Determination of Retinyl Palmitate (Vitamin A) in Fortified Fluid Milk by Liquid Chromatography: Collaborative Study

Abstract

A liquid chromatographic (LC) method was developed for fast and simple measurement of retinyl palmitate (vitamin A) in fortified milk. Retinyl acetate internal standard was added to a test portion of milk followed by extraction into hexane. The hexane extract was analyzed by LC using a normal-phase silica gel column equilibrated with mobile phase (conditioned hexane–isopropanol, 99.85 + 0.15, v/v) about 1 h before injections. The retinyl palmitate concentration was calculated by using a relative response factor determined with calibration standards. In the collaborative study, 11 laboratories analyzed 13 pairs of fluid milk materials in blind duplicate. Twelve of the materials were composed of skim milk (<0.5% fat), 1% fat milk, 2% fat milk, and 1% fat chocolate milk. Each material was fortified at 3 concentrations of retinyl palmitate of approximately 581 μg/L (1000 IU/qt), 1163 μg/L (2000 IU/qt), and 2236 μg/L (4000 IU/qt). The 13th material, unfortified skim milk, served as a matrix blank. Repeatability standard deviations (RSDr) without outliers ranged from 1.5 to 5.7% and reproducibility standard deviations (RSDR) without outliers ranged from 5.0 to 22.7%. cis-Isomers co-eluted with the predominant trans-retinyl palmitate isomer and were included in the results reported by all the collaborative laboratories. Endogenous long-chain esters from milk fat were also measured with the retinyl palmitate additive. The Study Director recommends that this method for determination of retinyl palmitate in fluid milk by LC be adopted First Action.