A sustainable spectrofluorimetric method for ivabradine quantification based on erythrosin B quenching with quantum mechanical modeling and Box–Behnken design optimization

Figures

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  Fig. 1. Spectral characterization of the erythrosin B-ivabradine interaction system: (A) UV-visible absorption spectra of erythrosin B (25 µg mL−1, green), ivabradine (1.0 µg mL−1, blue), and their complex (red) in Britton–Robinson buffer (pH 5.6), showing hypsochromic shift and hypochromic effect upon complex formation; (B) fluorescence excitation (blue, λem = 555 nm) and emission (red, λex = 528 nm) spectra of erythrosin B (25 µg mL−1, pH 5.6) showing a Stokes shift of 27 nm; (C) progressive fluorescence quenching spectra upon incremental ivabradine addition (0–2.0 µg mL−1, arrow indicates increasing concentration), showing systematic emission decrease with preserved spectral shape consistent with ground-state complex formation. All measurements in triplicate at 25 ± 2 °C.

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  Fig. 2. Fluorescence quenching mechanism and thermodynamic studies of the erythrosin B-ivabradine system: (A) Stern–Volmer plots of F0/F versus ivabradine molar concentration at 298 K (●), 303 K (■), and 313 K (▲) in Britton–Robinson buffer (pH 5.6); the decreasing Ksv values with increasing temperature confirm static quenching; error bars represent standard deviation of triplicate measurements; (B) Van't Hoff plot of ln(Ka) versus 1/T (r2 = 0.999) yielding ΔH = −24.54 kJ mol−1 and ΔS = 36.71 J mol−1 K−1, confirming spontaneous exothermic complex formation; (C) Job's method continuous variation plot of ΔF versus mole fraction of ivabradine (total concentration = 1.0 × 10−5 M, pH 5.6), with maximum at 0.5 confirming 1 : 1 binding stoichiometry.

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  Fig. 3. Quantum mechanical calculations using PM3 semi-empirical methodology (Gaussian 09, gas phase): (A) energy-minimized structure of erythrosin B (E = −0.049215 Hartree, dipole = 12.77 Debye) showing the planar xanthene framework with peripheral iodine substituents and carboxylate groups; (B) optimized geometry of ivabradine (E = 0.057156 Hartree, dipole = 10.17 Debye) showing the benzofuran–benzazepine framework with protonated tertiary amine; (C) optimized complex geometry (ΔE = −0.184157 Hartree) showing three interaction sites: primary electrostatic ion-pair (3.6 Å), secondary electrostatic interaction involving iodine substituent (2.9 Å), and hydrogen bonding (1.7 Å). Calculations performed in gas phase; binding energies may be overestimated relative to solution-phase values due to absence of solvent screening effects.

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  Fig. 4. Individual factor effects on fluorescence quenching efficiency (QE%) from Box–Behnken design, with all other factors at central values: (A) effect of pH (3.0–8.0) showing bell-shaped response with optimum at pH 5.6, consistent with complete erythrosin B deprotonation (pKa ≈ 3.8, 4.6) and preserved ivabradine cationic character (pKa ≈ 9.3); (B) effect of buffer volume (0.5–1.5 mL) with optimum at 1.1 mL; (C) effect of erythrosin B concentration (10–30 µg mL−1) showing plateau above 25 µg mL−1 indicating binding site saturation; (D) effect of reaction time (3.0–10.0 min) showing flat response confirming rapid equilibrium formation. Dashed lines represent 95% confidence intervals derived from the quadratic model.

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  Fig. 5. Factor interaction plots and response surface analysis from Box–Behnken optimization (R2 = 0.9727, F = 75.22, p < 0.0001): (A) two-dimensional interaction plot between pH and buffer volume showing moderate curvature; (B) three-dimensional response surface for pH-buffer volume interaction displaying broad optimal region; (C) pH-reagent concentration interaction plot exhibiting pronounced curvature; (D) three-dimensional surface for pH-reagent concentration interaction revealing distinct maximum at intermediate pH and higher concentrations; (E) buffer volume-reagent concentration interaction showing strong synergistic effects; (F) three-dimensional surface for buffer volume-reagent concentration displaying sharp maximum confirming significant interaction coefficient.

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  Fig. 6. Green analytical chemistry assessment of the developed method: (A) AGREE pictogram (overall score = 0.76) showing excellent performance for waste generation, energy consumption, reagent toxicity, and operator safety, with instrumentation accessibility identified as the primary area for improvement; (B) BAGI star diagram (overall score = 77.5) confirming method suitability for routine implementation through simple instrumentation, minimal sample preparation, and low reagent costs; (C) RGB12 whiteness assessment (overall score = 90.1) with dimensional scores of 96.3 (analytical), 97.5 (environmental), and 76.7 (economic), confirming excellent balance between analytical performance, environmental friendliness, and economic viability.