Browsing by Author "Afonso, Carlos"
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- Avaliação da utilização em rações da biomassa algal crescida em efluente de uma industria cervejeiraPublication . Raposo, M. F. J; Oliveira, S.; Castro, Paula M. L.; Bandarra, N. M.; Monteiro, M.; Afonso, Carlos; Morais, R. M.
- Biodegradation of Beta-Blockers and Fluoxetine followed by a Chiral HPLC-FDPublication . Ribeiro, Ana R.; Castro, Paula M. L.; Afonso, Carlos; Tiritan, Maria E.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.
- Biodegradation of chiral pharmaceuticals by an activated sludge consortium followed by a Chiral HPLC-FDPublication . Ribeiro, Ana R.; Castro, P. M. L.; Afonso, Carlos; Tiritan, MariaBiodegradation tends to be enantioselective in contrast to abiotic degradation and it is necessary enantioselective analytical methods to quantify the enantiomeric fraction of chiral pharmaceuticals in the environment for correct risk assessment. In this work, we developed HPLC-FD methods to follow the biodegradation of four beta-blockers: alprenolol , propranolol , metoprolol and atenolol and the antidepressant fluoxetine during 15 days in batch mode. The biodegradation assays were performed using AS from the aerated tanks of a municipal wastewater treatment plant with a singly compound supplementation and a mixture compound supplementation similar to those found in wastewater influents. Abiotic degradation in the presence of light and in the dark was evaluated. Either the low concentration or the mixture effects are situations closer to those found in the environment. The results indicate the higher degradation extents for the S-enantiomer forms, as is shown in Figure 1.
- Chemical reactivity of paraquat with the previously validated antidote, sodium salicylatePublication . Dinis-Oliveira, Ricardo; Pinho, Paula Guedes de; Ferreira, António César Silva; Silva, Artur; Afonso, Carlos; Bastos, Maria de Lourdes; Remião, Fernando; Duarte, José Alberto
- Chiral HPLC-FD method validation for determination of several Beta-Blockers and Fluoxetine in biodegradation assaysPublication . Ribeiro, Ana R.; Castro, Paula M.L.; Afonso, Carlos; Tiritan, Maria E.Chiral pharmaceuticals and the fate and effects of their enantiomers in the environment are still largely unknown [1, 2]. Enantiomers have different interactions with enzymes, receptors and any chiral molecules leading to different biological activities and affecting organisms in a different manner. Thus, biodegradation tends to be enantioselective in contrast to abiotic degradation. The methods developed to quantify the enantiomeric fraction in the environment and to follow biodegradation are scarce [3]. Thus in this work we describe the development and validation of HPLC methods that allow the enantiomeric separation of widely used drugs namely four beta-blockers: alprenolol (ALP), propranolol (PHO), metoprolol (MET) and atenolol (ATE) and the antidepressant fluoxetine (FX). The macrocyclic antibiotic vancomycin CSP (ASTEC Chirobiotic V 5µm) was used under polar organic mode (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 a matrix which is the condition used in the biodegradation studies. The vancomycin CSP was able to resolve ALP and PHO as well as MET, ATE and FX in two chromatographic runs. The chromatographic parameters obtained have shown the separation factor (α) between 1.12 and 1.34 and resolution (Rs) between 1.30 and 4.35. The methods demonstrated to be selective and linearity with r2 higher than 0,999 for the range selected. The method detection limits were between 2.5 to 10ng/mL. These methods were applied to follow the biodegradation of the target chiral compounds during 15 days. The biodegradation assays were performed using activated sludge from a WWTP and the results indicate the higher degradation extents for the S- enantiomer forms.
- Enantiomeric separation of tramadol and Its metabolites: method validation and application to environmental samplesPublication . Silva, Cátia; Ribeiro, Cláudia; Maia, Alexandra S.; Gonçalves, Virgínia; Tiritan, Maria Elizabeth; Afonso, CarlosThe accurate assessment of racemic pharmaceuticals requires enantioselective analytical methods. This study presents the development and validation of an enantioselective liquid chromatography with a fluorescence detection method for the concomitant quantification of the enantiomers of tramadol and their metabolites, N-desmethyltramadol and O-desmethyltramadol, in wastewater samples. Optimized conditions were achieved using a Lux Cellulose-4 column 150 × 4.6 mm, 3 μm isocratic elution, and 0.1% diethylamine in hexane and ethanol (96:4, v/v) at 0.7 mL min-1. The samples were extracted using 150 mg Oasis® mixed-mode cation exchange (MCX) cartridges. The method was validated using a synthetic effluent of a laboratory-scale aerobic granular sludge sequencing batch reactor. The method demonstrated to be selective, accurate, and linear (r2 > 0.99) over the range of 56 ng L-1 to 392 ng L-1. The detection and the quantification limits of each enantiomer were 8 ng L-1 and 28 ng L-1 for tramadol and N-desmethyltramadol, and 20 ng L-1 and 56 ng L-1 for O-desmethyltramadol. The feasibility of the method was demonstrated in a screening study in influent and effluent samples from a wastewater treatment plant. The results demonstrated the occurrence of tramadol enantiomers up to 325.1 ng L-1 and 357.9 ng L-1, in the effluent and influent samples, respectively. Both metabolites were detected in influents and effluents.
- 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 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
- Fármacos quirais em diferentes matrizes ambientais: ocorrência, remoção e toxicidadePublication . Ribeiro, Ana R.; Afonso, Carlos; Castro, Paula M. L.; Tiritan, Maria E.In recent decades, the occurrence of pharmaceuticals in the environment has been widely reported due to their high frequency and recalcitrance in many cases. Concerning the chiral pharmaceuticals (CPs) in environmental matrices, the stereochemistry is often neglected and enantiomers are determined together as unique molecules. However, it is well known that CPs might have enantioselective toxicity, rendering important to assess the occurrence and degradation processes of single enantiomers in the environment, namely during biological treatment in wastewater treatment plants (WWTPs). The development of analytical methods to qualitatively and quantitatively evaluate the enantiomers of CPs is crucial for determining enantiomeric fraction (EF). The EF is the most important parameter in studies involving enantiomers and enantioselective processes and fundamental in biodegradation studies and wastewater monitoring. This review summarizes the analytical methods used to determine EF of CPs in environmental matrices and/or during biodegradation processes. The occurrence of CPs in the environment and their biodegradation are reviewed and future trends in the area outlined.
- Pé diabéticoPublication . Afonso, Carlos; Carvalho, P.; Xavier, A.; Dias, Vanessa; Alves, Paulo