Development and Validation of a Rapid Liquid Chromatography–Tandem Mass Spectrometry Method for the Quantitation of Vitamin K Metabolites in Different Matrices

ABSTRACT Adequate vitamin K is crucial for optimal health. Although vitamin K detection methods have been established using liquid chromatography–tandem mass spectrometry (LC–MS/MS), some limitations remain. Therefore, we aimed to establish a stable and rapid LC–MS/MS method that can quantify phylloquinone (VK1), menaquinones‐4 (MK‐4), and menaquinones‐7 (MK‐7) in serum and cerebrospinal fluid and explore its clinical applications. We developed an LC–MS/MS method with atmospheric pressure chemical ionization to quantify and validate its performance according to Clinical Laboratory and Standard Institution standards (C62‐Ed2). Serums from 50 healthy individuals and cerebrospinal fluid from 15 patients were collected for clinical application. Sample preparation involved lipase incubation, protein precipitation with ethanol, and liquid–liquid extraction with hexane/ethyl; optimization was performed for sample preparation and LC separation. Linearity was 50–10 000 pg/mL for VK1, MK‐4, and MK‐7. The total coefficient of variation (%) for VK1, MK‐4, and MK‐7 ranged from 8.5% to 10.4%, 8.0% to 10.4%, and 7.0% to 11.1%, respectively. Recovery of VK1, MK‐4, and MK‐7 was 82.3%–110.6%, 92.3%–110.6%, and 89.5%–117.8%, respectively. VK1 and MK‐7 were detected in the serum of all 50 healthy subjects, whereas MK‐4 was detected in only 13 (26%) subjects. Approximately 53.3% (8/15) had no detectable vitamin K in their cerebrospinal fluid. The developed method exhibited satisfactory performance and was applicable for detecting VK1, MK‐4, and MK‐7 in serum and cerebrospinal fluid.