A Validated HPLC Method for the Determination of Vitamin K in Human Serum – First Application in a Pharmacological Study

Milka Marinova#, 1, 2, Dieter Lütjohann*, #, 2, Philipp Westhofen1, Matthias Watzka1, Olof Breuer2, Johannes Oldenburg1
1 Institute of Experimental Hematology and Transfusion Medicine, University of Bonn, Germany
2 Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Germany

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© 2011 Marinovaet al;

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Institute of Clinical Chemistry and Clinical Pharmacology, University Clinic Bonn, Bonn, Germany, Sigmund- Freud-Str. 25, D-53127 Bonn, Germany; Tel: +4922828714027; Fax: +4922828716047; E-mail:
# both authors contributed equally


Dietary phylloquinone (vitamin K1) is considered to be a major determinant of human vitamin K status. For this reason, measurements of plasma vitamin K concentrations provide a useful tool of vitamin K status in man. There is a growing interest in the role, biochemical function, and metabolism of vitamin K in vivo.

A modified reversed phase-HPLC method with fluorescence detection after post-column zinc reduction in serum samples was validated for vitamin K analysis. Two human volunteers were investigated after p.o. and i.v. administration of 2 mg Konakion® MM. Blood samples were collected periodically after administration and subjected to pharmacokinetic evaluation. High sensitivity, analytical recoveries, accuracy and calibration curves linearities could be reached. Our results showed an overall coefficient of variation of less than 10% for the intra- and interday reproducibility, while the recovery ranged from 91% to 114 %. The limit of detection and quantification were 0.015 ng mL-1 and 0.15 ng mL-1, respectively. Long-term stability was verified over a period of six months. The accuracy was proven by good results from external quality assurance. The observed pharmacokinetic differences indicate significant intra- and inter-individual variance of vitamin K fate in the human body.

This highly accurate, robust and reliable method is appropriate for the evaluation of vitamin K status in human serum. The ability to determine vitamin K has the potential to improve pharmacokinetic studies.

Keywords: Chromatography, coagulation, Konakion, lipids, menaquinone-4, pharmacokinetics, phylloquinone.