LC/MS/MS Quantitative Analysis Of Vitamin D: Hidden Analytical Errors Overlooked By Validation | 10239
Journal of Analytical & Bioanalytical Techniques
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Vitamin D deficiency is a widespread clinical problem and has been associated with many adverse health outcomes. Analysis
of Vitamin D2 (ergocalciferol) and D3 (cholecalciferol) and their major metabolites 25(OH)D2 and 25(OH)D3 has become
a high priority topic in clinical analysis. Since water loss is a favored process, in-source self dehydration of vitamin D can decrease
S/N and such an undesirable process was tightly controlled. In-source water loss was found to be insignificant (~8%) within
the assay linear range, using a methanol/water mobile phase under our experimental conditions. In contrast, if acetonitrile/
water was used, severe in-source water loss was observed and ions m/z 395.2 and m/z 383.2 resulting from analyte in-source
dehydration became predominant. When acetonitrile was used as mobile phase, and if only the transitions m/z 395.2?→?377.2 for
25(OH)D2 and m/z 383.2?→?377.2 for 25(OH)D3 were used for quantitation, the assay standard curve was still acceptably linear;
however the area ratio between analyte and its in-source water loss product was non-linear at different analyte concentrations.
The concentration-dependent water loss varied by 20%, resulting in a significant quantitation error in this provisionally valid
assay. For an acetonitrile-based mobile phase, both transitions (for analyte and for its in-source dehydrated product) should be
summed to maintain true full range assay linearity.
Eduard Rogatsky completed his M.Sc in physical chemistry from Belarus State University, Ph.D. in Bioanalytical chemistry from Bar-Ilan University
(Israel) in 1999, and postdoctoral studies at Albert Einstein College of Medicine, NY. He joined the faculty there in 2001, and is currently a Senior
Associate Scientist and Director of Mass Spectrometry in the Biomarker Analytical Resource Core Laboratory, Einstein-Montefiore Institute for
Clinical and Translational Research, Bronx, NY, USA
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