A sensitive and rapid method is described for determination of clopidogrel carboxylic acid (CCA), the inactive metabolite of the antiplatelet agent clopidogrel in human serum. The analytical procedure involves liquid-liquid extraction of the analyte and an internal standard (phenytoin) with ethyl acetate. Amobile phase consisting of 0.05 M phosphate buffer containing triethylamine (0.5 ml/l; pH 5.7)and acetonitrile (56:44; v/v) was used and chromatographic separation was achieved using a C18analytical column at detector wavelength of 220 nm. The calibration curves were linear over a concentration range of 0.05-10 μg/ml of CCAin human serum. The total run time of analysis was 5.5 min. and the lower limits of detection (LOD) and quantification (LOQ) were 0.02 and 0.05 μg/ml, respectively. The method validation was carried out in terms of specificity, sensitivity, linearity,precision, accuracy and stability. The validated method was applied in a randomized cross-over bioequivalence study of two different clopidogrel preparations in 24 healthy volunteers

Amir Farshchi

Gholamreza Bahrami

Golbarg Ghiasi

English

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ROriginal ArticleIranian Journal of Pharmaceutical Sciences Autumn 2009: 5(4): 231-238ijps.sums.ac.ir High Performance Liquid Chromatographic Determination ofInactive Carboxylic Acid Metabolite of Clopidogrel in HumanSerum: Application to a Bioequivalence StudyGolbarg Ghiasia,b, Amir Farshchia,b,*, Gholamreza BahramiaaSchool of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran bDepartment of Pharmocoeconomy and Pharmaceutical Management, Shcool of Pharmacy,Tehran University of Medical Sciences, Tehran, IranAbstractA sensitive and rapid method is described for determination of clopidogrelcarboxylic acid (CCA), the inactive metabolite of the antiplatelet agent clopidogrelin human serum. The analytical procedure involves liquid-liquid extraction of theanalyte and an internal standard (phenytoin) with ethyl acetate. A mobile phaseconsisting of 0.05 M phosphate buffer containing triethylamine (0.5 ml/l; pH 5.7)and acetonitrile (56:44; v/v) was used and chromatographic separation was achievedusing a C18 analytical column at detector wavelength of 220 nm. The calibrationcurves were linear over a concentration range of 0.05-10 μg/ml of CCA in humanserum. The total run time of analysis was 5.5 min. and the lower limits of detection(LOD) and quantification (LOQ) were 0.02 and 0.05 μg/ml, respectively. Themethod validation was carried out in terms of specificity, sensitivity, linearity,precision, accuracy and stability. The validated method was applied in a randomizedcross-over bioequivalence study of two different clopidogrel preparations in 24 healthyvolunteers. Keywords: Bioequivalence study; Clopidogrel; HPLC; Metabolites.Received: December 8, 2008; Accepted: February 14, 2009.1. IntroductionClopidogrel hydrogen sulfate, methyl (+)-(S)-α-(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-acetate hydrogensulfate, is athienopyridine derivative that irreversiblyblocks ADP receptor. Clopidogrel ischemically related to ticlopidine with superiorside effects profile and dosing requirements.The drug which reduces platelet aggregationis extensively used for prevention ofthrombosis in patients undergoing placementof a coronary stent [1]. The drug is rapidly, butincompletely, absorbed after oraladministration and extensively metabolized toan active metabolite. The parent drug or itsactive metabolite remains undetectable inplasma. The major circulating compound,however, is an inactive carboxylic derivative,which its blood concentration is used todocument the pharmacokinetic profile of*Corresponding author: Amir Farshchi, School of Pharmacy,Kermanshah University of Medical Sciences, Kermanshah, IranTel (+98)831-9188563290; Fax (+98)831-8369850 E-mail:farshchi_a@razi.tums.ac.irG Ghiasi et al / IJPS Autumn 2009; 5(4): 231-238232clopidogrel [2]. A few analytical methodsincluding gas chromatography-massspectrometry (GC-MS) [3], liquidchromatography-mass spectrometry (LC-MS)[4, 5] and high-performance liquidchromatography (HPLC) with UV detection[6, 7] have been published for determinationof the inactive metabolite of clopidogrel in thebiological fluids. In these methods, however,complex two steps extraction methods [3, 4]or long analytical run time (10 min. [4], 16min. [6] and 12 min. [7]) are required. Whilelimit of quantification (LOQ) of 0.01 μg/ml[5], 0.125 μg/ml [6] and 0.2 μg/ml [7] havebeen reported in published papers, in humansingle dose pharmacokinetic studies a LOQof less than 0.1 μg/ml is required formeasuring the analyte up to three half livespost-dose. The present paper which has beenapplied in a bioequivalence study of twodifferent clopidogrel preparations, describesa simple and fast method for analysis of theinactive carboxylic metabolite of clopidogrelin human serum, with sensitivity of 0.05μg/ml and analytical run time of 5.5 min. 2. Materials and methods2.1. Chemicals and standard solutionsClopidogrel carboxylic acid (CCA) (purity99.5%) was synthesized by Ind-SwiftLaboratories Ltd. (Punjab, India). Phenytoin(I.S.) was from Sigma (St. Louis, MO, USA).All reagents used were of analytical gradeexcept acetonitrile which was HPLC grade.Water was glass double-distilled and furtherpurified for HPLC with a Maxima purificationsystem (USF ELGA, England).A stock solution of CCA (1000 μg/ml)was prepared in methanol. Working standardsof the drug (0.5-100 μg/ml) were prepared byserial dilution of the stock solution inmethanol. Working standard solution of theI.S. (7.5 μg/ml) was prepared in methanol. A2N hydrochloric acid solution was preparedFigure 1. Typical chromatograms obtained from an extract of (A) human blank serum spiked with phenytoin as the I.S., (B)human blank serum spiked with 0.05 μg/mL CCA and phenytoin as the I.S., (C and D) serum samples obtained at 6 and 12 hafter a single oral dose of 150 mg clopidogrel from a healthy volunteer containing 1.06 and 0.30 μg/mL of CCA, respectively.Peaks eluted at 3.4 and 4.5 min. correspond to CCA and the I.S., respectively.Quantitative determination of clopidogrel in human serum233in distilled water. All solutions were stored at4 °C and were stable for at least 3 weeks.2.2. Chromatographic conditionsThe HPLC system used consisted of twopumps of Shimadzu LC-10A solvent deliverysystem, a system controller (SCL 10AD), aUV-Vis spectrophotometer detector (SPD-10AD) operated at wavelength of 220 nm, acolumn oven (CTO-10A), a degasser (DGU-3A) and a data processor (C-R4A) all fromShimadzu, Kyoto, Japan. The analyticalcolumn was a Shimpack CLC-ODS(Shimadzu, Kyoto, Japan), 150 mm×4.6 mmI.D., 5 μm particle size which was protectedby a Shim-pack G-ODS guard column (1cm×4.0 mm I.D., 5 μm particle size). Amixture of 0.05 M sodium phosphate buffer(pH 5.7; adjusted with phosphoric acid) andacetonitrile (56:44 v/v) was used as the mobilephase. The column oven temperature was setat 50 °C and the mobile phase was filtered,degassed and pumped at a flow rate of 1.7ml/min.2.3. Sample preparationAliquots of blank, calibration standard orunknown human serum samples (1 ml) werepipetted into 100 mm×16 mm disposableglass tubes, containing 100 μl of the workinginternal standard solution. The samples weremixed with 200 μl of the HCl solution (2.0 N)and extracted with 5 ml of ethyl acetate. Aftervortex mixing for 30 s and centrifugation (5min. at 6000×g) the organic phase wasremoved and evaporated to dryness understream of nitrogen at 50 °C. The residue wasreconstituted in 100 μl mobile phase andfollowing brief mixing a volume of 20 μlwas injected onto the HPLC system.2.4. Validation of the method2.4.1. Calibration curve and linearityCalibration curves samples were preparedwithin the concentration range of 0.05-10μg/ml using pooled human blank serumobtained from normal subjects. In disposableglass tubes (100 mm×16 mm), afterevaporation of 100 μl from each workingsolutions of the analyte, under a gentle streamof nitrogen at 50 °C, the residues werereconstituted in 1 ml of drug-free humanserum, mixed for 10 s on a vortex mixer andsubjected to extraction and analysis asdescribed above. The linearity of the methodwas checked in the same day (n=6) and in 6consecutive days. Calibration curves(weighted regression line) were obtained bylinear least-squares regression analysis ofplots of peak-area ratio to I.S. versus drugconcentrations.2.4.2. Accuracy, precision, sensitivity andspecificityQuality control samples used in methodvalidation were prepared with the drugworking solutions to make low (0.05 μg/ml),medium (0.5 μg/ml) and high (5 μg/ml)concentrations of the analyte. Intra- and inter-day variations were calculated by repeatedanalysis (n=6) of different concentrations ofCCA in a single analytical run and in tenTable 1. HPLC precision and accuracy results of the validation of clopidogrel carboxylic acid calibration curve.Known concentration Concentration found Coefficient of Accuracy (μg/ml) (mean±S.D.) variation (%) (%mean deviation)Inter-day0.05 0.047±0.005 10.6 -3.90.5 0.495±0.017 3.4 -1.95 5.071±0.075 1.5 1.2Intra-day0.05 0.051±0.006 11.7 1.10.5 0.512±0.016 3.1 1.85 5.135±0.082 1.6 1.4Note: Accuracy has been calculated as a percentage of the nominal concentration.G Ghiasi et al / IJPS Autumn 2009; 5(4): 231-238234analytical runs performed on different days,respectively. Limit of detection (LOD) andLOQ were defined as the concentrations of thedrug giving a signal-to-noise ratio of 3:1 and10:1, respectively. The specificity of themethod was examined by presence ofdisturbing endogenous peaks in 24 humanserum samples from different volunteers.These samples were pretreated according tothe sample preparation procedure except fromthe addition of the internal standard (I.S.).Selectivity of the assay was examined byanalysis of several potentially co-administrated drugs with clopidogrel.2.4.3. Recovery, selectivity and stabilityThe extraction efficiencies of CCA at theabove mentioned concentrations as well as theI.S. at applied concentration were calculatedin replicates (n=5) by comparing therespective peak areas obtained by extractionof the samples from serum, with thoseobtained from the same amounts of un-extracted solutions in methanol. The stabilityof the analyte in blood specimen duringsample handling was also verified bysubjecting the samples to three freeze-thawcycles and up to 60 days maintenance at -40°C. Stability of the solutions of CCA and theI.S. were studied over a period of 3 weeks bycomparing of the peak areas at different times.2.5. Application of the methodThe present method was applied in arandomized crossover bioequivalence studyof two different clopidogrel preparations in 24male healthy volunteers aged 24.8±6.5 yearsand weighing 75.3±4.2 kg with normalbiochemical parameters. After an overnightfasting, all the subjects received a single oraldose of 150 mg clopidogrel from either Exir(Tehran, Iran) or Sanofi-Synthelabo (Plavix;Ambares, France) pharmaceutical companieson 2 working days separated by a wash-outperiod of 2 weeks. Blood sampling wascarried out at suitable intervals up to 24 husing disposable glass tubes (100 mm×16mm) without any additives. All the sampleswere allowed to clot at room temperature for30 min. Serum was separated bycentrifugation at 2000×g for 10 min. andwere stored at -40 °C until analysis. Pharma-cokinetic parameters including maximumconcentration (Cmax), area under theconcentration time curve from zero to thetime of last sampling (AUC0-t) and area underthe concentration time curve from zero toinfinity (AUC0-t) were compared. Student's t-test was used for statistical analysis of the dataand statistical significance was defined at thelevel of p<0.05.Figure 2. Mean serum concentrations-time profiles of clopidogrel carboxylic acid in 24 human volunteers following single150 mg oral dose administration of two different clopidogrel preparations.Quantitative determination of clopidogrel in human serum2353. Results3.1. Chromatography, sensitivity and linearityUnder the chromatographic conditionsdescribed, no endogenous components fromserum were found to interfere with the elutionof CCA or the I.S. Representativechromatograms of human blank serum spikedwith the I.S. and human blank serum spikedwith the I.S. and CCA at the concentration of0.05 μg/ml are shown in Figure 1A and 1B,respectively. Figure 1C and 1D show thechromatograms of serum samples obtained at6 and 12 h after a single oral dose of 150 mgclopidogrel from a healthy volunteer,respectively. The calibration curves werelinear over the concentration range of 0.05-10μg/ml and the LOD and LOQ were found tobe 0.02 and 0.05 μg/ml, respectively. Thecorrelation coefficients for calibration curveswere equal to or better than 0.9982. Intra-and inter-day reproducibility for calibrationcurves were determined on the same day inreplicate (n=6) and on different days (n=6),respectively, using the same pooled serumsample. The intra-day average slope of thefitted straight lines was 12.85±0.542 μg/ml(CV=4.2%) and the mean intercept of thecalibration curves was 1.97±0.188(CV=9.5%). The corresponding mean (±SD)coefficient of the linear regression analysiswas 0.9982±0.013 (CV=0.1%). Forcalibration curves prepared on different days(n=6), the mean±SD of results were asfollows: Slope 13.64±0.521 μg/ml(CV=3.8%), coefficient of the linearregression analysis=0.9985±0.012(CV=0.1%) and intercept=1.75±0.162(CV=9.2%).3.2. Recovery, accuracy, precision, selectivityand stabilityThe inter- and intra-day accuracy andprecision values of the assay method havebeen presented in Table 1. The coefficientvariation values of both inter- and intra-dayanalysis were less than 11.7% whereas thepercentage error was less than 3.9. The meanextraction efficacies of CCA and I.S. fromserum were found to be 99±2% and 97±4%,respectively. The results of the selectivitystudy showed that using the same analyticalconditions there were no interfering peaksfrom any of the following drugs:Acetaminophen, naproxen, indometacin,celecoxib, ibuprofen, diclofenac, theophylline,fursemide, amiodarone, cinnarizine,ticlopidine, glybenclamide, verapamil,propranolol diltiazem, aspirin, salicylic acid,omeprazole, cimetidine, codeine and caffeine.All of the drugs were tested at concentrationrange of 10-1000 ng/ml. The stock solutionsof CCA and the I.S. were stable for at least 21days when stored at 4 °C. The stability ofthe drug was found to be 100% from theinitial value, after 60 days maintenance ofthe serum at -40 °C and following three thaw-freeze cycles.3.3. Application of the methodThis method has been successfully used forthe determination of serum concentrations ofCCA in a randomized cross-overTable 2. Mean (±S.D.) pharmacokinetic parameters of CCA in 24 healthy volunteers after administration of a single150 mg oral dose of clopidogrel in 24 healthy volunteers.Parameter prep. Test Reference p valueaTmax (h) 1.35 (0.33) 1.11 (0.42) NSCmax (μg/mL) 5.11 (1.85) 5.46 (1.97) NSAUC0-24 (μg h/mL) 19.75 (9.08) 20.62 (10.32) NSAUC0-∞ (μg h/mL) 23.05 (9.4) 24.70 (10.3) NST1/2 (h) 8.84 (4.2) 9.05 (4.75) NSTmax, time to maximum concentration; Cmax, maximum concentration; AUC, area under the concentration time curve; T1/2,elimination half-life. NS, no significant difference. a p<0.05. G Ghiasi et al / IJPS Autumn 2009; 5(4): 231-238236bioequivalence study following single oraladministration of two different clopidogrelpreparations in 24 healthy volunteers. Typicalserum concentration-time profiles of CCAhave been shown in Figure 2 and resultedpharmacokinetic parameters have beensummarized in Table 2.4. DiscussionA few analytical methods have beenpublished for determination of the inactivemetabolite of clopidogrel in the biologicalmatrix. A sensitive gas chromatography-massspectrometric method with LOQ of 0.005μg/ml has been published [3]. In thistechnique however, a complex two stepsextraction method using both liquid-liquidand solid phase extraction procedures as wellas derivatization of the analyte are required.Two LC-MS methods for the determinationinactive metabolite of clopidogrel have beenpublished [4, 5]. In the method described byMitakos and Panderi [5] extraction of theanalyte from the serum has been achievedusing single step solid phase extraction,however, their method is not sensitive enough(LOQ 0.1 μg/ml) to be used in single-dosepharmacokinetic studies of the drug. In theother published LC-MS method [4] two stepstime-consuming extraction procedure usingboth liquid-liquid and solid phase extractiontechniques have been used however, themethod is sensitive enough (LOQ 0.02 μg/ml)for measuring of the analyte up to at least threehalf lives post-dose. The inactive metaboliteof the drug has been measured in Wistar ratplasma using HPLC and UV detection [6]. Inthis method, LOQ of 0.125 μg/ml has beenreported using 50 μl injection. Furthermore,this method involves long run time of analysis(16 min.) and gradient elution of the mobilephase. In HPLC-UV technique described bySouri et al. [7] ticlopidine has been used asinternal standard. To overcome close retentiontimes of CCA and ticlopidine, they used amobile phase with low flow rate (0.9 ml/min.)and high percent of aqueous phase whichleads to long analytical run time (12 min.) andreduction of sensitivity (LOQ 0.2 μg/ml). Toimprove run time and sensitivity of theanalysis, the flow rate should be increased andit is preferred to select a mobile phase withhigher proportion of organic solvent. Thus,comparing to their method [7], we used amobile phase with higher flow rate and moreproportion of the organic solvent. Due tosimilar retention behavior of CCA andticlopidine, a number of chemical agents weretested to choose I.S. and phenytoin wasselected considering its UV spectrum,retention time, recovery and resolution fromthe drug and endogenous peaks. While theirmethod has been applied in a human singledose study, poor sensitivity of the method,does not allow measurement of the analyteconcentrations for more than 12 h followingsingle dose administration of the drug.Furthermore, estimated elimination half-lifeof the inactive metabolite in their study is2.25±0.84 h. This does not agree with reportedvalues in the literature, using the same singledose of the drug [4, 8]. Determination ofclopidogrel in human plasma by liquidchromatography/tandem mass spectrometryhas been reported [9]. As low blood levels ofthe intact drug is achieved following singledose administration [8, 9] and consideringdifference in polarity between the drug and itsinactive metabolite, like other publishedpapers [3-7], our method failed to detect peakof clopidogrel in the samples. However, themajor advantages of the present method arereduction in run time of analysis (5.5 min.)and improvement of sensitivity (LOQ 0.05μg/ml) which allows determination of theanalyte up to three half-lives post-dose moreprecisely, so that the extent of absorptionuntil the last sampling time is more closer toits extrapolated value (AUC0-∞).Quantitative determination of clopidogrel in human serum237AcknowledgmentsThis work was supported by ExirPharmaceutical Company and in part byKermanshah University of Medical Sciences.References[1] Majerus PW, Tollefsen DM. Blood coagulationand anticoagulant, thrombolytic, and antiplateletdrugs. In: Brunton LL, (editor). The pharmaco-logical basis of therapeutics. 11th ed. New York:The McGraw-Hill Companies, 2006; p. 1483.[2] Herbert JM, Frehel D, Vallee E, Kieffer G, GouyD, Berger Y, Defreyn G, Maffrand JP. Clopidogrel,a novel antiplatelet and antithrombotic agent.Cardiovascular Drug Rev 1993; 2: 180-98. [3] Lagorce P, Perez Y, Ortiz J, Necciari J, BressolleF. Assay method for the carboxylic acid metaboliteof clopidogrel in human plasma by gaschromatography-mass spectrometry. JChromatogr B 1998; 720: 107-17. [4] Ksycinska H, Rudzki P, Kiliszek MB.Determination of clopidogrel metabolite(SR26334) in human plasma by LC-MS. J PharmBiomed Anal 2006; 41: 533-9.[5] Mitakos A, Panderi I. Determination of thecarboxylic acid metabolite of clopidogrel inhuman plasma by liquid chromatography-electrospray ionization mass spectrometry. AnalChim Acta 2004; 505:107-14.[6] Singh SS, Sharma K, Barot D, Mohan PR, LohrayVB. Estimation of carboxylic acid metabolite ofclopidogrel in Wistar rat plasma by HPLC and itsapplication to a pharmacokinetic study. JChromatogr B 2005; 821: 173-80.[7] Souri E, Jalalizadeh H, Kebriaee-Zadeh A,Shekarchi M, Dalvandi A. Validated HPLCmethod for determination of carboxylic acidmetabolite of clopidogrel in human plasma and itsapplication to a pharmacokinetic study. BiomedChromatogr 2006; 20: 1309-14. [8] Caplain H, Francois MD, Gaud C, Necciari J.Pharmacokinetics of clopidogrel. Semin ThrombHemost 1999; 25: 25-8. [9] Shin BS, Yoo SD. Determination of clopidogrelin human plasma by liquid chromatography/tandem mass spectrometry: Application to aclinical pharmacokinetic study. BiomedChromatogr 2007; 21: 883-9.238ONLINE SUBMISSIONijps.sums.ac.ir

High Performance Liquid Chromatographic Determination ofInactive Carboxylic Acid Metabolite of Clopidogrel in HumanSerum: Application to a Bioequivalence Study

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High Performance Liquid Chromatographic Determination ofInactive Carboxylic Acid Metabolite of Clopidogrel in HumanSerum: Application to a Bioequivalence Study

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