Application of gel permeation chromatography and nonaqueous reverse phase chromatography to high pressure liquid chromatographic determination of retinyl palmitate in fortified breakfast cereals.

A high pressure liquid chromatographic (HPLC) method was developed for determing 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 (RP-HPLC). HP-GPC fractionation was accomplished using 2 microStyragel (100A) 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. RP-HPLC quantitation was achieved on microBondapak C18 (10 micrometers) 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 micrograms/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.