Measurement of vitamin K and its derivatives in human plasma through enzyme-assisted liquid chromatography-tandem mass spectrometry assay

Demirel M., Selek Ş.

EUROPEAN JOURNAL OF MASS SPECTROMETRY, vol.29, no.3, pp.200-206, 2023 (SCI-Expanded)

  • Publication Type: Article / Article
  • Volume: 29 Issue: 3
  • Publication Date: 2023
  • Doi Number: 10.1177/14690667231183046
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, BIOSIS, Chemical Abstracts Core, Compendex, EMBASE, INSPEC, MEDLINE
  • Page Numbers: pp.200-206
  • Bezmialem Vakıf University Affiliated: Yes


Vitamin K is an essential lipophilic vitamin that acts as a coenzyme in several metabolic pathways. Accurate measurement of apolar metabolites transported by lipoproteins in serum matrices requires high-recovery extractions of vitamin K and its derivatives following standardized protocols. Conventionally developed methods in this field have predominantly employed solid-phase extraction for the measurement of vitamin K and its derivatives. In this study, our objective was to develop an enzyme-assisted extraction method for the precise measurement of vitamin K and its derivatives. Our methodology involved mixing 450 µL of serum samples with 50 µL of an internal standard and 50 µL of a lipase enzyme solution. Following vortexing, the mixture was incubated at 37°C for 15 min to activate the enzymes. The enzyme reaction was subsequently quenched with a mixture of 250 µL of methanol and 1 mL of hexane, followed by centrifugation at 12,000 g for 5 min. The upper phase was collected, concentrated using a concentrator device, and dissolved in a 100 µL solution of methanol/acetone/isopropanol (7:1:1, v/v/v) for analysis. Spectrum analysis was performed using the open-source MZmine 3 software, and a reference interval was established using the Python programming language on the Google Colab platform. The developed method for measuring vitamin K and its derivatives exhibited limit of detection and limit of quantitation values of 0.005 and 0.01 ng/mL, respectively. In conclusion, our study presents a precise and reliable method for the measurement of vitamin K and its derivatives using enzyme-assisted extraction.