Abstract Background Nitroxynil and clorsulon are commonly co-formulated in veterinary injectable formulations, where benzyl alcohol is used as a preservative, necessitating selective and reliable analytical methods for their simultaneous determination. Objective This study aimed to develop and validate sensitive, selective, and eco-friendly TLC and HPLC methods for the simultaneous determination of nitroxynil, clorsulon, and benzyl alcohol in their pharmaceutical formulation. Methods TLC separation was performed on silica gel 60 F254 plates using a mobile phase of ethyl acetate–ammonia–methanol (9.5:0.5:0.05, by volume), with densitometric detection at 254 nm. The HPLC method employed a C8 column with gradient elution using 3 mM tetrabutylammonium hydrogen sulfate (pH 6.3) and a mixture of acetonitrile and methanol, with UV detection at 215 nm. Method validation was conducted in accordance with ICH guidelines. Greenness assessment was performed using MoGAPI, AGREE, and BAGI tools. Results The TLC method showed linearity over concentration ranges of 0.5–3.5 and 0.2–2 µg/band for nitroxynil and clorsulon, respectively, with correlation coefficients of 0.9996 and 0.9994. The HPLC method exhibited linearity over ranges of 1–24 and 1–21 µg/mL for the respective drugs. Both methods demonstrated high precision, accuracy, selectivity, and reproducibility, enabling effective separation of the analytes in the presence of benzyl alcohol. Greenness evaluation confirmed the low environmental impact of the proposed methods. Conclusion The developed TLC and HPLC methods are reliable, accurate, and environmentally sustainable for the simultaneous determination of nitroxynil, clorsulon, and benzyl alcohol in veterinary injectable formulations. Highlights Simultaneous determination of nitroxynil, clorsulon, and benzyl alcohol; validated TLC-densitometric and HPLC methods; successful application to Flulock® veterinary injectable formulation; high accuracy, precision, and selectivity; greenness evaluated using MoGAPI, AGREE, and BAGI tools.
Farid et al. (Tue,) studied this question.