Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD.

Abstract

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 (r(2) = 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.

• Authors: Martin Kaufmann, J Christopher Gallagher, Munro Peacock, Karl-Peter Schlingmann, Martin Konrad, Hector F DeLuca, Rita Sigueiro, Borja Lopez, Antonio Mourino, Miguel Maestro, René St-Arnaud, Joel S Finkelstein, Donald P Cooper, Glenville Jones
• Journal: The Journal of clinical endocrinology and metabolism (J Clin Endocrinol Metab) Vol. 99 Issue 7 Pg. 2567-74 (Jul 2014) ISSN: 1945-7197 [Electronic] United States
• PMID: 24670084 (Publication Type: Evaluation Study, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Quinoxalines
• Triazoles
• 4-(2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl)-1,2,4-triazoline-3,5-dione
• Vitamin D
• 24,25-Dihydroxyvitamin D 3
• 25-hydroxyvitamin D

Topics

• 24,25-Dihydroxyvitamin D 3 (analysis, blood)
• Animals
• Blood Chemical Analysis (instrumentation, methods)
• Chromatography, Liquid (methods)
• Dietary Supplements
• Female
• Humans
• Hypercalcemia (blood, diagnosis)
• Mice
• Mice, Knockout
• Predictive Value of Tests
• Quinoxalines (chemistry)
• Tandem Mass Spectrometry (methods)
• Triazoles (chemistry)
• Vitamin D (administration & dosage, analogs & derivatives, analysis, blood)
• Vitamin D Deficiency (blood, diagnosis)