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  3. Studies on the analysis of 25-hydroxyvitamin D(3) by isotope-dilution liquid chromatography-tandem mass spectrometry using enzyme-assisted derivatisation

Studies on the analysis of 25-hydroxyvitamin D(3) by isotope-dilution liquid chromatography-tandem mass spectrometry using enzyme-assisted derivatisation

  • Biochem Biophys Res Commun. 2014 Apr 11;446(3):745-50. doi: 10.1016/j.bbrc.2014.01.088.
Jonas Abdel-Khalik 1,  Peter J Crick 2,  Graham D Carter 3,  Hugh L Makin 4,  Yuqin Wang 2,  William J Griffiths 5
Affiliations

Affiliations

  • 1 Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK. Electronic address: J.A.F.A.ABDEL-KHALIK.744116@swansea.ac.uk.
  • 2 Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK.
  • 3 DEQAS, Imperial College Healthcare NHS Trust, Clinical Biochemistry Department, Charing Cross Hospital, Fulham Palace Rd, London W6 8RF, UK.
  • 4 Barts and the Royal London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AD, UK.
  • 5 Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK. Electronic address: w.j.griffiths@swansea.ac.uk.
PMID: 24486315 DOI: 10.1016/j.bbrc.2014.01.088
Abstract

The total serum concentration of 25-hydroxyvitamins D (25-hydroxyvitamin D3 and 25-hydroxyvitamin D2) is currently used as an indicator of Vitamins D status. Vitamins D insufficiency is claimed to be associated with multiple diseases, thus accurate and precise reference methods for the quantification of 25-hydroxyvitamins D are needed. Here we present a novel enzyme-assisted derivatisation method for the analysis of Vitamins D metabolites in adult serum utilising 25-[26,26,26,27,27,27-(2)H6]hydroxyvitamin D3 as the internal standard. Extraction of 25-hydroxyvitamins D from serum is performed with acetonitrile, which is shown to be more efficient than ethanol. Cholesterol oxidase is used to oxidize the 3β-hydroxy group in the Vitamins D metabolites followed by derivatisation of the newly formed 3-oxo group with Girard P reagent. 17β-Hydroxysteroid dehydrogenase type 10 is shown to oxidize selectively the 3α-hydroxy group in the 3α-hydroxy epimer of 25-hydroxyvitamin D3. Quantification is achieved by isotope-dilution liquid chromatography-tandem mass spectrometry. Recovery experiments for 25-hydroxyvitamin D3 performed on adult human serum give recovery of 102-106%. Furthermore in addition to 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3 and other uncharacterised dihydroxy metabolites, were detected in adult human serum.

Keywords

17βHSD10; Cholesterol oxidase; Girard P reagent; LC–MS(n); Serum; Vitamins D.

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