Eugênia Azevedo Vargas

Advances in Sampling and Analysis for Aflatoxins in Food and Animal Feed

Advances in analytical methods and novel detection systems are reviewed from publications from 1995 onwards. The review covers aflatoxins B1, B2, G1, G2, and their metabolites in food, animal feed and biological matrices such as blood and urine. Improved extraction techniques, new clean‐up methods and optimized methods for specific matrices are summarized. This review highlights methods such as thin layer chromatography (TLC) and high performance‐TLC (HPTLC), which are particularly suited to developing countries, and advances that have been made in TLC quantification through low cost detection and scanning systems. Novel developments in detection of aflatoxins are assessed such as the application of surface plasmon resonance biosensors, flow injection monitoring, fibre optic sensors, capillary electrokinetics, electrochemical transduction, and immunological‐based rapid test kits. Recent advances in confirmatory techniques such as liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) for aflatoxins are also covered. This review summarizes performance data from recent collaborative studies, and assesses the overall quality of analytical data for aflatoxins being produced worldwide, as evidenced by results from various proficiency testing schemes. Although there are only a few recent studies on sampling for aflatoxins, the recent progress in this area is also assessed.

Growth of aflatoxigenic moulds and aflatoxin formation in Brazil nuts

The present study aimed at gaining more knowledge of the growth of aflatoxigenic moulds and aflatoxin production in Brazil nuts in relation to humidity conditions and storage time. For this purpose, the growth of aflatoxigenic moulds and the increase in aflatoxin levels in Brazil nuts was studied in the laboratory at temperature and humidity conditions that are relevant for the Amazon region. Fresh unprocessed Brazil nuts in shell were inoculated with an aflatoxin producing strain of Aspergillus nomius previously isolated from Brazil nuts. The nuts were stored at 27 °C in combination with 97, 90 or 80% surrounding relative humidity in a respirometer for up to 3 months. The General Linear Model (GLM) was used for evaluation of the effect of water activity and time on aflatoxigenic mould levels and on aflatoxin levels, as well as the relationship between mould and aflatoxin levels. During storage at the highest relative humidity (97%) aflatoxin formation occurred rapidly, whereas storage at 90% relative hum...

Development and validation of a methodology to qualitatively screening veterinary drugs in porcine muscle via an innovative extraction/clean-up procedure and LC-MS/MS analysis

A qualitative multiresidue method that facilitates rapid monitoring of veterinary drugs in porcine muscle is described. The method comprises the application of an innovative extraction/clean-up procedure, namely liquid–liquid extraction with partition at very low temperature (LLE-FPVLT), and analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Besides the high selectivity, sensitivity and specificity, this high-throughput method proved to be quite general as 34 veterinary drugs (from six distinct classes: tetracyclines, sulfonamides, penicillins, quinolones, macrolides and benzimidazoles) could be successfully detected. The whole screening procedure was validated according to the directives from European Commission Decision 2002/657/EC and guidelines for the validation of screening methods. Acceptable values for the evaluation parameters were achieved for all analytes (except for ampicillin, clindamycin and erythromycin). Finally, these very promising results have strengthened the possibility of inclusion of such a methodology as an integral part of the National Residue Control Plan scope of the Ministry of Agriculture, Livestock and Food Supply of Brazil.

Development and validation (according to the 2002/657/EC regulation) of a method to quantify sulfonamides in porcine liver by fast partition at very low temperature and LC-MS/MS

A novel multi-residue method for the quantification of 15 sulfonamides in porcine liver is described. It involves the application of a liquid-liquid extraction with fast partition at very low temperature (LLE-FPVLT) procedure followed by HPLC-MS/MS (high performance liquid chromatography coupled to tandem mass spectrometry) analysis. By this innovative method, acetonitrile is added to a minced porcine liver sample and the resulting suspension centrifuged and immersed in a container with liquid nitrogen for 15 s. The acetonitrile phase, which remains liquid under these conditions, is isolated, evaporated to dryness, recomposed with formic acid 0.1% v/v, and injected into the liquid chromatograph. The whole analytical procedure was validated according to the European Commission Decision 2002/657/EC and acceptable values (except for sulfanilamide) were obtained for the following parameters: linearity (0.97 < R2 < 0.99), decision limit (107.70 µg kg-1 < CCα < 128.65 µg kg-1), detection capability (115.40 µg kg-1 < CCβ < 157.29 µg kg-1), limit of detection (5.58 µg kg-1 < LOD < 16.75 µg kg-1), limit of quantification (18.41 µg kg-1 < LOQ < 55.26 µg kg-1), accuracy (recovery rates), precision (repeatability, intermediate precision, and measurement uncertainty tests), selectivity, and robustness. Recoveries higher than 70%, at three concentration levels (0.5, 1.0 and 1.5 of the maximum residue limit, MRL), were attained for the majority of the sulfonamides. These very promising results point to the inclusion of the present methodology into the National Residue Control Plan scope of the Ministry of Agriculture, Livestock and Food Supply of Brazil.

Validation of an LC-MS/MS method for malachite green (MG), leucomalachite green (LMG), crystal violet (CV) and leucocrystal violet (LCV) residues in fish and shrimp

A quantitative liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous analyses of malachite green (MG), crystal violet (CV) and its major metabolites, leucomalachite green (LMG) and leucocrystal violet (LCV) residues in fish and shrimp samples has been validated. Fish and shrimp samples were extracted with citrate buffer/acetonitrile, and the extracts were purified on strong cation-exchange (SCX) solid-phase extraction (SPE) cartridge. After conversion of LMG into MG using a post column oxidation reactor containing lead (IV) oxide (PbO2), the effluents were analysed. Residues were analysed using positive-ion electrospray ionisation (ESI). Identification and quantification of analytes were based on the ion transitions monitored by multiple reaction monitoring (MRM). Validation of the method was carried out in accordance with the Decision 2002/657/EC, which establishes criteria and procedures for the validation of methods. The following parameters were determined: decision limit (CCα), detection capability (CCβ), linearity, accuracy, precision, selectivity, specificity and matrix effect. The decision limits (CCα) for MG, LMG, CV and LCV were 0.164, 0.161, 0.248 and 0.860 µg kg–1. The respective detection capabilities (CCβ) were 0.222, 0.218, 0.355 and 1.162 µg kg–1. Typical recoveries (intermediate precision) in shrimp, for MG, CV, LMG and LCV for 2.0 µg kg–1 level fortified samples using the optimised procedure were in the range 69%, 97%, 80.3% and 71.8%, respectively. The findings demonstrate the suitability of the method to detect simultaneously MG, CV and its metabolite (LMG and LCV) in fish and shrimp.

Eficiência de um kit de ELISA na detecção e quantificação de aflatoxina M1 em leite e investigação da ocorrência no estado de Minas Gerais

Procedures for intralaboratory validation of an ELISA kit were adopted to verify its efficiency in aflatoxin M1 detection and quantification in milk. Assays were accomplished with standard solutions provided with the kit, spiked samples and naturally contaminated samples. The mean recovery values obtained for the kit standard solutions were between 85.6 and 114.8%, with coefficient of variation ranging from 11.6 to 23.1%. Working with spiked samples, a concentration range for quantitative analysis (0.019 to 0.090µg/L) was defined. The occurrence of aflatoxin M1 in 110 milk samples from the State of Minas Gerais was investigated. Five of the 27 positive samples in the screening for ELISA were confirmed by thin layer chromatography.

Heavy metals investigation in bovine tissues in Brazil.

The aim of this study was to investigate the presence of arsenic, lead, and cadmium residues in samples of liver, kidney, and muscle of cattle during the years of 2002 to 2008. A total of 1017 samples from 20 Brazilian States were used. The samples were analyzed at the National Agricultural Laboratory using the atomic absorption spectrometry technique. Arsenic residues were detected in 15.7% of liver samples and 28.7% of kidney samples although no results have exceeded the MRL. With regard to lead, 16 samples of liver and 74 samples of kidney were contaminated (5.2 and 10.9%, respectively). Among these samples, only one liver and two of kidney samples had lead levels above the MRL. Cadmium was found with levels below the MRL in 12.5% of the liver samples, and only 3 samples (1%) were quantified above the MRL. Among the kidney samples, 420 (60.8% of the total tested) had cadmium residues, and five of them exceeded the limits established by legislation. It is concluded that the Brazilian meat meets the legislation requirements without putting consumer’s healthy at risk since as it satisfies the national and international food-safety conditions.

Determining aflatoxins B1, B2, G1 and G2 in maize using florisil clean up with thin layer chromatography and visual and densitometric quantification

A method for determining aflatoxins B1 (AFB1), B2 (AFB2),G1 (AFG1) andG2 (AFG2) in maize with florisil clean up was optimised aiming at one-dimensional thin layer chromatography (TLC) analysis with visual and densitometric quantification. Aflatoxins were extracted with chloroform: water (30:1, v/v), purified through florisil cartridges, separated on TLC plate, detected and quantified by visual and densitometric analysis. The in-house method performance characteristics were determined by using spiked, naturally contaminated maize samples, and certified reference material. The mean recoveries for aflatoxins were 94.2, 81.9, 93.5 and 97.3% in the range of 1.0 to 242 µg/kg for AFB1, 0.3 to 85mg/kg for AFB2, 0.6 to 148mg/kg for AFG1 and 0.6 to 140mg/kg for AFG2, respectively. The correlation values between visual and densitometric analysis for spiked samples were higher than 0.99 for AFB1, AFB2, AFG1 and 0.98 for AFG2. The mean relative standard deviations (RSD) for spiked samples were 16.2, 20.6, 12.8 and 16.9% for AFB1, AFB2, AFG1 and AFG2, respectively. The RSD of the method for naturally contaminated sample (n = 5) was 16.8% for AFB1 and 27.2% for AFB2. The limits of detection of the method (LD) were 0.2, 0.1, 0.1 and 0.1mg/kg and the limits of quantification (LQ) were 1.0, 0.3, 0.6 and 0.6mg/kg for AFB1, AFB2, AFG1 and AFG2, respectively.

Development and validation of an efficient and innovative method for the quantification of multiclass veterinary drugs in milk by using LC–MS/MS analysis

An efficient analytical method for the simultaneous quantification of 27 veterinary drugs from five different classes (benzimidazoles, betalactams, quinolones, sulfonamides and tetracyclines) in bovine milk using a novel extraction procedure (liquid–liquid extraction with fast partition at very low temperature) and liquid chromatography coupled to tandem mass spectrometry is described. The full analytical method was validated according to Brazilian legislation (normative instruction SDA/MAPA 24/2009) and suitable values were achieved for all the parameters evaluated. Hence, the recoveries were within the established ranges (>70%), except for sulfathiazole (67.4% at the 0.5 × MRL level) and marbofloxacin and thiabendazole (112.5 and 114.6% respectively, at the 1.0 × MRL level). The repeatability and intermediate precision for all the analytes, expressed as relative standard deviations, were lower than 20 and 25%, respectively. The decision limits (CCα), detection capabilities (CCβ) and expanded uncertainties were also calculated and satisfactory results were obtained. Finally, the method was applied to 15 actual samples and traces of oxytetracycline and sulfamethazine were detected in three of them.

Assessment of heavy metal residues in Brazilian poultry and swine tissue

Objetivou-se investigar a presenca de residuos de arsenico, chumbo e cadmio em amostras de figado, rins e musculo de aves e suinos, durante os anos de 2002 a 2008. Um total de 1.978 amostras foi analisado: 1031 de aves e 947 de suinos provenientes de matadouros brasileiros. As amostras foram analisadas no Laboratorio Nacional Agropecuario (Lanagro), sendo utilizada a tecnica de espectrometria de absorcao atomica. Em aves, os residuos de arsenico foram detectados em 53,6% das amostras de figado, embora sem exceder o LMR. Nos rins, 39,7% das amostras mostraram residuos mensuraveis e nao houve violacao dos limites. Em relacao ao chumbo, cinco amostras de figado e 24 de amostras de rins estavam contaminadas (1,5 e 3,6%, respectivamente). No tecido muscular, houve apenas uma amostra com residuos. Para o cadmio, 3,8% das amostras apresentavam valores abaixo do LMR. Residuos de cadmio foram encontrados em 110 amostras de rins (16,3% testados), mas apenas uma ultrapassou o limite maximo permitido pela legislacao nacional. Em suinos, residuos de arsenico foram detectados em 15,3% das amostras de figado, sem exceder o LMR. Nos rins, 14,2% das amostras apresentaram residuos mensuraveis, sem, no entanto, haver violacao dos limites. No que diz respeito aos niveis de chumbo, sete amostras de figado e 18 amostras de rins estavam contaminadas (2,6 e 2,7%, respectivamente). O cadmio foi encontrado abaixo do LMR em 14,9% das amostras analisadas. Nos rins, 448 amostras (67,8% do total analisado) tinham residuos de cadmio e quatro amostras excederam o LMR. Durante os sete anos de estudo (2002-2008), apenas cinco (0,25%) das 1.978 amostras analisadas (de aves e suinos) violaram a lei brasileira. Entretanto, mesmo com baixos niveis de violacao, o controle e essencial, uma vez que o numero de amostras com residuos foi muitas vezes maior do que o numero de violacoes e, em alguns casos, como o cadmio nos rins, a maioria das amostras continha residuos quantificaveis e com niveis, muitas vezes, perto do LMR.