Determination of Vitamins A and E in Infant Formula and Adult/Pediatric Nutritional Formula by HPLC with UV and Fluorescence Detection: First Action 2012.09

The method presented is for quantification of alpha-tocopherol (vitamin E), vitamin E acetate, vitamin A acetate, and vitamin A palmitate in infant formula and adult/pediatric nutritionals. The entire lipid fraction, including vitamins A and E, is extracted from product with iso-octane after products are mixed with methanol, which precipitates proteins and disrupts micelles freeing lipids for extraction. Vitamin A palmitate, vitamin A acetate, and vitamin E acetate are separated from alpha-tocopherol on a 3 cm silica column with a 1% methylene chloride, 0.06% isopropanol in iso-octane mobile phase; eluted onto a 20 cm silica column; and, after a column switch, further separated on the 20 cm column before UV detection at 325 nm (vitamin A palmitate and vitamin A acetate) and 285 nm (vitamin E acetate). alpha-Tocopherol is further separated from other extraneous compounds on the 3 cm silica column and detected by fluorescence at excitation and emission wavelengths of 295 and 330 nm, respectively. Quantification limits in ready-to-feed products were estimated to be 80 IU/L for vitamin A palmitate, 207 International Units (IU)/L for vitamin A acetate, 2.4 mg/L for vitamin E acetate, and <0.15 mg/L for alpha-tocopherol. Over-spike recoveries and intermediate precision averaged 100.4 and 2.09% RSD for vitamin A palmitate, 100.4 and 1.52% RSD for vitamin E acetate, and 99.6 and 3.02% RSD for alpha-tocopherol. Vitamin A acetate spike recovery data averaged 96.6%, and the intermediate precision for the only product fortified with vitamin A acetate was 2.75% RSD.