Endocrine Abstracts

Vitamin D exists as two forms; D3 (UV) and D2 (plant derived). Measuring the metabolite 25-hydroxyvitamin D (25OHD) is routinely applied in research and clinical laboratories to assess vitamin D status. The Institute of Medicine and Society for Endocrinology have previously set recommended vitamin D guidelines based on combined 25OHD3 and 25OHD2 serum concentrations. In order to achieve accurate quantitation of these metabolites, the respective C3 epimers must be separately quantified owing to varying activities between isoforms. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is considered the gold standard approach for measuring 25OHD metabolites, owing to potential interferences of the C3 epimers in other analytical techniques such as immunoassays. To account for the role of C3 epimers and accurately measure 25OHD in high throughput research and clinical analysis, we have developed an LC-MS/MS method to quantify 25OHD3 and 25OHD3 along with their C3 epimers in a total run time of three minutes. Method development and analysis was performed on a Waters AQUITY UPLC coupled to a Waters TS-MS mass spectrometer. A Phenomenex Lux cellulose-3 chiral column (100 mm, 2 mm, 3 μm) was used for separation, the mobile phase was water and methanol 0.1% formic acid. The optimised method achieved retention times of the following analytes; 25OHD3 – 1.51 min, 3-epi-25OHD3 – 1.82 min, 25OHD2 – 1.55 min, 3-epi-25OHD2 – 1.97 min. Further accuracy of 25OHD3 was achieved through separating the isobar 7αC4. Post column infusion was performed during method validation to ensure no matrix interference. Regression analysis was performed with this method comparing two previously developed LC-MS/MS methods for measuring multiple vitamin D metabolites with longer run times using a cohort of human serum samples. A strong regression was observed between methods (R²=0.987 and 0.929) and no significant bias was observed between 25OHD3 measurements (0.76% [95% CI 1.888–3.403, t=0.937, P=0.768] and 1.38% [95% CI 2.864–5.614, t=0.935, P=0.995]). Application of this method could significantly enhance throughput of 25OHD measurements in research and clinical laboratories.