M.O. Leite

Optimisation and validation of a quantitative and confirmatory LC-MS method for multi-residue analyses of β-lactam and tetracycline antibiotics in bovine muscle

A multi-residue method for the determination of the β-lactam antibiotics ampicillin, cefazolin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V and the tetracyclines chlotetracycline, tetracycline and oxytetracycline was optimised and validated in bovine muscle. The method is based on the extraction of the residues from muscle using water/acetonitrile (2/8, v/v) with subsequent use of dispersive solid-phase C18 and hexane for purification. Extracts were analysed using ultra-performance liquid chromatography (UPLC-MS/MS) coupled with the mass spectrometer in positive electrospray ionisation mode (ESI+) for all analytes. The method was validated according to the requirements of European Commission Decision 2002/657/EC. The validation results were obtained within the MRL range of 0–1.5 of the MRL, with recoveries varying from 90% to 110% and CV < 20% (n = 54), except for cloxacillin, dicloxacillin and nafcillin. However, matrix interference was observed. The decision limit (CCα) ranged from 10% to 15% of the MRL. The uncertainty measurement was estimated based on both bottom-up and top-down strategies and the uncertainty values were found to be lower than 20% of the MRL. The method has a simple extraction procedure whereby analytes are separated with reasonable resolutions in a single 11-min chromatographic run. According to the validation results, this method is suitable for monitoring β-lactams and tetracyclines according to National Program for Residue and Contaminant Control – Brazil (NPRC-Brazil) in bovine muscle.

Optimization and validation method to evaluate the residues of β-lactams and tetracyclines in kidney tissue by UPLC–MS/MS

Methods are validated by a process that defines the analytical requirements and confirms that the investigated method is capable of performing consistently. A quantitative and confirmatory method for determining the presence of β-lactam and tetracycline multiresidues in avian, bovine, equine, and swine kidney tissues using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed, optimized, and validated. Analytes were extracted from the kidneys by a mixture of water and acetonitrile, and the extract was then purified with hexane and C18 (dispersive phase). The method was evaluated by the following parameters: linearity, matrix effect, specificity, decision limits (CCα), detection capability (CCβ), accuracy, precision, trueness, limits of detection (LOD), limits of quantification (LOQ), and robustness. The validated method presented a broad linear study range and significant matrix effect. The limit of detection (LOD) was defined from 2.5 to 25.0 µg kg(-1), and the limit of quantification (LOQ) was defined from 5.0 to 50.0 µg kg(-1) for individual analytes. The resultant recovery values ranged from 98.1% to 107.3% in repeatability conditions and from 95.2% to 106% under intralaboratory reproducibility conditions for the studied analytes. It was concluded that the performance parameters demonstrated total method adequacy for detecting and quantifying β-lactam and tetracycline residues in swine, equine, bovine, and avian kidneys.

Detection of cheese whey in raw milk preserved with bronopol® through high performance liquid chromatography

High performance liquid chromatography was used in order to detect cheese whey in samples of raw milk preserved with Bronopol®. Six samples were collected and divided in 45 aliquots of 40mL. From these, 15 were used as control and stored frozen, 15 were added with Bronopol® and stored at 7oC, and the other 15 were added with Bronopol® and stored at 30oC. In all groups, five levels of cheese whey addition (0, 2, 5, 10, and 20%) were tested. The samples were submitted to high performance liquid chromatography on the 2nd, 4th, and 8th days of storage. A completely random design was used, following the factorial scheme (5x3x3) and the results were compared through the non-parametric Kruskal-Wallis test. There was no difference among the treatments (P>0.05), which allows the conclusion that raw milk preserved with Bronopol® may be used for the determination of cheese whey addition in milk through high performance liquid chromatography.

A Lactobacillus rhamnosus strain induces protection in different sites after Salmonella enterica subsp. enterica serovar Typhimurium challenge in gnotobiotic and conventional mice

The ability of a Lactobacillus rhamnosus strain isolated from a healthy breast-fed human newborn to reduce the pathological consequences for the host due to an experimental oral infection with Salmonella enterica subsp. enterica serov. Typhimurium in vivo was determined using gnotobiotic and conventional mice. Conventional mice received 0.1mL probiotic milk (8.0 log colony-forming unit) daily for 10 days before the oral pathogenic challenge (5.0 log colony-forming unit). Then probiotic treatment was continued until the end of the experiment. Probiotic treatment in germ-free mice consisted of a single dose of the probiotic milk at the beginning of the experiment and a challenge with S. Typhimurium 10 days later (3.0 log colony-forming unit). A protective effect was observed in both gnotobiotic and conventional animals in terms of histopathologic and morphometric data, but in different anatomical sites. This protection was observed in liver and intestines, respectively, for gnotobiotic and conventional mice. However, S. Typhimurium populations were similar in the feces of both treated and control gnotobiotic mice. We conclude that a protective effect of L. rhamnosus against experimental S. Typhimurium was observed. This protection was not due to the reduction of the population of pathogenic bacteria in the intestine.

Application of Scharer's quantitative method for the determination of residual alkaline phosphatase activity in standard Minas

Milk pasteurization is a critical issue in the dairy industry, and failures in this process can affect final product safety. Scharers enzymatic method is still traditionally used to verify pasteurization efficiency compliance, and it is based on screening for residual alkaline phosphatase in milk. Although several methods are used to quantify enzymatic activity to assess milk pasteurization efficiency, there is a small amount of published data regarding the use of these methods to quantify alkaline phosphatase in cheese. In this study, the Scharers modified method was used to determine the levels of residual alkaline phosphatase in standard minas cheese, before and after 20 days of ripening. The cheeses were made using raw or pasteurized milk with the addition of different concentrations of raw milk (0; 0.05%; 0.10%; 0.20%; and 0.50%). In the fresh cheese samples, the method showed a sensitivity of only 0.50% with the addition of raw milk to the pasteurized milk used to make cheese. In addition, levels of up 0.20% of raw milk in pasteurized milk, the concentrations of phenol was inferior to 1μg phenol/g of dairy product which is the preconized indicator value for adequate pasteurization.

Fourier Transform Infrared Spectroscopy (FTIR) for MUN analysis in normal and adulterated Milk

O objetivo deste estudo foi realizar a avaliacao do CombiScopeTM FTIR (Delta Instruments), um equipamento baseado na espectroscopia de infravermelho por metodologia em Transformada Fourier (FTIR) para a avaliacao do teor de nitrogenio ureico no leite (NUL) cru produzido no Brasil. A repetibilidade e reprodutibilidade do CombiScopeTM FTIR (Delta Instruments) e a comparacao com um metodo enzimatico automatizado (ChemSpec® 150; Bentley Instruments) foram testados para a medicao do nitrogenio ureico no leite (NUL) cru. Adicionalmente, os niveis de NUL apos armazenamento das amostras de leite a 4oC e 20oC por ate 15 dias, e a capacidade e precisao para detectar ureia adicionada de forma fraudulenta ao leite foram avaliados por FTIR. Houve alta correlacao entre os metodos FTIR e enzimatico automatizado para a analise de ureia, sem diferenca significativa entre ambos (p>0,05). A concentracao de ureia no leite cru manteve-se estavel durante o armazenamento das amostras a 4oC por ate 15 dias. No entanto, apos tres dias a temperatura de 20oC houve um aumento nos niveis de ureia. Os resultados obtidos evidenciam que o equipamento CombiScopeTM FTIR e um metodo confiavel para a analise do teor de ureia no leite cru. Entretanto, a deteccao de ureia adicionada de forma fraudulenta ao leite cru nao foi linearmente proporcional, com diferenca significativa para adicao de ureia em niveis de 40mg/dL.

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.

Partial budget analysis of prepartum antimicrobial therapy and Escherichia coli J5 vaccination of dairy heifers and their effect on milk production and milk quality parameters

This study aimed to determine whether prepartum antimicrobial and/or Escherichia coli J5 vaccination in dairy heifers influence the milk production, milk quality, and estimate their economic benefit. Thus, 33 dairy heifers were enrolled in four groups using a split-splot design. Groups were: (G1) prepartum antimicrobial infusion and vaccination with an E. coli J5 bacterin, (G2) prepartum antimicrobial infusion, (G3) vaccination with an E. coli J5 bacterin, and (G4) control heifers. Composite milk samples for somatic cell count, total bacteria count and milk composition were collected 15 days after calving and every 15 days until the end of the experiment. Bacteriological analysis was carried out at the end of study. The milk production and the incidence of clinical cases of mastitis, as well as the costs associated with them were recorded. The results demonstrate a reduction on clinical mastitis rates by preventive strategies, which implicated in lower volume of discarded milk (0.99, 1.01, 1.04 and 3.98% for G1, G2, G3 and G4, respectively) and higher economic benefit. Thus, in well-managed dairy herds the prevention of heifer mastitis by vaccination or antimicrobial therapy can reduce the amount of antimicrobials needed to treat clinical mastitis cases and the days of discarded milk.

Detection of cheese whey and caseinomacropeptide in fermented milk beverages using high performance liquid chromatography

O presente trabalho teve como objetivos quantificar o teor de soro e o indice de caseinomacropeptideo (CMP) de bebidas lacteas fermentadas preparadas em laboratorio, adicionadas de diferentes concentracoes de soro (0, 10, 20 e 40%), fermentadas e armazenadas em refrigeracao (8-10oC) por tempos distintos (zero, sete, 14 e 21 dias), por cromatografia liquida de alta eficiencia-filtracao em gel (CLAE-FG), bem como verificar a interferencia da cultura utilizada no preparo das bebidas lacteas fermentadas e do tempo de armazenamento na deteccao de soro lacteo e CMP. Quando os teores de soro lacteo e os indices de CMP obtidos por CLAE-FG de bebidas lacteas fermentadas foram analisados ao longo do tempo de armazenamento, verificou-se que nao houve diferenca (p>0,05) para o leite (0% de soro) e as bebidas lacteas com 10 e 20% de soro nos tempos de zero, sete, 14 e 21 dias de armazenamento. No entanto, para a bebida lactea fermentada adicionada de 40% de soro, foi observada diferenca para o tempo de armazenamento de 21 dias (p 0,05) para os tempos de zero, sete e 14 dias.

Caseinomacropeptide index in UHT whole milk stored under different conditions of temperature and time

Caseinomacropeptide (CMP) index is a method used to detect adulteration of milk by addition of cheese whey, since CMP is a glycopeptide characteristic produced during cheesemaking, and soluble in the whey phase. The objective of this work was to evaluate the caseinomacropeptide index of UHT milk stored under different temperatures. Six batches of recently processed UHT milk were collected and stored under three temperatures (21°C, 6°C, and -12°C) and analyzed by HPLC in the day of the milk collection (day 0) and at 30, 60, 90, and 120 days of storage. The experiment was run as a randomized block design with a 3x5 factorial arrangement, and the Student-Newman-Keuls (SNK) method was used as the post- hoc test (p = 0.05). There was a progressive increase of the CMP index during the storage period of 120 days, and this indicates the possibility of false positive results if the CMP index is used as an adulteration test for long term stored UHT milk. The validity of the CMP index as an adulteration indicator is only possible soon after packaging, and sample freezing is the only alternative when immediate analysis is not possible. The method was found to be precise, with robust CV of 1.9% even with high CMP levels.