Fast separation of 25-hydroxyvitamin D 3 from its C3-epimer in human serum by liquid chromatography-tandem mass spectrometry showing variable C3-epimer prevalence in infants and adults

Measurement of 25-hydroxyvitamin D (25(OH)D) is accepted as a reliable clinical indicator of the vitamin D status in humans, which is important in the diagnosis of vitamin D deficiency and for monitoring supplementation therapy. The number of laboratories that use liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurement of 25(OH) D is steadily increasing. LC-MS/MS allows measurement of both 25(OH)D3 and 25(OH)D2 independently with high sensitivity. A limitation of most LC-MS/MS methods is the potential interference from co-eluting isomeric compounds having identical elemental composition but different structure, leading to overestimation of true 25(OH)D concentrations. It was recently shown that 25(OH)D3 is metabolized through the C3-epimerization pathway like 1,25(OH)2D3 and 24,25(OH)2D3 (1). However, the biological relevance of 3-epi-25(OH)D remains to be elucidated. The C3-epimer has received attention from its detection in 23% of infant sera less than 1 year old with 3-epi-25(OH) D contributing 9-61% of the total 25(OH)D by using a modified LC-MS/MS method which partially sepa rated 25(OH)D from its C3-epimer (2). There was an inverse relationship between patient age in days and the percentage of 3-epi-25(OH)D. The 3-epi-25(OH) D metabolite was not detected in children from 1-18 years of age or in adults. This led the authors to postulate that C3-epimerisation originates from immature vitamin D metabolism. A limitation of their study was that 3-epi-25(OH)D was only partially separated from the major 25(OH)D peak which did not allow them to quantify low (<8.5%) levels of 3-epi-25(OH)D. It was recently shown that by using techniques achieving baseline separation between the 25(OH)D3 and epi25(OH)D3, the C3-epimer can also be detected in sera from adults, albeit at much lower concentrations when compared to infants (3,4). To separate 3-epi-25(OH) D from 25(OH)D, current procedures require lengthy chromatographic run times varying from 12 to 40 min (5), which makes these methods unsuitable for clinical laboratories that must deal with increasing numbers