1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16215:2012Animal feeding stuffs Determination of dioxins and dioxin-like PCBs by GC/HRMS and of indicator PCBs by GC/HRMSBS EN 16215:2012 BRITISH STANDARDNational forewordT
2、his British Standard is the UK implementation of EN 16215:2012. The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport
3、 to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2012. Published by BSI Standards Limited 2012ISBN 978 0 580 67003 9 ICS 65.120 Compliance with a British Standard cannot confer immunity from legal obligations
4、.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 May 2012.Amendments issued since publicationDate T e x t a f f e c t e dBS EN 16215:2012EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16215 April 2012 ICS 65.120 English Version Anima
5、l feeding stuffs - Determination of dioxins and dioxin-like PCBs by GC/HRMS and of indicator PCBs by GC/HRMS Aliments des animaux - Dosage des dioxines, des PCB de type dioxine et des PCB indicateurs par GC/HRMS Futtermittel - Bestimmung von Dioxinen und dioxin-hnlichen PCBs mittels GC/HRMS und von
6、Indikator-PCBs mittels GC/HRMS This European Standard was approved by CEN on 9 March 2012. 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 l
7、ists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC 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 und
8、er the responsibility of a CEN member into its own language and notified to the CEN-CENELEC 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,
9、 Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMIT
10、EE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16215:2012: EBS EN 16215:2012EN 16215:2012 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 63 Ter
11、ms and definitions . 64 Principle 75 Reagents . 76 Apparatus . 97 Sampling . 98 Preparation of test sample 99 Procedure . 910 Extraction .1011 Clean up 1312 Module D: Gas chromatograph-high resolution mass spectrometer (GC-HRMS) 2713 Calculation and expression of results .2914 Precision .3415 Test r
12、eport 36Annex A (informative) Description of PTV injection system .37Annex B (informative) Description of standards and concentration of the standard solutions .39Annex C (informative) Example of automated procedure .46Annex D (informative) Mass spectrometer (MS) .48Annex E (informative) Use of addi
13、tional clean up after fractionation using a small multi-layer silica column 52Bibliography 53BS EN 16215:2012EN 16215:2012 (E) 3 Foreword This document (EN 16215:2012) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”, the secretariat of which is held by NEN. This European
14、Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by October 2012, and conflicting national standards shall be withdrawn at the latest by October 2012. Attention is drawn to the possibility that some of the elements
15、 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. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this Europe
16、an 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, Turkey
17、 and the United Kingdom. BS EN 16215:2012EN 16215:2012 (E) 4 1 Scope This European Standard is applicable to the determination of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), (together termed dioxins (PCDD/Fs) and dioxin-like PCBs and non dioxin-like PCBs (dl-PCB
18、s and ndl-PCBs) in animal feeding stuffs. Collaborative studies have been carried out. The method is suitable for the determination of dioxins, dl-PCBs and ndl-PCBs at the appropriate MRL in compound feed and ingredients e.g. oil, mineral clay. The method is applicable to samples containing residues
19、 of one or more of the following dioxins, dioxin-like PCBs and indicator PCBs. The limit of quantification (LOQ) for the relevant individual congeners of dioxins/furans is 0,05 pg/g (OCDD/F = 0,1 pg/g), of non-ortho PCBs 0,05 pg/g, of mono-ortho PCBs 10 pg/g and of indicator PCBs 100 pg/g. For deter
20、mination of dioxins and dioxin-like PCBs, the procedure can be used as confirmatory method as defined by Commission Regulation (EC) No 152/2009 for dioxins and dl-PCB in feed 6. Confirmatory methods are high-resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS) methods. If only
21、 the analysis of indicator PCBs is required, a GC-LRMS method can be used (e.g. EN 15741 Animal feeding stuffs - Determination of OC-pesticides and PCBs by GC/MS 1 and EN 15742 Animal feeding stuffs - Determination of OC-pesticides and PCBs by GC/ECD 2) provided that appropriate analytical performan
22、ce criteria are met in the relevant range for the matrix of interest. This European Standard is split into four modules each describing a part of the whole procedure (see Figure 1 and Figure 2) to be followed: a) Module A: Description of standards which might be used; b) Module B: Description of ext
23、raction procedures; c) Module C: Description of clean up procedures; d) Module D: GC/HRMS determination. Each module describes a part of the whole method as well as, when applicable, alternatives which should be equivalent. Each module has to be regarded as an example. Combining modules and/or alter
24、natives gives a highly flexible procedure which is “performance based“. It is permitted to modify the method if all performance criteria laid down in Commission Regulation (EC) No 152/2009 6 are met. Any deviation of the described method, combination of modules needs to be recorded as part of the QA
25、/QC procedures of accredited laboratories and should be available on request. BS EN 16215:2012EN 16215:2012 (E) 5 = optional Figure 1 Flow scheme for the determination of Dioxins, dl-PCBs and Indicator PCBs in feed BS EN 16215:2012EN 16215:2012 (E) 6 = optional Figure 2 Flow scheme for the determina
26、tion of Dioxins, dl-PCBs and Indicator PCBs in oil / fat 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the lates
27、t edition of the referenced document (including any amendments) applies. prEN ISO 6498, Animal feeding stuffs Guidelines for sample preparation (ISO/DIS 6498) 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 limit of detection smallest measure
28、d content, from which it is possible to deduce the presence of the analyte with reasonable statistical certainty Note 1 to entry: The limit of detection is numerically equal to three times the standard deviation of the mean of blank determinations (n 10). BS EN 16215:2012EN 16215:2012 (E) 7 3.2 limi
29、t of quantification lowest content of the analyte that can be measured with reasonable statistical certainty Note 1 to entry: If both accuracy and precision are constant over a concentration range around the limit of detection, then the limit of quantification is numerically equal to six times the s
30、tandard deviation of the mean of blank determinations (n 10). Note 2 to entry: For dl-PCBs and PCDD/F: Use correct definition for limit of quantification for each congener as in Commission Regulation (EC) No 152/2009 6. Note 3 to entry: Limit of quantification should be in the range of about one fif
31、th of the level of interest. 3.3 feed additives substances that comply with the definition of feed additives given in the Commission Regulation (EC) No 1831/2003 7 3.4 upper, middle and lower bound upper, middle and lower bound results for WHO-PCDD/F-TEQ and WHO-PCB-TEQ are defined as follows: upper
32、 bound concentrations are calculated assuming that all values of the different congeners less than the limit of quantification are equal to the limit of quantification; for lower bound: LOQ = 0 and middle bound: LOQ is used 4 Principle A test portion of animal feeding stuff or ingredient is fortifie
33、d with 13C labelled internal standards (dioxins, furans, dioxin-like PCBs and indicator PCBs) and extracted using a manual or an automated method. After automated or manual clean up an aliquot of the extract is concentrated and injected into a GC-HRMS using a split less injector (an alternative here
34、 is PTV injection (Programmed Temperature Vaporizer injection) see NOTE. Quantification is based on isotope dilution. If only indicator PCBs are required, they can be determined with GC-LRMS (e.g. according to EN 15741 Animal feeding stuffs - Determination of OC-pesticides and PCBs by GC/MS 1 and EN
35、 15742 Animal feeding stuffs - Determination of OC-pesticides and PCBs by GC/ECD 2) Preconditions of combining modules for extraction and clean-up are: a) for each extraction module an equal sample intake of 10 g for feed or feed ingredients with a fat content 25 % or 2,5 g fat or oil is required; b
36、) in order to achieve the required LOQ for dioxins a final volume of 10 l in combination with an injection volume of 2 l is required. If a different injection volume is applied, the final volume has to be adjusted directly proportional. NOTE In case more sensitivity is necessary or less volume reduc
37、tion is wanted, injection of a larger volume by PTV (an example is described in Annex A) or higher sample intake is possible (see also 9.2.2 NOTE). 5 Reagents Use only reagents of recognized analytical grade and with purity suitable for dioxin and PCB residue analysis. Check the purity of the reagen
38、ts and reference materials (e.g. standard solutions) by performing a blank test under the same conditions as used in the method. The chromatogram should not show any interfering impurity at the retention time of compounds of interest. BS EN 16215:2012EN 16215:2012 (E) 8 WARNING The use of this Europ
39、ean 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 European Standard to establish appropriate safety and health practices and to determine the
40、applicability of regulatory limitations prior to use. 5.1 Dioxins, furans, non-ortho PCBs, mono-ortho PCBs and indicator PCBs and their labelled analogues: 13C-spiking solution for PCDD/F (internal standard); 13C-spiking solution for PCB (internal standard); calibration solutions PCDD/F; calibration
41、 solutions PCB; recovery standard PCDD/F; recovery standard PCB. See Annex B: Description of standards and concentration of the standard solutions. BS EN 16215:2012EN 16215:2012 (E) 9 6 Apparatus All technical descriptions are examples of possible system setups and parameters and have to be scaled o
42、r adopted to the users equipment. 6.1 Evaporator, suitable for volumes up to 200 ml and inlet for nitrogen gas. 6.2 Evaporator tubes, endpoint about 0,5 ml. 6.3 Homogeniser 6.4 Pasteur pipette, borosilicate glass, 150 mm. 6.5 Vortexmixer 6.6 Measuring cylinder, borosilicate glass, 100 ml, 2 ml gradu
43、ations with a precision of 0,5 ml. 6.7 Measuring cylinder, borosilicate glass, glass-stoppered, 25 ml, 1 ml graduation with a precision of 0,5 ml graduation and 50 ml, 2 ml graduation with a precision of 0,5 ml graduation. 7 Sampling The sample should be truly representative and not damaged or chang
44、ed during transport or storage. Sampling is not part of the method specified in this European Standard. A recommended sampling method is given in EN ISO 6497 3. 8 Preparation of test sample Prepare the test sample in accordance with prEN ISO 6498. Dry or low moisture products such as cereals and cer
45、eal products, mixed feeds, and hay should be ground carefully so that it passes completely through a sieve with 1 mm apertures. Mix thoroughly. High moisture products such as grasses and silages and liquid feed should be (freeze-)dried and after that ground carefully so that it passes completely thr
46、ough a sieve with 1 mm apertures. Mix thoroughly. Oil / fat are directly dissolved in n-hexane. 9 Procedure 9.1 General Analyse the following samples in each series: procedure blank (n = 1); (certified) reference material at appropriate level or a home made reference sample; all samples (maximum 20)
47、. The procedure blank should be free of contaminants at or above the limits of quantification. BS EN 16215:2012EN 16215:2012 (E) 10 9.2 Animal feed stuff sample and oil/fat sample 9.2.1 10 g animal feed stuff sample (12 % moisture content) Weigh an appropriate amount, e.g. 10,0 g ( 0,10 g) of the pr
48、epared test sample into a 100 ml glass vial. Sample amount is based on 12 % moisture content. If extracting by Pressurized Fluid Extraction (PFE), add 3 g diatomaceous earth and mix thoroughly. Fortify the sample with 500 l 13C-DIOXNOP-2 (Annex B, B.2.31) and 500 l 13C-MOPIP-2 (Annex B, B.2.33) and
49、incubate until solvent has been evaporated and continue at 10.2 (module BI) or 10.3 (module BII). For samples with more than 25 % fat, the sample intake has to be reduced proportionally. 9.2.2 2,5 g oil/fat sample Weigh an appropriate amount, e.g. 2,5 g ( 0,10 g) of the oil/fat sample into graduated cylinder of 25 ml (6.7). Fortify the sample with 500 l 13C-DIOXNOP-2 (Annex B, B.2.31) and 500 l 13C-MOPIP-2 (Annex B, B.2.33). Fill the graduat
