Rapid Separation of 25-OH-vitamin D3 and 3-epi-25-OH-vitamin D3 in Human Serum Under RP-LC Conditions and Tandem Mass Spectrometry Detection

Faulty vitamin D metabolism in children less than 12 months of age can lead to formation of the inactive 3-epi-25 monohydroxy form. The resolution of 3-epimer from the active monohydroxy form by tandem mass spectrometry is not possible due to the mostly identical fragmentation pattern of the two species. As a result, the two isomers should be separated chromatographically. This application note describes an assay that utilizes the Kinetex 2.6 μm PFP column that can accurately quantitate 25-OH-D2, 25-OH-D3, and the 25-OHD3 epimer with a total analysis time of less than 5 minutes.