Measurement of fortification levels for vitamins A and D in fluid milk products is required by the U.S. Food and Drug Administration (FDA)... Show moreMeasurement of fortification levels for vitamins A and D in fluid milk products is required by the U.S. Food and Drug Administration (FDA) according to Pasteurized Milk Ordinance. In the current study, a high performance liquid chromatographic (HPLC) method was developed for simultaneous determination of retinyl palmitate (vitamin A) and cholecalciferol (vitamin D3) in fortified milk, which will enhance the efficiency of routine monitoring of fortification for processors as well as laboratories. Saponification conditions and extraction solution compositions were studies for best recovery and simplification of the extraction procedures. Saponification conditions studied included temperature (20, 40 and 60℃), time (0.5 to 16 hour) and concentration of KOH (20, 30, 40 and 50%). The variables studied for extraction included solvent compositions (hexane, diethyl ether, ethyl acetate and dichloromethane) and corresponding polarities (0.1, 0.25, 0.5 and 0.75), and number of extractions (once, twice and three times). Freezing and centrifuging steps were also investigated to compare to SPE clean-up, as an alternative method to clean-up before injection. The optimum parameters found were: saponification conditions at 60℃ for 2 hours with 40% (w/v) KOH liquid-liquid extraction with hexane: diethyl ether (80:20, v:v). Vitamins A and D3 were quantitated using an HPLC system with fluorescence detector (FLD) and diode array detector (DAD) respectively within the same injection. Spike recoveries from three different fat levels (skim, 2% and whole) of commercial pasteurized milk samples were in the range of 94 to 110% for both vitamins A and D3 at three spike concentration levels. In addition, at National Institute of Standards and Technology (NIST) Standard Reference Material (SRM 1849a) was used xii for method accuracy evaluation. With eight replicates, the average vitamin A (expressed as retinol equivalents) concentration was 7.52 ± 0.23 μg/g, vitamin D3 concentration was 0.114 ± 0.0062 μg/g, an excellent agreement with the certified value of 7.68 ± 0.23 μg/g for vitamin A and 0.11 ± 0.017 μg/g for vitamin D3. In summary, an HPLC method for simultaneous determination of vitamins A and D3 in fluid milk products has been developed. The method showed high efficiency and accuracy of analysis during a single laboratory validation. M.S. in Food Safety and Technology, December 2013 Show less
