A novel spectrofluorimetric method for argatroban determination using Rhodamine B as an “on-off” fluorescent probe with Box-Behnken optimization and sustainability assessment

A novel, sensitive, and environmentally sustainable spectrofluorimetric method was developed for the determination of argatroban using Rhodamine B as an “on-off” fluorescent probe. The interaction mechanism between argatroban and Rhodamine B was thoroughly investigated through spectroscopic studies, Stern-Volmer analysis, and Job's method. Results revealed that argatroban effectively quenched the fluorescence of Rhodamine B through static quenching with a Stern-Volmer constant (Ksv) of 7.07 × 10⁵ M⁻¹, indicating a strong static quenching process. Response surface methodology based on Box-Behnken design was employed to identify the critical factors affecting quenching efficiency. Analysis of variance revealed that pH and Rhodamine B concentration significantly influenced the response, while reaction time showed minimal impact. Through systematic investigation of the response surfaces and model validation, optimal analytical conditions were determined to be pH 7.5, Rhodamine B volume 2.6 mL, and reaction time 3 min. The developed method exhibited a linear response across the concentration range 0.1–3.0 μg/mL with detection limit of 0.0324 μg/mL. Validation studies confirmed the method's precision, accuracy, and robustness in accordance with ICH guidelines. Application to complex matrices demonstrated the method's selectivity and practical utility, with recoveries from 95.03 to 104.98 % in spiked plasma and environmental water samples. Notably, sustainability assessment using AGREE (score: 0.64), BAGI (score: 70), and RGB12 (whiteness: 90.1 %) tools demonstrated that this method offers significant environmental advantages over conventional chromatographic techniques. By combining analytical reliability with environmental sustainability, this simple and cost-effective approach provides a viable alternative for argatroban determination in pharmaceutical quality control and environmental monitoring applications.