| In LC-MS analysis of 25OH-Vitamin D, dehydration (water loss) is the major side reaction. We found that quantitation of the 25(OH)D from its M+H+, is temperature invariant. In contrast, ionization efficiency of the in-source dehydrated precursor is temperature dependent. Since actual temperature of droplets can vary with mass spectrometer hardware, flow rate, and mobile phase composition, fluctuations of these factors may contribute additional variability to the assay, which requires particular care during method development and validation, while intact M+H Vit D metabolite quantitation is more robust in terms of instrumental/assay conditions. |
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