Quantitative analysis of phylloquinone (vitamin K1) and menaquinone (vitamin K2) in serum of Russians by liquid chromatography-tandem mass spectrometry
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Mikhail Vokuev
,
Anastasia Frolova
,
Stepan Makarkin
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.
Funding source: Lomonosov Moscow State University
Award Identifier / Grant number: â„– 23-SCH-04-34
Award Identifier / Grant number: â„– 124041900012-4
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Research ethics: The residual serum samples used in this study were fully anonymised prior to use and the research team had no access to any identifiable patient information. In accordance with the ethical principles outlined in the national research guidelines, the secondary use of this anonymised material was deemed exempt from full ethical review and qualified for a waiver of informed consent by the local ethics committee.
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Informed consent: Not applicable.
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Author contributions: Mikhail Vokuev: Investigation, Validation, Conceptualisation, Writing - Original draft preparation, Writing - Review & Editing. Stepan Makarkin: Validation. Anastasia Frolova: Writing - Original draft preparation, Resources. Daria Prosuntsova: Validation. Timur Baygildiev: Writing - Original draft preparation. Lidia Nefedova: Sample acquisition, Funding acquisition. Oleg Klychnikov: Project administration. Igor Rodin: Supervision. All authors participated in discussions about developing the experimental workflow, interpreting the results, and editing the manuscript text.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: The authors state no conflict of interest.
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Research funding: This research was supported by the MSU Program of Development (№ 23-SCH-04-34) and the Ministry of Science and Higher Education of the Russian Federation (№ 124041900012-4).
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Data availability: Not applicable.
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Supplementary Material
This article contains supplementary material (https://doi.org/10.1515/cclm-2025-0719).
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