Identification and structural characterization of in vivo metabolites of balofloxacin in rat plasma, urine and feces samples using Q-TOF/LC/ESI/MS/MS : In silico toxicity studies.

Balofloxacin is a fluroquinolone antibiotic drug which has been used for the treatment of urinary tract infections (UTIs). Identification and structural characterization of metabolites is a critical component of both drug discovery and drug development research. In vivo metabolites of balofloxacin have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI-HR-MS/MS) experiments. To identify in vivo metabolites, blood, urine and feces samples were collected after oral administration of the drug to the female Sprague-Dawley rats (n = 3 per group). Protein precipitation, freeze liquid separation followed by solid-phase extraction methods were used for sample preparation. The extracted samples were subjected to LC-ESI/HRMS/MS analysis. The chromatographic separation of the drug and its metabolites were achieved on a XDB, C18 (50, 4.6 mm, 5 mm) column using gradient elution method in combination with 0.1% formic acid and acetonitrile at a flow rate of 0.4 mL/min. A total of 13 phase I and phase II metabolites of balofloxacin have been identified in plasma, urine and feces samples. Most of metabolites were observed in plasma and urine samples including dealkylated, desmethylated, decarbonylated, decarboxylated, hydroxylated, methylated, carboxylated, cysteine conjugated metabolites and high abundance glucuronidated metabolite. The structures of metabolites have been elucidated based on fragmentation patterns, accurate mass measurements and LC/MS/MS experiments. The main phase I metabolites of balofloxacin, decarbonylated, decarboxylated and desmethylated metabolites and phase II methylated metabolite undergo subsequent phase II glucuronidation pathways. In silico toxicity of the drug and its metabolites was determined using ProTox-II. Metabolites B-1, B-2, B-5, B-6, B-7, and B-8 to B-13 were predicted to possess immunotoxicity with high probability score. Additionally, Amine Oxidase A and Prostaglandin G/H Synthase 1 are predicted for metabolites B-1, B-3 to B-6 as toxicity targets with binding probability.