title: A sensitive and rapid ultra HPLC-MS/MS method for the simultaneous detection of clopidogrel and its derivatized active thiol metabolite in human plasma.
abstract: A sensitive, selective, and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) was developed for the simultaneous quantification of clopidogrel (Plavix(®)) and its derivatized active metabolite (CAMD) in human plasma. Derivatization of the active metabolite in blood with 2-bromo-3'-methoxy acetophenone (MPB) immediately after collection ensured metabolite stability during sample handling and storage. Following addition of ticlopidine as an internal standard and simple protein precipitation, the analytes were separated on a Waters Acquity UPLC™ sub-2 μm-C(18) column via gradient elution before detection on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. The method was validated across the clinically relevant concentration range of 0.01-50 ng/mL for parent clopidogrel and 0.1-150 ng/mL (r(2)=0.99) for CAMD, with a fast run time of 1.5 min to support pharmacokinetic studies using 75, 150, or 300 mg oral doses of clopidogrel. The analytical method measured concentrations of clopidogrel and CAMD with accuracy (%DEV) <±12% and precision (%CV) of <±6%. The method was successfully applied to measure the plasma concentrations of clopidogrel and CAMD in three subjects administered single oral doses of 75, 150, and 300 mg clopidogrel. It was further demonstrated that the derivatizing agent (MPB) does not affect clopidogrel levels, thus from one aliquot of blood drawn clinically, this method can simultaneously quantify both clopidogrel and CAMD with sensitivity in the picogram per mL range.
authors.affiliation: Clinical Pharmacology Program, Office of the Clinical Director, National Cancer Institute, Bethesda, MD 20892, United States.
authors.forename: Cody J
authors.initials: CJ
authors.lastname: Peer
authors.forename: Shawn D
authors.initials: SD
authors.lastname: Spencer
authors.forename: Dustin A H
authors.initials: DA
authors.lastname: VanDenBerg
authors.forename: Michael A
authors.initials: MA
authors.lastname: Pacanowski
authors.forename: Richard B
authors.initials: RB
authors.lastname: Horenstein
authors.forename: William D
authors.initials: WD
authors.lastname: Figg
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000077144:Clopidogrel; D004355:Drug Stability; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000097:blood* / Q000493:pharmacokinetics; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D013438:Sulfhydryl Compounds; D000077144:Clopidogrel; D013988:Ticlopidine