Application of NIR Spectroscopy for the Valorisation of Cork By-Products: A Feasibility Study over the Screening and Discrimination of Chemical Compounds of Interest.

Quercus suber is considered a sustainable tree mainly due to its outer layer (cork) capacity to regenerate after each harvesting cycle. Cork bark is explored for several application; however, its industrial transformation generates a significant amount of waste. Recently, cork by-products have been studied as a supplier of bioactive ingredients. This work aimed to explore whether near infrared spectroscopy (NIRS), a non-destructive analysis, can be employed as a screening device for selecting cork by-products with higher potential for bioactives extraction. A total of 29 samples of cork extracts were analysed regarding their qualitative composition. Partial least squares (PLS) models were developed for quantification purposes, and R2P and RER values of 0.65 and above 4, respectively, were obtained. Discrimination models, performed through PLS-DA, yielded around 80% correct predictions, revealing that four out of five of samples were correctly discriminated, thus revealing that NIR can be successfully applied for screening purposes.

Microfluidic mixing system for precise PLGA-PEG nanoparticles size control.

In this study, a microfluidic device was employed to produce polymeric nanoparticles (NPs) with well-controlled sizes. The influence of several parameters in the synthesis process, namely, polymer concentration, flow rate and flow rate ratio between the aqueous and organic solutions was investigated. To evaluate the NPs size effect, three diameters were selected (30, 50 and 70 nm). Their cytocompatibility was demonstrated on endothelial cells and macrophages. Additionally, their efficacy to act as drug carriers was assessed in an in vitro inflammatory scenario. NPs loaded and released diclofenac (DCF) in a size-dependent profile (smaller sizes presented lower DCF content and higher release rate). Moreover, 30 nm NPs were the most effective in reducing prostaglandin E2 concentration. Therefore, this study demonstrates that microfluidics can generate stable NPs with controlled sizes, high monodispersity and enhanced batch-to-batch reproducibility. Indeed, NPs size is a crucial parameter for drug encapsulation, release and overall biological efficacy.

Chiral Separations in Preparative Scale: A Medicinal Chemistry Point of View.

Enantiomeric separation is a key step in the development of a new chiral drug. Preparative liquid chromatography (LC) continues to be the technique of choice either during the drug discovery process, to achieve a few milligrams, or to a scale-up during the clinical trial, needing kilograms of material. However, in the last few years, instrumental and technical developments allowed an exponential increase of preparative enantioseparation using other techniques. Besides LC, supercritical fluid chromatography (SFC) and counter-current chromatography (CCC) have aroused interest for preparative chiral separation. This overview will highlight the importance to scale-up chiral separations in Medicinal Chemistry, especially in the early stages of the pipeline of drugs discovery and development. Few examples within different methodologies will be selected, emphasizing the trends in chiral preparative separation. The advantages and drawbacks will be critically discussed.

Monitoring of the 17 EU Watch List contaminants of emerging concern in the Ave and the Sousa Rivers.

The occurrence of micropollutants in the environment is a matter of high concern. Some regulations have been published in the last years and a Watch List of contaminants of emerging concern (CECs) for European Union monitoring of surface water was launched in the Decision 2015/495, including three estrogens (estrone, E1; 17-ß-estradiol, E2; and 17-α-ethinylestradiol, EE2), four pharmaceuticals (diclofenac and the macrolide antibiotics azithromycin, clarithromycin and erythromycin), an anti-oxidant (2,6-ditert-butyl-4-methylphenol, BHT), an UV filter (2-ethylhexyl 4-methoxycinnamate, EHMC), some pesticides (methiocarb and the neonicotinoids imidacloprid, thiacloprid, thiamethoxam, clothianidin and acetamiprid) and two herbicides (oxadiazon and triallate). This study provides the first spatial and seasonal monitoring campaign in the Ave and the Sousa Rivers for the all set of the 17 Watch List CECs (not reported yet for any country), in order to assess their occurrence, distribution, frequency and risk assessment. It also highlights the need of extend the study to other regions and environmental matrices to investigate the occurrence and possible sources of contamination of CECs, aiming to give insights for decision makers to define mitigation strategies for a more sustainable water policy.

Enantiomeric ratios: Why so many notations?

The correct quantification of enantiomers is pivotal in a variety of fields, such as pharmacokinetic studies, enantioselective syntheses, chemical characterization of natural products, authentication of fragrance and food, biodegradation behavior, accurate evaluation of environmental risk, and it can also provide information for sentencing guidance in forensic field. Enantioselective chromatography is the first choice to assess the composition of an enantiomeric mixture. Different notations have been used to express the measured enantiomeric ratios, which compromise the results and represent a challenge for data comparison. This manuscript critically discusses the currently used notations and exemplifies with applications in different fields indicating the advantages and disadvantages of one of the adopted systems. In order to simplify the notations, the use of enantiomeric ratio (e.r.%) as standardization for nonchiroptical methods is proposed.

Lipophilicity assessement in drug discovery: Experimental and theoretical methods applied to xanthone derivatives.

For the last several years, searching of new xanthone derivatives (XDs) with potential pharmacological activities has remained one of the main areas of interest of our group. The optimization of biological activity and drug-like properties of hits and leads is crucial at early stage of the drug discovery pipeline. Lipophilicity is one of the most important drug-like properties having a great impact in both pharmacokinetics and pharmacodynamics processes. In this work, we describe the lipophilicity of a small library of bioactive XDs, previously synthesized by our group, using different methods: computational, vortex-assisted liquid-liquid microextraction coupled with high-performance liquid chromatography (VALLME-HPLC), reversed-phase high-performance thin layer chromatography (RP-HPTLC), reversed-phase high-performance liquid chromatography (RP-HPLC), and biomembrane model by the partition between micelles and aqueous phase. The different results obtained by the used methods were compared and discussed. The methodologies and data gathered in this study will expand the investigation of lipophilicity of XDs, an important class of compounds in medicinal chemistry.

Quantification of alprenolol and propranolol in human plasma using a two-dimensional liquid chromatography (2D-LC).

A new two-dimensional liquid chromatography (2D-LC) method using a column switching valve, with a restricted-access media (RAM) column in the first dimension was developed and validated for the quantification of two ß-blockers in human plasma. Several parameters, such as sample collection, mobile phase composition and flow rate for sample cleanup, transference and analytical separation of the ß-blockers were investigated and optimized. The developed method allowed for the simultaneous pre-treatment and quantification of alprenolol (ALP) and propranolol (PRO) in human plasma in less than 25min. The method consisted in the injection of 100µL of plasma samples on the RAM alkyl-diol-silica column (Lichrospher® RP-18 ADS, 25µm) with water/acetonitrile (98:2, v/v; at a flow rate of 2.0mL/min) and then transferred (via a six-port valve) to the analytical column (Luna PFP (2), 150×4.6mm ID, 100Å, 3µm) with 0.1% (v/v) triethylamine in water acidified with trifluoroacetic acid (pH=3)/acetonitrile (74:26, v/v) at a flow rate of 0.6mL/min in a back-flush mode. The column oven temperature was optimized to 42°C and the fluorescence detector set at 280nm and 310nm (excitation and emission, respectively). The method was validated according to the European Medicines Agency's guidelines and was linear (r2>0.999) over a dynamic range of 5.0 - 200ng/mL. Recoveries ranged from 90.2 and 107% and the lower limit of quantification was 5.0ng/mL for both compounds. Precision was expressed as a percentage of relative standard deviation and did not exceed 11%. Finally, the method was successfully applied to determine the plasma concentration of PRO in four healthy volunteers.

Resolution, determination of enantiomeric purity and chiral recognition mechanism of new xanthone derivatives on (S,S)-whelk-O1 stationary phase.

The enantioresolution and determination of the enantiomeric purity of 32 new xanthone derivatives, synthesized in enantiomerically pure form, were investigated on (S,S)-Whelk-O1 chiral stationary phase (CSP). Enantioselectivity and resolution (α and RS ) with values ranging from 1.41-6.25 and from 1.29-17.20, respectively, were achieved. The elution was in polar organic mode with acetonitrile/methanol (50:50 v/v) as mobile phase and, generally, the (R)-enantiomer was the first to elute. The enantiomeric excess (ee) for all synthesized xanthone derivatives was higher than 99%. All the enantiomeric pairs were enantioseparated, even those without an aromatic moiety linked to the stereogenic center. Computational studies for molecular docking were carried out to perform a qualitative analysis of the enantioresolution and to explore the chiral recognition mechanisms. The in silico results were consistent with the chromatographic parameters and elution orders. The interactions between the CSP and the xanthone derivatives involved in the chromatographic enantioseparation were elucidated.

Anthropogenic pressure in a Portuguese river: Endocrine-disrupting compounds, trace elements and nutrients.

Natural organic compounds such as phytoestrogens and phytosterols found in various plants, as well as mycotoxins produced by fungi, can be found in aquatic environments. The aim of this study was to investigate the occurrence of three different classes of natural estrogenic compounds, i.e., phytoestrogens, phytosterols and mycotoxins, in estuarine water samples from the Ave River estuary. For that, water samples were collected at five sampling points distributed along the estuary at low tide, during 1 year, processed by solid-phase extraction (SPE) and analyzed by gas chromatography coupled to mass spectrometry (GC-MS). To correlate the presence of phytoestrogens and phytosterols in the estuarine water, local flora was collected on riverside. Trace elements content and physicochemical parameters such as nutrients and dissolved oxygen were also determined seasonally at each sampling point, to give insights for the evaluation of water quality and anthropogenic pressure. Both phytoestrogens and phytosterols showed a seasonal variation, with the highest values observed in spring and summer and the lowest in winter. Daidzein (DAID) was found up to 404.0 ng L(-1) in spring and coumestrol (COUM) was found up to 165.0 ng L(-1) in summer. The mycotoxin deoxynivalenol (DON) was ubiquitously determined with values ranging from 59.5 to 642.4 ng L(-1). Nutrients and metals distribution and concentration varied among sampling stations and seasons. This study revealed for the first time the presence of mycotoxins, various classes of phytoestrogens and stigmasterol (STG) in estuarine water from the Ave River (Portugal), and the evaluation of the water quality confirmed that this estuary is still highly impacted by anthropogenic activities.

Removal of fluoxetine and its effects in the performance of an aerobic granular sludge sequential batch reactor.

Fluoxetine (FLX) is a chiral fluorinated pharmaceutical mainly indicated for treatment of depression and is one of the most distributed drugs. There is a clear evidence of environmental contamination with this drug. Aerobic granular sludge sequencing batch reactors constitute a promising technology for wastewater treatment; however the removal of carbon and nutrients can be affected by micropollutants. In this study, the fate and effect of FLX on reactor performance and on microbial population were investigated. FLX adsorption/desorption to the aerobic granules was observed. FLX shock loads (≤4µM) did not show a significant effect on the COD removal. Ammonium removal efficiency decreased in the beginning of first shock load, but after 20 days, ammonia oxidizing bacteria became adapted. The nitrite concentration in the effluent was practically null indicating that nitrite oxidizing bacteria was not inhibited, whereas, nitrate was accumulated in the effluent, indicating that denitrification was affected. Phosphate removal was affected at the beginning showing a gradual adaptation, and the effluent concentration was <0.04mM after 70 days. A shift in microbial community occurred probably due to FLX exposure, which induced adaptation/restructuration of the microbial population. This contributed to the robustness of the reactor, which was able to adapt to the FLX load.

Enantioselective biodegradation of fluoxetine by the bacterial strain Labrys portucalensis F11.

Fluoxetine (FLX) is a chiral fluorinated pharmaceutical indicated mainly for the treatment of depression and is one of the most dispensed drugs in the world. There is clear evidence of environmental contamination with this drug and its active metabolite norfluoxetine (NFLX). In this study the enantioselective biodegradation of racemic FLX and of its enantiomers by Labrys portucalensis strain F11 was assessed. When 2µM of racemic FLX was supplemented as sole carbon source, complete removal of both enantiomers, with stoichiometric liberation of fluoride, was achieved in 30d. For racemic FLX concentration of 4 and 9µM, partial degradation of the enantiomers was obtained. In the presence of acetate as an additional carbon source, at 4, 9 and 21µM of racemic FLX and at 25µM of racemic FLX, (S)-FLX or (R)-FLX, complete degradation of the two enantiomers occurred. At higher concentrations of 45 and 89µM of racemic FLX, partial degradation was achieved. Preferential degradation of the (R)-enantiomer was observed in all experiments. To our knowledge, this is the first time that enantioselective biodegradation of FLX by a single bacterium is reported.

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.

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.

Biodegradation of ofloxacin, norfloxacin, and ciprofloxacin as single and mixed substrates by Labrys portucalensis F11.

Fluoroquinolone (FQ) antibiotics are extensively used both in human and veterinary medicine, and their accumulation in the environment is causing an increasing concern. In this study, the biodegradation of the three most worldwide used FQs, namely ofloxacin, norfloxacin, and ciprofloxacin, by the fluoroorganic-degrading strain Labrys portucalensis F11 was assessed. Degradation occurred when the FQs were supplied individually or as mixture in the culture medium, in the presence of an easily degradable carbon source. Consumption of individual FQs was achieved at different extents depending on its initial concentration, ranging from 0.8 to 30 µM. For the lowest concentration, total uptake of each FQ was observed but stoichiometric fluoride release was not achieved. Intermediate compounds were detected and identified by LC-MS/MS with a quadrupole time of flight detector analyzer. Biotransformation of FQs by L. portucalensis mainly occurred through a cleavage of the piperazine ring and displacement of the fluorine substituent allowing the formation of intermediates with less antibacterial potency. FQ-degrading microorganisms could be useful for application in bioaugmentation processes towards more efficient removal of contaminants in wastewater treatment plants.

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.

Microbial degradation of pharmaceuticals followed by a simple HPLC-DAD method.

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.

Microbial degradation of 17beta -estradiol and 17alpha -ethinylestradiol followed by a validated HPLC-DAD method.

This work aimed at studying the biodegradation of two estrogens, 17alpha -estradiol (E2) and 17beta -ethinylestradiol (EE2), and their potential metabolism to estrone (E1) by microbial consortia. The biodegradation studies were followed by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) using a specifically developed and validated method. Biodegradation studies of the estrogens (E2 and EE2) were carried out with activated sludge (consortium A, CA) obtained from a Wastewater Treatment Plant (WWTP) and with a microbial consortium able to degrade recalcitrant compounds, namely fluorobenzene (consortium B, CB). E2 was more extensively degraded than EE2 by CA whereas CB was only able to degrade E2. The addition of acetate as a supplementary carbon source led to a faster biodegradation of E2 and EE2. E1 was detected as a metabolite only during the degradation of E2. The 16S rRNA gene sequence analyses of strains recovered from the degrading cultures revealed the presence of the genera Pseudomonas, Chryseobacterium and Alcaligenes. The genera Pseudomonas and Chryseobacterium were retrieved from cultures supplied with E2 and EE2, while the genus Alcaligenes was found in the presence of E2, suggesting that they might be involved in the degradation of these compounds.