Quality by design optimization of a liquid chromatographic-tandem mass spectrometric method for the simultaneous analysis of structurally heterogeneous pharmaceutical compounds and its application to the rapid screening in wastewater and surface water samples by large volume direct injection.

This study focused on the Analytical Quality by Design (AQbD) optimization of the chromatographic separation and mass spectrometric detection of a wide group of structurally heterogeneous model pharmaceutical compounds (PhCs) and transformation products (TPs), chosen to cover the challenging issues of the co-presence of compounds characterized by (i) a wide range of physicochemical properties, (ii) the same mass transitions, and (iii) different ionisation modes. Italian consumption of PhCs were also considered as election criteria of target analytes. Octadecyl and pentafluorophenyl stationary phases, acetonitrile/methanol ratios and acidity of the eluents, column temperature, initial organic phase percentage, and elution gradient were investigated by AQbD, aiming at optimizing critical resolutions, sensitivities, and analysis time. Statistically significant models were obtained in most cases with fitting and cross-validation coefficients in the ranges of 0.681-0.998 and 0.514-0.967, respectively. After optimization, the analysis of target analytes was performed in a single chromatographic run, adopting a mixed acquisition mode based on scheduled acquisition windows comprising both single polarity and continuous polarity switching. For most investigated analytes the method provided detection limits in the sub-ng/L to low ng/L range, meeting for macrolides the sensitivity requested by the "Watch List" 2018/840/EU. The optimized method was applied to the direct injection analysis of PhCs and TPs in four wastewater treatment plant (WWTP) effluents and surface water (SW) samples collected in the receiving water bodies. Absolute values of matrix effect were found to be far higher than 20% for most target analytes in most samples. Seventeen PhCs and two TPs were quantified in at least one sample, at the wide concentration range of about 1-3200 ng/L. The most occurring PhCs in both WWTP effluents and SWs were levofloxacin (202-1239 and 100-830 ng/L), furosemide (865-3234 and 230-880 ng/L), ketoprofen (295-1104 and 270-490 ng/L), and ibuprofen (886-3232 and 690-1440 ng/L).

Liquid Chromatographic Quadrupole Time-of-Flight Mass Spectrometric Untargeted Profiling of (Poly)phenolic Compounds in Rubus idaeus L. and Rubus occidentalis L. Fruits and Their Comparative Evaluation.

This study provided a detailed profiling of the antioxidant and bioactive compounds occurring in three varieties of Rubus idaeus L. fruits ("Fall Gold", "Glen Ample" and "Tulameen") compared to Rubus occidentalis L. black raspberry ("Jewel" cultivar), adopting a comprehensive untargeted metabolomics approach developed with UHPLC analysis coupled with quadrupole/time-of-flight high resolution mass spectrometry, using the SWATH® acquisition protocol. The feature selection and annotation workflow, applied to the analysis of raspberry extracts in both polarities, allowed identifying 68 bioactive compounds mainly belonging to the classes of (poly)phenolic compounds. Interestingly, some of these identifications (e.g., ferulic acid glycosides and the ellagitannin-like nobotanin/malabathrin) represent the first report in raspberry fruits. Principal component analysis made possible highlighting the features more related to the expression of a genotype effect within the R. idaeus species or between the two raspberry species herein investigated. Overall, flavanols were the most discriminating features for the Fall Gold variety, whereas ellagitannins and flavonol glycosides represent more distinctive metabolic traits in Glen Ample and Tulameen fruits. Moreover, R. occidentalis Jewel variety was strongly characterized by the occurrence of anthocyanins, such as cyanidin, pelargonidin and delphinidin glycosides.

Vaccinium myrtillus L. extract and its native polyphenol-recombined mixture have anti-proliferative and pro-apoptotic effects on human prostate cancer cell lines.

Vaccinium myrtillus berry extract (VME) and a recombined standard mixture (RSM) of its main native phenolic compounds were investigated for cell growth inhibition and pro-apoptotic activity on hormone-dependent (LNCaP) and hormone-independent (PC3 and DU-145) prostate cancer (PCa) cell lines. Normal prostate epithelial cells (PrEC) were also studied in comparison. VME hindered anchorage-dependent PCa cell proliferation in a dose-dependent manner, that is, at 1/800 (v/v) dilution for LNCaP and PC3, and 1/100 (v/v) dilution for DU-145 (corresponding to 14.15 and 113.2 µg cyanidin-3-O-glucoside equivalents per ml of culture medium), respectively. VME had a growth inhibitory effect towards PrEC at the same dilution of DU-145 cells although the IC50 values indicated that PrEC are more resistant than PCa cell lines. VME also reduced the anchorage-independent growth of PCa cells. The study of the apoptotic profile (i.e., non-apoptotic, early apoptotic, late apoptotic and necrotic cells) evidenced that the apoptotic rate (early+late) was statistically higher in all three cell lines exposed to VME compared to control. Anchorage-dependent and anchorage-independent growth inhibition of RSM was very similar to that displayed by VME. Moreover, RSM exerted its growth inhibitory effect also under hypoxia, the latter representing a biological condition known to sustain PCa proliferation and aggressiveness.

Productivity and nutritional and nutraceutical value of strawberry fruits (Fragaria x ananassa Duch.) cultivated under irrigation with treated wastewaters.

BACKGROUND: Agriculture represents a productive sector typically characterized by a high water demand, whereas freshwater (FW) availability is a problem of increasing concern in the world and FW resources are becoming insufficient for sustaining agricultural irrigation. The reuse of treated wastewaters (TWWs) for crop irrigation could be an efficient tool for reducing water shortage. Hence, the present study evaluated the food quality of Fragaria x ananassa (cultivar Camarosa) fruits irrigated with four types of treated wastewaters (TWWs). Strawberries were analysed for yield, sucrose, fructose, glucose, total soluble polyphenols (TSP), total monomeric anthocyanins (TMA), and antiradical and antioxidant capacity. In addition, a targeted quantification of the most representative phenolic compounds of strawberry was performed. RESULTS: TWWs complied the Italian ministerial decree 185/2003 for wastewater reuse, with very few exceptions, mainly represented by chloride concentrations (258-643 mg L-1 versus a legal threshold of 250 mg L-1 ). The reuse of TWWs reduced fruit yield (10-26%) compared to irrigation with tap water as a control. Irrigation with TWWs gave also rise to the decrease of total sugars (14-26%), TSP (2-10%) and TMA (29-49%). Individual phenolic acids, flavonols and flavanols were quite stable in response to the irrigation with TWWs, whereas anthocyanidins decreased significantly. CONCLUSION: Although TWWs negatively affected fruit quality, the nutritional and nutraceutical parameters determined in the present study were in line with data previously reported for strawberries purchased in the market or cultivated in research orchards, thus suggesting that the use of TWWs does not prevent the fruit marketability. © 2020 Society of Chemical Industry.

Improving the efficiency of wastewater treatment plants: Bio-removal of heavy-metals and pharmaceuticals by Azolla filiculoides and Lemna minuta.

In this study, we investigated the removal of Fe(III), Cr(VI), Al(III), diclofenac, and levofloxacin from treated wastewater in the presence of the free-floating plants Azolla filiculoides and Lemna minuta, to understand whether these species can be effectively used in a surface flow constructed wetland as wastewater refining treatment. Fe and Al were selected owing to their wide use as coagulant agents in wastewater treatment plants for promoting clariflocculation processes, whilst Cr was chosen due to its common use in industry. Diclofenac and levofloxacin, two molecules belonging to the most widely used pharmaceutical classes in the world, were studied as representative anti-inflammatory drugs and antibiotics, respectively. The study was performed at laboratory scale, exposing the plants separately to each individual contaminant at the concentrations of 5 mg L-1 for the metals (i.e. 2.5-5 times higher than the European limits concerning discharge into surface water), and 1 µg L-1 for the pharmaceuticals (concentration levels commonly found in wastewater). Depending on the plant species and contaminant tested, the range of different effects observed included low toxicity (i.e. Cr, Fe and diclofenac in L. minuta) and even a stimulatory effect on plant growth (i.e. for A. filiculoides with Al and for L. minuta with Al and levofloxacin). Moreover, both species proved to be very effective in the removal of Fe, Al and levofloxacin, with A. filiculoides showing the best performance (removal efficiency of 92%, 96%, and 60%, respectively), whereas for Cr and diclofenac the removal was always less than 10%. The higher removal capacity of A. filiculoides compared to L. minuta can be attributed to its superior tolerance of the contaminants, probably in turn related to the presence of nitrogen-fixing microorganism in its fronds.

Optimization and validation of a method based on QuEChERS extraction and liquid chromatographic-tandem mass spectrometric analysis for the determination of perfluoroalkyl acids in strawberry and olive fruits, as model crops with different matrix characteristics.

A QuEChERS method was optimized and validated for the LC-MS/MS analysis of perfluoro-n-pentanoic acid (PFPeA), perfluoro-1-butanesulfonic acid (PFBuS), perfluoro-n-hexanoic acid (PFHxA), perfluoro-n-heptanoic acid (PFHpA), perfluoro-1-hexanesulfonic acid (PFHxS), perfluoro-n-octanoic acid (PFOA), perfluoro-n-nonanoic acid (PFNA), perfluoro-1-octanesulfonic acid (PFOS) and perfluoro-n-decanoic acid (PFDA) in freeze-dried strawberry and olive, as model fruits characterized by very different chemical compositions. The method was evaluated for apparent recovery, intra-day and inter-day precision, matrix effect and recovery. The method optimized for strawberry provided for most compounds absolute values of matrix effect (|ME%|) ≤ 11%, except for PFHxA, which showed a signal suppression of 22%. The extraction efficiency was tested at the spike levels 500-5000 pg/g d.w. for PFPeA, PFBuS, and PFHxA, and 100-1000 pg/g d.w. for the other target analytes, evidencing as a whole recoveries in the range of 65-89%. For olive fruits, due to their high fat content, an ultrasound-assisted extraction was necessary to obtain an efficient sample disgregation so as to increase the extraction yield and its precision. Moreover, a d-SPE clean-up with GCB allowed to achieve |ME%| ≤ 8% (except for PFBuS, which showed a signal enhancement of 19%) and recoveries calculated at the aforementioned spike levels were in the range 75-97%. The two methods provided very good linearity (R2 ≥ 0.9984) from 10,000 pg/g down to compound specific quantification limits, which were included in the ranges of 2.9-393 pg/g and 2.6-127 pg/g for strawberry and olive fruit, respectively. The methods were applied to the analysis of PFAAs in strawberry and olive fruits commercially available in two Italian supermarkets, as well as obtained under irrigation with various treated wastewaters (TWWs), evidencing in both cases a higher PFAAs occurrence in olives than in strawberry. However, PFAAs concentrations determined in the investigated fruit matrixes were quite low, being their sum 1.9 ng/g d.w. in the worst case (i.e. olive fruits grown under irrigation with TWWs).

Removal efficiency and mass balance of polycyclic aromatic hydrocarbons, phthalates, ethoxylated alkylphenols and alkylphenols in a mixed textile-domestic wastewater treatment plant.

In this work the occurrence and fate of polycyclic aromatic hydrocarbons (PAHs), phthalic acid esters (PAEs), mono and diethoxylate alkylphenols (AP1-2EOs) and alkylphenols (APs) have been investigated during a two-weeks period in a facility treating mixed textile-domestic wastewater (Prato, Italy). The wastewater treatment plant (WWTP) consists of primary sedimentation, activated sludge biological oxidation, secondary sedimentation, clariflocculation and ozonation. The sludge is treated within the facility by thickening, dewatering and final incineration, thus providing the almost quantitative removal of the adsorbed micropollutants. Naphthalene (50%), di(2-ethylhexyl) phthalate (74%) and branched 4-nonylphenols (59%) were the individual main representative compounds of each class in the influent wastewater, which showed concentration ranges of 5.6-66, 85-290 and 21-133µg/L for PAHs, PAEs and APs+AP1-2EOs, respectively. The WWTP efficiently removed PAHs, PAEs and APs+AP1-2EOs, providing effluent concentrations of 0.075-0.16ng/L 0.38-9.9µg/L and 0.53-1.4µg/L. All targeted priority and priority-hazardous micropollutants showed effluent concentrations in line with the European environmental quality standards (EQS), even though for di(2-ethylhexyl) phthalate and benzo(a)pyrene after correction for the dilution factor of the recipient. The WWTP performance was evaluated by mass balance, verifying its accuracy by monitoring Pb and Cd as conservative species. The biological treatment sections provided mass losses of 85.5%, 74.5% and 56.8% for APs+AP1-2EOs, PAEs and PAHs, highlighting efficient biotransformation performances of the activated sludge process. However, for the more volatile PAHs (e.g. naphthalene), a significant contribution of stripping cannot be excluded. A remarkable mass loss was also determined in the ozonation stage for PAEs (72.9%) and especially PAHs (97.0%), whereas a lower efficiency was observed for APs+AP1-2EOs (41.3%). The whole plant allowed for obtaining an almost quantitative removal (96.7-98.4%) for all targeted compounds.