Application of XCMS Online and toxicity bioassays to the study of transformation products of levofloxacin

We studied the nature and antimicrobial activity of ozonolysis transformation products (OTPs) of levofloxacin (LEV), a frequently detected fluoroquinolone antimicrobial in environmental waters. Two bioassays, the Kirby–Bauer test and the broth microdilution assay, were used to measure changes in the antimicrobial activity of solutions at low LEV to O₃ molar ratios (2:1, 2:3 and 1:3) compared to solutions without added O₃ (LEV:O₃ 1:0). The Kirby–Bauer test was not sensitive enough to detect significant differences in the growth inhibition zones in samples LEV:O₃ 2:1 and LEV:O₃ 1:0; however, the broth microdilution assay showed that bacterial growth inhibition was significantly lower (P < 0.001) in the solutions exposed to O₃. Loss of antimicrobial activity in LEV:O₃ 2:1 solutions of (48 ± 16)% was in agreement with the concentration decrease of LEV of (36 ± 3)% in those same samples. A method of identification of OTPs using XCMS Online was applied to LEV:O₃ 2:1 and 1:0 samples and indicated the presence of an OTP of LEV of formula C₁₈H₂₀O₅N₃F, which was identified as LEV-N-oxide. The molecular structure of this compound was partially confirmed by tandem mass spectrometry experiments. This study showed that even at sub-optimal ozone doses, OTPs of higher antimicrobial activity than LEV were not formed.