skip to main content

Title: Studies on the analysis of 25-hydroxyvitamin D{sub 3} by isotope-dilution liquid chromatography–tandem mass spectrometry using enzyme-assisted derivatisation

Highlights: • New method for the analysis of 25-hydroxyvitamin D{sub 3} exploiting Girard P derivatisation. • Method also applicable to vitamin D{sub 3}, 1α,25- and 24,25-dihydroxyvitamin D{sub 3}. • By modification of the method 3-epi-25-hydroxyvitamin D{sub 3} can also be analysed. - Abstract: The total serum concentration of 25-hydroxyvitamins D (25-hydroxyvitamin D{sub 3} and 25-hydroxyvitamin D{sub 2}) 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-{sup 2}H{sub 6}]hydroxyvitamin D{sub 3} 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 D{sub 3}. Quantification is achieved by isotope-dilution liquid chromatography–tandem mass spectrometry. Recovery experiments for 25-hydroxyvitamin D{submore » 3} performed on adult human serum give recovery of 102–106%. Furthermore in addition to 25-hydroxyvitamin D{sub 3}, 24,25-dihydroxyvitamin D{sub 3} and other uncharacterised dihydroxy metabolites, were detected in adult human serum.« less
Authors:
 [1] ;  [1] ;  [2] ;  [3] ;  [1] ;  [1]
  1. Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP (United Kingdom)
  2. DEQAS, Imperial College Healthcare NHS Trust, Clinical Biochemistry Department, Charing Cross Hospital, Fulham Palace Rd, London W6 8RF (United Kingdom)
  3. Barts and the Royal London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AD (United Kingdom)
Publication Date:
OSTI Identifier:
22416369
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 446; Journal Issue: 3; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ACETONITRILE; CALIBRATION STANDARDS; CHOLESTEROL; CONCENTRATION RATIO; DILUTION; DISEASES; ETHANOL; INDICATORS; LIQUID COLUMN CHROMATOGRAPHY; MASS SPECTROSCOPY; METABOLITES; NAD; OXIDASES; PYRIDINE; REAGENTS; VITAMIN D