Development of Vitamin D Determination in Infant Formula by Column-Switching HPLC with UV Detector

This study was carried out to develop an analytical method for the determination of vitamin D in infant formula. Vitamin D was determined by column-switching high-performance liquid chromatography (HPLC) equipped with a reversed phase column and UV detector after saponification and extraction of the formula with an organic solvent. A preseparation column (C?), focusing column (C18), analytical column (C18) and UV-Vis detector (254 nm) were used. The limits of detection (LOD) and the limits of quantification (LOQ) for vitamin D were estimated to be 1.51 μg/kg and 4.95 μg/kg, respectively. The linearity, recovery, precision and accuracy of the analytical method for vitamin D were evaluated through the application of a SRM (Standard Reference Material) 1846 (National Institute of Standard & Technology, USA). The linearity of this method was calculated with a value of the coefficient of determination (r²) ≥0.9999. The recovery of vitamin D was 85.20±3.00%. The intra-assay precision for vitamin D was between 1.68±0.03% and 5.75±0.33%, and the inter-assay precision for vitamin D ranged from 1.73 0.03% to 2.96±0.09%. The intra-assay accuracy for vitamin D was between 100.03±2.77% and 102.01±0.59%, and the inter-assay accuracy for vitamin D ranged from 99.00±1.53% to 102.01±3.04%. The proposed method is optimal for the separation and quantification of vitamin D from infant formula.