Aflatoxin standard

Manufactured by Merck Group

Sourced in United States

Aflatoxin standards are certified reference materials used to calibrate and validate analytical methods for the detection and quantification of aflatoxins in various matrices, such as food and feed. They provide a known concentration of aflatoxins, which allows for accurate measurement and comparison of sample results.

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Lab products found in correlation

Chloroform, by Merck Group (1 mentions) Methanol, by Merck Group (1 mentions) Anhydrous sodium sulfate, by Merck Group (1 mentions) S1125 isocratic pump, by Sykam (1 mentions) Rf 20a, by Sykam (1 mentions) Kh2po4, by Merck Group (1 mentions) Dc 290 zoom digital camera, by Kodak (1 mentions) 1d 3.6 image analysis software, by Kodak (1 mentions)

9 protocols using aflatoxin standard

Oregano Essential Oil: GC-MS Analysis

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O. vulgare essential oil (OEO) was obtained from Elhawag Company for Natural Oils, Nasr City, and Cairo, Egypt (LOT#: 150/2/159). The bioactive components of OEO were identified by gas chromatography-mass spectroscopy (GC–MS) analysis [44 (link)] and are represented in Table 4.
AFB1 preparation and selection of the used concentration were based on the previous observations [6 (link)]. To obtain AFB1, Aspergillus flavus MD 341 was retrieved from the Central Lab. of Residues of Agricultural Products, Dokki, Egypt and incubated for 8 days in liquid media with 2% of yeast extract and 20% of sucrose. A reversed-phase column was utilized for the extraction, filtration, and quantitative HPLC analysis of aflatoxins. 45% methanol served as the mobile phase and was inoculated into the apparatus at a flow rate of 1 mL per minute. The analyses were performed using a fluorescence detector, and the column temperature was set to 40 °C. Aflatoxin standard was bought from Sigma-Aldrich (St. Louis, MO, USA). The media was found to contain only AFB1.

Hassan M.A., Abo-Elmaaty A.M., Zaglool A.W., Mohamed S.A., Abou-Zeid S.M., Farag M.R., Alagawany M., Di Cerbo A., Azzam M.M., Alhotan R, & EL-Hady E. (2023). Origanum vulgare Essential Oil Modulates the AFB1-Induced Oxidative Damages, Nephropathy, and Altered Inflammatory Responses in Growing Rabbits. Toxins, 15(1), 69.

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Aflatoxin Standard and Chemical Synthesis

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Aflatoxin standard was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and solvents were purchased from Sigma-Aldrich, Kanto Chemical (Tokyo, Japan), Tokyo Chemical Industry (Tokyo, Japan), and Nacalai Tesque (Kyoto, Japan), unless otherwise specified. Cyclo(l-Ala-l-Pro) was synthesized according to the method reported previously [12 (link)].

Iimura K., Furukawa T., Yamamoto T., Negishi L., Suzuki M, & Sakuda S. (2017). The Mode of Action of Cyclo(l-Ala-l-Pro) in Inhibiting Aflatoxin Production of Aspergillus flavus. Toxins, 9(7), 219.

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Aflatoxin B1 Extraction and Quantification

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AFB₁ was extracted from a 2 mL liquid fungal and yeast co-culture by adding 1 mL of acetonitrile into the conical tube, vortex for 10 min, and 0.5 mL of the supernatant was filtered through SINGLE StEP^TM eXtreme/FV 0.45 mm Nylon (Thomson Instrument Company; Oceanside, CA). Filtered samples were analyzed by high performance liquid chromatography (HPLC) on an Agilent model 1260 Infinity ChemStation (Agilent, Palo Alto, California, USA). HPLC was performed on a Supelcosil LC-18 reversed-phase column (150 mm × 4.6 mm i.d., 5 μm particle size) at a flow rate of 1 mL/min.
The mobile phase was methanol/acetonitrile/H₂O (20:20:60). Aflatoxins were quantified by a fluorescent detector with excitation at 365 nm and emission at 455 nm and quantified by peak areas relative to a standard curve of authentic AFB₁ [55 (link)]. Aflatoxin standards were purchased from Sigma-Aldrich (St. Louise, MO, USA).

Hua S.S., Sarreal S.B., Chang P.K, & Yu J. (2019). Transcriptional Regulation of Aflatoxin Biosynthesis and Conidiation in Aspergillus flavus by Wickerhamomyces anomalus WRL-076 for Reduction of Aflatoxin Contamination. Toxins, 11(2), 81.

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Aflatoxin Resistance Evaluation in Maize

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To evaluate the preharvest aflatoxin contamination resistance of the selected lines, they were inoculated with the A. flavus isolate NRRL3357 (Norton, 1999; Scarpari et al., 2014). Briefly, the isolate was cultured on V8 agar (20% V8 juice, 2% agar, 0.5% CaCO₃) in the dark at 37 °C (Fountain et al., 2015; Guo et al., 1996). After 5 days, conidia were harvested in sterile 0.1% (v/v) Tween‐20. Conidial concentration of the suspension was determined using a hemocytometer, and the concentration was adjusted to 4.0 × 10⁶ conidia/mL. The resulting spore suspension (3 mL) was used to inoculate ears at 14 DAP using the side injection method with a modified tree marking gun fitted with a needle (Guo et al., 2017; Windham et al., 2003). Inoculated ears with or without drought treatments were collected at 60 DAP for use in aflatoxin analysis and quantification. Aflatoxin extraction and analysis were conducted according to Guo et al. (1996) using thin‐layer chromatography (TLC; Park et al., 1994; Robertson et al., 1967; Trucksess et al., 1984). The aflatoxin standards were purchased from Sigma‐Aldrich (St. Louis, MO), and all the samples were analysed in three replicates.

Yang L., Fountain J.C., Ji P., Ni X., Chen S., Lee R.D., Kemerait R.C, & Guo B. (2018). Deciphering drought‐induced metabolic responses and regulation in developing maize kernels. Plant Biotechnology Journal, 16(9), 1616-1628.

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Aflatoxin Quantification Protocol

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Aflatoxin standards (in acetonitrile 1 μg/mL) and chemicals like methanol (HPLC grade), chloroform (HPLC grade), cupric carbonate (HPLC grade), anhydrous sodium sulfate (HPLC grade) were acquired from (Sigma-Aldrich, Steinheim, Germany). The other chemicals and reagents used in the current research were of high purity grade (≥90%), and the double-distilled water was used for analysis.

Razis A.F., Shehzad M.M., Usman S., Ali N.B., Iqbal S.Z., Naheed N, & Asi M.R. (2020). Seasonal Variation in Aflatoxin Levels in Edible Seeds, Estimation of Its Dietary Intake and Vitamin E Levels in Southern Areas of Punjab, Pakistan. International Journal of Environmental Research and Public Health, 17(23), 8964.

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HPLC-based Quantification of Aflatoxins

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A High-Performance Liquid Chromatography (HPLC) system S 500 routine series equipped with an S1125 isocratic pump (Sykam, Eresing, Germany) and coupled to a fluorescence detector as described previously [39 (link)] with some adjustments was used to quantify aflatoxins. The mobile phase was prepared using water/methanol/acetonitrile (65/25/15, v/v/v) at a flow rate of 1 mL/min. The column oven temperature was adjusted to 37 °C. The excitation and emission wavelengths of the fluorescence detector (Sykam, RF-20A) were set at 365 nm and 440 nm, respectively. A reverse phase C18 column (Welch Material, Inc., Austin, TX, USA) (4.6 × 250 mm) was used as the stationary phase. Aflatoxin standards—AFB₁ (0.5 µg/mL), AFB₂ (0.25 µg/mL), AFG₁ (0.5 µg/mL), and AFG₂ (0.25 µg/mL)—of HPLC-grade purity (≥98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Each standard and sample were injected at a volume of 20 µL with a run time of 22 min. The retention time for AFB₁ was 5.09 min, 8.78 min for AFB₂, 4.28 min for AFG₁, and 6.66 min for AFG₂.

Naeem I., Ismail A., Rehman A.U., Ismail Z., Saima S., Naz A., Faraz A., de Oliveira C.A., Benkerroum N., Aslam M.Z, & Aslam R. (2022). Prevalence of Aflatoxins in Selected Dry Fruits, Impact of Storage Conditions on Contamination Levels and Associated Health Risks on Pakistani Consumers. International Journal of Environmental Research and Public Health, 19(6), 3404.

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Aflatoxin Quantification Protocol

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All aflatoxin standards were prepared from Sigma (MO, USA). High-performance liquid chromatography (HPLC) grade of methanol and acetonitrile supplied from Caledon Co. (Caledon laboratories Ltd., Canada). All materials used in the preparation of phosphate-buffered saline [pH 7.4; 0.20 g KCl, 0.20 g KH2PO4, 1.16 g anhydrous Na₂HPO₄ (or 2.92 g Na₂HPO₄,12H₂O) and 8.0 g NaCl dissolved in 900 ml water and pH adjusted to 7.4 with 0.1 M HCl or 0.1 M NaOH and diluted to 1 L with water] were procured from Merck Co. (Merck, Darmstadt, Germany).

Dini A., Esmaeili Nadimi A, & Behmaram K. (2022). The Effect of Monitoring System on Risk Assessment of Aflatoxins in Iran's Pistachio Nuts Exported to the E.U. During 2012 - 2018. Iranian Journal of Pharmaceutical Research : IJPR, 21(1), e123951.

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Quantifying Lichen Extract Inhibition of NA

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To assess the inhibitory effects of lichen extracts on the accumulation of NA, cultures from treated and control cell wells were extracted with chloroform twice followed by acetone twice. Extracts were combined and evaporated and resuspended in 70% methanol to 1 ml. Culture extracts (5 µl sample per lane) and known quantities of aflatoxin standards (Sigma) were spotted on Partisils LHPKD silica gel TLC plates (60Å, 10 × 10 cm, 200 µm thick, plates; Whatman Inc., Clifton, NJ). Plates were resolved using a 95% chloroform in acetone (v/v) solvent system and analyzed under UV light. TLC plates were photographed using a Kodak DC 290 Zoom Digital Camera and band intensity was calculated using Kodak 1D 3.6 Image Analysis software. Band intensity and Rf values for bands were compared with values for the aflatoxin standards to establish relative concentrations and identity. Background intensity was subtracted from each band to obtain a true measure of band intensity (Roze et al. 2007) .

Roze L.V., Laivenieks M., Gdanetz K., Linz J.E., Fryday A.M., & Trail F. (2020). Activity throughout the lichen phylogeny indicates a focus on regulation of specialized metabolites.

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Aflatoxin Quantification in Peanuts

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Peanut and peanut products were obtained from different vendors and shops. Consumers of these products are generally low income and middle class groups. The low-income group also consumes the low quality of peanuts in the homemade products. Chemicals and solvents used in the analysis were of analytical grades. Aflatoxin standards were purchased from Sigma Chemical Company, St. Louis, MO, and USA. Precoated TLC plates of Silica gel 60 thickness 0.25mm, 20cm x 20cm) on glass or aluminum, without fluorescent indicator were purchased from E. Merck, Dramstadt, Germany. The microwave oven used was of Frigidaire R model RCM5130 with temperature probe. Detoxification procedure was used for the microwave cooking at the heating set approximately up to 92 o C with maximum time of 5 minutes. Aflatoxins were determined as described in the Methods of the A.O.A.C. official method 975.36/968.22 [16] , except that the defatting of the acetone extract of the sample was performed. TLC plates were observed under long wavelength UV light in an enclosed viewing cabinet and samples were quantified by visual comparison with standards. All positive findings of aflatoxin B 1 , naturally present were confirmed by spraying the TLC plates with 50% sulphuric acid and making the derivative with trifluoroacetic acid. The method is ISO-17025 accredited dually by national and international accredited bodies.

Mobeen A., Aftab A., Asif A., & Zuzzer A. (2022). Aflatoxins B1 and B2 Contamination of Peanut and Peanut Products and Subsequent Microwave Detoxification. Journal of Pharmacy and Nutrition Sciences, 1(1), 1-3.

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