Application of Gel Permeation Chromatography and Nonaqueous Reverse Phase Chromatography to High Pressure Liquid Chromatographic Determination of Retinyl Palmitate in Fortified Breakfast Cereals

Abstract

A high pressure liquid chromatographic (HPLC) method was developed for determining retinyl palmitate in cereals. Retinyl palmitate is fractionated from other methylene chloride-soluble components by using high pressure gel permeation chromatography (HP-GPC) followed by quantitation with nonaqueous reverse phase HPLC (RPHPLC). HP-GPC fractionation was accomplished using 2 µStyragel (100Å) columns connected in series on sample extracts in methylene chloride with methylene chloride as the mobile phase. A valve designed to facilitate the collection of the vitamin A fraction was installed in-line between the refractive index and absorbance detectors. RPHPLC quantitation was achieved on µBondapak C₁₈ (10 µm) using methylene chloride–acetonitrile (30+70). Based on 23 repetitive analyses, recovery of vitamin A as retinyl palmitate in cereal products was 95.4±4.2% with spiking levels between 21.4 and 140 µg/g. Vitamin A in 15 cereals, representing bran, corn, oats, rice, and wheat, ranged from 34 to 194% of the declared level. The HPLC procedure was compared with the AOAC official colorimetric method and with an ultraviolet spectrophotometric method.