1、BSI Standards PublicationBS EN 16802:2016Foodstuffs Determination of elements and their chemical species Determination of inorganic arsenic in foodstuffs of marine and plant origin by anion-exchange HPLC-ICP-MSBS EN 16802:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implemen
2、tation of EN 16802:2016.The UK participation in its preparation was entrusted to TechnicalCommittee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessa
3、ryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 79938 9ICS 67.050; 67.060; 67.120.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This British
4、Standard was published under the authority of theStandards Policy and Strategy Committee on 30 April 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS EN 16802:2016EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16802 March 2016 ICS 67.050; 67.060; 67.120.30 Engl
5、ish Version Foodstuffs - Determination of elements and their chemical species - Determination of inorganic arsenic in foodstuffs of marine and plant origin by anion-exchange HPLC-ICP-MS Produits alimentaires - Dtermination des lments et de leurs espces chimiques - Dtermination de la teneur en arseni
6、c inorganique dans les produits alimentaires dorigines marine et vgtale, par CLHP avec change danions et spectromtrie de masse plasma induit par haute frquence (ICP-SM) Lebensmittel - Bestimmung von Elementen und ihren Verbindungen - Bestimmung von anorganischem Arsen in Lebensmitteln marinen Urspru
7、ngs und pflanzlichen Lebensmitteln mit Anionenaustausch-HPLC-ICP-MS This European Standard was approved by CEN on 8 February 2016. 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 stand
8、ard without any alteration. Up-to-date lists 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 an
9、y 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 status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Repu
10、blic, Denmark, Estonia, Finland, Former 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 United Kingdom. EUROPEAN
11、 COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2016 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16802:2016 EBS EN 16
12、802:2016EN 16802:2016 (E) 2 Contents Page European foreword . 3 1 Scope 4 2 Normative references 4 3 Principle . 4 4 Reagents . 4 5 Apparatus and equipment . 6 6 Procedure. 7 6.1 General 7 6.2 Waterbath extraction 7 6.3 Determination of inorganic arsenic by HPLC-ICP-MS 7 6.3.1 Preparation of HPLC-IC
13、P-MS for analysis . 7 6.3.2 Calibration . 8 6.3.3 Determination of samples and blank solution . 8 6.3.4 HPLC sequence . 8 6.3.5 Typical HPLC-ICP-MS settings . 8 6.4 Quality control . 9 7 Calculation . 9 7.1 Integration of peaks . 9 7.2 Inorganic arsenic in test solutions 9 7.3 Calculation of inorgan
14、ic arsenic in the samples 9 8 Precision 10 8.1 General . 10 8.2 Repeatability 10 8.3 Reproducibility . 10 9 Test report 10 Annex A (informative) Precision data . 11 Annex B (informative) Supplementary information about chromatographic conditions 12 Bibliography . 13 BS EN 16802:2016EN 16802:2016 (E)
15、 3 European foreword This document (EN 16802:2016) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or
16、 by endorsement, at the latest by September 2016, and conflicting national standards shall be withdrawn at the latest by September 2016. 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 responsi
17、ble for identifying any or all such patent rights. This document has been prepared under mandate M 422 given to CEN by the European Commission and the European Free Trade Association. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries a
18、re bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, P
19、ortugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16802:2016EN 16802:2016 (E) 4 1 Scope This European Standard describes a procedure for the determination of inorganic arsenic in foodstuffs of marine and plant origin by anion-exchange HPLC-ICP-MS
20、following waterbath extraction. This method has been validated in an interlaboratory test on white rice, wholemeal rice, leek, blue mussels, fish muscle and seaweed with an inorganic arsenic mass fraction in the range 0,073 mg/kg to 10,3 mg/kg 1. 2 Normative references The following documents, in wh
21、ole 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 latest edition of the referenced document (including any amendments) applies. EN 13804, Foodstuffs Determination of
22、 elements and their chemical species General considerations and specific requirements EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696) 3 Principle This standard describes a method for the determination of inorganic arsenic. Inorganic arsenic consists of arse
23、nite, As(III) and arsenate, As(V). A representative test portion of the sample is treated with a diluted nitric acid and hydrogen peroxide solution in a heated waterbath. Hereby the arsenic species are extracted into solution and As(III) is oxidized to As(V). The inorganic arsenic is selectively sep
24、arated from other arsenic compounds using anion exchange HPLC (High Performance Liquid Chromatography) coupled online to the element-specific detector ICP-MS (Inductively Coupled Plasma Mass Spectrometry) for the determination of the mass fraction of inorganic arsenic. External calibration with solv
25、ent matrix-matched standards is used for quantification of the amount of inorganic arsenic. WARNING The use of this 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
26、of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to its use. 4 Reagents 4.1 General The concentration of the arsenic species in the reagents and water used shall be low enough to not affect the results o
27、f the determination. When using a method of high sensitivity like ICP-MS, the control of the blank levels of water, acid and other reagents is very important. Generally ultra-pure water complying with ISO 3696 grade 1 (i.e. electrical conductivity below 0,1 S/cm at 25 C) and acid of high purity, e.g
28、. cleaned by sub-boiling distillation, are recommended. Reagents should be of minimum p.a. quality where possible. Special facilities can be used in order to avoid contamination during the steps of preparation and measurement (e.g. uses of laminar flow benches or comparable clean room facilities). 4
29、.2 Nitric acid concentrated, mass fraction w(HNO3), 65 %, mass concentration of approximately (HNO3) = 1,4 g/ml. Use only nitric acid available with high purity or perform a clean-up by a sub-boiling distillation in order to avoid potential contamination. BS EN 16802:2016EN 16802:2016 (E) 5 4.3 Hydr
30、ogen peroxide, w(H2O2) 30 %. High purity is essential to avoid potential contamination. Commercially available hydrogen peroxide for analysis should be tested for contamination of arsenic. 4.4 Extraction solution 1, c(HNO3) = 0,1 mol/l in 3 %(V/V) H2O2.Pour 800 ml of H2O and then 6,5 ml of HNO3(4.2)
31、 and thereafter 100 ml of H2O2(4.3) into a 1 000 ml volumetric flask. Fill it up to the mark with H2O. This solution should be prepared on the same day of use. It is recommended that the total volume needed for the analysis is estimated and only this amount is produced. 4.5 Extraction solution 2, c(
32、HNO3) = 0,2 mol/l in 6 % (V/V) H2O2.Pour 70 ml of H2O, 1,3 ml of nitric acid (4.2) and 20 ml of hydrogen peroxoide (4.3) into a 100 ml volumetric flask. Fill up to the mark at 100 ml with H2O. This solution should be prepared on the same day of use. It is recommended that the total volume needed for
33、 the analysis is estimated and only this amount is produced. 4.6 Ammonium carbonate, w(NH4)2CO3 99,999 %, for production of mobile phase solution. 4.7 Aqueous ammonia, wNH3(aq.) 25 %, for adjustment of pH in the mobile phase. 4.8 Methanol, (CH3OH), HPLC grade, for production of mobile phase. 4.9 Mob
34、ile phase, e.g. 50 mmol/l ammonium carbonate in 3 % methanol at pH 10,3. Dissolve e.g. 4,80 g of ammonium carbonate (4.6) in approximately 800 ml of water. Adjust the pH to 10,3 with aqueous ammonia (4.7), add 30 ml of methanol (4.8) and fill up to 1 000 ml with water. Filter the mobile phase soluti
35、on through a 0,45 m filter prior to use. The optimal concentration of ammonium carbonate in the mobile phase depends on the analytical column used (e.g. brand, particle size and dimensions). The appropriate concentration of ammonia carbonate is at the discretion of the analyst and should fulfil the
36、criteria for sufficient resolution of the arsenate peak as stated in 5.10. Methanol is added to the mobile phase in order to enhance the signal intensity for arsenic (carbon enhancement effect 2). The concentration of methanol for maximum signal depends on the instrument used and should be identifie
37、d by the analyst. 4.10 Diarsenic trioxide, w(As2O3) 99,5 %, optional. 4.11 Potassium hydroxide solution, (KOH) = 20 g/100 ml, optional. 4.12 Sulfuric acid solutions, w(H2SO4) = 20 % and w(H2SO4) = 1 %, optional. 4.13 Phenolphthalein, optional. 4.14 Standard solutions, with an arsenic mass concentrat
38、ion of 1 000 mg/l. The use of commercial standards of arsenic, arsenic III and/or V, with a mass concentration of 1 000 mg/l is recommended. BS EN 16802:2016EN 16802:2016 (E) 6 Otherwise proceed as follows: Dissolve e.g. 1,320 g of diarsenic trioxide (4.10) in 25 ml of potassium hydroxide solution (
39、4.11), neutralize with 20 % sulfuric acid solution (4.12) with phenolphthalein (4.13) as indicator and dilute to 1 000 ml in a volumetric flask with 1 % sulfuric acid solution (4.12). NOTE By preparing the standard in the extraction solution 1 (4.4) all arsenite will be completely oxidized to arsena
40、te. 4.15 Calibration solutions. Prepare a range of standards including a blank calibration solution that covers the linear range of the analyte to be determined by diluting the analyte stock solution with extraction solution 1 (4.4). Appropriate matrix matching of the calibration solutions shall be
41、performed by using the extraction solution 1 (4.4) for the final dilution step, which furthermore will prevent reduction of arsenate to arsenite. Transfer an aliquot of the calibration solutions to HPLC vials prior to analysis (6.3.2). The quantitative oxidation of arsenite to arsenate in the standa
42、rd solutions should be verified (e.g. visual inspection of chromatogram by looking for an additional peak or a reduced intensity of the arsenate peak). 4.16 Solution for checking chromatographic separation, containing the organic arsenic compounds (e.g. 10 g/l) monomethylarsenous acid (MMA), dimethy
43、larsinic acid (DMA) and arsenobetaine (AB), as well as arsenate (e.g. 10 g/l) and chloride (e.g. 100 mg/l). 5 Apparatus and equipment 5.1 General To minimize the contamination, all apparatus and equipment that come into direct contact with the sample and the solutions shall be carefully pre-treated.
44、 It is recommended to avoid the use of glassware, since this may cause contamination with arsenate, see 3. WARNING Some autosampler systems use syringes made of glass. In this case, it is only possible to check for contamination and to minimize it. 5.2 Laboratory grinder, capable of grinding to a pa
45、rticle size of less than 0,5 mm. 5.3 Analytical balance, accuracy of 1 mg. 5.4 Filtering device, for filtration of mobile phase, maximum pore size 0,45 m. 5.5 Waterbath, adjustable to 90 C. 5.6 Centrifuge, minimum 2 010 g (4 000 min-1). 5.7 Single use syringe filters (0,45 m) or HPLC vials with filt
46、ers, compatible with acidic solutions for filtering of test solutions prior to analysis. 5.8 Plastic volumetric flasks, for preparation of mobile phase, blank and calibration solutions. NOTE If calibration standards are prepared by weighing, plasticware without marks can be used. 5.9 High Performanc
47、e Liquid Chromatographic System (HPLC). 5.10 Strong anion exchange column (SAX), suitable for selective separation of arsenate from other arsenic compounds present in the sample extracts. It is advisable to use a guard column to prolong the life-time of the analytical column. BS EN 16802:2016EN 1680
48、2:2016 (E) 7 Usually, the minimum acceptable retention time for the analyte is twice the retention time corresponding to the void volume of the column. Furthermore the nearest peak in the chromatogram should be separated from the analyte peak by at least one full peak width at 10 % of the analyte pe
49、ak height. It is recommended to verify sufficient separation of the analyte peak using a solution of organic arsenic compounds (e.g. monomethylarsenous acid (MMA), dimethylarsinic acid (DMA) and arsenobetaine (AB) and arsenate. Make sure that the HPLC run is long enough for chloride (m/z 35) and for arsenic compounds with longer retention times than arsenate, to elute from the column prior to injection of the next sample. It should furthermore be ensured that the arsenate and chloride peaks do not co-elute in order to avoid
