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

Blood levels of the vitamin D(3) (D(3)) metabolites 25-hydroxyvitamin D(3) (25(OH)D(3)), 24R,25-dihydroxyvitamin D(3), and 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) are recognized indicators for the diagnosis of bone metabolism-related diseases, D(3) 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(3) metabolites, such as 20-hydroxyvitamin D(3) and lactone D(3), 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(3) metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D(3) (2), 25(OH)D(3)-23,26-lactone (6), and 1,25(OH)(2)D(3)-23,26-lactone (7) 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)(2)D(3)-23,26-lactone (7) in human serum.