1、August 2014 Translation by DIN-Sprachendienst.English price group 11No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS
2、 67.050!%:;English version EN 14164:2014,English translation of DIN EN 14164:2014-08Lebensmittel Bestimmung von Vitamin B6 mit Hochleistungs-Flssigchromatographie;Englische Fassung EN 14164:2014,Englische bersetzung von DIN EN 14164:2014-08Produits alimentaires Dtermination de la teneur en vitamine
3、B6 par chromatographie liquide hauteperformance;Version anglaise EN 14164:2014,Traduction anglaise de DIN EN 14164:2014-08SupersedesDIN EN 14164:2008-08www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 18 pages07.14 DIN EN 14164:2014-08 2
4、 A comma is used as the decimal marker. National foreword This document (EN 14164:2014) has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und
5、 landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-01-13 AA Vitamine und Carotinoide. The DIN Standards corresponding to the International Standards referred to in this document are as follows, whereby EN ISO Standards are only listed bel
6、ow if these have not been published as DIN EN ISO Standards with the same number: EN ISO 3696 DIN ISO 3696 ISO 5725 (all parts) DIN ISO 5725 (all parts) Amendments This standard differs from DIN EN 14164:2008-08 as follows: a) the standard has been editorially revised. Previous editions DIN V ENV 14
7、164: 2002-05 DIN EN 14164: 2008-08 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725 (all parts), Accuracy (trueness and precision) of measurement methods and results EN 14164June 2014 ICS 67.050 Supersedes EN 1
8、4164:2008English Version Foodstuffs - Determination of vitamin B6 by high performance chromatography Produits alimentaires - Dtermination de la teneur en vitamine B6 par chromatographie liquide haute performanceLebensmittel - Bestimmung von Vitamin B6 mit Hochleistungs-Flssigchromatographie This Eur
9、opean Standard was approved by CEN on 17 April 2014. 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 co
10、ncerning 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 CEN member
11、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 Macedonia, France
12、, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2014 CEN All rights o
13、f exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14164:2014 EEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGEN 14164:2014 (E) 2 Contents PageForeword
14、3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus .8 6 Procedure .8 7 Calculation 9 8 Test report . 10 Annex A (informative) Example of a chromatogram . 11 Annex B (informative) Precision data . 12 Annex C (informative) Sample treatment option without acid hydrolysis 14 An
15、nex D (informative) Examples for molar absorption coefficients . 15 Bibliography . 16 DIN EN 14164:2014-08EN 14164:2014 (E) 3 Foreword This document (EN 14164:2014) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This
16、 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 December 2014 and conflicting national standards shall be withdrawn at the latest by December 2014. Attention is drawn to the possibility that some of t
17、he elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 14164:2008. The Annexes A, B, C and D are informative. According to the CEN-CENELEC Internal Regulations, the
18、national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
19、 Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. WARNING The use of this European Standard can involve hazardous materials, operations and equipment. This European Standard does not purport t
20、o address all the safety problems associated with its use. It is the responsibility of the user of this European Standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. DIN EN 14164:2014-08EN 14164:2014 (E) 4 1 Scope This
21、European Standard specifies a method for the determination of vitamin B6in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B6is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives determined as pyridoxine. The -glycosyla
22、ted forms are not taken into account. These can be determined with the method given in EN 14663 1 by which the different vitamins of vitamin B6(pyridoxal, pyridoxamine and pyridoxine) are separated and individually quantified. A third European Standard, EN 14166 2, determines the total vitamin B6by
23、microbiological assay. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced docume
24、nt (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696) 3 Principle Pyridoxal, pyridoxamine and pyridoxine are extracted from food by acid hydrolysis and dephosphorylated enzymatically using acid phosphatase. By reaction wit
25、h glyoxylic acid in the presence of Fe2+as a catalyst, pyridoxamine is transformed into pyridoxal, which is then reduced to pyridoxine by the action of sodium borohydride in alkaline medium. Pyridoxine is then quantified in the sample solution by HPLC with a fluorometric detection 3, 4. 4 Reagents D
26、uring the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at least grade 1 according to EN ISO 3696, or double distilled water. 4.1 Acid phosphatase, (CAS 9001-77-8), from potatoes, enzyme activity is 33 nkat/mg1)with substrate p-nitrophenyl phosphate
27、 at pH = 4,8 and T = 37 C, for example from Boehringer or Sigma2). 33 nkat/mg corresponds to 2 U/mg. 4.1.1 Acid phosphatase solution Prepare a solution of 20 mg/ml acid phosphatase in sodium acetate solution (4.14). It is necessary to use acid phosphatase rather than Taka-diastase to obtain a comple
28、te hydrolysis of phosphorylated forms of vitamin B6. Where 300 mg of Taka-diastase is needed to obtain good dephosphorylation, only 0,5 mg of acid phosphatase is needed, see 5. 1)Katal (symbol kat) is a derived SI unit of enzyme activity. One katal is that catalytic activity which will raise the rat
29、e of reaction by one mol/s in a specified assay system. 2)This information is given for the convenience of users of this European standard and does not constitute an endorsement by CEN of the supplier. Equivalent products may be used if they can be shown to lead to the same results. DIN EN 14164:201
30、4-08EN 14164:2014 (E) 5 4.1.2 Activity check of acid phosphatase The activity of acid phosphatase can be checked as proposed below. Prepare a stock solution of approximately 0,1 mg/ml of pyridoxal phosphate (4.9) in water. Mix 3,0 ml of the PLP stock solution and 10 ml of hydrochloric acid (4.21) in
31、 a 20 ml volumetric flask and fill up to the mark with water. Check the concentration of PLP by measuring the absorbance at 293 nm in a 1 cm cell using a UV-spectrometer (5.1) against a hydrochloric acid solution (4.20) as reference. The molar absorption coefficient () of PLP in 0,1 mol/l HCl is 7 2
32、00. Calculate the mass concentration PLPof the stock solution, in milligram per millilitre, according to Formula (1): FMA=PLP293PLP(1) where A293is the absorption value of the solution at 293 nm; MPLPis the molar mass of vitamin B6standard substance, in gram per mol (MPLP= 247,14); F is the dilution
33、 factor (here F = 20/3); is the molar absorption coefficient of PLP in 0,1 mol/l of hydrochloric acid at 293 nm, in l mol1cm1, (here: = 7 200), see 6. Take 1,0 ml of the PLP stock solution for extraction and proceed with 6.2.1, 6.2.2, 6.2.3 and 6.2.4. Calculate the pyridoxine (PN) conversion rate fr
34、om the dephosphorylated pyridoxal phosphate solution according to Formula (2): PNPLPSTPLPSHClPN0001100822,01002(%) rate ConversionMAMA=(2) where PN HCLis the mass concentration of pyridoxine hydrochloride in the standard test solution, in micrograms per millilitre; ASis the peak area or peak height
35、for pyridoxine obtained with the sample test solution, in units of area or height; 2 is the factor of dilution of the reaction with sodium borohydride if acetic acid is added, otherwise the dilution factor is 1,9; 100 is the total volume of the sample test solution, in millilitre; 0,822 is the facto
36、r to convert pyridoxine hydrochloride to pyridoxine; 100 is the conversion factor for %; MPLPis the molar mass of pyridoxal phosphate (PLP), in gram per mol (MPLP= 247,14); ASTis the peak area or peak height for pyridoxine obtained with the standard test solution, in units of area or height; 1 000 i
37、s the factor to convert microgram to milligram; PLPis the mass concentration of pyridoxal phosphate (PLP) in the stock solution, in milligrams per millilitre; MPNis the molar mass of pyridoxine (PN), in gram per mol (MPN= 169,1). DIN EN 14164:2014-08EN 14164:2014 (E) 6 4.2 Sodium acetate, trihydrate
38、, mass fraction w(CH3COONa 3H2O) 99,0 %. 4.3 Glacial acetic acid, w(CH3COOH) 99,8 %. 4.4 Glyoxylic acid, w(C2H2O3 H2O) 97,0 %. 4.5 Ferrous sulfate II, heptahydrate, w(FeSO4 7H2O) 99,5 %. 4.6 Sodium hydroxide, w(NaOH) 99,0 %. 4.7 Sodium borohydride, w(NaBH4) 97,0 %. 4.8 Potassium dihydrogen phosphate
39、, w(KH2PO4) 99,0 %. 4.9 Pyridoxal phosphate (PLP), w 99,0 %. 4.10 Orthophosphoric acid, w(H3PO4) 84,0 %. 4.11 Sodium octanesulfonate, w(C8H17NaO3S) 98,0 %, or sodium heptanesulfonate, w (C7H15NaO3S) 98,0 %. 4.12 Acetonitrile (HPLC grade), w(C2H3N) 99,8 %. 4.13 Sodium acetate solution, substance conc
40、entration c(CH3COONa 3H2O) = 2,5 mol/l. Dissolve 170,1 g of sodium acetate, trihydrate (4.2) in 500 ml of water. 4.14 Sodium acetate solution, c(CH3COONa 3H2O) = 0,05 mol/l (pH = 4,5). Dissolve 6,8 g of sodium acetate, trihydrate (4.2) in 1 l of water. Adjust the pH to 4,5 with glacial acetic acid (
41、4.3). 4.15 Ferrous sulfate solution, c(FeSO4 7H2O) = 0,0132 mol/l. Dissolve 36,6 mg of ferrous sulfate II, heptahydrate (4.5) in 10 ml of sodium acetate solution (4.14). Prepare fresh each day of use. NOTE In a study described by Mann et al., see 7, a ferrous sulfate solution of 10 g/l was used. Thi
42、s concentration was based on the completion of the conversion of pyridoxamine to pyridoxal at pyridoxamine levels up to 8 times the minimum level of vitamin B6required by the infant formula Act in the US, see Mann et al. 8. This concentration seems not to be necessary for the European situation. 4.1
43、6 Sodium hydroxide solution, c(NaOH) = 0,2 mol/l. Dissolve 800 mg of sodium hydroxide (4.6) in 100 ml of water. 4.17 Sodium hydroxide solution, c(NaOH) = 6,0 mol/l. Dissolve 24 g of sodium hydroxide (4.6) in 100 ml of water. 4.18 Sodium borohydride solution, c(NaBH4) = 0,1 mol/l. Dissolve 378 mg of
44、sodium borohydride (4.7) in 100 ml of sodium hydroxide solution (4.16). Prepare fresh on day of use. 4.19 Glyoxylic acid solution, c(C2H2O3 H2O) = 1 mol/l (pH = 4,5). DIN EN 14164:2014-08EN 14164:2014 (E) 7 Dissolve 4,7 g of glyoxylic acid monohydrate (4.4) in 30 ml of sodium acetate solution (4.13)
45、. Adjust the pH to 4,5 with the sodium hydroxide solution (4.17) and dilute to 50 ml with water in a volumetric flask. Prepare fresh on day of use. 4.20 Hydrochloric acid, c(HCl) = 0,1 mol/l. 4.21 Hydrochloric acid, c(HCl) = 0,2 mol/l. 4.22 HPLC mobile phase In a beaker add 940 ml of water, 40 ml of
46、 acetonitrile (4.12), 160 mg of sodium octanesulfonate or sodium heptanesulfonate (4.11) and 6,8 g of potassium dihydrogen phosphate (4.8). After dissolving sodium octanesulfonate or sodium heptanesulfonate and potassium dihydrogen phosphate by stirring, adjust the pH to 2,5 with orthophosphoric aci
47、d (4.10). Transfer the solution in a 1 l volumetric flask. Adjust to the mark with water. Filter through a 0,45 m filter. 4.23 Pyridoxine hydrochloride (vitamin B6standard substance), w(C8H11NO3HCI) 99 %. 4.24 Pyridoxine hydrochloride stock solution, mass concentration 0,5 mg/ml. Dissolve an accurat
48、ely weighed amount of pyridoxine hydrochloride (4.23), e.g. 50 mg, in a defined volume, e.g. 100 ml, of water. The stock solution is stable for 4 weeks if stored at 4 C in the dark. For the concentration test, dilute 0,5 ml of pyridoxine hydrochloride stock solution (4.24) to 20 ml with 0,1 mol/l HC
49、I (4.20) and measure the absorbance at 290 nm in a 1 cm cell using a UV-spectrometer (5.1) against 0,1 mol/l HCl solution as reference. Calculate the mass concentration , in microgram per millilitre of the stock solution according to Formula (3): FMA=0001PNHCl290PNHCl(3) where A290is the absorption of the value of the solution at 290 nm; MPNHClis the molar mass of vitamin B6standard substance, i
