title: High throughput LC-MS/MS method for the simultaneous analysis of multiple vitamin D analytes in serum.
abstract: Recent studies suggest that vitamin D-deficiency is linked to increased risk of common human health problems. To define vitamin D 'status' most routine analytical methods quantify one particular vitamin D metabolite, 25-hydroxyvitamin D3 (25OHD3). However, vitamin D is characterized by complex metabolic pathways, and simultaneous measurement of multiple vitamin D metabolites may provide a more accurate interpretation of vitamin D status. To address this we developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyse multiple vitamin D analytes, with particular emphasis on the separation of epimer metabolites. A supportive liquid-liquid extraction (SLE) and LC-MS/MS method was developed to quantify 10 vitamin D metabolites as well as separation of an interfering 7α-hydroxy-4-cholesten-3-one (7αC4) isobar (precursor of bile acid), and validated by analysis of human serum samples. In a cohort of 116 healthy subjects, circulating concentrations of 25-hydroxyvitamin D3 (25OHD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3), 24,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), and 25-hydroxyvitamin D2 (25OHD2) were quantifiable using 220μL of serum, with 25OHD3 and 24R,25(OH)2D3 showing significant seasonal variations. This high-throughput LC-MS/MS method provides a novel strategy for assessing the impact of vitamin D on human health and disease.
authors.affiliation: Institute for Metabolism and Systems Research, The University of Birmingham, Birmingham B15 2TT, UK.
authors.forename: Carl
authors.initials: C
authors.lastname: Jenkinson
authors.affiliation: Institute for Metabolism and Systems Research, The University of Birmingham, Birmingham B15 2TT, UK.
authors.forename: Angela E
authors.initials: AE
authors.lastname: Taylor
authors.affiliation: Institute for Metabolism and Systems Research, The University of Birmingham, Birmingham B15 2TT, UK.
authors.forename: Zaki K
authors.initials: ZK
authors.lastname: Hassan-Smith
authors.affiliation: Orthopaedic Surgery, Medicine and Molecular, Cell & Developmental Biology, UCLA 615 Charles E. Young Dr. South, Rm. 410E, Los Angeles 90095, CA, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Adams
authors.affiliation: Faculty of Medicine and Health, University of Leeds, Leeds LS2 9NL.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Stewart
authors.affiliation: Institute for Metabolism and Systems Research, The University of Birmingham, Birmingham B15 2TT, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Hewison
authors.affiliation: Department of Clinical Biochemistry, University Hospital South Manchester NHS Foundation Trust, Manchester, UK Manchester Academic Health Science Centre, University Hospital South Manchester, The University of Manchester, Manchester, M13 9NT, UK.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Keevil
mesh_terms: D000328:Adult; D000368:Aged; D002853:Chromatography, Liquid / Q000379:methods; D015331:Cohort Studies; D005260:Female; D006801:Humans; D059625:Liquid-Liquid Extraction / Q000379:methods; D008297:Male; D008875:Middle Aged; D012621:Seasons; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014807:Vitamin D / Q000032:analysis / Q000097:blood* / Q000378:metabolism; D014815:Vitamins / Q000032:analysis / Q000097:blood* / Q000378:metabolism; D055815:Young Adult
chemical_list: D014815:Vitamins; D014807:Vitamin D
keywords: Chiral separation; LC–MS/MS; Method validation; Serum analysis; Vitamin D