EN 12014-2:2017

SLOVENSKI STANDARD
01-februar-2018
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Foodstuffs - Determination of nitrate and/or nitrite content - Part 2: HPLC/IC method for
the determination of nitrate content of vegetables and vegetable products
Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes - Teil 2: HPLC/IC-
Verfahren für die Bestimmung des Nitratgehaltes in Gemüse und Gemüseerzeugnissen
Produits alimentaires - Détermination de la teneur en nitrates et/ou en nitrites - Partie 2:
Méthode de détermination par CLHP/CI de la teneur en nitrates des légumes et des
produits à base de légumes
Ta slovenski standard je istoveten z: EN 12014-2:2017
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.080.20 Zelenjava in zelenjavni Vegetables and derived
proizvodi products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 12014-2
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2017
EUROPÄISCHE NORM
ICS 67.080.20 Supersedes EN 12014-2:1997
English Version
Foodstuffs - Determination of nitrate and/or nitrite
content - Part 2: HPLC/IC method for the determination of
nitrate content of vegetables and vegetable products
Produits alimentaires - Détermination de la teneur en Lebensmittel - Bestimmung des Nitrat- und/oder
nitrates et/ou en nitrites - Partie 2: Méthode de Nitritgehaltes - Teil 2: HPLC/IC-Verfahren für die
détermination par CLHP/CI de la teneur en nitrates des Bestimmung des Nitratgehaltes in Gemüse und
légumes et des produits à base de légumes Gemüseerzeugnissen
This European Standard was approved by CEN on 15 October 2017.

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, Serbia, 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
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 12014-2:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Principle . 5
4 Reagents . 5
5 Apparatus and equipment . 6
6 Procedure. 7
6.1 Sample preparation . 7
6.1.1 Liquid samples, e.g. vegetable juice . 7
6.1.2 Solid samples, e.g. leaf vegetable . 7
6.1.3 Pasty samples, e.g. mashed vegetables . 7
6.2 Hot water extraction of samples (solid and pasty samples). 7
6.3 Preparation of sample test solutions . 7
6.4 Preparation of the calibration curve. 7
6.5 Determination . 8
6.5.1 High-performance liquid chromatography . 8
6.5.2 Ion chromatography . 8
7 Evaluation . 8
7.1 Identification and quantitative determination . 8
7.2 Calculation . 8
7.3 Precision . 8
7.3.1 General . 8
7.3.2 Repeatability . 9
7.3.3 Reproducibility . 9
8 Test report . 9
Annex A (informative) Examples of HPLC/IC chromatograms . 10
Annex B (informative) Precision data. 13
Annex C (informative) Examples of suitable HPLC conditions . 15
Annex D (informative) Examples of suitable IC conditions . 16
Bibliography . 18
European foreword
This document (EN 12014-2:2017) 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 June 2018, and conflicting national standards shall be
withdrawn at the latest by June 2018.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 12014-2:1997.
The following changes have been made to the former edition:
a) the scope has been reduced from 50 mg/kg nitrate to 25 mg/kg nitrate and the upper limit has
been deleted;
b) the purification method 1 and 2 used in the preparation of sample test solutions has been deleted;
c) a new matrix (iceberg lettuce) has been verified in an interlaboratory test;
d) update of the HPLC/IC-conditions and chromatograms in Annex A;
e) the procedure has been extensively revalidated and precision data in Annex B have been revised;
f) editorially revised.
According to the CEN-CENELEC Internal Regulations, the national standards organisations 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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Introduction
The method described in this standard has been developed and validated for investigations regarding
the European legislation for nitrate in vegetables and vegetable products. Laboratory experience has
shown that this analytical method is also suitable for the determination of nitrite in other matrices;
however, this has not been validated in the interlaboratory test scheme cited here.
1 Scope
This European Standard specifies a high-performance liquid chromatographic (HPLC) and an ion
chromatographic (IC) method for determination of the nitrate level in vegetables and vegetable
products. This method is applicable for samples with a content of 25 mg/kg or greater.
It has been validated on naturally contaminated and spiked samples as beetroot juice with nitrate mass
fractions of 194 mg/kg and 691 mg/kg, pureed carrots with nitrate mass fractions of 26 mg/kg and
222 mg/kg and with iceberg lettuce with nitrate mass fractions of 623 mg/kg and 3 542 mg/kg.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Principle
The nitrate is extracted from the foodstuff with hot water. The determination is performed either by
reverse-phase HPLC and UV detection, or by IC and conductivity or UV detection.
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified. Water shall be nitrate-free
and shall comply with grade 1 of ISO 3696.
4.1 Potassium nitrate.
4.2 Sodium nitrate.
1)
4.3 Sulfuric acid, 96 % (ρ = 1,84 g/ml ).
2)
4.4 Regenerator for suppressor, e.g. sulfuric acid (c = 0,0125 mol/l ).
Carefully pipette 20 ml of sulfuric acid (4.3) into a 1 000 ml volumetric flask containing 800 ml of water.
Mix the solution, dilute to the mark with water and mix again. Transfer 33 ml of this solution to a
1 000 ml volumetric flask containing 500 ml of water. Dilute to the mark with water and mix.
Alternatively, use a method with electrolytic suppressor.
-
4.5 Nitrate stock solution, ρ = 1 000 mg/l (expressed as ion NO ).
Weigh 1,630 7 g of potassium nitrate (4.1) to the nearest 0,1 mg and dissolve in water in a 1 000 ml
volumetric flask.
Alternatively, 1,370 9 g of sodium nitrate (4.2) can be used. If required, 15 ml of sulfuric acid (4.3) can
be added as a preservative. Mix the solution, dilute to the mark with water and mix again. This solution
is stable for at least two months if stored at +4 °C in the dark (refrigerator).
Alternatively, commercially available standard solutions can also be used.

1) ρ = mass concentration
2) c = substance concentration

4.6 Nitrate standard solutions.
Prepare suitable dilutions from the nitrate stock solution (4.5), preferably in the range of 1 mg/l to
50 mg/l. Pipette an appropriate volume of nitrate stock solution into a 100 ml volumetric flask, dilute to
the mark with water, mix and filter through a membrane filter (5.4), if required.
4.7 Mobile phase, for the HPLC.
Examples of suitable eluents are given in Annex C.
4.8 Mobile phase, for the IC.
Examples of suitable eluents are given in Annex D.
5 Apparatus and equipment
Usual laboratory equipment and, in particular, the following:
5.1 Commercially available laboratory mixer, e.g. a laboratory cutter.
5.2 Homogenizer.
5.3 Fluted filter paper (nitrogen-deficient).
5.4 Membrane filter, for aqueous solutions, with a pore size of 0,45 µm (e.g. regenerated cellulose).
5.5 Filter holder for membrane filter, with suitable syringe.
5.6 HPLC equipment.
5.6.1 High-performance liquid chromatograph, consisting of an eluent reservoir, a pump, a sample
applicator, a UV detector adjustable from 190 nm to 800 nm and an evaluation unit.
5.6.2 Analytical (reversed-phase) separating column.
Examples of suitable separating columns are given in Annex C.
Use a pre-column with filling material of the same type to protect the analyti
...