Validated Erythrosin B spectrofluorimetric method for ganciclovir bioanalysis in rabbit plasma following valganciclovir bioconversion and Pharmacokinetic application

A novel, environmentally sustainable spectrofluorimetric method was developed and validated for sensitive determination of ganciclovir in rabbit plasma following valganciclovir administration. The method is based on fluorescence quenching of Erythrosin B probe through static complex formation with ganciclovir, as confirmed by comprehensive spectroscopic characterization using UV-visible absorption and fluorescence spectroscopy. Furthermore, temperature-dependent Stern-Volmer analysis revealed static quenching mechanism with Stern-Volmer constants decreasing from 3.77 × 10⁵ to 2.79 × 10⁵ M^{- 1} upon temperature increase from 298 to 313 K. Moreover, thermodynamic studies demonstrated spontaneous, exothermic interactions with negative Gibbs free energy and enthalpy changes, while positive entropy indicated favorable complex formation. Additionally, Job's method confirmed 1:1 stoichiometry, while semiempirical PM3 quantum mechanical calculations identified halogen bonding and electrostatic interactions as primary binding mechanisms. Experimental conditions were systematically optimized to achieve maximum quenching efficiency at pH 5.0 with 10 µg/mL Erythrosin B concentration and 5-minute reaction time. The method demonstrated excellent analytical performance with good linearity (r² = 0.9991) over 0.05-3.0 µg/mL range and adequate sensitivity for therapeutic monitoring applications. Furthermore, comprehensive validation according to ICH M10 guidelines confirmed method reliability with accuracy ranging from 96.04% to 104.76% and precision below 4.61% RSD across all quality control levels. Subsequently, successful pharmacokinetic application in New Zealand white rabbits provided key parameters including C_max of 4.25 µg/mL, half-life of 2.94 h, and AUC_₀₋∞ of 21.6 µg·h/mL, consistent with published clinical data. Finally, environmental sustainability assessment using four complementary evaluation tools confirmed favorable green analytical chemistry and analytical practicality traits with scores of 0.71, 76%, 70.0, and 65% for AGREE, MoGAPI, BAGI, and CACI, respectively. In conclusion, the developed spectrofluorimetric method offers significant advantages including cost-effectiveness, minimal waste generation, and elimination of derivatization steps, positioning it as an attractive alternative to conventional methods.