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-H-2(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 3 beta-hydroxy group in the vitamins D metabolites followed by derivatisation of the newly formed 3-oxo group with Girard P reagent. 17 beta-Hydroxysteroid dehydrogenase type 10 is shown to oxidize selectively the 3 alpha-hydroxy group in the 3 alpha-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. (C) 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).