Validation of a high-performance liquid chromatographic method for the simultaneous assessment of vitamin D isoforms

of [OH] discussion: Good linearity was achieved for 25 OHD2, 25 OHD3 and 24,25 (OH)2D with r 2 ≥ 0.99, with limits of detection ≤ 5.0 ng/ml and lower limits of quantitation ≤ 10.0 ng/m. The 1,25 (OH) 2 D metabolites, present in the blood at pg/ml levels, could not be quantified within the required ranges. Comparing total 25 OH Vit D immunoassay and HPLC results showed a poor correlation (r 2 = 0.073), possibly due to cross-reactivity of the antibodies in the immunoassay with other Vit D isoforms. Conclusion: This method provides a precise and accurate, with a potential for high throughput, determination of Vit D isoforms in the routine laboratory.