Simultaneous measurement of plasma vitamin D3 metabolites, including 4β,25-dihydroxyvitamin D3, using liquid chromatography-tandem mass spectrometry

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 1 alpha,25-dihydroxyvitamin D-3 [1 alpha,25(OH)(2)D-3] concentrations. This interfering metabolite has been identified as 4 beta,25-dihydroxyvitamin D-3 [4 beta,25(OH)(2)D-3] and was found at concentrations comparable to 1 alpha,25(OH)(2)D-3. Quantification of 1 alpha,25(OH)(2)D-3 in plasma required complete chromatographic separation of 1 alpha,25(OH)(2)D-3 from 4 beta,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, 1 alpha,25(OH)(2)D-3, and 4 beta,25(OH)(2)D-3 in healthy individuals. The LC-MS/MS method developed and described here could result in considerable improvement in quantifying 1 alpha,25(OH)(2)D-3 as well as monitoring the newly identified circulating metabolite, 4 beta,25(OH)(2)D-3. (C) 2011 Elsevier Inc. All rights reserved.