Andreia Freitas

Multi-residue and multi-class method for the determination of antibiotics in bovine muscle by ultra-high-performance liquid chromatography tandem mass spectrometry.

A multi-residue quantitative screening method covering 41 antibiotics from 7 different families, by ultra-high-performance-liquid-chromatography tandem mass spectrometry (UHPLC-MS/MS), is described. Sulfonamides, trimethoprim, tetracyclines, macrolides, quinolones, penicillins and chloramphenicol are simultaneously detected after a simple sample preparation of bovine muscle optimized to achieve the best recovery for all compounds. A simple sample treatment was developed consisting in an extraction with a mixture of acetonitrile and ethylenediaminetetraacetic acid (EDTA), followed by a defatting step with n-hexane. The methodology was validated, in accordance with Decision 2002/657/EC by evaluating the required parameters: decision limit (CCα), detection capability (CCβ), specificity, repeatability and reproducibility. Precision in terms of relative standard deviation was under 20% for all compounds and the recoveries between 91% and 119%. CCα and CCβ were determined according the maximum residue limit (MRL) or the minimum required performance limit (MRPL), when required.

Detection, accumulation, distribution, and depletion of furaltadone and nifursol residues in poultry muscle, liver, and gizzard.

Nitrofurans were broadly used as an extremely effective veterinary antibiotic especially in pig and poultry production farms. Because of fears of the carcinogenic effects on humans, the nitrofurans were banned from use in livestock production in many countries, including the European Union. The present study examines the accumulation, distribution, and depletion of furaltadone and nifursol and of their tissue-bound metabolites [3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) and 3,5-dinitro-salicylic acid hydrazine (DNSAH), respectively, in poultry edible tissues (muscle, liver, and gizzards) following administration to chickens of therapeutic and subtherapeutic concentrations of both compounds. Nitrofurans determination was performed by high-performance liquid chromatography-diode array detection and liquid chromatography-tandem mass spectrometry, respectively, for feeds and for poultry tissues. Furaltadone and nifursol, in very low concentrations, were found in samples of muscle, liver, and chickens gizzard collected from slaughtered animals after 5 weeks of treatment and no withdrawal time period. When a withdrawal time period of 3 weeks was respected, no detectable nitrofuran parent compounds was observed in all of the studied matrices. For AMOZ, concentrations of 270 μg/kg in meat, 80 μg/kg in liver, and 331 μg/kg in gizzard were determined after administration of a medicated feed with furaltadone (132 mg/kg), 3 weeks after withdrawal of treatment. For DNSAH, the concentration values obtained are much lower than those observed for AMOZ. For meat, liver, and gizzard, DNSAH concentrations of 2.5, 6.4, and 10.3 μg/kg, respectively, were determined, after administration of a medicated feed with nifursol (98 mg/kg), 3 weeks after withdrawal of treatment. The gizzard could be considered a selected matrix for nitrofuran residues evaluation in poultry, due to its capacity of retaining either nitrofuran parent compounds or metabolites in higher concentrations, regardless of the administered dose or of the respected withdrawal time period.

Multi-residue and multi-class determination of antibiotics in gilthead sea bream (Sparus aurata) by ultra high-performance liquid chromatography-tandem mass spectrometry

This paper describes a method for the determination of 41 antibiotics from seven different classes in gilthead sea bream (Sparus aurata) by ultra-high-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). Sulfonamides, trimethoprim, tetracyclines, macrolides, quinolones, penicillins and chloramphenicol were simultaneously determined. Fourteen procedures for sample treatment were tested and an extraction with acetonitrile and ethylenediaminetetra acetic acid was found to be the best option. The methodology was validated in accordance with Decision 2002/657/EC. Precision in terms of relative standard deviation (RSD) was under 17% for all compounds, and the recoveries ranged from 92% to 111%. CCα and CCβ were determined according to the maximum residue limit or the minimum required performance limit, when necessary. The validation provided evidence that the method was suitable for application in routine analysis for the detection and confirmation of antibiotics in muscle of gilthead sea bream, an important and intensively produced fish in aquaculture. Graphical Abstract

Determination of furaltadone and nifursol residues in poultry eggs by liquid chromatography-electrospray ionization tandem mass spectrometry.

The use of nitrofurans as veterinary drugs has been banned from intensive animal production in the European Union (EU) since 1993. The objective of the present study was to evaluate the accumulation and depletion of furaltadone and nifursol and their side-chain metabolites 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) and 3,5-dinitrosalicylic acid hydrazide (DNSAH) in eggs after administration of therapeutic and subtherapeutic doses of the drugs to laying hens during three consecutive weeks. LC-MS/MS, with positive and negative electrospray ionization methods, was used for the determination of parent compounds and metabolites in yolk and egg white and was validated according to criteria established by Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) of the analytical methodology for metabolites were 0.1 and 0.5 μg/kg for AMOZ and 0.3 and 0.9 μg/kg for DNSAH, respectively. For the parent compounds, CCα and CCβ were 0.9 and 2.0 μg/kg for furaltadone and 1.3 and 3.1 μg/kg for nifursol, respectively. The data obtained show that the parent compounds are much less persistent than their side-chain metabolites in either yolk or egg white. Between the studied metabolites, AMOZ is the most persistent and could be detected in either yolk or egg white three weeks following withdrawal from treatment.

Development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry for determination of amanitins in urine and liver samples

Amanitins, highly toxic cyclopeptides isolated from various Amanita species, are the most potent poisons accounting for the hazardous effects on intestinal epithelium cells and hepatocytes, and probably the sole cause of fatal human poisoning. The present study was focused on the development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), following urine and liver sample preparation by protein precipitation with organic solvents, and solid phase extraction (SPE) procedure, for the determination of the amatoxins, α- and β-amanitin. Linearity, detection and quantification limits, selectivity, sensitivity, intra and inter-assay precision and recovery were studied, in order to guarantee reliability in the analytical results. The developed method proved to be specific and selective, with LOD (Limit of Detection) values for α- and β-amanitin of 0.22 and 0.20 ng mL(-1) in urine and 10.9 and 9.7 ng g(-1) in liver, respectively. LOQ (Limit of Quantification) values ranged from 0.46 to 0.57 ng mL(-1) in urine and 12.3-14.7 ng g(-1) in tissue, for both amanitins. Linearity, in the range of 10.0-200.0 ng mL(-1) or ng g(-1), shows that coefficients of correlation were greater than 0.997 for α-amanitin and 0.993 for β-amanitin. Precision was checked at three levels during three consecutive days with intra-day and inter-day coefficients of variation not greater than 15.2%. The extraction recovery presents good results for the concentrations analyzed, with values ranging from 90.2 to 112.9% for both matrices. Thus, the proposed analytical method is innovative, presents a high potential in the identification, detection and determination of α- and β-amanitins in urine and tissue samples, as well as in other biological samples, such as kidney and mushrooms.

The effects of the nitrofuran furaltadone on Ulva lactuca

The use of pharmaceuticals in the food production industry as prophylatic and therapeutic agents is necessary to promote animal health, but may entail significant consequences to natural ecosystems, especially in the cases of overdosing and use of banned pharmaceuticals. The vast effects that antibiotics released into the environment have on non-target organisms are already under the scope of researchers but little attention has been given to primary producers such as macroalgae. The present study assessed furaltadones, an antibacterial agent illegally used for veterinary purposes, uptake capacity by Ulva lactuca and its effect in the growth of this cosmopolitan macroalgae. Differences in macroalgal growth were shown when submitted to prophylactic and therapeutic concentrations of furaltadone in the water (16 and 32 μg mL⁻¹, respectively). The therapeutic concentration caused higher growth impairment than the prophylactic treatment did, with 87.5% and 58% reductions respectively. Furthermore, together with data collected from the accumulation assays, with values of internal concentrations as high as 18.84 μg g⁻¹ WW, suggest that the macroalgae U. lactuca should be included in field surveys as a biomonitor for the detection of nitrofurans.

Matrix Effects in Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry Antibiotic Multi-Detection Methods in Food Products with Animal Origins

The efficiency of multi-detection screening methods, based on liquid chromatography coupled with tandem mass spectrometry, has been proven in recent years. However, when simultaneously analyzing different groups of compounds with different physicochemical properties, the specificity of sample preparation has to be minimized. In mass spectrometry, this situation can lead to ion suppression or enhancement of signals due to interferences coming from the matrices. This phenomenon was studied to understand the real impact in routine analysis. Matrix interferences were monitored in extracts of milk, muscle, liver, and fish for 40 antibiotics using recently developed and validated multi-detection methods with ultra-high pressure liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Although a significant dispersion in the results was observed, for most of compounds, ion suppression is the major problem that, although it does not compromise the screening methods, can prevent the use of multi-detection for confirmation and quantification of antibiotic residues in food matrices.

Multidetection of antibiotics in liver tissue by Ultra-High-Pressure-Liquid-Chromatography tandem Mass Spectrometry

A multiresidue quantitative screening method covering 39 antibiotics from 7 different families by ultra-high-pressure-liquid-chromatography-tandem mass spectrometry (UHPLC-MS/MS) is described. Sulfonamides, trimethoprim, tetracyclines, macrolides, quinolones, penicillins and chloramphenicol are simultaneously detected in liver tissue. A simple sample treatment method consisting of extraction with a mixture of acetonitrile and ethylenediaminetetraacetic acid (EDTA) followed by solid-phase extraction (SPE) with a hydrophilic-lipophilic balanced (HLB) cartridge was developed. The methodology was validated, in accordance with Decision 2002/657/EC, by evaluating the following required parameters: decision limit (CCα), detection capability (CCβ), specificity, repeatability and reproducibility. The precision, in terms of the relative standard deviation, was under 22% for all of the compounds, and the recoveries were between 80% and 110%. The CCα and CCβ were determined according to the maximum residue limit (MRL) or the minimum required performance limit (MRPL), when established.

A LC-MS/MS methodology to determine furaltadone residues in the macroalgae Ulva lactuca.

Presently, the rise of new contaminants in the environment has widened the scope of pharmaceutical analyses as to face the demanding new challenges. An increasing tendency for the interconnection and overlap of research fields, such as ecology and biochemistry, is intensifying the demand for new methodologies to be applied to the survey of drugs in unconventional matrices. Integrated in this group are macrophytes, such as the green macroalgae Ulva lactuca, which are under study as to ascertain their ability as indicators of contamination for many substances. Nonetheless, methodologies for extraction and determination of drugs in such matrices are scarce and new studies on the subject are pressing. A new methodology for the determination of the antibiotic furaltadone in U. lactuca by liquid chromatography-tandem mass spectrometric (LC-MS/MS) procedure was developed, optimized and validated following the guidelines of the EC Decision 2002/657. The calibration curves showed linearity above 0.99 (R(2)). The relative standard deviations obtained for repeatability, expressed as CV, were between 15.3 and 20.5 and for reproducibility 25.3 and 28.2 whereas accuracy was in the interval of 88.9-95.5 (%). The limit of decision (CCα) and the detection capability (CCβ) were respectively 5.57 μg kg(-1) and 10.97 μg kg(-1). The method was successfully applied to experimental samples.

Ecotoxicological effects of pig manure on Folsomia candida in subtropical Brazilian soils

The effects of pig manure, from diets incorporating veterinary pharmaceuticals, on survival and reproduction of Folsomia candida were evaluated. Manures derived from the following diets: corn and soymeal (CS); 85% CS diet+15% wheat meal (TR); CS diet+100ppm doxycycline+50ppm colistin+2500ppm Zn oxide (CSa); TR diet+100ppm doxycycline+50ppm colistin+2500ppm Zn oxide (TRa). Manures were tested in two subtropical soils representative of southern (Oxisol and Entisol). Despite the antibiotics no significant differences were found between the four manures within each soil. However, strong differences were found on the toxicity between soils. In Oxisol, LC50 values were around 100m(3)ha(-1), and EC50 values around 80m(3)ha(-1). In Entisol these were much lower, with LC50 values oscillating around 20m(3)ha(-1) and EC50 values between 10-15m(3)ha(-1). The observed toxicity on both soils was attributed to excess of nitrogen, Cu and Zn in the highest doses. The strong difference between soils could be explained by soil properties, namely CEC, organic matter, and clay contents that were lower in Entisol, indicating a poor ability to retain contaminants increasing their availability in soil. Results suggest that the application of these residues should be regulated not only using a volume-based criterion, but should incorporate data on soil properties, complemented by an ecotoxicological assessment.