Determination Of Aflatoxins In Food Research Articles

Study on Molecularly Imprinted Polymers Obtained Sonochemically for the Determination of Aflatoxins in Food

Aflatoxins (AFs) are fungi secondary metabolites produced by the Aspergillus family. These compounds can enter the food chain through food contamination, representing a risk to human health. Commercial immunoaffinity columns are widely used for the extraction and cleanup of AFs from food samples; however, their high cost and large solvent consumption create a need for alternative strategies. In this work, an alternative strategy for producing molecularly imprinted polymers (MIPs) was proposed to extract aflatoxins AFB1, AFB2, AFG1, and AFG2 from complex food samples, using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The MIPs were synthesized via a low-cost and rapid (5 min) sonochemical free-radical polymerization, using 1-hydroxy-2-naphthoic acid as a dummy template. MIPs-based solid phase extraction performance was tested on 17 dietary supplements (vegetables, fruits, and cereals), obtaining appreciable recovery rates (65-90%) and good reproducibility (RSD ≤ 6%, n = 3); the selectivity towards other mycotoxins was proved and the data obtained compared with commercial immunoaffinity columns. The proposed strategy can be considered an alternative affordable approach to the classical immunoaffinity columns, since it is more selective and better performing.

Optimisation, validation and comparison of methods for aflatoxin M1 determination in cheese

Accurate and reliable determination of aflatoxins in food is important for human health. There is no standard method for the determination of aflatoxin M1 (AFM1) in cheese, a more complex matrix, compared with milk. In this research, we applied two analytical detection techniques (a standard accredited HPLC method for AFM1 in milk and a commercial ELISA method), with three extraction methods, on two different cheese matrices, with repeated tests and appropriate statistical analysis. The examined methods were sensitive, with detection limits from 10 to 25 ng/kg. The validation parameters were in accordance with current regulations and therefore could be successfully used for the determination of AFM1 in cheese.

Determination of aflatoxins in food using liquid chromatography coupled with electrospray ionization quadrupole time of flight mass spectrometry (LC-ESI-QTOF-MS/MS)

A rapid, reliable and confirmatory method has been developed to determine the levels of aflatoxins B1, B2, G1, and G2 in barley, wheat, soybean, corn, peanut and peanut butter. The method is based on a single extraction step followed by liquid chromatography coupled with electrospray ionization quadrupole time of flight mass spectrometry (LC-ESI-QTOF-MS/MS). The extraction, dilution, chromatographic and MS conditions were optimized in order to increase the sample throughput and sensitivity. The quantification of the analytes was measured by performing low-energy collision induced tandem mass spectrometry (CID-MS/MS) using the product ion scan mode. The calibration curves were linear over the range from 0.195 to 50.000 μg/kg for aflatoxin B1, G1 and from 0.029 to 15.000 μg/kg for aflatoxin B2, G2. The correlation coefficients (r2) were higher than 0.998. The overall recoveries for aflatoxins were in the range of 57.1–108.7% at spike levels of 0.6–15.0 μg/kg with relative standard deviations lower than 16%. The limit of quantification (LOQ) for aflatoxins is less than 0.8 μg/kg.

A neutral red desiccated coconut agar for rapid detection of aflatoxigenic fungi and visual determination of aflatoxins

Desiccated coconut agar is the conventional medium used for the detection of aflatoxigenic fungi and direct visual determination of aflatoxins. In this study, an improved medium was developed by the incorporation of 0.2% (v/v) neutral red dye into desiccated coconut agar. The medium was formulated by a 2×3 factorial design of neutral red and phenol red stains at three concentration levels. The formulated medium was evaluated for performance by screening for the minimal time required by each Aspergillus species to produce pigments and fluorescence of agar. The medium was also employed for detection of aflatoxigenic fungi and direct visual determination of aflatoxins in foods and fish-meal. The neutral red desiccated coconut agar (NRDCA) as compared to the conventional desiccated coconut agar (DCA) had a light pink background as opposed to the white background of the DCA which often interferes with the visibility of fluorescence. The time of pigmentation and fluorescence production on NRDCA was 28 and 38 h respectively as compared with 33 and 44 h of DCA and 41 and 48 h of palm kernel agar (PKA: an alternative culture medium for cultivation of aflatoxigenic fungi with a reddish pink background). Furthermore, aflatoxigenic moulds were detected in all food commodities and fish-meal after 60 hours of incubation. The highest percentage of aflatoxigenic moulds (62.5%) was detected in yam flour with NRDCA while the lowest percentage (4.46%) was detected with PKA on rice. In addition, aflatoxins were produced in high amounts in food commodities in which aflatoxigenic moulds were detected and there was a significant positive correlation (r=0.4, P<0.05) between the isolates and aflatoxin concentration of the food samples. Rice (a major staple food for Nigerians) had the highest total aflatoxin concentration of 140, 220 and 205 µg/kg on DCA, NRDCA and PKA respectively, while ‘gari’ had the least concentration of 45, 50 and 40 µg/kg. These values were far above the NAFDAC recommended level of 10 µg/kg for unprocessed foods in Nigeria and therefore a source of concern. In addition the study also reveals that Aspergillus nomius can produce aflatoxins B1 in copious amounts on NRDCA, contrary to previous reports of its production in minute quantities on laboratory media. The benefit of this study lies in the rapid analysis and simplified technique for the detection of aflatoxigenic fungi and visual determination of aflatoxins.

Adaptation of glass columns for clean-up RP-HPLC determination of aflatoxins with post-column derivatisation with bromine and fluorescence detection.

Consumption of aflatoxin-contaminated foods in developing countries may not only cause cancer in the general population, but is also associated with stunting and immune suppression in children. Accurate determination of aflatoxins in foods is important for the prevention of the above health risks. This study aimed to evaluate the possibility of using cheap florisil-packed glass columns instead of relatively complex and expensive pre-packed cartridges for afltoxin clean-up prior to HPLC analysis. Additionally, effects of aflatoxin extract storage temperature, and themoisture content of florisil on aflatoxin recovery were assessed. Results showed that the standard curve was highly linear as depicted by the high correlation coefficient for all four aflatoxins [0.99, 0.99, 0.97 and 0.98, respectively for AFG1, AFB2 and AFB1 and AFG2. The retention times over a total run time of 20 minutes were 14.8 (SD 0.4), 12.4 (SD 0.1), 10.7 (SD 0.3) and 9.1 (SD 0.3) minutes, respectively for AFB1, AFB2, AFG1 and AFG2. Florisil powder that had been dried for 12 hours at 100 oC, then hydrated with water at 5% (w/v) before packing on glass columns gave slightly higher recovery for all aflatoxins (100.3% AFB1, 93.1% AFB2, 83.8% AFG1 and 71.1% AFG2) compared to the pre-packed cartridges (96.4% AFB1, 91.5% AFB2, 80.6% AFG1 and 66.9% AFG2). There was 10% and 20% aflatoxin loss at 4 oC and 22 oC, respectively. After 6 hours of storage, no aflatoxin degradation was observed at – 18 oC. There was 0-5% aflatoxin loss at –18 oC after 12 hours, while at 4 oC and 22oC, the loss range was 20-50% and 30-55%, respectively. Aflatoxin recoveries ranged from 120-160%, 90-110%, 80-98%, and 55-74% for florisil which had been hydrated with water at 2.5%, 5% and 10%, respectively. Glass columns are cheap and can be used many times and are a feasible and sustainable alternative to pre-packed florisilcartridges for aflatoxin clean up to ensure food safety, especially in resource poor settings.

LC–MS/MS multi-method for mycotoxins after single extraction, with validation data for peanut, pistachio, wheat, maize, cornflakes, raisins and figs

Mycotoxin analysis is usually carried out by high performance liquid chromatography after immunoaffinity column cleanup or in enzyme-linked immunosorbent assay tests. These methods normally involve determination of single compounds only. EU legislation already exists for the aflatoxins, ochratoxin A and patulin in food, and legislation will come into force for deoxynivalenol, zearalenone and the fumonisins in 2007. To enforce the various legal limits, it would be preferable to determine all mycotoxins by routine analysis in different types of matrices in one single extract. This would also be advantageous for HACCP control purposes. For this reason, a multi-method was developed with which 33 mycotoxins in various products could be analysed simultaneously. The mycotoxins were extracted with an acetonitrile/water mixture, diluted with water and then directly injected into a LC–MS/MS system. The mycotoxins were separated by reversed-phase HPLC and detected using an electrospray ionisation interface (ESI) and tandem MS, using MRM in the positive ion mode, to increase specificity for quality control. The following mycotoxins could be analysed in a single 30-min run: Aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin, α-zearalenol, α-zearalanol, β-zearalanol, sterigmatocystin, cyclopiazonic acid, penicillic acid, fumonisins B1, B2 and B3, diacetoxyscirpenol, 3- and 15-acetyl-deoxynivalenol, zearalanone, ergotamin, ergocornin, ergocristin, α-ergocryptin, citrinin, roquefortin C, fusarenone X, nivalenol, mycophenolic acid, alternariol and alternariol monomethyl ether. The limit of quantification for the aflatoxins and ochratoxin A was 1.0 µg kg−1 and for deoxynivalenol 50 µg kg−1. The quantification limits for the other mycotoxins were in the range 10–200 µg kg−1. The matrix effect and validation data are presented for between 13 and 24 mycotoxins in peanuts, pistachios, wheat, maize, cornflakes, raisins and figs. The method has been compared with the official EU method for the determination of aflatoxins in food and relevant FAPAS rounds. The multi-mycotoxin method has been proven by the detection of more than one mycotoxin in maize, buckwheat, figs and nuts. The LC–MS/MS technique has also been applied to baby food, which is subject to lower limits for aflatoxin B1 and ochratoxin A, ergot alkaloids in naturally contaminated rye and freeze-dried silage samples.

Use of Isotope-Labeled Aflatoxins for LC-MS/MS Stable Isotope Dilution Analysis of Foods

Aflatoxins are a group of very carcinogenic mycotoxins that can be found on a wide range of food commodities including nuts, cereals, and spices. In this study, the first LC-MS/MS stable isotope dilution assay (SIDA) for the determination of aflatoxins in foods was developed. The development of this method was enabled by easily accessible isotope-labeled (deuterated) aflatoxins B2 and G2, which were synthesized by catalytic deuteration of aflatoxin B1 and G1, purified, and well-characterized by NMR and MS. All four aflatoxins of interest (B1, B2, G1, and G2) were quantified in food samples by using these two labeled internal standards. The response factors (RF) of the linear calibrations were revealed to be matrix independent for labeled aflatoxin B2/aflatoxin B2 and labeled aflatoxin G2/aflatoxin G2. For labeled aflatoxin B 2/aflatoxin B 1 and labeled aflatoxin B2/aflatoxin G1 matrix-matched calibration was performed for the model matrices almonds and wheat flour, showing significant differences of the RFs. Limits of detection (LOD) were determined by applying a statistical approach in the presence of the two model matrices, yielding 0.31 microg/kg (aflatoxin B1), 0.09 microg/kg (aflatoxin B2), 0.38 microg/kg (aflatoxin G1), and 0.32 microg/kg (aflatoxin G2) for almonds (similar LODs were obtained for wheat flour). Recovery rates were between 90 and 105% for all analytes. Coefficients of variation (CV) of 12% (aflatoxin B1), 3.6% (aflatoxin B2), 14% (aflatoxin G1), and 4.8% (aflatoxin G2) were obtained from interassay studies. For further validation, a NIST standard reference food sample was analyzed for aflatoxins B1 and B2. The method was successfully applied to determine trace levels of aflatoxins in diverse food matrices such as peanuts, nuts, grains, and spices. Aflatoxin contents in these samples ranged from about 0.5 to 6 microg/kg.

Atmospheric pressure photo-ionization liquid chromatography/mass spectrometric determination of aflatoxins in food

A comparison between electrospray ionization (ESI) and the recent introduced atmospheric pressure photo-ionization (APPI) technique is described for the liquid chromatography/mass spectrometric (LC/MS) determination of aflatoxin B1, B2, G1 and G2 in food. For the optimization of APPI, several APPI ion source parameters were examined. Furthermore, the chemical noise and signal suppression of ESI and APPI by sample matrix interference were investigated. The results indicated that APPI has the low chemical noise and the little signal suppression in comparison with ESI. The mean recovery of each aflatoxin from peanuts, corn, nutmeg and red pepper sample spiked at 1 ng g−1 ranged from 89 to 105% with relative standard deviations of 3.3–5.1%. The limit of detection (S/N = 3) in all food was in the range 0.11–0.5 ng g−1. From these studies, it is suggested that APPI can be used as an additional tool in food residue analysis.

New strategies for the screening and determination of aflatoxins and the detection of aflatoxin-producing moulds in food and feed

This article describes the current analytical approaches for the detection and determination of aflatoxins in food. Emphasis is on the development of simplified and cost-effective but efficient analytical methods for fast screening or for precise determination in order to comply with legislative requirements. An overview is also given of methods for the detection of aflatoxin-producing moulds.

Determination of aflatoxins in food by overpressured-layer chromatography

Legislative measures for monitoring and control of aflatoxin levels in foods and foodstuffs have been introduced in many countries. The aim of the present work was to make developments in the field of aflatoxin analysis, focusing upon the use of overpressured-layer chromatography (OPLC) for quantitative determination. OPLC methods have been developed for the determination of aflatoxins B 1, B 2, G 1, G 2 in different foods. These methods are suitable for sample clean-up and separation as well. Using OPLC we could analyze 10 samples simultaneously. The methods were investigated with fish, corn and wheat samples spiked with 2–10 ng/g aflatoxins. Quantitative evaluation of aflatoxins was accomplished by densitometry. Average recoveries from each food were greater than 73%. The OPLC technique seems to be a rapid, reproducible and cost-effective analysis for quantitative determination of aflatoxins in foods.

Determination of aflatoxins in food using LC/MS/MS

A liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometric method is described for the determination of aflatoxins B1, B2, G1 and G2 in food with the use of aflatoxin M1 as an internal standard. The method works well with matrices such as those of figs and peanuts, but there are problems with spices, due to limitations of the clean-up method used.

Determination of aflatoxins in food by use of an automatic work station

An automated HPLC method with postcolumn derivatization is described for the determination of aflatoxins B 1, B 2, G 1 and G 2 after an immunoaffinity column clean-up. It can be used for the determination of aflatoxins in a variety of foodstuffs such as nuts, nut-like products (pistachios, almonds, etc.) and dried fruit. The aflatoxins are extracted with methanol-water, followed by a filtration step. Dilution of the extract, mixing, immunoaffinity column clean-up, elution of the aflatoxins and optional on-line HPLC are performed by an automatic work station (Zymark BenchMate). The subsequent HPLC analysis includes a postcolumn derivatization step with iodine solution and fluorimetric detection. The method compared well with manual techniques and another automated method.

Determination of Aflatoxins in Foods Using HPLC and a Commercial ELISA System

A commercially available enzyme linked immunosorbent assay (ELISA) system and a high performance liquid chromatography (HPLC) method were used simultaneously to analyze 178 samples of foodstuffs for total aflatoxins. High correlation coefficient values between results of the two methodologies were obtained (>0.96) with nuts, nut products, peanuts, and peanut butter. However, poor correlation was obtained from results of cereals and grain samples. The ELISA system demonstrated a high degree of reproducibility between wells (p<0.01). Of the 35 samples of nut and nut products examined, aflatoxins were detected in five samples at levels ranging from 40 to 276 μg/kg. However, of 73 peanut and peanut butter samples only one peanut sample contained 61 μg/kg, and of 70 samples of processed cereal and grain, three samples contained 6–10 μg/kg total aflatoxins. Both methods were also applied to six peanut meal samples, with known aflatoxin concentrations, provided in an interlaboratory collaborative trial. The results obtained by the HPLC and ELISA methods were in close agreement with the mean results obtained by the participating laboratories.

Reverse Phase High Pressure Liquid Chromatographic Determination of Aflatoxins in Foods

A method for determining aflatoxins by high pressure liquid chromatography (HPLC) with fluorescence detection after CB extraction and cleanup has been applied to various foods. Recoveries at 1--15 ppb levels from green coffee and peanut butter was 72--85 and 74--104%, respectively. Precision of the method has been tested for peanut butter. Other products to which the method has been successfully applied include tree nuts, seeds, grains, chocolate-covered peanut butter candy, and roasted, salted-in-shell peanuts. High levels of aflatoxins found in several samples of nuts by this method have been verified by the official thin layer chromatographic (TLC) method. The advantages of this HPLC method are speed, precision, sensitivity, selectivity, and immediate chemical confirmation of aflatoxins B1 and G1. None of the products analyzed required special cleanup procedures. Preparative-scale HPLC was used to isolate purified B1 for toxicity testing.

Determination of aflatoxins in food by absorption spectrometry

A method has been developed for the determination of aflatoxins B1 and G1 in laboratory animal food. The procedure involves extraction of the food with acetone and removal of interfering substances from the extract by chromatography on columns of silica gel, followed by thin-layer chromatography on silicic acid. The separated aflatoxins B1 and G1 are estimated spectrophotometrically. Recoveries of added aflatoxins range from 40 to 67% at a sensitivity of 0·03 ppm.