Browsing by Author "Ribeiro, Ana Rita Lado Teixeira"
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- Chiral pharmaceuticals in the environment : enantiomeric fraction and biodegradation studiesPublication . Ribeiro, Ana Rita Lado Teixeira; Castro, Paula Maria Lima; Tiritan, Maria Elizabeth; Afonso, Carlos Manuel MagalhãesThe importance of the stereochemistry in medicinal chemistry and in pharmacology is well recognized and the diverse behaviour of enantiomers in the presence of chiral entities is fully documented. Therefore biodegradation in biotic media, as occurs in secondary treatment of wastewater treatment plants (WWTPs) can be enantioselective. The significance of chirality in the environment is comparable to the magnitude in medicinal chemistry and pharmacology since enantiomers of chiral contaminants can significantly differ in environmental fate as well as in effects. The need to evaluate the occurrence and biodegradation processes of single enantiomers of chiral pharmaceuticals (CPs) in the environment is imperative by enantiomeric fraction (EF) monitoring of biodegradation processes, wastewaters and surface waters. Assessing EF is only possible through enantioselective validated methods, which are currently scarce. The work described in this thesis comprises the development of new enantioselective analytical methodologies to accurately measure the EFs of CPs in environmental matrices and/or during biodegradation processes, especially for application to monitoring studies and designed biodegradation. The enantioselective analytical methods developed allowed the studies of biodegradation of several CPs in mineral growth medium (MM) inoculated with activated sludge; with the bacterial strain Labrys portucalensis F11; and in wastewater effluents. Additionally an enantioselective method for EFs quantification of multi-residue CPs was developed to monitor effluents of WWTPs and laboratory-scale bioreactors. Thus, this thesis allows increasing the knowledge on EF variation during biodegradation studies and also in environmental matrices. A vancomycin-based chiral stationary phase ChirobioticTM V column (4.6 mm internal diameter (i.d.)) was used to develop and validate two enantioselective analytical methods by High Performance Liquid Chromatography with Fluorescence Detector (HPLC-FD), allowing the analysis of enantiomers of alprenolol (ALP), atenolol (ATE), metoprolol (MET), propranolol (PHO) and fluoxetine (FLX) in biodegradation assays using MM inoculated with activated sludge. Biodegradation of the racemic mixtures was assessed in MM inoculated with activated sludge. The developed HPLC-FD methods under polar ionic mode had several advantages such as a simple sample preparation method; short chromatographic run; low flow rate; no need of solvents for sample preparation or derivatization and the use of an easy to operate, selective and sensitive detector. Biodegradation by activated sludge had slightly higher rates for the (S)-enantiomeric forms of ALP, PHO and MET, with the enhanced biodegradation by acetate as another growth substrate. Biodegradation of ATE and FLX was non-enantioselective. The effect of simultaneous supplementation decreased the biodegradation rates of all enantiomers perhaps due to a competitive mechanism. An enantioselective analytical method by HPLC-FD was optimized and validated using the ChirobioticTM V column (4.6 mm i.d.) to analyse individual enantiomers of FLX and the metabolite norfluoxetine (NFLX). This method preconcetrated 50 mL of wastewater samples through solid phase extraction (SPE) with Oasis® Mixed mode Cation eXchange (MCX) cartridges and allowed the simultaneous analysis of FLX and NFLX enantiomers under reversed mode using an eco-friendly mobile phase constituted by ethanol and ammonium acetate buffer. The method demonstrated to be easy to operate, selective, sensitive and easy to adapt to other matrices. Biodegradation of racemic FLX in microcosms mimicking aquatic environments (WWTP effluents) was followed by the validated method during 46 days with no observation of enantioselectivity in degradation process. The biodegradation of each enantiomer of FLX separately was also evaluated in MM inoculated by the bacterial strain Labrys portucalensis F11. Degradation of the two enantiomers with preferential degradation of the (R)-enantiomer was observed, with no observation of enantiomerization during the monitorization. The metabolite NFLX was not detected during degradation. An enantioselective SPE-HPLC with tandem Mass Spectrometry method was developed with a triple quadrupole mass analyser using the ChirobioticTM V column with 2.1 mm i.d. in reversed mode to quantify enantiomers of seven CPs belonging to different therapeutic classes (ALP, bisoprolol (BSP), MET, PHO, FLX, venlafaxine (VNF) and salbutamol (SBT)) and one metabolite (NFLX). This method used effluent of a laboratory-scale bioreactor for matrix validation and Oasis® MCX cartridges to preconcentrate 250 mL of water samples, being useful for monitoring wastewater effluents and also effluents of laboratory-scale bioreactors. The limits of detection were between 0.65 and 11.5 ng L-1. The use of a selective and sensitive detector allowed the unequivocal identification and quantification of the target enantiomers in complex matrices. The effluents of three municipal WWTPs in Portugal were analysed. Enantiomers of FLX, VNF, BSP, MET and PHO were found. VNF and FLX were found with EFs different from 0.5.