Effects of Dietary Exposure to Sulfamethazine on the Hematological Parameters and Hepatic Oxidative Stress Biomarkers in Nile Tilapia (Oreochromis niloticus).

Sulfamethazine (SMZ) is one of the most commonly used sulfonamide compounds in fish farming, and its physiological effects on fish are unknown. SMZ was administered to juvenile Nile tilapia (Oreochromis niloticus) at a dose level of 422 mg kg(-1) body weight, for a period of 11 days, via medicated feed. Fish were divided into two groups, the control group (CG) and the group fed with SMZ in feed. The administration of SMZ did not alter the erythrograms and leukograms of the Nile tilapia. The SMZ-fed group showed the same hepatic lipid peroxidation (LPO) concentration as the CG. Nonetheless, the oral administration of SMZ raised the hepatic catalase (CAT) and glutathione S-transferase (GST) activities, the increase probably being sufficient to prevent hepatic LPO production. The oral administration of SMZ affects the hepatic GST and CAT activities of Nile tilapia.

Elimination of the artefact peaks in capillary electrophoresis determination of glutamate by using organic solvents in sample preparation.

Focusing on the demand from the food industry for fast and reliable alternative methods to control the quality of food products, we present in this paper a method for amino acid separation and glutamic acid quantification in complex matrices employing capillary electrophoresis with capacitively coupled contactless conductivity detection. We demonstrate by simulation and experimentally the use of organic solvents in sample preparation to prevent peak splitting and increase stacking in capillary electrophoretic separations of amino acids. Additionally, we obtained results for glutamic acid quantification comparable to those obtained via traditional methods used at industrial sites. We tested premium and low-cost samples with large variations in their glutamic acid content, which demonstrated the wide range of applicability of the method presented herein. The results of the proposed capacitively coupled contactless conductivity detection based capillary electrophoresis method agreed with those obtained by an enzymatic detector and ultra high performance liquid chromatography coupled to tandem mass spectrometry, considering a confidence level of 95%.

Exploring miniaturized sample preparation approaches combined with LC-QToF-MS for the analysis of sulfonamide antibiotic residues in meat- and/or egg-based baby foods.

Miniaturized and simplified sample preparation methods with reduced consumption of chemicals and non-halogenated solvents are presented for the determination of 12 sulfonamides in baby foods. Hydrophilic interaction liquid chromatography coupled to quadrupole time-of-flight mass spectrometry was used for the identification and quantification of the compounds based on the acquisition of full spectrum at high resolution with accurate mass for precursor and its fragment ions. Three miniaturized protocols based on QuEChERS, salting-out assisted liquid-liquid extraction or low-temperature cleanup were evaluated regarding the extraction efficiency and removal capability of matrix co-extractives. All approaches achieved satisfactory recoveries (70.0-120.0%); however, the miniaturized QuEChERS distinguished by lower co-extractives content in the final extract providing lower matrix effects. Thus, the performance characteristics of the miniaturized QuEChERS were established using different matrices: beef-, egg yolk- and vegetable-based baby food or chicken- and vegetable-based baby food, in compliance with the Codex Alimentarius Commission guidelines. The target compounds were investigated in 30 commercial baby foods.

Occurrence of antimicrobial residues in tilapia (Oreochromis niloticus) fillets produced in Brazil and available at the retail market.

This study was carried out to assess the occurrence of antimicrobial residues in samples of tilapia (Oreochromis niloticus) fillets collected at the State of São Paulo retail market and produced from fish farmed in Brazil. For this purpose, a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was validated and used to quantify residues of 25 antibacterial drugs (2 ß-lactams, 8 quinolones, 2 macrolides, 5 sulfonamides, 4 tetracyclines, 3 amphenicols and 1 sulfonamide potentiator). For the sample preparation step the QuEChERS approach was performed. Chromatographic separation was conducted using a Zorbax SB C18 column. Method validation was performed based on European and Brazilian guidelines. The validation parameters (linearity, intra- and inter-day precision, accuracy, decision limit, detection capability and robustness) attended the adopted validation guidelines. Limits of detection and quantitation were also determined. Antimicrobial drug residues were quantitated in the incurred samples by using matrix-matched analytical curves. Oxytetracycline, florfenicol and, for the first time, enrofloxacin residues are reported in tilapia fillet samples from Brazil, though, in accordance with the European and Brazilian regulatory framework. Thus, our results draw attention to the use of veterinary products in fish farming in Brazil. Monitoring of veterinary drug residues is essential to ensure the safety of fish products available to the consumer, as well as to keep fish as a food commodity.

Effect of food processing (fish burger preparation and frying) on residual levels of enrofloxacin and ciprofloxacin.

The influence of fish burger preparation and frying on residual levels of enrofloxacin (ENR) and ciprofloxacin (CIP) was evaluated. For this purpose, a high-throughput liquid chromatography-mass spectrometry analytical method for the quantitation of ENR and CIP residues in tilapia products (fillet, raw fish burger and fried fish burger) was developed and validated based on European and Brazilian guidelines. Sample preparation was accomplished by extraction with acidified acetonitrile followed by clean-up with hexane. Chromatographic analysis was performed on a C18 column using isocratic elution with 0.1% formic acid and acetonitrile (85:15 v:v). The analytical method showed suitable performance to quantify the residual levels of ENR and CIP in the studied matrices. No reduction in the residual levels of ENR and CIP was observed during fish burger preparation and only a 10% reduction occurred as a consequence of frying, indicating that both compounds were stable to the preparation of the fish burger and to frying conditions.

Glyphosate and aminomethylphosphonic acid (AMPA) residues in Brazilian honey.

Glyphosate (GLY) is the most widely used herbicide in the world and studies have shown that its exposure at sublethal doses has led to reduced sensitivity and decreased associative memory in bees. Thus, a high-performance liquid chromatography-fluorescence detector with derivatisation reaction was used to determine residues of GLY and aminomethylphosphonic acid (AMPA) in Brazilian honey. The method met the validation criteria of the European Union (EU) SANTE 11813/2017 guideline and showed a limit of quantitation of 0.04 µg g-1 for both GLY and AMPA. Residues of these compounds were quantitated in honey samples from five Brazilian States. Six samples showed GLY levels above the EU maximum residue limit (0.05 µg g-1) and one sample showed AMPA at 0.10 µg g-1. This study indicates the presence of GLY residues in honey from regions that have had high losses of bee colonies and at the same time, frequent use of GLY in agriculture.

Hydrophilic interaction liquid chromatography coupled to quadrupole time-of-flight mass spectrometry as a potential combination for the determination of sulfonamide residues in complex infant formula matrices.

An accurate, sensitive and selective analytical method is proposed for sulfonamide residues analysis in infant formulas based on hydrophilic interaction liquid chromatography (HILIC) and quadrupole time-of-flight mass spectrometry in full scan mode. The sample preparation approach involves low-temperature lipid precipitation followed by dispersive solid-phase extraction with PSA and C18 sorbents, which was successfully optimized using Plackett-Burman design. In order to achieve high analytical sensitivity, the influence of HILIC conditions on sulfonamide ionization was investigated, such as the mobile phase composition, buffer concentration, and sample diluent for injection. The method performance characteristics, including linearity (range 5-120 µg kg-1), reliable limits of quantification (between 5 and 20 µg kg-1), recovery (72.9-109.2%) and precision (coefficient of variation values ≤ 19.8%) under repeatability and within-laboratory reproducibility conditions, were in accordance with the Codex Alimentarius Commission CAC/GL 71-2009 for quantitative analytical methods for veterinary drug residues in foods. Moreover, adequate identification of the compounds was provided with accurate mass measurement of both precursor and fragment ions in one single run. Finally, the developed method was applied to thirty-five powdered milk-based infant formula samples available in the Brazilian market.

Quantitation and identity confirmation of residues of quinolones in tilapia fillets by LC-ESI-MS-MS QToF.

A method for simultaneous determination of flumequine (FLM), oxolinic acid (OXO), sarafloxacin (SAR), danofloxacin (DAN), enrofloxacin (ENR), and ciprofloxacin (CIP) in tilapia (Orechromis niloticus) fillets, using liquid chromatography-tandem mass spectrometry (LC-ESI-MS-MS QToF) is presented. The quinolones were extracted from the food matrix with a solution of 10% trichloroacetic acid-methanol (80:20 v/v) with ultrasonic assistance. Clean-up of the extract solution was performed by using polymeric solid-phase extraction cartridges. The LC separation was carried out on an octadecyl hybrid silica column (C18, 150 mm x 3 mm, 5 microm). The column temperature was set at 30 degrees C, and gradient elution (0.2 mL min(-1)) was performed using water and acetonitrile, both containing 0.1% of acetic acid, as mobile phase components. The analytes were ionized using electrospray in the positive polarity mode. The following analytical results were obtained: linearity was about 0.99 for all the quinolones; intra and inter-assay precision (RSD%) were lower than 12.7 and 20%, respectively; and recoveries were from 89 to 112%. The quantitation limits were below the maximum residue limits established for the analytes. The method is suitable for the determination of quinolone residues in fish fillets and the QToF technique made it possible to obtain m/z ratios with less than 10 ppm of error for each analyte.

A high-throughput method for determining chloramphenicol residues in poultry, egg, shrimp, fish, swine and bovine using LC-ESI-MS/MS.

A LC-ESI-MS/MS method for determining chloramphenicol residues in fish, shrimp, poultry, eggs, bovine and swine was developed and validated. The samples were extracted with a phosphate extraction solution followed by liquid-liquid extraction with ethyl acetate. The LC was performed on a C18 column at room temperature. For all the matrices, the analytical curves showed r values and recoveries higher than 0.99 and 50%, respectively. The accuracy values lay between 85 and 120% and the precision was lower than 20%. The LOQ was 0.1 ng/g. The method was employed to analyze samples collected in Brazil.

A simple and high-throughput method for multiresidue and multiclass quantitation of antimicrobials in pangasius (Pangasionodon hypophthalmus) fillet by liquid chromatography coupled with tandem mass spectrometry.

The development and validation of a throughput method for the determination of 25 antibacterial drugs (two ß-lactams, eight quinolones, two macrolides, five sulfonamides, trimethoprim, four tetracyclines and three amphenicols) in pangasius fish muscle by LC-MS/MS were performed. A simple, efficient and fast extraction procedure was developed using acetonitrile and a 0.1 M EDTA solution as solvents for extraction. All compounds were determined in a single run, and chromatographic separation was achieved using a Zorbax SB C18 column with a mobile phase comprised of purified water +0.1% formic acid (A) and acetonitrile +0.1% formic acid (B) in a linear gradient program. The method was validated aαording to the requirements of European Decision 2002/657/EC. To quantify the analytes, matrix-matched analytical curves were constructed with spiked blank tissues and showed linearity (r2) higher than 0.99. For all analytes, the precision and accuracy were determined at the levels of 3 ng/g (low), 10 ng/g (low-middle), 50 ng/g (high-middle) and 100 ng/g (high). The precision (CV%) was lower than 18.6% and the accuracy (determined as recovery) was between 65% and 119%. The limit of quantitation was 3.0 ng/g, with the exception of chloramphenicol, which was 0.3 ng/g, and amoxicillin and doxycycline, which were 10 ng/g. The method was successfully applied to analyze pangasius muscle samples from Vietnam available at the Brazilian retail market, and 5 out of 40 samples showed the presence of low-residue levels of enrofloxacin and, consequently, must be considered out of conformity. It is recommended that competent authorities should avoid the commercialization of pangasius fillet contaminated with residues of this veterinary drug.

Determination of oxytetracycline in tomatoes by HPLC using fluorescence detection.

An analytical method for the determination of oxytetracycline (OTC) in tomatoes was developed and validated. Liquid-liquid extraction (LLE) and a solid-phase extraction (SPE) were used for sample preparation. Reversed-phase high performance liquid chromatography (RP-HPLC) using a C18 column and a mobile phase containing MeOH: calcium chloride, disodium ethylenediaminetetraacetate (EDTA) and sodium acetate, pH 7.3 (30:70, v/v), with fluorescence detection at 390nm excitation and 512nm emission, was used for separation and quantitation of OTC. The method was validated through the following performance criteria: linearity and linear range, sensitivity, selectivity, intra-day and inter-day precision, detection and quantitation limits and accuracy. Limit of quantitation show that the method developed is suitable for the determination of OTC at a level below the maximum residue limits established by the Brazilian legislations (250µgkg(-1)). Of 40 samples analyzed, none contained OTC above the limit of quantitation.

Simultaneous determination of streptomycin and oxytetracycline in agricultural antimicrobials by CZE after an experimental design.

A capillary zone electrophoresis (CZE) method was developed and validated for the simultaneous determination of both streptomycin (STP) and oxytetracycline (OTC) in bactericidal products to be used in agriculture. Using fused-silica capillaries, the influence of the electrolyte composition, pH and concentration, as well as temperature and applied voltage were investigated using a central composite design to optimize the method. The optimized electrophoretic conditions were as follows: 0.10 M sodium phosphate, pH 2.5, 7.0 kV and 20.0 degrees C. The method was validated for STP and OTC determination in agricultural formulations through the following performance criteria: linearity and linear range, sensitivity, selectivity, intra-day and inter-day precision, detectability, accuracy and ruggedness. This optimized CZE-method for the identification and quantification of STP and OTC is a potential alternative method to the HPLC methods described by the US Pharmacopeia, with the advantage that the same method could be used for the simultaneous determination of these different antibiotics.

Moxidectin residues in tissues of lambs submitted to three endoparasite control programs.

The indiscriminate and continuous use of anthelmintic drugs has promoted the selection of resistant parasites population, the presence of drug residues in food products, and heavy environmental contamination. The aim of the present study was to determine the presence of antiparasitic drug residues in 42-days old lamb serum and tissues, submitted to three endoparasite control programs: preventive treatment (PT) using moxidectin (MOX) at every 28days; selective treatment (FEC) using MOX when fecal egg count was greater than or equal to 700; and selective treatment (FMC), using MOX when FAMACHA/FMC score was 3 and above. For this purpose, MOX residues were quantified in serum, muscle, fat, liver and kidney. Lambs were slaughtered when reaching 30kg of body weight, and after a 28-day MOX withdrawal period. Before slaughter, blood was collected to determine the concentration of MOX in serum. Tissues and organ samples were collected at slaughter. The quantitation of MOX residues was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS). From the 756 tissue samples analyzed, only one sample of fat from the PT group showed residue levels (586.3µg/kg) above the maximum residue limit (MRL) of 500µg/kg. No treated lambs presented traces of MOX residues in fat and liver, suggesting possible environmental contamination. In conclusion, all weaned lambs, produced in continuous grazing and subjected to gastrointestinal parasite control programs via selective (FEC and FMC) or preventive (PT) treatment, displayed a low risk (<1%) of MOX residues above the MRL in muscle, fat, kidney, and liver.

Electric field-assisted solid phase extraction and cleanup of ionic compounds in complex food matrices: Fluoroquinolones in eggs.

The use of electric fields as additional driving forces in sample preparation techniques is an innovative approach that is environmentally friendly, straightforward, and able to overcome several limitations of conventional sample preparation procedures. In this work, the advantages of electric field-assisted solid phase extraction (E-SPE) using syringe-type cartridges were demonstrated for the extraction of four fluoroquinolones (FQs) in their anionic forms. The FQs were extracted from eggs and subsequently determined by UHPLC-MS/MS. The use of electric fields during the washing and final elution steps resulted in a significant improvement of the extraction efficiencies for almost all FQs when compared to conventional SPE. Intra- and inter-day assays showed coefficients of variation below 10%. The better cleanup also resulted in the appearance of less precipitated matter in the final eluate, as well as reduced matrix effects. The results showed that the electrophoretic forces derived from electric fields are a promising way of significantly increasing the extraction efficiency of ionic analytes, while minimizing matrix effects associated with complex samples.

Avaliação de risco ecológico do óleo essencial de Piper aduncum em organismos não alvo / Ecological risk assessment of Piper aduncum essential oil in non-target organisms

Uma possível alternativa ao uso de fármacos veterinários no tratamento e prevenção de doenças na piscicultura é o uso do óleo essencial de Piper aduncum. No entanto, são necessários dados ecotoxicológicos para garantir seu uso apropriado sem causar efeitos adversos a organismos não alvo. Esta informação é relevante, pois esse óleo essencial é descrito como tendo atividades inseticidas, moluscicidas e citotóxicas, possivelmente associadas à sua composição química. Assim, o objetivo deste estudo foi avaliar a ecotoxicidade do óleo essencial de P. aduncum para cinco organismos-teste, usando o método estatístico da Distribuição da Sensibilidade das Espécies (SSD). A composição química do óleo essencial foi caracterizada por cromatografia gasosa acoplada a espectrometria de massa (GC-MS) e cromatografia gasosa com detector de ionização de chama (GC-FID), para fins de identificação e quantificação, respectivamente. O principal componente (75,5%) do óleo essencial foi o dilapiol. A concentração perigosa para 5% de espécies biológicas (HC5) foi calculada com um nível de proteção de 95%, resultando em um valor de 0,47 mg L-1 (com intervalo de confiança de 50% = 0,028 - 1,19 mg L-1). A faixa de concentração relacionada aos níveis de proteção para comunidades aquáticas (concentração sem efeito previsto - PNEC) foi calculada através da aplicação de fatores de segurança ao valor de HC5. Os parâmetros de ecotoxicidade indicaram que o óleo essencial de P. aduncum pode ser usado com segurança em corpos d'água se a concentração for igual ou inferior a 0,09 mg L-1.(AU)

Considerations on the aquaculture development and on the use of veterinary drugs: special issue for fluoroquinolones--a review.

Aquaculture has become an important source of fish available for human consumption. In order to achieve greater productivity, intensive fish cultivation systems are employed, which can cause greater susceptibility to diseases caused by viruses, bacteria, fungi, and parasites. Antimicrobial substances are compounds used in livestock production with the objectives of inhibiting the growth of microorganisms and treatment or prevention of diseases. It is well recognized that the issues of antimicrobial use in food animals are of global concern about its impact on food safety. This paper present an overview of the aquaculture production in the whole world, raising the particularities in Brazil, highlighting the importance of the use of veterinary drugs in this system of animal food production, and address the potential risks arising from their indiscriminate use and their impacts on aquaculture production as they affect human health and the environment. The manuscript also discusses the analytical methods commonly used in the determination of veterinary drug residues in fish, with special issue for fluroquinolones residues and with emphasis on employment of LC-MS/MS analytical technique.

Decolorization and biodegradation of reactive sulfonated azo dyes by a newly isolated Brevibacterium sp. strain VN-15.

Azo dyes constitute the largest and most versatile class of synthetic dyes used in the textile, pharmaceutical, food and cosmetics industries and represent major components in wastewater from these industrial dying processes. Biological decolorization of azo dyes occurs efficiently under low oxygen to anaerobic conditions. However, this process results in the formation of toxic and carcinogenic amines that are resistant to further detoxification under low oxygen conditions. Moreover, the ability to detoxify these amines under aerobic conditions is not a wide spread metabolic activity. In this study we describe the use of Brevibacterium sp. strain VN-15, isolated from an activated sludge process of a textile company, for the sequential decolorization and detoxification of the azo dyes Reactive Yellow 107 (RY107), Reactive Black 5 (RB5), Reactive Red 198 (RR198) and Direct Blue 71 (DB71). Tyrosinase activity was observed during the biotreatment process suggesting the role of this enzyme in the decolorization and degradation process, but no-activity was observed for laccase and peroxidase. Toxicity, measured using Daphnia magna, was completely eliminated.

Determination of volatile organic compounds in recycled polyethylene terephthalate and high-density polyethylene by headspace solid phase microextraction gas chromatography mass spectrometry to evaluate the efficiency of recycling processes.

A method for the determination of volatile organic compounds (VOCs) in recycled polyethylene terephthalate and high-density polyethylene using headspace sampling by solid-phase microextraction and gas chromatography coupled to mass spectrometry detection is presented. This method was used to evaluate the efficiency of cleaning processes for VOC removal from recycled PET. In addition, the method was also employed to evaluate the level of VOC contamination in multilayer packaging material containing recycled HDPE material. The optimisation of the extraction procedure for volatile compounds was performed and the best extraction conditions were found using a 75 µm carboxen-polydimethylsiloxane (CAR-PDMS) fibre for 20 min at 60 °C. The validation parameters for the established method were linear range, linearity, sensitivity, precision (repeatability), accuracy (recovery) and detection and quantification limits. The results indicated that the method could easily be used in quality control for the production of recycled PET and HDPE.

Development and validation of an LC-APCI-MS-MS analytical method for the determination of streptomycin and dihydrostreptomycin residues in milk.

An analytical method for the quantification and identity confirmation of streptomycin and dihydrostreptomycin residues in pasteurized milk using liquid chromatography (LC)-atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry (MS-MS) was developed and validated. Method validation was performed according to the recommendations of the international agencies European Community and IUPAC, and the following parameters were evaluated: analytical curve, linearity, sensitivity, precision (intra- and inter-day repeatability), accuracy, and the limit of detection (LOD) and limit of quantification (LOQ). Simple sample preparation was followed by the LC-APCI-MS-MS analysis. The method presented adequate linearity with correlation coefficients above 0.99 for both analytes in the dynamic range of 50-400 microg/kg, and average accuracies between 84-110%. The LOD and LOQ were, respectively, 25 microg/kg and 50 microg/kg for both analytes. Method selectivity was verified by the absence of interfering peaks in the retention regions of the analytes and the internal standard when a blank sample was tested. The results qualified the method for the quantification and confirmation of the analytes in milk at concentrations inferior to the established maximum residue limits (200 microg/kg).

Use of experimental design and effective mobility calculations to develop a method for the determination of antimicrobials by capillary electrophoresis.

A capillary zone electrophoresis (CZE) method for the determination of chloramphenicol (CLP), danofloxacin (DANO), ciprofloxacin (CIPRO), enrofloxacin (ENRO), sulfamethazine (SMZ), sulfaquinoxaline (SQX) and sulfamethoxazole (SMX) is described. For the development, the effective mobilities were estimated and a central composite design was performed. The method was in-house validated for CLP, CIPRO, ENRO and SMX determination in pharmaceuticals. In comparison with the HPLC method recommended by the United States Pharmacopoeia, this CZE method exhibited the same performance, with the advantage that seven different antimicrobials in pharmaceutical formulations could be simultaneously determined.