1、July 2012 Translation by DIN-Sprachendienst.English price group 20No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 6
2、5.120!$,h“1900969www.din.deDDIN EN 16215Animal feeding stuffs Determination of dioxins and dioxin-like PCBs by GC/HRMS and ofindicator PCBs by GC/HRMSEnglish translation of DIN EN 16215:2012-07Futtermittel Bestimmung von Dioxinen und dioxin-hnlichen PCBs mittels GC/HRMS und vonIndikator-PCBs mittels
3、 GC/HRMSEnglische bersetzung von DIN EN 16215:2012-07Aliments des animaux Dosage des dioxines, des PCB de type dioxine et des PCB indicateurs par GC/HRMSTraduction anglaise de DIN EN 16215:2012-07www.beuth.deDocument comprises 55 pagesIn case of doubt, the German-language original shall be considere
4、d authoritative.06.12 DIN EN 16215:2012-07 2 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs Methods of sampling and analysis” (Secretariat: NEN, Netherlands). The responsible German body involved in it
5、s preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-03-03 AA Futtermittel. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trad
6、e Association, and supports essential requirements of EU Directive(s). EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16215 April 2012 ICS 65.120 English Version Animal feeding stuffs - Determination of dioxins and dioxin-like PCBs by GC/HRMS and of indicator PCBs by GC/HRMS Aliments des anima
7、ux - 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 Indikator-PCBs mittels GC/HRMS This European Standard was approved by CEN on 9 March 2012. CEN members are bound to comply with
8、 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-CENELEC Manage
9、ment 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-CENELEC Management Centre has the same stat
10、us 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
11、, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any m
12、eans reserved worldwide for CEN national Members. Ref. No. EN 16215:2012: EEN 16215:2012 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 63 Terms and definitions . 64 Principle 75 Reagents . 76 Apparatus . 97 Sampling . 98 Preparation of test sample 99 Procedure . 910 Extraction .1011
13、Clean up 1312 Module D: Gas chromatograph-high resolution mass spectrometer (GC-HRMS) 2713 Calculation and expression of results .2914 Precision .3415 Test report 36Annex A (informative) Description of PTV injection system .37Annex B (informative) Description of standards and concentration of the st
14、andard solutions .39Annex C (informative) Example of automated procedure .46Annex D (informative) Mass spectrometer (MS) .48Annex E (informative) Use of additional clean up after fractionation using a small multi-layer silica column 52Bibliography 53DIN EN 16215:2012-07 EN 16215:2012 (E) 3 Foreword
15、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 Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by
16、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 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 pa
17、tent rights. 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, Ice
18、land, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 16215:2012-07 EN 16215:2012 (E) 4 1 Scope This European Standard is applicable to the determination of pol
19、ychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), (together termed dioxins (PCDD/Fs) and dioxin-like PCBs and non dioxin-like PCBs (dl-PCBs and ndl-PCBs) in animal feeding stuffs. Collaborative studies have been carried out. The method is suitable for the determination o
20、f 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 of one or more of the following dioxins, dioxin-like PCBs and indicator PCBs. The limit of quantification (LOQ) for the relevant ind
21、ividual 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 determination of dioxins and dioxin-like PCBs, the procedure can be used as confirmatory method as defined by Commission Regulation (EC) N
22、o 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 the analysis of indicator PCBs is required, a GC-LRMS method can be used (e.g. EN 15741 Animal feeding stuffs - Determination of OC-
23、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 performance criteria are met in the relevant range for the matrix of interest. This European Standard is split into four modules each describi
24、ng 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 extraction procedures; c) Module C: Description of clean up procedures; d) Module D: GC/HRMS determination. Each module describes a part
25、 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 alternatives gives a highly flexible procedure which is “performance based“. It is permitted to modify the method if all performance crite
26、ria 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/QC procedures of accredited laboratories and should be available on request. DIN EN 16215:2012-07 EN 16215:2012 (E) 5 = optional Fig
27、ure 1 Flow scheme for the determination of Dioxins, dl-PCBs and Indicator PCBs in feed DIN EN 16215:2012-07 EN 16215:2012 (E) 6 = optional Figure 2 Flow scheme for the determination of Dioxins, dl-PCBs and Indicator PCBs in oil / fat 2 Normative references The following documents, in whole or in par
28、t, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. prEN ISO 6498, Animal feeding stuffs Guidelines fo
29、r 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 measured content, from which it is possible to deduce the presence of the analyte with reasonable statistical certainty Note 1 to
30、entry: The limit of detection is numerically equal to three times the standard deviation of the mean of blank determinations (n 10). DIN EN 16215:2012-07 EN 16215:2012 (E) 7 3.2 limit of quantification lowest content of the analyte that can be measured with reasonable statistical certainty Note 1 to
31、 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 standard deviation of the mean of blank determinations (n 10). Note 2 to entry: For dl-PCBs and PCDD/F: Use correct def
32、inition 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 fifth of the level of interest. 3.3 feed additives substances that comply with the definition of feed additives given in
33、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 bound concentrations are calculated assuming that all values of the different congeners less than the limit of quanti
34、fication 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 fortified with 13C labelled internal standards (dioxins, furans, dioxin-like PCBs and indicator PCBs) and extracted using a ma
35、nual 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 is PTV injection (Programmed Temperature Vaporizer injection) see NOTE. Quantification is based on isotope dilution.
36、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 15742 Animal feeding stuffs - Determination of OC-pesticides and PCBs by GC/ECD 2) Preconditions of combining modules
37、 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) in order to achieve the required LOQ for dioxins a final volume of 10 l in combination with an injection volume of 2
38、 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 reduction is wanted, injection of a larger volume by PTV (an example is described in Annex A) or higher sample intake is po
39、ssible (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 reagents and reference materials (e.g. standard solutions) by performing a blank test under the same conditions as used in t
40、he method. The chromatogram should not show any interfering impurity at the retention time of compounds of interest. DIN EN 16215:2012-07 EN 16215:2012 (E) 8 WARNING The use of this European Standard can involve hazardous materials, operations and equipment. This standard does not purport to address
41、 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 applicability of regulatory limitations prior to use. 5.1 Dioxins, furans, non-ortho PCBs, mono-ortho PCBs and in
42、dicator 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 solutions PCB; recovery standard PCDD/F; recovery standard PCB. See Annex B: Description of standards and concen
43、tration of the standard solutions. DIN EN 16215:2012-07 EN 16215:2012 (E) 9 6 Apparatus All technical descriptions are examples of possible system setups and parameters and have to be scaled or adopted to the users equipment. 6.1 Evaporator, suitable for volumes up to 200 ml and inlet for nitrogen g
44、as. 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 graduations with a precision of 0,5 ml. 6.7 Measuring cylinder, borosilicate glass, glass-stoppered, 25 ml, 1 ml
45、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 changed during transport or storage. Sampling is not part of the method specified in this European Standard. A re
46、commended 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 cereal products, mixed feeds, and hay should be ground carefully so that it passes completely through a sieve w
47、ith 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 through a sieve with 1 mm apertures. Mix thoroughly. Oil / fat are directly dissolved in n-hexane. 9 Procedure
48、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). The procedure blank should be free of contaminants at or above the limits of quantification. DIN EN 16215:2012-07 EN 16215:2012 (E) 10 9.2 Animal feed stuff sample and oil/fat sample 9.2.1 10 g animal feed stuff sample (12 % moistu
