Studies on the analysis of 25-hydroxyvitamin D(3) by isotope-dilution liquid chromatography-tandem mass spectrometry using enzyme-assisted derivatisation.
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.
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.
Jonas
J
Abdel-Khalik
Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK.
Peter J
PJ
Crick
DEQAS, Imperial College Healthcare NHS Trust, Clinical Biochemistry Department, Charing Cross Hospital, Fulham Palace Rd, London W6 8RF, UK.
Graham D
GD
Carter
Barts and the Royal London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AD, UK.
Hugh L
HL
Makin
Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK.
Yuqin
Y
Wang
Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK. Electronic address: w.j.griffiths@swansea.ac.uk.
William J
WJ
Griffiths
D001622:Betaine / Q000031:analogs & derivatives / Q000737:chemistry; D002112:Calcifediol / Q000097:blood*; D002789:Cholesterol Oxidase / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001622:Betaine; D002789:Cholesterol Oxidase; C012332:Girard's reagent T; D002112:Calcifediol
17βHSD10; Cholesterol oxidase; Girard P reagent; LC–MS(n); Serum; Vitamins D