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

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 D-2 (25(OH)D-2) and 25-hydroxyvitamin D-3 (25(OH)D-3). A set of 50 single-donor samples was assigned target values for concentrations of 25(OH)D-2, 25(OH)D-3, 3-epi-25-hydroxyvitamin D-3 (3-epi-25(OH)D-3), and 24R,25-dihydroxyvitamin D-3 (24R,25(OH)(2)D-3) 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)D-3. 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)D-3 and 25(OH)D-3 on assay performance, particularly with regard to mean % bias.

Automated summary

The Vitamin D Standardization Program (VDSP) conducted an interlaboratory comparison study to evaluate the performance of liquid chromatography - tandem mass spectrometry (LC-MS/MS) assays for determining serum total 25-hydroxyvitamin D. Results showed that only 53% of the assays met the VDSP criterion, with four assays having significant performance issues due to the lack of separation between 3-epi-25(OH)D-3 and 25(OH)D-3.