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

• Published in: Journal of Chromatography A 2024-01, Vol.1714, p.464548, Article 464548
• Main Authors: Mrštná, Kristýna, Matoušová, Kateřina, Krčmová, Lenka Kujovská, Carazo, Alejandro, Pourová, Jana, Mladěnka, Přemysl, Matysová, Ludmila, Švec, František
• Format: Article
• Language: English
• Subjects: Bioanalysis; blood serum; chemical species; chromatography; coagulation; electrospray ionization mass spectrometry; high density lipoprotein; humans; liquid-liquid extraction; Menaquinone; methanol; Phylloquinone; rats; UHPLC-MS/MS; Ultracentrifugation
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2023.464548

•A novel UHPLC-MS/MS method was developed for the analysis vitamin K.•A simple extraction technique based on miniaturized LLE was used.•Ultracentrifugation for the separation of lipoprotein fractions was included.•Four forms of vit. K were analyzed in human and rat serum and lipoprotein fractions. 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 K2 (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™ 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 K1, 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.