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1、a Date: 21 January 2009 Origin: European Latest date for receipt of comments: 30 April 2009 Project no.: 2008/04335 Responsible committee: AW/-/3 Food analysis - Horizontal methods Interested committees: Title: Draft BS EN 15890 Foodstuffs - Determination of patulin in fruit juice and fruit based pu
2、ree for young children - HPLC method with liquid/liquid partition cleanup and solid phase extraction and UV detection Supersession information: If this document is published as a standard, the UK implementation of it will supersede NONE and partially supersede. NONE If you are aware of a current nat
3、ional standard which may be affected, please notify the secretary (contact details below). WARNING: THIS IS A DRAFT AND MUST NOT BE REGARDED OR USED AS A BRITISH STANDARD. THIS DRAFT IS NOT CURRENT BEYOND 30 April 2009. This draft is issued to allow comments from interested parties; all comments wil
4、l be given consideration prior to publication. No acknowledgement will normally be sent. See overleaf for information on commenting. No copying is allowed, in any form, without prior written permission from BSI except as permitted under the Copyright, Designs and Patent Act 1988 or for circulation w
5、ithin a nominating organization for briefing purposes. Electronic circulation is limited to dissemination by e-mail within such an organization by committee members. Further copies of this draft may be purchased from BSI Customer Services, Tel: +44(0) 20 8996 9001 or email . British, International a
6、nd foreign standards are also available from BSI Customer Services. Information on the co-operating organizations represented on the committees referenced above may be obtained from the responsible committee secretary. Cross-references The British Standards which implement International or European
7、publications referred to in this draft may be found via the British Standards Online Service on the BSI web site http:/. Direct tel: 020 8996 7009 Responsible Committee Secretary: Committee Service Centre (BSI) E-mail: Draft for Public Comment Head Office 389 Chiswick High Road London W4 4AL Teleph
8、one: +44(0)20 8996 9000 Fax: +44(0)20 8996 7001 Form 36 Version 8.0 DPC: 09/30195609 DC Licensed Copy: London South Bank University, South Bank University, 02/02/2009 04:33, Uncontrolled Copy, (c) BSI b Introduction This draft standard is based on European discussions in which the UK has taken an ac
9、tive part. Your comments on this draft are welcome and will assist in the preparation of the consequent British Standard. Comment is particularly welcome on national, legislative or similar deviations that may be necessary. Even if this draft standard is not approved by the UK, if it receives the ne
10、cessary support in Europe, the UK will be obliged to publish the official English Language text unchanged as a British Standard and to withdraw any conflicting standard. UK Vote Please indicate whether you consider the UK should submit a negative (with reasons) or positive vote on this draft. Submis
11、sion The guidance given below is intended to ensure that all comments receive efficient and appropriate attention by the responsible BSI committee. Annotated drafts are not acceptable and will be rejected. All comments must be submitted, preferably electronically, to the Responsible Committee Secret
12、ary at the address given on the front cover. Comments should be compatible with Version 6.0 or Version 97 of Microsoft Word for Windows, if possible; otherwise comments in ASCII text format are acceptable. Any comments not submitted electronically should still adhere to these format requirements. Al
13、l comments submitted should be presented as given in the example below. Further information on submitting comments and how to obtain a blank electronic version of a comment form are available from the BSI web site at: http:/ Template for comments and secretariat observations Date: xx/xx/200x Documen
14、t: ISO/DIS xxxxx 1 2 (3) 4 5 (6) (7) MB Clause No./ Subclause No./ Annex (e.g. 3.1) Paragraph/ Figure/Table/ Note (e.g. Table 1) Type of com- ment Comment (justification for change) by the MB Proposed change by the MB Secretariat observations on each comment submitted 3.1 Definition 1 ed Definition
15、is ambiguous and needs clarifying. Amend to read . so that the mains connector to which no connection . 6.4 Paragraph 2 te The use of the UV photometer as an alternative cannot be supported as serious problems have been encountered in its use in the UK. Delete reference to UV photometer. Microsoft a
16、nd MS-DOS are registered trademarks, and Windows is a trademark of Microsoft Corporation. Licensed Copy: London South Bank University, South Bank University, 02/02/2009 04:33, Uncontrolled Copy, (c) BSI EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM DRAFT prEN 15890 January 2009 ICS 67.080.10; 67
17、.160.20 English Version Foodstuffs - Determination of patulin in fruit juice and fruit based puree for young children - HPLC method with liquid/liquid partition cleanup and solid phase extraction and UV detection Denres alimentaires - Dosage de la patuline dans le jus de fruit et la compote de fruit
18、s 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 Kleinkinder - HPLC-Verfahren mit Reinigung durch Flssig/Flssig-Verteilung, Festphasenextrakt
19、ion und UV-Detektion This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee CEN/TC 275. If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions f
20、or giving this European Standard the status of a national standard without any alteration. This draft European Standard was established by CEN 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 o
21、wn 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, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithu
22、ania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to provide supportin
23、g documentation. Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and shall not be referred to as a European Standard. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NOR
24、MUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2009 CENAll rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. prEN 15890:2009: E Licensed Copy: London South Bank University, South Bank University, 02/02/2009 04:33, Uncontrolled Copy,
25、(c) BSI prEN 15890:2009 (E) 2 Contents Page Foreword3 1 Scope 3 2 Normative references3 3 Principle3 4 Reagents.3 5 Apparatus .6 6 Procedure .7 7 HPLC analysis8 8 Calculation8 9 Precision.9 10 Test report10 Annex A (informative) Typical chromatograms 11 Annex B (informative) Precision data12 Bibliog
26、raphy14 Licensed Copy: London South Bank University, South Bank University, 02/02/2009 04:33, Uncontrolled Copy, (c) BSI prEN 15890:2009 (E) 3 Foreword This document (prEN 15890:2009) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which i
27、s held by DIN. This document is currently submitted to the CEN Enquiry. 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
28、standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 1 Scope This European Standard specifies a method for the determination of patulin in fruit juices and fruit based purees such as baby food purees using high performa
29、nce liquid chromatography (HPLC). The method has been validated for the determination of patulin via the analysis of naturally contaminated and spiked samples in apple juice at levels ranging from 3,0 g/kg up to 15,5 g/kg and in baby food based fruit purees at levels ranging from 3,4 g/kg up to 17,9
30、 g/kg. Baby food fruit purees used in this study contained mainly the following ingredients: blueberry, apple, banana, lemon, wheat biscuits, wheat syrup, whole milk and vegetable oils as ingredience as commercially available products on the European market. A detailed listing including the fraction
31、s of each product used in this study is given in 1. 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
32、 any amendments) applies. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987). 3 Principle Patulin is extracted from juice or puree with a mixture of ethyl-acetate and hexane in the presence of sodium sulfate and sodium hydrogen carbonate. An aliquot of
33、the solvent is purified by solid-phase extraction and evaporated. The purified extract 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 wat
34、er complying with grade 1 of EN ISO 3696, 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. Licensed Copy: London South Bank University, South Bank Un
35、iversity, 02/02/2009 04:33, Uncontrolled Copy, (c) BSI prEN 15890:2009 (E) 4 4.2 Perchloric acid, volume concentration (HClO4) = 60 % 4.3 Seasand 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 Water o
36、f pH = 4 Adjust water to pH = 4 with acetic acid. 4.8 Absolute ethanol, (CH3CH2OH) 99,7 % 4.9 Acetonitrile 4.10 Ethyl acetate 4.11 Glacial acetic acid, (CH3COOH) 98 % 4.12 n-Hexane 4.13 Extraction solvent Add 60 ml of ethyl acetate (4.10) to 40 ml of n-hexane (4.12). 4.14 Mixture of acetic acid and
37、ethyl acetate Add 3 ml of glacial acetic acid (4.11) to 97 ml of ethyl acetate (4.10). 4.15 HPLC mobile phase Mix 990 parts per volume of water with 0 parts to 10 parts per volume of acetonitrile (4.9) and 1 part per volume of perchloric acid (4.2). The amount of acetonitrile will depend upon the pu
38、rification degree of the sample extract and the HPLC column chosen for analysis. Degas this solution before use. 4.16 Patulin 4.17 Patulin stock solution WARNING Patulin is a suspect mutagen and has been reported to have immunotoxic and neurotoxic properties. Gloves and safety glasses should be worn
39、 at all times and all standard and sample preparation stages should be carried out in a fume cupboard. Dissolve 5 mg of patulin or the contents of 1 ampoule (if patulin has been obtained as a film) in ethyl acetate (4.10). Transfer the solution to a 25 ml volumetric flask and dilute volume with ethy
40、l acetate to produce a solution containing approximately 200 g/ml of patulin. Store the stock solution in a freezer. A solution stored this way is stable for several months. Ensure the standard solution is allowed to reach room temperature before use to avoid incorporation of water by condensation.
41、Licensed Copy: London South Bank University, South Bank University, 02/02/2009 04:33, Uncontrolled Copy, (c) BSI prEN 15890:2009 (E) 5 4.18 Patulin standard solution Evaporate 1000 l of the stock solution (4.17) to dryness under nitrogen and then immediately dissolve it in 20 ml of ethanol (4.8) to
42、obtain a mass concentration of approximately 10 g/ml of patulin. To determine the exact 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, p
43、at, in micrograms per millilitre, using equation 1: b MA = 100 max pat (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 mol, of patulin (M = 154 g/mol); is the molar absorption coefficient, in square
44、 metres per mol, 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 at less than 0 C. A solution stored in this way is stable for several months. Ensure that the standard solution is allowed to reach room temperat
45、ure before use to avoid incorporation of water by condensation. 4.19 Patulin calibration solutions Pipette amounts of 60 l, 120 l, 180 l, 240 l, 300 l, 360 l and 420 l of the patulin standard solution (4.18) into different 50 ml volumetric flasks. Fill the flasks up to the mark with water of pH = 4
46、(4.7) and shake. This will result in patulin solutions with concentrations of 12 ng/ml, 24 ng/ml, 36 ng/ml, 48 ng/ml, 60 ng/ml, 72 ng/ml and 84 ng/ml. These concentrations reflect contamination levels of 4,8 g/kg, 9,6 g/kg, 14,4 g/kg, 19,2 g/kg, 24,0 g/kg, 28,8 g/kg and 33,6 g/kg patulin in the samp
47、le based on the assumption that a patulin standard solution of 10 g/ml (4.18) was used for preparation and they can be used directly for injection into the HPLC system. Store this solution in a refrigerator at 4 C. A solution stored in this way is stable for at least eight weeks. 4.20 Spiking soluti
48、ons For spiking experiments at levels of 10 ng/ml and 25 ng/ml patulin in the sample, prepare solutions of patulin in water of pH = 4 (4.7) at concentrations of 100 ng/ml and 250 ng/ml, respectively. These solutions can be obtained by evaporating exactly 50 l, respectively 125 l of the stock solutio
49、n (4.17) to dryness under nitrogen in a 100 ml volumetric flask, followed by immediate dissolution in water of pH = 4 (4.7) to obtain a mass concentration of 100 ng/ml, respectively 250 ng/ml of patulin. Make sure that the patulin is completely dissolved in the water of pH = 4 before the volumetric bottle is filled up to the mark. Store this solution in a refrigerator at 4 C. A solution stored