Validation and Greenness Evaluation of TLC and HPLC Methods for Analysis of Nitroxynil, Clorsulon, and Benzyl Alcohol in Flulock® Veterinary Injectable Formulation.

BACKGROUND

Nitroxynil (NIT) and clorsulon (CLO) 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 NIT, CLO, 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 International Conference on Harmonization (ICH) guidelines. Greenness assessment was performed using Modified Green Analytical Procedure Index (MoGAPI), Analytical Greenness Metric (AGREE), and Blue Applicability Grade Index (BAGI) tools.

RESULTS

The TLC method showed linearity over concentration ranges of 0.5-3.5 µg/band and 0.2-2 µg/band for NIT and CLO, respectively, with correlation coefficients of 0.9996 and 0.9994. The HPLC method exhibited linearity over ranges of 1-24 µg/mL 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 NIT, CLO, and benzyl alcohol in veterinary injectable formulations.

HIGHLIGHTS: Simultaneous determination of NIT, CLO, 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.