Foodstuffs - Determination of nitrate and/or nitrite content - Part 2: HPLC/IC method for the determination of nitrate content of vegetables and vegetable products

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.

Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes - Teil 2: HPLC/IC-Verfahren für die Bestimmung des Nitratgehaltes in Gemüse und Gemüseerzeugnissen

Diese Europäische Norm legt ein hochleistungs-flüssigchromatographisches (HPLC) und ein ionenchromatographisches (IC) Verfahren zur Bestimmung des Nitratgehaltes in Gemüse und Gemüseerzeugnissen fest. Dieses Verfahren ist auf Proben mit einem Gehalt von 25 mg/kg oder größer anwendbar.
Es wurde mit natürlich kontaminierten und aufgestockten Proben in Rote-Beete-Saft mit Nitrat-Massenanteilen von 194 mg/kg und 691 mg/kg, auf pürierte Karotten mit Nitrat-Massenanteilen von 26 mg/kg und 222 mg/kg sowie auf Eisbergsalat mit Nitrat-Massenanteilen von 623 mg/kg und 3 542 mg/kg validiert.

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

La présente Norme européenne spécifie une méthode de chromatographie en phase liquide à haute performance (CLHP) et une méthode de chromatographie par échange d'ions (Cl) pour déterminer la teneur en nitrates des légumes et des produits à base de légumes. Ces méthodes sont applicables aux échantillons dont la teneur est supérieure ou égale à 25 mg/kg.
Elles ont été validées sur des échantillons dopés et sur des échantillons contaminés naturellement de jus de betterave ayant une teneur en nitrates (fraction massique) de 194 mg/kg et 691 mg/kg, de purée de carottes ayant une teneur en nitrates (fraction massique) de 26 mg/kg et 222 mg/kg, et de laitue iceberg ayant une teneur en nitrates (fraction massique) de 623 mg/kg et 3 542 mg/kg.

Živila - Določevanje vsebnosti nitratov in/ali nitritov - 2. del: Metoda HPLC/IC za določevanje vsebnosti nitratov v zelenjavi in zelenjavnih proizvodih

Ta evropski standard določa metodo tekočinske kromatografije visoke ločljivosti (HPLC) in ionsko kromatografsko metodo (IC) za določevanje ravni nitrata v zelenjavi in zelenjavnih proizvodih. Ta metoda se uporablja za vzorce z vsebnostjo 25 mg/kg ali več.
Metoda je bila potrjena na naravno kontaminiranih in primešanih vzorcih, na primer soku rdeče pese z masnimi deleži nitrata 194 mg/kg in 691 mg/kg, korenjevim pirejem z masnimi deleži nitrata 26 mg/kg in 222 mg/kg ter ledenki z masnimi deleži nitrata 623 mg/kg in 3542 mg/kg.

General Information

Status
Withdrawn
Publication Date
30-Mar-2018
Withdrawal Date
29-Jun-2018
Current Stage
9020 - Submission to 2 Year Review Enquiry - Review Enquiry
Start Date
15-Jan-2023
Completion Date
15-Jan-2023

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SLOVENSKI STANDARD
SIST EN 12014-2:2018
01-februar-2018
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SIST EN 12014-2:1999

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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
SIST EN 12014-2:2018 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 12014-2:2018
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SIST EN 12014-2:2018
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.
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SIST EN 12014-2:2018
EN 12014-2:2017 (E)
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

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SIST EN 12014-2:2018
EN 12014-2:2017 (E)
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.
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SIST EN 12014-2:2018
EN 12014-2:2017 (E)
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.

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SIST EN 12014-2:2018
EN 12014-2:2017 (E)
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.
4.3 Sulfuric acid, 96 % (ρ = 1,84 g/ml ).
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
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SIST EN 12014-2:2018
EN 12014-2:2017 (E)
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 analytical separating column.

5.7 IC apparatus.

5.7.1 Ion-exchange chromatography unit, consisting of an eluent reservoir, a pump, a pulsation

damper, a dialysis unit (if required), a suppressor (if required), a sample applicator with a sample loop,

a conductivity detector or UV detector adjustable from 190 nm to 800 nm and an evaluation unit.

5.7.2 Analytical separating column.
Examples of suitable separating columns are given in Annex D.

Use a pre-column with filling material of the same type to protect the analytical separating column.

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SIST EN 12014-2:2018
EN 12014-2:2017 (E)
6 Procedure
6.1 Sample preparation
6.1.1 Liquid samples, e.g. vegetable juice

Vigorously shake a representative sample until it is thoroughly mixed. If required, filter the sample

through a fluted filter paper (5.3) or, if necessary, predilute it with water to achieve a nitrate

concentration of approximately 25 mg/l in the solution.
6.1.2 Solid samples, e.g. leaf vegetable

Take a representative sample and remove adhering animals, soil or dirty particles. If required, remove

any inedible or damaged external leaves. Shred the sample prepared in this way, e.g. in a laboratory

cutter (5.1) and thoroughly homogenize it using a homogenizer (5.2). Analyse the sample immediately

after homogenization.

The requirements of the European legislation shall be taken into account, e.g. [1], [2] . If immediate

testing of the sample homogenate is not possible, immediately freeze the sample. For analysis, thaw the

sample as gently as possible (e.g. overnight in the refrigerator).

If it is not possible to homogenize a fresh produced sample within 24 h, the whole sample should be

frozen and analysed within a maximum of 6 weeks.
6.1.3 Pasty samples, e.g. mashed vegetables
Repeatedly stir a representative sample, e.g. by using a ho
...

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