liquid chromatography–tandem mass spectrometry for quantification of lacosamide, an antiepileptic...

Research article

Received: 2 March 2011, Revised: 4 May 2011, Accepted: 15 May 2011 Published online in Wiley Online Library: 7 July 2011

(wileyonlinelibrary.com) DOI 10.1002/bmc.1668

Liquid chromatography–tandem massspectrometry for quantification of lacosamide,an antiepileptic drug, in rat plasma and itsapplication to pharmacokinetic studySoo‐Jin Kima, Tae‐Sung Kooa*, Dong‐Jin Haa, Myoungki Baeka,Sang‐Kil Leeb, Dong‐Soo Shinb and Hongsik Moona

ABSTRACT: A rapid, simple and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) was developed forthe determination of an antiepileptic drug, lacosamide, in rat plasma. The method involves the addition of acetonitrile andinternal standard solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with wa-ter and directly injected into the LC/MS/MS system. The separations were performed on column packed with octadecylsilica(5 µm, 2.0 × 50mm) with 0.1% formic acid and acetonitrile as mobile phase, and the detection was performed on tandemmass spectrometry by the multiple‐reaction monitoring via an electrospray ionization source. The standard curve was linearover the concentration range from 0.3 to 1000ng/mL. The lower limit of quantification was 0.3 ng/mL using 50μL of ratplasma sample. The intra‐ and inter‐assay precision and accuracy were found to be less than 11.7 and 8.8%, respectively.The developed analytical method was successfully applied to the pharmacokinetic study of lacosamide in rats. Copyright ©2011 John Wiley & Sons, Ltd.

Keywords: lacosamide; antiepileptic drug; pharmacokinetics; LC/MS/MS; rat plasma

* Correspondence to: T.‐S. Koo, Life Science R&D Park, SK Holdings Co. Ltd,Daejeon 305–712, Korea. E‐mail: [email protected]

a Life Science R&D Park, SK Holdings Co. Ltd, Daejeon 305‐712, Korea

b Department of Chemistry, Changwon National University, Changwon,641‐773, Korea

37

Introduction

Lacosamide (Vimpat®, UCB Pharma, Brussels, Belgium) is a newantiepileptic drug that was licensed in Europe 2008 and in theUSA in 2009 for the adjunctive treatment of partial‐onset sei-zures with or without secondary generalization in patients withepilepsy aged 16 years and older. Based on clinical trials, lacosa-mide is expected to have low potential for drug interactions andis available in an intravenous formulation that may be used forreplacement therapy in patients temporarily unable to take oralmedication. However, lacosamide has some disadvantages thatinclude the need for slow dose titration and caution before usein patients with pre‐existing cardiac disease because of potentialelectrocardiogram changes, and an apparently narrow effectivedose range (Ben‐Menachem et al., 2007; Cross and Curran,2009; Halasz et al., 2009). Lacosamide is a functionalized aminoacid that acts by selectively enhancing the slow inactivation ofvoltage‐gated sodium channels, resulting in stabilization of hy-perexcitable neuronal membranes and inhibition of repetitiveneuronal firing without exhibiting effects on fast inactivation(Beyreuther et al., 2007; Errington et al., 2008). Lacosamide doesnot affect α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate,kainate, N‐methyl‐D‐aspartate, gamma‐aminobutyric acid A orB, or a variety of dopaminergic, serotonergic, adrenergic, musca-rinic or cannabinoid receptors, and does not block potassium orcalcium currents (Errington et al., 2006).

Generally, dose individualization is thought to be essential inepilepsy therapy. The monitoring of lacosamide is useful foravoiding adverse effects owing to overdose and to establish an

Biomed. Chromatogr. 2012; 26: 371–376 Copyright © 2011 John

individual patient’s therapeutic range (Patsalos et al., 2008).However, only high‐performance liquid chromatography (HPLC)methods for the determination of lacosamide have beenreported (Greenaway et al., 2010, 2011). Greenaway and cowor-kers used HPLC with ultraviolet detection for monitoring thelacosamide in patients with epilepsy, but their method is limitedto application in various pharmacokinetic and pharmacologystudies because of its long analytical run time (27min) and lowsensitivity (lower limit of quantification, LLOQ, 250 ng/mL;Greenaway et al., 2010, 2011). The purpose of this paper wasto develop and validate a simple, rapid and sensitive liquidchromatography‐tandem mass spectrometry (LC/MS/MS) forthe quantitative analysis of lacosamide in rat plasma. Thismethod was successfully applied to a pharmacokinetic studyafter intravenous and oral administration of lacosamide in rats.

Experimental

Reagents and materials

Lacosamide and LCD001 (lacosamide analog, internal standard, IS;Fig. 1) were obtained from ChangWon National University (ChangWon,

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1

HN

NH

OO

O

(b) LCD001

HN

NH

OO

O

(a) Lacosamide

Figure 1. Chemical structures of (a) lacosamide and (b) IS.

S.‐J. Kim et al.

372

Korea). Acetonitrile and methanol (HPLC grade) were purchased fromBurdick & Jackson Inc. (Muskegon, MI, USA) and the other chemicalswere of HPLC grade or the highest quality available. Rat plasma containingsodium heparin as the anticoagulant was prepared in our laboratory.

Preparation of calibration standards and quality controlsolutions

Primary stock solutions of lacosamide (1mg/mL) were prepared inmethanol. The stock solution of lacosamide was serially diluted withacetonitrile to achieve working standard solutions at concentrationsof 0.3, 0.5, 1, 3, 10, 30, 100, 300, 500 and 1000ng/mL. Quality control (QC)solutions at 0.8, 40 and 800 ng/mL were prepared in bulk by dilutingother primary stock solutions. The IS working solution (500 ng/mL)was prepared by diluting an aliquot of stock solution (1mg/mL) ofLCD001 with acetonitrile. All lacosamide and IS solutions were storedat −20°C in glass bottles in the dark.

Sample preparation

Aliquots of 50 μL of blank plasma and calibration standards (or QCsample) or aliquots of 50 μL of rat plasma and acetonitrile weremixed with 100 μL of IS working solution and 300 μL of acetonitrileto induce the precipitation of plasma proteins. The mixture was vigorouslymixed for 10min, followed by centrifugation at 10,000 g for 10minand the aliquot (50 μL) of the supernatant was transferred to well plate.After dilution with 150μL of distilled water, an aliquot (5 μL) was directlyinjected into the LC/MS/MS system.

LC/MS/MS analysis

HPLC was performed using an Agilent 1200 HPLC series (Agilent Tech-nologies, Santa Clara, CA, USA), which consisted of an autosampler, abinary pump, a column oven and a system controller. The compounds

were separated on a Gemini® C18 column (5.0 µm, 2.0mm i.d. × 50mm,

Penomenex, Torrance, CA, USA) with 0.1% formic acid and acetonitrile.The mobile phase was delivered using a gradient elution program:0.1% formic acid (A):acetonitrile (B) = 95:5 (v/v) from 0 to 0.5min,A:B = 20:80 from 1.5 to 2.5min and A:B = 95:5 from 3.0 to 4.5min.The flow rate was 0.3mL/min. The column and autosampler traywere maintained at 25 and 4°C, respectively. The analytical run timewas 4.5min. The eluent was introduced directly into the tandemquadrupole mass spectrometer (API 4000, Applied Biosystems/MDSSCIEX, Foster City, CA, USA) through the turbo ionspray source with

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typical settings as follows: curtain gas, 20 psi; nebulizer gas, 40 psi;turbo gas, 40 psi; ionspray voltage, 5500 V; temperature, 500°C inpositive mode. The declustering potentials of lacosamide and ISwere 61 and 66 V, respectively, and their protonated molecules werefragmented at collision energy of 15 and 37V by collision‐activated disso-ciation with nitrogen gas. Multiple reaction monitoring (MRM) mode wasemployed for the quantification: m/z 251.2→ 108.1 for lacosamide andm/z 265.2→ 74.1 for IS. The analytical data were processed with Analystsoftware (version 1.42; Applied Biosystems/MDS SCIEX).

Method validation

Batches, consisting of duplicate calibration standards at each concentra-tion, were analyzed on three different days to complete the methodvalidation. In each batch, QC samples at 0.3, 0.8, 40 and 800ng/mL wereassayed in sets of six replicates to evaluate the intra‐ and inter‐dayprecision and accuracy. The percentage deviation of the mean from truevalues, expressed as relative error (RE) and the relative standard deviation(RSD), serves as the measure of accuracy and precision, respectively.To investigate the effect of diluting over‐range samples into the calibrationrange, the accuracy and precision of dilution control samples at20 µg/mL (n=4) were assessed by performing a 100‐fold dilution. Accep-tance criteria werewithin ± 15% of accuracy and precision except the LLOQwithin ± 20%.

The matrix effects, extraction recovery and process efficiency wereassessed by analyzing three sets of standards at three concentrations(0.8, 40 and 800 ng/mL) according to the approach of Matuszewskiet al. (2003). The matrix effects for lacosamide and IS were assessed bycomparing mean peak areas of the analyte at three concentrationsspiked after extraction into blank plasma extracts (set 2) with mean peakareas for neat solutions of the analytes in 50% acetonitrile (set 1).The extraction recoveries of lacosamide and IS were determined bycomparing mean peak areas of analytes spiked before extraction intoblank plasma (set 3) with set 2. The process efficiency of lacosamideand ISwas determined by comparing set 3 with set 1 (Buhrman et al., 1996;Koo et al., 2011).

To evaluate the three freeze–thaw cycle stability and room tempera-ture matrix stability, the six replicates of QC samples at each of the lowand high concentrations (0.8 and 800 ng/mL) were subjected to threefreeze–thaw cycles or were stored at room temperature for 4 h beforeprocessing, respectively. The long‐term stability was tested by deter-mining the six aliquots of the low and high concentration QC samplesstored at −70°C for 2weeks. Six replicates of QC samples at each of thelow and high concentrations were processed and stored under auto-sampler conditions at 4°C for 24h, and assayed to assess post‐preparativestability.

Application

The developed LC/MS/MS assay method was used in a pharmacokineticstudy after an intravenous and oral administration of lacosamide to maleSprague–Dawley rats (8weeks of age, body weight 215–239 g, OrientBio, Seongnam, Korea). This study was approved by the InstitutionalAnimal Care and Use Committee of the SK Life Science (Daejeon, Korea).Animals were kept in plastic cages with free access to standard ratdiet (PMI Nutrition International, Brentwood, MO, USA) and water.The animals were maintained at a temperature of 20–26°C with a12 h light/dark cycle and relative humidity of 40–60%. Before thetest, animals were fasted prior to dosing by withholding food over-night, but not water. After dosing, food was withheld for a further4 h. Lacosamide dissolved in vehicle (dimethly sulfoxide:polyethylene gly-col 300:saline=10:27:63, v/v/v) was given as either a single intravenous bo-lus dose via the tail vein or an oral gavage dose at 1mg/kg in the rats(n=4× 2, 4 rats were used for each administration group). Blood samples(200 μL) were collected from the jugular vein at 0.05 (i.v. only), 0.25, 0.5,1, 2, 4, 6, 8 and 24 h after dosing. Blood samples were immediately

Biomed. Chromatogr. 2012; 26: 371–376Wiley & Sons, Ltd.

Bioanalysis of lacosamide in rat plasma with LC/MS/MS

centrifuged at 10,000g for 2min and harvested plasma samples werestored at −70°C until analysis.

The plasma concentration–time profile for each rat was analyzedwith WinNonlin 4.2 (Pharsight, Cary, NC, USA) software to obtainpharmacokinetic parameters. The elimination rate constant (ke) wasdetermined by linear regression of the log–linear portion of the terminalphase. The terminal elimination half‐life (T1/2) was calculated by dividing0.693 by ke. To determine the elimination clearance (Cl ) and steady‐statevolume of distribution (Vss) for lacosamide, a moment analysis was carriedout. The area under the lacosamide concentration in the plasma vsthe time curve from time zero to infinity (AUC0–∞) and the area underthe respective first moment–time curve from time zero to infinity(AUMC0–∞) were calculated using the linear trapezoidal rule and the stan-dard area extrapolation method using WinNonlin 4.2. The Cl, Vss, andMRT (mean residence time) for lacosamide were calculated using the fol-lowing equations:

Cl ¼ doseAUC0−∞

Vss ¼ MRT⋅Cl

MRT ¼ AUMC0−∞

AUC0−∞

Values of Cmax (maximum plasma concentration observed) and Tmax

(Time to Cmax) were directly compiled from the plasma concentration–time curves.

a

b

Figure 2. Product ion mass spect


Results and discussion

LC/MS/MS

The electrospray ionization of lacosamide and IS produced theabundant protonated molecule [MH+]+ at m/z 251.0 and265.2, respectively, under positive ionization conditions, withoutany evidence of fragmentation. [MH+]+ from lacosamide and ISwere selected as the precursor ions and subsequently fragmen-ted in MS/MS mode to obtain the product ion spectra, yieldinguseful structural information (Fig. 2). The fragment ions at m/z107.9 and m/z 74.0 were produced as the prominent productions for lacosamide and IS, respectively. In order to obtainmaximum response and stable product ions, the MS para-meters and collision energies were optimized. Quantitativeanalysis was carried out by MRM at m/z 251.2→ 108.1 for laco-samide and m/z 265.2→ 74.1 for IS.Several stationary and mobile phases were tested and

among them a combination of a C18 column and a mobilephase consisting of 0.1% formic acid and acetonitrile gavethe optimum separation of lacosamide and better sensitivity.Figure 3 shows the representative LC/MS/MS MRM chromato-grams obtained from the analysis of blank rat plasma, plasmaspiked with lacosamide (0.3 ng/mL) and a plasma sampleobtained at 2 h after oral administration of lacosamide to maleSprague–Dawley rat. The analysis of blank plasma samples fromsix different sources did not show any interference at the reten-tion times of lacosamide (3.15min) and IS (3.19min), confirming

HN

NH

O

O

O

m/z 108

HN

NH

O

O

O

m/z 74

ra of (a) lacosamide and (b) IS.

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b

c

a

Figure 3. MRM LC/MS/MS chromatograms of lacosamide (left) and IS (right) obtained by extraction of (a) blank rat plasma, (b) rat plasma samplespiked with lacosamide (0.3 ng/mL) and IS (500 ng/mL), and (c) a plasma sample obtained at 2 h after oral administration of lacosamide at dose of1mg/kg in rat.

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the selectivity of the present method. Using 70% methanol asneedle washing solution for 2 s, the sample carryover effectwas not observed.
Method validation

Calibration curves were obtained over the concentration range0.3–1000 ng/mL of lacosamide in rat plasma. Linear regressionanalysis with a weighting of 1/concentration2 gave the optimumprecision (RSD, 1.9–9.0%) and accuracy (RE, 0.7–8.1%) of thecorresponding calculated concentrations at each level (Table 1).

Table 2 shows a summary of intra‐ and inter‐assay precisionand accuracy data for QC samples containing lacosamide. Inboth intra‐ and inter‐assay, the precision was <11.7% and the


accuracy was <8.8% at four QC levels. These results indicatedthat the present method has acceptable accuracy and precision.The LLOQ was set at 0.3 ng/mL for lacosamide using 50μL of ratplasma. A representative chromatogram of an LLOQ is shown inFig. 3(b) and the signal‐to‐noise ratio for lacosamide is morethan 5 times at 0.3 ng/mL. The back‐calculated concentrationsfor dilution control samples (20 µg/mL) were in good agreementwith the theoretical concentrations. After 100‐fold dilution of di-lution control samples, the RSD and RE for lacosamide were 1.9and 10.2%, respectively, indicating the acceptability of 100‐folddilution prior to analysis.

The matrix effect, the ratio of mean peak areas of set 2 to thoseof set 1, was 101.0 and 101.4% for lacosamide and IS, respectively.This value indicates that lacosamide and IS have practically no


Table 2. Precision and accuracy of the method for the de-termination of lacosamide in rat plasma

Spiked concentration(ng/mL)

Found concentration(ng/mL)

RE(%)

RSD(%)

Intra‐day (n = 6)0.30 0.30 0.9 11.70.80 0.87 8.8 4.740.0 42.5 6.1 1.8800 797 0.4 2.2Inter‐day (n = 3)0.30 0.29 2.1 8.10.80 0.84 5.2 3.540.0 43.5 8.8 3.4800 807 0.9 1.1

Table 3. Stability of lacosamide in rat plasma (n= 6)

Stability conditions Spiked concentration (ng/mL) Found concentration (ng/mL) RE (%) RSD (%)

Post‐operative (24 h, 4°C) 0.80 0.76 4.5 6.4800 829 3.6 1.9

Three freeze–thaw cycles 0.80 0.79 1.2 6.3800 791 1.1 1.1

Bench‐top (4 h, roomtemperature)

0.80 0.78 2.2 5.0800 798 0.3 2.1

Long‐term (2weeks, −20°C) 0.80 0.81 1.0 2.4800 790 1.2 1.0

Time (h)

0 4 8 12 16 20 24Pla

sma

Con

cent

ratio

n of

Lac

osam

ide

(ng/

mL)

1

10

100

1000

10000IntravenousOral

Figure 4. Mean plasma concentration–time curves of lacosamide afterintravenous (●) and oral (○) administration of lacosamide at 1mg/kg torats. Each point represents the mean± SD (n=4).

Table 1. Back‐calculated concentrations of lacosamide in calibration standards prepared in rat plasma (n= 8)

Theoretical concentration (ng/mL)

0.30 0.50 1.00 3.00 10.0 30.0 100 300 500 1000

Mean 0.30 0.48 1.04 3.06 9.91 31.6 103 305 497 919RE (%) 1.3 3.6 3.5 1.9 0.9 5.3 2.8 1.7 0.7 8.1RSD (%) 9.0 6.8 5.5 4.5 3.0 2.4 3.4 3.2 3.4 1.9

y= ax+ b (1/x2, a= 0.00161 ± 0.00002, b= 0.00018 ± 0.00002, r= 0.9975± 0.0005).

Bioanalysis of lacosamide in rat plasma with LC/MS/MS

37

effect on the determination of lacosamide spiked into ratplasma. The mean extraction recoveries determined at low,medium and high QC concentrations were 100.7% for lacosamideand 100.0% for the IS. The overall process efficiency of lacosamidefrom rat plasma was 100.4–101.7%, and consistent at three con-centration levels, and that of IS was 100.3%. The protein precipita-tion with acetonitrile has been successfully applied to theextraction of lacosamide from rat plasma.

The stability of lacosamide during sample handling(three freeze–thaw cycles, short‐term temperature storage andlong‐term stability) and the stability of processed sampleswere evaluated (Table 3). Three freeze–thaw cycles, long‐termstorage for 2weeks at −70°C and short‐term (4 h) storageat room temperature of the QC samples at the low andhigh concentrations before analysis had little effect on the


quantification. Extracted QCs and calibration standards wereallowed to stand at 4°C for 24 h prior to injection withoutaffecting the quantification.

Application of method

The developed assay was applied to a pharmacokinetic study af-ter intravenous and oral administration of lacosamide at a doseof 1mg/kg to eight rats. The mean concentration–time profilesof lacosamide are shown in Fig. 4. After intravenous injection,the systemic clearance (Cl), steady‐state volume of distribution(Vss), area under the curve (AUC0–∞) and the terminal elimi-nation half life (T1/2) were 0.23 ± 0.02 L/h/kg, 0.73 ± 0.04 L/kg,4390 ± 302 ngh/mL and 3.53 ± 0.32 h, respectively. In case oforal gavage dose, the apparent terminal elimination half‐life

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Table 4. Pharmacokinetic parameters after a single intrave-nous (i.v.) and oral (p.o.) administration of lacosamide 1mg/kg in rats, respectively (n=4)

PK parameters i.v. p.o.

Tmax (h) 0.05 0.56Cmax (ng/mL) 1553 975T1/2 (h) 3.53 3.42AUC0–24h (ng h/mL) 4371 4398AUC0–∞ (ng h/mL) 4390 4419Cl (L/h/kg) 0.23 —Vss (L/h/kg) 0.73 —BA (%)a — 100.66aBA=AUCpo/AUCiv

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was 3.42 ± 0.02 h and the AUC0–∞ was 4419 ± 218 ngh/mL. Theabsolute oral bioavailability calculated as AUCpo/AUCiv was100.66% (Table 4).

ConclusionsA rapid, simple and sensitive LC/MS/MS method was developedand validated for the determination of lacosamide concentra-tion in rat plasma, utilizing a protein precipitation and gradientelution monitored in the MRM mode. The developed assay pro-vided a linear dynamic range from 0.3 to 1000 ng/mL andafforded an LLOQ of 0.3 ng/mL using 0.05mL of rat plasma. Toour knowledge, this is the first report of a LC/MS/MS methodfor lacosamide quantification in rat plasma and this methodwas successfully applied for single dose pharmacokinetic study.Therefore it may be suitable for various exploratory and otherpreclinical studies.


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