Quantitation of chromophore-free analytes is always a challenge. To this purpose, derivatization of the analyte constitutes a common strategy, leading to a product with a strong signal. In the current study, a novel xanthone analogue was utilized for the first time for the derivatization of pregabalin, a model analyte with a primary amine moiety that lacks a chromophore. The fact that only the xanthene-based derivative, formed after the derivatization reaction fluoresces, enables avoiding its chromatographic separation from the reagent and thus reducing the analysis time of a series of samples in 1-2 min via a plate reader. The reaction conditions were optimized via a central composite design (CCD), with fluorescence signal as the measure of the yield. The following factors that affect the derivatization reaction were chosen: (a) temperature, (b) reaction time, and (c) triethylamine solution volume used to drive the reaction to completion. After the identification of the optimal conditions, the method was validated according to ICH guidelines, using a fluorescence plate reader for signal measurement (λex = 540, λem = 615 nm). Finally, the newly developed high-throughput method was applied to the determination of drug content in pregabalin bulk.
Keywords: central composite design; derivatization reaction; experimental design; fluorescence; plate reader; pregabalin; xanthone.