Determination of clopidogrel metabolite (SR26334) in human plasma by LC–MS

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Abstract

A new, sensitive, specific and reproducible method for determination of clopidogrel metabolite (SR26334) in human plasma has been developed. After liquid–liquid extraction on Chem Elut cartridges with dichloromethane, samples were quantified using reversed-phase high performance liquid chromatography with mass detection. The determination was performed on a Luna C18, 3 μm (75 mm × 4.6 mm i.d.) column with an acetonitrile-water-formic acid mixture (60:40:0.1, v/v/v) as a mobile phase. The flow rate was set at 0.2 mL/min. Repaglinide was chosen as an internal standard and the time of analysis was 12 min. For SR26334 the limits of detection and quantification were 7.5 ng/mL and 20 ng/mL, respectively, and the calibration curve was linear up to 3000 ng/mL. The extraction recovery of SR26334 from plasma was within the range of 85–90%. The method has been successfully used to study clopidogrel metabolite pharmacokinetics in healthy volunteers.

Introduction

Clopidogrel, methyl (+)-(S)-2-(2-chlorophenyl)2-{6,7-dihydrothiene[3,2-c]pyridine-5(4H)}-acetate (SR25990C, Fig. 1A), inhibits platelet aggregation by selective preventing of the binding of adenosine diphosphate (ADP) to its platelet receptor [1]. The drug reduces thrombotic events in a broad range of patients (e.g. recent myocardial infarction, recent stroke, established peripheral arterial disease, or acute coronary syndrome). Clopidogrel is an inactive prodrug, and a biotransformation by the liver is necessary to induce expression of its antiaggregating activity [2]. It is rapidly absorbed and undergoes extensive metabolism after oral administration and its plasma concentration goes down very fast [3]. Moreover, the compound belongs to a family of eight stereoisomers with chemical structure of 2-{1-[1-(2-chlorophenyl)-2-methoxy-2-oxoethyl]-4-sulfanyl-3-piperidinylidene}acetic acid, that is believed to be the active metabolite of clopidogrel and is highly labile [4]. The carboxylic acid derivative of clopidogrel (SR26334, Fig. 1B), which is its inactive metabolite, is the major circulating compound and information on the absorption and elimination of clopidogrel is derived from the pharmacokinetics of SR26334 [3]. It is formed by hydrolysis of the ester function by carboxylesterase [5].

In general, two methods for determination of SR26334 in plasma have been reported in literature: HPLC with UV detection and GC–MS. The former was used for multiple-dose pharmacokinetics study [3], the latter—for single-dose pharmacokinetics of SR26334 [3], [6]. Recently, several LC–MS–MS methods for the study of pharmacokinetics of clopidogrel and SR26334 have been published [7], [8], [9].

The purpose of the study described below was to develop and validate a specific, simple and reproducible method for determination of clopidogrel metabolite (SR26334) in human plasma using a liquid–liquid extraction procedure on Chem Elut cartridges followed by HPLC analysis on reversed phase with single mass detection. In the study, repaglinide (Fig. 2) was used as the internal standard. The method was subsequently applied to bioavailability studies.

Section snippets

Reagents and chemicals

The metabolite of clopidogrel was supplied by Adamed Ltd. (Pieńków, Poland). Repaglinide (internal standard, I.S.) was synthesized in Pharmaceutical Research Institute (Warsaw, Poland). HPLC-grade acetonitrile and dichloromethane were purchased from LabScan (Dublin, Ireland), acetic acid and HPLC-grade ammonium acetate were from T.J. Baker (Deventer, Netherlands) and formic acid was from Riedel-de Haën (Seelze, Germany). Distilled water was purified by a Millipore System Milli Q (Molsheim,

Specificity

Drug-free human plasma from six different lots was tested for endogenous interference. In Fig. 5 representative chromatograms of drug-free human plasma, spiked plasma and plasma sample from a volunteer who received clopidogrel as a single 75 mg tablet are shown. The extraction procedure and chromatographic conditions make it possible to separate both compounds. The chromatogram of drug-free human plasma shows that any interfering endogenous substances did not extract from the plasma. The

Discussion and conclusion

The method of clopidogrel metabolite (SR26334) determination was developed. Repaglinide was selected as the internal standard because some of its physicochemical properties are similar to those of the clopidogrel metabolite: this compound was extracted with similar recovery to that of CLM (about 90%) under the method conditions and retained good separation from the target compound (retention time for CLM was about 4.2 min and for I.S. about 5.9 min).

It was important that the relative short run

Acknowledgements

We are grateful to Dr. Mariusz K. Piskula and Wieslaw Wiczkowski, M.Sc. (Institute of Animal Production and Food Research of the Polish Academy of Sciences, Olsztyn, Poland) for help in solving our problem with LC–MS.

References (12)

  • P. Savi et al.

    Biochem. Pharmacol.

    (1992)
  • P. Lagorce et al.

    J. Chromatogr. B

    (1998)
  • T.L. Lenz et al.

    Clin. Pharmacokinet.

    (2003)
  • H. Caplain et al.

    Semin. Thromb. Hemost.

    (1999)
  • J.-M. Pereillo et al.

    Drug Metab. Disp.

    (2002)
  • J.M. Herbert et al.

    Cardiovasc. Drug Rev.

    (1993)
There are more references available in the full text version of this article.

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