EN 12014-3:2005

• Abstract:

  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).

• 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: 17-Sep-2026
• 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

• 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
• Refers: EN ISO 3696:1995 - Water for analytical laboratory use - Specification and test methods (ISO 3696:1987)
• Effective Date: 28-Jan-2026

Overview

EN 12014-3:2005 is a European Standard developed by CEN that specifies a spectrometric method for the determination of nitrate and nitrite content in meat products. The method is based on enzymatic reduction of nitrate to nitrite followed by spectrometric measurement and is validated for content ranging from 10 mg/kg up to 125 mg/kg (as nitrite ion). This standard ensures reliable measurement for food safety testing and regulatory compliance concerning nitrates and nitrites in processed meat products.

Key Topics

• Spectrometric analysis: Utilizes absorbance measurements at 540 nm after color-forming reactions specific to nitrites.
• Enzymatic reduction: Employs nitrate reductase to convert nitrate in meat products to nitrite prior to measurement.
• Sample preparation: Includes homogenization, pH adjustment, clarification, and filtration to ensure analytical accuracy.
• Calibration and evaluation: Provides procedures for preparing calibration graphs and calculating the concentrations of nitrite and nitrate.
• Precision and repeatability: The method demonstrates high precision, with well-defined repeatability and reproducibility limits, making it suitable for laboratory use.
• Test reporting: Defines essential elements for laboratory reporting, such as sample identification, reference to the standard, results, and procedural notes.

Applications

EN 12014-3:2005 is vital for:

• Food safety laboratories: Ensuring meat products comply with legal limits for nitrate and nitrite content set by food safety authorities.
• Quality control in meat processing: Allowing producers to monitor and control nitrite and nitrate levels during manufacturing.
• Regulatory enforcement: Enabling government agencies to inspect and verify compliance with food safety regulations.
• Research and development: Supporting studies on the impact of curing agents on meat products and consumer health.
• International trade: Providing a harmonized method accepted across Europe, facilitating the movement and certification of meat products.

The analytical range (10–125 mg/kg as nitrite ion) encompasses the most relevant concentration levels found in a wide variety of meat products, covering both raw and cooked samples including sausages, hams, and specialty items.

Related Standards

EN 12014-3:2005 is part of a series of standards dedicated to the determination of nitrate and nitrite in foodstuffs, including:

• EN 12014-1: General considerations for nitrate and nitrite determination in foodstuffs.
• EN 12014-2: HPLC/IC method for nitrate content in vegetables and related products.
• EN 12014-4: Ion-exchange chromatographic method for nitrate and nitrite in meat products.
• EN 12014-5: Enzymatic determination of nitrate in vegetable-containing food for babies and infants.
• EN 12014-7: Continuous flow method for nitrate in vegetables after cadmium reduction.

These related standards provide alternative and complementary analytical techniques, broadening the applicability of nitrate and nitrite testing in various food matrices, thus supporting a comprehensive approach to food quality assurance and consumer protection.

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Keywords: EN 12014-3:2005, nitrate content determination, nitrite content measurement, spectrometric method, meat products, enzymatic reduction, food safety, food analysis standards, CEN, nitrite in foods, nitrate in processed meat, laboratory testing method.

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
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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
Management Centre: rue de Stassart, 36
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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