Analysis of vitamin K₁ and major K₂ variants in rat/human serum and lipoprotein fractions by a rapid, simple, and sensitive UHPLC-MS/ MS method

Determination of the various forms of vitamin K, which are involved in coagulation and other physiological processes in humans, is challenging and no standardized method is yet available. Therefore, a reliable and practical method was developed to quantify vitamin K levels in serum and additionally in lipoprotein fractions to clarify its distribution. The LC-MS/MS method for the determination of vitamin K1 and the three main isoforms of vitamin K-2 (MK-4, MK-7, MK-9) was combined with a gradient ultracentrifugation technique to allow the separation of lipoprotein fractions. The chromatographic separation was carried out on a Kinetex (TM) C18 column using a mobile phase consisting mainly of methanol. The target analytes were detected by electrospray ionization mass spectrometry. The separation of all four substances was achieved after a simple sample preparation technique based on miniaturized liquid-liquid extraction. Our method of only 8.5 min revealed the levels of the major forms of vitamin K in 59 human and 12 rat sera and confirmed our hypothesis that vitamin K is primarily (about 50 %) found in the high-density lipoprotein fraction. The median concentrations of vitamin K-1, MK-4, MK-7, and MK-9 were found to be 1.19, 2.98, 0.43, and < 0.71 nmol/L in human serum and 1.74, 6.75, less than 0.2, and less than 0.5 nmol/L in rat serum, respectively.