The discovery of hypercalcemic diseases due to loss-of-function mutations in 25-hydroxyvitamin D-24-hydroxylase has placed a new demand for sensitive and precise assays for 24,25-dihydroxyvitamin D [24,25-(OH)₂D].

We describe a novel liquid chromatography and tandem mass spectrometry-based method involving derivatization with DMEQ-TAD {4-[2-(6,7-dimethoxy-4-methyl-3,4-dihydroquinoxalinyl)ethyl]-1,2,4-triazoline-3,5-dione} to simultaneously assay multiple vitamin D metabolites including 25-hydroxyvitamin D (25-OH-D) and 24,25-(OH)₂D using 100 μL of serum with a 5-minute run time.

The assay uses a newly synthesized internal standard d₆-24,25-(OH)₂D₃ enabling the quantitation of 24,25-(OH)₂D₃ as well as the determination of the ratio of 25-OH-D₃ to 24,25-(OH)₂D₃, a physiologically useful parameter.

We report data on more than 1000 normal and disease samples involving vitamin D deficiency or hypercalcemia in addition to studies involving knockout mouse models.

The assay showed good correlation with samples from quality assurance schemes for 25-OH-D (25-OH-D₂ and 25-OH-D₃) determination (−2% to −5% bias) and exhibited low inter- and intraassay coefficients of variation (4%–7%) and lower limits of quantitation of 0.25–0.45 nmol/L. In clinical studies, we found a strong correlation between serum levels of 25-OH-D₃ and 24,25-(OH)₂D₃ (r² = 0.80) in subjects over a broad range of 25-OH-D₃ values and a marked lack of production of 24,25-(OH)₂D₃ below 25 nmol/L of 25-OH-D. The ratio of 25-OH-D₃ to 24,25-(OH)₂D₃, which remained less than 25 in vitamin D-sufficient subjects (serum 25-OH-D < 50 nmol/L) but was greatly elevated (80–100) in patients with idiopathic infantile hypercalcemia.

The new method showed good utility in clinical settings involving vitamin D deficiency; supplementation with vitamin D and idiopathic infantile hypercalcemia, as well as in animal models with ablation of selected cytochrome P450-containing enzymes involved in vitamin D metabolism.