Measurement of Deuterium-Labeled Phylloquinone in Plasma by High-Performance Liquid Chromatography/Mass Spectrometry

• Published in: Analytical chemistry (Washington) Vol. 81; no. 13; pp. 5421 - 5425
• Main Authors: Fu, Xueyan, Peterson, James W, Hdeib, Mona, Booth, Sarah L, Grusak, Michael A, Lichtenstein, Alice H, Dolnikowski, Gregory G
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.07.2009
• Subjects: Analytical chemistry; Bioavailability; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Chromatography, High Pressure Liquid - methods; Correlation coefficient; Deuterium; Deuterium - chemistry; Exact sciences and technology; Humans; Hydrogen; Ionization; Isotope Labeling; Liquid chromatography; Mass spectrometry; Mass Spectrometry - methods; Other chromatographic methods; Plasma; Spectrometric and optical methods; Stable isotopes; Vitamin K 1 - blood; Vitamin K 1 - chemistry; Vitamins; Correlation coefficient; Deuterium; Solid phase extraction; Atmospheric pressure; Coupled method; Vitamin; HPLC chromatography; Liquid chromatography; Bioavailability; Chemical ionization; Fluorescence spectrometry; Chemical enrichment; Lipophilic compound; Dilution; Sample preparation; Simultaneous measurement; Hexane; Standard deviation; Phylloquinone; Mass spectrometry
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/ac900732w

Phylloquinone (vitamin K1) is a lipophilic compound present in plasma at low concentrations, which presents technical challenges for determining its bioavailability or metabolic fate using stable isotopes. We developed a method to simultaneously measure unlabeled and deuterium-labeled phylloquinone concentrations in plasma specimens using high-performance liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization (LC−APCI/MS). Phylloquinone was extracted from plasma using hexane, further purified by solid-phase extraction, and then quantified using high-performance liquid chromatography with an APCI/MS as a detector. Plotting the expected versus the measured amount of serial dilutions of either unlabeled or labeled phylloquinone gave correlation coefficients (R) of 0.999 for both compounds. The minimum detectable concentrations of unlabeled and labeled phylloquinone were 0.05 and 0.08 pmol/injection, respectively. Pooled plasma samples spiked with between 0.5 and 32 nmol phylloquinone/L gave average recoveries of 96.7% with 5.4% relative standard deviation (RSD) for unlabeled phylloquinone and 96.2% with 6.6% RSD for labeled phylloquinone. Plasma phylloquinone concentrations determined by LC-fluorescence and LC−APCI/MS methods from healthy subjects (n = 17) were not statistically different (P = 0.13). The LC−APCI/MS method is a sensitive technique for simultaneous determination of both unlabeled and labeled phylloquinone and can be applied to bioavailability studies.