RESUMO
Levels of essential metals in human breast milk (HBM) have been determined by different analytical techniques, but there is few woks about human whey milk fractions. However, the current trend lies in metalloproteomic and identification of different metalloproteins. In this sense, native separative techniques (N-PAGE and SEC) coupled to ICP-MS provide us with valuable information. Besides it is necessary the development of new methodologies in order to determine with accuracy and precision the profile of such metals and metalloproteins in the different whey protein fractions of HBM. Thus, the aim of this work was to develop a new method for metals and metalloproteins determination by SEC-ICP-MS in whey protein fractions of HBM. Human whey fractions were obtained of HBM samples by ultracentrifugation. Then, protein fractions of whey milk were separated by SEC coupled to ICP-MS for metalloproteins and Mn, Co, Cu and Se quantification. Besides, protein profile of whey milk was determined by N-PAGE and computer assisted image analysis. SEC-ICP-MS results indicated that first and second protein fractions showed detectable levels of the Mn, Co, Cu, and Se. Protein profile determined by N-PAGE and image analysis showed that molecular weight of protein fractions ranged between 68,878-1,228.277â¯Da. In this work, metalloproteins were analyzed by SEC coupled to ICP-MS, with adequate sensitivity and accuracy. Our study has shown the presence of Mn, Co, Cu and Se bound to two protein fractions in whey milk of HBM. Metals levels analyzed were within the ranges reported in the literature.
Assuntos
Metaloproteínas/análise , Metais/análise , Micronutrientes/análise , Leite Humano/química , Adulto , Cromatografia em Gel/instrumentação , Cromatografia em Gel/métodos , Estudos de Viabilidade , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Eletroforese em Gel de Poliacrilamida Nativa/instrumentação , Eletroforese em Gel de Poliacrilamida Nativa/métodos , Sensibilidade e Especificidade , Proteínas do Soro do Leite/análiseRESUMO
A novel, simple, easy and cheap sample treatment strategy based on salting-out assisted liquid-liquid extraction for ochratoxin A (OTA) ultra-trace analysis in beer samples using ultra-high-performance liquid chromatography-tandem mass spectrometry determination was developed. The factors involved in the efficiency of pre-treatment were studied employing factorial design in the screening phase and the optimal conditions of the significant variables on the analytical response were evaluated using a central composite face-centred design. Consequently, the amount of salt ((NH4)2SO4), together with the volumes of sample, hydrophilic (acetone) and nonpolar (toluene) solvents, and times of vortexing and centrifugation were optimised. Under optimised conditions, the limits of detection and quantification were 0.02 µg l-1 and 0.08 µg l-1 respectively. OTA extraction recovery by SALLE was approximately 90% (0.2 µg l-1). Furthermore, the methodology was in agreement with EU Directive requirements and was successfully applied for analysis of beer samples.
Assuntos
Cerveja/análise , Extração Líquido-Líquido/métodos , Micotoxinas/análise , Ocratoxinas/análise , Cloreto de Sódio/química , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em TandemRESUMO
BACKGROUND: The assessment of ochratoxin A (OTA) in wine is relevant for food safety and its continuous control allows to reduce the risk of intake. Thus, a novel sensitive QuEChERS-SPE (Quick, Easy, Cheap, Effective, Rugged and Safe - Solid Phase Extraction) pretreatment prior to liquid chromatography coupled to tandem mass spectrometry was developed for the determination of OTA in red wine samples from different grape-growing regions in Argentine. RESULTS: A sensitive methodology was achieved and thus the limits of detection and quantification were 0.02 and 0.05 µg L-1 , respectively. Recoveries ranged from 89.0% to 105.3%. The method was applied to 136 red wine samples (Argentina's flagship varieties: Malbec and Cabernet Sauvignon) from ten grape-growing regions, during vintages 2013-2015. Although all of the samples investigated were contaminated with OTA (concentrations ranged from 0.02 to 0.98 µg L-1 ), the levels detected were lower than the maximum allowable concentration limit of 2.0 µg L-1 established by international regulations. CONCLUSION: The methodology proposed is suitable for reliable OTA analysis in red wines. Similarly, the values obtained from the samples analyzed were in accordance with the current regulations and, as a consequence, preventive actions to reduce this mycotoxin incidence can be undertaken. © 2016 Society of Chemical Industry.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Micotoxinas/análise , Micotoxinas/isolamento & purificação , Ocratoxinas/análise , Ocratoxinas/isolamento & purificação , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Vinho/análise , Argentina , Contaminação de Alimentos/análiseRESUMO
A novel and advanced technology on solid phase extraction column prior to liquid chromatography coupled to tandem mass spectrometry has been used for the determination of ochratoxin A in red wine samples. Due to the need of a reliable and rugged method according to current regulations and with the aim of minimize heuristic efforts associated with analytical method development, the statistical design of experiment was employed. On other hand, the method validation according to European Commission 2002/657/EC was achieved. The values obtained for decision limit (CCα), detection capability (CCß), limits of detection and quantification were 0.07 µg L(-1), 0.14 µg L(-1), 0.13 µg L(-1) and 0.41 µg L(-1), respectively. The recoveries values were ranged from 95.7% to 107.2%. These values were compatible with the 2.0 µg L(-1) maximum allowable concentration limit established by different international regulations.