Tânia Mara Pizzolato

A liquid-liquid extraction procedure followed by a low temperature purification step for the analysis of macrocyclic lactones in milk by liquid chromatography-tandem mass spectrometry and fluorescence detection.

In this work a method is proposed and demonstrated for the analysis of the macrocyclic lactones abamectin, doramectin, eprinomectin, ivermectin and moxidectin in bovine milk by liquid chromatography coupled to mass spectrometry (LC-MS/MS) and liquid chromatography with fluorescence detection (LC-FL). The method is based on liquid-liquid extraction followed by a low temperature purification (LLE-LTP) step. Moreover, the proposed method was validated according to the Commission Decision 2002/657/EC, using LC-MS/MS and LC-FL for confirmatory and quantitative analysis, respectively. For LC-MS/MS the recovery rates observed ranged from 101.2 to 141.6% with coefficient of variation from 2.6 to 19.8%. For LC-FL the recovery rates observed ranged from 100.2 to 105% and coefficient of variations from 2.9 to 8.8%. Matrix effects were negligible due to the low temperature purification step. The quantification limits were far below the maximum limits established by regulations of all countries consulted. The proposed method proved to be simple, easy, and adequate for high-throughput analysis of a large number of samples per day at low cost.

Analytical quality assurance in veterinary drug residue analysis methods: Matrix effects determination and monitoring for sulfonamides analysis

In residue analysis of veterinary drugs in foodstuff, matrix effects are one of the most critical points. This work present a discuss considering approaches used to estimate, minimize and monitoring matrix effects in bioanalytical methods. Qualitative and quantitative methods for estimation of matrix effects such as post-column infusion, slopes ratios analysis, calibration curves (mathematical and statistical analysis) and control chart monitoring are discussed using real data. Matrix effects varying in a wide range depending of the analyte and the sample preparation method: pressurized liquid extraction for liver samples show matrix effects from 15.5 to 59.2% while a ultrasound-assisted extraction provide values from 21.7 to 64.3%. The matrix influence was also evaluated: for sulfamethazine analysis, losses of signal were varying from -37 to -96% for fish and eggs, respectively. Advantages and drawbacks are also discussed considering a workflow for matrix effects assessment proposed and applied to real data from sulfonamides residues analysis.

Determination of sulfonamide antibiotics and metabolites in liver, muscle and kidney samples by pressurized liquid extraction or ultrasound-assisted extraction followed by liquid chromatography–quadrupole linear ion trap-tandem mass spectrometry (HPLC–QqLIT-MS/MS)

Sulfonamides are widely used in human and veterinary medicine. The presence of sulfonamides residues in food is an issue of great concern. Throughout the present work, a method for the targeted analysis of 16 sulfonamides and metabolites residue in liver of several species has been developed and validated. Extraction and clean-up has been statistically optimized using central composite design experiments. Two extraction methods have been developed, validated and compared: i) pressurized liquid extraction, in which samples were defatted with hexane and subsequently extracted with acetonitrile and ii) ultrasound-assisted extraction with acetonitrile and further liquid-liquid extraction with hexane. Extracts have been analyzed by liquid chromatography-quadrupole linear ion trap-tandem mass spectrometry. Validation procedure has been based on the Commission Decision 2002/657/EC and included the assessment of parameters such as decision limit (CCα), detection capability (CCβ), sensitivity, selectivity, accuracy and precision. Method׳s performance has been satisfactory, with CCα values within the range of 111.2-161.4 µg kg(-1), limits of detection of 10 µg kg(-1) and accuracy values around 100% for all compounds.

Detection and confirmation of milk adulteration with cheese whey using proteomic-like sample preparation and liquid chromatography-electrospray-tandem mass spectrometry analysis

Caseinomacropeptide (CMP) is a peptide released by chymosin in cheese production, remaining in whey. Thus, CMP can be used as a biomarker to fluid milk adulteration through whey addition. Commonly, CMP is analyzed by reversed phase (RP-HPLC) or size-exclusion chromatography (SEC). However, some psychrotropic microorganisms - specially Pseudomonas fluorescens - when present in storaged milk, can produce, by enzymatic pathway, a CMP-like peptide generally called pseudo-CMP. These two peptides differ from each other only by one amino acid. RP-HPLC and SEC methods are unable to distinguish these two peptides, which demand development of a confirmatory method with high selectivity. Considering the several degrees of glycosilation and phosphorylation sites in CMP, allied with possible genetic variation (CMP A and CMP B), analytical methods able to differentiate these peptides are extremely complex. In the present work, we developed a proteomic-like technique for separation and characterization of these peptides, using liquid chromatography coupled to mass spectrometry with electrospray ionization able to differentiate and subsequently quantify CMP and pseudo-CMP in milk samples in order to identify adulteration or contamination of these products. The method shows satisfactory precision (<11%) with a detection limit of 1.0 µg mL(-1) and quantification limit of 5.0 µg mL(-1). Specificity, matrix effects and applicability to real samples analysis were also performed and discussed.

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 (CCβ) 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 CCβ 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.

Structural Elucidation of Sulfaquinoxaline Metabolism Products and Their Occurrence in Biological Samples Using High-Resolution Orbitrap Mass Spectrometry

Four previously unreported metabolism products of sulfaquinoxaline (SQX), a widely used veterinary medicine, were isolated and analyzed using liquid chromatography coupled to high-resolution Orbitrap mass spectrometry. Metabolites were structurally elucidated, and a fragmentation pathway was proposed. The combination of high-resolution MS(2) spectra, linear ion trap MS(2), in-source collision-induced dissociation (CID) fragmentation, and photolysis were used to analyze SQX and its metabolites. All metabolism products identified showed a similar fragmentation pattern to that of the original drug. Differential product ions were produced at m/z 162 and 253 which contain the radical moiety with more 16 Da units than sulfaquinoxaline. This occurs by a hydroxyl attachment to the quinoxaline moiety. With the exception of two low-intensity compounds, all the mass errors were below 5.0 ppm. The distribution of these metabolites in some animal species are also presented and discussed.

β-lactam antibiotics residues analysis in bovine milk by LC-ESI-MS/MS: a simple and fast liquid–liquid extraction method

This study presents the development and validation of a simple method for the detection and quantification of six β-lactam antibiotics residues (ceftiofur, penicillin G, penicillin V, oxacillin, cloxacillin and dicloxacillin) in bovine milk using a fast liquid–liquid extraction (LLE) for sample preparation, followed by liquid chromatography-electrospray-tandem mass spectrometry (LC-MS/MS). LLE consisted of the addition of acetonitrile to the sample, followed by addition of sodium chloride, centrifugation and direct injection of an aliquot into the LC-MS/MS system. Separation was performed in a C18 column, using acetonitrile 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. Limits of detection ranged from 0.4 (penicillin G and penicillin V) to 10.0 ng ml−1 (ceftiofur), and linearity was achieved. The decision limit (CCα), detection capability (CCβ), accuracy, inter- and intra-day repeatability of the method are reported.

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 (CCβ) 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.

Scope extension validation protocol: inclusion of analytes and matrices in an LC-MS/MS sulfonamide residues method

Validation is a required process for analytical methods. However, scope extension, i.e. inclusion of more analytes, other matrices and/or minor changes in extraction procedures, can be achieved without a full validation protocol, which requires time and is laborious to the laboratory. This paper presents a simple and rugged protocol for validation in the case of extension of scope. Based on a previously reported method for analysis of sulfonamide residues using LC-MS/MS, inclusion of more analytes, metabolites, matrices and optimisation for the extraction procedure are presented in detail. Initially, the method was applied only to liver samples. In this work, milk, eggs and feed were also added to the scope. Several case-specific validation protocols are proposed for extension of scope. Graphical Abstract

Simultaneous determination of eight antibiotics from distinct classes in surface and wastewater samples by solid-phase extraction and high-performance liquid chromatography–electrospray ionisation mass spectrometry

A reliable multiclass method has been developed and validated for the determination of eight antibiotics from distinct classes (sulfonamides, macrolides, fluoroquinolones, tetracyclines, cephalosporins and dihydrofolate reductase inhibitors) in wastewater – influent and effluent – and surface water from Porto Alegre, Brazil. The pre-concentration and clean-up was conducted with a simple and fast protocol using solid-phase extraction allowing a 100-fold concentration factor. The proposed method was validated by using spiked blank wastewater samples in terms of linearity, repeatability, reproducibility, recovery, matrix effects and limits of detection and quantification. Recovery was obtained in the range of 66–149%. Method limit of quantification ranged between 1.6 and 61.7 ng L−1. Samples (n = 16) were taken from January to August 2011 in one wastewater treatment plant, which uses conventional biological treatment. Sulfamethoxazole and trimethoprim show higher concentration, ranging from >10 to <6500 ng L−1, whereas erythromycin presented the lower amount. Differences between influent and effluent profiles were discussed. Surface water samples (n = 8) were collected in Arroio Diluvio, in four sampling points, in February 2012. From the eight antibiotics analysed, five were detected: sulfamethoxazole, trimethoprim, azythromicyn, ciprofloxacin and norfloxacin, in a concentration range of 376–572 ng L−1, 27–94 ng L−1, 24–40 ng L−1, 16–66ng L−1 and 30–54 ng L−1, respectively.