3-Epimer-25-hydroxyvitamin D3 interference in assays for the measurement of 25-hydroxyvitamin D3

In chromatographic measurements of vitamin D, such as liquid chromatography tandem mass spectrometry (LC-MS/MS), 3-epi-25hydroxyvitamin D3 (3-epi-25OHD3) can interfere with 25-hydroxyvitamin D3 (25OHD3) quantification due to their identical mass. Chromatographic separation is therefore required to overcome this issue. In October 2024, the Vitamin D External Quality Assessment Scheme (DEQAS) distributed two serum samples: 664 (without exogenous 3-epi-25OHD3) and 665 (with 50 nmol/L of added 3-epi-25OHD3). Sample 664 yielded consistent results across 445 labs, giving an Trimmed Mean (ALTM) = 45.6 ± 6.1 nmol/L (±SD, CV = 13.5%), aligning with the Centers for Disease Control and Prevention (CDC) reference value of 46.7 nmol/L. In contrast, sample 665 showed a higher ALTM of 62.1 ± 27.2 nmol/L (±SD, CV = 43.9%) when compared to the CDC target value, with significant variability observed. Most automated immunoassays did not detect 3-epi-25OHD3, except for Roche Gen III (91% cross-reactivity) and Beckman DxI (39.8%). Among 90 LC-MS/MS laboratories reporting #665, 18 routinely measured 3-epi-25OHD3; 15 of these excluded it from their reported total 25OHD (method mean = 47 nmol/L) and 3 included 3-epi-25-OHD3, despite measuring both metabolites separately. The LC-MS/MS results showed a bimodal distribution, with 40 LC-MS/MS laboratories (44%) resolving the epimer chromatographically and 50 LC-MS/MS laboratories (56%) not effectively separating the epimer, leading to overestimation. This positive interference may have clinical implications, especially in neonates who naturally have high 3-epi-25OHD3 levels. Although its biological role remains unclear, current consensus advises excluding 3-epi-25OHD3 from total 25OHD when assessing vitamin D status.