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Licensed Unlicensed Requires Authentication Published by De Gruyter April 18, 2022

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

  • Andreas Meinitzer EMAIL logo , Dietmar Enko , Sieglinde Zelzer , Florian Prüller , Nerea Alonso , Eva Fritz-Petrin and Markus Herrmann



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.


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.


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.


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.

Corresponding author: Andreas Meinitzer, Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria, Phone: +43 316 385 83988, Fax: +43 316 385 13419, E-mail:

  1. Research funding: None declared.

  2. Author contributions: AM and DE wrote the manuscript and AM, supervised the study. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  3. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

  4. Informed consent: Informed consent was obtained from all individuals included in this study.

  5. Ethical approval: The ethical approval was provided by the Ethics Comitee of Upper Austria (Linz, Austria).


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Received: 2021-12-22
Accepted: 2022-03-31
Published Online: 2022-04-18
Published in Print: 2022-06-27

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