UPLC-MS/MS Determination of Deuterated 25-Hydroxyvitamin D (d-25OHD) and Other Vitamin D Metabolites for the Measurement of 25OHD Half-Life.

Plasma 25-hydroxyvitamin D (25OHD) half-life (25OHDt ) is a dynamic marker of vitamin D metabolism that can be used to assess vitamin D expenditure and help inform vitamin D requirements. Our group recently established an approach to determine the 25OHDt as an alternative biomarker of 25OHD expenditure in humans. The approach uses a small oral dose of stable isotope labeled 25OHD [3-H-25OHD (6,19,19-d)] (d-25OHD) (tracer), which is distinguishable from endogenous 25OHD by liquid chromatography tandem-mass spectrometry (LC-MS/MS). We report here the method, which relies on protein precipitation, purification with solid phase extraction, derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and determination of the compounds by isotope-dilution UPLC-MS/MS. The method proved to be rapid and sensitive (LOQ 0.2 nmol/L) for the quantification of this tracer as well as the other vitamin D metabolites: 25OHD, 25OHD, and 24,25(OH)D in human plasma.