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Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 — Part 1 liquid chromatography – tandem mass spectrometry (LC-MS/MS) assays — impact of 3-epi-25-hydroxyvitamin D3 on assay performance

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Abstract

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of liquid chromatography – tandem mass spectrometry (LC-MS/MS) assays used for the determination of serum total 25-hydroxyvitamin D (25(OH)D), which is the sum of 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3). A set of 50 single-donor samples was assigned target values for concentrations of 25(OH)D2, 25(OH)D3, 3-epi-25-hydroxyvitamin D3 (3-epi-25(OH)D3), and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) using isotope dilution liquid chromatography – tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 1 includes results from 14 laboratories using 14 custom LC-MS/MS assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 53% of the LC-MS/MS assays met the VDSP criterion of mean % bias ≤ |±5%|. For the LC-MS/MS assays not meeting the ≤ |±5%| criterion, four assays had mean % bias of between 12 and 21%. Based on multivariable regression analysis using the concentrations of the four individual vitamin D metabolites in the 50 single-donor samples, the performance of several LC-MS/MS assays was found to be influenced by the presence of 3-epi-25(OH)D3. The results of this interlaboratory study represent the most comprehensive comparison of LC-MS/MS assay performance for serum total 25(OH)D and document the significant impact of the lack of separation of 3-epi-25(OH)D3 and 25(OH)D3 on assay performance, particularly with regard to mean % bias.

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Acknowledgements

The authors acknowledge David L. Duewer (NIST) for his suggestions and discussions regarding multivariable linear regression analysis.

Funding

The Office of Dietary Supplements at the National Institutes of Health (NIH-ODS) provided partial funding for this study to the National Institute of Standards and Technology (NIST). MWC is affiliated to Metabolomics Australia, University of Western Australia, Perth, Western Australia, Australia and was supported by infrastructure funding from the Western Australian State Government in partnership with the Australian Federal Government, through Bioplatforms Australia and the National Collaborative Research Infrastructure Strategy (NCRIS).

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The National Institute of Standards and Technology Research Protections Office reviewed the protocol for this project and determined that it is “not human subjects research” as defined in 15 CFR 27, the Common Rule for the Protection of Human Subjects. The laboratory study participants agreed to the publication of their measurements data, laboratory identification, and measurement assay platform identification.

Conflict of interest

S.A. Wise is an Editor of the journal Analytical and Bioanalytical Chemistry and was not involved in peer reviewing this manuscript. Several of the coauthors are employees of companies that produce assays that were evaluated in this study. There are no financial or nonfinancial competing interest for any of the coauthors.

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Wise, S.A., Camara, J.E., Burdette, C.Q. et al. Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 — Part 1 liquid chromatography – tandem mass spectrometry (LC-MS/MS) assays — impact of 3-epi-25-hydroxyvitamin D3 on assay performance. Anal Bioanal Chem 414, 333–349 (2022). https://doi.org/10.1007/s00216-021-03576-1

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