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Enantioselective quantification of fluoxetine and norfluoxetine by HPLC in wastewater effluents

Publication . Ribeiro, Ana R.; Maia, Alexandra S.; Moreira, Irina S.; Afonso, Carlos M.; Castro, Paula M.L.; Tiritan, Maria E.

Microbial degradation is the most important process to remove organic pollutants in Waste Water Treatment Plants. Regarding chiral compounds this process is normally enantioselective and needs the suitable analytical methodology to follow the removal of both enantiomers in an accurate way. Thus, this paper describes the development and validation of an enantioselective High Performance Liquid Chromatography with Fluorescence Detection (HPLC-FD) method for simultaneous analysis of fluoxetine (FLX) and norfluoxetine (NFLX) in wastewater effluents. Briefly, this method preconcentrated a small volume of wastewater samples (50 mL) on 500 mg Oasis MCX cartridges and used HPLC-FD with a vancomycin-based chiral stationary phase under reversed mode for analyses. The optimized mobile phase was EtOH/aqueous ammonium acetate buffer (92.5/7.5, v/v) at pH 6.8. The effect of EtOH percentage, buffer concentration, pH, column oven temperature and flow rate on chromatographic parameters was systematically investigated. The developed method was validated within the wastewater effluent used in microcosms laboratory assays. Linearity (R2 > 0.99), selectivity and sensitivity were achieved in the range of 4.0–60 ng mL 1 for enantiomers of FLX and 2.0–30 ng mL 1 for enantiomers of NFLX. The limits of detection were between 0.8 and 2.0 ng mL 1 and the limits of quantification were between 2.0 and 4.0 ng mL 1 for both enantiomers of FLX and the enantiomers of its demethylated metabolite NFLX. The validated method was successfully applied and proved to be robust to follow the degradation of both enantiomers of FLX in wastewater samples, during 46 days.

2014Journal article

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Enantiomeric fraction evaluation of pharmaceuticals in environmental matrices by liquid chromatography-tandem mass spectrometry

Publication . Ribeiro, Ana Rita; Santos, Lúcia H. M. L. M.; Maia, Alexandra S.; Delerue-Matos, Cristina; Castro, Paula M. L.; Tiritan, Maria Elizabeth

The interest for environmental fate assessment of chiral pharmaceuticals is increasing and enantioselective analytical methods are mandatory. This study presents an enantioselective analytical method for the quantification of seven pairs of enantiomers of pharmaceuticals and a pair of a metabolite. The selected chiral pharmaceuticals belong to three different therapeutic classes, namely selective serotonin reuptake inhibitors (venlafaxine, fluoxetine and its metabolite norfluoxetine), beta-blockers (alprenolol, bisoprolol, metoprolol, propranolol) and a beta2-adrenergic agonist (salbutamol). The analytical method was based on solid phase extraction followed by liquid chromatography tandem mass spectrometry with a triple quadrupole analyser. Briefly, Oasis® MCX cartridges were used to preconcentrate 250 mL of water samples and the reconstituted extracts were analysed with a Chirobiotic™ V under reversed mode. The effluent of a laboratory-scale aerobic granular sludge sequencing batch reactor (AGS-SBR) was used to validate the method. Linearity (r2 > 0.99), selectivity and sensitivity were achieved in the range of 20–400 ng L−1 for all enantiomers, except for norfluoxetine enantiomers which range covered 30–400 ng L−1. The method detection limits were between 0.65 and 11.5 ng L−1 and the method quantification limits were between 1.98 and 19.7 ng L−1. The identity of all enantiomers was confirmed using two MS/MS transitions and its ion ratios, according to European Commission Decision 2002/657/EC. This method was successfully applied to evaluate effluents of wastewater treatment plants (WWTP) in Portugal. Venlafaxine and fluoxetine were quantified as non-racemic mixtures (enantiomeric fraction ≠ 0.5). The enantioselective validated method was able to monitor chiral pharmaceuticals in WWTP effluents and has potential to assess the enantioselective biodegradation in bioreactors. Further application in environmental matrices as surface and estuarine waters can be exploited.

2014Journal article

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Performance of aerobic granular sludge in a sequencing batch bioreactor exposed to ofloxacin, norfloxacin and ciprofloxacin

Publication . Amorim, Catarina L.; Maia, Alexandra S.; Mesquita, Raquel B. R.; Rangel, António O.S.S.; Loosdrecht, Mark C.M. van; Tiritan, Maria Elizabeth; Castro, Paula M.L.

A granular sludge sequencing batch reactor (SBR) was operated for 340 days for treating a synthetic wastewater containing fluoroquinolones (FQs), namely ofloxacin, norfloxacin and ciprofloxacin. The SBR was intermittently fed with FQs, at concentrations of 9 and 32 mM. No evidence of FQ biodegradation was observed but the pharmaceutical compounds adsorbed to the aerobic granular sludge, being gradually released into the medium in successive cycles after stopping the FQ feeding. Overall COD removal was not affected during the shock loadings. Activity of ammonia oxidizing bacteria and nitrite oxidizing bacteria did not seemto be inhibited by the presence of FQs (maximum of 0.03 and 0.01 mM for ammonium and nitrite in the effluent, respectively). However, during the FQs feeding, nitrate accumulation up to 1.7 mM was observed at the effluent suggesting that denitrificationwas inhibited. The activity of phosphate accumulating organismswas affected, as indicated by the decrease of P removal capacity during the aerobic phase. Exposure to the FQs also promoted disintegration of the granules leading to an increase of the effluent solid content, nevertheless the solid content at the bioreactor effluent returned to normal levelswithin ca. 1month after removing the FQs in the feed allowing recovery of the bedvolume. Denaturing gradient gel electrophoresis revealed a dynamic bacterial community with gradual changes due to FQs exposure. Bacterial isolates retrieved from the granules predominantly belonged to a- and g-branch of the Proteobacteria phylum.

2014Journal article

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Bacterial degradation of moxifloxacin in the presence of acetate as a bulk substrate

Publication . Carvalho, M. F.; Maia, A. S.; Tiritan, M. E.; Castro, P. M. L.

Fluoroquinolones constitute a group of emerging pollutants and their occurrence in different environmental compartments is becoming object of increasing public concern due to their ecotoxicological effects and the potential to develop resistant bacteria. This study aimed to investigate the biodegradation of moxifloxacin (MOX), for which studies in the literature are very scarce. An activated sludge (AS) consortium and three bacterial strains able to degrade fluoroaromatic compounds e strains F11, FP1 and S2 e were tested. Biodegradation studies were conducted using acetate as a bulk carbon source. Strain F11 showed the highest biodegradation capacity, being able to completely consume and dehalogenate 7.5 mM of the target antibiotic when daily co-supplemented with acetate present as a readily degradable organic substrate in wastewaters. MOX could be used by strain F11 as a sole nitrogen source but the presence of an external nitrogen source in the culture medium was essential for complete biodegradation. Strain F11 was capable of completely consuming MOX in a range between 2 and 11 mM, although stoichiometric fluoride release was not obtained for the highest tested concentration. The antibacterial activity of residual MOX and of the metabolic products potentially resultant from the biodegradation process was investigated by agar diffusion tests, demonstrating that MOX biodegradation is associated with the elimination of the antibacterial properties of the target antibiotic and of the produced metabolites, which is an important result, as the activity of antibiotics and/or their metabolites in the environment, even at low levels, may lead to the development of resistant bacterial strains. Overall, the results obtained in this study suggest that strain F11 is a promising microorganism for the treatment of waters contaminated with MOX, where it could be used for bioaugmentation/bioremediation purposes. To the best of our knowledge, this is the first study reporting complete removal and dehalogenation of MOX by a single microorganism.

2016Journal article

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Enantioselective degradation of ofloxacin and levofloxacin by the bacterial strains Labrys portucalensis F11 and Rhodococcus sp. FP1

Publication . Maia, Alexandra S.; Tiritan, Maria Elizabeth; Castro, Paula M.L.

Fluoroquinolones are a class of antibiotics widely prescribed in both human and veterinary medicine of high environmental concern and characterized as environmental micropollutants due to their ecotoxicity and persistence and antibacterial resistance potential. Ofloxacin and levofloxacin are chiral fluoroquinolones commercialized as racemate and in enantiomerically pure form, respectively. Since the pharmacological properties and toxicity of the enantiomers may be very different, understanding the stereochemistry of these compounds should be a priority in environmental monitoring. This work presents the biodegradation of racemic ofloxacin and its (S)-enantiomer levofloxacin by the bacterial strains Labrys portucalensis F11 and Rhodococcus sp. FP1 at a laboratory-scale microcosm following the removal and the behavior of the enantiomers. Strain F11 could degrade both antibiotics almost completely when acetate was supplied regularly to the cultures. Enrichment of the (R)-enantiomer was observed in FP1 and F11 cultures supplied with ofloxacin. Racemization was observed in the biodegradation of the pure (S)-ofloxacin (levofloxacin) by strain F11, which was confirmed by liquid chromatography - exact mass spectrometry. Biodegradation of ofloxacin at 450 μg L−1 by both bacterial strains expressed good linear fits (R2> 0.98) according to the Rayleigh equation. The enantiomeric enrichment factors were comprised between −22.5% to −9.1%, and −18.7% to −9.0% in the biodegradation of ofloxacin by strains F11 and FP1, respectively, with no significant differences for the two bacteria under the same conditions. This is the first time that enantioselective biodegradation of ofloxacin and levofloxacin by single bacteria is reported.

2018Journal article

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