Multiresponse optimization of a UPLC method for the simultaneous determination of tryptophan and 15 tryptophan-derived compounds using a Box-Behnken design with a desirability function

- UPLC allowed for a full separation of tryptophan and 15 derivatives in 3 min.
- Two stationary phases were compare to reach enough chromatographic resolution for very similar compounds.
- Resulting LOD and LOQ allowed for the determination of tryptophan and its derivatives in rice grain extracts.

A Box-Behnken design was used in conjunction with multiresponse optimization based on the desirability function to carry out the simultaneous separation of tryptophan and 15 derivatives by Ultra Performance Liquid Chromatography. The gradient composition of the mobile phase and the flow rate were optimized with respect to the resolution of severely overlapping chromatographic peaks and the total run time. Two different stationary phases were evaluated (hybrid silica and a solid-core-based C18 column). The methods were validated and a suitable sensitivity was found for all compounds in the concentration range 1-100 μg L-1 (R2 > 0.999). High levels of repeatability and intermediate precision (CV less than 0.25% and 1.7% on average for the retention time and the signal area, respectively) were obtained. The new method was applied to the determination tryptophan and its derivatives in black pigmented glutinous and non-glutinous rice grain samples.