1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16278:2012Animal feeding stuffs Determination ofinorganic arsenic by hydridegeneration atomic absorptionspectrometry (HG-AAS) aftermicrowave extraction andseparation by sol
2、id phaseextraction (SPE)BS EN 16278:2012 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16278:2012.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can
3、 beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2012. Published by BSI StandardsLimited 2012ISBN 978 0 580 66999 6ICS 65.120Compliance w
4、ith a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 July 2012.Amendments issued since publicationDate Text affectedBS EN 16278:2012EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE
5、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 inorganique
6、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 Standard was
7、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 such nationa
8、l 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 own language
9、 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, Greece, Hu
10、ngary, 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 NORMUNG Manage
11、ment 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: EBS EN 16278:2012EN 16278:2012 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .
12、45 Apparatus 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-A
13、AS) after microwave extraction and separation by solid phase extraction (SPE) . 13Bibliography . 18BS EN 16278:2012EN 16278:2012 (E) 3 Foreword This document (EN 16278:2012) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”, the secretariat of which is held by NEN. This Eur
14、opean 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. Attention is drawn to the possibility that some of the el
15、ements 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 Commission and the European Free Trade Association. According to t
16、he 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
17、Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16278:2012EN 16278:2012 (E) 4 1 Scope This European Standard describes a procedure for the dete
18、rmination 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 with a working range from 0,19 mg/kg to 2,7 mg/kg (HORRATvalues 4
19、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 Washing of SPE cartridges Wash the SPE cartridges with 3 ml 0,5 mol/l
20、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 with 1,25 ml of 0,4 mol/l hydrochloric acid (4.2.1). IMPORTANT Coll
21、ect 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 until analysis. 6.4 Determination of inorganic arsenic 6.4.1 Gener
22、al 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 dependent on the hydride system used. Therefore, please follow the
23、 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). BS EN 16278:2012EN 16278:2012 (E) 9 Table 2 Example of a procedure for the pre-reduction of test solutions prior to HG-AAS measurement Aliq
24、uot 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 thoroughly Incubation 60 min at room temperature All samples including the ca
25、libration 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 the Atomic absorption spectrometer (HG-AAS procedure) To devise a test
26、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 settings are listed in Table 3. Table 3 Typical settings of HG-AAS for ars
27、enic 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-reduced calibration solutions are measured directly with an atomic a
28、bsorption 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 acid (4.2.4) and borohydride solution (4.13). The resulting gas/liquid
29、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 solutions are measured first; then the test solutions are measured.
30、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, the reference samples being subjected to all the steps in the metho
31、d starting from microwave extraction. Blank solutions prepared by subjecting them to all the steps in the method shall also be determined. BS EN 16278:2012EN 16278:2012 (E) 10 7 Calculation In general, the calibration curve and the element concentration of the test solution are calculated by the AAS
32、 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 test solution, in g/l; V1is the total volume of the pre-reduction solution,
33、 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 solution, in ml, (6 ml); V6is the volume of supernatant for the buffered sample
34、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 in parenthesis are used, Formula (1) can be simplified as follows: Simpli
35、fied 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, Technical University of Denmark (DTU Food) in collaboration with the Institute
36、 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. BS EN 16278:2012EN 16278:2012 (E) 11 Details of an interlaboratory test on the
37、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 absolute difference between two independent single test results, obtained with the
38、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 limit)given in Table 4. 8.3 Reproducibility The absolute difference between two sin
39、gle 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 (reproducibility limit) given in Table 4. Table 4 Precision data Fish feed Fish meal Fis
40、h 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 limit; R is the reproducibility limit. 9 Test report The test report shall specify
41、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 specified; d) date of sampling and sampling procedure (if known); e) date when the an
42、alysis 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 result(s). BS EN 16278:2012EN 16278:2012 (E) 12 Annex A (informative) Results of t
43、he 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 9 8 8 9 Remaining data after outlier elimination 31 24 32 28 26 31 Outliers % 3,1 14
44、,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,
45、363 1,397 0,189 0,467 HORRAT value 0,93 1,61 0,76 1,30 0,85 1,7 NOTE r is the repeatability limit; Sris the repeatability standard deviation; CVris the repeatability variation coefficient; HORRAT value is Horwitz Ratio value; R is the reproducibility limit; SRis the reproducibility standard deviatio
46、n; CVRis the reproducibility variation coefficient. BS EN 16278:2012EN 16278:2012 (E) 13 Annex B (informative) Flowchart Animal feeding stuffs Determination of inorganic arsenic by hydride generation atomic absorption spectrometry (HG-AAS) after microwave extraction and separation by solid phase ext
47、raction (SPE) * The solutions can be stored (at 4 C) for later continuation of the analysis. BS EN 16278:2012EN 16278:2012 (E) 14 BS EN 16278:2012EN 16278:2012 (E) 15 The following Table B.1 gives an overview of which chemicals, solutions and equipment are used for the three different steps in the a
48、nalytical procedure Table B.1 Solutions and equipment Step Chemicals and solutions Equipment -wave Water 30 % Hydrochloric acid (HCl) (4.1) 0,055 mol/l HCl (4.2.2) 30 % Hydrogen peroxide (H2O2) (4.3) 3 % H2O2, 0,055 mol/l HCl (4.4) for sample extraction Microwave oven (5.3) Closed vessels (5.3) Bala
49、nce (5.2) Centrifuge (5.5) Centrifuge containers (5.5)Volumetric flasks (5.8) SPE 100 % methanol (4.11) for conditioning of SPE columns Water 99,999 % ammonium carbonate (4.9) 40 mmol/l ammonium carbonate (4.10) 30 % Hydrogen peroxide (H2O2) (4.3) 3 % H2O2, 0,055 mol/l HCl (4.4) prepared earlier for the -wave step SPE equilibration solution (4.17) Acetic acid (4.5) 0,5 mol/l acetic acid (4.6) for washing of SPE columns 30 % HCl (4.1) 0,4 mol/l HCl (4.2.1) for sample elution of S
