Reversed phase UPLC/APCI-MS determination of Vitamin K1 and menaquinone-4 in human plasma: Application to a clinical study

Yuning Zhang, Yashpal S. Chhonker, Veenu Bala, Alex Hagg, Linda G. Snetselaar, Terry L. Wahls, Daryl J. Murry

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


A rapid and sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated to simultaneously quantitate vitamin K1 (PK) and vitamin K2 (MK-4) concentrations in human plasma to evaluate nutritional interventions. Charcoal-stripped human plasma was used for standard and calibration preparation with vitamin E-d6 as the internal standard. Patient plasma samples were extracted using n-hexane and analytes separated using an ACE –PFP C18 column (50 × 2.1 mm, 3 μ) equipped with standard C18 guard. The mobile phase consisted of 0.1 % formic acid in water and 0.1 % formic acid in methanol at a flow rate of 0.5 mL/min. Analyte quantification was achieved by using MS/MS with a positive atmospheric pressure chemical ionization in multiple reaction monitoring (MRM) mode. The lower limit of quantification was 0.01 ng/mL for both PK and MK-4. The assay was linear over the concentration range from 0.01–50 ng/mL for all analytes with a determination coefficient (r2) of 0.998 or better. The intra-and inter-day accuracy and precision were within the acceptable limits per FDA guidance. The validated method was successfully applied to a clinical study for quantification of PK and MK-4 in human plasma to assess nutritional status and dietary interventions in patients with neurodegenerative diseases. Baseline plasma concentrations of PK and MK-4 ranged from 0.23 to 1.81 ng/mL and 0.33–0.57 ng/mL, respectively.

Original languageEnglish (US)
Article number113147
JournalJournal of Pharmaceutical and Biomedical Analysis
StatePublished - May 10 2020


  • Charcoal-stripped human plasma
  • Liquid chromatography-tandem mass spectrometry
  • Liquid-liquid extraction
  • Menaquinones
  • Phylloquinone
  • Vitamin K

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry


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