1、BRITISH STANDARDBS EN ISO 17375:2006Animal feeding stuffs Determination of aflatoxin B1The European Standard EN ISO 17375:2006 has the status of a British StandardICS 65.120g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g5
2、1g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN ISO 17375:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2006 BSI 2006ISBN 0 580 48655 9National forewordThis British Standard is the official English
3、 language version of EN ISO 17375:2006. It is identical with ISO 17375:2006.The UK participation in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to
4、its secretary.Cross-referencesThe British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic
5、 Catalogue or of British Standards Online.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to underst
6、and the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK.Summary of pagesThis document comprises a fron
7、t cover, an inside front cover, the EN ISO title page, the EN ISO foreword page, the ISO title page, pages ii to iv, pages 1 to 11 and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date Comment
8、sEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 17375June 2006ICS 65.120English VersionAnimal feeding stuffs - Determination of aflatoxin B1 (ISO17375:2006)Aliments des animaux - Dosage de laflatoxine B1 (ISO17375:2006)Futtermittel - Bestimmung von Aflatoxin B1 (ISO17375:2006)This European St
9、andard was approved by CEN on 12 May 2006.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning suc
10、h nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and
11、 notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Nethe
12、rlands, Norway, Poland, Portugal, Romania,Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2006 CEN All rights of exploitation in
13、any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 17375:2006: EForeword This document (EN ISO 17375:2006) has been prepared by Technical Committee ISO/TC 34 “Agricultural food products“ in collaboration with Technical Committee CEN/TC 327 “Animal feeding stuffs - M
14、ethods of sampling and analysis“, the secretariat of which is held by NEN. 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 December 2006, and conflicting national standards shall be withdrawn at t
15、he latest by December 2006. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland
16、, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Endorsement notice The text of ISO 17375:2006 has been approved by CEN as EN ISO 17375:2006 without any modifications. EN ISO 173
17、75:2006Reference numberISO 17375:2006(E)INTERNATIONAL STANDARD ISO17375First edition2006-06-01Animal feeding stuffs Determination of aflatoxin B1Aliments des animaux Dosage de laflatoxine B1EN ISO 17375:2006ii iiiContents Page Foreword iv 1 Scope . 1 2 Normative references . 1 3 Principle. 1 4 Reage
18、nts 1 5 Apparatus 4 6 Procedures 6 6.1 Conditioning of IACs 6 6.2 Extraction 6 6.3 Immunoaffinity clean up 6 6.4 Post-column derivatization 7 6.5 Calibration curve. 7 6.6 Calculation. 7 6.7 Spiking procedures for recovery determination .8 7 Precision 8 7.1 Interlaboratory test . 8 7.2 Repeatability
19、8 7.3 Reproducibility 9 8 Test report . 9 Annex A (informative) Results of an interlaboratory test. 10 Bibliography . 11 EN ISO 17375:2006iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of prepa
20、ring International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in l
21、iaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task o
22、f technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention i
23、s drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 17375 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal feeding stuff
24、s. EN ISO 17375:20061Animal feeding stuffs Determination of aflatoxin B11 Scope This International Standard specifies a method for the determination of aflatoxin B1in animal feeding stuffs using high-performance liquid chromatography with post-column derivatization. It is applicable to animal feedin
25、g stuffs with a fat content of up to 50 %. The limit of quantification of this method has been demonstrated to be better than 0,5 g/kg for aflatoxin B1for a signal-to-noise ratio of 6. NOTE The method is based on that given in Reference 1. 2 Normative references The following referenced documents ar
26、e 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. ISO 3696:1987, Water for analytical laboratory use Specification and test methods 3 Pr
27、inciple A test portion is extracted with a solvent solution (acetone/water). The sample extract is filtered, diluted with water or phosphate-buffered saline to a specified solvent concentration. A test portion is applied on an immunoaffinity column (IAC) containing antibodies specific to aflatoxin B
28、1. The aflatoxin B1is removed from the IAC with neat methanol, and then quantified by reverse-phase high-performance liquid chromatography (RP-HPLC) with post-column derivatization (PCD) involving bromination. The PCD is achieved with either electrochemically generated bromine or with pyridinium hyd
29、robromide perbromide (PBPB) followed by fluorescence detection. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified. WARNING This method requires the use of toxic inflammable liquids such as acetone, methanol and acetonitrile. Avoid contact and keep away from heat
30、, sparks or open flames. NOTE Decontamination procedures for laboratory wastes2,3have been developed and validated by the International Agency for Research on Cancer (WHO). 4.1 Water, complying with grade 3 in accordance with ISO 3696:1987. 4.2 Phosphate buffer saline (PBS), pH 7,4. EN ISO 17375:200
31、62 PBS may be prepared from potassium chloride (0,20 g), potassium dihydrogen phosphate (0,20 g), anhydrous disodium hydrogen phosphate (1,16 g) or disodium hydrogen phosphate dodecahydrate (2,92 g) and sodium chloride (8,00 g) added to 900 ml purified water. Adjust the pH to pH 7,4 (with 0,1 mol/l
32、HCl or 0,1 mol/l NaOH as appropriate) and dilute the solution to 1,0 l. Alternatively, commercially available phosphate-buffered saline tablets with equivalent properties may be used. PBS is not microbiologically stable and should be prepared fresh at least once a week. 4.3 Pyridinium hydrobromide p
33、erbromide (PBPB, CAS: 39416-48-3). This reagent is not required in the case of using electrochemically generated bromine. 4.4 Potassium bromide. This reagent is not required in the case of using the PBPB reagent. 4.5 HPLC-grade acetonitrile. 4.6 HPLC-grade methanol. 4.7 Acetone, pure. 4.8 HPLC grade
34、 water, complying with grade 1 of ISO 3696:1987. 4.9 Extraction solvent, solution of acetone (4.7) and water (4.8) 85+15 (by volume). 4.10 Nitric acid, c(HNO3) = 4 mol/l. This reagent is not required in the case of using the PBPB reagent. 4.11 Immunoaffinity column (IAC) The IAC should contain antib
35、odies raised against aflatoxin B1. The IAC should have a capacity of not less than 40 ng of aflatoxin B1and should give a recovery of not less than 80 % for aflatoxin B1when applied as a standard solution in acetone/water containing 0,25 ng of aflatoxin B1. 4.12 HPLC mobile phase solvent A, for use
36、with PBPB post column reagent only. Use a solution of water (4.8)/acetonitrile (4.5)/methanol (4.6) 6+2+3 (by volume). The ratio of solvents may be adjusted to give optimum separation parameters. 4.13 HPLC mobile phase solvent B, for use with electrochemically generated bromine only. Use a solution
37、of water (4.8)/acetonitrile (4.5)/methanol (4.6) 6+2+3 (by volume) containing 120 mg potassium bromide (4.4) and 350 l nitric acid at 4 mol/l (4.10) per litre of mobile phase. The ratio of solvents may be adjusted to give optimum separation parameters. The mobile phase solvents (4.12 and 4.13) shoul
38、d be degassed. 4.14 Post-column reagent, for use with PBPB post column reagent only. Dissolve 25 mg of PBPB (4.3) in 500 ml of water. This solution may be used for up to 4 days if stored in a dark place at room temperature. This post-column reagent shall be used only in combination with HPLC mobile
39、phase solvent A (4.12) but not with HPLC mobile phase solvent B (4.13). NOTE Post column reagent is only stable for 3 days. 4.15 Toluene/acetonitrile, 98 + 2 (by volume). EN ISO 17375:200634.16 Aflatoxin B1standard material, in form of crystals or a dry film for analytical purposes. WARNING 1 This m
40、ethod requires the use of solutions of aflatoxin B1. Aflatoxins are carcinogenic to humans. Attention is drawn to the statement made by the International Agency for Research on Cancer (WHO)2. WARNING 2 Aflatoxins are subject to light degradation. Protect analytical work adequately from the daylight,
41、 and keep aflatoxin standard solutions protected from light by using amber vials or aluminium foil. 4.17 Calibration stock solutions for HPLC 4.17.1 General stock solution Prepare an aflatoxin B1(4.16) stock solution containing 10,0 g/ml in toluene/acetonitrile (4.15). NOTE The toluene/acetonitrile
42、stock solution is stable for at least one year provided it is stored in acid-washed glassware and kept for storage at 18 C in the dark. If stock solutions are used up in a much shorter period (maximum of 3 months), methanol (4.6) might be used as an alternative. Note that methanolic solutions are mo
43、re sensitive to an alkaline ambient of the glass surface and to day light than toluene/acetonitrile solutions. Wrap the flasks tightly in aluminium foil and store them at less than 4 C. To determine the exact concentration of aflatoxins in this stock solution, record the absorption curve between a w
44、avelength of 330 nm and 370 nm in 1 cm quartz glass cells (5.21) in a spectrometer (5.20), with the stock solution solvent in the reference cell. Calculate the mass concentration of each aflatoxin, ca, in micrograms per millilitre, using Equation (1): aamaxa100McAd=(1) where Amaxis the absorbance de
45、termined at the maximum of the absorption curve; Mais the molar mass of aflatoxin B1, in grams per mole (312 g/mol); ais the molar absorptivity of aflatoxin B1, in square metres per mole (1930 m2/mol for toluene/acetonitrile and 2150 m2/mol for methanol solutions); d is the optical path length of th
46、e cell, in centimetres. 4.17.2 Calibration stock solution Prepare an aflatoxin B1(4.16) calibration solution containing 50,0 ng/ml in either toluene/acetonitrile (4.15) or methanol (4.6) from the stock solution (4.17.1). 4.17.3 Option A (see 6.3) Pipette from the calibration stock solution (4.17.2)
47、the volumes as listed in Table 1 (Option A) into a set of 20 ml calibrated volumetric flasks. Evaporate the toluene/acetonitrile solution just to dryness under a stream of nitrogen at room temperature. If methanol is used in the preparation of the stock solution, evaporation is not required. To each
48、 flask, add 7 ml of methanol. Allow the aflatoxins to dissolve, then dilute to the mark with water, and shake well. NOTE Remember that methanol and water are subject to volume contraction when mixed. EN ISO 17375:20064 4.17.4 Option B (see 6.3) Pipette from the calibration stock solution (4.17.2) th
49、e volumes as listed in Table 1 (Option B) into a set of at least 5 different 20 ml volumetric flasks. Evaporate the toluene/acetonitrile solution just to dryness under a stream of nitrogen at room temperature. If methanol is used in preparation of the stock solution, evaporation is not required. To each flask, add approximately 10 ml of methanol. Allow the aflatoxins to dissolve, then dilute further with neat methanol (not with methanol/water) to the mark and shake well. Then transfer exactly 1 ml of this calibration working solution to
