Method for simultaneous analysis of eight analogues of vitamin D using liquid chromatography tandem mass spectrometryReport as inadecuate




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Chemistry Central Journal

, 6:112

Analytical Chemistry

Abstract

BackgroundDespite considerable global investigation over several decades, the roles of vitamin D in health and disease development remains convoluted. One recognised issue is the difficulty of accurately measuring the active forms of vitamin D. Advances made include some new methods addressing the potential interference by excluding epimers and isobars. However, there is no evidence that epimers are without function. Therefore, the aim of this study was to develop and validate, for the first time, a new assay to simultaneously measure levels of six forms of vitamin D along with two epimers. The assay was applied to multilevel certified reference material CRM and 25 pooled human sera samples, obtained from the Vitamin D External Quality Assessment Scheme DEQAS, to demonstrate its efficiency.

ResultsThe assay is capable of simultaneously measuring eight vitamin D analogues over the calibration ranges and LODs in nmol-L of: 1α25OH2D2 0.015-1; 0.01, 1α25OH2D3 0.1-100; 0.01, 25OHD3 0.5-100, 0.025, 3-epi-25OHD3 0.1-100, 0.05, 25OHD2 0.5-100, 0.025, 3-epi-25OHD2 0.1-100, 0.05, vitamin D3 0.5-100, 0.05 and vitamin D2 0.5-100, 0.05, using stanozolol-d3 as internal standard. Certified reference material and external quality control samples DEQAS were analysed to meet the standards outlined by National Institute of Standards and Technology NIST. Validation steps included recovery and both precision and accuracy under inter- and intra-day variation limit of detection, and analysis of each analyte over a linear range. All validation parameters were in line with acceptable Food and Drug Administration FDA guidelines. All eight analogues were quantified with the 25OHD levels being commensurate with DEQAS data.

ConclusionsThis report details the application of a new LC-MS-MS based assay for the efficient analysis of eight analogues of vitamin D over a range of samples, which is a significant advance over the existing methods. Simultaneous measure of eight vitamin D analogues does not compromise the analytical capability of the assay to quantify the commonly used biomarker 25OHD for vitamin D status. The results demonstrate the feasibility of applying the assay in research and clinical practice that i excludes misleading measures owing to epimers and isobars and ii is able to quantify the excluded component to facilitate further in vivo investigation into the roles of ubiquitous epimers.

KeywordsVitamin D Epimer Analyses LC-MS-MS 25-hydroxyvitamin-D3 25-hydroxyvitamin-D2 3-epi-25OHD3 3-epi-25OHD2 Electronic supplementary materialThe online version of this article doi:10.1186-1752-153X-6-112 contains supplementary material, which is available to authorized users.

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Author: Iltaf Shah - Andrea Petroczi - Declan P Naughton

Source: https://link.springer.com/







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