SIST EN 12014-4:2005

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.YRGLKLebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes - Teil 4: Ionenchromatographisches Verfahren (IC) für die Bestimmung des Nitrat- und Nitritgehaltes in FleischerzeugnissenProduits alimentaires - Détermination de la teneur en nitrates et/ou en nitrites - Partie 4 : Détermination par chromatographie ionique (CI) de la teneur en nitrates et en nitrites dans les produits carnésFoodstuffs - Determination of nitrate and/or nitrite content - Part 4: Ion-exchange chromatographic (IC) method for the determination of nitrate and nitrite content of meat products67.120.10Meso 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 12014-4:2005SIST EN 12014-4:2005en01-september-2005SIST EN 12014-4:2005SLOVENSKI
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SIST EN 12014-4:2005



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 12014-4
May 2005 ICS 67.120.10 Supersedes ENV 12014-4:1998 English version
Foodstuffs - Determination of nitrate and/or nitrite content - Part 4: Ion-exchange chromatographic (IC) method for the determination of nitrate and nitrite content of meat products
Produits alimentaires - Détermination de la teneur en nitrates et/ou en nitrites - Partie 4: Détermination par chromatographie ionique (CI) de la teneur en nitrates et en nitrites dans les produits à base de viande
Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes - Teil 4: Ionenchromatographisches Verfahren (IC) für die Bestimmung des Nitrat- und Nitritgehaltes in Fleischerzeugnissen 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 12014-4:2005: ESIST EN 12014-4:2005



EN 12014-4:2005 (E) 2 Contents Page Foreword.3 1 Scope.4 2 Normative references.4 3 Principle.4 4 Reagents.4 5 Apparatus and equipment.6 6 Procedure.6 7 Precision.8 Annex A (informative)
Example for an IC chromatogram of corned beef
(Sample from the interlaboratory test).10 Annex B (informative)
Precision data.11 Annex C (informative)
Further applicability.13 Bibliography.16
SIST EN 12014-4:2005



EN 12014-4:2005 (E) 3 Foreword This European Standard (EN 12014-4:2005) 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 November 2005, and conflicting national standards shall be withdrawn at the latest by November 2005. This document supersedes ENV 12014-4:1998. This series Foodstuffs - Determination of nitrate and/or nitrite content consist of the following parts: Part 1: General considerations; Part 2: HPLC/IC method for the determination of nitrate content of vegetables and vegetable products; Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymatic reduction of nitrate to nitrite; Part 4: Ion-exchange chromatographic (IC) method for the determination of nitrate and nitrite content of meat products; Part 5: Enzymatic determination of nitrate content of vegetable-containing food for babies and infants; Part 7: Continuous flow method for the determination of nitrate content of vegetables and vegetable products after Cadmium reduction. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: 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. SIST EN 12014-4:2005



EN 12014-4:2005 (E) 4 1 Scope This European Standard specifies an ion-exchange chromatographic method for the determination of the nitrate and nitrite contents of meat products and has been validated for different meat products with a nitrate content of 50 mg/kg to 300 mg/kg as nitrate ion and a nitrite content of approximately 40 mg/kg as nitrite ion. NOTE Validation data obtained from interlaboratory studies show that this method may also be applied to the determination of nitrate in vegetables and baby food, see [1], [2]. Furthermore, the method may be applied for the determination of nitrite in meat products having a nitrite content greater than 40 mg/kg. 2 Normative references The following referenced documents are indispensable for the application 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. EN ISO 3696, Water for analytical laboratory use – Specification and test methods (IS0 3696:1987) 3 Principle Nitrate and nitrite are extracted from the test sample with hot water. The aqueous solution is treated with acetonitrile to remove any interfering substance. The nitrate and nitrite contents of the solution are then determined by ion-exchange chromatography (IC) and ultraviolet (UV) detection at a wavelength of 205 nm. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents 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 Acetonitrile 4.3 Glycerol 4.4 Lithium hydroxide, anhydrous, or lithium hydroxide monohydrate 4.5 Boric acid, having a mass fraction of 99 % 4.6 Hydrochloric acid, c (HCl) = 1,8 mol/l1)
Dilute 15 ml of hydrochloric acid of 20 (HCl) = 1,18 g/ml in a 100 ml volumetric flask to the mark with water and mix. 4.7 Hydrochloric acid, c (HCl) ≈ 0,1 mol/l Dilute 5 ml of hydrochloric acid (4.6) in a 100 ml volumetric flask to the mark with water and mix.
1) c is the substance concentration SIST EN 12014-4:2005



EN 12014-4:2005 (E) 5 4.8 Stock solution of nitrate and nitrite Dissolve 1,500 g of sodium nitrite and 3,258 g of potassium nitrate in a 1000 ml volumetric flask in water, dilute to the mark with water and mix well. The stock solution may be used for 2 weeks if stored in a refrigerator at 4 °C. Sodium nitrite is a hygroscopic substance. 4.9 Standard solutions of nitrate and nitrite Pipette 0 ml, 0,1 ml, 0,5 ml, 1,0 ml, 1,5 ml and 2,0 ml, respectively, of the stock solution (4.8) into six 200 ml volumetric flasks, dilute to the mark with water and mix. 1 l of these solutions contains 0 mg, 0,5 mg, 2,5 mg, 5,0 mg, 7,5 mg and 10 mg of nitrite ion and 0 mg, 1 mg, 5 mg, 10 mg, 15 mg and 20 mg of nitrate ion, respectively. It is recommended to prepare the standard solutions on the day of use. 4.10 Gluconic acid solution2), optionally clarified on solid phase extraction columns (5.9) Dilute 50 g of gluconic acid in a 100 ml volumetric flask to the mark with demineralized water and mix.
NOTE Gluconic acid sometimes has a dark brown colour. In this case it is recommended to clarify it until a slight yellow colour is obtained. Generally two cartridges activated with methanol are necessary. 4.11 Lithium borate gluconate buffer solution To 500 ml of water in a 1000 ml volumetric flask add 34,00 g of boric acid (4.5) and 19,6 ml of gluconic acid solution (4.10). Dissolve completely in the solution 11,00 g of anhydrous lithium hydroxide or 19,26 g of lithium hydroxide monohydrate (4.4). Add 125 ml of glycerol (4.3), dilute to the mark with water and mix well. This solution is stable for 6 months if stored in a refrigerator at approximately 4 °C. 4.12 Mobile phase To 500 ml of water in a 1000 ml volumetric flask, add 17 ml of buffer solution (4.11) and 125 ml of acetonitrile (4.2). Dilute to the mark with water. Mix well. Adjust the pH to 6,5 ± 0,1 by adding hydrochloric acid (4.6 then 4.7). Filter through a membrane filter of pore size of approximately 0,22 µm (5.6). This solution is stable for not longer than one week and the pH value shall be checked every day before use. If the pH value falls outside the stated range, prepare a new solution.
2) This solution is commercially available. SIST EN 12014-4:2005



EN 12014-4:2005 (E) 6 5 Apparatus and equipment 5.1 General Usual laboratory apparatus and, in particular, the following: 5.2 Homogenizing equipment, mechanical or electrical, capable of homogenizing the test sample, this includes a high-speed rotational cutter, or a mincer fitted with a plate with holes not exceeding 4,5 mm in diameter, and a homogenizer.
5.3 Magnetic stirrer (optional) 5.4 Fluted filter paper (nitrate/nitrite free), care is needed in the choice of filter paper as some can absorb nitrate. 5.5 Membrane filter, for aqueous and acetonitrile solutions, with a pore size of approximately 0,45 µm. 5.6 Membrane filter, for aqueous and acetonitrile solutions, with a pore size of approximately 0,22 µm. 5.7 Filter holder for membrane filter, with suitable syringe. 5.8 pH meter
5.9 Solid phase extraction column RP C18, with suitable syringe (optional). 5.10 IC apparatus, comprising the following 5.10.1 Isocratic high performance liquid chromatography unit, with a UV detector, capable of measuring at a wavelength of 205 nm and an evaluation unit (e.g an integrator with plotter). 5.10.2 Analytical separating column, Anion exchanger, 4,6 mm × 150 mm, packing material; polymethacrylate resin with a quarternary ammonium functional group, particle size of 10 µm, capacity (30 ± 3) µ eq/ml with a precolumn, e.g. 20 mm, having the same packing to protect the analytical column. 6 Procedure
6.1 Sample preparation Homogenize the laboratory sample with the appropriate equipment (5.2). Take care that the temperature of the sample material does not rise above 25 °C. If a mincer is used, pass the sample at least twice through the equipment. Weigh, to the nearest 10 mg, 10 g of the homogenized sample into e.g. a 150 ml wide neck conical flask (test portion).
6.2 Extraction and clarification
The analysis should be performed in a single working day. Add 50 ml of water at a temperature of 50 °C to 60 °C to the test portion in the wide neck flask. Mix thoroughly with the homogenizer (5.2). Rinse the homogenizer with water and add the washings to the flask. Quantitatively transfer this slurry into a 200 ml volumetric flask by rinsing the wide neck flask with water.
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