EN 15851:2010

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Aflatoxin B1 in Säuglings- und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und FluoreszenzdetektionProduits alimentaires - Dosage de l'aflatoxine B1 dans les produits pour nourrissons et jeunes enfants à base de céréales - Méthode de chromatographie liquide haute performance avec purification sur colonne d'immunoaffinité et détection par fluorescenceFoodstuffs - Determination of aflatoxin B1 in cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection67.230Predpakirana in pripravljena hranaPrepackaged and prepared foods67.060QMLKCereals, pulses and derived productsICS:Ta slovenski standard je istoveten z:EN 15851:2010SIST EN 15851:2010en,fr,de01-september-2010SIST EN 15851:2010SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15851
April 2010 ICS 67.060 English Version
Foodstuffs - Determination of aflatoxin B1 in cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection
Produits alimentaires - Dosage de l'aflatoxine B1 dans les produits pour nourrissons et jeunes enfants à base de céréales - Méthode de chromatographie liquide haute performance avec purification sur colonne d'immunoaffinité et détection par fluorescence
Lebensmittel - Bestimmung von Aflatoxin B1 in Säuglings- und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion This European Standard was approved by CEN on 27 February 2010.
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Typical chromatogram . 13Annex B (informative)
Precision data . 14Bibliography . 15
37 % in water. 4.11 Hydrochloric acid solution, substance concentration c(HCl) = 0,1 mol/l. Dilute 8,28 ml of hydrochloric acid solution (4.10) to 1 l with water. 4.12 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l. Dissolve 4 g of sodium hydroxide (4.9) in 1 l of water. 4.13 Phosphate buffered saline (PBS) solution, c(NaCl) = 120 mmol/l, c(KCl) = 2,7 mmol/l, c(phosphate buffer) = 10 mmol/l, pH = 7,4. Dissolve 8,0 g of sodium chloride (4.8), 1,2 g of anhydrous disodium hydrogen phosphate or 2,9 g of Na2HPO4·12 H2O (4.2), 0,2 g of potassium dihydrogen phosphate (4.7) and 0,2 g of potassium chloride (4.6) in 900 ml of water. After dissolution, adjust the pH to 7,4 with hydrochloric acid solution (4.11) or sodium hydroxide solution (4.12) as appropriate, then dilute to 1 l with water. Alternatively, a PBS solution with equivalent properties can be prepared from commercially available PBS material. 4.14 Pyridinium hydrobromide perbromide (PBPB), [CAS: 39416-48-3]. 4.15 Acetonitrile. WARNING — Acetonitrile is hazardous and samples shall be blended using an explosion proof blender which is housed within a fume cupboard. After blending, samples shall be filtered inside a fume cupboard. 4.16 Methanol, HPLC grade. 4.17 Methanol,
technical grade. 4.18 Toluene. 4.19 Extraction solvent. Mix eight parts per volume of methanol (4.17) with two parts per volume of water. 4.20 Nitric acid, c(HNO3) = 4 mol/l. 4.21 HPLC mobile phase A, for use with PBPB. Mix six parts per volume of water with two parts per volume of acetonitrile (4.15) and three parts per volume of methanol (4.16). Degas mobile phase A with for example helium (4.2). 4.22 HPLC mobile phase B, for use with electrochemically generated bromine. Mix six parts per volume of water with two parts per volume of acetonitrile (4.15) and three parts per volume of methanol (4.16). Add 120 mg of potassium bromide (4.5) and 350 µl of nitric acid (4.20) per litre of mobile phase. Degas mobile phase B with for example helium (4.2). 4.23 Post-column reagent. Dissolve 50 mg of PBPB (4.14) in 1 l of water. To be used with mobile phase solvent A (4.21). The solution may be used up to four days if stored in a dark place at room temperature. SIST EN 15851:2010

aflatoxin B1 are applied as a standard solution in a mixture of ten parts per volume of methanol and 90 parts per volume of water. 4.26 Aflatoxin B1, in crystal form or as a film in ampoules or in form of commercially available aflatoxin B1 solution. WARNING — Aflatoxins are subject to light degradation. Protect the laboratory, where the analyses are done, adequately from daylight. This can be achieved effectively by using ultraviolet (UV) absorbing foil on the windows in combination with subdued light (no direct sunlight) or curtains or blinds in combination with artificial light (fluorescent tubes are acceptable). Protect aflatoxin containing solutions from light as much as possible (keep in the dark, use aluminium foil or amber-coloured glassware). 4.27 Aflatoxin B1 stock solution, c ≈ 10 µg/ml. Prepare a solution of aflatoxin B1 in the mixture of toluene and acetonitrile (4.24) to give a solution with a mass concentration of approximately 10 µg/ml. To determine the exact mass concentration, record the absorption curve between 330 nm and 370 nm in 1 cm quartz cells in a spectrometer (5.14) with the mixture of toluene and acetonitrile (4.24) as reference. Identify the wavelength for maximum absorption (between 330 nm and 370 nm). Calculate the mass concentration of aflatoxin B1, afl, in µg/ml, using Equation (1):
bMA×××=ερ100maxafl (1) where
Amax is the absorption determined at the maximum of the absorption curve (between 330 nm and 370 nm);
M is the molar mass, in g/mol, of aflatoxin B1 (M = 312 g/mol);
0 is the molar absorption coefficient, in square metres per mole, of aflatoxin B1 in the mixture of toluene and acetonitrile (4.24) (1 930 m2/mol, see [5]);
b is the optical path length, in centimetres, of the quartz cell. Store this solution in a freezer at approximately - 18 °C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the concentration of the solution if it is older than 12 months. 4.28 Aflatoxin B1 standard solution,
= 5,00 ng/ml. Pipette a volume of aflatoxin B1 stock solution (4.27) containing exactly 1,00 µg aflatoxin B1 into a 200 ml calibrated volumetric flask and dilute to the mark with the mixture of toluene and acetonitrile (4.24). This solution contains 5,00 ng/ml aflatoxin B1. SIST EN 15851:2010

= 2 µg/ml. Pipette a volume of aflatoxin B1 stock solution (4.27) containing exactly 20 µg aflatoxin B1 into a 10 ml calibrated volumetric flask.
Evaporate the mixture of toluene and acetonitrile solution just to dryness under a stream of nitrogen at room temperature. Make up to volume with methanol (4.16) and shake well. The concentration of this spiking solution is 2 µg/ml for aflatoxin B1. Wrap the flask tightly in aluminium foil and store it at less than 4 °C. Before use, do not remove the aluminium foil until the contents have reached room temperature to avoid incorporation of water by condensation. A solution stored in this way is stable for at least three months. 5 Apparatus WARNING — All glassware coming into contact with aqueous solutions of aflatoxins shall be washed with acid solution before use. Many laboratory washing machines do this as part of the washing programme. Otherwise soak laboratory glassware coming into contact with aqueous solutions of aflatoxins in sulfuric acid (c = 2 mol/l) for several hours (e.g. 15 h overnight), then rinse well (e.g. at least three times) with water to remove all traces of acid. Check the absence of acid with pH paper.
This treatment is necessary, because the use of non-acid washed glassware can cause losses of aflatoxins. In practice, the treatment is necessary for round bottomed flasks, volumetric flasks, measuring cylinders, vials or tubes used for calibration solutions and final extracts (particularly autosampler vials), and Pasteur pipettes, if these are used to transfer calibration solutions or extracts. Usual laboratory glassware and equipment and, in particular, the following. 5.1 Analytical balance, capable of weighing to 0,000 1 g. 5.2 Laboratory balance, capable of weighing to 0,01 g. 5.3 Adjustable vertical or horizontal shaker. 5.4 Filter paper, e.g. 24 cm diameter, prefolded. 5.5 Conical flask, with screw top or glass stopper of 500 ml capacity. 5.6 Glass microfibre filter, retention size 1,6 µm or smaller. 5.7 Reservoir, of 75 ml capacity with luer tip connector and attachments for immunoaffinity column (IAC). 5.8 Hand pump, 20 ml syringe with luer lock or rubber stopper for IAC. 5.9 Volumetric flasks, of 5 ml, 10 ml, 20 ml, 150 ml, and 200 ml capacity with an accuracy of at least 0,5 %. 5.10 Disposable syringe filter unit, with pore size of 0,45 µm. Prior to usage, verify that no aflatoxin losses occur during filtrat
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