# Sustainable spectrofluorimetric determination of berberine in dietary supplements via Erythrosin B Ion-Pair complexation with mechanistic investigation, Box-Behnken optimization, and green chemistry assessment

## TL;DR

A new, eco-friendly method was developed to accurately measure berberine in dietary supplements using fluorescence and ion-pair complexation.

## Contribution

The study introduces a sustainable spectrofluorimetric method for berberine analysis with mechanistic and green chemistry insights.

## Key findings

- The method achieved high sensitivity (LOD = 0.032 µg/mL) and accuracy (99.83%).
- Environmental assessment showed the method is more sustainable than traditional techniques.
- Optimal conditions were determined using Box-Behnken design for efficient fluorescence quenching.

## Abstract

A novel spectrofluorimetric method was developed for berberine determination in dietary supplements based on ion-pair complex formation with Erythrosin B. Spectroscopic characterization of Erythrosin B, berberine, and their complex revealed hypsochromic shift and hypochromic effect in UV-visible absorption upon complexation, with concentration-dependent fluorescence quenching at 555 nm (λex = 530 nm). Mechanistic investigations confirmed static quenching through ground-state complex formation, evidenced by decreasing Stern-Volmer constants with increasing temperature (3.68 × 105 to 2.77 × 105 M− 1 from 298 to 313 K) and bimolecular quenching rate constants exceeding the diffusion-controlled limit. Thermodynamic analysis indicated spontaneous, exothermic complexation driven by both enthalpic and entropic contributions. Moreover, Job’s method established 1:1 stoichiometry, while PM3 quantum mechanical calculations revealed multiple stabilizing interactions including hydrogen bonding (1.7 Å), electrostatic interaction (3.2 Å), and halogen bonding (3.9 Å). Subsequently, Box-Behnken design optimization elucidated the influence of pH, reagent concentration, buffer volume, and incubation time on fluorescence quenching efficiency and yielded optimal conditions (pH 6.4, Erythrosin B 13.0 µg/mL, buffer volume 1.1 mL, incubation time 6.0 min). The method was then validated according to ICH Q2(R2) guidelines, demonstrating excellent linearity (0.1–3.0 µg/mL, r2 = 0.9997), high sensitivity (LOD = 0.032 µg/mL), satisfactory accuracy (99.83%), and precision (RSD < 1.4%). Selectivity studies revealed cross-reactivity with structurally related quaternary alkaloids, rendering the method suitable for standardized berberine supplements but not crude botanical extracts. Furthermore, statistical comparison with HPLC reference methods confirmed method equivalence. Finally, environmental assessment using AGREE (0.75), MoGAPI (78), CaFRI (82), and BAGI (77.5) demonstrated the method superior sustainability through minimal solvent consumption, reduced waste generation, and elimination of toxic reagents, offering a practical and environmentally friendly alternative for routine berberine quality control.

The online version contains supplementary material available at 10.1038/s41598-026-36903-6.