Abstract:

A rapid method for the simultaneous determination of vitamins A, D₃ and E in infant formula and adult nutritions has been developed using online two-dimensional liquid chromatography (2D-LC). First of all, C8 and polar embedded C18 columns were chosen as the first and second dimensional column respectively according to hydrophobic-subtraction model, which constituted excellent orthogonal separation system. The detection wavelengths were set at 263 nm for vitamin D₃, 296 nm for vitamin E and 325 nm for vitamin A. The purification of vitamin D₃ and quantifications of vitamins A and E were completed simultaneously in the first dimensional separation using the left pump of Dual Gradient LC (DGLC) with methanol, acetonitrile and water as mobile phases. The heart-cutting time window of vitamin D₃ was confirmed according to the retention time of vitamin D₃ in the first dimensional separation. The elute from the first dimensional column (1-D column) which contained vitamin D₃ was collected by a 500 μL sample loop and then taken into the second dimensional column (2-D column) by the right pump of DGLC with methanol, acetonitrile and water as mobile phases. The quantification of vitamin D₃ was performed in the second dimensional separation with vitamin D₂ as internal standard. At last, this method was applied for the analysis of the three vitamins in milk powder, cheese and yogurt. The injected sample solution with no further purification was pre-treated by hot-saponification using 1.25 kg/L KOH solution and extracted by petroleum ether solvent. The recoveries of vitamin D₃ spiked in all samples were 75.50%-85.00%. There was no statistically significant difference for the results between this method and standard method through t-test. The results indicate that vitamins A, D₃ and E in infant formula and adult fortified dairy can be determined rapidly and accurately with this method.

Key words: adult nutritions, infant formula, online separation, two-dimensional liquid chromatography (2D-LC), vitamin A, vitamin D₃, vitamin E