Maria Elizabeth Tiritan

Spatiotemporal distribution of pharmaceuticals in the Douro River estuary (Portugal)

The amount and distribution of six pharmaceutical compounds belonging to distinct therapeutic classes were investigated along the navigation channel of the Douro River estuary. Distinct spatial and temporal trends were considered and a total of 87 water samples were pre-concentrated by solid-phase extraction (SPE) and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with an ion trap (IT) analyzer and electrospray ionization (ESI). The maximum concentrations found were 178ng/L for carbamazepine, 3.65ng/L for diazepam, 70.3ng/L for fenofibric acid, 3.18ng/L for propranolol, 15.7ng/L for trimethoprim and 53.3ng/L for sulfamethoxazole. Carbamazepine was the most ubiquitous compound with 100% positive detection frequency followed by propranolol (38%), trimethoprim (34%) and sulfamethoxazole (33%). The pharmaceutical compounds were quantified at higher levels in the lower stretch of the estuary, especially near the wastewater treatment plant (WWTP). The data proves that pollution of the Douro River estuary by pharmaceuticals is consistent and is occurring in a fairly constant manner in time, covering a wide area and displaying hot-spots. Individually, the concentration levels are not likely to cause acute effects, based on reference experimental data. However, the fact that complex mixtures exist gives cause for concern as regards potentially relevant toxicological risks. The study points out the need for continuous monitoring of contamination levels not only in the Douro River estuary but also in other major estuaries. Finally, the scenario supports the need for experimental studies on toxicological impacts on aquatic organisms at environmentally relevant concentrations.

Enantioseparation of chiral pharmaceuticals in biomedical and environmental analyses by liquid chromatography: an overview.

This review aims to present the issues associated to enantioseparation of chiral pharmaceuticals in biological and environmental matrices using chiral stationary phases (CSP). Thus, it related some enantioselective methods in liquid chromatography (LC) and compares the importance given to chiral separation in biomedical and environmental fields. For that the most used CSP, the enantioselective chromatographic methods, their advantages and drawbacks were swiftly revised and compared. The recent advances and the limitations of chiral analytical methods in LC were also discussed.

New Trends in Sample Preparation Techniques for Environmental Analysis

Environmental samples include a wide variety of complex matrices, with low concentrations of analytes and presence of several interferences. Sample preparation is a critical step and the main source of uncertainties in the analysis of environmental samples, and it is usually laborious, high cost, time consuming, and polluting. In this context, there is increasing interest in developing faster, cost-effective, and environmentally friendly sample preparation techniques. Recently, new methods have been developed and optimized in order to miniaturize extraction steps, to reduce solvent consumption or become solventless, and to automate systems. This review attempts to present an overview of the fundamentals, procedure, and application of the most recently developed sample preparation techniques for the extraction, cleanup, and concentration of organic pollutants from environmental samples. These techniques include: solid phase microextraction, on-line solid phase extraction, microextraction by packed sorbent, dispersive liquid-liquid microextraction, and QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe).

Pharmaceutical trace analysis in aqueous environmental matrices by liquid chromatography–ion trap tandem mass spectrometry

An analytical method based on solid-phase extraction followed by liquid chromatography tandem mass spectrometry with an ion trap analyser was developed and validated for the quantification of a series of pharmaceutical compounds with distinct physical-chemical characteristics in estuarine water samples. Method detection limits were between 0.03 and 16.4 ng/L. The sensitivity and the accuracy obtained associated with the inherent confirmatory potential of ion trap tandem mass spectrometry (IT-MS/MS) validates its success as an environmental analysis tool. Two MS/MS transitions were used to confirm compound identity. Almost all pharmaceuticals were detected at ng/L level in at least one sampling site of the Douro River estuary, Portugal.

Degradation of fluoroquinolone antibiotics and identification of metabolites/transformation products by liquid chromatography–tandem mass spectrometry

Antibiotics are a therapeutic class widely found in environmental matrices and extensively studied due to its persistence and implications for multi-resistant bacteria development. This work presents an integrated approach of analytical multi-techniques on assessing biodegradation of fluorinated antibiotics at a laboratory-scale microcosmos to follow removal and formation of intermediate compounds. 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 LC with fluorescence detection (LC-FD) using a Luna Pentafluorophenyl (2) 3 μm column. The validated method demonstrated good selectivity, linearity (r(2)>0.999), intra-day and inter-day precisions (RSD<2.74%) and accuracy. The quantification limits were 5 μg L(-1) for OFL, NOR and CPF and 20 μg L(-1) for MOX. The optimized conditions allowed picturing metabolites/transformation products formation and accumulation during the process, stating an incomplete mineralization, also shown by fluoride release. OFL and MOX presented the highest (98.3%) and the lowest (80.5%) extent of degradation after 19 days of assay, respectively. A representative number of samples was selected and analyzed by LC-MS/MS with triple quadrupole and the molecular formulas were confirmed by a quadruple time of flight analyzer (QqTOF). Most of the intermediates were already described as biodegradation and/or photodegradation products in different conditions; however unknown metabolites were also identified. The microbial consortium, even when exposed to high levels of FQ, presented high percentages of degradation, never reported before for these compounds.

Chiral pharmaceuticals in the environment

Many pharmaceutical pollutants are chiral, existing in the environment as a single enantiomer or as mixtures of the two enantiomers. In spite of their similar physical and chemical properties, the different spatial configurations lead the enantiomers to have different interactions with enzymes, receptors or other chiral molecules, which can give diverse biological response. Consequently, biodegradation process and ecotoxicity tend to be enantioselective. Despite numerous ongoing research regarding analysis and monitorization of pharmaceutical ingredients in the environment, the fate and effects of single enantiomers of chiral pharmaceuticals (CP) in the environment are still largely unknown. There are only few chiral analytical methods to accurately measure the enantiomeric fraction (EF) in environmental matrices and during biodegradation processes. Furthermore, the ecotoxicity studies usually consider the enantiomeric pair as unique compound. We reviewed the current knowledge about CP in the environment, as well as the chiral analytical methods to determine the EF in environmental matrices. The degradation and removal processes of CP of important therapeutic classes, usually detected in the environment, and their toxicity to aquatic organisms were also reviewed. On the other hand, this review demonstrate that despite the great importance of the stereochemistry in pharmaceutical science, pharmacology and organic chemistry, this is normally neglected in environmental studies. Therefore, CP in the environment need much more attention from the scientific community, and more research within this subject is required.

Enantioselective biodegradation of pharmaceuticals, alprenolol and propranolol, by an activated sludge inoculum.

Biodegradation of chiral pharmaceuticals in the environment can be enantioselective. Thus quantification of enantiomeric fractions during the biodegradation process is crucial for assessing the fate of chiral pollutants. This work presents the biodegradation of alprenolol and propranolol using an activated sludge inoculum, monitored by a validated enantioselective HPLC method with fluorescence detection. The enantioseparation was optimized using a vancomycin-based chiral stationary phase under polar ionic mode. The method was validated using a minimal salts medium inoculated with activated sludge as matrix. The method was selective and linear in the range of 10-800 ng/ml, with a R²>0.99. The accuracy ranged from 85.0 percent to 103 percent, the recovery ranged from 79.9 percent to 103 percent, and the precision measured by the relative standard deviation (RSD) was <7.18 percent for intra-batch and <5.39 percent for inter-batch assays. The limits of quantification and detection for all enantiomers were 10 ng/ml and 2.5 ng/ml, respectively. The method was successfully applied to follow the biodegradation of the target pharmaceuticals using an activated sludge inoculum during a fifteen days assay. The results indicated slightly higher biodegradation rates for the S-enantiomeric forms of both beta-blockers. The presence of another carbon source maintained the enantioselective degradation pattern while enhancing biodegradation extent up to fourteen percent.

A column-switching method for quantification of the enantiomers of omeprazole in native matrices of waste and estuarine water samples.

This work reports the use of a two-dimensional liquid chromatography (2D-LC) system for quantification of the enantiomers of omeprazole in distinct native aqueous matrices. An octyl restricted-access media bovine serum albumin column (RAM-BSA C(8)) was used in the first dimension, while a polysaccharide-based chiral column was used in the second dimension with either ultraviolet (UV-vis) or ion-trap tandem mass spectrometry (IT-MS/MS) detection. An in-line configuration was employed to assess the exclusion capacity of the RAM-BSA columns to humic substances. The excluded macromolecules had a molecular mass in the order of 18 kDa. Good selectivity, extraction efficiency, accuracy, and precision were achieved employing a very small amount (500 microL or 1.00 mL) of native water sample per injection, with detection limits of 5.00 microg L(-1), using UV-vis, and 0.0250 microg L(-1), using IT-MS/MS. The total analysis time was only 35 min, with no time spent on sample preparation. The methods were successfully applied to analyze a series of waste and estuarine water samples. The enantiomers were detected in an estuarine water sample collected from the Douro River estuary (Portugal) and in an influent sample from the wastewater treatment plant (WWTP) of São Carlos (Brazil). As far as we are concerned, this is the first report of the occurrence of (+)-omeprazole and (-)-omeprazole in native aqueous matrices.

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

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 (r(2)>0.99), selectivity and sensitivity were achieved in the range of 20-400 ngL(-1) for all enantiomers, except for norfluoxetine enantiomers which range covered 30-400 ngL(-1). The method detection limits were between 0.65 and 11.5 ngL(-1) and the method quantification limits were between 1.98 and 19.7 ngL(-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.

Enantioselective quantification of fluoxetine and norfluoxetine by HPLC in wastewater effluents

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 (R(2)>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.