 16
OTHER SUBSTANCES
Extracted: buprenorphine, caffeine, cocaine, codeine, diamorphine, ethylmorphine, methaqua-
lone, morphine, naloxone, noscapine, papaverine, pentazocine, procaine
Also analyzed: bromazepam, clonazepam, diazepam, flunitrazepam, flurazepam, medazepam,
nitrazepam, oxazepam
KEYWORDS
whole blood
REFERENCE
by high performance liquid chromatography, Chromatographia, 1994, 38, 617623.
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Plasma + 10 IJLL 15 mg/mL bupivacaine, mix, add 100 |xL 2 M
NaOH, vortex briefly, add 5 mL anhydrous ethyl ether, vortex for 30 s, rotate for 10 min,
centrifuge at 1000 g for 5 min. Remove 4.5 mL ether and add to 250 |xL 12.5 mM sulfuric acid,
vortex for 30 s, rotate for 10 min, centrifuge for 5 min, inject a 50 |xL aliquot of the lower
aqueous phase.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Octyl IB (Keystone)
Mobile phase: MeCN:50 mM Na2HPO4 27:73 pH adjusted to 5.8 with 50% phosphoric acid
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Internal standard: bupivacaine (9.81)
OTHER SUBSTANCES
Extracted: prilocaine, o-toluidine
KEYWORDS
plasma; pig; pharmacokinetics
REFERENCE
Klein,J.; Fernandes,D.; Gazarian,M.; Kent,G.; Koren,G. Simultaneous determination of lidocaine, prilocaine
and the prilocaine metabolite o-toluidine in plasma by high-performance liquid chromatography,
J.Chromatogr.B, 1994, 655, 83-88.
SAMPLE
Matrix: blood
Sample preparation: 100 JJLL Plasma + 100 (xL 20 jxg/mL caffeine + 8 mL dichloromethane,
shake for 20 min, centrifuge at 2500 rpm for 20 min. Remove 7 mL of the organic layer and
evaporate it to dryness under nitrogen or at 60. Dissolve residue in 200 |xL mobile phase,
inject a 20 JXL aliquot.
HPLC VARIABLES
Column: 150 X 6 Shimpack CLS-ODS (Shimadzu)
Mobile phase: MeCN:MeOH:0.5 mM phosphoric acid 7.5:3:89.5
Flow rate: 1.5
Detector: UV 210
CHROMATOGRAM
Internal standard: caffeine
KEYWORDS
plasma; rat
REFERENCE
Lee,C.K.; Uchida,T.; Kitagawa,K.; Yagi,A.; Kim,N.-S.; Goto,S. Skin permeability of various drugs with different
lipophilicity, J.Pharm.Sci., 1994, 83, 562-565.
SAMPLE
Matrix: blood
|xL 0.1 M phosphoric acid (pH 2.5), mix, separate, inject a 100 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Flow rate: 2
Detector: UV 228
CHROMATOGRAM
Retention time: 1.8
OTHER SUBSTANCES
norfluoxetine, nortriptyline, norverapamil, promazine, propafenone, propoxyphene, protripty-
line, temazepam, trazodone, trimipramine, verapamil
warfarin
Interfering: encainide, mexiletine, pentazocine, propranolol, quinidine
KEYWORDS
plasma; SPE
REFERENCE
Clin.Chem., 1994, 40, 1312-1316.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 (JLL 10 |xg/mL bupivacaine in 25 mM sulfuric acid +
1 mL 1 M NaOH + 5 mL diethyl ether, shake or rotate for 15 min, centrifuge at 1000 rpm for
5 min, freeze at -20. Remove the organic layer and add it to 250 JJLL 25 mM sulfuric acid,
shake for 15 min, centrifuge at 1000 rpm for 5 min, freeze, discard the organic layer. Thaw
the aqueous layer, pass air over the aqueous phase at room temperature to remove traces of
ether, adjust pH to 5.0-6.5 by adding 10 |xL 1 M NaOH, inject a 50 |xL aliquot.
HPLCVARIABLES
Guard column: 4 X 4 4 |xm LiChroCART Superspher 60 RP Select B (Merck)
Column: 125 X 4 4 pjn LiChroCART Superspher 60 RP Select B (Merck)
Mobile phase: MeCN:buifer 30:70 (Buffer was 7.0 g/L K2HPO4 in water adjusted to pH 5.8 with
1 M NaOH.)
Flow rate: 1
Detector: UV 202
CHROMATOGRAM
Retention time: 5
Internal standard: bupivacaine (10)
KEYWORDS
plasma
REFERENCE
Sattler,A.; Kramer,!; Jage,J.; Vrana,S.; Kleemann,P.P.; Dick,W. Development of a HPLC-system for quantitative
measurement of lidocaine and bupivacaine in patients plasma during postoperative epidural pain therapy,
Pharmazie, 1995, 50, 741-744.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform.isopropanol:
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 264
CHROMATOGRAM
KEYWORDS
profen; cicletanine; moclobemide; chloroquine; cocaine; timolol; nomifensine; ticlopidine; ace-
nocoumarol; vindesine; mexiletine; dipyridamole; trazodone; pipamperone; pyrimethamine;
cainide; phencyclidine; thiopental; fennuramine; metipranolol; triprolidine; naproxen; bupren-
amitriptyline; nortiiptyline; tioclomarol; diclofenac; mefloquine; trimipramine; chlorambucil;
REFERENCE
254-262.
SAMPLE
Matrix: blood
water. Add 1 mL serum or 300-500 |xL ultrafiltrate to the SPE cartridge, wash with 5 mL
water, wash with 2 mL MeOH:water 5:95, wash with 2 mL EtOH:water 2.5:97.5, wash with 2
mL MeCN:water 10:90, elute with 1 mL MeCN:50 mM pH 2.4 phosphate buffer 25:75, inject
a 100 |xL aliquot of the eluate.
HPLC VARIABLES
Column: 300 X 4.6 ixBondapak
Flow rate: 1.5
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
serum; ultrafiltrate; SPE; lidocaine is IS
REFERENCE
Mazoit,J.X.; Cao,L.S.; Samii,K. Binding of bupivacaine to human serum proteins, isolated albumin and isolated
a-l-acid glycopotein. Differences between the two enantiomers are partly due to cooperativity,
J.Pharmacol.Exp.Ther., 1996, 276, 109-115.
SAMPLE
Sample preparation: 200 u,L Serum, urine, CSF, or gastric fluid + 300 |xL reagent. Flush column
A to waste with 500 |JLL 500 mM ammonium sulfate, inject sample onto column A, flush column
A to waste with 500 |xL 500 mM ammonium sulfate, backflush the contents of column A onto
HPLCVARIABLES
Column: A 40 u,m preparative grade C18 (Analytichem); B 75 X 2.1 pellicular C18 (Whatman)
Mobile phase: Gradient. A was 50 mM pH 4.5 KH2PO4. B was MeCN.isopropanol 80:20. A:B 90:
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Retention time: 8.0
OTHER SUBSTANCES
phenhydramine, dipipanone, ethylbromphos, flufenamic acid, formothion, griseofulvin, indo-
methacin, lorazepam, malathion, medazepam, midazolam, oxazepam, paraoxon, penicillin G,
pentobarbital, prazepam, propoxyphene, prothiophos, quinine, salicylic acid, secobarbital,
KEYWORDS
REFERENCE
Kruger,P.B.; Albrecht,C.F.De V.; Jaarsveld,RP. Use of guanidine hydrochloride and ammonium sulfate in com-
SAMPLE
Sample preparation: 10 g Whole blood or tissue + 10 mL 100 mM HCl + 100 (xL 200 fjig/mL
procaine in ethyl acetate, homogenize, shake for 10 min, centrifuge at 6000 g for 10 min, add
the supernatant to an Extrelut-20 SPE cartridge, let stand for 15 min, pass ammonia gas
through the column, elute with 40 mL chloroform. Evaporate the eluate to dryness, reconstitute
the residue in 1 mL mobile phase, dilute 10 times with mobile phase, inject a 50 |xL aliquot.
(Ammonia gas was generated by placing concentrated ammonia under reduced pressure and
pulling the evolved ammonia through the column.)
HPLC VARIABLES
Column: 250 X 4.6 6 |xm normal phase silica (BST)
Mobile phase: MeCN: 100 mM pH 2 KH2PO4:water:THF:concentrated phosphoric acid 5.4:90:4.6:
1:1
Flow rate: 2
Detector: UV 230
CHROMATOGRAM
Internal standard: procaine (4.13)
OTHER SUBSTANCES
Simultaneous: benzocaine, bupivacaine, cocaine, tetracaine
KEYWORDS
whole blood; SPE; brain; liver
REFERENCE
Benko,A.; Kimura,K. Toxicological analysis of lidocaine in biological materials by using HPLC, Forensic ScLInL,
1991, 49, 65-73.
SAMPLE
Sample preparation: 2 mL Whole blood, plasma, or urine + 1 mL saturated sodium carbonate
+ 10 JULL 100 jxg/mL etidocaine, add to a 3 mL Extrelut SPE cartridge, elute with 15 mL di-
chloromethane. Evaporate eluate to dryness under a stream of nitrogen at 40, reconstitute in
100 |xL 10 mM HCl, add 3 mL diethyl ether, vortex for 20 s, centrifuge at 2800 g for 5 min,
inject a 40 |xL aliquot of the aqueous layer.
HPLC VARIABLES
Guard column: 5 x 6 ixBondapak Guard Pak
Column: 300 X 3.9 10 |xm fjuBondapak C18
Flow rate: 1.5
Detector: UV 230
CHROMATOGRAM
Retention time: 8
Internal standard: etidocaine (14)
OTHER SUBSTANCES
Extracted: prilocaine, bupivacaine, dibucaine
Also analyzed: procaine, butacaine, tetracaine, p-aminobenzoic acid, articaine, o-toluidine, caf-
feine, amphetamine, ephedrine, epinephrine, morphine, monoacetylmorphine, diamorphine,
ethylmorphine, codeine, acetylcodeine
KEYWORDS
REFERENCE
Rop,P.R; Grimaldi,R; Bresson,M.; Fornaris,M.; Viala,A. Liquid chromatographic analysis of cocaine, benzo-
ylecgonine, local anaesthetic agents and some of their metabolites in biologicalfluids,J.Liq.Chromatogr.,
1993, 16, 2797-2811.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Prepare a 10 mg/mL solution in 500 mM sodium bicarbonate solutions,
extract a 10 mL aliquot twice with 15 mL portions of dichloromethane. Combine the extracts
and add 10 |xL phenylisothiocyanate, evaporate to dryness under a stream of air, reconstitute
with 10 mL MeOH, inject a 10 jxL aliquot.
HPLC VARIABLES
Guard column: 70 X 2.1 COiPELL ODS
Column: 300 X 3.9 |jiBondapak C18
Mobile phase: MeOH:water:acetic acid 45:54:1
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Simultaneous: ephedrine, phenylpropanolamine, pseudoephedrine
KEYWORDS
derivatization
REFERENCE
Noggle,F.T.,Jr.; Clark,C.R. Liquid chromatographic analysis of samples containing cocaine, local anesthetics,
and other amines, J.Assoc.Off.Anal.Chem., 1983, 66, 151-157.
SAMPLE
Matrix: bulk
Sample preparation: Prepare a 750 (xg/mL solution in 10 mM pH 2.5 orthophosphoric acid,
sonicate for 10 min, filter (0.2 jjim), inject a 15 |xL aliquot.
HPLC VARIABLES
Guard column: 4 x 4 5 |xm LiChrospher 100
Column: 125 X 4 3 urn Spherisorb ODS-I
Mobile phase: Gradient. A was water containing 5 mL/L 85% orthophosphoric acid and 0.56 mL/
L hexylamine. B was MeCN:water 90:10 containing 5 mL/L 85% orthophosphoric acid and 0.56
mL/L hexylamine. A:B from 91:9 to 86:14 over 4 min, maintain at 86:14 for 13 min, to 55:45
Flow rate: 0.7
Detector: UV 210
CHROMATOGRAM
Retention time: 9.4
OTHER SUBSTANCES
Simultaneous: acetaminophen, acetylcodeine, benzocaine, caffeine, cocaine, codeine, diamor-
phine, 6-monoacetylmorphine, morphine, noscapine, papaverine, procaine
REFERENCE
Grogg-Sulser,K.; Helmlin,H.-J.; Clerc,J.-T. Qualitative and quantitative determination of illicit heroin street
samples by reversed-phase high-performance liquid chromatography: method development by CARTAGO-
S, J.ChromatogrA, 1995, 692, 121-129.
SAMPLE
Sample preparation: Dilute with MeOH:water 500 mM pH 7 sodium borate 35:65:2, inject an
aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Whatman PXS ODS-3 C18
Mobile phase: MeCN:buffer 20:80 (Buffer was water glacial acetic acid 93:5 adjusted to pH 3.0
with 1 M NaOH.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 5
OTHERSUBSTANCES
Simultaneous: milrinone
KEYWORDS
REFERENCE
SAMPLE
Matrix: perfusate
HPLC VARIABLES
Column: 100 X 8 4 |xm Novapak C18
Mobile phase: MeCNrO.092%phosphoric acid + 0.2% triethylamine 26:74
Flow rate: 2
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: diphenhydramine, diltiazem, metabolites
Also analyzed: bupivacaine
KEYWORDS
lidocaine is IS; rat; liver
REFERENCE
Hussain,M.D.; Tam,Y.K; Gray,M.R.; Coutts,K.T. Kinetic interactions of lidocaine, diphenhydramine, and ve-
rapamil with diltiazem: A study using isolated perfused rat liver, Drug Metab.Dispos., 1994, 22, 530-536.
SAMPLE
Matrix: perfusate
HPLC VARIABLES
Column: 125 X 4 5 |xm LiChrospher 60 RP-Select B C18
Mobile phase: Gradient. MeCNibuffer 9:91 for 5 min, to 19:81 over 10 min, maintain at 19:81
for 11.5 min, return to initial conditions over 1 min, re-equilibrate for 7.5 min. (Buffer was
6.66 g/L KH2PO4, 150 jxL/L phosphoric acid, and 5 mM sodium n-heptanesulfonate, pH 3.5.)
Flow rate: 1
Detector: UV 214
CHROMATOGRAM
KEYWORDS
rat; liver
REFERENCE
Ngo,L.Y.; Tam,Y.K; Coutts,R.T. Lack of residual effects of diethyl ether, methoxyflurane, and sodium pento-
barbital on lidocaine metabolism in a single-pass isolated rat liver perfusion system, Drug Metab.Dispos.,
1995, 23, 525-528.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 262
OTHER SUBSTANCES
Also analyzed: disopyramide, metoprolol
REFERENCE
Sugawara,M.; Takekuma,Y; Yamada,H.; Kobayashi,M.; Iseki,K.; Miyazaki,K A general approach for the pre-
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 300 X 4 10 |xm u,Bondapak C18
Mobile phase: MeCNiMeOH:water 20:20:60 containing 0.06% sulfuric acid, 0.5% sodium sulfate,
and 0.02% sodium heptanesulfonate, pH 2.6
Flow rate: 2
Injection volume: 5
Detector: UV 305
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Simultaneous: benzocaine, butamben, pramoxine, procaine, tetracaine
REFERENCE
Menon,G.N.; Norris,B.J. Simultaneous determination of tetracaine and its degradation product, p-n-butyl-
aminobenzoic acid, by high-performance liquid chromatography, J.Pharm.Sci., 1981, 70, 569-570.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 JiL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.4
OTHER SUBSTANCES
isoxsuprine, ketanserin, laudanosine, lofepramine, loxapine, maprotiline, mecamylamine, me-
clophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine,
thipendyl, protriptyline, proxymetacaine, pseudoephedrine, pjrrimethamine, quinidine, qui-
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 |JLg/mL, inject a 10
jxL aliquot.
HPLCVARIABLES
Column: 300 X 3.9 10 fxm ixBondapak C18
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 6
OTHERSUBSTANCES
Simultaneous: butacaine, bupivacaine, benzocaine, tetracaine
REFERENCE
separation of drugs using triethylamine as a competing base, J.Chromatogr., 1986, 370, 403418.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:buffer 35:65 (Buffer was 0.1% phosphoric acid containing 5 mM sodium 1-
hexanesulfonate.)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Internal standard: ethyl p-hydroxybenzoate
REFERENCE
Cheng,Y.H.; Hosoya,O.; Sugibayashi,K; Morimoto,Y. Effect of skin surface lipid on the skin permeation of
lidocaine from pressure sensitive adhesives, Biol.Pharm.BulL, 1994, 17, 1640-1644.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
triptyline, amobarbital, amoxapine, amphetamine, amylocaine, antipyrine, aprobarbital, as-
pirin, atenolol, atropine, avermectin, barbital, benzocaine, benzoic acid, benzotropine, benz-
nuorouracil, fluoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide,
isoxsuprine, ivermectin, ketamine, ketoprofen, kûrenic acid, lorazepam, lormetazepam, lox-
apine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, me-
pivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene,
methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone,
naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitra-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ketorolac, labetalol, levorphanol, loratadine, lorazepam, lovastatin, loxapine, mazindol, mefen-
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 JJLL aliquot of a 100-500 u,g/mL solution in mobile phase.
HPLC VARIABLES
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
operidol, hydroxyzine, imipramine, indomethacin, megestrol acetate, metoprolol, nabumetone,
KEYWORDS
REFERENCE
Hanna,M.; de Biasi,V.; Bond,B-*> Salter,C; Hutt,A.J.; Camilleri,P. Estimation of the partitioning characteristics
SAMPLE
Matrix: urine
Sample preparation: 500 |xL Urine + N-ethylnordiazepam + chlorpheniramine + 100 |xL buffer,
IxL mobile phase B, with 200 jxL mobile phase C, and with 1.15 mL mobile phase D. Elute
HPLC VARIABLES
3.2 11 [Lm Aminex A-28 (Bio-Rad); C 25 X 3.2 5 jjum C8 (Phenomenex) + 150 X 4.6 5 jxm silica
(Macherey-Nagel)
phoric acid; C MeCN.buffer 40:60 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylam-
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: methamphetamine, desipramine, nortriptyline, diphenhydramine, methadone, imip-
ramine, flurazepam, amitriptyline, morphine, codeine, hydromorphone, hydrocodone, caffeine,
cotinine, benzoylecgonine, secobarbital, oxazepam, phenobarbital, nordiazepam, diazepam,
phenylpropanolamine
Interfering: phentermine, amphetamine, phenmetrazine, ephedrine, pentazocine
KEYWORDS
column-switching
REFERENCE
341.
SAMPLE
Matrix: urine
min, filter (0.2 |xm), inject 20 u,L aliquot
HPLC VARIABLES
over 15 min.
Flow rate: 1.5
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Extracted: morphine, ephedrine, phenylpropanolamine, diphenhydramine, nortriptyline, co-
caine, benzoylecgonine, norpropoxyphene, nordiazepam
Also analyzed: amitriptyline, amphetamine, meperidine, codeine, (different gradient)
REFERENCE
Li,S.; Gemperline,P.J.; Briley,K.; Kazmierczak,S. Identification and quantitation of drugs of abuse in urine
Lidoflazine
Molecular formula: C30H35F2N3O
Merck Index: 5507
Lednicer No.: 1 279
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 265
CHROMATOGRAM
KEYWORDS
diazepam; mianserin; ramipril; haloperidol; tetracaine; alprenolol; aceprometazine; glibencla-
mide; chlorophenacinone; doxepin; nimodipine; diphenhydramine; cyclizine; histapyrrodine;
mine; pro trip tyline; flurbiprofen; tetrazepam; zorubicin; prazepam; alimemazine; loperamide;
REFERENCE
Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J,Forensic ScL, 1995, 4O7
254-262.
Limaprost
CAS Registry No.: 74397-12-9, 88852-12-4
Merck Index: 5514
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut Certify C18 SPE cartridge with water, MeCN, and
20 mM citric acid. Add 1 mL plasma to SPE cartridge, wash with 1 mL 20 mM citric acid,
wash with 2 mL MeOH:water 10:90, wash with 2 mL cyclohexane, elute with 3 mL 3% am-
monia in MeOH. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the
residue in 500 |xL MeCN, add 200 |JLL 10 mM DBD-PZ in MeCN, add 300 |xL 10 mM 2,2'-
dipyridyl disulfide and 10 mM triphenylphosphine in MeCN, let stand at room temperature
for 30 min, inject an aliquot. (DBD-PZ prepared from 123 mg 4-(N,N-dimethylaminosulfonyl)-
7-fluoro-2,l,3-benzoxadiazole in 20 mL MeCN added dropwise to 129 mg piperazine in 20 mL
MeCN at room temperature, stir for 30 min, evaporate under reduced pressure, dissolve residue
in 50 mL 5%. HCl, extract three times with 20 mL ethyl acetate, discard ethyl acetate extracts,
adjust pH of aqueous solution to 13-14 with 5% NaOH, extract five times with 50 mL ethyl
acetate, combine extracts, wash with 20 mL water, dry over anhydrous sodium sulfate, evap-
orate under vacuum to give 4-(N,N-dimethylaminosulfonyl)-7-(l-piperazinyl)-2,l,3-benzoxadi-
azole (DBD-PZ) as orange crystals, mp 121-2 (J. Chromatogr. 1991, 588, 61).)
HPLC VARIABLES
Mobile phase: Gradient. MeCNiwater from 35:65 to 60:40 over 1 h
Flow rate: 1
CHROMATOGRAM
Limit of detection: 1.7-5 fmole
OTHER SUBSTANCES
Extracted: alprostadil (prostaglandin El), dinoprost (prostaglandin F2a), dinoprostone (prosta-
glandin E2), 6-ketoprostaglandin FIa, prostaglandin FIa, prostaglandin D2, prostaglandin Al,
prostaglandin Bl
KEYWORDS
plasma; rat; SPE; derivatization
REFERENCE
Toyo'oka,T.; Ishibashi,M.; Terao,T.; Imai,K. Sensitive fluorometric detection of prostaglandins by high perfor-
mance liquid chromatography after precolumn labelling with 4-(iV,iV-dimethylaminosulphonyl)-7-(l-piper-
azinyl)-2,l,3-benzoxadiazole (DBD-PZ), Biomed.Chromatogr., 1992, 6, 143-148.
Lincomycin
CAS Registry No.: 154-21-2, 7179-49-9 (HCI monohydrate),
859-18-7(HCI)
Merck Index: 5525
SAMPLE
Matrix: solutions
Sample preparation: Centrifuge and filter cell solutions (0.22 |xm), inject an aliquot.
HPLCVARIABLES
Column: 150 X 3.9 5 jim NOVA PAK C18
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 3.9
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 250 (xg/mL solution of clindamycin in mobile phase, inject a 25
|xL aliquot.
HPLC VARIABLES
Mobile phase: MeCNiwater 12:88 containing 0.25 g/L tetrabutylammonium perchlorate and 2
mL/L 70% perchloric acid, apparent pH adjusted to 2.5 with 50% NaOH
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: benzyl alcohol, clindamycin, pirlimycin
REFERENCE
Theis,D.L. Ion-pairing liquid chromatographic method for the determination of pirlimycin hydrochloride,
J.Chromatogr., 1987, 402, 335-343.
SAMPLE
Matrix: tissue
Next Page
MeOH and 5 mL water. Homogenize 5 g blended fish tissue with 20 mL 10 mM pH 4.5
KH2P04buffer at 8000 rpm for 2 min. Centrifuge the homogenized sample at 4000 g for 10 min.
Decant and collect the supernatant, extract the residue with 20 mL buffer. Filter the combined
extracts through glass wool. Add 1 mL 10% sodium tungstate solution and 1 mL 34 mM sulfuric
acid, mix. Centrifuge at 4000 rpm for 15 min, filter the supernatant and discard the precipi-
tated protein. Add ImL 3% 1-pentanesulfonic acid and mix well. Add the fish extract to the
SPE cartridge. Wash with 4 mL MeOH.water 10:90 and 2 mL water at 1 mL/min. Elute with
2 mL MeCN:water 50:50. Add 200 |xL 1 M KOH and 500 mg NaCl to the eluate. Extract with
three 3 mL portions of ethyl acetate by agitating on a Vortex mixer and centrifuging at 800 g
for 3-5 min. Combine the extracts and filter through 3 g anhydrous sodium sulfate. Wash
sodium sulfate with 2 mL ethyl acetate. Evaporate the filtrate to dryness under vacuum at
35. Reconstitute the residue in 1 mL mobile phase, filter (0.45 |xm). Inject a 50 |AL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Spherisorb S5 ODS2
Mobile phase: MeCN:20 mM pH 4.5 potassium phosphate buffer containing 20 mM sodium 1-
octanesulfonate 22:88
Flow rate: 1.0
Detector: E, ESA Model 5100A, Model 5010 analytical cell, first electrode +0.65 V, second elec-
trode +0.9 V, Model 5020 guard cell +0.95 V
CHROMATOGRAM
Retention time: 22
Limit of detection: 7 ng/g (muscle), 12 ng/g (skin)
Limit of quantitation: 17 ng/g (muscle), 24 ng/g (skin)
KEYWORDS
salmon; muscle; skin; SPE
REFERENCE
Luo,W.; Hansen,E.B.,Jr.; Ang,C.Y.W.; Thompson,H.C.,Jr. Determination of lincomycin residue in salmon tissues
by ion-pair reversed-phase liquid chromatography with electrochemical detection, J.AOAC Int., 1996, 79,
839-843.
SAMPLE
Matrix: tissue
Sample preparation: Condition a C18 Sep-Pak SPE cartridge with 20 mL MeOH and 40 mL
distilled water. Homogenize 5 g chopped kidney with 50 mL MeOH, 5 g sodium sulfate, and 2
mL 1 M NaOH. Centrifuge at 1200 rcf for 10 min, decant the supernatant, repeat the extraction
with another 50 mL portion of MeOH. Adjust the MeOH phases to pH 4 with 1 M HCl, filter
(Whatman No.4 filter paper), transfer filtrate to a separatory funnel, add 50 mL hexane, shake.
Remove the lower MeOH phase and reduce its volume to approximately 5 mL on a rotary
evaporator. Transfer concentrated extract to a centrifuge tube with four 2 mL portions of water,
add 50 mg sodium tungstate, centrifuge at 1200 rcf for 10 min. Add the supernatant to the
SPE cartridge, wash with 4 mL water, elute with 5 mL MeOH, evaporate the eluate to dryness
under nitrogen, redissolve the residue in 200 |xL mobile phase inject an aliquot.
HPLC VARIABLES
Guard column: 50 X 5 30-35 |jim Co Pell ODS
Column: 250 X 5 5 |jim Spherisorb ODS
Flow rate: 1
Detector: UV 214
KEYWORDS
cow; pig; kidney; SPE
REFERENCE
Farrington,W.H.H.; Cass,S.D.; Patey,A.L.; Shearer,G. A method for the analysis of lincomycin in porcine and
bovine kidneys, Food Addit.Contam., 1987, 5, 67-76.
Previous Page
Linezolid
SAMPLE
HPLCVARIABLES
Column: Zorbax RX-8
Mobile phase: MeOH:THF:water:trifluoroacetic acid 25:73.7:1.2:0.1
Detector: UV 251
CHROMATOGRAM
Retention time: 7
Internal standard: PNU-101145 (10)
KEYWORDS
linezolid is PNU-100766
REFERENCE
Johnson,R.A.; Haan,D.E.; James,C.A.; Hopkins,N.K. Determination of linezolid, PNU-100766, in human
plasma and urine using high-performance liquid chromatography with ultraviolet detection (Abstract 2487),
Pharm.Res., 1997, 14, S374.
Linoleic acid
Merck Index: 5529
SAMPLE
Matrix: beverages
Sample preparation: Heat 240 mL beer at 60, cool. Remove a 200 mL aliquot and add it to 10
mL 3 M sulfuric acid, add 100 u,L 100 |xg/mL n-heptadecanoic acid in MeOH, add 200 mL ether:
pentane 50:50, add 60 g NaCl, shake for 30 min, centrifuge at 5000 rpm for 5 min. Remove a
150 mL aliquot of the organic layer and wash it with 150 mL 5% sodium bicarbonate, evaporate
to dryness under reduced pressure, reconstitute the residue in 1 mL 6 mM tetramethylam-
monium hydroxide in DMF, add 1 mL 7.5 mM 9-(chloromethyl)anthracene in DMF, heat at 75
for 20 min, inject a 50 JULL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Kaseisorb LC ODS-60-5S C18 (Tokyo Kasei)
Mobile phase: Gradient. MeCN:water 90:10 for 5 min, to 95:5 over 40 min, maintain at 95:5 for
20 min, to 100:0 over 5 min, maintain at 100:0 for 15 min.
Flow rate: 1.1
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: n-heptadecanoic acid, lauric acid, linolenic acid, myristic acid, oleic acid, palmitic
acid, palmitoleic acid, stearic acid
KEY WpRDS
derivatization; beer
REFERENCE
Kaneda,H.; Kano,Y.; Kamimura,M.; Osawa,T.; Kawakishi,S. Analysis of long-chain fatty acids in beer by HPLC-
fluorescence detection method, J.Agric.Food Chem., 1990, 38, 1363-1367.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 20 mL chloroform:MeOH 2:1, vortex for 30 s, add 4 mL
water, shake for 5 min, centrifuge at 2500 rpm for 5 min, extract the aqueous layer twice more
with 10 mL portions of chloroform:MeOH 2:1. Combine the organic layers and dry them over
anhydrous sodium sulfate, evaporate to dryness under a stream of nitrogen at 40, reconstitute
the residue in 1 mL dry dichloromethane, add 1 mg p-aminophenol, add 150 |JLL triethylamine,
add at least a 3-fold molar excess of 2-bromo-l-methylpyridinium iodide, heat at 60 for 30
min, cool, concentrate under a stream of nitrogen, add 1 mL 100 mM HCl, add 1 mL ethyl
acetate, shake vigorously, centrifuge at 2500 rpm for 5 min, inject an aliquot of the supernatant.
(Prepare 2-bromo-l-methylpyridinium iodide by analogy with the preparation of 2-chloro-l-
methylpyridinium iodide. Add 15 g methyl iodide to 13.9 g 2-bromopyridine in 3 mL acetone
at 0, stir at room temperature for 3 days. Filter the precipitate and wash it with 50 mL dry
ether, dry under reduced pressure to give 2-bromo-l-methylpyridinium iodide (Bull. Chem. Soc.
Japan 1977, 50, 1863).)
HPLCVARIABLES
Column: 250 X 4.6 10 |xm Nucleosil C-18
Mobile phase: MeOH:water:perchloric acid 88:12:0.1 containing 50 mM sodium perchlorate
Flow rate: 1.2
Detector: E, 0.75 V, Ag/AgCl reference electrode
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: linolenic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid
KEYWORDS
derivatization; guinea pig; plasma
REFERENCE
Ikenoya,S.; Hiroshima,O.; Ohmae,M.; Kawabe,K. Electrochemical detector for high performance liquid chro-
matography. IV. Analysis of fatty acids, bile acids and prostaglandins by derivatization to an electrochem-
ically active form, Chem.Pharm.Bull.(Tokyo), 1980, 28, 2941-2947.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 100 |xL 100 |xg/mL margaric acid in MeOH + 1.4 mL 1/
15 M pH 7.0 phosphate buffer, shake, add to a 45 X 12 glass column packed with 1 g Extrelut,
let stand for 20 min, elute with 10 mL chloroform. Evaporate the eluate to dryness under
reduced pressure and reconstitute the residue with 600 |ULL benzene (Caution! Benzene is a
carcinogen!), add 600 |xL 2% oxalyl chloride in benzene, heat at 70 for 30 min, evaporate to
dryness under reduced pressure, add 100 JJLL 40 mM 1-naphthylamine in benzene, add 10 (xL
400 (xM triethylamine in benzene, heat at 30 for 15 min, inject a 40 |xL aliquot.
HPLC VARIABLES
Column: 300 X 4 8-10 fjum jxBondapak C18
Flow rate: 2
Detector: UV 280
CHROMATOGRAM
Retention time: 12
Internal standard: margaric acid
OTHER SUBSTANCES
Extracted: myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid
KEY WpRDS
REFERENCE
Ikeda,M.; Shimada,K.; Sakaguchi,T. High-performance liquid chromatographic determination of free fatty acids
with 1-naphthylamine, J.Chromatogr., 1983, 272, 251-259.
SAMPLE
Matrix: blood
Sample preparation: 10 |xL Plasma + 200 |xL 500 mM pH 6.5 phosphate buffer + 50 |xL 20 (JLM
IS in MeOH + 2 mL n-heptane:chloroform 50:50, vortex for 2 min, centrifuge at 1000 g for 10
min. Remove the lower organic layer and evaporate it to dryness, reconstitute the residue in
two 100 |xL aliquots of acetone. Evaporate the acetone solution to dryness, add 2-3 mg finely
powdered potassium bicarbonate:sodium sulfate 50:50, add 50 jxL 800 jxM dibenzo-18-crown-6
in acetone, add 50 JJLL 2 mM 4-bromomethyl-7-acetoxycoumarin in acetone, heat at 50 in the
dark for 30 min, inject a 50 |xL aliquot. (Prepare 4-bromomethyl-7-acetoxycoumarin as follows.
Reflux 50 g 7-hydroxy-4-methylcoumarin (p-methylumbelliferone) and 100 mL acetic anhydride
for 1 h, cool, pour into 500 mL cold water, filter, dry the solid, recrystallize from EtOH to give
4-methyl-7-acetoxycoumarin. Reflux 10 g 4-methyl-7-acetoxycoumarin, 9 g N-bromosuccinim-
ide, a little 2,2'-(azobis(2-methylpropionitrile) (a,a'-azobisisobutyronitrile, Eastman), and 100
mL carbon tetrachloride for 20 h, cool, evaporate under reduced pressure to remove the solvent,
wash the residue with water, filter, dry, recrystallize from ethyl acetate/cyclohexane to give 4-
bromomethyl-7-acetoxycoumarin (mp 184-185). (J. Chromatogr. 1982, 234, 121).)
HPLC VARIABLES
Mobile phase: Gradient. MeCN:MeOH:water from 35:35:30 to 0:90:10 over 70 min (convex
gradient).
Flow rate: 1.2
mM NaOH in MeOH:water 80:20 pumped at 0.4 mL/min and the mixture flowed through a
3.5 m X 0.5 mm ID stainless steel coil at 50 to the detector.
CHROMATOGRAM
Retention time: 42
Internal standard: margaric acid (57)
Limit of quantitation: 5 pmole
OTHER SUBSTANCES
Extracted: arachidonic acid, capric acid, caproic acid, caprylic acid, heptanoic acid, lauric acid,
linoleic acid, linolenic acid, myristic acid, myristoleic acid, nonanoic acid, oleic acid, palmitic
acid, palmitoleic acid, stearic acid, tridecanoic acid, undecanoic acid
KEY WpRDS
derivatization; post-column reaction; plasma
REFERENCE
Tsuchiya,H.; Hayashi,T.; Sato,M.; Tatsumi,M.; Takagi,N. Simultaneous separation and sensitive determination
of free fatty acids in blood plasma by high-performance liquid chromatography, J.Chromatogr., 1984, 309,
43-52.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak silica SPE cartridge with chloroform. 500 jxL Serum
+ 1 mL 20 |JLM tridecanoic acid in chloroform containing 9.53 (xM 2-heptenoic acid + 100 (xL
500 mM sulfuric acid + 5 mL chloroform + 20 mL MeCN, shake slowly, add 1.5 g anhydrous
calcium chloride, invert slowly, let stand for 30 min, filter (No 5A paper) the upper layer. Add
500 |xL 45 mM dansyl semipiperazide in chloroform and 150 mg dicyclohexylcarbodiimide to
the filtrate, let stand for 30 min, remove the solvent by evaporation under reduced pressure,
dissolve the residue in the minimum amount of chloroform, add to the SPE cartridge, elute
with two 1 mL portions of chloroform. Evaporate the eluate to dryness under reduced pressure,
reconstitute with the minimum amount of MeOH, inject an aliquot. (Prepare dansyl semipi-
perazide as follows. Add 8 g dansyl chloride to 50 g piperazine dissolved in 500 mL acetone
with stirring at room temperature over 30 min, stir overnight, evaporate to dryness under
reduced pressure. Dissolve the residue in 300 mL chloroform, filter (5A paper), wash the filtrate
three times with 5% sodium bicarbonate, wash with water. Extract the organic layer three
times with 100 mL portions of 1 M HCl, combine the extracts and wash them repeatedly with
chloroform, make alkaline with a slight excess of powdered sodium carbonate, extract with
benzene (Caution! Benzene is a carcinogen!). Wash the benzene layer twice with 5% sodium
bicarbonate, dry over anhydrous sodium sulfate, evaporate to dryness under reduced pressure.
Dissolve the residue in the minimum amount of chloroform, chromatograph on a 200 X 50
column of Wako Gel C-200 silica gel with chloroform. When the first eluting yellow band
reaches the bottom change the eluent to MeOH, continue eluting until all the yellow band is
collected. Evaporate the eluate to dryness to obtain dansyl semipiperazide, store in the dark
at 5. Determine the concentration of dansyl semipiperazide by spectrophotometry, extinction
coefficient at 340 nm in MeOH or EtOH is 4300 M1Cm1.)
HPLC VARIABLES
Column: 250 X 4.6 Hitachi C18 3056
Mobile phase: Gradient. MeCN.water 45:55 for 43 min, 60:40 for 32 min, 75:25 for 28 min, 85:
15 for 100 min, 100:0 for rest of run (step gradient).
Flow rate: 0.8
CHROMATOGRAM
Retention time: 145
Internal standard: tridecanoic acid (139), 2-heptenoic acid (77)
OTHER SUBSTANCES
Extracted: acetic acid, arachidic acid, arachidonic acid, butyric acid, capric acid, caproic acid,
caprylic acid, docosahexaenoic acid, eicosatrienoic acid, eicosenoic acid, elaidic acid, erucic acid,
isobutyric acid, isocaproic acid, isovaleric acid, lactic acid, lauric acid, linoleic acid, linolenic
acid, margaric acid, myristic acid, myristoleic acid, nonanoic acid, palmitic acid, palmitoleic
acid, pentadecanoic acid, petroselenic acid, propionic acid, stearic acid, undecanoic acid, vac-
cenic acid, valeric acid
KEY WpRDS
derivatization; serum; SPE
REFERENCE
Yanagisawa,L; Yamane,M.; Urayama,T. Simultaneous separation and sensitive determination of free fatty acids
in blood plasma by high-performance liquid chromatography, J.Chromatogr., 1985, 345, 229-240.
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Plasma + 3 mL 3.9 |xg/mL margaric acid in chloroform:n-heptane:
MeOH 28:21:1, vortex for 2 min, centrifuge at 2000 g for 20 min. Remove a 2 mL aliquot of
the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute with 50
IJLL 2 mg/mL 9-anthracenemethanol (9-(hydroxymethyl)anthracene) in dichloromethane, add 50
JJLL reagent solution, add 10 |xL triethylamine, sonicate for 15 s, vortex for 15 s, heat at 50 for
30 min, evaporate to dryness under a stream of nitrogen, reconstitute with 1 mL mobile phase,
inject an aliquot. (Prepare the reagent, 2-bromo-l-methylpyridinium iodide, as follows. Reflux
5 mL 2-bromopyridine and 7 mL iodomethane in 20 mL dry diethyl ether for 1 h, wash the
precipitate of 2-bromo-l-methylpyridinium iodide with ether. The reagent solution was a 20
mg/mL suspension of 2-bromo-l-methylpyridinium iodide in dichloromethane.)
HPLC VARIABLES
Mobile phase: Gradient. MeCN:water 93:7 for 12 min, 86:14 for 5 min, 100:0 for 23 min (step
gradient?).
Flow rate: 1
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Extracted: arachidic acid, arachidonic acid, behenic acid, lauric acid, lignoceric acid, linolenic
acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid
KEYWORDS
REFERENCE
Baty,J.D.; Pazouki,S.; Dolphin,J. Analysis of fatty acids as their anthrylmethyl esters by high-performance
liquid chromatography with fluorescence detection, J.Chromatogr., 1987, 395, 403411.
SAMPLE
Matrix: blood
Sample preparation: 15 JJLL Plasma + 485 |JLL 6.2 |xM IS in water, mix, add 1 mL MeOH, vortex,
add 3 mL 50 mg/mL BHT in chloroform, vortex for 1 min, centrifuge at 2000 g for 10 min,
remove the organic layer, extract the aqueous layer with 4 mL chlorofornr.MeOH 75:25. Com-
with 1 mL chloroform, add to a Bond Elut aminopropyl SPE cartridge, elute with diethyl ether:
acetic acid 98:2. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute with
acetone, evaporate to dryness (?), add 50 JJLL 1 mg/mL 4-bromomethyl-7-acetoxycoumarin in
acetone, add 50 JJLL 800 \iM dibenzo-18-crown-6 in acetone, add 2-3 mg of a finely powdered
mixture of potassium bicarbonate and sodium sulfate (50:50 ?), shake at 50 for 30 min, inject
a 5-50 |JLL aliquot. (Prepare 4-bromomethyl-7-acetoxycoumarin as follows. Reflux 50 g 7-hy-
droxy-4-methylcoumarin (p-methylumbelliferone) and 100 mL acetic anhydride for 1 h, cool,
pour into 500 mL cold water, filter, dry the solid, recrystallize from EtOH to give 4-methyl-7-
acetoxycoumarin. Reflux 10 g 4-methyl-7-acetoxycoumarin, 9 g N-bromosuccinimide, a little
2,2'-(azobis(2-methylpropionitrile) (a,ot'-azobisisobutyronitrile, Eastman), and 100 mL carbon
tetrachloride for 20 h, cool, evaporate under reduced pressure to remove the solvent, wash the
residue with water, filter, dry, recrystallize from ethyl acetate/cyclohexane to give 4-bromo-
methyl-7-acetoxycoumarin (mp 184-185) (J. Chromatogr. 1982, 234, 121).)
HPLC VARIABLES
Column: 100 X 5 5 jim Nova Pak radial compression
Mobile phase: Gradient. A was MeCN:MeOH:water 35:35:30. B was MeOH:water 90:10. A:B
from 100:0 to 0:100 over 70 min (convex gradient).
Flow rate: 2
mM NaOH in MeOH:water 80:20 pumped at 0.4 mL/min and the mixture flowed through a 9
m X 0.23 mm ID stainless steel coil at 80 to the detector.
CHROMATOGRAM
Retention time: 50
OTHER SUBSTANCES
Extracted: arachidonic acid, eicosatrienoic acid, lauric acid, linolenic acid, myristic acid, myris-
toleic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid
KEY WpRDS
derivatization; post-column reaction; rat; plasma; SPE
REFERENCE
Kelly,R.A.; O'Hara,D.S.; Kelley,V. High-performance liquid chromatographic separation of femtomolar quanti-
ties of endogenous carboxylic acids, including arachidonic acid metabolites, as 4-bromomethyl-7-acetoxy-
coumarin derivatives, J.Chromatogr., 1987, 416, 247-254.
SAMPLE
Matrix: blood
Sample preparation: 25 (xL Serum + 25 uL 80 (xM margaric acid in EtOH, mix, add 100 jxL 20
mM 2-nitrophenylhydrazine hydrochloride in EtOH:40 mM HCl 25:75, add 200 (xL 125 mM 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in EtOH containing 6.5% pyridine,
heat at 60 for 20 min, add 2 mL 33.3 mM pH 6.4 phosphate buffer:500 mM HCl 3.8:0.4, add
1.5 mL n-hexane, vortex for 30 s, centrifuge at 1500 g for 5 min. Remove the n-hexane layer
and evaporate it to dryness under a stream of nitrogen at room temperature, reconstitute the
residue in 50 |xL MeOH, inject a 5-10 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm YMC-C8 (Yamamura, Kyoto)
Mobile phase: MeCN:water 85:15 adjusted to pH 4.5 with MeCN:100 mM HCl 85:15
Flow rate: 1.2
CHROMATOGRAM
Retention time: 7.6
Internal standard: margaric acid (11.2)
Limit of detection: 0.4-1 pmole (UV 400), 100-200 fmole (UV 230)
OTHER SUBSTANCES
Extracted: arachidonic acid, capric acid, dihomo-gamma-linolenic acid, docosahexaenoic acid, ei-
cosapentaenoic acid, lauric acid, linolenic acid, myristic acid, myristoleic acid, oleic acid, pal-
mitic acid, palmitoleic acid, stearic acid
KEY WpRDS
REFERENCE
Miwa,H.; Yamamoto,M.; Nishida,T.; Nunoi,K; Kikuchi,M. High-performance liquid chromatographic analysis
of serum long-chain fatty acids by direct derivatization method, J.Chromatogr., 1987, 416, 237-245.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL rat platelets suspended in pH 7.2 Tyrode-HEPES buffer con-
taining 2 mM calcium chloride and 2.5 U/mL thrombin with 50 |xL 2 |iM margaric acid in
MeOH, filter (0.45 |xm), add 10 (xL 20% HCl to the filtrate, extract with 2 mL chloroform.MeOH
50:50, centrifuge at 880 g for 10 min, remove the organic layer, extract with 1 mL chloroform,
centrifuge at 800 g for 10 min. Combine the organic layers and evaporate them to dryness
under a stream of nitrogen, reconstitute the residue in 50 JJLL DMF, add 50 |JLL 12 mM mono-
dansylcadaverine in DMF, add 2 |xL diethyl cyanophosphonate (diethylphosphorocyanidate),
stir for 10 s, let stand at room temperature for 15 min, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 TSKgel ODS-80TM (Tosoh)
Mobile phase: Gradient. A was MeOH:200 mM pH 7.8 Tris-HCl buffer 50:50. B was MeCN. A:
B from 50:50 to 10:90 over 1 h.
Flow rate: 1
CHROMATOGRAM
Retention time: 32
OTHER SUBSTANCES
Extracted: lauric acid, linolenic acid, myristic acid, myristoleic acid, oleic acid, palmitic acid,
palmitoleic acid, stearic acid
KEYWORDS
derivatization; rat; platelets
REFERENCE
Lee,Y.M.; Nakamura,H-; Nakajima,T. Rapid determination by high-performance liquid chromatography of free
fatty acids released from rat platelets after derivatization with monodansylcadaverine, J.Chromatogr., 1990,
515, 467-473.
SAMPLE
Matrix: blood
Sample preparation: 50 JJLL Plasma + 10 |xL IS solution + 450 |JLL micelle solution, vortex for
10 s, add 25 jxL 28 mg/mL 9-bromomethylacridine in acetone, mix. Remove a 50 (JLL aliquot
and heat it to 60 for 6 min, inject the whole amount through a 2 (xm stainless steel filter of 8
sq mm area onto column A, wash to waste with 400 jxL mobile phase A, back flush the contents
of column A onto column B with the mobile phase B, monitor the effluent from column B. After
each injection backflush the stainless steel filter with 1 mL buffer. (Micelle solution was 25
mM Arkopal N-130 (a polyoxyethylene(13)nonylphenol, Hoechst Holland, Amsterdam) in 10
mM pH 7.0 phosphate buffer containing 6 mM tetrakis(decyl)ammonium bromide. Synthesize
9-bromomethylacridine as follows. Heat 10 g diphenylamine, 10 mL glacial acetic acid, and 40
g anhydrous zinc chloride to 220, evaporate excess acetic acid with stirring, heat at 220-230
for 6 h, digest with hot 10% sulfuric acid, make strongly alkaline with 25% ammonia to dissolve
the zinc chloride. Extract the insoluble residue with toluene. Extract the organic layer with
10% sulfuric acid, make the aqueous layer alkaline with aqueous ammonia. Collect the yellow
precipitate that separates and recrystallize it twice from petroleum ether to give 9-methyl
acridine as pale yellow needles (Chromatographia 1989, 28, 267). Reflux 560 mg 9-methyl-
acridine, 445 mg N-bromosuccinimide, and 10 mg benzoyl peroxide in 30 mL carbon tetrachlo-
ride for more than 2 h, cool, chromatograph on silica gel with benzene:ethyl acetate 30:1 (Cau-
tion! Benzene is a carcinogen!) to obtain 9-bromomethylacridine as yellow crystals (mp
147-151) (Anal. Lett. 1987, 20, 1581).)
HPLC VARIABLES
Column: A 10 X 2.1 40 |xm Chromsep C18 (Chrompack); B 100 X 3 5 jxm Chromspher C18
(Chrompack)
Mobile phase: A 10 mM pH 7.0 phosphate buffer; B Gradient. MeOHiwater 75:25 for 3 min, to
100:0 over 12 min (concave gradient).
CHROMATOGRAM
Retention time: 8.9
Internal standard: heptadecanoic acid (11.8)
OTHER SUBSTANCES
Extracted: arachidonic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic
acid, stearic acid
KEY WpRDS
derivatization; column-switching; plasma
REFERENCE
van der Horst,F.A.L.; Post,M.H.; Holthuis,J.J.M.; Brinkman,U.A.T. Automated high-performance liquid chro-
matographic determination of plasma free fatty acids using on-line derivatization with 9-bromomethylacri-
dine based on micellar phase-transfer catalysis, J.Chromatogr., 1990, 500, 443-452.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Serum + 10 |xL 100 |xM margaric acid in DMF + 200 |xL 500 mM
pH 6.5 phosphate buffer, mix, add 2 mL chloroform.n-heptane 50:50, vortex for 1 min, centri-
fuge at 1500 g for 5 min. Remove a 1.5 mL aliquot of the organic layer and evaporate it to
dryness, reconstitute the residue in 200 (xL DMF. Remove a 100 |xL aliquot and add it to 50
jxL 1 M l-(3-dimethylaminopropyl)-3-ethylcarbodiimide in water, 50 |xL 10% pyridine in water,
and 100 (xL 30 mM reagent in DMF, mix well, let stand at room temperature for 45 min, inject
a 20 (xL aliquot. (Synthesis of the reagent, 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimida-
zole, is as follows. Add 3.15 g benzil and 2.46 g methyl 4-formylbenzoate (terephthalaldehydic
acid methyl ester) to 10 g ammonium acetate in 30 mL acetic acid, stir at 80 for 9 h, cool to
room temperature, pour into cold water, filter. Wash the precipitate with water and recrystal-
lize it from EtOH to give 4-(4,5-diphenyl-lH-imidazol-2-yl)benzoic acid methyl ester as pale
yellow crystals (mp 245-248). Reflux 1.47 g 4-(4,5-diphenyl-lH-imidazol-2-yl)benzoic acid
methyl ester and 15 mL 80% hydrazine hydrate (Caution! Hydrazine hydrate is a carcinogen!)
in 100 mL EtOH for 4 h, cool, to room temperature, pour into cold water, filter. Wash the
precipitate with water and recrystallize it from EtOHibenzene 50:50 (Caution! Benzene is a
carcinogen!) to give 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole as a colorless powder
(mp >300).)
HPLC VARIABLES
Column: 150 X 6 5 jxm Shim-pack CLC-ODS
Mobile phase: Gradient. MeOH:water 90:10 for 5 min, to 100:0 over 25 min, maintain at 100:0
for 5 min.
Flow rate: 1
CHROMATOGRAM
Limit of detection: 7-57 fmole
OTHER SUBSTANCES
Extracted: lauric acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid,
stearic acid
Interfering: arachidonic acid
KEY WpRDS
REFERENCE
Nakashima,K; Taguchi,Y; Kuroda,N.; Akiyama,S.; Duan,G. 2-(4-Hydrazinocarbonylphenyl)-4,5-diphenylimi-
dazole as a versatile fluorescent derivatization reagent for the high-performance liquid chromatographic
analysis of free fatty acids, J.Chromatogr., 1993, 619, 1-8.
SAMPLE
Matrix: blood
Sample preparation: 100 JJLL Serum + 20 JJLL 5 mM IS in isopropanol, mix, add 500 |xL isopro-
panol:n-heptane:2 M phosphoric acid 40:10:1, mix, let stand at room temperature for 5-10 min,
add 200 JJLL n-heptane, add 300 ^xL water, vortex thoroughly, centrifuge at 1000 g for 5 min.
Remove a 200 |xL aliquot of the upper organic layer and evaporate it to dryness under a stream
of nitrogen, reconstitute the residue in 6 JJLL reagent, 500 |xL MeCN, and ca. 1 mg potassium
bicarbonate, flush the tube with nitrogen, close the PTFE-lined cap tightly, heat at 85 with
vigorous stirring for 45 min (weigh vial before and after heating to check for leakage), cool,
remove stir bar, centrifuge, inject a 10-25 JJLL aliquot of the supernatant. (Reagent was 50 mM
p-bromophenacyl bromide in MeCN containing 5 mM 18-crown-6, store protected from light.)
HPLCVARIABLES
Guard column: 4 X 4 5 jxm CN
Column: 25 X 4 3 jim Spherisorb C6
Flow rate: 1.3
Detector: UV 254
CHROMATOGRAM
Internal standard: heptadecanoic acid (margaric acid) (14.7)
OTHER SUBSTANCES
Extracted: arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid, elaidic acid, lauric
acid, linolenic acid, myristic acid, myristoleic acid, oleic acid, palmitic acid, palmitoleic acid,
stearic
KEYWORDS
REFERENCE
Puttmann,M.; Krug,H.; von Ochsenstein,E.; Kattermann,R. Fast HPLC determination of serum free fatty acids
in the picomole range, Clin.Chem., 1993, 39, 825-832.
SAMPLE
Matrix: blood
Sample preparation: 10 |JLL Serum + 44 |xL MeOH + 1 JJLL pyridine, sonicate for 5 min, add 25
JJLL 100 mM reagent in DMF, add 20 fxL 400 mM l-ethyl-3-(3-dimethylaminopro-
pyDcarbodiimide in MeOH, let stand at 25 for 2 h, centrifuge, inject an aliquot. (Reagent was
2-(5-hydrazinocarbonyl-2-furyl)-5,6-dimethoxybenzothiazole which was synthesized as follows.
Pass dry hydrogen chloride into a mixture of 12.6 g methyl 2-furoate, 4.5 g paraformaldehyde,
and 3.4 g anhydrous zinc chloride in 50 mL dry chloroform for 3 h while holding the reaction
temperature at 30. After cooling pour the contents of the flask into 100 mL cold water, remove
the chloroform layer, extract the aqueous layer with chloroform (cf Coll. Czech. Chem. Com-
mun. 1960, 25, 1058). Combine the chloroform layers, neutralize, dry over anhydrous calcium
chloride, evaporate, distil to give 5-chloromethyl furyl-2-carboxylic acid methyl ester (bp 1087
4 mm Hg). Reflux 10 g 5-chloromethyl furyl-2-carboxylic acid methyl ester and 25 g silver
carbonate in 100 mL THF:water 70:30 for 5 h, filter through Celite, concentrate the filtrate
under reduced pressure, chromatograph the product on silica gel with chloroform to give 5-
hydroxymethyl furyl-2-carboxylic acid methyl ester as a light yellow oil. Add a solution of 2.9
g 5-hydroxymethyl furyl-2-carboxylic acid methyl ester in 30 mL dichloromethane to 12 g pyr-
idinium chlorochromate in 100 mL, dichloromethane, stir at room temperature for 4 h, evap-
orate to dryness under reduced pressure, chromatograph on silica with dichloromethane to give
5-formyl furyl-2-carboxylic acid methyl ester as a light yellow powder. Add 10 mL concentrated
nitric acid dropwise to 20 g 4-bromoveratrole in 60 mL acetic acid while keeping the temper-
ature at 10-30 with occasional cooling, when the addition is complete pour the reaction mixture
into ice-water. Collect the precipitate and dissolve it in 500 mL hot EtOH, add activated char-
coal, filter, add 40 mL water to the filtrate to give 4,5-dimethoxy-2-nitrobromobenzene as a
light yellow crystalline solid (mp 121-122). Prepare sodium sulfide by melting together 5 g
sodium sulfide nonahydrate and 700 mg sulfur, add this mixture to 5 g 4,5-dimethoxy-2-ni-
trobromobenzene in 50 mL EtOH:water 95:5, reflux for 30 min, pour into ice-water, collect the
solid, recrystallize from dichloromethane to give di(4,5-dimethoxy-2- nitrophenyDsulfide as yel-
low needles (mp 231-232). Add 15 mL concentrated HCl dropwise to 1.5 g di(4,5-dimethoxy-2-
nitrophenyl)sulfide and 4.5 g tin powder stirred at 40-50 in 150 mL EtOH, reflux for 1 h, cool
to room temperature, filter, add 1.17 g 5-formyl furyl-2-carboxylic acid methyl ester to the
filtrate, reflux for 1 h, cool, filter, chromatograph the solid on silica gel with dichloromethane,
recrystallize from EtOH to give 5-(5',6>-dimethoxybenzothiazolyl)-N-furan-2-carboxylic acid
methyl ester as a yellow powder (mp 192-202).>Add 2 mL hydrazine hydrate (Caution! Hydra-
zine hydrate is a carcinogen!) to 800 mg 5-(5',6 -dimethoxybenzothiazolyl)-N-furan-2-carboxylic
acid methyl ester in 20 mL EtOH, reflux for 30 min, collect the solid, wash with MeOH, dry
under vacuum over phosphorus pentoxide to give 2-(5-hydrazinocarbonyl-2-furyl)-5,6-dime-
thoxybenzothiazole as a light yellow solid (mp 226-228).)
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Wakosil-II 5C18 HG
Mobile phase: Gradient. MeCN:water from 70:30 to 75:25 over 25 min, to 100:0 over 15 min,
maintain at 100:0.
Flow rate: 1
CHROMATOGRAM
Retention time: 25
OTHER SUBSTANCES
Extracted: alprostadil, arachidonic acid, dinoprost, dinoprostone, lauric acid, linolenic acid, mar-
garic acid, myristic acid, myristoleic acid, oleic acid, palmitic acid, palmitoleic acid, prosta-
glandin F la , stearic acid
KEYWORDS
REFERENCE
Saito,M.; Ushijima,T.; Sasamoto,K; Ohkura,Y.; Ueno,K. 2-(5-Hydrazinocarbonyl-2-furyl)-5,6-dimethoxybenzo-
thiazole as a precolumn fluorescence derivatization reagent for carboxylic acids in high-performance liquid
chromatography and its application to the assay of fatty acids in human serum, Anal.ScL, 1995,11, 103-
107.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Serum + 100 |xL isopropanol:heptane:0.5 M sulfuric acid 80:20:2,
extract. Remove 50 |xL of the organic layer and evaporate it to dryness under reduced pressure,
reconstitute the residue in 50 jxL 15 mM reagent in DMSO, 50 |xL 100 mM l-ethyl-3-(3-di-
methylaminopropyl)carbodiimide in MeOH, 50 |xL 1% pyridine in MeOH, and 50 |xL 100 |xM
nonadecanoic acid, heat at 37 for 1 h, inject a 10 |xL aliquot. (Reagent was 2-(5-hydrazinocar-
bonyl-2-oxazolyl)-5,6-dimethoxybenzothiazole which was synthesized as follows. Add 10 mL
concentrated nitric acid dropwise to 20 g 4-bromoveratrole in 60 mL acetic acid while keeping
the temperature at 10-30 with occasional cooling, when the addition is complete pour the
reaction mixture into ice-water. Collect the precipitate and dissolve it in 500 mL hot EtOH,
add activated charcoal, filter, add 40 mL water to the filtrate to give 4,5-dimethoxy-2-nitro-
bromobenzene as a light yellow crystalline solid (mp 121-122). Prepare sodium sulfide by
melting together 5 g sodium sulfide nonahydrate and 700 mg sulfur, add this mixture to 5 g
4,5-dimethoxy-2-nitrobromobenzene in 50 mL EtOH:water 95:5, reflux for 30 min, pour into
ice-water, collect the solid, recrystallize from dichloromethane to give di(4,5-dimethoxy-2-nitro-
phenyDsulfide as yellow needles (mp 231-232) (Anal. Sci. 1995, 11, 103). Add ethyl oxalyl
chloride in ether to a solution of diazomethane in ether at 0 to give ethyl diazopyruvate
(Caution! Diazo compounds are explosive and toxic!) (cf. Buehler,C.A.; Pearson,D.E. Survey of
Organic Syntheses, Wiley, New York, 1970, p. 179). Heat 100 mg ethyl diazopyruvate, a few
mg copper(II) acetylacetonate, and 400 |xL chloroacetonitrile in benzene at 60 overnight (Cau-
tion! Benzene is a carcinogen!), cool, add to sodium bicarbonate solution, extract with ether,
dry the organic layer, evaporate, chromatograph on silica with petroleum ether:ethyl acetate
90:10, distil the product at 90712 mm Hg to give ethyl 2-chloromethyl-5-oxazolecarboxylate as
an oil in 18% yield (US Patent 4 603 209 (July 29, 1986)). Reflux 5.0 g ethyl 2-chloromethyl-
5-oxazolecarboxylate and 11.7 g NaI in 80 mL acetone for 1 h, partition the reaction mixture
between ethyl acetate and water. Wash the organic layer with water and dry it over anhydrous
sodium sulfate, evaporate to give ethyl 2-iodomethyl-5-oxazolecarboxylate as a reddish-brown
oil. Reflux 7.4 g ethyl 2-iodomethyl-5-oxazolecarboxylate and 21.5 g silver carbonate in 100 mL
THFrwater 70:30 for 4 h, filter through Celite, evaporate under reduced pressure, chromato-
graph on silica gel using benzene:ethyl acetate 95:5 to give ethyl 2-hydroxymethyl-5-oxazole-
carboxylate (mp 60.5-62). Stir 2.04 g oxalyl chloride in 15 mL dichloromethane at -50 under
nitrogen, add 1.54 g DMSO in 3 mL dichloromethane, after 5 min add 1.4 g ethyl 2-hydroxy-
methyl-5-oxazolecarboxylate in 6 mL dichloromethane, stir for 15 min at -50, add 5.7 mL
triethylamine, allow to warm to room temperature, dilute with dichloromethane, wash with
water, dry over anhydrous sodium sulfate, concentrate under reduced pressure, chromatograph
on silica gel using benzene:ethyl acetate 95:5 to give ethyl 2-carboxaldehyde-5-oxazolecarbox-
ylate (mp 71.5-73). Add 11.3 mL concentrated HCl to 750 mg di(4,5-dimethoxy-2-nitro-
phenyDsulfide stirred in 100 mL EtOH, add 3.3 g tin powder at 40-45, stir for 1 h at 40-45,
dilute with 100 mL water, pass hydrogen sulfide gas through this solution (Caution! Hydrogen
sulfide is highly toxic!), filter, concentrate the filtrate under reduced pressure to give 4,5-di-
methoxy-2-aminothiophenol. Take up this compound in 30 mL EtOH:acetic acid 2:1 and add
750 mg ethyl 2-carboxaldehyde-5-oxazolecarboxylate, reflux for 1 h, collect the precipitate and
recrystallize it from EtOH to give 2-(5-ethoxycarbonyl-2-oxazolyl)-5,6-dimethoxybenzothiazole
as yellow needles (mp 200-201). Add 381 mg 2-(5-ethoxycarbonyl-2-oxazolyl)-5,6-dimethoxy-
benzothiazole to 20 mL EtOH containing 3 mL DMF and 5 mL hydrazine hydrate, reflux for
1 h, collect the precipitate and wash it with EtOH, dry under vacuum to give 2-(5-hydrazino-
carbonyl-2-oxazolyl)-5,6-dimethoxybenzothiazole as a yellow powder (mp 255.5-280 (d)).)
HPLCVARIABLES
Column: 250 X 4.6 5 ^m Wakosil-II 5C18 HG
Flow rate: 1
CHROMATOGRAM
Retention time: 10
Internal standard: nonadecanoic acid (19)
OTHER SUBSTANCES
Extracted: lauric acid, linolenic acid, margaric acid, myristic acid, myristoleic acid, oleic acid,
palmitic acid, palmitoleic acid, stearic acid
KEY WpRDS
REFERENCE
Saito,M.; Ushijima,T.; Sasamoto,K; Ohkura,Y.; Ueno,K. 2-(5-Hydrazmocarbonyl-2-oxazolyl)-5,6-dimethoxyben-
zothiazole as a precolumnfluorescencederivatization reagent for carboxylic acids in high-performance liquid
chromatography and its application to the assay of fatty acids in human serum, J.Chromatogr.B, 1995,674,
167-175.
SAMPLE
Matrix: blood
Sample preparation: 10 u,L Serum or plasma + 10 |xL 100 |xM pentadecanoic acid in DMF +
200 fxL 500 mM pH 6.5 phosphate buffer, mix, add 2 mL n-heptane:chloroform 50:50, vortex
thoroughly, centrifuge at 1500 g for 10 min. Remove 750 |xL of the organic layer and evaporate
it to dryness under reduced pressure, reconstitute the residue in 375 u-L DMF, add 25 JJLL 4 M
l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in pyridine:water 40:60, add 100
|JLL 5 mM DBD-ProCZ in DMF, let stand at room temperature in the dark for 1.5 h, inject an
aliquot. (Synthesis of DBD-ProCZ is as follows. Dissolve 0.5 g magnesium sulfate heptahydrate
and 6 g NaOH in 60 mL water, throughout the reaction keep the flask at about 20 with cold
water cooling, add 15 mL 30% hydrogen peroxide, add 75 mL MeOH, add 12.1 g powdered
benzoyl peroxide in one go, stir for 10 min, pour into 150 mL 20% sulfuric acid, extract three
times with 50 mL portions of chloroform, determine peroxybenzoic acid concentration by io-
dometric titration (Tetrahedron 1967, 23, 3327). Slowly add 110 mL 1 M peroxybenzoic acid in
chloroform to 7 g 2,6-difluoroaniline dissolved in 100 mL chloroform, stir at room temperature,
when reaction is complete (iodometric titration) wash with 2% sodium thiosulfate, wash with
5% sodium carbonate, wash with water, dry over anhydrous sodium sulfate, evaporate to dry-
ness under reduced pressure, recrystallize 2,6-difluoronitrosobenzene form EtOH (mp 108.5-
109.5). Stir 8.5 g 2,6-difluoronitrosobenzene in 85 mL DMSO at room temperature and add a
solution of 3.91 g sodium azide in 85 mL DMSO dropwise, let stand for about 1 h, add to a
large volume of water, extract with ether, dry the extracts over anhydrous sodium sulfate,
evaporate to dryness under reduced pressure and distil to give 4-fluoro-2,l,3-benzoxadiazole
as a colorless oil (bp 83712 mm Hg) (J.Chem.Soc.(C) 1970, 1433). Add 11 mL chlorosulfonic
acid dropwise to 3 g 4-fluoro-2,l,3-benzoxadiazole in 10 mL chloroform at 0-10 (use a calcium
chloride drying tube), stir at room temperature for 1 h, reflux for 2 h, cool, slowly pour into
ice water, remove the organic layer, extract the aqueous layer with chloroform, combine the
organic layer, wash, dry over anhydrous magnesium sulfate, evaporate under reduced pressure,
take up the residue in 5 mL benzene (Caution! Benzene is a carcinogen!), chromatograph on a
150 X 30 column of silica gel (100-200 mesh Kanto Chemical) with n-hexane:benzene 50:50,
evaporate the appropriate fractions to give 4-(chlorosulfonyl)-7-fluoro-2,l,3-benzoxadiazole
(CBD-F) as pale yellow needles (mp 64-66) (Anal. Chem. 1984. 56, 2461). Stir 0.76 g CBD-F
in 70 mL MeCN at 0-10 and add 1 g dimethylamine hydrochloride in 10 mL 100 mM pH 10
borax dropwise, adjust pH to 5 with 1 M HCl, concentrate to about 10 mL under reduced
pressure, extract three times with 200 mL portions of diethyl ether, wash with water, dry over
anhydrous magnesium sulfate, evaporate under reduced pressure, chromatograph on a 500 X
20 column of silica gel with chloroform, isolate the appropriate fraction and re-chromatograph
on the same column with ethyl acetate:benzene 1:2 to give 4-(N,N-dimethylaminosulfonyl)-7-
fluoro-2,l,3-benzoxadiazole (DBD-F) as white needles (mp 124-125) (yield = 1% !). On a Merck
no. 5714 60F254 tic plate eluted with chloroform DBD-F has Rf 0.32 and lies between two other
reaction products (Analyst 1989, 114, 413). It is also reported that DBD-F can be purchased
from Tokyo Kasei. Add 100 mg DBD-F in 10 mL MeCN to 47 mg proline in 20 mL 250 mM pH
11.5 sodium carbonate solution, stir at room temperature for 30 min, wash with ethyl acetate,
adjust the pH of the aqueous layer to 1-2 with 2 M HCl, extract three times with 30 mL ethyl
acetate. Combine the extracts and evaporate them under reduced pressure, recrystallize from
benzene/ethyl acetate to give 4-(N,N-dimethylaminosulfonyl)-7-(2- carboxypyrrolidin-1-yl)-
2,1,3-benzoxadiazole (DBD-Pro) as yellow needles (mp 187-9 d) (Analyst 1989, 114, 1233).
Suspend 55 mg (S)-(-)-DBD-Pro in 55 mL anhydrous diethyl ether at 0, add 110 mg phosphorus
pentachloride, stir at 5 for 1 h, filter quickly, evaporate to dryness under reduced pressure,
dry under vacuum over phosphorus pentoxide for 12 h to give 4-(N,N-dimethylaminosulfonyl)-
7-(2-chloroformylpyrrolidin-l-yl)-2,l,3-benzoxadiazole (DBD-Pro-Cl) as yellow crystals (mp 116-
17) (Analyst 1993, 118, 759). DBD-Pro-Cl is also available from Tokyo Kasei (TCI America,
Portland OR). Add 130 mg DBD-Pro-Cl dissolved in 25 mL anhydrous benzene dropwise to 100
mL MeOH containing 70 mg hydrazine hydrate, stir for 30 min at room temperature, evaporate
under reduced pressure, recrystallize from ethyl acetate:MeOH 90:10 to give 4-(2-carbazolyl-
pyrrolidin-l-yl)-7-(N,N-dimethylaminosulfonyl)-2,l,3-benzoxadiazole (DBD-ProCZ) as orange
crystals (mp 107-109) (Anal.Proc. 1994, 31, 265).)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Inertsil ODS-80A
Flow rate: 1
CHROMATOGRAM
Retention time: 118
Internal standard: pentadecanoic acid (105)
OTHER SUBSTANCES
Extracted: arachidonic acid, linolenic acid, myristic acid, palmitoleic acid
KEYWORDS
rat; human; derivatization; serum; plasma
REFERENCE
Toyo'oka,T.; Takahashi,M.; Suzuki,A.; Ishii,Y. Determination of free fatty acids in blood, tagged with 4-(2-
carbazoylpyrrolidin-l-yl)-7-(N,N-dimethylaminosulfonyl)-2,l,3-benzoxadiazole, by high-pressure liquid chro-
matography with fluorescence detection, Biomed.Chromatogr., 1995, 9, 162-170.
SAMPLE
Matrix: blood
Sample preparation: Dilute serum 20 times with water. 100 |xL Diluted serum + 100 (xL 1 M
l-(3-dimethylaminopropyl)-3-ethylcarbodiimide in MeOH + 100 |xL pyridine.MeOH 20:80 + 100
|xL 15 mM reagent in DMF, heat at 37 for 20 min, inject a 20 |xL aliquot. (Prepare reagent as
follows. Stir 20 g 4-bromoveratrole in 60 mL acetic acid and add 10 mL concentrated nitric
acid drop wise, keep at 10-30 with occasional cooling, pour in to ice-water, filter. Take up the
precipitate in 500 mL hot EtOH and add activated charcoal, filter, add 40 mL water to the
nitrate, collect 4-bromo-5-nitroveratrole as light yellow crystals (mp 121-2). Melt 5 g sodium
sulfide nonahydrate and 0.7 g sulfur together, add this mixture to 5 g 4-bromo-5-nitroveratrole
in 50 mL 95% EtOH, reflux for 30 min, pour into ice-water, filter, recrystallize the product
from dichloromethane to give the nitro disulfide as yellow needles (mp 231-2). Add 8 g tin
powder to 2 g of the nitro disulfide in 300 mL EtOH, add 30 mL concentrated HCl dropwise,
basify with 4 M NaOH, filter, dilute the filtrate with 200 mL water. Extract the diluted filtrate
twice with 100 mL portions of benzene (Caution! Benzene is a carcinogen!). Combine the ex-
tracts and evaporate them under reduced pressure, take up the residue in 10 mL benzene and
add 2 mL 10% hydrogen peroxide, stir for 30 min, recrystallize the precipitate from EtOH to
give 2,2'-dithiobis(l-ammo-4,5-dimethoxybenzene) (mp 155-6) (Anal.Chim.Acta 1994, 291,
189). Add a mixture of 3.7 g 2,2'-dithiobis(l-amino-4,5-dimethoxybenzene) and 1.2 g tri-n-bu-
tylphosphine in 40 mL 800 mM disodium hydrogen phosphite in MeOH to 1.2 g 4-carboxybenz-
aldehyde in 20 mL MeOH, stir at 37 for 2 h, filter the precipitate. Wash the precipitate with
MeOH:water 70:30 and dry it under reduced pressure. Dissolve in 50 mL MeOH and treat
with ethereal diazomethane (e.g., prepared as Anal. Chem. 1960, 32, 1412), evaporate to dry-
ness under reduced pressure, chromatograph the residue on a 250 X 35 column of 130 g 70-
230 mesh silica gel 60 (Merck) with n-hexane:ethyl acetaterchloroform 50:25:25, collect the
main fraction and evaporate to obtain the product, 5,6-dimethoxy-2-(4-carbomethoxy-
phenyl)benzothiazole, as pale yellow needles (mp 223-224). Add 100 mL aqueous 45% hydra-
zine hydrate (Caution! Hydrazine hydrate is a carcinogen and may explode on distillation!) to
500 mg of the product in 50 mL EtOH, reflux for 1 h, collect the precipitate and recrystallize
it from EtOH to give the reagent, 5,6-dimethoxy-2-(4-hydrazmocarbonylphenyl)benzothiazole,
as colorless needles (mp 252-254).)
HPLC VARIABLES
Column: 250 X 4.6 5 [Jim TSK gel ODS 120T (Tosoh)
Mobile phase: Gradient. MeCN:water 40:60 for 15 min, to 70:30 over 20 min, maintain at 70:
Flow rate: 1
CHROMATOGRAM
Retention time: 69
OTHER SUBSTANCES
Extracted: arachidic acid, capric acid, caprylic acid, docosahexaenoic acid, lauric acid, linolenic
acid, margaric acid, myristic acid, myristoleic acid, oleic acid, palmitic acid, palmitoleic acid,
stearic acid
KEYWORDS
REFERENCE
Yamaguchi,M.; Hara,S.; Obata,K. 5,6-Dimethoxy-2-(4'-hydrazinocarbonylphenyl)benzothiazole as a highly sen-
sitive and stable fluorescence derivatization reagent for carboxylic acids in high performance liquid chro-
matography, J.Liq.Chromatogr., 1995, 18, 2991-3006.
SAMPLE
Matrix: blood
Sample preparation: 50 JJLL Serum + 200 |xL ethyl acetate + 100 |xL dilute HCl (pH 3.0), vortex
for 5 min. Remove the supernatant and add it to 50 |xL 4 mM 4-aminomethyl-6,7-dimethoxy-
coumarin in DMF containing 25 mM 1-hydroxybenzotriazole, add 40 |xL 500 mM pH 7.0 phos-
phate buffer, add 100 ^L 2 M l-(3-dimethylaminopropyl)-3-ethylcarbodiimide in water, add 10
(JLL 1 mM nonadecanoic acid in MeOH, vortex at 25 for 5 min, inject a 10 |JLL aliquot of the
organic layer. (Preparation of 4-aminomethyl-6,7-dimethoxycoumarin is as follows. Heat 1 g 4-
bromomethyl-6,7-dimethoxycoumarin and 1.3 g potassium phthalimide in 40 mL DMF at 80
for 1 h, cool, add 100 mL chloroform, wash with 50 mL 100 mM NaOH, wash with 100 mL
water. Dry the organic layer over anhydrous sodium sulfate, concentrate to a small volume,
add ether, collect 4-phthalimidylmethyl-6,7-dimethoxycoumarin as a crystalline solid (mp
240.5-242). Reflux 950 mg 4-phthalimidylmethyl-6,7-dimethoxycoumarin and 520 mg hydra-
zine hydrate in 40 mL THFrMeOH 50:50 for 1.5 h (Caution! Hydrazine hydrate is a carcinogen
and explodes on distillation in air!), filter, evaporate the filtrate to dryness, add 20 mL dilute
HCl, evaporate to dryness, recrystallize from EtOHrether 50:50 to give 4-aminomethyl-6,7-
dimethoxyeoumarin hydroehloride as slightly yellow crystals (mp 240-242.5). Dissolve 370 mg
4-aminomethyl-6,7-dimethoxycoumarin hydroehloride in 20 mL 5% NaOH, extract 5 times with
20 mL portions of ethyl acetate, evaporate to dryness under reduced pressure, chromatograph
on silica with chloroform:MeOH 98:2 to give 4-aminomethyl-6,7-dimethoxycoumarin as a
slightly yellow powder (mp 137-147).)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Wakosil-II 5C18
Mobile phase: Gradient. MeCN:water from 40:60 to 100:0 over 35 min, maintain at 100:0.
Flow rate: 1
CHROMATOGRAM
Retention time: 27
Internal standard: nonadecanoic acid (42)
OTHER SUBSTANCES
Extracted: arachidonic acid, lauric acid, linolenic acid, margaric acid, myristic acid, myristoleic
acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid
KEYWORDS
REFERENCE
Sasamoto,K.; Ushijima,T.; Saito,M.; Ohkura,Y. Precolumn fluorescence derivatization of carboxylic acids using
4-aminomethyl-6,7-dimethoxycoumarinin a two-phase medium, Anal.ScL, 1996, 12, 189-193.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 10 ixmoles linoleic acid, 20 jjimoles a-bromo-2'-acetonaphthone,
and 40 ixmoles N,N-diisopropylethylamine in 1 mL DMF, heat at 60 for 10 min, inject an
aliquot.
HPLC VARIABLES
Column: 914 X 1.8 Corasil C18
Flow rate: 0.2
Detector: UV 254
CHROMATOGRAM
Retention time: 55
OTHER SUBSTANCES
Simultaneous: arachidonic acid, dihomolinolenic acid, linolenic acid, oleic acid
KEYWORDS
derivatization
REFERENCE
Cooper,M.J.; Anders,M.W. Determination of long chain fatty acids as 2-naphthacyl esters by high pressure
liquid chromatography and mass spectrometry, A/iaZ. Chem., 1974, 46, 1849-1852.
SAMPLE
Matrix: bulk
Sample preparation: Add 3-5 equivalents of anhydrous potassium carbonate to the acid, add
0.5-1.5 mL MeCN containing an excess of a,p-dibromoacetophenone:dicyclohexyl-18-crown-6
10:1, reflux with vigorous stirring for 45 min, evaporate to dryness, take up in chloroform,
filter, dilute the filtrate with chloroform, inject an aliquot.
HPLC VARIABLES
Column: 250 X 5 5 |xm HI-EFF Micropart C18 (Applied Science Laboratories)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
Simultaneous: elaidic acid, linoleaidic acid, linolenic acid, stearic acid
Also analyzed: oleic acid, palmitelaidic acid, palmitoleic acid, petroselinic acid
KEY WORDS
derivatization
REFERENCE
Pei,P.T.-S.; Kossa,W.C; Ramachandran,S.; Henly,R.S. High pressure reverse phase liquid chromatography of
fatty acid p-bromophenacyl esters, Lipids, 1976, 11, 814-816.
SAMPLE
Matrix: bulk
Sample preparation: Mix 10 u-moles fatty acid, 40 ixmoles N,N-diisopropylethylamine or lith-
ium carbonate, and 10-20 ixmoles 2-bromo-4'-chloroacetophenone (p-chlorophenacyl bromide)
in 1 mL DMF, heat at 65 for 15 min, inject an aliquot.
HPLC VARIABLES
Column: two 300 X 3.9 ixBondapak C18 columns in series
Mobile phase: Gradient. MeCN:water from 40:60 to 100:0 (Waters convex curve 5) over 3 h.
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 95
OTHER SUBSTANCES
Simultaneous: acetic acid, arachidic acid, arachidonic acid, behenic acid, butyric acid, capric
acid, caproic acid, caprylic acid, elaidic acid, lactic acid, lauric acid, lignoceric acid, linolenic
acid, myristic acid, nervonic acid, oleic acid, palmitic acid, palmitoleic acid, propionic acid,
stearic acid, vaccenic acid
KEYWORDS
derivatization
REFERENCE
Jordi,H.C. Separation of long and short chain fatty acids as naphthacyl and substituted phenacyl esters by
high performance liquid chromatography, J.Liq.Chromatogr., 1978,1, 215-230.
SAMPLE
Matrix: bulk
Sample preparation: 10-50 mg Linoleic acid + 4 mL hydrazine hydrate:THF:EtOH:cyclopentene
1:3:3:1 (Caution! Hydrazine is carcinogenic and may explode when distilled!), heat at 50 under
nitrogen in a tightly closed tube for 2 h, add 5 mL acetone, heat at 50 for 30 min, cool. Remove
a 100 jxL aliquot and evaporate it to dryness under a stream of nitrogen at 50, reconstitute
the residue in 1 mL MeOH, evaporate to dryness under a stream of nitrogen, repeat, recon-
stitute in 100 |xL MeOH, inject a 1-5 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Hypersil RP-18 ODS
Mobile phase: MeOH:water 85:15 (Wash with 30 mL MeOH after each use.)
Flow rate: 1
Detector: UV 229
CHROMATOGRAM
Retention time: 17
OTHER SUBSTANCES
Simultaneous: elaidic acid, linolenic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid
KEYWORDS
derivatization
REFERENCE
Agrawal,V.R; Schulte,E. High-performance liquid chromatography of fatty acid isopropylidene hydrazides and
its application in lipid analysis, Anal.Biochem., 1983, 131, 356-359.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve up to 1 mg fatty acid in 1 mL dichloromethane, add 1 mL of a
solution containing 100 u,moles tetrabutylammonium hydrogen sulfate and 200 jxmoles NaOH,
add 20 |JLL pentafluorobenzyl bromide, shake vigorously at room temperature for 30 min. Re-
move the dichloromethane layer and evaporate it to dryness, reconstitute with hexane, add to
a 20 X 10 silica Sep-Pak SPE cartridge, elute with hexane idichloromethane 85:15, inject an
aliquot.
HPLC VARIABLES
Column: 300 X 7.8 ixPorasil
Mobile phase: Hexane.dichloromethane 85:15, half saturated with water
Flow rate: 4
Detector: UV 254
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Simultaneous: linolenic acid, oleic acid, stearic acid
KEYWORDS
derivatization; SPE; normal phase; semi-preparative
REFERENCE
Netting,A.G.; Duffield,A.M. Pentafluorobenzyl esters as derivatives for the semi-preparative high-performance
liquid chromatography of fatty acids, J.Chromatogr., 1983, 257, 174-179.
SAMPLE
Matrix: bulk
Sample preparation: 100 |xg Fatty acid mixture + 25 jiL 10 mg/mL a-bromoacetophenone in
acetone + 25 (xL 10 mg/mL triethylamine in acetone, heat in a boiling water bath for 15 min,
add 35 |xL 2 mg/mL acetic acid in acetone, heat for 5 min, evaporate to dryness under a stream
of nitrogen at 40, reconstitute with 100 |xL MeCN, inject a 5-10 |xL aliquot.
HPLCVARIABLES
Guard column: octadecyl
Column: 250 X 4.5 5 |xm octadecyl-bonded spherical silica (IBM)
Mobile phase: Gradient. MeCN:water 80:20 for 25 min, to 85:15 over 15 min
Flow rate: 2
CHROMATOGRAM
Retention time: 21
OTHER SUBSTANCES
Simultaneous: arachidonic acid, all-cis-delta981114-eicosatrienoic acid, cis-deltan-eicosenoic acid,
elaidic acid, heptadecanoic acid, trans-delta -hexadecenoic acid, lauric acid, linolenic acid, my-
ristic acid, myristoleic acid, oleic acid, palmitic acid, pentadecanoic acid, cis-deltalo-pentadece-
noic acid, stearic acid, tridecanoic acid
KEYWORDS
derivatization
REFERENCE
Wood,R.; Lee,T. High-performance liquid chromatography of fatty acids: Quantitative analysis of saturated,
monoenoic, polyenoic and geometrical isomers, J.Chromatogr., 1983, 254, 237-246.
SAMPLE
Matrix: bulk
Sample preparation: React 4 mg fatty acid, 3 mg dicyclohexylcarbodiimide, and 120 |xL 40 mg/
mL dansyl ethanolamine in chloroform in the dark at room temperature overnight, add water,
filter, inject an aliquot of the organic layer of the filtrate. (Prepare dansyl ethanolamine by
adding dansyl chloride to a large excess of stirred ethanolamine. Collect the precipitate of
dansyl ethanolamine and recrystallize it from MeOH.)
HPLC VARIABLES
Column: 250 X 4.65 |xm Ultrasphere ODS
Mobile phase: MeCN:MeOH:20 mM silver nitrate in water 45:45:10
Flow rate: 2
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Simultaneous: eicosadienoic acid, elaidic acid, linolenic acid, margaric acid, oleic acid, palmitic
acid, palmitoleic acid
KEYWORDS
derivatization
REFERENCE
Ryan,P.J.; Honeyman,T.W. Determination of fatty acids by high-performance liquid chromatography of Dns-
ethanolamine derivatives, J.Chromatogr., 1984, 312, 461-466.
SAMPLE
Matrix: bulk
Sample preparation: Mix 5-10 mg fatty acid with 1 mL 5% triethylamine in acetone and 1.5
mL acetone containing 1 equivalent of p-phenylazophenacyl bromide, shake for 30 min, purify
by TLC on silica gel G with benzene (Caution! Benzene is a carcinogen!), scrape off the appro-
priate band and elute it with benzene, evaporate the eluate to dryness under a stream of
nitrogen, reconstitute with mobile phase, inject an aliquot. (Prepare p-phenylazophenacyl bro-
mide as follows. React equimolar quantities of p-aminoacetophenone and nitrosobenzene in
glacial acetic acid at room temperature, recrystallize the crude product from EtOH to give p-
phenylazoacetophenone as orange crystals (mp 114.5-116) (Anal. Chem. 1954, 26,1228). React
3.5 g bromine in 25 mL chloroform with 5 g p-phenylazoacetophenone in 50 mL chloroform at
room temperature to obtain p-phenylazophenacyl bromide as orange red prisms after recrys-
tallization from petroleum ether (mp 103) (J. Chem. Soc. Japan, Pure Chem. Sect. 1951, 72,
152; Chem. Abs. 1952, 46, 3447i).)
HPLC VARIABLES
Guard column: 0DS-5S (Bio-Rad)
Column: 250 X 4 5 [Lin Bio-Sil ODS (Bio-Rad)
Flow rate: 1.5
Detector: UV 330
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Simultaneous: linolenic acid, oleic acid
KEY WORDS
derivatization
REFERENCE
Vioque,E.; Maza,M.R; Millan,F. High-performance liquid chromatography of fatty acids as their p-phenylazo-
phenacyl esters, J.Chromatogr., 1985, 331, 187-192.
SAMPLE
Matrix: butter, oil, margarine, solutions
Sample preparation: Butter, oil, margarine. Dissolve 20-40 mg in 5 mL chloroform. Remove a
100 |xL aliquot and add it to 1.5 mL 25 mM tetraethylammonium carbonate in MeOH, heat in
a capped tube at 70 for 5 min, remove the cap, heat at 70 for 30 min, evaporate to dryness
under a stream of nitrogen, reconstitute with 1 mL DMF. Remove a 100 |JLL aliquot and add it
to 200 JJLL 5 mM 9-bromomethylacridine in DMF, let stand at room temperature for at least 10
min, inject an aliquot. Solutions. Mix 100 (xL of a solution in DMF with 100 (xL 5 mM 9-
bromomethylacridine in DMF in 100 |xL 2.5 mM tetraethylammonium carbonate in DMF, let
stand at room temperature for 10 min, inject a 10 JJLL aliquot. (Prepare tetraethylammonium
carbonate by adding dry ice to an aqueous solution of tetraethylammonium hydroxide, evapo-
rate to dryness under reduced pressure at 56 over phosphorus pentoxide to give tetraethylam-
monium carbonate as a white hygroscopic powder (mp 294-288 d). Prepare 9-bromomethyl-
acridine as follows. Reflux 560 mg 9-methylacridine, 445 mg N-bromosuccinimide, and 10 mg
benzoyl peroxide in 30 mL carbon tetrachloride for more than 2 h, cool, chromatograph on a
column of silica gel with benzene.ethyl acetate 30:1 to obtain 9-bromomethylacridine as yellow
crystals (mp 147-151).)
HPLC VARIABLES
Column: 150 X 4.6 TSK-gel ODS 120A (Toyo Soda)
Mobile phase: Gradient. MeOHiwater from 90:10 to 97:3 over 32 min (JASCO concave curve 2),
Flow rate: 0.8
Detector: F ex 365 em 425, UV 252
CHROMATOGRAM
Retention time: 36
OTHER SUBSTANCES
Extracted: arachidonic acid, capric acid, caprylic acid, docosahexaenoic acid, eicosapentaenoic
acid, eicosatrienoic acid, lauric acid, linolenic acid, margaric acid, myristic acid, oleic acid,
palmitic acid, palmitoleic acid, stearic acid
KEYWORDS
derivatization
REFERENCE
Akasaka,K; Suzuki,T.; Ohrui,H.; Meguro,H.; Shindo,Y.; Takahashi,H. 9-Bromomethylacridine a novel fluores-
cent labeling reagent of carboxylic group for HPLC, Anal.Lett, 1987, 20, 1581-1594.
SAMPLE
Sample preparation: 50 JJLL Enzyme incubation + 200 |xL n-heptane:isopropanol:l M sulfuric
acid 20:80:2, add 120 JJLL heptane, add 70 jxL water, add 5 nmoles margaric acid, vortex for 20
s. Remove a 30-50 |xL aliquot of the heptane layer and evaporate it to dryness under reduced
pressure, add 50 |xL 500 |xg/mL 9-anthryldiazomethane in MeOH:ethyl acetate 90:10 (prepare
immediately before use), let stand at room temperature for 15 min, inject a 3-10 jxL aliquot.
(9-Anthryldiazomethane is available from Funakoshi Co., Tokyo or it may be synthesized as
follows. Stir 8.8 g 9-anthraldehyde and 8.5 g 80% hydrazine hydrate in 150 mL EtOH at room
temperature for 3 h, filter off the solid 9-anthraldehyde hydrazone and dry under vacuum (mp
124-6) (Bull. Chem. Soc. Jpn. 1967, 40, 691). Dissolve 220 mg 9-anthraldehyde hydrazone in
100 mL anhydrous ether, add 800 mg activated manganese dioxide, follow the reaction by
reverse-phase HPLC using MeCN at 0.4 mL/min and UV 254. At the end of the reaction filter
off the manganese and wash it with 20 mL ether, evaporate the filtrate to obtain 9-anthryl-
diazomethane (mp 64-6) (Anal.Biochem. 1980, 107, 116 and 1983, 132 456). Prepare activated
manganese dioxide as follows. Stir a solution of 20 g potassium permanganate in 250 mL water
at room temperature, add 10 g activated carbon (Nuchar C-190 or C-190N), stir for 16 h, filter
(Buchner funnel), wash 4 times with 50 mL portions of water, dry in air, dry in an oven at
105-110 for 8-24 h (J.Org.Chem. 1970, 35, 3971).)
HPLC VARIABLES
Column: 50 X 4 Superspher RP-18 (Merck)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Extracted: arachidonic acid, linolenic acid, oleic acid, palmitic acid, stearic acid
KEYWORDS
derivatization
REFERENCE
Tojo,H.; Ono,T.; Okamoto,M. Reverse-phase high-performance liquid chromatographic assay of phospholipases:
application of spectrophotometric detection to rat phospholipase A2 isozymes, J.Lipid Res., 1993, 34, 837
844.
SAMPLE
Matrix: fat, oil
Sample preparation: Prepare a 0.5-1 mg/mL solution of fat or oil in chloroform containing
0.005% BHT. Remove a 100 |JLL aliquot and add it to 40 nmole margaric acid, evaporate to
dryness under a stream of nitrogen at room temperature, add 100 |xL 2.5 M KOH:EtOH 20:
80, heat at 90 for 10 min, cool to room temperature, add 400 JJLL reagent solution, add 200 jxL
20 mM 2-nitrophenylhydrazine hydrochloride in 250 mM HCl, heat at 60 for 20 min, add 100
JJLL 15% KOH in MeOH:water 80:20, heat at 60 for 15 min, cool, inject a 1-5 |JLL aliquot.
(Prepare reagent by mixing equal volumes of 3% pyridine in EtOH and 250 mM l-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride in EtOH.)
HPLC VARIABLES
Guard column: 30 X 4.6 ODS
Column: 250 X 4.6 5 |xm YMC-C8 (Yamamura Chemical Institute, Kyoto)
Mobile phase: MeCN:water 85:15 adjusted to pH 4.5 with 100 mM HCl in MeCN
Flow rate: 1.2
Detector: UV 230
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Extracted: arachidonic acid, capric acid, docsahexenoic acid, eicosapentaenoic acid, eicosatrienoic
acid, lauric acid, linolenic acid, myristic acid, myristoleic acid, oleic acid, palmitic acid, pal-
mitoleic acid, stearic acid
KEYWORDS
derivatization
REFERENCE
Miwa,H.; Yamamoto,M. Improved method of determination of biologically important Ci010-C22^ fatty acids as
their 2-nitrophenylhydrazides by reversed-phase high-performance liquid chromatography, J.Chromatogr.,
1986, 351, 275-282.
SAMPLE
Matrix: fat, oil
Sample preparation: Dissolve 1 mg fat or oil in 200 |xL 2 mM margaric acid in EtOH, add 100
IJLL 400 mM KOH:EtOH 50:50, heat at 80 for 20 min, add 200 JJLL 20 mM 2-nitrophenylhydra-
zine hydrochloride in 300 mM HCIiEtOH 50:50, add 200 |xL 250 mM l-ethyl-3-(3-dimethylam-
inopropyl)carbodiimide hydrochloride in EtOH:pyridine 97:3, heat at 80 for 5 min, add 200 |xL
10% KOH in MeOH:water 50:50, heat at 80 for 5 min, cool, inject a 5-10 jxL aliquot.
HPLC VARIABLES
Guard column: J'sphere ODS-M 80 (Yamamura Chemical Laboratories, Kyoto)
Column: 250 X 4.6 4 |xm J'sphere ODS-M 80 (Yamamura Chemical Laboratories, Kyoto)
Mobile phase: MeCN:water 86:14, adjusted to pH 4-5 with 100 mM HCl
Flow rate: 2
Detector: UV 400
CHROMATOGRAM
Retention time: 5.8
Internal standard: margaric acid (9.3
OTHER SUBSTANCES
Extracted: arachidic acid, arachidonic acid, capric acid, caprylic acid, dihomo-gamma-linolenic
acid, docosadienoic acid, docosahexaenoic acid, docosatetraenoic acid, docosatrienoic acid, ei-
cosadienoic acid, eicosapentaenoic acid, eicosatrienoic acid, eicosenoic (omega-12) acid, eicosen-
oic (omega-9) acid, eicosenoic (omega-15) acid, elaidic acid, erucic acid, lauric acid, linoelaidic
acid, a-linolenic acid, gamma-linolenic acid, myristic acid, myristoleic acid, octadecatetraenoic
KEY WpRDS
derivatization
REFERENCE
Miwa,H-; Yamamoto,M. Rapid liquid chromatographic determination of fatty acids as 2-nitrophenylhydrazine
derivatives, JAOAC Int., 1996, 79, 493-497.
SAMPLE
Matrix: food
Sample preparation: Saponify margarine with KOH/EtOH/water, slowly acidify with concen-
trated HCl, extract with diethyl ether. Allow the ether to evaporate in a hood overnight. Dis-
solve 300 mg of the residue in 1 mL MeCN, add 3-4 drops THF, mix, make up to 5 mL with
MeCN (add additional THF if the solution is cloudy), inject a 25 JULL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:THF:water 67:3:30 containing 0.1% acetic acid (Place an 88.5 X 4.6 37-75
jxm Porasil A column between pump and injector.)
Flow rate: 1.3
Detector: RI
CHROMATOGRAM
Retention time: 21
OTHER SUBSTANCES
Extracted: elaidic acid, linoledaic acid, linolenic acid, myristic acid, oleic acid, palmitic acid,
stearic acid
KEYWpRDS
margarine; protect from light
REFERENCE
BailieA.G.,Jr.; Wilson,T.D.; O'Brien,R.K; Beebe,J.M.; Stuart,J.D.; McCosh-Lilie,E.; Hill,D.W. HPLC analysis
of underivatized fatty acids in margarines, J.Chromatogr.Sci., 1982, 20, 466-470.
SAMPLE
Matrix: food
Sample preparation: Dissolve 20-40 mg oil, butter, or margarine in 5 mL chloroform. Remove
a 100 |JLL aliquot and add it to 1.5 mL 25 mM tetraethylammonium carbonate in MeOH, heat
in a capped tube at 70 for 5 min, heat at 70 for 30 min in an open tube, evaporate to dryness
under a stream of nitrogen, reconstitute with 1 mL DMF. Remove a 100 |xL aliquot and add it
to 200 |JLL 5 mM 9-bromomethylacridine in DMF, let stand at room temperature for at least 10
min, inject a 10 (xL aliquot. (Synthesize 9-bromomethylacridine as follows. Heat 10 g diphen-
ylamine, 10 mL glacial acetic acid, and 40 g anhydrous zinc chloride to 220, evaporate excess
acetic acid with stirring, heat at 220-230 for 6 h, digest with hot 10% sulfuric acid, make
strongly alkaline with 25% ammonia to dissolve the zinc chloride. Extract the insoluble residue
with toluene. Extract the organic layer with 10% sulfuric acid, make the aqueous layer alkaline
with aqueous ammonia. Collect the yellow precipitate that separates and recrystallize it twice
from petroleum ether to give 9-methyl acridine as pale yellow needles (Chromatographia 1989,
28, 267). Reflux 560 mg 9-methylacridine, 445 mg N-bromosuccinimide, and 10 mg benzoyl
peroxide in 30 mL carbon tetrachloride for more than 2 h, cool, chromatograph on silica gel
with benzene:ethyl acetate 30:1 (Caution! Benzene is a carcinogen!) to obtain 9-bromomethyl-
acridine as yellow crystals (mp 147-151). Prepare tetraethylammonium carbonate by adding
dry ice to an aqueous solution of tetraethylammonium hydroxide, evaporate to dryness under
reduced pressure over phosphorus pentoxide at 56 to obtain tetraethylammonium carbonate
as a white hygroscopic powder (decomposes 284-288).)
HPLC VARIABLES
Column: 150 X 4.6 TSK-gel ODS 120A (Toyo Soda)
Mobile phase: Gradient. MeOH:water from 90:10 to 97:3 over 32 min (JASCO concave curve 2),
Flow rate: 0.8
CHROMATOGRAM
Retention time: 36
OTHER SUBSTANCES
Extracted: arachidonic acid, capric acid, caprylic acid, docosahexaenoic acid, eicosapentaenoic
acid, eicosatrienoic acid, heptadecanoic acid, lauric acid, linoleic acid, linolenic acid, myristic
KEY WpRDS
derivatization; oil; butter; margarine
REFERENCE
Akasaka,K; Suzuki,T.; Ohrui,H-l Meguro,H.; Shindo,Y.; Takahashi,H. 9-Bromomethylacridine a novel fluores-
cent labeling reagent of carboxylic group for HPLC, Anal.Lett, 1987, 20, 1581-1594.
SAMPLE
Matrix: food
Sample preparation: Orange juice. 10 mL Orange juice + 70 mL water + 8 g NaCl, mix until
homogeneous, add 50 mL dichloromethane, shake for 1 min, centrifuge at 400 g for 5 min,
repeat extraction twice more. Combine the organic layers and evaporate them to dryness under
reduced pressure at 40, dissolve the residue in five 3 mL portions of MeCN. Combine the
MeCN extracts and evaporate them to 1 mL under a stream of nitrogen at 40, filter (0.45 |xm),
inject an aliquot. Butter, margarine. Warm 1 g butter of margarine at 40 until it melts, add
10 mL MeCN, vortex for 5 min, filter (0.45 |xm), inject an aliquot.
HPLC VARIABLES
Mobile phase: Gradient. MeCN:buffer from 79:21 to 87:13 over 10 min, to 99:1 over 5 min,
maintain at 99:1 for 5 min, return to initial conditions over 2 min, re-equilibrate for 6 min.
(Buffer was water adjusted to pH 4.0 with 5% orthophosphoric acid.)
Flow rate: 0.8
Detector: UV 651 following post-column extraction. The column effluent mixed with 2 |xg/mL
methylene blue in 20 mM Na2HPO4 pumped at 2 mL/min and the mixture flowed through a
45 cm X 0.5 mm ID stainless steel coil. The effluent from this coil mixed with chloroform
pumped at 1 mL/min and the mixture flowed through a 50 cm X 1 mm ID stainless steel coil
to a CTFE polymer phase separator fitted with a polyethylene filter disc from a SPE cartridge
(construction details in paper). The organic phase flowed downwards to the detector at 0.5 mL/
min and the aqueous phase flowed to waste. Every 2-3 injections rinse detector cell with 2-3
mL MeCN.
CHROMATOGRAM
Retention time: 9.7
OTHER SUBSTANCES
Extracted: capric acid, caprylic acid, trans-elaidic acid, lauric acid, linoledaidic acid, linolenic
acid, myristic acid, cis-oleic acid, palmitic acid, palmitoleic acid, stearic acid
KEYWORDS
post-column extraction; butter; margarine; orange juice
REFERENCE
Lawrence,J.F.; Charbonneau,C.F. Direct, sensitive and selective detection of free fatty acids by high-perfor-
mance liquid chromatography with post-column ion-pair extraction and absorbance detection,
J.Chromatogr., 1988, 445, 189-197.
SAMPLE
Matrix: milk, milk products
Sample preparation: Free fatty acids. Measure out 100 |xL milk, 20 mg butter, 20 mg cheese,
50 mg condensed milk, 50 mg ice cream, or 50 mg yogurt, add 100 jxL water, add 200 |xL 100
IxM 2-ethylbutyric acid in EtOH containing 100 |xM margaric acid, add 200 JJLL 20 mM 2-
nitrophenylhydrazine hydrochloride in EtOH:100 mM HCl 50:50, add 200 |xL 250 mM 1-ethyl-
3-(3-dimethylammopropyl)carbodiimide hydrochloride in EtOH:pyridine 97:3, heat at 80 for 5
min, add 200 ^L 10% KOH in MeOH:water 50:50, heat at 80 for 5 min, cool, add 4 mL 33
mM pH 6.4 phosphate buffer:500 mM HCl 7:1, extract with 5 mL n-hexane. Remove the organic
layer and evaporate it to dryness under a stream of nitrogen at room temperature, reconstitute
the residue in 200 jiL MeOH, filter (0.45 |xm), inject a 10-20 |xL aliquot of the filtrate. Total
fatty acids. Measure out 10 |xL milk, 1 mg butter, 1 mg cheese, 2 mg condensed milk, 2 mg ice
cream, or 10 mg yogurt, add 200 |xL 2 mM 2-ethylbutyric acid in EtOH containing 1 mM
margaric acid, add 100 |xL EtOH:400 mM KOH 50:50, heat at 80 for 20 min, add 200 jxL 20
mM 2-nitrophenylhydrazine hydrochloride in EtOH:300 mM HCl 50:50, add 200 |xL 250 mM
l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in EtOH:pyridine 97:3, heat at
80 for 5 min, add 200 |xL 10% KOH in MeOH:water 50:50, heat at 80 for 5 min, cool, add 4
mL 33 mM pH 6.4 phosphate buffer:500 mM HCl 7:1, extract with 5 mL n-hexane. Remove
the organic layer and evaporate it to dryness under a stream of nitrogen at room temperature,
reconstitute the residue in 200 (xL MeOH, filter (0.45 |xm), inject a 2-10 |JLL aliquot of the
filtrate.
HPLC VARIABLES
Guard column: 10 X 4 5 |xm BBC-4-C8 (Yamamura Chemical Labs)
Column: 250 X 6 5 |xm YMC-FA C8 (Yamamura Chemical Labs)
Mobile phase: MeCN:MeOH:water 75:11:14, adjusted to pH 4.5 with 100 mM HCl
Flow rate: 1.2
Detector: UV 400
CHROMATOGRAM
Internal standard: 2-ethylbutyric acid (17.5 (A)), margaric acid (19.5 (B))
Limit of detection: 0.5-2 pmole
OTHER SUBSTANCES
Extracted: arachidic acid, arachidonic acid, capric acid, caprylic acid, docosadienoic acid, doco-
sahexaenoic acid, docosatetraenoic acid, docosatrienoic acid, eicoatrienoic acid, eicosadienoic
acid, eicosaenoic acid, eicosapentaenoic acid, elaidic acid, erucic acid, lauric acid, linoelaidic
acid, linolenic acid, myristic acid, myristoleic acid, oleic acid, palmitic acid, palmitoleic acid,
stearic acid
KEYWORDS
derivatization; milk; butter; cheese; condensed milk; ice cream; yogurt
REFERENCE
Miwa,H.; Yamamoto,M. Liquid chromatographic determination of free and total fatty acids in milk and milk
products as their 2-nitrophenylhydrazides, J.Chromatogr., 1990, 523, 235-246.
SAMPLE
Matrix: oil
Sample preparation: Mix oil with mobile phase, centrifuge, inject a 20 JJLL aliquot of the
supernatant.
HPLC VARIABLES
Mobile phase: MeCNiEtOH 90:10
Flow rate: 1.1
Detector: E, Jasco Model EC-840, glassy carbon working electrode -415 mV, saturated calomel
reference electrode, stainless steel auxiliary electrode following post-column reaction. The col-
umn effluent mixed with 76 mM lithium perchlorate in MeCN:EtOH 90:10 containing 6 mM
2-methyl-l,4-naphthoquinone (menadione, vitamin K3) pumped at 1.1 mL/min and the mixture
flowed through a 50 cm X 0.5 mm ID coil to the detector.
CHROMATOGRAM
Retention time: 5.5
OTHER SUBSTANCES
Extracted: oleic acid, palmitic acid, stearic acid
KEYWORDS
post-column reaction; camellia oil; corn oil; rapeseed oil; olive oil; soy bean oil
REFERENCE
Fuse,T.; Kusu,R; Takamura,K. Determination of higher fatty acids in oils by high-performance liquid chro-
matography with electrochemical detection, J.Chromatogr.A, 1997, 764, 177-182.
SAMPLE
Matrix: solutions
Sample preparation: Mix 100 (xg fatty acid, 10 |xL 12 mg/mL phenacyl bromide in acetone, and
10 |xL 10 mg/mL triethylamine in acetone, let stand at room temperature overnight, inject an
aliquot.
HPLC VARIABLES
Mobile phase: Gradient. MeCNiwater 67:33 for 125 min, 74:26 for 50 min, 80:20 for 40 min, 97:
3 for 35 min (step gradients).
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 120
OTHER SUBSTANCES
Simultaneous: arachidic acid, arachidonic acid, behenic acid, eicosadienoic acid, eicosaenoic acid,
eicosatrienoic acid, elaidic acid, erucic acid, heneicosanoic acid, heptadecanoic acid, lauric acid,
lignoceric acid, linolelaidic acid, linolenic acid, myristic acid, myristoleic acid, nervonic acid,
nonadecanoic acid, oleic acid, palmitic acid, palmitoleic acid, pentadecanoic acid, petroselinic
acid, stearic acid, vaccenic acid
KEYWORDS
derivatization
REFERENCE
Borch,RR Separation of long chain fatty acids as phenacyl esters by high pressure liquid chromatography,
Anal.Chem., 1975, 47, 2437-2439.
SAMPLE
Matrix: solutions
Sample preparation: Neutralize a solution of the acid in MeOH or water with KOH in MeOH
(phenolphthalein endpoint). Evaporate to dryness under reduced pressure, dissolve in 0.5-1.5
mL MeCN containing a,p-dibromoacetophenone:18-crown-6 10:1, stir at 80 for 15 min, cool,
inject an aliquot.
HPLCVARIABLES
Column: 250 X 4 Corasil II C9
Mobile phase: MeOH:water 62.5:37.5, after 132 min 75:25
Flow rate: 0.3
Detector: UV 254
CHROMATOGRAM
Retention time: 100
OTHER SUBSTANCES
Simultaneous: linolenic acid, oleic acid, stearic acid
KEYWORDS
derivatization
REFERENCE
Durst,H.D.; Milano,M.; Kikta,E.J.,Jr.; Connelly,S.A.; Grushka,E. Phenacyl esters of fatty acids via crown ether
catalysts for enhanced ultraviolet detection in liquid chromatography, AraaZ.CJiem., 1975, 47, 1797-1801.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve sample in 2 mL carbon tetrachloride, add 1.4 g triphenylphos-
phine, polymer supported, heat at 80 with gentle shaking for 5 min, cool to room temperature,
add 8 mL 62.5 mg/mL p-methoxyaniline in ethyl acetate, heat at 80 for 10 min, inject a 2 JJLL
aliquot.
HPLC VARIABLES
Mobile phase: Gradient. MeCNrwater from 0:100 to 100:0 over 40 min (Waters curve 2).
Flow rate: 1
Injection volume: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: arachidonic acid, decanoic acid, docosanoic acid, 4,7,10,13,16,19-docoshexaenoic
acid, dodecanoic acid, eicosanoic acid, erucic acid, heptadecanoic acid, hexadecanoic acid, hex-
anoic acid, linolenic acid, nervonic acid, octanoic acid, oleic acid, palmitoleic acid, pentadecanoic
acid, stearic acid, tetracosanoic acid, tetradecanoic acid
KEY WpRDS
derivatization
REFERENCE
Hoffman,N.E.; Liao,J.C. High pressure liquid chromatography of p-methoxyanilides of fatty acids, Anal.Chem.,
1976, 48, 1104-1106.
SAMPLE
Matrix: solutions
Sample preparation: Shake 200-400 |xL of a solution of fatty acids in benzene with an equal
volume of 2% oxalyl chloride in benzene (Caution! Benzene is a carcinogen!), heat at 70 for 30
min, evaporate to dryness under a stream of nitrogen, add 100 |xL 7.76 mM 9-aminophenan-
threne in benzene, add 100 (xL 0.1% triethylamine in benzene, heat at 70 for 45 min. (Purify
9-aminophenanthrene by dissolving 50 mg in 30 mL EtOH, filter, add hydrochloric acid satu-
rated ether to the filtrate until precipitate no longer appears. Filter off the precipitate and
wash it with ether, dry in a desiccator under vacuum, dissolve in hot water, basify with am-
monia, filter to obtain pure 9-aminophenanthrene as white crystals (mp 134).)
HPLCVARIABLES
Column: 300 X 4 8-10 |xm ixBondapak C18
Flow rate: 2
CHROMATOGRAM
Internal standard: margaric (26)
OTHER SUBSTANCES
Simultaneous: arachidonic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic
acid
KEYWORDS
derivatization; protect from light; paper contains some details for analysis of fatty acids in serum
REFERENCE
Ikeda,M.; Shimada,K.; Sakaguchi,T.; Matsumoto,U. Fluorometric high-performance liquid chromatography of
9-aminophenanthrene-derivatizedfree fatty acids, J.Chromatogr., 1984, 305, 261-270.
SAMPLE
Matrix: solutions
Sample preparation: Mix 100 |xL of a solution in EtOH, EtOH/water, or water with 400 JJLL
reagent solution and 200 |xL 20 mM 2-nitrophenylhydrazine hydrochloride in water, heat at
60 for 20 min, add 100 |xL 15% KOH in MeOH:water 80:20, heat at 60 for 15 min, cool, inject
a 1-2 |xL aliquot. (Prepare reagent by mixing equal volumes of 3% pyridine in EtOH and 250
mM l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in EtOH.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm YMC-C8 (Yamamuta Chemical Research, Kyoto)
Mobile phase: MeOH.water 86:14 (B) adjusted to pH 4.5 with 100 mM HCl
Flow rate: 1.2
CHROMATOGRAM
Retention time: 8.5
Limit of detection: 2.5-5 pmole (UV 230), 10-15 pmole (UV 400)
OTHER SUBSTANCES
Simultaneous: capric acid, lauric acid, myristic acid, linolenic acid, palmitoleic acid, palmitic
acid, oleic acid, stearic acid
KEYWORDS
derivatization
REFERENCE
Miwa,H.; Hiyama,C; Yamamoto,M. High-performance liquid chromatography of short- and long-chain fatty
acids as 2-nitrophenylhydrazides, J. Chromatogr., 1985, 321, 165-174.
SAMPLE
Matrix: solutions
Sample preparation: Add 500 ^xL of a solution in MeCN to 100 mg finely powdered potassium
carbonate, add 250 |xL 3.8 mM 18-crown-6 in MeCN, add 250 |xL 0.8 mM reagent in MeCN,
heat at 80 in the dark for 20 min, cool, inject a 5 |xL aliquot. (Synthesize the reagent, 3-
bromomethyl-6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone, as follows. Stir 483 g veratrole in
1.45 L acetic acid at 15 for 1 h, add 683 g concentrated nitric acid (d 1.05) over 1 h (maintain
the temperature below 40 by cooling and regulating the rate of addition of the nitric acid).
Continue stirring and add 2.127 L fuming nitric acid (d 1.50) over 1 h while maintaining the
temperature below 30, let stand for 2 h, pour into a large volume of cold water, filter, wash
the solid with water until the washings are neutral, recrystallize from EtOH to give 4,5-dini-
troveratrole (mp 129.5-130.5) (J. Am. Chem. Soc. 1946, 68, 1536). Reflux 5 g 4,5-dinitrovera-
trole in 200 mL benzene (Caution! Benzene is a carcinogen!), add 100 g 60 mesh iron powder
and 20 mL concentrated HCl in small portions over 1 h, reflux for 4 h, add 10 mL water, reflux
for 2 h, cool, make alkaline with 2.5 M NaOH, extract several times with 200 mL portions of
benzene. Combine the organic layers and evaporate them to dryness, add 10 mL concentrated
HCl, recrystallize from EtOH to give l,2-diamino-4,5-dimethoxybenzene monohydrochloride as
very slightly pink needles (mp 240) (Anal. Chim. Acta 1982, 134, 39). Heat 2.5 mmoles 1,2-
diamino-4,5-dimethoxybenzene hydrochloride and 2.4 mmoles pyruvic acid in 30 mL 500 mM
HCl on a boiling water bath for 2 h, cool with ice-water, filter. Wash the precipitate with water
and dry it under vacuum, recrystallize from MeOH:water 90:10 to give 6,7-dimethoxy-3-methyl-
2(lH)-quinoxalinone as yellow needles (mp 255) (Chem. Pharm. Bull. 1985, 33, 3493). Treat 1
g 6,7-dimethoxy-3-methyl-2(lH)-quinoxalinone dissolved in 50 mL anhydrous MeOH with a
solution of diazomethane in ether, evaporate to dryness under reduced pressure, dissolve the
residue in 5 mL ethyl acetate, chromatograph on a 250 X 35 column filled with 130 g 70-230
mesh silica gel 60 (Merck) using n-hexane:ethyl acetate 25:75 to give 6,7-dimethoxy-l,3-di-
methyl-2(lH)-quinoxalinone as yellow needles (mp 170-171). Dissolve 350 mg 6,7-dimethoxy-
l,3-dimethyl-2(lH)-quinoxalinone in 3 mL acetic acid, add 350 mg anhydrous sodium acetate,
add 2 mL 1.5 M bromine in acetic acid, heat at 100 for 15 min, cool, add 10 mL ether, filter,
wash the solid 2 or 3 times with small portions of ether. Combine the filtrate and washings
and evaporate them to dryness, dissolve the residue in 5 mL ethyl acetate, chromatograph on
a 250 X 35 column filled with 130 g 70-230 mesh silica gel 60 (Merck) using ether, evaporate
the main fraction to dryness, recrystallize the residue from n-hexane:ethyl acetate 50:50 to
give 3-bromomethyl-6,7-dimethoxy-l-methyl-2(lH)-quinoxalinone as yellow needles (mp 161-
163).)
HPLCVARIABLES
Mobile phase: Gradient. MeOH:water from 57:43 to 100:0 over 20 min, maintain at 100:0 for 12
min
Flow rate: 2
Injection volume: 5
CHROMATOGRAM
Limit of detection: 0.3-1 fmole
OTHER SUBSTANCES
Simultaneous: p-aminobenzoic acid, arachidic acid, benzoic acid, butyric acid, capric acid, ca-
proic acid, caprylic acid, deoxyuridine, glucuronic acid, imidazole-4-acetic acid, lauric acid, mar-
garic acid, l-methyl-4-imidazoleacetic acid, myristic acid, myristoleic acid, oleic acid, palmitic
acid, propionic acid, salicylic acid, stearic acid, thymidine, uridine, valeric acid
Interfering: arachidonic acid, linolenic acid, palmitoleic acid
KEYWORDS
derivatization
REFERENCE
Yamaguchi,M.; Hara,S.; Matsunaga,R.; Nakamura,M.; Ohkura,Y. 3-Bromomethyl-6,7-dimethoxy-l-methyl-
2(lH)-quinoxalinone as a new fluorescence derivatization reagent for carboxylic acids in high-performance
SAMPLE
Matrix: solutions
Sample preparation: Mix 100 |xL of a 0.01-10 (xg/mL solution in MeOH with 100 JJLL 1 mg/mL
1-pyrenyldiazomethane in ethyl acetate, let stand at room temperature for 1.5 h, inject a 5 |xL
aliquot. (Synthesis of 1-pyrenyldiazomethane is as follows. Suspend 5 g 1-pyrenecarboxalde-
hyde in 80 mL EtOH, add 3.4 g hydrazine monohydrate (Caution! Hydrazine monohydrate is
a carcinogen!), stir at room temperature for 3 h, filter off the product and wash it with 50 mL
cold EtOH, recrystallize from EtOH to obtain 1-pyrenecarboxaldehyde hydrazone as yellow
crystals (mp 186-194 d). Add 6.55 g activated manganese dioxide to 2 g 1-pyrenecarboxalde-
hyde hydrazone in 300 mL diethyl ether, sonicate at room temperature for about 80 min (mon-
itor by HPLC), filter, wash the solid with a little ether, evaporate the filtrate to obtain 1-
pyrenyldiazomethane as red crystals. Prepare activated manganese dioxide as follows. Stir a
solution of 20 g potassium permanganate in 250 mL water at room temperature, add 10 g
activated carbon (Nuchar C-190 or C-190N), stir for 16 h, filter (Buchner funnel), wash 4 times
with 50 mL portions of water, dry in air, dry in an oven at 105-110 for 8-24 h (J.Org.Chem.
1970, 35, 3971). 1-Pyrenyldiazomethane is also available from Molecular Probes, Eugene OR.)
HPLC VARIABLES
Column: 150 X 4 5 jxm TSK-GEL-120A ODS (TOSOH)
Mobile phase: Gradient. MeCN:water 85:15 for 30 min, to 100:0 over 30 min.
Flow rate: 1
Injection volume: 5
CHROMATOGRAM
Retention time: 20
OTHER SUBSTANCES
Simultaneous: arachidic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic
acid, stearic acid
KEYWORDS
derivatization
REFERENCE
Nimura,N.; Kinoshita,T.; Yoshida,T.; Uetake,A.; Nakai,C. 1-Pyrenyldiazomethane as a fluorescent labeling re-
agent for liquid chromatographic determination of carboxylic acids, Anal. Chem., 1988, 60, 2067-2070.
SAMPLE
Matrix: solutions
Sample preparation: Mix 100 jxL of a 0.01-10 u,g/mL solution with 100 |xL of a 9-anthryldiazo-
methane solution, vortex, let stand at room temperature for 1 h, inject a 5 |xL aliquot. (Prepare
9-anthryldiazomethane solution as follows. Stir 8.8 g 9-anthraldehyde and 8.5 g 80% hydrazine
hydrate in 150 mL EtOH at room temperature for 3 h, filter off the solid 9-anthraldehyde
hydrazone and dry under vacuum (mp 124-6) (Bull. Chem. Soc. Jpn. 1967, 40, 691). Add 500
IxL 69 mM quinuclidine in ethyl acetate and 500 JJLL 6.9 mM N-chlorosuccinimide in ethyl
acetate to 500 JJLL 6.9 mM 9-anthraldehyde hydrazone in ethyl acetate, vortex, let stand for 30
min, use immediately.)
HPLC VARIABLES
Column: 150 X 4 5 |xm TSK-GEL-120T (Tosoh)
Mobile phase: Gradient. MeCN:water 84:0 for 30 min, to 100:0 over 25 min, maintain at 100:0
for 15 min
Flow rate: 1
Injection volume: 5
Detector: F ex 255 or 365 em 412
CHROMATOGRAM
Retention time: 26
Limit of quantitation: 125 fmole
OTHER SUBSTANCES
Simultaneous: arachidic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic
acid, stearic acid
KEYWORDS
derivatization
REFERENCE
Yoshida,T.; Uetake,A.; Yamaguchi,H.; Nimura,N.; Kinoshita,T. New preparation method for 9-anthryldiazo-
methane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives, Anal.Biochem., 1988,
173, 70-74.
SAMPLE
Matrix: solutions
Sample preparation: Mix 100 |xL of a 0.3 |xM solution in MeCN with 50 |xL 100 |xM tetraethyl-
ammonium carbonate in MeCN, add 50 |xL freshly-prepared 200 |xM reagent in MeCN, vortex
for 10 s, let stand at room temperature for 10 min, inject a 1 jxL aliquot. (Prepare tetraethyl-
ammonium carbonate by adding dry ice to an aqueous solution of tetraethylammonium hy-
droxide, evaporate to dryness under reduced pressure at 56 over phosphorus pentoxide to give
tetraethylammonium carbonate as a white hygroscopic powder (mp 294-288 d) (Anal. Lett.
1987, 20, 1581). The reagent was 2,3-(anthracenedicarboximido)ethyl trifluoromethanesulfo-
nate, prepared as follows. Add 11.7 g benzoyl chloride dropwise over 30 min to 10 g 1,2,4-
trimethylbenzene and 11.7 g aluminum trichloride in 10 mL dichloromethane stirred at 0, stir
at room temperature for 6 h, pour into a mixture of 25 mL concentrated HCl and 50 g ice,
remove the organic layer, extract the aqueous layer with dichloromethane. Combine the organic
layers and wash them with 5% sodium bicarbonate, dry over anhydrous magnesium sulfate,
evaporate to dryness under reduced pressure, distil through a Vigreux column to give 2,4,5-
trimethylbenzophenone (bp 13070.15 mm Hg). Reflux 12.5 g 2,4,5-trimethylbenzophenone in
75 mL 20% nitric acid for 5 days, cool, decant the aqueous layer, wash the solid with 75 mL
water. Dissolve the solid in 125 mL 10% NaOH, reflux this solution while stirring it mechan-
ically, add 35 g potassium permanganate in portions (Caution! Frothing may occur!) over 40
min, reflux for 3 h, allow to cool somewhat, filter. Add the solid that is collected to water, reflux
for 6 h, filter while hot. Combine the filtrates and evaporate them to half volume under reduced
pressure, cool, acidify slowly with concentrated HCl, filter, dry the solid in air to give benzo-
phenone-2,4,5-tricarboxylic acid as white crystals (mp 281-283). Stir 2.1 g benzophenone-2,4,5-
tricarboxylic acid in 21 g concentrated sulfuric acid at 120 for 3 h, pour onto 30 g of ice, filter,
wash the solid with water, dry in air to give anthraquinone-2,3-dicarboxylic acid as a pale
yellow solid (mp 342). Add 1 g anthraquinone-2,3-dicarboxylic acid to 50 mL 20% ammonium
hydroxide then add 3.75 g activated zinc dust, reflux, as soon as the blood-red color disappears
filter while hot. Add the solid that is collected it to 50 mL 20% ammonium hydroxide, reflux
for 2 h, filter while hot. Combine the filtrates and cool them to 0, acidify to pH 1 with 6 M
HCl, let stand at room temperature for 1 day, filter to give 2,3-anthracenedicarboxylic acid as
a bright yellow solid (mp 345) (J. Org. Chem. 1991, 56, 6243). Reflux 2,3-anthracenedicarbox-
ylic acid in acetic anhydride for 2 h to give 2,3-anthracenedicarboxylic anhydride. Vigorously
reflux 200 mg 2,3-anthracenedicarboxylic anhydride, 200 mg 2-aminoethanol, 60 mL dry tol-
uene, and 30 mL butanol under a Dean-Stark trap for 1.5 h, evaporate the solvent under
reduced pressure until crystallization starts, dissolve these crystals by warming, cool to obtain
crystals, recrystallize from toluene/butanol to give N-(hydroxyethyl)-2,3- anthracenedicarbox-
imide as orange crystals (mp 292-294). Suspend 200 mg N-(hydroxyethyl)-2,3-anthracenedi-
carboximide in 50 mL dichloromethane and 1 mL pyridine, add this mixture to a solution of
400 mg trifluoromethanesulfonic anhydride in 30 mL dichloromethane at such a rate as to keep
the temperature below -5, stir for 3 h below -5, add 200 mL cold water. Remove the organic
layer and dry it over anhydrous magnesium sulfate, evaporate to dryness under reduced pres-
sure, recrystallize from dichloromethane at -20 to give 2,3-(anthracenedicarboximido)ethyl tri-
fluoromethanesulfonate as pale yellow crystals (mp >300).)
HPLC VARIABLES
Column: 100 X 4.6 3 jim Develosil ODS-K3
Mobile phase: MeCN:MeOH:water 22.5:67.5:10
Flow rate: 0.8
Injection volume: 1
CHROMATOGRAM
Retention time: 14
Limit of detection: 1.4-3.8 pinole
OTHER SUBSTANCES
Simultaneous: arachidonic acid, capric acid, caprylic acid, cis-4,7,10,13,16,19-docosahexaenoic
acid, cis-ll,14-eicosadienoic acid, cis-5,8,ll,14,17-eicosapentaenoic acid, cis-8,ll,14-eicosatri-
enoic acid, gondoic acid, heptadecanoic acid, lauric acid, linolenic acid, myristic acid, myristoleic
KEY WpRDS
derivatization
REFERENCE
Akasaka,K.; Ohrui,H.; Meguro,H. Determination of carboxylic acids by high-performance liquid chromatogra-
phy with 2-(2,3-anthracenedicarboximido)ethyl trifluoromethanesulfonate as highly sensitive fluorescent
labelling reagent, Analyst, 1993, 118, 765-768.
SAMPLE
Matrix: solutions
Sample preparation: Mix 25 |xL of an aqueous solution of fatty acids with 100 JJLL DMF:pyridine
93:7, add 50 JJLL 5 mM MPIB-hydrazide in DMF, add 100 |xL 4 M (sic) l-(3-dimethylaminopro-
pyl)-3-ethylcarbodiimide, heat at 40 for 20 min, inject a 10 (xL aliquot. (Synthesis of MPIB-
hydrazide, 4-(l-methylpnenanthro[9,10-d]imidazol-2-yl)benzohydrazide, is as follows. Stir 1 g
9,10-diaminophenanthrene and 800 mg methyl 4-formylbenzoate (terephthaldehydic acid
methyl ester) in 200 mL EtOH at room temperature for 1 h, add 5 mL MeOH saturated with
HCl, reflux under an inert gas for 2 h, cool, concentrate to 50 mL under reduced pressure,
chromatograph the precipitate on a 200 X 35 column of 70-230 mesh silica gel (ca. 100 g;
Merck) with chloroform, recrystallize from MeOH to give methyl 4-(phenanthro[9,10-
d]imidazol- 2-yl)benzoate as colorless needles (mp 312-315). Dissolve 500 mg methyl 4-(phen-
anthro[9,10-d]imidazol-2-yl)benzoate in 100 mL anhydrous MeOH, treat with a solution of di-
azomethane in ether, evaporate to dryness under reduced pressure, dissolve the residue in 20
mL chloroform, chromatograph on a 200 X 60 column of about 250 g 100 mesh silica gel with
chloroform to give methyl 4-(l-methylphenanthro[9,10-d]imidazol-2-yl)benzoate as colorless
needles (mp 199-201). Dissolve 2 g methyl 4-(l-methylphenanthro[9,10-d]imidazol-2-
yl)benzoate in 100 mL aqueous hydrazine hydrate (45%) (Caution! Hydrazine hydrate is a
carcinogen and explodes on distillation in air!), heat at 100 for 1 h, recrystallize the precipitate
from 95% EtOH to give MPIB-hydrazide (4-(l-methylphenanthro[9,10-d]imidazol-2-yl)-
benzohydrazide) (mp 291-293).)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm TSKgel ODS-80Ts (Tosoh)
Flow rate: 1
Detector: F ex 360 em 460, F ex 325 (10 mW He-Cd laser) em 460
CHROMATOGRAM
Retention time: 8.0
Limit of detection: 2.2-12.5 fmole (F), 0.4-2.3 fmole (laser F)
OTHER SUBSTANCES
Simultaneous: arachidic acid, arachidonic acid, margaric acid, oleic acid, palmitic acid, stearic
acid
KEY WpRDS
derivatization
REFERENCE
Iwata,T.; Hirose,T.; Nakamura,M.; Yamaguchi,M. 4-(l-Methylphenanthro[9,10-d]imidazol-2-yl)benzohydrazide
as derivatization reagent for carboxylic acids in high-performance liquid chromatography with conventional
and laser-inducedfluorescencedetection, Analyst, 1994, 119, 1747-1751.
Next Page
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 0.17-1.7 mg/mL solution in MeOH, inject a 30 |xL aliquot.
HPLC VARIABLES
Mobile phase: Gradient. A was MeOH containing 0.05% acetic acid. B was water containing
0.05% acetic acid. A:B 85:15 to 100:0 over 40 min, maintain at 100:0.
Flow rate: 1
Detector: evaporative light scattering (ELSD, MK VIII, Varex), drift tube 75, nitrogen flow 1 L/
min, nitrogen pressure 22 psi or UV 205
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: ricinoleic acid, fatty acids
REFERENCE
Lin,J.-T.; McKeon,T.A.; Stafford,AE. Gradient reversed-phase high-performance liquid chromatography of sat-
urated, unsaturated and oxygenated free fatty acids and their methyl esters, J.Chromatogr.A, 1995, 699,
85-91.
SAMPLE
Matrix: tissue
Sample preparation: 50 mg Fat + 1 mL 25% KOH in 96% EtOH, heat in a boiling water bath
for 1 h, cool, adjust pH to 2 with 3 M HCl, extract three times with 2 mL portions of n-hexane:
diethyl ether 50:50. Combine the organic layers and evaporate them to dryness under a stream
of nitrogen, reconstitute the residue with 25 (xL 10 mg/mL a-bromoacetophenone in acetone
and 25 |xL 10 mg/mL triethylamine in acetone, heat in a boiling water bath for 5 min, add 40
IL 2 mg/mL acetic acid in acetone, heat for 5 min, evaporate to dryness under a stream of
nitrogen at 40, reconstitute with 100 |xL MeOH, inject a 10 |xL aliquot.
HPLC VARIABLES
Guard column: 50 X 4 7 |xm Separon SGX C18 (Tessek, Prague)
Column: 250 X 4 5 |xm Separon SGX C18 (Tessek, Prague)
Mobile phase: Gradient. MeCN:MeOH:water from 40.5:40:19.5 to 0:81.5:18.5 over 25 min, to 0:
90:10 over 45 min, to 0:100:0 over 20 min.
Flow rate: 1
Detector: UV 242
CHROMATOGRAM
Retention time: 31
Limit of detection: 0.8-1.2 ng
OTHER SUBSTANCES
Extracted: arachidonic acid, capric acid, caproic acid, caprylic acid, docosahexaenoic acid, eico-
sadienoic acid, eicosatrienoic acid, eicosenoic acid, elaidic acid, erucic acid, heptadecanoic acid,
lauric acid, linoelaidic acid, linoleic acid, linolenic acid, myristic acid, myristoleic acid, oleic
acid, palmitic acid, palmitoleic acid, pentadecanoic acid, stearic acid
KEY WORDS
derivatization; rat; fat
REFERENCE
Hanis,T.; Smrz,M.; Klir,R; Macek,K.; Klima,J.; Base,J.; Deyl,Z. Determination of fatty acids as phenacyl esters
in rat adipose tissue and blood vessel walls by high-performance liquid chromatography, J.Chromatogr.,
1988, 452, 443-457.
Previous Page
Linuron
Merck Index: 5534
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 209.9
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve in 1 mL 2 M NaOH, heat at 75 for 45 min, cool, add 2 drops
methyl isobutyl ketone, add 2 drops 0.1% dansyl chloride in acetone, shake well, heat at 65
for 30 min, cool, acidify with 10% HCl, add 300 |xL benzene (Caution! Benzene is a carcinogen!),
extract, inject a 1-10 |xL aliquot.
HPLC VARIABLES
Column: 1000 X 2.4 Zipax coated with 0.5% p,p'-oxydipropionitrile
Mobile phase: Hexane:MeOH 95:5
Flow rate: 0.78
Detector: F ex Turner filter no. 811 em Turner filter no. 817
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Simultaneous: chloropropham, propham
KEYWORDS
REFERENCE
Frei,R.W.; Lawrence,J.F. Fluorigenic labelling in high-speed liquid chromatography, J.Chromatogr., 1973, 83,
321-330.
Liothyronine
Molecular formula: C15H12I3NO4
CAS Registry No.: 6893-02-3, 66-06-1 (Na salt)
Merck Index: 5535
Lednicer No.: 1 97
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 1 mg compound in 1 mL EtOH and 10 |xL 1 M KOH in EtOH:
water 50:50 (freshly prepared). Remove a 10 |xL aliquot and evaporate it to dryness under
<0.05 Torr at 45 for 30 min, add 50 jxL 80 mM 4-dimethylaminopyridine in dry MeCN, add 5
(LJLL EtOH, vortex thoroughly, add 50 (xL trimethylacetic anhydride, vortex for 10 s, heat at 65-
70 for 50 min, add 100 |xL EtOH, heat at 65-70 for 10 min, evaporate to dryness, add 100 |xL
toluene, add 100 jxL 100 mM pH 6 phosphate buffer, vortex, centrifuge. Remove the organic
layer and evaporate it to dryness, add 100 uX MeCN, sonicate for 2 min, add 100 (xL pH 2.1
phosphate buffer, mix, inject an aliquot. (Pass MeCN through an aluminum oxide column be-
fore use.)
HPLCVARIABLES
Column: 150 X 4.6 Supelcosil LC-8
Mobile phase: Gradient. MeCN: 10 mM KH2PO4 adjusted to pH 2.1 with phosphoric acid from
30:70 to 87:13 over 10 min.
Flow rate: 2
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: 3,5-diiodothyronine, thyroxine
KEYWORDS
derivatization
REFERENCE
Joppich,M.; Joppich-Kuhn,R.; Sentissi,A-; Giese,R.W. Single-step, quantitative derivatization of amino, car-
boxyl, and hydroxyl groups in iodothyronine amino acids with ethanolic pivalic anhydride containing 4-
dimethylaminopyridine, Anal.Biochem., 1986, 153, 159-165.
SAMPLE
Sample preparation: Weigh out powder equivalent to about 65 mg thyroid, add 5 mL enzyme
solution, mix well, incubate at 37 for 28 h, agitate after 4-8 h and after 20-24 h, add 2 mL
deactivating solution, mix well, centrifuge at 2000 rpm for 5-10 min, if necessary filter (0.45
ixm). (The enzyme solution was about 150 protease units/mL of bacterial protease from Strep-
tomyces griseus in 110 mM NaCL + 40 mM Tris buffer + 50 mM methimazole (pH adjusted to
8.4 0.05 with 6 M HCl) reducing buffer. Deactivating solution was 1:100 phosphoric acid:
MeCN.)
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 225
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Simultaneous: levothyroxine, L-3,3',5'-triiodothyronine
KEYWORDS
tablets
REFERENCE
Richheimer,S.L.; Jensen,C.B. Determination of liothyronine and levothyroxine in thyroid preparations by liquid
SAMPLE
Sample preparation: Grind a tablet, add 50 |xg 3,3',5'-triiodothyronine, add 20 mL solvent A,
stir for 10 min, add 40 mL solvent B, stir for 30 min, filter. Remove the upper layer and wash
it six times with 15 mL portions of water saturated with butanol, evaporate under vacuum at
40-42, reconstitute in 2.5 mL 3% ammonium hydroxide in MeOH, inject an aliquot (Anal.Lett.
1979, 12, 1201). (Prepare the solvents by mixing 1.8 L 1-butanol, 1.35 L water and 450 mL
concentrated HCl, shake vigorously for 20 min, allow to separate. The lower layer was solvent
A and the upper layer was solvent B.)
HPLC VARIABLES
Guard column: 25 X 2.5 CorPel ODS
Column: 300 X 3.9 10 ^m ^Bondapak C18
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Internal standard: S^'^'-triiodothyronine
OTHER SUBSTANCES
KEYWORDS
protect from light; tablets
REFERENCE
Rapaka,R.S.; Knight,P.W.; Prasad,V.K. Reversed-phase high-performance liquid chromatographic analysis of
liothyronine sodium and levothyroxine sodium in tablet formulations: preliminary studies on dissolution
and content uniformity, J.Pharm.ScL, 1981, 70, 131-134.
SAMPLE
Sample preparation: Powder tablets, weigh out amount equivalent to about 200 |xg sodium
levothyroxine, add 10 mL mobile phase, sonicate for 5 min, centrifuge. Filter (0.45 |xm, 25 mm
Acrodisc CR, Gelman) the supernatant, inject a 200 |xL aliquot.
HPLC VARIABLES
Guard column: 40 X 4 40 |jim RP 201SC pellicular (Vydac)
Column: 300 X 4 (xBondapak C18
Mobile phase: MeCN:buffer 60:40 (Buffer was pH 3.0 containing 5 mM 1-octanesulfonic acid and
5 mM tetramethylammonium chloride.)
Flow rate: 2
Detector: UV 230
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Simultaneous: levothyroxine, 3,5-diiodo-L-thyronine
KEYWORDS
REFERENCE
Richheimer,S.L.; Amer,T.M. Stability-indicating assay, dissolution, and content uniformity of sodium levothy-
roxine in tablets, J.Pharm.ScL, 1983, 72, 1349-1351.
SAMPLE
Matrix: solution
Sample preparation: Inject a 10 |xL aliquot of a solution in MeOH:water 50:50.
HPLC VARIABLES
Mobile phase: Gradient. A was MeCN:water 5:95 containing 0.05% acetic acid. B was MeCN:
water 80:20 containing 0.05% acetic acid. A:B from 100:0 to 70:30 over 12 min, to 45:55 over
Flow rate: 1
Detector: UV 231; MS5 HP Model 5987, thermospray, tip temperature 250, negative ion mode
or filament mode, source 320, m/z 200-900
CHROMATOGRAM
Retention time: 25
OTHER SUBSTANCES
Simultaneous: degradation products, levothyroxine
REFERENCE
Andre,M.; Domanig,R.; Riemer,E.; Moser,H.; Groeppelin,A. Identification of the thermal degradation products
of G-triiodothyronine sodium (liothyronine sodium) by reversed-phase high-performance liquid chromatog-
raphy with photodiode-array UV and mass spectrometric detection, J.Chromatogr.A, 1996, 725, 287-294.
SAMPLE
Matrix: solution
Sample preparation: Inject a 10 |xL aliquot of a solution in MeOH:water 50:50
HPLC VARIABLES
Mobile phase: Gradient. A was 50 mM pH 3.0 triethylammonium phosphate. B was MeCN. A:
B from 95:5 to 30:70 over 70 min.
Flow rate: 1
Detector: UV 231
CHROMATOGRAM
Retention time: 23
OTHER SUBSTANCES
REFERENCE
Andre,M.; Domanig,R-; Riemer,E.; Moser,H.; Groeppelin,A- Identification of the thermal degradation products
of G-triiodothyronine sodium (liothyronine sodium) by reversed-phase high-performance liquid chromatog-
raphy with photodiode-array UV and mass spectrometric detection, J.Chromatogr.A, 1996, 725, 287294.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Guard column: 45 X 4.6 10 |xm, 300 A Nucleosil C8 (Bio-Rad)
Column: 250 X 4.6 5 |xm, 300 A Nucleosil C8 (Bio-Rad)
Mobile phase: Gradient. A was 0.1% pH 2.0 trifluoroacetic acid. B was MeCNrO. 1% trifluoroacetic
acid 90:10. A:B maintain at 80:20 (initial equilibration), from 80:20 to 20:80 over 40 min, from
20:80 to 0:100 over 10 min, from 0:100 to 80:20 over 10 min (re-equilibration)
Flow rate: 0.5
Detector: UV 280
CHROMATOGRAM
Retention time: 37
OTHER SUBSTANCES
REFERENCE
de Ia Vieja,A.; Calero,M.; Santisteban,R; Lamas,L. Identification and quantitation of iodotyrosines and iodo-
thyronines in proteins using high-performance liquid chromatography by photodiode-array ultraviolet-vis-
ible detection, J.Chromatogr.B, 1997, 688, 143-149.
SAMPLE
Matrix: solutions
Sample preparation: Take up 1.5 mg liothyronine in 200 |xL 100 mM sodium bicarbonate and
400 |xL reagent, stir in an ice bath for 30 min, evaporate to dryness below 30, add 100 |xL
trifluoroacetic acid to the dry residue, let stand for 30 min at room temperature, add 2 mL 1
M sodium bicarbonate, centrifuge. Remove the precipitate and dissolve it in 600 JJLL MeOH:20
mM NaOH 50:50, inject a 30 jxL aliquot. Reagent was 7 mg/mL BOC-L-Leu-SU (tert-butyloxy-
L-leucine-N-hydroxysuccinimide ester) in MeOH, prepared immediately before use.)
HPLC VARIABLES
Column: 250 X 3.2 7 |jim LiChrosorb RP-18
Mobile phase: MeOH:buffer 60:40 (Buffer was 85 mM pH 6.4 phosphate-citrate buffer obtained
by mixing 200 mM pH 8.0 phosphate buffer with 100 mM pH 2.2 citrate buffer.)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Simultaneous: dextrothyroxine, levothyroxine, D-isomer, impurities
KEYWORDS
chiral
REFERENCE
Lankmayr,E.R; Budna,K.W.; Nachtmann,F. Separation of enantiomeric iodinated thyronines by liquid chro-
matography of diastereomers, J.Chromatogr., 1980, 198, 471-479.
SAMPLE
Matrix: solutions
Sample preparation: 100 [iL Solution + 100 |JLL 500 mM pH 7.7 borate buffer + 100 JJLL 2.5 mM
9-fluorenylmethyl chloroformate in dry acetone, mix, let stand at room temperature for 45 s,
add 200 (xL 12 mM 1-adamantamine in MeCN, inject an aliquot.
HPLCVARIABLES
Guard column: 10 X 3 30 |xm Chromspher C18 (Chrompack)
Column: 100 X 3 5 |xm Chromspher C18 (Chrompack)
Mobile phase: Gradient. A was MeOH:50 mM pH 4.2 sodium acetate buffer 40:60. B was MeCN:
MeOH:50 mM pH 4.2 sodium acetate buffer 20:60:20. A:B from 100:0 to 0:100 over 40 min.
Flow rate: 0.7
Detector: UV 260
CHROMATOGRAM
Retention time: 30
OTHER SUBSTANCES
Simultaneous: 3,5-diiodothyronine, 3,5-diiodotyrosine, levothyroxine, 3-monoiodotyrosine, thy-
ronine, 3,3',5-triiodothyronine
KEYWORDS
REFERENCE
553, 135-142.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 u,m Phenomenex cyano-bonded silica
Mobile phase: MeCN:water:phosphoric acid 400:600:1
Flow rate: 1.5
Detector: UV 225
CHROMATOGRAM
Retention time: 6.6
OTHER SUBSTANCES
REFERENCE
9, 131-137.
SAMPLE
Matrix: tissue
Sample preparation: 100 (JLL Thyroid tissue + 200 jxL MeCN, mix, centrifuge. Remove a 100
u-L aliquot of the supernatant and add it to 100 jxL 4 nM dabsyl chloride in MeCN, heat at 70
for 10 min, add 400 pX, MeOH:50 mM pH 7.0 phosphate buffer 50:50, inject a 20 jxL aliquot.
HPLC VARIABLES
at 0:100 for 2 min.
Flow rate: 1
Detector: UV 436
CHROMATOGRAM
Retention time: 17
OTHER SUBSTANCES
Extracted: diiodothyronine (T2), levothyroxine (T4)
KEY WpRDS
REFERENCE
Lisinopril
CAS Registry No.: 76547-98-3 (anhydrous),
83915-83-7 (dihydrate)
Merck Index: 5540
Lednicer No.: 4 83
SAMPLE
Sample preparation: Add MeOH: 100 mM pH 2.8 phosphate buffer 25:75 to powdered capsules
or tablets so as to give a lisinopril concentration of ca. 1.8 mg/mL, stir for 15 min, inject a 20
IxL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:20 mM pH 2.5 sodium heptanesulfonate 35.15:1.85:63
Flow rate: 1
Detector: UV 215
CHROMATOGRAM
Retention time: 5.0
KEYWORDS
capsules; tablets
REFERENCE
Bonazzi,D.; Gotti,R.; Andrisano,V.; Cavrini,V. Analysis of ACE inhibitors in pharmaceutical dosage forms by
derivative UV spectroscopy and liquid chromatography (HPLC), J.Pharm.Biomed.Anal., 1997,16,431438.
Lisofylline
SAMPLE
Sample preparation: Add 6 mL ice-cold dichloromethane to 500 |xL microsomal incubation, add
100 |xL 10 |xg/mL CT-2410 R, shake on a reciprocal shaker for 10 min, centrifuge at 3000 g for
10 min, evaporate the organic layer to dryness under a stream of nitrogen at 50, reconstitute
the residue in 100 |xL mobile phase, inject 30-60 |xL aliquot.
HPLCVARIABLES
Guard column: Opti-Guard (Optimize Technologies, Inc.)
Column: 100 X 4.6 3 |xm Microsorb-MV C18
Mobile phase: MeOHrbuffer 35:65 (Buffer was 25 mM ammonium phosphate containing 0.25%
acetic acid, pH adjusted to 4.5 with ammonium hydroxide.)
Flow rate: 0.7
Detector: UV 273
CHROMATOGRAM
Internal standard: CT-2410 R (20.0)
OTHER SUBSTANCES
Extracted: pentoxifylline
KEYWORDS
human; liver
REFERENCE
Lee,S.H.; Slattery,J.T. Cytochrome P450 isozymes involved in lisofylline metabolism to pentoxifylline in human
liver microsomes, Drug Metab.Dispos., 1997, 25, 1354-1358.
Lisuride
Merck Index: 5541
SAMPLE
residue with 50 |xL MeCN.water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 209.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Lobeline
Molecular weight: 337.46 L
CAS Registry No.: 90-69-7, 134-65-6 (), 134-63-4 (HCI), 134-64-5 (sulfate)
Merck Index: 5580
SAMPLE
residue with 50 JULL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
a 10 (urine) or 30 (blood) fxL aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Lobenzarit
Merck Index: 5581
LednicerNo.: 4 43
SAMPLE
Matrix: blood
Sample preparation: Add 50 JJLL 35 |JL g/mL IS in MeOH to 200 pX. plasma, shake, add 300 jxL
MeCN, shake. Vortex for 2 min, centrifuge at 25 at 460 g for 10 min, inject a 20 fxL aliquot
of supernatant.
HPLC VARIABLES
Guard column: 4 X 4 5 |xm LiChroCART
Column: 125 X 4 5 jxm LichrospherlOO RP-18
Mobile phase: MeCN:water:glacial acetic acid 50:50:0.2
Flow rate: 1.0
Injection volume: 20 RT 4.48
Detector: UV 308
CHROMATOGRAM
Internal standard: diphenylamine (9.20)
Limit of quantitation: 2 jx g/L
KEYWORDS
plasma
REFERENCE
Castillo,B-; Alberto,N.; Nuevas,L.; Peris,J.E. Determination of lobenzarit disodium in human plasma by high-
performance liquid chromatography, J.Liq.Chromatogr.Rel.Technol, 1998, 21, 1063-1072.
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Plasma + 50 |xL 200 mM HCl + 50 |xL water, mix, add 50 fxL 100
|xg/mL diphenylamine in MeCN, add 550 (xL MeCN, centrifuge at 4 at 1400 g for 10 min,
HPLCVARIABLES
Column: 250 X 4.6 5 (xm Zorbax phenyl
Flow rate: 1
Detector: UV 308
CHROMATOGRAM
Retention time: 10
Internal standard: diphenylamine (18.8)
OTHER SUBSTANCES
Simultaneous: acetaminophen, aspirin, flufenamic acid, hydrochlorothiazide, indomethacin, ke-
toprofen, meclofenamic acid, mefenamic acid, naproxen, phenylbutazone, piroxicam, sulindac,
tolmetin
Noninterfering: fenoprofen, ibuprofen
KEYWORDS
REFERENCE
Schwende,F.J.; Turner,S.W. High-performance liquid chromatographic (HPLC) determination of lobenzarit in
plasma and its application to a bioavailability study in beagle dogs, Pharm.Res., 1991, 8, 523-526.
Lodoxamide
CAS Registry No.: 53882-12-5, 63610-09-3 (tromethamine salt)
Merck Index: 5585
Lednicer No.: 3 57
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 10 |xL 115 jxg/mL p-nitrocinnamic acid in MeOH:water
50:50 + 100 JJLL concentrated HCl, vortex for 30 s, heat on a steam bath for 1 min, cool to room
temperature, add 5 mL ethyl acetate, vortex for 45 s, centrifuge at 860 g for 10 min. Remove
the residue in 100 |xL MeOH:water 50:50 containing 50 mM tris(hydroxymethyl)aminomethane
adjusted to pH 6, if necessary centrifuge at 1239 g for 5 min, inject a 25 |xL aliquot.
HPLC VARIABLES
Column: 300 X 4 10 fxm ixBondapak C18
Mobile phase: MeOH:50 mM tris(hydroxymethyl)aminomethane 10:90 adjusted to pH 6 with
concentrated phosphoric acid
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 10
Internal standard: p-nitrocinnamic acid (19)
KEYWORDS
plasma
REFERENCE
Honigberg,I.L.; Stewart,J.T.; Ewing,B-J.| Taylor,W.D. Determination of lodoxamide in plasma using ion-pairing
and reversed-phase high-performance liquid chromatography, J.Chromatogr., 1983, 276, 213-217.
SAMPLE
Sample preparation: Spray aerosol (weigh can before and after) into 10 mL 8 mM pH 7.0
phosphate buffer and 5 mL 22 mM salicylic acid in MeOH:8 mM pH 7.0 phosphate buffer 10:
90, wash container with 75 mL water, inject an aliquot of this mixture.
HPLC VARIABLES
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Retention time: 7
Internal standard: salicylic acid (10)
KEYWORDS
aerosol
REFERENCE
Havel,H.A.; Beaubien,L.J.; Haaland,P.D. Analysis of the variance components in a pharmaceutical aerosol prod-
uct: lodoxamide tromethamine, J.Pharm.Sci., 1985, 74, 978-982.
Lofepramine
Merck Index: 5587
Lednicer No.: 4 201
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
OTHER SUBSTANCES
isoxsuprine, ketanserin, laudanosine, lidocaine, loxapine, maprotiline, mecamylamine, meclo-
REFERENCE
Jane,L; McKinnon,A.; Flanagan,R.J- High-performance liquid chromatographic analysis of basic drugs on silica
Lomefloxacin
Molecular formula: Ci 7 H 19 F 2 N 3 O 3
Merck Index: 5592
LednicerNo.: 5 125
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL serum with 20 |xL 20 |xg/mL IS in MeOH and 100 u.L 20 mM
NaOH. Vortex for 1 min with 4 mL dichloromethanerdiethylether 80:20, centrifuge at 2000 g
organic layers, evaporate to dryness under a stream of nitrogen. Add 100 (xL 20 mM NaOH to
the residue, shake for 5 min, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 10 jxm Vydac AXGU
Flow rate: 1.2
Detector: UV 280
CHROMATOGRAM
Retention time: 4.3
OTHER SUBSTANCES
Extracted: felbinac, fenbufen
KEYWORDS
plasma
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: 500 uX. Serum + 250 |xL 10% trichloroacetic acid, vortex for 10 s, centri-
fuge at >700 g for 10 min, inject a 20 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: 100 X 8 ixBondapak C18 Radial-PAK
CHROMATOGRAM
Retention time: 8.0
KEYWORDS
serum
REFERENCE
J.Antimicrob.Chemother., 1989, 24, 437-445.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 100 JJLL 2 jxg/mL IS + 400 JXL 200 mM pH 7.0 sodium
phosphate buffer, vortex for 5 s, add 5 mL chloroform:isoamyl alcohol 95:5, rotate for 5 min,
stream of nitrogen, reconstitute the residue in 500 |xL mobile phase, inject a 40 |xL aliquot.
HPLCVARIABLES
Mobile phase: MeCN:50 mM citric acid:l M ammonium acetate 22:77:1
Flow rate: 1.5
CHROMATOGRAM
Retention time: 6.2
Internal standard: E-1608 (G.D.Searle) (12.6)
KEYWORDS
REFERENCE
Healy,D.R; Schoenle,J.R.; Stotka,J.; Polk,R.E. Lack of interaction between lomefloxacin and caffeine in normal
volunteers, Antimicrob.Agents Chemother., 1991, 35, 660-664.
SAMPLE
Matrix: blood
Sample preparation: 500 yiL Plasma + 25 |xL 100 |xg/mL acebutolol in MeOH:water 10:90 +
100 JJLL 70 mM pH 7 phosphate buffer + 4 mL chloroformdsopentyl alcohol:diethyl ether 71.25:
3.75:25, vortex for 30 s, centrifuge at 1800 g for 5 min. Remove the organic layer and evaporate
it to dryness under reduced pressure, reconstitute the residue in 100 |xL chloroformitriethyl-
amine 100:1, add 100 JJLL 1% (S)-(+)-l-(l-naphthyl)ethyl isocyanate in chloroform, after 1 min
add 50 JJLL 2% ethylchloroformate in chloroform, after 30 s add 50 jxL 2.5% ethanolamine in
chloroform, inject a 20-125 |JLL aliquot.
HPLC VARIABLES
Column: 100 X 8 4 |xm Nova-Pak silica Radial Pak
Mobile phase: Hexane:chloroform:MeOH 64.5:33:2.5
Flow rate: 2
Detector: F ex 245 em 420 for 12 min then ex 280 em 470
CHROMATOGRAM
Retention time: 21 ((SM-)), 22 ((RM+))
Internal standard: acebutolol (8.5, 9.5 enantiomers)
KEYWORDS
plasma; derivatization; chiral; normal phase
REFERENCE
Foster,R.T.; Carr,R.A.; Pasutto,F.M.; Longstreth,J.A. Stereospecific high-performance liquid chromatographic
assay of lomefloxacin in human plasma, J.Pharm.Biomed.Anal., 1995, 13', 12431248.
SAMPLE
Sample preparation: Adjust pH of 500 (JLL plasma or urine to 7, extract with 5 mL chloroform:
isoamyl alcohol 95:5. Remove the organic layer and evaporate it to dryness under a stream of
nitrogen at 40, reconstitute the residue in 500 jxL mobile phase, inject a 30 jxL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 jjim ixBondapak C18
Mobile phase: MeCN:50 mM citric acid containing 1% 1 M ammonium acetate 22:78
Flow rate: 1.3
CHROMATOGRAM
Retention time: 6.5
Internal standard: KK-123 (Searle) (12.5)
Limit of quantitation: 3 |ig/mL (urine), 20 ng/mL (plasma)
KEYWORDS
REFERENCE
Hooper,W.D.; Dickinson,R.G.; Eadie,M.J. Effect of food on absorption of lomefloxacin, Antimicroh.Agents Chem-
other., 1990, 34, 1797-1799.
SAMPLE
Sample preparation: Centrifuge an erythrocyte suspension at 15000 g for 3 min, inject an al-
iquot of the supernatant.
HPLCVARIABLES
Guard column: Perisorb RP-18 (P. J. Cobert)
Column: 150 X 4.6 5 jxm MOS Hypersil C8
Mobile phase: MeCN:THF:100 mM phosphoric acid:triethylamine:water 50:10:10:0.03:30
Flow rate: 1.5
CHROMATOGRAM
Limit of detection: <1 [LgImL
KEYWORDS
protect from light; erythrocytes
REFERENCE
Knaub,S.R.; Chang,M.R; Lunte,C.E.; Topp,E.M.; Riley,C.M. Automated analytical systems for drug develop-
ment studies. Part IV. A microdialysis system to study the partitioning of lomefloxacin across an erythrocyte
membrane in vitro, J.Pharm.Biomed.Anal., 1996, 14, 121129.
SAMPLE
Matrix: cells
HPLCVARIABLES
droxide 25:75
Flow rate: 1.5
OTHER SUBSTANCES
Also analyzed: ciprofloxacin, fleroxacin, norfloxacin, ofloxacin, temafloxacin
REFERENCE
Pascual,A.; Garcia,!; Conejo,M.C; Perea,E.J. Fluorometric and high-performance liquid chromatographic mea-
SAMPLE
Matrix: erythrocytes
Sample preparation: Centrifuge erythrocyte suspension at 15000 g for 3 min. inject an aliquot
of the supernatant
HPLC VARIABLES
Guard column: Perisorb RP-18 (P.J. Cobert)
Column: 150 X 4.6 5 |xm MOS Hypersil C8
Mobile phase: MeCN:THF:100 mM phosphoric acid:triethylamine:water 30:10:10:0.03:50
Flow rate: 1.5
KEYWORDS
REFERENCE
Knaub,S.R.; Priston,M.J.; Morton,M.D.; Slechta,J.D.; Vander Velde,D.G.; Riley,C.M. A 19F NMR study of
lomefloxacin in human erythrocytes and its interaction with hemoglobin, J.Pharm.Biomed.Anal., 1995,13,
1225-1233.
SAMPLE
Matrix: urine
Sample preparation: 50 |xL Urine + 100 |xL IS + 3.85 mL water, mix, inject a 10 u,L aliquot.
HPLC VARIABLES
Mobile phase: MeCN:50 mM citric acidrl M ammonium acetate 22:77:1
Flow rate: 1.5
CHROMATOGRAM
Retention time: 3.6
Internal standard: KK-123 (G.D.Searle) (6.3)
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Stuht,H.; Lode,H.; Koeppe,R; Rost,K.L.; Schaberg,T. Interaction study of lomefloxacin and ciprofloxacin with
omeprazole and comparative pharmacokinetics, Antimicrob.Agents Chemother., 1995, 39, 1045-1049.
Lometrexol
CAS Registry No.: 106400-81 -8,
120408-07-3 (disodium salt)
Lednicer No.: 5 151
SAMPLE
Matrix: blood
Sample preparation: Condition a 10 mm Bond-Elut C8 SPE cartridge with 5 mL MeOH and 5
mL buffer. Dilute plasma with an equal volume of 100 ng/mL IS in buffer, vortex, centrifuge
at 4 at 1000 g for 20 min, add a 2 mL aliquot of the supernatant to the SPE cartridge, wash
with 5 mL buffer, elute with 1.5 mL MeCN:buffer 20:80. Centrifuge the eluate at 4 at 1000 g
for 5 min, evaporate the supernatant to dryness under reduced pressure, reconstitute with 20
IxL 13% formic acid in water, add 100 |xL of a freshly prepared 200 jxg/mL suspension of
activated manganese dioxide (Sigma) in water, vortex, heat at 37 for 1.5 h, cool to 4, add 30
JJLL 5 M NaOH: 1% pH 5 ammonium carbonate 50:50, cool on ice, centrifuge at room temperature
at 13000 g for 10 min, inject a 100 |xL aliquot of the supernatant. (Buffer was 1% aqueous
formic acid adjusted to pH 3.7 with 5 M NaOH.)
HPLC VARIABLES
Column: 150 X 4.6 3 |xm Apex II C18 (Jones Chromatography)
Mobile phase: MeCN:buffer 12:88 containing 171 (xg/mL tetramethylammonium hydrogen sul-
fate (Buffer was 1% aqueous acetic acid adjusted to pH 5 with strong ammonia. Wash column
with MeOH for 10 min between experiments.)
Flow rate: 1
CHROMATOGRAM
Retention time: 4.2
Internal standard: ClO-desmethylene lometrexol (5.3)
KEYWORDS
derivatization; plasma; SPE; analytical procedures that do not involve derivatization are also
given in the paper.; pharmacokinetics
REFERENCE
Wedge,S.R.; Laohavinij,S.; Taylor,G.A.; Newell,D.R. Measurement of 5,10-dideaza-5,6,7,8-tetrahydrofolate(lom-
etrexol) in human plasma and urine by high-performance liquid chromatography, J.Chromatogr.B, 1995,
663, 327-335.
Lomustine
Merck Index: 5594
Lednicer No.: 2 12
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg CBA Bond Elut SPE cartridge with 1 mL MeOH and
1 mL water. Centrifuge blood at 5000 g for 2-3 min, freeze plasma in dry ice/hexane within 1
min. Thaw within 3 min by immersion in a 50 water bath. 1 mL Thawed plasma + 500 |xL
2.5 |xg/mL IS in 100 mM citric acid, vortex for 5 s, centrifuge for 5 min, add a 1 mL aliquot of
the supernatant to the SPE cartridge, wash with 1 mL water, elute with 200 fxL MeOH into a
vial containing 50 jxL 100 mM acetic acid, inject a 25 |xL aliquot.
HPLC VARIABLES
Column: 125 X 5 5 fjim Spherisorb ODS
acid
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Internal standard: l-methyl-3-isobutyl-8-vinyl-2,6-dioxopurine (S 10338) (7.2)
OTHER SUBSTANCES
Extracted: carmustine, fotemustine
KEYWORDS
plasma; SPE
REFERENCE
Gordon,B.H.; Richards,R.R; Hiley,M.R; Gray,A.J.; Ings,R-M.; Campbell,D.B. A new method for the measure-
ment of nitrosoureas in plasma: an h.p.l.c. procedure for the measurement of fotemustine kinetics, Xeno-
biotica, 1989, 19, 329-339.
SAMPLE
a 10 (urine) or 30 (blood) u,L aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
mL/min)
Detector: UV 229.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: If necessary, remove oxidizing power of solution by adding sodium meta-
bisulfite, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 15 X 4.6 5 |xm Microsorb C8
Mobile phase: MeOH:0.4 g/L (NH4)H2PO4 (pH 4.7) 75:25
Flow rate: 1
Detector: UV 228
CHROMATOGRAM
Retention time: 6.4
REFERENCE
Lunn,G.; Rhodes,S.W.; Sansone,E.B.; Schmuff,N.R. Photolytic destruction and polymeric resin decontamination
of aqueous solutions of Pharmaceuticals, J.Pharm.Sci., 1994, 83, 1289-1293.
Lonidamine
Merck Index: 5598
SAMPLE
Matrix: blood
Sample preparation: Condition an AASP C8 SPE cartridge (Varian) with 500 |xL MeOH and
500 |xL 10 mM HCl. 10 jxL Serum + 990 |xL 10 mM HCl containing IS, add to the SPE cartridge,
wash with 500 |xL 10 mM HCl, elute the contents of the SPE cartridge on to the analytical
column with the mobile phase for 1 min.
HPLC VARIABLES
Column: 150 X 4 4.5 urn MicroPak SP-C18-5
Mobile phase: Gradient. MeCN: 100 mM pH 3.5 acetate buffer 40:60 for 2.5 min, to 60:40 over
1 min, to 70:30 over 6.5 min, return to initial conditions over 5 min, re-equilibrate for 10 min.
Flow rate: 1
Detector: UV 300
CHROMATOGRAM
Retention time: 8
Internal standard: l-[4-chlorobenzyl]-lH-indazole-3-carboxylicacid (AF1312/TS) (6.5)
OTHER SUBSTANCES
KEYWORDS
serum; SPE
REFERENCE
Bottalico,C; Micelli,G.; Guerrieri,A.; Palmisano,F.; Lorusso,V.; De Lena,M. An on-line semi-automated solid-
phase extraction procedure for high-performance liquid chromatographic determination of lonidamine in
serum, J.Pharm.Biomed.Anal., 1995, 13, 1349-1353.
SAMPLE
Sample preparation: 1 mL Plasma or urine + 50 uL 100 u,g/mL IS in MeOH + 400 jxL 1 M
HCl, vortex for 15 s, add 5 mL anhydrous diethyl ether, shake on a reciprocal shaker for 10
min, centrifuge at 800 g for 10 min, repeat extraction with 3 mL anhydrous diethyl ether.
reconstitute the residue in 200 |xL MeCN, inject a 20 JJLL aliquot.
HPLCVARIABLES
Mobile phase: MeCNrIOO mM pH 3.50 acetate buffer 50:50
Flow rate: 2
Detector: UV 300
CHROMATOGRAM
Retention time: 5.5
Internal standard: l-(4-chlorobenzyl)-lH-indazole-3-carboxylicacid (AF 1312/TS) (4)
KEYWORDS
REFERENCE
Leclaire,R.; Besner,J.-G.; Band,R; Mailhot,S.; Gervais,R; De Sanctis,A.; Deschamps,M.; Liverani,L. High-per-
formance liquid chromatography of lonidamine in human plasma and urine, J.Chromatogr., 1983,277,427
432.
Loperamide
Merck Index: 5601
LednicerNo.: 2 334
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 260
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: blood, cell incubations, cell lysates, feces, urine
Sample preparation: Urine, plasma. Inject an aliquot directly. Feces. Extract with MeOH, evap-
orate extract to dryness under a stream of nitrogen, reconstitute in DMSO, inject an aliquot.
Cell incubates. Centrifuge, inject an aliquot of the supernatant. Cell lysates. Dilute with an
equal volume of DMSO, centrifuge, inject an aliquot of the supernatant.
HPLCVARIABLES
Mobile phase: Gradient. A was 100 mM ammonium acetate containing 0.2% diethylamine, pH
7.88. B was MeCN:MeOH:l M ammonium acetate containing 2% diethylamine (pH 7.88) 65:
25:10 (?). A:B from 100:0 to 65:35 over 2 min, to 35:65 over 30 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 41
OTHER SUBSTANCES
Extracted: loperamide oxide (prodrug)
KEYWORDS
plasma; prodrug; pharmacokinetics
REFERENCE
Lavrijsen,K; Van Dyck,D.; Van Houdt,J.; Hendrickx,J.; Monbaliu,J.; Woestenborghs,R.; Meuldermans,W.; Hey-
kants,K. Reduction of the prodrug loperamide oxide and its active drug loperamide in the gut of rats, dogs,
and humans, Drug Metab.Dispos., 1995, 23, 354-362.
SAMPLE
Sample preparation: Capsules, tablets. Remove contents of capsules and powder tablets. Weigh
out an amount equivalent to about 10 mg loperamide hydrochloride, add 80 mL chloroform,
shake for 15 min, make up to 100 mL with chloroform, filter and discard first 10-20 mL of
filtrate. 5 mL Filtrate + 1 mL 400 (xg/mL cyclizine hydrochloride in chloroform, make up to 25
mL with chloroform, inject an aliquot. Syrups. Add a quantity of syrup corresponding to about
10 mg loperamide hydrochloride to 30 mL water, extract four times with 20 mL portions of
chloroform and filter each extract through glass wool, combine the extracts and make up to
100 mL with chloroform. Remove a 5 mL aliquot and add it to 1 mL 400 jxg/mL cyclizine
hydrochloride in chloroform, make up to 25 mL with chloroform, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 jxm Perkin-Elmer Analytical silica
Mobile phase: Chloroform:MeOH:ammonia 95.5:4.5:0.05
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 6
Internal standard: cyclizine (4)
OTHER SUBSTANCES
Noninterfering: propylene glycol
KEYWORDS
capsules; tablets; syrups; normal phase
REFERENCE
Leung,C.R; Au-Yeung,C.Y. High-performance liquid chromatographic determination of loperamide hydrochlo-
ride in pharmaceutical preparations, J.Chromatogr., 1988, 449, 341-344.
Loprazolam
Merck Index: 5604
SAMPLE
Matrix: blood
the residue in 100 JJLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of
HPLC VARIABLES
Mobile phase: MeOH.THF.buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted
Flow rate: 0.8
Detector: UV 329
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Loratadine
Merck Index: 5608
SAMPLE
residue with 50 JULL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
R e t e n t i o n t i m e : 22.943
KEYWORDS
whole blood
REFERENCE
163.
Lorazepam
Molecular formula: C15H10CI2N2O 2
Merck Index: 5609
Lednicer No.: 1 368
SAMPLE
M a t r i x : blood
Sample preparation: 500 \xL Serum + 20 |xL 20 |xg/mL IS + 200 |xL 1 M potassium carbonate
mobile phase, inject a 20 fxL aliquot. (Caution! Chloroform is a carcinogen!)
HPLC VARIABLES
Column: 100 X 4.6 2 [im TSK gel Super-ODS (A) or 100 X 4.6 5 jjim Hypersil ODS-C18 (B)
Flow rate: 0.65
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: bromazepam, chlordiazepoxide, clonazepam, estazolam, etizolam, flutazolam, halox-
azolam, nitrazepam, oxazolam, triazolam
KEYWORDS
serum
REFERENCE
Tanaka,E.; Terada,M.; Misawa,.; Wakasugi,C. Simultaneous determination of twelve benzodiazepines inhuman
serum using a new reversed-phase chromatographic column on a 2-jxm porous microspherical silica gel,
J.Chromatogr.B, 1996, 682, 173-178.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 228.7
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
163.
SAMPLE
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
304.
SAMPLE
Matrix: solutions
Sample preparation: Dilute a 2 mg/mL sample 1:100 with mobile phase, inject an aliquot.
HPLCVARIABLES
Mobile phase: MeCN:80 mM pH 2.0 orthophosphoric acid in water 46:54
Flow rate: 1.2
Detector: UV 240
CHROMATOGRAM
Retention time: 6.6
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Lormetazepam
Merck Index: 5611
Lednicer No.: 3 196
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, loxapine,
puromycin, pyrilamine, pjîthyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
Hill,D.W.; Kind,A-J- Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnaLToxicoL, 1994,
18, 233-242.
Lornoxicam
Merck Index: 5612
SAMPLE
Matrix: blood
Sample preparation: Mix plasma with 800 uL 5 mM pH 4 phosphate buffer and 400 JJLL 1 |xg/
mL tenoxicam in water. Centrifuge at 2500 rpm for 5 min. Add the supernatant to Extrelut-1
cartridge (Merck, Darmstadt, Germany). Elute with 10 mL dichloromethane, dry eluate under
a stream of nitrogen at 35, dissolve the residue in 300 uL mobile phase, centrifuge and inject
HPLC VARIABLES
Mobile phase: MeOH:50 mM pH 6 phosphate buffer 50:50
Flow rate: 1.3
Detector: UV 371
CHROMATOGRAM
Retention time: 4.4
Internal standard: tenoxicam (2.2)
KEYWORDS
REFERENCE
Bareggi,S.R.; Gambaro,V.; Valenti,M.; Benvenuti,C. Absorption of oral lornoxicam in healthy volunteers using
a granular formulation in comparison with standard tablets, Arzneimittelforschung, 1997, 47, 755-757.
SAMPLE
Matrix: blood, synovial fluid
Sample preparation: Plasma. 500 |xL Plasma + 100 [xL 4 |xg/mL IS in 200 mM NaOH + 500
U-L 500 mM pH 4 phosphate buffer, mix, homogenize, centrifuge at 900 g. Add 1 mL of the
mixture to an Extrelut-1 SPE cartridge. Elute with two 5 mL portions of dichloromethane.
Evaporate the eluate under a stream of nitrogen at 35. Dissolve the residue in 120 jxL mobile
phase by vortexing, inject an aliquot. Plasma, synovial fluid. Condition a 100 mg C18 SPE
cartridge (Phenomenex) with 1 mL MeOH, 1 mL water, and 500 |xL 500 mM pH 2 phosphate
buffer. 500 JJLL Plasma or synovial fluid + 100 |xL 2 ixg/mL IS in 200 mM NaOH + 500 |xL 500
mM pH 2 phosphate buffer. Mix, homogenize, centrifuge at 2500 rpm. Add 1 mL of the clean
supernatant to the SPE cartridge, wash with 1 mL water, dry with 2 mL air, elute with 1.25
mL MeCN:25% ammonia 90:10. Evaporate the eluate under reduced pressure at 35. Recon-
stitute the residue in 100 uL MeOH: 100 mM pH 8 phosphate buffer 50:50, inject an aliquot.
Next Page
HPLC VARIABLES
Column: 250 x 4.6 5 |xm Hypersil ODS
Mobile phase: MeOH: 100 mM pH 6 sodium dihydrogen phosphate buffer 50:50
Flow rate: 1.5
Detector: UV 372
CHROMATOGRAM
Retention time: 5.8-6.1 (Extrelut), 6.7 (C18 SPE)
Internal standard: tenoxicam (2.6-2.8)
OTHER SUBSTANCES
KEYWORDS
plasma; pharmacokinetics; mouse; rat; rabbit; dog; monkey; human; SPE
REFERENCE
Radhofer-Welte,S.; Dittrich,P. Determination of the novel non-steroidal anti-inflammatory drug lornoxicam and
its main metabolite in plasma and synovial fluid, J.Chromatogr.B, 1998, 707, 151-159.
Losartan
Merck Index: 5613
Lednicer No.: 5 73
SAMPLE
Matrix: bile, blood, tissue, urine
Sample preparation: Plasma, urine. Condition a Varian Bond Elut non-endcapped CN SPE
cartridge with two 1 mL portions of MeOH and two 1 mL portions of water. Vortex 500 |xL
plasma + 300 ng IS + 500 |ULL 200 mM HCl or 500 \xL urine + 300 ng IS + 500 JULL water + 200
JJLL 1.0 M HCl and add to the SPE cartridge. Wash with 500 |xL water, with 500 |xL MeOH:
water 10:90, and with 50 JJLL MeOH, elute with five 200 |xL portions of pH 8 MeOH:ammonia
99:1. Evaporate the combined eluates to dryness. Reconstitute the residue in 100 |xL mobile
phase adjusted to pH 1.9 with 85% phosphoric acid, inject an 80 |xL aliquot. Elute with mobile
phase A. Whole blood. Freeze at -30, centrifuge at 2000 g for 10 min at -10, dilute 500 |xL
supernatant with 500 |xL water, add 300 ng IS and 200 ^xL 1 M HCl, vortex, extract with 7
mL MTBE by shaking at low speed for 30 min, centrifuge at 2000 g at -10 for 15 min. Freeze
the aqueous layer in a dry ice-acetone bath and discard it, evaporate the organic layer to
dryness. Reconstitute the residue in 120 |xL mobile phase adjusted to pH 3.1 with 85% phos-
phoric acid (if necessary centrifuge at 0 at 2000 g for 5 min), inject a 90 jxL aliquot. Elute
with mobile phase B. Bile. Mix 100 parts bile with 20 parts 1.0 M HCl, centrifuge at 5000 g
for 15 min at 15, dilute 120 |xL of the supernatant (=100 |xL bile) with 500 |xL water, add IS
and 100 |xL 1 M HCl, vortex, extract with 7 mL MTBE by shaking for 30 min, centrifuge at
2000 g for 15 min at -10. Freeze the aqueous layer in a dry ice-acetone bath and discard it,
evaporate the organic layer to dryness. Reconstitute the residue in 120 JJLL mobile phase ad-
justed to pH 3.1 by 85% phosphoric acid, inject a 90 |xL aliquot. Elute with mobile phase B.
Liver, kidney, heart, lung, small intestine. Homogenize (Ultra Turrax T25) tissue in 2 volumes
of pH 7.4 phosphate buffer at highest speed and filter through cotton gauze with a Potter S
with 10-20 hubs at 450 g. (All steps were carried out on ice.) 1 mL Homogenate (for Ii ver 300
IxL) + IS + 400 jxL 500 mM HCL (for liver 600 \xL 100 mM HCl), vortex, extract with 7 mL
MTBE by shaking at low speed for 30 min, centrifuge at 2000 g for 15 min at 4, freeze in dry
ice/acetone. Remove the organic layer and evaporate it to dryness. Reconstitute the residue in
Previous Page
HPLC VARIABLES
Column: 250 x 4.6 5 |xm Hypersil ODS
Mobile phase: MeOH: 100 mM pH 6 sodium dihydrogen phosphate buffer 50:50
Flow rate: 1.5
Detector: UV 372
CHROMATOGRAM
Retention time: 5.8-6.1 (Extrelut), 6.7 (C18 SPE)
Internal standard: tenoxicam (2.6-2.8)
OTHER SUBSTANCES
KEYWORDS
plasma; pharmacokinetics; mouse; rat; rabbit; dog; monkey; human; SPE
REFERENCE
Radhofer-Welte,S.; Dittrich,P. Determination of the novel non-steroidal anti-inflammatory drug lornoxicam and
its main metabolite in plasma and synovial fluid, J.Chromatogr.B, 1998, 707, 151-159.
Losartan
Merck Index: 5613
Lednicer No.: 5 73
SAMPLE
Matrix: bile, blood, tissue, urine
Sample preparation: Plasma, urine. Condition a Varian Bond Elut non-endcapped CN SPE
cartridge with two 1 mL portions of MeOH and two 1 mL portions of water. Vortex 500 |xL
plasma + 300 ng IS + 500 |ULL 200 mM HCl or 500 \xL urine + 300 ng IS + 500 JULL water + 200
JJLL 1.0 M HCl and add to the SPE cartridge. Wash with 500 |xL water, with 500 |xL MeOH:
water 10:90, and with 50 JJLL MeOH, elute with five 200 |xL portions of pH 8 MeOH:ammonia
99:1. Evaporate the combined eluates to dryness. Reconstitute the residue in 100 |xL mobile
phase adjusted to pH 1.9 with 85% phosphoric acid, inject an 80 |xL aliquot. Elute with mobile
phase A. Whole blood. Freeze at -30, centrifuge at 2000 g for 10 min at -10, dilute 500 |xL
supernatant with 500 |xL water, add 300 ng IS and 200 ^xL 1 M HCl, vortex, extract with 7
mL MTBE by shaking at low speed for 30 min, centrifuge at 2000 g at -10 for 15 min. Freeze
the aqueous layer in a dry ice-acetone bath and discard it, evaporate the organic layer to
dryness. Reconstitute the residue in 120 |xL mobile phase adjusted to pH 3.1 with 85% phos-
phoric acid (if necessary centrifuge at 0 at 2000 g for 5 min), inject a 90 jxL aliquot. Elute
with mobile phase B. Bile. Mix 100 parts bile with 20 parts 1.0 M HCl, centrifuge at 5000 g
for 15 min at 15, dilute 120 |xL of the supernatant (=100 |xL bile) with 500 |xL water, add IS
and 100 |xL 1 M HCl, vortex, extract with 7 mL MTBE by shaking for 30 min, centrifuge at
2000 g for 15 min at -10. Freeze the aqueous layer in a dry ice-acetone bath and discard it,
evaporate the organic layer to dryness. Reconstitute the residue in 120 JJLL mobile phase ad-
justed to pH 3.1 by 85% phosphoric acid, inject a 90 |xL aliquot. Elute with mobile phase B.
Liver, kidney, heart, lung, small intestine. Homogenize (Ultra Turrax T25) tissue in 2 volumes
of pH 7.4 phosphate buffer at highest speed and filter through cotton gauze with a Potter S
with 10-20 hubs at 450 g. (All steps were carried out on ice.) 1 mL Homogenate (for Ii ver 300
IxL) + IS + 400 jxL 500 mM HCL (for liver 600 \xL 100 mM HCl), vortex, extract with 7 mL
MTBE by shaking at low speed for 30 min, centrifuge at 2000 g for 15 min at 4, freeze in dry
ice/acetone. Remove the organic layer and evaporate it to dryness. Reconstitute the residue in
120 |JLL mobile phase adjusted to pH 3.1 with 85% phosphoric acid (if necessary centrifuge at
15 at 4000 g for 30 min), inject an 80 |xL aliquot. Elute with mobile phase C. Brain. Homog-
enize (Ultra Turrax T25) tissue in 2 volumes of pH 7.4 phosphate buffer at highest speed. 1
mL Homogenate + IS + 400 (xL 1 M HCL + (?) mL MeCN:MeOH 1:2, let stand on ice for 1 h,
centrifuge at 2000 g for 15 min at 0. Freeze in dry ice/acetone, remove the organic layer, and
evaporate it to dryness. Reconstitute the residue in 120 |JLL mobile phase adjusted to pH 3.1
with 85% phosphoric acid (if necessary centrifuge at 0 at 2000 g for 5 min), inject a 90 |xL
aliquot. Elute with mobile phase C.
HPLCVARIABLES
Guard column: 30 X 4.6 3 |xm Ultremex 5 CN
Column: 250 X 4.6 3 jxm Ultremex 3 CN
Mobile phase: MeCN:MeOH:THF:4.5 mM sodium dihydrogen phosphate buffer:85% phosphoric
acid 21:5:4:69.9:0.1 (A), MeCN:THF:7.5 mM sodium dihydrogen phosphate buffer:85% phos-
phoric acid 26:5:68.9:0.1 (B), MeCN:THF:7.5 mM sodium dihydrogen phosphate buffer:85%
phosphoric acid 29:3:67.9:0.1 (C)
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
Retention time: 17-18 (A), 14-15 (B,C)
Internal standard: L-158.854 (DuPont Merck Co.,USA)(20-21 (A), 18-19 (B,C))
Limit of quantitation: 5 ng/mL (plasma), 10 ng/mL (urine), 12.5 ng/mL (blood and bile), 10-15
ng/100 mg (tissue)
OTHER SUBSTANCES
KEYWORDS
SPE; plasma; urine; whole blood; rat; brain; liver; kidney; heart; lung; spleen; small intestine
REFERENCE
Soldner,A.; Spahn-Langguth,H.; Mutschler,E. HPLC assays to simultaneously determine the angiotensin-ATl
antagonist losartan as well as its main and active metabolite EXP 3174 in biological material of humans
and rats, J.Pharm.Biomed.Anal, 1998, 16, 863-873.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 200 ng IS, adjust pH to 9.5 with 1 M KOH, make up to
2.5 mL with tetrabutylammonium hydrogen sulfate in water so that the final concentration of
tetrabutylammonium hydrogen sulfate is 10 mM, add 10 mL dichloromethane, extract for 1 h,
centrifuge. Remove the organic layer and evaporate it to dryness under a stream of nitrogen
at room temperature, reconstitute the residue in 100 |JLL mobile phase, inject an 80 JJLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |jim Zorbax C8
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 14
Internal standard: 4-chloro-l-[((lH-tetrazoyl-5-yl)biphenyl-4-yl)methyl]-5-methal-2-n-propyl
imidazole
OTHER SUBSTANCES
Noninterfering: metabolites
KEYWORDS
dog; plasma; pharmacokinetics
REFERENCE
Christ,D.D.; Wong,RC; Wong,Y.N.; Hart,S.D.; Quon,C.Y.; Lam,G.N. The pharmacokinetics and pharmacody-
namics of the angiotensin II receptor antagonist losartan potassium (DuP 753/MK 954) in the dog,
SAMPLE
Matrix: blood
Sample preparation: 150 |xL Plasma + 150 |xL mobile phase A, mix, centrifuge at 3000 rpm for
2 min, inject a 250 JJLL aliquot of the supernatant on to column A and elute to waste with mobile
phase A, after 5 min backflush the contents of column A on to column B with mobile phase B,
monitor the effluent from column B. Re-equilibrate column A with mobile phase A and column
B with mobile phase B for 5 min before next injection.
HPLC VARIABLES
Column: A 20 X 3.9 25-40 ^m LiChroprep RP-8; B 10 X 4 Nova-Pak C8 + 250 X 4.6 5 (xm Inertsil
ODS-2
Mobile phase: A MeCN:50 mM phosphoric acid 5:95; B MeCN:50 mM pH 3.5 ammonium acetate
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
column-switching; plasma; rat; pharmacokinetics
REFERENCE
Lee,H; Shim,H-O.; Lee,H.S. Simultaneous determination of losartan and active metabolite EXP3174 in rat
plasma by HPLC with column switching, Chromatographia, 1996, 42, 39-42.
SAMPLE
Sample preparation: Blood. 150 |JLL Plasma + 150 JULL MeCN, vortex for 15 s, centrifuge at 13000
g for 10 min, inject a 10 |xL aliquot of the supernatant. Dialysate. Inject a 10 (xL aliquot of the
dialysate. Urine. 50 JXL Urine + 450 |xL water, vortex for 10 s, inject a 10 JJLL aliquot.
HPLC VARIABLES
Guard column: 30 X 4.6 40-50 |xm C18 (Alltech)
Column: 150 X 3.2 3 |xm Hypersil Phenyl
Mobile phase: Gradient. MeCN:25 mM pH 2.2 potassium phosphate 35:65 for 5 min, to 60:40
over 4 min, maintain at 60:40 for 3 min, return to initial conditions over 1 min (plasma) or
MeCN:25 mM pH 2.2 potassium phosphate 40:60 (dialysate, urine)
Flow rate: 0.75
CHROMATOGRAM
Retention time: 7.3 (plasma), 5.5 (urine, dialysate)
Limit of quantitation: 10 ng/mL (plasma, dialysate), 50 ng/mL (urine)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Farthing,D.; Sica,D.; Fakhry,L; Pedro ,A.; Gehr,T.W.B. Simple high-performance liquid chromatographic method
for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate, J.Chromatogr.B,
1997, 704, 374-378.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 125 |xL 1 M phosphoric acid + 100 |xL IS solution
+ 10 mL MTBE, shake at 60 rpm for 20 min, centrifuge at 2060 g for 5 min, freeze in dry ice/
acetone. Remove the organic layer and add it to 200 JJLL 50 mM NaOH, shake at 60 rpm for
15 min, centrifuge at 2060 g for 5 min, freeze in dry ice/acetone. Discard the organic layer and
allow the aqueous layer to thaw, add 75 |xL 200 mM phosphoric acid to the aqueous layer, add
6 mL hexane, vortex for 2 min, centrifuge, freeze in dry ice acetone. Discard the hexane layer
and allow the aqueous layer to thaw, remove residual hexane using a stream of nitrogen, add
75 |xL isopropanol, inject a 110 |xL aliquot. Urine. 500 u,L Urine + 500 |xL water + 50 u,L 1 M
phosphoric acid + 100 |xL IS solution + 10 mL MTBE.hexane 80:20, shake at 60 rpm for 20
min, centrifuge at 2060 g for 5 min, freeze in dry ice/acetone. Remove the organic layer and
add it to 200 |xL 50 mM NaOH, shake at 60 rpm for 15 min, centrifuge at 2060 g for 5 min,
freeze in dry ice/acetone. Discard the organic layer and allow the aqueous layer to thaw, add
75 |xL 200 mM phosphoric acid to the aqueous layer, add 6 mL hexane, vortex for 2 min,
centrifuge, freeze in dry ice acetone. Discard the hexane layer and allow the aqueous layer to
thaw, remove residual hexane using a stream of nitrogen, add 75 |xL isopropanol, inject a 65
|xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultremex CN
Mobile phase: MeCN:buffer 23:77 (Buffer was 100 mL 200 mM NaH2PO4 and 2 mL 85% phos-
phoric acid in 2 L water, pH adjusted to 2.5 with 2 M NaOH.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 13
Internal standard: 2-n-butyl-4-(2-chlorophenyl)-5-carboxy-1-[(2'-(lH-tetrazol-5-yl)biphenyl-4-
yl)methyl]iimdazole (L-158,854, Merck) (15)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Furtek,C.L; Lo,M.-W. Simultaneous determination of a novel angiotensin II receptor blocking agent, losartan,
and its metabolite in human plasma and urine by high-performance liquid chromatography, J.Chromatogr.,
1992, 573, 295-301.
SAMPLE
Sample preparation: Add IS to plasma or urine, buffer with 100 mM pH 2.8 citrate buffer, add
to a Bond Elut C8 or NH2/C18 SPE cartridge, elute with MeOH. Evaporate the eluate, recon-
stitute with EtOH:water 50:50, inject a 25-50 u-L aliquot.
HPLC VARIABLES
Guard column: 15 X 3.2 New Guard RP18 (Biosystem Inc.)
Column: 250 X 4.6 Capcell Pak C18-ODS (Shiseido)
Mobile phase: Gradient. MeCN:25 mM pH 2.8 phosphate buffer from 35:65 to 50:50 over 10 min,
to 65:35 over 10 min
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: 2-n-propyl-4-chloro-l-[2'-(tetrazol-5-yl)-l,l>-biphenyl-4-ylmethyl]-lH-imidaz-
ole-5-aldehyde (Dup 167)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Ohtawa,M.; Takayama,R; Saitoh,K; Yoshinaga,T.; Nakashima,M. Pharmacokinetics and biochemical efficacy
after single and multiple oral administration of losartan, an orally active nonpeptide angiotensin II receptor
antagonist, in humans, Br.J.Clin.PharmacoL, 1993, 35, 290-297.
SAMPLE
Sample preparation: Stir ten 50 mg tablets with 1 L 10 mM pH 8 NaH2PO4 for 30 min, filter
(0.45 |xm PVDF), inject a 4 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Lichrosorb 10 RP-8 [C8]
Mobile phase: MeCN.10 mM pH 2.5 NaH2PO4 40:60
Flow rate: 1
Injection volume: 4
Detector: UV 230
CHROMATOGRAM
Retention time: 11
Internal standard: butyl paraben (17.5)
KEYWORDS
tablets; comparison with capillary electrophoresis and packed column SFC
REFERENCE
Williams,R.C; Alasandro,M.S.; Fasone,V.L.; Boucher,R.J.; Edwards,J.F. Comparison of liquid chromatography,
capillary electrophoresis and super-critical fluid chromatography in the determination of Losartan Potas-
sium drug substance in Cozaar tablets, J.Pharm.BiomedAnal., 1996, 14, 1539-1546.
SAMPLE
Sample preparation: Condition a 3 mL BondElut C18 SPE cartridge with 5 mL MeOH and 5-
10 mL 0.2% trifluoroacetic acid. 0.5-1 mL Microsomal incubation + 2 mL 0.2% trifluoroacetic
acid, add to the SPE cartridge, wash with 2 mL 0.2% trifluoroacetic acid, elute with 4-5 mL
MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 35-45 or under reduced
pressure, reconstitute the residue in 100-200 |xL MeCN:MeOH:2 mM ammonium acetate:tri-
fluoroacetic acid 20:40:40:0.1, inject a 25-50 |xL aliquot.
HPLC VARIABLES
Mobile phase: Gradient. A was MeCN:MeOH:100 mM ammonium acetate:acetic acid 15:30:54:
1. B was MeCN:MeOH:2 mM ammonium acetate trifluoroacetic acid 20:40:40:0.1. A:B 100:0 for
20 min, to 0:100 over 10 min, maintain at 0:100 for 10 min.
Flow rate: 1
Detector: UV 250, UV 280, or MS, SCIEXAPI III tandem MS, nebulizer interface 400, positive-
ion detection, orifice 65 V, m/z 423
CHROMATOGRAM
Retention time: 30
OTHER SUBSTANCES
KEY WORDS
human; liver; SPE
REFERENCE
Stearns,R.A.; Chakravarty,P.K; Chen,R.; Chiu,S.-H.L. Biotransformation of losartan to its active carboxylic
acid metabolite in human liver microsomes: Role of cytochrome P4502C and 3A sub-family members, Drug
Metab.Dispos., 1995, 23, 207-215.
SAMPLE
Sample preparation: 500 JJLL Microsomal incubation + 50 |xL 43% aqueous phosphoric acid + 1
mL dichloromethane, vortex, centrifuge. Remove a 700 (JLL aliquot of the organic layer and
evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 50 |JLL MeOH,
HPLC VARIABLES
Mobile phase: MeCN:50 mM pH 7.4 ammonium acetate 26:74
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
human; liver
REFERENCE
Yun,C.-H.; Lee,H.S.; Lee,H.; Rho,J.K.; Jeong,H.G.; Guengerich,F.P. Oxidation of the angiotensin II receptor
antagonist losartan (DuP 753) in human liver microsomes: role of cytochrome P4503A(4) in formation of
the active metabolite EXP3174, Drug Metab.Dispos., 1995, 23, 285-289.
Loxapine
CAS Registry No.: 1977-10-2, 27833-64-3 (succinate)
Merck Index: 5617
Lednicer No.: 2 427
SAMPLE
Matrix: blood
Sample preparation: 500 (xL Plasma or 1 mL diluted red blood cells (red blood cells .water 50:
50), add 6 mL ethyl acetate, vortex for 2 min, centrifuge at 2000 g for 5 min. Remove the
organic layer, add 250 |xL 100 mM HCl, vortex for 2 min, centrifuge at 2000 g for 5 min. Remove
a 200 |JLL volume of the acid layer, evaporate to dryness at 40. Reconstitute the residue in 200
IxL mobile phase and inject a 100 \xL aliquot.
HPLCVARIABLES
Guard column: 20 X 4.6 5 jjim Hypersil ODS
Column: 250 X 4.6 5 |xm Kromasil Ultrabase C 18
Mobile phase: MeCN:buffer 48:52 (Buffer was 716 mg disodium hydrogen phosphate and 2 g
cetrimide in 1 L water, adjusted to pH 7.0 with phosphoric acid.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Internal standard: loxapine
OTHER SUBSTANCES
Extracted: amitriptyline, clonazepam, clorazepate, clozapine, droperidol
Noninterfering: clomipramine, diazepam, hydroxyzine
KEYWORDS
plasma; red blood cells; loxapine is IS
REFERENCE
Guitton,C; Kinowski,J.-M.; Aznar,R.; Bressolle,F. Determination of clozapine and its major metabolites in hu-
man plasma and red blood cells by high-performance liquid chromatography with ultraviolet absorbance
SAMPLE
Matrix: blood
Sample preparation: Add 200 jxL 330 mM NaOH to 1 mL plasma, vortex, add 6 mL hexane:
isoamyl alcohol 98.5:1.5, shake for 30 min, centrifuge at 4000 rpm for 5 min. Collect the organic
layer, add 150 JJLL 100 mM HCl, vortex for 1 min. Collect the acidic aqueous phase and inject
a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 C8 (Bischoff, Germany)
Column: 125 X 4.6 5 |xm Ecotube Nucleosil C8 (Bischoff, Germany)
Mobile phase: MeCN:Pic B5:water:diethylamine 37:2.5:63:0.04 (Pic B5 is a mixture of water,
MeOH, 1-pentanesulfonic acid, and acetic acid and is available from Waters.)
Flow rate: 1.7
Detector: UV 245
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: clozapine
Noninterfering: alprazolam, bromazepam, chlordiazepoxide, clorazepate, diazepam, flunitraze-
pam, haloperidol, lorazepam, nitrazepam, oxazepam, paroxetine, prazepam, temazepam,
triazolam
KEYWORDS
plasma; loxapine is IS
REFERENCE
Edno,L.; Combourieu,I.; Cazenave,M.; Tignol,J. Assay for quantitation of clozapine and its metabolite N-des-
methylclozapine in human plasma by high-performance liquid chromatography with ultraviolet detection,
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 |JLL isopropanol:diethylamine 99.9:0.1 + 250 JJLL 25%
potassium carbonate containing 0.1% diethylamine + 5 mL hexanedsoamyl alcohol 97:3, vortex
for 30 s, centrifuge at 500 g for 3 min. Remove the organic layer and add it to 100 JULL 250 mM
HCl, vortex for 30 s, inject a 50 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
desmethylchlordiazepoxide, desmethyldoxepin, diazepam, doxepin, fluphenazine, haloperidol,
imipramine, nortriptyline, oxazepam, thiothixene
KEYWORDS
plasma; loxapine is IS
REFERENCE
Riel,J.S.; Abramson,R.K.; Morgan,S.L.; Voris,J.C. A rapid high performance liquid chromatographic method for
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 |JLL 1 |xg/mL 2-hydroxydesmethylimipramine, mix,
add 300 jxL 2 M pH 9.7 carbonate buffer, mix, add 4 mL heptanerisopentyl alcohol 93:7, shake
mechanically for 20 min, centrifuge at 1600 g for 5 min. Remove the organic layer and add it
to 250 IJLL 7 mM orthophosphoric acid, vortex vigorously, centrifuge at 1600 g for 10 min, inject
a 100 |xL aliquot of the aqueous layer.
HPLC VARIABLES
Mobile phase: 105 |xM nonylamine in MeCN:5 mM KH2PO4 + 14 mM orthophosphoric acid 23:
77
Flow rate: 2.2
Detector: UV 210
CHROMATOGRAM
Retention time: 30
Internal standard: 2-hydroxydesmethylimipramine
OTHER SUBSTANCES
Extracted: metabolites, amoxapine
Simultaneous: propranolol, doxepin, desmethylimipramine, haloperidol, protriptyline, imipra-
mine, amitriptyline
Noninterfering: chlorpromazine, clomipramine, maprotiline, nortriptyline, thioridazine, trimi-
pramine, trifluoperazine
KEYWORDS
plasma
REFERENCE
Cheung,S.W.; Tang,S.W.; Remington,G. Simultaneous quantitation of loxapine, amoxapine and their 7- and 8-
hydroxy metabolites in plasma by high-performance liquid chromatography, J.Chromatogr., 1991,564,213-
221.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Serum + 75 |xL MeCN containing 4 mg/mL amoxapine + 2 mL 250
mM NaOH + 400 JJLL isoamyl alcohol, vortex vigorously, let stand for 5 min, add 10 mL heptane,
shake vigorously for 1 h, centrifuge at >2000 g for 30 min. Remove the upper heptane layer
and add it to 1 mL 100 mM pH 3 glycylglycine buffer, shake vigorously for 1 h, centrifuge at
>2000 g for 30 min. Discard the heptane layer, add 1 mL 250 mM NaOH to the aqueous layer,
add 5 mL n-pentane, shake for 1 h, centrifuge at 2000 g for 30 min. Remove the organic layer
and evaporate it to dryness under educed pressure, reconstitute the residue in 70 |xL mobile
phase, vortex vigorously, centrifuge at 2000 g for 2-3 min, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 40 |jim pellicular silica (CEL Associates, Houston)
Column: 100 X 6 3 |xm silica 80 A (CEL Associates, Houston)
Mobile phase: MeCN:buffer 20:80 containing 21 mM n-nonylamine, pH 7.4-7.8 (Buffer was 25
mM Na2HPO4 adjusted to pH 3 with concentrated phosphoric acid.)
Flow rate: 1.6
Detector: UV (wavelength not specified)
CHROMATOGRAM
Internal standard: amoxapine (9.63)
OTHER SUBSTANCES
Extracted: doxepin
Simultaneous: amitriptyline, chlorpromazine, clomipramine, desipramine, fluoxetine, imipra-
mine, mianserin, nortriptyline, thioridazine, trimipramine
Noninterfering: diazepam
KEYWORDS
serum; loxapine is IS
REFERENCE
Adamczyk,M.; Fishpaugh,J.R.; Harrington,C. Quantitative determination of E- and Z-doxepin and E- and Z-
desmethyldoxepin by high-performance liquid chromatography, Ther.Drug Monit., 1995, 17, 371-376.
SAMPLE
Matrix: blood
HPLC VARIABLES
Column: 300 X 3.9 4 |im NovaPack C18
Flow rate: 0.8
Detector: UV 299
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
JJIL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the organic
layer and inject a 30 |xL aliquot of the aqueous layer. Liver homogenate. 0.5 mL Liver homo-
genate + 10 |xg cianopramine + 500 |xL 2% sodium tetraborate + 8 mL hexane:l-butanol 95:5,
gently agitate for 30 min, centrifuge at 2500 g for 5 min. Remove the organic layer and add it
to 400 jxL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the
HPLC VARIABLES
Guard column: 15 X 3.2 7 jxm RP-18 Newguard (Applied Biosystems)
Column: 100 X 4.6 5 jjim Brownlee Spheri-5 RP-18
Mobile phase: MeCN:100 mM NaH2PO4:diethylamine 40:57.5:2.5
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
doxepin, fluoxetine, haloperidol, imipramine, maprotiline, meperidine, mesoridazine, metha-
done, metoclopramide, mianserin, moclobemide, nomifensine, nordoxepin, norfluoxetine, nor-
propoxyphene, northiaden, nortriptyline, pentobarbital, pheniramine, promethazine, propoxy-
phene, propranolol, protriptyline, quinidine, quinine, sulforidazine, thioridazine, thiothixene,
tranylcypromine, trazodone, trihexyphenidyl, trimipramine, triprolidine
Noninterfering: dextromethorphan, norphetidine, phenoxybenzamine, prochlorperazine, tri-
fluoperazine
KEYWORDS
REFERENCE
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 209.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.8
OTHER SUBSTANCES
isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, maprotiline, mecamylamine, me-
clophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine,
thipendyl, protriptyline, proxjmaetacaine, pseudoephedrine, pjndmethamine, quinidine, qui-
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:MeOH:40 mM ammonium acetate 24:40:36 containing 0.04% triethyl-
amine, pH adjusted to 7.3 with glacial acetic acid
Flow rate: 1.2
Detector: UV 237
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: alprazolam, amitriptyline, desipramine, diltiazem, imipramine, nortriptyline
Noninterfering: clomipramine
REFERENCE
Yeung,P.K.R; Montague/!. J.; Tsui,B.; McGregor,C. High-performance liquid chromatographic assay of diltiazem
and six of its metabolites in plasma: application to a pharmacokinetic study in healthy volunteers,
J.Pharm.Sci., 1989, 78, 592-597.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN: 10 mM pH 2.5 KH2PO4 60:40
Flow rate: 2.5
Detector: E, Environmental Science Associates Coulochem Model 5100A, Model 5100 guard cell
+0.85 V (between pump and injector), Model 5010 analytical cell +0.8 V, preanalytical cell +0.3
V
CHROMATOGRAM
Retention time: 4.6
OTHER SUBSTANCES
Simultaneous: amitriptyline, amoxapine, chlorpromazine, fluphenazine, haloperidol, imipra-
mine, loxapine, mesoridazine, perphenazine, phenylephrine, prochlorperazine, thioridazine,
thiothixene, trazodone, triflupromazine, trimeprazine, tripelennamine
Noninterfering: diazepam, diphenhydramine, ethopropazine, fluoxetine, nordiazepam, oxaze-
pam, phenylpropanolamine, pseudoephedrine, trifluoperazine
Interfering: desipramine, desmethyldoxepin, doxepin, nortriptyline, pheniramine, promazine,
promethazine
REFERENCE
Hariharan,M.; VanNoord,T.; Kindt,E.K; Tandon,R. A simple, sensitive liquid chromatographic assay of cis-
thiothixene in plasma with coulometric detection, Ther.Drug MoniL, 1991,13, 79-85.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Adsorbosphere C18 (PEEK column) (retention times are longer and
Flow rate: 1.2
Detector: UV 214
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: amitriptyline, desipramine, desmethyldoxepin, doxepin, imipramine, maproti-
line, nortriptyline, trazodone
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
pam, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid, me-
meth3rprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, na-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
oxylate, dipyridamole, disopâmide, dobutamine, doxapram, doxepin, droperidol, encainide,
ketorolac, labetalol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, mazindol, mefen-
dine, norepinephrine, nortriptyline, oxazepam, oxycodone, ox5nnetazoline, paroxetine, pemo-
phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenjrtoin, pimo-
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Loxoprofen
Merck Index: 5619
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 JJLL 1 M HCl, extract with 5 mL hexane:ethyl acetate
75:25, wash the organic layer with 1 mL 100 mM HCl. Remove a 4 mL aliquot of the organic
layer and evaporate it to dryness under reduced pressure, reconstitute the residue in 100 |xL
1 mg/mL (lS)-l-(4-dimethylaminonaphthalen-l-yl)ethylamine in dichloromethane:pyridine 10:
1, add 20 JJLL 10 mg/mL 1-hydroxybenzotriazole in dichloromethane:pyridine 10:1, add 100 |JLL
2 mg/mL N,N'-dicyclohexylcarbodiimide, mix, let stand at room temperature for 1 h, evaporate
to dryness, add 500 |xL hexane:ethyl acetate 75:25, add 500 jxL 100 mM HCl, vortex for 1 min.
Remove the organic layer and dry it over anhydrous sodium sulfate, inject a 20 |xL aliquot.
(Synthesis of (lS)-l-(4-dimethylaminonaphthalen-l-yl)ethylamine is as follows. Mix 9.6 g (S)-
(-)-l-(l-naphthyDethylamine ((lS)-l-(naphthalen-l-yl)ethylamine) with 37 mL acetic anhy-
dride, let stand at room temperature for 30 min, cool to 12, add 7.1 mL concentrated nitric
acid dropwise, stir at room temperature for 1 h, adjust pH to 9 with 1 M NaOH, extract with
ethyl acetate. Dry the organic layer over anhydrous sodium sulfate and evaporate to dryness
under reduced pressure, chromatograph on a column of silica gel with ethyl acetate:benzene
10:1 (Caution! Benzene is a carcinogen!). Evaporate the eluate to dryness under reduced pres-
sure to give (lS)-l-(4-nitronaphthalen-l-yl)-N-acetylethylamine as colorless needles. Stir 100
mg 10% PdC and 2.5 g (lS)-l-(4-nitronaphthalen-l-yl)-N-acetylethylamine in 15 mL EtOH, add
1.5 mL hydrazine hydrate in portions (Caution! Hydrazine hydrate is a carcinogen and explodes
on distillation in air!), add 100 mg 10% PdC, reflux for 1 h, cool, filter. Evaporate the filtrate
to dryness under reduced pressure and chromatograph the residue on a column of silica gel
with ethyl acetate, evaporate the eluate to dryness under reduced pressure to give (lS)-l-(4-
aminonaphthalen-l-yl)-N-acetylethylamine as a colorless powder. Stir 1.5 g (lS)-l-(4-aminon-
aphthalen-l-yl)-N-acetylethylamine and 1.7 g sodium bicarbonate in 5 mL water at 0, add 2
mL dimethyl sulfate (Caution! Dimethyl sulfate is highly toxic!), stir at room temperature for
3 h, distil to remove excess reagent. Concentrate the aqueous layer under reduced pressure,
purify the residue by TLC using ethyl acetate (Rf 0.30) to obtain (lS)-l-(4-dimethylaminona-
phthalen-l-yl)-N-acetylethylamine as a colorless powder. Reflux 1.2 g (lS)-l-(4-dimethylami-
nonaphthalen-l-yl)-N-acetylethylamine in 15 mL concentrated HCl for 35 h, adjust the pH to
9 with 2 M NaOH (Caution! Exothermic!), cool, extract with ethyl acetate. Dry the organic
layer over anhydrous sodium sulfate and evaporate it to dryness under reduced pressure, purify
by TLC using ethyl acetate to obtain (lS)-l-(4-dimethylaminonaphthalen-l-yl)ethylamine as a
slightly yellow oil ([a]D +19.75 (c = 2.0, EtOH).)
HPLC VARIABLES
Column: 300 X 3.9 jjiPorasil
Mobile phase: n-Hexane:ethyl acetate 68:32
Flow rate: 1.7
CHROMATOGRAM
OTHER SUBSTANCES
KEY WORDS
derivatization; normal phase; chiral; rat; plasma; pharmacokinetics
REFERENCE
Nagashima,H.; Tanaka,Y.; Watanabe,H.; Hayashi,R.; Kawada,K. Optical inversion of (2R)-to (2S)-isomers of 2-
[4-(2-oxocyclopentylmethyl)phenyl]propionic acid (loxoprofen), a new anti-inflammatory agent, and its mo-
nohydroxy metabolites in the rat, Chem.Pharm.Bull., 1984, 32, 251-257.
SAMPLE
Sample preparation: Dilute urine nine-fold with water. 500 JJLL Plasma or diluted urine + 200
JXL 2 M HCl + 500 |xL 2 |xg/mL 1-naphthoic acid, extract with 6 mL benzene (Caution! Benzene
is a carcinogen!), centrifuge at 900 g for 10 min, repeat extraction. Combine the upper organic
layers and evaporate them to dryness under reduced pressure, reconstitute the residue in 500
JJLL freshly prepared 250 (xg/mL 4-bromomethyl-6,7-methylenedioxycoumarin in MeCN and 500
|xL freshly prepared 1.5 mg/mL 18-crown-6 in MeCN (saturated with finely powdered potas-
sium carbonate), sonicate briefly, heat at 40 for 30 min, add 100 /ULL MeCNracetic acid 90:10,
inject a 5 |xL aliquot. (To hydrolyze glucuronides add 500 |xL 500 mM NaOH to 500 |xL diluted
urine, let stand for 30 min, neutralize with 500 mM HCl. Prepare 4-bromomethyl-6,7-methyl-
enedioxycoumarin as follows. Add 250 mmole finely powdered crystalline citric acid step wise
to 67.5 mL concentrated sulfuric acid at 70 (Caution! Carbon monoxide is evolved!), cool in
ice, add 250 mmoles finely ground sesamol (3,4-methylenedioxyphenol) keeping the tempera-
ture below 5, add 29 mL concentrated sulfuric acid, stir gently overnight, dilute with 50 mL
chilled water, filter. Wash the brown solid with 10 mM sulfuric acid, with 2 M NaOH, and with
2 M HCl. Recrystallize from MeCN to give white prisms, mp 170. Suspend 10 mL of this solid
in 7.5 mL acetic acid, slowly add 7.5 mL acetic acid containing 10 mmoles bromine, reflux for
1 h, cool, chromatograph the solid on a silica gel column, elute with acetone, recrystallize 4-
bromomethyl-6,7-methylenedioxycoumarin from MeOH to give yellow prisms, mp 241
(J.Chromatogr. 1989, 478, 149).)
HPLC VARIABLES
Flow rate: 1.2 (plasma), 1.5 (urine)
Injection volume: 5
CHROMATOGRAM
Internal standard: 1-naphthoic acid (20 (plasma), 14 (urine))
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Naganuma,H.; Kawahara,Y. High-performance liquid chromatographic determination of loxoprofen and its
diastereomeric alcohol metabolites in biological fluids by fluorescence labelling with 4-bromomethyl-6, 7-
methylenedioxycoumarin, J.Chromatogr., 1990, 530, 387-396.
SAMPLE
Matrix: solutions
Sample preparation: Add 500 ng IS, evaporate to dryness, add 100 |xL 200 |xg/mL (+)-(R)-l-(l-
naphthyl)ethylamine in dichloromethane:pyridine 98:2 + 20 |xL 2 mg/mL 1-hydroxybenzotria-
zole in dichloromethaneipyridine 98:2 + 100 |xL 400 |xg/mL N,N'-dicyclohexylcarbodiimide in
dichloromethane:pyridine 98:2, let stand at room temperature for 1 h, evaporate to dryness,
reconstitute in 500 |xL ethyl acetate and 500 |xL HCl, vortex for 1 min. Remove the organic
phase and dry it over anhydrous sodium sulfate, inject a 50 |xL aliquot.
HPLCVARIABLES
Column: 250 X 6 ERC-Silica-1282 (ERMA CR, Saitama, Japan)
Flow rate: 2
CHROMATOGRAM
Retention time: 7 (S), 8.5 (R)
Internal standard: (+)-(S)-2-[2-(2-hydroxyprop-2-yl)indan-5-yl]propionicacid (16)
OTHER SUBSTANCES
KEYWORDS
chiral; derivatization; normal phase; plasma is purified on an immobilized antibody column (full
preparative details given) before analysis
REFERENCE
Takasaki,W.; Tanaka,Y. Application of antibody-mediated extraction for the stereoselective determination of
the active metabolite of loxoprofen in human and rat plasma, Chirality, 1992, 4, 308-315.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xM solution of the compound in MeCN containing 2 mM L-
DBD-APy, 3 mM 2,2'-dipyridyl disulfide, and 3 mM triphenylphosphine, let stand at room
temperature for 2 h, inject a 5 |xL aliquot. (Synthesis of L-DBD-APy, (R)-(-)-4-(3-aminopyrrol-
idin-l-yl)-7-(N,N-dimethylaminosulfonyl)-2,l,3-benzoxadiazole, is as follows. Cool a solution of
16.4 g (S)-(-)-l-benzyl-3-pyrrolidinol in 164 mL pyridine to +5, add 19.35 g p-toluenesulfonyl
chloride, stir at +10 for 48 h, evaporate to dryness, chromatograph using dichloromethane:
acetone 95:5 to obtain (3S)-3-[(4-tolylsulfonyl)oxy]-l-(phenylmethyl)pyrrolidine (mp 68). Heat
a solution of (3S)-3-[(4-tolylsulfonyl)oxy]-l-(phenylmethyl)pyrrolidine in 200 mL anhydrous
DMF to 65, add 33.5 g sodium azide (Caution! Sodium azide is highly toxic!), stir at 60 for 7
h, filter, evaporate the filtrate to dryness under reduced pressure, dissolve the residue in ethyl
acetate, wash twice with water, dry over anhydrous magnesium sulfate, evaporate to obtain
(3R)-3-azido-l-(phenylmethyl)pyrrolidine as an oil. Add 3.5 g 10% palladium on carbon under
nitrogen to a solution of 7.05 g (3R)-3-azido-l-(phenylmethyl)pyrrolidine in 34.8 mL 1 M HCl
in water and 245 mL EtOH, hydrogenate at atmospheric pressure for 30 min, add 3.5 g catalyst,
hydrogenate for 2 h, filter, add 34.8 mL 1 M HCl to the filtrate, evaporate to dryness under
reduced pressure, take up the residue in 70 mL EtOH, filter, evaporate the filtrate to dryness
under reduced pressure, repeat this operation twice, crystallize with the minimum amount of
EtOH to obtain (3R)-3-aminopyrrolidine dihydrochloride (J. Med. Chem. 1992, 35, 4205). 3R-
(+)-aminopyrrolidine is also reported to be available from Tokyo Kasei. Dissolve 0.5 g magne-
sium sulfate heptahydrate and 6 g NaOH in 60 mL water, throughout the reaction keep the
flask at about 20 with cold water cooling, add 15 mL 30% hydrogen peroxide, add 75 mL
MeOH, add 12.1 g powdered benzoyl peroxide in one go, stir for 10 min, pour into 150 mL 20%
sulfuric acid, extract three times with 50 mL portions of chloroform, determine peroxybenzoic
acid concentration by iodometric titration (Tetrahedron 1967, 23, 3327). Slowly add 110 mL 1
M peroxybenzoic acid in chloroform to 7 g 2,6-difluoroaniline dissolved in 100 mL chloroform,
stir at room temperature, when reaction is complete (iodometric titration) wash with 2% so-
dium thiosulfate, wash with 5% sodium carbonate, wash with water, dry over anhydrous so-
dium sulfate, evaporate to dryness under reduced pressure, recrystallize 2,6-difluoronitroso-
benzene form EtOH (mp 108.5-109.5). Stir 8.5 g 2,6-difluoronitrosobenzene in 85 mL DMSO
at room temperature and add a solution of 3.91 g sodium azide in 85 mL DMSO dropwise, let
stand for about 1 h, add to a large volume of water, extract with ether, dry the extracts over
anhydrous sodium sulfate, evaporate to dryness under reduced pressure and distil to give 4-
fluoro-2,l,3-benzoxadiazole as a colorless oil (bp 83712 mm Hg) (J.Chem.Soc.(C) 1970, 1433).
Add 11 mL chlorosulfonic acid dropwise to 3 g 4-fluoro-2,l,3-benzoxadiazole in 10 mL chloro-
form at 0-10 (use a calcium chloride drying tube), stir at room temperature for 1 h, reflux for
2 h, cool, slowly pour into ice water, remove the organic layer, extract the aqueous layer with
chloroform, combine the organic layer, wash, dry over anhydrous magnesium sulfate, evaporate
under reduced pressure, take up the residue in 5 mL benzene (Caution! Benzene is a carcin-
ogen!), chromatograph on a 150 X 30 column of silica gel (100-200 mesh Kanto Chemical) with
n-hexane:benzene 50:50, evaporate the appropriate fractions to give 4-(chlorosulfonyl)-7-nuoro-
2,1,3-benzoxadiazole (CBD-F) as pale yellow needles (mp 64-66) (Anal. Chem. 1984. 56, 2461).
Stir 0.76 g CBD-F in 70 mL MeCN at 0-10 and add 1 g dimethylamine hydrochloride in 10
mL 100 mM pH 10 borax dropwise, adjust pH to 5 with 1 M HCl, concentrate to about 10 mL
under reduced pressure, extract three times with 200 mL portions of diethyl ether, wash with
water, dry over anhydrous magnesium sulfate, evaporate under reduced pressure, chromato-
graph on a 500 X 20 column of silica gel with chloroform, isolate the appropriate fraction and
re-chromatograph on the same column with ethyl acetate:benzene 1:2 to give 4-(N,N-dimethy-
laminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (DBD-F) as white needles (mp 124-125) (yield =
1% !). On a Merck no. 5714 60F254 TLC plate eluted with chloroform DBD-F has Rf 0.32 and
lies between two other reaction products (Analyst 1989,114, 413). It is also reported that DBD-
F can be purchased from Tokyo Kasei. Add 100 mg 4-(N,N-dimethylaminosulfonyl)-7- fluoro-
2,1,3-benzoxadiazole in 20 mL MeCN dropwise to a stirred solution of 200 mg 3R-(+)-amino-
pyrrolidine in 20 mL MeCN at 0-10, stir at room temperature for 30 min, remove the MeCN
by evaporation under reduced pressure, dissolve the residue in 50 mL 5% HCl, wash 3 times
with 50 mL portions of ethyl acetate, adjust the pH of the aqueous solution to 13-14 with 5%
NaOH, extract 6 times with 50 mL portions of ethyl acetate. Combine the organic layers and
wash them with 20 mL water, dry over anhydrous sodium sulfate, evaporate to dryness under
reduced pressure, recrystallize from hexane to obtain (R)-(-)-4-(3-aminopyrrolidin-l-yl)-7-(N,N-
dimethylaminosulfonyl)-2,l,3-benzoxadiazole as orange crystals (mp 96-98).)
HPLC VARIABLES
Flow rate: 1
Injection volume: 5
CHROMATOGRAM
Retention time: k' 12.75 (L), k' 15.42 (D)
KEYWORDS
REFERENCE
Toyo'oka,T.; Ishibashi,M.; Terao,T. Fluorescent chiral derivatization reagents for carboxylic acid enantiomers
in high-performance liquid chromatography, Analyst, 1992, 117, 727-733.
SAMPLE
Matrix: urine
by aspiration of air. Evaporate an aliquot of 100 ^xL 20 |xg/mL IS in MeOH to dryness at 37.
mobile phase, inject a 10-30 u,L aliquot.
HPLC VARIABLES
Flow rate: 0.9
Detector: UV 230
CHROMATOGRAM
Retention time: 5.5
OTHERSUBSTANCES
Extracted: diclofenac, ibuprofen, felbinac, fenbufen, fiurbiprofen, ketoprofen, mefenamic acid,
KEYWORDS
SPE
REFERENCE
Hirai,T.; Matsumoto,S.; KishiJ. Simultaneous analysis of several non-steroidal anti-inflammatory drugs in
J.Chromatogr.B, 1997, 692, 375-388.
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 1 mL 1 M NaOH, mix, let stand at room temperature for
1 h, add 1.5 mL M HCl, add 10 mL mobile phase, shake for 5 min, centrifuge at 1000 g for 5
min. Remove a 1 mL aliquot of the organic layer and evaporate it to dryness under reduced
pressure at 40, reconstitute the residue in 100 |xL 2 mg/mL (lS)-l-(4-dimethylaminonaph-
thalen-l-yl)ethylamine in dichloromethane:pyridine 10:1, add 20 JJLL 10 mg/mL 1-hydroxyben-
zotriazole in dichloromethanerpyridine 10:1, add 100 jxL 2 mg/mL N,N'-dicyclohexylcarbodi-
imide, mix, let stand at room temperature for 1 h, evaporate to dryness, add 500 |xL mobile
phase, add 500 |xL 100 mM HCl, vortex for 1 min. Remove the organic layer and dry it over
anhydrous sodium sulfate, inject a 20 |xL aliquot. (Synthesis of (lS)-l-(4- dimethylamino-
naphthalen-l-yl)ethylamine is as follows. Mix 9.6 g (S)-(-)-l-(l-naphthyl)ethylamine ((IS)-I-
(naphthalen-l-yl)ethylamine) with 37 mL acetic anhydride, let stand at room temperature for
30 min, cool to 12, add 7.1 mL concentrated nitric acid dropwise, stir at room temperature for
1 h, adjust pH to 9 with 1 M NaOH, extract with ethyl acetate. Dry the organic layer over
anhydrous sodium sulfate and evaporate to dryness under reduced pressure, chromatograph
on a column of silica gel with ethyl acetate:benzene 10:1 (Caution! Benzene is a carcinogen!).
Evaporate the eluate to dryness under reduced pressure to give (lS)-l-(4-nitro-naphthalen-l-
yl)-N-acetylethylamine as colorless needles. Stir 100 mg 10% PdC and 2.5 g (lS)-l-(4-nitro-
naphthalen-l-yl)-N-acetylethylamine in 15 mL EtOH, add 1.5 mL hydrazine hydrate in por-
tions (Caution! Hydrazine hydrate is a carcinogen and explodes on distillation in air!), add 100
mg 10% PdC, reflux for 1 h, cool, filter. Evaporate the filtrate to dryness under reduced pressure
and chromatograph the residue on a column of silica gel with ethyl acetate, evaporate the
eluate to dryness under reduced pressure to give (lS)-l-(4-aminonaphthalen-l-yl)-N-acetyl-
ethylamine as a colorless powder. Stir 1.5 g (lS)-l-(4-aminonaphthalen-l-yl)-N-acetylethylam-
ine and 1.7 g sodium bicarbonate in 5 mL water at 0, add 2 mL dimethyl sulfate (Caution!
Dimethyl sulfate is highly toxic!), stir at room temperature for 3 h, distil to remove excess
reagent. Concentrate the aqueous layer under reduced pressure, purify the residue by TLC
using ethyl acetate (Rf 0.30) to obtain (lS)-l-(4-dimethylaminonaphthalen-l-yl)-N-acetylethy-
lamine as a colorless powder. Reflux 1.2 g (lS)-l-(4-dimethylaminonaphthalen-l-yl)-N-acety-
lethylamine in 15 mL concentrated HCl for 35 h, adjust the pH to 9 with 2 M NaOH (Caution!
Exothermic!), cool, extract with ethyl acetate. Dry the organic layer over anhydrous sodium
sulfate and evaporate it to dryness under reduced pressure, purify by TLC using ethyl acetate
to obtain (lS)-l-(4-dimethylaminonaphthalen-l-yl)ethylamine as a slightly yellow oil ([a]D
+19.75 (c = 2.0, EtOH) (Chem. Pharm. Bull. 1984, 32, 251).)
HPLC VARIABLES
Column: 300 X 3.9 10 |xm jxPorasil
Flow rate: 1.7
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
derivatization; normal phase; chiral
REFERENCE
Nagashima,H.; Tanaka,Y.; Hayashi,R. Column liquid chromatography for the simultaneous determination of
the enantiomers of loxoprofen sodium and its metabolites in human urine, J.Chromatogr., 1985,345, 373
379.
Lufenuron
Molecular formula: Ci7H8CI2F8N2O3
Merck Index: 6524
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 209.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Lypressin
Molecular formula: C46H65N13Oi2S2
Merck Index: 5661
SAMPLE
Sample preparation: Condition a Sep-Pak ODS SPE cartridge with MeOH. Homogenize 500
mg tissue with 6 mL 100 mM pH 7.4 Tris buffer. Acidify a 2 mL aliquot of plasma or tissue
homogenate with 200 |xL 1 M HCl, add to the SPE cartridge, elute with 3 mL MeOH over 3
min, elute with 2 mL over 1 min. Evaporate the eluate to dryness under a stream of air at
37, reconstitute the residue in 200 |xL mobile phase, inject a 20 (JLL aliquot.
HPLCVARIABLES
Column: 280 X 5 Dynamax 300-A C8 (Rainin)
Mobile phase: MeCN:water 20:80 containing 0.1% trifluoroacetic acid, 50 mM heptanesulfonic
acid and 30 mM triethylamine, pH adjusted to 2.5 with Na2HPO4
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: arginine vasopressin, oxytocin
KEYWORDS
pig; plasma; SPE; heart
REFERENCE
Rao,P.S.; Weinstein,G.S.; Wilson,D.W.; Rujikarn,N.; Tyras,D.H. Isocratic high-performance liquid chromatog-
raphy-photodiode-array detection method for determination of lysine- and arginine-vasopressins and oxy-
tocin in biological samples, J.Chromatogr., 1991, 536, 137-142.
SAMPLE
HPLC VARIABLES
Column: 250 X 3 10 |xm RP 8 (Merck)
Mobile phase: MeCN:pH 7 phosphate buffer 15:80
Flow rate: 1.58
|xg/mL fluorescamine in MeCN pumped at 0.16 mL/min and the mixture flowed through a 4.4
m X 0.25 mm ID coil to the detector.
CHROMATOGRAM
Retention time: 8
KEYWORDS
post-column reaction; injections
REFERENCE
Frei,R.W.; Michel,L.; Santi,W. Post-column fluorescence derivatization of peptides. Problems and potential in
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Nucleosil 5 C18
Flow rate: 1.2
Detector: UV 206
CHROMATOGRAM
Retention time: 18
KEYWORDS
tritium labelled
REFERENCE
Janaky,T.; Laczi,R; Laszl6,F.A. Biological half-lives and organ distribution of tritiated 8-lysine-vasopressin and
l-deamino-8-D-arginine-vasopressinin Brattleboro rats, Ann.N.Y.Acad.ScL, 1982, 394, 116127.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 125 X 5 Nova-Pak C18
Mobile phase: Gradient. MeCN:10 mM pH 5.0 acetate buffer 15:85 for 10 min, then to 18:82
over 5 min, maintain at 18:82 for 25 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 8.7
OTHER SUBSTANCES
Simultaneous: oxytocin, arginine vasopressin
KEYWORDS
hippopotamus
REFERENCE
Rouille,Y.; Chauvet,M.T.; Chauvet,J.; Acher,R.; Hadley,M.E. The distribution of lysine vasopressin (lysipressin)
in placental mammals: a reinvestigation of the Hippopotamidae (Hippopotamus amphibius) and Tayassui-
dae {Tayassu angulatus) families, Gen.Comp.Endocrinol., 1988, 71, 475-483.
Lysozyme
Molecular weight: about 14400
Merck Index: 5671
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 2 mg/mL solution in mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 7 jim PoIyCAT A WCX polyaspartate (Custom LC, Houston)
Mobile phase: 40 mM calcium acetate buffer containing 8 M urea, pH 5.0
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 19
REFERENCE
Parente,E.S.; Wetlaufer,D.B. Influence of urea on the high-performance cation-exchange chromatography of
hen egg white lysozyme, J.Chromatogr., 1984, 288, 389-398.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 50 X 4.6 C4 wide pore (300 A) (Supelco)
Mobile phase: Gradient. A was MeOH:isopropanol:water:trifluoroacetic acid 8:2:90:0.05 contain-
ing 2.8 g/L NaCl. B was MeOH:isopropanol:water:trifluoroacetic acid 70:10:20:0.05 containing
4.2 g/L NaCl. A:B from 95:5 to 0:100
Flow rate: 1.5
Detector: E, Bioanalytical systems Model LC-4B, dual glassy-carbon working electrode used in
parallel mode, +0.65 V and +0.80 V (monitored), stainless steel auxiliary electrode, Ag/AgCl
reference electrode following post-column reaction. The column effluent flowed at 0-5 through
a 2 mL knitted coil of 0.5 mm i.d. PTFE tubing irradiated with a low pressure mercury lamp
(Photronix Model 816) to the detector.
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
Simultaneous: b-lactoglobulin A, ribonuclease A
KEYWORDS
REFERENCE
Dou,L.; KrullJ.S. Determination of aromatic and sulfur-containing amino acids, peptides, and proteins using
high-performance liquid chromatography with photolytic electrochemical detection, Anal. Chem., 1990, 62,
2599-2606.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a solution in 50 mM pH 7.5 phosphate buffer.
HPLC VARIABLES
Column: 300 X 7.5 TSKgel-G3000SW (Tosoh)
Mobile phase: 100 mM pH 7.5 Phosphate buffer containing 100 mM sodium sulfate
Flow rate: 0.8
oxidant pumped at 0.2 mL/min and this mixture flowed through a 3 m X 0.5 mm ID PTFE
coil at 70. The effluent from this coil mixed with the reagent pumped at 0.2 mL/min and this
mixture flowed through a 5 m X 0.5 mm ID PTFE coil at 70 and a 1 m X 0.5 mm ID PTFE
cooling coil to the detector. (Prepare oxidant by diluting 10% sodium hypochlorite solution with
50 mM NaH2PO4 containing 0.1% Brij-35 and 50 mM Na2HPO4 containing 0.1% Brij-35 to a
final chlorine concentration of 0.8% and final pH of 7.5 . Prepare reagent by dissolving 8 g
sodium nitrite and 40 mg thiamine hydrochloride in 100 mL 50 mM pH 7.5 phosphate buffer,
adjust to pH 7.5 with 50 mM Na2HPO4 or 50 mM NaH2PO4, make up to 200 mL with 50 mM
pH 7.5 phosphate buffer. The chlorinated proteins oxidize thiamine to fluorescent thiochrome.)
CHROMATOGRAM
Retention time: 16
OTHER SUBSTANCES
Simultaneous: bovine serum albumin, cytochrome c, myoglobin, ovalbumin, thyroglobulin
KEYWORDS
REFERENCE
Yokoyama,T.; Kinoshita,T. High-performance liquid chromatographic determination of proteins by post-column
fluorescence derivatization with thiamine reagent, J.Chromatogr., 1990, 518, 141-148.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.1 10 |xm PRP-3 (Hamilton)
Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in 50 mM NaOH. B was 0.1% trifluo-
roacetic acid in MeCN. A:B from 100:0 to 40:60 over 30 min.
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
Retention time: 20
OTHER SUBSTANCES
Simultaneous: cytochrome C, insulin, myoglobin, ribonuclease A, trypsin
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 PLRP-S IOOOA (Polymer Labs)
in 95% MeCN. A:B from 80:20 to 40:60 over 22 min.
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Simultaneous: bovine serum albumin, cytochrome C, insulin, myoglobin, ovalbumin, ribonu-
clease
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.5 5 |xm Kromasil C8 (Eka-Nobel)
Mobile phase: Gradient. A was MeCN:water 10:90 containing 0.1% trifluoroacetic acid. B was
MeCN:water 90:10 containing 0.1% trifluoroacetic acid. A:B from 0:100 to 75:25 over 8 min, to
25:75 over 12 min.
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 12
OTHERSUBSTANCES
Simultaneous: angiotensin I, angiotensin II, bradykinin, insulin, leucin enkephalin, melittin,
methionine enkephalin, oxytocin
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 SynChropak C4 (SynChrom)
in MeOH. A:B from 95:5 to 0:100 over 30 min.
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 22
OTHER SUBSTANCES
Simultaneous: conalbumin, cytochrome C, p-lactoglobulin, ovalbumin
REFERENCE
Scientific Products Chromatography Catalog, Baxter Diagnostics, 1992, p. 187.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm 300 A Rexchrom ODS (Regis)
in MeCN. A:B from 75:25 to 0:100 over 30 min.
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: insulin, myoglobin, ovalbumin, ribonuclease
REFERENCE
Regis Technologies, Inc. Catalog, 1993, p. 29.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Vydac 228TP
Mobile phase: Gradient. A was 0.25% trifluoroacetic acid in water. B was 0.25% trifluoroacetic
acid in MeCN:water 70:30. A:B from 95:5 to 0:100 over 30 min.
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Retention time: 17
OTHER SUBSTANCES
Simultaneous: angiotensin I, angiotensin II, bradykinin, eledosin, insulin, myoglobin, neuroten-
sin, ovalbumin, oxytocin
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Spherisorb 300A C18
Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in water. B MeCN. A:B from 80:20 to
20:80 over 30 min.
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: insulin, myoglobin, ovalbumin, ribonuclease
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 75 X 4.6 5 jjim Hypersil WP 300-octyl
Mobile phase: Gradient. A was 0.1% trifluoroacetic in water. B was 0.1% trifluoroacetic acid in
n-propanol. A.B 100:0 for 2.5 min, to 90:10 over 2.5 min, to 10:90 over 112 min
Flow rate: 1
Detector: UV 225
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: angiotensin II, bombesin, bovine growth hormone, bovine serum albumin, cata-
lase, L-tryptophan
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.5 Kromasil-5-C8 (Eka Nobel)
Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in MeCN:water 10:90. B was 0.1% tri-
fluoroacetic acid in MeCN:water 90:10. A:B from 100:0 to 75:25 over 8 min, to 25:75 over 12
min
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Simultaneous: angiotensin I, angiotensin II, bradykinin, insulin, leukin enkephalin, melittin,
methionine enkephalin, oxytocin
REFERENCE
Bodman Chromatography Catalog, 1994, p. 62.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 5 400VHP552 (Vydac)
Mobile phase: Gradient. A was pH 7.21 Tris-HCl. B was pH 7.21 Tris HCl containing 500 mM
NaCl. A:B from 100:0 to 0:100 over 5 min
Flow rate: 4
CHROMATOGRAM
Retention time: 2.4
OTHER SUBSTANCES
Simultaneous: a-chymotrypsinogen A, conalbumin, cytochrome, myoglobin
REFERENCE
Bodman Chromatography Catalog, 1994, p. 193.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 jxL aliquot of a 5 mg/mL solution.
HPLC VARIABLES
Column: 250 X 4.6 Dynamax-300A C8 (Rainin)
in MeCN. A:B from 85:15 to 45:55 over 30 min
Flow rate: 0.75
Detector: UV 230
CHROMATOGRAM
Retention time: 25
OTHER SUBSTANCES
Simultaneous: insulin, ribonuclease A, bovine serum albumin, ovalbumin
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 50 X 4.6 (3-cyclodextrin sulfate immobilized on TSKgel AF-Epoxy TOYOPEARL 650M
(Tosoh) (preparation details in paper)
Mobile phase: Gradient. A was 20 mM Tris-HCl buffer containing 60 mM NaCl. B was 20 mM
Tris-HCl buffer containing 3 M NaCl. A:B 100:0 for 10 min, to 0:100 over 5 min, maintain at
0:100 for 5 min.
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Simultaneous: bovine serum albumin, basic fibroblast growth factor
REFERENCE
Ishimura,K.; Fukunaga,K.; Ohta,T.; Nakamura,H.; Irie,T.; Uekama,K. Preparation of p-cyclodextrin sulfate-
immobilized hydrophilic vinyl-polymer gel as a selective, high recovery and stable adsorbent for high-per-
formance affinity chromatography of heparin-binding substances, Chromatographia, 1995, 41, 349-352.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 jxm Zorbax 300 A SB-C3
Mobile phase: Gradient. A was MeCN:water:trifluoroacetic acid 5:95:0.1. B was MeCN.water:
trifluoroacetic acid 5:95:0.085. A:B from 85:15 to 47:53 over 20 min.
Flow rate: 1
Detector: UV 215
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Simultaneous: angiotensin II, carbonic anhydrase, cytochrome C, insulin, leucine enkephalin,
myoglobin, RNAase
REFERENCE
Ricker,R.D.; Sandoval,L.A.; Permar,B.J.; Boyes,B.E. Improved reversed-phase high performance liquid chro-
matography columns for biopharmaceutical analysis, J.Pharm.Biomed.Anal., 1996, 14, 93-105.
Mabuterol
Merck Index: 5674
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 250 mM NaOH + 100 |xL 50 ng/mL IS in water + 5
mL diethyl ether:2-butanol 90:10, shake for 10 min, centrifuge at 1500 g for 10 min. Remove
the residue in 100 |xL mobile phase, vortex for 2 min, store at 4 overnight, warm tyo room
temperature, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 4 x 4 7 jxm LiChrosorb (select B) C18
Column: 125 X 4 7 jxm LiChrosorb (select B) C18
Mobile phase: MeCN:buffer 23:77 containing 0.2 mM sodium 1-heptanesulfonate (Buffer was
pH 4.0-4.1 phosphate buffer, ionic strength 0.1.)
Detector: E, ESA Coulochem Model 5100A, Model 5011 detector, +0.75 V
CHROMATOGRAM
Internal standard: 4-amino-3,5-dichloro-a-[[( 1,l-dimethylpropyl)amino]methyl]benzenemetha-
nol (NAB 760) (10)
OTHER SUBSTANCES
Extracted: clenbuterol
KEYWORDS
horse; plasma
REFERENCE
Qureshi,G.A.; Eriksson,A. Determination of clenbuterol and mabuterol in equine plasma by ion-pair liquid
chromatography with electrochemical detection. Chromatographic and electrochemical characteristics,
J.Chromatogr., 1988, 441, 197-205.
Maduramicin
Molecular formula: C 47 H 83 NOi 7
Merck Index: 5682
SAMPLE
Matrix: bulk, feed, premix
Sample preparation: Bulk, premix. Dissolve in MeCN to give a maduramicin concentration of
3 |xg/mL (sonicate premix). Remove a 1 mL aliquot and mix it with 30 3 mg calcium car-
bonate, 100 |JLL 7.5 mg/mL dansyl hydrazine in MeCN, and 100 JJLL 150 mg/mL trichloroacetic
acid in MeCN, shake vigorously by hand for 1 min, centrifuge for 1 min, immediately inject an
aliquot. Feed. Add 50 g feed to 250 mL MeCN, shake for 30 min, clarify. Remove a 2 mL aliquot
and add it to 600 |xL 7.5 mg/mL dansyl hydrazine in MeCN and 600 fxL 150 mg/mL trichlo-
roacetic acid in MeCN, shake by hand for 10 s, add to a Florisil Sep-Pak at 1 drop/s, wash with
three 5 mL aliquots of MeCN at 1 drop/s, purge with air, elute with MeCN:water 90:10, collect
5 mL eluate, mix eluate, inject an aliquot.
HPLC VARIABLES
Column: 50 X 4.6 5 jjum C18 (IBM)
Mobile phase: MeCN:water 75:25 containing 250 mg/L tetrabutylammonium hydrogen sulfate,
pH3.5
Flow rate: 2
CHROMATOGRAM
Retention time: 5.8, 7.7 (two possible derivatives are formed)
Limit of quantitation: 3 ppm
KEYWORDS
derivatization; SPE
REFERENCE
Markantonatos,A- Derivatization and HPLC/fluorescence quantitation of maduramicin ammonium in feed and
premixes at levels down to 5 ppm, J.Liq.Chromatogr., 1988,11, 877-890.
Mafenide
CAS Registry No.: 138-39-6, 130099-9-9 (acetate), 138-37-4 (HCI)
Merck Index: 5683
Lednicer No.: 2 114
SAMPLE
Sample preparation: Shake 25-30 mg cream with 25 mL mobile phase at 40 for 30 min, make
up to 50 mL with mobile phase, filter (0.45 fxm Nylon), inject an aliquot of the filtrate.
HPLC VARIABLES
Column: 250 X 4.6 Spherisorb C18
Mobile phase: MeOH:buffer 65:35 containing 0.44 g/L sodium 1-heptanesulfonate, pH 4.2 (Buffer
was 992 mL 9.073 g/L KH2PO4 and 8 mL 9.48 g/L Na2HPO4, pH 5.0.)
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 2.6
Internal standard: lidocaine (5.3)
Limit of quantitation: 1.15 fxg/mL
KEYWORDS
cream
REFERENCE
Dash,A.K.; Harrison,J.S. Ion-pair chromatographic method for the analysis of mafenide acetate,
J.ChromatogrA, 1995, 705, 83-88.
Malachite green
CAS Registry No.: 569-64-2 (chloride)
Merck Index: 5739
SAMPLE
Sample preparation: Plasma. 200 |xL Plasma + 1 mL MeCN, vortex for 20 s, centrifuge for 5
min. Add the supernatant to 2 mL MeOH, evaporate to dryness, reconstitute with 500 JULL
diluting solvent with sonication, centrifuge for 5 min, filter (0.2 |xm), inject a 50 |xL aliquot of
the nitrate. Muscle. Condition a 6 mL Bakerbond neutral alumina SPE cartridge with 1 column
volume MeCN and a 3 mL Bond Elut LRC propylsulfonic acid (PRS) SPE cartridge with 1
column volume MeCN, place the alumina cartridge on top of the PRS cartridge. 5 g Muscle +
1.5 mL 250 mg/mL hydroxylamine hydrochloride in water + 2.5 mL 1 M p-toluenesulfonic acid
in water + 5 mL buffer, mix, homogenize (Tekmar SDT Tissuemizer) at medium speed for 30
s, remove contents of container, add 45 mL MeCN to container and shake vigorously for 30 s,
add 1Og alumina (activated 80-200 mesh, Alcoa type F-20) to the container and shake vigor-
ously for 30 s. Combine all these mixtures and centrifuge at 4 at 2700 g for 10 min, remove
the supernatant, add 45 mL MeCN to the pellet, shake vigorously for 30 s. Combine the su-
pernatants and add them to 100 mL water and 2 mL diethylene glycol, add 50 mL dichloro-
methane, shake vigorously for 30 s, allow to separate for 15 min, repeat extraction. Combine
the organic layers and evaporate to about 10 mL under reduced pressure at 40, add 15 mL
dichloromethane and evaporate to about 10 mL, add 10 mL MeCN and evaporate to about 1
mL, add 2 mL dichloromethane, mix, add 5 mL MeCN, mix, add to the SPE cartridges, rinse
container 3 times with MeCN and add the rinses to the SPE cartridges, discard the alumina
cartridge. Elute the PRS cartridge with two 1 mL portions of mobile phase then 1 mL 2.5 mg/
mL hydroxylamine hydrochloride in MeOH, make up the eluate to 3 mL with mobile phase (if
necessary), mix, inject a 50 (JLL aliquot. (Diluting solvent was MeCNrbuffer 60:40 containing
700 JJLL/L 250 mg/mL hydroxylamine hydrochloride in water. Prepare buffer by dissolving 8.2
g sodium acetate trihydrate and 0.95 g p-toluenesulfonic acid monohydrate in 900 mL water,
adjust pH to 4.5 with glacial acetic acid, make up to 1 L with water.)
HPLC VARIABLES
Column: 250 X 4.6 5 fjim Ultremex 5 CN
Mobile phase: MeCNrbuffer 50:50 (Prepare buffer by dissolving 8.2 g sodium acetate trihydrate
and 0.95 g p-toluenesulfonic acid monohydrate in 900 mL water, adjust pH to 4.5 with glacial
acetic acid, make up to 1 L with water.)
Flow rate: 1
Detector: UV 618 following post-column reaction. The column effluent passed through a column
(Alltech direct-connect rfefillable guard column) packed with lead(IV) oxide:Celite 545-AW (25:
75) to the detector.
CHROMATOGRAM
Retention time: 16.2 (malachite green), 6.3 (leucomalachite green)
Limit of detection: 2 ppb (muscle), 10 ppb (plasma)
OTHER SUBSTANCES
Extracted: metabolites, leucomalachite green
KEYWORDS
plasma; muscle; fish; catfish; SPE; post-column reaction
REFERENCE
Plakas,S.M.; El Said,KR.; Stehly,G.R.; Roybal,J.E. Optimization of a liquid chromatographic method for de-
termination of malachite green and its metabolites in fish tissues, J.AOAC Int., 1995, 78, 1388-1393.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 6 mL neutral alumina SPE cartridge (J.T. Baker) and a 2.8
mL 500 mg Bond Elut PRS SPE cartridge with 5 mL MeCN. Place the alumina SPE cartridge
on the top of the PRS SPE cartridge using a Bond Elut adapter. Mix 3 mL 250 mg/mL hy-
droxylamine hydrochloride in water with 5 mL 50 mM p-toluene sulfonic acid, 20 mL 100 mM
ammonium acetate adjusted to pH 4.5 with glacial acetic acid, and 20 g fish tissue. Homogenize
at 20000 rpm for 1 min (Ultra-Turrax T25 Tissuemizer, Tekmar, USA). Add 90 mL MeCN to
the sample and homogenize for 10 s. Shake vigorously by hand for 1 min. Add 20 g basic
alumina (Brockman activity I), shake for 1 min. Centrifuge, decant the supernatant. Add 30
mL MeCN to the residue, extract, combine the supernatants. Add 100 mL water, 50 mL dich-
loromethane, and 20 mL diethylene glycol to the supernatants, shake vigorously, separate the
bottom layer. Again add 50 mL dichloromethane, shake for 1 min, combine the separated di-
chloromethane layers. Concentrate to 2-3 mL under reduced pressure at 65. Add 2 mL di-
chloromethane and 5 mL MeCN then add the mixtures to the SPE cartridges. Rinse the flask
twice with 5 mL portions of MeCN and add the rinses to the SPE cartridges. Wash with 5 mL
MeCN to waste. Remove the alumina cartridge. Wash the PRS cartridge with 2 mL water and
with 1 mL MeCNrIOO mM ammonium acetate buffer 50:50 adjusted to pH 4.5 with glacial
acetic acid. Elute with 2 mL MeCNrIOO mM ammonium acetate buffer 50:50 adjusted to pH
4.5 with glacial acetic acid and collect in a tube containing 500 jxL 2.5 mg/mL hydroxylamine
hydrochloride in water. Inject a 100 |xL aliquot.
HPLCVARIABLES
Guard column: 20 X 2.0 pellicular C18
Column: 150 X 4.6 5 |xm SynChropak SCD-100 (SynChrom, USA)
Mobile phase: MeCN:buffer 55:45 (Buffer was 400 mg ammonium acetate and 1 mL triethylam-
ine in 400 mL water, adjusted to pH 3.0 with glacial acetic acid, and made up to 450 mL with
water)
Flow rate: 2.0
Detector: E, ESA Coulochem Model, oxidative electrochemical cell (EC) +900 mV; UV 588; F ex
265 em 360
CHROMATOGRAM
Retention time: 5.2
OTHER SUBSTANCES
Extracted: metabolites, gentian violet
KEYWORDS
SPE; catfish; trout,
REFERENCE
Rushing,L.G.; Hansen,E.B.,Jr. Confirmation of malachite green, gentian violet and their leuco analogs in catfish
and trout tissue by high-performance liquid chromatography utilizing electrochemistry with ultraviolet-
visible diode array detection and fluorescence detection, J.Chromatogr.B, 1997, 700, 223-231.
Malotilate
Molecular formula: Ci2H16O4S2
Merck Index: 5751
Lednicer No.: 5 75
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 1 M pH 5.0 acetate buffer + 1 mL water + 5 mL
200 ng/mL IS in chloroform; shake horizontally for 10 min, centrifuge. Remove 4 mL of the
organic layer and evaporate it to dryness, reconstitute the residue in 100 |xL MeOH, inject a
10 JLJLL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 360
CHROMATOGRAM
Retention time: 8
Internal standard: di-sec-butyl l,3-dithiol-2-ylidenemalonate (15)
OTHER SUBSTANCES
KEYWORDS
plasma; dog
REFERENCE
Takasugi,N.; Mafune,E.; Yokokawa,S.; Toriyama,K.; Tsuchiya,K.; Sugimoto,T. Determination of malotilate and
its metabolites in plasma and urine, J.Chromatogr., 1985, 342, 349358.
Manidipine
Merck Index: 5786
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 1 mL 20 mM pH 10 Na2HPO4, extract twice with 5 rtiL n-
hexane:diethyl ether 2:1. Combine the organic layers and evaporate them to dryness under a
stream of nitrogen, reconstitute the residue in 200 jxL mobile phase A, inject a 150 |xL aliquot
into column A and elute to waste with mobile phase A. Divert the eluate from column A con-
taining manidipine onto column B. When all the manidipine has passed onto column B elute
column B with mobile phase B and monitor the effluent from column B. (Elute column A with
mobile phase A to waste.)
HPLC VARIABLES
Column: A 100 X 2.1 3 |xm Develosil ODS-3K; B 100 X 2.1 5 |xm Develosil ODS-5K
Mobile phase: A MeCN:20 mM KH2PO4 46:54 containing 5 mM sodium nonanesulfonate, ad-
justed to pH 3.0 with 85% orthophosphoric acid; B MeCN:20 mM KH2PO4 46:54 adjusted to
pH 3.0 with 85% orthophosphoric acid
Flow rate: 0.3
Detector: UV 230
CHROMATOGRAM
Retention time: 20
OTHER SUBSTANCES
KEYWORDS
serum; protect from light; column-switching; heart-cut; pharmacokinetics
REFERENCE
Miyabayashi,T.; Yamashita,K; Aoki,L; Motohashi,M.; Yashiki,T.; Yatani,K. Determination of manidipine and
its pyridine metabolite in human serum by high-performance liquid chromatography with ultraviolet de-
tection and column switching, J.Chromatogr., 1989, 494, 209-217.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 1 mL 20 mM pH 10 Na2HPO4, extract twice with 5 mL n-
hexane:diethyl ether 2:1. Combine the organic layers and evaporate them to dryness under a
stream of nitrogen, reconstitute the residue in 200 |xL EtOH, inject a 150 |xL aliquot. Collect
the eluate for each enantiomer, evaporate to dryness under a stream of nitrogen, reconstitute
in 200 |xL mobile phase A, inject a 150 |xL aliquot, and analyze further as in J.Chromatogr.
1989, 494, 209. This resolves the enantiomers from interfering endogenous serum peaks.
HPLC VARIABLES
Mobile phase: n-Hexane:EtOH:MeOH 80:15:5
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 10 (R-(-)), 14 (S-(+))
KEYWORDS
serum; protect from light; chiral
REFERENCE
Yamaguchi,M.; Yamashita,K; Aoki,L; Tabata,T.; Hirai,S.-L; Yashiki,T. Determination of manidipine enantio-
mers in human serum using chiral chromatography and column-switching liquid chromatography,
J.Chromatogr., 1992, 575, 123-129.
Mannitol
Merck Index: 5788
SAMPLE
Matrix: beverages, juice, milk
Sample preparation: Orange juice. Dilute orange juice 100-fold with water, filter (Millipore HV,
0.45 (xm), dilute filtrate 10-fold, inject an aliquot. Beverages. Dilute soft drinks 1000-fold with
water, inject an aliquot. Milk. Dilute 5 mL milk to 100 mL with mobile phase, filter (Millipore
HV, 0.45 |xm), dilute nitrate 50-fold, inject an aliquot.
HPLC VARIABLES
Guard column: 30 X 4.6 Cation H (Bio-Rad)
Column: 300 X 3.8 9 |xm HPX 87-H Aminex (Bio-Rad)
Mobile phase: 10 mM Sulfuric acid
Flow rate: 0.5
Detector: E following post-column reaction, Hewlett-Packard 1049A programmable electrochem-
ical detector, Metrohm detector cell, cuprous oxide working electrode +550 mV, glassy carbon
auxiliary electrode, Ag/AgCl (3 M KCl) reference electrode. The column effluent mixed with
200 mM NaOH pumped at 0.4 mL/min, the mixture flowed through a 220 X 0.8 single-bead
string reactor packed with 0.6 mm glass beads to the detector. (Prepare cuprous oxide electrode
as follows. Stir 300 mg conductive carbon cement (Gerhard Neubauer, Miinster), 60 mg cuprous
oxide (Fluka), and 300 |JLL acetone until a thick paste forms as the acetone evaporates. Pack
conductive carbon cement into the base of a 3 mm diameter cavity carbon paste electrode base
(Metrohm), allow to dry, polish with dry emery paper (grade 2/0, Oakey), remove surface layer
with an acetone-soaked tissue, pack the paste into the cavity, allow to dry overnight, polish
with dry emery paper (grade 2/0), 3 |xm imperial micro finishing film sheet (3M), 0.3 |xm
imperial micro finishing film sheet (3M), and 0.05 |xm alumina particles on a Buehler pad,
sonicate for 2 min in water (Anal. Chim. Acta 1995, 300, 5).)
CHROMATOGRAM
Limit of detection: 0.8 (xM
OTHER SUBSTANCES
Also analyzed: arabinose, cellobiose, dextrose, fructose, fucose, galactitol, galactose, galacturonic
acid, lactose, lactulose, lyxose, maltose, mannose, myo-inositol, raffinose, rhamnose, ribose,
sorbose, sucrose, xylose
KEYWORDS
orange juice; soft drinks; post-column reaction; fruit
REFERENCE
Huang,X.; Pot,J.J.; Kok,W.T. Determination of sugars by liquid chromatography and amperometric detection
with a cuprous oxide modified electrode, Chromatographia, 1995, 40, 684-689.
SAMPLE
Matrix: blood
Sample preparation: 500 jxL Serum + 25 |xg adonitol + 1 mL EtOH, centrifuge at 1000 g for
10 min. Remove the organic layer and evaporate it to 500 |xL under a stream of nitrogen. Apply
the residue to a Bond Elut SCX SPE cartridge (100 mg/mL), elute with 2 mL water. Apply the
eluate to a Bond Elut SAX SPE cartridge (100 mg/mL), elute with 2 mL water. Freeze-dry the
eluate, reconstitute in 100 |JLL MeOH:water 50:50, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 6 10 jxm Gelpack GL-C64Z sulfonated styrene-divinylbenzene copolymer with
zinc ions (Hitachi-Kasei)
Flow rate: 1
Detector: MS, Hitachi M-1000S quadrupole, negative-ion APCI, drift voltage -25 V, vaporizer
250, desolvator 399, m/z 217, [M + Cl]", SIM The column effluent was mixed with MeOH:
chloroform 99:1 pumped at 0.5 mL/min, the combined effluent then flowed to the MS detector.
CHROMATOGRAM
Internal standard: adonitol (m/z 187) (9)
OTHER SUBSTANCES
Extracted: 1,5-anhydroglucitol, arabinose, arabitol, dextrose, erythritol, fructose, galactose,-
myoinositol, sorbitol, xylitol
KEYWORDS
serum; SPE
REFERENCE
Niwa,T.; Tohyama,K; Kato,Y. Analysis of polyols in uremic serum by liquid chromatography combined with
atmospheric pressure chemical ionization mass spectrometry, J.Chromatogr., 1993, 613, 9-14.
SAMPLE
Matrix: blood
Sample preparation: 70 jxL Serum + 10 |xL 1 mM D-glucosamine.HCl + 20 |xL 1 M K2HPO4 +
10 |JLL benzoyl chloride + 25 |xL 8 M NaOH, vortex at 2500 vibrations/min for 5 min, add 10
IxL 1.4 M phosphoric acid and 100 |xL ethyl acetate, vortex at 2500 vibrations/min for 1 min.
Remove 25 fxL of the ethyl acetate phase and add it to 100 |xL MeCN:water 70:30, inject an
aliquot.
HPLC VARIABLES
Guard column: 5 jxm Kromasil 100 C18
Column: 250 X 4 5 ^m Kromasil 100 C18
Mobile phase: Gradient. MeCNrwater from 70:30 to 95:5 over 30 min.
Flow rate: 1
Detector: UV 228 or MS, electrospray, Finnigan MAT, TSQ 700, flow rate 1 |xL/min, 2.8 kV,
drying gas 140
CHROMATOGRAM
Retention time: 11.3, 11.5 (pentabenzoyl), 18.7 (hexabenzoyl)
Internal standard: D-glucosamine (9.7)
Limit of detection: 1-5 pmol
OTHER SUBSTANCES
Extracted: benzyl alcohol, adenosine, dextrose, 2-desoxy-D-glucose, cytidine, myoinositol,
sucrose
KEYWORDS
serum; derivatization; fetal bovine serum
REFERENCE
Oehlke,J.; Brudel,M.; Blasig,I.E. Benzoylation of sugars, polyols and amino acids in biological fluids for high-
performance liquid chromatographic analysis, J.Chromatogr.B, 1994, 655, 105111.
SAMPLE
Matrix: blood, erythrocytes, tissue
Sample preparation: Homogenize lens tissue in 4 (human) or 1 (rat) mL 2 mg/mL sodium
fluoride. Dilute 600 |xL frozen and thawed erythrocytes with 400 |xL water. Filter (Amicon
Centrifree) 1 mL homogenate, plasma, or diluted erythrocytes while centrifuging at 2400 g for
30 min. 200 jxL Filtrate + 10 jxL 1 mg/mL IS, mix, lyophilize, add 200 |xL 100 mg/mL p-
nitrobenzoyl chloride in pyridine, heat at 60 for 1 h, add 1 drop of water, add 2 mL chloroform.
Wash mixture twice with 2 mL 5% sodium bicarbonate and twice with 3 mL 1 M HCl by
vortexing for 1 min and centrifuging for 30 s, inject a 50 |xL aliquot of the organic layer.
HPLC VARIABLES
Column: 250 X 4.6 6 |xm Zorbax SIL
Mobile phase: Hexane:chloroform:MeCN 10:3:1.9 containing 0.1% water
Flow rate: 1.5
Detector: UV 260
CHROMATOGRAM
Retention time: 18
Internal standard: perseitol (a-mannoheptitol) (26)
Limit of detection: 1-2 ng
OTHER SUBSTANCES
Extracted: fructose, myo-inositol, sorbitol, dextrose
KEYWORDS
plasma; lens; human; rat; normal phase; derivatization
REFERENCE
Petchey,M.; Crabbe,M.J.C. Analysis of carbohydrates in lens, erythrocytes, and plasma by high-performance
liquid chromatography of nitrobenzoate derivatives, J.Chromatogr., 1984, 307, 180-184.
SAMPLE
Sample preparation: Urine. Dilute urine 1:10 to 1:40 with water, add a 1 mL aliquot to 1 mL
250 |xg/mL melibiose, add Amberlite IR-120 H+ to occupy one third of the volume, inject a 25
jxL aliquot of the supernatant. Plasma. 200 |xL Plasma + 200 |xL 250 |xg/mL melibiose, mix,
add 200 jxL ice cold 35 mg/mL 5-sulfosalicylic +acid, let stand on ice for 20 min, centrifuge at
9000 g for 5 min, mix with Amberlite IR-120 H :Amberlite IRA 400 Cl" 40:60, centrifuge, inject
a 25 |xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: Carbopac PA-I (Dionex)
Column: 250 X 40 Carbopac PA-I (Dionex)
Mobile phase: 120 mM NaOH containing 0.5 mM zinc acetate (urine) or 160 mM NaOH con-
taining 0.675 mM zinc acetate (plasma) (At the end of each plasma sample wash with 1 M
NaOH for 4 min.)
Flow rate: 1
Detector: E, Dionex pulsed electrochemical detector, detection potential -0.01 V (0-0.5 s), oxi-
dation potential +0.75 V (0.51-0.64 s), reduction potential -0.75 V (0.65-0.75 s), integration
period 0.05-0.5 s
CHROMATOGRAM
Internal standard: melibiose (4.0 (plasma), 4.6 (urine))
OTHER SUBSTANCES
Extracted: lactulose, 3-O-methylglucose, dextrose
KEYWORDS
plasma
REFERENCE
Fleming,S.C; Kynaston,J.A.; Laker,M.E; Pearson,A.D.J.; Kapembwa,M.S.; Griffin,G.E. Analysis of multiple
sugar probes in urine and plasma by high-performance anion-exchange chromatography with pulsed elec-
trochemical detection. Application in the assessment of intestinal permeability in human immunodeficiency
virus infection, J.Chromatogr., 1993, 640, 293-297.
SAMPLE
Matrix: food
Sample preparation: Freeze chewing gum, pulverize. Sonicate 1 g with 80 mL EtOH:water 96:
4 at 60 for 20 min, cool, filter, rinse the filter, make up the filtrate to 100 mL. Remove a 5 mL
aliquot and evaporate it to dryness under reduced pressure, add 4 mL pyridine, add 500 JXL
benzoyl chloride, sonicate at 60 for 1 h with swirling every 15 min, add 500 |xL MeOH, swirl,
let stand for 10 min, add 50 mL water (Z. Lebensm. Unters. Forsch. 1984, 178, 199), shake,
add to a Sep-Pak RP-18 SPE cartridge (conditioning of cartridge is not necessary), rinse flask
four times with 5 mL portions of water, add the rinses to the SPE cartridge, push 5 mL volumes
of air through cartridge 3 times, elute with five 10 mL portions of isooctane:ether:MeCN 60:
32:8, make up the volume of the eluate to 50 mL, inject an aliquot.
HPLC VARIABLES
Column: 300 X 3 5 [xm. LiChrosorb Si 60 (glass column)
Mobile phase: Isooctane:ether:MeCN 150:60:10
Flow rate: 0.9
Detector: UV 230
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: dextrose, saccharose, sorbitol, xylitol
KEYWORDS
derivatization; SPE; chewing gum; mayonnaise; normal phase
REFERENCE
Galensa,R. Hochleistungs-flussigchromatographische Bestimmung von Zuckeralkoholen mit UV-Detektion im
ppm-Bereich in Lebensmitteln. I. [High-performance liquid chromatographic determination of sugar alco-
hols with UV-detection in the ppm-range in food. L], Z.Lebensm.Unters.Forsch., 1983, 176, 417-420.
SAMPLE
Matrix: intestinal mucosal homogenate
Sample preparation: Homogenate mixture + 100 JJLL 250 mM NaCN, mix, centrifuge at 4 at
34000 g for 10 min, filter (0.45 |xm) the supernatant, inject an aliquot of the filtrate.
HPLC VARIABLES
Column: Supelco Gel C610-H
Mobile phase: 0.1% phosphoric acid in water
Flow rate: 0.5
Detector: UV 190
KEYWORDS
rat
REFERENCE
Sinko,RJ.; Hu,P. Determining intestinal metabolism and permeability for several compounds in rats. Impli-
cations on regional bioavailability in humans, Pharm.Res., 1996, 13, 108-113.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: water
Flow rate: 0.5
Detector: RI
CHROMATOGRAM
Retention time: 34
OTHER SUBSTANCES
Simultaneous: arabinose, dextrose, fructose, galactose, glycerol, lactose, lactulose, pullulan P-
REFERENCE
SAMPLE
Matrix: urine
Sample preparation: Condition a 600 mg Maxi-Clean C18 SPE cartridge with 5 mL MeOH and
5 mL water. Add 2-3 mL urine to the SPE cartridge and pass it through the cartridge. Discard
the first 1 mL, collect the residual volume and dilute it 1:1 with water. Dilute a 200 jxL aliquot
with 1.8 mL water containing 75 (xm/mL cellobiose, add 400 g/L Amberlite IRA-400 resin Cl"
form (Fluka). Vortex for 10 s, centrifuge at 3000 g for 2 min. Filter (Micro-spin centrifuge
cartridge, Nylon 66, 0.2 |xm, Alltech) a 400 |xL aliquot of the supernatant while centrifuging
at 3000 g for 5 min. Inject a 50 (JLL aliquot.
HPLC VARIABLES
Guard column: Benson Carbohydrate BC-100 Ca2+ (Alltech)
Column: 300 X 6.5 10 |xm Alltech 700 CH Carbohydrate (Alltech)
Mobile phase: water
Flow rate: 0.5
Detector: ELSD, Varex MKIII (Alltech), drift tube temperature 120, carrier gas flow (air) 41.67
mL/s
CHROMATOGRAM
Internal standard: cellobiose (7.55)
Limit of detection: 650 |xg/L
OTHER SUBSTANCES
Extracted: dextrose, lactulose
Simultaneous: fructose, galactose
KEYWORDS
REFERENCE
Marsilio,R.; D'Antiga,L.; Zancan,L.; Dussini,N.; Zaccello,F. Simultaneous HPLC determination with light-scat-
tering detection of lactulose and mannitol in studies of intestinal permeability in pediatrics, Clin.Chem.,
1998, 44, 1685-1691.
SAMPLE
Matrix: urine
Sample preparation: Dilute urine 2.5-20 fold with water, add a 1 mL aliquot to 1 mL water
containing 250 |xg/mL arabinose and 25 |xg/mL cellobiose, add 0.5 g washed Amberlite IR-120
H:Amberlite IRA400 Cl 40:60, vortex, centrifuge, filter (0.2 |xm), inject a 50 |xL aliquot of the
supernatant.
HPLC VARIABLES
Column: 250 X 40 HPIC-AS6 (Dionex)
Mobile phase: 15OmMNaOH
Flow rate: 1
Detector: E, Dionex pulsed electrochemical detector, gold working electrode, detection potential
-0.05 V, oxidation potential +0.6 V, reduction potential -0.95 V, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 3
Internal standard: arabinose (4), cellobiose (9)
OTHER SUBSTANCES
Extracted: lactulose
REFERENCE
Fleming,S.C; Kapembwa,M.S.; Laker,M.R; Levin,G.E.; Griffin,G.E. Rapid and simultaneous determination of
lactulose and mannitol in urine, by HPLC with pulsed amperometric detection, for use in studies of intes-
tinal permeability, Clin.Chem., 1990, 36, 797-799.
SAMPLE
Matrix: urine
Sample preparation: Centrifuge, dilute 10-20 fold with water, inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 300 X 6.5 Sugar Pak I cation-exchange in calcium form
Mobile phase: Water containing 1 mL/L of a 50 g/L calcium EDTA solution
Flow rate: 0.5
Detector: RI
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Extracted: lactulose
REFERENCE
Willems,D.; Cadranel,S.; Jacobs,W. Measurement of urinary sugars by HPLC in the estimation of intestinal
permeability: evaluation in pediatric clinical practice, Clin.Chem., 1993, 39, 888-890.
SAMPLE
Matrix: urine
Sample preparation: 2 mL Urine + 500 mg washed and mixed Duolite ion-exchange resin
(BDH), vortex for 10 s, centrifuge at 3000 g for 10 min, filter (0.2 |xm) the supernatant, inject
an aliquot.
HPLC VARIABLES
Guard column: Direct-Connect polymeric guard column (Alltech)
Column: 250 X 4.6 5 |xm Kromasil NH2 (Alltech)
Flow rate: 1
Detector: RI
CHROMATOGRAM
Retention time: 9.4
Limit of detection: 50 jxM
OTHER SUBSTANCES
Extracted: lactulose, L-rhamnose, urea
REFERENCE
Miki,K; Butler,R-l Moore,D.; Davidson,G. Rapid and simultaneous quantification of rhamnose, mannitol, and
lactulose in urine by HPLC for estimating intestinal permeability in pediatric practice, Clin.Chem., 1996,
42, 71-75.
Maprotiline
CAS Registry N a : 10262-69-8, 10347-81-6 (HCI)
Merck Index: 5792
Lednicer No.: 2 220
SAMPLE
Matrix: blood
Sample preparation: Add 250 |xL 2 M sodium carbonate to 500 |xL plasma. Add 100 |xL 1 |xg/
10 min. Cool in a dry ice-acetone bath. Add 200 jxL 0.3% phosphoric acid to upper organic layer.
|xL aliquot of the acidic aqueous layer.
HPLCVARIABLES
Column: 250 X 4.6 5 ^m C18 Symmetry (Waters Millipore, USA)
50:50.)
Flow rate: 1.2
CHROMATOGRAM
Limit of quantitation: 5 ng/mL (UV 226, UV 400 nm); 7 ng/mL (UV 254)
OTHER SUBSTANCES
Extracted: metabolites, amitriptyline clomipramine desipramine fluoxetine, imipramine, nor-
triptyline
Simultaneous: amineptine, carbamazepine, chlordiazepoxide, chlorpromazine, clonazepam,
clorazepate, clozapine, cyamemazine, desmethylmaproptiline, desmethylvenlafaxine, doxepin,
fiunitrazepam, fluvoxamine, haloperidol, levomepromazine, lorazepam, loxapine, mianserine,
sulpiride, trimipramine, venlafaxine, viloxazine, zolpidem, zopiclone
KEYWORDS
plasma
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 400 |xL 700 mM pH 9.7 bicarbonate buffer, vortex
for 1 min, add 1.5 mL isoamyl alcohol:hexane 7.5:92.5, shake at 300 rpm for 30 min. Centrifuge
at 1500 g for 15 min, freeze at -20 overnight, decant the organic layer into a tube containing
200 JJLL 0.05% orthophosphoric acid. Shake vigorously at 300 rpm for 45 min, centrifuge at
1500 g for 15 min, inject a 100 (xL aliquot of the aqueous phase.
HPLC VARIABLES
Guard column: 13 X 4.3 5 |xm C4/E butyl-bonded reversed-phase (MetaChem Technologies)
Column: 150 X 4.6 5 jxm C4/E butyl-bonded reversed-phase(MetaChem Technologies)
Flow rate: 1.5
CHROMATOGRAM
Retention time: 10
Internal standard: maprotiline
OTHER SUBSTANCES
Extracted: venlafaxine
KEYWORDS
plasma; maprotiline is IS
REFERENCE
Vu,R-L.; Helmeste,D.; Albers,L.; Reist,C. Rapid determination of venlafaxine and O-desmethylvenlafaxine in
human plasma by high-performance liquid chromatography with fluorimetric detection, J.Chromatogr.B,
1997, 703, 195-201.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 800 ng clomipramine in MeOH + 2 mL 1 M NaOH + 5
mL hexanerisoamyl alcohol 99:1, shake mechanically for 15 min, centrifuge at 1686 g for 5
min. Remove the organic phase and add it to 200 JJLL 0.05% orthophosphoric acid, shake for 15
min, centrifuge for 5 min, inject a 50 |xL aliquot of the aqueous phase (J.Liq.Chromatogr. 1981,
4, 849).
HPLC VARIABLES
Guard column: 23 X 3.9 Bondapak/Corasil C 18
Mobile phase: MeCN.buffer 40:60 (Buffer was 13.68 g KH2PO4 in 2 L water, adjusted to pH 4.7
with dilute KOH.)
Flow rate: 2
Detector: UV 214
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Simultaneous: amoxapine, amitriptyline, chlordiazepoxide, chlorpromazine, cimetidine, desipra-
mine, diazepam, doxepin, flurazepam, imipramine, lorazepam, oxazepam, pentobarbital, per-
phenazine, phenobarbital, phenytoin, prochlorperazine, propoxyphene, secobarbital, thiorida-
zine, trifluoperazine
Noninterfering: acetaminophen, codeine, meperidine
KEYWORDS
plasma
REFERENCE
Wong,S.H.Y.; Waugh,S.W. Determination of the antidepressants maprotiline and amoxapine, and their metab-
olites, in plasma by liquid chromatography, Clin.Chem., 1983, 29, 314-318.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 500 |xL 10% potassium carbonate + 3 mL freshly-distilled
n-hexane, shake for 30 min, centrifuge. Remove 2 mL of the organic layer and evaporate it to
dryness under reduced pressure, reconstitute the residue in 500 |xL 1 mg/mL benoxaprofen
chloride in dry dichloromethane, heat at 50 for 30 min, inject a 10 |xL aliquot. (Prepare be-
noxaprofen chloride as follows. Dissolve 600 mg benoxaprofen in 50 mL dry toluene, slowly
add 5 mL thionyl chloride (freshly distilled from linseed oil), reflux for 30 min, evaporate to
dryness, recrystallize from dichloromethane (if necessary) to give benoxaprofen chloride (mp
91.5).)
HPLCVARIABLES
Column: 250 X 4.6 7 jim Zorbax-sil
Mobile phase: Cyclohexane:dichloromethane:THF 5:1:1
Flow rate: 1
CHROMATOGRAM
Retention time: 8.6
OTHER SUBSTANCES
Simultaneous: amphetamine, benzylamine, methamphetamine, a-methylbenzylamine, phenyl-
butylamine, p-phenylethylamine, tolylethylamine, tranylcypromine
Interfering: procaine
KEYWORDS
derivatization; normal phase; plasma
REFERENCE
Spahn,H.; Weber,H.; Mutschler,E.; Mohrke,W. a-Alkyl-a-arylacetic acid derivatives as fluorescence markers for
thin-layer chromatographic and high-performance liquid chromatographic assay of amines and alcohols,
J.Chromatogr., 1984, 310, 167-178.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 37 |xL 2 |xg/mL IS in MeOH + 500 |xL pH 10 borate
buffer +1.5 mL hexane:isoamyl alcohol 95:5, shake for 10 min. Evaporate the organic layer to
dryness under a stream of nitrogen, reconstitute in 100 |xL MeOH, inject a 50 u-L aliquot. (The
borate buffer was prepared as follows. Prepare a solution of 61.8 g boric acid and 74.6 g KCl
in 1 L water. Add 630 mL of this solution to 370 mL 106 g/L sodium carbonate solution. Adjust
pH to 10.0 with 6 M NaOH and store at 35-37.)
HPLC VARIABLES
Mobile phase: MeOHrammonium hydroxide 998:2
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 14
Internal standard: desipramine hydrochloride (12)
OTHER SUBSTANCES
Extracted: desmethylclomipramine, clomipramine, protriptyline, metabolites
Also analyzed: doxepin, desmethyldoxepin, amitriptyline, nortriptyline, imipramine, 2-hydroxy-
imipramine, 2-hydroxydesipramine
Noninterfering: chlordiazepoxide, diazepam, flurazepam, oxazepam, thioridazine
KEYWORDS
plasma
REFERENCE
Sutfin,T.A.; D'Ambrosio,R.; Jusko,W.J. Liquid-chromatographic determination of eight tri- and tetracyclic an-
tidepressants and their major active metabolites, Clin.Chem., 1984, 30, 471-474.
SAMPLE
Matrix: blood
Sample preparation: Inject 200 |xL serum onto column A and elute with mobile phase A for 10
min then back-flush column A onto column B with mobile phase B for 4 min. Elute column B
with mobile phase B and monitor the effluent. Remove column A from circuit and wash with
MeCN:water 60:40 for 6 min then with mobile phase A for 10 min.
HPLC VARIABLES
Column: A 40 x 4 TSKprecolumn PW (Tosoh); B 150 X 4 TSKgel ODS-80TM (Tosoh)
Mobile phase: A 50 mM pH 7.5 potassium phosphate; B MeCNiIOO mM pH 2.7 potassium phos-
phate 32.5:67.5, containing 0.2 g/L sodium 1-heptanesulfonate
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Simultaneous: amitriptyline, amoxapine, clomipramine, desipramine, doxepin, imipramine, nor-
triptyline, trimipramine
KEYWORDS
serum; column-switching; use gradient to determine metabolites
REFERENCE
Matsumoto,K; Kanba,S.; Kubo,H.; Yagi,G.; Iri,H.; Yuki,H. Automated determination of drugs in serum by col-
umn-switching high-performance liquid chromatography. IV. Separation of tricyclic and tetracyclic antide-
pressants and their metabolites, Clin.Chem., 1989, 35, 453-456.
SAMPLE
Matrix: blood
Sample preparation: Add 10 |xL 20 |xg/mL oxaprotiline in MeOH to 990 |xL plasma or serum.
Inject 100 |xL plasma or serum onto column A with mobile phase A and elute to waste, after
mobile phase B.
HPLC VARIABLES
Column: A 20 X 4.6 10 |xm Hypersil MOS C8; B 20 X 4.6 5 jxm Hypersil CPS CN + 250 X 4.6
5 (xm Nucleosil 100 CN
Mobile phase: A MeOHrwater 5:95; B MeOH:MeCN:10 mM pH 6.8 potassium phosphate buffer
188:578:235
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: clozapine, fluvoxamine, metoclopramide, fluoxetine, norfluoxetine, imipramine,
nortriptyline, desipramine, doxepin, clomipramine, amitriptyline
pam, carbamazepine
KEYWORDS
REFERENCE
Hartter,S.; Wetzel,H.; Hiemke,C. Automated determination of fluvoxamine in plasma by column-switching high-
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 0.6 M pH 9.8 carbonate buffer + 40 jxL 5 jig/mL
min, centrifuge at 1500 g for 10 min. Remove organic layer and add it to 150 JJLL 100 mM HCl,
layer at 45 in a vacuum centrifuge for 1 h. Take up residue in 50 |xL 1 M pH 10.3 carbonate
buffer and 25 fxL 10 mg/mL dansyl chloride in MeCN, vortex, allow to react at room temper-
ature for 45 min, evaporate at 45 in a vacuum centrifuge for 20 min, reconstitute in 125 jxL
HPLC VARIABLES
Column: 250 X 4.6 5 |im Supelcosil LC-18
Mobile phase: MeCN:25 mM KH2PO4 75:25 + 500 jxL/L orthophosphoric acid + 600 JJLL/L n-
butylamine
Flow rate: 2
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: fluoxetine, propranolol, clovoxamine, fluvoxamine, fenfluramine, amoxapine, pro-
triptyline, nortriptyline, norfluoxetine, desipramine, sertraline
loxapine
KEYWORDS
plasma; maprotiline is IS
REFERENCE
Suckow,R-F.; Zhang,M.R; Cooper,T.B. Sensitive and selective liquid-chromatographic assay of fluoxetine and
norfluoxetine in plasma withfluorescencedetection after precolumn derivatization, Clin.Chem., 1992, 38,
1756-1761.
SAMPLE
Matrix: blood
MeOH, 1 mL water, and 1 mL 1% potassium carbonate. 700 |xL Serum + 50 |xL 5 |jig/mL
trimipramine in 5% potassium bicarbonate + 700 JJLL MeCN, vortex, centrifuge at 1500 g for 5
MeCN, elute with 250 (xL MeOH:35% perchloric acid 20:1 by gravity (10 min) then centrifuge
for 20 s to remove rest of eluant, inject a 50 |xL aliquot of the eluate.
HPLC VARIABLES
Guard column: 15 mm 7 jxm Brownlee RP-8
Mobile phase: MeCN:water 37.5:62.5 containing 0.5 g/L tetramethylammonium perchlorate and
Flow rate: 1.5
Detector: UV 215
CHROMATOGRAM
Retention time: 8.0
OTHER SUBSTANCES
Extracted: clomipramine, desipramine, doxepin, fluoxetine, fluvoxamine, imipramine, nortrip-
tyline, protriptyline
Interfering: amitriptyline, desmethyltrimipramine
KEYWORDS
serum; SPE
REFERENCE
Gupta,R.N. An improved solid phase extraction procedure for the determination of antidepressants in serum
by column liquid chromatography, J.Liq.Chromatogr., 1993, 16, 2751-2765.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 1 mL 2 M sodium bicarbonate + 6 mL hexane, extract,
centrifuge at 2000 g for 10 min. Remove 5 mL of the hexane layer and add it to 150 |xL 100
mM phosphoric acid, mix, centrifuge at 2000 g for 10 min, inject a 100 |xL aliquot of the aqueous
layer.
HPLC VARIABLES
Column: 250 X 4 4 |xm Supersphere Select B (Merck)
Mobile phase: MeCN:pH 5.8 phosphate buffer 25:75 (Buffer was 2 mL 85% phosphoric acid and
4 mL triethylamine in 1 L water.)
Flow rate: 0.3-1
Detector: F ex 275 em 315 following post-column photolysis. The effluent from the column flowed
through a Beam Boost photochemical reactor equipped with a 20 m coil and then to the
detector.
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
serum; post-column reaction; pharmacokinetics; post-column photochemical derivatization
REFERENCE
Kuss,H.-J.; Sirch,S.; Zhao,D.Y. Assay for maprotiline in human serum with improved sensitivity and selectivity,
J.Chromatogr.B, 1994, 656, 245-249.
SAMPLE
Matrix: blood
with 3 mL 750 mL/L methanol. Elute with three aliquots of 300 uJL 0.1 M ammonium acetate
IxL 0.1 M phosphoric acid (pH 2.5), mix, separate, inject a 100 JXL aliquot of the aqueous phase.
HPLC VARIABLES
Flow rate: 2
Detector: UV 228
CHROMATOGRAM
Retention time: 5.0
OTHER SUBSTANCES
desipramine, dextromethorphan, diazepam, diphenhydramine, doxepin, encainide, fentanyl,
flecainide, fluoxetine, flurazepam, haloperidol, hydroxyethylflurazepam, ibuprofen, lidocaine,
methadone, methaqualone, mexiletine, midazolam, norchlorimipramine, nordoxepin, norfluox-
etine, norverapamil, pentazocine, promazine, propafenone, propranolol, protriptyline, quini-
dine, trazodone, trimipramine
ethosuximide, furosemide, glutethimide, hydrochlorothiazide, hydrocodone, hydroflumethi-
azide, hydromorphone, lorazepam, mephentermine, meprobamate, methamphetamine, meth-
arbital, methoxsalen, methoxyphenteramine, methsuximide, methylcyclothiazide, metoprolol,
warfarin
Interfering: nordiazepam, propoxyphene, temazepam, imipramine, nortriptyline, verapamil
KEYWORDS
plasma; SPE
REFERENCE
Clin.Chem., 1994, 40, 1312-1316.
SAMPLE
Matrix: blood
Sample preparation: 100 |JLL Plasma + 100 |xL 100 ng/mL desipramine in 2-methoxyethanol
containing 75 mM triethylamine + 500 jxL 6 M NaOH + 3 mL n-hexane:isoamyl alcohol 95:5,
vortex for 5 min, centrifuge at 1000 g for 5 min. Remove a 2 mL aliquot of the organic layer
and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 100 |xL 75
mM triethylamine in 2-methoxyethanol, add 100 |xL 1 mM 6-isothiocyanato-
benzo[g]phthalazine-l,4(2H,3H)-dione in DMSO, add 10 |xL 75 mM triethylamine in 2-meth-
oxyethanol, heat at 80 for 10 min, inject a 20 JAL aliquot. (Prepare 6-isothiocyanato-
benzo[g]phthalazine-l,4(2H,3H)-dione as follows. Dissolve 2.2 g 2,3-naphthalenedicarboxylic
acid in 6.6 mL MeOH.concentrated sulfuric acid 10:1, reflux for 3 h, pour into ice-water, filter.
Dissolve the precipitate in 20 mL ethyl acetate and purify on a 250 X 35 column containing
120 g 70-230 mesh silica gel 60 (Merck) using n-hexane:ethyl acetate 50:50 to give dimethyl
2,3-naphthalenedicarboxylate as a brownish crystalline solid (mp 46-49). Stir 8 mL concen-
trated nitric acidxoncentrated sulfuric acid 3:5 at 4, add 2.5 g dimethyl 2,3-naphthalenedi-
carboxylate, stir at 4 for 4-6 h until the reaction is complete. (Check by TLC using Merck
Kieselgel 60 F254 with n-hexane:ethyl acetate 50:50.) Pour the reaction mixture into ice-water
and extract with 350 mL dichloromethane. Wash the organic layer with water, with 5% sodium
bicarbonate, and with water. Evaporate to dryness under reduced pressure, recrystallize from
EtOH to give dimethyl 5-nitro-2,3- naphthalenedicarboxylate as yellow needles (mp 146-148).
Dissolve 1.25 g dimethyl 5-nitro-2,3-naphthalenedicarboxylate in 60 mL MeOH, add 500 mg
5% platinum on activated carbon, hydrogenate, filter, evaporate to dryness under reduced pres-
sure, recrystallize from n-hexane/ethyl acetate to give dimethyl 5-amino-2,3-naphthalenedicar-
boxylate as pale yellow-green crystals (mp 129-131). Add 2.7 mL triethylamine and 2.7 mL
hydrazine hydrate (Caution! Hydrazine hydrate is a carcinogen!) to 1.2 g dimethyl 5-amino-
2,3-naphthalenedicarboxylate dissolved in 18 mL MeOH, reflux for 3 h, evaporate to remove
the solvent, rinse the residue with EtOH, dry to give 6-aminobenzo[g]phthalazine-l,4(2H,3H)-
dione as a yellow powder (mp 280 d). Add 1 mL thiophosgene drop wise to a solution of 1 g 6-
aminobenzo[g]phthalazine-l,4(2H,3H)-dione in 20 mL water stirred at room temperature, stir
for 1 h, filter, wash the precipitate with MeCN, dry to give 6-isothiocyanatobenzo[g]-
phthalazine-l,4(2H,3H)-dione as a pale brownish-yellow powder (mp 360 d) (Anal. Chim. Acta
1995, 302, 61).)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm TSKgel ODS-80 (Tosoh)
Mobile phase: MeCN: 100 mM pH 3.2 acetate buffer 40:60
Flow rate: 1
20 mM hydrogen peroxide in water pumped at 1 mL/min and with 40 mM potassium ferricy-
anide in 1.5 M NaOH pumped at 2 mL/min and the mixture flowed to the detector.
CHROMATOGRAM
Internal standard: desipramine (39.5)
OTHER SUBSTANCES
Simultaneous: amoxapine, metoprolol, nortriptyline, propranolol
Noninterfering: betamethasone, diazepam, phenobarbital, phenytoin, prednisolone, triazolam
KEY WpRDS
derivatization; plasma; pharmacokinetics
REFERENCE
Ishida,J.; Horike,N.; Yamaguchi,M. Determination of maprotiline in plasma by high-performance liquid chro-
matography with chemiluminescence detection, J.Chromatogr.B, 1995, 669, 390-396.
SAMPLE
Matrix: blood
the residue in 100 (xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 272
CHROMATOGRAM
KEYWORDS
REFERENCE
Tracqui,A.; Kintz,R; Mangin,R Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
Sample preparation: Dilute urine ten-fold. 1 mL Plasma, serum, CSF, or diluted urine + 250
(xL 100 mM diethylamine in 4 M HCl + 1 mL 1 |JLM desmethyldoxepin hydrochloride in water,
mix briefly, add 8 mL n-hexane, shake horizontally at 150 rpm for 30 min, centrifuge, discard
the hexane layer. Add 250 jxL 6 M NaOH to the aqueous layer, add 8 mL hexane, shake for 30
min, centrifuge. Remove the organic layer and evaporate it to 0.5 mL under a stream of nitro-
gen at 45, add 100 |JLL 100 mM HCl to the residue , vortex for 2 min, inject an aliquot of the
aqueous layer.
HPLC VARIABLES
Flow rate: 2
Detector: UV 205
CHROMATOGRAM
Internal standard: desmethyldoxepin (k' 4.56)
Limit of detection: H n M
OTHER SUBSTANCES
Simultaneous: amitriptyline, chlordiazepoxide, chlorpromazine, desmethyldiazepam, desmethyl-
imipramine, desmethylmaprotiline, diazepam, doxepin, fluphenazine, haloperidol, imipramine,
lorazepam, mianserin, nomifensine, oxazepam, perphenazine, trifluoperazine
Noninterfering: sulpiride, thioridazine
KEYWORDS
plasma; serum
REFERENCE
Salonen,J.S.; Scheinin,M. Determination of maprotiline and N-desmethylmaprotiline from biological fluids by
HPLC, J.Anal.ToxicoL, 1983, 7, 175-177.
SAMPLE
vortex, add 1 mL 200 mM sodium carbonate, vortex, add 6 mL hexane: 1-butanol 95:5, gently
layer and inject a 30 JXL aliquot of the aqueous layer. Liver homogenate. 0.5 mL Liver ho-
mogenate + 10 |xg cianopramine + 500 (xL 2% sodium tetraborate + 8 mL hexane: 1-butanol 95:
it to 400 |JLL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the
HPLC VARIABLES
Guard column: 15 X 3.2 7 (xm RP-18 Newguard (Applied Biosystems)
Column: 100 X 4.6 5 (xm Brownlee Spheri-5 RP-18
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
chlorpromazine, clomipramine, cyproheptadine, diphenhydramine, dothiepin, doxepin, fluoxe-
tine, haloperidol, imipramine, loxapine, meperidine, mesoridazine, methadone, metoclopra-
mide, mianserin, moclobemide, nomifensine, nordoxepin, norfluoxetine, norpropoxyphene, nor-
thiaden, nortriptyline, pentobarbital, pheniramine, promethazine, propoxyphene, propranolol,
protriptyline, quinidine, quinine, sulforidazine, thioridazine, thiothixene, tranylcypromine, tra-
zodone, trihexyphenidyl, trimipramine, triprolidine
trinuoperazine
Interfering: desipramine
KEYWORDS
REFERENCE
SAMPLE
Sample preparation: Serum, urine. 500 |xL Serum or urine + 100 u,L 2 |xg/mL diazepam + 200
gentle stream of nitrogen at about 40. Dissolve the residue in 100 uL mobile phase, inject a
10 jxL aliquot. Tissue. Homogenize 1 g sample with 9 mL 100 mM HCl and 100 |xL 20 jig/mL
diazepam, centrifuge at 15000 g for 10 min. Add 500 |xL 20% sodium carbonate and 4 mL n-
hexane:isoamyl alcohol 98.5:1.5 to 1 mL of the supernatant, mix for 5 min. Remove the organic
100 |xL mobile phase, filter by microconcentrator (Microcon-30, Grace). Inject a 10 JJLL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 2 ^m TSK gel Super-Octyl (A) or 100 X 4.6 5 |xm Hypersil MOS-C8 (B),
(Yokogawa, Japan)
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: amitriptyline, clomipramine, dothiepin, doxepin, imipramine, melitracen, mianserin,
nortriptyline
Interfering: amoxapine, desipramine
KEYWORDS
serum; brain; liver
REFERENCE
in human biological samples using a new reversed-phase chromatographic column of 2 fxm porous micro-
SAMPLE
Sample preparation: Plasma. 0.5-2 mL Plasma + 2 mL pH 10 phosphate buffer + 6 mL diethyl
ether:hexane 50:50, shake for 15 min, centrifuge at 3750 g for 5 min. Remove the organic phase
and add it to 2 mL 250 mM sulfuric acid, shake for 10 min, centrifuge for 5 min, discard the
organic layer. Adjust the pH of the aqueous phase to 9.5-10.5 with 500 mM NaOH containing
1 M K2HPO4, add 6 mL diethyl ether:hexane 50:50, shake for 10 min, centrifuge for 10 min.
Remove the organic layer and evaporate it to dryness under vacuum and a stream of nitrogen
at 45, reconstitute the residue in 100 |xL 100 mM sodium carbonate, add 10 |xL 1% dansyl
chloride in acetone, vortex for 20-30 s, heat at 45 for 30 min. Evaporate the solvent, reconsti-
tute in 100 |JLL mobile phase, inject a 10-50 |JLL aliquot. Urine. 0.5-2 mL Urine + 2 mL pH 10
phosphate buffer + 6 mL diethyl ether:hexane 50:50, shake for 15 min, centrifuge at 3750 g
for 5 min. Remove the organic phase and evaporate it to dryness under vacuum and a stream
of nitrogen at 45, reconstitute the residue in 100 |xL 100 mM sodium carbonate, add 10 |xL
1% dansyl chloride in acetone, vortex for 20-30 s, heat at 45 for 30 min. Evaporate the solvent,
reconstitute in 100 JJLL mobile phase, inject a 10-50 (JLL aliquot.
HPLC VARIABLES
Flow rate: 2
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: metapramine
KEYWORDS
plasma; derivatization; maprotiline is IS
REFERENCE
Sommadossi,J.R; Lemar,M.; Necciari,J.; Sumirtapura,Y; Cano,J.R; Gaillot,J. High-performance liquid chro-
matographic method for the determination of plasma and urine metapramine after dansylation,
J.Chromatogr., 1982, 228, 205-213.
SAMPLE
a 10 (urine) or 30 (blood) JXL aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jjig/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.9
OTHER SUBSTANCES
isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, mecamylamine, meclo-
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 20 mm long Pelliguard LC-8 40 |xm
Column: 150 X 4.6 C8 5 |xm (Supelco)
Mobile phase: MeCNibuffer 50:50 (Buffer was obtained by adding 1.2 mL of butylamine to 1 L
10 mM NaH2PO4 then adjusting pH to 3 with phosphoric acid.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: clobazam (k' 1.344)
OTHER SUBSTANCES
Simultaneous: diazepam, desipramine, haloperidol, nortriptyline, imipramine, amitriptyline,
clomipramine
REFERENCE
Segatti,M.R; Nisi,G.; Grossi,R; Mangiarotti,M.; Lucarelli,C. Rapid and simple high-performance liquid chro-
matographic determination of tricyclic antidepressants for routine and emergency serum analysis,
J.Chromatogr., 1991, 536, 319-325.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 (xm Adsorbosphere C18 (PEEK column) (retention times are longer and
Flow rate: 1.2
Detector: UV 214
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Simultaneous: amitriptyline, desmethyldoxepin, desipramine, doxepin, imipramine, loxapine,
nortriptyline, trazodone
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 Econosil C8
Mobile phase: MeCN:buffer 30:70 (Buffer was 20 mM KH2PO4 and 14 mM triethylamine ad-
justed to pH 3.0 with phosphoric acid.)
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: doxepin, desipramine, protriptyline, cyclobenzaprine
Also analyzed: amitriptyline, amoxapine, carbamazepine, imipramine, nortriptyline
KEYWORDS
UV spectra given
REFERENCE
Ryan,T.W. Identification and quantification of tricyclic antidepressants by UV-photodiode array detection with
multicomponent analysis, J.Liq.Chromatogr., 1993, 16, 1545-1560.
Mazindol
Merck Index: 5801
Lednicer No.: 2 462
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: methylphenidate, phenelzine, epinephrine, pipradol, phenylpropanolamine, fen-
camfamin, chlorphentermine, norpseudoephedrine, phentermine, fenfluramine, methylenedi-
oxyamphetamine, amphetamine, normetanephrine, 4-hydroxyamphetamine, bromo-STP, STP,
prolintane, 2-phenethylamine, tyramine, trimethoxyamphetamine, phenylephrine, pseudo-
ephedrine, ephedrine, methylephedrine, dimethylamphetamine, methamphetamine, mescaline,
mephentermine, levallorphan, hydroxypethidine, normethadone, meperidine, dipipanone, dia-
morphine, pentazocine, acetylcodeine, monoacetylmorphine, thebacon, oxycodone, thebaine,
norlevorphanol, methadone, benzylmorphine, ethylmorphine, morphine-N-oxide, codeine, co-
deine-N-oxide, morphine, ethoheptazine, morphine-3-glucuronide, pholcodeine, norpethidine,
hydrocodone, dihydrocodeine, dihydromorphine, levorphanol, norcodeine, normorphine
Interfering: pemoline, benzphetamine, diethylpropion, tranylcypromine, caffeine, fenethyline,
phendimetrazine, buprenorphine, dextromoramide, phenoperidine, fentanyl, etorphine, piri-
tramide, noscapine, papaverine, naloxone, dextropropoxyphene, nalorphine, phenazocine,
norpipanone
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
pam, lormetazepam, mebendazole, meclizine, meclofenamic acid, medazepam, mefenamic acid,
megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital,
mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, meth-
aqualone, methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate,
methyldopa, methyldopamine, methylphenidate, methylprednisolone, methyltestosterone,
methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone,
naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitra-
REFERENCE
Hill,D.W.; Kind ,A. J- Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 |m LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ketorolac, labetalol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mefen-
talol, spironolactone, sulnnpjrrazone, sulindac, temazepam, terbutaline, terfenadine, tetra-
tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupro-
mazine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yo-
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatognA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Next Page
Sample preparation: Condition a 3 mL Bond-Elut strong cation exchange SPE cartridge packed
with benzenesulfonic acid material with two 3 mL portions of MeOH, two 3 mL portions of
water, and 3 mL 7 mM phosphoric acid (pH 3.5), do not allow column to dry. Adjust pH of 10
mL urine to 3.5 with 5 mL phosphoric acid, add to column, allow column to dry for 30 s, wash
with 3 mL dilute phosphoric acid (pH 3.5), wash with 1 mL 1 M acetic acid, wash with 1 mL
MeOH, elute with 1 mL 1% ammonium hydroxide in MeOH (pH 10), inject a 20 |JLL aliquot of
the eluate.
HPLCVARIABLES
Mobile phase: MeCN:5 mM pentanesulfonic acid:85% phosphoric acid 25:75:5
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
KEYWORDS
horse; SPE; details of GC-MS procedure given
REFERENCE
Moore,C.M.; TebbettJ.R.; Kalita,S.; Artememko,M. Rapid extraction and detection of mazindol in horse urine,
J.Pharm.Biomed.Analy 1990, 8, 445-448.
Mebendazole
Merck Index: 5807
Lednicer No.: 2 353
SAMPLE
dryness, reconstitute in 50 |xL MeOH, sonicate, inject a 5 |xL aliquot.
HPLCVARIABLES
Flow rate: 1.5
Injection volume: 5
Detector: UV 292
CHROMATOGRAM
Retention time: 5
Previous Page
Sample preparation: Condition a 3 mL Bond-Elut strong cation exchange SPE cartridge packed
with benzenesulfonic acid material with two 3 mL portions of MeOH, two 3 mL portions of
water, and 3 mL 7 mM phosphoric acid (pH 3.5), do not allow column to dry. Adjust pH of 10
mL urine to 3.5 with 5 mL phosphoric acid, add to column, allow column to dry for 30 s, wash
with 3 mL dilute phosphoric acid (pH 3.5), wash with 1 mL 1 M acetic acid, wash with 1 mL
MeOH, elute with 1 mL 1% ammonium hydroxide in MeOH (pH 10), inject a 20 |JLL aliquot of
the eluate.
HPLCVARIABLES
Mobile phase: MeCN:5 mM pentanesulfonic acid:85% phosphoric acid 25:75:5
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
KEYWORDS
horse; SPE; details of GC-MS procedure given
REFERENCE
Moore,C.M.; TebbettJ.R.; Kalita,S.; Artememko,M. Rapid extraction and detection of mazindol in horse urine,
J.Pharm.Biomed.Analy 1990, 8, 445-448.
Mebendazole
Merck Index: 5807
Lednicer No.: 2 353
SAMPLE
HPLCVARIABLES
Flow rate: 1.5
Injection volume: 5
Detector: UV 292
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Extracted: albendazole, oxfendazole, cambendazole, thiabendazole, oxibendazole, fenbendazole,
parbendazole
KEYWORDS
plasma; sheep
REFERENCE
J.Pharm.Sci., 1980, 69, 422-423.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 40 jxL MeOHiDMF 99:1 + 1 mL 50 mM pH 8 borate buffer
+ 5 mL ethyl acetate, vortex for 20 s, centrifuge at 2500 g for 5 min. Remove 4 mL of the
organic layer and evaporate it to dryness under a stream of nitrogen at 50 for about 50 min,
reconstitute the residue in 200 |xL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 125 X 4.6 5 fxm Hypersil SAS
tetraethylammonium hydroxide.)
Flow rate: 2
Detector: UV 300
CHROMATOGRAM
Retention time: 3.2
Internal standard: mebendazole
OTHER SUBSTANCES
Extracted: fenbendazole
KEYWORDS
plasma; sheep; mebendazole is IS
REFERENCE
Lehr,K.H.; Damm,P. Simultaneous determination of fenbendazole and its two metabolites and two triclaben-
dazole metabolites in plasma by high-performance liquid chromatography, J.Chromatogr., 1986, 382, 3 5 5 -
360.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 5 mL MeOH and 5 mL 20
mM KH2PO4. Add 1 mL serum to the SPE cartridge, wash with 20 mL water, wash with 500
|JLL MeOH:water 40:60, elute with 2 mL MeOH. Evaporate the eluate to dryness under a stream
of nitrogen at room temperature, reconstitute the residue in 200 |xL MeOH, inject an aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Hypersil SAS
Mobile phase: MeCN: 10 mM KH2PO4 20:80 adjusted to pH 3 with orthophosphoric acid
Flow rate: 0.8
Detector: E, ESA Coulochem 5100 A, Model 5020 guard cell +0.70 V, Model 5010 A analytical
cell, first electrode +0.60 V, second electrode -0.10 V (monitored)
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
serum; SPE
REFERENCE
Betto,R; Gianbenedetti,M.; Ponti,R; Ferretti,R.; Settimj,G.; Gargiulo,M.; Lorenzini,R. Application of a high-
performance liquid chromatography coulometric method for the estimation of mebendazole and its metab-
olites in human sera, J.Chromatogr., 1991, 563, 115-123.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Whole blood + 10 |xL 10 (xg/mL flubendazole + 2 mL 150 mM saline,
vortex for 15 s, add 6 mL diethyl ether, vortex for 1.5 min, centrifuge at 1000 g for 10 min.
Remove the organic layer and evaporate it to dryness under a stream of nitrogen at room
temperature, reconstitute the residue in 100 |xL DMSO, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 150 X 3.9 5 |jim Nova Pak
Mobile phase: MeOH:50 mM (NH4)H2PO4 50:50 adjusted to pH 4.0 with 17.2 M orthophosphoric
acid
Flow rate: 1
Detector: UV 291
CHROMATOGRAM
Retention time: 6.8
Internal standard: flubendazole (8.5)
OTHER SUBSTANCES
Extracted: UMF-058
KEYWORDS
whole blood; monkey; pharmacokinetics
REFERENCE
Ramanathan,S.; Nair,N.K; Mansor,S.M.; Navaratnam,V. Determination of a new antifilarial drug, UMF-058,
and mebendazole in whole blood by high-performance liquid chromatography, J.Chromatogr., 1993, 615,
303-307.
SAMPLE
Matrix: blood, CSF
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 5 mL MeOH and 5 mL 17
mM KH2PO4 adjusted to pH 5.5 with 800 mM NaOH. 2 mL Plasma or CSF + 2 mL 10 mM
KH2PO4 adjusted to pH 7.4 with 800 mM NaOH, vortex for 30 s, add to the SPE cartridge,
wash with 20 mL 10 mM KH2PO4 adjusted to pH 7.4 with 800 mM NaOH, wash with 1 mL
MeOHrwater 20:80, elute with 3 mL MeOH. Evaporate the eluate to dryness under a stream
of nitrogen at 40, reconstitute the residue in 100 |xL MeOH, inject a 20 (xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |un ODS C18
Flow rate: 0.8
Detector: UV 295
CHROMATOGRAM
Retention time: 8.0
OTHER SUBSTANCES
Extracted: albendazole
KEYWORDS
plasma; mebendazole is IS; SPE
REFERENCE
Hurtado,M.; Medina,M.T.; Sotelo,J.; Jung,H. Sensitive high-performance liquid chromatographic assay for al-
bendazole and its main metabolite albendazole sulphoxide in plasma and cerebrospinal fluid, J. Chromatogr.,
1989, 494, 403-407.
SAMPLE
Sample preparation: Homogenize lung tissue in 5 volumes 100 mM pH 6.0 Na2HPO4 and cen-
trifuge at 1500 g for 15 min. 500 |xL Serum or 1 mL lung tissue homogenate supernatant + 1
mL 100 mM potassium carbonate + 4 mL dichloromethane, mix on an Eberbach shaker for 10
min, centrifuge at 1500 g for 10 min, repeat extraction. Combine the organic layers and evap-
orate them to dryness under a stream of nitrogen, reconstitute the residue in 100 (JLL MeOH,
HPLC VARIABLES
Mobile phase: MeOH:MeCN:70 mM monochloroacetic acid 27:18:55
Flow rate: 1.2
Detector: UV 290
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
serum; lung; mouse; mebendazole is IS
REFERENCE
Bartlett,M.S.; Edlind,T.D.; Lee,C.H.; Dean,R.; Queener,S.R; Shaw,M.M.; Smith,J.W. Albendazole inhibits Pneu-
mocystis carinii proliferation in inoculated immunosuppressed mice, Antimicrob.Agents Chemother., 1994,
38, 1834-1837.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 209.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Place 1.5 mL microsomal incubation in a boiling water bath for 2 min,
add to a Sep-Pak C18 SPE cartridge, wash with water, elute with MeOH, inject an aliquot of
the eluate.
HPLC VARIABLES
Column: Nucleosil C18
Mobile phase: Gradient. MeCN:0.5% acetic acid 35:65 for 5 min, 70:30 for 5 min, re-equilibrate
at initial conditions for 5 min.
Flow rate: 1 for 5 min, 1.5 for 5 min, re-equilibrate at 1
Detector: UV 292
CHROMATOGRAM
Retention time: 7.3
OTHER SUBSTANCES
KEYWORDS
rat; intestine; SPE; mebendazole is IS
REFERENCE
Villaverde,C; Alvarez,A.L; Redondo,R; Voces,J.; del Estal,J.L.; Prieto,J.G. Small intestinal sulphoxidation of
albendazole, Xenobiotica, 1995, 25, 433-441.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
fluorouracil, fiuoxymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide,
isoxsuprine, ivercnectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, loraze-
pam, lormetazepam, loxapine, meclizine, meclofenamic acid, medazepam, mefenamic acid, me-
methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naph-
azoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitraze-
pam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone,
REFERENCE
18, 233-242.
SAMPLE
Matrix: tissue
Sample preparation: Activate amino propyl SPE cartridge (containing 1 g 40 |xm Bakerbond
Amino aminopropylsilane (J.T. Baker) for flash chromatography) with 5 mL MeOH. Condition
SPE cartridge with 2 mL ethyl acetate and 2 mL ethyl acetate:hexane 4:5. Add 1 mL ethyl
acetate:hexane 4:5 to SPE cartridge. Attach SPE reservoir to SPE cartridge. 5 g tissue + 3 mL
buffer, vortex for 10 s. Add 10 mL ethyl acetate, vortex for 10 s, shake mechanically at 500
rpm for 10 min, centrifuge at 3800 g for 10 min. Decant the supernatant, add 10 mL ethyl
acetate to pellet, vortex for 10 s, shake mechanically at 500 rpm for 10 min, centrifuge at 3800
g for 10 min. Combine the supernatants, add 25 mL hexane and mix well. Centrifuge at 3800
g for 5 min. Add combined centrifuged supernatants into SPE reservoir and aspirate through
SPE cartridge at flow rate of ca 2 mL/min. Add 5 mL ethyl acetate:hexane 4:5 to remaining
pellet, vortex for 10 s, centrifuge at 3800 g for 5 min. Add centrifuged liquid into SPE reservoir
and aspirate through SPE cartridge at ca. 2 mL/min. Remove SPE reservoir, wash SPE car-
tridge twice with 2 mL isooctane, let dry, dry under a stream of nitrogen for 20 min. Elute
twice with 2 mL portions of MeOH. Evaporate eluate to dryness under a stream of nitrogen
at 37. Reconstitute the residue in 1.0 mL mobile phase, vortex for 10 s, centrifuge at 3800 g
for 5 min. Inject a 50 |xL aliquot of the supernatant. (Prepare buffer as follows. Dissolve 44.5
g Na2HPO4. 2H2O in 450 mL water, adjust to pH 7.8 with 5 M NaOH and make up to 500 mL
with water.)
HPLC VARIABLES
Guard column: 10 X 2.1 20 jjim nonporous nonspherical C18 (Chrompack)
Column: 100 X 3.0 5 |xm ChromSpher B porous spherical C18 (Chrompack)
Mobile phase: MeCN:buffer 30:70 (Buffer was 10 mM NaH2PO4 adjusted to pH 6.2 with 500
mM NaOH.)
Flow rate: 0.5
Detector: UV 289
CHROMATOGRAM
Retention time: 6
Limit of detection: 1.4 (xg/kg
Limit of quantitation: 2.3 |xg/kg
OTHER SUBSTANCES
KEYWORDS
muscle; eel; SPE
REFERENCE
Hajee,C.A.J.; Haagsma,N. Liquid chromatographic determination of mebendazole and its metabolites, amino-
mebendazole and hydroxymebendazole, in eel muscle tissue, J.AOAC Int., 1996, 79, 645-651.
SAMPLE
Matrix: tissue
MeOH and dry under vacuum aspiration. Gently blend 2 g C18 material and 0.5 g liver in a
glass pestle for 1 min until homogeneous in appearance. Place in a 10 mL syringe barrel
plugged with filter paper (Whatman No. 1), cover with filter paper, compress to 4.5 mL, place
a 100 |xL pipette tip on the barrel to restrict flow, wash with 8 mL hexane, elute with 8 mL
MeCN. Pass the eluate through 0.5 g activated alumina (EM Science Type F-20 80-200 mesh)
between filter paper in a 10 mL syringe barrel (wash column with 4 mL MeCN just before use).
Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue in 100
fjuL MeOH and 400 JJLL 17 mM phosphoric acid, sonicate for 5-10 min, centrifuge at 17000 g for
5 min, filter the supernatant (0.45 (xm), inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 300 X 4 10 (Jim Micro Pak ODS (Varian)
Flow rate: 1
Detector: UV 290
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Extracted: albendazole, thiafendazole, oxfendazole, fenbendazole
KEYWORDS
matrix solid-phase dispersion; liver; mebendazole is IS
REFERENCE
Long,A.R.; Malbrough,M.S.; Hsieh,L.C; Short,C.R.; Barker,S.A. Matrix solid phase dispersion isolation and
JAssoc.OffAnalChem., 1990, 73, 860-863.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 2.8 mL 500 mg 40 jxm 60 A Bond Elut silica SPE cartridge
filter it (No. 41 paper), add 150 mL acetone to material remaining in blender, blend at low
speed for 2-3 min, filter, wash solid with 10 mL EtOH. Combine all the filtrates and evaporate
them to dryness under vacuum at 30-35 (beware of bumping). Rinse out flask with two 10 mL
portions of hexane and two 10 mL portions of 1 M phosphoric acid, combine rinses, shake
vigorously for 2 min, allow to separate for 10 min, extract the hexane layer twice more with
10 mL portions of 1 M phosphoric acid. Combine all the aqueous layers and wash them with
10 mL hexane, adjust the pH of the aqueous layer to 8.5 1.0 by slowly adding about 9 mL
10 M KOH while using an ice bath. Extract twice with 50 mL ethyl acetate (2 min shaking),
25 mL ethyl acetate. Combine the ethyl acetate layers, add 200 JJLL 10 mg/mL BHT in ethyl
filter (0.2 |xm), inject a 50 (JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jim C18 (Alltech)
Flow rate: 1
Detector: UV 298
CHROMATOGRAM
Retention time: 20
OTHER SUBSTANCES
Extracted: oxfendazole, thiabendazole
KEYWORDS
cow; liver; SPE
REFERENCE
LeVan,L.W.; Barnes,C. J. Liquid chromatographic method for multiresidue determination of benzimidazoles in
beef liver and muscle: collaborative study, JAssoc.Off.Anal.Chem., 1991, 74, 487-493.
SAMPLE
Matrix: tissue
Sample preparation: Condition a J.T.Baker 3 mL 500 mg silica SPE cartridge with 6 mL n-
hexane:ethyl acetate 2.5:1. 5 g Ground eel muscle tissue + 5 g sodium sulfate + 500 |xL 4 M
potassium carbonate solution + 10 mL ethyl acetate, vortex for 10 s, shake mechanically at
500 rpm for 10 min, centrifuge at 1300 g for 5 min, decant the supernatant, repeat the ex-
traction with 10 mL ethyl acetate. Combine the extracts, add 50 mL hexane (ratio of n-hexane
to ethyl acetate should be 2.5:1), add 5 g anhydrous sodium sulfate, shake, allow to stand until
solution becomes transparent, filter (S & S 589.1 paper), add the filtrate to the SPE cartridge,
rinse flask with 5 mL n-hexane:ethyl acetate 2.5:1, add the rinse to the SPE cartridge. Dry the
SPE cartridge in a stream of nitrogen for 10 min, elute with 3 mL MeOH:acetic acid 97:3.
Evaporate the eluate to dryness under a stream of nitrogen at 37, reconstitute the residue in
1 mL mobile phase, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 2.1 40 fxm ChromSep pellicular reversed-phase (stainless steel)
Column: 100 X 3 5 jim LiChrosorb RP-8 glass column
Mobile phase: MeCN:buffer 30:70, apparent pH 6.7 (Buffer was 50 mM (NH4)H2PO4 adjusted to
pH 6.2 with 10 M NaOH.)
Flow rate: 0.5
Detector: UV 311
CHROMATOGRAM
Retention time: 8
KEYWORDS
eel; muscle; SPE
REFERENCE
Steenbaar,J.G.; Hajee,C.A.J.; Haagsma,N. High-performance liquid chromatographic determination of the ant-
helmintic mebendazole in eel muscle tissue, J.Chromatogr., 1993, 615, 186-190.
Mecamylamine
Molecular formula: CnH21N
Merck Index: 5814
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.4
OTHER SUBSTANCES
isoxsuprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, meclophen-
oxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine, mesor-
idazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene, metho-
trimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine,
REFERENCE
Mechlorethamine
Molecular formula: C5H11CI2N
Merck Index: 5815
SAMPLE
Matrix: blood
Sample preparation: Condition a 2.4 mL 500 mg Bond Elut phenyl SPE cartridge with 2 mL
MeOH and 2 mL water. 1 mL Plasma + 100 JJLL freshly prepared 100 mg/mL diethyldithiocar-
bamic acid in 100 mM NaOH, heat at 37 for 30 min, add to the SPE cartridge, wash with 5
mL water, allow to dry in the dark at room temperature for 1 h, elute with 2 mL MeCN.
Evaporate the eluate to dryness under reduced pressure at 40, reconstitute in 200 |xL MeOH,
HPLC VARIABLES
Mobile phase: Gradient. MeCN:5 mM pH 3.0 orthophosphoric acid 30:70 for 4 min, to 100:0 over
9 min, return to initial conditions over 7 min, re-equilibrate for 5 min.
Flow rate: 1
Detector: UV 276
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: galamustine
KEYWORDS
REFERENCE
Cummings,J.; MacLellan,A.; Smyth,J.F.; Farmer,P.B. Determination of reactive nitrogen mustard anticancer
drugs in plasma by high-performance liquid chromatography using derivatization, Anal.Chem., 1991, 63,
1514-1519.
SAMPLE
Sample preparation: 0.1 g Ointment + 8 mL chloroform + 1.9 mL isopropanol + 100 |xL water,
shake until homogeneous, add 10 mL 10 mM HCl, invert twice for 2-3 min with a 10 min
interval. Remove the aqueous phase and neutralize it with 1 mL 100 mM NaOH, add 1 mL
100 mg/mL diethyldithiocarbamic acid in 100 mM NaOH, heat at 37 for 1 h. Remove a 1 mL
aliquot and add it to 1 mL MeOH, inject a 100 |xL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 276
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
ointment; derivatization; chromatographic procedure as (Anal.Chem. 1991; 63; 1514)
REFERENCE
Cummings,J.; MacLellan,A.; Langdon,S.J.; Smyth,J.F. The long term stability of mechlorethamine hydrochlo-
ride (nitrogen mustard) ointment measured by HPLC, J.Pharm.Pharmacol., 1993, 45, 6-9.
SAMPLE
HPLC VARIABLES
Flow rate: 1
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: granisetron (UV 300), morphine (UV 300)
KEY WORDS
REFERENCE
304.
Meclizine
CAS Registry No.: 569-65-3, 31884-77-2
(di HCI monohydrate), 1104-22-9 (di HCI)
Merck Index: 5817
Lednicer No.: 1 59
SAMPLE
Matrix: blood
Sample preparation: 4 mL Plasma + 15 mL toluene, shake by hand, centrifuge, discard organic
layer, add 3 mL 1 M NaOH to the aqueous layer, add 15 mL diethyl ether, centrifuge. Remove
the ether layer and evaporate it to dryness under a stream of nitrogen. Reconstitute in 230 |xL
MeCN:40 mM sodium dodecyl sulfate + 2.5% glacial acetic acid adjusted to 4.3 with ammonium
hydroxide 80:20, inject a 200 |JLL aliquot.
HPLC VARIABLES
Guard column: 30 X 3.9 Corasil
Mobile phase: Gradient. A was 8 mM sodium dodecyl sulfate in water with 0.5% glacial acetic
acid, pH adjusted to 4.3 with ammonium hydroxide. B was MeCN:40 mM sodium dodecyl
sulfate + 2.5% glacial acetic acid adjusted to 4.3 with ammonium hydroxide 80:20. A:B 22:78
until meclizine eluted then to 0:100 for 8 min
Flow rate: 1.2
Detector: UV 232
CHROMATOGRAM
Retention time: 12
KEYWORDS
plasma; rat; dog
REFERENCE
Chovan,J.R; Klett,R.R; Rakieten,N. Comparison of meclizine levels in the plasma of rats and dogs after in-
tranasal, intravenous, and oral administration, J.Pharm.ScL, 1985, 74, 1111-1113.
SAMPLE
Sample preparation: Blood. Centrifuge 200 |xL fresh blood at 3000 rpm for 10 min. Inject an
aliquot of the plasma. Formulations. Completely dissolve 50 mg sample in 20 mL MeOH, son-
icate. Filter insoluble material and adjust filtrate to 50 mL with MeOH. Inject an aliquot of
the filtrate.
HPLC VARIABLES
Column: 150 X 4.6 Cosmosil 5C18 (Nacalai Tesque, Japan)
Mobile phase: MeOH: 100 mm pH 6.2 acetate buffer 90:10
Flow rate: 1.5
Detector: UV 232
CHROMATOGRAM
Internal standard: pyrene
KEYWORDS
freeze-dried formulations; plasma; rat; egg albumin; olive oil
REFERENCE
Tsuji,Y; Kakegawa,H.; Miyataka,H.; Nishiki,M.; Matsumoto,H.; Satoh,T. Pharmaceutical properties of freeze-
dried formulations of egg albumin, several drugs and olive oil, Biol.Pharm.BulL, 1996, 19, 636-640.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 3.9 5 |xm Symmetry C8
Mobile phase: MeCN:water:triethylamine:100 mM citrate buffer 40:55:0.05:5
Detector: UV(?)
REFERENCE
Lambropoulos,J.; Spanos,G.A.; Lazaridis,N.V. Method development and validation for the HPLC assay in pa-
roxetine 20 mg strength tablets (Abstract 3391), Pharm.Res., 1997, 14, S591.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.5
OTHER SUBSTANCES
amine, meclophenoxate, medazepam, mephentermine, mepivacaine, meptazinol, mepyramine,
nine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyl-
diamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine,
REFERENCE
Jane,L; McKinnon,A-; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica
SAMPLE
Matrix: solutions
uL aliquot.
HPLCVARIABLES
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
pam, lormetazepam, loxapine, mazindol, meclofenamic acid, medazepam, mefenamic acid, me-
pivacaine, mescaline, mesoridazine, methadone, methamphetamine, methapjrrilene,
oxyphenbutazone, oxêtracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbi-
puromycin, pîlamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
faethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapîdine, sul-
REFERENCE
Hill,D.W.; Kind A-J- Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Vydac 201HS54 C18
Mobile phase: Gradient MeCN:25 mM pH 3.6 phosphate buffer from 20:80 to 70:30 over 20 min
Flow rate: 1.5
Detector: UV 220 (from Vydac Applications Brochure)
CHROMATOGRAM
Retention time: 16
OTHER SUBSTANCES
Simultaneous: chlorcyclizine, tripelennamine, triprolidine, methaphenilene, pyrrobutamine, cy-
clizine, buclizine
REFERENCE
Vydac HPLC Catalog, 1994-5, p. 26.
Meclocycline
CAS Registry No.: 2013-58-3, 73816-42-9 (sulfosalicylate)
Merck Index: 5818
SAMPLE
Sample preparation: Weigh out cream containing 5 mg meclocycline, add 20 mL MeOH, add
20 mL 12.5 mM sulfuric acid, sonicate for 1 h, vortex for 2 min until cream is thoroughly
dispersed, make up to 50 mL with MeOH, centrifuge at 2000 g for 10 min. Remove a 5 mL
aliquot of the supernatant and make it up to 50 mL with mobile phase, filter (0.5 fxm, Millipore
Fluorophore FHL P04700), inject a 10 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 3.2 10 |xm 201 TP reverse phase (Vydac)
Mobile phase: THF:buffer 15:85 (Prepare buffer by dissolving 0.6 g EDTA in 2 mL MeOH in 15
mL concentrated ammonium hydroxide, add 1.8 L water, adjust pH to 6.6 with glacial acetic
acid, make up to 2 L with water. Periodically purge column with 95% EtOH.)
Flow rate: 0.8
Detector: UV 340
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Simultaneous: methacycline, C-4-epimeclocycline
KEYWORDS
cream
REFERENCE
Depaolis,A.M.; Britt,T.E.; Holman,A.J.; McGonigle,E.J.; Kaplan,G.; Davies,W.C. Determination of meclocycline,
a tetracycline analogue, in cream formulations by liquid chromatography, J.Pharm.Sci., 1984, 73, 1650-
1651.
Meclofenamic acid
Merck Index: 5819
Lednicer No.: 1 110
SAMPLE
Sample preparation: 100 |xL Aqueous humor + 500 |xL MeCN + 30 |xL 400 ng/mL (+)-naproxen
and dry it under nitrogen at room temperature, dissolve the residue in 50 fxL mobile phase by
swirl-mixing for 1 min, centrifuge at 3000 g for 20 s, reduce volume to 20-30 jxL, inject an
aliquot.
HPLCVARIABLES
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diclofenac, flurbiprofen, indomethacin
KEYWORDS
human; rabbit
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: 1.5 mL Plasma + 2 mL 150 ng/mL diclofenac sodium in 100 mM pH 4.6
sodium citrate buffer, mix, add 5 mL dichloromethane, rotate for 30 min, centrifuge. Remove
3 mL of the organic layer and evaporate it to dryness, reconstitute the residue in 200 jxL eluent,
HPLC VARIABLES
Column: 100 X 3 10 jxm Spherisorb ODS
Mobile phase: MeOH: 100 mM pH 6.1 sodium phosphate buffer 40:60
Flow rate: 0.8
Detector: UV 280
CHROMATOGRAM
Retention time: 10
Internal standard: diclofenac sodium (5.5)
OTHER SUBSTANCES
Simultaneous: flunixin, mefenamic acid
KEYWORDS
plasma; horse; pharmacokinetics
REFERENCE
JohanssonJ.M.; Eklund,M.-L. Liquid chromatographic determination of meclofenamic acid in equine plasma,
J.Liq.Chromatogr., 1984, 7, 1609-1626.
SAMPLE
Matrix: blood
Sample preparation: 500 (xL Plasma + 7.5 mL chloroform + 200 |xL 6 M HCl + 50 |xL 100 jxg/
mL (?) diclofenac in MeOH, shake for 2 min. Remove the organic layer and evaporate it to
dryness under vacuum at 45, reconstitute the residue in 500 |xL mobile phase, vortex for 1
min, inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeOH:water 60:40, pH adjusted to 3.0 with acetic acid
Flow rate: 1.5
Detector: UV 270
CHROMATOGRAM
Retention time: 5.9
Internal standard: diclofenac (3.6)
KEYWORDS
REFERENCE
Niazy,E.M.; Khidr,S.H.; El-Sayed,Y.M. High-performance liquid chromatographic determination of meclofena-
mate in plasma, J.Liq.Chromatogr., 1994, 17, 2331-2341.
SAMPLE
Sample preparation: Dilute urine with an equal volume of water. 1 mL Plasma or diluted urine
+ 500 JXL 6 M HCl + 10 mL toluene:EtOH 80:20, shake horizontally at slow speed for 15 min,
centrifuge at 2000 rpm for 10 min. Remove 8 mL of the organic layer and evaporate it to
dryness under a stream of nitrogen at 50, reconstitute the residue in 200 (plasma) or 500
(urine) |xL MeOH:water:acetic acid 70:30:0.5, inject a 20 (plasma) or 30 (urine) JJLL aliquot. (To
assay total concentration add 1 mL 2 M pH 5.2 acetate buffer and 25 |xL p-glucuronidase (Helix
pomatia, Type H-I, 500000 units, Sigma) to 1 mL plasma or diluted urine, heat at 37 for 24
h, add 6 M HCl and proceed as above.)
HPLC VARIABLES
Mobile phase: MeOH:water 70:30 (plasma) or 64:36 (urine) containing 5 mM tetrabutylammon-
ium phosphate
Flow rate: 0.6
Detector: UV 340
CHROMATOGRAM
Retention time: 25 (plasma)
Internal standard: 3,5-dichloroanthranilic acid (11.3)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Koup,J.R.; Tucker,E.; Thomas,D.J.; Kinkel,A.W.; Sedman,A.J.; Dyer,R.; Sharoky,M. A single and multiple dose
pharmacokinetic and metabolism study of meclofenamate sodium, Biopharm.Drug Dispos., 1990,11, 1-15.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
tilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, iso-
xsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam,
lormetazepam, loxapine, mazindol, mebendazole, medazepam, mefenamic acid, megestrol, me-
REFERENCE
18, 233-242.
Meclofenoxate
Merck Index: 5620
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 4 X 4 5 |xm LiChrospherlOORP-18
Column: 125 X 4 5 |xm Spherisorb ODS 2
Mobile phase: Gradient. MeCN:buffer from 15:85 to 30:70 over 5 min. (Buffer was 20 mM sodium
acetate containing 0.28% triethylamine, adjusted to pH 4.5 with acetic acid.)
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: 4-chlorophenoxyacetic acid
REFERENCE
Yang,H-l Thyrion,F.C. Determination of six pharmaceuticals and their degradation products in reversed-phase
high performance liquid chromatography by using amine additives, J.Liq.Chromatogr.Rel.TechnoL, 1998,
21, 1347-1357.
SAMPLE
Matrix: solutions
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.4
OTHER SUBSTANCES
amine, meclozine, medazepam, mepnentermine, mepivacaine, meptazinol, mepyramine, me-
soridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene, meth-
otrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine,
REFERENCE
Medazepam
Merck Index: 5829
Lednicer No.: 1 368
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 255
CHROMATOGRAM
KEYWORDS
penfluridol; bepridil; terfenadine; trifiuoperazine
REFERENCE
TracquiA-J Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.0
OTHER SUBSTANCES
mine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, fiurazepam, hal-
amine, meclophenoxate, meclozine, mephentermine, mepivacaine, meptazinol, mepyramine,
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
pam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, mefenamic acid, megestrol,
santine, phenacetin, phenazocine, phenazopyiidine, phencyclidine, phendimetrazine,
puromycin, pyrilamine, p5rrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
metin, tranylc5rpromine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine,
trihexj^phenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tâmine, verapa-
REFERENCE
18, 233-242.
Medifoxamine
Merck Index: 5834
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 266
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Sample preparation: 1 mL Plasma or urine + 50 JJLL 100 (plasma) or 1000 (urine) (xg/mL 5,6-
benzoquinoline in water + 50 (xL 4 M NaOH + 5 mL diethyl ether, shake gently for 10 min,
centrifuge at 350 g for 5 min. Remove the organic layer and evaporate it to dryness at 70,
HPLC VARIABLES
Column: 150 X 5 5 jxm Spherisorb silica
Mobile phase: MeOH:60% perchloric acid 100:0.02
Flow rate: 2
Detector: UV 266
CHROMATOGRAM
Retention time: 3
Internal standard: 5,6-benzoquinoline (6)
KEYWORDS
REFERENCE
Saleh,S.; Johnston A-I Chanon,M.; Turner,R Determination of medifoxamine in plasma and urine by high-
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 1 mL pH 10 borate-NaOH buffer + 50 |xL 1 jxg/L (sic) 5,6-
benzoquinoline in water + 6 mL ethyl acetate:diethyl ether, shake gently for 10 min, centrifuge
at 350 g for 5 min. Remove the organic layer and evaporate it to dryness at 70, reconstitute
the residue in 120 |xL mobile phase, inject an aliquot. Hydrolyze conjugates as follows. 1 mL
Urine + 1 mL buffer + 100 |xL 1000 U/mL p-glucuronidase (from Helix pomatia, Sigma) in
0.2% NaCl, shake gently, heat at 37 for 24 h. (Buffer was 681 mg sodium acetate trihydrate
in 40 mL water, equilibrate at 37, adjust pH to 5 with 1 M HCl, make up to 50 mL with
water.)
HPLCVARIABLES
Column: 150 X 5 5 |xm Hypersil silica 5 ODS
Mobile phase: MeOH:buffer 56:40 (Buffer was 2.375 g Na2HPO4 and 0.135 g KH2PO4 in 400 mL
water, pH 8.)
Flow rate: 2
Detector: UV 266
CHROMATOGRAM
Internal standard: 5,6-benzoquinoline (5.7)
OTHER SUBSTANCES
REFERENCE
Saleh,S.; Johnston,A.; Turner,P. Measurement of medifoxamine metabolites in urine by high-performance liquid
Medroxyprogesterone
Merck Index: 5838
Lednicer No.: 1 180, 2 165
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 1 mL 0.5 |xg/mL 16p-methylprogesterone in 200 mM pH
7.0 phosphate buffer, vortex for 3 s, add 7 mL hexane, mix on a rolling mixer for 30 min.
Remove the hexane layer and evaporate it to dryness at 30 under nitrogen. Dissolve residue
in 200 |xL mobile phase, inject a 150 (xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherisorb 5-ODS2
Mobile phase: MeOH: 20 mM pH 4 acetate buffer 79:21
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: 5.3 (medroxyprogesterone acetate)
Internal standard: 16p-methylprogesterone (9.0)
OTHER SUBSTANCES
Extracted: progesterone, 17a-hydroxyprogesterone, cortisone, corticosterone, aldosterone, tes-
tosterone, androstenedione, estradiol
Noninterfering: cholesterol
Interfering: lignocaine
KEYWORDS
plasma
REFERENCE
Read,J.; Mould,G.; Stevenson,D. Simple high-performance liquid chromatographic method for the determina-
tion of medroxyprogesterone acetate in human plasma, J.Chromatogr., 1985, 341, 437-444.
SAMPLE
Matrix: blood
Sample preparation: Extract 0.1-1 mL plasma twice with 40 volumes of diethyl ether, evaporate
the organic solvent, dissolve the residue in 100 |xL MeOH.water 80:20, inject a 30 (JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |jim Beckman RP-ODS
Mobile phase: Gradient. MeCNiwater from 40:60 to 70:30 over 20 min, then at 70:30 for 5 min
Detector: UV 238
CHROMATOGRAM
OTHER SUBSTANCES
KEY WORDS
plasma
REFERENCE
Sturm,G.; Haberlein,H-; Bauer,T.; Plaum,T.; Stalker,D.J. Mass spectrometric and high-performance liquid chro-
matographic studies of medroxyprogesterone acetate metabolites in human plasma, J.Chromatogr., 1991,
562, 351-362.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 241.7
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Suspensions. Dilute 2 mL suspension to 1 L with EtOH, remove a 2.5 mL
aliquot and add it to 1 mL 1 mg/mL hydrocortisone in EtOH. Dilute this mixture to 50 mL
with EtOH, inject an aliquot. Tablets. Grind tablets to a fine powder, stir with 50 mL EtOH,
make up to 100 mL with EtOH, filter (Whatman No. 1 paper), reject the first 20 mL of the
filtrate. Mix 2 mL of the filtrate with 1 mL 1 mg/mL hydrocortisone in EtOH, make this mixture
up to 50 mL with EtOH, inject an aliquot.
HPLC VARIABLES
Column: 300 x 4 ixBondapak CN
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Internal standard: hydrocortisone (2.5)
OTHER SUBSTANCES
Simultaneous: progesterone, benzyl benzoate
Noninterfering: polyethylene glycol 4000, mystristyl-gamma-picolinium chloride, methylcellu-
lose, thimerosal
REFERENCE
Das Gupta,V. Quantitation of hydroxyprogesterone caproate, medroxyprogesterone acetate, and progesterone
by reversed-phase high-pressure liquid chromatography, J.Pharm.Sci., 1982, 71, 294-297.
SAMPLE
Sample preparation: Grind tablets to a fine powder, weigh out an amount equivalent to about
10 mg medroxyprogesterone acetate, add 10 mL MeOH, sonicate for 5 min, dilute to 50 mL
with MeOH. Dilute 25 mL of this solution to 50 mL with MeOH, filter (0.45 |xm), inject a 20
IxL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 5 p-m Novapak C18
Mobile phase: MeOH: 10 mM (NHJ 2 HPO 4 80:20, adjust pH to 7.2 0 . 1 with 85% phosphoric
acid
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
KEYWORDS
REFERENCE
Fatmi,A.A.; Williams,G.V.; Hickson,E.A. Liquid chromatographic determination of medroxyprogesterone ace-
tate in tablets, J.Assoc.Off.Anal.Chem., 1988, 71, 528-530.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 jxg/mL, inject a 10
IxL aliquot.
HPLC VARIABLES
Flow rate: 1.5
Next Page
Detector: UV
CHROMATOGRAM
Retention time: k' 3.12 (medroxyprogesterone acetate)
REFERENCE
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Waring blender) tissue at full speed for 2 min, lyophilize,
grind. Extract with supercritical carbon dioxide at 60 at 400 atmospheres with a 20 cm X 21
|xm restrictor for 1 h, collect the extract in 1 mL MeOH cooled to 5. Evaporate the MeOH to
dryness under a stream of nitrogen, reconstitute the residue in 100 |xL MeCN:MeOH:20 mM
ammonium formate 15:15:70, inject an aliquot. Alternatively, vortex 5 g ground tissue with 10
mL 40 mM sodium acetate, adjust pH to 4.2-4.7 with glacial acetic acid, add 100 u,L p-glucu-
ronidase (Sigma), heat at 37 for 8 h, add 20 mL MeCN, vortex for 30 s, centrifuge at 5000
rpm for 20 min. Remove a 30 mL aliquot of the supernatant and add it to 8 mL hexane and 2
mL dichloromethane, rotate for 3 min, centrifuge at 2000 rpm for 2 min. Remove a 15 mL
aliquot of the middle layer and evaporate it to dryness under a stream of nitrogen, reconstitute
the residue in 1 mL dichloromethane, inject an aliquot.
HPLC VARIABLES
Column: 50 X 4.6 5 jxm Supelcosil
Mobile phase: Gradient. MeCN:MeOH:20 mM ammonium formate from 2.5:2.5:95 to 47.5:47.5:
5 over 19 min.
Flow rate: 1
Detector: UV 245 or MS, Sciex TAGA 6000E tandem triple quadrupole, APCI
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: dexamethasone, diethylstilbestrol, melengestrol acetate, trenbolone, triamcinolone
acetonide, zeranol
KEYWORDS
cow; muscle; liver; SFE
REFERENCE
Huopalahti,R.P.; Henion,J.D. Application of supercritical fluid extraction and high performance liquid chro-
matography/mass spectrometry for the determination of some anabolic agents directly from bovine tissue
samples, J.Liq.Chromatogr.Rel.Technol, 1996, 19, 69-87.
Mefenamic acid
Merck Index: 5842
Lednicer No.: 1 10
SAMPLE
Matrix: blood
Previous Page
Detector: UV
CHROMATOGRAM
Retention time: k' 3.12 (medroxyprogesterone acetate)
REFERENCE
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Waring blender) tissue at full speed for 2 min, lyophilize,
grind. Extract with supercritical carbon dioxide at 60 at 400 atmospheres with a 20 cm X 21
|xm restrictor for 1 h, collect the extract in 1 mL MeOH cooled to 5. Evaporate the MeOH to
dryness under a stream of nitrogen, reconstitute the residue in 100 |xL MeCN:MeOH:20 mM
ammonium formate 15:15:70, inject an aliquot. Alternatively, vortex 5 g ground tissue with 10
mL 40 mM sodium acetate, adjust pH to 4.2-4.7 with glacial acetic acid, add 100 u,L p-glucu-
ronidase (Sigma), heat at 37 for 8 h, add 20 mL MeCN, vortex for 30 s, centrifuge at 5000
rpm for 20 min. Remove a 30 mL aliquot of the supernatant and add it to 8 mL hexane and 2
mL dichloromethane, rotate for 3 min, centrifuge at 2000 rpm for 2 min. Remove a 15 mL
aliquot of the middle layer and evaporate it to dryness under a stream of nitrogen, reconstitute
the residue in 1 mL dichloromethane, inject an aliquot.
HPLC VARIABLES
Column: 50 X 4.6 5 jxm Supelcosil
Mobile phase: Gradient. MeCN:MeOH:20 mM ammonium formate from 2.5:2.5:95 to 47.5:47.5:
5 over 19 min.
Flow rate: 1
Detector: UV 245 or MS, Sciex TAGA 6000E tandem triple quadrupole, APCI
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: dexamethasone, diethylstilbestrol, melengestrol acetate, trenbolone, triamcinolone
acetonide, zeranol
KEYWORDS
cow; muscle; liver; SFE
REFERENCE
Huopalahti,R.P.; Henion,J.D. Application of supercritical fluid extraction and high performance liquid chro-
matography/mass spectrometry for the determination of some anabolic agents directly from bovine tissue
samples, J.Liq.Chromatogr.Rel.Technol, 1996, 19, 69-87.
Mefenamic acid
Merck Index: 5842
Lednicer No.: 1 10
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 1 M hydrochloric acid + 6 mL dichloromethane,
shake for 20 min. Centrifuge at 2000 g for 5 min, evaporate organic phase to dryness under a
gentle stream of nitrogen at 40. Reconstitute the residue with 50 |xL MeOH, inject a 20 u,L
aliquot.
HPLC VARIABLES
Column: 5 |jim Nucleosil C18
Flow rate: 0.8
Detector: UV 280
CHROMATOGRAM
Retention time: 8.6
KEYWORDS
plasma; rat; mefenamic acid is IS
REFERENCE
Cerretani,D.; Micheli,L.; Fiaschi,L.; Giorgi,G. High-performance liquid chromatography of flufenamic acid in
rat plasma, J.Chromatogr.B, 1996, 678, 365-368.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 50 |JLL 1 M HCl, add 4 mL hexane:acetone 85:15, vortex
for 30 s, centrifuge at 2000 g for 5 min, repeat extraction. Combine the organic layers and
IxL MeCN:20 mM pH 5 phosphate buffer 35:65, inject a 200 JJLL aliquot onto column A with
mobile phase A, collect the eluate containing mefenamic acid in a sample loop and inject it
onto column B with mobile phase B. Collect the eluate containing mefenamic acid in a sample
loop and inject it onto column C with mobile phase C, monitor the effluent from column C.
HPLC VARIABLES
Column: A 70 x 4.6 5 ^m YMC ODS A type (Yakamura Chemical); B 70 X 4.6 5 [im. YMC ODS
A type (Yakamura Chemical); C 150 X 4.6 5 |xm TSK gel ODS 80 TM (Tosoh)
Mobile phase: A MeCN:20 mM pH 5 phosphate buffer 35:65; B MeCN:20 mM pH 3.5 phosphate
buffer 45:55; C MeCN:20 mM pH 6 phosphate buffer 45:55
Flow rate: 1
Detector: UV 219
CHROMATOGRAM
Retention time: 39
KEYWORDS
serum; column-switching; heart-cut
REFERENCE
Yamashita,K; Motohashi,M.; Yashiki,T. Column-switching techniques for high-performance liquid chromatog-
raphy of ibuprofen and mefenamic acid in human serum with short-wavelength ultraviolet detection,
J.Chromatogr., 1991, 570, 329-338.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 100 u-L 4 |xg/mL methylclonazepam in 100 mM HCl +
3 mL diethyl ether, vortex for 30 s, centrifuge at 4 at 2000 g for 10 min. Remove the organic
layer and evaporate it to dryness under a stream of air at room temperature, reconstitute the
residue in 100 u-L MeOH, inject a ^10 |xL aliquot.
HPLC VARIABLES
Guard column: 10 mm long 10 jxm CN (Merck)
Column: 250 X 4 10 jxm CN (Merck)
Mobile phase: MeCN:MeOH:water:17 M acetic acid 15:15:69:1
Flow rate: 1.5
Injection volume: ^10
Detector: UV 290
CHROMATOGRAM
Retention time: 6.9
Internal standard: methylclonazepam (4.8)
OTHER SUBSTANCES
Noninterfering: carbamazepine, clonazepam, ketoprofen, phenobarbital, phenytoin, salicylic
acid, valproic acid
Interfering: indomethacin
KEYWORDS
REFERENCE
Poirier,J.-M.; Lebot,M.; Cheymol,G. Rapid and sensitive liquid chromatographic assay of mefenamic acid in
plasma, Ther.Drug Monit, 1992, 14, 322-326.
SAMPLE
Matrix: blood
Sample preparation: 0.5 mL Plasma + 10 |JLL 50 jig/mL mefenamic acid + 50 JJLL 85% phosphoric
acid, vortex 10 sec, add 3 mL chloroform, vortex 1 min, centrifuge at 6000 rpm for 5 min.
Remove organic layer and evaporate it to dryness at 45 under a stream of nitrogen. Vortex
residue with 200 JJLL mobile phase for 10 s, inject 50 |xL aliquot.
HPLCVARIABLES
Guard column: 30-40 |xm C18 pellicular
Column: 150 X 3.9 Novapak C18
Mobile phase: MeCNiwater 50:50 adjusted to pH 3.5 with glacial acetic acid
Flow rate: 1.5
Detector: UV 278
CHROMATOGRAM
Retention time: 6.3
OTHER SUBSTANCES
Simultaneous: diclofenac
KEYWORDS
plasma; dog; mefenamic acid is IS
REFERENCE
Mohamed,F.A.; Jun,H.W.; Elfaham,T.H.; Sayed,H.A.; Hafez,E. An improved HPLC procedure for the quanti-
tation of diclofenac in plasma, J.Liq.Chromatogr., 1994, 17, 1065-1088.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Plasma + 250 |xL 0.6 u-g/mL indomethacin in MeCN + 50 uL MeCN,
vortex, centrifuge at 9000 g for 3 min. Remove 250 uL of the supernatant and evaporate it to
dryness under vacuum, dissolve the residue in 50 (xL mobile phase, inject a 20 (xL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 Alltech 5 fim C18 bonded-phase silica
Column: 250 X 4.6 Vydac column packed with Merck 5 |xm C18 bonded-phase silica
Mobile phase: MeCNrIO mM phosphoric acid 60:40, pH 2.6
Flow rate: 0.9
Detector: UV 280
CHROMATOGRAM
Retention time: 9.2
KEYWORDS
plasma
REFERENCE
NiopasJ.; Mamzoridi,K. Determination of indomethacin and mefenamic acid in plasma by high-performance
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 220
CHROMATOGRAM
KEYWORDS
lidoflazine; ibuprofen; fioctafenine; alpidem; loratadine; chlorpromazine; clomipramine; carpi-
penfiuridol; bepridil; terfenadine; trifluoperazine
REFERENCE
254-262.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 200 |xL 2 M HCl + 5 mL dichloromethane, rotate for 10
min, centrifuge at 5000 rpm for 8 min. Remove 4 mL of the organic layer and evaporate it to
dryness under a stream of nitrogen, reconstitute the residue in 250 |xL plasma mobile phase,
inject a 100 |AL aliquot.
HPLC VARIABLES
Guard column: 4 x 4 4 |xm Lichrocart C18 (Merck)
Column: 50 X 4 4 ^m Lichrocart C18 (Merck)
Mobile phase: MeCN:100 mM pH 7.4 phosphate buffer:triethylamine 25:75:0.02
Flow rate: 1
Detector: UV 282
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: fluvastatin
KEYWORDS
plasma; mefenamic acid is IS
REFERENCE
Transon,C; Leemann,T.; Vogt,N.; Dayer,P. In vivo inhibition profile of cytochrome P450TB (CYP2C9) by ()-
fluvastatin, Clin.Pharmacol.Ther., 1995, 58, 412-417.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 50 |xL 500 ng/mL indomethacin + 1 mL 100 mM
HCl + 10 mL dichloromethane, rotate for 10 min, centrifuge at 1500 g for 15 min. Remove the
organic layer and evaporate it to dryness under a stream of nitrogen at 45. Redissolve the
residue in mobile phase, inject a 20 (xL aliquot. Urine. 50 JJLL Urine + 1 mL mobile phase, inject
a 20 |xL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 235
CHROMATOGRAM
Retention time: 8
Internal standard: indomethacin (5)
OTHER SUBSTANCES
Simultaneous: naproxen, flunixin, thiosalicylic acid, ethacrynic acid, phenylbutazone
KEYWORDS
plasma
REFERENCE
Singh,A.K; Jang,Y.; Mishra,U.; Granley,K. Simultaneous analysis of flunixin, naproxen, ethacrynic acid, in-
mance liquid chromatography and gas chromatography-mass spectrometry, J.Chromatogr., 1991,568, 351-
361.
SAMPLE
Sample preparation: Add 150 |xL MeCN to 250 |xL microsomal incubation, vortex vigorously,
filter (0.45 |xm), inject an aliquot
HPLC VARIABLES
Column: 250 X 5 Spherisorb C8
Mobile phase: Gradient. A was MeCN. B was 50 mM pH 4.5 ammonium acetate buffer. A:B from
20:80 to 60:40 over 20 (?) min.
Detector: UV 280
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
Extracted: metabolites, mefenamic acid 1-O-acyl glucuronide
REFERENCE
McGurk,K.A.; Remmel,R.R; Hosagrahara,V.R; Tosh,D.; Burchell,B. Reactivity of mefenamic acid 1-O-acyl glu-
curonide with proteins in vitro and ex vivo, Drug Metab.Dispos., 1996, 24, 842-849.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Spherisorb S10-ODS2
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
REFERENCE
Galia,E.; Nicolaides,E.; H6rter,D.; L6benberg,R.; Reppas,C; Dressman,J.B. Evaluation of various dissolution
SAMPLE
Matrix: solutions
Sample preparation: Dissolve compounds in MeOH, inject a 1 |xL aliquot.
HPLC VARIABLES
Column: 150 X 1 3 jxm Hitachi-Gel 3011 porous polymer (Hitachi)
Mobile phase: MeOH.ammonia 99:1
Flow rate: 0.03
Injection volume: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: acetaminophen, caffeine, bucetin (3-hydroxy-p-butyrophenetidine), phenacetin,
dipyrone (sulpyrin), aspirin, salicylamide, salicylic acid, ethenzamide (o-ethoxybenzamide),
theobromine, theophylline
KEYWORDS
semi-micro; porous polymer
REFERENCE
Matsushima,Y.; Nagata,Y.; Niyomura,M.; Takakusagi,K; Takai,N. Analysis of antipyretics by semimicro liquid
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 500 |xg/mL solution in MeOH:water 50:50, inject a 5 |xL aliquot.
HPLC VARIABLES
column.)
Flow rate: 2
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
Retention time: 27
OTHER SUBSTANCES
Simultaneous: acetophenone, amphetamine, desipramine, ethylmorphine, imipramine, meth-
amphetamine, morphine, phenylbutazone, salicylic acid
KEYWORDS
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Inject a 50 |xL aliquot of a solution in mobile phase.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Spheri-5 RP-8
Mobile phase: MeOH:buffer 30:70 (Prepare buffer by mixing 4 mM Na2HPO4 and 7 mM KH2PO4
to achieve pH 7.)
Flow rate: 1
Detector: F ex 355 em 460 (408 nm cutoff filter) following post-column extraction. The column
effluent mixed with 50 jxg/mL reagent in mobile phase pumped at 0.5 mL/min and then with
chloroform pumped at 1 mL/min and the mixture flowed through a 1.8 m X 0.3 mm ID knitted
PTFE coil to a 50 |xL membrane phase separator using a polyethylene-backed 0.5 |xm Fluo-
ropore membrane filter (design in paper). The organic phase flowed to the detector. (Synthesize
the reagent, a-(3,4-dimethoxyphenyl)-4>-trimethylammoniummethylcinnamonitrile methosul-
fate, as follows. Stir 20 mmoles 3,4-dimethoxyphenylacetonitrile and 20 mmoles p-toluamide
in 50 mL EtOH at 50, add 5 mL 50% aqueous KOH slowly, stir at 50 for 5 min, cool to room
temperature, filter, dry the precipitate of a-(3,4-dimethoxyphenyl)-4'- methylcinnamonitrile.
Dissolve 20 mmoles a-(3,4-dimethoxyphenyl)-4>-methylcinnamonitrile, 20 mmoles N-bromosuc-
cinimide, and 20 mg benzoyl peroxide in 100 mL carbon tetrachloride (Caution! Carbon tet-
rachloride is a carcinogen!), reflux with stirring for 1.5 h , cool, filter, evaporate to dryness
under reduced pressure, recrystallize from MeOH to give a-(3,4-dimethoxyphenyl)-4'-bromo-
methylcinnamonitrile. Vigorously stir 30 mmoles anhydrous dimethylamine in 100 mL dry
benzene (Caution! Benzene is a carcinogen!) at 0, very slowly add 10 mmoles a-(3,4-dime-
thoxyphenyl)-4'-bromomethylcinnamonitrile while stirring at 0, stir at room temperature over-
night, add 150 mL water, remove the organic phase, extract the aqueous phase twice with 100
mL portions of diethyl ether, wash the organic layers with saturated NaCl solution, dry over
anhydrous magnesium sulfate, evaporate under reduced pressure to give a-(3,4-dimethoxy-
phenyl)-4'-dimethylaminomethylcinnamonitrile (J.Chem.Eng.Data 1987, 32, 387). Reflux 10
mmoles a-(3,4-dimethoxyphenyl)-4'-dimethylaminomethylcinnamonitrile, 20 mmoles dimethyl
sulfate (Caution! Dimethyl sulfate is a carcinogen and acutely toxic!), and 5 g potassium car-
bonate in 50 mL acetone for 1 h, cool to room temperature, filter, dry the precipitate under
vacuum at room temperature overnight, recrystallize from chloroform containing 2-3 drops of
95% EtOH to give a-(3,4-dimethoxyphenyl)-4>-trimethylammoniummethylcinnamonitrilemeth-
osulfate (mp 212-215). Protect solutions from light.)
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: ibuprofen, ketoprofen, naproxen, probenecid, salicylic acid, valproic acid
KEYWORDS
post-column extraction; post-column reaction
REFERENCE
Kim,M.; Stewart,J.T. HPLC post-column ion-pair extraction of acidic drugs using a substituted a-phenylcin-
namonitrile quaternary ammonium salt as a new fluorescent ion-pair reagent, J.Liq.Chromatogr., 1990,13,
213-237.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
chlorpromazine, clenbuterol, cortisone, danazol, diflunisal, doxapram, estrone, fluoxymester-
one, methyltestosterone, nicotine, oxazepam, phentermine, phenylpropanolamine, progester-
one, sulfamethazine, sulfanilamide, testosterone, testosterone propionate, tranylcypromine,
tripelennamine
KEYWORDS
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHERSUBSTANCES
pam, lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, meges-
trol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepi-
vacaine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene,
REFERENCE
Hill,D.W.; Kind,A-<L Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 50 |xg/mL solution in the mobile phase, inject a 10 JJIL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 7 (xm Lichrosorb RP 18
Mobile phase: MeOH:water 75:25 containing 1% acetic acid
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: flufenamic acid
REFERENCE
Nivaud-Guernet,E.; Guernet,M.; Ivanovic,D.; Medenica,M. Effect of eluent pH on the ionic and molecular forms
of the non-steroidal anti-inflammatory agents in reversed-phase high-performance liquid chromatography,
J.Liq.Chromatogr., 1994, 17, 2343-2357.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fiuoxetine, fluphenazine, flur-
ketorolac, labetalol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazin-
dol, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone, meth-
ylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam,
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
by aspiration of air. Evaporate an aliquot of 100 |xL 20 jig/mL IS in MeOH to dryness at 37.
Add 1 mL urine, vortex, add 250 JJLL 1 M pH 5.0 acetate buffer, vortex. Add 250 JJIL of the
the eluate to dryness under a stream of nitrogen at 37, reconstitute the residue in 200 (xL
mobile phase, inject a 10-30 (JLL aliquot.
HPLC VARIABLES
Flow rate: 0.9
Detector: UV 230
CHROMATOGRAM
Retention time: 33
OTHER SUBSTANCES
Extracted: diclofenac, ibuprofen, felbinac, fenbufen, flurbiprofen, ketoprofen, loxoprofen, na-
KEYWORDS
SPE
REFERENCE
J.Chromatogr.B, 1997, 692, 375-388.
Mefenorex
Molecular formula: C12H18CIN
Merck Index: 5843
Lednicer No.: 2 47
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 \im Symmetry C8 (Waters)
mL/min)
Detector: UV 207.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Mefloquine hydrochloride
CAS Registry No.: 51773-92-3, 53230-10-7 (free base)
Merck Index: 5845
SAMPLE
Matrix: blood
Sample preparation: Add 150 |xL 100 mM zinc sulfate to 600 JJLL plasma while vortexing over
15 s, add 700 |xL MeCN containing 4 (xm WR 184806 and 75 JJLM sulfadimethoxine while
vortexing over 15 s, , let stand for 15 min, centrifuge at 10000 g for 10 min. Remove the
supernatant and add it to 2 mL pH 9.0 phosphate buffer, add 2 mL 60 mM tetrabutylammo-
nium hydroxide, add 5 mL MTBE, shake for 10 min, centrifuge at 1200 g for 5 min. Remove
the upper organic layer and evaporate it to dryness at 50, reconstitute the residue in 200 |xL
HPLCVARIABLES
Column: 150 X 4 3 |xm Spherisorb S3-ODS-1
Mobile phase: MeCN: 100 mM phosphate buffer 48:52, adjusted to pH 3.5
Flow rate: 0.5
Detector: UV 229
CHROMATOGRAM
Internal standard: sulfadimethoxine (6.22), 2,8-bis(trifluoromethyl)-4-[l-hydroxy-3-(N-tert-bu-
tylamino)propyl]quinoline phosphate (WR 184806) (Walter Reed) (21.00)
OTHER SUBSTANCES
Extracted: metabolites, pyrimethamine, sulfadoxine
KEYWORDS
plasma
REFERENCE
Bergqvist,Y.; Eckerbom,S.; Larsson,H.; Malekzadeh,M. Reversed-phase liquid chromatographic method for the
simultaneous determination of the antimalarial drugs sulfadoxine, pyrimethamine, mefloquine and its ma-
jor carboxylic metabolite in plasma, J.Chromatogr., 1991, 571, 169-177.
SAMPLE
Matrix: blood
Sample preparation: 300 u,L Plasma + 75 (xL 100 mM zinc sulfate, vortex for 15 s, add 750 |xL
3 mM quinine in MeCN, vortex for 15 s, let stand for 15 min, centrifuge at 10000 g for 10 min.
Remove the supernatant and add it to 2 mL 0.1% ammonium (?) solution (also specified on p.
222 as 1% ammonia) and 6 mL MTBE, extract for 30 min, centrifuge at 3000 g for 5 min.
Remove the organic layer and evaporate it to dryness at 80 under a stream of air. Reconstitute
the residue in 150 JJLL mobile phase, inject a 100 |xL aliquot.
HPLCVARIABLES
Column: 100 X 4.6 7 |xm Hypercarb-S
Mobile phase: MeCN:MeOH:60 mM pH 4.6 acetate buffer 48:20:32 containing 5 mM N-benzy-
loxycarbonylglycyl-L-proline (L-ZGP), pH adjusted to 4.6 with NaOH
Flow rate: 0.8
Detector: UV 278
CHROMATOGRAM
Retention time: 6 (SR), 7 (RS)
Internal standard: quinine (10)
KEYWORDS
plasma; chiral
REFERENCE
Bergqvist,Y.; Al Kabbani,J.; Pettersson,C; Huynh,N.-H. Enantioselective high-performance liquid chromato-
graphic determination of (SR)- and (RS)-mefloquine in plasma using N-benzyloxycarbonyl-glycyl-L-proline
as chiral counter ion, J.Chromatogr., 1993, 620, 217-224.
SAMPLE
Matrix: blood
Sample preparation: Allow 100 |JLL whole blood to dry on chromatographic paper (Whatman 31
ET Chroma). Cut paper into small pieces, add to 2 mL ammonia:water 10:90, incubate for 30-
60 min at room temperature, sonicate for 30 min at 37, add 5 mL MTBE, add 2 mL 500 mM
pH 9.0 phosphate buffer, add 2 mL 20 |xM IS in 60 mM tetrabutylammonium hydroxide, shake
at 80, reconstitute the residue in 150 JJLL mobile phase, inject a 125 (JLL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:100 mM phosphate buffer:l M sodium perchlorate 137:173:15 containing
7 mM N,N-dimethyloctylamine, adjust pH to 4.0 with 5 M NaOH
Flow rate: 1.4
Detector: UV 227
CHROMATOGRAM
Retention time: 7.8
Internal standard: 2,8-bis(trifluoromethyl)-4-[ l-hydroxy-3-(N-t-butylamino)propyl]quinoline
(WR 184,806) (14.6)
OTHER SUBSTANCES
KEYWORDS
whole blood
REFERENCE
Bergqvist,Y.; Al Kabbani,J.; Krysen,B; PalmeJ.B.; Rombo,L. High-performance liquid chromatographic method
for the simultaneous determination of mefloquine and its carboxylic metabolite in 100-|xl blood samples
dried on paper, J.Chromatogr., 1993, 615, 297-302.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 500 |xL 20% Na3PO4 + 2 mL MTBE5 vortex for 30 s,
centrifuge at 2000 g for 10 min, freeze in dry ice/acetone. Remove the organic phase, repeat
the extraction with 2 mL MTBE. Combine the organic phases and evaporate them under a
stream of nitrogen, reconstitute the residue in 70 jxL mobile phase, inject a 50 IJLL aliquot onto
column A with mobile phase A. Three min after mefloquine had left column A and entered
column B (use a detector between column A and column B to ascertain this) backflush contents
of column B onto column C with mobile phase B, elute column C with mobile phase B and
monitor the effluent for the enantiomers.
HPLC VARIABLES
Column: A 250 X 4.6 5 u,m Nucleosil cyanopropyl; B 60 X 4 5 |xm Kromasil (Informatiques &
Technologies); C 250 X 4.6 5 ^m chiral (S)-naphthylurea (S.F.C.C./Shandon)
Mobile phase: A Hexane:isopropanol:MeOH 82:4:14 modified with 125 jxL triethylamine in
MeOH (1:40); B Hexane:isopropanol:MeOH 50:5:45 modified with 125 jxL triethylamine in
MeOH (1:40)
Detector: UV 285
CHROMATOGRAM
KEYWORDS
plasma; chiral; column-switching
REFERENCE
Gimenez,R; Dumartin,C; WainerJ.W.; Farinotti,R. Improved column-switching liquid chromatographic method
for the determination of the enantiomers of mefloquine, J.Chromatogr., 1993, 619, 161166.
SAMPLE
Matrix: blood
Sample preparation: 500 IJLL Plasma + 50 |xL 10 |xg/mL quinidine in MeOH + 500 |xL 20%
Na3PO4 + 2 mL MTBE, vortex for 30 s, centrifuge at 2000 g for 10 min, freeze in dry ice/
acetone. Remove the organic phase, repeat the extraction with 2 mL MTBE. Combine the
organic phases and evaporate them under a stream of nitrogen, reconstitute the residue in 70
|xL mobile phase, inject a 50 jxL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |jim Nucleosil cyanopropyl
Mobile phase: Hexane:isopropanol:MeOH 82:4:14 modified with 125 |JLL triethylamine in MeOH
(1:40)
Flow rate: 2
Detector: UV 285
CHROMATOGRAM
Internal standard: quinidine (k' 13.65)
KEYWORDS
plasma
REFERENCE
Gimenez,R; Dumartin,C; Wainer,I.W.; Farinotti,R. Improved column-switching liquid chromatographic method
for the determination of the enantiomers of mefloquine, J.Chromatogr., 1993, 619, 161-166.
SAMPLE
Matrix: blood
Sample preparation: Whole blood. 100 u.L Whole blood was allowed to dry on Whatman 31 ET
Chroma chromatographic paper. Cut paper into small pieces and add to 150 |xL 5 (xM IS in
water, add 2 mL ammonia:water 90:10, incubate at room temperature for 30-60 min, sonicate
at 37 for 30 min, add 4 mL 10 mM pH 10.0 borate buffer and 5 mL MTBE, shake for 20 min,
centrifuge at 3000 g for 5 min. Remove the organic layer and evaporate it to dryness at 65,
add 125 |xL 0.4 mM (-)-l-(9-fluorenyl)ethyl chloroformate in water-free MeCN, add 50 |iL 15
mM pH 8.5 borate buffer, vortex, leave at room temperature for 40 min, centrifuge at 3000 g
for 10 min, inject a 100 JJLL aliquot. Plasma. 100 JXL Plasma + 25 JJLL 100 mM zinc sulfate,
vortex for 15 s, add 750 |xL 1 jxM IS in MeCN, vortex for 15 s, let stand for 15 min, centrifuge
at 10000 g for 10 min. Remove the supernatant and add it to 4 mL 10 mM pH 10.0 borate
buffer and 5 mL MTBE, shake for 20 min, centrifuge at 3000 g for 5 min. Remove the organic
layer and evaporate it to dryness at 65, add 125 jxL 0.4 mM (-)-l-(9-fluorenyl)ethyl chlorofor-
mate in water-free MeCN, add 50 |xL 15 mM pH 8.5 borate buffer, vortex, leave at room
temperature for 40 min, centrifuge at 3000 g for 10 min, inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere octadecylsilica
Flow rate: 1
CHROMATOGRAM
Retention time: 13 (RS), 15 (SR)
Internal standard: D,L-erythro-a-(2-piperidyl)-2-trifluoromethyl-6,8-dichloro-4-quinolineme-
thanol (18 and 22)
Limit of detection: 50 nM (SR), 10 nM (RS)
OTHER SUBSTANCES
Noninterfering: chloroquine, quinine, pyrimethamine, sulfadoxine
KEYWORDS
whole blood; plasma; chiral; derivatization
REFERENCE
Bergqvist,Y.; Doverskog,M.; Al Kabbani,J. High-performance liquid chromatographic determination of (SR)-
and (i?S)-enantiomers of mefloquine in plasma and capillary blood sampled on paper after derivatization
with (-)-l-(9-fluorenyl)ethylchloroformate, J.Chromatogr.B, 1994, 652, 73-81.
SAMPLE
Matrix: blood
the residue in 100 |JLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 JJLL aliquot of
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 222
CHROMATOGRAM
KEYWORDS
dralazine; omeprazole; strychnine; acebutolol; glutethimide; chlorpropamide; glipizide; tri-
azolam; prazosin; flunitrazepam; clonazepam; metoclopramide; melphalan; estazolam;
REFERENCE
Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 4O1
254-262.
SAMPLE
Sample preparation: 1 mL Plasma or urine + 100 JJLL 1 M NaOH + 2 mL MTBE, extract by
rotating slowly for 15 min, centrifuge at 3000 g for 5 min. Remove the organic phase and add
it to 500 fjuL 500 mM phosphoric acid, extract for 15 min, centrifuge. Remove the aqueous phase
and add it to 1 mL 1 M NaOH and 1.5 mL MTBE, extract for 15 min, centrifuge. Remove the
organic phase and evaporate it under nitrogen, dissolve the residue in 300 |xL mobile phase,
inject an aliquot.
HPLC VARIABLES
Guard column: Chiral-AGP guard column
Column: 150 X 4 Chiral-AGP (ChromTech)
Mobile phase: n-Propanol:50 mM pH 4.85 sodium phosphate buffer 10:90
Flow rate: 0.9
Detector: UV 222
CHROMATOGRAM
Retention time: 10 (SR), 13 (RS)
OTHER SUBSTANCES
Noninterfering: atenolol, cimetidine, cycloguanil, diazepam, flunitrazepam, hydroxychloro-
quine, proguanil, propranolol, pyrimethamine, ranitidine, sulfadoxine, verapamil
KEYWORDS
plasma; chiral
REFERENCE
Walln,L.; Ericsson,.; WikstromJ.; Hellgren,U. High-performance liquid chromatographic method for the en-
antioselective analysis of mefloquine in plasma and urine, J.Chromatogr.B, 1994, 655, 153-157.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 222.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Megazol
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 50 |xL 10 |xg/mL tinidazole in mobile phase and
50 |xL 1 M NaOH. Add 7 mL dichloromethane, mix on a rotary agitator for 20 min and cen-
trifuge at 1636 g for 10 min. Remove 5 mL of the organic layer and evaporate it to dryness
under a stream of nitrogen at 45. Reconstitute the dry residue by vortex agitation with 200
jxL mobile phase, inject a 50 (xL aliquot.
HPLC VARIABLES
Column: 250 X 4 10 |xm Kromasil C8 (Bischoff Chromatography, Germany)
Mobile phase: MeCN:MeOH:68 mM pH 3 phosphate buffer 15:20:65
Flow rate: 0.7
Detector: UV 360
CHROMATOGRAM
Internal standard: tinidazole (6.10)
KEYWORDS
human; rat; plasma; pharmacokinetics
REFERENCE
Enanga,B.; Labat,C; Boudra,H.; Chauviere,G.; Keita,M.; Bouteille,B.; Dumas,M.; Houin,G. Simple high-per-
formance liquid chromatographic method to analyse megazol in human and rat plasma, J.Chromatogr.B,
1997, 696, 261-266.
Megestrol acetate
Molecular formula: G24H32O4
Merck Index: 5849
Lednicer No.: 1 180
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 500 |xL water + 50 |xL 3 |xg/mL 2,3-diphenyl-l-indenone
in MeOH + 7 mL hexane, rotate at 40 rpm for 10 min, centrifuge at 800 g for 7 min. Remove
5 mL of the organic layer and evaporate it to dryness under a stream of nitrogen at 30-35,
reconstitute the residue in 100 |xL MeOH, vortex thoroughly, inject a 50 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:water:acetic acid 41:23:36:1
Flow rate: 2
Detector: UV 280
CHROMATOGRAM
Retention time: 6-7 (megestrol acetate)
Internal standard: 2,3-diphenyl-l-indenone (12-14)
OTHER SUBSTANCES
Extracted: megestrol (4-5 min)
KEYWORDS
REFERENCE
Gaver,R.C; Movahhed,H.S.; Farmen,R.H.; Pittman,K.A. Liquid chromatographic procedure for the quantitative
analysis of megestrol acetate in human plasma, J.Pharm.ScL, 1985, 74, 664-667.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL 14 |xg/mL cyproterone acetate + 100 JJLL MeOH,
mix, let stand for 1 h, add to a pre-washed Sep-Pak C18 SPE cartridge, wash with 5 mL water,
elute with 2 mL MeOH. Evaporate the eluate to 500 |JLL under a stream of nitrogen, add 5 mL
water, extract with 5 mL dichloromethane. Remove the organic layer and evaporate it to dry-
ness, reconstitute the residue in 100 |xL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 220 X 4.6 5 jim Spheri RP18 (Brownlee)
Flow rate: 1
Detector: UV 260
CHROMATOGRAM
Retention time: 14 (megestrol acetate)
Internal standard: cyproterone acetate (12)
KEYWORDS
serum; SPE
REFERENCE
Dikkeschei,L.D.; Wolthers,B.G.; de Ruyter-BuitenhuisAW.; Nagel,G.T.; Sleijfer,D.T.; Willemse,RH.; van der
SHk,W. Determination of megestrol acetate and cyproterone acetate in serum of patients with advanced
breast cancer by high-performance liquid chromatography, J.Chromatogr., 1990, 529, 145-154.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 290.2
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
camfamine, fenoprofen, fenproporex, fentanyl, flubendazole, fiufenamic acid, flunitrazepam, 5-
pam, mepacrine, meperidine, mephentermine, mephenytoin, mephesin, mephobarbital, mepi-
REFERENCE
Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.ToxicoL, 1994,
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 |xL aliquot of a 100-500 (xg/mL solution in mobile phase.
HPLC VARIABLES
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
idine, codeine, desipramine, diphenhydramine, dipyridamole, ephedrine, fiufenamic acid, hal-
operidol, hydroxyzine, imipramine, indomethacin, lidocaine, metoprolol, nabumetone, nadolol,
phenobarbital, phenol, promazine, propranolol, pyrilamine, quinidine, ropinirole, testosterone,
thioridazine, tolfenamic acid, verapamil
KEY WORDS
REFERENCE
Meglutol
Merck Index: 5852
SAMPLE
Sample preparation: Powder tablets, weigh out amount equivalent to 100 mg meglutol, suspend
in 10 mL water, make up to 100 mL with MeOH, stir for 10 min, dilute an aliquot 1:10 with
water. 200 |xL Sample + 150 |xL 20 mM tetrahexylammonium bromide in 100 mM pH 7.0
phosphate buffer + 100 |xL 4.2 mg/mL 2-bromoacetyl-6-methoxynaphthalene in acetone, stir at
65 for 43 min, add 150 JJLL 7.5 |jig/mL IS in MeCN, sonicate for 1 min, inject a 50 JLJLL aliquot.
(Synthesis of 2-bromoacetyl-6-methoxynaphthalene is as follows. Stir equimolar amounts of 2-
acetyl-6-methoxynaphthalene (6'-methoxy-2'-acetonaphthone, Aldrich) and methyltriphenyl-
phosphonium tribromide in anhydrous THF under nitrogen at room temperature for 1 h, dilute
the reaction mixture with ether, wash with sodium bisulfite solution, wash with water (Phos-
phorus and Sulfur 1985, 25, 357). [Bromination can also be achieved with phenyltrimethylam-
monium tribromide over 3 h but the reaction is less selective.] Purify the crude product by
column chromatography on silica gel using chloroform:petroleum ether 50:50 to give 2-bro-
moacetyl-6-methoxynaphthalene (mp 109-112) (Chromatographia 1992, 33, 13).)
HPLC VARIABLES
Column: 250 X 4.6 Hypersil 5 ODS
Mobile phase: MeCN:MeOH:THF:water 35.75:26:3.25:35
Flow rate: 1.2
CHROMATOGRAM
Retention time: 12
Internal standard: n-hexanoic acid 6-methoxynaphthacylester (15) [Prepare by dissolving 2
mmoles n-hexanoic acid and 1 mmole 2-bromoacetyl-6-methoxynaphthalene in 10 mL anhy-
drous MeCN, add 0.5 mL triethylamine, heat at 60 for 30 min, cool, dilute with 30 mL water,
extract three times with 10 mL portions of diethyl ether. Combine the extracts, wash with 5%
sodium bicarbonate, wash three times with 10 mL portions of water, dry over anhydrous so-
dium sulfate, evaporate under reduced pressure, recrystallize from MeOH/water.]
KEY WORDS
tablets; derivatization
REFERENCE
Gatti,R.; Andrisano,V.; Di Pietra,A.M.; Cavrini,V. Analysis of aliphatic dicarboxylic acids in Pharmaceuticals
and cosmetics by liquid chromatography (HPLC) withfluorescencedetection, J.Pharm.Biomed.Anal., 1995,
13, 589-595.
Melitracen
CAS Registry No.: 5118-29-6, 10563-70-9(HCI)
Merck Index: 5866
SAMPLE
Sample preparation: Serum, urine. 500 |xL Serum or urine + 100 JJLL 2 |xg/mL diazepam + 200
jxL 20% sodium carbonate + 500 |xL water + 3 mL n-hexane:isoamyl alcohol 98.5:1.5, mix for
gentle stream of nitrogen at about 40. Dissolve the residue in 100 (xL mobile phase, inject a
10 JULL aliquot. Tissue. Homogenize 1 g sample with 9 mL 100 mM HCl and 100 JJLL 20 |xg/mL
diazepam, centrifuge at 15000 g for 10 min. Add 500 jxL 20% sodium carbonate and 4 mL n-
hexane:isoamyl alcohol 98.5:1.5 to 1 mL of the supernatant, mix for 5 min. Remove the organic
100 |xL mobile phase, filter by microconcentrator (Microcon-30, Grace). Inject a 10 JJLL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 2 fjim TSK gel Super-Octyl (A) or 100 X 4.6 5 ^m Hypersil MOS-C8 (B),
(Yokogawa, Japan)
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: amitriptyline, amoxapine, clomipramine, desipramine, dothiepin, doxepin, imipra-
mine, maprotiline, mianserin, nortriptyline
tal, phenobarbital, phenytoin, primidone, sulphide, trimethadione, trimipramine
KEYWORDS
serum; brain; liver
REFERENCE
in human biological samples using a new reversed-phase chromatographic column of 2 |xm porous micros-
pherical silica gel, J.Chromatogr.B, 1997, 692, 405-412.
Meloxicam
Merck Index: 5869
SAMPLE
M a t r i x : blood
Sample preparation: Inject 50 (xL plasma onto column A and elute to waste with mobile phase
A, after 4 min backflush the contents of column A onto column B with mobile phase B, elute
with mobile phase B, monitor the effluent from column B.
HPLC VARIABLES
Column: ABondapak C18 Corasil; B 125 X 4.6 Hypersil ODS
Mobile phase: A 5 mM sulfuric acid; B MeCN:MeOH:water:glacial acid 5:60:50:2 containing 0.86
g/L heptanesulfonic acid
Detector: UV 355
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
baboon; dog; human; kidney; liver; mouse; pharmacokinetics; pig; plasma; rat; column-switching
REFERENCE
Busch,U.; Schmid,J.; Heinzel,G.; Schmaus,H.; Baierl,J.; Huber,C; Roth,W. Pharmacokinetics of meloxicam in
animals and the relevance to humans, Drug Metab.Dispos., 1998, 26, 576-584.
Melphalan
Merck Index: 5871
Lednicer No.: 2 120
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 5 jxg dansylproline + 2 mL ice-cold MeOH, vortex for 20
s, freeze in dry ice/acetone for 3 min, centrifuge at 3000 rpm for 3 min, inject an aliquot of the
supernatant.
HPLC VARIABLES
Column: micro C18 (Waters)
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 4
Internal standard: dansylproline (8)
KEYWORDS
plasma; rat; human; pharmacokinetics
REFERENCE
Chang,S.Y.; Alberts,D.S.; Melnick,L.R.; Walson,P.D.; Salmon,S.E. High-pressure liquid chromatographic anal-
ysis of melphalan in plasma, J.Pharm.ScL, 1978, 67, 679-681.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 500 |xL 5% trichloroacetic acid, vortex, centrifuge, filter
(0.45 |xm) the supernatant, inject a 10 |xL aliquot of the filtrate.
HPLC VARIABLES
Column: 250 X 2.6 10 jim HCODS/SIL X-C18 (Perkin-Elmer)
Mobile phase: Gradient. MeCN: 17.5 mM acetic acid from 12:88 to 80:20 over 14 min (concave
gradient).
Flow rate: 1.5
Detector: UV 263
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
KEYWORDS
plasma; human; rat; pharmacokinetics
REFERENCE
Ahmed,A.E.; Hsu,T.-F. Quantitative analysis of melphalan and its major hydrolysate in patients and animals
by reversed-phase high-performance liquid chromatography, J.Chromatogr., 1981, 222, 453-460.
SAMPLE
Matrix: blood
Sample preparation: Wash Amberlite XAD-2 thoroughly with acetone and then MeOH until
absorbance of washings was less than 0.1 a.u. at 254 nm. Prepare a 25 X 5 column of washed
Amberlite XAD-2 in a 5 mL pipette and wash it with 10 mL acetone, with 10 mL of MeOH,
and with 10 mL of water. 1 mL Plasma + 500 ng IS, mix, add to the column, wash with 10 mL
water, elute with 1.5 mL MeOH. Centrifuge the eluate at 12000 g for 3 min (or filter (0.45 jxm
PTFE) and inject a 200 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeOH:675 |xg/mL sodium dodecyl sulfate 80:20 adjusted to pH 3.0 with concen-
trated sulfuric acid
CHROMATOGRAM
Retention time: 9
Internal standard: Dns-arginine (Na(5-dimethylaminonaphthalene)-l-sulfonyl)-L-arginine)
. (1.L5)
KEYWORDS
REFERENCE
Bosanquet,A.G.; Gilby,E.D. Measurement of plasma melphalan at therapeutic concentrations using isocratic
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 44 |xL cold concentrated perchloric acid, mix vigorously
for 10 s, centrifuge at 13000 g for 5 min, inject an aliquot of the supernatant.
HPLC VARIABLES
Column: Reversed-phase C18 (Waters or Bio-Rad)
Mobile phase: MeOH:2% acetic acid (pH 3.6) 22:78
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 18
KEYWORDS
REFERENCE
Davis,T.R; Peng,Y.-M.; Goodman,G.E.; Alberts,D.S. HPLC, MS, and pharmacokinetics of melphalan, bisantrene
and 13-cis retinoic acid, J.Chromatogr.Sci., 1982, 20, 511-516.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 250 mM trichloroacetic acid, mix at 4, let stand at
4 for 30 min, centrifuge at 1500 g for 10 min. Remove a 1 mL aliquot of the supernatant and
add it to 200 |xL 1 M N-acetylcysteine, 200 |xL 2.65 M NaOH, and 200 |JLL 500 mM pH 11.0
phosphate buffer, mix, heat at 70 for 15 min, add 200 JJLL 2 M citric acid, filter (0.2 jjLin), inject
a 100 |xL aliquot of the filtrate.
HPLC VARIABLES
Column: 150 X 4 5 fjum Nova-Pak C18 Radial-Pak
Mobile phase: EtOH:36 mM pH 4.25 citrate buffer containing 50 JAM octanesulfonic acid 4:96
(The mobile phase flowed through a 100 X 4 column of ixBondapak C18 before the injector.)
CHROMATOGRAM
Retention time: 13
KEYWORDS
REFERENCE
Ehrsson,H.; Eksborg,S.; Lindfors A- Quantitative determination of melphalan in plasma by liquid chromatog-
raphy after derivatization with N-acetylcysteine, J.Chromatogr., 1986, 380, 222-228.
SAMPLE
Matrix: blood
Sample preparation: Condition a 2.4 mL 500 mg Bond Elut phenyl SPE cartridge with 2 mL
MeOH and 2 mL water. 1 mL Plasma + 100 |xL freshly prepared 100 mg/mL diethyldithiocar-
bamic acid in 100 mM NaOH, heat at 50 for 90 min, add to the SPE cartridge, wash with 5
mL water, allow to dry in the dark at room temperature for 1 h, elute with 2 mL MeCN.
Evaporate the eluate to dryness under reduced pressure at 40, reconstitute in 200 |xL MeOH,
HPLC VARIABLES
Flow rate: 1
Detector: UV 276
CHROMATOGRAM
Retention time: 14.6 (derivatized), 7.6 (underivatized)
KEYWORDS
REFERENCE
Cummings,J.; MacLellan,A.; Smyth,J.E; Farmer,P.B. Determination of reactive nitrogen mustard anticancer
drugs in plasma by high-performance liquid chromatography using derivatization, Anal.Chem., 1991, 63,
1514-1519.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL 60% perchloric acid, vortex for 30 s, centrifuge at
4 at 2000 g for 10 min. Remove 700 |xL of the supernatant and add it to 2 mL chloroform,
vortex for 30 s, centrifuge at 4 at 2000 g for 10 min, inject a 50 |iL aliquot of the supernatant.
HPLCVARIABLES
Column: 250 X 4 5 |xm Spheri 5 phenyl
Mobile phase: MeCNiIOO mMpH 4.5 sodium phosphate buffer 12:88
Flow rate: 1.2
Detector: E, ESA Coulochem 5100A, ESA 5011 analytical cell, upstream electrode 0.1 V, down-
stream electrode 0.6 V monitored
CHROMATOGRAM
KEYWORDS
REFERENCE
Silvestro,L.; Viano,L; Baiocchi,C; Saini,G.; Marmont,R; Ferro,R. Quantitation of melphalan in plasma of pa-
tients by reversed-phase high-performance liquid chromatography with electrochemical detection,
J.Chromatogr., 1991, 563, 443-450.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform.isopropanol:
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 261
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: blood, perfusate, tissue
Sample preparation: Plasma, perfusate. 100 |xL Plasma or perfusate + 200 |xL cold 38 |xg/mL
dansylarginine in MeOH, vortex at 4 for 30 s, centrifuge at 10000 g for 15 min, inject a 20 JJLL
aliquot of the supernatant. (Keep all samples and reagents on ice.) Tissue. Sonicate (3 mm,
Sonics and Materials, Danbury, CT) 100 mg minced tissue and 400 JJLL 38 |xg/mL dansylarginine
in MeOH on ice for 1 min, centrifuge at 10000 g for 15 min, inject a 20 |JLL aliquot of the
supernatant.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Alltima phenyl (Alltech)
Mobile phase: MeOH:water:glacial acetic acid 25:75:2, pH 2.7 containing 500 |xg/mL 1-octane-
sulfonic acid
Flow rate: 2
CHROMATOGRAM
Retention time: 8.4
Internal standard: dansylarginine (F ex 265 em 575) (12.6)
Limit of quantitation: 7.2 ng (tissue), 1.4 ng (plasma, perfusate)
OTHER SUBSTANCES
KEYWORDS
plasma; human; rat; fat; skin; muscle; pharmacokinetics
REFERENCE
Wu,Z.-Y.; Thompson,M.J.; Roberts,M.S.; Addison,R.S.; Cannell,G.R.; Grabs,A.J-; Smithers,B.M. High-perfor-
mance liquid chromatographic assay for the measurement of melphalan and its hydrolysis products in
perfusate and melphalan in tissues from human and rat isolated limb perfusions, J.Chromatogr.B, 1995,
673, 267-279.
SAMPLE
Sample preparation: Allow whole blood to clot. Homogenize <500 mg mouse tissue or clotted
blood with 500 |xL 10% perchloric acid in 2-methoxyethanol:0.1% acetic acid 55:45. Homogenize
>500 mg mouse tissue or clotted blood with 1 mL 10% perchloric acid in 2-methoxyethanol:
0.1% acetic acid 55:45. Homogenize 1 g dog tissue with 1 mL 10% perchloric acid in 2-meth-
oxyethanol:0.1% acetic acid 55:45. Centrifuge homogenate at 2000 g and mix 2 volumes of the
supernatant with 1 volume 1.0 M KH2PO4. Centrifuge, filter (Millipore Pellicon molecular filter
with 25000 dalton cut-off) the supernatant under 3.7 atmospheres of nitrogen, inject a 5 |xL
HPLCVARIABLES
Column: u, Bondapak C18
Mobile phase: Gradient. A:B 85:15 for 1 min, to 15:85 (step gradient), maintain at 15:85. A was
2-methoxyethanol:0.1% acetic acid 30:70. B was 2-methoxyethanol.0.1% acetic acid 55:45.
Flow rate: 1.5
Injection volume: 5
CHROMATOGRAM
KEYWORDS
whole blood; serum; bile; brain; testes; muscle; kidney; liver; heart; spleen; stomach; intestine;
lung; ovary; adrenal gland; pancreas; fat; bladder; dog; mouse; pharmacokinetics
REFERENCE
Furner,R.L.; Mellett,L.B.; Brown,R.K; Duncan,G. A method for the measurement of L-phenylalanine mustard
in the mouse and dog by high-pressure liquid chromatography, Drug Metab.Dispos., 1976, 4, 577-583.
SAMPLE
Sample preparation: Vortex up to 3 mL plasma or tissue homogenate with 2 volumes of chilled
MeOH at 4 for 20 s, centrifuge at 1500 g for 15 min, repeat the extraction with another volume
of MeOH. Combine the supernatants and add them to 4 volumes of ice cold water (i.e., MeOH
concentration is 15%). Pass this mixture through a Sep-Pak C18 SPE cartridge, wash with 10
mL ice-cold MeOH:water 15:85, elute with 2 mL MeOH, discard the first 400 |xL, collect the
rest, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.9 5 |xm Spherisorb S5 ODS
Flow rate: 3
CHROMATOGRAM
KEYWORDS
plasma; rat; kidney; liver; SPE
REFERENCE
Egan,C.M.; Jones,C.R; McCluskey,M. Method for the measurement of melphalan in biological samples by high-
performance liquid chromatography with fluorescence detection, J.Chromatogr., 1981, 224, 338-342.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 201.7
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Mix an aliquot with an equal volume of 20 mM pH 4.4 KH2PO4, centrifuge,
inject a 20 |JLL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 X 4.6 5 (xm Microsorb C8
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 7.8
REFERENCE
Lunn,G.; Sansone,E.B.; Andrews ,A-W.; Hellwig,L.C. Degradation and disposal of some antineoplastic drugs,
J.Pharm.Sci., 1989, 78, 652-659.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in mobile phase, inject a 50 JJLL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:triethylamine:200 mM ammonium acetate 18:2:0.5:79.5 pH ad-
justed to 3.8 with acetic acid
Flow rate: 1.4
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Simultaneous: pentamidine
REFERENCE
Yeh,T.-K.; Dalton,J.T.; Au,J.L.-S. High-performance liquid chromatographic determination of pentamidine in
plasma, J.Chromatogr., 1993, 622, 255-261.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 500 |xL aliquot of a solution in 10 mM pH 7.57 potassium phos-
phate buffer.
HPLC VARIABLES
Column: 100 X 10 15 |xm Pharmacia PepRPC C18
in MeCN. A:B 100:0 for 4 min, to 80:20 over 32 min, to 0:100 (step gradient).
Flow rate: 5
Detector: UV 260
REFERENCE
Awasthi,S.; Bajpai,K.K; Piper,J.T.; Singhal,S.S.; Ballatire,A; Seifert,W.E.,Jr.; Awasthi,Y.C; Ansari,GA.S. In-
teractions of melphalan with glutathione and the role of glutathione S-transferase, Drug Metab.Dispos.,
1996, 24, 371-374.
Menadione
Molecular formula: CnH 8 O 2
Merck Index: 5874
SAMPLE
Matrix: blood
Sample preparation: 800 jxL Plasma + 100 JJLL 35 |xg/mL carbazole in mobile phase + 6 mL n-
hexane, rotate for 45 min, centrifuge at 1080 g for 15 min, freeze at -76 for 30 min. Remove
the organic layer and add it to 200 |xL MeOHrwater 70:30, evaporate under a stream of nitro-
gen, inject an aliquot.
HPLC VARIABLES
Guard column: 10 jxm Bondapak C18
Column: 250 X 4.6 |xm Ultrasphere ODS
Flow rate: 0.8
Detector: UV 265
CHROMATOGRAM
Retention time: 8.7
Internal standard: carbazole (12.5)
OTHER SUBSTANCES
Extracted: menadiol, metabolites
KEYWORDS
plasma; protect from light; rabbit; pharmacokinetics
REFERENCE
Hu,0.Y.-R; Wu,C.-Y.; Chan,W.-K; Wu,F.Y.-H. Determination of anticancer drug vitamin K3 in plasma by high-
performance liquid chromatography, J.Chromatogr.B, 1995, 666, 299-305.
SAMPLE
Matrix: feed
Sample preparation: Grind feed to pass 20 mesh sieve. Feed + 100 mL chloroform, shake for 3
min, add 5 mL 25% ammonium hydroxide, shake for 3 min, add 10 g Celite:anhydrous sodium
sulfate 3:10 w/w, shake for 20 min, neutralize with acetic acid, shake mechanically for 20 min,
centrifuge at 2500 rpm for 10 min. Either dilute chloroform layer with chloroform and inject
a 40-100 (JiL aliquot or evaporate chloroform layer to dryness under reduced pressure at 40,
reconstitute in 1,2-dichloroethane, inject a 40 -100 |xL aliquot.
HPLC VARIABLES
Guard column: Guard-Pak Resolve Si (waters)
Column: 250 X 4 5 |xm Lichrosorb Si 60
Mobile phase: 1,2-Dichloroethane
Flow rate: 1.8
Detector: UV 251
CHROMATOGRAM
KEYWORDS
normal phase; protect from light
REFERENCE
Laffi,R.; Marchetti,S.; Marchetti,M. Normal-phase liquid chromatographic determination of menadione in an-
imal feeds, JAssoc.Off.Anal.Chem., 1988, 71, 826-828.
SAMPLE
Matrix: feed
Sample preparation: Extract 500 mg feed in a 152 X 19 stainless steel tube with supercritical
carbon dioxide at 65 at 8000 psi for 20 min, the effluent is passed through a 152 X 6.35 column
of silica gel. Elute the silica gel with 10 mL dichloromethane and evaporate the eluate to
dryness under reduced pressure, reconstitute in 1 mL MeCN, inject a 20 |JLL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:25 mM sodium perchlorate 90:10
Flow rate: 2
Detector: E, polished silver working electrode -0.75 V, calomel reference electrode
CHROMATOGRAM
Retention time: 2.5
KEYWORDS
SFE
REFERENCE
Schneiderman,M.A.; Sharma,A.K.; Locke,D.C. Determination of menadione in an animal feed using supercrit-
ical fluid extraction and HPLC with electrochemical detector, J.Chromatogr.ScL, 1988, 26, 458-462.
SAMPLE
Matrix: feed
Sample preparation: 1 g Ground feed + 10 mL MeOH:water 40:60, shake for 30 min on a
mechanical shaker, centrifuge at 1000 g for 10 min. Remove 5 mL of the supernatant and add
it to 10 mL 5% sodium carbonate and 10 mL n-pentane, shake for 1 min, centrifuge at 1000 g
for 1 min, repeat extraction twice. Combine the n-pentane layers and evaporate to dryness
under reduced pressure at room temperature, reconstitute the residue in 10 mL MeOH, inject
a 10 jxL aliquot.
HPLC VARIABLES
Flow rate: 0.9
Detector: F ex 325 em 425 following post-column reduction. The effluent from the column flowed
through a 20 X 2 column packed with powdered zinc (^ 38 |xm) and a 0.5 |xm filter and then
to the detector.
CHROMATOGRAM
Retention time: 6
KEYWORDS
post-column reaction; also for menadione sodium bisulfite
REFERENCE
Billedeau,S.M. Fluorimetric determination of vitamin K3 (menadione sodium bisulfite) in synthetic animal feed
by high-performance liquid chromatography using a post-column zinc reducer, J.Chromatogr., 1989, 472,
371-379.
SAMPLE
Sample preparation: 1-10 g Ground feed or premix + 96 mL EtOH:water 40:60, shake for 10
min, add 4 mL 10% tannin solution, shake for 1 min, centrifuge at 2000 g for 5 min, filter
through a fine-pore glass filter. Add a 40 mL portion of the filtrate to 50 mL n-hexane and 20
mL 10% sodium carbonate, shake for 1 min, discard the lower layer. Wash the n-hexane layer
twice with 100 mL portions of water and dry it with strips of blue-ribbon filter paper. Evaporate
an aliquot to dryness under nitrogen with a rotary evaporator, reconstitute in mobile phase,
sonicate for 1 min, filter (0.2 jxm) (if necessary), inject a 100 JJLL aliquot. (Use a brown glass
separatory funnel.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm ODS-Hypersil
Mobile phase: EtOH.water 60:40
Flow rate: 0.6
Detector: F ex 325 em 425 following post-column reduction. The reagent passed through a de-
bubbler was mixed with the effluent at 0.27 mL/min, the combined solution flowed through a
1.4 m long stainless steel reaction coil to the detector. (Reagent was 0.8 g/L sodium borohydride
in EtOH, sonicate for 5 min, stable for 2 h.)
CHROMATOGRAM
Retention time: 12
KEYWORDS
protect from light; post-column reaction
REFERENCE
Speek,A.J.; Schrijver,J.; Schreurs,W.H.P. Fluorimetric determination of menadione sodium bisulphite (vitamin
K3) in animal feed and premixes by high-performance liquid chromatography with post-column derivati-
zation, J.Chromatogr., 1984, 301, 441-447.
SAMPLE
Sample preparation: Weigh out 500 mg ground tablets, extract with water, make up to 50 or
100 mL with water, filter, inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Nucleosil 10 C18
Mobile phase: MeOH: 1% acetic acid 25:75
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 12.5 (menadione hydrogen sulfite)
OTHER SUBSTANCES
Simultaneous: niacinamide, pyridoxine, riboflavin, thiamine, ascorbic acid
KEYWORDS
tablets; multi-vitamin
REFERENCE
Sadlej-Sosnowska,N.; Blitek,D.; Wilczynska-WojtulewiczJ. Determination of menadione sodium hydrogen sul-
phite and nicotinamide in multivitamin formulations by high-performance liquid chromatography,
J.Chromatogr., 1986, 357, 227-232.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in EtOH, inject a 20 (xL aliquot.
HPLC VARIABLES
Column: 150 X 6 5 |xm Shim-pack CLC-ODS (Shimadzu)
Mobile phase: MeOH:water 85:15 containing 50 mM sodium perchlorate
Flow rate: 1
Detector: E, EICOM ECD 100, glassy carbon working electrode +0.7 V, Ag/AgCl reference elec-
trode following post-column catalytic reduction. The column effluent flowed through a 10 X 4.6
column packed with 10 |xm 5% platinum on alumina catalyst (Toa Electronics) to the detector.
(Purge catalyst column with water at 10 mL/min for 5 min before use.)
CHROMATOGRAM
Retention time: 4.9
OTHER SUBSTANCES
Simultaneous: idebenone
KEYWORDS
REFERENCE
Wakabayashi,H; Nakajima,M.; Yamato,S.; Shimada,K. Determination of idebenone in rat serum and brain by
high-performance liquid chromatography using platinum catalyst reduction and electrochemical detection,
J.Chromatogr., 1992, 573, 154-157.
Menotropins
Merck Index: 4299
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in 100 mM NaH2PO4 adjusted to pH 2.1 with orthophosphoric
acid, inject a 100 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4 Aquapore RP 300 (Kontron)
Mobile phase: Gradient. A was 100 mM NaH2PO4 adjusted to pH 2.1 with orthophosphoric acid.
B was MeOH. A:B from 90:10 to 35:65 over 180 min.
Flow rate: 1
Detector: UV 225
CHROMATOGRAM
Retention time: 4 (LH), 18 (FSH)
OTHER SUBSTANCES
Simultaneous: adrenocorticotropin hormone and fragments, lipotropic hormone and fragments,
melanotropin, endorphins, prolactin, somatropin
REFERENCE
Richter,W.O.; Schwandt,P. Separation of neuropeptides by HPLC: evaluation of different supports, with ana-
lytical and preparative applications to human and porcine neurophysins, (3-lipotropin, adrenocorticotropic
hormone, and (3-endorphin, J.Neurochem., 1985, 44, 1697-1703.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1 mg/mL solution in 100 mM pH 7.0 sodium phosphate buffer,
HPLC VARIABLES
Column: 100 X 4.6 6.5 |jim 300 A Cl SynChropak RP-I + 250 X 10 5 fjim 300 A Vydac C4
Mobile phase: Gradient. A was 100 mM triethylamine phosphate in water adjusted to pH 6.5
with triethylamine. B was a 100 mM triethylamine phosphate in MeCN:water 60:40 adjusted
to pH 6.5 with triethylamine. A:B from 80:20 to 0:100 over 60 min.
Flow rate: 0.8
Detector: UV 226
CHROMATOGRAM
Retention time: 32
OTHER SUBSTANCES
Simultaneous: luteinizing hormone, thyroid-stimulating hormone
REFERENCE
Hiyama,J.; Renwick,A.G.C. Separation of human glycoprotein hormones and their subunits by reversed-phase
Menthol
Molecular formula: C10H20O
CAS Registry No.: 1490-04-6, 89-78-1
Merck Index: 5882
SAMPLE
Sample preparation: Powder, granules. Dissolve the powder or granules in a small volume of
MeCN, sonicate for 30 min, centrifuge at 2500 rpm for 5 min, dilute 2-fold with water, pass
through a Sep-pak C18 SPE cartridge, filter(0.45 |xm), inject a 100 jxL aliquot. Lotion. Dilute
lotion 2-fold with water, filter(0.45 jxm), inject a 100 JAL aliquot. Plaster. Extract plasters by
sonication with MeCN for 30 min., filter (0.45 |jim), inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 J'sphere ODS-H80 (YMC)
Flow rate: 1
Detector: UV 210; Polarized photometric detector, SPD-10 AV UV-VIS detector with HN32 po-
larizers (Polaroid, MA), 530 nm, angle+1.0 rad
CHROMATOGRAM
Retention time: 7.5
KEYWORDS
chiral; comparison with GC; SPE; polarized photometric detection; powders; granules; lotion;
plasters
REFERENCE
Hamasaki,K; Kato,K; Watanabe,T.; Yoshimura,Y; Nakazawa,H.; Yamamoto,A.; Matsunaga,A. Determination
of 1-menthol in pharmaceutical products by high performance liquid chromatography with polarized pho-
tometric detection, J.Pharm.Biomed.AnaL, 1998, 16, 1275-1280.
SAMPLE
Sample preparation: Shake 2 g powder with 10 mL EtOH for 1 h, directly inject 1 mL of this
solution using a syringe-coupled nylon filter (Teknokroma).
HPLC VARIABLES
Column: 125 X 4 5 jxm Aluspher RP Select B (E. Merck) (alumina particles bonded with
polybutadiene)
Mobile phase: MeOH:water:diammonium phosphate 30:70:0.2 (v:v:w), pH 8.2
Flow rate: 1
Detector: UV 191
CHROMATOGRAM
Retention time: 8
Next Page
OTHER SUBSTANCES
Simultaneous: benzocaine, tyrothricin
Noninterfering: gramicidin
REFERENCE
Caraballo,L; Fernandez-Arevalo,M.; Holgado,M.-A.; Vela,M.-T.; Rabasco,A.-M. A rapid HPLC method for the
quantification of tyrothricin, menthol, and benzocaine in pharmaceutical formulations, J.Pharm.ScL, 1994,
83, 1147-1149.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 Zorbax SAX
Mobile phase: MeOH:buffer 50:50 (Buffer was 180 mM Na2HPO4 adjusted to pH 3.00 0.05
with 180 mM orthophosphoric acid. Pass mobile phase through a 250 X 4.6 25-40 |xm silica
(HPLC Technology) column to saturate it with silica.)
Flow rate: 1
Detector: UV 253
CHROMATOGRAM
Retention time: 2.8
OTHER SUBSTANCES
Simultaneous: cromolyn, minocromil, nedocromil, quinoline yellow, saccharin, salicylic acid
Interfering: acetaminophen, albuterol, aspartame, aspirin, beclomethasone dipropionate, caf-
feine, isoproterenol, reproterol, ribofiavin, sorbitan trioleate, terbutaline, theophylline
REFERENCE
Baker,P.R.; Gardner,J.J.; Wilkinson,D. Automated high-performance liquid chromatographic method for the
determination of nedocromil sodium in human urine using bimodal column switching, J.Chromatogr.B,
1995, 668, 59-65.
Mepazine
Merck Index: 5892
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jjug/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 4.4
Previous Page
OTHER SUBSTANCES
Simultaneous: benzocaine, tyrothricin
Noninterfering: gramicidin
REFERENCE
Caraballo,L; Fernandez-Arevalo,M.; Holgado,M.-A.; Vela,M.-T.; Rabasco,A.-M. A rapid HPLC method for the
quantification of tyrothricin, menthol, and benzocaine in pharmaceutical formulations, J.Pharm.ScL, 1994,
83, 1147-1149.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 Zorbax SAX
Mobile phase: MeOH:buffer 50:50 (Buffer was 180 mM Na2HPO4 adjusted to pH 3.00 0.05
with 180 mM orthophosphoric acid. Pass mobile phase through a 250 X 4.6 25-40 |xm silica
(HPLC Technology) column to saturate it with silica.)
Flow rate: 1
Detector: UV 253
CHROMATOGRAM
Retention time: 2.8
OTHER SUBSTANCES
Simultaneous: cromolyn, minocromil, nedocromil, quinoline yellow, saccharin, salicylic acid
Interfering: acetaminophen, albuterol, aspartame, aspirin, beclomethasone dipropionate, caf-
feine, isoproterenol, reproterol, ribofiavin, sorbitan trioleate, terbutaline, theophylline
REFERENCE
Baker,P.R.; Gardner,J.J.; Wilkinson,D. Automated high-performance liquid chromatographic method for the
1995, 668, 59-65.
Mepazine
Merck Index: 5892
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jjug/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 4.4
OTHER SUBSTANCES
ine, pargyline, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone,
REFERENCE
Mepenzolate bromide
Molecular formula: C2i H26BrNO3
Merck Index: 5893
SAMPLE
Matrix: urine
Sample preparation: Condition a 500 mg 14 mL 40 |xm CCX-2 cation-exchange SPE cartridge
(Worldwide Monitoring) with two 2.5 mL aliquots of MeOH, two 2.5 mL aliquots of water, and
two 2.5 mL aliquots of 100 mM pH 7.00 phosphate buffer, do not allow to dry. 5 mL Urine + 3
mL 100 mM pH 7.00 phosphate buffer + 5 mL water, centrifuge at 800 g for 5 min, add to the
SPE cartridge, wash with 5 mL MeOH, wash with 5 mL water, dry under vacuum for 5 min,
elute with 4 mL MeOH:500 mM pH 3.00 ammonium acetate 95:5 (all flow rates were 1-2 mL/
min). Evaporate the eluate under a stream of nitrogen at 60, reconstitute in 100 |xL MeOH,
HPLC VARIABLES
Column: 150 X 4.1 10 |xm LiChroma (Chromatographic Specialties)
Flow rate: 0.8
Detector: MS, Sciex API III triple quadrupole, ion spray interface, split column effluent 95:5
before entering detector, nebulizing gas air at 550 kPa, collision gas argon, curtain gas nitrogen,
positive-ion mode, m/z 340 and 130
CHROMATOGRAM
Retention time: 2.1
Internal standard: mepenzolate
OTHER SUBSTANCES
Extracted: glycopyrrolate
KEYWORDS
SPE; horse; mepenzolate is IS
REFERENCE
Matassa,L.C; Woodard,D.; Leavitt,R.K.; Firby,R; Beaumier,P. Solid-phase extraction techniques for the deter-
mination of glycopyrrolate from equine urine by liquid chromatography-tandem mass spectrometry and gas
chromatography-mass spectrometry, J.Chromatogr., 1992, 573, 4348.
Meperidine
Merck Index: 5894
Lednicer No.: 1 300
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 200 ng doxepin or desipramine + 100 |xL 1 M NaOH + 9
mL freshly prepared hexanedsoamyl alcohol 99:1, shake vigorously for 5 min, centrifuge. Re-
move 8.5 mL of the organic phase and add it to 200 JJLL 50 mM HCl, shake well for 1 min,
centrifuge, inject a 50 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Column: 300 X 4 jjuBondapak phenyl
Mobile phase: MeCN:0.01% phosphoric acid containing 0.01% NaCl 35:65, final pH 2.8
Flow rate: 1.5
Detector: UV 210
CHROMATOGRAM
Internal standard: doxepin (12.2), desipramine (14.2)
OTHER SUBSTANCES
Extracted: cocaine, dextromoramide, methadone, normeperidine, norpropoxyphene, pentazocine,
propoxyphene
Simultaneous: amitriptyline, buprenorphine, chlorpromazine, codeine, desmethyldoxepin, di-
phenhydramine, ephedrine, imipramine, nortriptyline, oxazepam, oxycodone, pericyazine,
pheniramine, propranolol, quinine, thiopropazate, thioridazine
KEY WORDS
serum
REFERENCE
Hackett,L.R; Dusci,L.J.; Ilett,K.F. The analysis of several nonopiate narcotic analgesics and cocaine in serum
using high-performance liquid chromatography, J.Anal.ToxicoL, 1987, 11, 269271.
SAMPLE
Matrix: blood
250 (xL IS solution and 250 |xL serum to the column at 1 mL/min, wash twice with water and
once with MeCN draining the column completely after each wash, elute with 250 JJLL eluting
solution, centrifuge for 20 s to remove last of eluate, inject a 5 JJLL aliquot of the eluate. (Prepare
IS solution by adding 40 JJIL 1 mg/mL N-pentyl-2,6-pipecoloxylidide (l-pentyl-N-(2,6-dimethyl-
phenyl)-2-piperidinecarboxamide, pentyl-PPX) in MeOH to 10 mL 100 mM NaH2PO4. Eluting
solution was 2.5 mL 35% perchloric acid in 100 mL MeOH.)
HPLC VARIABLES
Guard column: 15 X 3.2 7 u,m RP-8 (Applied Biosystems)
Column: 150 X 4.6 5 imm Ultrasphere octyl
Mobile phase: MeCNiIO mM KH2PO4 25:80, pH 5.2
Flow rate: 1.5
Injection volume: 5
Detector: UV 205
CHROMATOGRAM
Retention time: 4.3
Internal standard: N-pentyl-2,6-pipecoloxylidide (l-pentyl-N-(2,6-dimethylphenyl)-2-piper-
idinecarboxamide, pentyl-PPX) (14.5)
OTHER SUBSTANCES
Extracted: bupivacaine, mepivacaine, fentanyl
KEYWORDS
serum; SPE
REFERENCE
Gupta,R.N.; Dauphin,A. Column liquid chromatographic determination of bupivacaine in human serum using
SAMPLE
Matrix: blood
HPLC VARIABLES
Column: 300 X 3.9 4 jjim NovaPack C18
Flow rate: 0.8
Detector: UV 259
CHROMATOGRAM
KEYWORDS
benazepril; vincristine; metapramine; chlordiazepoxide; oxprenolol; warfarin; clorazepate; fie-
REFERENCE
TracquiA-J Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
Matrix: blood tissue
jxL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the organic
mogenate + 10 |xg cianopramine + 500 |JLL 2% sodium tetraborate + 8 mL hexane:l-butanol 95:
it to 400 JJLL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the
HPLC VARIABLES
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: amitriptyline, amoxapine, benztropine, bromphenirainine, chlorpheniramine,
doxepin, fluoxetine, imipramine, loxapine, maprotiline, mesoridazine, methadone, metoclo-
pramide, mianserin, moclobemide, nomifensine, nordoxepin, norfluoxetine, nortriptyline, pen-
tobarbital, pheniramine, promethazine, propoxyphene, propranolol, protriptyline, quinidine,
quinine, sulforidazine, thioridazine, thiothixene, tranylcypromine, trazodone, trihexyphenidyl,
trimipramine, triprolidine
Noninterfering: dextromethorphan, norphetidine, phenoxybenzamine, prochlorperazine, triflu-
operazine
Interfering: haloperidol, norpropoxyphene, northiaden
KEYWORDS
REFERENCE
MclntyreJ.M.; King,C.V.; Skafidis,S.; Drummer,0.H. Dual ultraviolet wavelength high-performance liquid chro-
SAMPLE
Sample preparation: Serum. 1 mL Serum + 100 |xL 1 |xg/mL diphenhydramine and 1 |xg/mL
nordiphenhydramine in 1 mM orthophosphoric acid + 100 |xL 1 M NaOH + 5 mL hexane, rotate
at 60 rpm for 15 min, centrifuge. Remove 4 mL of the organic layer and add it to 80 |xL 1 mM
orthophosphoric acid, vortex vigorously for 30 s, inject a 50 (JLL aliquot of the aqueous phase.
Urine. Dilute 1:10 with drug-free urine. 1 mL Diluted urine + 100 (xL 1 u,g/mL diphenhydra-
mine and 1 |xg/mL nordiphenhydramine in 1 mM orthophosphoric acid + 1 mL saturated so-
dium borate (pH 10.2) + 5 mL hexane, rotate at 60 rpm for 15 min, centrifuge. Remove 4 mL
of the organic layer and add it to 80 |xL 1 mM orthophosphoric acid, vortex vigorously for 30
s, inject a 50 jxL aliquot of the aqueous phase.
HPLCVARIABLES
Guard column: 20 X 4.6 5 jxm Supelguard LC-CN cyanopropyl (Supelcosil)
Column: 150 X 4.6 5 |xm Supelcosil LC-PCN cyanopropyl
Mobile phase: MeCNrMeOHrbuffer 55:20:25 (Buffer was 2.6 g K2HPO4 in 1 L water, pH adjusted
to 7.0 with 900 mM orthophosphoric acid.) (Optimize the separation by adjusting the pH of the
mobile phase with a few drops of 1 M NaOH or 900 mM orthophosphoric acid.)
Flow rate: 2.5
Detector: UV 205
CHROMATOGRAM
Retention time: 1.5
Internal standard: diphenhydramine (1.9), nordiphenhydramine (2.9)
OTHER SUBSTANCES
Extracted: metabolites, normeperidine
KEYWORDS
REFERENCE
Meatherall,R.C; Guay,D.R.R; Chalmers,J.L. Analysis of meperidine and normeperidine in serum and urine by
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
HPLC VARIABLES
Column: 150 X 3.9 5 |xm NovaPak phenyl
Flow rate: 1.5
Detector: UV 232
CHROMATOGRAM
Retention time: 6.7
OTHER SUBSTANCES
Noninterfering: cefazolin
KEYWORDS
REFERENCE
Lee,D.K.T.; Wong,C.-Y.; Wang,D.-P. Stability of cefazolin sodium and meperidine hydrochloride, Am. J.Health-
Syst.Pharm., 1996, 53, 1608-1610.
SAMPLE
Matrix: solutions
Sample preparation: Weigh 95.6 mg meperidine hydrochloride and 15.0 mg ondansetron hy-
drochloride in a 10 mL volumetric flask, add 0.9% sodium chloride, shake vigorously for 2 min,
add 0.9% sodium chloride to volume. Dilute 1:2.5, 1:7.5, and 1:15, inject a 20 |ULL aliquot.
HPLC VARIABLES
Column: 220 X 4.6 5 |xm underivatized silica column (Brownlee Silica Applied Biosystems,
Inc.,San Jose)
Mobile phase: MeOH: 10 mM pH 4.0 aqueous monobasic potassium phosphate (adjusted with
10% phosphoric acid) 40:60
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 8.8
OTHER SUBSTANCES
REFERENCE
Venkateshwaran,T.G.; Stewart,J.T.; King,D.T. HPLC determination of morphine-ondansetron and meperidine-
ondansetron mixtures in 0.9% sodium chloride injection, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 1329-
1338.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: acetylcodeine, monoacetylmorphine, thebacon, oxycodone, thebaine, norlevor-
phanol, methadone, benzylmorphine, ethylmorphine, morphine-N-oxide, codeine, codeine-N-ox-
ide, morphine, ethoheptazine, morphine-3-glucuronide, pholcodeine, norpethidine, hydroco-
done, dihydrocodeine, dihydromorphine, levorphanol, norcodeine, normorphine pemoline,
benzphetamine, diethylpropion, mazindol, tranylcypromine, caffeine, fenethyline, phendimet-
razine, methylphenidate, phenelzine, fencamfamin, chlorphentermine, norpseudoephedrine,
phentermine, fennuramine, methylenedioxyamphetamine, amphetamine, normetanephrine, 4-
hydroxyamphetamine, bromo-STP, STP, prolintane, 2-phenethylamine, tyramine, trimethox-
yamphetamine, phenylephrine, pseudoephedrine, ephedrine, methylephedrine, dimethylam-
phetamine, methamphetamine, mescaline, mephentermine, buprenorphine, dextromoramide,
phenoperidine, fentanyl, etorphine, piritramide, noscapine, papaverine, naloxone, dextropro-
poxyphene, nalorphine, phenazocine, norpipanone
Interfering: epinephrine, pipradol, phenylpropanolamine, levallorphan, hydroxypethidine, nor-
methadone, dipipanone, diamorphine, pentazocine
REFERENCE
Law,B.; Gill,R; Moffat,A.C. High-performance liquid chromatography retention data for 84 basic drugs of fo-
rensic interest on a silica column using an aqueous methanol eluent, J.Chromatogr., 1984, 301, 165172.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 1
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: buprenorphine, nalbuphine, ethylmorphine, morphine, codeine, fentanyl,
butorphanol
Interfering: tramadol
REFERENCE
J.Chromatogr., 1991, 570, 339-350.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
feine, cocaine, codeine, doxylamine, fluoxetine, glutethimide, hexobarbital, hypoxanthine,
levorphanol, LSD, mephobarbital, methadone, methylphenidate, methyprylon, N-norcodeine,
oxazepam, oxycodone, phenylpropanolamine, prilocaine, procaine, terfenadine
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
pam, mefenamic acid, megestrol, mephentermine, mephenytoin, mephesin, mephobarbital, me-
meth^rylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, na-
ox5^phenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbi-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
dol, mefenamic acid, mephenytoin, mepivacaine, mesoridazine, metaproterenol, methadone,
zopiclone
KEY WORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
min, filter (0.2 jxm), inject 20 |xL aliquot
HPLC VARIABLES
Column: 250 X 4 Lichrospher 5fxm 60 RP-select B
over 7 min, hold at 75:25 for 3 min, return to 10:90 over 5 min, equilibrate at 10:90 for 5 min
Flow rate: 1.5
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Extracted: amitriptyline, morphine, codeine, benzoylecgonine, amphetamine, norpropoxyphene,
nordiazepam
Also analyzed: phenylpropanolamine, lidocaine, diphenhydramine, nortriptyline, ephedrine (dif-
ferent gradient).
Interfering: cocaine
REFERENCE
Li,S.; Gemperline,P.J.; Briley,K; Kazmierczak,S. Identification and quantitation of drugs of abuse in urine
Mephenesin
CAS Registry No.: 59-47-2, 533-06-2 (carbamate)
Merck Index: 5895
Lednicer No.: 1 118
SAMPLE
Matrix: blood
the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 uL aliquot of
HPLC VARIABLES
Column: 300 x 3.9 4 jxm NovaPack C18
Flow rate: 0.8
Detector: UV 272
CHROMATOGRAM
KEYWORDS
REFERENCE
Tracqui,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic Set, 1995, 40,
254-262.
SAMPLE
Sample preparation: Serum. 2 mL Serum, adjust pH to 14 with 2 M NaOH, extract twice with
6 mL diethyl ether. Combine the organic layers, dry under nitrogen, dissolve the residue in
200 ^L MeOH. Inject a 20 |xL aliquot. Urine. 5 mL Urine, adjust pH to 5 with 1 mL 2 M pH
5 sodium acetate buffer. Add 100 JJLL p-glucuronidase (Helix pomatia 98400 U/mL), incubate
at 37 overnight, cool, wash with 8 mL diethyl ether, discard the organic layer. Adjust to pH
14 with 25% NaOH, extract twice with 8 mL diethyl ether. Combine the organic layers, dry
under nitrogen, reconstitute the residue in 200 |xL MeOH. Inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 4 X 4 7 jxm LiChrospher 100 RP-18
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 19
Internal standard: mephenesin
OTHER SUBSTANCES
Extracted: methocarbamol
KEYWORDS
horse; serum; mephenesin is IS
REFERENCE
Koupai-Abyazani,M.R.; Esaw,B.; Laviolette,B. Determination of methocarbamol in equine serum and urine by
high-performance liquid chromatography with ultraviolet detection and atmospheric pressure ionization-
mass spectrometric confirmation, J.Anal.ToxicoL, 1997, 21, 301-305.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
pam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephobarbital, me-
puromycin, pyrilamine, pîthyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
18, 233-242.
Mephentermine
CAS Registry No.: 100-92-5, 6190-60-9 (sulfate dihydrate)
Merck Index: 5897
Lednicer No.: 1 72
SAMPLE
Matrix: blood
the residue in 100 jxL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 JJLL aliquot of
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 259
CHROMATOGRAM
KEYWORDS
cainide; phencyclidine; thiopental; fennuramine; metipranolol; triprolidine; naproxen; bupren-
REFERENCE
254-262.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 |JLL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
OTHER SUBSTANCES
amine, meclophenoxate, meclozine, medazepam, mepivacaine, meptazinol, mepyramine, me-
soridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene, meth-
otrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine,
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
pam, mefenamic acid, megestrol, mepacrine, mephenytoin, mephesin, mephobarbital, mepi-
REFERENCE
18, 233-242.
Mephenytoin
Molecular formula: Ci2H14N2O2
Merck Index: 5898
SAMPLE
Matrix: blood
|xL 10 jjLg/mL tolylphenobarbital in 200 mM HCl to the SPE cartridge, let stand for 2 min,
elute with 1 mL chloroform.-isopropanol 6:1. Evaporate the eluate to dryness under a stream
of nitrogen at 30, reconstitute the residue in 100 jxL mobile phase, inject a 15 |JLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Supelcosil-LC-8
Flow rate: 3.3
Detector: UV 208
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: theophylline, caffeine, barbital, ethosuximide, primidone, carbamazepinediol, phen-
acemide, methyprylon, nirvanol, phenobarbital, chloramphenicol, butabarbital, carbamazepine
epoxide, pentobarbital, amobarbital, carbamazepine, glutethimide, phenytoin, secobarbital,
methaqualone
Noninterfering: acetaminophen, amikacin, amitriptyline, clonazepam, cyclosporine, desi-
pramine, diazepam, digoxin, disopyramide, gentamicin, imipramine, lidocaine, methotrexate,
N-acetylprocainamide, netilmicin, nortriptyline, procainamide, quinidine, salicylic acid, sulfa-
methoxazole, tobramycin, trimethoprim, valproic acid, p-hydroxyphenobarbital, vancomycin
KEYWORDS
plasma; SPE
REFERENCE
ration, Clin.Chem., 1989, 35, 1615-1618.
SAMPLE
Matrix: blood, milk
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 5 mL water and MeOH:
water 20:80. Add 5 mL 0.5% pH 6.0 KLH2PO4 to 1 mL human breast milk or plasma, mix briefly,
add the sample to the SPE cartridge, elute with 5 mL MeOH, evaporate the eluate to dryness,
dissolve the residue in 200 jxL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Develosil C8-5 (Nomura Chemicals)
Mobile phase: MeCN:0.5% KH2PO4 buffer 30:70 (The pH of mobile phase was adjusted to 4.5
with 50% H3PO4.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 13
Internal standard: mephenytoin
OTHER SUBSTANCES
Extracted: phenytoin
KEYWORDS
cord blood plasma; human breast milk; maternal plasma; mephenytoin is IS; human; plasma; SPE
REFERENCE
Shimoyama,R.; Ohkubo,T.; Sugawara,K; Ogasawara,T.; Ozaki,T.; Kagiya,A.; Saito,Y. Monitoring of phenytoin
in human breast milk, maternal plasma and cord blood plasma by solid-phase extraction and liquid chro-
matography, J.Pharm.Biomed.Anal., 1998, 17, 863-869.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 |xm LiChrosorb RP18
Mobile phase: EtOH:water 10:90 containing 10 mM ct-cyclodextrin and 0.5 mM tri-O-methyl-p-
cyclodextrin
Flow rate: 0.95
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: morsuximide
KEYWORDS
chiral
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeOH, inject a 5 fxL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 |xm RP C18 (Beckman)
Mobile phase: Gradient. Isopropanol:water 20:80, to 25:75 after 7 min (step gradient).
Flow rate: 1.4
Injection volume: 5
Detector: UV 225
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Simultaneous: phenytoin
REFERENCE
Lum,J.T.; Vassanji,N.A.; Wells,RG. Analysis of the toxicologically relevant metabolites of phenytoin in biological
samples by high-performance liquid chromatography, J.Chromatogr., 1985, 338, 242-248.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 6-10 JJLL aliquot.
HPLC VARIABLES
Column: 250 X 4,6 5 |jim Supelcosil LC-I (Supelco)
Flow rate: 2
Detector: UV 204
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: acetaminophen, acetanilide, N-acetylcysteine, N-acetylprocainamide, amobarbi-
tal, ampicillin, aspirin, barbital, butabarbital, butalbital, caffeine, carbamazepine, chloram-
phenieol, chlorpropamide, codeine, cyheptamide, diazoxide, diflunisal, diphylline, disopyram-
ide, ethchlorvynol, gentisic acid, glutethimide, heptabarbital, hexobarbital, ibuprofen,
indomethacin, ketoprofen, mefenamic acid, mephobarbital, methaqualone, methsuximide,
methyl salicylate, methyprylon, morphine, naproxen, nirvanol, oxphenylbutazone, pentobar-
bital, phenacetin, phenobarbital, phensuximide, phenytoin, procainamide, salicylamide, sali-
cylic acid, secobarbital, sulfamethoxazole, sulindac, theophylline, thiopental, tolmetin, trimeth-
oprim, vancomycin
Interfering: ethosuximide, cimetidine, primidone, phenylbutazone
REFERENCE
ramphenicol, caffeine, anticonvulsants, and barbiturates in serum, Ther.Drug Monit., 1988, 10, 101-115.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 u.m Chiralcel OJ
Mobile phase: MeOH
Flow rate: 0.5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: mephobarbital (flow rate 1 mL/min)
KEYWORDS
chiral
REFERENCE
Aboul-Enein,H.Y.; Serignese,V.; Bojarski,J. Simple chiral liquid chromatographic enantioseparation of some
racemic antiepileptic drugs, J.Liq.Chromatogr., 1993, 16, 2741-2749.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 1.5
CHROMATOGRAM
Retention time: 5.4
OTHER SUBSTANCES
Simultaneous: barbital, carbamazepine, diazepam, ethotoin, methsuximide, phenacemide, phe-
nobarbital, phensuximide
REFERENCE
Vydac HPLC Catalog, 1994-5, 1994, p. 26.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
pam, mefenamic acid, megestrol, mepacrine, meperidine, mephesin, mephobarbital, mepiva-
caine, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene, methaqualone,
methazolamide, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa,
nephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxjnnorphone,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 ^m Supelcosil LC-DP (A) or 250 X 4 5 |jim LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ethidium bromide, ethopropazine, fenoprofen, fentanyl, fiavoxate, fluoxetine, fluphenazine, flur-
dol, mefenamic acid, meperidine, mepivacaine, mesoridazine, metaproterenol, methadone,
zide, pindoloj, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide, procaine,
tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifiuoperazine, triflupromazine, tri-
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Sample preparation: 200 \xL Urine + 125 |xL 200 |xg/mL IS, extract twice with 3.0 mL portions
of ethyl acetaterdiethyl ether 67:33. Dry combined organic phases over 200 mg anhydrous
magnesium sulfate for 30 min, centrifuge. Evaporate supernatant under a stream of the nitro-
gen at 35. Reconstitute residue in 500 |xL MeCN:water 20:80, inject a 5 |xL aliquot.
HPLC VARIABLES
Injection volume: 5
Detector: UV 220
CHROMATOGRAM
Retention time: 21
Internal standard: 5-(4-hydroxyphenyl)-5-phenylhydantoin(12.2)
OTHER SUBSTANCES
KEY WORDS
metabolism
REFERENCE
Sarich,T.; Kalhorn,T.; Magee,S.; Al-sayegh,R; Adams,S.; Slattery,J.; Goldstein,J.; Nelson,S.; Wright,J. The effect
of omeprazole pretreatment on acetaminophen metabolism in rapid and slow metabolizers of S-mephe-
nytoin, Clin.Pharmacol.Ther., 1997, 62, 21-28.
SAMPLE
Matrix: urine
Sample preparation: Condition a Sep-Pak silica SPE cartridge with 2 mL chloroform. Adjust
pH of urine to 6 with 100 mM HCl or 100 mM NaOH, centrifuge. Remove a 10 mL aliquot of
the supernatant and add it to 15 mL chloroform, shake for 30 s, filter (Whatman IPS phase
separating paper), add the organic nitrate to the SPE cartridge, elute with 2 mL MeOH. Evap-
orate the eluate to dryness under a stream of nitrogen, reconstitute the residue in 50 (xL MeOH,
shake for 30 s, inject a 20 |JLL aliquot.
HPLC VARIABLES
Column: 75 X 4.5 3 \xm Supelcosil LC-8
Mobile phase: MeOH:100 mM pH 5.0 acetate buffer containing 10 mM p-cyclodextrin 20:80
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
KEYWORDS
chiral; SPE
REFERENCE
R6na,K.; Szab6,I. Determination of mephenytoin stereoselective oxidative metabolism in urine by chiral liquid
chromatography employing p-cyclodextrin as a mobile phase additive, J.Chromatogr., 1992, 573, 173-177.
Mephobarbital
Merck Index: 5899
SAMPLE
Matrix: blood
Sample preparation: 100 JJLL Serum + 100 |xL buffer +1.5 mL IS in 5% isopropanol in chloro-
a 6-10 JJLL aliquot. (Buffer was 13.6 g KH2PO4 in 90 mL water, pH adjusted to 6.8 with about
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-I (Supelco)
Flow rate: 2
Detector: UV 204
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: acetaminophen, amobarbital, barbital, caffeine, carbamazepine, chloramphenicol,
ethosuximide, methsuximide, pentobarbital, phenobarbital, phenytoin, primidone, secobarbital,
theophylline, thiopental
Simultaneous: acetanilide, N-acetylcysteine, N-acetylprocainamide, ampicillin, aspirin, buta-
barbital, butalbital, chlorpropamide, cimetidine, codeine, cyheptamide, diazoxide, diflunisal,
diphylline, disopyramide, gentisic acid, glutethimide, heptabarbital, hexobarbital, ibuprofen,
indomethacin, ketoprofen, mefenamic acid, mephenytoin, methaqualone, methsuximide,
methyl salicylate, methyprylon, morphine, naproxen, nirvanol, oxphenylbutazone, phenacetin,
phensuximide, phenylbutazone, procainamide, salicylamide, salicylic acid, sulfamethoxazole,
sulindac, tolmetin, trimethoprim, vancomycin
Interfering: ethchlorvynol
KEYWORDS
serum
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 600 JJLL allobarbital in 75 mM pH 6.8 buffer, add 200
units p-glucuronidase (Type VII-A from E. coli), incubate at 37 for 30 min, add 1 mL of the
sample to an Extrelut-1 SPE cartridge, after 10 min elute with 2.5 mL MTBE, dry the eluate
under a stream of nitrogen, dissolve the residue in 50 jxL MeOH:water 1:1, inject a 10 |xL
aliquot.
HPLC VARIABLES
Column: 25 X 4 4 |jim Superspher RP-18e (Merck)
Mobile phase: MeOH:11.2 mM p-cyclodextrin in 20 mM KH2PO4 5:95
Flow rate: 0.8
Detector: UV 210
CHROMATOGRAM
Retention time: 19 (R), 21 (S)
Internal standard: allobarbital (16)
OTHER SUBSTANCES
Simultaneous: phenobarbital, zonisamide
KEYWORDS
serum; SPE; chiral
REFERENCE
Eto,S.; Noda,H.; Noda,A. Simultaneous determination of antiepileptic drugs and their metabolites, including
chiral compounds, via p-cyclodextrin inclusion complexes by a column-switching chromatographic tech-
nique, J.Chromatogr.B, 1994, 658, 385-390.
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Serum + 600 uL allobarbital in 75 mM pH 6.8 phosphate buffer,
add 200 units p-glucuronidase, heat at 37 for 30 min, add 1 mL of this solution to an Extrelut-
1 SPE cartridge, let stand for 10 min, elute with 2.5 mL MTBE. Evaporate the eluate to dryness
under a stream of nitrogen, reconstitute the residue in 50 JJLL MeOH:water 50:50, inject a 10
IxL aliquot onto columns A and B in series with mobile phase A. After 12 min elute column A
with mobile phase B, continue to elute column B with mobile phase A. Carbamazepine diol,
carbamazepine epoxide, phenytoin, and carbamazepine elute from column A and the enantio-
mers of 5-(p-hydroxyphenyl)-5-phenylhydantoin and mephobarbital, phenobarbital, zonisam-
ide, and allobarbital elute from column B. Re-equilibrate columns A and B with mobile phase
A for 5 min before the next injection.
HPLC VARIABLES
Column: A 250 X 4 4 |xm Superspher RP-18e (E. Merck); B 250 X 4 4 |xm Superspher RP-18e
(E. Merck)
Mobile phase: A MeOH:11.2 mM p-cyclodextrin in 20 mM KH2PO4 5:95; B MeCN:20 mM KH2PO4
16:84
Flow rate: 0.8
Detector: A UV 210; B UV 210
CHROMATOGRAM
Retention time: 19 (R), 21 (S) (column B)
Internal standard: allobarbital (17, from column B)
Limit of detection: 12.& ng/mL (S), 11.6 ng/mL (R)
OTHER SUBSTANCES
Extracted: carbamazepine diol, carbamazepine epoxide, carbamazepine, 5-(p-hydroxyphenyl)-5-
phenylhydantoin, zonisamide, phenytoin, phenobarbital, metabolites
KEYWORDS
serum; column-switching; SPE; chiral
REFERENCE
Eto,S.; Noda,H.; Noda,A. Simultaneous determination of antiepileptic drugs and their metabolites, including
chiral compounds, via p-cyclodextrin inclusion complexes by a column-switching chromatographic tech-
nique, J.Chromatogr.B, 1994, 658, 385-390.
SAMPLE
Matrix: solutions
HPLC VARIABLES
cyclodextrin
Flow rate: 0.04
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: glutethimide
KEYWORDS
chiral
REFERENCE
chromatography with joint use of two cyclodextrin additives, J.Chromatogr.A, 1997, 782, 111.
SAMPLE
Matrix: solutions
Sample preparation: Mix 50 JJLL of a 20-200 |xg/mL solution in acetone with 50 |xL of a 0.4-1.6
mg/mL solution of 2-bromo-2'-acetonaphthone in acetone, add 5-10 mg cesium carbonate, heat
at 30 for 30 min, add 50 |xL glacial acetic acid, mix, inject an aliquot.
HPLC VARIABLES
Flow rate: 2
Detector: UV 249
CHROMATOGRAM
Retention time: 3.1
OTHER SUBSTANCES
Simultaneous: amobarbital, barbital, butobarbital, heptobarbital, pentobarbital, phenobarbital,
secobarbital
Interfering: hexobarbital
KEYWORDS
derivatization
REFERENCE
Hulshoff,A.; Roseboom,H.; Renema,J. Improved detectability of barbiturates in high-performance liquid chro-
matography by pre-column labelling and ultraviolet detection, J.Chromatogr., 1979, 186, 535-541.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: Gradient. A was MeCN:5 mM sodium carbonate 9:81. B was MeCN:20 mM sodium
carbonate 20:80. A:B from 100:0 to 0:100 over 10 min.
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: allobarbital, amobarbital, barbital, barbituric acid, butabarbital, methabarbital,
methohexital, phenobarbital, phenytoin, secobarbital, thiamylal
REFERENCE
Slingsby,R.W.; Rey,M. Determination of pharmaceuticals by multi-phase chromatography: Combined reversed
phase and ion exchange in one column, J.Liq.Chromatogr., 1990, 13, 107-134.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 ^m Chiralcel OJ
Mobile phase: MeOH
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: mephenytoin (flow rate 0.5 mL/min)
KEYWORDS
chiral
REFERENCE
Aboul-Enein,H.Y; Serignese,V.; Bojarski,J. Simple chiral liquid chromatographic enantioseparation of some
racemic antiepileptic drugs, J.Liq.Chromatogr., 1993, 16, 2741-2749.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
feine, cocaine, codeine, doxylamine, fluoxetine, glutethimide, hexobarbital, hypoxanthine, Ie-
vorphanol, LSD, meperidine, methadone, methylphenidate, methyprylon, N-norcodeine, oxa-
zepam, oxycodone, phenylpropanolamine, prilocaine, procaine, terfenadine
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: MeCNrIO mm KH2PO4 + 5 mM 1-decanesulfonic acid 30:70, adjusted to pH 3.2
with 85% phosphoric acid
Flow rate: 1
Detector: UV 214
CHROMATOGRAM
Internal standard: methyl paraben (7.0)
OTHER SUBSTANCES
Simultaneous: allobarbital, barbital, butalbital, aprobarbital, pentobarbital, phenobarbital,
secobarbital, talbutal, vinbarbital
KEYWORDS
REFERENCE
Ibrahim,F.B. Simultaneous determination and separation of several barbiturates and analgesic products by
ion-pair high-performance liquid chromatography, J.Liq.Chromatogr., 1993, 16, 2835-2851.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4 5 |xm LiChroCART ChiraDex (p-cyclodextrin chemically bonded to silica)
(Merck)
Flow rate: 0.5
Detector: UV 220
CHROMATOGRAM
KEYWORDS
chiral
REFERENCE
Cabrera,K; Lubda,D. Influence of temperature on chiral high-performance liquid chromatographic separations
of oxazepam and Prominal on chemically bonded p-cyclodextrin as stationary phase, J.Chromatogr.A, 1994,
666, 433-438.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
REFERENCE
Hill,D.W.; Kind,AJ- Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.Toxicol, 1994,
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in mobile phase at a concentration of 100 jjig/mL, inject a 5 JAL
aliquot.
HPLC VARIABLES
Column: 300 X 2 (juBondapak C18
Mobile phase: MeCN:water 30:70 adjusted to pH 3.0 with formic acid
Flow rate: 0.27
Injection volume: 5
Detector: MS, VG TRIO 2000 single quadrupole MS with EI or CI or UV 270
KEYWORDS
mass spectra given
Next Page
REFERENCE
Ryan,T.W. Identification of barbiturates using high performance liquid chromatography-particle beam EI/CI
mass spectrometry, J.Liq.Chromatogr., 1994, 17, 867-881.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 62 X 2 packed with chiral packing (Prepare packing by dissolving 3,5-dimethylphen-
ylcarbamate amylose in DMF, coat on Nucleosil 1000-7, dry at 60 for 3 h under reduced
pressure.)
Mobile phase: Hexane:isopropanol 90:10
Flow rate: 0.1
Detector: UV 254
CHROMATOGRAM
KEYWORDS
narrow-bore; chiral; a 1.80
REFERENCE
Chankvetadze,B; Chankvetadze,L.; Sidamonidze,S.; Yashima,E.; Okamoto,Y. Enantioseparation of some chiral
Pharmaceuticals using narrow-bore liquid chromatography, J.Pharm.Biomed.Anal., 1995, 13, 695-699.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 (xL aliquot of an 8 |xg/mL solution.
HPLC VARIABLES
Column: 250 X 4 4 |xm Superspher 100 RP-18
Mobile phase: EtOH:buffer containing 25 mM p-cyclodextrin substituted with 2-hydroxy-3-tri-
methylammoniumpropyl groups (Roquette Freres, Lestrem, France) (Buffer was 1.776 g/L
NaH2PO4 adjusted to pH 2.5 with orthophosphoric acid.)
Column temperature: 22.5
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
Retention time: 16.75 (-), 18.09 (+)
OTHER SUBSTANCES
Interfering: hexobarbital
KEYWORDS
chiral
REFERENCE
Roussel,C; Favrou,A. Cationic (3-cyclodextrin: a new versatile chiral additive for separation of drug enantio-
mers by high-performance liquid chromatography, J.Chromatogr.A, 1995, 704, 67-74.
Previous Page
Mepivacaine
Merck Index: 5905
Lednicer No.: 1 17
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 25 |xL EtOH + 25 JJLL 17.6 |xg/mL ropivacaine in EtOH,
mix. Add 5 mL diethyl ether, vortex for 3 min, centrifuge at 2000 g for 10 min at 4. Add 500
JULL 100 mM hydrochloric acid to organic phase, back extract for 3 min. Discard organic phase,
add 5 mL n-pentane containing 100 (xL isoamyl alcohol, wash for 3 min, discard organic phase
again. Add 50 |xL 2 M NaOH, extract aqueous phase with 5 mL n-pentane containing 100 JJLL
isoamyl alcohol, centrifuge at 2000 g for 10 min at 4. Evaporate organic phase to dryness
under a gentle stream of nitrogen at 40. Reconstitute the residue in 100 |xL mobile phase,
HPLC VARIABLES
Guard column: 10 X 4.0 40 |xm al-AGP (J.T.Baker)
Column: 100 X 4.0 5 |xm al-AGP (J.T.Baker)
Mobile phase: 2-Propanol:buffer 6.8:93.2 (Buffer was 3,6 g/L Na2HPO4.12 H2O adjusted to pH
6.8 with phosphoric acid.)
Flow rate: 1.1
Detector: UV 210
CHROMATOGRAM
Internal standard: S-(-)-ropivacaine (10.5)
OTHER SUBSTANCES
Noninterfering: bupivacaine
Interfering: lidocaine, prilocaine
KEYWORDS
plasma; chiral; pharmacokinetics
REFERENCE
Vletter,A.A.; 01ieman,W.; Burm,A.G.L.; Groen,K.; van Kleef,J.W. High-performance liquid chromatographic as-
say of mepivacaine enantiomers in human plasma in the nanogram per milliliter range, J.Chromatogr.B,
1996, 678, 369-372.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond-Elut C18 SPE cartridge with 1 mL 100 mM HCl,
1 mL MeOH and 1 mL water. Add 350 |xL 100 mM pH 4.5 phosphate buffer and 40 |xL 200
u,g/mL S-bupivacaine in MeOH to 1 mL serum, vortex. Add the mixture to the SPE cartridge
and pass it through the cartridge attached to a vacuum manifold, wash 4 times with 250 (xL
water, then wash with 500 JJLL MeCN and allow to air dry for 1 min between each wash. Elute
with four 250 |xL aliquots of MeOH containing 2% HCl, evaporate the eluate to dryness under
a stream of nitrogen, reconstitute the residue in 1 mL mobile phase and inject a 100 jxL aliquot.
HPLCVARIABLES
Column: 250 X 4 5 |xm Sumichiral OA-4700 (YMC, Wilmington, NC, USA)
Mobile phase: Hexane:dichloroethane:absoluteMeOH 85:10:5
Flow rate: 0.8
Detector: UV 220
CHROMATOGRAM
Retention time: 9.3 (R-(+)), 11.4 (S-(-))
Internal standard: S-bupivacaine (7.7)
KEYWORDS
chiral; SPE; serum
REFERENCE
Siluveru,M.; Stewart,J.T. Stereoselective determination of mepivacaine in human serum using a bush-type
chiral stationary phase and solid-phase extraction, J.Chromatogr.B, 1997, 690, 359-362.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 |xL 40 (xg/mL etidocaine hydrochloride in water + 100
IxL 1 M NaOH, vortex for 15 s, add 5 mL diethyl ether, shake on a reciprocating shaker for 10
min, centrifuge. Remove the organic layer and evaporate it to dryness under a stream of ni-
trogen, reconstitute the residue in 100 |JLL mobile phase, inject a 80 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.2 10 |xm uEondapak C18
Flow rate: 0.9
Detector: UV 210
CHROMATOGRAM
Retention time: 3.7
Internal standard: etidocaine (12.0)
OTHER SUBSTANCES
Extracted: 2,6-pipecolylxylidine, bupivacaine, lidocaine
Noninterfering: metabolites, 2,3-chloroprocaine, theophylline, mexiletine, quinidine, disopyr-
KEYWORDS
plasma
REFERENCE
Ha,H.-R.; Funk,B-l Gerber,H.R.; Follath,F. Determination of bupivacaine in plasma by high-performance liquid
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL 1 M NaOH + 3 mL heptane:ethyl acetate 90:10,
|JLL 50 mM sulfuric acid, shake for 2 min, centrifuge at 1200 g for 5 min. Remove the aqueous
phase and add it to 820 |xg sodium acetate, inject a 40 |xL aliquot. (The sodium acetate was
measured out by adding 50 |xL 200 mM sodium acetate in MeOH to the tube and evaporating
the MeOH.)
HPLC VARIABLES
Column: 250 X 4 10 |xm (xBondapak C18
Mobile phase: MeCN:10 mM NaH2PO4 7:93, adjusted to pH 2.1
Flow rate: 1
Detector: UV 205
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Extracted: articaine
KEYWORDS
plasma; rabbit
REFERENCE
Le Gue"vello,R; Le Corre,R; Chevanne,R; Le Verge,R. High-performance liquid chromatographic determination
SAMPLE
Matrix: blood
250 jxL bupivacaine in 100 mM NaH2PO4 and 250 jxL serum to the column at 1 mL/min, wash
twice with water and once with MeCN draining the column completely after each wash, elute
with 250 |xL eluting solution, centrifuge for 20 s to remove last of eluate, inject a 5 jxL aliquot
of the eluate. (Eluting solution was 2.5 mL 35% perchloric acid in 100 mL MeOH.)
HPLC VARIABLES
Guard column: 15 X 3.2 7 |xm RP-8 (Applied Biosystems)
Column: 150 X 4.6 5 (xm Ultrasphere octyl
Mobile phase: MeCN: 10 mM KH2PO4 25:80, pH 5.2
Flow rate: 1.5
Injection volume: 5
Detector: UV 205
CHROMATOGRAM
Retention time: 2.6
Internal standard: bupivacaine (7.2)
OTHER SUBSTANCES
Extracted: bupivacaine, meperidine, fentanyl
KEYWORDS
serum; SPE
REFERENCE
Gupta,R-N.; Dauphin,A. Column liquid chromatographic determination of bupivacaine in human serum using
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 264
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.7
OTHER SUBSTANCES
mine, fenoterol, fentanyl, fiavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, hal-
amine, meclophenoxate, meclozine, medazepam, mephentermine, meptazinol, mepyramine,
thipendyl, protriptyline, proxjnnetacaine, pseudoephedrine, p3nrimethamine, quinidine, qui-
REFERENCE
Jane,L; McKinnonA-; Flanagan,R-J- High-performance liquid chromatographic analysis of basic drugs on silica
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
phesin, mescaline, mesoridazine, methadone, methamphetamine, methapyrilene,
meth^rylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, na-
phazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, nifiumic acid, nitra-
oxyphenbutazone, oxjrtetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbi-
puromycin, pyrilamine, p5ndthyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 200 (xM solution in MeOH.
HPLC VARIABLES
Column: 100 X 4.7 7 |xm Hypercarb (Shandon)
Mobile phase: MeOH containing 5 mM N-benzyloxycarbonylglycyl-L-proline and 4.5 mM NaOH
Detector: UV 270
CHROMATOGRAM
KEYWORDS
chiral; a = 1.11
REFERENCE
Huynh,N.-H.; Karlsson,A.; Pettersson,C. Enantiomeric separation of basic drugs using N-benzyloxycarbonyl-
glyclyl-L-proline as counter ion in methanol, J.Chromatogr.A, 1995, 705, 275287.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ethidium bromide, ethopropazine, fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine,flur-
dol, mefenamic acid, meperidine, mephenytoin, mesoridazine, metaproterenol, methadone,
talol, spironolactone, sulfinpâzone, sulindac, temazepam, terbutaline, terfenadine, tetra-
tolbutamide, tolmetin, trazodone, triamterene, triazolam, trinuoperazine, triflupromazine, tri-
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Meprobamate
Merck Index: 5908
Lednicer No.: 1 218
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 100 |xL 200 mM HCl + 200 |xL 100 |xg/mL carisoprodol
+ 1 mL chloroform, shake for 2 min, sonicate for 1 min, centrifuge for 1 min. Remove the
organic layer and add it to 200 mM NaOH, shake for 1 min, sonicate for 1 min, centrifuge for
1 min. Remove 750 (xL of the organic layer and evaporate it to dryness, dissolve the residue
in 50 |xL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 100 X 3 octyl CP-tm-Spher C8 glass column (Chrompack)
Mobile phase: MeCN:50 mM NaH2PO4 35:65 adjusted to pH 2.2 with phosphoric acid
Flow rate: 0.8
Detector: UV 190
CHROMATOGRAM
Retention time: 1.8
Internal standard: carisoprodol (5.5)
KEYWORDS
serum
REFERENCE
Van Damme,M.; Molle,L.; Abi Khalil,F. Useful sample handlings for reversed phase high performance liquid
chromatography in emergency toxicology, J.Toxicol.Clin.Toxicol., 1985, 23, 589-614.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 100 jxL 200 mM HCl + 200 u,L 100 jxg/mL carisoprodol
+ 1 mL chloroform, shake for 2 min, sonicate for 1 min, centrifuge for 1 min. Remove the
organic layer and add it to 200 mM NaOH, shake for 1 min, sonicate for 1 min, centrifuge for
1 min. Remove 750 |xL of the organic layer and evaporate it to dryness, dissolve the residue
in 50 uL mobile phase, inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 100 X 3 octyl CP-tm-Spher C8 glass column (Chrompack)
Flow rate: 0.8
Detector: UV 190
CHROMATOGRAM
Retention time: 1.8
Internal standard: carisoprodol (5.5)
KEYWORDS
serum
REFERENCE
Hormazabal,V.; Steffanak,L; Yndestad,M. Simultaneous extraction and determination of sulfadiazine and tri-
methoprim in medicated fish feed by high-performance liquid chromatography, J.Chromatogr., 1993, 648,
183-186.
SAMPLE
Sample preparation: Powder tablets, weigh out amount equivalent to 100 mg meprobamate,
dissolve in 50 mL mobile phase, sonicate for 10 min, filter, inject an aliquot.
HPLCVARIABLES
Column: 125 X 4 5 |xm Lichrospher RP-18
Mobile phase: MeOH:50 mM pH 1.9 phosphoric acid 30:70 containing 1 mM benzoic acid
Flow rate: 0.9
Detector: UV 273
CHROMATOGRAM
KEY WORDS
tablets; indirect UV detection
REFERENCE
Bechet,L; Ceccato,A.; Hubert,R; Herne,R; Crommen,J. Determination of meprobamate in pharmaceutical dos-
age forms also containing carbromal by liquid chromatography and indirect photometric detection,
Meptazinol
Merck Index: 5910
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 20 |xL 2.5 |xg/mL IS in MeOH + 100 (xL buffer + 3 mL
ethyl acetate, vortex for 2 min, centrifuge at 2000 rpm for 5 min. Remove the organic layer
|xL MeOH, add 50 |xL water, inject an aliquot. (Buffer was 1 M Na2HPO4 adjusted to pH 9.5
with 5 M NaOH.)
HPLCVARIABLES
Mobile phase: MeOH:0.5% ammonium acetate 45:55
Flow rate: 2
CHROMATOGRAM
Retention time: 5.8
Internal standard: m-(l-cyclopropylmethyl-3-ethylhexahydro-lH-azepin-3-yl)phenol (7.0)
KEYWORDS
plasma
REFERENCE
Frost,T. Determination of meptazinol in plasma by high-performance liquid chromatography with fluorescence
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Plasma + 100 |xL 40 ng/mL fenethazine in 2 M aqueous Tris +
200 (xL MTBE, vortex for 30 s, centrifuge at 9950 g for 2 min, inject a 100 |xL aliquot of the
organic phase.
HPLC VARIABLES
Column: 125 X 5 5 |xm Spherisorb S5W silica
Mobile phase: MeOH:glacial acetic acid:ammonia 996:3:1 (Ammonia was 0.88 g/mL.)
Flow rate: 2
Detector: E, EDT Research LCA 15, glassy carbon electrode +1.2 V, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 3
Internal standard: fenethazine (4)
KEYWORDS
plasma; cow; human
REFERENCE
Storey,G.C.A.; Schootstra,R.; Henry,J.A. Measurement of meptazinol in plasma by high-performance liquid
chromatography with electrochemical detection, J.Chromatogn, 1985, 341, 223-227.
SAMPLE
Matrix: microsomal incubations, urine
Sample preparation: Microsomal incubations. 2 mL Microsomal incubation + IS + 1 mL 1 M
pH 10.0 ammonia/ammonium chloride buffer + 5 mL n-hexane:ethyl acetate 50:50, extract.
Remove 4 mL of the organic layer and evaporate it to dryness under a stream of nitrogen,
reconstitute the residue in 50 pX water, inject a 20 fxL aliquot. Urine. Untreated or deconju-
gated (with 500-2000 IU/mL p-glucuronidase/sulfatase from Helix pomatia for 24 h) urine + IS
+ 1 mL 1 M pH 10.0 ammonia/ammonium chloride buffer + 5 mL n-hexane:ethyl acetate 30:
70, stir vigorously for 10 min. Remove 4 mL of the organic layer and evaporate it to dryness
under a stream of nitrogen, reconstitute the residue in 50 |xL water, inject a 20 JJLL aliquot.
HPLCVARIABLES
Guard column: 30 X 4 10 |xm LiChrospher 60 CN
Column: 250 X 4 5 |xm LiChrospher 100 CN
Mobile phase: MeCN:MeOH:l% triethylammonium acetate buffer (pH 5.5) 15:5:80
Flow rate: 0.5
Detector: UV 220
CHROMATOGRAM
Retention time: 31
Internal standard: 3-(3-ethylhexahydro-l-ethyl-lH-azepin-3-yl)phenol (35)
OTHER SUBSTANCES
KEYWORDS
rabbit; liver; human; pharmacokinetics; rat
REFERENCE
Rudolphi,C; Blaschke,G. Determination of the stereoselective aspects in in-vitro and in-vivo metabolism of the
analgesic meptazinol by high-performance liquid chromatography, J.Chromatogr.B, 1995, 663, 315-326.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 3.6
OTHER SUBSTANCES
amine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, mepyramine,
REFERENCE
Jane,I.; McKinnonÂ..; Flanagan,R.J- High-performance liquid chromatographic analysis of basic drugs on silica
Mercaptobenzothiazole
Molecular formula: C7H5NS2
Merck Index: 5916
SAMPLE
Sample preparation: Urine. 1 mL Urine + 4 mL EtOH, centrifuge at 3000 rpm for 10 min.
Dilute the supernatant with EtOH, filter (0.2 |m, Aero LC13), inject a 20 jxL aliquot. (Hydro-
lyze conjugates by mixing 100 |xL urine with 900 jxL 100 mM pH 5 acetate buffer and 33 units
sulfatase or 5000 units p-glucuronidase, heat at 37 for 30 min, proceed as above.) Tissue.
Homogenize liver or kidney in 3 volumes of cold 100 mM pH 5 acetate buffer containing 2 mM
aminooxyacetic acid, add 4 volumes of EtOH, centrifuge at 3000 rpm for 20 min. Filter (0.2
|xm, Aero LC13) the supernatant and inject a 20 |xL aliquot. Plasma. Add 4 volumes of EtOH
to plasma, centrifuge at 3000 rpm for 20 min. Filter (0.2 |xm, Aero LC13) the supernatant and
HPLC VARIABLES
Column: 250 X 4.6 Partisil 10 ODS 3
Mobile phase: MeCN:water 50:50 adjusted to pH 4.5 with trifluoroacetic acid
Flow rate: 1
Detector: UV 321
CHROMATOGRAM
Retention time: 7.0
KEYWORDS
rat; hamster; guinea pig; mouse; plasma; liver; kidney
REFERENCE
Elfarra,A.A.; HwangJ.Y. In vivo metabolites of <S-(2-benzothiazolyl)-L-cysteine as markers of in vivo cysteine
conjugate p-lyase and thiol glucuronosyl transferase activities, Drug Metab.Dispos., 1990, 18, 917-922.
SAMPLE
Sample preparation: Bulk. Weigh out 100 mg morphine, dissolve in 25 mL MeOH:water:acetic
acid 24:72:1, dilute with MeOH:water:acetic acid 24:72:1 to a final concentration of 240 |xg/mL
morphine, filter (0.45 |jim), inject a 20 |xL aliquot. Injections. Dilute with MeOH:water:acetic
acid 24:72:1 to a final concentration of 240 |xg/mL morphine, filter (0.45 |mm), inject a 20 \LL
aliquot.
HPLC VARIABLES
Mobile phase: MeOH:5 mM sodium l-heptanesulfonate:acetic acid 24:72:1
Flow rate: 1.5
Detector: UV 230
CHROMATOGRAM
Retention time: 16
OTHER SUBSTANCES
Simultaneous: morphine (UV 284), phenol (UV 284), pseudomorphine (UV 230)
KEYWORDS
injections
REFERENCE
Bello,A.C; Jhangiani,R.K. Liquid chromatographic determination of morphine sulfate and some contaminants
in injections and bulk drug material: collaborative study, J.Assoc.Off.Anal.Chem., 1988, 71, 1046-1048.
SAMPLE
Sample preparation: Directly inject a 20 |xL aliquot of a 250 |xg/mL digoxin injection.
HPLC VARIABLES
Flow rate: 2
Detector: UV 218
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Simultaneous: digoxin
KEYWORDS
injections
REFERENCE
Reepmeyer,J.C; Juhl,Y.H. Contamination of injectable solutions with 2-mercaptobenzothiazole leached from
rubber closures, J.Pharm.ScL, 1983, 72, 1302-1305.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 100 X 3 5 u,m ChromSpher C18 (Chrompack)
Flow rate: 0.4
Detector: UV 321
CHROMATOGRAM
Retention time: 5.5
REFERENCE
Stijntjes,G.J.; te Koppele,J.M.; Vermeulen,N.P.E. High-performance liquid chromatography-fluorescence assay
of pyruvic acid to determine cysteine conjugate (3-lyase activity: application to S-l,2-dichlorovinyl-L-cysteine
and S-2-benzothiazolyl-L-cysteine, Anal.Biochem., 1992, 206, 334-343.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeCN, inject a 50 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 10 ^m Spheri-10 RP-18
Flow rate: 1
Detector: UV 328
CHROMATOGRAM
Retention time: 2
REFERENCE
Gaind,V.S.; Jedrzejczak,K. HPLC determination of rubber septum contaminants in the iodinated intravenous
contrast agent (sodium iothalamate), JAnal.Toxicol., 1993, 17, 34-37.
SAMPLE
Matrix: solutions
Sample preparation: Mix 500 u-L of a solution in MeOH:water 90:10 with 500 (xL 50 |xM
CY5.4a-IA in MeOH:water 70:30, pass dry nitrogen through the mixture for 1 min, heat at 65
for 1.5 h, inject a 25 jxL aliquot. (Some details for the synthesis of CY5.4a-IA are given in the
paper.)
HPLC VARIABLES
Column: 150 X 3.1 5 pm LiChrosorb RP-8
Mobile phase: MeOH:10 mM pH 6.8 phosphate buffer 65:35 containing 1 mM triethylamine
Flow rate: 0.75
Detector: F ex 670 (9.5 mW Lasermax LAS200-670-10 diode laser)
CHROMATOGRAM
Retention time: 7
KEYWORDS
derivatization
REFERENCE
Mank,A.J.G.; Molenaar,E.J.; Lingeman,H.; Gooijer,C; Brinkman,U.A.T.; Velthorst,N.H. Visible diode laser in-
duced fluorescence detection in liquid chromatography after precolumn derivatization of thiols, Anal.Chem.,
1993, 65, 2197-2203.
Mercaptopurine
CAS Registry No.: 50-44-2,6112-76-1 (monohydrate)
Merck Index: 5919
SAMPLE
Matrix: blood
Sample preparation: Condition a 500 mg Bakerbond SPE cartridge packed with LiChrosorb
RP-18 with 2 mL MeOH, 2 mL water and 1 mL MeOH. Make up 500 |xL plasma to 3 mL with
MeOH. Centrifuge at 1100 g for 15 min, add 1.5 mL supernatant to the SPE cartridge, elute
at 50 (jiL/min flow-rate, inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeOH: pH 4.15 phosphate buffer 20:80
Flow rate: 1
Detector: UV 262
CHROMATOGRAM
Internal standard: mercaptopurine
KEY WORDS
plasma; SPE; mercaptopurine is IS
REFERENCE
Misztal,G.; Paw,B. Determination of fludarabine phosphate in human plasma using reversed phase high-per-
formance liquid chromatography, Pharmazie, 1996, 51, 733-734.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Blood + 120 jig dithiothreitol (to prevent oxidation of 6-mercapto-
purine), centrifuge at 2000 g for 5 min. Cool plasma in ice, add a volume of ice-cold 50%
trichloroacetic acid equal to 10% of the plasma volume, mix vigorously, keep on ice for 10 min,
centrifuge at 2000 g for 10 min. Remove the supernatant and adjust the pH to 6-7 with 4 M
KOH, inject a 460 |xL aliquot.
HPLC VARIABLES
Column: Two 250 X 4.6 10 (xm Spherisorb 10-ODS columns in series
Mobile phase: 50 mM pH 6.35 potassium phosphate buffer
Flow rate: 1.5
Detector: UV 312
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
KEYWORDS
plasma; pharmacokinetics; dog
REFERENCE
De Abreu,R.A.; van Baal,J.M.; Schouten,T.J.; Schretlen,E.D.A.M.; de Bruyn,C.H.M.M. High-performance liquid
chromatographic determination of plasma 6-mercaptopurine in clinically relevant concentrations,
J.Chromatogr., 1982, 227, 526-533.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 25 jxL 4 |xg/mL 6-thioguanine in water + 100 |xL 400 mM
NaOH + 1 mL 0.3% phenylmercuric acetate in ethyl acetate + 3 mL diethyl ether, shake on a
tumble mixer for 10 min, centrifuge for 5 min. Remove the organic layer and add it to 500 |xL
100 mM HCl, whirlmix for 2 min, centrifuge for 5 min, discard the organic layer, evaporate
traces of organic solvent under a stream of nitrogen at room temperature for 15 min, add 10
IxL 3 mg/mL dithioerythritol in water, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 LiChrosorb 10 RP-18
Mobile phase: Isopropanol:water 3:97 containing 13.80 g/L NaH2PO4.H2O, 200 |xL/L 85% phos-
phoric acid, 60 mg/L dithioerythritol, and 500 mg/L sodium octanesulfonate, pH 3.6-3.7
Flow rate: 1.5
mM potassium chromate in 500 mM HCl pumped at 0.16 mL/min and with air flowing at 0.32
mL/min and the mixture flowed through a single mixing coil. The effluent from this coil mixed
with 1.6% sodium metabisulfite pumped at 0.16 mL/min and this mixture flowed through a
single mixing coil. The effluent from this coil mixed with 4 M ammonium hydroxide pumped
at 0.23 mL/min and this mixture flowed through a double mixing coil to a debubbler. The liquid
effluent from the debubbler flowed to the detector.
CHROMATOGRAM
Retention time: 6
Internal standard: 6-thioguanine (8)
KEY WORDS
post-column reaction; plasma; pharmacokinetics
REFERENCE
Jonkers,R.E.; Oosterhuis,B.; ten Berge,R.J.M.; van Boxtel,C.J. Analysis of 6-mercaptopurine in human plasma
with a high-performance liquid chromatographic method including post-column derivatization and fluori-
metric detection, J.Chromatogr., 1982, 233, 249-255.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 80 JJLL 10 jxg/mL 6-thioguanine in water + 10 JULL 1 M
dithiothreitol, vortex for 10 s, add 2 mL MeCN, vortex for 30 s, centrifuge at 2000 g for 5 min.
Remove the supernatant and add it to 2 mL dichloromethane, shake on a reciprocating shaker
for 5 min, centrifuge at 2000 g for 5 min. Remove 750 |xL from the top aqueous layer and
evaporate it to dryness under a stream of nitrogen at 37, reconstitute the residue in 150 JJLL
distilled water, vortex for 1 min, inject a 15 JULL aliquot.
HPLC VARIABLES
Guard column: 70 X 2.2 30-38 jxm Co:Pell ODS
Mobile phase: MeCN.acetic acid.water 3.5:0.2:96.3
Flow rate: 1.4
Detector: UV 322
CHROMATOGRAM
Retention time: 4.8
Internal standard: 6-thioguanine (6.5)
OTHERSUBSTANCES
Noninterfering: caffeine, cytarabine, 5-fluorouracil, prednisone, theophylline, vinblastine,
vincristine
KEYWORDS
plasma; protect from light; monkey; pharmacokinetics; human
REFERENCE
Narang,P.K; Yeager,R.L.; Chatterji,D.C. Quantitation of 6-mercaptopurine in biologic fluids using high-perfor-
mance liquid chromatography: a selective and novel procedure, J.Chromatogr., 1982, 230, 373-380.
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Serum + 5 |xL 50 (xg/mL 2-ethyl-4-oxoquinazoline in EtOH + 100
|xL reagent, let stand at room temperature for 1 h, add 1.8 mL ethyl acetate, mix, centrifuge
at 1800 g for 5 min, remove 1.5 mL of the supernatant, repeat the extraction. Combine the
organic layers and evaporate them to dryness under reduced pressure below 30, reconstitute
the residue in 100 |xL initial mobile phase, inject a 90 (xL aliquot. (Reagent was 30 mg N-
ethylmaleimide in 2 mL 50 mM pH 7.0 phosphate buffer, prepare fresh daily.)
HPLC VARIABLES
Column: 10 jxm jxBondapak C18
Mobile phase: Gradient. MeCNrIO mM KH2PO4 9:91 for 26 min, then 50:50 for 1 min (step
gradient).
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Internal standard: 2-ethyl-4-oxoquinazoline (28)
OTHER SUBSTANCES
Extracted: azathioprine
KEYWORDS
REFERENCE
Tsutsumi,K; Otsuki,Y.; Kinoshita,T. Simultaneous determination of azathioprine and 6-mercaptopurine in se-
rum by reversed-phase high-performance liquid chromatography, J.Chromatogr., 1982, 231, 393-399.
SAMPLE
Matrix: blood
Sample preparation: 250 |xL Plasma + 5 |xL 2.5 |xg/mL 6-thioguanine + 25 |xL 1 M aluminum
perchlorate in water, let stand at room temperature for 15 min, chill in ice water for 15 min,
centrifuge at 15600 g for 15 min, discard the supernatant. Suspend the precipitate in 500 |xL
50 mM aluminum perchlorate in water by stirring to break up the precipitate, vortex for 20 s,
centrifuge at 15600 g for 15 min, discard the supernatant. Suspend the precipitate in 150 u,L
400 mM perchloric acid, add 5 jxL freshly prepared 200 mM aqueous sodium hydrosulfite, mix,
let stand at room temperature for 30 min, chill in ice water, centrifuge at 15600 g for 15 min,
inject a 50 JJLL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 45 X 2 40 |xm ODS
Mobile phase: Water:85% phosphoric acid 99.32:0.68 containing 154.3 mg/L dithiothreitol
Flow rate: 1
Detector: UV 340
CHROMATOGRAM
Internal standard: 6-thioguanine (4.73)
OTHER SUBSTANCES
Extracted: metabolites, 6-thiouric acid
KEYWORDS
REFERENCE
Lin,K.T.; Varin,R; Rivard,G.E.; Leclerc,J.M. Isolation of 6-mercaptopurine in human plasma by aluminum ion
complexation for high-performance liquid chromatographic analysis, J.Chromatogr., 1991, 536, 349-355.
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Plasma + 800 |xL MeOH, stir for 15 s, let stand for 20 min, cen-
trifuge at 5 at 3000 rpm for 10 min. Remove 800 JJLL of the supernatant and evaporate it to
dryness under a stream of nitrogen, reconstitute the residue in 100 jxL mobile phase, inject a
40 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 ODS-II (Shimadzu)
Mobile phase: MeOH:water 7:100 containing 0.2% glacial acetic acid and 0.1% sodium 1-
heptanesulfonate
Flow rate: 1
Detector: UV 325
CHROMATOGRAM
Retention time: 6.8
KEYWORDS
plasma; pharmacokinetics; rat
REFERENCE
Takeichi,Y.; Kimura,T. Improvement of aqueous solubility and rectal absorption of 6-mercaptopurine by addi-
tion of sodium benzoate, Biol.Pharm.Bull., 1994, 17, 1391-1394.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 |JLL 2.8 |xg/mL 6-mercaptopurine arabinoside + 2-3 mg
mercurial cellulose, vortex for 30 s, centrifuge at 550 g for 5 min. Resuspend the pellet in 2
mL phosphate buffered saline, centrifuge, repeat this process twice. Suspend the pellet in 250
JJLL 20 mM 2-mercaptoethanol, centrifuge, inject a 100 |xL aliquot of the supernatant. (Prepare
mercurial cellulose as follows. Add 40 g Sigmacell Type 20 cellulose (20 (xm) to 175 mL 40%
NaOH at 5, let stand at 0 for 2 h, add 40 mL allyl glycidyl ether dropwise, add 100 mL water,
heat at 70-80 for 2 h. Suspend the cellulose in 4 L water, allow to settle, decant the water,
wash several more times, filter, wash with water until the pH of the filtrate is about 8, wash
with 1 L 10% acetic acid, wash 1 L with EtOH:water 95:5, wash with 1 L MeOH, wash with
1 L diethyl ether, air dry. Add to 400 mL 10% acetic acid containing 2.3 g mercuric acetate,
stir at 60 for 1 h, filter, wash with 2 L 10% acetic acid, wash with 1.5 L water, wash with 300
mL EtOH.water 95:5, wash with 700 mL MeOH, wash with 1 L diethyl ether, air dry in the
dark, store in the dark (Anal.Biochem. 1985, 144, 514).)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm ODS (Beckman)
Flow rate: 1.2
Detector: UV 323
CHROMATOGRAM
Retention time: 7.6
Internal standard: 6-mercaptopurine arabinoside (13.6)
OTHER SUBSTANCES
Noninterfering: aspirin, chloroquine, cyclosporin, diltiazem, nifedipine, prednisolone
KEYWORDS
REFERENCE
Albertioni,E; Pettersson,B; Ohlman,S.; Peterson,C. Analysis of azathioprine and 6-mercaptopurine in plasma
in renal transplant recipients after administration with oral azathioprine, J.Liq.Chromatogr., 1995, 18,
3991-4005.
SAMPLE
Matrix: blood
Sample preparation: Add 1 volume ice-cold 8 M perchloric acid to 20 volumes plasma, mix,
keep on ice for 10 min, centrifuge at 10 000 g for 15 min, remove the supernatant. Adjust the
pH of the supernatant to 6-7 with 10 volumes ice-cold 4 M K2KHPO4, keep on ice for 10 min,
centrifuge at 10 000 g for 5 min, inject a 100 (xL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 X 4.6 5 ^m Supelcosil LC-18-DB
Mobile phase: Gradient. A was 25 mM KH2PO4. B was MeOH:50 mM KH2PO4 25:75. A:B from
98:2 to 96:4 over 5 min, to 85:15 over 3 min, to 80:20 over 2 min, to 40:60 over 10 min, maintain
at 40:60 over 2 min, to 20:80 over 3 min, maintain at 20:80 for 20 min, return to initial
conditions over 3 min, re-equilibrate for 12 min.
Flow rate: 1.25
Detector: UV 320
CHROMATOGRAM
Limit of detection: 20-50 nM
OTHER SUBSTANCES
Extracted: metabolites, thioguanine (UV 342)
KEYWORDS
plasma
REFERENCE
Keuzenkamp-Jansen,S.W.; De Abreu,R.A.; B6kkerink,J.P.M.; Trijbels,J.M.F. Determination of extracellular
and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography,
J.Chromatogr.B, 1995, 672, 53-61.
SAMPLE
Matrix: blood
Sample preparation: Treat whole blood with EDTA, freeze a 2 mL aliquot at -20, thaw, add 2
mL PBS, centrifuge at 3000 g for 15 min, suspend the pellet in 200 |xL PBS, treat with pro-
teinase K at 70 for 10 min, add to a disposable spin column (Diagen QIAamp Blood Kit, Hilden,
Germany), centrifuge for 1 min, wash twice with buffer for 1 min, elute with 200 |xL 10 mM
pH 9.0 Tris-HCl buffer containing 0.1 mM EDTA, heat at 100 for 5 min, cool in ice. Remove
a 100 (xl aliquot and add it to 10 |xL buffer, add 20 JXL 25 |xg/mL Pi nuclease (Boehringer
Mannheim) and 12.5 U/mL acid phosphatase (Sigma) in bufferiwater 10:90, heat at 42 for 1
h, add 10 |xL 400 mM formic acid, add 60 |xL MeOH, add 1 JJLL 5 mM N-[6-(7-amino-4-meth-
ylcoumarin-3-acetamido)hexyl]-3'-(2>-pyridyldithio)propionamide (AMCA-HPDP, Pierce) in
DMF, inject a 25 JJLL aliquot. (Buffer was 500 mM pH 4.5 sodium acetate buffer containing 10
mM magnesium chloride.)
HPLC VARIABLES
Guard column: 20 mm long Supelguard (Supelco)
Mobile phase: MeOH:buffer 37:63 (Between analyses wash column with MeOH:buffer 80:20 for
3 min. Buffer was 200 mM formic acid adjusted to pH 4.0 with 10 M NaOH.)
Flow rate: 1
CHROMATOGRAM
Retention time: 11.5 (as 2'-deoxy-6-thioguanosine metabolite)
Limit of detection: 60 pmole/g DNA
KEY WpRDS
derivatization; whole blood
REFERENCE
Warren,D.J.; Andersen,A.; Slordal,L. Quantitation of 6-thioguanine residues in peripheral blood leukocyte DNA
obtained from patients receiving 6-mercaptopurine-based maintenance therapy, Cancer Res., 1995, 55,
1670-1674.
SAMPLE
Sample preparation: Blood, CSF. Collect 2 mL blood in tubes containing heparin and 120 |xg
dithiothreitol (DTT). Mix, centrifuge at 2000 g for 5 min, remove plasma. CSF. Collect 0.5 mL
CSF in tubes containing 30 iLg DTT. Cool CSF and plasma samples on ice, add freshly prepared
ice-cold 50% trichloroacetic acid equal to 10% of sample volume. Urine. Collect 2 mL urine in
tubes containing 120 |xg DTT, filter (0.22 |xm), inject an aliquot.
HPLC VARIABLES
Column: two 250 X 4.6 10|xm Nucleosil 10 C18 columns in series
Mobile p h a s e : Gradient. A was 25 mM pH 2.75 phosphoric acid. B was MeOHiwater 50:50. C
was 100 mM pH 6.6 KH2PO4. A:B:C from 100:0:0 to 98:2:0 over 5 min, to 30:3.5:66.5 over 5
min, maintain at 30:3.5:66.5 for 10 min, re-equilibrate at initial conditions for 10 min.
Flow r a t e : 1.7
Injection volume: 195, 500
Detector: UV 312
CHROMATOGRAM
R e t e n t i o n t i m e : 14
OTHER SUBSTANCES
Extracted: metabolites, 6-mercaptopurine riboside, thioguanine (UV 342), 6-thioguanosine
KEYWORDS
plasma; goat; pharmacokinetics
REFERENCE
van Baal,J.M.; van Leeuwen,M.B.; Schouten,T.J.; De Abreu,R.A. Sensitive high-performance liquid chromato-
graphic determination of 6-mercaptopurine, 6-thioguanine, 6-mercaptopurine riboside and 6-thioguanosine
in biological fluids, J.Chromatogr., 1984, 336, 422-428.
SAMPLE
Sample preparation: To 237 |AL enzyme incubation add 850 jxL ice-cold 3.5 mM DL-dithiothre-
itol immediately followed by 500 |JLL 1.5 M sulfuric acid, place tubes on ice. Equilibrate tubes
to room temperature, heat at 100 for 2 h. Cool, add 500 JJLL 3.4 M NaOH immediately followed
by 8 mL toluene:amyl alcoholiphenyl mercury acetate mixture. Shake gently for 10 min, cen-
trifuge at 10 at 900 g for 5 min. Transfer 6 mL toluene to another tube and add 200 ^xL 100
mM HCl. Vortex for four 20 s periods, centrifuge at 10 at 900 g for 5 min, inject a 50 jxL
aliquot of the aqueous layer. (Prepare toluene:amyl alcohohphenyl mercury acetate mixture by
adding enough phenyl mercury acetate to toluene containing 170 mM amyl alcohol so as to
form a solution containing 1.3 mM phenyl mercury acetate, mix gently for 1 h, store in the
dark (J.Chromatogr. 1992, 583, 83).)
HPLC VARIABLES
G u a r d column: 5 x 4 5 |jim Resolve C18
Column: 100 X 8 5jjum Resolve C18 radial compression
Mobile p h a s e : MeOHiwater 20:80 containing 100 mM triethylamine and 0.5 mM dithiothreitol,
pH adjusted to 3.2 with orthophosphoric acid (Add dithiothreitol immediately prior to use.)
Flow r a t e : 1.5
Detector: UV 303
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: methylmercaptopurine
REFERENCE
Lennard,L.; Singleton,H-J- High-performance liquid chromatographic assay of human red blood cell thiopurine
methyltransferase activity, J.Chromatogr.B, 1994, 661, 25-33.
SAMPLE
Sample preparation: Dissolve crushed tablets or the freeze-dried compound for injection in 20
mM NaOH. Add a 10 mL aliquot of this solution (or a saline injection) to 10 mL 3 mg/mL
theophylline in 20 mM NaOH, inject a 1.5 |xL aliquot.
HPLC VARIABLES
Column: 100 X 5 5 |xm ODS-Hypersil
Mobile phase: MeOH:25 mM KH2PO4:glacial acetic acid 20:79:5 adjusted to pH 4.50 (Flush col-
umn with MeOH:water 60:40 at the end of each day.)
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: 1.7
Internal standard: theophylline (3.5)
OTHER SUBSTANCES
Simultaneous: azathioprine, impurities, degradation products
KEYWORDS
stability-indicating; injections; tablets
REFERENCE
Fell,A.F.; Plag,S.M.; Neil,J.M. Stability-indicating assay for azathioprine and 6-mercaptopurine by reversed-
phase high-performance liquid chromatography, J.Chromatogr., 1979, 186, 691-704.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 40 X 4 C18 Corasil II
Column: 300 X 4 10 |xm ixBondapak phenyl
Mobile phase: Propanol:6 mM Ci2 DAPS (Fluka) 3:97 (C12 DAPS is 3-(dimethyldodecyl-
ammonio)propanesulfonate.)
Detector: UV 273
CHROMATOGRAM
Retention time: 3.3
OTHER SUBSTANCES
Simultaneous: albendazole, aminophylline, antipyrine, caffeine, dipropyline, flubendazole,
metronidazole, nimorazole, procaine, p-hydroxytheophylline, theophylline, tinidazole
Interfering: albendazole sulfoxide, amyleine, theobromine
KEYWORDS
micellar chromatography
REFERENCE
Habel,D.; Guermouche,S.; Guermouche,M.H. Direct determination of theophylline in human serum by high-
performance liquid chromatography using zwitterionic micellar mobile phase. Comparison with an enzyme
multiplied immunoassay technique, Analyst, 1993, 118, 1511-1513.
Meropenem
Merck Index: 5960
SAMPLE
Matrix: bile, blood
Sample preparation: Condition a 100 mg Bond Elut C8 SPE cartridge with two 1 mL portions
of MeOH and two 1 mL portions of 500 mM KH2PO4. 100 uL Plasma or 50 uX, bile + 900 |JLL
water, mix, add to the SPE cartridge, wash with 1 mL 50 mM KH2PO4, elute with 800 (plasma)
or 600 (bile) |xL MeOH:50 mM pH 6.0 phosphate buffer 10:90, inject a 100 jxL aliquot of the
eluate.
HPLC VARIABLES
Mobile phase: MeOH:buffer 15:85 (Buffer was 50 (plasma) or 57.5 (bile) mM pH 7.0 phosphate
buffer.)
Flow rate: 1
Detector: UV 296 (plasma), UV 310 (bile)
CHROMATOGRAM
Limit of detection: 280 ng/mL (bile), 100 ng/mL (plasma)
KEYWORDS
plasma; SPE
REFERENCE
Granai,R; Smart,H.L.; Triger,D.R. A study of the penetration of meropenem into bile using endoscopic retro-
grade cholangiography, J.Antimicrob.Chemother., 1992, 29, 711-718.
SAMPLE
Matrix: blood
Sample preparation: Dilute 50 JJLL plasma with 75 |xL 5OmM pH 7.0 phosphate buffer, inject a
100 JJLL aliquot onto column A with mobile phase A, elute to waste with mobile phase A, after
4 min backflush the contents of column A onto column B with mobile phase B, monitor the
effluent from column B. After 11 min re-equilibrate column A with mobile phase A and column
B with mobile phase B for 5 min.
HPLC VARIABLES
Column: 20 X 3.9 25-40 (xm LiChroprep RP-8; B 4.0 X 10 Nova -Pak C8 + 150 X 4.6 5 |xm
Inertsil ODS
Mobile phase: A 50 mM pH 7.0 phosphate buffer; B MeCN:50 mM pH 7.0 phosphate buffer 6:
94
Flow rate: 1
Detector: UV 300
CHROMATOGRAM
KEYWORDS
rat; plasma; column-switching; pharmacokinetics
REFERENCE
Lee,H.S.; Shim,H.O.; Yu,S.R. High-performance liquid chromatographic determination of meropenem in rat
plasma using column-switching, Chromatographia, 1996, 42, 405-408.
SAMPLE
Matrix: blood
Sample preparation: Dilute serum with an equal volume of water, inject 20 |xL diluted serum
onto column A, elute to waste with mobile phase A, after 1.1 min elute the contents of column
A to column B with mobile phase B, elute with mobile phase B, monitor the effluent from
column B.
HPLCVARIABLES
Column: A 50 X 2.1 40 jxm Supelclean LC-NH2 (Supelco); B 150 X 4.6 3 jxm C18 (Supelco)
Mobile phase: A MeOH: 10 mM pH 7.0 phosphate buffer 5:95; B MeOH: 10 mM pH 7.0 phosphate
buffer containing 5 mM tetrabutylammonium hydrogen sulfate 30:70
Detector: UV 298
CHROMATOGRAM
Retention time: 4.2
OTHER SUBSTANCES
Noninterfering: acetaminophen, acyclovir, digoxin, fluconazole, theophylline, vancomycin
KEYWORDS
column-switching; serum
REFERENCE
Bompadre,S.; Ferrante,L.; de Martinis,M.; Leone,L. Determination of meropenem in serum by high-perfor-
mance liquid chromatography with column switching, J.ChromatogrA, 1998, 812, 249-253.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 900 u-L water, add to a conditioned Bond Elut C18 SPE
cartridge, wash with 1 mL 50 mM KH2PO4, elute with 800 |xL mobile phase, inject an aliquot
of the eluate.
HPLCVARIABLES
Column: 100 mm long 3 |xm Hypersil ODS
Mobile phase: MeOH:5 mM tetrabutylammonium dihydrogen phosphate 12:88
Flow rate: 1
Detector: UV 296
CHROMATOGRAM
Retention time: 9
KEYWORDS
REFERENCE
Harrison,M.P.; Haworth,S.J.; Moss,S.R.; Wilkinson,D.M.; Featherstone,A. The disposition and metabolic fate
of uC-meropenem in man, Xenobiotica, 1993, 23, 1311-1323.
SAMPLE
Matrix: blood
Sample preparation: Dilute plasma with water, prepare using a 1 mL C18 SPE cartridge.
HPLC VARIABLES
Mobile phase: MeOH:5 mM tetrabutylammonium dihydrogen phosphate 15:85
Detector: UV 296
CHROMATOGRAM
KEYWORDS
REFERENCE
Bedikian,A.; Okamoto,M.P.; Nakahiro,R.K; Farino,J.; Heseltme,RN.R.;Appleman,M.D.;Yellm,A.E.;Berne,T.V.;
Gill,M.A. Pharmacokinetics of meropenem in patients with intra-abdominal infections, Antimicrob.Agents
Chemother., 1994, 38, 151-154.
SAMPLE
Sample preparation: Dilute plasma 1:4, urine 1:10, and injections 1:100 with water, filter (0.6
|xm), inject a 5-50 |xL aliquot of the filtrate.
HPLC VARIABLES
Mobile phase: MeOH: 10 mM pH 7.4 potassium phosphate buffer 18:82 (plasma, injections) or
25:75 (urine)
Flow rate: 1
Detector: UV 296
CHROMATOGRAM
Limit of detection: 5 (xg/mL (urine), 500 ng/mL (plasma)
KEYWORDS
plasma; injections; saline; pharmacokinetics
REFERENCE
Burman,L.A.; Nilsson-Ehle,L; Hutchison,M.; Haworth,S.J.; Norrby,S.R. Pharmacokinetics of meropenem and
its metabolite ICI 213,689 in healthy subjects with known renal metabolism of imipenem,
SAMPLE
Sample preparation: Plasma. Condition a Bond Elut C18 SPE cartridge. Add 100 \LL plasma
to the SPE cartridge, wash with 50 mM KH2PO4, elute with 800 |xL eluent, inject an aliquot
of the eluate. Urine. Dilute urine 1:10 with water, inject an aliquot directly. (Eluent was MeOH:
5 mM tetrabutylammonium dihydrogen phosphate 12:88.)
HPLC VARIABLES
Column: 100 X 4 3 jim Hypersil ODS
Mobile phase: MeOH:5 mM tetrabutylammonium dihydrogen phosphate 12:88 (plasma) or
MeCN:10 mM pH 7.4 phosphate buffer 6:100 (urine)
Detector: UV 296
CHROMATOGRAM
Limit of detection: 60 ng/mL (plasma), 10 |xg/mL (urine)
KEYWORDS
REFERENCE
Bax,R.R; Bastain,W.; Featherstone,A.; Wilkinson,D.M.; Hutchison,M. Haworth,S.J. The pharmacokinetics of
meropenem in volunteers, J.Antimicrob.Chemother., 1989, 24, 311-320.
SAMPLE
Sample preparation: Add 2 volumes of MeOH, mix well, centrifuge at 3000 g for 15 min, inject
a 10 JJLL aliquot of the supernatant.
HPLCVARIABLES
Column: Inertsil ODS-2
Mobile phase: MeOH:5 mM tetrabutylammonium phosphate 30:70
Flow rate: 1
Detector: UV 300
CHROMATOGRAM
REFERENCE
Hikida,M.; Kawashima,K.; Yoshida,M.; Mitsuhashi,S. Inactivation of new carbapenem antibiotics by dehydro-
peptidase-I from porcine and human renal cortex, J.Antimicrob.Chemother., 1992, 30, 129-134.
SAMPLE
Matrix: urine
Sample preparation: Dilute urine 1:5 with water, inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 50 mm long 3 |xm Hypersil ODS
Flow rate: 1
Detector: UV 296
CHROMATOGRAM
Retention time: 5.5
KEYWORDS
pharmacokinetics
REFERENCE
Harrison,M.R; Haworth,S.J.; Moss,S.R.; Wilkinson,D.M.; Featherstone,A. The disposition and metabolic fate
of 14C-meropenem in man, Xenobiotica, 1993, 23, 1311-1323.
SAMPLE
Matrix: urine
Sample preparation: Dilute urine with water, prepare using a 1 mL C18 SPE cartridge.
HPLC VARIABLES
Column: 150 X 4.6 5 |im Partisil silica
Mobile phase: 0.1% Phosphoric acid in water
Detector: UV 313
CHROMATOGRAM
KEYWORDS
REFERENCE
BedikianA; Okamoto,M.R;Nakahiro,R.K; Farino,J.;Heseltine,P.N.R.;Appleman,M.D.;Yellin,AE.;Berne,T.V.;
Gill,M.A. Pharmacokinetics of meropenem in patients with intra-abdominal infections, Antimicrob.Agents
Chemother., 1994, 38, 151-154.
Mesalamine
Merck Index: 5964
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 4 mL MeOH, let stand for 15 min at 4, centrifuge at 3000
g for 15 min. Remove 1 mL of the supernatant and add it to 1 mL water, mix, inject an aliquot.
HPLC VARIABLES
Column: 40 X 4.6 3 |xm Hypersil
Mobile phase: MeOH:water:200 mM pH 6.5 potassium phosphate buffer 40:40:20 containing 4
mM hexadecyltrimethylammonium bromide
Flow rate: 1.7
CHROMATOGRAM
Retention time: 2.5
KEY WORDS
plasma
REFERENCE
Tjornelund,J.; Hansen,S.H. Stability of 5-aminosalicylic acid and its metabolites in plasma at -200C. Formation
of N-(3-D-glucopyranosyl-5-aminosalicylie acid, J.Chromatogr., 1991, 570, 224-228.
SAMPLE
Matrix: blood, feces, urine
Sample preparation: Plasma. 1 mL Plasma + 2 mL MeOH, vortex for 20 s, let stand for 15 min
at -20 and 10 min at room temperature, mix, centrifuge at 2000 g for 15 min. Remove the
supernatant and add it to 5 mL 1,1,1-trichloroethane, shake for 2 min, centrifuge at 2000 g
for 15 min, inject a 20 |xL aliquot of the supernatant. Urine. 1 mL Urine + 4 mL MeOH, mix,
let stand at -20 for 15 min, centrifuge at 2000 g for 15 min. Dilute an aliquot of the supernatant
with an equal volume of water, inject a 20 |xL aliquot. Feces. Extract with 500-1000 mL MeOH,
centrifuge at 10000 g for 4 min. Dilute an aliquot of the supernatant with an equal volume of
water, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 40 X 4.6 3 jxm Hypersil
Mobile phase: MeOH:water:200 mM pH 6.5 potassium phosphate buffer 40:40:20 containing 4
mM hexadecyltrimethylammonium bromide
Flow rate: 1.7
CHROMATOGRAM
Retention time: 2.5
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Tjornelund,J.; Hansen,S.H. High-performance liquid chromatographic assay of 5-aminosalicylic acid (5-ASA)
and its metabolites N-p-D-glucopyranosyl-5-ASA, N-acetyl-5-ASA, N-formyl-5-ASA and N-butyryl-5-ASAin
biological fluids, J.Chromatogr., 1991, 570, 109-117.
SAMPLE
Sample preparation: Serum. Add disodium-3,5'-azo-bis-(6-hydroxybenzoate) to serum, treat
with proteinase K (0.5 mg/mL protein) for 10 min, add tetrabutylammonium hydrogen sulfate
buffered to pH 6.5, add dichloromethane, agitate for 30 min, centrifuge. Remove the organic
layer and evaporate it to dryness, reconstitute the residue in mobile phase, inject an aliquot.
Urine. Add 2,4-dihydroxybenzoic acid to urine, react with propionic anhydride for 5 min, add
perchloric acid, add diethyl ether, shake for 10 min, freeze. Remove the organic layer and add
it to pH 7.4 phosphate buffer, extract, inject an aliquot of the aqueous phase.
HPLCVARIABLES
Guard column: 30 X 4 30-40 |xm Perisorb RP-18
Mobile phase: MeOHrbuffer 30:70 (Buffer was pH 7.4 phosphate buffer containing 20 mM te-
trabutylammonium hydrogen sulfate.)
CHROMATOGRAM
Retention time: k' 2.4 (propionyl derivative)
Internal standard: disodium-3,5'-azo-bis-(6-hydroxybenzoate), 2,4-dihydroxybenzoic acid
Limit of quantitation: 6.1 |xM urine, 0.4 |xM (serum)
OTHER SUBSTANCES
Extracted: acetylaminosalicylic acid
KEYWORDS
serum; pharmacokinetics; derivatization
REFERENCE
Ryde,E.M.; Ahnfelt,N.-O. The pharmacokinetics of olsalazine sodium in healthy volunteers after a single i.v.
dose and after oral doses with and without food, Eur.J.Clin.PharmacoL, 1988, 34, 481-488.
SAMPLE
Sample preparation: Plasma. 500 |xL Plasma + 500 |xL 100 mg/mL (sic) 2,4-dihydroxybenzoic
acid in MeOH, mix, centrifuge, inject an aliquot. Urine. 100 |xL Urine + 500 jxL 600 mg/mL
(sic) 2,4-dihydroxybenzoic acid in MeOH, mix, centrifuge, inject an aliquot.
HPLCVARIABLES
Guard column: ixBondapak C18 Guard Pak
Column: 250 x 4.1 10 ^m Caulab CSC PRP-I
Mobile phase: MeCN:MeOH:50 mM pH 7.9 potassium phosphate buffer 10:10:80
Flow rate: 1
CHROMATOGRAM
Retention time: 5.1
Internal standard: 2,4-dihydroxybenzoic acid (3.3)
OTHER SUBSTANCES
Extracted: N-acetyl-5-aminosalicylic acid
KEYWORDS
REFERENCE
Corey,A.E.; Rose,G.M.; Conklin,J.D. Bioavailability of single and multiple doses of enteric-coated mesalamine
and sulphasalazine, J.Int.Med.Res., 1990, 18, 441-453.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 205.2
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve in dilute HCl (pH 2), sonicate if necessary, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Hypersil-BDS C18
Mobile phase: MeOH:THF:buffer 11:4:85 (Buffer was 8.6 g NaH2PO4-H2O, 8.2 g NaCl, 2.2 g
sodium 1-heptanesulfonate, and 5.75 mL 85% phosphoric acid in 2 L water, pH 2.0.)
Flow rate: 1.5
CHROMATOGRAM
Retention time: 3.6
OTHER SUBSTANCES
Simultaneous: impurities, salicylic acid
REFERENCE
Kersten,B.S.; Catalano,T.; Rozenman,Y. Ion-pairing high-performance liquid chromatographic method for the
determination of 5-aminosalicylic acid and related impurities in bulk chemical, J.Chromatogr., 1991, 588,
187-193.
SAMPLE
Sample preparation: 75 JJLL Sample + 6 mL mobile phase, vortex for 2 min, add 300 |xL 300
|xg/mL acetaminophen, make up to 10 mL with mobile phase, mix for 2 min, inject a 5 JJLL
aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Spheri-5 ODS (Applied Biosystems)
Mobile phase: MeOH:buffer 30:70 (Buffer was 900 mL 50 mM Na2HPO4 + 18.75 mL tetrabutyl-
ammonium phosphate, pH adjusted to 6.8 with 1 N phosphoric acid.)
Flow rate: 1.2
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 6.0
Internal standard: acetaminophen (3.8)
KEY WORDS
stability-indicating; rectal suspension; enema
REFERENCE
Henderson,L.M.; Johnson,C.E.; Berardi,R.R. Stability of mesalamine in rectal suspension diluted with distilled
water, Am. J.Hosp.Pharm., 1994, 51, 2955-2957.
SAMPLE
Sample preparation: 250 |xL Microsomal incubation + 500 |xL MeOH, centrifuge, inject an al-
iquot of the supernatant.
HPLC VARIABLES
Column: 120 X 4.6 5 urn RP-18
Mobile phase: MeCN:200 mM pH 7.5 phosphate buffer:1.25 mM cetyltrimethylammonium bro-
mide 30:5:65
Flow rate: 1
KEYWORDS
rat; for 5-aminosalicylic acid-O-sulfate and 5-aminosalicylic acid
REFERENCE
Herzog,R.; Leuschner,J. Experimental studies on the pharmacokinetics and toxicity of 5-aminosalicylic acid-O-
sulfate following local and systemic application, Arzneimittelforschung, 1995, 45, 300-303.
SAMPLE
Matrix: tissue
Sample preparation: Freeze mucosal intestinal biopsy (ca. 5 mg) in liquid nitrogen and crush
the sample, allow to warm to room temperature, add 20 |xL propionic anhydride, add 100 (JLL
57.6 jiig/mL N-propionyl-4-aminosalicylic acid (purified by HPLC) in mobile phase, add 500 jxL
50 mM pH 7.4 phosphate buffer, wash grinding/mixing rod with 500 |xL 50 mM pH 7.4 phos-
phate buffer, sonicate (80 W) by immersing microprobe tip in mixture for 1 min, vortex, let
stand at 37 for 1 h, add 500 |xL 10% NaCl, add 6 mL MeCN, extract, cool at 4 for 1 h,
a stream of air, reconstitute the residue in 500 |xL mobile phase, filter (0.45 |xm), inject a 100
jxL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 5 jxm Spherisorb ODS-2
Column: 150 X 4.6 3 jxm Spherisorb ODS-2
Mobile phase: MeCN: 100 mM acetic acid:triethylamine 8:92:0.2
Flow rate: 1.5
CHROMATOGRAM
Retention time: 5.7
Internal standard: N-propionyl-4-aminosalicylic acid (7.3)
OTHER SUBSTANCES
Extracted: N-acetyl-5-aminosalicylic acid
KEYWORDS
mucosal intestinal biopsy; derivatization
REFERENCE
De Vos,M.; Verdievel,H-l Schoonjans,R.; Beke,R.; De Weerdt,G.A.; Barbier,F. High-performance liquid chromat-
ographic assay for the determination of 5-aminosalicylic acid and acetyl-5-aminosalicylic acid concentrations
in endoscopic intestinal biopsy in humans, J.Chromatogr., 1991, 564, 296-302.
SAMPLE
Matrix: tissue
Sample preparation: Sonicate tissue in 2 mL 5 ng/mL 3,4-dihydroxybenzylamine in MeOH for
two 30 s cycles (W = 60), centrifuge at 1800 g for 10 min. Filter (0.5 |xm) the supernatant,
evaporate the filtrate to dryness under a stream of nitrogen under reduced pressure, reconsti-
tute with 100 uL mobile phase, vortex, inject a 5 |xL aliquot.
HPLC VARIABLES
Guard column: 50 X 4.6 40 |xm Pelliguard (Supelco)
Column: 250 X 4.6 10 |xm Erbasil S C18 (Carlo Erba)
Mobile phase: MeOHrbuffer 15:85, pH adjusted to 3 with 100 mM NaOH. (Buffer was 10 mM
Na2HPO4 containing 0.1 mM EDTA, 100 mM citric acid, and 0.1 mM heptanesulfonic acid.)
Flow rate: 1
Injection volume: 5
Detector: E, ESA Model 5100A, Model 5021 conditioning cell +0.35 V (between column and an-
alytical cell), Model 5011 analytical cell, first electrode +0.05 V, second electrode -0.50 V
(monitored)
CHROMATOGRAM
Retention time: 3.2
OTHER SUBSTANCES
REFERENCE
Palumbo,G.; Carlucci,G.; Mazzeo,R; Frieri,G.; Pimpo,M.T.; Fanini,D. Simultaneous determination of 5-amino-
salicylic acid, acetyl-5-aminosalicylic acid and 2,5-dihydroxybenzoic acid in endoscopic intestinal biopsy
samples in humans by high-performance liquid chromatography with electrochemical detection,
Mescaline
Merck Index: 5965
SAMPLE
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
camfamine, fenoprofen, fenproporex, fentanyl, flubendazole, flufenamic acid, fiunitrazepam, 5-
phesin, mephobarbital, mesoridazine, methadone, methamphetamine, methapyrilene,
REFERENCE
18, 233-242.
Mesna
Molecular formula: C2H5NaO3S2
CAS Registry No.: 19767-45-4, 3375-50-6 (free acid)
Merck Index: 5969
SAMPLE
Matrix: blood
Sample preparation: 50 JJLL Plasma (within 3 min of collection) + penicillamine + 10 JULL 25 mM
monobromobimane in MeCN, let stand for 5 min at room temperature, add 20 JJLL 20% per-
chloric acid. (For total mesna add 100 |xL 20 mM dithiothreitol in 200 mM pH 8.5 Tris/HCl
buffer to 50 |xL plasma, let stand for 40 min at room temperature, add 50 jxL 15% sulfosalicylic
acid, centrifuge. Remove 200 |xL of the supernatant and wash it three times with ethyl acetate.
Remove 60 \iL of the aqueous phase and add it to 300 |xL 200 mM pH 8.5 Tris/HCl buffer and
10 JJLL 15 mM monobromobimane in MeCN, let stand at room temperature for 5 min, add 20
|xL 20% perchloric acid.)
HPLC VARIABLES
Column: 150 X 4.6 7 *Jim Nucleosil RP-18
Mobile phase: Gradient. A was MeCN. B was 1% aqueous acetic acid containing 1 g/L octane-
sulfonic acid. A:B from 5:95 to 8:92 over 2 min, to 10:90 over 13 min (Waters convex), to 30:70
over 20 min (Waters convex), maintain at 30:70 for 6 min, re-equilibrate at initial conditions
for 9 min.
Flow rate: 1.4
CHROMATOGRAM
Internal standard: penicillamine
KEYWORDS
REFERENCE
Stofer-Vogel,B.; Cerny,T.; Borner,M.; Lauterburg,B.H. Oral bioavailability of mesna tablets, Cancer Chem-
other.Pharmacol., 1993, 32, 78-81.
SAMPLE
Sample preparation: Plasma. 400 JJLL Plasma + 400 |xL 333 mM sulfuric acid + 400 JJLL 5%
sodium hexametaphosphate, vortex for 5 s, centrifuge at 15000 g for 2 min, inject a 20 |xL
aliquot of the supernatant. (Process plasma as rapidly as possible after collection.) (For total
mesna mix 100 |xL plasma, 100 |JLL EDTA solution, and 100 jxL 4% sodium borohydride, heat
to 50 for 30 min, cool, add 200 jxL 9.7% acetic acid, store in the dark for 3-4 days, inject an
aliquot. (EDTA solution was 1% disodium EDTA in a mixture of 50 mL 500 mM Na2HPO4 and
22 mL 1 M NaOH.)) Urine. Dilute 1:10 to 1:400 with mobile phase, inject an aliquot. (For total
mesna dilute 1:10 to 1:1000 then proceed as above.)
HPLCVARIABLES
Mobile phase: MeCN:buffer 3:97 (Buffer was 100 mM pH 7.0 phosphate buffer containing 5 mM
n-tetrabutylammonium hydroxide.)
Flow rate: 1
Detector: E, Coulochem Model 5100A, Model 5011 analytical cell, detector 1 not used, detector
2 -200 mV
CHROMATOGRAM
Retention time: 4.5
Limit of quantitation: 6.1 |JLM
KEYWORDS
plasma
REFERENCE
James,C.A.; Rogers,H.J. Estimation of mesna and dimesna in plasma and urine by high-performance liquid
chromatography with electrochemical detection, J.Chromatogr., 1986, 382, 394-398.
SAMPLE
Sample preparation: Plasma. 100 (xL Plasma + 25 |xL 330 mM sulfuric acid + 25 |JLL 5% sodium
hexametaphosphate + 10 JJLL 40 jxg/mL p-aminobenzoic acid, make up to 200 |xL, vortex for 2
s, centrifuge at 3400 g for 6 min, inject a 50-100 >L aliquot of the supernatant. Urine. Dilute
1:50 with water. 100 |xL Diluted urine + 100 |xL 150 |xg/mL p-aminobenzoic acid in 1.25%
sodium hexametaphosphate, vortex for 2 s, inject a 50 |xL aliquot. (Reduce dimesna to mesna
as follows. 100 JJLL Plasma or diluted urine + 100 |xL 1% EDTA in buffer + 100 |xL 1.06 M
sodium borohydride in water, mix thoroughly, heat at 50 for 30 min, cool to room temperature,
add 200 |xL 1.74 M acetic acid, vortex for 20 s, centrifuge for 10 min, inject a 50-100 |xL aliquot
of the supernatant. Buffer was 50 mL 500 mM Na2HPO4 + 22 mL NaOH.)
HPLC VARIABLES
Guard column: Guard PAK C18 (Waters)
Column: 100 X 8 10 |xm Resolve C18 (Waters)
Mobile phase: 100 mM Sodium citrate containing 1 mM tetrabutylammonium phosphate and
0.71 mM triethylamine, pH adjusted to 5 with 85% phosphoric acid
Flow rate: 2
Detector: E, ESA Coulochem II model 5100, model 5011 analytical cell +450 mV, model 5021
conditioning cell +500 mV
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: acetaminophen, 5-fluorouracil, procarbazine, prochlorperazine
Noninterfering: aspirin, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide,
cyclosporine A, cytarabine, doxorubicin, etoposide, hydrocortisone, ifosfamide, lomustine, meth-
otrexate, mitomycin, mitoxantrone, teniposide, thiotepa, vincristine
KEYWORDS
REFERENCE
el-Yazigi,A.; Yusuf,A.; Al-Rawithi,S. Liquid chromatographic analysis of mesna and dimesna in plasma and
urine of patients treated with mesna, Ther.Drug Monit., 1995,17, 153-158.
SAMPLE
Sample preparation: Dilute formulation with 15 |xg/mL disodium EDTA solution to a mesna
concentration of 3.2 |xg/mL. Remove a 1 mL aliquot and add it to 300 |xL reagent solution, let
stand at room temperature for 20 min, add 500 |xL 300 mM phosphoric acid solution, add 3
mL 4 |xg/mL IS solution, make up to 10 mL with water, inject a 50 |xL aliquot. (Prepare the
reagent solution by dissolving 3.5 mg methyl 4-(6-methoxynaphthalen-2-yl)-4-oxo-2-butenoate
in 10 mL THF, make up to 25 mL with pH 7.5 borate buffer. Prepare methyl 4-(6-methoxy-
naphthalen-2-yl)-4-oxo-2-butenoate as follows. Dissolve 5 g 6'-methoxy-2'-acetonaphthone in
warm glacial acetic acid and add 2.5 g glyoxylic acid, reflux for 24 h, evaporate to dryness
under reduced pressure. Take up the residue in chloroform and extract it three times with 5%
sodium carbonate solution. Combine the aqueous layers and acidify them with concentrated
HCl, collect the product by filtration, recrystallize from MeOH/water or acetic acid to give 4-
(6-methoxynaphthalen-2-yl)-4-oxo-2-butenoic acid (mp 167-9) (Farmaco, Ed. Sci. 1982, 37,
171). Reflux 0.5 g 4-(6-methoxynaphthalen-2-yl)-4-oxo-2-butenoic acid, 2.5 mL MeOH, and 2-3
drops sulfuric acid in 25 mL anhydrous benzene (Caution! Benzene is a carcinogen!) for 1 h,
add 20 mL water, wash the organic layer with 10 mL 5% sodium bicarbonate solution, wash
the organic layer with 20 mL water. Dry the organic layer over anhydrous sodium sulfate,
evaporate to dryness under reduced pressure, purify by flash chromatography on silica gel
using ethyl acetate:light petroleum (bp 40-70) 40:60 to give methyl 4-(6-methoxynaphthalen-
2-yl)-4-oxo-2-butenoate as a pale yellow compound (mp 116-120).)
HPLC VARIABLES
Column: 150 X 4 5 jmi Spherisorb RP-8
Mobile phase: MeOH:50 mM pH 3.0 triethylammonium phosphate 53:47
Flow rate: 1
CHROMATOGRAM
Retention time: 6
Internal standard: 4-(6-methoxynaphthalen-2-yl)-4-oxobutanoic acid (8)
OTHER SUBSTANCES
Simultaneous: acetylcysteine, cysteamine, cysteine, glutathione, homocysteine
Noninterfering: bacitracin, biotin, calcium pantothenate, cystine, glycine, magnesium oxide, ne-
omycin, starch, threonine, vitamin E, pyridoxine, riboflavin phosphate
KEYWORDS
solutions; derivatization
REFERENCE
Gatti,R.; Cavrini,V.; Roveri,R; Pinzauti,S. High-performance liquid chromatographic determination of aliphatic
thiols with aroylacrylic acids as fluorogenic precolumn derivatization reagents, J.Chromatogr., 1990, 507,
451-458.
SAMPLE
HPLCVARIABLES
Flow rate: 2.5
Next Page
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: diphenhydramine, granisetron (UV 300)
KEYWORDS
REFERENCE
304.
SAMPLE
Matrix: urine
Sample preparation: Dilute urine 1:3 to 1:39, inject a 50 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4 5 ^m Hypersil ODS
Mobile phase: MeOH:250 mM pH 7.4 phosphate buffer 5:95 containing 5 mM tetrabutylam-
monium phosphate
Flow rate: 1
Detector: UV 412 following post-column reaction. The column effluent mixed with the reagent
pumped at 0.5 mL/min and the mixture flowed through a 200 X 4 column packed with 100-
120 mesh glass beads (dichlorodimethylsilane treated) to the detector. (Prepare reagent by
diluting 0.2% 5,5'-dithiobis(2-nitrobenzoic acid) in 250 mM pH 7.4 phosphate buffer containing
10% tripotassium citrate 1:10 with water.)
CHROMATOGRAM
Retention time: 7.5
KEY WORDS
post-column reaction; comparison with electrochemical detection without derivatization
REFERENCE
Sidau,B.; Shaw,I.C. Determination of sodium 2-mercaptoethanesulphonate by high-performance liquid chro-
matography using post-column reaction colorimetry or electrochemical detection, J.Chromatogr., 1984,311,
234-238.
Mesoridazine
Molecular formula: C21H26N2OS2
CAS Registry No.: 5588-33-0, 32672-69-8 (besylate)
Merck Index: 5970
Lednicer No.: 1 389
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 uX 1 M HCl, vortex for 30 s, add 4 mL isopropanol,
mix for 5 min, centrifuge at 5000 rpm at 0 for 20 min. Remove the supernatant and adjust
Previous Page
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: diphenhydramine, granisetron (UV 300)
KEYWORDS
REFERENCE
304.
SAMPLE
Matrix: urine
Sample preparation: Dilute urine 1:3 to 1:39, inject a 50 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4 5 ^m Hypersil ODS
Mobile phase: MeOH:250 mM pH 7.4 phosphate buffer 5:95 containing 5 mM tetrabutylam-
monium phosphate
Flow rate: 1
pumped at 0.5 mL/min and the mixture flowed through a 200 X 4 column packed with 100-
120 mesh glass beads (dichlorodimethylsilane treated) to the detector. (Prepare reagent by
diluting 0.2% 5,5'-dithiobis(2-nitrobenzoic acid) in 250 mM pH 7.4 phosphate buffer containing
10% tripotassium citrate 1:10 with water.)
CHROMATOGRAM
Retention time: 7.5
KEY WORDS
post-column reaction; comparison with electrochemical detection without derivatization
REFERENCE
Sidau,B.; Shaw,I.C. Determination of sodium 2-mercaptoethanesulphonate by high-performance liquid chro-
matography using post-column reaction colorimetry or electrochemical detection, J.Chromatogr., 1984,311,
234-238.
Mesoridazine
Molecular formula: C21H26N2OS2
CAS Registry No.: 5588-33-0, 32672-69-8 (besylate)
Merck Index: 5970
Lednicer No.: 1 389
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 uX 1 M HCl, vortex for 30 s, add 4 mL isopropanol,
mix for 5 min, centrifuge at 5000 rpm at 0 for 20 min. Remove the supernatant and adjust
the pH to 12.5 with 200 |xL 5 M NaOH, mix for 10 s, add 4 mL n-heptane, mix for 10 min,
centrifuge at 2500 rpm. Remove the organic layer and evaporate it to dryness under a stream
of nitrogen, reconstitute the residue in 200 |xL MeCN, mix for 2 min, inject a 75-100 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:15 mM pH 6.5 acetate buffer 90:10
Flow rate: 1.6
Detector: UV 254
CHROMATOGRAM
Retention time: 7
Internal standard: mesoridazine
OTHER SUBSTANCES
Extracted: chlorpromazine
KEYWORDS
plasma; meosridazine is IS
REFERENCE
Midha,K.K; Cooper,J.K.; McGilverayJ.J.; Butterfield,A.G.; Hubbard,J.W. High-performance liquid chromato-
graphic assay for nanogram determination of chlorpromazine and its comparison with a radioimmunoassay,
J.Pharm.Sci., 1981, 70, 1043-1046.
SAMPLE
Matrix: blood
reconstitute the residue in 100 JJLL mobile phase, inject a 50 |xL aliquot. (It is implied, but not
HPLC VARIABLES
Column: 10 u,m Micropak CN (Varian)
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
promazine, fluphenazine, haloperidol, imipramine, nortriptyline, orphenadrine, piperacetazine,
promazine, promethazine, thioridazine, thiothixene, trifluoperazine, triflupromazine, trihexy-
phenidyl, trimeprazine
KEYWORDS
plasma; whole blood
REFERENCE
SAMPLE
|xL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the organic
layer and inject a 30 |xL aliquot of the aqueous layer. Liver homogenate. 0.5 mL Liver homog-
enate + 10 |xg cianopramine + 500 |xL 2% sodium tetraborate + 8 mL hexane:l-butanol 95:5,
gently agitate for 30 min, centrifuge at 2500 g for 5 min. Remove the organic layer and add it
to 400 |xL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the
HPLCVARIABLES
Column: 100 X 4.6 5 ^m Brownlee Spheri-5 RP-18
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
doxepin, fluoxetine, haloperidol, imipramine, loxapine, maprotiline, meperidine, methadone,
metoclopramide, mianserin, moclobemide, nomifensine, nordoxepin, norfluoxetine, norpropox-
yphene, northiaden, nortriptyline, pentobarbital, pheniramine, promethazine, propranolol, pro-
triptyline, quinidine, quinine, sulforidazine, thioridazine, thiothixene, tranylcypromine, trazo-
done, trihexyphenidyl, trimipramine, triprolidine
fluoperazine
Interfering: propoxyphene
KEYWORDS
REFERENCE
MclntyreJ.M.; King,C.V.; Skafidis,S.; Drummer,O.H. Dual ultraviolet wavelength high-performance liquid chro-
SAMPLE
add 75 mL mobile phase, sonicate for 20 min, dilute to 100 mL with mobile phase, mix, filter
(0.45 u,m) (discard first 10 mL of filtrate), inject a 20 |xL aliquot of the filtrate. Syrups, elixirs,
injectables. Measure out amount equivalent to about 10 mg, add 75 mL mobile phase, sonicate
for 20 min, dilute to 100 mL with mobile phase, mix, inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 urn ixBondapak CN
Mobile phase: MeOH:3 mM ammonium acetate 90:10
Flow rate: 1.3
Detector: UV 254
CHROMATOGRAM
Retention time: 5.2
OTHER SUBSTANCES
Also analyzed: chlorpheniramine, cyclizine, doxylamine, pentazocine, promethazine, protripty-
line, pyrilamine, pyrimethamine, tripelennamine
KEYWORDS
tablets; syrups; elixirs; injections
REFERENCE
Walker,S.T. Liquid chromatographic determination of organic nitrogenous bases in dosage forms: a progress
report, JAssoc.Off.Anal.Chem., 1985, 68, 539-542.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 |AL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 5.3
OTHER SUBSTANCES
mepyramine, metaraminol, methadone, methamphetamine, methapyrilene, methdilazene,
REFERENCE
SAMPLE
Matrix: solutions
|JLL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 urn jxBondapak C18
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Simultaneous: promethazine, acetophenazine, chlorpromazine, thioridazine, prochlorperazine,
butaperazine, thiethylperazine
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2.5
Detector: E, Environmental Science Associates Coulochem Model 5100A, Model 5100 guard-
cell +0.85 V (between pump and injector), Model 5010 analytical cell +0.8 V, preanalytical cell
+0.3V
CHROMATOGRAM
Retention time: 4.0
OTHER SUBSTANCES
Simultaneous: amitriptyline, chlorpromazine, desipramine, doxepin, fluphenazine, haloperidol,
imipramine, loxapine, nortriptyline, perphenazine, pheniramine, phenylephrine, prochlorper-
azine, promazine, promethazine, thioridazine, thiothixene, triflupromazine, trimeprazine,
tripelennamine
Noninterfering: diazepam, diphenhydramine, ethopropazine, fluoxetine, nordiazepam, oxaze-
pam, phenylpropanolamine, pseudoephedrine, trifiuoperazine
Interfering: amoxapine, reduced haloperidol, desmethyldoxepin, trazodone
REFERENCE
Hariharan,M.; VanNoord,T.; Kindt,E.K.; Tandon,R. A simple, sensitive liquid chromatographic assay of cis-
thiothixene in plasma with coulometric detection, Ther.Drug Monit, 1991, 13, 79-85.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 0.5
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: promazine, thiothixene, chlorpromazine, trifiuoperazine, thioridazine
Also analyzed: amitriptyline, amphetamine, chlordiazepoxide, desalkylflurazepam, desipra-
mine, desmethyldoxepin, diazepam, diethylpropion, doxepin, ephedrine, fenfluramine, flura-
zepam, imipramine, methamphetamine, norchlordiazepoxide, nordiazepam, nortriptyline, ox-
azepam, phentermine, phenylpropanolamine, prazepam
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
phesin, mephobarbital, mepivacaine, methadone, methamphetamine, methapyrilene,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ehlorprothixene, chlorthalidone, chlorzoxazone, cimetidine, cisapride, clomipramine, clonaze-
dol, mefenamic acid, meperidine, mephenytoin, mepivacaine, metaproterenol, methadone,
zopiclone
KEYWORDS
REFERENCE
J.Chromatogr.A, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Sample preparation: Perform all procedures in subdued light. 10 mL Urine + 10 |xL 100 ^g/
mL IS, lyophilize, extract residue with 3 mL MeOH by shaking for 15 min, repeat extraction
twice, combine extracts and evaporate them to dryness under vacuum below 45, dissolve res-
idue in 2 mL 300 mM pH 7.2 phosphate buffer, extract three times with 3 mL dichloromethane,
wash the combined organic layers twice with 2 mL phosphate buffer, twice with 2 mL water,
dry over anhydrous sodium sulfate, evaporate to dryness, reconstitute with 100 |xL MeCN,
HPLC VARIABLES
Column: 150 X 4.6 3 |jnn Spherisorb cyano
Mobile phase: MeCN:50 mM ammonium acetate:diethylamine 92:8:0.05
Flow rate: 1.1
Detector: UV 254
CHROMATOGRAM
Internal standard: prochlorperazine ring sulfoxide (14)
OTHER SUBSTANCES
KEYWORDS
human; rat; dog
REFERENCE
Lin,G.; Hawes,E.M.; McKay,G.; Korchinski,E.D.; Midha,K.K. Metabolism of piperidine-type phenothiazine an-
tipsychotic agents. IV. Thioridazine in dog, man and rat, Xenobiotica, 1993, 23, 1059-1074.
SAMPLE
Matrix: urine
Sample preparation: Perform all procedures in subdued light. 10 mL Urine + 10 |xL 100 |xg/
mL IS, lyophilize, extract residue with 3 mL MeOH by shaking for 15 min, repeat extraction
twice, combine extracts and evaporate them to dryness under vacuum below 45, dissolve res-
idue in 2 mL 300 mM pH 7.2 phosphate buffer, extract three times with 3 mL dichloromethane,
wash the combined organic layers twice with 2 mL phosphate buffer, twice with 2 mL water,
dry over anhydrous sodium sulfate, evaporate to dryness, reconstitute with 100 |xL MeCN,
inject a 10 (xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 (xm Serva Si 100 polyol (Terochem Laboratories)
Flow rate: 1
Detector: MS plasmaspray, VG 7OSQ, discharge 320 V, ion source 250, plasmaspray probe tip
200-300
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: metabolites, sulforidazine, thioridazine
KEYWORDS
human; rat; dog
REFERENCE
Lin,G.; Hawes,E.M.; McKay,G.; Korchinski,E.D.; Midha,K.K. Metabolism of piperidine-type phenothiazine an-
tipsychotic agents. IV. Thioridazine in dog, man and rat, Xenobiotica, 1993, 23, 1059-1074.
SAMPLE
Matrix: urine
Sample preparation: Human urine. 10 mL Urine + 1 mL 10 |xg/mL IS, lyophilize, add 3 mL
MeOH, shake for 15 min, repeat extraction twice more. Combine the extracts and evaporate
them to dryness under reduced pressure below 45, reconstitute the residue in 2 mL 300 mM
pH 7.2 phosphate buffer, extract three times with 3 mL dichloromethane. Combine the organic
extracts and wash them twice with 2 mL phosphate buffer, wash twice with 2 mL water, dry
over anhydrous sodium sulfate, evaporate to dryness, reconstitute in 100 /xL MeCN, inject a
10 |xL aliquot. Dog, rat urine. 1 mL Urine + 50 (xL 100 |xg/mL IS, lyophilize, add 3 mL MeOH,
shake for 15 min, repeat extraction twice more. Combine the extracts and evaporate them to
dryness under reduced pressure below 45, reconstitute the residue in 2 mL 300 mM pH 7.2
phosphate buffer, extract three times with 3 mL dichloromethane. Combine the organic extracts
and wash them twice with 2 mL phosphate buffer, wash twice with 2 mL water, dry over
anhydrous sodium sulfate, evaporate to dryness, reconstitute in 100 JULL MeCN, inject a 10 L
| JLL
aliquot.
HPLC VARIABLES
Column: 150 X 4.6 3 (xm Spherisorb cyano
Mobile phase: MeCN:50 mM ammonium acetaterdiethylamine 92:8:0.05
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: prochlorperazine ring sulfoxide
OTHER SUBSTANCES
KEYWORDS
dog; rat; human; protect from light
REFERENCE
Lin,G.; Hawes,E.M.; McKay,G.; Korchinski,E.D.; Midha,K.K. The metabolism of piperidine-type phenothiazine
antipsychotic agents. III. Mesoridazine in dog, human and rat, Xenobiotica, 1993, 23, 37-52.
Mestranol
Merck Index: 5976
Lednicer No.: 1 162
SAMPLE
Sample preparation: Powder tablets (60 mesh), weigh out amount equivalent to one tablet, add
2 mL 50 |xg/mL BHT in MeCNrwater 80:20, shake 30 min, centrifuge
HPLC VARIABLES
Column: 250 X 3.2 Altex RP-2 express series
Flow rate: 1.75
CHROMATOGRAM
Internal standard: BHT (butylated hydroxytoluene) (k' 16.54)
OTHER SUBSTANCES
Simultaneous: ethynodiol diacetate, ethinyl estradiol, degradation products
KEYWORDS
tablets
REFERENCE
Carignan,G.; Lodge,B-A.; Skakum,W. Quantitative analysis of ethynodiol diacetate and ethinyl estradiol/mes-
tranol in oral contraceptive tablets by high-performance liquid chromatography, J.Pharm.Sci., 1982, 71,
264-266.
SAMPLE
Sample preparation: Dissolve 6 tablets in 600 mL dissolution medium (water:isopropanol 97:
3), remove 5 mL samples, centrifuge at 1500 rpm for 10 min, inject a 50-200 JLJLL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 |xm Bondapak C18
Flow rate: 1
Detector: UV 200
OTHER SUBSTANCES
Simultaneous: norethindrone
KEYWORDS
tablets; modified USP method
REFERENCE
Nguyen,H-T.; Shiu,G.K; Worsley,W.N.; Skelly,J.P. Dissolution testing of norethindrone:ethinyl estradiol, no-
rethindroneimestranol, and norethindrone acetate:ethinyl estradiol combination tablets, J.Pharm.Sci.,
1990, 79, 163-167.
SAMPLE
Sample preparation: 500 |xL Microsomal incubation + 100 |xL MeCN + IS, centrifuge at 16000
g for 5 min. Inject an aliquot of the supernatant.
HPLC VARIABLES
Column: 300 X 3.9 ixBondapack C18
Flow rate: 1.5
Detector: UV 208
CHROMATOGRAM
Internal standard: estradiol-3-acetate (13-14)
OTHER SUBSTANCES
Noninterfering: fluconazole, itraconazole, miconazole, a-naphthoflavone, quinidine, sulfaphen-
azole, troleandomycin
Interfering: ketoconazole
KEYWORDS
liver
REFERENCE
Schmider,J.; Greenblatt,D.J.; von Moltke,L.L.; Karsov,D.; Vena,R.; Friedman,H.L.; Shader,R.I. Biotransforma-
tion of mestranol to ethinyl estradiol in vitro: The role of cytochrome P-450 2C9 and metabolic inhibitors,
J.Clin.PharmacoL, 1997, 37, 193-200.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOHrwater 1:1 at a concentration of 50 |xg/mL, inject a 10
(JLL aliquot.
HPLCVARIABLES
Column: 300 X 3.9 10 |xm fxBondapak C18
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
REFERENCE
separation of drugs using triethylamine as a competing base, J.Chromatogn, 1986, 370, 403-418.
SAMPLE
Matrix: solutions
Sample preparation: Extract 15 mL water with dichloromethane, evaporate organic layer, take
up residue in 3 mL mobile phase, inject 50 u,L aliquot.
HPLC VARIABLES
Column: reverse phase
CHROMATOGRAM
Internal standard: mestranol
OTHER SUBSTANCES
Simultaneous: ethinylestradiol
KEYWORDS
mestranol is IS
REFERENCE
de Leede,L.G.J.; Govers,C.P.M.; de Nijs,H. A multi-compartment vaginal ring system for independently ad-
justable release of contraceptive steroids, Contraception, 1986, 34, 589-602.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Hexane:dioxane:isopropanol 95:3:2
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Simultaneous: estrone, ethinyl estradiol, norethindrone, norethindrone acetate, norgestrel
KEYWORDS
normal phase
REFERENCE
Gazdag,M.; Szepesi,G.; Fabian-Varga,K. Selection of high-performance liquid chromatographic methods in
pharmaceutical analysis. II. Optimization for selectivity in normal-phase systems, J.Chromatogr., 1988,
454, 95-107.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:THF:water 12.9:22.4:64.7
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 37
OTHER SUBSTANCES
Simultaneous: estrone, ethinyl estradiol, norethindrone, norethindrone acetate, norgestrel
REFERENCE
Gazdag,M.; Szepesi,G.; Szeleczki,E. Selection of high-performance liquid chromatographic methods in phar-
maceutical analysis. I. Optimization for selectivity in reversed-phase chromatography, J.Chromatogr., 1988,
454, 83-94.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 12 X 6 Zorbax Reliance
Mobile phase: MeCNipH 3.8 acetate buffer 70:30 containing 0.1 mM tetrabutylammonium
phosphate
Flow rate: 0.9
Detector: UV 280
CHROMATOGRAM
Retention time: 4
REFERENCE
Patel,J.U.; Prankerd,R.J.; Sloan,K.B. A prodrug approach to increasing the oral potency of a phenolic drug. 1.
Synthesis, characterization, and stability of an O-(imidomethyl) derivative of 17(3-estradiol, J.Pharm.ScL,
1994, 83, 1477-1481.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 |xL aliquot of a solution in MeOH:water 50:50.
HPLC VARIABLES
Column: 250 X 4 7 [xm LichroCART RP-8 (Merck)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 11
OTHER SUBSTANCES
Simultaneous: fluoxymesterone, medrogestone, norethindrone, progesterone, testosterone
propionate
REFERENCE
Gau,Y.S.; Sun,S.W.; Chem,R.R.-L. Optimization of high-performance liquid chromatographic separation for pro-
gestogenic, estrogenic, and androgenic steroids using factorial design, J.Liq.Chromatogr., 1995, 18, 2373-
2382.
Metapramine
Merck Index: 5991
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 |xL 10 |xg/mL chlorohaloperidol in MeOH + 50 |xL 1 M
NaOH + 6 mL distilled ether, shake mechanically for 10 min, centrifuge for 5 min. Remove the
organic layer and add it to 100 |xL 100 mM HCl, shake for 10 min, centrifuge for 5 min. Remove
the aqueous layer, centrifuge for 2 min, inject a 60 JLJLL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:buffer 13:35:52 (Buffer was 6.5 g/L (?) KH2PO4 adjusted to pH 3
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Internal standard: chlorohaloperidol (6.83)
OTHER SUBSTANCES
Simultaneous: amitriptyline, bromazepam, chlorpromazine, clobazam, clomipramine, desipra-
mine, diazepam, flunitrazepam, haloperidol, imipramine, indalpine, levomepromazine, loraze-
pam, maprotiline, mianserin, oxazepam, prochlorperazine, triazolam
Noninterfering: meprobamate
KEYWORDS
plasma
REFERENCE
Rouquette,C; Hecquet,D.; Pommereau,X.; Gardere,J.J.; Brachet-Liermain,A. Metapramine overdose: report of
two cases and analytical determinations, J.Anal.ToxicoL, 1985, 9, 275-277.
SAMPLE
Matrix: blood
Sample preparation: Evaporate 200 |xL 1 |xg/mL citalopram in MeOH into a tube, add 2 mL
plasma, add 2 mL pH 10 Titrisol buffer (Merck), add 8 mL diethyl ether, shake for 15 min,
centrifuge at 2800 g for 5 min. Remove the organic phase and shake it with 100 |xL 50 mM
phosphoric acid for 15 min, centrifuge at 2800 g for 10 s. Remove the aqueous layer and vortex
it with 2 mL diethyl ether for 10 s, centrifuge at 2800 g. Discard the organic layer and inject
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
Retention time: 8.6
Internal standard: citalopram (10.8)
OTHER SUBSTANCES
Noninterfering: diazepam, amitriptyline, clobazam, levomepromazine, norclobazam, triazolam,
monodesmethyltrimipramine, flunitrazepam, alimemazine, alprazolam, amineptine, caffeine,
carbamazepine, desmethylflunitrazepam, diazepam, dibenzepine, estazolam, ethyl loflazepate,
loprazolam, lorazepam, meprobamate, nitrazepam, nordiazepam, nortriptyline, oxazepam,
viloxazine
Interfering: indalpine
KEYWORDS
plasma
REFERENCE
Pok Phak,R.; Conquy,T.; Gouezo,R; Viala,A.; Grimaldi,F. Determination of metapramine, imipramine, trimi-
pramine and their major metabolites in plasma by reversed-phase column liquid chromatography,
J.Chromatogr., 1986, 375, 339-347.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 200 (xL MeOH + 2 mL 1 M pH 10.0 phosphate buffer + 6
mL diethyl ether:hexane 50:50, shake for 15 min, centrifuge at 4000 g for 5 min. Remove the
organic layer and add it to 2 mL 62.5 mM sulfuric acid, vortex for 5 min, centrifuge at 4000 g
for 5 min. Remove the aqueous phase and add it to 1 mL 500 mM NaOH, vortex, add 6 mL
hexane:diethyl ether 50:50, shake for 10 min, centrifuge at 4000 g for 5 min. Remove the
residue in 100 mM sodium carbonate, add 10 JXL 10 mg/mL dansyl chloride in acetone, vortex
for 1 min, heat at 45 for 30 min, evaporate under a stream of nitrogen at 50. Reconstitute
the residue in 200 JJLL MeCN:water 45:55, inject a 100 |xL aliquot.
HPLC VARIABLES
min
Flow rate: 1.5
Detector: F ex Fluorichrom 7.54 and 7.60 filters, em 3.71 and 4.76 filters
CHROMATOGRAM
Retention time: 28
Internal standard: metapramine
OTHER SUBSTANCES
Extracted: fluvoxamine
Noninterfering: alimemazine, alprazolam, amineptine, amitriptyline, caffeine, clobazam, clom-
ipramine, clorazepate, cyamemazine, diazepam, demethyldiazepam, flunitrazepam, levome-
promazine, loprazolam, lorazepam, meprobamate, nitrazepam, oxazepam, triazolam, viloxazine
KEYWORDS
plasma; protect from light; derivatization; metapramine is IS
REFERENCE
Pommery,J.; Lhermitte,M. High performance liquid chromatographic determination of fluvoxamine in human
plasma, Biomed.Chromatogr., 1989, 3, 177-179.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chlorofornr.isopropanol:
the residue in 100 |ULL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 jxL aliquot of
HPLC VARIABLES
Column: 300 X 3.9 4 |im NovaPack C18
Flow rate: 0.8
Detector: UV 269
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Sample preparation: Plasma. 0.5-2 mL Plasma + 10-1000 ng maprotiline in water + 2 mL pH
10 phosphate buffer + 6 mL diethyl ether:hexane 50:50, shake for 15 min, centrifuge at 3750
g for 5 min. Remove the organic phase and add it to 2 mL 250 mM sulfuric acid, shake for 10
min, centrifuge for 5 min, discard the organic layer. Adjust the pH of the aqueous phase to 9.5-
10.5 with 500 mM NaOH containing 1 M K2HPO4, add 6 mL diethyl ether:hexane 50:50, shake
for 10 min, centrifuge for 10 min. Remove the organic layer and evaporate it to dryness under
vacuum and a stream of nitrogen at 45, reconstitute the residue in 100 |xL 100 mM sodium
carbonate, add 10 |xL 1% dansyl chloride in acetone, vortex for 20-30 s, heat at 45 for 30 min.
Evaporate the solvent, reconstitute in 100 ^L mobile phase, inject a 10-50 JJLL aliquot. Urine.
0.5-2 mL Urine + 10-1000 ng maprotiline in water + 2 mL pH 10 phosphate buffer + 6 mL
diethyl ether:hexane 50:50, shake for 15 min, centrifuge at 3750 g for 5 min. Remove the
organic phase and evaporate it to dryness under vacuum and a stream of nitrogen at 45,
reconstitute the residue in 100 |JLL 100 mM sodium carbonate, add 10 JJLL 1% dansyl chloride
in acetone, vortex for 20-30 s, heat at 45 for 30 min. Evaporate the solvent, reconstitute in
100 |JLL mobile phase, inject a 10-50 (xL aliquot.
HPLC VARIABLES
Flow rate: 2
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Sommadossi,J.P.; Lemar,M.; Necciari,J.; Sumirtapura,Y.; Cano,J.R; Gaillot,J. High-performance liquid chro-
matographic method for the determination of plasma and urine metapramine after dansylation,
J.Chromatogr., 1982, 228, 205-213.
SAMPLE
Sample preparation: 1 mL Microsomal incubation + 100 \xL 1 M NaOH + 25 (xL 4 mM desi-
pramine in MeOH, extract twice with diethyl ether. Combine the organic layers and evaporate
them to dryness under a stream of nitrogen, reconstitute the residue in 200 (JLL, inject an
aliquot.
HPLC VARIABLES
Column: 250 X 4 Lichrosorb RP18
Mobile phase: Gradient. A was MeCN:20 mM sulfuric acid 20:80. B was MeCN:20 mM sulfuric
acid 90:10. A:B from 100:0 to 0:100 over 20 min.
Flow rate: 1
Detector: UV 275
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
rat; liver
REFERENCE
Barret,R-l Jaussaud,R; Pautet,R; Guyot,J.L.; Daudon,M. Metabolism of metapramine in vitro by chemical
model systems and rat liver microsomes, Arzneimittelforschung, 1989, 39, 1574-1576.
Metaproterenol
Merck Index: 5992
Lednicer No.: 1 64
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond-Elut C18 SPE cartridge with MeCN and water. 1 mL
Plasma + 100 |xL 2 |xg/mL terbutaline sulfate + 1 mL 20 mM pH 9.0 Na2HPO4, mix, add to
the SPE cartridge, wash with 5 column volumes of water, dry the SPE cartridge for 5 min,
wash with 3 mL dichloromethanern-butanol 97:3, elute with two 1 mL portions of 0.09% HCl
in MeCN, evaporate the eluate to dryness under a stream of nitrogen at 37, dissolve the
residue in 300 |xL mobile phase, inject a 200 JJLL aliquot. (To hydrolyze 3-O-metaproterenol
sulfate mix 1 mL plasma and 200 |xL 2 |xg/mL terbutaline sulfate, add 1 mL 6% trichloroacetic
acid, vortex, centrifuge at 1000 g for 15 min. Remove the supernatant and add it to 200 |xL 2
M HCl, heat at 65 for 90 min, cool, adjust pH to 10 with 400 |xL 2 M carbonate buffer, proceed
as above.)
HPLCVARIABLES
Mobile phase: MeCN:buffer:water 4:1.5:94.5
Flow rate: 1.8
CHROMATOGRAM
Retention time: 6.9
Internal standard: terbutaline (14.8)
KEYWORDS
REFERENCE
Selinger,K.; Hill,H.M.; Matheou,D.; Dehelean,L. Determination of free and total metaproterenol in human
plasma by high-performance liquid chromatography with fluorimetric detection, J.Chromatogr., 1989,493,
230-238.
SAMPLE
residue with 50 |iL MeCNiwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
Sample preparation: Evaporate an aliquot of a solution in MeCN containing 625 ng drug to
dryness under a stream of nitrogen at room temperature, add 200 (xL saturated sodium car-
bonate, add 200 |xL 4% (-)-menthyl chloroformate in MeCN, vortex for 30 s, add an excess
amount of 4-hydroxy-L-proline, vortex for 30 s, centrifuge for 3 min, inject a 10-25 jxL aliquot
of the upper layer.
HPLC VARIABLES
Guard column: 50 X 4.6 Pellicular ODS (Whatman)
Column: 100 X 4.6 5 fxm Partisil 5 ODS3
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: sotalol
KEY WpRDS
REFERENCE
Mehvar,R. Stereospecific liquid chromatographic analysis of racemic adrenergic drugs utilizing precolumn de-
rivatization with (-)-menthyl chloroformate, J.Chromatogr., 1989, 493, 402-408.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Hexane:l,2-dichloroethane:EtOH/trifluoroacetic acid 55:35:10 (EtOH/trifluoro-
acetic acid was premixed 20:1.)
Flow rate: 0.7-1
Detector: UV 278
KEY WORDS
REFERENCE
J.Liq.Chromatogr., 1995, 18, 649-671.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 fim Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ethidium bromide, ethopropazine, fenoprofen, fentanyl, fiavoxate, fluoxetine, fluphenazine, flur-
dol, mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, methadone, meth-
ylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam,
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Sample preparation: Condition a Bond Elut C18 SPE cartridge with 3 volumes of MeOH and
2 volumes of water, dry under vacuum. 500 (xL Urine + 5 |xg terbutaline, add to the SPE
cartridge, wash with 5 volumes of water, elute with 200 |xL MeOH:50 mM pH 6 potassium
phosphate buffer 50:50, add 50 JJLL 50 mM Na3PO4 to the eluate, pass argon through the mix-
ture, inject a 25 |xL aliquot.
HPLC VARIABLES
Column: 300 mm long jxBondapak phenyl
Flow rate: 2.8
CHROMATOGRAM
Retention time: 2.7
Internal standard: terbutaline (4.1)
KEYWORDS
SPE; protect from light; pharmacokinetics
REFERENCE
MacGregor,T.R.; Nastasi,L.; Farina,P.R.; Keirns,J.J. Isolation and characterization of metaproterenol-3-O-sul-
fate: a conjugate of metaproterenol in human urine, Drug Metab.Dispos., 1983, 11, 568-573.
Metaraminol
CAS Registry No.: 54-49-9, 33402-03-8 (bitartrate)
Merck Index: 5993
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 950 mg metaraminol bitartrate in 50 mL water. Remove a 2 mL
aliquot and add it to 5 mL 100 jjug/mL butylparaben in water, make up to 100 mL with water,
inject a 20 |JLL aliquot.
HPLC VARIABLES
Column: 300 X 4 10 ^m ixBondapak C18
Mobile phase: MeOH:water:acetic acid 60:40:1 containing 2.2 g/L dioctyl sodium sulfosuccinate
(Dissolve dioctyl sodium sulfosuccinate in MeOH then add water and acetic acid.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: butylparaben (8.2)
OTHER SUBSTANCES
Simultaneous: methylparaben, propylparaben
REFERENCE
Martin,C.J.; Saxena,S.J. High-performance liquid chromatographic determination of metaraminol bitartrate in
the presence of parabens, J.Pharm.Sci., 1980, 69, 1459-1461.
SAMPLE
HPLC VARIABLES
acid.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: norepinephrine, epinephrine, levonordefrin, isoproterenol, phenylephrine, impu-
rities
KEYWORDS
REFERENCE
forms, J.Assoc.Off.Anal.Chem., 1991, 74, 289-291.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 u-L aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.7
OTHER SUBSTANCES
mepyramine, mesoridazine, methadone, methamphetamine, methapyrilene, methdilazene,
prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol,
prothipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine,
quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyl-
diamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothix-
ene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylc^romine, trazodone, tri-
fluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim,
trimipramine, tripelennamine, triprolidine, tryptamine, verapamil, xylometazoline
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: epinephrine, isoproterenol, levonordefrin, phenylephrine
REFERENCE
Metformin
Merck Index: 6001
SAMPLE
Matrix: blood
Sample preparation: Add 10 |xL 60% perchloric acid to 250 (JLL plasma. Vortex for 1 min, cen-
trifuge at 12800 g for 3 min. Inject a 50 |xL aliquot of the supernatant.
HPLCVARIABLES
Guard column: 5 |xm Spherisorb CN
Column: 250 X 4.6 5 |xm Supelcosil LC-CN
Mobile phase: MeCN.buffer 40:60 (Buffer was 10 mM KH2PO4 adjusted to pH 3.5 with glacial
acetic acid.)
Flow rate: 1
Detector: UV 234
CHROMATOGRAM
KEYWORDS
REFERENCE
Yuen,K.H.; Peh,K.K. Simple high-performance liquid chromatographic method for the determination of met-
formin in human plasma, J.Chromatogr.B, 1998, 710, 243-246.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Plasma + 50 |xL 100 g/L trichloroacetic acid in water, mix, centri-
fuge, inject a 20-50 jxL aliquot of the supernatant
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Whatman SCX
Mobile phase: 100 mM pH 4.42 (NH4)H2PO4
Flow rate: 3
Detector: UV 232
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Noninterfering: glibornuride, gliclazide, glipizide, glyburide (glibenclamide)
KEYWORDS
plasma
REFERENCE
Lacroix,C; Danger,R; Wojciechowski,F. Microdosage de Ia metformine plasmatique et intra-6rythrocytaire par
chromatographie en phase liquide [Microassay of plasma and erythrocyte metformin by high performance
liquid chromatography], Ann.Biol.Clin.(Paris), 1991, 49, 98-101.
SAMPLE
Matrix: blood
Sample preparation: Condition an Amprep 100 mg C8 SPE cartridge (Amersham) with 2 mL
MeOH and 1 mL water, do not allow to run dry. 500 jxL Plasma + 1 |xg phenformin, add to the
SPE cartridge, wash with 2 mL diethyl ether, elute with 500 |iL MeCN:10 mM pH 3.5 KH2PO4
70:30. Centrifuge for 5 min or filter (Millipore HV) the eluate, inject an aliquot.
HPLC VARIABLES
Guard column: Guard-pak C18 (Waters)
Mobile phase: MeCNrIO mM KH2PO4 40:60 adjusted to pH 7 with diethylamine
Flow rate: 1.35
Detector: UV 236
CHROMATOGRAM
Retention time: 2.8
Internal standard: phenformin (5.6)
KEYWORDS
plasma; SPE; pharmacokinetics; human; rat
REFERENCE
Huupponen,R.; Ojala-Karlsson,R; Rouru,J.; Koulu,M. Determination of metformin in plasma by high-perfor-
SAMPLE
Matrix: blood
the residue in 100 JJLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |JLL aliquot of
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 231
CHROMATOGRAM
KEYWORDS
REFERENCE
Tracqui,A-; Kintz,R; Mangin,R Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
Sample preparation: Plasma. 500 |xL Plasma + 200 JJLL 30 |xg/mL 1-propylbiguanide sulfate in
1.2 mM trichloroacetic acid, vortex for 5 s, let stand for 10 min, centrifuge at 5000 g for 5 min,
inject a 100 |xL aliquot of the supernatant. Urine. Dilute urine with water, if necessary. 500
|JLL Urine + 200 |xL 30 |xg/mL 1-propylbiguanide sulfate in water, vortex for 5 s, let stand for
10 min, centrifuge at 5000 g for 5 min, inject a 100 JAL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 X 4.6 Partisil-10 SCX
Mobile phase: 30 mM (NH4)H2PO4 adjusted to pH 2.4 with orthophosphoric acid
Flow rate: 3
Detector: UV 230
CHROMATOGRAM
Retention time: 8
Internal standard: 1-propylbiguanide sulfate (Synthesis of 1-propylbiguanide is as follows. Heat
9 g propylamine, 7.5 g cyanoguanidine, 11.6 g copper sulfate pentahydrate, 75 mL water in a
sealed tube at 100 for 12 h, cool, dilute with 350 mL water, heat to 80, pass a stream, of
hydrogen sulfide (Caution! Highly toxic!) through the solution to precipitate copper salts, filter.
Evaporate the filtrate under reduced pressure at 100. Recrystallize the product from hot EtOH
and dry it at 100, mp 194-6.) (10)
OTHER SUBSTANCES
Noninterfering: acetaminophen, albuterol, allopurinol, amiloride, amitriptyline, ampicillin,
amobarbital, aspirin, caffeine, carbamazepine, chlorpropamide, chlorothiazide, clonidine, dan-
thron, dextropropoxyphene, diazepam, digoxin, dioctyl sodium sulfosuccinate, doxepin, ephed-
rine, ethylestranol, furosemide, glyburide (glibenclamide), hydrochlorothiazide, insulin, isosor-
bide dinitrate, methyclothiazide, methyldopa, metoprolol, nitrazepam, nitroglycerin,
nortriptyline, oxazepam, phenylbutazone, phenytoin, pindolol, prazosin, prochlorperazine, pro-
poxyphene, propranolol, quinine, sodium chromoglycate, sulfamethoxazole, theophylline, thy-
roxine, tolbutamide, trimethoprim, verapamil, vitamin B, warfarin
KEYWORDS
plasma
REFERENCE
Charles,B-G.; Jacobsen,N.W.; Ravenscroft,P.J. Rapid liquid-chromatographic determination of metformin in
plasma and urine, Clin.Chem., 1981, 27, 434-436.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 250 |xL water + 1 mL bromothymol blue solution
+ 50 |xL 2 |xg/mL propyl bigiianide in water, vortex briefly, adjust pH to 7.6-7.8 with a few
drops of 0.1% orthophosphoric acid, add 5 mL diethyl ether:dichloromethane 2:1, vortex for 1
min, centrifuge at 2000 g for 5 min. Remove 4 mL of the upper organic phase and add it to
100 |xL 0.1% tetrabutylammonium hydroxide adjusted to pH 7 with 0.1% orthophosphoric acid,
vortex for 1 min, centrifuge at 2000 g for 5 min. Discard the organic phase, heat the aqueous
phase at 70-90 for 10 min, inject a 25 jxL aliquot of the aqueous phase. Urine. 20 |xL Urine +
2 mL bromothymol blue solution + 100 |xL 20 (xg/mL propyl biguanide in water, vortex briefly,
adjust pH to 7.6-7.8 with a few drops of 0.1% orthophosphoric acid, add 5 mL diethyl ether:
dichloromethane 2:1, vortex for 1 min, centrifuge at 2000 g for 5 min. Remove 4 mL of the
upper organic phase and add it to 100 |JIL 1% tetrabutylammonium hydroxide adjusted to pH
7 with 0.1% orthophosphoric acid, vortex for 1 min, centrifuge at 2000 g for 5 min. Discard the
organic phase, heat the aqueous phase at 70-90 for 10 min, inject a 25 |xL aliquot of the
aqueous phase. (Bromothymol blue solution was 1.48 g bromothymol blue in 20 mL 200 mM
NaOH, make up to 200 mL with water, let stand at room temperature with occasional mixing
for 2 days before use.)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Spherisorb ODS 2
Mobile phase: MeCN:buffer 8:92 adjusted to pH 4.0 with orthophosphoric acid (Buffer was 50
mM K2HPO4 containing 3 mM heptanesulfonic acid.)
Flow rate: 1
Detector: UV 234
CHROMATOGRAM
Retention time: 3.3
Internal standard: propyl biguanide (Synthesis of 1-propylbiguanide is as follows. Heat 9 g
propylamine, 7.5 g cyanoguanidine, 11.6 g copper sulfate pentahydrate, 75 mL water in a sealed
tube at 100 for 12 h, cool, dilute with 350 mL water, heat to 80, pass a stream, of hydrogen
sulfide (Caution! Highly toxic!) through the solution to precipitate copper salts, filter. Evaporate
the nitrate under reduced pressure at 100. Recrystallize the product from hot EtOH and dry
it at 100, mp 194-6 (Clin.Chem. 1981, 27, 434).) (7.80)
KEYWORDS
plasma
REFERENCE
Keal,J.; Somogyi,A. Rapid and sensitive high-performance liquid chromatographic assay for metformin in
plasma and urine using ion-pair extraction techniques, J.Chromatogr., 1986, 378, 503-508.
SAMPLE
Sample preparation: Blood. Add plasma or whole blood to MeCN containing propylbiguanide,
wash the supernatant with dichloromethane, inject an aliquot. Urine. Mix urine with MeCN
containing propylbiguanide, inject an aliquot.
HPLC VARIABLES
Column: 250 X 3.6 5 fxm Si
Mobile phase: MeCNiwater 20:80 containing 10 mM (NH4)2HPO4, pH adjusted to 7.5 with phos-
phoric acid
Detector: UV 235
CHROMATOGRAM
Internal standard: propylbiguanide
Limit of detection: 4 |jig/mL (urine), 10 ng/mL (plasma, whole blood)
KEY WORDS
REFERENCE
Sambol,N.C; Chiang,J.; Lin,.T.; Goodman,A.M.; Liu,C.Y.; Benet,L.Z.; Cogan,M.G. Kidney function and age are
both predictors of pharmacokinetics of metformin, J.Clin.Pharmacol., 1995, 35, 1094-1102.
SAMPLE
residue with 50 u>L MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 233.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 /Jim Supelcosil LC-DP (A) or 250 X 4 5 fjim LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
dol, mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol,
methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine,
methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, mida-
zolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedi-
pine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxe-
tine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine,
phenobarbital, phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine,
phenytoin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procain-
amide, procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, pro-
pantheline, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine,
racemethorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital,
sertraline, sotalol, spironolactone, sulfinp3rrazone, sulindac, temazepam, terbutaline, terfena-
dine, tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol,
tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupro-
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Sample preparation: Mix 2 mL urine with 1 mL 20% NaOH and saturate the mixture with
NaCl, add 1 mL MeCN, add 10 mg p-nitrobenzoyl chloride, let stand at room temperature for
1 h, add 10 mg p-nitrobenzoyl chloride, let stand for 1 h, inject a 10 (xL aliquot of the upper
MeCN phase.
HPLC VARIABLES
Column: 914 X 2.2 37-50 fjim Bondapak phenyl/Corasil
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 6
Limit of detection: 200 ng/mL (using more urine and less MeCN)
KEYWORDS
deerivatization
REFERENCE
Ross,M.S.R Determination of metformin in biological fluids by derivatization followed by high-performance
Methacholine chloride
Merck Index: 6003
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in saline, inject a 20 jxL aliquot.
HPLC VARIABLES
Mobile phase: MeOHibuffer 25:75 (Buffer was 20 mL Low-UV PIC B-7 (Waters) diluted with
480 mL water (10 mM 1-heptanesulfonic acid).)
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 9.6
Next Page
REFERENCE
Woodman,T.E; Johnson,B.; Marwaha,R.K. Determination of methacholine chloride by ion-pair high-pressure
Methacycline
Merck Index: 6007
Lednicer No.: 2 227
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 242.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y; Pe"pin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
163.
Methadone
Merck Index: 6008
SAMPLE
Matrix: blood
Previous Page
REFERENCE
Woodman,T.E; Johnson,B.; Marwaha,R.K. Determination of methacholine chloride by ion-pair high-pressure
Methacycline
Merck Index: 6007
Lednicer No.: 2 227
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 242.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y; Pe"pin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
163.
Methadone
Merck Index: 6008
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 50 (JLL 2 (xg/mL methadone in MeOH + 500 jxL pH 9.6
carbonate buffer + 6 mL butyl chloride, shake on a mechanical shaker at 100 rpm for 15 min,
centrifuge at 2000 g for 5 min. Remove the organic layer and add it to 3 mL 200 mM HCl.
Shake for 15 min, centrifuge, remove aqueous layer. Add aqueous layer to 3 drops 60% NaOH
and 6 mL butyl chloride, shake for 15 min, centrifuge. Remove organic layer and evaporate it
to dryness at 50 under a stream of nitrogen, reconstitute residue in 60 JJLL mobile phase, inject
a 40 |xL aliquot.
HPLC VARIABLES
Column: 10 |xm Brownlee RP-8
Mobile phase: MeCNrMeOH: 10 mM KH2PO4 50:30:20
Flow rate: 1
Detector: E, Bioanalytical Systems, glassy carbon working electrode 1.20 V, Ag/AgCl reference
electrode
CHROMATOGRAM
Retention time: 16
Internal standard: methadone
OTHER SUBSTANCES
Simultaneous: oxycodone, fentanyl, meperidine, phenoperidine
KEYWORDS
plasma; methadone is IS
REFERENCE
Schneider,J.J.; Triggs,E.J.; Bourne,D.W.; StephensJ.D.; Haviland,A.M. Determination of oxycodone in human
308, 359-362.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 200 ng doxepin or desipramine + 100 |xL 1 M NaOH + 9
mL freshly prepared hexane.isoamyl alcohol 99:1, shake vigorously for 5 min, centrifuge. Re-
move 8.5 mL of the organic phase and add it to 200 |AL 50 mM HCl, shake well for 1 min,
centrifuge, inject a 50 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Column: 300 X 4 jxBondapak phenyl
Mobile phase: MeCN:0.01% phosphoric acid containing 0.01% NaCl 35:65, final pH 2.8
Flow rate: 1.5
Detector: UV 210
CHROMATOGRAM
Retention time: 20
Internal standard: doxepin (12.2), desipramine (14.2)
OTHER SUBSTANCES
Extracted: cocaine, dextromoramide, meperidine, normeperidine, norpropoxyphene, pentazocine,
propoxyphene
Simultaneous: amitriptyline, buprenorphine, chlorpromazine, codeine, desmethyldoxepin, di-
phenhydramine, ephedrine, imipramine, nortriptyline, oxazepam, oxycodone, pericyazine,
pheniramine, propranolol, quinine, thiopropazate, thioridazine
KEYWORDS
serum
REFERENCE
Hackett,L.R; Dusci,L.J.; Ilett,K.F. The analysis of several nonopiate narcotic analgesics and cocaine in serum
using high-performance liquid chromatography, J.Anal.ToxicoL, 1987, 11, 269271.
SAMPLE
Matrix: blood
Sample preparation: 900 jxL Plasma + 100 |xL 10 jxg/mL difenoxin in mobile phase, add to a
C18 Sep Pak SPE cartridge at 1 mL/min, wash with 4 mL MeCN:buffer 10:90 at 1 mL/min,
elute with 4 mL MeCN:buffer 40:60 at 5 mL/min, inject a 100 \xL aliquot of the eluate. (Buffer
was 0.08% diethylamine in water adjusted to pH 2.3 with orthophosphoric acid.)
HPLCVARIABLES
Guard column: C18
Column: 150 X 4.6 30 |xm Ultracarb ODS (Phenomenex)
Mobile phase: MeCN:buffer 25:75 (Buffer was 0.08% diethylamine in water adjusted to pH 2.3
Flow rate: 1.5
Detector: UV 210
CHROMATOGRAM
Retention time: 18
Internal standard: difenoxin (32)
OTHER SUBSTANCES
KEYWORDS
plasma; SPE; rat
REFERENCE
Pierce,T.L.; Murray,A.G.W.; Hope,W. Determination of methadone and its metabolites by high performance
liquid chromatography following solid-phase extraction in rat plasma, J.Chromatogr.Sci., 1992, 30, 443
447.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 1 mL 160 nM dextropropoxyphene in 1 M sodium carbonate
+ 6 mL n-hexane, shake horizontally for 15 min, centrifuge at 1300 g for 5 min, freeze in dry
ice/acetone for 10 min. Remove the organic layer and evaporate it to dryness under a stream
of nitrogen, reconstitute the residue in 100 JJLL mobile phase, inject a 10-80 JULL aliquot.
HPLC VARIABLES
Guard column: reversed-phase (Chrompack)
Column: 100 X 3 5 |xm Spherisorb CN + 100 X 4 Chiralcel-AGP (in series)
Mobile phase: MeCN: 10 mM pH 5.0 sodium phosphate buffer:dimethyloctylamine 10:90:0.05
Flow rate: 0.9
Detector: UV 200
CHROMATOGRAM
Internal standard: dextropropoxyphene (15.2)
OTHER SUBSTANCES
Noninterfering: benzodiazepines, carbamazepine, ketobemidone, morphine, piroxicam, tenoxi-
cam, valproic acid
KEYWORDS
serum; chiral; pharmacokinetics
REFERENCE
Kristensen,K.; Angelo,H.R.; Blemmer,T. Enantioselective high-performance liquid chromatographic method for
the determination of methadone in serum using an AGP and a CN column as chiral and analytical column,
respectively, J.ChromatogrA, 1994, 666, 283-287.
SAMPLE
Matrix: blood
(JLL 0.1 M phosphoric acid (pH 2.5), mix, separate, inject a 100 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Flow rate: 2
Detector: UV 228
CHROMATOGRAM
Retention time: 5.4
OTHER SUBSTANCES
Extracted: acetazolamide, chlordiazepoxide, chlorimipramine, chlorpromazine, desipramine,
dextromethorphan, diazepam, diphenhydramine, doxepin, encainide, fentanyl, flecainide, fluox-
etine, flurazepam, haloperidol, hydroxyethylflurazepam, ibuprofen, imipramine, lidocaine, ma-
protiline, methaqualone, mexiletine, midazolam, norchlorimipramine, nordoxepin, nortripty-
line, norverapamil, pentazocine, promazine, propafenone, propranolol, protriptyline, quinidine,
trazodone, trimipramine, verapamil
warfarin
Interfering: amitriptyline, nordiazepam, norfluoxetine, propoxyphene, temazepam
KEYWORDS
plasma; SPE
REFERENCE
Clin.Chem., 1994, 40, 1312-1316.
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 261
CHROMATOGRAM
KEYWORDS
pramine; thioridazine; fentiazac; clemastine; mefenamic acid; nuphenazine; prochlorperazine;
penfluridol; bepridil; terfenadine; trinuoperazine
REFERENCE
Tracqui,A.; Kintz,P.; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
Matrix: blood
2 mL 100 mM pH 6.0 phosphate buffer, do not allow to dry. 1 mL Blood + 6 mL 100 mM pH
6.0 phosphate buffer, vortex, sonicate, centrifuge, add the supernatant to the SPE cartridge,
wash with water, wash with 1 mM pH 3.3 acetic acid, dry by suction, wash with 2 mL acetone:
chloroform 50:50, elute with 3 mL ethyl acetate:ammonia 98:2. Evaporate the eluate under a
stream of nitrogen at 40, reconstitute the residue in 50 |xL MeOH, inject a 10 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeOH:50 mM ammonium acetate 75:25 (Mix column effluent with 50 mM am-
monium acetate pumped at 0.5 mL/min.)
Flow rate: 0.6
Detector: MS, Finnigan MAT TSQ 700 tandem quadrupole, MAT TSP-2 interface, thermospray,
selective reaction monitoring m/z 310-265, collision offset -10 V, repeller 100 V, vaporizer 130,
source 200, filament on 200 jxA, argon 2.5 mTorr, multiplier 1500 V, dynode 15 kV, scan time
1.20 s, MSMSC factor 10
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: benperidol, dextromoramide, droperidol, haloperidol, penfluridol, pimozide, pipam-
peridone, propoxyphene (dextropropoxyphene)
KEYWORDS
SPE; LC/MS
REFERENCE
Verweij,A.M.; Hordijk,M.L.; Lipman,P.J. Quantitative liquid chromatographic thermospray-tandem mass spec-
trometric analysis of some analgesics and tranquilizers of the methadone, butyrophenone, or diphenylbu-
tylpiperidine groups in whole blood, J.Anal.Toxicol., 1995, 19, 65-68.
SAMPLE
Matrix: blood, CSF
Sample preparation: 200 |xL Serum, plasma, or CSF + 300 |xL reagent. Flush column A to
waste with 500 |xL 500 mM ammonium sulfate, inject sample onto column A, flush column A
to waste with 500 |xL 500 mM ammonium sulfate, elute the contents of column A onto column
HPLC VARIABLES
Column: A 30 X 2.1 40 jxm preparative grade C18 (Analytichem); B 250 X 4.6 10 fjim Partisil
C8
Mobile phase: Gradient. A was 50 mM pH 4.5 KH2PO4. B was MeCNiisopropanol 80:20. A:B 90:
Flow rate: 1.5
CHROMATOGRAM
OTHER SUBSTANCES
chlorothiazide, diazepam, droperidol, ethionamide, furosemide, isoniazid, penicillin G, pheno-
trimethoprim
KEYWORDS
REFERENCE
Seifart,H.I.; Kruger,P.B.; Parkin,D.R; van Jaarsveld,P.P.; Donald,P.R. Therapeutic monitoring of antitubercu-
1993, 619, 285-290.
SAMPLE
Sample preparation: 2 mL Blood or 250 mg liver (homogenized with 3 parts water) or 500 mg
brain (homogenized with 3 parts water) + 2 |xg SKF-525-A + 1.5 mL pH 9.5 ammonium car-
bonate/ammonium hydroxide buffer + 10 mL hexaneiisopropanol 99:1, rotate at 10 rpm for 10
min, centrifuge at 3500 rpm for 10 min. Remove the organic layer and add it to 2.5 mL 0.25
M sulfuric acid, rotate for 5 min, centrifuge at 1500 rpm for 5 min. Remove the aqueous layer
and add concentrated ammonium hydroxide to make the pH 9.5, add 1.5 mL chloroform, vortex
for 15 s, centrifuge at 1500 rpm for 5 min. Remove the organic layer and add 1 drop of 1% HCl
in MeOH, evaporate to dryness at 50 under vacuum, reconstitute with 200 |JLL mobile phase,
HPLC VARIABLES
Guard column: 30 X 2.1 Whatman C18 pellicular
Column: 250 X 4.6 Spherisorb S-5-ODS
Mobile phase: MeCN:MeOH:buffer 48:4:48 (Buffer was 1980 mL water + 20 mL 85% phosphoric
acid + 3.7 mL methanesulfonic acid adjusted to pH 3.0 with 5 M NaOH.)
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
Retention time: 9.8
Internal standard: SKF-525-AU1.2)
OTHER SUBSTANCES
Simultaneous: norpropoxyphene, propoxyphene, diazepam, N-desmethyldiazepam
KEYWORDS
liver; brain
REFERENCE
Rio,J.; Hodnett,N.; Bidanset,J.H. The determination of propoxyphene, norpropoxyphene, and methadone in
postmortem blood and tissues by high-performance liquid chromatography, J.Anal.Toxicol., 1987,11, 222-
224.
SAMPLE
Sample preparation: Blood or serum. 1 mL Blood or serum + 1 jxg cianopramine + 1 mL water,
(xL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the organic
layer and inject a 30 (JLL aliquot of the aqueous layer. Liver homogenate. 0.5 mL Liver ho-
mogenate + 10 |xg cianopramine + 500 |xL 2% sodium tetraborate + 8 mL hexane:l-butanol 95:
it to 400 JJIL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the
organic layer and inject a 30 |JLL aliquot of the aqueous layer.
HPLC VARIABLES
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: amitriptyline, amoxapine, benztropine, chlorpheniramine, chlorpromazine, clom-
ipramine, cyproheptadine, desipramine, diphenhydramine, dothiepin, doxepin, fluoxetine, hal-
operidol, imipramine, loxapine, maprotiline, meperidine, mesoridazine, metoclopramide, mian-
serin, moclobemide, nomifensine, nordoxepin, norfluoxetine, norpropoxyphene, northiaden,
nortriptyline, pentobarbital, pheniramine, promethazine, propoxyphene, propranolol, protrip-
tyline, quinidine, quinine, sulforidazine, thioridazine, thiothixene, tranylcypromine, trazodone,
trihexyphenidyl, trimipramine, triprolidine
fluoperazine
Interfering: brompheniramine
KEYWORDS
REFERENCE
SAMPLE
Sample preparation: Serum. Mix 100 |xL serum with 100 |xL. 10 |xg/mL estazolam in EtOH.
Add 300fiL 10% anhydrous sodium carbonate in water, 4 mL hexane, and 500 jxL 2-propanol.
Vortex for 2 min. Centrifuge at 1500 g for 10 min and evaporate the upper organic layer to
dryness under a stream of nitrogen at room temperature. Add 100 (JiL mobile phase, vortex for
30 s, centrifuge at 3000 g or filter (0.22 (xm). Inject a 50 |xL aliquot. Urine. Mix 100 |xL urine
with 50 |xL. 10 (jLg/mL estazolam in EtOH. Add 300|xL 10% anhydrous sodium carbonate in
water and 4 mL hexane. Vortex for 2 min. Centrifuge at 1500 g for 10 min and evaporate the
upper organic layer to dryness under a stream of nitrogen at room temperature. Add 100 |xL
mobile phase, vortex for 30 s. Inject a 50 JJLL aliquot.
HPLCVARIABLES
Guard column: 10 X 3.2 5 |xm Cyclobond 1-200 RSP
Column: 250 X 4.6 5 |xm Cyclobond 1-200 RSP
Mobile phase: MeCN:buffer:water 19:8:73 (Prepare buffer by adding 1 mL triethylamine to 70
mL water, adjusting the pH to 4.5 with glacial acetic acid, and making up to 100 mL with
water.)
Flow rate: 0.4
Detector: UV 210
CHROMATOGRAM
Retention time: 15.5 (R-(-)), 17 (S-(+))
Internal standard: estazolam (22)
OTHER SUBSTANCES
Simultaneous: amphetamine, benzoylecgonine, caffeine, clonazepam, cocaine, codeine, dextro-
propoxyphene, diamorphine, diazepam, dionine, lorazepam, morphine, monoacetylmorphine,
nalorphine, narcotine, nitrazepam, oxazepam, theophylline
Noninterfering: aspirin, barbiturates, clomipramine, imipramine, phenytoin, salicylic acid, val-
proic acid
KEYWORDS
serum; chiral
REFERENCE
Pham-Huy,C; Chikhi-Chorfi,N.; Galons,H.; Sadeg,N.; Laqueille,X.; Aymard,N.; Massicot,F.; Warner,J.-M.;
Claude,J.-R. Enantioselective high-performance liquid chromatography determination of methadone enan-
tiomers and its major metabolite in human biological fluids using a new derivatized cyclodextrin-bonded
phase, J.Chromatogr.B, 1997, 700, 155-163.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 |im Symmetry C8 (Waters)
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in mobile phase, inject 75-100 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelco
Mobile phase: EtOH:MeCN:t-butylamine 98:2:0.05 (Prepared from 1 gal EtOH + 77 mL MeCN
+ 1.9 mL t-butylamine.)
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 2.9
Internal standard: promazine (5.2)
OTHER SUBSTANCES
Simultaneous: N-acetylprocainamide, amitriptyline, amoxapine, amphetamine, buprion, chlor-
diazepoxide, chlorpheniramine, chlorpromazine, cocaine, codeine, demoxepam, desipramine,
desmethylchlordiazepoxide, desmethyldisopyramide, desmethyldoxepin, dextropropoxyphene,
diazepam, disopyramide, doxepin, hydroxyamoxapine (7- and 8-), 2-hydroxydesipramine, 2-hy-
droxyimipramine, 10-hydroxynortriptyline, iminostilbene, imipramine, iprindole, maprotiline,
meperidine, mianserin, morphine, nortriptyline, norzimeldine, oxapam, oxaprotiline, perphen-
azine, procainamide, prochlorperazine, prolixin, promethazine, propoxyphene, protriptyline,
pyrilamine, quinidine, thioridazine, trifluoperazine, trimeprazine, trimipramine, zimeldine
Noninterfering: thiopropazine
Interfering: chlorimipramine, fluphenazine, loxepin, phentermine, triflupromazine
KEYWORDS
normal phase
REFERENCE
Beierle,F.A.; Hubbard,R.W. Liquid chromatographic separation of antidepressant drugs: I. Tricyclics, Ther.Drug
Monit, 1983, 5, 279-292.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: codeine, codeine-N-oxide, morphine, ethoheptazine, morphine-3-glucuronide,
pholcodeine, norpethidine, hydrocodone, dihydrocodeine, dihydromorphine, levorphanol, nor-
codeine, normorphine, pemoline, benzphetamine, diethylpropion, mazindol, tranylcypromine,
caffeine, fenethyline, phendimetrazine, methylphenidate, phenelzine, epinephrine, pipradol,
phenylpropanolamine, fencamfamin, chlorphentermine, norpseudoephedrine, phentermine,
mephentermine, buprenorphine, dextromoramide, phenoperidine, fentanyl, etorphine, piri-
tramide, noscapine, papaverine, naloxone, dextropropoxyphene, nalorphine, phenazocine, nor-
pipanone, levallorphan, hydroxypethidine, normethadone, meperidine, dipipanone, diamor-
phine, pentazocine, acetylcodeine, monoacetylmorphine, thebacon, oxycodone
Interfering: fenfluramine, methylenedioxyamphetamine, amphetamine, normetanephrine, 4-hy-
droxyamphetamine, bromo-STP, STP, thebaine, norlevorphanol, benzylmorphine, ethylmor-
phine, morphine-N-oxide
REFERENCE
Law,B; Gill,R.; Moffat,A-C. High-performance liquid chromatography retention data for 84 basic drugs of fo-
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 u,L aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.9
OTHER SUBSTANCES
mepyramine, mesoridazine, metaraminol, methamphetamine, methapyrilene, methdilazene,
phenoxybenzamine, phentolamine, phenylepnrine, phenyltoloxamine, physostigmine, pimino-
thipendyl, protriptyline, proxymetacaine, pseudoephedrine, pjrrimethamine, quinidine, qui-
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: Pelliguard LC-CN (Supelco)
Column: 150 X 4.6 5 ^m Supelcosil LC-PCN
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Internal standard: N-propionylprocainamide (6)
OTHER SUBSTANCES
Simultaneous: amitriptyline, atropine, butalbital, chlorpromazine, desipramine, desmethylma-
protiline, doxepin, imipramine, maprotiline, norpropoxyphene, nortriptyline, phenylpropanol-
amine, procainamide, prochlorperazine, promethazine, propranolol, protriptyline, quinidine,
trifluoperazine, trimeprazine, trimipramine
Noninterfering: acetaminophen, allopurinol, amikacin, amoxapine, amytal, bretylium, caffeine,
carbamazepine, carisoprodol, chloramphenicol, chlordiazepoxide, chlorpropamide, clonazepam,
codeine, diazepam, disopyramide, droperidol, ethinamate, ethinamate, ethosuximide, fluphen-
azine, flurazepam, furosemide, gentamicin, haloperidol, hydrochlorothiazide, hydroxyzine, ibu-
profen, kanamycin, lidocaine, loxapine, meperidine, mephobarbital, meprobamate, methaqua-
lone, methotrexate, morphine, nafcillin, naloxone, neomycin, perphenazine, phenacetin,
phenobarbital, phenytoin, prazepam, primidone, procaine, propoxyphene, reserpine, salicylam-
ide, salicylic acid, secobarbital, spironolactone, theophylline, thiopental, thioridazine, tobra-
mycin, valproic acid, verapamil
REFERENCE
Lin,W.-N.; Frade,P.D. Simultaneous quantitation of eight tricyclic antidepressants in serum by high-perfor-
mance liquid chromatography, Ther.Drug Monit, 1987, 9, 448-455.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 4.6 10 |xm PRP-I (Hamilton)
Mobile phase: Gradient. MeCN:20 mM ammonium hydroxide from 15:85 to 100:0 over 17 min
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 16
OTHER SUBSTANCES
Simultaneous: cocaine, codeine, reserpine, thebaine, yohimbine
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
feine, cocaine, codeine, doxylamine, fluoxetine, glutethimide, hexobarbital, hypoxanthine,
levorphanol, LSD, meperidine, mephobarbital, methylphenidate, methyprylon, N-norcodeine,
oxazepam, oxycodone, phenylpropanolamine, prilocaine, procaine, terfenadine
REFERENCE
column, Supelco Reporter, 1993,12(3), 18-21.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
phesin, mephobarbital, mepivacaine, mescaline, methamphetamine, methapyrilene,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
metformin, methdilazine, methocarbamol, methotrexate, methotrimeprazine, methoxamine,
sertraline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfena-
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Sample preparation: 500 |xL Urine + N-ethylnordiazepam + chlorpheniramine + 100 JJLL buffer,
IxL mobile phase B, with 200 jxL mobile phase C, and with 1.15 mL mobile phase D. Elute
HPLC VARIABLES
3.2 11 jxm Aminex A-28 (Bio-Rad); C 25 X 3.2 5 jxm C8 (Phenomenex) + 150 X 4.6 5 |xm silica
(Macherey-Nagel)
CHROMATOGRAM
OTHER SUBSTANCES
imipramine, flurazepam, amitriptyline, morphine, codeine, hydromorphone, hydrocodone
KEYWORDS
column-switching
REFERENCE
341.
SAMPLE
Matrix: urine
Sample preparation: Extract 1 mL urine.
HPLCVARIABLES
Guard column: Lichrospher 100 RP-18
Column: CHIRAL-AGP (Chrom-Tech)
Mobile phase: MeCN: 10 mM pH 5.0 sodium phosphate buffer:dimethyloctylamine 10:90:0.05
Flow rate: 0.7
Detector: UV 200
CHROMATOGRAM
Internal standard: imipramine (26.9)
OTHER SUBSTANCES
Next Page
KEYWORDS
chiral
REFERENCE
Kristensen,K.; Angelo,H.R. A stereoselective HPLC method for the determination of methadone and is main
metabolite in urine (using an AGP column) (Abstract 39), Ther.Drug Monti., 1995, 17, 393-393.
Methamphetamine
Merck Index: 6015
Lednicer No.: 1 37
SAMPLE
Matrix: blood
Sample preparation: Inject a 5 JJLL aliquot of serum directly.
HPLC VARIABLES
Column: 100 X 4.6 5-10 |jim Silicalite (by sieving Silicalite, 3M Co.(?))
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ethosuximide, sulfamethoxazole, primidone
KEYWORDS
serum
REFERENCE
89-96.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 100 mM NaOH + 3 mL n-hexane, shake for 20 min,
centrifuge for 10 min. Remove 2 mL of the organic layer and evaporate it to dryness using a
vacuum centrifuge, reconstitute the residue in 500 JJLL 100 jjig/mL (S)-(+)-benoxaprofen chloride
in dried dichloromethane, let stand at room temperature for 30 min, inject a 10 JJLL aliquot.
(Synthesis of benoxaprofen chloride is as follows. Dissolve 600 mg benoxaprofen in 50 mL
toluene, slowly add 5 mL freshly-distilled thionyl chloride, reflux for 30 min, evaporate to
dryness, recrystallize benoxaprofen chloride from dichloromethane.)
HPLC VARIABLES
Column: 250 X 4.6 7 |xm Zorbax-Sil
Flow rate: 1
Previous Page
KEYWORDS
chiral
REFERENCE
Kristensen,K.; Angelo,H.R. A stereoselective HPLC method for the determination of methadone and is main
metabolite in urine (using an AGP column) (Abstract 39), Ther.Drug Monti., 1995, 17, 393-393.
Methamphetamine
Merck Index: 6015
Lednicer No.: 1 37
SAMPLE
Matrix: blood
HPLC VARIABLES
Column: 100 X 4.6 5-10 |jim Silicalite (by sieving Silicalite, 3M Co.(?))
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ethosuximide, sulfamethoxazole, primidone
KEYWORDS
serum
REFERENCE
89-96.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 100 mM NaOH + 3 mL n-hexane, shake for 20 min,
centrifuge for 10 min. Remove 2 mL of the organic layer and evaporate it to dryness using a
vacuum centrifuge, reconstitute the residue in 500 JJLL 100 jjig/mL (S)-(+)-benoxaprofen chloride
in dried dichloromethane, let stand at room temperature for 30 min, inject a 10 JJLL aliquot.
(Synthesis of benoxaprofen chloride is as follows. Dissolve 600 mg benoxaprofen in 50 mL
toluene, slowly add 5 mL freshly-distilled thionyl chloride, reflux for 30 min, evaporate to
dryness, recrystallize benoxaprofen chloride from dichloromethane.)
HPLC VARIABLES
Column: 250 X 4.6 7 |xm Zorbax-Sil
Flow rate: 1
CHROMATOGRAM
Retention time: 10.5 (IK-)), 11.5 (S-(+
OTHER SUBSTANCES
Extracted: amphetamine
Interfering: tranylcypromine
KEYWORDS
plasma; derivatization; normal phase; chiral
REFERENCE
Weber,H.; Spahn,H.; Mutschler,E.; Mohrke,W. Activated ot-alkyl-a-arylacetic acid enantiomers for stereoselec-
tive thin-layer chromatographic and high-performance liquid chromatographic determination of chiral
amines, J.Chromatogr., 1984, 307, 145-153.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Serum + 1 mL acetone, vortex for 2 min, centrifuge at 2000 g for
10 min. Remove a 500 JJLL aliquot of the supernatant and add it to 500 jxL reagent, add 20 |xL
0.5% triethylamine in MeOH, vortex, heat at 80 for 30 min, cool to room temperature, inject
a 20 |JLL aliquot. (Prepare reagent by mixing 2 mL 2.5 mM N-(4-aminobutyl)-N-ethylisoluminol
in MeOH with 2 mL 2.5 mM N,N'-disuccinimidyl carbonate in MeCN, let stand for 2 h.)
HPLC VARIABLES
Guard column: 30 X 4.6 TSKm Guardgel ODS-80TM (Toyo Soda)
Column: 150 X 6 5 |xm Shimpack CLC C18 (Shimadzu)
Mobile phase: MeOH:water 54:46 containing 30 mM sodium 1-octanesulfonate
Flow rate: 1
15 mM potassium ferricyanide in 2.5 M NaOH pumped at 1 mL/min and this mixture flowed
through a 200 mm X 0.5 mm ID stainless steel coil. The effluent from this coil mixed with 300
mM hydrogen peroxide containing 10 mM p-cyclodextrin pumped at 1 mL/min and this mixture
flowed through a 100 mm X 0.5 mm ID stainless steel coil to the detector.
CHROMATOGRAM
Retention time: 30
KEY WpRDS
REFERENCE
Nakashima,K; Suetsugu,K.; Akiyama,S.; Yoshida,M. High-performance liquid chromatography-chemilumines-
cence determination of methamphetamine in human serum using N-(4-aminobutyl)-N-ethylisoluminol as a
chemiluminogen, J.Chromatogr., 1990, 530, 154159.
SAMPLE
Matrix: blood
Sample preparation: 100 |JLL Serum + 50 (xL 100 ng/mL aniline sulfate in water + 200 (xL 20
mM pH 10.6 carbonate buffer + 2 mL ethyl acetate, shake for 15 min, centrifuge at 1200 g for
5 min. Remove the organic layer and add it to 200 |xL 50 mM HCl, shake for 15 min, centrifuge
at 1200 g for 5 min. Remove the aqueous layer and add it to 40 JJLL 250 mM NaOH, add 50 |JLL
330 mM pH 7.8 phosphate buffer, add 250 |JLL MeCN, add 25 (JLL 1 mM (-)-l-(9-fluorenyl)ethyl
chloroformate in acetone, let stand overnight at room temperature, add 30 JAL 100 mM glycine
in water, add 750 |JLL n-pentane, vortex for 2 min, centrifuge at 1200 g for 5 min. Remove the
organic layer and evaporate it to dryness under vacuum, reconstitute the residue in MeCN:
water 50:50, inject a 100 jxL aliquot.
HPLC VARIABLES
Guard column: Direct-Connect column prefilter (Alltech)
Column: 150 X 4.6 3 |xm Adsorbosphere HS C18 (Alltech)
Mobile phase: MeCN:THF:20 mM pH 3.6 acetate buffer 39:15:46
Flow rate: 1
CHROMATOGRAM
Internal standard: aniline (21.0)
OTHER SUBSTANCES
KEYWORDS
serum; rat; chiral; derivatization
REFERENCE
Hutchaleelaha,A.; Walters,A.; Chow,H.-H.; Mayersohn,M. Sensitive enantiomer-specific high-performance liq-
uid chromatographic analysis of methamphetamine and amphetamine from serum using precolumn fluo-
rescent derivatization, J.Chromatogr.B, 1994, 658, 103-112.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 500 mM pH 11 borate buffer, mix, add 2.5 mL diethyl
ether, vortex for 5 min, centrifuge at 1200 g for 5 min, remove organic layer, repeat extraction.
Combine the organic layers and add them to 200 jxL 100 mM HCl, vortex for 2 min, centrifuge
at 1200 g for 5 min. Remove the aqueous phase and add it to 150 |xL 1 M pH 8 borate buffer
and 100 |xL 4 mM 9-fluorenylmethyl chloroformate in MeCN, shake, allow to react at 50 for
5 min, add 20 |xL 20 mM proline in water, allow to react at 50 for 2 min, inject a 200 (xL
aliquot.
HPLCVARIABLES
Column: 150 X 3.9 4 |xm Nova-Pak phenyl
Flow rate: 1
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Extracted: amphetamine, desmethyldeprenyl
KEYWORDS
plasma
REFERENCE
La Croix,R-; Pianezzola,E.; Strolin Benedetti,M. Sensitive high-performance liquid chromatographic method
for the determination of the three main metabolites of selegiline (L-deprenyl) in human plasma,
J.Chromatogr.B, 1994, 656, 251-258.
SAMPLE
Sample preparation: Adjust pH of 10 mL plasma or 20 mL urine to 11.4 with 5 M NaOH, add
to a column containing 1.5 g Amberlite XAD-2, wash with 10 mL water, elute with 20 (plasma)
or 40 (urine) mL chlorofornv.isopropanol 75:25, add 100 |JLL 6 M HCl in EtOH to the eluate,
evaporate to dryness under reduced pressure, reconstitute with 1 mL 8% sodium bicarbonate,
add 1 mL 0.5% sodium l,2-naphthoquinone-4-sulfonate, heat at 70 for 20 min, add an equal
volume of chloroform, vortex for 1 min, inject a 50 |xL aliquot of the organic layer.
HPLC VARIABLES
Column: 150 X 5 Partisil 5
Mobile phase: Hexane:chloroform:ethyl acetate:EtOH 50:25:35:1
Flow rate: 2.5
Detector: UV 248
CHROMATOGRAM
Retention time: 3
Internal standard: phenylethylamine (6)
OTHER SUBSTANCES
Extracted: amphetamine, hydroxyamphetamine, norephedrine
KEYWORDS
derivatization; plasma; normal phase; SPE; comparison with other derivatization reagents and
with ion-pair chromatography
REFERENCE
Farrell,B.M.; Jefferies,T.M. An investigation of high-performance liquid chromatographic methods for the anal-
ysis of amphetamines, J.Chromatogr., 1983, 272, 111-128.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 206.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr A, 1997, 763,149-
163.
SAMPLE
Matrix: bulk
Sample preparation: Mix a 1 mg/mL solution in 1 M sodium carbonate with 2 mL 5 mg/mL 8-
quinolinesulfonyl chloride in acetone, heat at 65 for 20 min, cool, extract twice with 30 mL
portions of chloroform. Combine the extracts and dry them over anhydrous magnesium sulfate,
evaporate to dryness under a stream of air, reconstitute, inject an aliquot.
HPLC VARIABLES
Guard column: 70 X 2.1 CorPell ODS
Column: 300 X 3.9 jjiBondapak C18
Flow rate: 1.5
CHROMATOGRAM
Retention time: 25
OTHER SUBSTANCES
Simultaneous: amphetamine, ephedrine, phenmetrazine, phentermine, phenylpropanolamine,
pseudoephedrine
KEY WORDS
derivatization
REFERENCE
Noggle,F.T.,Jr.; Clark,C.R. Liquid chromatographic determination of primary and secondary amines as 8-quin-
olinesulfonyl chloride derivatives, J.Assoc.Off.Anal.Chem., 1984, 67, 687-691.
SAMPLE
Matrix: bulk
Sample preparation: Prepare an aqueous solution. Adjust pH of 500 uX aqueous solution to 12
with 1 M NaOH, add 200 |xL (+)-l-(9-fluorenyl)ethyl chloroformate:dichloromethane 1:100, let
stand for 20 min, add 500 |xL dichloromethane, shake for 30 min. Remove the organic phase
and wash it twice with 500 (JLL aliquots of water, evaporate the organic phase to dryness under
a stream of nitrogen, reconstitute the residue in 3 mL MeOH, filter (0.45 jxm), inject a 25 (xL
aliquot.
HPLC VARIABLES
Column: 250 X 3.9 5 |xm 5C18-AR (Waters)
Flow rate: 1
CHROMATOGRAM
Retention time: 44 ((SM+)), 47 ((RM-))
OTHER SUBSTANCES
Extracted: ephedrine, pseudoephedrine
KEY WpRDS
REFERENCE
Chen,Y.-R; Hsu,M.-C; Chien,C.S. Analysis of forensic samples using precolumn derivatization with (+)-l-(9-
fluorenyl)ethyl chloroformate and liquid chromatography withfluorimetricdetection, J.Chromatogr.A, 1994,
672, 135-140.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 10 jxinole compound (as free base or hydrochloride) in 500 jxL
MeCN, add 250 |xL 5% sodium carbonate (for hydrochlorides only), add 500 |xL 100 mM reagent
in MeCN, vortex for 1 min, heat at 60 for 2 h, add 100 jxmole L-proline, heat at 60 for 30
min. Remove a 100 |xL aliquot and dilute it with mobile phase, neutralize with acetic acid,
inject a 10 jxL aliquot. Prepare the reagent ((R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexyl-
isothiocyanate) as follows. Add 0.7 mL carbon disulfide to 6 mL (lR,2R)-(-)-l,2-diaminocyclo-
hexane, 12 mL water, and 12 mL EtOH, heat the oil bath to 80, add 2.8 mL carbon disulfide
reflux for 1 h, acidify with 500 jxL 5 M HCl, reflux for 12 h, cool, filter, wash the solid with a
dichloroethane, reflux for 16 h to obtain more (R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohex-
ylisothiocyanate (mp >250, Ia]546 = -133 (c = 1) in MeCN).
HPLCVARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: acebutolol, alprenolol, atenolol, carazolol, carvedilol, formoterol, metipranolol,
metoprolol, nifenanol, nitrilo atenolol, oxprenolol, pindolol, propranolol, xamoterol
KEY WpRDS
REFERENCE
KLeidernigg,O.R; Posch,K; Lindner,W. Synthesis and application of a new isothiocyanate as a chiral derivatiz-
ing agent for the indirect resolution of chiral amino alcohols and amines, J.Chromatogr.A, 1996, 729, 3 3 -
42.
SAMPLE
Sample preparation: Powder tablet and add 50 mg to 50 mL MeCN:20 mM pH 3.8 phosphate
buffer 3:97, sonicate for 5 min, filter (0.5 |xm), inject a 20 |xL aliquot of the filtrate.
HPLCVARIABLES
Guard column: Supelguard pre-column containing 5 (xm Suplex pKblOO (Supelco)
Column: 150 X 4.6 5 |xm Suplex pKblOO (Supelco)
Mobile phase: Gradient. MeCN:20 mM pH 3.8 phosphate buffer at 3:97 for 3 min, to 15:85 over
5 min, stay at 15:85 for 4 min, re-equilibrate for 8 min.
Flow rate: 1.5
CHROMATOGRAM
Retention time: 4.3
OTHER SUBSTANCES
Simultaneous: ephedrine, amphetamine, caffeine, 3,4-methylenedioxyamphetamine, N-methyl-
3,4-methylenedioxyamphetamine, N-ethyl-3,4-methylenedioxyamphetamine
KEYWORDS
tablets
REFERENCE
Longo,M.; Martines,C; Rolandi,L.; Cavallaro,A- Simple and fast determination of some phenethylamines in
illicit tablets by base-activated reversed phase HPLC, J.Liq.Chromatogr., 1994, 17, 649-658.
SAMPLE
Matrix: solutions
Sample preparation: Mix 1 mL 20-300 jjug/mL amine solution in water with 2 mL 50 mg/mL 4-
nitrobenzoyl chloride in THF (freshly prepared) and 1 mL 1 M NaOH, heat at 65 for 1 h, cool,
adjust pH to 12 with 1 M NaOH, extract with two 10 mL portions of chloroform. Combine the
extracts and wash them with two 20 mL portions of 10% potassium carbonate, wash with water,
dry over anhydrous magnesium sulfate. Evaporate to dryness under a stream of air, reconsti-
tute the residue in MeOH, inject a 5 JJLL aliquot.
HPLCVARIABLES
Mobile p h a s e : MeCNiwater 35:65
Flow r a t e : 1.5
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
R e t e n t i o n t i m e : 15
OTHER SUBSTANCES
Simultaneous: amphetamine, benzylamine, a-methylbenzylamine, n-propylamphetamine
KEY WpRDS
derivatization
REFERENCE
Clark,R-C; Teague,J.D.; Wells,M.M.; Ellis,J.H. Gas and high-pressure liquid chromatographic properties of
some 4-nitrobenzamides of amphetamines and related arylalkylamines, A/iaZ.Chem., 1977, 49, 912-915.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH at a concentration of 1 mg/mL, inject a 20 JLJLL aliquot.
HPLC VARIABLES
Mobile p h a s e : MeOH:bufifer 90:10 (Buffer was 94 mL 35% ammonia and 21.5 mL 70% nitric
Flow r a t e : 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: dihydrocodeine, dihydromorphine, levorphanol, norcodeine, normorphine, pemo-
line, benzphetamine, diethylpropion, mazindol, tranylcypromine, caffeine, fenethyline, phen-
dimetrazine, methylphenidate, phenelzine, epinephrine, pipradol, phenylpropanolamine,
fencamfamin, chlorphentermine, norpseudoephedrine, fenfluramine, methylenedioxyamphet-
amine, amphetamine, normetanephrine, 4-hydroxyamphetamine, bromo-STP, STP, prolintane,
ephedrine, methylephedrine, mephentermine, buprenorphine, dextromoramide, phenoperidine,
fentanyl, etorphine, piritramide, noscapine, papaverine, naloxone, dextropropoxyphene, nalor-
phine, phenazocine, norpipanone, levallorphan, hydroxypethidine, normethadone, meperidine,
dipipanone, diamorphine, pentazocine, acetylcodeine, monoacetylmorphine, thebacon, oxy-
codone, thebaine, norlevorphanol, methadone, benzylmorphine, ethylmorphine, morphine-N-
oxide, codeine, codeine-N-oxide, morphine, ethoheptazine, morphine-3-glucuronide, pholcodine
Interfering: dimethylamphetamine, mescaline, norpethidine, hydrocodone
REFERENCE
Law,B-; Gill,R-; Moffat,A.C. High-performance liquid chromatography retention data for 84 basic drugs of fo-
SAMPLE
Matrix: solutions
water, 1 mL 20% aqueous ammonia, and 5 mL chloroform:carbon disulfide 98:2, shake vigor-
1 mL mobile phase, inject a 10 jxL aliquot. (Copper may also be used with electrochemical
detection or TJV detection at 270 nm.)
HPLC VARIABLES
Guard column: 30 X 4 40 jxm LiChrosorb RP-18
perchlorate
Flow rate: 1.5
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: ephedrine
Also analyzed: acebutolol, alprenolol, flecainide, propranolol
KEYWORDS
REFERENCE
Leroy,R; Nicolas ,A- Determination of secondary amino drugs as their metal dithiocarbamate complexes by
1984, 317, 513-521.
SAMPLE
Matrix: solutions
Sample preparation: 2 mL THF + 1 mL 33.5 mM reagent in THF (freshly prepared) + 1 mL 1
mg/mL amphetamine in water + 700 |xL 10% sodium bicarbonate in water, heat at 65 for 1 h,
cool, extract three times with 10 mL aliquots of chloroform. Combine the extracts and wash
them with 10 mL water, dry over anhydrous magnesium sulfate, evaporate to dryness, recon-
stitute with 2.5 mL mobile phase, inject a 5 |xL aliquot. (Prepare reagent (l-[(4-nitro-
phenyl)sulfonyl]prolyl chloride) as follows. Mix 40-45 mmoles L-(-)-proline, 40 mL THF, and
200 mL 10% potassium carbonate, add 37-43 mmoles 4-nitrobenzenesulfonyl chloride in 40 mL
THF dropwise, heat at 50 and maintain at pH 8 or above for 3 h, cool, acidify to pH 2, extract
with chloroform. Extract the organic layers with potassium carbonate in water. Acidify the
aqueous layer and extract it with chloroform. Dry the chloroform layer and evaporate it to
dryness, recrystallize the resulting l-[(4-nitrophenyl)sulfonyl]proline from petroleum ether and
benzene (Caution! Benzene is a carcinogen!). Stir 15 mmoles l-[(4-nitrophenyl)sulfonyl]proline
in 100 mL benzene and add 75 mmoles thionyl chloride in 50 mL benzene dropwise, heat at
35-40 until the reaction is complete (about 48 h; monitor by IR), evaporate to dryness, recrys-
tallize from n-heptane to give l-[(4-nitrophenyl)sulfonyl]prolyl chloride (Anal.Chem. 1984, 56,
958) (mp 110-110.5).)
HPLC VARIABLES
Flow rate: 1.5
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
KEYWORDS
REFERENCE
Barksdale,J.M.; Clark,C.R. Liquid chromatographic determination of the enantiomeric composition of amphet-
amine and related drugs by diastereomeric derivatization, J.Chromatogr.ScL, 1985, 23, 176-180.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.7
OTHER SUBSTANCES
clizine, cyclopentamine, eyproheptadine, deserpidine, desipramine, dextromoramide, dextro-
mepyramine, mesoridazine, metaraminol, methadone, methapyrilene, methdilazene, methotri-
meprazine, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, methyl-
ergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone,
nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nortriptyline,
noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline, pecazine,
penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenampro-
mide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, pheninda-
mine, pheniramine, phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxy-
benzamine, phentolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine,
pimozide, pindolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenze-
pine, piritramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, prima-
quine, proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolin-
tane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol,
prothipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine, qui-
amine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothix-
ene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone,
trifluoperazine, trifiuperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim,
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH:water 1:1 at a concentration of 50 fjig/mL, inject a 10
IxL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 ^m ^Bondapak C18
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Simultaneous: phenylpropanolamine, ephedrine, hydroxyamphetamine, amphetamine, phenter-
mine, mephentermine
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: 100 |JLL 100 |JLM Solution + 300 |xL buffer + 500 |JLL 1 mM dansyl chloride
in acetone, mix, heat at 45 in the dark for 1 h, dilute with MeCN:water 50:50, inject a 20 |xL
aliquot. (Prepare buffer by adjusting 10 mM sodium bicarbonate to pH 9.0 with NaOH.)
HPLC VARIABLES
acid
Flow rate: 1
CHROMATOGRAM
Limit of detection: 4 fmole (chemiluminescence), 50 fmole (F)
OTHER SUBSTANCES
Simultaneous: benzylamine, ephedrine, N-isopropylbenzylamine, N-methylphenethylamine,
phenylbutylamine, phenylethylamine, phenylpropanolamine, phenylpropylamine
KEY WpRDS
REFERENCE
Hayakawa,K; Hasegawa,K; Imaizumi,N.; Wong,O.S.; Miyazaki,M. Determination of amphetamine-related
SAMPLE
Matrix: solutions
Sample preparation: 100 (JLL 100 JJLM Solution + 400 |xL buffer + 500 (JLL 80 mM 4-fluoro-7-
nitrobenzoxadiazole in EtOH, mix, heat at 60 in the dark for 1 min, dilute with MeCN:water
50:50, inject a 20 u,L aliquot. (Prepare buffer by adjusting 100 mM boric acid to pH 8.0 with
NaOH.)
HPLC VARIABLES
acid
Flow rate: 1
CHROMATOGRAM
Retention time: 17
Limit of detection: 20 nmole (chemiluminescence), 30 nmole (F)
OTHER SUBSTANCES
Simultaneous: benzylamine, ephedrine, N-isopropylbenzylamine, N-methylphenethylamine,
phenylbutylamine, phenylethylamine, phenylpropanolamine, phenylpropylamine
KEYWORDS
REFERENCE
Hayakawa,K; Hasegawa,K; Imaizumi,N.; Wong,O.S.; Miyazaki,M. Determination of amphetamine-related
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 500 |xg/mL solution in MeOH:water 50:50, inject a 5 |xL aliquot.
HPLC VARIABLES
column.)
Flow rate: 2
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Simultaneous: acetophenone, amphetamine, desipramine, ethylmorphine, imipramine, mefen-
amic acid, morphine, phenylbutazone, salicylic acid
KEY WORDS
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Mix 50 |JLL of a 200 ppm solution in MeCN:500 mM pH 9.0 borate buffer
50:50 with 25 mg reagent, after 1 min elute with 1 mL hexanerTHF 75:25, inject a 5 JJLL aliquot.
(Reagent was dinitrophenyl carbamate benzotriazole polymeric reagent, synthesized as follows.
(Caution! Chloroform, dichloromethane, dioxane, and hydrazine are carcinogenic in experi-
mental animals! DMF may be carcinogenic! 3,5-dinitrobenzoyl chloride and aluminum chloride
are corrosive! Nitrobenzene is toxic!) 10 g Dried macroporous polystyrene (Xe-305, Rohm and
Haas) + 1Og 3-nitro-4-chlorobenzyl alcohol + 1Og anhydrous aluminum chloride + 50 mL
nitrobenzene, heat at 65-70 for 3 days, cool, filter, wash polymer with three 50 mL portions
of 1 M HCl in dioxane, with three 50 mL portions of DMF,with three 50 mL portions of MeOH,
and with three 50 mL portions of dichloromethane, dry under vacuum at 100. Reflux 19 g of
this polymer in 60 mL hydrazine hydrate:ethylene glycol monoethyl ether 40:60 for 20 h, cool
to room temperature, filter off the polymer and wash it thoroughly with water. Suspend the
polymer in 100 mL concentrated HCl:dioxane 50:50, reflux for 20 h, filter the polymer and
wash it with five 100 mL portions of water, with three 100 mL portions of MeOH, and with
three 50 mL portions of ether, dry under vacuum at 80. Functionalization was 1.17 mmoles/g
(Eur.J.Biochem. 1975, 59, 55). Dissolve 3,5-dinitrobenzoyl chloride in the minimum amount of
glacial acetic acid, add an equimolar amount of sodium azide, stir for 30 min, dilute with water,
filter to obtain 3,5-dinitrobenzoyl azide (Caution! Azides are toxic and potentially explosive!)
(J. Liq. Chromatogr. 1986,9, 443). Heat 71 mg 3,5-dinitrobenzoyl azide in 15 mL toluene (dried
over calcium hydride) at ??? for 30 min, cool using an ice bath, add 200 mg polymer, allow to
warm to room temperature with stirring for 1 h, filter, wash the polymer with four 10 mL
portions of warm (40) dichloromethane, dry under high vacuum for 1 h.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm LC-(R)-naphthylurea (Supelco)
Mobile phase: Hexane:EtOH:MeCN 93:7:0.5
Flow rate: 2
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: amphetamine
KEY WORDS
REFERENCE
Bourque,A.J.; Krull,I.S. Immobilized isocyanates for derivatization of amines for chiral recognition in liquid
chromatography with UV detection, J.Pharm.Biomed.AnaL, 1993, 11, 495-503.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.5
Detector: UV 200
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Simultaneous: diethylpropion, phenylpropanolamine, ephedrine, amphetamine, phentermine,
fenfluramine
Also analyzed: amitriptyline, chlordiazepoxide, chlorpromazine, desalkylfiurazepam, desipra-
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
phesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methapyrilene, methaqualone,
methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methjrprylon, me-
toprolol, mibolerone, morphine, nadolol, nalorphine, naloxone,, naltrexone, naphazoline, na-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4 5 jxm Chiradex (immobilized p-cyclodextrin) (Merck)
Mobile phase: MeOH: 100 mM pH 7 ammonium acetate 5:95
Flow rate: 0.5
Detector: UV 254
CHROMATOGRAM
Retention time: 30.28 (S(+)), 31.93 (R(-))
KEY WORDS
chiral
REFERENCE
Rizzi,A.M.; Hirz,R-5 Cladrowa-Runge,S.; Jonsson,H. Enantiomeric separation of amphetamine, methampheta-
mine and ring substituted amphetamines by means of a p-cyclodextrin-chiral stationary phase, Chroma-
tographia, 1994, 39, 131-137.
SAMPLE
Matrix: solutions
Sample preparation: 1 mL Solution + 500 JJLL 0.5% sodium l,2-naphthoquinone-4-sulfonate in
water + 500 |xL buffer, let stand for 10 min, extract three times with 2 mL aliquots of n-hexane:
ethyl acetate 50:50. Combine the organic layers and evaporate them to dryness at 80, recon-
stitute with 2 mL MeCN:water 50:50, inject a 50 JJLL aliquot. (Buffer was 4% sodium bicarbonate
HPLCVARIABLES
Column: 250 X 4 5 jxm Hypersil ODS C18
Mobile p h a s e : Gradient. MeCN:0.5% propylamine hydrochloride in water from 40:60 to 50:50
over 2.5 min, to 70:30 over 1 min, maintain at 70:30 for 4.5 min.
Flow r a t e : 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
derivatization
REFERENCE
Herr&ez-Hernndez,R-; Campins-Falc6,R; Sevillano-Cabeza,A. On-line derivatization into precolumns for the
determination of drugs by liquid chromatography and column switching: Determination of amphetamines
in urine, Anal.Chem., 1996, 68, 734-739.
SAMPLE
Matrix: solutions
Sample preparation: 500 u,L Solution + 250 |xL buffer + 250 (JLL 20 mM 9-fluorenylmethyl
chloroformate in MeCN, mix, add 1 mL MeCN, inject a 50 |xL aliquot. (Prepare buffer by
adjusting the pH of 4% sodium bicarbonate to 10 with 10% NaOH.)
HPLCVARIABLES
Column: 125 X 4 5 |xm LiChrospher 100 RP 18
Mobile phase: Gradient. MeCN:water from 40:60 to 50:50 over 2.5 min, to 70:30 over 2.5 min,
to 100:0 over 5 min.
Flow rate: 1.5
CHROMATOGRAM
Retention time: 8.8
OTHER SUBSTANCES
KEYWORDS
derivatization
REFERENCE
Herraez-Hernndez,R.; Campins-Falco,P.; Sevillano-Cabeza,A. On-line derivatization into precolumns for the
in urine, Anal. Chem., 1996, 68, 734-739.
SAMPLE
Matrix: urine
HPLC VARIABLES
min.
Flow rate: 1
170 to 150 over 20 min. SIM, m/z 150
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Extracted: 6-acetylmorphine, amphetamine, benzoylecgonine, cocaine, ephedrine, methylephed-
rine, morphine, morphine-3-glucuronide, morphine-6-glucuronide
KEYWORDS
SPE
REFERENCE
Tatsuno,M.; Nishikawa,M.; Katagi,M.; Tsuchihashi,H. Simultaneous determination of illicit drugs in human
urine by liquid chromatography-mass spectrometry, J.Anal.Toxicol., 1996, 20, 281286.
SAMPLE
Matrix: urine
Sample preparation: 500 u,L Urine + N-ethylnordiazepam + chlorpheniramine + 100 jxL buffer,
centrifuge at 11000 g for 30 s, inject a 500 jxL aliquot onto column A with mobile phase A, after
|JLL mobile phase B, with 200 |JLL mobile phase C, and with 1.15 mL mobile phase D. Elute
HPLCVARIABLES
3.2 11 |jim Aminex A-28 (Bio-Rad); C 25 X 3.2 5 |jim C8 (Phenomenex) + 150 X 4.6 5 |xm silica
(Macherey-Nagel)
D MeCNrbuffer 33:67 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylammonium
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: methadone, imipramine, flurazepam, amitriptyline, morphine, codeine, hydromor-
phone, hydrocodone, caffeine, cotinine, benzoylecgonine, secobarbital, oxazepam, phenobarbi-
tal, nordiazepam, diazepam, phenylpropanolamine, phentermine, amphetamine, phenmetra-
zine, lidocaine, ephedrine
Interfering: pentazocine, desipramine, nortriptyline, diphenhydramine
KEYWORDS
column-switching
REFERENCE
341.
SAMPLE
Matrix: urine
Sample preparation: 500 |xL Urine + 100 fxL 25 jxg/mL N-n-propylaniline + 6 mL pH 10.0
carbonate buffer + 15 mL water, add mixture to an Extrelut SPE cartridge, let stand for 20
min, elute with 40 mL hexane:ethyl acetate 90:10. Add the eluate to 3 mL 100 mM sulfuric
acid and 500 mg NaCl, stir for 20 min, centrifuge at 1000 g for 5 min. Remove the lower layer
and add it to 3 mL 2.5 M NaOH and 20 |xL benzoyl chloride, stir vigorously for 30 min. Extract
the mixture with 1.5 mL chloroform. Wash the chloroform layer twice with 5 mL water and
evaporate it to dryness at 40, reconstitute the residue in 200 jxL hexane.isopropanol 90:10,
inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Chiralcel OB + 250 X 4.6 Chiralcel OJ
Mobile phase: Hexane:isopropanol 90:10
Flow rate: 1-1.4
Detector: UV 220
CHROMATOGRAM
Retention time: 13 (d), 15 (1)
Internal standard: N-n-propylaniline (11)
OTHER SUBSTANCES
KEY WORDS
rat; SPE; derivatization; chiral
REFERENCE
Nagai,T.; Kamiyama,S. Assay of the optical isomers of methamphetamine and amphetamine in rat urine using
high-performance liquid chromatography with chiral cellulose-based columns, J.Chromatogr., 1990, 525,
203-209.
SAMPLE
Matrix: urine
Sample preparation: 200-500 JJLL Rat urine + 200-500 |JLL pH 3.8 acetate buffer + 25 |JLL 40 (xg/
mL p-glucuronidase and 20 |xg/mL arylsulfatase (Merck), heat at 37 for 24 h, add 100 |xL 25
lig/mL 3-methoxytyramine in water, add 100 |xL water, adjust pH to 9.0 with 1.9 M sodium
carbonate, add to an Extrelut SPE cartridge, let stand for 20 min, elute with 6 mL ethyl acetate.
Add the eluate to 1 mL 100 mM sulfuric acid, extract. Add the aqueous layer to 3 mL 2.5 M
NaOH, add 25 JJLL benzoyl chloride, extract with 5 mL ethyl acetate. Wash the ethyl acetate
layer with water, evaporate to dryness under a stream of nitrogen at 40, reconstitute the
residue in 50 |xL EtOH, 3.5 mL 50 mM pH 8.0 Tris/HCl buffer, and 35 JJLL esterase (Type 1
porcine liver, Sigma). Heat at 25 for 45 min, add to an activated Sep-Pak C18 SPE cartridge,
wash with 5 mL water, elute with 5 mL acetone. Evaporate the eluate to dryness under a
stream of nitrogen at 40, reconstitute the residue in 200 (xL hexane:EtOH 89:11, inject a 20
(xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Chiralcel OB + 250 X 4.6 Chiralcel OJ
Mobile phase: n-Hexane:EtOH 89:11
Flow rate: 1.4
Detector: UV 220
CHROMATOGRAM
Retention time: 10 (D), 11.5 (L)
OTHER SUBSTANCES
Extracted: metabolites, amphetamine
KEYWORDS
rat; SPE; derivatization; chiral
REFERENCE
Nagai,T.; Kamiyama,S. Simultaneous HPLC analysis of optical isomers of methamphetamine and its metab-
olites, and stereoselective metabolism of racemic methamphetamine in rat urine, J.Anal.ToxicoL, 1991,15,
299-304.
SAMPLE
Matrix: urine
Sample preparation: Adjust pH of 3 mL urine to 11 with 10 M KOH, add to an Extrelut 3
column, let stand for 10 min, elute with 15 mL n-hexane into a tube containing one drop of 3
M HCl. Evaporate the eluate to dryness under a stream of nitrogen at 35. Add 1.5 mL 8%
sodium bicarbonate in water and 1 mL 0.5% sodium naphthoquinone-4-sulfonate in water to
the residue, heat at 70 for 20 min, cool, extract with 5 mL carbon tetrachloride. Evaporate
mobile phase, inject a 20 uL aliquot.
HPLC VARIABLES
Guard column: 4 x 4 5 u-m Lichrospher 100 RP8
Column: 250 X 4 5 fxm Lichrospher 100 RP8
Mobile phase: MeCN:buffer 55:45 (Buffer was 1.361 g KH2PO4 in 950 mL, add 1.3 mL metha-
nesulfonic acid, adjust pH to 3 with 5 M KOH, make up to 1 L with water.)
Flow rate: 1
Detector: UV 460
CHROMATOGRAM
Retention time: 7.8
OTHER SUBSTANCES
Noninterfering: acetaminophen, aspirin, amitriptyline, buprenorphine, caffeine, carbamaze-
pine, chlorpromazine, desipramine, dextromethorphan, doxepin, ephedrine, fenfiuramine, imip-
ramine, lidocaine, loxapine, meperidine, methadone, methaqualone, naloxone, naltrexone, nic-
otine, orphenadrine, oxycodone, papaverine, pentazocine, phendimetrazine, phenmetrazine,
phentermine, phenylpropanolamine, phenytoin, primidone, procaine, promethazine, propoxy-
phene, propyphenazone, theobromine, theophylline, trazodone, triflupromazine, trimethoprim,
trimipramine
KEYWORDS
SPE; derivatization
REFERENCE
Ferrara,S.D.; Tedeschi,L.; Frison,G.; Castagna,F. Solid-phase extraction and HPLC-UV confirmation of drugs
of abuse in urine, JAnal.ToxicoL, 1992, 16, 217-222.
SAMPLE
Matrix: urine
Sample preparation: Condition a 100 mg Adsorbex SCX cation-exchange SPE cartridge (Merck)
with 2 mL MeOH, 1 mL water, and 1 mL 17 mM KH2PO4, do not allow to dry. Centrifuge urine
at 2000 g for 5 min. 1 mL Urine + 500 |xL 50 mM KH2PO4, sonicate for 1 min, add to the SPE
cartridge, rinse vial with 50 JJLL 50 mM KH2PO4 and add to cartridge, dry cartridge for 1 min,
wash with three 500 |xL portions of 17 mM KH2PO4, wash with 1 mL MeOH, dry under vacuum
for 1 min, elute with four 500 |xL portions of MeOH:7.3% HCl (97.5:2.5) at a flow rate of 0.5
mL/min, inject a 10 JJLL aliquot.
HPLC VARIABLES
Column: 125 X 4 3 jjim Spherisorb ODS-I
Mobile phase: Gradient. A was water containing 5 mL (8.5 g) 85% orthophosphoric acid and 280
|xL (0.22 g) hexylamine per liter. B was MeCN containing 100 mL water, 5 mL (8.5 g) 85%
orthophosphoric acid, and 280 JJLL (0.22 g) hexylamine per liter. A:B 94.5:5.5 for 10.6 min, then
to 61:39 over 11 min.
Flow rate: 0.8
Detector: UV 198
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Extracted: 3,4-methylenedioxyamphetamine, amphetamine, 4-methoxyamphetamine, phenter-
mine, 3,4-methylenedioxymethamphetamine, 5-methoxy-3,4-methylenedioxyamphetamine,
3,4,5-trimethoxyamphetamine, 3,4-methylenedioxyethylamphetamine, 2,5-dimethoxyam-
phetamine, 4-bromo-2,5-dimethoxyphenylethylamine, 2,5-dimethoxy-4-methylamphetamine,
4-bromo-2,5-dimethoxyamphetamine, 2,5-dimethoxy-4-ethylamphetamine, mescaline, methox-
amine
KEYWORDS
SPE
REFERENCE
Helmlin,H.-J.; Brenneisen,R. Determination of psychotropic phenylalkylamine derivatives in biological matri-
ces by high-performance liquid chromatography with photodiode-array detection, J.Chromatogr., 1992,593,
87-94.
SAMPLE
Matrix: urine
Sample preparation: 5 mL Urine + 4.5 mL MeCN + 500 |xL 1 M KOH, centrifuge at 2500 rpm
for 10 min, filter (45 jxm) the supernatant. Inject on to column A at 180 |xL/min 25 JJLL 50 mM
KOH in MeCN:water 20:80, 50 JJLL filtrate, 25 JJLL 50 mM KOH in MeCN:water 20:80, and 200
JULL MeCN:water 20:80, backflush the contents of column A on to column B with mobile phase,
after 18 s remove column A from the circuit, elute column B with mobile phase, monitor the
effluent from column B. Wash column A with 400 jxL MeCN.
HPLC VARIABLES
Column: A 20 X 2 polymeric reagent; B 250 X 4.6 5 jxm Supelcosil LC-18-DB (with a guard
column) (Prepare polymeric reagent as follows. Prepare a porous rigid resin using a divinyl-
benzene:ethylstyrene:styrene 24:6:70 mixture with trimethylsilyl modified silica (102 A average
pore size, 1.08 mL/g pore volume, 366 m2/g surface area, 16-20 jutm irregular particle shape,
IMPAQ RG 1020 Si silica, PQ Co., Valley Forge PA). Further preparation details are not given
but a typical procedure given in the cited reference is as follows. Aerate a mixture of 10 g
modified silica in 100 mL water with nitrogen for 15 min, add 10 mL styrene:80% divinylben-
zene:t-butyl peroxybenzoate 49:49:2 (remove preservative by passing through a butylcatechol
remover (Scientific Polymer, Ontario NY), shake vigorously at room temperature for 4 h, add
150 mL 0.75% polyvinyl alcohol, shake for 4 h, heat at 120 for 24 h while shaking on a Parr
instrument, cool to room temperature, filter, wash with 100 mL water, wash with 50 mL MeOH.
Add the solid to 500 mL 3 M NaOH in MeOH:water 40:60, shake at room temperature for 14
h (to dissolve the silica), filter, wash with water until the washings are neutral, wash with 100
mL MeOH, dry at 60. The polymer has similar properties to the template silica (US Pat. 4
933 372 (1990)). Soxhlet extract the resin with dioxane for 8 h (Caution! Dioxane is a carcin-
ogen!). Add 25 g aluminum trichloride in 300 mL dry nitrobenzene to 50 g resin and 100 g 4-
chloro-3-nitrobenzoyl chloride, stir mechanically at 60 for 5 h, pour into a mixture of 150 mL
DMF, 100 mL concentrated HCl, and 150 g ice, filter. Wash the solid with 300 mL portions of
DMF:water 75:25 until the washings are colorless, wash with warm (60) DMF, wash with six
300 mL portions of dichloromethane:MeOH 2:1. Stir the product in 130 mL 40% benzyltri-
methylammonium hydroxide in water, 130 mL water, and 260 mL dioxane at 90 for 8 h, filter,
repeat the process. Wash the product with four portions of warm (60) dioxane. Stir the solid
with 30 mL acetic acid for 15 min, filter. Wash the solid with dioxane until the washings are
neutral, wash with six 300 mL portions of dichloromethane :MeOH 2:1 to give a nitrobenzo-
phenol-substituted polymer (J. Org. Chem. 1984, 49, 924). Heat 4 g 9-fluoreneacetic acid, 3.9
mL oxalyl chloride, 30 mL benzene (dried over anhydrous sodium sulfate, Caution! Benzene is
a carcinogen!), and 3 drops of triethylamine at 55 for 1 h, evaporate under reduced pressure
to remove oxalyl chloride, dissolve the product in 35 mL dichloromethane to give a 120 mg/mL
solution of 9-fluoreneacetyl chloride, dilute to obtain a 2 mM solution. Stir 1.3 g nitrobenzo-
phenol-substituted polymer, 4.2 mL 2 mM 9-fluoreneacetyl chloride solution, 300 |JLL triethyl-
amine, and 20 mL dichloromethane at room temperature for 1 h, filter, wash with three 20 mL
portions of MeCN to obtain the reagent, polymer-bound nitrobenzophenol 9-fluoreneacetate (J.
Chromatogr. 1992, 609, 103).)
Mobile phase: Gradient. MeCN:water 50:50 for 3.5 min, to 70:30 over 12 min, maintain at 70:
30 for 2.5 min, return to initial conditions over 1 min. (Place a 100 X 4.6 30-40 |jim silica
column before the injector.)
Column temperature: 60 (column A only)
Detector: F ex 254 em 305-395
CHROMATOGRAM
KEYWORDS
derivatization; column-switching
REFERENCE
Bourque,A.J.; Krull,I.S.; Feibush,B. Automated HPLC analyses of drugs of abuse via direct injection of biolog-
ical fluids followed by simultaneous solid-phase extraction and derivatization with fluorescence detection,
SAMPLE
Matrix: urine
Sample preparation: Condition a 100 mg Bond-Elut C18 SPE cartridge with 500 |xL MeOH
and 500 JJLL water. Adjust pH of urine to 10, centrifuge at 1500 g. 2 mL Supernatant + 100 |xL
75 |jLg/mL p-phenylethylamine hydrochloride in water, add to the SPE cartridge, wash with 2.5
mL water, elute with 2 mL MeOH, evaporate the eluate to dryness. Reconstitute in water, add
500 |xL 8% sodium bicarbonate, add 500 |xL 0.5% l,2-naphthoquinone-4-sulfonic acid sodium
salt, make up to 1.5 mL with water, heat at 70 for 20 min, cool, add an equal volume of
chloroform, shake for 2 min, centrifuge at 1500 g for 5 min. Remove the organic layer and dry
it over anhydrous sodium sulfate, filter (0.45 |xm), inject a 25 |xL aliquot of the filtrate.
HPLC VARIABLES
Column: 125 X 4 5 |xm LiChrospher Si-60
Mobile phase: EtOH:chloroform:ethyl acetate:n-hexane 1:22:32:45
Flow rate: 2
Detector: UV 280
CHROMATOGRAM
Retention time: 2.6
Internal standard: p-phenylethylamine hydrochloride (4.9)
OTHER SUBSTANCES
KEYWORDS
SPE; normal phase; derivatization
REFERENCE
Campins Falc6,R; Molins Legua,C; Herraez Hernandez,R-; Sevillano Cabeza,A. Improved amphetamine and
methamphetamine determination in urine by normal-phase high-performance liquid chromatography with
sodium 1,2-naphthoquinone 4-sulphonate as derivatizing agent and solid-phase extraction for sample clean-
up, J.Chromatogr.B, 1995, 663, 235-245.
SAMPLE
Matrix: urine
Sample preparation: Condition a Bond Elut C18 SPE cartridge with 1 mL MeOH and 1 mL 50
mM pH 11 phosphate buffer. 500 |xL Urine + 500 |JLL 2000 U/mL p-glucuronidase with sulfatase
activity (Type H-I, Sigma) in 100 mM pH 5 acetate buffer, heat at 37 overnight, add 500 mg
NaCl, add 500 jxL 50 mM pH 11 potassium phosphate buffer, adjust pH to 11 with ammonium
hydroxide, mix. Add the mixture to the SPE cartridge, wash with 1 mL 50 mM pH 11 potassium
phosphate buffer, wash with 1 mL freshly prepared MeOH:water 30:70, wash with 1 mL MeCN,
elute with 1 mL freshly prepared MeCN:acetic acid 98:2, elute with 1 mL MeCN:HCl 98:2.
Combine the eluates and evaporate them to dryness under a stream of air at room temperature,
reconstitute the residue in mobile phase (?), inject a 10 fxL aliquot.
HPLC VARIABLES
Guard column: phenyl
Column: Microsorb phenyl
Mobile phase: MeCN:MeOH:50 mM pH 3 potassium phosphate 5:10:85
Flow rate: 1
Detector: UV 215
CHROMATOGRAM
Internal standard: methamphetamine
OTHER SUBSTANCES
KEYWORDS
SPE; rat; methamphetamine in IS
REFERENCE
Law,M.Y.L.; Moody,D.E. Simultaneous quantitation of amphetamine and 4'-hydroxyamphetamine by high per-
formance liquid chromatography, J.Liq.Chromatogr., 1995, 18, 2029-2043.
SAMPLE
Matrix: urine
Sample preparation: Condition an Extra-Sep C18 SPE cartridge (Teknokroma) with 1 mL
MeOH and 1 mL buffer. Adjust pH of 2 mL urine to ca. 10 with 100 jxL concentrated ammonium
hydroxide, add 5 \xg p-phenylethylamine, add to the SPE cartridge, wash with 5 mL water,
wash with 1 mL MeCN, elute with 2 mL MeOH. Add 100 |xL EtOHxoncentrated HCl 6:1 to
the eluate, evaporate to dryness. Reconstitute with 1 mL buffer and 1 mL 0.5% 1,2-naphtho-
quinone-4-sulfonic acid sodium salt, let stand at room temperature for 10 min, add 2 mL n-
hexane.ethyl acetate 50:50, shake for 2 min, centrifuge at 1500 g for 5 min. Remove the organic
layer and evaporate it to dryness, reconstitute the residue in 500 |xL MeCN:water 50:50, inject
a 50 |JLL aliquot. (Buffer was 1% aqueous sodium bicarbonate adjusted to pH 10 with 5 M
NaOH.)
HPLC VARIABLES
Column: 250 X 4 5 |xm Hypersil ODS-C18
Mobile phase: Gradient. MeCN:0.5% propylamine in water from 40:60 to 50:50 over 2.5 min, to
70:30 over 1 min, maintain at 70:30.
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 6.3
Internal standard: p-phenylethylamine (4.1)
OTHER SUBSTANCES
KEYWORDS
SPE; derivatization
REFERENCE
Molins Legua,C; Campins Falc6,R; Sevillano Cabeza,A. Amphetamine and methamphetamine determination
in urine by reversed-phase high-performance liquid chromatography with sodium 1,2-naphthoquinone 4-
sulfonate as derivatizing agent and solid-phase extraction for sample clean-up, J.Chromatogr.B, 1995, 672,
81-88.
SAMPLE
Matrix: urine
Sample preparation: 100-300 |xL Urine + 100 JJLL 1.5 M NaOH + 5 |ig IS, make up to 1 mL
with water, add to an Extrelut 1 SPE cartridge, let stand for 20 min, elute with 6 mL benzene
(Caution! Benzene is a carcinogen!). Add the eluate to 1 mL 100 mM sulfuric acid, extract.
Remove the aqueous layer and add it to 3 mL 1.5 M NaOH, add 5 JLJLL benzoyl chloride, vortex
vigorously, extract twice with 3 mL portions of n-hexane. Combine the organic layers and wash
them twice with 3 mL portions of water, evaporate the organic to dryness under a stream of
nitrogen at 40, reconstitute the residue in 200 |xL mobile phase, inject a 20 jxL aliquot.
HPLCVARIABLES
Column: Chiralcel OB-H
Mobile phase: n-Hexane:isopropanol 90:10
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 6.5 (D), 10 (L)
Internal standard: 1-p-methoxyamphetamine (12)
OTHER SUBSTANCES
Extracted: amphetamine, ethylamphetamine
KEY WORDS
rat; derivatization; SPE; chiral
REFERENCE
Nagai,T.; Kamiyama,S.; Matsushima,K. Analysis of time-lapse changes of d- and 1-enantiomers of racemic
ethylamphetamine and stereoselective metabolism in rat urine by HPLC determination, JAnal.Toxicol.,
1995, 19, 225-228.
SAMPLE
Matrix: urine
Sample preparation: Dilute urine 20-fold or more. 500 JJLL Diluted urine + 50 |xL 500 ng/mL
(+)-2,5-dimethoxyamphetamine hydrochloride + 100 |xL 500 mM NaOH + 2 mL benzene (Cau-
tion! Benzene is a carcinogen!), shake for 15 min, centrifuge at 1200 g for 5 min. Remove 1.8
mL of the organic phase and add it to 220 JJLL 50 mM HCl, shake for 15 min, centrifuge at
1200 g for 5 min. Remove 200 |xL of the aqueous phase and add it to 40 |JLL 250 mM NaOH,
add 50 IJLL 330 mM pH 7.8 phosphate buffer, add 250 |xL MeCN, add 25 |JLL 3 mM (-)-l-(9-
fluorenyl)ethyl chloroformate, let stand at room temperature for 24 h, add 30 |xL 100 mM
glycine in water, let stand for 30 min, add 750 |xL pentane, vortex for 2 min, centrifuge at 1200
g for 5 min. Remove the organic layer and evaporate it to dryness in a centrifugal evaporator
at room temperature, reconstitute the residue in 300 |xL MeCN:water 50:50, inject a 100 jxL
aliuot.
HPLC VARIABLES
Mobile phase: MeCN:THF:20 mM pH 3.6 sodium acetate buffer 25:21:54
Flow rate: 1
CHROMATOGRAM
Retention time: 42 (L), 44 (D)
Internal standard: (+)-2,5-dimethoxyamphetamine (33)
OTHER SUBSTANCES
KEYWORDS
rat; derivatization; chiral
REFERENCE
Sukbuntherng,J.; Hutchaleelaha,A.; Chow,H.-H.; Mayersohn,M. Separation and quantitation of the enantio-
mers of methamphetamine and its metabolites in urine by HPLC: Precolumn derivatization and fluores-
cence detection, JAnal.Toxicol., 1995, 19, 139-147.
SAMPLE
Matrix: urine
1 mL 1% pH 10 sodium bicarbonate buffer. 2 mL Urine + 400 jxL 8% pH 10 sodium bicarbonate
buffer, mix, centrifuge at 1500 g for 2 min, add a 2 mL aliquot of the supernatant to the SPE
cartridge, wash with 3 mL water, pass 500 JJLL 2% sodium 1,2-naphthoquinone 4-sulfonate
through the cartridge, pass 500 |xL 1% pH 10 sodium bicarbonate buffer through the cartridge,
let stand at room temperature for 15 min, wash with 3 mL water, elute with 1 mL MeCN:
water 50:50, inject a 20 |JLL aliquot of the eluate.
HPLC VARIABLES
Column: 250 X 4 5 |jim Hypersil ODS
Mobile phase: Gradient. MeCNibuffer from 40:60 to 50:50 over 2.5 min, to 70:30 over 0.5 min,
maintain at 70:30 for 1.5 min, to 85:15 over 1 min, maintain at 85:15 for 1.5 min. (Buffer was
5 mL/L propylamine in water.)
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 5.6
Internal standard: p-phenylethylamine (3.6)
OTHER SUBSTANCES
KEYWORDS
derivatization; SPE
REFERENCE
Campins-Falc6,R; Sevillano-Cabeza,A.; Molins-Legua,C; Kohlmann,M. Amphetamine and methamphetamine
determination in urine by reversed-phase high-performance liquid chromatography with simultaneous sam-
ple clean-up and derivatization with 1,2-naphthoquinone 4-sulphonate on solid-phase cartridges,
J.Chromatogr.B, 1996, 687, 239-246.
SAMPLE
Matrix: urine
Sample preparation: Inject 15 (JLL urine, inject a mixture of 5 JJLL 20 mM 9-fluorenylmethyl
chloroformate in MeCN and 45 |xL water, and inject 10 |xL buffer on to column A and elute to
waste with mobile phase A. After 2.8 min backflush the contents of column A on to column B
with mobile phase B and start the gradient, monitor the effluent from column B. At the end
of the run condition column A with 1 mL mobile phase A. (Buffer was 4% sodium bicarbonate
HPLC VARIABLES
Column: A 20 X 2.1 30 |xm Hypersil ODS-C18; B 125 X 4 5 |xm LiChrospher 100 PR-C18
Mobile phase: A water; B Gradient. MeCN:water from 40:60 to 70:30 over 15 min. to 100:0 over
5 min.
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: amphetamine, ephedrine, norephedrine, 3-phenylpropylamine, pseudoephedrine
KEYWORDS
REFERENCE
Herraez-Hernandez,R; Campins-Falc6,P; Sevillano-Cabeza,A. Determination of amphetamine and related
compounds in urine using on-line derivatization in octadecyl silica columns with 9-fluorenylmethyl chlo-
roformate and liquid chromatography, J.Chromatogr.B, 1996, 679, 69-78.
SAMPLE
Matrix: urine
Sample preparation: Inject 50 JJLL urine on to column A and elute to waste with mobile phase
A, after 2 min inject a mixture of 25 /JLL 0.5% sodium l,2-naphthoquinone-4-sulfonate in water
and 25 (JLL buffer on to column A, stop the flow of mobile phase A, after 10 min start pump A,
after 5 min backflush the contents of column A on to column B with mobile phase B and start
the gradient, monitor the effluent from column B. After each run flush column A with ethyl
acetate for 1 min, n-hexane for 1 min, and ethyl acetate for 1 min, re-equilibrate with mobile
phase A. (Buffer was 4% sodium bicarbonate adjusted to pH 10 with 10% NaOH.)
HPLC VARIABLES
Column: A 20 X 2.1 30 |xm Hypersil ODS-C18; B 250 X 4 5 [xm Hypersil ODS C18
Mobile phase: A water; B Gradient. MeCN:0.5% propylamine hydrochloride in water from 40:
60 to 50:50 over 2.5 min, to 70:30 over 1 min, maintain at 70:30 for 4.5 min.
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Herraez-Hernandez,R.; Campins-Falc6,R; Sevillano-Cabeza,A. On-line derivatization into precolumns for the
in urine, Anal.Chem., 1996, 68, 734-739.
SAMPLE
Matrix: urine
Sample preparation: Condition a Bond Elut SCX SPE cartridge with 10 mL MeOHiaqueous
ammonia 98:2, 10 mL MeOH, and 30 mL water. 10 mL Urine + 10 mL 50 mM pH 7.8 potassium
phosphate buffer, mix, add to the SPE cartridge, wash with 2 mL water, wash with 10 mL
MeOH, elute with 3 mL MeOH:2% aqueous ammonia 98:2. Add 30 |JLL glacial acetic acid and
30 JJLL 1 mg/mL N-ethylaniline in MeOH to the eluate, inject a 5-50 |xL aliquot.
HPLC VARIABLES
Column: 150 X 6 ULTRON ES-PhCD p-cyclodextrin phenylcarbamate-bonded silica (Shinwa
Chemical Industries, Kyoto)
Mobile phase: MeCN:MeOH:buffer 10:30:60 (Buffer was 50 mM pH 6.0 potassium phosphate for
UV detection or 100 mM pH 6.0 ammonium acetate for MS detection.)
Flow rate: 1
Detector: UV 220, MS, Shimadzu LCMS-QP1100EX, thermospray, positive ion mode, filament
off, vaporizer 230, ion source 300, m/z 150
CHROMATOGRAM
Retention time: 12 (D, UV), 15 (L, UV), 15 (D, MS), 17.5 (L, MS)
Internal standard: N-ethylaniline (17, UV only)
Limit of detection: 50 ng/mL (D, UV detection), 100 ng/mL (L, UV detection), 10 ng/mL (D,
scan MS), 20 ng/mL (L, scan MS), 0.8 ng/mL (D, SIM MS), 1.0 ng/mL (L, SIM MS)
OTHER SUBSTANCES
Extracted: metabolites, amphetamine, p-hydroxymethamphetamine
KEYWORDS
SPE; chiral
REFERENCE
Katagi,M.; Nishioka,H.; Nakajima,K.; Tsuchihashi,H.; Fujima,H.; Wada,H.; Nakamura,K; Makino,K. Direct
high-performance liquid chromatographic and high-performance liquid chromatographic-thermospray-mass
spectrometric determination of enantiomers of methamphetamine and its main metabolites amphetamine
and p-hydroxymethamphetamine in human urine, J.Chromatogr.B, 1996, 676, 3543.
SAMPLE
Matrix: urine
Sample preparation: 2 mL Urine + 20 u,L 100 JXM 1-phenylethylamine in water + 400 |xL
concentrated HCl, heat at 80 for 1 h, cool, neutralize with 600 iiL 25% ammonia, add 5 mL
10% sodium carbonate solution, add 2 mL 500 mM pH 10.5 sodium borate buffer, add 2 mL
chloroform:isopropanol 75:25, vortex for 1 min, centrifuge at 12.5 at 1500 g for 10 min, repeat
the extraction. Combine the organic layers and remove a 100 jxL aliquot, add 10 uL acetic acid
to the aliquot, evaporate it to dryness under a stream of nitrogen at room temperature, recon-
stitute the residue in 50 (JLL carbonate buffer, add 50 u,L 10 mM fluorescein-4-isothiocyanate
in EtOH, mix, heat in the dark at 80 for 15 min, inject a 20 u,L aliquot. (Prepare carbonate
buffer by adjusting the pH of 200 mM sodium bicarbonate to 9.0 with 200 mM sodium
carbonate.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Daisopak SP-120-5-ODS (Daiso, Osaka)
Mobile phase: Gradient. MeCN:20 mM pH 7.9 sodium phosphate buffer 20:80 for 16 min then
24:76 (step-gradient).
Flow rate: 0.8
CHROMATOGRAM
Internal standard: 1-phenylethylamine (26.6)
OTHER SUBSTANCES
Extracted: metabolites, amphetamine, norepinephrine
KEYWORDS
derivatization
REFERENCE
Al-Dirbashi,O.; Kuroda,N.; Akiyama,S.; Nakashima,K. High-performance liquid chromatography of metham-
phetamine and its related compounds in human urine following derivatization with fluorescein isothio-
cyanate, J.Chromatogr.B, 1997, 695, 251-258.
Methapyrilene
CAS Registry No.: 91-80-5,33032-12-1 (fumarate), 135-23-9 (HCI)
Merck Index: 6027
Lednicer No.: 1 54
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 (JLL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 4.6
OTHER SUBSTANCES
mepyramine, mesoridazine, metaraminol, methadone, methamphetamine, methdilazene,
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
phesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methaqualone,
methazolamide, methocarbamol, methoxamine, methsnximide, methyl salicylate, methyldopa,
REFERENCE
18, 233-242.
Methaqualone
Merck Index: 6028
LednicerNo.: 1 353
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 226
CHROMATOGRAM
KEYWORDS
REFERENCE
Traequi,A.; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
Next Page

REFERENCE
Hill,D.W.; Kind ,A. J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnaLToxicol., 1994,
18, 233-242.
Methazolamide
Merck Index: 6031
Lednicer No.: 1 250
SAMPLE
Sample preparation: To 500 (xL whole blood, plasma, or urine add 20 |xL 1 mg/mL acetazolam-
ide solution and 2.5 mL 50% ammonium sulfamate, vortex for 30 s. (Place the tube containing
whole blood in boiling water for 30 s and then quickly in cold water.) Add 5 mL ethyl acetate,
vortex, centrifuge at 3000 g for 10 min, transfer the organic layer to 5 mL pH 8.0 phosphate
buffer, vortex, centrifuge at 3000 g for 10 min, transfer the organic layer to 500 JJLL pH 10.0
glycine buffer, vortex for 30 s, centrifuge at 3000 g for 5 min. Aspirate and discard the organic
layer, add 500 |xL ether to the remaining glycine buffer layer, vortex for 1 min, discard the
ether phase. Vent the aqueous layer for about 30 min and inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Altima C18 (Alltech)
Flow rate: 1
Detector: UV 285
CHROMATOGRAM
Internal standard: acetazolamide (4.55)
KEYWORDS
plasma; whole blood
Previous Page

REFERENCE
Hill,D.W.; Kind ,A. J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnaLToxicol., 1994,
18, 233-242.
Methazolamide
Merck Index: 6031
Lednicer No.: 1 250
SAMPLE
Sample preparation: To 500 (xL whole blood, plasma, or urine add 20 |xL 1 mg/mL acetazolam-
ide solution and 2.5 mL 50% ammonium sulfamate, vortex for 30 s. (Place the tube containing
whole blood in boiling water for 30 s and then quickly in cold water.) Add 5 mL ethyl acetate,
vortex, centrifuge at 3000 g for 10 min, transfer the organic layer to 5 mL pH 8.0 phosphate
buffer, vortex, centrifuge at 3000 g for 10 min, transfer the organic layer to 500 JJLL pH 10.0
glycine buffer, vortex for 30 s, centrifuge at 3000 g for 5 min. Aspirate and discard the organic
layer, add 500 |xL ether to the remaining glycine buffer layer, vortex for 1 min, discard the
ether phase. Vent the aqueous layer for about 30 min and inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Altima C18 (Alltech)
Flow rate: 1
Detector: UV 285
CHROMATOGRAM
Internal standard: acetazolamide (4.55)
KEYWORDS
plasma; whole blood
REFERENCE
Iyer,G.R.; Taft,D.R. Determination of methazolamide concentrations in human biological fluids using high per-
formance liquid chromatography, J.Pharm.Biomed.Anal., 1998, 16, 1021-1027.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4 RP-18 (Merck)
Flow rate: 0.7
Detector: UV 300
OTHER SUBSTANCES
Simultaneous: glutathione conjugate
REFERENCE
Kishida,K; Akaki,Y.; Sasabe,T.; Yamamoto,C; Manabe,R. Glutathione conjugation of methazolamide and sub-
sequent reactions in the ciliary body in vitro, J.Pharm.ScL, 1990, 79, 638-642.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
methapyrilene, methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa,
REFERENCE
18, 233-242.
Methdilazine
Merck Index: 6034
Lednicer No.: 1 387
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 \Lg/mL solution in MeOH, inject a 20 JJLL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 6.1
OTHER SUBSTANCES
none, diprenorphine, dip3rridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine,
prolintane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol,
prothipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine,
quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyl-
diamine, theophylline, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothix-
ene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazodone, tri-
fluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim,
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
oxylate, dipyridamole, disopâmide, dobutamine, doxapram, doxepin, droperidol, encainide,
dol, mefenamic acid, meperidine, mephen5rtoin, mepivacaine, mesoridazine, metaproterenol,
metformin, methadone, methocarbamol, methotrexate, methotrimeprazine, methoxamine,
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Methicillin sodium
Molecular formula: C17H19N2NaO6S
CAS Registry No.: 132-92-3, 7246-14-2 (monohydrate), 61-32-5 (free acid)
Merck Index: 6047
LednicerNo.: 1 412
SAMPLE
Matrix: blood
Sample preparation: 400 uL Serum + 400 |xL MeCN, vortex for 10 s, shake slowly for 15 min,
centrifuge at 3000 g for 10 min. Remove the supernatant and add it to 4 mL dichloromethane,
vortex for 10 s, shake for 15 min, centrifuge at 3000 g for 10 min, inject a 50 jxL aliquot of the
upper aqueous layer.
HPLCVARIABLES
Mobile phase: MeCN:water:200 mM ammonium acetate 28:62:10, pH 5.6
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 4.0
OTHER SUBSTANCES
Extracted: cloxacillin, dicloxacillin, nafcillin, oxacillin
Noninterfering: amdinocillin (mecillinam), amikacin, amoxicillin, ampicillin, carbenicillin,
cefamandole, cefazolin, ceforanide, cefatoxamine, cefoxitin, cephalexin, cephaloridine, cephal-
othin, cephradine, cepharin, chloramphenicol, clindamycin, co-trimoxazole, fluorocytosine, gen-
tamicin, metronidazole, moxalactam, penicillin, piperacillin, sulfamethoxazole, theophylline,
ticarcillin, tobramycin, trimethoprim, vancomycin
KEYWORDS
serum
REFERENCE
Rudrik,J.T.; Bawdon,R.E. Determination of penicillinase-resistant penicillins in serum using high-pressure
liquid chromatography, J.Liq.Chromatogr., 1981, 4, 15251545.
SAMPLE
Matrix: blood
Sample preparation: 500 jxL Serum + 2 mL MeCN, vortex for 1 min, centrifuge at 3000 g for
10 min. Remove the supernatant and add it to 5 mL dichloromethane, vortex, centrifuge at
3000 g for 10 min, inject a 15 |xL aliquot of the aqueous phase.
HPLCVARIABLES
Column: 300 mm long (xBondapak C18
Flow rate: 2.5
Detector: UV 229
CHROMATOGRAM
Internal standard: methicillin
OTHER SUBSTANCES
Extracted: piperacillin, mezlocillin
KEYWORDS
serum; pharmacokinetics; methicillin is IS
REFERENCE
Martens,M.G.; Faro,S.; Feldman,S.; Cotton,D.B.; Dorman,K; Riddle,G.D. Pharmacokinetics of the acyclure-
idopenicillins piperacillin and mezlocillin in the postpartum patient, Antimicrob.Agents Chemother., 1987,
31, 2015-2017.
SAMPLE
Matrix: blood
Sample preparation: Condition a 55 X 5 100-200 mesh AG 50W-X8 (H+) column (Bio-Rad) with
10 mL MeCNrwater 50:50. 600 ^L Serum + 600 |xL MeCN, vortex for 1 min, centrifuge at 2000
g for 5 min, add a 1 mL aliquot of the supernatant to the column, discard the first 200 |xL
effluent, collect the rest of the effluent. Remove a 450 |xL aliquot and add it to 50 JXL 10%
sodium carbonate solution, heat at 60 for 1 h (to hydrolyse the (5-lactam ring), cool in an ice
bath. Remove a 100 |JLL aliquot and add it to 15 |xL 200 mM pH 6.0 phosphate buffer, add 35
JXL 80 mM 7-fluoro-4-nitrobenzo-2-oxa-l,3-diazole in MeCN, heat at 60 for 10 min, cool in an
ice bath, add 30 |xL 1 M HCl, inject a 5-10 |iL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 ODS-80TM (Tosoh)
Flow rate: 1
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Extracted: penicillin G, piperacillin
KEYWORDS
REFERENCE
Iwaki,K; Okumura,N.; Yamazaki,M.; Nimura,N.; Kinoshita,T. Precolumn derivatization technique for high-
performance liquid ehromatographie determination of penicillins withfluorescencedetection, J.Chromatogr.,
1990, 504, 359-367.
SAMPLE
Sample preparation: Adjust pH of fermentation broth to 7, centrifuge at 8000 g for 10 min,
add MeCN, centrifuge, add dichloromethane to the supernatant, vortex for 10 s, shake for 15
min, centrifuge at 8000 g for 15 min. Add 1 mL of the aqueous layer to 100 |xL reagent, heat
at 50 for 50 min, cool in an ice bath, inject a 20 |xL aliquot. (Prepare reagent by dissolving
4.125 g imidazole in 2.5 mL water, add 1 mL HCl, add 500 |xL 110 mM mercury(II) chloride,
add 1.5 mL HCl. Recrystallize imidazole twice from isopropanol.)
HPLCVARIABLES
Guard column: 10 X 4 5 |xm Spherisorb C18
Column: 20 X 4.6 5 |xm Spherisorb C18 S5ODS2
Mobile phase: Gradient. MeCNibuffer from 16.5:83.5 to 31.5:68.5 over 17 min (Buffer was 10
mM NaH2PO4 containing 10 mM EDTA, adjusted to pH 6.5 with 2 M NaOH.)
Flow rate: 2
Detector: UV 325
CHROMATOGRAM
Retention time: 12
OTHER SUBSTANCES
Extracted: penicillin G, penicillin V, penicillin X
KEYWORDS
derivatization
REFERENCE
Rogers,M.E.; Adlard,M.W.; Saunders,G.; Holt,G. High-performance liquid chromatographic determination of
penicillins following derivatization to mercury-stabilized penicillenic acids, J.Liq.Chromatogr., 1983, 6,
2019-2031.
SAMPLE
Sample preparation: Blend tablets and capsules with water in a high-speed blender for 5 min,
filter, dilute with mobile phase, inject a 20 |xL aliquot. Dilute oral suspensions and injections
with mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 70 mm long Co:Pell ODS
Column: 300 X 4.6 10 |xm Chromegabond C18 (E.S. Industries)
Mobile phase: MeCN:MeOH:10 mM KH2PO4 19:11:70
Flow rate: 1
Detector: UV 225
CHROMATOGRAM
Retention time: 5.2
OTHER SUBSTANCES
Simultaneous: amoxicillin, ampicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G,
penicillin V
KEYWORDS
tablets; capsules; oral suspensions; injections
REFERENCE
Briguglio,G.T.; Lau-Cam,C.A. Separation and identification of nine penicillins by reverse phase liquid chro-
matography, JAssoc.Off.Anal.Chem., 1984, 67, 228-231.
SAMPLE
Matrix: milk
Sample preparation: 50 g Milk + 2 drops penicillinase (Difco Laboratories), let stand 1 h at
37, add 50 MeCN, shake vigorously for 1 min, centrifuge at 9000 g for 10 min, decant, add 5
g NaCl, swirl to dissolve, add 100 mL dichloromethane, shake for 1 min, centrifuge at 1000 g
for 10 min. Remove top aqueous layer and extract organic layer with 25 mL 10% NaCl by
shaking and centrifuging as before. Combine aqueous layers, add 1 mL 0.3% mercuric chloride
in water, let stand 30 min, add 1 mL 2 M HCl, extract with three 50 mL portions of dichlo-
romethane by shaking each portion for 1 min and centrifuging at 1000 g for 10 min, filter
dichloromethane extracts through 30 g anhydrous sodium sulfate, evaporate to dryness under
reduced pressure at 35, if water remains add 5-10 mL MeOH to flask and complete evapora-
tion. Dissolve residue in 1 mL 10% acetic acid, add 0.5 mL 0.08% dansyl hydrazine in 10%
acetic acid, let stand 90 min to overnight in the dark, transfer reaction mixture to a separatory
funnel with three 25 mL portions of dichloromethane, add 5 mL 2 M HCl, shake for 1 min,
wash organic layer with 5 mL 5% NaHCO3 solution, filter through 10-20 g anhydrous sodium
sulfate. Extract acid aqueous layer again with 25 mL dichloromethane. Combine dichloro-
methane layers and evaporate to dryness at 35 under reduced pressure. Dissolve residue in 2
mL IS solution, inject a 20 |xL aliquot. (Prepare IS solution by dissolving 10 |xL benzaldehyde
in 100 mL dichloromethane, evaporate 1 mL to dryness under reduced pressure, dissolve res-
idue in 1 mL 10% acetic acid, add 0.5 mL 0.08% dansyl hydrazine in 10% acetic acid, let stand
90 min to overnight in the dark, transfer reaction mixture to a separatory funnel with three
25 mL portions of dichloromethane, add 5 mL 2 M HCl, shake for 1 min, wash organic layer
with 5 mL 5% NaHCO3 solution, filter through 10-20 g anhydrous sodium sulfate. Extract acid
aqueous layer again with 25 mL dichloromethane. Combine dichloromethane layers and evap-
orate to dryness at 35 under reduced pressure. Dissolve residue in 100 mL MeCN then dilute
an aliquot 1:4 with MeCN.)
HPLC VARIABLES
Flow rate: 1
Detector: F ex 254 em 500 filter
CHROMATOGRAM
Internal standard: benzaldehyde (derivatized) (12.18)
OTHER SUBSTANCES
Extracted: penicillin V, phenethicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin
Interfering: penicillin G
KEYWORDS
derivatization
REFERENCE
Munns,R.K; Shimoda,W.; Roybal,J.E.; Vieira,C. Multiresidue method for determination of eight neutral |3-
lactam penicillins in milk byfluorescence-liquidchromatography, J.Assoc.Off.Anal.Chem., 1985, 68, 968-
971.
SAMPLE
Matrix: milk
Sample preparation: Add 2 volumes MeCN to milk, stand 5 min, decant aqueous portion, suc-
tion filter, extract with an equal volume of 1:1 methylene chloride:hexane, centrifuge aqueous
phase at 3000 rpm for 10 min. Dilute 3:1 with 20 mM sodium acetate buffer and filter (0.2 |xm
nylon). Inject 50 |xL onto column with mobile phase A, run mobile phase A for 30 min and elute
to waste. After 30 min switch to mobile phase B and elute through detector.
HPLCVARIABLES
Column: 100 X 8 Radial-Pak 10 fxm ixBondapak C18
Mobile phase: A 20 mM sodium acetate buffer; B Gradient. MeCN:MeOH:20 mM sodium acetate
buffer from 15:10:75 to 30:0:70 over 15 min and hold at 30:0:70
Flow rate: A 3; B 2
Detector: E5 Waters 464 pulsed electrochemical detector using a thin layer cell with a Ag/AgCl
reference electrode. El = 1300 mV for 0.166 s, E2 = 1500 mV for 0.166 s, E3 = -200 mV for
0.333 s.
CHROMATOGRAM
Retention time: 7.2
OTHER SUBSTANCES
Simultaneous: penicillin V, ampicillin, penicillin G, oxacillin, cloxacillin, nafcillin, dicloxacillin.
REFERENCE
Kirchmann,E.; Earley,R.L.; Welch,L.E. The electrochemical detection of penicillins in milk, J.Liq.Chromatogr.,
1994, 17, 1755-1772.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 |xL aliquot of an aqueous solution.
HPLC VARIABLES
Column: 250 X 4.6 10 jjim jjiBondapak C18
Mobile phase: Gradient. A was 1.2 mM triethylamine and 42 mM acetic acid. B was MeCN:
water 24:76 containing 1.2 mM triethylamine and 42 mM acetic acid. A:B from 75:25 to 60:40
using Waters Model 660 curve select-9 over 15 min then stay at 60:40.
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 22
OTHER SUBSTANCES
Simultaneous: ampicillin, cefoperazone
Noninterfering: gentamicin, kanamycin, tobramycin
Interfering: penicillin G
REFERENCE
Dokladalova,J.; Quercia,G.T.; Stankewich,J.P. High-performance liquid chromatographic determination of
cefoperazone in human serum and urine, J.Chromatogr., 1983, 276, 129-137.
SAMPLE
Matrix: solutions
Sample preparation: Prepare an aqueous solution, inject a 200 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 4 |xm Micropak SPC-18 C18
Mobile phase: Gradient. MeCN: 10 mM orthophosphoric acid from 15:85 to 60:40 over 20 min
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Simultaneous: dicloxacillin, penicillin G, penicillin V, cloxacillin, nafcillin, carbenicillin
REFERENCE
Moats, W. A. Effect of the silica support of bonded reversed-phase columns on chromatography of some antibiotic
SAMPLE
Matrix: solutions
Sample preparation: React the antibiotic, triethylamine, and l-(2,5-dihydroxyphenyl)-2-brom-
oethanone in a 1:2:4 molar ratio in DMF at 45 for 2 h (use dibenzo-18-crown-6 to make the
sodium salt soluble), inject a 10 JJLL aliquot. (Preparation of l-(2,5-dihydroxyphenyl)-2-bro-
moethanone is as follows. Stir 27.6 g 1,4-dimethoxybenzene and 28 mL bromoacetyl bromide
at 0, add 53.4 g aluminum bromide over 10 min (an exothermic reactions ensues), let stand
at room temperature for 12 h, add 100 mL 48% HBr, add 100 g ice, stir for 1 h, extract twice
with 200 mL portions of diethyl ether. Combine the extracts and wash them 3 times with 200
mL portions of water, dry over 40 g anhydrous magnesium sulfate, evaporate to dryness, re-
crystallize the product 3 times from EtOH to yield l-(2,5-dihydroxyphenyl)-2- bromoethanone
monobromoacetate (mp 105-107). Dissolve H g l-(2,5-dihydroxyphenyl)-2-bromoethanone
monobromoacetate in 200 mL warm dry MeOH saturated with HBr, stir for 18 h, add 200 mL
water, cool to -10. Collect the yellow solid and dry it under vacuum at 50 for 48 h, recrystallize
from toluene:heptane 50:50 then toluene to obtain l-(2,5-dihydroxyphenyl)-2-bromoethanone
as yellow needles (mp 117-119).)
HPLC VARIABLES
Mobile phase: MeOH.100 mM pH 6.5 sodium acetate 58:42
Flow rate: 1
electrode
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: carbenicillin, cephapirin, cloxacillin, dicloxacillin, hetacillin, nafcillin, oxacillin, pen-
icillin G
KEYWORDS
derivatization
REFERENCE
Munns,R.K; Roybal,J.E.; Shimoda,W.; Hurlbut,J.A. l-(4-Hydroxyphenyl)-, l-(2,4-dihydroxyphenyl)- and l-(2,5-
SAMPLE
Matrix: solutions
Sample preparation: Separate buffer containing drug from human serum albumin by centri-
fuging at 37 at 700 g for 3 min using a Micropartition System MPS-I (Amicon) unit, inject a
10-20 |xL aliquot of the ultrafiltrate.
HPLC VARIABLES
Guard column: C18/Corasil (Waters)
Column: 300 X 3.9 u-Bondapak C18
Flow rate: 1.5
Detector: UV 220
OTHER SUBSTANCES
Also analyzed: penicillin G, cefoperazone, cephalothin
REFERENCE
Terasaki,T.; Nouda,H.; Tsuji,A. Relationship between lipophilicity and binding affinity with human serum al-
bumin for penicillin and cephem antibiotics, J.Pharmacobiodyn., 1992, 15, 99-106.
Methimazole
Merck Index: 6049
Lednicer No.: 1 240
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 4 mL buffer + 200 (xL 100 mg/mL 2,6-dichloroquinone-4-
chloroimide in MeOH, vortex for 1 min, let stand at room temperature for 5 min, add 2 mL
chloroform, vortex for 5 min, centrifuge at 1000 g for 10 min, filter (Whatman IPS phase-
separating paper) the lower organic layer, evaporate the filtrate to dryness, reconstitute with
50 |xL chloroform, inject a 20 |xL aliquot. (Prepare buffer by dissolving 3.1g boric acid, 3.75 g
KCl, and 100 g NaCl in water, add 40 mL 100 mM NaOH, make up to 1 L with water, adjust
pH to 8 with 6 M NaOH.)
HPLC VARIABLES
Column: 300 X 3.9 |xBondapak-NH2
Mobile phase: chloroform
Flow rate: 1.5
Detector: UV 405
CHROMATOGRAM
Retention time: 5
KEYWORDS
REFERENCE
Meulemans,A.; Manuel,C; Ferriere,C; Vulpillat,M. Determination of methimazole in plasma by high perfor-
mance liquid chromatography, J.Liq.Chromatogr., 1980, 3, 287-298.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Serum + 300 |xL 160 ng/mL p-hydroxyanisole in MeOH, vortex
for 1 min, let stand for 30 min at room temperature, centrifuge at 10000 g for 10 min, inject
a 10-20 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: 100 X 8 10 ^m radial Pak C18 (Waters)
Mobile phase: MeOH: 10 mM ammonium phosphate containing 1 mM disodium EDTA 8:92, pH
Flow rate: 3
Detector: E, Bioanalytical Systems, glassy carbon working electrode +0.70 V, Ag/AgCl reference
electrode, detector temp 20
CHROMATOGRAM
Retention time: 4
Internal standard: p-hydroxyanisole (28)
KEYWORDS
REFERENCE
Tatsuhara,T.; Tabuchi,R; Unate,M.; Okamura,Y; Shigemasa,C; Abe,K; Mashiba,H. Determination of thia-
mazole in serum by high-performance liquid chromatography with electrochemical detection, J.Chromatogr.,
1985, 339, 149-156.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 100 mg disodium EDTA + 3 mL ethyl acetate, vortex
under a stream of nitrogen at 50, reconstitute the residue in 200 |xL MeOH, inject a 10 jxL
aliquot.
HPLCVARIABLES
Guard column: 25 X 3 LiChroprep RP-18 (Merck)
Mobile phase: Gradient. MeOH:25 mM pH 3 phosphate buffer from 10:90 to 70:30 over 30 min
Flow rate: 1
Detector: UV 258
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: methylthiouracil (UV 276), propylthiouracil (UV 276), phenylthiouracil (UV 276), thi-
ouracil (UV 276)
KEYWORDS
cow; plasma
REFERENCE
Moretti,G.; Betto,R; Cammarata,R; Fracassi,R; Giambenedetti,M.; Borghese,A- Determination of thyreostatic
residues in cattle plasma by high-performance liquid chromatography with ultraviolet detection,
J.Chromatogr., 1993, 616, 291-296.
Methocarbamol
Merck Index: 6060
LednicerNo.: 1 118
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Plasma + 100 |xL 200 |xg/mL mephenesin in water, mix, add 5 mL
ethyl acetate, shake for 15 min. Remove 3 mL of the organic layer and evaporate it to dryness
under a stream of nitrogen. Reconstitute in 500 |xL of water, inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 3 (xm Microsorb C18
Mobile phase: MeOH:100 mM KH2PO4:water 35:10:55
Flow rate: 0.8
Detector: UV 272
CHROMATOGRAM
Retention time: 5.2
Internal standard: mephenesin (10.5)
OTHER SUBSTANCES
Simultaneous: guaifenesin
Noninterfering: acetaminophen, ibuprofen
KEYWORDS
plasma
REFERENCE
Weng,N.; Lee,J.W.; Hulse,J.D. Development and validation of a high-performance liquid chromatographic
method for the determination of methocarbamol in human plasma, J.Chromatogr.B, 1994, 654, 287-292.
SAMPLE
Matrix: blood
the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 (JLL aliquot of
HPLC VARIABLES
Column: 300 X 3.9 4 \jjm NovaPack C18
Flow rate: 0.8
Detector: UV 223
CHROMATOGRAM
KEYWORDS
teolol; procainamide; carbamazepine; bromazepam; nalbuphine; nadolol; procarbazine; di-
hydralazine; omeprazole; strychnine; acebutolol; glutethimide; chlorpropamide; glipizide; tri-
azolam; prazosin; nunitrazepam; clonazepam; metoclopramide; melphalan; estazolam;
REFERENCE
254-262.
SAMPLE
Sample preparation: Serum. 2 mL Serum + 20 |xg IS, adjust pH to 14 with 2 M NaOH, extract
twice with 6 mL diethyl ether. Combine the organic layers, dry under nitrogen, dissolve the
residue in 200 JJLL MeOH. Inject a 20 (xL aliquot. Urine. 5 mL Urine + 75-1000 u-g IS, adjust
pH to 5 with 1 mL 2 M pH 5 sodium acetate buffer. Add 100 |xL p-glucuronidase (Helix pomatia
98400 U/mL), incubate at 37 overnight, cool, wash with 8 mL diethyl ether, discard the organic
layer. Adjust the aqueous layer to pH 14 with 25% NaOH, extract twice with 8 mL diethyl
ether. Combine the organic layers, dry under nitrogen, reconstitute the residue in 200 |xL
MeOH. Inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4 7 jxm LiChrospher 100 RP-18
Flow rate: 1
Detector: UV 280; MS, API III LC-MS-MS (PE Sciex, Canada), triple quadrupole, nebulizer 400
and 75 psi, auxiliary nitrogen gas, nitrogen-curtain gas, positive mode ionization, CID tech-
nique, collision gas argon, m/z 242
CHROMATOGRAM
Internal standard: mephenesin (19)
Limit of detection: 700 ng/mL (urine), 200 ng/mL (serum)
Limit of quantitation: 1.09 |xg/mL (urine), 550 ng/mL (serum)
OTHER SUBSTANCES
KEYWORDS
horse; serum; pharmacokinetics
REFERENCE
Koupai-Abyazani,M.R.; Esaw,B.; Laviolette,B. Determination of methocarbamol in equine serum and urine by
high-performance liquid chromatography with ultraviolet detection and atmospheric pressure ionization-
mass spectrometric confirmation, JAnalToxicoL, 1997, 21, 301-305.
SAMPLE
Sample preparation: Dilute urine 1:100. Plasma or diluted urine + 10 |xg/mL (RM-)-flecainide
acetate in water + 1 mL 1 M NaOH + 5 mL ethyl acetate, shake mechanically for 20 min,
stream of nitrogen, reconstitute the residue in 1 mL distilled ethyl acetate, add 80 |xL 0.2%
(S)-(+)-l-(l-naphthyl)ethyl isocyanate in ethyl acetate, vortex for 5 s, heat at 85 for 12 h,
evaporate, reconstitute in 200 |xL ethyl acetate, inject a 30-150 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Partisil 5 silica
Flow rate: 1.6
Detector: UV 280
CHROMATOGRAM
Internal standard: (RM-)-flecainide (12)
OTHER SUBSTANCES
Simultaneous: guaifenesin
KEYWORDS
rat; human; plasma; normal phase; chiral; derivatization; pharmacokinetics
REFERENCE
Alessi-Severini,S.; Coutts,R.T.; Jamali,R; Pasutto,F.M. High-performance liquid chromatographic analysis of
methocarbamol enantiomers in biological fluids, J.Chromatogr., 1992, 582, 173-179.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 3.2 7 jxm SI 100 ODS (not commercially available)
Mobile phase: MeCNrbuffer 31.2:68.8 (Buffer was 6.66 g KH2PO4 and 4.8 g 85% phosphoric acid
Flow rate: 0.5-1
CHROMATOGRAM
Retention time: 2.0
OTHER SUBSTANCES
diazepam, doxylamine, ethosuximide, furosemide, haloperidol, hydrochlorothiazide, methotri-
meprazine, nicotine, oxazepam, procaine, promazine, propafenone, propranolol, salicylamide,
temazepam, tetracaine, thiopental, triamterene, verapamil, zolpidem, zopiclone
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
methapyrilene, methaqualone, methoxamine, methsuximide, methyl salicylate, methyldopa,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: 450 |xL Buffer solution + 50 |JLL 4 mg/mL acetanilide, cool in ice, inject a
10 |xL aliquot.
HPLC VARIABLES
Column: Perkin-Elmer 3 X 3 CR C-18
Mobile phase: MeCN:water 10:90 containing 1% acetic acid
Flow rate: 2.5
Detector: UV 274
CHROMATOGRAM
Retention time: 4.7
Internal standard: acetanilide (2.1)
OTHERSUBSTANCES
Simultaneous: guaifenesin, degradation products
KEYWORDS
buffers; stability indicating
REFERENCE
Pouli,N.; Antoniadou-Vyzas,A-; Foscolos,G.B. Methocarbamol degradation in aqueous solution, J.Pharm.Sci.,
1994, 83, 499-501.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Hexane:l,2-dichloroethane:EtOH/trifluoroacetic acid 60:35:5 (EtOH/trifluoro-
acetic acid was premixed 20:1.)
Flow rate: 0.7-1
Detector: UV 274
KEYWORDS
REFERENCE
J.Liq.Chromatogr., 1995, 18, 649-671.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 jim Supelcosil LC-DP (A) or 250 X 4 5 jmi LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
metformin, methadone, methdilazine, methotrexate, methotrimeprazine, methoxamine, meth-
yldopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam,
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Methohexital
CAS Registry No.: 18652-93-2, 151-83-7, 309-36-4 (Na salt)
Merck Index: 6061
Lednicer No.: 1 269
SAMPLE
Matrix: blood
Sample preparation: Mix 250 JJLL plasma with 400 |xL MeCN, vortex, centrifuge. Inject a 20 \xL
aliquot of the clear supernatant.
HPLC VARIABLES
Column: C18 reverse phase
Mobile phase: MeOH:0.5% tetrabutylammonium phosphate in water 50:50
Detector: UV 265
CHROMATOGRAM
KEYWORDS
sheep; plasma; methohexital is IS
REFERENCE
Upton,R-N.; Huang,Y.F.; Grant,C; Gray,E.C; Ludbrook,G.L. Myocardial pharmacokinetics of thiopental in
sheep after short-term administration: Relationship to thiopental-induced reductions in myocardial con-
tractility, J.Pharm.ScL, 1996, 85, 863-867.
SAMPLE
Matrix: blood
Sample preparation: Buffer serum to pH 5.6 with 100 mM acetate buffer, add thiopental, ex-
tract with hexane. Remove the hexane and extract it with 250 mM NaOH. Neutralize the
aqueous layer with phosphoric acid and inject an aliquot.
HPLC VARIABLES
Mobile phase: MeCNrO.l mM pH 4.2 phosphate buffer 43:57
Flow rate: 1
Detector: UV 195
CHROMATOGRAM
Internal standard: thiopental
KEYWORDS
REFERENCE
Hudson,R.J.; Stanski,D.R.; Burch,P.G. Pharmacokinetics of methohexital and thiopental in surgical patients,
Anesthesiology, 1983, 59, 215-219.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma or hemolyzed (frozen and thawed) whole blood + 500 |xL 2
|xg/mL hexobarbital in water + 500 |xL 250 mM HCl + 40 mg NaCl + 3 mL toluene, rotate,
centrifuge. Remove the organic layer and evaporate it to dryness under a stream of air at 50
5, reconstitute the residue in 200 JXL MeCN and 200 [xh 50 mM NaH2PO4, inject a 20 |xL
aliquot.
HPLC VARIABLES
Guard column: 30 X 4 Perisorb RP-18 (Merck)
Column: 250 X 4 7 (xm LiChroCart RP-18 (Merck)
Mobile phase: MeCN:50 mM pH 4.6 NaH2PO4 50:50
Flow rate: 1
Detector: UV 195
CHROMATOGRAM
Retention time: 7.3
OTHER SUBSTANCES
Extracted: thiopental
Simultaneous: barbital, caffeine, indomethacin, pentobarbital, phenobarbital
Noninterfering: aspirin, salicylic acid
KEYWORDS
REFERENCE
Bjorkman,S.; Idvall,J. A high-performance liquid chromatographic method for methohexital and thiopental in
plasma or whole blood, J.Chromatogr., 1984, 307, 481-487.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond-Elut C8 SPE cartridge with 2 volumes of MeOH,
2 volumes of water, and 1 volume of 100 mM pH 5.59 Sorensen's phosphate buffer. 500 JJLL
Plasma + 10 |xL 1 mg/mL sodium secobarbital in EtOH, add to the SPE cartridge, wash with
2 volumes of 100 mM pH 5.59 Sorensen's phosphate buffer, wash with 1 volume of water, elute
with 500 |xL MeOH. Evaporate the eluate to dryness under vacuum, reconstitute in 50 |xL
MeOH, inject an aliquot.
HPLCVARIABLES
Guard column: 10 |jim Guard-Pak C18 (Waters)
Mobile phase: MeOH:THF:100 mM pH 7.72 Sorensen's phosphate buffer 28:16:52
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Internal standard: secobarbital (6.39)
OTHER SUBSTANCES
Extracted: pentobarbital, thiopental
Noninterfering: ketamine
KEYWORDS
plasma; dog; pharmacokinetics; SPE
REFERENCE
Avram,M.J.; Krejcie,T.C. Determination of sodium pentobarbital and either sodium methohexital or sodium
thiopental in plasma by high-performance liquid chromatography with ultraviolet detection, J.Chromatogr.,
1987, 414, 484-491.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 |xL aliquot of a solution in EtOH.
HPLC VARIABLES
Column: 250 X 4.6 Hypersil
Mobile phase: Isooctane:acetic acid:isopropanol 100:1.5:1
Flow rate: 2
Detector: UV 250
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: allobarbital, amobarbital, aprobarbital, barbital, brallobarbital, butabarbital, bu-
talbital, butobarbital, cyclobarbital, cyclopentobarbital, enallylpropymal, heptabarbital, hex-
ethal, hexobarbital, ibomal, idobutal, metharbital, methylphenobarbital, nealbarbital, pento-
barbital, phenobarbital, phenylmethylbarbituric acid, probarbital, secobarbital, sigmodal,
talbutal, vinbarbital
KEYWORDS
normal phase
REFERENCE
Gill,R.; Stead,A.H.; Moffat,A.C. Identification of drugs by the effective combination of gas-liquid, high-perfor-
mance liquid and thin-layer chromatographic techniques, J.Chromatogr., 1981, 204, 275-284.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeCN.water 25:75, inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 a-m C18 (Alltech)
Mobile phase: MeCN:4 mM potassium phosphate buffer 50:50, pH 4.0
Flow rate: 1.2
Detector: UV 290
CHROMATOGRAM
Retention time: 6.4
OTHER SUBSTANCES
Simultaneous: amobarbital, heptabarbital, hexobarbital, pentobarbital, phenobarbital, secobar-
bital, thiamylal, thiopental
REFERENCE
Ebling,W.R; Mills-Williams,L.; Harapat,S.R.; Stanski,D.R. High-performance liquid chromatographic method
for determining thiopental concentrations in twelve rat tissues: application to physiologic modeling of dis-
position of barbiturate, J.Chromatogr., 1989, 490, 339-353.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Gradient. A was MeCN:5 mM sodium carbonate 9:81. B was MeCN:20 mM sodium
carbonate 20:80. A:B from 100:0 to 0:100 over 10 min.
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: allobarbital, amobarbital, barbital, barbituric acid, butabarbital, mephobarbital,
methabarbital, phenobarbital, phenytoin, secobarbital, thiamylal
REFERENCE
Slingsby,R.W.; Rey,M. Determination of Pharmaceuticals by multi-phase chromatography: Combined reversed
phase and ion exchange in one column, J.Liq.Chromatogr., 1990,13, 107-134.
Methoprene
Merck Index: 6063
SAMPLE
Matrix: solutions
Sample preparation: 5 mL Water + 100 JJLL 22.7 |xg/mL dipentyl phthalate in MeCN + 3 mL
MTBE, vortex for 1 min, centrifuge at 2500 g for 10 min. Remove the organic layer and evap-
orate it to dryness under a stream of nitrogen at 45, reconstitute the residue in 100 |xL mobile
phase, vortex for 30 s, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 70 X 4.6 3 jxm Ultrasphere XL-ODS
Flow rate: 1
Detector: UV 255
CHROMATOGRAM
Retention time: 6.5
Internal standard: dipentyl phthalate (4)
KEYWORDS
water; groundwater
REFERENCE
Allen,C.R.; Dickinson,C.M. Determination of methoprene in water samples by high performance liquid chro-
matography, J.Liq.Chromatogr., 1990, 13, 371-381.
Methotrexate
Merck Index: 6065
SAMPLE
M a t r i x : blood
Sample preparation: Mix 20 |xL serum with 20 |xL 2 M perchloric acid, vortex for a few seconds,
HPLC VARIABLES
Column: 150 X 4.6 5 jim Inertsil ODS-2 (GL Sciences, Japan)
Mobile phase: MeCN:25 mM pH 7.0 sodium phosphate buffer 8:92 containing 25 mM hydrogen
peroxide
Flow rate: 1
a 5 m X 0.5 mm i.d. stainless-steel reaction coil at 160 and a 3 m X 0.5 mm i.d. stainless-
steel coil at 15 to the detector.
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Extracted: metabolites, leucovorin
KEYWORDS
serum; post-column reaction; comparison with immunoassay
REFERENCE
Kubo,H.; Umiguchi,Y; Fukumoto,M.; Kinoshita,T. Fluorometric determination of methotrexate in serum by
high-performance liquid chromatography using in-line oxidation with hydrogen peroxide, Anal. ScL, 1992,
8, 789-792.
SAMPLE
Matrix: blood
Sample preparation: Condition a C18 Sep-Pak Classic SPE cartridge with 20 mL MeOH and
3 mL 200 mM pH 6 phosphate buffer. Gently vortex a 200-500 |xL aliquot of serum with 1 ml
4 jxg/mL IS in 200 mM pH 6 phosphate buffer. Add to the SPE cartridge, wash with 1 mL
water, dry under vacuum for 5 min, elute with 2 mL MeOH. Dry the eluate under nitrogen at
60. Reconstitute the residue in 200-300 jxL 5 mM HCl, inject a IOOJXL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Beckmann Ultrasphere ODS
Mobile phase: MeCN.buffer 10:90 (Buffer was 10 mM pH 2.5 KH2PO4 containing 20 mM tetra-
methylammonium chloride.)
Flow rate: 1
Detector: UV 313
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
serum; SPE
REFERENCE
Aboleneen,H.; Simpson, J.; Backes,D. Determination of methotrexate in serum by high-performance liquid chro-
SAMPLE
Matrix: blood
Sample preparation: Inject 100 |xL plasma onto column A and elute to waste with mobile phase
A, after 4 min backflush the contents of column A onto column B with mobile phase B, after 3
min remove column a from the circuit, elute column B with mobile phase B, monitor the
effluent from column B.
HPLC VARIABLES
Column: A 25 X 4 25 |xm C8-alkyl-diol silica (ADS); B 125 X 4 5 |xm LiChrospher RP-18
Mobile phase: A MeCNrpH 7.4 phosphate buffer 2:98 containing 2 mM tetrabutylammonium
hydrogen sulfate; B MeCNipH 7.4 phosphate buffer 18:82 containing 5 mM tetrabutylammo-
nium hydrogen sulfate
Flow rate: 1
Detector: UV 307
CHROMATOGRAM
Retention time: 9.5-10
KEYWORDS
column-switching; plasma
REFERENCE
Yu,Z.; Westerlund,D. Ion-pair chromatography of methotrexate in a column-switching system using an alkyl-
diol silica precolumn for direct injection of plasma, J.Chromatogr.A, 1996, 742, 113-120.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 500 |xL 2.5 |xg/mL sulfafurazole in 10% trichloroacetic
acid in 100 mM HCl, vortex for 20 s, centrifuge at 8300 g for 5 min, inject a 100 JJLL aliquot of
the supernatant.
HPLC VARIABLES
Column: 250 X 4 5 fxm LiChrosorb RP18
Mobile phase: MeCN: 150 mM pH 4.85 ammonium phosphate buffer 12.6:87.4
Flow rate: 1.5
Detector: UV 313
CHROMATOGRAM
Retention time: 5
Internal standard: sulfafurazole (UV 254) (23)
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Van der Steuijt,K; Sonneveld,P. Concurrent analysis of methotrexate, trimethoprim, sulfamethoxazole and
their major metabolites in plasma by high-performance liquid chromatography, J.Chromatogr., 1987, 422,
328-333.
SAMPLE
Matrix: blood
Sample preparation: Add 2 mg ascorbic acid to each 1 mL blood, centrifuge at 800 g in the
cold. 1 mL Plasma +1.5 mL MeOH, vortex, centrifuge at 80Og. Remove the supernatant and
evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 200 |xL water,
inject the whole amount.
HPLCVARIABLES
Column: 300 X 3.9 10 jxm jxBondapak phenyl
Mobile phase: Gradient. A was 250 mM pH 5.0 phosphate buffer. B was MeOH:250 mM pH 5.0
phosphate buffer 1:1. A:B 100:0 for 15 min, to 50:50 over 15 min, to 0:100 over 5 min, maintain
at 0:100 for 5 min.
Flow rate: 2
Detector: UV 310
CHROMATOGRAM
Retention time: 29
Internal standard: methotrexate
OTHER SUBSTANCES
Extracted: leucovorin
KEYWORDS
plasma; methotrexate is IS
REFERENCE
WainerJ.W.; Stiffin,R.M. Direct resolution of the stereoisomers of leucovorin and 5-methyltetrahydrofolate us-
ing a bovine serum albumin high-performance liquid chromatographic chiral stationary phase coupled to
an achiral phenyl column, J.Chromatogr., 1988, 424, 158-162.
SAMPLE
Matrix: blood
Sample preparation: 1 Volume plasma + 2 volumes MeCN, mix thoroughly, centrifuge at 2000
g for 3 min. Remove the supernatant and add it to 4 volumes chloroform, vortex briefly, inject
a 30 |xL aliquot of the aqueous supernatant.
HPLC VARIABLES
Column: 250 X 4 5 |xm LiChrospher 100RP8
Flow rate: 1.5
Detector: UV 307
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Cociglio,M.; Hillaire-Buys,D.; Alric,C. Determination of methotrexate and 7-hydroxymethotrexate by liquid
chromatography for routine monitoring of plasma levels, J.Chromatogr.B, 1995, 674, 101-110.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond-Elut SPE cartridge with 1 mL MeOH and 1.5
mL 50 mM pH 2.7 phosphate buffer. 1 mL Plasma + 1 mL 50 mM pH 6.5 phosphate buffer,
mix, add to the SPE cartridge at 2 mL/min, wash with 2 mL 50 mM pH 2.7 phosphate buffer,
wash with 1 mL 100 mM NaOH, wash with 1 mL 50 mM pH 2.7 phosphate buffer, elute with
1.5 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the
residue in 200 uL mobile phase, inject a 100 JJLL aliquot.
HPLC VARIABLES
Guard column: 30 X 3.2 30 |xm Spherisorb C18
Column: 150 X 3.9 5 jjun Novapak C18
Mobile phase: DMF:MeCN:3% hydrogen peroxide:14 mM pH 6.5 phosphate buffer 4:3.3:0.5:92.2
Flow rate: 1
Detector: F ex 350 em 465, following post-column photolytic oxidation in a 3 m long X 0.38 mm
i.d. length of polyethylene (PE-20) tubing with a Spectroline pencil UV lamp (UV 254 nm)
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
KEYWORDS
plasma; SPE; human; dog; pharmacokinetics; post-column photochemical derivatization; post-col-
umn reaction
REFERENCE
Lu,G.; Jun,H-W. Determination of trace methotrexate and 7-OH-methotrexate in plasma by high-performance
liquid chromatography with fluorimetric detection, J.Liq.Chromatogr., 1995, 18, 155-171.
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 301
CHROMATOGRAM
KEYWORDS
teolol; procainamide; carbamazepine; bromazepam; nalbuphine; nadolol; procarbazine; di-
hydralazine; omeprazole; strychnine; acebutolol; glutethimide; chlorpropamide; glipizide; tria-
zolam; prazosin; flunitrazepam; clonazepam; metoclopramide; melphalan; estazolam;
amine; pimozide; daunorabicin; indomethacin; maprotiline; tropatenine; etodolac; fluoxetine;
pramine; thioridazine; fentiazac; clemastine; mefenamic acid; fluphenazine; proehlorperazine;
REFERENCE
254-262.
SAMPLE
Matrix: blood
Sample preparation: Condition an Isolute HAX 200 mg SPE cartridge (International Sorbent
Technology) with 3 mL MeOH and 3 mL 100 mM phosphoric acid. Dilute 0.01-1 mL plasma
with 2 mL 100 mM phosphoric acid, add to the SPE cartridge at 2.5 mL/min, wash with 2 mL
MeOH:100 mM phosphoric acid 5:95, wash with 3 mL pH 8.6 Na2HPO^, wash with MeOH:
water 5:95, air dry for 2 min, elute with 2 mL 20 g/L trifluoroacetic acid in MeOH. Evaporate
the eluate to dryness under a stream of nitrogen at 40, reconstitute the residue in 100 |xL 100
mM phosphoric acid, inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 80 X 4.6 3 |xm C18 (Perkin-Elmer)
Mobile phase: MeCN:100 mM pH 6.5 phosphate buffer:30% hydrogen peroxide 6.5:93.3:0.2
Flow rate: 1
a 10 m X 0.3 mm coil irradiated with a 254 nm UV lamp at 37 and flowed to the detector.
CHROMATOGRAM
KEYWORDS
post-column photochemical derivatization; SPE; plasma; comparison with immunoassays; post-
column reaction
REFERENCE
Albertioni,E; Rask,C; Eksborg,E.; Poulsen,J.H.; Pettersson,B.; Beck,O.; Schroeder,H.; Peterson,C. Evaluation
of clinical assays for measuring high-dose methotrexate in plasma, Clin.Chem., 1996, 42, 39-44.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 500 uX acetone, vortex vigorously for 1 min, centrifuge
at 3000 rpm for 4 min. Remove a 600 |xL aliquot of the supernatant and add it to 600 |xL
butanol and 800 |xL diethyl ether, mix vigorously, centrifuge at 2000 rpm for 2 min. Remove a
300 |JLL aliquot of the lower aqueous layer and evaporate it to dryness under reduced pressure,
reconstitute the residue with 150 |xL 0.9% NaCl in water, filter (0.45 |xm), inject a 50 uX aliquot
onto column A and column B in series and elute with mobile phase A, after 5 min remove
column A from the circuit, elute column B with mobile phase B, monitor the effluent from
column B. (Methotrexate is oxidized on column A to the fluorescent compounds 2,4-diaminop-
teridine-6-carboxylic acid and 2,4-diaminopteridine-6-carboxaldehyde.)
HPLC VARIABLES
Column: A 70 X 4.5 cerium(IV) trihydroxyperoxide; B 250 X 4.6 5 juum ODS/TM silica (Prepa-
ration of cerium(IV) trihydroxyperoxide is as follows. Add 15 mL 35% ammonia solution with
stirring to a cooled solution of 11.18 g cerium(III) chloride heptahydrate in 100 mL water, add
20 mL 30% hydrogen peroxide with stirring over 15 min, collect the orange-red precipitate by
filtration, wash the solid several times with water, wash with acetone, dry under vacuum at
room temperature overnight to obtain cerium(IV) trihydroxyperoxide, discard after 6 months.
Suspend 1 g cerium(IV) trihydroxyperoxide in chloroform, degas under vacuum with stirring
for 10 min, add the suspension to a 100 X 7 reservoir on top of the column, pump the slurry
into the column using acetone as a purge solvent at 5 mL/min for 20 min, wash the column
with acetoneipH 3.5 phosphate buffer 50:50 at 1 mL/min for 20 min, equilibrate with mobile
phase A at 0.5 mL/min for 1 h (Analyst 1996, 121, 183).)
Mobile phase: A 40 mM pH 3.5 phosphate buffer; B MeCN:50 mM pH 6.6 phosphate buffer 10:
90
CHROMATOGRAM
Retention time: 5 (2,4-diaminopteridine-6-carboxylic acid), 6 (2,4-diaminopteridine-6-
carboxaldehyde)
KEYWORDS
derivatization; plasma; column-switching
REFERENCE
Emara,S.; Razee,S.; Khedr,A-; Masujima,T. On-line precolumn derivatization for HPLC determination of meth-
otrexate using a column packed oxidant, Biomed.Chromatogr., 1997, 11, 42-46.
SAMPLE
Matrix: blood, CSF
Sample preparation: 250 |xL Plasma or CSF + 25 |xL 10 mg/mL ascorbic acid + 250 |xL ice-cold
1.5 M perchloric acid, vortex, let stand in ice-water for 5 min, centrifuge at 4 at 3000 g for 5
min. Remove 350 |xL of the supernatant with 50 ^L 8 M potassium acetate, keep in ice-water
for 2 min, centrifuge at 4 at 3000 g for 2 min, inject a 100 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: 200 X 3 5 |xm Hypersil ODS glass column
Mobile phase: Gradient. A was 10 mM ammonium formate adjusted to pH 3.5 with HCl. B was
MeCN:10 mM ammonium formate 25:75 adjusted to pH 3.5 with HCl. A:B from 85:15 to 5:95
Flow rate: 0.4
Detector: UV 305
CHROMATOGRAM
Retention time: 20
OTHER SUBSTANCES
Extracted: leucovorin, N5-methyltetrahydrofolate
KEYWORDS
plasma
REFERENCE
van Tellingen,O.; van der Woude,H.R.; Beijnen,J.H.; van Beers,C.J.T.; Nooyen,W.J. Stable and sensitive method
for the simultaneous determination of N5-methyltetrahydrofolate, leucovorin, methotrexate and 7-hydroxy-
methotrexate in biological fluids, J.Chromatogr., 1989, 488, 379-388.
SAMPLE
Sample preparation: Condition a 3 mL Isolute C8 SPE cartridge (International Sorbent Tech-
nology) with 3 mL MeOH and 3 mL 100 mM phosphoric acid at about 3 mL/min, do not allow
to dry. 1 mL Plasma, 10 |xL urine, or 100 |xL saliva + 2 mL 100 mM phosphoric acid, wash
with 2 mL MeOHiwater 5:95, dry with air, elute with 2 mL MeOH:trifluoroacetic acid 98:2.
100 |xL water, inject a 10 JJLL aliquot.
HPLC VARIABLES
Column: 80 X 4.6 3 jxm C18 (Perkin-Elmer)
Mobile phase: MeCN:10 mM pH 6.5 phosphate buffer:30% hydrogen peroxide 6:93.8:0.2
Flow rate: 1
Detector: F ex 350 em 435 following post-column photochemical reaction. The column effluent
flowed through a 10 m X 0.3 mm i.d. reaction coil, illuminated with a UV 254 lamp at 37,
and then to the detector.
CHROMATOGRAM
Retention time: 10
Limit of detection: 10 nM (urine), 0.1 nM (plasma, saliva)
OTHER SUBSTANCES
Noninterfering: acetaminophen, allopurinol, amoxicillin, ara-C, aspirin, azathioprine, chloro-
quine, ciprofloxacin, F-ara-A, ibuprofen, 6-mercaptopurine, naproxen, sulfamethoxazole,
trimethoprim
KEYWORDS
post-column photochemical derivatization; plasma; pharmacokinetics; SPE; post-column reaction
REFERENCE
Albertioni,R; Pettersson,B.; Beck,O.; Rask,C; Seideman,R; Peterson,C. Simultaneous quantitation of metho-
trexate and its two main metabolites in biological fluids by a novel solid-phase extraction procedure using
SAMPLE
Sample preparation: Centrifuge heparinized blood at 16000 g for 15 min, inject a 10-80 JJLL
aliquot. Inject a 10 |xL aliquot of urine.
HPLC VARIABLES
Guard column: 40 |xm LC-18 (Supelco)
Column: 250 X 4.6 10 jxm LiChrosorb RP-18
Mobile phase: MeCN:DMF:50 mM pH 6.2 phosphate buffer:30% hydrogen peroxide 7:5.6:100:
0.15 (Prepare and sonicate 1 day before use. Degas with helium during use.)
Flow rate: 1
Detector: F 370 em 417 (cut-off filter following post-column reaction. The column effluent flowed
through a 1.59 m X 0.25 mm ID PTFE coil irradiated by a Sylvania G8T5 germicidal lamp at
254 nm to the detector.
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
KEYWORDS
post-column reaction; post-column photochemical derivatization; whole blood
REFERENCE
Salamoun,J.; Smrz,M.; Kiss,F.; Salamounova,A. Column liquid chromatography of methotrexate and its me-
tabolites using a post column photochemical reactor and fluorescence detection, J.Chromatogr., 1987, 419,
213-223.
SAMPLE
Sample preparation: Mix plasma or urine with 2.5 volumes of MeCN, vortex, centrifuge, inject
a 50 (xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 30 X 2.1 7 |xm CX-300 (Applied Biosystems)
Column: 250 X 2.9 10 |xm SCX (Whatman)
Mobile phase: MeCN:20 mM (NH4)H2PO4 30:70, adjust pH to 1.6 with phosphoric acid
Flow rate: 2
Detector: UV 313
KEYWORDS
REFERENCE
Park,J.M.; Ahn,B.-N.; Yoon,E.J.; Lee,M.G.; Shim,C.-K; Kim,C.-K The pharmacokinetics of methotrexate after
intravenous administration of methotrexate-loaded proliposomes to rats, Biopharm.Drug Dispos., 1994,15,
391-407.
SAMPLE
HPLC VARIABLES
Column: 300 X 4.6 5 jim C18
Mobile phase: MeCNrIOO mM NaH2PO4 20:80 adjusted to pH 4.2 with phosphoric acid
Flow rate: 1.2
Detector: UV 300
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: granisetron
KEYWORDS
REFERENCE
liquids and during simulated Y-site injection with selected drugs, Am.J.Health-Syst.Pharm., 1996,53,294-
304.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10 jxL aliquot of an aqueous solution.
HPLC VARIABLES
Guard column: 5 |xm Lichrospher 60 RP-select B
Column: 125 X 3 3 /mm Hypersil BDS
Mobile phase: Gradient. A was MeCN. B was 5 mM monobasic potassium phosphate adjusted
to pH 2.3 with phosphoric acid. A:B 7:93 for 5 min, to 13:87 over 15 min, to 21:79 over 6 min,
maintain at 21:79 for 1 min, back to 7:93 over 2 min, re-equilibrate at initial conditions for 5
min.
Flow rate: 0.5
Detector: UV 310
CHROMATOGRAM
Retention time: 21
OTHER SUBSTANCES
Simultaneous: leucovorin
REFERENCE
Mandl,A.; Lindner,W. Improved detection of leucovorin in mixed Mates and antifolates by reversed-phase liquid
chromatography and on-line post-column UV irradiation, Chromatographia, 1996, 43, 327-330.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a saline solution.
HPLC VARIABLES
Mobile phase: MeOH:buffer 35:65, pH 3.0 (Buffer was 4.88 mM KH2PO4 containing 1.623 mM
phosphoric acid.)
Flow rate: 1
Detector: UV 303
CHROMATOGRAM
Retention time: 5
KEYWORDS
saline
REFERENCE
Chatelut,E.; Suh,R; Kim,S. Sustained-release methotrexate for intracavitary chemotherapy, J.Pharm.Sci.,
1994, 83, 429-432.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
metformin, methadone, methdilazine, methocarbamol, methotrimeprazine, methoxamine,
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatognA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 JULL aliquot.
HPLC VARIABLES
Column: 5 fjim Lichrospher 100 RP-18
Mobile phase: MeOH: 100 mM pH 7.3 phosphate buffer 20:80 to 32:68
Flow rate: 0.8
Detector: UV 300
REFERENCE
Smal,M.A.; Dong,Z.; Cheung,H.T.A.; Asano,Y.; Escoffier,L.; Costello,M.; Tattersall,M.H.N. Activation and cyto-
toxicity of 2-a-aminoacyl prodrugs of methotrexate, Biochem.Pharmacol., 1995, 49, 567-574.
SAMPLE
Matrix: urine
Sample preparation: Add 250 |xL 1 M pH 5.0 acetate buffer to a 1.0 mL urine, vortex and add
a 250 |AL aliquot of the mixture to a Sep-Pak silica cartridge and dry by aspiration of air. Wash
the cartridge with 5 mL ethyl acetate and 5 mL EtOH:MeOH 60:40 and dry by aspiration of
air. Elute with 3 mL of 1% ammonia in MeOH, dry eluate under a stream of nitrogen in a
water bath at 37. Reconstitute dried residue with 100 |xL mobile phase and inject a 20 jxL
aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 (xm Inertsil ODS-2 (GL Science, Tokyo, Japan)
Flow rate: 1.1
Detector: UV 303
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Next Page
Noninterfering: diclofenac, felbinac, fenbufen, indomethacin, loxoprofen, naproxen, piroxicam,

sulindac
KEYWORDS
SPE
REFERENCE
Hirai,T.; Matsumoto,S.; Kishi,I. Determination of methotrexate and its main metabolite 7-hydroxymethotrexate
in human urine by high-performance liquid chromatography with normal solid-phase extraction,
J.Chromatogr.B, 1997, 690, 267-273.
SAMPLE
Matrix: urine
Sample preparation: Condition a 100 mg Bond Elut phenyl SPE cartridge with 1 mL MeOH
and 1 mL water. Centrifuge urine at 1000 g. Adjust pH of 1 mL supernatant to 5.0 0.1 with
2 M trichloroacetic acid then with 0.6 M trichloroacetic acid, add to the SPE cartridge, wash
with 2 mL water, wash with 1 mL ethyl acetate, elute with 2 mL MeOH. Evaporate the eluate
to dryness under a stream of nitrogen at 37, reconstitute the residue in 100 |xL water, inject
a 10-50 uL aliquot onto column A and elute to waste with mobile phase A, after 9.5 min divert
the effluent from column B onto column A, after another 5 min remove column A from the
circuit and start the gradient, elute column B with mobile phase B and monitor the effluent
from column B. Re-equilibrate both columns with mobile phase A for 15 min. Wash both col-
umns separately with MeCN at the end of the day.
HPLC VARIABLES
Column: A 250 X 4 10 u.m Nucleosil 100 SB strong anion-exchange (maintained at 25 1); B
250 x 4 10 |xm LiChrospher 100 RP-18e
Mobile phase: A MeCN.buffer 1:99; B Gradient. MeCN:buffer from 1:99 to 25:75 over 24 min.
(Buffer was 314 mM formic acid adjusted to pH 2.7 with 25% ammonia.)
Flow rate: 1
Detector: UV 310
CHROMATOGRAM
Retention time: 34
KEYWORDS
column-switching; heart-cut; SPE
REFERENCE
Mader,R.M.; Rizovski,B.; Steger,G.G.; Rainer,H.; Proprentner,R.; Kotz,R. Determination of methotrexate inhu-
man urine at nanomolar levels by high-performance liquid chromatography with column switching,
J.Chromatogr., 1993, 613, 311-316.
Previous Page
Methotrimeprazine
Molecular formula: C19H24N2OS
Merck Index: 6066
SAMPLE
Matrix: blood
Sample preparation: 2-5 mL Plasma + 1 mL 1 M NaOH + 8 mL diethyl etherxhloroform 4:1,
shake for 10 min, centrifuge. Remove the upper organic layer and evaporate it to dryness at
50 under a stream of nitrogen. Dissolve the residue in 50 JULL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 fjim Spherisorb nitrile-bonded silica
Mobile phase: MeCNiMeOH: 100 mM K2HPO4 adjusted to pH 6.5 with orthophosphoric acid 6:
4:7
Detector: E, Model LCA 15-EDT Research, glassy carbon electrode +0.85 V
CHROMATOGRAM
Retention time: 4
Internal standard: methotrimeprazine
OTHER SUBSTANCES
Simultaneous: prochlorperazine
Noninterfering: chlorpromazine
KEYWORDS
plasma; methotrimeprazine is IS
REFERENCE
Sankey,M.G.; Holt,J.E.; Kaye,C.M. A simple and sensitive H.P.L.C. method for the assay of prochlorperazine
in plasma, Br.J.Clin.PharmacoL, 1982, 23, 578-580.
SAMPLE
Matrix: blood
Sample preparation: Work under yellow light. 1 mL Serum + 2 mL water + 2 mL 2 M NaOH,
vortex for 10 s, add 5 mL water-saturated n-heptane:isoamyl alcohol 99:1, shake gently for 20
min, centrifuge at 4 at 2800 g, remove organic layer and repeat the extraction. Combine the
organic layers and evaporate them to dryness under reduced pressure. Dissolve the residue in
500 |JLL MeCN, inject a 30 |xL aliquot (store at 5).
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Nucleosil 100 CN
Mobile phase: MeCNipyridine: 140 mM sodium acetate pH 3.1 698:2:300
Flow rate: 0.9
Detector: E, Environmental Sciences Assoc. Coulochem II, Model 5011 detector cell, oxidative
screen mode, screen electrode +0.35 V, sample electrode +0.65 V
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: chlorprothixene
Interfering: promethazine
KEYWORDS
serum; recirculate mobile phase
REFERENCE
Bagli,M.; Rao,M.L.; Hdflich,G. Quantification of chlorprothixene, levomepromazine and promethazine in human
serum using high-performance liquid chromatography with coulometric electrochemical detection,
J.Chromatogr.B, 1994, 657, 141-148.
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 252
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: blood, milk
1 mL Serum or milk + 5 mL 0.5 (serum) or 1 (milk) M HCl, mix, add to the SPE cartridge,
wash with 5 mL water, wash with 5 mL MeOH:water 20:80, elute with 5 mL MeOH:water 60:
40, evaporate eluate to dryness under vacuum at 60, dissolve residue in 100 |xL mobile phase,
inject whole amount.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Develosil C8-5 (Nomura)
Mobile phase: MeCN:0.5% KH2PO4 adjusted to pH 4.5 with 50% phosphoric acid 35:65
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 10
Internal standard: methotrimeprazine (levomepromazine)
OTHER SUBSTANCES
KEYWORDS
serum; SPE; methotrimeprazine is IS
REFERENCE
Ohkubo,T.; Shimoyama,R-; Sugawara,K. Determination of chlorpromazine in human breast milk and serum by
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 4 mL water + 1 mL 160 ng/mL thioridazine + 0.8
mL 1 M NaOH + 15 mL n-heptane:isoamyl alcohol 98.5:1.5, shake for 10 min, centrifuge at
700 g, remove the organic layer, repeat the extraction twice more. Combine the organic layers
and evaporate them to dryness, reconstitute the residue in 10 mL 50 mM HCl, add 20 mL
diethyl ether, shake for 3 min. Remove the aqueous layer and make it alkaline with 1 mL 5
M NaOH, add 10 mL n-heptane:isoamyl alcohol 98.5:1.5, shake for 10 min, centrifuge at 700
g. Remove the organic layer and evaporate it to dryness, dissolve the residue in 1 mL MeCN,
inject a 50 jxL aliquot. Urine. 20 mL Urine + 3 mL 1 M HCl + 1 mL 1 |xg/mL thioridazine,
wash with diethyl ether, make the aqueous layer alkaline with 5 M NaOH, add n-heptane:
isoamyl alcohol 98.5:1.5, shake for 10 min, centrifuge at 700 g. Remove the organic layer and
evaporate it to dryness, dissolve the residue in 1 mL MeCN, inject a 10 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:pyridine:THF:100 mM pH 3.5 acetate buffer 68.9:0.1:1:30 containing 20
mM sodium perchlorate
Flow rate: 0.7
Detector: E, Yanaco Model VMD-101, glassy carbon working electrode 0.95 V, Ag/AgCl reference
electrode
CHROMATOGRAM
Retention time: 7.0
Internal standard: thioridazine (9.5)
Limit of detection: 0.5 ng/mL (urine), 2 ng/mL (plasma)
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Murakami,K.; Ueno,T.; Hijikata,J.; Shirasawa,K.; Muto,T. Simultaneous determination of chlorpromazine and
levomepromazine in human plasma and urine by high-performance liquid chromatography using electro-
chemical detection, J.Chromatogr., 1982, 227y 103-112.
SAMPLE
residue with 50 u,L MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 251.1
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 3.7
OTHER SUBSTANCES
mine, fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, fiurazepam, hal-
methdilazene, methoxamine, methoxyphenamine, methoxypromazine, methylephedrine, meth-
ylergonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone,
ene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylc^romine, trazodone,
trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, trimethoprim,
REFERENCE
SAMPLE
Matrix: solutions
JJLL aliquot.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 20 X 4.6 5 jxm Supelguard LC 18-DB (Supelco)
Column: 250 X 4.6 5 |xm Supelcosil C 18-DB
Mobile phase: MeCN:THF:buffer 47.5:2.5:50 (Buffer was 500 mM pH 5.0 ammonium acetate
containing 25 mM sodium dodecyl sulfate.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Internal standard: methoxypromazine (20.41)
OTHER SUBSTANCES
REFERENCE
Loennechen,T.; Dahl,S.G. High-performance liquid chromatography of levomepromazine (methotrimeprazine)
and its main metabolites, J.Chromatogr., 1990, 503, 205-215.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN.buffer 31.2:68.8 (Buffer was 6.66 g KH2PO4 and 4.8 g 85% phosphoric acid
Flow rate: 0.5-1
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
diazepam, doxylamine, ethosuximide, furosemide, haloperidol, hydrochlorothiazide, methocar-
bamol, nicotine, oxazepam, procaine, promazine, propafenone, propranolol, salicylamide, tem-
azepam, tetracaine, thiopental, triamterene, verapamil, zolpidem, zopiclone
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1 mg/mL solution in MeOH, inject a 5 JJLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Lichrosphere cyanopropyl
Mobile phase: Carbon dioxide:MeOH:isopropylamine 94:6:0.03
Flow rate: 3
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 3.4
OTHER SUBSTANCES
Simultaneous: triflupromazine, carphenazine, promazine, perphenazine, chlorprothixene, deser-
pidine, thiothixene, reserpine
Also analyzed: acetophenazine, ethopropazine, promethazine, propiomazine
KEYWORDS
REFERENCE
Berger,T.A.; Wilson,W.H. Separation of drugs by packed column supercritical fluid chromatography. 1. Pheno-
thiazine antipsychotics, J.Pharm.Sci., 1994, 83, 281-286.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 (xm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
metformin, methadone, methdilazine, methocarbamol, methotrexate, methoxamine, methyl-
dopa, methylphenidate, metoclopramide, metolazone, metoprolol, metronidazole, midazolam,
phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phenjrtoin, pimo-
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 |xL aliquot of a 25 ng/mL solution in pH 4.0 acetate/citrate
buffer.
HPLC VARIABLES
Mobile phase: MeCN:pH 4.0 acetate/citrate buffer 45:55
Detector: UV
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: chlorpromazine, thioridazine
KEYWORDS
microcolumn
REFERENCE
Streel,B.; Ceccato,A.; Chiap,R; Hubert,R; Crommen,J. Injection-generated solvent and pH gradients for sample
enrichment on injection of large volumes in microcolumn liquid chromatography, Biomed.Chromatogr., 1995,
9, 254-256.
Methoxamine
Merck Index: 6067
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 50 |JLL water + 100 (xL 1 M NaOH + 4 mL ethyl
acetate, vortex for 30 s, centrifuge at 3000 rpm for 10 min. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen at room temperature, reconstitute the
residue in 200 |xL saturated sodium carbonate and 200 |xL 187 mM (-)-menthyl chloroformate
in MeCN, vortex for 30 s, add 1 mL water, add 2 mL chloroform, vortex for 30 s, centrifuge for
room temperature, reconstitute the residue in 200 (JLL mobile phase, vortex for 5 s, centrifuge
for 5 min, inject a 20-60 |xL aliquot of the supernatant. Urine. Dilute urine 10 times with
water. 100 ^xL Diluted urine + 50 JJLL water + 100 jxL saturated sodium carbonate + 200 (JLL
187 mM (-)-menthyl chloroformate in MeCN, vortex for 30 s, add 1 mL water, add 2 mL chlo-
roform, vortex for 30 s, centrifuge for 3 min. Remove the organic layer and evaporate it to
dryness under a stream of nitrogen at room temperature, reconstitute the residue in 200 (JLL
mobile phase, vortex for 5 s, centrifuge for 5 min, inject a 20-60 |xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 50 mm long pellicular ODS (Whatman)
Column: 100 X 4.6 5 jxin Partisil 5 0DS3
Flow rate: 1.2
CHROMATOGRAM
Internal standard: methoxamine
OTHER SUBSTANCES
Extracted: atenolol
KEYWORDS
plasma; derivatization; methoxamine is IS; chiral
REFERENCE
Mehvar,R. Liquid chromatographic analysis of atenolol enantiomers in human plasma and urine, J.Pharm.Sci.,
1989, 78, 1035-1039.
SAMPLE
Matrix: perfusate
Sample preparation: 30 |xL Perfusate (artificial CSF) + 10 jxL 200 mM perchloric acid. Mix a
25 |JLL aliquot with 12.5 |xL reagent, let stand for 2 min, inject an aliquot. (Prepare a stock
solution by dissolving 27 mg o-phthalaldehyde in 1 mL MeOH, add 5 |xL (3-mercaptoethanol,
10 |JLM EDTA, allow to stand for 24 h before use.)
HPLCVARIABLES
Column: two columns 150 X 4.6 5 jxm M.S. Gel C18 (ESA)
Mobile phase: MeOH:buffer 8:92 adjusted to pH 3.0 with phosphoric acid (Buffer was 54 mM
Flow rate: 1.2
potential 900 mV
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: apomorphine, dopamine, hydralazine, isoproterenol, morphine, norepinephrine,
phenylephrine
KEYWORDS
rat
REFERENCE
Acworth,I.N.; Yu,J.; Ryan,E.; Gariepy,K.C; Gamache,P.; Hull,K; Maher,T. Simultaneous measurement of
monoamine, amino acid, and drug levels, using high performance liquid chromatography and coulometric
array technology: application to in vivo microdialysis perfusate analysis, J.Liq.Chromatogr., 1994,17, 685-
705.
SAMPLE
Matrix: solutions
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.7
OTHER SUBSTANCES
methdilazene, methotrimeprazine, methoxyphenamine, methoxypromazine, methylephedrine,
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Evaporate an ethyl acetate solution to dryness under a stream of nitrogen
at 37, reconstitute the residue in 50 |xL 100 mM NaOH, vortex briefly, add 200 uL 200 mM
(-)-menthyl chloroformate in MeCN, vortex for 30 s, let stand at room temperature for 10 min,
HPLC VARIABLES
Column: 150 X 4.6 5 (xm Hypersil ODS
Flow rate: 1.2
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: atenolol
KEYWORDS
REFERENCE
Miller,R.B.; Guertin,Y. High-performance liquid chromatographic assay for the derivatized enantiomers of aten-
olol in whole blood, J.Liq.Chromatogr., 1992, 15, 1289-1302.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
methapyrilene, methaqualone, methazolamide, methsuximide, methyl salicylate, methyldopa,
oxyphenbutazone, ox5rtetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbi-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.7-1
Detector: UV 290
KEYWORDS
REFERENCE
J.Liq.Chromatogr., 1995,18, 649-671.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
metformin, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine,
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Methoxsalen
Merck Index: 6068
Lednicer No.: 1 333
SAMPLE
Matrix: blood
Sample preparation: 1 mL Whole blood + 2.5 mL 1 M pH 9 borate buffer, mix well, add 8 mL
n-hexane:isopropanol 95:5, shake at 80-100 strokes/min for 10 min, centrifuge at 5 at 1500 g.
Remove 7.0 mL of the organic layer and evaporate it to dryness under a stream of nitrogen at
60, reconstitute the residue in 100 JJLL mobile phase, inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Partisil silica
Mobile phase: DichloromethaneiMeCN 95:5
Flow rate: 2.2
Detector: UV 254
CHROMATOGRAM
Retention time: 3.8
KEYWORDS
normal phase; whole blood; dog; human; pharmacokinetics
REFERENCE
Puglisi,C.V.; de Silva,J.A.R; Meyer,J.C. Determination of 8-methoxypsoralen, a photoactive compound, in blood
by high pressure liquid chromatography, Anal.Lett., 1977, 20, 39-50.
SAMPLE
Matrix: blood
mL water. Add 500 jxL plasma to the SPE cartridge, wash with 9 mL water, elute with 500 (xL
500 ng/mL 5-methoxypsoralen in MeOH, vortex the eluate, inject a 20 |xL aliquot of the eluate.
HPLC VARIABLES
Column: 100 mm long Spheri-5 RP-18
Mobile phase: MeCN.MeOHrwater 10:25:65
Flow rate: 0.7
Detector: UV 300
CHROMATOGRAM
Retention time: 5.0
Internal standard: 5-methoxypsoralen (bergapten) (8.5)
OTHER SUBSTANCES
Noninterfering: acetaminophen, alprazolam, amikacin, amobarbital, caffeine, carbamazepine,
chloramphenicol, diazepam, disopyramide, gentamicin, lidocaine, lithium, lorazepam, mepro-
bamate, mexiletine, phenobarbital, phenytoin, procainamide, propranolol, quinidine, salicylic
acid, theophylline, tobramycin, valproic acid, vancomycin
KEYWORDS
SPE; plasma
REFERENCE
Ketchum,C.H.; Robinson,C.A.,Jr.; Huang,S.T. Analysis of 8-methoxypsoralen by high-performance liquid chro-
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 60 |xm Silicacart C18 SPE cartridge (Tessek) with 5 mL
MeOH and 5 mL water. 2 mL Plasma + 2 mL MeCN + 1 mL 6.25 |jig/mL griseofulvin in MeOH,
shake by hand for 1-2 s, centrifuge at 500 g for 10 min, add 4 mL of the supernatant to the
SPE cartridge, wash with 5 mL water, elute with 2 mL MeOH. Evaporate the eluate to dryness
under vacuum at 38, reconstitute in 1 mL mobile phase, inject a 50 (JLL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Spherisorb 5 ODS
Mobile phase: MeCN.lO mM phosphoric acid 34:66, pH 2.82
Flow rate: 1
Detector: UV 248
CHROMATOGRAM
Internal standard: griseofulvin (12.13)
KEYWORDS
plasma; SPE
REFERENCE
Kucovd,D.; Maryskov&,D.; Davidkova,P.; Gasparic,J. High-performance liquid chromatographic determination
of methoxsalen in plasma after liquid-solid extraction, J.Chromatogr., 1993, 614, 340-344.
SAMPLE
Matrix: blood
Sample preparation: Inject 25 JJLL plasma onto column A with mobile phase A, elute column A
with mobile phase A to waste, after 8 min backflush the contents of column A onto column B
with mobile phase B, monitor the effluent from column B. (Re-equilibrate column A with mobile
phase A for 2 min before next injection.)
HPLC VARIABLES
Column: A 250 X 4 25 |xm C8-Alkyl-Diol Silica (some preparation details given); B 125 X 4 5
jxm LiChrospher RP-18
Mobile phase: A water; B MeOH:water 60:40
CHROMATOGRAM
Retention time: 4
KEYWORDS
plasma; column-switching; protect from light
REFERENCE
Vielhauer,S.; Rudolphi,A.; Boos,K.-S.; Seidel,D. Evaluation and routine application of the novel restricted-access
precolumn packing material Alkyl-Diol Silica: coupled-column high-performance liquid chromatographic
analysis of the photoreactive drug 8-methoxypsoralen in plasma, J.Chromatogr.B, 1995, 666, 315-322.
Methoxyphenamine
Merck Index: 6077
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.3
OTHER SUBSTANCES
methdilazene, methotrimeprazine, methoxamine, methoxypromazine, methylephedrine, meth-
REFERENCE
Methoxypromazine
Molecular formula: C18H22N2OS
Merck Index: 6080
Lednicer No.: 1 387
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 5.3
OTHER SUBSTANCES
methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methylephedrine, meth-
REFERENCE
Methscopolamine bromide
Molecular formula: Ci8H24BrNO4
Merck Index: 6084
SAMPLE
Sample preparation: Crush 10 tablets, add 250 mL 50 mM HCl in EtOH:water 50:50, heat for
15 min on a steam bath, shake mechanically for 2 h, filter (glass fiber GF/A, Whatman), inject
a 30 |xL aliquot of the filtrate.
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Partisil-10-ODS
Mobile phase: MeCN:buffer 50:50 (Buffer was 2.85 mM ethylenediamine sulfate adjusted to pH
7.44 0.02 with 1 M ammonium hydroxide.)
Flow rate: 3.8
Detector: UV 216.5
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: aposcopolamine, pheniramine, phenylpropanolamine, pyrilamine, tropic acid
KEYWORDS
tablets
REFERENCE
Heidemann,D.R. High-pressure liquid chromatographic determination of methscopolamine nitrate, phenylpro-
panolamine hydrochloride, pyrilamine maleate, and pheniramine maleate in tablets, J.Pharm.Sci., 1981,
70, 820-822.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1.5 mg/mL solution, inject a 10 |xL aliquot.
HPLCVARIABLES
Guard column: Supelguard LC-8-DB (Supelco)
Mobile phase: MeCNibuffer 10:90 containing 0.02% triethylamine (Buffer was KH2PO4 adjusted
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Simultaneous: chlorpheniramine, phenylpropanolamine, pseudoephedrine, triprolidine
REFERENCE
Methsuximide
Merck Index: 6085
Lednicer No.: 1 228
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Serum + 100 |xL buffer + 1.5 mL IS in 5% isopropanol in chloro-
a 6-10 JJLL aliquot. (Buffer was 13.6 g KH2PO4 in 90 mL water, pH adjusted to 6.8 with about
HPLC VARIABLES
Column: 250 X 4.6 5 u,m Supelcosil LC-I (Supelco)
Flow rate: 2
Detector: UV 204
CHROMATOGRAM
Internal standard: 3-isobutyl-l-methylxanthine(3.15)
OTHER SUBSTANCES
Extracted: acetaminophen, amobarbital, barbital, caffeine, carbamazepine, chloramphenicol,
ethosuximide, mephobarbital, pentobarbital, phenobarbital, phenytoin, primidone, secobarbi-
tal, theophylline, thiopental
Also analyzed: acetanilide, acetylcysteine, acetylprocainamide, ampicillin, aspirin, butabarbital,
butalbital, chlorpropamide, cimetidine, codeine, cyheptamide, diazoxide, diflunisal, diphylline,
disopyramide, ethchlorvynol, gentisic acid, glutethimide, heptabarbital, hexobarbital, ibupro-
fen, indomethacin, ketoprofen, mefenamic acid, mephenytoin, methaqualone, methsuximide,
methyl salicylate, methyprylon, morphine, naproxen, nirvanol, oxphenylbutazone, phenacetin,
phensuximide, phenylbutazone, procainamide, salicylamide, salicylic acid, sulfamethoxazole,
sulindac, tolmetin, trimethoprim, vancomycin
KEYWORDS
serum
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeOH, inject a 3 |xL aliquot.
HPLC VARIABLES
Column: 125 X 4.5 5 |xm SAS Hypersil
Mobile phase: MeCNrbuffer 20:80 (Buffer was 5 mM tetrabutylammonium hydroxide adjusted
Flow rate: 1.6
Injection volume: 3
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: amobarbital, barbital, butabarbital, carbamazepine, ethosuximide, ethotoin,
ethylphenacemide, glutethimide, heptabarbital, pheneturide, phenobarbital, phenylethyl-
malonamide, phenytoin, primidone, secobarbital, sulfamethoxazole, sulthiame
Interfering: cyclobarbital, pentobarbital
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Inject a 30-100 JJLL aliquot.
HPLCVARIABLES
5:95 for 5 min.
Flow rate: 3
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: acetaminophen, amitriptyline, amobarbital, butabarbital, butalbital, caffeine,
chlordiazepoxide, clomipramine, codeine, desipramine, diazepam, ethchlorvynol, ethotoin, flur-
azepam, glutethimide, hexobarbital, imipramine, lidocaine, mesantoin, methaqualone, methy-
prylon, nirvanol, nitrazepam, nortriptyline, oxazepam, pentobarbital, phenobarbital, phenyl-
propanolamine, phenytoin, primidone, procainamide, propranolol, quinidine, salicylic acid,
secobarbital, theophylline
REFERENCE
tography, JAnal.Toxicol, 1981, 5, 177-182.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1.5
CHROMATOGRAM
Retention time: 6.6
OTHER SUBSTANCES
Simultaneous: barbital, carbamazepine, diazepam, ethotoin, mephenytoin, phenacemide, phen-
obarbital, phensuximide
REFERENCE
Vydac HPLC Catalog, 1994-5, 1994, p. 26.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
methap5rrilene, methaqualone, methazolamide, methocarbamol, methyl salicylate, methyldopa,
methyldopamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon,
metoprolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline,
trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tjâmine, verapa-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 62 X 2 packed with chiral packing (Prepare packing by dissolving 4-chloro-3-methyl-
phenylcarbamate cellulose in THF, coat on Nucleosil 1000-7, dry at 60 for 3 h under reduced
pressure.)
Flow rate: 0.1
Detector: UV 254
CHROMATOGRAM
KEYWORDS
REFERENCE
Chankvetadze,B.; Chankvetadze,L.; Sidamonidze,S.; Yashima,E.; Okamoto,Y. Enantioseparation of some chiral
Pharmaceuticals using narrow-bore liquid chromatography, J.Pharm.BiomedAnaL, 1995, 13, 695-699.
Methyclothiazide
Molecular formula: C 9 H 1 , GI2N3O4S2
Merck Index: 6086
Lednicer No.: 1 360
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 2 mL 100 mM NaOH + 8 mL ethyl acetate, vortex vig-
orously for 30 s, centrifuge at 10 at 2000 g for 10 min. Remove the organic layer and pass it
through a 35 X 6 column of anhydrous magnesium sulfate, rinse column with 2 mL ethyl
acetate. Evaporate the eluate to dryness under a stream of nitrogen at 50, reconstitute the
residue in 100 |xL 8 jxg/mL phenacetin in MeOH, inject a 20 \LL aliquot.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 225
CHROMATOGRAM
Retention time: 7.5
Internal standard: phenacetin (11)
OTHER SUBSTANCES
Noninterfering: clonidine, methyldopa, prazosin, propranolol
KEYWORDS
plasma
REFERENCE
Hartman,C.A.; Kucharczyk,N.; Sofia,R.D.; Perhach,J.L.,Jr. Determination of methyclothiazide in human
plasma by high-performance liquid chromatography, J.Chromatogr., 1981, 226, 510-513.
SAMPLE
residue with 50 JULL MeCNiwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 226.3
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: urine
Sample preparation: 2 mL Urine + 2 mL 1 M pH 4.1 NaH2PO4 + 4 mL ethyl acetate, vortex
for 2 min, centrifuge at 1500 g for 5 min. Remove the organic phase and add it to 5 mL 100
mM pH 7.5 Na2HPO4, vortex for 2 min, centrifuge at 1500 g for 5 min. Remove the organic
in 100 JJLL MeCNrIO mM pH 3.0 phosphate buffer, inject a 5 |xL aliquot.
HPLC VARIABLES
Column: 125 X 4 5 jxm LiCHrosorb RP-18
Mobile phase: Gradient. MeCN: 10 mM pH 3.0 phosphate buffer 10:90 for 1.5 min then to 35:65
over 2 min
Flow rate: 1.5
Injection volume: 5
Detector: UV 271
CHROMATOGRAM
Retention time: 4.7
OTHER SUBSTANCES
Extracted: chlorothiazide, hydrochlorothiazide, quinethazone, chlorthalidone, furosemide, me-
tolazone, mefruside, bendroflumethiazide, cyclopenthiazide, bumetanide
Simultaneous: indapamide, clorexolone, ethacrynic acid
Noninterfering: aspirin, albuterol, allopurinol, alprenolol, atenolol, captopril, carbimazole, clo-
nidine, coloxyl, danthron, diazepam, digoxin, doxepin, glibenclamide, hydralazine, indometh-
acin, labetalol, metformin, methyldopa, metoprolol, mianserin, minoxidil, nifedipine, nitraze-
pam, oxazepam, oxprenolol, pindolol, prazosin, propranolol, senokot, theophylline,
trifluoperazine
Interfering: clopamide
REFERENCE
Fullinfaw,R.O.; Bury,R.W.; Moulds,R.F.W. Liquid chromatographic screening of diuretics in urine,
J.Chromatogr., 1987, 415, 347-356.
SAMPLE
Matrix: urine
300 |xL 50 |xg/mL p-hydroxyethyltheophylline in MeOH, inject 5 ^xL aliquot. (Solid buffer I was
HPLC VARIABLES
Column: 250 X 4.6 5 jjim HP Hypersil ODS (A) or HP LiChrosorb RP-18 (B)
Flow rate: 1
Injection volume: 5
CHROMATOGRAM
OTHERSUBSTANCES
Extracted: furosemide, metolazone, amiloride, acetazolamide, chlorothiazide, hydrochlorothi-
azide, quinethazone, triamterene, hydroflumethiazide, chlorthalidone, dichlorphenamide, tri-
chloromethiazide, benzthiazide, cyclothiazide, polythiazide, bendroflumethiazide, ethacrynic
acid, bumetanide, probenecid, spironolactone, canrenone, flumethiazide
REFERENCE
Cooper,S.R; Mass6,R-", Dugal,R. Comprehensive screening procedure for diuretics in urine by high-performance
Methyl salicylate
Merck Index: 6200
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood + 100 jxL 2 M NaOH + 10 mL dichloromethane, rotate
for 10 min, centrifuge for 5 min. Remove 6 mL of the organic layer and evaporate it to dryness,
reconstitute the residue in 1 mL 100 mM NaOH, heat at 60 for 2 h, cool, add 200 JJLL 1 M
HCl, add 200 |xL 1 mg/mL p-hydroxyethyltheophylline in MeOH, add 5 mL dichloromethane,
rotate for 10 min, centrifuge for 5 min. Remove the organic layer and evaporate it to dryness,
reconstitute the residue in 200 yJL MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 jjim ODS (Altex)
Mobile phase: MeOH:water:glacial acetic acid 40:66:1
Flow rate: 1.5
Detector: UV 250
CHROMATOGRAM
Retention time: 6.4 (salicylic acid), 13 (unhydrolyzed methyl salicylate)
Internal standard: p-hydroxyethyltheophylline (2)
OTHER SUBSTANCES
Simultaneous: theophylline
Noninterfering: acetaminophen, amobarbital, butalbital, caffeine, carbamazepine, glutethimide,
ibuprofen, indomethacin, meprobamate, methaqualone, pentobarbital, phenobarbital, phenyl-
butazone, phenytoin, primidone, secobarbital
KEYWORDS
whole blood; methyl salicylate is hydrolyzed to salicylic acid
REFERENCE
Levine,B; Caplan,Y.H. Liquid chromatographic determination of salicylate and methyl salicylate in blood and
application to a postmortem case, JAnal.ToxicoL, 1984, 8, 239-241.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 6-10 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-I (Supelco)
Flow rate: 2
Detector: UV 204
CHROMATOGRAM
Retention time: 7.17 (tails)
OTHER SUBSTANCES
Simultaneous: acetaminophen, acetanilide, N-acetylcysteine, N-acetylprocainamide, amobarbi-
tal, ampicillin, aspirin, barbital, butabarbital, butalbital, caffeine, carbamazepine, chloram-
phenicol, chlorpropamide, codeine, cyheptamide, diazoxide, diflunisal, diphylline, disopyram-
ide, ethchlorvynol, gentisic acid, glutethimide, heptabarbital, hexobarbital, ibuprofen,
indomethacin, ketoprofen, mefenamic acid, mephenytoin, mephobarbital, methaqualone, meth-
suximide, methyprylon, morphine, naproxen, nirvanol, oxphenylbutazone, pentobarbital, phe-
nacetin, phenobarbital, phensuximide, phenytoin, procainamide, salicylamide, salicylic acid,
secobarbital, sulfamethoxazole, sulindac, theophylline, thiopental, tolmetin, trimethoprim,
vancomycin
Interfering: ethosuximide, cimetidine, primidone, phenylbutazone
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Inertsil C8
Mobile phase: MeCN:water 55:45 containing 10 mM sodium dodecanesulfonate
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: diphenhydramine
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
ethaciynic acid, ethosuximide, etonitazene, etorphine, eugenol, famotidine, fenbendazole, fen-
stilbene, imipramine, indomethacin, isocarbostjrril, isocarboxazid, isoniazid, isoproterenol,
methapyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methyldopa,
oxjrphenbutazone, oxjrtetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbi-
puromycin, pj/rilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
faethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapjrridine, sul-
REFERENCE
18, 233-242.
Methyldopa
CAS Registry No.: 555-30-6,41372-08-1 (sesquihydrate)
Merck Index: 6132
Lednicer No.: 1 95
SAMPLE
Matrix: blood
Sample preparation: Add 50 ^xL 2 M HCl to 50 |xL plasma, make up to 170 JJLL with water,
centrifuge at 1400 rpm for 5 min, inject an aliquot of the supernatant.
HPLCVARIABLES
Column: 100 X 8 4 jxm NovaPak C18
Mobile phase: MeCN:MeOH:10 mM pH 3 sodium acetate buffer 47.25:15.75:37
Flow rate: 2
CHROMATOGRAM
Internal standard: methyldopa
OTHER SUBSTANCES
Extracted: propofol
Noninterfering: albuterol, ampicillin, amoxicillin, amphotericin B, bleomycin, ceftazidime, cef-
oxitin, cephalexin, ciprofloxacin, dobutamine, dopamine, epinephrine, erythromycin, esmolol,
fluconazole, gentamicin, labetalol, metoclopramide, miconazole, nitroglycerin, nitroprusside,
norepinephrine, paclitaxel, penicillin G benzathine, ranitidine, streptomycin, tetracycline
KEYWORDS
plasma; methyldopa is IS
REFERENCE
el-Yazigi,A.; Hussein,R-F. Microdetermination of propofol in plasma by a rapid and sensitive liquid chromato-
graphic method, J.Pharm.Biomed.Anal., 1996, 15, 99-104.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 fxL 10 (xg/mL dopa, vortex, add 50 |xL perchloric acid,
vortex for 5 s, let stand at -20 for 15 min, centrifuge at 4 at 4000 rpm for 15 min, inject a 17
IxL aliquot of the supernatant.
HPLC VARIABLES
Column: 125 X 4 5 jxm Hypersil ODS
Mobile phase: MeOH:buffer 16:130 (Buffer contained 23.8 g/L phosphoric acid, 15.4 g/L KH2PO4,
and 3.4 g/L PIC B8.)
Flow rate: 1
Detector: E, Metrohm 656, +0.75 V
CHROMATOGRAM
Internal standard: dopa
KEYWORDS
REFERENCE
Dilger,C; Salama,Z.; Jaeger,H. Determination of methyldopa in plasma using high-performance liquid chro-
matography with electrochemical detection. Application to pharmacokinetic/bioavailability studies, Arz-
neimittelforschung, 1987, 37, 1399-1401.
SAMPLE
2, centrifuge at 4 at 4000 g for 3 min. Remove 200 |xL of the supernatant and add it to 100
IxL 80 ng/mL N-methyldopamine, evaporate to dryness under vacuum, reconstitute in 200 |xL
mobile phase, inject a 5-20 |xL aliquot. Urine. 1 mL Urine + 50 mL water, inject a 10 JJLL aliquot.
with 50 mL water, inject a 10 (xL aliquot.
HPLC VARIABLES
Flow rate: 1
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Extracted: norepinephrine, epinephrine, dopamine, dihydroxyphenylacetic acid, 3-O-methyl-
methyldopa, homovanilic acid
KEYWORDS
REFERENCE
541, 285-296.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Powder tablets or contents of capsules, weigh out an amount equivalent
to about 100 mg levodopa, add 30 mL 0.1 M HCl, sonicate, make up to 50 mL with 0.1 M HCl,
mix, filter (0.45 |xm), discard first 5 mL filtrate. 5 mL Filtrate + 10 mL 2 mg/mL methyldopa
in 0.1 M HCl, make up to 100 mL with mobile phase, mix, inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: 3% aqueous acetic acid
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Retention time: 4.5
Internal standard: methyldopa
OTHER SUBSTANCES
Simultaneous: levodopa
KEYWORDS
capsules; tablets; methyldopa is IS
REFERENCE
Ting,S. Liquid chromatographic determination of levodopa and levodopa-carbidopa in solid dosage forms: col-
laborative study, J.Assoc.Off.Anal.Chem., 1987, 70, 987-990.
SAMPLE
Sample preparation: Grind beads from sustained-release microcapsules containing 500 mg
methyldopa to a fine powder, add 50 mL 50 mM sulfuric acid, sonicate for 15 min, make up to
100 mL with 50 mM sulfuric acid. Filter, reject the first 10 mL of the filtrate, make up a 10
mL aliquot to 100 mL with 50 mL sulfuric acid, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jjum CN (Phenomenex)
Mobile phase: MeOH:water:acetic acid 20:80:2 containing 5 mM sodium 1-heptanesulfonate, pH
adjusted to 2.60
Flow rate: 1.6
Detector: UV 280
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
Simultaneous: degradation products, 3-O-methylmethyldopa
KEYWORDS
microcapsules; stability-indicating
REFERENCE
Metwally,M.E.-S. Stability-indicating high-performance liquid chromatographic assay for a-methyldopa in sus-
tained-release capsules, J.Chromatogr., 1991, 549, 221-228.
SAMPLE
Sample preparation: Dissolve in mobile phase, filter, inject an aliquot.
HPLC VARIABLES
Mobile phase: MeOH:50 mM ammonium acetate adjusted to pH 4.1 with 0.6 M acetic acid 1:99
Flow rate: 0.9
Detector: E, Coulochem model 5100A, screen electrode +0.3 V, sample electrode +0.6 V and UV
280
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: hydroxydopa, carbidopa, levodopa, methoxytyrosine, methylcarbidopa, impu-
rities
KEYWORDS
stability-indicating; tablets
REFERENCE
Kafil,J.B.; Dhingra,B.S. Stability-indicating method for the determination of levodopa, levodopa-carbidopa and
related impurities, J.ChromatogrA, 1994, 667, 175-181.
SAMPLE
HPLC VARIABLES
Mobile phase: Isopropanolibuffer 5:95 (Buffer was 100 mM sodium dodecyl sulfate containing
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
Simultaneous: carbidopa, dopamine, epinephrine, hydrochlorothiazide, isoproterenol, levodopa,
KEYWORDS
REFERENCE
Villanueva Camanas,R-M.; Sanchis Mallols,J.M.; Torres Lapasi6,J.R.; Ramis-Ramos,G. Analysis of pharmaceu-
tical preparations containing catecholamines by micellar liquid ehromatography with spectrophotometric
detection, Analyst, 1995, 12O7 1767-1772.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1
Detector: UV 201
REFERENCE
mans, J.Pharm.ScL, 1996, 85, 1070-1076.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
faethidole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapîdine, sul-
REFERENCE
18, 233-242.
Methyldopate
SAMPLE
Matrix: solutions
Sample preparation: Dilute with 5% dextrose, inject a 15 jxL aliquot.
HPLC VARIABLES
Flow rate: 1.6-2.0
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: theophylline, terbutaline
Interfering: isoproterenol
REFERENCE
catecholamines with aminophylline in intravenous solutions, J.Pharm.Sci., 1982, 71, 956-958.
SAMPLE
Matrix: solutions
|xL aliquot.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
REFERENCE
Methylene blue
Molecular formula: C16H18CIN3S
CAS Registry No.: 61-73-4, 7220-79-3 (trihydrate)
Merck Index: 6137
SAMPLE
Matrix: blood
Sample preparation: Condition a 40 |xm Sepralyte non-bonded silica SPE cartridge (Analyti-
chem) with I m L l M HCl, with 3 mL water, and with 500 |xL 2 M (NH4)H2PO4. Centrifuge 1
mL plasma at 4 at 12000 g for 1 min, add the supernatant to the SPE cartridge at 1 mL/min,
vortex the precipitate in the centrifuge tube with 500 jxL 0.3% ascorbic acid in 12 mM pH 3.0
citrate buffer, add the mixture to the SPE cartridge, wash with 500 |xL water, wash with 500
JULL MeCN saturated with tetramethylammonium chloride, apply suction for 5 min, elute with
300 JJLL elution solvent by centrifuging at 4 at 2000 g for 5 min, inject an aliquot of the eluate.
(The elution solvent was MeOH: 1 M tetramethylammonium chloride: 1 M citric acid:water 32:
10:20:38.)
HPLC VARIABLES
Column: 75 X 3.9 4 ^m Nova-pak ODS
Mobile phase: MeOH:THF:triethylamine phosphate solution: 1 M tetramethylammonium chlo-
ride:water 30:1:10:2:57 (Triethylamine phosphate was 67.8 mL orthophosphoric acid in 800 mL
water, adjust pH to 3.0 with triethylamine, make up to 1 L with water.) (Filter mobile phase
but do not degas.)
Flow rate: 1
Detector: UV 658
CHROMATOGRAM
Retention time: 7
Internal standard: methylene blue
OTHER SUBSTANCES
Extracted: mitoxantrone
KEYWORDS
plasma; SPE; use polypropylene containers; methylene blue is IS
REFERENCE
Lin,K.T.; Rivard,G.E.; Leclerc,J.-M. High-performance liquid chromatographic determination of mitoxantrone
in plasma utilizing non-bonded silica gel for solid-phase isolation to reduce adsorptive losses on glass during
sample preparation, J.Chromatogr., 1989, 465, 75-86.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6, 5 |xm TSKgel ODS 80 TM (TOSOH, Japan)
Mobile phase: MeCN:25 mM pH 6.5 imidazole buffer 60:40 containing 10 mM sodium 1-
propanesulfonate
Flow rate: 1.0
0.25 mM bis(4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (Wako) and 25 mM hydro-
gen peroxide in MeCN pumped at 1.3 mL/min and the mixture flowed to the detector (a red
sensitive photomultiplier).
CHROMATOGRAM
Retention time: 3.5
Limit of detection: 120 fmol
OTHER SUBSTANCES
Simultaneous: pyridine 1, oxazine 1, 3,3'-diethylthiadicarbocyanine iodide (DTDCI)
KEYWORDS
peroxyoxalate chemiluminescence (PO-CL) detection
REFERENCE
Kimoto,K.; Gohda,R-; Murayama,K; Santa,R; Fukushima,T.; Homma,H.; Imai,K. Sensitive detection of near-
infrared fluorescent dyes using high-performance liquid chromatography with peroxyoxalate chemilumi-
nescence detection system, Biomed.Chromatogr., 1996, 10, 189-190.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 100 (JLL aliquot.
HPLC VARIABLES
Mobile phase: MeCN.buffer 40:60 (Prepare buffer by mixing 400 mL MeCN, 300 mL water, 5
mL glacial acetic acid, and a vial of low-UV PIC B5 (pentanesulfonic acid, Waters), make up
to 1 L with water.)
Flow rate: 1
Detector: UV 664
CHROMATOGRAM
Retention time: 4.6
REFERENCE
Haddad,P.R.; Heckenberg,A.L. Trace determination of sulfide by reversed-phase ion-interaction chromatogra-
phy using pre-column derivatization, J.Chromatogr., 1988, 447, 415-420.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm CN (Alltech)
Next Page
Mobile phase: MeOH: 100 mM sodium acetate 60:40 containing 50 mg/L disodium EDTA, pH
adjusted to 4.5 with aldehyde free glacial acetic acid
Flow rate: 0.8
Detector: E, Bioanalytical Systems LC-4B, glassy carbon working electrode +1.000 V, Ag/AgCl
reference electrode
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: metabolites, leucogentian violet
Interfering: gentian violet
REFERENCE
Roybal,J.E.; Munns,R.K; Hurlbut,J.A.; Shimoda,W. High-performance liquid chromatography of gentian violet,
its demethylated metabolites, leucogentian violet and methylene blue with electrochemical detection,
J.Chromatogr., 1989, 467, 259-266.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 20 X 4 Keystone ODS/H
Mobile phase: MeCNrIO mM KH2PO4 40:60 containing 15 mM sodium dodecyl sulfate, pH 2.8
Flow rate: 2
Detector: UV 280
CHROMATOGRAM
Retention time: 1
OTHER SUBSTANCES
Simultaneous: thionin
REFERENCE
SAMPLE
Matrix: tissue
Sample preparation: 100-300 mg minced tissue + 500 (xL MeCN:MeOH:water, mix, filter (10000
molecular weight cut-ofD, inject a 20 |xL aliquot of the ultrafiltrate.
HPLC VARIABLES
Flow rate: 0.4
Detector: MS, Hewlett-Packard 5988A, particle beam, EI, 70 eV, source 300, desolvation cham-
ber 35 or Finnigan MAT 4500 quadrupole, thermospray, vaporizer 180-195, source 250
CHROMATOGRAM
Retention time: 6
Limit of detection: 1.3 ppm (particle beam)
KEYWORDS
cow; muscle
REFERENCE
Voyksner,R.D.; Smith,C.S.; Knox,P.C. Optimization and application of particle beam high-performance liquid
chromatography/mass spectrometry to compounds of pharmaceutical interest, Bio-
med.Environ.Mass.Spectrom., 1990, 19, 523-534.
Previous Page
Methylephedrine
Merck Index: 6145
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: MeOH containing 10 mM ammonium perchlorate and 1 mL'li 100 mM NaOH in
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.9
OTHER SUBSTANCES
idine, cinchonidine, cinnarizine, clemastine, clomipramine, clonidine, cocaine, cyclazocine,
cyclizine, cyclopentamine, cyproheptadine, deserpidine, desipramine, dextromoramide, dex-
tropropoxyphene, dicyclomine, diethylcarbamazine, diethylpropion, diethylthiambutene, dihy-
droergotamine, dimethindene, dimethothiazine, diphenhydramine, diphenoxylate, dipipanone,
diprenorphine, dipyridamole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, dro-
peridol, ephedrine, ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergo-
sine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine,
fenoterol, fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloper-
idol, hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isoxsu-
prine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamylamine,
meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, meptazinol, mepyra-
mine, mesoridazine, metaraminol, methadone, methamphetamine, methapyrilene, methdila-
zene, methotrimeprazine, methoxamine, methoxyphenamine, methoxypromazine, methyler-
gonovine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, morazone,
penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phenamprom-
ide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide, pheninda-
prothipendyl, protriptyline, proxymetacaine, pseudoephedrine, pjrrimethamine, quinidine, qui-
REFERENCE
Methylergonovine
Merck Index: 6147
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 1 mL 500 mM potassium carbonate, mix for 5 s, add 10
mL ethyl acetate, shake horizontally for 15 min, centrifuge at 700-1000 g for 5 min. Remove
8 mL of the upper organic phase and add it to 700 |xL 50 mM phosphoric acid, shake horizon-
tally for 15 min, centrifuge at 750 g for 2 min, discard the organic phase. Heat the aqueous
phase at 50 in a vortex evaporator to remove residual organic solvent, inject a 400 (xL aliquot.
HPLCVARIABLES
Mobile phase: MeCN: 10 mM pH 7 potassium phosphate buffer 30:70
Flow rate: 1
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
Extracted: methysergide
KEYWORDS
plasma; protect from sunlight; pharmacokinetics
REFERENCE
Smith,H.T.; Molinaro,N.C. High-performance liquid chromatographic method for the determination of meth-
ysergide and methylergonovine in human plasma, J.Chromatogr., 1988, 424, 416423.
SAMPLE
Matrix: blood
Sample preparation: 0.1-0.7 mL Plasma or whole blood + 2.5 mL HCl/ammonia solution + 200
fjuL 100 nM ergometrine in water, mix, add to an Extrelut silica SPE cartridge, let stand for
20 min, elute with two 5 mL portions of ethyl acetate. Evaporate the eluate to dryness under
a stream of nitrogen below 40, reconstitute the residue in mobile phase, inject a 25-150 |xL
aliquot. (HCl/ammonia solution was 2 M HCl and 1 M ammonia mixed to a pH of 9.0.)
HPLC VARIABLES
Column: 250 mm long 5 jjim Hypersil C18 ODS
Flow rate: 2
CHROMATOGRAM
Retention time: 3.0
Internal standard: ergonovine (ergometrine) (2.4)
OTHER SUBSTANCES
Extracted: methysergide
KEYWORDS
rat; plasma; whole blood; SPE; pharmacokinetics
REFERENCE
DrugMetab.Dispos., 1990, 18, 338-343.
SAMPLE
Sample preparation: Injections. 1 mL Injection (200 |xg/mL) + 300 mg NaCl + 200 |xL 10%
stitute the residue in 4 mL water, mix an aliquot with an equal volume of 30 |xg/mL 17a-
hydroxyprogesterone in MeOH, inject a 20 |xL aliquot. Tablets. Weigh out amount of powdered
of 30 |xg/mL 17a-hydroxyprogesterone in MeOH, inject a 20 |xL aliquot.
HPLCVARIABLES
Column: 150 X 4 5 ^m LiChrosorb RP-18
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 9
Internal standard: 17a-hydroxyprogesterone (12)
OTHER SUBSTANCES
Simultaneous: benzyl alcohol, ergonovine
KEYWORDS
injections; tablets
REFERENCE
1983, 31, 3988-3993.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.2
OTHER SUBSTANCES
methylephedrine, methysergide, metoclopramide, metopimazine, metoprolol, mianserin, mor-
thipendyl, protriptyline, proxymetacaine, pseudoephedrine, p5rrimethamine, quinidine, qui-
REFERENCE
Methylprednisolone
CAS Registry No.: 83-43-2, 53-36-1 (acetate),
5015-36-1 (sodium phosphate), 2375-03-3 (sodium succinate),
2921-57-5 (hemisuccinate), 90350-40-6 (suleptanate)
Merck Index: 6189
Lednicer No.: 1 193, 196
SAMPLE
MeOH and 3 mL water. 1 mL Serum or urine + 500 uX 200 mM pH 3.85 acetate buffer (serum
only) + 400 (xL 2.5 jxM IS in mobile phase, mix, centrifuge. Add the supernatant to the SPE
cartridge, wash with 3 mL acetone:water 20:80, 3 mL water, and 3 mL hexane. Elute with 3
mL diethyl ether into tubes containing 1 mL 200 mM NaOH, vortex, centrifuge. Dry the organic
layer under a stream of nitrogen. Reconstitute the residue in 250 |xL mobile phase, mix for 5
min. Inject a 60 u-L aliquot.
HPLC VARIABLES
Flow rate: 1.0
Detector: UV 254
CHROMATOGRAM
Internal standard: fludrocortisone (15.9)
OTHER SUBSTANCES
Extracted: 11-deoxycortisol, dexamethasone, hydrocortisone, prednisolone
KEYWORDS
serum; SPE
REFERENCE
McWhinney,B-C.; Ward,G.; Hickman,P.E. Improved HPLC method for simultaneous analysis of cortisol, 11-
deoxycortisol, prednisolone, methylprednisolone, and dexamethasone in serum and urine, Clin.Chem., 1996,
42, 979-981.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 245.2
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.ChromatognA, 1997, 763,149
163.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 4.6 5 |xm Inertsil Ph (GL Science, Japan)
Mobile phase: MeCN:THF:buffer 21:4:75 (Buffer was 11.6 g ammonium acetate in 750 mL water,
adjusted to pH 5.8 with acetic acid.)
Flow rate: 1.6
Detector: UV 254
OTHER SUBSTANCES
Simultaneous: methylprednisolone 17-suleptanate, PNU-675004
REFERENCE
Okamoto,H.; Mori,K; Ohtsuka,K; Ohuchi,H.; Ishii,H. Effect of ionic strength on solution of PNU-67590A, a
micellar prodrug of methylprednisolone, Pharm.Res., 1997, 14, 1181-1185.
SAMPLE
Matrix: urine
Sample preparation: Activate 3-mL 500 mg Bakerbond C18 cartridge with 2 mL MeOH and 2
mL water. Filter sample. Add 2 mL urine to the SPE cartridge, wash with two 2 mL portions
of 25 mM borate buffer and with 200 mL/L acetone in water. Add 1 mL hexane and air-dry
under reduced pressure for 4 min. Elute with two 1 mL portions of ethyl acetate. Dry the
eluate under a stream of nitrogen and dissolve in 75 |xL 400 mL/L MeOH, inject a 25 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm LiChrospher 100 C18
Flow rate: 1
Detector: UV 242
CHROMATOGRAM
Internal standard: 6a-methylprednisolone
OTHER SUBSTANCES
Extracted: hydrocortisone
Simultaneous: metabolites, alprazolam, amlodipine, aspirin, carbamazepine, citalopram, corti-
sone, dexamethasone, digoxin, enalapril, ferrous sulfate, fluoxetine, furosemide, gabapentin, 5-
hydroxyindoleacetic acid, lamotrigine, metyrapone, naproxen, oxazepam, oxcarbazepine, oxy-
butynin, phenobarbital, phenytoin, prednisone, prednisolone, spironolactone, valproic acid,
Noninterfering: octreotide
Interfering: corticosterone
KEYWORDS
SPE; methylprednisolone is IS
REFERENCE
Turpeinen,U.; Markkanen,H.; Valimaki,M.; Stenman,U.-H. Determination of urinary free cortisol by HPLC,
Clin.Chem., 1997, 43, 1386-1391.
Methyltestosterone
Merck Index: 6206
Lednicer No.: 1 172, 4 11
SAMPLE
Sample preparation: 1 Tablet + 4 mL 50 mM KH2PO4, rotate 15 min, add 2 mL 1 |xg/mL o-
phenylphenol in mobile phase, add 4 mL MeOH, rotate 15 min, centrifuge. Remove superna-
tant, extract residue twice with 5 mL mobile phase (10 min rotation), combine supernatants,
inject 50 (xL aliquot.
HPLC VARIABLES
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: norgestrel, norethindrone, norethindrone acetate, ethinyl estradiol
KEYWORDS
REFERENCE
Strusiak,S.H.; Hoogerheide,J.G.; Gardner,M.S. Determination of ethinyl estradiol in solid dosage forms by high-
SAMPLE
min, centrifuge, inject a 10 JJLL aliquot of the supernatant. Tablets. Grind a tablet to a fine
powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 jim), discard first 5 mL of filtrate,
remaining filtrate.
HPLC VARIABLES
Mobile phase: MeOHiwater 75:25
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: 8.0
OTHER SUBSTANCES
Simultaneous: calusterone, mesterolone (UV 210), norethandrolone, trenbolone acetate, benzyl
benzoate, nandrolone acetate, testosterone acetate, stanozolol, oxymetholone, nandrolone pro-
pionate, methenolone acetate, testosterone propionate, aspirin, caffeine, formebolone, benzyl
alcohol, testolactone, cortisone, fluoxymesterone, norethindrone, oxandrolone (UV 210), bolden-
one, ethisterone, methandrostenolone, nandrolone, norgestrel, testosterone, dehydroepiandros-
terone (UV 210), mibolerone
Interfering: methandriol (UV 210), norethindrone acetate
KEYWORDS
REFERENCE
Walters,M.J.; Ayers,R-L; Brown,D.J. Analysis of illegally distributed anabolic steroid products by liquid chro-
JAssoc.OffAnaLChem., 1990, 73, 904-926.
SAMPLE
25 uL aliquot.
HPLC VARIABLES
Mobile phase: Gradient. MeOHiwater from 70:30 to 100:0 over 15 min, maintain at 100:0 for 15
min.
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
OTHER SUBSTANCES
(UV 280), fluoxymesterone, methandriol, methandriol-3-acetate, methandriol dipropionate,
methandrostenolone, nandrolone, nandrolone decanoate, nandrolone phenylpropionate, nan-
drolone propionate, stanolone, stanozolol, testosterone, testosterone acetate, testosterone cy-
pionate, testosterone enanthate, testosterone isobutyrate, testosterone propionate, testosterone
undecanoate
KEYWORDS
tablets
REFERENCE
LurieJ.S; Sperling,A.R.; Meyers,R.R The determination of anabolic steroids by MECC, gradient HPLC, and
capillary GC, J.Forensic ScL, 1994, 39, 74-85.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Guard column: 4 x 4 5 jxm Spherisorb ODS-2
Flow rate: 1
Detector: UV 238
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: spironolactone
REFERENCE
Kaukonen,A.M.; Vuorela,P.; Vuorela,H.; Mannermaa,J.-R High-performance liquid chromatography methods
for the separation and quantitation of spironolactone and its degradation products in aqueous formulations
and of its metabolites in rat serum, J.ChromatogrA, 1998, 797, 271-281.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOHiwater 1:1 at a concentration of 50 u.g/mL, inject a 10
|AL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 |xm uEondapak C18
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
Simultaneous: prednisone, prednisolone, prednisolone succinate, hydrocortisone acetate, noreth-
indrone, progesterone
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 u.m Hypersil ODS
Detector: UV
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Simultaneous: diethylstilbestrol, trenbolone, nandrolone, zeranol, dienestrol, medroxypro-
gesterone
Interfering: hexestrol
REFERENCE
Jansen,E.H.J.M.; Both-Miedema,R.; van den Berg,R-H. Application of optimization procedures for the separa-
tion of anabolic compounds by high-performance liquid chromatography, J.Chromatogr., 1989, 489, 57-64.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 100 |xg/mL solution in MeOH.
HPLC VARIABLES
Guard column: 70 X 2.1 Whatman COrPeIl ODS
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Simultaneous: boldenone, nandrolone, methandrostenolone, testosterone, danazol, fluoxymes-
terone
REFERENCE
bolic steroids, J.Chromatogr.ScL, 1990, 28, 162-166.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 0.5 mg/mL solution in MeOH, inject a 5 JJLL aliquot.
HPLC VARIABLES
Flow rate: 2
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
one, mefenamic acid, nicotine, oxazepam, phentermine, phenylpropanolamine, progesterone,
sulfamethazine, sulfanilamide, testosterone, testosterone propionate, tranylcypromine,
tripelennamine
Interfering: danthron
KEYWORDS
REFERENCE
Hill,D.W.; Kind A-J- The effects of type B silica and triethylamine on the retention of drugs in silica based
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
methyl salicylate, methyldopa, methyldopamine, methylphenidate, methyprylon, metoprolol,
mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, naproxen, ne-
fopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, norepinephrine,
nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone, ox-
ytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phe-
nacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, phenira-
mine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone,
phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, pro-
benecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromy-
cin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recin-
namine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine,
scopoletin, secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethi-
dole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasox-
izole, sulindac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, the-
baine, theobromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid,
trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tjnâmine, verapa-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 25 (xg/mL solution in mobile phase, inject an aliquot.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
OTHER SUBSTANCES
240), hydroxyprogesterone (UV 240), lynestrenol (UV 210), medroxyprogesterone acetate (UV
240), medroxyprogesterone (UV 240), methandienone (UV 240), methylandrostenediol (UV
210), methylprednisolone acetate (UV 240), methylprednisolone (UV 240), nandrolone (UV
240), norethisterone (UV 240), prednisolone acetate (UV 240), prednisolone (UV 240), predni-
sone (UV 240), pregnenolone (UV 210), progesterone (UV 240), testosterone (UV 240)
REFERENCE
SAMPLE
Matrix: tissue
Sample preparation: 1 g Tissue + 10 mL Chloroform:MeOH 2:1, homogenize for 1 min (Polytron
setting 5), filter, rinse tube with an additional 10 mL chloroformiMeOH, filter, combine filtrates,
add 4 mL water, vortex for 1 min, centrifuge at 600 g for 10 min. Remove organic layer and
dry it under air at 40. Reconstitute with 200 jxL MeOH, add to an activated Sep-Pak C18
cartridge, wash tube onto cartridge with 200 fxL MeOH, elute with 5 mL each 0, 25, 50, 75,
100% MeOH, collect 5 mL fractions, inject a 100 |xL aliquot of each fraction. (Elutes in 75%
MeOH fraction.)
HPLCVARIABLES
Guard column: in line guard column
Column: 80 mm long 10 jmm jmBondapak C18 radially compressed
Flow rate: 3
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: testosterone
KEYWORDS
fish; muscle; tilapia aurea; SPE
REFERENCE
Goudie,C.A. Extraction of a synthetic androgen from fish muscle and quantitation by high performance liquid
chromatography, Steroids, 1984, 44, 241-252.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize 1-5 g tissue in 10-50 mL chloroform:MeOH 2:1, add 0.2 vol-
ume water, centrifuge at 1800 g for 10 min, extract aqueous phase again with 2 volumes of
chloroform, evaporate organic phases under vacuum, dissolve residues in MeCN, pass through
a Lipidex 5000 column and a Sep Pak silica cartridge, inject an aliquot.
HPLC VARIABLES
Guard column: 25 X 3.8 5 (xm ODS 2
Column: 150 X 4.6 5 |xm Hypersil C8 plus 150 X 4.6 5 jjim Nucleosil C18
Flow rate: 0.8
Detector: F ex 340 em 470 following post-column reaction. The eluate from the column mixed
with freshly prepared 0.6 mM nicotinamide-adenine dinucleotide (NAD) in 20 mM pH 9 py-
rophosphate buffer pumped at 1.2 mL/min and flowed into a 950 |xL static mixing chamber
then through an immobilized enzyme reactor into the fluorescence detector. The Chrom Sep
immobilized enzyme reactor was prepared by pumping 10 mM pH 7.8 KH2PO4 through it at
0.8 mL/min. Ten 100 |JLL samples of a solution of 25 U/mL 3a-hydroxysteroid dehydrogenase
in 10 mM pH 7.8 KH2PO4 were injected onto the reactor which was then equilibrated with 10
mM pH 7.8 KH2PO4 for 20 min. The reactor was then attached to the chromatographic system.
CHROMATOGRAM
Retention time: 21.2 (5p-17-methyltestosterone), 22.6 (5a-17-methyltestosterone)
KEYWORDS
immobilized enzyme reactor; fish; muscle; SPE
REFERENCE
Cravedi,J.R; Delous,G. Use of an immobilized enzyme reactor for the analysis of residues of 17a-methyltestos-
terone in trout by high-performance liquid chromatography, J.Chromatogr., 1991, 564, 461-467.
SAMPLE
Matrix: urine
Sample preparation: 10 mL Urine + glucuronidase/sulfatase (Helix pomatia), incubate at 37
for 1 h, extract twice with 5 mL diethyl ether, add 225 |xL water and evaporate ether under
nitrogen, add 400 |xL MeOH, inject a 250 |xL aliquot of this mixture.
HPLC VARIABLES
Guard column: 75 X 2.1 Corasil C18
Flow rate: 2
Detector: UV 240
CHROMATOGRAM
Retention time: 9
Limit of detection: about 6 ng/mL
OTHER SUBSTANCES
Simultaneous: 17 p-trenbolone, zeranol, trans-diethylstilbestrol, medroxyprogesterone,
nandrolone
KEYWORDS
cow
REFERENCE
Jansen,E.H.; Both-Miedema,R.; van Blitterswijk,H.; Stephany,R.W. Separation and purification of several an-
abolics present in bovine urine by isocratic high-performance liquid chromatography, J.Chromatogr., 1984,
299, 450-455.
SAMPLE
Matrix: urine
Sample preparation: Add 10 mL urine to a Supelclean LC-18 SPE tube at a flow rate of 2 mL/
min, wash with 4 mL 25 mM sodium borate buffer, wash with 4 mL 40% MeOH, wash with 4
mL 20% acetone, elute with two 500 |xL aliquots of 73% MeOH, evaporate under nitrogen at
40, reconstitute with 1 mL mobile phase, inject a 200 JULL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Microsorb silica
Mobile phase: Cyclohexane:ethyl acetate 40:60
Detector: F ex 247 em 547, after post-column reaction with 30 mM Tb(NO3)3 in ethyl acetate
using a 50 cm tightly coiled capillary tube to ensure mixing
CHROMATOGRAM
Retention time: 16
OTHER SUBSTANCES
Extracted: testosterone acetate, progesterone, bolasterone, testosterone
KEYWORDS
SPE; normal phase; post-column reaction
REFERENCE
Amin,M.; Harrington,K; von Wandruszka,R. Determination of steroids in urine by micellar HPLC with detec-
tion by senstized terbiumfluorescence,Anal Chem., 1993, 65, 2346-2351.
SAMPLE
Matrix: urine
Sample preparation: 5 mL Urine + 1 mL 200 mM pH 7.0 sodium phosphate buffer + 50 |xL (3-
glucuronidase (E. coli K12, Boehringer Mannheim), heat at 55 for 1 h, cool to room tempera-
ture, add 1 g sodium bicarbonate .'potassium carbonate 1:2, add 1 g sodium sulfate, add 5 mL
n-pentane, shake for 20 min, centrifuge at 1500 g for 5 min. Remove the organic layer and
evaporate it to dryness, reconstitute the residue in 50 |xL MeOH, inject a 20 juiL aliquot.
HPLCVARIABLES
Column: 250 X 4 5 |xm Hypersil BDS-C18
Mobile phase: Gradient. MeCNrI mM phosphoric acid from 25:75 to 30:70 over 10 min, to 35:
65 over 6.5 min, maintain at 35:65 for 11.5 min.
Flow rate: 1.2
Detector: UV 240
CHROMATOGRAM
Retention time: 25
Internal standard: methyltestosterone
OTHER SUBSTANCES
Extracted: epitestosterone, testosterone
KEYWORDS
methyltestosterone is IS
REFERENCE
Navajas,R.; Imaz,C; Carreras,D.; Garcia,M.; Prez,M.; Rodriguez,C; Rodriguez,A.F.; Cort6s,R. Determination
of epitestosterone and testosterone in urine by high-performance liquid chromatography, J.Chromatogr.B,
1995, 673, 159-164.
Methyprylon
Molecular formula: G10H17NO2
Merck Index: 6216
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
methyl salicylate, methyldopa, methyldopamine, methylphenidate, methylprednisolone, meto-
prolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline, na-
puromycin, pjndlamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine,
REFERENCE
18, 233-242.
Methysergide
Merck Index: 6217
Lednicer No.: 2 477
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 1 mL 500 mM potassium carbonate, mix for 5 s, add 10
mL ethyl acetate, shake horizontally for 15 min, centrifuge at 700-1000 g for 5 min. Remove
8 mL of the upper organic phase and add it to 700 |xL 50 mM phosphoric acid, shake horizon-
tally for 15 min, centrifuge at 750 g for 2 min, discard the organic phase. Heat the aqueous
phase at 50 in a vortex evaporator to remove residual organic solvent, inject a 400 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jutm Supelcosil LC-8
Mobile phase: MeCN: 10 mM pH 7 potassium phosphate buffer 30:70
Flow rate: 1
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Extracted: methylergonovine
KEYWORDS
plasma; protect from sunlight; pharmacokinetics
REFERENCE
Smith,H.T.; Molinaro,N.C. High-performance liquid chromatographic method for the determination of meth-
ysergide and methylergonovine in human plasma, J. Chromatogr., 1988, 424, 416423.
SAMPLE
Matrix: blood
Sample preparation: 0.1-0.7 mL Plasma or whole blood + 2.5 mL HCl/ammonia solution + 200
IxL 100 nM ergometrine in water, mix, add to an Extrelut silica SPE cartridge, let stand for
20 min, elute with two 5 mL portions of ethyl acetate. Evaporate the eluate to dryness under
a stream of nitrogen below 40, reconstitute the residue in mobile phase, inject a 25-150 |xL
aliquot. (HCl/ammonia solution was 2 M HCl and 1 M ammonia mixed to a pH of 9.0.)
HPLC VARIABLES
Column: 250 mm long 5 (xm Hypersil C18 ODS
Flow rate: 2
CHROMATOGRAM
Retention time: 7.0
Internal standard: ergonovine (ergometrine) (2.4)
OTHER SUBSTANCES
Extracted: methylergonovine (methylergometrine)
KEYWORDS
rat; plasma; whole blood; SPE; pharmacokinetics
REFERENCE
Drug Metab.Dispos., 1990, 18, 338-343.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.2
OTHER SUBSTANCES
droperidol, ephedrine, ergocornine, ergocristine, ergocristinine, ergociyptine, ergometrine, er-
methylephedrine, methylergonovine, metoclopramide, metopimazine, metoprolol, mianserin,
morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nor-
triptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline,
pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone, phen-
ampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutarimide,
REFERENCE
Jane,L; McKinnonA-l Flanagan,R-J- High-performance liquid chromatographic analysis of basic drugs on silica
Metiamide
Molecular formula: C9H16N4S2
LednicerNo.: 2 252
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 12 urn l-myristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine
Mobile phase: MeCN:100 rnM pH 7.0 phosphate buffer 20:80
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
famotidine, isothipendyl, ketotifen, metoprolol, moxonidine, nadolol, naphazoline, nifenalol, ni-
REFERENCE
Kaliszan,R-l Nasal,A.; Turowski,M. Binding site for basic drugs on ax-acid glycoprotein as revealed by chemo-
Metipranolol
Merck Index: 6221
Lednicer No.: 5 17
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform dsopropanol:
the residue in 100 |JLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 (xL aliquot of
HPLC VARIABLES
Mobile phase: MeOH:THF.buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) KH2PO4 adjusted
Flow rate: 0.8
Detector: UV 281
CHROMATOGRAM
KEYWORDS
REFERENCE
Tracqui,A.; Kintz,P.; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
Metoclopramide
Molecular formula: G14H22CIN3O2
CAS Registry No.: 364-62-5, 54143-47-6 (HCI monohydrate),
7232-21-5 (HCI)
Merck Index: 6226
Lednicer No.: 4 41
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 JLLL 10 [LgJmL quinidine sulfate in water + 100 JJLL 1
M NaOH + 5 mL chlorobutane:MeCN 90:10, shake thoroughly for 1 min, centrifuge at 2500 g
for 5 min. Remove the organic phase and add it to 100 |xL 100 mM HCl, mix for 1 min,
centrifuge at 2500 g for 5 min, inject a 20 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Column: 250 X 4.55 5 jjim Spherisorb CN
Flow rate: 2
Detector: UV 275
CHROMATOGRAM
Retention time: 4.6
Internal standard: quinidine sulfate (5.8)
KEYWORDS
plasma
REFERENCE
Buss,D.C; Hutchings,A.D.; Scott,S.; Routledge,P.A. A rapid liquid chromatographic method for the determi-
nation of metoclopramide in human plasma, Ther.Drug Monit, 1990, 12, 293-296.
SAMPLE
Matrix: blood
Sample preparation: Add 10 |xL 20 jxg/mL oxaprotiline in MeOH to 990 |xL plasma or serum.
Inject 100 |JLL plasma or serum onto column A with mobile phase A and elute to waste, after
mobile phase B.
HPLC VARIABLES
Column: A 20 X 4.6 10 jxm Hypersil MOS C8; B 20 X 4.6 5 urn Hypersil CPS CN + 250 X 4.6
5 iim Nucleosil 100 CN
Mobile phase: A MeOH:water 5:95; B MeOHiMeCN: 10 mM pH 6.8 potassium phosphate buffer
188:578:235
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 5.7
OTHERSUBSTANCES
Simultaneous: clozapine, fluvoxamine, doxepin, amitriptyline, clomipramine, fluoxetine, imip-
ramine, norfluoxetine, nortriptyline, desipramine, maprotiline
diazepoxide, clobazam, diazepam, nordiazepam, nurazepam, lorazepam, nitrazepam, oxaze-
pam, carbamazepine
KEYWORDS
REFERENCE
Hartter,S.; Wetzel,H>; Hiemke,C. Automated determination offluvoxaminein plasma by column-switching high-
SAMPLE
Matrix: blood
Sample preparation: 2 mL Whole blood or plasma + 2 mL buffer + 5 mL chloroform risopropanol:
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 275
CHROMATOGRAM
KEYWORDS
REFERENCE
Tracqui,A-; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J,Forensic ScL, 1995, 40,
254-262.
SAMPLE
vortex, add 1 mL 200 mM sodium carbonate, vortex, add 6 mL hexaneil-butanol 95:5, gently
mogenate + 10 |xg cianopramine + 500 |JLL 2% sodium tetraborate + 8 mL hexanerl-butanol 95:
it to 400 |xL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the
organic layer and inject a 30 |JLL aliquot of the aqueous layer.
HPLC VARIABLES
Guard column: 15 X 3.2 7 fun RP-18 Newguard (Applied Biosystems)
Mobile phase: MeCN.100 mM NaH2PO4:diethylamine 40:57.5:2.5
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
doxepin, fluoxetine, haloperidol, imipramine, loxapine, maprotiline, meperidine, mesoridazine,
methadone, mianserin, nomifensine, nordoxepin, norfluoxetine, norpropoxyphene, northiaden,
nortriptyline, pheniramine, promethazine, propoxyphene, propranolol, protriptyline, quinidine,
quinine, sulforidazine, thioridazine, thiothixene, trazodone, trihexyphenidyl, trimipramine,
triprolidine
fluoperazine
Interfering: moclobemide, tranylcypromine, pentobarbital
KEYWORDS
REFERENCE
SAMPLE
Sample preparation: Plasma. Condition a 3 mL C8 Analytichem SPE cartridge with 1 volume
(2.7 mL) MeOH and 1 volume buffer A, do not allow to dry. Mix 1 mL plasma + 100 |xL 10 |xg/
mL amisulpride and 10 |xg/mL alpiropride in 10 mM HCl + 1 mL buffer A, add to SPE cartridge,
rinse the sample container with 1 mL buffer A and add the rinse to the SPE cartridge, wash
with 1 volume water, wash with 2 mL buffer B, dry the column for 1 min, wash with 200 |xL
acetone, dry for 30 s, elute with 1 mL buffer C, add 50 |xL buffer D, evaporate to dryness under
a stream of air, reconstitute in 200 (xL mobile phase, sonicate for 1 min, inject an aliquot.
Urine. Connect a Baker 3 mL ion exchange quaternary aminesilicane-bonded silica gel SPE
cartridge on top of a 3 mL Baker carboxylic acid-bonded silica gel SPE cartridge, condition
with 1 volume (2.7 mL) buffer D, 1 volume of water, 1 volume of MeOH, and 1 volume of water.
Mix 1 mL urine + 100 |xL 10 |xg/mL amisulpride and 10 fxg/mL alpiropride in 10 mM HCl + 1
mL water, add to SPE cartridges, rinse the sample container with 2 mL water and add the
rinse to the SPE cartridges, wash with 1 mL water, remove the top column, wash the bottom
column with 1 volume of water and 2 volumes of MeOH, dry the column for 1 min, elute with
1 mL buffer D, evaporate the eluate to dryness under a stream of air at 45, reconstitute in
200 JJLL mobile phase, sonicate for 1 min, inject an aliquot. (Buffer A was 10 mL triethylamine
in 1 L water, pH adjusted to 7.00 with acetic acid. Buffer B was MeOHrwater 20:80. Buffer C
was 10 mL triethylamine + 7 mL acetic acid in 1 L MeOH. Buffer D was 2.10 mL concentrated
HCl in 250 mL MeOH (100 mM).)
HPLC VARIABLES
Guard column: 10 cm long Chrompack reverse-phase pellicular material
Mobile phase: MeCN:MeOH:buffer 160:80:760 (Buffer was 10 mL triethylamine + 760 mL water
adjusted to pH 6.8 with acetic acid (about 4.2 mL).)
Flow rate: 2
Detector: UV 230
CHROMATOGRAM
Retention time: 7.1
Internal standard: amisulpride (4.9), alpiropride (6.0)
OTHER SUBSTANCES
Simultaneous: alizapride, aspirin, theophylline, acetaminophen, caffeine, isosorbide-5-mononi-
trate, nitrofurantoin, codeine, acenocoumarol, carbamazepine, nitrazepam, clonazepam
Noninterfering: indomethacin, orphenadrine, furosemide, cisplatin, amitriptyline, isosorbide
dinitrate, propranolol
KEYWORDS
plasma; SPE
REFERENCE
de Jong,A.P.; Wittebrood,A.J.; du CMtinier,W.M.; Bron,J. Liquid chromatographic analysis of alizapride and
metoclopramide in human plasma and urine using solid-phase extraction, J.Chromatogr., 1987, 419, 233-
242.
SAMPLE
Sample preparation: Plasma. 250 |xL Plasma + 300 (xL 2 M NaOH, vortex for 30 s, add 5 mL
dichloromethane, mix for 2 min, centrifuge at 10000 rpm for 15 min. Remove the organic layer
and evaporate to dryness under nitrogen. Reconstitute the residue in 150 JJLL mobile phase,
inject a 50 JJLL, aliquot. Urine. Dilute 100 |xL urine + 75 |xL 2 mg/mL metoclopramide to 10 mL
with water, inject a 50 JULL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Spherisorb S 5 ODS II
Mobile phase: MeCN:MeOH:buffer 60:30:5 pH adjusted to 3.8 with glacial acetic acid (Buffer
was 50 mM ammonium acetate containing 10 mM sodium octane sulfonate.)
Flow rate: 1.2
Detector: UV 330
CHROMATOGRAM
Retention time: 8
Internal standard: metoclopramide
OTHER SUBSTANCES
Simultaneous: oxazepam, desmethyldiazepam, chlorodiazepam, diazepam, theophylline, theo-
bromine, acetaminophen, chlorpromazine, ranitidine
KEYWORDS
plasma; metoclopramide is IS
REFERENCE
Shiekh Salem,M.; Gharaibeh,A.M.; Alkaysi,H.N.; Badwan,A. High-performance liquid chromatographic anal-
ysis of ranitidine in plasma and urine, J.Clin.Pharm.Ther., 1988, 13, 351-357.
SAMPLE
Sample preparation: Condition a 100 mg Isolute MF C18 (International Sorbent Technology)
SPE cartridge with 2 mL MeCN, 2 mL water, and 500 JJLL buffer. Mix 1 mL urine and 25 mL
water. Mix 1 mL diluted urine or plasma with 1 mL buffer, vortex, add to the SPE cartridge,
wash with 1 mL buffer, wash with 1 mL MeCN:water 30:70, wash with 80 |xL MeOH, air dry
for 30 s, elute with 500 |xL MeOH. Evaporate the eluate to dryness under a stream of nitrogen
at 40, reconstitute the residue in 250 JAL mobile phase, inject a 100 (JLL aliquot. (Prepare buffer
by dissolving 6.18 g boric acid and 7.46 g KCl in 1 L water. Mix 500 mL of this solution with
185 mL 100 mM NaOH, pH 9.)
HPLC VARIABLES
Guard column: 20 X 4.6 40 |jim Pelliguard Si
Column: 250 X 4.6 Chiralpak AS amylose carbamate (J.T. Baker)
Mobile phase: n-Heptane:EtOH:diethylamine 70:29.8:0.2
Flow rate: 0.5
CHROMATOGRAM
Internal standard: metoclopramide
OTHER SUBSTANCES
Extracted: amisulpride
KEY WORDS
mobile phase reservoir at 34; plasma; SPE; metoclopramide is IS
REFERENCE
Ascalone,V.; Ripamonti,; Malavasi,B. Stereospecific determination of amisulpride, a new benzamide derivative,
in human plasma and urine by automated solid-phase extraction and liquid chromatography on a chiral
column, application to pharmacokinetics, J.ChromatognB, 1996, 676, 95-105.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 213.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y.; Pepin,G. Use of high-performance liquid ehromatography with photodiode-array UV detection for
163.
SAMPLE
Sample preparation: Dilute with water.
HPLC VARIABLES
Column: Waters C18 column (PN 86344)
Mobile phase: Sodium acetate 2.7 g in 500 mL water, 500 mL MeCN, 1 mL glacial acetic acid,
and 2 mL tetramethylammonium hydroxide in methanol (1:5)
Flow rate: 1.5
Detector: UV 270
CHROMATOGRAM
Retention time: 5.5
KEYWORDS
saline; injections; stability-indicating
REFERENCE
Stiles,M.L.; Allen,L.V.,Jr.; Prince,S.J.; Holland,J.S. Stability of dexamethasone sodium phosphate, diphenhy-
dramine hydrochloride, lorazepam, and metoclopramide hydrochloride in portable infusion-pump reservoirs,
Am.J.Hosp.Pharm., 1994, 51, 514-517.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 (xm Zorbax Rx-C8 base deactivated octylsilane (Mac-Mod Analytical)
Flow rate: 1
Detector: UV 273
CHROMATOGRAM
Retention time: 7.9
OTHER SUBSTANCES
KEYWORDS
saline; injections
REFERENCE
Venkateshwaran,T.G.; King,D.T.; Stewart, J.T. HPLC determination of a metoclopramide and ondansetron mix-
ture in 0.9% sodium chloride injection, J.Liq.Chromatogr., 1995, 18, 117-126.
SAMPLE
Sample preparation: Dilute 1 mL formulation to 10 mL with mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 150 X 4.6 fiBondapak phenyl
Mobile phase: MeCN:MeOH:0.01% acetic acid:0.005 N sulfonic acid 20:15:40:25
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: nuorouracil
KEYWORDS
REFERENCE
Wang,D.-R; Chang,L.-C; Lee,D.K.T.; Wong,C.-Y. Stability of fluorouracil-metoclopramide hydrochloride admix-
ture, Am.J.Health-Syst.Pharm., 1995, 52, 98-99.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 25-75 JJLL aliquot of an aqueous solution.
HPLC VARIABLES
Guard column: 25 X 4 5 |xm Bondapak C18
Column: 150 X 3.9 5 |jim Bondapak C18
Mobile phase: MeCN: 10 mM pH 5.5 sodium phosphate buffer containing 5 mM triethylamine
17:83
Flow rate: 1.2
Detector: UV 230
CHROMATOGRAM
Retention time: 4.4
OTHER SUBSTANCES
Simultaneous: tramadol
REFERENCE
Zaghloul,I.Y.; Radwan,M.A. High performance liquid chromatographic determination of tramadol in pharma-
ceutical dosage forms, J.Liq.Chromatogr.Rel.Technol., 1997, 20, 779-787.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 5.3
OTHER SUBSTANCES
methylephedrine, methylergonovine, methysergide, metopimazine, metoprolol, mianserin, mor-
thipendyl, protriptyline, proxymetacaine, pseudoephedrine, pjnrimethamine, quinidine, qui-
nine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine, thenyidi-
thiothixene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylc^romine, tra-
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Centrifuge.
HPLC VARIABLES
Column: 100 X 5 4 |jim Novapack radial compression RCM 8x10
Mobile phase: MeCN:buffer 60:40 (Buffer was 10 mM KH2PO4, pH 4.5.)
Flow rate: 1.5
Detector: UV 274
OTHER SUBSTANCES
Also analyzed: alizapride, bromopride, clebopride, domperidone, metopimazine
REFERENCE
Calpena,A.C; Blanes,C; Moreno,J.; Obach,R.; Domenech,J. A comparative in vitro study of transdermal ab-
sorption of antiemetics, J.Pharm.ScL, 1994, 83, 29-33.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 \xm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
methoxamine, methyldopa, methylphenidate, metolazone, metoprolol, metronidazole, midazo-
lam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nifedi-
himbine, zopiclone
KEYWORDS
REFERENCE
J.Chromatogr.A, 1995, 692, 103-119.
Metocurine iodide
Merck Index: 6227
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond Elut C18 SPE cartridge with 2 column volumes
of THF, 2 volumes of MeOH, and 2 volumes of water. 1 mL Plasma + 100 o.L 5 |xg/mL D-
tubocurarine chloride in 10 mM HCl, add to the SPE cartridge, wash with 2 volumes of water,
elute with 250 (xL mobile phase. Evaporate the eluate and reconstitute with 100 |xL mobile
phase, vortex, centrifuge at 12800 g for 5 min, inject an aliquot.
HPLC VARIABLES
Guard column: 10 |xm CN Guard-Pak (Waters)
Column: 10 |xm Radial-Pak CN (Waters)
Mobile phase: MeCN:MeOH:water:l M pH 2.5 dibutylamine phosphate 40:10:10:1
Flow rate: 2.4
Detector: UV 204
CHROMATOGRAM
Internal standard: D-tubocurarine chloride (5.9)
KEYWORDS
REFERENCE
Avram,M.J.; Shanks,C.A. Determination of D-tubocurarine chloride or metocurine iodide in human plasma by
high-performance liquid chromatography with ultraviolet detection, J.Chromatogr., 1984, 306, 398-404.
SAMPLE
Matrix: blood
Sample preparation: 250 |xL Plasma + 250 |xL picric acid (1:50 dilution of saturated picric acid
solution) + 250 |xL tubocurarine solution + 250 jxL water + 2.5 mL dichloromethane:isopropanol
85:15, vortex for 15 s, centrifuge at 1500 g for 10 min. Remove the organic phase and evaporate
it to dryness at 40 under a stream of nitrogen, reconstitute the residue in 150-250 jxL MeCN:
water 40:60, centrifuge at 1500 g for 4 min, inject a 20-100 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 jxPorasil
Mobile phase: MeCN:2 mM sulfuric acid 50:50
Flow rate: 2
Detector: UV 210
CHROMATOGRAM
Retention time: 5.3
Internal standard: tubocurarine (3.7)
OTHER SUBSTANCES
Also analyzed: atracurium, alcuronium
KEYWORDS
plasma
REFERENCE
Bjorksten,A.R.; Beemer,G.H.; Crankshaw,D.P. Simple high-performance liquid chromatographic method for the
analysis of the non-depolarizing neuromuscular blocking drugs in clinical anaesthesia, J.Chromatogr., 1990,
533, 241-247.
Metolazone
Merck Index: 6231
Lednicer No.: 2 384
SAMPLE
Matrix: blood
Sample preparation: Clean an Amalytichem C2 ethyl sorbent cartridge with 1 mL MeCN and
1 mL buffer. 25 fxL Plasma + 1 mL buffer, add to cartridge, wash with 1 mL buffer, blow dry
with nitrogen for 1 min, elute cartridge directly on to column (Varian AASP system). (Buffer
was 10 mM KH2PO4 adjusted to pH 3.0 with concentrated phosphoric acid.)
HPLC VARIABLES
Guard column: 3 X 4.6 30 (Jim C18 Alltech pellicular packing
Mobile phase: MeCNrIO mM KH2PO4 adjusted to pH 3.0 with concentrated phosphoric acid 30:
70
Flow rate: 1.5
CHROMATOGRAM
Retention time: 8.3
Internal standard: metolazone
OTHER SUBSTANCES
Noninterfering: bumetanide, chlorothiazide, hydrochlorothiazide, chlorthalidone, ibuprofen,
acetaminophen, salicylic acid
KEYWORDS
plasma; SPE; metolazone is IS
REFERENCE
Farthing,D.; Karnes,T.; Gehr,T.W.; March,C; Fakhry,L; Sica,D.A. External-standard high-performance liquid
chromatographic method for quantitative determination of furosemide in plasma by using solid-phase ex-
traction and on-line elution, J.Pharm.ScL, 1992, 81, 569-571.
SAMPLE
Matrix: blood
Sample preparation: Condition a C2 ethyl SPE cartridge (Analytichem) with 1.5 mL MeCN:
MeOH 50:50 and 1.5 mL buffer. 500 |xL Whole blood + 1.5 mL water, vortex for 15 s, centrifuge
at 13000 g for 5 min. 250 JJLL Plasma or diluted whole blood + 25 JULL 1 |mg/mL IS in water +
250 jiL buffer, add to the SPE cartridge, wash with 1 mL MeOH:buffer 10:90, wash with 1 mL
hexane, purge with nitrogen for 1 min, elute the contents of the SPE cartridge onto the column
with mobile phase. (Buffer was 25 mM (?) K2HPO4 adjusted to pH 7.0 with concentrated phos-
phoric acid.)
HPLC VARIABLES
Guard column: 30 mm long 40-50 |xm pellicular C18
Column: 100 X 4.6 3 |xm Spherisorb ODS C18
Mobile phase: MeCN-KH2PO4 30:70 adjusted to pH 3.0 with concentrated phosphoric acid
Flow rate: 1
CHROMATOGRAM
Internal standard: 7-chloro-1,2,3,4-tetrahydro-2-methyl-3-(3-methylphenyl)-4-oxo-6-quinazo-
linesulfonamide (7.40)
OTHER SUBSTANCES
Noninterfering: bumetanide, furosemide, hydrochlorothiazide, ibuprofen, indomethacin
KEYWORDS
plasma; whole blood; protect from light; SPE
REFERENCE
Farthing,D.; Sica,D.A.; Fakhry,L; Gehr,T.W.B. Novel high-performance liquid chromatographic method using
solid-phase on-line elution for determination of metolazone in plasma and whole blood, J.Chromatogr.B,
1994, 653, 171-176.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in mobile phase, inject a 30 JJLL aliquot.
HPLCVARIABLES
Flow rate: 1.4
Detector: UV 254
CHROMATOGRAM
Retention time: 7.8
OTHER SUBSTANCES
Simultaneous: acetaminophen, aldosterone, allopurinol, amitriptyline, caffeine, calcitriol,
cephalothin, chlordiazepoxide, chlorothiazide, corticosterone, cortisone, dexamethasone, di-
azepam, ephedrine, ethinyl estradiol, furosemide, hydrocortisone, ibuprofen, imipramine,
indomethacin, mechlorethamine, methylprednisone, nandrolone, naproxen, phenacetin, phen-
obarbital, phenytoin, prednisolone, prednisone, probenecid, progesterone, propranolol, sulfas-
alazine, testosterone, theophylline, vincristine
REFERENCE
Cheng,M.H.; Huang,W.Y.; Lipsey,A.I. Simultaneous liquid-chromatographic determination of prednisone and
prednisolone in plasma, Clin.Chem., 1988, 34, 1897-1899.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
methoxamine, methyldopa, methylphenidate, metoclopramide, metoprolol, metronidazole, mid-
azolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, nife-
dipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, parox-
etine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine,
sertraline, sotalol, spironolactone, sulfinpjrrazone, sulindac, temazepam, terbutaline, terfena-
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Sample preparation: 2 mL Urine + 2 mL 1 M pH 4.1 NaH2PO4 + 4 mL ethyl acetate, vortex
for 2 min, centrifuge at 1500 g for 5 min. Remove the organic phase and add it to 5 mL 100
mM pH 7.5 Na2HPO4, vortex for 2 min, centrifuge at 1500 g for 5 min. Remove the organic
in 100 |xL MeCN: 10 mM pH 3.0 phosphate buffer, inject a 5 |xL aliquot.
HPLC VARIABLES
Column: 125 X 4 5 ^m LiCHrosorb RP-18
Mobile phase: Gradient. MeCN:10 mM pH 3.0 phosphate buffer 10:90 for 1.5 min then to 35:65
over 2 min
Flow rate: 1.5
Injection volume: 5
Detector: UV 271
CHROMATOGRAM
Retention time: 5.3
OTHER SUBSTANCES
Extracted: chlorothiazide, hydrochlorothiazide, quinethazone, chlorthalidone, clopamide, meth-
yclothiazide, furosemide, mefruside, bendroflumethiazide, cyclopenthiazide, bumetanide
Simultaneous: indapamide, clorexolone, ethacrynic acid
Noninterfering: aspirin, albuterol, allopurinol, alprenolol, atenolol, captopril, carbimazole, clo-
nidine, coloxyl, danthron, diazepam, digoxin, doxepin, glibenclamide, hydralazine, indometh-
acin, labetalol, metformin, methyldopa, metoprolol, mianserin, minoxidil, nifedipine, nitraze-
pam, oxazepam, oxprenolol, pindolol, prazosin, propranolol, senokot, theophylline,
trifluoperazine
REFERENCE
Fullinfaw,R.O.; Bury,R.W.; Moulds,R.F.W. Liquid chromatographic screening of diuretics in urine,
J.Chromatogn, 1987, 415, 347-356.
SAMPLE
Matrix: urine
300 |xL 50 |xg/mL p-hydroxyethyltheophylline in MeOH, inject 5 |xL aliquot. (Solid buffer I was
HPLC VARIABLES
Flow rate: 1
Injection volume: 5
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: amiloride, acetazolamide, chlorothiazide, hydrochlorothiazide, quinethazone, triam-
terene, flumethiazide, hydroflumethiazide, chlorthalidone, dichlorphenamide, trichloromethi-
azide, methyclothiazide, benzthiazide, cyclothiazide, polythiazide, bendroflumethiazide, etha-
crynic acid, bumetanide, probenecid, spironolactone, canrenone
Next Page

Interfering: furosemide
REFERENCE
Cooper,S.R; Mass6,R-J Dugal,R- Comprehensive screening procedure for diuretics in urine by high-performance
SAMPLE
Matrix: urine
Sample preparation: Mix urine by inversion, vortex for 15 s. 100 |xL Urine + 300 |xL water,
mix, inject a 40 |xL aliquot.
HPLC VARIABLES
Guard column: 30 mm long 40-50 jxm pellicular C18
Mobile phase: MeCNiKH2PO4 35:65 adjusted to pH 3.0 with concentrated phosphoric acid
Flow rate: 1
CHROMATOGRAM
Retention time: 7.1
OTHER SUBSTANCES
Simultaneous: bumetanide, furosemide, salicylic acid
Noninterfering: acetaminophen, chlorothiazide, hydrochlorothiazide, ibuprofen
KEYWORDS
REFERENCE
Farthing,D.; Fakhry,L; Gehr,T.W.; Sica,D.A. Quantitation of metolazone in urine by high-performance liquid
chromatography with fluorescence detection, J.Chromatogr., 1990, 534, 228-232.
Metopimazine
Merck Index: 6233
Lednicer No.: 1 153
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
Previous Page

Interfering: furosemide
REFERENCE
Cooper,S.R; Mass6,R-J Dugal,R- Comprehensive screening procedure for diuretics in urine by high-performance
SAMPLE
Matrix: urine
Sample preparation: Mix urine by inversion, vortex for 15 s. 100 |xL Urine + 300 |xL water,
mix, inject a 40 |xL aliquot.
HPLC VARIABLES
Guard column: 30 mm long 40-50 jxm pellicular C18
Mobile phase: MeCNiKH2PO4 35:65 adjusted to pH 3.0 with concentrated phosphoric acid
Flow rate: 1
CHROMATOGRAM
Retention time: 7.1
OTHER SUBSTANCES
Simultaneous: bumetanide, furosemide, salicylic acid
Noninterfering: acetaminophen, chlorothiazide, hydrochlorothiazide, ibuprofen
KEYWORDS
REFERENCE
Farthing,D.; Fakhry,L; Gehr,T.W.; Sica,D.A. Quantitation of metolazone in urine by high-performance liquid
chromatography with fluorescence detection, J.Chromatogr., 1990, 534, 228-232.
Metopimazine
Merck Index: 6233
Lednicer No.: 1 153
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.1
OTHER SUBSTANCES
methylephedrine, methylergonovine, methysergide, metoclopramide, metoprolol, mianserin,
morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine, nor-
REFERENCE
Metoprolol
CAS Registry No.: 37350-58-6, 56392-17-7 (tartrate),
119637-66-0 (fumarate), 98418-47-4 (succinate), 37350-58-6 (tartrate)
Merck Index: 6235
Lednicer No.: 2 109
SAMPLE
Matrix: blood
Sample preparation: Add 1.5 mL MeCN to 500 |xL serum, centrifuge, evaporate the superna-
tant to dryness, redissolve the residue in 200 |xL water. Inject onto column A, wash with MeCN:
water 10:90 or MeOHrwater 20:80 for 20 min, backflush the contents of column A onto column
B with mobile phase, elute with mobile phase, monitor the effluent from column B.
HPLC VARIABLES
Column: A 25 X 4 25 |xm pore diameter 6 nm LiChrospher RP-18 ADS (Merck); B 125 X 4 5 ^m
endcapped LiChroCART HPLC-cartridge RP-18 (Merck)
Mobile phase: MeCN:50 mM pH 4 K3PO4 buffer 27:73
Flow rate: 1
CHROMATOGRAM
Retention time: 3.0
OTHER SUBSTANCES
Extracted: celiprolol, tiracizine, talinolol, oxprenolol, metabolites
KEYWORDS
REFERENCE
Oertel,R.; Richter,K; Gramatt,T.; Kirch,W. Determination of drugs in biological fluids by high-performance
liquid chromatography with on-line sample processing, J.Chromatogr.A, 1998, 797, 203-209.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 |xL 200 fjig/mL (R)-(-)-flecainide in water + 1 mL pH
11 borate buffer, mix, add 5 mL diisopropyl ether (Caution! Diisopropyl ether readily forms
explosive peroxides!), shake on a reciprocal shaker for 15 min, centrifuge at -10 at 4000 rpm,
freeze in dry ice/acetone. Remove the organic layer and evaporate it to dryness under reduced
pressure, reconstitute the residue in 100 |xL 10 mM HCl, adjust pH to 11 with 200 |xL borate
buffer, add 50 JJLL 100 (xg/mL (+)-4-(6-methoxy-2-naphthyl)-2-butyl chloroformate in MeCN, mix,
let stand at room temperature for 1 h, add 3 mL dichloromethane, swirl mix for 30 s. Remove
the organic layer and evaporate it to dryness under reduced pressure, reconstitute the residue
in 100 fxL n-hexane:MeOH 100:6, inject a 20 JJLL aliquot. (Synthesis of (+)-4-(6-methoxy-2-
naphthyl)-2-butyl chloroformate is as follows. Slowly add 2 mmoles 4-(6-methoxy-2-naphthyl)-
2-butanone (nabumetone) in 20 mL anhydrous diethyl ether to 0.55 mmoles lithium aluminum
hydride in anhydrous diethyl ether, when the reaction is complete (monitor by TLC) cautiously
add water, add 6 M HCl, remove the organic layer. Extract the aqueous layer 3 times with 10
mL portions of diethyl ether. Combine the organic layers and dry them over anhydrous sodium
sulfate, evaporate to dryness to obtain racemic 4-(6-methoxy-2-naphthyl)-2-butanol. Dissolve
0.5 mmole 4-(6-methoxy-2-naphthyl)-2-butanol in 20 mL dry dichloromethane, add 0.55 mmole
dry pyridine, stir, add 1 mmole (lS)-(-)-camphanoyl chloride ((lSM-)-camphanic chloride) in
small portions, stir at room temperature overnight, wash 3 times with 1 M HCl, wash with
water, wash with saturated sodium bicarbonate solution, wash with water until neutral. Dry
the organic layer over anhydrous magnesium sulfate, evaporate to dryness to obtain the cam-
phanoyl ester. Purify by preparative HPLC (250 X 50 (R)-DNBPG (J.T. Baker), n-hexane:iso-
propanol 70:30, 65 mL/min, UV 220) to obtain diastereomerically pure camphanoyl esters. Add
5 mmole diastereomerically pure camphanoyl ester dissolved in 30 mL dry diethyl ether drop-
wise under argon to 2.5 mmoles lithium aluminum hydride stirred in diethyl ether at 0, stir
at room temperature for 1 h, stir in an ice bath, cautiously add 10% aqueous ammonium
chloride, remove the organic layer, extract the aqueous layer three times with diethyl ether.
Combine the organic layers and dry them over anhydrous magnesium sulfate, evaporate to
dryness. Dissolve 1 g in 10 mL dichloromethane, purify by flash chromatography on a 500 X
40 column filled with 200 g 40-63 |xm silica gel 60 using dichloromethane:MeOH 99.5:0.5 at
45 mL/min to obtain enantiomerically pure 4-(6-methoxy-2-naphthyl)-2-butanol. Dissolve 0.5
mmole (+)-4-(6-methoxy-2-naphthyl)-2-butanol and 0.5 mmole triethylamine in 10 mL dry tol-
uene, cool to 0, stir, add 1 mL 20% phosgene in dry toluene, stir for 4 h, filter, evaporate under
reduced pressure to obtain (+)-4-(6-methoxy-2-naphthyl)-2-butyl chloroformate as an oil.)
HPLC VARIABLES
Guard column: 20 X 4 6 |xm Ultrasep ES 100 (Bischoff, Germany)
Column: 125 X 3 3 |xm Nucleosil 120
Mobile phase: n-Hexane:MeOH 100:0.4
Flow rate: 1
CHROMATOGRAM
Retention time: 24.5 ((SM-)), 26.5 ((RM+))
Internal standard: (RM-)-flecainide (37.5)
OTHER SUBSTANCES
Also analyzed: acebutolol, alprenolol, flecainide, metoprolol, mexiletine, propafenone, propran-
olol, tocainide
KEY WpRDS
derivatization; plasma; chiral; pharmacokinetics; normal phase; protect from light
REFERENCE
Buschges,R.; Devant,R.; Mutschler,E.; Spahn-Langguth,H. 4-(6-Methoxy-2-naphthyl)-2-butyl chloroformate en-
antiomers: New reagents for the enantiospecific analysis of amino compounds in biogenic matrices,
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 10 ixmole compound (as free base or hydrochloride) in 500 |xL
MeCN, add 250 JJLL 5% sodium carbonate (for hydrochlorides only), add 500 |xL 100 mM reagent
in MeCN, vortex for 1 min, heat at 60 for 2 h, add 100 ixmole L-proline, heat at 60 for 30
min. Remove a 100 fiL aliquot and dilute it with mobile phase, neutralize with acetic acid,
inject a 10 |xL aliquot. Prepare the reagent ((R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohexyl-
isothiocyanate) as follows. Add 0.7 mL carbon disulfide to 6 mL (lR,2R)-(-)-l,2-diaminocyclo-
hexane, 12 mL water, and 12 mL EtOH, heat the oil bath to 80, add 2.8 mL carbon disulfide
reflux for 1 h, acidify with 500 jxL 5 M HCl, reflux for 12 h, cool, filter, wash the solid with a
dichloroethane, reflux for 16 h to obtain more (R,R)-N-(3,5-dinitrobenzoyl)-2-aminocyclohex-
ylisothiocyanate (mp >250, [a]546 = -133 (c = 1) in MeCN).
HPLC VARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: acebutolol, alprenolol, atenolol, carazolol, carvedilol, formoterol, methampheta-
mine, metipranolol, nifenanol, nitrilo atenolol, oxprenolol, pindolol, propranolol, xamoterol
KEY WORDS
REFERENCE
Kleidernigg,O.R; Posch,K.; Lindner,W. Synthesis and application of a new isothiocyanate as a chiral derivatiz-
ing agent for the indirect resolution of chiral amino alcohols and amines, J.Chromatogr.A, 1996, 729, 3 3 -
42.
SAMPLE
Matrix: perfusate
Sample preparation: Mix 1 mL perfusate with 50 |xL 6.46 |xM nadolol in water and inject a 50
|JLL aliquot.
HPLC VARIABLES
Column: 100 X 8 4 |xm NovaPak C18
Mobile phase: MeCN:triethylamine:water 9:0.3:91, adjusted to pH 3.0 with orthophosphoric acid
Flow rate: 3.0
CHROMATOGRAM
Internal standard: nadolol (5.55)
OTHER SUBSTANCES
KEYWORDS
liver; rat; pharmacokinetics
REFERENCE
Wang,B.; Semple,H.A. Inhibition of metoprolol metabolism by amino acids in perfused rat livers. Insights into
the food effect?, Drug Metab.Dispos., 1997, 25, 287-295.
SAMPLE
Matrix: perfusate
Sample preparation: Dilute perfusate 101 times with water and inject a 10 JULL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:water:acetic acid 19:80:1 containing 625 nM 1-heptane-sulfonic acid
Flow rate: 2
Detector: F ex 225 em300
CHROMATOGRAM
Retention time: 8.1
OTHER SUBSTANCES
Extracted: atenolol
KEYWORDS
rat
REFERENCE
Lindahl,A.; Krondahl,E.; Grud6n,A.-C; Ungell,A.-L.; Lennernas,H. Is the jejunal permeability in rats age-
dependent?, Pharm.Res., 1997, 14, 1278-1281.
SAMPLE
Matrix: solutions
Sample preparation: Inject 15 or 50 |xL aliquot.
HPLC VARIABLES
Guard column: 5 |xm LiChrospher 60 RP-Select B
Column: 125 X 4 5 fim LiChrospher 60 RP-Select B
Mobile phase: MeCN:MeOH:30 mM pH 4.5 phosphate buffer 10:20:70 (A) or 20:47:33 (B)
Flow rate: 1.5(A), 2 (B)
Injection volume: 50 (A), 15 (B)
Detector: UV 223
REFERENCE
Galia,E.; Nicolaides,E.; H6rter,D.; Lobenberg,R.; Reppas,C; Dressman,J.B. Evaluation of various dissolution
media for predicting in vivo performance of class I and II drugs, Pharm.Res., 1998, 15, 698705.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 bidentate C18 silane column (Preparation is as follows. Reflux 60 g 7 |xm
Zorbax PSM300 silica in 600 mL 75 ppm HF in water for 72 h (Caution! HF is highly toxic!),
wash with 1.5 L water, wash with 500 mL acetone, dry overnight under vacuum (30 in. Hg).
Add to 570 mL water boil for 10 h, cool to room temperature, wash with 500 mL acetone, dry
overnight at 110 under vacuum (30 in. Hg). Heat 6 g of this material at 110 under vacuum
(30 in. Hg) and place it in a dry nitrogen atmosphere. Add 60 mL dry xylene, 240 JJLL pyridine,
and 4.9 mL dichlorodimethyldioctadecyldisiloxane (?) (Petrarch Systems, Bristol, PA). Reflux
under nitrogen for 80 h, cool, wash with 300 mL toluene, 300 mL dichloromethane, 300 mL
MeOH, 300 mL MeOH:water 50:50, and 300 mL acetone. Dry at 110 under vacuum (30 in.
Hg overnight) (cf. US Pat. 4 746 572).)
Mobile phase: MeCN:17 mM pH 11 K3PO4 buffer 50:50
Flow rate: 1
Injection volume: 5
Detector: not given
CHROMATOGRAM
Retention time: 2.4
OTHER SUBSTANCES
Simultaneous: pindolol
REFERENCE
Kirkland,J.J.; van Straten,M.A.; Claessens,H.A. Reversed-phase high-performance liquid chromatography of
basic compounds at pH 11 with silica-based column packings, J.Chromatogr.A, 1998, 797, 111120.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 275
OTHER SUBSTANCES
Also analyzed: disopyramide, lidocaine
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Mix 300 |xL of a 30 |xM solution in dichloromethane with 10 JJLL 20 mM
l-(6-methoxy-2-naphthyl)ethyl isothiocyanate in anhydrous dichloromethane and 50 |xL 0.1%
triethylamine in dichloromethane, vortex thoroughly, heat at 50 for 1.5 h, inject an aliquot.
(Synthesize l-(6-methoxy-2-naphthyl)ethyl isothiocyanate as follows (protect from light). Dis-
solve 500 mg (S)-(+)-naproxen in 50 mL dry toluene, slowly add 5 mL freshly distilled thionyl
chloride, reflux for 1 h, evaporate to dryness under vacuum, dry the acyl chloride (mp 87.5)
under vacuum over KOH for 2 days. Dissolve 0.5 mmoles acyl chloride in 5 mL acetone, stir
at 0, add 0.6 mmoles sodium azide dissolved in ice water, stir at 0 for 30 min, add 10 mL ice-
cold water, filter, dry solid in a desiccator under vacuum. Dissolve the solid in 1 mL toluene
or dichloromethane (dried over 3 A molecular sieve), reflux for 10 min, evaporate, store re-
sulting isocyanate (mp 51) under vacuum over a desiccant. Dissolve 0.5 mmole isocyanate in
5 mL acetone, add 20 mL 8.5% phosphoric acid, heat to 80 for 1.5 h, adjust to pH 13, extract
with diethyl ether:dichloromethane 4:1. Wash the organic layer twice with water, dry over
anhydrous sodium sulfate, evaporate to dryness, dissolve in 1 mL toluene, evaporate to give
the amine from naproxen as crystals (mp 53) (Pharm.Res. 1990, 7, 1262). Dissolve 1 mmole
1,1-thiocarbonyldiimidazole in 15 mL ice-cold chloroform, stir at 0, add dropwise 1 mmole of
the amine dissolved in 10 mL chloroform, stir at room temperature for 1.5 h, evaporate to
dryness, reconstitute with carbon tetrachloride (Caution! Carbon tetrachloride is a carcinogen!),
filter, evaporate the filtrate to dryness, store the resulting oil in a desiccator, purify on a short
silica gel column with dichloromethane:light petroleum 50:50 to give l-(6-methoxy-2-
naphthyDethyl isothiocyanate as a slightly yellow liquid (store in the freezer under argon).)
HPLC VARIABLES
Column: 250 X 4 5 |xm Zorbax ODS
Flow rate: 1
CHROMATOGRAM
Retention time: k' 20.4 (S-(-)), 25.4 (BrM)
KEYWORDS
derivatization; chiral; F not much more sensitive than UV; a = 1.25
REFERENCE
Buschges,R.; Linde,H.; Mutschler,E.; Spahn-Langguth,H. Chloroformates and isothiocyanates derived from 2-
J.ChromatogrA, 1996, 725, 323-334.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 CSP-4 (Prepare as follows. Add a solution of 1.07 g L-valyl-L-valyl-L-valine
isopropylester (Bunseki Kagaku 1079, 28, 125) in 30 mL dry dioxane (Caution! Dioxane is a
carcinogen!) dropwise to a mixture of 2.2 g 2,4,6-trichloro-l,3,5-triazine (cyanuric chloride) in
20 mL dry dioxane stirred at 0, add 3 g anhydrous sodium carbonate at room temperature,
stir, filter, evaporate to give a colorless solid. Dissolve 8.3 g of this solid in 30 mL dry dioxane,
add 2 g N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, add 1.5 g anhydrous sodium carbon-
ate, reflux with stirring for 40 h, filter, add 3 g dried 10 am LiChrosorb Si 100, reflux with
slow stirring for 10 h, cool, filter. Wash the solid with dioxane, MeOH, and diethyl ether, dry
under reduced pressure (J.Chromatogr. 1984, 292, 427).)
Mobile phase: Hexane:l,2-dichloroethane:EtOH:trifluoroaceticacid 62.5:35:0.625:0.25
Detector: UV
CHROMATOGRAM
KEYWORDS
chiral; a = 1.05
REFERENCE
Oi,N.; Kitahara,H.; Matsushita,Y.; Kisu,N. Enantiomer separation by gas and high-performance liquid chro-
matography with tripeptide derivatives as chiral stationary phases, J.Chromatogr.A, 1996, 722, 229-232.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 10 X 3.2 5 jmm Partisil ODS3
Column: 100 X 4.6 5 |xm Partisil ODS3
Mobile phase: MeCN:buffer 25:75 (Buffer was 60 mM KH2PO4 adjusted to pH 3.0 with phos-
phoric acid.)
Flow rate: 0.6-1
Detector: UV 270
OTHER SUBSTANCES
Also analyzed: oxprenolol
REFERENCE
Palm,K; Luthman,K; Ungell,A.-L.; Strandlund,G.; Artursson,P. Correlation of drug absorption with molecular
surface properties, J.Pharm.Sci., 1996, 85, 32-39.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 jxL aliquot of a 100-500 fjig/mL solution in mobile phase.
HPLC VARIABLES
Column: 100 X 4.6 5 |jim Hypersil C8 MOS 100A coated with phosphatidylcholine (95% pure
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
nidine, codeine, desipramine, diphenhydramine, dipyridamole, ephedrine, flufenamic acid, hal-
operidol, hydroxyzine, imipramine, indomethacin, lidocaine, megestrol acetate, nabumetone,
KEYWORDS
REFERENCE
Metronidazole
CAS Registry No/. 443-48-1, 69198-10-3 (HCI), 13182-89-3 (benzoate), 73334-05-1 (phosphate)
Merck Index: 6242
Lednicer No.: 1 240
SAMPLE
Matrix: blood
Sample preparation: Prepare a 250 X 5 column filled with 1.5 g Extrelut. 1 mL Serum + 100
|xL 100 |jLg/mL ornidazole in MeOH:water 1:1 + 1 mL 1 M Na2HPO4, shake for 5 s, add to
column, After 15 min elute with 10 mL dichloromethane, evaporate the eluate to dryness in a
stream of nitrogen, redissolve residue in 100 JJLL MeOH:water 1:1, shake for 30 s, inject a 5 |xL
aliquot.
HPLC VARIABLES
Column: 100 X 2.1 5 |xm MOS-Hypersil RP-8
Flow rate: 0.2
Injection volume: 5
Detector: UV 318
CHROMATOGRAM
Internal standard: ornidazole (3.36)
KEYWORDS
serum; SPE
REFERENCE
R6na,K.; Gachalyi,B- Simple liquid chromatographic method for the determination of ornidazole and metron-
idazole in human serum, J.Chromatogr., 1987, 420, 228-230.
SAMPLE
Matrix: blood, eggs, tissue
Sample preparation: Serum. 2 mL Serum + 1 mL 20% trichloroacetic acid in MeOH, vortex for
1 min, centrifuge at 3500 rpm. Filter (0.45 |xm) the supernatant, inject a 20 |xL aliquot of the
nitrate. Eggs. Condition a 3 mL Bakerbond octadecyl SPE cartridge with 5 mL MeOH, 10 mL
water, and 2 mL 1% trichloroacetic acid. Homogenize 5 g blended whole egg and 5 mL pH 4.5
phosphate buffer, add 25 mL MeCN, homogenize, centrifuge at 3500 rpm for 10 min. Remove
the supernatant and evaporate it to 2 mL under reduced pressure at 40, add 10 mL 1%
trichloroacetic acid to the residue, add the mixture to the SPE cartridge, wash with 10 mL
water, dry under vacuum, elute with 2 mL 0.1% triethylamine in MeCN. Evaporate the eluate
to dryness, reconstitute with 2 mL mobile phase, inject a 20 |xL aliquot. Tissue. Condition a 3
mL Bakerbond octadecyl SPE cartridge with 5 mL MeOH, 10 mL water, and 2 mL 1% tri-
chloroacetic acid. Homogenize 5 g tissue and 25 mL MeCN, centrifuge at 3500 rpm for 10 min,
remove the supernatant, extract the residue with 15 mL MeCN. Combine the supernatants,
add 20 mL n-hexane, shake. Remove the lower layer and evaporate it to 2 mL, add 10 mL 1%
trichloroacetic acid to the residue, add the mixture to the SPE cartridge, wash with 10 mL
water, dry under vacuum, elute with 2 mL 0.1% triethylamine in MeCN. Evaporate the eluate
to dryness, reconstitute with 2 mL mobile phase, inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 200 X 4.6 5 jxm Bakerbond BDS-C18
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Limit of quantitation: 5 ng/mL (serum), 2 ng/g (tissue, eggs)
OTHER SUBSTANCES
Extracted: dimetronidazole
Noninterfering: chloramphenicol, nitrofurans, sulfonamides
KEYWORDS
hen; serum; liver; muscle; SPE
REFERENCE
Semeniuk,S.; Posyniak,A.; Niedzielska,J.; Zmudski,J. Determination of nitroimidazole residues in poultry tis-
sues, serum and eggs by high-performance liquid chromatography, Biomed.Chromatogr., 1995, 9, 238-242.
SAMPLE
Matrix: blood, gastric juice, saliva
Sample preparation: Plasma, saliva. Mix 500 JLJLL plasma or saliva with 50 |JLL 50% perchloric
acid (w/v), vortex briefly, add 1.5 g solid anhydrous potassium carbonate to neutral pH. Add
300 jxL MeCN, mix, centrifuge at 11600 g for 6 min., remove a 180 jxL aliquot of the super-
natant, evaporate under a stream of nitrogen at 50, reconstitute the residue with 500 |iL
mobile phase, inject a 100 |JLL aliquot. Gastric juice. Mix 500 (JLL gastric juice with 20 |xL 10
|xg/mL IS in water and 50 |JLL 50% perchloric acid, vortex briefly, add 1.5 g solid anhydrous
potassium carbonate to neutral pH. Add 300 (JLL MeCN, mix, centrifuge at 11600 g for 6 min,
remove a 180 fxL aliquot of the supernatant, evaporate under a stream of nitrogen at 50,
reconstitute the residue with 500 JJLL mobile phase, inject a 100 |JLL aliquot.
HPLC VARIABLES
Guard column: 20 X 2 5 jxm Hypersil ODS
Mobile phase: MeCNrbuffer 10:90 (Buffer was 50 mM KH2PO4 containing 0.1% triethylamine,
Flow rate: 1
Detector: UV 317
CHROMATOGRAM
Retention time: 6.7
Limit of quantitation: 250 ng/mL (plasma, gastric juice), 100 ng/mL (saliva)
OTHER SUBSTANCES
Extracted: active metabolite
KEYWORDS
plasma
REFERENCE
Jessa,M.J.; Barrett,D.A.; Shaw,P.N.; Spiller,R-CJ. Rapid and selective high-performance liquid chromatographic
method for the determination of metronidazole and its active metabolite in human plasma, saliva and
gastric juice, J.Chromatogr.B, 1996, 677, 374-379.
SAMPLE
Sample preparation: 100 |xL Urine or 1 mL plasma + 100 ng IS + 1 mL MeCN, centrifuge.
Remove supernatant and add it to 1 mL buffer, add 6 mL dichloromethaneipetroleum ether:
isopropanol 45:45:10, rotate for 10 min. Remove the upper organic layer and evaporate it under
nitrogen at 60. Dissolve residue in 100 |xL mobile phase, inject. (Buffer contained 80 g NaHCO3
and 30 g K2CO3 per liter, pH 9.5.)
HPLC VARIABLES
Column: 466 X 5 5 |xm Spherisorb ODS
Mobile phase: MeCN:MeOH:45 mM pH 4.5 KH2PO4 40:3:57, containing 40 g/L NaClO4 and 40
g/L trimethylammonium chloride
Flow rate: 1
Detector: UV 338
CHROMATOGRAM
Retention time: 3.8
Internal standard: 4-(3-dimethylaminopropyl)-4-chloroquinoline (5)
OTHER SUBSTANCES
Simultaneous: chloroquine
KEYWORDS
plasma
REFERENCE
Okonkwo,P.O.; Eta,E.I. Simultaneous determination of chloroquine and metronidazole in human biological fluid
by high-pressure liquid chromatography, Life ScL, 1988, 42, 539-545.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 320
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Grind tablets to a fine powder and weigh out powder equivalent to about
80 mg metronidazole, add 90 mL water, heat to 60 with stirring, cool to room temperature,
make up to 100 mL with water, filter, reject first 20 mL of filtrate. Mix 12.5 mL filtrate with
7 mL 10 mg/mL phenylpropanolamine hydrochloride in water, make up to 50 mL with water,
HPLCVARIABLES
Column: 300 X 4 10 (xm (xBondapak phenyl
Mobile phase: 20 mM KH2PO4, pH 4.2
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Retention time: 7
Internal standard: phenylpropanolamine hydrochloride (3.5)
OTHER SUBSTANCES
Noninterfering: mannitol, excipients
KEYWORDS
REFERENCE
Das Gupta,V. Quantitation of metronidazole in pharmaceutical dosage forms using high-performance liquid
chromatography, J.Pharm.Sci., 1984, 73, 1331-1333.
SAMPLE
Sample preparation: Dissolve 5 g suspension (containing about 200 mg metronidazole benzoate)
in 100 mL MeOH. Dilute 2.5 mL of this solution and 2 mL 200 fxg/mL ethyl paraben in MeOH
to 10 mL with MeOH, filter, inject a 10 |AL aliquot.
HPLCVARIABLES
Column: 300 X 4 Varian MicroPak MCH-10 reverse phase
Mobile phase: 20 mM ammonium acetate in MeCNrwater 47:53, adjusted to pH 4.5 using glacial
acetic acid
Flow rate: 2
Detector: UV 270
CHROMATOGRAM
Retention time: 5.56 (metronidazole benzoate), 2.9 (metronidazole with 12% MeCN in mobile
phase)
Internal standard: ethyl paraben (2.78)
OTHER SUBSTANCES
Simultaneous: 2-methyl-5-nitroimidazole, misonidazole, tinidazole, benzoic acid, methyl para-
ben, propyl paraben
KEYWORDS
suspensions; stability-indicating
REFERENCE
Sa'sa',S.L; Khalil,H.S.; Jalal,I.M. Determination of metronidazole benzoate in liquid preparations by reversed
phase HPLC, J.Liq.Chromatogr., 1986, 9, 3617-3631.
SAMPLE
Sample preparation: Weigh out 2 g metronidazole benzoate suspension containing 3.5%
metronidazole benzoate, add 80 mL MeOHrwater 80:20, sonicate for a few minutes, make up
to 100 mL with MeOH water 80:20, centrifuge at 900 g for 10 min, inject a 10 jxL aliquot.
HPLCVARIABLES
Guard column: 50 X 2 30-38 urn Whatman Co:Pell
Column: 250 X 4.6 10 |xm Perkin-Elmer C18
Mobile phase: MeOH:water 60:40 containing 5 mM acetate buffer and 50 mM KNO3, pcH* 5.2
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 5 (metronidazole), 12 (metronidazole benzoate)
OTHER SUBSTANCES
Simultaneous: benzoic acid, methylparaben, propylparaben
KEYWORDS
suspensions
REFERENCE
Pashankov,P.R; Kostova,L.L. Reversed-phase high-performance liquid chromatography of metronidazole ben-
zoate in suspension dosage form, J.Chromatogr., 1987, 394, 382-387.
SAMPLE
Sample preparation: Dilute 1:5 with water, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 4 jxm jxBondapak C18
Mobile phase: MeCN:buffer 7:93 (Buffer was 20 mM KH2PO4 and 5 mM triethylamine adjusted
to pH 4.8 with NaOH.)
Flow rate: 1.5
Detector: UV 270
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: desacetylcefotaxime, cefotaxime
KEYWORDS
stability-indicating; injections; water
REFERENCE
Rivers,T.E.; McBride,H.A.; Trang,J.M. Stability of cefotaxime sodium and metronidazole in an i.v. admixture
at 8C, Am. J.Hosp.Pharm., 1991, 48, 2638-2640.
SAMPLE
Sample preparation: Mix an aliquot with an equal volume of 5 mg/mL cefoxitin, dilute with
water, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 5 |xm Resolv (Waters)
Mobile phase: MeCN:buffer 18:86 (Buffer was 2.46 g anhydrous sodium acetate, 8 mL glacial
acetic acid, and 200 mg tetrabutylammonium hydrogen sulfate in 1 L water, pH 3.0.)
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Retention time: 1.8
Internal standard: cefoxitin (3.0)
OTHER SUBSTANCES
Simultaneous: cefotaxime
KEY WORDS
REFERENCE
Belliveau,RR; Nightingale,C.H.; Quintiliani,R. Stability of cefotaxime sodium and metronidazole in 0.9% so-
dium chloride injection or in ready-to-use metronidazole bags, Am.J.Health-Syst.Pharm., 1995, 52, 1561-
1563.
SAMPLE
Sample preparation: Dilute 50-fold with water, inject an aliquot.
HPLCVARIABLES
Mobile phase: MeCN:20 mM KH2PO4 7:93 containing 10 mM triethylamine, adjusted to pH 4.8
with HCl
Flow rate: 1.5
Detector: UV 270
CHROMATOGRAM
Retention time: 3.1
OTHER SUBSTANCES
Simultaneous: ceftazidime, ceftizoxime, ceftriaxone
KEYWORDS
saline; injections
REFERENCE
Rivers,T.E.; Webster,A.A. Stability of ceftizoxime sodium, ceftriaxone sodium, and ceftazidime with metroni-
dazole in ready-to-use metronidazole bags, Am.J.Health-Syst.Pharm., 1995, 52, 2568-2570.
SAMPLE
Sample preparation: If necessary, remove oxidizing power of solution by adding sodium meta-
bisulfite, inject a 20 |xL aliquot.
HPLCVARIABLES
Guard column: 15 X 4.6 5 |xm Microsorb C8
Flow rate: 1
Detector: UV 325
CHROMATOGRAM
Retention time: 7.1
REFERENCE
Lunn,G.; Rhodes,S.W.; Sansone,E.B.; Schmuff,N.R. Photolytic destruction and polymeric resin decontamination
of aqueous solutions of Pharmaceuticals, J.Pharm.Sci., 1994, 83, 1289-1293.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCNibuffer 15:85 (Buffer was 10 mM NaH2PO4 adjusted to pH 8 with
trimethylamine.)
Flow rate: 1.5
Detector: UV 250
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Simultaneous: miconazole
REFERENCE
Faouzi,M.E.A.; Dine,T.; Luyckx,M.; Brunet,C; Mallevais,M.-L.; Goudaliez,E; Gressier,B.; Cazin,M.; Kablan,J.;
Cazin,J.C. Stability, compatibility and plasticizer extraction of miconazole injection added to infusion so-
lutions and stored in PVC containers, J.Pharm.Biomed.Anal., 1995, 13, 1363-1372.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 Jim LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol, mida-
himbine, zopiclone
KEYWORDS
REFERENCE
J.Chromatogr.A, 1995, 692, 103-119.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 1 mL Accubond C18 SPE cartridge (J&W Scientific) with three
1 mL portions of MeOH and three 1 mL portions of water. Homogenize (Tekmar tissuemiser)
250 ng vaginal tissue, 1 mL water, and 1 mL 400 ng/mL tinidazole in water in ice for 30 s,
rinse homogenizer. Combine homogenate and rinse and add 100 |JLL 5% trichloroacetic acid,
mix, centrifuge at 1500 g for 30 min, remove the supernatant, wash the precipitate with 1 mL
water. Add the supernatant and the wash to the SPE cartridge, wash with two 1 mL portions
of water, dry under vacuum for 10 min, elute with four 250 JJLL aliquots of MeOH. Evaporate
the eluate to dryness under a stream of nitrogen, reconstitute the residue in 1 mL mobile
HPLC VARIABLES
Column: 150 X 4.6 5 jxm Zorbax SB-phenyl
Mobile phase: MeOHrbuffer 15:85 (Buffer was 10 mM KH2PO4 adjusted to pH 4.0 with 10%
phosphoric acid
Flow rate: 1
Detector: UV 313
CHROMATOGRAM
Retention time: 9.2
OTHER SUBSTANCES
Simultaneous: cefazolin, cefmetazole, cephalexin
KEYWORDS
SPE; human; dog; vaginal tissue
REFERENCE
Venkateshwaran,T.G.; Stewart,J.T. Determination of metronidazole in vaginal tissue by high-performance liq-
uid chromatography using solid-phase extraction, J.Chromatogr.B, 1995, 672, 300-304.
SAMPLE
Matrix: urine
Sample preparation: Dilute urine 1:2 with MeOH, let stand for 1 h, centrifuge at 5000 g for 1
h. Dilute the supernatant 1:1 with water, inject a 20 (xL aliquot.
HPLC VARIABLES
Column: 120 X 4.6 5 fxm LiChrosorb Si 60
Mobile phase: MeOH:200 mM pH 7.0 potassium phosphate:water 25:30:45 containing 2.5 mM
N-cetyl-N,N,N-trimethylammonium bromide (Use a 150 X 4.6 column of 15-25 |xm LiChroprep
Si 60 between pump and injector.)
Flow rate: 0.5 for 9 min, then 1
Detector: UV 312
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
REFERENCE
Thomsen,U.G.; Cornett,C; Tjornelund,J.; Hansen,S.H. Separation of metronidazole, its major metabolites and
their conjugates using dynamically modified silica, J.Chromatogr.A, 1995, 697, 175-184.
Metyrapone
CAS Registry No.: 54-36-4, 908-35-0 (ditartrate)
Merck Index: 6246
SAMPLE
Matrix: blood
Sample preparation: 250 |xL Whole blood + 500 |xL 1 M NaOH + 100 |JLL 500 |xM 2,3'-dipyridyl
in water + I g NaCl, extract twice with 1 mL portions of dichloromethane. Combine the organic
layers and evaporate them to dryness at 40, reconstitute the residue in 25 |xL MeOH, inject
an aliquot.
HPLCVARIABLES
Mobile phase: MeCN:67 mM pH 7.4 phosphate buffer 25:75 (Place a 100 X 4.6 column of 40-60
ixm silica between pump and injector.)
Detector: UV 261
CHROMATOGRAM
Internal standard: 2,3'-dipyridyl (7)
Limit of detection: 4.4 jiM
OTHER SUBSTANCES
KEYWORDS
rat; whole blood
REFERENCE
Usansky,J.L; Damani,L.A. Assay of metyrapone, metyrapol and the isomeric mono-N-oxides of metyrapone in
biological fluids by high-performance liquid chromatography, J.Chromatogr., 1991, 563, 283-298.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 100 |xL 1 M NaOH + 250 mg NaCl + 100 |xL 249
jxg/mL oxprenolol in MeOH, vortex for 30 s, add 5 mL diethyl ether.dichloromethane 80:20,
vortex for 2 min, centrifuge at 1500 g for 20 min, freeze in dry ice-acetone. Remove the organic
in 500 |xL mobile phase, inject a 100 |xL aliquot. Urine. 1 mL Urine + 100 |xL 1 M NaOH +
250 mg NaCl + 100 (xL 249 jjig/mL oxprenolol in MeOH, vortex for 30 s, add 5 mL diethyl
ether.dichloromethane 80:20, vortex for 2 min, centrifuge at 1500 g for 20 min, freeze in dry
ice-acetone. Remove the organic layer and evaporate it to dryness under a stream of nitrogen
at 40, reconstitute the residue in 1 mL water, add 100 |xL 1 M HCl, vortex for 30 s, add 5 mL
hexane:diethyl ether 50:50, vortex for 1 min, centrifuge at 1500 g for 10 min, freeze in dry ice-
acetone. Discard the organic phase, thaw the aqueous phase and add it to 200 u-L 1 M NaOH
and 250 mg NaCl, vortex for 30 s, add 5 mL diethyl ether.dichloromethane 80:20, vortex for 2
min, freeze in dry ice-acetone. Remove the organic layer and evaporate it to dryness under a
stream of nitrogen at 40, reconstitute the residue in 500 fxL mobile phase, inject a 100 |xL
aliquot.
HPLC VARIABLES
Guard column: 75 X 4.6 Ultremex 3 silica
Column: 250 X 4.6 10 jjim Chiralcel OJ-CSP cellulose tris(4-methylbenzoate)
Mobile phase: Hexane:EtOH 92:8 containing 0.1% diethylamine (plasma) or hexane:EtOH 94:6
containing 0.2% diethylamine (urine)
Flow rate: 1
Detector: UV 261
CHROMATOGRAM
Internal standard: oxprenolol (10)
OTHER SUBSTANCES
KEYWORDS
plasma; chiral (for metabolites)
REFERENCE
Chiarotto,J.A.; Wainer,I.W. Determination of metyrapone and the enantiomers of its chiral metabolite metrapol
in human plasma using coupled achiral-chiral liquid chromatography, J.Chromatogr.B, 1995,665,147-154.
SAMPLE
Matrix: microsomal incubation
Sample preparation: 2 mL Microsomal incubation + 500 |xL 1 M NaOH + 500 |xL 500 |xM 2,3'-
dipyridyl in water + I g NaCl, extract twice with 1 mL portions of dichloromethane. Combine
the organic layers and evaporate them to dryness at 40, reconstitute the residue in 25 |xL
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Partisil ODS-I
Mobile phase: MeCN:67 mM pH 7.4 phosphate buffer 20:80 (Place a 100 X 4.6 column of 40-60
(xm silica between pump and injector.)
Flow rate: 2
Detector: UV 261
CHROMATOGRAM
Retention time: 16
Internal standard: 2,3'-dipyridyl (10)
OTHER SUBSTANCES
KEYWORDS
rat; liver
REFERENCE
Usansky,J.L; Damani,L.A. Assay of metyrapone, metyrapol and the isomeric mono-N-oxides of metyrapone in
biological fluids by high-performance liquid chromatography, J.Chromatogr., 1991, 563, 283-298.
SAMPLE
Matrix: urine
Sample preparation: Buffer urine to 4.9 by mixing with an equal volume of pH 4.9 200 mM
sodium phosphate buffer. Inject a 40 jxL aliquot onto column A with mobile phase A, after 3
min backflush the contents of column A onto column B with mobile phase B and start the
gradient. At the end of the run re-equilibrate for 10 min.
HPLC VARIABLES
Column: A 20 X 4 5 |xm Hypersil octadecylsilica ODS; B 200 X 4.6 5 jxm Shiseido SG-120 poly-
mer-based C18
Mobile phase: A water; B Gradient. MeCN:buffer from 7:93 to 15:85 over 3.5 min, to 50:50 over
8.5 min, maintain at 50:50 for 11 min (Buffer was 6.9 g NaH2PO4.H2O in 1L water, pH adjusted
to 3.1 with phosphoric acid.)
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: acetazolamide, amiloride, bendroflumethiazide, benzthiazide, bumetanide, caffeine,
carbamazepine, chlorothiazide, chlorthalidone, clopamide, dichlorfenamide, ethacrynic acid, fu-
rosemide, hydrochlorothiazide, probenecid, spironolactone, triamterene, trichlormethiazide
KEYWORDS
column-switching; optimum detection wavelengths vary for each drug
REFERENCE
Saarinen,M.; Sir6n,H.; Riekkola,M.-L. A column switching technique for the screening of diuretics in urine by
high performance liquid chromatography, J.Liq.Chromatogr., 1993, 16, 4063-4078.
Mexiletine
Merck Index: 6257
SAMPLE
Matrix: blood
Sample preparation: Mix 400 JJLL serum with 20 |xL 10 |xg/mL IS in MeOH and 50 |JLL 10%
sodium carbonate. Add 4 mL diisopropyl ether, shake vigorously for 4 min, centrifuge, freeze
at -20. Mix the organic layer with 100 |xL 10 mM HCl, vortex carefully for 45 s using a
microshaker, centrifuge, evaporate the aqueous phase to dryness under a stream of argon in a
56 water bath. Reconstitute the residue in 100 |xL mobile phase, inject a 50 jxL aliquot. (Cau-
tion! Diisopropyl ether readily forms explosive peroxides!)
HPLC VARIABLES
Guard column: 20 X 4.6 5 jxm Supelguard LC-CN
Column: 150 X 4.6 5 (xm Supelcosil LC-CN
Mobile phase: MeCN:water:500 mM KH2PO4 36:62:2
Flow rate: 1.8
Detector: UV 210
CHROMATOGRAM
Retention time: 3.5
Internal standard: LU41616 (2>-[2-hydroxy-3-(3"-hydroxy-3"-methylbutylamino)propoxy]-3-
phenylpropiophenone hydrochloride) (7.7)
OTHER SUBSTANCES
Extracted: metabolites, diltiazem, propafenone
Simultaneous: acebutolol, amiodarone, aprobarbital, atenolol, bupranolol, celiprolol, clobazam,
debrisoquine, diazepam, flecainide, gallopamil, hexobarbiltal, lidocaine, mephenytion, meto-
polol, nadolol, pentobarbital, phenacetin, prazosin, procainamide, progesterone, propranolol,
quinidine, sotalol, theophylline, verapamil
KEYWORDS
serum
REFERENCE
Kunicki,P.K.; Sitkiewicz,D. High performance liquid chromatographic analysis of some antiarrhythmic drugs
in human serum using cyanopropyl derivatized silica phase, J.Liq.Chromatogr.Rel.Technol., 1996,19,1169-
1181.
SAMPLE
Matrix: blood
Sample preparation: 1 mL serum + 250 JJLL triethylamine + 100 |xL 1 M NaOH + 100 |xL 2 |xg/
mL IS in 10 mM HCl + 1.75 mL dichloromethane, rotate slowly for 20 min, centrifuge at 3600
g for 10 min. Remove 1 mL of the organic layer and evaporate it to dryness under a stream of
nitrogen at room temperature, reconstitute the residue in 500 |xL 10 mM HCl, inject a 200 (xL
aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Hypersil CPS (CN)
Mobile phase: MeCN:buffer 5:95 (Buffer was 973.5 mL water, 25 mL PIC B-8 low-UV reagent
+ 1 mL butylamine + 0.5 mL PIC D-4 reagent.)
Flow rate: 2
Detector: UV 215
CHROMATOGRAM
Retention time: 21
Internal standard: l-(2,4-dimethylphenoxy)-2-aminopropane hydrochloride (KOE 768-CL) (29)
OTHER SUBSTANCES
Noninterfering: acebutolol, acenocoumarol, amiodarone, aspirin, atenolol, betaxolol, digoxin, di-
pyridamole, flecainide, furosemide, hydrochlorothiazide, metoprolol, nifedipine, phenprocou-
mon, pindolol, propafenone, quinidine, sotalol, spironolactone, sulfinpyrazone, triamterene,
verapamil
KEYWORDS
serum
REFERENCE
Kramer,B.K.; Ress,K.M.; Mayer,R; Kuhlkamp,V.; Liebich,H.M.; Risler,T.; Seipel,L. Rapid high-performance liq-
uid chromatographic method for the quantification of mexiletine and its metabolites in serum,
J.Chromatogr., 1989, 493, 414-420.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum or plasma + 100 |xL 500 mM sodium carbonate + 100 jxL
5 |xg/mL N-propionylprocainamide in water, mix gently, add 5 mL dichloromethane, shake
gently for 5 min, centrifuge. Remove the lower organic layer and add it to 200 |xL 10 mM HCl,
vortex for 15 s, centrifuge, inject a 20 JAL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 100 X 5 NovaPak cyano HP radial compression
Mobile phase: MeCN:buffer 10:90, final pH adjusted to 6.0 (Buffer was 5 mM acetate buffer
containing 0.05% triethylamine.)
Flow rate: 1.5
Detector: UV 210
CHROMATOGRAM
Retention time: 6.8
Internal standard: N-propionylprocainamide (5.9)
OTHER SUBSTANCES
Simultaneous: N-acetylprocainamide, disopyramide, lidocaine, procainamide, quinidine,
tocainide
Noninterfering: carbamazepine, desmethyldoxepin, digoxin, doxepin, ethosuximide, lithium,
phenobarbital, phenytoin, primidone, propranolol, theophylline, valproic acid
KEY WORDS
serum; plasma
REFERENCE
vasBinder,E.; Annesley,T. Liquid chromatographic analysis of mexiletine in serum, with alternate application
to tocainide, procainamide, and N-acetylprocainamide, Biomed.Chromatogr., 1991, 5, 1922.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL 0.4 |xg/mL methylmexiletine hydrochloride in
water + 1 mL 150 mM barium hydroxide + 1 mL 170 mM zinc sulfate, vortex, add 500 JJLL 2
M NaOH, extract twice with 5 mL portions of diethyl ether. Combine the organic layers and
evaporate them to dryness at 45, reconstitute the residue in 20 jxL 30 mM HCl, add 60 jxL
100 mM sodium borate, add 60-80 JJLL reagent, keep at 4, inject an aliquot. (Reagent was 40
mg o-phthalaldehyde and 50 mg N-acetyl-L-cysteine in 5 mL MeOH, prepare daily.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Apex C18 (Jones Chromatography)
Mobile phase: MeOH:50 mM sodium acetate 65:35, pH 7.3
Flow rate: 1
CHROMATOGRAM
Retention time: 14 (S-(+)), 15 (R-(-))
Internal standard: methylmexiletine (23, 24 (enantiomers))
OTHER SUBSTANCES
KEYWORDS
chiral; plasma; pharmacokinetics; derivatization
REFERENCE
Abolfathi,Z.; Belanger,R-M.; Gilbert,M.; Rouleau,J.R.; Turgeon,J. Improved high-performance liquid chromat-
ographic assay for the stereoselective determination of mexiletine in plasma, J.Chromatogr., 1992, 579,
366-370.
SAMPLE
Matrix: blood
Sample preparation: 200 |JLL Serum + 30 |xL 1 |xg/mL IS in water + 200 |xL 300 mM barium
hydroxide, vortex, add 200 JJLL 300 mM zinc sulfate, add 200 (xL 2 M NaOH, add 5 mL diethyl
ether, vortex, centrifuge at 2000 g for 5 min, repeat extraction. Combine the organic layers
and evaporate them to 1 mL under a stream of nitrogen at 37, add 300 |xL 100 mM HCl,
vortex, discard the ether layer. Wash the aqueous layer with 2 mL diethyl ether, add 300 |xL
2 M NaOH to the aqueous layer, add 10 JULL 1 mg/mL 2-anthroyl chloride in dry dichloro-
methane, vortex for 3 min, extract with 1 mL dichloromethane. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen at 37, reconstitute the residue in 200 (xL
mobile phase, inject an aliquot. (Prepare 2-anthroyl chloride as follows. Reflux 500 mg anthra-
quinone-2-carboxylic acid with 30 mL 14% aqueous ammonia, add 2 g zinc dust in small por-
tions over 30 min, reflux with stirring for 1 h, filter, acidify the filtrate to pH 3 with dilute
HCl, filter, recrystallize from hot EtOH to give 2-anthracenecarboxylic acid (mp 278-279, yield
230%). Add 100 mg 2-anthracenecarboxylic acid in 100 mL dry benzene (Caution! Benzene is
a carcinogen!) to 500 jxL oxalyl chloride with stirring, heat slowly on a water bath for 30 min,
add 500 JJLL oxalyl chloride, heat for 30 min, cool, filter. Evaporate the filtrate to obtain 40 mg
pale yellow 2-anthroyl chloride. Purify by dissolving in dichloromethane and precipitating with
hexane. Purify further by HPLC by injecting 100 |xL aliquots of a 50 mg/mL solution in MeCN
onto a 250 X 9.4 10 |xm ODS-2 Magnum-9 (Whatman) column and eluting with MeCN at 0.7
mL/min. Using a UV 230 detector collect the appropriate fraction (about 11 min) and evaporate
to dryness to obtain pure compound.)
HPLC VARIABLES
Guard column: 150 X 4.6 5 fjim silica (Alltech)
Column: 250 X 4.6 5 |xm Pirkle 1-A phenylglycine (Regis)
Mobile phase: Hexane:isopropanol:chloroform 78:7:15
Flow rate: 0.8
CHROMATOGRAM
Internal standard: l-(2',6'-dimethylphenoxy)-2-ethanamine hydrochloride (Boehringer Ingel-
heim KPE-2963) (14.5)
KEYWORDS
derivatization; serum; chiral; pharmacokinetics
REFERENCE
Kwok,D.K.W.; Igwemezie,L.; Kerr,C.R.; McErlane,K.M. High-performance liquid chromatographic analysis us-
ing a highly sensitive fluorogenic reagent, 2-anthroyl chloride, and stereoselective determination of the
enantiomers of mexiletine in human serum, J.Chromatogr.B, 1994, 661, 271-280.
SAMPLE
Matrix: blood
with 3 mL 750 mL/L methanol. Elute with three aliquots of 300 (xL 0.1 M ammonium acetate
(xL 0.1 M phosphoric acid (pH 2.5), mix, separate, inject a 100 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Flow rate: 2
Detector: UV 228
CHROMATOGRAM
Retention time: 1.4
OTHER SUBSTANCES
norfluoxetine, nortriptyline, norverapamil, pentazocine, promazine, propafenone, propoxy-
phene, propranolol, protriptyline, temazepam, trazodone, trimipramine, verapamil
warfarin
Interfering: encainide, lidocaine, quinidine
KEYWORDS
plasma; SPE
REFERENCE
Clin.Chem., 1994, 40, 1312-1316.
SAMPLE
Matrix: blood
Sample preparation: Make serum alkaline with 10% sodium carbonate, extract with diisopropyl
ether (Caution! Diisopropyl ether readily forms explosive peroxides!). Remove the organic layer
and extract it with 10 mM HCl, inject an aliquot of the aqueous layer.
HPLCVARIABLES
Column: 150 X 4.6 5 jxm Supelcosil LC-CN
Mobile phase: MeCN:water:500 mM KH2PO4 36:62:2
Flow rate: 1.8
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diltiazem, propafenone
KEYWORDS
serum
REFERENCE
Kunicki,P.K.; Sitkiewicz,D. High-performance liquid chromatographic determination of some antiarrhythmic
drugs using cyanopropyl derivatized silica phase (Abstract 43), Ther.Drug Monit, 1995, 17, 394-394.
SAMPLE
Matrix: blood
HPLC VARIABLES
Column: 300 X 3.9 4 ^m NovaPack C18
Flow rate: 0.8
Detector: UV 262
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: blood
Sample preparation: 500 JXL Plasma + 50 |xL 300 mM NaOH, mix, add 4 mL diisopropyl ether
(Caution! Diisopropyl ether readily forms explosive peroxides!), vortex for 2 min, centrifuge at
1800 g for 10 min, repeat the extraction. Combine the organic layers and add them to 50 |xL
100 mM HCl in MeOH, evaporate to dryness under a stream of nitrogen, reconstitute with 100
fxL aqueous 100 mM HCl, add 100 |JLL 2 M NaOH, add 200 |JLL water, vortex for 15 s, let stand
for 15 min, add 100 JLJLL 4 mg/mL 2-naphthoyl chloride in dichloromethane, vortex for 2 min,
add 2 mL hexane:isopropanol 90:10, vortex for 2 min, centrifuge at 1800 g for 5 min. Remove
the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the
residue in 200 JJIL hexanerisopropanol 90:10, inject an aliquot. (The 300 mM NaOH, 2 M NaOH,
and aqueous 100 mM HCl solutions should be washed with diisopropyl ether before use.)
HPLC VARIABLES
Guard column: 50 mm long Chiralcel OJ
Mobile phase: Hexane:EtOH 71:29
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: amiodarone, captopril, clobazam, clonazepam, disopyramide, ergotamine, nitra-
zepam, nordisopyramide, procainamide, propafenone, sotalol
KEYWORDS
plasma; derivatization; chiral
REFERENCE
Lanchote,V.L.; Bonato,RS.; Dreossi,S.A.C; Gon?alves,P.V.B.; Cesarino,E.J.; Bertucci,C. High-performance liq-
uid chromatographic determination of mexiletine enantiomers in plasma using direct and indirect enantio-
selective separations, J.Chromatogr.B, 1996, 685, 281-289.
SAMPLE
Sample preparation: Serum, saliva. 1 mL Serum or saliva + 30 ng IS + 200 |xL 150 mM barium
hydroxide solution, vortex, add 200 JXL 150 mM zinc sulfate solution, mix, add 200 |xL 2 M
NaOH, extract twice with 5 mL portions of diethyl ether. Combine the organic layers and
evaporate them to 1 mL under a stream of nitrogen at 37, add 300 |xL 100 mM HCl, extract.
Remove the aqueous layer and wash it twice with 2 mL portions of diethyl ether, add 300 |xL
2 M NaOH to the aqueous layer, add 10 jxL 1 mg/mL 2-anthroyl chloride, vortex for 3 min,
extract with 1 mL dichloromethane. Remove the organic layer and evaporate it to dryness
under a stream of nitrogen at 37, reconstitute the residue in 200 jxL mobile phase, inject an
aliquot. Urine. Make urine alkaline with 2 M NaOH, extract twice with 5 mL portions of diethyl
ether. Combine the organic layers and evaporate them to 1 mL under a stream of nitrogen at
37, add 300 |JLL 100 mM HCl, extract. Remove the aqueous layer and wash it twice with 2 mL
portions of diethyl ether, add 300 |xL 2 M NaOH to the aqueous layer, add 10 |xL 1 mg/mL 2-
anthroyl chloride, vortex for 3 min, extract with 1 mL dichloromethane. Remove the organic
in 200 JJLL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Pirkle 1-A phenylglycine (Regis) + 150 X 4.6 5 |xm silica (Alltech) in
series
Mobile phase: Hexane:isopropanol:chloroform 39:3.5:75.5
Flow rate: 0.8
CHROMATOGRAM
Internal standard: l-(2>,6>-dimethylphenoxy)-2-ethanamine (KOE 2963)
KEYWORDS
chiral; serum; derivatization; pharmacokinetics
REFERENCE
Kwok,D.W.; Kerr,C.R.; McErlane,KM. Pharmacokinetics of mexiletine enantiomers in healthy human subjects.
A study of the in vivo serum protein binding, salivary excretion and red blood cell distribution of the
enantiomers, Xenobiotica, 1995,25, 1127-1142.
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 50 |xL 20 |xg/mL 4-methylmexiletine in MeOH +
1 mL 200 mM pH 9 borate buffer + 7 mL diethyl ether, shake on a reciprocating shaker for 10
min, centrifuge at 3000 g for 5 min, freeze in MeOH/dry ice. Remove the organic layer and
MeOH: 10 mM phosphoric acid 48:52 containing 300 mg/L sodium 1-octanesulfonate, inject a
100 |xL aliquot. Urine. 100 uL Urine + 300 uL 1 M pH 5.5 sodium acetate buffer + 2500 U p-
glucuronidase (Helix pomatia Type H-5, Sigma), vortex, heat at 37 for 4 h, add 100 u,L 20 (xg/
mL 4-methylmexiletine in MeOH, add 1 mL 200 mM pH 10 borate buffer, add 7 mL diethyl
ether, shake on a reciprocating shaker for 10 min, centrifuge at 3000 g for 5 min, freeze in
MeOH/dry ice. Remove the organic layer and evaporate it to dryness under a stream of nitrogen
at 40, reconstitute the residue in 250 |xL MeOH: 10 mM phosphoric acid 48:52 containing 300
mg/L sodium 1-octanesulfonate, inject a 100 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 Wakosil ODS (Wako)
Column: 250 X 4.6 Wakosil ODS (Wako)
Mobile phase: Gradient. A was MeOH: 10 mM phosphoric acid 16:84 containing 300 mg/L sodium
1-octanesulfonate. B was MeOH: 10 mM phosphoric acid 80:20 containing 300 mg/L sodium 1-
octanesulfonate. A:B from 65:35 to 20:80 over 10 min, maintain at 20:80 for 4 min, return to
initial conditions over 1 min, re-equilibrate for 10 min.
Flow rate: 1
Detector: F ex 345 em 445 following post-column derivatization. The column effluent was mixed
with reagent pumped at 0.6 mL/min and the mixture flowed through a 5 m X 0.5 mm i.d.
tefzel tube to the detector. (Reagent was 300 mg o-phthalaldehyde in 100 mL EtOH, add 900
mL 200 mM pH 10 borate buffer, add 500 |xL 2-mercaptoethanol.)
CHROMATOGRAM
Internal standard: 4-methylmexiletine (14)
Limit of detection: 2 ng/mL (plasma)
OTHER SUBSTANCES
KEYWORDS
plasma; post-column reaction; pharmacokinetics
REFERENCE
Tateishi,T.; Harada,K; Ebihara,A. Fluorescence detection of mexiletine and its p-hydroxylated and hydroxy-
methylated metabolites in human plasma and urine by high-performance liquid chromatography using post-
column derivatization with o-phthalaldehyde, J.Liq.Chromatogr., 1994, 17, 659-671.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Reflux a 10% excess of reagent in toluene for 10 min, add the drug, let
stand at room temperature for 10 min, cool, dilute, inject an aliquot. (The reagent was N-(p-
toluenesulfonyDprolyl azide and was prepared as follows. Mix 40-45 mmoles L-(-)-proline, 40
mL THF, and 200 mL 10% potassium carbonate, add 37-43 mmoles p-toluenesulfonyl chloride
in 40 mL THF dropwise, heat at 50 and maintain at pH 8 or above for 3 h, cool, acidify to pH
2, extract with chloroform. Extract the organic layers with potassium carbonate in water. Acid-
ify the aqueous layer and extract it with chloroform. Dry the chloroform layer and evaporate
it to dryness, recrystallize the resulting l-[(p-toluene)sulfonyl]proline from petroleum ether and
benzene (Caution! Benzene is a carcinogen!) (Anal.Chem. 1984, 56, 958). Suspend 86 mmoles
l-[(p-toluene)sulfonyl]proline in 15 mL water and add sufficient acetone to give a clear solution,
cool to 0, add 10.2 g triethylamine in 175 mL acetone, slowly add 12.5 g ethyl chloroformate
in 45 mL acetone while maintaining the temperature at 0, stir at 0 for 30 min, add dropwise
8.6 g sodium azide in 30 mL water, stir at 0 for 1 h, pour into ice water, extract with ether,
dry over anhydrous magnesium sulfate, evaporate under reduced pressure at room tempera-
ture to give N-(p-toluenesulfonyl)prolyl azide (cf J.Org.Chem. 1961, 26, 3511).)
HPLC VARIABLES
Column: 300 X 4 7-9 |xm silica gel
Mobile phase: Petroleum ether.isopropanol 97:3
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
KEY WpRDS
REFERENCE
Zhou,Y.; Sun,Z.R; Lin,D.K. Liquid chromatographic evaluation of a new chiral derivatizing agent for enantio-
meric resolution of amine and alcohol drugs, J.Liq.Chromatogr., 1990, 13, 875-885.
SAMPLE
Matrix: microbial broth
Sample preparation: Dilute microbial broth 1:20. 250 (xL Diluted microbial broth + 5 |xg IS +
100 jxL 200 mM sodium carbonate, add 2 mL diethyl ether, vortex for 15 s, centrifuge at 1800
g for 4 min, remove 1.5 mL of the ether layer, repeat the extraction. Combine the organic layers
and evaporate them to dryness under a stream of nitrogen, reconstitute the residue in 300 |xL
chloroform, add 75 |xL 0.1% S-(+)-l-(l-naphthyl)ethyl isocyanate in chloroform, vortex for 10 s,
evaporate to dryness under a stream of nitrogen. Reconstitute the residue in 220 |xL chloro-
form, add 30 |xL 0.33% n-butylamine in chloroform, inject a 10-65 (xL aliquot.
HPLC VARIABLES
Column: 250 X 4 Partisil 5
Mobile phase: Hexane:chloroform:MeOH 65:34:1
Flow rate: 0.8 for 12 min then 2.5 for 23 min
CHROMATOGRAM
Internal standard: prenalterol (()-l-(4-hydroxyphenoxy)-3-isopropylaminopropan-2-ol) (27.7,
32.4 (enantiomers))
OTHER SUBSTANCES
KEYWORDS
chiral; derivatization; normal phase
REFERENCE
Freitag,D.G.; Foster,R.T.; Coutts,R.T.; Pasutto,F.M. High-performance liquid chromatographic method for re-
solving the enantiomers of mexiletine and two major metabolites isolated from microbial fermentation
media, J.Chromatogr., 1993, 616, 253-259.
SAMPLE
Matrix: solutions
Sample preparation: Mix a 50 jxL aliquot of a solution in MeOH:triethylamine 99:1 with 20 jxL
0.1% FLOPIC in dry toluene, vortex briefly, let stand at room temperature in the dark for 30
min, add 50 |xL 1% ethanolamine in MeOH, let stand at room temperature for 15 min, evap-
orate to dryness under reduced pressure, reconstitute with 100 |xL mobile phase, sonicate for
30 s, inject a 20 |xL aliquot. (FLOPIC is (-MS)-flunoxaprofen isocyanate; synthesis is as follows.
Dissolve 1 g (+)-(S)-flunoxaprofen in 30 mL acetone, cool to 0, add a solution of 500 |xL tri-
ethylamine in 2 mL acetone dropwise, add a solution of 370 jxL ethyl chloroformate in 2 mL
acetone dropwise, stir at 0 for 15 min, add a solution of 250 mg sodium azide in 1 mL water
dropwise (Caution! Sodium azide is highly toxic!), stir for 1 h, pour into 60 mL ice water, stir
for 10 min, filter, wash the solid with two 50 mL aliquots of ice-water, dry under reduced
pressure to obtain flunoxaprofen azide. Dissolve 100 mg flunoxaprofen azide in 3 mL dry tol-
uene, reflux for 10-15 min, cool to room temperature, filter. Evaporate the filtrate to dryness
under reduced pressure and dry under reduced pressure to obtain FLOPIC as a crystalline
solid (mp 93-94), store in a desiccator under reduced pressure.)
HPLC VARIABLES
Mobile phase: MeOH:water:THF 62:35:3
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: flecainide
KEY WpRDS
REFERENCE
Martin,E.; Quinke,K.; Spahn,H.; Mutschler,E. (-MS)-Flunoxaprofen and (-)-(S)-naproxen isocyanate: two new
fluorescent chiral derivatizing agents for an enantiospecific determination of primary and secondary amines,
Chirality, 1989, 1, 223-234.
SAMPLE
Matrix: solutions
Sample preparation: 50 |xL 5 mg/mL Mexiletine in 100 mM HCl + 50 |xL buffer + 100 |xL
reagent, swirl for 1 min, place on ice for 5 min, add 2 mL mobile phase, inject a 5 |JLL aliquot.
(Buffer was 100 mM sodium borate adjusted to pH 9.50 with 2 M NaOH. Reagent was 13.40
g o-phthaldialdehyde and 16.3 mg N-acetyl-L-cysteine in 1 mL MeOH, protect from light, keep
on ice.)
HPLC VARIABLES
Mobile phase: MeOH:MeCN:buffer 60:1.5:40 (Buffer was 3 mL/L glacial acetic acid in water, pH
adjusted to 7.20 with 2 M NaOH.)
Flow rate: 1
Injection volume: 5
CHROMATOGRAM
Retention time: 12.38 (S-(+)) (a = 1.11)
Limit of detection: 75 pg (F)
KEY WpRDS
derivatization; protect from light; chiral; a = 1.11
REFERENCE
Desai,D.M.; Gal,J. Enantiospecific drug analysis via the orô-phthalaldehyde/homochiral thiol derivatization
method, J.Chromatogr., 1993, 629, 215-228.
SAMPLE
Matrix: solutions
Sample preparation: 10 JXL 100 |xM Mexiletine hydrochloride in 200 mM pH 8.0 borate buffer
containing 4 mM disodium EDTA + 30 |xL 50 mM 4-fluoro-7-nitro-2,l,3-benzoxadiazole in
MeCN, mix, heat at 60 for 5 min, add 960 jxL MeOH:acetic acid 99:1, inject a 10 |JLL aliquot.
HPLC VARIABLES
Column: 150 X 60 (sic) 5 |jon Ultron ES-phCD phenylcarbamylated p-cyclodextrin (Sinwa Kakou)
Flow rate: 0.8
CHROMATOGRAM
Retention time: 60 (R-(-)), 65 (S-(+))
KEYWORDS
REFERENCE
Fukushima,T.; Kato,M.; Santa,T.; Imai,K. Enantiomeric separation and spectrofluorometric detection of the
racemic drugs, ()-l-(2,5-dimethylphenoxy)-2-propamine (mexiletine) and (3RS)-4-amino-3-hydroxybuta-
noic acid (GABOB), derivatized with 4-fluoro-7-nitro-2,l,3-benzoxadiazole on a phenylcarbamylated cyclo-
dextrin bonded stationary phase, Analyst, 1995, 120, 381-383.
SAMPLE
Matrix: solutions
Sample preparation: Mix 20 JULL of a 1 mM solution in MeOH or water with 50 |xL pH 8 borate
buffer and 50 JULL 18 mM 2-(6-methoxy-2-naphthyl)-l-propyl chloroformate in acetone, vortex,
let stand at room temperature for 30 min, add 100 |xL 10 mM trans-4-hydroxy-L-proline in
water, mix, let stand for 2 min, add 2 mL dichloromethane, vortex for 30 s. Remove the organic
layer and evaporate it to dryness under reduced pressure, reconstitute the residue in 100 |xL
mobile phase, inject an aliquot. Prepare 2-(6-methoxy-2-naphthyl)-l-propyl chloroformate as
follows. Stir 1.5 mmoles lithium aluminum hydride in THF, slowly add 2 mmoles (S)-naproxen
in 20 mL anhydrous THF, reflux for 1 h, evaporate most of the solvent, cautiously add water
with stirring, acidify with 6 N HCl, extract three times with diethyl ether. Combine the organic
layers and dry them over anhydrous sodium sulfate, evaporate to dryness, chromatograph on
silica gel with dichloromethane:MeOH 100:2 (flash chromatography), evaporate eluate to dry-
ness, dry under vacuum over KOH to give 2-(6-methoxy-2-naphthyl)propanol as a white solid
(mp 92-3). Stir 0.5 mmoles 2-(6-methoxy-2-naphthyl)propanol and 0.5 mmoles triethylamine
in 10 mL dry toluene at 0, add 1 mL 20% phosgene in toluene (Caution! Phosgene is highly
toxic, perform reaction in a chemical fume hood!) (Fluka), stir for 4 h, filter, evaporate to
dryness under reduced pressure, dry under vacuum to give 2-(6-methoxy-2-naphthyl)-l-propyl
chloroformate (mp 60). Store under vacuum over phosphorus pentoxide at room temperature.)
HPLC VARIABLES
Column: 250 X 4 5 |jim Zorbax-SIL
Mobile phase: n-Hexane:isopropanol 100:0.25
Flow rate: 1.5
CHROMATOGRAM
Retention time: k' 22.5 (S-(+)), k' 23.3 (R-(-))
KEY WORDS
REFERENCE
Biischges,R.; Linde,H-; Mutschler,E.; Spahn-Langguth,H- Chloroformates and isothiocyanates derived from 2-
J.ChromatogrA, 1996, 725, 323-334.
Mezlocillin
Merck Index: 6259
Led nicer No.: 3 206
SAMPLE
Sample preparation: Serum. 0.5 mL serum + 0.5 mL MeCN mix in 7 mL tube on vortex mixer;
shake by rotation (20 rpm) 10 min; centrifuge 10 min 1000 g; transfer supernatant to another
tube, add 7 aliquots dichloromethane; equilibrate 10 min; shake by rotation (20 rpm) 10 min;
centrifuge 10 min 1000 g; inject aliquot of upper aqueous layer. Urine. Centrifuge urine and
dilute 1:20. Bile. Centrifuge bile and dilute 1:10
HPLC VARIABLES
Mobile phase: 24:76 MeCN:20 mM ammonium acetate adjusted to pH 5 with glacial acetic acid
Flow rate: 1
Detector: UV 214
CHROMATOGRAM
Retention time: 5.2
OTHER SUBSTANCES
Also analyzed: ampicillin, azlocillin, aztreonam, cefmenoxime, cefoperazone, cefsulodin, cefotax-
ime, ceftazidime, ceftriaxone, cloxacillin, desacetylcefotaxime, penicillin G, piperacillin,
ticarcillin
KEYWORDS
serum
REFERENCE
Jehl,F.; Birckel,R; Monteil,H. Hospital routine analysis of penicillins, third-generation cephalosporins and az-
treonam by conventional and high-speed high-performance liquid chromatography, J.Chromatogr., 1987,
413, 109-119.
SAMPLE
Matrix: blood
Sample preparation: 500 jxL Serum + methicillin + 2 mL MeCN, vortex for 1 min, centrifuge
at 3000 g for 10 min. Remove the supernatant and add it to 5 mL dichloromethane, vortex,
centrifuge at 3000 g for 10 min, inject a 15 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Column: 300 mm long u.Bondapak C18
Mobile phase: MeCNiIOO mM pH 6.1 sodium phosphate buffer 25:75
Flow rate: 2.5
Detector: UV 229
CHROMATOGRAM
Internal standard: methicillin
OTHER SUBSTANCES
Extracted: piperacillin
KEY WORDS
REFERENCE
Martens,M.G.; Faro,S.; Feldman,S.; Cotton,D.B.; Dorman,K.; Riddle,G.D. Pharmacokinetics of the acyclurei-
dopenicillins piperacillin and mezlocillin in the postpartum patient, Antimicrob.Agents Chemother., 1987,
31, 2015-2017.
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Serum + 200 jxL 400 mM HCl + 3.5 mL dichloromethane, extract.
Extract the organic phase with 200 (xL 20 mM pH 6.2 phosphate buffer, inject a 30-60 |xL
aliquot.
HPLC VARIABLES
Column: C18
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: mezlocillin
OTHER SUBSTANCES
Extracted: piperacillin
KEYWORDS
serum; mezlocillin is IS
REFERENCE
Klepser,M.E.; Patel,K.B.; Nicolau,D.R; Quintiliani,R.; Nightingale,C.H. Comparison of the bactericidal activi-
ties of ofloxacin and ciprofloxacin alone and in combination with ceftazidime and piperacillin against clinical
strains of Pseudomonas aeruginosa, Antimicrob.Agents Chemother., 1995, 39, 2503-2510.
SAMPLE
Sample preparation: Serum, plasma. Dilute serum or plasma 1:2 to 1:10 with buffer, centrifuge,
inject a 20 jxL aliquot of supernatant. Urine. Dilute urine 1:10 to 1:100 with buffer, centrifuge,
inject a 20 |JLL aliquot of supernatant. Tissue (lung, gut). Cut tissue with a scalpel, homogenize
with 1-3 mL buffer, centrifuge at 9600 g for 5 min three times, inject a 20 JJLL aliquot. Tissue
(chondral). Cut tissue with a scalpel, homogenize with 3-6 mL buffer in an ice bath for 2-3
min, centrifuge at 9600 g for 5 min four or five times, inject a 100 JULL aliquot. Dilute human
pleural samples with buffer, centrifuge, inject a 20 |xL aliquot. (Buffer was 66.6 mM K2HPO4
adjusted to pH 7.40 with KH2PO4.)
HPLCVARIABLES
Column: 200 X 4 5 |jim Nucleosil C18
Mobile phase: MeCN:buffer 24:76, adjusted to pH 4.0 with phosphoric acid (Buffer was 57.4 mM
K2HPO4 adjusted to pH 4.0 with phosphoric acid.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 16
KEYWORDS
serum; plasma; lung; gut; pleural; chondral
REFERENCE
Knoller,J.; K6nig,W.; Schonfeld,W.; Bremm,K.D.; K6ller,M. Application of high-performance liquid chromatog-
raphy of some antibiotics in clinical microbiology, J.Chromatogr., 1988, 427, 257-267.
SAMPLE
Sample preparation: Condition a Bondelut SPE cartridge (cat. no. 607101) with two 1 mL por-
tions of MeCN and 1 mL buffer. Dilute urine 1:10 with buffer. 100 (JLL Serum or diluted urine
+ 100 JJLL 190 |xg/mL nafcillin in buffer, vortex for 10 s, add to the SPE cartridge, wash with 1
mL buffer, wash with 500 JJLL MeCN:buffer 15:85, elute with 500 JJLL MeCN.buffer 70:30, inject
a 10 |xL portion of the eluate. (Buffer was 70 mM KH2PO4 adjusted to pH 5.0 with 5 M NaOH.)
HPLCVARIABLES
Guard column: 45 X 4 10 |xm Spherisorb C-18
Column: 300 X 4 jxBondapak C-18
Flow rate: 1.1
Detector: UV 220
CHROMATOGRAM
Retention time: 4.2
Internal standard: nafcillin (7.5)
Limit of detection: 1.6 jxg/mL
OTHER SUBSTANCES
Noninterfering: acetaminophen, dexamethasone, diazepam, furosemide, morphine
KEYWORDS
serum; SPE
REFERENCE
Fiore,D.; Auger,F.A.; Drusano,G.L.; Dandu,V.R.; Lesko,L.J. Improved micromethod for mezlocillin quantitation
in serum and urine by high-pressure liquid chromatography, Antimicrob.Agents Chemother., 1984,26, lib-
Ill.
SAMPLE
HPLC VARIABLES
Flow rate: 2.5
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
liquids and during simulated Y-site injection with selected drugs, Am.J.Health-Syst.Pharm., 1996,53,294-
304.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10-20 jxL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 231.1
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: azlocillin
KEYWORDS
mezlocillin is IS
REFERENCE
Barriere,S.L.; Catlin,D.H.; Orlando,P.L.; Noe,A.; Frost,R.W. Alteration in the pharmacokinetic disposition of
ciprofloxacin by simultaneous administration of azlocillin, Antimicrob.Agents Chemother., 1990, 34, 823-
826.
Next Page
SAMPLE
Matrix: solutions
Sample preparation: Inject a 40 JJLL aliquot of an aqueous solution.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm LiChrospher C18
Mobile phase: MeCNiKH2PO4 20:80, pH 5.5
Flow rate: 1.7
Detector: UV 220
CHROMATOGRAM
Retention time: 7.7
OTHER SUBSTANCES
Simultaneous: piperacillin
KEYWORDS
mezlocillin is IS
REFERENCE
Kinzig,M.; S6rgel,R; Brismar,B.; Nord,C.E. Pharmacokinetics and tissue penetration of tazobactam and piper-
acillin in patients undergoing colorectal surgery, Antimicrob.Agents Chemother., 1992, 36, 1997-2004.
Mianserin
Merck Index: 6260
Lednicer No.: 2 451
SAMPLE
Matrix: Blood, tissue, urine
Sample preparation: Serum, urine. 500 JJLL Serum or urine + 100 |xL 2 |xg/mL diazepam + 200
gentle stream of nitrogen at about 40. Dissolve the residue in 100 |xL mobile phase, inject a
10 |JLL aliquot. Tissue. Homogenize 1 g sample with 9 mL 100 mM HCl and 100 |xL 20 |xg/mL
diazepam, centrifuge at 15000 g for 10 min. Add 500 |JLL 20% sodium carbonate and 4 mL n-
hexaneiisoamyl alcohol 98.5:1.5 to 1 mL of the supernatant, mix for 5 min. Remove the organic
100 uL mobile phase, filter by microconcentrator (Microcon-30, Grace). Inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 2 |xm TSK gel Super-Octyl (A) or 100 X 4.6 5 |xm Hypersil MOS-C8 (B),
(Yokogawa, Japan)
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
Previous Page
SAMPLE
Matrix: solutions
Sample preparation: Inject a 40 JJLL aliquot of an aqueous solution.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm LiChrospher C18
Mobile phase: MeCNiKH2PO4 20:80, pH 5.5
Flow rate: 1.7
Detector: UV 220
CHROMATOGRAM
Retention time: 7.7
OTHER SUBSTANCES
Simultaneous: piperacillin
KEYWORDS
mezlocillin is IS
REFERENCE
Kinzig,M.; S6rgel,R; Brismar,B.; Nord,C.E. Pharmacokinetics and tissue penetration of tazobactam and piper-
acillin in patients undergoing colorectal surgery, Antimicrob.Agents Chemother., 1992, 36, 1997-2004.
Mianserin
Merck Index: 6260
Lednicer No.: 2 451
SAMPLE
Matrix: Blood, tissue, urine
Sample preparation: Serum, urine. 500 JJLL Serum or urine + 100 |xL 2 |xg/mL diazepam + 200
10 |JLL aliquot. Tissue. Homogenize 1 g sample with 9 mL 100 mM HCl and 100 |xL 20 |xg/mL
diazepam, centrifuge at 15000 g for 10 min. Add 500 |JLL 20% sodium carbonate and 4 mL n-
hexaneiisoamyl alcohol 98.5:1.5 to 1 mL of the supernatant, mix for 5 min. Remove the organic
100 uL mobile phase, filter by microconcentrator (Microcon-30, Grace). Inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 2 |xm TSK gel Super-Octyl (A) or 100 X 4.6 5 |xm Hypersil MOS-C8 (B),
(Yokogawa, Japan)
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
Limit of quantitation: 50 ng/mL (serum, urine), 500 ng/mL (tissue)
OTHER SUBSTANCES
mine, maprotiline, melitracen, nortriptyline
KEYWORDS
serum; brain; liver
REFERENCE
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 279
CHROMATOGRAM
KEYWORDS
REFERENCE
Tracqui,A-; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic Sd., 1995, 40,
254-262.
SAMPLE
residue with 50 JJLL MeCN.water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr~A, 1997, 763,149-
163.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 2 mg mianserin in 1 mL EtOH, dilute with 4 mL mobile phase,
HPLC VARIABLES
Column: 250 X 4.6 Chiralcel OD or 250 X 4.6 Chiralpak AD
Mobile phase: EtOH.n-hexane 95:5 or n-hexane:2-propanol 90:10
Flow rate: 0.5 or 1
Detector: UV 250
KEYWORDS
chiral
REFERENCE
Selditz,U.; Liao,Y; Franke,J.R; de Zeeuw,R.A.; Wikstrom,H. Direct enantiomeric separation of mianserin and
6-azamianserin derivatives using chiral stationary phases, J.Chromatogr.A, 1998, 803, 169-177.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 fig/mL solution in MeOH, inject a 20 JULL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.4
OTHER SUBSTANCES
lol, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine,
nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, par-
gyline, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadox-
one, phenampromide, phenazocine, phenbutrazate, phendimetrazine, phenelzine, phenglutar-
imide, phenindamine, pheniramine, phenmetrazine, phenomorphan, phenoperidine,
pranolol, prothipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quin-
REFERENCE
Mibefradil
Merck Index: 6261
SAMPLE
M a t r i x : blood
Sample preparation: Add 100-30 ng/mL IS, extract either using dichloromethane or using a
Bond-Elut C2 SPE cartridge.
HPLC VARIABLES
Column: 3(xm C6 (Gromsil or Spherisorb)
Mobile phase: MeCN:9.5 mM pH 3.9 phosphate buffer 55:45 to 70:30
Flow rate: 1
CHROMATOGRAM
Internal standard: Ro 40-6792
OTHER SUBSTANCES
KEYWORDS
rat; marmoset; cynomolgus monkey; human; plasma; pharmacokinetics; SPE
REFERENCE
Wiltshire,H.R.; Sutton,B.M.; Heeps,G.; Betty,A.M.; Angus,D.W.; Harris,S.R.; Worth,E.; Welker,H.A. Metabolism
of the calcium antagonist, mibefradil (POSICOR, Ro 40-5967). Part III. Comparative pharmacokinetics of
mibefradil and its major metabolites in rat, marmoset, cynomolgus monkey and man, Xenobiotica, 1997,
27, 557-571.
Mibolerone
Merck Index: 6262
Lednicer No.: 2 144
SAMPLE
Sample preparation: Oils. 1 mL Sample + 25 mL MeOHrwater 90:10, shake vigorously for 5
powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 |xm), discard first 5 mL of filtrate,
mL with MeOH, filter (0.45 jim), discard first 5 mL of filtrate, inject a 10 |xL aliquot of the
remaining filtrate.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: 7.3
Limit of detection: 5 jjig/mL
OTHER SUBSTANCES
Simultaneous: methyltestosterone, methandriol (UV 210), norethindrone acetate, calusterone,
mesterolone (UV 210), norethandrolone, trenbolone acetate, benzyl benzoate, nandrolone ace-
tate, testosterone acetate, stanozolol, oxymetholone, nandrolone propionate, methenolone ac-
etate, testosterone propionate, aspirin, caffeine, formebolone, benzyl alcohol, testolactone, cor-
tisone, fluoxymesterone, norethindrone, oxandrolone (UV 210), boldenone, ethisterone,
methandrostenolone, nandrolone, norgestrel, testosterone
Interfering: dehydroepiandrosterone (UV 210)
KEYWORDS
REFERENCE
Walters,M.J.; Ayers,RJ-; Brown,D.J. Analysis of illegally distributed anabolic steroid products by liquid chro-
JAssoc.OffAnaLChem., 1990, 73, 904-926.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
yltestosterone, methyprylon, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoline,
REFERENCE
18, 233-242.
Miconazole
Merck Index: 6266
Lednicer No.: 2 249
SAMPLE
Matrix: blood
Sample preparation: 400 |xL Plasma + 400 |xL water + 50 |xL MeOH + 100 |xL 1 M KOH + 6
mL hexane:dichloromethane 50:50, shake for 3 min, centrifuge at 4000 rpm for 6 min. Remove
the upper organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute
the residue in 100 JJLL MeOH, inject a 25 |xL aliquot.
HPLC VARIABLES
Guard column: 80 X 4 CoPeIl ODS
Mobile phase: MeCN: 10 mM pH 8.0 NaH2PO4 buffer 66:34
Flow rate: 2
Detector: UV 229
CHROMATOGRAM
Retention time: 10
Internal standard: miconazole
OTHER SUBSTANCES
Extracted: sulconazole
KEYWORDS
plasma; dog; miconazole is IS
REFERENCE
Fass,M.; Zaro,B.; Chaplin,M.; Matin,S. Reversed-phase high-pressure liquid chromatographic analysis of sul-
conazole in plasma, J.Pharm.Sci., 1981, 70, 1338-1340.
SAMPLE
Sample p r e p a r a t i o n : Add 1 mL whole blood or urine to Toxi-Tube A (Toxi-Lab, Irvine CA), add
HPLC VARIABLES
G u a r d column: 20 mm long Symmetry C18
Mobile p h a s e : Gradient. A was 50 mM pH 3.8 sodium phosphate buffer. B was MeCN. A:B 85:
Flow r a t e : 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5
mL/min)
Detector: UV 202.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample p r e p a r a t i o n : Powders. Extract a sample equivalent to about 10 mg miconazole twice
with 20 mL portions of MeOH with magnetic stirring, filter extracts, combine, dilute to 50 mL
with MeOH. Remove a 2.5 mL aliquot and add it to 2 mL 200 |xg/mL econazole in MeOH,
dilute to 10 mL with MeOH, inject a 10 |xL aliquot. Creams. Condition a Baker diol SPE
cartridge with 6 mL dichloromethane. Add a sample equivalent to 10 mg miconazole to 30 mL
dichloromethane, sonicate for 2 min, make up to 50 mL with dichloromethane, filter, add 2 mL
of the filtrate to the SPE cartridge. Wash with two 3 mL portions of n-hexane:dichloromethane
4:1, aspirate to dryness, elute with three 1 mL portions of MeOH: 100 mM triethylamine ad-
justed to pH 7.0 with acetic acid 4:1. Combine the eluates and dilute them to 5 mL with MeOH.
Remove a 2.5 mL aliquot and add it to 1 mL 200 |jLg/mL econazole in MeOH, dilute to 5 mL
with MeOH, inject a 10 |xL aliquot.
HPLCVARIABLES
Column: 150 X 3.9 5 |xm Nova-Pak RP-18
Mobile p h a s e : MeOH:THF:100 mM triethylamine adjusted to pH 7.0 with acetic acid 70:12:18
Flow r a t e : 0.8
Detector: UV 230
CHROMATOGRAM
Retention time: 3.5
Internal standard: econazole (2.5)
KEYWORDS
creams; powders; SPE
REFERENCE
Cavrini,V.; Di Pietra,A.M.; Gatti,R- Analysis of miconazole and econazole in pharmaceutical formulations by
derivative UV spectroscopy and liquid chromatography (HPLC), J.Pharm.Biomed.AnaL, 1989, 7, 1535-
1543.
SAMPLE
Sample preparation: Dilute peritoneal dialysis fluid with an equal volume of 4 |xg/mL p-di-
chlorobenzene in MeOH5 inject a 20 (xL aliquot.
HPLCVARIABLES
Guard column: 37-50 |xm Bondapak C18/Corasil
Flow rate: 1
Detector: UV 229
CHROMATOGRAM
Retention time: 8.5
Internal standard: p-dichlorobenzene (4.5)
KEYWORDS
stability-indicating; peritoneal dialysis fluid
REFERENCE
Holmes,S.E.; Aldous,S. Stability of miconazole in peritoneal dialysis fluid, Am.J.Hosp.Pharm., 1991, 48, 286-
290.
SAMPLE
add 100 mL MeOH, sonicate for 5 min, filter. Add a 2 mL aliquot of filtrate to 5 mL of 100 |xg/
mL ketoconazole in MeOH, make up to 25 mL with MeOH, inject 20 JJLL aliquot. Cream. Con-
mL with dichloromethane, filter. Add a 2 mL aliquot to the cartridge, wash with 2 mL di-
chloromethane rmethanol 4:1, wash with 2 mL dichloromethane, elute with 3 mL MeOH:buffer
inject 20 jxL aliquot. (Buffer was 50 mM triethylamine adjusted to pH 7.0 with phosphoric
acid.)
HPLC VARIABLES
Mobile phase: THF:buffer 30:70 (Buffer was 50 mM triethylamine adjusted to pH 3.0 with phos-
phoric acid.)
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
Simultaneous: clotrimazole, ketoconazole, bifonazole, tioconazole, isoconazole, econazole,
fenticonazole
KEYWORDS
tablets; creams
REFERENCE
Di Pietra,A.M.; Cavrini,V.; Andrisano,V.; Gatti,R. HPLC analysis of imidazole antimycotic drugs in pharma-
ceutical formulations, J.Pharm.Biomed.Anal., 1992, 10, 873-879.
SAMPLE
Sample preparation: Dilute with mobile phase, add metronidazole, inject a 10 |xL aliquot.
HPLCVARIABLES
Mobile phase: MeCN:buffer 15:85 (Buffer was 10 mM NaH2PO4 adjusted to pH 8 with
trimethylamine.)
Flow rate: 1.5
Detector: UV 250
CHROMATOGRAM
Internal standard: metronidazole (2)
KEYWORDS
injections; stability-indicating; 5% dextrose; saline
REFERENCE
Faouzi,M.E.A.; Dine,T.; Luyckx,M.; Brunet,C; Mallevais,M.-L.; Goudaliez,E; Gressier,B.; Cazin,M.; Kablan,J.;
Cazin,J.C. Stability, compatibility and plasticizer extraction of miconazole injection added to infusion so-
lutions and stored in PVC containers, J.Pharm.Biomed.Anal., 1995, 13, 1363-1372.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: RCSS Guard-Pak (Waters)
Column: 100 X 8 C18 Radial Pak (Waters)
Mobile phase: MeOH:0.75% acetic acid 30:70, pH adjusted to 5.5 with triethylamine
Flow rate: 3
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: acetaminophen, N-acetylprocainamide, cefaclor, cefamandole, cefazolin, cefotax-
ime, cefoxitin, cephalexin, cephalothin, cephapirin, chloramphenicol, cimetidine, moxalactam,
procainamide, sulfamethoxazole, theophylline, tobramycin, vancomycin
REFERENCE
Danzer,L.A. Liquid-chromatographic determination of cephalosporins and chloramphenicol in serum,
Clin.Chem., 1983, 29, 856-858.
SAMPLE
Matrix: solutions
Sample preparation: Centrifuge at 10000 rpm, dilute the supernatant with mobile phase, inject
an aliquot.
HPLC VARIABLES
Mobile phase: MeCN:50 mM (NH4)H2PO4 85:15
Detector: UV 230
REFERENCE
Okimoto,K; Rajewski,R.A.; Uekama,K; Jona,J.A.; Stella,V.J. The interaction of charged and uncharged drugs
with neutral (HP-p-CD) and anionically charged (SBE7-p-CD) p-cyclodextrins, Pharm.Res., 1996,13, 256-
264.
SAMPLE
Matrix: tissue
Sample preparation: Skin sample extracted with 500 |xL mobile phase, vortex 1 min, centrifuge
at 8000 rpm for 10 min, inject 40 fxL aliquot.
HPLC VARIABLES
Column: 125 X 4.5 Whatman 5 |xm reverse-phase C18
Flow rate: 0.7
Detector: UV 214
CHROMATOGRAM
Retention time: 5.5
KEYWORDS
skin
REFERENCE
Pershing,L.K; Corlett,J.; Jorgensen,C. In vivo pharmacokinetics and pharmacodynamics of topical ketoconazole
and miconazole in human stratum corneum, Antimicrob.Agents Chemother., 1994, 38, 90-95.
Midazolam
Molecular formula: C18H13CIFN3
CAS Registry No.: 59467-70-8, 59467-96-8 (HCI), 59467-94-6 (maleate)
Merck Index: 6270
Lednicer No.: 3 197
SAMPLE
Matrix: blood
Sample preparation: 100 fxL Plasma + 100 jxL MeOH + 500 |xL 1 M NaOH + 3 mL hexane,
shake for 5 min, centrifuge at 3000 rpm for 5 min. Evaporate 2 mL of the organic phase to
dryness under a stream of nitrogen. Dissolve residue in 200 |xL mobile phase, inject a 75 |xL
aliquot.
HPLC VARIABLES
Guard column: 30 X 4.6 5 |xm YMC-Guardpak ODS-AM (GL Sciences)
Column: 250 X 4.6 5 ^m Inertsil ODS (GL Sciences)
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Limit of detection: 50 ng/mL (sic)
KEYWORDS
REFERENCE
Takedomi,S.; Matsuo,H.; Yamano,K.; Yamamoto,K.; Iga,T.; Sawada,Y. Quantitative prediction of the interaction
of midazolam and histamine H2 receptor antagonists in rats, Drug Metab.Dispos., 1998, 26, 318-323.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 100 jxL MeOH + 500 |iL 1 M NaOH + 3 mL hexane,
shake for 5 min, centrifuge at 3000 rpm for 5 min. Evaporate 2 mL of the organic phase to
dryness under a stream of nitrogen. Dissolve residue in 200 |xL mobile phase, inject a 75 |xL
aliquot.
HPLC VARIABLES
Guard column: 30 X 4.6 5 |xm YMC-Guardpack ODS-AM (GL Sciences, Japan)
Column: 250 X 4.6 5 \xm Inertsil ODS
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
KEYWORDS
REFERENCE
Takedomi,S.; Matsuo,H.; Yamano,K; Yamamoto,K.; Iga,T.; Sawada,Y. Quantitative prediction of the interaction
of midazolam and histamine H2 receptor antagonists in rats, Drug Metab.Dispos., 1998, 26, 318323.
SAMPLE
Matrix: blood
Sample preparation: 100 jxL Plasma + 100 |xL 1 M NaOH + 2 mL 10% (v/v) isopropanol in
dichloromethane containing 25 ng/mL IS, vortex for 60 s, centrifuge at 2000 g for 45 s. Evap-
orate organic phase under a gentle stream of air at 60 for 10 min. Dissolve residue in 1 mL
hexane.MTBE 2:1, add 200 |xL 20 mM orthophosphoric acid, vortex for 30 s, centrifuge at 3200
g for 45 s. Carefully aspirate upper organic layer, add 7 |xL 1 M NaOH to acidic aqueous phase
to adjust pH to 6.6-6.9, inject an 80 jxL aliquot.
HPLCVARIABLES
Column: 150 X 3.9 5 |xm C8 Symmetry (Waters)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 7.4
Internal standard: climazolam (8.4)
OTHER SUBSTANCES
Noninterfering: amoxicillin, caffeine, carbamazepine, cisapride, dexamethasone, dopamine, fu-
rosemide, gentamicin, indomethacin, morphine, phenobarbital, ranitidine, theophylline
KEYWORDS
REFERENCE
Lee,T.C; Charles,B- Measurement by HPLC of midazolam and its major metabolite, 1-hydroxymidazolam in
plasma of very premature neonates, Biomed.Chromatogr., 1996, 10, 65-68.
SAMPLE
Matrix: blood
Sample preparation: Add 25 |xL 40 jxg/mL diazepam in MeOH, 40 JJLL 2% NaOH, and 3.5 mL
cyclohexane:diethyl ether 31:69 to 1 mL plasma. Extract on a rotary mixer at 4 for 10 min,
centrifuge at 4 at 2000 g for 10 min. Remove a 3.3 mL aliquot of the organic layer and evap-
orate it to dryness under a stream of nitrogen at 40. Dissolve the residue in 300 |xL MeCN:
water 5:95, inject a 20 (xL aliquot.
HPLCVARIABLES
Guard column: 5 X 0.8 |x-Precolumn cartridge C18 (LC Packings)
Column: 150 X 0.8 3 |xm Hypersil C18 BDS
Mobile phase: Gradient. MeCN: 10 mM pH 7.0 sodium phosphate buffer 35:65 for 16 min, to 60:
40 over 1 min, maintain at 60:40.
Flow rate: 0.016
Detector: UV 240 for 17.6 min then UV 300
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: metabolites, l'-hydroxymidazolam
KEYWORDS
pharmacokinetics; capillary HPLC; plasma
REFERENCE
Eeckhoudt,S.L.; Desager,J.-R; Horsmans,Y.; De Winne,A.J.; Verbeeck,R.K. Sensitive assay for midazolam and
its metabolite l'-hydroxymidazolam in human plasma by capillary high-performance liquid chromatography,
J.Chromatogr.B, 1998, 710, 165-171.
SAMPLE
Matrix: blood
Sample preparation: Add 100 |xL 1 M pH 9.0 borate buffer to 50 jxL serum, mix well, add 1
mL chloroform:diethyl ether 95:5 (Caution! Chloroform is a carcinogen!), vortex for 1 min,
centrifuge at 1100 for 5 min, evaporate the organic layer to dryness under nitrogen at 40,
resuspend the residue in 50 |xL mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 150 X 2.1 C18 symmetry column (Waters)
Mobile phase: MeCNrMeOH: 14.9 mM pH 3 sodium acetate 23:10:67
Detector: UV 230
CHROMATOGRAM
Retention time: 4.7
OTHER SUBSTANCES
KEYWORDS
pharmacokinetics; rat; serum
REFERENCE
Ma,R; Lau,C.E. Determination of midazolam and its metabolites in serum microsamples by high-performance
liquid chromatography and its application to pharmacokinetics in rats, J.Chromatogr.B, 1996, 682, 109-
113.
SAMPLE
residue with 50 jxL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Cool microsomal incubation on ice, add 100 JXL MeCN and IS, centrifuge,
inject an aliquot of the supernatant.
HPLCVARIABLES
Mobile phase: MeCN:MeOH:10 mM phosphate buffer 22.5:37.5:40
Flow rate: 0.8
Detector: UV 220
CHROMATOGRAM
Internal standard: phenacetin (5.4)
OTHER SUBSTANCES
KEYWORDS
liver
REFERENCE
von Moltke,L.L.; Greenblatt,D.J.; Schmider,J.; Duan,S.X.; Wright,C.E.; Harmatz,J.S.; Shader,R.I. Midazolam
hydroxylation by human liver microsomes in vitro: Inhibition by fluoxetine, norfluoxetine, and by azole
antifungal agents, J.Clin.Pharmacol., 1996, 36, 783-791.
SAMPLE
Sample preparation: Cool incubation mixture on ice, add 100 u,L MeCN and IS, centrifuge,
inject an aliquot.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:10 mM phosphate buffer 22.5:37.5:40
Flow rate: 0.8
Detector: UV 220
CHROMATOGRAM
Internal standard: phenacetin (5.4)
OTHER SUBSTANCES
KEYWORDS
liver
REFERENCE
von Moltke,L.L.; Greenblatt,D.J.; Schmider,J.; Duan,S.X.; Wright,C.E.; Harmatz,J.S.; Shader,R.I. Midazolam
hydroxylation by human liver microsomes in vitro: Inhibition by fluoxetine, norfluoxetine, and by azole
antifungal agents, J.Clin.Pharmacol, 1996, 36, 783-791.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 2 mg/mL solution in 0.9% sodium chloride, dilute 1:1000 with
HPLC VARIABLES
Column: Bakerbond phenyl column
Flow rate: 2
Detector: UV 240
CHROMATOGRAM
Retention time: 9.2
OTHER SUBSTANCES
KEYWORDS
REFERENCE
SAMPLE
Matrix: tissue
Sample preparation: Homogenize brain in 100 mM NaOH (5 mL/g) by sonication. Add 60 |xL
1 |xg/mL IS in MeOH to a tube, dry in a water bath under a stream of nitrogen at 50. Add
250 |xL homogenate (ca. 50 mg tissue equivalent) and 250 |xL 100 mM pH 13 NaOH, vortex
thoroughly. Add 2 mL toluene, mix (50 inversions), centrifuge at 15 000 rpm at 4 for 20 min.
Dry the organic phase under a stream of nitrogen. Add 2 mL toluene to the aqueous phase and
repeat the extraction. Combine the organic layers and dry under a stream of nitrogen. Recon-
stitute the residue in 100 |AL mobile phase, inject a 40 (xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 3 |xm Rainin C8 Microsorb
Mobile phase: MeCN:MeOH:25 mM potassium phosphate buffer 18.5:16.5:65
Flow rate: 0.9
Detector: UV 240
CHROMATOGRAM
Retention time: 4.2
Internal standard: halazepam (6.2)
KEYWORDS
brain; rat
REFERENCE
Jiang,Q.; Walton,N.Y; Gunawan,S.; Treiman,D.M. High-performance liquid chromatographic determination of
midazolam in rat brain, J.Chromatogr.B, 1996, 683, 276-280.
Mifepristone
Merck Index: 6273
Lednicer No.: 5 53
SAMPLE
Matrix: blood
Sample preparation: Add 200-400 |xL plasma or serum to a Pasteur pipette packed with 3 mL
60-80 mesh Chromosorb W-NAW:ethylene glycol 80:20 (w/w), let stand for 30 min, elute with
5 mL n-hexane:ethyl acetate 95:5. Evaporate the eluate and reconstitute the residue in mobile
HPLCVARIABLES
Column: Lichrosorb RP-18
Mobile phase: MeOH:water:triethanolamine 90:10:0.05
Flow rate: 1.5
Detector: UV 304
CHROMATOGRAM
KEYWORDS
plasma; serum; SPE; pharmacokinetics
REFERENCE
Heikinheimo,O.; Tevilin,M.; Shoupe,D.; Croxatto,H.; Lahteenmaki,P. Quantitation of RU 486 in human plasma
by HPLC and RIA after column chromatography, Contraception, 1986, 34, 613-624.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Serum + 50 |JLL 12.6 |xg/mL IS in MeOH + 650 |xL water, mix,
inject a 400 |xL aliquot into column A and elute to waste with mobile phase A for 7 min, elute
the contents of column A onto column B with mobile phase B, after 10 min remove column A
from the circuit, elute column B with mobile phase B, monitor the effluent from column B.
Wash column A with MeOH:water 70:30 for 5 min then re-equilibrate with water.
HPLC VARIABLES
Column: A 30 X 4 Serumout (Sekisui); B 300 X 3.9 ixBondapak C18
Mobile phase: A water; B MeCN:MeOH:50 mM pH 3.1 phosphate buffer 27:18:60
Detector: UV 305 or E, Sekisui ECD-120, +1.0 V, Ag/AgCl reference electrode
CHROMATOGRAM
Internal standard: 13-ethyl-17-hydroxy-18,19-dinorpregna-4,9,ll-trien-20-yn-3-one (R2323)
(35.3)
Limit of detection: 0.9 ng (E), 6.6 ng (UV)
OTHER SUBSTANCES
KEYWORDS
serum; pharmacokinetics; column-switching
REFERENCE
Nagoshi,K; Hayashi,N.; Sekiba,K. Automated direct assay system for RU38486, an antiprogesterone-antiglu-
cocorticoid agent, and its metabolites using high performance liquid chromatography, Ada Med.Okayama.,
1991, 45, 81-87.
Miloxacin
Molecular formula: Ci2H9NO6
Merck Index: 6283
SAMPLE
Sample preparation: Blood. Filter serum through a 0.45 |xm syringe filter with a cellulose
acetate membrane, inject a 50 |xL aliquot of the filtrate. Tissue. Add 1 mL MeCNiTHF 95:5 to
1 g muscle, homogenize with a Pencil Mixer (Iuchi, Japan) for 2 min, centrifuge at 1500 g for
5 min, filter the supernatant through a syringe filter unit, inject a 20 jxL aliquot of the filtrate.
HPLC VARIABLES
Guard column: 20 X 4.6 5 |xL Hisep shielded hydrophobic phase precolumn (Supelco)
Column: 150 X 4.6 5 jxL Hisep shielded hydrophobic phase (Supelco)
Mobile phase: MeCN:buffer 15:85 (Buffer was 50 mM citric acid:200 mM pH 2.5 Na2HPO4 buffer
containing 10 mM tetra-n-butyl ammonium bromide 85:15.)
Flow rate: 1
Detector: UV 265
CHROMATOGRAM
Limit of detection: 50 ng/mL (serum), 100 ng/mL (muscle)
OTHER SUBSTANCES
Extracted: sulfamonomethoxine, oxolinic acid
KEYWORDS
fish; muscle; serum
REFERENCE
Ueno,R.; Aoki,T. High-performance liquid chromatographic method for the rapid and simultaneous determi-
nation of sulfamonomethoxine, miloxacin and oxolinic acid in serum and muscle of cultured fish,
J.Chromatogr.B, 1996, 682, 179-181.
Milrinone
Molecular formula: Ci2H9N3O
Merck Index: 6284
SAMPLE
Sample preparation: Homogenize (Ystral D7801) tissue with 2 volumes of 10 mM sodium bi-
carbonate containing 2.5% acetic acid (final pH 3), centrifuge at 20000 g for 30 min. 100 JJLL
Plasma or tissue homogenate + 375 |xL water, mix, add 25 (xL 50% acetic acid, vortex, centrifuge
at 7000 g for 5 min. Filter (Amicon Centricon-30 microconcentrator) while centrifuging in a
refrigerated centrifuge at 5500 g for 35 min, inject an aliquot of the filtrate.
HPLC VARIABLES
Guard column: 10 X 3 RP-2 (Chrompack)
Column: 250 X 4 RP Select B RT (Merck)
Mobile phase: MeOH:25 mM pH 3.75 ammonium acetate containing 0.01% tetramethylammo-
nium hydroxide
Flow rate: 0.75
Detector: UV 331
CHROMATOGRAM
Retention time: 5
KEYWORDS
rat; liver; heart; brain; lung; plasma
REFERENCE
Verrijk,R.; van Rooij,H.H.; Werner,J.; Porsius,A.J. High-performance liquid chromatographic determination of
milrinone in biological tissues and fluids, J.Chromatogr., 1989, 491, 265-268.
SAMPLE
Sample preparation: Urine. Acidify urine to pH 3.75 with 50% acetic acid, filter (Amicon Cen-
tricon-30 microconcentrator), inject an aliquot. Plasma. 100 |xL Plasma + 375 |JLL water, mix,
add 25 |xL 50% acetic acid, vortex, centrifuge at 7000 g. Filter (Amicon Centricon-30 microcon-
centrator) while centrifuging in a refrigerated centrifuge at 5500 g for 35 min, inject an aliquot
of the filtrate.
HPLC VARIABLES
Guard column: 10 X 3 RP-2 (Chrompack)
Column: 250 X 4 RP Select B RT (Merck)
Mobile phase: MeOH:25 mM pH 3.75 ammonium acetate containing 0.01% tetramethylammo-
nium hydroxide
Flow rate: 0.75
Detector: UV 331
CHROMATOGRAM
KEYWORDS
REFERENCE
Verrijk,R.; Vleeming,W.; van Rooij,H.H.; Werner,J.; Porsius,A.J. Plasma elimination of milrinone in rats in
relation to its hemodynamic effects, J.Pharm.Sci., 1990, 79, 236-239.
SAMPLE
Sample preparation: 1 mL Sample + 99 mL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Partisil ODS III
Mobile phase: MeOH:500 mM pH 7 borate buffer:water 40:2:58
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 2.5
OTHER SUBSTANCES
Noninterfering: digoxin
KEY WORDS
REFERENCE
Riley,C.M. Stability of milrinone and digoxin, fiirosemide, procainamide hydrochloride, propranolol hydrochlo-
45, 2079-2091.
SAMPLE
Sample preparation: 1 mL Sample + 9 mL mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 [un. Spherisorb phenyl
Mobile phase: MeCN:500 mM KH2PO4:water 22:10:68 adjusted to pH 7.1 with 10 M NaOH
Flow rate: 2
Detector: UV 268
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
Simultaneous: procainamide, degradation products
KEYWORDS
REFERENCE
Riley,C.M. Stability of milrinone and digoxin, furosemide, procainamide hydrochloride, propranolol hydrochlo-
45, 2079-2091.
SAMPLE
Sample preparation: Dilute with water, inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 10 ^m Whatman PXS ODS-3 C18
Mobile phase: MeCN:Buffer 25:75 (Buffer is 1.08 g sodium octanesulfonate in 900 mL of water
adjusted to pH 3.5 with glacial acetic acid and diluted to 1 L with water.)
Flow rate: 2
Detector: UV 229
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Simultaneous: atropine
KEYWORDS
REFERENCE
Miltefosine
Molecular formula: C21H46NO4P
Merck Index: 6285
SAMPLE
Matrix: blood
Sample preparation: Lyophilize 1 mL serum, extract residue 3 times with 1 mL portions of
MeOH. Combine the extracts, add 500 jxL of a saturated solution of potassium tert-butoxide
in MeOH, heat at 50 for 15 min, add 1 mL 3 M HCl, heat at 50 for 15 min, neutralize with
250 JXL 2 M NaOH, wash twice with 2 mL portions of n-hexane. Extract the MeOH/water phase
with chloroform. Evaporate the chloroform layer to dryness under a stream of nitrogen, recon-
stitute the residue in 500 \LL MeCN.MeOH.water 20:57:23, inject an aliquot.
HPLC VARIABLES
Guard column: 70 X 3.2 30-40 |xm SC-201 RP (Vydac)
Column: 250 X 4.6 5 |xm Nucleosil 120-5 C18
Mobile phase: MeCN:MeOH:water 20:57:23 containing 20 mM choline chloride
Flow rate: 2
CHROMATOGRAM
Retention time: 22
KEYWORDS
rat; serum; pharmacokinetics; tritium-labeled
REFERENCE
Unger,C; Fleer,E.; Damenz,W.; Hilgard,R; Nagel,G.; Eibl,H. Hexadecylphosphocholine: determination of serum
concentrations in rats, J.Lipid Mediat., 1991, 3, 71-78.
Minaprine
Merck Index: 6287
Lednicer No.: 4 120
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 204
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Minocycline
Merck Index: 6289
Lednicer No.: 1 214
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 400 |xL 500 mM pH 5 KH2PO4 and 5 mL ethyl
acetate, shake for 1 min, centrifuge at ^=2000 g for 2 min, mix 4 mL of the upper organic phase
with 500 |xL 20 mM HCl, shake for 1 min, centrifuge at 2*2000 g for 2 min, inject a 20 uL
aliquot of the lower aqueous phase.
HPLC VARIABLES
Column: 125 X 4.0 Nucleosil 5-CN
Mobile phase: MeOH:20 mM perchloric acid containing 4 mM triethylamine 20:80
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Retention time: 2.8
KEYWORDS
plasma
REFERENCE
Mascher,H.J. Determination of minocycline in human plasma by high-performance liquid chromatography with
UV detection after liquid-liquid extraction, J.ChromatognA, 1998, 812, 339-342.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 20 |xL trifluoroacetic acid, mix 30 s in a whirl mixer,
centrifuge at 5400 g for 5 min, inject supernatant (80 |xL).
HPLCVARIABLES
Guard column: 10 |xm Waters RP phenyl
Column: 150 X 3.9 10 urn Waters RP phenyl
Mobile phase: Gradient. A 0.1 M diammonium EDTA: 1 M diethanolamine (to pH 7.3 with 85%
phosphoric acid):isopropanol:water 10:50:185:755. B 0.1 M diammonium EDTA:1 M dietha-
nolamine (to pH 7.3 with 85% phosphoric acid)dsopropanol:water 10:50:400:540. From 100% A
to 100% B over 6 min
Flow rate: 2
Detector: UV 340
CHROMATOGRAM
Retention time: 4.1
KEYWORDS
plasma
REFERENCE
Kramer-Horaczynska,F. High-performance liquid chromatographic procedures for the quantitative analysis of
15 tetracycline derivatives in small blood samples, J.Chromatogr.Sci., 1991, 29, 107-113.
SAMPLE
Matrix: blood
Sample preparation: 200 JJLL Serum + 50 JJLL 20 |xg/mL oxytetracycline in water + 250 |xL 500
mM trichloroacetic acid, vortex for 30 s, centrifuge at 10000 g for 10 min, inject a 50 jiL aliquot
of the supernatant.
HPLC VARIABLES
Guard column: Nova-Pak phenyl guard
Column: 4 jim Nova-Pak phenyl radial compression
Mobile phase: MeCN:MeOH:100 mM oxalic acid 11:9:80 adjusted to pH 2.7 with 1 M HCl
Flow rate: 2
Detector: UV 350
CHROMATOGRAM
Retention time: 8-9
Internal standard: oxytetracycline (13.5-14)
KEYWORDS
REFERENCE
Birmingham,K.; Vaughan,L.M.; Strange,C. Rapid serum minocycline assay for pleurodesis monitoring using
high-performance liquid chromatography with radial compression, Ther.Drug MoniL, 1995, 17, 268-272.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y; PSpin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
the creation of a 600-compound library. Application to forensic toxicology, J.ChromatogrA, 1997, 763, 149-
163.
SAMPLE
Sample preparation: Bulk. Dissolve 25 mg minocycline hydrochloride in solvent, make up to
25 mL with solvent, inject an aliquot. Capsules, tablets. Add an amount equivalent to 25 mg
minocycline hydrochloride to 25 mL solvent, sonicate for 5 min, centrifuge at 2500 g for 5 min,
filter (1.5 |xm), inject an aliquot of the filtrate. (Solvent was 10 mM NaOH containing 0.1%
sodium sulfite.)
HPLCVARIABLES
Column: 250 X 4.6 8 |jun 100 A PLRP-S poly(styrene-divinylbenzene) (Polymer Labs)
Mobile phase: t-Butanol:200 mM pH 10.5 phosphate buffer:TBAS solution:EDTA solution:water
9:10:10:10:61 (TBAS solution was 20 mM tetrabutylammonium sulfate adjusted to pH 10.5
with NaOH solution. EDTA solution was 10 mM EDTA adjusted to pH 10.5 with NaOH
solution.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 20
OTHER SUBSTANCES
KEYWORDS
capsules; tablets
REFERENCE
Weng,N.; Thuranira,J.; Vermeulen,K; Roets,E.; Hoogmartens,J. Quantitative analysis of minocycline by liquid
chromatography on poly(styrene-divinylbenzene), J.Lig. Chromatogr., 1992, 15, 2529-2549.
SAMPLE
Sample preparation: Bulk. Prepare a 10-100 fjug/mL solution in buffer, inject an aliquot. Cap-
sules, tablets. Prepare a 1 mg/mL solution of capsule contents or crushed tablets in buffer,
sonicate for 10 min, filter (0.45 |xm), dilute with buffer, inject an aliquot. (Buffer was 20 mM
sodium perchlorate adjusted to pH 2.0 with perchloric acid.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm 100 A PLRP-S polystyrene-divinylbenzene (Polymer Laboratories)
Mobile phase: MeCNibuffer 20:80 (Buffer was 20 mM sodium perchlorate adjusted to pH 2.0
with perchloric acid.)
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
KEY WORDS
capsules; tablets
REFERENCE
Bryan,P.D.; Stewart,J.T. Chromatographic analysis of selected tetracyclines from dosage forms and bulk drug
substance using polymeric columns with acidic mobile phases, J.Pharm.Biomed.Anal., 1994, 12, 675-692.
SAMPLE
Matrix: cells
Sample preparation: 100 JJLL Cell suspension + 100 uX. cefoperazone solution + 100 |xL Hanks
balanced salt solution, sonicate 30 min, add 800 JJLL MeCN, centrifuge at 13000 g for 5 min,
remove supernatant. Dry supernatant under air, dissolve in 100 (xL mobile phase, inject 75
HL.
HPLC VARIABLES
Column: |j,Bondapak C18
Flow rate: 1
Detector: UV 353
CHROMATOGRAM
Retention time: 3
Internal standard: tetracycline
REFERENCE
Darouiche,R.O.; Hamill,R.J. Antibiotic penetration of and bactericidal activity within endothelial cells, Anti-
microb.Agents Chemother., 1994, 38, 1059-1064.
SAMPLE
Sample preparation: Inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Vydac 208TP54 C8
Mobile phase: MeCNrbuffer 22:78 (Buffer was 50 mM K2HPO4 adjusted to pH 6.5 with phos-
phoric acid.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 7.2
OTHER SUBSTANCES
Noninterfering: rifampin
KEYWORDS
REFERENCE
Pearson,S.D.; Trissel,L.A. Stability and compatibility of minocycline hydrochloride and rifampin in intravenous
solutions at various temperatures, Am. J.Hosp.Pharm., 1993, 50, 698-702.
SAMPLE
Matrix: honey
Sample preparation: Condition a 500 mg Baker-10 C18 SPE cartridge with 10 mL MeOH, 10
mL water, and 10 mL saturated aqueous disodium EDTA. Condition a 500 mg Baker-10 COOH
cartridge with MeOH:ethyl acetate 10:90. Dissolve 25 g honey in 50 mL 100 mM pH 4.0 di-
sodium EDTA-Mcllvaine buffer, filter. Add the filtrate to the C18 SPE cartridge, wash with 20
mL water, wash with 400 |xL ethyl acetate, air dry under vacuum for 5 min, elute with 50 mL
MeOH:ethyl acetate 10:90. Add a 5 mL aliquot to the COOH SPE cartridge, wash with 5 mL
MeOH (?), elute with 10 mL mobile phase, inject a 100 |xL aliquot.
HPLCVARIABLES
Column: 75 X 4.6 3 |xm Chemcosorb 3C8 (Chemco)
Mobile phase: MeCN:MeOH:10 mM aqueous oxalic acid 3:2:16, pH 3.0
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Extracted: chlortetracycline, demeclocycline (demethylchlortetracycline), doxycycline, methacyc-
line, oxytetracycline, tetracycline
KEYWORDS
SPE
REFERENCE
Oka,H.; Ikai,Y.; Kawamura,N.; Uno,K; Yamada,M.; Harada,K; Suzuki,M. Improvement of chemical analysis
of antibiotics. XII. Simultaneous analysis of seven tetracyclines in honey, J.Chromatogr., 1987, 400, 253
261.
SAMPLE
Matrix: milk
Sample preparation: Fill a disposable polypropylene column (Bio-Rad Econo-Pac column) with
Chelating Sepharose Fast Flow (Pharmacia) and condition it with 10 mL water, 1.5 mL 100
mM copper sulfate, and 100 mL water. Condition a 6 mL SupelClean ENVI-Chrom P SPE
cartridge with 2 mL MeOH and 5 mL water. Homogenize 10 g tissue with 20-30 mL 100 mM
pH 4 succinic acid buffer. Centrifuge the homogenate at 2000 g at 10 for 15-20 min. Add the
supernatant to the metal chelate affinity column, wash sequentially with 5 mL 500 mM NaCl,
10 mL water, 10 mL MeOH, 10 mL water, and 3 mL Mcllvaine buffer, discard the clear effluent.
Elute with 8 mL Mcllvaine-EDTA-NaCI buffer. Add the eluate to the SPE cartridge under
gravity, rinse the column with 2.5 mL water, add the rinse to the SPE cartridge. Wash the
SPE cartridge with 2.5 mL water. Dry the SPE cartridge by drawing air through it for 2-3 min.
Elute with 5 mL MeOH. Evaporate the eluate to dryness under nitrogen at 40-50, dissolve
the residue in 1 mL water. Inject a 100 |xL aliquot. (Mcllvaine buffer was 500 mM NaCl and
100 mM EDTA (Carson, M.C. J. AOAC Int. 1993, 76, 329).)
HPLC VARIABLES
Column: 150 X 3.9 5 (xm PLRP-S (Polymer Labs, USA)
Mobile phase: MeOH:5 mM oxalic acid 58:42
Flow rate: 0.5
Detector: MS, HP 5989, NICI, high energy dynode, HP 59980B particle beam interface 60,
helium sheath 40-45 p.s.i., source 250, quadrupole 100, source pressure 1 Torr with methane
reagent gas, m/z 378-483
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: chlortetracycline, demeclocycline, doxycycline, oxytetracycline, tetracycline
KEYWORDS
metal chelate affinity chromatography; cow; SPE
REFERENCE
Carson,M.C; Ngoh,M.A.; Hadley,S.W. Confirmation of multiple tetracycline residues in milk and oxytetracy-
cline in shrimp by liquid chromatography-particle beam mass spectrometry, J.Chromatogr.B, 1998, 712,
113-128.
SAMPLE
Matrix: milk
Sample preparation: Prepare a column as follows. Swirl Chelating Sepharose Fast Flow resin
(Pharmacia) in its bottle, add it to a polypropylene column to give a bed volume of 1.0-1.2 mL,
wash 3 times with 2 mL portions of water, wash with 2 mL 10 mM copper sulfate, wash with
two 2 mL portions of water. Centrifuge 5 mL milk at 10 at 1500 g for 15 min, remove the
lower layer and add it to 10 mL succinate buffer, mix, centrifuge at 1500 g for 30 min, add the
supernatant to the column. Wash with 2 mL succinate buffer, wash with 2 mL water, wash
with 2 mL MeOH, wash with 2 mL water, wash with 700 |xL citrate/phosphate buffer (be careful
not to disturb bed), elute with 2.5 mL citrate/phosphate buffer (column is white and eluate is
blue). Filter (Amicon Centricon 30, MW 30000 cut-off; pre-washed by centrifuging with 2 mL
water) while centrifuging at 5000 g for 30-90 min, inject a 600 JJLL aliquot of the ultrafiltrate.
(Prepare succinate buffer by dissolving 11.8 g succinic acid in 980 mL water, adjust pH to 4.0
with 10 M NaOH, make up to 1 L. Prepare the citrate/phosphate buffer by dissolving 12.9 g
citric acid monohydrate, 10.9 g Na2HPO4, 37.2 g disodium EDTA dihydrate, and 29.2 g NaCl
in 1 L water.)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm PLRP-S (Polymer Labs)
Mobile phase: Gradient. MeCN.MeOH.lO mM oxalic acid 0:0:100 for 1 min, to 22:8:70 over 5
min, maintain at 22:8:70 for 11 min, return to initial conditions.
Flow rate: 1
Detector: UV 355
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: chlortetracycline, demeclocycline, doxycycline, methacycline, oxytetracycline,
tetracycline
Noninterfering: chloramphenicol, gentian violet, hydromycin B, ivermectin, spectinomycin,
sulfa drugs
KEY WORDS
cow; SPE; ultrafiltrate
REFERENCE
Carson,M.C. Simultaneous determination of multiple tetracycline residues in milk using metal chelate affinity
chromatography, JAOAC Int., 1993, 76, 329-334.
SAMPLE
Matrix: urine
Sample preparation: Adjust the pH of 80 mL urine to 6.5 by adding 5.6 g NaH2PO4 and 10 g
sodium sulfite, extract with 100 mL ethyl acetate. Remove the organic layer and evaporate it
to dryness under reduced pressure at room temperature, reconstitute the residue in 1 mL
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Nucleosil SA (strong cation-exchange)
Mobile phase: EtOH:buffer 48:52 (Buffer was 100 mM pH 4.6 citrate containing 0.05% disodium
EDTA.)
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
REFERENCE
Nelis,H.J.C.R; De Leenheer,A.P- Metabolism of minocycline in humans, Drug Metab.Dispos., 1982, 10, 142-
146.
Minoxidil
Merck Index: 6290
Lednicer No.: 1 262
SAMPLE
Matrix: blood
Sample preparation: Condition a Fisher Prepsep C18 SPE column with 2 volumes MeOH then
2 volumes water. 1 mL Plasma + 50 jxL 0.056 |xg/mL IS in water, vortex for 15 s, add to SPE
column, wash with 2 volumes water, wash with 6 mL acetone, elute with ten 200 |xL fractions
of MeOH, dry at 50 under a stream of nitrogen, reconstitute in 100 JJIL mobile phase, vortex
for 10 s, inject a 10-60 jxL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 jmm juiBondapak C18
Mobile phase: MeCN:water 11:98, adjusted to pH 3.0 with phosphoric acid
Flow rate: 1.5
Detector: E, Environmental Sciences Assoc. Model 5100 A, guard cell ESA Model 5020 +0.90 V,
detector ESA model 5011 with detector 1 +0.30 V and detector 2 +0.80 V (UV 280
J.Pharm.Sci.1990, 79, 483)
CHROMATOGRAM
Retention time: 12 (minoxidil), 18 (minoxidil sulfate)
Internal standard: 2,4-diamino-6-diethylaminopyrimidine 3-oxide (10)
KEYWORDS
plasma; SPE
REFERENCE
Carrum,G.; Abernethy,D.R.; Sadhukhan,M.; Wright,C.E. Minoxidil analysis in human plasma using high-per-
formance liquid chromatography with electrochemical detection. Application to pharmacokinetic studies,
J.Chromatogr., 1986, 381, 127-135.
SAMPLE
Matrix: blood
HPLC VARIABLES
Column: 300 X 3.9 4 (xm NovaPack C18
Flow rate: 0.8
Detector: UV 231
CHROMATOGRAM
KEYWORDS
REFERENCE
Tracqui,A; Kintz,R; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 231.1
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
an aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 |xm Spherisorb SCX
Mobile phase: MeOH:water 80:20 containing 20 mM ammonium formate and 2.3 mIVL trifluo-
roacetic acid
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 4.7
OTHER SUBSTANCES
Simultaneous: cimetidine, clomipramine, halofantrine, haloperidol, reserpine, verapamil
REFERENCE
the analysis of basic drugs, J.ChromatogrA, 1996, 725, 335-341.
Miokamycin
Merck Index: 6291
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 60 mg of bulk compound in 50 mL MeCN, add 5 mL 1.2 mg/mL
josamycin in MeCN, mix, inject an aliquot.
HPLC VARIABLES
Column: 150 X 2.6 5 |xm Spheri ODS-2
Mobile phase: MeCN:buffer 75:25 (Buffer was 10 mM ammonium acetate containing 1 mM
K2HPO4 adjusted to pH 6.5 with 10 mM phosphoric acid.)
Flow rate: 1.5
Detector: UV 232
CHROMATOGRAM
Retention time: 7.0
Internal standard: josamycin (3.2)
REFERENCE
Marini,D.; Balestrieri,R; Pollino,G. Dosaggio mediante HPLC della miocamicina e delle sue principali impur-
ezze [HPLC analysis of miokamycin and its principle impurities], Boll.Chim.Farm., 1986, 125, 193-196.
Mirtazepine
Merck Index: 6295
LednicerNo.: 5 177
SAMPLE
Sample preparation: Extract 300 mg powdered tablet with 100 mL MeCN:water 50:50, inject
a 10 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 urn Hypersil ODS
Mobile phase: MeCN:MeOH:THF:buffer 14.875:12.67:7.455:65 (Buffer was 18 g/L tetramethyl-
ammonium hydroxide pentahydrate in water adjusted to pH 7.4 with 85% phosphoric acid.)
Flow rate: 1.5
Detector: UV 210
CHROMATOGRAM
Limit of detection: 0.01-0.04%
Limit of quantitation: 0.02-0.12%
OTHER SUBSTANCES
KEYWORDS
tablets; comparison with capillary electrophoresis
REFERENCE
Wynia,G.S.; Windhorst,G.; Post,P.C; Maris,F.A. Development and validation of a capillary electrophoresis
method within a pharmaceutical quality control environment and comparison with high-performance liquid
chromatography, J.ChromatogrA, 1997, 773, 339-350.
Misoprostol
Merck Index: 6297
SAMPLE
Sample preparation: Extract polymeric controlled-release formulations under supercritical
fluid conditions using carbon dioxide:formic acid 95:5 at 330 atmospheres at 75 in a 500 |xL
cell, restrictor temperature 100, collection solvent 15 mL hexane:EtOH 2:1, collection solvent
temperature 0, extraction time 1 h. Evaporate the collection solvent to dryness, reconstitute
with 1 mL mobile phase, inject a 10 fxL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Supelco ODS
Flow rate: 1.5
Detector: UV 280 following post-column reaction. The column effluent mixed with 4 M KOH
pumped at 0.5 mL/min and the mixture flowed at 80 to the detector.
CHROMATOGRAM
Retention time: 7.5
Limit of detection: <0.1 ppt
OTHER SUBSTANCES
KEYWORDS
polymeric controlled-release formulations; SFE; post-column reaction
REFERENCE
Roston,D.A.; Sun,J.J.; Collins,P.W.; Perkins,W.E.; Tremont,S.J. Supercritical fluid extraction-liquid chromatog-
raphy method development for a polymeric controlled-release drug formulation, J.Pharm.Biomed.Anal.,
1995, 13, 1513-1520.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1 mg/mL solution in MeOH:water 60:40, inject a 20 u,L aliquot
onto column A with mobile phase A. When the 11R,16S and 11S,16R enantiomers elute (as one
peak) switch effluent into the 250 uL sample loop of an injection valve. When the whole peak
has been collected inject the contents of the sample loop onto column B with mobile phase B.
HPLCVARIABLES
Column: A 150 X 4.6 3 |xm Supelcosil ODS; B 100 X 4.6 LKB EnantioPac
Mobile phase: A MeOH:water 60:40; B Isopropanol:20 mM pH 5.7 phosphate buffer 4:96
Flow rate: A 0.7; B 0.4
Detector: A UV 205; B UV 205
CHROMATOGRAM
Retention time: 43 (11R,16S and 11S,16R from column A), 37 (11S,16R from column B), 55
(11R,16S from column B)
KEYWORDS
chiral; column-switching
REFERENCE
Roston,D.A.; Wijayaratne,R. Two-dimensional liquid chromatographic method for resolution of prostaglandin
enantiomers, ArcaZ. Chem., 1988, 60, 948-950.
Mitoguazone
Merck Index: 6299
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 |xL aliquot of a 10 |xg/mL solution in mobile phase.
HPLC VARIABLES
Column: 250 X 4.6 5 (xm Hypersil BDS C8
Mobile phase: MeCN:buffer 11:89 (Buffer was 50 mM KH2PO^ containing 1 g/L sodium hep-
tanesulfonate, adjusted to pH 3.0 with concentrated phosphoric acid.)
Flow rate: 2
Detector: UV 283
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
KEYWpRDS
comparison with capillary electrophoresis and TLC
REFERENCE
Thomson,C.E.; Gray,M.R.; Baxter,M.P. The use of capillary electrophoresis as part of a specificity testing strat-
egy for mitoguazone dihydrochloride HPLC methods, J.Pharm.Biomed.AnaL, 1997, 15, 1103-1101.
Mitolactol
Molecular formula: C6H12Br2O4
Merck Index: 6300
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 1 mL ice-cold MeOH, let stand at -20 for 20 min, cen-
trifuge at 1500 g for 5 min. Remove a 1 mL aliquot of the supernatant and add it to 1 mL 5%
diethyldithiocarbamate and 2 mL 50 mM pH 7.4 potassium phosphate buffer, heat at 50 for
1 h, extract with 5 mL chloroform. Remove 4 mL of the organic layer and evaporate it to dryness
under a stream of nitrogen at 40, reconstitute, inject an aliquot.
Next Page
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Alltech CN
Mobile phase: Heptane:isopropanol:acetic acid 74.4:21.6:4
Flow rate: 1.3
Detector: UV 254
CHROMATOGRAM
Retention time: 9.9
KEYWORDS
derivatization; plasma; human; mouse; pharmacokinetics
REFERENCE
Henner,W.D.; Furlong,E.A.; Kelley,S.L.; Rosowsky,A. Assay for mitolactol and its bifunctional alkylating me-
tabolites in plasma, J.Pharm.Sci., 1985, 74, 983-986.
Mitomycin
Merck Index: 6301
SAMPLE
Sample preparation: 100 |xL Aqueous humor + 25 jxL 5 jxg/mL 4-aminoacetophenone in water,
vortex, inject a 100 (xL aliquot.
HPLCVARIABLES
Guard column: Microsorb C18
Column: 50 X 4.6 3 urn Microsorb C18 (Short-One)
Mobile phase: MeOHrwater 28:72 containing (NH4)H4PO4 adjusted to pH 7.0 with dilute am-
monium hydroxide
Flow rate: 1.5
Detector: UV 365
CHROMATOGRAM
Retention time: 2.3
Internal standard: 4-aminoacetophenone (2.7)
REFERENCE
Li,W.Y.; Seah,S.K.L.; Koda,R.T. Determination of mitomycin C in human aqueous humor and serum by high-
SAMPLE
Matrix: aqueous humor, tissue
Sample preparation: Tissue. Add 5 mL cold MeCN to 200 mg conjunctiva or sclera, homogenize,
sonicate for 30 min, centrifuge at 4 at 3000 rpm for 10 min, evaporate the organic layer to
dryness under reduced pressure at 40, reconstitute with 400 |xL mobile phase, sonicate, filter,
inject an aliquot. Aqueous humor. Add 4 mL ethyl acetate to 200 |xL aqueous humor, stir.
Sonicate for 30 min, centrifuge at 4 at 3000 rpm for 10 min, evaporate the organic layer to
dryness under reduced pressure at 40, reconstitute with 400 jxL mobile phase, sonicate, filter,
inject an aliquot.
Previous Page
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Alltech CN
Mobile phase: Heptane:isopropanol:acetic acid 74.4:21.6:4
Flow rate: 1.3
Detector: UV 254
CHROMATOGRAM
Retention time: 9.9
KEYWORDS
derivatization; plasma; human; mouse; pharmacokinetics
REFERENCE
Henner,W.D.; Furlong,E.A.; Kelley,S.L.; Rosowsky,A. Assay for mitolactol and its bifunctional alkylating me-
tabolites in plasma, J.Pharm.Sci., 1985, 74, 983-986.
Mitomycin
Merck Index: 6301
SAMPLE
Sample preparation: 100 |xL Aqueous humor + 25 jxL 5 jxg/mL 4-aminoacetophenone in water,
vortex, inject a 100 (xL aliquot.
HPLCVARIABLES
Column: 50 X 4.6 3 urn Microsorb C18 (Short-One)
Mobile phase: MeOHrwater 28:72 containing (NH4)H4PO4 adjusted to pH 7.0 with dilute am-
monium hydroxide
Flow rate: 1.5
Detector: UV 365
CHROMATOGRAM
Retention time: 2.3
REFERENCE
SAMPLE
Matrix: aqueous humor, tissue
Sample preparation: Tissue. Add 5 mL cold MeCN to 200 mg conjunctiva or sclera, homogenize,
sonicate for 30 min, centrifuge at 4 at 3000 rpm for 10 min, evaporate the organic layer to
dryness under reduced pressure at 40, reconstitute with 400 |xL mobile phase, sonicate, filter,
inject an aliquot. Aqueous humor. Add 4 mL ethyl acetate to 200 |xL aqueous humor, stir.
Sonicate for 30 min, centrifuge at 4 at 3000 rpm for 10 min, evaporate the organic layer to
dryness under reduced pressure at 40, reconstitute with 400 jxL mobile phase, sonicate, filter,
inject an aliquot.
HPLC VARIABLES
Column: 250 X 4 5 |xm Zorbax ODS
Flow rate: 1
Detector: UV 360
CHROMATOGRAM
Retention time: 5.6
Limit of detection: 20 ng/g (conjunctiva and sclera), 20 ng/mL (aqueous humor)
KEYWORDS
rabbit; ocular tissue; conjunctiva; sclera
REFERENCE
Chen,B.-M.; Xia,L.-W.; Xia,X.-B. Determination of mitomycin C in rabbit ocular tissue after topical adminis-
tration by high performance liquid chromatography, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 2659-2667.
SAMPLE
Matrix: blood
Sample preparation: Add 50 JJLL plasma to 50 JJLL 2 |xg/mL porfiromycin in MeCN. Vortex the
mixture for 0.5 min and centrifuge at 2200 g at 4 for 10 min. Mix 50 |xL supernatant with 50
IxL water. Inject a 50 u,L aliquot.
HPLC VARIABLES
Column: 150 X 6 AM-312 ODS (YMC, Japan)
Mobile phase: Gradient. A was MeCN:50 mM pH 7.0 phosphate buffer 10:90 containing 5 mM
sodium 1-octanesulphonate. B was MeCN:50 mM pH 7.0 phosphate buffer 50:50 containing 5
mM sodium 1-octanesulphonate. A:B from 100:0 to 70:30 over 20 min, to 0:100 over 1 min,
maintain at 0:100 for 5 min, to 100:0 over 1 min, maintain at 100:0 for 10 min.
Flow rate: 1
Detector: UV 360
CHROMATOGRAM
Internal standard: porfiromycin
KEYWORDS
rat; plasma
REFERENCE
Fuse,E.; Takai,K.; Okuno,K.; Kobayashi,S. Hepatic extraction ratio of 5-fluorouracil in rats. Dose dependence
and effect of uracil and interleukin-2, Biochem.PharmacoL, 1996, 52, 561-568.
SAMPLE
Matrix: blood
Sample preparation: Centrifuge plasma at 15800 g at 4 for 5 min. 500 JJLL Plasma + 50 |xL 4
fjig/mL IS in MeOH:water 50:50 + I m L MeCN, stir for 1 min, centrifuge at 15800 g at 4 for
10 min. Lyophilize the supernatant in a vacuum centrifuge (Hetovac VR-I, Allerod, Denmark).
Reconstitute the residue in 150 fxL MeOH:10 mM pH 6.5 NaH2PO4 30:70. Inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4 5 |xm Hypersil ODS
Mobile phase: MeOH:10 mM pH 6.5 NaH2PO4 30:70
Flow rate: 1.3
Detector: UV 365
CHROMATOGRAM
Internal standard: porfiromycin (6.64)
OTHER SUBSTANCES
Simultaneous: dexamethasone, lorazepam, mezlocillin, ondansetron, meperidine
KEY WORDS
plasma
REFERENCE
Joseph,G.; Biederbick,W.; Wosch6e,U.; Theisohn,M.; Klaus,W. Sensitive and convenient high-performance liquid
chromatographic method for the determination of mitomycin C in human plasma, J.Chromatogr.B, 1997,
698, 261-267.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 10 mL chloroformiisopropanol 50:50, shake for 1 min,
centrifuge at 2500 g for 5 min. Remove the supernatant and evaporate it to dryness under a
stream of nitrogen at 30-40, reconstitute the residue in 100 |xL MeOH, inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 jim ixBondapak C18
Flow rate: 1
Detector: UV 365
CHROMATOGRAM
Retention time: 7.5
KEYWORDS
REFERENCE
Den Hartigh,J.; van Oort,W.J.; Bocken,M.C.Y.M.; Pinedo,H.M. High-performance liquid chromatographic de-
termination of the antitumor agent mitomycin C in human blood plasma, Anal. Chim.Acta, 1981,127, 47-
53.
SAMPLE
Matrix: blood
Sample preparation: Condition a 500 mg Baxter C18 SPE cartridge with 1 volume of MeOH
and 2 volumes of water. 500 |xL Serum + 100 |xL 5 |xg/mL 4-aminoacetophenone in water, mix,
add to the SPE cartridge, wash with two 500 |xL aliquots of water, elute with three 500 |xL
aliquots of MeOH. Combine the eluates and evaporate them to dryness under a stream of air
at 40-50, reconstitute the residue in 150 |xL water, centrifuge at 13700 g for 5 min, inject a
50 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm C18
Mobile phase: MeOHiwater 28:72 containing (NH4)H4PO4 adjusted to pH 7.0 with dilute am-
monium hydroxide
Flow rate: 1.5
Detector: UV 365
CHROMATOGRAM
Retention time: 7.5
KEYWORDS
serum; SPE
REFERENCE
performance liquid chromatography, J.Chromatogr., 1993, 619, 148153.
SAMPLE
Matrix: blood
Sample preparation: 100 (xL Serum + 400 |xL MeCN, vortex, centrifuge at 12000 g for 2 min,
HPLCVARIABLES
Guard column: 15 X 3.2 NewGuard RP18
Column: 220 X 4.6 5 |xm Spheri-5 RP18
Flow rate: 0.8
Detector: UV 340
CHROMATOGRAM
Internal standard: mitomycin C
OTHER SUBSTANCES
Extracted: novobiocin
KEYWORDS
serum; mitomycin C is IS
REFERENCE
Zuhowski,E.G.; Gutheil,J.C; Egorin,M.J. Rapid and sensitive high-performance liquid chromatographic assay
for novobiocin in human serum, J.Chromatogr.B, 1994, 655, 147-152.
SAMPLE
Matrix: blood
Sample preparation: Centrifuge plasma at 2000 g for 15 min, inject a 25 |xL aliquot of the
supernatant.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm CAPCELL PAK MF Ph-I internal surface reversed-phase (Shiseido,
Tokyo)
Mobile phase: Water
Flow rate: 1
Detector: UV 365
CHROMATOGRAM
Retention time: 5.0
KEYVVORDS
direct injection; plasma
REFERENCE
Song,D.; Au,J.L.-S. Direct injection isocratic high-performance liquid chromatographic analysis of mitomycin
C in plasma, J.Chromatogr.B, 1996, 676, 165-168.
SAMPLE
Sample preparation: Prepare an SPE column of 100 mg 100-200 (xm Amberlite XAD-2 in a
Pasteur pipette, wash with three 3 mL portions of MeOH, wash with 10 mL water. 2 mL
plasma, serum, or urine + 100 |xL 1 |xg/mL porfiromycin in water, add to the SPE column,
wash with three 2 mL portions of water, elute with three 2 mL aliquots of MeOH. Evaporate
the eluate to dryness under reduced pressure at 57, reconstitute the residue in 200 yJL mobile
phase, mix on a whirlmixer for 2 min, inject a 10-100 |xL aliquot.
HPLC VARIABLES
Column: 100 X 3 5 jxm Hypersil-MOS
Flow rate: 2
Detector: UV 360 or E, E.G. & G. Par 310, hanging mercury drop electrode -600 mV, Ag/AgCl
reference electrode
CHROMATOGRAM
Retention time: 3
Internal standard: porfiromycin (5)
Limit of detection: 250 pg (E), 150 pg (UV)
KEYWORDS
SPE; serum; plasma; pharmacokinetics; human; rat
REFERENCE
Tjaden,U.R.; Langenberg,J.R; Ensing,K; Van Bennekom,W.P.; de Bruijn,E.A.; van Oosterom,A.T. Determina-
tion of mitomycin C in plasma, serum and urine by high-performance liquid chromatography with ultra-
violet and electrochemical detection, J.Chromatogr., 1982, 232, 355-367.
SAMPLE
Sample preparation: Plasma. Condition a 40 X 8 polypropylene SPE column containing 1 mL
Porapak Q (Waters) with 20 volumes MeOH and 40 volumes water. 0.2-2.5 mL Plasma or
thawed red blood cells + 100-200 ng porfiromycin + 5 volumes water, centrifuge red blood cell
ghosts at 8000 g for 90 min, add to the SPE column, wash with 20 mL water, elute with 6 mL
MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 37, reconstitute the
residue in 200 |xL water, inject a 100 |xL aliquot. Urine. Dilute urine 100 (rabbit) or 10 (human)
fold, add porfiromycin, inject an aliquot.
HPLCVARIABLES
Column: 10 |xm C8 (Hewlett-Packard)
Mobile phase: Gradient. MeCN:water from 5:95 to 41:59 over 8 min. Wash with MeCN for 5
min, re-equilibrate at initial conditions for 5 min.
Flow rate: 2
Detector: UV 360
CHROMATOGRAM
Retention time: 6.2
KEYWORDS
pharmacokinetics; plasma; rabbit; human; red blood cells; SPE
REFERENCE
van Hazel,G.A.; Kovach,J.S. Pharmacokinetics of mitomycin C in rabbit and human, Cancer Chem-
other.PharmacoL, 1982, 8, 189-192.
SAMPLE
1 mL Plasma or urine + 100 jxL 100 |xg/mL desipramine chloride, add to the SPE cartridge,
wash with 3 mL water, elute with 4 mL MeOH. Evaporate the eluate to dryness under a stream
of nitrogen, reconstitute the residue in 200 |xL mobile phase, vortex, centrifuge, inject a 100
IxL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 70 mm long 5 |xm LiChrosorb RP-8
Column: 150 X 4 5 urn LiChrosorb RP-8
Mobile phase: MeOH:buffer:water 25:10:65 (plasma) or 20:10:70 (urine) (Buffer was pH 7.0 phos-
phate buffer, |x = 0.1.)
Flow rate: 0.7
Detector: UV 365
CHROMATOGRAM
Retention time: 7
KEYWORDS
plasma; SPE; desipramine prevents adsorption on glass; pharmacokinetics
REFERENCE
Eksborg,S.; Ehrsson,H.; Lindfors,A. Liquid chromatographic determination of mitomycin C in human plasma
and urine, J.Chromatogr., 1983, 274, 263-270.
SAMPLE
Sample preparation: Emulsion. 500 fxL Emulsion + 10 mL 400 |xg/mL hydroquinone in MeOH
+ 40 mL 0.1% Tween 80, shake until homogeneous, inject a 10 jxL aliquot. Drug release me-
dium. 1 mL Drug release medium + 200 (xL 100 |xg/mL hydroquinone, mix, inject a 50 |xL
aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Cosmosil 10 C18 (Nacalai Tesque)
Mobile phase: Gradient. MeCN: 10 mM pH 3.0 phosphate buffer 2:98 for 1 min, to 45:55 over
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
Retention time: 10
Internal standard: hydroquinone (4.2)
OTHER SUBSTANCES
Simultaneous: carboplatin, epirubicin, iomeprol
KEYWORDS
emulsions; drug release medium; injections
REFERENCE
Yamazoe,K.; Horiuchi,T.; Sugiyama,T.; Katagiri,Y. Simultaneous high-performance liquid chromatographic de-
J.ChromatogrA, 1996, 726, 241-245.
SAMPLE
HPLC VARIABLES
Guard column: 15 X 3.2 7 |xm NewGuard RP-18
Column: 250 X 4 5 u.m Nucleosil C18
Mobile phase: MeOHrIO mM pH 6.0 phosphate buffer 7:13
Flow rate: 0.6
Detector: UV 364
REFERENCE
Ishiki,N.; Onishi,H.; Machida,Y. Conversion characteristics of the conjugates of mitomycin C with estradiol
benzoate in various pH media, Chem.Pharm.Bull., 1997, 45, 1345-1349.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Silverson) tumor tissue with 2 volumes 154 mM KCl. 1 mL
Homogenate + 10 |xL 0.5-5 |xg/mL porfiromycin in MeOH + 5 mL chloroform:isopropanol:ethyl
acetate 40:40:20, vortex vigorously for 15 min, centrifuge at 4 at 1000 g for 15 min, repeat
trogen, reconstitute the residue in 300 (xL mobile phase, vortex for 2 min, centrifuge at 15000
g, filter, inject a 100 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeOH: 18 mM pH 5.8 sodium phosphate buffer 26:74
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
tumor; rat
REFERENCE
Cummings,J.; Chirrey,L.; Willmott,N.; Halbert,G.W.; Smyth,J.F. Determination of mitomycin C, 2,7-diamino-
mitosene, 1,2-cis- and l,2-rans-l-hydroxy-2,7-diaminomitosene in tumour tissue by high-performance liquid
chromatography, J. Chromatogr., 1993, 612, 105-113.
Mitotane
Molecular formula: C14H10CI4
Merck Index: 6302
SAMPLE
Matrix: blood
Sample preparation: 200 jxL Plasma + 300 |xL acetone, vortex, centrifuge at 26 000 g for 5 min,
inject a 50 |xL aliquot of the supernatant
HPLCVARIABLES
Guard column: 20 mm long Supelguard (Supelco)
Column: 150 X 4.6 3 |im Supelcosil LC-18
Mobile phase: MeOH:buffer 80:20 (Buffer was 50 mM KH2PO4 adjusted to pH 7.0 with KOH.)
Flow rate: 1.25
Detector: UV 230
CHROMATOGRAM
Retention time: 6.2
OTHER SUBSTANCES
Extracted: metabolites, o,p'-(dichlorodiphenyl)-2,2-dichloroethene
KEYWORDS
plasma
REFERENCE
Andersen,A.; Warren,D.J.; Nome,O.; Vesterhus,L.; Slordal,L. A high-pressure liquid chromatographic method
for measuring mitotane [l,l-(o,p'-dichlorodiphenyl)-2,2-dichloroethane] and its metabolite l,l-(o,p'-dichlo-
rodiphenyl)-2,2-dichloroethenein plasma, Ther.Drug Monti., 1995, 17, 526[94]531.
SAMPLE
Sample preparation: Tablets. Powder tablet, add 10 mg DDE, extract four times with 10 mL
isooctane. Combine extracts, filter, make up to 50 mL with isooctane. Dilute an aliquot 1:10,
inject an aliquot. Emulsions. 5 mL Emulsion + 10 mg IS, extract with 40 mL isooctane:EtOH
98:2 for 15 min. Make up isooctane layer to 50 mL with isooctane. Remove a 1 mL aliquot and
make up to 10 mL with isooctane, inject an aliquot.
HPLC VARIABLES
Guard column: 100 X 4.6 30-38 ^m HC Pellosil (Whatman)
Column: 250 X 4.6 Partisil PxS 5/25
Mobile phase: Isooctanexyclohexane 80:20 (Flush column with isooctane:EtOH 98:2 at 2 mL/
min for 15 min to remove corn oil from emulsions, re-equilibrate with 120 mL mobile phase.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 9
Internal standard: l,l-bis-(p-chlorophenyl)-2,2-dichloroethylene(p,p'-DDE) (4.5)
KEYWORDS
tablets; emulsions; normal phase
REFERENCE
Musial,S.R; Freeman,C.J.; Sinsheimer,J.E. Mitotane (o,p'-DDD) emulsion and tablet analysis by high-perfor-
SAMPLE
Matrix: solutions
HPLCVARIABLES
Guard column: 30 X 4.6 10 |xm spherical C-18 MCP (Brownlee)
Column: 100 X 5 Nova-Pak C-18
Mobile phase: Gradient. MeOH:water:acetic acid 50:50:0.2 for 40 min then to MeCN:water:acetic
acid 55:45:0.2 over 10 min
CHROMATOGRAM
OTHER SUBSTANCES
REFERENCE
Cai,W.; Counsell,R.E.; Djanegara,T.; Schteingart,D.E.; Sinsheimer,J.E.; Wotring,L.L. Metabolic activation and
binding of mitotane in adrenal cortex homogenates, J.Pharm.ScL, 1995, 84, 134-138.
Mitoxantrone
CAS Registry No.: 65271-80-9, 70476-82-3 (di HCI)
Merck Index: 6303
Lednicer No.: 3 75
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond-Elut C18 SPE cartridge with 10 mL MeOH and
5 mL water. Add 1-2 mL plasma to the SPE cartridge, wash with 5 mL water, elute with 300
IxL 500 mM methanolic HCl, inject an aliquot.
HPLCVARIABLES
Guard column: 70 X 2.1 Co:Pell ODS (Whatman)
Flow rate: 1.5
Detector: UV 658
CHROMATOGRAM
Retention time: 4
KEYWORDS
plasma; SPE
REFERENCE
Peng,Y.-M.; Ormberg,D.; Alberts,D.S.; Davis,T.P. Improved high-performance liquid chromatography of the new
antineoplastic agents bisantrene and mitoxantrone, J.Chromatogr., 1982, 233, 235-247.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 jxL 100 |ig/mL bisantrene, adjust to pH 11 with 50 |xL
1 M NaOH, add 5 mL dichloromethane, extract. Remove the organic layer and evaporate it to
dryness under a stream of nitrogen, reconstitute the residue in 250 |xL mobile phase, centrifuge
at 15600 g for 1 min, inject a 150 |xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 37-50 |xm C18/Corasil
Mobile phase: MeCN:80 mM pH 3.0 sodium formate 28:72
Flow rate: 1
Detector: E, BAS LC-4B detector, TL-5 glassy carbon electrode at +0.75 V, Ag/AgCl reference
electrode or UV 660
CHROMATOGRAM
Retention time: 5.5
Internal standard: bisantrene (8)
Limit of detection: 0.1 ng/mL (electrochemical)
KEYWORDS
REFERENCE
Choi,K.E.; Sinkule,J.A.; Han,D.S.; McGrath,S.C; Daly,K.M.; Larson,R.A. High-performance liquid chromato-
graphic assay for mitoxantrone in plasma using electrochemical detection, J.Chromatogr., 1987, 420, 81
88.
SAMPLE
Matrix: blood
Sample preparation: Condition a 40 |xm Sepralyte non-bonded silica SPE cartridge (Analyti-
chem) with I m L l M HCl, with 3 mL water, and with 500 JULL 2 M (NH4)H2PO4. 1 mL Plasma
+ 5 |xL 5 (xg/mL methylene blue, centrifuge at 4 at 12000 g for 1 min, add the supernatant to
the SPE cartridge at 1 mL/min, vortex the precipitate in the centrifuge tube with 500 |xL 0.3%
ascorbic acid in 12 mM pH 3.0 citrate buffer, add the mixture to the SPE cartridge, wash with
500 JJLL water, wash with 500 JJLL MeCN saturated with tetramethylammonium chloride, apply
suction for 5 min, elute with 300 |xL elution solvent by centrifuging at 4 at 2000 g for 5 min,
inject an aliquot of the eluate. (The elution solvent was MeOH: 1 M tetramethylammonium
chloride:l M citric acid:water 32:10:20:38.)
HPLC VARIABLES
Column: 75 X 3.9 4 |xm Nova-pak ODS
Mobile phase: MeOHrTHF: triethylamine phosphate solution: 1 M tetramethylammonium chlo-
ride:water 30:1:10:2:57 (Triethylamine phosphate was 67.8 mL orthophosphoric acid in 800 mL
water, adjust pH to 3.0 with triethylamine, make up to 1 L with water.) (Filter mobile phase
but do not degas.)
Flow rate: 1
Detector: UV 658
CHROMATOGRAM
Retention time: 3.5
Internal standard: methylene blue (7)
KEYWORDS
plasma; SPE; use polypropylene containers
REFERENCE
Lin,K.T.; Rivard,G.E.; Leclerc,J.-M. High-performance liquid chromatographic determination of mitoxantrone
in plasma utilizing non-bonded silica gel for solid-phase isolation to reduce adsorptive losses on glass during
sample preparation, J.Chromatogr., 1989, 465, 75-86.
SAMPLE
Sample preparation: Add 1 mL solution containing 10 (xg/mL hexanesulfonic acid, 500 |xg/mL
ascorbic acid and 8 |xg/mL ametantrone to 1 mL whole blood or tissue homogenate, vortex for
30 s. Add 1 mL 100 mM pH 9.5 borate buffer, 300 |xL 1 M NaOH, vortex for 30 s. Extract using
a horizontal linear shaker (Infors HT, Infors, Switzerland) with 5 mL dichloromethane at 150
rpm for 60 min. Centrifuge at 2800 g for 15 min, evaporate organic layer to dryness under
reduced pressure, dissolve the residue in 150 |xL mobile phase. Inject a 92 |xL aliquot. (Silanize
all glassware.)
HPLC VARIABLES
Guard column: 11 X 4 5 |xm Nucleosil C18
Mobile phase: MeCN:160 mM pH 2.7 ammonium formate buffer 33:67 containing 250 mM (sic)
hexanesulfonic acid
Flow rate: 1
Detector: UV 658
CHROMATOGRAM
Internal standard: ametantrone (6.9-8.3)
KEYWORDS
mouse; liver; heart; spleen; kidney; whole blood
REFERENCE
Rentsch,K.M.; Schwender,R.A.; Hanseler,E. Determination of mitoxantrone in mouse whole blood and different
tissues by high-performance liquid chromatography, J.Chromatogr.B, 1996, 679, 185-192.
SAMPLE
Matrix: cells
Sample preparation: 1 mL Bone marrow suspension (20000 cells/|xL) + 50 ng ametantrone + 1
mL 100 mM pH 10.0 borate buffer + 5 mL dichloromethane, extract, centrifuge at 1000 g for
40, reconstitute the residue in 1 mL mobile phase, inject a 100 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN: 160 mM pH 2.7 ammonium formate buffer 30:70 containing 25 mM hex-
anesulfonic acid, 2 mM NaCl, and 1.34 mM EDTA
Flow rate: 1.6
Detector: E, AMOR/Spark, glassy carbon electrode +0.75 V, Ag/AgCl reference electrode
CHROMATOGRAM
Internal standard: ametantrone (4.99)
KEYWORDS
rat; bone marrow; pharmacokinetics
REFERENCE
de Vries,A.J.; Nooter,K. Quantification of mitoxantrone in bone marrow by high-performance liquid chroma-
tography with electrochemical detection, J.Chromatogr., 1991, 563, 435-442.
SAMPLE
Sample preparation: Condition a 3 mL Supelclean LC-18 SPE cartridge (Supelco) with 1 mL
MeOH and 1 mL water. Dissolve one volume liposome preparation in 3 volumes EtOH, add
500 Uj LLf to the SPE cartridge, wash with 1 mL water, elute with 1 mL 500 mM HCl in MeOH,
inject an aliquot of the eluate.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm LiChrospher RP-18
Mobile phase: MeCNiIO mM KH2PO4 40:60, pH adjusted to 3.0 with orthophosphoric acid
Flow rate: 1
Detector: UV 242
CHROMATOGRAM
Retention time: 4.2
Internal standard: propyl paraben (11.4)
OTHER SUBSTANCES
KEYWORDS
liposomes; SPE; stability-indicating
REFERENCE
Law,S.-L.; Jang,T.-F. High-performance liquid chromatographic determination of mitoxantrone in liposome
preparations using solid-phase extraction and its application in stability studies, J.Chromatogr.A, 1994,
670, 234-238.
Mivacurium chloride
Merck Index: 6305
SAMPLE
Sample preparation: Condition a Bond Elut Cl SPE cartridge with 3 mL MeOH and 3 mL
water, do not allow to dry. Dilute bile 1:3 with water. Add 50 JJLL 70 mg/mL phenylmethane-
sulfonyl fluoride (an enzyme inhibitor) in DMF to 5 mL blood before centrifuging at 3000 g for
10 min to prepare plasma. 1 mL Plasma, urine, or diluted bile, add to the SPE cartridge, wash
with 3 mL water, wash with 3 mL MeCN, wash with 3 mL MeOH, wash with 3 mL water,
elute with 1 mL eluant, mix eluate thoroughly, inject a 20-100 |JLL aliquot. (Eluant was MeOH:
50 mM pH 3 KH2PO4 80:20.)
HPLC VARIABLES
Guard column: 5 |xm Spherisorb S5C1 methylsilyl
Column: 150 X 4.6 5 jim Spherisorb S5C1 methylsilyl
Mobile phase: MeCN:MeOH:50 mM pH 3 KH2PO4 70:0.35:30
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 5.7
Internal standard: mivacurium chloride
Limit of quantitation: 30 ng/mL (bile), 10 ng/mL (plasma, urine)
OTHER SUBSTANCES
Extracted: doxacurium
Simultaneous: pancuronium, atracurium, tubocurarine succinylcholine, metocurine
KEYWORDS
plasma; dog; human; SPE; mivacurium is IS
REFERENCE
DeAngelis,R.; Loebs,R; Maehr,R.; Savarese,J.; Welch,R- High-performance liquid chromatographic analysis of
doxacurium, a new long-acting neuromuscular blocker, J.Chromatogr., 1990, 525, 389-400.
SAMPLE
Matrix: blood
Condition a QMA anion-exchange SPE cartridge with 5 mL MeOH and 8 mL water. 1 mL
Plasma containing 330 jxg phospholine iodide (esterase inhibitor) + 1 mL 100 ng/mL IS in
saline, add to the C18 SPE cartridge, wash with 4 mL water, wash with 4 mL MeOH, wash
with 4 mL MeCN, elute with 2.8 mL MeCN:6 M HCl 99.67:0.33. Add the eluate to the anion-
exchange SPE cartridge, elute with 900 jxL MeCN:6 M HCl 99.67:0.33. Evaporate the eluate
to dryness under vacuum at 55, reconstitute the residue in 200 |xL mobile phase, inject a 50
fxL aliquot.
HPLC VARIABLES
Guard column: 5 |xm LiChrosphere 60 RP Select B
Column: 125 X 4.6 5 fxm LiChrosphere 60 RP Select B
Mobile phase: MeCN:water 40 60 containing 5 mM octanesulfonic acid (PIC B-8, low UV)
Flow rate: 1
CHROMATOGRAM
Retention time: 8.4 (trans-trans), 9.3 (cis-trans), 10.2 (cis-cis)
Internal standard: bis-[3-[trans-l,2,3,4-tetrahydro-6,7-dimethoxy-N-methyl-l-(3,4,5-trimethoxy-
benzyl)isoquinolinium]propyl]-l,3-phenylenedipropionatedihydrochloride (BW 785U77) (13.1)
KEYWORDS
plasma; SPE
REFERENCE
Brown,A.R.; James,C.D.; Welch,R-M.; Harrelson,J.C. Stereoselective high-performance liquid chromatographic
assay with fluorometric detection for the isomers of mivacurium in human plasma, J.Chromatogr., 1992,
578, 302-308.
SAMPLE
Matrix: blood
Sample preparation: Condition a 300 mg PrepSep C18 SPE cartridge with 3 mL MeOH and 3
mL water. 1 mL Plasma + 100 |iL 4 |xg/mL IS in physiological saline (adjusted to pH 3 with
100 mM HCl) + 1 mL water, mix, add to SPE cartridge, wash with 3 mL water, wash with 3
mL MeCN, wash with 3 mL water, elute with two 750 |xL portions of MeOH:50 mM pH 3
ammonium diphosphate 80:20. Evaporate the eluates to dryness under vacuum, reconstitute
the residue in 300 |xL mobile phase, inject a 150 JJLL aliquot.
HPLC VARIABLES
Guard column: 4 X 4.6 5 |xm LiChrosphere 60 RP select B
Column: 125 X 4.6 5 |xm LiChrosphere 60 RP select B
Mobile phase: MeCNrwater 37.5:62.5 containing 5.2 mM octanesulfonic acid (PIC B-8)
Flow rate: 2
CHROMATOGRAM
Retention time: 7.1 (trans-trans), 7.9 (cis-trans), 8.9 (cis-cis)
Internal standard: bis-3-[trans-1,2,3,4-tetrahydro-6,7-dimethoxy-N-methyl-1-(3,4,5-trimethoxy-
benzyl)isoquinolinium] propyl-l,3-phenylenedipropionate dichloride (BW785U77) (12)
OTHER SUBSTANCES
KEY WORDS
REFERENCE
Lacroix,M.; Tu,T.M.; Donati,R; Varin,R High-performance liquid chromatographic assays with fluorometric
detection for mivacurium isomers and their metabolites in human plasma, J.Chromatogr.B, 1995,663,297-
307.
Mizoribine
Merck Index: 6306
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 50 JJLL 65 jjig/mL 3-methylxanthine in water + 50 |xL
70% perchloric acid, mix, add 50|xL saturated potassium chloride, mix well, centrifuge. Remove
a 350 (xL aliquot of the supernatant and add it to 35 uL 10 M NaOH, wash with 1.5 mL
dichloromethane. Remove a 250 |xL aliquot of the aqueous phase and neutralize it with 20 (JLL
2 M HCl, inject an aliquot.
HPLCVARIABLES
Mobile phase: Gradient. MeOH:20 mM Na2HPO4 2:98, pH 3, containing 0.04% octanesulfonic
acid, after 1.1 min MeOH:20 mM Na2HPO4 6:94, pH 3, containing 0.04% octanesulfonic acid
(step gradient).
Flow rate: 1.3
Injection volume: 2
Detector: UV 275
CHROMATOGRAM
Retention time: 2.5
Internal standard: 3-methylxanthine (5)
KEY WORDS
dog; plasma; human
REFERENCE
Erdmann,G.R.; Gruber,S.A.; McGuiggan,M.M.; Cipolle,R.J.; Canafax,D.M. Determination of mizoribine in
plasma using ion-pair high-performance liquid chromatography, J.Chromatogr., 1989, 494, 354-360.
Moclobemide
Merck Index: 6309
Lednicer No.: 4 39
SAMPLE
Matrix: bile, blood, tissue, vitreous humor
Sample preparation: Bile, blood, vitreous humor. Mix 1 mL sample with 2 jxg IS, 1 mL deionized
water, and 1 mL 200 mM sodium carbonate. Add 6 mL hexane:butanol 95:5, extract for 30 min,
centrifuge. Remove the organic layer and add it to 100 |xL 0.2% orthophosphoric acid, extract
for 30 min, centrifuge. Inject a 30 |xL aliquot of the aqueous layer. Tissue. Mix 500 |xL liver
homogenate with 2 fig IS, 1 mL deionized water, and 1 mL 200 mM sodium carbonate. Add 6
mL hexane:butanol 95:5, extract for 30 min, centrifuge. Remove the organic layer and add it
to 400 (JLL 0.2% orthophosphoric acid, extract again for 30 min, centrifuge. Inject a 30 |xL aliquot
of the aqueous layer.
HPLCVARIABLES
Column: 100 X 4.6 Spheri-5RP-18 (Applied Biosystems)
Mobile phase: MeCN:100 mM NaH2PO4.2H2O containing 1% diethylamine 15:85, adjusted to pH
5.8
Flow rate: 0.9
CHROMATOGRAM
Internal standard: R011-9900 (Roche Australia)
KEYWORDS
liver; use silanized glassware
REFERENCE
MclntyreJ.M.; King,C.V.; Staikos,V; Gall,J.; Drummer,O.H. A fatality involving moclobemide, sertraline, and
pimozide, J.Forensic ScL, 1997, 42, 951-953.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 200 (xL 5 (xg /mL IS in MeOH:water 30:70 + 1 mL 500
mM Na2HPO4 + 10 mL distilled n-butyl chloride, extract for 10 min, centrifuge. Remove the
organic layer and evaporate it to dryness, reconstitute the residue in 400 |xL mobile phase,
centrifuge at 1000 g for 5 min, inject a 40 |xL aliquot.
HPLC VARIABLES
Column: 125 X 3.2 7 jxm Lichrosorb RP-18
Mobile phase: MeCN:water:25% ammonia 25:75:0.15 (Wash column with MeCN:water 50:50 at
the end of the day.)
Flow rate: 1.5
Detector: UV 237
CHROMATOGRAM
Retention time: 4.3
Internal standard: p-methyl-N-(2-morpholinoethyl)benzamide (2.9)
KEY WORDS
REFERENCE
Raaflaub,J.; Haefelfinger,R; Trautmann,KH. Single-dose pharmacokinetics of the MAO-inhibitor moclobemide
in man, Arzneimittelforschung, 1984, 34, 80-82.
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 239
CHROMATOGRAM
KEYWORDS
tarabine; benzoylecgonine; acetaminophen; diazoxide; dacarbazine; sulfinpyrazole; numazenil;
REFERENCE
254-262.
SAMPLE
Sample preparation: Blood or serum. 1 mL Blood or serum + 1 (xg cianopramine + 1 mL water,
(xL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the organic
layer and inject a 30 JJLL aliquot of the aqueous layer. Liver homogenate. 0.5 mL Liver ho-
mogenate + 10 [xg cianopramine + 500 |xL 2% sodium tetraborate + 8 mL hexane:l-butanol 95:
it to 400 fjuL 0.2% phosphoric acid, agitate gently for 30 min, centrifuge for 5 min. Remove the
HPLCVARIABLES
Column: 100 X 4.6 5 jxm Brownlee Spheri-5 RP-18
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
doxepin, fluoxetine, haloperidol, imipramine, loxapine, maprotiline, meperidine, mesoridazine,
methadone, mianserin, nomifensine, nordoxepin, norfluoxetine, norpropoxyphene, northiaden,
nortriptyline, pheniramine, promethazine, propoxyphene, propranolol, protriptyline, quinidine,
quinine, sulforidazine, thioridazine, thiothixene, trazodone, trihexyphenidyl, trimipramine,
triprolidine
fluoperazine
Interfering: metoclopramide, tranylcypromine, pentobarbital
KEYWORDS
REFERENCE
SAMPLE
Sample preparation: Plasma. 500 |xL Plasma + 400 |xL saturated sodium phosphate solution
(pH 11) + 100 |xL 8 |ig/mL IS in water, vortex for a few s, add to a glass Extrelut 1 SPE
cartridge, let stand for 10 min, elute with two 6 mL portions of freshly distilled dichlorome-
thane. Evaporate the eluate to dryness under a stream of nitrogen at 37, reconstitute the
residue in 250 |xL mobile phase, vortex for 30 s, inject a 75 |xL aliquot. Urine. 200 |xL Urine +
700 jxL saturated sodium phosphate solution (pH 11) + 100 |xL 20 |xg/mL IS in water, vortex
for a few s, add to a glass Extrelut 1 SPE cartridge, let stand for 10 min, elute with two 6 mL
portions of freshly distilled dichloromethane. Evaporate the eluate to dryness under a stream
of nitrogen at 37, reconstitute the residue in 750 (xL mobile phase, vortex for 30 s, inject a 50
JXL aliquot.
HPLC VARIABLES
Column: 125 X 4 Spherisorb S5 C6
Mobile phase: MeCN:67 mM KH2PO4 30:320 adjusted to pH 3.9 with dilute HCl
Flow rate: 1.3
Detector: UV 240
CHROMATOGRAM
Retention time: 7.2
Internal standard: p-iodo-N-(2-morpholinoethyl)benzamide (Ro 11-9900) (12.7)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Geschke,R.; Korner,J.; Eggers,H. Determination of the new monoamine oxidase inhibitor moclobemide and
three of its metabolites in biological fluids by high-performance liquid chromatography, J. Chromatogr., 1987,
420, 111-120.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: hepatocytes
Sample preparation: Add an equal volume of MeOH to the hepatocytes, centrifuge at 5000 g
for 15 min, inject an aliquot of the supernatant.
HPLCVARIABLES
Guard column: 20 mm long 5 |jim Nucleosil C18
Mobile phase: Gradient. MeOH:buffer 12:88 for 45 min, then 80:20 for 15 min (step gradient).
(Buffer was 25 mM pH 3.9 sodium phosphate buffer containing 0.2% N-hexylamine.)
Flow rate: 1
Detector: UV 250
CHROMATOGRAM
Retention time: 34
OTHER SUBSTANCES
KEYWORDS
rat; human
REFERENCE
Vall&s,B.; Coassolo,R; De Sousa,G.; Aubert,C; Rahmani,R. In vitro hepatic biotransformation of moclobemide
(Ro 11-1163) in man and rat, Xenobiotica, 1993, 23, 1101-1111.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 ^m Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol, met-
ronidazole, midazolam, morphine, nadolol, nalbuphine, naloxone, naphazoline, naproxen, ni-
fedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, pa-
roxetine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine,
himbine, zopiclone
KEYWORDS
REFERENCE
J.Chromatogr.A, 1995, 692, 103-119.
Modafinil
Molecular formula: C15H15NO2S
Merck Index: 6311
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + I m L 100 mM HCl + 100 |xL MeOH + 100 |xL 40 |xg/mL
IS in MeOH + 10 mL diethyl ether, shake for 10 min, centrifuge at 0-4 at 4000 g for 10 min.
Remove the organic layer and evaporate it to dryness under a stream of nitrogen at room
temperature, reconstitute the residue in 200 JXL mobile phase, vortex for 30 s, inject a 25 |xL
aliquot.
HPLCVARIABLES
Flow rate: 1.4
Detector: UV 236
CHROMATOGRAM
Internal standard: [bis-(4-nuorophenyl)methylsulfinyl]acetic acid (24.0)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Moachon,G.; Matinier,D. Simultaneous determination of modafinil and its acid metabolite by high-performance
liquid chromatography in human plasma, J.Chromatogr.B, 1994, 654, 91-96.
Molindone
Merck Index: 6315
Lednicer No.: 2 455
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut CN SPE cartridge with MeOH and then with wa-
ter. Add 1 mL plasma to the SPE cartridge, wash with 200 |xL water, elute with five 200 |xL
portions of MeOH. Evaporate the eluate to dryness under a stream of air at 35, reconstitute
the residue in 200 (xL 750 ng/mL p-aminobenzonitrile in mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Zorbax CN
Mobile phase: Cyclopentane:MeOH:THF:morpholine 90:7:2.9:0.1
Detector: UV 254
CHROMATOGRAM
Internal standard: p-aminobenzonitrile
KEYWORDS
REFERENCE
Zetin,M.; Cramer,M.; Garber,D.; Plon,L.; Paulshock,M.; Hoffinan,H.E.; Schary,W.L. Bioavailability of oral and
intramuscular molindone hydrochloride in schizophrenic patients, Clin.Ther., 1985, 7, 169175.
Molsidomine
Merck Index: 6316
Lednicer No.: 3 140
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 312.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Monensin
Merck Index: 6329
SAMPLE
Sample preparation: 5 g Pulverized frozen tissue or 5 g homogenized whole eggs + 2 mL water
+ 13 mL MeOH, homogenize for 30 s. Sonicate for 10 min and centrifuge at 2000 g for 10 min.
Add 4 mL 100 mM NaOH to a 2 mL aliquot of the supernatant, extract with 2 mL and 1 mL
hexane:toluene 50:50 (v/v) for 30 s by inversion, centrifuge at 1500 g for 10 min. Evaporate the
combined extracts to dryness under a stream of nitrogen at 60. Dissolve the residue in 200
|xL MeCN:water 75:25. Inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Inertsil ODS-2 (GL Sciences, Japan)
Mobile phase: MeCN:MeOH:THF:water:trifluoroaceticacid 67:10:10:13:0.1
Flow rate: 1
Detector: MS, VG Platform, Megaflow electrospray probe, positive ion mode, source at 125, cone
voltage 25 V, m/z 693
CHROMATOGRAM
Retention time: 7
Limit of detection: 0.5-1 ng/g
OTHER SUBSTANCES
Extracted: narasin, salinomycin
KEYWpRDS
domestic fowl; muscle; liver
REFERENCE
Blanchflower,W.J.; Kennedy,D.G. Determination of monensin, salinomycin, and narasin in muscle, liver and
eggs from domestic fowl using liquid chromatography-electrospray mass spectrometry, J.Chromatogr.B,
1996, 675, 225-233.
SAMPLE
Matrix: feed
Sample preparation: 20 g Ground feed + 50 mL MeOH, shake on a reciprocating shaker for 1
h, allow to settle, centrifuge an aliquot of the supernatant at 2500 rpm for 5 min, inject a 50
jxL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 X 4.6 Partisil 5ODS-3
Mobile phase: MeOH:water:glacial acetic acid 94:5.9:0.1
Flow rate: 0.7
Detector: UV 520 following post-column reaction with the reagent pumped at 0.7 mL/min. The
mixture passed through a 7.6 m X 0.05 mm i.d. coil of stainless steel tubing maintained at 70
to the detector. (Prepare reagent by stirring 900 mL MeOH and slowly and cautiously adding
20 mL concentrated sulfuric acid (Caution! Corrosive! Exothermic reaction!), cool in an ice bath,
add 100 g vanillin, mix to dissolve, make up to 1 L with MeOH. Degas under vacuum with
sonication for 2 min.)
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: narasin, salinomycin
KEYWORDS
REFERENCE
Blanchflower,W.J.; Rice,D.A.; Hamilton,J.T.G. Simultaneous high-performance liquid chromatographic deter-
mination of monensin, narasin and salinomycin in feeds using post-column derivatisation, Analyst, 1985,
110, 1283-1287.
SAMPLE
Matrix: feed
Sample preparation: 20 g Ground Feed + 200 mL hexane:ethyl acetate 90:10, stir at high speed
for 2 h, let stand. Remove an aliquot equivalent to 1 g feed and evaporate it to dryness under
reduced pressure at 40, reconstitute with 2 mL MeOH, filter (0.45 |xm), inject an aliquot of
the nitrate.
HPLCVARIABLES
Column: 60 X 4.6 3 |xm C18 (Hewlett-Packard)
Mobile phase: MeOH:5% acetic acid 90:10
Flow rate: 0.5
pumped at 1 mL/min and the mixture flowed through a 1.5 mL reaction coil (Kratos Model
510) at 95 to the detector. (Reagent was 40 g/L vanillin in MeOHisulfuric acid 100:2. Keep in
an ice bath and prepare fresh daily.)
CHROMATOGRAM
Retention time: 5.5
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Lapointe,M.R.; Cohen,H. High-speed liquid chromatographic determination of monensin, narasin, and salino-
mycin in feeds, using post-column derivatization, J.Assoc.Off.Anal.Chem., 1988, 71, 480-484.
SAMPLE
Sample preparation: Feed. Shake 5 g feed with 15 mL MeOH for 2 h, filter, evaporate the
filtrate to 3 mL and make up to 10 mL with MeOH, inject a 3 JJLL aliquot. Premix. Shake 0.5
g premix with 15 mL MeOH for 2 h, filter, make up the filtrate to 50 mL with MeOH, inject a
3 (JLL aliquot.
HPLCVARIABLES
Column: 150 X 1 5 |xm Separon SGX C18 glass column (Tessek Prague)
Flow rate: 0.02
Injection volume: 3
Detector: UV 592 following post-column derivatization. The column effluent mixed with the re-
agent pumped at 0.015 mL/min and the mixture flowed through a 150 X 1 reactor containing
40-70 (Jim acid-washed glass beads at 75 to the detector. (The reagent was 500 mM 4-di-
methylaminobenzaldehyde in 1.2 M sulfuric acid in MeOH.)
CHROMATOGRAM
Retention time: 11 (monensin B), 14 (monensin A)
Limit of detection: 1.7 |xg/mL (monensin A)
OTHER SUBSTANCES
KEY WORDS
microbore; post-column reaction
REFERENCE
Fejglova,Z.; Dolezal,J.; Hrdlicka,A.; Frgalova,K. Microbore HPLC determination of polyether antibiotics using
postcolumn derivatization with benzaldehyde reagents, J.Liq.Chromatogr., 1994, 17, 359-372.
SAMPLE
Sample preparation: Homogenize (mechanical grinder) 5 mL fermentation broth with 45 mL
MeOH, allow to settle at 5 for 1 h, filter (0.45 |xm) the supernatant, dilute the filtrate with
MeOH (if necessary), inject a 10 |xL aliquot.
HPLCVARIABLES
Column: Little Champ C18 (Regis)
Mobile phase: MeOH:buffer 85:15 (Buffer was 1 g/L (?) (NH4)H2PO4 adjusted to pH 3.0 with
phosphoric acid.)
Flow rate: 1.5
pumped at 1.5 mL/min and the mixture flowed through a 3 m X 0.25 mm ID stainless steel
coil at 120 to the detector. (Prepare reagent by making a 3% solution of vanillin in MeOH,
chill solution, slowly add concentrated sulfuric acid to a final concentration of 3%. Store at 0,
discard after 2 weeks.)
CHROMATOGRAM
Retention time: 2 (monensin B), 2.7 (monensin A)
KEY WORDS
REFERENCE
Neely,F.L. Determination of monensin in fermentation broth by HPLC with post-column derivatization,
J.Liq.Chromatogr., 1992, 15, 1513-1522.
SAMPLE
Sample preparation: 100 |xL Liposome + 50 |xL Tween 20, vortex for 5 min, add 600 |xL MeOH,
centrifuge at 10000 rpm for 30 min, dilute the supernatant 10-fold with MeOH, inject an
aliquot.
HPLC VARIABLES
Column: 70 X 4.6 3 (xm Ultrasphere XL-ODS (Beckman)
Mobile phase: MeCN:MeOH:dichloromethane:water:acetic acid 20:45:25:9.5:0.5
Flow rate: 0.33
pumped at 0.67 mL/min and the mixture flowed through a Beckman 231 reactor at 70 to the
detector. (Prepare the reagent by dissolving 8 g vanillin in 100 mL cooled MeOH, add 4 g
concentrated sulfuric acid, mix, keep in an ice bath.)
CHROMATOGRAM
KEYWORDS
post-column reaction; liposomes
REFERENCE
Ferdous,A.J.; Bennefield,S.D.; Singh,M. A modified HPLC method for monensin analysis in liposomes and
nanocapsules and its comparison with spectrophotometric and radioactive methods, J.Pharm.Biomed.Anal.,
1997, 15, 1775-1780.
SAMPLE
Matrix: milk, tissue
Sample preparation: Tissue. Condition a Sep-Pak silica gel SPE cartridge (No. 51900) with 3
mL dichloromethane, do not allow it to go dry. Sonicate or blend 10 g ground or minced tissue
and 75 mL MeOH:water 85:15, centrifuge at 2000 rpm for 10 min. Remove the supernatant
and add it to 50 (muscle, liver), 40 (kidney) or 60 (fat) mL 100 mg/mL NaCl, extract twice with
35 mL portions of dichloromethane. Combine the organic layers and evaporate them to dryness
under vacuum at 45, reconstitute the residue in 7 mL dichloromethane, add it to the SPE
cartridge at no more than 5 mL/min, rinse out the flask with 3 mL dichloromethane, add the
rinse to the SPE cartridge, wash with 10 mL dichloromethane, elute with 5 mL dichloro-
methane.MeOH 95:5. Evaporate the eluate to dryness under a stream of nitrogen or air at 45,
reconstitute the residue in 1 mL MeOH:water 90:10, filter (Gelman Acrodisc CR PTFE 0.45
ixm), inject a 100 |xL aliquot. Milk. Condition a Sep-Pak silica gel SPE cartridge (No. 51900)
with 3 mL dichloromethane, do not allow it to go dry. Sonicate (ultrasonic cell disrupter) or
blend 40 mL milk and 160 mL MeOH for 30 s, let stand for 10-15 min, centrifuge at 2000 rpm
for 10 min. Decant the supernatant and add it to 60 mL 100 mg/mL NaCl, extract twice with
70 mL portions of dichloromethane. Combine the organic layers and evaporate them to dryness
under vacuum at 45, reconstitute the residue in 7 mL dichloromethane, add it to the SPE
cartridge at no more than 5 mL/min, rinse out the flask with 3 mL dichloromethane, add the
rinse to the SPE cartridge, wash with 10 mL dichloromethane, elute with 5 mL dichloro-
methane:MeOH 95:5. Evaporate the eluate to dryness under a stream of nitrogen or air at 45,
reconstitute the residue in 1 mL MeOH:water 90:10, filter (Gelman Acrodisc CR PTFE 0.45
ixm), inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Partisil 5 ODS-3 25 LC
Mobile phase: MeOH.water.acetic acid 94:6:0.1
Flow rate: 0.7
pumped at 0.7 mL/min and flowed through a 0.5 mm i.d. X 6 m stainless steel tube held at
98 to the detector. (The reagent was prepared by slowly adding 20 mL concentrated sulfuric
acid to 950 mL MeOH (Caution! Exothermic!), allow to cool to room temperature, add 30 g
vanillin while stirring.)
CHROMATOGRAM
Retention time: 8
Limit of quantitation: 25 ppb (tissue), 5 ppb (milk)
OTHER SUBSTANCES
Noninterfering: bacitracin, bambermycin, lasalocid, lincomycin, nicarbazin, tylosin
KEYWORDS
cow; muscle; liver; fat; kidney; post-column reaction; SPE
REFERENCE
Moran,J.W.; Turner,J.M.; Coleman,M.R. Determination of monensin in edible bovine tissues and milk by liquid
chromatography, JAOAC Int., 1995, 78, 668-673.
SAMPLE
Matrix: premix
Sample preparation: 5 g Feed premix + 75 mL MeOHrwater 90:10, let stand overnight with
periodic swirling, make up to 100 mL with MeOH:water 90:10, dilute an aliquot to a monensin
concentration of 2 mg/mL, inject a 40 fxL aliquot.
HPLCVARIABLES
Column: 300 X 4 Zorbax C8
Detector: RI
CHROMATOGRAM
Retention time: retention volume 12-14 mL
OTHER SUBSTANCES
Noninterfering: arprinocid, lasalocid, narasin, salinomycin
KEYWORDS
premix
REFERENCE
Macy,T.D.; Loh,A. High pressure liquid chromatographic determination of monensin in feed premixes,
SAMPLE
Matrix: solutions
Sample preparation: Condition a Mega Bond Elut silica gel SPE cartridge with benzene (Cau-
tion! Benzene is a carcinogen!). Evaporate a solution in MeOH to dryness, add 5 mL 5.28 mg/
mL 1-bromoacetylpyrene in MeCN, add 5 mL 1.28 mg/mL Kryptofix 222 in MeCN, heat at 50
for 1.5 h, cool. Either inject this solution directly or evaporate it to dryness, dissolve the residue
in 5 mL benzenerchloroform 50:50, rinse out the flask with two 5 mL portions of benzene:
chloroform 50:50, filter, add the filtrate to the SPE cartridge, elute with two 5 mL portions of
benzene-.acetone 70:30. Evaporate the eluate to dryness, reconstitute the residue in 10 mL
MeCN, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 (xm Develosil 5C18
Flow rate: 1
CHROMATOGRAM
Internal standard: 18,19-dihydrosalinomycin(25), 18,19-dihydro-20-ketosalinomycin(16.5)
OTHERSUBSTANCES
Simultaneous: lasalocid, narasin, salinomycin
KEYWORDS
derivatization; SPE
REFERENCE
Asukabe,H-; Murata,H.; Harada,K.-I.; Suzuki,M.; Oka,H-; Ikai,Y. Improvement of chemical analysis of antibi-
otics. XX. Basic study on high-performance liquid chromatographic determination of four polyether anti-
biotics pre-derivatized with 1-bromoacetylpyrene, J.Chromatogr.A, 1993, 657, 349-356.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Tissuemizer) 10 g tissue and 25 mL solvent for 1 min, wash
blades with 3-4 mL solvent, combine with homogenate, shake for 30 min, centrifuge at 800 g
for 15 min, decant. Add 25 mL solvent to residue, mix thoroughly, shake vigorously for 1 min,
centrifuge at 800 g for 15 min, add supernatants to a 75 X 20 column of 80-200 mesh alumina
(Fisher), rinse container onto column with 25 mL solvent, add 100 mL solvent to the column.
Combine all the eluates and add 100 mL 5% NaCl, shake vigorously, let stand 2-3 min, add 30
mL dichloromethane, shake vigorously for 30 s, repeat extraction twice. Combine the dichlo-
romethane layers and evaporate them to dryness under reduced pressure at 48-50, reconsti-
tute the residue in 1 mL solvent, add to a 75 X 20 column of 25-100 jxm Sephadex LH-20,
rinse flask with two 3.5 mL portions of solvent and add the rinses to the column, add 10 mL
solvent to the column, discard the first 18 mL of eluate, add 10 mL solvent to the column,
collect this fraction, evaporate to dryness under a stream of nitrogen at 48-50, reconstitute
the residue in 1 mL dichloromethane, evaporate to dryness under a stream of nitrogen at 48-
50, repeat twice, reconstitute in MeOH, evaporate to dryness under a stream of nitrogen at
48-50, reconstitute in 100 jxL pyridine and 100 JJLL acetic anhydride, let stand overnight at
room temperature, add to 35 mL water, rinse vial with 5 mL water. Combine the aqueous
layers and add 40 mL petroleum ether, shake vigorously for 1 min, separate and preserve the
organic layer. Add 40 mL anhydrous ethyl ether to the aqueous phase, shake for 1 min. Com-
bine the organic phases and wash with 20 mL 10% HCl, twice with 20 mL water, and with 40
mL saturated NaCl (shake for 1 min each time). Filter the organic layer through a glass fiber
filter containing 20-30 g anhydrous sodium sulfate, wash the sodium sulfate with 10-15 mL
petroleum ether, evaporate the filtrate to dryness under a stream of nitrogen at 48-50. Take
up the residue in 1 mL solvent and put it in another tube, wash flask into tube with three 2
mL portions of solvent, evaporate almost to dryness under a stream of nitrogen at 48-50, make
up to 1 mL with solvent, mix, add 500 |xL 9-anthryldiazomethane solution, let stand in the
dark for 30 min, evaporate to dryness under a stream of nitrogen at 48-50, reconstitute in 1
mL hexane. Condition a Baker-10 silica SPE cartridge with 5-10 mL hexane, do not allow to
dry. Add the hexane solution to the SPE cartridge, rinse tube with 9 mL hexane, add rinse to
the SPE cartridge. Wash SPE cartridge with 10 mL hexane.dichloromethane 50:50, with 10
mL hexane:dichloromethane 20:80, with 10 mL dichloromethane, and with 1 mL MeOH. Elute
with 1 mL MeOH, inject a 20 juiL aliquot of the eluate. (Solvent was MeOH.water 80:20. Prepare
9-anthryldiazomethane solution as follows. Add 1100 g manganous sulfate tetrahydrate in 1.5
L water and 1170 mL 40% NaOH over 1 h to a hot stirred solution of 960 g potassium per-
manganate in 6 L water, stir for 1 h, centrifuge, wash solid with water until washings are
colorless, dry solid at 100-120, grind the activated manganese dioxide to a fine powder. Add
8.5 g 85% hydrazine hydrate (Caution! Hydrazine hydrate is a carcinogen!) to 8.8 g 9-anthral-
dehyde dissolved in 150 mL EtOH, stir at room temperature for 3 h, filter off solid, dry under
vacuum, recrystallize from EtOH to give 9-anthraldehyde hydrazone as red-yellow crystals,
mp 124-6. Dissolve 220 mg 9-anthraldehyde hydrazone in 100 mL anhydrous ethyl ether, add
800 mg activated manganese dioxide, add 600 |xL EtOH saturated with KOH, stir vigorously
for 30 min, filter (glass fiber), wash solid with 20 mL anhydrous ethyl ether, evaporate to reduce
volume, make up to 100 mL with anhydrous ethyl ether, store in a dark flask in the dark in a
refrigerator. Discard after 30 days (J.Assoc.Off.Anal.Chem. 1985, 68, 1149).)
HPLC VARIABLES
Column: 200 X 4.6 5 jxrn RP-C8 (Hewlett-Packard)
Mobile phase: Gradient. A was MeCN. B was MeCN:water 10:90. A:B 20:80 for 9 min, 10:90 for
7 min, 20:80 for 1 min.
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: salinomycin, narasin
KEYWORDS
cow; liver; SPE; derivatization
REFERENCE
Martinez,E.E.; Shimoda,W. Liquid chromatographic determination of multiresidue fluorescent derivatives of
ionophore compounds, monensin, salinomycin, narasin, and lasalocid, in beef liver tissue,
SAMPLE
Matrix: tissue
Sample preparation: Blend 20 g ground or minced tissue with 150 mL MeOH:water 85:15 for
30 s or until homogeneous, centrifuge at 2500 rpm for 15 min. Remove the supernatant and
mix it with 40 mL 10% NaCl in water, extract three times with 25 mL portions of carbon
tetrachloride (Caution! Carbon tetrachloride is a carcinogen!), evaporate the extracts to dryness
under reduced pressure at ^40, reconstitute with 5 mL chloroform, add to a SepPak silica
SPE cartridge at 2 mL/min, rinse flask with 3 mL chloroform, add the rinse to the SPE car-
tridge, wash with 10 mL hexane.chloroform 10:90, elute with 10 mL chloroform.MeOH 95:5,
evaporate the eluate to dryness under reduced pressure or a stream of air at ^40, reconstitute
with 2 mL MeOH:water 90:10, filter (0.45 |xm), inject a 200 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeOH:water:acetic acid 94:6:0.1
Flow rate: 0.7
pumped at 0.7 mL/min and the mixture flowed through a 6.1 m X 0.5 mm ID stainless steel
coil at 98 to the detector. (Prepare reagent by cautiously adding 20 mL concentrated sulfuric
acid to 950 mL MeOH, allow to cool to room temperature, allow 30 g vanillin while stirring.)
CHROMATOGRAM
Retention time: 11
OTHER SUBSTANCES
Noninterfering: bacitracin, bambermycin, lasalocid, lincomycin, nicarbazin, tylosin
KEYWORDS
post-column reaction; SPE; chicken; muscle; liver; skin
REFERENCE
Moran,J.W.; Rodewald,J.M.; Donoho,A.L.; Coleman,M.R. Determination of monensin in chicken tissues by liq-
uid chromatography with postcolumn derivatization, JAOAC Int., 1994, 77, 885-890.
SAMPLE
Matrix: tissue
Sample preparation: Condition a 500 mg Bond Elut silica SPE cartridge with 5 mL isooctane:
ethyl acetate 90:10. Homogenize (Polytron) 5 g tissue with 10 mL iso-octane.ethyl acetate 90:
10 for 30 s, shake mechanically for 5 min, centrifuge at 1500 g for 5 min, remove the super-
natant, extract twice more with 10 mL portions of isooctane:ethyl acetate 90:10. Combine the
supernatants and dry them over 1 g anhydrous sodium sulfate, add to the SPE cartridge at 3
mL/min, wash with 10 mL dichloromethane, dry under vacuum for 30 s, elute with two 3 mL
portions of dichloromethane:MeOH 90:10. Evaporate the eluate to dryness under a stream of
nitrogen at 40, reconstitute the residue in 500 ^xL MeOH:water 90:10, vortex for 30 s, sonicate
for 5 min, inject a 100 |xL aliquot.
HPLCVARIABLES
Column: 250 X 3.2 5 fjim Inertsil ODS-2
Flow rate: 0.5
pumped at 0.3 mL/min and the mixture flowed through a 150 X 4.6 column packed with 75
|xm glass beads at 100 to the detector. (Prepare reagent by adding 2.5 mL sulfuric acid to 125
mL cold MeOH, add 9.5 g vanillin, prepare fresh each day.)
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: bacitracin, chlortetracycline, furazolidone, lasalocid, lincomycin, melengestrol
acetate, oxytetracycline, penicillin G, roxarsone, tetracycline, tilmicosin, tylosin, virginiamycin
KEYWORDS
post-column reaction; cow; chicken; muscle; fat; kidney; liver; SPE
REFERENCE
Gerhardt,G.C; Salisbury,C.D.C; Campbell,H-M. Determination of ionophores in the tissues of food animals by
liquid chromatography, Food Addit. Con tarn., 1995, 12, 731-737.
Montelukast
Molecular formula: C35H36CINO3S,
C35H35CINNaO3S (monosodium salt)
CAS Registry No.: 158966-92-8,151767-02-1 (monosodium salt)
Merck Index: 6340
SAMPLE
Matrix: blood
Sample preparation: Mix 200 |xL plasma with 40 |xL 5 |xg/mL IS and 400 |xL MeCN, vortex,
centrifuge. Inject an aliquot of the supernatant.
HPLC VARIABLES
Column: 50 X 4.6 Apex C18
Flow rate: 1.5
CHROMATOGRAM
Internal standard: montelukast analog containing a dimethylmethylene group instead cyclo-
propyl group
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Balani,..; Xu,X.; Pratha,V.; Koss,M.A.; Amin,R.D.; Dufresne,C; Miller,R.R.; Arison,B.H.; Doss,G.A.; Chiba,M.;
Freeman,A.; Holland,S.D.; Schwartz,J.L; Lasseter,K.C; Gertz,B.J.; Isenberg,J.L; Rogers,J.D.; Lin,J.H.;Bail-
lie,T.A. Metabolic profiles of montelukast sodium (Singulair), a potent cysteinyl leukotrienel receptor an-
tagonist, in human plasma and bile, Drug Metab.Dispos., 1997, 25, 1282-1287.
SAMPLE
Matrix: blood
Sample preparation: Mix 300 |xL plasma with 20 |xL 3 |xg/mL IS in MeOH:water 70:30 and
400 JJLL MeCN. Vortex, centrifuge at 10000 rpm for 10 min. Inject 60 |xL of the supernatant
onto column A, elute to waste with mobile phase A for 5 min, elute to waste for 5 min with
MeCNimobile phase A 12:88. Backflush the contents of column A onto column B with mobile
phase B, after 3.5 min remove column A from the circuit, elute column B with mobile phase
B, monitor the effluent from column B. (Wash column A with MeCN:mobile phase A 80:20 for
5 min then re-equilibrate with mobile phase A.)
HPLC VARIABLES
Column: A 10 X 3 5 |xm Chromspher 5 Biomatrix + 50 X 4.6 5 |xm Chromspher 5 Biomatrix; B
10 X 3 5 um Chiral AGP + 100 X 4 5 |xm Chiral AGP
Mobile phase: A MeOH:10 mM pH 3.6 ammonium acetate 10:100; B MeCN:10 mM pH 5.8 am-
monium acetate 32.5:100.
Flow rate: 1
CHROMATOGRAM
Internal standard: l-(((l(R)-(3-(2-(7-chloro-2-quinolinyl)-(E)-ethenyl)-phenyl) (3-(2-(l-hydroxy-
l-methylethyl)phenyl)propyl)thio)-methyl)-isopropane)acetate(30)
KEYWORDS
plasma; column-switching; chiral
REFERENCE
Liu,L.; Cheng,H.; Zhao,J.J.; Rogers,J.D. Determination of montelukast (MK-0476) and its S-enantiomer in
human plasma by stereoselective high-performance liquid chromatography with column-switching,
SAMPLE
Matrix: blood, bile
Sample preparation: Plasma. Deproteinize plasma with MeCN and evaporate under a stream
of nitrogen at 37. Reconstitute with MeCNrI mM pH 3.5 ammonium acetate 90:10. Inject an
aliquot. Bile. Centrifuge bile and inject an aliquot directly.
HPLC VARIABLES
Column: 250 X 4.6 Beckman C18
35:65 to 45:55 over 5 min, to 55:45 over 35 min, to 87:13 over 20 min, to 95:5 over 0.3 min
Flow rate: 1.1
Detector: MS, PE-SCIEX III tandem mass, turbo-ion spray interface, positive ion mode, m/z 586.
The column effluent mixed with MeCN: 1% trifluoroacetic acid in water 90:10 pumped at 50-
100 |xL/min and the mixture flowed to the detector.
OTHER SUBSTANCES
KEYWORDS
plasma; pharmacokinetics; radiolabeled
REFERENCE
Balani,S.K; Xu,X.; Pratha,V.; Koss,M.A.; Amin,R.D.; Dufresne,C.; Miller,R.R.; Arison,B.H.; Doss,G.A.; Chiba,M.;
Freeman A ; Holland,S.D.; Schwartz,J.L; Lasseter,K.C; Gertz,B.J.; Isenberg,J.L; Rogers,J.D.; Lin,J.H.;Bail-
SAMPLE
Matrix: blood, bile
Sample preparation: Plasma. Deproteinize plasma with MeCN. Evaporate under a stream of
nitrogen at 37. Reconstitute the residue with MeCNrI mM pH 3.5 ammonium acetate 90:10.
Inject an aliquot. Bile. Centrifuge bile and inject an aliquot directly.
HPLC VARIABLES
Column: 250 X 4.6 Beckman C18
35:65 to 45:55 over 5 min, to 55:45 over 35 min, to 87:13 over 20 min, to 95:5 over 0.3 min
Flow rate: 1.1
Detector: Radioactivity, Ramona 5 (Raytest) following post-column reaction. The column effluent
mixed with Packard Flow -Scint III cocktail pumped at 2.2 mL/min and this mixture flowed
to the detector.
CHROMATOGRAM
Internal standard: montelukast analog containing dimethylmethylene group instead cyclopro-
pyl group
OTHER SUBSTANCES
KEYWORDS
plasma; pharmacokinetics; radiolabeled
REFERENCE
Balani,S.K; Xu,X.; Pratha,V.; Koss,M.A.; Amin,R.D.; Dufresne,C; Miller,R.R.; Arison,B.H.; Doss,G.A; Chiba,M.;
Freeman,A; Holland,S.D.; Schwartz,J.I.; Lasseter,K.C; Gertz,B. J.; Isenberg,J.L; Rogers,J.D.; Lin,J.H.; Bail-
Moperone
Merck Index: 6343
SAMPLE
Matrix: blood
the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 jxL aliquot of
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 247
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
Moprolol
CAS Registry No.: 5741-22-0, 27058-84-0 (HCI),
77164-20-6 (l-form), 113482-87-4 (l-form HCI)
Merck Index: 6345
Lednicer No.: 2 109
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond Elut CN SPE cartridge with 1 volume dichloro-
methane, 1 volume MeOH, and 1 volume pH 10 carbonate buffer, do not allow to go dry. 1 mL
Plasma + 10 JJLL 100 ng/mL IS in water + 500 |JLL pH 10 carbonate buffer, mix, add to the SPE
cartridge, wash with two volumes of pH 10 carbonate buffer, centrifuge at 4000 rpm for 15 s,
elute with two volumes of dichloromethane while centrifuging at 1000 rpm for 3 min. Evaporate
the eluate to dryness under a stream of nitrogen at room temperature, reconstitute the residue
in 1 mL diethyl ether, add 250 yJb trifluoroacetic anhydride, let stand at room temperature for
45 min, evaporate to dryness under a stream of nitrogen, reconstitute with 500 jxL mobile
HPLC VARIABLES
Column: 250 X 4 5 |xm LiChrosorb CN
Mobile phase: n-Pentane:diethyl ether 55:45
Flow rate: 1
Detector: UV 223
CHROMATOGRAM
Retention time: 4.7
Internal standard: l-(3-chloroisoxazol-5-yl)-2-(tert-butylamino)ethanol (Zambon Group, Milan)
(5)
KEYWORDS
derivatization; SPE; plasma; an aliquot of the effluent can be further analyzed by GC using an
electron capture detector
REFERENCE
Gianesello,V.; Brenn,E.; Figini,G.; Gazzaniga,A. Determination by coupled high-performance liquid chromatog-
raphy-gas chromatography of the |3-blocker levomoprolol in plasma following ophthalmic administration,
J.Chromatogr., 1989, 473, 343-352.
Morantel
Merck Index: 6348
Lednicer No.: 1 266
SAMPLE
Matrix: feed
Sample preparation: 5 g Ground feed + 100 mL methanolic HCl, shake on a reciprocating
shaker for 30 min. Remove a 40 mL aliquot and centrifuge at 2000 rpm for 10 min. Remove 5
mL of the supernatant and make up to 100 mL with MeCN, mix, centrifuge at 2000 rpm for
10 min, inject an aliquot of the supernatant. (Prepare methanolic HCl by slowly adding 8.5
mL concentrated HCl to 992 mL MeOH:water 50:50.)
HPLCVARIABLES
Guard column: 400 mg Corasil II
Mobile phase: MeCN:acetic acid:water:diethylamine 95:2:2:1
Flow rate: 1.6
Detector: UV 313
CHROMATOGRAM
Retention time: 7.3
OTHER SUBSTANCES
Simultaneous: cis-isomer, degradation products, pyrantel
KEYWORDS
protect from light
REFERENCE
Goras,J.T.; Gauthier,A.R. Liquid chromatographic determination of morantel tartrate in cattle feed,
SAMPLE
Matrix: milk
Sample preparation: 20 mL Milk + 1.5 mL HCl + 1 mL 37.4 ng/mL pyrantel in water, heat at
95 with stirring for 2 h, cool in an ice bath, basify with 2.5 mL 12 M KOH, add 25 mL toluene,
shake, centrifuge, repeat extraction. Combine the toluene layers and extract them vigorously
twice with 4 mL portions of 100 mM HCl. Combine the aqueous layers and basify them with
2.5 mL 12 M KOH, heat at 95 for 5 h, acidify with 3 mL HCl, cool in an ice bath, extract twice
with 5 mL portions of chloroform. Combine the organic layers, add 500 (xL 100 mM NaOH,
mix, centrifuge. Remove the aqueous layer and place it on a vortex evaporator for 5 min to
remove residual chloroform, inject an aliquot. (Prepare 12 M KOH by cautiously dissolving 790
g KOH pellets in enough water to make 1 L in a fume hood, use an ice bath.)
HPLC VARIABLES
Guard column: 30 mm long |xC18 Corasil Bondapak
Column: 100 X 5 Radial-Pak A (Waters)
Mobile phase: MeOH:MeCN:water:acetic acid 40:10:49:1
Flow rate: 1
Detector: UV 313
CHROMATOGRAM
Retention time: 6.6 (as 3-(3-methyl-2-thienyl)-acrylic acid, the hydrolysis product)
Internal standard: pyrantel (as 3-(2-thienyl)acrylic acid, the hydrolysis product) (4.2)
KEYWORDS
protect from light
REFERENCE
Lynch,M.J.; Mosher,F.R.; Brunner,L.A.; Bartolucci,S.R. Liquid chromatographic determination and identifica-
tion of morantel-related residues as precursors of 3-(3-methyl-2-thienyl) acrylic acid (CP-20,107) in bovine
milk, J.Assoc.Off.Anal.Chem., 1986, 69, 931-935.
Morazone
Merck Index: 6349
Lednicer No.: 2 261
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |jig/mL solution in MeOH, inject a 20 |JLL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.5
OTHER SUBSTANCES
lol, mianserin, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine,
REFERENCE
Moricizine
Merck Index: 6351
Lednicer No.: 4 200
SAMPLE
Sample preparation: 1 mL Bile, plasma, or urine + 20 |xL 40 (xg/mL clozapine in EtOH + 200
JXL 2.5 mM pH 6.0 sodium pentanesulfonate, mix for 3 s, add 6 mL diethyl ether, shake vig-
orously for 2 min, centrifuge at 2500 g for 5 min. Remove the organic layer and evaporate it
to dryness under a stream of nitrogen at 40, reconstitute the residue in 100 |xl mobile phase,
vortex for 30 s, inject a 25 JJLL aliquot.
HPLC VARIABLES
Guard column: 25 X 4.6 30 |xm C18 (Merck)
Column: 100 X 8 10 urn ^Bondapak C18
Mobile phase: MeOH:water:triethylamine 65:35:0.5, adjust pH to 6-7 with glacial acetic acid
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 6.2
Internal standard: clozapine (9.7)
OTHER SUBSTANCES
KEYWORDS
plasma; rat
REFERENCE
Yang,J.M.; Chan,K. Simultaneous determination of moricizine and its sulphoxidation metabolites in biological
fluids by high-performance liquid chromatography, J.Chromatogr.B, 1995, 663, 172-176.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL 200 mM NaOH + 5 mL 100 mM pH 9.0 boric acid/
KCl/NaOH buffer + 10 mL dichloromethane, shake for 30 min, centrifuge. Remove the organic
layer and wash it with 5 mL water, evaporate the organic layer to dryness under a stream of
nitrogen, reconstitute the residue in 200 u,L mobile phase, inject an aliquot.
HPLCVARIABLES
Column: 10 u,m uPorasil
Mobile phase: Hexane:THF:MeOH:water 60:27:6.3:0.7
Flow rate: 2
Detector: UV 268
CHROMATOGRAM
Retention time: 4.6
KEYWORDS
serum; normal phase; pharmacokinetics
REFERENCE
Rice,P.J.; LeClairJ.O.; Stone,W.L.; Mehta,A.V. Pharmacokinetics of moricizine in young patients,
J.Clin.PharmacoL, 1995, 35, 1016-1019.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN.buffer 42:58 (Buffer was 5 JAM sodium octanesulfonate, 0.2% glacial acetic
acid, and 0.1% triethylamine.)
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Retention time: 5
Internal standard: N-butyl-p-aminobenzoate (7)
OTHER SUBSTANCES
KEY WORDS
buffer; protect from light
REFERENCE
King,S.-Y.R; Sigvardson,K.W.; Dudzinski,J.; Tbrosian,G. Degradation kinetics and mechanisms of moricizine
hydrochloride in acidic medium, J.Pharm.ScL, 1992, 81, 586-591.
Moroxydine
Merck Index: 6354
SAMPLE
HPLC VARIABLES
Next Page
Column: 250 X 4.6 5 Jim Symmetry C8 (Waters)

mL/min)
Detector: UV 236.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Morphine
CAS Registry No.: 57-27-2, 52-26-6 (HCI), 630-81-9 (HBr),
64-13-3 (sulfate)
Merck Index: 6359
Lednicer No.: 1 286
SAMPLE
Matrix: bile, blood
Sample preparation: 0.5 mL Blood or bile + 10 (blood) or 15 (bile) fxL 100 |xg/mL nalorphine in
MeOH + 300 jxL 1.1 M pH 5.0 sodium acetate buffer + 3000-3500 U of Patella vulgata glucu-
ronidase, incubate at 55 overnight, add 0.5 mL borate buffer to achieve a pH of 8.3-8.5. Add
8 mL chlorofornv.isopropanol 90:10, gently rotate for 30 min, centrifuge at 3500 rpm for 10
min, remove aqueous layer. Wash organic layer (twice for blood, three times for bile) with 3
mL 100 mM pH 9.9 sodium phosphate buffer with gentle rotation for 10 min and centrifugation
each time. Add organic layer to 200 (blood) or 400 (bile) |JLL 0.2% phosphoric acid, gently rotate
for 30 min, discard organic layer, inject 50 |xL of the acid layer. (Borate buffer was 50 mM boric
acid and 43 mM sodium tetraborate, adjusted to pH 9.8.)
HPLC VARIABLES
Guard column: Nova-Pak phenyl guard column
Flow rate: 1.2
Detector: UV 210 and F ex 220 em 370 (cut-off)
CHROMATOGRAM
Retention time: 5.4
Limit of detection: 200 ng/mL (bile), 100 ng/mL (blood)
OTHER SUBSTANCES
Simultaneous: hydrocodone, dihydrocodeine, codeine, oxycodone, 6-monoacetylmorphine
Previous Page
Column: 250 X 4.6 5 Jim Symmetry C8 (Waters)

mL/min)
Detector: UV 236.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Morphine
CAS Registry No.: 57-27-2, 52-26-6 (HCI), 630-81-9 (HBr),
64-13-3 (sulfate)
Merck Index: 6359
Lednicer No.: 1 286
SAMPLE
Matrix: bile, blood
Sample preparation: 0.5 mL Blood or bile + 10 (blood) or 15 (bile) fxL 100 |xg/mL nalorphine in
MeOH + 300 jxL 1.1 M pH 5.0 sodium acetate buffer + 3000-3500 U of Patella vulgata glucu-
ronidase, incubate at 55 overnight, add 0.5 mL borate buffer to achieve a pH of 8.3-8.5. Add
8 mL chlorofornv.isopropanol 90:10, gently rotate for 30 min, centrifuge at 3500 rpm for 10
min, remove aqueous layer. Wash organic layer (twice for blood, three times for bile) with 3
mL 100 mM pH 9.9 sodium phosphate buffer with gentle rotation for 10 min and centrifugation
each time. Add organic layer to 200 (blood) or 400 (bile) |JLL 0.2% phosphoric acid, gently rotate
for 30 min, discard organic layer, inject 50 |xL of the acid layer. (Borate buffer was 50 mM boric
acid and 43 mM sodium tetraborate, adjusted to pH 9.8.)
HPLC VARIABLES
Flow rate: 1.2
CHROMATOGRAM
Retention time: 5.4
Limit of detection: 200 ng/mL (bile), 100 ng/mL (blood)
OTHER SUBSTANCES
Simultaneous: hydrocodone, dihydrocodeine, codeine, oxycodone, 6-monoacetylmorphine
Noninterfering: acetylcodeine, amitriptyline, amphetamine, diamorphine, diazepam, dothiepin,
doxepin, ephedrine, ephedrine, hydromorphone, mesoridazine, methadone, methamphetamine,
3-monoacetylmorphine, nordiazepam, norpropoxyphene, nortriptyline, oxazepam, propoxy-
phene, pseudoephedrine, quinidine, quinine, sulfamethoxazole, sulforidazine, thioridazine
KEYWORDS
UV and F detection used together
REFERENCE
Crump,K.L.; MclntyreJ.M.; Drummer,O.H. Simultaneous determination of morphine and codeine in blood and
bile using dual ultraviolet andfluorescencehigh-performance liquid chromatography, J.Anal.Toxicol., 1994,
18, 208-212.
SAMPLE
Sample preparation: 250 jxL Bile, 3 mL blood, or 5 mL tissue homogenate + 1 mL 200 jxg/mL
nalorphine in water + 2 mL 200 mM pH 8.9 sodium borate buffer + 5 (bile) or 10 (blood, tissue)
mL chloroformdsopropanol 90:10, rotate gently for 20 min, centrifuge at 2000 rpm for 10 min.
Remove the organic layer and add it to 2 mL 500 mM HCl, rotate for 20 min, centrifuge for 5
min. Remove 1.8 mL of the upper aqueous phase, adjust to pH 8.6 0.2 by carefully adding
powdered ammonium carbonate until the solution was saturated, add 5 mL ethyl acetate:
isopropanol 90:10, rotate for 20 min, centrifuge for 5 min. Remove 4.8 mL of the upper organic
in 50 (xL MeOH, vortex for 30 s, inject a 20 uL aliquot.
HPLC VARIABLES
Guard column: 30 X 4.6 5 |xm RP-18 Spheri-5
Column: 250 X 4.6 5 fim Ultrasphere ODS
Mobile phase: MeOH:50 mM pH 7 phosphate buffer 40:60 Place a 70 X 2 30-38 |xm Co-Pell ODS
Flow rate: 2
CHROMATOGRAM
Retention time: 4.2
OTHER SUBSTANCES
Extracted: codeine, hydromorphone, norcodeine, normorphine
secobarbital
REFERENCE
Hepler,B.R.; Sutheimer,C; Sunshine,I.; Sebrosky,G.F. Combined enzyme immunoassay-LCEC method for the
identification, confirmation, and quantitation of opiates in biological fluids, J.Anal.Toxicol., 1984,8, 78-90.
SAMPLE
Matrix: bile, perfusate
Sample preparation: Dilute bile 100-fold with water. Dilute venous perfusate 1:4 with water.
Inject an aliquot.
HPLC VARIABLES
Column: 300 mm long 10 (xm Econosil C18
Mobile phase: MeCN:0.1% trifluoroacetic acid 4:96, adjusted to pH 2.4 with 1 M NaOH
Flow rate: 0.8
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
KEYWORDS
liver; pig; pharmacokinetics
REFERENCE
Milne,R.W.; Jensen,R.H.; Larsen,C; Evans,A.M.; Nation,R.L. Comparison of the disposition of hepatically-gen-
erated morphine-3-glucuronide and morphine-6-glucuronide in isolated perfused liver from the guinea pig,
Pharm.Res., 1997, 14, 1014-1018.
SAMPLE
Matrix: blood
Sample preparation: Condition a 500 mg Bond Elut LRC C2 SPE cartridge with 2 mL MeOH,
2 mL MeCN:water:trifluoroacetic acid 80:20:0.1, and 5 mL Tris buffer. Mix 100 |xL 100 ng/mL
IS with 2 mL 50 mM Tris buffer adjusted to pH 7.5 with concentrated HCl and 500 |xL plasma.
Add sample to the SPE cartridge and draw through by vacuum at 2 mL/min. Wash with 20
mL Tris buffer, dry with vacuum for 1 min. Elute with 2 mL MeCN:water:trifluoroacetic acid
80:20:0.1, evaporate the eluate to dryness under a stream of nitrogen at 40. Reconstitute the
residue in 1 mL MeOH, vortex for 40 s, centrifuge at 3000 rpm for 15 min, decant the super-
natant, evaporate to dryness. Reconstitute the residue in 200 |xL mobile phase, vortex for 20
s. Inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 4 |xm Nova-Pak phenyl Guard-Pak
Column: two 75 X 3.9 4 |xm Nova-Pak phenyl columns in series
Mobile phase: MeOH: 10 mM potassium phosphate monobasic 10:90, adjusted to pH 4.0 with
85% phosphoric acid
Flow rate: 1
Detector: F ex 210 em 335 or E, ESA Coulochem II, Model 5200, Model 5011 high-sensitivity
analytical cell, cell 1 -300 mV, cell 2 -450 mV, Model 5020 guard cell -750 mV
CHROMATOGRAM
Internal standard: noroxymorphone (12)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Rotshteyn,Y.; Weingarten,B. A highly sensitive assay for the simultaneous determination of morphine, mor-
phine-3-glucuronide, and morphine-6-glucuronide in human plasma by high-performance liquid chromatog-
raphy with electrochemical and fluorescence detection, Ther.Drug Monti., 1996, 18, 179-188.
SAMPLE
Matrix: blood
Sample preparation: Activate a Sep-Pak SPE cartridge by rinsing successively with 10 mL
MeOH, 5 mL MeCNiIO mM pH 2.1 NaH2PO4 buffer 25:75, and 5 mL water. 100 uL Plasma +
3 mL 500 mM pH 3 sodium bicarbonate/sodium carbonate buffer, mix, add to the SPE cartridge.
Wash with 20 mL 5 mM pH 9.3 sodium bicarbonate/sodium carbonate buffer, 0.5 mL water,
and 0.35 mL MeCN:10mM pH 2.1 NaH2PO4 buffer 25:75, elute with an additional 0.8 mL of
MeCN:10 mM pH 2.1 NaH2PO4 buffer 25:75, inject an aliquot of the eluate.
HPLC VARIABLES
Guard column: 15 X 3.2 C18
Column: 150 X 4.6 C18
Mobile phase: MeCN:0.8 mM 1-dodecylsulfate sodium in 10 mM pH 2.1 NaH2PO4 buffer 26.5:
73.5
Flow rate: 0.8
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: metabolites, morphine-3-P-D-glucuronide, morphine-6--D-glucuronide
KEYWORDS
REFERENCE
Wu,D.; Kang,Y.-S.; Bickel,U.; Pardridge,.M. Blood-brain barrier permeability to morphine-6-glucuronide is
markedly reduced compared with morphine, Drug Metab.Dispos., 1997, 25, 768-771.
SAMPLE
Matrix: blood
Sample preparation: Activate a Sep-Pak Light C18 SPE cartridge with 3 mL MeOH and 3 mL
water. Mix 1 mL serum with 1 mL 500 mM pH 9.3 potassium carbonate. Add to the SPE
cartridge, wash with 5 mL 5 mM pH 9.3 potassium carbonate and 250 |xL water. Dry the SPE
cartridge with air for 30 s, wash with 200 |xL MeCN:30 mM pH 2 potassium phosphate buffer
16:84. Elute with 600 fiL MeCN:30 mM pH 2 potassium phosphate buffer 16:84. Dilute the
eluate with an equal volume of water, inject a 100 (JLL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 YMC ODS-AL
Mobile phase: Gradient. A. 3 mM formic acid in MeCN. B. 3 mM formic acid in water. A:B from
96:4 to 30:70 over 3.5 min (sic).
Flow rate: 1
Detector: MS, Fisons VG Platform, electrospray, spray voltage 2.4 kV, sampling cone voltage 40
V, SIM m/z 286.2
CHROMATOGRAM
Retention time: 3.8
OTHER SUBSTANCES
Extracted: metabolites (m/z 462.2)
KEYWORDS
serum; SPE
REFERENCE
Tyrefors,N.; Hyllbrant,B.; Ekman,L.; Johansson,M.; Langstrom,B. Determination of morphine, morphine-3-glu-
curonide and morphine-6-glucuronide in human serum by solid-phase extraction and liquid chromatogra-
phy-mass spectrometry with electrospray ionisation, J.ChromatognA, 1996, 729, 279-285.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg ethyl SPE cartridge (J.T.Baker) with 2 mL
MeOH, 1 mL water, and 2 mL 1 mM pH 9.3 ammonium hydrogen carbonate buffer. Mix 1 mL
serum with 200 |xL 1 |xg/mL IS in water. Add to the SPE cartridge, wash with 1 mL 1 mM pH
9.3 ammonium hydrogen carbonate buffer, elute with 1 mL MeOH. Evaporate the eluate to
dryness, reconstitute the residue in 100 |xL mobile phase, inject a 5 jxL aliquot.
HPLC VARIABLES
Column: 250 X 2.1 5 |xm Supelcosil LC-Si (Supelco)
Mobile phase: MeCN:MeOH:water:formic acid 5.2:59.8:34.65:0.35
Flow rate: 0.23
Injection volume: 5
Detector: MS, API I MS single quadrupole, ionspray, capillary tip 5000 V, interface plate 650 V,
source 60, positive ion mode, SIM, m/z 286
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: metabolites, codeine, diamorphine
KEYWORDS
serum; pharmacokinetics; SPE; mouse
REFERENCE
Zuccaro,R; Ricciarello,R.; Pichini,S.; Pacifici,R.; Altieri,L; Pellegrini,M.; D'Ascenzo,G. Simultaneous determi-
nation of heroin, 6-monoacetylmorphine, morphine, and its glucuronides by liquid chromatography-atmo-
spheric pressure ionspray-mass spectrometry, JAnaLToxicoL, 1997, 21, 268-277.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Whole blood + 1 mL 1 M pH 9 borate buffer + 100 (xL 1 jxg/mL
nalorphine + 5 mL ethyl acetate, vortex thoroughly, centrifuge, repeat the extraction. Combine
the extracts and add them to 4 mL 50 mM sulfuric acid, extract. Discard the organic layer and
saturate the aqueous layer with freshly ground ammonium carbonate, extract with 8 mL ethyl
acetate, filter (phase-separating paper). Evaporate the organic layer and to dryness under a
stream of nitrogen at 50, reconstitute the residue in 100 |xL mobile phase, inject a 20 jxL
aliquot.
HPLC VARIABLES
Column: 250 X 5 ODS Hypersil
Mobile phase: MeCNrIO mM KH2PO4 20:80 containing 20 mM octanesulfonic acid, adjusted to
Detector: E, BAS LC-4B, TL-5A thin-layer flow cell +0.870 V
CHROMATOGRAM
Retention time: 9
Internal standard: nalorphine (19)
KEYWORDS
whole blood
REFERENCE
Logan,B.K.; Oliver,J.S.; Smith,H. The measurement and interpretation of morphine in blood, Forensic Sci.Int,
1987, 35, 189-195.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut SPE cartridge with 1 mL MeOH, 1 mL water, and
1 mL 100 mM pH 9.0 sodium bicarbonate buffer, do not allow to go dry. 100 JJLL Plasma + 20
(xL 1 |xg/mL nalorphine in MeOH:water 30:70 + 750 |xL 100 mM pH 9.0 sodium bicarbonate
buffer, vortex for 10 s, add to the SPE cartridge, wash with 1 mL 100 mM pH 9.0 sodium
bicarbonate buffer, wash with 1 mL water, wash with 100 |xL MeOH:water 50:50, dry under
vacuum for 10 min, elute with three 250 |xL aliquots of MeOH. Evaporate the eluate to dryness
at 45 in a vacuum centrifuge for 1 h, reconstitute with 25 |xL 100 mM pH 11.4 sodium car-
bonate or 25 |xL pH 9.5 sodium bicarbonate, add 25 |xL 1 mg/mL dansyl chloride in acetone,
vortex, let stand in the dark at 45 for 20 min, add 250 |xL toluene, vortex for 2 min, centrifuge
at 12500 g for 1 min, inject a 100-200 |xL aliquot of the upper organic layer.
HPLCVARIABLES
Guard column: 10 X 4.6 3 |xm silica
Column: 150 X 4.6 3 |xm Spherisorb 3CN
Mobile phase: n-Hexane:isopropanol:ammonia 95:5:0.25 (Place a silica column between the
pump and the injector.)
Flow rate: 1.5
CHROMATOGRAM
Retention time: 5.7
OTHER SUBSTANCES
Extracted: 6-acetylmorphine
KEYWORDS
plasma; derivatization; pharmacokinetics; SPE; normal phase
REFERENCE
Barrett,D.A.; Shaw,P.N.; Davis,S.S. Determination of morphine and 6-acetylmorphine in plasma by high-per-
SAMPLE
Matrix: blood
3 mL MeCN: 10 mM NaH2PO4 10:90, and 5 mL water. 1 mL Plasma + 0.5 |xg nalorphine in
water + 1 mL 500 mM ammonium sulfate, add to the SPE cartridge, wash with 6 mL 5 mM
ammonium sulfate, elute with 1 mL MeCN: 10 mM NaH2PO4 10:90, inject a 100 |JLL aliquot of
the eluate.
HPLC VARIABLES
Column: 300 X 3.9 10 fim jxBondapak C18
Mobile phase: MeCN:buffer 26:74 (Buffer was 10 mM NaH2PO4 containing 1 mM sodium dodecyl
sulfate, pH adjusted to 2.1 with orthophosphoric acid.)
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: metabolites, glucuronides, normorphine
KEYWORDS
REFERENCE
Glare,RA.; Walsh,T.D.; Pippenger,C.E. A simple, rapid method for the simultaneous determination of morphine
and its principal metabolites in plasma using high-performance liquid chromatography and fluorometric
detection, Ther.Drug Monit., 1991, 13, 226-232.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Baxter C18 SPE cartridge with 3 mL MeOH and 3 mL
water. 1 mL Plasma + 2 mL 500 mM pH 9.3 ammonium sulfate + 30 |xL 1 |xg/mL naltrexone,
add to the SPE cartridge, wash with 3 mL 5 mM pH 9.3 ammonium sulfate, wash with 3
mL water, dry under vacuum, elute with 1 mL MeOHitriethylamine 99.5:0.5. Evaporate the
eluate to dryness under a stream of nitrogen at room temperature, reconstitute the residue in
100 |xL mobile phase, inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 100 X 3.2 5 (Jim Spherisorb C8
Mobile phase: MeOH:50 mM Na2HPO4 15:85 containing 3 mM 1-heptanesulfonic acid, pH ad-
Flow rate: 0.8
(monitored)
CHROMATOGRAM
Retention time: 5.2
OTHER SUBSTANCES
Extracted: hydromorphone
KEYWORDS
plasma; SPE
REFERENCE
Bouquillon,A.I.; Freeman,D.; Moulin,D.E. Simultaneous solid-phase extraction and chromatographic analysis
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 40 |xm bonded silica Clean Screen SPE cartridge (World-
wide monitoring) with 3 mL MeOH, 3 mL water, and 1 mL pH 3 phosphate buffer. 1 mL Plasma
+ 2 mL 10 mM phosphoric acid, mix, add to the SPE cartridge, air dry for 30 s, wash with 1
mL pH 3 phosphate buffer, wash with 1 mL MeOH, air dry for 30 s, elute with 3 mL 2%
ammoniacal MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 45, recon-
stitute the residue in 50 |xL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 200 X 4.5 5 jjim LiChrosphere diol
Mobile phase: MeCN:50 mM NaH2PO4 80:20 pH adjusted to 3 with orthophosphoric acid
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Extracted: metabolites, glucuronides, normorphine, codeine
KEYWORDS
plasma; SPE
REFERENCE
Wielbo,D.; Bhat,R-> Chari,G.; Vidyasagar,D.; Tebbett,I.R.; Gulati,A. High-performance liquid chromatographic
determination of morphine and its metabolites in plasma using diode-array detection, J.Chromatogr., 1993,
615, 164-168.
SAMPLE
Matrix: blood
stoppered tube for 1 min, add 6 mL NiCl2, rock for 5 min, add 15 mL dichloromethane:isobutyl
mL porcelain dish at a moderate temperature in a sand bath. Take up residue in 500 (xL MeCN:
water.)
HPLC VARIABLES
min
Detector: UV 230
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Extracted: buprenorphine, caffeine, cocaine, codeine, diamorphine, ethylmorphine, lidocaine,
methaqualone, naloxone, noscapine, papaverine, pentazocine, procaine
KEYWORDS
whole blood
REFERENCE
by high performance liquid chromatography, Chromatographia, 1994, 38, 617-623.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 200 \xL ethyl p-hydroxybenzoate in MeOH, stir thor-
oughly, centrifuge at 15000 rpm for 10 min, inject a 20 [LL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 X 4 5 |xm LiChrospher 100RP-8(e)
Mobile phase: MeCN:buffer 23:77 (Buffer was 50 mM NaH2PO4 containing 5 mM sodium do-
decylsulfonate, pH adjusted to 2.1 with phosphoric acid.)
Flow rate: 1
Detector: E, Shimadzu L-ECD-6A, +0.80 V
CHROMATOGRAM
Internal standard: ethyl p-hydroxybenzoate (F ex 280 em 330)
OTHER SUBSTANCES
Extracted: metabolites, glucuronides (F ex 280 em 330)
KEYWORDS
REFERENCE
Matsuzawa,T.; Wada,Y.; Shimoyama,M.; Nakajima,K; Seki,T.; Sugibayashi,K.; Morimoto,Y. The effect of dif-
ferent routes of administration on the metabolism of morphine: The disposition of morphine and its me-
tabolites after topical application, Biopharm.Drug Dispos., 1994, 15, 665-678.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 50 mg Bond Elut C8 SPE cartridge with 1 mL MeOH
and 1 mL water. Add 200 |JLL buffer to the SPE cartridge, add 200 |xL serum, add 200 jxL buffer,
wash with 1 mL buffer, elute with two 150 (xL portions of MeOH. Evaporate the eluate to
dryness under a stream of nitrogen, reconstitute the residue in 50 jxL mobile phase or water,
inject a 20 |xL aliquot. (Buffer was 1 mM pH 9 ammonium bicarbonate.)
HPLCVARIABLES
Mobile phase: Gradient. MeCN:25 mM tetraethylammonium phosphate buffer 1:99 for 8 min,
3:97 for 15 min (step gradient).
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
serum; SPE
REFERENCE
Aderjan,R.; Hofmann,S.; Schmitt,G.; Skopp,G. Morphine and morphine glucuronides in serum of heroin con-
sumers and in heroin-related deaths determined by HPLC with native fluorescence detection,
JAnaLToxicoL, 1995, 29, 163-168.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Baker CN SPE cartridge with 2 column volumes of
MeOH, 2 column volumes of water, and 1 mL 500 mM pH 9.3 diammonium sulfate. 600 |xL
Serum + 500 |xL 500 mM pH 9.3 diammonium sulfate buffer, mix, add to the SPE cartridge,
wash with 2 mL 50 mM pH 9.3 diammonium sulfate buffer, elute with 2 mL chloroformdso-
propanol 90:10. Evaporate the eluate to dryness under a stream of nitrogen at 37, reconstitute
the residue in 200 (xL water, inject a 20 jxL aliquot.
HPLC VARIABLES
Column: Cp-Sper C8 (Chrompack)
Mobile phase: MeCN:10 mM pH 2.1 KH2PO4 11:89 containing 0.4 g/L heptanesulfonic acid
Flow rate: 2
Detector: E, ESA, model 5010 analytical cell, detector 1 0.3 V, detector 2 0.4 V (monitored)
CHROMATOGRAM
Retention time: 3.5
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Koopman-Kimenai,P.M.; Vree,T.B.; Booij,L.H.D.J.; Hasenbros,M.A.W.M. Pharmacokinetics of epidurally admin-
istered nicomorphine with its metabolites and glucuronide conjugates in patients undergoing pulmonary
surgery during combined epidural local anaesthetic block and general anaesthesia, Biopharm.Drug Dispos.,
1995, 16, 507-520.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 50 |xL 50 |xg/mL nalorphine hydrochloride, add to a Sep-
Pak tC18 SPE cartridge, wash with 2 mL water, wash with 3 mL acetone:water 5:95, elute
with 4 mL MeOH.water 70:30. Evaporate the eluate to dryness under a stream of nitrogen at
60, reconstitute the residue in 200 |xL mobile phase, inject a 50 |JLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 TSK-gel ODS-120T (Tosoh)
sulfate, adjusted to pH 2.1 with phosphoric acid.)
Flow rate: 1
Detector: E, Shimadzu L-ECD-6A, 0.8 V
CHROMATOGRAM
Internal standard: nalorphine
Limit of detection: 0.5 ng/mL (1 mL plasma)
KEYWORDS
plasma; rabbit; SPE; pharmacokinetics
REFERENCE
Matsumoto,Y.; Yamamoto,L; Watanabe,Y.; Matsumoto,M. Enhancing effect of viscous sodium hyaluronate so-
lution on the rectal absorption of morphine, Biol.Pharm.Bull., 1995, 18, 1744-1749.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg ethyl SPE cartridge (J.T.Baker) with 2 volumes
of MeOH, 1 volume of water, and 2 volumes of 10 mM pH 9.3 ammonium hydrogen carbonate
buffer. 1 mL Serum + 100 |xL 1 jig/mL IS in water, add to the SPE cartridge, wash with 1
volume of 10 mM ammonium hydrogen carbonate buffer, elute with 1 volume of MeOH. Evap-
orate the eluate to dryness under a stream of nitrogen, reconstitute the residue in 100 (JLL
mobile phase, inject a 20 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil ABZ
Mobile phase: Gradient. MeOH:water from 15:85 to 60:40 over 10 min. (Convex gradient where
MeOH% = -0.46exp(-x/1.18) + 0.6 where X = time in min.)
Flow rate: 0.8 (0.018 mL/min entered MS)
Detector: MS, Fisons TRIO 2, electrospray, capillary tip 2.97 kV, counter electrode 390 V, sam-
pling cone voltages 66 V, -106 V, -17 V, source 60, SIM m/z 286
CHROMATOGRAM
Internal standard: codeine (7.03 min, m/z 300), naltrexone (7.27 min, m/z 342)
OTHER SUBSTANCES
KEY WORDS
serum; human; mouse; SPE; pharmacokinetics; LC-MS
REFERENCE
Pacifici,R.; Pichini,S.; AltieriJ.; Caronna,A.; Passa,A.R.; Zuccaro,P. High-performance liquid chromatographic-
electrospray mass spectrometric determination of morphine and its 3- and 6-glucuronides: application to
pharmacokinetic studies, J.Chromatogr.B, 1995, 664, 329-334.
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 287
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: blood, CSF
Sample preparation: Prepare 500 mg 3 mL Bond Elut C2 cartridges by rinsing with 2 mL
MeOH then 2 mL 50 mM pH 7.5 Tris-HCl buffer. Apply 1 mL serum or CSF + 1 mL 50 mM
pH 7.5 Tris-HCl buffer to the column and wash column with 10 mL 50 mM pH 7.5 Tris-HCl
buffer. Elute with 2 mL 50% MeCN containing 0.1% trifluoroacetic acid. Freeze dry eluent or
dry an aliquot at 40 under a stream of nitrogen, dissolve residue in 2560 |JLL mobile phase,
inject 20-200 |xL aliquot.
HPLCVARIABLES
Guard column: Hexyl
Column: 150 X 4.6 Spherisorb S5 C6
Mobile phase: Gradient. A 0.1% trifluoroacetic acid in water; B 0.1% trifluoroacetic acid in 40%
MeCN. 16% B for 2 min then to 50% B over 10 min then to 100% B over 2 min, after 7 min
return to original conditions over 2 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Extracted: codeine, normorphine, metabolites
Simultaneous: diamorphine, dihydrocodeine
KEYWORDS
serum; SPE
REFERENCE
Venn,R.R; Michalkiewicz,A. Fast reliable assay for morphine and its metabolites using high-performance liquid
chromatography and native fluorescence detection, J.Chromatogr., 1990, 525, 379-388.
SAMPLE
Matrix: blood, CSF
Sample preparation: Condition 4 X 4 10 |xm LiChrosphere 60 RP-select B SPE cartridge with
buffer at 1 mL/min for 3 min and 0.5 mL/min for 4.5 min. Inject a 100-400 |JLL aliquot on to
column A and elute to waste with buffer for 10 min, elute the contents of column A on to
column B with the mobile phase and start the gradient. (Buffer was 100 mM ammonium sulfate
adjusted to pH 9.3 with 25% ammonia in water.)
HPLC VARIABLES
Guard column: 4 x 4 5 |xm LiChrosphere 60 RP-select B
Column: 250 X 4 5 |xm LiChrosphere 60 RP-select B
Mobile phase: Gradient. A was 200 mM pH 3.0 potassium phosphate buffer. B was MeCN:200
mM pH 3.0 potassium phosphate buffer 20:80. A:B from 80:20 to 40:60 over 12.5 min. After
each run wash with MeCN:MeOH:water 40:40:20 for 6 min at 1.2 mL/min, re-equilibrate at
initial conditions at 1.2 mL/min for 6 min.
Flow rate: 0.8
CHROMATOGRAM
Retention time: 9.5
OTHER SUBSTANCES
KEYWORDS
plasma; SPE
REFERENCE
Huwyler,J.; Rufer,S.; Kusters,E.; Drewe,J. Rapid and highly automated determination of morphine and mor-
phine glucuronides in plasma by on-line solid-phase extraction and column liquid chromatography,
J.Chromatogr.B, 1995, 674, 57-63.
SAMPLE
Matrix: blood, CSF, urine, vitreous humor
Sample preparation: Condition a 200 mg Bond Elut SPE cartridge with 1 mL MeOH, 1 mL
water, and 2 mL buffer. Centrifuge 1.5 mL serum, CSF, urine, or vitreous humor at 14000 g
for 5 min, vortex 1 mL supernatant with 2 mL buffer and 100 ng internal standard. Centrifuge
at 5000 g for 10 min, slowly add 2 mL supernatant to the SPE cartidge, wash with 2 mL buffer,
dry under vacuum for 5 min. Elute with 500 jxL MeOH:500 mM acetic acid 90:10 under gravity.
Dry the eluate under a stream of nitrogen, reconstitute in 100 |xL mobile phase, centrifuge at
14000 g for 4 min, inject a 10-20 JJLL aliquot. (Prepare buffer by adjusting pH of 900 mL 960
mg/L ammonium carbonate to 9.3 with concentrated ammonium hydroxide and 1M ammonium
hydroxide, make up to 1 L.)
HPLC VARIABLES
Column: 125 X 3 4 |xm Superspher RP 18
Mobile phase: MeCN:50 mM pH 3 ammonium formate buffer 5:95
Flow rate: 0.6 for 4 min, to 1.1 over 3 min, maintain at 1.1 for 10 min
Detector: MS, Finnigan MAT SSQ 7000 single quadrupole, 100-50Ou, 10 V, positive ion, sheath
gas nitrogen pressure 70 p.s.i., auxiliary gas nitrogen 20 mL/min; heated vaporizer temperature
400, heated capillary temperature 170, corona current, 5 |x A, m/z 286
CHROMATOGRAM
Retention time: 4.1
Internal standard: morphine-d3
OTHER SUBSTANCES
Extracted: metabolites, codeine, 6-monoacetylmorphine
KEYWORDS
serum; SPE
REFERENCE
Bogusz,M.J.; Maier,R.-D.; Erkens,M.; Driessen,S. Determination of morphine and its 3- and 6-glucuronides,
codeine, codeine glucuronide and 6-monoacetylmorphine in body fluids by liquid chromatography atmo-
spheric pressure chemical ionization mass spectrometry, J.Chromatogr.B, 1997, 703, 115-127.
SAMPLE
Sample preparation: 50-100 mg Brain tissue + 2.6 ng/mL nalbuphine hydrochloride in MeOH,
homogenize (PTFE pestle), sonicate (Soniprep 150) at 23 jutm amplitude for 1 min, centrifuge
at 4 at 15000 g for 30 min. Evaporate the supernatant in a centrifugal evaporator, reconstitute
with 3 mL 10 mM di-(2-ethylhexyl)phosphoric acid in ethyl acetate, add 1 mL 50 mM pH 7.0
sodium phosphate buffer, wash, centrifuge at 4 at 3000 g for 10 min. Remove 2.8 mL of the
organic phase and add it to 500 JJIL 170 mM orthophosphoric acid, vortex for 30 s, centrifuge
at 4 at 3000 g for 10 min. Remove the aqueous phase and adjust the pH to 2.5-3.0 with 24
(xL 11.2 M ammonia solution, inject a 100 (JLL aliquot.
HPLC VARIABLES
Guard column: SCX Newguard
Column: 250 X 4.6 10 jxm Partisil 10SCX
Mobile phase: MeCNibuffer 15:85 (Buffer was 76 mM orthophosphoric acid adjusted to pH 3.0
with ammonia.)
Flow rate: 3
Detector: E, Bioanalytical Systems, TL-5A glassy carbon working electrode +0.95 V, Ag/AgCl
reference electrode
CHROMATOGRAM
Retention time: 4.5
Internal standard: nalbuphine (6)
KEYWORDS
rat; plasma; brain; pharmacokinetics
REFERENCE
Borg,RJ.; Sitaram,B-R; Taylor,D.A. Ion-pair extraction and liquid chromatographic analysis of morphine in rat
brain and plasma, J.Chromatogr., 1993, 621, 165-172.
SAMPLE
Sample preparation: 10 mL Serum, plasma, whole blood, or urine + 10 mL nalorphine in water
+ 25 mL saturated ammonium sulfate solution + 500 |xL concentrated HCl, heat at 120 for 30
min, filter (Whatman No. 1 paper), adjust the pH of the nitrate to 9.0 with 25% NaOH, extract
with 125 mL chlorofornrisopropanol 80:20, repeat extraction with 50 mL chloroformdsopro-
panol 80:20. Combine the organic phases and wash them twice with 15 mL portions of 50 mM
sodium borate solution, extract the organic phase twice with 10 mL portions of 1 M sulfuric
acid. Combine the aqueous extracts and add 4 mL saturated ammonium sulfate, adjust pH to
9.0 with 25% NaOH, extract twice with 5 mL portions of chloroform:isopropanol 80:20. Combine
the organic layers and evaporate them to dryness under a stream of nitrogen, reconstitute the
residue in 50 |xL water, add 100 |xL 0.1% dansyl chloride in acetone, add 50 |xL 200 mM sodium
carbonate, let stand at room temperature in the dark for 3 h, add 1 mL toluene, vortex for 2
min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, recon-
stitute the residue in mobile phase, inject a 20 fiL aliquot.
HPLC VARIABLES
Column: 150 X 4.5 3 |xm Spherisorb S3W silica
Mobile phase: n-Hexane:isopropanol:ammonia 97:2.7:0.3
Flow rate: 1.5
Detector: F ex 330-380 (filters) em 410-500 (filters)
CHROMATOGRAM
Retention time: 9.5
OTHER SUBSTANCES
Noninterfering: acetaminophen, amitriptyline, amphetamine, atropine, benzoylecgonine, caf-
feine, carbamazepine, carisoprodol, chlorpromazine, chlorprothixene, cimetidine, cocaine, co-
deine, cyclizine, dextromethorphan, diazepam, dihydrocodeine, diphenoxilate, disopyramide,
doxepin, doxylamine, emetine, erythromycin, ethylmorphine, flurazepam, glutethimide, hydro-
codone, hydrocortisone, hydromorphone, hydroxyzine, imipramine, lidocaine, loxapine, meper-
idine, metapyrilene, methadone, methamphetamine, methocarbamol, methylphenidate, nal-
oxone, nicotine, nordiazepam, nortriptyline, orphenadrine, oxycodone, papaverine, pentazocine,
phenacetin, phencyclidine, phenmetrazine, phenolphthalein, phentermine, phenytoin, praze-
pam, procainamide, propoxyphene, propranolol, protriptyline, pyrilamine, quinine, spironolac-
tone, strychnine, terpin hydrate, thioridazine, thiothixene, triamterene, trifluoperazine, triflu-
promazine, trihexyphenidyl, trimethoprim, trimetobenzamide, tripelennamine
KEYWORDS
derivatization; serum; plasma; whole blood; normal phase
REFERENCE
Tagliaro,E; Frigerio,A-; Dorizzi,R.; Lubli,G.; Marigo,M. Liquid chromatography with pre-column dansyl deriv-
atisation and fluorimetric detection applied to the assay of morphine in biological samples, J.Chromatogr.,
1985, 330, 323-331.
SAMPLE
Sample preparation: Condition two 130 mg Sep-Pak Light C18 SPE cartridges with 1 mL
MeOH and 1 mL water. Dilute urine, if necessary, 20-fold with water. 1 mL Plasma, urine, or
diluted urine + 1 mL 500 mM pH 9.3 ammonium sulfate buffer, mix, add 1.9 mL of this mixture
to a SPE cartridge at 0.75 mL/min, wash with 4 mL 5 mM pH 9.1 ammonium sulfate buffer
at 1.5 mL/min, wash with 200 |xL MeCN:30 mM pH 2.1 potassium phosphate buffer 15:85 at
0.75 mL/min, elute with 600 |xL MeCN:30 mM pH 2.1 potassium phosphate buffer 15:85 at
0.75 mL/min. Mix the eluate with 1 mL 500 mM pH 9.3 ammonium sulfate buffer, add to a
second SPE cartridge at 0.75 mL/min, wash with 4 mL 5 mM pH 9.1 ammonium sulfate buffer
at 1.5 mL/min, wash with 200 |xL MeCN:30 mM pH 2.1 potassium phosphate buffer 15:85 at
0.75 mL/min, elute with 600 uL MeCN:30 mM pH 2.1 potassium phosphate buffer 15:85 at
0.75 mL/min, inject a 400 |xL aliquot of the eluate.
HPLC VARIABLES
Column: 100 X 4 3 fjim Spherisorb S3 ODS2
Mobile phase: MeCNrbuffer 22:78 (Buffer was 30 mM KH2PO4 containing 3 mM dodecyl sulfate,
adjusted to pH 2.1 with phosphoric acid.)
Flow rate: 1.5
Detector: E, ESA Model 5100 A Coulochem, Model 5010 detector cell, first electrode 0.25 V,
second electrode 0.35 V
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Extracted: metabolites, codeine (UV 214), norcodeine (UV 214), normorphine
KEY WORDS
plasma; SPE
REFERENCE
Svensson,J.O.; Yue,Q.Y.; Sawe,J. Determination of codeine and metabolites in plasma and urine using ion-pair
SAMPLE
Sample preparation: Plasma. Condition a Toxiclean SPE cartridge (Alltech) with 3 mL MeOH,
two 3 mL portions of water, and 2 mL buffer. 100 JJLL Plasma or serum + 100 |xL MeOH + 200
IxL MeCN + 100 |xL buffer, vortex for 1 min, centrifuge at 4000 rpm for 15 min, add the
supernatant to the SPE cartridge, wash with two 3 mL portions of water, dry under vacuum
for 10 min, elute with 2 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen
at 45, reconstitute the residue in 100 (xL 2.5 |xg/mL flufenamic acid in MeOH (?), inject an
aliquot. Urine. Condition a Bond Elut C8 SPE cartridge with 3 mL MeOH, two 3 mL portions
of water, and 2 mL buffer. 100 |xL Urine + 100 |xL MeOH + 200 JULL MeCN + 500 \xL buffer,
vortex for 1 min, centrifuge at 2000 rpm for 5 min, add the supernatant to the SPE cartridge,
wash with two 3 mL portions of water, dry under vacuum for 10 min, elute with 2 mL MeOH.
100 |JLL 2.5 |xg/mL flufenamic acid in MeOH (?), inject an aliquot. (Buffer was 250 mL 25 mM
sodium borate and 18 mL 100 mM NaOH, pH 9.2.)
HPLC VARIABLES
Mobile phase: MeCN:MeOH:1.2% ammonium acetate 15:40:45
Flow rate: 0.8
Detector: UV 239
CHROMATOGRAM
Retention time: 5.6
Internal standard: flufenamic acid (24.39)
Limit of quantitation: 300 ng/mL (urine), 100 ng/mL (plasma, serum)
OTHER SUBSTANCES
Extracted: codeine, monoacetylmorphine, papaverine
KEYWORDS
SPE; plasma; serum
REFERENCE
Theodoridis,G.; Papadoyannis,L; Tsoukali-Papadopoulou,H.; Vasilikiotis,G. A comparative study of different
solid phase extraction procedures for the analysis of alkaloids of forensic interest in biological fluids by RP-
HPLC/Diode array, J.Liq.Chromatogr., 1995, 18, 1973-1975.
SAMPLE
residue with 50 |ULL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLCVARIABLES
mL/min)
Detector: UV 211.1
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Dilute with mobile phase, filter (0.45 |xm)
HPLC VARIABLES
Column: 300 X 3.9 10 |im ixBondapak phenyl
Mobile phase: MeOH:7 mM pH 3.1 triethylammonium phosphate buffer 20:80
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
Simultaneous: codeine, meconic acid, O6-codeine methyl ether, a-codeimethine
REFERENCE
Ayyangar,N.R.; Bhide,S.R.; Kalkote,U.R. Assay of semi-synthetic codeine base with simultaneous determination
of a-codeimethine and 06-codeine methyl ether as by-product impurities by high-performance liquid chro-
SAMPLE
Matrix: bulk
Sample preparation: Prepare a 750 (xg/mL solution in 10 mM pH 2.5 orthophosphoric acid,
sonicate for 10 min, filter (0.2 |xm), inject a 15 u,L aliquot.
HPLC VARIABLES
Guard column: 4 x 4 5 jjum LiChrospher 100
Column: 125 X 4 3 fxm Spherisorb ODS-I
Mobile phase: Gradient. A was water containing 5 mL/L 85% orthophosphoric acid and 0.56 mIV
Flow rate: 0.7
Detector: UV 210
CHROMATOGRAM
Retention time: 2.9
OTHER SUBSTANCES
phine, lidocaine, 6-monoacetylmorphine, noscapine, papaverine, procaine
REFERENCE
Grogg-Sulser,K; Helmlin,H.-J.; Clerc,J.-T. Qualitative and quantitative determination of illicit heroin street
S, J.Chromatogr.A, 1995, 692, 121-129.
SAMPLE
Sample preparation: Bulk. Weigh out 100 mg, dissolve in 25 mL MeOH:water:acetic acid 24:
72:1, dilute with MeOH:water:acetic acid 24:72:1 to a final concentration of 240 (xg/mL, filter
(0.45 |xm), inject a 20 |xL aliquot. Injections. Dilute with MeOH:water:acetic acid 24:72:1 to a
final concentration of 240 |xg/mL, filter (0.45 (xm), inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeOH:5 mM sodium l-heptanesulfonate:acetic acid 24:72:1
Flow rate: 1.5
Detector: UV 284
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Simultaneous: phenol, 2-mercaptobenzothiazole (UV 230), pseudomorphine (UV 230)
KEYWORDS
injections
REFERENCE
Bello,A.C; Jhangiani,R.K. Liquid chromatographic determination of morphine sulfate and some contaminants
in injections and bulk drug material: collaborative study, J.Assoc.Off.Anal.Chem., 1988, 71, 1046-1048.
SAMPLE
Sample preparation: Dilute a 5% bupivacaine hydrochloride injection and 25 mg/mL morphine
sulfate injection with 0.9% NaCl injections to a bupivacaine concentration of 625 jmg/mL and a
morphine concentration of 100 |xg/mL, inject a 15 |xL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 2.2
OTHER SUBSTANCES
Noninterfering: bupivacaine
KEYWORDS
REFERENCE
Johnson,C.E.; Christen,C; Perez,M.M.; Ma,M. Compatibility of bupivacaine hydrochloride and morphine sul-
fate, Am.J.Health-Syst.Pharm., 1997, 54, 61-64.
SAMPLE
Sample preparation: Dilute with MeOH:water 500 mM pH 7 sodium borate 35:65:2, inject an
aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Whatman PXS ODS-3 C18
Mobile phase: MeCN:MeOH:water:85% phosphoric acid: sodium octanesulfonate 7.5:12.5:80:0.5:
0.108 (v/v/v/v/w)
Flow rate: 1.5
Detector: UV 254
OTHER SUBSTANCES
Simultaneous: milrinone, phenol
KEYWORDS
REFERENCE
SAMPLE
Sample preparation: Filter (0.22 |xm), dilute to 25 jxg/mL with mobile phase, inject a 15 |xL
aliquot.
HPLCVARIABLES
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 3.2
OTHER SUBSTANCES
Noninterfering: midazolam
KEYWORDS
REFERENCE
Johnson,C.E.; Bhatt-Mehta,V.; Mancari,S.C; McKown,J.A. Stability of midazolam hydrochloride and morphine
sulfate during simulated intravenous coadministration, Am. J.Hosp.Pharm., 1994, 51, 2812-2815.
SAMPLE
Sample preparation: Dilute 1:10, inject a 10 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:20 mM KH2PO4 50:50, pH adjusted to 5.40 with 1 M NaOH
Flow rate: 1.5
Detector: UV 216
CHROMATOGRAM
Retention time: 6.7
OTHER SUBSTANCES
Simultaneous: hydromorphone, ondansetron
KEYWORDS
REFERENCE
Trissel,L.A.; Xu,Q.; Martinez,J.F.; Fox,J.L. Compatibility and stability of ondansetron hydrochloride with mor-
phine sulfate and with hydromorphone hydrochloride in 0.9% sodium chloride injection at 4, 22, and 32
(SC, Am.J.Hosp.Pharm., 1994, 51, 2138-2142.
SAMPLE
Sample preparation: Dilute formulation 1:100 with water, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 5 x 4 35-60 |xm Perisorb RP18
Column: 250 X 4 10 ^m LiChrosorb RP18
Mobile phase: MeOH:MeCN:2.72 g/L KH 2 PO 4 12:2:86
Detector: UV 220
CHROMATOGRAM
KEY WORDS
injections; water
REFERENCE
medication pump, Arzneimittelforschung, 1995, 45, 9398.
SAMPLE
Sample preparation: Inject a 20 \xL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 |xm (xBondapak phenyl
Flow rate: 1
Detector: UV 235
CHROMATOGRAM
Retention time: 6.6
OTHER SUBSTANCES
Simultaneous: hydromorphone, bupivacaine
KEYWOTDS
saline; injections
REFERENCE
565-578.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 |jim cyano
Flow rate: 1
Detector: UV 300
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: granisetron, mechlorethamine (UV 200)
KEYWORDS
REFERENCE
304.
SAMPLE
Matrix: hair
Sample preparation: Incubate 20-200 mg hair in 2 mL 250 mM HCL at 45 overnight. Extract
and neutralize with a commercial liquid-liquid method (Toxi-Tubes A, Analytical systems, La-
guna Hills). Reconstitute the residue with mobile phase A, inject a 10 JJLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 3 |xm C18 (NBS, ESA)
Mobile phase: Gradient. A:B 93:7 for 11.5 min, to 50:50 over 8 min, maintain at 50:50 for 5 min,
to 10:90 over 10 min, maintain at 10:90 for 15.5 min, re-equilibrate at initial conditions for 7.5
min. A was MeCN:20 mM pH 7.0 monobasic sodium phosphate 10:90. B was MeCN:20 mM pH
7.0 monobasic sodium phosphate 50:50.
Flow rate: 0.8
Detector: E, Two ESA CoulArray modules, each containing four electrochemical cells, 450 mV
at electrode 1, 550 mV at electrode 2, 650 mV at electrode 3, 750 mV at electrode 4, 850 mV
at electrode 5, 900 mV at electrode 6, 950 mV at electrode 7, 1000 mV at electrode 8, solid-
state palladium reference electrode built in the coulometric cells. Increase all cell potentials to
1200 for 60 s at the end of each analysis. Allow the electrodes to stabilize for 7.5 min before
the next injection.
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: 6-acetylmorphine, 3,4-methylenedioxymetamphenamine, buprenorphine, co-
caine, codeine, diamorphine, dihydrocodeine, ethylmorphine, hydrocodone, lidocaine, metha-
done, naloxone, procaine, thebaine
KEYWORDS
hair
REFERENCE
Achilli,G.; Cellerino,G.R; Melzi d'Eril,G.V.; Tagliaro,F. Determination of illicit drugs and related substances by
high-performance liquid chromatography with an electrochemical coulometric-array detector,
J.ChromatogrA, 1996, 729, 273-277.
SAMPLE
Matrix: hair
Sample preparation: Wash 100-200 mg hair with 10 mL ethyl ether and 12 mL 10 mM HCl,
add 3 mL 100 mM HCl, heat at 45 for 12 h, neutralize with 100 |xL 3 M NaOH, add to an
extraction tube containing sodium carbonate, sodium bicarbonate, and organic solvents (Toxi-
Tubes A, Analytical Systems), add 2 mL water, vortex for 2 min, centrifuge at 759 g for 10 min,
remove the organic layer, add dichloromethane:dichloroethane:heptane 18:18:64 to the aqueous
layer, extract. Combine the organic layers and evaporate them to dryness, reconstitute with
50 (xL water, add 50 |xL 1 mg/mL dansyl chloride in acetone, add 50 |xL 100 mM sodium
carbonate, let stand in the dark at room temperature for at least 1.5 h, add 1 mL toluene,
vortex for 2 min. Remove the organic layer and evaporate it to dryness under a stream of
nitrogen, reconstitute the residue in mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherisorb silica
Mobile phase: Hexane:isopropanol:ammonia 95:4.5:0.5
Flow rate: 2
Detector: F ex 330-380 (bandpass filter) em 410-500 (bandpass filter)
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Noninterfering: codeine
KEYWORDS
derivatization; derivatization at C3 hydroxy group; normal phase
REFERENCE
Marigo,M.; Tagliaro,R; Poiesi,C; Lafisca,S.; Neri,C. Determination of morphine in the hair of heroin addicts
by high performance liquid chromatography with fluorimetric detection, J.Anal.Toxicol., 1986,10,158161.
SAMPLE
Matrix: meconium
Sample preparation: 500 mg Meconium + 5 mL water + 1 drop 500 mM HCl, vortex for 1 min,
sonicate for 5 min, vortex for 1 min, centrifuge at 2683 g for 10 min. Remove the supernatant
and add it to 5 mL water and 1 drop 500 mM HCl, vortex for 1 min, sonicate for 5 min, vortex
for 1 min, centrifuge at 2683 g for 10 min. Make up the supernatant to 20 mL with pH 9.0
borax buffer, add it to an Extrelut SPE cartridge, after 10 min elute with 60 mL dichlorome-
thane.isopropanol 80:20. Add 1 drop 2% tartaric acid in water and evaporate the eluate under
a stream of nitrogen at 40, reconstitute the residue in 100 |xL mobile phase, inject a 20 |xL
aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Supelcosil LC-18 DB
Mobile phase: MeOH:MeCN:THF:triethylamine:water 100:25:7:1.5:600 containing 77 mM
KH2PO4
Flow rate: 1
Detector: UV 204
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: caffeine, benzoylecgonine, cocaine, codeine
KEYWORDS
SPE
REFERENCE
Franssen,R.M.E.; Stolk,L.M.L.; van den Brand,W.; Smit,B.J. Analysis of morphine and amphetamine in me-
conium with immunoassay and HPLC-diode-array detection, J.Anal.ToxicoL, 1994, 18, 294-295.
SAMPLE
Sample preparation: Prepare a 1 mL 100 mg C18 Bond Elut by washing with 1 mL MeOH, 1
mL water, 1 mL 5 mM pH 9.0 carbonate buffer. Mix 100 |xL microsomal incubation, 20 |xL 25
jjug/mL 10,11-dihydrocarbamazepine in MeCN:water 25:75, 600 |xL 200 mM pH 10.2 carbonate
buffer, 80 JAL 20 mM tetrabutylammonium hydrogen sulfate in water with vortex mixing after
each addition. Add to SPE cartridge, wash with 1 mL 5 mM pH 9.0 carbonate buffer, elute
with 0.5 mL MeCN:mobile phase buffer 40:60.
HPLC VARIABLES
Guard column: 20 X 2 Phase Separations pellicular ODS
Column: 250 X 4.6 5|xm Hypersil CPS (cyanopropyl)
Mobile phase: MeCN:buffer 24:76 (Buffer was 50 mM potassium hydrogen phosphate containing
1 mM sodium dodecyl sulfate adjusted to pH 2.5 with orthophosphoric acid.)
Flow rate: 1
Detector: UV 210; E, ESA Coulochem II with a 5020 guard cell (+0.60 V) and a 5011 analytical
cell (cell 1 +0.22 V, cell 2 +0.45 V)
CHROMATOGRAM
Retention time: 11
Internal standard: 10,11-dihydrocarbamazepine
OTHER SUBSTANCES
Simultaneous: codeine, norcodeine, metabolites
KEYWORDS
SPE
REFERENCE
Pawula,M.; Shaw,P.N.; Barrett,D.A. Determination of codeine and its metabolites in mirosomal incubates by
SAMPLE
Matrix: perfusate
Sample preparation: 30 |xL Perfusate (artificial CSF) + 10 |xL 200 mM perchloric acid. Mix a
25 |xL aliquot with 12.5 |xL reagent, let stand for 2 min, inject an aliquot. (Prepare a stock
10 |xM EDTA, allow to stand for 24 h before use.)
HPLC VARIABLES
Column: two columns 150 X 4.6 5 u,m M.S. Gel C18 (ESA)
Mobile phase: MeOHibuffer 8:92 adjusted to pH 3.0 with phosphoric acid (Buffer was 54 mM
Flow rate: 1.2
potential 280 mV
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: apomorphine, dopamine, hydralazine, isoproterenol, methoxamine, norepinephrine,
phenylephrine
KEYWORDS
rat; derivatization
REFERENCE
Acworth,I.N.; Yu,J.; Ryan,E.; Gariepy,K.C; Gamache,R; Hull,K; Maher,T. Simultaneous measurement of
705.
SAMPLE
Matrix: perfusate, urine
Sample preparation: Perfusate. Add 200 |xL 1 u,g/mL IS and 500(xL 200 mM pH 9.0 potassium
buffer to 500 |xL perfusate, add 6 mL chloroform:n-butyl alcohol 80:20, rotate at 33 rpm for 10
min, centrifuge at 1800 g for 10 min. Add 200 |xL 0.05% sulfuric acid to 5 mL organic phase.
Mix for 10 min and centrifuge at 1800 g for 10 min. Inject a 100 fxL aliquot of the acidic phase.
(Caution! Chloroform is a carcinogen!) Urine. Directly inject a 100 |xL aliquot of diluted urine.
HPLCVARIABLES
Guard column: Nova-Pak C18 Guard-Pak
Column: 4 |xm Nova-Pak C18 Radial-Pak cartridge
Mobile phase: MeCN:MeOH:70 mM pH 3.0 potassium dihydrogen orthophosphate buffer 1.5:1:
97.5
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 6.2
Internal standard: normorphine (4.9)
Limit of quantitation: 13 pM (perfusate), 26 pM (urine)
KEYWORDS
pharmacokinetics; rat; kidney
REFERENCE
Disposition of morphine in the rat isolated perfused kidney: Concentration ranging studies,
J.PharmacoLExp.Ther., 1997, 282, 1518-1525.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 4.6 5 (xm Adsorbosphere C18 (Alltech)
Mobile phase: MeCN:water:20 mM pH 3.0 KH2PO4 25:55:20, containing 10 |xL triethylamine per
10OmL
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 6.4
OTHER SUBSTANCES
Also analyzed: codeine, diamorphine, fentanyl, meperidine
REFERENCE
Lichtman,A.H.; Meng,Y.; Martin,B-R- Inhalation exposure to volatilized opioids produces antinociception in
mice, J.Pharmacol.Exp.Ther., 1996, 279, 69-76.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: Et 280/4 Nucleosil 100 C18
Mobile phase: 1 M triethylammonium phosphate buffer (Fluka):water 1:40
OTHER SUBSTANCES
Simultaneous: morphine glucuronides
REFERENCE
Skopp,G.; Lutz,R.; P6tsch,L.; Gan|3mann,B.; Klinder,K.; Schmidt,A.; Aderjan,R.; Mattem,R. An in vitro exper-
iment for postmortem vascular permeation. The passage of morphine and morphine glucuronides across a
vascular wall, J.Forensic ScL, 1997, 42, 486-491.
SAMPLE
Matrix: solutions
Sample preparation: Weigh 35.5 mg morphine sulfate and 15.0 mg ondansetron hydrochloride
in a 10 mL volumetric flask, add 0.9% sodium chloride, shake vigorously for 2 min, add 0.9%
sodium chloride to volume. Dilute 1:5, 1:7.5, and 1:20, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 220 X 4.6 5 |xm underivatized silica (Brownlee Silica Applied Biosystems, Inc.,San Jose)
Mobile phase: MeOH: 10 mM pH 4.0 aqueous monobasic potassium phosphate (adjusted with
10% phosphoric acid) 40:60
Flow rate: 1
Detector: UV 233
CHROMATOGRAM
Retention time: 6.7
OTHER SUBSTANCES
REFERENCE
ondansetron mixtures in 0.9% sodium chloride injection, J.Liq.Chromatogr.Rel.TechnoL, 1996, 19, 1329-
1338.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 jim IB-SIL C8 (Phenomenex)
Flow rate: 1
Detector: UV 285
CHROMATOGRAM
Retention time: 4.8
OTHER SUBSTANCES
Simultaneous: doxorubicin (7.3)
REFERENCE
Zhang,H.; Ye,L.; Stewart,J.T. HPLC determinations of doxorubicin with selected medications in 0.9% sodium
chloride injection USP, J.Liq.Chromatogr.Rel.Technol., 1998, 21, 2375-2385.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: Supelco LC-8
Mobile phase: MeOH:water:acetic acid 40:59:1 containing 100 mM potassium nitrate, 10 mM
tetramethylammonium bromide, and 2.5 mM heptanesulfonic acid
Flow rate: 1
Detector: E, Metrohm 1096/2, platinum working electrode +0.4 V, Ag/AgCl reference electrode
following post-column reaction. The column effluent passed through an electrochemical cell
(construction details in paper) and the bromide was oxidized to bromine at 1.5 JJLA. The mixture
flowed through a 20 s reaction coil (3.9 m (?) X 0.33 mm ID) to the detector.
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
Simultaneous: codeine, noscapine, papaverine
KEYWORDS
REFERENCE
Kok,W.T.; Brinkman,U.A.T.; Frei,R.W. On-line electrochemical reagent production for detection in liquid chro-
matography and continuous flow systems, Anal.Chim.Acta, 1984, 162, 19-32.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: ethoheptazine, morphine-3-glucuronide, pholcodeine, norpethidine, hydrocodone,
dihydrocodeine, dihydromorphine, levorphanol, norcodeine, normorphine, pemoline, benzphet-
amine, diethylpropion, mazindol, tranylcypromine, caffeine, fenethyline, phendimetrazine,
methylphenidate, phenelzine, epinephrine, pipradol, phenylpropanolamine, fencamfamin,
chlorphentermine, norpseudoephedrine, phentermine, fenfluramine, methylenedioxyamphet-
amine, amphetamine, normetanephrine, 4-hydroxyamphetamine, bromo-STP, STP, tyramine,
trimethoxyamphetamine, phenylephrine, pseudoephedrine, ephedrine, methylephedrine, di-
methylamphetamine, methamphetamine, mescaline, mephentermine, buprenorphine, dextro-
moramide, phenoperidine, fentanyl, etorphine, piritramide, noscapine, papaverine, naloxone,
dextropropoxyphene, nalorphine, phenazocine, norpipanone, levallorphan, hydroxypethidine,
morphine, thebacon, oxycodone, thebaine, norlevorphanol, methadone, benzylmorphine,
ethylmorphine
Interfering: prolintane 2-phenethylamine, morphine-N-oxide, codeine, codeine-N-oxide
REFERENCE
Law,B-; Gill,R-; Moffat,A-C. High-performance liquid chromatography retention data for 84 basic drugs of fo-
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 500 |xg/mL solution in MeOH:water 50:50, inject a 5 uX aliquot.
HPLC VARIABLES
column.)
Flow rate: 2
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Simultaneous: acetophenone, amphetamine, desipramine, ethylmorphine, imipramine, mefen-
amic acid, methamphetamine, phenylbutazone, salicylic acid
KEYWORDS
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 100 X 2 10 fun ixPorasil
Flow rate: 1
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: buprenorphine, butorphanol, ethylmorphine, codeine, nalbuphine, nalorphine,
meperidine, tramadol, fentanyl
REFERENCE
J.Chromatogn, 1991, 570, 339-350.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
methapjndlene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide,
yltestosterone, methyprylon, metoprolol, nadolol, nalorphine, naloxone, naltrexone, naphazo-
line, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam,
norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxy-
phenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital,
persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phe-
nelzine, pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbu-
tazone, phenylephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone,
probenecid, progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puro-
mycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quiiiidine, quinine, ranitidine, re-
cinnamine, reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ronidazole, midazolam, moclobemide, nadolol, nalbuphine, naloxone, naphazoline, naproxen,
nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, pa-
himbine, zopiclone
KEYWORDS
REFERENCE
J.Chromatogr.A, 1995, 692, 103-119.
SAMPLE
Matrix: urine
HPLC VARIABLES
min.
Flow rate: 1
170 to 150 over 20 min. SIM, m/z 286
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Extracted: 6-acetylmorphine, amphetamine, benzoylecgonine, cocaine, ephedrine, methamphet-
amine, methylephedrine, morphine-3-glucuronide, morphine-6-glucuronide
KEY WORDS
SPE
REFERENCE
Tatsuno,M.; Nishikawa,M.; Katagi,M.; Tsuchihashi,H. Simultaneous determination of illicit drugs in human
urine by liquid chromatography-mass spectrometry, J.Anal.Toxicol., 1996, 20, 281286.
SAMPLE
Matrix: urine
Sample preparation: Pack a 110 X 10 mm polypropylene disposable column having support
frits with 830 mg aldehyde-activated silica (Clifmar Associates, UK), wash the packed column
with 50 mL phosphate buffered saline. Add 5 mL phosphate buffered saline to the column, add
500 |xL unpurified antisera (J.Pharm.Biomed.Anal. 1994, 12, 353), close the column, place on
a rotamixer for 2 h. Wash the immunocolumn with 10 mL phosphate buffered saline, carefully
add 5 mL 1 M pH 6 glycine buffer, rotate the column overnight, wash with 10 mL 0.3% HCl
and 20 mL phosphate buffered saline. Dilute 100 |xL urine with 900 |xL phosphate buffered
saline, add 1 mL of the diluted urine to the immunocolumn, wash with 15-20 mL phosphate
buffered saline, elute with two 1 mL portions of EtOH:phosphate buffered saline 40:60 (pH 4),
collect the second eluted fraction, inject an aliquot of the fraction. (Phosphate buffered saline
(pH 7.2-7.4) was 8 g NaCl, 200 mg KH2PO4, 200 mg KCl, and 2.9 g K2HPO4 dissolved in 1 L
water.)
HPLCVARIABLES
Column: 250 X 5 5 |jim Hypersil CPS (Jones Chromatography, UK)
Mobile phase: MeCN:65 mM pH 2.5 phosphate buffer containing 1.5 mM sodium lauryl sulfate
13:87
Flow rate: 1
Detector: E, ESA Coulochem model 5100A, +450 mV
CHROMATOGRAM
Retention time: 8
KEYWORDS
immunoafnnity; SPE
REFERENCE
Rashid,B.A.; Aherne,G.W.; Katmeh,M.R; Kwasowski,R; Stevenson,D. Determination of morphine in urine by
solid-phase immunoextraction and high-performance liquid chromatography with electrochemical detection,
J.ChromatogrA, 1998, 797, 245-250.
SAMPLE
Matrix: urine
Sample preparation: 500 |JLL Urine + N-ethylnordiazepam + chlorpheniramine + 100 JJLL buffer,
|JLL mobile phase B, with 200 |xL mobile phase C, and with 1.15 mL mobile phase D. Elute
HPLC VARIABLES
Column: A 10 X 2.1 12-20 jim PRP-I spherical poly(styrene-divinylbenzene) (Hamilton); B 10 X
3.2 11 |jim Aminex A-28 (Bio-Rad); C 25 X 3.2 5 |xm C8 (Phenomenex) + 150 X 4.6 5 |xm silica
(Macherey-Nagel)
D MeCN.buffer 33:67 (Buffer was 6 mM KH2PO4 containing 5 mM tetramethylammonium
CHROMATOGRAM
OTHER SUBSTANCES
methadone, imipramine, flurazepam, amitriptyline, codeine, hydromorphone, hydrocodone
KEYWORDS
column-switching
REFERENCE
341.
SAMPLE
Matrix: urine
Sample preparation: 10 mL Urine + 500 |xL 100 jxg/mL nalorphine hydrobromide in MeOH +
1 mL concentrated HCl, heat at 100 for 1 h, cool, add 500 |xL saturated ammonium sulfate
solution, adjust pH to 9 with 25% NaOH, dilute to 20 mL with water, add mixture to an
Extrelut 20 column, let stand for 10 min, elute with 40 mL dichloromethanerisopropanol 85:
15. Add the eluate to 3 mL 200 mM HCl, extract, repeat extraction. Combine the aqueous
phases and add them to 500 JULL saturated ammonium sulfate solution, adjust pH to 9.2 with
25% NaOH, dilute to 20 mL with water, add to another Extrelut 20 column, let stand for 10
min, elute with 40 mL dichloromethaneiisopropanol 85:15. Evaporate the eluate to dryness
|iL aliquot.
HPLC VARIABLES
Guard column: 4 x 4 5 (xm Lichrosorb
Column: 250 X 4 5 |xm Lichrospher Si 100
Mobile phase: n-Hexane:dichloromethane:MeOH containing 0.75% diethylamine 72.5:20:7.5
Flow rate: 1.35
Detector: UV 225
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
Extracted: codeine
pine, chlorpromazine, desipramine, dextromethorphan, doxepin, ephedrine, fenfluramine, imip-
trimipramine
KEYWORDS
SPE; normal phase
REFERENCE
of abuse in urine, JAnal.ToxicoL, 1992, 16, 217-222.
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 10 |xL nalorphine solution + 3000-3500 U glucuronidase
(Patella vulgata, Sigma) + 300 jxL 1.1 M pH 5 sodium acetate buffer, heat overnight at 55,
add 500 |xL buffer, add 8 mL chloroformdsopropanol 90:10, rotate gently for 30 min, centrifuge
at 2500 g for 10 min. Remove the organic layer and add it to 3 mL pH 9.9 NaH2PO4 buffer,
rotate gently for 10 min, centrifuge, discard the aqueous layer, repeat the wash. Remove the
organic layer and add it to 200 JJLL 0.2% phosphoric acid, rotate gently for 30 min, inject a 50
IxL aliquot of the aqueous layer. (Buffer was 50 mM boric acid and 43 mM sodium tetraborate,
pH adjusted to 9.9.)
HPLC VARIABLES
Guard column: Nova-Pak phenyl
Mobile phase: MeCNiIO mM pH 6.6 NaH2PO4 10:90
Flow rate: 1.2
Detector: E, ESA Coulochem, Model 5010 analytical cell, detector cell 1 +0.20 V, detector cell 2
+ 0.55 V, model 5020 guard cell + 0.75 V or UV 210
CHROMATOGRAM
Retention time: 5.9
OTHER SUBSTANCES
Extracted: codeine, 6-monoacetylmorphine
Simultaneous: dihydrocodone, hydrocodone, oxycodone
Noninterfering: 7-aminoclonazepam, 7-aminoflunitrazepam, amitriptyline, amphetamine, di-
azepam, dothiepin, doxepin, ephedrine, mesoridazine, methadone, methamphetamine, nordi-
azepam,norpropoxyphene, nortriptyline, oxazepam, propoxyphene, quinidine, quinine, sulfa-
methoxazole, sulforidazine, thioridazine, trimethoprim
REFERENCE
Gerostamoulos,J.; Crump,K; MclntyreJ.M.; Drummer,O.H. Simultaneous determination of 6-monoacetylmor-
phine, morphine and codeine in urine using high-performance liquid chromatography with combined ultra-
SAMPLE
Matrix: urine
min, filter (0.2 jjum), inject 20 |xL aliquot
HPLC VARIABLES
over 7 min, hold at 75:25 for 3 min, return to 10:90 over 5 min, equilibrate at 10:90 for 5 min
Flow rate: 1.5
CHROMATOGRAM
Retention time: 2.5
OTHER SUBSTANCES
Extracted: amitriptyline, codeine, amphetamine, benzoylecgonine, meperidine, norpropoxy-
phene, nordiazepam
Also analyzed: phenylpropanolamine, lidocaine, diphenhydramine, nortriptyline, ephedrine, co-
caine (different gradient).
REFERENCE
Li,S.; Gemperline,P.J.; Briley,K; Kazmierczak,S. Identification and quantitation of drugs of abuse in urine
SAMPLE
Matrix: urine
Sample preparation: Condition a 300 mg Bond Elut Certify SPE cartridge with 2 mL MeOH
and 2 mL water. 5 mL Urine + 1 mL concentrated HCl, vortex, heat at 120 for 30 min, cool,
adjust pH to between 7.0 and 8.0 with 10 M KOH. 5 mL Urine or hydrolysed urine + nalor-
phine, add to the SPE cartridge, wash with 2 mL water, wash with 1 mL pH 4 acetate buffer,
wash with 2 mL MeOH, elute with 2 mL dichloromethane:isopropanol 80:20 containing 2%
ammonia. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue
in 0.5-1 mL pentanerdichloromethane 90:10. (Use unhydrolysed urine to determine diamor-
phine and unconjugated compounds.)
HPLC VARIABLES
Column: 200 X 2 3 |xm Hypersil
Mobile phase: Pentane:dichloromethane:MeOH containing 0.5% diethylamine 65:29.8:5.2
Flow rate: 0.4
Detector: UV 280
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Extracted: diamorphine, 6-monoacetylmorphine, pholcodine, dihydrocodeine, codeine
Simultaneous: diphenhydramine, ephedrine, hydrocodone
Noninterfering: aspirin, caffeine, chlordiazepoxide, dextropropoxyphene, diazepam, lignocaine,
naloxone, norcodeine, normorphine, papaverine, procaine, quinine, theobromine, theophylline
KEYWORDS
normal phase; SPE
REFERENCE
Low,A-S.; Taylor,R-B. Analysis of common opiates and heroin metabolites in urine by high-performance liquid
chromatography, J.Chromatogr.B, 1995, 663, 225-233.
SAMPLE
Matrix: urine
HPLC VARIABLES
Column: A 10 X 4.6 5 |xm Spherisorb cyanopropyl; B 250 X 4.6 Capcell Pak C18 UG-120
(Shiseido)
Mobile phase: A water; B Gradient. MeCN:buffer from 3:97 to 30:70 over 30 min, to 40:60 over
8 min (Buffer was 3.4 mL/L phosphoric acid adjusted to pH 3.0 with 5 M NaOH.)
Detector: UV 220
CHROMATOGRAM
Retention time: 6.3
OTHER SUBSTANCES
Extracted: acebutolol, alprenolol, amphetamine, atenolol, bopindolol, codeine, ephedrine, labe-
talol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, timolol
KEYWORDS
column-switching
REFERENCE
Saarinen,M.T.; Siren,H.; Riekkola,M.-L. Screening and determination of p-blockers, narcotic analgesics and
1995, 664, 341-346.
Morsuximide
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: EtOH:water 10:90 containing 10 mM p-cyclodextrin and 0.5 mM tri-O-methyl-p-
cyclodextrin
Flow rate: 0.95
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: mephenytoin
KEY WORDS
chiral
REFERENCE
Nowakowski,R; Bielejewska,A; Duszczyk,K; Sybilska,D. Chiral discrimination by high-performance liquid
Moxidectin
Merck Index: 6373
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg Supelclean LC18 SPE cartridge with 5 mL
MeOH and 5 mL water at 6 mL/min. 1 mL Plasma + 1 mL MeCN + 250 (JLL water, mix for 20
min, centrifuge at 2000 g for 2 min, add 2.2 mL supernatant to the SPE cartridge at 3 mL/
min, wash with 2 mL water, wash with 1 mL MeOH:water 25:75 at 3 mL/min, dry under
nitrogen at 6 mL/min for 10 s, elute with 1.2 mL MeOH at 3 mL/min. Evaporate the eluate to
dryness under a stream of nitrogen at 50, reconstitute with 100 JJLL N-methylimidazole:MeCN
1:1, add 150 |xL trifluoroacetic anhydride :MeCN 1:2, let stand for <30 s, inject a 100 |xL aliquot.
HPLC VARIABLES
Column: Supelcosil C18
Mobile phase: MeCN:MeOH:0.2% acetic acid 62:30:8
Flow rate: 1.5
CHROMATOGRAM
Retention time: 9.2
KEY WORDS
plasma; SPE; cow; derivatization; pharmacokinetics
REFERENCE
Alvinerie,M.; Sutra,J.F.; Badri,M.; Galtier,P. Determination of moxidectin in plasma by high-performance liquid
chromatography with automated solid-phase extraction andfluorescencedetection, J.Chromatogr.B, 1995,
674, 119-124.
Moxonidine
Merck Index: 6375
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 4.6 12 ^m l-m3n*istoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
famotidine, isothipendyl, ketotifen, metiamide, metoprolol, nadolol, naphazoline, nifenalol, ni-
REFERENCE
Kaliszan,R.; Nasal,A.; Turowski,M. Binding site for basic drugs on o^-acid glycoprotein as revealed by chemo-
Muzolimine
Merck Index: 6397
Lednicer No.: 3 137
SAMPLE
Matrix: blood
Sample preparation: 500 |JLL Plasma + 2 mg cysteine + 100 |xL 200 jig/mL IS in MeOH + 5 mL
hexane.dichloromethane 60:40, vortex for 30 s, centrifuge at 550 g for 5 min. Remove the
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 272
CHROMATOGRAM
Retention time: 4.0
Internal standard: 3-amino-l-(l-(3,4-dichlorophenyl)ethyl)pyrazol-5-one (7.2)
KEYWORDS
plasma; dog; human; pharmacokinetics
REFERENCE
Osman,M.A.; Dunning,L.K; Bhavnagri,V.R; Cheng,L.K. Determination of muzolimine in plasma and urine by
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 4 mg cysteine + 100 jxL 200 |xg/mL IS in MeOH + 5 mL
hexane:dichloromethane 60:40, vortex for 30 s, centrifuge at 550 g for 5 min. Remove the
HPLC VARIABLES
Column: 150 X 3.9 5 |xm Nova Pak phenyl
Mobile phase: MeCN:50 mM pH 4.2 phosphate buffer 28>72
Flow rate: 1.5
Detector: UV 272
CHROMATOGRAM
Retention time: 4.4
Internal standard: N-ethyl-3-(3-methylphenoxy)azetidine-l-carboxamide (5.6)
KEYWORDS
dog; human
REFERENCE
Osman,M.A.; Dunning,L.K; Bhavnagri,V.R; Cheng,L.K. Determination of muzolimine in plasma and urine by
Mycophenolic acid
Merck Index: 6408
SAMPLE
Matrix: blood
Sample preparation: Mix 50 |xL plasma with 100 |xL MeCN containing 100 mM phosphoric
acid. Inject a 5 (JLL aliquot of the supernatant.
HPLC VARIABLES
Column: 100 X 4.6 3.5 fxm Zorbax SB-C18
Flow rate: 1.5
Injection volume: 5
Detector: UV 215
CHROMATOGRAM
Retention time: 9.7
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Svensson,J.O.; Brattstrom,C; Sawe, J. A simple HPLC method for simultaneous determination of mycophenolic
acid and mycophenolic acid glucuronide in plasma (Abstract 75), Ther.Drug Monit., 1997, 19, 566.
SAMPLE
Matrix: blood
Sample preparation: Add 100 |xL MeCN containing 15 |xg/mL IS, 20 |JLL 150 g/L perchloric acid,
and 20 u-L 250 g/L sodium tungstate to 200 |xL plasma. Mix and centrifuge at 10 000 g for 5
min. Inject a 50 u,L aliquot of the supernatant.
HPLC VARIABLES
Mobile phase: Gradient. A was MeCN:20 mM pH 3.0 phosphate buffer 25:75. B was MeCN:20
mM pH 6.5 phosphate buffer 70:30. A:B 94:6 for 4.5 min, to 62:38 over 7 min, to 0:100 over 2
min.
Flow rate: 1.2
CHROMATOGRAM
Internal standard: carboxybutoxy ether of mycophenolic acid
Limit of detection: 10 ng/mL (UV 215); 30 ng/mL (UV 254)
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Andreeva,M.; Niedmann,D.; Schiitz,E.; Armstrong,V.W.; Oellerich,M. An HPLC procedure for simultaneous de-
termination of mycophenolic acid and its glucuronide in human plasma (Abstract 73), Ther.Drug Monit.,
1997, 19, 565.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 2.1 Hypersil C18
Mobile phase: MeCN:0.1% acetic acid 43:57
Flow rate: 0.15
Detector: MS, Hewlett Packard 5989, electrospray, selected ion monitoring m/z=319
CHROMATOGRAM
Retention time: 8
Internal standard: (E)-6-[l,3-dihydro-4-(4-carboxybutoxy)-6-methoxy-7-methyl-3-oxo-5-isoben-
zofuranyl-4-methyl-4-hexenoic acid (m/z=419)
REFERENCE
Korecka,M.; Van Breemen,R.B.; Nikolic,D.; Nowak,L; Shaw,L.M. Mycophenolic acid measurement in plasma:
Validation of an HPLC method by comparison with an HPLC-mass spectrometry procedure (Abstract 60),
Ther.DrugMonti., 1997, 19, 562.
Nabumetone
Merck Index: 6428
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 uX aliquot of a 100-500 |xg/mL solution in mobile phase.
HPLC VARIABLES
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
operidol, hydroxyzine, imipramine, indomethacin, lidocaine, megestrol acetate, metoprolol, na-
KEYWORDS
REFERENCE
Nadolol
Merck Index: 6431
Lednicer No.: 2 110
REFERENCE
Korecka,M.; Van Breemen,R.B.; Nikolic,D.; Nowak,L; Shaw,L.M. Mycophenolic acid measurement in plasma:
Validation of an HPLC method by comparison with an HPLC-mass spectrometry procedure (Abstract 60),
Ther.DrugMonti., 1997, 19, 562.
Nabumetone
Merck Index: 6428
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 uX aliquot of a 100-500 |xg/mL solution in mobile phase.
HPLC VARIABLES
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Nadolol
Merck Index: 6431
Lednicer No.: 2 110
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Serum + 80 fxL 250 ng/mL atenolol in EtOH + 500 jxL 10 M NaOH
+ 300 mg NaCl + 5 mL diethyl ether, shake for 10 min, centrifuge at 1500 g for 5 min. Remove
4 mL of the organic layer and evaporate it to dryness under a stream of nitrogen, dissolve the
residue in 200 JJLL mobile phase, inject a 50 jxL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 jxm STR ODS-M (Shimadzu)
Mobile phase: MeCN:50 mM ammonium acetate adjusted to pH 4.5 with acetic acid 15:85
Flow rate: 0.8
CHROMATOGRAM
Retention time: 6.5
Internal standard: atenolol (4)
KEYWORDS
serum
REFERENCE
Noguchi,H.; Yoshida,K; Murano,M.; Naruto,S. Determination of nadolol in serum by high-performance liquid
chromatography with nuorimetric detection, J.Chromatogr., 1992, 573, 336-338.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 100 |xL 1 M NaOH, vortex gently for 1 min, add to an
Extrelut-1 SPE cartridge, rinse out the tube with 300 JJLL water, add the rinse to the SPE
cartridge, let stand for 15 min, elute with 10 mL diethyl ether. Evaporate the eluate to dryness
under a stream of nitrogen at 40, reconstitute the residue in 100 |xL water and 400 |xL MeOH,
evaporate to dryness under a stream of nitrogen at 50, reconstitute with 50 |xL MeOH, add
10 |xL 5 mg/mL (R)-(-)-l-(l-naphthyl)ethylisocyanate in MeCN, vortex for 30 s, heat at 45 for
5 min, evaporate to dryness under a stream of nitrogen at room temperature, reconstitute with
100 /ULL MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 fim YMC-AM-303 ODS (YMC)
Flow rate: 1
CHROMATOGRAM
Retention time: 31 (SR), 33 (RS), 35 (RR), 37 (SS)
KEYWORDS
derivatization; pharmacokinetics; dog; plasma; chiral; SPE
REFERENCE
Hoshino,M.; Yajima,K; Suzuki,Y.; Okahira,A. Determination of nadolol diastereomers in dog plasma using
chiral derivatization and reversed-phase high-performance liquid chromatography with fluorescence detec-
tion, J.Chromatogr.B, 1994, 661, 281-289.
SAMPLE
Matrix: blood
Sample preparation: Condition a 500 mg non-end-capped cyanopropyl SPE cartridge (Varian)
with 1 mL MeOH and 1 mL water. Condition a 100 mg phenylboronic acid SPE cartridge
(Varian) with 1 mL MeOH, 2 mL 100 mM HCl, 4 mL 0.3% ammonium hydroxide, and 2 mL
100 mM pH 8.5 ammonium sulphate. 1 mL Plasma + 80 ng IS, add to the cyanopropyl SPE
cartridge, wash with 3 mL water, wash with 3 mL MeCN, elute with 2.5 mL alkaline MeOH.
Evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue in 1 mL
MeOH, evaporate to dryness under a stream of nitrogen, reconstitute the residue in 50 |xL
distilled 1,2-dimethoxyethane, add 20 |xL reagent, let stand at room temperature for 1 h, add
300 IJLL water, extract with 1 mL dichloromethane. Wash the organic layer with 300 (xL water,
evaporate the organic layer to dryness under a stream of nitrogen, reconstitute the residue in
1 mL MeOHrwater 40:60. Add this solution to the phenylboronic SPE column, wash with 2 mL
water, wash with 100 |xL MeOH, wash with 1 mL hexane:ethyl acetate 80:20, wash with 1 mL
hexane, elute with 5 mL MeOH:triethylamine 95:5. Evaporate the eluate to dryness under a
stream of nitrogen, reconstitute the residue in 100 |xL MeOH:2% tetramethylethylenediamine
50:50, inject an 80 |JLL aliquot. (Reagent was 0.1% R(-)-l-(naphthyl)ethylisocyanate in 1,2-
dimethoxyethane.)
HPLC VARIABLES
Column: 250 X 4.6 5 jxm C18 (Beckman)
Mobile phase: MeOH:THF'.buffer 52:7:41 (Buffer was 0.4% tetramethylethylenediamine ad-
justed to pH 3.0 with trifluoroacetic acid.)
Flow rate: 1
CHROMATOGRAM
Retention time: 20.6 (SRS), 21.8 (RSR), 26.2 (SSR), 28.2 (RRS)
Internal standard: 5-(3-[methylethylamino]-2-hydroxypropoxy)-l,2,3,4-tetrahydro-2,3-naphtha-
lenediol (SQ 11559, Bristol-Myers Squibb) (Racemate A was separated by HPLC using the
above system with MeOH:100 mM ammonium acetate 18:82 mobile phase and UV 230.) (13.5,
15.5 (enantiomers))
KEYWORDS
chiral; plasma; SPE; pharmacokinetics; derivatization
REFERENCE
Belas,F.J.; Phillips,M.A.; Srinivas,N.R.; Barbhaiya,RH.; Blair,I.A. Simultaneous determination of nadolol en-
antiomers in human plasma by high-performance liquid chromatography using fluorescence detection, Bio-
med.Chromatogr., 1995, 9, 140-145.
SAMPLE
Matrix: blood
Sample preparation: 200 IJLL Plasma + 50 |xL 4 |jig/mL IS + 250 |xL 10 M NaOH, vortex for 10
s, add 5 mL MTBE, rotate for 15 min, centrifuge at 2600 rpm for 5 min. Remove the organic
in 150 IJLL mobile phase, inject a 100 uL aliquot (maintain samples at 5 until injection).
HPLC VARIABLES
Column: 250 X 4.6 C18 octadecylsilane (Beckmann)
Mobile phase: MeCN:water 16:84 containing 5.75 g/L (NH4)H2PO4, pH 4.2
Flow rate: 1.4
CHROMATOGRAM
Retention time: 3.3
Internal standard: desmethyl nadolol (4.3)
KEYWORDS
REFERENCE
Srinivas,N.R.; Shyu,W.C; Shah,V.R.; Campbell,D.A.; Barbhaiya,R.H. High-performance liquid chromatographic
assay for the quantitation of nadolol in human plasma using fluorescence detection, Biomed.Chromatogr.,
1995, 9, 75-79.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 jxL 2 jjLg/mL desmethylnadolol + 750 JJLL 1 M NaOH,
vortex for 10 s, add 5 mL dichloromethane, rotate for 15 min, centrifuge at 2600 rpm for 10
reconstitute the residue in 50 |JLL 0.1% R-(-)-naphthylethylisocyanate in dichloromethane, let
stand for 1 h, reconstitute with 150 |xL mobile phase, inject a 125 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm C18 ODS (Beckman)
Mobile phase: MeOH:THF:water:phosphoric acid 52:7:41:0.001 adjusted to pH 3 with tetra-
methylene diamine
Flow rate: 1.4
CHROMATOGRAM
Retention time: 21 (SRS), 23 (RSR), 28 (SSR), 30 (RRS)
Internal standard: desmethylnadolol (13, 15 (enantiomers))
KEYWORDS
plasma; chiral; derivatization
REFERENCE
Srinivas,N.R.; Shyu,W.C; Dhah,V.R.; Campbell,D.A.; Barbhaiya,R.H. Stereoselective analysis of nadolol in hu-
man plasma, Biomed.Chromatogr., 1995, 9, 226-228.
SAMPLE
Matrix: blood
HPLC VARIABLES
Column: 300 X 3.9 4 (xm NovaPack C18
Flow rate: 0.8
Detector: UV 270
CHROMATOGRAM
KEYWORDS
REFERENCE
Tracqui,A.; Kintz,R; Mangin,R Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
a 10 (urine) or 30 (blood) JLJLL aliquot. (The detector wavelength shown is the wavelength of
HPLC VARIABLES
Column: 250 X 4.6 5 |im Symmetry C8 (Waters)
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 10 ^m Chiralcel OD
Mobile phase: EtOH:hexane:diethylamine 15:85:0.4 (A) or EtOH:hexane:diethylamine 20:80:0.4
(B)
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Retention time: 6.94 (RSR , RRS (A)), 9.30 (SRS (A)), 10.96 (SSR (A)) or 6.51 (RRS (B)), 6.98
(RSR (B)), 9.19 (SRS (B)), 10.83 (SSR (B))
KEYWORDS
chiral
REFERENCE
Aboul-Enein,H.Y.; Abou-Basha,L.I. HPLC separation of nadolol and enantiomers on Chiralcel OD column,
SAMPLE
Sample preparation: Grind tablet equivalent to about 50 mg nadolol, add 200 mL mobile phase,
sonicate for 15 min, make up to 250 mL with mobile phase, filter or centrifuge, to 20 mL
solution add 5 mL 1.2 mg/mL atenolol in mobile phase, mix, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 |xm LiChrosorb C2
Mobile phase: MeCN:buffer 35:65 (1 mL 100 mM HCl + 1200 mL water + 5.84 g NaCl, mix to
dissolve, add 700 mL MeOH, make up to 2 L, apparent pH 4.5.)
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Retention time: 6
Internal standard: atenolol (4.5)
OTHER SUBSTANCES
Simultaneous: impurities, acebutolol, alprenolol, metoprolol, oxprenolol, pindolol, practolol, pro-
pranolol, sotalol, timolol
KEY WORDS
REFERENCE
Patel,B.R.; Kirschbaum,J.J.; Poet,R.B. High-pressure liquid chromatography of nadolol and other p-adrenergic
blocking drugs, J.Pharm.ScL, 1981, 70, 336-338.
SAMPLE
Matrix: saliva
Sample preparation: Condition a 100 mg 1 mL Bond-Elut C2 SPE cartridge with 1 mL MeOH,
1 mL water, and 1 mL pH 9.0 borate buffer. Centrifuge a cotton roll soaked with saliva at 1000
g for 5 min, remove the liquid supernatant. 1 mL Supernatant + 50 (xL 10 fjig/mL (S)-alprenolol,
add to the SPE cartridge, wash with 500 (xL water, wash with 500 |xL MeCN, elute with two
500 JJLL portions of acidified MeOH. Evaporate the eluate to dryness under a stream of nitrogen
at 60, reconstitute the residue in 50 |xL mobile phase, mix for 15 s, inject a 40 |xL aliquot.
(Acidified MeOH was 50 mL MeOH + 300 |xL 96% acetic acid.)
HPLC VARIABLES
Guard column: RCSS silica guard-pack (Waters)
Column: 250 X 4.6 Chiralcel OD-H
Mobile phase: n-Hexane:EtOH:diethylamine 50:50:1
Flow rate: 1
Detector: F ex 225 em 290 cut-off filter
CHROMATOGRAM
Internal standard: (S)-alprenolol
KEYWORDS
SPE; chiral
REFERENCE
H6ld,K.M.; de Boer,D.; Zuidema,J.; Maes,R.A.A. Evaluation of the Salivette as sampling device for monitoring
p-adrenoceptor blocking drugs in saliva, J.Chromatogr.B, 1995, 663, 103-110.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 100 X 8 4 |xm NovaPak C18
Mobile phase: MeCN:triethylamine:water 9:0.3:91, adjusted to pH 3.0 with orthophosphoric acid
Flow rate: 3.0
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: metoprolol
REFERENCE
Wang,B.; Semple,H.A. Inhibition of metoprolol metabolism by amino acids in perfused rat livers. Insights into
the food effect?, Drug Metab.Dispos., 1997, 25, 287-295.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jig/mL solution in MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.9
OTHER SUBSTANCES
lol, mianserin, morazone, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine,
pranolol, prothipendyl, protriptyline, prox3nnetacaine, pseudoephedrine, pyrimethamine, quin-
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 0.5-2 mg/mL solution in mobile phase, add a 5-fold molar excess
of 1-naphthylisoeyanate, mix, let stand for 15 min, inject a 20 JULL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm CHM-LEU (R)-N-(3,5-dinitrobenzoyl)-L-leucine covalently bound to
aminopropyl silica (Hichrom)
Mobile phase: n-Hexane:EtOH:MeCN 90:10:2
Flow rate: 2
Detector: UV 239
CHROMATOGRAM
Retention time: 26 (SS), 28 (RS), 32 (SR), 36 (RR)
KEYWORDS
REFERENCE
Dyas,A.M.; Robinson,M.L.; Fell,A.F. Direct separation of nadolol enantiomers on a Pirkle-type chiral stationary
phase, J.Chromatogr., 1991, 586, 351-355.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 1 mg/mL solution in mobile phase, add 1 |xL 1-naphthyliso-
cyanate for each mL of solution, let stand for 20 min, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 3 fjim aminopropylsilica (HPLC Technology) (Prepare column by passing
through a solution of 5 g (R)-N-(3,5)-dinitrobenzoyl-L-leucine and 5 g 2-ethoxy-l-ethoxycar-
bonyl-l,2-dihydroquinoline in THF, wash with THF, wash with dichloromethane, wash with
100 mL dichloromethane containing 10 g trifluoroacetic anhydride, wash with dichloromethane,
equilibrate with mobile phase. [J.Chromatogr.,1991,586,351])
Mobile phase: n-Hexane:isopropanol 80:20
Flow rate: 2
Detector: UV 239
CHROMATOGRAM
Retention time: 26, 28, 31, 36 (different enantiomers)
KEYWORDS
chiral
REFERENCE
Dyas,A.M.; Robinson,M.L.; Fell,A.F. Influence of the structure of the alcoholic modifier on the enantioselective
separation of nadolol, J.Chromatogr.A, 1994, 660, 249-253.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
yltestosterone, methyprylon, metoprolol, mibolerone, nalorphine, naloxone, naltrexone, napha-
zoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam,
mycin, pyrilamine, pjn*ithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, re-
REFERENCE
Hill,D.W.; Kind,AJ- Reversed-phase solvent gradient HPLC retention indexes of drugs, J.Anal.ToxicoL, 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 ^m YMC-Pack ODS-AM-303 end-capped (YMC)
Flow rate: 1
Injection volume: 5
CHROMATOGRAM
Retention time: 14 (racemate A), 15.5 (racemate B)
KEYWORDS
diastereomers
REFERENCE
Hoshino,M.; Matsui,E.; Yajima,K.; Okahira,A. Direct high-performance liquid chromatographic separation of
the racemates and diastereomers of nadolol, J.Chromatogr.A, 1994, 664, 104-110.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 12 ^m l-m3nistoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
famotidine, isothipendyl, ketotifen, metiamide, metoprolol, moxonidine, naphazoline, nifenalol,
REFERENCE
Kaliszan,R.; Nasal,A.; Turowski,M. Binding site for basic drugs on ai-acid glycoprotein as revealed by chemo-
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 fxm Supelcosil LC-DP (A) or 250 X 4 5 ixm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ronidazole, midazolam, moclobemide, morphine, nalbuphine, naloxone, naphazoline, naproxen,
tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, trifluprom-
azine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohim-
bine, zopiclone
KEYWORDS
REFERENCE
J.Chromatogr.A, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 cellulose 3,5-dimethylphenylcarbamate/10-undecenoate bonded to allylsilica
Mobile phase: Heptane:isopropanol:diethylamine 80:20:0.1
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
KEYWORDS
chiral; a 1.36
REFERENCE
Oliveros,L.; Lopez,R; Minguillon,C; Franco,P. Chiral chromatographic discrimination ability of a cellulose 3,5-
dimethylphenylcarbamate/10-undecenoate mixed derivative fixed on several chromatographic matrices,
J.Liq.Chromatogr., 1995, 18, 1521-1532.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.5-2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
bumetone, phenobarbital, phenol, promazine, propranolol, pyrilamine, quinidine, ropinirole,
KEYWORDS
REFERENCE
Hanna,M.; de Biasi,V.; Bond,B-; Salter,C; Hutt,A.J.; Camilleri,P. Estimation of the partitioning characteristics
SAMPLE
Matrix: urine
Sample preparation: 3 mL Urine + 500 JJLL 5 M NaOH + I g anhydrous sodium sulfate + 2 mL
diethyl ether, shake mechanically for 15 min, centrifuge at 734 g for 5 min. Remove the organic
in 10 mL mobile phase, inject a 20 (xL aliquot.
HPLC VARIABLES
Guard column: (xBondapak C18
Mobile phase: MeCNiwater 40:60 containing 5 mM KH2PO4ZK2HPO4 adjusted to pH 6.5 with
phosphoric acid or 1 M KOH
Flow rate: 1.3
Detector: E, EG&G Princeton Applied Research PAR Model 400, glassy carbon cell +1300 mV,
d.c. mode, Ag/AgCl reference electrode (At the end of each day clean electrode with MeOH as
mobile phase and potential -600 mV for 1 min and +1500 mV for 10 min, repeat 3 times. If
necessary, wipe with a tissue wetted with water then a tissue wetted with MeOH.)
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: alprenolol, metoprolol, oxprenolol, timolol
Simultaneous: atenolol
REFERENCE
Maguregui,M.L; Alonso,R.M.; Jimenez,R.M. High-performance liquid chromatography with amperometric de-
tection applied to the screening of 0-blockers in human urine, J.Chromatogr.B, 1995, 674, 85-91.
SAMPLE
Matrix: urine
HPLC VARIABLES
Column: A 10 X 4.6 5 jjim Spherisorb cyanopropyl; B 250 X 4.6 Capcell Pak C18 UG-120
(Shiseido)
Mobile phase: A water; B Gradient. MeCN:buffer from 3:97 to 30:70 over 30 min, to 40:60 over
8 min (Buffer was 3.4 mIVL phosphoric acid adjusted to pH 3.0 with 5 M NaOH.)
Detector: UV 220
CHROMATOGRAM
Retention time: 9.5
OTHER SUBSTANCES
Extracted: acebutolol, alprenolol, amphetamine, atenolol, bopindolol, codeine, ephedrine, labe-
talol, metoprolol, morphine, oxprenolol, pindolol, propranolol, timolol
KEYWORDS
column-switching
REFERENCE
Saarinen,M.T.; Siren,H.; Riekkola,M.-L. Screening and determination of 0-blockers, narcotic analgesics and
1995, 664, 341-346.
Nadoxolol
Merck Index: 6432
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 211.1
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
163.
Nafarelin
86220-42-0 (acetate)
Merck Index: 6437
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 200 X 3 Spherisorb S5ODS-2
Mobile phase: Gradient. A was 0.05% phosphoric acid containing 0.5% (NHJ2SO4. B was MeCN.
A:B from 82:18 to 64:36 over 25 min, maintain at 64:36 for 2.5 min, return to initial conditions
over 1 min, re-equilibrate for 6.5 min. or Isocratic MeCN:0.05% phosphoric acid containing
0.5% (NHJ2SO4 24:76
Flow rate: 0.5
Detector: UV 210
CHROMATOGRAM
Retention time: 29 (gradient), 64 (isocratic)
OTHER SUBSTANCES
Simultaneous: buserelin, deslorelin, gonadorelin, goserelin, leuprolide
KEYWORDS
REFERENCE
Corran,P.H.; Sutcliffe,N. Identification of gonadorelin (LHRH) derivatives: comparison of reversed-phase high-
performance liquid chromatography and micellar electrokinetic chromatography, J.Chromatogr., 1993,636,
87-94.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Detector: UV 225
REFERENCE
Nerenberg,C; Foreman,J.; Chu,N.; Chaplin,M.D.; Kushinsky,S. Radioimmunoassay of nafarelin ([6-(3-(2-
naphthyl)-D-alanine)]-luteinizing hormone-releasing hormone) in plasma or serum, Anal.Biochem., 1984,
141, 10-16.
Nafcillin
Molecular formula: C2IH22N2O5S
CAS Registry No.: 147-52-4, 985-16-0 (Na salt),
7177-50-6 (Na salt monohydrate)
Merck Index: 6438
Lednicer No.: 1 412
SAMPLE
Matrix: blood
Sample preparation: 400 (xL Serum + 400 (xL MeCN, vortex for 10 s, shake slowly for 15 min,
vortex for 10 s, shake for 15 min, centrifuge at 3000 g for 10 min, inject a 50 |xL aliquot of the
HPLC VARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 9.0
OTHER SUBSTANCES
Extracted: cloxacillin, dicloxacillin, methicillin, oxacillin
Noninterfering: amdinocillin (mecillinam), amikacin, amoxicillin, ampicillin, carbenicillin, ce-
famandole, cefazolin, ceforanide, cefatoxamine, cefoxitin, cephalexin, cephaloridine, cephal-
othin, cephradine, cepharin, chloramphenicol, clindamycin, co-trimoxazole, fluorocytosine, gen-
KEYWORDS
serum
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Serum + 50 |xL 1 M sulfuric acid, mix for 30 s, add 2 mL dichlo-
romethane, mix for 2 min, centrifuge at 1130 g for 2 min. Remove 1.8 mL of the organic layer
and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 200 u-L
HPLC VARIABLES
Column: 300 X 4 10 urn jxBondapak C18
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 4
Internal standard: nafcillin
OTHER SUBSTANCES
Extracted: cloxacillin
KEYWORDS
serum; nafcillin is IS
REFERENCE
Jamaluddin,A.B.M.; Sarwar,G.; Rahim,M.A.; Rahman,M.K. Assay for cloxacillin in human serum utilising high-
performance liquid chromatography with ultraviolet detection, J.Chromatogr., 1989, 490, 243-246.
SAMPLE
Sample preparation: 500 |xL Serum or urine + 50 |xL 1 M sulfuric acid, mix for 5 s, add 2 mL
dichloromethane, shake for 2 min, centrifuge at 2000 rpm for 2 min. Remove the organic layer
and add it to 1 mL 40 mM pH 6.8 NaH2PO4, shake for 2 min, centrifuge at 2000 rpm for 2
min. Place the aqueous layer in a separate tube, agitate to remove traces of dichloromethane,
HPLC VARIABLES
Guard column: Co.Pell ODS
Column: 100 X 4.6 10 |xm MPLC Concept RP-8 (Brownlee)
Mobile phase: MeCN:40 mM NaH2PO4 6.2:20, pH 4.5
Flow rate: 1.6
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
KEY WORDS
serum; nafcillin is IS
REFERENCE
Teare,F.W.; Kwan,R.H.; Spino,M.; MacLeod,S.M. High-pressure liquid chromatographic assay of cloxacillin in
serum and urine, J.Pharm.ScL, 1982, 7I7 938-941.
SAMPLE
Sample preparation: Condition a Bondelut SPE cartridge (cat. no. 607101) with two 1 mL por-
tions of MeCN and 1 mL buffer. Dilute urine 1:10 with buffer. 100 (JLL Serum or diluted urine
+ 100 U-L buffer, vortex for 10 s, add to the SPE cartridge, wash with 1 mL buffer, wash with
500 |JLL MeCN.buffer 15:85, elute with 500 jxL MeCN:buffer 70:30, inject a 10 |JLL portion of
the eluate. (Buffer was 70 mM KH2PO4 adjusted to pH 5.0 with 5 M NaOH.)
HPLC VARIABLES
Guard column: 45 X 4 10 u,m Spherisorb C-18
Column: 300 X 4 (xBondapak C-18
Flow rate: 1.1
Detector: UV 220
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
Extracted: mezlocillin
Noninterfering: acetaminophen, dexamethasone, diazepam, furosemide, morphine
KEY WORDS
serum; SPE; nafcillin is IS
REFERENCE
Fiore,D.; Auger,F.A.; Drusano,G.L.; Dandu,V.R.; Lesko,L.J. Improved micromethod for mezlocillin quantitation
in serum and urine by high-pressure liquid chromatography, Antimicrob.Agents Chemother., 1984,26,775-
777.
SAMPLE
Matrix: cells
Sample preparation: 100 |xL Cell suspension + 100 u,L cefoperazone solution + 100 |xL Hanks
balanced salt solution, sonicate 30 min, add 800 |JLL MeCN, centrifuge at 13000 g for 5 min,
remove supernatant. Dry supernatant under air, dissolve in 100 uX mobile phase, inject 75
uL.
HPLCVARIABLES
Column: u,Bondapak C18
Flow rate: 1.5
Detector: UV 224
CHROMATOGRAM
Retention time: 8.0
Internal standard: 5-(p-Hydroxyphenyl)-5-phenylhydantoin
REFERENCE
microb. Agents Chemother., 1994, 38, 1059-1064.
SAMPLE
Sample preparation: Dilute with water (if necessary), inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 300 X 4 ixBondapak phenyl
Mobile phase: MeOH:water 50:50 containing 10 mM ammonium acetate
Flow rate: 2
Detector: UV 280
CHROMATOGRAM
Retention time: 4.5
KEYWORDS
saline; 5% dextrose; stability-indicating
REFERENCE
Das Gupta,V.; Stewart,K.R. Quantitation of carbenicillin disodium, cefazolin sodium, cephalothin sodium, naf-
cillin sodium, and ticarcillin disodium by high-pressure liquid chromatography, J.Pharm.ScL, 1980, 69,
1264-1267.
SAMPLE
HPLC VARIABLES
Flow rate: 1
Detector: UV 225
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: amoxicillin, ampicillin, cloxacillin, dicloxacillin, methicillin, oxacillin, penicillin
G, penicillin V
KEYWORDS
REFERENCE
matography, J.Assoc.Off.Anal.Chem., 1984, 67, 228-231.
SAMPLE
Sample preparation: Dilute 1:100 with water, inject a 10 JJLL aliquot.
HPLC VARIABLES
Flow rate: 0.5
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: lidocaine (broad tailing peak)
KEYWORDS
REFERENCE
Hagan,R.L.; Carr-Lopez,S.M.; Strickland, J.S. Stability of nafcillin sodium in the presence of lidocaine hydro-
chloride, Am. J.Health-Syst.Pharm., 1995, 52, 521-523.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Bakerbond C18
Mobile phase: MeCN:50 mM sodium acetate 30:70
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
KEYWORDS
injections; saline; water; stability-indicating
REFERENCE
Stiles,M.L.; Allen,L.V.,Jr.; Prince,S.J. Stability of various antibiotics kept in an insulated pouch during admin-
istration via portable infusion pump, Am.J.Health-Syst.Pharm., 1995, 52, 70-74.
SAMPLE
Matrix: milk
Sample preparation: Mix 10 mL milk with 2 mL 100 mM tetraethylammonium chloride, add
40 mL MeCN slowly with continual stirring, let stand for 10 min, decant the supernatant
through a plug of glass wool. Collect 40 mL nitrate, add 2 mL buffer, evaporate to 1-2 mL
under reduced pressure at 40-50, dilute to 4 mL with water, filter (0.45 |xm PVDF). Inject a
2 mL aliquot onto a 150 X 4.6 5 |xm Supelcosil LC-18 column, elute with MeCNrIO mM KH2PO4
0:100 for 3 min, to 60:40 over 37 min at 1 mL/min, collect a 1.5-2 mL aliquot containing the
compound (ca. 30.3 min), evaporate to <1 mL under reduced pressure, make up to 1 mL with
water, inject an aliquot. (Prepare the buffer by mixing 10 mM KH2PO4 and 10 mM Na2HPO4
in a 5:1 ratio, pH 6.)
HPLC VARIABLES
Mobile phase: MeCN:buffer 38:62 (Buffer was 2 mM phosphoric acid containing 8 mM potassium
dihydrogen phosphate.)
Flow rate: 1
Detector: UV 215
REFERENCE
Moats,W.A.; Romanowski,R.D. Multiresidue determination of p-lactam antibiotics in milk and tissues with the
aid of high-performance liquid chromatographic fractionation for clean up, J.Chromatogr.A, 1998,812,237-
247.
SAMPLE
Matrix: milk
Sample preparation: Condition a 3 mL 500 mg Baker-10 C18 SPE cartridge (J.T. Baker) with
3 mL MeOH and 3 mL distilled water. Add 20 mL MeCN to 10 mL milk, vortex for 1 min,
centrifuge at 1500 g for 10 min, concentrate the supernatant to 2-3 mL on a rotary evaporator
at 40, add to the SPE cartridge, dry the cartridge under reduced pressure for 3 min, elute
with 1 mL MeOH, filter (0.45 jjim) the eluate, inject a 10 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Kaseisorb LC ODS-300-5 (Tokyo Kasei)
Mobile phase: MeCN:MeOH:50 mM KH2PO4 buffer 20:10:80 containing 5 mM sodium 1-deca-
nesulfonate, adjusted to pH 3.5 with concentrated phosphoric acid
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 24
OTHER SUBSTANCES
Extracted: ampicillin, cloxacillin, dicloxacillin, penicillin G
KEYWORDS
SPE
REFERENCE
Takeba,K; Fujinuma,K; Miyazaki,T.; Nakazawa,H. Simultaneous determination of (Hactam antibiotics in milk
by ion-pair liquid chromatography, J.ChromatogrA, 1998, 812, 205-211.
SAMPLE
Matrix: milk
37, add 50 MeCN, shake vigorously for 1 min, centrifuge at 9000 g for 10 min, decant, add 5
g NaCl, swirl to dissolve, add 100 mL dichloromethane, shake for 1 min, centrifuge at 1000 g
for 10 min. Remove top aqueous layer and extract organic layer with 25 mL 10% NaCl by
shaking and centrifuging as before. Combine aqueous layers, add 1 mL 0.3% mercuric chloride
in water, let stand 30 min, add 1 mL 2 M HCl, extract with three 50 mL portions of dichlo-
romethane by shaking each portion for 1 min and centrifuging at 1000 g for 10 min, filter
sulfate. Extract acid aqueous layer again with 25 mL dichloromethane. Combine dichlorome-
thane layers and evaporate to dryness at 35 under reduced pressure. Dissolve residue in 2
mL IS solution, inject a 20 JJLL aliquot. (Prepare IS solution by dissolving 10 |xL benzaldehyde
with 5 mL 5% NaHCO3 solution, filter through 10-20 g anhydrous sodium sulfate. Extract acid
HPLC VARIABLES
Flow rate: 1
CHROMATOGRAM
Retention time: 9.0
OTHER SUBSTANCES
Extracted: penicillin V, phenethicillin, penicillin G, oxacillin, cloxacillin, dicloxacillin, methicillin
KEYWORDS
derivatization
REFERENCE
Munns,R.K.; Shimoda,W.; Roybal,J.E.; Vieira,C. Multiresidue method for determination of eight neutral (3-
971.
SAMPLE
Matrix: milk
tion filter, extract with an equal volume of 1:1 methylene chlorideihexane, centrifuge aqueous
nylon). Inject 50 u-L onto column with mobile phase A, run mobile phase A for 30 min and elute
HPLC VARIABLES
Column: 100 X 8 Radial-Pak 10 urn ^Bondapak C18
Flow rate: A 3; B 2
Detector: E, Waters 464 pulsed electrochemical detector using a thin layer cell with a Ag/AgCl
0.333 s.
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
Simultaneous: penicillin V, ampicillin, methicillin, penicillin G, oxacillin, dicloxacillin, cloxacillin
KEY WORDS
column-switching
REFERENCE
Kirchmann,E.; Earley,R-L-; Welch,L.E. The electrochemical detection of penicillins in milk, J.Liq.Chromatogr.,
1994, 17, 1755-1772.
SAMPLE
Matrix: milk
Sample preparation: Condition a Bond Elut C8 SPE cartridge with 5 mL MeOH and 5 mL water.
20 mL Milk + 20 mL buffer, heat at 60 for 20 min or until milk curdles, centrifuge for 10 min,
add the supernatant to the SPE cartridge, wash with two 2.5 mL portions of water, elute with
2.5 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen, extract the residue
with three 100 |xL portions of 50 mM pH 6.0 potassium phosphate buffer, filter (0.2 |xm), inject
an aliquot of the nitrate. (Buffer was 545 mL 100 mM citric acid, 455 mL 200 mM Na2HPO4,
and 74.4 g EDTA, adjust to pH 4.5 with ammonium hydroxide, make up to 2 L with water.)
HPLC VARIABLES
Mobile phase: MeOH:50 mM pH 6.0 potassium phosphate buffer 35:65
Flow rate: 1
Detector: UV 210 or Charm II assay
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ampicillin, ceftiofur, cephapirin, cloxacillin, dicloxacillin, oxacillin, penicillin G
Simultaneous: amoxicillin
KEYWORDS
SPE
REFERENCE
Zomer,E.; Quintana,J.; Saul,S.; Charm,S.E. LC-Receptograms: A method for identification and quantitation of
(3-lactams in milk by liquid chromatography with microbial receptor assay, J.AOAC Int., 1995, 78, 1165-
1172.
SAMPLE
Matrix: solutions
Sample preparation: Prepare an aqueous solution, inject a 200 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 4 |xm Micropak SPC-18 C18
Mobile phase: Gradient. MeCN: 10 mM orthophosphoric acid from 15:85 to 60:40 over 20 min
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 19
OTHER SUBSTANCES
Simultaneous: dicloxacillin, methicillin, penicillin G, penicillin V, cloxacillin, carbenicillin
REFERENCE
Moats ,W. A. Effect of the silica support of bonded reversed-phase columns on chromatography of some antibiotic
SAMPLE
Matrix: solutions
moethanone in a 1:2:4 molar ratio in DMF at 45 for 2 h (use dibenzo-18-crown-6 to make the
sodium salt soluble), inject a 10 jxL aliquot. (Preparation of l-(2,5-dihydroxyphenyl)-2-bro-
HPLC VARIABLES
Mobile phase: MeOH: 100 mM pH 6.5 sodium acetate 58:42
Flow rate: 1
electrode
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: carbenicillin, cephapirin, cloxacillin, dicloxacillin, hetacillin, methicillin, oxacillin,
penicillin G
KEYWORDS
derivatization
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 2.5-5 |xg/mL solution, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 80 X 4.6 3.65 |xm Zorbax Rx-SIL (similar to Zorbax SB-C8 (Mac-Mod Analytical))
Flow rate: 1
Detector: UV 235
CHROMATOGRAM
REFERENCE
Kirkland,K.M.; McCombs,D.A.; Kirkland,J.J. Rapid, high-resolution high-performance liquid chromatographic
analysis of antibiotics, J.ChromatogrA, 1994, 660, 327-337.
Nafronyl
CAS Registry No.: 31329-57-4, 3200-06-4 (acid oxalate)
Merck Index: 6440
Lednicer No.: 2 213
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 p,m Symmetry C8 (Waters)
mL/min)
Detector: UV 225.2
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 4 X 4 5 (xm LiChrospherlOORP-18
Column: 250 X 4 5 |xm Spherisorb ODS 2
Mobile phase: MeCN.buffer 60:40. (Buffer was 20 mM sodium acetate containing 0.28% triethyl-
amine, adjusted to pH 4.5 with acetic acid.)
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
REFERENCE
Yang,H.; Thyrion,F.C. Determination of six pharmaceuticals and their degradation products in reversed-phase
high performance liquid chromatography by using amine additives, J.Liq.Chromatogr.Rel.Technol., 1998,
21, 1347-1357.
Naftifine
Merck Index: 6442
Lednicer No.: 4 55
SAMPLE
Matrix: blood, lymph, tissue
Sample preparation: Grind skin in a mortar under liquid nitrogen. Homogenize blood, lymph,
or skin with 10 volumes of EtOH, centrifuge at 6000 g for 10 min, wash the sediment twice
with EtOH. Evaporate, reconstitute, inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 10 |xm Lichrosrb RP8
Mobile phase: Gradient. MeOHrIO mM pH 7.2 Sorensen buffer from 50:50 to 100:0 over 30 min
Detector: UV 260
CHROMATOGRAM
Retention time: 30
OTHER SUBSTANCES
KEYWORDS
rat; skin
REFERENCE
Grimus,R-C.; Schuster,! The role of the lymphatic transport in the enteral absorption of naftifine by the rat,
Xenobiotica, 1984, 14, 287-294.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 100 |xg/mL solution in MeOH, inject an aliquot.
HPLC VARIABLES
Column: 125 X 4 5 |xm LiChrospher 60 RP-Select-B
Mobile phase: MeCN: pH 2 trifluoroacetic acid buffer 35:65.
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: cloxyquin, chlorphenesin, sulbentine, tolnaftate
KEYWORDS
photodegradation; kinetics
REFERENCE
Thoma,K.; Kiibler,N.; Reimann,E. Untersuchung der Photostabilitat von Antimykotica. 3. Mitteilung: Photo-
stabilitat lokal wirksamer Antimykotica [Photodegradation of antimycotic drugs. 3. Communication: Pho-
todegradation of topical antimycotics], Pharmazie, 1997, 52, 362-373.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 400 jxg/mL solution in MeOH, inject an aliquot.
HPLCVARIABLES
Column: 125 X 2 4 fxm Superspher 60 RP-18
Mobile phase: Gradient. A was MeOH:water 90:10. B was MeOH:pH 2 trifluoroacetic acid buffer
15:85. A:B 0:100 for 10 min, 20:80 for 15 min, then A:B 80:20 (step gradient).
Flow rate: 0.5
Detector: UV 225
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: cloxyquin, chlorphenesin, sulbentine, tolnaftate
KEYWORDS
photodegradation; kinetics
REFERENCE
Thoma,K.; Kiibler,N.; Reimann,E. Untersuchung der Photostabilitat von Antimykotica. 3. Mitteilung: Photos-
tabilitat lokal wirksamer Antimykotica [Photodegradation of antimycotic drugs. 3. Communication: Pho-
todegradation of topical antimycotics], Pharmazie, 1997, 52, 362-373.
Nalbuphine
Merck Index: 6444
Lednicer No.: 2 319
SAMPLE
Matrix: blood
Sample preparation: Add 1 mL of pH 9 borate buffer to 500 jxL plasma, extract with 10 mL
chloroformdsopropanol 98:2, shake mechanically for 15 min, centrifuge at 2000 rpm for 10 min.
Remove the organic layer using phase-separating filter paper and add it to 200 jxL 17 mM
phosphoric acid, shake mechanically for 2 min, centrifuge at 2000 rpm for 5 min. Inject a 50
IxL aliquot of the aqueous layer.
HPLC VARIABLES
Guard column: 20 X 4.6 5 ixm Hypersil ODS
Mobile phase: MeOHrpH 3.4 phosphate buffer 20:80
Flow rate: 1.0
Detector: E, ESA Coulochem II Model 5200, Model 5020 guard cell 550 mV, Model 5021 analyt-
ical cell in oxidation screening mode E 1=60 mV, E2=450 mV
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: metabolites, caffeine, codeine, naloxone, theobromine, theophylline
KEYWORDS
plasma; recycle mobile phase; pharmacokinetics
REFERENCE
de Cazanove,E; Kinowski,J.-M.; Audran,M.; Rochette,A-; Bressolle,F. Determination of nalbuphine in human
plasma by high-performance liquid chromatography with electrochemical detection. Application to a phar-
macokinetic study, J.Chromatogr.B, 1997, 690, 203-210.
SAMPLE
Matrix: blood
Sample preparation: Condition a 10 mL 130 mg Bond Elut Certify SPE cartridge with 2 mL
MeOH and 2 mL 200 mM pH 8 Tris buffer without column drying. Dilute 500 |xL plasma with
2 mL 200 mM pH 8 Tris buffer, add 50 ng IS, pass slowly through the column. Wash with 2
mL water, 2 mL 10 mM pH 3.3 acetic acid and dry completely. Wash with 3 mL MeOH and
elute with two 2 mL portions of dichlormethane:2-propanol:ammonia 79:20:1. Evaporate the
eluate to dryness under a stream of nitrogen at 40, reconstitute the residue in 200 |xL mobile
phase containing 2% of isopropanol, inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 LD. 5 jxm LiChrospher C18
Mobile phase: MeOH:water 20:80 containing 24 mM KH2PO4 and 60 mM EDTA, pH 3.4
Flow rate: 0.8
Detector: E, ESA Coulochem, Model 5020 guard cell +0.5 V, Model 5011 dual electrode analytical
cell operating in the oxidation screening mode, El +0.06 V, E2 +0.4 V
CHROMATOGRAM
Internal standard: 6-monoacetylmorphine (8.4)
OTHER SUBSTANCES
Noninterfering: morphine, nalorphine, naloxone, naltrexone, norbuprenorphine
KEYWORDS
REFERENCE
Nicolle,E.; Veitl,S.; Guimier,C; Bessard,G. Modified method of nalbuphine determination in plasma: validation
and application to pharmacokinetics of the rectal route, J.Chromatogr.B, 1997, 690, 89-97.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 500 mM pH 9.25 sodium carbonate buffer + 3 mL
hexanedsoamyl alcohol 9:1, mix on a rotary shaker for 30 min, centrifuge at 1880 g for 20 min,
freeze at -20 for 1 h (for rabbit plasma perform on half-scale). Remove the organic layer and
evaporate it to dryness under a stream of air, reconstitute in 250 |xL mobile phase, inject a
200 JJIL aliquot.
HPLC VARIABLES
Column: 100 X 2 10 jjim ixPorasil
Flow rate: 1
CHROMATOGRAM
Internal standard: nalbuphine
OTHER SUBSTANCES
Simultaneous: butorphanol, morphine, ethylmorphine, codeine, fentanyl, meperidine, tramadol,
buprenorphine
Interfering: nalorphine
KEYWORDS
plasma; human; pig; dog; rabbit; nalbuphine is IS
REFERENCE
J.Chromatogr., 1991, 570, 339-350.
SAMPLE
Matrix: blood
Sample preparation: 500 (xL Plasma + 100 |xL 200 ng/mL 6-monoacetylmorphine in water + 2
mL buffer + 10 mL hexane:dichloromethane:isopropanol 69:30:1, shake mechanically at 60 rpm
for 15 min, centrifuge at 2000 g at 4 for 5 min. Remove the upper organic layer and add it to
200 |JLL 17 mM phosphoric acid, shake for 2 min, centrifuge at 2000 g at 4 for 5 min, inject a
50 |xL aliquot of the aqueous layer. (Buffer was 100 mM boric acid and 100 mM KCl adjusted
to pH 9 with 100 mM NaOH.)
HPLCVARIABLES
Column: 150 X 4.6 5 |xm Lichrospher C18
Mobile phase: MeOH:water 20:80 containing 24 mM KH2PO4 and 0.06 mM disodium EDTA
adjusted to pH 3.4 with orthophosphoric acid
Flow rate: 0.8
Detector: E, ESA Model 5100A Coulochem, Model 5020 guard cell 0.50 V, Model 5011 analytical
cell in oxidation screening mode, first electrode 0.06 V, second electrode 0.40 V
CHROMATOGRAM
Retention time: 8.5
OTHER SUBSTANCES
Extracted: naloxone
Noninterfering: buprenorphine, codeine, dextromoramide, dextropropoxyphene, ethylmorphine,
fentanyl, methadone, morphine, nalorphine, norcodeine, pholcodine
KEYWORDS
plasma
REFERENCE
Nicolle,E.; Michaut,S.; Serre-Debeauvais,F.; Bessard,G. Rapid and sensitive high-performance liquid chromat-
ographic assay for nalbuphine in plasma, J.Chromatogr.B, 1995, 663, 111-117.
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 259
CHROMATOGRAM
KEY WORDS
imipramine; desipramine; levomepromazine; hydroxyzine; ninumic acid; penbutolol; fluvox-
REFERENCE
254-262.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 5 jxm Biophase ODS
Mobile phase: MeCN:buffer:water 20:10:70 containing 3 mM sodium 1-heptanesulfonate, pH 3.7
(Buffer was 2.1 M acetic acid containing 400 mM ammonium acetate.)
Flow rate: 1
Detector: E, Bioanalytical Systems Model 4B, glassy carbon electrode 0.95 V, Ag/AgCl reference
electrode
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: ritodrine
REFERENCE
Kuhnert,P.; Erhard,P.; Dixon,A.; Kuhnert,B.; Gross,T. Determination of ritodrine in plasma using HPLC,
J.Liq.Chromatogr., 1983, 6, 2775-2783.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ronidazole, midazolam, moclobemide, morphine, nadolol, naloxone, naphazoline, naproxen, ni-
fedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymetazoline, pa-
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Sample preparation: 1.25 mL (?) Urine + 250 |xL 20000 U/mL p-glucuronidase + 500 |xL 1 M
pH 5.2 sodium acetate buffer, heat at 56 for 12 h, add 1 mL 40% pH 9.2 phosphate buffer, add
50 |xL 10 |xg/mL levallorphan, add 10 mL chloroform:isopropanol:n-heptane 50:17:33, agitate,
centrifuge at 2000 g for 10 min. Remove the aqueous phase and add it to 5 mL 200 mM HCl,
extract. Remove the aqueous phase and add it to 1 mL 40% pH 9.2 phosphate buffer, add 500
IxL concentrated ammonia solution, extract with 5 mL chloroform. Remove the organic layer
and evaporate it to dryness under vacuum at 45, reconstitute the residue in 100 |xL mobile
phase, inject a 70 JJLL aliquot.
HPLCVARIABLES
Mobile phase: MeOH:THF:10 mM pH 2.6 KH2PO4 65:5:30
Flow rate: 0.8
Detector: UV 215-238 (scan mode)
CHROMATOGRAM
REFERENCE
Kintz,R; Tracqui,A.; Mangin,P. Determination of nalbuphine using high-performance liquid chromatography
coupled to photodiode-array detection and gas chromatography coupled to mass spectrometry,
J.Chromatogn, 1992, 579, 172-176.
Nalidixic acid
15769-77-4 (Na salt monohydrate)
Merck Index: 6446
Lednicer No.: 1 429
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond-Elut C18 SPE cartridge with two 3 mL portions
of MeOH and 3 mL of 0.05% orthophosphoric acid. Centrifuge plasma at 500 g for 5 min. Mix
200 jxL centrifuged plasma with 3.2 mL buffer, vortex for 1 min. Add the mixture to the SPE
cartridge under low vacuum, wash with 1 mL water and 500 (xL 0.05% orthophosphoric acid
solution, dry under vacuum. Elute with six 250 |xL portions of MeCN. Evaporate the combined
eluates to dryness under a stream of nitrogen at 40. Reconstitute the residue with 200 |xL
buffer, vortex, sonicate, let stand for 30 min, vortex again. Inject a 50 |xL aliquot. (Buffer was
1/15 M pH 8.0 phosphate buffer.)
HPLC VARIABLES
Guard column: 4 x 4 5 iim LiChroSpher 100 RP-18 end-capped
Column: 125 X 4 5 |xm LiChroSpher 100 RP-18 end-capped
Mobile phase: MeCN:20 mM pH 2.3 orthophosphoric acidrdimethylformamide 10:60:30
Flow rate: 0.8
Detector: UV 340
CHROMATOGRAM
Internal standard: nalidixic acid
OTHER SUBSTANCES
Extracted: oxolinic acid
Noninterfering: 2-phenoxyethanol
KEY WORDS
plasma; sea bass; SPE; nalidixic acid is IS; fish
REFERENCE
Loussouarn,S.; Pouliquen,H.; Armand,F. High-performance liquid chromatographic determination of oxolinic
acid in the plasma of seabass (Dicentrarchus labrax) anaesthetized with 2-phenoxyethanol, J. Chromatogr.B,
1997, 698, 251-259.
SAMPLE
Matrix: blood
HPLC VARIABLES
Mobile phase: MeCN:MeOH:buffer 16:34:50 containing 5 mM tetrabutylammonium sulfate, ad-
justed to pH 2.5 with 1 NaOH (Buffer was 100 mM citric acid containing 200 mM ammonium
perchlorate.)
Flow rate: 0.8
Detector: UV 258
CHROMATOGRAM
Internal standard: cinoxacin (6.26)
KEY WORDS
REFERENCE
Zlotos,G.; Biicker,A.; Kinzig-Schippers,M.; Sorgel,R; Holzgrabe,U. Plasma protein binding of gyrase inhibitors,
J.Pharm.ScL, 1998, 87, 215-220.
SAMPLE
residue with 50 |JLL MeCN.water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 258.2
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Measure out powdered tablets or suspension containing 150 mg nalidixic
acid, add 400 mL MeOH, sonicate for 30 min, shake for 30 min, sonicate for 30 min. Make up
to 500 mL with MeOH, mix , filter. Dilute 3.0 mL filtrate and 1.0 mL 800 |xg/mL sulfanilic acid
in mobile phase to 25 mL with MeOH. Inject a 20 |xL aliquot.
HPLCVARIABLES
Mobile phase: MeOH:buffer 67.5:32.5 (Prepare mobile phase by dissolving 784 mg K2HPO4 in
325 mL water. Dissolve 2.62 g hexadecyltrimethylammonium bromide in 350 mL MeOH. Com-
bine solutions, add 325 mL MeOH, mix.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 4.9
Internal standard: sulfanilic acid (3.5)
KEYWORDS
tablets; suspensions
REFERENCE
Walker,S.T. Liquid chromatographic determination of nalidixic acid in pharmaceutical preparations, J.AOAC
Int., 1996, 79, 431-433.
SAMPLE
Sample preparation: Powder tablets, add 40 mg trimethoprim, dissolve in 70 mL MeOH, filter
(paper), wash filter with MeOH, make up filtrate to 100 mL with MeOH. Dilute a 1 mL aliquot
to 10 mL, inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4 10 /xm Nucleosil C18
Mobile phase: MeOH:MeCN:water:triethylamine 20:20:60:0.15, pH adjusted to 3.0 with phos-
phoric acid
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: 10
Internal standard: trimethoprim (3)
OTHER SUBSTANCES
Simultaneous: phenazopyridine
KEYWORDS
tablets
REFERENCE
Sane,R.T.; Ghadge,J.K; Jani,A.B.; Vaidya,A.J.; Kotwal,S.S. Simultaneous high-performance liquid chromato-
graphic determination of haloperidol with propantheline bromide, nalidixic acid with phenazopyridine hy-
drochloride, and dipyridamole with aspirin in combined dosage (forms), Indian Drugs, 1992, 29, 240244.
SAMPLE
Sample preparation: Pulverize tablets, add 10 mL water, swirl for 2-3 min, make up to 250 mL
with MeOH, shake mechanically for 5 min. Remove a 5 mL aliquot and centrifuge it at 3000
rpm for 5 min. Remove 100 |xL of the supernatant and add it to 200 |xL 1 mg/mL methyl p-
hydroxybenzoate, make up to 10 mL with mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:50 mM ammonium acetate 30:5:65, pH 5
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Internal standard: methyl p-hydroxybenzoate (4.0)
KEYWORDS
tablets
REFERENCE
Foda,N.H. High-performance liquid chromatographie determination of nalidixic acid in tablets,
J.Liq.Chromatogr., 1995, 18, 4135-4147.
SAMPLE
Matrix: solutions
HPLC VARIABLES
acid
Flow rate: 0.6
Detector: UV 275
CHROMATOGRAM
Retention time: 5
Internal standard: nalidixic acid
OTHER SUBSTANCES
Simultaneous: ciprofloxacin, fenbufen, felbinac
KEY WORDS
nalidixic acid is IS
REFERENCE
Naora,K.; Katagiri,Y.; Ichikawa,N.; Hayashibara,M.; Iwamoto,K. Simultaneous high-performance liquid chro-
matographie determination of ciprofloxacin, fenbufe and felbinac in rat plasma, J.Chromatogr., 1990, 530,
186-191.
SAMPLE
Matrix: tissue
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 10 mL MeOH and 20 mL
water. Homogenize 5 g tissue with 100 mL MeOH:0.1% metaphosphoric acid 40:60 at high
speed for 2 min, filter homogenate through a 2 mm layer of Hyflo Super-Cel. Evaporate the
filtrate to about 20 mL under reduced pressure at 40, add the residue to the SPE cartridge at
5 mL/min, wash with 20 mL water, wash with 10 mL MeOH:water 5:95, elute with 10 mL
MeOH. Evaporate the eluate to dryness under reduced pressure, reconstitute the residue in 1
mL mobile phase, inject a 10 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Kaseisorb LC ODS 300-5 (Tokyo Kasei Kogyo)
Flow rate: 0.5
CHROMATOGRAM
Retention time: 11
OTHER SUBSTANCES
Extracted: oxolinic acid, piromidic acid (UV 280)
Noninterfering: ampicillin, chlortetracycline, colistin, doxycycline, erythromycin, oxytetracy-
cline, sodium nifulstyrate, spiramycin, sulfamdimethoxine, sulfamonomethoxine, sulfisozole,
tetracycline, thiamphenicol
KEY WORDS
eel; fish; trout; carp; muscle; SPE
REFERENCE
Horie,M.; Saito,K; Hoshino,Y.; Nose,N.; Mochizuki,E.; Nakazawa,H. Simultaneous determination of nalidixic
acid, oxolinic acid and piromidic acid in fish by high-performance liquid chromatography with fluorescence
and UV detection, J.Chromatogr., 1987, 402, 301-308.
SAMPLE
Matrix: tissue
10 mL water. Homogenize 5 g tissue with 100 mL MeOH:0.2% metaphosphoric acid 40:60 for
2 min, filter through a 1 mm layer of Hyflo Super-Cel. Evaporate the filtrate under reduced
pressure at 40 to 10 mL, add the residue to the SPE cartridge, wash with 10 mL water, elute
with 10 mL MeOH. Evaporate the eluate to dryness under reduced pressure, take up the
residue in 1 mL mobile phase, inject a 10 |xL aliquot.
HPLC VARIABLES
Guard column: 15 X 3.2 Newguard RP-8
Column: 150 X 4.6 5 |xm Inertsil ODS
Mobile phase: MeCN:5 mM oxalic acid 45:55
Flow rate: 0.5
Detector: UV 265
CHROMATOGRAM
Retention time: 11
OTHER SUBSTANCES
Extracted: sulfadimethoxine, sulfamonomethoxine, sulfisozole, oxolinic acid, piromidic acid, so-
dium nifurstyrenate, furazolidone
KEYWORDS
fish; SPE
REFERENCE
Horie,M.; Saito,K.; Hoshino,Y.; Nose,N.; Nakazawa,H.; Yamane,Y. Simultaneous determination of residual syn-
thetic antibacterials in fish by high-performance liquid chromatography, J.Chromatogr., 1991, 538, 484-
491.
SAMPLE
Matrix: tissue
10 mL water. Homogenize 5 g tissue with 100 mL MeOH:0.2% metaphosphoric acid 1:2 at high
speed for 2 min, filter homogenate through a 2 mm layer of Hyflo Super-Cel. Evaporate the
filtrate to about 30 mL under reduced pressure at 50, add the residue to the SPE cartridge,
wash with 20 mL water, elute with 10 mL MeOH. Evaporate the eluate to dryness under
reduced pressure, reconstitute the residue in 1 mL mobile phase, inject a 20 u,L aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |jim Inertsil ODS-2
Mobile phase: MeCN: 100 mM pH 4.5 ammonium acetate buffer 40:60
Flow rate: 0.8
Detector: MS, Shimadzu LCMS-QP 1000 quadrupole, Vestec thermospray interface, filament-on
mode (ionization by electron beam), vaporizer 165, ion source block 260, repeller potential 0
V, electron multiplier 3000 V, ionization potential 1000 eV, positive ion mode, scan m/z 150-
400; SIM m/z 233
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
Extracted: oxolinic acid, piromidic acid
KEYWORDS
fish; muscle; thermospray; SPE
REFERENCE
Horie,M.; Saito,K; Nose,N.; Tera,M.; Nakazawa,H. Confirmation of residual oxolinic acid, nalidixic acid and
piromidic acid in fish by thermospray liquid chromatography-mass spectrometry, J.Liq.Chromatogr., 1993,
16, 1463-1472.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Oster model 54841) 10 g catfish tissue with 50 mL acetone
for 10-20 s, centrifuge at 3000 rpm for 2 min, filter (paper) supernatant. Repeat extraction
twice more, combine extracts, add 15 mL n-propanol, evaporate to 15 mL under reduced pres-
sure at 40. Transfer residue to another container using 20 mL acetone, 30 mL hexane, and 60
mL 3% NaCl. Shake vigorously for 10 s, centrifuge at 3000 rpm for 1 min, discard hexane layer,
wash with another 30 mL hexane. Extract the remaining mixture twice with 25 mL portions
of chloroform by shaking vigorously for 1 min. Combine the chloroform extracts and add them
to 25 mL 100 mM NaOH, shake vigorously for 1 min. Discard the chloroform layer and wash
the aqueous layer with 25 mL chloroform with gentle rocking (10 rocks). Add the aqueous layer
to 25 mL 75 mM phosphoric acid (pH 6 0.5), extract twice with 25 mL portions of chloroform
(vigorous shaking for 1 min). Filter (paper) extracts and evaporate them to dryness under
reduced pressure at 40, add 2 mL MeCN, evaporate to dryness, reconstitute in 1 mL mobile
phase, sonicate briefly, filter (0.45 (xm), inject a 100 uL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 |xm PLRP-S polymer (Polymer Laboratories)
Mobile phase: MeCN:THF:20 mM phosphoric acid 16:12:72
CHROMATOGRAM
Retention time: 9.9
OTHER SUBSTANCES
Extracted: flumequine, oxolinic acid, piromidic acid (UV 280)
KEYWORDS
catfish; fish
REFERENCE
Munns,R.K; Turnipseed,S.B.; Pfenning,A.R; Roybal,J.E.; Holland,D.C.; LongAR-; Plakas,S.M. Determination
of residues of flumequine and nalidixic, oxolinic, and piromidic acids in catfish by liquid chromatography
withfluorescenceand UV detection, J.AOAC Int., 1995, 78, 343-352.
SAMPLE
Matrix: urine
Sample preparation: Make up 1 mL urine to 25 mL with mobile phase, filter (0.45 (xm). Inject
a 20 u.L aliquot.
HPLC VARIABLES
Mobile phase: MeCN:400 |xM oxalic acid in water 28:72
Flow rate: 2.0
Detector: UV 265
Next Page
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: metabolites, cinoxacin, oxolinic acid, pipemidic acid, piromidic acid
KEYWORDS
antibiotics
REFERENCE
Dur&n Mer,L; Galeano Diaz,T.; Rodriguez Caceres,M.L; Salinas L6pez,F. Determination of the chemothera-
peutic quinolonic and cinolonic derivatives in urine by high-performance liquid chromatography with ul-
traviolet andfluorescencedetection in series, J.Chromatogr.A, 1997, 787, 119-127.
SAMPLE
Matrix: urine
Sample preparation: Make up 1 mL urine to 25 mL with deionized water. Adjust to pH 2.5-3
with HCl, extract with 25 mL chloroform. Separate the organic layer, dry the organic phase
with sodium sulfate, evaporate it to dryness. Dissolve the residue in 3 mL MeCN, dilute to
1OmL with water, filter (0.45 jxm). Inject a 20 |xL aliquot.
HPLC VARIABLES
Flow rate: 2.0
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: metabolites, oxolinic acid
REFERENCE
Durn Mer,L; Galeano Diaz,T.; Rodriguez Caceres,M.I.; Salinas L6pez,F. Determination of the chemothera-
traviolet andfluorescencedetection in series, J.ChromatogrA, 1997, 787, 119-127.
Nalmefene
Merck Index: 6447
SAMPLE
Matrix: blood
Sample preparation: Make plasma alkaline and extract with MTBE. Evaporate the organic
layer to dryness under nitrogen, reconstitute the residue in 100 u.L MeOH:water 90:10, inject
an aliquot.
HPLC VARIABLES
Column: Zorbax SB-C18
Previous Page
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: metabolites, cinoxacin, oxolinic acid, pipemidic acid, piromidic acid
KEYWORDS
antibiotics
REFERENCE
Dur&n Mer,L; Galeano Diaz,T.; Rodriguez Caceres,M.L; Salinas L6pez,F. Determination of the chemothera-
SAMPLE
Matrix: urine
1OmL with water, filter (0.45 jxm). Inject a 20 |xL aliquot.
HPLC VARIABLES
Flow rate: 2.0
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: metabolites, oxolinic acid
REFERENCE
Durn Mer,L; Galeano Diaz,T.; Rodriguez Caceres,M.I.; Salinas L6pez,F. Determination of the chemothera-
traviolet andfluorescencedetection in series, J.ChromatogrA, 1997, 787, 119-127.
Nalmefene
Merck Index: 6447
SAMPLE
Matrix: blood
Sample preparation: Make plasma alkaline and extract with MTBE. Evaporate the organic
layer to dryness under nitrogen, reconstitute the residue in 100 u.L MeOH:water 90:10, inject
an aliquot.
HPLC VARIABLES
Mobile phase: MeOHiIOmM pH 4.0 ammonium formate 40:60
Flow rate: 1
Detector: MS, Sciex API IIIplus tandem mass, positive ion mode, 316.0/298.0 parent/product ions
CHROMATOGRAM
Retention time: 6.9
Internal standard: nalmefene
OTHER SUBSTANCES
Extracted: oxycodone
KEYWORDS
plasma; nalmefene is IS
REFERENCE
Kaisershot,C; Pierce,A.; Talaat,R. Quantitative analysis of oxycodone and its metabolites noroxycodone and
oxymorphone in human plasma by high-performance liquid chromatography with ionspray tandem mass
spectrometry (Abstract 2129), Pharm.Res., 1997, 14, S262.
Nalorphine
Merck Index: 6448
SAMPLE
Matrix: bile, blood
Sample preparation: 0.5 mL Blood or bile 300 fxL 1.1 M pH 5.0 sodium acetate buffer + 3000-
3500 U of Patella vulgata glucuronidase, incubate at 55 overnight, add 0.5 mL borate buffer
to achieve a pH of 8.3-8.5. Add 8 mL chlorofornr.isopropanol 90:10, gently rotate for 30 min,
centrifuge at 3500 rpm for 10 min, remove aqueous layer. Wash organic layer (twice for blood,
three times for bile) with 3 mL 100 mM pH 9.9 sodium phosphate buffer with gentle rotation
for 10 min and centrifugation each time. Add organic layer to 200 (blood) or 400 (bile) ^L 0.2%
phosphoric acid, gently rotate for 30 min, discard organic layer, inject 50 |xL of the acid layer.
(Borate buffer was 50 mM boric acid and 43 mM sodium tetraborate, adjusted to pH 9.8.)
HPLC VARIABLES
Mobile phase: MeCNiIO mM pH 6.6 NaH2PO4 10:90
Flow rate: 1.2
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: hydrocodone, oxycodone, dihydrocodeine, morphine, codeine, 6-monoacetylmor-
phine
Noninterfering: acetylcodeine, amitriptyline, amphetamine, diamorphine, diazepam, dothiepin,
doxepin, ephedrine, ephedrine, hydromorphone, mesoridazine, methadone, methamphetamine,
3-monoacetylmorphine, nordiazepam, norpropoxyphene, nortriptyline, oxazepam, propoxy-
phene, pseudoephedrine, quinidine, quinine, sulfamethoxazole, sulforidazine, thioridazine, na-
lorphine is IS
KEYWORDS
UV and F detection used together
REFERENCE
Crump,K.L.; McIntyre,I.M.; Drummer,O.H. Simultaneous determination of morphine and codeine in blood and
bile using dual ultraviolet and fluorescence high-performance liquid chromatography, J.Anal.Toxicol., 1994,
18, 208-212.
SAMPLE
Sample preparation: 250 |xL Bile, 3 mL blood, or 5 mL tissue homogenate + 1 mL water + 2
mL 200 mM pH 8.9 sodium borate buffer + 5 (bile) or 10 (blood, tissue) mL chloroformrisopro-
panol 90:10, rotate gently for 20 min, centrifuge at 2000 rpm for 10 min. Remove the organic
layer and add it to 2 mL 500 mM HCl, rotate for 20 min, centrifuge for 5 min. Remove 1.8 mL
of the upper aqueous phase, adjust to pH 8.6 0.2 by carefully adding powdered ammonium
carbonate until the solution was saturated, add 5 mL ethyl acetateiisopropanol 90:10, rotate
for 20 min, centrifuge for 5 min. Remove 4.8 mL of the upper organic layer and evaporate it
to dryness under a stream of nitrogen at 40, reconstitute the residue in 50 |xL MeOH, vortex
for 30 s, inject a 20 |xL aliquot.
HPLCVARIABLES
Guard column: 30 X 4.6 5 \xm RP-18 Spheri-5
Column: 250 X 4.6 5 urn Ultrasphere ODS
Mobile phase: MeOH:50 mM pH 7 phosphate buffer 40:60 (Place a 70 X 2 30-38 jxm CoPeIl ODS
Flow rate: 2
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: codeine, hydromorphone, morphine, norcodeine, normorphine
secobarbital
KEYWORDS
nalorphine is IS
REFERENCE
Hepler,B.R.; Sutheimer,C; Sunshine,!; Sebrosky,G.F. Combined enzyme immunoassay-LCEC method for the
identification, confirmation, and quantitation of opiates in biological fluids, JAnaLToxicoL, 1984, 8, 78-90.
SAMPLE
Matrix: blood
Sample preparation: 200 uX. Plasma + 200 u,L 3.5% sodium carbonate, extract with 4 mL dry
ether. Extract the organic layer with 200 u,L 20 mM phosphoric acid. Inject an aliquot of the
phosphoric acid solution.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:EDTA:70 mM KH2PO4 6:10:0.02:83.98
Flow rate: 1
Detector: E, LCB4, oxidation potential 900 mV
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: oxymorphone
KEY WORDS
plasma; rat; nalorphine is IS
REFERENCE
Hussain,M.A.; Aungst,B-J- Intranasal absorption of oxymorphone, J.Pharm.ScL, 1997, 86, 975-976.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg ethyl SPE cartridge (J.T.Baker) with 2 mL
MeOH, 1 mL water, and 2 mL 1 mM pH 9.3 ammonium hydrogen carbonate buffer. Add 1 mL
serum to the SPE cartridge, wash with 1 mL 1 mM pH 9.3 ammonium hydrogen carbonate
buffer, elute with 1 mL MeOH. Evaporate the eluate to dryness, reconstitute the residue in
100 JJLL mobile phase, inject a 5 JJIL aliquot.
HPLC VARIABLES
Column: 250 X 2.1 5 |xm Supelcosil LC-Si (Supelco)
Mobile phase: MeCN:MeOH:water:formic acid 5.2:59.8:34.65:0.35
Flow rate: 0.23
Injection volume: 5
Detector: MS, API I MS single quadrupole, ionspray, capillary tip 5000 V, interface plate 650 V,
source 60, positive ion mode, orifice 70 V, SIM, m/z 312
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: codeine, diamorphine, morphine
KEYWORDS
serum; SPE; nalorphine is IS; mouse
REFERENCE
Zuccaro,R; Ricciarello,R-; Pichini,S.; Pacifici,R.; Altieri,L; Pellegrini,M.; D'Ascenzo,G. Simultaneous determi-
nation of heroin, 6-monoacetylmorphine, morphine, and its glucuronides by liquid chromatography-atmo-
spheric pressure ionspray-mass spectrometry, JAnaLToxicol., 1997, 21, 268-277.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Whole blood + 1 mL 1 M pH 9 borate buffer + 5 mL ethyl acetate,
vortex thoroughly, centrifuge, repeat the extraction. Combine the extracts and add them to 4
mL 50 mM sulfuric acid, extract. Discard the organic layer and saturate the aqueous layer
with freshly ground ammonium carbonate, extract with 8 mL ethyl acetate, filter (phase-sep-
arating paper). Evaporate the organic layer and to dryness under a stream of nitrogen at 50,
HPLC VARIABLES
Column: 250 X 5 ODS Hypersil
Mobile phase: MeCN: 10 mM KH2PO4 20:80 containing 20 mM octanesulfonic acid, adjusted to
Detector: E, BAS LC-4B, TL-5A thin-layer flow cell +0.870 V
CHROMATOGRAM
Retention time: 19
OTHER SUBSTANCES
Extracted: morphine
KEYWORDS
whole blood; nalorphine is IS
REFERENCE
Logan,B.K; Oliver, J.S.; Smith,H. The measurement and interpretation of morphine in blood, Forensic Sci.Int,
1987, 35, 189-195.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut SPE cartridge with 1 mL MeOH, 1 mL water, and
1 mL 100 mM pH 9.0 sodium bicarbonate buffer, do not allow to go dry. 100 (JLL Plasma + 750
|xL 100 mM pH 9.0 sodium bicarbonate buffer, vortex for 10 s, add to the SPE cartridge, wash
with 1 mL 100 mM pH 9.0 sodium bicarbonate buffer, wash with 1 mL water, wash with 100
JJLL MeOH:water 50:50, dry under vacuum for 10 min, elute with three 250 jxL aliquots of
MeOH. Evaporate the eluate to dryness at 45 in a vacuum centrifuge for 1 h, reconstitute
with 25 IJLL 100 mM pH 11.4 sodium carbonate or 25 (xL pH 9.5 sodium bicarbonate, add 25
JULL 1 mg/mL dansyl chloride in acetone, vortex, let stand in the dark at 45 for 20 min, add
250 |xL toluene, vortex for 2 min, centrifuge at 12500 g for 1 min, inject a 100-200 |JLL aliquot
of the upper organic layer.
HPLC VARIABLES
Guard column: 10 X 4.6 3 |xm silica
Column: 150 X 4.6 3 |xm Spherisorb 3CN
Mobile phase: n-Hexane:isopropanol:ammonia 95:5:0.25 (Place a silica column between the
pump and the injector.)
Flow rate: 1.5
CHROMATOGRAM
Retention time: 2.8
OTHER SUBSTANCES
Extracted: morphine, 6-acetylmorphine
KEYWORDS
plasma; derivatization; SPE; nalorphine is IS
REFERENCE
Barrett,D.A.; Shaw,P.N.; Davis,S.S. Determination of morphine and 6-acetylmorphine in plasma by high-per-
SAMPLE
Matrix: blood
3 mL MeCN: 10 mM NaH2PO410:90, and 5 mL water. 1 mL Plasma + 1 mL 500 mM ammonium
sulfate, add to the SPE cartridge, wash with 6 mL 5 mM ammonium sulfate, elute with 1 mL
MeCN: 10 mM NaH2PO4 10:90, inject a 100 JULL aliquot of the eluate.
HPLC VARIABLES
Column: 300 X 3.9 10 /xm jxBondapak C18
sulfate, pH adjusted to 2.1 with orthophosphoric acid.)
Flow rate: 1
CHROMATOGRAM
Retention time: 22
OTHER SUBSTANCES
Extracted: morphine, normorphine
KEYWORDS
plasma; SPE; nalorphine is IS
REFERENCE
Glare,P.A.; Walsh,T.D.; Pippenger,C.E. A simple, rapid method for the simultaneous determination of morphine
and its principal metabolites in plasma using high-performance liquid chromatography and fluorometric
detection, Ther.Drug Monit, 1991, 13, 226-232.
SAMPLE
Matrix: blood
Sample preparation: Add 500 |xL plasma to a Sep-Pak tC18 SPE cartridge, wash with 2 mL
water, wash with 3 mL acetoneiwater 5:95, elute with 4 mL MeOH:water 70:30. Evaporate the
eluate to dryness under a stream of nitrogen at 60, reconstitute the residue in 200 u,L mobile
HPLC VARIABLES
Column: 150 X 4.6 TSK-gel ODS-120T (Tosoh)
sulfate, adjusted to pH 2.1 with phosphoric acid.)
Flow rate: 1
Detector: E, Shimadzu L-ECD-6A, 0.8 V
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: morphine
KEYWORDS
plasma; rabbit; SPE; nalorphine is IS
REFERENCE
Matsumoto,Y.; Yamamoto,L; Watanabe,Y.; Matsumoto,M. Enhancing effect of viscous sodium hyaluronate so-
lution on the rectal absorption of morphine, Biol.Pharm.BulL, 1995, 18, 1744-1749.
SAMPLE
Matrix: blood
the residue in 100 |JLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 287
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Matrix: blood
2 mL water. 1 mL Plasma + 1 mL water, add to the SPE cartridge at 2 mL/min, wash with 2
mL water, wash with 2 mL MeCN, dry under vacuum for 1 min, elute with 2 mL dichloro-
methane:isopropanol:ammonium hydroxide 80:20:2. Evaporate the eluate to dryness under a
stream of nitrogen at 40, reconstitute in 100 |xL mobile phase, inject a 60 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 3 |xm Basic C8 (YMC)
Mobile phase: MeCN:5 mM (NHJ2HPO4 8:92 adjusted to pH 5.8 with phosphoric acid
Flow rate: 1
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
Extracted: codeine
Simultaneous: morphine, norcodeine
KEYWORDS
plasma; SPE; nalorphine is IS
REFERENCE
Weingarten,B-; Wang,H.Y.; Roberts,D.M. Determination of codeine in human plasma by high-performance liq-
uid chromatography withfluorescencedetection, J.Chromatogr.A, 1995, 696, 83-92.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 211.1
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: levallorphan, hydroxypethidine, normethadone, meperidine, dipipanone, diamor-
phine, pentazocine, acetylcodeine, monoacetylmorphine, thebacon, oxycodone, thebaine, nor-
levorphanol, methadone, benzylmorphine, ethylmorphine, morphine-N-oxide, codeine, codeine-
N-oxide, morphine, ethoheptazine, morphine-3-glucuronide, pholcodine, norpethidine,
hydrocodone, dihydrocodeine, dihydromorphine, levorphanol, norcodeine, normorphine, epi-
nephrine, pipradol, phenylpropanolamine, fencamfamin, chlorphentermine, norpseudoephed-
rine, phentermine, fenfiuramine, methylenedioxyamphetamine, amphetamine, normetane-
phrine, 4-hydroxyamphetamine, bromo-STP, STP, prolintane, 2-phenethylamine, tyramine,
trimethoxyamphetamine, phenylephrine, pseudoephedrine, ephedrine, methylephedrine, di-
methylamphetamine, methamphetamine, mescaline, mephentermine, buprenorphine, dextro-
moramide, phenoperidine, fentanyl, etorphine
Interfering: pemoline, benzphetamine, diethylpropion, mazindol, tranylcypromine, caffeine, fen-
ethyline, phendimetrazine, methylphenidate, phenelzine, piritramide, noscapine, papaverine,
naloxone, dextropropoxyphene, phenazocine, norpipanone
REFERENCE
Law,B-5 Gill,R-l Moffat,A.C. High-performance liquid chromatography retention data for 84 basic drugs of fo-
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.8
OTHER SUBSTANCES
mepyramine, mesoridazine, metaraminol, methadone, methamphetamine, methapjîlene,
lol, mianserin, morazone, nadolol, naloxone, naphazoline, nicotine, nifedipine, nomifensine,
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 15 X 3.2 7 (xm Applied Biosystems pre-column
Column: 100 X 2 10 fjim ixPorasil
Flow rate: 1
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: buprenorphine, butorphanol, ethylmorphine, fentanyl, morphine, codeine, me-
peridine, tramadol
Interfering: nalbuphine
REFERENCE
J.Chromatogr., 1991, 570, 339-350.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
yltestosterone, methyprylon, metoprolol, mibolerone, morphine, naloxone, naltrexone, napha-
zoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam,
mycin, pyrilamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, re-
thioridazine, thiosalicylic acid, thiothixene, thjrmol, tolazamide, tolazoline, tobutamide, tol-
REFERENCE
18, 233-242.
SAMPLE
Matrix: urine
Sample preparation: 10 mL Urine + 1 mL concentrated HCl, heat at 100 for 1 h, cool, add 500
fxL saturated ammonium sulfate solution, adjust pH to 9 with 25% NaOH, dilute to 20 mL
with water, add mixture to an Extrelut 20 column, let stand for 10 min, elute with 40 mL
dichloromethanedsopropanol 85:15. Add the eluate to 3 mL 200 mM HCl, extract, repeat ex-
traction. Combine the aqueous phases and add them to 500 |JLL saturated ammonium sulfate
solution, adjust pH to 9.2 with 25% NaOH, dilute to 20 mL with water, add to another Extrelut
20 column, let stand for 10 min, elute with 40 mL dichloromethaneiisopropanol 85:15. Evap-
IxL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 4 x 4 5 fxm Lichrosorb
Column: 250 X 4 5 jxm Lichrospher Si 100
Mobile phase: n-Hexane:dichloromethane:MeOH containing 0.75% diethylamine 72.5:20:7.5
Flow rate: 1.35
Detector: UV 225
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Extracted: morphine, codeine
trimipramine
KEYWORDS
SPE; normal phase; nalorphine is IS
REFERENCE
of abuse in urine, JAnaLToxicoL, 1992, 16, 217-222.
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 3000-3500 U glucuronidase (Patella vulgata, Sigma) + 300
(JLL 1.1 M pH 5 sodium acetate buffer, heat overnight at 55, add 500 |xL buffer, add 8 mL
chloroformdsopropanol 90:10, rotate gently for 30 min, centrifuge at 2500 g for 10 min. Remove
the organic layer and add it to 3 mL pH 9.9 NaH2PO4 buffer, rotate gently for 10 min, centri-
fuge, discard the aqueous layer, repeat the wash. Remove the organic layer and add it to 200
IJLL 0.2% phosphoric acid, rotate gently for 30 min, inject a 50 |xL aliquot of the aqueous layer.
(Buffer was 50 mM boric acid and 43 mM sodium tetraborate, pH adjusted to 9.9.)
HPLCVARIABLES
Guard column: Nova-Pak phenyl
Mobile phase: MeCNrIO mM pH 6.6 NaH2PO4 10:90
Flow rate: 1.2
Detector: E, ESA Coulochem, Model 5010 analytical cell, detector cell 1 +0.20 V, detector cell 2
+ 0.55 V, model 5020 guard cell + 0.75 V or UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: codeine, morphine, 6-monoacetylmorphine
Simultaneous: dihydrocodone, hydrocodone, oxycodone
Noninterfering: 7-aminoclonazepam, 7-aminoflunitrazepam, amitriptyline, amphetamine, di-
azepam, dothiepin, doxepin, ephedrine, mesoridazine, methadone, methamphetamine, nordi-
azepam,norpropoxyphene, nortriptyline, oxazepam, propoxyphene, quinidine, quinine, sulfa-
methoxazole, sulforidazine, thioridazine, trimethoprim
KEYWORDS
nalorphine is IS
REFERENCE
Gerostamoulos,J.; Crump,K; MclntyreJ.M.; Drummer,O.H. Simultaneous determination of 6-monoacetylmor-
phine, morphine and codeine in urine using high-performance liquid chromatography with combined ultra-
Naloxone
51481-60-8 (HCI dihydrate)
Merck Index: 6449
Lednicer No.: 1 289
SAMPLE
Matrix: blood
Sample preparation: 500 JXL Plasma + 50 |xL 2 M NaOH + 4.5 mL chloroformiisopropanol 90:
10 (Caution! Chloroform is a carcinogen!), vortex for 1 min. Centrifuge at 2000 g for 20 min.
Evaporate the organic phase to dryness under a stream of nitrogen at 40. Reconstitute the
residue with 70 |xL mobile phase. Inject a 50 |xL aliquot.
HPLCVARIABLES
Guard column: 30-40 |xm Perisorb RP-18 (Upchurch Scientific, USA)
Column: 250 X 4.6 5 jim KR100-5-C18 (Biosains, Malaysia)
Mobile phase: MeCN:buffer 85.5; 14.5 (Buffer was 100 mM disodium hydrogen orthophosphate
adjusted to pH 3.5 with 85% phosphoric acid.)
Flow rate: 0.8
Detector: E, DECADE (Antec Leyden), glassy carbon working electrode, oxidative mode +0.95
V, Ag/AgCl reference electrode saturated with LiCl
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: naltrexone
KEYWORDS
naloxone is IS; plasma
REFERENCE
Peh,K.K; Billa,N.; Yuen,K.H. Simple liquid chromatographic method for the determination of naltrexone in
human plasma using amperometric detection, J.Chromatogr.B, 1997, 701, 140-145.
SAMPLE
Matrix: blood
Sample preparation: Buffer plasma to pH 8.6, extract with MTBE, evaporate under a stream
of nitrogen, reconstitute the residue with 50 |JLL MeOH:water 50:50, inject an aliquot.
HPLCVARIABLES
Column: 5 u,m Hypersil ODS-5
Mobile phase: MeOH:20mM pH 4.0 ammonium acetate 90:10
Detector: MS, Sciex API IIIplus tandem mass, positive ion mode, scan 328.2/310.0 parent prod-
uct ion
CHROMATOGRAM
Retention time: 3.2
Internal standard: naloxone
KEYWORDS
plasma; naloxone is IS
REFERENCE
Selenka,J.M.; Talaat,R.E. Quantitative analysis of naltrexone in human plasma by high-performance liquid
chromatography with ionspray tandem mass spectrometry (Abstract APQ 1239), Pharm.Res., 1996, 13,
S60.
SAMPLE
Matrix: blood
Sample preparation: Condition a 50 mg Isolute CBA SPE cartridge (Hengoed, UK) with full
column volumes of MeOH and water. 1 mL plasma + 15 ng IS, add to the SPE cartridge, wash
with two column volumes of water. Dry SPE cartridge under vacuum. Elute with one column
volume of 1% ammonia in MeOH. Evaporate eluate to dryness under vacuum at 50, reconsti-
tute the residue in 150 JJLL mobile phase, vortex, inject an aliquot.
HPLC VARIABLES
Guard column: Brownlee Newguard C2 (Anachem, UK)
Column: 100 X 4 5 |xm cyano-propyl column (Capitol HPLC, UK)
Flow rate: 1
Detector: E, ESA Coulochem 2, Model 5020 guard cell, Model 5011 analytical cell, detector 1 +
450 mV, detector 2 + 850 mV, guard cell + 900 mV
CHROMATOGRAM
Retention time: 5.3
Internal standard: sumatriptan succinate (6.1)
KEYWORDS
plasma; SPE
REFERENCE
Franklin,M.; Odontiadis,J. Determination of naloxone in human plasma by high-performance liquid chroma-
tography with coulometric determination, J.Chromatogr.B, 1996, 679, 199-203.
SAMPLE
Matrix: blood
Sample preparation: Condition a C18 SPE cartridge (J.T. Baker) with 2 mL MeOH and 2 mL
10 mM pH 7 potassium phosphate buffer. Add 1 mL plasma to the SPE cartridge, wash with
2 mL water, dry under a stream of nitrogen, elute with 3 mL benzene:butanol 85:15 (Caution!
Benzene is a carcinogen!). Evaporate the eluate to dryness under a stream of nitrogen at room
temperature, reconstitute the residue in 100 jxL mobile phase, inject a 20 JULL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Supelcosil LC-18 DB
Flow rate: 0.8
Detector: E, ESA Coulochem 5100A, 5011A analytical cell, first electrode +0.20 V, second elec-
trode +0.70 V (monitored), palladium reference electrode
CHROMATOGRAM
Internal standard: naloxone
OTHER SUBSTANCES
Extracted: naltrexone
KEYWORDS
plasma; SPE; naloxone is IS
REFERENCE
Zuccaro,R; Altieri,L; Betto,R; Pacinci,R-l Ricciarello,G.; Pini,L.A.; Sternieri,E.; Pichini,S. Determination of nal-
trexone and 6@-naltrexol in plasma by high-performance liquid chromatography with coulometric detection,
J.Chromatogr., 1991, 567, 485-490.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 2 mL MeOH and with 2 mL
109 mM sodium octyl sulfate. 250 |xL Plasma + 80 ng naltrexone + 750 |xL water + 1 mL buffer
+ 200 |JLL 0.3 mM sodium octyl sulfate, add to the SPE cartridge, wash four times with 2.5 mL
water, wash twice with 2.5 mL portions of MeOH:water 50:50, elute with 2 mL MeOH. Evap-
JULL mobile phase, inject a 20 JULL aliquot. (Buffer was 83.5 mL 100 mM HCl and 17.5 mL boric
acid solution, pH 9.0. Boric acid solution was 1.237 g boric acid + 90 mL water + 10 mL 1 M
HCl.)
HPLCVARIABLES
Column: 150 X 4.6 5 |xm Econosphere C18
Mobile phase: MeCN:buffer 15:85 (Buffer was 100 mM monochloroacetic acid containing 2.59
mM sodium octyl sulfate and 2.39 mM EDTA, pH adjusted to 4.5 with NaOH.)
Flow rate: 1
Detector: E, Bioanalytical Systems LC-4A, glassy carbon electrode +0.89 V
CHROMATOGRAM
Retention time: 7.0
KEYWORDS
plasma; wash polypropylene tubes with 6.3 M nitric acid; SPE; pharmacokinetics
REFERENCE
Reid,R.W.; Deakin,A-; Leehey,D.J. Measurement of naloxone in plasma using high-performance liquid chro-
matography with electrochemical detection, J.Chromatogr., 1993, 614, 117-122.
SAMPLE
Matrix: blood
stoppered tube for 1 min, add 6 mL NiCl2, rock for 5 min, add 15 mL dichloromethanerisobutyl
water.)
HPLC VARIABLES
Column: 200 X 4.6 5 (xm Hypersil C8
min
Detector: UV 230
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
methaqualone, morphine, noscapine, papaverine, pentazocine, procaine
Also analyzed: bromazepam, clonazepam, diazepam, flunitrazepam, fiurazepam, medazepam,
KEYWORDS
whole blood
REFERENCE
Bernal,J.L.; Del Nozal,M. J.; Rosas,V.; Villarino,A. Extraction of basic drugs from whole blood and determination
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 100 |xL 200 ng/mL 6-monoacetylmorphine in water + 2
mL buffer + 10 mL hexane:dichloromethane:isopropanol 69:30:1, shake mechanically at 60 rpm
for 15 min, centrifuge at 2000 g at 4 for 5 min. Remove the upper organic layer and add it to
200 JJLL 17 mM phosphoric acid, shake for 2 min, centrifuge at 2000 g at 4 for 5 min, inject a
50 |xL aliquot of the aqueous layer. (Buffer was 100 mM boric acid and 100 mM KCl adjusted
to pH 9 with 100 mM NaOH.)
HPLC VARIABLES
Column: 150 X 4.6 5 (Jim Lichrospher C18
Mobile phase: MeOH:water 20:80 containing 24 mM KH2PO4 and 0.06 mM disodium EDTA
adjusted to pH 3.4 with orthophosphoric acid
Flow rate: 0.8
Detector: E, ESA Model 5100A Coulochem, Model 5020 guard cell 0.50 V, Model 5011 analytical
cell in oxidation screening mode, first electrode 0.06 V, second electrode 0.40 V
CHROMATOGRAM
Retention time: 3.6
OTHER SUBSTANCES
Extracted: nalbuphine
Noninterfering: buprenorphine, codeine, dextromoramide, dextropropoxyphene, ethylmorphine,
fentanyl, methadone, morphine, nalorphine, norcodeine, pholcodine
Interfering: naltrexone
KEY WORDS
plasma
REFERENCE
Nicolle,E.; Michaut,S.; Serre-Debeauvais,F.; Bessard,G. Rapid and sensitive high-performance liquid chromat-
ographic assay for nalbuphine in plasma, J.Chromatogr.B, 1995, 663, 111-117.
SAMPLE
Matrix: blood
the residue in 100 |xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 u-L aliquot of
HPLC VARIABLES
Column: 300 X 3.9 4 fjun NovaPack C18
Flow rate: 0.8
Detector: UV 283
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeOH, inject a 20 |xL aliquot.
HPLCVARIABLES
Guard column: 30 X 4.6 5 |xm RP-18 Spheri-5
Mobile phase: MeOH:50 mM pH 7 phosphate buffer 40:60 (Place a 70 X 2 30-38 |xm CoPeIl ODS
Flow rate: 2
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: acetaminophen, atropine, codeine, epinephrine, ethylmorphine, hydrocodone, hydro-
morphone, hydroxyzine, morphine, nalorphine, norcodeine, normorphine, oxycodone, pentazo-
cine, phenylpropanolamine, pseudomorphine, scopolamine, secobarbital
REFERENCE
Hepler,B.R.; Sutheimer,C; Sunshine,L; Sebrosky,G.F. Combined enzyme immunoassay-LCEC method for the
identification, confirmation, and quantitation of opiates in biological fluids, J.Anal.ToxicoL, 1984,8, 78-90.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH at a concentration of 1 mg/mL, inject a 20 JJLL aliquot.
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHERSUBSTANCES
Simultaneous: methylphenidate, phenelzine, epinephrine, pipradol, phenylpropanolamine, fen-
camfamin, chlorphentermine, norpseudoephedrine, phentermine, fenfluramine, methylenedi-
oxyamphetamine, amphetamine, normetanephrine, 4-hydroxyamphetamine, bromo-STP, STP,
mephentermine, norpipanone, levallorphan, hydroxypethidine, normethadone, meperidine, di-
pipanone, diamorphine, pentazocine, acetylcodeine, monoacetylmorphine, thebacon, oxycodone,
thebaine, norlevorphanol, methadone, benzylmorphine, ethylmorphine, morphine-N-oxide, co-
deine, codeine-N-oxide, morphine, ethoheptazine, morphine-3-glucuronide, pholcodine, nor-
pethidine, hydrocodone, dihydrocodeine, dihydromorphine, levorphanol, norcodeine,
normorphine
Interfering: pemoline, benzphetamine, diethylpropion, mazindol, tranylcypromine, caffeine,
fenethyline, phendimetrazine, buprenorphine, dextromoramide, phenoperidine, fentanyl, etor-
phine, piritramide, noscapine, papaverine, dextropropoxyphene, nalorphine, phenazocine
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 (xg/mL solution in MeOH, inject a 20 |JLL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.2
OTHER SUBSTANCES
methdilazene, methotrimeprazine, methoxamine, methoxyphenamine, methoxj^promazine,
lol, mianserin, morazone, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nomifensine,
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
yltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, naltrexone, naphazo-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 fxm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHERSUBSTANCES
oxylate, dip5nridamole, disopâmide, dobutamine, doxapram, doxepin, droperidol, encainide,
ronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naphazoline, naproxen,
tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifiuoperazine, triflupro-
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: urine
Sample preparation: Buffer the urine sample at pH 9.7. Extract with ethyl acetateiheptane (4:
1). Dry the organic phase and reconstitute with mobile phase.
HPLC VARIABLES
Column: Hypersil silica
Detector: MS, Sciex API III, heated nebulizer, positive ion mode
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: buprenorphine, norbuprenorphine
REFERENCE
Johnson,R.A.; Haan,D.E.; James,C.A.; Hopkins,N.K. Determination of linezolid, PNU-100766, in human
plasma and urine using high-performance liquid chromatography with ultraviolet detection (Abstract 2487),
Pharm.Res., 1997, 14, S374.
Naltrexone
Merck Index: 6450
Lednicer No.: 2 319
SAMPLE
Matrix: blood
Sample preparation: Buffer plasma to pH 8.6, add IS, extract with MTBE, evaporate under a
stream of nitrogen, reconstitute the residue with 50 |xL MeOH:water 50:50, inject an aliquot.
HPLC VARIABLES
Column: 5 |xm Hypersil ODS-5
Mobile phase: MeOH:20mM pH 4.0 ammonium acetate 90:10
Detector: MS, Sciex API IIIplus tandem mass, positive ion mode, scan 342.0/324.0 parent prod-
uct ion
CHROMATOGRAM
Retention time: 3.7
Internal standard: naloxone (3.2)
KEYWORDS
plasma
REFERENCE
Selenka,J.M.; Talaat,R.E. Quantitative analysis of naltrexone in human plasma by high-performance liquid
chromatography with ionspray tandem mass spectrometry (Abstract APQ 1239), Pharm.Res., 1996, 13,
S60.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 50 |xL 250 ng/mL IS + 50 JLJLL 2 M NaOH + 4.5 mL
chloroform.isopropanol 90:10 (Caution! Chloroform is a carcinogen!), vortex for 1 min. Centri-
fuge at 2000 g for 20 min. Evaporate the organic phase to dryness under a stream of nitrogen
at 40. Reconstitute the residue in 70 jxL mobile phase. Inject a 50 JXL aliquot.
HPLC VARIABLES
Guard column: 30-40 |xm Perisorb RP-18 (Upchurch Scientific, USA)
Column: 250 X 4.6 5 |xm KR100-5-C18 (Biosains, Malaysia)
Mobile phase: MeCN:buffer 85.5:14.5 (Buffer was 100 mM disodium hydrogen orthophosphate
adjusted to pH 3.5 with 85% phosphoric acid.)
Flow rate: 0.8
Detector: E, DECADE (Antec Leyden), glassy carbon working electrode, oxidative mode +0.95
V, Ag/AgCl reference electrode saturated with LiCl
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Peh,K.K.; Billa,N.; Yuen,K.H. Simple liquid chromatographic method for the determination of naltrexone in
human plasma using amperometric detection, J.Chromatogr.B, 1997, 701, 140-145.
SAMPLE
Matrix: blood
Sample preparation: Condition a C18 SPE cartridge (J.T. Baker) with 2 mL MeOH and 2 mL
10 mM pH 7 potassium phosphate buffer. 1 mL Plasma + 100 |xL 100 ng/mL naloxone, add to
the SPE cartridge, wash with 2 mL water, dry under a stream of nitrogen, elute with 3 mL
benzenerbutanol 85:15 (Caution! Benzene is a carcinogen!). Evaporate the eluate to dryness
under a stream of nitrogen at room temperature, reconstitute the residue in 100 |JLL mobile
phase, inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 jun Supelcosil LC-18 DB
Flow rate: 0.8
Detector: E, ESA Coulochem 5100A, 5011A analytical cell, first electrode +0.20 V, second elec-
trode +0.70 V (monitored), palladium reference electrode
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Zuccaro,R; Altieri,L; Betto,R; Pacifici,R.; Ricciarello,G.; Pini,L.A.; Sternieri,E.; Pichini,S. Determination of nal-
trexone and 6p-naltrexol in plasma by high-performance liquid chromatography with coulometric detection,
J.Chromatogr., 1991, 567, 485-490.
SAMPLE
Matrix: blood
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 2 mL MeOH and with 2 mL
109 mM sodium octyl sulfate. 250 |xL Plasma + 80 ng naltrexone + 750 |xL water + 1 mL buffer
+ 200 |xL 0.3 mM sodium octyl sulfate, add to the SPE cartridge, wash four times with 2.5 mL
water, wash twice with 2.5 mL portions of MeOHiwater 50:50, elute with 2 mL MeOH. Evap-
(xL mobile phase, inject a 20 |xL aliquot. (Buffer was 83.5 mL 100 mM HCl and 17.5 mL boric
acid solution, pH 9.0. Boric acid solution was 1.237 g boric acid + 90 mL water + 10 mL 1 M
HCl.)
HPLCVARIABLES
Column: 150 X 4.6 5 fxm Econosphere C18
Mobile phase: MeCNrbuffer 15:85 (Buffer was 100 mM monochloroacetic acid containing 2.59
mM sodium octyl sulfate and 2.39 mM EDTA, pH adjusted to 4.5 with NaOH.)
Flow rate: 1
Detector: E, Bioanalytical Systems LC-4A, glassy carbon electrode +0.89 V
CHROMATOGRAM
Internal standard: naltrexone
OTHER SUBSTANCES
Extracted: naloxone
KEYWORDS
plasma; wash polypropylene tubes with 6.3 M nitric acid; SPE; naltrexone is IS
REFERENCE
Reid,R-\V.; Deakin,A.; Leehey,D.J. Measurement of naloxone in plasma using high-performance liquid chro-
matography with electrochemical detection, J.Chromatogr., 1993, 614, 117-122.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 100 mg ethyl SPE cartridge (J.T.Baker) with 2 volumes
of MeOH, 1 volume of water, and 2 volumes of 10 mM pH 9.3 ammonium hydrogen carbonate
buffer. 1 mL Serum + 100 |xL water, add to the SPE cartridge, wash with 1 volume of 10 mM
ammonium hydrogen carbonate buffer, elute with 1 volume of MeOH. Evaporate the eluate to
dryness under a stream of nitrogen, reconstitute the residue in 100 jxL mobile phase, inject a
20 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil ABZ
Mobile phase: Gradient. MeOH:water from 15:85 to 60:40 over 10 min. (Convex gradient where
MeOH% = -0.46exp(-x/1.18) + 0.6 where x = time in min.)
Flow rate: 0.8 (0.018 mL/min entered MS)
Detector: MS, Fisons TRIO 2, electrospray, capillary tip 2.97 kV, counter electrode 390 V, sam-
pling cone voltages 66 V, -106 V, -17 V, source 60, SIM m/z 342
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: morphine (m/z 286)
KEYWORDS
serum; human; mouse; SPE; LC-MS; naltrexone is IS
REFERENCE
Pacifici,R-5 Pichini,S.; Altieri,L; Caronna,A.; Passa,A.R; Zuccaro,P. High-performance liquid chromatographic-
electrospray mass spectrometric determination of morphine and its 3- and 6-glucuronides: application to
pharmacokinetic studies, J.Chromatogr.B, 1995, 664, 329-334.
SAMPLE
Matrix: blood
the residue in 100 |JLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 (JLL aliquot of
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 225
CHROMATOGRAM
KEYWORDS
penmiridol; bepridil; terfenadine; trifluoperazine
REFERENCE
254-262.
SAMPLE
HPLCVARIABLES
Column: 250 X 4.6 5 fjim Symmetry C8 (Waters)
mL/min)
Detector: UV 205.2
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeOH:50 mM Na2HPO^ 15:85 containing 3 mM 1-heptanesulfonic acid, pH ad-
Flow rate: 0.8
(monitored)
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: hydromorphone, morphine
KEYWORDS
naltrexone is IS
REFERENCE
Bouquillon,A.L; Freeman,D.; Moulin,D.E. Simultaneous solid-phase extraction and chromatographic analysis
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
pam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenjrtoin, me-
methap}nilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide,
yltestosterone, methyprylon, metoprolol, mibolerone, morphine, nadolol, nalorphine, naphazo-
REFERENCE
18, 233-242.
Nandrolone
CAS Registry No/. 434-22-0, 360-70-3 (decanoate),
52279-57-9 (p-hydroxyphenylpropionate), 62- 90-8 (phenpropionate),
7207-92-3 (propionate), 22263-51-0 (cyclotate)
Merck Index: 6452
Lednicer No.: 1 164
SAMPLE
Matrix: bile, tissue, urine
Sample preparation: Urine. 50 mL Urine + 6 g Amberlite XAD-2, mix for 15 min, transfer
settled Amberlite XAD-2 to a 100 X 9 glass column, wash with 10 mL water, dry under a
stream of nitrogen, elute with 25 mL MeOH.ethyl acetate 50:50. Evaporate eluate to dryness
at 50 and take up residue in 2 mL 250 mM pH 4.8 acetate buffer. Add 50 |JLL Helix pomatia
juice containing a minimum 40 U/mL p-glucuronidase and 20 U/mL arylsulfatase and incubate
at 37 for 2 h. Add 4 drops 6 M HCl and 20 mL ethyl acetate, mix for 15 min, remove water
layer, incubate ethyl acetate layer at 37 for 1 h, wash with two 3 mL portions of 10% NaHCO3,
wash with 3 mL water. Evaporate to dryness, dissolve in 70 mL MeCN:water 5:95, analyze a
53 mL aliquot. Bile. 3 mL Bile + 2 mL 250 mM pH 4.8 acetate buffer + 50 jxL Helix pomatia
juice containing a minimum 40 U/mL p-glucuronidase and 20 U/mL arylsulfatase, incubate at
37 for 2 h. Add 4 drops 6 M HCl and 20 mL ethyl acetate, mix for 15 min, remove water layer,
incubate ethyl acetate layer at 37 for 1 h, wash with two 3 mL portions of 10% NaHCO3, wash
with 3 mL water. Evaporate to dryness, dissolve in 70 mL MeCN:water 5:95, analyze a 53 mL
aliquot. Liver, kidney. Add 80 mL 100 mM pH 9.5 Tris buffer containing 20 mg subtilopeptidase
A (11.6 U/mg) to 20 g minced sample, incubate at 60 for 3.5 h, filter over glass wool, add 6 g
Amberlite XAD-2 to the filtrate, mix for 15 min, transfer settled Amberlite XAD-2 to a 100 X
9 glass column, wash with 10 mL water, dry under a stream of nitrogen, elute with five 10 mL
portions of MeOH. Evaporate eluate to dryness and take up residue in 2 mL 250 mM pH 4.8
acetate buffer. Add 50 |JLL Helix pomatia juice containing a minimum 40 U/mL p-glucuronidase
and 20 U/mL arylsulfatase and incubate at 37 for 2 h. Add 4 drops 6 M HCl and 20 mL ethyl
acetate, mix for 15 min, remove water layer, incubate ethyl acetate layer at 37 for 1 h, wash
with two 3 mL portions of 10% NaHCO3, wash with 3 mL water. Evaporate to dryness, dissolve
in 70 mL MeCN:water 5:95, analyze a 53 mL aliquot. Meat. Add 80 mL 100 mM pH 9.5 Tris
buffer containing 20 mg subtilopeptidase A (11.6 U/mg) to 20 g minced sample, incubate at 60
for 3.5 h, filter over glass wool, add 6 g Amberlite XAD-2 to the filtrate, mix for 15 min, transfer
settled Amberlite XAD-2 to a 100 X 9 glass column, wash with 10 mL water, dry under a
stream of nitrogen, elute with five 10 mL portions of MeOH. Evaporate eluate to dryness and
take up residue in 70 mL MeCN:water 5:95, analyze a 53 mL aliquot. Condition column A with
20 mL water then add 53 mL sample, flush column A with 10 mL water. Condition column B
with 10 mL water. Elute column A onto column B with 20 mL water containing 250 jxg/mL
norgestrel and 5% MeCN. Switch column B into circuit with column C and elute with mobile
phase. Recondition column A with MeOHiwater 70:30.
HPLC VARIABLES
Column: A 10 X 10 immuno precolumn (with immunoglobulin G immobilized on cyanogen bro-
mide-activated Sepharose 4B, prepared as J.Chromatogr. 1988, 452, 419-433); B 10 X 2 Chrom-
pack reverse phase column; C Chromsep reverse phase guard column (Chrompack) + 100 X 3
5 |xm Chromspher glass column
Flow rate: 0.4
Detector: UV 247
CHROMATOGRAM
Retention time: 9 (a), 6 (0)
Limit of detection: 50 ng/L
OTHER SUBSTANCES
Simultaneous: trenbolone (at UV 340)
KEYWpRDS
meat; liver; kidney; SPE; column-switching
REFERENCE
Haasnoot,W.; Schilt,R.; Hamers,A.R.; Huf,F.A.; FarjamA; Frei,R.W.; Brinkman,U.A. Determination of (3-19-
nortestosterone and its metabolite a-19-nortestosterone in biological samples at the sub parts per billion
level by high-performance liquid chromatography with on-line immunoaffinity sample pretreatment,
J.Chromatogr., 1989, 489, 157-171.
SAMPLE
Sample preparation: Oils. 1 mL Sample + 25 mL MeOHiwater 90:10, shake vigorously for 5
min, centrifuge, inject a 10 (JLL aliquot of the supernatant. Tablets. Grind a tablet to a fine
powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 |jim), discard first 5 mL of filtrate,
remaining filtrate.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: 5.8 (nandrolone), 13.5 (nandrolone acetate)
Limit of detection: 5 ixg/mL
OTHER SUBSTANCES
Simultaneous: dehydroepiandrosterone (UV 210), mibolerone, methyltestosterone, methandriol
(UV 210), norethindrone acetate, calusterone, mesterolone (UV 210), norethandrolone, tren-
bolone acetate, benzyl benzoate, testosterone acetate, stanozolol, oxymetholone, nandrolone
propionate, methenolone acetate, testosterone propionate, aspirin, caffeine, formebolone, ben-
zyl alcohol, testolactone, cortisone, fluoxymesterone, norethindrone, oxandrolone (UV 210),
boldenone
Interfering: ethisterone, methandrostenolone, norgestrel, testosterone
KEYWORDS
REFERENCE
Walters,M.J.; Ayers,R-L; Brown,D.J. Analysis of illegally distributed anabolic steroid products by liquid
chromatography with identity confirmation by mass spectrometry or infrared spectrophotometry,
JAssoc.Off.Anal.Ckem., 1990, 73, 904-926.
SAMPLE
25 |xL aliquot.
HPLC VARIABLES
Mobile phase: Gradient. MeOHrwater from 70:30 to 100:0 over 15 min, maintain at 100:0 for 15
min.
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 7.7 (nandrolone), 26.4 (nandrolone decanoate), 20.7 (nandrolone phenylpropio-
nate), 17.3 (nandrolone propionate)
OTHER SUBSTANCES
(UV 280), fluoxymesterone, methandriol, methandriol-3-acetate, methandriol dipropionate,
methandrostenolone, methyltestosterone, stanolone, stanozolol, testosterone, testosterone ac-
etate, testosterone cypionate, testosterone enanthate, testosterone isobutyrate, testosterone
propionate, testosterone undecanoate
KEYWORDS
tablets
REFERENCE
Lurie,I.S.; Sperling,A.R.; Meyers,R.P. The determination of anabolic steroids by MECC, gradient HPLC, and
capillary GC, J.Forensic ScL, 1994, 39, 74-85.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Detector: UV
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
Simultaneous: diethylstilbestrol, trenbolone, zeranol, dienestrol, hexestrol, 17a-methyltestos-
terone, medroxyprogesterone
REFERENCE
Jansen,E.H.J.M.; Both-Miedema,R.; van den Berg,R.H. Application of optimization procedures for the separa-
tion of anabolic compounds by high-performance liquid chromatography, J.Chromatogr., 1989, 489, 57-64.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH at a concentration of 100 jxg/mL, inject a 5 |xL aliquot.
HPLC VARIABLES
Guard column: 70 X 2.1 CO:Pell ODS
Column: 300 X 3.9 Bondex C18 (Phenomenex)
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 4 (nandrolone), 10 (nandrolone propionate), 22 (nandrolone phenylpropionate)
OTHER SUBSTANCES
Also analyzed: boldenone, boldenone acetate, boldenone benzoate
REFERENCE
Noggle,F.T.,Jr.; Clark,C.R.; DeRuiter,J. Liquid chromatographic and mass spectral analysis of the anabolic 17-
hydroxy steroid esters, J.Chromatogr.Sci., 1990, 28, 263-268.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 100 |jig/mL solution in MeOH.
HPLC VARIABLES
Guard column: 70 X 2.1 Whatman CO:Pell ODS
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Simultaneous: methyltestosterone, danazol, boldenone, testosterone
Interfering: methandrostenolone, fluoxymesterone
REFERENCE
bolic steroids, J.Chromatogr.Sci., 1990, 28, 162-166.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 25 jxg/mL solution in mobile phase, inject an aliquot.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
OTHER SUBSTANCES
240), hydroxyprogesterone (UV 240), lynestrenol (UV 210), medroxyprogesterone acetate (UV
240), medroxyprogesterone (UV 240), methandienone (UV 240), methylandrostenediol (UV
210), methylprednisolone acetate (UV 240), methylprednisolone (UV 240), methyltestosterone
(UV 240), norethisterone (UV 240), prednisolone acetate (UV 240), prednisolone (UV 240),
prednisone (UV 240), pregnenolone (UV 210), progesterone (UV 240), testosterone (UV 240)
REFERENCE
SAMPLE
Matrix: tissue
Sample preparation: Condition a 500 mg Bond Elut silica SPE cartridge with 5 mL EtOH and
5 mL cyclohexane. Homogenize 5 g tissue with 11 mL 40 mM pH 4.1 sodium acetate buffer.
Add 500 (xL 17 mg/mL p-glucuronidase (type H-5) in acetate buffer, mix. Incubate at 37 over-
night. Mix with 5 mL 2 M aqueous tris(hydroxymethyl)aminomethane. Fill an Extrelut car-
tridge with 25% of an Extrelut 20 sachet, mix the remainder of the sachet with the sample
and add it to the cartridge. Rinse the sample tube with 15 mL butan-2-ol:hexane 5:95 and add
the rinse to the cartridge. Elute with 100 mL butan-2-ol:hexane 5:95. Extract the eluate with
two 10 mL portions of MeCN. Evaporate the combined MeCN extracts to dryness after adding
two 2 mL portions of heptane. Reconstitute the residue with 2 mL cyclohexane. Add to the SPE
cartridge Wash the tube with two 2 mL and two 1 mL portions of cyclohexane. Add to the SPE
cartridge. Wash with 2 mL cyclohexane. Elute with 5 mL acetone rcyclohexane 25:75. Evaporate
the eluate and redissolve the residue in 400 |xL MeOH and 3.6 mL 85 water. Cool to room
temperature and add to an immunoaffinity column (Randox Laboratories, UK). Complete the
transfer with three 1 mL portions of hot water, cooled before adding to the column. Wash with
2 mL 1 mM pH 10 carbonate buffer, elute with 4 mL MeOH:water 70:30. Dilute the eluate
with 12 mL water. Inject a 4 mL aliquot onto column A and then backflush the contents of
column A onto column B with mobile phase. Monitor the effluent from column B.
HPLC VARIABLES
Column: A Chromspher (type R2) (Chrompack, UK); B 200 X 3 5 urn Chromspher C18 (Chrom-
pack, UK)
Flow rate: 0.5
Detector: UV 247
CHROMATOGRAM
Retention time: 12 ( epimer), 9 (a epimer)
Limit of detection: 500 pg/g
OTHER SUBSTANCES
Extracted: trenbolone
KEYWORDS
pig; cow; liver; corned beef; SPE; column-switching
REFERENCE
Stubbings,G.W.; Cooper,A.D.; Shepherd,M.J.; Croucher,M.; Airs,D.; Farrington/W.H.H.H.; Shearer,G. Determi-
nation of 19-nortestosterone and trenbolone in animal tissues by high-performance liquid chromatography
with immunoaffinity clean-up, Food Addit.Contam., 1998, 15, 293-301.
SAMPLE
Matrix: urine
Sample preparation: 10 mL Urine + glucuronidase/sulfatase (Helix pomatia), incubate at 37
for 1 h, extract twice with 5 mL diethyl ether, add 225 |xL water and evaporate ether under
nitrogen, add 400 |xL MeOH, inject a 250 |xL aliquot of this mixture.
Next Page
HPLC VARIABLES
Flow rate: 2
Detector: UV 240
CHROMATOGRAM
Retention time: 5.5
OTHER SUBSTANCES
Simultaneous: 17a-methyltestosterone, 17p-trenbolone, zeranol, trans-diethylstilbestrol, me-
droxyprogesterone
KEYWORDS
cow
REFERENCE
299, 450-455.
Naphazoline
Merck Index: 6455
Lednicer No.: 1 241
SAMPLE
Matrix: blood
Sample preparation: 500 jxL Plasma + 1 mL 600 mM perchloric acid, vortex, let stand for 10
min, centrifuge at 2000 g for 15 min. Remove 800 |xL of the supernatant and add it to 500 |xL
1.2 M potassium hydrogen carbonate in a 10 mL tube, mix, let stand for a few min, add 500
IxL 2 M NaOH, add 4 mL dichloromethane, shake gently for 15 min, centrifuge at 500 g for 10
min. Remove 3.5 mL of the organic layer and evaporate it to dryness under a stream of nitrogen
at 40, reconstitute the residue in 100 |xL 25 mM pH 5 sodium phosphate buffer, inject a 20
|xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Hypersil phenyl
Mobile phase: MeOH:buffer 44:56 (Buffer was 25 mM pH 5 sodium phosphate buffer containing
5 mM sodium 1-heptanesulfonate.)
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 11
KEYWORDS
rat; plasma
Previous Page
HPLC VARIABLES
Flow rate: 2
Detector: UV 240
CHROMATOGRAM
Retention time: 5.5
OTHER SUBSTANCES
Simultaneous: 17a-methyltestosterone, 17p-trenbolone, zeranol, trans-diethylstilbestrol, me-
droxyprogesterone
KEYWORDS
cow
REFERENCE
299, 450-455.
Naphazoline
Merck Index: 6455
Lednicer No.: 1 241
SAMPLE
Matrix: blood
Sample preparation: 500 jxL Plasma + 1 mL 600 mM perchloric acid, vortex, let stand for 10
min, centrifuge at 2000 g for 15 min. Remove 800 |xL of the supernatant and add it to 500 |xL
1.2 M potassium hydrogen carbonate in a 10 mL tube, mix, let stand for a few min, add 500
IxL 2 M NaOH, add 4 mL dichloromethane, shake gently for 15 min, centrifuge at 500 g for 10
min. Remove 3.5 mL of the organic layer and evaporate it to dryness under a stream of nitrogen
at 40, reconstitute the residue in 100 |xL 25 mM pH 5 sodium phosphate buffer, inject a 20
|xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Hypersil phenyl
Mobile phase: MeOH:buffer 44:56 (Buffer was 25 mM pH 5 sodium phosphate buffer containing
5 mM sodium 1-heptanesulfonate.)
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 11
KEYWORDS
rat; plasma
REFERENCE
Chabenat,C; Boucly,P. Determination of naphazoline in rat plasma using column liquid chromatography with
ultraviolet detection, Biomed.Chromatogr., 1992, 6, 241-243.
SAMPLE
Sample preparation: 50-100 uL Serum or urine + 250 |xL buffer + 5 mL dichloromethane, shake
mechanically for 20 min, centrifuge at 1500 g for 5 min. Remove the organic layer and evap-
orate it to dryness under a stream of nitrogen at 35, reconstitute the residue in 100 (serum)
or 500 (urine) uL 20 mM pH 3.75 KH2PO4, vortex for 15 s, inject a 10 (urine) or 90 (serum)
uL aliquot. (Buffer was 100 mM potassium hydrogen carbonate and 100 mM potassium car-
bonate, pH 10.0.)
HPLC VARIABLES
Guard column: 50 x 2 Co:Pell ODS
Column: Resolve C18 (Waters)
phosphoric acid.)
Flow rate: 1.2
Detector: UV 210
CHROMATOGRAM
Retention time: 10
Internal standard: naphazoline
OTHER SUBSTANCES
Extracted: tolazoline
Noninterfering: acetaminophen, N-acetylprocainamide, carbamazepine, chloramphenicol, desi-
pramine, digoxin, disopyramide, dopamine, ethosuximide, gentamicin, imipramine, lidocaine,
methotrexate, phenobarbital, phenytoin, primidone, procainamide, propranolol, quinidine, sal-
icylic acid, theophylline, valproic acid
KEYWORDS
naphazoline is IS; serum
REFERENCE
Cwik,M.J.; Chiu,G.R; Fischer,J.H.; Chow-Tung,E.; Currie,B-L. Quantitative determination of tolazoline in se-
rum and urine, J.Chromatogr., 1985, 338, 123-130.
SAMPLE
Sample preparation: 5 mL Ophthalmic solution + 5 mL 1.5 mg/mL tetrahydrozoline hydro-
chloride in water, inject a 20 JJLL aliquot.
HPLC VARIABLES
Mobile phase: MeOHibuffer 30:70 (Buffer was 6 g sodium citrate dihydrate and 4 g anhydrous
citric acid in 700 mL water, add 7 mL perchloric acid, adjust pH to 2.2 0.2 with perchloric
acid.)
Flow rate: 2
Detector: UV 265
CHROMATOGRAM
Internal standard: tetrahydrozoline (5.31)
OTHER SUBSTANCES
KEYWORDS
ophthalmic solutions; stability-indicating
REFERENCE
Bauer,J.; Krogh,S. High-performance liquid chromatographic stability-indicating assay for naphazoline and
tetrahydrozoline in ophthalmic preparations, J.Pharm.ScL, 1983, 72, 1347-1349.
SAMPLE
Sample preparation: 2 mL Sample + 1 mL 200 jxg/mL emetine hydrochloride in water, make
up to 10 mL with mobile phase, filter (0.45 jxm), inject a 50-100 |xL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 5 (xm Technosphere RP C-8 (HPLC Technology)
Mobile phase: MeCN:40 mM tetramethylammonium bromide: 1 M acetic acid 80:15:5 (apparent
pH 4.5)
Flow rate: 1.5
Detector: UV 260
CHROMATOGRAM
Internal standard: emetine (1.75)
OTHER SUBSTANCES
Simultaneous: benzalkonium (C 12, C14, C16)
Interfering: tetrahydrozoline
KEYWORDS
nasal; ophthalmic
REFERENCE
Santoni,G.; Medica,A.; Gratteri,R; Furlanetto,S.; Pinzauti,S. High-performance liquid chromatographic deter-
mination of benzalkonium and naphazoline or tetrahydrozoline in nasal and ophthalmic solutions, Farmaco,
1994, 49, 751-754.
SAMPLE
Sample preparation: Dilute nasal solution 10-fold with water, filter (0.45 |xm), inject a 10 |xL
HPLC VARIABLES
Flow rate: 1.2
CHROMATOGRAM
Retention time: 9.5
Limit of detection: 0.5 ng (UV 224)
OTHER SUBSTANCES
Simultaneous: ephedrine, oxymetazoline, xylometazoline
KEYWORDS
nasal solution
REFERENCE
3233-3242.
SAMPLE
Sample preparation: Dilute ophthalmic solution 1:10 with water,inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeOHiwater 57:43 containing 22 mM heptanesulfonic acid and 1% acetic acid
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 8.5
OTHER SUBSTANCES
Simultaneous: antazoline, degradation products
KEYWORDS
ophthalmic solutions; stability-indicating
REFERENCE
Ruckmick,S.C; Marsh,D.R; Duong,S.T. Synthesis and identification of the primary degradation product in a
commercial ophthalmic formulation using NMR, MS, and a stability-indicating HPLC method for antazoline
and naphazoline, J.Pharm.Sci., 1995, 84, 502-507.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:buffer 75:25 (Buffer was 12 g KH2PO4 in 1800 mL water, adjust pH to 3.0
with 1:3 phosphoric acid, make up to 2 L.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 5.5
Internal standard: naphazoline hydrochloride
OTHER SUBSTANCES
Simultaneous: cyclobenzaprine, amitriptyline
Interfering: desipramine
KEYWORDS
naphazoline is IS
REFERENCE
Heinitz,M.L. Determination of cyclobenzaprine in tablets by high-performance liquid chromatography,
J.Pharm.ScL, 1982, 71, 656-658.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 3.1
OTHER SUBSTANCES
lol, mianserin, morazone, nadolol, nalorphine, naloxone, nicotine, nifedipine, nomifensine, nor-
REFERENCE
Jane,L; McKinnon,A-; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 300 X 3.9 10 jxm LiChrosorb Si-60
Mobile phase: MeOH:water 60:40 containing 4 mM disodium citrate and 4 mM tetrabutylam-
monium bromide, pH 5.9
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Simultaneous: atropine, codeine, dansylamide, dansylcadaverine, doxorubicin, methylatropine,
noscapine, xylometazoline
REFERENCE
Lingeman,H.; van Munster,H.A.; Beynen,J.H.; Underberg,W.J.; Hulshoff,A. High-performance liquid chromat-
ographic analysis of basic compounds on non-modified silica gel and aluminium oxide with aqueous solvent
mixtures, J.Chromatogr., 1986, 352, 261-274.
SAMPLE
Matrix: solutions
|AL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 jjim uBondapak C18
Flow rate: 1.5
Detector: UV
CHROMATOGRAM
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeOH:water 40:60, inject a 10 JJLL aliquot.
HPLCVARIABLES
Column: 150 X 4.1 RSIL C18 (RSL, Eke, Belgium)
Mobile phase: MeOH:water 40:60 containing 20 mM sodium 1-octanesulfonate and 10 mM N,N-
dimethyloctylamine, pH adjusted to 3.0 with orthophosphoric acid
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Simultaneous: degradation products, antazoline, coumazoline, lidocaine, oxymetazoline, pred-
nisolone, sulfadimidine, sulfanilamide, sulfathiazole, tenaphtoxaline, tetrahydrozoline, tolazo-
line, tramazoline, xylometazoline
REFERENCE
De Schutter,J.A.; Van den Bossche,W.; De Moerloose,P. Stability-indicating analysis of tetryzoline hydrochloride
in pharmaceutical formulations by reversed-phase ion-pair liquid chromatography, J.Chromatogr., 1987,
391, 303-308.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 12 |xm l-myristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
famotidine, isothipendyl, ketotifen, metiamide, metoprolol, moxonidine, nadolol, nifenalol, ni-
REFERENCE
Kaliszan,R.; Nasal,A; Turowski,M. Binding site for basic drugs on c^-acid glycoprotein as revealed by chemo-
metric analysis of biochromatographic data, Biomed.Chromatogr., 1995, 9, 211215.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 jxin LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naproxen, ni-
fedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, ox5maetazoline, pa-
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Naproxen
Merck Index: 6504
LednicerNo.: 1 86
SAMPLE
Matrix: blood
Sample preparation: Precipitate plasma with phosphoric acid, extract with dichloromethane
containing IS. Centrifuge at 3000 rpm for 10 min. Remove a 4 mL aliquot of the upper layer
and evaporate it to dryness under a stream of nitrogen. Reconstitute the residue with 500 JJLL
MeOH, inject a 20 |xL aliquot.
HPLC VARIABLES
Flow rate: 2.5
Detector: UV 232
CHROMATOGRAM
Retention time: 6.5
Internal standard: ibuprofen (15.5)
KEYWORDS
REFERENCE
Niazi,S.K.; Alam,S.M.; Ahmad,S.I. Partial-area method in bioequivalence assessment: Naproxen, Bio-
pharm.DrugDispos., 1997, 18, 103-116.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Whole blood + 20 IJLL 500 mM pH 2.5 NaH2PO4 + 1 mL ethyl
acetate, vortex for 1 min. Centrifuge at 10000 rpm for 10 min, evaporate the organic layer to
dryness under a stream of nitrogen at 50. Dissolve the residue in 200 JJLL 75 mM pH 7 Na2HPO4
buffer:MeOH 50:50. Inject a 100 |xL aliquot.
HPLCVARIABLES
Column: 300 X 4 10 |xm MicroPak C18
Mobile phase: MeCN:buffer 45:55 (Buffer was 75 mM sodium acetate adjusted to pH 3.3 with
acetic acid.)
Flow rate: 2
Detector: E, BAS LC-4B, glassy carbon working electrode 1.1 V, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 3.5
Internal standard: naproxen
OTHER SUBSTANCES
KEY WORDS
rat; whole blood; pharmacokinetics; naproxen is IS
REFERENCE
Torres-L6pez,J.E.; L6pez-Mufioz,J.; Castaiieda-Hern&ndez,G.; Flores-Murrieta,F.J.; Granados-Soto,V. Pharma-
cokinetic-pharmacodynamic modeling of the antinociceptive effect of diclofenac in the rat,
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 10 |xL 2.5 |xg/mL 6-methoxy.2-acetonaphthone in MeCN
+ 500 IJLL pH 7.4 phosphate buffer + 100 |xL 37% HCl + 5 mL ethyl acetate, stir for 5 min,
centrifuge at 1500 g for 10 min. Remove the supernatant into another test tube and extract
the residue again. Evaporate the combined organic phases to dryness under a gentle nitrogen
flow at 40. Reconstitute the residue in 500 |xL MeCN, inject a 10 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4 2.5 |xm Viosfer ODS
Flow rate: 1.8
Detector: UV 238
CHROMATOGRAM
Internal standard: 6-methoxy-2-acetonaphthone (3.75)
KEYWORDS
REFERENCE
Marzo,A.; Monti,N.; Ripamonti,M.; Marzo,R; Wool,C; Cerutti,R.; Maggi,G.C. Comparative bioavailability study
on naproxen betainate sodium salt monohydrate and naproxen sodium salt in healthy volunteers, Arzneim-
ittelforschung, 1997, 47, 385-389.
SAMPLE
Matrix: blood
Sample preparation: Acidify 100 |xL whole blood with 20 |xL 500 mM pH 2.5 NaH2PO4 buffer,
add 1 mL ethyl acetate, vortex at maximal speed for 1 min, centrifuge at 10000 rpm for 10
min, evaporate the organic layer to dryness under nitrogen stream at 50, reconstitute the
residue with 200 |xL MeOH:75 mM pH 7 Na2HPO4 buffer 1:1, inject a 100 JJLL aliquot.
HPLC VARIABLES
Guard column: 40 X 4 37-50 |xm Corasil C18 (Waters)
Column: 300 X 4 10 ^m Micro Pak C18 (Varian)
Mobile phase: MeCN:75 mM sodium acetate adjusted to pH 3.3 with glacial acetic acid 45:55
Flow rate: 2
Detector: E, LC-4B, glassy carbon electrode +1100 mV, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 3.5
OTHER SUBSTANCES
KEYWORDS
rat; whole blood; naproxen is IS
REFERENCE
Torres-L6pez,J.E.; Robles,M.B.; Perez-Urizar,J.; Flores-Murrieta,F.J.; Granados-Soto,V. Determination of diclo-
fenac in micro-whole blood samples by high-performance liquid chromatography with electrochemical de-
tection. Application in a pharmacokinetic study, Arzneimittelforschung, 1997, 47, 10401043.
SAMPLE
Matrix: blood
Sample preparation: 500 uX Plasma + 500 uL 500 mM HCl, vortex for 1 min, add 5 mL ethyl
acetate, extract for 20 min, centrifuge at 2500 rpm for 10 min. Remove the organic layer and
evaporate it to dryness, reconstitute the residue in 200 u,L MeCN, inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 250 X 5 5 |xm C18 (Machery & Nagel)
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
plasma; naproxen is IS
REFERENCE
Ramakrishna,S.; Fadnavis,N.W.; Diwan,P.V. Comparative pharmacokinetic evaluation of compressed supposi-
tories of diclofenac sodium in humans, Arzneimittelforschung, 1996, 46, 175-177.
SAMPLE
Sample preparation: 500 |xL Plasma or tissue (homogenized in phosphate buffer in a ratio 1:
10), 10 |xL 5 (xg/|xL 6-methoxy-2-acetonaphthone in MeCN, 500 |xL pH 7.4 phosphate buffer,
100 u,L 37% HCl, and 5 mL ethyl acetate stir for 5 min, centrifuge at 1500 g for 10 min. Pipette
the supernatant into another test tube and extract the residue again. Evaporate combined
organic layer to dryness under a gentle nitrogen flow at 40, reconstitute the residue in 500
IxL MeCN and inject an aliquot.
HPLCVARIABLES
Flow rate: 1.5
Detector: UV 238
CHROMATOGRAM
Internal standard: 6-methoxy-2-acetonaphthone (7.22)
Limit of quantitation: 1 |xg/mL (plasma), 10 |xg/g (tissue)
KEYWORDS
gastric wall; heart; kidney; liver; lung; pharmacokinetics; plasma; rat
REFERENCE
Marzo,A.; Ripamonti,M.; Benatti,R; Marzo,R; Wool,C; Cerutti,R.; Reiner,V. Absorption and distribution of na-
proxen in rats orally treated with naproxen betainate sodium salt monohydrate. Comparison with naproxen,
Arzneimittelforschung, 1997, 47, 381-384.
SAMPLE
Matrix: dialysate
Sample preparation: Inject a 10 |xL aliquot of dialysate (pH 7.4 isotonic phosphate buffer).
HPLC VARIABLES
Guard column: 37-50 |xm Corasil C18
Flow rate: 1.1
CHROMATOGRAM
Retention time: 2.5
OTHER SUBSTANCES
Extracted: flurbiprofen (F ex 258 em 310)
KEYWORDS
mouse; rat; naproxen is IS
REFERENCE
Evrard,RA.; Deridder,G.; Verbeeck,R.K. Intravenous microdialysis in the mouse and the rat: Development and
pharmacokinetic application of a new probe, Pharm.Res., 1996, 13, 1217.
SAMPLE
Matrix: perfusate
Sample preparation: Mix 1 mL perfusate with 100 |xL 1 M HCl and 8 mL diethyl ether. Vortex
for 1 min, centrifuge at 3000 rpm for 5 min. Remove the organic layer and evaporate it to
dryness under nitrogen. Reconstitute the residue with 2 mL mobile phase. Inject a 30 JJLL
aliquot.
HPLCVARIABLES
Detector: UV 330
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
KEYWORDS
naproxen is IS
REFERENCE
Takamatsu,N.; Welage,L.S.; Idkaidek,N.M.; Liu,D.Y.; Lee,P.I.-D.; Hayashi,Y.; Rhie,J.K; Lennernas,H.; Bar-
nett,J.L.; Shah,V.R; Lesko,L.; Amidron,G.L. Human intestinal permeability of piroxicam, propranolol, phen-
ylalanine, and PEG 400 determined by jejunal perfusion, Pharm.Res., 1997, 14, 1127-1132.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |jim Ultrasphere C18
Mobile phase: MeCN:water:l% glacial acetic acid 50:49:1
Flow rate: 1
Detector: UV 243
REFERENCE
mans, J.Pharm.Sci., 1996, 85, 1070-1076.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:50 mM phosphoric acid 40:60adjusted to pH 5.5 with NaOH
Flow rate: 0.6
Detector: UV 230
OTHER SUBSTANCES
Also analyzed: carbamazepine, fenbufen, indomethacin, ketoprofen, a.-naphthoquinone,
tolmetin
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Filter (0.45 |jim), dilute the nitrate with mobile phase, inject an aliquot.
HPLC VARIABLES
Detector: UV 250
REFERENCE
Okimoto,K.; Rajewski,R.A.; Uekama,K.; Jona,J.A.; Stella,V.J. The interaction of charged and uncharged drugs
with neutral (HP-p-CD) and anionically charged (SBE7-(S-CD) p-cyclodextrins, Pharm.Res., 1996,13, 256-
264.
SAMPLE
Matrix: urine
Add 1 mL urine, vortex, add 250 |xL 1 M pH 5.0 acetate buffer, vortex. Add 250 JJLL of the
the eluate to dryness under a stream of nitrogen at 37, reconstitute the residue in 200 |JLL
mobile phase, inject a 10-30 JJLL aliquot.
HPLC VARIABLES
Flow rate: 0.9
Detector: UV 230
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Extracted: diclofenac, ibuprofen, felbinac, fenbufen, flurbiprofen, ketoprofen, loxoprofen, mefen-
amic acid, piroxicam, sulindac
KEYWORDS
SPE
REFERENCE
J.Chromatogr.B, 1997, 692, 375-388.
Narasin
Molecular formula: C43H72On
Merck Index: 6506
SAMPLE
Matrix: bulk, feed, premix
Sample preparation: 100 mg Bulk or 5 g premix or feed + 200 mL MeOH:water 90:10, shake
on a gyratory shaker at 200 rpm for 1 h, allow to settle. Dilute an aliquot to 20 jjig/mL with
MeOH:water 90:10, filter (0.45 jxm), inject a 200 |JLL aliquot.
HPLCVARIABLES
Guard column: C18
Mobile phase: MeOH:water:acetic acid 94:6:0.1
Flow rate: 0.7
pumped at 0.7 mL/min and the mixture flowed through a 6.1 m X 0.5 mm ID stainless steel
coil at 98 to the detector. (Prepare reagent by cautiously adding 20 mL concentrated sulfuric
acid to 950 mL MeOH, allow to cool to room temperature, add 30 g vanillin while stirring.)
CHROMATOGRAM
Retention time: 14
Limit of quantitation: 2.5 ppm
OTHER SUBSTANCES
Extracted: monensin
Noninterfering: bacitracin, bambermycin, lincomycin, nicarbazin, tylosin
KEYWORDS
REFERENCE
Rodewald,J.M.; Moran,J.W.; Donoho,A.L.; Coleman,M.R. Determination of narasin in raw material, premix,
and animal feeds by liquid chromatography and correlation to microbiological assay, J.AOAC Int., 1994,
77, 821-828.
SAMPLE
Sample preparation: 5 g Pulverized frozen tissue or 5 g homogenized whole eggs + 2 mL water
+ 13 mL MeOH, homogenize for 30 s. Sonicate for 10 min and centrifuge at 2000 g for 10 min.
Add 4 mL 100 mM NaOH to a 2 mL aliquot of the supernatant, extract with 2 mL and 1 mL
hexane:toluene 50:50 (v/v) for 30 s by inversion, centrifuge at 1500 g for 10 min. Evaporate the
combined extracts to dryness under a stream of nitrogen at 60. Dissolve the residue in 200
yJu MeCNrwater 75:25. Inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 fim Inertsil ODS-2 (GL Sciences, Japan)
Mobile phase: MeCN:MeOH:THF:water:trifluoroaceticacid 67:10:10:13:0.1
Flow rate: 1
Detector: MS, VG Platform, Megaflow electrospray probe, positive ion mode, source at 125, cone
voltage 25 V, m/z 787
CHROMATOGRAM
Retention time: 8.3
Limit of detection: 0.5-1 ng/g
OTHER SUBSTANCES
KEYWpRDS
domestic fowl; muscle; liver
REFERENCE
Blanchflower,W.J.; Kennedy,D.G. Determination of monensin, salinomycin, and narasin in muscle, liver and
eggs from domestic fowl using liquid chromatography-electrospray mass spectrometry, J.Chromatogr.B,
1996, 675, 225-233.
SAMPLE
Matrix: feed
Sample preparation: 20 g Ground Feed + 200 mL hexane:ethyl acetate 90:10, stir at high speed
for 2 h, let stand. Remove an aliquot equivalent to 1 g feed and evaporate it to dryness under
reduced pressure at 40, reconstitute with 2 mL MeOH, filter (0.45 fim), inject an aliquot of
the filtrate.
HPLC VARIABLES
Column: 60 X 4.6 3 |xm C18 (Hewlett-Packard)
Mobile phase: MeOH:5% acetic acid 90:10
Flow rate: 0.5
pumped at 1 mL/min and the mixture flowed through a 1.5 mL reaction coil (Kratos Model
510) at 95 to the detector. (Reagent was 40 g/L vanillin in MeOH:sulfuric acid 100:2. Keep in
an ice bath and prepare fresh daily.)
CHROMATOGRAM
Retention time: 8.2
Limit of detection: 1 ppm
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Lapointe,M.R.; Cohen,H. High-speed liquid chromatographic determination of monensin, narasin, and salino-
mycin in feeds, using post-column derivatization, J.Assoc.Off.Anal.Chem., 1988, 71, 480-484.
SAMPLE
Sample preparation: Feed. Shake 5 g feed with 15 mL MeOH for 2 h, filter, evaporate the
nitrate to 3 mL and make up to 10 mL with MeOH, inject a 3 |xL aliquot. Premix. Shake 0.5
g premix with 15 mL MeOH for 2 h, filter, make up the filtrate to 50 mL with MeOH, inject a
3 |JLL aliquot.
HPLC VARIABLES
Column: 150 X 1 5 |xm Separon SGX C18 glass column (Tessek Prague)
Flow rate: 0.02
Injection volume: 3
Detector: UV 592 following post-column derivatization. The column effluent mixed with the re-
agent pumped at 0.015 mL/min and the mixture flowed through a 150 X 1 reactor containing
40-70 (Jim acid-washed glass beads at 75 to the detector. (The reagent was 500 mM 4-di-
methylaminobenzaldehyde in 1.2 M sulfuric acid in MeOH.)
CHROMATOGRAM
Retention time: 18
Limit of detection: 2.3 |xg/mL
OTHER SUBSTANCES
KEYWORDS
microbore; post-column reaction
REFERENCE
Fejglova,Z.; Dolezal,J.; HrdlickaÂ..; Frgalova,K. Microbore HPLC determination of polyether antibiotics using
postcolumn derivatization with benzaldehyde reagents, J.Liq.Chromatogr., 1994, 17, 359-372.
SAMPLE
Sample preparation: Homogenize 5 mL fermentation broth with 25 mL MeOH, filter (0.45 jxm)
the supernatant, inject a 20 |xL aliquot of the filtrate.
HPLC VARIABLES
Column: 50 X 4.6 3 ^m Little Champ ODS (Regis)
Mobile phase: MeOH:100 mM pH 4 KH2PO4 buffer 93:7
Flow rate: 2
Detector: UV 520 following post-column reaction. The column effluent mixed with 10% sulfuric
acid in MeOH pumped at 1 mL/min and with 6% vanillin in MeOH pumped at 1 mL/min and
the mixture flowed through a 1.5 m X 0.25 mm ID stainless steel coil at 120 to the detector.
CHROMATOGRAM
Retention time: 2
KEYWORDS
REFERENCE
Neely,F.L. A rapid HPLC determination of narasin in fermentation broth, Chromatographia, 1991, 31, 277-
280.
Naratriptan
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL Lichrolut C18-Select B SPE cartridge with 3 mL MeOH
and 3 mL water. Mix 100 or 300 jxL plasma with 500 |xL 40 ng/mL IS in MeOH:water 50:50,
add 800 |xL MeOH, vortex, centrifuge at 15000 rpm for 10 min. Evaporate the supernatant to
dryness under a stream of nitrogen at 40, reconstitute the sample in 1 mL MeOH:water 10:
90. Add 1 mL of the sample to the SPE cartridge, wash with 5 mL water, wash with 3 mL
MeOH:water 20:80, elute with 2 mL MeCN containing 10% 1 M HCl, evaporate the eluate to
HPLCVARIABLES
Column: 150 X 2 4 ^m Nova-Pak C8
Mobile phase: Gradient. A was MeCN:20 mM ammonium acetate 10:90. B was MeCN:20 mM
ammonium acetate 80:20. A:B from 80:20 to 20:80 over 20 min, re-equilibrate at initial con-
ditions for 10 min
Flow rate: 0.5
Detector: MS, Finnigan MAT SSQ-700 or TSQ-7000, API, ESI, 4.8 kV needle, +5.8 V to the
capillary, +44.6 V to the tube lens, source 230, m/z 339
CHROMATOGRAM
Internal standard: MDL 74,967
OTHER SUBSTANCES
Noninterfering: MDL 74,721, sumatriptan
KEYWORDS
plasma; rabbit; SPE; pharmacokinetics
REFERENCE
Dulry,B.D.; Petty,M.A.; Schoun,J.; David,M.; Huebert,N.D. A method using a liquid chromatographic-electro-
spray-mass spectrometric assay for the determination of antimigraine compounds: preliminary pharmaco-
kinetics of MDL 74,721, sumatriptan and naratriptan, in rabbit, J.Pharm.Biomed.Anal., 1997, 15, 1009-
1020.
Natamycin
Merck Index: 6513
SAMPLE
Sample preparation: Serum. 50-150 |xL Serum + 2 mL MeOH:acetic acid 90:10, vortex for 30
s, leave in the dark for 1 h, centrifuge at 1000 g for 10 min, decant, filter (0.45 |xm), inject a
100 |xL aliquot. Tissue. 100-200 mg Tissue + 500 |xL 1 mM pH 7.4 phosphate buffer, vortex,
homogenize using a manual glass homogenizer, add 2 mL MeOHiacetic acid 90:10, vortex for
30 s, leave in the dark for 1 h, centrifuge at 1000 g for 10 min, decant, filter (0.45 /xm), inject
a 100 |xL aliquot.
HPLC VARIABLES
Guard column: 30 X 2 Alltech C18
Column: 300 X 3.9 10 jxm fxBondapak RP-C18
Flow rate: 1 for 6 min, then 2
Detector: UV 303
CHROMATOGRAM
Retention time: 6
Internal standard: natamycin
OTHER SUBSTANCES
Extracted: amphotericin (UV 383)
KEYWORDS
serum; lung; liver; natamycin is IS
REFERENCE
Polikandritou Lambros,M.; Abbas,S.A.; Bourne,D.W.A. New high-performance liquid chromatographic method
for amphotericin B analysis using an internal method, J.Chromatogr.B, 1996, 685, 135-140.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 303.3
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
the creation of a 600-compound library. Application to forensic toxicology, J.Chromatogr~A, 1997, 763,149-
163.
SAMPLE
Matrix: cheese
Sample preparation: Cut cheese into small pieces, weigh out 5 g, add 50 mL MeOH (100 mL
for cheese rind), stir or shake for 1.5 h, add 25 mL water (50 mL for cheese rind), let stand at
-15 to -20 for 1 h, filter (paper) while cold, discard first 5 mL filtrate, filter (0.45 |xm), filter
(0.20 |xm) again, dilute with two volumes of water, add 75-150 mL to an activated Sep-Pak
C18 SPE cartridge at 25 mL/min, wash with 10 mL water, elute with 3 mL MeOH, make up
eluate to 5 mL with MeOH, inject a 20 jxL aliquot.
HPLC VARIABLES
Guard column: 100 X 2.1 30-40 |xm Perisorb RP-8
Column: 150 x 4.6 5 |xm Lichrosorb RP-8
Flow rate: 1
Detector: UV 303
CHROMATOGRAM
KEYWORDS
SPE
REFERENCE
de Ruig,W.G.; van Oostrom,J.J.; Leenheer,K. Spectrometric and liquid chromatographic determination of na-
tamycin in cheese and cheese rind, J.Assoc.Off.Anal.Chem., 1987, 70, 944-948.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH at a concentration of 140 mM, inject a 10 (xL aliquot.
HPLCVARIABLES
Mobile phase: MeOH:buffer 50:50 (Buffer was 6.3 g NaH2PO4 in 1 L water, adjust pH to 2.6
Flow rate: 1
Detector: UV 318
CHROMATOGRAM
Retention time: 10
REFERENCE
Backes,B.J.; Rychnovsky,S.D. A reverse-phase HPLC assay for measuring the interaction of polyene macrolide
antifungal agents with sterols, Anal.Biochem., 1992, 205, 96-99.
Nedocromil
101626-68-0 (Ca salt)
Merck Index: 6524
Lednicer No.: 4 209
SAMPLE
Matrix: urine
Sample preparation: Condition a 100 mg Isolute phenyl SPE cartridge with 2 mL MeOH and
2 mL 100 mM HCl. Mix 5 mL urine with 1 mL water. Adjust pH to 1 with 4 mL 250 mM
hydrochloric acid, vortex for 30 s. Remove 2 mL aliquot, add it to the SPE cartridge, allow to
elute through the bed over 2-3 min, dry under full vacuum for 3 min. Wash with 2 mL 100
mM HCl, 2 mL MeOH:100 mM HCl 30:70 and dry under full vacuum for 3 min. Elute with 2
mL MeOH, evaporate to dryness under a stream of nitrogen, reconstitute the residue in 1 mL
mobile phase, vortex for 30 s and inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 LD. 5 |xm Spherisorb S5C8
Column: 250 X 4.6 LD. 5 |xm Spherisorb S5C8
Mobile phase: MeOH:45 mM phosphate buffer:500 mM dodecyl triethyl ammonium phosphate
55:44.76:0.24 adjusted to pH 2.3 with orthophosphoric acid.
Flow rate: 0.85
Detector: UV 238
CHROMATOGRAM
Internal standard: nedocromil
OTHER SUBSTANCES
Extracted: cromolyn disodium
KEYWORDS
SPE; nedocromil is IS
REFERENCE
Aswania,O.A.; Corlett,S.A.; Chrystyn,H. Development and validation of an ion-pair liquid chromatographic
method for the quantitation of sodium cromoglycate in urine following inhalation, J.Chromatogr.B, 1997,
690, 373-378.
SAMPLE
Matrix: urine
Sample preparation: Inject 200 JJLL urine through an in-line filter on to column A and elute to
waste with mobile phase A, after 1.5 min backflush the contents of column A onto column B
with mobile phase B, after 4 min remove column A from the circuit, elute column B with mobile
HPLC VARIABLES
Column: A 30 X 4 Hypersil 5-ODS; B 250 X 4.6 Zorbax SAX
Mobile phase: A MeOH:9.2 mM sulfuric acid (Pass mobile phase through a 250 X 4.6 25-40 fxm
silica (HPLC Technology) column to saturate it with silica.); B MeOH:buffer 50:50 (Buffer was
180 mM Na2HPO4 adjusted to pH 3.00 0.05 with 180 mM orthophosphoric acid. Pass mobile
phase through a 250 X 4.6 25-40 /xm silica (HPLC Technology) column to saturate it with
silica.)
Flow rate: A 5 (0.2 when no sample is being chromatographed); B 1
Detector: UV 253
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Simultaneous: acetaminophen, albuterol, aspartame, aspirin, beclomethasone dipropionate, caf-
feine, cromolyn, isoproterenol, menthol, minocromil, quinoline yellow, reproterol, riboflavin,
saccharin, salicylic acid, sorbitan trioleate, terbutaline, theophylline
KEYWORDS
column-switching
REFERENCE
Baker,RR.; Gardner,J.J.; Wilkinson,D. Automated high-performance liquid chromatographic method for the
1995, 668, 59-65.
Nefazodone
Merck Index: 6527
Lednicer No.: 4 98
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 500 JJIL saturated sodium bicarbonate solution + 50 |xL 4
ixg/mL aprindine in EtOH, vortex for 20 s, add 5 mL butyl chloride, tumble mix for 17 min,
centrifuge at 600 g for 2 min. Remove 4.5 mL of the upper organic layer and evaporate it to
dryness at 42 over 17 min, cool for 30 s, reconstitute the residue in 200 (xL 10 mM (NHJ2HPO4,
vortex for 20 s, inject an aliquot
HPLCVARIABLES
Mobile phase: MeCN:MeOH:buffer 45:10:45 (Buffer was 20 mL 1 M (NHJ2HPO4 adjusted to pH
3.0 with phosphoric acid, 10 mL 1 M tetramethylammonium hydroxide adjusted to pH 3.0 with
phosphoric acid, and 860 mL water.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: aprindine (15.3)
OTHER SUBSTANCES
Simultaneous: diazepam, haloperidol
KEYWORDS
REFERENCE
Franc,J.E.; Duncan,G.R; Farmen,R-H.; Pittman,K.A. High-performance liquid chromatographic method for the
determination of nefazodone and its metabolites in human plasma using laboratory robotics, J.Chromatogr.,
1991, 570, 129-138.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond Elut cyanopropyl SPE cartridge with one vol-
ume MeOH and one volume water, do not allow to dry. 1 mL Plasma + 30 ng gepirone, add to
the SPE cartridge, wash with 2 column volumes of water, dry under vacuum, elute with one
column volume of MeOH:ammonia 90:10. Evaporate the eluate to dryness under vacuum, re-
constitute the residue in 150 u,L mobile phase, vortex, inject an aliquot.
HPLCVARIABLES
Guard column: 5 |xm C2 (Capital HPLC, Edinburgh)
Column: 150 X 4.6 5 |xm cyanopropyl (Capital HPLC, Edinburgh)
Mobile phase: MeCN:MeOH:40 mM pH 6 potassium phosphate buffer 22.5:17.5:60
Flow rate: 1.4
Detector: E, ESA coulometric Model 5100A, Model 5020 guard cell 0.80 V, detector 1 0.75 V,
detector 2 0.5 V
CHROMATOGRAM
Retention time: 3.2
Internal standard: gepirone (5.6)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Franklin,M. Determination of nefazodone and its metabolites in plasma by high-performance liquid chroma-
tography with coulometric detection, J.Pharm.BiomedAnal., 1993, 11, 1109-1113.
SAMPLE
Sample preparation: Plasma. Add 5 mL plasma to a conditioned Sep-Pak C8 SPE cartridge,
wash with 2 mL 10 mM pH 5.0 ammonium acetate buffer, elute with 2 mL MeOH. Evaporate
the eluate to dryness under a stream of nitrogen, reconstitute the residue in 800 (xL 10 mM
pH 5.0 ammonium acetate buffer, add 400 |xL MeOH, filter (0.5 |xm), inject a 200 |xL aliquot.
Urine. Hydrolyze urine with p-glucuronidase/arylsulfatase (Helix pomatia, Sigma) at 37 over-
night, add a 5 mL aliquot to a conditioned Sep-Pak C8 SPE cartridge, wash with 2 mL 10 mM
pH 5.0 ammonium acetate buffer, elute with 2 mL MeOH. Evaporate the eluate to dryness
under a stream of nitrogen, reconstitute the residue in 800 |xL 10 mM pH 5.0 ammonium
acetate buffer, add 400 |xL MeOH, filter (0.5 |xm), inject a 200 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Zorbax Rx C8
Mobile phase: Gradient. MeOH: 10 mM pH 5.0 ammonium acetate buffer from 60:40 to 90:10
over 20 min, to 100:0 (step-gradient).
Flow rate: 1
CHROMATOGRAM
Retention time: 22
OTHER SUBSTANCES
KEYWORDS
human; dog; plasma; SPE
REFERENCE
Mayol,R.F.; Cole,C.A.; Luke,G.M.; Colson,K.L.; Kerns,E.H. Characterization of the metabolites of the antide-
pressant drug nefazodone in human urine and plasma, Drug Metab.Dispos., 1994, 22, 304-311.
SAMPLE
Sample preparation: 1 mL Plasma or urine + 100 /xL 1 |xg/mL aprindine + 100 JXL 1 M sodium
carbonate + 100 |xL saturated ammonium sulfate + 5 mL n-butyl chloride, extract, centrifuge,
freeze the aqueous phase in isopropanol/dry ice. Remove the organic layer and evaporate it to
dryness under a stream of nitrogen, reconstitute the residue in 30 |xL EtOH, add 90 (JLL mobile
phase, inject a 70 u.L aliquot.
HPLCVARIABLES
Guard column: 37-50 jim Corasil Type II
Column: 300 X 3.9 7-8 jxm Zorbax silica
Mobile phase: MeCN: 10 mM ammonium acetate 58:42, pH 5.0
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 8.4
Internal standard: aprindine (11.2)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Barbhaiya,R.H.; Marathe,P.H.; Greene,D.S.; Mayol,R.R; Shukla,U.A.; Gammans,R.R.; Pittman,K.A.; Robin-
son,D. Safety, tolerance, and preliminary pharmacokinetics of nefazodone after administration of single and
multiple oral doses to healthy adult male volunteers: A double blind, Phase I study, J.Clin.Pharmacol.,
1995, 35, 974-984.
Nefopam
Merck Index: 6529
Lednicer No.: 2 447
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
naltrexone, naphazoline, niacinamide, nicotine, niacin, nifedipine, niflumic acid, nitrazepam,
REFERENCE
18, 233-242.
Nelfinavir
CAS Registry No.: 159989-64-7, 159989-65-8 (mesylate)
SAMPLE
Sample preparation: Dissolve oral powder granules in MeCN:MeOH:water 70:20:10. Heat at
40 with shaking, inject a 10 JJLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Capcell Pak UG120A C18
Mobile phase: Gradient. MeCN:5 mM pH 6.0 tetrabutylammonium sulfate from 50:50 to 65:35
over 7.0 min, maintain at 65:35 for 2 min, to 90:10 over 9 min.
Flow rate: 1.1
Detector: UV 210
OTHER SUBSTANCES
Simultaneous: coupling reagents, degradation products, excipients, synthetic intermediates
KEYWORDS
oral powder; stability-indicating
REFERENCE
Tsai,W.-C; Liu,J.; TyIe,P.; Wilke,T.L. Development of a stability-indicating HPLC method for nelfinavir mes-
ylate (agl343) oral powder formulation (Abstract 3347), Pharm.Res., 1997, 14, S580.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 3.9 5 |xm Delta-pak C4 (Waters)
Mobile phase: MeCN:buffer 35:65 (Buffer was 10 mM ammonium dihydrogen phosphate and 1
mM 1-heptanesulfonic acid sodium salt, pH adjusted to 4.8 with ammonium hydroxide.)
Flow rate: 0.6
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: indinavir, ritonavir, saquinavir
Noninterfering: didanosine, lamivudine, stavudine, zalcitabine, zidovudine
REFERENCE
Iayewardene,A-L-; Zhu,R; Aweeka,F.T.; Gambertoglio,J.G. Simple high-performance liquid chromatographic de-
termination of the protease inhibitor indinavir in human plasma, J.Chromatogr.B, 1998, 707, 203-211.
Nemonapride
Molecular formula: C2IH36CIN3O2
Merck Index: 6533
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 212.2
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Neomycin
Molecular formula: C23H46N6O13 (neomycin B)
Molecular weight: 614.65 (neomycin B)
CAS Registry No.: 1404-04-2, 1406-04-8 (undecylenate),
1405-10-3 (sulfate), 1405-12-5 (palmitate)
Merck Index: 6542
SAMPLE
Sample preparation: Condition a 3 mL 100 mg Isolute carboxypropyl CBA-bonded silica SPE
cartridge (Jones Chromatography) with 1 mL MeOH and 1 mL pH 7.4 phosphate buffer. Add
1 mL plasma or Iso-sensitest broth to the SPE cartridge, wash with 2 mL pH 7.4 phosphate
buffer and 4 mL 200 mM pH 9 borate buffer. Dry the cartridge with approximately 30 mL of
air, elute with 1 mL MeCN:200 mM pH 10.5 borate buffer 50:50. Adjust the pH value of a 1
mL portion of the eluate to 8.9 by the addition of 200 juiL 800 mM boric acid, add 200 |xL 7
mM fluorenylmethyl chloroformate in MeCN, let stand at ambient temperature for 15 min,
add 50 |xL 100 mM glycine, inject a 20 |xL aliquot of the mixture. (Prepare pH 7.4 phosphate
buffer by mixing appropriate volumes of 20 mM NaH2PO4 and Na2HPO4 solutions. Prepare
borate buffer by adjusting the pH of boric acid solution with 45% KOH.)
HPLC VARIABLES
Flow rate: 1
CHROMATOGRAM
Retention time: 10
Internal standard: sisomycin (14)
OTHER SUBSTANCES
KEY WpRDS
REFERENCE
Stead,D.A.; Richards,R.M.E. Sensitive high-performance liquid chromatographic assay for aminoglycosides in
biological matrices enables the direct estimation of bacterial drug uptake, J.Chromatogr.B, 1997, 693,415-
421.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 8 |xm PLRP-S 1000 A poly(styrene-divinylbenzene) (Polymer Laboratories)
Mobile phase: Water containing 70 g/L sodium sulfate, 1.4 g/L sodium 1-octanesulfonate, and
50 mL/L 200 mM pH 3.0 phosphate buffer
Flow rate: 1
Detector: E, Dionex PED-I pulsed electrochemical detector at 35, 3 mm dia. gold working elec-
trode, E 1 +0.05 V, E 2 +0.75 V, E 3 -0.15 V, tx 0-0.40 s, t2 0.41-0.60 s, t3 0.61-1.00 s, measure signal
between 0.2 and 0.4 s, stainless steel counter electrode, Ag/AgCl reference electrode, following
post-column reaction. The column effluent mixed with 500 mM NaOH pumped at 0.3 mL/min
and the mixture flowed through a 1.2 m long 500 |xL coil to the detector. (Prepare 500 mM
NaOH solution by diluting 50% NaOH with helium-degassed water. Clean gold electrode after
each 60 analyses.)
CHROMATOGRAM
Retention time: 23 (neomycin B)
OTHER SUBSTANCES
Simultaneous: neamine, neomycin C, neomycin LP-A, neomycin LP-B, paromamine, paromo-
mycin I, paromomycin II
KEYWORDS
REFERENCE
Adams,E.; Schepers,R.; Roets,E.; Hoogmartens,J. Determination of neomycin sulfate by liquid chromatography
with pulsed electrochemical detection, J.ChromatognA, 1996, 741, 233-240.
Neostigmine bromide
Molecular formula: C12H19BrN2O2
CAS Registry No.: 114-80-7, 59-99-4 (neostigmine),
51-60-5 (neostigmine methylsulfate)
Merck Index: 6553
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL 500 ng/mL edrophonium in water + 500 |xL 100
mM picric acid in 100 mM NaOH (pH adjusted to 7) + 400 |xL 100 mM NaH2PO4 + 12 mL
water saturated dichloromethane, shake vigorously for 5 min, centrifuge at 2000 g for 10 min.
Remove 10 mL of the organic phase and add it to 200 fiL 1 mM tetrabutylammonium hydrogen
sulfate, shake vigorously for 30 s, centrifuge at 2000 g for 2 min, discard most of the organic
layer, centrifuge at 2000 g for 1 min, inject a 50 fxL aliquot of the aqueous layer. (Store glass-
ware in 100 mM tetramethylammonium chloride solution and wash 5 times with water before
use.)
HPLC VARIABLES
Guard column: 50 X 3.2 30-40 ^m Perisorb RP-2 (Merck)
Mobile phase: MeCN:water 20:80 containing 10 mM sodium heptanesulfonate, 10 mM NaH2PO4,
and 2.5 mM tetramethylammonium chloride, pH adjusted to 3 with concentrated sulfuric acid
Flow rate: 2
Detector: UV 214
CHROMATOGRAM
Retention time: 5
Internal standard: edrophonium (3.5)
KEYWORDS
REFERENCE
De Ruyter,M.G.M.; Cronnelly,R.; Castagnoli,N.,Jr. Reversed-phase, ion-pair liquid chromatography of quater-
nary ammonium compounds: determination of pyridostigmine, neostigmine and edrophionium in biological
fluids, J.Chromatogr., 1980, 183, 193-201.
SAMPLE
Matrix: blood
Sample preparation: Extract serum.
HPLC VARIABLES
Column: 150 X 3.3 7 |xm Separon SGX
Mobile phase: 80 mM ammonium perchlorate in MeOH
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: nicotine, strychnine, amrinone
KEYWORDS
serum
REFERENCE
Eigendorf,H.G.; Nagel,S. Zur Analytik von Amrinone (Cordemcura). 2. Mitteillung: Hochdruckflussigchroma-
tographie [The analysis of amrinone (Cordemcura). 2. High pressure liquid chromatography], Pharmazie,
1987, 42, 631.
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 200 mg octadecyl SPE cartridge with 2 column volumes
of MeOH and 2 column volumes of 100 mM pH 4 phosphate buffer. 1 mL Serum + 5 |xL 100
mM pH 4 NaH2PO4, vortex for 15 s, add 1 mL 1 mM reagent in 100 mM pH 4 phosphate buffer,
mix for 30 s, add to the SPE cartridge, wash with 4 mL water, elute with 200 |xL MeOH:water
95:5. Evaporate the eluate to dryness under a stream of nitrogen at 30, reconstitute the res-
idue in 100 |xL mobile phase, inject a 50 jxL aliquot. (Synthesize reagent, sodium a-(3,4-di-
methoxyphenyl) cinnamonitrile-2'-sulfonate, as follows. Add 5 mL 10% KOH in water to a
stirred solution of 20 mmoles 3,4-(dimethoxyphenyl)acetonitrile and 20 mmoles 2-formylben-
zenesulfonic acid, sodium salt hydrate (sodium benzaldehyde-2-sulfonate) in 50 mL EtOH at
50, stir at 50 for 5 min, cool (evaporate to near dryness, if necessary), filter to obtain sodium
a-(3,4-dimethoxyphenyl) cinnamonitrile-2'-sulfonate (mp of p-toluidine salt is 218-223) (J.
Chem. Eng. Data 1975, 20, 215).)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm diol (ES Industries, Marlton NJ)
Mobile phase: MeOH:50 mM pH 4 NaH2PO4 20:80 containing 500 JJLM sodium a-(3,4-dimethox-
yphenyl) cinnamonitrile-2'-sulfonate
Flow rate: 1
Detector: F ex 243 em 418 (cutoff filter) following post-column extraction. The column effluent
mixed with dichloromethane pumped at 1 mL/min and the mixture flowed through a 90 cm X
0.3 mm ID knitted PTFE coil to a 50 (xL membrane phase separator using a polyethylene-
backed 0.5 jxm Fluoropore membrane filter (design in paper). The organic phase flowed to the
detector.
CHROMATOGRAM
Internal standard: neostigmine
OTHER SUBSTANCES
Extracted: physostigmine, eseroline
Also analyzed: amantadine, amphetamine, atropine, chlorpheniramine, clidinium bromide, N,N-
dimethyl-N-benzyltetradecylammonium chloride, guanethidine, hydralazine, imipramine, mal-
achite green, promazine, propantheline bromide
Noninterfering: chlordiazepoxide
KEYWORDS
post-column extraction; SPE; neostigmine is IS; serum; silanize glassware; post-column reaction
REFERENCE
Quinn,K.D.; Stewart,J.T. A high performance liquid chromatographic post-column fluorescent ion pair extrac-
tion system: application to physostigmine and its metabolite eseroline in human serum, Biomed.
Chromatogr., 1991, 5, 8-13.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y; Pe*pin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
the creation of a 600-compound library. Application to forensic toxicology, J. Chromatogr.A, 1997, 763,149-
163.
SAMPLE
Matrix: perfusate
Sample preparation: Direct injection of a 100 \LL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 octyl Spherisorb S5-ODS
Mobile phase: MeCNrbuffer 30:70 (Buffer was 10 mM sodium octanesulfonate, 10 mM NaH2PO4,
2.5 mM tetramethylammonium chloride, adjust to pH 3 with sulfuric acid.)
Flow rate: 1.1
Detector: UV 214
KEYWORDS
calibration range 2-10 |xM
REFERENCE
Michael-Baruch,E.; Shiri,Y.; Cohen,S. Alkali halide-assisted penetration of neostigmine across excised human
skin: A combination of structured water disruption and a Donnan-like effect, J.Pharm.ScL, 1994,83,1071-
1076.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 50 X 4.6 5 |xm Suplex pKb-100 (Supelco)
Mobile phase: MeCN-.buffer 30:70 (Buffer was 10 mM pH 7.0 sodium phosphate containing 5
mM sodium dodecyl sulfate.)
Flow rate: 2
Detector: UV 210
CHROMATOGRAM
Retention time: 2.2
OTHER SUBSTANCES
Simultaneous: 4'-methylphenazone, edrophonium chloride
REFERENCE
Netilmicin
Merck Index: 6563
SAMPLE
Matrix: blood
Sample preparation: 50 JJLL Serum + 20 |xL 10 |xg/mL gentamicin CIa in water + 50 |xL buffer,
vortex for 15 s, add 200 |xL MeCN, vortex for 15 s, centrifuge at 2000 g for 5 min. Filter (0.45
ixm, Millex-HV4) the supernatant and add 300 |xL of the nitrate to 20 jxL 250 mg/mL 1-fluoro-
2,4-dinitrobenzene in MeCN. Heat at 80 for 2 h, cool rapidly to room temperature, filter (0.45
fxm, Millex-HV4), inject a 50 fiL aliquot of the filtrate. (Buffer was prepared by dissolving 3.81
g disodium tetraborate decahydrate in water, adjusting pH to 10 with NaOH, and making up
to 100 mL with water.)
HPLCVARIABLES
Guard column: 33 X 4.6 5 |xm C18 (Perkin-Elmer)
Flow rate: 2.2
Detector: UV 365
CHROMATOGRAM
Internal standard: gentamicin CIa (11.0)
KEYWORDS
serum; guinea pig; human; derivatization
REFERENCE
Dionisotti,S.; Bamonte,R; Gamba,M.; Ongini,E. High-performance liquid chromatographic determination of
netilmicin in guinea-pig and human serum by fluorodinitrobenzene derivatization with spectrophotometric
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 100 |xL trichloroacetic acid + 100 uL 25 |xg/mL genta-
micin, shake for 30 s, centrifuge at 3200 rpm for 5 min, add 100 (xL 1 M NaOH to the super-
natant, shake (?) for 30 s, add 1 mL pH 11 KH2PO4, add 2 mL dichloromethane, shake for 10
s, centrifuge at 3200 rpm for 5 min. Remove the aqueous layer and add it to 1 mL reagent,
shake for 30 s, add 500 mg anhydrous sodium carbonate, shake for 30 s, add 500 ^xL isopro-
panol, shake, centrifuge for 5 min, inject an aliquot of the supernatant. (Reagent was 5 mg o-
phthaldialdehyde, 500 |xL MeOH, 300 |xL 2-mercaptoethanol, and 5 mL 100 mM pH 10.4 borate
buffer. Prepare fresh each day.)
HPLC VARIABLES
Column: 300 X 4 10 |xm RP-18
Mobile phase: Gradient. A was water:acetic acid: 100 mM heptanesulfonic acid 80:10:10. B was
MeCN. A:B from 60:40 to 50:50 over 10 min, re-equilibrate at initial conditions for 10 min.
Flow rate: 2
CHROMATOGRAM
Retention time: 6.5
Internal standard: gentamicin (11)
KEYWORDS
REFERENCE
Santos,M.; Garcia,E.; L6pez,F.G.; Lanao,J.M.; Dominguez-Gil,A. Determination of netilmicin in plasma by
HPLC, J.Pharm.Biomed.AnaL, 1995, 13, 1059-1062.
SAMPLE
Sample preparation: Condition a 3 mL 100 mg Isolute carboxypropyl CBA-bonded silica SPE
cartridge (Jones Chromatography) with 1 mL MeOH and 1 mL pH 7.4 phosphate buffer. Add
1 mL plasma or Iso-sensitest broth to the SPE cartridge, wash with 2 mL pH 7.4 phosphate
buffer and 4 mL 200 mM pH 9 borate buffer. Dry the cartridge with approximately 30 mL of
air, elute with 1 mL MeCN:200 mM pH 10.5 borate buffer 50:50. Adjust the pH value of a 1
mL portion of the eluate to 8.9 by the addition of 200 JULL 800 mM boric acid, add 200 JULL 7
mM fluorenylmethyl chloroformate in MeCN, let stand at ambient temperature for 15 min,
add 50 IJLL 100 mM glycine, inject a 20 |JLL aliquot of the mixture. (Prepare pH 7.4 phosphate
buffer by mixing appropriate volumes of 20 mM NaH2PO4 and Na2HPO4 solutions. Prepare
borate buffer by adjusting the pH of boric acid solution with 45% KOH.)
HPLC VARIABLES
Column: 200 X 4.6 3 ^m ODS Hypersil
Flow rate: 1
CHROMATOGRAM
Retention time: 18
Internal standard: sisomycin (14)
OTHER SUBSTANCES
Extracted: neomycin
KEY WORDS
REFERENCE
Stead,D.A.; Richards,R-M.E. Sensitive high-performance liquid chromatographic assay for aminoglycosides in
biological matrices enables the direct estimation of bacterial drug uptake, J.Chromatogr.B, 1997, 693, 415-
421.
SAMPLE
with 2 mL MeOH, 2 mL water, and 2 mL buffer. 1 mL Plasma + 100 |xL 100 u-g/mL dibekacin
add to SPE cartridge, wash with 500 uL water, wash with 250 |xL mobile phase, elute to
dryness. Elute with 250 |xL mobile phase, inject an aliquot of the eluate. Urine, dialysate.
Dilute 1:100 with water, add 100 |xL 100 |jig/mL dibekacin per 1 mL of sample, mix well, inject
a 100 |JLL aliquot. (Buffer was 0.94 g sodium hexanesulfonate in 300 mL water, add 500 |xL
HPLC VARIABLES
Guard column: 10 X 4.6 5 |xm Hypersil C18
Mobile phase: MeOHrbuffer 15:85 (Buffer was 3.48 g sodium hexanesulfonate + 28.4 g sodium
Flow rate: 1.1
CHROMATOGRAM
Retention time: 16
OTHER SUBSTANCES
Simultaneous: kanamycin, isepamicin, tobramycin, gentamicin
KEYWORDS
REFERENCE
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 206.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve sample in mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 8 jxm 1000 A poly(styrene-divinylbenzene) PLRP-S column (Polymer Labs.,
UK)
Mobile phase: THF:buffer 1:99 (Mobile phase was 35 g sodium sulfate, 500 mg sodium 1-oc-
tanesulfonate, 10 mL THF, and 50 mL 200 mM pH 3.0 phosphate buffer diluted to 1 L with
water.)
Flow rate: 1
Detector: E, Dionex PED-I, pulsed electrochemical detection, gold working electrode, E l +50
mV, E2 +750 mV, E3 -150 mV, t l 0-400 ms, t2 410-600 ms, t3 610-1000 ms, signal acquired
200-400 ms, Ag/AgCl reference electrode, stainless-steel counter electrode following post-col-
umn reaction. The column effluent mixed with 500 mM NaOH pumped at 0.3 mL/min and
flowed through a 1.2 m (500 |xL) reaction coil to the detector.
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
KEYWORDS
Next Page
REFERENCE
Adams,E.; Puelings,D.; Rafiee,M.; Roets,E.; Hoogmartens,J. Determination of netilmicin sulfate by liquid chro-
matography with pulsed electrochemical detection, J.Chromatogr.A, 1998, 812, 151-157.
SAMPLE
Sample preparation: 50 |xL Buffered reaction mixture + 50 o,L isopropanol + 50 u,L reagent,
heat at 60 for 10 min, centrifuge at 1000 g for 2 min, immediately inject a 50 jxL aliquot of
HPLC VARIABLES
Flow rate: 2
Detector: UV 330
CHROMATOGRAM
Retention time: 19
KEY WpRDS
derivatization
REFERENCE
liquid chromatographic determination of their reaction products, Antimicrob.Agents Chemother., 1984,26,
10-12.
Niacin
Merck Index: 6612
SAMPLE
Matrix: blood
Sample preparation: 0.5 mL Plasma + 50 |xL 100 jxg/mL anthranilic acid, mix, add 3 mL ace-
tone.water 2:1, centrifuge. Remove the supernatant and evaporate it to dryness, reconstitute
the residue in 500 jutL 100 mM HCl, add 1 mL, evaporate to dryness, reconstitute in 350 JULL
HPLC VARIABLES
Column: 250 X 4 5 |xm ODS-2 (Knauer)
Mobile phase: MeCN: 10 mM pH 5.4 MOPS (3-(N-morpholino)propanesulfonic acid) buffer 25:75
containing 1 mM n-dodecylamine
Flow rate: 1
Detector: UV 262
CHROMATOGRAM
Internal standard: anthranilic acid (13)
Previous Page
REFERENCE
Adams,E.; Puelings,D.; Rafiee,M.; Roets,E.; Hoogmartens,J. Determination of netilmicin sulfate by liquid chro-
matography with pulsed electrochemical detection, J.Chromatogr.A, 1998, 812, 151-157.
SAMPLE
Sample preparation: 50 |xL Buffered reaction mixture + 50 o,L isopropanol + 50 u,L reagent,
heat at 60 for 10 min, centrifuge at 1000 g for 2 min, immediately inject a 50 jxL aliquot of
HPLC VARIABLES
Flow rate: 2
Detector: UV 330
CHROMATOGRAM
Retention time: 19
KEY WpRDS
derivatization
REFERENCE
liquid chromatographic determination of their reaction products, Antimicrob.Agents Chemother., 1984,26,
10-12.
Niacin
Merck Index: 6612
SAMPLE
Matrix: blood
Sample preparation: 0.5 mL Plasma + 50 |xL 100 jxg/mL anthranilic acid, mix, add 3 mL ace-
tone.water 2:1, centrifuge. Remove the supernatant and evaporate it to dryness, reconstitute
the residue in 500 jutL 100 mM HCl, add 1 mL, evaporate to dryness, reconstitute in 350 JULL
HPLC VARIABLES
Column: 250 X 4 5 |xm ODS-2 (Knauer)
Mobile phase: MeCN: 10 mM pH 5.4 MOPS (3-(N-morpholino)propanesulfonic acid) buffer 25:75
containing 1 mM n-dodecylamine
Flow rate: 1
Detector: UV 262
CHROMATOGRAM
Internal standard: anthranilic acid (13)
KEYWORDS
plasma
REFERENCE
Zarzycki,P.K.; Kowalski,R; Nowakowska,J.; Lamparczyk,H. High-performance liquid chromatographic and cap-
illary electrophoretic determination of free nicotinic acid in human plasma and separation of its metabolites
by capillary electrophoresis, J.ChromatogrA, 1995, 709, 203-208.
SAMPLE
Sample preparation: Tablets. Powder tablets, dissolve in water, inject a 10 |JLL aliquot. Injec-
tions. Dilute with water, inject a 10 |xL aliquot. Plasma, urine. Condition a Lichrolut RP-18
(Merck) SPE cartridge with 3 mL MeOH and 3 mL water. Mix 40 |xL plasma or 100 uL urine
with twice the volume of MeCN for 2 min, add 100 |xL water, centrifuge at 3500 rpm for 15
at 45. Reconstitute the residue with 500 |xL MeOH containing 4.2 |xg/mL IS. Inject a 10 jxL
aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ascorbic acid, folic acid, niacinamide, ribofiavin, vitamin B12
KEY WORDS
REFERENCE
Papadoyannis,I.N.; Tsioni,G.K.; Samanidou,V.F. Simultaneous determination of nine water and fat soluble vi-
SAMPLE
residue with 50 |JLL MeCNiwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 209.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Dilute injections with water, inject a 50 JJLL aliquot. Dissolve tablets or
capsule contents in water (warm if necessary), filter (0.5 (xm PTFE), inject a 50 \LL aliquot of
the filtrate. (Dissolve tablets or other formulations containing proteinaceous material in water
at 60, add 5% trichloroacetic acid (to pH 4.4), filter, inject a 50 |xL aliquot.)
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: folic acid (UV 280), niacinamide, pyridoxamine (UV 280), thiamine, riboflavin,
pyridoxine (UV 280), ascorbic acid (UV 280)
KEYWORDS
REFERENCE
SAMPLE
HPLC VARIABLES
Column: 100 X 4 3 jxm Hypersil BDS-C18
Flow rate: 0.5
Detector: UV 220
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Simultaneous: biotin, caffeine, citric acid, folic acid, niacinamide, pantothenic acid, riboflavin,
saccharin, thiamine, pyridoxine, vitamin B12, ascorbic acid
KEYWORDS
tablets
REFERENCE
Hewlett Packard Leaflet 12-5091-7351 EUS, 1993.
SAMPLE
Sample preparation: Dilute liquid multivitamin formulations, filter (0.45 |xm), inject an aliquot.
HPLC VARIABLES
Flow rate: 1
Injection volume: 5
Detector: UV 272
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: folic acid, niacinamide, thiamine, riboflavin, pyridoxine (UV 290)
KEYWORDS
REFERENCE
Blanco,D.; Sanchez,L.A.; Gutierrez,M.D. Determination of water soluble vitamins by liquid chromatography
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Gradient. A was 100 mM pH 4.7 acetate buffer. B was MeCN:100 mM pH 4.7
acetate buffer 25:75.
Flow rate: 4
Detector: UV 254
CHROMATOGRAM
Retention time: 1
OTHER SUBSTANCES
Simultaneous: pyridoxine, riboflavin, thiamine, niacinamide, ascorbic acid
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeOH:buffer 15:85 (Buffer was 4.3 mM sodium hexanesulfonate containing 0.1%
triethylamine, adjusted to pH 2.8 with phosphoric acid.)
Flow rate: 1
Detector: UV 200
CHROMATOGRAM
Retention time: 0.6
OTHER SUBSTANCES
Simultaneous: pantothenic acid, pyridoxine, riboflavin, thiamine, niacinamide, ascorbic acid
REFERENCE
Rainin Catalog, Cl-94, 1994, p. 780.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
pam, mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenôin, me-
naltrexone, naphazoline, naproxen, nefopam, niacinamide, nifedipine, niflumic acid, nitraze-
pam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 jjim Inertsil ODS-2
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 1.5
OTHER SUBSTANCES
Simultaneous: biotin, folic acid, pantothenic acid, riboflavin, niacinamide
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.16 (xm PolyEncap ODS (n-octadecylacrylate copolymerized with vinyl silica in
heptane, carrier Ultrasep ES 100; preparation described in paper)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Simultaneous: aspirin, diazepam, diphenhydramine, o-hydroxyhippuric acid, MPPH, toluene
REFERENCE
Engelhardt,H-J Cufiat-Walter,M.A. Polymer encapsulated stationary phases with improved efficiency, Chro-
matographia, 1995, 40, 657-661.
SAMPLE
Matrix: tissue, food
Sample preparation: Condition a Sep-Pak Plus C18 SPE cartridge with 10 mL EtOH and 10
niL water. Weigh out 0.5-15 g cheese, semolina, or beef, make up to 200 mL with water, add
10 g calcium hydroxide, blend at high speed for 30 s, autoclave at 121 for 15 min, cool in an
ice bath for at least 30 min, filter (Whatman 2V fluted paper). Add 100 mL filtrate to 300-320
mg oxalic acid, mix well, adjust pH to 6.5-7.0 with oxalic acid, filter (1-2 pieces Whatman No
42 paper) slowly, add 10 mL filtrate to the SPE cartridge, discard the first 6.5 mL effluent,
collect the next 6.5 mL and add one drop 85% phosphoric acid, mix well, inject a 200 (JLL aliquot.
HPLC VARIABLES
Guard column: C18 Guard-Pak
Column: 150 X 4.6 5 |xm C18 LC-18-DB (Supelco)
Mobile phase: MeCN:water:24 mM phosphoric acid 23:17:60 containing 1 g/L sodium dodecyl
sulfate
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 9
KEYWORDS
cow; muscle; semolina; cheese; SPE
REFERENCE
Tyler,T.A.; Genzale,J.A. Liquid chromatographic determination of total niacin in beef, semolina, and cottage
cheese, J.Assoc.Off.Anal.Chem., 1990, 73, 467-469.
SAMPLE
Matrix: water
Sample preparation: Adjust 50 mL wastewater to pH 6.6 with acetic acid, add 50 mL ethyl
acetate, shake vigorously for 5 min, let stand for 1 min, transfer the ethyl acetate layer to a
flask, extract the residual aqueous layer with two 20 mL portions of ethyl acetate. Combine
the organic layers and evaporate them at 90 to about 500 jxL, dissolve the residue in 5 mL 10
mM HCl, make up to 50 mL with water, inject an aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 |xm Inertsil ODS-2 (Vercopak)
Mobile phase: MeOHrbuffer 20:80 (Buffer was 100 mM sodium acetate adjusted to pH 6.6 with
10 mM acetic acid.)
Flow rate: 1
Detector: UV 260
CHROMATOGRAM
Retention time: 3.3
Internal standard: niacin
OTHER SUBSTANCES
Extracted: sulfathiazole, sulfamethazine, sulfacetamide, sulfadiazine, sulfamerazine, sulfa-
methoxazole, sulfamonomethoxine
KEYWORDS
wastewater; niacin is IS
REFERENCE
Jen,J.-R; Lee,H--L.; Lee,B.-N. Simultaneous determination of seven sulfonamide residues in swine wastewater
by high-performance liquid chromatography, J.ChromatogrA, 1998, 793, 378-382.
Niacinamide
Merck Index: 6574
SAMPLE
tions. Dilute with water, inject a 10 |JLL aliquot. Plasma, urine. Condition a Lichrolut RP-18
(Merck) SPE cartridge with 3 mL MeOH and 3 mL water. Mix 40 (JLL plasma or 100 |xL urine
with twice the volume of MeCN for 2 min, add 100 |xL water, centrifuge at 3500 rpm for 15
at 45. Reconstitute the residue with 500 |xL MeOH containing 4.2 |xg/mL IS. Inject a 10 |xL
aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ascorbic acid, folic acid, niacin, ribonavin, vitamin B12
KEYWORDS
REFERENCE
PapadoyannisJ.N.; Tsioni,G.K.; Samanidou,V.F. Simultaneous determination of nine water and fat soluble vi-
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 214.6
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: formula, milk
Sample preparation: Mix 8.0 g powdered infant milk with 10 mL water to it. Mix the diluted
powder or 10.5 g liquid infant milk with 1 g solid trichloroacetic acid, shake thoroughly with
magnetic stirring for 10 min, centrifuge at 1250 g for 10 min, add 3 mL 4% trichloroacetic acid
to the solid residue, mix thoroughly for 10 min, centrifuge, discard the solid phase. Combine
the two acid extracts and make up to 10 mL with 4% trichloroacetic acid, filter (0.45 |xm), inject
HPLC VARIABLES
Guard column: 5 |xm Tracer Spherisorb ODS 2 C18 (Teknokroma, Spain)
Column: 250 X 4.6 5 |xm Tracer Spherisorb ODS 2 C18 (Teknokroma, Spain)
Mobile phase: MeOH:buffer 15:85 (Buffer was 5 mM octanesulfonic acid and 0.5% triethylamine,
pH 3.6.)
Flow rate: 1
Detector: UV 261 for 6 min, UV 287 for 2 min, UV 290 for 5 min, UV 282 for 3 min, UV 268 for
3.5 min, UV 361 for 20.5 min, UV 246 for 20 min
CHROMATOGRAM
Retention time: 5
Limit of quantitation: ^50 ng/mL
OTHER SUBSTANCES
Extracted: thiamine, riboflavin, pyridoxine, vitamin B12, folic acid, pyridoxal, pyridoxamine
REFERENCE
Albal&-Hurtado,S.; Veciana-Nogus,M.; Izquierdo-Pulido,M.; Marin6-Font,A. Determination of water-soluble vi-
tamins in infant milk by high-performance liquid chromatography, J.Chromatogr.A, 1997, 778, 247-253.
SAMPLE
Sample preparation: Pulverize tablets if necessary. Add tablets to 100 mL 5 mM pH 4.5 potas-
sium phosphate buffer, sonicate at 75 W for 2 min, cool to room temperature, make up to 200
mL with buffer, filter (0.45 |xm), inject a 10 |xL aliquot of the nitrate.
HPLC VARIABLES
Column: 250 X 4.6 10 |xm LiChrosorb NH2 aminopropyl
Mobile phase: MeCN:5 mM KH2PO4 87:13 (Wash column with MeCN:water 10:90 at the end of
the day.)
Flow rate: 2
Detector: UV 210
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Simultaneous: pantothenic acid, thiamine, riboflavin, pyridoxine
KEYWORDS
tablets
REFERENCE
Hudson,T.J.; Allen,R.J. Determination of pantothenic acid in multivitamin pharmaceutical preparations by
reverse-phase high-performance liquid chromatography, J.Pharm.Sci., 1984, 73, 113115.
SAMPLE
Sample preparation: Tablets without iron. Grind 5 tablets to a fine powder, add 10 mL mono-
thioglycerol and 800 mL buffer, sonicate for 30 min, add 150 mL MeOH, make up to 1 L with
buffer, filter (GF/C paper), discard first few mL, remove a 10 mL aliquot, make up to 25 mL
with mobile phase, inject an aliquot. Tablets with dioctyl sodium sulfosuccinate. Grind 5 tablets
to a fine powder, add 10 mL 2-monothioglycerol and 1 g barium chloride, make up to 1 L with
buffer, stir vigorously for 30 min, filter (GF/C paper), discard first few mL, inject an aliquot.
Capsules with iron. Contents of one capsule + 5 mL 2-monothioglycerol + 2 mL glacial acetic
acid + 75 mL buffer, sonicate for 5 min, make up to 100 mL with buffer, stir vigorously for 30
min, filter (GF/C paper), add 300 mg cupferron, stir for 10 min, let stand for 1 h at room
temperature, filter (GF/C paper), let stand for 30 min, filter again (if necessary), discard first
few mL, inject an aliquot. (Buffer was 48 mL glacial acetic acid and 10 mL trietnylamine in 1
L water, adjust pH to 3.6 0.05 with acetic acid or triethylamine, make up to 1.7 L with
water.)
HPLCVARIABLES
Column: 100 X 8 Radial Pak A C18 (Waters)
Mobile phase: MeOHibuffer 15:85 (Buffer was 2.20 g sodium heptanesulfonate, 100 mg EDTA,
48 mL glacial acetic acid, and 10 mL triethylamine made up to 1.7 L with water, adjust pH to
3.6 0.05 with acetic acid or triethylamine.)
Flow rate: 2
Detector: UV 280
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Simultaneous: thiamine, riboflavin, pyridoxine, ascorbic acid (UV 254)
KEYWpRDS
multi-vitamin; protect from light; tablets; capsules
REFERENCE
Lam,F.-L.; Holcomb,I.J.; Fusari,S.A. Liquid chromatographic assay of ascorbic acid, niacinamide, pyridoxine,
thiamine, and riboflavin in multivitamin-mineral preparations, J.Assoc.Off.Anal.Chem., 1984, 67, 1007-
1011.
SAMPLE
Sample preparation: Dilute injections with water, inject a 50 JJIL aliquot. Dissolve tablets or
capsule contents in water (warm if necessary), filter (0.5 |xm PTFE), inject a 50 |xL aliquot of
the nitrate. (Dissolve tablets or other formulations containing proteinaceous material in water
at 60, add 5% trichloroacetic acid (to pH 4.4), filter, inject a 50 (xL aliquot.)
HPLC VARIABLES
Column: 10 (xm (xBondapak C18
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: folic acid (UV 280), niacin, pyridoxamine (UV 280), thiamine, riboflavin, pyri-
doxine (UV 280), ascorbic acid (UV 280)
KEYWORDS
REFERENCE
SAMPLE
Sample preparation: Weigh out 500 mg ground tablets, extract with water, make up to 50 or
100 mL with water, filter, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Nucleosil 10 C18
Mobile phase: MeOH:l% acetic acid 25:75
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
Simultaneous: menadione hydrogen sulfite, pyridoxine, riboflavin, thiamine, ascorbic acid
KEYWORDS
tablets; multi-vitamin
REFERENCE
Sadlej-Sosnowska,N.; Blitek,D.; Wilczynska-Wojtulewicz,I. Determination of menadione sodium hydrogen sul-
phite and nicotinamide in multivitamin formulations by high-performance liquid chromatography,
J.Chromatogr., 1986, 357, 227-232.
SAMPLE
Sample preparation: Dilute liquid multivitamin formulations, filter (0.45 jxm), inject an aliquot.
HPLC VARIABLES
Flow rate: 1
Injection volume: 5
Detector: UV 272
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: folic acid, niacin, thiamine, riboflavin, pyridoxine (UV 290)
KEYWORDS
REFERENCE
Blanco,D.; Snchez,L.A.; Gutirrez,M.D. Determination of water soluble vitamins by liquid chromatography
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Accubond Amino (J & W)
Mobile phase: MeCNrbuffer 10 :90 (Buffer was 20 mM phosphoric acid adjusted to pH 3.0 with
20 mM NaOH.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 1.8
OTHER SUBSTANCES
Simultaneous: p-aminobenzoic acid, pyridoxal, pyridoxamine, thiamine, riboflavin, pyridoxine,
vitamin B12
REFERENCE
J & W Catalog, 1992-3, p. 277.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: Gradient. A was 100 mM pH 4.7 acetate buffer. B was MeCNrIOO mM pH 4.7
acetate buffer 25:75.
Flow rate: 4
Detector: UV 254
CHROMATOGRAM
Retention time: 1.8
OTHER SUBSTANCES
Simultaneous: niacin, pyridoxine, riboflavin, thiamine, ascorbic acid
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
F l o w rate: 1
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, ascorbic acid
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
naltrexone, naphazoline, naproxen, nicotine, niacin, nifedipine, niflumic acid, nitrazepam, nor-
REFERENCE
Hill,D.W.; Kind,A-J- Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.Toxicol., 1994,
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Simultaneous: biotin, folic acid, niacin, pantothenic acid, riboflavin
REFERENCE
SAMPLE
Matrix: tissue
Sample preparation: Homogenize rat intestinal tissue in physiological saline. Removal a 3 mL
aliquot and add it to 18 mL acetone, centrifuge at 10000 g for 10 min. Add the supernatant to
14 mL chloroform, mix intensively, centrifuge at 1000 g for 5 min. Filter (0.45 (xm) a 500 (xL
aliquot of the upper aqueous phase, inject a 100 |xL aliquot.
HPLC VARIABLES
Guard column: 10 |xm ODS Hypersil in an Upchurch C-135 B pre-column kit
Mobile phase: Gradient. MeOH:5 mM pH 7.0 tetrabutylammonium phosphate from 10:90 to 70:
30 over 10 min.
Flow rate: 1.2
Detector: UV 410 following post-column derivatization. The column effluent mixed with 2% chlo-
ramine-T pumped at 0.5 mL/min and flowed through a 2 m X 0.5 mm i.d. coil of PTFE tubing
kept at 60. This mixture was mixed with 0.25% KCN containing 25 mM Tris and 40 mM HCl
pumped at 0.5 mL/min. The mixture flowed through an 8 m X 0.5 mm i.d. coil of PTFE tubing
kept at 60 to the detector.
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: niacin
KEYWORDS
post-column reaction; rat; intestine
REFERENCE
Stein,J.; Hahn,A.; Rehner,G. High-performance liquid chromatographic determination of nicotinic acid and
nicotinamide in biological samples applying post-column derivatization resulting in bathmochrome absorp-
tion shifts, J.Chromatogr.B, 1995, 665, 71-78.
Nialamide
Merck Index: 6575
Lednicer No.: 1 254
SAMPLE
Matrix: blood
the residue in 100 (xL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 JXL aliquot of
HPLC VARIABLES
Column: 300 X 3.9 4 jmi NovaPack C18
Flow rate: 0.8
Detector: UV 264
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
Niaprazine
Molecular formula: C20H25FN4O
Merck Index: 6576
SAMPLE
Sample preparation: Add 1 mL whole blood or urine to Toxi-Tube A (Tbxi-Lab, Irvine CA), add
residue with 50 |AL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Nicarbazin
Merck Index: 6578
SAMPLE
Matrix: eggs
Sample preparation: Condition a 100 mg Bond Elut SPE cartridge (no. 601101) with two 1 mL
portions of chloroform:MeCN 80:20 and with 1 mL chloroform, air dry. Stir 1 g homogenized
egg and 50 |xL 10% acetic acid and slowly add 2.5 mL MeCN, 4 min after the MeCN addition
is complete add 0.25 g anhydrous sodium sulfate, stir for 2 min, centrifuge at 1400 g for 2 min,
filter the supernatant through glass wool. Re-extract the sediment with 2.5 mL by stirring for
3 min, centrifuge at 1400 g for 2 min, filter the supernatant through glass wool, rinse the glass
wool with 1 mL MeCN. Combine the filtrates and evaporate them to dryness under a stream
of nitrogen at 60, reconstitute the residue in 1 mL hexanexhloroform 50:50, add to the SPE
cartridge, rinse the tube with 1 mL hexanexhloroform 50:50, add the rinse to the SPE car-
tridge, dry the SPE cartridge under vacuum for 1 min, elute with chloroform:MeCN 80:20.
Collect the first 2 mL of eluate and evaporate it to dryness under a stream of nitrogen at room
temperature, reconstitute with 1 mL mobile phase, vortex for 4 min, inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 2.1 30-40 |xm pellicular RP (Chrompack)
Column: two 100 X 3 5 |xm ChromSpher C18 columns in series (Chrompack)
Mobile phase: MeCN:20 mM pH 4.8 acetate buffer:water 54:10:36
Flow rate: 0.6
Detector: UV 360
CHROMATOGRAM
Retention time: 4.5 (dinitrocarbanilide)
KEYWORDS
SPE
REFERENCE
Vertommen,M.H.; van der Laan,A.; Veenendaal-Hesselman,H-M. High-performance liquid chromatographic
screening method for low levels of nicarbazin in eggs with off-line cartridge sample clean-up, J.Chromatogr.,
1989, 481, 452-457.
SAMPLE
Matrix: eggs
Sample preparation: 1 g Homogenized egg + 2.5 mL MeCN, vortex, stir for 4 min, add 300 mg
anhydrous sodium sulfate, stir for 2 min, centrifuge at 1860 g for 2 min, decant the superna-
tant. Add 2.5 mL MeCN to the solid residue, vortex, stir for 3 min, centrifuge at 1860 g for 2
min, decant the supernatant. Combine the organic layers and evaporate them to dryness under
a stream of nitrogen at 50-60, reconstitute the residue in 1 mL mobile phase A, vortex for 15
s, sonicate for 3 min, filter (0.45 fim), inject a 250 |xL aliquot onto column A with mobile phase
A and elute to waste, after 5 min elute the contents of column A onto column B with mobile
phase B, after 1 min remove column A from the circuit, elute column B with mobile phase B
and monitor the effluent from column B. (Clean column A with mobile phase A for 1 min at 1
mL/min and with MeCN at 2 mIVmin for 13 min. Re-equilibrate with mobile phase A at 2 mL/
min for 5 min.)
HPLC VARIABLES
Column: A10 X 2.1 reverse-phase (Chrompack); B 10 X 2.1 pellicular reverse-phase (Chrompack)
+ 100 X 3 5 |xm Chromspher C18 (Chrompack)
Mobile phase: A MeCNiwater 20:80; B MeCN:10 mM pH 4.0 KH2PO4 50:50
Flow rate: 0.4
Detector: UV 343
CHROMATOGRAM
Retention time: 6 (dinitrocarbanilide)
KEYWORDS
column-switching
REFERENCE
Tarbin,J.A.; Shearer,G. High-performance liquid chromatographic method for the determination of the dinitro-
carbanilide component of nicarbazin in eggs with on-line clean-up, J.Chromatogr., 1993, 613, 354-358.
SAMPLE
Matrix: eggs, feed, litter, tissue
Sample preparation: Tissue, eggs, litter. 25 g Homogenized sample + 50 mL MeCN, blend
(Biiler H04) at low speed for 2-3 min, centrifuge at 1000 g for 10 min, remove the supernatant,
re-extract the sediment with 30 mL MeCN, centrifuge at 1000 g for 10 min, combine the
supernatants, shake well, discard the lower layer, add 80 mL dichloromethane, add 3 g NaCl,
discard the lower layer, add 3 g anhydrous sodium sulfate, filter (paper). Evaporate the filtrate
to dryness under reduced pressure at 45-50, transfer to a smaller tube with 2 mL MeOH:
MeCN:water 50:30:20 and 1 mL n-hexane, rinse in with four 1 mL portions of MeCN, evaporate
to dryness under a stream of nitrogen at 50. Reconstitute the residue in 2 mL MeOH:MeCN:
water 50:30:20 and 0.5 mL n-hexane, centrifuge at 4000 rpm for 10 min, inject an aliquot of
the MeOH layer (usually, but not always, the lower layer). Feed. 5 g Ground sample + 25 mL
MeCN, blend (Btiler H04) at low speed for 2-3 min, centrifuge at 1000 g for 10 min, remove
the supernatant, re-extract the sediment with 25 mL MeCN, centrifuge at 1000 g for 10 min,
combine the supernatants, evaporate to dryness under reduced pressure at 45-50, transfer to
a smaller tube with 2 mL MeOH:MeCN:water 50:30:20 and 1 mL n-hexane, rinse in with four
1 mL portions of MeCN, evaporate to dryness under a stream of nitrogen at 50. Reconstitute
the residue in 2 mL MeOH:MeCN:water 50:30:20 and 0.5 mL n-hexane, centrifuge at 4000
rpm for 10 min, inject an aliquot of the MeOH layer (usually, but not always, the lower layer).
HPLC VARIABLES
Column: 300 X 1 5 |xm Supelcosil LC-18
Flow rate: 0.03
Detector: UV 340
CHROMATOGRAM
Retention time: 9.5 (4,4'-dinitrocarbanilide)
KEYWORDS
chicken; narrow bore; muscle; liver; nicarbazin is an equimolar mixture of 4,4'-dinitrocarbanilide
and 4,6-dimethylpyrimidine
REFERENCE
Draisci,R.; Lucentini,L.; Boria,R; Lucarelli,C. Micro high-performance liquid chromatography for the deter-
mination of nicarbazin in chicken tissues, eggs, poultry feed and litter, J.Chromatogr.A, 1995, 697, 407-
414.
SAMPLE
Matrix: tissue
Sample preparation: Prepare a 12.5-15 mm bed of alumina (80-200 mesh, Brockman Activity
I, Fisher) in a 5 mL pipette tip, condition with two 2 mL portions of chloroform:ethyl acetate
50:50. Blend (Polytron) 2.5 g ground tissue with 20 mL chloroform:ethyl acetate:DMSO 50:50:
0.8 for 45 s, centrifuge at 3500 rpm for 5 min, discard the aqueous layer. Filter the organic
layer through glass wool, add 2 mL nitrate to the alumina column, wash with 3.25 mL chlo-
roform, dry with air pressure (column is dry when moisture no longer condenses on the outside,
continue for another 3 min), elute with MeOH:pH 6.0 phosphate buffer 50:50, collect first 2
mL of eluate, purge eluate with helium for 2 min, inject a 50 |xL aliquot.
HPLCVARIABLES
Mobile phase: MeOH:buffer 65:35 (Buffer was 50 mM KH2PO4 containing 1 mM EDTA, adjusted
to pH 6.0 with NaOH.)
Flow rate: 1
Detector: E, Bioanalytical Systems LC-45, glassy carbon electrode -0.8 V, Ag/AgCl reference
electrode
CHROMATOGRAM
Retention time: 8.5 (dinitrocarbanilide)
Limit of detection: 0.1-0.2 ppm
KEYWORDS
chicken; muscle; liver; SPE
REFERENCE
Parks,O.W. Rapid procedure for determination of nicarbazin residues in chicken tissues, JAssoc.Off.
Anal.Chem., 1988, 71, 778-780.
SAMPLE
Matrix: tissue
Sample preparation: Connect two Sep-Pak alumina B SPE cartridges in series and condition
with 4 mL DMF. Homogenize (Sorvall Omni-Mixer, do not substitute) 10 g ground liver or
muscle with 25 mL ethyl acetate at half speed, decant supernatant, repeat homogenization
twice more. Filter extracts, evaporate to an oily residue under vacuum, take up the residue in
10 mL MeCN, extract with 50 mL hexane. Extract the hexane layer twice with 10 mL portions
of MeCN. Combine all the MeCN layers and wash with 50 mL hexane. Evaporate the MeCN
layer to dryness under vacuum, reconstitute in 2 mL DMF, add to the SPE cartridges, wash
with 14 mL DMF, eliminate as much DMF as possible, elute with 10 mL MeOH, evaporate the
eluate to 2-3 mL under vacuum, make up to 10 mL with MeOH:water 75:25, inject a 50 |xL
aliquot.
HPLCVARIABLES
Column: 250 X 4.6 3 |xm IB-SIL C18 (Phenomenex)
Mobile phase: MeOH:water 75:25 containing 3.85 g/L ammonium acetate
Flow rate: 0.8-1
Detector: UV 340 or MS, Finnigan 4600, negative ion thermospray, SIM m/z 164, 272, 302,
filament on
CHROMATOGRAM
Retention time: 5 (4,4'-dinitrocarbanilide)
Limit of quantitation: 4 ppm
KEYWORDS
chicken; liver; muscle; SPE
REFERENCE
Leadbetter,M.G.; Matusik,J.E. Liquid chromatographic determination and liquid chromatographic-thermo-
spray mass spectrometric confirmation of nicarbazin in chicken tissues: interlaboratory study, J.AOAC Int.,
1993, 76, 420-423.
Nicardipine
Merck Index: 6579
Lednicer No.: 3 150
SAMPLE
Matrix: blood
Mix 1 mL plasma with 10 |xL 10 |xg/mL IS in MeOH, dilute with 5 mL 1 M NaCl, mix briefly.
Add the mixture to the SPE cartridge, wash with 10 mL water and 10 ml MeOHrwater 40:60,
elute with 5 mL MeOH:water 80:20. Dry the eluate under vacuum at 60 and dissolve the
residue in 200 |iL chloroform (Caution! Chloroform is a carcinogen!). Inject a 50 JLL aliquot
onto column A, wash with (?) for 30 s, elute the contents of column A onto column B with mobile
phase, elute with mobile phase, monitor the effluent from column B.
HPLCVARIABLES
Column: A 10 X 4.6 5 |xm TMS; B 250 X 4.6 5 |xm Sumichiral OA-4500 (Sumika Chemical
Analysis Service, Japan)
Mobile phase: MeOH:n-hexane:l,2-dichloroethane:trifluoroaceticacid 10:250:40:1
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 33.7 (+), 35.8 (-)
Internal standard: (+)-barnidipine (18)
KEYWORDS
plasma; SPE; chiral; column-switching
REFERENCE
Uno,T.; Ohkubo,T.; Sugawara,K. Enantioselective high-performance liquid chromatographic determination of
nicardipine in human plasma, J.Chromatogr.B, 1997, 698, 181-186.
SAMPLE
Matrix: blood
Sample preparation: 200 jxL Plasma or serum + 50 |xL buffer + 50 |xL 5 jxg/mL butriptyline in
water + 20 jxL MTBE, vortex for 30 s, centrifuge at 9950 g for 2 min, inject an aliquot of the
organic layer. (Buffer was 2 M Iris adjusted to pH 11.0 with 1 M NaOH.)
HPLCVARIABLES
Column: 125 X 5 5 |xm Spherisorb S5SCX
Mobile phase: MeOH containing 20 mM ammonium perchlorate
Flow rate: 2
Detector: E, Thames Chromatography TClOO, V25-grade glassy-carbon electrode in a wall-jet
assembly 1.1 V, Ag/AgCl reference electrode, range 100 nA
CHROMATOGRAM
Retention time: 4.5
Internal standard: butriptyline (7.5)
OTHER SUBSTANCES
Simultaneous: metabolites, acebutolol, acetylprocainamide, ajamaline, amiodarone, amitripty-
line, bupivacaine, chlorpromazine, desipramine, dextropropoxyphene, disopyramide, dothiepin,
doxepin, fenethazine, fluazepam, hydroxypropafenone, imipramine, lignocaine, maprotiline,
mepivacaine, metoprolol, mianserin, nortriptyline, norverapamil, phenazone, pindolol, prilo-
caine, propafenone, propranolol, protriptyline, quinidine, quinine, timolol, tocainide, trimipra-
mine, verapamil, metabolites
Noninterfering: diazepam, mexiletine, nitrazepam
Interfering: oxprenolol, nadolol
KEYWORDS
plasma; serum
REFERENCE
Eastwood,R.J.; Galustian,C; Bhamra,R.K; Holt,D.W. High-performance liquid chromatographic method for the
measurement of nicardipine in plasma or serum, J.Chromatogr., 1990, 530, 463-468.
SAMPLE
Matrix: blood
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 238
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 205.2
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Dilute with mobile phase, (if necessary), inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCNrMeOHrbuffer 50:10:40 (Buffer was 20 mM KH2PO4 adjusted to pH 4.4-5.0
Flow rate: 1.5
Detector: UV 237
CHROMATOGRAM
Retention time: 7.4
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Baaske,D.M.; DeMay,J.F.; Latona,C.A.; Mirmira,S.; Sigvardson,K.W. Stability of nicardipine hydrochloride in
intravenous solutions, Am.J.Health-Syst.Pharm., 1996, 53, 1701-1705.
SAMPLE
Sample preparation: Filter (0.45 fxm), dilute, and inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 Partisil ODS-3
Mobile phase: MeCN:MeOH:20 mM KH2PO4 50:10:40, adjusted to pH 4.7
Flow rate: 1
Detector: UV 237
KEYWORDS
suspensions
REFERENCE
Maurin,M.B.; Rowe,S.M.; Koval,C.A.; Hussain,M.A. Solubilization of nicardipine hydrochloride via complexa-
tion and salt formation, J.Pharm.Sci., 1994, 83, 1418-1420.
SAMPLE
Matrix: perfusate
HPLC VARIABLES
Column: 100 X 8 5 |xm Novapak C18 radial compression
Flow rate: 2
Detector: UV 237
OTHER SUBSTANCES
Also analyzed: felodipine, nifedipine, nimodipine, nitrendipine
REFERENCE
SAMPLE
Matrix: perfusate
Sample preparation: Add perfusate to an equal volume of nitrendipine in MeOH, vortex, cen-
trifuge, inject an aliquot.
HPLC VARIABLES
Detector: UV 240
CHROMATOGRAM
Internal standard: nitrendipine
KEYWORDS
protect from light
REFERENCE
Kobayashi,D.; Matsuzawa,T.; Sugibayashi,K; Morimoto,Y.; Kobayashi,M.; Kimura,M. Feasibility of use of sev-
eral cardiovascular agents in transdermal therapeutic systems with Z-menthol-ethanol system on hairless
rat and human skin, Biol.Pharm.Bull., 1993, 16, 254-258.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 cellulose tris(4-tert-butylphenylcarbamate)
Flow rate: 0.5
Detector: UV
CHROMATOGRAM
KEYWORDS
chiral
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 237
OTHER SUBSTANCES
Also analyzed: nifedipine, nimodipine, nitrendipine, felodipine
KEYWORDS
buffers
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Direct injection of a MeOH solution containing 200-1000 ng.
HPLCVARIABLES
Column: 250 X 4.6 Sumchiral OA-4500 (Sumika Chemical Analysis Service)
Mobile phase: n-Hexane:l,2-dichloroethane:MeOH:trifluoroaceticacid 250:140:10:1
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 25 (+), 26 (-) (a = 1.05)
KEYWORDS
chiral
REFERENCE
Ohkubo,T.; Uno,T.; Sugawara,K. Enantiomer separation of dihydropyridine derivative calcium antagonists by
high-performance liquid chromatography with chiral stationary phases, J.Chromatogr.A, 1994, 659, 467-
471.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 100 u-M solution in buffer, inject a 20 jxL aliquot.
HPLC VARIABLES
panohwater 1:2, pack in a 100 X 4.6 column.) (Buffer was 100 mM pH 5.3 sodium acetate.)
Mobile phase: EtOH:50 mM pH 5.5 KH2PO4 5:95
Flow rate: 0.8
Detector: UV
CHROMATOGRAM
OTHERSUBSTANCES
Simultaneous: bepridil, lorazepam, manidipine, oxazepam
KEYWORDS
chiral; a = 1.32
REFERENCE
protein as a new chiral stationary phase in high-performance liquid chromatography, J.Chromatogr.A, 1995,
704, 55-65.
Nicergoline
Molecular formula: C24H26BrN3O3
Merck Index: 6580
Lednicer No.: 2 478
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 202.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Niclosamide
Merck Index: 6602
Lednicer No.: 2 94
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 205.2
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 (xm Hypersil C18
Mobile phase: MeOH.buffer 19:81, pH 3.9 (Buffer was prepared by dissolving 6.6 g dibasic am-
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: albendazole, fenbendazole, oxyclozanide
KEYWORDS
tablets; powder; liquid formulations
REFERENCE
lations, J.Chromatogr.A, 1996, 729, 267-272.
Nicorandil
Merck Index: 6608
Lednicer No.: 3 148
SAMPLE
Matrix: blood
Sample preparation: Condition two Bond Elut C18 SPE cartridges with 2 volumes of MeOH
and 2 volumes of water. 1 mL Plasma + 50 JJLL 2 |xg/mL IS in water, add to the SPE cartridge,
wash with two 1 mL portions of water, elute with MeCN. Evaporate the eluate to less than
200 |xL under a stream of nitrogen, add 1 mL water, add the mixture to another SPE cartridge,
wash with two 1 mL portions of water, dry under vacuum for 10-15 min, wash with 2 mL
MeCN, elute with 1 mL MeOHrwater 95:5. Evaporate the eluate to dryness under a stream of
nitrogen, reconstitute the residue in 200 (xL mobile phase, inject a 160 |xL aliquot.
HPLC VARIABLES
Guard column: 15 X 3.2 phenyl (Brownlee)
Mobile phase: MeCN:isopropanol:water 12:2:86
Flow rate: 1
Detector: UV 254 or photoconductivity (Tracor 965)
CHROMATOGRAM
Retention time: 15
Internal standard: N-[2-(nitrooxy)propyl]-3-pyridine carboxamide (24)
Limit of detection: 10 ng/mL, 2 ng/mL (photoconductivity)
KEYWORDS
plasma; dog; pharmacokinetics; SPE
REFERENCE
Schwende,F.J.; Lewis,R.C. Determination of nicorandil in plasma using high-performance liquid chromatog-
raphy with photoconductivity and ultraviolet detection. Application to pre-clinical pharmacokinetics in bea-
gle dogs, J.Chromatogr., 1990, 525, 151-160.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond-Elut PH SPE cartridge with two 1 mL portions of MeOH
and two 1 mL portions of water. 20 |xL Plasma + 50 JJLL 10 |xg/mL IS, add to the SPE cartridge,
elute with 250 JJLL MeOH, inject a 40 JJLL aliquot of the eluate.
HPLC VARIABLES
Column: 10 |jim ixBondapak C18
Flow rate: 1.2
Detector: UV 254
CHROMATOGRAM
Retention time: 4.5
Internal standard: N-(2-hydroxypropyl)nicotinamide nitrate (5.5)
KEYWORDS
rat; plasma; pharmacokinetics; SPE
REFERENCE
Gomita,Y.; Eto,K; Furuno,K; Mimaki,Y.; Araki,Y. Influences of exposure to cigarette smoke on concentration
of nicorandil in plasma of rats, J.Pharm.Sci., 1992, 81, 228-231.
SAMPLE
Matrix: blood
Sample preparation: 180 (xL Plasma + 10 |xL water + 10 |xL 210 (xg/mL IS + 1 drop 100 mM
NaOH + 1 mL ethyl acetate, vortex for 30 s, centrifuge at 2000 g for 10 min, repeat the
trogen, immediately reconstitute the residue in 200 JULL water, vortex for 10 s, inject a 30-50
(JLL aliquot.
HPLCVARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 5.5
Internal standard: N(3-hydroxypropyl) nicotinamide nitrate ester (6.7)
OTHER SUBSTANCES
Extracted: metabolites, metabolites
KEYWORDS
REFERENCE
Bachert,E.L.; Fung,H.L. High performance liquid chromatographic method for stability and pharmacokinetic
studies of nicorandil, J.Chromatogr., 1993, 619, 336-341.
SAMPLE
Matrix: blood
Sample preparation: Condition a Bond Elut PH SPE cartridge with 2 mL MeOH and 2 mL
water. 500 |xL Plasma + 50 |xL 2.5 jxg/mL, mix, add to the SPE cartridge, wash with 2 mL
water, wash with 1 mL MeOH.water 8:92, elute with 500 JULL MeOH.water 50:50. Evaporate
the eluate to dryness under a stream of nitrogen at room temperature, reconstitute the residue
in 1 mL water, add 100 |JLL 2 M HCl, add 4 mL ethyl acetate, shake for 10 min, centrifuge at
2800 g for 10 min, discard the organic layer. Adjust the pH of the aqueous layer to 9-10 with
300 |xL 1 M sodium carbonate, add 4 mL dichloromethane, shake for 10 min, centrifuge at
nitrogen at room temperature, reconstitute the residue in 100 |JLL water, inject a 45 |xL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 10 |xm jjiBondapak C18
Mobile phase: MeCN:water:100 mM pH 8 borate buffer 15:70:15
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 8
Internal standard: N-[2-(nitroxy)propyl]-3-pyridinecarboxamide(SG-89) (11)
KEYWORDS
Next Page
REFERENCE
Tanikawa,M.; Uzu,M.; Ohsawa,Y.; Fukushima,M. Sensitive method for determination of nicorandil in human
plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection,
J.Chromatogr., 1993, 617, 163-167.
SAMPLE
Matrix: blood
Sample preparation: 200 u,L Plasma + 100 |xL 1.5 M perchloric acid, vortex, centrifuge at 9600
g for 1 min, add to 100 |xL 3 M potassium carbonate, vortex, centrifuge for 1 min, inject a 100-
200 |JLL aliquot of the supernatant.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Capcell Pak C8 AG-120 endcapped (Shiseido)
Mobile phase: MeOHibuffer 20:80 (Prepare by mixing 60 mL 500 mM acetic acid and 940 mL
500 mM pH 6.0 sodium acetate containing 20% MeOH and 20 mM hydrogen peroxide.)
Flow rate: 0.8
a 3 m X 0.25 mm ID Tefzel coil irradiated by two 4 W germicidal lamps (Nippon Denki, Tokyo)
at about 40, a 0.4 m X 0.1 mm ID stainless steel coil, and a 2 m X 0.25 mm ID PTFE coil to
the detector.
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Noniriterfering: histidine, kynurenic acid, niacin, phenylalanine, pyridoxine, thiamine, tyrosine
KEYWORDS
post-column reaction; post-column photochemical derivatization; plasma; pharmacokinetics
REFERENCE
Mawatari,K.-i.; Nakamura,Y.; Shimizu,R-; Sate,S.; Iinuma,R; Watanabe,M. Fluorimetric determination of
nicorandil in human plasma by a high-performance liquid chromatographic-postcolumn ultraviolet detection
system equipped with on-line back-pressure tubing, J.Chromatogr.B, 1996, 679, 155-159.
Nicotine
CAS Registry No.: 54-11-5, 96055-45-7 (nicotine polacrilex)
Merck Index: 6611
SAMPLE
Matrix: blood
Sample preparation: Add 100 u,L 100 ng/mL nicotine-methyl-d3 to 1 mL heparinized plasma,
vortex briefly, add 100 |xL 5 M ammonium hydroxide and 8 mL dichloromethane, shake for 5
min on a horizontal shaker, centrifuge at 2500 rpm for 5 min, evaporate the organic phase to
dryness under nitrogen at 37, reconstitute the residue in 100 |xL mobile phase, inject 10 u,L
aliquot.
HPLC VARIABLES
Guard column: 10 X 2.0 BDS C18
Column: 100 X 3.0 3 |jLm BDS Hypersil C18
Flow rate: 1.4
Previous Page
REFERENCE
Tanikawa,M.; Uzu,M.; Ohsawa,Y.; Fukushima,M. Sensitive method for determination of nicorandil in human
plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection,
J.Chromatogr., 1993, 617, 163-167.
SAMPLE
Matrix: blood
Sample preparation: 200 u,L Plasma + 100 |xL 1.5 M perchloric acid, vortex, centrifuge at 9600
g for 1 min, add to 100 |xL 3 M potassium carbonate, vortex, centrifuge for 1 min, inject a 100-
200 |JLL aliquot of the supernatant.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Capcell Pak C8 AG-120 endcapped (Shiseido)
Mobile phase: MeOHibuffer 20:80 (Prepare by mixing 60 mL 500 mM acetic acid and 940 mL
500 mM pH 6.0 sodium acetate containing 20% MeOH and 20 mM hydrogen peroxide.)
Flow rate: 0.8
a 3 m X 0.25 mm ID Tefzel coil irradiated by two 4 W germicidal lamps (Nippon Denki, Tokyo)
at about 40, a 0.4 m X 0.1 mm ID stainless steel coil, and a 2 m X 0.25 mm ID PTFE coil to
the detector.
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Noniriterfering: histidine, kynurenic acid, niacin, phenylalanine, pyridoxine, thiamine, tyrosine
KEYWORDS
post-column reaction; post-column photochemical derivatization; plasma; pharmacokinetics
REFERENCE
Mawatari,K.-i.; Nakamura,Y.; Shimizu,R-; Sate,S.; Iinuma,R; Watanabe,M. Fluorimetric determination of
nicorandil in human plasma by a high-performance liquid chromatographic-postcolumn ultraviolet detection
system equipped with on-line back-pressure tubing, J.Chromatogr.B, 1996, 679, 155-159.
Nicotine
CAS Registry No.: 54-11-5, 96055-45-7 (nicotine polacrilex)
Merck Index: 6611
SAMPLE
Matrix: blood
Sample preparation: Add 100 u,L 100 ng/mL nicotine-methyl-d3 to 1 mL heparinized plasma,
vortex briefly, add 100 |xL 5 M ammonium hydroxide and 8 mL dichloromethane, shake for 5
min on a horizontal shaker, centrifuge at 2500 rpm for 5 min, evaporate the organic phase to
dryness under nitrogen at 37, reconstitute the residue in 100 |xL mobile phase, inject 10 u,L
aliquot.
HPLC VARIABLES
Guard column: 10 X 2.0 BDS C18
Column: 100 X 3.0 3 |jLm BDS Hypersil C18
Flow rate: 1.4
Detector: MS, Perkin-Elmer Sciex API-Ill, APCI interface, nebulizer 480, pressure 551 kPa,
nitrogen 1.2 L/min, interface heater 55, electron multiplier 4000 V, m/z 163.2 to 84.0
CHROMATOGRAM
Internal standard: nicotine-methyl-d3 (0.57)
OTHER SUBSTANCES
Extracted: cotinine
KEYWORDS
REFERENCE
Xu,A.S.; Peng,L.L.; Havel, J.A.; Petersen,M.E.; Fiene,J.A.; Hulse,J.D. Determination of nicotine and cotinine in
human plasma by liquid chromatography-tandem mass spectrometry with atmospheric-pressure chemical
ionization interface, J.Ckromatogr.B, 1996, 682, 249-257.
SAMPLE
Matrix: blood
Sample preparation: Condition a Merck Extrelut-3 glass extraction column with 12 mL di-
chloromethane the day before. 1.5 mL Serum + 100 |xL 3 u-g/mL N-ethylnorcotinine in water
+ 1.4 mL 0.5 M NaOH. Add to column, after 15 min elute under gravity with dichloromethane:
isopropanol 9:1. Add 300 |xL 25 mM HCl in MeOH to the organic phase and evaporate it to
dryness under nitrogen. Redissolve in 100 |xL water and inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC8DB
Mobile phase: Gradient. A was MeCN:water 3.6:96.4 containing 2 mL/L triethylamine, 12 mM
sodium heptanesulfonate, 12 mM K2HPO4, 12 mM citric acid adjusted to pH 4.7 with citric
acid. B was MeCN:water 19.7:80.3 containing 2 mL/L triethylamine, 12 mM sodium heptane-
sulfonate, 12 mM K2HPO4, 12 mM citric acid adjusted to pH 5.2 with citric acid. A:B 100:0 for
15 min then to 50:50 over 20 min using a concave gradient. Re-equilibrate for 15 min before
next injection.
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 14
Internal standard: N-ethylnorcotinine (33)
OTHER SUBSTANCES
Simultaneous: metabolites, cotinine-N-oxide, trans-3'-hydroxycotinine, norcotinine, cotinine,
caffeine
KEYWORDS
serum; SPE
REFERENCE
Zuccaro,R; Altieri,L; Rosa,M.; Passa,A.R.; Pichini,S.; Ricciarello,G.; Pacifici,R. Determination of nicotine and
four metabolites in the serum of smokers by high-performance liquid chromatography with ultraviolet de-
tection, J.Chromatogr., 1993, 621, 257-261.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 |xL 70% perchloric acid, centrifuge at 2000 g for 10
min. Remove the supernatant and evaporate it to dryness under a stream of nitrogen at 40,
reconstitute the residue in 100 \xL water, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 fjim Supelcosil LC8DB
Mobile phase: MeCN:MeOH:THF:buffer 4.3:2.3:0.3:93.1 (Buffer was 2 mL/L triethylamine con-
taining 12 mM sodium heptanesulfonate, 12 mM K2HPO4, and 12 mM citric acid, adjusted to
pH 4.7 with citric acid.)
Flow rate: 1.4
Detector: UV 254
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
Extracted: metabolites, cotinine, caffeine
KEYWORDS
serum
REFERENCE
Pichini,S.; AltieriJ.; Passa,A.R.; Rosa,M.; Zuccaro,R; Pacifici,R. Use of solvent optimization software for rapid
selection of conditions for reversed-phase high-performance liquid chromatography of nicotine and its me-
tabolites, J.ChromatogrA, 1995, 697, 383-388.
SAMPLE
Sample preparation: Wash a C18 Sep Pak cartridge with 2 mL MeOH then 2 mL water. Blood.
1 mL Blood + 2 mL water, add 2 mL MeCN, cool to 5, centrifuge. Tissue. 1 g Tissue + 4 mL
water, homogenize, add 4 mL MeOH, centrifuge, extract pellet again with 2 mL MeOH. Com-
bine supernatants, evaporate, take up in 50 |JLL 5 M NaOH, exctract with ethyl acetate, discard
organic phase, add 100 |xL 6 M HCl, extract with ethyl acetate, discard organic phase, add 8
mg of dodecyl sodium sulfate (at pH 2.0-2.1), add to cartridge, wash with MeOH/water, elute
with MeOH, concentrate, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 ixm Alltech NH2
Mobile phase: Isopropanol:water 75:25
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Extracted: cotinine, cotinine N-oxide, nicotine N-oxide
KEYWORDS
SPE; liver
REFERENCE
Thompson,J.A.; Norris,K.J.; Petersen,D.R. Isolation and analysis of N-oxide metabolites of tertiary amines:
quantitation of nicotine-l'-N-oxide formation in mice, J.Chromatogr., 1985, 341, 349-359.
SAMPLE
Matrix: dialysate, saliva
Sample preparation: Saliva. Incubate 500 mg smokeless tobacco and 10 mL artificial saliva at
37 for 0.5-60 min, filter (0.45 HVLP), inject an aliquot. Dialysate (Method 1). Place 1.5 g
tobacco (without sachet) in dialysis tube and tie ends. Press dialysis bag with a glass rod for
30 s , immerse it in 50 mL isotonic pH 7.4 phosphate buffer, incubate at 37, inject an aliquot.
(Method 2). Place 500 mg tobacco (with or without sachet) in dialysate tube, tie the ends,
immerse the dialysis bag in 25 mL artificial saliva and incubate at 37. Remove an aliquot
from the saliva surrounding the bag. Inject an aliquot.
HPLCVARIABLES
Column: 300 X 3.9 10 |xm Bondapak C18
Mobile phase: MeOH:buffer 40:60 (Buffer was 0.2% phosphoric acid adjusted to pH 7.25 with
triethylamine.)
Flow rate: 1.5
Detector: UV 245
KEYWORDS
artificial saliva; tobacco
REFERENCE
Nasr,M.M.; Reepmeyer,J.C; Tang,Y. In vitro study of nicotine release from smokeless tobacco, J.AOAC Int.,
1998, 81, 540-543.
SAMPLE
Matrix: meconium
Sample preparation: Condition a 3 mL C8 SPE cartridge (Backer-Bond, Gross Gerau, Germany)
with 5 mL MeOH and 5 mL water. Condition a 3 mL silica SPE cartridge (Backer-Bond) with
5 mL chloroform. Place the C8 SPE cartridge on top of the silica SPE cartridge. Thaw and mix
meconium, weigh a 2 g aliquot, add IS, and emulsify in 20 mL 100 mM pH 8.0 phosphate
buffer. Vortex, centrifuge, filter the supernatant. Extract the supernatant 3 times with 2 mL
portions of chloroform. Evaporate chloroform extracts to dryness and dissolve in 10 mL pH 9.0
boric buffer. Add to the SPE cartridges, remove the C8 column and elute the silica column with
3 mL MeOH:dichloromethane:ammonium hydroxide 30:70:1. Evaporate the eluate to dryness
under nitrogen and redissolved in 100 |xL water. Inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm LiChrosorb C18
Mobile phase: MeCN:MeOH:water:pH 4.66 acetate buffer: acetic acid 2:29:50:20:1, pH adjusted
to 4.3 with diethylamine
Flow rate: 1.0
Detector: UV
CHROMATOGRAM
Internal standard: ephedrine
OTHER SUBSTANCES
Extracted: caffeine, cotinine
KEYWORDS
SPE
REFERENCE
Baranowski,J.; Pochopien,G.; BaranowskaJ. Determination of nicotine, cotinine and caffeine in meconium us-
ing high-performance liquid chromatography, J.Chromatogr.B, 1998, 707, 317-321.
SAMPLE
Sample preparation: Extract microsomal incubation with isopropanol/dichloromethane, evap-
orate the extracts to a minimum volume, reconstitute with MeOH, inject an aliquot.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:60% perchloric acid 50:50:0.03
Detector: UV 255
CHROMATOGRAM
Retention time: 16.4 mL (retention volume)
OTHER SUBSTANCES
Extracted: metabolites, cotinine
KEYWORDS
pig; human; liver
REFERENCE
Berkman,C.E.; Park,S.B.; Wrighton,S.A.; Cashman,J.R. In vitro-in vivo correlations of human (S)-nicotine me-
tabolism, Biochem.PharmacoL, 1995, 50, 565-570.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 35 X 4 Dionex Ion Pac NGI
Mobile phase: MeCN:water 7:93 containing 500 IJLM hexanesulfonic acid
Flow rate: 1.0
Detector: UV 262
CHROMATOGRAM
Retention time: 2.4
OTHERSUBSTANCES
Simultaneous: cotinine, 4-ethenylpyridine
REFERENCE
Bertoni,G.; Di Palo,V.; Tappa,R.; Possanzini,M. Fast determination of nicotine and 3-ethenylpyridine in indoor
environments, Chromatographia, 1996, 43, 296-300.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 jxg/mL solution in MeOH, inject a 20 (xL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.9
OTHER SUBSTANCES
mine, fenoterol, fentanyl, flavoxate, fluopromazine, fiupenthixol, fluphenazine, flurazepam, hal-
lol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nifedipine, nomifensine,
pranolol, prothipendyl, protriptyline, proxjnnetacaine, pseudoephedrine, pyrimethamine, quin-
REFERENCE
Jane,I.; McRinnon,A.; Flanagan,R.J- High-performance liquid chromatographic analysis of basic drugs on silica
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 3.3 7 |jim Separon SGX
Mobile phase: 80 mM ammonium perchlorate in MeOH
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: amrinone, strychnine, nicotine, neostigmine
REFERENCE
Eigendorf,H.G.; Nagel,S. Zur Analytik von Amrinone (Cordemcura). 2. Mitteillung: Hochdruckflussigchroma-
tographie [The analysis of amrinone (Cordemcura). 2. High pressure liquid chromatography], Pharmazie,
1987, 42, 631.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 0.5 mg/mL solution in water, inject a 5 |xL aliquot.
HPLC VARIABLES
Flow rate: 2
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
Retention time: 1.3
OTHER SUBSTANCES
one, mefenamic acid, methyltestosterone, oxazepam, phentermine, phenylpropanolamine, pro-
gesterone, sulfamethazine, sulfanilamide, testosterone, testosterone propionate, tranylcypro-
mine, tripelennamine
KEYWORDS
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCNibuffer 31.2:68.8 (Buffer was 6.66 g KH2PO4 and 4.8 g 85% phosphoric acid
Flow rate: 0.5-1
Detector: UV 254
CHROMATOGRAM
Retention time: 0.7
OTHER SUBSTANCES
bamol, methotrimeprazine, oxazepam, procaine, promazine, propafenone, propranolol, salicyl-
amide, temazepam, tetracaine, thiopental, triamterene, verapamil, zolpidem, zopiclone
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
naltrexone, naphazoline, naproxen, nefopam, niacin, nifedipine, nifiumic acid, nitrazepam, nor-
epinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxj^phen-
trihexyphenidyl, trimetnoprim, tripelennamine, triprolidine, tropacocaine, tâmine, verapa-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 0.7-1
Detector: UV 260
KEYWORDS
REFERENCE
J.Liq.Chromatogr., 1995, 18, 649-671.
SAMPLE
Matrix: urine
Sample preparation: 500 |xL Urine + 200 |xL 4 M pH 4.7 acetate buffer + 100 |xL 1.5 M KCN
in water + 100 |xL 400 mM chloramine T in water + 500 |xL 50 mM l,3-diethyl-2-thiobarbituric
acid in acetoneiwater 50:50, mix, let stand at room temperature for 15 min, inject a 5 jxL aliquot
immediately.
HPLCVARIABLES
Guard column: 10 X 4.6 10 jjum reversed-phase (Whatman)
Mobile phase: MeOH:water 2:1 containing 20 mM pentanesulfonic acid
Flow rate: 2
Injection volume: 5
Detector: UV 546
CHROMATOGRAM
Retention time: 6
Limit of detection: 10 ng/mL (20 |xL injection)
OTHER SUBSTANCES
Extracted: metabolites, cotinine
KEY WpRDS
derivatization
REFERENCE
Barlow,R.D.; Thompson,P.A.; Stone,R.B. Simultaneous determination of nicotine, cotinine and five additional
nicotine metabolites in the urine of smokers using pre-column derivatization and high performance liquid
SAMPLE
Matrix: urine
Sample preparation: Centrifuge urine at 10000 g for 5 min, filter (Millipore Ultrafree-MC, 30
000 NMWL). Dilute a 10-150 JJLL aliquot to 150 jxL with water, add 60 |xL 5 |xg/mL IS in buffer,
add 30 ILL 1.5 M KCN, add 15 uL 1 M chloramine T, add 75 jxL 75 mM in l,3-diethyl-2-
thiobarbituric acid in waterracetone 50:50, mix vigorously for 30 s, centrifuge for 2 min, let
stand for 11-12 min, inject a 50-250 |JLL aliquot. (Buffer was 6 M sodium acetate adjusted to
pH 4.7 with 6 M trichloroacetic acid. The chloramine T and l,3-diethyl-2-thiobarbituric acid
solutions should be stored at 40.)
HPLC VARIABLES
Guard column: 25 X 4 5 |xm Nucleosil 100 C18
Column: 150 X 3.9 4 |xm Nova Pak RP18
Mobile phase: Gradient. A was 60 mM sodium 1-pentanesulfonate adjusted to pH 3.8 with 1%
orthophosphoric acid. B was MeOHiTHF 95:5. C was MeCNrTHF 97:3. A:B:C from 80:10:10 to
74:15:11 over 5 min, to 65:20:15 over 5 min, to 65:15:20 over 5 min, to 65:10:25 over 5 min, to
60:0:40 over 10 min, to 35:0:65 over 3 min.
Flow rate: From 1.55 to 1.45 over 15 min, maintain at 1.45.
Detector: UV 529
CHROMATOGRAM
Retention time: 26
Internal standard: N'-ethylnorcotinine (21)
Limit of quantitation: 1.5 jxM
OTHER SUBSTANCES
KEY WpRDS
derivatization; human; hamster; rat
REFERENCE
Rustemeier,K; Demetriou,D.; Schepers,G.; Voncken,P. High-performance liquid chromatographic determination
of nicotine and its urinary metabolites via their l,3-diethyl-2-thiobarbituric acid derivatives, J. Chromatogr.,
1993, 613, 95-103.
SAMPLE
Matrix: urine
Sample preparation: 200 |xL Urine + 100 |xL 4 M pH 4.7 sodium acetate buffer + 40 |xL 1.5 M
KCN in water + 40 |xL 400 mM chloramine T in water + 200 |xL 78 mM barbituric acid in
acetonerwater 50:50, mix for 10 s, let stand at room temperature for 15 min, add 40 fxL 1 M
sodium metabisulfite in water (Clin. Chim. Acta 1987, 165, 45), inject a 50 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 10 |xm reversed-phase (Whatman)
Column: 250 X 4.6 5 |xm Spherisorb S 5ODS2
Flow rate: 1.5
Detector: UV 490
CHROMATOGRAM
Retention time: 4.1
OTHER SUBSTANCES
Extracted: cotinine
KEYWORDS
derivatization
REFERENCE
Ubbink,J.B.; Lagendijk,J.; Vermaak,W.H.H. Simple high-performance liquid chromatographic method to verify
the direct barbituric acid assay for urinary cotinine, J.Chromatogr., 1993, 620, 254-259.
SAMPLE
Matrix: urine
Sample preparation: Acidify urine to pH 2.00 with phosphoric acid, filter (Acrodisc 0.20 |xm),
inject a 20 u,L aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Econosphere CN
Mobile phase: Isopropanol:20 mM sodium dodecyl sulfate 3:97, pH 4.60
Flow rate: 1
Detector: UV 260
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: cotinine
REFERENCE
Reynolds,J.; Albazi,S.J. Simultaneous determination of nicotine and cotinine in untreated human urine by
micellar liquid chromatography, J.Liq.Chromatogr., 1995, 18, 537-552.
SAMPLE
Matrix: urine
Sample preparation: Condition a 700 mg Extrelut-1 diatomaceous earth glass SPE cartridge
with 6 mL dichloromethane, let stand for 1 day. 200 |xL Urine + 200 |xL 10 |xg/mL N-ethylnor-
nicotine in water + 600 |xL 500 mM NaOH, add to the SPE cartridge, let stand for 10 min,
elute with 5 mL dichloromethane risopropanol 90:10. Add the eluate to 100 |xL 25 mM HCl in
MeOH and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 200
jxL water, inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm LC8DB (Supelchem)
Mobile phase: MeCN:water 9:80 containing 5 mL/L triethylamine, 670 mg/L sodium heptane-
sulfonate, 34 mM K2HPO4, and 34 mM citric acid, pH 4.4.
Flow rate: 1.6
Detector: UV 254
CHROMATOGRAM
Retention time: 4
Internal standard: N-ethylnornicotine (8.5)
OTHER SUBSTANCES
Extracted: metabolites, caffeine, cotinine
KEYWORDS
SPE
REFERENCE
Zuccaro,R; Altieri,L; Rosa,M.; Passa,A.R.; Pichini,S.; Pacifici,R- Solid-phase extraction of nicotine and its me-
tabolites for high-performance liquid chromatographic determination in urine, J.Chromatogr.B, 1995, 668,
187-188.
Nifedipine
Merck Index: 6617
Lednicer No.: 2 283; 4 106
SAMPLE
Matrix: blood
HPLCVARIABLES
Column: 100 X 4.6 5-10 |xm Silicalite (by sieving Silicalite, 3M Co.(?))
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
KEY WORDS
serum
REFERENCE
89-96.
SAMPLE
Matrix: blood
Sample preparation: Add 200 |xL 1 M NaOH and 50 |JLL 2 |xg/mL IS to 1 mL plasma, vortex
for 10 s, add 3 mL hexaneidichloromethane 70:30, vortex for 5 min, centrifuge at 1400 g for
15 min, evaporate the organic layer under nitrogen at 50, dissolve the dried sample in 250
|xL mobile phase, inject a 50 |xL aliquot onto column A in series with column B and elute with
mobile phase. After 1 min remove column A from the circuit, elute column B with mobile phase,
monitor the effluent from column B. While column A is removed from the circuit flush it sep-
arately with mobile phase to remove strongly retained plasma components.
HPLC VARIABLES
Column: A 20 X 4.0 5 |xm Hypersil BST ODS; B 200 X 4.6 5 |xm Hypersil ODS
Mobile phase: MeOH: 10 mM pH 4 acetate buffer 75:25
Flow rate: 0.8
Detector: E, Bioanalytical Systems Model BAS LC-3C equipped with BAS LC-44 thin-layer cell,
glassy carbon electrode 1 V, Ag/AgCl/3 M KCl reference electrode
CHROMATOGRAM
Retention time: 4.7
Internal standard: dimethyl-1,4-(3-nitrophenyl)-3,5-pyridine-dicarboxylate (5.3)
KEYWORDS
dog; pharmacokinetics; plasma; column-switching
REFERENCE
Horvth,V.; Hrab6czy-PU,A.; Niegreisz,Z.; Kocsi,E.; Horvai,G.; God6rhzy,L.; Tolokan,A.; KlebovichJ.; Balogh-
Nemes,K. Sensitive high-performance liquid chromatographic determination of nifedipine in dog plasma
using an automated sample preparation system with laboratory robot, J.Chromatogr.B, 1996, 686, 211-
219.
SAMPLE
Matrix: blood
Sample preparation: Add 200 |xL 1 M NaOH solution and 50 fiL 2 |xg/mL IS to 1 mL plasma,
mix for 10 s, add 3 mL dichloromethane:n-hexane 30:70, vortex for 5 min, centrifuge. Evaporate
a 2 mL aliquot of the organic layer to dryness in a stream of nitrogen at 50, dissolve the
HPLC VARIABLES
Guard column: 20 X 4 5 |xm Hypersil BST ODS (Bio Separation Technologies, Hungary)
Column: 200 X 4.6 5 |xm Hypersil ODS (Hewlett-Packard, USA)
Mobile phase: MeOH:10 mM pH 4 acetate buffer 75:25
Flow rate: 0.8
Detector: E, Bioanalytical Systems BAS LC-3C equipped with BAS LC-44 thin-layer cell, glassy
carbon electrode 1 V, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 4.9
Internal standard: dimethyl-l,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxy-
late (5.5)
KEYWORDS
plasma
REFERENCE
Tolokn,A-; God6rhzy,L.; Horvth,V.; Hrab6czy-P11,A.; Niegreisz,Z.; Kocsi,E.; Horvai,G.; Klebovich,!; Balogh-
Nemes,K. Economic approach to robotic sample pretreatment in high-performance liquid chromatography,
J.Chromatogr.A, 1997, 771, 35-43.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 juim Symmetry C8 (Waters)
mL/min)
Detector: UV 236.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y; Ppin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
163.
SAMPLE
Sample preparation: Weigh 10 tablets, powder finely. Weigh accurately powder containing 10
mg nifedipine, dissolve in MeOH and make up to 50 mL with MeOH. Add 1.6 mg IS, filter
through a 45 jxm membrane filter. Inject a 10 |xL aliquot.
HPLCVARIABLES
Column: 250 X 4.6 10 pjn Lichrosorb RP C18
Mobile phase: MeOHrwater 55:45 pH 4.5
Flow rate: 1.0 for 4 min, then 2.0
Detector: UV 260
CHROMATOGRAM
Internal standard: oxprenolol (4.35)
OTHER SUBSTANCES
Simultaneous: acebutolol, nifedipine oxidation products
KEYWORDS
comparison with GC and first-derivative spectroscopy; tablets
REFERENCE
el Walily,A.F.M. Analysis of nifedipine-acebutolol hydrochloride binary combination in tablets using UV-deriv-
ative spectroscopy, capillary gas chromatography and high performance liquid chromatography,
SAMPLE
Sample preparation: Crush five 10 mg tablets and mix with lactose to a nifedipine concentra-
tion 1 mg/500 mg of powder. Sonicate 500 mg aliquot of the powder in 10 mL MeOH at 20 for
10 min, dilute 1 mL of this solution to 10 mL with MeOH, inject a 20 |xL aliquot.
HPLCVARIABLES
Mobile phase: MeOHrIOO mM pH 5.8 ammonium acetateitriethylamine 70:30:0.1, pH adjusted
to 5.8 with acetic acid
Flow rate: 1
Detector: UV 238
CHROMATOGRAM
Retention time: 5.0
OTHER SUBSTANCES
Simultaneous: photodegradation products
KEYWORDS
powder; stability-indicating
REFERENCE
Helin,M.M.; Kontra,K.M.; Naaranlahti,T.J.; Wallenius,K.J. Content uniformity and stability of nifedipine in
extemporaneously compounded oral powders, Am. J.Health-Syst.Pharm., 1998, 55, 1299-1301.
Nifenalol
Molecular formula: CnH 16 N 2 O 3
Merck Index: 6618
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 12 ^m l-m5n*istoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
famotidine, isothipendyl, ketotifen, metiamide, metoprolol, moxonidine, nadolol, naphazoline,
REFERENCE
Kaliszan,R.; Nasal,A.; Turowski,M. Binding site for basic drugs on c^-acid glycoprotein as revealed by chemo-
Niflumic acid
CAS Registry No.: 4394-00-7, 65847-85-0 (p-morpholinoethyl ester)
Merck Index: 6620
Lednicer No.: 1 256
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 288
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Gradient. A was 10 inL concentrated orthophosphoric acid and 7 mL triethylamine
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nitrazepam, nor-
REFERENCE
18, 233-242.
Nifuroxazide
Merck Index: 6626
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Nikethamide
Merck Index: 6635
Lednicer No.: 1 253
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Nilutamide
Merck Index: 6636
SAMPLE
Matrix: perfusate, tissue
Sample preparation: Homogenize (Polytron) rat lung with 8 mL saline using an ice bath. 250
|JLL Perfusate or lung homogenate + 40 ng norandrostenedione + 500 |xL pH 7.0 phosphate
buffer, vortex, add 4 mL dichloromethane, shake at 250 cycles/min for 15 min, centrifuge at 4
nitrogen at 40, reconstitute the residue in 100 |xL MeOH:water 50:50, inject a 10-50 (xL aliquot.
HPLC VARIABLES
Mobile phase: Gradient. A was MeCNrMeOH:water 18.2:18.2:63.6. B was MeCN:MeOH:water
31:13:53. C was MeCN. A:B:C 100:0:0 for 3 min, to 0:85:15 over 5 min, maintain at 0:85:15 for
12 min, to 100:0:0 over 1 min, maintain at 100:0:0 for 14 min.
Detector: UV 260
CHROMATOGRAM
Internal standard: norandrostenedione (22.4)
KEYWORDS
lung; rat
REFERENCE
Camus,R; Coudert,B.; d'Athis,P.; Foucher,R; Delchambre,J.; Dupront,A.; Jeannin,L. Pharmacokinetics and me-
tabolism of nilutamide in the isolated rat lung, J.Pharmacol.Exp.Ther., 1991, 259, 1247-1255.
Nilvadipine
Merck Index: 6637
SAMPLE
Matrix: perfusate
Sample preparation: 2 mL Perfusate + 100 |xL 200 |xg/mL nitrendipine in MeOH + 2 mL pH
9.0 phosphate buffer + 2 mL benzene:hexane 50:50 (Caution! Benzene is a carcinogen!), shake
for 15 min, centrifuge. Remove the organic layer and evaporate it to dryness under a stream
of nitrogen, reconstitute the residue in mobile phase, inject an aliquot.
HPLC VARIABLES
Mobile phase: MeCN:water 60:40 containing 5.25 mL/L 1% phosphoric acid, 2.5 g/L ammonium
phosphate, and 10 mL/L 10% tetra-n-butylammonium hydroxide
Detector: UV 254
CHROMATOGRAM
KEYWORDS
protect from light
REFERENCE
Kobayashi,D.; Matsuzawa,T.; Sugibayashi,K; Morimoto,Y; Kobayashi,M.; Kimura,M. Feasibility of use of sev-
rat and human skin, Biol.Pharm.Bull., 1993, 16, 254-258.
SAMPLE
Matrix: solutions
Sample preparation: Direct injection of a MeOH solution containing 200-1000 ng.
HPLC VARIABLES
Column: 250 X 4.6 Sumchiral OA-4500 (Sumika Chemical Analysis Service)
Mobile phase: n-Hexane:l,2-dichloroethane:MeOH:trifluoroaceticacid 250:140:2:1
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 14 (-), 18.5 (+) (a = 1.39)
KEYWORDS
chiral
REFERENCE
471.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution of nilvadipine in a solution of human serum albumin
in sodium phosphate buffer (I = 0.17), inject a 667-1330 (JLL aliquot onto column A and elute
with mobile phase A, monitor the effluent from column A and divert the fraction containing
the nilvadipine onto column B, elute the contents of column B onto column C with mobile
phase B. (Wash column B with water for 4 min.)
HPLC VARIABLES
Column: A 300 X 8 Develosil 100Diol5 (Nomura Chemical); B 10 X 4 Wakosil 5C4; C 150 X 6
Ultron ES-OVM
Mobile phase: A pH 7.4 sodium phosphate buffer (I = 0.17); B Isopropanol:pH 6.5 sodium phos-
phate buffer (I = 0.04)
Flow rate: 1
Detector: UV 244
CHROMATOGRAM
KEYWORDS
column-switching; heart-cut; chiral; human serum albumin
REFERENCE
Shibukawa,A.; Nakao,C; Sawada,T.; Terakita,A.; Morokoshi,N.; Nakagawa,T. Determination of the unbound
concentration of hydrophobic drugs in albumin solutions by high-performance frontal analysis using a diol-
silica column, J.Pharm.Sci., 1994, 83, 868-873.
Nimesulide
Merck Index: 6640
SAMPLE
Matrix: blood
Sample preparation: 1 mL PLasma or urine + 10 |xL 250 |xg/mL tolbutamide in MeOH + 10
jxL concentrated HCl + 8 mL toluene, extract for 15 min, centrifuge at 1500 g for 10 min, repeat
trogen at room temperature, reconstitute the residue in 200 fxL mobile phase, inject a 5 JJLL
aliquot. (To hydrolyze conjugates adjust pH of 2 mL urine to 5.5 with 50 mM sodium acetate
buffer, add 200 JJLL 200 U/mL p-glucuronidase, heat at 37 for 3 h, add 10 |xL concentrated HCl,
extract as above using twice the volume of IS solution.)
HPLC VARIABLES
Column: 300 X 4.6 10 |jim C18 (Merck)
Flow rate: 1
Injection volume: 5
Detector: UV 230
CHROMATOGRAM
Retention time: 9
Internal standard: tolbutamide (6)
OTHER SUBSTANCES
Simultaneous: acetaminophen, aspirin, bezafibrate, doxepin, glibenclamide, salicylic acid,
theophylline
Noninterfering: digoxin, flurazepam, tiadenol
KEYWORDS
REFERENCE
Castoldi,D.; Monzani,V.; Tofanetti,O. Simultaneous determination of nimesulide and hydroxynimesulide in hu-
man plasma and urine by high-performance liquid chromatography, J.Chromatogr., 1988, 425, 413418.
SAMPLE
Sample preparation: Tissue. Homogenize 2 g tissue in 10 mL MeCN, wash twice with 20 mL
n-hexane. Evaporate the MeCN layer to dryness under reduced pressure at 50, reconstitute
in 100 (JLL 150 |xg/mL 2-tert-butyl-4-methoxyphenol, inject a 20 |xL aliquot. Serum. 1 mL Serum
+ 1 mL MeOH + 600 |xL 1 M HCl, extract twice with 5 mL portions of benzene (Caution!
Benzene is a carcinogen!). Combine the organic layers and extract twice with 2 mL portions of
200 mM NaOH. Combine the aqueous layers and acidify them with 2 M HCl, extract twice
with 3 mL portions of benzene. Combine the organic layers and evaporate them to dryness
under a stream of nitrogen, reconstitute the residue in 100 |xL 150 jxg/mL 2-tert-butyl-4-meth-
oxyphenol, inject a 20 |xL aliquot.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 313
CHROMATOGRAM
Internal standard: 2-tert-butyl-4-methoxyphenol
KEYWORDS
muscle; serum; ovary; uterus; cervix
REFERENCE
Pulkkinen,M.O.; Vuento,M.; Macciocchi,A.; Monti,T. Distribution of oral nimesulide in female genital tissues,
Biopharm.Drug Dispos., 1991, 12, 113-117.
Nimodipine
Merck Index: 6643
Lednicer No.: 3 149
SAMPLE
Matrix: blood
Sample preparation: 2 mL Serum + 2 mL water, mix, add 1 mL 1 M NaOH, add 12 mL hexane:
ethyl ether 50:50, extract, centrifuge at 300 g for 10 min. Remove the organic layer and evap-
orate it to dryness under a stream of nitrogen, reconstitute the residue in 250 (xL mobile phase,
add 250 |ULL water, inject a 50 JULL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 3 |jim Spherisorb ODS
Flow rate: 0.5
Detector: UV 238
CHROMATOGRAM
Internal standard: nimodipine
OTHER SUBSTANCES
Extracted: nitrendipine
KEYWORDS
serum; nimodipine is IS
REFERENCE
Janis,R.A.; Krol,G.J.; Noe,A.J.; Pan,M. Radioreceptor and high-performance liquid chromatographic assays for
the calcium channel antagonist nitrendipine in serum, J.Clin.PharmacoL, 1983, 23, 266-273.
SAMPLE
Matrix: blood
Sample preparation: 100 JXL Plasma + 10 u,L 10 |xg/mL nitrendipine + 1 mL ethyl acetate,
shake horizontally for 3 min, centrifuge at 10000 g for 10 min. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen at 40-50, reconstitute the residue in 200
|xL mobile phase, inject a 100 |xL aliquot.
HPLC VARIABLES
Guard column: 20 X 2 30-40 |xm Perisorb RP-18 (Upchurch)
Flow rate: 1
Detector: UV 238
CHROMATOGRAM
Retention time: 9
Internal standard: nitrendipine (7.5)
KEYWORDS
monkey; plasma; pharmacokinetics; protect from light
REFERENCE
Qian,M.; Gallo,J.M. High-performance liquid chromatographic determination of the calcium channel blocker
nimodipine in monkey plasma, J.Chromatogr., 1992, 578, 316320.
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Serum + 200 |xL 1 M NaOH + n-hexane:ethyl acetate 50:50, rotate
for 10 min, centrifuge at 2000 g for 10 min, repeat extraction. Combine the organic layers and
JULL mobile phase, inject a 100 |xL aliquot.
HPLC VARIABLES
Guard column: 50 X 4.6 10 |xm Daicel OJ
Column: 250 X 4.6 10 |xm Daicel OJ
Flow rate: 0.3
Detector: UV 238
CHROMATOGRAM
KEYWORDS
protect from light; serum; chiral; confirmation by GC/MS
REFERENCE
Fischer,C; Schonberger,R; Miick,W.; Heuck,K.; Eichelbaum,M. Simultaneous assessment of the intravenous
and oral disposition of the enantiomers of racemic nimodipine by chiral stationary-phase high-performance
liquid chromatography and gas chromatography/mass spectroscopy combined with a stable isotope tech-
nique, J.Pharm.Sci., 1993, 82, 244-250.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 30 |xL 800 ng/mL IS in MeCN + 300 |JLL 1 M NaOH + 5
mL diethyl ether.n-heptane 50:50, shake at 400 rpm for 5 min, centrifuge at 4 at 3000 g for
40, reconstitute the residue in 200 |xL mobile phase, inject a 50 (xL aliquot.
HPLCVARIABLES
Guard column: 10 X 2 8 |xm Chira OJ MOD (Grom)
Column: 250 X 2 8 |xm Chira OJ MOD (Grom)
Mobile phase: EtOH.n-heptane 20:80 containing 2 mM ammonium acetate
Flow rate: 0.3
Detector: MS, Sciex API III+, electrospray +4.2 kV, flow rate into MS was +reduced to 0.05 mL/
min, nebulizer gas nitrogen, m/z 436 (nimodipine), m/z 443 (IS) (M+NH4)
CHROMATOGRAM
Internal standard: heptadeuteronimodipine (deuterium in isopropyl group)
KEYWORDS
protect from light; plasma; pharmacokinetics; chiral
REFERENCE
Miick,W.M. Enantiospecific determination of nimodipine in human plasma by liquid chromatography-tandem
mass spectrometry, J.Chromatogr.A, 1995, 712, 45-53.
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 238
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Sample preparation: 500 |xL Microsomal incubation + 500 |xL ice-cold MeOH, vortex for 15 s,
let stand in ice, centrifuge at 0 at 10000 g for 5 min, inject a 40 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: 100 X 4 Spherisorb S30DS II
Flow rate: 0.5
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: hydroxymidazolam, midazolam, quinidine, quinine, verapamil
Noninterfering: cyclosporin A, erythromycin, naphthoflavone, naringenin, troleandomycin
KEYWORDS
rat; liver
REFERENCE
Leuenberger,P.M.; Ha,H.R.; Pletscher,W.; Meier,P.J.; Sticher,O. Measurement of nimodipine metabolism in rat
liver microsomes by high-performance liquid chromatography, J.Liq.Chromatogn, 1995, 18, 2243-2255.
SAMPLE
Matrix: perfusate
HPLCVARIABLES
Column: 100 X 8 5 (xm Novapak C18 radial compression
Flow rate: 2
Detector: UV 237
OTHER SUBSTANCES
Also analyzed: felodipine, nicardipine, nifedipine, nitrendipine
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 2
Detector: UV 237
OTHER SUBSTANCES
Also analyzed: nicardipine, nifedipine, nitrendipine, felodipine
KEYWORDS
buffers
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 100 |xM solution in buffer, inject a 20 jxL aliquot.
HPLCVARIABLES
Flow rate: 0.8
Detector: UV
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: flurbiprofen, isradipine, ketoprofen, suprofen
KEY WORDS
chiral; a = 1.11
REFERENCE
protein as a new chiral stationary phase in high-performance liquid chromatography, J.Chromatogr.A, 1995,
704, 55-65.
Nisoldipine
Merck Index: 6658
SAMPLE
Matrix: blood
Sample preparation: Wrap tubes in Al foil. 1 mL Serum + 50 jxL mobile phase, adjust to pH 9
with 1 M KOH, add 0.5 g KCl, add 3 mL diethyl ether, shake gently (Fisher Roto-Rack) for 10
min, centrifuge at 1000 rpm for 15 min. Separate and retain ether layer, extract aqueous layer
as before. Evaporate all ether layers to dryness under dry nitrogen at 20. Reconstitute the
HPLCVARIABLES
Column: 250 X 4 Brownlee 10 |xm RP8
Mobile phase: MeOH:50 mM buffer 30:70 (Buffer was 12 g NaH2PO4 in 2 L water adjusted to
pH 4 0.5 with 80% phosphoric acid.)
Flow rate: 0.75
Detector: UV 350
CHROMATOGRAM
Retention time: 13
Internal standard: nisoldipine
OTHER SUBSTANCES
Extracted: nifedipine
Simultaneous: photodegradation products of nifedipine
Noninterfering: aspirin, acetaminophen, caffeine, chlorthalidone, diazepam, methyldopa, ox-
prenolol, propranolol
KEYWORDS
serum; furosemide; hydrochlorothiazide; timolol; triamterene; nisoldipine is IS
REFERENCE
Snedden,W.; Fernandez,P.G.; Galway,B.A.; Kim,B.K. Specific HPLC assay for serum nifedipine, Clin.Invest.
Med., 1984, 7, 173-178.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 |xL MeOH + 100 |xL 1 M NaOH, vortex for 3 s, add 5
mL 75:25 MTBErisooctane, vortex for 30 s, centrifuge at 1800 g for 5 min. Transfer upper layer
to a 100 X 13 mm glass tube, evaporate to dryness without heating using a Speed Vac concen-
trator evaporator. Reconstitute in 200 |xL mobile phase, vortex for 15 s, inject a 150 |JLL aliquot.
HPLC VARIABLES
Column: 100 X 8 4|xm C8 Nova-Pak radial pack
Mobile phase: MeOHrwater 65:35 adjusted to approx. pH 4 with 1% acetic acid and 0.03%
triethylamine
Flow rate: 1.1
Detector: UV 350
CHROMATOGRAM
Retention time: 16
Internal standard: nisoldipine
OTHER SUBSTANCES
Extracted: nifedipine
KEYWORDS
plasma; analysis conducted under sodium lamps; nisoldipine is IS
REFERENCE
Grundy,J.S.; Kherani,R.; Foster,R.T. Sensitive high-performance liquid chromatographic assay for nifedipine in
human plasma utilizing ultraviolet detection, J.Chromatogr.B, 1994, 654, 146-151.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 |xL saturated NaCl + 100 jxL 1 M NaOH + 1.5 mL
toluene, shake at 300 rpm for 15 min, centrifuge at 10 at 3000 rpm for 10 min. Remove the
organic layer and evaporate it to dryness under reduced pressure for 25 min, reconstitute the
HPLC VARIABLES
Guard column: 10 X 2 10 |xm aminopropylsilica coated with tris(4-methylbenzoate)-modined
cellulose (Daicel)
Column: 250 X 2 10 |xm aminopropylsilica coated with tris(4-methylbenzoate)-modined cellulose
(Daicel)
Mobile phase: n-Heptane:isopropanol containing 0.2% trifluoroacetic acid 88:12
Flow rate: 0.2
Detector: UV 230
CHROMATOGRAM
Retention time: 10.5 (+), 14.6 (-)
KEYWORDS
dog; rat; mouse; plasma; chiral; microbore; confirmation by GC/MS
REFERENCE
Zimmer,D.; Muschalek,V. Enantioselective assay for the determination of nisoldipine in dog, rat and mouse
plasma by chiral microbore high-performance liquid chromatography combined with gas chromatography-
mass spectrometry, J.ChromatogrA, 1994, 666, 241-248.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 Sumchiral OA-4600
Mobile phase: n Hexane:l,2-dichloroethane:MeOH:trifluoroaceticacid 250:140:0.5:1
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
KEY WORDS
chiral; a = 1.12
REFERENCE
471.
SAMPLE
Matrix: solutions
HPLCVARIABLES
pressure.)
Mobile phase: Hexanerisopropanol 90:10
Flow rate: 0.1
Detector: UV 254
CHROMATOGRAM
KEYWORDS
REFERENCE
Pharmaceuticals using narrow-bore liquid chromatography, J.Pharm.Biomed.Anal., 1995,13, 695-699.
Nitrazepam
Merck Index: 6667
Lednicer No.: 1 366
SAMPLE
Matrix: bile, blood, gastric contents, tissue, urine
Sample preparation: Chop 5-g tissue and homogenize (Ultra Turrax T25) at 8500, 9500,13500,
20500, and 24000 rpm for 1 min each. Add homogenate to 20 mL water. Dilute blood, urine,
gastric contents, and bile four times with water. Mix 4 mL sample with 10 |xL 1 mg/mL pra-
zepam and 1 mL pH 7.4 phosphate buffer, vortex briefly, add 4 mL diethyl ether and mix for
15 min (Spiramix 10, Denley, UK). Separate the organic layer, add 4 mL diethyl ether to
extraction sample, mix. Evaporate combined organic layers to dryness under a stream of dry
air at 50. Purify extracts by partition between 1 mL MeCN and 2 mL heptane, separate MeCN
layer, evaporate it to dryness, reconstitute the residue in 100 jxL MeOH and inject a 20 JJLL
aliquot of the solution.
HPLC VARIABLES
Guard column: 20 X 4.6 5 |jim Apex II ODS
Column: 150 X 4.6 5 |xm Apex II ODS
Mobile phase: MeCNrMeOH: 10 mM phosphoric acid: 10 mM Na2HPO4 40:20:36:4
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 3.5
Internal standard: prazepam (14.5)
OTHER SUBSTANCES
Extracted: diazepam, oxazepam, temazepam
KEYWORDS
liver; lung; muscle; urine; pericardial fluid
REFERENCE
Pounder,D. J.; Adams,E.; Fuke,C; Langford,A.M. Site to site variability of postmortem drug concentrations in
liver and lung, J.Forensic ScL, 1996, 41, 927-932.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond-Elut C18 SPE cartridge with 2 mL MeOH and
2 mL water. Mix 1 mL plasma or serum with 200 uX 512 nM IS in MeOH:water 5:95, add to
the SPE cartridge, wash with 2 mL water, wash with 50 JAL MeOH. Elute with 200 |xL and
100 |xL MeOH, evaporate the eluate to dryness under a stream of air at 37, reconstitute the
residue with 100 /JLL mobile phase, inject a 20 JULL aliquot.
HPLC VARIABLES
Column: 4 jxm Novapak C18
Mobile phase: MeCN:MeOH:10 mM pH 3.7 K2HPO4 30:2:10
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: 6.2
Internal standard: flunitrazepam (9.8)
OTHER SUBSTANCES
Extracted: alprazolam, clonazepam
Simultaneous: amobarbital, carbamazepine, citalopram, clobazam, clozapine, diazepam, doxe-
pin, ethosuximide, norclobazam, oxazepam, oxcarbamazepine, pentobarbital, phenobarbital,
phenytoin, primidone, valproic acid, zopiclone
Interfering: medazepam, midazolam, nordiazepam, temazepam
KEYWORDS
SPE; plasma; serum
REFERENCE
Akerman,K.K; Jolkkonen,J.; Parviainen,M.; Penttila,I. Analysis of low-dose benzodiazepines by HPLC with
automated solid-phase extraction, Clin.Chem., 1996, 42, 1412-1416.
SAMPLE
Matrix: blood
Sample preparation: 500 JJLL Serum + 20 ^L 20 |xg/mL IS + 200 |xL 1 M potassium carbonate
HPLC VARIABLES
Column: 100 X 4.6 2 jjum TSK gel Super-ODS (A) or 100 X 4.6 5 |xm Hypersil ODS-C18 (B)
Mobile phase: MeCN:5 mM pH 6 NaH 2 PO 4 45:55
Flow rate: 0.65
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
azolam, lorazepam, oxazolam, triazolam
KEYWORDS
serum
REFERENCE
J.Chromatogr.B, 1996, 682, 173-178.
SAMPLE
Matrix: blood
Sample preparation: Add 500 JULL 200 mM pH 9.0 glycine buffer to 1 mL plasma, add 5 mL
ethyl acetate, extract. Centrifuge at 700 g for 10 min, evaporate 4 mL of the organic phase to
dryness under nitrogen at 60, reconstitute the residue in 200 |xL mobile phase, inject a 20 JXL
aliquot.
HPLCVARIABLES
Mobile phase: MeCN:buffer 69:31 (Mobile phase was 690 mL MeCN, 310 mL water, and 9 mL
glacial acetic acid, adjusted to pH 5.0 with 5 M NaOH.)
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Internal standard: nitrazepam
OTHER SUBSTANCES
Extracted: furosemide
KEYWORDS
plasma; nitrazepam is IS
REFERENCE
Jankowski,A; Skorek-Jankowska,A; Lamparczyk,H. Determination and pharmacokinetics of a furosemide-
amiloride drug combination, J.Chromatogr.B, 1997, 693, 383-391.
SAMPLE
Matrix: blood
the residue in 100 jxL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 JJLL aliquot of
HPLC VARIABLES
Column: 300 X 3.9 4 pjn NovaPack C18
Flow rate: 0.8
Detector: UV 260
CHROMATOGRAM
KEYWORDS
REFERENCE
TracquiA-; Kintz,P.; Mangin,P. Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
HPLCVARIABLES
F l o w rate: 1 for 6.5 min, to 1.5 over 18.5 min, maintain at 1.5 for 3 min (re-equilibrate at 1.5
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
naltrexone, naphazoline, naproxen, nefopam, niacinamide, nicotine, niacin, nifedipine, norep-
inephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbu-
tazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persan-
tine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine,
pheniramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: Nova-Pak C18
Next Page
Mobile phase: MeOH:buffer 85:15 (Buffer was 90.7 mL 66.7 mM Na2HPO4 and 9.3 mL 66.7 mM
Flow rate: 5 (sic)
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: chlordiazepoxide, diazepam, mirazepam
KEYWORDS
REFERENCE
J.Chromatogr.A, 1996, 735, 237-247.
Nitrendipine
Merck Index: 6669
SAMPLE
Matrix: blood
Sample preparation: 2 mL Serum + 2 mL 20 ng/mL nimodipine in water, mix, add 1 mL 1 M
NaOH, add 12 mL hexane:ethyl ether 50:50, extract, centrifuge at 300 g for 10 min. Remove
residue in 250 u,L mobile phase, add 250 |xL water, inject a 50 |xL aliquot.
HPLC VARIABLES
Flow rate: 0.5
Detector: UV 238
CHROMATOGRAM
Internal standard: nimodipine (13.08)
OTHER SUBSTANCES
KEYWORDS
serum
REFERENCE
Janis,R-A.; Krol,G.J.; Noe,A.<L; Pan,M. Radioreceptor and high-performance liquid chromatographic assays for
Previous Page
Mobile phase: MeOH:buffer 85:15 (Buffer was 90.7 mL 66.7 mM Na2HPO4 and 9.3 mL 66.7 mM
Flow rate: 5 (sic)
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: chlordiazepoxide, diazepam, mirazepam
KEYWORDS
REFERENCE
J.Chromatogr.A, 1996, 735, 237-247.
Nitrendipine
Merck Index: 6669
SAMPLE
Matrix: blood
Sample preparation: 2 mL Serum + 2 mL 20 ng/mL nimodipine in water, mix, add 1 mL 1 M
NaOH, add 12 mL hexane:ethyl ether 50:50, extract, centrifuge at 300 g for 10 min. Remove
residue in 250 u,L mobile phase, add 250 |xL water, inject a 50 |xL aliquot.
HPLC VARIABLES
Flow rate: 0.5
Detector: UV 238
CHROMATOGRAM
Internal standard: nimodipine (13.08)
OTHER SUBSTANCES
KEYWORDS
serum
REFERENCE
Janis,R-A.; Krol,G.J.; Noe,A.<L; Pan,M. Radioreceptor and high-performance liquid chromatographic assays for
SAMPLE
Matrix: blood
Sample preparation: 100 JJLL Plasma + 1 mL ethyl acetate, shake horizontally for 3 min, cen-
trifuge at 10000 g for 10 min. Remove the organic layer and evaporate it to dryness under a
stream of nitrogen at 40-50, reconstitute the residue in 200 |JLL mobile phase, inject a 100 |xL
aliquot.
HPLC VARIABLES
Guard column: 20 X 2 30-40 |xm Perisorb RP-18 (Upchurch)
Column: 150 X 4.6 5 jim Hypersil ODS
Flow rate: 1
Detector: UV 238
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
Extracted: nimodipine
KEYWORDS
monkey; plasma; protect from light; nitrendipine is IS
REFERENCE
Qian,M.; Gallo,J.M. High-performance liquid chromatographic determination of the calcium channel blocker
nimodipine in monkey plasma, J.Chromatogr., 1992, 578, 316-320.
SAMPLE
Matrix: blood
the residue in 100 (JLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 (xL aliquot of
HPLC VARIABLES
Mobile phase: MeOH:THF:buffer 65:5:30 (Buffer was 0.68 g/L (10 mM (sic)) ICH2PO4 adjusted
Flow rate: 0.8
Detector: UV 236
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Sample preparation: 1 mL Blood, urine, or gastric contents or 1 g tissue homogenate + 500 |xL
buffer + 8 mL n-hexane:ethyl acetate 70:30, mix on a rotary mixer for 10 min, centrifuge at
nitrogen, reconstitute the residue in 100 JXL 12.5 mM NaOH in MeOHrwater 50:50, inject a 50
|JLL aliquot. (Buffer was 13.8 g potassium carbonate in 100 mL water, pH adjusted to 9.5 with
concentrated HCl.)
HPLC VARIABLES
Guard column: 4 X 4 30 |xm LiChrocart Aluspher RP-select B (Merck)
Column: 125 X 4 5 |jim Aluspher RP-select B (Merck)
Mobile phase: Gradient. A was 12.5 mM NaOH in MeOH. B was 12.5 mM NaOH in water. A:B
10:90 for 5 min, to 90:10 over 15 min, maintain at 90:10 for 5 min, return to initial conditions
over 1 min, re-equilibrate for 5 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 17
OTHER SUBSTANCES
Extracted: alprenolol, amitriptyline, bromazepam, carbamazepine, chlordiazepoxide, chlorpro-
mazine, clonazepam, desipramine, diazepam, flunitrazepam, haloperidol, nordiazepam, nor-
triptyline, pindolol, zolpidem
Also analyzed: acebutolol, acetaminophen, alprazolam, amphetamine, atenolol, betaxolol, bro-
tizolam, caffeine, camazepam, captopril, chloroquine, clobazam, clomipramine, clothiapine, clo-
tiazepam, cloxazolam, cocaine, codeine, diclofenac, dihydralazine, dihydrocodeine, dihydroer-
gotamine, diphenhydramine, domperidone, doxepin, droperidol, ergotamine, ethyl loflazepate,
fenethylline, fluoxetine, flupentixol, flurazepam, furosemide, gliclazide, hydrochlorothiazide,
hydroxyzine, ibuprofen, imipramine, ketazolam, loprazolam, lorazepam, lormetazepam, ma-
protiline, medazepam, mepyramine, methadone, methaqualone, methyldopa, methylphenidate,
metoclopramide, metoprolol, mexiletine, mianserin, midazolam, minoxidil, morphine, nadolol,
nitrazepam, oxprenolol, papaverine, pentazocine, phenprocoumon, phenylbutazone, pipampe-
rone, piritramide, practolol, prazepam, prazosin, promazine, promethazine, propoxyphene, pro-
pranolol, prothipendyl, quinine, sotalol, sulpride, thioridazine, trazodone, triazolam, trimipra-
mine, tripelennamine, tyramine, verapamil, yohimbine
REFERENCE
Lambert,W.E.; Meyer,E.; De Leenheer,A.P. Systematic toxicological analysis of basic drugs by gradient elution
of an alumina-based HPLC packing material under alkaline conditions, J.Anal.ToxicoL, 1995, 19, 73-78.
SAMPLE
residue with 50 u,L MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLCVARIABLES
mL/min)
Detector: UV 236.9
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: cytosol incubations
Sample preparation: 2 mL Incubation + 5 mL chlorobutane:l,2-dichloroethane 80:20, shake for
15 min, centrifuge at 4000 g for 10 min. Remove the upper organic layer and evaporate it to
inject an 80 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeOH: 10 mM ammonium phosphate buffer 66:34, pH 5.8
Flow rate: 1.5
Detector: UV 230
OTHER SUBSTANCES
Extracted: diphenyl sulfoxide
KEYWORDS
rat; rabbit; nitrendipine is IS
REFERENCE
Lee,S.C; Renwick,A-G- Sulphoxide reduction by rat and rabbit tissues in vitro, Biochem.Pharmacol, 1995,49,
1557-1565.
SAMPLE
1 mL 1% aqueous formic acid. Add the microsomal incubation to the SPE cartridge, wash with
1 mL 1% aqueous formic acid, elute with 1 mL MeCN. Evaporate the eluate to dryness under
a stream of nitrogen in the dark at room temperature, reconstitute the residue in 1 mL MeCN:
0.5% phosphoric acid 10:90, inject a 50 jxL aliquot.
HPLCVARIABLES
Column: 250 X 4 10 |xm Nucleosil RP C8
Mobile phase: Gradient. MeCN:0.5% phosphoric acid from 40:60 to 60:40 over 12 min.
Flow rate: 1.4
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
KEYWORDS
rat; liver; SPE
REFERENCE
B6cker,R.H.; Preuss,E.; Peter,R. High-performance liquid chromatography of the metabolites of nitrendipine
and investigation into the metabolic pathways of this dihydropyridine, J.Chromatogr., 1990, 530, 206-211.
SAMPLE
Matrix: microsomal incubations, perfusate
Sample preparation: Extract using a 100 mg 1 mL Bond Elut C2 SPE cartridge, elute with
MeOH, evaporate eluate to dryness under a stream of nitrogen, reconstitute the residue in 100
fxL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 250 X 5 Hichrom HiRPB deactivated reverse-phase
Mobile phase: MeOH:25 mM pH 5.0 N,N,N\N'-tetramethylethylenediamine phosphate buffer
75:25
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Internal standard: felodipine
KEYWORDS
rat; liver; SPE
REFERENCE
Walker,D.K.; Humphrey,M.J.; Smith,D.A. Importance of metabolic stability and hepatic distribution to the
pharmacokinetic profile of amlodipine, Xenobiotica, 1994, 24, 243250.
SAMPLE
Matrix: perfusate
HPLC VARIABLES
Column: 100 X 8 5 (xm Novapak C18 radial compression
Flow rate: 2
Detector: UV 237
OTHER SUBSTANCES
Also analyzed: felodipine, nicardipine, nifedipine, nimodipine
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in mobile phase, inject a 20 (xL aliquot.
HPLCVARIABLES
Column: 75 X 4.6 3 |im XL octyl (Beckman)
Mobile phase: MeCN:water 44:56 containing 5 mM heptanesulfonic acid
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 6.2
REFERENCE
Greiner,P.O.; Angignard,D.; Cahn,J. High performance liquid chromatography of a new 1,4-dihydropyridine:
applications to pharmacokinetic study in dogs, J.Pharm.Sci., 1988, 77, 387-389.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 10 jxL aliquot of a solution in MeCN.
HPLCVARIABLES
Column: 100 X 4.6 CS-MP Spheri-5 cyano
Mobile phase: Gradient. MeCN:buffer from 10:90 to 40:60 over 10 min, re-equilibrate for 5 min.
(Buffer was 50 mM KH2PO4 adjusted to pH 3 with phosphoric acid.)
Flow rate: 1.5
Detector: UV 200, 272, 276, 280, 314
CHROMATOGRAM
Retention time: 11
OTHER SUBSTANCES
Simultaneous: nifedipine
REFERENCE
Logan,B.K.; Patrick,K.S. Photodegradation of nifedipine relative to nitrendipine evaluated by liquid and gas
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCNrIO mM pH 4.5 phosphate buffer 70:30
Flow rate: 2
Detector: UV 237
OTHER SUBSTANCES
Also analyzed: nicardipine, nifedipine, nimodipine, felodipine
KEYWORDS
buffers
REFERENCE
SAMPLE
Matrix: solutions
HPLCVARIABLES
Mobile phase: MeCN:water 60:40 containing 5.25 mIVL 1% phosphoric acid, 2.5 g/L ammonium
phosphate, and 10 mL/L 10% tetra-n-butylammonium hydroxide
Detector: UV 254
OTHER SUBSTANCES
Simultaneous: nilvadipine
KEYWORDS
protect from light
REFERENCE
Kobayashi,D.; Matsuzawa,T.; Sugibayashi,K; Morimoto,Y.; Kobayashi,M.; Kimura,M. Feasibility of use of sev-
rat and human skin, BioLPharm.BulL, 1993, 16, 254-258.
Nitrofurantoin
Merck Index: 6696
Lednicer No.: 1 230
SAMPLE
Sample preparation: Centrifuge cell culture at 4000 g for 30 min, filter (0.2 |xm Acrodisk), inject
an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 urn Prodigy ODS-3
Mobile phase: Gradient. A was MeCN containing 2 g/L ammonium acetate. B was water. A:B 5:
95 for 2 min, to 80:20 over 30 min, 80:20 for 5 min
Flow rate: 1
Detector: UV 376
CHROMATOGRAM
REFERENCE
Rafii,R; Hansen,E.B.,Jr. Isolation of nitrofurantoin-resistant mutants of nitroreductase-producing Clostridium
sp. Strains from the human intestinal tract, Antimicrob.Agents Chemother., 1998, 42, 11211126.
SAMPLE
Matrix: milk
HPLCVARIABLES
Mobile phase: MeCN: 10OmM aqueous sodium perchlorate:glacial acetic acid 28:72:0.5
Flow rate: 1
Detector: E, ESA Coulochem II, Model 5011 analytical cell, porous carbon electrode -600 V, Model
CHROMATOGRAM
Retention time: 2.2
OTHER SUBSTANCES
Extracted: furaltadone, furazolidone
KEYWORDS
cow; SPE
REFERENCE
Galeano Diaz,T.; Guiberteau CabanillasA-; Acedo Valenzuela,M.L; Correa,C.A.; Salinas,F. Determination of
Nitrofurazone
Merck Index: 6697
Lednicer No.: 1 229
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 fjim Symmetry C8 (Waters)
mIVmin)
Detector: UV 260.6
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil L C 18-DB
Mobile phase: MeCN:water 50:50 containing 1 mM ammonium acetate and 0.025% acetic acid
Flow rate: 0.6
Detector: MS, PESCIEX API I, ionspray interface 5500 V, OR 60 V, m/z 199, split the column
effluent so that 0.03 mL/min enters the MS
CHROMATOGRAM
Retention time: 5.2
KEYWORDS
REFERENCE
Draisci,R.; Giannetti,L.; Lucentini,L.; Palleschi,L.; Brambilla,G.; Serpe,L.; Gallo,P. Determination of nitrofuran
chromatography-ionspray mass spectrometry, J.Chromatogr.A, 1997, 777, 201-211.
SAMPLE
HPLCVARIABLES
Guard column: 10 X 4.6 ixBondapak C18
Column: 150 X 4.6 5 |xm Spherisorb ODS2 S5
Flow rate: 1
Detector: UV 362
CHROMATOGRAM
Retention time: 5.3
OTHER SUBSTANCES
Extracted: furazolidone, furaltadone
KEYWORDS
REFERENCE
Draisci,R.; Giannetti,L.; Lucentini,L.; Palleschi,L.; Brambilla,G.; Serpe,L.; Gallo,R Determination of nitrofuran
chromatography-ionspray mass spectrometry, J.ChromatognA, 1997, 777, 201-211.
SAMPLE
Sample preparation: Blend (Stomacher) 10 g homogenized tissue with 30 mL saline for 3 min,
centrifuge at 2000 g, mix 20 mL of the supernatant with 2 mL 1% sodium azide. Dilute 10 mL
homogenized egg with 10 mL saline, add 3 mL 10% sodium azide solution. Dialyze sample
using a Cuprophan membrane (10000-15000 dalton cut-off) against water pumped at 0.36-1.44
mL/min for 3-9 min, pass the water through column A, flush the column with pure water for
8 min, backflush the contents of column A onto column B with mobile phase, after 5 min remove
column A from the circuit, elute column B with mobile phase, monitor the effluent from column
B. To increase sensitivity a number of sample batches can be dialyzed before the contents of
column A are analyzed. (Caution! Sodium azide is carcinogenic, mutagenic, and highly toxic!
Do not discharge to the sink!)
HPLC VARIABLES
Column: A 60 X 4.6 37-50 |xm Bondapak C18/Corasil; B 250 X 4.6 5 |xm Hypersil ODS
Mobile phase: MeCN:100 mM pH 5 acetate buffer 20:80
Flow rate: 1
Detector: UV 365
CHROMATOGRAM
Retention time: 6
Limit of detection: 2 ng/g (tissue), 1 ng/g (eggs)
OTHER SUBSTANCES
Extracted: furaltadone, furazolidone, nitrofurantoin
KEYWORDS
protect from light; cow; muscle; dialysis; column-switching
REFERENCE
Aerts,M.M.; Beek,W.M.; Brinkman,U.A. On-line combination of dialysis and column-switching liquid chroma-
tography as a fully automated sample preparation technique for biological samples. Determination of ni-
trofuran residues in edible products, J.Chromatogn, 1990, 500, 453-468.
SAMPLE
Matrix: feed
Sample preparation: Grind feed to pass 20 mesh. 10 g Feed + 5 mL water, swirl, let stand for
5 min, add 50 mL DMF:water 95:5, shake vigorously for 15 s, let stand in the dark at room
temperature overnight, filter (paper). Add 15 mL of the filtrate to 5 g alumina (Alcoa F-20, 80-
200 mesh) in a 300 X 10 glass column, discard first several mL of eluate, collect remaining
eluate, inject an aliquot
HPLC VARIABLES
Guard column: 100 X 2 ixBondapak C18/Corasil
Column: 300 X 4 jxBondapak C18
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Extracted: furazolidone, carbadox
KEYWORDS
protect from light
REFERENCE
Thorpe,V.A. Sample preparation of carbadox, furazolidone, nitrofurazone, and ethopabate in medicated feeds
for high pressure liquid chromatography, J.Assoc.Off.Anal.Chem., 1980, 63, 981-984.
SAMPLE
Matrix: food
mL water. Homogenize (Tissuemizer) 5 g finely-chopped (by hand) shrimp and 20 mL MeCN
at medium speed for 45 s, centrifuge at 3000 rpm for 5 min, decant the supernatant. Add 30
mL hexane saturated with MeCN to the supernatant, shake for 30 s, discard the hexane layer.
Add 10 mL EtOH to the MeCN layer, evaporate under reduced pressure at 45 to 2-5 mL (until
liquid looks milky), add 2 mL EtOH, continue evaporation until there is 2 mL of a thick liquid,
add 2 mL EtOH, evaporate to dryness. Add 2 mL water to the residue, sonicate for 5 min, add
to the SPE cartridge, wash with 4 mL water, elute at ^ 3 mL/min with 5 mL MeCN. Evaporate
the eluate to dryness under a stream of nitrogen at ^45 (remove promptly when dry), recon-
stitute the residue in 1 mL mobile phase, filter (0.45 |xm), inject a 50 JJLL aliquot.
HPLC VARIABLES
Guard column: 20 X 4 5 |xm ODS Hypersil C18
Column: 200 X 4.6 5 |xm ODS Hypersil C18
Mobile phase: MeCN: 1% aqueous acetic acid 25:75
Flow rate: 1
Detector: UV 375
CHROMATOGRAM
Retention time: 3.7
OTHER SUBSTANCES
Extracted: furazolidone
KEYWORDS
SPE; shrimp
REFERENCE
Rupp,H.S.; Munns,R.K.; Long,A.R. Simultaneous determination of nitrofurazone and furazolidone in shrimp
(Penaeus vannamei) muscle tissue by liquid chromatography with UV detection, J.AOAC Int., 1993, 76,
1235-1239.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeOH:water 30:70, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 5 |xm Resolve spherical C18 (Waters)
Flow rate: 1
Detector: UV 305
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Simultaneous: carbadox, furazolidone
Noninterfering: pyrantel
KEYWORDS
protect from light
REFERENCE
Roybal,J.E.; Munns,R.K; Shimoda,W. Liquid chromatographic determination of carbadox residues in animal
feed, JAssoc.Off.Anal.Chem., 1985, 68, 653-657.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in chloroform at a concentration of 1 |xg/mL, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4 5 |xm Lichrospher RP-18
Mobile phase: MeCN: 10 mM sodium acetate 20:80, pH 5
Flow rate: 1.6
Detector: UV 365
CHROMATOGRAM
Retention time: 7.5
OTHER SUBSTANCES
Simultaneous: degradation products, carbadox, nitrofurantoin, furazolidone, furaltadone
REFERENCE
KaniouJ.; Zachariadis,G.; Kalligas,G.; Tsoukali,H.; Stratis,J. Separation and determination of carbadox, nitro-
furazone, nitrofurantoin, furazolidone, and furaltadone in their mixtures by thin layer and high perfor-
mance liquid chromatography, J.Liq.Chromatogr., 1994,17, 1385-1398.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Waring blender) 10 g muscle, liver, or kidney in 100 mL
EtOH for 5 min, let stand for 5 min, filter through 10 g Celite 545 on top of a sintered glass
filter, rinse blender with 100 mL EtOH and filter rinse. Add 25 mL 3.6% aqueous metaphos-
phoric acid to the combined filtrates, evaporate to 25 mL under reduced pressure at 45. Re-
move residue, rinse out flask with 5 mL hexane and 3 mL water, combine, centrifuge at 0 at
27000 g for 30 min, discard hexane, rinse surface with 5 mL hexane, discard hexane. Remove
aqueous layer, rinse out tube twice with 3 mL portions of water, combine, add 10 mL 1 M
KH2PO4, make up to 100 mL with water, extract three times for 5 min with 50 mL ethyl acetate.
Combine the extracts and dry them over 15 g anhydrous sodium sulfate, filter through glass
wool, evaporate to dryness under reduced pressure at 45. Take up residue in 3 mL ethyl
acetate and add to alumina column, rinse flask with 2 mL ethyl acetate and add rinse to
column. Elute with 20 mL EtOH:MeOH:ethyl acetate 10:10:80 and combine all the eluate.
Evaporate to dryness under reduced pressure at 45, reconstitute in 500 |xL mobile phase, inject
a 100 |xL aliquot. (Prepare alumina column by slurrying 1 g aluminum oxide (Baker) in 20 mL
ethyl acetate and adding to a 200 X 6 glass chromatographic column.)
HPLC VARIABLES
Guard column: Brownlee 10 ^m RP-GU MPLC C-8
Column: 250 X 4.6 Brownlee RP-10A C-8
Mobile phase: MeCN:EtOH:10 mM ammonium acetate 25:5:70, pH 6.8
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Retention time: 6.6
OTHER SUBSTANCES
Extracted: quinoxaline-2-carboxylic acid, furazolidone, carbadox, desoxycarbadox
KEYWORDS
protect from light; pig; muscle; liver; kidney
REFERENCE
MacIntosh,A.L; Neville,G.A. Liquid chromatographic determination of carbadox, desoxycarbadox, and nitro-
furazones in pork tissues, J.Assoc.Off.Anal.Chem., 1984, 67, 958-962.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Ultra-Turrax TP 18/2) 3 g ground muscle + 6.8 mL MeCN
for 6 s, centrifuge at 5000 rpm for 5 min. Remove 6.5 mL of the supernatant and add it to 2
mL 5 M NaCl, shake vigorously for 10 s, centrifuge at 3000 rpm for 2 min. Remove the organic
in 250 JJLL MeCNibuffer 20:80, add 1 mL hexane, mix (Whirlimixer), centrifuge for 4 min,
discard the hexane layer, filter (Costar Spin-X 0.22 |xm cellulose acetate) while centrifuging at
5600 g for 4 min, inject a 20 |xL aliquot of the nitrate. (Buffer was 20 mM sodium 1-heptane-
sulfonate and 10 mM Na2HPO4, pH adjusted to 6.0 with phosphoric acid.)
HPLC VARIABLES
Guard column: 20 X 4.6 5 |xm Supelcosil LC-ABZ
Column: 250 X 4.6 5 |xm Supelcosil LC-ABZ
Mobile phase: MeCNibuffer 25:75 (Buffer was 4.45 g sodium 1-heptanesulfonate and 9.5 g
Na3PO4.12H2O in 750 mL water, adjust pH to 2.5 with 5 M phosphoric acid, make up to 1 L
with water.)
Flow rate: 1
Detector: UV 365
CHROMATOGRAM
Retention time: 5.5
Internal standard: nitrofurazone
OTHER SUBSTANCES
Extracted: furazolidone
KEYWORDS
cow; muscle; nitrofurazone is IS
REFERENCE
Hormazabal,V.; Yndestad,M. Simple and rapid method of analysis for furazolidone in meat tissues by high-
performance liquid chromatography, J.Liq.Chromatogr., 1995, 18, 1871-1877.
Nizatidine
Merck Index: 6758
Lednicer No.: 4 95
SAMPLE
Sample preparation: Plasma. 100 jxL Plasma + 100 jxL 75 uM IS + 100 jxL 5 M NaOH + 5 mL
dichloromethane, shake for 10 min, centrifuge at 2000 rpm for 10 min. Evaporate 4 mL of the
organic phase to dryness under a stream of nitrogen. Dissolve residue in 100 |xL mobile phase,
inject a 50 |JLL aliquot. Tissue. Homogenize 500 mg liver with saline on ice for 1 min. Add 100
IxL 75 |AM IS, 100 |xL 0.5 M NaOH, and 5 mL dichloromethane, shake for 10 min, centrifuge
at 3000 rpm for 10 min. Evaporate 3 mL of the organic phase to dryness under a stream of
nitrogen. Dissolve residue in 100 |xL mobile phase, pass through a Ministar-RC 15 cartridge
(Sartorius, Germany), inject a 50 JULL aliquot.
HPLCVARIABLES
Guard column: 30 X 4.6 Senshu Pak ODS-1031 (Senshu Sciences, Japan)
Column: 250 X 4.6 Senshu Pak ODS -1251 (Senshu Sciences, Japan)
Mobile phase: MeCN:water 5:95 containing 5 mM NaH2PO4 and 5 mM tetramethylammonium
chloride
Flow rate: 1.5
Detector: UV 228
CHROMATOGRAM
Internal standard: cimetidine
Limit of detection: 50-100 |xg/mL (sic)
KEY WORDS
REFERENCE
Takedomi,S.; Matsuo,H-; Yamano,K; Yamamoto,K.; Iga,T.; Sawada,Y. Quantitative prediction of the interaction
of midazolam and histamine H2 receptor antagonists in rats, Drug Metab.Dispos., 1998, 26, 318-323.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 316.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Nomifensine
Merck Index: 6768
Lednicer No.: 4 146
SAMPLE
Matrix: blood
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 240
CHROMATOGRAM
KEYWORDS
done; amoxapine; quinupramine; opipramol; C3rproheptadine; brompheniramine; mefenidra-
REFERENCE
254-262.
SAMPLE
Matrix: bulk
Sample preparation: Mix 8 mg nomifensine maleate with 200 |xL trifluoroacetic anhydride, stir
at room temperature for 10 min, evaporate to dryness under a stream of nitrogen at 40,
reconstitute the residue in 2 mL mobile phase, inject a 5 |xL aliquot.
HPLC VARIABLES
Column: 250 X 3.6 5 jxm Spherisorb silica
Mobile phase: Chloroform:MeOH:water 100:3:0.15 containing 2 mM (+)-camphor-10-sulfonic
acid
Flow rate: 1
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 16 (D), 18 (L)
KEYWORDS
REFERENCE
Tsujiyama,T.; Tsuchiya,M.; Hamachi,Y.; Kuriki,T.; Fukunaga,T.; Suzuki,N. Ion-pair chromatographic separation
of nomifensine maleate enantiomers, Anal.Sci., 1989, 5, 285-288.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.7
OTHER SUBSTANCES
lol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, nor-
thipendyl, protriptyline, proxymetacaine, pseudoephedrine, p5n*imethamine, quinidine, qui-
REFERENCE
Nonoxynol-9
Molecular formula: C33H60Oi0
CAS Registry Na: 26027-38-3
Merck Index: 6772
SAMPLE
Matrix: blood, urine, vaginal fluid
Sample preparation: Urine. Centrifuge at 4000 rpm for 5 min, inject a 20 |xL aliquot of the
supernatant. Serum. Mix serum with an equal volume of THF, centrifuge at 4000 rpm for 5
min, remove an aliquot of the supernatant and mix it with an equal volume of THF, centrifuge
at 4000 rpm for 5 min, inject a 20 |xL aliquot of the supernatant. Vaginal fluid. Vortex swab
with 1 mL MeCN, let stand at room temperature for 15 min, weigh, vortex, remove swab,
centrifuge at 4000 rpm for 5 min, inject a 20 JJLL aliquot of the supernatant.
HPLCVARIABLES
Column: 250 X 4.6 10 |xm R-SIL-amine (Alltech)
Mobile phase: THF.MeCN 95:5
Flow rate: 1
CHROMATOGRAM
Retention time: 4
Limit of detection: 460 ng (vaginal fluid), 230 ng/mL (urine), 1.01 |xg/mL (serum)
KEYWORDS
place a 2 |xm filter at the column inlet
REFERENCE
Beck,G.J.; Kossak,D.; Saxena,S.J. A simple, sensitive assay for the spermicide nonoxynol-9 in biological fluids
by high-performance liquid chromatography, J.Pharm.ScL, 1990, 79, 1029-1031.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 0.2% solution, inject a 10 IJLL aliquot.
HPLC VARIABLES
Column: 300 X 75 PLgel 5 mixed-C (polymer Laboratories) in series with 300 X 78 Ultrastyragel
100 A styrene-divinylbenzene(Waters)
Mobile phase: THF
Flow rate: 1
Detector: RI
KEYWORDS
SEC
REFERENCE
Ysambertt,R; Cabrera,W.; Marquez,N.; Salager,J.L. Analysis of ethoxylated nonylphenol surfactants by high-
performance size-exclusion chromatography (HPSEC), J.Liq.Chromatogr., 1995, 18, 1157-1171.
SAMPLE
Matrix: vaginal lavage fluid
Sample preparation: 500 |xL Vaginal lavage fluid (water) + 500 jxL 25 |xg/mL 4-octylphenol in
mobile phase, vortex briefly, inject a 20 \LL aliquot.
Next Page
HPLC VARIABLES
Column: 250 X 4.6 10 |xm R-SIL-Amine (NH2) (Alltech) (Change 2 (xm filter in front of column
before each run.)
Mobile phase: MeCNrTHF 2:98
Flow rate: 1
CHROMATOGRAM
Retention time: 5.7
Internal standard: 4-octylphenol (4.0)
Limit of quantitation: 3.125 |xg/mL
REFERENCE
McPherson,J.L.; Nichols, J.H.; Barditch-Crovo,R; Hamzeh,F.M. Determination of the spermicide nonoxynol-9 in
vaginal lavage by high-performance liquid chromatography, J.Chromatogr.B, 1996, 677, 204-208.
Norepinephrine
CAS Registry No.: 51-41-2, 69815-49-2 (bitartrate monohydrate),
Merck Index: 6788
SAMPLE
Matrix: blood
Sample preparation: 20 mL Whole blood + 1 mL 20 mg/mL EDTA solution containing 10 mg/
mL sodium metabisulfite, mix, centrifuge at 4 at 4000 g for 10 min. Remove the plasma and
add concentrated perchloric acid until the concentration of perchloric acid is 400 mM, mix, let
stand in the cold for 15 min, centrifuge at 4 at 20000 g for 20 min. Adjust pH of 2 mL super-
natant to 7.0 0.2 with 500 mM KOH, add 400 |JLL 875 |xg/mL o-phthalaldehyde in pH 10.40
0.02 buffer (containing 2-mercaptoethanol ?), add 2 g NaCl, add 2 mL ethyl acetate, shake
for 1 min, centrifuge at 3400 g, repeat the extraction. Combine the organic layers and add
them to 2 mL 35 mM pH 10.0 0 . 1 Na2HPO4 buffer, shake for 1 min, centrifuge at 3400 g,
discard the aqueous layer, wash the ethyl acetate layer again with phosphate buffer. Reduce
the ethyl acetate volume to 100 jxL under a stream of nitrogen, inject a 10-50 JJLL aliquot.
HPLCVARIABLES
Column: 300 X 4 10 jxm ixBondapak phenyl
Mobile phase: Gradient. MeCN:25 mM pH 5.10 NaH2PO4 buffer 25:75 for 15 min then MeOH:
25 mM pH 5.10 NaH2PO4 buffer 45:55 (step gradient).
Flow rate: 1.5
CHROMATOGRAM
Retention time: 11
Internal standard: tyramine (44)
OTHER SUBSTANCES
Extracted: dopamine, serotonin
Previous Page
HPLC VARIABLES
Column: 250 X 4.6 10 |xm R-SIL-Amine (NH2) (Alltech) (Change 2 (xm filter in front of column
before each run.)
Mobile phase: MeCNrTHF 2:98
Flow rate: 1
CHROMATOGRAM
Retention time: 5.7
Internal standard: 4-octylphenol (4.0)
Limit of quantitation: 3.125 |xg/mL
REFERENCE
McPherson,J.L.; Nichols, J.H.; Barditch-Crovo,R; Hamzeh,F.M. Determination of the spermicide nonoxynol-9 in
vaginal lavage by high-performance liquid chromatography, J.Chromatogr.B, 1996, 677, 204-208.
Norepinephrine
CAS Registry No.: 51-41-2, 69815-49-2 (bitartrate monohydrate),
Merck Index: 6788
SAMPLE
Matrix: blood
Sample preparation: 20 mL Whole blood + 1 mL 20 mg/mL EDTA solution containing 10 mg/
mL sodium metabisulfite, mix, centrifuge at 4 at 4000 g for 10 min. Remove the plasma and
add concentrated perchloric acid until the concentration of perchloric acid is 400 mM, mix, let
stand in the cold for 15 min, centrifuge at 4 at 20000 g for 20 min. Adjust pH of 2 mL super-
natant to 7.0 0.2 with 500 mM KOH, add 400 |JLL 875 |xg/mL o-phthalaldehyde in pH 10.40
0.02 buffer (containing 2-mercaptoethanol ?), add 2 g NaCl, add 2 mL ethyl acetate, shake
for 1 min, centrifuge at 3400 g, repeat the extraction. Combine the organic layers and add
them to 2 mL 35 mM pH 10.0 0 . 1 Na2HPO4 buffer, shake for 1 min, centrifuge at 3400 g,
discard the aqueous layer, wash the ethyl acetate layer again with phosphate buffer. Reduce
the ethyl acetate volume to 100 jxL under a stream of nitrogen, inject a 10-50 JJLL aliquot.
HPLCVARIABLES
Column: 300 X 4 10 jxm ixBondapak phenyl
Mobile phase: Gradient. MeCN:25 mM pH 5.10 NaH2PO4 buffer 25:75 for 15 min then MeOH:
25 mM pH 5.10 NaH2PO4 buffer 45:55 (step gradient).
Flow rate: 1.5
CHROMATOGRAM
Retention time: 11
Internal standard: tyramine (44)
OTHER SUBSTANCES
Extracted: dopamine, serotonin
KEYWORDS
plasma; whole blood; pig; derivatization
REFERENCE
Davis,T.R; Gehrke,C.W.,Jr.; Williams,C.H.; Gehrke,C.W.; Gerhardt,K.O. Pre-column derivatization and high-
SAMPLE
Matrix: blood
Sample preparation: Chill 20 mL whole blood in ice water, add 1 mL reagent, invert several
time, centrifuge at 4 at 4000 g for 10 min. Remove the plasma and make it 0.4 M in perchloric
acid by adding concentrated perchloric acid, mix, let stand at 4 for 15 min, centrifuge at 4 at
20000 g for 20 min. Remove a 2 mL aliquot and adjust the pH to 7.0 0.2 with 0.5 M KOH,
add 400 jxL reagent, add 2 g NaCL, add 2 mL ethyl acetate, shake for 1 min, centrifuge at
3400 g, repeat the extraction. Combine the organic layers, add 2 mL 35 mM pH 10.0 0 . 1
Na2HPO4 buffer, shake for 1 min, centrifuge at 3400 g, repeat the wash, evaporate the ethyl
acetate layer to 100 |xL with a stream of nitrogen, inject a 10-50 |JLL aliquot. (Reagent was 875
jutg/mL o-phthalaldehyde and 2-mercaptoethanol in pH 10.40 0.2 potassium borate buffer.)
HPLCVARIABLES
Column: 300 X 4 10 |xm jxBondapak phenyl
Mobile phase: Gradient. MeCN:25 mM pH 5.10 NaH2PO4 25:75 for 15 min, then MeOH:25 mM
pH 5.10 NaH2PO4 45:55 for 35 min (step gradient).
Flow rate: 1.5
CHROMATOGRAM
Retention time: 12
Limit of detection: <0.5 ng/mL
OTHER SUBSTANCES
Extracted: dopamine, histamine, octopamine, serotonin, tyramine
KEYWORDS
pig; whole blood; derivatization
REFERENCE
Davis,T.R; Gehrke,C.W.,Jr.; Williams,C.H.; Gehrke,C.W.; Gerhardt,K.O. Pre-column derivatization and high-
SAMPLE
Matrix: blood
Sample preparation: Plasma. Prepare a SPE column by adding 500 |xL of a 20% suspension of
19-40 |xm Toyopak SP (strong cation-exchange sulfopropyl resin, Na+ (Toyo Soda)) in water to
of water, wash with three 1 mL portions of buffer. 500 jxL Plasma + 25 |xL 10 nM isoproterenol
+ 500 |xL buffer, mix, add to the SPE column, wash with two 5 mL portions of water, wash
with 1 mL MeCN:water 50:50, elute with 300 |xL 600 JJLM potassium ferricyanide in 600 mM
KChMeCN 50:50, add 50 |xL reagent to the eluate, heat at 37 for 40 min, cool in ice-water,
inject a 100 JJLL aliquot. Urine. 10 jxL Urine + 1 mL MeCN:500 mM KCl 60:40 + 10 |xL 500 nM
isoproterenol + 10 |xL 75 mM potassium hexacyanoferrate(III) + 100 |xL reagent, heat at 37
for 40 min, inject a 100 |xL aliquot (J. Chromatogr. 1986, 380, 229). (Prepare buffer by mixing
8 volumes 250 mM LiOH in 200 mM phosphoric acid with 1 volume 200 mM phosphoric acid,
pH 5.8. Prepare reagent by dissolving 212 mg 1,2-diphenylethylenediamine in 10 mL 100 mM
HCl, pH 6.7.)
HPLCVARIABLES
Column: 150 X 4.6 5 juim TSK-gel ODS-120T (Tbyo Soda)
Flow rate: 1
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Extracted: dopamine, epinephrine
KEY WpRDS
REFERENCE
Mitsui,A.; Nohta,H.; Ohkura,Y. High-performance liquid chromatography of plasma catecholamines using 1,2-
diphenylethylenediamine as precolumn fluorescence derivatization reagent, J.Chromatogr., 1985, 344, 6 1 -
70.
SAMPLE
Matrix: blood
mM pH 7 phosphate buffer. 1 mL Plasma + 30 |xL 80 ng/mL N-methyldopamine, apply to
column, wash with 5.5 mL water (A), elute with 3 mL 0.5 M perchloric acid. Collect eluate,
add 2 mL 1.5 M pH 9.3 Tris buffer containing 60 mM EDTA, add 20 mg alumina, vortex for 2
min, discard supernatant, add 2 mL water to alumina, mix, centrifuge at 3000 g for 3 min,
repeat water wash, remove as much water as possible, elute catecholamines from alumina with
100 jxL 100 mM acetic acid with vortexing for 2 min, centrifuge, inject 25 jxL aliquot of super-
natant. (Wash water A contains levodopa, carbidopa, DOPAC, and O-methyldopa.)
HPLCVARIABLES
Flow rate: 0.9
trode -0.30 V
CHROMATOGRAM
Retention time: 8
OTHERSUBSTANCES
Simultaneous: epinephrine, dopamine
KEYWORDS
plasma; SPE
REFERENCE
plasma of parkinsonian patients under L-dopa therapy, J.Chromatogr., 1988, 459, 341-349.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 250 (xL 1 ng/mL a-methylnorepinephrine + 1 mL buffer
+ 5 mL n-heptane containing 4.6 mM tetraoctylammonium bromide and 10 mL/L 1-octanol,
organic phase and add it to 2 mL 1-octanol and 200 (JLL 80 mM acetic acid, shake, centrifuge
in dry ice/acetone. Remove the organic layer and add it to 2 mL 1-octanol and 150 (xL 80 mM
organic layer. Thaw the aqueous layer and add it to 250 JJLL MeCN, 50 |xL 1.75 M pH 7.05
bicine, and 100 |xL 100 mM 1,2-diphenylethylenediamine in 100 mM HCl, add 20 |JLL 20 mM
a 100 jxL aliquot. (Buffer was 2 M pH 8.6 ammonia/ammonium chloride buffer containing 8.9
diphenylethylenediamine from toluene:light petroleum (bp 60-80) 10:90, dry overnight at 60.)
HPLC VARIABLES
Column: 100 X 4.6 3 (xm Cp MicroSpher C18 (Chrompack)
Flow rate: 1
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Extracted: dihydroxybenzylamine, dopamine, epinephrine, isoproterenol
KEYWORDS
REFERENCE
van der Hoorn,F.A.J.; Boomsma,R; Man in 't Veld,A.J.; Schalekamp,M.A.D.H. Determination of catecholamines
17-28.
SAMPLE
Matrix: blood
Sample preparation: Pack a 65 X 15 SPE column with 50 mg WA-4 alumina (Sigma). Add 500
|xL plasma to the SPE column, add 1 mL buffer, rotate for 15 min, wash three times with water
(aspirating to dryness each time), centrifuge to dryness, add 200 fxL 100 mM pH 1.2 perchloric
acid, mix, let stand for 15 min, centrifuge the SPE column at 1000 g for 3 min, inject an aliquot
of the effluent. (Buffer was 45 g Tris and 5 g EDTA in 200 mL water, pH adjusted to 8.6 with
concentrated HCl.)
HPLCVARIABLES
Guard column: 50 X 4.6 5 |xm reversed-phase
Column: 250 X 4.6 5 |xm ODS Spherisorb
Mobile phase: Buffer contained 1.4% monochloroacetic acid, 0.47% NaOH, and 0.075% EDTA,
finally pH adjusted to 3.0 with NaOH or monochloroacetic acid and 6 mg% sodium octylsulfate
added.
Flow rate: 1
Detector: E, Bioanalytical Systems LC-4B, TL-5 transducer with a glassy carbon electrode, +650
mV, 1 nA, Ag/AgCl reference electrode
OTHER SUBSTANCES
Extracted: epinephrine, dopamine
KEY WORDS
plasma; rabbit; human; SPE
REFERENCE
Ganhao,M.E; Hattingh,J.; Hurwitz,M.L.; Pitts,N.I. Evaluation of a simple plasma catecholamine extraction
procedure prior to high-performance liquid chromatography and electrochemical detection, J.Chromatogr.,
1991, 564, 55-66.
SAMPLE
Matrix: blood
Sample preparation: Plasma. 1 mL Plasma + 125 (xL 2 ng/mL a-methylnorepinephrine + 1 mL
buffer + 5 mL n-heptane containing 4.6 mM tetraoctylammonium bromide and 10 mL/L 1-
octanol, shake for 2 min, centrifuge at 1000 g for 5 min, freeze in dry ice/acetone. Remove the
organic phase and add it to 2 mL 1-octanol (saturated with 80 mM acetic acid) and 200 |xL 80
mM acetic acid, shake, centrifuge at 1000 g for 5 min. Freeze the aqueous layer and remove
the organic layer. Add 1 mL 10 mM HCl, 1 mL buffer, and 5 mL n-heptane containing 4.6 mM
tetraoctylammonium bromide and 10 mL/L 1-octanol to the aqueous phase. Shake, centrifuge,
freeze, remove the organic layer and add it to 2 mL 2 M pH 8.6 ammonia-ammonium chloride
buffer containing 13.4 mM EDTA (but no complex). Freeze, remove the organic layer and add
it to 2 mL 1-octanol and 150 |xL 80 mM acetic acid, shake, centrifuge at 1000 g for 5 min.
Freeze, remove the organic layer and add the aqueous layer to 200 |xL MeCN, 50 |xL 1.75 M
pH 6.95 bicine buffer containing 1% EDTA, 100 |xL 100 mM 1,2-diphenylethylenediamine in
100 mM HCl, and 20 |xL 20 mM potassium ferricyanide in water. Heat at 37 in the dark for
1 h, inject a 75 jxL aliquot (keep it in the dark in the autosampler). Urine. 100 |xL Urine + 1
mL 10 mM HCl + 125 jxL 40 ng/mL a-methylnorepinephrine + 1 mL buffer + 5 mL n-heptane
containing 4.6 mM tetraoctylammonium bromide and 10 mL/L 1-octanol, shake for 2 min,
centrifuge at 1000 g for 5 min, freeze in dry ice/acetone. Remove the organic phase and add it
to 2 mL 1-octanol (saturated with 80 mM acetic acid) and 200 |xL 80 mM acetic acid, shake,
centrifuge at 1000 g for 5 min. Freeze the aqueous layer and remove the organic layer. Add 1
mL 10 mM HCl, 1 mL buffer, and 5 mL n-heptane containing 4.6 mM tetraoctylammonium
bromide and 10 mL/L 1-octanol to the aqueous phase. Shake, centrifuge, freeze, remove the
organic layer and add it to 2 mL 1-octanol and 150 |xL 80 mM acetic acid, shake, centrifuge at
1000 g for 5 min. Freeze, remove the organic layer and add the aqueous layer to 200 JJLL MeCN,
50 |xL 1.75 M pH 6.95 bicine buffer containing 1% EDTA, 100 jxL 100 mM 1,2-diphenylethyl-
enediamine in 100 mM HCl, and 20 |xL 20 mM potassium ferricyanide in water. Heat at 37
in the dark for 1 h, inject a 50 (xL aliquot (keep it in the dark in the autosampler). (Buffer was
a 2 M pH 8.6 ammonia-ammonium chloride buffer containing 8.9 mM diphenyl borate-etha-
nolamine complex and 13.4 mM EDTA.)
HPLC VARIABLES
Column: 100 X 4.6 3 |xm PhaseSep C18 ODS2
100 over 0.5 min, stay at 0:100 for another 4.5 min. (After the last sample flush column with
60 mL MeCN:MeOH:water 70:10:20.)
Flow rate: 1
CHROMATOGRAM
Retention time: 2
Internal standard: a-methylnorepinephrine (2.5)
OTHER SUBSTANCES
Simultaneous: epinephrine, dopamine, epinine
KEYWORDS
REFERENCE
Boomsma,R; Alberts,G.; van der Hoorn,F.A.J.; Man in 't Veld,A.J.; Schalekamp,M.A.D.H. Simultaneous deter-
mination of free catecholamines and epinine and estimation of total epinine and dopamine in plasma and
urine by high-performance liquid chromatography with fluorimetric detection, J.Chromatogr., 1992, 574,
109-117.
SAMPLE
Matrix: blood
Sample preparation: 100 \LL Plasma + 5 mg alumina + 10 JJLL 10 \xM IS in 10 mM perchloric
acid + 100 jxL pH 8.7 Tris-HCl buffer, stir for 10 min, centrifuge at 3000 g for 1 min, discard
the supernatant. Wash the alumina with two 500 |xL portions of water, add 100 ^L 100 mM
perchloric acid, mix for 1 min, centrifuge at 3000 g for 1 min, inject a 50 jxL aliquot of the
supernatant. (Heat 20 g alumina (WA-4, Sigma) with 200 mL 2 M HCl at 100 for 1 h with
gentle mixing, decant the supernatant, wash with twenty 200 mL portions of water, filter (Toyo
Roshi No. 2 paper), dry at 120 overnight.)
HPLC VARIABLES
Column: 150 X 4.6 catecholpak (JASCO)
Mobile phase: MeCN:50 mM pH 3.20 potassium acetate:50 mM pH 3.20 potassium phosphate
buffer 3:92.15:4.85 containing 1 mM sodium hexanesulfonate
Flow rate: 0.5
Detector: Chemiluminescence (Kenko filter Y-46) following post-column reaction. The column
effluent mixed with reagent 1 pumped at 0.25 mL/min and the mixture flowed through a 15
m x 0.5 mm i.d. knitted PTFE coil at 80. The effluent from the coil mixed with reagent 2
pumped at 1.4 mL/min and this mixture flowed to the detector. (Reagent 1 was 105 mM ethyl-
enediamine (semiconductor grade) and 175 mM imidazole in MeCNiEtOH 90:10. Reagent 2
was 0.25 mM bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (Wako), 150 mM hy-
drogen peroxide, and 110 mM trifluoroacetic acid in dioxane:ethyl acetate 50:50 (Caution! Di-
oxane is a carcinogen!).)
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
KEYWORDS
human; rat; plasma; SPE; post-column reaction
REFERENCE
Higashidate,S.; Imai,K. Determination of femtomole concentrations of catecholamines by high-performance
liquid chromatography with peroxyoxalate chemiluminescence detection, Analyst, 1992, 117, 1863-1868.
SAMPLE
Matrix: blood
Sample preparation: Filter (Ultrafree-MC with 10000 molecular mass cut-off, Millipore) 100
IxL plasma while centrifuging at 15000 g for 15 min. Mix 50 JULL ultrafiltrate and 10 JJLL 140
ng/mL 3-methoxytyramine in Ringer solution, inject a 5 (xL aliquot.
HPLC VARIABLES
Column: 150 X 1 5 |xm Inertsil-2 ODS
Mobile phase: MeCN:THF:water 6:0.8:93.2 containing 0.48 g/L sodium 1-octanesulfonate, 2 g/L
NaH2PO4, 8.82 g/L sodium citrate, 10 mg/L EDTA, and 1 mL/L diethylamine, pH adjusted to
3.2 with concentrated orthophosphoric acid.
Flow rate: 0.06
Injection volume: 5
Detector: E, Bioanalytical Systems BAS-4C, glassy carbon working electrodes, upstream +0.75
V, downstream +0.05 V (measuring electrode), Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 2.5
OTHER SUBSTANCES
Extracted: dopamine, epinephrine, 3,4-dihydroxyphenylacetie acid, serotonin, 5-hydroxyindole-
acetic acid, homovanillic acid
KEYWORDS
plasma; microbore; rat; ultrafiltrate
REFERENCE
Cheng,F.-C; Yang,L.-L.; Kuo,J.-S.; Yang,M.C.M.; Yu,P.-C. Rapid assay of the monoamine content in small vol-
umes of rat plasma, J.Chromatogr.B, 1994, 653, 9-16.
SAMPLE
at 110 for 24 h, evaporate to dryness, reconstitute with 50-200 ^L 0.1% HCl containing 0.2%
3,3'-thiodipropionic acid at 20000 rpm for 2 min, sonicate for ^30 min, centrifuge at 5000 g for
with 200 |xL 100 mM HCl containing 0.2% 3,3'-thiodipropionic acid) 3 mL supernatant while
centrifuging at 3500 g for 1 h. Mix 20 jxL deproteinized sample (or 10 jxL peptide hydrolysate)
with 180 JJLL buffer, vortex, add 200 JJLL reagent, mix, heat at 70 for 15 min with mixing at 1
min and 12 min, cool in an ice bath for 5 min, centrifuge at 10000 g for 10 s, add 400 |xL
diluent, mix thoroughly, centrifuge at 15000 g for 5 min, inject a 10 |xL aliquot of the super-
HPLCVARIABLES
Column: 150 X 3.9 4 ^m Novapak C18
to pH 6.55 with phosphoric acid. B was MeCN:water 80:20. A:B 92:8 for 2 min, to 80:20 over
Flow rate: 1
Detector: UV 436
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: amino acids dopamine, epinephrine, histamine, taurine
KEYWORDS
REFERENCE
Krause,L; Bockhardt,A.; Neckermann,H-; Henle,T.; Klostermeyer,H- Simultaneous determination of amino
SAMPLE
Sample preparation: Serum. Add 1 mL serum to 100 mg activated aluminum oxide suspended
in 1 mL pH 8.7 Tris-HCl buffer, stir, let stand for 10 min. Discard the supernatant and wash
the solid three times with 5 mL portions of water, wash the solid with 3 mL MeOH, dry under
reduced pressure, elute with 3 mL 4 M acetic acid. Evaporate the eluate to dryness under
reduced pressure, reconstitute the residue with 90 |xL water, inject a 10 (xL aliquot. Urine. 5
mL Urine + 5.3 mL 2 M HCl, heat at 100 for 20 min, cool to room temperature, add 1 mL 50
mM disodium EDTA, adjust the pH to 8.5 with dilute ammonia, add 500 mg 200 mesh alu-
minum oxide (Wako), shake for 10 min, filter, wash the solid with 10 mL water, elute with 5
mL 300 mM acetic acid, inject an aliquot of the eluate.
HPLC VARIABLES
Column: 250 X 3.6 10-25 |xm Hitachi 3011 C resin
Mobile phase: 50 mM K2HPO4 containing 0.05% phosphoric acid
Flow rate: 0.6
2-cyanoacetamide in water pumped at 0.5 mL/min and with buffer pumped at 1 mL/min and
the mixture flowed through a 5 m X 0.5 mm ID PTFE coil at 100 1 to the detector. (Buffer
was 600 mM boric acid containing 750 mM KOH.)
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
KEYWORDS
post-column reaction; serum; SPE
REFERENCE
Honda,S.; Takahashi,M.; Araki,Y; Kakehi,K. Postcolumn derivatization of catecholamines with 2-cyanoacetam-
ide for fluorimetric monitoring in high-performance liquid chromatography, J.Chromatogr., 1983,274, 45-
52.
SAMPLE
cartridge (Tosoh) with 10 mL water. Plasma. 700 |xL Plasma + 50 JJLL 700 nM 3,4-dihydroxy-
benzylamine + 350 |JLL 2 M perchloric acid, mix, centrifuge at 4 at 1000 g for 15 min. Remove
a 700 jxL aliquot of the supernatant and add it to 30 (xL 2 M potassium carbonate, centrifuge
at 4 at 1000 g for 5 min. Add a 500 jxL aliquot of the supernatant to the SPE cartridge, wash
with 1 mL water, wash with 500 |xL EtOH:water 50:50, wash with 5 mL water, elute with 500
(JLL 2 M sodium perchlorate, filter (0.2 |xm), inject a 50 jxL aliquot of the nitrate. Urine. Acidify
urine collected over 24 h with 10 mL 6 M HCl. 500 |xL Urine + 25 |xL 10 jxM 3,4-dihydroxy-
benzylamine + 25 |xL 40 |xM ferulic acid + 500 |JLL 1 M perchloric acid, mix, centrifuge at 4 at
1000 g for 15 min. Remove a 700 (xL aliquot of the supernatant and add it to 30 |xL 2 M
potassium carbonate, centrifuge at 4 at 1000 g for 5 min, add a 500 |xL aliquot of the super-
wash with 5 mL water, elute with 500 (JLL 2 M sodium perchlorate, filter (0.2 (xm), inject a 50
(JLL aliquot of the filtrate.
HPLCVARIABLES
Column: 150 X 4.6 5 Jim TSK-gel ODS-80TM (Tbsoh)
Mobile phase: Gradient. A was buffer. B was MeCN:MeOH:buffer 8:12:80, pH 3.1. A:B 100:0 for
Chromatogr. 1989, 467, 237).
Flow rate: 1
237).
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Extracted: dopamine, epinephrine, levodopa, metanephrine, 3-methoxytyramine
KEYWORDS
REFERENCE
Nohta,H.; Yamaguchi,E.; Ohkura,Y.; Watanabe,H. Measurement of catecholamines, their precursor and metab-
SAMPLE
2, centrifuge at 4 at 4000 g for 3 min. Remove 200 |xL of the supernatant and add it to 100
IxL 80 ng/mL N-methyldopamine, evaporate to dryness under vacuum, reconstitute in 200 |xL
mobile phase, inject a 5-20 JJLL aliquot. Urine. 1 mL Urine + 50 mL water, inject a 10 |JLL aliquot.
with 50 mL water, inject a 10 |xL aliquot.
HPLC VARIABLES
Flow rate: 1
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Extracted: methyldopa, epinephrine, dopamine, dihydroxyphenylacetic acid, 3-O-methylmethyl-
dopa, homovanilic acid
KEYWORDS
REFERENCE
541, 285-296.
SAMPLE
(Tosoh) with 10 mL water and 2 mL 200 mM pH 5.0 sodium phosphate buffer. Plasma. 700 \xL
Plasma + 30 JJIL 700 nM isoproterenol + 50 jxL 7 jxM 3,4-dihydroxyphenylpropanoic acid + 350
|xL 2 M perchloric acid, mix, centrifuge at 4 at 1000 g for 15 min. Remove a 700 |xL aliquot
of the supernatant and adjust the pH to 1.5-2.0 with about 150 (xL 2 M potassium carbonate,
mL water, elute with 300 |xL MeOH:2 M sodium perchlorate 7:93, filter (cellulose acetate mem-
brane), inject a 100 |xL aliquot of the nitrate. Urine. Collect human urine for 24 h in the
presence of 10 mL 6 M HCl. 500 |xL Urine + 10 |xL 15 JXM isoproterenol + 25 JJLL 800 |xM 3,4-
for 15 min. Remove a 700 jxL aliquot of the supernatant and adjust the pH to 1.5-2.0 with
about 130 |JLL 2 M potassium carbonate, centrifuge at 4 at 1000 g for 5 min, add the super-
wash with 5 mL water, elute with 500 |xL 1.5 M KCl in MeOH: 100 mM HCl 7:93, filter (cellulose
acetate membrane), inject a 100 JJLL aliquot of the filtrate.
HPLC VARIABLES
Flow rate: 0.8
meso-l,2-diphenylethylenediamine in EtOH:water 70:30 containing 130 mM sodium
methylate.)
CHROMATOGRAM
Retention time: 16
Limit of detection: 0.4-0.5 nM
OTHER SUBSTANCES
Extracted: dopamine, epinephrine, levodopa, metanephrine, 3-methoxytyramine, normetanep-
hrine
KEYWORDS
REFERENCE
Jeon,H.-K.; Nohta,H.; Ohkura,Y. High-performance liquid chromatographic determination of catecholamines
chemical oxidation followed byfluorescencereaction, Anal.Biochem., 1992, 200, 332-338.
SAMPLE
alumina + 200 |xL 1 M pH 8.6 Tris-EDTA buffer, mix for 10 min, discard plasma. Wash the
alumina three times with 3 mL water and dry it. Add 125 |xL 500 mM phosphoric acid, after
1 min inject a 100 |JLL aliquot. (Ann. CHn. Biochem. 1985, 22, 194-203)
HPLC VARIABLES
Column: 250 X 4.5 5 |jim Ultratechsphere
mL MeOH
Flow rate: 1
-0.35 V
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: levodopa, metanephrine, epinephrine, 3-methoxytyrosine, normetanephrine, di-
hydroxyphenylacetic acid, dopamine
KEYWORDS
plasma
REFERENCE
Clin.Chem., 1993, 39, 629-634.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
Retention time: 2.8
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Centrifuge at 20000 g for 5 min, dilute the supernatant 10-50-fold, inject
a 30 uL aliquot.
HPLC VARIABLES
Column: 125 X 3 3 |xm Nucleosil 100C18
Mobile phase: MeOH:buffer 5:95, pH adjusted to 3.0 with 5 M NaOH (Buffer was 50 mM citric
acid containing 30 mM phosphoric acid, 0.75 mM octylsulfate, and 0.5 mM EDTA.)
Flow rate: 0.7
Detector: E, Waters 460, glassy carbon electrode
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: epinephrine
REFERENCE
Ghindilis,A.L.; Michael,N.; Makower,A. A new sensitive and simple method for detection of catecholamines
from adrenal chromamn cells, Pharmazie, 1995, 50, 599-600.
SAMPLE
Matrix: dialysate
Sample preparation: Mix 30 uL dialysate + 5 JJLL 200 mM perchloric acid, inject a 20 (xL aliquot.
HPLC VARIABLES
Column: 80 X 4.6 3 |xm HR-80 C18 (ESA)
Mobile phase: MeCN:MeOH:buffer 15:13:72 (Buffer was 75 mM NaH2PO4, 1.5 mM sodium do-
decyl sulfate, 100 |xL/L triethylamine, and 20 |xm EDTA, adjusted to pH 5.6.)
Flow rate: 1
Detector: E, ESA Coulochem II, Model 5014 Microdialysis Cell with El -175 mV and E2 +175
mV, Pd reference electrode, Model 5020 Guard Cell EGC +300 mV
CHROMATOGRAM
Limit of detection: 400 fg
OTHER SUBSTANCES
Extracted: dopamine, epinephrine, serotonin
KEYWORDS
rat; brain; pharmacokinetics; use PEEK tubing and sample loop
REFERENCE
Gariepy,K.C; Bailey,B.; Yu,J.; Maher,T.; AcworthJ.N. Simultaneous determination of norepinephrine, dopa-
mine, and serotonin in hippocampal microdialysis samples using normal bore high performance liquid chro-
matography: Effects of dopamine receptor agonist stimulation and euthanasia, J.Liq.Chromatogr., 1994,
17, 1541-1556.
SAMPLE
Matrix: dialysate
Sample preparation: 20 JJLL Dialysate (Ringer's solution) + 50 |xL 80 pg/mL 3,4-dihydroxyben-
zylamine in 2% acetic acid + 5 mg acid-washed alumina + 1 mL 1 M pH 8.6 Iris buffer con-
taining 0.2% disodium EDTA, shake for 15 min, wash the alumina 3 times with water. Place
the alumina in an Ultrafree C3 microfilter tube (Millipore), dry by centrifuging at 600 g for 5
min, elute with 60 |xL 2% acetic acid, inject a 50 (xL aliquot of the eluate.
HPLC VARIABLES
Guard column: 5 x 4 AC-ODS (Eicom)
Column: 150 X 2.1 Eicompak CA-50DS (Eicom)
Mobile phase: MeOH: 100 mM pH 6.1 phosphate buffer 10:90 containing 600 jxg/mL sodium 1-
octanesulfonate
Flow rate: 0.25
Detector: E, Eicom ECD-300, graphite electrode + 400 mV, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 7
KEYWORDS
SPE; cat
REFERENCE
Yamazaki,T.; Akiyama,T.; Shindo,T. Routine high-performance liquid chromatographic determination of myo-
cardial interstitial norepinephrine, J.Chromatogr.B, 1995, 670, 328-331.
SAMPLE
HPLCVARIABLES
acid.)
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: epinephrine, levonordefrin, isoproterenol, phenylephrine, metaraminol, impuri-
ties
KEYWORDS
REFERENCE
SAMPLE
HPLC VARIABLES
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: carbidopa, dopamine, epinephrine, hydrochlorothiazide, isoproterenol, levodopa,
methyldopa, phenylephrine
KEYWORDS
REFERENCE
Villanueva Camanas,RM; Sanchis Mallols,J.M.; Torres Lapasi6,J.R.; Ramis-Ramos,G. Analysis of pharmaceu-
SAMPLE
Matrix: perfusate
Sample preparation: 30 |xL Perfusate (artificial CSF) + 10 JXL 200 mM perchloric acid. Mix a
25 |xL aliquot with 12.5 |xL reagent, let stand for 2 min, inject an aliquot. (Prepare a stock
10 fjiM EDTA, allow to stand for 24 h before use.)
HPLC VARIABLES
Column: two columns 150 X 4.6 5 jim M.S. Gel C18 (ESA)
Mobile phase: MeOH:buffer 8:92 adjusted to pH 3.0 with phosphoric acid (Buffer was 54 mM
Flow rate: 1.2
potential 70 mV
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: apomorphine, dopamine, hydralazine, isoproterenol, methoxamine, morphine,
phenylephrine
KEYWORDS
rat; derivatization
REFERENCE
AcworthJ.N.; Yu,J.; Ryan,E.; Gariepy,K.C; Gamache,P.; Hull,K; Maher,T. Simultaneous measurement of
705.
SAMPLE
Matrix: perfusate
Sample preparation: Perfusate + 20 ^L 25 pM dihydroxybenzylamine + 15 mg activated alu-
mina + pH 8.3-8.5 Tris buffer containing 1% EDTA, mix. Remove the alumina and wash it with
water, add 100 |xL 200 mM perchloric acid, centrifuge, store supernatant on ice, inject a 20 |xL
aliquot.
HPLCVARIABLES
Column: Lichrospher 60 RP-select B C18
Mobile phase: MeCN:buffer 1.5:98.5 (Buffer was 100 mM NaH2PO4 containing 1.4 mM sodium
1-octanesulfonate and 0.2 mM EDTA, pH 2.5.)
Flow rate: 0.8
Detector: E, BAS LC3A, 0.8 V
CHROMATOGRAM
Retention time: 10
KEYWORDS
SPE
REFERENCE
Forray,M.L; Andre"s,M.E.; Bustos,G.; Gysling,K. Regulation of endogenous noradrenaline release from the bed
nucleus of stria terminalis, Biochem.Pharmacol., 1995, 49, 687-692.
SAMPLE
Matrix: solutions
Sample preparation: Dilute a few JJLL of a <1 mM solution to 20 (xL with 50 mM pH 8.0 phos-
phate or borate buffer, add 10 u>L 2 mg/mL (?) fluorescamine in acetone with vigorous shaking,
inject an aliquot.
HPLC VARIABLES
Column: 500 X 3 Hitachi 3011 gel glass column
Mobile phase: MeOH:100 mM pH 8.0 Tris-HCl buffer 70:30
Flow rate: 0.72
Detector: F primary filter Corning No. 7-51, secondary filter No. 4-7116
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Simultaneous: dopamine
Also analyzed: 3-methoxytyramine, normetanephrine
KEY WpRDS
derivatization
REFERENCE
Imai,K. Fluorimetric assay of dopamine, norepinephrine and their 3-O-methyl metabolites by using fluores-
camine, J.Chromatogr., 1975, 105, 135-140.
SAMPLE
Matrix: solutions
Sample preparation: 200 |xL 5 jxg/mL Amine solution + 300 |xL 100 mM pH 8.0 phosphate
buffer + 300 |xL 200 |xg/mL fluorescamine in acetone + 200 |xL water, mix, saturate with NaCl,
add 500 jxL ethyl acetate, shake for 1 min, inject a 50 |xL aliquot of the organic phase.
HPLC VARIABLES
Column: 250 X 2 10 |xm LiChrosorb Si 60-10
Mobile phase: Benzene:dioxane:acetic acid 76:22:2 (Caution! Benzene and dioxane are
carcinogens!)
Flow rate: 0.5
Detector: F ex 325-385 (filter) em 451
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Simultaneous: dopamine
KEY WpRDS
REFERENCE
Schwedt,G. Hochdruck-Flussigkeits-chromatographische Analyse der Katecholamine Dopamin und Noradren-
alin als Fluorescaminderivate, J.Chromatogr., 1976, 118, 429-432.
SAMPLE
Matrix: solutions
Sample preparation: Dilute with 5% dextrose, inject a 15 |xL aliquot.
HPLC VARIABLES
Flow rate: 1.6-2.0
CHROMATOGRAM
OTHER SUBSTANCES
Interfering: dopamine
REFERENCE
catecholamines with aminophylline in intravenous solutions, J.Pharm.Sci., 1982, 71, 956-958.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Nucleosil 5-C18
Mobile phase: 50 mM Potassium perchlorate containing 250 |xL/L 3% copper acetate in water
and 10 g/L sodium acetate, pH adjusted to 4.45 with acetic acid
Flow rate: 1
the reactor then through a 3.5 m X 0.8 mm ID coil of PTFE tubing at 30. The effluent from
the coil mixed with reducing solution pumped at 1 (?) mL/min and this mixture flowed through
a 2 m X 0.8 mm ID PTFE coil at 30 to the detector. (Prepare the reactor as follows. Dissolve
75.3 g manganese nitrate in 500 mL water, add 50 g 18-35 mesh silica gel (Macherey-Nagel),
stir vigorously, slowly add 31.6 g potassium permanganate in 500 mL water, stir for 30 min,
filter (500 |xm sieve), wash until no permanganate color is left, dry in a desiccator, pack in a
50 X 2.1 stainless steel tube. The reducing solution was 266 g NaOH, 13.4 g anhydrous sodium
sulfite, and 9 mL 2-mercaptoethanol in 1 L water. Note that some Nucleosil 5-C18 column
packings do not give separation at pH 4.45. In this case it is necessary to use 50 mM perchloric
acid as mobile phase and mix the column effluent with pH 4.4 sodium acetate buffer before it
enters the reactor.)
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
KEY WORDS
REFERENCE
Riiter,J.; Kurz,U.R; Neidhart,B- Solid phase reactors as an analytical tool in the determination of urinary
noradrenaline and adrenaline, J.Liq.Chromatogr., 1985, 8, 2475-2496.
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
acid, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbutazone,
oxjrtetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phe-
mine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenyle-
phrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid,
progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyril-
amine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine,
reserpine, resorcinol, saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin,
secobarbital, strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfa-
merazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulin-
dac, tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, the-
obromine, theophylline, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine,
thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcy-
promine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine, trihexyphenidyl,
trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, verapamil, vincamine,
warfarin, yohimbine, zoxazolamine
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP
Mobile phase: MeCN:0.025% phosphoric acid:buffer 25:10:5 (Buffer was 9 rnL concentrated phos-
phoric acid and 10 mL triethylamine in 900 mL water, adjust pH to 3.4 with dilute phosphoric
acid, make up to 1 L.)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
ronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, naphazoline,
naproxen, nifedipine, nizatidine, nortriptyline, oxazepam, oxycodone, oxymetazoline, paroxe-
tine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, pheno-
barbital, phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, pheny-
toin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide,
procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, propanthe-
line, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, race-
methorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, ser-
traline, sotalol, spironolactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine,
tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocain-
ide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine,
trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine,
zopiclone
KEYWORDS
REFERENCE
J.Chromatogr.A, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 100 fxg/mL solution in mobile phase.
HPLC VARIABLES
Column: 150 X 4 5 |xm Crownpak CR(+) immobilized crown ether
Mobile phase: 0.1% pH 1.9 Perchloric acid
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 3.88,4.18
OTHER SUBSTANCES
Simultaneous: octopamine
KEY WORDS
REFERENCE
Nishi,H.; Nakamura,K.; Nakai,H.; Sato,T. Separation of enantiomers and isomers of amino compounds by
J.ChromatogrA, 1997, 757, 225-235.
SAMPLE
Matrix: tissue
Sample preparation: Prepare a 70 X 5 SPE column of Sephadex G 10 in a Pasteur pipette,
wash with 3 mL 20 mM ammonia and 3 mL 10 mM formic acid. Homogenize up to 150 mg rat
brain in 1 mL 100 mM perchloric acid, centrifuge at 4000 g at 4 for 15 min, add 500 ^xL of
the supernatant to the SPE column, wash with 2.5 mL 10 mM formic acid, elute with 1 mL
10 mM formic acid followed by 1.5 mL 5 mM Na2HPO4, inject an aliquot of the eluate.
HPLCVARIABLES
Column: Nucleosil 5 C18
Mobile phase: pH 6.5 Buffer prepared from 200 mM Na2HPO4 and 100 mM citric acid
Flow rate: 0.7
Detector: E, rotating disc electrode, 500 mV
CHROMATOGRAM
Retention time: 4
Limit of detection: 0.05 nmole/g
OTHER SUBSTANCES
Extracted: uric acid
KEYWORDS
rat; brain; SPE
REFERENCE
Westerink,B.H.C; Mulder,T.B.A. Determination of picomole amounts of dopamine, noradrenaline, 3,4-dihy-
droxyphenylacetic acid, homovanillic acid, and 5-hydroxyindolacetic acid in nervous tissue after one-step
purification on Sephadex G-IO, using high-performance liquid chromatography with a novel type of elec-
trochemical detection, J.Neurochem., 1981, 36, 1449-1462.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (glass potters) with 5 volumes of 100 mM perchloric acid
containing 1.9 mM sodium bisulfite, centrifuge at 10000 g at 4 for 30 min. Filter (0.22 |xm)
the supernatant, add dihydroxybenzylamine, inject a 5-20 (JLL aliquot.
HPLCVARIABLES
Column: 100 X 4.6 Hypersil H5 ODS
Mobile phase: EtOHibuffer 2:98 (Buffer was 13.8 g NaH2PO4, 60 mg disodium EDTA, and 20
mg 1-octanesulfonic acid in 1 L water, pH adjusted to 3.70 with phosphoric acid.)
Flow rate: 1
Detector: E, Unicam PU 4022, 70 mV
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
KEYWORDS
adrenal; fetal
REFERENCE
Garcia,J.C; Blanco,L.; McPherson,M.; Leiva,A.; Macis,R. High-performance liquid chromatographic determi-
nation of norepinephrine, epinephrine and dopamine in human foetal adrenal gland, J.Chromatogr.B, 1994,
656, 77-80.
SAMPLE
Matrix: urine
Sample preparation: Acidify urine with 1% (v/v) 6 M HCl. 6-10 mL Swine urine or 1-2.5 mL
rat urine, centrifuge at 4000 g for 30 min, add 200 ng 3,4-dihydroxybenzylamine hydrobromide
and 15 mL lg/L EDTA, adjust to pH 6.45-6.55 with HCl or NaOH. Add the mixture to a cation-
exchange resin SPE cartridge (Bio-Rad), wash twice with 10 mL water and with 5 ml water,
elute with 8 mL 10 g/L boric acid. Dilute boric acid eluate with an equal volume of mobile
phase, inject a 60 |xL aliquot. (Procedure for determining methoxycatecholamines is also
described.)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Kromasil C8
Mobile phase: MeOH.buffer 15:85 (Mobile phase was 300 mL MeOH, 1.5 mL 200 mg/mL 1-
octanesulfonic acid, 100 mL 1 M sodium acetate, and about 1 L water. The pH was adjusted
to pH 3.8 with citric acid and made up to 2 L with water.)
Flow rate: 0.6
Detector: E, Bioanalytical Systems, glassy carbon electrode + 650 mV, Ag/AgCl reference
electrode
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
pig; rat; SPE; pharmacokinetics
REFERENCE
Hay,M.; Mormede,P. Determination of catecholamines and methoxycatecholamines excretion patterns in pig
and rat urine by ion-exchange liquid chromatography with electrochemical detection, J.Chromatogr.B, 1997,
703, 15-23.
SAMPLE
Matrix: urine
Sample preparation: Acidify urine by adding 1% 6 M HCl. 5 mL Acidified urine + 1 mL 7.5%
disodium EDTA, adjust pH to 8.5 with 1 M NaOH, add 250 mg alumina (previously treated
with 2 M HCl), shake for 5 min, decant the supernatant, wash the alumina three times with
5 mL portions of water. Place the alumina in a 4 mm ID glass column, elute with 250 mM
acetic acid in water, collect 2.5 mL eluate, inject a 100 (xL aliquot.
HPLC VARIABLES
Column: 1000 X 2.1 Zipax SCX (DuPont)
Flow rate: 0.8
reagent pumped at 0.4 mL/min and the mixture flowed through a 10 m X 0.5 mm PTFE coil
at 75 0.1 to the detector. (Reagent was 500 mM borate buffer adjusted to pH 9.7 with NaOH.)
CHROMATOGRAM
Retention time: 5.5
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Nimura,N.; Ishida,K.; Kinoshita,T. Novel post-column derivatization method for the fluorimetric determination
of norepinephrine and epinephrine, J.Chromatogr., 1980, 221, 249-255.
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 2 mL water + 1 mL 500 |JLM EDTA in 1 mM HCl + 1 mL 2
M pH 8.5 phosphate buffer + 20 |xL 100 |xM 3,4-dihydroxybenzylamine + 50 mg alumina, vortex
for 3 min, discard the supernatant, wash the alumina 3 times with 5 mL portions of water,
elute by washing the alumina twice with 200 |xL portions of 100 mM phosphoric acid for 30 s
each time. Combine the acidic layers and add them to 560 (JLL 400 mM pH 9.0 borate buffer,
add 20 |xL 50 mM NaCN, add 20 |xL 5 mM naphthalene-2,3-carboxaldehyde in MeOH, mix
thoroughly, let stand at room temperature for 20 min, inject a 5 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm ODS-120T (Toyo Soda)
Mobile phase: MeCNrTHF: 10 mM pH 2.5 phosphate buffer 38:6:56
Flow rate: 1
Injection volume: 5
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
Extracted: dopamine
KEYWORDS
derivatization; SPE
REFERENCE
Kawasaki,T.; Higuchi,T.; Imai,K; Wong,O.S. Determination of dopamine, norepinephrine, and related trace
amines by prechromatographic derivatization with naphthalene-2,3-dicarboxaldehyde, AnaLBiochem.,
1989, 180, 279-285.
SAMPLE
Matrix: urine
Sample preparation: Add disodium EDTA and sodium metabisulfite to urine. 100 JJLL Urine +
2 mL water, vortex, add 1 mL reagent 1, add 5 mL reagent 2, shake vigorously for 2 min,
centrifuge at 2000 g for 2 min, freeze in dry ice/acetone. Remove the organic layer and add it
to 200 |xL 80 mM acetic acid and 2 mL n-octanol saturated with acetic acid, shake vigorously
for 2 min, centrifuge at 2000 g for 2 min, freeze in dry ice/acetone until the aqueous layer is
just solid, remove the organic layer. Thaw out the aqueous layer and add it to 1 mL reagent 1
and 5 mL reagent 2, shake vigorously for 2 min, centrifuge at 2000 g for 2 min, freeze in dry
ice/acetone. Remove the organic layer and add it to 200 JJLL 80 mM acetic acid and 2 mL n-
octanol saturated with acetic acid, shake vigorously for 2 min, centrifuge at 2000 g for 2 min,
freeze in dry ice/acetone until the aqueous layer is just solid, remove the organic layer. Thaw
out the aqueous layer and add 100 u-L Bicine buffer, 250 JJLL MeCN, and 100 jxL 100 mM 1,2-
diphenylethylenediamine in 100 mM HCl, vortex, add 20 |JLL 20 mM potassium ferricyanide,
vortex, heat at 37 for 40 min, cool to room temperature, inject a 100 |xL aliquot. (Prepare
reagent 1 by dissolving 214 g ammonium chloride and 10 g disodium EDTA in 2 L water, adjust
pH to 8.3-8.5 with concentrated ammonium hydroxide, add 4.0 g diphenylborate-ethanolamine
complex, stir for several hours until a clear solution is obtained. Prepare reagent 2 by dissolving
2.5 g tetraoctylammonium bromide and 10 mL n-octanol (saturated with acetic acid) in 1 L n-
heptane. Prepare Bicine buffer by dissolving 14.3 g Bicine (N,N-bis(2-hydroxyethyl)glycine)and
359 mg anhydrous sodium acetate in 45 mL water, stir overnight until dissolved, adjust pH to
7.30 with concentrated NaOH, make up to 50 mL with water. Note that concentration of 1,2-
diphenylethylenediamine is not given in paper. Other authors have used 100 mM
(J.Chromatogr. 1989, 487, 17; 1992, 574, 109; 1992, 583, 236).)
HPLC VARIABLES
was MeCN:MeOH:50 mM pH 7.0 sodium acetate buffer 50:10:40. A:B 75:25 for 1 min, to 10:
90 over 7 min, return to initial conditions over 1 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 3
Limit of detection: <0.4 nM
OTHER SUBSTANCES
Simultaneous: isoproterenol
KEY WpRDS
REFERENCE
Moleman,R; van Dijk,J. Determination of urinary norepinephrine and epinephrine by liquid chromatography
with fluorescence detection and pre-column derivatization, Clin.Chem., 1990, 36, 732-736.
SAMPLE
Matrix: urine
Sample preparation: Add 10-15 mL 6 M HCl to a 24 h volume of urine. 2 mL 3 M Tris buffer
containing 30 mM EDTA + 500 JJLL 10 |xM dihydrobenzylamide in 100 mM perchloric acid + 2
mL 3 M tris buffer containing 30 mM EDTA + 100 |xL 5 M NaOH + 4 mL acidified urine, mix,
add to a 1 mL SPE column containing 200 mg alumina (70-230 mesh-ASTM (Touzart et Ma-
tignon) at 3 mL/min, wash with 9 mL water at 12 mL/min, force through 0.5 mL air, elute
with 1 mL 150 mM perchloric acid at 0.75 mL/min, mix eluate, inject a 100 (xL aliquot. (The
pH of the Tris buffer is such that the pH of the mixture applied to the SPE column is 7.75-
8.0.)
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Nucleosil 100/C18
Mobile phase: MeOHrbuffer 36:64 (Buffer was 75 mM NaH2PO4, 0.15 mM EDTA, and 6 mM
sodium heptanesulfonate, pH 3.96.)
Flow rate: 1.25
CHROMATOGRAM
Retention time: 6.7
Internal standard: dihydrobenzylamide hydrobromide (9.6)
OTHER SUBSTANCES
Simultaneous: levodopa, methyldopa
KEYWORDS
SPE
REFERENCE
Said,R.; Robinet,D.; Barbier,C; Sartre,J.; Huguet,C. Fully automated high-performance liquid chromatographic
assay for the analysis of free catecholamines in urine, J.Chromatogr., 1990, 530, 11-18.
SAMPLE
Matrix: urine
Sample preparation: 100 |xL Urine + 125 jxL 218.6 nM a-methylnorepinephrine in 10 mM HCl
+ 1 mL 10 mM HCl + 1 mL reagent + 5 mL 4.6 mM tetraoctylammonium bromide in n-heptane:
1-octanol 99:1, shake for 2 min, centrifuge at 20 at 1000 g for 5 min, freeze in dry ice/acetone.
Remove the organic phase and add it to 2 mL 1-octanol saturated with 80 mM acetic acid and
200 IJLL 80 mM acetic acid, shake, centrifuge at 20 at 1000 g for 5 min, freeze in dry ice/
acetone. Discard the organic layer and add 1 mL 10 mM HCl to the aqueous layer, add 2 mL
1-octanol saturated with 80 mM acetic acid, add 150 jxL 80 mM acetic acid, shake, centrifuge
at 20 at 1000 g for 5 min, freeze in dry ice/acetone. Discard the organic layer and add 200 JJLL
MeCN and 50 u,L buffer to the aqueous layer, add 100 |xL 100 mM 1,2-diphenylethylenediamine
in 100 mM HCl, add 20 JJLL 20 mM potassium ferricyanide in water, heat at 37 in the dark
for 1 h, inject a 50 |xL aliquot. (Reagent was 8.9 mM diphenylborate-ethanolamine complex in
2 M pH 8.6 ammonia/ammonium chloride buffer containing 13.4 mM EDTA. Buffer was 1.75
M pH 7.05 bicine in water containing 1% EDTA. Recrystallize 1,2-diphenylethylenediamine
from toluene:light petroleum (bp 60-80) 10:90, dry overnight at 60.)
HPLC VARIABLES
Column: 100 X 4.6 3 ^m Cp MicroSpher C18 (Chrompack)
Mobile phase: MeCN:MeOH:50 mM pH 7.0 sodium acetate 40:8:50 (At the end of the day flush
column with 60 mL MeCN:MeOH:water 70:10:20.)
Flow rate: 1
CHROMATOGRAM
Retention time: 2
Internal standard: a-methylnorepinephrine (Janssen, Beerse, Belgium) (3)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
van der Hoorn,F.A.J.; Boomsma,E; Man in 't VeIdA-J-; Schalekamp,M.A.D.H. Improved measurement of uri-
nary catecholamines by liquid-liquid extraction, derivatization and high-performance liquid chromatogra-
phy with fluorimetric detection, J.Chromatogn, 1991, 563, 348-355.
SAMPLE
Matrix: urine
Sample preparation: Condition a 100 mg Bakerbond C-18 SPE cartridge with 2 mL MeOH and
2 mL buffer I. Heat urine at 50, centrifuge, remove a 500 (xL aliquot and add it to 1 mL buffer
II (?), add 20 (JLL 860 ng/mL dihydroxybenzylamine, shake for 5 min, add a 1 mL aliquot to the
SPE cartridge, wash with 2 mL buffer I, wash with 1 mL MeOHrbuffer I 50:50, wash with 500
IxL water, elute with 2 mL 1 M acetic acid, inject a 20 |xL aliquot. (Buffer I was 200 mM
ammonium chloride containing 0.05% EDTA and 0.4% tetrabutylammonium iodide, pH ad-
justed to 8.0 0.1. Buffer II was 2 M ammonium chloride containing 0.5% EDTA and 1.2%
tetrabutylammonium iodide, pH adjusted to 8.0 0.1.)
HPLCVARIABLES
Column: 150 X 3 5 |xm Separon SGX C-18 (Tessek)
Mobile phase: MeOHrbuffer 5:95-7:93 (Buffer was 50 mM pH 3.0 0 . 1 phosphate buffer con-
taining 50 mM EDTA, 1 mM sodium octanesulfonate, and 1 mM NaCl.)
Detector: E, AMOR 400 mV (SunChrom)
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
SPE
REFERENCE
Brandsteterova,E.; Krajnak,K; Skacani,I. HPLC analysis of urinary catecholamines using affinity SPE pro-
cedure, Pharmazie, 1995, 50, 825-826.
SAMPLE
Matrix: urine
Sample preparation: Adjust urine to pH 3.0 with 6 M HCl, centrifuge at 1600 g for 10 min.
900 (JLL Supernatant + 100 |xL 2.5 |xM N-methyldopamine, mix, inject a 100 |xL aliquot on to
column A and elute to waste with mobile phase A, after 5 min elute the contents of column A
on to column B with mobile phase B, monitor the effluent from column B.
HPLC VARIABLES
Column: A 35 X 4 TSK-precolumn-CA 2 (Tosoh); B 150 X 4.6 mixed mode (C18/cation exchange)
(ALltech)
Mobile phase: A 15 mM pH 6.0 citric acid/trisodium citrate buffer; B 200 mM pH 6.0 citric acid/
trisodium citrate buffer
Flow rate: 0.7
Detector: E, Mitsubishi 8 channel, 150 mV
CHROMATOGRAM
Internal standard: N-methyldopamine (30 mV) (20)
OTHER SUBSTANCES
Extracted: dopamine (30 mV), epinephrine (150 mV), metanephrine (380 mV), 3-methoxytyra-
mine (340 mV), normetanephrine (380 mV), serotonin (150 mV)
KEY WORDS
column-switching
REFERENCE
Mashige,R; Matsushima,Y.; Miyata,C; Yamada,R.; Kanazawa,H.; Sakuma,L; Takai,N.; Shinozuka,N.; Ohk-
ubo,A.; Nakahara,K. Simultaneous determination of catecholamines, their basic metabolites and serotonin
in urine by high-performance liquid chromatography using a mixed-mode column and an eight-channel
electrochemical detector, Biomed.Chromatogr., 1995, 9, 221-225.
SAMPLE
Matrix: urine
Sample preparation: Condition a 10 X 2 20 mg 15-25 |xm PLRP-S polymer-based SPE cartridge
(Spark Holland) with 1 mL MeOH, with 0.5 mL water, and with 1.5 mL 200 mM pH 8.5
ammonia/ammonium chloride buffer containing 0.05% EDTA. Collect 24 h urine with 10 mL 6
M HCl, final pH 1-3. Dilute 4-fold with buffer. Inject a 200 uL aliquot onto the SPE cartridge,
wash with 1 mL 200 mM pH 8.5 ammonia/ammonium chloride buffer containing 0.05% EDTA,
wash with 250 |JLL MeOH:200 mM pH 8.5 ammonia/ammonium chloride buffer 20:80, wash
with water at 1 mL/min for 2.25 min. Elute the contents of the SPE cartridge onto column A
with the mobile phase for 30 s then remove the SPE cartridge from the circuit, elute column
A with mobile phase onto column B, after 1.25 min elute column A to waste with mobile phase
and elute column B with mobile phase, monitor the effluent from column B. (Buffer was 2 M
pH 8.5 ammonia/ammonium chloride containing 0.5% EDTA, 0.1% diphenylborate, and 18 ng/
mL dihydroxybenzylamine.)
HPLC VARIABLES
Column: A 30 X 4.6 C18 (Brownlee); B 250 X 4.6 5 |xm Ultrasphere IP C18
Mobile phase: MeCN:MeOH:buffer 15:8:100, apparent pH adjusted to 3.2 with 1.5 M ortho-
phosphoric acid (Buffer was 50 mM KH2PO4 containing 1 mM sodium heptane sulphate and
0.07 mM EDTA.)
Flow rate: 0.8
Detector: E, ESA Model 5100A, Model 5021 conditioning cell, Model 5011 analytical cell, oxidiz-
ing electrode +350 mV, screen electrode +100 mV, quantifying electrode -300 mV
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Extracted: epinephrine, dopamine
KEYWORDS
SPE; column-switching
REFERENCE
Pastoris,A; Cerutti,L.; Sacco,R.; De Vecchi,L.; SbaffiÂ. Automated analysis of urinary catecholamines by high-
performance liquid chromatography and on-line sample pretreatment, J.Chromatogr.B, 1995, 664, 287
293.
SAMPLE
Matrix: urine
Sample preparation: Acidify urine to pH 2.0-3.5 with 5 M HCl, centrifuge at 7000 g for 10 min,
inject a 10-500 |xL aliquot on to column A and elute to waste with mobile phase A, after 10
min backflush the contents of column A on to column B with mobile phase B, elute with mobile
HPLC VARIABLES
Column: A nitrophenylboronic acid modified copolymer (U.S. Patent 4 767 529 (Chem.Abs. 1988,
108, 716980); B 53 X 4.6 1.5 |xm MICRA NPS RP-18 (MICRA Scientific, Northbrook)
Mobile phase: A 20 mM (NH4)2HPO4 containing 10 mM EDTA, adjusted to pH 8.7 with 25%
ammonia solution; B 10 mM NaH2PO4 containing 0.1 mM dodecanesulfonic acid, adjusted to
Flow rate: A 0.5; B from 0.2 to 0.5 over 2 min, maintain at 0.5
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
KEYWORDS
column-switching
REFERENCE
Rudolphi,A.; Boos,K.-S.; Seidel,D. Coupled-column HPLC analysis of free urinary catecholamines using re-
stricted access affinity precolumn and micro-particulate nonporous silica analytical column, Chromatogra-
phia, 1995, 41, 645-650.
Norethindrone
Merck Index: 6790
Lednicer No.: 1 164, 165; 2 145
SAMPLE
HPLC VARIABLES
Next Page
mL/min)
Detector: UV 240.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Norethynodrel
Merck Index: 6791
Lednicer No.: 1 168
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
Retention time: 6.2 (norethynodrel acetate)
OTHER SUBSTANCES
Simultaneous: ethinyl estradiol, norethindrone, norethindrone acetate, norgestrel
REFERENCE
Norfloxacin
CAS Registry No.: 70458-96-7, 100587-52-8 (norfloxacin succinil)
Merck Index: 6793
SAMPLE
Matrix: aqueous humor, blood, tissue
Previous Page
mL/min)
Detector: UV 240.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Norethynodrel
Merck Index: 6791
Lednicer No.: 1 168
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
Retention time: 6.2 (norethynodrel acetate)
OTHER SUBSTANCES
Simultaneous: ethinyl estradiol, norethindrone, norethindrone acetate, norgestrel
REFERENCE
Norfloxacin
CAS Registry No.: 70458-96-7, 100587-52-8 (norfloxacin succinil)
Merck Index: 6793
SAMPLE
Matrix: aqueous humor, blood, tissue
Sample preparation: Homogenize sample in 1 M HCl, add MeCN, centrifuge, add an equal
volume of dichloromethane, centrifuge, inject a 50-100 JJIL aliquot of the aqueous supernatant.
HPLC VARIABLES
Guard column: Corasil C18
Column: jjiBondapak C18
droxide 9.5:90.5
Flow rate: 1.5
CHROMATOGRAM
Retention time: 6.5
KEYWORDS
serum; rabbit; human; cornea
REFERENCE
Bron,A.M.; Pe"chinot,A-; Garcher,C; Guyonnet,G.; Kazmierczak,A. Ocular penetration of topically applied nor-
floxacin 0.3% in the rabbits and in humans, J.Ocul.PharmacoL, 1992, 8, 241-246.
SAMPLE
Matrix: blood
Sample preparation: 200 JJLL Serum + 50 |xL 400 |xg/mL IS in water, vortex for 30 s. Add 500
fxL MeCN, vortex for 1 min. Centrifuge at 6000 rpm for 10 min. Evaporate the supernatant to
200 |xL at 40 under a stream of nitrogen, vortex for 30 s. Inject a 30-80 u,L aliquot.
HPLCVARIABLES
Column: 100 X 8.0 4 pm Radial-pak Novapak C18
Mobile phase: MeCN:buffer 14:86 (Buffer was 2 g citric acid, 2 g sodium acetate, and 1 mL
triethylamine in 1 L water.)
Flow rate: 2.5
CHROMATOGRAM
Retention time: 4.0
Internal standard: acebutolol (7.4)
OTHER SUBSTANCES
Extracted: pefloxacin
Simultaneous: ciprofloxacin, lomefloxacin, ofloxacin
KEYWORDS
REFERENCE
Abanmi,N.; Zaghloud,L; El Sayed,N.; al-Khamis,K.I. Determination of pefloxacin and its main active metabolite
in human serum by high-performance liquid chromatography, Ther.Drug Monit., 1996, 18, 158163.
SAMPLE
Matrix: blood
Sample preparation: Add 20 |xL MeOH:0.1% trifluoroacetic acid 15:85 and 5 |xL (sic) MeCN to
300 |xL plasma. Centrifuge at 600 g for 10 min. Evaporate the supernatant under nitrogen at
40 for 30 min. Reconstitute the residue in 200 |xL MeOH:0.1% trifluoroacetic acid 15:85. Inject
a 50 |xL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 fjim Zorbax SB-C8
Mobile phase: MeCN:water:trifluoracetic acid 19:81:0.02
Flow rate: 1
Detector: UV 279
CHROMATOGRAM
Internal standard: norfloxacin
OTHER SUBSTANCES
Extracted: ciprofloxacin, enrofloxacin
KEYWORDS
cat; plasma; norfloxacin is IS
REFERENCE
Kordick,D.L.; Papich,M.G.; Breitschwerdt,E.B. Efficacy of enrofloxacin or doxycycline for treatment of Barto-
nella henselae or Bartonella clarridgeiae infection in cats, Antimicrob.Agents Chemother., 1997, 41, 2448-
2455.
SAMPLE
Matrix: blood
HPLC VARIABLES
perchlorate.)
Flow rate: 1.2
Detector: UV 272
CHROMATOGRAM
Internal standard: pipemidic acid (4.14)
OTHER SUBSTANCES
Simultaneous: pefloxacin
KEYWORDS
REFERENCE
Zlotos,G.; Biicker,A.; Kinzig-Schippers,M.; Sorgel,E; Holzgrabe,U. Plasma protein binding of gyrase inhibitors,
J.Pharm.ScL, 1998, 87, 215-220.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Plasma + 1 mL 100 mM pH 7.0 K2HPO4 adjusted to pH 7.0 with
85% orthophosphoric acid + 100 jxL 300 |xg/mL nalidixic acid in water + 3 mL dichloromethane:
stitute residue in 100 |xL MeOH:50 mM NaOH 2:1, vortex, inject a 10 |xL aliquot.
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 300
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Extracted: fenbufen, felbinac
Interfering: ofloxacin, enoxacin
KEYWORDS
plasma; rat
REFERENCE
Katagiri,Y.; Naora,K.; Ichikawa,N.; Hayashibara,M.; Iwamoto,K. Simultaneous determination of ofloxacin, fen-
135-142.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 250 (xL 10% trichloroacetic acid, vortex for 10 s, centri-
fuge at >700 g for 10 min, inject a 20 |xL aliquot of the supernatant.
HPLC VARIABLES
Column: 100 X 8 ixBondapak C18 Radial-PAK
CHROMATOGRAM
Retention time: 5.6
KEYWORDS
serum
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 100 IJLL pH 7.4 phosphate buffer + 50 jxL 10 |xg/mL -
hydroxypropyltheophylline in pH 7.4 phosphate buffer + 5 mL chloroform.isopropanol 80:20,
shake on a rotary mixer for 15 min, centrifuge at 800 g for 5 min. Evaporate organic layer
under nitrogen at 45, sonicate residue with 100 |xL mobile phase, inject 25 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.9 Spherisorb ODS
Column: 250 X 4.9 Sherisorb S5 ODS2
Mobile phase: MeCN:buffer 15:85 adjusted to pH 3.0 with 85% phosphoric acid immediately
before use (Buffer was 4.54 g KH2PO4 + 5.94 g Na2HPO4.2H2O + 1.49 g tetrabutylammonium
hydrogen sulfate per L.)
Flow rate: 1.3
Detector: UV 280
CHROMATOGRAM
Retention time: 5.5
Internal standard: p-hydroxypropyltheophylline
OTHER SUBSTANCES
Simultaneous: theophylline, enoxacin, ciprofloxacin
KEYWORDS
plasma; rat
REFERENCE
Davis,J.D.; Aarons,L.; Houston,J.B. Simultaneous assay of fluoroquinolones and theophylline in plasma by
SAMPLE
Matrix: blood
Sample preparation: 150 JXL Plasma + 10 JJLL 100 /xg/mL enoxacin in water + 75 JJLL 10% tri-
chloroacetic acid + 600 |xL chloroform, vortex for 5 min, centrifuge at 13800 g for 10 min.
Remove 500 ^L of the organic layer and evaporate it to dryness under reduced pressure, re-
constitute the residue in 150 jxL mobile phase, inject a 50 JULL aliquot.
HPLC VARIABLES
Mobile phase: MeOH:0.01% trifluoroacetic acid 25:75
Flow rate: 1.2
CHROMATOGRAM
Retention time: 6.9
Internal standard: enoxacin (5.8)
KEYWORDS
REFERENCE
Hussain,M.S.; Chukwumaeze-Obiajunwa,V.; Micetich,R.G. Sensitive high-performance liquid chromatographic
assay for norfloxacin utilizing fluorescence detection, J.Chromatogr.B, 1995, 663, 379-384.
SAMPLE
Matrix: blood
Sample preparation: 100 (xL Serum + 1 mL 2.6 |xg/mL N-ethylnorfloxacin in chloroform, vortex,
centrifuge at 11000-12000 g for 1 min. Remove the organic layer and evaporate it to dryness
under a stream of air at 60, reconstitute the residue in 200 JXL mobile phase, inject a 25-50
U1L aliquot.
HPLC VARIABLES
Column: 40 X 3.2 3 |xm RP-18 Spheri-3
Mobile phase: MeCN:10 mM pH 2.5 NaH2PO4 containing 1 mM triethylamine 11:89
Flow rate: 1
Detector: UV 279
CHROMATOGRAM
Retention time: 1.9
Internal standard: N-ethylnorfloxacin (Heat 3.2 g norfloxacin, 1.5 g triethylamine, and 12-20
mmoles ethyl iodide in 40 mL DMF at 80-90 with stirring for 2 h, concentrate to dryness,
recrystallize from chloroform/benzene to give N-ethylnorfloxacin (mp 251-3) (J.Med.Chem.
1980, 23, 1358)) (2.9)
KEYWORDS
serum
REFERENCE
Wallis,S.C; Charles,B.G.; Gahan,L.R. Rapid and economical high-performance liquid chromatographic method
for the determination of norfloxacin in serum using a microparticulate C18 guard cartridge, J.Chromatogr.B,
1995, 674, 306-309.
SAMPLE
Sample preparation: 100 |xL Plasma or dialysate + 400 |xL MeOH, vortex, centrifuge, inject an
HPLC VARIABLES
Column: strong cation exchange
Mobile phase: MeCN: 100 mM pH 3 citrate buffer 20:80
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Rose,T.R; Bremner,D.A.; Collins,J.; Ellis-Pegler,R.; Isaacs,R.; Richardson,R.; Small,M. Plasma and dialysate
levels of pefloxacin and its metabolites in CAPD patients with peritonitis, J.Antimicrob.Chemother., 1990,
25, 657-664.
SAMPLE
Sample preparation: Blood. Centrifuge 200 |xL fresh blood at 3000 rpm for 10 min. Inject an
aliquot of the plasma. Formulations. Completely dissolve 50 mg sample in 20 mL MeOH, son-
icate. Filter insoluble material and adjust filtrate to 50 mL with MeOH. Inject an aliquot of
the filtrate.
HPLC VARIABLES
Column: 150 X 4.6 Cosmosil 5C18 (Nacalai Tesque, Japan)
Mobile phase: MeCN:solution 1:6.5 (Solution was 1 g sodium acetate trihydrate, 2 g citric acid
monohydrate and 1 mL triethylamine in 1 L water (?).)
Flow rate: 1.5
Detector: UV 277
CHROMATOGRAM
Internal standard: p-nitrophenylacetic acid
KEYWORDS
freeze-dried formulations; plasma; rat; egg albumin; olive oil
REFERENCE
Tsuji,Y.; Kakegawa,H.; Miyataka,H.; Nishiki,M.; Matsumoto,H.; Satoh,T. Pharmaceutical properties of freeze-
dried formulations of egg albumin, several drugs and olive oil, Biol.Pharm.Bull., 1996, 19, 636640.
SAMPLE
Sample preparation: Plasma. Mix 500 |xL plasma with 5 (xg IS, add 4 mL dichloromethane and
100 |xL pH 7.4 phosphate buffer, agitate for 10 min, centrifuge at 5300 g for 10 min. Collect
3.5 mL organic phase. Add 4 mL dichloromethane to the aqueous phase again, agitate, centri-
fuge. Combine the organic phases, evaporate at 60, reconstitute the residue in 100 |xL mobile
phase, inject a 20 |xL aliquot. Tissue. Mix 500 |xL epiploic-fat and 4 mL dichloromethane and
keep at 4, add 5 jig IS, mix by using an automatic grinder (Ultra Turrax, Ika-Werk, Stauffen,
Germany). Collect the mixture, centrifuge at 5300 g for 10 min. Add 4 mL 100 mM NaOH to
the dichloromethane, agitate for 10 min, centrifuge at 5300 g for 5 min. Eliminate the organic
phase, adjust the aqueous phase to pH 7.4 with concentrated trichloroacetic acid, add 4 mL
dichloromethane, agitate for 10 min and centrifuge at 5300 g for 5 min. Evaporate the organic
phase at 60. Reconstitute the residue in 100 |xL mobile phase, inject a 20 |xL aliquot.
HPLCVARIABLES
Guard column: 25 X 4 5 jjim 100 RP-18 Lichrosphere
Column: 125 X 4 5 |xm 100 RP-18 endcapped Lichrosphere
Mobile phase: MeCN:pH 4.8 citrate buffer 85:15
Flow rate: 1
CHROMATOGRAM
Internal standard: 4844P (pefloxacin analog)
OTHER SUBSTANCES
KEYWORDS
epiploic-fat; plasma; pharmacokinetics; fat
REFERENCE
Jacoberger,B.; Ubeaud,G.; Freys,G.; Pottecher,T.; Jung,L.; Koffel,J.C. Concentrations of pefloxacin in plasma
and tissue after administration as surgical prophylaxis, Antimicrob.Agents Chemother., 1998,42, 425-427.
SAMPLE
Sample preparation: Tissue. Homogenize 100-250 mg tissue with 5 mL 500 mM pH 7.0 phos-
phate buffer, remove a 1 mL aliquot, add 100 jxL 10 |xg/mL IS, mix, add 10 mL chloroform:
isopentanol 90:10, shake for 15 min, centrifuge , repeat extraction. Combine the organic layers
and evaporate them to dryness under a stream of air at 60, reconstitute the residue in 100
JXL 1% ammonia, inject a 25 |xL aliquot. Plasma. 250-500 JJLL Plasma + 100 (xL 10 jjug/mL IS +
1 mL 500 mM pH 7.0 sodium phosphate buffer, mix, add 10 mL chloroform:isopentanol 90:10,
shake for 15 min, centrifuge, repeat extraction. Combine the organic layers and evaporate them
to dryness under a stream of air at 60, reconstitute the residue in 100 jxL 1% ammonia, inject
a 25 JJLL aliquot.
HPLC VARIABLES
Mobile phase: MeCN.water 15:85 containing 2 g/L sodium acetate trihydrate, 2 g/L citric acid
monohydrate, and 1 mIVL triethylamine
Flow rate: 2
CHROMATOGRAM
Retention time: 2.8
Internal standard: l-allyl-6-fluoro-l,4-dihydro-4-oxo-7-(4-methyl-l-piperazinyl)quinoline-3-car-
boxylic acid (Roger Bellon Laboratories) (6.6)
OTHER SUBSTANCES
Extracted: metabolites, pefloxacin
KEYWORDS
plasma; prostate
REFERENCE
Montay,G.; Tassel, J.P. Improved high-performance liquid chromatographic determination of pefloxacin and its
metabolite norfloxacin in human plasma and tissue, J.Chromatogr., 1985, 339, 214218.
SAMPLE
Sample preparation: Serum, plasma. Dilute serum or plasma 1:2 to 1:10 with 30 mM phos-
phoric acid, centrifuge, inject a 20 JULL aliquot of supernatant. Urine. Dilute urine 1:10 to 1:100
with 30 mM phosphoric acid, centrifuge, inject a 20 |xL aliquot of supernatant. Tissue (lung,
gut). Cut tissue with a scalpel, homogenize with 1-3 mL buffer, centrifuge at 9600 g for 5 min
three times, inject a 20 |xL aliquot. Tissue (chondral). Cut tissue with a scalpel, homogenize
with 3-6 mL buffer in an ice bath for 2-3 min, centrifuge at 9600 g for 5 min four or five times,
inject a 100 |xL aliquot. Dilute human pleural samples with buffer, centrifuge, inject a 20 (JLL
aliquot. (Buffer was 66.6 mM K2HPO4 adjusted to pH 7.40 with KH2PO4.)
HPLC VARIABLES
Mobile phase: MeOH:MeCN:buffer 13:7:80, adjusted to pH 3.0 with phosphoric acid (Buffer was
15 mM phosphoric acid adjusted to pH 3.0 with tetrabutylammonium hydroxide.)
Flow rate: 1
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
Simultaneous: ofloxacin, ciprofloxacin
KEYWORDS
serum; plasma; lung; gut; pleural; chondral
REFERENCE
Knoller,J.; Konig,W.; Schonfeld,W.; Bremm,K.D.; Koller,M. Application of high-performance liquid chromatog-
raphy of some antibiotics in clinical microbiology, J.Chromatogr., 1988, 427, 257-267.
SAMPLE
Sample preparation: Add 500 |xL MeCN to 500 |xL plasma or urine diluted with water, vortex
vigorously for 20 s, centrifuge at 4000 rpm for 2 min, mix a 250 |xL aliquot of the supernatant
with 500 jxL 100 mM perchloric acid containing 20 mM triethylamine, inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 80 X 4.0 3 |xm Nucleosil 3C18
Mobile phase: MeOH: 100 mM perchloric acid containing 20 mM triethylamine 30:70
Flow rate: 1
CHROMATOGRAM
OTHERSUBSTANCES
Noninterfering: acyclovir, ampicillin, amoxicillin, clavulanic acid, doxycycline, erythromycin,
lansoprazole, metronidazole, minocycline, omeprazole, penicillin V, trimethoprim
KEYWORDS
plasma
REFERENCE
Mascher,H-J-; Kikuta,C. Determination of norfioxacin in human plasma and urine by high-performance liquid
chromatography and fluorescence detection, J.Chromatogr.A, 1998, 812, 381-385.
SAMPLE
Sample preparation: Plasma. Wash a C18 Sep-Pak cartridge with 1 mL 4% methanolic phos-
phoric acid and 10 mL water. Add 1 mL plasma to the cartridge, wash with 6 mL water, elute
with 1 mL 4% methanolic phosphoric acid then with 1 mL water. Combine the eluates, make
up to 2 mL with water, inject a 50 |xL aliquot. Urine. Dilute 100 |xL urine with 900 JJLL mobile
phase and directly inject a 10 jxL aliquot.
HPLCVARIABLES
Guard column: jxBondapak C18-Corasil
Column: 300 X 3.9 uBondapak C18
Mobile phase: MeOH:MeCN:water 300:50:700 + 1.74 g K2HPO4 + 20 mg sodium heptanesulfon-
ate, pH adjusted to 3 with phosphoric acid
Flow rate: 2
Injection volume: 10 (urine), 50 (plasma)
CHROMATOGRAM
Retention time: 4
KEYWORDS
plasma; SPE
REFERENCE
Gutzler,R; de Vries,J.X. Bestimmung von Norfioxacin in Plasma und Urin durch Hochdruckfliissigkeitschro-
matographie, Fortschr.Antimikr.Antineoplast.Chemother., 1984, 3, 673-677.
SAMPLE
Sample preparation: Dilute with one or more volumes of water, filter (0.6 |xm)
HPLC VARIABLES
Column: 200 X 4 5 fjim Nucleosil C18
Mobile phase: MeCN:25 mM orthophosphoric acid adjusted to pH 3.0 with tetrabutylammonium
hydroxide 11:89
Flow rate: 1.5
CHROMATOGRAM
Retention time: 2.8
OTHER SUBSTANCES
Noninterfering: trimethoprim, sulfamethoxazole, netilmicin, metronidazole, penicillin G, clox-
acillin, doxycycline, cefuroxime, erythromycin, salicylic acid, digoxin, furosemide, acetamino-
phen, prednisolone, warfarin, dextropropoxyphene
Interfering: ciprofloxacin
KEYWORDS
serum
REFERENCE
Nilsson-Ehle,I. Assay of ciprofloxacin and norfloxacin in serum and urine by high-performance liquid chro-
matography, J.Chromatogn, 1987, 416, 207-211.
SAMPLE
Sample preparation: Plasma. 250 JAL Plasma + 250 \LL 10 jxg/mL IS in water + 750 jxL MeOH,
stir, centrifuge at 2000 rpm for 5 min, inject a 50 |ULL aliquot of the supernatant. Urine. 500
JJLL Urine + 3.5 mL 8 u-g/mL IS in water, inject a 50 jxL aliquot.
HPLC VARIABLES
Column: 200 X 4.6 Nucleosil C8
Mobile phase: MeCN:water:triethylamine:formic acid 11:87.5:0.1:1 containing 0.2% sodium ac-
etate and 0.1% formic acid
Flow rate: 1
CHROMATOGRAM
Internal standard: l-ethyl-6-chloro-l,4-dihydro-7-(4-methyl-l-piperazinyl)-4-oxo-3-quinoline-
carboxylic acid (RP 41983)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Humbert,G.; BrumptJ.; Montay,G.; Le Liboux,A.; Frydman,A.; Borsa-Lebas,F.; Moore,N. Influence of rifampin
on the pharmacokinetics of pefloxacin, Clin.Pharmacol.Ther., 1991, 50, 682-687.
SAMPLE
Matrix: cells
HPLC VARIABLES
droxide 25:75
Flow rate: 1.5
OTHER SUBSTANCES
Also analyzed: ciprofloxacin, fleroxacin, lomefloxacin, ofloxacin, temafloxacin
REFERENCE
Pascual,A-; Garcia,!; Conejo,M.C; Perea,E.J. Fluorometric and high-performance liquid chromatographic mea-
SAMPLE
Matrix: hair
Sample preparation: Wash hair successively with 0.1% sodium dodecyl sulfate and water for
30 min, repeat twice, blot between 2 sheets of paper towel, allow to dry at room temperature.
Take a 1 cm fragment of hair, add 500 JXL 1 M NaOH, heat at 80 for 30 min, cool, add 500
IJLL 1 M HCl, add 1 mL 100 mM pH 4.6 potassium hydrogen citrate buffer, add 50 |xL 1 |xg/mL
IS in water. Add the mixture to a Bond-Elut C8 cartridge, elute with 2 mL THF:25 mM ortho-
phosphoric acid 20:80, evaporate eluate to dryness in vacuum, dissolve residue in 150 |xL mo-
bile phase, vortex, inject a 60 JJLL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Tosoh 5 |xm TSKgel ODS-80Ts
Mobile phase: MeCN:25 mM orthophosphoric acid adjusted to pH 3.0 with 0.5 M tetra-n-butyl-
amine hydroxide 5:95
Flow rate: 1
CHROMATOGRAM
Internal standard: (R)-9-fluoro-2,3-dihydro-3-methyl- 10-(4-ethyl-l-piperazinyl)-7-oxo-7H-pyr-
ido[l,2,3-de][l,4]benzoxazine-6-carboxylic acid (DS-4632) (10.2)
OTHER SUBSTANCES
Simultaneous: ciprofloxacin, ofloxacin (determine at F ex 295 em 490)
KEYWORDS
SPE
REFERENCE
Mizuno,A.; Uematsu,T.; Nakashima,M. Simultaneous determination of ofloxacin, norfloxacin and ciprofloxacin
in human hair by high-performance liquid chromatography and fluorescence detection, J.Chromatogr.B,
1994, 653, 187-193.
SAMPLE
Matrix: perfusate
HPLC VARIABLES
Column: 250 X 4.6 Spheris C18 (Phase Separations)
Mobile phase: MeCN: 15 mM tetrabutylammonium iodide 5:95
Flow rate: 1
Detector: UV 275
CHROMATOGRAM
REFERENCE
Lin,H.-H.; Hsu,L.-R.; Wu,P.-C; Tsai,Y.-H. Increased norfloxacin skin permeability for fatty alcohol propylene
glycol (FAPG) ointment by optimized process of preparation: Behavior of stearic acid in stratum corneum
lipids, Biol.Pharm.Bull, 1995, 18, 1560-1565.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 450 |xg/mL solution in MeCN:water 50:50. 5 mL Solution + 5
mL THF + 200 molar excess of acetic anhydride + 3 molar excess of 1 M NaOH, sonicate for
15 min, add 15 mL mobile phase, sonicate for 15 min, cool to room temperature, make up to
50 mL with mobile phase, inject a 50 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:buffer 35:65 (Buffer was prepared by mixing equal volumes of 20 mM citric
acid and 20 mM sodium citrate, pH adjusted to 2.4 with perchloric acid.)
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 5.9
OTHER SUBSTANCES
Simultaneous: ciprofloxacin, sarafloxacin, temafloxacin
KEYWORDS
derivatization
REFERENCE
Morley,J.A.; Elrod,L.,Jr. Determination of fluoroquinolone antibacterials as N-Acyl derivatives, Chromatogra-
phia, 1993, 37, 295-299.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 20 |xg/mL solution in MeCN:water 10:90, filter (0.45 |xm), inject
an aliquot.
HPLCVARIABLES
Column: 250 X 4 5 ^m LiChrospher 100 RP-18
Mobile phase: MeCN:25 mM phosphoric acid 7:93, adjusted to pH 3.09 with 100 mM tetrabu-
tylammonium hydroxide
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 8.6
OTHER SUBSTANCES
Simultaneous: ciprofloxacin, ofloxacin (UV 295), pipemidic acid
REFERENCE
Barbosa,J.; Berg6s,R.; Sanz-Nebot,V. Linear solvation energy relationships in reversed-phase liquid chroma-
tography. Prediction of retention of several quinolones, J.Liq.Chromatogr., 1995, 18, 3445-3463.
SAMPLE
Matrix: solutions
Sample preparation: Filter (0.45 u,m) a solution in MeCN:water 10:90, inject an aliquot of the
filtrate.
HPLC VARIABLES
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 8.8
OTHER SUBSTANCES
Simultaneous: ciprofloxacin, enoxacin, neroxacin, ofloxacin (UV 295), pipemidic acid
REFERENCE
Barbosa,J.; Berges,R.; Sanz-Nebot,V. Solvatochromic parameter values and pH in aqueous-organic mixtures
27-36.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Ultra-Turrax) eye tissue with 3 mL 50 mM pH 5.8 sodium
phosphate-citrate buffer and IS, centrifuge. Add the supernatant to 7 mL chloroform, agitate,
centrifuge at 1000 g for 10 min. Remove the lower organic layer and evaporate it to dryness
under a stream of nitrogen at 37, reconstitute the residue in 100 jxL mobile phase, inject a 5-
20 (JLL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:water 26:74 containing 2 g/L sodium acetate trihydrate, 2 g/L citric acid
monohydrate, 4 mL/L triethylamine, and 2 mIVL formic acid, pH 4.8
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Internal standard: l-allyl-6-fl uoro-1,4-dihydro-4-oxo-7-(4-methyl- l-piperazinyl)quinoline-3-car-
boxylic acid (?) 4662 P (Roger-Bellon Laboratories) (2.95)
OTHER SUBSTANCES
KEYWORDS
rabbit; eye; pharmacokinetics
REFERENCE
Cochereau-Massin,L; Bauchet,J.; Faurisson,E; Vallois,J.M.; Lacombe,R; Pocidalo,J.J. Ocular kinetics of peflox-
acin after intramuscular administration in albino and pigmented rabbits, Antimicrob.Agents Chemother.,
1991, 35, 1112-1115.
SAMPLE
Matrix: urine
HPLC VARIABLES
Mobile phase: MeOH:MeCN:100 mM pH 5.75 phosphate buffer 24.1:2.6:73.3
Flow rate: 1
CHROMATOGRAM
Limit of quantitation: 3.13 jxg/mL
OTHER SUBSTANCES
KEYWORDS
pharmacokinetics
REFERENCE
Jaehde,U.; Sorgel,E; Stephan,U.; Schunack,W. Effect of an antacid containing magnesium and aluminum on
absorption, metabolism, and mechanism of renal elimination of pefloxacin in humans, Antimicrob.Agents
Chemother., 1994, 38, 1129-1133.
Norgestimate
Merck Index: 6796
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 3 mL MTBE, vortex for 1 min, centrifuge at 1500 rpm for
room temperature, reconstitute the residue in 100 (JLL MeOH, inject an aliquot.
HPLCVARIABLES
Guard column: 18 mm long (Brownlee)
Flow rate: 1
CHROMATOGRAM
Retention time: 9.3, 9.8 (syn and anti)
OTHER SUBSTANCES
Extracted: metabolites, norgestrel
KEYWORDS
serum
REFERENCE
Wong,F.A.; Juzwin,S.J.; Tischio,N.S.; Flor,S.C. Determination of norgestimate in serum by automated high-
performance liquid chromatography and subsequent radioimmunoassay, J.Liq.Chromatogr., 1995,18,1851-
1861.
SAMPLE
Sample preparation: Extract culture medium twice with 2 volumes of ether, combine the ex-
tracts and evaporate them to dryness, reconstitute with MeOH, inject an aliquot.
HPLC VARIABLES
Column: 300 X 3.9 Techopak 10 C18 (HPLC Technology)
Flow rate: 1.5
Detector: UV 240, radioactivity
CHROMATOGRAM
Retention time: 13.5, 15.5 (racemate)
OTHER SUBSTANCES
Extracted: metabolites, norgestrel
KEYWORDS
tritium labeled
REFERENCE
Wild,M.J.; Rudland,P.S.; Back,D.J. Metabolism of the oral contraceptive steroids ethynylestradiol and norges-
timate by normal (Huma 7) and malignant (MCF-7 and ZR-75-1) human breast cells in culture, J.Steroid
Biochem.Mol.BioL, 1991, 39, 535-543.
SAMPLE
Sample preparation: 5 Tablets + 2 glass beads + 25 mL 50 |xg/mL dibutyl phthalate in MeOH,
vortex 15 min or until tablets have completely disintegrated, sonicate 5 min, filter (2 |xm),
HPLC VARIABLES
Column: 50 X 4.5 5|xm IBM C18
Flow rate: 2.1
Detector: UV 230
CHROMATOGRAM
Retention time: 3.5
Internal standard: dibutyl phthalate
OTHER SUBSTANCES
Simultaneous: ethinylestradiol, degradation products
KEYWORDS
REFERENCE
Lane,P.A.; Mayberry,D.O.; Young,R.W. Determination of norgestimate and ethinyl estradiol in tablets by high-
SAMPLE
Matrix: microsomal incubations, mucosal fluid
Sample preparation: Mucosal fluid. Extract 1 mL mucosal fluid twice with 5 mL diethyl ether,
evaporate extracts to dryness, resuspend residue in 100 |xL MeOH, inject an aliquot. Micro-
somal incubations. Extract 2.5 mL microsomal incubation with 5 mL diethyl ether, proceed as
before.
HPLC VARIABLES
Guard column: on-line guard column
Column: 100 X 8 ixBondapak radial compression module
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: 3-ketonorgestimate, 17-deacetylnorgestimate, norgestrel
REFERENCE
Madden,S.; Back,D.J. Metabolism of norgestimate by human gastrointestinal mucosa and liver microsomes in
vitro, J.Steroid Biochem.Mol.BioL, 1991, 38, 497-503.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Dioxane:water 50:50 (CAUTION! Dioxane is a carcinogen!)
Flow rate: 1.4
Detector: UV 254
OTHER SUBSTANCES
Simultaneous: levonorgestrel
REFERENCE
Killinger,J.; Hahn,D.W.; Phillips,A-; Hetyei,N.S.; McGuire,J.L. The affinity of norgestimate for uterine proges-
togen receptors and its direct action on the uterus, Contraception, 1985, 32, 311319.
SAMPLE
Matrix: tissue
Sample preparation: Incubate endometrial tissue with buffer, remove tissue, extract medium
twice with 2 volumes of diethyl ether, evaporate to dryness, reconstitute in a small volume of
HPLC VARIABLES
Column: 300 X 3.9 Technopak 10 C18
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Retention time: 15, 18 (syn and anti)
OTHER SUBSTANCES
Simultaneous: norgestrel, metabolites
KEYWORDS
endometrial tissue
REFERENCE
Wild,M.J.; Rudland,P.S.; Back,D.J. Metabolism of the oral contraceptive steroids ethynylestradiol, norgestimate
and 3-ketodesogestrel by a human endometrial cancer cell line (HEC-IA) and endometrial tissue in vitro,
J.Steroid Biochem.Mol.Biol., 1993, 45, 407-420.
Norgestrel
CAS Registry No.: 797-63-7, 797-64-8 ((-) form), 6533-00-2
Merck Index: 6797
LednicerNo.: 1 167, 2 151, 3 84
SAMPLE
residue with 50 uL MeCN.water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 241.7
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Centrifuge oil formulation at 30 at 2000 rpm for 30 min, filter (Whatman
No. 1 paper), collect the last 4 mL of the filtrate. Dilute a 10 |xL aliquot to 10 mL with MeCN:
water 60:40 containing 0.3% Tween 80, add a 2 mL aliquot and add it to 1 mL 3.33 (xg/mL
progesterone, vortex for 10 s, inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 300 X 3.9 Novapak C18
Flow rate: 2
Detector: UV 248
CHROMATOGRAM
Internal standard: progesterone
KEY WORDS
oils
REFERENCE
Gao,Z.-H.; Shukla,A.J.; Johnson,J.R.; Crowley,W.R. Controlled release of a contraceptive steroid from biode-
gradable and injectable gel formulations: In vitro evaluation, Pharm.Res., 1995, 12, 857-863.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2.0
Detector: UV 243
CHROMATOGRAM
REFERENCE
Kim,D.-D.; Kim,J.L.; Chien,Y.W. Mutual hairless rat skin permeation-enhancing effect of ethanol/water system
and oleic acid, J.Pharm.Sci., 1996, 85, 1191-1195.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Dioxane.-water 50:50 (CAUTION! Dioxane is a carcinogen!)
Flow rate: 1.4
Detector: UV 254
OTHER SUBSTANCES
Simultaneous: norgestimate
REFERENCE
KillingeiyJ.; Hahn,D.W.; Phillips,A.; Hetyei,N.S.; McGuire,J.L. The affinity of norgestimate for uterine proges-
togen receptors and its direct action on the uterus, Contraception, 1985, 32, 311-319.
SAMPLE
Matrix: solutions
Sample preparation: Direct injection
HPLCVARIABLES
Column: 250 X 4.6 10 \xm Partisil C-18 ODS-3
Flow rate: 2
Detector: UV 243
CHROMATOGRAM
Retention time: 6.0
KEYWORDS
see also J.Pharm.Sci. 1989; 78; 477
REFERENCE
Catz,R; Friend,D.R. In vitro evaluations of transdermal levonorgestrel, Drug Des.Deliv., 1990, 6, 49-60.
Nortriptyline
Merck Index: 6812
Lednicer No.: 1 151
SAMPLE
gastric contents, and bile four times with water. Mix 4 mL sample with 100 |xL 400 |xg/mL IS
and 2 mL 500 mM NaOH, vortex briefly, add 4 mL heptanerisoamyl alcohol 98.5:1.5 and mix
for 15 min (Spiramix 10, Denley, UK). Separate the organic layer, add 4 mL heptaneiisoamyl
alcohol 98.5:1.5 to extraction sample, mix. Combine the organic layers and extract them with
2 mL 50 mM sulfuric acid. Make the acid layer alkaline with 1 mL 1.0 M pH 9.0 carbonate/
bicarbonate buffer and mix with 2 mL toluene:isoamyl alcohol 85:15 for 15 min. Evaporate the
organic layer to dryness, reconstitute the residue in 100 |xL MeOH and inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 5 |xm Apex II ODS
Column: 150 X 4.6 5 |xm Apex II OD
Mobile phase: MeCN:pH 3 phosphate buffer:n-nonylamine 40-50:60:0.12
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Internal standard: doxepin (2.99)
OTHER SUBSTANCES
Extracted: amitriptyline
KEYWORDS
REFERENCE
Pounder,D.J.; Adams,E.; Fuke,C; Langford,A.M. Site to site variability of postmortem drug concentrations in
SAMPLE
Matrix: blood
Sample preparation: Add 250 |xL 2 M sodium carbonate to 500 jxL plasma. Add 100 |xL 1 |xg/
10 min. Cool in a dry ice-acetone bath. Add 200 (xL 0.3% phosphoric acid to upper organic layer.
jxL aliquot of the acidic aqueous layer.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm C18 Symmetry (Waters Millipore, USA)
50:50.)
Flow rate: 1.2
CHROMATOGRAM
Limit of quantitation: 5 ng/mL (UV 226, UV 400 nm); 7 ng/mL (UV 254)
OTHER SUBSTANCES
Extracted: metabolites, amitriptyline, clomipramine, desipramine, fluoxetine imipramine,
maprotiline
Simultaneous: amineptine, carbamazepine, chlordiazepoxide, chlorpromazine, clonazepam,
clorazepate, clozapine, cyamemazine, desmethylmaproptiline, desmethylvenlafaxine, doxepin,
flunitrazepam, fluvoxamine, haloperidol, levomepromazine, lorazepam, loxapine, mianserine,
sulphide, trimipramine, venlafaxine, viloxazine, zolpidem, zopiclone
KEYWORDS
plasma
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 30 mg Oasis HLB SPE cartridge with 1 mL MeOH and
1 mL water. Acidify (?) mL serum with 20 |xL phosphoric acid, vortex for 5 s, add to the SPE
cartridge, wash with 1 mL MeOH :water 5:95, elute with 1 mL MeOH. Evaporate the eluate
to dryness at 40 under a stream of nitrogen. Reconstitute the residue with 200 yiL MeOH:20
mM pH 7 phosphate buffer 20:80, inject a 20 |xL aliquot.
HPLCVARIABLES
Guard column: 20 X 3.9 Sentry
Mobile phase: MeOH:20 mM pH 7 potassium phosphate 70:30
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 8
Internal standard: nordoxepin (4.9)
OTHER SUBSTANCES
Simultaneous: amitriptyline, doxepin
KEYWORDS
pig; serum; SPE
REFERENCE
Cheng,Y.-R; Phillips,D.J.; Neue,U.; Bean,L. Solid-phase extraction for the determination of tricyclic antide-
pressants in serum using a novel polymeric extraction sorbent, J.Liq.Chromatogr.Rel.Technol., 1997, 20,
2461-2473.
SAMPLE
Sample preparation: Vortex 1 mL plasma or microsomal incubation with 200 jxL 1 jxg/mL
desipramine and 100 |iL 5 M NaOH for 10 s, add 5 mL butan-l-ol:hexane 2:98, vortex for 1
min, centrifuge at 2000 g and 4 for 5 min, evaporate the organic phase to dryness at 40 using
a vacuum vortex evaporator, reconstitute the residue in 200 JULLI mobile phase, inject a 50 JJLL
aliquot.
HPLCVARIABLES
Column: 5 (xm Nova-Pak C18
Mobile phase: MeCN:buffer 30:70 (Buffer was water containing 1% triethylamine, adjusted to
pH 3 with orthophosphoric acid.)
Flow rate: 2
Detector: UV 240
CHROMATOGRAM
Retention time: 8.5
OTHER SUBSTANCES
Extracted: amitriptyline
Noninterfering: diazepam, furafylline, hydroxyamitriptyline, hydroxynortriptyline, quinidine,
mephenytoin, triacetyloleandomycin
Interfering: ketoconazole
KEYWORDS
human; liver; rat; plasma
REFERENCE
Ghahramani,R; Lennard,M.S. Quantitative analysis of amitriptyline and nortriptyline in human plasma and
liver microsomal preparations by high-performance liquid chromatography, J.Chromatogr.B, 1996, 685,
307-313.
SAMPLE
Sample preparation: Serum, urine. 500 |xL Serum or urine + 100 |xL 2 |xg/mL diazepam + 200
JJLL 20% sodium carbonate + 500 |xL water + 3 mL n-hexane:isoamyl alcohol 98.5:1.5, mix for
10 JJLL aliquot. Tissue. Homogenize 1 g sample with 9 mL 100 mM HCl and 100 |xL 20 ixg/mL
diazepam, centrifuge at 15000 g for 10 min. Add 500 |xL 20% sodium carbonate and 4 mL n-
hexanedsoamyl alcohol 98.5:1.5 to 1 mL of the supernatant, mix for 5 min. Remove the organic
100 |xL mobile phase, filter by microconcentrator (Microcon-30, Grace). Inject a 10 jxL aliquot.
HPLC VARIABLES
Column: 100 X 4.6 2 jim TSK gel Super-Octyl (A) or 100 X 4.6 5 jjum Hypersil MOS-C8 (B),
(Yokogawa, Japan)
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
Limit of quantitation: 50 ng/mL(serum, urine), 500 ng/mL (tissue)
OTHER SUBSTANCES
mine, maprotiline, melitracen, mianserin
KEYWORDS
serum; brain; liver
REFERENCE
SAMPLE
Sample preparation: Add 500 fiL 3 M ammonia solution and 7 mL n-pentane:isopropanol 95:5
to 2 mL plasma or urine, shake in an overhead shaker for 20 min, let stand for 10 min. Transfer
the upper organic layer to a tube containing 1 mL 100 mM HCl, shake for 20 min, let stand
for 5 min. Aspirate the organic phase to waste, wash the remaining aqueous layer with 3 mL
pentane by shaking for 10 min. Add 500 jxL 3 M ammonia solution and 6 mL a-pentane:
isopropanol 95:5 to the washed aqueous layer, shake for 20 min, evaporate the organic layer
under a stream of nitrogen at 65, reconstitute the residue with 160 |xL mobile phase, inject
60 jxL aliquot.
HPLC VARIABLES
Column: 150 X 4.5 3 |xm Spherisorb silica
Mobile phase: MeOH:hexane:nonylamine 5:95:0.3
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 10
Internal standard: nortriptyline
OTHER SUBSTANCES
Extracted: N-desmethyldoxepin, doxepin
KEYWORDS
dog; human; plasma; normal phase; nortriptyline is IS
REFERENCE
Yan,J.; Hubbard,J.W.; McKay,G.; Midha,KK. Stereoselective and simultaneous measurement of cis- and trans-
isomers of doxepin and N-desmethyldoxepin in plasma or urine by high-performance liquid chromatography,
J.Chromatogr.B, 1997, 691, 131-138.
SAMPLE
residue with 50 /xL MeCNrwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLCVARIABLES
mL/min)
Detector: UV 206.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: serum
Sample preparation: 1 mL Serum + 500 JJIL 750 mM pH 10 sodium bicarbonate/carbonate buffer
+ 50 |xL IS in EtOH:water 50:50 + 8 mL heptane:isoamyl alcohol 98:2, shake at 250 cycles/min
for 5 min, centrifuge at 1500 g for 10 min, freeze in dry ice/EtOH. Remove the organic layer
and add it to 150 JULL 22 mM pH 2.5 KH^PCVphosphoric acid buffer, shake at 250 cycles/min
for 5 min, centrifuge at 1500 g for 10 min, freeze in dry ice/EtOH. Discard the organic layer,
inject a 65 |xL aliquot of the aqueous layer.
HPLC VARIABLES
Column: 250 X 4.6 Supelco C18
Mobile phase: MeCNibuffer 45:55 (Buffer was 44 mM KH2PO4 containing 1.5 mL/L triethylam-
ine, adjusted to pH 2.5 with phosphoric acid.)
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
Internal standard: l-(3-(dimethylamino)propyl)- l-(p-chlorophenyl)-1,3-dihydroisobenzofuran-5-
carbonitrile (LU 10-202) (Lundbeck, Copenhagen) (8.33)
OTHER SUBSTANCES
Extracted: metabolites, citalopram, amitriptyline
Simultaneous: chlorprothixene, clomipramine, clozapine, flupenthixol, haloperidol, levomepro-
mazine, perphenazine, zuclopenthixol
Noninterfering: benzodiazepines
Interfering: desmethyllevomepromazine
KEYWORDS
serum
REFERENCE
Olesen,O.V.; Linnet,K. Simplified high-performance liquid chromatographic method for the determination of
citalopram and desmethylcitalopram in serum without interference from commonly used psychotropic drugs
and their metabolites, J.Chromatogr.B, 1996, 675, 83-88.
Noscapine
CAS Registry No.: 128-62-1,6035-40-1 (dl-form), 912-60-7 (HCI)
Merck Index: 6815
SAMPLE
Matrix: blood
Sample preparation: Condition a C18 SPE cartridge with 2 mL MeOH, 2 mL water, and 1 mL
50 mM pH 7.4 sodium phosphate buffer at 1.5 mL/min. Centrifuge rapidly thawed plasma
samples. Combine 1 mL plasma with 40 |xL 903 ng/mL papaverine free base and 2 mL 50 mM
pH 7.4 sodium phosphate buffer, vortex. Add 2.75 mL diluted plasma at a rate of 0.36 ml/min
to the SPE cartridge, wash with 1 mL 50 mM pH 7.4 sodium phosphate buffer and 2 mL water.
Dry by passing 5 mL of air through the SPE cartridge at 5 mL/min. Elute with 1.5 mL MeCN,
evaporate the eluate to dryness under nitrogen at 30. Reconstitute with 200 |xL MeCN, inject
a 50 |xL aliquot.
HPLCVARIABLES
Column: 125 X 4.0 5 jxm Nucleosil 120-5-C18
Flow rate: 1.0
Detector: UV 211
CHROMATOGRAM
Internal standard: papaverine (3.98)
KEYWORDS
plasma; SPE
REFERENCE
Chollet,D.E; Ruols,C; Arnera,V. Determination of noscapine in human plasma using solid-phase extraction
and high-performance liquid chromatography, J.Chromatogr.B, 1997, 701, 81-85.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 213.4
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.2
OTHER SUBSTANCES
gosine, ergosinine, ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfiura-
ifensine, nortriptyline, orphenadrine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargy-
line, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone,
REFERENCE
Jane,L; McRinnon,A-; Flanagan,R-J- High-performance liquid chromatographic analysis of basic drugs on silica
SAMPLE
Matrix: solutions
HPLCVARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
acid, nitrazepam, norepinephrine, nylidrin, oxazepam, oxycodone, oxymorphone, oxyphenbu-
tazone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persan-
dole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapjridine, sulfasox-
REFERENCE
Hill,D.W.; Kind,AJ- Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnalToxicol., 1994,
IS, 233-242.
Novobiocin
Molecular formula: C3i H36N2Oi 1
CAS Registry No.: 303-81 - 1 ,
1476-53-5 (Na salt), 4309-70-0 (Ca salt)
Merck Index: 6818
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL buffer + 4.5 mL MTBE.isopropanol 97.5:2.5, shake
at 70 strokes/min for 5 min, centrifuge at 10-15 at 1100 g for 10 min, repeat extraction.
Combine the organic layers and evaporate them to dryness under a stream of nitrogen at 20-
25, reconstitute the residue in 0.5 mL mobile phase, vortex, sonicate for 5 min, inject a 100
jxL aliquot. (Buffer was 400 mL 1 M KH2PO4, 400 mL K2HPO4 solution, and 250 g KCl, adjust
pH to 6.5.)
HPLC VARIABLES
Column: 100 X 8 5 jxm Nova-Pak C18 Radial-Pak
Mobile phase: MeOH:2-methoxyethanol:buffer 80:5:15 (Buffer was 4.33 g sodium lauryl sulfate
and 2 mL 1 M phosphoric acid in 150 mL water, pH 2.8.)
Flow rate: 1.8
Detector: UV 330
CHROMATOGRAM
Retention time: 3.0
Internal standard: novobiocin
OTHER SUBSTANCES
Extracted: coumermycin A l
KEY WORDS
dog; plasma; novobiocin is IS
REFERENCE
Strojny,N.; Conzentino,R; de Silva,J.A. Determination of coumermycin Al in plasma by reversed-phase high-
performance liquid chromatographic analysis, J.Chromatogr., 1985, 342, 145-158.
SAMPLE
Matrix: blood
Sample preparation: 0.5-1 mL Plasma + 10 |xL 5 mM prednisone in MeOH + 5 mL MeOH,
vortex for 10 s, centrifuge at 2000 g for 10 min, inject a 15 JJLL aliquot of the supernatant.
HPLC VARIABLES
Guard column: CN Guard-Pak
Column: 150 X 4.6 5 fjim APEX octyl EC C8 (Jones Chromatography)
Mobile phase: Gradient. MeOH:buffer from 55:45 to 20:80 over 20 min. (Buffer was water acid-
ified to pH 3.0 with trifluoroacetic acid.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Internal standard: prednisone (5.5)
KEYWORDS
plasma
REFERENCE
Chen,T.-L.; Kennedy,M.J.; Dunlap,V.M.; Colvin,O.M. Determination of plasma novobiocin levels by a reversed-
phase high-performance liquid chromatographic assay, J.Chromatogr.B, 1994, 652, 109-113.
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Serum + 5 |xL 4 mM mitomycin C in MeCN + 400 |xL MeCN,
vortex, centrifuge at 12000 g for 2 min, inject a 50 |xL aliquot of the supernatant.
HPLC VARIABLES
Guard column: 15 X 3.2 NewGuard RP18
Column: 220 X 4.6 5 |xm Spheri-5 RP18
Flow rate: 0.8
Detector: UV 340
CHROMATOGRAM
Internal standard: mitomycin C (6.66)
KEYWORDS
serum
REFERENCE
Zuhowski,E.G.; Gutheil,J.C; Egorin,M.J. Rapid and sensitive high-performance liquid chromatographic assay
for novobiocin in human serum, J.Chromatogr.B, 1994, 655, 147152.
SAMPLE
Matrix: blood, milk, tissue
Sample preparation: Blend 10 g tissue with 30 mL 200 mM (NH4)H2PO4. Dilute each 1 mL of
milk or serum with 3 mL 200 mM (NH4)H2PO4. Add 10 mL Tissue homogenate, diluted milk,
or diluted serum to 10 (muscle, milk, serum) or 20 (liver, kidney) mL MeOH, swirl vigorously,
let stand for 5 min, filter (paper), refilter if not clear, to each 3 mL of filtrate from liver or
kidney add 1 mL water, inject a 200 |xL aliquot.
HPLC VARIABLES
Guard column: Supelcosil LC-18-DB
Mobile phase: Gradient. MeCNiMeOH: 10 mM phosphoric acid 0:50:50 for 1 min, to 80:0:20 over
19 min, return to initial conditions. (At the end of each day flush the column with the final
mobile phase, store in this mobile phase.)
Flow rate: 1 (at end of run set flow rate to 2 mL/min for 5 min then 1 mL/min for 5 min)
Detector: UV 340
CHROMATOGRAM
Retention time: 21
KEYWORDS
serum; cow; muscle; liver; kidney
REFERENCE
Moats,W.A.; Leskinen,L. Determination of novobiocin residues in milk, blood, and tissues by liquid chromatog-
raphy, JAssoc.Off.Anal.Chem., 1988, 71, 776-778.
SAMPLE
Sample preparation: Add 10 mL THF to the peanut oil formulation, make up to 100 mL with
mobile phase, sonicate, shake at high speed for 5 min, centrifuge at 2000 g for 5 min, inject a
20 |xL aliquot of the clear supernatant.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm LiChrosorb SM00
Mobile phase: Butyl chloride:THF:MeOH:acetic acid 88:5:4:3 (Butyl chloride was 50% water sat-
urated prepared by mixing equal volumes of water-saturated butyl chloride and anhydrous
butyl chloride.)
Flow rate: 1
Detector: UV 340
CHROMATOGRAM
Retention time: 16
Internal standard: prednisone (UV 254) (10)
OTHER SUBSTANCES
KEYWORDS
stability-indicating; normal phase; oils
REFERENCE
Tsuji,K; Rahn,P.D.; Kane,M.P. High-performance liquid chromatographic method for the determination of no-
vobiocin, J.Chromatogr., 1982, 235, 205-214.
SAMPLE
Matrix: milk
Sample preparation: 10 g Milk + 30 mL 200 mM ammonium phosphate, shake. Remove a 10
mL aliquot and add it to 10 mL MeOH, swirl vigorously, swirl occasionally for 5 min, filter
(paper), refilter if necessary, inject a 1 mL aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 5 |xm Supelcosil LC-18-DB
Mobile phase: Gradient. MeOH:5 mM phosphoric acid 50:50 for 1 min, to MeCN:MeOH:5 mM
phosphoric acid 80:0:20 over 20 min, maintain at MeCN:MeOH:5 mM phosphoric acid 80:0:20
for 5 min, re-equilibrate at initial conditions for 6 min.
Flow rate: 1 (re-equilibrate at 2 mL/min for 5 min and 1 mL/min for 1 min)
Detector: UV 340
CHROMATOGRAM
Limit of detection: <0.05 ppm
KEYWORDS
cow
REFERENCE
Reeves,V.B. Liquid chromatographic procedure for the determination of novobiocin residues in bovine milk:
Interlaboratory study, JAOAC Int., 1995, 78, 55-58.
Nylidrin
Merck Index: 6830
Lednicer No.: 1 69
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Plasma + 500 IJLL buffer, mix briefly, add 50-75 mg resin, mix at
10-25 rpm for 30 min, discard the supernatant, wash twice with 1 mL buffer, add 500 |xL 50
mg/mL KOH in MeOH: water 50:50, mix for 30 min, inject a 20 |xL aliquot of the eluate. (Buffer
was 100 mM citric acid:200 mM Na2HPO4 29:71, pH 6.5 (Mcllvaine buffer) Wash 20-50 mesh
Dowex HCR-S resin twice with water and allow it to equilibrate in buffer).
HPLCVARIABLES
Guard column: 25 X 4.6 5 |xm Spherisorb ODS-I
Column: 250 X 4.6 5 fim Spherisorb ODS-I
Mobile phase: MeCN:MeOH:buffer 30:18:52 containing 1.8 mM octanesulfonic acid (Buffer was
30 mM KH2PO4 adjusted to pH 3.0 with concentrated orthophosphoric acid.)
Flow rate: 1
Detector: E, Gynotek M20, glassy carbon working electrode 950 mV, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 11
Internal standard: nylidrin
OTHERSUBSTANCES
Extracted: isoxsuprine
KEYWORDS
horse; plasma; nylidrin is IS; SPE
REFERENCE
Hashem,A.; Lubczyk,B. Determination of isoxsuprine in equine plasma by high-performance liquid chromatog-
raphy with electrochemical detection, J.Chromatogr., 1991, 563, 216-223.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
acid, nitrazepam, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymorphone, oxy-
dole, sulfamerazine, sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapjrridine, sulfasox-
REFERENCE
18, 233-242.
Nystatin
Merck Index: 6834
SAMPLE
Matrix: CSF
Sample preparation: Condition a BakerBond C18 SPE cartridge with 3 mL MeOH and 3 mL
100 mM pH 9 carbonate buffer. Add 1 mL CSF to the SPE cartridge, wash with 2 mL 100 mM
pH 9 carbonate buffer, air dry for 2 min, elute with two 500 |xL aliquots of MeOH. Evaporate
the eluate to dryness under a stream of nitrogen, reconstitute with 200 |xL MeOH, inject a 100
IxL aliquot.
HPLCVARIABLES
Mobile phase: MeCN: 10 mM pH 5 EDTA 35:65
Flow rate: 0.5
Detector: UV 410
CHROMATOGRAM
Retention time: 8.5
Internal standard: nystatin
OTHER SUBSTANCES
Extracted: amphotericin B
KEYWORDS
dog; human; SPE; nystatin is IS
REFERENCE
Liu,H.; Davoudi,H.; Last,T. Determination of Amphotericin B in cerebrospinal fluid by solid-phase extraction
and liquid chromatography, J.Pharm.Biomed.AnaL, 1995, 23, 1395-1400.
SAMPLE
Matrix: bulk
HPLCVARIABLES
Column: 500 X 1 7-8 |xm Zorbax B.P. SiI
Mobile phase: MeOH:DMF:water:acetic acid 72:25:3:0.4
Flow rate: 0.125
Injection volume: 1
Detector: UV 308
CHROMATOGRAM
Retention time: 13
KEYWORDS
microbore; normal phase
REFERENCE
Milhaud,J.; Gareil,P.; Rosset,R. Separation of filipin and nystatin complexes by semi-preparative and microbore
SAMPLE
Matrix: bulk
Sample preparation: Make up a 10% solution in DMF then dilute to 20 mg/mL with MeOH:
water 66:34, inject a 200 |xL aliquot.
HPLCVARIABLES
Column: 500 X 9 10 |xm Partisil ODS 2
Flow rate: 8
Detector: UV 308
CHROMATOGRAM
Retention time: 22
KEYWORDS
semi-preparative
REFERENCE
Milhaud,J.; Gareil,R; Rosset,R. Separation of filipin and nystatin complexes by semi-preparative and microbore
SAMPLE
Matrix: bulk
HPLC VARIABLES
Column: 300 X 4.5 jjiBondapak C18
Flow rate: 2
Detector: UV 280
CHROMATOGRAM
Retention time: 10
REFERENCE
Mehta,R.T.; Hopfer,R.L.; Gunner,L.A.; Juliano,R.L.; Lopez-Berestein,G. Formulation, toxicity, and antifungal
activity in vitro of liposome-encapsulated nystatin as therapeutic agent for systemic candidiasis, Antimi-
crob.Agents Chemother., 1987, 31, 1897-1900.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve in mobile phase to a concentration of 1 mg/mL.
HPLCVARIABLES
Mobile phase: MeOH:water 90:10 + 1% formic acid, pH 3.5
Flow rate: 1
Detector: UV 340
CHROMATOGRAM
Retention time: 6.3
OTHER SUBSTANCES
REFERENCE
Sauer,B.; Matusch,R. High-performance liquid chromatographic separations of nystatin and their influence on
the antifungal activity, J.ChromatogrA, 1994, 672, 247-253.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4 5 jjum ODS-Hypersil
Mobile phase: MeOH:DMF:10 mM pH 7.0 Tris in water 56:9.6:34.4
Detector: UV 305
OTHER SUBSTANCES
REFERENCE
Egodage,K.L.; Haslam,J.S.; Rajewski,R.A.; Stella,V.J. Correlation and validation to the USP bioassay of a RP-
HPLC assay for nystatin (Abstract 3373), Pharm.Res., 1997, 14, S587.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in DMSO to 10 mg/mL, dilute 1:20 with MeOH.
HPLC VARIABLES
Mobile phase: MeCN:50 mM phosphate buffer (pH 3.5-8.1) 30:70 to 35:65
Flow rate: 0.4-2
Detector: UV 313
OTHER SUBSTANCES
Simultaneous: amphotericin A
REFERENCE
Aszalos,A.; Bax,A; Burlinson,N.; Roller,R; McNeal,C. Physico-chemical and microbiological comparison of nys-
tatin, amphotericin A and amphotericin B, and structure of amphotericin A, J.Antibiot.(Tokyo), 1985, 38,
1699-1713.
Octhilinone
Merck Index: 6853
SAMPLE
Mobile phase: MeOH:water 90:10 + 1% formic acid, pH 3.5
Flow rate: 1
Detector: UV 340
CHROMATOGRAM
Retention time: 6.3
OTHER SUBSTANCES
REFERENCE
Sauer,B.; Matusch,R. High-performance liquid chromatographic separations of nystatin and their influence on
the antifungal activity, J.ChromatogrA, 1994, 672, 247-253.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4 5 jjum ODS-Hypersil
Mobile phase: MeOH:DMF:10 mM pH 7.0 Tris in water 56:9.6:34.4
Detector: UV 305
OTHER SUBSTANCES
REFERENCE
Egodage,K.L.; Haslam,J.S.; Rajewski,R.A.; Stella,V.J. Correlation and validation to the USP bioassay of a RP-
HPLC assay for nystatin (Abstract 3373), Pharm.Res., 1997, 14, S587.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in DMSO to 10 mg/mL, dilute 1:20 with MeOH.
HPLC VARIABLES
Mobile phase: MeCN:50 mM phosphate buffer (pH 3.5-8.1) 30:70 to 35:65
Flow rate: 0.4-2
Detector: UV 313
OTHER SUBSTANCES
Simultaneous: amphotericin A
REFERENCE
Aszalos,A.; Bax,A; Burlinson,N.; Roller,R; McNeal,C. Physico-chemical and microbiological comparison of nys-
tatin, amphotericin A and amphotericin B, and structure of amphotericin A, J.Antibiot.(Tokyo), 1985, 38,
1699-1713.
Octhilinone
Merck Index: 6853
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Octopamine
CAS Registry No.: 104-14-3, 876-04-0 (D-(-)), 770-05-8 (DL HCI)
Merck Index: 6856
LednicerNo.: 5 23
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 100 |xg/mL solution in mobile phase.
HPLCVARIABLES
Column: 150 X 4 5 (Jim Crownpak CR(+) immobilized crown ether
Mobile phase: 0.1% pH 1.9 Perchloric acid
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 4.64, 4.95
OTHER SUBSTANCES
Simultaneous: norepinephrine
KEYWORDS
REFERENCE
Nishi,H.; Nakamura,K; Nakai,H.; Sato,T. Separation of enantiomers and isomers of amino compounds by
J.ChromatogrA, 1997, 757, 225-235.
Octreotide
Molecular formula: G49H66N1OO10S2
Merck Index: 6859
SAMPLE
Matrix: bile, blood, feces, urine
Sample preparation: Hydrolyze pooled plasma with 5 mg Subtilisin A at 50 for 1 h. Lyophilize
feces and extract twice with 4 volumes MeOH. Lyophilize urine and bile and reconstitute with
water.
HPLC VARIABLES
Guard column: 30 X 4.6 5 jxm RP-18 (Brownlee)
Column: 100 X 4.6 5 |xm RP-18 (Brownlee)
Mobile phase: Gradient. MeCN:water:trifluoroacetic acid from 0:99.8:0.2 to 99.8:0:0.2 over 40
min.
Flow rate: 1.5
CHROMATOGRAM
Retention time: 45
KEYWORDS
rat; plasma
REFERENCE
Lemaire,M.; Azria,M.; Dannecker,R.; Marbach,R; Schweitzer,A.; Maurer,G. Disposition of sandostatin, a new
synthetic somatostatin analogue, in rats, Drug Metab.Dispos., 1989, 17, 699-703.
SAMPLE
HPLC VARIABLES
Column: 250 X 2.6 5 |xm Bakerbond C18
Mobile phase: Gradient. A was MeCN:water: 1 M tetramethylammonium hydroxide 10:88:2 ad-
justed to pH 4.5 with concentrated orthophosphoric acid. B was MeCN:water:1 M tetramethyl-
ammonium hydroxide 60:38:2 adjusted to pH 4.5 with concentrated orthophosphoric acid. A:B
from 100:0 to 0:100 over 14 min.
Flow rate: 1.3
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: degradation products, des-threninol
KEYWORDS
protect from light; injections
REFERENCE
Stiles,M.L.; Allen,L.V.,Jr.; Resztak,K.E.; Prince,S.J. Stability of octreotide acetate in polypropylene syringes,
Am.J.Hosp.Pharm., 1993, 50, 2356-2358.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherisorb RP18
Mobile phase: MeCN:buffer 31:69, pH 4.7 (Buffer was 20 mM tetramethylammonium hydroxide
adjusted to pH 4.7 with phosphoric acid.)
Flow rate: 1.2
Detector: UV 210
CHROMATOGRAM
Retention time: 18
OTHER SUBSTANCES
Simultaneous: derivatives
REFERENCE
Kuhn,R.; Morin,C; Erni,F. A simple model describing the retention behavior of octreotide and its glycosylated
derivatives in reversed phase HPLC, Chromatographia, 1995, 41, 516-520.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 250 X 4.6 Spheri-5 C18
Mobile phase: MeCN:water:1 M tetramethylammonium hydroxide pentahydrate 33:65:2, ad-
justed to pH 4.5 with phosphoric acid
Flow rate: 0.8
Detector: UV 280
CHROMATOGRAM
Retention time: 6.2
OTHER SUBSTANCES
Simultaneous: degradation products (UV 210)
KEYWORDS
injections
REFERENCE
Ripley,R.G.; Ritchie,D.J.; Holstad,S.G. Stability of octreotide acetate in polypropylene syringes at 5 and -200C,
Octyl methoxycinnamate
Merck Index: 6864
SAMPLE
Sample preparation: 1-1.5 g sun-screen lotion + 50 mL isopropanol, dissolve. Remove a 5 mL
aliquot and make up to 50 mL with mobile phase, filter (0.45 |xm) inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 10 |xm C8 Hypersil
Mobile phase: Isopropanol:buffer 10:90 (Buffer was 100 mM sodium dodecyl sulfate (electropho-
resis grade) containing 0.3% triethylamine adjusted to pH 3.0 with phosphoric acid.)
Flow rate: 1.5
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: 2-ethylhexyl p-dimethylaminobenzoate, oxybenzone, methyl paraben, propyl
paraben
KEYWORDS
lotion
REFERENCE
Tomasella,F.R; Zuting,P.; Love,L.J. Determination of sun-screen agents in cosmetic products by micellar liquid
SAMPLE
Sample preparation: Weigh out lotion, add 20 mL EtOH, heat to 60, stir for 30 min at room
temperature, make up to 25 mL, stir for 5 min, centrifuge at 14500 g for 5 min, inject an
HPLCVARIABLES
Column: 200 X 4.6 5 \xm Ultrasphere C8
Mobile phase: Gradient. MeOH: 1% aqueous acetic acid from 80:20 to 100:0 over 10 min, main-
tain at 100:0 for 2 min, re-equilibrate for 4 min.
Flow rate: 1
Detector: UV 325
CHROMATOGRAM
Retention time: 8.7
OTHER SUBSTANCES
Simultaneous: benzophenone-3, butylmethoxydibenzoylmethane, impurities
KEYWORDS
lotion; sunscreen; an isomer forms on exposure to light
REFERENCE
Meijer,J.; Lod6n,M. Stability analysis of three UV-filters using HPLC, J.Liq.Chromatogr., 1995,18,1821-1832.
Odapipam
SAMPLE
Sample preparation: Condition a 500 mg C18 Bond-Elut SPE cartridge with MeOH and water.
Mix 2 mL microsomal incubation with 4.5 mL ice-cold MeOH, centrifuge at 4000 g for 10 min,
dilute the supernatant with 15 mL water. Add a 2 mL aliquot to the SPE cartridge, wash with
2 mL water, elute with 2 mL MeOH:25% aqueous ammonia 96:4, evaporate the eluate to dry-
ness under reduced pressure, reconstitute with mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 100 X 4.6 3 |xm ChromSpher Si (Chrompack)
Mobile phase: n-Heptane:2-propanol:water:25% ammonia 90:10:0.2:0.1
Detector: UV 280; MS, VG TRIO 1000, particle beam interface at 50, helium at 25-30 psi, ion
source 200, positive ionization mode, electron current 150 |xA, electron energy 70 eV
CHROMATOGRAM
Retention time: 2.3
OTHERSUBSTANCES
KEYWORDS
normal phase; rat; liver; SPE
REFERENCE
Andersen,J.V.; Hansen,KT. Normal-phase liquid chromatography-particle-beam mass spectrometry in drug
metabolism studies of the dopamine receptor antagonist Odapipam and the muscarine Ml receptor agonist
Xa.nom.elme, Xenobiotica, 1997, 27, 901-912.
Ofloxacin
Merck Index: 6865
Lednicer No.: 4 141-145
SAMPLE
Matrix: aqueous humor, blood
Sample preparation: Aqueous humor. Inject a 10 |xL aliquot directly. Plasma. Condition a 3 mL
C18 SPE cartridge (Varian) with two 3 mL portions of MeCN and 3 mL buffer. Add 2 mL 625
ng/mL ciprofloxacin in buffer to 500 |xL of plasma, mix, add to the SPE cartridge. Wash with
3 mL buffer. Remove moisture with vacuum (200 mbar) for 10 min. Elute with two 500 |xL
portions of MeCN:buffer 40:60. Vortex the eluate, inject a 10 |xL aliquot. (Buffer was 100 mM
Tris adjusted to pH 5.0 with HCl).
HPLC VARIABLES
Column: 300 X 4.6 5 |xm endcapped ODS-Hypersil
Mobile phase: MeCNiDMF: 10 mM NaH2PO4 15:6:79, adjusted to pH 3.0 with 85% phosphoric
acid
Flow rate: 1
Detector: UV 285
CHROMATOGRAM
Limit of detection: 80 ng/mL (aqueous humor), 310 ng/mL (plasma)
OTHER SUBSTANCES
Extracted: cefotaxime
KEYWORDS
plasma; SPE
REFERENCE
Kraemer,H.-L; Gehrke,R.; Breithaupt,A.; Breithaupt,H. Simultaneous quantification of cefotaxime, desacetyl-
cefotaxime, ofloxacine and ciprofloxacine in ocular aqueous humor and in plasma by high-performance liquid
chromatography, J. Chromatogr.B, 1997, 700, 147-153.
SAMPLE
Matrix: bile, blood, perfusate
Sample preparation: Intestinal perfusate. Centrifuge before analysis. Plasma. Mix 200 |xL
plasma with 200 |xL 100 mM pH 6.8 phosphate buffer, add 4 mL dichloromethane, shake at
100 cycles/min for 10 min. Remove the organic layer and dry it under nitrogen at 40, recon-
stitute with 200 uX mobile phase, inject a 20 jxL aliquot (J. Chromatogr. 1988, 434, 320). Bile.
Mix 100 JJLL bile with 900 |xL pH 7.0 phosphate buffer, add 500 |xL MeOH. Shake for 1 min
and centrifuge at 1000 g for 5 min. Inject a 20 JXL aliquot of the supernatant.
HPLC VARIABLES
Column: Bovine Serum Albumin (Macherey Nagel)
Mobile phase: 200 mM Potassium dihydrogen phosphate containing 5 mM N,N-dimethylocty-
lamine, adjusted to pH 8.0 with KOH
Flow rate: 1
Detector: F ex 298 em458
CHROMATOGRAM
Retention time: 6 (S-(-)), 7.8 (R-M)
OTHER SUBSTANCES
Also analyzed: cefoperazone, ciprofloxacin, ofloxacin, quinidine, verapamil
KEY WORDS
chiral; intestinal efflux; pharmacokinetics; plasma
REFERENCE
Rabbaa,L.; Dautrey,S.; Colas-Linhart,N.; Carbon,C; Farinotti,R. Intestinal elimination of ofloxacin enantio-
mers in the rat: Evidence of a carrier-mediated process, Antimicrob.Agents Chemother., 1996, 40, 2126-
2130.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL blood with 1 mL 0.25 mM Triton, vortex for 30 s, add 4 mL 6%
trichloroacetic acid. Vortex for 30 s, centrifuge at 2000 g for 10 min, inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 (xm C18
Mobile phase: MeCN:buffer 10:90 (Buffer was 1 L 25 mM phosphoric acid and 15 mL 40%
tetrabutyl ammonium hydrogen sulfate, adjusted to pH 3.0 with 66.6 mM phosphate buffer.)
Flow rate: 2
CHROMATOGRAM
Retention time: 5
Limit of detection: 30 ng/mL (plasma), 50 ng/mL (blood)
KEYWORDS
plasma; pharmacokinetics; rabbit
REFERENCE
ColinOjC.L; Garcia Turino,A.; Sanchez Navarro,A.; Lanao,J.M. A comparative study of ofloxacin and ciproflox-
acin erythrocyte distribution, Biopharm.Drug Dispos., 1998, 19, 71-77.
SAMPLE
Matrix: blood
Sample preparation: Mix equal volumes serum and MeOH. Centrifuge sample, inject a 20 u-L
HPLCVARIABLES
Column: 250 X 4 Spherisorb 5 ODS II
Mobile phase: MeCN:water:phosphoric acid 5:94.6:0.32 adjusted to pH 3.0 with tetrabutylam-
monium hydroxide
Flow rate: 2
CHROMATOGRAM
KEYWORDS
REFERENCE
Bethell,D.B.; Day,N.R; Dung,N.M.; McMullin,C; Loan,H.T.; Tam,D.T.; Minh,L.T.; Linh,N.T.; Dung,N.Q.;
Vinh,H.; MacGowan,A.P.; White,L.O.; White,N.J. Pharmacokinetics of oral and intravenous ofloxacin in
children with multidrug-resistant typhoid fever, Antimicrob.Agents Chemother., 1996, 40, 2167-2172.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Whole blood or 500 |xL plasma, vortex for 30 s with 1 mL 250 JXM
Triton, add 4 mL 6% trichloroacetic acid. Vortex for 30 s, centrifuge at 2000 g for 10 min. Inject
a 100 fxL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 X 4.6 10 |xm C18
Mobile phase: MeCN:buffer 10:90 (Buffer was mixture of 1 L 25 mM phosphoric acid and 15 mL
40% tetrabutylamonium hydrogen sulfate, adjusted to pH 3 with 66.6 mM phosphate buffer.)
Flow rate: 2
CHROMATOGRAM
Retention time: 5
KEYWORDS
REFERENCE
Colino,C.L; Garcia Turino,A.; Sanchez Navarro,A.; Lanao,J.M. A comparative study of ofloxacin and ciproflox-
SAMPLE
Matrix: blood
Sample preparation: 1 mL Blood or 500 JJLL plasma, vortex for 30 s with 1 mL 250 |JLM Triton,
add 4 mL 6% trichloroacetic acid. Vortex for 30 s, centrifuge at 2000 g for 10 min. Inject a 100
JJIL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 X 4.6 10 fun C18
Mobile phase: MeCN:buffer 10:90 (Buffer was mixture of 1 L 25 mM phosphoric acid and 15 mL
40% tetrabutylamonium hydrogen sulfate, adjusted to pH 3 with 66.6 mM phosphate buffer.)
Flow rate: 2
CHROMATOGRAM
Retention time: 9
KEYWORDS
REFERENCE
Colino,C.I.; Garcia Turino,A.; Sanchez Navarro,A.; Lanao,J.M. A comparative study of ofloxacin and ciproflox-
SAMPLE
Matrix: blood
HPLC VARIABLES
perchlorate.)
Flow rate: 1
Detector: UV 295
CHROMATOGRAM
Internal standard: pipemic acid (4.67)
KEY WORDS
REFERENCE
Zlotos,G.; Biicker,A.; Kinzig-Schippers,M.; Sorgel,E; Holzgrabe,U. Plasma protein binding of gyrase inhibitors,
J.Pharm.ScL, 1998, 87, 215-220.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 295
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Dilute a 25 mg/mL levofloxacin injection with an infusion fluid to a con-
centration of 100 |xg/mL. Inject a 20 |xL aliquot. (Infusion fluids were 0.9% NaCl, 5% dextrose,
5% dextrose and 0.9% NaCl, 5% dextrose and lactated Ringer's injections, 5% sodium bicar-
bonate, Plasma-Lyte 56 and 5% dextrose, 5% dextrose, 0.45% NaCl, and 0.15% KCl, 1/6 M
sodium lactate, sterile water, and 20% mannitol.)
HPLC VARIABLES
Column: 250 X 4.6 Zorbax SB-Phenyl
Mobile phase: MeCN:MeOH:94 mM KH2PO4:trifluoroacetic acid 15:5:80:0.3
Flow rate: 1
Detector: UV 294
CHROMATOGRAM
Retention time: 15
OTHER SUBSTANCES
KEYWORDS
injections; stability indicating; for levofloxacin
REFERENCE
Williams,N.A.; Bornstein,M.; Johnson,K. Stability of levofloxacin in intravenous solutions in polyvinyl chloride
bags, Am.J.Health-SystPharm., 1996, 53, 2309-2313.
SAMPLE
Matrix: growth medium
Sample preparation: 500 |xL Sample + 500 |xL 100 |xg/mL IS in cold (4) MeCN, vortex, cen-
trifuge at 3000 g for 5 min. Remove a 500 JJLL aliquot of the supernatant, filter (0.45 |xm Acrodisc
syringe filter), inject a 30 |xL aliquot. (Protect all specimens from light.)
HPLC VARIABLES
Guard column: C18 5U (Alltech)
Column: 150 X 4.6 7 |xm Adsorbosphere HS C18 7U
Mobile phase: MeCN:20 mM pH 3.0 phosphate buffer 35:65 containing 0.2% triethylamine and
0.2% sodium dodecyl sulfate, adjusted to pH 3.0 with 85% phosphoric acid
Flow rate: 1.75
Detector: UV 280
CHROMATOGRAM
Internal standard: sparfloxacin (7.09)
OTHER SUBSTANCES
Also analyzed: ciprofloxacin, clinafloxacin, levofloxacin, sparfloxacin, temafloxacin, trovafloxacin
KEYWORDS
Mueller-Hinton broth
REFERENCE
Wright,D.H.; Herman,V.K.; Konstantinides,F.N.; Rotschafer,J.C. Determination of quinolone antibiotics in
growth media by reversed-phase high-performance liquid chromatography, J.Chromatogr.B, 1998, 709, 97
104.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 4 (xm NovaPak C18
Mobile phase: MeCN:MeOH:buffer:acetic acid 2.5:10:86.5:1 containing 20 mM triethylamine
(The pH 2.7 buffer was 0.4% diammonium hydrogen phosphate in water containing 0.4% (?)
Flow rate: 1
Detector: UV 279
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ciprofloxacin, enrofloxacin
REFERENCE
Cester,C.C; Toutain,P.L. A comprehensive model for enrofloxacin to ciprofloxacin transformation and disposi-
tion in dog, J.Pharm.Sci., 1997, 86, 1148-1155.
SAMPLE
Matrix: solutions
filtrate.
HPLCVARIABLES
Column: 250 X 4 5 juim LiChrospher 100 RP-18
Flow rate: 1
Detector: UV 295
CHROMATOGRAM
Retention time: 8.8
OTHER SUBSTANCES
Simultaneous: ciprofloxacin (UV 280), enoxacin (UV 280), fieroxacin (UV 280), norfioxacin (UV
280), pipemidic acid (UV 280)
REFERENCE
Barbosa,J.; Berg6s,R; Sanz-Nebot,V. Solvatochromic parameter values and pH in aqueous-organic mixtures
27-36.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: 20 mm long Supelguard LC-18S (Supelco)
Column: 250 X 4.6 Suplecosil LC-18S
Mobile phase: MeCN:buffer:water 10:3.5:86.5 (Buffer was 400 mM tetrabutylammonium hy-
droxide adjusted to pH 2.85.)
Flow rate: 1.8
Detector: UV 280
REFERENCE
Sinko,P.J.; Hu,P. Determining intestinal metabolism and permeability for several compounds in rats. Impli-
cations on regional bioavailability in humans, Pharm.Res., 1996, 13, 108-113.
Olanzapine
Merck Index: 6959
SAMPLE
Matrix: blood
Sample preparation: Condition a Certify mixed bed (RP and ion exchange) SPE cartridge with
MeOH and phosphate buffer. Add serum, wash with 1 M acetic acid, wash with MeOH, elute
with 3% ammonium hydroxide in ethyl acetate. Evaporate the eluate, reconstitute, inject an
aliquot.
HPLCVARIABLES
Column: 100 X 4.6 Microsorb CN
Mobile phase: MeCN:MeOH:50 mM pH 6.5 sodium phosphate buffer 5:28:67
Flow rate: 1.5
CHROMATOGRAM
Retention time: 9.6
Internal standard: clozapine (12.5)
OTHER SUBSTANCES
Simultaneous: imipramine
Also analyzed: haloperidol, risperidone
Interfering: paroxetine
KEYWORDS
SPE; serum
REFERENCE
Prieto,I.V.; Hoffman,D.W. HPLC monitoring of olanzapine (Abstract 131), Ther.Drug Monit, 1997, 19,
580.
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL Plasma with 100 JXL 100 ng/mL IS in MeCN, add 500 |xL
saturated sodium carbonate, mix. Add 7 mL pentane:dichloromethane 85:15, shake for 10 min,
centrifuge at 18 for 10 min. Evaporate the supernatant to dryness under a slow stream of
nitrogen at 60. Dissolve the residue in 150 \LL MeCN, inject a 30-120 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere CN
Mobile phase: MeCNiMeOH: 130 mM pH 6.8 ammonium acetate 86:6:8
Flow rate: 0.8
Detector: E, ESA Coulochem model 5100A, model 5020 guard cell +1 V, model 5011 dual elec-
trode analytical cell, electrode 1 +300 mV, electrode 2 +930 mV
CHROMATOGRAM
Internal standard: 2-ethyl analog of olanzapine LY170222 (Lilly Research Labs) (13)
OTHER SUBSTANCES
Simultaneous: acetaminophen, benztropine, chlorpromazine, clonazepam, clozapine, fluphena-
zine, ibuprofen, lorazepam, perphenazine, pseudoephedrine, risperidone, spiperone, sulphide,
trifluoperazine, trihexyphenidyl
KEYWORDS
REFERENCE
Aravagiri,M.; Ames,D.; Wirshing,W.C; Marder,S.R. Plasma level monitoring of olanzapine in patients with
schizophrenia: Determination by high-performance liquid chromatography with electrochemical detection,
Ther.Drug Monit, 1997, 19, 307-313.
SAMPLE
Sample preparation: 200 |xL Microsomal incubation + 200 JXL cold MeCN, mix, centrifuge in a
microfuge at maximum speed for 5 min, inject an aliquot of the supernatant.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm YMC basic column (YMC, USA)
Mobile phase: MeCN:MeOH:75 mM pH 7 sodium phosphate buffer 20:30:50
Flow rate: 1
Detector: E, ESA Coulochem Dual Electrode Detector, guard cell model 5020 + 300 mV, analyt-
ical cell model 5014, detector 1 + 0.0 V, detector 2 + 300 mV
CHROMATOGRAM
Retention time: 12
Internal standard: LY170222 (2-ethylolanzapine)
OTHER SUBSTANCES
KEYWORDS
liver; human
REFERENCE
Ring,B.J.; Catlow,J.; Lindsay,T.J.; Gillespie,T.; Roskos,L.K; Cerimele,B.J.; Swanson,S.R; Hamman,M.A.;
Wrighton,S.A. Identification of the human cytochromes P450 responsible for the in vitro formation of the
major oxidative metabolites of the antipsychotic agent olanzapine, J.Pharmacol.Exp.Ther., 1996,276, 658-
666.
Olaquindox
Merck Index: 6960
SAMPLE
Matrix: feed
Sample preparation: Grind and sieve feed with a Moulinex blender. Weigh out 10 g and add it
to 20 mL DMF and 60 mL carbon tetrachloride, stir magnetically at 500 rpm at 60 for 30 min,
cool, filter (100 jim glass), wash the residue with a little carbon tetrachloride. Remove 25 mL
of the filtrate and add it to 45 mL water, stir vigorously for 2 min, centrifuge at 320 g for 5
min, inject an aliquot of the aqueous layer.
HPLC VARIABLES
Column: 250 X 4.1 10 juim Versapack C18 (Alltech)
Mobile phase: Gradient. MeOH:water 15:85 for 4 min, to 50:50 over 2 min, maintain at 50:50
for 4 min, return to initial conditions over 2 min.
Flow rate: 1.5
Detector: UV 262
CHROMATOGRAM
Retention time: 4
OTHER SUBSTANCES
Extracted: carbadox (UV 305 nm)
KEYWORDS
protect from light
REFERENCE
dos Ramos,F.J.; da Silveira,I.N.; de Graaf,G. Column liquid chromatographic determination of carbadox and
olaquindox in feeds, J.Chromatogr., 1991, 558, 125-130.
Oleandomycin
7060-74-4 (phosphate)
Merck Index: 6962
SAMPLE
Sample preparation: Homogenize (Physcotron) liver with 4 volumes of ice-cold saline. Put 200
(xL plasma or liver homogenate into the tube. Add 2 mL MTBE and 5 (xL 1 M NaOH and shake
mechanically for 5 min. Centrifuge at 1500 g for 10 min, transfer the upper layer into a glass
tube and evaporate it to dryness under dry nitrogen. Rinse the inner wall of the tube with 200
|JLL MeOH and evaporate to dryness. Dissolve the residue in 30 |xL MeOH and inject a 10 |xL
aliquot.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Cosmosil 5-C18 (Nacalai Tesque)
Mobile phase: MeCN: 100 mM pH 6.6 sodium acetate buffer 50:50
Flow rate: 0.6
Detector: E, BAS LC-4C, 1.1 V, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 8.5
OTHER SUBSTANCES
Extracted: erythromycin
KEY WORDS
rat; plasma; liver; pharmacokinetics; oleandomycin is IS
REFERENCE
Hanada,E.; Ohtani,H.; Kotaki,H.; Sawada,Y.; Iga,T. Determination of erythromycin concentrations in rat
plasma and liver by high-performance liquid chromatography with amperometric detection,
J.Chromatogr.B, 1997, 692, 478-482.
Olsalazine
Merck Index: 6976
Lednicer No.: 4 42
SAMPLE
Sample preparation: Serum. Add disodium-3,5'-azo-bis-(6-hydroxybenzoate) to serum, treat
with proteinase K (0.5 mg/mL protein) for 10 min, add tetrabutylammonium hydrogen sulfate
buffered to pH 6.5, add dichloromethane, agitate for 30 min, centrifuge. Remove the organic
layer and evaporate it to dryness, reconstitute the residue in mobile phase, inject an aliquot.
Urine. Add 2,4-dihydroxybenzoic acid to urine, add perchloric acid, add diethyl ether, shake for
10 min, freeze. Remove the organic layer and add it to pH 7.4 phosphate buffer, extract, inject
an aliquot of the aqueous phase.
HPLCVARIABLES
Guard column: 30 X 4 30-40 juim Perisorb RP-18
Mobile phase: MeOHrbuffer 52:48 (Buffer was pH 7.4 phosphate buffer containing 20 mM
Detector: UV 365
CHROMATOGRAM
Internal standard: disodium-3,5'-azo-bis-(6-hydroxybenzoate), 2,4-dihydroxybenzoic acid
Limit of quantitation: 1 |xM urine, 0.5 JJLM (serum)
OTHER SUBSTANCES
Extracted: olsalazine sulfate
KEYWORDS
REFERENCE
Ryde,E.M.; Ahnfelt,N.-O. The pharmacokinetics of olsalazine sodium in healthy volunteers after a single i.v.
dose and after oral doses with and without food, Eur.J.Clin.PharmacoL, 1988, 34, 481-488.
Omeprazole
Merck Index: 6977
LednicerNo.: 4 133
SAMPLE
Matrix: blood
Sample preparation: Extract 500 |xL plasma with dichloromethane containing 50 |xL 1.0 M
dibasic sodium phosphate and 100 |xL 25 (xg/mL IS. Centrifuge and aspirate aqueous layer to
waste. Evaporate organic layer under a stream of nitrogen, reconstitute residue in 200 |xL
MeCN:20 mM pH 8.0 phosphate buffer 25:75. Inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 Zorbax SB C18
Mobile phase: Gradient. MeCN:20 mM pH 7.5 phosphate buffer from 30:70 to 40:60 over 10 min.
(Mobile phase was contained 0.1% triethylamine.)
Detector: UV 302, to UV 280 at 6.4 min
CHROMATOGRAM
Retention time: 5.7
Internal standard: carbamazepine (7.3)
KEYWORDS
plasma
REFERENCE
Sarich,T.; Kalhorn,T.; Magee,S.; Al-sayegh,R; Adams,S.; Slattery,J.; Goldstein,J.; Nelson,S.; Wright,J. The effect
of omeprazole pretreatment on acetaminophen metabolism in rapid and slow metabolizers of S-mephe-
nytoin, Clin.Pharmacol.Ther., 1997, 62, 21-28.
SAMPLE
Matrix: blood
Sample preparation: Add 10 |xL 72 (xg/mL flunitrazepam in MeOH to 1 mL plasma, shake
briefly, add 3 mL toluenedsoamyl alcohol 95:5, vortex at 1000 rpm for 90 s, centrifuge at 2600
g for 10 min. Evaporate a 2.5 mL aliquot of the upper organic layer to dryness under nitrogen
at 40, reconstitute with 100 JJLL mobile phase, inject a 25 (xL aliquot.
HPLC VARIABLES
Guard column: 10 X 4.6 5 (xm Nucleosil 120-5 C18
Column: 250 X 4 5 |xm Nucleosil 120-5 C 18
Mobile phase: MeOHibuffer 47:53 (Buffer was 100 mM Na2HPO4 adjusted to pH 7.8 with or-
thophosphoric acid.)
Flow rate: 1.2
Detector: UV 302
CHROMATOGRAM
Internal standard: flunitrazepam (11.4)
OTHER SUBSTANCES
KEY WORDS
plasma
REFERENCE
Macek,J.; Ptacek,R; Klima,J. Determination of omeprazole in human plasma by high-performance liquid chro-
SAMPLE
residue with 50 /xL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Ondansetron
CAS Registry No.: 99614-02-5, 116002-70-1, 99614-01 -4
(HCI dihydrate), 103639-04-9 (HCI dihydrate)
Merck Index: 6979
LednicerNo.: 5 164
SAMPLE
HPLCVARIABLES
Column: 250 X 4.6 5 jxm Spherisorb ODS-2
Flow rate: 1.8
Detector: UV 254
CHROMATOGRAM
Retention time: 9.6
OTHER SUBSTANCES
Simultaneous: idarubicin
KEYWORDS
REFERENCE
Zhang,H.; Ye,L.; Stewart, J.T. HPLC determination of idarubicin-etoposide and idarubicin-ondansetron mixtures
in 0.9% sodium chloride injection USP, J.Liq.Chromatogr.ReLTechnoL, 1998, 21, 979-988.
SAMPLE
Sample preparation: If necessary, dilute injection 1:9 with mobile phase (for 50 mL admixtures)
and 1:4 (for 100 mL admixtures), inject a 20 jxL aliquot.
HPLCVARIABLES
Column: 125 X 4 5 jxm LiChrospher 60 RP-Select B
Mobile phase: MeCNibuffer 25:75 (Buffer was 20 mM KH2PO4 adjusted to pH 6.0 with NaOH
solution.)
Detector: UV 241
CHROMATOGRAM
OTHERSUBSTANCES
Simultaneous: dexamethasone
KEYWORDS
5% dextrose; 0.9% sodium chloride; injections; stability-indicating
REFERENCE
Evrard,B.; Ceccato,A.; Gaspard,O.; Delattre,L.; Delporte,J.-P. Stability of ondansetron hydrochloride and dex-
amethasone sodium phosphate in 0.9% sodium chloride injection and in 5% dextrose injection, Am.J.Health-
Syst.Pharm., 1997, 54, 1065-1068.
SAMPLE
Matrix: solutions
Sample preparation: Add 380 |xg propofol and 83 |xg ondansetron hydrochloride to 0.9% sodium
chloride, shake vigorously for 2 min, make up to 10 mL with 0.9% sodium chloride, inject a 20
IxL aliquot.
HPLCVARIABLES
Column: 300 X 4.6 10 |xm T-Bondapak phenyl
phosphoric acid.)
Flow rate: 1
Detector: UV 268
CHROMATOGRAM
Retention time: 8.5
OTHER SUBSTANCES
Simultaneous: propofol
REFERENCE
King,D.T.; Stewart,J.T.; Venkateshwaran,T.G. HPLC determination of propofol-thiopental sodium and propofol-
ondansetron mixtures, J.Liq.Chromatogr.RelTechnoL, 1996, 19, 2285-2294.
SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 |xL aliquot of a solution in 0.9% sodium chloride.
HPLCVARIABLES
Column: 220 X 4.6 5 |xm underivatized silica (Brownlee Silica Applied Biosystems, Inc.,San Jose)
Mobile phase: MeOH:buffer 40:60 (Buffer was 10 mM KH2PO4 adjusted to pH 4.0 with 10%
phosphoric acid.)
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 6.7
OTHER SUBSTANCES
Simultaneous: meperidine, morphine (UV 233)
REFERENCE
ondansetron mixtures in 0.9% sodium chloride injection, J.Liq.Chromatogr.Rel.Technol., 1996, 19, 1329-
1338.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Partisil ODSl
Mobile phase: MeOH:50 mM pH 3.0 phosphoric acid 40:60
Flow rate: 1.5
Detector: radioactivity detection
KEYWORDS
14C labeled
REFERENCE
CollettA; Sims,E.; Walker,D.; He,Y.-L.; Ayrton,J.; Rowland,M.; Warhurst,G. Comparison of HT29-18-d and
Caco-2 cell lines as models for studying intestinal paracellular drug absorption, Pharm.Res., 1996,13,216
221.
Ontazolast
Molecular formula: C2IH25N3O
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Zorbax LC-8
Mobile phase: MeCN:MeOH:50 mM pH 6 phosphate buffer 1:3:8
Flow rate: 2
Detector: UV 245
CHROMATOGRAM
Retention time: 7
Internal standard: BIRM 390 BS (10.5)
REFERENCE
Hauss,D.J.; Fogal,S.E.; Ficorilli,J.V.; Price,C.A.; Roy,T.; Jayaraj,A.A.; Keirns,J.J. Lipid-based delivery systems
for improving the bioavailability and lymphatic transport of a poorly water-soluble LTB4 inhibitor,
J.Pharm.ScL, 1998, 87, 164-169.
Opipramol
Merck Index: 6985
SAMPLE
M a t r i x : blood
Sample preparation: Deproteinize 100 |xL plasma with 100 jxL MeCN containing 2.0 mg/L IS.
Inject a 50 |xL aliquot of the supernatant.
HPLCVARIABLES
Guard column: 4 x 4 Superspher
Column: 125 X 4 Superspher 60 select B
Mobile phase: MeCN:0.07% orthophosphoric acid 20:80
Flow rate: 1.2
CHROMATOGRAM
Retention time: 4.1
Internal standard: methylphenyl-phenylhydantion (7.8)
Limit of detection: 50 |xg/L
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Lappenberg-Pelzer,M. Identification and determination of opipramol metabolites in plasma and urine,
JAnal.ToxicoL, 1998, 22, 215-219.
SAMPLE
Matrix: blood
the residue in 100 |JLL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 |xL aliquot of
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 256
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
residue with 50 juiL MeCN:water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLCVARIABLES
mL/min)
Detector: UV 255.8
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Ormetoprim
Lednicer No.: 2 302
SAMPLE
Sample preparation: Plasma. 200 |xL Plasma + 90 |xL 24% trichloroacetic acid in MeOH + 10
IxL 10 |xg/mL sulfamethoxazole in MeOH, vortex for 30 s, centrifuge at 14000 g for 5 min, inject
a 50 |xL aliquot of the supernatant. Muscle. Homogenize 1 g muscle in 1.5 mL MeOH:buffer
20:80, add 50 jxL 10 |xg/mL sulfamethoxazole in MeOH, mix thoroughly for 1 min, centrifuge
at 14000 g for 5 min, inject a 50 JXL aliquot of the supernatant. (Buffer was 25 mM NaH2PO4
containing 15 mM sodium 1-heptanesulfonate adjusted to pH 2.8 with 5 M phosphoric acid.)
HPLC VARIABLES
Guard column: 20 X 4.6 40 |xm ODS-Hypersil
Column: 150 X 4.6 3 |xm ODS-Hypersil C18
Mobile phase: MeCN:buffer:triethylamine 20:80:0.02 (Buffer was 25 mM NaHgPO4 containing
15 mM sodium 1-heptanesulfonate adjusted to pH 2.8 with 5 M phosphoric acid.)
Flow rate: 1
Detector: UV 270
CHROMATOGRAM
Internal standard: sulfamethoxazole (8)
Limit of detection: 50 ng/g (muscle), 30 ng/mL (plasma)
OTHER SUBSTANCES
Extracted: sulfadimethoxine
KEYWORDS
plasma; muscle; fish; salmon
REFERENCE
Samuelsen,O.B. Simultaneous determination of ormethoprim and sulphadimethoxine in plasma and muscle of
Atlantic salmon (Salmo salar), J.Chromatogr.B, 1994, 660, 412-417.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeCNiMeOH: 100 mM pH 4.5 acetate buffer 12.5:
12.5:75, inject an aliquot.
HPLC VARIABLES
Column: C18 (Rainin)
Mobile phase: MeCN:MeOH:50 mM phosphate buffer 12.5:12.5:75, pH 3.0
Flow rate: 1.5
Detector: UV 280
CHROMATOGRAM
Retention time: 8
OTHER SUBSTANCES
Simultaneous: sulfamethoxazole, trimethoprim
REFERENCE
Brown,M.R; Gronwall,R.; Castro,L. Pharmacokinetics and body fluid and endometrial concentrations of tri-
methoprim-sulfamethoxazole in mares, Am.J.Vet.Res., 1988, 49, 918-922.
SAMPLE
Matrix: tissue
5 g Fish + I m L 100 |xg/mL carbamazepine diol in MeOH + 15 mL MeCN + 500 \LL 50%
trichloroacetic acid, homogenize (Brinkmann Polytron PT 10/35) at medium speed for 30 s,
centrifuge at 4 at 7800 g for 25 min. Remove the supernatant and evaporate it to dryness
under a stream of nitrogen at 40, reconstitute the residue in 5 mL water, vortex for 30 s, filter
(13 mm dia. 8 |xm Membra-Fil (Nucleopore)), add the filtrate to the SPE cartridge, elute with
5 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen at 40, reconstitute
the residue in 1 mL mobile phase, vortex for 30 s, inject a 20 |xL aliquot. (Flush injection valve
with 1 mL mobile phase between analyses.)
HPLCVARIABLES
Guard column: 15 X 3.2 NewGuard RP-18
Column: 250 X 4.6 5 |xm Ultrasphere
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Retention time: 5.5
Internal standard: carbamazepine diol (10.5)
OTHER SUBSTANCES
Extracted: sulfadimethoxine
Simultaneous: sulfacetamide, sulfadiazine, sulfamerazine, sulfamethazine, sulfisoxazole
KEYWORDS
fish; salmon; SPE; pharmacokinetics
REFERENCE
Walisser,J.A.; Burt,H.M.; Valg,T.A.; Kitts,D.D.; McErlane,K.M. High-performance liquid chromatographic anal-
ysis of Romet-30 in salmon following administration of medicated feed, J.Chromatogr., 1990,518,179-188.
Orphenadrine
CAS Registry No.: 83-98-7, 4682-36-4 (citrate)
Merck Index: 7007
Lednicer No.: 1 42
SAMPLE
Matrix: blood
Sample preparation: Add 300 |xL MeCN to 100 |JLL plasma, vortex, centrifuge for 2 min. Remove
the supernatant and add it to 300 JJLL pH 5.9 sodium phosphate buffer and 3 mL hexane and
vortex for 45 s. Centrifuge at 2500 g for 3 min. Evaporate the supernatant under a stream of
nitrogen and reconstitute with mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 100 X 4.6 ODS
Mobile phase: MeCN:MeOH:25 mM potassium phosphate 25:25:50 containing 0.75 mL/L 2 M
sulfuric acid and 0.25 mL/L triethylamine
Flow rate: 1.0
Detector: UV 250
CHROMATOGRAM
Retention time: 5.7
Internal standard: orphenadrine
OTHER SUBSTANCES
Extracted: ethopropazine
KEYWORDS
plasma; rat; pharmacokinetics; orphenadrine is IS
REFERENCE
Padovani,RK; Timby,D.M.; Wright,M.R.; Kapil,R.P. Quantitative analysis of DMP 851 in rat and dog plasma
SAMPLE
Matrix: blood
reconstitute the residue in 100 fxL mobile phase, inject a 50 fxL aliquot. (It is implied, but not
HPLC VARIABLES
Column: 10 ijum Micropak CN (Varian)
Flow rate: 2.5
Detector: UV 254
CHROMATOGRAM
Retention time: 8.2
OTHER SUBSTANCES
Simultaneous: acetophenazine, amitriptyline, benztropine, butaperazine, carphenazine, flu-
phenazine, haloperidol, imipramine, mesoridazine, nortriptyline, piperacetazine, promazine,
promethazine, thioridazine, thiothixene, trifluoperazine, triflupromazine, trihexyphenidyl,
trimeprazine
Interfering: chlorpromazine
KEYWORDS
plasma; whole blood
REFERENCE
compression columns and UV and electrochemical detection, J.Chromatogr., 1982, 231, 361376.
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 3.6
OTHER SUBSTANCES
ifensine, nortriptyline, noscapine, oxeladin, oxprenolol, oxymetazolin, papaverine, pargyline,
thipendyl, protriptyline, proxymetacaine, pseudoephedrine, p5nimethamine, quinidine, qui-
REFERENCE
Jane,I.; McKinnon,A.; Flanagan,R.J. High-performance liquid chromatographic analysis of basic drugs on silica
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 0.5
Detector: UV
CHROMATOGRAM
KEYWORDS
chiral; a 1.89
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:buffer 60:40 (Buffer was 1.5 mL triethylamine in 1 L water adjusted to
pH 3.0 with 85% phosphoric acid.)
Flow rate: 1.5
Detector: UV 199
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: hyoscyamine, bromocriptine, benztropine, biperiden
Noninterfering: amantadine, carbidopa, levodopa
REFERENCE
Selinger,K; Lebel,G.; Hill,H.M.; Discenza,C. High-performance liquid chromatographic method for the analysis
of benztropine in human plasma, J.Chromatogr., 1989, 491, 248-252.
Oryzalin
Merck Index: 7015
SAMPLE
water. Plasma. Add 500 |xL plasma to the SPE cartridge, dry with vacuum. Elute with three
200 |xL portions of MeCN. Inject a 10-50 |xL aliquot of the eluate. Microsomal incubations.
Centrifuge microsomal incubation at 12000 g. Add 900 |xL portion of the supernatant to the
SPE cartridge, dry with vacuum. Elute with three 200 JJLL portions of MeCN. Inject a 10-50 jxL
aliquot of the eluate.
HPLCVARIABLES
Flow rate: 2
Detector: UV 254; MS, Finnigan TSQ tandem mass, APCI, vaporizer 450, corona discharge
current 5 |xA, CID, neutral argon -31 eV
CHROMATOGRAM
Retention time: 4.7
OTHER SUBSTANCES
KEYWORDS
SPE; plasma; mouse; rat; liver; pharmacokinetics
REFERENCE
Dvorakova,K.; Dorr,R.T.; GallegosÂ..; McClure,T.; Powis,G. Pharmacokinetic studies of the herbicide and an-
titumor compound oryzalin in mice, J.Chromatogr.B, 1997, 696, 275-281.
Oxacillin
CAS Registry No.: 66-79-5, 7240-38-2 (Na salt monohydrate),
1173-88-2 (Na salt)
Merck Index: 7036
LednicerNo.: 1 413
SAMPLE
Matrix: blood
Sample preparation: 400 |xL Serum + 400 (xL MeCN, vortex for 10 s, shake slowly for 15 min,
vortex for 10 s, shake for 15 min, centrifuge at 3000 g for 10 min, inject a 50 (xL aliquot of the
HPLCVARIABLES
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: 5.7
OTHER SUBSTANCES
Extracted: cloxacillin, dicloxacillin, methicillin, nafcillin
Noninterfering: amdinocillin (mecillinam), amikacin, amoxicillin, ampicillin, carbenicillin, cefa-
chmandole, cefazolin, ceforanide, cefatoxamine, cefoxitin, cephalexin, cephaloridine, cephalo-
thin, cephradine, cepharin, chloramphenicol, clindamycin, co-trimoxazole, fluorocytosine, gen-
KEYWORDS
serum
REFERENCE
SAMPLE
Matrix: blood, milk
Sample preparation: Milk. Adjust 5 mL milk to pH 6.3 with 100 mM HCl, deproteinize with
10 mL MeCN. Centrifuge at 1932 g for 20 min and extract the aqueous phase with two 5 mL
portions of chloroform for 20 min (Caution! Chloroform is a carcinogen!). Centrifuge at 1932 g
for 20 min. Evaporate the organic phase to dryness, reconstitute the residue in 200 |xL mobile
phase, inject a 100 (xL aliquot. Serum. Adjust 2.5 mL serum to pH 6.3 with 100 mM HCl,
deproteinize with 10 mL MeCN. Centrifuge at 1932 g for 20 min and extract the aqueous phase
with two 5 mL portions of dichloromethane for 20 min. Centrifuge at 1932 g for 20 min. Evap-
orate the organic phase to dryness, reconstitute the residue in 200 jxL mobile phase, inject a
100 |xL aliquot
HPLC VARIABLES
Column: 15 X 3.9 4 |xm Nova Pack C18
Mobile phase: MeCN:20 mM KH2PO4 21:79, pH 5
Flow rate: 1.2
Detector: UV 225
CHROMATOGRAM
Retention time: 3.8
Internal standard: oxacillin
OTHER SUBSTANCES
KEYWORDS
serum; cow; oxacillin is IS
REFERENCE
Perez,B.| Prats,C; Castells,E.; Arboix,M. Determination of cloxacillin in milk and blood of dairy cows by high-
performance liquid chromatography, J.Chromatogr.B, 1997, 698, 155-160.
SAMPLE
Sample preparation: Plasma, serum. 100 |iL Plasma or serum + cloxacillin + 100 |xL 500 mM
pH 2.2 citric acid buffer + 20 |xL 500 mM HCl + 2.5 mL dichloromethane, extract. Remove the
residue in mobile phase, inject an aliquot. Urine. Dilute urine with water, inject an aliquot.
HPLC VARIABLES
Guard column: 50 X 2.1 ODS pellicular
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 7
Internal standard: cloxacillin (9)
OTHER SUBSTANCES
Extracted: metabolites, dicloxacillin, flucloxacillin
KEYWORDS
plasma; serum
REFERENCE
Thijssen,H.H.W. Analysis of isoxazolyl penicillins and their metabolites in body fluids by high-performance
SAMPLE
Sample preparation: Plasma. 1 mL Plasma + 20 |xL 4% aqueous sodium dodecyl hydrogen
sulfate solution, shake for 30 min, filter (Amicon MPS-I micropartition system, YMT mem-
brane) while centrifuging, adjust the pH of the ultrafiltrate to 6.3-6.5 with pH 4 citrate buffer,
inject a 500 jxL aliquot onto column A with mobile phase A and elute to waste, after 10 min
elute the contents of column A onto column B with mobile phase B, elute with mobile phase
B, monitor the effluent from column B. Urine. Make up 5-100 ^xL urine to 500 |xL with water,
inject onto column A with mobile phase A and elute to waste, after 10 min elute the contents
of column A onto column B with mobile phase B, elute with mobile phase B, monitor the effluent
from column B.
HPLCVARIABLES
Column: A 50 X 4 Nucleosil 5-C18; B 250 X 5 Nucleosil 5-C18
Mobile phase: A MeCN:33 mM NaH2PO4 5:95; B MeCN:33 mM NaH2PO4 20:80
Detector: UV 210
CHROMATOGRAM
Internal standard: oxacillin
OTHERSUBSTANCES
Extracted: penicillin V
KEYWORDS
plasma; column-switching; ultrafiltrate; oxacillin is IS
REFERENCE
Lintz,W.; Hirsch,L; Osterloh,G.; Schmidt-Bothelt,E.; Sous,H. Bioverfiigbarkeit von Penicillin V in einer
wajMgen Zubereitungsform [Bioavailability of penicillin V in aqueous dosage forms], Arzneimittelforschung,
1984, 34, 66-71.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
HPLC VARIABLES
Flow rate: 1
Detector: UV 225
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: amoxicillin, ampicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, penicillin
G, penicillin V
KEYWORDS
REFERENCE
matography, J.Assoc.Off.Anal.Chem., 1984, 67, 228-231.
SAMPLE
Matrix: milk
through a plug of glass wool. Collect 40 mL filtrate, add 2 mL buffer, evaporate to 1-2 mL
2 mL aliquot onto a 150 X 4.6 5 |xm Supelcosil LC-18 column, elute with MeCNiIO mM KH2PO4
compound (ca. 27.0 min), evaporate to <1 mL under reduced pressure, make up to 1 mL with
water, inject an aliquot. (Prepare the buffer by mixing 10 mM KH2PO4 and 10 mM Na2HPO4
in a 5:1 ratio, pH 6.)
HPLC VARIABLES
Mobile phase: MeCN.buffer 38:62 (Buffer was 2 mM phosphoric acid containing 8 mM potassium
Flow rate: 1
Detector: UV 215
REFERENCE
Moats,W.A.; Romanowski,R.D. Multiresidue determination of (3-lactam antibiotics in milk and tissues with the
aid of high-performance liquid chromatographic fractionation for clean up, J.Chromatogr.A, 1998,812,237-
247.
SAMPLE
Matrix: milk
Sample preparation: Condition a 6 mL 500 mg Bond Elut C18 SPE cartridge with 10 mL
MeOH, 10 mL water, 5 mL 2% NaCl, and 5 mL 100 mM pH 8 phosphate extraction buffer. Add
30 mL 100 mM pH 8 phosphate extraction buffer to 5 mL milk, add 1.65 mL 1 M sulfuric acid
to reach pH 4.0-4.5, vortex for 30 s, centrifuge at 2400 g for 10 min, add 600 |xL 5 M NaOH
to the supernatant to reach pH 8, vortex, centrifuge at 2400 g for 5 min. Add the supernatant
to a reservoir attached to the SPE cartridge, pull through the SPE cartridge at 3 mL/min,
remove the reservoir and elute with 1 mL MeCN:water 40:60. Add 500 |xL derivatizing reagent
to the eluate, vortex, heat at 65 for 10 min, cool to room temperature (protect from light),
inject a 100 JJLL aliquot of the derivatized sample. (Prepare the 100 mM pH 8 phosphate ex-
traction buffer as follows. Dissolve 15.6 g K2HPO4 dihydrate in 800 mL water, adjust pH to 8
with 10 M NaOH, make up to 1 L. Prepare the derivatizing reagent as follows. Weigh out 13.78
g 1,2,4-triazole, add 60 mL water, stir, add 10 mL 100 mM mercuric chloride solution, mix,
adjust pH to 9.0 0.5 with 5 M NaOH, dilute to 100 mL with water.)
HPLC VARIABLES
Mobile phase: MeCN:MeOH:buffer 37:5:58 (Prepare the 100 mM pH 6.5 phosphate buffer con-
taining 15 mM thiosulfate and 30 mM tetrabutylammonium hydrogen sulfate as follows. Weigh
4.969 g anhydrous NaH2PO4, 10.139 g Na2HPO4 dihydrate, 3.894 g sodium thiosulfate penta-
hydrate, and 10.186 g tetrabutylammonium hydrogen sulfate, dissolve in 800 mL water, adjust
pH to 6.5 with 5 M NaOH, dilute to 1 L with water, mix thoroughly, filter under vacuum (0.45
u-m).)
Flow rate: 1
Detector: UV 340
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Extracted: cloxacillin, dicloxacillin
KEY WpRDS
derivatization; SPE
REFERENCE
Verdon,E.; Couedor,P. Determination of isoxazolylpenicillins residues in milk by ion-pair reversed-phase high-
performance liquid chromatography after precolumn derivatization, J.Chromatogr.B, 1998, 705, 71-78.
SAMPLE
Matrix: milk
37, add 50 mL MeCN, shake vigorously for 1 min, centrifuge at 9000 g for 10 min, decant,
add 5 g NaCl, swirl to dissolve, add 100 mL dichloromethane, shake for 1 min, centrifuge at
1000 g for 10 min. Remove top aqueous layer and extract organic layer with 25 mL 10% NaCl
by shaking and centrifuging as before. Combine aqueous layers, add 1 mL 0.3% mercuric chlo-
ride in water, let stand 30 min, add 1 mL 2 M HCl, extract with three 50 mL portions of
dichloromethane by shaking each portion for 1 min and centrifuging at 1000 g for 10 min, filter
sulfate. Extract acid aqueous layer again with 25 mL dichloromethane. Combine dichlorometh-
ane layers and evaporate to dryness at 35 under reduced pressure. Dissolve residue in 2 mL
IS solution, inject a 20 |xL aliquot. (Prepare IS solution by dissolving 10 uL benzaldehyde in
100 mL dichloromethane, evaporate 1 mL to dryness under reduced pressure, dissolve residue
in 1 mL 10% acetic acid, add 0.5 mL 0.08% dansyl hydrazine in 10% acetic acid, let stand 90
min to overnight in the dark, transfer reaction mixture to a separatory funnel with three 25
mL portions of dichloromethane, add 5 mL 2 M HCl, shake for 1 min, wash organic layer with
5 mL 5% NaHCO3 solution, filter through 10-20 g anhydrous sodium sulfate. Extract acid
HPLC VARIABLES
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: penicillin G, phenethicillin, methicillin, cloxacillin, dicloxacillin, nafcillin
Interfering: penicillin V, phenethicillin
KEYWORDS
derivatization
REFERENCE
Munns,R.K; Shimoda,W.; Roybal,J.E.; Vieira,C. Multiresidue method for determination of eight neutral p-
lactam penicillins in milk by fluorescence-liquid chromatography, J.Assoc.Off.Anal.Chem., 1985, 68, 968-
971.
SAMPLE
Matrix: milk
nylon). Inject 50 (xL onto column with mobile phase A, run mobile phase A for 30 min and elute
HPLC VARIABLES
Column: 100 X 8 Radial-Pak 10 |xm ixBondapak C18
Flow rate: A 3; B 2
0.333 s.
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: penicillin V, ampicillin, methicillin, penicillin G, cloxacillin, nafcillin, dicloxacillin
KEYWORDS
column-switching
REFERENCE
1994, 17, 1755-1772.
SAMPLE
Matrix: milk
Sample preparation: Condition a Bond Elut C8 SPE cartridge with 5 mL MeOH and 5 mL
water. 20 mL Milk + 20 mL buffer, heat at 60 for 20 min or until milk curdles, centrifuge for
10 min, add the supernatant to the SPE cartridge, wash with two 2.5 mL portions of water,
elute with 2.5 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen, extract
the residue with three 100 |JLL portions of 50 mM pH 6.0 potassium phosphate buffer, filter (0.2
|xm), inject an aliquot of the filtrate. (Buffer was 545 mL 100 mM citric acid, 455 mL 200 mM
Na2HPO4, and 74.4 g EDTA, adjust to pH 4.5 with ammonium hydroxide, make up to 2 L with
water.)
HPLC VARIABLES
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ampicillin, ceftiofur, cephapirin, cloxacillin, dicloxacillin, nafcillin, penicillin G
KEYWORDS
SPE
REFERENCE
Al-Obaidy,S.S.; Po,A.L.W.; McKiernan,RJ.; Glasgow,J.F.T.; Millership,J. Assay of paracetamol and its metabo-
lites in urine, plasma and saliva of children with chronic liver disease, J.Pharm.Biomed.Anal., 1995, 13,
1033-1039.
SAMPLE
Matrix: milk
the residue with three 100 (JLL portions of 50 mM pH 6.0 potassium phosphate buffer, filter (0.2
jjim), inject an aliquot of the filtrate. (Buffer was 545 mL 100 mM citric acid, 455 mL 200 mM
water.)
HPLCVARIABLES
Column: 250 X 4.6 10 (xm Lichrosorb RP-8
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ampicillin, ceftiofur, cephapirin, cloxacillin, dicloxacillin, nafcillin, penicillin G
KEYWORDS
SPE
REFERENCE
(3-lactams in milk by liquid chromatography with microbial receptor assay, J.AOAC Int., 1995, 78, 1165-
1172.
SAMPLE
Matrix: milk
Sample preparation: Condition a 500 mg tC18 SPE cartridge (Waters) with 20 mL MeOH, 20
mL water, and 10 mL 2% NaCl. Centrifuge 30 mL milk at 1500 g for 10 min. Dilute a 10 mL
portion of the defatted milk with 20 mL water, add 200 |xL 2 |xg/mL penicillin V in pH 9.0
buffer, add 6 mL 170 mM sulfuric acid, add 5.6 mL 5% sodium tungstate, shake vigorously for
1 min, allow to stand for 5 min, check that the pH is in the range 4.6-4.8 (if it is outside this
range start again using a different volume of sodium tungstate solution), centrifuge at 1500 g
for 10 min, adjust the pH of the supernatant to 8.1-8.2 with 5 M and 0.1 M NaOH, filter (glass
fiber) the clear liquid phase. Pass the filtrate through the SPE cartridge at 2 mL/min, wash
with 2 mL water, dry by pulling air through the cartridge for 1 min, elute with 2 mL MeCN.
Add 150 |xL pH 9.0 buffer to the eluate and evaporate to about 100 JJLL under a stream of
nitrogen at 45-50, add 400 |xL pH 9.0 buffer, add 75 JJLL reagent I, vortex for 30 s, let stand
at room temperature for 10 min, use 500 |xL water to transfer the mixture to a separatory
funnel, add 20 mL dichloromethane, add 5 mL pH 2.45 buffer, shake for 1 min, let stand for
no more than 5 min. Remove the organic layer and evaporate it to dryness under reduced
pressure at 35-40, dissolve the residue in 500 |xL pH 9.0 buffer, add 75 |xL reagent I, vortex
for 30 s, let stand at room temperature for 10 min, add 450 jxL reagent II, vortex for 1 min,
heat at 55 1 for 30 min, cool, filter (0.45 |xm), inject a 150 |xL aliquot. (Prepare pH 9.0 buffer
by dissolving 0.34 g KH2PO4 in water, adjusting the pH to 9.0 with NaOH, and making up to
100 mL with water. Prepare pH 2.45 buffer by dissolving 2.72 g KH2PO4 in water, adjusting
the pH to 2.45 with phosphoric acid, and making up to 100 mL with water. Prepare reagent 1
by dissolving 1.13 g benzoic anhydride in MeCN, make up to 25 mL with MeCN. Prepare
reagent II by dissolving 6.905 g 1,2,4-triazole in 30 mL water and adding 5 mL 26 mM mercuric
chloride in water, adjust pH to 9.0 0.05 with 5 M NaOH, make up to 50 mL. Prepare reagents
I and II 1-4 h before use. Silanize glassware with dichlorodimethylsilane.)
HPLCVARIABLES
Mobile phase: Gradient. A as MeCN:buffer 10:90. B was MeCNrbuffer 30:70. A:B from 100:0 to
0:100 over 30 min, maintain at 0:100 for 13 min, return to initial conditions over 2 min, re-
equilibrate at initial conditions for 5 min. (Prepare buffer by dissolving 9.938 g Na2HPO4,
17.938 g NaH2PO4-H2O, and 4.964 g sodium thiosulfate in water, make up to 2 L with water,
pH 6.5.)
Flow rate: 1
Detector: UV 323
CHROMATOGRAM
Retention time: 34
Internal standard: penicillin V (28.5)
OTHER SUBSTANCES
Extracted: amoxicillin, ampicillin, cloxacillin, dicloxacillin, penicillin G
KEY WpRDS
derivatization; cow; SPE
REFERENCE
Sorensen,L.K.; Rasmussen,B.M.; Boison,J.O.; Keng,L. Simultaneous determination of six penicillins in cows'
raw milk by a multiresidue high-performance liquid chromatographic method, J.Chromatogr.B, 1997, 694,
383-391.
SAMPLE
Matrix: perfusate
Sample preparation: 200 JJLL Perfusate + 300 |xL MeCN, mix, centrifuge for 5 min, inject a 15
IxL aliquot.
HPLC VARIABLES
Guard column: 50 X 3.6 LiChrosorb RP-2
Column: Chemcosorb 5-ODS-H (Chemco, Japan)
Mobile phase: MeOH:pH 5.2 acetate buffer 50:50
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
KEYWORDS
rat; liver; pharmacokinetics
REFERENCE
Yasui,H.; Yamaoka,K; Fukuyama,T.; Nakagawa,T. Effect of liver intoxication by carbon tetrachloride in hepatic
local disposition of oxacillin using moment characteristics as index, Drug Metab.Dispos., 1995, 23, 779-
785.
SAMPLE
Matrix: perfusate
HPLC VARIABLES
Column: 150 X 4.6 Chemcosorb 5-ODS-H (Chemco, Osaka)
Mobile phase: MeOH:acetate buffer 50:50, pH 5.2
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
KEYWORDS
rat
REFERENCE
Ohata,Y.; Yamaoka,K; Yasui,H.; Nakagawa,T. Consideration on moments of outflow profile in liver perfusion
system with change in perfusate flow rate using oxacillin as model drug, Biol.Pharm.Bull., 1996, 19, 8 3 -
87.
SAMPLE
Matrix: solutions
sodium salt soluble), inject a 10 |xL aliquot. (Preparation of l-(2,5-dihydroxyphenyl)-2-bro-
HPLC VARIABLES
Column: 250 X 4 7 urn RP-18 LiChrocart (Merck)
Mobile phase: MeOH:100 mM pH 6.5 sodium acetate 58:42
Flow rate: 1
electrode
CHROMATOGRAM
Retention time: 19
OTHER SUBSTANCES
Extracted: carbenicillin, cephapirin, cloxacillin, dicloxacillin, hetacillin, methicillin, nafcillin,
penicillin G
KEY WpRDS
derivatization
REFERENCE
Munns,R.K; Roybal,J.E.; Shimoda,W.; Hurlbut,J.A. l-(4-Hydroxyphenyl)-, l-(2,4-dihydroxyphenyl)-and l-(2,5-
SAMPLE
Matrix: solutions
10-20 |xL aliquot of the ultrafiltrate.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 220
OTHER SUBSTANCES
Also analyzed: penicillin V
REFERENCE
bumin for penicillin and cephem antibiotics, J.Pharmacobiodyn.y 1992, 15, 99106.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Ultra-Turrax) 25 g tissue with 25 mL MeCN for 1 min, add
5 mL 500 mM pH 2.2 phosphate buffer while the homogenizer is still running, add 65 mL
MeCN, homogenize for 1 min, centrifuge at 4000 g for 10 min. Remove the supernatant and
add it to 7 g NaCl and 50 mL dichloromethane, shake for 2 min, allow to stand for 30 min.
Remove the upper organic layer and add it to 5 g anhydrous sodium sulfate, shake for 30 s,
filter through a cotton-wool plug, evaporate to about 4 mL under reduced pressure at 30, add
3 mL dichloromethane, evaporate to about 4 mL, add 3 mL light petroleum, evaporate to about
0.5 mL, Suspend this residue with sonication in three 3 mL portions of light petroleum and
place these fractions in a separate tube, rinse the original tube with 2 mL pH 7 phosphate
buffer. Add the phosphate buffer rinse to the light petroleum extracts, vortex for 30 s, centri-
fuge, remove the aqueous layer. Extract the light petroleum layer with 2 mL pH 7 phosphate
buffer and with two 1.5 mL portions of pH 7 phosphate buffer, combine all the aqueous phase,
centrifuge, inject a 200 |xL aliquot on to column A and elute to waste with mobile phase B,
after 15 min elute to waste with mobile phase C at 2 mL/min, after 10 min elute the contents
of column A on to column B with mobile phase D, after 2 min remove column A from the circuit,
elute column B with mobile phase D, monitor the effluent from column B. (Wash column A
with mobile phase A at 2 mL/min for 7 min, with mobile phase A at 1 mL/min for 5 min, with
mobile phase B at 2 mL/min for 8 min, and with mobile phase B at 1 mL/min for 6 min.)
Next Page
HPLC VARIABLES
Column: A 4 X 4 5 jxm LiChrospher 100 RP- 18e; B 250 X 4 5 |xm LiChrospher 100 RP-18e
Mobile phase: A MeCNrwater 50:50; B 20 mM pH 7 phosphate buffer; C MeCN:20 mM pH 3
phosphate buffer 10:90; D MeCN:200 mM pH 3.0 phosphate buffer 35:65 containing 2 mM
disodium EDTA
Flow rate: 1 (except where indicated)
Detector: E, Merck Model L3500, glassy carbon working electrode +0.65 V, stainless-steel aux-
iliary electrode, Ag/AgCl reference electrode following post-column reaction. The column efflu-
ent flowed through a 10 m X 0.3 mm ID woven PTFE coil illuminated by a UV 254 low-pressure
mercury lamp to the detector.
CHROMATOGRAM
Retention time: 7.1
OTHER SUBSTANCES
Extracted: cloxacillin, dicloxacillin, penicillin V, penicillin G
KEYWORDS
post-column reaction; post-column photochemical derivatization; cow; muscle; column-switching
REFERENCE
Lihl,S.; Rehorek,A.; Petz,M. High-performance liquid chromatographic determination of penicillins by means
of automated solid-phase extraction and photochemical degradation with electrochemical detection,
J.Chromatogr.A, 1996, 729, 229-235.
SAMPLE
Matrix: urine
Sample preparation: Filter (0.45 |xm), inject a 5 JJLL aliquot.
HPLCVARIABLES
Guard column: 50 X 5 LiChrosorb RP-2
Column: 250 X 4.6 LiChrosorb RP-18
Mobile phase: MeOH:30 mM pH 5.6 acetate buffer 33:66
Flow rate: 1.5
Injection volume: 5
Detector: UV 254
CHROMATOGRAM
Retention time: 28
OTHER SUBSTANCES
KEYWORDS
rat; human; pharmacokinetics
REFERENCE
Murai,Y.; Nakagawa,T.; Yamaoka,K; Uno,T. High performance liquid chromatographic analysis and pharma-
cokinetic investigation of oxacillin and its metabolites in man, Chem.Pharm.Bull.(Tokyo), 1981, 29, 3290
3297.
Previous Page
Oxamniquine
Molecular formula: Ci 4 H 2 IN 3 O 3
Merck Index: 7051
Lednicer No.: 2 372
SAMPLE
Sample preparation: Basify 1 mL microsomal incubation with 2 drops of 5 M NaOH, add 4 mL
diethyl ether, vortex for 30 s, centrifuge at 3000 rpm for 5 min, repeat extraction. Combine the
organic layers and evaporate them to dryness under a stream of nitrogen at 30, reconstitute
the residue in 200-500 |xL mobile phase, filter (0.45 u-m), inject an aliquot.
HPLC VARIABLES
Column: 100 X 4 Chiral-AGP al-acid glycoprotein (ChromTech)
Mobile phase: MeCN: 10 mM pH 5.80 sodium phosphate buffer 0.6:99.4
Flow rate: 0.9
Detector: UV 246
CHROMATOGRAM
Retention time: 2.3 (L), 3.4 (D)
Limit of detection: 2.3 ng (d), 0.3 ng (1)
KEYWORDS
chiral; rat; mouse; liver
REFERENCE
Noctor,T.A.G.; Fell,A.E; Kaye,B. High-performance liquid chromatographic resolution of oxamniquine enanti-
omers: application to in vitro metabolism studies, Chirality, 1990, 2, 269-274.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 100 X 5 Enantiopac al-glycoprotein (AGP) (LKB)
Mobile phase: IsopropanolrlO mM phosphate buffer containing 100 mM NaCl 0.5:99.5, pH 5.85
Detector: UV 260
CHROMATOGRAM
Retention time: 8 (L), 12.5 (D)
KEYWORDS
chiral
REFERENCE
Fell,A.R; Noctor,T.A.G.; Mama,J.E.; Clark,B.J. Computer-aided optimisation of drug enantiomer separation in
chiral high-performance liquid chromatography, J.Chromatogr., 1988, 434, 377-384.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 100 X 4.6 5 jxm Chiral-AGP (ChromTech)
Mobile phase: Isopropanohbuffer 99.5:0.5 (Buffer was 10 mM NaH2PO4 containing 100 mM
NaCl, pH 5.85.)
Flow rate: 1
Detector: UV 246
CHROMATOGRAM
KEYWORDS
chiral
REFERENCE
Abushoffa,A.M.; Clark,B.L Resolution of the enantiomers of oxamniquine by capillary electrophoresis and high-
performance liquid chromatography with cyclodextrins and heparin as chiral selectors, J.Chromatogr.A,
1995, 700, 51-58.
Oxandrolone
Merck Index: 7054
Lednicer No.: 1 174
SAMPLE
min, centrifuge, inject a 10 jxL aliquot of the supernatant. Tablets. Grind a tablet to a fine
powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 jim), discard first 5 mL of filtrate,
inject a 10 jxL aliquot of the remaining filtrate. Suspensions (aqueous). Make up 5 mL to 50
remaining filtrate.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 210
CHROMATOGRAM
Retention time: 4.7
OTHER SUBSTANCES
Simultaneous: methandrostenolone, nandrolone, norgestrel, testosterone, dehydroepiandroste-
rone (UV 210), mibolerone, methyltestosterone, methandriol (UV 210), norethindrone acetate,
calusterone, mesterolone (UV 210), norethandrolone, trenbolone acetate, benzyl benzoate, nan-
drolone acetate, testosterone acetate, stanozolol, oxymetholone, nandrolone propionate, meth-
enolone acetate, testosterone propionate, aspirin, caffeine, formebolone, benzyl alcohol, testo-
lactone, cortisone
Interfering: fluoxymesterone, norethindrone, boldenone, ethisterone
KEY WORDS
REFERENCE
Walters,M. J.; Ayers,R.J.; Brown,D.J. Analysis of illegally distributed anabolic steroid products by liquid chro-
Oxatomide
Merck Index: 7058
Lednicer No.: 3 173
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 205.2
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
Gaillard,Y.; Pe*pin,G. Use of high-performance liquid chromatography with photodiode-array UV detection for
163.
Oxazepam
Merck Index: 7059
Lednicer No.: 1 366
SAMPLE
gastric contents, and bile four times with water. Mix 4 mL sample with 10 (xL 1 mg/mL pra-
zepam and 1 mL pH 7.4 phosphate buffer, vortex briefly, add 4 mL diethyl ether and mix for
15 min (Spiramix 10, Denley, UK). Separate the organic layer, add 4 mL diethyl ether to
extraction sample, mix. Evaporate combined organic layers to dryness under a stream of dry
air at 50. Purify extracts by partition between 1 mL MeCN and 2 mL heptane, separate MeCN
layer, evaporate it to dryness, reconstitute the residue in 100 jxL MeOH and inject a 20 |xL
aliquot of the solution.
HPLC VARIABLES
Guard column: 20 X 4.6 5 |xm Apex II ODS
Column: 150 X 4.6 5 ^m Apex II ODS
Mobile phase: MeCNiMeOH: 10 mM phosphoric acid: 10 mM Na2HPO4 40:20:36:4
Flow rate: 1
Detector: UV 240
CHROMATOGRAM
Retention time: 3.8
OTHER SUBSTANCES
Extracted: diazepam, nitrazepam, temazepam
KEYWORDS
REFERENCE
Pounder,D.J.; Adams,E.; Fuke,C; Langford,A.M. Site to site variability of postmortem drug concentrations in
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 |xL 1 (xg/mL loxapine in isopropanol:diethylamine
and add it to 100 |xL 250 mM HCl, vortex for 30 s, inject a 50 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: amitriptyline, chlorpromazine, desipramine, desmethldiazepam, desmethylchlordi-
azepoxide, diazepam, doxepin, haloperidol, imipramine, nortriptyline, thiothixene
Noninterfering: molindone, perphenazine, trifiuoperazine
Interfering: chlordiazepoxide, desmethyldoxepin, fluphenazine
KEYWORDS
plasma
REFERENCE
Kiel,J.S.; Abramson,R-K.; Morgan,S.L.; Voris,J.C. A rapid high performance liquid chromatographic method for
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 2 mL water + 50 |xL 3.2 ixg/mL estazolam in MeOH + 2
mL 100 mM NaOH, mix gently, add 8 mL diethyl ether, shake for 15 min, centrifuge at 2500
rpm for 5 min. Remove 4 mL of the organic layer and evaporate it to dryness under a stream
of nitrogen at 40, reconstitute the residue in 100 u,L mobile phase, vortex for 30 s, inject a 50
|JLL aliquot.
HPLC VARIABLES
Column: 50 X 4.6 Shim-pack FLC-C8 (Shimadzu)
Mobile phase: MeOHibuffer 53:47 (Buffer was 5 mM Na2HPO4 adjusted to pH 6.0 with phos-
phoric acid.)
Flow rate: 0.6
Detector: UV 254
CHROMATOGRAM
Retention time: 5
Internal standard: estazolam (4)
OTHER SUBSTANCES
Extracted: diazepam, temazepam, nordiazepam, triazolam
Simultaneous: sulpride, bromazepam, nitrazepam, flunitrazepam
Noninterfering: haloperidol, trihexyphenidyl
Interfering: clorazepate
KEY WORDS
REFERENCE
Tada,K; Moroji,T.; Sekiguchi,R.; Motomura,H.; Noguchi,T. Liquid-ehromatographic assay of diazepam and
its major metabolites in serum, and application to pharmacokinetic study of high doses of diazepam in
schizophrenics, Clin.Chem., 1985, 31, 1712-1715.
SAMPLE
Matrix: blood
Sample preparation: Filter (0.5 |xm) serum, inject 200 u-L directly onto column A with mobile
phase A, run with mobile phase A for 1.5 min then change to mobile phase B over 0.1 min,
wash column A with mobile phase B for 10.5 min, backflush column A onto column B with
mobile phase C for 7.5 min then switch column B out of circuit, elute column B with mobile
phase C and monitor the eluant, re-equilibrate column A with mobile phase A for at least 5
min.
HPLC VARIABLES
Column: A 15 X 3.2 5 (xm Brownlee ODS; B 250 X 1 5 |xm Adsorbosphere ODS
Mobile phase: A 10 mM sodium dodecyl sulfate; B water; C MeOH:water 65:35
Flow rate: A 1; B 1; C 0.06
Detector: UV 242
CHROMATOGRAM
Retention time: 25
OTHER SUBSTANCES
Simultaneous: temazepam, nordiazepam, diazepam
KEYWORDS
serum; column-switching; microbore
REFERENCE
Koenigbauer,M.J.; Curtis,M.A. Use of micellar mobile phases and microbore column switching for the assay of
drugs in physiological fluids, J.Chromatogr., 1988, 427, 277-285.
SAMPLE
Matrix: blood
Sample preparation: Inject 100-200 |xL plasma onto column A with mobile phase A and elute
to waste, after 5 min backflush the contents of column A onto column B with mobile phase B,
after 5 min remove column A from the circuit, elute column B with mobile phase B, monitor
the effluent from column B. Wash column A with MeCN:water 60:40 at 1 mL/min for 6 min
then re-equilibrate with pH 7.5 buffer for 10 min.
HPLC VARIABLES
Column: A 45 X 4 12 fjum TSK-gel G 3 PW (Tosohass); B 75 X 4.6 Ultrasphere ODS C18 3 jxm
Mobile phase: A 50 mM pH 7.5 phosphate buffer; B Gradient. A was MeCN. B was 65 mM
KH2PO4 + 1% diethylamine adjusted to pH 5.4 with phosphoric acid. A:B 22:78 for 5 min, to
25:75 over 10 min, to 60:40 over 15 min.
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: alprazolam, bromazepam, chlordiazepoxide, clobazam, clonazepam, clorazepate, clo-
tiazepam, desmethylclobazam, desmethyldiazepam, diazepam, flunitrazepam, loflazepate, lor-
azepam, medazepam, nitrazepam, prazepam, temazepam, tetrazepam, tofisopam, triazolam
Noninterfering: carbamazepine, phenytoin, ethosuximide, phenobarbital, primidone, valproic
acid
Interfering: estazolam
KEYWORDS
REFERENCE
Lacroix,C; Wojciechowski,R; Danger,P. Monitoring of benzodiazepines (clobazam, diazepam and their main
active metabolites) in human plasma by column-switching high-performance liquid chromatography,
J.Chromatogr., 1993, 617, 285-290.
SAMPLE
Matrix: blood
stoppered tube for 1 min, add 6 mL NiCl2, rock for 5 min, add 15 mL l-chlorobutane:isobutyl
alcohol.THF 40:40:20, centrifuge at 2500 g for 15 min. Remove organic phase and repeat the
water 80:20, inject a 20 |ULL aliquot. (Na2WO4 prepared by mixing 10 g Na2WO4.2H2O in 38 mL
water.)
HPLC VARIABLES
Mobile phase: A = MeCN; B = 20 mM n-hexylamine adjusted to pH 4 with 85% phosphoric acid.
A:B from 25:75 to 40:60 over 25 min to 50:50 over another 5 min
Detector: UV 230
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Extracted: bromazepam, clonazepam, diazepam, flunitrazepam, flurazepam, medazepam,
nitrazepam
Also analyzed: buprenorphine, caffeine, cocaine, codeine, diamorphine, ethylmorphine, lido-
caine, methaqualone, morphine, naloxone, noscapine, papaverine, pentazocine, procaine
KEYWORDS
whole blood
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: 200 u.L Plasma + 500 \LL 23 u-g/mL acetophenone in MeCN, vortex for 20
s, decant, filter (13 mm diameter, 0.2 |xm), inject a 100 |xL aliquot of the filtrate.
HPLC VARIABLES
Guard column: 20 X 2 Pellicular ODS (Whatman)
Column: 250 X 4.6 Ultracarb 7 ODS 30 (Phenomenex)
Mobile phase: MeCN:waterrglacial acetic acid 45:55:0.5
Flow rate: 1
Detector: UV 238
CHROMATOGRAM
Retention time: 6.8
Internal standard: acetophenone (9.1)
KEYWORDS
rat; mouse; plasma; pharmacokinetics
REFERENCE
Yuan,J.; Goehl,T.J.; Hong,L.; Clark,J.; Murrill,E.; Moore,R. Toxicokinetics of oxazepam in rats and mice,
J.Pharm.ScL, 1994, 83, 1373-1379.
SAMPLE
Matrix: blood
HPLC VARIABLES
Column: 300 X 3.9 4 fxm NovaPack C18
Flow rate: 0.8
Detector: UV 229
CHROMATOGRAM
KEYWORDS
ide; chlorophenacinone; doxepin; nimodipine; diphenhydramine; cyclizine; histap^rodine;
REFERENCE
TVacqui,A-; Kintz,R; Mangin,R Systematic toxicological analysis using HPLC/DAD, J.Forensic ScL, 1995, 40,
254-262.
SAMPLE
Matrix: blood
Sample preparation: Make 200 |xL serum alkaline with borate buffer, extract with cyclohexane:
dichloromethane 60:40. Remove the organic layer and evaporate it to dryness, reconstitute the
residue in mobile phase, inject an aliquot
HPLC VARIABLES
Column: C18 DB (Supelco)
Detector: UV 254
OTHER SUBSTANCES
Extracted: bromazepam, clobazam, diazepam, fluvoxamine, lorazepam
KEYWORDS
serum
REFERENCE
Vandenberghe,H.; MacDonald,J.C. Analysis of fluvoxamine, clobazam and other benzodiazepines on the same
HPLC system (Abstract 40), Ther.Drug Monit, 1995, 17, 393.
SAMPLE
HPLCVARIABLES
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
methacin, lidocaine, lorazepam, malathion, medazepam, midazolam, paraoxon, penicillin G,
pentobarbital, prazepam, propoxyphene, prothiophos, quinine, salicylic acid, secobarbital,
KEYWORDS
REFERENCE
Kruger,RB.; Albrecht,C.F.De V.; Jaarsveld,RR Use of guanidine hydrochloride and ammonium sulfate in com-
SAMPLE
Sample preparation: Condition a 100 mg C18 Bond Elut SPE cartridge with 2 mL MeOH then
2 mL water. 1 mL Plasma or 1 mL urine or 10 mL dialysate + 1 mL MeCN:water 30:70, vortex
for 10 s, centrifuge at 4000 g for 5 min, add to SPE cartridge, wash with 2 mL MeCN:water
20:80, dry for 3 to 4 min, elute with four 200 |xL aliquots of MeOH, evaporate combined eluates
under nitrogen and take up residue in 100 (xL MeOH, inject a 20 jxL aliquot.
HPLC VARIABLES
Guard column: 4 x 4 Lichrocart Lichrosorb RP 18-5
Column: 5 |xm Lichrocart 125-4 Lichrospher 100 RP 18 endcapped
Mobile phase: MeCNrIO mM pH 5.6 buffer 40:60 (Prepare 1 M buffer from 94.8 mL 1M KH2PO4
+ 5.2 mL 1 M K2HPO4, dilute to 10 mM with water.)
Flow rate: 1.6
Detector: UV 254
CHROMATOGRAM
Internal standard: climazolam (5.62)
Limit of quantitation: 5ng/mL
OTHERSUBSTANCES
Simultaneous: temazepam
KEYWORDS
plasma; SPE
REFERENCE
Chopineau,J.; Rivault,R; Sautou,V.; Sommier,M.F. Determination of temazepam and its active metabolite, ox-
azepam in plasma, urine and dialysate using solid-phase extraction followed by high performance liquid
chromatography, J.Lig. Chromatogr., 1994, 17, 373-383.
SAMPLE
Sample preparation: Whole blood, stomach contents. 1 mL Whole blood or stomach contents +
IS + 500 JULL 1 M potassium carbonate + 8 mL n-hexane:ethyl acetate 70:30, vortex for 1 min,
centrifuge at 2000 rpm for 5 min. Remove the organic layer and evaporate it to dryness under
a stream of nitrogen, reconstitute the residue in 100 |xL initial mobile phase, vortex, centrifuge,
inject a 50 |xL aliquot. Tissue. Cut 1 g tissue into small pieces, make up to 5 mL with water,
homogenize (Ultraturrax). 1 mL Homogenate + IS + 500 jxL 1 M potassium carbonate + 8 mL
n-hexane:ethyl acetate 70:30, vortex for 1 min, centrifuge at 2000 rpm for 5 min. Remove the
in 100 |xL initial mobile phase, vortex, centrifuge, inject a 50 JAL aliquot. Urine. 1 mL Urine +
IS + 250 JJLL concentrated HCl, heat at 100 for 1 h, cool, adjust pH to 9 with NaOH pellets
and 1 M potassium carbonate. Add 8 mL n-hexane:ethyl acetate 70:30, vortex for 1 min, cen-
trifuge at 2000 rpm for 5 min. Remove the organic layer and evaporate it to dryness under a
stream of nitrogen, reconstitute the residue in 100 |xL initial mobile phase, vortex, centrifuge,
HPLCVARIABLES
Guard column: 10 X 2.1 pellicular reverse phase (Chrompack)
Column: 100 X 3 5 |xm Chromspher C8 (Chrompack)
Mobile phase: Gradient. MeOH containing 0.03% isopropylamine:water containing 0.03% iso-
propylamine 20:80 for 2 min, to 30:70 over 0.2 min, maintain at 30:70 for 1.8 min, to 40:60
over 0.2 min, maintain at 40:60 for 0.3 min, to 43:57 over 0.5 min, to 45:55 over 1 min, to 52:
48 over 1 min, to 58:42 over 2.5 min, to 75:25 over 1 min, maintain at 75:25 for 4.5 min, return
to initial conditions over 0.3 min, re-equilibrate for 3.7 min before next injection.
Flow rate: 0.7
Detector: UV 230
CHROMATOGRAM
Retention time: 9
Internal standard: camazepam (10.5), clotiazepam (11)
OTHER SUBSTANCES
Extracted: nitrazepam, bromazepam, flunitrazepam, diazepam, nordiazepam
Simultaneous: clonazepam, triazolam, alprazolam, brotizolam, chlordiazepoxide, loprazolam,
cloxazolam, prazepam, flurazepam, medazepam, lormetazepam
KEYWORDS
whole blood; liver; kidney
REFERENCE
Lambert,W.E.; Meyer,E.; Xue-Ping,Y; De Leenheer,A.P. Screening, identification, and quantitation of benzodi-
azepines in postmortem samples by HPLC with photodiode array detection, J.Anal.Toxicol., 1995,19, 3 5 -
40.
SAMPLE
Matrix: blood, milk
Sample preparation: 500 |xL Plasma or milk + 25 |xL 5 (xg/mL flurazepam in water:MeCN 2.5:
97.5 + 500 fxL 67 mM pH 7.4 phosphate buffer + 7 mL diethyl ether, extract for 15 min (A).
Remove ether layer and add it to 1 mL 1.5 M HCl, shake for 15 min. Freeze and discard ether
phase. Basify aqueous phase with 1 mL 2 M NaOH, extract with 7 mL diethyl ether for 15
min. Evaporate ether at 37 under a stream of nitrogen and take up residue in mobile phase,
inject an aliquot. (For plasma ONLY ether at (A) can be evaporated at 37 under a stream of
nitrogen, take up residue in mobile phase, inject an aliquot.)
HPLC VARIABLES
Guard column: 25 X 4 5 (xm LiChrospher 60 RP-select B
Column: 125 X 4 5 |jim LiChrospher 60 RP-select B
Mobile phase: MeCN: 10 mM KH2PO4 31:69, adjusted to pH 2.80 with phosphoric acid
Flow rate: 2
Detector: UV 241
CHROMATOGRAM
Retention time: 3.8
OTHER SUBSTANCES
Extracted: diazepam, nordiazepam, temazepam
KEYWORDS
plasma; human; rabbit
REFERENCE
Stebler,T.; Guentert,T.W. Determination of diazepam and nordazepam in milk and plasma in the presence of
oxazepam and temazepam, J.Chromatogr., 1991, 564, 330-337.
SAMPLE
Sample preparation: Blood. Condition a Bond-Elut C18 SPE cartridge with 2 mL MeOH and
30 mL water. Mix 1 mL serum with 4 mL water and 10 |JLL 40 |xM IS in EtOH. Adjust to pH
4.0 with 250 mM sulfuric acid. Add to the SPE cartridge, wash with 10 mL water, dry under
vacuum, elute with 5 mL MeOH, evaporate the eluate to dryness under a stream of nitrogen,
dissolve the residue in 200 |xL MeOH, inject a 20 JAL aliquot. Urine. Mix 100 |xL urine with 50
JULL 40 |xM IS in EtOH, dilute to 1 mL with water, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm LiChrospher 100 RP 18
Mobile phase: MeCN:isopropanol:25% orthophosphoric acid:water 18:7.5:1.2:73.3 (?), pH 2.05
Detector: UV 230
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
SPE; serum; sheep; pharmacokinetics
REFERENCE
Mawa,R.; Mis,D.; Gagnieu,M.C; Grancher,D.; Petit-Ramel,M.; Bressolle,R; Vallon,J.J. Simple high-perfor-
mance liquid chromatographic separation of oxazepam and its diastereoisomeric glucuronides in serum.
Applications in a pharmacokinetic study in sheep, J.Chromatogr.B, 1996, 677, 331-338.
SAMPLE
Sample preparation: Wash a C2 Bond-Elut SPE cartridge with 1 column volume methanol and
1 column volume buffer. Add 1 mL of urine buffered with pH 6 100 mM phosphate buffer or
plasma buffered with pH 8 100 mM phosphate bufferto the SPE cartridge, wash with 3 column
volumes of water, wash with 1 mL of MeOH:water 30:70, elute with 1 mL of MeOH:water 60:
40. Evaporate to the eluate to dryness and take up the residue in 200 jxL mobile phase, inject
an aliquot.
HPLC VARIABLES
Column: 35 X 4.6 5 |xm ultrabase C18 (Scharlau)
Flow rate: 1
Detector: UV 228
CHROMATOGRAM
Retention time: 2
Internal standard: prazepam (7)
OTHER SUBSTANCES
Also analyzed: diazepam, temazepam, nordazepam, brotizolam, adinazolam, midazolam
KEYWORDS
plasma; SPE.
REFERENCE
Casas,M.; Berrueta,L.A.; Gallo,B; Vicente,F. Solid-phase extraction of 1,4-benzodiazepines from biological flu-
ids, J.Pharm.Biomed.AnaL, 1993, 11, 277-284.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 228.7
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: feces, urine
Sample preparation: Urine. Centrifuge at 1000 rpm for 10 min, inject an aliquot of the super-
natant. Feces. Homogenize (Kinematica polytron) feces in 100 mM pH 6.8 sodium acetate
buffer. 1 mL Homogenate + 3 mL MeCN, vortex vigorously, centrifuge at 1000 rpm for 10 min,
pass through a C18 Sep-Pak.
HPLC VARIABLES
Column: 250 X 4.6 5 ^m Microsorb C18
Mobile phase: Gradient. MeOH:10 mM pH 6.5 dibutylamine phosphate from 15:85 to 100:0 over
23 min.
Flow rate: 1.2
CHROMATOGRAM
KEYWORDS
rat; radiolabeled
REFERENCE
Griffin,R.J.; Burka,L.T. Metabolism and elimination of oxazepam in F344 rats, Drug Metab.Dispos., 1995,23,
232-239.
SAMPLE
Matrix: hair
hair + 1 mL MeOH, heat at 55 for 18 h, adjust pH to 9.5-10. 1 mL Extract + 1 jxg protriptyline
+ 1 mL water + 1 mL 200 mM sodium carbonate buffer, mix, extract with hexane:butanol 95:
5 for 20 min. Remove the organic layer and add it to 100 |xL 0.2% orthophosphoric acid, mix
for 20 min, inject a 30 |xL aliquot of the aqueous layer.
HPLC VARIABLES
diethylamine.)
Flow rate: 2
Detector: UV 214
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: amitriptyline, desipramine, diazepam, dothiepin, flunitrazepam, haloperidol, imip-
ramine, imipramine, nitrazepam, nortriptyline, temazepam
KEYWORDS
REFERENCE
Couper,F.J.; McIntyre,I.M.; Drummer,0.H. Extraction of psychotropic drugs from human scalp hair, J.Forensic
ScL, 1995, 40, 83-86.
SAMPLE
ness under reduced pressure at 40, reconstitute the residue in 100 jxL mobile phase, inject an
aliquot.
HPLC VARIABLES
Column: 250 X 6.2 7 \xm Zorbax silica
Mobile phase: Hexane:dichloromethane:isopropanol 77:20:3
Flow rate: 2
Detector: UV 232
CHROMATOGRAM
Retention time: 37
Internal standard: diazepam (12)
OTHER SUBSTANCES
Extracted: halazepam
KEYWORDS
human; liver; normal phase; pharmacokinetics
REFERENCE
Lu,X.-L.; Guengerich,F.R; Yang,S.K. Stereoselective metabolism of prazepam and halazepam by human liver
microsomes, Drug Metab.Dispos., 1991, 19, 637-642.
SAMPLE
Sample preparation: Irradiate MeCN solutions with UV light, inject a 20-100 JXL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 265
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: desmethylchlordiazepoxide, demoxepam, desmethyldiazepam, chlordiazepoxide
REFERENCE
Soentjens-Werts,V.; Dubois,J.G.; Atassi,G.; Hanocq,M. High-performance liquid chromatographic determination
of chlordiazepoxide, its metabolites and oxaziridines generated after UV irradiation, J.Chromatogr.A, 1994,
662, 255-262.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4 5 |xm ChiraDex, LichroCART
Flow rate: 0.5
Detector: UV 220
CHROMATOGRAM
KEYWORDS
chiral
REFERENCE
Cabrera,K; Jung,M.; Fluck,M.; Schurig,V. Determination of enantiomerization barriers by computer simulation
of experimental elution profiles obtained by high-performance liquid chromatography on a chiral stationary
phase, J.ChromatogrA, 1996, 731, 315-321.
SAMPLE
Matrix: solutions
HPLCVARIABLES
% for 2 min, to 0:100 over 15 min, maintain at 0:100 for 5 min.
Flow rate: 0.5
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: chlordiazepoxide, desalkylflurazepam, diazepam, flurazepam, norehlordiazepox-
ide, nordiazepam, prazepam
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
feine, cocaine, codeine, doxylamine, fiuoxetine, glutethimide, hexobarbital, hypoxanthine,
levorphanol, LSD, meperidine, mephobarbital, methadone, methylphenidate, methyprylon, N-
norcodeine, oxycodone, phenylpropanolamine, prilocaine, procaine, terfenadine
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 2
Injection volume: 5
Detector: UV 210
CHROMATOGRAM
OTHERSUBSTANCES
one, mefenamic acid, methyltestosterone, nicotine, phentermine, phenylpropanolamine, pro-
gesterone, sulfamethazine, sulfanilamide, testosterone, testosterone propionate, tranylcypro-
mine, tripelennamine
KEYWORDS
REFERENCE
Hill,D.W.; Kind>A.J. The effects of type B silica and triethylamine on the retention of drugs in silica based
reverse phase high performance chromatography, J.Liq.Chromatogr., 1993, 16, 39413964.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Flow rate: 0.5
Detector: UV 200
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: norchlordiazepoxide, chlordiazepoxide, nordiazepam, desalkylflurazepam, diaze-
pam, flurazepam, prazepam
Also analyzed: amitriptyline, amphetamine, chlorpromazine, desipramine, desmethyldoxepin,
diethylpropion, doxepin, ephedrine, fenfluramine, imipramine, mesoridazine, methampheta-
mine, nortriptyline, phentermine, phenylpropanolamine, promazine, thioridazine, thiothixene,
trifluoperazine
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Dissolve the compound, S-trolox methyl ether (Fluka), dicyclohexylcar-
bodiimide, and 4-dimethylaminopyridine in dichloromethane, stir at room temperature for 1 h,
filter (0.45 |xm), inject an aliquot.
HPLC VARIABLES
Column: 300 X 0.32 5 |xm LiChrosorb Diol
Mobile phase: Carbon dioxide:MeOH 90:10
Detector: UV 254
CHROMATOGRAM
Retention time: 19.1 (second peak)
KEYWORDS
derivatization; subcritical fluid chromatography; chiral; density of mobile phase 0.65 g/mL; reso-
lution (Rs) 1.2
REFERENCE
Almquist,S.R.; Petersson,R; Walther,W.; Markides,K.E. Direct and indirect approaches to enantiomeric sepa-
ration of benzodiazepines using micro column techniques, J.Chromatogr.A, 1994, 679, 139-146.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.5-1
CHROMATOGRAM
Retention time: 5.2
OTHER SUBSTANCES
bamol, methotrimeprazine, nicotine, procaine, promazine, propafenone, propranolol, salicylam-
ide, temazepam, tetracaine, thiopental, triamterene, verapamil, zolpidem, zopiclone
REFERENCE
in toxicological analysis, J.Liq.Chromatogr., 1994,17, 4131-4144.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4 5 jxm LiChroCART ChiraDex O-cyclodextrin chemically bonded to silica)
(Merck)
Mobile phase: MeOH: 10 mM pH 2.8 NaH2PO4 40:60
Flow rate: 0.5
Detector: UV 220
CHROMATOGRAM
KEYWORDS
chiral
REFERENCE
Cabrera,K; Lubda,D. Influence of temperature on chiral high-performance liquid chromatographic separations
of oxazepam and Prominal on chemically bonded p-cyclodextrin as stationary phase, J.Chromatogr.A, 1994,
666, 433-438.
SAMPLE
Matrix: solutions
Sample preparation: Dilute in MeOH to a concentration of 10-80 mg/mL, inject an aliquot
HPLCVARIABLES
Column: 150 X 3.9 5 |xm Nova pak RP 18
Flow rate: 0.82
Detector: UV 254
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: bromazepam, nitrazepam, flunitrazepam, clobazam, chlordiazepoxide, loraze-
pam, tofisopam, chlorazepate, diazepam
KEYWORDS
conditions are optimized
REFERENCE
Guillaume,Y.; Guinchard,C. Study and optimization of column efficiency in HPLC: Comparison of two methods
for separating ten benzodiazepines, J.Liq.Chromatogr., 1994, 17, 1443-1459.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
acid, nitrazepam, norepinephrine, nortriptyline, noscapine, oxycodone, oxymorphone, oxy-
persantine, phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine,
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Chiralpak OD (Daicel)
Mobile phase: Carbon dioxide:EtOH:diethylamine 79.5:20:0.5
Flow rate: 2
Detector: UV 220
CHROMATOGRAM
Retention time: 8, 8.9 (enantiomers)
KEYWORDS
SFC; chiral; pressure 350 bar
REFERENCE
Kot,A.; Sandra,R; Venema,A. Sub- and supercritical fluid chromatography on packed columns: A versatile tool
for the enantioselective separation of basic and acidic drugs, J.Chromatogr.ScL, 1994, 32, 439-448.
SAMPLE
Matrix: solutions
HPLC VARIABLES
pressure.)
Flow rate: 0.1
Detector: UV 254
CHROMATOGRAM
KEYWORDS
REFERENCE
Pharmaceuticals using narrow-bore liquid chromatography, J.Pharm.BiomedAnaL, 1995, 13, 695-699.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Guard column: 10 X 3 not otherwise specified
Column: 100 X 4 Chiral-AGP (ChromTech)
Flow rate: 0.9
Detector: UV 220
CHROMATOGRAM
KEYWORDS
chiral
REFERENCE
FitosJ.; Visy,J.; Simonyi,M.; Hermansson,J. Separation of enantiomers of benzodiazepines on the Chiral-AGP
column, J.ChromatogrA, 1995, 709, 265-273.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 3.9 4 |xm Nova pak C18
Flow rate: 1.1
Detector: UV 254
CHROMATOGRAM
Retention time: 7.2
OTHER SUBSTANCES
Simultaneous: bromazepam, chlordiazepoxide, clobazam, clorazepate, diazepam, flunitrazepam,
lorazepam, nitrazepam, tofisopam
REFERENCE
Guillaume,Y.; Guinchard,C. Marked difference between acetonitrile/water and methanol/water mobile phase
systems on the thermodynamic behavior of benzodiazepines in reversed phase liquid chromatography, Chro-
matographia, 1995, 41, 84-87.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 3.9 4 |xm Nova pack C18
Flow rate: 0.8
Detector: UV 254
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
Simultaneous: bromazepam, chlordiazepoxide, clobazam, clorazepate, diazepam, flunitrazepam,
lorazepam, nitrazepam, tofisopam
REFERENCE
Guillaume,Y.; Guinchard,C. Thermodynamic behavior of mixed benzodiazepines by a new liquid chromato-
graphic method, Chromatographia, 1995, 40, 193-196.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxycodone, oxymetazoline, pa-
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 100 \xM solution in buffer, inject a 20 JJIL aliquot.
HPLCVARIABLES
Mobile phase: EtOH:50 mM pH 5.5 KH2PO4 5:95
Flow rate: 0.8
Detector: UV
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: bepridil, manidipine, nicardipine
Interfering: lorazepam
KEYWORDS
chiral; a = 4.55
REFERENCE
protein as a new chiral stationary phase in high-performance liquid chromatography, J.Chromatogr.A> 1995,
704, 55-65.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 4.6 cellulose 3,5-dimethylphenylcarbamate/10-undecenoate bonded to allylsilica
Mobile phase: Heptanedsopropanol 90:10
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
KEYWORDS
chiral; a 1.27
REFERENCE
Oliveros,L.; Lopez,P.; Minguillon,C; Franco,P. Chiral chromatographic discrimination ability of a cellulose 3,5-
dimethylphenylcarbamate/10-undecenoate mixed derivative fixed on several chromatographic matrices,
J.Liq.Chromatogr., 1995, 18, 1521-1532.
SAMPLE
Matrix: urine
Sample preparation: 500 (xL Urine + N-ethylnordiazepam + chlorpheniramine + 100 |xL buffer,
|xL mobile phase B, with 200 |iL mobile phase C, and with 1.15 mL mobile phase D. Elute
HPLC VARIABLES
3.2 11 (Jim Aminex A-28 (Bio-Rad); C 25 X 3.2 5 |xm C8 (Phenomenex) + 150 X 4.6 5 jjim silica
(Macherey-Nagel)
Mobile phase: A 0.1% pH 8.0 potassium borate buffer; B 6 mM KH 2 PO 4 containing 5 mM tetra-
phoric acid; C MeCNibuffer 40:60 (Buffer was 6 mM KH 2 PO 4 containing 5 mM tetramethylam-
D MeCN:buffer 33:67 (Buffer was 6 mM KH 2 PO 4 containing 5 mM tetramethylammonium
buffer 70:30 (Buffer was 6 mM KH 2 PO 4 containing 5 mM tetramethylammonium hydroxide,
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: diazepam, phenylpropanolamine, phentermine, amphetamine, phenmetrazine, lido-
caine, ephedrine, pentazocine, methamphetamine, desipramine, nortriptyline, diphenhydra-
mine, methadone, imipramine, flurazepam, amitriptyline, morphine, codeine, hydromorphone,
hydrocodone, caffeine
Interfering: cotinine, benzoylecgonine, secobarbital, phenobarbital, nordiazepam
KEYWORDS
column-switching
REFERENCE
341.
SAMPLE
Matrix: urine
Sample preparation: Adjust pH of 2.5 mL urine to 5 with dilute HCl, add 400 |xL 200 mM pH
5 acetate buffer, add 50 |xL (3-glucuronidase, heat at 45 for 5 h, cool, adjust pH to 8-9 with
25% NaOH, add to an Extrelut 3 SPE cartridge, let stand for 10 min, elute with 15 mL chlo-
roform:isopropanol 90:10. Evaporate the eluate to dryness under a stream of nitrogen at 40,
reconstitute the residue in 2 mL 100 mM pH 9.2 borate buffer. Extract this solution twice with
3 mL diethyl ether, evaporate the combined organic phases to dryness under a stream of ni-
trogen at 40, reconstitute the residue in 100 jxL mobile phase, inject a 20 jxL aliquot.
HPLC VARIABLES
Guard column: 4 X 4 5 jutm Lichrospher 100 RP8
Column: 250 X 4 5 |xm Lichrospher 100 RP8
Mobile phase: MeCN.bufifer 45:55 (Buffer was 1.361 g KH2PO4 in 950 mL, add 1.3 mL meth-
anesulfonic acid, adjust pH to 3.5 with 5 M KOH, make up to 1 L with water.)
Flow rate: 1
Detector: UV 234
CHROMATOGRAM
Retention time: 5.2
OTHER SUBSTANCES
Extracted: desmethyldiazepam
trimipramine
KEYWORDS
SPE
REFERENCE
of abuse in urine, JAnal.Toxicol, 1992,16, 217-222.
SAMPLE
Matrix: urine
Sample preparation: Condition a 100 mg Bond-Elut C2 SPE cartridge with MeOH and 10 mM
pH 6.0 phosphate buffer. 5 mL Urine + 1250 U p-glucuronidase, adjust pH to 5.0 with HCl,
heat at 37 for 24 h. Add 5 |xg nordiazepam, buffer to pH 6.0 with 500 |xL 100 mM pH 6.0
phosphate buffer, add to the SPE cartridge, wash with 3 volumes of water, wash with 1 mL
MeOH:water 25:75, wash with 1 mL water, elute with 1 mL MeOHrwater 60:40. Evaporate the
eluate to dryness, reconstitute in 200 |xL mobile phase, inject an aliquot.
HPLC VARIABLES
Column: 35 X 4.6 5 |xm Ultrabase C18 (Scharlau)
Flow rate: 0.5
Detector: F ex 364 em 469 following post-column reaction. The effluent from the column mixed
with acetic acid pumped at 1.1 mL/min and the mixture flowed through a 15 m X 0.5 mm i.d.
coil of PTFE tubing at 100 to the detector
CHROMATOGRAM
Retention time: 6
Internal standard: nordiazepam (8.5)
KEYWORDS
REFERENCE
Berrueta,L.A.; Gallo,B-; Vicente,F. Analysis of oxazepam in urine using solid-phase extraction and high-perfor-
mance liquid chromatography with fluorescence detection by post-column derivatization, J.Chromatogr.,
1993, 616, 344-348.
SAMPLE
Matrix: urine
Sample preparation: 1 mL Urine + 100 |xL 5 mM pH 5.5 acetate buffer + 25 |xL p-glucuronidase/
arylsulfatase (0.235/0.065 U, Calbiochem), mix, heat at 37 for 16 h, add 50 \LL 5-50 |xg/mL
prazepam in MeOH, add 1 mL saturated trisodium phosphate, add 3 mL dichloromethane,
vortex for 2 min, centrifuge at 1610 g for 5 min. Remove a 2 mL aliquot of the organic layer
and add it to 2 mL hexane and 2 mL 6 M HCl, vortex for 2 min, centrifuge at 1610 g for 5
min. Remove 1 mL of the aqueous phase and adjust pH to 6 with 1 mL 6 M NaOH and 1 mL
saturated trisodium phosphate, add 3 mLL dichloromethane, vortex for 2 min, centrifuge at
nitrogen at 50, reconstitute the residue in 150 |xL mobile phase, inject a 60 uX aliquot.
HPLC VARIABLES
Column: 250 X 4 5 |xm LiChrospher 100 RP-18(e)
Mobile phase: MeOH:water:triethylamine 30:70:0.1 adjusted to pH 5.5 with phosphoric acid
Flow rate: 0.7
Detector: UV 240
CHROMATOGRAM
Retention time: 7.0
OTHER SUBSTANCES
Extracted: desmethyldiazepam, diazepam, temazepam
Simultaneous: amitriptyline, caffeine, carbamazepine, chlordiazepoxide, chlorpromazine, clon-
azepam, desipramine, flunitrazepam, flurazepam, haloperidol, imipramine, levomepromazine,
maprotiline, nitrazepam, nortriptyline, perphenazine, phenobarbital, phenytoin, sulpride,
thioridazine, triazolam
Interfering: mianserin
REFERENCE
Chiba,K; Horii,H.; Chiba,T.; Kato,Y; Hirano,T.; Ishizaki,T. Development and preliminary application of high-
performance liquid chromatographic assay of urinary metabolites of diazepam in humans, J.Chromatogr.B,
1995, 668, 77-84.
Oxazolam
Merck Index: 7061
SAMPLE
Matrix: blood
Sample preparation: 500 (xL Serum + 20 ^L 20 |xg/mL IS + 200 (xL 1 M potassium carbonate
Evaporate the organic phase under a stream of nitrogen at 40. Dissolve the residue in 100 uL
HPLCVARIABLES
Column: 100 X 4.6 2 |xm TSK gel Super-ODS (A) or 100 X 4.6 5 jxm Hypersil ODS-C18 (B)
Flow rate: 0.65
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
azolam, lorazepam, nitrazepam, triazolam
KEYWORDS
serum
REFERENCE
J.Chromatogr.B, 1996, 682, 173-178.
Oxcarbazepine
Merck Index: 7063
SAMPLE
Matrix: blood
Sample preparation: 250 |xL Plasma + 2 |xg 10-methoxycarbamazepine + 25 (xL 1 M NaOH +
1.2 mL dichloromethane, mix for 15 min, centrifuge. Remove the organic layer and evaporate
it to dryness under a stream of nitrogen at 40, reconstitute the residue in 20 |xL MeCN, inject
a 10 JLJLL aliquot.
HPLC VARIABLES
Flow rate: 1.8
Detector: UV 215
CHROMATOGRAM
Retention time: 4.8
Internal standard: 10-methoxycarbamazepine (9.3)
OTHER SUBSTANCES
Extracted: metabolites, carbamazepine, phenobarbital, primidone
Noninterfering: clobazam, clonazepam, diazepam, ethosuximide, phenytoin, valproic acid
KEYWORDS
plasma
REFERENCE
Elyas,A.A.; Goldberg,V.D.; Patsalos,P.N. Simple and rapid micro-analytical high-performance liquid chromat-
ographic technique for the assay of oxcarbazepine and its primary active metabolite 10-hydroxycarbazepine,
J.Chromatogr., 1990, 528, 473-479.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL Bond Elut C18 SPE cartridge with 2 column volumes
of MeCN and 2 column volumes of water. Dilute plasma with two volumes 2 |xg/mL 10,11-
dihydrocarbamazepine in 67 mM pH 5 Sorensen's phosphate buffer. Add 500 |xL water then
300 |xL diluted plasma to the SPE cartridge, let stand for 2 min, wash with 1 column volume
of water, wash with 1 column volume of MeCN:water 5:95, elute with 250 jxL acetone. Evap-
orate the eluate to dryness, reconstitute the residue in 500 |xL mobile phase, inject a 75 JXL
aliquot.
HPLC VARIABLES
Guard column: 5 jxm LiChrospher C18
Column: 100 X 5 4 |xm Nova-Pak C18 radial compression
Flow rate: 1.2
Detector: UV 214
CHROMATOGRAM
Retention time: 6.2
Internal standard: 10,11-dihydrocarbamazepine (13.8)
OTHER SUBSTANCES
Simultaneous: carbamazepine, chlormethiazole edisylate, clonazepam, diazepam, ethoxyzolam-
ide, nitrazepam, phenobarbital, phenytoin, prednisolone, prednisone
Noninterfering: acetaminophen, acetazolamide, ampicillin, aspirin, caffeine, cefuroxime, chlo-
rothiazide, theophylline, vitamin K
KEYWORDS
REFERENCE
Hartley,R.; Green,M.; Lucock,M.D.; Ryan,S.; Forsythe,W.I. Solid phase extraction of oxcarbazepine and its me-
tabolites from plasma for analysis by high performance liquid chromatography, Biomed.Chromatogr., 1991,
5, 212-215.
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL 50 mg Bond-Elut C18 SPE cartridge with 1 mL MeOH
and 1 mL water. 100 |xL Plasma + 100 \xL water + 10 jxL 198 IJLM IS in MeOH:water 50:50,
vortex for a few s, add to the SPE cartridge, wash with 2 mL 20 mM K2HPO4, wash with 2
mL MeOH:water 5:95, elute with 250 |xL MeOH, add 950 |xL water to the eluate, mix, inject
a 500 |xL aliquot.
HPLCVARIABLES
Guard column: 33 X 4.6 37-53 |xm pellicular ODS (Whatman)
Mobile phase: MeCN:MeOH:10 mM pH 5.0 KH2PO4 9:11:80
Flow rate: 2
Detector: UV 210
CHROMATOGRAM
Retention time: 9.5
Internal standard: 5,6-dmydro-ll-oxo-llH-dibenz[b,e]azepme-5-carboxamide (Ciba-Geigy)
(14.0)
OTHER SUBSTANCES
Simultaneous: carbamazepine, phenobarbital, phenytoin
Noninterfering: valproic acid
KEYWORDS
REFERENCE
Rouan,M.C; Decherf,M.; Le Clanche,V.; Lecaillon,J.B.; Godbillon,J. Automated microanalysis of oxcarbazepine
and its monohydroxy and transdiol metabolites in plasma by liquid chromatography, J.Chromatogr.B, 1994,
658, 167-172.
SAMPLE
Matrix: blood
Sample preparation: Condition an Extrelut-1 glass SPE cartridge with 5 mL dichloromethane:
isopropanol 90:10, dry under nitrogen. 1 mL Serum + 100 fiL 2 |xg/mL carbamazepine in EtOH,
vortex for 30 s, add to the SPE cartridge, let stand for 10 min, elute with 5 mL dichloromethane:
isopropanol 90:10. Evaporate the eluate to dryness under a stream of nitrogen, reconstitute
the residue in 100 (xL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 fjim Chiralcel OD + 250 X 4.6 10 |xm Chiralcel ODH
Column temperature: 40 (second column only)
Flow rate: 0.9
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: phenytoin, valproic acid
Interfering: phenobarbital
KEYWORDS
serum; chiral (for metabolites)
REFERENCE
Pichini,S.; Altieri,L; Passa,A-R-; Zuccaro,P.; Pacifici,R. Stereoselective bioanalysis of oxcarbazepine and the
enantiomers of its metabolites by high-performance liquid chromatography, J.Liq.Chromatogr., 1995, 18,
1533-1541.
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 6 mL MTBE, vortex for 30 s, shake for 5 min, centrifuge
nitrogen in a warm water bath, reconstitute the residue in 40 |xL MeOH:water 5:2, inject a 20
jxL aliquot.
HPLCVARIABLES
Column: 125 X 4 4 |xm Superspher 60 RP-select B (Merck)
Mobile phase: MeCN:20 mM KH2PO4 20:80 containing 0.05% triethylamine, pH 6.30
Flow rate: 1
Detector: UV 212
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: metabolites, carbamazepine
KEYWORDS
serum
REFERENCE
Pienimaki,R; Fuchs,S.; Isojarvi,J.; Vahakangas,K. Improved detection and determination of carbamazepine and
oxcarbazepine and their metabolites by high-performance liquid chromatography, J.Chromatogr.B, 1995,
673, 97-105.
SAMPLE
Matrix: blood
Sample preparation: 50 |xL Plasma + 100 |xL MeCN, centrifuge, inject a 5 jxL aliquot of the
supernatant.
HPLC VARIABLES
Column: 100 X 4.6 3.5 jim Zorbax SB
Mobile phase: MeCNiMeOH: 10 mM pH 7.1 phosphate buffer 7:34:59
Flow rate: 1.5
Injection volume: 5
Detector: UV 220
CHROMATOGRAM
Limit of detection: <1 fxM
OTHER SUBSTANCES
Extracted: carbamazepine, carbamazepine epoxide, hydroxycarbamazepine, lamotrigine, phe-
nobarbital, phenytoin
Also analyzed: ibuprofen, naproxen, trimethoprim
KEYWORDS
plasma
REFERENCE
Lessing,U.; Vielemeyer,O.; Heilmann,R; Schoneshofer,M. Routine determination of serum primidone levels with
a fully automated liquid chromatographic method: Comparison with an immuno-assay-technique (Abstract
100), Ther.DrugMonit, 1995, 17, 408.
SAMPLE
Sample preparation: Dialyze with artificial CSF (brain) or physiological saline (blood, liver)
using a 20 kDa cut-off membrane (CMA-10). Mix 40 |xL dialysate with 25 JJLL 500 ng/mL car-
bamazepine in artificial CSF, inject a 50 jxL aliquot.
HPLC VARIABLES
Guard column: 30 X 2.1 Spheri 5-ODS
Column: 220 X 2.1 5 \xm Spheri-5 ODS
Mobile phase: MeCN:buffer 22:78 (Buffer was 50 mM KH2PO4 adjusted to pH 6.5 with 5 M
NaOH.)
Flow rate: 0.4
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
rat; brain; liver; narrow-bore; dialysate
REFERENCE
Van Belle,K.; De Koster,V.; Sarre,S.; Ebinger,G.; Michotte,Y. Narrow-bore liquid chromatographic assay for
oxcarbazepine and its major metabolite in rat brain, liver and blood microdialysates, J.Chromatogr.B, 1994,
657, 149-154.
SAMPLE
Matrix: dialysate
Sample preparation: 10 |xL Dialysate + 2.5 |xL 1 jxg/mL carbamazepine-10,11-epoxide in water,
HPLC VARIABLES
Column: 150 X 0.8 3 |xm Hypersyl C18 BDS
Flow rate: 0.025
Detector: UV 220
CHROMATOGRAM
Retention time: 4.5
Internal standard: carbamazepine-10,11-epoxide (4)
OTHER SUBSTANCES
KEYWORDS
rat; microcolumn
REFERENCE
Van Belle,K; Verfaillie,L; Ebinger,G.; Michotte,Y. Liquid chromatographic assay using a microcolumn coupled
to a U-shaped optical cell for high-sensitivity ultraviolet absorbance detection of oxcarbazepine and its major
metabolite in microdialysates, J.Chromatogr.B, 1995, 672, 97-102.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 0.8 3 |xm Hypersil C18 BDS
Flow rate: 0.025
Detector: UV 220 (8 mm path length, quartz ball lens)
CHROMATOGRAM
Retention time: 4.5
OTHER SUBSTANCES
KEYWORDS
microcolumn
REFERENCE
Van Belle,K; Ebinger,G.; Michotte,Y. An LC assay using a microcolumn coupled to a U-shaped optical cell for
high sensitivity UV absorbance detection of oxcarbazepine and its major metabolite in microdialysates,
Oxeladin
CAS Registry No.: 468-61-1, 52432-72-1
Merck Index: 7064
Lednicer No.: 1 90
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
OTHER SUBSTANCES
ifensine, nortriptyline, noscapine, orphenadrine, oxprenolol, oxymetazolin, papaverine, pargy-
line, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone,
REFERENCE
Oxfendazole
Merck Index: 7069
Lednicer No.: 3 353
SAMPLE
dryness, reconstitute in 50 |L MeOH, sonicate, inject a 5 |xL aliquot.
HPLCVARIABLES
Flow rate: 1.5
Injection volume: 5
Detector: UV 292
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Extracted: albendazole, thiabendazole, cambendazole, mebendazole, oxibendazole, fenbendazole,
parbendazole
KEYWORDS
plasma; sheep
REFERENCE
J.Pharm.ScL, 1980, 69, 422-423.
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 |xL 10 (xg/mL albendazole in MeOH + 200 |xL 500
the residue in 60 |xL MeOH, sonicate for 2 min, inject a 20 |xL aliquot.
HPLC VARIABLES
Flow rate: 0.9
Detector: UV 292
CHROMATOGRAM
Retention time: 0.9
OTHER SUBSTANCES
Extracted: febantel, fenbendazole, oxfendazole sulfone
KEY WORDS
plasma; sheep
REFERENCE
SAMPLE
Matrix: feed
Sample preparation: 20 g Pulverized feed + 100 mL MeOH.glacial acetic acid 90:10, shake on
a gyrorotary shaker at 45 for 30 min, cool to room temperature,, centrifuge an aliquot at 1000
rpm for 5 min. 10 mL Supernatant + 4 mL reagent, mix thoroughly, let stand for 5 min, make
up to 25 mL with water, mix thoroughly, centrifuge at 1000 rpm. 15 mL Supernatant + 100
mL phosphate solution, adjust pH to 8 with 3.2 mL 1 M NaOH, mix well, add 50 mL dichlo-
romethane, shake gently, repeat extraction twice more with 40 mL portions of dichloromethane.
Filter extracts through anhydrous sodium sulfate, add 15 mL 20 |xg/mL 2-guanadinobenzimi-
dazole in MeOH to the filtrate, evaporate to dryness under a stream of nitrogen using a steam
bath, reconstitute the residue in 10 mL mobile phase, sonicate for 5 min, filter (Millipore polyvic
2 ^,m), inject 100 fxL of the filtrate. (Reagent was 100 g zinc acetate dihydrate in 300 mL water,
stir until dissolved, add 1.5 mL glacial acetic acid, make up to 500 mL with water. Phosphate
solution was 35 g K2HPO4 + 5Og NaCl in 1 L water.)
HPLCVARIABLES
Column: 250 X 4.6 10 |xm Partisil PSX-SCX
Mobile phase: MeCNiIO mM phosphoric acid:10 mM KH2PO4 50:25:25
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 10
Internal standard: 2-guanadinobenzimidazole (39)
Limit of detection: 0.012%
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Shah,G.; Bradley,D.; Shek,E. Liquid chromatographic determination of oxfendazole in swine feeds,
SAMPLE
Sample preparation: 1 g Paste + 75 mL MeOH, sonicate for 15 min, shake for 1 min, sonicate
for 5 min, shake for 30 s, cool to room temperature, make up to 100 mL with MeOH, centrifuge
an aliquot at 2000 rpm for 10 min. Mix 2 mL supernatant with 2 mL 200 |xg/mL methyl paraben
in MeOH, make up to 25 mL with MeCN:water:phosphoric acid 20:80:1, filter (Millipore 0.6
|xm polyvic), inject a 10 fxL aliquot of the filtrate.
HPLCVARIABLES
Guard column: 70 X 2.1 CO:PELL ODS
Column: 250 X 4.6 Partisil-5 ODS-3
Mobile phase: MeCN:buffer 20:80 (Buffer was 720 mL 1.38 g/L NaH2PO4-H2O + 80 mL 1.42 g/
L Na2HPO4.)
Flow rate: 1.5
Detector: UV 200
CHROMATOGRAM
Internal standard: methyl paraben (15.71)
OTHER SUBSTANCES
Simultaneous: degradation products, trichlorfon
KEYWORDS
horse; paste
REFERENCE
Fleitman,J.; Neu,D.; Benjamin,E. Analysis of pharmaceutical dosage forms for oxfendazole: II. Simultaneous
liquid chromatographic determination of oxfendazole and trichlorfon in equine paste, J.Assoc.Off.AnaL
Chem., 1986, 69, 24-28.
SAMPLE
Matrix: milk
Sample preparation: 50 g Milk + 1 mL 1 (uig/mL IS in 100 mM HCl, swirl, add 150 mL acetone,
shake mechanically for 10 min, centrifuge. Remove the acetone extract and add it to 10 mL
pH 8 phosphate buffer and 300 mL chloroform, shake for 10 min, let stand for 10 min. Dry the
chloroform layer briefly over 80 g anhydrous sodium sulfate, filter through glass wool, rinse
flask with 50 mL chloroform:acetone 2:1, filter rinse through glass wool. Combine filtrates and
evaporate them to an oily residue under reduced pressure at 37, take up the residue in 50 mL
hexane and 50 mL MeCN, shake for 5 min, wash the MeCN layer twice with 25 mL portions
of hexane. Evaporate the MeCN layer and take up the residue in 50 mL ethyl acetate. Extract
the ethyl acetate layer with three 10 mL portions of 200 mM HCl. Combine the aqueous layers
and wash them with two 10 mL portions of hexane. Add 6 mL 1 M NaOH to the aqueous layer,
adjust the pH to 8 with 300 mg sodium bicarbonate, extract twice with 25 mL portions of ethyl
acetate. Dry the ethyl acetate extracts over 5 g anhydrous sodium sulfate, evaporate to dryness,
reconstitute in 10 mL ethyl acetate, filter (0.5 |xm Millipore fluoropore FHLP 1300), evaporate
the filtrate to dryness, reconstitute in 200 jxL MeOH, inject a 25 JXL aliquot.
HPLC VARIABLES
Guard column: Co.Pell ODS
Mobile phase: MeCN:water 24.5:75.5
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 8.5
Internal standard: [5-(4-methylsulfinylphenoxy)-lH-benzimidazol-2-yl]carbamate(Syntex) (11)
KEYWORDS
pharmacokinetics
REFERENCE
TsinaJ.W.; Matin,S.B. Determination of oxfendazole in cow milk by reversed-phase high-performance liquid
chromatography, J.Pharm.Scl, 1981, 70, 858-860.
SAMPLE
Matrix: milk
Sample preparation: Condition a 3 mL Bond Elut silica SPE cartridge with 2 mL dichlorometh-
ane. 10 g Milk + 5 mL 1 M sodium carbonate, mix, add 150 mL ethyl acetate, add 1 mL 10
mg/mL BHT in ethyl acetate, blend (tissuemizer) at high speed for 5 min, add 10 g anhydrous
sodium sulfate, blend for 1 min, let settle for 2-3 min, filter (No. 41 paper), add another 150
mL ethyl acetate to the sodium sulfate, blend for 2 min, filter. Combine the filtrates and evap-
orate them to dryness under vacuum. Rinse out flask with two 10 mL portions of hexane and
two 10 mL portions of 1 M phosphoric acid. Combine rinses, shake vigorously for 2 min, extract
the hexane layer with two 10 mL portions of 1 mL phosphoric acid. Combine all the aqueous
layers and wash them with 5 mL hexane, adjust the pH to 8-9 by slowly adding about 9 mL
10 M KOH (use an ice bath), add 50 mL ethyl acetate, shake vigorously for 2 min, repeat
extraction. Filter the organic layers through 40 g anhydrous sodium sulfate, wash the sodium
sulfate with 25 mL ethyl acetate. Combine the organic layers, add 200 |xL 10 mg/mL BHT in
ethyl acetate, evaporate to dryness under vacuum. Take up the residue in two 3 mL portions
of dichloromethane and add them to the SPE cartridge, wash with 5 mL dichloromethane,
elute with 5 mL dichloromethane:MeOH 75:25. Evaporate the eluate to dryness under nitrogen,
reconstitute in 1 mL mobile phase, vortex, filter (0.2 |xm), inject an aliquot.
HPLCVARIABLES
Guard column: 30 X 4.6 pellicular C18 (Alltech)
Flow rate: 1
Detector: UV 298
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Extracted: thiabendazole
KEYWORDS
cow; SPE
REFERENCE
Tai,S.S.; Cargile,N.; Barnes,C.J. Determination of thiabendazole, 5-hydroxythiabendazole, fenbendazole, and
oxfendazole in milk, J.Assoc.Off.AnaLChem., 1990, 73, 368-373.
SAMPLE
Matrix: premix
Sample preparation: Weigh out premix containing 67.5 mg oxfendazole, add 50 mL acetone,
shake gently on a mechanical shaker for 2 h, make up to about 190 mL with MeOH, sonicate
for 15 min, cool to room temperature, shake well, make up to 250 mL with MeOH, centrifuge
an aliquot at 2000 rpm for 10 min. Mix 5 mL supernatant with 2 mL 240 |xg/mL methyl paraben
in MeOH, make up to 25 mL with MeCN:water:phosphoric acid 20:80:1, filter (Millipore 0.6
ixm polyvic), inject a 10 |xL aliquot of the filtrate.
HPLCVARIABLES
Guard column: 70 X 2.1 CO:PELL ODS
Column: 250 X 4.6 Partisil-5 ODS-3
Mobile phase: MeCN.buffer 20:80 (Buffer was 720 mL 1.38 g/L NaH2PO4.H2O + 80 mL 1.42 g/
L Na2HPO4.)
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 20
Internal standard: methyl paraben (15)
OTHER SUBSTANCES
REFERENCE
Fleitman,J.; Neu,D.; Visor,G. Analysis of pharmaceutical dosage forms for oxfendazole: I. Reverse phase liquid
chromatographic determination of oxfendazole in swine premix, J.Assoc.Off.Anal.Ckem., 1986, 69, 20-24.
SAMPLE
Matrix: tissue
MeOH and dry under vacuum aspiration. Gently blend 2 g C18 material, 0.5 g liver, and 10
IJLL 40 u-g/mL mebendazole in DMF in a glass pestle for 1 min until homogeneous in appearance.
Place in a 10 mL syringe barrel plugged with filter paper (Whatman No. 1), cover with filter
paper, compress to 4.5 mL, place a 100 |xL pipette tip on the barrel to restrict flow, wash with
8 mL hexane, elute with 8 mL MeCN. Pass the eluate through 0.5 g activated alumina (EM
Science Type F-20 80-200 mesh) between filter paper in a 10 mL syringe barrel (wash column
with 4 mL MeCN just before use). Evaporate the eluate to dryness under a stream of nitrogen,
reconstitute the residue in 100 JJLL MeOH and 400 |xL 17 mM phosphoric acid, sonicate for 5-
10 min, centrifuge at 17000 g for 5 min, filter the supernatant (0.45 |xm), inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 300 X 4 10 |xm Micro Pak ODS (Varian)
Flow rate: 1
Detector: UV 290
CHROMATOGRAM
Retention time: 6
Internal standard: mebendazole (9)
OTHER SUBSTANCES
Extracted: albendazole, thiafendazole, fenbendazole
KEYWORDS
matrix solid-phase dispersion; liver
REFERENCE
Long,A.R.; Malbrough,M.S.; Hsieh,L.C; Short,C.R.; Barker,S.A. Matrix solid phase dispersion isolation and
SAMPLE
Matrix: tissue
Sample preparation: Condition a 2.8 mL 500 mg 40 |xm 60 A Bond Elut silica SPE cartridge
filter it (No. 41 paper), add 150 mL acetone to material remaining in blender, blend at low
speed for 2-3 min, filter, wash solid with 10 mL EtOH. Combine all the filtrates and evaporate
them to dryness under vacuum at 30-35 (beware of bumping). Rinse out flask with two 10 mL
portions of hexane and two 10 mL portions of 1 M phosphoric acid, combine rinses, shake
vigorously for 2 min, allow to separate for 10 min, extract the hexane layer twice more with
10 mL portions of 1 M phosphoric acid. Combine all the aqueous layers and wash them with
10 mL hexane, adjust the pH of the aqueous layer to 8.5 1.0 by slowly adding about 9 mL
10 M KOH while using an ice bath. Extract twice with 50 mL ethyl acetate (2 min shaking),
25 mL ethyl acetate. Combine the ethyl acetate layers, add 200 JJLL 10 mg/mL BHT in ethyl
filter (0.2 jjim), inject a 50 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm C18 (Alltech)
Mobile phase: MeOHibuffer 53:47 (Buffer was 1.15 g (NH4)H2PO4 in 950 mL water, adjust pH
Flow rate: 1
Detector: UV 298
CHROMATOGRAM
Retention time: 8.5
OTHER SUBSTANCES
Extracted: thiabendazole, mebendazole
KEYWORDS
cow; liver; SPE
REFERENCE
LeVan,L.W.; Barnes,C.J. Liquid chromatographic method for multiresidue determination of benzimidazoles in
beef liver and muscle: collaborative study, J.Assoc.Off.Anal.Chem., 1991, 74, 487-493.
SAMPLE
Matrix: tissue
Sample preparation: 3 g Pulverized tissue + 7 mL water, homogenize (Silverson) for 1 min, add
20 mL MeOH, sonicate for 15 min, centrifuge at 2000 g at 5 for 10 min. Remove 10 mL of the
supernatant and add it to 8 mL light petroleum (bp 40-60), shake gently for 30 s, centrifuge.
Add the aqueous phase to 14 mL 500 mM NaH2PO4 and 8 mL diethyl etherrethyl acetate 60:
40, shake gently for 30 s, centrifuge, repeat the extraction with 5 mL and 3 mL portions of
diethyl ether:ethyl acetate 60:40. Combine the upper organic layers and evaporate them to
dryness under a stream of nitrogen at 60, reconstitute the residue in 200 u,L MeCN:water 50:
50, sonicate for 5 min, inject a 50 (xL aliquot.
HPLC VARIABLES
Column: 125 X 4 LiChrosorb RP18
Mobile phase: MeCN:THF:water 30:10:60 containing 50 mM ammonium acetate
Flow rate: 1
Detector: MS, Vestec Model 20IA, thermospray, positive-ion chemical ionization, electron beam
250 (JiA, electron multiplier 2000 V, source block 250, tip heater 250, lens assembly 150,
vaporizer probe 10 below take-off point, m/z 316
CHROMATOGRAM
Retention time: 3
KEYWORDS
sheep; muscle; liver; pharmacokinetics; LC-MS
REFERENCE
Blanchflower,W.J.; Cannavan,A.; Kennedy,D.G. Determination of fenbendazole and oxfendazole in liver and
muscle using liquid chromatography-mass spectrometry, Analyst, 1994, 119, 1325-1328.
Oxibendazole
Merck Index: 7070
Lednicer No.: 2 352
SAMPLE
Next Page
HPLC VARIABLES
Flow rate: 1.5
Injection volume: 5
Detector: UV 292
CHROMATOGRAM
Retention time: 5.3
OTHER SUBSTANCES
Extracted: albendazole, oxfendazole, cambendazole, thiabendazole, mebendazole, fenbendazole,
parbendazole
KEYWORDS
plasma; sheep
REFERENCE
J.Pharm.ScL, 1980, 69, 422-423.
Oxiracetam
Merck Index: 7076
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL 35% perchloric acid, vortex for 15 s, sonicate for
10 min, centrifuge at 12000 rpm for 10 min. Remove a 500 JJLL aliquot of the supernatant and
add it to 500 IJLL mobile phase, vortex for 15 s, centrifuge at 12000 rpm for 10 min, inject an
HPLC VARIABLES
Guard column: 15 X 3.2 PRP (Brownlee)
Column: 300 X 7.8 Aminex Ion-Exclusion HPX 874 (Bio-Rad)
Mobile phase: MeCN:0.05% sulfuric acid 12:88
Flow rate: 0.6
Detector: UV 210
CHROMATOGRAM
Internal standard: oxiracetam
OTHER SUBSTANCES
Extracted: pidotimod
KEYWORDS
plasma; oxiracetam is IS
Previous Page
HPLC VARIABLES
Flow rate: 1.5
Injection volume: 5
Detector: UV 292
CHROMATOGRAM
Retention time: 5.3
OTHER SUBSTANCES
Extracted: albendazole, oxfendazole, cambendazole, thiabendazole, mebendazole, fenbendazole,
parbendazole
KEYWORDS
plasma; sheep
REFERENCE
J.Pharm.ScL, 1980, 69, 422-423.
Oxiracetam
Merck Index: 7076
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 100 jxL 35% perchloric acid, vortex for 15 s, sonicate for
10 min, centrifuge at 12000 rpm for 10 min. Remove a 500 JJLL aliquot of the supernatant and
add it to 500 IJLL mobile phase, vortex for 15 s, centrifuge at 12000 rpm for 10 min, inject an
HPLC VARIABLES
Guard column: 15 X 3.2 PRP (Brownlee)
Column: 300 X 7.8 Aminex Ion-Exclusion HPX 874 (Bio-Rad)
Mobile phase: MeCN:0.05% sulfuric acid 12:88
Flow rate: 0.6
Detector: UV 210
CHROMATOGRAM
Internal standard: oxiracetam
OTHER SUBSTANCES
Extracted: pidotimod
KEYWORDS
plasma; oxiracetam is IS
REFERENCE
Dal Bo,L.; Broccali,G.P.; Silingardi,S.; Coppi,G. A new HPLC method for pidotimod plasma levels determina-
tion, Boll Chim.Farm., 1993, 132, 126-128.
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg phenylboronic acid SPE cartridge (Analytichem) with
one column volume of 100 mM pH 8.5 potassium phosphate buffer. Condition a silica SPE
cartridge with one column volume of MeCN. 200 jxL Plasma + 50 ûL water + 50 JJLL 25 jxg/mL
IS in water + 500 |xL MeCN, vortex, centrifuge at 8800 g for 10 min. Remove the supernatant
and add it to 500 |xL 100 mM pH 8.5 potassium phosphate buffer, vortex, add to the phenyl-
boronic acid SPE cartridge. Collect the eluate and evaporate it to dryness under a stream of
nitrogen at 50, reconstitute with 1 mL MeOH, sonicate, vortex, centrifuge at 1500 g for 5 min.
Evaporate the supernatant to dryness under a stream of nitrogen at 50, reconstitute the
residue in 200 |xL anhydrous pyridine, add 100 fxL n-propyl isocyanate, vortex, heat at 50 for
1 h, evaporate to dryness under a stream of nitrogen. Reconstitute the residue in two 500 (xL
aliquots of MeCN, add to the silica SPE cartridge, wash with 1 mL MeCN, elute with 1 mL
MeOHiMeCN 50:50. Evaporate the eluate to dryness under a stream of nitrogen at 50, recon-
stitute the residue in 200 jxL water, inject a 10-25 jxL aliquot.
HPLC VARIABLES
Column: 250 X 2 5 jxm Ultrasphere octadecylsilica
Mobile phase: Gradient. MeOH:50 mM pH 6.0 acetate buffer 10:90 for 5 min, to 20:80 over 4
min, maintain at 20:80 for 1 min, to 50:50 over 1 min, maintain at 50:50 for 5 min, return to
initial conditions over 1 min, re-equilibrate for 14 min.
Flow rate: 0.3
reagent pumped at 0.2 mL/min and the mixture flowed through a reaction coil (1 mL volume,
ABI Analytical PCRS Model 520) at 90 to the detector. (Reagent was prepared by adding 2
mL 2 mg/mL o-phthalaldehyde in MeOH and 80 |xL 3-mercaptopropionic acid to 1 L 2 g/L
NaOH in water, filter (0.45 |xm), use within 48 h.)
CHROMATOGRAM
Internal standard: 4-hydroxy-2-oxo-l-pyrrolidinepropionamide(ISF 2839) (12.9)
KEYWORDS
plasma; SPE; derivatization; post-column reaction
REFERENCE
Simpson,R.C; Boppana,V.K; Hwang,B.Y.-H.; Rhodes,G.R. Determination of oxiracetam in human plasma by
reversed-phase high-performance liquid chromatography with fluorimetric detection, J.Chromatogr., 1993,
631, 227-232.
SAMPLE
Sample preparation: Plasma. 250 |xL Plasma + 4.2 mL MeCN.water 100:0.4, mix, add 10 (xL
50 |xg/mL IS in water, add 800 |xL dichloromethane, vortex for a few s, centrifuge at 2000 g
for 5 min, inject a 1 mL aliquot of the supernatant. Urine. 10 |xL Urine + 4.2 mL MeCN:water
100:0.4, mix, add 40 jxL 50 |xg/mL IS in water, add 800 |xL dichloromethane, vortex for a few
s, inject a 1 mL aliquot of the supernatant onto column A and elute to waste with mobile phase
A, after 4 min divert effluent containing oxiracetam and IS onto column B, after 1.5 min remove
column A from the circuit, elute column B with mobile phase B, monitor the effluent from
column B. (Flush column A with MeCN:water 50:50 for 7.5 min, re-equilibrate with mobile
phase A for 5 min.)
HPLCVARIABLES
Column: A 100 X 4.7 5 jim Lichrosorb NH2; B 300 X 4.7 5 |xm Nucleosil NH2
Mobile phase: A MeCN.water 95:5; B MeCNrwater 90:10
Flow rate: A 2; B 1
Detector: UV 200
CHROMATOGRAM
Retention time: 15
Internal standard: 2-(2-oxo-l-pyrrolidine)acetamidoacetamide (CGP 14 998) (13)
Limit of quantitation: 12 |xg/mL (urine), 240 ng/mL (plasma)
KEYWORDS
plasma; column-switching; heart-cut; pharmacokinetics
REFERENCE
Lecaillon,J.B.; Souppart,C; Le Duigou,R; Dubois,J.P. Determination of oxiracetam in plasma and urine by
column-switching high-performance liquid chromatography, J.Chromatogr., 1989, 497, 223-230.
Oxolinic acid
Merck Index: 7079
Lednicer No.: 2 370
SAMPLE
Matrix: blood
Sample preparation: Condition a 100 mg Bond-Elut C18 SPE cartridge with two 3 mL portions
of MeOH and 3 mL of 0.05% orthophosphoric acid. Centrifuge plasma at 500 g for 5 min. Mix
200 JJLL centrifuged plasma with 200 |xL 10 jxg/mL IS in buffer, let stand for 1 h. Add 3 mL
buffer, vortex for 1 min. Add the mixture to the SPE cartridge under low vacuum, wash with
1 mL water and 500 jxL 0.05% orthophosphoric acid solution, dry under vacuum. Elute with
six 250 |xL portions of MeCN. Evaporate the combined eluates to dryness under a stream of
nitrogen at 40. Reconstitute the residue with 200 |xL buffer, vortex, sonicate, let stand for 30
min, vortex again. Inject a 50 jxL aliquot. (Buffer was 1/15 M pH 8.0 phosphate buffer.)
HPLCVARIABLES
Guard column: 4 x 4 5 fxm LiChroSpher 100 RP-18 end-capped
Column: 125 X 4 5 |xm LiChroSpher 100 RP-18 end-capped
Mobile phase: MeCN:DMF:20 mM pH 2.3 orthophosphoric acid 10:30:60
Flow rate: 0.8
Detector: UV 340
CHROMATOGRAM
OTHER SUBSTANCES
Noninterfering: 2-phenoxyethanol
KEYWORDS
plasma; sea bass; SPE; fish
REFERENCE
Loussouarn,S.; Pouliquen,H.; Armand,F. High-performance liquid chromatographic determination of oxolinic
acid in the plasma of seabass (Dicentrarchus labrax) anaesthetized with 2-phenoxyethanol, J. Chromatogr.B,
1997, 698, 251-259.
SAMPLE
Matrix: blood
HPLC VARIABLES
perchlorate.)
Flow rate: 0.9
Detector: UV 263
CHROMATOGRAM
KEYWORDS
REFERENCE
Zlotos,G.; Bucker,A.; Kinzig-Schippers,M.; Sorgel,F.; Holzgrabe,U. Plasma protein binding of gyrase inhibitors,
J.Pharm.ScL, 1998, 87, 215-220.
SAMPLE
Sample preparation: Blood. Filter serum through a 0.45 |xm syringe filter with a cellulose
acetate membrane, inject a 50 jxL aliquot of the filtrate. Tissue. Add 1 mL MeCN:THF 95:5 to
1 g muscle, homogenize with a Pencil Mixer (Iuchi, Japan) for 2 min, centrifuge at 1500 g for
5 min, filter the supernatant through a syringe filter unit, inject a 20 jxL aliquot of the filtrate.
HPLC VARIABLES
Guard column: 20 X 4.6 5 |xL Hisep shielded hydrophobic phase precolumn (Supelco)
Column: 150 X 4.6 5 |xL Hisep shielded hydrophobic phase (Supelco)
Mobile phase: MeCN.buffer 15:85 (Buffer was 50 mM citric acid:200 mM pH 2.5 Na2HPO4 buffer
containing 10 mM tetra-n-butyl ammonium bromide 85:15.)
Flow rate: 1
Detector: UV 265
CHROMATOGRAM
Retention time: 14
Limit of detection: 50 ng/mL (serum), 100 ng/mL (muscle)
OTHER SUBSTANCES
Extracted: sulfamonomethoxine, miloxacin
KEYWORDS
fish; muscle; serum
REFERENCE
Ueno,R.; Aoki,T. High-performance liquid chromatographic method for the rapid and simultaneous determi-
nation of sulfamonomethoxine, miloxacin and oxolinic acid in serum and muscle of cultured fish,
J.Chromatogr.B, 1996, 682, 179-181.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize 1 g tissue with 5 mL acetone, centrifuge, decant and save
the supernatant, repeat the extraction. Add 2 mL acetone, 3 mL hexane, and 6 mL 3% NaCl
to the combined supernatants, extract, centrifuge, discard the hexane layer. Add the extract to
25 mL chloroform (Caution! Chloroform is a carcinogen!), mix, separate the phases, add 2.5
mL 100 mM pH 9.0 Na3PO4 buffer and one drop 1 M NaOH to the chloroform phase, mix,
separate the phases, discard the chloroform layer. Wash the aqueous layer with 2.5 mL chlo-
roform, centrifuge the aqueous layer. Dialyze a 740 jxL aliquot of the supernatant (in 2 portions)
against 3.9 mL 20 mM pH 5.0 sodium phosphate buffer pumped at 0.6 mL/min using a Cu-
prophan 15000 MW cut-off cellulose acetate membrane. After washing column A with 500 (xL
MeCN:water 50:50 and 500 JJLL 20 mM pH 5.0 Na3PO4 buffer the dialysate flowed through
column A to waste. Wash column A with 500 |j,L 20 mM pH 5.0 sodium phosphate buffer, elute
the contents of column A onto column B with mobile phase, elute with mobile phase, monitor
the effluent from column B. (Wash the donor channel with 2 mL 0.01% Triton X-100 in 20 mM
pH 5.0 sodium phosphate buffer. Wash the acceptor channel with 3 mL 20 mM pH 5.0 sodium
phosphate buffer. Regenerate the membrane with 2 mL 100 mM pH 9.0 sodium phosphate
buffer (donor channel) and 3 mL 20 mM pH 5.0 sodium phosphate buffer (acceptor channel).)
HPLC VARIABLES
Column: A 5.8 X 4.6 Hypersil ODS; B 150 X 4.6 PLRP-S (Polymer Labs., UK)
Mobile phase: MeCN:THF:20 mM pH 5.0 Na3PO4 buffer 20:15:65
Flow rate: 0.6
CHROMATOGRAM
Retention time: 7
OTHER SUBSTANCES
Extracted: flumequine
KEYWORDS
column switching; chicken; liver; dialysis
REFERENCE
Eng,G.Y; Maxwell,R-J-; Cohen,E.; Piotrowski,E.G.; Fiddler,W. Determination of flumequine and oxolinic acid
in fortified chicken tissue using on-line dialysis and high-performance liquid chromatography with fluores-
cence detection, J.ChromatogrA, 1998, 799, 349-354.
SAMPLE
Matrix: urine
1OmL with water, filter (0.45 |xm). Inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:400 fxM oxalic acid in water 28:72
Flow rate: 2.0
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: nalidixic acid
REFERENCE
Dur&n Mer,L; Galeano Diaz,T.; Rodriguez Cceres,M.L; Salinas L6pez,F. Determination of the chemothera-
SAMPLE
Matrix: urine
Sample preparation: Make up 1 mL urine to 25 mL with mobile phase, filter (0.45 fim). Inject
a 20 JJLL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:400 IJLM oxalic acid in water 28:72
Flow rate: 2.0
Detector: UV 265
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: cinoxacin, nalidixic acid, pipemidic acid, piromidic acid
REFERENCE
Duran Mera,L; Galeano Diaz,T.; Rodriguez Cceres,M.L; Salinas L6pez,F. Determination of the chemothera-
Oxprenolol
Merck Index: 7086
Lednicer No.: 1 117
SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 25 |xL 25 ng/mL propranolol + 100 uX 1 M NaOH + 5
mL dichloromethane, shake, centrifuge at 1000 g for 10 min. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen at room temperature, reconstitute the
residue in 100 JULL dichloromethane, vortex for 5 s, add 10 |xL 0.01% S-(+)-l-(l-naphthyl)ethyl
isocyanate, heat at 37 for 2 h, add 20 (xL tert-butylamine, evaporate under a stream of nitro-
gen, reconstitute with 50 |xL mobile phase, inject a 20 JXL aliquot.
HPLC VARIABLES
Column: 240 X 4.6 5 jmrn Spherisorb C18 ODS
Mobile phase: MeOH:THF:200 mM pH 3.6 acetate buffer 51:14:35
Flow rate: 1
CHROMATOGRAM
Retention time: 15.8 (S-(-)), 17.8 (R-(+))
Internal standard: propranolol (25.2 (R-(+)), 28.3 (S-(-))
OTHER SUBSTANCES
KEYWORDS
plasma; chiral; derivatization; pharmacokinetics
REFERENCE
Laethem,M.E.; Rosseel,M.T.; Wijnant,R; Belpaire,F.M. Chiral high-performance liquid chromatographic deter-
mination of oxprenolol in plasma, J.Chromatogr., 1993, 621, 225-229.
SAMPLE
Matrix: blood
HPLCVARIABLES
Flow rate: 0.8
Detector: UV 273
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
Sample preparation: 100 |xL Plasma or urine + 25 JJLL 25 ng/mL propranolol + 100 |xL 1 M
NaOH + 5 mL dichloromethane, shake, centrifuge at 1000 g for 10 min (J.Chromatogr. 1993,
621, 225). Remove the organic layer and evaporate it to dryness under a stream of nitrogen at
room temperature, reconstitute the residue in 50 |xL 0.02% S(+)-l-(l-naphthyl)ethylisocyanate
in hexanerchloroform 50:50, vortex for 30 min, evaporate, reconstitute with 50 IULL mobile phase,
HPLC VARIABLES
Column: Lichrosorb DIOL
Mobile phase: Hexane:chloroform:MeOH 90:10:0.38
CHROMATOGRAM
Internal standard: propranolol
KEYWORDS
plasma; chiral; derivatization; pharmacokinetics
REFERENCE
Laethem,M.E.; Lefebvre,R.A.; Belpaire,F.M.; Vanhoe,H.L.; Bogaert,M.G. Stereoselective pharmacokinetics of
oxprenolol and its glucuronides in humans, Clin.Pharmacol.Ther., 1995, 57, 419-424.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 10 |xm Lichrosorb RP C18
Mobile phase: MeOH:water 55:45 pH 4.5
Flow rate: 1.0 for 4 min, then 2.0
Detector: UV 260
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: acebutolol, nifedipine
REFERENCE
el Walily,A.F.M. Analysis of nifedipine-acebutolol hydrochloride binary combination in tablets using UV-deriv-
ative spectroscopy, capillary gas chromatography and high performance liquid chromatography,
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 yJL aliquot.
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.1
OTHER SUBSTANCES
ifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxymetazolin, papaverine, pargyline,
REFERENCE
Jane,L; McKinnon,A.; Flanagan,R-J High-performance liquid chromatographic analysis of basic drugs on silica
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 150 X 4.6 12 jxm l-myristoyl-2-[(13-carboxyl)-tridecoyl]-sn-3-glycerophosphocholine
Mobile phase: MeCNrIOO mM pH 7.0 phosphate buffer 20:80
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
Retention time: k'3.85
OTHERSUBSTANCES
famotidine, isothipendyl, ketotifen, metiamide, metoprolol, moxonidine, nadolol, naphazoline,
nifenalol, nizatidine, pheniramine, phentolamine, pindolol, pizotyline (pizotifen), practolol, pra-
REFERENCE
Kaliszan,R.; Nasal,A-; Turowski,M. Binding site for basic drugs on ai-acid glycoprotein as revealed by chemo-
SAMPLE
Matrix: solutions
Sample preparation: Mix a 100 |JLL of a 10 (xM solution in MeCN:water:triethylamine 50:50:0.1
with 100 |xL 1 mM (R)-(-)-4-(3-isothiocyanatopyrrolidin-l-yl)-7-(N,N-dimethylaminosulfonyl)-
2,1,3-benzoxadiazole in MeCN, heat in the dark at 65 for 1.5 h, inject an aliquot. (Synthesis
of (R)-(-)-4-(3-isothiocyanatopyrrolidin-l-yl)-7-(N,N-dimethylaminosulfonyl)-2,l,3-benzoxadiaz-
ole is as follows. Dissolve 0.5 g magnesium sulfate heptahydrate and 6 g NaOH in 60 mL
water, throughout the reaction keep the flask at about 20 with cold water cooling, add 15 mL
30% hydrogen peroxide, add 75 mL MeOH, add 12.1 g powdered benzoyl peroxide in one go,
stir for 10 min, pour into 150 mL 20% sulfuric acid, extract three times with 50 mL portions
of chloroform, determine peroxybenzoic acid concentration by iodometric titration (Tetrahedron
1967, 23, 3327). Slowly add 110 mL 1 M peroxybenzoic acid in chloroform to 7 g 2,6-difluo-
roaniline dissolved in 100 mL chloroform, stir at room temperature, when reaction is complete
(iodometric titration) wash with 2% sodium thiosulfate, wash with 5% sodium carbonate, wash
with water, dry over anhydrous sodium sulfate, evaporate to dryness under reduced pressure,
recrystallize 2,6-difluoronitrosobenzene from EtOH (mp 108.5-109.5). Stir 8.5 g 2,6-difluoroni-
trosobenzene in 85 mL DMSO at room temperature and add a solution of 3.91 g sodium azide
in 85 mL DMSO dropwise, let stand for about 1 h, add to a large volume of water, extract with
ether, dry the extracts over anhydrous sodium sulfate, evaporate to dryness under reduced
pressure and distil to give 4-fluoro-2,l,3-benzoxadiazole as a colorless oil (bp 83712 mm Hg)
(J.Chem.Soc.(C) 1970, 1433). Add 11 mL chlorosulfonic acid dropwise to 3 g 4-fluoro-2,l,3-
benzoxadiazole in 10 mL chloroform at 0-10 (use a calcium chloride drying tube), stir at room
temperature for 1 h, reflux for 2 h, cool, slowly pour into ice water, remove the organic layer,
extract the aqueous layer with chloroform, combine the organic layer, wash, dry over anhydrous
magnesium sulfate, evaporate under reduced pressure, take up the residue in 5 mL benzene
(Caution! Benzene is a carcinogen!), chromatograph on a 150 X 30 column of silica gel (100-
200 mesh Kanto Chemical) with n-hexane:benzene 50:50, evaporate the appropriate fractions
to give 4-(chlorosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (CBD-F) as pale yellow needles (mp 64-
66) (Anal. Chem. 1984. 56, 2461). Stir 0.76 g CBD-F in 70 mL MeCN at 0-10 and add 1 g
dimethylamine hydrochloride in 10 mL 100 mM pH 10 borax dropwise, adjust pH to 5 with 1
M HCl, concentrate to about 10 mL under reduced pressure, extract three times with 200 mL
portions of diethyl ether, wash with water, dry over anhydrous magnesium sulfate, evaporate
under reduced pressure, chromatograph on a 500 X 20 column of silica gel with chloroform,
isolate the appropriate fraction and re-chromatograph on the same column with ethyl acetate:
benzene 1:2 to give 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (DBD-F) as
white needles (mp 124-125) (yield = 1% !). On a Merck no. 5714 60F254 TLC plate eluted with
chloroform DBD-F has Rf 0.32 and lies between two other reaction products (Analyst 1989,
114, 413). It is also reported that DBD-F can be purchased from Tokyo Kasei. Cool a solution
of 16.4 g (S)-(-)-l-benzyl-3-pyrrolidinol in 164 mL pyridine to +5, add 19.35 g p-toluenesulfonyl
chloride, stir at +10 for 48 h, evaporate to dryness, chromatograph using dichloromethane:
acetone 95:5 to obtain (3S)-3-[(4-tolylsulfonyl)oxy]-l-(phenylmethyl)pyrrolidine (mp 68). Heat
a solution of (3S)-3-[(4-tolylsulfonyl)oxy]-l-(phenylmethyl)pyrrolidine in 200 mL anhydrous
DMF to 65, add 33.5 g sodium azide (Caution! Sodium azide is highly toxic!), stir at 60 for 7
h, filter, evaporate the filtrate to dryness under reduced pressure, dissolve the residue in ethyl
acetate, wash twice with water, dry over anhydrous magnesium sulfate, evaporate to obtain
(3R)-3-azido-l-(phenylmethyl)pyrrolidine as an oil. Add 3.5 g 10% palladium on carbon under
nitrogen to a solution of 7.05 g (3R)-3-azido-l-(phenylmethyl)pyrrolidine in 34.8 mL 1 M HCl
in water and 245 mL EtOH, hydrogenate at atmospheric pressure for 30 min, add 3.5 g catalyst,
hydrogenate for 2 h, filter, add 34.8 mL 1 M HCl to the filtrate, evaporate to dryness under
reduced pressure, take up the residue in 70 mL EtOH, filter, evaporate the filtrate to dryness
under reduced pressure, repeat this operation twice, crystallize with the minimum amount of
EtOH to obtain (3R)-3-aminopyrrolidine dihydrochloride (J. Med. Chem. 1992, 35, 4205). 3R-
(+)-aminopyrrolidine is also reported to be available from Tokyo Kasei. Add 100 mg 4-(N,N-
dimethylaminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole in 20 mL MeCN dropwise to a stirred
solution of 200 mg 3R-(+)-aminopyrrolidine in 20 mL MeCN at 0-10, stir at room temperature
for 30 min, remove the MeCN by evaporation under reduced pressure, dissolve the residue in
50 mL 5% HCl, wash 3 times with 50 mL portions of ethyl acetate, adjust the pH of the aqueous
solution to 13-14 with 5% NaOH, extract 6 times with 50 mL portions of ethyl acetate. Combine
the organic layers and wash them with 20 mL water, dry over anhydrous sodium sulfate,
evaporate to dryness under reduced pressure, recrystallize from hexane to obtain (R)-(-)-4-(3-
aminopyrrolidin-l-yl)-7-(N,N-dimethylaminosulfonyl)-2,l,3-benzoxadiazole as orange crystals
(mp 96-98) (Analyst 1992, 117, 727). Add 100 |xL thiophosgene in 10 mL benzene (Caution!
Benzene is a carcinogen!) to 100 mg (R)-(-)-4-(3-aminopyrrolidin-l-yl)-7-(N,N-dimethylamino-
sulfonyl)-2,l,3-benzoxadiazole in 100 mL acetone, reflux for 1 h, remove the solvent by evap-
oration under reduced pressure, suspend the residue in 100 mL water, extract 4 times with 25
mL portions of benzene. Combine the extracts and wash them with 20 mL water, dry over
anhydrous sodium sulfate, evaporate to dryness under reduced pressure, recrystallize from
hexane:benzene 1:2 to obtain (R)-(-)-4-(3-isothiocyanatopyrrolidin-l-yl)-7-(N,N-dimethylamino-
sulfonyl)-2,l,3-benzoxadiazole as yellow crystals (mp 160-170 d) (Analyst 1995, 120, 385).)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Inertsil ODS-80A
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: alprenolol, propranolol
KEYWORDS
REFERENCE
Toyo'oka,T.; Toriumi,M.; Ishii,Y. Enantioseparation of {3-blockers labelled with a chiral fluorescent reagent,
R(-)-DBD-PyNCS, by reversed-phase liquid chromatography, J.Pharm.BiomedAnal, 1997,15, 1467-1476.
Oxybenzone
Merck Index: 7088
SAMPLE
Sample preparation: Plasma. Add 200 |xL MeCN to 100 |xL plasma or 2% bovine serum albumin
in pH 7.2 phosphate buffer, centrifuge at 10000 g for 10 min, inject an aliquot of the super-
natant. Formulations. Keep about 5 mL aliquot of lotion or cream in full sunlight at 17-34
during the day, dilute a 100 mg sample to 100 mL with MeOHrwater 50:50, dilute a 200 |xL
aliquot of the diluted sample to 10 mL with MeOH, inject an aliquot.
HPLC VARIABLES
Column: 100 X 8 4 |xL Nova Pak C18 RCM
Flow rate: 1
Detector: UV 315
CHROMATOGRAM
Retention time: 4.8
OTHER SUBSTANCES
Simultaneous: Escalol 507, Parsol MCX, Parsol 1789, octylsalieylate
KEYWORDS
cream; bovine serum albumin; lotion; plasma; spray
REFERENCE
Jiang,R.; Hayden, C. G.; Prankerd,R.J.; Roberts,M.S.; Benson,H.A. High-performance liquid chromatographic
assay for common sunscreening agents in cosmetic products, bovine serum albumin solution and human
plasma, J.Chromatogr.B, 1996, 682, 137-145.
SAMPLE
Sample preparation: Lotion. Weigh out lotion equivalent to about 90 mg oxybenzone, add 7 mL
water, add MeOH slowly with vigorous shaking until total volume was 100 mL. Remove a 10
mL aliquot and make up to 100 mL with MeOH, filter (paper), discard first 5 mL of filtrate.
Mix 4 mL filtrate and 1 mL 200 |xg/mL sulfathiazole in MeOH, make up to 10 mL with MeOH,
filter (0.45 |xm), inject a 20 |xL aliquot. Lipstick. Weigh out an amount equivalent to 25-90 mg
oxybenzone, add 10 mL chloroform, add MeOH slowly with vigorous shaking until total volume
was 100 mL. Remove a 10 mL aliquot and make up to 100 mL with MeOH, filter (paper),
discard first 5 mL of filtrate. Mix 4 mL filtrate and 1 mL 200 |xg/mL sulfathiazole in MeOH,
make up to 10 mL with MeOH, filter (0.45 |xm), inject a 20 |xL aliquot.
HPLCVARIABLES
Guard column: 40 X 4.6 25-37 urn Co:Pell ODS
Column: 250 X 4.6 10 |xm Partisil PXS ODS-2
Mobile phase: MeCNrMeOH 10:90
Flow rate: 0.7
Detector: UV 254
CHROMATOGRAM
Retention time: 5.7
OTHER SUBSTANCES
Simultaneous: padimate-O, propyl paraben
KEYWORDS
lotion; lipstick; sun-screen
REFERENCE
Tan,H.S.L; Sih,R.; Moseley,S.E.; Lichtin,J.L. Assay of mixtures of padimate-O and oxybenzone in sunscreen
formulations by high-performance liquid chromatography, J.Chromatogr., 1984, 291, 275-282.
SAMPLE
Sample preparation: 1-1.5 g sun-screen lotion + 50 mL isopropanol, dissolve. Remove a 5 mL
aliquot and make up to 50 mL with mobile phase, filter (0.45 |xm) inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 10 jxm C8 Hypersil
Mobile phase: Isopropanol rbuffer 10:90 (Buffer was 100 mM sodium dodecyl sulfate (electropho-
resis grade) containing 0.3% triethylamine adjusted to pH 3.0 with phosphoric acid.)
Flow rate: 1.5
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: 2-ethylhexyl p-dimethylaminobenzoate, octyl methoxycinnamate, methyl para-
ben, propyl paraben
KEYWORDS
lotion
REFERENCE
Tomasella,F.R; Zuting,R; Love,L.J. Determination of sun-screen agents in cosmetic products by micellar liquid
Oxybutynin chloride
CAS Registry No.: 1508-65-2, 5633-20-5 (free base)
Merck Index: 7089
LednicerNo.: 1 93
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 100 u.L 1 jjug/mL dicyclomine in water + 1 mL MeCN,
vortex, allow to stand for 10 min, add 200 |xL 1 M pH 9.4 tris(hydroxymethyl)methylamine
(TRIS), add 5 mL hexane, shake horizontally for 10 min, centrifuge at 2000 g for 5 min. Remove
the aqueous layer and add it to 3 mL hexane, shake horizontally for 10 min, centrifuge at 2000
g for 5 min. Combine the hexane layers and add them to 1 mL 100 mM HCl, shake for 10 min,
centrifuge. Remove the aqueous layer and evaporate it to dryness under vacuum, reconstitute
in 250 jxL mobile phase, inject a 100 jxL aliquot.
HPLC VARIABLES
Column: 100 mm long 5 |xm Techsil CN
Mobile phase: MeOH:20 mM pH 6.2 orthophosphoric acid buffer 40:60
Flow rate: 0.6
Detector: E, ESA Coulochem 5100-A, guard cell 1.0 V, dual porous graphite electrode 0.85 and
0.95 V
CHROMATOGRAM
Retention time: 17
Internal standard: dicyclomine (23)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Hughes,K.M.; Lang,J.C.T.; Lazare,R.; Gordon,D.; Stanton,S.L.; Malone-Lee,J.; Geraint,M. Measurement of
oxybutynin and its N-desethyl metabolite in plasma, and its application to pharmacokinetic studies in
young, elderly and frail elderly volunteers, Xenobiotica, 1992, 22, 859-869.
Oxyclozanide
Merck Index: 7092
SAMPLE
HPLC VARIABLES
Mobile phase: MeOHrbuffer 19:81, pH 3.9 (Buffer was prepared by dissolving 6.6 g dibasic am-
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: albendazole, fenbendazole, niclosamide
KEYWORDS
tablets; powder; liquid formulations;
REFERENCE
lations, J.ChromatognA, 1996, 729, 267-272.
Oxycodone
CAS Registry No.: 76-42-6, 124-90-3 (HCI), 64336-55-6 (terephthalate)
Merck Index: 7093
Lednicer No.: 1 290
SAMPLE
Matrix: blood
Sample preparation: Make plasma containing IS alkaline and extract it with MTBE. Evaporate
the organic layer to dryness under nitrogen, reconstitute the residue in 100 |xL MeOH:water
90:10, inject an aliquot.
HPLCVARIABLES
Mobile phase: MeOH: 1OmM pH 4.0 ammonium formate 40:60
Flow rate: 1
Detector: MS, Sciex API IIIplus tandem mass, positive ion mode, 316.0/298.0 parent/product ions
CHROMATOGRAM
Retention time: 5
Internal standard: nalmefene (6.9)
OTHER SUBSTANCES
KEYWORDS
plasma
REFERENCE
Kaisershot,C; Pierce,A; Talaat,R. Quantitative analysis of oxycodone and its metabolites noroxycodone and
oxymorphone in human plasma by high-performance liquid chromatography with ionspray tandem mass
spectrometry (Abstract 2129), Pharm.Res., 1997, 14, S262.
SAMPLE
Matrix: blood
Sample preparation: Condition a Certify SPE cartridge (Varian) with 2 mL MeOH and 2 mL
100 mM pH 8.0 phosphate buffer. Add 50 fxL 5.96 |xM IS in water to 500 ^L plasma. Add 1
mL 100 mM pH 8.0 phosphate buffer, vortex for 10 min, add to the SPE cartridge using a
gentle vacuum. Wash with 5 mL 100 mM pH 8.0 phosphate buffer, 1 mL water, 2 mL 100 mM
pH 4.0 acetate buffer, and 2 mL MeOH. Dry cartridge under vacuum, wash with 200 JJLL butyl
chloridedsopropanol 80:20, dry under vacuum. Elute with 1.2 mL butyl chloridedsopropanol
80:20, evaporate the eluate to dryness under a stream of nitrogen, reconstitute the residue in
200 |JLL mobile phase. Inject a 150 |xL aliquot.
HPLC VARIABLES
Guard column: GuardPak C18 jxBondapak
Column: 100 X 8 NovaPak C18
Mobile phase: MeCN:MeOH:13.3 mM pH 7.5 phosphate buffer 2:23:75 containing 40 mg/L
cetyltrimethylammonium bromide
Flow rate: 1
Detector: E, Waters Model 460, working electrode 1.10-1.25 V, reference electrode KCl
CHROMATOGRAM
Retention time: 9.6
Internal standard: codeine hydrochloride (14.7)
KEYWORDS
plasma; SPE
REFERENCE
Wright,A.W.E.; Lawrence,J.A.; Iu,M.; Cramond,T.; Smith,M.T. Solid-phase extraction method with high-perfor-
mance liquid chromatography and electrochemical detection for the quantitative analysis of oxycodone in
human plasma, J.ChromatognB, 1998, 712, 169-175.
Oxymetazoline
Merck Index: 7100
Lednicer No.: 1 242
SAMPLE
Matrix: blood
Sample preparation: 300 |xL Whole blood + 50 ng IS, vortex, let stand for 30 min, add 900 JJLL
100 mM sodium carbonate, extract with 3 mL diethyl ether. Remove the organic layer and
evaporate it to dryness under a stream of nitrogen at 35, reconstitute the residue in 100 |JLL
MeCN:5 mM ammonium acetate 50:50, inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 150 X 2.1 5 |xm 8 nm pore size Zorbax Rx-C 18
Flow rate: 0.4
Detector: MS, PE-Sciex API III tandem quadrupole, articulated ionspray, 20:1 post-column split,
SIM, m/z 261, 270, nebulizer gas air at 42 psi, curtain gas nitrogen 1.2 L/min, orifice 70 V,
open collision cell (Q2)
CHROMATOGRAM
Retention time: 1.5
Internal standard: nonadeutero oxymetazoline (deuterium all on t-butyl group)
KEYWORDS
whole blood; LC-MS; rat; pharmacokinetics
REFERENCE
Hayes,F.J.; Baker,T.R.; Dobson,R.L.M.; Tsueda,M.S. Rapid liquid chromatographic-mass spectrometric assay
for oxymetazoline in whole rat blood, J.Chromatogr.A, 1995, 692, 73-81.
SAMPLE
Sample preparation: Dilute nasal solution 10-fold with water, filter (0.45 fxm), inject a 10 jxL
HPLC VARIABLES
Column: 125 X 4 5 jxm Aluspher RP-select B (Merck)
Flow rate: 1.2
CHROMATOGRAM
Limit of detection: 1 ng (UV 224)
OTHER SUBSTANCES
Simultaneous: ephedrine, naphazoline, xylometazoline
KEY WORDS
nasal solution
REFERENCE
3233-3242.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 fxg/mL solution in MeOH, inject a 20 (xL aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 2.3
OTHER SUBSTANCES
mine, fenoterol, fentanyl, flavoxate, fluopromazine, fiupenthixol, fluphenazine, fiurazepam, hal-
lol, mianserin, morazone, nadolol, nalorphine, naloxone, naphazoline, nicotine, nifedipine, no-
mifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, papaverine, pargyline,
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in MeOHrwater 40:60, inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.1 RSIL C18 (RSL, Eke, Belgium)
Mobile phase: MeOH.water 40:60 containing 20 mM sodium 1-octanesulfonate and 10 mM N,N-
dimethyloctylamine, pH adjusted to 3.0 with orthophosphoric acid
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 30
OTHER SUBSTANCES
Simultaneous: degradation products, antazoline, coumazoline, lidocaine, naphazoline, predni-
solone, sulfadimidine, sulfanilamide, sulfathiazole, tenaphtoxaline, tetrahydrozoline, tolazo-
line, tramazoline, xylometazoline
REFERENCE
De Schutter,J.A.; Van den Bossche,W.; De Moerloose,P. Stability-indicating analysis of tetryzoline hydrochloride
in pharmaceutical formulations by reversed-phase ion-pair liquid chromatography, J.Chromatogr., 1987,
391, 303-308.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Supelcosil LC-DP (A) or 250 X 4 5 ixm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, paroxe-
tine, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine, pheno-
barbital, phenol, phenolphthalein, phentolamine, phenylbutazone, phenyltoloxamine, phen^-
oin, pimozide, pindolol, piroxicam, pramoxine, prazepam, prazosin, probenecid, procainamide,
procaine, prochlorperazine, procyclidine, promazine, promethazine, propafenone, propanthe-
line, propiomazine, propofol, propranolol, protriptyline, quazepam, quinidine, quinine, race-
methorphan, ranitidine, remoxipride, risperidone, salicylic acid, scopolamine, secobarbital, ser-
traline, sotalol, spironolactone, sulfinpjrrazone, sulindac, temazepam, terbutaline, terfenadine,
tetracaine, theophylline, thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocain-
ide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine,
trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine,
zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Oxymetholone
Merck Index: 7101
Lednicer No.: 1 173
SAMPLE
powder, add 25 mL MeOH, sonicate for 5-10 min, filter (0.45 jxm), discard first 5 mL of filtrate,
mL with MeOH, filter (0.45 |xm), discard first 5 mL of filtrate, inject a 10 JJLL aliquot of the
remaining filtrate.
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 240
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: nandrolone propionate, methenolone acetate, testosterone propionate, aspirin,
caffeine, formebolone, benzyl alcohol, testolactone, cortisone, fluoxymesterone, norethindrone,
oxandrolone (UV 210), boldenone, ethisterone, methandrostenolone, nandrolone, norgestrel,
testosterone, dehydroepiandrosterone (UV 210), mibolerone, methyltestosterone, methandriol
(UV 210), norethindrone acetate, calusterone, mesterolone (UV 210), norethandrolone, benzyl
benzoate, trenbolone acetate, nandrolone acetate
Interfering: testosterone acetate, stanozolol
KEYWORDS
REFERENCE
Walters,M.J.; Ayers,R.J.; Brown,D.J. Analysis of illegally distributed anabolic steroid products by liquid
chromatography with identity confirmation by mass spectrometry or infrared spectrophotometry,
SAMPLE
Matrix: solutions
Sample preparation: Inject a 5 |xL aliquot of a 10 |jig/mL solution in MeOH.
HPLC VARIABLES
Column: 75 X 4.6 3 urn Ultrasphere ODS
Mobile phase: MeCN: 10 mM ammonium acetate buffer 45:55
Flow rate: 0.5
Injection volume: 5
Detector: UV 280
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: boldenone (UV 254), epimethandienone (UV 254), epitestosterone (UV 254),
fluoxymesterone (UV 254), 6p-hydroxymethandienone (UV 254), methandienone (UV 254),
norethindrone (UV 254), trenbolone (UV 254)
REFERENCE
Barr6n,D.; Pascual,J.A.; Segura,J.; Barbosa,J. Prediction of LC retention of steroids using solvatochromic pa-
rameters, Chromatographia, 1995, 41, 573-580.
Oxymorphone
Merck Index: 7103
Lednicer No.: 1 290
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Plasma + 20 JJLL 1 |xg/mL IS + 200 |xL 3.5% sodium carbonate,
extract with 4 mL dry ether. Extract the organic layer with 200 |xL 20 mM phosphoric acid.
Inject an aliquot of the phosphoric acid solution.
HPLC VARIABLES
Mobile phase: MeCN:MeOH:EDTA:70 mM KH2PO4 6:10:0.02:83.98
Flow rate: 1
Detector: E, LCB4, oxidation potential 900 mV
CHROMATOGRAM
Retention time: 6.5
KEYWORDS
plasma; rat
REFERENCE
Hussain,M.A.; Aungst,B.J. Intranasal absorption of oxymorphone, J.Pharm.ScL, 1997, 86, 975-976.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 1 mL 1 M sodium bicarbonate + 200 |xL 400 ng/mL hy-
dromorphone hydrochloride + 15 mL diethyl ether, rotate for 20 min, centrifuge at 800 g for
15 min. Remove the organic phase and add it to 200 |xL 17 mM phosphoric acid, mix vigorously
for 15 s, centrifuge for 5 min. Remove the aqueous phase and evaporate it to dryness under a
stream of air at 45, reconstitute the residue in 200 JULL 17 mM phosphoric acid, inject a 100
|JLL aliquot.
HPLCVARIABLES
Column: 25 X 4.6 5 |jim Hi-Chrom reversible octyl (Regis)
Mobile phase: MeCN:30 mM pH 4 KH2PO4:0.3% sodium octanesulfonate 15:75:9
Flow rate: 1.5
Detector: E, BAS LC4B, glassy carbon working electrode 0.9 V, Ag/AgCl reference electrode
CHROMATOGRAM
Retention time: 7.0
Internal standard: hydromorphone hydrochloride (8.9)
KEYWORDS
REFERENCE
Lam,G.; Williams,R.M.; Whitney,C.C. Electrochemical determination of oxymorphone in rat plasma by ion-pair
reversed-phase high-performance liquid chromatography, J.Chromatogr., 1987, 413, 309-314.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: Gradient. A was 10 mL concentrated orthophosphorie acid and 7 mL triethylamine
in 1 L water. B was 10 mL concentrated orthophosphorie acid and 7 mL triethylamine in 200
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
acid, nitrazepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxyphenbuta-
zone, oxytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine,
phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, phen-
iramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, phenyl-
ephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid,
progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyri-
lamine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine,
thiosalicylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcy-
REFERENCE
Hill,D.W.; Kind,A.J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnal.ToxicoL, 1994,
18, 233-242.
Oxyphenbutazone
CAS Registry No.: 129-20-4,7081-38-1 (H2O)
Merck Index: 7106
Lednicer No.: 1 236
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
tilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, iso-
xsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam,
lormetazepam, loxapine, mazindol, mebendazole, meclizine, meclofenamic acid, medazepam,
mefenamic acid, megestrol, mepacrine, meperidine, mephentermine, mephenytoin, mephesin,
mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine, meth-
apyrilene, methaqualone, methazolamide, methocarbamol, methoxamine, methsuximide,
Next Page
ytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phe-

mine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone,
REFERENCE
Hill,D.W.; Kind,A-J. Reversed-phase solvent gradient HPLC retention indexes of drugs, JAnaLToxicoL, 1994,
18, 233-242.
Oxyquinoline
Molecular formula: G9H7NO
Merck Index: 4890
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 PRP-I in a PEEK column (Hamilton)
Mobile phase: Gradient. MeCN:10 mM pH 5.0 acetate buffer 5:95 for 3 min, to 90:10 over 27
min (Waters curve no. 5). (At the beginning of each day flush column with 0.01% EDTA for 30
min, equilibrate at initial conditions for 30 min.)
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: metabolites, glucuronide, quinoline, 5-hydroxyquinoline
KEYWORDS
use PEEK tubing; metal-free system
REFERENCE
Nagiel-Ostaszewski,L; Vavrek,M.T.; Weisburger,J.H. Separation of hydroxyquinolines by high-performance liq-
uid chromatography, Xenobiotica, 1991, 21, 751-754.
Paclitaxel
Molecular formula: C47H51NOi4
Merck Index: 7117
SAMPLE
Matrix: blood
Sample preparation: Add 100 \xL 10 jig/mL docetaxel in MeOH:water 50:50 and 5 mL MeCN:
n-butyl chloride 20:80 to 1 mL plasma, vortex for 5 min, centrifuge at 4000 g for 5 min, evap-
orate the organic layer to dryness under a stream of nitrogen at 60, reconstitute the residue
with MeOH:water 50:50 with sonication for 1 min, inject a 100 jxL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 jxm Inertsil ODS-80A (GL Science, Japan)
Mobile phase: MeOH:THF:water:ammonium hydroxide 60:2.5:37.5:0.1, pH adjusted to 6.0 with
formic acid
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 7.5
Internal standard: docetaxel (8.5)
OTHER SUBSTANCES
Noninterfering: acetaminophen, alizapride, codeine, dexamethasone, domperidone, lorazepam,
metoclopramide, morphine, ranitidine
Interfering: paroxetine
KEYWORDS
REFERENCE
Sparreboom,A.; de Bruijn,R; Nooter,K; Loos,W.J.; Stoter,G.; Verweij,J. Determination of paclitaxel in human
plasma using single solvent extraction prior to isocratic reversed-phase high-performance liquid chroma-
tography with ultraviolet detection, J.Chromatogr.B, 1998, 705, 159-164.
SAMPLE
Sample preparation: Condition a 1 mL Baker Bond cyanopropyl SPE cartridge with 1 mL
MeCN, 1 mL mobile phase, and 1 mL 50 mM KH2PO4. Free paclitaxel. Add 500 |JLL plasma or
urine to the SPE cartridge, wash with 1 mL water, 1 mL MeCN:water 30:70, and 1 mL 50 mM
KH2PO4, elute with two 300 |xL portions of mobile phase, inject a 200 |xL aliquot of the eluate.
Total paclitaxel. To 500 |xL plasma or urine add 500 |xL MeOH: 100 mM KH2PO4 50:50 (pH
7.5), hydrolyze at 22 for 20 h, stop the hydrolysis by adding an equal volume of 500 mM
KH2PO4. Add the hydrolyzed sample to the SPE cartridge, wash with 1 mL water, 1 mL MeCN:
water 30:70, and 1 mL 50 mM KH2PO4, elute with two 300 (xL portions of mobile phase, inject
a 200 jxL aliquot of the eluate.
HPLC VARIABLES
Guard column: 4 X 4 4 (xm LiChrospher 100 RP-8e
Column: 250 X 4 4 |xm Superspher 60 RP-8e
Mobile phase: MeCN:175 mM pH 4.6 KH2PO4 buffer 55:45
Flow rate: 0.45
Detector: UV 229
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
KEYWORDS
dog; plasma; SPE
REFERENCE
Fraier,D.; Cenacchi,V.; Frigerio,E. Determination of a new polymer-bound paclitaxel derivative (PNU 166945),
free paclitaxel and 7-epipaclitaxel in dog plasma and urine by reversed-phase high-performance liquid
chromatography with UV detection, J.ChromatogrA, 1998, 797, 295-303.
SAMPLE
Sample preparation: Bulk. Dilute sample with MeOH containing 0.1% acetic acid. Injections.
Dilute sample with MeCN:water:acetic acid 70:30:0.1. Method 1. Column A, gradient A. Method
2. Column B, gradient B. Method 3. Column A, gradient C. Method 4. Column B, gradient D.
Methods 1 and 2 are for bulk drug potency determination. Methods 3 and 4 are for the deter-
mination of the degradation profile for bulk and injections, potency and content uniformity of
the injections and chromatographic purity profile of the bulk drug.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Curosil PFP (Phenomenex) (A), 250 X 4.6 5 jxm Whatman TAC-I (PFP)
(Whatman) (B)
Mobile phase: Gradient A. MeCN:water from 40:60 by linear gradient at 0.444%/min until the
paclitaxel peak elutes, return rapidly to initial composition and equilibrate. Gradient B. MeCN:
water 38:62 for 12 min, by linear gradient at 4%/min until paclitaxel peak elutes, return to
initial composition and equilibrate. Gradient C. MeCN:water from 40:60 to 60:40 over 45 min,
to 80:20 over 5 min, hold for 5 min, return to initial composition and equilibrate. Gradient D.
MeCN:water 38:62 for 12 min, to 70:30 over 8 min, hold for 15 min, return to initial composition
and equilibrate.
Flow rate: 1 (column A), 1.5 (column B)
Detector: UV 230
CHROMATOGRAM
Retention time: 24 (Method 1), 22.5 (Method 3), 17 (Method 4)
Limit of detection: 370 ng/mL (method 1), 310 ng/mL (method 2)
Limit of quantitation: 1.11 |jig/mL (method 1), 930 ng/mL (method 2)
OTHER SUBSTANCES
Simultaneous: 13-acetyl-9-dihydrobaccatin, baccatin III, cephalomannine, 10-deacetyl baccatin
III, N-debenzoyl-N-phenylacetyl taxol, 10-deacetyl-7-epi-taxol, 10-deacetyl taxol, 10-deacetyl-
7-xylosyl taxol, 10-deacetyl-7-xylosyl taxol B, 10-deacetyl-7-xylosyl taxol C, 7-epi-taxol, taxinine
M, taxol C, 7-xylosyl taxol
Noninterfering: degradation products, Cremophor EL
KEYWORDS
injections
REFERENCE
Shao,L.K; Locke,D.C. Determination of paclitaxel and related taxanes in bulk drug and injectable dosage forms
by reversed phase liquid chromatography, Anal.Chem., 1997, 69, 2008-2016.
SAMPLE
Matrix: cell culture
Sample preparation: Pulverize dry callus cell culture, pass through 40-mesh sieve. Extract
ultrasonically with MeOH:dichloromethane 10:1 for 30 min. Evaporate extract to dryness, dis-
solve residue in MeOH. Add an aliquot to a Sep-Pak C18 SPE cartridge, wash with water,
wash with MeOH:water 30:70, elute with MeOH:water 85:15. Evaporate the eluate to dryness
and redissolve in a minimum amount of MeOH, inject a 10 (xL aliquot.
HPLC VARIABLES
Flow rate: 1.0
Detector: UV 227
CHROMATOGRAM
Retention time: 27
OTHER SUBSTANCES
Extracted: baccatin, cephalomannine, 10-deacetyltaxol, 10-deacetylcephalomannine, baccatin,
10-deacetylbaccatin
KEYWORDS
SPE
REFERENCE
Wu,Y.; Zhu,W. High performance liquid chromatographic determination of taxol and related taxanesfromTaxus
callus cultures, J.Liq.Chromatogr., 1997, 20, 3147-3154.
SAMPLE
Sample preparation: Add paclitaxel injection to 0.9% NaCl injection or 5% dextrose injection
to make a paclitaxel concentration of 0.3 or 1.2 mg/mL, mix thoroughly. Inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 ^m Lichrosphere RP18
Flow rate: 1.3
Detector: UV 273
CHROMATOGRAM
Retention time: 2.1
OTHER SUBSTANCES
Simultaneous: polyoxyethylated castor oil, diethylhexyl phthalate
KEYWORDS
injections
REFERENCE
Mazzo,D.J.; Nguyen-Huu,J.-J.; Pagniez,S.; Denis,R Compatibility of docetaxel and paclitaxel in intravenous
solutions with polyvinyl chloride infusion materials, Am.J.Health-Syst.Pharm., 1997, 54, 566569.
SAMPLE
HPLCVARIABLES
Column: 250 X 4.6 5 |xm C18
Flow rate: 2.25
Detector: UV 254
CHROMATOGRAM
KEYWORDS
REFERENCE
Mayron,D.; Gennaro,A-R- Stability and compatibility of granisetron hydrochloride in i.v. solutions and oral
304.
SAMPLE
Matrix: plant
Sample preparation: Bark. Condition a Supelclean LC-18 SPE cartridge with MeOH and water.
Homogenize (Ystral) 20 g dried bark with 100 mL MeOH. Sonicate the homogenate in a bath
for 10 min, shake at 110 rpm for 30 min, filter through a glass filter. Re-extract residue with
50 mL MeOH, combine two MeOH extracts, evaporate to dryness at 40-45, reconstitute the
residue with 5 mL MeOH. Add a 500 (JLL aliquot to the SPE cartridge. Needles, clippings. Add
3 g sample to 100 mL chloroform:EtOH 50:50 (Caution! Chloroform is a carcinogen!), sonicate,
filter though a glass filter, evaporate the filtrate to dryness, reconstitute the residue in 5 mL
MeOH. Add a 500 JJLL aliquot to the SPE cartridge. Wash the cartridge twice with 2 mL portions
of water, with 2 mL MeOH:water 20:80, and with 2 mL MeOH:water 50:50. Elute with 2 mL
MeOH, evaporate the fractions to dryness in a speed vac, reconstitute the residue with two
100 |xL aliquots of MeCN, inject 10 |xL aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 10 |xm Lichrosorb RP-18
Column: 150 X 3.9 4 |xm Novapak Phenyl
Mobile phase: Gradient. A was MeCN:50 mM ammonium acetate 30:70. B was MeCN:50 mM
ammonium acetate 90:10. A:B 100:0, to 66:34 over 30 min, return to initial conditions over 2
min.
Flow rate: 0.8 (UV), 1 (MS)
Detector: UV 227; MS, Finnigan MAT TSQ-70, electrospray, positive ion mode 150 V, sheath
flow MeOH:water:acetic acid 80:20:1, mobile phase split 19:1 before MS
CHROMATOGRAM
Retention time: 22 (UV), 20 (MS)
OTHER SUBSTANCES
Extracted: baccatin III, cephalomannine, 10-DAB, 10-deacetyltaxol, 7-epi-10-deacetyltaxol, 7-
epi-taxol, taxinine M, taxol C, 7-xylosyl-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol B, 7-xylo-
syl-10-deacetyltaxol C, 7-xylosyl-taxol
KEYWORDS
bark; needles; clippings; SPE
REFERENCE
Theodoridis,G.; Laskaris,G.; de Jong,C.R; Hofte,A-J-P; Verpoorte,R. Determination of paclitaxel and related
diterpenoids in plant extracts by high-performance liquid chromatography with UV detection in high-per-
formance liquid chromatography-mass spectrometry, J.ChromatognA, 1998, 802, 297-305.
SAMPLE
Matrix: plants
Sample preparation: Extract needles with MeOH, concentrate the extract under reduced pres-
sure at <30, partition concentrate with 0.8 volumes of water and 0.8 volumes of chloroform,
repeat the extraction with 0.6 volumes chloroform then 0.4 volumes chloroform, concentrate
under reduced pressure, dry under vacuum at 35-40, dissolve in MeCN/water, inject an aliquot.
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Partisil C8
Mobile phase: MeCNiMeOH:water 50:10:40
Flow rate: 0.5
Detector: UV 254
KEYWORDS
needles; details of preparative HPLC in paper
REFERENCE
Rao,K.V.; Bhakuni,R.S.; Juchum,J.; Davies,R.M. A large scale process for paclitaxel and other taxanes from the
needles of Taxus x media Hicksii and Taxus floridana using reverse phase column chromatography,
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 (xm LiChrospher diol
Mobile phase: Gradient. MeOH.carbon dioxide 8:92 for 3 min, to 28:72 over 25 min, to 35:65
over 5.7 min, maintain at 35:65 for 4 min.
Flow rate: 2
Detector: UV 227
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Jagota,N.K.; Nair,J.B.; Frazer,R.; Klee,M.; Wang,M.Z. Supercritical fluid chromatography of paclitaxel,
J.ChromatogrA, 1996, 721, 315-322.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Guard column: C-18 Guard Pak
Column: 100 X 8 10 (xm Resolve C-18 radial compression (Waters)
Mobile phase: Gradient. MeCN:water from 45:55 to 100:0 over 20 min (exponential gradient),
maintain at 100:0 for 5 min, re-equilibrate at initial conditions for 5 min.
Flow rate: 2.5
Detector: UV 227
CHROMATOGRAM
REFERENCE
Wenk,M.R.; Fahr,A.; Reszka,R.; Seelig,J. Paclitaxel partitioning into lipid bilayers, J.Pharm.Sci., 1996, 85,
228-231.
Padimate O
Molecular formula: Ci 7 H 27 NO 2
Merck Index: 3282
SAMPLE
Sample preparation: Lotion. Weigh out lotion equivalent to about 90 mg oxybenzone, add 7 mL
water, add MeOH slowly with vigorous shaking until total volume was 100 mL. Remove a 10
mL aliquot and make up to 100 mL with MeOH, filter (paper), discard first 5 mL of filtrate.
Mix 4 mL filtrate and 1 mL 200 |xg/mL sulfathiazole in MeOH, make up to 10 mL with MeOH,
filter (0.45 (xm), inject a 20 jxL aliquot. Lipstick. Weigh out an amount equivalent to 25-90 mg
oxybenzone, add 10 mL chloroform, add MeOH slowly with vigorous shaking until total volume
was 100 mL. Remove a 10 mL aliquot and make up to 100 mL with MeOH, filter (paper),
discard first 5 mL of filtrate. Mix 4 mL filtrate and 1 mL 200 (xg/mL sulfathiazole in MeOH,
make up to 10 mL with MeOH, filter (0.45 |xm), inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 40 X 4.6 25-37 |xm Co:Pell ODS
Column: 250 X 4.6 10 |xm Partisil PXS ODS-2
Mobile phase: MeCN:MeOH 10:90
Flow rate: 0.7
Detector: UV 254
CHROMATOGRAM
Retention time: 7.4
OTHER SUBSTANCES
Simultaneous: oxybenzone, propyl paraben
KEYWORDS
lotion; lipstick; sun-screen
REFERENCE
Tan,H-S.L; Sih,R; Moseley,S.E.; Lichtin,J.L. Assay of mixtures of padimate-0 and oxybenzone in sunscreen
formulations by high-performance liquid chromatography, J.Chromatogr., 1984, 291, 275-282.
Palinavir
CAS Registry No/. 154612-39-2
SAMPLE
M a t r i x : blood
Sample preparation: 500 |xL Plasma + 50 |xL 1.5 M NaOH, extract three times with 2 mL
portions of diethyl ether, vortex for 30 s, centrifuge at 3000 rpm for 10 min at 4. Evaporate
the ether extract under a stream of nitrogen. Reconstitute the residue with 100 JJLL mobile
phase. Inject an 80 |xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 C-8 Nova-Pak
Mobile phase: MeCN:50 mM pH 3.0 potassium phosphate buffer 50:50 containing 0.1%
dimethyloctylamine
Flow rate: 1.5
Detector: UV 237
CHROMATOGRAM
KEYWORDS
plasma; pharmacokinetics; rat
REFERENCE
Liard,R; Jaramillo,J.; Paris,W.L.; Yoakim,C. Pharmacokinetic aspects of palinavir, an HIV protease inhibitor,
in Sprague-Dawley rats, J.Pharm.Sci., 1998, 87, 782-785.
Pamidronate
Molecular formula: C3H9NNa2O7P2 o o
Molecular weight: 279.01 Na+ -o Il OH || JQ- Na+
CAS Registry No.: 57248-88-1, 109552-15-0 ^ p \ ^ p \
(pentahydrate), 40391-99-9 (free acid) HCT ^ ' T)H
Merck Index: 7135
H2N
\ ^
SAMPLE
Matrix: blood
Sample preparation: Condition a 3 mL 500 mg Bakerbond quaternary amine SPE cartridge
(J.T.Baker) with 2.5 mL water, 1 mL 1 mg/mL etidronate solution, 2.5 mL 100 mM nitric acid,
and 5 mL water. Add 20 jxL 5 mg/mL etidronate solution, 35 |JLL 100 mg/mL citric acid (to
serum only), and 30 JJLL 2050 ng/mL neridronic acid solution to 1 mL serum or citrated plasma.
Add 200 |xL 20% trichloroacetic acid, vortex vigorously, centrifuge at 14000 g for 5 min. Add
30 |xL 1 M calcium chloride, 40 |JLL 100 mM NaH2PO4, and 200 |xL 1 M NaOH to the clear
supernatant, vortex, centrifuge at 3900 g for 2 min, discard the liquid phase. Dissolve the
residue in 25 |JLL 1 M HCl, dilute with 2.5 mL water, add to the SPE cartridge, wash with 1
mL water at 2 mL/min, wash with 4 mL water and 2.5 mL 10 mM nitric acid at 3 mL/min.
Elute with 2.5 mL 100 mM nitric acid at 2.5 mL/min, evaporate the eluate to dryness under
0.6 bar nitrogen at 80 for 50-75 min, reconstitute the residue in 500 |JLL water, vortex. Add 50
fjiL 1 mg/mL etidronate, 75 |JLL triethylamine, and 500 |xL 20 mg/mL 1-naphthylisothiocyanate
in pyridine, mix by air bubbling to form a clear yellow solution. Heat the mixture at ca.80 for
15 min, extract twice with 2 mL 10 mg/mL tetrabutylammonium bromide in chloroform (Cau-
tion! Chloroform is a carcinogen!), mix the two phases by bubbling 3 mL air through the liquid,
let the phases separate for 1 min, remove the lower organic layer. Mix 300 jxL of the resulting
sample and 90 |xL 3% hydrogen peroxide, heat at ca.80 for 5 min, inject a 100 jxL aliquot.
HPLC VARIABLES
Guard column: 10 X 2 reversed-phase R2 (Chrompack)
Column: 100 X 4.6 3 ^m Microspher C18 (Chrompack)
Mobile phase: MeCN: 10 mM phosphate buffer containing 10 mM tetraoctylammonium bromide
and 2 mM etidronate 62:38, pH 8.0
Flow rate: 0.8
CHROMATOGRAM
Retention time: 12
Internal standard: neridronic acid (10.5)
KEY WpRDS
derivatization; plasma; serum; SPE
REFERENCE
Sparidans,R.W.; Den Hartigh,J.; Beijnen,J.H.; Vermeij,P. Semi-automatic liquid chromatographic analysis of
pamidronate in serum and citrate plasma after derivatization with 1-naphthylisothiocyanate,
J.Chromatogr.B, 1998, 705, 331-339.
SAMPLE
Sample preparation: Add 10 |JLL 300 |xg/mL IS to 100 |xL 30 |xL aqueous solution prepared from
injections or tablets, vortex with 50 |JLL EtOH, 40 |xL pyridine, 10 JJLL triethylamine, and 2 |JLL
phenylisothiocyanate to yield a clear solution. Heat at 80 for 5 min and evaporate under
nitrogen at 80. Reconstitute the dry residue in 1 mL water, wash twice by vortexing with 1
mL 2 g/L tetrabutylammonium bromide in chloroform (Caution! Chloroform is an carcinogen!),
centrifuge at 3900 g for 2 min, discard the organic layer. Add 90 jxL 0.06% hydrogen peroxide
to 900 |JLL of the aqueous phase, heat at 80 for 2 min, evaporate to dryness at 80, reconstitute
the residue in 100 |xL mobile phase. Inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 10 X 2 R2 (Chrompack, Netherlands)
Column: 100 X 4.6 3 |xm Microspher C18 (Chrompack, Netherlands)
Mobile phase: MeCNibuffer 22.5:77.5 (Buffer was 30 mM pH 7.0 phosphate buffer containing 5
mM tetrabutylammonium hydroxide and 2 mM etidronate.)
Flow rate: 0.8
Detector: UV 240
CHROMATOGRAM
Retention time: 6.8
Internal standard: neridronic acid (9.0)
KEYWORDS
tablets; injections; derivatization
REFERENCE
Sparidans,R.W.; Den Hartigh,J.; Ramp-Koopmanschap,W.M.; Langebroek,R.H.; Vermeij,P. The determination
of pamidronate in pharmaceutical preparations by ion-pair liquid chromatography after derivatization with
phenylisothiocyanate, J.Pharm.Biomed.Anal., 1997, 16, 491-497.
SAMPLE
Matrix: solutions
Sample preparation: Vortex 100 |xL 0.5 mg/mL pamidronate disodium in water, 15 JJLL triethyl-
amine, and 100 |xL 20 mg/mL naphthylisothiocyanate in pyridine to yield a clear solution. Heat
in a sealed tube at 80 for 15 min. Wash twice with 1 mL 10 mg/ml tetrabutyl ammonium
bromide in chloroform, discard the organic solvent. Add 10 |JLL 0.2% hydrogen peroxide solution
and heat at 80. Inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 100 X 3.0 5|xm Chromspher C18 (Chrompack)
Mobile phase: MeCN:buffer 60:40 (Buffer was 20 mM phosphate buffer containing 5 mM tet-
raoctylammonium bromide + 500 |xM etidronate (adsorption supressor).)
Flow rate: 0.4
Detector: UV 285; F ex 285 em 390
CHROMATOGRAM
KEYWORDS
derivatization
REFERENCE
Sparidans,R.W.; Den Hartigh,J.; Beijnen,J.H.; Vermeij,P. Derivatization of pamidronate and other
amino(bis)phosphonates with different isothiocyanates prior to ion-pair liquid chromatography,
J.ChromatogrA, 1997, 782, 211-217.
SAMPLE
Sample preparation: 2 mL Plasma or urine + 100 |xL 25 juig/mL IS in 100 mM NaOH, adjust
pH to 3 with concentrated HCl, filter (0.45 fxm fluoro-membrane, Skan Model Aero LC 13). Add
0.5 mL 1.5 M trichloroacetic acid to the nitrate, centrifuge at 1500 g for 15 min. Remove the
supernatant, add 20 |xL 2.5 M calcium chloride solution, add 40 |xL 500 mM NaH2PO4, adjust
pH to 12.0 with 6.25 M NaOH then with 0.5 M NaOH, centrifuge at 1500 g for 15 min. Wash
the precipitate with water, dissolve the precipitate in 200 IJLL 130 mM disodium EDTA adjusted
to pH 10 with 6.25 M NaOH, add 100 |xL 3 mg/mL fluorescamine in MeCN while vortexing
vigorously, add 200 JJLL dichloromethane, extract, centrifuge at 1000 g for 3 min, remove the
aqueous phase, inject a 10 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Mobile phase: MeOHrI mM disodium EDTA adjusted to pH 6.5 with 1 M NaOH 3:97
Flow rate: 1
CHROMATOGRAM
Retention time: 3.9
Internal standard: 6-amino-l-hydroxypentilidenebisphosphonate (CGP 33 637) (4.8)
Limit of detection: 10 nM (plasma, urine)
Limit of quantitation: 800 nM (plasma), 700 nM (urine)
KEYWORDS
derivatization; pharmacokinetics; plasma
REFERENCE
Flesch,G.; Tominaga,N.; Degen,P. Improved determination of the bisphosphonate pamidronate disodium in
plasma and urine by pre-column derivatization with fluorescamine, high-performance liquid chromatogra-
phy and fluorescence detection, J.Chromatogr., 1991, 568, 261-266.
SAMPLE
Matrix: bone
Sample preparation: Dissolve 25 mg ground (<20 |jim) bone in 2 mL 200 mM HCl, add 5 |xg
IS, vortex, let stand overnight at room temperature, centrifuge. Remove a 500 |xL aliquot and
add it to 1 mL 10 mM NaOH and 50 |xL 1 M NaOH, centrifuge at 1000 g for 10 min, wash the
pellet with 1 mL water, centrifuge, discard the supernatant. Dissolve the pellet in 200 |xL 200
mM phosphoric acid, add 250 |JLL 200 mM pH 10.3 EDTA in 200 mM NaOH, add 200 |JLL resin,
vortex, centrifuge, filter (0.2 jjum) a 550 |xL aliquot of the supernatant, add 10 JJLL 10 M NaOH
to the filtrate. Remove a 50 |xL aliquot and add it to 50 (xL 1 M pH 10.7 carbonate buffer, add
10 JJLL 1 mg/mL 2,3-naphthalenedicarboxaldehyde, add 10 |xL 1 mg/mL N-acetyl-D-penicilla-
mine, mix, let stand for 2 min, inject a 20-50 JJLL aliquot. (The resin was AG 50W-X8 (K+ form)
resin. Prepare the+ resin by adding 3 volumes of 1 M KOH to 200-400 mesh AG 50W-X8 cation-
exchange resin H form (Bio-Rad), stir for 30 s, decant the supernatant, repeat the procedure
twice, wash five times with 3 volumes of water, store at 4, before use wash 2 or 3 times with
three volumes of water (J. Chromatogr. 1992, 584, 213).)
HPLC VARIABLES
Column: 150 X 4.6 PLRP-S (Phenomenex)
Mobile phase: MeCN:25 mM pH 6.5 citrate-phosphate buffer 16:94
Flow rate: 1
CHROMATOGRAM
Retention time: 5.0
Internal standard: l-hydroxypentanylidene-l,l-bisphosphonate(Ciba-Geigy) CGP 38146 (13.5)
Limit of quantitation: 7.5 jxg/g
KEYWORDS
derivatization
REFERENCE
King,L.E.; Vieth,R. Extraction and measurement of pamidronate from bone samples using automated pre-
column derivatization, high-performance liquid chromatography and fluorescence detection, J.Chro-
matogr.B, 1996, 678, 325-330.
SAMPLE
Sample preparation: Dilute injections 100-fold, inject a 20 |xL aliquot. Disintegrate a 5 mg
tablet in 100 mL water, sonicate for 5 min, centrifuge an aliquot at 3600 g for 4 min, inject a
HPLC VARIABLES
Column: 150 X 4.6 10 |xm IC-PAK Anion HC (Waters)
Mobile phase: 1.5 mM Nitric acid containing 0.5 mM copper(II) nitrate (Prepare column by
pumping ILC Regenerant A (Waters) and 100 mM nitric acid for 30 min.)
Flow rate: 1
Detector: UV 245
CHROMATOGRAM
Retention time: 2
OTHER SUBSTANCES
Simultaneous: alendronate, clodronate, etidronate, neridronate, olpadronate
KEYWORDS
derivatization; complexation; injections; tablets
REFERENCE
Sparidans,R.W.; Den Hartigh,J.; Vermeij,P. High-performance ion-exchange chromatography with in-line
complexation of bisphosphonates and their quality control in pharmaceutical preparations,
SAMPLE
Matrix: urine
Sample preparation: Filter dog urine through filter paper. 2 mL Urine + 100 |xL IS in water,
adjust pH to 3 with concentrated HCl, filter (0.45 |jim fluoro-membrane, Skan Model Aero LC
13). Add 0.5 mL 1.5 M trichloroacetic acid to the filtrate, centrifuge at 1500 g for 15 min.
Remove the supernatant, add 20 |xL 2.5 M calcium chloride solution, add 40 |JLL 500 mM
NaH2PO^, adjust pH to 12.0 with 6.25 M NaOH then with 0.5 M NaOH, centrifuge at 1500 g
for 15 min. Wash the precipitate with water, dissolve the precipitate in 200 |xL 130 mM diso-
dium EDTA adjusted to pH 9 with 6.25 M NaOH, add 100 |JLL 1 mg/mL fluorescamine in MeCN
while vortexing vigorously, add 200 |xL dichloromethane, extract, centrifuge at 1000 g for 3
min, remove the aqueous phase, inject a 10 |xL aliquot of the aqueous phase.
HPLC VARIABLES
Mobile phase: MeOH:l mM disodium EDTA adjusted to pH 6.5 with 1 M NaOH 3:97
Flow rate: 1
CHROMATOGRAM
Retention time: 4
Internal standard: 6-amino-l-hydroxyhexylidenebisphosphonate(CGP 38 146) (8)
KEY WpRDS
derivatization; human; dog; pharmacokinetics
REFERENCE
Flesch,G.; Hauffe,S.A. Determination of the bisphosphonate pamidronate disodium in urine by pre-column
derivatization with fluorescamine, high-performance liquid chromatography and fluorescence detection,
J.Chromatogr., 1989, 489, 446-451.
SAMPLE
Matrix: urine
Sample preparation: Condition a 3 mL 500 mg Bakerbond quaternary amine SPE cartridge
with 2.5 mL water, 1 mL 1 mg/mL etidronate, 2.5 mL 100 mM nitric acid, and two 2.5 mL
portions of water. 2.5 mL Urine + 100 JULL 1 mg/mL etidronate + 100 JULL 2.16 jjLg/mL IS, mix,
add 30 jxL 1 M calcium chloride, vortex, add 25 jxL portions of 1 M NaOH (vortexing after each
addition) until a precipitate is clearly present, add 25 |xL 1 M NaOH, vortex, centrifuge at
3900 g for 2 min. Remove the pellet and dissolve it in 50 uX. 1 M HCl, add 2.5 mL water, add
50 |xL 1 M NaOH, centrifuge. Remove the pellet and dissolve it in the minimum amount of 1
M HCl (30-50 (xL), add 2.5 mL water, add 30 |xL 1 M NaOH, centrifuge. Remove the pellet and
dissolve it in the minimum amount of 1 M HCl (30-50 |iL), add 2.5 mL water, add to the SPE
cartridge, wash with two 2.5 mL portions of water, wash with 2.5 mL 10 mM nitric acid, elute
with 2.5 mL 100 mM nitric acid. Evaporate the eluate to dryness under 0.8 bar nitrogen at
60 for 1.65-2 h, add 500 JJLL water, vortex. Remove a 250 JXL aliquot and add it to 25 jxL 1 mg/
mL etidronate, add 40 |JLL triethylamine, add 250 jxL 20 mg/mL 1-naphthylisothiocyanate in
pyridine, vortex, heat at 80 for 15 min, cool, add 2 mL 10 mg/mL tetrabutylammonium bromide
(?) in chloroform, vortex, centrifuge, discard the lower organic phase, repeat the procedure.
Remove a 250 uL aliquot of the aqueous phase and add it to 75 jxL 3% hydrogen peroxide (to
convert the thiourea derivative to the corresponding urea), mix, heat at 80 for 5 min, inject a
100 (JLL aliquot.
HPLC VARIABLES
Guard column: 10 X 2 R2 (Chrompack)
Column: 100 X 4.6 3 jjim Microspher C18 (Chrompack)
Mobile phase: MeCN:buffer 65:35 (Buffer was 10 mM phosphate containing 10 mM tetraoctyl-
ammonium bromide and 2 mM etidronate (monosodium (l-hydroxyethylidene)bisphosphonate)
(as adsorption suppressor), pH 7.6-7.9.)
Flow rate: 0.8
CHROMATOGRAM
Retention time: 10
Internal standard: (3-amino-3-phenyl-l-hydroxypropylidene)bisphosphonicacid (17)
KEYWpRDS
derivatization; SPE
REFERENCE
Sparidans,R-W.; Den Hartigh,J.; Beijnen,J.H.; Vermeij,P. Determination of pamidronate in urine by ion-pair
liquid chromatography after derivatization with 1-naphthylisothiocyanate, J.Chromatogr.B, 1997,696,137-
144.
Pancreatin
Merck Index: 7137
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 Rexchrom 5 |xm 300A C8 (Regis)
in MeCN. A:B 100:0 to 0:100 over 40 min.
Flow rate: 1.5
Detector: UV 214
CHROMATOGRAM
Retention time: 18-27 (multiple peaks)
REFERENCE
Baxter Scientific Products Catalog, 1990-1, p. 154.
Pancuronium bromide
Molecular formula: C35H60Br2N2O4
Merck Index: 7139
Lednicer No.: 2 163
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma + 250 (xL picric acid (1:10 dilution of saturated picric acid
solution) + 250 |xL vecuronium solution + 250 |xL water + 5 mL dichloromethane:isopropanol
85:15, vortex for 15 s, centrifuge at 1500 g for 10 min. Remove the organic phase and evaporate
it to dryness at 40 under a stream of nitrogen, reconstitute the residue in 150-250 |xL MeCN:
water 40:60, centrifuge at 1500 g for 4 min, inject a 20-100 (xL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 jxPorasil
Mobile phase: MeCN:2 mM sulfuric acid 50:50
Flow rate: 2
Detector: conductivity 2500 nS full scale
CHROMATOGRAM
Retention time: 5.7
Internal standard: vecuronium (4.7)
KEYWORDS
plasma
REFERENCE
Bjorksten,A.R.; Beemer,G.H.; Crankshaw,D.P. Simple high-performance liquid chromatographic method for the
analysis of the non-depolarizing neuromuscular blocking drugs in clinical anaesthesia, J.Chromatogr., 1990,
533, 241-247.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Prepare a 0.5% solution in the mobile phase, inject a 20 JULL aliquot.
HPLC VARIABLES
Column: 250 X 4 5 |xm SI 100 (Bio Separation Technologies)
Mobile phase: MeCNrIOO mM sodium perchlorate 96:4
Flow rate: 1
Detector: UV 213
CHROMATOGRAM
Retention time: 4.4
OTHER SUBSTANCES
Simultaneous: pipecuronium
Interfering: vecuronium
REFERENCE
Gazdag,M.; Babjak,M.; Kemenes-Bakos,R; Gorog,S. Analysis of steroids. XLI. Ion-pair high-performance liquid
chromatographic separation of quaternary ammonium steroids on silica, J.Chromatogr., 1991, 550, 639-
644.
Panomifene
Molecular formula: C25H24F3NO2
SAMPLE
Matrix: blood
Sample preparation: Condition a 1 mL phenyl SPE cartridge with five 1 mL portions of MeCN,
1 mL water, and 1 mL 2.5 mL/L triethylamine in 50 mM pH 3.0 phosphate buffer. 980 JJLL
Plasma + 20 |xL MeOH:water 50:50 + 10 |xL 2 |xg/mL tamoxifen in MeOH:water 50:50 + 1 mL
MeCN, vortex for 1 min, centrifuge at -10 at 2500 g for 1 h. Remove a 1.6 mL aliquot of the
supernatant and add it to 400 |xL 2% heptanesulfonic acid in 50 mM pH 3.0 KHsPCVphosphoric
acid buffer, add to the SPE cartridge, wash with two 100 JJLL portions of MeCN:buffer 80:20,
wash with 50 fjuL 25 mM sulfuric acid, elute with five 100 |xL portions of MeCN.buffer 80:20.
Evaporate the eluate to dryness under a stream of nitrogen at room temperature, reconstitute
the residue in 100 jxL MeCN:buffer 70:30, inject a 10-30 |xL aliquot. (Buffer was 5 mM hep-
tanesulfonic acid in 50 mM pH 3.0 phosphate buffer.)
HPLC VARIABLES
Guard column: 20 X 4.6 10 jim Si-IOO-S Phenyl (BST, Budapest)
Column: 250 X 4.6 10 |xm Si-IOO-S Phenyl (BST, Budapest)
Mobile phase: MeCN:buffer 75:25 (Buffer was 50 mM pH 3.0 KHgPCVphosphoric acid buffer
containing 5 mM heptanesulfonic acid and 300 jxL/L triethylamine. Temperature of MeCN was
60 and temperature of buffer was 80.)
Flow rate: 1.2
a 10 m X 0.3 mm ID knitted PTFE coil irradiated by a mercury lamp at 254 nm to the detector.
CHROMATOGRAM
Internal standard: tamoxifen (7.03)
KEYWORDS
post-column reaction; post-column photochemical derivatization; plasma; pharmacokinetics; SPE
REFERENCE
Erdlyi-T6th,V.; Pap,E.; Kralovdnszky,J.; Bojti,E.; Klebovich,I. Determination of panomifene in human plasma
by high-performance liquid chromatography, J.ChromatogrA, 1994, 668, 419-425.
Pantoprazole
Molecular formula: C16H15F2N3O4S
Merck Index: 7146
Lednicer No.: 5 115
SAMPLE
Matrix: blood
Sample preparation: Centrifuge plasma at 2000 g for 10 min, inject a 100 JJLL aliquot on to
column A and elute to waste with mobile phase A, after 2 min backnush the contents of column
A on to column B and start the gradient, monitor the effluent from column B.
HPLC VARIABLES
Column: A 10 X 4.6 25-40 jxm LiChroprep RP-2; B 12 X 4.6 5 jxm Hypersil RP-18 + 125 X 4.6
5 |xm Hypersil RP-18
Mobile phase: A MeCN:50 mM pH 5 sodium acetate (Merck Extra Pure) buffer 10:90; B Gra-
dient. MeOH:10 mM pH 6.5 ammonium phosphate buffer from 43:57 to 83:17 over 2 min, after
17 min flush at 100:0 for 2 min, re-equilibrate at initial conditions for 7 min.
Detector: UV 290
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Doyle,E.; McDowall,R.D.; Murkitt,G.S.; Picot,V.S.; Rogers,S.J. Two systems for the automated analysis of drugs
in biological fluids using high-performance liquid chromatography, J.Chromatogr., 1990, 527, 67-77.
SAMPLE
Matrix: blood
Sample preparation: Centrifuge serum or plasma at 2000 g for 10 min, inject a 200 jxL aliquot
on to column A and elute to waste with mobile phase A, after 2 min backflush the contents of
column A on to column B with mobile phase B and start the gradient, monitor the effluent
from column B. Between runs flush column A with mobile phase A.
HPLC VARIABLES
Column: A 25-40 jxm LiChroprep RP-2; B 12 X 4.6 5 (xm Hypersil RP-18 + 125 X 4.6 5 |xm
Hypersil RP-18
Mobile phase: A 100 mM pH 5 sodium acetate buffer; B Gradient. MeOHrIO mM pH 6.5
(NH4)2HPO4 43:57 for 2 min, to 83:17 over 17 min, to 100:0 over 2 min, re-equilibrate for 7
min.
Detector: UV 290
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
serum; plasma; dog; pharmacokinetics; column-switching
REFERENCE
Huber,R-; Muller,W.; Banks,M.C; Rogers,S.J.; Norwood,P.C; Doyle,E. High-performance liquid chromato-
graphic determination of the H+/K+ ATPase inhibitor (BY 1023/SK&F 96,022) and its sulphone metabolite
in serum or plasma by direct injection and fully automated pre-column sample clean-up, J.Chromatogr.,
1990, 529, 389-401.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a solution in EtOH, inject a 10-20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Chiralpak AD
Mobile phase: Hexane:EtOH 80:20
Flow rate: 1
Detector: UV 302
CHROMATOGRAM
Retention time: k' 6.10 (of first enantiomer)
OTHER SUBSTANCES
Simultaneous: lansoprazole, omeprazole, timoprazole
KEYWORDS
chiral; a = 1.19
REFERENCE
Balmer,K; Persson,B--A.; Lagerstrom,R-O. Stereoselective effects in the separation of enantiomers of omepra-
zole and other substituted benzimidazoles on different chiral stationary phases, J.ChromatogrA, 1994,660,
269-273.
Pantothenic acid
CAS Registry No.: 79-83-4 (D), 137-08-6 (Ca salt D),
6381-63-1 (Ca salt racemic), 599-54-2 (racemic)
Merck Index: 7147
SAMPLE
residue with 50 JULL MeCN.water 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: food
Sample preparation: Completely dissolve 20 g of an elemental diet (Elental, Ajinomoto Co.,
Kawasaki) in 60 mL water at 50, add 10 g sodium chloride, let stand at room temperature for
30 min. Dilute to 100 mL with water, wash with 10 mL hexane for 3 min to remove any oils.
Inject a 20 |xL aliquot of the aqueous layer onto column A and elute to waste with mobile phase
A, after 4 min elute the contents of column A onto column B with mobile phase B, when the
compounds of interest have moved onto column B remove column A from the circuit, elute
column B with mobile phase B, monitor the effluent from column B.
HPLC VARIABLES
Column: A 150 X 4.6 5 (xm Capcellpak C18 (Shiseido, Tokyo); B 250 X 4.6 5 ^m Capcellpak C18
(Shiseido, Tokyo)
Mobile phase: A MeCNrbuffer 5:95; B MeCN:buffer 9:91 containing 1.5 mM sodium 1-heptane-
sulfonate (Buffer was water adjusted to pH 2.1 with phosphoric acid.)
Flow rate: 1.2
Detector: UV 210
CHROMATOGRAM
Limit of detection: ca. 5 ng
KEYWORDS
elemental diet; column switching
REFERENCE
Iwase,H. Determination of pantothenic acid in an elemental diet by column-switching high-performance liquid
chromatography with ultraviolet detection, Anal.ScL, 1993, 9, 149-151.
SAMPLE
Sample preparation: Sonicate tablet in 40 mL mobile phase until it dissolves, make up to 50
mL with mobile phase, filter (0.45 (Jim), inject a 100 |xL aliquot.
HPLC VARIABLES
Column: 200 mm long Nucleosil 7 C18
Mobile phase: Waterrglacial acetic acid 95:5
Flow rate: 2
Detector: RI
CHROMATOGRAM
KEYWORDS
tablets; detector temp 35
REFERENCE
Jonvel,R; Andermann,G.; Barthelemy,J.F. Determination of calcium pantothenate in multivitamin preparations
SAMPLE
Sample preparation: Pulverize tablets if necessary. Add tablets containing 50 mg calcium pan-
tothenate to 100 mL 5 mM pH 4.5 potassium phosphate buffer, sonicate at 75 W for 2 min,
cool to room temperature, make up to 200 mL with buffer, filter (0.45 fxm), inject a 10 JJLL
HPLC VARIABLES
Column: 250 X 4.6 10 |xm LiChrosorb NH2 aminopropyl
Mobile phase: MeCN:5 mM KH2PO4 87:13 (Wash column with MeCN:water 10:90 at the end of
the day.)
Flow rate: 2
Detector: UV 210
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: thiamine, riboflavin, niacinamide, pyridoxine
KEYWORDS
tablets
REFERENCE
Hudson,T.J.; Allen,R-J- Determination of pantothenic acid in multivitamin pharmaceutical preparations by
reverse-phase high-performance liquid chromatography, J.Pharm.Sci., 1984, 73, 113-115.
SAMPLE
Sample preparation: Grind tablet, add 80 mL water, shake at 240 oscillations/min for 30 min,
make up to 100 mL with water, mix well, centrifuge at 1500 rpm, dilute supernatant with
water to a concentration of 45 |xg/mL, filter (0.45 |xm), inject an aliquot of the nitrate.
HPLC VARIABLES
Column: 150 X 4.6 5 um Hypersil ODS
Mobile phase: MeCN:buffer 3:97 (Buffer was 250 mM NaH2PO4 adjusted to pH 2.5 with phos-
phoric acid.) (Flush column with MeCN:water 5:95 at the end of each day.)
Flow rate: 2
Detector: UV 205
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Simultaneous: saccharin, pantoyllactone, degradation products
Interfering: panthenol
KEYWORDS
REFERENCE
Timmons,J.A.; Meyer,J.C; Steible,D.J.; Assenza,S.P. Reverse phase liquid chromatographic assay for calcium
pantothenate in multivitamin preparations and raw materials, J.Assoc.Off.Anal.Chem., 1987, 70, 510-513.
SAMPLE
HPLC VARIABLES
Column: 100 X 4 3 ^m Hypersil BDS-C18
Flow rate: 0.5
Detector: UV 220
CHROMATOGRAM
Retention time: 6.3
OTHER SUBSTANCES
Simultaneous: biotin, caffeine, citric acid, folic acid, niacinamide, niacin, riboflavin, saccharin,
thiamine, pyridoxine, vitamin B12, ascorbic acid
KEYWORDS
tablets
REFERENCE
Hewlett Packard Leaflet 12-5091-7351 EUS,, 1993.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1
Detector: UV 200
CHROMATOGRAM
Retention time: 0.9
OTHER SUBSTANCES
Simultaneous: niacin, pyridoxine, riboflavin, thiamine, niacinamide, ascorbic acid
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 2.5
OTHERSUBSTANCES
Simultaneous: biotin, folic acid, niacin, riboflavin, niacinamide
REFERENCE
Papaverine
Merck Index: 7151
Lednicer No.: 1 347
SAMPLE
Matrix: blood
Sample preparation: 2 mL Plasma +15 jxL MeOH + 200 jxL 4 M NaOH + 5 mL diethyl ether,
vortex for 5 min, centrifuge at 2000 g for 10 min, remove the organic layer, extract the aqueous
layer with 2 mL diethyl ether, centrifuge. Combine the organic layers and add them to 500 |xL
1 M HCl, vortex for 1 min, centrifuge at 2000 g for 10 min. Remove the aqueous layer and add
it to 500 jiL 4 M NaOH, vortex for 1 min, centrifuge at 2000 g for 10 min. Remove the organic
in 25 |JLL MeOH, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 ^m Partisil 10 ODS
Mobile phase: MeOH:0.1% KH2PO4 65:35
Flow rate: 2
Detector: UV 238
CHROMATOGRAM
Retention time: 3.5
Internal standard: papaverine
OTHER SUBSTANCES
Extracted: ethaverine
KEYWORDS
plasma; papaverine is IS
REFERENCE
Brodie,R-R; Chasseaud,L.R; Walmsley,L.M.; Soegtrop,H.H.; Darragh,A.; O'Kelly,D.A. Determination of the an-
tispasmodic agent ethaverine in human plasma by high-performance liquid chromatography, J. Chromatogr.,
1980, 182, 379-386.
SAMPLE
Matrix: blood
Sample preparation: 4 mL Whole blood + 100 |xL 8 (xg/mL mepyramine maleate in water (pre-
pare fresh daily), vortex, add 10 mL pH 10.0 phosphate buffer (|x = 0.4), vortex, add 5 mL
chloroform:hexane 40:60, shake gently horizontally for 30 min, centrifuge. Remove 3 mL of the
residue in 250 |JLL dichloromethane, inject a 100 JJIL aliquot.
HPLC VARIABLES
Column: 300 X 4 10 |xm Micropak CN-10
Mobile phase: n-Hexane:dichloromethane:MeCN:propylamine 50:25:25:0.1
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
Retention time: 3.4
Internal standard: pyrilamine maleate (mepyramine maleate) (4.0)
OTHER SUBSTANCES
Simultaneous: carbetapentane, cocaine, diamorphine, dioxyline, ethaverine, fluphenazine, imip-
ramine, papaveraldine, promethazine, strychnine, thonzylamine
Interfering: methapyrilene, procaine, yohimbine
KEYWORDS
whole blood; pharmacokinetics
REFERENCE
Hoogewijs,G.; Michotte,Y.; Lambrecht,J.; Massart,D.L. High-performance liquid chromatographic determina-
tion of papaverine in whole blood, J.Chromatogr., 1981, 226, 423-430.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 50 |xL 100 |xg/mL noscapine + 1 mL 100 mM HCl + 6 mL
dichloromethane, extract, centrifuge. Remove the organic layer and evaporate it to dryness
JJLL aliquot.
HPLC VARIABLES
Column: Lichrosorb Si-60
Mobile phase: Hexane:dichloromethane:MeOH:diethylamine 95:4:1:0.03
CHROMATOGRAM
Internal standard: noscapine
KEYWORDS
plasma; normal phase; pharmacokinetics
REFERENCE
Berg,G.; Jonsson,K.-A.; Hammar,M.; Norlander,B- Variable bioavailability of papaverine, Pharmacol.ToxicoL,
1988, 62, 308-310.
SAMPLE
Matrix: blood
stoppered tube for 1 min, add 6 mL NiCl2, rock for 5 min, add 15 mL dichloromethane:isobutyl
water.)
HPLC VARIABLES
Mobile phase: Gradient A was MeCN. B was 20 mM n-propylamine adjusted to pH 5 with 85%
phosphoric acid. A:B from 15:85 to 20:80 over 5 min to 45:55 over another 15 min to 65:35 over
another 5 min.
Detector: UV 230
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
methaqualone, morphine, naloxone, noscapine, pentazocine, procaine
KEYWORDS
whole blood
REFERENCE
SAMPLE
Sample preparation: 1 mL Plasma or urine + 300 |JLL 200 |xg/mL laudanosine, mix, add 10 mL
chloroformiisopropanol 95:5, vortex for 2 min, centrifuge for 30 min. Remove 8 mL of the or-
ganic layer and evaporate it to dryness under a stream of air at room temperature, reconstitute
the residue in 100 (xL MeOH, inject a 20 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 10 |jim C8 (Brownlee)
Mobile phase: MeOH:15 mM pH 8.5 sodium borate buffer 58:42
Flow rate: 2.7
Detector: UV 239
CHROMATOGRAM
Retention time: 5.0
Internal standard: laudanosine (9.5)
OTHER SUBSTANCES
Noninterfering: caffeine, chlorothiazide, gentamicin, hydrochlorothiazide, oxytetracycline, tet-
racycline, theobromine, theophylline
KEYWORDS
plasma
REFERENCE
Gautam,S.R.; Nahum,A.; Baechler,J.; Bourne,D.W. Determination of papaverine in plasma and urine by high-
SAMPLE
Sample preparation: Plasma. Condition a Toxiclean SPE cartridge (Alltech) with 3 mL MeOH,
two 3 mL portions of water, and 2 mL buffer. 100 |JLL Plasma or serum + 100 |xL MeOH + 200
(JLL MeCN + 100 |JLL buffer, vortex for 1 min, centrifuge at 4000 rpm for 15 min, add the
supernatant to the SPE cartridge, wash with two 3 mL portions of water, dry under vacuum
for 10 min, elute with 2 mL MeOH. Evaporate the eluate to dryness under a stream of nitrogen
at 45, reconstitute the residue in 100 |xL 2.5 jxg/mL flufenamic acid in MeOH (?), inject an
aliquot. Urine. Condition a Bond Elut C8 SPE cartridge with 3 mL MeOH, two 3 mL portions
of water, and 2 mL buffer. 100 |xL Urine + 100 (xL MeOH + 200 |xL MeCN + 500 |xL buffer,
vortex for 1 min, centrifuge at 2000 rpm for 5 min, add the supernatant to the SPE cartridge,
wash with two 3 mL portions of water, dry under vacuum for 10 min, elute with 2 mL MeOH.
100 (xL 2.5 |xg/mL flufenamic acid in MeOH (?), inject an aliquot. (Buffer was 250 mL 25 mM
sodium borate and 18 mL 100 mM NaOH, pH 9.2.)
HPLC VARIABLES
Column: 250 X 4.6 5 um Adsorbosphere HS C18
Mobile phase: MeCN:MeOH:1.2% ammonium acetate 15:40:45
Flow rate: 0.8
Detector: UV 239
CHROMATOGRAM
Internal standard: flufenamic acid (24.39)
Limit of quantitation: 60 ng/mL (urine), 20 ng/mL (plasma, serum)
OTHER SUBSTANCES
Extracted: codeine, monoacetylmorphine, morphine
KEYWORDS
SPE; plasma; serum
REFERENCE
Theodoridis,G.; Papadoyannis,L; Tsoukali-Papadopoulou,H.; Vasilikiotis,G. A comparative study of different
solid phase extraction procedures for the analysis of alkaloids of forensic interest in biological fluids by RP-
HPLC/Diode array, J.Liq.Chromatogr., 1995, 18, 1973-1975.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 251.1
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: bulk
Sample preparation: Prepare a 750 ixg/mL solution in 10 mM pH 2.5 orthophosphoric acid,
sonicate for 10 min, filter (0.2 |xm), inject a 15 |xL aliquot.
HPLC VARIABLES
Guard column: 4 x 4 5 |xm LiChrospher 100
Column: 125 X 4 3 ^m Spherisorb ODS-I
Mobile phase: Gradient. A was water containing 5 mL/L 85% orthophosphoric acid and 0.56 mL/
Flow rate: 0.7
Detector: UV 210
CHROMATOGRAM
OTHER SUBSTANCES
phine, lidocaine, 6-monoacetylmorphine, morphine, noscapine, procaine
REFERENCE
Grogg-Sulser,K; Helmlin,H.-J.; Clerc,J.-T. Qualitative and quantitative determination of illicit heroin street
S, J.ChromatogrA, 1995, 692, 121-129.
SAMPLE
Matrix: cells
Sample preparation: 100 |xL Cell suspension + 100 |xL cefoperazone solution + 100 jxL Hanks
balanced salt solution, sonicate 30 min, add 800 |xL MeCN, centrifuge at 13000 g for 5 min,
remove supernatant. Dry supernatant under air, dissolve in 100 |xL mobile phase, inject 75
JIL.
HPLC VARIABLES
Mobile phase: MeCN:50 mM pH 4.7 KH2PO440:60
Flow rate: 1
Detector: UV 340
CHROMATOGRAM
Retention time: 9.5
Internal standard: rifampin
REFERENCE
microb,Agents Chemother., 1994, 38, 1059-1064.
SAMPLE
Sample preparation: Ampules. Dilute a 2 mL aliquot to 100 mL with buffer, dilute further with
water to a papaverine concentration of 60 jjig/mL, inject a 20 JJLL aliquot. Tablets. Crush a
tablet, shake with 70 mL buffer for 10 min, make up to 100 mL with buffer, filter, dilute a 10
mL aliquot to 50 mL with water, inject a 20 |xL aliquot. (The buffer was 1.248% NaH2PO4
adjusted to pH 3 with orthophosphoric acid.)
HPLC VARIABLES
Column: 250 X 4.6 5 jjum Supelco C18
Mobile phase: MeCN:buffer 70:30 (Buffer contained 2.88% sodium lauryl sulfate and 1.248%
NaH2PO4 adjusted to pH 3 with orthophosphoric acid.)
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: moxaverine, drotaverine, ethaverine, codeine
KEYWORDS
ampules; tablets
REFERENCE
Girgis,E.H. Ion-pair reversed-phase liquid chromatographic identification and quantitation of papaverine con-
geners, J.Pharm.ScL, 1993, 82, 503-505.
SAMPLE
Sample preparation: Dilute syrup with mobile phase to a concentration of 5-100 jJig/mL, shake,
filter, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 jxm 80 A Ultrasphere CN
Mobile phase: MeCN:water:EtOH 60:38:2 containing 1 mM perchloric acid
Flow rate: 1
Detector: Conductivity, zero suppression 2, range 1 or 10
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: bromhexine, chlorpheniramine, codeine, dextromethorphan, diphenhydramine,
ephedrine, phenylephrine
KEYWORDS
syrup; indirect conductometric detection; presence of compound causes a decrease in mobile phase
conductivity
REFERENCE
Lau,0.-W.; Mok,C.-S. High-performance liquid chromatographic determination of active ingredients in cough-
cold syrups with indirect conductometric detection, J.Chromatogr.A, 1995, 693, 45-54.
SAMPLE
Matrix: solutions
Sample preparation: Dissolve in MeOH at a concentration of 1 mg/mL, inject a 20 (xL aliquot.
HPLC VARIABLES
Flow rate: 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: phendimetrazine, methylphenidate, phenelzine, epinephrine, pipradol, phenyl-
propanolamine, fencamfamin, chlorphentermine, norpseudoephedrine, phentermine, fenflura-
mine, methylenedioxyamphetamine, amphetamine, normetanephrine, 4-hydroxyamphetamine,
amine, mescaline, mephentermine, norpipanone, levallorphan, hydroxypethidine, normetha-
done, meperidine, dipipanone, diamorphine, pentazocine, acetylcodeine, monoacetylmorphine,
thebacon, oxycodone, thebaine, norlevorphanol, methadone, benzylmorphine, ethylmorphine,
morphine-N-oxide, codeine, codeine-N-oxide, morphine, ethoheptazine, morphine-3-glucuron-
ide, pholcodine, norpethidine, hydrocodone, dihydrocodeine, dihydromorphine, levorphanol,
norcodeine, normorphine
Interfering: pemoline, benzphetamine, diethylpropion, mazindol, tranylcypromine, caffeine, fen-
ethyline, buprenorphine, dextromoramide, phenoperidine, fentanyl, etorphine, piritramide,
noscapine, naloxone, dextropropoxyphene, nalorphine, phenazocine
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.2
OTHER SUBSTANCES
ifensine, nortriptyline, noscapine, orphenadrine, oxeladin, oxprenolol, oxymetazolin, pargyline,
zodone, trinuoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide, tri-
REFERENCE
columns using non-aqueous ionic eluents. IL Application of UV, fluorescence and electrochemical oxidation
SAMPLE
Matrix: solutions
HPLC VARIABLES
Flow rate: 1.5
Detector: UV 254
CHROMATOGRAM
Retention time: 8.9
OTHER SUBSTANCES
Simultaneous: phentolamine
REFERENCE
Wang,D.-R; Tu,Y.-H.; Allen,L.V.,Jr. Degradation kinetics of phentolamine hydrochloride in solution,
J.Pharm.ScL, 1988, 77, 972-976.
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
stilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproterenol, isox-
suprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, lidocaine, lorazepam,
ymorphone, oxyphenbutazone, pargyline, pemoline, pentazocine, pentobarbital, persantine,
phenacetin, phenazocine, phenazopyridine, phencyclidine, phendimetrazine, phenelzine, phen-
iramine, phenobarbital, phenothiazine, phensuximide, phentermine, phenylbutazone, pheny-
lephrine, phenylpropanolamine, piperocaine, prazepam, prednisolone, primidone, probenecid,
progesterone, propiomazine, propranolol, propylparaben, pseudoephedrine, puromycin, pyril-
amine, pyrithyldione, quazepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine,
thiosalicylic acid, thiothixene, th3rmol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcy-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.16 |xm PolyEncap ODS (n-octadecylacrylate copolymerized with vinyl silica in
heptane, carrier Ultrasep ES 100; preparation described in paper)
Mobile phase: MeCNrpH 2.2 phosphate buffer 20:80
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
Simultaneous: atropine, barbituric acid, codeine, diphenhydramine, noscapine
REFERENCE
Engelhardt,H.; Cunat-Walter,M.A. Polymer encapsulated stationary phases with improved efficiency, Chro-
matographia, 1995, 40, 657-661.
SAMPLE
Matrix: solutions
Sample preparation: Centrifuge at 10000 rpm, dilute the supernatant with mobile phase, inject
an aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 |xm Hypersil CPS
Detector: UV 254
REFERENCE
Okimoto,K; Rajewski,R.A.; Uekama,K; Jona,J.A.; Stella,V.J. The interaction of charged and uncharged drugs
with neutral (HP-p-CD) and anionically charged (SBE7-|S-CD) (3-cyclodextrins, Pharm.Res., 1996,13, 256-
264.
Paramethadione
Merck Index: 7161
Lednicer No.: 1 232
SAMPLE
Matrix: blood
Sample preparation: 500 |xL Serum + 50 JJLL 7 |xg/mL IS in water + 1 mL buffer, vortex for 10
s, add 5 mL n-hexane:ether:n-propanol 49:49:2, shake gently for 20 min, centrifuge at 1000 g
for 5 min, repeat the extraction. Combine the organic layers and evaporate them to dryness
50-100 |xL aliquot. (Buffer was 10 mM sodium acetate:10 mM acetic acid 88.5:11.5, pH 5.5.)
[Note: Extraction of paramethadione is implied but not explicitly stated.]
HPLCVARIABLES
Mobile phase: MeCN.buffer 28:72 (Buffer was 300 jxL 1 M KH2PO4 and 50 |xL 900 mM phos-
phoric acid in 1.8 L water, pH 4.4.)
Flow rate: 2.8
Detector: UV 195
CHROMATOGRAM
Retention time: 3.8
OTHER SUBSTANCES
Extracted: carbamazepine, ethosuximide, phenytoin, secobarbital
Simultaneous: mephobarbital, phenobarbital, primidone
Noninterfering: chlorazepate, clonazepam, diazepam, thioridazine, valproic acid
KEYWORDS
serum
REFERENCE
Levine,H.L.; Cohen,M.E.; Duffner,P.K.; Kustas,K.A.; Shen,D.D. An improved high-pressure liquid chromato-
graphic assay for secobarbital in serum, J.Pharm.ScL, 1982, 71, 1281-1283.
Paramethasone
Merck Index: 7162
Lednicer No.: 1 200
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 100 JJLL water containing 5 |jig/mL 2,3-diaminonaphthalene
orate them to dryness under a stream of nitrogen at 30-40, reconstitute the residue in 70 |JLL
MeOH:100 mM perchloric acid 50:50, inject a 20 JJLL aliquot.
HPLC VARIABLES
Column: 150 X 3.9 4 jim Nova-Pak C18
min.
Flow rate: 1
Detector: UV 245, 256, 343
CHROMATOGRAM
OTHER SUBSTANCES
ide, fluocinolone acetonide, fluorometholone, hydrocortisone, hydroxychloroquine, 178-hydrox-
yprogesterone, meprednisone, methylprednisolone, methylprednisolone acetate, prednisolone,
prednisone, progesterone, triamcinolone
Interfering: fluprednisolone
KEYWORDS
serum
REFERENCE
multaneous determination of serum hydroxychloroquine, chloroquine and some corticosteroids.
J,Chromatogr.B, 1995, 666, 347-353.
Parathyroid hormone
Molecular weight: ca. 9500
Merck Index: 7168
SAMPLE
Sample preparation: Prepare a 40 JULM solution in 100 mM pH 10.0 borate/NaOH/HCl buffer,
add hydrogen peroxide to a concentration of 1 mM, inject an aliquot.
HPLC VARIABLES
Column: 150 X 6.4 YMC-Pack ODS-A A312 (YMC)
in MeCN:water 60:40. A:B from 60:40 to 50:50 over 10 min, to 40:60 over 40 min.
Flow rate: 1
Detector: UV 215
CHROMATOGRAM
Retention time: 41
OTHER SUBSTANCES
KEY WORDS
recombinant
REFERENCE
Nabuchi,Y.; Fujiwara,E.; Ueno,K.; Kuboniwa,H.; Asoh,Y.; Ushio,H. Oxidation of recombinant human parathy-
roid hormone: Effect of oxidized position on the biological activity, Pharm.Res., 1995, 12, 2049-2052.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 150 X 4.6 5 |jim ODS-Hypersil
Mobile phase: Gradient. A was 155 mM NaCl containing 10 mM HCl, pH 2.1. B was MeCN. A:
B 100:0 for 2.5 min, to 90:10 over 2.5 min, to 40:60 over 67 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 40.5 (human), 43 (cow)
REFERENCE
Zanelli,J.M.; Kent,J.C; Rafferty,B.; Nissenson,R.A.; Nice,E.C; Capp,M.W.; O'Hare,M.J. High-performance liq-
uid chromatographic methods for the analysis of human parathyroid hormone in reference standards,
parathyroid tissue and biological fluids, J.Chromatogr., 1983, 276, 55-68.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 RP C18 (Vydac)
Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in water. B was MeCN:0.1% aqueous
trifluoroacetic acid 70:30. A: from 65:35 to 45:55 in 48 min, to 0:100 (step gradient), after 10
min return to initial conditions.
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 32
KEYWORDS
human; recombinant
REFERENCE
Hogset,A.; Blingsmo,O.R.; Saether,O.; Gautvik,V.T.; Holmgren,E.; Hartmanis,M.; Josephson,S.;Gabrielsen,O.S.;
Gordeladze,J.O.; Alestrom,R; Gautvik,K.M. Expression and characterization of a recombinant human
parathyroid hormone secreted by Escherichia coli employing the staphylococcal protein A promoter and
signal sequence, J.Biol.Chem., 1990, 265, 7338-7344.
Parbendazole
Merck Index: 7169
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 207.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Parconazole
LednicerNo.: 3 133
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 202.8
CHROMATOGRAM
KEY WORDS
whole blood
REFERENCE
163.
Pargyline
Molecular formula: CnH13N
Merck Index: 7172
SAMPLE
Matrix: solutions
HPLCVARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.0
OTHER SUBSTANCES
ine, pecazine, penbutolol, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone,
REFERENCE
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
ymorphone, oxyphenbutazone, oxytetracycline, pemoline, pentazocine, pentobarbital, persan-
REFERENCE
18, 233-242.
Paromomycin
Merck Index: 7173
SAMPLE
Sample preparation: Plasma. 300 JJLL Plasma + 30 |xL 101.4 pug/mL kanamycin B in water +
100 |JLL 2 M perchloric acid, vortex for 2-3 s, centrifuge at 1000 g for 5 min. Remove the
supernatant and neutralize it with 1.5 M NaOH, add 300 fxL buffer, add 400 |xL DMSO, add
100 |JLL 2% 2,4-dinitrofluorobenzene in EtOH, vortex, heat at 64 for 30 min, add 3 mL toluene,
vortex, centrifuge, discard the upper toluene layer, add 3 mL MeCN:toluene 50:50, vortex for
5-10 s. Remove the upper organic layer and evaporate it to dryness under a stream of nitrogen
at 30-40, reconstitute the residue in 1 mL MeCN:water 50:50, inject a 20 jxL aliquot. Urine.
Dilute urine 100-fold with water. 300 |JLL Diluted urine + 30 |JLL 101.4 |xg/mL kanamycin B in
water + 300 JJLL buffer + 400 |JLL DMSO + 100 |JLL 2% 2,4-dinitrofluorobenzene in EtOH, vortex,
heat at 64 for 30 min, add 3 mL toluene, vortex, centrifuge, discard the upper toluene layer,
add 3 mL MeCN:toluene 50:50, vortex for 5-10 s. Remove the upper organic layer and evaporate
it to dryness under a stream of nitrogen at 30-40, reconstitute the residue in 1 mL MeCN:
water 50:50, inject a 20 uL aliquot. (Prepare buffer by mixing 80 mL 100 mM Na2HPO4 and
20 mL 100 mM NaH2PO4, adding 1 g Tris HCl, and adjusting the pH to 7.8 with 6 M HCl.)
HPLC VARIABLES
Mobile phase: MeOH:water 64:36, adjusted to pH 3.0 with phosphoric acid
Flow rate: 2
Detector: UV 350
CHROMATOGRAM
Internal standard: kanamycin B (24.0)
Limit of detection: 200 ng/mL (plasma), 500 ng/mL (urine)
Limit of quantitation: 500 ng/mL (plasma), 1 |xg/mL (urine)
KEY WpRDS
REFERENCE
Lu, J.; Cwik,M.; Kanyok,T. Determination of paromomycin in human plasma and urine by reversed-phase high-
performance liquid chromatography using 2,4-dinitrofluorobenzene derivatization, J.Chromatogr.B, 1997,
695, 329-335.
SAMPLE
Matrix: bulk
aqueous phase, inject a 20 (JLL aliquot of the lower organic phase.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm LiChrosorb SMOO
Flow rate: 1
Detector: UV 350
CHROMATOGRAM
Retention time: 12,25
KEY WORDS
REFERENCE
Tsuji,K; Goetz,J.R; VanMeter,W.; Gusciora,K.A. Normal-phase high-performance liquid chromatographic de-
141-152.
SAMPLE
Sample preparation: Mix 2 g cream with 3 mL n-butanol, add 5 mL 2% sulfuric acid, mix
thoroughly. Separate lower aqueous layer and re-extract the organic layer with another portion
of sulfuric acid. Combine the aqueous layers and make up to 100 mL with water. Filter a
portion of the extract through a 0.45 \xm Nylon 66 syringe filter. Dilute filtrate with water,
inject a 10 |xL. aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Metachem Inertsil C8
Mobile phase: 200 mM Sodium sulfate containing 1.2 mM sodium 1-heptanesulfonate and 0.1%
acetic acid
Flow rate: 1
agent pumped at 1.0 mL/min and this mixture flowed through a 9 m X 0.25 mm LD. stainless
steel coil to the detector. (Reagent was 800 mg o-phthalaldehyde and 1 mL mercaptoethanol
in 10 mL MeOH diluted to 1 L with 2.5% boric acid and adjusted to pH 10 with 2.5% KOH.)
CHROMATOGRAM
OTHER SUBSTANCES
Also analyzed: gentamicin
KEYWORDS
cream; post-column reaction
REFERENCE
Pick,J.; Olson,L.L.; Ellis,W.Y; Lim,P. Development and validation of a method to extract and quantitate
paromomycin and gentamicin from an Aquaphilic cream formulation, J.Pharm.Biomed.Anal., 1997, 16,
131-137.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile phase: MeCN:water 17:83 containing 16 mM sodium 1-hexanesulfonate 20 mM Na3PO4,
pH 3.5 (Connect a 250 X 4.6 column of Bondapak C18/Corasil or Co:Pell ODS between pump
and injector. Flush column with MeOH:water 50:50 at the end of the day.)
Flow rate: 1.5
Detector: RI
CHROMATOGRAM
Retention time: 6
OTHER SUBSTANCES
Simultaneous: neomycin
REFERENCE
Whall,T. J. Determination of streptomycin sulfate and dihydrostreptomycin sulfate by high-performance liquid
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 8 jjim PLRP-S 1000 A poly(styrene-divinylbenzene) (Polymer Laboratories)
Mobile phase: Water containing 70 g/L sodium sulfate, 1.4 g/L sodium 1-octanesulfonate, and
50 mIVL 200 mM pH 3.0 phosphate buffer
Flow rate: 1
Detector: E, Dionex PED-I pulsed electrochemical detector at 35, 3 mm dia. gold working elec-
trode, E1 +0.05 V, E2 +0.75 V, E3 -0.15 V, ti 0-0.40 s, t2 0.41-0.60 s, t3 0.61-1.00 s, measure signal
between 0.2 and 0.4 s, stainless steel counter electrode, Ag/AgCl reference electrode, following
post-column reaction. The column effluent mixed with 500 mM NaOH pumped at 0.3 mL/min
and the mixture flowed through a 1.2 m long 500 |xL coil to the detector. (Prepare 500 mM
NaOH solution by diluting 50% NaOH with helium-degassed water. Clean gold electrode after
each 60 analyses.)
CHROMATOGRAM
Retention time: 9 (paromomycin II), 11 (paromomycin I)
OTHER SUBSTANCES
Simultaneous: neamine, neomycin B, neomycin C, neomycin LP-A, neomycin LP-B, paromamine
KEY WORDS
REFERENCE
Adams,E.; Schepers,R.; Roets,E.; Hoogmartens,J. Determination of neomycin sulfate by liquid chromatography
with pulsed electrochemical detection, J.Chromatogr.A, 1996, 741, 233-240.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Tissumizer) 1 g Ground tissue + 4 mL buffer at medium
speed for 1 min, centrifuge at 3600 g for 20 min, remove the supernatant, re-homogenize pellet
in 4 mL buffer for 10 min, centrifuge. Combine the supernatants, heat in a boiling water bath
with occasional mixing for 5 min, centrifuge at 2000 g for 20 min, remove the supernatant,
vortex the precipitate with 2 mL buffer for 30 s, centrifuge at 2000 g for 10 min. Combine the
supernatants, acidify to pH 3.5-4 with 50-60 jxL sulfuric acid, centrifuge at 2000 g for 10 min,
Next Page
inject an aliquot of the supernatant. (Buffer was 33.46 g K2HPO4 and 1.046 g KH2PO4 in 1 L
water, pH 8.0.)
HPLC VARIABLES
Guard column: 10 |xm RP-18
Column: 150 X 4.6 5 |jim Supelcosil LC-8-DB
Mobile phase: MeOH:buffer 1.5:98.5 (Buffer was 10 mM sodium 1-pentanesulfonate, 56 mM
sodium sulfate, and 7 mM acetic acid.)
Flow rate: 1.5
Detector: F ex 340 em 455 following post-column reaction with derivatization reagent pumped
at 0.9 mL/min. (Derivatization reagent was commercially available (Pierce) or prepared by
adding 2.5 mL 2-mercaptoethanol and 2.5 mL Brij-35 to 850 mg o-phthalaldehyde in 10 mL
MeOH, mix until decolorization is complete, add 1 L buffer, filter (0.45 ixm), and refrigerate
until used. Buffer was prepared by adjusting pH of 250 mM boric acid to 9.5 with 5 M KOH.)
CHROMATOGRAM
Retention time: 19
OTHER SUBSTANCES
Extracted: neomycin
Simultaneous: dihydrostreptomycin, streptomycin
KEY WORDS
kidney; muscle; cow; pig; post-column reaction
REFERENCE
Shaikh,B.; Allen,E.H.; Gridley,J.C. Determination of neomycin in animal tissues, using ion-pair liquid chro-
matography with fluorometric detection, J.Assoc.Off.Anal.Chem., 1985, 68, 29-36.
Paroxetine
Merck Index: 7175
Lednicer No.: 5 87
SAMPLE
Matrix: blood
Sample preparation: Condition a 50 mg Carboxymethyl Isolute SPE cartridge with 1 mL MeOH
and 1 mL 25 mM pH 6.8 phosphate buffer, dry under vacuum. Add 500 |xL plasma to the SPE
cartridge, wash with two 1 mL portions of 25 mM pH 6.8 phosphate buffer, dry under vacuum,
elute with 1 mL 1% ammonia in MeOH, evaporate to dryness under vacuum at 40, reconstitute
the residue in 100 (JLL MeOH, vortex, inject a 25 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherisorb ODS/CN
Flow rate: 1
Detector: E, ESA, Model 5100 A, Model 5010 analytical cell +650 mV on channel 1, +950 mV on
channel 2, Model 5020 guard cell +980 mV
CHROMATOGRAM
Retention time: 9.6
Internal standard: paroxetine
Previous Page
inject an aliquot of the supernatant. (Buffer was 33.46 g K2HPO4 and 1.046 g KH2PO4 in 1 L
water, pH 8.0.)
HPLC VARIABLES
Guard column: 10 |xm RP-18
Column: 150 X 4.6 5 |jim Supelcosil LC-8-DB
Mobile phase: MeOH:buffer 1.5:98.5 (Buffer was 10 mM sodium 1-pentanesulfonate, 56 mM
sodium sulfate, and 7 mM acetic acid.)
Flow rate: 1.5
Detector: F ex 340 em 455 following post-column reaction with derivatization reagent pumped
at 0.9 mL/min. (Derivatization reagent was commercially available (Pierce) or prepared by
adding 2.5 mL 2-mercaptoethanol and 2.5 mL Brij-35 to 850 mg o-phthalaldehyde in 10 mL
MeOH, mix until decolorization is complete, add 1 L buffer, filter (0.45 ixm), and refrigerate
until used. Buffer was prepared by adjusting pH of 250 mM boric acid to 9.5 with 5 M KOH.)
CHROMATOGRAM
Retention time: 19
OTHER SUBSTANCES
Extracted: neomycin
Simultaneous: dihydrostreptomycin, streptomycin
KEY WORDS
kidney; muscle; cow; pig; post-column reaction
REFERENCE
Shaikh,B.; Allen,E.H.; Gridley,J.C. Determination of neomycin in animal tissues, using ion-pair liquid chro-
matography with fluorometric detection, J.Assoc.Off.Anal.Chem., 1985, 68, 29-36.
Paroxetine
Merck Index: 7175
Lednicer No.: 5 87
SAMPLE
Matrix: blood
Sample preparation: Condition a 50 mg Carboxymethyl Isolute SPE cartridge with 1 mL MeOH
and 1 mL 25 mM pH 6.8 phosphate buffer, dry under vacuum. Add 500 |xL plasma to the SPE
cartridge, wash with two 1 mL portions of 25 mM pH 6.8 phosphate buffer, dry under vacuum,
elute with 1 mL 1% ammonia in MeOH, evaporate to dryness under vacuum at 40, reconstitute
the residue in 100 (JLL MeOH, vortex, inject a 25 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Spherisorb ODS/CN
Flow rate: 1
Detector: E, ESA, Model 5100 A, Model 5010 analytical cell +650 mV on channel 1, +950 mV on
channel 2, Model 5020 guard cell +980 mV
CHROMATOGRAM
Retention time: 9.6
Internal standard: paroxetine
OTHER SUBSTANCES
Extracted: desipramine, venlafaxine
KEYWORDS
plasma; SPE; paroxetine is IS
REFERENCE
Clement,E.M.; Odontiadis,J.; Franklin,M. Simultaneous measurement of venlafaxine and its major metabolite,
oxydesmethylvenlafaxine, in human plasma by high-performance liquid chromatography with coulometric
detection and utilisation of solid-phase extraction, J.Chromatogr.B, 1998, 705, 303-308.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 250 u-L buffer + 200 u-L water + 100 |xL 25 ng/mL ma-
protiline in water + 4 mL toluene, extract on a tumble mixer for 15 min, centrifuge at 1500 g
55, reconstitute the residue in 50 \LL acetone, add 25 |xL 100 mM sodium bicarbonate, add 10
IxL 1 mg/mL dansyl chloride in acetone (prepare fresh daily), vortex for 15 s, heat at 55 for 1
min, centrifuge for 1 min, let stand at room temperature for 30 min, add 25 |JLL 25 mg/mL L-
proline in water (prepare fresh daily), vortex briefly, centrifuge for 1 min, let stand at room
temperature for 5 min, add 500 |xL water, add 2 mL toluene, agitate on a tumble mixer for 10
min, centrifuge at 1500 g for 5 min. Remove the organic layer and evaporate it to dryness
under a stream of nitrogen at 55, reconstitute the residue in 100 u-L mobile phase, vortex for
30 s, inject an aliquot. (Buffer was 8.6 g Na2HPO4.12H2O in 100 mL water, adjusted to pH 12.0
with 4 M NaOH, made up to 200 mL with water.)
HPLC VARIABLES
Guard column: 30 mm long 5 u,m Spherisorb ODS
Column: 200 X 4 5 jxm Spherisorb ODS
Mobile phase: MeOH:50 mM pH 4.5 sodium acetate buffer 84:16 (At the end of the day wash
column with 95:5.)
Flow rate: 1.5
CHROMATOGRAM
Retention time: 5.8
Limit of quantitation: 0.5-1 ng/mL
OTHER SUBSTANCES
Noninterfering: cimetidine, digoxin, methyldopa, phenobarbital, phenytoin, procyclidine,
tranylcypromine
KEYWORDS
REFERENCE
Brett,M.A.; Dierdorf,H.-D.; Zussman,B.D.; Coates,RE. Determination of paroxetine in human plasma, using
high-performance liquid chromatography withfluorescencedetection, J.Chromatogr., 1987, 419, 438444.
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 75 jxL 1.3 jxg/mL norfemoxetine in water + 200 JJLL 1 M
perchloric acid + 5 mL toluene, extract in a tumble mixer for 20 min, centrifuge at 1500 g for
10 min, let stand at -20 for 20 min. Remove the aqueous phase and add it to 750 JXL 25 mM
pH 12 phosphate buffer and 200 u,L 1% lauryl sulfate, add 5 mL heptane:toluene 80:20, extract
on a tumble mixer for 20 min, centrifuge at 2000 g for 10 min. Remove the upper organic layer
and evaporate it to dryness under a stream of nitrogen at 50, reconstitute the residue in 220-
250 |xL MeOH:pH 4.5 acetate buffer 30:70, mix thoroughly, inject a 180-200 |JLL aliquot.
HPLC VARIABLES
Column: 250 X 4 RP-Select B
Mobile phase: MeCN:EtOH:buffer 21:14:65 (Buffer was 50 mM acetic acid adjusted to pH 4.5
with 1 M NaOH containing 2 g/L (?) tetrabutylammonium hydrogen sulfate.)
Flow rate: 1
Detector: UV 295
CHROMATOGRAM
Internal standard: norfemoxetine (9.5)
KEY WORDS
plasma
REFERENCE
Knoeller,J.; Vogt-Schenkel,R.; Brett,M.A. A simple and robust HPLC method for the determination of paroxetine
in human plasma, J.Pharm.Biomed.Anal., 1995, 13, 635-638.
SAMPLE
HPLC VARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: dialysate
Sample preparation: Inject dialysate onto column A and column B in series, elute with mobile
phase, monitor the effluent from column B, after 15.3 min remove column B from the circuit,
continue to elute column A with mobile phase, monitor the effluent from column A. (Serotonin
elutes from column A and column B. The more highly retained paroxetine is eluted from the
shorter column A.)
HPLC VARIABLES
Column: A 50 X 2.0 |xm Nucleosil C18; B 250 X 2.1 5 |xm Supelcosil LC-18-DB (Supelco, USA)
Mobile phase: MeCN:buffer 33:67 (Buffer was 0.23 mM 1-octanesulfonic acid sodium salt in 65
mM acetic acid, adjusted to pH 2.8 with glacial acetic acid.)
Flow rate: 0.127 for 13.7 min then 0.4
CHROMATOGRAM
Limit of detection: 300 fmol
Limit of quantitation: 4.2 pmol
OTHER SUBSTANCES
Extracted: serotonin
KEYWORDS
brain; column switching; rat
REFERENCE
Ramaiya,A.; Karnes,H.T. Simultaneous measurement of serotonin and paroxetine in rat brain dialysate by a
single-pump column-switching technique, J.Chromatogr.B, 1997, 691, 119-129.
SAMPLE
Sample preparation: Extract tablets with mobile phase so as to give a paroxetine concentration
of 400 |x g/mL.
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Inertsil C18
Mobile phase: MeCN:buffer 40:60 (Buffer was 10 mM 1-decanesulfonic acid sodium salt con-
taining 10 mM NaH2PO4, pH 3.0.)
Detector: UV 235
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Lambropoulos,J.; Spanos,G.A.; Lazaridis,N.V. Method development and validation for the HPLC assay in
paroxetine 20 mg strength tablets (Abstract 3391), Pharm.Res., 1997, 14, S591.
SAMPLE
Sample preparation: Weight out powdered tablets equivalent to 20 mg paroxetine. Suspend the
powder in three 5 mL portions of MeOH, stir for 30 min, filter, dry under a gentle stream of
nitrogen. Reconstitute the residue in 20 mL 2-propanol. Inject a 20 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 Chiralpak AD
Mobile phase: n-Hexane:EtOH:diethylamine 94:6:0.5 (At the end of each day wash column with
ca. 100 mL n-hexane:2-propanol 90:10.)
Flow rate: 0.5
Detector: UV 296
CHROMATOGRAM
KEYWORDS
chiral; tablets
REFERENCE
Ferretti,R.; Gallinella,B.; La Torre,E; Turchetto,L. Validated chiral high-performance liquid chromatographic
method for the determination of trans-(-)-paroxetine and its enantiomer in bulk and pharmaceutical for-
mulations, J.Chromatogr.B, 1998, 710, 157-164.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |jim Supelcosil LC-DP (A) or 250 X 4 5 |xm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymet-
azoline, pemoline, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine,
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Pefloxacin
CAS Registry No.: 70458-92-3, 70458-95-6 (mesylate)
Merck Index: 7197
Lednicer No.: 4 141
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma with 20 |xL 20 |xg/mL IS in MeOH. Vortex for 1 min
with dichloromethaneidiethyl ether 80:20, centrifuge at 1000 g for 10 min, separate the organic
twice, evaporate the organic phase to dryness under a stream of nitrogen, add 100 |JLL 10 mM
NaOH to the residue, shake for 5 min, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 10 ^m Vydac AXGU
Flow rate: 1.2
Detector: UV 280
CHROMATOGRAM
Retention time: 9.2
Internal standard: 2-[4-(2-furoyl)phenyl]propionic acid (3.9)
OTHER SUBSTANCES
Extracted: felbinac, fenbufen
KEY WORDS
plasma
REFERENCE
1115.
SAMPLE
Matrix: blood
Sample preparation: 200 jxL Serum + 50 jxL 400 jxg/mL IS in water, vortex for 30 s. Add 500
JJLL MeCN, vortex for 1 min. Centrifuge at 6000 rpm for 10 min. Evaporate the supernatant to
200 |xL at 40 under a stream of nitrogen, vortex for 30 s. Inject a 30-80 |xL aliquot.
HPLC VARIABLES
Column: 100 X 8.0 4 |xm Radial-pak Novapak C18
Mobile phase: MeCN:buffer 14:86 (Buffer was 2 g citric acid, 2 g sodium acetate, and 1 mL
triethylamine in 1 L water.)
Flow rate: 2.5
CHROMATOGRAM
Retention time: 5.2
Internal standard: acebutolol (7.4)
OTHER SUBSTANCES
Simultaneous: ciprofloxacin, lomefloxacin, ofloxacin
KEYWORDS
REFERENCE
Abanmi,N.; ZaghloudJ.; El Sayed,N.; al-Khamis,K.I. Determination of pefloxacin and its main active metabolite
in human serum by high-performance liquid chromatography, Ther.Drug Monti., 1996, 18, 158-163.
SAMPLE
Matrix: blood
HPLC VARIABLES
perchlorate.)
Flow rate: 1.2
Detector: UV 272
CHROMATOGRAM
Internal standard: pipemic acid (4.14)
OTHER SUBSTANCES
Simultaneous: norfloxacin
KEYWORDS
REFERENCE
J.Pharm.Sci., 1998, 87, 215-220.
SAMPLE
Matrix: blood
Sample preparation: 500 jxL Serum + 250 JJLL 10% trichloroacetic acid, vortex for 10 s, centri-
fuge at >700 g for 10 min, inject a 20 jxL aliquot of the supernatant.
HPLC VARIABLES
CHROMATOGRAM
Retention time: 6.5
OTHER SUBSTANCES
KEYWORDS
serum
REFERENCE
SAMPLE
Matrix: blood
Sample preparation: Add two volumes of MeCN to plasma, centrifuge, inject an aliquot of the
supernatant.
HPLC VARIABLES
Column: 5 (xm Nucleosil C18
Mobile phase: MeOHrIOO mM pH 4.9 phosphate buffer 50:50
Flow rate: 1.2
CHROMATOGRAM
KEYWORDS
REFERENCE
Jaehde,U.; Sorgel,R; Stephan,U.; Schunack,W. Effect of an antacid containing magnesium and aluminum on
Chemother., 1994, 38, 1129-1133.
SAMPLE
Sample preparation: 100 |xL Plasma or dialysate + 400 |xL MeOH, vortex, centrifuge, inject an
HPLC VARIABLES
Column: strong cation exchange
Mobile phase: MeCN: 100 mM pH 3 citrate buffer 20:80
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: metabolites, norfloxacin
KEYWORDS
REFERENCE
Rose,T.R; Bremner,D.A.; Collins,J.; Ellis-Pegler,R.; Isaacs,R.; Richardson,R.; Small,M. Plasma and dialysate
levels of pefloxacin and its metabolites in CAPD patients with peritonitis, J.Antimicrob.Chemother., 1990,
25, 657-664.
SAMPLE
Sample preparation: Plasma. Mix 500 |xL plasma with 5 |xg IS, add 4 mL dichloromethane and
100 (JiL pH 7.4 phosphate buffer, agitate for 10 min, centrifuge at 5300 g for 10 min. Collect
3.5 mL organic phase. Add 4 mL dichloromethane to the aqueous phase again, agitate, centri-
fuge. Combine the organic phases, evaporate at 60, reconstitute the residue in 100 jxL mobile
phase, inject a 20 |JLL aliquot. Tissue. Mix 500 |JLL epiploic-fat and 4 mL dichloromethane and
keep at 4, add 5 jxg IS, mix by using an automatic grinder (Ultra Turrax, Ika-Werk, Stauffen,
Germany). Collect the mixture, centrifuge at 5300 g for 10 min. Add 4 mL 100 mM NaOH to
the dichloromethane, agitate for 10 min, centrifuge at 5300 g for 5 min. Eliminate the organic
phase, adjust the aqueous phase to pH 7.4 with concentrated trichloroacetic acid, add 4 mL
dichloromethane, agitate for 10 min and centrifuge at 5300 g for 5 min. Evaporate the organic
phase at 60. Reconstitute the residue in 100 JJLL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Guard column: 25 X 4 5 |xm 100 RP-18 Lichrosphere
Column: 125 X 4 5 |xm 100 RP-18 endcapped Lichrosphere
Mobile phase: MeCNipH 4.8 citrate buffer 85:15
Flow rate: 1
CHROMATOGRAM
Internal standard: 4844P (pefloxacin analog)
OTHER SUBSTANCES
KEYWORDS
epiploic-fat; plasma; pharmacokinetics; fat
REFERENCE
Jacoberger,B.; Ubeaud,G.; Freys,G.; Pottecher,T.; Jung,L.; Koffel,J.C. Concentrations of pefloxacin in plasma
and tissue after administration as surgical prophylaxis, Antimicrob.Agents Chemothen, 1998,42, 425-427.
SAMPLE
Sample preparation: Blood. Briefly vortex 500 |xL plasma and 500 |JLL pH 7.4 phosphate buffer,
add 5 mL dichloromethane:isopropanol 80:20, vortex for 30 s, shake gently for 15 min on an
electric shaker, centrifuge at 1500 g for 10 min. Separate the lower layer using phase-sepa-
rating filter paper (Whatman 1 PS), evaporate the organic phase under nitrogen at 25, dissolve
the residue in 100 JLJLL mobile phase, inject a 20JJLL aliquot. Tissue. Pulverize prostatic tissue
under liquid nitrogen, weigh a 200 mg aliquot, add 500 |JLL pH 7.4 phosphate buffer and 5 mL
dichloromethanerisopropanol 80:20, vortex for 30 s, shake gently for 15 min on an electric
shaker, centrifuge at 1500 g for 10 min. Separate the lower layer using phase-separating filter
paper (Whatman 1 PS), evaporate the organic phase under nitrogen at 25, dissolve the residue
in 100 |JIL mobile phase, inject a 20 |xL aliquot. (The pH 7.4 phosphate buffer was 28.2 g K2HPO4
and 5.17 g KH2PO4 in 1 L water.)
HPLC VARIABLES
Column: 250 X 4 5 jxm Nucleosil C18
Mobile phase: MeCN:pH 2.1 buffer 20.9:79.1 (Prepare the mobile phase by dissolving 18.1 g
citric acid and 4.1g ammonium perchlorate in about 300 mL distilled water, add 209 mL MeCN,
dilute to 1 L with water, and add 3 mL tetrabutylammonium hydroxide. Filter through a 0.45
ixm HV Millipore filter.)
Flow rate: 0.9
Detector: UV 340
CHROMATOGRAM
Retention time: 6.8
Internal standard: pefloxacin
OTHER SUBSTANCES
Extracted: enoxacin, 4-oxo-enoxacin
Noninterfering: amikacin, ciprofloxacin, fosfomycin, ofloxacin, rifampicin, roxithromycin, tobra-
mycin, vancomycin
KEYWORDS
plasma; prostatic tissue; prostate; pefloxacin is IS
REFERENCE
Hamel,B.; Audran,M.; Costa,R; Bressolle,F. Reversed-phase high-performance liquid chromatographic deter-
mination of enoxacin and 4-oxo-enoxacin in human plasma and prostatic tissue. Application to a pharma-
cokinetic study, J.ChromatognA, 1998, 812, 369-379.
SAMPLE
Sample preparation: Tissue. Homogenize 100-250 mg tissue with 5 mL 500 mM pH 7.0 phos-
phate buffer, remove a 1 mL aliquot, add 100 JJLL 10 jig/mL IS, mix, add 10 mL chloroform:
isopentanol 90:10, shake for 15 min, centrifuge , repeat extraction. Combine the organic layers
and evaporate them to dryness under a stream of air at 60, reconstitute the residue in 100
IxL 1% ammonia, inject a 25 |xL aliquot. Plasma. 250-500 |xL Plasma + 100 jiL 10 jxg/mL IS +
1 mL 500 mM pH 7.0 sodium phosphate buffer, mix, add 10 mL chloroformdsopentanol 90:10,
shake for 15 min, centrifuge, repeat extraction. Combine the organic layers and evaporate them
to dryness under a stream of air at 60, reconstitute the residue in 100 |JLL 1% ammonia, inject
a 25 jxL aliquot.
HPLC VARIABLES
monohydrate, and 1 mL/L triethylamine
Flow rate: 2
CHROMATOGRAM
Retention time: 4.8
Internal standard: l-allyl-6-fluoro-1,4-dihydro-4-oxo-7-(4-methyl- l-piperazinyl)quinoline-3-car-
boxylic acid (Roger Bellon Laboratories) (6.6)
OTHER SUBSTANCES
KEY WORDS
prostate
REFERENCE
Montay,G.; Tassel, J.P. Improved high-performance liquid chromatographic determination of pefloxacin and its
metabolite norfloxacin in human plasma and tissue, J.Chromatogr., 1985, 339, 214-218.
SAMPLE
Sample preparation: Plasma. 250 |xL Plasma + 250 |xL 10 |xg/mL IS in water + 750 |xL MeOH,
stir, centrifuge at 2000 rpm for 5 min, inject a 50 |xL aliquot of the supernatant. Urine. 500
IxL Urine + 3.5 mL 8 |xg/mL IS in water, inject a 50 |xL aliquot.
HPLC VARIABLES
Column: 200 X 4.6 Nucleosil C8
Mobile phase: MeCN:water:triethylamine:formic acid 11:87.5:0.1:1 containing 0.2% sodium ac-
etate and 0.1% citric acid
Flow rate: 1
CHROMATOGRAM
Internal standard: l-ethyl-6-chloro-l,4-dihydro-7-(4-methyl-l-piperazinyl)-4-oxo-3-quinoline-
carboxylic acid (RP 41983)
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Humbert,G.; BrumptJ.; Montay,G.; Le Liboux,A.; Frydman,A.; Borsa-Lebas,R; Moore,N. Influence of rifampin
on the pharmacokinetics of pefloxacin, Clin.Pharmacol.Ther., 1991, 50, 682-687.
SAMPLE
residue with 50 JJLL MeCNiwater 50:50, vortex for 10 s, centrifuge at 7500 g for 2 min, inject
HPLC VARIABLES
mL/min)
Detector: UV 278.3
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Sample preparation: Dissolve 40 mg freeze-dried nanoparticles in 25 mL acetone:MeOH 90:10
containing a few drops of 100 mM HCl, filter (0.45 (xm), inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Partisil 10-ODS-l C18
Mobile phase: MeCN: 10 mM KH2PO4:triethylamine 14:86:0.2
Flow rate: 1.5
Detector: UV 275
OTHER SUBSTANCES
Simultaneous: ofloxacin (UV 292)
KEY WORDS
nanoparticles
REFERENCE
Fresta,M.; Puglisi,G.; Giammona,G.; Cavallaro,G.; Micali,N.; Furneri,P.M. Pefloxacine mesilate and ofloxacin-
loaded polyethylcyanoacrylate nanoparticles: Characterization of the colloidal drug carrier formulation,
J.Pharm.ScL, 1995, 84, 895-902.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Ultra-Turrax) eye tissue with 3 mL 50 mM pH 5.8 sodium
phosphate-citrate buffer and IS, centrifuge. Add the supernatant to 7 mL chloroform, agitate,
centrifuge at 1000 g for 10 min. Remove the lower organic layer and evaporate it to dryness
under a stream of nitrogen at 37, reconstitute the residue in 100 jxL mobile phase, inject a 5-
20 JJLL aliquot.
HPLC VARIABLES
monohydrate, 4 mL/L triethylamine, and 2 mL/L formic acid, pH 4.8
Flow rate: 1
Detector: UV 280
CHROMATOGRAM
Internal standard: l-allyl-6-fluoro-l,4-dihydro-4-oxo-7-(4-methyl-l-piperazinyl)quinoline-3-car-
boxylic acid (?) 4662 P (Roger-Bellon Laboratories) (2.95)
OTHER SUBSTANCES
KEYWORDS
rabbit; eye; pharmacokinetics
REFERENCE
Cochereau-Massin,L; Bauchet,J.; Faurisson,E; Vallois,J.M.; Lacombe,R; Pocidalo,J.J. Ocular kinetics of peflox-
acin after intramuscular administration in albino and pigmented rabbits, Antimicrob.Agents Chemother.,
1991, 35, 1112-1115.
SAMPLE
Matrix: urine
HPLCVARtABLES
Column: (juBondapak C18
Mobile phase: MeOH:MeCN:100 mM pH 5.75 phosphate buffer 24.1:2.6:73.3
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
KEYWORDS
pharmacokinetics
REFERENCE
Jaehde,U.; Sdrgel,E; Stephan,U.; Schunack,W. Effect of an antacid containing magnesium and aluminum on
Chemother., 1994, 38, 1129-1133.
Pemirolast
CAS Registry No.: 69372-19-6, 100299-08-9 (potassium salt)
Merck Index: 7205
LednicerNo.: 5 150
SAMPLE
Matrix: blood
Sample preparation: 500 |JLL Plasma + 25 |JLL 2.5 jig/mL IS in water + 1 mL MeOH, mix,
centrifuge at 13000 g for 5 min, inject a 50 (xL aliquot of the supernatant.
HPLC VARIABLES
Column: 100 X 9.4 5 ^m ODS-3 RAC (Whatman)
Mobile phase: MeOH.water 45:55 containing 0.31% acetic acid
Flow rate: 1.5
CHROMATOGRAM
Retention time: 8.5
Internal standard: 7,9-dimethyl-3-(lH-tetrazol-5-yl)-4H-pyrido[l,2-a]pyrimidin-4-one (BL 5609)
(Mead Johnson) (13.3)
KEYWORDS
REFERENCE
Cheng,H.; Pittman,K.A.; Dandekar,K.A. Liquid chromatographic determination of 9-methyl-3-(lH-tetrazol-5-
yl)-4H-pyrido[l,2-a]pyrimidin-4-one in human plasma with fluorescence detection, J.Pharm.Sci., 1987, 76,
918-919.
Pemoline
Molecular formula: G9H8N2O2
Merck Index: 7206
SAMPLE
Sample preparation: Plasma. 100 JULL Plasma + 500 |xL 1 M pH 10 carbonate buffer + 4 mL
dichloromethane, shake for 10 min, centrifuge at 1680 g for 5 min. Remove 3 mL of the organic
layer and add it to 1 mL 1 |xg/mL 5-methyl-5-phenylhydantoin in dichloromethane, evaporate
to dryness under reduced pressure at room temperature, reconstitute the residue in 50 |xL
mobile phase, inject a 20 |xL aliquot. Urine. 100 (xL Urine + 500 |xL 1 M pH 10 carbonate
buffer + 4 mL dichloromethane, shake for 10 min, centrifuge at 1680 g for 5 min. Remove 1
mL of the organic layer and add it to 1 mL 4 |jig/mL 5-methyl-5-phenylhydantoin in dichloro-
methane, evaporate to dryness under reduced pressure at room temperature, reconstitute the
residue in 50 fxL mobile phase, inject a 5 JXL aliquot. Liver, kidney, lung, spleen, muscle. Ho-
mogenize on ice with 2 (liver, kidney, lung, spleen) or 3 (muscle) volumes ice-cold saline. 200
IxL Homogenate + 500 JJLL 1 M pH 10 carbonate buffer + 4 mL dichloromethane, shake for 10
min, centrifuge at 1680 g for 5 min. Remove 3 mL of the organic layer and add it to 1 mL 1
|xg/mL 5-methyl-5-phenylhydantoin in dichloromethane, evaporate to dryness under reduced
pressure at room temperature, reconstitute the residue in 50 |xL mobile phase, inject a 20 |xL
aliquot. Brain. Homogenize on ice with 2 volumes ice-cold saline. 200 JJLL Homogenate + 500
fxL 1 M pH 10 carbonate buffer + 4 mL dichloromethane, shake for 10 min, centrifuge at 1680
g for 5 min. Remove 3 mL of the organic layer and add it to 1 mL 1 |jig/mL 5-methyl-5-phen-
ylhydantoin in dichloromethane, evaporate to dryness under reduced pressure at room tem-
perature, reconstitute the residue in 50 |xL mobile phase, centrifuge at 15300 g for 5 min, inject
a 20 jxL aliquot of the supernatant.
HPLC VARIABLES
Column: 250 X 4 5 |xm Kaseisorb LC C8-60-5 (Tokyo Kasei Kogo)
Mobile phase: MeCN:buffer 20:80 (Buffer was water adjusted to pH 5 with 15 mM phosphoric
acid.)
Flow rate: 0.7
Detector: UV 215
CHROMATOGRAM
Retention time: 7
Internal standard: 5-methyl-5-phenylhydantoin(ll)
Limit of detection: 5 ng (urine), 2 ng (tissue), 0.5 ng (plasma)
KEYWORDS
rat; plasma; brain; liver; kidney; lung; spleen; muscle; pharmacokinetics
REFERENCE
Aoyama,T.; Kotaki,H.; Saitoh,Y.; Nakagawa,F. Determination of pemoline in plasma, urine and tissues by re-
versed-phase high-performance liquid chromatography, J.Chromatogr., 1988, 430, 351-360.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Mobile p h a s e : MeOH.buffer 90:10 (Buffer was 94 mL 35% ammonia and 21.5 mL 70% nitric
Flow r a t e : 2
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: phendimetrazine, methylphenidate, phenelzine, epinephrine, pipradol, phenyl-
propanolamine, fencamfamin, chlorphentermine, norpseudoephedrine, phentermine, fenflura-
mine, methylenedioxyamphetamine, amphetamine, normetanephrine, 4-hydroxyamphetamine,
amine, mescaline, mephentermine, phenazocine, norpipanone, levallorphan, hydroxypethidine,
morphine, thebacon, oxycodone, thebaine, norlevorphanol, methadone, benzylmorphine, ethyl-
morphine, morphine-N-oxide, codeine, codeine-N-oxide, morphine, ethoheptazine, morphine-3-
glucuronide, pholcodeine, norpethidine, hydrocodone, dihydrocodeine, dihydromorphine,
levorphanol, norcodeine, normorphine
Interfering: benzphetamine, diethylpropion, mazindol, tranylcypromine, caffeine, fenethyline,
buprenorphine, dextromoramide, phenoperidine, fentanyl, etorphine, piritramide, noscapine,
papaverine, naloxone, dextropropoxyphene, nalorphine
REFERENCE
Law,B-; Gill,R.; Moffat,A.C. High-performance liquid chromatography retention data for 84 basic drugs of fo-
SAMPLE
Matrix: solutions
HPLC VARIABLES
100 for 5 min.
Flow rate: 2
Detector: UV 210
OTHER SUBSTANCES
ymorphone, oxyphenbutazone, oxytetracycline, papaverine, pentazocine, pentobarbital, persan-
REFERENCE
18, 233-242.
SAMPLE
Matrix: solutions
HPLC VARIABLES
Column: 250 X 4.6 5 |jim Supelcosil LC-DP (A) or 250 X 4 5 jxm LiChrospher 100 RP-8 (B)
Flow rate: 0.6
Detector: UV 229
CHROMATOGRAM
OTHER SUBSTANCES
naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, oxycodone, oxymet-
azoline, paroxetine, pentazocine, pentobarbital, pentoxifylline, perphenazine, pheniramine,
tocainide, tolbutamide, tolmetin, trazodone, triamterene, triazolam, trifluoperazine, trifluprom-
azine, trimeprazine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yo-
himbine, zopiclone
KEYWORDS
REFERENCE
J.ChromatogrA, 1995, 692, 103-119.
Penbutolol
Merck Index: 7209
SAMPLE
Matrix: blood
Sample preparation: 200 |xL Plasma or serum + 50 |xL 100 ng/mL protriptyline hydrochloride
in 2 M pH 10.6 aqueous Tris buffer + 200 ^LL MTBE, vortex for 30 s, centrifuge at 9950 g for
2 min, inject a 100 JJLL aliquot of the organic layer.
HPLC VARIABLES
Column: 250 X 5 5 juim Spherisorb S5W
Mobile phase: Isooctane:MeOH:buffered MeOHrMTBE 55:15:10:20, apparent pH 5.7 (Buffered
MeOH was 1 L 100 mM ammonium perchlorate in MeOH to which was added 10 mL 100 mM
NaOH in MeOH, apparent pH 6.5.)
Flow rate: 2
Detector: F ex 200 em no filter
CHROMATOGRAM
Retention time: 5.3
OTHER SUBSTANCES
Simultaneous: N-acetylprocainamide, ajmaline, atenolol, betaxolol, chlorpromazine, desipra-
mine, dipyridamole, doxazosin, flecainide, gallopamil, imipramine, labetalol, metoprolol, mian-
serin, nadolol, norverapamil, orphenadrine, oxprenolol, pindolol, prajmaline, prazosin, procain-
amide, propranolol, quinidine, quinine, terazosin, trazodone, triamterene, verapamil
Noninterfering: acebutolol, amiodarone, desethylamiodarone, disopyramide, lidocaine, lorcain-
ide, methyldopa, nifedipine, propafenone, sotalol, timolol, tocainide
Interfering: mexiletine, pyrimethamine
KEYWORDS
plasma; serum; normal phase
REFERENCE
Bhamra,R.K; Flanagan,R.J.; Holt,D.W. Measurement of penbutolol and 4-hydroxypenbutolol in plasma or se-
rum by HPLC, Biomed.Chromatogr., 1986, I9 140-142.
SAMPLE
Matrix: blood
Sample preparation: 0.5-1 mL Plasma or 0.5 mL plasma water + 1 mL 1 M NaOH + 12 mL n-
heptanedsoamyl alcohol 98.5:1.5, shake mechanically for 10 min, centrifuge at 1680 g for 5
min. Remove the organic layer and evaporate it to dryness under reduced pressure at 40,
reconstitute the residue in 50 |xL EtOH, inject a 10 |xL aliquot.
HPLC VARIABLES
Column: 250 X 4 5 |xm Hitachi Gel 3013 spherical styrene-divinylbenzene
Mobile phase: EtOH:buffer 65:35 (Buffer was 20 mM pH 2.0 perchloric acid/sodium perchlorate.)
Flow rate: 0.2
CHROMATOGRAM
Retention time: 18
Internal standard: penbutolol
OTHER SUBSTANCES
Simultaneous: quinidine, reserpine
Noninterfering: allopurinol, benzbromarone, diazepam, digoxin, diltiazem, dipyridamole, diso-
pyramide, furosemide, isosorbide dinitrate, maprotiline, nifedipine, nitrazepam, trichlorme-
thiazide, verapamil
KEYWORDS
penbutolol is IS; plasma; plasma water
REFERENCE
Yamamura,Y.; Uchino,K.; Kotaki,H.; Isozaki,S.; Saitoh,Y. Quantitative determination of propranolol in plasma
and plasma water from normal subjects and patients with angina pectoris by high-performance liquid
SAMPLE
Matrix: blood
Sample preparation: 100-500 (JLL Plasma + 5 ng bufarolol, mix, add 4 mL 500 mM pH 7.0
potassium phosphate buffer, add to a Sep-Pak C18 SPE cartridge, wash with 5 mL water, wash
with 5 mL EtOH:water 30:70, elute with 5 mL EtOH:methylamine 99.9:0.1, evaporate to dry-
ness, add 100 |JLL 2 mg/mL (aS)-2'-methoxy-l,l'-binaphthalene-2-carbonyl cyanide in MeCN
containing 0.1% quinuclidine, heat at 60 for 20 min, add 50 |JLL MeOH, evaporate to dryness
under a stream of nitrogen, reconstitute with 1 mL in EtOH:water 90:10, add to an 18 X 6
column packed with 100 mg carboxymethyl Sephadex LH-200, wash with EtOH:water 90:10
at 0.2 mL/min, elute with 5 mL 100 mM methylamine in EtOH:water 90:10. Evaporate the
eluate to dryness, reconstitute with 50-100 |xL mobile phase, inject an aliquot. (Derivatization
occurs on the alcohol. Preparation of (aS)-2'-methoxy-l,l?-binaphthalene-2-carbonyl cyanide is
as follows. Treat l-bromo-2-naphthol with sodium hydride in DMF, add iodomethane, stir at
room temperature overnight to obtain l-bromo-2-methoxynaphthalene (mp 85-86). Add a so-
lution of 37.7 g l-bromo-2-methylnaphthalene in 200 mL ether over 1 h to a sonicated mixture
of 7 g magnesium turnings in 50 mL ether, the mixture should reflux rapidly (Caution! There
may be in an induction period!), sonicate for 2 h after addition is complete, add 200 mL benzene
(Caution! Benzene is a carcinogen!), add this mixture dropwise to a stirred mixture of 100
mmoles l-bromo-2-methoxynaphthalene and 655 mg bis(triphenylphosphine)nickel(II) chloride
(NiCl2(PPh3)2) in 150 mL benzene at room temperature over 1 h, stir at room temperature
overnight, reflux for 3 h, remove the ether by distillation through a short Vigreux column,
remove the solvent by evaporation under reduced pressure, remove excess l-bromo-2-methyl-
naphthalene by heating at 15070.1 mm Hg, cool, dissolve the residue in hexane, pass through
silica gel, evaporate to dryness, recrystallize from hexane to obtain l-methoxy-2'-methylbi-
naphthalene (mp 118-121). Reflux 10 mmoles l-methoxy-2'-methyl-binaphthalene, 1.96 g N-
bromosuccinimide, and 100 mg benzoyl peroxide in 70 mL carbon tetrachloride for 3 h, filter,
evaporate the filtrate to obtain crude l-bromomethyl-2'-methoxy-binaphthalene. Dissolve the
crude l-bromomethyl-2'-methoxy-binaphthalene in 60 mL DMSO under nitrogen, slowly add a
sodium ethoxide/nitropropane mixture, stir at room temperature for 3 h, stir at 60 for 3 h,
pour into 300 mL ice-water, extract with dichloromethane, wash with 2 M HCl, wash with 1
M sodium carbonate, wash with water, dry over anhydrous sodium sulfate, evaporate to obtain
crude 2'-methoxy-l,l'-binaphthalene-2-carboxaldehyde. (Prepare the sodium ethoxide/nitropro-
pane mixture by dissolving 580 mg sodium in 35 mL EtOH, add 3.25 g 2-nitropropane.) Reflux
the crude 2'-methoxy-l,l'-binaphthalene-2-carboxaldehyde in 60 mL acetone, add a solution of
2.36 g potassium permanganate in 60 mL hot water dropwise over 1 h, heat for an additional
hour, pass sulfur dioxide through the solution until it becomes clear (sodium metabisulfite may
work). Filter off the precipitate and dissolve it in 200 mL hot toluene, add a small amount of
activated charcoal, filter while hot, concentrate to about a third of the volume, recrystallize
from EtOH:water 1:2 to obtain 2'-methoxy-l,r-binaphthalene-2-carboxylic acid (mp 258.5-260)
(Bull. Chem. Soc. Japan 1986, 59, 2044). Reflux 9.15 g racemic 2'-methoxy-l,l'-binaphthalene-
2-carboxylic acid in 55 mL freshly distilled thionyl chloride for 5 h, evaporate under reduced
pressure, add a little benzene, evaporate under reduced pressure, repeat the benzene evapo-
ration twice more to obtain 2>-methoxy-l,l>-binaphthalene-2-carbonyl chloride as a brown solid.
Dissolve the acid chloride in 70 mL benzene, add dropwise to 12.8 g (-)-menthol in 100 mL
benzene containing 1 g 4-dimethylaminopyridine and 5 mL pyridine, stir overnight at room
temperature, heat at 70 for 3 h, cool, dilute with benzene, wash with 2 M HCl, wash with 1
M sodium carbonate, wash with water, dry over anhydrous magnesium sulfate in the presence
of activated charcoal, evaporate to dryness, remove as much menthol as possible by sublimation
under vacuum, chromatograph twice on a column of silica gel with toluene to obtain the (aS,R)
menthol ester (mp 145-146 from hexane) and the (aR,R) menthol ester (mp 126-129 from
hexane) as well as a mixture of diastereomers. Reflux the (aS,R) menthol ester with KOH in
aqueous EtOH for 8-10 h to obtain (aS)-2>-methoxy-l,l'-binaphthalene-2-carboxylic acid (Bull.
Chem. Soc. Japan 1989, 62, 1528). Add 1.5 mL oxalyl chloride to a solution of (aS)-2'-methoxy-
l,r-binaphthalene-2- carboxylic acid in 10 mL anhydrous benzene, reflux for 10 h, evaporate
to dryness under reduced pressure. Take up the residue in 10 mL anhydrous benzene, add 1
mL trimethylsilyl cyanide, add 1 mg zinc iodide, stir at room temperature for 5 h, evaporate
to dryness, recrystallize from hexane/acetone to obtain (aS)-2'-methoxy-l,l'-binaphthalene-2-
carbonyl cyanide as orange-yellow needles (mp 143-146).)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Cosmosil 5SL (Nacalai Tesque, Kyoto)
Mobile phase: Hexane:ethyl acetate:triethylamine 83.3:16.7:0.005
Flow rate: 2
CHROMATOGRAM
Internal standard: bufarolol (8.5 (R), 12 (S))
KEYWORDS
derivatization; plasma; chiral; normal phase; dog; SPE; pharmacokinetics
REFERENCE
Goto,J.; Shao,G.; Ito,M.; Kuriki,T.; Nambara,T. High-performance liquid chromatographic determination of pen-
butolol enantiomers in plasma with fluorescence detection, Anal.ScL, 1991, 7, 723-726.
SAMPLE
Matrix: blood
the residue in 100 jxL mobile phase, centrifuge at 2800 g for 5 min, inject a 50 JULL aliquot of
HPLC VARIABLES
Column: 300 X 3.9 4 u,m NovaPack C18
Flow rate: 0.8
Detector: UV 270
CHROMATOGRAM
KEYWORDS
pride; phenol; chlormezanone; aspirin; metfdrmin; ritodrine; codeine; sultopride; amisulpride;
REFERENCE
254-262.
SAMPLE
Sample preparation: Evaporate 0.5 mL 100 ng/mL propranolol in MeOH to dryness in a glass
tube under a stream of nitrogen at 37, add 1 mL plasma or urine, add 500 |xL 1 M NaOH,
add 8 mL freshly distilled diethyl ether, shake on a rotating shaker at 60 rpm for 15 min,
centrifuge at 1200 g for 15 min. Remove 6.5 mL of the organic layer and pass it through a 20
X 4 column filled with glass wool, evaporate to dryness under a stream of nitrogen at 37,
reconstitute the residue in 200 |xL mobile phase, inject a 20 |xL aliquot. For conjugated com-
pounds proceed as follows. Evaporate 0.5 mL 100 ng/mL propranolol in MeOH to dryness in a
glass tube under a stream of nitrogen at 37, add 1 mL plasma or urine, add 1 mL 100 mM
pH 5 acetate buffer, add 100 |xL solution containing 10000 U/mL p-glucuronidase and 0.6 U/
mL sulfatase (Sigma), heat at 37 for 48 h, cool to room temperature, add 500 |xL 1 M NaOH,
add 8 mL freshly distilled diethyl ether, shake on a rotating shaker at 60 rpm for 15 min,
centrifuge at 1200 g for 15 min. Remove 6.5 mL of the organic layer and add it to 6 mL 100
mM HCl, shake at 60 rpm on a rotating shaker for 15 min, centrifuge at 1200 g for 10 min.
Remove 5.5 mL of the aqueous layer and add it to 700 |JLL 1 M NaOH, add 6 mL diethyl ether,
shake on a rotating shaker at 60 rpm for 15 min, centrifuge at 1200 g for 10 min, remove 5
mL of the organic layer, evaporate to dryness under a stream of nitrogen at 37, reconstitute
the residue in 200 |xL mobile phase, inject a 20 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:buffer 48:52 containing 1 g/L sodium heptanesulfonate (Buffer was 100
mM citric acid-sodium citrate buffer adjusted to pH 2.85 with 1 M HCl.)
Flow rate: 1.7
CHROMATOGRAM
Internal standard: propranolol (F ex 290 em 330) (4)
OTHER SUBSTANCES
Extracted: metabolites, 4-hydroxypenbutolol (F ex 290 em 330)
KEYWORDS
REFERENCE
Bernard,N.; Cuisinaud,G.; Sassard,J. Determination of penbutolol and its hydroxylated metabolite in biological
fluids by reversed-phase high-performance liquid chromatography, J.Chromatogr., 1982, 228, 355-361.
SAMPLE
Sample preparation: 1 mL Plasma + 100 |xL 400 ng/mL propranolol in water + 20 jxL 25 mg/
mL ascorbic acid in water (prepare fresh daily) + 500 |xL buffer, vortex, add 6 mL hexane.n-
butanol 96:4, shake vigorously for 2 min, centrifuge briefly. Remove 5 mL of the organic layer
IxL MeOH:21 mM pH 5.5 ammonium acetate buffer 50:50, vortex, inject a 50 )JLL aliquot. To
deconjugate samples proceed as follows. 300 JJLL Plasma or urine + 50 jxL 200 mg/mL sodium
bisulfite in water (prepare fresh daily) + 600 JJLL glucuronidase solution, vortex, flush tube with
nitrogen, heat at 45 for 2 h, add 100 |JLL 400 ng/mL propranolol in water, add 20 uL 25 mg/mL
ascorbic acid in water (prepare fresh daily), add 500 \LL buffer, vortex, add 6 mL hexane:n-
butanol 96:4, shake vigorously for 2 min, centrifuge briefly. Remove 5 mL of the organic layer
and wash it with 2 mL buffer:25 mg/mL ascorbic acid 100:1. Remove 4 mL of the organic layer
u,L MeOH:21 mM pH 5.5 ammonium acetate buffer 50:50, vortex, inject a 50 jxL aliquot. (Buffer
was prepared by mixing saturated sodium carbonate and saturated sodium bicarbonate to pH
9.4. Glucuronidase solution was 50000 U type G-0258 (abalone entrails) and 40000 U type L-
II (limpet) glucuronidases (Sigma) in 10 mL 50 mM pH 5 acetate buffer.)
HPLC VARIABLES
Column: 250 X 4.6 Zorbax CN
Mobile phase: MeOH:THF:buffer 60:6:34 (Buffer was 0.8 g ammonium acetate in 340 mL water,
pH adjusted to 5.5 with acetic acid (if necessary).)
Flow rate: 1.5
Detector: F ex 275 (slit width 6 nm) em 324 (slit width 10 nm)
CHROMATOGRAM
Retention time: 10
Internal standard: propranolol (8)
OTHER SUBSTANCES
Simultaneous: carazolol, metoprolol, physostigmine
Noninterfering: acetaminophen, aspirin, atenolol, bromocriptine, chloroquine, doxorubicin, hy-
drochlorothiazide, indomethacin, 17-methyltestosterone, nadolol, nandrolone, practolol, quini-
dine, salicyluric acid, sulfadiazine, sulfamerazine, sulfamethazine, timolol, triamterene, vin-
zolidine, warfarin
Interfering: pergolide
KEYWORDS
REFERENCE
Miner,D.J.; Binkley,D.A.; Bechtol,L.D. Liquid-chromatographic determination of penbutolol and its principal
metabolites in plasma and urine, CHn.Chem., 1984, 30, 717-723.
SAMPLE
HPLCVARIABLES
mL/min)
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
SAMPLE
Matrix: solutions
Sample preparation: Prepare a 10 |xg/mL solution in MeOH, inject a 20 ^L aliquot.
HPLC VARIABLES
MeOH, pH 6.7
Flow rate: 2
CHROMATOGRAM
Retention time: 1.9
OTHER SUBSTANCES
ine, pargyline, pecazine, pentazocine, penthienate, pericyazine, perphenazine, phenadoxone,
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Dissolve 100 ng penbutolol and 200 |xg(-)-2-niethyl-l,l'-binaphthalene-2'-
carbonyl cyanide in 200 |JLL 0.01% quinuclidine in MeCN, heat at 60 for 10 min, inject an
aliquot. (Synthesis of (-)-2-methyl-l,l'-binaphthalene-2'-carbonyl cyanide is as follows. Reflux
21Og l-bromo-2-methylnaphthalene, 160 g N-bromosuccinimide, 1 g benzoyl peroxide, and 250
mL carbon tetrachloride for 2.5 h, add 250 mL carbon tetrachloride, filter while warm, wash
the residue several times with solvent. Concentrate and cool the nitrate to give l-bromo-2-
bromomethylnaphthalene (mp 230-240) (J. Org. Chem. 1949, 14, 375). Dissolve 90 g 1-bromo-
2-bromomethylnaphthalene in 400 mL chloroform, reflux, add 46.5 g powdered hexamine in
portions, remove the hexaminium salt by filtration. Reflux this salt in 650 mL 50% acetic acid
for 1 h, add 105 mL concentrated HCl, reflux for 5 min, cool, obtain l-bromo-2-naphthaldehyde
(mp 119-120) by filtration. Heat 11 g l-bromo-2-naphthaldehyde in 275 mL acetone at 60-68,
add a hot solution of 14 g potassium permanganate in 330 mL water over 30 min, heat for
another 30 min, pass in sulfur dioxide (sodium metabisulfite ?) until the solution is clear, pour
into water to give l-bromo-2-naphthoic acid, purify by forming the ammonium salt and repre-
cipitating. Reflux l-bromo-2-naphthoic acid in MeOH in the presence of sulfuric acid to give
methyl l-bromo-2-naphthoate. Heat methyl-l-bromo-2-naphthoate with copper bronze at 270-
280 for 20 min, while still hot extract with toluene, cool to obtain dimethyl l,l'-binaphthalene-
2,2'- dicarboxylate, obtain more crystals by evaporating some of the solvent, recrystallize from
EtOH to give dimethyl l,l'-binaphthalene-2,2'-dicarboxylate (mp 158) (J. Chem. Soc. 1955,
1242). Add 8 g lithium tri-tert-butoxyaluminohydride in portions to 2.8 g dimethyl l,l'-bi-
naphthalene-2,2'-dicarboxylate in 150 mL anhydrous benzene:ether 50:50 (Caution! Benzene
is a carcinogen!), heat at 80 for 2 h, acidify with 5% HCl. Remove the organic layer and dry
it over anhydrous sodium sulfate, evaporate to dryness, chromatograph on 50 g silica gel with
hexane:ethyl acetate 80:20, recrystallize the product from hexane/acetone to give methyl 2-
hydroxymethyl-l,r-binaphthalene-2'-dicarboxylate (mp 117.5-118.5). Add 5 mL 30% hydrogen
bromide in acetic acid to 2 g methyl 2-hydroxymethyl-l,r- binaphthalene-2'-dicarboxylate in
10 mL acetic acid, stir at 50 for 10 min, pour into ice-water, filter, chromatograph the solid on
40 g silica gel with hexane:ethyl acetate 30:1 to give methyl 2-bromomethyl-l,l'-binaphthalene-
2'-dicarboxylate as pale yellow needles (mp 137-138). Add 400 mg sodium borohydride to 1.9
g methyl 2-bromomethyl-l,l>-binaphthalene-2'-dicarboxylate in 10 mL DMSO, stir at 60 for
15 min, pour into ice-water, acidify with concentrated HCl, chromatograph the crude product
on 40 g silica gel with hexane:ethyl acetate 10:1, recrystallize from MeOH to give methyl 2-
methyl-l,r-binaphthalene-2'-carboxylate as colorless needles mp 97-98. Add 30 mL 10% KOH
to 1.2 g methyl 2-methyl-l,l>-binaphthalene-2'-carboxylate in 50 mL MeOH, reflux for 3 h, pour
into ice-water, filter, recrystallize from hexane/ethyl acetate to give 2-methyl-l,l'-binaphthal-
ene-2'-carboxylic acid as colorless needles (mp 232-233). Add 4.1g (-)-brucine in 20 mL EtOH
to 3.3 g 2-methyl-l,l'-binaphthalene-2'-carboxylic acid dissolved in 60 mL EtOH, allow to stand
overnight, filter, recrystallize the precipitate several times from EtOH. Add 5% HCl to the salt
and extract with ethyl acetate, wash the organic layer with water, dry over anhydrous sodium
sulfate, evaporate to dryness, recrystallize from hexane/acetone to give (-)-2-methyl-l,l'-bina-
phthalene-2'-carboxylic acid as colorless needles (mp 229-229.5; >
[a]D20 -41.3 (c> = 0.58 in chlo-
roform). Add 3 mL oxalyl chloride to 500 mg (-)-2-methyl-l,l -binaphthalene-2 -carboxylicacid
in 30 mL anhydrous dichloromethane, stir at room temperature for 2 h, evaporate to give an
oily residue, take up in 10 mL dichloromethane, add 2 mL trimethylsilyl cyanide, add 1 mg
zinc iodide, stir at room temperature for 2 h, evaporate to dryness, chromatograph on 5 g silica
gel with hexane to give (-)-2-methyl-l,l>-binaphthalene-2'-carbonyl cyanide as a yellow oil ([a]D20
-42.8 (c = 1.05 in chloroform).)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm spherical silica (Waters)
Mobile phase: Hexane:chloroform:MeOH 100:5:0.3
CHROMATOGRAM
KEYWORDS
REFERENCE
Goto,J.; Goto,N.; Shao,G.; Ito,M.; Hongo,A.; Nakamura,S.; Nambara,T. Fluorescence chiral derivatization rea-
gents for high performance liquid chromatographic resolution of enantiomeric hydroxyl compounds,
Anal.ScL, 1990, 6, 261-264.
Penciclovir
Merck Index: 7210
SAMPLE
Sample preparation: Filter (0.45 |xm) a reaction mixture containing activated sludge, inject a
20 |xL aliquot of the nitrate.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm YMC-AQ C18 (YMC)
Flow rate: 1
Detector: UV 260
CHROMATOGRAM
Retention time: 17
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Hsu,L.C; Constable,D.J.C; Orvos,D.R.; Hannah,R.E. Comparison of high-performance liquid chromatography
and capillary zone electrophoresis in penciclovir biodegradation kinetic studies, J.Chromatogr.B, 1995,669,
85-92.
Penclomedine
SAMPLE
Sample preparation: Plasma, red cells. Add 1 mL plasma or red cells to 2.8 mL ethyl acetate
and 200 JJLL 700 mM pH 2.7 ammonium phosphate buffer, homogenize, centrifuge. Draw off
the organic layer, add 50 |ULL dimethyl sulfoxide, evaporate to approximately 100 |JLL under
nitrogen, add 50 |xL MeCN, inject an aliquot. Liver slices. Add 1 mL incubation to 500 JJLL
Krebs-Henseleit buffer containing 2.25% bovine serum albumin, homogenize, centrifuge. Draw
off the organic layer, add 50 (xL dimethyl sulfoxide, evaporate to approximately 100 |JLL under
nitrogen, add 50 |xL MeCN, inject an aliquot. Bile. Dilute bile with an equal volume mobile
HPLC VARIABLES
Column: 250 X 4.6 5 jxm Adsorbosphere HS C18 (Alltech)
Mobile phase: Gradient. A was MeCN. B was 10 mM pH 2.7 ammonium phosphate buffer. A:B
from 0:100 to 100:0 in 25 min, maintain at 100:0 for 15 min.
Flow rate: 1
Detector: UV 240; Radioactivity, Radiomatic Flo-One/Beta A140, with 500 |xL cell, using FIo-
Scint VI scintillant at ratio 2:1
CHROMATOGRAM
Retention time: 32
OTHER SUBSTANCES
KEYWORDS
human; liver; mouse; 14C labeled; plasma; red cells; erythrocytes
REFERENCE
Hartman,N.R.; Leo,K.U.; Brewer,T.G.; Strong,J.M. The in vitro metabolism of penclomedine in mouse, rat, and
human systems, Drug Metab.Dispos., 1998, 26, 513-519.
SAMPLE
Sample preparation: Microsomes. Add 1 mL microsomal incubation to 2.8 mL ethyl acetate and
200 |xL 700 niM pH 2.7 ammonium phosphate buffer, vortex, centrifuge. Draw off the organic
layer, add 50 jxL dimethyl sulfoxide, evaporate to approximately 100 (JLL under nitrogen, add
50 fxL MeCN to the residue, inject an aliquot. Liver slices. Mix 1 mL microsomal incubation
with 500 jxL Krebs-Henseleit buffer containing 2.25% bovine serum albumin, homogenize, cen-
trifuge. Draw off the organic layer, add 50 |JLL dimethyl sulfoxide, evaporate to approximately
100 jxL under nitrogen. Reconstitute the residue with 50 |xL MeCN, inject an aliquot.
HPLC VARIABLES
Column: 250 x 4.6 5 |xm Adsorbosphere HS C18 (Alltech)
Mobile phase: Gradient. A was MeCN. B was 10 mM pH 2.7 ammonium phosphate buffer. A:B
from 0:100 to 100:0 over 25 min, maintain at 100:0.
Flow rate: 1
Detector: UV 240; Radioactivity, Radiomatic Flo-One/Beta A140, with 500 JJLL cell, using FIo-
Scint VI scintillant at ratio 2:1
CHROMATOGRAM
Retention time: 32
OTHER SUBSTANCES
KEYWORDS
human; liver; mouse; 14C labeled
REFERENCE
Hartman,N.R.; Leo,K.U.; Brewer,T.G.; Strong,J.M. The in vitro metabolism of penclomedine in mouse, rat, and
human systems, Drug Metab.Dispos., 1998, 26, 513-519.
Penfluridol
Molecular formula: C28H27CIF5NO
Merck Index: 7213
Lednicer No.: 2 334
SAMPLE
Matrix: blood
HPLC VARIABLES
Flow rate: 0.8
Detector: UV 266
CHROMATOGRAM
KEYWORDS
REFERENCE
254-262.
SAMPLE
HPLC VARIABLES
Column: 250 X 4.6 5 imm Symmetry C8 (Waters)
mL/min)
Next Page
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Penicillamine
Merck Index: 7214
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 150 |xL 25% trichloroacetic acid, vortex, cool on ice for 10
min, centrifuge at 6500 g for 2 min. Remove a 500 jxL aliquot of the supernatant and add it
to 200 jxL 1% NaOH in water, add 250 |xL buffer, add 1 mL 1 mM N-[p-(2-benzoxazolyl)phenyl]
maleimide (Eastman) in EtOH, heat at 37 overnight, inject a 50 JULL aliquot. (Buffer was 500
mM sodium citrate adjusted to pH 5.0 with perchloric acid.)
HPLCVARIABLES
Column: 300 X 3.9 10 |xm jiBondapak C18
Mobile phase: MeOH: 100 jxM sodium acetate 48:52
Flow rate: 2
CHROMATOGRAM
Retention time: 5.5
Limit of quantitation: 1 \xM
KEY WpRDS
REFERENCE
Miners,J.O.; Fearnley,L; Smith,K.J.; Birkett,D.J.; Brooks,P.M.; Whitehouse,M.W. Analysis of D-penicillamine
in plasma by fluorescence derivatisation with N-[p-(2-benzoxazolyl)-phenyl] maleimide and high-perfor-
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma and 400 |xL 18% (w/v) trichloroacetic acid in a 100 X
15 polypropylene tube, let stand at 0 for 5 min, centrifuge at 4 at 1700 g for 10 min, remove
the supernatant as completely as possible. Suspend the precipitate in 1 mL 5% (w/v) trichlo-
roacetic acid by stirring magnetically, centrifuge at room temperature at 2000 g for 5 min,
discard the supernatant, repeat this washing step. Air-dry the precipitate then blanket it with
nitrogen, add 2 mL 200 mM pH 8.0 Tris buffer, pass nitrogen over the mixture for 1 h, add
100 |JLL 250 mM EDTA, add 50 |xL octanol, add 100 mg solid sodium borohydride, remove the
Previous Page
Detector: UV 200.5
CHROMATOGRAM
KEYWORDS
whole blood
REFERENCE
163.
Penicillamine
Merck Index: 7214
SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 150 |xL 25% trichloroacetic acid, vortex, cool on ice for 10
min, centrifuge at 6500 g for 2 min. Remove a 500 jxL aliquot of the supernatant and add it
to 200 jxL 1% NaOH in water, add 250 |xL buffer, add 1 mL 1 mM N-[p-(2-benzoxazolyl)phenyl]
maleimide (Eastman) in EtOH, heat at 37 overnight, inject a 50 JULL aliquot. (Buffer was 500
mM sodium citrate adjusted to pH 5.0 with perchloric acid.)
HPLCVARIABLES
Column: 300 X 3.9 10 |xm jiBondapak C18
Mobile phase: MeOH: 100 jxM sodium acetate 48:52
Flow rate: 2
CHROMATOGRAM
Retention time: 5.5
Limit of quantitation: 1 \xM
KEY WpRDS
REFERENCE
Miners,J.O.; Fearnley,L; Smith,K.J.; Birkett,D.J.; Brooks,P.M.; Whitehouse,M.W. Analysis of D-penicillamine
in plasma by fluorescence derivatisation with N-[p-(2-benzoxazolyl)-phenyl] maleimide and high-perfor-
SAMPLE
Matrix: blood
Sample preparation: Mix 1 mL plasma and 400 |xL 18% (w/v) trichloroacetic acid in a 100 X
15 polypropylene tube, let stand at 0 for 5 min, centrifuge at 4 at 1700 g for 10 min, remove
the supernatant as completely as possible. Suspend the precipitate in 1 mL 5% (w/v) trichlo-
roacetic acid by stirring magnetically, centrifuge at room temperature at 2000 g for 5 min,
discard the supernatant, repeat this washing step. Air-dry the precipitate then blanket it with
nitrogen, add 2 mL 200 mM pH 8.0 Tris buffer, pass nitrogen over the mixture for 1 h, add
100 |JLL 250 mM EDTA, add 50 |xL octanol, add 100 mg solid sodium borohydride, remove the
hydrogen line, allow hydrogen to vent through a pin-hole in the cap, stir slowly, after 10 min
cool in ice and slowly add 1 mL ice-cold 2 M perchloric acid, stir briefly, centrifuge an aliquot
at room temperature at 8000 g for 30 s, remove 200 |xL of the supernatant and add it to 10
(JLL 1 mM L-cysteine in water, inject an aliquot.
HPLC VARIABLES
Column: 100 X 4.6 3 |jim C18 "Short One" (Rainin)
Mobile p h a s e : MeCNibuffer 6:94 (Buffer was 100 mM pH 3.0 monochloroacetic aeid/NaOH con-
taining 1 g/L heptanesulfonic acid.) (Rigorously degas mobile phase by refluxing, negative-
pressure nitration, and passing helium through it for 3 h before starting the assay.)
Flow r a t e : 0.6
Detector: E, Bioanalytical Systems BAS LC-4B/19, BAS TL-6A Au/Hg working electrode + 150
mV, glassy carbon auxiliary electrode, Ag/AgCl reference electrode
CHROMATOGRAM
Limit of detection: 1.2 |JLM
KEY WORDS
plasma
REFERENCE
Joyce,D.A.; Wade,D.N. Assay for D-penicillamine-protein conjugate in human plasma utilising chemical reduc-
tion followed by high-performance liquid chromatography with gold/mercury electrochemical detection,
J.Chromatogr., 1988, 430, 319-327.
SAMPLE
Matrix: blood
Sample p r e p a r a t i o n : 1 Volume plasma + 0.4 volume 10 mM monobromobimane (Calbiochem;
Molecular Probes, Eugene OR) + 0.01 volume 1 M acetic acid + 2.5 volume MeCN, mix, cen-
trifuge at 4 at 1000 g for 10 min, filter (5 |xm) the supernatant, inject a 100 u-L aliquot.
HPLC VARIABLES
Column: ixBondapak C18 radial compression
Mobile p h a s e : MeOH:water:glacial acetic acid 22:77.75:0.25, pH adjusted to 3.9 with NaOH.
(After each injection wash column with MeOH at 4 mL/min for 10 min.)
Flow r a t e : 1.5
CHROMATOGRAM
I n t e r n a l s t a n d a r d : d-penicillamine
OTHER SUBSTANCES
E x t r a c t e d : cysteine, glutathione, homocysteine
KEYWORDS
derivatization; plasma; penicillamine is IS
REFERENCE
Velury,S.; Howell,S.B. Measurement of plasma thiols after derivatization with monobromobimane,
J.Chromatogr., 1988, 424, 141-146.
SAMPLE
Matrix: blood
Sample p r e p a r a t i o n : 50 JULL Plasma + 10 |xL 25 mM monobromobimane in MeCN, let stand for
5 min at room temperature, add 20 JJLL 20% perchloric acid.
HPLC VARIABLES
Column: 150 X 4.6 7 |xm Nucleosil RP-18
Mobile phase: Gradient. A was MeCN. B was 1% aqueous acetic acid containing 1 g/L octane-
sulfonic acid. A:B from 5:95 to 8:92 over 2 min, to 10:90 over 13 min (Waters convex), to 30:70
over 20 min (Waters convex), maintain at 30:70 for 6 min, re-equilibrate at initial conditions
for 9 min.
Flow rate: 1.4
CHROMATOGRAM
Internal standard: penicillamine
OTHER SUBSTANCES
Extracted: mesna
KEYWORDS
plasma; derivatization; penicillamine is IS
REFERENCE
Stofer-Vogel,B.; Cerny,T.; Borner,M.; Lauterburg,B.H. Oral bioavailability of mesna tablets, Cancer Chem-
other.Pharmacol, 1993, 32, 78-81.
SAMPLE
Sample preparation: Blood. Mix 9 mL whole blood with 1 mL 3.8% sodium citrate, centrifuge
at 4 at 150 g for 15 min, wash the erythrocytes three times with isotonic saline. Suspend 100
|JLL erythrocytes in 700 |JLL 6 mM EDTA, mix gently for 1 min, add 200 jxL 25% metaphosphoric
acid, mix for 10 min, centrifuge at 5000 g for 15 min, filter (0.45 |xm) the supernatant, inject
a 10 |xL aliquot of the filtrate. Cells. Wash 500 mg (wet weight) E. coli cells with water, add 2
mL 5% metaphosphoric acid, sonicate, centrifuge at 4 at 15000 g for 15 min, filter (0.45 (xm)
the supernatant, inject an aliquot of the filtrate.
HPLC VARIABLES
Column: 250 X 4.6 Fine SiI C18-10 (Japan Spectroscopic)
Mobile phase: 33 mM KH2PO4 adjusted to pH 2.2 with phosphoric acid
Flow rate: 1
Detector: UV 344 following post-column reaction. The column effluent mixed with the 1.5 mM
6,6'-dithiodinicotinic acid in 200 mM pH 7.0 sodium phosphate buffer pumped at 1 mL/min
and the mixture flowed through a 60 cm X 0.5 mm ID stainless steel coil to the detector.
CHROMATOGRAM
Retention time: 8.2
Limit of detection: 0.1 nmole
OTHER SUBSTANCES
Extracted: cysteamine, cysteine, glutathione, homocysteine
KEYWORDS
post-column reaction; whole blood; erythrocytes
REFERENCE
Nishiyama,J.; Kuninori,T. Assay of biological thiols by a combination of high-performance liquid chromatog-
raphy and postcolumn reaction with 6,6'-dithiodinicotinic acid, Anal.Biochem., 1984, 138, 9598.
SAMPLE
Matrix: blood, sea water, urine
Sample preparation: Blood. Centrifuge blood at 550 g for 30 min. Mix the clear solution with
MeOH and centrifuge at 500 g for 30 min. Filter (0.22 mm) and centrifuge at 2500 g. Dilute
the clear liquid with an equal amount of mobile phase. Centrifuge 2500 g for 5 min inject an
aliquot of the supernatant. Sea water, urine. Filter (0.22 mm), dilute with an equal amount of
mobile phase, store at -5, inject an aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 fxm Zorbax ODS
Mobile p h a s e : MeOH:50 mM pH 2.2 trichloroacetic acid 1:99
Flow r a t e : 0.3
Detector: E, PAR-174, 1.0 mm tungsten wire electrode at -0.3 V, Ag/AgCl reference electrode, 1.0
mm dia. Pt wire auxiliary electrode (construction details for cell in paper) following post-column
reaction. The column effluent mixed with 100 mM pH 3.1 phosphate buffer containing 25 jxM
or 25 mM Hg2+ (both quantities in paper) pumped at 1.0 mL/min and the mixture flowed to
the detector.
CHROMATOGRAM
OTHER SUBSTANCES
E x t r a c t e d : cysteine, glutathione, homocysteine, mercaptopropionic acid, thiourea
KEYWORDS
plasma
REFERENCE
Hidayat,A.; Hibbert,D.B.; Alexander,P.W. Amperometric detection of organic thiols at a tungsten wire electrode
following their separation by liquid chromatography, J.Chromatogr.B, 1997, 693, 139-146.
SAMPLE
Sample p r e p a r a t i o n : Plasma. 1 mL Plasma + 1 mL ice-cold 2 M perchloric acid containing 4
mM EDTA, vortex, centrifuge at 4 at 4000 g for 10 min. Neutralize an aliquot of the super-
natant with cold 2 M LiOH solution, adjust to 10% with pH 8.4 borate buffer. Remove a 100
IJLL aliquot and add it to 100 |xL 50 mM N-acetylcysteine solution, 100 |xL reagent solution,
and 700 uL pH 8.4 borate buffer, vortex for a few s, let stand at room temperature for 1 h, add
an equal volume of the mobile phase, inject a 10 jxL aliquot. Tissue. Powder tissue at low
temperature. Add 4 volumes ice-cold 2 M perchloric acid containing 25 mM EDTA to 0.1-0.5 g
powdered tissue, vortex quickly, homogenize (Brinkman). Neutralize an aliquot with cold 2 M
LiOH solution, adjust to 1% (brain, lung, liver, kidney, testes, small intestine) or 2.5% (aorta,
heart, spleen) with pH 8.4 borate buffer. Remove a 100 |xL aliquot and add it to 100 |xL 50 mM
N-acetylcysteine solution, 100 uX reagent solution, and 700 |JLL pH 8.4 borate buffer, vortex for
a few s, let stand at room temperature for 1 h, add an equal volume of the mobile phase, inject
a 10 JULL aliquot. (Prepare reagent, 2-(4-N-maleimidephenyl)-6-methylbenzothiazole, as follows.
Recrystallize 2-(4-aminophenyl)-6-methylbenzothiazole from chloroform before use. Add 500 mg
maleic anhydride in 2 mL chloroform dropwise to 1.2 g 2-(4-aminophenyl)-6-methylbenzothia-
zole in 10 mL DMF, stir at room temperature for 2 h, filter, wash with 30 mL chloroform,
recrystallize from DMF to give 2-(4-N-phenylmaleamic acid)-6-methylbenzothiazole as yellow
crystals (mp 242). Reflux 2 g 2-(4-N-phenylmaleamic acid)-6-methylbenzothiazole, 100 mg an-
hydrous sodium acetate, and 25 mL acetic anhydride for 2 h, cool on ice, filter, wash the solid
with water. Neutralize the filtrate with cold 10% NaOH, extract with chloroform. Dry the
organic layer over anhydrous magnesium sulfate and evaporate it to dryness under reduced
pressure. Combine this product with the solid obtained earlier and recrystallize from isopro-
panol to give 2-(4-N-maleimidephenyl)-6-methylbenzothiazole as yellow needles (mp 254-6).
Prepare the reagent solution by dissolving 50 jjumoles of this compound in 10 mL DMF and
diluting 25-fold with MeCN.)
HPLC VARIABLES
Column: 250 X 4.6 5 jmm Ultrasphere ODS
Mobile p h a s e : MeCNrbuffer 35:65, pH 4.5 (Buffer was 10 mM KH 2 PO 4 containing 0.1% sodium
hexanesulfonate.)
Flow r a t e : 1.5
CHROMATOGRAM
Retention time: 17
Internal standard: N-acetylcysteine (7)
OTHER SUBSTANCES
Extracted: N-acetylpenicillamine, coenzyme A, cysteine, glutathione, homocysteine
KEY WpRDS
derivatization; plasma; rat; aorta; heart; lung; liver; kidney; testes; spleen; brain; small intestine
REFERENCE
Haj-Yehia,A.I.; Benet,L.Z. Determination of aliphatic thiols by fluorometric high-performance liquid chroma-
tography after precolumn derivatization with 2-(4-N-maleimidophenyl)-6-methylbenzothiazole, Pharm.Res.,
1995, 12, 155-160.
SAMPLE
Matrix: bulk
Sample preparation: Dissolve 5 mg amino acids in 10 mL MeCN:water:triethylamine 50:50:
0.55. Remove a 50 |xL aliquot and add it to 50 |xL 0.66% 2,3,4,6-tetra-O-benzoyl-p-D-glucopy-
ranosyl isothiocyanate (Fluka) in MeCN, shake mechanically for 30 min, add 10 |xL 0.26%
ethanolamine in MeCN, shake for 10 min, make up to 1 mL with MeCN, inject a 10 fxL aliquot.
HPLC VARIABLES
Column: 25 X 4 (sic) 5 |jiin LiChrospher 100 RP-18
Mobile phase: MeOH:water:67 mM pH 7.0 phosphate buffer 70:25:5
Flow rate: 0.45
Detector: UV 231
CHROMATOGRAM
Retention time: k' 7.37 (L), 10.05 (D)
OTHER SUBSTANCES
Simultaneous: amino acids
KEY WpRDS
REFERENCE
Lobell,M.; Schneider,M.R 2,3,4,6-Tetra-O-benzoyl-p-D-glucopyranosyl isothiocyanate: an efficient reagent for
the determination of enantiomeric purities of amino acids, |3-adrenergic blockers and alkyloxiranes by high-
performance liquid chromatography using standard reversed-phase columns, J.Chromatogr., 1993, 633,
287-294.
SAMPLE
Sample preparation: Dissolve about 1 mg of the contents of a capsule in 10 mL water and
dilute with MeCN to a penicillamine concentration of 8 mM. 50 jxL Solution + 200 JJLL buffer
+ 150 JJIL reagent, heat at 60 for 30 min, cool, inject a 10 (xL aliquot. (Buffer was prepared by
adjusting the pH of a solution containing 100 mM boric acid and 100 mM KCl to 8.5 with 100
mM sodium carbonate. Reagent was 200 |JLM N-[4-(6-dimethylamino-2-benzofu-
ranyl)phenyl]maleimide (DBPM) in MeCN. Synthesis of N-[4-(6-dimethylamino-2-benzofu-
ranyl)phenyl]maleimide is as follows. Add 8.8 g aluminum trichloride to 12.50 g 3-dimethyl-
aminophenol in 185 mL chloroform and 84 g triethyl orthoformate, mix at room temperature
for 10 min, when the exothermic reaction ceases add 50 mL 10% HCl, stir to hydrolyze the
acetal, neutralize with 10% NaOH, filter through a short column of Celite, wash through with
chloroform, wash the filtrate with saturated aqueous NaCl, dry over magnesium sulfate, con-
centrate under reduced pressure, recrystallize from chloroform to give 4-(dimethylam-
ino)salicylaldehyde (mp 78-79). Add 400 mg KOH in 3 mL EtOH to a solution of 1 g 4-(di-
methylamino)salicylaldehyde and 1.3 g (?) 4-nitrobenzylbromide in 12 mL EtOH, reflux for 7
h, cool, filter to recover the crystals, wash with water, dry under vacuum, recrystallize from
EtOH to give 4-dimethylamino-2-(4-nitrobenzyloxy)benzaldehyde (mp 179-180). Add a solution
of 900 mg 4-dimethylamino-2-(4-nitrobenzyloxy)benzaldehyde in 6 mL DMF to a sodium meth-
oxide solution (prepared from 69 mg sodium in 1 mL MeOH), reflux for 20 min, add 1 mL
MeOH, filter the crystals, recrystallize from EtOH to give 6-dimethylamino-2-(4-nitro-
phenyDbenzofuran as red needles (mp 209.5-210.5). Reflux 1 g 6-dimethylamino-2-(4-nitro-
phenyDbenzofuran in 20 mL benzene (Caution! Benzene is a carcinogen!) and 18 mL MeOH
containing 80 mg active carbon and a catalytic amount of ferric chloride hexahydrate for 10
min, add 2.30 g 98% hydrazine hydrate (Caution! Hydrazine hydrate is a carcinogen!) dropwise,
reflux for 7 h, filter, concentrate the filtrate, recrystallize from cyclohexane to give 6-dimeth-
ylamino-2-(4-aminophenyl)benzofuran as orange needles (mp 198.5-200). Stir 605 mg 6-di-
methylamino-2-(4-aminophenyl)benzofuran and 230 mg maleic anhydride in 5 mL chloroform
at room temperature for 3 h, filter the crystals, wash with a small amount of chloroform,
recrystallize from EtOH to obtain N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleamic
acid (mp 219.5-221). Reflux a mixture of 1.17 g N-[4-(6-dimethylamino-2-benzofu-
ranyl)phenyl]maleamic acid and 30 mg sodium acetate in 18 mL acetic anhydride, cool in an
ice bath, collect the crystals of product, wash with water. Neutralize the filtrate with 20%
NaOH, extract twice with 30 mL portions of chloroform, wash the organic layers with saturated
aqueous NaCl, dry over anhydrous magnesium sulfate, evaporate to give more product. Com-
bine the products and recrystallize them from acetone to give N-[4-(6-dimethylamino-2-
benzofuranyl)phenyl]maleimide as reddish purple crystals (mp 203-204) (Bull.Chem.Soc.Jpn.
1985, 58, 2192).)
HPLC VARIABLES
Column: 250 X 4.6 5 (xm Sumichiral OA-2500S Pirkle-type (Sumika Chemical Analysis Service)
Mobile phase: MeOH:water 75:25 containing 150 mM ammonium acetate and 50 mM tetra-n-
butylammonium bromide
Flow rate: 1
CHROMATOGRAM
Retention time: 27 (D), 31 (L) (Each enantiomer gives 2 peaks, the later peaks are used for
quantitation.)
Limit of detection: 350 fmole (L), 290 fmole (D)
KEYWORDS
capsules; chiral; derivatization
REFERENCE
Nakashima,K.; Ishimaru,T.; Kuroda,N.; Akiyama,S. High-performance liquid chromatographic separation of
penicillamine enantiomers labelled with iV-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide on a chi-
ral stationary phase, Biomed.Chromatogr., 1995, 9, 90-93.
SAMPLE
Sample preparation: Dissolve about 1 mg of the contents of a capsule in 10 mL water and
dilute with MeCN to a penicillamine concentration of 8 mM. 50 jxL Solution + 200 |JLL buffer
+ 150 jxL reagent, heat at 60 for 30 min, cool, inject a 10 (xL aliquot. (Buffer was prepared by
adjusting the pH of a solution containing 100 mM boric acid and 100 mM KCl to 8.5 with 100
mM sodium carbonate. Reagent was 200 juiM N-[4-(6-dimethylamino-2-benzofu-
ranyl)phenyl]maleimide (DBPM) in MeCN. Synthesis of N-[4-(6-dimethylamino-2-benzofu-
ranyl)phenyl]maleimide is as follows. Add 8.8 g aluminum trichloride to 12.50 g 3-dimethyl-
aminophenol in 185 mL chloroform and 84 g triethyl orthoformate, mix at room temperature
for 10 min, when the exothermic reaction ceases add 50 mL 10% HCl, stir to hydrolyze the
acetal, neutralize with 10% NaOH, filter through a short column of Celite, wash through with
chloroform, wash the filtrate with saturated aqueous NaCl, dry over magnesium sulfate, con-
centrate under reduced pressure, recrystallize from chloroform to give 4-(dimethylam-
ino)salicylaldehyde (mp 78-79). Add 400 mg KOH in 3 mL EtOH to a solution of 1 g 4-(di-
methylamino)salicylaldehyde and 1.3 g (?) 4-nitrobenzylbromide in 12 mL EtOH, reflux for 7
h, cool, filter to recover the crystals, wash with water, dry under vacuum, recrystallize from
EtOH to give 4-dimethylamino-2-(4-nitrobenzyloxy)benzaldehyde (mp 179-180). Add a solution
of 900 mg 4-dimethylamino-2-(4-nitrobenzyloxy)benzaldehyde in 6 mL DMF to a sodium meth-
oxide solution (prepared from 69 mg sodium in 1 mL MeOH), reflux for 20 min, add 1 mL
MeOH, filter the crystals, recrystallize from EtOH to give 6-dimethylamino-2-(4-nitro-
phenyDbenzofuran as red needles (mp 209.5-210.5). Reflux 1 g 6-dimethylamino-2-(4-nitro-
phenyl)benzofuran in 20 mL benzene (Caution! Benzene is a carcinogen!) and 18 mL MeOH
containing 80 mg active carbon and a catalytic amount of ferric chloride hexahydrate for 10
min, add 2.30 g 98% hydrazine hydrate (Caution! Hydrazine hydrate is a carcinogen!) dropwise,
reflux for 7 h, filter, concentrate the filtrate, recrystallize from cyclohexane to give 6-dimethy-
lamino-2-(4-aminophenyl)benzofuran as orange needles (mp 198.5-200). Stir 605 mg 6-dime-
thylamino-2-(4-aminophenyl)benzofuran and 230 mg maleic anhydride in 5 mL chloroform at
room temperature for 3 h, filter the crystals, wash with a small amount of chloroform, recrys-
tallize from EtOH to obtain N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleamic acid (mp
219.5-221). Reflux a mixture of 1.17 g N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleamic
acid and 30 mg sodium acetate in 18 mL acetic anhydride, cool in an ice bath, collect the
crystals of product, wash with water. Neutralize the filtrate with 20% NaOH, extract twice
with 30 mL portions of chloroform, wash the organic layers with saturated aqueous NaCl, dry
over anhydrous magnesium sulfate, evaporate to give more product. Combine the products and
recrystallize them from acetone to give N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimideas
reddish purple crystals (mp 203-204) (Bull.Chem.Soc.Jpn. 1985, 58, 2192).)
HPLC VARIABLES
Column: 250 X 4.6 5 |jim Sumichiral OA-2500S Pirkle-type (Sumika Chemical Analysis Service)
Mobile phase: MeOH:water 75:25 containing 150 mM ammonium acetate and 50 mM tetra-n-
butylammonium bromide
Flow rate: 1
CHROMATOGRAM
Retention time: 27 (D), 31 (L) (Each enantiomer gives 2 peaks, the later peaks are used for
quantitation.)
Limit of detection: 350 fmole (L), 290 fmole (D)
KEYWORDS
capsules; chiral; derivatization
REFERENCE
Reichelova,V.; Liliemark,J.; Albertioni,F. Structure-activity relationships of 2-chloro-2'-ara&mo-fluoro-2'-deox-
yadenosine and related analogues: Protein binding, lipophilicity, and retention in reversed-phase LC,
J.Liq.Chromatogr., 1995, 18, 1123-1135.
SAMPLE
Matrix: solutions
Sample preparation: Add 1 mL 50 |xg/mL N-(4-anilinophenyl)maleimide in 33 mM pH 6.85
phosphate buffer to 0.1-4 jxg thiol, let stand at 0 for 90 min, wash twice with 2 mL portions
of ether, heat the aqueous phase to 50 for 20 min, inject an aliquot. (Prepare N-(4-anilino-
phenyl)maleimide as follows. Add dropwise 1.1 g maleic anhydride in 10 mL chloroform to 1 g
N-phenylphenylenediamine (4-aminodiphenylamine) stirred in 10 mL chloroform at 0, filter,
dry to give N-(4-anilinophenyl)maleamic acid. Heat 100 mg N-(4-anilinophenyl)maleamic acid
and 25 mg sodium acetate in 400 JJLL acetic anhydride on a water bath for 2 h, cool, pour into
ice-water, filter, recrystallize from ethyl acetate/hexane to give N-(4-anilinophenyl)maleimide
HPLC VARIABLES
Mobile phase: MeCN:0.5% pH 3.0 (NH4)H2PO4 4:7
Flow rate: 1
Detector: E, Yanagimoto model VMD-101, glassy carbon electrode +1.0 V, Ag/AgCl reference
electrode
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: N-acetyl-L-cysteine, L-cysteine, glutathione
KEYWORDS
derivatization
REFERENCE
Shimada,K; Tanaka,M.; Nambara,T. Sensitive derivatization reagents for thiol compounds in high-performance
liquid chromatography with electrochemical detection, Anal.Chim.Acta, 1983, 147, 375-380.
SAMPLE
Matrix: solutions
Sample preparation: Add 1.05-3 equivalents 2,3,4,6-tetra-O-acetyl-p-D-glucopyranosyl isothio-
cyanate to 10 mL of a 100 JJLM solution of the thiol in MeCN:water 50:50 containing 1-3 equiv-
alents triethylamine, vortex briefly, let stand at room temperature for 30 min, dilute with
mobile phase, inject a 20 fxL aliquot.
HPLC VARIABLES
Column: 150 x 4.6 5 jxm TSKgel ODS-80TM (Tosoh)
Flow rate: 1
Detector: UV 250
CHROMATOGRAM
Retention time: 3.99 (L), 5.24 (D)
OTHER SUBSTANCES
Simultaneous: cysteine, homocysteine
KEY WpRDS
REFERENCE
Ito,S.; Ota,A.; Yamamoto,K.; Kawashima,Y. Resolution of the enantiomers of thiol compounds by reversed-phase
liquid chromatography using chiral derivatization with 2,3,4,6-tetra-O-acetyl-p-D-glucopyranosyl isothio-
cyanate, J.Chromatogr., 1992, 626, 187-196.
SAMPLE
Matrix: solutions
Sample preparation: Mix 20 |xL of a 30 uM solution in 2 mM disodium EDTA containing 3%
triethylamine with 10 (JLL 12 mM R-(-)-4-(3-isothiocyanatopyrrolidin-l-yl)-7-nitro-2,l,3-benzox-
adiazole in MeCN, let stand for 40 min, inject a 10 JJLL aliquot. (Synthesis of (R)-(-)-4-(3-iso-
thiocyanatopyrrolidin-l-yl)-7-nitro-2,l,3-benzoxadiazole, (R)-O-NBD-PyNCS, is as follows. Cool
a solution of 16.4 g (S)-(-)-l-benzyl-3-pyrrolidinol in 164 mL pyridine to +5, add 19.35 g p-
toluenesulfonyl chloride, stir at +10 for 48 h, evaporate to dryness, chromatograph using di-
chloromethane:acetone 95:5 to obtain (3S)-3-[(4-tolylsulfonyl)oxy]-l-(phenylmethyl)pyrrolidine
(mp 68). Heat a solution of (3S)-3-[(4-tolylsulfonyl)oxy]-l-(phenylmethyl)pyrrolidine in 200 mL
anhydrous DMF to 65, add 33.5 g sodium azide (Caution! Sodium azide is highly toxic!), stir
at 60 for 7 h, filter, evaporate the nitrate to dryness under reduced pressure, dissolve the
residue in ethyl acetate, wash twice with water, dry over anhydrous magnesium sulfate, evap-
orate to obtain (3R)-3-azido-l-(phenylmethyl)pyrrolidine as an oil. Add 3.5 g 10% palladium on
carbon under nitrogen to a solution of 7.05 g (3R)-3-azido-l-(phenylmethyl)pyrrolidine in 34.8
mL 1 M HCl in water and 245 mL EtOH, hydrogenate at atmospheric pressure for 30 min,
add 3.5 g catalyst, hydrogenate for 2 h, filter, add 34.8 mL 1 M HCl to the filtrate, evaporate
to dryness under reduced pressure, take up the residue in 70 mL EtOH, filter, evaporate the
filtrate to dryness under reduced pressure, repeat this operation twice, crystallize with the
minimum amount of EtOH to obtain (3R)-3-aminopyrrolidine dihydrochloride (J. Med. Chem.
1992, 35, 4205). 3R-(+)-aminopyrrolidine is also reported to be available from Tokyo Kasei. Add
100 mg 4-fluoro-7-nitro-2,l,3-benzoxadiazole in 20 mL MeCN dropwise to a stirred solution of
200 mg 3R-(+)-aminopyrrolidine in 20 mL MeCN at 0-10, stir at room temperature for 30 min,
remove the MeCN by evaporation under reduced pressure, dissolve the residue in 50 mL water,
extract 4 times with 80 mL portions of ethyl acetate. Combine the organic layers and wash
them with 20 mL water, dry over anhydrous sodium sulfate, evaporate to dryness under re-
duced pressure, recrystallize from hexane to obtain (R)-(-)-4-(3-aminopyrrolidin-l-yl)-7-nitro-
2,1,3-benzoxadiazole as dark red crystals (mp 178-181) (Analyst 1992, 117, 727). Add 100 |xL
thiophosgene in 10 mL benzene (Caution! Benzene is a carcinogen!) to 100 mg (R)-(-)-4-(3-
aminopyrrolidin-l-yl)-7-nitro-2,l,3-benzoxadiazole in 100 mL acetone, reflux for 1 h, remove
the solvent by evaporation under reduced pressure, suspend the residue in 100 mL water,
extract 4 times with 25 mL portions of benzene. Combine the extracts and wash them with 20
mL water, dry over anhydrous sodium sulfate, evaporate to dryness under reduced pressure,
recrystallize from hexane:benzene 1:2 to obtain (R)-(-)-4-(3-isothiocyanatopyrrolidin-l-yl)-7-ni-
tro-2,l,3-benzoxadiazole as red crystals (mp 165-170) (Analyst 1995, 120, 385).)
HPLC VARIABLES
Column: 150 X 4.6 5 ^m Ultron VX-ODS (Shinwa, Kyoto)
Flow rate: 1
CHROMATOGRAM
KEYWORDS
REFERENCE
Jin,D.; Takehana,K.; Toyo'oka,T. Chiral separation of racemic thiols based on diastereomer formation with a
fluorescent chiral tagging reagent by reversed-phase liquid chromatography, Anal.ScL, 1997,13, 113-115.
SAMPLE
Matrix: solutions
Sample preparation: Mix a 200 |xM solution in buffer with three volumes of a 400 |JLM solution
of 5,5'-dithio-(bis-2-nitrobenzoic acid) in buffer, let stand at room temperature for 30 min, inject
a 75 IJLL aliquot. (Buffer was 125 mM NaH2PO4 containing 154 mM NaCl, pH adjusted to 7.4
with NaOH.)
HPLC VARIABLES
Column: 250 X 4.6 Hypersil ODSl
Mobile p h a s e : Gradient. MeCN:buffer 0:100 for 20 min, to 17.5:82.5 over 40 min. (Buffer was
125 mM NaH 2 PO 4 containing 154 mM NaCl, pH adjusted to 7.4 with NaOH.)
Flow r a t e : 0.25 for 20 min, to 1 over 40 min
Detector: UV 357
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: N-acetylcysteine, N-acetylpenicillamine, captopril, cysteine, glutathione, thio-
malic acid
KEYWORDS
derivatization
REFERENCE
Russell,J.; McKeown,J.A.; Hensman,C; Smith,W.E.; Reglinski,J. HPLC determination of biologically active
thiols using pre-column derivatization with 5,5'-dithio-(bis-2-nitrobenzoic acid), J.Pharm.Biomed.Anal.,
1997, 15, 1757-1763.
SAMPLE
Matrix: tissue
Sample preparation: Freeze tissue in liquid nitrogen and pulverize. Homogenize 50-100 mg
tissue in 1 mL MeCN:20 mM EDTA 30:70, centrifuge at 4 at 4000 g for 5 min, adjust to 1-
2.5% w/v with pH 8.4 borate buffer, keep on ice. 100 |xL Sample + 100 |xL 0.05 mM N-acetyl-
cysteine + 100 |JLL 0.25 mM reagent in MeCN:DMF 95:5 + 700 JULL pH 8.4 borate buffer, vortex
for a few s, let stand for 1 h at room temperature, dilute with an equal volume of mobile phase,
inject a 10 (xL aliquot. (Reagent was 2-(4-maleimidophenyl)-6-methoxybenzofuran, a partial
synthesis is given in the paper.)
HPLCVAWABLES
Column: 250 X 4.6 5 |xm Ultrasphere-ODS
Mobile phase: MeCN:buffer 35:65 adjusted to pH 4.5 (Buffer was 10 mM KH2PO4 containing
0.1% sodium hexanesulfonate.)
Flow rate: 1.5
CHROMATOGRAM
Retention time: 10
Internal standard: N-acetylcysteine (8)
OTHER SUBSTANCES
Extracted: glutathione, homomocysteine, acetylpenicillamine
KEYWORDS
rat; heart; lung; liver; kidney; testes; spleen; derivatization
REFERENCE
Haj-Yehia,A.L; Benet,L.Z. 2-(4-N-Maleimidophenyl)-6-methoxybenzofuran: a superior derivatizing agent for
fluorimetric determination of aliphatic thiols by high-performance liquid chromatography, J.Chromatogr.B,
1995, 666, 45-53.
SAMPLE
Matrix: urine
Sample preparation: For each 1 mL urine add 1-2 mg EDTA and 5 jxg homocysteine, inject a
20 fxL aliquot.
HPLC VARIABLES
Guard column: 45 X 4.6 5 |jim ODS Hypersil
Mobile phase: 1 g/L pH 4 Heptanesulfonic acid in water containing 150 mg/L sodium EDTA
Flow rate: 1
pumped at 0.5 mL/min and the mixture flowed through a 150 X 2 column filled with 40 |xm
glass beads to the detector. (Prepare reagent by dissolving 200 mg 5,5'-dithiobis(2-nitrobenzoic
acid) and 10 g tripotassium citrate in 100 mL 250 mM pH 7.4 phosphate buffer, dilute 10-fold
with water immediately before use.)
CHROMATOGRAM
Retention time: 1.3
Internal standard: homocysteine (0.8)
OTHER SUBSTANCES
Extracted: cysteine
KEYWORDS
REFERENCE
Beales,D.; Finch,R.; McLean,A.E.M.; Smith,M.; Wilson,I.D. Determination of penicillamine and other thiols by
combined high-performance liquid chromatography and post-column reaction with Ellman's reagent: appli-
cation to human urine, J.Chromatogr., 1981, 226, 498-503.
Penicillin G
CAS Registry No.: 113-98-4 (potasium salt), 61-33-6 (free acid), 69-57-8 (Na salt),
751-84-8 (penicillin G benethamine), 1538-09-6 (penicillin G benzathine), 41372-02-5
(penicillin G benzathine tetrahydrate), 1538-11-0 (penicillin G benzhydrylamine),
973-53-5 (Ca salt), 3344-16-9 (penicillin G hydrabamine), 6130-64-9
(penicillin G procaine monohydrate), 54-35-3 (penicillin G procaine)
Merck Index: 7225
SAMPLE
Sample preparation: Serum. 0.5 mL serum + 0.5 mL MeCN mix in 7 mL tube on vortex mixer;
shake by rotation (20 rpm) 10 min; centrifuge 10 min 1000 g; transfer supernatant to another
tube, add 7 aliquots dichloromethane; equilibrate 10 min; shake by rotation (20 rpm) 10 min;
centrifuge 10 min 1000 g; inject aliquot of upper aqueous layer. Urine. Centrifuge urine and
dilute 1:20. Bile. Centrifuge bile and dilute 1:10
HPLC VARIABLES
Mobile phase: 24:76 MeCN:20 mM ammonium acetate adjusted to pH 5 with glacial acetic acid
Flow rate: 1
Detector: UV 214
CHROMATOGRAM
Retention time: 4.8
OTHER SUBSTANCES
Also analyzed: ampicillin, azlocillin, aztreonam, cefmenoxime, cefoperazone, cefsulodin, cefotax-
ime, ceftazidime, ceftriaxone, cloxacillin, desacetylcefotaxime, mezlocillin, piperacillin,
ticarcillin
KEYWORDS
serum
REFERENCE
Jehl,F.; Birckel,R; Monteil,H. Hospital routine analysis of penicillins, third-generation cephalosporins and az-
treonam by conventional and high-speed high-performance liquid chromatography, J.Chromatogr., 1987,
413, 109-119.
SAMPLE
Matrix: blood
buffer. 1 mL Plasma + 2 mL buffer, vortex, add to SPE cartridge, wash with buffer, elute with
500 (xL MeOH: 10 mM pH 5.2 potassium phosphate buffer 90:10, inject a 25 fjiL aliquot. (Buffer
was 121 g Trizma base in 1 L water, adjust pH to 7.0 with concentrated HCl. Dilute 1:100 to
obtain the 10 mM buffer.)
HPLC VARIABLES
Column: 150 X 3.9 4 jxm Nova-Pak phenyl
Mobile phase: MeOH: 10 mM pH 5.2 potassium phosphate buffer 20:80 (Buffer was 1 M KH2PO4
adjusted to pH 5.2 with 5 M KOH, dilute 1:100 with water to give working buffer.)
Flow rate: 0.9
Detector: UV 225
CHROMATOGRAM
Retention time: 412
Internal standard: penicillin G
OTHER SUBSTANCES
Extracted: cefteran (ceftetrane)
KEYWORDS
plasma; SPE; method stated to be applicable to urine (no details); penicillin G is IS
REFERENCE
Hicks,C.M.; Powell,M.L. Rapid analysis of ceftetrame in human plasma using sorbent extraction and high-
SAMPLE
Matrix: blood
Sample preparation: Condition a 55 X 5 100-200 mesh AG 50W-X8 (H+) column (Bio-Rad) with
10 mL MeCN:water 50:50. 600 |JLL Serum + 600 |JLL MeCN, vortex for 1 min, centrifuge at 2000
g for 5 min, add a 1 mL aliquot of the supernatant to the column, discard the first 200 JJLL
effluent, collect the rest of the effluent. Remove a 450 |JLL aliquot and add it to 50 (xL 10%
sodium carbonate solution, heat at 60 for 1 h (to hydrolyse the p-lactam ring), cool in an ice
bath. Remove a 100 \LL aliquot and add it to 15 JJLL 200 mM pH 6.0 phosphate buffer, add 35
IJLL 80 mM 7-fluoro-4-nitrobenzo-2-oxa-l,3-diazole in MeCN, heat at 60 for 10 min, cool in an
ice bath, add 30 |xL 1 M HCl, inject a 5-10 |xL aliquot.
HPLC VARIABLES
Column: 150 X 4.6 ODS-80TM (Tosoh)
Flow rate: 1
CHROMATOGRAM
Retention time: 10
OTHER SUBSTANCES
Extracted: methicillin, piperacillin
KEY WpRDS
REFERENCE
Iwaki,K; Okumura,N.; Yamazaki,M.; Nimura,N.; Kinoshita,T. Precolumn derivatization technique for high-
performance liquid chromatographic determination of penicillins with fluorescence detection, J.Chromatogr.,
1990, 504, 359-367.
SAMPLE
Matrix: blood
Sample preparation: Condition a Baker C18 SPE cartridge with 5 mL water and 5 mL 2%
NaCl, do not allow to run dry. 2 mL Plasma + 120 IJLL 5 fxg/mL penicillin V + 30 mL water +
2 mL 170 mM sulfuric acid +2 mL 5% sodium tungstate solution, vortex for 30 s, centrifuge at
2200 g for 10 min, filter supernatant (GF/B glass fiber filter), add 10 mL 20% NaCL, mix, add
to SPE cartridge at 3 mL/min, wash with 5 mL 2% NaCl, wash with 5 mL water, draw air
through cartridge for 5 min, elute with 500 |xL elution solution. Add 500 IJLL derivatization
reagent to the eluate, vortex for 20 s, allow to react at 65 for 30 min, cool to room temperature,
vortex, filter (0.45 jjim), inject 50-100 JJLL aliquots. (Prepare derivatization reagent by dissolving
34.45 g 1,2,4-triazole in 150 mL water, add 25 mL 10 mM mercuric chloride solution, mix,
adjust the pH to 9.0 0.5 with 5 M NaOH, dilute to 250 mL with water. Prepare elution
solution by mixing 60 mL MeCN and 5 mL buffer and making up to 100 mL with water. The
buffer was 0.994 g Na2HPO4 + 1.794 g NaH2PO4.H2O in 100 mL water, pH 6.5.)
HPLC VARIABLES
Column: 150 X 3.9 4 jim Nova-Pak C18
Mobile phase: MeCN:buffer 25:75 (Buffer contained 4.969 g Na2HPO4 + 8.969 g NaH2PO4.H2O
+ 2.482 g anhydrous sodium thiosulfate per liter.)
Flow rate: 1
Detector: UV 325
CHROMATOGRAM
Retention time: 4.5
KEYWORDS
plasma; cow; SPE; derivatization
REFERENCE
Boison,J.O.; Korsrud,G.O.; MacNeil,J.D.; Keng,L.; Papich,M. Determination of penicillin G in bovine plasma
by high-performance liquid chromatography after pre-column derivatization, J.Chromatogr., 1992, 576,
315-320.
SAMPLE
Matrix: blood, CSF
Sample preparation: 200 |JLL Serum, plasma, or CSF + 300 |xL reagent. Flush column A to
waste with 500 JJLL 500 mM ammonium sulfate, inject sample onto column A, flush column A
to waste with 500 JJLL 500 mM ammonium sulfate, elute the contents of column A onto column
HPLC VARIABLES
Column: A 30 X 2.1 40 pm preparative grade C18 (Analytichem); B 250 X 4.6 10 juim Partisil
C8
Mobile phase: Gradient. A was 50 mM pH 4.5 KH2PO4. B was MeCNrisopropanol 80:20. A:B 90:
Flow rate: 1.5
CHROMATOGRAM
OTHER SUBSTANCES
chlorothiazide, diazepam, droperidol, ethionamide, furosemide, isoniazid, methadone, pheno-
trimethoprim
KEYWORDS
REFERENCE
Seifart,H.L; Kruger,P.B.; Parkin,D.R; van Jaarsveld,P.R; Donald,P.R. Therapeutic monitoring of antitubercu-
1993, 619, 285-290.
SAMPLE
Sample preparation: 200 u,L Serum, urine, CSF, or gastric fluid + 300 jxL reagent. Flush column
HPLCVARIABLES
Column: A 40 jjim preparative grade C18 (Analytichem); B 75 X 2.1 pellicular C18 (Whatman)
+ 250 X 4.6 5 fmi C8 end-capped (Whatman)
Mobile phase: Gradient. A was 50 mM pH 4.5 KH2PO4. B was MeCN:isopropanol 80:20. A:B 90:
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Retention time: 8.5
OTHER SUBSTANCES
methacin, lidocaine, lorazepam, malathion, medazepam, midazolam, oxazepam, paraoxon, pen-
KEYWORDS
REFERENCE
SAMPLE
Sample preparation: Plasma. Add 500 |xL MeCN to 500 JJLL plasma while mixing on a Whirl-
mixer, rotate for 10 min, centrifuge at 1000 g for 5 min. Remove a 700 |xL aliquot of the
supernatant and ad it to 3.5 mL dichloromethane, mix for 30 s, centrifuge at 1000 g for 1 min,
inject a 20 jxL aliquot of the aqueous layer. Urine. Dilute with Sorensen buffer, inject an aliquot.
CSF. Inject an aliquot directly.
HPLC VARIABLES
Column: 100 X 3 5 jxm MOS-Hypersil C8
Mobile phase: MeCN:MeOH:buffer 12:26:62 containing 3 mM tetrabutylammonium bromide
(Buffer was 5 mM pH 5.0 sodium acetate.)
Flow rate: 1
Detector: UV 231
CHROMATOGRAM
Retention time: 3
OTHER SUBSTANCES
Extracted: probenecid
KEYWORDS
plasma
REFERENCE
van Gulpen,C; Brokerhof,A.W.; van der Kaay,M.; Tjaden,U.R.; Mattie,H. Determination of benzylpenicillin and
probenecid in human body fluids by high-performance liquid chromatography, J.Chromatogr., 1986, 381,
365-372.
SAMPLE
Sample preparation: Plasma. 50 JJLL Plasma + 50 JJLL IS solution + 50 jxL MeCN, mix for 30 s,
centrifuge at 5000 g for 15 min. Inject an aliquot of the supernatant. Urine. Mix 100 |xL IS
solution with 200 |JLL MeCN and 100 |xL urine for 30 s, centrifuge at 5000 g for 15 min. Inject
an aliquot. Tissue. Weight out finely chopped tissue and suspend it in 200 JLJLL water. Add 100
fiL 100 jjLg/mL IS, sonicate for 60 s. Add 200 [xL MeCN, vortex for 30 s, centrifuge at 10000 g
for 15 min. Inject an aliquot.
HPLC VARIABLES
Guard column: 15 X 3.2 7 |xm Newguard C18 (Alltech)
Column: 250 X 4.6 5 |xm Alltima C18 (Alltech)
Mobile phase: MeCN:50 mM pH 5.0 sodium dihydrogen phosphate 30:70
Flow rate: 1.0
Detector: UV 214
CHROMATOGRAM
Retention time: 6.4
Limit of quantitation: 800 ng/mL (plasma), 1 |xg/mL (urine), 5 jxg/g (tissue)
OTHER SUBSTANCES
Extracted: flucloxacillin
KEYWORDS
plasma; muscle; rat; pharmaeokinetics
REFERENCE
Cross,S.E.; Thompson,M.J.; Roberts,M.S. Distribution of systemically administered ampicillin, benzylpenicillin,
and flucloxacillin in excisional wounds in diabetic and normal rats and effects of local topical vasodilator
treatment, Antimicrob.Agents Chemother., 1996, 40, 1703-1710.
SAMPLE
Sample preparation: Plasma, serum. 200 JJLL Plasma or serum + 200 |xL 50 mM pH 6.0 sodium
phosphate buffer + 800 (JLL MeCN, vortex for 30 s, centrifuge at 3000 g for 10 min. Remove the
clear supernatant and add it to 2 mL dichloromethane, mix for 30 s, centrifuge at 3000 g for
10 min, inject a 25 uL aliquot of the upper aqueous layer. Urine. 100 jxL Urine + 9.9 mL 50
mM pH 6.0 sodium phosphate buffer, inject a 25 (xL aliquot.
HPLC VARIABLES
Guard column: 15 X 3.2 7 jxm Brownlee C 18 guard column
Column: 250 X 4.6 5 (xm Hypersil ODS (Keystone)
Mobile phase: Gradient. A was MeCN: 10 mM sodium phosphate adjusted to pH 2.7 with con-
centrated phosphoric acid 3:97. B was MeCN: 10 mM sodium phosphate adjusted to pH 2.7 with
concentrated phosphoric acid 90:10. A:B from 95:5 to 50:50 over 9 min and then to 95:5 over 1
min.
Flow rate: 1.5
Detector: UV 220
CHROMATOGRAM
Internal standard: penicillin G
OTHER SUBSTANCES
Extracted: piperacillin, tazobactam
Simultaneous: amoxicillin, ampicillin, cefoperazine, cefometazole, cefotaxime, cefotetan, cefu-
roxime, mezlocillin
KEYWORDS
plasma; serum; penicillin G is IS
REFERENCE
Ocampo,A.R; Hoyt,K.D.; WadgaonkaiyN.; Carver,A.H.; Puglisi,C.V. Determination of tazobactam and pipera-
cillin in human plasma, serum, bile and urine by gradient elution reversed-phase high-performance liquid
SAMPLE
Sample preparation: 300 |xL Cell suspension + 300 |JLL MeCN, vortex, centrifuge, inject a 10
|xL aliquot.
HPLC VARIABLES
Column: 80 X 4 Nucleosil 120 3 C18
Detector: UV 210
CHROMATOGRAM
Retention time: 3.1
REFERENCE
Kersten,A.; Poitschek,C; Rauch,S.; Aberer,E. Effects of penicillin, ceftriaxone, and doxycycline on morphology
oiBorrelia burgdorferi,Antimicrob.Agents Chemother., 1995, 39, 1127-1133.
SAMPLE
Matrix: cheese, milk, yogurt
MeOH, 20 mL water, and 10 mL 2% NaCl, do not allow to go dry. 5 mL Milk (or 5 g yogurt or
cottage cheese + 4 mL 1 M pH 6 phosphate buffer) + 20 jxL 20 |xg/mL penicillin V in water +
25 mL water + 4 mL 170 mM sulfuric acid + 40 mL 5% sodium tungstate, vortex for 30 s,
centrifuge at 1500 g for 10 min, remove the supernatant, add 10 mL 20% NaCl to the residue,
vortex for 10 s, centrifuge. Combine the supernatants and add them to the SPE cartridge, wash
with 10 mL 2% NaCl, wash with 10 mL water, elute with 750 JJLL MeCN:200 mM ammonium
acetate:water 60:5:35, filter (Aero 0.45 jutm), inject a 50-100 JJLL aliquot of the filtrate.
HPLC VARIABLES
Mobile phase: Gradient. A was MeCN. B was MeCN: 150 mM ammonium acetate 10:90. A:B 0:
100 for 10 min, to 30:70 over 10 min, return to initial conditions over 10 min.
Flow rate: 0.9
Detector: MS, VG Trio II, probe tip 255, source 180, thermospray/plasmaspray, m/z 335, m/z
160
CHROMATOGRAM
Internal standard: penicillin V
KEYWORDS
cow; SPE
REFERENCE
Boison,J.O.K.; Keng,L.J.-Y.; MacNeil,J.D. Analysis of penicillin G in milk by liquid chromatography, J.AOAC
Int., 1994, 77, 565-570.
SAMPLE
Sample preparation: Adjust pH of fermentation broth to 7, centrifuge at 8000 g for 10 min,
add MeCN, centrifuge, add dichloromethane to the supernatant, vortex for 10 s, shake for 15
min, centrifuge at 8000 g for 15 min. Add 1 mL of the aqueous layer to 100 JJLL reagent, heat
at 50 for 50 min, cool in an ice bath, inject a 20 |JLL aliquot. (Prepare reagent by dissolving
4.125 g imidazole in 2.5 mL water, add 1 mL HCl, add 500 |xL 110 mM mercury(II) chloride,
add 1.5 mL HCl. Recrystallize imidazole twice from isopropanol.)
HPLC VARIABLES
Guard column: 10 X 4 5 |xm Spherisorb C18
Column: 20 X 4.6 5 |xm Spherisorb C18 S5ODS2
Mobile phase: Gradient. MeCNibuffer from 16.5:83.5 to 31.5:68.5 over 17 min (Buffer was 10
mM NaH2PO4 containing 10 mM EDTA, adjusted to pH 6.5 with 2 M NaOH.)
Flow rate: 2
Detector: UV 325
CHROMATOGRAM
Retention time: 13
OTHER SUBSTANCES
Extracted: methicillin, penicillin V, penicillin X
KEYWORDS
derivatization
REFERENCE
Rogers,M.E.; Adlard,M.W.; Saunders,G.; Holt,G. High-performance liquid chromatographic determination of
penicillins following derivatization to mercury-stabilized penicillenic acids, J.Liq.Chromatogr., 1983, 6,
2019-2031.
SAMPLE
Sample preparation: Filter (0.22 |xm) fermentation broth. Mix 980 JJLL nitrate with 20 JJLL 200
mM benzoic anhydride in MeCN at room temperature for 3 min, add 100 \LL reagent, mix, heat
at 45 for 1 h, cool to room temperature, inject a 40 |xL aliquot. (Prepare reagent by dissolving
6.76 g 1-hydroxybenzotriazole hydrate in 10 mL water, add 2.5 mL mercury(II) chloride solution
(no concentration given), adjust pH to 9.2 with 4 M NaOH, make up to 25 mL.)
HPLC VARIABLES
Guard column: 10 X 4 5 |xm Spherisorb S5ODS2
Column: 259 X 4.9 5 |xm Spherisorb S5ODS2
Mobile phase: Gradient. MeCN:20 mM pH 6.5 potassium phosphate buffer:20 mM sodium thi-
osulfate from 10:45:45 to 25:37.5:37.5 over 25 min, maintain at 25:37.5:37.5 for 10 min (The
mobile phase flowed through a 50 X 4 column of 5 fjim Spherisorb S5ODS2 before the injector.)
Flow rate: 1 for 25 min then 1.2
Detector: UV 328
CHROMATOGRAM
Retention time: 24
OTHER SUBSTANCES
Extracted: 6-aminopenicillanic acid, ampicillin, penicillin K, penicillin V, penicillin X
KEY WpRDS
derivatization
REFERENCE
Shah,A.J.; Adlard,M.W.; Holt,G. Determination of natural penicillins in fermentation media by high-perfor-
mance liquid chromatography using precolumn derivatization with 1-hydroxybenzotriazole, Analyst, 1988,
113, 1197-1200.
SAMPLE
Sample preparation: Centrifuge fermentation broth at 4 at 5000 g for 20 min, filter (0.45 jxm)
a 1 mL aliquot of the supernatant, add 50 |xL 1 M NaOH to the filtrate. Remove a 50 |JLL aliquot
and add it to 50 \xL 3 mM N-dansylaziridine (Sigma) in dioxane (Caution! Dioxane is a carcin-
ogen!), heat at 100 for 30 min, cool to room temperature, inject a 20 |JLL aliquot.
HPLC VARIABLES
Guard column: 50 X 4.6 5 |xm Spherisorb C18
Mobile phase: Gradient. MeCN:20 mM pH 4.4 acetate buffer containing 0.5 mM EDTA from 19:
81 to 23:77 over 15 min, to 40:60 over 10 min, to 65:35 over 2.5 min.
Flow rate: 1
CHROMATOGRAM
Retention time: 24
OTHER SUBSTANCES
Extracted: 8-(L-a-aminoadipyl)-L-cysteinyl-D-valine, 6-aminopenicillanc acid, isopenicillin N
KEY WpRDS
derivatization
REFERENCE
Orford,C.D.; Perry,D.; Adlard,M.W. The determination of naturally produced penicillins and their biosynthetic
precursors using pre-column derivatisation with dansylaziridine, J.Liq.Chromatogr., 1991, 14, 2665-2684.
SAMPLE
with mobile phase, inject a 20 JXL aliquot.
HPLC VARIABLES
Flow rate: 1
Detector: UV 225
CHROMATOGRAM
Retention time: 6.2
OTHER SUBSTANCES
Simultaneous: amoxicillin, ampicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin,
penicillin V
KEY WORDS
REFERENCE
matography, JAssoc.OffAnal.Chem., 1984, 67, 228-231.
SAMPLE
HPLC VARIABLES
Column: 150 X 3.9 5 jxm Nova Pak C18
Mobile phase: MeOH:buffer 50:50 (Buffer was 5 mM pH 7.5 containing 1.3 mM tetrabutylam-
monium hydroxide.)
Flow rate: 0.5
Detector: UV 254
CHROMATOGRAM
Retention time: 2.4
OTHER SUBSTANCES
KEYWORDS
injections; water; stability-indicating
REFERENCE
Stiles,M.L.; Tu5YH.; Allen,L.V.,Jr. Stability of cefazolin sodium, cefoxitin sodium, ceftazidime, and penicillin G
sodium in portable pump reservoirs, Am.J.Hosp.Pharm., 1989, 46, 1408-1412.
SAMPLE
Matrix: milk
37, add 50 mL MeCN, shake vigorously for 1 min, centrifuge at 9000 g for 10 min, decant,
add 5 g NaCl, swirl to dissolve, add 100 mL dichloromethane, shake for 1 min, centrifuge at
1000 g for 10 min. Remove top aqueous layer and extract organic layer with 25 mL 10% NaCl
by shaking and centrifuging as before. Combine aqueous layers, add 1 mL 0.3% mercuric chlo-
ride in water, let stand 30 min, add 1 mL 2 M HCl, extract with three 50 mL portions of
dichloromethane by shaking each portion for 1 min and centrifuging at 1000 g for 10 min, filter
sulfate. Extract acid aqueous layer again with 25 mL dichloromethane. Combine dichloro-
methane layers and evaporate to dryness at 35 under reduced pressure. Dissolve residue in 2
mL IS solution, inject a 20 JULL aliquot. (Prepare IS solution by dissolving 10 (JLL benzaldehyde
with 5 mL 5% NaHCOa solution, filter through 10-20 g anhydrous sodium sulfate. Extract acid
HPLC VARIABLES
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: penicillin V, phenethicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin
Interfering: methicillin
KEY WpRDS
derivatization
REFERENCE
Munns,R.K.; Shimoda,W.; Roybal,J.E.; Vieira,C. Multiresidue method for determination of eight neutral (3-
971.
SAMPLE
Matrix: milk
Sample preparation: Condition a Sep-Pak C18 SPE cartridge with 20 mL MeOH, 20 mL water,
and 2 mL 2% NaCl. Pass through 30 g filtered (glass-wool plug) milk at 2 mL/min, wash with
5 mL water, wash with 10 mL MeOH:water:20% NaCl 10:80:10 containing 20 mM 18-crown-
6, elute with 10 mL 15% (v/v) MeOH, inject a 100 |xL aliquot.
HPLC VARIABLES
Guard column: 50 X 2.1 Permaphase ETH (Du Pont)
Column: 150 X 4.3 LiChrosorb RP-18
Mobile phase: MeOH:water:0.2 M pH 4.0 phosphate buffer 25:65:10 containing 11 mM sodium
1-heptanesulfonate
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Extracted: ampicillin, penicillin V
KEYWORDS
cow; SPE
REFERENCE
Terada,H.; Sakabe,Y. Studies on residual antibacterials in foods. IV. Simultaneous determination of penicillin
G, penicillin V and ampicillin in milk by high-performance liquid chromatography, J.Chromatogr., 1985,
348, 379-387.
SAMPLE
Matrix: milk
Sample preparation: 500 jxL Milk + 500 |xL MeCN:MeOH:water 40:20:40, vortex for 10-15 s,
filter (Amicon Centricon-10, 10000 dalton cut-off) while centrifuging at 2677 g for 30 min, inject
a 10-60 fxL aliquot of the ultrafiltrate.
HPLC VARIABLES
Column: 220 X 2.1 5 |jim Spheri-5 phenyl
Mobile phase: MeCN'.buffer 25:75 (Buffer was 2.5 mM octanesulfonate, 2.5 mM dodecanesulfon-
ate, 0.5% 85% phosphoric acid, and 0.5% triethylamine.)
Flow rate: 0.3-0.5
Detector: UV 210
CHROMATOGRAM
Retention time: 6.3
KEY WORDS
cow; ultrafiltrate
REFERENCE
Tyczkowska,K.; Voyksner,R.D.; Aronson,A.L. Development of an analytical method for penicillin G in bovine
milk by liquid chromatography with ultraviolet-visible detection and confirmation by mass spectrometric
SAMPLE
Matrix: milk
MeOH, 20 mL water, and 10 mL 2% NaCl, do not allow to go dry. 5 mL Milk (or 5 g yogurt or
cottage cheese + 4 mL 1 M pH 6 phosphate buffer) + 20 |xL 20 |xg/mL penicillin V in water +
25 mL water + 4 mL 170 mM sulfuric acid + 40 mL 5% sodium tungstate, vortex for 30 s,
centrifuge at 1500 g for 10 min, remove the supernatant, add 10 mL 20% NaCl to the residue,
vortex for 10 s, centrifuge. Combine the supernatants and add them to the SPE cartridge, wash
with 10 mL 2% NaCl, wash with 10 mL water, elute with 1 mL MeCN:200 mM pH 6.5 sodium
phosphate buffer:water 60:5:35. Add 1 mL reagent to the eluate, vortex for 10 s, heat at 65
for 30 min, cool to room temperature, vortex, filter (Aero 0.45 jxm), inject a 50-100 jxL aliquot
of the filtrate. (Prepare reagent by dissolving 34.45 g 1,2,4-triazole in 150 mL water, add 25
mL 10 mM mercuric chloride solution, mix, adjust pH to 9.0 0.5 with 5 M NaOH, make up
to 250 mL with water.)
HPLC VARIABLES
Mobile phase: MeCN:buffer 25:75 (Buffer was 4.696 g Na2HPO4, 8.969 g NaH2PO4-H2O, and
2.482 g anhydrous sodium thiosulfate in 1 L water.)
Flow rate: 0.8
Detector: UV 325
CHROMATOGRAM
Retention time: 5.5
Internal standard: penicillin V (7)
KEYWORDS
REFERENCE
Boison,J.O.K.; Keng,L.J.-Y.; MacNeil,J.D. Analysis of penicillin G in milk by liquid chromatography, J.AOAC
/^.,1994,77,565-570.
SAMPLE
Matrix: milk
nylon). Inject 50 JJLL onto column with mobile phase A, run mobile phase A for 30 min and elute
HPLC VARIABLES
Column: 100 X 8 Radial-Pak 10 |xm ixBondapak C18
Flow rate: A 3; B 2
0.333 s.
CHROMATOGRAM
Retention time: 9
OTHER SUBSTANCES
Simultaneous: penicillin V, ampicillin, methicillin, oxacillin, cloxacillin, nafcillin, dicloxacillin.
REFERENCE
1994, 17, 1755-1772.
SAMPLE
Matrix: milk
and two 1 mL portions of water, suck dry for 5 s. 1 mL Milk + 200 |xL water + 10 mL acetone,
mix for 10 s, centrifuge at 3000 rpm for 3 min. Remove the organic layer and evaporate it to
600 |JLL under a stream of nitrogen at 45, add 1 mL hexane, shake vigorously for 5 s, centrifuge
for 2 min, discard the hexane layer, repeat the hexane wash, evaporate the aqueous layer to
dryness, reconstitute the residue in 350 |JLL MeCN:water 20:80, add slowly to the SPE cartridge,
suck dry for 5 s, wash with two 50 |xL portions of MeCN:water 10:90, suck dry for 5 s, elute
with four 100 |xL portions of MeCN:water 20:80. Centrifuge the eluate at 3000 rpm for 3 min,
inject a 125 pX aliquot.
HPLC VARIABLES
Guard column: 20 X 4.6 5 |xm Supelcosil LC-C18 DB
Column: 250 X 4.6 5 |xm Supelcosil LC-C18 DB
Mobile phase: MeCN:buffer 34:66 (Prepare buffer by dissolving 1.78 g Na2HPO4.2H2O and 4.45
g sodium 1-heptanesulfonate in 750 mL water, adjust pH to 2.15 with 5 M phosphoric acid,
make up to 1 L with water.)
Flow rate: 0.9 for 3 min, 0.6 for 10 min, 2 for 2 min
Detector: UV 200
CHROMATOGRAM
Retention time: 12
KEYWORDS
cow; SPE
REFERENCE
Hormazal,V.; Yndestad,M. Detection of benzylpenicillin in milk by HPLC, J.Liq.Chromatogr., 1995,18, 2469-
2474.
SAMPLE
Matrix: milk
Sample preparation: 10 mL Milk + 2 mL 200 mM tetraethylammonium chloride, stir, slowly
add 38 mL MeCN over 30 s, let stand for 5 min, decant the supernatant through a plug of
glass wool. 40 mL Filtrate + 1 mL water, evaporate under reduced pressure to 1-2 mL, make
up to 4 mL with water, filter (0.45 (xm polyvinylidene difluoride). Inject 2 mL into an LC system
(150 X 4.6 5 jxm Supelcosil LC-18; MeCNiIO mM KH2PO4 0:100 for 3 min, to 40:60 over 27
min, to 0:100 over 1 min; 1 mL/min; UV 210 and 295), collect a 1.5 mL fraction at retention
time for penicillin G (23 min), evaporate to 1 mL, inject a 200 jxL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:bufifer 29:71 (Buffer was 3.3 mM phosphoric acid and 6.7 mM KH2PO4.)
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Limit of quantitation: 2-5 ppb
OTHER SUBSTANCES
Also analyzed: ampicillin, amoxicillin, cephapirin, ceftiofur, penicillin V, cloxacillin
KEYWORDS
cow
REFERENCE
Moats,W.A.; Harik-Khan,R. Liquid chromatographic determination of p-lactam antibiotics in milk: A multires-
idue approach, JAOAC Int., 1995, 78, 49-54.
SAMPLE
Matrix: milk
the residue with three 100 |xL portions of 50 mM pH 6.0 potassium phosphate buffer, filter (0.2
IJim), inject an aliquot of the filtrate. (Buffer was 545 mL 100 mM citric acid, 455 mL 200 mM
water.)
HPLC VARIABLES
Flow rate: 1
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: ampicillin, ceftiofur, cephapirin, cloxacillin, dicloxacillin, nafcillin, oxacillin
KEYWORDS
SPE
REFERENCE
p-lactams in milk by liquid chromatography with microbial receptor assay, J.AOAC Int., 1995, 78, 1165
1172.
SAMPLE
Matrix: milk
inL water, and 10 mL 2% NaCl. Centrifuge 30 mL milk at 1500 g for 10 min. Dilute a 10 mL
portion of the defatted milk with 20 mL water, add 200 jxL 2 |xg/mL penicillin V in pH 9.0
buffer, add 6 mL 170 mM sulfuric acid, add 5.6 mL 5% sodium tungstate, shake vigorously for
1 min, allow to stand for 5 min, check that the pH is in the range 4.6-4.8 (if it is outside this
range start again using a different volume of sodium tungstate solution), centrifuge at 1500 g
for 10 min, adjust the pH of the supernatant to 8.1-8.2 with 5 M and 0.1 M NaOH, filter (glass
fiber) the clear liquid phase. Pass the filtrate through the SPE cartridge at 2 mL/min, wash
with 2 mL water, dry by pulling air through the cartridge for 1 min, elute with 2 mL MeCN.
Add 150 |JLL pH 9.0 buffer to the eluate and evaporate to about 100 |xL under a stream of
nitrogen at 45-50, add 400 |xL pH 9.0 buffer, add 75 |xL reagent I, vortex for 30 s, let stand
at room temperature for 10 min, use 500 (JLL water to transfer the mixture to a separatory
funnel, add 20 mL dichloromethane, add 5 mL pH 2.45 buffer, shake for 1 min, let stand for
no more than 5 min. Remove the organic layer and evaporate it to dryness under reduced
pressure at 35-40, dissolve the residue in 500 |xL pH 9.0 buffer, add 75 (xL reagent I, vortex
for 30 s, let stand at room temperature for 10 min, add 450 JJLL reagent II, vortex for 1 min,
heat at 55 1 for 30 min, cool, filter (0.45 |Jim), inject a 150 |JLL aliquot. (Prepare pH 9.0 buffer
by dissolving 0.34 g KH2PO4 in water, adjusting the pH to 9.0 with NaOH, and making up to
100 mL with water. Prepare pH 2.45 buffer by dissolving 2.72 g KH2PO4 in water, adjusting
the pH to 2.45 with phosphoric acid, and making up to 100 mL with water. Prepare reagent 1
by dissolving 1.13 g benzoic anhydride in MeCN, make up to 25 mL with MeCN. Prepare
reagent II by dissolving 6.905 g 1,2,4-triazole in 30 mL water and adding 5 mL 26 mM mercuric
chloride in water, adjust pH to 9.0 0.05 with 5 M NaOH, make up to 50 mL. Prepare reagents
I and II 1-4 h before use. Silanize glassware with dichlorodimethylsilane.)
HPLC VARIABLES
Mobile phase: Gradient. A as MeCNibuffer 10:90. B was MeCN:buffer 30:70. A:B from 100:0 to
pH 6.5.)
Flow rate: 1
Detector: UV 323
CHROMATOGRAM
Retention time: 27
OTHER SUBSTANCES
Extracted: amoxicillin, ampicillin, cloxacillin, dicloxacillin, oxacillin
KEYWORDS
REFERENCE
Sorensen,L.K.; Rasmussen,B-M.; Boison,J.O.; Keng,L. Simultaneous determination of six penicillins in cows'
383-391.
SAMPLE
Matrix: milk, tissue
Sample preparation: Milk. Mix 10 mL milk with 2 mL 100 mM tetraethylammonium chloride,
add 40 mL MeCN slowly with continual stirring, let stand for 10 min, decant the supernatant
2 mL aliquot onto a 150 X 4.6 5 jxm Supelcosil LC-18 column, elute with MeCN: 10 mM KH2PO4
compound in a tube containing 100 JJLL Na2HPO4 (ca. 24.5 min), evaporate to <1 mL under
reduced pressure, make up to 1 mL with water, inject an aliquot. Tissue. Blend 5 g tissue, 5
mL water, 2 mL 100 mM tetraethylammonium chloride (for liver and kidney 1 mL 200 mM
tetraethylammonium chloride and 1 mL 5 mM KH2PO4), and 40 mL MeCN at half power for
1 min, let stand for 10 min, decant the supernatant through a plug of glass wool. Collect 40
mL filtrate (20 mL for liver and kidney), add 2 mL buffer, add 5 mL water, add 5 mL t-butanol,
evaporate to 1-2 mL under reduced pressure at 40-50, dilute to 4 mL with water, filter (0.45
|xm PVDF). Proceed as above. (Prepare the buffer by mixing 10 mM KH2PO4 and 10 mM
Na2HPO4 in a 5:1 ratio, pH 6.)
HPLC VARIABLES
Column: 150 X 4.6 5 |xm Supelcosil LC-18-DB (milk) or Inertsil ODS-2 (tissue)
Mobile phase: MeCN:buffer 28:72 (milk) or 30:70 (tissue) (Buffer was 3.3 mM phosphoric acid
containing 6.7 mM potassium dihydrogen phosphate.)
Flow rate: 1
Detector: UV 215
KEYWORDS
muscle; liver; kidney
REFERENCE
aid of high-performance liquid chromatographic fractionation for clean up, J.Chromatogr.A, 1998,812,237
247.
SAMPLE
Matrix: solutions
HPLCVARIABLES
Column: 100 X 4 5 |xm ODS-Hypersil
Flow rate: 2
Detector: UV 227
OTHER SUBSTANCES
Also analyzed: imipenem (UV 300)
REFERENCE
Eley,A.; Greenwood,D. Beta-lactamases of type culture strains of the Bacteroides fragilis group and of strains
that hydrolse cefoxitin, latamoxef and imipenem, J.Med.Microbiol., 1986, 21, 49-57.
SAMPLE
Matrix: solutions
Sample preparation: Prepare an aqueous solution, inject a 200 u,L aliquot.
HPLC VARIABLES
Column: 150 X 4.6 4 jim Micropak SPC-18 C18
Mobile phase: Gradient. MeCN:10 mM orthophosphoric acid from 15:85 to 60:40 over 20 min
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
Retention time: 14
OTHER SUBSTANCES
Simultaneous: dicloxacillin, methicillin, penicillin V, cloxacillin, nafcillin, carbenicillin
REFERENCE
Moats,W.A. Effect of the silica support of bonded reversed-phase columns on chromatography of some antibiotic
SAMPLE
Matrix: solutions
sodium salt soluble), inject a 10 (xL aliquot. (Preparation of l-(2,5-dihydroxyphenyl)-2-bro-
HPLC VARIABLES
Mobile phase: MeOH:100 mM pH 6.5 sodium acetate 58:42
Flow rate: 1
electrode
CHROMATOGRAM
OTHER SUBSTANCES
Extracted: carbenicillin, cephapirin, cloxacillin, dicloxacillin, hetacillin, methicillin, nafcillin,
oxacillin
KEY WpRDS
derivatization
REFERENCE
SAMPLE
Matrix: solutions
Sample preparation: Mix a 200 (xL aliquot of an aqueous solution with 30 JJLL reagent, heat at
60 for 20 min, inject an aliquot. (Reagent was imidazole:water:mercury(II) chloride 40:59.9:
0.1, adjusted to pH 6.8 with phosphoric acid.)
HPLC VARIABLES
Guard column: 40 X 4 5 jim LiChrosorb RP-18
Mobile phase: MeOH:water 45:55 containing 2% imidazole and 50 jxM mercury(II) chloride, pH
adjusted to 6.6 with phosphoric acid (At the end of each day wash the column with 30 mL
MeOH:pH 3.0 phosphate buffer (JUL = 0.1) 45:55.)
Detector: UV 325
CHROMATOGRAM
Retention time: 10
KEYWORDS
derivatization
REFERENCE
Wiese,B.; Martin,K. Basic extraction studies of benzylpenicillin and its determination by liquid chromatography
with pre-column derivatisation, J.Pharm.Biomed.Anal., 1989, 7, 6778.
SAMPLE
Matrix: solutions
10-20 fxL aliquot of the ultrafiltrate.
HPLCVARIABLES
Flow rate: 1.5
Detector: UV 220
OTHER SUBSTANCES
Also analyzed: methicillin, cefoperazone, cephalothin
REFERENCE
bumin for penicillin and cephem antibiotics, J.Pharmacobiodyn., 1992, 15, 99-106.
SAMPLE
Matrix: solutions
Sample preparation: Inject an aliquot of a 1 mg/mL solution in water.
HPLC VARIABLES
Flow rate: 1
Detector: UV 225
CHROMATOGRAM
Retention time: 17
OTHER SUBSTANCES
KEY WORDS
REFERENCE
Yongxin,Z.; Dalle,J.; Van Schepdael,A.; Roets,E.; Hoogmartens,J. Analysis of benzylpenicillin by capillary elec-
trophoresis, J.ChromatogrA, 1997, 792, 83-88.
SAMPLE
Matrix: tissue
MeOH, 20 mL water, and 10 mL 2% NaCl, do not allow to go dry. 5 g Tissue + 75 JJLL 20 ^g/
mL penicillin V in water + 20 mL water, homogenize (Polytron, 20 mm probe), rinse probe with
water so that total volume is 35 mL, shake mechanically for 5 min, add 5 mL 170 mM sulfuric
acid, add 5 mL 5% sodium tungstate, vortex for 20 s, centrifuge at 2200 g for 10 min, remove
the supernatant, add 15 mL water to the residue, shake for 5 min, centrifuge at 2200 g for 10
min. Combine the supernatants and filter (Whatman GFfB) them, add 10 mL 20% NaCl to the
filtrate, mix thoroughly, add to the SPE cartridge at 3 mL/min, wash with 10 mL 2% NaCl,
wash with 10 mL water, draw air through the cartridge for 5 min, immediately elute with 1
mL MeCN:200 mM pH 6.5 sodium phosphate buffenwater 60:5:35. Add 1 mL reagent to the
eluate, vortex, heat at 65 for 30 min, cool rapidly to room temperature, vortex, filter (Aero
0.45 |xm), inject a 80-100 JJLL aliquot of the filtrate. (Prepare reagent by dissolving 34.45 g 1,2,4-
triazole in 150 mL water, add 25 mL 10 mM mercuric chloride solution, mix, adjust pH to 9.0
0.5 with 5 M NaOH, make up to 250 mL with water.)
HPLC VARIABLES
Mobile phase: MeCN:buffer 25:75 (Buffer was 4.969 g Na2HPO4, 8.969 g NaH2PO4-H2O, and
2.482 g anhydrous sodium thiosulfate in 1 L water.)
Flow rate: 0.8
Detector: UV 325
CHROMATOGRAM
Retention time: 5.8
OTHER SUBSTANCES
Simultaneous: ampicillin, chloramphenicol
KEYWORDS
muscle; liver; kidney; derivatization; cow; SPE
REFERENCE
Boison,J.O.; Salisbury,C.D.C; Chan,W.; MacNeil,J.D. Determination of penicillin G residues in edible animal
tissues by liquid chromatography, J.Assoc.Off.Anal.Chem., 1991, 74, 497501.
SAMPLE
Matrix: tissue
Sample preparation: Blend 15 g tissue with 45 mL (60 mL for liver and kidney) water in a 300
or 500 mL blender jar at half power (or less to control foaming) for 2 min. Add a 20 mL aliquot
of homogenate to 40 mL MeCN, mix, after 5 min decant supernatant through a plug of glass
wool, collect 30 mL. Shake vigorously 30 mL filtrate, 10 mL 200 mM phosphoric acid, and 20
mL dichloromethane, remove organic layer and extract aqueous layer with 10 mL dichloro-
methane (and 10 mL MeCN for liver and kidneys). Combine dichloromethane layers, add 15
mL MeCN, add 40 mL hexane, wash the mixture twice with 4 mL portions of water, extract
the organic layer four times with 1 mL 10 mM pH 7 buffer. Combine extracts and add 0.1-0.2
mL tert-butanol, place in a rotary evaporator without heating at first. When the flask becomes
cold warm to 50, concentrate to less than 1 mL, adjust to a final volume of 1 mL, filter (Gelman
Acrodisc LCPVDF), inject a 200 jxL aliquot.
HPLC VARIABLES
Guard column: Polymer Labs guard cartridge
Column: 150 X 4.6 5 |xm 100 A PLRP-S polystyrene-divinylbenzene (Polymer Labs)
Mobile phase: MeCN:buffer 15:85, after run was over flush at 35:65 for 5 min then re-equilibrate
with 15:85 for 9 min. (Buffer was 10 mM pH 7 phosphate buffer prepared from 1.36 KH2PO4
and 2.84 g Na2HPO4 in 3 L water.)
Flow rate: 1
Detector: UV 210
CHROMATOGRAM
Retention time: 9.5
KEYWORDS
cow; pig
REFERENCE
Moats,W.A. High-performance liquid chromatographic determination of penicillin G, penicillin V and cloxacillin
in beef and pork tissues, J.Chromatogr., 1992, 593, 1520.
SAMPLE
Matrix: tissue
MeOH, 20 mL water, and 20 mL 2% NaCl. Shake 10 g tissue and 20 mL MeCN on a mechanical
shaker for 30 min, centrifuge, remove the supernatant, repeat the extraction with 20 and 10
mL portions of MeCN. Combine the extracts and add them to 30 mL 4% NaCl, remove the
MeCN under reduced pressure at 40, filter (Whatman GF/C and Gelman 0.45 jjtm membrane)
the remaining aqueous mixture, add the filtrate to the SPE cartridge at <2 mL/min, wash with
15 mL 2% NaCl, elute with 5 mL MeCN. Add 100 |xL 20 jxg/mL penicillin V in MeCN to the
eluate, evaporate to dryness under a stream of nitrogen at 37, reconstitute the residue in 1
mL water, vortex, add 1 mL 2 M pH 9 1,2,4-triazole containing 1 mM mercuric chloride, vortex,
heat at 65 for 30 min, cool, filter (0.45 |xm), inject a 50 |xL aliquot.
HPLC VARIABLES
Mobile phase: MeCN:buffer 22.5:77.5 (Prepare buffer by dissolving 4.96 g Na2HPO4, 10.14 g
NaH2PO4.2H2O, and 3.90 g sodium thiosulfate in 1 L water.)
Flow rate: 1.2
Detector: UV 325
CHROMATOGRAM
Retention time: 5
OTHER SUBSTANCES
KEYWORDS
derivatization; cow; sheep; kidney; liver; muscle; SPE
REFERENCE
Gee,H.-E.; Ho,K.-B.; Toothill,J. Liquid chromatographic determination of benzylpenicillin and cloxacillin in
animal tissues and its application to a study of the stability at -2O0C of spiked and incurred residues of
benzylpenicillin in ovine liver, J.AOAC Int., 1996, 79, 640-644.
SAMPLE
Matrix: tissue
Sample preparation: Homogenize (Ultra-Turrax) 25 g tissue with 25 mL MeCN for 1 min, add
centrifuge, inject a 200 |JLL aliquot on to column A and elute to waste with mobile phase B,
HPLC VARIABLES
Column: A 4 X 4 5 (Jim LiChrospher 100 RP-18e; B 250 X 4 5 |xm LiChrospher 100 RP-18e
Mobile phase: A MeCN:water 50:50; B 20 mM pH 7 phosphate buffer; C MeCN:20 mM pH 3
disodium EDTA
Flow rate: 1 (except where indicated)
CHROMATOGRAM
Retention time: 5.2
OTHER SUBSTANCES
Extracted: cloxacillin, dicloxacillin, oxacillin, penicillin V
KEYWORDS
REFERENCE
J.ChromatognA, 1996, 729, 229-235.
Penicillin V
CAS Registry No.: 87-08-1, 132-98-9 (potassium salt),
5928-84-7 (benzathine), 63690-57-3 (benzathine tetrahydrate), 6591-72-6 (hydrabamine)
Merck Index: 7230
LednicerNo.: 7230
SAMPLE
Matrix: bulk
Sample preparation: Dissolve a 50 mg sample in 50 mL 50 mM pH 6.5 potassium dihydrogen
phosphate buffer. Inject a 20 JULL aliquot.
HPLC VARIABLES
Mobile phase: MeOH:water:500 mM pH 3.5 phosphate buffer 39:51.2:9.8
Flow rate: 1.0
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: 4-hydroxyphenoxymethylpenicillin
KEY WORDS
use gradient to determined impurities; details for HPLC
REFERENCE
Yongxin,Z.; Roets,E.; Trippen,B.; Christiansen,C.-P.; Arevalo,M.P.; Porqueras,E.; Maichel,B.; Inama,P; Soder-
holm,S.; Miller,J.H.MB.; Spieser,J.M.; Hoogmartens,J. Interlaboratory study of analysis of phenoxymethyl-
penicillin by liquid chromatography, Chromatographia, 1998, 47, 152156.
SAMPLE
Matrix: bulk
Sample preparation: Inject an aliquot of a 1 mg/mL solution.
HPLC VARIABLES
Column: 250 X 4.65 |xm Hypersil C18
Detector: UV 254
CHROMATOGRAM
Limit of detection: 11.8 pg
Limit of quantitation: 23.6 pg
OTHER SUBSTANCES
KEYWORDS
REFERENCE
Zhu,Y; Van Schepdael,A.; Roets,E.; Hoogmartens,J. Micellar electrokinetic capillary chromatography for the
separation of phenoxymethylpenicillin and related substances, J.Chromatogr.A, 1997, 781, 417-422.
SAMPLE
Matrix: milk
2 mL aliquot onto a 150 X 4.6 5 jxm Supelcosil LC-18 column, elute with MeCNiIO mM KH2PO4
compound, evaporate to <1 mL under reduced pressure, make up to 1 mL with water, inject
an aliquot. (Prepare the buffer by mixing 10 mM KH2PO4 and 10 mM Na2HPO4 in a 5:1 ratio,
pH6.)
HPLC VARIABLES
Mobile phase: MeCN:buffer 33:67 (Buffer was 5 mM phosphoric acid containing 5 mM potassium
Flow rate: 1
Detector: UV 215
REFERENCE
aid of high-performance liquid chromatographic fractionation for clean up, J.ChromatogrA, 1998,812,237-
247.
SAMPLE
Matrix: milk
mL water, and 10 mL 2% NaCl. Centrifuge 30 mL milk at 1500 g for 10 min. Dilute a 10 mL
portion of the defatted milk with 20 mL water, add 200 |xL pH 9.0 buffer, add 6 mL 170 mM
sulfuric acid, add 5.6 mL 5% sodium tungstate, shake vigorously for 1 min, allow to stand for
5 min, check that the pH is in the range 4.6-4.8 (if it is outside this range start again using a
different volume of sodium tungstate solution), centrifuge at 1500 g for 10 min, adjust the pH
of the supernatant to 8.1-8.2 with 5 M and 0.1 M NaOH, filter (glass fiber) the clear liquid
phase. Pass the filtrate through the SPE cartridge at 2 mL/min, wash with 2 mL water, dry
by pulling air through the cartridge for 1 min, elute with 2 mL MeCN. Add 150 |xL pH 9.0
buffer to the eluate and evaporate to about 100 (xL under a stream of nitrogen at 45-50, add
400 |xL pH 9.0 buffer, add 75 |xL reagent I, vortex for 30 s, let stand at room temperature for
10 min, use 500 (xL water to transfer the mixture to a separatory funnel, add 20 mL dichlo-
romethane, add 5 mL pH 2.45 buffer, shake for 1 min, let stand for no more than 5 min. Remove
the organic layer and evaporate it to dryness under reduced pressure at 35-40, dissolve the
residue in 500 |xL pH 9.0 buffer, add 75 |xL reagent I, vortex for 30 s, let stand at room
temperature for 10 min, add 450 jutL reagent II, vortex for 1 min, heat at 55 1 for 30 min,
cool, filter (0.45 jxm), inject a 150 |xL aliquot. (Prepare pH 9.0 buffer by dissolving 0.34 g
KH2PO4 in water, adjusting the pH to 9.0 with NaOH, and making up to 100 mL with water.
Prepare pH 2.45 buffer by dissolving 2.72 g KH2PO4 in water, adjusting the pH to 2.45 with
phosphoric acid, and making up to 100 mL with water. Prepare reagent 1 by dissolving 1.13 g
benzoic anhydride in MeCN, make up to 25 mL with MeCN. Prepare reagent II by dissolving
6.905 g 1,2,4-triazole in 30 mL water and adding 5 mL 26 mM mercuric chloride in water,
adjust pH to 9.0 0.05 with 5 M NaOH, make up to 50 mL. Prepare reagents I and II 1-4 h
before use. Silanize glassware with dichlorodimethylsilane.)
HPLCVARIABLES
Column: 150 X 3.9 4 jjum Nova-Pak C18
Mobile phase: Gradient. A as MeCN:buffer 10:90. B was MeCNibuffer 30:70. A:B from 100:0 to
pH 6.5.)
Flow rate: 1
Detector: UV 323
CHROMATOGRAM
Internal standard: penicillin V
OTHER SUBSTANCES
Extracted: amoxicillin, ampicillin, cloxacillin, dicloxacillin, oxacillin, penicillin G
KEYWORDS
derivatization; cow; SPE; penicillin V is IS
REFERENCE
Sorensen,L.K; Rasmussen,B-M.; Boison,J.O.; Keng,L. Simultaneous determination of six penicillins in cows'
383-391.
SAMPLE
Matrix: tissue
Sample p r e p a r a t i o n : Homogenize (Ultra-Turrax) 25 g tissue with 25 mL MeCN for 1 min, add
centrifuge, inject a 200 JJLL aliquot on to column A and elute to waste with mobile phase B,
HPLC VARIABLES
Column: A 4 X 4 5 |xm LiChrospher 100 RP-18e; B 250 X 4 5 |xm LiChrospher 100 RP-18e
Mobile p h a s e : A MeCN:water 50:50; B 20 mM pH 7 phosphate buffer; C MeCN:20 mM pH 3
disodium EDTA
Flow r a t e : 1 (except where indicated)
CHROMATOGRAM
OTHER SUBSTANCES
E x t r a c t e d : cloxacillin, dicloxacillin, oxacillin, penicillin G
KEY WORDS
REFERENCE
J.ChromatogrA, 1996, 729, 229-235.
Pentaerythritol tetranitrate
Merck Index: 7249
SAMPLE
Sample preparation: Weigh out amount of bulk drug or powdered tablets or capsules equivalent
to about 25 mg pentaerythritol tetranitrate, add 125 mL mobile phase, if clumping occurs
sonicate for 5 min, shake for 30 min, add 5 mL IS solution, dilute to 250 mL with mobile phase,
filter (0.45 jxm), inject a 20 |xL aliquot. (Prepare IS solution by adding 10 g 10% nitroglycerin
solution in lactose to 125 mL MeOH, sonicate for 5 min, shake mechanically for 30 min, dilute
to 200 mL with MeOH, let undissolved lactose settle, and filter through paper.)
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Ultrasphere ODS C18
Flow rate: 1
Detector: UV 230
CHROMATOGRAM
Retention time: 7.8
KEYWORDS
tablets; capsules; collaborative study
REFERENCE
Carlson,M. Liquid chromatographic determination of pentaerythritol tetranitrate in Pharmaceuticals: collab-
orative study, J.Assoc.Off.Anal.Chem., 1990, 73, 693-697.
SAMPLE
Sample preparation: Powder tablets, weigh out a portion equivalent to 2 mg pentaerythritol
tetranitrate, add to 10 mL 75 |xg/mL nitroglycerin in MeOH, sonicate for 2 min, shake me-
chanically for 30 min, filter, inject an aliquot
HPLC VARIABLES
Flow rate: 1
Detector: UV 220
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: isosorbide dinitrate, erythrityl tetranitrate
KEYWORDS
tablets
REFERENCE
J.Pharm.ScL, 1984, 73, 1303-1304.
SAMPLE
in MeOH, make up to 100 mL with buffer, filter (0.45 |xm), inject a 20 |xL aliquot. If the sample
200 mM ammonium acetate buffer:water 55:10:35.)
Next Page
HPLCVARIABLES
Guard column: 50 X 6.4 25-37 |jim Whatman Co-Pell ODS
Mobile phase: MeOH:200 mM ammonium acetate bufferrwater 55:10:35
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: isosorbide mononitrate, saccharin, isosorbide dinitrate
KEYWORDS
tablets; capsules
REFERENCE
Carlson,M.; Thompson,R-Eh; Snell,R.P. Determination of isosorbide dinitrate in pharmaceutical products by
Pentamidine
CAS Registry No.: 100-33-4, 6823-79-6 (dimethanesulfonate), 140-64-7 (isethionate)
Merck Index: 7254
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 50 |JLL 4.92 |jig/mL hexamidine, vortex briefly, add 500 u>L
2 M NaOH, vortex, add 500 |JLL 2 M HCl, vortex, add 1 mL pH 10 carbonate buffer, vortex,
add 4 mL 20 mM di(2-ethylhexyl)phosphoric acid in chloroform, vortex for 1 min, centrifuge at
700 g for 15 min. Remove the chloroform layer and add it to 1 mL 20 mM HCl, vortex for 1
min. Remove the aqueous layer and adjust the pH to 12 with 4 drops 2 M NaOH, add 2 mL
dichloromethane, vortex for 1 min. Remove the organic layer and evaporate it to dryness under
a stream of nitrogen at 35, reconstitute the residue in 100 (xL solvent, vortex for 30 s, inject
a 20 |JLL aliquot. (Solvent was MeOHibuffer 50:50. Buffer was 50 mM sodium heptanesulfonate
containing 0.4% triethylamine, pH adjusted to 3.0 with phosphoric acid.)
HPLC VARIABLES
Column: 150 X 2.1 5 jxm solvent miser C18 (Alltech)
Mobile phase: MeOHrbuffer 60:40 (Buffer was 50 mM sodium heptanesulfonate containing 14
mM triethylamine, pH adjusted to 3.0 with phosphoric acid.)
Flow rate: 0.3
Detector: UV 280
CHROMATOGRAM
Retention time: 5.7
Internal standard: hexamidine (8.2)
KEYWORDS
serum; dog; human; pharmacokinetics
Previous Page
HPLCVARIABLES
Guard column: 50 X 6.4 25-37 |jim Whatman Co-Pell ODS
Mobile phase: MeOH:200 mM ammonium acetate bufferrwater 55:10:35
Flow rate: 1
Detector: UV 254
CHROMATOGRAM
OTHER SUBSTANCES
Simultaneous: isosorbide mononitrate, saccharin, isosorbide dinitrate
KEYWORDS
tablets; capsules
REFERENCE
Carlson,M.; Thompson,R-Eh; Snell,R.P. Determination of isosorbide dinitrate in pharmaceutical products by
Pentamidine
CAS Registry No.: 100-33-4, 6823-79-6 (dimethanesulfonate), 140-64-7 (isethionate)
Merck Index: 7254
SAMPLE
Matrix: blood
Sample preparation: 1 mL Serum + 50 |JLL 4.92 |jig/mL hexamidine, vortex briefly, add 500 u>L
2 M NaOH, vortex, add 500 |JLL 2 M HCl, vortex, add 1 mL pH 10 carbonate buffer, vortex,
add 4 mL 20 mM di(2-ethylhexyl)phosphoric acid in chloroform, vortex for 1 min, centrifuge at
700 g for 15 min. Remove the chloroform layer and add it to 1 mL 20 mM HCl, vortex for 1
min. Remove the aqueous layer and adjust the pH to 12 with 4 drops 2 M NaOH, add 2 mL
dichloromethane, vortex for 1 min. Remove the organic layer and evaporate it to dryness under
a stream of nitrogen at 35, reconstitute the residue in 100 (xL solvent, vortex for 30 s, inject
a 20 |JLL aliquot. (Solvent was MeOHibuffer 50:50. Buffer was 50 mM sodium heptanesulfonate
containing 0.4% triethylamine, pH adjusted to 3.0 with phosphoric acid.)
HPLC VARIABLES
Column: 150 X 2.1 5 jxm solvent miser C18 (Alltech)
Mobile phase: MeOHrbuffer 60:40 (Buffer was 50 mM sodium heptanesulfonate containing 14
mM triethylamine, pH adjusted to 3.0 with phosphoric acid.)
Flow rate: 0.3
Detector: UV 280
CHROMATOGRAM
Retention time: 5.7
Internal standard: hexamidine (8.2)
KEYWORDS
serum; dog; human; pharmacokinetics
REFERENCE
Dickinson,C.M.; Navin,T.R.; Churchill,F.C. High-performance liquid chromatographic method for quantitation
of pentamidine in blood serum, J.Chromatogr., 1985, 345, 91-97.
SAMPLE
Matrix: blood
Sample preparation: Condition an SPE-C8 SPE cartridge (Jones Chromatography) with two 1
mL aliquots of MeOH, two 1 mL aliquots of MeCN: 1 M pH 3 ammonium acetate 75:25, and
five 1 mL aliquots of water, do not allow to dry. 100 |xL Plasma + 100 u,L water, mix, add to
the SPE cartridge, wash with three 1 mL aliquots of MeOH, add 80 |xL 500 ng/mL melphalan
in MeOH to the SPE cartridge, elute with 1 mL MeCN:l M pH 3 ammonium acetate 75:25.
Evaporate the eluate to dryness under reduced pressure, reconstitute in 200 JJLL mobile phase,
HPLC VARIABLES
Column: 150 X 3.9 5 (xm Resolve C18 (Waters)
Mobile phase: MeCN:MeOH:triethylamine:200 mM ammonium acetate 18:2:0.5:79.5, pH ad-
justed to 3.8 with acetic acid
Flow rate: 1.4
CHROMATOGRAM
Retention time: 5.5
Internal standard: melphalan (10)
KEYWORDS
plasma; rat; SPE; pharmacokinetics
REFERENCE
Yeh,T.-K; Dalton,J.T.; Au,J.L.-S. High-performance liquid chromatographic determination of pentamidine in
plasma, J.Chromatogr., 1993, 622, 255-261.
SAMPLE
Matrix: blood
Sample preparation: Let blood stand at 4 for 4 h, centrifuge at 15000 g for 10 min, add sodium
azide to a concentration of 0.01%, inject a 25 jxL aliquot.
HPLC VARIABLES
Column: 250 X 4.6 5 |xm Zorbax RK-C8
Mobile phase: Gradient. A was 4.2 mM phosphoric acid containing 10 mM sodium heptanesul-
fonate and 10 mM tetramethylammonium chloride. B was MeCN:water 75:25 containing 4.2
mM phosphoric acid, 10 mM sodium heptanesulfonate, and 10 mM tetramethylammonium
chloride. A:B from 90:10 to 10:90 over 30 min, return to initial conditions over 7 min, re-
equilibrate for 3 min.
Flow rate: 1.5
Detector: UV 265
CHROMATOGRAM
Retention time: 18
Internal standard: pentamidine
OTHER SUBSTANCES
Extracted: guanylhydrazones
KEYWORDS
serum; mouse; pentamidine is IS; rat
REFERENCE
CeramijC; Zhang,X.; Ulrich,R; Bianchi,M.; Tracey,K.J.; Berger,B.J. High-performance liquid chromatographic
method for guanylhydrazone compounds, J.Chromatogr.B, 1996, 675, 71-75.
SAMPLE
Matrix: blood, broncho-alveolar lavage fluid, cells, urine
Sample preparation: Plasma. 0.5 mL Plasma + 1 mL 30 ng/mL hexamidine in MeCN, vortex,
centrifuge at 1000 g for 10 min, add the supernatant to a Bond Elut C8 S

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Epidermal growth factor

Erythrityl tet ran it rate

Molecular formula: C18H18O2 (equilenin), C18H22O2 (17a-dihydroequilin), C18H20O2 (equilin), C18H22O2

Limit of detection: 5 ng/mL

Limit of detection: 5 ng/mL

stilbene, imipramine, indomethacin, isocarbostyril, isoproterenol, isoxsuprine, ivermectin, ke-

stilbene, imipramine, indomethacin, isocarbostyril, isoproterenol, isoxsuprine, ivermectin, ke-

Noninterfering: dopamine, levodopa, methyldopa, methyldopate, norepinephrine

Flow rate: 1.5

Flow rate: 1.5

phesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine,

phesin, mephobarbital, mepivacaine, mescaline, mesoridazine, methadone, methamphetamine,

Noninterfering: diclofenac, felbinac, fenbufen, indomethacin, loxoprofen, naproxen, piroxicam,

Noninterfering: acetaminophen, aspirin, caffeine, diflunisal, fenoprofen, ibuprofen, indometha-

Noninterfering: acetaminophen, aspirin, caffeine, diflunisal, fenoprofen, ibuprofen, indometha-

Column: 250 X 4.6 5 Jim Symmetry C8 (Waters)

Column: 250 X 4.6 5 Jim Symmetry C8 (Waters)

ytetracycline, papaverine, pargyline, pemoline, pentazocine, pentobarbital, persantine, phe-

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