Simultaneous measurement of 13 circulating vitamin D3 and D2 mono and dihydroxy metabolites using liquid chromatography mass spectrometry

Abstract

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)₂D3 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-epi-25(OH)D3, 20(OH)D3, 24,25(OH)₂D3, 1,25(OH)₂D3 and 1,20S(OH)₂D3) 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.

Keywords: liquid chromatography; mass spectrometry; metabolism; vitamin D.

Figure 1:

Chromatographic separation of 13 vitamin D metabolites in a single sample run from a vitamin D depleted serum spiked with known concentrations of vitamin D analytes, split into three panels. The concentration of 1,25(OH)₂D3 extracted in the chromatogram was 2 ng/mL. A further chromatogram of 1,25(OH)₂D3 at LLOQ (12.5 pg/mL) is displayed in Supplementary Figure S-3. *Indicates the analyte signal used for quantitation.

Figure 2:

Concentrations of vitamin D metabolites measured in a cohort of healthy control donor serum samples.