Biodegradation of Beta-Blockers and Fluoxetine followed by a Chiral HPLC-FD

Despite of the massive publications concerning pharmaceuticals in the environment, the problematic related with chiral compounds and enantioselective degradation are still largely unknown [1]. Enantiomers have different interactions with enzymes, receptors and other chiral molecules, leading to different biological activities. Thus, biodegradation tends to be enantioselective in contrast to abiotic degradation. However, biodegradation studies regarding enantioselectivity on the process are scarce [2].] MATERIALS AND METHODS [Four beta-blockers: alprenolol (ALP), propranolol (PHO), metoprolol (MET) and atenolol (ATE) and the antidepressant fluoxetine (FX) were enantiomerically separated by a macrocyclic antibiotic vancomycin CSP (ASTEC Chirobiotic V 5µm) under polar organic mode phase (methanol:ethanol:triethylamine:acetic acid.50:50 v/v) and fluorescence detection for enantiomeric fraction quantification. The developed methods were established using a minimal medium inoculated with activated sludge (AS) as a matrix.] RESULTS AND DISCUSSION [The Chirobiotic VTM was able to resolve ALP and PHO as well as MET, ATE and FX in two short runs. A separation factor (α) between 1.12 and 1.34 and resolution (Rs) between 1.30 and 4.35 were obtained. The methods demonstrated to be selective and linear within the range, with detection limits between 2.5 and 10ng/mL. These methods were applied to follow the biodegradation of the target compounds. The biodegradation assays were performed using AS from a municipal WWTP and the results indicate the higher degradation extents for the S- enantiomer forms at initial concentrations tenfold above those found in the environment (10ppm and 5ppm). The same assays were performed at an initial concentration of 1ppm for a singly supplementation and at 0,5ppm for a mixture of compounds, a closer situation to the real environment.] CONCLUSIONS [To our knowledge, chromatographic enantioseparations of the mixture of ALP and PHO and the mixture of MET, FX and ATE using the Chirobiotic™ V, have not been previously reported. The feasibility of this application was confirmed by two biodegradation studies using AS, with S-form being faster degraded, showing stereoselectivity

Solid Phase Extraction of Fluoroquinolone Antibiotics from Wastewaters – Assessment of Different Commercial Sorbents

Microbial degradation of fluorinated pharmaceuticals during wastewater treatment processes remains inadequate in most situations. Due to incomplete elimination, these residues are continually being introduced into the aquatic environments in which they settle throughout time since many of them are resistant to degradation. Fluoroquinolone antibiotics due to its persistence and implication on resistant-bacteria development, pose special interest in environmental analysis. Due to their zwitterionic characteristics, the extraction/pre-concentration process of fluoroquinolones prior analyses is an unquestionable challenge. This work compares the solid phase extraction efficiency of four fluoroquinolones (Ofloxacin, Norfloxacin, Ciprofloxacin and Moxifloxacin) from wastewater effluents by different commercial sorbents. Prior to wastewater analysis, preliminary tests were conducted in distilled water with a larger number of sorbents. Different experimental protocols and sorbents, namely OASIS® HLB, OASIS® WAX, OASIS® WCX (500 mg) and the molecularly imprinted polymer SupelMIP TM were applied to wastewater samples collected from a municipal wastewater treatment plant from the north of Portugal. The extracts were analyzed by a HPLC withFluorescence Detection validated method using a Luna PFP (2) 3µm column. Despite good results obtained with the molecularly imprinted polymer in distilled water, these cartridges did not perform efficiently when applied to wastewater effluents, probably due to the sample high complexity especially since their specific design for biological samples. Regarding OASIS® considered sorbents; HLB 500mg and WAX 500mg presented the best recovery rates of the fourstudied antibiotics, between 84-75% and 64-94%, respectively. Although the recoveries achievedwere not that dissimilar between the two mentioned sorbents, chromatograms of WAX extracts appear much cleaner in the antibiotics retention times while chromatograms of HLB extractsclearly show the presence of strong polar substances, probably matrix humic and fulvic acids,that behave as resilient interferences in the analysis, disturbing a proper identification of target compounds and reducing chromatographic resolution

Development and Optimization of an Online SPE-HPLC-FD Method for Quantification of Fluoroquinolones in Wastewater Effluents

Fluoroquinolones are antimicrobial agents widely found in environmental matrices and extensively studied due to their persistence and implications for multiresistant bacteria. The presence of fluoroquinolones in the environment is mainly due to the incapability of wastewater treatment plants (WWTPs) to completely remove those compounds. The amount of fluoroquinolones released through effluents depends on the type of treatment used by the WWTPs. So, accurate analytical methods to quantify those compounds on WWTPs process and in effluents are crucial. Solid phase extraction (SPE) coupled to liquid chromatography is a straightforward technique that provides analyte extraction, cleanup, separation and detection while providing a good reproducibility and efficiency. The purpose of this work was the establishment of a novel method for quantification of Ofloxacin, Norfloxacin, Ciprofloxacin and Moxifloxacin on WWTPs effluents using on-line SPE. Samples were injected directly on a restricted access material column LichroCart 25-4 Lichrospher® RP-18 ADS (25 μm) and then transferred to an analytical column Luna PFP (2) (150 x 4.6 mm ID, 100 Å, 3 μm) for separation in isocratic mode with a mixture of 0.1% triethylamine in water (acidified to pH = 2.2 with trifluoroacetic acid) and ethanol as mobile phase; column oven was set at 45ºC. The detection was performed by fluorescence with an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The injection volume of 100 μL of previous preconcentrated sample was compared with larger volume injection of only filtered effluent samples. The study was conducted with effluent samples collected from a municipal WWTP in the north of Portugal

Studies on enantioselective biodegradation of fluoxetine

Fluoxetine (FLX) is a chiral fluorinated pharmaceutical indicated mainly for treatment of depression and is one of the most dispensed drugs in the world. There is a clear evidence of environmental contamination with this drug. Granular sludge sequencing batch reactors (SBR) constitute a promising technology for the treatment of effluents containing micropollutants. In this study, a SBR was operated in order to assess its performance when treating a synthetic wastewater containing racemic FLX (rac-FLX), under continuous and intermittent feeding of the compound. The concentration of FLX enantiomers was followed using an enantioselective HPLC method. A removal of 70% of the total supplied FLX was observed in the first continuous feeding period. However, the subsequent feeding periods revealed a significant decrease in the FLX removal; FLX liberation occurred during periods when no compound was supplied. This can be probably explained by desorption of FLX previously adsorbed to the granules. No intermediate metabolites or fluoride release were detected, corroborating the hypothesis that adsorption of FLX to the aerobic granules occurred. Moreover, the absence of enantioselectivity in the decrease of FLX enantiomers concentration is also an indicator of an abiotic mechanism. In face of the incapacity of the aerobic granules to biodegrade FLX, the ability of Labrys portucalensis F11, a previously isolated microbial strain with the capacity to degrade a range of fluorinated aromatic compounds, to biodegrade this compound was investigated. In this study, the enantioselective biodegradation of rac-FLX and of its enantiomers was assessed. The results obtained revealed that this strain is able to degrade both enantiomers of FLX, when supplemented as a racemic mixture, as well as when supplemented as a single enantiomers. Preferential degradation of the (R)-enantiomer was observed. This feature makes L. portucalensis F11 a potential candidate for devising biodegradation technologies able to deal with contamination by this pharmaceutical. Acknowledgements: I.S. Moreira, A.R. Ribeiro and C.L. Amorim wish to acknowledge a research grant from Fundação para a Ciência e Tecnologia (FCT), Portugal (Ref. SFRH/BPD/87251/2012, SFRH/BD/64999/2009 and SFRH/BD/47109/2008 , 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, CEQUIMED-Pest-OE/SAU/UI4040/2011 and PEst-OE/EQB/LA0016/2011

Enantioselective Degradation of Enantiomers of Fluoxetine Followed by HPLC- FD

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 Determination of Fluoxetine and Norfluoxetine in Wastewater

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 wastewate

Strategies to enhance the removal of Fluoroquinolones

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