Journal
CLINICAL CHEMISTRY AND LABORATORY MEDICINE
Volume 59, Issue 10, Pages 1642-1652Publisher
WALTER DE GRUYTER GMBH
DOI: 10.1515/cclm-2021-0441
Keywords
liquid chromatography; mass spectrometry; metabolism; vitamin D
Categories
Funding
- European Union [840567]
- NIH [1R01AR073004-01A1, R01AR071189-01A1, R21 AI149267-01A1]
- VA merit grant [1I01BX004293-01A1]
- Marie Curie Actions (MSCA) [840567] Funding Source: Marie Curie Actions (MSCA)
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The study developed an LC-MS/MS method to measure 13 vitamin D metabolites simultaneously, providing new insights into the circulating vitamin D metabolite pathways.
Objectives: Clinical evaluation of vitamin D status is conventionally performed by measuring serum levels of a single vitamin D metabolite, 25-hydroxyvitamin D predominantly by immunoassay methodology. However, this neglects the complex metabolic pathways involved in vitamin D bioactivity, including two canonical forms D3 and D2, bioactive 1,25-dihydroxy metabolites and inactive 24-hydroxy and other metabolites. Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can measure multiple analytes in a sample during a single run with high sensitivity and reference level specificity. We therefore aimed to develop and validate a LC-MS/MS method to measure simultaneously 13 circulating vitamin D metabolites and apply it to 103 human serum samples. Results: The LC-MS/MS method using a Cookson-type derivatization reagent phenyl-1,2,4-triazoline-3,5-dione (PTAD) quantifies 13 vitamin D metabolites, including mono and dihydroxy-metabolites, as well as CYP11A1-derived D3 and D2 metabolites in a single run. The lower limit of quantitation was 12.5 pg/mL for 1,25(OH)(2)D3 with accuracy verified by analysis of National Institute of Standards and Technology (NIST) 972a standards. Quantification of seven metabolites (25(OH)D3, 25(OH)D2, 3-epi25(OH)D3, 20(OH)D3, 24,25(OH)(2)D3, 1,25(OH)(2)D3 and 1,20S(OH)(2)D3) was consistently achieved in human serum samples. Conclusions: This profiling method can provide new insight into circulating vitamin D metabolite pathways forming the basis for improved understanding of the role of vitamin D in health and disease.
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