Systematic development and comprehensive validation of a green spectrofluorimetric method for enalapril determination: response surface optimization, mechanistic investigation with application to pharmaceutical and bioanalytical samples

A sensitive, selective, and environmentally friendly spectrofluorimetric method was developed and validated for the determination of enalapril, an antihypertensive drug, using eosin Y as a fluorescent probe. At acidic pH, an ion-association complex is formed between enalapril and eosin Y, resulting in the quenching of the dye's fluorescence. The quenching mechanism was systematically investigated using temperature-dependent Stern-Volmer analysis (298–313 K), Job's method, thermodynamic characterization, and quantum mechanical calculations, revealing a static mode of interaction with Stern-Volmer constants decreasing from 8.22 × 10⁵ M⁻¹ (298 K) to 6.17 × 10⁵ M⁻¹ (313K). Comprehensive thermodynamic analysis provided negative enthalpy and negative entropy changes, indicating highly exothermic binding dominated by strong electrostatic interactions with loss of translational freedom upon complex formation. Job's ratio confirmed 1:1 stoichiometry with maximum at mole fraction 0.5 while PM3 quantum mechanical calculations identified the binding site between the protonated amino group of enalapril and carboxylate group of eosin Y, confirming 1:1 complex formation. Factors affecting the quenching process, including pH, buffer volume, and reagent volume, were optimized using response surface methodology with a central composite design. Significant modeling parameters were identified through ANOVA, and a reduced quadratic model was developed and validated, demonstrating good predictive ability. Numerical optimization was then performed to maximize the quenching efficiency. The proposed method was fully validated according to ICH guidelines, exhibiting excellent linearity in the range of 0.05–1.5 μg/mL, with a limit of detection of 0.0147 μg/mL. Moreover, accuracy, precision, and robustness were within acceptable limits. The developed method was successfully applied for the determination of enalapril in pharmaceutical formulations and spiked human plasma samples with satisfactory results. Statistical comparison to a reported HPLC method revealed comparable analytical performance. Furthermore, the environmental impact and analytical practicality of the method were evaluated using AGREE and BAGI tools, respectively, indicating that the proposed spectrofluorimetric technique offers a greener and practical alternative to traditional HPLC methods for routine quality control and therapeutic drug monitoring of enalapril showcasing its potential for widespread application in the pharmaceutical industry and clinical settings.