title: Direct measurement of active thiol metabolite levels of clopidogrel in human plasma using tris(2-carboxyethyl)phosphine as a reducing agent by LC-MS/MS.
abstract: A simple, robust, and rapid LC-MS/MS method has been developed and validated for the simultaneous quantitation of clopidogrel and its active metabolite (AM) in human plasma. Tris(2-carboxyethyl)phosphine (TCEP) was used as a reducing agent to detect the AM as a disulfide-bonded complex with plasma proteins. Mixtures of TCEP and human plasma were deproteinized with acetonitrile containing 10 ng/mL of clopidogrel-d4 as an internal standard (IS). The mixtures were separated on a C18 RP column with an isocratic mobile phase consisting of 0.1% formic acid in acetonitrile and water (90:10, v/v) at a flow rate of 0.3 mL/min. Detection and quantification were performed using ESI-MS. The detector was operated in selected reaction-monitoring mode at m/z 322.0→211.9 for clopidogrel, m/z 356.1→155.2 for the AM, and m/z 326.0→216.0 for the IS. The linear dynamic range for clopidogrel and its AM were 0.05-20 and 0.5-200 ng/mL, respectively, with correlation coefficients (r) greater than 0.9976. Precision, both intra- and interday, was less than 8.26% with an accuracy of 87.6-106%. The validated method was successfully applied to simultaneously analyze clinical samples for clopidogrel and its AM.
authors.affiliation: College of Pharmacy, The Catholic University of Korea, Gyeonggi-do, Korea.
authors.forename: Jung Bae
authors.initials: JB
authors.lastname: Park
authors.forename: Soo Hyeon
authors.initials: SH
authors.lastname: Bae
authors.forename: Su-Min
authors.initials: SM
authors.lastname: Jang
authors.forename: Won Jun
authors.initials: WJ
authors.lastname: Noh
authors.forename: Jang-Hee
authors.initials: JH
authors.lastname: Hong
authors.forename: Kee Dong
authors.initials: KD
authors.lastname: Yoon
authors.forename: Han Chang
authors.initials: HC
authors.lastname: Kang
authors.forename: Soo Kyung
authors.initials: SK
authors.lastname: Bae
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D000077144:Clopidogrel; D006801:Humans; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000097:blood* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism
chemical_list: D000077144:Clopidogrel; D010720:Phosphines; D013438:Sulfhydryl Compounds; D013988:Ticlopidine; C080938:tris(2-carboxyethyl)phosphine
keywords: Clopidogrel; Clopidogrel active metabolite; Human plasma; LC-MS/MS; Reducing agent; tris(2-Carboxyethyl)phosphine