Development of a Dual‐Regimen HPLC–UV Method for Simultaneous Analysis of Metformin and Donepezil: A Study on Diabetic Alzheimer's Patients and Pharmacokinetic Profiling of Metformin in Rabbit Plasma

ABSTRACT Combination therapy for Alzheimer's disease often pairs symptomatic agents with treatments that may influence metabolic pathways. Although previous analytical studies have quantified metformin (MTF) and donepezil (DNZ) either individually or in combination with other compounds, a clear HPLC–UV method enabling their simultaneous determination in pharmaceutical formulations and plasma, together with a structured assessment of environmental impact, has not been established. In this study, we developed, validated, and applied a reliable, solvent‐lean HPLC–UV method for the concurrent quantification of MTF and DNZ in dosage forms and rabbit plasma, and we examined the pharmacokinetics of MTF administered alone and in combination with DNZ. Chromatographic separation was performed on a C 18 column using acetonitrile and 0.05 M phosphate buffer (pH 4; 35:65, v/v) at a flow rate of 1.2 mL/min with detection at 235 nm, and plasma samples were processed using solid‐phase extraction. Validation demonstrated linearity across 10–1000 ng/mL for MTF and 20–1000 ng/mL for DNZ, with correlation coefficients of 0.9991 and 0.9992, respectively, and the method met all acceptance criteria for accuracy, precision, selectivity, stability, and system suitability. The procedure enabled quantification of MTF in rabbit plasma following single‐dose administration both alone and with DNZ, supporting non‐compartmental pharmacokinetic analysis, and its greenness assessment indicated a favorable environmental profile. Overall, the validated method provides accurate and precise simultaneous determination of MTF and DNZ and is suitable for evaluating potential pharmacokinetic interactions.