Laboratory of Pharmaceutical Analysis, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimioupoli Zografou, GR-157 71 Athens, Greece.
Division of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Panepistimioupoli Zografou, GR-157 71 Athens, Greece.
Molecules. 2022 Mar 17;27(6):1954. doi: 10.3390/molecules27061954.
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 (λ = 540, λ = 615 nm). Finally, the newly developed high-throughput method was applied to the determination of drug content in pregabalin bulk.
定量分析无发色团的分析物一直是一个挑战。为此，衍生化分析物是一种常见的策略，可得到信号较强的产物。在本研究中，首次将一种新型的蒽酮类似物用于普瑞巴林（一种缺乏发色团的伯胺结构模型分析物）的衍生化。只有在衍生化反应后形成的基于蒽环的衍生物才具有荧光，这一事实可避免其与试剂的色谱分离，从而通过板读数器在 1-2 分钟内减少一系列样品的分析时间。通过中心复合设计（CCD）优化了反应条件，以荧光信号作为产率的衡量标准。选择了以下影响衍生化反应的因素：（a）温度，（b）反应时间，以及（c）用于使反应完全进行的三乙胺溶液体积。在确定最佳条件后，根据 ICH 指南使用荧光板读数器进行信号测量（λ = 540、λ = 615nm）对方法进行了验证。最后，将新开发的高通量方法应用于普瑞巴林散装药物含量的测定。
