1、BSI Standards PublicationBS EN 16801:2016Foodstuffs Determinationof elements and their chemicalspecies Determination ofmethylmercury in foodstuffsof marine origin by isotopedilution GC-ICP-MSBS EN 16801:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16801:
2、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 necessaryprovisions of a c
3、ontract. Users are responsible for its correctapplication. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 79937 2ICS 67.120.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the au
4、thority 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 16801:2016EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16801 March 2016 ICS 67.120.30 English Version Foodstuffs - Determination of elements
5、and their chemical species - Determination of methylmercury in foodstuffs of marine origin by isotope dilution GC-ICP-MS Produits alimentaires - Dtermination des lments et de leurs espces chimiques - Dtermination de la teneur en mthylmercure dans les produits alimentaires dorigine marine par dilutio
6、n isotopique CG-ICP-SM Lebensmittel - Bestimmung von Elementen und ihren Verbindungen - Bestimmung von Methylquecksilber in Lebensmitteln marinen Ursprungs mit Isotopenverdnnung GC-ICP-MS This European Standard was approved by CEN on 8 February 2016. CEN members are bound to comply with the CEN/CENE
7、LEC 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 Management Centre o
8、r 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 status as the off
9、icial 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, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands
10、, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey andUnited Kingdom. EUROPEAN 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
11、exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16801:2016 EBS EN 16801:2016EN 16801:2016 (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 7 Calc
12、ulation 10 8 Precision 11 9 Test report 12 Annex A (informative) Precision data . 13 Bibliography . 14 BS EN 16801:2016EN 16801:2016 (E) 3 European foreword This document (EN 16801:2016) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of whic
13、h is held by DIN. 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 September 2016, and conflicting national standards shall be withdrawn at the latest by September 2016. Attention is drawn to the p
14、ossibility 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 mandate M 422 given to CEN by the European Commission and the European Free
15、 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, Former Yugoslav Republic of Ma
16、cedonia, 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. BS EN 16801:2016EN 16801:2016 (E) 4 1 Scope This European Standard
17、describes a method for the determination of monomethylmercury (MMHg) in foodstuffs of marine origin. The method has been validated in an interlaboratory test on mussel tissue, squid muscle, crab claw muscle, dog fish liver, whale meat, cod muscle and Greenland halibut muscle (all freeze-dried) with
18、mass fractions from 0,04 mg/kg to 3,6 mg/kg dry weight according to ISO 5725-2 1. Laboratory experiences have shown that this method is also applicable on fresh samples 2. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispe
19、nsable 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 elements and their chemical species General considerations and specific re
20、quirements EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696) 3 Principle The sample is spiked with an appropriate amount of Hg-isotope enriched MMHg and digested using tetramethylammonium hydroxide (TMAH). After pH adjustment, derivatisation and extraction, t
21、he organic phase is analysed using GC-ICP-MS. The GC separates the different mercury species before the derivatised species (ethylmethylmercury) is atomised and ionised in the high temperature by the ICP. The ions are extracted from the plasma by a set of sampler and skimmer cones and transferred to
22、 a mass spectrometer where the ions are separated by their mass/charge ratio and determined by a pulse-count and/or analogue detector. The result is calculated using the isotope dilution equation. WARNING The use of this method may involve hazardous materials, operations and equipment. This method d
23、oes not purport to address all the safety problems associated with its use. It is the responsibility of the user of this method to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 4 Reagents 4.1 General The concentration of mer
24、cury species in the reagents and water used shall be low enough to not affect the results of 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
25、 grade 1 (i.e. electrical conductivity below 0,1 S/cm at 25 C) and acid of high purity is recommended, e.g. cleaned by sub-boiling distillation. 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 a
26、nd measurement (e.g. uses of laminar flow benches or comparable clean room facilities). BS EN 16801:2016EN 16801:2016 (E) 5 4.2 Monomethylmercury stock solutions. Commercially available MMHg standard enriched in the 201Hg-isotope with a mass fraction of 5,5 g/g (as Hg) is recommended, such as IES-MM
27、Hg2011). Other MMHg Hg-isotope enriched standards may also be available in suitable mass fractions from other suppliers or may be prepared in-house. In this case, the method shall be adjusted accordingly. The quality of the standards should be designed to be used by isotope dilution methods. Stock s
28、olutions in diluted acid are preferred. 4.3 Monomethylmercury standard solution. 4.3.1 General The mass fractions of the MMHg in the standard solutions shall be chosen in relation to the expected mass fraction of MMHg in samples. It is important that all dilutions are done by weighing so that their
29、accurate mass fractions can be calculated. The following descriptions are given as examples. 4.3.2 MMHg approximately 500 ng/g (as Hg). Dilute approximately 1 g, to the nearest milligram, of 201Hg enriched MMHg stock solution (4.2) with water up to 10 g. Calculate the exact mass fraction using the m
30、ass fraction of the stock solution and weight. 4.3.3 MMHg approximately 50 ng/g (as Hg). Dilute approximately 1 g, to the nearest milligram, of the 500 ng/g 201Hg enriched MMHg solution (4.3.2) with water up to 10 g. Calculate the exact mass fraction using the exact mass fraction of the 500 ng/g sol
31、ution and weights. 4.3.4 MMHg approximately 5 ng/g (as Hg). Dilute approximately 1 g, to the nearest milligram, of the 50 ng/g 201Hg enriched MMHg solution (4.3.3) with water up to 10 g. Calculate the exact mass fraction using the exact mass fraction of the 50 ng/g solution and weights. 4.4 Tetramet
32、hylammonium hydroxide (TMAH), mass fraction w = 25 % in water, minimum synthesis quality. 4.5 Acetic acid, concentrated, mass concentration = 1,05 g/ml, minimum p.a. quality. 4.6 Sodium hydroxide, minimum p.a. quality. 4.7 Sodium hydroxide solution, substance concentration c(NaOH) = 0,1 mol/l. Trans
33、fer 0,4 g of sodium hydroxide to a 100 ml volumetric flask and add water to the mark. 4.8 Sodium acetate, minimum p.a. quality. 4.9 Sodium acetate/acetic acid buffer (pH 5). Dissolve 41 g of sodium acetate in approximately 0,5 l of water. Adjust the pH of the solution to 5 by adding concentrated ace
34、tic acid (4.5) dropwise by using a pH-meter (5.4). Finally, dilute the solution to 1 l with water. 1) IES-MMHg201 is available from e.g. Innovative Solutions in Chemistry S.L., Edificio Cientfico-Tecnolgico, Campus de “El Cristo“, 33006, Oviedo, Spain http:/www.isc- or Qmx Laboratories, bolford Stre
35、et, Thaxted, Essex, CM6 2PY, UK http:/ This is an example of a suitable product available commercially. This information is given for the convenience of the users of this International Standard and does not constitute an endorsement by CEN of this product. BS EN 16801:2016EN 16801:2016 (E) 6 4.10 Ni
36、tric acid, 65 %, of approximately 1,4 g/ml. Hg-free quality. Other acid concentrations may be used if the volume added in 6.2 is adjusted accordingly. 4.11 Sodium tetraethyl borate, minimum synthesis quality (98 %). 4.12 Sodium tetraethyl borate solution (2 %). Dissolve 1 g of sodium tetraethyl bora
37、te in 0,1 mol/l of sodium hydroxide solution (4.7), transfer to a 50 ml-volumetric flask and fill up to the mark with 0,1 mol/l sodium hydroxide solution. Prepare freshly at each day of analysis or divide the solution into smaller amounts and store in the freezer at approximately 20 C. The solution
38、may be stored at approximately 20 C for at least three months. The solution shall be used within the day after removal from the freezer. 4.13 Hexane, minimum HPLC-quality. 4.14 Optimising solution for the ICP-MS. The optimising solution should contain elements that cover the whole mass range giving
39、a high rate of oxides and doubly charged ions. Use the solutions recommended by the manufacturer of the ICP-MS instrument. A solution containing e.g. Li, Ce and Tl is suitable for those purposes. In this case, choose the concentration of these elements in order to achieve a count rate of 10 000 cps
40、(counts per second). 5 Apparatus and equipment All pieces of equipment described here are examples of suitable equipment and may be replaced by equivalent equipment unless otherwise stated. Generally, clean and rinse the vessels carefully according to the procedure in EN 13804. In addition to standa
41、rd laboratory equipment, use the following: 5.1 Analytical balance, accuracy of 0,5 mg. 5.2 Orbital/overhead rotator, capable of approximately 0,04 g (20 min1). 5.3 Centrifuge, capable of 1 200 g (4 000 min1). 5.4 pH-meter. 5.5 Laboratory ware, volumetric flasks of glass, polypropylene tubes (10 ml)
42、 for samples, GC-vials, pH paper. 5.6 Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Mass spectrometer with inductively coupled argon plasma operating in a mass range from 5 amu (atomic mass units) to 240 amu. Using routine settings the mass spectrometer shall be capable to resolve 1 amu pea
43、k width at 5 % peak height or better (resolution 300) with sufficient sensitivity to achieve the detection limits suitable for the analytical purpose. 5.7 Argon, purity 99,99 %. 5.8 Gas chromatograph (GC), with injector heating, programmable column heating and heating of transfer line to ICP-MS. 5.9
44、 GC-column, capillary or preparative column capable of separating ethylmethylmercury from other mercury species (e.g. 30 m x 0,32 mm, analytical column with 5 % phenyl methyl siloxane; film thickness: 0,25 m). BS EN 16801:2016EN 16801:2016 (E) 7 5.10 Helium, purity 99,99 %. 5.11 Helium, (5.10) with
45、1 % to 2 % added xenon for tuning of the GC-ICP-MS interface or some other tuning configuration capable of optimising the instrument parameters, optional. 5.12 Oxygen, optional, to prevent carbon deposition, according to manufacturers instructions, e.g. 5 %. 6 Procedure 6.1 Calculation of optimal sp
46、ike amount The following description is given for the 201Hg enriched MMHg spike solution (4.2). To ensure that the measurement is within acceptable error limits, the abundance of the isotopes 200Hg, 201Hg and 202Hg in the spiked sample should be as close as possible 3. This is achieved when the amou
47、nt of spike to analyte is 1 to 7 for the given isotope abundances in Table 1. The ratio of 1 to 7 applies for the specified spike solution with the given isotopic composition. If another spike solution with another isotope composition is used, the factor should be adjusted accordingly. Table 1 Isoto
48、pic composition of natural Hg and recommended 201Hg enriched spike Hg-isotope % of isotope in natural Hg % of isotope in enriched Hg 200 23,10 0,89 201 13,18 96,50 202 29,86 2,37 Calculate the appropriate spike amount in gram, mSp, to be added from either 4.3.2, 4.3.3 or 4.3.4 to the sample using Fo
49、rmula (1): =Se SSpSp7wmmw(1) where wSeis the estimated mass fraction of MMHg in the sample, in ng/g or g/kg; mSis the mass of the sample to be analysed, in g; 7 is the factor needed to achieve approximate abundance matching of the isotopes in the spiked sample; wSp is the exact mass fraction from the diluted MMHg stock solutions 4.3.2, 4.3.3 or 4.3.4, in ng/g. The amount of spike to be added should not be below 0,1 g and should not exceed 1 g. This is to ensure enough significant figures from the wei
