Simultaneous measurement of plasma vitamin D(3) metabolites, including 4β,25-dihydroxyvitamin D(3), using liquid chromatography-tandem mass spectrometry.

Abstract Simultaneous and accurate measurement of circulating vitamin D metabolites is critical to studies of the metabolic regulation of vitamin D and its impact on health and disease. To that end, we have developed a specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method that permits the quantification of major circulating vitamin D 3 metabolites in human plasma. Plasma samples were subjected to a protein precipitation, liquid–liquid extraction, and Diels–Alder derivatization procedure prior to LC–MS/MS analysis. Importantly, in all human plasma samples tested, we identified a significant dihydroxyvitamin D 3 peak that could potentially interfere with the determination of 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ] concentrations. This interfering metabolite has been identified as 4β,25-dihydroxyvitamin D 3 [4β,25(OH) 2 D 3 ] and was found at concentrations comparable to 1α,25(OH) 2 D 3 . Quantification of 1α,25(OH) 2 D 3 in plasma required complete chromatographic separation of 1α,25(OH) 2 D 3 from 4β,25(OH) 2 D 3 . An assay incorporating this feature was used to simultaneously determine the plasma concentrations of 25OHD 3 , 24R,25(OH) 2 D 3 , 1α,25(OH) 2 D 3 , and 4β,25(OH) 2 D 3 in healthy individuals. The LC–MS/MS method developed and described here could result in considerable improvement in quantifying 1α,25(OH) 2 D 3 as well as monitoring the newly identified circulating metabolite, 4β,25(OH) 2 D 3 .