SENSITIVE AND SIMPLE DETERMINATION OF VITAMIN K1 IN HUMAN PLASMA BY ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY COUPLED WITH A FLUORESCENCE DETECTOR

Abstract

We developed a sensitive and simple method for the determination of vitamin K1 (VK1) in human plasma using ultra-performance liquid chromatography with post-column reduction. VK1 was extracted with single-step liquid–liquid partitioning. As an analytical column, we used an ACQUITY CSH C18 (1.7 µm, 2.1 × 50 mm). The mobile phase consisted of a mixture of distilled water/methanol delivered using a linear gradient, starting with a composition of 94% methanol and ending at 100% methanol with a flow rate of 0.6 mL/min. Commonly, synthetic vitamin K analogs are used as the internal standards (IS); however, we selected menaquinone-8, which is undetectable in human plasma. The peaks were detected by a fluorescence detector (excitation at 244 nm, emission at 430 nm). The lower limit of quantification was 0.1 ng/mL. The inter- and intra-assay precision varied between 2.7–16.1%, and the accuracy varied between −10.3–13.4%. This method was simple, rapid, and sensitive for the detection of VK1 and is suitable for the determination of VK1 in small volumes of human serum. We believe that this method is useful for determining the concentration of VK1 in human plasma and in pharmacodynamic studies of warfarin, a VK antagonist, with clinical samples.