An easy and short-time TLC-densitometric method was developed for the separation of metronidazole, secnidazole, ornidazole, tinidazole, and 2-methyl-5-nitroimidazole and for the determination of metronidazole and tinidazole in Metronidazole Polpharma and Tinidazolum Polpharma tablets. Analyzes were performed on chromatographic plates precoated with silica gel 60F254 using the optimal mobile phase: chloroform + methanol + diethylamine in a volume ratio of 9:1:1. The method has been validated. The intraday and interday precision values for the three different concentrations ranged from 0.99% to 1.48% and 0.89% to 1.76%, and the precision values ranged from 1.13% to 2.48% and 0.95% to 2.49% for metronidazole and tinidazole, respectively. The limit of quantification (LOQ) was 0.036 and 0.066 µg/spot for metronidazole and tinidazole, respectively. The mean recovery was 103.1% and 100.6% for metronidazole and tinidazole, respectively. The content of metronidazole and tinidazole in tablets in relation to the content declared by the manufacturer was 101.3% and 99.8%, respectively. The obtained results were verified using the pharmacopoeial method. The presented method is fast, sensitive, precise, selective, accurate and robust. It allows for the analysis of several samples on one chromatography plate at the same time.

Bacterial nitroreductase enzymes that convert prodrugs to cytotoxins are valuable tools for creating transgenic targeted ablation models to study cellular function and cell-specific regeneration paradigms. We recently engineered a nitroreductase (“NTR 2.0”) for substantially enhanced reduction of the prodrug metronidazole, which permits faster cell ablation kinetics, cleaner interrogations of cell function, ablation of previously recalcitrant cell types, and extended ablation paradigms useful for modelling chronic diseases. To provide insight into the enhanced enzymatic mechanism of NTR 2.0, we have solved the X-ray crystal structure at 1.85 Angstroms resolution and compared it to the parental enzyme, NfsB from Vibrio vulnificus. We additionally present a survey of reductive activity with eight alternative nitroaromatic substrates, to provide access to alternative ablation prodrugs, and explore applications such as remediation of dinitrotoluene pollutants. The predicted binding modes of four key substrates were investigated using molecular modelling.