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- Enantioselective Degradation of Enantiomers of Fluoxetine Followed by HPLC- FDPublication . Ribeiro, Ana R.; Maia, Alexandra S.; Moreira, Irina S.; Afonso, Carlos; Castro, Paula M.L.; Tiritan, Maria E.Environmental fate assessment of chiral pharmaceuticals is an important issue and little information is known about enantioselectivity in the environment. This kind of information is important for regulamentation of pharmaceutical industry and chiral switching processes. Fluoxetine (FLX), an anti-depressant worldwide used, is a chiral pharmaceutical prescribed in racemic form, and its main metabolite norfluoxetine (NFLX) is also chiral. In this study, enantioselective degradation of rac-FLX and degradation of its enantiomers separately, in a minimal salts medium inoculated by a bacterium consortium was examined both at light and dark conditions. Theassays were performed in a shaker at aerobic and ambient temperature conditions. The analytical method used was an enantioselective HPLC-FD method using a vancomycin-based chiral stationary phase in reversed mode to monitor enantiomers of FLX and NFLX. No degradation of enantiomers of FLX in the abiotic controls was observed. In theall assays (R)-FLX was degraded faster and totally until day 24th while (S)-FLX remained up to 20% of its initial concentration until the end of the experiment (38 days). NFLX wasdetected in all biotic experiments.
- Enantioselective quantification of fluoxetine and norfluoxetine by HPLC in wastewater effluentsPublication . 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.
- Strategies to enhance the removal of FluoroquinolonesPublication . Amorim, Catarina L.; Maia, Alexandra S.; Moreira, Irina S.; van Loosdrecht, Mark C.M.; Tiritan, Maria E.; Castro, Paula M.L.Fluoroquinolones (FQs) are broad-spectrum antibiotics that play an important role in the treatment of serious bacterial infections. Currently, several FQs are available but ciprofloxacin (CPF), ofloxacin (OFL) and norfloxacin (NOR) are amongst the most worldwide prescribed antibiotics. Antibiotics can reach wastewater treatment plants (WWTP) from different routes. Thus removal of these contaminants during the biotreatment process is of major importance in order to avoid their release to other environmental matrices. Granular sludge sequencing batch reactors (SBR) constitute a novel biofilm technology for wastewater treatment extremely promising for the treatment of effluents containing toxic compounds. Therefore, in this study a granular sludge SBR, established with activated sludge from a WWTP, was operated for the treatment of an aqueous stream containing FQs. No evidence of FQ biodegradation followed by HPLC with Fluorescence Detection was observed but FQs adsorbed to the aerobic granular sludge, being gradually released into the medium after withdrawal of the FQs in the inlet stream. In a previous study, Labrys portucalensis F11 demonstrated to be able to degrade FQs, namely OFL, NOR and CPF, when supplied individually or as a mixture, in the presence of an easy degradable carbon source. Different removal extents were obtained for the tested concentrations (ranging from 0.8 to 30 μM), but overall the uptake capacity of strain F11 for individual FQs decreased with increasing the initial FQ concentration. When supplied with a mixture FQs, strain F11 concomitantly removed each target antibiotic but a decrease on the biodegradability of FQs was observed which could be explained by competition mechanisms. The ability of Labrys portucalensis F11 to grow using the readily available carbon source while maintain its ability to degrade FQs reinforce the potential of this strain in bioaugmentation processes. As the indigenous microbial communities in biotreatment processes rarely are able to remove such contaminants, using this promising FQ-degrading strain, bioaugmentation strategies such as inoculation of the degrading strain, as a suspension or immobilized on carrier material, or using a plasmid donor strain carrying the degradative genes, could be assessed to improve FQ removal. Acknowledgments: C.L. Amorim, A.S. Maia and I.S. Moreira wish to acknowledge the research grants from Fundação para a Ciência e Tecnologia (FCT), Portugal (Ref. SFRH/BD/47109/2008, SFRH/BD/86939/2012 and SFRH/BPD/87251/2012, respectively) and Fundo Social Europeu (Programa Operacional Potencial Humano (POPH), Quadro de Referência Estratégico Nacional (QREN))). This work was supported by FCT through the projects PTDC/EBB-EBI/111699/2009 and PEst-OE/EQB/LA0016/2011.
- Microbial degradation of pharmaceuticals followed by a simple HPLC-DAD methodPublication . Ribeiro, Ana R.; Goncalves, Virgínia M.F.; Maia, Alexandra S.; Carvalho, Maria F.; Castro, Paula M.L.; Tiritan, Maria E.The biodegradation of five pharmaceutical ingredients (PIs) of different therapeutic classes, namely antibiotics (trimethoprim, sulfametoxazole and ciprofloxacin), anti-inflammatory (diclofenac) and anti-epileptic (carbamazepine), by two distinct microbial consortia, was investigated. For the monitoring of biodegradation assays, a simple HPLC-DAD (High Performance Liquid Chromatography – Diode Array Detector) method was developed and validated. The separation of the target pharmaceuticals was performed using an environmental friendly mobile phase in a gradient mode of 0.1% triethylamine (TEA) in water acidified at pH 2.23 with trifluoroacetic acid (TFAA) and ethanol as organic solvent. The method revealed to be selective, linear and precise in the range of 1.0 to 30.0 μg/mL for all PIs. Biodegradation assays were performed using activated sludge and a bacterial consortium (able to degrade fluoroaromatic compounds) supplemented with the target PIs at a final concentration of 25 μg/mL. The results revealed that activated sludge removed the target compounds more efficiently than the bacterial consortium.
- Performance of aerobic granular sludge in a sequencing batch bioreactor exposed to ofloxacin, norfloxacin and ciprofloxacinPublication . 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.
- Enantioselective Determination of Fluoxetine and Norfluoxetine in WastewaterPublication . Ribeiro, Ana R.; Maia, Alexandra S.; Moreira, Irina S.; Afonso, Carlos; Castro, Paula M.L.; Tiritan, Maria E.Microbial degradation of chiral compounds during wastewater treatment processes can be enantioselective and needs chiral analytical methodology to discriminate the biodegradation of both enantiomers. An enantioselective HPLC-FD method was developed and validated to monitor the degradation of fluoxetine (FLX) enantiomers by wastewater and the possible formation of its metabolite norfluoxetine (NFLX). The Solid Phase Extraction (SPE) of 50 mL of wastewater samples on 500 mg Oasis MCX cartridges was followed by the HPLC analysis using a Chirobiotic V chiral stationary phase under reversed mode. The developed method wasvalidated within the wastewater effluent used in microcosms laboratory assays. The chiral SPE-HPLC-FD method demonstrated to be selective, linear, sensitive, accurate and precise to quantify the enantiomers of FLX and of its metabolites NFLX in wastewaters. The limits of detection (0.8-2.0 ng mL -1 ) and quantification (2.0 – 4.0 ng mL -1 ) were adequate to monitoring the degradation assays at environmental level. The method proved to be robust to follow the biodegradation assays using real wastewater samplesspiked with FLX, during 46 days. To the best of our knowledge, this is the first reportof simultaneous separation of FLX and NFLX enantiomers using a Chirobiotic V and the application of the validated method to the enantioselective degradation by wastewater
- Enantioselective degradation of ofloxacin and levofloxacin by the bacterial strains Labrys portucalensis F11 and Rhodococcus sp. FP1Publication . 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.
- Degradation of Fluoroquinolone Antibiotics and Identification of Metabolites/Transformation Products by LC-MS/MSPublication . Maia, Alexandra S.; Ribeiro, Ana R.; Amorim, Catarina L.; Barreiro, Juliana C.; Cass, Quezia B.; Castro, Paula M.L.; Tiritan, Maria E.Antibiotics are a therapeutic class widely found inenvironmental matrices and extensively studied due to its persistence and implications for multi-resistant bacteria development. Degradation of four fluoroquinolone antibiotics, namely Ofloxacin (OFL), Norfloxacin (NOR), Ciprofloxacin (CPF) and Moxifloxacin (MOX), at 10 mg L-1 using a mixed bacterial culture, was assessed for 60 days. The assays were followed by a developed and validated analytical method of HPLC with Fluorescence Detection using a Luna PFP (2) 3µm column. The optimized conditions allowed picturing metabolites/transformation products formation and accumulation during the process, stating an incomplete mineralization, also shown byfluoride release. OFL and MOX presented the highest (98.3%) and the lowest (80.5%) extent of degradation after 19 days of assay, respectively. Some of these intermediate compounds were identified by LCMS/MS in selected degradation samples. Most of the intermediates were already described as biodegradation and/or photodegradationproducts in different conditions, but new and/or unknown metabolites were also present.