Analysis Of Alpha-Tocopherol Stereoisomers In Fortified Infant Formula By Chiral Chromatography

Background: Direct measurement of the bioavailable alpha-tocopherol content presents a significant analytical challenge and requires chiral separation of the alpha-tocopherol stereoisomers.Objective: The objective of the study was to validate an analytical method for the analysis of alpha-tocopherol stereoisomers in infant formulas and dairy products.Method: Samples were saponified at elevated temperature and lipophilic components were extracted into an organic solvent, with subsequent chromatographic separation of the alpha-tocopherol stereoisomers achieved by HPLC with a chiral column and fluorescence detection.Results: The method was shown to be accurate, with spike recoveries of 91.9-108.8% for RRR-alpha-tocopherol and 90.1-104.7% for alpha-tocopherol, with no statistical bias against NIST 1849a certified reference material (P-value = 0.54) and an HPLC-UV analytical method (P-value = 0.48). Acceptable precision was confirmed, with repeatabilities estimated at 3.5% RSDr (HorRat = 0.6) for RRR-alpha-tocopherol and 4.6% RSDr (HorRat = 0.4) for alpha-tocopherol.Conclusions: A straightforward chiral chromatographic method for the analysis of stereoisomeric forms of alpha-tocopherol is described. In a single analytical run, the method can quantify: (i) the total alpha-tocopherol content; (ii) the nutritionally important RRR-alpha-tocopherol and/or 2 R, 4-ambo, 8'-ambo-alpha-tocopherol contents; (iii) the amount of all-rac-alpha-tocopherol, all-rac-alpha-tocopheryl acetate, or all-rac-alpha-tocopheryl succinate fortified into the product.Highlights: An accurate and precise chiral chromatographic method for the analysis of isomeric forms of alpha-tocopherol is described. The method is able to distinguish between natural and synthetic tocopherol sources. The method is accurate and precise and is suitable either for routine product compliance testing during product manufacture or as a possible reference method.