eNS-active Drugs· Hypnotics Psychotropics· Sedatives
A sensitive and selective HPLC method for estimation
of lamotrigine in human plasma and saliva: application to plasma-saliva correlation in epileptic patients Suruli\-;el Rajan Mallayasamyl, Karthik Arumugam l , Tarun faint, Thiyagu Rajakannanl, Krishnamurthy Bhatl, Padma Gurumadhavrao l , Ramakrishna Devarakonda 2 I
2
Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, India
Covidien/Mallinckrodt, Inc, Clinical Research Division, Hazelwood, Missouri, USA
Correspondence to: Surulivel Rajan Mallayasamy, M Pharm, Department of Pharmacy Practice, Manipa! College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, 576104, India; e-mail:
[email protected]
Abstract A sensitive and selective high perfor
mance liquid chromatographic (HPLC) method was developed and validated for estimation oflamotrigine (CAS 84057 -84-1) in human plasma and saliva. The chro matographic separation was achieved with a reversed phase column and a mo bile phase consisting of acetonitrile and 20 mM ammonium acetate buffer pH 6.5 (30: 70) with a flow rate of 1 mLimin. The calibration curve was linear within the working range for both plasma and sali va. The validated method has been suc
cessfully applied for a study of lamotri gine in human plasma and saliva to establish the correlation between these two matrices. A scatter plot of plasma versus salivary lamotrigine concentra tions showed a good linear relationship between them (Pearson correlation co efficient, r = 0.6832, P < 0.001).
1. Introduction Lamotrigine (CAS 84057-84-1) is an antiepileptic drug from the phenyl triazine class, chemically unrelated to existing antiepileptic drugs. Its chemical name is 3,5 diamino-6- (2,3 -dichlorophenyJ) -as- triazine. Lamotrigine is indicated as adjunctive therapy for partial seizures, the generalized seizures of Lennox-Gastaut syndrome, and primary generalized tonic-clonic seizures in adult and pediatric patients = 2 years of age [1, 2] It is also in dicated for conversion to monotherapy in adults with partial seizures who are receiving treatment with phe nobarbital, primidone, or valproate as the single AED [3]. The therapeutic range of lamotrigine is not clearly established. Earlier clinical trials reported a range of 1 4 !1g/mL which did not appear to correlate well "'lith clinical outcome [4]. Hirsch et al. reported that concen trations up to 10 !1g/mL were tolerated and concentra tions beyond 20 I-tg/mL were associated with adverse ef fects of the dll.lg [5]. Due to differences in available reports, there is a need to assess the therapeutic range of lamotrigine. There were reports on the usefulness of
Keywords • • • •
Antiepileptic drug CAS 84057-84-1 HPLC method Lamotrigine, pharmaco kinetics, plasma-saliva correlation
Arzneimittelforschung 20 I 0;60 (10) :599-606
salivary monitoring of lamotrigine and correlation of saliva and plasma lamotrigine concentrations [6-10]. Lamotrigine is extensively used in the pediatric popula tion for seizure conditions. Salivary monitoring of lamo trigine might serve as a non-invasive method for drug level monitoring in these patients. A study was planned to establish the correlation between salivary and plasma lamotrigine concentrations in patients undergoing treatment with this drug. There was a need for the de velopment of a simple HPLC method for the purpose of the study. Several methods have been reported in the literature for determination of lamotrigine in serum and plasma using HPLC methods which have their own advantages and disadvantages. In some reported HPLC methods for the determination of lamotrigine, a .solid phase extrac tion method was used for extraction of drug from the biological matrices [1l-13]. Use of solid phase extrac tion for sample processing makes these 'methods cost lier and cumbersome for routine situations like thera peutic drug monitoring. In a HPLC method reported
Arzneimittelforschung 2010;60(10) :599-606 © ECV . Edil io Cantor Verlag, Aulendorf (Germany)
Mallayasamy et al. - Lamotrigine
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eNS-active Drugs Hypnotics· Psychotropics . Sedatives
for lamotrigine analysis in the blood and brain of lab oratory animals, the working range reported was 0.1 15.0 mg/L [14J. Lamotrigine concentrations in plasma were reported above this range in some studies and therefore this method may not be suitable when plasma concentrations are beyond this level. In a method reported for simultaneous liquid chro matographic determination of lamotrigine, oxcarbazine and felbama!e in plasma, the processing volume was 500 f.lL [15). The sample processing volume appears to be greater. with this method. In case of studies where pediatric population is involved the sample available will be less. In such instances, if the processing volumes are in the order of 100-200 flL, it might allow collection of a minimwu volume of samples from the study sub jects. In some of the methods reported for rapid liquid chromatographic determination of lamotrigine in plas ma, tertiary and quaternary solvent systems were em ployed [16-17]. Use of a binary solvent system wherever possible instead of a tertiary or quaternary solvent sys tem makes the analytical process simple and cost effec tive as it reduces solvents consumption. In a HPLC method reported for the determination of lamotrigine in small volume of samples, the retention time for lamo trigine and the internal standard was 6 and 7 min re spectively [18]. The separation achieved was inadequate for the lamotrigine and the internal standard. In a method for determination of lamotrigine in hu man plasma where protein precipitation technique was employed for extraction of the drug from matrix, the lower limit of quantification (LLOQ) was reported as 1000 ng/mL. This method may not be applicable to all situations as there are reports of plasma concentrations of lamotrigine below 1000 ng/mL [19].
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In a yet another reported method. lamotrigine was quantified using HPLC. This method involved extraction with chloroform-isopropanol after alkalinization with a carbonate buffer. The drug was back extracted to 0.05 % phosphoric acid and chromatographic separation was achieved. [20] An easier extraction procedure with the least number of steps is preferable over the extrac tion procedure with many intermediate steps. As the number of steps increases the chance for variability also increases. A few more reports are also available in the literature for quantification of lamotrigine in biological matrices [21-24]. The present work was aimed to devel op a simple, precise and cost effective HPLC method for quantifying lamotrigine in plasma and saliva. The ad vantages of the present method are its specificity, sim ple one step liquid-liquid extraction procedure, low sample processing volume (200 flL) and the wide quan tification range which would cover the whole range of therapeutic concentrations reported in various clinical studies [25 - 26].
2. Experimental 2.1 Chemicals and reagents Lamotrigine and oxcarbamazepine (CAS 28721-07-5) (internal standard, IS) (Fig. 1) were kindly donated by Torrent Pharma ceuticals limited (Ahmedabad, India) and Amoli Organics (Mumbai, India), respectively. HPLC grade acetonitrile, metha nol and analytical grade ammonium acetate was obtained from Merck Chemicals (Mumbai, India). Acetic acid was obtained from SO Fine Chemicals (Mumbai. India). Water was purified through a Milli-Q UV plus system procured from Millipore (Bedford, MA, USA). All other chemicals used were of analytical grade.
2.2 Chromatographic conditions The analysis was carried out on a Waters HPLC system - 2695 separation Module (Waters Corporation, Wichita, KS, USA) equipped with a 2487 dual UV detector and Millennium Soft ware (Version 4). The column temperature was maintained at 25°C. The chromatographic separation was achieved using In tersil ODS 3V (250 x 4.6 mm) 5 11m column (GL Sciences Inc., Tokyo, Japan). The mobile phase consisting of a mixture of 20 mM ammonium acetate buffer (pH 6.5) and acetonitrile (70: 30 v/v) was filtered through a 0.45 11m membrane filter and used. The flow rate of the mobile phase was maintained at 1.0 mLi min and the detection wavelength was 260 nm with a run time of 15 min.
2.3 Preparation of stock and standard solutions
Fig. 1: Formula of lamotrigine (top) and oxcarbamazepine (bottom).
600
The stock solutions of lamotrigine and the IS, oxcarbamazepine were prepared separately in methanol to yield primary stan dard solutions with a concentration of 1 mg/mL. Working stock solutions were prepared in the range of 1, 2,.5, 10, 20, 50, 100, 200 Jlg/mL and 1,2,4,6, 10,20,50, 100 Jlg/mL for plasma and saliva, respectively, using methanol as the diluent Similarly control stocks were prepared at 3, 80, 160 ~lg/mL and 3, 40, 80 Jlg/mL for plasma and saliva, respectively. The IS working stock solution (l00 Jlg/ml) was prepared by diluting the stock solution with methanol. Arzneimittelforschung 2010;60(10):599-606
Mallayasamy et al. - Lamotrigine
© ECV . Edilio Canror V(,r1ag, Aulendorf (Germany)
eNS-active Drugs· Hypnotics· Psychotropics . Sedatives
2.4 Extraction of lamotrigine from human plasma and saliva 2.4.1 Extraction from human plasma
Institutional ethics committee approval was obtained for col
lecting blood and salivary samples from patients who were on
lamotrigine therapy. Blood samples from patients were col
lected and plasma was separated and stored at -70 °C until ana
lysis. Extraction of lamotrigine from the human plasma sam
ples was carried out using a liquid-liquid extraction (LLE)
technique. 200 ,uL of the human plasma sample was mixed with
10 flL of [S working stock solution (l00 flg/mL) and vortexed for
60 s. Then 2 mL of tertiary methyl butyl ether (TBME) was
added and vortexed for 5 min then centrifuged at 10 000 rpm
for 5 min. The clear 1.8 mL organic layer was separated and
evaporated in a Turbo Vap LV Evaporator (Zymark, Hopkinton,
Ivlf\, USA) at 50°C under a stream of nitrogen. The dried resi
due was then reconstituted with ISO >IL of mobile phase and
100 flL was injected for the HPLC analysis.
2.4.2 Extraction from human saliva
100 flL of blank human saliva was taken in a 1.5 mL Eppendorf
centrifuge tube. 10 >IL of IS working stock solution (l00 flg/mL
of oxcarbamazepine) was added and vortexed for 30 seconds.
Then 300 flL of acetonitrile was added and vortexed for 5 min.
then centrifuged at 10000 rpm for 5 min. 200 flL of clear super
natant was transferred into a vial and 100 flL was subjected to
HPLC analysis.
2.5 Bioanalytical method validation 2.5.1 Selectivity and specificity
The selectivity of the assay method was established using a
minimum of six independent sources (human plasma and sali
va) of the same matrix. The effect of anticoagulants, hemolysis
and lipolysis on the selectivity of the plasma matrix was tested.
The saliva samples were used for analysis without pH adjust
ment. The specificity of the method was evaluated by checking
for interferences by some of the co-administered drugs like car
bamzepine and phenytoin. These drugs were also analyzed
using the same chromatographic method and checked for in
terferences at the retention time of lamotrigine and the internal
standard,
2.5.3 Accuracy and precision Intra-day and inter-day accuracy and precision were deter mined by duplicate analysis of six sets of spiked plasma and saliva samples. Plasma samples were spiked with four different concentrations of lamotrigine viz., 0.1, 0.3, 8, 16 flg/mL for LLOQ, low, medium and high quality control samples. Similarly saliva samples were spiked with four concentrations of lamotri gine viz., 0.1, 0.3, 4, 8 flg/mL. These samples were prepared and analyzed on day one and this procedure was repeated for 3 consecutive days. Acceptance criteria for intra-day, inter-day accuracy should be within 85 -llS % of nominal concentration and precision should be less than 15 % of relative standard de viation (% RSD). At the LLOQ level accuracy should be between 80 and 120 % and % RSD should not exceed 20 %.
2.5.4 Recovery Recovery of lamotrigine in plasma and saliva was evaluated by comparing the mean peak areas of three extracted low, med ium and high quality control samples to the mean peak areas of three neat reference solutions (unextracted). Recovery of ox carbamazepine (IS) was evaluated by comparing the mean peak areas of the extracted samples to the mean peak areas of neat reference solutions (unextracted) of the same concentra tion.
2.5.5 Stability In order to determine the stability of lamotrigine in plasma and saliva samples, they were studied at four different stability con ditions including bench top, freeze-thaw, auto injector and long term stability. Stability samples were examined by repli cate analysis of the low and high plasma QC samples. Bench top stability was carried out by keeping replicates of the low and high plasma QC samples for approximately 12 h at ambi ent temperature. Freeze-thaw stability of the samples was ob tained over three freeze-thaw cycles, by thawing at room tem perature for 2-3 h and refreezing for 12 h for each cycle. Auto sampler stability was tested by analysis of processed and recon stituted QC samples which were stored in the auto sampler tray for 24 h. Long term stability of lamotrigine in human plasma and saliva were tested after storage at -70°C for 30 days. For each concentration and each storage condition, six replicates were analyzed in one set of the batch. The results were com pared to the data for a freshly prepared and processed set of QC samples.
2.5.2 Calibration curve The linearity of the method was evaluated by use of a calibra tion curve in the range of 0.1-20 ,ug/mL and 0.1-10 flg/mL in human plasma and saliva, respectively. The calibration curve was plotted using the peak area ratios of lamotrigine and the internal standard versus the concentration of lamotrigine by least-squares linear regression analysis. The calibration curve requires a coefficient determination (r2) of > 0.98 as per US FDA guidance for bioanalytical method validation [27]. The ac ceptance criteria for each back calculated standard concentra tion should be within 15 % of the nominal concentration except at the LLOQ level where it should not exceed 20 %. Each validation run consisted of a double blank, system suit ability sample, zero standard, calibration curve consisting of eight non-zero samples covering the total range and quality control (QC) samples at three concentrations (n =6, at each concentration). Validation runs were generated on four conse cutive days. Arznei";tte!forschung 2010;60(10) :599-606 © ECV . Edilio Cantor Verlag, Aulendod (Germany)
3. Results 3.1 Selectivity and specificity
Both lamotrigine and the IS were well separated from the endogenous components like plasma and salivary proteins. There were no interferences at the retention time of both lamotrigine and the IS. The peaks were of good shape, completely resolved from the plasma com ponents. A matrix peak was observed in aU the blank human plasma samples tested at the retention time of internal standard. It could be an endogenous interfer ence. The peak area of matrix peak was almost similar in all the plasma samples. The matrix peak was less than 5 % of the peak area of the internal standard which is an acceptable limit as per the US FDA guidance on bioana lyrical method validation [27]. The matrix peak did not
Mallayasamy el aL - Lamolrigine
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eNS-active Drugs· Hypnotics Psychotropics Sedatives
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Mallayasamy et aJ. - Lamolrigine
Arzneimittelforschung 2010;60(lO):599~06 © ECV . Editio Cantor Vedag. Aulendorf (Germany)
eNS-active Drugs Hypnotics· Psychotropics . Sedatives
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