EN 12014-4-2005 en Foodstuffs - Determination of nitrate and or nitrite content - Part 4 Ion-exchange chromatographic (IC) method for the determination of nitrate and nitrite conte.pdf

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1、BRITISH STANDARD BS EN 12014-4:2005 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 The European Standard EN 12014-4:2005 has the status of a British Standard ICS 67.120.

2、10 BS EN 12014-4:2005 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2005 BSI 30 June 2005 ISBN 0 580 46248 X National foreword This British Standard is the official English language version of EN 12014-4:2005. It supersedes DD ENV 1

3、2014-4:1998 which is withdrawn. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Food analysis horizontal methods, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references

4、The British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Stand

5、ards Online. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the

6、responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside fron

7、t cover, the EN title page, pages 2 to 16, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORMEN 12014-4 May

8、 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 - Dtermination de la teneur en n

9、itrates et/ou en nitrites - Partie 4: Dtermination par chromatographie ionique (CI) de la teneur en nitrates et en nitrites dans les produits base de viande Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes - Teil 4: Ionenchromatographisches Verfahren (IC) fr die Bestimmung des Nitrat- u

10、nd 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

11、-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 und

12、er 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, Ic

13、eland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassa

14、rt, 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 12014-4:2005: EEN 12014-4:2005 (E) 2 Contents Page Foreword3 1 Scope 4 2 Normative references 4 3 Principle4 4 Reagents.4 5 Apparatus and equipment 6 6 Pro

15、cedure .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 data11 Annex C (informative) Further applicability13 Bibliography 16EN 12014-4:2005 (E) 3 Foreword This European Standard (EN 12014-4:

16、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 20

17、05, 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

18、 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 c

19、ontent 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

20、 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, Netherlan

21、ds, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. 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

22、 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 a

23、nd 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, onl

24、y 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 wi

25、th 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 an

26、alysis, 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 l

27、ithium hydroxide monohydrate 4.5 Boric acid, having a mass fraction of 99 % 4.6 Hydrochloric acid, c (HCl) = 1,8 mol/l 1) 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 hydr

28、ochloric acid (4.6) in a 100 ml volumetric flask to the mark with water and mix. 1) c is the substance concentration 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 t

29、he 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)

30、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 th

31、e day of use. 4.10 Gluconic acid solution 2) , 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

32、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 c

33、ompletely 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 50

34、0 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 m (5.6)

35、. 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. EN 12014-4:2005 (E) 6 5 Apparatus and equipment 5.1 General Usual laborat

36、ory 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 s

37、tirrer (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 m. 5.6 Membrane filter, for aqueous and acetonitrile solution

38、s, with a pore size of approximately 0,22 m. 5.7 Filter holder for membrane filter, with suitable syringe. 5.8 pH meter 5.9 Solid phase extraction column RP C 18 , with suitable syringe (optional). 5.10 IC apparatus, comprising the following 5.10.1 Isocratic high performance liquid chromatography un

39、it, 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

40、 10 m, capacity (30 3) 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

41、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

42、 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 wa

43、ter. Add 50 ml of acetonitrile (4.2). Mix gently. Allow to cool to room temperature. Dilute to the mark with water. EN 12014-4:2005 (E) 7 Filter first through the fluted filter paper (5.4) and then through a membrane filter of pore size of approximately 0,45 m (5.5). If this solution is clear, it ma

44、y be injected. If the solution is still not clear, filter through a membrane filter of pore size of approximately 0,22 m (5.6) (Sample test solution). Prepare a blank replacing the test portion by 10 ml of water. 6.3 Preparation of the calibration graph To plot a calibration graph, inject equal volu

45、mes of the standard solutions (4.9) first and then the blank solution (prepared according to 6.2) under the conditions as described in 6.4.1. Check the linearity of the calibration graph. 6.4 Determination 6.4.1 IC operating conditions If the column specified in 5.10.2 is used, it has been found sat

46、isfactory to adopt the following parameters. Mobile phase: as described in 4.12 UV: 205 nm Injection volume: 40 l (minimum) Flow rate: 1 ml/min If other columns than the one described in 5.10.2 are used, adjust chromatographic conditions. 6.4.2 IC measurement Inject the standard solutions (4.9) firs

47、t and then the blank and the sample test solutions under the conditions as described in 6.4.1. One of the standard solutions should be injected every five sample test solutions when performing a series of analyses. If the peak obtained for the sample falls outside the range of the calibration graph,

48、 dilute the sample test solution in the mobile phase and repeat the measurement step. Identify the nitrate or nitrite peak by comparing the retention times for the standard solutions (4.9) and the sample test solutions (6.2). Read off the content of nitrate or nitrite of the sample test solution fro

49、m the calibration graph. Check the blank value to ensure that there was no nitrate and/or nitrite contamination during the sample preparation. 6.5 Expression of results Calculate the mass fraction of nitrite, ) (NO - 2 w , expressed in milligrams of ion per kilogram with Equation (1): F m A w = ) NO ( ) NO ( - 2 - 2 200 (1) where: EN 12014-4:2005 (E) 8 ) (NO - 2 A is the value for nitrite, read off the calibration graph, in milligrams per litre; 200 is the volume