Quantification Of Aflatoxin M1 Research Articles

Simultaneous Detection and Quantification of Aflatoxin M1, Eight Microcystin Congeners and Nodularin in Dairy Milk by LC-MS/MS

Dairy milk holds a prominent position as a widely consumed food, particularly among infants and children. However, it is crucial to address the presence of multiple natural toxic compounds that may co-occur in dairy milk to ensure its safety prior to consumption. Aflatoxin M1 (AFM1), an emerging mycotoxin of interest, is a potential contaminant in the milk of animals who ingest aflatoxin B1 (AFB1). The toxin is regulated in the European Union under Commission Regulation No 1881/2006. Unfortunately, there is a notable lack of data concerning the transfer of various emerging microbial contaminants into dairy milk and, therefore, their natural occurrences. In this study, a simple and sensitive LC-MS/MS method was developed and validated for the quantification of multiple cyanotoxins (microcystin congeners and nodularin) and AFM1 by the main analytical guidelines. Toxins are extracted with methanol 80%, followed by an SPE clean-up step before LC-MS/MS analysis. The LOQ was fixed at 1 µg/L for the nine cyanotoxins and 0.05 µg/L for AFM1. Recoveries were measured between 82.67% and 102%. To the best of our knowledge, there are no other LC-MS/MS methods available for the simultaneous quantification of cyanotoxins and mycotoxins in milk.

Monitoring of Aflatoxin M1 in Various Origins Greek Milk Samples Using Liquid Chromatography Tandem Mass Spectrometry

Aflatoxin M1(AFM1), a major metabolite of Aflatoxin B1(AFB1), has been identified as a potential contaminant in dairy products. Because of its possible carcinogenicity, the legislation limits as set by Commission Regulation (EC) No. 1881/2006 are very strict, namely 0.050 μg kg−1 in milk and 0.025 μg kg−1 in infant formulas. To meet these requirements, a sensitive and accurate method was developed, employing liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Ιmmunoaffinity columns (R-Biopharm) were used for sample purification and preconcentration of the analyte of interest. The quantification of AFM1 was conducted using fortified milk samples, while Aflatoxin B2 (AFB2) was used as an internal standard (IS). The method was validated in terms of linearity, precision, trueness, limits of detection and quantification and uncertainty. The performance criteria for the method were evaluated based on European Commission Regulation (EC) No. 401/2006 and its most recent amendment, as well as the suggested criteria for revision by the EU Reference Laboratory for Mycotoxins and Plant Toxins. The recovery was in the range of 77.9–81.0% for all fortification levels (0.025–0.050–0.075 μg kg−1), with RSDR values (Relative Standard Deviation of intermediate precision) ranging from 6.1% to 12%. The method’s detection and quantification limits were 0.0027 μg kg−1 and 0.0089 μg kg−1, respectively. The occurrence of AFM1 was investigated in 40 samples of different animal origin (cow, goat and sheep milk) provided by Greek producers.

Design and Evaluation of a Competitive Phosphorescent Immunosensor for Aflatoxin M1 Quantification in Milk Samples Using Mn:ZnS Quantum Dots as Antibody Tags

Aflatoxin M1 (AFM1) is one of the most widespread aflatoxins that can be present in the milk of lactating mammals. It can cause carcinogenicity, mutagenesis, teratogenesis, genotoxicity and immunosuppression. The WHO recommends reducing the AFM1 concentration in food products, so the European Commission has set a maximum allowable limit of 0.05 µg L−1 in milk and its products. Thus, there is a need to develop new methodologies to satisfy the demand for reliable, cost-effective, robust and sensitive AFM1 routine controls. In the present work, a competitive phosphorescent immunosensor for AFM1 quantification in milk, based on antibody–antigen recognition and Mn:ZnS quantum dots (d-QDs) as photoluminescent labels, has been developed. Two different assay strategies based on the use of d-QDs as labels of secondary antibodies (direct assay), or of a derivative species of the antigen AFM1-Bovine Serum Albumin (indirect assay) were compared in terms of analytical performance for AFM1 quantification. The best analytical results were obtained with the immunoassay format that uses d-QDs as tags of secondary antibodies (direct assay), and said design was finally selected. The selected immunosensor provided a detection limit for AFM1 quantification of only 0.002 µg L−1, which greatly satisfied the maximum tolerable limit of AFM1 in milk of 0.05 µg L−1. The accuracy, calculated as recovery of AFM1 in fortified skimmed milk samples, ranged from 81 to 90%, with relative standard deviations from 3% to 14%. These results bring to light the good performance of such phosphorescent biosensors as simple and fast alternatives to conventional chromatographic analytical methods.

Analysis of aflatoxin M1 contamination in milk and cheese produced in Mexico: a review

Due to the carcinogenic character of aflatoxins when present in foods, these compounds are considered a risk to human health. This systematic review aimed at compiling the available research data on detection and quantification of aflatoxin M1 (AFM1) in milk and common types of cheese produced in Mexico in the past two decades. A limited number of studies were found that matched the purpose of our review. Only ten research works focused on the evaluation of AFM1 content in milk while three studies analysed the occurrence of this mycotoxin in oaxaca and panela cheeses. HPLC-FD and ELISA were the methods of choice utilised to detect AFM1. Concentrations higher than 0.5 μg AFM1/kg, a maximum limit set in current food regulation in Mexico, were found in major dairy brands consumed in Mexico. Analysis of raw milk produced during the rainy season in the states of Jalisco (2007) and Chiapas (2013) showed mycotoxin levels within the regulation limits while milk samples obtained during the dry season in the Mexico City and the State of Mexico (2008) exceeded that threshold. For cheeses, 33% of the artisanal produced oaxaca type samples from Veracruz (2016) and 55% of those acquired in Mexico City (2019) were found above the limit set for milk. In contrast, the panela cheese samples obtained in Baja California and Guanajuato (2009) complied with the AFM1 regulation. Additionally, the presence of AFB1 and its hydroxylated metabolites other than AFM1 were determined in the major milk brands at concentrations that could be of high risk for human health. Similar results were reported for both artisan and industrially produced oaxaca cheese. Finally, mycotoxins enter human food chain through animals fed with contaminated fodder. Our systematic review demonstrated the urgent need to amend the existing food regulation in Mexico to include mycotoxins as potent contaminants in cheese.

TOTAL BACTERIAL COUNT, SOMATIC CELL COUNT AND PRESENCE OF AFLATOXIN M1 IN RAW MILK FROM THE “OVČE POLE” REGION, REPUBLIC OF NORTH MACEDONIA

Dairy product quality monitoring begins at the farm and ends in the hands of the consumer. Raw milk must also meet other quality standards; it should be free of drug residues, free of added water and free of sediment, contaminants and other abnormalities. In our researches, is taken somatic cells count, the number of bacteria and Aflatoxins as indicators of the quality of raw milk from the Ovče Pole region in the period January-June, 2018. For the needs of this research, an analysis was made of 1320 samples for the presence of bacteria in raw milk, determination of somatic cell count in 478 samples as well as identification and quantification of aflatoxin M1 in 60 samples.  The results from this study indicated determination acceptable count of somatic cells in 95.5 % of the samples from raw milk while in 2 samples of raw milk, the amount of aflatoxin M1 was above limits with highest amount of 0.58 mg/kg raw milk. According to European milk quality standards, in the biggest part of the samples (89.55 %), presence of bacteria does not meet the standard. From the analyzes made by the milk producers that were the subject of analysis in this research, it can be concluded that they do not adhere to good agricultural practice, the level of milk contamination is high due to poor hygiene, improper handling of milk after milking and insufficient education of farmers for hygiene in primary production.

Application of a modified lighter than water organic solvent-based air-assisted liquid–liquid microextraction method for the efficient extraction of aflatoxin M1 in unpasteurized milk samples

ABSTRACT An efficient analytical method has been developed for the extraction and quantification of aflatoxin M1 in milk samples using a modified air-assisted liquid-liquid microextraction method combined with high-performance liquid chromatography-fluorescence detector. Firstly, defatted milk is transferred into a glass test tube and its proteins are participated with zinc acetate solution. After vortexing and centrifugation of the mixture, the obtained supernatant is removed and transferred into a conical bottom glass test tube. Then, the adsorbed analyte onto the precipitated proteins is eluted with acetonitrile and after centrifugation, the eluant is mixed with the supernatant obtained from the previous step. A few microlitres of toluene (as an extraction solvent) is added into the solution and the mixture is sucked into a glass syringe and pushed out in the tube for four times. The obtained cloudy solution is centrifuged to collect the extraction solvent. Then, the lower aqueous phase is withdrawn by a glass syringe to transfer the extraction solvent to the conical section of the tube. The method was validated and under optimal conditions, it was found that the method has low limits of detection (0.9 ng L−1) and quantification (3 ng L–1), good linearity (r2 = 0.9976), and satisfactory repeatability (relative standard deviation ≤9% for intra- (n = 8) and inter-day (n = 6) precisions). The extraction recovery and enrichment factor of AFM1 were 87% and 87, respectively. Finally, the proposed method was applied on different milk samples and concentration of aflatoxin M1 was determined in the studied samples.

Comparison of Immunoassay and High Performance Liquid Chromatography Methods for Determining Aflatoxin M1 in Raw Milk

The objective of this study was to compare methods of immunoassay with High Efficiency Liquid Chromatography methods (HPLC) of processing plants of dairy products. Aflatoxins are a group of organic chemical non-protein compounds of low molecular weight, mainly produced by fungi <em>Aspergillus flavus</em>, <em>Aspergillus parasiticus</em> and <em>Aspergillus nomius</em>. The Aflatoxin M1 (AFM1) is an oxidative metabolite of Aflatoxin B1 (AFB1), produced by animals after the ingestion of this, which appears in milk and urine. The negative impact of aflatoxins in human health is particularly due to their carcinogenicity, by what it is important the detection and quantification of AFM1 in raw milk used as raw material. In the dairy industry it is common to use rapid tests for the determination of the AFM1, but how reliable are these tests? The study discussed 28 raw milk samples taken in times of drought and rain in the South of Cesar, to determine the AFM1 using an immunoassay method with sensitivity of 0.35 &mug/kg and for Liquid Chromatography (HPLC) method. The first method data were analyzed with basic descriptive statistics and with the results obtained by chromatography it was estimated the average level of aflatoxin by intervals, AFM1 average estimate was slightly higher in the dry season. It was also noted that results in dry season coincide with the results of the two methods by 42.9% and in the rainy season by 64.3%. The use of immunoassay method has as a disadvantage: the appearance of cross reactions with mycotoxins from the same group.

Quality of the Serrano Cheese from Santa Catarina for the fungal count, aflatoxin M1 and light filth

The Serrano Artisanal Cheese is a traditional product from the south of Brazil, produced by family farming from raw milk. The growth of the consumption of this cheese requires attention to its quality, since the only measures of control of this product are the good practices of manufacture and maturation. The objective of this work was to evaluate some parameters of contamination of Serrano Artisanal Cheese over 4 ripening periods (14, 21, 28 and 35 days), such as total fungal count, quantification of aflatoxin M1, and light filth. The efficacy of the ripening period in relation to the loss of water was evaluated by determining the moisture and water activity. The fungal count and water activity (aw) did not change during the maturation periods, with averages between 4 - 7 log CFU g -1 and 0.97-0.98, respectively. The humidity was different only between the periods of 14 and 21 days of maturation, but the average values still remained between 40.39 and 43.66%, classifying the cheese as a semi-hard mass. AFM1 was detected in only one property, in all periods, but in values below that established by Brazilian legislation. Light filth were present in 100% of the analyzed samples, with a high prevalence of synthetic fibers, besides the presence of insects and hairs. The analyzed cheeses were safe for consumption in relation to fungal quantification and aflatoxin M1, but the light filth were identified in a number above that allowed by the legislation, although it did not alter the counting of the microorganisms analyzed.

Detection of aflatoxin M1 in milk using spectroscopy and multivariate analyses

Aflatoxin M1 (AFM1), a potentially carcinogenic compound, is found in milk obtained from animals that consume contaminated feed. Spectra of bovine milk, spiked with AFM1 (0, 0.02, 0.04, 0.06, 0.08 and 0.1μg/l) were acquired using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrometer. Spectra revealed significant differences among pure and AFM1 spiked samples in spectral regions 1800–650cm−1 and 3689–3499cm−1, which may be attributed to complex chemical structure of AFM1. Principal component analysis (PCA) showed clear clustering of samples (p⩽0.05). The models could successfully classify (>86%) and detect even 0.02μg/l AFM1 in milk (p⩽0.05) using SIMCA. AFM1 was best predicted in wavenumber range of 1800–650cm−1 with coefficient of determination (R2) of 0.99 and 0.98, for calibration and validation, respectively, using partial least square (PLS) regression. The study indicated feasibility of ATR-FTIR spectroscopy and chemometrics in rapid detection and quantification of AFM1 in milk.

Highly sensitive and simultaneous detection of melamine and aflatoxin M1 in milk products by multiplexed planar waveguide fluorescence immunosensor (MPWFI)

Mycotoxins and industrial chemicals, such as aflatoxin M1 and melamine, now commonly exist in milk and cause potential health risks. This study presents an indirect competitive immunoassay through multiplex planar waveguide fluorescence immunosensor (MPWFI) for rapid, sensitive, and simultaneous detection and quantification of aflatoxin M1 and melamine by applying the principle of immunoreaction and total internal reflect fluorescent.Double-channel standard curves with appropriate logistic correlation (R2>0.99) were plotted, respectively. The working ranges (0.073–0.400ng/mL and 26.38–270.00ng/mL, respectively) were calculated, as well as the limit of detection (0.045 and 13.37ng/mL, respectively), when two analytes were simultaneously detected. Both results satisfied the requirements for the maximum amount set by the WHO, which illustrated that the current method was better than some other standard methods. The recovery rates in the actual samples ranged from 85% to 103%, with relative standard deviations between 1.3% and 6.5%, which indicated high accuracy and repeatability.

Quantification of aflatoxin M1 in raw milk by a core-shell column on a conventional HPLC with large volume injection and step gradient elution

The immunoaffinity column cleanup method coupled with HPLC separation and fluorescence detection is still one of the most frequently used quantitative methods for routine analyses of AFM1. In this study, aflatoxin M1 (AFM1) was quantified with a chromatography column packed with 2.6 μm core-shell particles on a conventional HPLC. A large volume of solvent (100 μL) was injected into the highly efficient column without any noticeable reduction in separation performance with the help of stepwise gradient elution. The instrumental conditions were optimized by response surface analysis methodology (RSM) with a three-level three-factor Box–Behnken design.The use of core-shell columns on conventional HPLC for AFM1 analysis under the optimized instrumental conditions leads to increased analytical performance compared with traditional totally porous columns without the heavy costs associated ultra-high-pressure instruments. Moreover, the improved instrumental sensitivity enables simplified sample preparation by avoiding any solvent replacement. The method could be easily applied to enhance the sensitivity of HPLC-FLD for AFM1 analysis that is based on isocratic elution and is more widely used. The method was successfully applied to 40 raw milk samples collected in summer from 20 cattle ranches located in two different provinces in southwestern China.

Pattern of partitioning of aflatoxins from feed to urine and its effect on serum chemistry in Nili-Ravi buffalo heifers

The objectives of the present study were (1) to monitor the pattern of excretion of aflatoxinM1 in urine after its conversion from aflatoxinB1 and (2) to observe the effects of different levels of aflatoxinB1 in feed on serum concentrations of key metabolites glucose, total protein, cholesterol and urea as indicators of metabolic status. Nili-Ravi buffalo heifers (n = 12) of similar age and weight were randomly distributed to four groups. Animals in Groups A, B and C were offered a contaminated cottonseed cake-based concentrate ration at 0.5%, 1.0% and 1.5% of bodyweight, respectively. Control animals in Group D were fed with aflatoxinB1-free green fodder. Based on the level of contamination of the concentrate ration with aflatoxinB1 (554 µg/kg), Groups A, B and C consumed 953, 2022, 3202 µg of aflatoxinB1 daily. Feed samples were analysed at Romer Laboratories Pty Ltd, Singapore by high performance liquid chromatography. AflatoxinM1 quantification in urine samples was conducted using a competitive enzyme-linked immunosorbent assay with kits supplied by Helica Biosystems, Inc., USA. Serum samples were analysed for concentrations of glucose, total protein, cholesterol and urea using clinical chemistry kits provided by Human diagnostics (HUMAN, Biochemica und Diagnostica mbH, Germany). Carry-over rate of aflatoxinM1 in urine for Groups A, B and C was 15.51%, 15.44% and 14.04% of aflatoxinB1 while there was no detectable aflatoxinM1 in the urine of the control group (D). There was no significant difference in the concentrations of serum glucose, total protein and cholesterol between treatment groups. However, the concentration of serum urea was significantly higher (P &lt; 0.05) in the group offered the highest level of aflatoxinB1-contaminated concentrate. This result suggests that mycotoxicosis may compromise protein metabolism and accretion in affected animals. This leaves open the possibility that high concentrations of aflatoxins in milk may ultimately affect the health status of human milk consumers.

Distribution and stability of aflatoxin M1 during processing and storage of Minas Frescal cheese

Aflatoxin M1 (AFM1) is a hepatocarcinogen found in milk of animals that have consumed feeds with aflatoxin B1. The carry-over of AFM1 from milk to Minas Frescal cheese produced with or without starter cultures was determined. 40 L of milk were divided into 10 L each and assigned to the following treatments for cheese manufacture: 0.250 ng AFM1 mL−1, 0.500 ng AFM1 mL−1, 0.250 ng AFM1 mL−1 + starter, 0.500 ng AFM1 mL−1 + starter. Quantification of AFM1 was achieved by high performance liquid chromatography. The carry-over of AFM1 from milk to cheese ranged from 30.64% to 42.26%. There was no effect of storage time on AFM1. Milk with AFM1 in levels studied may concentrate the toxin in Minas Frescal cheese, but at concentrations below the Brazilian tolerance limit. The addition of starter cultures did not influence concentration or stability of the AFM1 in cheese over 30 days storage.

Use of immunoaffinity chromatography as a purification step for the determination of aflatoxin M1 in cheeses.

A method using immunoaffinity as a purification step for the determination of aflatoxin M1 in cheese is described. A simple solvent extraction with dichloromethane followed by a washing step with N-hexane gives a prepurified extract. A comparison between two ways of aflatoxin M1 purification, by solid-phase extraction clean-up and by immunoaffinity, was carried out. The use of immunoaffinity columns containing monoclonal antibodies against aflatoxin M1 gives the best result, i.e. a very clean preparation containing purified aflatoxin M1. The quantification of aflatoxin M1 is then performed by high performance liquid chromatography using fluorometric detection. This method was successfully carried out on naturally-contaminated and spiked cheeses. Recoveries are about 75%. The limit of quantification is 0.020 microgram of aflatoxin M1 per kg of cheese. This method seems suitable for use in monitoring programmes for aflatoxin M1 contamination in dairy products such as cheese.