EN 12014-2-2017 en Foodstuffs - Determination of nitrate and or nitrite content - Part 2 HPLC IC method for the determination of nitrate content of vegetables and vegetable product.pdf

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1、BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06Foodstuffs - Determination of nitrate and/or nitrite contentPart 2: HPLC/IC method for the determination of nitrate content of vegetables and vegetable productsBS EN 12014-2:2017EUROPEAN STANDARD NORME EUROPENNE EUROPIS

2、CHE NORM EN 12014-2 December 2017 ICS 67.080.20 Supersedes EN 12014-2:1997English 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 - Dtermination de la ten

3、eur en nitrates et/ou en nitrites - Partie 2 : Mthode de dtermination par CLHP/CI de la teneur en nitrates des lgumes et des produits base de lgumes Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes - Teil 2: HPLC/IC-Verfahren fr die Bestimmung des Nitratgehaltes in Gemse und Gemseerzeug

4、nissen 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 bibliographi

5、cal 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

6、 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

7、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 COMIT EUROPEN DE NORMA

8、LISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12014-2:2017 ENational forewordThis British Standard is the UK implem

9、entation of EN 12014-2:2017. It supersedes BS EN 12014-2:1997, which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained on request to its secretary.

10、This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2018 Published by BSI Standards Limited 2018ISBN 978 0 580 94297 6ICS 67.080.20Compliance with a British Standard cannot confe

11、r immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 January 2018.Amendments/corrigenda issued since publicationDate Text affectedBRITISH STANDARDBS EN 12014-2:2017EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE N

12、ORM EN 12014-2 December 2017 ICS 67.080.20 Supersedes EN 12014-2:1997English 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 - Dtermination de la teneur e

13、n nitrates et/ou en nitrites - Partie 2 : Mthode de dtermination par CLHP/CI de la teneur en nitrates des lgumes et des produits base de lgumes Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes - Teil 2: HPLC/IC-Verfahren fr die Bestimmung des Nitratgehaltes in Gemse und Gemseerzeugnisse

14、n 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 r

15、eferences 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

16、 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 Maced

17、onia, 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 COMIT EUROPEN DE NORMALISAT

18、ION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12014-2:2017 EBS EN 12014-2:2017EN 12014-2:2017 (E) 2 Contents Page Europe

19、an 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

20、 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 quantitativ

21、e 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 .

22、15 Annex D (informative) Examples of suitable IC conditions 16 Bibliography . 18 BS EN 12014-2:2017EN 12014-2:2017 (E) 3 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

23、. 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 th

24、e 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

25、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 Ann

26、ex 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,

27、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, Sw

28、eden, Switzerland, Turkey and the United Kingdom. BS EN 12014-2:2017EN 12014-2:2017 (E) 4 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

29、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. BS EN 12014-2:2017EN 12014-2:2017 (E) 5 1 Scope This European Standard specifies a high-performance liquid ch

30、romatographic (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 w

31、ith 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

32、 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 tes

33、t 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 sh

34、all 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 % ( = 1,84 g/ml1). 4.4 Regenerator for suppressor, e.g. sulfuric acid (c = 0,0125 mol/l2). Carefully pipette 20 ml of sulfuric acid (4.3) into a 1 000 ml volumetric flask c

35、ontaining 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 solu

36、tion, = 1 000 mg/l (expressed as ion NO3-). 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. M

37、ix 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) = mass concentration2) c = substance concentrationBS EN 12014-2:20

38、17EN 12014-2:2017 (E) 6 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 filte

39、r 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:

40、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 m (e.g. regenerated cellulose). 5.5 Filter holder for membrane filter, with suitable syringe. 5.6

41、 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

42、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 sampl

43、e 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 an

44、alytical separating column. BS EN 12014-2:2017EN 12014-2:2017 (E) 7 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

45、, 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 sa

46、mple 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 3). If immediate testing of the sample homogen

47、ate 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 Pas

48、ty samples, e.g. mashed vegetables Repeatedly stir a representative sample, e.g. by using a homogenizer until it is thoroughly mixed. 6.2 Hot water extraction of samples (solid and pasty samples) From the sample prepared in accordance with 6.1.2 or 6.1.3, weigh approximately 10 g of material to the

49、nearest of 1 mg into a 500 ml conical flask. Add approximately 400 ml of hot water (approximately 80 C) and place the conical flask into a boiling water bath. When the water bath has reached boiling point temperature again, keep the flask there for 15 min. Cool to room temperature, transfer the solution into a 500 ml volumetric flask, dilute to the mark, shake thoroughly and filter the contents through a fluted filter paper (5.3). For sample material with low nitrate content, make appropriate adjustments, if necessary, to the initial test