A systematic stepwise optimization framework for rapid multi-analyte UPLC–MS/MS plasma analysis with integrated sustainability assessment

Abstract A stepwise optimization guidance model for rapid multi-analyte UPLC–MS/MS is proposed to report recurring limitations in bioanalytical method development. The model involves six stages: (1) physicochemical profiling, (2) extraction strategy selection, (3) chromatographic optimization, (4) mobile phase selection, (5) method validation, and (6) greenness evaluation. The applicability of this framework was demonstrated by determining Rifaximin, Ciprofloxacin, and Fluconazole in spiked human plasma, using Ibuprofen as an internal standard. Physicochemical profiling guided the selection of ionization mode and prediction of solubility behavior. Based on the model recommendations, liquid–liquid extraction using dichloromethane was selected to achieve effective analyte recovery. Chromatographic optimization and mobile phase selection through the proposed framework supported the use of rapid isocratic separation on a C18 column using a methanol/water mixture (95:5, v/v) as mobile phase. The method validation, performed as an integral stage of the workflow according to regulatory guidelines, demonstrated exceptional linearity (r² ≥ 0.999). The final stage of the model involved evaluating environmental applicability using AGSA and EPPI analyses. The proposed sequential framework is intended to serve as a methodological reference for analysts employing LC–MS/MS platforms, to establish a structured and reproducible analytical workflow that advances ecological sustainability in multi-analyte LC–MS/MS bioanalytical applications.