Journal
MOLECULES
Volume 27, Issue 6, Pages -Publisher
MDPI
DOI: 10.3390/molecules27061954
Keywords
fluorescence; xanthone; derivatization reaction; plate reader; pregabalin; experimental design; central composite design
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In this study, a novel xanthone analogue was used for the first time to derivatize the colorless analyte pregabalin, enabling the reduction of analysis time and achieving high yield and accuracy through optimized reaction conditions.
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 (lambda(ex) = 540, lambda(em) = 615 nm). Finally, the newly developed high-throughput method was applied to the determination of drug content in pregabalin bulk.
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