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 ), 24 R ,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.