Development of a liquid chromatography mass spectrometry method for the determination of vitamin K1, menaquinone-4, menaquinone-7 and vitamin K1-2,3 epoxide in serum of individuals without vitamin K supplements.

ABSTRACT

OBJECTIVES: Vitamin K and metabolites have a beneficial role in blood coagulation, bone metabolism and growth. However, the determination of vitamin K concentrations in the blood in patients consuming a diet with naturally occurring vitamin K is currently challenging. We aim to develop a cost-effective and rapid method to measure vitamin K metabolites with potential application for clinics and research. METHODS: We developed a simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of vitamin K1, menaquinone-4 (MK-4), menaquinone-7 (MK-7) and vitamin K1-2,3 epoxide in human serum and validated the method in a study cohort of 162 patients tested for carbohydrate malabsorption and in 20 patients with oral phenprocoumon intake. RESULTS: The overall precision (CVs) ranged between 4.8 and 17.7% in the specified working range (0.06-9.0 nmol/L for all analytes except for MK-7 with 0.04-6.16 nmol/L). In the malabsorption cohort samples, measured values were obtained for all different vitamin K metabolites except for vitamin K1-2,3 epoxide. This metabolite could be detected only in patients with phenprocoumon intake. The good performance of the method is especially achieved by the interaction of three factors the use of lipase in the sample preparation, the use of an atypical fluorinated reversed phase column, and a logarithmic methanol gradient. CONCLUSIONS: The described method is able to determine the concentration of four vitamin K metabolites in a time-efficient, simple and cost-effective manner. It can be suitable for both routine clinics and research.