title: Quantitative determination of clopidogrel active metabolite in human plasma by LC-MS/MS.
abstract: A quantitative method for the determination of clopidogrel active metabolite (AM) in human plasma was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Clopidogrel AM contains a thiol group, thus requiring stabilization in biological samples. The alkylating reagent 2-bromo-3'-methoxyacetophenone was used to stabilize clopidogrel AM in blood. An analog of the derivatized clopidogrel AM was used as the internal standard (IS). The derivatized samples were subjected to solid-phase extraction with a C2 disk plate and the overall procedure exhibited good reaction (more than 90%) and recovery efficiencies (from 85% to 105%). The derivative of clopidogrel AM (MP-AM) and IS were separated on an ODS column and quantified by tandem mass spectrometry with electrospray ionization. No significant endogenous peaks corresponding to MP-AM or IS were detected in blank human plasma samples, and no significant matrix effect was observed for MP-AM and IS in human plasma samples (from 102% to 121%). The calibration curve ranged from 0.5 to 250 ng/mL with good linearity, and extended by validation of a 50-fold dilution. In the intra- and inter-assay reproducibility tests, the accuracy and precision were within 12% relative error and 6% coefficient of variation, respectively. The derivatized MP-AM was stable in human plasma for 4 months at -80 degrees C. The validated method was successfully used to analyze clinical samples and determine the pharmacokinetics of clopidogrel AM.
authors.affiliation: Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan. takahashi.makoto.zx@daiichisankyo.co.jp
authors.forename: Makoto
authors.initials: M
authors.lastname: Takahashi
authors.forename: Henrianna
authors.initials: H
authors.lastname: Pang
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Kawabata
authors.forename: Nagy A
authors.initials: NA
authors.lastname: Farid
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kurihara
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D019540:Area Under Curve; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D000077144:Clopidogrel; D004355:Drug Stability; D005260:Female; D005615:Freezing; D006801:Humans; D008297:Male; D008657:Metabolic Clearance Rate; D008875:Middle Aged; D015394:Molecular Structure; D010975:Platelet Aggregation Inhibitors / Q000097:blood* / Q000378:metabolism* / Q000493:pharmacokinetics; D012015:Reference Standards; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics; D013997:Time Factors; D055815:Young Adult
chemical_list: D010975:Platelet Aggregation Inhibitors; D000077144:Clopidogrel; D013988:Ticlopidine