DIN EN 16278-2012 Animal feeding stuffs - Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation.pdf

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1、September 2012 Translation by DIN-Sprachendienst.English price group 11No 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).

2、ICS 65.120!$Vg“1915168www.din.deDDIN EN 16278Animal feeding stuffs Determination of inorganic arsenic by hydride generation atomicabsorption spectrometry (HG-AAS) after microwave extraction andseparation by solid phase extraction (SPE);English version EN 16278:2012,English translation of DIN EN 1627

3、8:2012-09Futtermittel Bestimmung von anorganischem Arsen mit Atomabsorptionsspektrometrie-Hydridtechnik(HD-AAS) nach Mikrowellen-Extraktion und Trennung durch Festphasenextraktion (SPE);Englische Fassung EN 16278:2012,Englische bersetzung von DIN EN 16278:2012-09Aliments des animaux Dosage de larsen

4、ic inorganique par spectromtrie dabsorption atomique par gnrationdhydrures aprs extraction par micro-ondes;Version anglaise EN 16278:2012,Traduction anglaise de DIN EN 16278:2012-09www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 20 page

5、s 08.12 DIN EN 16278:2012-09 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 its preparation

6、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 Trade Association,

7、 and supports essential requirements of EU Directive(s). The International Standards referred to in this document have been published as the corresponding DIN EN or DIN EN ISO Standards with the same number, except for those below, which correspond as follows: EN ISO 3696 DIN ISO 3696 ISO 5725-1 DIN

8、 ISO 5725-1 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions EUROPEAN STANDARD NORME EUROPENNE

9、EUROPISCHE NORM EN 16278 July 2012 ICS 65.120 English Version Animal feeding stuffs - Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation by solid phase extraction (SPE) Aliments des animaux - Dosage de larsenic i

10、norganique par spectromtrie dabsorption atomique par gnration dhydrures aprs extraction par micro-ondes Futtermittel - Bestimmung von anorganischem Arsen mit Atomabsorptionsspektrometrie-Hydridtechnik (HD-AAS) nach Mikrowellen-Extraktion und Trennung durch Festphasenextraktion (SPE) This European St

11、andard was approved by CEN on 17 May 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 lists and bibliographical references concerning s

12、uch 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 under the responsibility of a CEN member into its o

13、wn 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, Former Yugoslav Republic of Macedonia, France, Germany,

14、 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 KOMITEE FR NOR

15、MUNG 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 16278:2012: EEN 16278:2012 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Ap

16、paratus and equipment 66 Procedure .77 Calculation . 108 Precision 109 Test report . 11Annex A (informative) Results of the interlaboratory tests 12Annex B (informative) Flowchart Animal feeding stuffs Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) a

17、fter microwave extraction and separation by solid phase extraction (SPE) . 13Bibliography . 18DIN EN 16278:2012-09 EN 16278:2012 (E) 3 Foreword This document (EN 16278:2012) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffss , t h e s e c r e t a r i a t o f w h i c h i s h

18、e l d b y N E N . Methods of sampling and analysis”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 January 2013, and conflicting national standards shall be withdrawn at the latest by January 2013

19、. 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 rights. This document has been prepared under a mandate given to CEN by the European Commiss

20、ion 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: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Forme

21、r Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 16278:2012-09 EN 16278:2012 (E) 4 1

22、 Scope This European Standard describes a procedure for the determination of inorganic arsenic in animal feeding stuffs of marine origin by Solid Phase Extraction (SPE) and Hydride Generation Atomic Absorption Spectrometry (HG-AAS). The method has been successfully tested in a collaborative trial wi

23、th a working range from 0,19 mg/kg to 2,7 mg/kg (HORRATvalues 4 000 rpm (2 100 g) for at least 10 min to remove particles. 6.3.4 Loading of sample solution 4 ml of the centrifuged buffered sample solution (6.3.3) is loaded to the SPE cartridges. IMPORTANT Let the SPE cartridges run dry. 6.3.5 Washin

24、g of SPE cartridges Wash the SPE cartridges with 3 ml 0,5 mol/l acetic acid (4.6). IMPORTANT Let the SPE cartridges run dry, using vacuum (50 kPa to 70 kPa) for at least 5 min before the final elution step (6.3.6). 6.3.6 Elution of SPE cartridges The retained As(V) on the SPE cartridge is eluted wit

25、h 1,25 ml of 0,4 mol/l hydrochloric acid (4.2.1). IMPORTANT Collect all sample solvent in suitable plastic containers by applying suitable vacuum (usually in the range of 50 kPa to 70 kPa) for at least 5 min. NOTE The eluate (6.3.6) can usually be stored at maximum 4 C for a maximum of three days un

26、til analysis. 6.4 Determination of inorganic arsenic 6.4.1 General The inorganic arsenic is determined as the total amount of arsenic in the SPE eluate. Carry out pre-reductions of test solutions (6.3.6) and calibration solutions (4.20) prior to the determination. 6.4.2 Pre-reduction This step is de

27、pendent on the hydride system used. Therefore, please follow the optimised hydride generation procedure of the equipment used and note that it may be necessary to use larger or smaller volumes than described below (Table 2). DIN EN 16278:2012-09 EN 16278:2012 (E) 9 Table 2 Example of a procedure for

28、 the pre-reduction of test solutions prior to HG-AAS measurement Aliquot of test solution (6.3.6) 1 ml Potassium iodide/ascorbic acid solution with hydrochloric acid (4.16) Add 7 ml and mix thoroughly Incubation 60 min at room temperature 2,6 mol/l hydrochloric acid (4.2.3) Add 6 ml and mix thorough

29、ly Incubation 60 min at room temperature All samples including the calibration solutions should be treated in the same way. The hydrochloric acid (4.2.3) and reducing-agent (4.16) concentrations shall be the same in all the test solutions (see Annex B, Table B.2 for an example). 6.4.3 Settings of th

30、e Atomic absorption spectrometer (HG-AAS procedure) To devise a test schedule, first adjust the apparatus as specified in the operating manual of the manufacturer. Then optimise the settings, paying particular attention to gas flow times and the amount of sodium borohydride introduced. Typical setti

31、ngs are listed in Table 3. Table 3 Typical settings of HG-AAS for arsenic measurement Temperature of the cell 900 C Wave length 193,7 nm Slit width 0,5 nm Signal processing Peak height with background correction Measurement time 4,0 s 6.4.4 HG-AAS determination The pre-reduced test solutions and pre

32、-reduced calibration solutions are measured directly with an atomic absorption spectrometer with electrically heated quartz cell coupled to an automated flow system (5.11). The apparatus should be programmed to mix appropriate amounts of test- or calibration solution (6.4.2), diluted hydrochloric ac

33、id (4.2.4) and borohydride solution (4.13). The resulting gas/liquid mixture is separated by an argon-flow separator. The argon stream separates and transports the arsenic hydrides to the quartz cell for atomisation reaction and measuring the atomic absorption of arsenic. The pre-reduced calibration

34、 solutions are measured first; then the test solutions are measured. Reanalyse the calibration solutions at the end of each analytical series. As an analytical control, reference samples having reliable known inorganic arsenic contents can be analysed parallel with all the series of samples analysed

35、, the reference samples being subjected to all the steps in the method starting from microwave extraction. Blank solutions prepared by subjecting them to all the steps in the method shall also be determined. DIN EN 16278:2012-09 EN 16278:2012 (E) 10 7 Calculation In general, the calibration curve an

36、d the element concentration of the test solution are calculated by the AAS system software. The mass fraction w in milligram inorganic arsenic per kilogram sample is calculated as follows: 1000642531=textrtmVVVmVVVcw(1) where (typical values in parenthesis) ctis the concentration of arsenic in the t

37、est solution, in g/l; V1is the total volume of the pre-reduction solution, in ml (14 ml); V2 is the sample volume for pre-reduction step, in ml, (1 ml); V3is the volume from SPE elution, in ml, (1,25 ml); V4is the volume put on the SPE, in ml, (4 ml); V5 is the total volume of buffered sample soluti

38、on, in ml, (6 ml); V6is the volume of supernatant for the buffered sample solution, in ml, (3 ml); mextris the mass of extractant solution after microwave extraction step, in g, (10 g); is the density of extractant solution (g/ml), (1 g/ml); mtis the mass of test portion, in g. If the typical values

39、 in parenthesis are used, Formula (1) can be simplified as follows: Simplified formula:ttmcw0875,0= (2) If necessary, subtract the concentration of inorganic arsenic in the reagent blank from ct. 8 Precision 8.1 General An interlaboratory comparison was organized by the National Food Institute, Tech

40、nical University of Denmark (DTU Food) in collaboration with the Institute for Reference Materials and Measurements (IRMM) in 2010. The results of the main method protocol are given in Annex A. Further details regarding the outcome of the collaborative study can be found in the final report 2. DIN E

41、N 16278:2012-09 EN 16278:2012 (E) 11 Details of an interlaboratory test on the precision of the method are summarized in Annex A. The values derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than those given in Annex A. 8.2 Repeatability The abso

42、lute difference between two independent single test results, obtained with the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in no more than 5 % of the cases exceed the values of r (repeatability limi

43、t)given in Table 4. 8.3 Reproducibility The absolute difference between two single test results, obtained with the same method on identical test material in different laboratories by different operators using different equipment, will in no more than 5 % of the cases exceed the values of R (reproduc

44、ibility limit) given in Table 4. Table 4 Precision data Fish feed Fish meal Fish meal Fish fillet Fish meal Lobster HepatopancreasMean, mg/kg 0,725 0,194 1,083 2,705 0,439 0,555 r, mg/kg 0,138 0,042 0,307 0,490 0,068 0,272 R, mg/kg 0,315 0,179 0,363 1,397 0,189 0,467 NOTE r is the repeatability limi

45、t; R is the reproducibility limit. 9 Test report The test report shall specify at least the following: a) information necessary for the complete identification of the sample; b) test method used with reference to this European standard; c) test results obtained and the units in which they are specif

46、ied; d) date of sampling and sampling procedure (if known); e) date when the analysis was finished; f) all operating details not specified in this document, or regarded as optional, together with details of any incidents that occurred when performing the method which may have influenced the test res

47、ult(s). DIN EN 16278:2012-09 EN 16278:2012 (E) 12 Annex A (informative) Results of the interlaboratory tests Table A.1 Precision data Matrix Units Fish feed Fish meal Fish meal Fish fillet Fish meal Control SampleNumber of labs 9 9 9 9 9 9 Number of outlier labs 0 1 0 1 1 0 Number of valid labs 9 8

48、9 8 8 9 Remaining data after outlier elimination 31 24 32 28 26 31 Outliers % 3,1 14,3 0,0 12,5 13,3 0,0 Overall mean Xobsmg/kg 0,725 0,194 1,083 2,705 0,439 0,555 srmg/kg 0,049 0,015 0,110 0,175 0,024 0,097 CVr% 6,8 7,7 10,1 6,5 5,5 17,5 r mg/kg 0,138 0,042 0,307 0,490 0,068 0,272 sRmg/kg 0,113 0,064 0,130 0,499 0,068 0,167 CVR% 15,5 33,0 12,0 18,4 15,4 30,1 R mg/kg 0,315 0,179 0,363 1,397 0,189 0,467 HORRAT value 0,93 1,61 0,76