A dual-function chromogenic and fluorogenic benzofurazan probe for plazomicin and its innovative utility for development of two microwell assays with high throughput for analysis of drug substance and pharmaceutical formulations

Abstract

Plazomicin (PLZ) is a novel aminoglycoside which has been recently approved by The US Food and Drug Administration for the treatment of complicated urinary tract infections including acute pyelonephritis, caused by certain Enterobacteriaceae, in adult patients with limited or no options for alternative treatment. This study focuses on the development of microwell-based photometric and fluorometric assays for the quantitative determination of PLZ in its bulk drug substance and commercial pharmaceutical formulations (Zemedri® injections). Both assays utilize the dual-function chromogenic and fluorogenic properties of the 4-fluoro-7-nitrobenzofurazan (NBD-F) probe. The reaction between PLZ and NBD-F, conducted in a borate buffer at pH 8, resulted in the formation of a colored and fluorescent reaction product. The product exhibited maximum light absorption at 473 nm and emitted fluorescence at 541 nm when excited at 473 nm. Factors influencing the reaction between PLZ and NBD-F were thoroughly investigated, and optimal conditions were determined. Under the optimized reaction conditions, calibration curves were generated to establish the relationship between absorbance and fluorescence intensities of the reaction product with the corresponding PLZ concentrations. The absorbance–concentration relation was linear in a PLZ concentration range of 20–800 μg mL⁻¹ with a limit of quantitation of 25 μg mL⁻¹, while the fluorescence–concentration relation was linear in the concentration range of 0.05–1.5 μg mL⁻¹ with a limit of quantitation of 0.08 μg mL⁻¹. Both assays underwent validation and were successfully applied to the quantitation of PLZ in its bulk drug substance and pharmaceutical formulations (injections) with satisfactory accuracy and precision. The eco-friendliness/greenness assessment of the assays demonstrated that both assays comply with the requirements of green analytical chemistry approaches. Furthermore, the proposed microwell assay plates allowed for the simultaneous handling of numerous samples with micro-volumes, enabling high-throughput analysis. In conclusion, this study represents the first evaluation of NBD-F as a dual-function probe for the microwell-based photometric and fluorometric determination of PLZ. The developed assays serve as valuable analytical tools for the quality control of PLZ's bulk drug substance and pharmaceutical formulations.