Clin Chem Lab Med. 2025 Dec 16. doi: 10.1515/cclm-2025-0719. Online ahead of print.
ABSTRACT
OBJECTIVES: Vitamin K homologues are essential to human health, and their concentrations in biological samples serve as valuable diagnostic biomarkers. This study was aimed to develop a method for determining vitamins K1 (phylloquinone, VK1) and K2 (menaquinone, MK-4) in human serum. The proposed method was validated and applied to the serum of a cohort of 20 Russian individuals.
METHODS: High-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was used to analyse the content of VK1 and MK-4 in serum. Atmospheric pressure chemical ionisation (APCI) in negative mode was applied to ionise VK1 and MK-4. Protein precipitation and solid-phase extraction (SPE) on polystyrene-divinylbenzene resin were combined to isolate and preconcentrate the analytes from serum.
RESULTS: The HPLC-MSMS method was developed and validated for the determination of vitamins VK1 and MK-4 in human serum. The method demonstrated a lower limit of quantification (LLOQ) of 0.05 μg/L, with more than 71 % recoveries and precision within 17 %. To demonstrate the applicability of the method to real samples, serum from 20 healthy adults was analyzed. VK1 was detected in four individuals (0.094-0.96 μg/L), whereas MK-4 concentrations were below 0.22 μg/L in all cases.
CONCLUSIONS: The validated HPLC-MS/MS workflow provides a reliable and sensitive approach for the quantification of VK1 and MK-4 in minimal serum volumes. The method demonstrates robustness, reproducibility, and suitability for large-scale analytical applications. The proposed LC-MS/MS protocol successfully applied to native human serum samples, illustrating its applicability for future clinical and biochemical studies involving vitamin K.
PMID:41397160 | DOI:10.1515/cclm-2025-0719