Determination Of Aflatoxin M1 Research Articles

Exposure assessment on aflatoxin M1 from milk and dairy products-relation to public health

Aflatoxin M1 (AFM1) contaminates milk and dairy products that are widely consumed foods. Therefore, there is increased concern for human exposure to the toxic action and the negative health effects that AFM1 presents. The aim of this study was to assess the contamination levels with AFM1, of dairy products available in the Greek market (Thessaly), and also to estimate the relevant nutritional exposure. A total of 52 infant milk, 25 Feta cheese and 32 pasteurized milk samples were randomly selected from the market. The determination of AFM1 was based on indirect immunoenzymatic ELISA method with two analytical packages; namely Tecna (Italy) and Prognosis Biotech (Greece), for comparison purposes, strictly following the manufacturer's instructions. The estimated daily intake (EDI) of AFM1 through dairy consumption and the relevant hazard index (HI) were calculated. The levels of AFM1, in all samples tested, were below the tolerable maximum level of 50ng/L for milk and 25ng/L for infant formulas (EC Regulation 1881/2006), while concentrations were very low (0.23-9.38ng/L for infant formula, 0.54-4.09ng/L for cheese and 0.20-17.84ng/L for pasteurized milk). The exposure assessment revealed that infants, toddlers and children were the most exposed groups in AFM1 via milk and formula consumption (0.65, 0.35 and 0.35 respectively). The HI was also noticed higher in those ages, with the maximum value to reach 1.90. Though, for feta cheese the exposure was calculated as very low (EDI: 0.01 and HI: 0.05). Comparing the two analytical packages used, no statistically significant difference was found (p-value >0.05). Milk and dairy products tested were found safe for consumption, regarding the AFM1 levels. From a public health perspective, the results are satisfactory, given that the population exposure to AFM1, based on the nutrient profile, was rather limited.

Correction: Determination of aflatoxin M1 using an aptamer-based biosensor immobilized on the surface of dendritic fibrous nano-silica functionalized by amine groups

Correction for ‘Determination of aflatoxin M1 using an aptamer-based biosensor immobilized on the surface of dendritic fibrous nano-silica functionalized by amine groups’ by Houman Kholafazad kordasht et al., Anal. Methods, 2019, 11, 3910–3919, DOI: 10.1039/C9AY01185D.

A novel electrochemical aptasensor based on layer-by-layer assembly of DNA-Au@Ag conjugates for rapid detection of aflatoxin M1 in milk samples

Aflatoxin M1 (AFM1) is a common toxin in dairy products that causes acute and chronic human health disorders. Thus, the development of a rapid and accurate AFM1 detection method is of vital importance for food safety monitoring. This work was to develop a novel electrochemical aptasensor for sensitive and specific determination of AFM1. The dendritic-like nanostructure was formed on the gold electrode surface by layer-by-layer assembly of gold-silver core-shell nanoparticles modified with DNA conjugates. In the presence of AFM1, the specific recognition between AFM1 and Apt caused the disassociation of the DNA controlled dual Au@Ag conjugates from the surface of the electrode, causing less methylene blue to bind to the surface and weakening the electrochemical signal. The more AFM1 there is, the weaker the electrochemical signal. Transmission electron microscope results showed that the successfully synthesized Au@Ag nanoparticles exhibited a core-shell structure with Au as core and Ag as shell, and their average diameter was about 30 nm. Under optimal conditions, the electrochemical aptasensor showed a wide detection ranging from 0.05 ng mL-1 to 200 ng mL-1, and a low detection limit of 0.02 ng mL-1. Moreover, the proposed strategy has been successfully applied to the detection of AFM1 in cow, goat, and sheep milk samples with satisfactory recoveries ranging from 91.10% to 104.05%. This work can provide a novel rapid detection method for AFM1, and also provide a new sensing platform for the detection of other toxins.

Development of Gold Nanoparticles Decorated Molecularly Imprinted–Based Plasmonic Sensor for the Detection of Aflatoxin M1 in Milk Samples

Aflatoxins are a group of extremely toxic and carcinogenic substances generated by the mold of the genus Aspergillus that contaminate agricultural products. When dairy cows ingest aflatoxin B1 (AFB1)−contaminated feeds, it is metabolized and transformed in the liver into a carcinogenic major form of aflatoxin M1 (AFM1), which is eliminated through the milk. The detection of AFM1 in milk is very important to be able to guarantee food safety and quality. In recent years, sensors have emerged as a quick, low–cost, and reliable platform for the detection of aflatoxins. Plasmonic sensors with molecularly imprinted polymers (MIPs) can be interesting alternatives for the determination of AFM1. In this work, we designed a molecularly–imprinted–based plasmonic sensor to directly detect lower amounts of AFM1 in raw milk samples. For this purpose, we prepared gold–nanoparticle–(AuNP)−integrated polymer nanofilm on a gold plasmonic sensor chip coated with allyl mercaptan. N−methacryloyl−l−phenylalanine (MAPA) was chosen as a functional monomer. The MIP nanofilm was prepared using the light–initiated polymerization of MAPA and ethylene glycol dimethacrylate in the presence of AFM1 as a template molecule. The developed method enabled the detection of AFM1 with a detection limit of 0.4 pg/mL and demonstrated good linearity (0.0003 ng/mL–20.0 ng/mL) under optimized experimental conditions. The AFM1 determination was performed in random dairy farmer milk samples. Using the analogous mycotoxins, it was also demonstrated that the plasmonic sensor platforms were specific to the detection of AFM1.

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.

Preparation of matrix reference material of aflatoxin M1 in milk powder

The preparation and characterization of a matrix reference material for aflatoxin M1 in milk powder is described. Milk powder samples containing aflatoxin M1 were found in the screened natural milk samples. The preparation of matrix milk powder samples was developed through steps by vacuum concentration, spray-drying, cooling, mixing, weighing and packaging, and irradiation. This paper has developed a method for the determination of aflatoxin M1 in milk powder by isotope dilution liquid chromatography–tandem mass spectrometry (ID-LC-MS/MS) with immunoaffinity extraction as the pretreatment method, which was validated and determined the content of aflatoxin M1 in milk powder accurately. The homogeneity was studied by analyzing in triplicate for their aflatoxin M1 contents by ID-LC-MS/MS using random sampling schemes. The short-term stability was evaluated at three different storage temperatures (4, 25 and 50 °C) for 0, 3, 5, 7, 14 days, the long-term stability study comprised of measurement after 0, 1, 3, 6, 9, 12 months after storage at 4 °C, and then, analyses were investigated by ID-LC-MS/MS. After the combination determination of 8 laboratories, the final quantity value and expanded uncertainty of matrix reference material of aflatoxin M1in milk powder were: 0.49 ± 0.11 μg/kg, k = 2, P = 0.95, and the matrix reference material is homogenous and stable at 4 °C for at least 12 months, which can provide quality assurance for the later detection accuracy.

Determination of Aflatoxin M1 in Raw Milk Using an HPLC-FL Method in Comparison with Commercial ELISA Kits-Application in Raw Milk Samples from Various Regions of Greece.

The highly toxic Aflatoxin M1 (AFM1) is most often detected in milk using an Enzyme-Linked-Immunosorbent Assay (ELISA) for screening purposes, while High-Performance Liquid Chromatography with Fluorescence Detector (HPLC-FL) is the reference method used for confirmation. The aim of the present study was the comparison between three commercially available ELISA kits and a newly developed HPLC-FL method for the determination of the AFM1 in milk samples. The developed HPLC-FL method was validated for the AFM1 and Aflatoxin M2 (AFM2), determining the accuracy, precision, linearity, decision limit, and detection capability with fairly good results. All three ELISA kits were also validated and showed equally good performance with high recovery rates. Moreover, the Limit Of Detection (LOD) and Limit Of Quantification (LOQ) values were found to be significantly lower than the Maximum Residue Limit (MRL) (50 ng kg−1). After the evaluation of all three commercial kits, the ELISA kit with the optimum performance along with the HPLC method was used for the determination of AFM1 in raw cow’s, goat’s, and sheep’s milk samples (396) obtained from producers in different regions of Greece. The evaluation of both methods showed that this ELISA kit could be considered as a faster and equally reliable alternative method to HPLC in routine analysis for the determination of AFM1 in milk.

Combination of solvent extraction with deep eutectic solvent based dispersive liquid-liquid microextraction for the analysis of aflatoxin M1 in cheese samples using response surface methodology optimization.

A new extraction procedure based on combination of a solvent extraction and deep eutectic solvent based dispersive liquid-liquid microextraction has been introduced for the extraction of aflatoxin M1 from cheese samples. In this method, acetonitrile, deionized water, and n-hexane are added onto the sample and vortexed. Owning to different affinities of the substances in cheese toward the mentioned solvents, an efficient and selective extraction of the analyte is done in the acetonitrile phase. After centrifugation, the acetonitrile phase is removed and mixed with a new hydrophobic deep eutectic solvent prepared from N,N-diethanol ammonium chloride and carvacrol as an extraction solvent. The mixture is injected into deionized water, and a cloudy solution is obtained. Eventually, an aliquot of the organic phase is injected into high-performance liquid chromatography-fluorescence detection. After optimizing the effective parameters with the response surface methodology and a quadratic model, limits of detection and quantification were 0.74 and 2.56ng/kg, respectively. The obtained extraction recovery and enrichment factor were 94% and 94, respectively. Also, intra- (n=6) and interday (n=4) precisions were less than or equal to 8.6% at a concentration of 5ng/kg. The suggested method was applied to determine aflatoxin M1 in different cheese samples.

Encapsulation of hemin in Fe-based metal-organic frameworks and its application for the direct determination of aflatoxin M1

A label-free electrochemical aptasensor was constructed for the sensitive and selective determination of AFM1. For preparation of the aptasensor, the AFM1 aptamer was immobilised on the surface of a glassy carbon electrode modified with hemin encapsulated in Fe-based metal-organic frameworks (hemin@Fe-MIL-101). The morphology and the structure of Fe-MIL-101 and hemin@Fe-MIL-101 were evaluated by scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray powder diffraction and Brunauer-Emmett-Teller-N2 sorption methods. Electrochemical impedance spectroscopy and cyclic voltammetry were performed to monitor the fabrication process of the electrochemical aptasensor. The electrochemical reduction current of hemin encapsulated in Fe-MIL-101 serves as a signal for the quantitative determination of AFM1. Differential pulse voltammetry was done to determine the AFM1 concentration in the linear range of 1.0×10-1-100.0 ng/ml. The detection limit of AFM1 was estimated to be 4.6×10-2 ng/ml. Finally, the fabricated aptasensor was applied to determine AFM1 in raw and boiled milk samples.

Determination of Aflatoxin M1 in Liquid Milk, Cheese, and Selected Milk Proteins by Automated Online Immunoaffinity Cleanup with Liquid Chromatography‒Fluorescence Detection.

Aflatoxins are secondary metabolites produced by a number of species of Aspergillus fungi. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 and is found in the milk of cows fed with feed spoilt by Aspergillus species. AFM1 is carcinogenic, especially in the liver and kidneys, and mutagenic, and is also an immunosuppressant in humans. A high-throughput method for the quantitative analysis of AFM1 that is applicable to liquid milk, cheese, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), and whey powder (WP) was developed and validated. AFM1 in cheese, milk, and protein products is extracted using 1% acetic acid in acetonitrile with citrate salts. The AFM1 in the resulting extract is concentrated using RIDA®CREST/IMMUNOPREP® ONLINE cartridges followed by quantification by HPLC‒fluorescence. The method was shown to be accurate for WP, WPC, WPI, MPC, liquid milk, and cheese, with acceptable recovery (81-112%) from spiked samples. Acceptable precision for WP, WPC, WPI, MPC, liquid milk, and cheese was confirmed, with repeatabilities of 4-12% RSD and intermediate precisions of 5-13% RSD. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. An international proficiency scheme (FAPAS) cheese sample showed that this method gave results that were comparable with those from other methods. A method for high-throughput, routine testing of AFM1 is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit-for-purpose. An automated online immunoaffinity cleanup HPLC‒fluorescence method for milk proteins, cheese, and milk was developed and single-laboratory validated. It allows for high-throughput analysis of AFM1 and can be used for the analysis of AFM1 in whey protein products.

In situ growth of covalent organic frameworks TpBD on electrode for electrochemical determination of aflatoxin M1

An in situ growth strategy for construction of covalent organic frameworks (COFs) TpBD on glassy carbon electrode using p-aminobenzoic acid and ethylenediamine as connector is introduced. The fabricated electrode was used for electrochemical biosensing of aflatoxin M1 in milk samples combined with magnetic nanoparticles, which acted as probe carriers. The developed electrochemical biosensor exhibited high selectivity and sensitivity toward aflatoxin M1 with a detection limit of 0.15 ng/mL (S/N = 3). Moreover, the covalent bonding of TpBD on the glassy carbon electrode has good stability, which retained 76% of the initial signal after 15 days storage at room temperature. This work provides a valuable case for in situ growth of COFs TpBD on the electrode, and the proposed synthetic route is expected to provide a powerful tool for the wide application of other functional COFs in electrochemical biosensor.

Determination of aflatoxin M1 and deoxynivalenol biomarkers in infants and children urines from Bangladesh

The mycotoxins aflatoxin B1 (AFB1) and deoxynivalenol (DON) are found worldwide in crops and dietary staples. The prevalence and levels of these contaminants can vary greatly, and data in Bangladeshi food commodities are scarce. To characterize human exposure, we have conducted biomonitoring, analyzing AFM1 (a metabolite of AFB1) and DON levels in urines of adult cohorts in Bangladesh. Yet, AFM1 and DON occurrence has not been studied in the very young population of this country. Thus, the same methods, HPLC-FD for AFM1 and LC–MS/MS for DON analysis, were now applied to determine these biomarkers in urines of infants (n = 49) and young children (n = 105) in Rajshahi and Dhaka district. Overall, AFM1 and DON detection frequency was 43.5% and 33.4%, with 34.7% and 11.5% in infant and 47.6% and 39.4% in children urines, respectively. The mean AFM1 levels in all infants (9.1 ± 14.3, max 55.6 pg/mL) and children (8.8 ± 12.9, max 75.3 pg/mL) were not significantly different. The AFM1 mean level was slightly higher in Dhaka (9.4 ± 12.4) compared to Rajshahi (8.5 ± 13.9 pg/mL) district. The average DON level was about 2-fold higher in infant (3.8 ± 2.9, max 6.8 ng/mL) than children urines (1.6 ± 1.8, max 8.6 ng/mL), and higher in Rajshahi (2.1 ± 2.3 ng/mL) than Dhaka (1.4 ± 1.6 ng/mL) district. The biomarker-based estimated average daily DON intake (29.6 ± 108.3 ng/kg bw in infants and 36.4 ± 81.8 ng/kg bw in children) or the maximum exposure (560 ng/kg bw) do not exceed the current maximum provisional tolerable daily intake value of 1 µg/kg bw for DON, although DON exposure in infants and children is higher than that of Bangladeshi adults. The AFM1 urine levels in young children are somewhat lower than those found previously in adult cohorts in Bangladesh, but the frequent detection of this biomarker for AFB1 exposure raises further concerns, also for this vulnerable part of the population. Therefore, continuous surveillance for aflatoxins in Bangladeshi food commodities is clearly required, first to identify major sources of intake and then to reduce exposure.

Determination of Aflatoxin M1 in Raw Milk from Different Provinces of Ecuador.

Aflatoxin M1 (AFM1) is a mycotoxin from Aspergillus flavus and A. parasiticus, classified as carcinogenic and hepatotoxic. The objective of the present investigation was to determine its presence in raw milk from north-central Ecuador, constituted by the provinces of Pichincha, Manabí, and Santo Domingo de los Tsáchilas. These areas represent approximately 30% of Ecuadorian milk production. By the end of the investigation, a total of 209 raw milk samples were collected, obtained both during the dry (June and August) and rainy seasons (April and November) of 2019. AFM1 concentrations were measured with lateral flow immunochromatographic assays, and 100% of the samples were positive for this mycotoxin, presenting a mean value of 0.0774 μg/kg with a range of 0.023 to 0.751 μg/kg. These AFM1 levels exceeded the European Union regulatory limit of 0.05 μg/kg in 59.3% (124/209) of samples, while only 1.9% (4/209) exceeded the Ecuadorian legal limit of 0.5 μg/kg. By using non-parametric tests, significant differences were determined (p ≤ 0.05) between the provinces for months of study, climatic season (being higher in the dry season), and climatic region (greater in the coast region). On the other hand, there were no significant differences (p ≥ 0.05) between the types of producers or between production systems. Therefore, AFM1 contamination in raw milk does not present a serious public health problem in Ecuador, but a monitoring and surveillance program for this mycotoxin in milk should be developed to prevent consumer health problems.

Determination of aflatoxin M1 in milk and milk powder using immuno-affinity column and fluorescence measurement

The method using immuno-affinity column clean-up and fluorescence measurement was evaluated for detection of aflatoxin M1 in milk and milk powder. Two kinds of commercial immuno-affinity columns used were Afla Test-P Column and AFLAPREP M. The results are shown below : (1) By using Afla Test-P Column and Fluorometer, the average recoveries of aflatoxin M1 from defatted milk spiked at the levels of 0.5 and 1.0ppb were 84.4±7.0% and 94.2±9.5%. The detection limit was 0.1ppb. This method is easy and suitable for multi-sample treatment. (2) By using AFLAPREP M, the average recoveries of aflatoxin M1 from defatted milk spiked at the levels of 0.1, 0.5 and 1.0ppb were 84.7±2.7 %, 88.7±1.9%and 89.2±4.2%. The detection limit was 0.05ppb. This method is more sensitive and can be used at the laboratory equipped with HPLC. (3) 25 samples of milk and 25 samples of milk powder were perchased from supermarkets in Taipei from July 1994 to June 1995 and tested by AFLAPREP M. Aflatoxin M1 was detected in 3 samples of milk. The toxin contents were 0.33, 0.12, 0.08ppb respectively. Milk powders were reconstituted and tested, no aflatoxin M1 was detected in 25 samples.

Bimodal porous silica nanomaterials as sorbents for an efficient and inexpensive determination of aflatoxin M1 in milk and dairy products

An extraction procedure was developed for the determination of aflatoxin M1 in milk and dairy products. A sorbent based on UVM-7 mesoporous silica was used as solid phase for the sample clean-up, and the analyte determination was carried out by HPLC coupled to a fluorescence detector. The material architecture was characterized by transmission electronic microscopy, X-ray diffraction, 29Si NMR and nitrogen adsorption-desorption. After the optimization of extraction parameters, the influence of the matrix has been evaluated, obtaining recoveries in the range 78–105% for whole and skimmed milk and yogurt matrix. The reusability of the material was also proved. The sensitivity of the method was also evaluated, and a LOQ (0.015 μg kg−1) below the European legislation limit was obtained. The procedure was successfully applied for the determination of aflatoxin M1 in real samples. The results were compared with those obtained with a reference method, being the results statistically comparable.

Health risk assessment of aflatoxin M1 in infant formula milk in IR Iran

In this study, two accurate, precise, selective and sensitive methods were developed for determining aflatoxin M1 (AFM1) in infant formula milk using immunoaffinity column clean-up followed by high performance liquid chromatography (HPLC) with fluorescence detection. The validated methods were used for determination of AFM1 in 29 samples of 6 different infant formula milk brands and the risk of AFM1 in infants aged zero to 6 months old was assessed using cancer risk, Margin of Exposure (MOE) and Hazard Index (HI). Only one sample (3.4%) was contaminated with AFM1. Although the results showed that MOE values for the mean and median exposure to AFM1 was <10,000 in infants, the additional cancer risk due to mean and median exposure to AFM1 in infant <6 months were 0.00010 and 0.00012 additional cases per year per 105 individuals, respectively, which indicates no health concern. In addition, HI values for the mean and median exposure to AFM1 for infants were quite below one which indicates no health concern. To the best of our knowledge, this is the first report on risk assessment of AFM1 in infant formula milk consumed by Iranian infants <6 months old, presenting a low risk for the evaluated groups.

Aflatoxin M1 Determination in Infant Formulae Distributed in Monterrey, Mexico.

The occurrence of aflatoxin M1 (AFM1) in infant formulae commercialized in the metropolitan area of Monterrey (Nuevo León, Mexico) was determined by using immunoaffinity column clean-up followed by HPLC determination with fluorimetric detection. For this, 55 infant formula powders were classified in two groups, starter (49 samples) and follow-on (6 samples) formulae. Eleven of the evaluated samples (20%) presented values above the permissible limit set by the European Union for infant formulae (25 ng/L), ranging from 40 to 450 ng/L. The estimated daily intake (EDI) for AFM1 was determined employing the average body weight (bw) of the groups of age in the ranges of 0–6 and 6–12 months, and 1–2 years. The results evidenced high intake values, ranging from 1.56 to 14 ng/kg bw/day, depending on the group. Finally, with the EDI value, the carcinogenic risk index was determined, presenting a high risk for all the evaluated groups. Based on these results, it is a necessary extra effort by the regulatory agencies to reduce the AFM1 presence in infant formulae consumed in Mexico.

Rapid and ultrasensitive method for determination of aflatoxin M1 in milk

ABSTRACT Aflatoxin M1 (AFM1) in milk is a problem of great concern. Current methods of detection require large instruments and need specific test sites. Therefore, it is necessary to establish a fast, convenient, and accurate detection method for AFM1. We established a system based on fluorescent microspheres containing a Eu3+ chelate named AFM1-POCT. These components comprised the AFM1-POCT kits. After refrigeration at 4°C for 12 months, the intra and inter assay coefficients of variability (CVs) for the kits were 4% and 5%, respectively. AFM1-POCT compared well with ultra-high-performance liquid chromatography (UHPLC) determination in the range 0.0121–2 μg/kg (paired samples test, P > 0.05). Aflatoxin B1 and G1 do not react with aflatoxin M1. By using the AFM1-POCT method, the detection time is shortened to 5 min, the accuracy is comparable to that of UHPLC, and convenience and range of application are improved.

An electrochemical immunosensor based on a combined amplification strategy with the GO–CS/CeO2–CS nanocomposite for the detection of aflatoxin M1

Herein, a sensitive electrochemical immunosensor modified with graphene oxide–chitosan (GO–CS) and cerium oxide–chitosan (CeO2–CS) using screen-printed electrodes (SPEs) was developed for the determination of aflatoxin M1(AFM1) in milk.

Electrochemical Aptasensor with Layer‐by‐layer Deposited Polyaniline for Aflatoxin M1 Voltammetric Determination

AbstractMycotoxins are highly toxic metabolites of some fungi that frequently contaminate water, food and feed and hence cause several human and animal diseases. In this work, a new approach to the fast and reliable determination of aflatoxin M1 (AFM1) in water and milk has been proposed with reagent free protocol of signal measurement. For this purpose, DNA aptamer selective to AFM1 was entrapped between two thin layers of polyaniline (PANI) electrodeposited on glassy carbon electrode. The incubation of the aptasensor in the AFM1 solution results in remarkable decrease of the PANI intrinsic activity monitored by direct current voltammetry or electrochemical impedance spectroscopy. Appropriate calibration curves were linear in the range from 3 to 90 ng/L with limit of detection (LOD) 1–5 ng/L depending on the measurement mode. Mechanism of signal generation involves shielding electrostatic interactions between the PANI and aptamer in the surface layer and variation of its redox activity attributed to the emeraldine form of PANI. Selectivity of the response was proved by similar experiments with aflatoxin B1 and ochratoxin A and by comparison of the results with those obtained with non‐specific aptamer in the sensing layer. Simple protocol for milk pretreatment has been proposed for reliable detection of AFM1 on the level of its threshold limited values (20 ng/L).