Method development for the simultaneous determination of vitamin A, vitamin E and beta-carotene in various foods

Several direct solvent extraction methods coupled with high performance liquid chromatography (HPLC) separation and quantitation have been studied for the determination of vitamin A, vitamin E and beta-carotene in various foods. A comparative study for the column performance between narrow-bore and standard-bore was also completed. A normal-phase HPLC system with fluorescence and photodiode array detectors attached to a Waters Millennium 2010 Chromatography Manager were used throughout this study. The analytical method validation parameters including accuracy, precision, limit of detection, limit of quantitation, and specificity were evaluated to ensure validity of the HPLC methods developed.;A liquid chromatographic method was described for the analysis of vitamin E in margarine and vegetable oil products (spreads). The tocopherol homologs (alpha-, gamma-, delta, tocopherol) were extracted in hexane with anhydrous MgSO4 added to remove water. Based on five repetitive assays the mean recoveries were greater than 97% for all analytes. The method is rapid, specific, and accurate for the measurement of vitamin E in margarine and vegetable spreads.;A simple, rapid direct solvent extraction procedure and HPLC system were developed for the simultaneous determination and identification of alpha-tocopheryl acetate, beta-carotene and tocopherols in reduced mayonnaise. The method used a zero control reference material (ZRM) (made in house from olive oil and egg) for accuracy determination. The sample was mixed with hot water (80°C), sonicated,MgS04 Was added, and the mixture was extracted with hexane/ethyl acetate (90/10, v/v) with the use of PolytronRTM homogenizer. The vitamins and beta-carotene were quantitated by fluorescence and photodiode array detectors, respectively. The analytes; were monitored and identified using an on-line, three-dimensional photodiode array detector. The direct solvent extraction was compared with saponification. Three brands of low fat mayonnaise were analyzed by both direct solvent extraction and saponification. The analytical values obtained from the direct extraction method were significantly higher than those from saponification (P<0.01).;A liquid chromatographic method was developed for the simulaneous analysis of natural vitamin E homologs, all-rac-alpha-tocopheryl acetate, retinyl palmitate (encapsulated and non-encapsulated) and beta-carotene in various fortified foods. The vitamins were extracted in isopropanol and hexane without saponification and quantitated by normalphase chromatography with fluorescence and visible detection. This method differentiates between natural and synthetic forms of vitamin E.;The results of validation parameters were reliable and satisfactory for the methods described above.;A comparative study on the performance of narrow-bore (2.1 mm I.D.) and standard-bore (4.6 mm I.D.) HPLC analytical silica columns having the same length was conducted. The studies were performed on HPLC equipment with minimum extra column contribution. Tocopherols (alpha-, beta-, gamma- and delta-) were used as test components. Both columns were tested under the same HPLC system with same mobile phase and different flow rates. The narrow-bore column gave up to a Mold increase in sensitivity compared with standard-bore column at equivalent running times for the tocopherols. About one third solvent savings can be achieved when a narrow-bore column was used.