Analysis of mycotoxin fumonisins in corn products by high-performance liquid chromatography coupled with evaporative light scattering detection

Fumonisins are mycotoxins produced by the fungus Fusarium verticillioides, which is a widespread pathogen of corn. The mycotoxins are known to cause fatal diseases in some domestic animals and have been linked to human esophageal cancer in China and South Africa. Here, we describe a simple method for direct and quantitative analysis of the toxins in food products. The method is based on high-performance liquid chromatography coupled with an evaporative laser scattering detector (HPLC–ELSD) without any prior derivatization of the samples. Using this method, we have analyzed corn-based food samples from central markets in eastern China. The results showed that FB₁ was the main contaminant in the samples. The overall level of fumonisin contamination was relatively low, with a range of 0.25–1.80 μg/g (mean 0.74 μg/g) in 66.7% (16 of 24) of corn samples from Middle-eastern Area, 0.21–0.29 μg/g (mean 0.24 μg/g) in 28.6% (6 of 21) of corn samples from Northeastern Area, and 0.30–3.13 μg/g (mean 0.47 μg/g) in 30.0% (6 of 20) of corn samples from Southeastern Area.     

Distribution of fungi and aflatoxins in a stored peanut variety

The objective of the present study was to evaluate the mycoflora and occurrence of aflatoxins in stored peanut samples (hulls and kernels) from Tupã, State of São Paulo, Brazil. The samples were analyzed monthly over a period of one year. The results showed a predominance of Fusarium spp. (67.7% in hulls and 25.8% in kernels) and Aspergillus spp. (10.3% in hulls and 21.8% in kernels), and the presence of five other genera. The growth of Aspergillus flavus was mainly influenced by temperature and relative humidity. Analysis of hulls showed that 6.7% of the samples were contaminated with AFB₁ (mean levels = 15–23.9 μg/kg) and AFB₂ (mean levels = 3.3–5.6 μg/kg); in kernels, 33.3% of the samples were contaminated with AFB₁ (mean levels = 7.0–116 μg/kg) and 28.3% were contaminated with AFB₂ (mean levels = 3.3–45.5 μg/kg). Analysis of the toxigenic potential revealed that 93.8% of the A. flavus strains isolated were producers of AFB₁ and AFB₂.     

Determination of mycotoxins in cereals by liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC–MS/MS) method is described for simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB₁ and FB₂), T2 and HT2-toxin in cereals. One-step extraction using solvent mixtures of acetonitrile:water:acetic acid (79:20:1) without any clean-up was employed for extraction of these mycotoxins from cereals. The mean recoveries of mycotoxins in spiked cereals ranged from 76.8% to 108.4%. Limits of detection (LOD) and quantification (LOQ) ranged 0.01–20 and 0.02–40 ng/g, respectively. The developed method has been applied for the determination of mycotoxins in 100 cereal samples collected from Malaysian markets. A total of 77 cereal samples (77%) contaminated with at least one of these mycotoxins. Occurrence of mycotoxins in commercial cereal samples were 70%, 40%, 25%, 36%, 19%, 13%, 16, and 16% for aflatoxins, OTA, ZEA, DON, FB₁, FB₂, T2 and HT2-toxin, respectively. The results demonstrated that the procedure was suitable for the determination of mycotoxins in cereals and could be implemented for the routine analysis.     

The determination of vitamin D3 in bovine milk by liquid chromatography mass spectrometry

A renewed international interest in vitamin D status has revealed significant deficiencies in several populations, including Australia. Vitamin D exists in two forms, cholcalciferol (D₃) and ergocalciferol (D₂). The main source of vitamin D₃ is from exposure of 7-dehydrocholesterol present in the skin to UV irradiation. However, there is an absolute requirement for vitamin D through proper dietary intake if humans live in the absence of sunlight or exclusively indoors. Bovine milk is considered to be a good dietary source of vitamin D₃, even though the levels are quite low. This paper describes robust methods using liquid chromatography–linear ion trap mass spectrometry (LC–MSⁿ) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to measure the levels of vitamin D₃ in fresh bovine milk (0.05 μg/100 ml), commercial (natural and fortified) milk samples (0.01–2 μg/100 ml) and a dairy based infant formula (8 μg/100 g), without the need for extensive clean-up procedures. The limits of quantification (LOQ) are 0.01 μg/100 ml and 0.02 μg/100 ml for LC–MSⁿ and LC–MS/MS, respectively. Recoveries of vitamin D₃ added to the samples prior to saponification were satisfactory (range 60–90%). 25-Hydroxyvitamin D₃ was not present in any of the samples analysed (LOQ = 0.01 μg/100 ml, recovery range 30–40%).     

Folate content in foods commonly consumed in Egypt

The folate content in some Egyptian foods was determined using RP-HPLC-FL. Trienzyme treatment was used for legumes, dienzyme treatment for cereals and starchy vegetables, and monoenzyme treatment for vegetables and fruits. The highest folate content (633 μg/100 g) was found in dried Jew’s mellow due to low water content, followed by legumes (e.g. 150 μg/100 g for chick peas) and leafy vegetables (100 μg/100 g). For other foods, folate content ranged from 10–90 μg/100 g. In all foods, the predominant folate form was 5-CH₃–H₄folate, except for dried Jew’s mellow, which contained more than 80% 10-HCO–PteGlu. Using folate data from our own analyses and food tables and food consumption data, the dietary folate intake per capita in Egypt was estimated. However, representative and validated food composition data for folate in Egyptian foods are needed for estimating and evaluating the adequacy of the population’s folate intake.     

Determination of aflatoxin levels in nuts and their products consumed in South Korea

A total of 85 nuts and their products marketed in South Korea were assessed for aflatoxins using a monitoring scheme consisting of enzyme-linked immunosorbent assay (ELISA) for rapid screening, high performance liquid chromatography (HPLC) for quantification and LC–mass spectrometry (MS) for confirmation. Thirty-one out of 85 samples gave ELISA readings above 0.06 and were screened as possible positive samples. Aflatoxin contents of possible positive samples were determined using HPLC with a detection limit of 0.08–1.25 μg/kg and a quantification limit of 0.15–2.50 μg/kg. Nine samples including 1 raw peanut, 4 roasted peanuts, 2 peanut butters, 1 pistachio and 1 seasoned assorted nut were contaminated with aflatoxins (10.6% of incidence), ranging in various levels up to 28.2 μg/kg. LC–MS analysis on contaminated samples revealed that peaks eluting at 4.4, 5.2, 9.1 and 11.9 min were confirmed as aflatoxin G₁, aflatoxin B₁, aflatoxin G₂ and aflatoxin B₂, respectively.     

Determination of trace metals in canned fish marketed in Turkey

The levels of trace metals of canned fish samples collected from markets in Turkey were determined by flame and graphite furnace atomic absorption spectrometry after microwave digestion. The accuracy of the method was corrected by standard reference material (NRCC-DORM-2 Dogfish Muscle). The contents of investigated trace metals in canned fish samples were found to be in the range 1.10–2.50 μg/g for copper, 7.57–34.4 μg/g for zinc, 0.90–2.50 μg/g for manganese, 10.2–30.3 μg/g for iron, 0.96–3.64 μg/g for selenium, 0.45–1.50 μg/g for aluminium, 0.97–1.70 μg/g for chromium, 0.42–0.85 μg/g for nickel, 0.09–0.40 μg/g for lead and 0.06–0.25 μg/g for cadmium. The results were compared with the literature values.     

Development of polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for sodium saccharin residue in food samples

A sensitive and specific polyclonal antibody (PcAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) for sodium saccharin is described. 6-Amino saccharin was coupled to carrier protein for artificial antigen by diazotisation. New Zealand white rabbits were immunised to obtain anti-sodium saccharin PcAb and then icELISA was developed. The assay showed high sensitivity and specificity to sodium saccharin, with the 50% inhibition value (IC₅₀) of 0.243 μg mL⁻¹, workable range (IC₃₀–IC₇₀) of 0.050–12.8 μg mL⁻¹ and limit of detection (LOD, IC₂₀) of 0.021 μg mL⁻¹. The average recoveries of sodium saccharin in spiked food samples were estimated ranging from 70.7% to 98.8%. A statistically significant correlation of results was obtained between this new ELISA and previously established HPLC approaches with the food-relevant sodium saccharin concentration range 0–320 μg mL⁻¹ (R² = 0.9887–0.9975). These results indicated that the established ELISA was a potential and useful analytical tool for rapid determination of sodium saccharin residue in food samples.     

Determination of Allura red in food samples after cloud point extraction using mixed micelles

A cloud point extraction process using mixed micelles of CTAB, Triton X-114 and Triton X-100 for preconcentration and spectrophotometric determination of trace amounts of Allura red in food samples was developed. The effect of different parameters such as concentration of surfactants, electrolyte concentration, temperature and pH on the cloud point extraction of Allura red was studied in detail and a set of optimum conditions were established. A linear calibration curve in the range of 20–1400 μg/L of Allura red was obtained. Detection limit based on 3S_b was 7.8 μg/L and the relative standard deviation for 75 μg/L of Allura red was 3.87 (n = 10). The method was applied to the determination of Allura red in food samples such as candy, soft drink and jellies.     

Co-occurrence of mycotoxins in corn samples from the Northern region of Paraná State,Brazil

Natural mycoflora and co-occurrence of fumonisins and aflatoxins were evaluated in 300 freshly-harvested corn samples (2003 and 2004 crops) collected at two points of the production chain in the Northern region of Paraná State, Brazil. In the 2003 crop, fumonisins were detected in 100% of samples and the mean levels were 2.54 μg/g in the reception and 3.12 μg/g in the pre-drying samples. On the other hand, in the 2004 crop fumonisins were detected in 98.9% and 95% of the reception and pre-drying samples, respectively. The mean levels were 1.31 μg/g in the reception, and 1.36 μg/g in the pre-drying samples. Aflatoxins were not detected in 92% of the samples analysed. The maximum probable daily intake (PDI_M) estimated for the Brazilian population (0.95 μg/kg body weight/day) is below the tolerable daily intake of 2.0 μg/kg body weight/day for fumonisin B₁.     

Monitoring and risk assessment of pesticide residues in yuza fruits (Citrus junos Sieb. ex Tanaka) and yuza tea samples produced in Korea

The objective of this study was to establish an analytical method to measure pesticides used to cultivate yuza (Citrus junos Sieb. ex Tanaka) and to analyze pesticide residue levels of yuza and yuza tea samples. Risk assessments were also performed by calculating estimated daily intake (EDI) and acceptable daily intake (ADI). An excellent linear correlation was achieved with coefficient correlation values of 0.9750–0.9999. Percent recoveries were 80.4–109.9% for most pesticides with a <6.9% relative standard deviation (RSD). The limits of quantification for the method were 0.10–0.67 μg/ml. The RSD of intra-day and inter-day variability was <15.3%. Seven pesticides in yuza (n = 80) and yuza tea (n = 75) were analyzed with the optimized analytical method. Acequinocyl, spirodiclofen and carbendazim were detected in yuza samples in the concentration range of 0.07–0.15 μg/g, 0.11–1.89 μg/g, and 0.03–5.15 μg /g, respectively, whereas chlorpyrifos, prothiofos, phosalone, and deltamethrin were not detected in yuza or yuza tea. The concentrations of acequinocyl, spirodiclofen and carbendazim ranged from 0.18–1.05 μg/g, 0.13–0.29 μg/g, and 0.17–2.36 μg/g, respectively, in yuza tea samples. The percent ratios of EDI to ADI for acequinocyl, spirodiclofen, and carbendazim were 24.6%, 22.7%, and 58.5%, respectively.     

Natural occurrence of fumonisins B1 and B2 in corn in four provinces of China

Fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) are the most abundant fumonisins (FBs) occurring worldwide in maize, infected mainly by Fusarium verticillioides and F. proliferatum. A total of 307 corn kernel samples were collected from 45 districts of Gansu, Shandong, Ningxia and the Inner Mongolia provinces of the north and northwest China. The samples were analysed for FB₁ and FB₂ by high-performance liquid chromatography. The FBs (FB₁+ FB₂) incidence rate in samples from Gansu, Shandong, Ningxia and Inner Mongolia were 31.5%, 81.1%, 46.2% and 53.6%, respectively. Average FBs concentration was 703 μg/kg and the concentrations ranged from ≤11 to 13,110 μg kg^?1. Results were compared with the European Commission (EC) regulation for FB₁+ FB₂ in unprocessed maize for human consumption of 4 mg kg^?1. Contamination in 17 samples was higher than these levels. More than 80% of the samples from Liaocheng county, which is located in the Shandong province, were contaminated with FBs, with a mean total FB concentration of 2496 μg/kg. The result was significantly different from that of the Inner Mongolia (1399 μg/kg), Ningxia (373 μg/kg) and Gansu (175 μg/kg). Average exposure to FBs (0.12 μg/kg body weight/day) is within the provisional maximum tolerable daily intake of 2.0 mg/kg of body weight set by the Joint Food and Agriculture Organization and World Health Organization Expert Committee on Food Additives.     

Short communication: Investigation of aflatoxin M1 levels in infant follow-on milks and infant formulas sold in the markets of Ankara,Turkey

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.     

Rhus verniciflua Stokes flavonoid extracts have anti-oxidant,anti-microbial and α-glucosidase inhibitory effect

We isolated four compounds, fustin, gallic acid, 3′,4′,7-trihydroxyflavone, and fisetin from Rhus verniciflua. These compounds showed electron donation ability (87–94%) that was stronger than butylated hydroxyanisole (52%). Gallic acid (OD₇₀₀ = 1.98) showed the highest reducing power, and the other isolated compounds (OD₇₀₀ = 0.66–1.31) showed stronger activity than α-tocopherol (OD₇₀₀ = 0.21). The ethyl-acetate fraction had the highest phenolic content (723 mg GAE/g), followed by the 80% ethanolic extract (597 mg GAE/g). For Gram-negative bacteria, fisetin had the most potent anti-bacterial activity (MIC = 8 μg/ml) against Escherichia coli. 3′,4′,7-Trihydroxyflavone (106%), the 80% ethanolic extract (101%), and ethyl-acetate (98%) had the most powerful α-glucosidase inhibitory effect at 50 μg/ml. R. verniciflua extracts have potential as functional food additives.     

Determination of ochratoxin A in organic and non-organic cereals and cereal products from Spain and Portugal

The objective of this work was to know the occurrence of OTA in organic and non-organic cereals and cereal products from Spain and Portugal. A method based on extraction with matrix solid phase dispersion (MSPD) using octylsilica (C₈) followed by liquid chromatography coupled with fluorescence detection (LC–FD) was used to determine OTA from the selected samples. Recoveries of OTA from the studied samples spiked at 10 ng/g level ranged from 78% to 89% with a standard deviation of 3.66. The limits of detection and quantification of this method were 0.05 and 0.19 ng/g, respectively. Furthermore, LC–FD after OTA methylation was used to confirm the identity of OTA in all positive samples. This procedure was applied to 83 organic and non-organic samples including rice, wheat, barley, rye, oats and maize from Spain and Portugal. OTA was detected in 22% of the samples, with concentrations ranging from 0.20 to 27.10 ng/g. From the total OTA contaminated samples (n = 18), 72% were organic cereal and 28% were non-organic cereal samples, with mean concentrations of 1.64 and 0.05 ng/g, respectively. The 66% and 34% of contaminated samples were from Spain and Portugal, respectively, with mean concentrations of 0.93 and 0.64 ng/g for each country. Six contaminated samples exceeded the maximum limits (ML) for OTA fixed by European Commission Regulation (5 μg/kg), among them three were from Spain and three from Portugal.     

Aflatoxins contamination in spices and processed spice products commercialized in Korea

A survey for total aflatoxins (aflatoxins B₁, B₂, G₁, and G₂) was conducted on 88 spices and processed spice products commercialized in Korea. The presence of aflatoxins was determined by high-performance liquid chromatography (HPLC) with fluorescence detector using immunoaffinity column clean-up. Total aflatoxins (AFs) are detected in 12 samples (13.6% of incidence) including seven red pepper powder, two red pepper pastes (Kochujang), two curry and one ginger product. The contamination levels are 0.08–4.45 μg/kg as aflatoxin B₁ and 0.08–4.66 μg/kg as AFs. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis on contaminated samples was conducted for the confirmation of detected aflatoxins. The 12 samples which showed aflatoxins by HPLC/FLD were confirmed as aflatoxins by LC–MS/MS.     

Free aromatic amino acids in egg yolk show antioxidant properties

Laying hens are known to be able to “bio-convert” health-promoting components from the diet into eggs. Wheat and corn are important sources of antioxidant phenolics in the human diet. Given the fact that wheat and corn are major feed ingredients of laying hens in Canada, the objectives of this study were to characterise the presence of novel phenolic compounds in egg yolk and to determine the effect of cooking methods on their content and activity. The total phenolic content of yolk extracts was 3.83 and 3.49 μmol gallic acid equivalents/g dry yolk for wheat-based and corn-based yolks, respectively. Antioxidant properties of yolk extracts were measured by the ORAC, DPPH and ABTS assays. ORAC values were 151.1 ± 26.1 and 155.9 ± 9.5 μmol TE/g for wheat-based and corn-based yolks, respectively. All cooking methods significantly reduced (p < 0.05) the antioxidant values. Ferulic acid was detected in trace amounts and no other phenolic compounds were found. However, tryptophan and tyrosine were found to be two main contributors to the antioxidant property of egg yolk. The contents of total free amino acids were 10081.0 and 10009.5 μg/g in wheat-based and corn-based yolks, respectively; cooking methods were found to reduce significantly the amount of free amino acids.     

Development and validation of a new LC–MS/MS method for the simultaneous determination of six major ergot alkaloids and their corresponding epimers. Application to some food and feed commodities

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of six major ergot alkaloids, ergometrine, ergosine, ergotamine, ergocornine, ergokryptine and ergocristine, as well as their corresponding epimers in food and feed samples. The method involves extraction under alkaline conditions and subsequent clean-up by applying a simple and rapid liquid–liquid partitioning procedure prior to LC–MS/MS analysis. Evaluation of the method revealed good linearity, accuracy and precision. The limits of quantification varied from 0.1 to 1 μg/kg depending on the analyte and matrix. The average extraction and clean-up recoveries in different matrices were between 45 (only for ergometrine in biscuit) and 90%. The uncertainty associated with the analytical method was not higher than 51% and 30%, at concentration levels of 2.5 and 150 μg/kg respectively. Analyte epimerization proved to be minimal during the analytical procedure. The method has been successfully applied to the determination of ergot alkaloids in some Belgian food and feed commodities. Ergot alkaloids were found in 104 out of 122 samples investigated. Ergosine was the most frequently occurring alkaloid, while the highest levels were observed for ergotamine, ergocristine or ergosine, depending on the product type. The total alkaloid content in positive samples varied from 1 to 1145 μg/kg.     

Trace metals in raw cows’ milk and assessment of transfer to Comté cheese

As part of a program to assess the transfer of metals from soil to dairy products, the transfer of metal trace elements to milk and cheese was studied. Concentrations of non-essential (Cd and Pb) and essential elements (Cu and Zn) were determined by atomic absorption spectrometry in 61 samples of raw milk and 21 of the corresponding cheese. While metal concentrations (dry weight) in raw milk were very low (Cd: 0.34–1.01 ng/g; Pb: 0.009–0.126 μg/g; Cu: 0.28–1.71 μg/g; Zn: 20.62–30.96 μg/g), concentrations in the corresponding cheese were significantly higher (Cd: 0.68–11.37 ng/g; Pb: 0.020–0.925 μg/g; Cu: 5.35–21.34 μg/g; Zn: 33.66–63.41 μg/g). The retention factor R_t suggests a concentration effect during the cheese making process, especially in the case of Cu, due to the use of large copper vats. Concentrations of non-essential elements (Cd and Pb) in cheese largely remained below those considered as dangerous for consumers. Finally, Comté cheese may constitute a useful source of Cu and Zn in human diet.     

Fumonisins B1 and B2 in the corn-milling process and corn-based products,and evaluation of estimated daily intake

The distribution of fumonisins (FBs: FB₁ and FB₂) in the corn-milling process and in corn-based products, as well as daily intake estimates for the Brazilian population were evaluated. Among corn fractions samples, corn meal had the highest mean concentration of FB₁ (1305 µg kg^?1) and FB₂ (651 µg kg^?1) and a distribution factors of 452% and 256% in relation to corn grain, respectively. On the other hand, the distribution factor of FB₁ and FB₂ in corn flour was found to be 144% and 88% respectively, which demonstrates that fumonisins in this fraction were reduced compared with corn grain. As a result, almost half the corn meal samples (47%) would be non-compliant with future Brazilian regulation (2017) for fumonisins. However, corn-based products, such as corn flakes and popcorn, were in compliance with the regulation. The average probable daily intake and maximum probable daily intake of fumonisins estimated for the Santa Catarina state (Brazil) population were below the provisional maximum tolerable daily intake of 2 µg kg^?1 body weight day^?1 for all corn samples. Despite this, the adoption of practices to control the occurrence of fumonisins should be applied to the corn-milling fractions that may contain a higher concentration of this toxin, such as corn meal, often used for animal feed in Brazil.