J. Sep. Sci. 2008, 31, 1 8 L. Budakova et al.
2008, 31, 1 8
Lucie Budakova1 Original Paper
Hana Brozmanova1
Milan Grundmann1
Jan Fischer2 Simultaneous determination of antiepileptic drugs
1
Department of Clinical
and their two active metabolites by HPLC
Pharmacology, University
Hospital and Medico-Social An HPLC procedure for the determination of lamotrigine (LAM) simultaneously
Faculty, University of Ostrava, with other antiepileptic drugs, primidone (PD), phenobarbital (PB), phenytoin
Ostrava, Czech Republic (DPH), carbamazepine (CMZ), and two active metabolites 2-phenyl-2-ethyl-malon-
2
Department of Analytical amide (PEMA) and 10,11-dihydro-10,11-epoxycarbamazepine (EPO) was developed
Chemistry, Faculty of Chemical
and validated. The method involves an ordinary RP system and a liquid liquid
Technology, University of
Pardubice, Pardubice, Czech
extraction. The mobile phase consisting of water/ACN/methanol/triethylamine in
Republic the ratio 72 : 23 : 5 : 0.1 with pH 7.0 was selected as the best one after the assays test-
ing both pH and triethylamine contents. UV detection was carried out at a wave-
length of 220 nm and the whole analysis took 15 min. The method was linear in the
range of 0.5 25 mg/L for PEMA and LAM; 1.25 25 mg/L for PD and CMZ; 0.625
12.5 mg/L for EPO; 1.5 60 mg/L for PB; and 1.25 50 mg/L for DPH, respectively.
Within-day CV% and between-day CV% were within 10%. The developed HPLC
method can be used for routine therapeutic drug monitoring both in children and
adults.
Keywords: Antiepileptics / HPLC / Lamotrigine /
Received: June 11, 2007; revised: August 24, 2007; accepted: August 24, 2007
DOI 10.1002/jssc.200700253
1 Introduction as the simultaneous determination of the other concom-
itantly administered AEDs and their primary metabolites
Therapeutic drug monitoring (TDM) of antiepileptic in a single run, is of a great relevance.
drugs (AEDs) is necessary to optimize the patient's clini- LAM (3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine)
cal outcome by managing their medication regimen is a broad spectrum AED of the third generation used in
with the assistance of measured drug concentration. The the general treatment of the epilepsy in monotherapy or
concept based on the assumption that drug concentra- in combinations with other AEDs [2]. When introduced
tion correlates better with clinical effects than the dose. to clinical praxis, the TDM of LAM was not considered
TDM is more important for drugs with a narrow thera- necessary and the target therapeutic range was not
peutic range, where a correlation has been established defined. Nowadays, it is well known that the estimation
between drug concentration and its therapeutic and of drug level enables controlling the compliance and
toxic effects [1]. serves as a useful tool in co-medicated patients, especially
Primidone (PD), phenobarbital (PB), phenytoin (DPH), in the cases when enzyme-inducing drugs such as CMZ,
and carbamazepine (CMZ) are AEDs of the first and sec- DPH, or PB are withdrawn, or added. Co-medication with
ond generation, which have been widely used. Lamotri- valproic acid (VPA) may influence the elimination of LAM
gine (LAM) is a newer AED, which was recently intro- due to the inhibition of hepatic glucuronic enzymes and
duced in therapeutic practice. Its determination as well significant increase in serum concentrations was
observed [3 5].
Correspondence: Lucie Budakova, Department of Clinical Phar- Different studies reported a wide range of serum con-
macology, University Hospital of Ostrava, 17. listopadu 1790, centrations associated with a seizure control. Based on
708 52 Ostrava, Czech Republic preclinical data, the therapeutic serum concentration
E-mail: lucie.pavlikova@fnspo.cz
range was originally stated to be 1 4 mg/L. However,
Fax: 420-59-698-4399
many patients require higher concentrations. The range
Abbreviations: AED, antiepileptic drug; CMZ, carbamazepine; of 3 14 mg/L was proposed by Morris et al. [6].
DPH, phenytoin; EPO, 10,11-dihydro-10,11-epoxycarbamaze- Therapeutic serum concentration range of other AEDs
pine; IS, internal standard; PEMA, 2-phenyl-2-ethyl-malonamide; has been precisely documented anywhere and is pre-
LAM, lamotrigine; PB, phenobarbital; PD, primidone;TCA, tri-
chloroacetic acid; TDM, therapeutic drug monitoring; TEA, trie- sented in Table 1 [6, 7]. CMZ and PD metabolites were
thylamine; VPA, valproic acid studied according to their contribution to both the seiz-
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
1 2 L. Budakova et al.
Table 1. Therapeutic drug concentration range of AEDs
(mg/L) [6, 7]
Drug Therapeutic drug concentra-
tion range (mg/L)
PEMA Not available
PD 5 15
PB 15 35
LAM 3 14
DPH 10 20
CMZ 49
CMZ + EPO a12
ure control and toxicity. The effect of 10,11-dihydro-
10,11-epoxycarbamazepine (EPO) as the active metabo-
lite of CMZ is well documented by many authors [8 17];
pointed out, that in most of the cases the sum of
CMZ + EPO plasma level better correlates with therapeu-
tic and toxic effects than plasma level of the parent drug
measured alone. Effective metabolites of PD, PB, and
mainly 2-phenyl-2-ethyl-malonamide (PEMA) have not
been studied so extensively [18 20]. In a group of
patients on long-term combination therapy interrela-
tions of PD with other co-medicated drugs were observed.
While the share of PD and PB is highly influenced by
enzyme inductor drugs such as DPH and CMZ, PEMA usu-
ally represents one third of the sum (PD + PB + PEMA)
and its share is relatively stable and unchanged in spite
of various co-medication [21].
LAM was measured in biological fluids by the GC
method [22 26] and by the immunoanalytical methods
[27]. HPLC procedures were used for LAM analyses in bio-
logical fluids and brain tissue with increasing frequency
[28 34]. Various HPLC techniques for simultaneously
determining the plasma concentrations of other AEDs as
CMZ, DPH, PB, and VPA have been also reported [35 42].
Some HPLC methods even permit simultaneous deter-
mination of LAM, other AEDs and their principal metabo-
lites in one run [37, 43 47]. Some problems connected
with different chemical properties of AEDs can occur.
Although most of them are weak acids, LAM represents
the base.
HPLC method for the determination of LAM with other
AEDs (PD, PB, CMZ, DPH) and two active metabolites,
PEMA and EPO, extends previously used method [21].
In this study, the conditions were tested for the simul-
taneous analysis of LAM and other AEDs (PD, PB, DPH,
and CMZ) and their two active metabolites PEMA and
EPO by changing pH of mobile phase and by using differ-
ent triethylamine (TEA) concentrations in the mobile
phase. The analysis with the optimal conditions was fur-
ther tested and validated for routine analysis according
to ref. [48].
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci.
2 Experimental
2.1 Chemicals and standards
Water for HPLC and TEA (analytical grade) was obtained
from Fluka (Buchs, Switzerland). ACN and methanol for
HPLC (gradient grade) were purchased from Merck
(Darmstadt, Germany), diethyl ether, phosphoric acid,
and sodium dihydrogenphosphate, all of analytical
grade, were purchased from Lachema (Brno, Czech
Republic).
LAM was kindly provided by GlaxoSmithKline (Prague,
Czech Republic). Other reference materials as PB, DPH,
CMZ, PD, EPO, PEMA, and 5-ethyl-5-p-tolylbarbituric acid
(used as internal standard (IS)) were obtained from Fluka
(Buchs). All substances are stable from 208C to 1008C.
2.2 Preparation of stock solutions and standard
solutions
The stock solutions of all analyzed compounds (parent
drugs and metabolites) were prepared in methanol at the
following concentration levels: 1 g/L of PB and DPH and
0.5 g/L of CMZ, PD, LAM, EPO, and PEMA, respectively.
The concentration of methanol stock solution of IS 5-
ethyl-5-p-tolylbarbituric acid was 10 mg/mL and it was
diluted for ten times to prepare the working solution.
Standard solutions for all drugs and metabolites were
prepared at seven concentration levels from the stock sol-
utions by diluting with methanol. Concentration ranges
were as follows (0.5 25 mg/L for PEMA and LAM; 1.25
25 mg/L for PD and CMZ; 0.625 12.5 mg/L for EPO; 3
60 mg/L for PB; and 2.5 50 mg/L in the case of DPH). All
stock solutions, standard solutions and working solu-
tions of IS were kept at 208C.
2.3 Equipment and chromatographic conditions
The chromatographic equipment consisting of isocratic
pump P 1500, autosampler AS 1000, and variable wave-
length detector UV 1000 (all Spectra Physic, San Jose,
USA) was used. The glass column (36150 mm) with sta-
tionary phase Separon SGX C18, 5 lm (Tessek, Prag,
Czech Republic) was used. The analyses were performed
at laboratory temperature. The injected volume was
20 lL and the UV signal was registered at 220 nm.
As the mobile phase was used ternary mixtures of
water/ACN/methanol in ratio 72 : 23 : 5 (by volume) with
addition of TEA as a modificator in the concentrations
10, 40, 100, and 300 lL TEA/100 mL. The pH of final
mobile phase was then adjusted by phosphoric acid in
the range of 3.5 7.0 by 0.5 units. The mobile phase flow
rate was set at 1 mL/min. For the HPLC method optimiza-
tion, retention factors of each AED and their resolution
were calculated.
www.jss-journal.com
J. Sep. Sci. 2008, 31, 1 8
For the optimization of separation, the mixture of IS
and AEDs was used as described as follows: 50 lL of both
IS working solution and standard solutions (third con-
centration level in the calibration series) were put into
glass test tubes and evaporated to dryness at 808C. The
residue was dissolved in 20 lL methanol for HPLC and
200 lL water for HPLC.
2.4 Validation of method
Both the 50 lL of IS working solution and standard solu-
tions in tested levels (all seven levels were used for the
linearity testing and three concentration levels, respec-
tively, for accuracy and precision testing) were evapo-
rated to dryness at 808C in glass test tubes. Then 50 lL of
drug-free serum from a nonmedicated volunteer and the
same volume of 1 mol/L NaH2PO4 were added. The mix-
ture was extracted with 1 mL diethylether. The upper
ether layer was, after centrifugation, transferred to a
small clean tube (Eppendorf) and evaporated to dryness.
The residue was reconstituted in 20 lL methanol and
200 lL water for HPLC.
The accuracy and precision of the method were eval-
uated by analyzing drug-free serum with different con-
centrations of AEDs. The within-day reproducibility was
determined using drug-free serum spiked with three con-
centrations of AEDs (1, 10, 25 mg/L for PEMA and LAM;
2.5, 10, 25 mg/L for PD and CMZ; 1.25, 5, 12.5 mg/L for
EPO; 3, 24, 60 mg/L for PB; and 2.5, 20, 50 mg/L for DPH)
which were analyzed six times on the same day. The
between-day reproducibility was determined by analyz-
ing three independent samples in duplicate during six
different days. LOD was determined as the concentra-
tions corresponding at an S/N of 3:1. Limit of quantifica-
tion (LLOQ) was verified on the basis of the measurement
the lowest concentrations of each analyte with the
defined accuracy (testing for the systematic error) and
precision (less than 20%).
2.5 Preparation of patient samples
The IS working solution (50 lL) was mixed with 50 lL of
patient serum and with 50 lL of 1 mol/L NaH2PO4. The
mixture was extracted with 1 mL diethylether. After cen-
trifugation, the upper ether layer was transferred to a
small Eppendorf tube and evaporated to dryness. The res-
idue was reconstituted in 20 lL methanol and 200 lL
water for HPLC.
3 Results and discussion
3.1 Optimization of separation
Both the pH and concentration of TEA in the mobile
phase play an important role in the retention of LAM and
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Liquid Chromatography 3 4 L. Budakova et al. J. Sep. Sci. 2008, 31, 1 8
other AEDs. Therefore, careful optimization of the
mobile phase composition is necessary. Four different
concentrations of TEA in the mobile phase (10, 40, 100,
and 300 lL/100 mL) were tested (Figs. 1A D). Graphs of
Figs. 1A D demonstrate the dependences of the reten-
tion factors of all AEDs and IS on pH in the mobile phases
with different concentrations of TEA. TEA may saturate
free silanol groups of the stationary phase. This leads to a
decrease in both the asymmetry of the peaks and the
sorption of basic compounds. In the mobile phases with
a low TEA concentration (10 and 40 lL/100 mL), the
retention factor of LAM shows big differences caused
even by small changes of pH (Figs. 1A and B). In the
mobile phases with higher TEA contents (100 and 300 lL/
100 mL) the retention of LAM shows smaller changes.
These mobile phases are more convenient and can be
used for the quantitative measurements.
As shown in the Fig. 1, the migration order of LAM is
also highly affected by pH. The pH of the mobile phase is
another parameter, which must be optimized precisely.
For this purpose, the resolution of critical pairs of LAM
peak and its nearest peak was calculated and plots of res-
olution versus pH were constructed. The recommended
value of this parameter considering more than 90% sep-
aration of two peaks is 1.5 and higher. Figures 2A and B
show the dependence of the resolution for LAM and the
nearest peak on the pH of the mobile phase with differ-
ent TEA contents. Their well-done separation is the cru- Figure 2. Dependencies of resolution of LAM and the near-
est peak on the mobile phase pH for the selected different
cial point of the whole analysis.
concentrations of TEA in the mobile phase. Symbols: (A) G
Based on the series of measurements in mobile phases 10 lL TEA/100 mL, f 40 lL TEA/100 mL; (B) g 100 lL TEA/
with different pH and concentration of TEA, the mobile 100 mL, 9 300 lL TEA/100 mL.
phase consisting of water/ACN/methanol mixture
72 : 23 : 5, with 100 lL TEA/100 mL and pH = 7 was
selected as an optimal for validation of method and for lyzed AEDs calculated by means of S/N parameter S/N = 3
quantitative measurements. In this mobile phase all were in the range of 0.087 0.402 mg/L. LLOQs of AEDs
AEDs are adequately separated and the analysis time is and metabolites for this purpose were given as the lowest
acceptable. Figure 3 illustrates chromatogram of the analyzed concentrations and varied between 0.5 and
drug-free serum spiked with 10 mg/L for PEMA; 20 mg/L 1.5 mg/L with CV% below 20% for all analytes (Table 2)
for PD and CMZ; 48 mg/L for PB; 10 mg/L for LAM; and met the criteria for the therapeutic ranges listed in
1.25 mg/L for EPO; and 40 mg/L for DPH. Table 1. Testing of accuracy of LLOQ did not detect the
Figure 1. Dependencies of the retention factors of AEDs presence of systematic error.
and metabolites and IS on the mobile phase pH for the As shown in Table 3, the within-day and between-day
3.2 Validation protocol of method selected different concentrations of TEA in the mobile
CV% were below 10% at all concentration levels and var-
phase. Concentrations of TEA in the mobile phase: (A) 10 lL
Validation parameters such as selectivity, accuracy, pre- TEA/100 mL, (B) 40 lL TEA/100 mL, (C) 100 lL TEA/ ied between 1.6 and 10%. The accuracy, expressed by the
cision, linearity, and detection and quantification limits 100 mL, (D) 300 lL TEA/100 mL. Analytes: g PEMA, 0 LAM, bias, varied between 9.8% and +9.6%.
were tested. The series of calibration solutions were pre- + DPH, CMZ, IS, 6 EPO, f PD, h PB. The procedure of extraction of the analytes from
pared as described in Section 2 and separated under opti- plasma was described in the Section 2.4. Diethylether
mal conditions described above. The concentrations of square method for all AEDs and metabolites. The y-sec- was selected because of its good extraction capacity and
AEDs responding to their concentrations in serum were tion of each AED was tested by means of QC Expert (Trilo- low boiling point, which affords rapid evaporation even
as follows: 0.5 25 mg/L for PEMA and LAM; 1.25 25 mg/ bit, Pardubice, Czech Republic) and data analysis shows, at room temperature. In contrast to the deproteinization
L for PD and CMZ; 0.625 12.5 mg/L for EPO; 1.5 60 mg/L that the y-section of each AED is statistically nonsignifi- agents like ACN, perchloric acid, or TCA, where the
for PB; and 1.25 50 mg/L for DPH, respectively. cant. Regression equations and correlation coefficients serum protein cannot be entirely removed, this kind of
for each AED are summarized in Table 2. Good correla- extraction enables isolation of drugs with negligible
The plots of ratio of peak area of each drug and IS peak
area versus AED concentration were constructed and tion (r A0.99) between the peak area ratio and concentra- amounts of soluble proteins. To obtain the extraction
regression straight lines were calculated using least tion over all the tested ranges was found. LODs of ana- yield values, the concentrations corresponding to the
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J. Sep. Sci. 2008, 31, 1 8 Liquid Chromatography 5 6 L. Budakova et al. J. Sep. Sci. 2008, 31, 1 8
Table 2 Calibration parameters of the analytes Table 4. Recovery data for the L L extraction step and for the freeze-thaw cycles (n = 6)
Analyte Calibration parameters LOD LLOQ measurement, n = 6 L L extraction Freeze-thaw, two times Freeze-thaw, five times
(mg/L)
Range Equation cali- r Added Found l SD CV% Accuracy Analyte Concentration Recovery R CV% Concentration Recovery Concentration Recovery
(mg/L) bration curve (mg/L) (mg/L) test added (mg/L) (%) found (mg/L) (%) found (mg/L) (%)
PEMA 0.5 25 y = 0.028x 0.9994 0.159 0.5 0.51 l 0.09 16.8 Passed PEMA 10 70.4 3.9 9.2 92 9.1 91
PD 1.25 25 y = 0.059x 0.9993 0.197 1.25 1.24 l 0.18 14.5 Passed PD 10 70.6 3.7 9.1 91 9.2 92
PB 1.5 60 y = 0.076x 0.9999 0.268 1.5 1.55 l 0.21 13.3 Passed PB 24 84.3 5.4 23.4 97.5 22.9 95.4
LAM 0.5 25 y = 0.129x 0.9962 0.087 0.5 0.53 l 0.07 13.6 Passed LAM 10 97.0 5.3 9.7 97 9.3 93
EPO 0.625 12.5 y = 0.244x 0.995 0.106 0.625 0.62 l 0.07 11.3 Passed EPO 5 66.3 8.3 4.7 94 4.6 92
DPH 1.25 50 y = 0.105x 0.9997 0.402 1.25 1.23 l 0.18 14.4 Passed IS 0.5 86.8 2.1
CMZ 1.25 25 y = 0.225x 0.9996 0.132 1.25 1.24 l 0.23 18.3 Passed DPH 20 75.6 3.8 18.7 93.5 19.1 95.5
CMZ 10 80.7 5.3 9.4 94 9.3 93
Table 3: Within-day and between-day accuracy and precision of the HPLC method for the determination of AEDs in drug-free
serum (n = 6) Table 5. Drugs potentially co-administered with the five anti-
epileptics drugs and two metabolites that were examined for
Concentration Within-day Between-day possible interference with this method. The drugs analyzed
added (mg/L) by the HPLC method not detected within 30 min from injec-
Found concentra- CV% Bias% Found concentra- CV% Bias% tion
tion l SD (mg/L) tion l SD (mg/L)
Effective substance
PEMA
1 1.03 l 0.08 7.8 2.5 1.06 l 0.10 9.6 5.8 Acetylsalicyl acid
10 9.72 l 0.80 8.2 2.8 9.68 l 0.95 9.8 3.2 Baclofene
25 22.6 l 1.27 5.6 9.6 22.9 l 2.1 9.1 8.3 Chlorpromazine
PD Clonazepam
2.5 2.55 l 0.23 9.0 2 2.51 l 0.24 9.5 0.3 Dexamethasone
10 10.44 l 1.05 10.0 4.4 10.36 l 0.65 6.3 3.6 Digoxin
25 25.6 l 0.89 3.5 2.5 26.2 l 2.5 9.5 4.9 Diltiazem
Doxepin
PB Gabapentin
3 3.3 l 0.3 9.0 9.8 3.3 l 0.13 3.9 8.9 Haloperidol
24 24.68 l 1.92 7.8 2.9 24.98 l 0.41 1.6 4.1 Hydrochlorothiazide
60 65.5 l 6.17 9.4 9.1 56.6 l 3.9 6.9 5.8 Ibuprofen
Indomethacin
LAM Imipramine
1 1.02 l 0.08 7.8 1.8 1.02 l 0.1 9.4 2 Indomethacin
10 9.67 l 0.36 3.8 3.3 10.52 l 0.65 6.2 5.2 Levomepromazine
25 24.3 l 2.2 9.1 3.0 25.6 l 2.4 9.5 2.3 Naproxen
Nifedipine
EPO Omeprazol
1.25 1.24 l 0.05 4.1 0.7 1.28 l 0.09 6.9 2.7 Paracetamol
5 5.23 l 0.14 2.6 4.7 4.70 l 0.45 9.6 5.9 Pentoxifylin
12.5 12.5 l 0.27 2.2 0.3 12.7 l 0.8 6.0 1.3 Risperidone
Salbutamol
Figure 3. Chromatogram of the drug-free serum spiked with VPA
DPH
10 mg/L for PEMA, 20 mg/L for PD and CMZ; 48 mg/L for Topiramate
2.5 2.42 l 0.20 8.4 3.2 2.55 l 0.19 7.4 2.2
PB; 10 mg/L for LAM; 1.25 mg/L for EPO; and 40 mg/L for
20 20.82 l 0.62 3.0 4.1 20.89 l 1.77 8.5 4.4
DPH. Chromatographic conditions: Separon SGX C 18,
50 54.0 l 3.48 6.5 8.0 54.9 l 5.5 10.0 10.0
5 lm (36150 mm); UV detection at 220 nm; mobile phase:
water/ACN/methanol/TEA 72 : 23 : 5 : 0.1 (by volume), pH 7.0; from the other no AED co-medicated drugs. Twelve fre-
CMZ
flow rate 1 mL/min. quently prescribed drugs were tested and no interfer-
2.5 2.58 l 0.18 6.8 3.3 2.43 l 0.2 8.1 2.7
10 9.73 l 0.51 5.3 2.7 10.97 l 0.66 6.0 9.7 ences were found (Table 5).
25 23.8 l 0.52 2.2 4.7 26.1 l 2.1 8.2 4.3 This method was used in practice for the determina-
number of freeze-thaw steps. The found recoveries were tion AEDs and their metabolites in the routine TDM in
higher than 90% and are almost not dependent on the epileptic patients. About 3000 patient samples have been
middle point of calibration curve were analyzed (10 mg/L Because of the possible freezing of patient's serum number of freeze-thaw steps. Table 4 summarizes results so far analyzed by this method and about half of all sam-
for PEMA, PD, LAM, and CMZ, 5 mg/L for EPO, 24 mg/L for before its processing and analysis, the recovery of freeze- of these measurements. ples are infant samples. The results of analyses of ten
PB, 20 mg/L for DPH, and 0.5 mg/L for IS). Extraction thaw procedure was calculated. The serum sample When the drug-free serum originating from non- infant samples with different medication scheme (No. 1,
yield, expressed by the recovery R (%) was estimated on spiked at a middle concentration level (10 mg/L for treated volunteer was analyzed, no interfering peak PD + PB + LAM; No. 2, LAM + DPH; Nos. 3, 6, 7, 9, and 10,
the basis of six determinations and the recoveries varied PEMA, PD, LAM, and CMZ, 5 mg/L for EPO, 24 mg/L for PB, from serum could be seen on the chromatogram (Fig. 4). CMZ + LAM; No. 4, PD + PB + DPH; Nos. 5 and 8,
between 66.3 and 97% (Table 4). and 20 mg/L for DPH) was two or five times treated by the The method was tested for the possible interferences LAM + DPH) are presented in Table 6. Figure 5 shows the
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J. Sep. Sci. 2008, 31, 1 8 Liquid Chromatography 7 8
Figure 5. Typical chromatogram of serum of patient treated
by combination of AEDs (PD, LAM, DPH, and CMZ). The
Figure 4. Typical chromatogram of drug-free serum. Chro-
found levels of AEDs are 5.1 mg/L PD, 6.2 mg/L LAM,
matographic conditions: Separon SGX C 18, 5 lm
4.9 mg/L, 1.7 mg/L EPO CMZ, and 22.4 mg/L DPH. Chroma-
(36150 mm); UV detection at 220 nm; mobile phase: water/
tographic conditions: Separon SGX C18, 5 lm,
ACN/methanol/TEA 72 : 23 : 5 : 0.1 (by volume), pH 7.0; flow
(36150 mm); UV detection at 220 nm; mobile phase: water/
rate 1 mL/min.
ACN/methanol/TEA 72 : 23 : 5 : 0.1 (by volume), pH 7.0; flow
rate 1 mL/min.
Table 6. Results of analysis of individual infant samples
Sample Concentration of AEDs and metabolites (mg/L)
(patient) hand, requirements of a routine laboratory, where a
No PEMA PD PB EPO LAM DPH CMZ large number of samples have to be analyzed every day,
1 2.1 2.1 2.2 17.7 are short analysis times, simple instrumentation, and
2 3.1 10.7 chromatographic conditions, as well as an easily applica-
3 1.3 3.7 5.8 ble technique, without affecting the accuracy and repro-
4 8.4 6.2 19.1 12.0
5 1.0 5.5
ducibility of the analytical methods.
6 0.9 3.9 8.0 The tested method involves an ordinary RP HPLC sys-
7 1.3 7.1 8.6 tem and the liquid liquid extraction with diethylether.
8 7.6 4.2
No problems concerning the contamination of the col-
9 1.1 5.5 6.4
10 0.8 10.4 5.0 umn were observed. The concentration of TEA in the
mobile phase and its pH was carefully optimized. It was
found that only higher TEA concentrations (above 40 lL/
100 mL) in the mobile phase lead to sufficient saturation
chromatogram of the serum sample of the patient of silanol groups and thus to stable position of LAM on
treated with PD, LAM, CMZ, and DPH. the chromatogram over a broad range of pH, when pH of
the mobile phase was optimized. The mixture consisting
of water/ACN/methanol/TEA in the ratio 72 : 23 : 5 : 0.1
4 Concluding remarks
with pH = 7 was selected as the universal mobile phase
The aim of this study was to develop and validate a new for both basic drugs and acid drugs, which were ana-
analytical HPLC method for the determination of LAM lyzed.
simultaneously with other AEDs PD, PB, DPH, and CMZ Preparation of patient samples is rapid, simple, accu-
and two of their active metabolites PEMA and EPO for rate, and reproducible. A low volume of a sample, only
routine TDM in patients. The therapeutic efficacy of 50 lL of blood serum which is needed for the whole anal-
AEDs depends on the achievement of well-defined ysis, is highly convenient namely for TDM in children.
plasma concentrations. A validated analytical method is The developed HPLC method allows simultaneous anal-
essential to yield results that satisfactorily allow the ysis of five AEDs with their two active metabolites and
monitoring of patients during therapy. On the other now is used for the routine TDM of epileptic patients.
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
L. Budakova et al.
Jan Fischer thanks the Ministry of Education, Youth and Sports of
the Czech Republic for support by the project No.
MSM0021627502.
5 Reference
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