Application of gel permeation chromatography and nonaqueous reverse phase chromatography to high performance liquid chromatographic determination of retinyl palmitate and alpha-tocopheryl acetate in infant formulas.

A high performance liquid chromatographic (HPLC) method was developed for determining retinyl palmitate and alpha-tocopheryl acetate in infant formulas. The lipid-soluble components were extracted from the aqueous phase by homogenizing in a solvent mixture of isopropanol and methylene chloride with magnesium sulfate added to remove water. The vitamins were fractionated from the lipid material by using high pressure gel permeation chromatography (HP-GPC) followed by quantitation using non-aqueous reverse phase (RP)-HPLC. Three muStyragel (100A) columns connected in series were used for HP-GPC fractionation of sample extracts in methylene chloride. A Zorbax ODS (6 micron) column and methylene chloride-acetonitrile-methanol (30 + 70 + 0.2) were used for RP-HPLC quantitation. The 32 commercial infant formulas that were analyzed represent a wide variety of formulations manufactured at various fortification levels. Vitamin A results ranged from 89 to 242% of the declared levels. Vitamin E values, determined as the supplemental form alpha-tocopheryl acetate, ranged from 83 to 272% of the declared levels. Determination of vitamin A in 6 samples and vitamin E in one sample by this method and the official AOAC method gave comparable results. This method, which requires no saponification, was successfully used to determine vitamins A and E in ready-to-use, liquid concentrated, and powdered infant formulas.