Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 953, Issue -, Pages 62-67Publisher
ELSEVIER
DOI: 10.1016/j.jchromb.2014.01.045
Keywords
Liquid chromatography; Tandem mass spectrometry; 1,25 Dihydroxyvitamin D; Derivatization method
Funding
- Institute for Clinical and Translational Research at the University of Wisconsin-Madison as the Clinical and Translational Science Award (CTSA) program
- National Center for Advancing Translational Sciences (NCATS) [UL1TR000427]
- Wisconsin National Primate Research Center [NIH P51RR000167/P51OD011106]
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Active vitamin D metabolites 1,25-dihydroxyvitamin D-2 [1,25-(OH)(2)-D-2; derived from ergocalciferol] and D-3 [1,25-(OH)(2)-D-3; derived from cholecalciferol] are found in low levels in the circulation and require a very sensitive method for measurement. Radioimmunoassay (RIA) has been the method of choice, but it lacks the specificity needed to distinguish between 1,25-(OH)(2)-D-2 and -D-3, whereas liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have the advantage of high specificity and sensitivity. Here, we compare a new derivative for ionizing 1,25-(OH)(2)-D to enhance the signal and provide the most sensitive assay for measuring vitamin D. We used the Amplifex diene method of derivatizing prior to LC/MS/MS and compared it to the standard RIA method and the 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) method of derivatizing prior to LC/MS/MS. In the evaluation of 20 human serum samples, all methods correlated strongly across the upper levels of the standard 1,25-(OH)(2)-D-2 and -D-3 ranges (Amplifex and R1A, p(c) = 0.97; Amplifex and PTAD, p(c) = 0.96) but less strongly on the lower levels of the standard range (Amplifex and RIA, pc = 0.81; Amplifex and PTAD, pc = 0.65) suggesting differences in the sensitivities between the assays. The Amplifex method was determined to be more sensitive than the PTAD method, as peak areas were significantly higher for the Amplifex method and provided for a 10 fold higher signal-to-noise ratio than PTAD. Therefore, the Amplifex LC/MS/MS method is the most sensitive and specific method available for measuring 1,25-(OH)(2)-D-2 and -D-3 while using the smallest sample volume. (C) 2014 Elsevier B.V. All rights reserved.
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