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.

Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry

“…The challenge in accurately quantifying glutathione is to measure both its oxidized and reduced states, and to do this, it is necessary to both quench any artifactual oxidation of GSH to GSSH by direct in situ derivatization as illustrated above, and to exhaustively reduce GSSG. For the latter, commonly used reducing agents include TCEP·HCl [55, 56], dithiothreitol (DTT) [57], trialkylphosphines [58], NaBH 4 [59], and occasionally sulfites, cyanides, or some enzymes. We selected the TCEP·HCl reagent because it eliminates the complication of added thiol reagents, as with the use of DTT, and the possibility of overreduction, as often the case with using NaBH 4 .…”

The impact of P2Y12 promoter DNA methylation on the recurrence of ischemic events in Chinese patients with ischemic cerebrovascular disease

“…The supernatants were taken and stored in a refrigerator (−80 °C) for the analysis of the clopidogrel active metabolite concentrations. The drug concentration was analyzed using the HPLC-MS/MS assay method as reported in the literature 34 .…”

Chemical cytometry of thiols using capillary zone electrophoresis-laser induced fluorescence and TMPAB-o-M, an improved fluorogenic reagent

“…9, 10 Other analytical methods employ separations using high performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) coupled with fluorescence, 11, 12 mass spectrometry, 13, 14 and electrochemical detection. 15 Most of these studies focus on the quantification of thiols in biological fluids, tissues, or cell lysates, which provides information on the average composition of the ensemble.…”

“…15 Most of these studies focus on the quantification of thiols in biological fluids, tissues, or cell lysates, which provides information on the average composition of the ensemble. 7, 8, 11, 12, 14–16 …”