Integrated liquid chromatography method in enantioselective studies: Biodegradation of ofloxacin by an activated sludge consortium

Ofloxacin is a chiral fluoroquinolone commercialized as racemate and as its enantiomerically pure form levofloxacin. This work presents an integrated liquid chromatography (LC) method with fluorescence detection (FD) and exact mass spectrometry (EMS) developed to assess the enantiomeric biodegradation of ofloxacin and levofloxacin in laboratory-scale microcosms. The optimized enantioseparation conditions were achieved using a macrocyclic antibiotic ristocetin A-bonded CSP (150 × 2.1 mm i.d.; particle size 5 μm) under reversed-phase elution mode. The method was validated using a mineral salts medium as matrix and presented selectivity and linearity over a concentration range from 5 μg L−1 (quantification limit) to 350 μg L−1 for each enantiomer. The method was successfully applied to evaluate biodegradation of ofloxacin enantiomers at 250 μg L−1 by an activated sludge inoculum. Ofloxacin (racemic mixture) and (S)-enantiomer (levofloxacin) were degraded up to 58 and 52%, respectively. An additional degradable carbon source, acetate, enhanced biodegradation up to 23%. (S)-enantiomer presented the highest extent of degradation (66.8%) when ofloxacin was supplied along with acetate. Results indicated slightly higher biodegradation extents for the (S)-enantiomer when supplementation was done with ofloxacin. Degradation occurred faster in the first 3 days and proceeded slowly until the end of the assays. The chromatographic results from LC-FD suggested the formation of the (R)-enantiomer during levofloxacin biodegradation which was confirmed by LC–MS with a LTQ Orbitrap XL. © 2016 Elsevier

Authors wish to thank Fundação para a Ciência e Tecnologia – FCT for financial support under the project Fluoropharma PTDC/EBB-EBI/111699/2009, PhD grant attributed to Alexandra S. Maia SFRH/BD/86939/2012, QREN-POPH, European Social Fund, MCTES, PEst (FCOMP-01-0124-FEDER-022718; PEst-OE/EQB/LA0016/2011, PEst-OE/SAU/UI4040/2014). This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by Fundação para a Ciência e Tecnologia – FCT and European Regional Development Fund (ERDF), in the framework of the programme PT2020 and by CESPU (PHARMADRUGS-CESPU-2014). The authors thank Parada WWTP for supplying the activated sludge, Virgínia Gonçalves for her collaboration, and also CEMUP – Materials Centre of the University of Porto and the Laboratory for Structural Elucidation for the mass spectrometry analysis.