Evaluating the persistence of malachite green residues in tilapia and pacu fish.

Although banned in food-producing animals, residues of malachite green (MG) and its primary metabolite, leucomalachite green (LMG), have been found in fish due to illegal use in aquaculture and the release of industrial wastewater, which represent a serious risk to food and environmental securities. This study aimed to investigate the residue depletion profile of MG and LMG in edible tissues of Nile tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus) cultured simultaneously under the same environmental conditions to support control measures in case of abuse. An analytical method involving QuEChERS sample preparation and liquid chromatography coupled to tandem mass spectrometry was developed, validated, and applied to quantify MG and LMG residues in fish fillets from two depletion experiments after treatment by immersion bath (MG at 0.10 mg L-1 for 60 min). During the experiment, the average water temperature was 30 ºC, while the pH was 6.9. The method is selective, precise (CV = 0.4 - 22%) and accurate (recovery 92 - 114%). The limits of detection and quantification are 0.15 and 0.5 ng g-1, respectively. In both species, the sum of MG and LMG residues were quantified up to the 32nd day post-exposure, and the concentrations were significantly higher in the pacu fillets (up to 3284 ng g-1) than in Nile tilapia (up to 432 ng g-1). The sums of MG and LMG residues were below 2 ng g-1 at 44 days and 342 days for Nile tilapia and pacu, respectively - the Minimum Required Performance Limit (MRPL) for analytical methods intended to monitor forbidden substances in food according to old European Commission guidelines. The persistence of MG residues in pacu may be attributed to its higher lipid content, which favors the accumulation of the non-polar metabolite LMG. These results provide insights into the concern about human, animal, and environmental health risks resulting from unauthorized use or aquatic contamination by industrial wastewater containing MG residues.

Levamisole incorporation in fish feed - Ensuring the medication dose and avoidance of leaching into the water.

Levamisole, an anthelmintic and immunostimulant drug, has been studied as a promising alternative for aquaculture use. While oral administration through feeding is the main route of administration in fish farming, no studies evaluating methods of levamisole incorporation into the feed have been reported so far. Therefore, this study aimed to evaluate potential procedures for levamisole incorporation in extruded fish feed using ethyl cellulose, gelatin, or vegetable oil, to avoid drug leaching to the water during the animal's medication. A suitable LC-MS/MS method was optimized (full factorial design), validated, and applied to evaluate the efficiency of the process, the homogeneity of the drug concentration, and the leaching rate. The method has been demonstrated to be selective, precise (RSD < 4.9%), accurate (recovery > 98.4%), and linear (r > 0.99, 125-750 mg kg-1). The incorporation procedures using the three coating agents showed high incorporation efficiency (70%) and a homogeneous drug concentration among the extruded feed pellets. A low levamisole leaching rate was verified in the feed prepared using the ethyl cellulose coating procedure (4.3% after 15 min of immersion in the water). On the other hand, fish feed coated with gelatin and oil resulted in a high leaching rate (30-35% after 15 min). Thus, this study shows that coating ethyl cellulose may be a promising procedure for levamisole incorporation in fish feed and with the potential to enhance its use in animal production while reducing environmental contamination.

Residue Depletion Profile and Estimation of Withdrawal Period for Sulfadimethoxine and Ormetoprim in Edible Tissues of Nile Tilapia (Oreochromis sp.) on Medicated Feed.

Sulfadimethoxine (SDM) and ormetoprim (OMP) are antimicrobials used in combination to treat bacterial infections in fish farming. The use of this drug combination is not yet regulated in some countries, such as Brazil. Due to the lack of regulated drugs for aquaculture in Brazil, this study investigated the residue depletion profile of SDM and OMP in Nile tilapia (Oreochromis sp.) after oral administration. Fish were treated with medicated feed containing a 5:1 ratio of SDM:OMP at the dose of 50 mg kg BW-1 for five consecutive days with an average water temperature of 28 °C. The drugs were incorporated into the feed by using a gelatin coating process which promoted homogeneity in drug concentration and prevented the drug leaching into the water during medication. The SDM and OMP determination in fish fillets (muscle plus skin in natural proportions) was performed using the QuEChERS approach followed by LC-MS/MS quantification. The analytical method was validated according to Brazilian and selected international guidelines. A withdrawal period of 9 days (or 252 °C days) was estimated for the sum of SDM and OMP residues at concentration levels below the maximum residue level of 100 µg kg-1.

Combination of extractive techniques followed by HPLC-MS/MS analysis to monitor ent-agathic acid in fish treated with Copaifera duckei Dwyer.

Plants are used as therapeutic alternatives in Veterinary Medicine, including therapies for food-producing animals. However, these medicinal resources can sometimes contain dangerous substances, and when used in animals that supply food, they stand out from the point of view of food safety. The diterpene ent-agathic acid, a component of Copaifera duckei oleoresin, is an example of substances already described with toxic activity in mammals. Thus, this study aimed to propose combining two extractive techniques followed by high-performance liquid chromatography coupled mass spectrometry analysis to monitor residues of ent-agathic acid in Piaractus mesopotamicus fillet treated in an immersion bath with Copaifera duckei oleoresin. An optimized combination of solid-liquid extraction (using acidified acetonitrile) and dispersive liquid-liquid microextraction (using acidified water and chloroform as dispersive and extracting solvent, respectively) was performed to recover the target analyte, added to the development of HPLC-MS/MS method with adequate validation parameters to quantify the ent-agathic acid present in the fish fillet. In vivo tests of residual persistence of ent-agathic acid in fishes treated with C. duckei oleoresin were performed, indicating the non-detection of the target diterpene (< 6.1 µg/mL). The combined extractive procedure followed by quantitative analysis in the in vivo test of residual persistence of the target analyte in fish indicated the absence of ent-agathic acid in all samples. Thus, the data found might contribute to understanding the use of oleoresins from C. duckei as an alternative to traditional veterinary products.

The association of modified QuEChERS and DLLME to offer high analytical detectability to assess residual depletion profile of erythromycin in fish.

An analytical method for the determination of erythromycin A (ERY) residues in fish fillet was developed, optimized, and validated employing a modified QuEChERS procedure associated to DLLME technique as a preconcentration step. The obtained LOD and the LOQ were 0.1 µg kg-1 and 1 µg kg-1, respectively. The validated method provides linearity in the range of 1 to 20 µg kg-1, precision (CV < 6.3 %) and accuracy (recovery ranging from 103 to 110 %). The procedure was applied in an experimental study to evaluate the residual depletion profile of ERY in fish (Piaractus mesopotamicus) after oral administration. The treatment was carried out at a daily dose of 100 mg (kg BW)-1 of ERY, for 7 consecutive days and with an average water temperature of 30 °C. A withdrawal time of 240°-day was estimated for eliminating ERY residues at concentration levels below the maximum residue limit considered (MRL 100 µg kg-1).

Ethnoveterinary for food-producing animals and related food safety issues: A comprehensive overview about terpenes.

Alternatives to the use of conventional veterinary drugs in food-producing animals have gained attention, such as the use of natural products (NPs), mainly to soften the risks to the animal, the environment, and consumer's health. Although NPs have consistent advantages over conventional drugs, they cannot be considered risk free under food safety matters. In this way, this document presents a comprehensive overview of the importance of considering both the pharmacological and toxicological properties of the constituents of a NP from plants intending the standardization and regulation of its use in food-producing animals. Terpenes are the most diverse class of natural substances present in NP of vegetal origin with a broad range of biological activities that can be explored in veterinary science; however, certain plants and terpenes also have significant toxic effects, a fact that can harm the health of animals and consequently generate economic losses and risks for humans. In this context, this review gathered scientific data of vegetal species of importance to ethnoveterinary for food-producing animals, which produce terpenes, its biological effects, and their implications on food safety issues for consumers. For this, more than 300 documents were selected from different online scientific databases. The present data and discussion may contribute to the rational commercial exploration of this class of NPs in veterinary medicine.

UPLC-ESI/Q-TOF MS/MS Method for Determination of Vildagliptin and its Organic Impurities.

Vildagliptin (VLG) corresponds to a drug used for the treatment of diabetes mellitus. This disease requires continuous treatment, and so the control of impurities present in it is important to assure the quality of this drug. Thus, it is necessary to use sensitive and selective detection techniques and the ultra-performance liquid chromatography is a better option compared with high-performance liquid chromatography because it enhances the separation efficiency with a shorter analysis time and an increased resolution. This research analysis was accomplished by using liquid chromatography/tandem mass spectrometry, and the quantification was performed by using an extracted ion from the VLG drug and its main organic impurities of synthesis. During the validation process, following international standards, the method proved to be linear for the tree substances (R2 = 0.997-0.998) and the analysis of variance showed a non-significant linearity deviation (P > 0.05). Three critical factors were selected to evaluate method robustness with a full factorial experimental design, and the changes in the parameters were found to be not significant for the quantification of VLG and its impurities. The ultra-performance liquid chromatography-tandem mass spectrometry for the determination of impurities in VLG was precise, accurate and robust proving to be effective for analysis in the pharmaceutical industry and to improve the quality, safety and effectiveness of the new drug developed.

Impact of Atrazine Exposure on the Microbial Community Structure in a Brazilian Tropical Latosol Soil.

Atrazine is a triazine herbicide that is widely used to control broadleaf weeds. Its widespread use over the last 50 years has led to the potential contamination of soils, groundwater, rivers, and lakes. Its main route of complete degradation is via biological means, which is carried out by soil microbiota using a 6-step pathway. The aim of the present study was to investigate whether application of atrazine to soil changes the soil bacterial community. We used 16S rRNA gene sequencing and qPCR to elucidate the microbial community structure and assess the abundance of the atrazine degradation genes atzA, atzD, and trzN in a Brazilian soil. The results obtained showed that the relative abundance of atzA and trzN, encoding triazine-initiating metabolism in Gram-negative and -positive bacteria, respectively, increased in soil during the first weeks following the application of atrazine. In contrast, the abundance of atzD, encoding cyanuric acid amidohydrolase-the fourth step in the pathway-was not related to the atrazine treatment. Moreover, the overall soil bacterial community showed no significant changes after the application of atrazine. Despite this, we observed increases in the relative abundance of bacterial families in the 4th and 8th weeks following the atrazine treatment, which may have been related to higher copy numbers of atzA and trzN, in part due to the release of nitrogen from the herbicide. The present results revealed that while the application of atrazine may temporarily increase the quantities of the atzA and trzN genes in a Brazilian Red Latosol soil, it does not lead to significant and long-term changes in the bacterial community structure.

In vitro toxic evaluation of two gliptins and their main impurities of synthesis.

BACKGROUND: The presence of impurities in some drugs may compromise the safety and efficacy of the patient's treatment. Therefore, establishing of the biological safety of the impurities is essential. Diabetic patients are predisposed to tissue damage due to an increased oxidative stress process; and drug impurities may contribute to these toxic effects. In this context, the aim of this work was to study the toxicity, in 3 T3 cells, of the antidiabetic agents sitagliptin, vildagliptin, and their two main impurities of synthesis (S1 and S2; V1 and V2, respectively). METHODS: MTT reduction and neutral red uptake assays were performed in cytotoxicity tests. In addition, DNA damage (measured by comet assay), intracellular free radicals (by DCF), NO production, and mitochondrial membrane potential (ΔψM) were evaluated. RESULTS: Cytotoxicity was observed for impurity V2. Free radicals generation was found at 1000 µM of sitagliptin and 10 µM of both vildagliptin impurities (V1 and V2). A decrease in NO production was observed for all vildagliptin concentrations. No alterations were observed in ΔψM or DNA damage at the tested concentrations. CONCLUSIONS: This study demonstrated that the presence of impurities might increase the cytotoxicity and oxidative stress of the pharmaceutical formulations at the concentrations studied.

Quantitative Assessment of Poorly Soluble Anticoagulant Rivaroxaban by Microemulsion Electrokinetic Chromatography.

Microemulsion electrokinetic chromatography (MEEKC) is an electrophoretic methodology based on the separation of compounds by a microemulsionated electrolyte. There are few options for the evaluation of the stability and content of the oral anticoagulant rivaroxaban (RIV) in pharmaceutical formulations. RIV has low water solubility and undergoes ionization only under restricted pH conditions (pH < 1 or pH > 13), thus, hindering the application of free zone capillary electrophoresis as an analytical method. Therefore, the work aimed at developing and validating a stability-indicating MEEKC method for the analysis of RIV in pharmaceutical formulations. Separation was performed in a fused-silica capillary applying a voltage of 30 kV. The microemulsion system consisted of 13 mM tetraborate, pH 9.75 + 1.2% SDS + 1.0% ethyl acetate + 2.4% butanol. The linearity range was 25-150 µg mL-1, with r = 0.9982. Drug degradations were performed in acid and basic media (HCl 1 M and NaOH 0.1 M, respectively), oxidation with 3%H2O2, 60°C temperature and exposure to UV-C radiation. No interferences with RIV or internal standard peaks were detected. Method robustness was accessed through Plackett-Burman experimental design, after evaluation of model validity. Trueness values between 100.49 and 100.68% were obtained with repeatability. The method developed was found appropriate for quality control of RIV tablets, as a consistent analytical technique that is considered less damaging to the environment due to its low consumption of organic reagents.

In vitro dissolution method fitted to in vivo absorption profile of rivaroxaban immediate-release tablets applying in silico data.

OBJECTIVE: This study aimed to develop and validate an in vitro dissolution method based on in silico-in vivo data to determine whether an in vitro-in vivo relationship could be established for rivaroxaban in immediate-release tablets. SIGNIFICANCE: Oral drugs with high permeability but poorly soluble in aqueous media, such as the anticoagulant rivaroxaban, have a major potential to reach a high level of in vitro-in vivo relationship. Currently, there is no study on scientific literature approaching the development of RIV dissolution profile based on its in vivo performance. METHODS AND RESULTS: Drug plasma concentration values were modeled using computer simulation with adjustment of pharmacokinetic properties. Those values were converted into drug fractions absorbed by the Wagner-Nelson deconvolution approach. Gradual and continuous dissolution of RIV tablets was obtained with a 30 rpm basket on 50 mM sodium acetate +0.2% SDS, pH 6.5 medium. Dissolution was conducted for up to 180 min. The fraction absorbed was plotted against the drug fraction dissolved, and a linear point-to-point regression (R2 = 0.9961) obtained. CONCLUSION: The in vitro dissolution method designed promoted a more convenient dissolution profile of RIV tablets, whereas it suggests a better relationship with in vivo performance.

Early hematological and immunological alterations in gasoline station attendants exposed to benzene.

INTRODUCTION: Elucidation of effective biomarkers may provide tools for the early detection of biological alterations caused by benzene exposure and may contribute to the reduction of occupational diseases. This study aimed to assess early alterations on hematological and immunological systems of workers exposed to benzene. METHODS: Sixty gasoline station attendants (GSA group) and 28 control subjects were evaluated. Environmental and biological monitoring of benzene exposure was performed in blood and urine. The potential effect biomarkers evaluated were δ-aminolevulinate dehydratase (ALA-D) activity, CD80 and CD86 expression in lymphocytes and monocytes, and serum interleukin-8 (IL-8). The influence of confounding factors and toluene co-exposure were considered. RESULTS: Although exposures were below ACGIH (American Conference of Governmental Industrial Hygienists) limits, reduced ALA-D activity, decreased CD80 and CD86 expression in monocytes and increased IL-8 levels were found in the GSA group compared to the control subjects. Furthermore, according to multiple linear regression analysis, benzene exposure was associated to a decrease in CD80 and CD86 expression in monocytes. CONCLUSIONS: These findings suggest, for the first time, a potential effect of benzene exposure on ALA-D activity, CD80 and CD86 expression, IL-8 levels, which could be suggested as potential markers for the early detection of benzene-induced alterations.

Are delta-aminolevulinate dehydratase inhibition and metal concentrations additional factors for the age-related cognitive decline?

Aging is often accompanied by cognitive impairments and influenced by oxidative status and chemical imbalances. Thus, this study was conducted to examine whether age-related cognitive deficit is associated with oxidative damage, especially with inhibition of the enzyme delta-aminolevulinate dehydratase (ALA-D), as well as to verify the influence of some metals in the enzyme activity and cognitive performance. Blood ALA-D activity, essential (Fe, Zn, Cu, Se) and non-essential metals (Pb, Cd, Hg, As, Cr, Ni, V) were measured in 50 elderly and 20 healthy young subjects. Cognitive function was assessed by tests from Consortium to Establish a Registry for Alzheimer's Disease (CERAD) battery and other. The elderly group presented decreased ALA-D activity compared to the young group. The index of ALA-D reactivation was similar to both study groups, but negatively associated with metals. The mean levels of essential metals were within the reference values, while the most toxic metals were above them in both groups. Cognitive function impairments were observed in elderly group and were associated with decreased ALA-D activity, with lower levels of Se and higher levels of toxic metals (Hg and V). Results suggest that the reduced ALA-D activity in elderly can be an additional factor involved in cognitive decline, since its inhibition throughout life could lead to accumulation of the neurotoxic compound ALA. Toxic metals were found to contribute to cognitive decline and also to influence ALA-D reactivation.