Response surface methodology-optimized microwell spectrophotometric method for saxagliptin quantitation: a sustainable, high-throughput approach for pharmaceutical quality control.

The growing demand for sustainable analytical methods in pharmaceutical quality control has driven the development of miniaturized, low-waste alternatives to conventional procedures. This study describes, for the first time, the development and validation of a simple, rapid, and sustainable microwell-spectrophotometric method (MW-SPM) for SAX quantitation in pharmaceutical tablets. The method is based on the reaction between SAX and the chromogenic reagent 1,2-naphthoquinone-4-sulfonate (NQS) in an aqueous medium (pH 9) at room temperature, producing a colored product with maximum absorbance at 472 nm. Critical reaction parameters were systematically optimized using response surface methodology (RSM), enabling efficient identification of optimal conditions with minimal experimental runs. The reaction was conducted in 96-well microplates using a total reaction volume of only 0.2 mL per test, representing a 50-fold reduction compared to conventional cuvette-based methods. The method was fully validated following ICH guidelines and exhibited a linear response over the concentration range of 8-500 µg mL-1, with limits of detection and quantitation of 5.9 µg mL-1 and 17.9 µg mL-1, respectively. The MW-SPM method demonstrated excellent accuracy (recovery: 98.7-101.4%), precision (RSD: 0.75-1.45%), and statistical agreement with a reference method, verified by student t- and F-tests. The proposed MW-SPM method was successfully applied to the assay of SAX in commercial pharmaceutical tablets and to content uniformity testing, demonstrating its reliability and practicality for routine quality control applications. A notable advantage of the proposed method is its exceptionally high effective throughput of 480 unknown samples per hour, achieved through parallel microwell processing, which is 60- to 80-fold higher than that of reported colorimetric methods for SAX. The method was evaluated using the Environmental, Practicality, and Performance Index (EPPI) tool, achieving a total score of 82.1%, classifying it as a "green method" with perfect or near-perfect performance in sample preparation, reagent consumption, and instrumentation. In summary, the proposed MW-SPM method offers an optimal balance of speed, simplicity, sensitivity, throughput, and sustainability, making it a highly attractive alternative for routine pharmaceutical quality control of SAX.