EN 12014-3:2005

Foodstuffs - Determination of nitrate and/or nitrite content - Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymatic reduction of nitrate to nitrite

This European Prestandard specifies a spectrometric method for the determination of nitrate and nitrite content of meat products and has been validated for a total nitrite and nitrate content of 25 mg/kg as nitrite ion. Note: Experiences have shown that the methods is also applicable for total nitrite and nitrate content for 10 mg/kg up to 50 mg/kg as nitrite ion. For further information on applicability, see (1).

Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes - Teil 3: Spektralphotometrische Bestimmung des Nitrat- und Nitritgehaltes in Fleischerzeugnissen nach enzymatischer Reduktion von Nitrat zu Nitrit

Diese Europäische Norm legt ein spektralphotometrisches Verfahren zur Bestimmung des Nitrat- und Nitritgehaltes von Fleischerzeugnissen fest und ist für verschiedene Fleischerzeugnisse mit Gehalten von 9 mg/kg bis 22 mg/kg an Nitrit, berechnet als Natriumnitrit, und 23 mg/kg bis 48 mg/kg an Nitrat, berechnet als Natriumnitrat, im Ringversuch geprüft worden.
ANMERKUNG   Erfahrungen haben gezeigt, dass das Verfahren auch auf Gesamtnitrat/-nitritgehalte von 5 mg/kg bis 125 mg/kg (berechnet als Natriumnitrit) angewendet werden kann. Weitere Informationen zur Anwendbarkeit siehe [1].

Produits alimentaires - Détermination de la teneur en nitrates et/ou en nitrites - Partie 3 : Détermination spectrométrique de la teneur en nitrates et en nitrites des produits carnés après réduction enzymatique des nitrates en nitrites

Cette Norme européenne spécifie une méthode de détermination spectrométrique de la teneur en nitrates et en nitrites dans les produits carnés, et a été validée pour différents produits carnés pour une teneur en nitrites comprise entre 9 mg/kg et 22 mg/kg, exprimée en nitrite de sodium et une teneur en nitrates comprise entre 23 mg/kg et 48 mg/kg, exprimée en nitrate de sodium.
NOTE   Des expériences ont montré que cette méthode est aussi applicable pour une teneur totale en nitrites et en nitrates allant de 5 mg/kg à 125 mg/kg, exprimée en nitrite de sodium. Pour de plus amples informations sur son applicabilité, se reporter au [1].

Živila - Določevanje nitratov in/ali nitritov - 3. del: Spektrometrijsko določevanje nitratov in nitritov v mesnih proizvodih po encimski redukciji nitratov v nitrite

General Information

• Status: Published
• Publication Date: 17-May-2005
• Withdrawal Date: 29-Nov-2005
• ICS: 67.120.10 - Meat and meat products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 7 - Nitrate, nitrite
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 24-Sep-2025
• Completion Date: 08-Oct-2025
• Ref Project: SIST EN 12014-3:2005 - Foodstuffs - Determination of nitrate and/or nitrite content - Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymatic reduction of nitrate to nitrite

Relations

• Replaces: ENV 12014-3:1998 - Foodstuffs - Determination of nitrate and/or nitrite content - Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymatic reduction of nitrate to nitrite
• Effective Date: 22-Dec-2008

Overview

EN 12014-3:2005 (CEN) defines a spectrometric (spectrophotometric) method for determining nitrate and nitrite in meat products by enzymatically reducing nitrate to nitrite and measuring the resulting coloured azo dye at 540 nm. The method uses nitrate reductase with NADPH to convert nitrate to nitrite, followed by the sulfanilamide / N-(1‑naphthyl)-ethylenediamine diazotization (Griess-type) reaction. Results for nitrate are obtained by calculating the difference between total nitrite after reduction and native nitrite.

The standard has been validated for typical meat matrices and reports specific validated ranges (examples in the standard include nitrite 9–22 mg/kg as sodium nitrite and nitrate 23–48 mg/kg as sodium nitrate). The document also notes validation at a total nitrite/nitrate level of 25 mg/kg (as nitrite ion) and experience-based applicability across a wider range (reported within the standard as approximately 5–125 mg/kg depending on expression and matrix). Annex A provides precision data.

Key topics and technical requirements

• Principle: Enzymatic reduction (nitrate → nitrite) followed by colour development and spectrophotometric measurement at 540 nm.
• Sample preparation: Homogenization, alkaline extraction (pH ~8.0–8.5), heating, Carrez clarification (Carrez solutions No.1 and No.2) and filtration to obtain a clear aqueous extract.
• Reagents: nitrate reductase (aspergillus sp.), NADPH, sulfanilamide and N‑(1‑naphthyl)ethylenediamine dihydrochloride colour reagents, sodium nitrite and potassium nitrate standards, and appropriate buffer systems (imidazole buffer).
• Calibration: Two calibration graphs - one for nitrite (direct) and one for nitrate after enzymatic reduction; quantification by comparing absorbance values and calculating nitrate by difference.
• Equipment: Spectrometer (540 nm), pH meter, homogenizer, water bath, fluted filter papers.
• Quality / traceability: Uses EN ISO 3696 water quality and includes precision/validation data and procedural details for reproducibility.

Applications and users

• Food testing laboratories performing regulatory or compliance testing of cured meats, sausages and other meat products for nitrate/nitrite content.
• Meat processors and QC teams monitoring additive levels for product safety and labeling.
• Regulatory authorities and public‑health laboratories enforcing legal limits and investigating nitrosation risks.
• Research laboratories studying nitrate/nitrite chemistry in food matrices.

Keywords: EN 12014-3:2005, nitrate determination, nitrite determination, meat products, spectrometric method, enzymatic reduction, nitrate reductase, spectrophotometry, food analysis, CEN.

Related standards

• EN 12014 series (Part 1: general; Part 2: HPLC/IC for vegetables; Part 4: IC for meat; Part 5: enzymatic for baby foods; Part 7: continuous flow after Cd reduction).
• EN ISO 3696 (Water for analytical laboratory use) - normative reference used in the method.

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes - Teil 3: Spektralphotometrische Bestimmung des Nitrat- und Nitritgehaltes in Fleischerzeugnissen nach enzymatischer Reduktion von Nitrat zu NitritProduits alimentaires - Détermination de la teneur en nitrates et/ou en nitrites - Partie 3 : Détermination spectrométrique de la teneur en nitrates et en nitrites des produits carnés apres réduction enzymatique des nitrates en nitritesFoodstuffs - Determination of nitrate and/or nitrite content - Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymatic reduction of nitrate to nitrite67.120.10Meso in mesni proizvodiMeat and meat products67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 12014-3:2005SIST EN 12014-3:2005en01-september-2005SIST EN 12014-3:2005SLOVENSKI
STANDARDSIST ENV 12014-3:19991DGRPHãþD

EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 12014-3
May 2005 ICS 67.120.10 Supersedes ENV 12014-3:1998 English version
Foodstuffs - Determination of nitrate and/or nitrite content - Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymatic reduction of nitrate to nitrite
Produits alimentaires - Détermination de la teneur en nitrates et/ou en nitrites des produits carnés - Partie 3: Détermination spectrométrique de la teneur en nitrates et en nitrites après réduction enzymatique des nitrates en nitrites
Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes -Teil 3: Spektralphotometrische Bestimmung des Nitrat- und Nitritgehaltes in Fleischerzeugnissen nach enzymatischer Reduktion von Nitrat zu Nitrit This European Standard was approved by CEN on 1 April 2005.
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 Central Secretariat 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 Central Secretariat has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
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B-1050 Brussels © 2005 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12014-3:2005: ESIST EN 12014-3:2005

Precision data.12 Bibliography.14
The nitrate content is calculated from the difference between the spectrometric measurements. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents and materials of recognized analytical grade and water of at least grade 3 according to EN ISO 3696. When preparing solutions, the purities of the reagents available shall be taken into account. 4.2 Sodium hydroxide solution c(NaOH) ≈ 1 mol/l1)
4.3 Carrez solution No. 1 Dissolve 150 g of potassium hexacyanoferrate(II), K4[Fe(CN)6] ⋅ 3 H2O in water and dilute to 1 000 ml. Store the solution in a brown bottle and replace it every two months. 4.4 Carrez solution No. 2 Dissolve 230 g of zinc acetate, Zn (CH3COO)2 ⋅ 2 H2O, in water and dilute to 1 000 ml.
1) c is the substance concentration SIST EN 12014-3:2005

Dissolve 125 mg of reduced ß-nicotinamide adenine dinucleotide phosphate and 2,5 mg of disodium salt of flavin adenin dinucleotide (mass fraction of at least 88 %) in 50 ml of the imidazole buffer solution (4.5.1). Prepare this solution immediately before use. 4.5.3 Nitrate reductase buffer solution
Dissolve 11 mg of nitrate reductase lyophilisate from aspergillus sp. (EC 1.6.6.2, approximately 0,4 U/mg3)) in 10 ml of imidazole buffer solution (4.5.1) and add 50 mg of ethylenedintrilotetraacetic acid disodium salt-dihydrate.
The solution will be stable for about 14 days if stored at 4 °C. 4.6 Colour reagents 4.6.1 Colour reagent No. 1 Dissolve 8 g of sulfanilamide in 500 ml of water while heating on a water bath (5.3). Cool the solution to room temperature and filter if necessary. Add 330 ml of hydrochloric acid (4.9) while stirring continuously and dilute the solution to 1 000 ml with water. The solution will be stable for several weeks at room temperature. 4.6.2 Colour reagent No. 2 Dissolve 0,330 g of N-(1-naphthyl)ethylenediamine dihydrochloride in 250 ml of water. Keep the solution in a brown bottle and replace it every week. 4.6.3 Colour reagent mixture Prepare the required amount of colour reagent mixture by mixing equal parts by volume of colour reagents No. 1 (4.6.1) and No. 2 (4.6.2) on the day of use. 4.7 Sodium nitrite solutions 4.7.1 General It is recommended to prepare sodium nitrite standard solutions of sodium nitrite on the day of use.
2) The ‘Nitrat’ test kit for the enzymatic determination of nitrate and nitrite is the trade name of a product produced by Boehringer Mannheim/Roche Diagnostics, Mannheim, supplied by r-biopharm, Darmstadt (Germany) This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. 3) U, this unit (often called the International unit or standard unit) is defined as the amount of enzyme which catalyses the transformation of 1 µmol substrate per minute under standard conditions. SIST EN 12014-3:2005
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