title: Development and validation of an HPLC-MS/MS method to determine clopidogrel in human plasma.
abstract: A quantitative method for clopidogrel using online-SPE tandem LC-MS/MS was developed and fully validated according to the well-established FDA guidelines. The method achieves adequate sensitivity for pharmacokinetic studies, with lower limit of quantifications (LLOQs) as low as 10 pg/mL. Chromatographic separations were performed on reversed phase columns Kromasil Eternity-2.5-C18-UHPLC for both methods. Positive electrospray ionization in multiple reaction monitoring (MRM) mode was employed for signal detection and a deuterated analogue (clopidogrel-d 4) was used as internal standard (IS). Adjustments in sample preparation, including introduction of an online-SPE system proved to be the most effective method to solve the analyte back-conversion in clinical samples. Pooled clinical samples (two levels) were prepared and successfully used as real-sample quality control (QC) in the validation of back-conversion testing under different conditions. The result showed that the real samples were stable in room temperature for 24 h. Linearity, precision, extraction recovery, matrix effect on spiked QC samples and stability tests on both spiked QCs and real sample QCs stored in different conditions met the acceptance criteria. This online-SPE method was successfully applied to a bioequivalence study of 75 mg single dose clopidogrel tablets in 48 healthy male subjects.
authors.affiliation: Central Laboratory, Shanghai Clinical Center, Chinese Academy of Sciences/Central Laboratory, Shanghai Xuhui Central Hospital, Shanghai 200031, China.
authors.forename: Gangyi
authors.initials: G
authors.lastname: Liu
authors.affiliation: Central Laboratory, Shanghai Clinical Center, Chinese Academy of Sciences/Central Laboratory, Shanghai Xuhui Central Hospital, Shanghai 200031, China.
authors.forename: Chunxia
authors.initials: C
authors.lastname: Dong
authors.affiliation: Central Laboratory, Shanghai Clinical Center, Chinese Academy of Sciences/Central Laboratory, Shanghai Xuhui Central Hospital, Shanghai 200031, China.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Shen
authors.affiliation: Central Laboratory, Shanghai Clinical Center, Chinese Academy of Sciences/Central Laboratory, Shanghai Xuhui Central Hospital, Shanghai 200031, China.
authors.forename: Xiaopei
authors.initials: X
authors.lastname: Lu
authors.affiliation: Central Laboratory, Shanghai Clinical Center, Chinese Academy of Sciences/Central Laboratory, Shanghai Xuhui Central Hospital, Shanghai 200031, China.
authors.forename: Mengqi
authors.initials: M
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai 200233, China.
authors.forename: Yuzhou
authors.initials: Y
authors.lastname: Gui
authors.affiliation: Central Laboratory, Shanghai Clinical Center, Chinese Academy of Sciences/Central Laboratory, Shanghai Xuhui Central Hospital, Shanghai 200031, China.
authors.forename: Qinyi
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: Central Laboratory, Shanghai Clinical Center, Chinese Academy of Sciences/Central Laboratory, Shanghai Xuhui Central Hospital, Shanghai 200031, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Yu
keywords: Back-conversion; Bioequivalence; Clopidogrel; Deuterated analogue; ESI, electrospray ionization; FDA guidelines; IS, internal standard; LC–MS/MS; LC–MS/MS, liquid chromatography tandem mass spectrometry; LLOQ, lower limit of quantification; MRM, multiple reaction monitoring; Online-SPE; QC, quality control; Real samples stability; SPE, solid phase extraction