Integrated analysis of marine biotoxins and contaminants of emerging concern in bivalve mollusks from Santa Catarina, Brazil.

Santa Catarina is the main producer state of oysters and mussels in Brazil, reaching 98 % of national production. To assure the safety of bivalve mollusks production, control programs of marine biotoxins (MBs) have been continuously performed. Herein, the co-occurrence of MBs and contaminants of emerging concern (CECs) in oyster and mussels from the main production sites of Santa Catarina was reported, covering 178 compounds. Samples of wild and non-cultivated oysters and mussels were also assessed. Chemometric tools were used to evaluate and optimize several sample preparation techniques such as solid-liquid, ultrasound assisted, and pressurized liquid extraction. The optimized protocol was based on ultrasound assisted extraction followed by liquid chromatography coupled to tandem mass spectrometry. The results showed the incidence of several CECs and MBs. In the case of MBs, all results were below the regulatory limits for both cultivated and non-cultivated samples. Wild mollusks have shown a higher number of compounds. Regarding CECs, the more frequent compounds were caffeine, diclofenac, meloxicam, and sertraline. Domoic acid and okadaic acid were the main toxins detected. The results highlighted the need of monitoring for MBs and the potential of oyster and mussels as sentinel organisms to risk analysis of CECs in coastal regions. To the best of our knowledge, this is the first method to describe a simultaneous sample preparation and analysis of CECs and MBs in bivalve mollusks, as well as the first report of meloxicam and florfenicol in mussels and oysters.

Optimization of the Green Chemistry-like Extraction of Phenolic Compounds from Grape (Vitis labrusca L.) and Blackberry (Rubus fruticosus L.) Seeds with Concomitant Biological and Antioxidant Activity Assessments.

The objective of this work was to determine the phenolic composition, chemical and cellular antioxidant activity, cytotoxicity in human cells, and peroxidative inhibition of the defatted fraction of grape (Vitis labrusca) and blackberry (Rubus fruticosus) seeds. Soxhlet extraction (Sox) was used to extract the fat and obtain the degreased material. A statistical optimization study was developed to maximize the extraction of bioactive compounds and antioxidant activity from defatted grape and blackberry seeds. Simultaneous optimization was applied with a combination of 35.9 min of extraction and a solid-to-solvent ratio of 1 g of defatted grape seed to 61.28 mL of an extracting solvent (60% ethanol) and 62.1 min of extraction and a solid-to-solvent ratio of 1 g of defatted blackberry seed to 64.1 mL of an extracting solvent (60% ethanol). In the cell viability assay, HepG2 cancer cells seemed more sensitive to grape and blackberry extracts, while Ea.hy926 hybrid cells showed more resistance to their effects. In general, the extracts presented low/no cytotoxicity, exhibited a protective effect against H2O2-induced ROS production, and demonstrated antioxidant activity and a protective effect on the erythrocytes when subjected to hypotonic and isotonic conditions not presenting hemolytic behavior (5.0 to 10.0 µg GAE/mL). Thus, the results provided a broad assessment of the bioactivity of the extracts obtained using a simple and low-cost process developed by employing non-toxic solvents and with the potential to be used in technological applications.

Sample pooling and incurred samples improve analytical throughput and quality control of lipophilic phycotoxins screening in bivalve mollusks.

Lipophilic marine biotoxins (LMBs) are one of the main risks associated with the consumption of mussels and oysters. Sanitary and analytical control programs are developed to detect the occurrence of these toxins in seafood before they reach toxic levels. To ensure quick results, methods must be easy and fast to perform. In this work, we demonstrated that incurred samples were a viable alternative to validation and internal quality control studies for the analysis of LMBs in bivalve mollusks. These samples were used to optimize, validate, and monitor a simple and fast ultrasound-assisted extraction (UAE) procedure. An internal quality control material containing okadaic acid (227 ± 46 µg kg-1) was produced and characterized. This material had its homogeneity and stability verified and was included as a quality control in all batches of analytical routine. Besides, a sample pooling protocol for extracts analysis was developed, based on tests for COVID-19. Up to 10 samples could be analyzed simultaneously, reducing the instrumental time of analysis by up to 80%. The UAE and sample pooling approaches were then applied to more than 450 samples, of which at least 100 were positive for the okadaic acid group of toxins.

Determination of amino acid content, fatty acid profiles, and phenolic compounds in non-conventional edible fruits of seven species of palm trees (Arecaceae) native to the southern half of South America.

The fruits of palm trees (Arecaceae) are a very important food source for fauna and for humans, despite the still incipient role of these fruits in conventional diet. Seven palm tree species (plus one variety) that are native to the southern half of South America located east of the Andes (Argentina, Paraguay, Uruguay, and the southern states of Brazil) were studied. For the first time, the fatty acid profiles of palm seeds and the content of both amino acids and phenolic compounds present in the pulp (yellowish fleshy mesocarps) of the fruits were determined. High-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was used to screen and quantify thirty-two phenols and HPLC was used to determine the amino acid contents and fatty acids profiles of the species. Twenty-five percent of the fatty acids in the seeds of B. lallemantii, Butia odorata (var. pulposa), B. witeckii, and Syagrus romanzoffiana were found to be unsaturated. One species (B. lallemantii) had a high and well-balanced amino acid content (28,900 mg/kg), while six species (B. odorata, B. catarinensis, B. witeckii, Euterpe edulis, and Syagrus romanzoffiana) contained more than 14,000 mg/kg of amino acids. A high content of the phenolic compounds ferulic acid, luteolin, quercetin-3-rutinoside, isoquercetin, and isorhamnetin were found in many species, most notably in B. odorata, B. catarinensis, and B. witeckii.

Phenolic compounds profile and main nutrients parameters of two underestimated non-conventional edible plants: Pereskia aculeata Mill. (ora-pro-nóbis) and Vitex megapotamica (Spreng.) Moldenke (tarumã) fruits.

Non-conventional edible plants are widely present in Brazil. Several fruits of spontaneous occurrence are underestimated and scarcely used by the population. The increase in the knowledge about these plants, especially regarding the bioactive compounds present in this species, is important to increase its use, cultivation, and propagation. Ora-pro-nóbis (Pereskia aculeata) and tarumã (Vitex megapotamica) fruits are two examples. In the present report, the phenolic compounds profile was determined, using liquid chromatography coupled to tandem mass spectrometry (UHPLC-ESI-MS/MS). Other assays were also performed such as protein content, fat, humidity, and calcium. The results show that tarumã fruits are rich in several phenolic compounds, especially p-coumaric acid (9.3-61.8 mg g-1), isoquercitrin (14.6-55.5 mg g-1), naringenin (7.5-30.1 mg g-1), and caffeic acid (12.7-18.3 mg g-1). Ora-pro-nóbis fruits have showed high contents of caffeic acid (4.9 mg kg-1), rutin (3.9 mg kg-1), and hesperidin (1.7 mg kg-1), and their seeds, specifically, have showed relevant content for galangin (1.1 mg kg-1) and ferulic acid (0.3 mg kg-1).

Residues of antibiotics in yeasts from ethanol production: a possible contamination route for feedingstuffs.

Sugarcane yeast and brewer's yeast from ethanol production are widely used as ingredients of animal feed formulations in Brazil. To avoid the contamination of the must in ethanol production refineries, the use of antibiotics is one of the main preventive treatments. Thus, there is a risk of antibiotic residues carry over from yeast to animal feed. This unintentional addition of antibiotics can produce non-compliant feed products, due to regulatory aspects and their toxicity for animals. The results of an exploratory program to assess the occurrence of over 60 antibiotics and other pharmaceuticals in 27 sugarcane yeast and brewer's yeast samples were described. Monensin was present in seven samples with concentrations ranging from 0.47 to 263.5 mg kg-1. Other antibiotics quantitated were virginiamycin (2.25 mg kg-1) and amprolium (0.25 mg kg-1). Monensin in sugarcane yeast may represent a risk for further feeds production, especially for those products intended for sensible species such as equines and rabbits, for which monensin has toxic effects.

Determination of 62 veterinary drugs in feedingstuffs by novel pressurized liquid extraction methods and LC-MS/MS.

A fast and simple method for the determination of 62 veterinary drugs in feedingstuffs was developed, optimized, validated, and applied to real samples. Sample preparation was based on a pressurized liquid extraction method using a hard cap coffee machine, which was compared to a commercial pressurized liquid extraction system. Extraction was performed with diatomaceous earth, acetonitrile (20%), and formic acid (0.1%). A central composite design was used to optimize the composition of the extraction solvent. The extracts were analyzed using two chromatographic modes (reversed phase with C18 and HILIC). Analytical limits were set to 25 (limit of detection) and 75 µg kg-1 (limit of quantitation). For banned substances, a salting-out step was included, achieving LOQ lower as 1 µg kg-1 for ractopamine. Other figures of merit such as precision, trueness, decision limit (CCα), method capability (CCß), matrix effects, stability, recovery, and measurement uncertainty were also reported for analytical validation. The method was successfully applied to hundreds of real samples demonstrating its fitness-for-purpose for the analysis of sulfonamides, tetracyclines, fluoroquinolones, avermectins, quinolones, beta-agonists, beta-lactams, amphenicols, benzimidazoles, coccidiostats, lincosamides, macrolides, nitrofurans, quinoxalines, melamine, and trimethoprim.

Effects of time and extraction temperature on phenolic composition and functional properties of red rooibos (Aspalathus linearis).

This work was aimed at assessing the time-temperature effects on the phenolic compounds and in vitro functional properties of aqueous extracts from red rooibos (Aspalathus linearis). The major phenolic composition (tannins, flavonoids, flavonols, ortho-diphenols, total phenolic content), antioxidant (ABTS and DPPH) and reducing capacities (FRAP and total reducing capacity), antimicrobial effects and inhibition of α-amylase/α-glucosidase were measured. Phenolic compounds were also determined by LC-ESI-MS/MS. Aqueous extracts did not inhibit the growth of Escherichia coli, Staphylococcus aureus, and Candida albicans between 7.81 and 1000mgL-1. Rooibos extracted at 85°C for 10min showed a beneficial interaction with the human erythrocytes, reducing the hemolysis. The correlation analysis showed that the phenolic compounds responsible for the inhibition of α-amylase (IC50) were isohrmanetin, isoquercitrin, luteolin, salicylic acid, and syringaldehyde, whereas the inhibition of α-glucosidase was correlated to syringaldehyde, isoquercitrin, and luteolin. Overall, rooibos extracted at 85°C had the highest antioxidant activity measured by all assays, higher contents of phenolic compounds (spectrophotometric and LC-ESI-MS/MS data), and lower IC50 values for the digestive enzymes. On the other hand, rooibos extracted at 65°C had the opposite behavior, while rooibos extracted at 75°C presented mean intermediate values for the responses. This result clearly indicates that the extraction temperature is the main factor leading to a higher extraction of bioactive compounds from red rooibos.