Utilizing Cooled Liquid Chromatography and Chemical Derivatization to Separate and Quantify C3-epimers of 25-hydroxy Vitamin D and Low Abundant 1α,25(OH)D3: Application in a Pediatric Population

There is need for a single assay able to quantify the most biologically active metabolite, 1α,25-dihydroxy-vitamin-D3, and the recently discovered biologically distinct C3-epimers of 25OHD, in addition to traditional vitamin D metabolites. We developed a method of chromatographic separation and absolute quantification of the following ten forms of vitamin D: 3-epi-25OHD3, 25OHD3, 3-epi-25OHD2, 25OHD2, 1α,25(OH)D3, 24R,25(OH)D3, 23R,25(OH)D3, 1a,25(OH)D2, D3, and D2 by single extraction and injection. Chemical derivatization followed by liquid chromatography using a charged surface hybrid C18 column and subsequent tandem mass spectrometry was utilized to detect and quantify each metabolite. This method is remarkable as a cooled column was required to achieve chromatographic resolution of epimers. Validation of each metabolite was performed at four concentrations and revealed inter- and intra-day precision and accuracy below 15% across three consecutive days of analysis. After validation, this method was applied to analyze the blood plasma from 739 samples from 352 subjects (8mo to 20 yr), 79 pooled plasma samples, and 10 NIST SRM972a samples. Healthy control samples (n = 357) were used to investigate developmentally associated changes in vitamin D metabolite concentrations during early life. This method yields excellent linearity (R ≥ 0.99) across concentrations encompassing the biological range of many metabolites including 1α,25(OH)D3. Concentrations of 25OHD2 and 24R,25(OH)D3 were significantly (q ≤0.05) lower in infants compared to both children and adolescents. The percentage of 3-epi-25OHD3 in total 25OHD3 was significantly lower (q ≤ 0.009) in post-puberty subjects. Here we present a single assay capable of separating and quantifying ten vitamin D metabolites including C3-epimers of 25OHD, and quantifying 1α,25-dihydroxy-vitamin-D3 at and below concentrations observed in human plasma (LLOQ < 10 pM).