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Abstract

A Box Behnken Design (BBD) in conjunction with multiple response optimizations was performed to develop the simultaneous separation of tryptophan and fifteen related compounds in rice grains by Ultrahigh Performance Liquid Chromatography (UPLC) coupled with Photodiode Array (PDA) detector. The gradient compositions of mobile phase (%B start and %B end) and flow rate were optimized with respect to the resolutions of severely overlapping chromatographic peaks between the phenolic compounds in addition to the total analysis time. Furthermore, two different stationary phases were also evaluated (Cortex C18 and BEH C18). A gradient program of only 4 min analysis run using Cortex C18 was found to provide the best resolution for the simultaneous separation of the 16 studied compounds. The method was validated resulting an adequate sensitivity in the range of 1–100 μg L-1 (R2=0.9993-0.9999) with high precisions (CV lower than 2% in average for both repeatability and intermediate precisions). The suitability of the method was checked by application to the analysis of pigmented and non-pigmented rice samples. Tryptophan and several related compounds were identified and quantified in the tested samples.