Spatial-temporal occurrence of contaminants of emerging concern in urban rivers in southern Brazil.

The widespread use and misuse of antibiotics and pesticides has been linked with several risks to the environment and human health. In the present report, the results of the monitoring of 64 pharmaceuticals and 134 pesticides occurrence in an urban river in Southern Brazil are presented and discussed. Sampling campaigns have covered the period 2016-2018. The identification and determination of the analytes were achieved by high-resolution mass spectrometry. The data were analyzed using chemometric tools to obtain spatial-temporal models. Toxicological evaluation was achieved using acute toxicity (zebrafish standardized protocol), and determination of risk quotient. Within the 198 analytes included in the targeted analysis method for surface water, 33 were identified in an urban river during 2 years of monitoring, being 20 pharmaceuticals and 13 pesticides. Using high-resolution mass spectrometry, a suspect screening approach was established in an un-target analysis. The evaluation was carried out using a data bank built from consumption data of drugs and pesticides, in the metropolitan region of Porto Alegre - RS and their respective metabolites. The suspect screening analysis done with a data bank with more than 1450 compounds results in 27 suspect findings. The target analysis results showed a continuous prevalence of non-steroidal anti-inflammatories, analgesics, antipyretics, beta-blockers, corticoids, and antibiotics. Regarding the pesticides, the main classes were fungicides, especially those from triazol and strobilurin classes.

In silico toxicity evaluation for transformation products of antimicrobials, from aqueous photolysis degradation.

This study investigates degradation processes of three antimicrobials in water (norfloxacin, ciprofloxacin, and sulfamethoxazole) by photolysis, focusing on the prediction of toxicity endpoints via in silico quantitative structure-activity relationship (QSAR) of their transformation products (TPs). Photolysis experiments were conducted in distilled water with individual solutions at 10 mg L-1 for each compound. Identification of TPs was performed by means of LC-TOF-MS, employing a method based on retention time, exact mass fragmentation pattern, and peak intensity. Ten main compounds were identified for sulfamethoxazole, fifteen for ciprofloxacin, and fifteen for norfloxacin. Out of 40 identified TPs, 6 have not been reported in the literature. Based on new data found in this work, and TPs already reported in the literature, we have proposed degradation pathways for all three antimicrobials, providing reasoning for the identified TPs. QSAR risk assessment was carried out for 74 structures of possible isomers. QSAR predictions showed that all 19 possible structures of sulfamethoxazole TPs are non-mutagenic, whereas 16 are toxicant, 18 carcinogenic, and 14 non-readily biodegradable. For ciprofloxacin, 28 out of the 30 possible structures for the TPs are mutagenic and non-readily biodegradable, and all structures are toxicant and carcinogenic. All 25 possible norfloxacin TPs were predicted mutagenic, toxicant, carcinogenic, and non-readily biodegradable. Results obtained from in silico QSAR models evince the need of performing risk assessment for TPs as well as for the parent antimicrobial. An expert analysis of QSAR predictions using different models and degradation pathways is imperative, for a large variety of structures was found for the TPs.

Presence of antibiotic resistance genes and its association with antibiotic occurrence in Dilúvio River in southern Brazil.

It is known that antibiotics are widely used in human and veterinary medicine. In some countries the use is controlled, however few restrictions to their use are enforced in many countries. Antibiotics and their metabolites can reach the water bodies through sewage systems, especially in those countries with partial or absent wastewater treatment systems. The overuse and misuse of antibiotics has been linked with the increase of antibiotic resistant bacteria. The relation between the occurrence of antibiotics and resistance genes in surface waters has been widely studied worldwide evincing the great importance of this subject. In this work, a methodology for quantification of 40 antibiotics of 5 different classes, in river water, by SPE-LC-MS/MS was validated. Samples were taken during a two-year period from Dilúvio River, a stream that crosses the city of Porto Alegre (RS - Brazil) and receives in nature domestic effluent. The methodology met the requirements of validation, with Limit of Quantification varying from 20 ng L-1 to 100 ng L-1. A total of 48 samples was analyzed for the presence of antibiotics for two years. From the 40 antibiotics analyzed, 8 of them (Azithromycin, Cephalexin, ciprofloxacin, clindamycin, norfloxacin, sulfadiazine, sulfamethoxazole and trimethoprim) were present in all sampling points in the range of

An LC-ESI-MS/MS method for residues of fluoroquinolones, sulfonamides, tetracyclines and trimethoprim in feedingstuffs: validation and surveillance.

An increasing concern about food safety has been observed over the years. The presence of drugs residues in food is one of the major subjects of research in food safety. Feedingstuffs can be responsible for carryover into the food chain of residues of several drugs. This paper describes the development, validation and application of a fast and simple method for analysis of 24 antibiotic residues in feedingstuffs for cattle, pigs and poultry. Analytes include compounds from different antimicrobials classes, such as sulfonamides (sulfadiazine, sulfamethazine, sulfamethoxazole, sulfaquinoxaline, sulfachlorpyridazine, sulfadoxine, sulfadimethoxine, sulfizoxazole, sulfamerazine and sulfathiazole), fluoroquinolones (ciprofloxacin, enrofloxacin, norfloxacin, danofloxacin, difloxacin, sarafloxacin, flumequine, nalidixic acid and oxolinic acid), tetracyclines (tetracycline, doxycycline, oxytetracycline and chlortetracycline) and trimethoprim. Samples were extracted with methanol:water (70:30) 0.1% formic acid, followed by clean-up steps using centrifugation, low-temperature purification (LTP) and ultracentrifugation. Instrumental analysis was performed using liquid chromatography coupled to tandem mass spectrometry. Chromatographic separation was achieved using a C18 column and a mobile phase composed of acetonitrile and water, both with 0.1% formic acid. Validation parameters such as limit of detection (LOD), limit of quantification (LOQ), selectivity, linearity, accuracy, precision, decision limit (CCα) and detection capability (CCß) were determined and meet the adopted criteria. LOD and LOQ were set to 30 and 75 µg kg-1, respectively. Inter-day precision were in the range from 4.0 to 11.1%, and linearity provides values of r2 above 0.95 for all analytes. The optimised method was applied to the analysis of more than 1500 real samples within the period 2012-2017. Non-compliant results were discussed and classified in terms of analytes, feed types and target species. Multivariate analysis of the data was performed using principal component analysis.

Transformation products of amoxicillin and ampicillin after photolysis in aqueous matrices: Identification and kinetics.

Antibiotics are widely used in human medicine and veterinary production. Residues of these compounds reach the water sources through waste or direct application (e.g. aquaculture). The constant input of the parent drugs and their transformation products into the environment leads these pharmaceuticals to be considered as emerging pollutants. For some molecules, the pathway of degradation and formation in products is less known. To assess the impact of these substances in the environment and in the human health, it is necessary to elucidate the transformation products and their kinetic of degradation to evaluate the possible risks. In the present report, the characterization and the degradation kinetic of two widely used ß-lactams antibiotics - amoxicillin and ampicillin - was evaluated. Surface water samples containing these antibiotics were submitted to photolysis and analyzed by liquid chromatography coupled to mass spectrometry with Orbitrap detection in order to establish the profile of degradation and the formation of transformation products. Results showed that the degradation of amoxicillin and ampicillin is almost complete and reach their maximum at 48 h in river water. Moreover, a database containing >65 transformation products of amoxicillin and ampicillin was build and real samples of industrial wastewater were analyzed to investigate the occurrence of amoxicillin, ampicillin and their transformation products.

Determination of aminoglycoside residues in milk and muscle based on a simple and fast extraction procedure followed by liquid chromatography coupled to tandem mass spectrometry and time of flight mass spectrometry.

Antibiotics are widely used in veterinary medicine mainly for treatment and prevention of diseases. The aminoglycosides are one of the antibiotics classes that have been extensively employed in animal husbandry for the treatment of bacterial infections, but also as growth promotion. The European Union has issued strict Maximum Residue Levels (MRLs) for aminoglycosides in several animal origin products including bovine milk, bovine, swine and poultry muscle. This paper describes a fast and simple analytical method for the determination of ten aminoglycosides (spectinomycin, tobramycin, gentamicin, kanamycin, hygromycin, apramycin, streptomycin, dihydrostreptomycin, amikacin and neomycin) in bovine milk and bovine, swine and poultry muscle. For sample preparation, an extraction method was developed using trichloroacetic acid and clean up with low temperature precipitation and C18 bulk. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to carry out quantitative analysis and liquid chromatography-quadrupole-time of flight-mass spectrometry (LC-QTOF-MS) was used to screening purposes. Both methods were validated according to the European Union Commission Directive 2002/657/EC. Good performance characteristics were obtained for recovery, precision, calibration curve, specificity, decision limits (CCα) and detection capabilities (CCß) in all matrices evaluated. The detection limit (LOD) and quantification limit (LOQ) were ranging from 5 to 100ngg(-1) and 12.5 to 250ngg(-1), respectively. Good linearity (r)-above 0.99-was achieved in concentrations ranging from 0.0 to 2.0×MRL. Recoveries ranged from 36.8% to 98.0% and the coefficient of variation from 0.9 to 20.2%, noting that all curves have been made into their own matrices in order to minimize the matrix effects. The CCß values obtained in qualitative method were between 25 and 250ngg(-1). The proposed method showed to be simple, easy, and adequate for high-throughput analysis of a large number of samples per day at low cost.

High-throughput method for macrolides and lincosamides antibiotics residues analysis in milk and muscle using a simple liquid-liquid extraction technique and liquid chromatography-electrospray-tandem mass spectrometry analysis (LC-MS/MS).

A fast and simple method for residue analysis of the antibiotics classes of macrolides (erythromycin, azithromycin, tylosin, tilmicosin and spiramycin) and lincosamides (lincomycin and clindamycin) was developed and validated for cattle, swine and chicken muscle and for bovine milk. Sample preparation consists in a liquid-liquid extraction (LLE) with acetonitrile, followed by liquid chromatography-electrospray-tandem mass spectrometry analysis (LC-ESI-MS/MS), without the need of any additional clean-up steps. Chromatographic separation was achieved using a C18 column and a mobile phase composed by acidified acetonitrile and water. The method was fully validated according the criteria of the Commission Decision 2002/657/EC. Validation parameters such as limit of detection, limit of quantification, linearity, accuracy, repeatability, specificity, reproducibility, decision limit (CCα) and detection capability (CCß) were evaluated. All calculated values met the established criteria. Reproducibility values, expressed as coefficient of variation, were all lower than 19.1%. Recoveries range from 60% to 107%. Limits of detection were from 5 to 25 µg kg(-1).The present method is able to be applied in routine analysis, with adequate time of analysis, low cost and a simple sample preparation protocol.

High-throughput method for the determination of residues of ß-lactam antibiotics in bovine milk by LC-MS/MS.

This study describes the development and validation procedures for scope extension of a method for the determination of ß-lactam antibiotic residues (ampicillin, amoxicillin, penicillin G, penicillin V, oxacillin, cloxacillin, dicloxacillin, nafcillin, ceftiofur, cefquinome, cefoperazone, cephapirine, cefalexin and cephalonium) in bovine milk. Sample preparation was performed by liquid-liquid extraction (LLE) followed by two clean-up steps, including low temperature purification (LTP) and a solid phase dispersion clean-up. Extracts were analysed using a liquid chromatography-electrospray-tandem mass spectrometry system (LC-ESI-MS/MS). Chromatographic separation was performed in a C18 column, using methanol and water (both with 0.1% of formic acid) as mobile phase. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Main validation parameters such as linearity, limit of detection, decision limit (CCα), detection capability (CCß), accuracy, and repeatability were determined and were shown to be adequate. The method was applied to real samples (more than 250) and two milk samples had levels above maximum residues limits (MRLs) for cloxacillin - CLX and cefapirin - CFAP.

Determination of quinolones and fluoroquinolones, tetracyclines and sulfonamides in bovine, swine and poultry liver using LC-MS/MS.

Antibacterials are widely used in veterinary medicine. Residues of these drugs can remain in food of animal origin, including bovine liver. This paper describes a fast and simple analytical method for the determination of quinolones and fluoroquinolones, tetracyclines and sulfonamides in bovine liver samples. Deuterated enrofloxacin, sulfapyridine and demeclocycline were used as internal standards. The homogenised liver samples were extracted with acidified acetonitrile. Steps of non-solid-phase extraction (SPE) clean-up and concentration were used in the presented method. The final extracts were analysed by sensitive and selective detection of all components in a single run using LC-MS/MS. Acceptable recoveries between 66% and 110% were obtained. Good linearity (r(2)) above 0.96, considering three different days, for all drugs was achieved in concentrations ranging from 0.0 to 2.0 × the maximum residue limit (MRL). Intraday precision with coefficient of variation (CV%) (n = 6) lower than 14.7% and inter-day precision lower than 18.8% in agreement with European Commission Decision 2002/657/EC were obtained in concentrations ranging from 0.5 to 1.5 MRL. Accuracy was between 86% and 110%. Limits of detection and quantitation, as well as decision limit (CCα) and detection capability (CCß), were also evaluated.

A simple, fast and cheap non-SPE screening method for antibacterial residue analysis in milk and liver using liquid chromatography-tandem mass spectrometry.

In routine laboratory work, screening methods for multiclass analysis can process a large number of samples in a short time. The main challenge is to develop a methodology to detect as many different classes of residues as possible, combined with speed and low cost. An efficient technique for the analysis of multiclass antibacterial residues (fluoroquinolones, tetracyclines, sulfonamides and trimethoprim) was developed based on simple, environment-friendly extraction for bovine milk, cattle and poultry liver. Acidified ethanol was used as an extracting solvent for milk samples. Liver samples were treated using EDTA-washed sand for cell disruption, methanol:water and acidified acetonitrile as extracting solvent. A total of 24 antibacterial residues were detected and confirmed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), at levels between 10, 25 and 50% of the maximum residue limit (MRL). For liver samples a metabolite (sulfaquinoxaline-OH) was also monitored. A validation procedure was conducted for screening purposes in accordance with European Union requirements (2002/657/EC). The detection capability (CCß) false compliant rate was less than 5% at the lowest level for each residue. Specificity and ruggedness were also discussed. Incurred and routine samples were analyzed and the method was successfully applied. The results proved that this method can be an important tool in routine analysis, since it is very fast and reliable.