EN 15890:2010

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Patulin in Fruchtsaft und Obstbrei für Säuglinge und Kleinkinder - HPLC-Verfahren mit Reinigung durch Flüssig/Flüssig-Verteilung, Festphasenextraktion und UV-DetektionDenrées alimentaires - Dosage de la patuline dans le jus de fruits et la compote de fruits en alimentation infantile - Méthode par CLHP avec purification par partition liquide-liquide et extraction en phase solide et détection UVFoodstuffs - Determination of patulin in fruit juice and fruit based purée for infants and young children - HPLC method with liquid/liquid partition cleanup and solid phase extraction and UV detection67.230Predpakirana in pripravljena hranaPrepackaged and prepared foods67.080.10Sadje in sadni proizvodiFruits and derived productsICS:Ta slovenski standard je istoveten z:EN 15890:2010SIST EN 15890:2011en,fr,de01-marec-2011SIST EN 15890:2011SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15890
September 2010 ICS 67.080.10; 67.160.20; 67.230 English Version
Foodstuffs - Determination of patulin in fruit juice and fruit based purée for infants and young children - HPLC method with liquid/liquid partition cleanup and solid phase extraction and UV detection
Denrées alimentaires - Dosage de la patuline dans le jus de fruits et la compote de fruits en alimentation infantile - Méthode par CLHP avec purification par partition liquide-liquide et extraction en phase solide et détection UV
Lebensmittel - Bestimmung von Patulin in Fruchtsaft und Obstbrei für Säuglinge und Kleinkinder - HPLC-Verfahren mit Reinigung durch Flüssig/Flüssig-Verteilung, Festphasenextraktion und UV-Detektion This European Standard was approved by CEN on 28 August 2010.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN Management Centre or to any CEN member.
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Typical chromatograms . 12Annex B (informative)
Precision data . 14Bibliography . 16 SIST EN 15890:2011

Specification and test methods (ISO 3696:1987) 3 Principle Patulin is extracted from apple juice, or fruit-based purée, with a mixture of ethyl-acetate and hexane in the presence of sodium sulfate and sodium hydrogen carbonate. An aliquot of the extract is purified by solid-phase extraction and evaporated. The residue is re-dissolved in water of pH = 4 and patulin is separated by reverse phase (RP)-HPLC and quantitatively determined by UV detection. 4 Reagents 4.1 General Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995, unless otherwise specified. Solvents shall be of quality for HPLC analysis, unless otherwise specified. Commercially available solutions with equivalent properties to the reagents listed may be used. 4.2 Perchloric acid, the mass fraction w(HClO4) ≥ 60 % in water. 4.3 Sand, 50 mesh to 70 mesh particle size.
4.4 Silicagel solid phase extraction (SPE) cartridges (500 mg SiO2). 4.5 Sodium sulfate anhydrous, Na2SO4. 4.6 Sodium hydrogen carbonate, NaHCO3. 4.7 Glacial acetic acid, w(CH3COOH) ≈ 98 % in water. SIST EN 15890:2011

NOTE A mobile phase of 990 part of water with one part of perchloric acid has been found to give sufficient separation between patulin and other interfering substances (in particular 5-hydroxymethylfurfural when used in combination with a Synergy® 1) column of 250 mm length and 4,6 mm diameter with a particle size of 4 µm and 8 nm porosity (see 5.13.4)). 4.16 Patulin. WARNING — Patulin is a suspect mutagen and has been reported to have immunotoxic and neurotoxic properties. Gloves and safety glasses should be worn at all times and all standard and sample preparation stages should be carried out in a fume cupboard. 4.17 Patulin stock solution. Dissolve 5 mg of patulin or the contents of one ampoule (if patulin has been obtained as a film) in ethyl acetate (4.11). Transfer the solution to a 25 ml volumetric flask and dilute to volume with ethyl acetate to produce a solution containing approximately 200 µg/ml of patulin.
1) Synergy® is a trade name of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 15890:2011

4.18 Patulin standard solution. Evaporate 1 000 µl of the stock solution (4.17) to dryness under nitrogen and then immediately dissolve it in 20 ml of ethanol (4.9) to obtain a mass concentration of approximately 10 µg/ml of patulin. To determine the exact mass concentration, record the absorption curve between 250 nm and 350 nm in a 1 cm quartz cell with ethanol as reference. Identify the wavelength for maximum absorption. Calculate the mass concentration of patulin, pat, in micrograms per millilitre, using Equation 1:
bMA×××=ερ100maxpat (1) where Amax is the absorption determined at the maximum of the absorption curve (here: at approximately 276 nm); M is the molar mass, in grams per mole, of patulin (M = 154 g/mol); 0 is the molar absorption coefficient, in square metres per mole, of patulin in ethanol (here: 1 460 m2/mol, see [2]); b is the optical path length, in centimetres, of the quartz cell. Store this solution in a freezer at approximately - 18 °C. A solution stored in this way is stable for several months. Ensure that the standard solution is allowed to reach room temperature before use to avoid incorporation of water by condensation. Confirm the concentration of the solution if it is older than six weeks. 4.19 Spiking solutions. For spiking experiments at levels of 10 ng/ml and 25 ng/ml patulin in the sample, prepare spiking solutions of patulin in water of pH = 4 (4.8) at mass concentrations of 200 ng/ml and 500 ng/ml, respectively.
These solutions can be obtained by evaporating exactly 100 µl and 250 µl respectively of the stock solution (4.17) to dryness under nitrogen in a 100 ml volumetric flask, followed by immediate dissolution in water of pH = 4 (4.8) to obtain a mass concentration of approximately 200 ng/ml and 500 ng/ml respectively of patulin, depending on the exact mass concentration of patulin in the stock solution. Make sure that the patulin is completely dissolved in the water of pH = 4 before the volumetric flask is filled up to the mark. In case the patulin standard solution (4.18) has a different mass concentration than 10 µg/ml, adjust spiking solutions by calculating the correct aliquots in order to take account of the actual mass concentration of the standard solution determined in 4.18. Store this solution in a refrigerator at 4 °C. A solution stored in this way is stable for at least eight weeks. 5 Apparatus 5.1 General Usual laboratory apparatus and, in particular, the following. SIST EN 15890:2011
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