An on-line normal-phase high performance liquid chromatography method for the rapid detection of radical scavengers in non-polar food matrixes

An on-line method for the rapid pinpointing of radical scavengers in non-polar mixtures like vegetable oils was developed. To avoid problems with dissolving the sample, normal-phase chromatography on bare silica gel was used with mixtures of hexane and methyl tert-butyl ether as the eluent. The high performance liquid chromatography-separated analytes are mixed post-column with a solution of stable free radicals in hexane. Reduced levels of the radical as a result of a reaction with a radical scavenger are detected as negative peaks by an absorbance detector. After investigating a number of different reagents, solvents, concentrations and solution flow rates an optimized instrumental set-up incorporating a superloop for pulse-free delivery of the reagent solution is presented. Both 2,2’-diphenyl-1-picrylhydrazyl (DPPH) and 2,6-di-tert-butyl-a-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy (galvinoxyl) were used as stable free radicals. The method is suitable for both isocratic and gradient HPLC operation. The method has a simple experimental protocol, uses standard instruments and inexpensive and stable reagents, and accepts any hexane-soluble sample. It can also be used for semi-quantitative analysis. The method was applied to several pure, non-polar natural antioxidants, vegetable oils and lipid-soluble rosemary extract. The limits of detection varied from 0.2 to 176 µg/ml, depending on the compound tested.