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 D(3) and 25-hydroxyvitamin D(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-(2)H(6)]hydroxyvitamin D(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(3). Quantification is achieved by isotope-dilution liquid chromatography–tandem mass spectrometry. Recovery experiments for 25-hydroxyvitamin D(3) performed on adult human serum give recovery of 102–106%. Furthermore in addition to 25-hydroxyvitamin D(3), 24,25-dihydroxyvitamin D(3) and other uncharacterised dihydroxy metabolites, were detected in adult human serum.