Spectrophotometric determination of cefepime and enmetazobactam in pharmaceutical formulations and human plasma using chemometric modeling, metaheuristic wavelength selection, and comprehensive sustainability assessment.

The recently introduced cefepime-enmetazobactam (CEF-ENM) combination requires reliable analytical methods for pharmaceutical quality control and proof-of-concept bioanalytical evaluation. Existing methods are predominantly chromatographic and rely on large volumes of organic solvents, costly instrumentation, and high energy consumption. In this work, a UV spectrophotometric approach integrated with chemometric analysis was established for the simultaneous quantification of CEF and ENM in pharmaceutical formulations and processed post-extraction-fortified human plasma. Calibration samples were prepared according to the Brereton multilevel experimental design, whereas an independent external validation set was constructed using the Maximin Distance Design (MMD) algorithm. The analytical performance of six chemometric models (CLS, PCR, PLS, GA-PLS, FA-PLS, and MCR-ALS) was evaluated to resolve the severe spectral overlap between both analytes. Matrix-specific calibration models were developed for processed plasma samples. The developed models showed excellent analytical performance, with MCR-ALS providing the highest predictive accuracy and robustness. Sustainability was comparatively evaluated using the integrated Multi-color Assessment (MA) Tool together with complementary greenness metrics. The proposed method demonstrated superior overall sustainability compared with previously reported chromatographic methods while maintaining reliable quantitative performance. The proposed approach offers a rapid, cost-effective, and environmentally sustainable alternative for routine pharmaceutical quality control and proof-of-concept evaluation in processed post-extraction-fortified human plasma. Further comprehensive bioanalytical validation is warranted to support routine clinical application.