Clinical Utility of Simultaneous Quantitation of 25-Hydroxyvitamin D and 24,25-Dihydroxyvitamin D by LC-MS/MS Involving Derivatization With DMEQ-TAD

• Published in: The journal of clinical endocrinology and metabolism Vol. 99; no. 7; pp. 2567 - 2574
• Main Authors: Kaufmann, Martin, Gallagher, J. Christopher, Peacock, Munro, Schlingmann, Karl-Peter, Konrad, Martin, DeLuca, Hector F., Sigueiro, Rita, Lopez, Borja, Mourino, Antonio, Maestro, Miguel, St-Arnaud, René, Finkelstein, Joel S., Cooper, Donald P., Jones, Glenville
• Format: Journal Article
• Language: English
• Published: United States Oxford University Press 01.07.2014; Copyright by The Endocrine Society; Endocrine Society
• Subjects: 24,25-Dihydroxyvitamin D 3 - analysis; 24,25-Dihydroxyvitamin D 3 - blood; 25-Hydroxyvitamin D; Animal models; Animals; Blood Chemical Analysis - instrumentation; Blood Chemical Analysis - methods; Chromatography, Liquid - methods; Cytochrome P450; Dietary Supplements; Endocrine Research; Female; Humans; Hypercalcemia; Hypercalcemia - blood; Hypercalcemia - diagnosis; Liquid chromatography; Mass spectroscopy; Mice; Mice, Knockout; Predictive Value of Tests; Quality assurance; Quantitation; Quinoxalines - chemistry; Serum levels; Tandem Mass Spectrometry - methods; Triazoles - chemistry; Vitamin D; Vitamin D - administration & dosage; Vitamin D - analogs & derivatives; Vitamin D - analysis; Vitamin D - blood; Vitamin D Deficiency - blood; Vitamin D Deficiency - diagnosis; Vitamin deficiency
• ISSN: 0021-972X; 1945-7197; 1945-7197
• DOI: 10.1210/jc.2013-4388

Context: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].Objective: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.Design: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.Setting: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.Results: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.Conclusions: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.