title: HPLC-MS/MS method for the simultaneous determination of clopidogrel, its carboxylic acid metabolite and derivatized isomers of thiol metabolite in clinical samples.
abstract: A fast and reproducible HPLC-MS/MS method was developed for the simultaneous determination of clopidogrel (CLP), its carboxylic acid derivative (CLPM), derivatized thiol metabolite isomers MP-H3 and the active MP-H4 in incurred human plasma. CLP, CLPM, MP-H3 and MP-H4 isomers together with the internal standard piroxicam were extracted from plasma samples using a simple protein precipitation with acetonitrile. The analytes were separated on HPLC Zorbax Plus C18 column via gradient elution with water and acetonitrile, both containing 0.1% (v/v) formic acid. Detection of the analytes were performed on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. Calibration curves of the analytes prepared in 250μL plasma were found to be linear in ranges: 0.25-5.00ng/mL for CLP, 0.25-50.00ng/mL for MP-H3 and MP-H4 isomers and 50-10,000ng/mL for CLPM. The lower limit of quantitation was 0.25ng/mL for CLP, MP-H3, MP-H4 and 50.00ng/mL for CLPM. Intra- and inter-assay precision, expressed as relative standard deviation, was ≤18.1% for CLP, ≤15.2% for CLPM, ≤10.1% for MP-H3 and ≤19.9% for MP-H4. Intra- and inter-day accuracy of the method, expressed as relative error, was ≤16%. The analytes were stable in samples stored for 6h in autosampler, in plasma samples for 24h at room temperature and for 3 months at -25°C. Resolution of CLP, CLPM and MP-H3 and MP-H4 isomers of thiol metabolite during one analytical run was reported in patient plasma. The HPLC-MS/MS method was applied for pharmacokinetic studies of CLP and its metabolites in patients treated with daily dose of 75mg CLP.
authors.affiliation: Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences, 6 Święcickiego Street, 60-781 Poznań, Poland. mkaraz@ump.edu.pl
authors.forename: Marta
authors.initials: M
authors.lastname: Karaźniewicz-Łada
authors.forename: Dorota
authors.initials: D
authors.lastname: Danielak
authors.forename: Artur
authors.initials: A
authors.lastname: Teżyk
authors.forename: Czesław
authors.initials: C
authors.lastname: Zaba
authors.forename: Gilles
authors.initials: G
authors.lastname: Tuffal
authors.forename: Franciszek
authors.initials: F
authors.lastname: Główka
mesh_terms: D000368:Aged; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000077144:Clopidogrel; D004355:Drug Stability; D005260:Female; D006801:Humans; D007536:Isomerism; D016014:Linear Models; D008297:Male; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D013438:Sulfhydryl Compounds; C528640:clopidogrel carboxylic acid; D000077144:Clopidogrel; D013988:Ticlopidine