Synthesis of Deuterium-Labeled Vitamin D Metabolites as Internal Standards for LC-MS Analysis.

Blood levels of the vitamin D (D) metabolites 25-hydroxyvitamin D (25(OH)D), 24,25-dihydroxyvitamin D, and 1α,25-dihydroxyvitamin D (1,25(OH)D) are recognized indicators for the diagnosis of bone metabolism-related diseases, D deficiency-related diseases, and hypercalcemia, and are generally measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using an isotope dilution method. However, other D metabolites, such as 20-hydroxyvitamin D and lactone D, also show interesting biological activities and stable isotope-labeled derivatives are required for LC-MS/MS analysis of their concentrations in serum. Here, we describe a versatile synthesis of deuterium-labeled D metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D (), 25(OH)D-23,26-lactone (), and 1,25(OH)D-23,26-lactone () were synthesized, and successfully applied as internal standards for the measurement of these compounds in pooled human serum. This is the first quantification of 1,25(OH)D-23,26-lactone () in human serum.