The identification and semi-quantitative assay of some fat-soluble vitamins and antioxidants in pharmaceutical products and animal feeds by thin-layer chromatography

Abstract

A thin-layer chromatographic method for the identification and semi-quantitative assay of vitamin A (alcohol), its acetate and palmitate, vitamin D, α-tocopherol, α-tocopheryl acetate, BHA (butylated hydroxyanisole; 2-t-butyl-4-methoxyphenol), BHT (butylated hydroxytoluene; 2,6-di-t-butyl-4-methyl-phenol) and ethoxyquin in vitamin preparations is described. The sample solutions are applied to thin layers of silica gel and the vitamins and antioxidants are separated by using n-hexane-ethyl methyl ketone-di-n-butyl ether (34 + 7 + 6) as the developing solvent. Decomposition of vitamins A and D when applied to the adsorbent layer is inhibited by the presence of triethylamine in the spotting solvent. The compounds are identified by means of their R_F values, their appearance in ultraviolet radiation of wavelengths 254 and 360 nm and their response to iron(III) chloride-bipyridyl and iron(III) chloride-potassium hexacyanoferrate(III) spray reagents; they are assayed by visual comparison with standards. The method has been applied to gelatin-protected vitamin beads, animal feed additives, multivitamin tablets, oily vitamin concentrates and halibut-liver oil samples.

A simple colour test for distinguishing vitamin D₂ from vitamin D₃ after removal of vitamin A and its esters is also described.