SIST EN 15890:2011

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



SIST EN 15890:2011



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.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
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Avenue Marnix 17,
B-1000 Brussels © 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15890:2010: ESIST EN 15890:2011



EN 15890:2010 (E) 2 Contents Page Foreword .31Scope .42Normative references .43Principle .44Reagents .45Apparatus .66Procedure .87HPLC analysis .98Calculation . 109Precision . 1010Test report . 11Annex A (informative)
Typical chromatograms . 12Annex B (informative)
Precision data . 14Bibliography . 16 SIST EN 15890:2011



EN 15890:2010 (E) 3 Foreword This document (EN 15890:2010) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by March 2011, and conflicting national standards shall be withdrawn at the latest by March 2011. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. WARNING — The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. SIST EN 15890:2011



EN 15890:2010 (E) 4 1 Scope This European Standard specifies a method for the determination of patulin in fruit juices and fruit-based purée, such as baby food purée, using high performance liquid chromatography with ultra-violet detection (HPLC-UV). Using naturally contaminated and spiked samples this method has been validated for the determination of patulin in apple juice, at levels ranging from 3,0 µg/kg to 15,5 µg/kg, and in fruit-based baby food purée, at levels ranging from 3,4 µg/kg to 17,9 µg/kg. Baby food fruit purée used in this study contained a mixture of the following ingredients which are commercially available on the European market: blueberry; apple; banana; lemon; wheat biscuits; wheat syrup; whole milk; and vegetable oil. A detailed listing, including the fractions, of each product used in this study is given in [1]. Further information on validation, see Clause 9 and Annex B. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use
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



EN 15890:2010 (E) 5 4.8 Water of pH = 4. Adjust water to pH = 4 with glacial acetic acid (4.7). 4.9 Absolute ethanol, w(CH3CH2OH) ≥ 99,7 % in water. 4.10 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. 4.11 Ethyl acetate. 4.12 n-Hexane. 4.13 Extraction solvent. Add 60 ml of ethyl acetate (4.11) to 40 ml of n-hexane (4.12). 4.14 Mixture of glacial acetic acid and ethyl acetate. Add 3 ml of glacial acetic acid (4.7) to 97 ml of ethyl acetate (4.11). 4.15 HPLC mobile phase. Mix 990 parts per volume of water with up to ten parts per volume of acetonitrile (4.10) and one part per volume of perchloric acid (4.2). The amount of acetonitrile will depend upon the type of samples analysed and their characteristic pattern of interferences after clean-up (see Annex A for typical chromatograms) and the HPLC column chosen for analysis. Degas this solution before use.
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



EN 15890:2010 (E) 6 Store this solution in a freezer at approximately - 18 °C. Confirm the mass concentration of the solution if it is older than six weeks. Ensure the solution is allowed to reach room temperature before use to avoid incorporation of water by condensation.
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



EN 15890:2010 (E) 7 5.2 Displacement pipettes, of e.g. 5 ml, 1 ml , 200 µl and 50 µl capacity with appropriate pipette tips. 5.3 Analytical balance, capable of weighing to 0,1 mg. 5.4 UV spectrometer, double beam and recording suitable for measurement at 250 nm to 350 nm. 5.5 Quartz cells, with an optical path length of 1 cm. 5
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