Development of an eco-friendly RP-HPLC method for simultaneous detection of toltrazuril and its degradation product: Greenness and blueness evaluation with applications in veterinary pharmaceuticals and cattle milk analysis

A novel, eco-friendly, validated RP-HPLC method was developed for the simultaneous detection and quantification of Toltrazuril (TZ) and its alkaline degradation product in pure form, synthetic mixtures, and pharmaceutical formulations. This method substitutes the traditionally hazardous solvent acetonitrile with ethanol, aligning with the principles of green chemistry. The analysis was performed using a C8 ODS column (250 x 4.6 mm, 5 mu m) with a mobile phase composed of ethanol and water (70:30 v/v) at a flow rate of 1 mL/min, with UV detection at 242 nm. The method was validated according to ICH guidelines, exhibiting high specificity, linearity, accuracy, and precision. Forced degradation studies confirmed the method's ability to detect TZ and its degradation product without interference, while stability tests demonstrated prolonged solution stability. The method exhibited excellent linearity, with a correlation coefficient of 0.999 for both TZ and its alkaline degradation product. This novel green approach presents a safer and more efficient alternative for veterinary pharmaceutical quality control, minimizing environmental impact and health risks while maintaining analytical accuracy. Comparative greenness assessment with two previously reported chromatographic techniques emphasized the superior eco-friendliness of the proposed method. A blueness assessment further confirmed the method's applicability. Additionally, a robust milk extraction protocol was developed for spiked cattle milk samples, utilizing effective protein precipitation followed by purification. This method facilitates the detection of TZ in milk post-extraction under the same chromatographic conditions.