Journal article 988 views
Studies on the analysis of 25-hydroxyvitamin D3 by isotope-dilution liquid chromatography–tandem mass spectrometry using enzyme-assisted derivatisation
,
William Griffiths 
,
Yuqin Wang
,
Yuqin Wang
Biochemical and Biophysical Research Communications
Swansea University Authors:
William Griffiths , Yuqin Wang
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DOI (Published version): 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 quantificatio...
| Published in: | Biochemical and Biophysical Research Communications |
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| Published: |
2014
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| URI: | https://cronfa.swan.ac.uk/Record/cronfa17818 |
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| 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-[26,26,26,27,27,27-2H6]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 |
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| College: |
Faculty of Medicine, Health and Life Sciences |