A simple, sensitive, and high-throughput LC-APCI-MS/MS method for simultaneous determination of vitamin K 1 , vitamin K 1 2,3-epoxide in human plasma and its application to a clinical pharmacodynamic study of warfarin

摘要

Warfarin exerts its anticoagulation activity by blocking the vitamin K-epoxide cycle. A quantitative understanding of how warfarin and related genes interact with the vitamin K-epoxide cycle and the associated change of coagulation activity in the human body may help study the pharmacodynamics of warfarin. The plasma concentration of vitamin K1(VK1) and vitamin K12,3-epoxide (VK1O) could reflect the status of vitamin K-epoxide cycle. However, their determination is a challenging task due to their extremely low concentrations in human plasma and the severe interferences caused by co-extracted lipids. In this study, we developed an LC-APCI-MS/MS method for the simultaneous determination of VK1and VK1O in human plasma using stable deuterium-labeled vitamin K1(vitamin K1-d7) as the internal standard (IS). Plasma samples were prepared through protein denaturation followed by one-step liquid extraction with cyclohexane. Chromatographic separation of analytes from isobaric interferences and endogenous ion suppressor was performed on a Synergi Hydro-RP column (150?mm?×?4.6?mm, 4?μm) under the reversed-phase condition with isocratic elution. The selective reaction monitoring (SRM) transitions were chosen asm/z?=?451.5→187.3 for VK1,m/z?=?467.5→161.2 for VK1O, andm/z?=?458.6→194.3 for IS in APCI positive mode. The assay was linear in the range of 100–10,000?pg/mL for the two analytes and achieved considerable extraction recoveries (87.8–93.3%, 91.0–96.9%, and 92.0% for VK1, VK1O, and IS, respectively), negligible matrix effects (93.6–96.0%, 96.3–100.1%, and 95.5%), and high selectivity with a small sample volume requirement (0.2?mL) and short run time (15?min). The validated method was successfully applied in a clinical pharmacodynamic study of warfarin, and the clotting activity was found to be negatively correlated with the plasma concentration ratio of VK1O to VK1.