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Abstract

A Box Behnken Design (BBD) in conjunction with multiple response optimizations was performed to develop the simultaneous separation of twenty phenolic compounds in rice grains by Ultrahigh Performance Liquid Chromatography (UPLC) coupled with Photodiode Array (PDA) detector. The gradient compositions of mobile phase and flow rate were optimized with respect to the resolutions of severely overlapping chromatographic peaks between the phenolic compounds and the analysis time. Additionally, two different stationary phases were also evaluated (Cortex C18 and BEH C18). A gradient program starts with 6.4% and rises to 32.6% of phase B in 3 minutes at flow rate of 0.6 mL min-1 using Cortex C18 was found to provide the best resolution for the separation of the 20 phenolic compounds. A complete validation of the method was accomplished reaching high precisions in terms of Coefficient of Variation (CV) lower than 2% for both repeatability and intermediate precision. The suitability of the method was checked by application to the analysis of pigmented and non-pigmented rice samples. The variations in the compositions and quantities of phenolics in rice samples analyzed by the developed method disclose the effect of varietal differences. Hence, this particular chromatographic method was demonstrated to be an excellent alternative for a fast and reliable determination of phenolic compounds in a wide range of rice variety.