title: Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  METHODS
  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.affiliation: Department of Biomedical and Molecular Sciences (M.Ka., G.J.), Queen's University, Kingston, Ontario, Canada K7L 3N6; Bone Metabolism Unit (J.C.G.), Creighton University School of Medicine, Omaha, Nebraska 68131; Indiana University School of Medicine (M.P.), Indianapolis, Indiana 46202-5250; Department of General Pediatrics, (K-P. S., M.Ko.) University Children's Hospital, D-48149, Münster, Germany; Department of Biochemistry (H.F.D.), University of Wisconsin-Madison, Madison, Wisconsin 53706; Departmento de Quimica Organica (R.S., B.L., A.M., M.M.), Laboratorio Ignacio Ribas, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; McGill University and Shriners Hospital for Children-Canada (R.S.), Montréal, Québec, Canada H3G 1A6; Endocrine Unit (J.S.F.), Massachusetts General Hospital, Boston Massachusetts 02114; and Health Sciences Research, Waters Corporation (D.P.C), Stamford Avenue, Altrincham Rd, Wilmslow, SK9 4AX United Kingdom.
authors.forename: Martin
authors.initials: M
authors.lastname: Kaufmann
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Gallagher
authors.forename: Munro
authors.initials: M
authors.lastname: Peacock
authors.forename: Karl-Peter
authors.initials: KP
authors.lastname: Schlingmann
authors.forename: Martin
authors.initials: M
authors.lastname: Konrad
authors.forename: Hector F
authors.initials: HF
authors.lastname: DeLuca
authors.forename: Rita
authors.initials: R
authors.lastname: Sigueiro
authors.forename: Borja
authors.initials: B
authors.lastname: Lopez
authors.forename: Antonio
authors.initials: A
authors.lastname: Mourino
authors.forename: Miguel
authors.initials: M
authors.lastname: Maestro
authors.forename: René
authors.initials: R
authors.lastname: St-Arnaud
authors.forename: Joel S
authors.initials: JS
authors.lastname: Finkelstein
authors.forename: Donald P
authors.initials: DP
authors.lastname: Cooper
authors.forename: Glenville
authors.initials: G
authors.lastname: Jones
mesh_terms: D015650:24,25-Dihydroxyvitamin D 3 / Q000032:analysis / Q000097:blood*; D000818:Animals; D001774:Blood Chemical Analysis / Q000295:instrumentation / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D019587:Dietary Supplements; D005260:Female; D006801:Humans; D006934:Hypercalcemia / Q000097:blood / Q000175:diagnosis*; D051379:Mice; D018345:Mice, Knockout; D011237:Predictive Value of Tests; D011810:Quinoxalines / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014230:Triazoles / Q000737:chemistry; D014807:Vitamin D / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood; D014808:Vitamin D Deficiency / Q000097:blood / Q000175:diagnosis*
chemical_list: D011810:Quinoxalines; D014230:Triazoles; C070204:4-(2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl)ethyl)-1,2,4-triazoline-3,5-dione; D014807:Vitamin D; D015650:24,25-Dihydroxyvitamin D 3; C104450:25-hydroxyvitamin D