Rodrigo Barcellos Hoff

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.

Analysis of sulfonamides by capillary electrophoresis

The methods of analysis of sulfonamides (SFAs) using CE are reviewed. Sulfonamides were the first antimicrobial group of drugs used in medical treatment. These compounds are still used today in medicine and widely are employed in veterinary medicine, also for their growth promoter effects. Improved methods of analysis of sulfonamides are a constant challenge for researchers. CE is a new trend in the fields of pharmaceutical and food analyses. Several methods for the determination of SFAs by CE have been published in recent years, and the present review considers applications in quality control of pharmaceutical dosage forms, food analysis, determinations in serum, and other biological fluids as well as in electrophoresis experiments which examine the behavior of this class of compounds for theoretical studies of the technique. This review covers studies ranging from the pioneering works on sulfonamide analyses using classical electrophoresis to the more recent CE methods coupled to tandem mass spectrometers. The sections are divided following the EC modes like CZE, MEKC, and hyphenated methods (CE-MS, CE-MS/MS). A brief compilation of theoretical findings of sulfonamides electrophoretic behavior is also included. Parameters such as recoveries, LOD and LOQ, among others, are examined, covering works published until August 2008. This review can contribute to further research aimed to improve the analysis of SFAs by CE.

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.

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 (CCβ) 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.

Multiclass analysis of antibacterial residues in milk using RP-liquid chromatography with photodiode array and fluorescence detection and tandem mass spectrometer confirmation

A simplified procedure for simultaneous quantification of ceftiofur (CEF), fluoroquinolone (FQ) and sulfonamide (SA) antibacterials in bovine milk was developed. The reverse-phase liquid chromatography (RP-LC) multiclass method for analysis of eleven distinct compounds, from three antibacterial classes, was validated in line with Commission Decision 2002/657/EC. Confirmation of the analytes identities was performed by electrospray mass spectrometry detection. The analytes were extracted from milk matrix by liquid-liquid extraction with acidified ultrapure water and directly analyzed in the chromatograph. The SA compounds were pre-column derivatized with fluorescamine for fluorescence detection. The method provided good results regarding the analytical parameters of linearity, selectivity, sensitivity, precision, recovery, decision limit (CCα), detection capability (CCβ), limit of detection (LOD), limit of quantification (LOQ), stability and robustness. Analytes were extracted by liquid-liquid extraction in the fortified matrix and the compounds identity was confirmed by their precursor ion and fragments through tandem mass spectrometry analysis. Additionally, milk samples from two state capitals in the South Region of Brazil were analyzed by both the quantitative and confirmatory methods. The validation process showed correlation coefficients (r(2)) greater than 0.98 for all the analytes, with recovery rates up to 98% for all the studied drugs. LOD and LOQ limits ranged from 8.0 to 20.0 ng mL(-1) and 10.0 to 32.0 ng mL(-1), demonstrating good specificity of the method. The intra-day and inter-day precisions for all the analytes were below or equal to 7.40 and 10.13, respectively. The studied antibacterials were not detected in milk samples. The developed method represents an efficient alternative for multi-residue analysis in milk, being suitable and especially viable for monitoring in developing countries.

Liquid Chromatography–Tandem Mass Spectrometry Multiclass Method for 46 Antibiotics Residues in Milk and Meat: Development and Validation

A screening method for analysis of 46 antibiotics residues, belonging to different classes, such as tetracyclines, sulfonamides, fluoroquinolones, β-lactams, cephalosporins, macrolides and other minority groups was developed and validated for application in bovine milk and bovine, swine, poultry, equine, fish and shrimp meat samples. Sample preparation consists in solvent extraction followed by clean up with C18 bulk and low temperature purification. Instrumental analysis was performed using liquid chromatography coupled to tandem mass spectrometry system. Chromatographic separation was achieved using a C18 column. Mobile phase was composed by methanol and water. The method was validated according to Commission Decision 2002/657/EC criteria. Validation parameters such as specificity and detection capability (CCβ) were determined and considered suitable to the established criteria. Values of CCβ ranged from 1.0 to 50.0xa0μgxa0L−1 or μg Kg−1, depending on the compound and the matrix. The proposed method has been applied into Brazilian National Residue Control Plan since 2013 for the determination of antibiotic residues. A total of 3833 samples were analyzed until the current date and 13 samples showed positive results with concentrations above the permitted. The method is fast, easy and adequate for high throughput analysis in routine laboratories.

Validation of a method for sedatives and β-blockers determination in swine, bovine and equine kidney using liquid chromatography coupled with tandem mass spectrometry.

ABSTRACT A method for the determination of five sedatives and 14 β-blocker residues in swine, bovine and equine kidney was validated. Samples were extracted with acetonitrile and purified using dispersive solid phase extraction (d-SPE) with Celite 545 with subsequent analysis by LC-MS/MS. A simplified protocol was applied to validate the method scope extension to include new matrices. Parameters such as recovery, trueness, linearity (r2), relative standard deviation (RSD), decision limit (CCα) and method capability (CCβ) were measured for the bovine and equine kidney matrices. The method was applied to the analysis of more than 300 real samples and is currently included in the Brazilian National Residue Control Plan. GRAPHICAL ABSTRACT

Resíduos de piretróides, lactonas macrocíclicas e antimicrobianos em amostras de leite de tanque no estado de Minas Gerais

Picinin L.C.A., Toaldo I.M., Hoff R.B., Souza F.N., Leite M.O., Fonseca L.M., Diniz S.A., Silva M.X., Cerqueira M.M.O.P. & Bordignon-Luiz M.T. 2017. Survey of pyrethroid, macrocyclic lactone and antibacterial residues in bulk milk tank in Minas Gerais State, Brazil. Pesquisa Veterinária Brasileira 37(2):97-104. Departamento de Ciência e Tecnologia de Alimentos, Universidade Federal de Santa Catarina, Rodovia Admar Gonzaga 1346, Bairro Itacorubi, Florianópolis, SC 88034-001, Brazil. E-mail: lidia.picinin@gmail.com A survey of veterinary drug residues in bulk milk tank from Minas Gerais State, Brazil, was carried out through a broad scope analysis. Here, 132 raw milk samples were collected at 45 dairy farms in Minas Gerais from August 2009 to February 2010, and analyzed for 42 analytes, comprising pyrethroids, macrocyclic lactones and antibacterials, using liquid chromatography coupled with mass spectrometry in tandem mode and gas chromatography with electron capture detection. Within all milk samples, at least one veterinary drug residue was identified in 40 milk samples (30.30%) by confirmatory tests, whereas 16 samples (12.12%) showed the presence of at least two residues. With regard to the Brazilian maximum residue levels, 11 milk samples (8.33%) were non-compliant according to Brazilian Legislation. The veterinary drugs detected in the non-compliant milk samples include penicillin V (one sample), abamectin (one sample) and cypermethrin (nine samples). Furthermore, the antibacterial screening methods failed to identify most of the positive samples that were detected by confirmatory tests, leading to a large discrepancy between the screening and confirmatory antimicrobial tests. Thus, the present study indicated that the veterinary drugs residues still represents a great concern for the milk production chain. INDEX-TERMS: Veterinary drug, pesticide, anthelmintic, antibiotic, raw milk. 1 Received on June 24, 2016. Accepted for publication on July 13, 2016. Part of the L.C.A. Picinin Doctoral thesis in Food Science, Universidade Federal de Santa Catarina, Florianópolis, Brazil. 2 Departamento de Ciência e Tecnologia de Alimentos, Universidade Federal de Santa Catarina (UFSC), Rodovia Admar Gonzaga 1346, Bairro Itacorubi, Florianópolis, SC 88034-001, Brazil. 3 Departamento de Produção Animal e Alimentos, Centro de Ciências Agroveterinárias, Universidade do Estado de Santa Catarina, Lages, SC 88520-000, Brazil. *Corresponding author: lidia.picinin@gmail.com 4 Laboratório Nacional Agropecuário (Lanagro), Ministério da Agricultura, Pecuária e Abastecimento, Porto Alegre, RS 91780-580, Brazil. 5 Departamento de Tecnologia e Inspeção de Produtos de Origem Animal, Escola de Medicina Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG 31270-901, Brazil. 6 Departamento de Medicina Veterinária Preventiva, Escola de Medicina Veterinária, UFMG, Belo Horizonte, MG 31270-901, Brazil.

Desenvolvimento de procedimento de extração para análise de sedativos e B-bloqueadores em rim suíno

A procedure was developed for determination of 5 sedatives and 14 β-blockers in swine kidney and subsequent analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Three different procedures for extraction were tested, evaluated through recovery studies. The procedure using acetonitrile for extraction and cleanup with freezing at low temperature and dispersive solid phase extraction using 500 mg celite® 545 before the concentration step presented the better results. The dried samples were redissolved with methanol and analyzed using a LC-MS/MS system with electrospray ionization (ESI) operating in positive MRM mode. The recovery values for this procedure were in the 75-88% range. The robustness of the method was tested against small variations. The method was used to analyze carazolol, azaperone and azaperol in collaborative assay, obtaining results close to designed value.

Evaluation of lipid removal from animal fats for the determination of organochlorine, pesticides, and polychlorinated biphenyl indicators by gas chromatography with electron capture detector

For cleanup of animal fat before GC analysis of organochlorine pesticides and polychlorinated biphenyls, freezing-lipid filtration with solid-phase extraction and matrix solid-phase dispersion were evaluated to replace the official Brazilian methodology that uses preparative alumina column chromatography. General drawbacks associated with this last technique, such as the use of large amounts of solvent, laborious and time-consuming procedure could be avoided by using these alternative approaches. Experiments were carried out to study the performance by using different combinations of sorbents and elution solvents. Efficiency of alternative extraction methods in terms of fat removal and recovery capability was monitored by gravimetry, TLC, and GC with electron capture detection. Freezing-lipid filtration with solid-phase extraction afforded better clean up efficiency with recoveries in a range of 54.5 to 103.6% with the relative standard deviation of less than 10% for all compounds under investigation.