1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 15890:2010Foodstuffs Determinationof patulin in fruit juice andfruit based pure for infantsand young children HPLCmethod with liquid/liquidpartition cleanup and solidphase
2、extraction and UVdetectionBS EN 15890:2010 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 15890:2010.The UK participation in its preparation was entrusted to TechnicalCommittee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on th
3、is committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2010ISBN 978 0 580 65880 8ICS 67.080.10; 67.160.20; 67.230Compliance with a British Standard cannot co
4、nfer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 October 2010.Amendments issued since publicationDate Text affectedBS EN 15890:2010EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15890 September 2010
5、ICS 67.080.10; 67.160.20; 67.230 English Version Foodstuffs - Determination of patulin in fruit juice and fruit based pure for infants and young children - HPLC method with liquid/liquid partition cleanup and solid phase extraction and UV detection Denres alimentaires - Dosage de la patuline dans le
6、 jus de fruits et la compote de fruits en alimentation infantile - Mthode par CLHP avec purification par partition liquide-liquide et extraction en phase solide et dtection UV Lebensmittel - Bestimmung von Patulin in Fruchtsaft und Obstbrei fr Suglinge und Kleinkinder - HPLC-Verfahren mit Reinigung
7、durch Flssig/Flssig-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 stan
8、dard 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
9、 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,
10、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 COMIT EUROPEN DE NORMALISATION
11、EUROPISCHES KOMITEE FR NORMUNG Management Centre: 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: EBS EN 15890:2010EN 15890:2010 (E) 2 Contents Page Foreword 31 Scope 42 Normative
12、references 43 Principle 44 Reagents .45 Apparatus .66 Procedure .87 HPLC analysis 98 Calculation . 109 Precision 1010 Test report . 11Annex A (informative) Typical chromatograms 12Annex B (informative) Precision data . 14Bibliography . 16BS EN 15890:2010EN 15890:2010 (E) 3 Foreword This document (EN
13、 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 Marc
14、h 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 rig
15、hts. 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: Aust
16、ria, 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.
17、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 determ
18、ine the applicability of regulatory limitations prior to use. BS EN 15890:2010EN 15890:2010 (E) 4 1 Scope This European Standard specifies a method for the determination of patulin in fruit juices and fruit-based pure, such as baby food pure, using high performance liquid chromatography with ultra-v
19、iolet 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 g/kg to 15,5 g/kg, and in fruit-based baby food pure, at levels ranging from 3,4 g/kg to 17,9 g/kg. Baby food fruit pu
20、re 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
21、 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 refer
22、enced 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 pure, with a mixture of ethyl-acetate and hexane in the presence of sodium sulfate
23、 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 o
24、f 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 a
25、cid, 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. BS EN 15890:
26、2010EN 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 cupb
27、oard. 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
28、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 interf
29、erences 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 substan
30、ces (in particular 5-hydroxymethylfurfural when used in combination with a Synergy1)column of 250 mm length and 4,6 mm diameter with a particle size of 4 m 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 prop
31、erties. 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)
32、. Transfer the solution to a 25 ml volumetric flask and dilute to volume with ethyl acetate to produce a solution containing approximately 200 g/ml of patulin. 1) Synergyis a trade name of a suitable product available commercially. This information is given for the convenience of users of this Europ
33、ean 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. BS EN 15890:2010EN 15890:2010 (E) 6 Store this solution in a freezer at approximately - 18 C. Confirm the mass concentration of the solut
34、ion 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 l of the stock solution (4.17) to dryness under nitrogen and then immediately dissolve it in 20 ml o
35、f ethanol (4.9) to obtain a mass concentration of approximately 10 g/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 con
36、centration of patulin, pat, in micrograms per millilitre, using Equation 1: bMA=100maxpat(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); is the molar absorption coe
37、fficient, 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 sol
38、ution 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 so
39、lutions 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 l and 250 l respectively of the stock solution (4.17) to dryness under nitrogen in a 100 ml volumetric flask, followed by immediate
40、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 volum
41、etric flask is filled up to the mark. In case the patulin standard solution (4.18) has a different mass concentration than 10 g/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. Stor
42、e 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. BS EN 15890:2010EN 15890:2010 (E) 7 5.2 Displacement pipettes, of e.g. 5 ml, 1 ml , 200 l and 50 l capacit
43、y 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. 5.6 Centrifuge, capable of operating at 400 g. 5.7 Centrifuge tu
44、bes, of 25 ml capacity with screw cap lids. 5.8 Mechanical shaker. 5.9 Evaporation block, capable of maintaining a temperature of 40 C, with nitrogen supply. 5.10 Glass vial, of 6 ml capacity with screw cap. 5.11 Syringe, gas tight with a polytetrafluoroethylene (PTFE) plunger and with a volume of 3
45、 ml to 5 ml. 5.12 Disposable syringe filters, of 0,2 m pore size (optional). Test each batch before use to ensure that patulin is not adsorbed onto the filter. 5.13 HPLC apparatus, comprising the following: 5.13.1 Injection system, a valve injection system with a 200 l injection loop. 5.13.2 Pump, i
46、socratic, pulse free, capable of maintaining a volume flow rate of 1 ml/min. 5.13.3 UV detector, fitted with an analytical flow cell and set at 276 nm. 5.13.4 Analytical reverse-phase HPLC separating column, capable to run with 100 % water as mobile phase such as a polar end-capped or polar embedded
47、 alkyl phases (for example columns of the type Synergy 2), Atlantis 2)or Luna 2)or similar). The column dimensions may vary dependingon the obtained peak separation of patulin from interfering peaks such as 5-hydroxymethylfurfural (5-HMF). The maximum height of overlapping peak shoulders shall be le
48、ss than 10 % of the maximum peak height. It could be necessary to adjust the mobile phase for sufficient baseline resolution. A suitable pre-column should be used. Columns with a length of 250 mm, an inner diameter of 4,6 mm and a particle size of approximately 4 m, with an 8 nm porosity have been s
49、hown to be suitable to meet these requirements when used in combination with the mobile phase proposed the Note under 4.15. 5.13.5 Data system. 2) Synergy, Atlantisand Lunaare trade names of suitable products 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. BS
