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

Abstract

Background: Asthma is one of the common conditions clinicians encounter in children. Various studies report the association of poor vitamin D (Vit D) status of mothers during pregnancy, with an increased risk of asthma in their children, especially in HIV-positive mothers. Simultaneous measurement of Vit D metabolites, rather than measuring total Vit D, would improve assessment of Vit D metabolism in these children. This study aimed to validate a high-performance liquid chromatographic (HPLC) method using ultraviolet detection for the measurement of five Vit D isoforms (25 OHD2 and D3; 1,25 [OH]₂D2 and D3 and 24,25 [OH]₂D) simultaneously.
Methodology: Serum samples from 80 paediatric patients with asthma were subjected to solid phase extraction and 4-phenyl-1,2,4-triazole-3,5-dione-derivatisation. Chromatographic separation was achieved with a pentafluoro-phenylpropyl (PFPP) analytical column. Total 25 OH Vit D was measured on an automated immunochemistry analyser for comparison purposes.
Results and discussion: Good linearity was achieved for 25 OHD2, 25 OHD3 and 24,25 (OH)₂D with r² ≥ 0.99, with limits of detection ≤ 5.0 ng/ml and lower limits of quantitation ≤ 10.0 ng/m. The 1,25 (OH)₂D 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 (r² = 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.