SIST EN 16801:2016

SLOVENSKI STANDARD
SIST EN 16801:2016
01-junij-2016
äLYLOD'RORþHYDQMHHOHPHQWRYLQQMLKRYLKNHPLMVNLKREOLN'RORþHYDQMHPHWLO
åLYHJDVUHEUDYåLYLOLKPRUVNHJDL]YRUD]L]RWRSQLPUD]UHGþHQMHP*&,&306
Foodstuffs - Determination of elements and their chemical species - Determination of
methylmercury in foodstuffs of marine origin by isotope dilution GC-ICP-MS
Lebensmittel - Bestimmung von Elementen und ihren Verbindungen - Bestimmung von
Methylquecksilber in Lebensmitteln marinen Ursprungs mit Isotopenverdünnung GC-ICP
-MS
Produits alimentaires - Détermination des éléments et de leurs espèces chimiques -
Détermination de la teneur en méthylmercure dans les produits alimentaires d'origine
marine par dilution isotopique CG-ICP-SM
Ta slovenski standard je istoveten z: EN 16801:2016
ICS:
67.120.30 Ribe in ribji proizvodi Fish and fishery products
SIST EN 16801:2016 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 16801:2016

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SIST EN 16801:2016


EN 16801
EUROPEAN STANDARD

NORME EUROPÉENNE

March 2016
EUROPÄISCHE NORM
ICS 67.120.30
English Version

Foodstuffs - Determination of elements and their chemical
species - Determination of methylmercury in foodstuffs of
marine origin by isotope dilution GC-ICP-MS
Produits alimentaires - Détermination des éléments et Lebensmittel - Bestimmung von Elementen und ihren
de leurs espèces chimiques - Détermination de la Verbindungen - Bestimmung von Methylquecksilber in
teneur en méthylmercure dans les produits Lebensmitteln marinen Ursprungs mit
alimentaires d'origine marine par dilution isotopique Isotopenverdünnung GC-ICP-MS
CG-ICP-SM
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 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 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 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, 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
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR 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 Ref. No. EN 16801:2016 E
worldwide for CEN national Members.

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SIST EN 16801:2016
EN 16801:2016 (E)
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 Calculation . 10
8 Precision . 11
9 Test report . 12
Annex A (informative) Precision data . 13
Bibliography . 14

2

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SIST EN 16801:2016
EN 16801:2016 (E)
European foreword
This document (EN 16801:2016) 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 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 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 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 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.
3

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SIST EN 16801:2016
EN 16801:2016 (E)
1 Scope
This European Standard 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 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
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 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
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, the
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 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 does 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 mercury 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 grade 1 (i.e. electrical conductivity below 0,1 µS/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 and measurement (e.g. uses of laminar flow benches or comparable clean room facilities).
4

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SIST EN 16801:2016
EN 16801:2016 (E)
4.2 Monomethylmercury stock solutions.
201
Commercially available MMHg standard enriched in the Hg-isotope with a mass fraction of 5,5 µg/g
1)
(as Hg) is recommended, such as IES-MMHg201 . 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 solutions 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
accurate mass fractions can be calculated. The following descriptions are given as examples.
4.3.2 MMHg approximately 500 ng/g (as Hg).
201
Dilute approximately 1 g, to the nearest milligram, of Hg enriched MMHg stock solution (4.2) with
water up to 10 g. Calculate the exact mass fraction using the mass fraction of the stock solution and
weight.
4.3.3 MMHg approximately 50 ng/g (as Hg).
201
Dilute approximately 1 g, to the nearest milligram, of the 500 ng/g Hg 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 solution and weights.
4.3.4 MMHg approximately 5 ng/g (as Hg).
201
Dilute approximately 1 g, to the nearest milligram, of the 50 ng/g Hg 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 Tetramethylammonium hydroxide (TMAH), mass fraction w = 25 % in water, minimum
synthesis quality.
4.5 Acetic acid, concentrated, mass concentration ρ = 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.
Transfer 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 acetic 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 Científico-Tecnológico, Campus de "El
Cristo", 33006, Oviedo, Spain http://www.isc-science.com/ or Qmx Laboratories, bolford Street, Thaxted, Essex, CM6 2PY, UK
http://www.qmx.com/. 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.
5

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SIST EN 16801:2016
EN 16801:2016 (E)
4.10 Nitric acid, 65 %, ρ 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 borate 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 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 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 (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 standard laboratory equipment, use the following:
5.1 Analytical balance, accuracy of 0,5 mg.
−1
5.2 Orbital/overhead rotator, capable of approximately 0,04 g (20 min ).
−1
5.3 Centrifuge, capable of 1 200 g (4 000 min ).
5.4 pH-meter.
5.5 Laboratory ware, volumetric flasks of glass, polypropylene tubes (10 ml) 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 peak 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 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 µm).
6

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SIST EN 16801:2016
EN 16801:2016 (E)
5.10 Helium, purity ≥ 99,99 %.
5.11 Helium, (5.10) with 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 manufacturer’s instructions, e.g. 5 %.
6 Procedure
6.1 Calculation of optimal spike amount
201
The following description is given for the Hg enriched MMHg spike solution (4.2). To ensure that the
200 201 202
measurement is within acceptable error limits, the abundance of the isotopes Hg, Hg and Hg in
the spiked sample should be as close as possible [3]. This is achieved when the amount 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.
201
Table 1 — Isotopic composition of natural Hg and recommended Hg 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, m , to be added from either 4.3.2, 4.3.3 or 4.3.4 to the
Sp
sample using Formula (1):
 w ×m 
Se S
 
7
 
m = (1)
Sp
w
Sp
where
w is the estimated mass fraction of MMHg in the sample, in ng/g or µg/k
...