A Sustainable UHPLC‐PDA/QTOF‐MS Approach for Comprehensive Profiling of Amoxicillin Degradation Impurities

Rationale Amoxicillin is a broad‐spectrum β‐lactam antibiotic that is prone to undergo degradation under various stress and storage conditions into multiple impurities, which may compromise drug stability, efficacy, and safety. This study established an eco‐efficient comprehensive analytical strategy for amoxicillin degradation impurities using UHPLC coupled with PDA detection and QTOF‐MS characterization, which has not been previously reported. Methods Chromatographic conditions were optimized through software‐assisted conversion from traditional chromatography and then validated for the simultaneous determination of degradation impurities. Separation was performed on a Cortecs C18 column (150 × 3 mm; 2.7 μm) at 254 nm with a gradient elution of acetonitrile and a pH 5.0 buffer solution. Degradation products were identified and characterized using UHPLC‐PDA/Q/TOF‐MS. The environmental impact of the method was evaluated using the Eco‐Scale, GAPI, AGREE, and BAGI tools. Results The validated method in accordance with ICH Q2(R2) (2023) demonstrates system suitability, specificity, linearity (3.003–22.519 μg/mL), accuracy (98.0%–102%, RSD < 2.0%), precision (RSD < 2.0%), and robustness. Limits of detection and quantification were 0.330 and 0.991 μg/mL, respectively, enabling the quantification of impurities at 0.066% relative to amoxicillin content in 500‐mg capsules. Four degradation impurities exceeding the identification thresholds were characterized, including amoxicillin diketopiperazine, penilloic acid, and two newly detected compounds not listed in the current international pharmacopoeias. Greenness evaluation tools confirm the environmental friendliness of the method, with high scores of 78 (Eco‐Scale), 80 (BAGI), 68 (GAPI), and 0.63 (AGREE). Conclusions The study delivers enhanced chromatographic resolution for understanding the degradation pathways of amoxicillin during short‐ and long‐term storage, enabling stability evaluation and routine drug quality control in accordance with sustainable development goals.