Influence of Vitamin D2 Percentage on Accuracy of 4 Commercial Total 25-Hydroxyvitamin D Assays

Abstract

To the Editor: 25-Hydroxyvitamin D (25-OH-D)1 is the most appropriate marker for monitoring vitamin D storage status. The 2 major forms of 25-OH-D [25-OH-D2 (D2) and 25-OH-D3 (D3)], differ slightly in molecular structures but have essentially the same physiological activity. Therefore, both D2 and D3 should be measured to assess vitamin D status. At our laboratory, 42% of patient samples submitted for vitamin D testing contain D2. Recognizing the ability of an assay to quantify both the D3 and D2 forms equally is therefore crucial. The analytical performance of several total 25-OH-D assays approved by the US Food and Drug Administration is well documented (1); however, information is limited on how D2 affects assay accuracies. Le Goff et al. (2) demonstrated the inconsistency between several assays in their cross-reactivities toward D2. The size of their study was small, however, and the range of D2 percentages (D2%) was unknown. We used a larger sample size than previous studies and a D2% interval of 1.3%–91.2% [3.4–95.1 ng/mL (8.5–238 nmol/L)] and evaluated the influence of D2% on the accuracies of the following assays: DiaSorin Liaison, Abbott Architect, Roche Cobas, and Siemens Centaur. We developed and validated a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay as the reference method. Samples were prepared for the LC-MS/MS assay by mixing with internal standard (D3- d 6/D2- d 6), ZnSO4 solution, and methanol; liquid–liquid …