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.