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Theophylline
Molecular formula: C7H8N4O2
Molecular weight: 180.2
CAS Registry No.: 58-55-9, 32156-80-2 (diethanolamine), 573-41-1 (ethanolamine),
5600-19-1 (isopropanolamine), 8002-89-9 (sodium acetate), 8000-10-1 (sodium
glycinate), 5967-84-0 (monohydrate)

SAMPLE
Matrix: bile, blood, tissue, urine
Sample preparation: Tissue. Macerate 2 g tissue with 5 mL water, add 15 mL MeCN, add
0.1 mL 1 mg/mL 2-acetamidophenol in ethanol, shake, centrifuge at 5200 g. Transfer
supernatant to 50 mL tube containing 8 mL diethyl ether + 12 mL dichloromethane + 1
mL citrate buffer, vortex, proceed as in (A). Bile. 2 mL Bile + 3 mL water, add 15 mL
MeCN, add 0.1 mL 1 mg/mL 2-acetamidophenol in ethanol, vortex, add 8 mL diethyl ether
+ 12 mL dichloromethane, vortex, centrifuge at 5200 g, proceed as in (A). Blood, urine.
5 mL Blood or urine + 15 mL acetone + 0.1 mL 1 mg/mL 2-acetamidophenol in ethanol,
vortex for a few s, add 8 mL diethyl ether, vortex, centrifuge 5200 g. Transfer supernatant
to 50 mL tube, add 12 mL dichloromethane, add 1 mL citrate buffer, vortex, proceed as
in (A). (A). Discard lower, aqueous layer. Filter the organic layer through 3 g Florisil +
8 g anhydrous sodium sulfate and wash through with 15 mL diethyl ether. Evaporate
nitrate to dryness under a stream of air at 40. Reconstitute in 5 mL MeCN: 0.1 N
NaH2PO4 60:40 + 3 mL hexane, vortex. Remove and discard upper hexane layer, add 8
mL 20% (v/v) isopropanol in chloroform to the aqueous layer, vortex. Remove and discard
the upper aqueous layer and evaporate lower layer. Reconstitute residue in MeCN: water
10:90, inject an aliquot. (Citrate buffer was saturated sodium citrate containing enough
sodium tunstate (sic) to bring pH to 8. To each 1 L of diethyl ether 10 mL of water and
1 mL of citrate buffer are added.)

HPLCVARIABLES
Column: 100 X 4.6 C18 microbore
Mobile phase: MeCN: dilute phosphoric acid (1 mL 85% phosphoric acid in 140 mL water)
7:93
Column temperature: 40
Flow rate: 0.3
Detector: UV 271

CHROMATOGRAM
Retention time: 3.2
Internal standard: 2-acetamidophenol (4.9)
Limit of detection: 50 (blood, urine); 200 (bile, tissue) ng/mL

OTHER SUBSTANCES
Extracted: acetaminophen

KEYWORDS
liver; whole blood

REFERENCE
Mathis, D.F.; Budd, R.D. Extraction of acetaminophen and theophylline from post-mortem tissues and
urine for high-performance liquid chromatographic analysis. J.Chromatogr., 1988, 439, 466-469

SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 10 |xL 40 |xg/mL p-hydroxyethyltheophylline + 10
imL perchloric acid, vortex for 10 s, centrifuge for 5 min, inject a 20 JULL aliquot of the
supernatant.

HPLCVARIABLES
Column: 5 |xm C18 Radpak (Waters)
Mobile phase: MeCN:MeOH: 10 mM KH2PO4:triethylamine 5:7:88:0.02, pH 4.5
Flow rate: 3
Injection volume: 20
Detector: UV 273

CHROMATOGRAM
Retention time: 5.5
Internal standard: p-hydroxyethyltheophylline (7.5)
Limit of quantitation: 625 ng/mL

KEYWORDS
plasma; rat; pharmacokinetics

REFERENCE
Angus, RW.; Ng, CY.; Ghabrial, H.; Morgan, D.J.; Smallwood, R.A. Effects of chronic left ventricular
failure on hepatic oxygenation and theophylline elimination in the rat. Drug Metab.Dispos., 1995,
23, 485-489

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma + 10 |xL 2 mg/mL IS in MeOH, mix, add 2 mL
dichloromethane: diethyl ether 80:20, vortex for 15 s, centrifuge at 1500 g for 5 min,
remove a 1.7 mL aliquot of the organic phase, repeat the extraction twice more with 2
mL portions of dichloromethane: diethyl ether 80:20. Combine the organic layers and
evaporate them to dryness under a stream of nitrogen at room temperature, reconstitute
the residue in 200 (xL mobile phase, inject a 20 |xL aliquot.

HPLCVARIABLES
Guard column: 20 X 4.6 40 |xm Pelliguard C18 (Supelco)
Column: 250 X 4.6 5 |xm Viosfer C18 (Violet, Rome)
Mobile phase: MeCN: buffer 15:85 (Buffer was 100 mM phosphate buffer containing 5 mM
tetrabutylammonium hydrogen sulfate, adjusted to pH 3 with orthophosphoric acid.)
Flow rate: 1.3
Injection volume: 20
Detector: UV 280

CHROMATOGRAM
Retention time: 4.8
Internal standard: 2-[4-(2'-furoyl)phenyl]propionicacid (7.3)
Limit of detection: 20 ng/mL

OTHER SUBSTANCES
Extracted: rufloxacin

KEYWORDS
plasma

REFERENCE
Carlucci, G.; Mazzeo, P.; Palumbo, G. Simultaneous determination of rufloxacin and theophylline by
high-performance liquid chromatography in human plasma. Analyst, 1995, 120, 2493-2495
SAMPLE
Matrix: blood
Sample preparation: Add 200 |xL whole blood to 400 |xL MeCN while mixing rapidly for
5 s, centrifuge at 1500 rpm for 2 min, mix 100 |xL supernatant with 1 mL water, inject
a 50 |xL aliquot.

HPLCVARIABLES
Column: 50 X 4.6 Little Champ reverse phase (Regis)
Mobile phase: MeCN: 10 mM pH 3 sodium phosphate buffer 4:96
Flow rate: 1
Injection volume: 50
Detector: UV 273

CHROMATOGRAM
Limit of quantitation: 500 nM

KEYWORDS
rat; whole blood

REFERENCE
Hoffman, D.J.; Seifert, T.; Borre, A.; Nellans, H.N. Method to estimate the rate and extent of intestinal
absorption in conscious rats using an absorption probe and portal blood sampling. Pharm.Res., 1995,
12, 889-894

SAMPLE
Matrix: blood
Sample preparation: 100 |xL Sample + 300 |xL 7-(2-hydroxyethyl)theophylline in
chloroform: isopropanol 50:50, extract. Remove 200 |xL of the organic layer and evaporate
it to dryness, reconstitute the residue in mobile phase, inject a 20 JJLL aliquot.

HPLCVARIABLES
Column: Bio-Sil ODS-5S
Mobile phase: MeCNrTHF: 10 mM pH 4.75 sodium acetate 2:1:97
Detector: UV 273

CHROMATOGRAM
Internal standard: 7-(2-hydroxyethyl)theophylline
Limit of detection: 500 ng/mL

OTHER SUBSTANCES
Extracted: paraxanthine
Noninterfering: cephalosporins

REFERENCE
Leonard, H.; Campbell, J.; Malliaros, D.; Berg, M.; Houser, S.; McLaughlin, L. Comparison of a theoph-
ylline HPLC reference method with an automated chemiluminescent immunoassay (Abstract 122).
Ther.Drug Monit, 1995, 17, 413

SAMPLE
Matrix: blood
Sample preparation: Extract 250 |xL plasma with chloroform: isopropanol 85:15. Remove
the organic layer and evaporate it to dryness, reconstitute the residue in mobile phase,
inject an aliquot.
HPLCVARIABLES
Column: 100 X 4.6 microsphere C18 (Chrompack)
Mobile phase: MeCN: 55 mM pH 4.0 sodium acetate buffer 6:94
Flow rate: 1.2
Detector: UV 278

CHROMATOGRAM
Internal standard: theophylline

OTHER SUBSTANCES
Extracted: caffeine

KEYWORDS
plasma; pig; theophylline is IS

REFERENCE
Monshouwer, M.; Witkamp, R.F.; Nijmeijer, S.M.; Pijpers, A.; Verheijden, J.H.M.; Van Miert,
A.S.J.P.A.M. Selective effects of a bacterial infection (Actinobacillus pleuropneumoniae) on the he-
patic clearance of caffeine, antipyrine, paracetamol, and indocyanine green in the pig. Xenobiotica,
1995,25,491-499

SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 50 |xL 20 |jig/mL p-hydroxyethyltheophylline in
MeOH + 200 |xL 67 mM pH 8 phosphate buffer + 5 mL chloroform, shake for 10 min,
centrifuge at 850 g 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 |xL mobile
phase, inject a 20 fxL aliquot.

HPLCVARIABLES
Column: reverse-phase
Mobile phase: MeCN: 20 mM pH 5 acetate buffer 7:93
Flow rate: 1.5
Injection volume: 20
Detector: UV 275

CHROMATOGRAM
Internal standard: p-hydroxyethyltheophylline

KEYWORDS
plasma; rat; pharmacokinetics

REFERENCE
Nagai, N.; Furuhata, M.; Ogata, H. Drug interactions between theophylline and H2-antagonists, roxa-
tidine acetate hydrochloride and cimetidine: Pharmacokinetic analysis in rats in vivo.
Biol.Pharm.BulL, 1995, 18, 1610-1613

SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma + 10 jxL 40 |xg/mL p-hydroxyethyltheophylline +10
IxL perchloric acid, vortex for 10 s, centrifuge for 5 min, inject a 20 |xL aliquot of the
supernatant.

HPLCVARIABLES
Column: 5 jim Radpak C18 (Waters)
Mobile phase: MeCN:MeOH:buffer 5:7:88 (Buffer was 10 mM KH2PO4 containing 0.02%
triethylamine, pH 4.5.)
Injection volume: 20
Detector: UV 273

CHROMATOGRAM
Retention time: 5.5
Internal standard: p-hydroxyethyltheophylline (7.5)
Limit of quantitation: 625 ng/mL

KEYWORDS
rat; plasma

REFERENCE
Ng, CY; Angus, RW.; Ghabrial, H.; Chou, S.T.; Arnolda, L.; Morgan, D.J.; Smallwood, R.A. Right heart
failure impairs hepatic oxygenation and theophylline clearance in rats. J.Pharm.Exp.Ther., 1995,
273, 1332-1336

SAMPLE
Matrix: blood
Sample preparation: Filter (0.22 |xm), inject a 20 jxL aliquot of the filtrate.

HPLCVARIABLES
Column: 250 X 4 5 |jim LiChrospher 100 Diol
Mobile phase: MeCN: 50 mM pH 6.9 phosphate buffer 1.8:98.2
Flow rate: 0.6
Injection volume: 20
Detector: UV 254

CHROMATOGRAM
Retention time: 5.5

OTHER SUBSTANCES
Extracted: caffeine

KEYWORDS
serum; direct injection

REFERENCE
Nimura, N.; Itoh, H.; Kinoshita, T. Diol-bonded silica gel as a restricted access packing forming a binary-
layered phase for direct injection of serum for the determination of drugs. J.Chromatogr.A, 1995,
689, 203-210

SAMPLE
Matrix: blood
Sample preparation: 25 |xL Serum + 100 JULL 250 ng/mL p-hydroxyethyltheophylline in
water + 200 p,L 200 mM pH 6.0 phosphate buffer + 3 mL dichloromethane, shake at 120
oscillations/min for 20 min, centrifuge at 174 g. Remove the organic layer and evaporate
it to dryness under a stream of nitrogen at 60, reconstitute the residue in 230 (JLL water,
heat at 90 for 6 min, vortex, inject a 180 JJLL aliquot. (No details given for milk extraction.)

HPLCVARIABLES
Guard column: Corasil Bondapak C18
Column: 5 \xm radial-compression C18 (Waters)
Mobile phase: MeOH:THF: 10 mM KH2PO4 9:1:90, adjusted to pH 3.5
Flow rate: 1.2
Injection volume: 180
Detector: UV 214

CHROMATOGRAM
Retention time: 9.4
Internal standard: p-hydroxyethyltheophylline (10.9)

OTHER SUBSTANCES
Extracted: caffeine, paraxanthine, theobromine

KEY WORDS
serum; pharmacokinetics

REFERENCE
Oo, CY; Burgio, D.E.; Kuhn, R.C.; Desai, N.; McNamara, RJ. Pharmacokinetics of caffeine and its
metabolites in lactation: Prediction of milk to serum concentration ratios. Pharm.Res., 1995, 12,
313-316

SAMPLE
Matrix: blood
Sample preparation: 200 |xL Serum + 10 |xL 500 ng/mL theophylline in water + 50 JULL
isoamyl alcohol, vortex for 30 s, add 2 mL chloroform, vortex for 1 min, centrifuge at 1000
rpm for 5 min. Remove 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 50 |xL aliquot.

HPLCVARIABLES
Column: 150 X 3.9 5 ^m Novapak C18
Mobile phase: MeCN: 0.2% acetic acid 7.5:92.5 (After 4 min increase flow to 1.8 mL/min
over 1 min, maintain at 1.8 mL/min for 2 min.)
Flow rate: 0.8
Injection volume: 50
Detector: UV 270

CHROMATOGRAM
Retention time: 3.4
Internal standard: zidovudine (6.0)
Limit of detection: 20 ng/mL

KEYWORDS
rat; serum; pharmacokinetics

REFERENCE
Radwan, M.A. HPLC assay of theophylline and zidovudine in rat serum. J.Liq.Chromatogr., 1995, 18,
3301-3309

SAMPLE
Matrix: blood
Sample preparation: Wash PCPure cartridge containing 0.4 g hydroxyapatite with 10 mL
MecN and remove MeCN by evaporation. 75 jxL Plasma + 25 |xL of 50 |xg/mL IS in 0.5%
aqueous MeCN injected onto PCPure cartridge, elute with MeCN: water 10:90. Use first
600 |xL of eluate, inject 20 |xL aliquots.

HPLCVARIABLES
Column: 150 X 4.6 5 \xm Inertsil ODS-2
Mobile phase: MeCN: water 5:95
Column temperature: 40
Flow rate: 1
Injection volume: 20
Detector: UV 280

CHROMATOGRAM
Retention time: 7
Internal standard: 7-(2-hydroxyethyl)theophylline (10)
Limit of detection: 2.9 ng

OTHER SUBSTANCES
Simultaneous: caffeine

KEYWORDS
plasma

REFERENCE
Iwase, H.; Gondo, K.; Koike, T.; Ono, I. Novel precolumn deproteinization method using a hydroxyapatite
cartridge for the determination of theophylline and diazepam in human plasma by high-performance
liquid chromatography with ultraviolet detection. J.Chromatogr.B, 1994, 655, 7381

SAMPLE
Matrix: blood
Sample preparation: 100 JJLL Plasma + 100 |xL 20 |xg/mL caffeine + 8 mL dichlorome-
thane, 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 jxL
mobile phase, inject a 20 |xL aliquot.

HPLCVARIABLES
Column: 150 X 6 Shimpack CLS-ODS (Shimadzu)
Mobile phase: MeCN: 0.5 mM phosphoric acid 10:90
Column temperature: 40
Flow rate: 1.5
Injection volume: 20
Detector: UV 273

CHROMATOGRAM
Internal standard: caffeine

KEYWORDS
plasma; rat

REFERENCE
Lee, CK.; 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 |xL Plasma + 100 JULL pH 7.4 phosphate buffer + 50 \xL 20 |xg/mL
p-hydroxypropyltheophylline in pH 7.4 phosphate buffer + 5 mL chloroform: isopropanol
95:5, 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 |JLL
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 immedi-
ately before use (Buffer was 4.54 g KH2PO4 + 5.94 g Na2HPO4.2H2O + 1.49 g tetrabu-
tylammonium hydrogen sulfate per L.)
Flow rate: 1.3
Injection volume: 25
Detector: UV 280

CHROMATOGRAM
Retention time: 3.3
Internal standard: p-hydroxypropyltheophylline
Limit of detection: 500 ng/mL

OTHER SUBSTANCES
Extracted: ciprofloxacin, enoxacin, 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: 200 |xL Serum + 200 JJLL 20 |xg/mL antipyrine in mobile phase, filter
(Millipore Millex 0.45 |xm), inject a 25 jxL aliquot.

HPLCVARIABLES
Guard column: 40 X 4 C18 Corasil II
Column: 300 X 4 10 |xm (xBondapak phenyl
Mobile phase: Propanol:6 mM C12 DAPS (Fluka) 3:97 (C12 DAPS is 3-(dimethyldodecylam-
monio) propanesulfonate.)
Injection volume: 25
Detector: UV 273

CHROMATOGRAM
Retention time: 5
Internal standard: antipyrine (8)
Limit of detection: 500 ng/mL

OTHER SUBSTANCES
Simultaneous: albendazole, albendazole sulfoxide, aminophylline, amyleine, caffeine, flu-
bendazole, p-hydroxytheophylline, mercaptopurine, nimorazole, procaine, theobromine
Interfering: dipropyline, metronidazole, tinidazole

KEYWORDS
serum; 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 immunoassa ytechnique. Analyst, 1993, 118, 1511-1513
SAMPLE
Matrix: blood
Sample preparation: Dilute serum with an equal volume 7.5 |xg/mL theobromine, inject
a 20 |xL aliquot directly.

HPLCVARIABLES
Column: 150 X 4.6 ChromSpher 5 BioMatrix (Chrompack)
Mobile phase: MeCN: water 5:95
Flow rate: 1
Injection volume: 20
Detector: UV 280

CHROIUIATOGRAM
Retention time: 2.4
Internal standard: theobromine (3.3)

OTHER SUBSTANCES
Simultaneous: caffeine

KEY WORDS
serum

REFERENCE
Helmsing, RJ.; Huisman, R.; van der Weele, A. HPLC determination of caffeine and theophylline by
direct serum injection. Clin.Chem., 1993, 39, 1348-1349

SAMPLE
Matrix: blood
Sample preparation: 10 \xL Plasma + 300 JJLL 100 mM pH 6.0 KH2PO4 buffer + 100 |xL
10 |xg/mL diprophylline + 2 mL chloroform: isopropanol 50:50, vortex for 30 s, centrifuge
at 2000 rpm for 10 min. Remove the organic layer and evaporate it to dryness under a
stream of air at 40, reconstitute the residue in 100 |xL mobile phase, inject a 25 |xL
aliquot.

HPLC VARIABLES
Column: NovaPak C18 radial compression
Mobile phase: MeOH: MeCN: 10 mM KH2PO4 9:2.5:90
Flow rate: 2
Injection volume: 25
Detector: E, ESA Coulochem Model 5100 A, Model 5010 analytical cell, first (screen) elec-
trode 0.68 V, second (measuring) electrode 0.98 V, Model 5020 guard cell (before injector)
1.0 V; UV 270

CHROMATOGRAM
Retention time: 8 (electrochemical detection)
Internal standard: diprophylline (UV detection) (10)
Limit of detection: 200 ng/mL

OTKER SUBSTANCES
Extracted: caffeine, 3-methylxanthine, theobromine

KEYWORDS
plasma
REFERENCE
Augustijns, P.; Verbeke, N. A microassay method for the determination of theophylline in biological
samples using HPLC with electrochemical detection. J.Liq.Chromatogr., 1992, 15, 1303-1313

SAMPLE
Matrix: blood
Sample preparation: 100 jxL Serum -I- 100 JJLL 20 jig/mL hydroxyethyltheophylline in 2
M perchloric acid, vortex, centrifuge 5 min, inject 50 |xL aliquot of supernatant.
HPLCVARIABLES
Column: 125 X 4 LiChroSpher RP-8 5 |xm
Mobile phase: MeOH: buffer 15:85 (Buffer was 5 mL 2 M sodium acetate + 845 mL water,
pH adjusted to 4.0 with acetic acid.)
Column temperature: 45
Flow rate: 1.5
Injection volume: 50
Detector: UV 282

CHROMATOGRAM
Retention time: 4.6
Internal standard: hydroxyethyltheophylline (5.6)
OTHER SUBSTANCES
Simultaneous: caffeine, chloramphenicol
KEYWORDS
serum
REFERENCE
Hannak, D.; Haux, P.; Scharbert, R; Kattermann, R. Liquid chromatographic analysis of phenobarbital,
phenytoin, and theophylline. Wien.Klin.Wochenschr.SuppL, 1992, 191, 27-31

SAMPLE
Matrix: blood
Sample preparation: 500 JXL Plasma + 50 ^xL 3 |xg/mL 8-chlorotheophylline in mobile
phase + 100 JULL 1 M HCl + 3 mL dichloromethane, vortex for 2 min, centrifuge at 1200
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 JULL mobile phase, inject a 20 |JLL aliquot.
HPLCVARIABLES
Column: 150 X 4.6 5 jxm TSK gel ODS-80TM (Tosoh)
Mobile phase: MeOH: 100 mM NaH2PO4 30:70
Flow rate: 0.8
Injection volume: 20
Detector: UV 274
CHROMATOGRAM
Retention time: 5.9
Internal standard: 8-chlorotheophylline (11.7)
Limit of quantitation: 10 ng/mL
OTHER SUBSTANCES
Extracted: metabolites, caffeine, paraxanthine, theobromine
Noninterfering: acetaminophen, aspirin, chlorpheniramine, ethenzamide, hydrocortisone,
phenacetin, phenobarbital, phenylbutazone, phenytoin, prednisolone, prednisone, salicylic
acid, trimethadione, vitamin Bl
KEYWORDS
plasma

REFERENCE
Tanaka, E. Simultaneous determination of caffeine and its primary demethylated metabolites in human
plasma by high-performance liquid chromatography. J.Chromatogr., 1992, 575, 311-314

SAMPLE
Matrix: blood
Sample preparation: Inject 20 jxL serum onto column A with mobile phase A and elute to
waste, after 1 min backflush 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,
monitor the effluent from column B. Re-equilibrate column A with mobile phase A.

HPLCVARIABLES
Column: A 30 X 4.6 IRSP silica (for preparation see Anal. Chem. 1989, 61, 2445); B 150 X
4.6 TSK gel ODS-80TM
Mobile phase: A 20 mM NaH2PO4; B MeCN: 100 mM NaH2PO4 10:90
Flow rate: A 0.8; B 1
Injection volume: 20
Detector: UV 275

CHROMATOGRAM
Retention time: 7.5
Limit of detection: 50 ng/mL

OTHER SUBSTANCES
Extracted: caffeine, theobromine

KEYWORDS
serum; column-switching

REFERENCE
Haginaka, J.; Wakai, J.; Yasuda, H.; Kimura, Y. Determination of anticonvulsant drugs and methyl
xanthine derivatives in serum by liquid chromatography with direct injection: column-switching
method using a new internal-surface reversed-phase silica support as a precolumn. J.Chromatogr.,
1990, 529, 455-461

SAMPLE
Matrix: blood
Sample preparation: 100 |xL Plasma or serum + 35 |xL 22 |mg/mL 3-ethylxanthine in 20
mM pH 4.0 acetate buffer, add to a Celute-MX SPE cartridge (Jones Chromatography),
let stand for 10 min, elute with two portions of isopropanol: dichloromethane 10:90, evap-
orate the eluate to dryness under a stream of nitrogen below 37, reconstitute the residue
in 100 |xL mobile phase B, centrifuge at 4400 rpm in a refrigerated centrifuge for 5 min,
inject a 20 |xL aliquot.

HPLCVARIABLES
Column: 250 X 4.5 3 |xm ODS Apex I (Jones Chromatography)
Mobile phase: Gradient. A was MeCN:THF: 10 mM pH 4.0 acetate buffer 25:2:73. B was
THF: 10 mM pH 4.0 acetate buffer 0.01:99.99. From A:B 0:100 increasing at 2.1% A/min.
Column temperature: 50
Flow rate: 0.8
Injection volume: 20
Detector: UV 273
CHROMATOGRAM
Retention time: 15.60
Internal standard: 3-ethylxanthine (13.64)

OTHER SUBSTANCES
Extracted: acetaminophen, caffeine, dimethyluric acids, methylxanthines, paraxanthine,
theobromine, trimethyluric acid

KEYWORDS
plasma; serum; SPE
REFERENCE
Leakey, T.E. Simultaneous analysis of theophylline, caffeine and eight of their metabolic products in
human plasma by gradient high-performance liquid chromatography J.Chromatogr., 1990, 507,
199-220

SAMPLE
Matrix: blood
Sample preparation: Centrifuge, filter (0.45 |Jim), inject an aliquot.

HPLCVARIABLES
Column: 150 X 4.6 5 |xm internal-surface, reversed-phase, Pinkerton-type, silica deriva-
tized 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: 10.88
Limit of detection: <1000 ng/mL

OTHER SUBSTANCES
Extracted: caffeine, doxofylline, dyphylline
Noninterfering: amitriptyline, amphetamine, atropine, benzoylecgonine, benztropine, caf-
feine, carbamazepine, carisoprodol, chlorpheniramine, chlorpromazine, chlorprothixene,
cimetidine, cocaine, codeine, dextromethorphan, diazepam, diphenhydramine, diphenox-
ilate, disopyramide, doxepin, doxylamine, emetine, erythromycin, flurazepam, gluteth-
imide, hydrocortisone, hydromorphone, hydroxyzine, imipramine, lidocaine, loxapine, me-
peridine, meprobamate, methadone, methamphetamine, methapyrilene, methaqualone,
methocarbamol, methylphenidate, nicotine, nordiazepam, nortriptyline, orphenadrine,
papaverine, pentazocine, phenacetin, phencyclidine, phenmetrazine, phenolphthalein,
phentermine, phenylpropanolamine, phenytoin, prazepam, procainamide, procaine, pro-
poxyphene, propranolol, protriptyline, pseudoephedrine, pyrilamine, quinine, salicylam-
ide, spironolactone, strychnine, terpin hydrate, thioridazine, thiothixene, triamterene,
trifluoperazine, triflupromazine, trihexyphenidyl, trimeprazine, trimethobenzamide, tri-
methoprim, tripelennamine
Interfering: acetaminophen

KEYWORDS
serum; plasma; direct injection
REFERENCE
Tagliaro, F.; Dorizzi, R.; Frigerio, A.; Marigo, M. Non-extraction HPLC method for simultaneous mea-
surement of dyphylline and doxofylline in serum. Clin.Chem., 1990, 36, 113-115
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 JJLL 10 |Jig/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 JULL aliquot.

HPLC VARIABLES
Column: 150 X 4.6 5 |xm Supelcosil-LC-8
Mobile phase: MeCN: water 20:80
Flow rate: 3.3
Injection volume: 15
Detector: UV 208

CHROMATOGRAM
Retention time: 0.77
Internal standard: tolylphenobarbital (7.57)
Limit of detection: 50-100 ng/mL

OTHER SUBSTANCES
Extracted: amobarbital, barbital, butabarbital, caffeine, carbamazepine epoxide, carba-
mazepine, carbamazepinediol, chloramphenicol, ethosuximide, glutethimide, mepheny-
toin, methaqualone, methyprylon, nirvanol, pentobarbital, phenacemide, phenobarbital,
phenytoin, primidone, secobarbital
Noninterfering: acetaminophen, N-acetylprocainamide, amikacin, amitriptyline, clonaze-
pam, cyclosporine, desipramine, diazepam, digoxin, disopyramide, gentamicin, p-hydrox-
yphenobarbital, imipramine, lidocaine, methotrexate, netilmicin, nortriptyline, procain-
amide, quinidine, salicylic acid, sulfamethoxazole, tobramycin, trimethoprim, valproic
acid, vancomycin

KEYWORDS
plasma; SPE

REFERENCE
Svinarov, D.A.; Dotchev, D.C. Simultaneous liquid-chromatographic determination of some bronchodi-
lators, anticonvulsants, chloramphenicol, and hypnotic agents, with Chromosorb P columns used for
sample preparation. Clin.Chem., 1989, 35, 1615-1618

SAMPLE
Matrix: blood
Sample preparation: 100 fxL Serum + 100 jxL buffer + 1 . 5 mL IS in 5% isopropanol in
chloroform, 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 jxL
mobile phase, inject a 6-10 |xL 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
Guard column: 20 X 4.6 Supelguard LC-I (Supelco)
Column: 250 X 4.6 5 |xm Supelcosil LC-I (Supelco)
Mobile phase: MeOH:MeCN:buffer 17.5:17.5:65 (Buffer was 2.72 g KH2PO4 in 1.9 L wa-
ter, pH adjusted to 6.3 with about 2 mL 1 M NaOH, made up to 2 L.)
Flow rate: 2
Injection volume: 6-10
Detector: UV 273
CHROMATOGRAM
Retention time: 1.95
Internal standard: 3-isobutyl-l-methylxanthine (3.15)

OTHER SUBSTANCES
Extracted: acetaminophen, amobarbital, barbital, caffeine, carbamazepine, chlorampheni-
col, ethosuximide, mephobarbital, methsuximide, pentobarbital, phenobarbital, pheny-
toin, primidone, secobarbital, thiopental
Also analyzed: acetanilide, N-acetylcysteine, N-acetylprocainamide, ampicillin, aspirin,
butabarbital, butalbital, chlorpropamide, cimetidine, codeine, cyheptamide, diazoxide, di-
fiunisal, diphylline, disopyramide, ethchlorvynol, gentisic acid, glutethimide, heptabar-
bital, hexobarbital, ibuprofen, indomethacin, ketoprofen, mefenamic acid, mephenytoin,
methaqualone, methsuximide, methyl salicylate, methyprylon, morphine, naproxen, nir-
vanol, oxphenylbutazone, phenacetin, phensuximide, phenylbutazone, procainamide, sal-
icylamide, salicylic acid, sulfamethoxazole, sulindac, tolmetin, trimethoprim, vancomycin
Noninterfering: amikacin, gentamicin, meprobamate, netilmicin, quinidine, tetracycldne,
tobramycin, valproic acid

KEYWORDS
serum

REFERENCE
Meatherall, R.; Ford, D. Isocratic liquid chromatographic determination of theophylline, acetaminophen,
chloramphenicol, caffeine, anticonvulsants, and barbiturates in serum. Ther.Drug Monit., 1988, 10,
101-115

SAMPLE
Matrix: blood
Sample preparation: 200 |xL Serum H- 400 |JLL 100 |xg/mL 8-chlorotheophylline in MeCN,
vortex for 10 s, centrifuge at 15000 rpm for 3 min, inject a 20 JJLL aliquot of the
supernatant.

HPLC VARIABLES
Column: 250 X 4.6 5 jxm Ultrasphere ODS
Mobile phase: MeCN: water: glacial acetic acid 4:84:12 containing 4.84 g/L Trizma, pH 2.3
Flow rate: 2
Injection volume: 20
Detector: UV 254

CHROMATOGRAM
Retention time: 3.00
Internal standard: 8-chlorotheophylline (5.29)
Limit of quantitation: 2 |xg/mL

OTHER SUBSTANCES
Extracted: acetaminophen, salicylic acid
Simultaneous: caffeine, cefazolin, cimetidine, ergotamine, glutethimide, heparin, meth-
amphetamine, propranolol, sulfamethoxazole, theobromine, tobutamide, trimethoprim
Noninterfering: amitriptyline, amobarbital, ampicillin, butabarbital, butalbital, celbenine,
chlordiazepoxide, chlorpromazine, clorazepate, desipramine, diazepam, doxepin, ethchlor-
vynol, fluphenazine, hydroxyzine, ibuprofen, imipramine, isoniazid, lidocaine, mepho-
barbital, mesoridazine, methaqualone, methyluric acid, naprotyline, nordiazepam,
nortriptyline, oxazepam, pentobarbital, perphenazine, phenelzine, phenmetrazine, phen-
obarbital, phenylbutazone, phenytoin, prednisolone, prednisone, procainamide, prochlor-
perazine, promazine, promethazine, propoxyphene, protriptyline, pyrilamine, secobarbi-
tal, thioridazine, thiothixene, timolol, trazodone, triazolam, trifluoperazine
KEYWORDS
serum

REFERENCE
Osterloh, J.; Yu, S. Simultaneous ion-pair and partition liquid chromatography of acetaminophen, the-
ophylline and salicylate with application to 500 toxicologic specimens. Clin.Chim.Acta, 1988, 175,
239-248

SAMPLE
Matrix: blood
Sample preparation: 100 (xL Serum + 1 mL reagent, vortex for 10 min, centrifuge for 2-
3 min. Remove the lower 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. (Reagent
was 1.5 mg p-hydroxyethyltheophylline and 25 |xL glacial acetic acid in 100 mL
chloroform: isopropanol 95:5.)

HPLCVARIABLES
Guard column: 30 mm long 5 |xm Ultrasphere ion-pair
Column: 150 X 4.6 5 ixm Ultrasphere ion-pair
Mobile phase: MeCN: MeOH: water: 1 M tetra-n-butylammonium hydroxide 3.5:3.5:91:2
containing 1.82 g Trizma base (Tris, tris(hydroxymethyl)aminomethane), pH adjusted to
7.50 0.03 with concentrated HCl
Flow rate: 1.2
Injection volume: 40
Detector: UV 280

CHROMATOGRAM
Retention time: k' 5.4
Internal standard: p-hydroxyethyltheophylline (k' 4.2)

OTHER SUBSTANCES
Extracted: caffeine, 1,7-dimethyl xanthine, theobromine
Simultaneous: acetaminophen, acetazolamide, allopurinol, dimethylurea, dyphylline, 3-
methylxanthine, oxypurinol, procainamide, sulfadiazine, sulfamethazine, uric acid
Noninterfering: ampicillin, cefazolin, cephalothin, cephapirin, chlorotheophylline, 1,3-di-
methyluric acid, gentamicin, lidocaine, methicillin, methylurea, 3-methyluric acid, quin-
idine, sulfamerazine, 1,3,7-trimethyluric acid

KEYWORDS
serum

REFERENCE
Lauff, J.J. Ion-pair high-performance liquid chromatographic procedure for the quantitative analysis of
theophylline in serum samples. J.Chromatogr., 1987, 417, 99-109

SAMPLE
Matrix: blood
Sample preparation: 200 |xL Serum + 200 |xL 4 |jtg/mL 8-chlorotheophylline in MeCN,
mix, centrifuge, evaporate the supernatant to dryness, reconstitute in 400 |xL mobile
phase, inject a 20 |xL aliquot.

HPLCVARIABLES
Column: 100 X 3 8 |jim octadecyl CP-tm-Spher C18 glass column (Chrompack)
Mobile phase: MeCN: 20 mM sodium acetate 20:80, adjusted to pH 4.4 with phosphoric
acid
Flow rate: 0.8
Injection volume: 20
Detector: UV 273

CHROMATOGRAM
Retention time: 3.8
Internal standard: 8-chlorotheophylline (8.8)
Limit of detection: 1 |xg/mL

OTHER SUBSTANCES
Simultaneous: caffeine

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 (JLL Serum or plasma + 200 |xL 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 u-L MeOH, inject a 10 u.L aliquot.

HPLCVARIABLES
Guard column: 50 X 3.2 30-38 |xm Co:Pell ODS
Column: 250 X 4.6 5 \xm Ultrasphere ODS
Mobile phase: MeCN:MeOH: 10 mM pH 5.2 sodium acetate buffer 6:3:91
Column temperature: 40
Flow rate: 1.5
Injection volume: 10
Detector: UV 274

CHROMATOGRAM
Retention time: 5.95
Internal standard: 8-chlorotheophylline (8.85)
Limit of detection: 100 ng/mL

OTHER SUBSTANCES
Extracted: caffeine, dyphylline, paraxanthine, proxyphylline
Simultaneous: cefoxitin
Noninterfering: carbenicillin, cefoperazone, cephacetril, heparin, penicillin G, phenytoin,
phenobarbital

KEYWORDS
serum; plasma; 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: 50 |JLL Serum H- 50 JULL 15 fxg/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 JJLL MeOH, inject a 20 JULL 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
Injection volume: 20
Detector: UV 254

CHROMATOGRAM
Retention time: 4.8
Internal standard: p-hydroxyethyltheophylline (5.8)
Limit of detection: 500 ng/mL

OTHER SUBSTANCES
Extracted: caffeine
Simultaneous: acetaminophen, N-acetylprocainamide, aspirin, procainamide, salicylic acid
Noninterfering: benzoic acid
Interfering: dyphylline (separated with MeCN-.buffer 8:92), ampicillin

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 JJLL Serum + p-hydroxypropyltheophylline, vortex, add 1.5 g an-
hydrous sodium sulfite, add 2.5 mL chloroform: MeOH 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 |xL chloroform, evaporate the filtrate under a stream of nitrogen at 40.
Take up the residue in 100 |xL dichloroethane and add it to 100 JULL 100 mM ammonium
carbonate, vortex for 10 s, centrifuge at 1000 g for 5 min, inject a 10 |xL aliquot of the
aqueous layer.

HPLCVARIABLES
Column: 250 X 4 10 fxm LiChrosorb RP-8
Mobile phase: MeOH: buffer 25:75 (Buffer was 1.5 mL 1 M KH2PO4 in 750 mL water,
adjust to pH 3.0 with 900 mM perchloric acid.)
Flow rate: 2
Injection volume: 10
Detector: UV 275
CHROMATOGRAM
Retention time: 4.5
Internal standard: p-hydroxypropyltheophylline (3.7)

OTHER SUBSTANCES
Simultaneous: caffeine, dyphylline, theobromine
Noninterfering: acetaminophen, acetazolamide, albuterol, amitriptyline, amobarbital, car-
bamazepine, diazoxide, disopyramide, ethosuximide, isoproterenol, nitrazepam, nortrip-
tyline, oxazepam, pentobarbital, phenobarbital, phenytoin, primidone, procainamide, sal-
icylic acid, sulthiame

KEYWORDS
serum
REFERENCE
Pater son, 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: 200 fxL Serum + 200 |xL 50 fxg/mL hexobarbital in MeCN + 25 JJLL
glacial acetic acid, vortex for 10 s, centrifuge for 1 min, inject a 30-100 JXL aliquot of the
supernatant.
HPLCVARIABLES
Column: u-Bondapak 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.
Column temperature: 50
Flow rate: 3
Injection volume: 30-100
Detector: UV 210
CHROMATOGRAM
Retention time: 4.3
Internal standard: hexobarbital (20.6)
Limit of detection: LOD 200-2000 ng/mL
OTHER SUBSTANCES
Extracted: acetaminophen, amobarbital, butabarbital, butalbital, chlordiazepoxide, diaze-
pam, ethchlorvynol, flurazepam, glutethimide, methaqualone, methyprylon, nitrazepam,
pentobarbital, phenobarbital, phenytoin, primidone, salicylic acid, secobarbital
Simultaneous: amitriptyline, caffeine, clomipramine, codeine, desipramine, ethotoin, imip-
ramine, lidocaine, mesantoin, methsuximide, nirvanol, nortriptyline, oxazepam, procain-
amide, 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
chromatography. J.Anal.ToxicoL, 1981, 5, 177-182

SAMPLE
Matrix: blood
Sample preparation: 1 mL Plasma H- 100 |xL 0.2 mg/mL p-hydroxyethyltheophylline in
buffer H- 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 JJLL aliquot of the supernatant. (Buffer was 10
mM sodium acetate adjusted to pH 4.0 with glacial acetic acid.)

HPLCVARIABLES
Column: 10 |xm ixBondapak C18
Mobile phase: MeCN: buffer 6:94 (Buffer was 10 mM sodium acetate adjusted to pH 4.0
with glacial acetic acid.)
Column temperature: 40
Flow rate: 2
Injection volume: 25
Detector: UV 274

CHROMATOGRAM
Retention time: 6.5
Internal standard: p-hydroxyethyltheophylline (9)
Limit of detection: 1 |xg/mL

OTHER SUBSTANCES
Extracted: caffeine, dyphylline, theobromine

KEYWORDS
plasma

REFERENCE
Valia, K.H.; Hartman, CA.; Kucharczyk, N.; Sofia, R.D. Simultaneous determination of dyphylline and
theophylline in human plasma by high-performance liquid chromatography. J.Chromatogr., 1980,
221, 170-175

SAMPLE
Matrix: blood, CSF, gastric fluid, urine
Sample preparation: 200 jxL Serum, urine, CSF, or gastric fluid + 300 u>L 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, backflush the con-
tents 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 40 |xm preparative grade C18 (Analytichem); B 75 X 2.1 pellicular C18 (What-
man) + 250 X 4.6 5 |xm C8 end-capped (Whatman)
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 20 min.
Column temperature: 50
Flow rate: 1.5
Detector: UV 220

CHROMATOGRAM
Retention time: 5
Internal standard: heptanophenone (19)

OTHER SUBSTANCES
Extracted: acetaminophen, allobarbital, azinphos, barbital, brallobarbitone, bromazepam,
butethal, caffeine, carbamazepine, carbaryl, cephaloridine, chloramphenicol, chlordiaze-
poxide, chlorothiazide, chlorvinphos, clothiapine, cocaine, coomassie blue, desipramine,
diazepam, diphenhydramine, dipipanone, ethylbromphos, flufenamic acid, formothion,
 griseofulvin, indomethacin, lidocaine, lorazepam, malathion, medazepam, midazolam, ox-
azepam, paraoxon, penicillin G, pentobarbital, prazepam, propoxyphene, prothiophos, qui-
nine, salicylic acid, secobarbital, strychnine, sulfamethoxazole, thiopental, thioridazine,
trimethoprim

KEYWORDS
serum; column-switching

REFERENCE
Kruger, RB.; Albrecht, CRDe V.; Jaarsveld, RR Use of guanidine hydrochloride and ammonium sulfate
in comprehensive in-line sorption enrichment of xenobiotics in biological fluids by high-performance
liquid chromatography. J.Chromatogr., 1993, 612, 191-198

SAMPLE
Matrix: blood, gastric juice, pancreatic juice
Sample preparation: 500 |xL Serum, pancreatic juice, or gastric juice + 25 |xL 200 |xg/mL
p-hydroxyethyltheophylline in water + 500 |xL MeCN, centrifuge at 6000 rpm for 10 min,
remove supernatant and add it to 1.8 mL chloroform, vortex, centrifuge at 6000 rpm for
10 min. Remove the lower organic phase and evaporate it to dryness at 60 under a stream
of air, dissolve the residue in 500 |JLL mobile phase, inject a 50 |xL aliquot.

HPLCVARIABLES
Guard column: C18 LichroCART
Column: 250 X 4.6 7 |xm Lichrosorb C18
Mobile phase: THF: MeOH: 10 mM pH 3.5 KH2PO4 1:20:79
Flow rate: 0.8
Injection volume: 50
Detector: UV 280

CHROMATOGRAM
Retention time: 7.03
Internal standard: p-hydroxyethyltheophylline (7.48)
Limit of quantitation: 250 ng/mL

OTHER SUBSTANCES
Extracted: caffeine, 1-methyluric acid, 1-methylxanthine, paraxanthine, theobromine

KEYWORDS
serum; dog

REFERENCE
Casoli, P.; Verine, H. High performance liquid chromatographic determination of methylxanthines in
canine serum, gastric and pancreatic juices. Biomed.Chromatogr., 1990, 4, 209-213

SAMPLE
Matrix: blood, saliva
Sample preparation: 500 \xL Plasma or saliva + 50 |xL 50 ng/mL difloxacin, vortex briefly,
add 500 \xL 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 organic layer and evaporate it to dryness under a stream of nitrogen
at 45, reconstitute the residue in 500 |JLL mobile phase, inject a 50-200 |xL aliquot.

HPLCVARIABLES
Guard column: jxBondapak C18 Guard-Pak
Column: 300 X 3.9 10 |xm jxBondapak C18
Mobile phase: MeOH-.buffer 35:100 (Buffer was 5.44 g KH2PO4 and 4 mL tetrabutylam-
monium hydroxide in 1 L water, adjust pH to 2.5 with 85% phosphoric acid.)
Flow rate: 2
Injection volume: 50-200
Detector: UV 268

CHROMATOGRAM
Retention time: 4.3
Internal standard: difloxacin (8.8)
Limit of detection: 100 ng/mL

OTHER SUBSTANCES
Extracted: ciprofloxacin, enoxacin
Simultaneous: caffeine
Noninterfering: 1,3-dimethyluric acid, hypoxanthine, 1-methyluric acid, 1-methylxan-
thine, 3-methylxanthine, 7-methylxanthine, theobromine
Interfering: 1,7-dimethylxanthine

KEYWORDS
plasma; pharmacokinetics

REFERENCE
Zhai, S.; Korrapati, M.R.; Wei, X.; Muppalla, S.; Vestal, R.E. Simultaneous determination of theophyl-
line, enoxacin and ciprofloxacin in human plasma and saliva by high-performance liquid chromatog-
raphy. J.Chromatogr.B, 1995, 669, 372-376

SAMPLE
Matrix: blood, saliva
Sample preparation: 100 |JLL Serum, plasma, or saliva + 100 |xL 15 |xg/mL 8-chlorotheo-
phylline in 6% perchloric acid, vortex for 10 s, centrifuge at 10800 g for 10 min, inject 20
jxL of the clear supernatant.

HPLC VARIABLES
Guard column: Whatman Co:Pell ODS
Column: 300 X 3.9 10 pirn Bondex ODS (Phenomenex)
Mobile phase: THF: buffer 6:94 (Buffer was pH 4.0 10 mM, prepared from 41 mL 200 mM
acetic acid and 9 mL 200 mM sodium acetate made up to 1 L with water, pH adjusted to
4.0 with glacial acetic acid or 100 mM NaOH if necessary.)
Flow rate: 1
Injection volume: 20
Detector: UV 273

CHROMATOGRAM
Retention time: 4.7
Internal standard: 8-chlorotheophylline (11)
Limit of quantitation: 70 ng/mL

OTHER SUBSTANCES
Simultaneous: caffeine, paraxanthine, theobromine

KEYWORDS
serum; plasma

REFERENCE
Blanchard, J.; Harvey, S.; Morgan, W.J. A rapid and specific high-performance liquid chromatographic
assay for theophylline in biological fluids. J.Chromatogr.Scl, 1990, 28, 303-306
SAMPLE
Matrix: blood, tissue
Sample preparation: Tissue. Homogenize brain in twenty volumes of water, centrifuge at
1500 g, freeze at -20 for 2 h, centrifuge. 200 |xL Supernatant + 100 JJLL pH 7.2 phosphate
buffer, mix, add 6 mL dichloromethane: propanol 95:5, mix for 2 min, centrifuge at 1500
g for 10 min. Remove the organic layer and evaporate it to dryness under a stream of
nitrogen at 35, reconstitute the residue in 200 \xL mobile phase, vortex for 1 min, inject
a 25 |xL aliquot. Serum. 200 |xL Serum + 100 |xL pH 7.2 phosphate buffer, mix, add 6
mL dichloromethane: propanol 95:5, mix for 2 min, centrifuge at 1500 g for 10 min. Re-
move the organic layer and evaporate it to dryness under a stream of nitrogen at 35,
reconstitute the residue in 200 |xL mobile phase, vortex for 1 min, inject a 25 |xL aliquot.

HPLCVARIABLES
Guard column: 10 |jim ixBondapak C18
Column: 250 X 4.6 10 |xm ixBondapak C18
Mobile phase: THF: 10 mM Na2HPO4 3:97, pH adjusted to 6.5 with phosphoric acid
Flow rate: 2.5
Injection volume: 25
Detector: UV 273

CHROMATOGRAM
Retention time: 5
Limit of detection: 62.5 ng/g (tissue); 62.5 ng/mL (serum)

OTHER SUBSTANCES
Extracted: caffeine, paraxanthine, theobromine

KEYWORDS
serum; rat; brain

REFERENCE
Parra, P.; Limon, A.; Ferre, S.; Guix, T.; Jane, F. High-performance liquid chromatographic separation
of caffeine, theophylline, theobromine and paraxanthine in rat brain and serum. J.Chromatogr., 1991,
570, 185-190

SAMPLE
Matrix: blood, urine
Sample preparation: Plasma. 500 |xL Plasma + 25 \xL 300 |xM IS in EtOH: water 30:70
+ 75 |JLL 1 M HCl, mix, add 5 mL ethyl acetate: isopropanol 90:10, shake for 10 min,
centrifuge at 1000 g for 10 min, freeze at -30. Remove the organic layer and evaporate
it to dryness under a stream of nitrogen at 55, reconstitute the residue in 300 |xL mobile
phase, sonicate until dissolved, centrifuge at 13000 g for 15 min, inject a 40 u,L aliquot.
Urine. Acidify 10 mL urine with 300 uX 1 M HCl. 40 u.L Acidified urine + 50 JJLL 300 |xM
IS in EtOH: water 30:70, mix, make up to 400 uX with 10 mM pH 4.0 acetate buffer, add
5 mL ethyl acetate: isopropanol 93:7, shake for 10 min, centrifuge at 1000 g for 10 min,
freeze at -30. Remove the organic layer and evaporate it to dryness under a stream of
nitrogen at 55, reconstitute the residue in 300 (JLL mobile phase, vortex for 10 s, inject a
40 |JLL aliquot.

HPLCVARIABLES
Column: 250 X 4.6 5 jxm Ultrasphere ODS
Mobile phase: MeOH: 10 mM pH 4.0 acetate buffer 9:91 (plasma) or 7:93 (urine)
Column temperature: 30
Flow rate: 1 for 11 min, 1.5 for 6 min, 2.5 for 13 min
Injection volume: 40
Detector: UV 273
CHROMATOGRAM
Retention time: 20.2 (plasma), 20.9 (urine)
Internal standard: p-hydroxyethyltheophylline (24.2 (plasma), 28.4 (urine))
Limit of detection: 200 nM (plasma); 2 jxM (urine)

OTHER SUBSTANCES
Extracted: metabolites, 1,3-dimethyluric acid, 1-methyluric acid, 3-methylxanthine

KEYWORDS
plasma

REFERENCE
Rasmussen, B.B.; Brosen, K. Determination of theophylline and its metabolites in human urine and
plasma by high-performance liquid chromatography. J.Chromatogr.B, 1996, 676, 169-174

SAMPLE
Matrix: formulations
Sample preparation: Inject a 20 uX aliquot.

HPLC VARIABLES
Guard column: 10 |xm Dynamax C18
Column: 250 X 4.6 10 jxm Dynamax C18
Mobile phase: MeCN:6.5 mM tetraheptylammonium bromide: 100 mM pH 7.0 phosphate
buffer: 100 mM pH 5.0 citrate buffer 40:55.2:4.4:0.4
Flow rate: 1
Injection volume: 20
Detector: UV 271

CHROMATOGRAM
Retention time: 3.0

OTHER SUBSTANCES
Simultaneous: degradation products, ceftriaxone

KEYWORDS
injections; use low actinic glassware; stability-indicating; saline; 5% dextrose

REFERENCE
Parrish, M.A.; Bailey, L.C.; Medwick, T. Stability of ceftriaxone sodium and aminophylline or theoph-
ylline in intravenous mixtures. Am.J.Hosp.Pharm., 1994, 51, 92-94

SAMPLE
Matrix: formulations
Sample preparation: Dilute to 0.8-13 |jig/mL, inject an aliquot.

HPLCVARIABLES
Column: 150 X 4.6 5 |xm Ultrasphere C18
Mobile phase: MeCN: 100 mM pH 3.4 acetate buffer 1:10 (Buffer was 50 mL 100 mM
sodium acetate diluted to 1 L with 100 mM acetic acid.)
Flow rate: 2
Injection volume: 20
Detector: UV 254

CHROMATOGRAM
Internal standard: 5-methylresorcinol
OTHER SUBSTANCES
Simultaneous: aminophylline, cefuroxime

KEYWORDS
injections; 5% dextrose

REFERENCE
Stewart, J.T.; Warren, RW.; Johnson, S.M. Stability of cefuroxime sodium and aminophylline or the-
ophylline. Am.J.Hosp.Pharm., 1994, 51, 809-811

SAMPLE
Matrix: formulations
Sample preparation: Shake, remove 2 mL of oral suspension, dilute to 40 mL with water,
vortex 1 min, centrifuge at 2000 rpm for 10 min. Dilute a 100 |xL aliquot of supernatant
with 100 fxL of 100 |xg/mL theophylline and add 800 JJLL water, inject 20 JAL aliquot.

HPLCVARIABLES
Column: 300 mm 10 jxm Waters reversed-phase C18
Mobile phase: MeCN: 50 mM sodium acetate buffer 8:92, adjusted to pH 6.5
Flow rate: 1.5
Injection volume: 20
Detector: UV 254

CHROMATOGRAM
Retention time: 4.3
Internal standard: theophylline

OTHER SUBSTANCES
Simultaneous: famotidine

KEYWORDS
stability-indicating; oral suspensions; theophylline is IS

REFERENCE
Quercia, R.A.; Jay, G.T.; Fan, C; Chow, M.S. Stability of famotidine in an extemporaneously prepared
oral liquid. Am.J.Hosp.Pharm., 1993, 50, 691-693

SAMPLE
Matrix: formulations
Sample preparation: Tablets. Weigh out powdered tablets containing 100 mg aminophyl-
line, add 50 mL water, sonicate for 15 min, make up to 100 mL with water, mix, filter.
Remove a 5 mL aliquot of the filtrate and add it to 10 mL 5 mg/mL dansyl chloride in
acetone and 5 mL buffer, mix gently, let stand in the dark for 12 h, make up to 50 mL
with acetone: water 50:50, mix, inject an aliquot. Injections, oral liquids. Measure out an
amount containing 100 mg aminophylline, make up to 100 mL with water, mix. Remove
a 5 mL aliquot and add it to 10 mL 5 mg/mL dansyl chloride in acetone and 5 mL buffer,
mix gently, let stand in the dark for 12 h, make up to 50 mL with acetone: water 50:50,
mix, inject an aliquot. (Prepare buffer by dissolving 550 mg anhydrous sodium carbonate
in 300 mL water, add 300 mL acetone, mix.)

HPLCVARIABLES
Guard column: 70 X 2.1 Co:Pell ODS
Column: 300 X 3.9 10 |xm ixBondapak C18
Mobile phase: MeOH: water: acetic acid: triethylamine 60:38:1.5:0.5 (A) or 65:33:1.5:0.5
(B)
Flow rate: 1.5
Injection volume: 20
Detector: UV 254

CHROMATOGRAM
Retention time: 6.0 (mobile phase B), 7.45 (mobile phase A)

OTHER SUBSTANCES
Simultaneous: ethylenediamine

KEYWORDS
derivatization; tablets; injections; oral solutions

REFERENCE
Lau-Cam, CA.; Roos, R.W. Simultaneous high performance liquid chromatographic determination of
theophylline and ethylenediamine in aminophylline dosage forms as their dansyl derivatives.
J.Liq.Chromatogn, 1991, 14, 1939-1956

SAMPLE
Matrix: microsomal incubations
Sample preparation: 500 \xL Microsomal incubation + 400 |xL 2% zinc sulfate, centrifuge
at 5000 rpm for 5 min. Remove the supernatant and add it to 30 mg ammonium sulfate
and 10 JULL 10 jmg/mL 1,7-dimethyluric acid, extract with dichloromethane: isopropanol
80:20. Remove the organic layer and evaporate it to dryness, reconstitute, inject an
aliquot.

HPLCVARIABLES
Guard column: C18
Column: 250 X 4.6 Spherex 5 C18 (Phenomenex)
Mobile phase: Gradient. MeCN: 25 |JLM pH 4.5 acetate buffer 2:98 for 20 min, 5:95 for 12
min, 9:91 for 8 min, re-equilibrate at initial conditions for 10 min.
Flow rate: 1
Detector: UV 276

CHROMATOGRAM
Retention time: 35.5
Internal standard: 1,7-dimethyluric acid (33.6)

OTHER SUBSTANCES
Extracted: metabolites, 1,3-dimethyluric acid, 1-methylxanthine, 3-methylxanthine

KEYWORDS
human; liver

REFERENCE
Tjia, J.R; Colbert, J.; Back, D.J. Theophylline metabolism in human liver microsomes: Inhibition stud-
ies. J.Pharmacol.Exp.Then, 1996, 276, 912-917

SAMPLE
Matrix: microsomal incubations
Sample preparation: 275 |xL Microsomal incubation + 50 JULL 30% perchloric acid, centri-
fuge at 2000 g for 10 min, inject a 60 JJLL aliquot of the supernatant.

HPLCVARIABLES
Guard column: 20 X 4.6 40 |xm Supelcosil LC-18
Column: 150 X 4.6 5 |xm Supelcosil LC-18
Mobile phase: MeCN: THF: 20 mM pH 3.5 sodium perchlorate 0.5:0.5:99
Flow rate: 1.5
Injection volume: 60
Detector: UV 280

CHROMATOGRAM
Retention time: 14

OTHER SUBSTANCES
Extracted: caffeine, paraxanthine, theobromine, trimethylurate

KEYWORDS
monkey; liver

REFERENCE
Bullock, P.; Pearce, R.; Draper, A.; Podval, J.; Bracken, W.; Veltman, J.; Thomas, P.; Parkinson, A.
Induction of liver microsomal cytochrome P450 in cynomolgus monkeys. Drug Metab.Dispos., 1995,
23, 736-748

SAMPLE
Matrix: milk
Sample preparation: Centrifuge at 12800 g for 10 min, remove top layer of fat with a
spatula. 450 |xL Supernatant + 50 |xL water + 150 JULL 21.65 jxg/mL proxyphylline in 6%
perchloric acid, vortex for 5 s, cool on ice for 10-15 min, centrifuge at 12800 g for 10 min,
inject a 50 JULL aliquot of the supernatant.

HPLCVARIABLES
Guard column: Co: Pell ODS glass beads (Whatman)
Column: 250 X 4.6 5 \xm Ultrasphere ODS
Mobile phase: Gradient. A was 10 mM sodium acetate + 5 mM tetrabutylammonium hy-
drogen sulfate, adjusted to pH 4.9 with 2 M NaOH. B was MeOH: water 50:50 containing
10 mM sodium acetate H- 5 mM tetrabutylammonium hydrogen sulfate, adjusted to pH
4.8 with glacial acetic acid. A:B 100:0 for 7.5 min, then to 85:15 over 7.5 min, then to
70:30 over 10 min, then to 68:32 over 4 min, then to 100:0 over 3 min.
Flow rate: 1.5
Injection volume: 50
Detector: UV 272

CHROMATOGRAM
Retention time: 22.5
Internal standard: proxyphylline (25)

OTHER SUBSTANCES
Simultaneous: caffeine, paraxanthine, theobromine

REFERENCE
Blanchard, J.; Weber, CW.; Shearer, L.E. HPLC analysis of methylxanthines in human breast milk.
J.Chromatogr.ScL, 1990, 28, 640-642

SAMPLE
Matrix: perfusate
Sample preparation: Inject a 100 |xL aliquot directly.

HPLCVARIABLES
Column: 250 X 4.6 octyl Spherisorb S5-ODS
Mobile phase: MeCN: 100 mM KH2PO4 10:90
Column temperature: 40
Flow rate: 1
Injection volume: 100
Detector: UV 272

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.Sci.,
1994,83, 1071-1076

SAMPLE
Matrix: saliva
Sample preparation: Centrifuge saliva at 10000 rpm for 2 min. 1 mL Supernatant + 50
IJLL 80 [Jug/mh p-hydroxyethyltheophylline + 100 jxL 100 mM HCl + 7 mL chlofoform:
isopropanol 95:5, shake for 10 min, centrifuge at 3000 rpm for 5 min. Remove 5 mL of
the organic layer and evaporate it to dryness under a stream of nitrogen at 60, recon-
stitute the residue in 200 u,L mobile phase, filter, inject a 10 u,L aliquot of the nitrate.

HPLCVARIABLES
Column: 150 X 4.6 ODS 120T (Tosoh)
Mobile phase: MeCN: 10 mM pH 4.0 acetate buffer 9:91
Flow rate: 1
Injection volume: 10
Detector: UV 270

CHROMATOGRAM
Internal standard: p-hydroxyethyltheophylline

KEYWORDS
pharmacokinetics

REFERENCE
Kanke, M.; Katayama, H.; Nakamura, M. Application of curdlan to controlled drug delivery. II. In vitro
and in vivo drug release studies of theophylline-containing curdlan tablets. Biol.Pharm.BulL, 1995,
18, 1104-1108

SAMPLE
Matrix: solutions
Sample preparation: Inject a 20 jxL aliquot.

HPLCVARIABLES
Column: 150 X 6 5 |xm 3-aminopropylsilyl silica gel with the amino group derivatized with
1,8-naphthalic anhydride (Bunseki Kagaku 1993, 42, 817)
Mobile phase: MeOH: buffer 50:50 (Prepare buffer by dissolving 6.183 g boric acid and
1.461 g NaCl in 500 mL water, adjust pH to 6.4 with sodium borate solution.)
Column temperature: 30
Flow rate: 1
Injection volume: 20
Detector: UV 270

CHROMATOGRAM
Retention time: 13.5
Internal standard: l,3-dimethyl-7-(2-hydroxyethyl)xanthine (12)
OTHER SUBSTANCES
Simultaneous: caffeine, hypoxanthine, pentoxifylline, propentofylline, theobromine, uric
acid, xanthine

REFERENCE
Nakashima, K.; Inoue, K.; Mayahara, K.; Kuroda, N.; Hamachi, Y; Akiyama, S. Use of 3-(l,8-naphthal-
imido)propyl-modified silyl silica gel as a stationary phase for the high-performance liquid chromat-
ographic separation of purine derivatives. J.Chromatogr.A, 1996, 722, 107-113

SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 250 X 4.6 5 \x.m 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 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
Injection volume: 25
Detector: UV 229

CHROMATOGRAM
Retention time: 4.52 (A), 3.45 (B)

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,
chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cimeti-
dine, cisapride, clomipramine, clonazepam, clonidine, clozapine, cocaine, codeine, colchi-
cine, cyclizine, cyclobenzaprine, dantrolene, desipramine, diazepam, diclofenac, diflunisal,
diltiazem, diphenhydramine, diphenidol, diphenoxylate, dipyridamole, disopyramide, do-
butamine, doxapram, doxepin, droperidol, encainide, ethidium bromide, ethopropazine,
fenoprofen, fentanyl, flavoxate, fluoxetine, fluphenazine, flurazepam, flurbiprofen, fluvox-
amine, furosemide, glutethimide, glyburide, guaifenesin, haloperidol, homatropine, hy-
dralazine, hydrochlorothiazide, hydrocodone, hydromorphone, hydroxychloroquine, hydro-
xyzine, ibuprofen, imipramine, indomethacin, ketoconazole, ketoprofen, ketorolac,
labetalol, levorphanol, lidocaine, loratadine, lorazepam, lovastatin, loxapine, mazindol,
mefenamic acid, meperidine, mephenytoin, mepivacaine, mesoridazine, metaproterenol,
metformin, methadone, methdilazine, methocarbamol, methotrexate, methotrimeprazine,
methoxamine, methyldopa, methylphenidate, metoclopramide, metolazone, metoprolol,
metronidazole, midazolam, moclobemide, morphine, nadolol, nalbuphine, naloxone, na-
phazoline, naproxen, nifedipine, nizatidine, norepinephrine, nortriptyline, oxazepam, ox-
ycodone, oxymetazoline, paroxetine, pemoline, pentazocine, pentobarbital, pentoxifylline,
perphenazine, pheniramine, phenobarbital, phenol, phenolphthalein, phentolamine,
phenylbutazone, phenyltoloxamine, phenytoin, pimozide, pindolol, piroxicam, pramoxine,
prazepam, prazosin, probenecid, procainamide, procaine, prochlorperazine, procyclidine,
promazine, promethazine, propafenone, propantheline, propiomazine, propofol, propran-
olol, protriptyline, quazepam, quinidine, quinine, racemethorphan, ranitidine, remoxi-
pride, risperidone, salicylic acid, scopolamine, secobarbital, sertraline, sotalol, spirono-
lactone, sulfinpyrazone, sulindac, temazepam, terbutaline, terfenadine, tetracaine,
thiethylperazine, thiopental, thioridazine, thiothixene, timolol, tocainide, tolbutamide,
tolmetin, trazodone, triamterene, triazolam, trifluoperazine, triflupromazine, trimepra-
zine, trimethoprim, trimipramine, verapamil, warfarin, xylometazoline, yohimbine,
zopiclone
KEY WORDS
some details of plasma extraction

REFERENCE
Koves, E.M. Use of high-performance liquid chromatography-diode array detection in forensic toxicology.
J.Chromatogr.A, 1995, 692, 103-119

SAMPLE
Matrix: solutions
Sample preparation: Inject a 50-200 |xL aliquot of a solution in pH 7.4 Tyrode's buffer.

HPLCVARIABLES
Column: 125 X 4 5 jxm LiChrospher 60 RP-select B
Mobile phase: MeCN: 10 mM pH 4 sodium acetate 12:88
Flow rate: 0.6
Injection volume: 50-200
Detector: UV 270

OTHER SUBSTANCES
Also analyzed: cefazolin

KEYWORDS
buffer

REFERENCE
Saitoh, H.; Aungst, B.J. Possible involvement of multiple P-glycoprotein-mediated efflux systems in the
transport of verapamil and other organic cations across rat intestine. Pharm.Res., 1995, 12, 1304-
1310

SAMPLE
Matrix: solutions

HPLCVARIABLES
Column: 250 X 4.6 Zorbax RX
Mobile phase: Gradient. A was 10 mL concentrated orthophosphoric acid and 7 mL trie-
thylamine in 1 L water. B was 10 mL concentrated orthophosphoric acid and 7 mL trie-
thylamine 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.
Column temperature: 30
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, diphenoxy-
 late, diprenorphine, dipyrone, disulfiram, dopamine, doxapram, doxepin, dronabinol,
ephedrine, epinephrine, epinine, estradiol, estriol, estrone, ethacrynic acid, ethosuximide,
etonitazene, etorphine, eugenol, famotidine, fenbendazole, fencamfamine, fenoprofen, fen-
proporex, fentanyl, flubendazole, flufenamic acid, flunitrazepam, 5-fluorouracil, fluo-
xymesterone, fluphenazine, furosemide, gentisic acid, gitoxigenin, glipizide, glunixin,
glutethimide, glybenclamide, guaiacol, halazepam, haloperidol, hydrochlorothiazide,
hydrocodone, hydrocortisone, hydromorphone, hydroxyquinoline, ibogaine, ibuprofen, im-
inostilbene, imipramine, indomethacin, isocarbostyril, isocarboxazid, isoniazid, isoproter-
enol, isoxsuprine, ivermectin, ketamine, ketoprofen, kynurenic acid, levorphanol, Ii-
docaine, lorazepam, lormetazepam, loxapine, mazindol, mebendazole, meclizine,
meclofenamic acid, medazepam, mefenamic acid, megestrol, mepacrine, meperidine, me-
phentermine, mephenytoin, mephesin, mephobarbital, mepivacaine, mescaline, mesori-
dazine, methadone, methamphetamine, methapyrilene, methaqualone, methazolamide,
methocarbamol, methoxamine, methsuximide, methyl salicylate, methyldopa, methyldo-
pamine, methylphenidate, methylprednisolone, methyltestosterone, methyprylon, meto-
prolol, mibolerone, morphine, nadolol, nalorphine, naloxone, naltrexone, naphazoljne,
naproxen, nefopam, niacinamide, nicotine, nicotinic acid, nifedipine, niflumic acid, nitra-
zepam, norepinephrine, nortriptyline, noscapine, nylidrin, oxazepam, oxycodone,
oxymorphone, oxyphenbutazone, oxytetracycline, papaverine, pargyline, pemoline, pen-
tazocine, pentobarbital, persantine, phenacetin, phenazocine, phenazopyridine, phency-
clidine, phendimetrazine, phenelzine, pheniramine, phenobarbital, phenothiazine, phen-
suximide, phentermine, phenylbutazone, phenylephrine, phenylpropanolamine,
piperocaine, prazepam, prednisolone, primidone, probenecid, progesterone, propiomazine,
propranolol, propylparaben, pseudoephedrine, puromycin, pyrilamine, pyrithyldione, qua-
zepam, quinaldic acid, quinidine, quinine, ranitidine, recinnamine, reserpine, resorcinol,
saccharin, albuterol, salicylamide, salicylic acid, scopolamine, scopoletin, secobarbital,
strychnine, sulfacetamide, sufadiazine, sulfadimethoxine, sulfaethidole, sulfamerazine,
sulfamethazine, sulfamethoxizole, sulfanilamide, sulfapyridine, sulfasoxizole, sulindac,
tamoxifen, temazepam, testosterone, tetracaine, tetracycline, tetramisole, thebaine, the-
obromine, thiabendazole, thiamine, thiamylal, thiobarbituric acid, thioridazine, thiosali-
cylic acid, thiothixene, thymol, tolazamide, tolazoline, tobutamide, tolmetin, tranylcy-
promine, triamcinolone, tribenzylamine, trichloromethiazide, trifluoperazine,
trihexyphenidyl, trimethoprim, tripelennamine, triprolidine, tropacocaine, tyramine, ver-
apamil, 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: Dissolve in MeOH: water 1:1 at a concentration of 50 (xg/mL, inject
a 10 |JLL aliquot.

HPLCVARIABLES
Column: 300 X 3.9 10 |xm ixBondapak C18
Mobile phase: MeOH: acetic acid: triethylamine: water 25:1.5:0.5:73
Flow rate: 1.5
Injection volume: 10
Detector: UV 254

CHROMATOGRAM
Retention time: 7

OTHER SUBSTANCES
Simultaneous: caffeine, 8-chlorotheophylline, diphylline, theobromine
REFERENCE
Roos, R.W.; Lau-Cam, CA. 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: Prepare a 10 |xg/mL solution in MeOH, inject a 20 \xL aliquot.

HPLCVARIABLES
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
Injection volume: 20
Detector: E, LeCarbone, V25 glassy carbon electrode, + 1.2 V

CHROMATOGRAM
Retention time: 1.04

OTHER SUBSTANCES
Also analyzed: acebutolol, acepromazine, acetophenazine, N-acetylprocainamide, albu-
terol, alprenolol, amethocaine, amiodarone, amitriptyline, antazoline, atenolol, azacy-
clonal, bamethane, 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, dipyrida-
mole, disopyramide, dothiepin, doxapram, doxepin, doxylamine, droperidol, ephedrine,
ergocornine, ergocristine, ergocristinine, ergocryptine, ergometrine, ergosine, ergosinine,
ergotamine, ethopropazine, etorphine, etoxeridine, fenethazine, fenfluramine, fenoterol,
fentanyl, flavoxate, fluopromazine, flupenthixol, fluphenazine, flurazepam, haloperidol,
hydroxyzine, hyoscine, ibogaine, imipramine, indapamine, iprindole, isothipendyl, isox-
suprine, ketanserin, laudanosine, lidocaine, lofepramine, loxapine, maprotiline, mecamy-
lamine, meclophenoxate, meclozine, medazepam, mephentermine, mepivacaine, mep-
tazinol, mepyramine, mesoridazine, metaraminol, methadone, methamphetamine,
methapyrilene, methdilazene, methotrimeprazine, methoxamine, methoxyphenamine,
methoxypromazine, methylephedrine, methylergonovine, methysergide, metoclopramide,
metopimazine, metoprolol, mianserin, morazone, nadolol, nalorphine, naloxone, napha-
zoline, nicotine, nifedipine, nomifensine, nortriptyline, noscapine, orphenadrine, oxeladin,
oxprenolol, oxymetazolin, papaverine, pargyline, pecazine, penbutolol, pentazocine, pen-
thienate, pericyazine, perphenazine, phenadoxone, phenampromide, phenazocine, phen-
butrazate, phendimetrazine, phenelzine, phenglutarimide, phenindamine, pheniramine,
phenmetrazine, phenomorphan, phenoperidine, phenothiazine, phenoxybenzamine, phen-
tolamine, phenylephrine, phenyltoloxamine, physostigmine, piminodine, pimozide, pin-
dolol, pipamazine, pipazethate, piperacetazine, piperidolate, pipradol, pirenzepine, piri-
tramide, pizotifen, practolol, pramoxine, prazosin, prenylamine, prilocaine, primaquine,
proadifen, procainamide, procaine, prochlorperazine, procyclidine, proheptazine, prolin-
tane, promazine, promethazine, pronethalol, properidine, propiomazine, propranolol, pro-
thipendyl, protriptyline, proxymetacaine, pseudoephedrine, pyrimethamine, quinidine,
quinine, ranitidine, rescinnamine, sotalol, tacrine, terazosin, terbutaline, terfenadine,
thenyldiamine, thiethylperazine, thiopropazate, thioproperazine, thioridazine, thiothix-
ene, thonzylamine, timolol, tocainide, tolpropamine, tolycaine, tranylcypromine, trazo-
 done, trifluoperazine, trifluperidol, trimeperidine, trimeprazine, trimethobenzamide,
trimethoprim, 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
Sample preparation: Dissolve in MeOH, inject a 1 |xL aliquot.

HPLCVARIABLES
Column: 150 X 1 3 jim Hitachi-Gel 3011 porous polymer (Hitachi)
Mobile phase: MeOH: ammonia 99:1
Flow rate: 0.03
Injection volume: 1
Detector: UV 254

CHROMATOGRAM
Retention time: 3.56

OTHER SUBSTANCES
Also analyzed: acetaminophen, aspirin, bucetin (3-hydroxy-p-butyrophenetidine), caffeine,
dipyrone (sulpyrin), ethenzamide (o-ethoxybenzamide), mefenamic acid, phenacetin, sal-
icylamide, salicylic acid, theobromine

KEYWORDS
semi-micro; porous polymer

REFERENCE
Matsushima, Y.; Nagata, Y.; Niyomura, M.; Takakusagi, K.; Takai, N. Analysis of antipyretics by sem-
imicro liquid chromatography. J.Chromatogr., 1985, 332, 269-273

SAMPLE
Matrix: urine
Sample preparation: Dilute urine 10-fold with 5 |xg/mL p-hydroxyethyltheophylline in
water, mix, centrifuge at 14000 rpm for 2 min, inject a 25 |JLL aliquot of the supernatant.

HPLCVARIABLES
Guard column: 40 X 2.5 10 |xm Lichrosorb RP-2
Column: 150 X 4.6 5 jxm Ultrasphere-ODS
Mobile phase: MeCN: THF: 10 mM sodium acetate 3:0.1:96.9 containing 5 mM tetrabu-
tylammonium hydrogen sulfate, pH 4.7
Flow rate: 1.5
Injection volume: 25
Detector: UV 280

CHROMATOGRAM
Retention time: 9
Internal standard: p-hydroxyethyltheophylline (11)
Limit of detection: 1 |xg/mL
OTHER SUBSTANCES
Extracted: metabolites, caffeine, 1,3-dimethyluric acid, 1-methyluric acid, 3-methylxan-
thine

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