Evaluation of 4-fluoro-7-nitrobenzofurazan as a dual-function chromogenic and fluorogenic probe for tulathromycin and its innovative utility for development of two eco-friendly and high-through microwell assays for analysis of pharmaceutical formulations

Abstract

Tulathromycin is a triamilide antibacterial drug which has been approved for use in the European Union and the United States for the treatment and prevention of bovine respiratory diseases. The aim of this study was the development of two innovative microwell spectrometric (photometric and fluorometric) assays for determination of tulathromycin in its pharmaceutical formulations. To achieve this goal, 4-fluoro-7-nitrobenzofurazan was investigated as a dual-function chromogenic and fluorogenic probe for tulathromycin. The reaction between tulathromycin and 4-fluoro-7-nitrobenzofurazan proceeded smoothly in an alkaline medium, resulting in the formation of a colored and fluorescent product. The product displayed a maximum light absorption at 475 nm and emitted fluorescence at 545 nm when excited at 475 nm. Extensive investigations were conducted to optimize the factors affecting the tulathromycin /4-fluoro-7-nitrobenzofurazan reaction, and the optimal conditions were established. Using these optimized conditions, both microwell-based photometric and fluorometric assays were developed. The calibration curves relating the absorbance and fluorescence intensities of the reaction product with the corresponding tulathromycin concentrations were generated. The absorbance-concentration relationship was found to be linear within a tulathromycin concentration range of 10–400 µg mL−1, with a limit of quantitation of 6.2 µg mL−1. On the other hand, the fluorescence-concentration relationship was linear within a concentration range of 0.04–1.2 µg mL−1, with a limit of quantitation of 0.06 µg mL−1. Rigorous validations of both assays’ procedures were performed, and both assays were successfully employed for the analysis of tulathromycin–containing pharmaceutical formulations (injections) with satisfactory accuracy and precision. The ecologically friendly assessment of both assays demonstrated their compliance with the principles of green analytical chemistry approaches. Moreover, the proposed microwell-based assays enabled the simultaneous analysis of multiple samples using small volumes, enabling high-throughput analysis. In conclusion, this study represents the first evaluation of 4-fluoro-7-nitrobenzofurazan as a probe with dual functionality for the microwell-based photometric and fluorometric analysis of tulathromycin. The developed assays serve as valuable analytical tools for ensuring the quality of tulathromycin ’s pharmaceutical formulations.