Modelling uncertainty estimation for the determination of aflatoxin M-1 in milk by visual and densitometric thin-layer chromatography with immunoaffinity column clean-up
Laboratory of Quality Control and Safety Food - LACQSA/LANAGRO-MG/MAPA, Av. Raja Gabaglia, 245, Cidade Jardim, CEP 30380-090 - Belo Horizonte, MG, Brazil.Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment (Impact Factor: 1.8). 01/2012; 29(4):679-93. DOI: 10.1080/19440049.2011.648959
The uncertainty of aflatoxin M(1) concentration in milk, determined by thin-layer chromatography (TLC) with visual and densitometric quantification of the fluorescence intensities of the spots, was estimated using the cause-and-effect approach proposed by ISO GUM (Guide to the expression of uncertainty in measurement) following its main four steps. The sources of uncertainties due to volume measurements, visual and densitometric TLC calibration curve, allowed range for recovery variation and intermediary precision to be taken into account in the uncertainty budget. For volume measurements the sources of uncertainties due to calibration, resolution, laboratory temperature variation and repeatability were considered. For the quantification by visual readings of the intensity of the aflatoxin M(1) in the TLC the uncertainty arising from resolution calibration curves was modelled based on the intervals of concentrations between pairs of the calibration standard solutions. The uncertainty of the densitometric TLC quantification arising from the calibration curve was obtained by weighted least square (WLS) regression. Finally, the repeatability uncertainty of the densitometric peak areas or of the visual readings for the test sample solutions was considered. For the test samples with aflatoxin M(1) concentration between 0.02 and 0.5 µg l(-1), the relative expanded uncertainties, with approximately 95% of coverage probability, obtained for visual TLC readings were between 60% and 130% of the values predicted by the Horwitz model. For the densitometric TLC determination they were about 20% lower. The main sources of uncertainties in both visual and densitometric TLC quantification were the intermediary precision, calibration curve and recovery. The main source of uncertainty in the calibration curve in the visual TLC analysis was due to the resolution of the visual readings, whereas in the densitometric analysis it was due to the peak areas of test sample solutions followed by the intercept and slope uncertainties of the calibration line.
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- [Show abstract] [Hide abstract] ABSTRACT: Among all mycotoxins, Aflatoxin B1 (AFB1) is considered to be the most carcinogenic, and it has been classified by the International Agency for Research on Cancer in Group 1 of human carcinogen. It signifies a high hazard because it contaminates a diversity of agricultural products such as nuts and derivatives, peanuts/hazelnuts, grains, seeds, cottonseed, milk, dairy food. In milk AFB1 is metabolized to aflatoxin M (AFM1) which is 4-hydroxy derivative of AFB1, it is formed in the liver and excreted in the milk into the mammary glands of both human and lactating animals which have been fed with AFB1 contaminated diet. After the food contamination, one part of the aflatoxin B1 which was present in the food is eliminated through the milk. At the molecular level aflatoxin biosynthesis involves several levels of transcriptional and post-transcriptional control, so the main stages subsequent biochemical and genetic constituents of aflatoxin biosynthesis have been demonstrated recently. Recent studies over the last few decades have shown that the metabolism of AFB is an essential component of hepatocarcinogenic, however it was shown that AFB1 is metabolized by cytochrome P450 oxidised to intermediates and other metabolites Therefore, the biotransformation process may also lead to the formation of carcinogenic metabolites.
- [Show abstract] [Hide abstract] ABSTRACT: A novel method to simultaneously detect 11 kinds of mycotoxins (aflatoxin B1 [AFB1], aflatoxin B2 [AFB2], aflatoxin G1 [AFG1], aflatoxin G2 [AFG2], aflatoxin M1 [AFM1], ochratoxin A [OTA], zearalenone [ZEN], deoxynivalenol [DON], 3-acetyldeoxynivalenol [3-AcDON], 15-AcDON, and fusarenon-X [Fus-X]) in aquatic products was developed, using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with multiple reaction monitoring (MRM). The samples were extracted with acetonitrile-water (84: 16, V/V), purified using a multifunctional clean-up column (MycoSpin 400), and separated by a chromatographic column (Waters Acquity UPLCTM BEH C18; 100 × 2.1 mm i.d., 1.7 μm) using (0.01% formic acid-0.05% ammonia-H2O)/methanol as mobile phase. The quantitative and qualitative analyses were conducted in MRM mode with a mass spectrometer and an external standard. The results indicated that 11 mycotoxins showed good linear relationship within their respective linear response ranges, the correlation coefficients were > 0.9918, the limit of quantitation (LOQ) was 0.1 to 20.0 μg/kg, and the average recoveries (n=3) were from 62.8% to 115.3%, with values of relative standard deviation (RSD) ranging between 2.6% and 19.0%. The developed method with the facile pretreatment, good purification effect, high sensitivity and reproducibility can be used for rapid detection of trace levels of multiple mycotoxins in aquatic products. ©, 2015, South China University of Technology. All right reserved.