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
2022
Wise, Stephen A. | Camara, Johanna E. | Burdette, Carolyn Q. | Hahm, Grace | Nalin, Federica | Kuszak, Adam J. | Merkel, Joyce | Durazo-Arvizu, Ramón A. | Williams, Emma L. | Hoofnagle, Andrew N. | Ivison, Fiona | Fischer, Ralf | van den Ouweland, Jody M. W. | Ho, Chung S. | Law, Emmett W. K. | Simard, Jean-Nicolas | Gonthier, Renaud | Holmquist, Brett | Meadows, Sarah | Cox, Lorna | Robyak, Kimberly | Creer, Michael H. | Fitzgerald, Robert | Clarke, Michael W. | Breen, Norma | Lukas, Pierre | Cavalier, Étienne | Sempos, Christopher T.
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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