Quantitative Liquid Chromatography/Mass Spectrometry/Mass Spectrometry Warfarin Assay for in Vitro Cytochrome P450 Studies

LC/MS/MS warfarin assay − An emerging tool for the early detection of cytochrome P450‐associated drug−drug interactions in drug discovery

“…Under negative ionization, the mass spectrometer (MS) was operated at the multiple reaction monitoring (MRM) mode for quantitative analysis, and synchronized with the HPLC. The curtain, nebulizer, turbo gas, and collision-associated dissociation (CAD) gas were typical, with 350 • C source temperature and −4500 V ion spray potential, and optimized MRM parameters [8]. Quantification was based on the peak area ratio of the metabolite over the internal standard against the respective concentration of the metabolite.…”

“…The assay performance was described in detail in the original publication [8]. It is herein briefly stated.…”

“…The monoclonal antibodies only recognized human CYP1A2, thus did not cross-react with CYP1A1 (data not shown). In addition to transformed cell lines, the assay has been successfully and frequently applied to study CYP induction in primary culture of human hepatocytes, which is the major advance of the assay and described extensively in the original publication [8].…”

“…A variety of detection, especially chromatography-based, methods have been developed during that long period, including the most recent LC/MS/MS method developed in our laboratory [8]. In this article, I will review the qualitative and quantitative characteristics of that newly developed LC/MS/MS technique, in comparison with other existing analytical methods for warfarin metabolism.…”

“…In this article, I will review the qualitative and quantitative characteristics of that newly developed LC/MS/MS technique, in comparison with other existing analytical methods for warfarin metabolism. Moreover, to explicitly demonstrate the advances of the LC/MS/MS method, especially the great potential for drug-drug interaction studies using human cells, the materials presented here combine what originally published and some of recent results [8].…”