Comprehensive analysis of vitamin D analogues: Rapid LC-MS/MS method for quantification of eight metabolites and two epimers

Despite numerous studies, little progress has been made in associating vitamin D levels or supplementation with the many diseases to which it is linked. One reason may be that current assays are ineffective and focus on a minority of metabolites. The aim of this study was to develop a comprehensive LC-MS/MS assay to rapidly quantify ten forms of vitamin D. The assay uses dynamic multiple reaction-monitoring (Dyn-MRM) technology to facilitate rapid analysis of 10 analogues of vitamin D with limits of detection and ranges (ng/mL) as follows: Vitamin D2 [0.019, 0.2-39.7], Vitamin D3 [0.019, 0.2- 38.5], 3-epi-25OHD2 [0.02, 0.04-40.2], 3-epi-25OHD3 [0.021, 0.04-41.3], 24R,25(OH)2D3 [0.021, 0.04-41.7], 25OHD2 [0.010, 0.2-41.3], 25OHD3 [0.010, 0.2-40.2], 1,α25(OH)2 D2 [0.004, 0.006-0.416], 1α25(OH)2 D3 [0.020, 0.04-40.2] and 23R,25(OH)2 D3 [0.004, 0.006-0.42]. The accuracy of the assay was assessed with Chromsystem and the vitamin D external quality assessment scheme (DEQAS) and it was applied to 20 healthy samples from Seralab. The results matched those of the DEQAS samples for key vitamin D analytes, whilst providing quantitative data for the additional vitamin D forms. The total run time of the analysis with the new assay is less than 7 minutes. In conclusion, this assay is the first to quantify ten forms of vitamin D simultaneously in human sera including the 23R,25(OH)2 D3 form. The significant improvement in run time, coupled with a single extraction protocol, makes this rapid assay more efficient for clinical use. This assay will be instrumental in future research fully exploring the roles of all forms of vitamin D in vivo