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

Blood levels of the vitamin D₃ (D₃) metabolites 25-hydroxyvitamin D₃ (25(OH)D₃), 24R,25-dihydroxyvitamin D₃, and 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are recognized indicators for the diagnosis of bone metabolism-related diseases, D₃ 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 D₃ metabolites, such as 20-hydroxyvitamin D₃ and lactone D₃, 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 D₃ metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D₃ (2), 25(OH)D₃-23,26-lactone (6), and 1,25(OH)₂D₃-23,26-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)₂D₃-23,26-lactone (7) in human serum.