Search

The aim of this study is to determine the level of contamination of aflatoxin M1 (AFM1) in raw and imported powdered milk in Khartoum state, Sudan. Thirty-five samples of fresh cow milk were collected from different farms, based on the source of concentrated feed introduced to the dairy cows (locally vs. commercially produced) and the size of the farm (≤ 50 vs. >50 cows/farm). Also 12 samples of powdered milk were obtained from repacking companies in Khartoum state. The samples were analysed by a fluorometer, using the Vicam method. AFM1 was detected in all raw and powdered milk samples. Almost 50% of the contaminated powdered milk samples and all the raw milk samples exceeded the European Union limit of 0.05 µg/kg whereas 33% of the contaminated powdered milk samples and 77% of the raw milk samples exceeded the limit of Codex regulations (0.5 µg/kg). The results revealed that the concentration of AFM1 is affected significantly (P < 0.05) by the source of concentrated feed (locally produced or purchased) but not by the farm size. It was concluded that the levels of AFM1 in the milk samples indicated that the feeds offered to the cows were contaminated with aflatoxin B1 to such a level that it might cause a serious health problem to the public. Therefore, there is a need to limit the exposure to aflatoxin by imposing regulatory limits, as well as further studies on large scale bases are needed to investigate the amount of AFM1 in milk and dairy products.

Tarhana chips are a popular traditional fermented food consumed widely in the Kahramanmaraş region of Turkey. Tarhana chips are different from many other types of fermented food in that they are produced in the form of tortilla chips. Cereal and yoghurt are the main ingredients in Tarhana chips. Aflatoxin M1 (AFM1) levels in dairy and dairy-based products are of concern for human health. To investigate AFM1 contamination, a total of 40 samples were collected from Kahramanmaraş region and AFM1 levels were determined by competitive enzyme-linked immunosorbent assay (ELISA). Furthermore, physicochemical characteristics of Tarhana chips were investigated and compared with classic fried chips in terms of nutritional value. Based on data obtained from enzyme-linked immunosorbent assay, 21 (52.5%) out of 40 samples contained AFM1 in the range 0.5-36.6 ng/kg, so AFM1 levels of all samples were below the legal limit.

Aflatoxin M₁ (AFM1) contamination in 21,969 milk samples taken in Italy during 2005-08 and 2010 provided the basis for designing an early warning self-control plan. Additionally, 4148 AFM1 data points from the mycotoxin crisis (2003-04) represented the worst case. No parametric function provided a good fit for the skewed and scattered AFM1 concentrations. The acceptable reference values, reflecting the combined uncertainty of AFM1 measured in consignments consisting of milk from one to six farms, ranged from 40 to 16.7 ng kg(-1), respectively. Asymmetric control charts with these reference values, 40 and 50 ng kg(-1) warning and action limits are recommended to assess immediately the distribution of AFM1 concentration in incoming consignments. The moving window method, presented as a worked example including 5 days with five samples/day, enabled verification of compliance of production with the legal limit in 98% of the consignments at a 94% probability level. The sampling plan developed assumes consecutive analyses of samples taken from individual farms, which makes early detection of contamination possible and also immediate corrective actions if the AFM1 concentration in a consignment exceeds the reference value. In the latter case different control plans with increased sampling frequency should be applied depending on the level and frequency of contamination. As aflatoxin B₁ increases in feed at about the same time, therefore a coordinated sampling programme performed by the milk processing plants operating in a confined geographic area is more effective and economical then the individual ones. The applicability of the sample size calculation based on binomial theorem and the fast response rate resulting from the recommended sampling plan were verified by taking 1000-10,000 random samples with replacement from the experimental databases representing the normal, moderately and highly contaminated periods. The efficiency of the control plan could be substantially enhanced if the dairy farms used feed with a tolerable level of AFB1.

Aflatoxin M1 (AFM1) was determined in 107 milk samples collected from urban and rural farmhouses from Punjab, Pakistan, by HPLC with fluorescence detection. An incidence rate of 64% (38/59) in milk samples from urban farmhouses was found, with a mean concentration of 0.064 ± 0.023 μg L(-1). In rural samples about 52% (25/48) of milk samples were contaminated with AFM1, with a mean of 0.04 ± 0.034 μg L(-1). About 42% of milk samples from urban and 27% from rural farmhouses were well above the limit permitted by the European Union (EU). However, only 15% and 8% of milk samples from urban and rural farmhouses, respectively, exceeded the limit of USDA/Codex regulations. The results showed that the contamination of milk with AFM1 from Punjab, Pakistan, when compared with Codex limit is present and needs continuous monitoring. The awareness and education among dairy farmers on the potential health risks associated with aflatoxins should be communicated.

Aflatoxin M₁ contamination in dairy products is a risk when feedstuff contaminated with aflatoxin B₁ produced by moulds is consumed by milk-producing animals. Milk can be screened for aflatoxin M₁ at the European Union maximum limit of 50 ng l⁻¹ by a lateral flow test, the MRLAFMQ (Aflatoxin M1) Test. The method takes 15 min with no milk dilution or a sample preparation step. The lateral flow assay was validated at the Technology and Food Science Unit of the Institute for Agricultural and Fisheries Research (ILVO-T&V) according to European Union guidelines using fortified raw milk samples. A detection capability of 50 ng l⁻¹ was demonstrated with a false negative rate lower than 2% at 50 ng l⁻¹ and a false positive rate of less than 0.3%. Quantitative readings had a mean bias of +2 to 6 ng l⁻¹ at 50 ng l⁻¹ with a standard deviation of 5-8 ng l⁻¹. Based on the validation results, the test could be considered appropriate for milk screening prior to milk unload at dairies.

Aflatoxins are fungal toxins known to be carcinogenic and are classified as food contaminants. This study was performed to investigate aflatoxin (AF) M1 levels in baby foods sold in Ankara (Turkey) and to evaluate the obtained results according to the Turkish Food Codex (TFC). For this purpose, a total of 84 baby food samples (50 follow-on milks and 34 infant formulas) were obtained from different markets in Ankara and the presence of AFM1 in the samples was analyzed by ELISA. In 32 (38.1%) of 84 infant food samples, the presence of AFM1 was detected in concentrations ranging between 0.0055 and 0.0201 µg/kg. The mean level (± standard error) of AFM1 was found to be 0.0089 ± 0.0006 µg/kg in positive infant follow-on milks. Aflatoxin M1 was detected in only 1 infant formula sample (2.94%) at a concentration of 0.0061 µg/kg. The extrapolated levels of AFB1 contamination in feedstuffs were calculated based on levels of AFM1 in baby food samples. The data estimating AFB1 contamination in dairy cattle feedstuff indicate that contamination may range from 0.3410 to 1.2580 µg/kg, with the mean level (± standard error) being 0.5499 ± 0.0385 µg/kg, which is lower than the level set by the TFC and European Union regulations (5 µg/kg). According to the obtained results, the levels of AFM1 in analyzed samples were within the allowed limit (0.025 µg/kg) set in the TFC. Low levels of AFM1 in infant follow-on milks and infant formula samples obtained during the study do not pose a health risk to infants., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The presence of aflatoxin M1 (AFM1) in milk was assessed in Italy in the framework of designing a monitoring plan actuated by the milk industry in the period 2005-10. Overall, 21,969 samples were taken from tankers collecting milk from 690 dairy farms. The milk samples were representative of the consignments of co-mingled milk received from multiple (two to six) farms. Systematic, biweekly sampling of consignments involved each of the 121 districts (70 in the North, 17 in the Central and 34 in the South regions of Italy). AFM1 concentration was measured using an enzyme-linked immunoassay method (validated within the range of 5-100 ng kg(-1)) whereas an HPLC method was used for the quantification of levels in the samples that had concentrations higher than 100 ng kg(-1). Process control charts using data collected in three processing plants illustrate, as an example, the seasonal variation of the contamination. The mean concentration of AFM1 was in the range between 11 and 19 ng kg(-1). The 90th and 99th percentile values were 19-34 and 41-91 ng kg(-1), respectively, and values as high as 280 ng kg(-1) were reached in 2008. The number of non-compliant consignments (those with an AFM1 concentration above the statutory limit of 50 ng kg(-1)) varied between 0.3% and 3.1% per year, with peaks in September, after the maize harvest season. The variability between different regions was not significant. The results show that controlling the aflatoxins in feed at farm level was inadequate, consequently screening of raw milk prior to processing was needed. The evaluation of the AFM1 contamination level observed during a long-term period can provide useful data for defining the frequency of sampling.

The aim of this study was to detect the presence of aflatoxin M₁ (AFM1) in samples of raw milk (n=38), UHT milk (n=12), white pickled cheese (n=50), and yogurt (n=50) collected from the Şanlıurfa city markets and locally produced dairy products by ELISA. The mean contamination rates were 56.74 ± 40.32, 43.1 ± 23.19, 103.2 ± 29.13, and 55.28 ± 12.68 ng/kg, respectively, for raw milk, UHT milk, white pickled cheese, and yogurt. According to the data, 21 (55%) raw milk, 3 (25%) UHT milk, 10 (20%) white pickled cheese, and 10 (20%) yogurt samples were contaminated with AFM1 over the acceptable levels (≥50 ng/kg), ranging from 0.82 to 130.89 ng/kg. None of the white pickled cheese samples contained AFM1 levels above the Turkish legal limit (250 ng/kg). Consequently, the AFM1 contamination levels determined in this study in white pickled cheese were not considered to pose a serious public health hazard. However, the AFM1 levels in raw and UHT milk and yogurt samples indicate an increased human health risk in Turkey related to high aflatoxin levels. Therefore, milk and dairy products have to be monitored by the Turkish public health authorities continuously to detect AFM1 contamination., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Contamination of milk and dairy products with aflatoxin M1 (AFM1) presents a risk for human health. The aim of this study was to investigate the presence of AFM1 in pasteurized milk samples in Fariman, located in the province of Khorasan Razavi, Iran, by enzyme-linked immunosorbent assay (ELISA). Forty-five samples of pasteurized milk from different supermarkets were collected during 3 months in summer (July to September, 2012). AFM1 contamination was detected in all of milk samples. The mean concentration of aflatoxin M1 was 27.2 ng/l. The range of AFM1 content was 8.8-64 ng/l. Thirteen (28.8 %) of the samples had AFM1 levels exceeding the maximum levels (50 ng/l) accepted by the European Union. Due to the fact that milk is used by all the age groups including infants and children in Fariman city, it is necessary to minimize the health risk from AFM1 contamination in milk. For this reason, the level of its precursor, aflatoxin B1 (AFB1), in dairy feeds must be reduced, requiring constant aflatoxin monitoring of relevant agricultural commodities.

A simple, rapid, and sensitive method based on simultaneous protein precipitation and extraction of aflatoxin M1 (AFM1) followed by dispersive liquid-liquid microextraction (DLLME) and ultrahigh pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis was developed for the determination of AFM1 in milk samples. In order to precipitate the proteins and extract AFM1 from milk, a sample pretreatment using acetonitrile and NaCl as the extraction/denaturant solvent and salting-out agent, respectively, was optimised. Subsequently, the acetonitrile (upper) phase, containing AFM1, was used as the disperser solvent in DLLME, and extractant (chloroform) and water were added in turn to the extract to perform the DLLME process. The main parameters affecting the extraction efficiency of the whole analytical procedure, such as acetonitrile volume, amount of salt, type and volume of extractant and water volume, were carefully optimised by experimental design. Under optimum conditions, the developed method provides an enrichment factor of 33 and detection and quantification limits (0.6 and 2.0 ng kg(-1), respectively) below the maximum levels imposed by current regulations for AFM1 in milk and infant milk formulae. Recoveries (61.3-75.3%) and repeatability (RSD < 10, n = 3), tested in different types of milk at four AFM1 levels, met the performance criteria required by EC Regulation No. 401/2006. Moreover, the matrix effect on the signal intensity of the analyte was negligible. The proposed method provides a rapid extraction and an accurate determination of AFM1 in milk and formula milk using a simple and inexpensive sample preparation procedure.

Objective: To assess the suitability of tolerance limit for aflatoxin M1 (AFM1) in foods and Chinese population dietary exposure to AFM1 from milk., Methods: Milk and dairy products consumption data combined with the tolerance limit for AFM1 in foods as well as the concentration of AFM1 in milk were employed in assessment of both the intakes of AFM1 and the Chinese population long-time exposure to AFM1. AFM1 intake and the dietary exposure to projected risks for liver cancer attributable to either use of the maximum levels of AFM1 of 0.05 microg/kg milk or consumption of milk contaminated with AFM1 were assessed., Results: Assuming that all milk and dairy products on sale contain AFM1 at the level equal to the tolerance limit of 0.5 microg/kg, the total average dietary exposure to AFM1 in Chinese population will be 0.20 ng per kilogram bodyweight per day, the increased risk of liver cancer in humans will be as 0.00062 cases per 100,000 individuals per year, if the incidence of HBs Ag carriers of 7.18% takes into account. As for the milk sample contaminated with AFM1 at the level of 1.2 microg/kg analyzed and issued by the General Adminstration of Quality Surpervision Inspection and Qarantine of China in 2011, the total average long-term exposure of the Chinese population to AFM1 from this milk sample will be 0.43 ng per kilogram bodyweight per day, the increased risk of liver cancer in humans might be 0.00133 cases per 100,000 individuals per year., Conclusion: The tolerance limit for AFM1 of 0.5 microg/kg implemented in China can effectively protect the whole population. In addition, although the health risk of short-term exposure to AFM1-contaminated milk is acceptable, the government still should take the effective measures to control the contamination of AFM1 in milk so as to reduce the Chinese long-time exposure to AFM1.

A quantitative fluorescence-labeled immunosorbent assay and qualitative on-site column tests were developed for the determination of aflatoxin M1 in milk products. The use of liposomes loaded with quantum dots as a label significantly increased the assay sensitivity by encapsulating multiple quantum dots in a single liposome and, therefore, amplifying the analytical signal. Two different techniques were compared to obtain aflatoxin-protein conjugates, used for further coupling with the liposomes. The influence of nonspecific interactions of the liposome-labeled conjugates obtained with the surface of microtiter plates and column cartridges was evaluated and discussed. The limit of detection for fluorescence-labeled immunosorbent assay was 0.014 μg kg(-1). For qualitative on-site tests, the cutoff was set at 0.05μg kg(-1), taking into account the EU maximum level for aflatoxin M1 in raw milk, heat-treated milk, and milk for the manufacture of milk-based products. The direct addition of labeled conjugate to the milk samples resulted in an additional decrease of analysis time. An intralaboratory validation was performed with sterilized milk and cream samples artificially spiked with aflatoxin M1 at concentrations less than, equal to and greater than the cutoff level. It is shown that milk products can be analyzed without any sample preparation, just diluted with the buffer. The rates for false-positive and false-negative results were below 5% (2.6% and 3.3%, respectively).

This study was conducted to investigate mycotoxin exposure in children (n=220, aged 1.5-4.5years) from high mycotoxin contamination regions of Cameroon and to examine the association between the mycotoxin levels (in total 18 analytes) and several socio-demographic factors and anthropometric characteristics. A cross-sectional study was conducted in six villages in Cameroon with 220 children. Mycotoxins and their metabolites were detected in 160/220 (73%) urine samples. There were significant differences in the mean contamination levels of ochratoxin A (p=0.01) and β-zearalenol (p=0.017) between the two agro-ecological zones investigated. Likewise significant differences were observed in the mean levels of aflatoxin M1 (p=0.001) across the weaning categories of these children. The mean concentration of aflatoxin M1 detected in the urine of the partially breastfed children (1.43ng/mL) was significantly higher (p=0.001) than those of the fully weaned children (0.282ng/mL). Meanwhile, the mean concentrations of deoxynivalenol (3.0ng/mL) and fumonisin B1 (0.59ng/mL) detected in the urine of the male children was significantly (p value 0.021 for deoxynivalenol and 0.004 for fumonisin B1) different from the levels detected in the urine of female children; 0.71ng/mL and 0.01ng/mL for deoxynivalenol and fumonisin B1 respectively. In this study, there was no association between the different malnutrition categories (stunted, wasting and underweight) and the mycotoxin concentrations detected in the urine of these children. However, there is sufficient evidence to suggest that children in Cameroon under the age 5 are exposed to high levels of carcinogenic substances such as fumonisin B1, aflatoxin M1 and ochratoxin A through breastfeeding. To the best of our knowledge, this is the first report of its kind carried out in West Africa to determine multi-mycotoxin exposure in infants., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aflatoxin M1 (AF M1), a hydroxylated metabolite of AF B1, is an important toxin that can contaminate the milk of lactating mothers. A correlation study was conducted to determine the relationship between AF M1 content of breast milk, dietary exposure to AF B1 and socioeconomic status of lactating mothers in the three Senatorial districts of Ogun State, Nigeria. Equal amounts of breast milk (20 ml) and food rations (40 kg) obtained from 50 volunteer lactating mothers and eighty-two frequently consumed food commodities in the preceding month were used for the study. The level of contamination of the foods by AF B1 was low (0.16-0.33 μg/kg) and differed significantly (p<0.05) across the state but did not exceed the EU limit of 2 μg/kg. The occurrence level of AF B1 was however high (93.75-94.45%) and was more pronounced in Ogun East Senatorial district (94.45%). Eighty-two percent of the breast milk was contaminated with AF M1 (3.49-35 ng/l) and 16% exceeded the EU limit of 25 ng/l while a 100% occurrence risk was recorded in Ogun Central Senatorial district. The socioeconomic status of the mothers also significantly influenced their dietary exposure and exposure risk of the sucklings to AF M1., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Occurrence of aflatoxin M1 (AFM1) was determined in 196 milk samples (conventional, organic and kids milk) from the Greek market during November 2009 to June 2010. AFM1 content was analyzed using an Enzyme-Linked Immunosorbent Assay (ELISA) commercial kit. Aflatoxin M1 was detected in 46.5% of the samples. 46.5% of the samples were found positive for AFM1. The most frequent range of detection was between 5 and 10 ng/l. Based on the EU regulation only 2 milk samples presented AFM1 levels higher than the maximum residue limits. Two different scenarios were used for the determination of hazard index: (a) scenario 1 using only positive (detected AFM1) samples and (b) scenario 2 when missing values where imputed with Limit of Detection (LOD) divided by 2. Significant statistical differences between different milk categories were presented only when the results were imputed with LOD/2 values. Exposure assessment scenarios were developed for ages 1, 3, 5, 7 and 12 and their respective estimated weights and daily milk consumption. Under the worst-case scenario all milk types presented a Hazard Index (HI) less than one. The highest HI values appear in the ages of 1-3., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This study aimed to develop a rapid and sensitive method for detection of aflatoxin M1 (AFM) by dynamic light scattering (DLS) coupled with superparamagnetic beads and gold nanoprobes. The nanoprobes were synthesized by the conjugate of AFM and bovine serum albumin (AFM-BSA), BSA, and gold nanoparticles. Magnetic beads-based immunosorbent assay (MBISA) was used to measure the concentration of AFM by direct competition between AFM and nanoprobes. DLS was used to determine the concentration of unattached nanoprobes that was positively proportional to the concentration of AFM in the sample. TEM images prove that the as-prepared nanoprobes were able to attach on the magnetic beads through the antibody-antigen reaction. Compared to conventional ELISA, MBISA could effectively reduce the incubation time to 15 min in buffer solution and completely eliminate the color development step, thus simplifying the analysis of AFM. A linear relationship was observed between the inhibition values and the concentrations of AFM in both buffer solution (0-1000 ng·L(-1)) and spiked milk samples (0-400 ng·L(-1)). The limit of detection was found to be 37.7 ng·L(-1) for AFM in buffer solution and 27.5 ng·L(-1) in milk samples. These results demonstrate that DLS coupled with magnetic beads and gold nanoprobes is a rapid and effective method to detect AFM. This method could also be easily extended to rapid detection of other mycotoxins and biological species.

This study was conducted to investigate the occurrence of aflatoxin M1 (AFM1) in milk products in China using the competitive enzyme-linked immunosorbent assay method and to estimate the dietary exposure to this toxin through a probabilistic approach. Based on the exposure assessment results, a quantitative cancer potency formula developed by the Joint Food and Agriculture Organization and World Health Organization Expert Committee on Food Additives was applied to assess the cancer risk. AFM1 was detected in 48.07% of the milk samples and 4.49% of the yoghurt samples. No samples contained AFM1 above the current regulatory limit in China. The simulated AFM1 intake (90% confidence interval) in various sex-age groups ranged from 0.023 (0.021 to 0.023) ng/kg of body weight per day for 30- to 45-year-old men to 0.382 (0.354 to 0.386) ng/kg of body weight per day for 2- to 4-year-old girls at the 99th percentile. The cancer risk of AFM1 to the general population of China was assessed to be 0.129 cancer cases per year per 10(8) persons at the 99th percentile. These results indicate that the health risk associated with AFM1 in milk in China is relatively low.

The selective detection of ultratrace amounts of aflatoxin M1 (AFM1) is extremely important for food safety since it is the most toxic mycotoxin class that is allowed to be present on cow milk with strictly low regulatory levels. In this work, Fe3O4 incorporated polyaniline (Fe3O4/PANi) film has been polymerized on interdigitated electrode (IDE) as sensitive film for AFM1 electrochemical biosensor. The immobilized aptamers as an affinity capture reagent and magnetic nanoparticles for signal amplification element have been employed in the sensing platform. Label-free and direct detection of the aptamer-AFM1 on Fe3O4/PANi interface were performed via electrochemical signal change, acquired by cyclic and square wave voltammetries. With a simplified strategy, this electrochemical aptasensor shows a good sensitivity to AFM1 in the range of 6-60 ng·L(-1), with the detection limit of 1.98 ng·L(-1). The results open up the path for designing cost effective aptasensors for other biomedical applications., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate aflatoxin M1 (AFM1) contamination in milk samples in Mashhad in Iran. A total of 60 milk samples were collected from retail stores of five regions in June 2011. The occurrence and concentration range of AFM1 in the samples were investigated by high-performance liquid chromatography method. AFM1 was found in 100% of the examined milk samples by average concentration of 16.16 ng/L and the contamination level ranging between 2 and 64 ng/L. The concentration of AFM1 in all the samples was lower than the Iranian national standard and Food and Drug Administration limit (500 ng/L), and only in one (1.6%) of the samples, the concentration of AFM1 was greater than the maximum tolerance limit (50 ng/L) accepted by European Union and Codex Alimentarius Commission. Statistical evaluation showed no significant difference between the mean concentrations of AFM1 in milk samples taken from different regions (p > 0.05).

An ultrasensitive electrochemiluminescent immunoassay (ECLIA) for aflatoxins M1 (ATM1) in milk using magnetic Fe3O4-graphene oxides (Fe-GO) as the absorbent and antibody-labeled cadmium telluride quantum dots (CdTe QDs) as the signal tag is presented. Firstly, Fe3O4 nanoparticles were immobilized on GO to fabricate the magnetic nanocomposites, which were used as absorbent to ATM1. Secondly, aflatoxin M1 antibody (primary antibody, ATM1 Ab1), was attached to the surface of the CdTe QDs-carbon nanotubes nanocomposite to form the signal tag (ATM1 Ab1/CdTe-CNT). The above materials were characterized. The optimal experimental conditions were obtained. Thirdly, Fe-GO was employed for extraction of ATM1 in milk. Results indicated that it can adsorb ATM1 efficiently and selectively within a large extent of pH from 3.0 to 8.0. Adsorption processes reached 95% of the equilibrium within 10 min. Lastly, the ATM1 with a serial of concentrations absorbed on Fe-GO was conjugated with ATM1 Ab1/CdTe-CNT signal tag based on sandwich immunoassay. The immunocomplex can emit a strong ECL signal whose intensity depended linearly on the logarithm of ATM1 concentration from 1.0 to 1.0 × 10(5) pg/mL, with the detection limit (LOD) of 0.3 pg/mL (S/N = 3). The method was more sensitive for ATM1 detection compared to the ELISA method. Finally, ten samples of milk were tested based on the immunoassay. The method is fast and requires very little sample preparation, which was suitable for high-throughput screening of mycotoxins in food.

A high sensitive immunoassay-based lateral flow device for semi-quantitatively determine aflatoxin M1 (AFM1) in milk was developed. Investigation and optimization of the competitor design and of the gold-labelling strategy allowed the attainment of the ultra-sensitive assessment of AFM1 contamination at nanograms per litre level (LOD 20 ng L(-1), IC50 99 ng L(-1)), as requested by European regulations. A one order of magnitude detectability enhancement in comparison to previously reported gold colloid immunochromatographic assays for this toxin was obtained. Direct detection of the target toxin in milk could be obtained by acquiring images of the strips and correlating intensities of the coloured lines with analyte concentrations. The one-step assay can be completed in 17 min, including a very simple and rapid sample preparation, which allowed the application of the assay to milk samples which differ in fat and protein contents. Although imprecise (mean RSD about 30%), the method proved to be accurate and sensitive enough to allow the correct attribution of sample as compliant or non-compliant according to EU legislation in force. Agreeing results to those of a reference ELISA were obtained on 40 milk samples by matrix-matched calibration in pasteurized milk., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The present study was aimed to determine the presence and levels of aflatoxin M(1) (AFM(1)) in 120 natural yoghurt samples consisting of 80 samples of commercial and 40 samples of traditional yoghurt in Guilan province in the north of Iran. The occurrence and concentration range of AFM(1) in samples were determined by immunoaffinity column extraction and high-performance liquid chromatography. Analysis of yoghurts showed that all the samples were contaminated with AFM(1) in concentration levels ranging from 4.2 to 78.9 ng/kg. In general, 16 samples (13.33%) had higher AFM(1) level than the maximum tolerance limit (50 ng/kg) accepted by European Union, but the contamination level was lower than 500 ng/kg in all the samples, which is accepted by Codex Alimentarius and National Standard. The concentration of AFM(1) in 26 samples (21.66) was lower than 10 ng/kg. The range of contamination of AFM(1) was comparatively higher in traditional yoghurt (average concentration of 32.9 ng/kg) than that in commercial yoghurt (average concentration of 21.6 ng/kg; p < 0.01). Because yoghurt is the most popular dairy product consumed in Iran, the AFM(1) contamination is a serious problem for public health. This study reports the data of a first survey on the presence of AFM1 in yoghurt in Guilan, Iran.

The potent hepatotoxin and carcinogen aflatoxin B1 (AFB1) is a common mycotoxin contaminant of grains used in animal feeds. Aflatoxin M1 (AFM1) is the major metabolite of AFB1 in mammals, being partially excreted into milk, and is a possible human carcinogen. The maximum permitted concentration of AFM1 in cows' milk is 0.05 μg/kg in Israel and the European Union. Since milk yield and the carry-over of AFB1 in the feed to AFM1 in the milk are highly correlated, it was considered important to determine the AFM1 carry-over in Israeli-Holstein dairy cows, distinguished by world record high milk production. Twelve such cows were used to determine AFM1 carry-over following daily oral administration of feed containing ~86 μg AFB1 for 7 days. The mean carry-over rate at steady-state (Days 3-7) was 5.8% and 2.5% in mid-lactation and late-lactation groups, respectively. The carry-over appears to increase exponentially with milk yield and could be described by the equation: carry-over% = 0.5154 e(0.0521 × milk yield), with r(2) = 0.6224. If these data truly reflect the carry-over in the national Israeli dairy herd, the maximum level of AFB1 in feed should not exceed 1.4 μg/kg, a value 3.6 times lower than the maximum residue level currently applied in Israel.

A new chromatographic method is proposed for the analysis of aflatoxin M(1) in milk. The method is based on liquid-liquid extraction followed by LC-MS/MS analysis. Liquid-liquid extraction (LLE) is performed on the defatted milk plus sodium chloride by using ethyl acetate as an extraction solvent. Accuracy and precision were evaluated at the LOQ (15 ng kg(-1)) spiked sample as well as with three other different naturally contaminated reference materials. The mean overall recovery (n = 24) was 95% with a confidence interval of 1.9% and a CV% of 4.5%. The performance of the proposed method was compared with that of the Official ISO Method based on the use of immunoaffinity chromatography columns (IAC): LLE protocol could be considered a valid alternative to the LC-IAC. In general it showed better accuracy with lower data dispersion. Moreover, the sample preparation is very simple and straightforward, potentially being applicable as a high-throughput method which, on account of its simplicity and low cost, may be applied to the analysis of a large number of samples in the occasion of outbreaks of large-scale contamination.

A survey on 73 milk samples from different animal breeds and 24 dairy products samples from Sicily, Italy, was carried out for the presence of aflatoxin M₁ (AFM1) by LC-fluorescence detection after immunoaffinity cleanup. AFM1 was detected in 48% and 42% of the milk and dairy samples at concentration ranges between <5.0-16.0 and <5.0-18.0 ng L⁻¹, respectively. Within the raw milk samples, 92% had an AFM1 content below 5.0 ng L⁻¹, in 7% of the cases it was in the range 5.0-10.0 ng L⁻¹ and 1% was contaminated between 10.0 and 20.0 ng L⁻¹. For the dairy products, ultra-high-temperature treated (UHT) milk, milk cream and cheese, the incidence was 42%, of which 83% contained less than 5.0 ng L⁻¹ and 17% contained 10.0-20.0 ng L⁻¹ AFM1. The levels of contamination found justify continuous monitoring for public health and to reduce consumer exposure.

Aflatoxin M1 (AFM1) is the only mycotoxin that has a legal limit in milk all over the world. In the present study, 360 raw milk samples were collected from Beijing, Hebei, Shanxi, Shanghai and Guangdong provinces in China in September 2010, and their AFM1 levels were determined by using enzyme-linked immunosorbent assay (ELISA). More than three-fourths (78.1%) of the 360 raw milk samples contained AFM1 at concentrations of 5-123 ng L⁻¹. AFM1 contents in all positive samples were far below the Chinese and US legal limit of 500 ng L⁻¹, but 10% of the raw milk samples exceeded the EU legal limit of 50 ng L⁻¹. Moreover, both incidence and content of AFM1 in milk collected from the southern provinces, including Shanghai and Guangdong, were higher than those collected from the northern provinces, including Beijing, Hebei and Shanxi.

Aflatoxin M(1) (AFM(1)) is a toxic metabolite found in the milk of lactating animals which have consumed feedstuffs contaminated with aflatoxin B(1). Ultra high temperature treated (UHT) milk is a product which is becoming popular in developing countries like India as there is a lack of proper cold storage or refrigeration facilities. In this study, 45 samples of UHT milk of popular brands prevalent in the market were analyzed for the presence of AFM(1) by reversed phase HPLC using fluorescent detector after cleanup of sample with immunoaffinity columns. All samples of plain UHT milk were positive for AFM(1) and 38% of these contained levels more than 0.5 μg/kg, the maximum permitted limit prescribed by the Codex Alimentarius Commission and by the mandatory regulations of the country, the FSSAI Regulations, 2011. In 62.5% of flavored UHT milk, AFM(1) was below detectable levels (0.02 μgL(-1)). However, 12.5% of these samples also contained levels exceeding the maximum permitted limits. AFM(1) was present in 61.6% of the 52 raw milk samples analyzed from the two states of Karnataka and Tamilnadu with a range of 0.1-3.8 μgL(-1). 17.3% of these samples also exceeded the regulatory limits of the country., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This project aimed to examine the effects of adding 2 doses of a montmorillonite-based mycotoxin adsorbent on milk aflatoxin M(1) (AFM(1)) concentrations and the performance and innate immune response of dairy cows fed an aflatoxin B(1) (AFB(1))-contaminated diet. Eight lactating cows were used in a duplicated 4×4 Latin square design with 12-d periods. Treatments included the following: (1) control diet (C), (2) aflatoxin diet (T) containing C and 75 µg of AFB(1)/kg, 3) low-clay (LC) diet containing T and Calibrin A (Amlan International, Chicago, IL) added at 0.2% of the diet dry matter (DM), and 4) high-clay diet (HC) containing T and Calibrin A added at 1% of the diet DM. Milk production and DM intake were recorded daily and milk was sampled twice daily on d 5, 9, 10, 11, and 12 in each period. Blood samples were collected on d 5 and 9 of each period. Dietary treatments did not affect DM intake, milk yield, or feed efficiency. Even though cows were limit fed, feeding T instead of C reduced milk fat yield (0.67 vs. 0.74 kg/d) and milk protein concentration (3.28 vs. 3.36%). Concentrations of AFM(1) in milk of cows fed the T and LC diets were similar (0.57 and 0.64 µg/kg) and greater than those of cows fed the HC diet (0.46 µg/kg). Haptoglobin concentration was greater (22.0 vs. 14.4) and β(2)-integrin expression (220 vs. 131) tended to be greater in cows fed diet T instead of C, but values for cows fed LC, HC, and C did not differ. In comparison to C, feeding T increased the innate immune response and decreased milk fat yield and milk protein concentration, but feeding LC and HC did not affect these measures. Only the HC diet reduced milk AFM(1) concentration., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

A survey of the presence of aflatoxin M1 (AFM1) was carried out on eight traditional dairies belonging to four sectors of Fez city situated in the northern center of Morocco. Raw milk samples were collected between October 2009 and September 2010, and analyzed by LC-fluorescence detection after immunoaffinity purification. AFM1 was detected in 13 out of 48 samples (27%) at concentrations ranged between 10 and 100 ng/l. Within these positive samples, four (∼8% of the total) were above the European legislation limit of 50 ng/l. This study revealed a variation of contamination from one sector to another with a higher incidence in milk samples collected in autumn compared to those collected in other seasons suggesting a link between feeding practices, such as the use of silage and AFM1 contamination. This is the first report on AFM1 contamination in raw milk directly collected from Moroccan traditional dairies. The levels of contamination found justify more detailed and continuous monitoring to assess the public health implication and reduce consumers' exposure to AFM1., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A total of 150 bovine (60), ovine (42), and caprine (48) bulk milk samples were analyzed using a commercially available competitive ELISA kit. Overall, AFM1 was found in 46.7 % of the analyzed samples by an average concentration of 40.3 ± 22.2 ng/L. The incidence rates of AFM1 contamination in bovine, ovine, and caprine bulk milk samples were 66.7, 31.0, and 35.4 %, respectively. The concentration of AFM1 in 37.5 % of AFM1-positive bovine milk samples and 5.9 % of AFM1-positive caprine milk samples were higher than 50 ng/L.

The presence of aflatoxin M1 (AFM1) in the breast milk of nursing Sudanese mothers was investigated using AOAC official method 980.21 as the extraction method and HPLC with fluorescence detector for separation and detection. Following informed consent, 94 breast milk samples of mothers were collected, and 51 samples were found to be positive for AFM1, with an average concentration of 0.401 ± 0.525 ng g(-1) and a maximum level of 2.561 ng g(-1). The volunteers completed a questionnaire concerning their dietary preferences. The data collected suggest that peanut butter, vegetable oils and rice are the main sources responsible for the AFM1 burden in breast milk. The toxin levels are alarmingly high, and indicate that Sudanese infants are exposed to high levels of AFM1. A wide range of harmful effects, and consequently health problems, can be expected due AFM1 toxicity.

The feedstuffs used in dairy animals must be able to give consumers confidence about the wholesomeness of milk with regard to aflatoxin contamination. The aim of this study was to determine the excretion patterns of aflatoxin M(1) (AFM1) in the milk of dairy goats fed a single dose of pure aflatoxin B(1) (AFB1), which can occasionally occur if feeds are infected by hot-spot growth of molds that produce aflatoxins. Five dairy goats in midlactation were administered 0.8 mg of AFB1 orally. Individual milk samples were collected for 84 h after AFB1 dosage. Aflatoxin M(1) was found in milk in the highest concentration. In all goats, AFM1 was not detected in milk before AFB1 administration, but was detected in the first milking following AFB1 administration. The excretion pattern of AFM1 concentration in milk was very similar in all goats even if the values of the concentration differed between animals. The peak values for AFM1 concentration in milk was observed in milk collected during the milking at 3 and 6h. After the peak, the AFM1 in milk disappeared with a trend that fitted well a monoexponential decreasing function, and the toxin was not detected after 84 h. Only about 0.17% of the amount of AFB1 administered was detected as AFM1 in milk, and about 50% of this was excreted in the first liter of milk yielded after AFB1 intake. Correct procedures to prevent growth of molds, and consequent AFB1 contamination, on the feedstuffs for lactating goats represent the key to providing consumers a guarantee that milk is not contaminated by AFM1., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The contamination of cow's milk at the farm level with aflatoxin M1 was investigated in South Africa. Samples of feeds, forage, maize and milk were taken at nine dairy farms, and at the same time samples of the processed milk (retail milk) were collected from the respective dairies to which the farms delivered their milk. The feeds were analysed for aflatoxin B1 and the milk samples for aflatoxin M1 using high performance liquid chromatography (HPLC) and fluorescence detection. All milk samples from the dairy farms were positive for aflatoxin M1, ranging from 0.02 μg/l to 1.5 μg/l. Retail milk was also frequently contaminated with AFM1, at levels of 0.01-3.1 μg/l. High AFB1 levels in feed materials on the farms supplying the raw milk indicate that various sources account for this contamination frequency in milk.

This study was undertaken to determine the presence of aflatoxin M1 (AFM1) in animal milk. In addition, exposure of infants to aflatoxin M1 (AFM1) and lactating mothers to aflatoxin B1 (AFB1) was examined using AFM1 in breast milk as a biomarker for exposure to AFB1. In total, 100 samples of fresh animal milk and fermented milk (buttermilk) and 80 samples of human breast milk were collected during the period 2011-2012. An enzyme-linked immunosorbent assay (ELISA) was used for the analysis of milk samples. AFM1 was detected in all animal fresh and fermented milk samples. The concentrations of AFM1 in 70 samples of fresh and fermented milk were higher than the maximum tolerance limit accepted by the European Union and the United States of 50 ng/kg. In human milk samples the average concentration of AFM1 was higher than the maximum tolerance limit accepted by the European Union and the United States of 25 ng/kg. Logistic regression analysis failed to show a correlation between AFM1 and type and amount of dairy consumption, vegetables, fruits, and meat. However, an association between AFM1 and cereal consumption was detected. This study is the first to report on the occurrence of AFM1 in milk consumed by the Jordanian population.

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.

Background: The major contaminants usually encountered in milk and milk products include pesticide residues, heavy metals, and aflatoxin M1 (AFM1). Primarily, milk get contaminated before milching, from the cattle feed, from sources/materials used during the processing of milk as well as improper handling of the milk during the pre- and postprocessing period., Objective: The purpose of this study was to evaluate the effect of household practices on milk contaminants., Materials and Methods: Samples of pasteurized as well as unpasteurized milk (Vendor's milk) were analyzed for AFM1, pesticide residues, and heavy metals. Simulating the household practices, the impact of boiling on these contaminants was assessed., Results: The contaminant Aflatoxin M1 (AFM1) was detected at a concentration ranging from 0.071-0.075 ppb in unpasteurized as well as pasteurized milk samples analyzed during the course of study. Moreover, boiling had no impact on the quantity of AFM1 present in the milk. Pesticides and heavy metal contents were found to be within acceptable limits in all the milk samples tested., Conclusion: Mycotoxins especially aflatoxins in cattle feed and their consequential presence in milk and milk products is a serious concern world over as they are reported carcinogens. These fungal toxins are resistant to high temperatures and may lead to various health hazards. Preventive steps must be taken at each stage to ensure good quality of milk and milk products free from these contaminants. Awareness programs and education for the dairy farmers and milk processors may be helpful in this regard.

Beja is an agricultural area in northwest Tunisia. It contributes to national needs by offering cereals and milk to the market for human and animal consumption. A small number of studies on mycotoxin occurrence in feedstuffs and raw milk from lactating dairy cows in this region are available. Therefore, 226 samples were collected from farms and local markets during November 2008 until April 2010. Samples consisted of 112 raw cow milk, 56 blood from lactating cows and 58 feed destined for dairy cows. Plasma and feed were analysed for aflatoxin B₁ (AFB₁). Milk samples were analysed for aflatoxin M₁ (AFM₁). All samples were treated using a simultaneous methanolic-aqueous extraction, followed by immunoaffinity column clean-ups and were investigated by competitive enzyme-linked immunoabsorbent assay (ELISA). Recoveries were 80%-95% and 81%-92% for AFB₁ and AFM₁, respectively, while the limit of detection (LOD) was 0.01 µg/kg or µg/l for both mycotoxins. Results revealed the presence of AFB₁ in 84.4% of the feed samples (mean 18.7 ± 1.4 µg/kg), and 39.2% of the plasma-examined samples (median 7.1 ± 1.0 µg/l) were found to be contaminated at levels higher than the Tunisian and the European Union (EU) limit for dairy animals, which are 20 and 5 µg/kg in animal feed, respectively. AFM₁ was detected in 60.7% of the cow raw milk samples examined (median 13.6 ± 1.4 µg/l). Contaminated levels were higher than the EU limit of 0.05 µg/l. It was concluded that more precaution should be taken on hygiene controls in order to prevent fungal contamination.

Aflatoxin M1 (AFM1) levels were determined in pasteurised milk from five commercial trademarks produced in different areas in Thailand. One hundred and twenty milk samples were collected from local markets in Chiang Mai province, Thailand, to evaluate AFM1 concentrations using immunoaffinity columns and high-performance liquid chromatography with fluorescence detection. The overall median AFM1 level was 0.023 µg L(-1) ranging from 0.004 to 0.293 µg L(-1). All trademarks had average AFM1 concentrations lower than 0.05 µg L(-1), with those in Trademarks 3 to 5 being higher than Trademarks 1 and 2 (P < 0.01). All trademarks had different seasonal patterns of AFM1, even though operating in the same area. However, only Trademark 3 showed significant differences of AFM1 levels between seasons. The results suggested that farm management factors, rather than environment factors, were likely to be the main cause of AFM1 contamination in dairy products.

A green enzyme-linked immunosorbent assay (ELISA) to measure aflatoxin M(1) (AFM(1)) in milk was developed and validated with a surrogate calibrator curve. Polyclonal anti-idiotype (anti-Id) antibody, used as an AFM(1) surrogate, was generated by immunizing rabbits with F(ab')(2) fragments from the anti-AFM(1) monoclonal antibody (mAb). The rabbits exhibited high specificity to the anti-AFM(1) mAb, and no cross-reactivity to either of the other anti-aflatoxin mAbs or the isotype matched mAb was observed. After optimizing the physicochemical factors (pH and ionic strength) that influence assay performance, a quantitative conversion formula was developed between AFM(1) and the anti-Id antibody (y=31.91x-8.47, r=0.9997). The assay was applied to analyze AFM(1) in spiked milk samples. The IC(50) value of the surrogate calibrator curve was 2.4 μg mL(-1), and the inter-assay and intra-assay variations were less than 10.8%; recovery ranged from 85.2 to 110.9%. A reference high-performance liquid chromatography method was used to validate the developed method, and a good correlation was obtained (y=0.81x+9.82, r=0.9922)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The aim of this work was to develop and validate a method to determine aflatoxin M1 (AFM1) in cheese, yogurt, and dairy beverages. The method consisted of aqueous methanol extraction, immunoaffinity column purification and isolation, RPLC separation, and fluorescence detection. The four types of cheese samples were classified according to moisture and fat content. The mean recoveries were 71% for cheese at spiked levels from 100 to 517 ng/kg, and 76% for yogurt and dairy beverages spiked at levels from 66 to 260 ng/kg. The mean RSDs were 5.9% for cheese, and 10% for yogurt and dairy beverages. The LOD was 3 ng/kg and the LOQ was 10 ng/kg for all test commodities. To test the applicability of the developed method, a small survey of the presence of AFM1 in cheese, yogurt, and dairy beverages purchased in Ribeirão Preto-SP, Brazil, was conducted. AFM1 was detected (> 3 ng/kg) in all samples. Twenty cheese samples (83%) were contaminated with AFM1 in the range of 13-304 ng/kg. In yogurt and dairy beverages, the contamination was lower (13-22 ng/kg) in five samples (42%). The results indicated that the method is adequate for the determination of AFM1 in these four types of cheese, as well as in yogurt and dairy beverages.

Yoghurt is a popular dairy product in Iran because of its beneficial influence on human health and nutritional value. Aflatoxin M1 (AFM1) is the metabolite of potential carcinogen aflatoxin B1, which can contaminate milk through the feed and is not eliminated by common processing heat treatment. An analytical method using immunoaffinity column for extraction and a high-performance liquid chromatography (HPLC) for quantification was developed for AFM1 in this study. An HPLC method with fluorimetric detection for the determination of AFM1 in yoghurt milk has been optimized and validated according to Commission Decision BS EN ISO 14501: 2007 by using the conventional validation approach. The procedure for determining selectivity, recovery, precision, decision limit (CCα) and detection capability (CCβ) of the method has been reported. The results of the validation process demonstrate the agreement of the method with the provisions of Commission Regulation 401: 2006:EC. A new HPLC method with fluorescence detection was developed to determine aflatoxin M1. The detection limit was 1 ng/kg for yoghurt. The calibration curve was linear from 0.1 to 3.0 μg l⁻¹ injected. The method includes a preliminary clean-up and the average recoveries determined on three different days at the concentration levels of 0.025, 0.050 and 0.075 μg kg⁻¹ were in the range of 72.57%-86.66% with RSD in the range of 2.56%-8.41%. The interday and interlevel mean recovery value, which has been used to correct routine analysis results, was 80%. The method is rapid, easily automatable and therefore useful for accurate and precise screening of aflatoxin M1 in yoghurt.

Aflatoxin M1 (AFM1) is a toxic undesirable compound in milk. AFM1 affinity for caseins causes a concentration effect during milk process for dairy transformation. In spite of this, no official method of analysis, nor maximum tolerance level for aflatoxin M1 in cheese have been established. Thus, the aim of this work was to test the suitability of different HPLC methods for the AFM1 quantification in soft cheese samples at three different contamination levels (low, medium and high, at respectively nearly 30, 100 and 250 ng/kg). Nine participants were selected among Italian laboratories accredited by the Italian accreditation body (ACCREDIA) for HPLC toxin analysis. They were asked to analyze samples applying the method routinely used. The different applied methods were compared, and precision and accuracy parameters were evaluated. The main differences among HPLC procedures were registered at the level of extraction step. The use of an enzymatic digestion for the extraction of the toxin from cheese seemed to be particularly advantageous and the use of immunoaffinity columns seemed to be determinant for the improvement of sensitivity at low contamination levels. In general, the applied methods well discriminated the 3 levels of contamination, even though they performed better at the medium and high concentration levels (100 and 250 ng/kg) than at the low one (30 ng/kg). In fact relative standard deviation for reproducibility at low level was higher (60.1%) than the same value at medium and high levels (22.8% and 28.9%, respectively)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The importance of ewe milk lies in the production of high quality cheeses, such as Manchego cheese with a Protected Designation of Origin, whose safety must be guaranteed. In a 2-yr study, 407 bulk tank milk samples from farms and 82 silo milk and curd samples from cheese factories were collected from southeast Spain and tested for aflatoxin M1 (AFM1) using 2 commercial ELISA tests. Of these, 99.3% of the bulk tank samples had AFM1 levels below the European Union (EU) legal limit for milk (50 ng/kg), and well below the limit adopted by the Codex Alimentarius (500 ng/kg). Moreover, 98.8% of the silo milk and curd samples from cheese factories had AFM1 levels below the EU limit for milk. When considering median AFM1 concentrations, an average 4-fold increase was found in the final curd in relation to the corresponding silo milk. Control of AFM1 in Manchega ewe milk would enhance dairy product safety by the possible detection of faults in the manufacture of Manchego cheese., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

A sensitive method was developed using ultra-high-performance liquid chromatography (UHPLC)/MS/MS with positive electrospray ionization for determining aflatoxin M1 (AFM1) in milk and milk powder. A 50 mL quantity of low-fat liquid milk containing 100 ng/L AFM1, was prepared using immunoaffinity columns with a mean recovery rate of 79% (n = 3). UHPLC columns (BEH C18, BEH HILIC, and HSS T3) greatly reduced the chromatographic time and lowered the instrumental detection limits (IDLs) 16 to 58 times compared to an HPLC column (Betabasic C18). The HSS T3 column was chosen because it provided a low IDL (0.11 pg) and the lowest ion suppression of signal intensity (63.4%) among the tested columns. Matrix-fortified calibration curves were used for quantification and showed good linearity (r > 0.997) at 0.05-500 ng/mL. The LOD was 0.18 ng/kg for milk and 2.08 nglkg for milk powder, based on the signal intensity of the confirmatory product ion (m/z 259.1), which was less abundant than the quantitative product ion (m/z 273.1). Certified reference materials of milk powder at three levels (<0.05, 0.111 +/- 0.018, and 0.44 +/- 0.06 microg/kg) were measured within a day and between days; the results were all close to the certified levels with low variations (RSDs < 15%), showing good precision and accuracy.

A reliable, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of chloramphenicol and aflatoxin M(1) in milk has been developed. This method includes simple extraction of sample with acetonitrile, separation on a MGIII-C(18) column using 5 mM ammonium acetate aqueous solution/methanol (60:40, v/v) as mobile phase, and MS/MS detection using multiple reaction monitoring mode. The method was validated according to Commission Decision 2002/657/EC. The limits of detection (LODs) were 0.05 μg/kg for chloramphenicol and 0.005 μg/kg for aflatoxin M(1.) The limits of quantification (LOQs) were 0.2 μg/kg for chloramphenicol and 0.02 μg/kg for aflatoxin M(1). The recovery values ranged from 88.8% to 100.6%, with relative standard deviation lower than 15% in all cases, when samples were fortified at three different concentrations. The decision limits (CCα) and detection capability (CCβ) of the method were also reported. This method has been successfully applied for simultaneous analysis of chloramphenicol and aflatoxin M(1) residues in milk from local supermarkets in China.

A rapid, sensitive convenient method for determination of aflatoxin M₁ (AFM₁) in cheese and butter by HPLC was developed and validated. The method employs a safe extraction solution (mixture of acetonitrile, methanol and water) and an immunoaffinity column (IAC) for clean-up. Compared with the widely used method employing chloroform and a Florisil column, the IAC method has a short analytical time and there are no interference peaks. The limits of quantification (LOQ) of the IAC method were 0.12 and 0.14 µg/kg, while those of the Florisil column method were 0.47 and 0.23 µg/kg in cheese and buffer, respectively. The recovery and relative standard deviation (RSD) for cheese (spiked at 0.5 µg/kg) in the IAC method were 92% and 7%, respectively, while for the Florisil column method the corresponding values were 76% and 10%. The recovery and RSD for butter (spiked at 0.5 µg/kg) in the IAC method were 97% and 9%, and those in the Florisil method were 74% and 9%, respectively. In the IAC method, the values of in-house precision (n=2, day=5) of cheese and butter (spiked at 0.5 µg/kg) were 9% and 13%, respectively. The IAC method is superior to the Florisil column method in terms of safety, ease of handling, sensitivity and reliability. A survey of AFM₁ contamination in imported cheese and butter in Japan was conducted by the IAC method. AFM₁ was not detected in 60 samples of cheese and 30 samples of butter.

A sensitive and reliable analytical method based on immunoaffinity chromatography cleanup followed by HPLC separation and fluorimetric detection is described for the quantitative determination of aflatoxin M(1) in milk. The chromatographic separation is accomplished by using a C18 column and a gradient elution with methanol, acetonitrile, and water. No extraction solvent process is required and a minimal milk sample cleanup is performed by a direct loading of the immunoaffinity columns and elution with methanol. The method has been successfully validated according to Decision EC No 657/2002 by using the conventional validation approach. The results of the validation process demonstrate the agreement of the method with the provisions of Regulation EC No 401/2006.

The aim of this study was to assess levels of aflatoxin M1 (AFM1) in milk samples from Kerman, Iran. AFM1 was detected in 72 samples, ranging in concentration from <0.01 to 0.41 µg l(-1). The samples were analyzed using immunoaffinity column for clean-up and HPLC for determining AFM1. Milk samples were collected from six dairy farms. AFM1 was found in ∼ 50% of the milk samples. The average level of AFM1 was below the tolerance limit (0.05 µg l(-1)), but 50% of the samples had greater levels than the maximum tolerance limit accepted by EU and the Iranian national standard. The method detection limit and limit of quantification were 0.01 and 0.03 µg l(-1), respectively, and recovery of the method was 87%. The results showed that AFM1 contamination is a serious problem for public health. To achieve a low level of AFM1 in milk, cattle feed must be monitored regularly for aflatoxin contamination and protected from fungal contamination as much as possible.

In the present study, 24 samples of Minas Frescal cheese and 24 samples of Minas Padrão cheese produced in the North-east region of the state of São Paulo, Brazil, were analysed for aflatoxin M₁ (AFM₁) by high-performance liquid chromatography (HPLC) between March and August 2008. AFM₁ was detected in 13 (27.1%) samples at concentrations ranging from 0.037 to 0.313 ng g⁻¹. The mean concentrations of AFM₁ in positive samples of Minas Frescal and Minas Padrão cheese were 0.142 ± 0.118 and 0.118 ± 0.054 ng g⁻¹, respectively. It is concluded that the incidence of AFM₁ in Minas cheese may contribute to an increase in the overall ingestion of aflatoxins in the diet, hence indicating the need for the adoption of a tolerance limit for AFM₁ in cheese in Brazil.

A novel method for aflatoxin B (AFB) determination is proposed. The AFB determination is based on acetylcholinesterase (AChE, from electric eel) inhibition, and the AChE residual activity is determined using the colorimetric method (Ellman's method). To select and optimize the analytical procedures, the investigation on type of AChE inhibition by AFB(1) was carried out. The AChE degree of inhibition by AFB(1) was independent of the incubation time and the enzyme concentrations, showing the reversibility of the inhibition. This reversibility of the inhibition permits a rapid analysis of AFB(1). In fact, only a 3-min analysis is required. For the development of AFB(1) assay, the pH, the reaction time, the temperature, and the substrate concentration were evaluated and optimized. The linear range of 10-60 ng/mL was assessed. To evaluate the selectivity of this method, the cross-reactivity with other aflatoxins, such as AFB(2) (aflatoxin B(2)), AFG(1) (aflatoxin G(1)), AFG(2) (aflatoxin G(2)), and AFM(1) (aflatoxin M(1)), was investigated. The suitability of the assay for AFB(1) quantification in barley was also evaluated. This study shows a new approach to detect aflatoxins based on enzyme inhibition with several advantages, such as the easiness of use, the rapidity, and the cost-effectiveness, demonstrating a possible use as screening method for this type of mycotoxins.