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J Chromatogr B Analyt Technol Biomed Life Sci. 2016 Mar 1;1014:56-63. doi: 10.1016/j.jchromb.2016.01.049. Epub 2016 Feb 2.

High throughput LC-MS/MS method for the simultaneous analysis of multiple vitamin D analytes in serum.

Author information

  • 1Institute for Metabolism and Systems Research, The University of Birmingham, Birmingham B15 2TT, UK. Electronic address:
  • 2Institute for Metabolism and Systems Research, The University of Birmingham, Birmingham B15 2TT, UK.
  • 3Orthopaedic Surgery, Medicine and Molecular, Cell & Developmental Biology, UCLA 615 Charles E. Young Dr. South, Rm. 410E, Los Angeles 90095, CA, USA.
  • 4Faculty of Medicine and Health, University of Leeds, Leeds LS2 9NL, UK.
  • 5Department 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.


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.


Chiral separation; LC–MS/MS; Method validation; Serum analysis; Vitamin D

[Available on 2017-03-01]
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