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

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

Research output: Contribution to journalArticle

91 Citations (Scopus)

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 (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)2D 3, 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.

Original languageEnglish
Pages (from-to)2567-2574
Number of pages8
JournalJournal of Clinical Endocrinology and Metabolism
Volume99
Issue number7
DOIs
StatePublished - 2014

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Dihydroxycholecalciferols
Vitamin D
Assays
Vitamin D Deficiency
Cytochrome P-450 Enzyme System
Serum
25-hydroxyvitamin D
4-(2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl)-1,2,4-triazoline-3,5-dione
hydroxide ion
Liquid chromatography
Hypercalcemia
Metabolites
Tandem Mass Spectrometry
Mixed Function Oxygenases
Ablation
Quality assurance
Metabolism
Knockout Mice
Liquid Chromatography
Mass spectrometry

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Endocrinology
  • Biochemistry, medical
  • Endocrinology, Diabetes and Metabolism

Cite this

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

In: Journal of Clinical Endocrinology and Metabolism, Vol. 99, No. 7, 2014, p. 2567-2574.

Research output: Contribution to journalArticle

Kaufmann, M, Gallagher, JC, Peacock, M, Schlingmann, KP, Konrad, M, DeLuca, HF, Sigueiro, R, Lopez, B, Mourino, A, Maestro, M, St-Arnaud, R, Finkelstein, JS, Cooper, DP & Jones, G 2014, 'Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD', Journal of Clinical Endocrinology and Metabolism, vol. 99, no. 7, pp. 2567-2574. https://doi.org/10.1210/jc.2013-4388
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. / Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD. In: Journal of Clinical Endocrinology and Metabolism. 2014 ; Vol. 99, No. 7. pp. 2567-2574.
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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 (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)2D 3, 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.",
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T1 - Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD

AU - Kaufmann, Martin

AU - Gallagher, J. Christopher

AU - Peacock, Munro

AU - Schlingmann, Karl Peter

AU - Konrad, Martin

AU - DeLuca, Hector F.

AU - Sigueiro, Rita

AU - Lopez, Borja

AU - Mourino, Antonio

AU - Maestro, Miguel

AU - St-Arnaud, René

AU - Finkelstein, Joel S.

AU - Cooper, Donald P.

AU - Jones, Glenville

PY - 2014

Y1 - 2014

N2 - 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)2D 3, 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.

AB - 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)2D 3, 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.

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