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

Published

August 27, 2021

Author(s)

Stephen Wise, Johanna Camara, Carolyn Burdette, Grace Hahm, Federica Nalin, Adam Kuszak, Joyce Merkel, Ramon Durazo-Arvizu, Emma Walker, Andrew Hoofnagle, Fiona Ivison, Ralf Fischer, Jody van den Ouweland, Chung Ho, Emmet Law, Jean-Nicolas Simard, Renaud Gonthier, Brett Holmquist, Sarah Meadows, Lorna Cox, Kimberly Robyak, Michael Creer, Robert Fitzgerald, Michael Clarke, Norma Breen, Pierre Lukas, Etienne Cavalier, Christopher Sempos

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, and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] using reference measurement procedures (RMPs) based on isotope dilution liquid chromatography – tandem mass spectrometry (ID LC-MS/MS) and for 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3] using a similar ID LC-MS/MS method. The 50 single-donor samples ranged in total 25(OH)D concentration from 16 nmol/L to 148 nmol/L and included eight samples with high concentrations of 25(OH)D2 (i.e., > 30 nmol/L). VDSP Intercomparison Study 2 Part 1 includes results from 15 laboratories using LC-MS/MS assays. Assay performance was evaluated using mean % bias compared to the NIST-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 seven LC-MS/MS assays not meeting the ≤ ±5 % criterion, four assays had mean % biases 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 the performance of various LC-MS/MS assays 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 LC-MS/MS assay performance, particularly with regard to mean % bias.

Citation

Analytical and Bioanalytical Chemistry

Pub Type

Journals

Download Paper

https://doi.org/10.1007/s00216-021-03576-1

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Keywords

25-hydroxyvitamin D3, 25-hydroxyvitamin D2, total 25-hydroxyvitamin D, 3-epi-25-hydroxyvitamin D3, 24R, 25-dihydroxyvitamin D3, liquid chromatography – tandem mass spectrometry (LC-MS/MS), immunoassay, reference measurement procedure (RMP), Vitamin D Standardization Program (VDSP)

Citation

Wise, S. , Camara, J. , Burdette, C. , Hahm, G. , Nalin, F. , Kuszak, A. , Merkel, J. , Durazo-Arvizu, R. , Walker, E. , Hoofnagle, A. , Ivison, F. , Fischer, R. , van den Ouweland, J. , Ho, C. , Law, E. , Simard, J. , Gonthier, R. , Holmquist, B. , Meadows, S. , Cox, L. , Robyak, K. , Creer, M. , Fitzgerald, R. , Clarke, M. , Breen, N. , Lukas, P. , Cavalier, E. and Sempos, C. (2021), 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, Analytical and Bioanalytical Chemistry, [online], https://doi.org/10.1007/s00216-021-03576-1, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=932139 (Accessed March 3, 2025)