Measurement of deuterium-labeled phylloquinone in plasma by high-performance liquid chromatography/mass spectrometry (LC/MS)

Authors: FU, XUEYAN - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; PETERSON, JAMES - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; HDEIB, MONA - Friedman School At Tufts; Booth, Sarah; GRUSAK, MICHAEL - US Department Of Agriculture (USDA); Lichtenstein, Alice; Dolnikowski, Gregory

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/22/2009
Publication Date: 7/1/2009
Citation: Fu, X., Peterson, J.W., Hdeib, M., Booth, S.L., Grusak, M.A., Lichtenstein, A.H., Dolnikowski, G. 2009. Measurement of deuterium-labeled phylloquinone in plasma by high-performance liquid chromatography/mass spectrometry (LC/MS). Analytical Chemistry. 81:5421-5425.

Interpretive Summary: Phylloquinone is a fat-soluble compound present in low concentrations in circulation, which presents technical challenges for its measurement. To study how much phylloquione is absorbed from food, we add deuterium (a naturally occurring isotope that contains extra hydrogens) to phylloquinone in the food of interest, and then measure the phylloquinone in plasma with and without deuterium. We developed a method to measure phylloquinone concentrations in plasma using high-performance liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization (LC-APCI/MS). Phylloquinone was extracted from plasma, purified by several analytical steps and then quantified using HPLC with an APCI/MS as a detector. We determined that the expected versus the measured amount of known amounts of phylloquinone added to plasma were highly correlated. The minimum detectable concentration of phylloquinone in plasma was 0.05 pmol/injection without deuterium added, and 0.08 pmol/injection when deuterium was added to the phylloquinone. We were able to recover 96.7 % of the phylloquinone added to the plasma without deuterium, and 96.2 % with phylloquinone with deuterium added. Plasma phylloquinone concentrations determined by our traditional HPLC method compared to our new method were not statistically different. The LC-APCI/MS method is a sensitive and accurate technique for simultaneous determination of phylloquinone with and without deuterium added, and can be applied to bioavailability studies.

Technical Abstract: 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 HPLC 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 concentration of unlabeled and labeled phylloquinone was 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 4.0% 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 and accurate technique for simultaneous determination of both unlabeled and labeled phylloquinone, and can be applied to bioavailability studies.