Dansk standard DS/EN 14663 1. udgave 2006-01-16 Fødevarer Bestemmelse af vitamin B6 (inklusive glykosylerede former) ved HPLC Foodstuffs Determination of vitamin B6 (including its glycosylated forms) by HPLC
DS/EN 14663 København DS projekt: 52724 ICS: 67.050 Første del af denne publikations betegnelse er: DS/EN, hvilket betyder, at det er en europæisk standard, der har status som dansk standard. Denne publikations overensstemmelse er: IDT med: EN 14663:2005. DS-publikationen er på engelsk. DS-publikationstyper Dansk Standard udgiver forskellige publikationstyper. Typen på denne publikation fremgår af forsiden. Der kan være tale om: Dansk standard standard, der er udarbejdet på nationalt niveau, eller som er baseret på et andet lands nationale standard, eller standard, der er udarbejdet på internationalt og/eller europæisk niveau, og som har fået status som dansk standard DS-information publikation, der er udarbejdet på nationalt niveau, og som ikke har opnået status som standard, eller publikation, der er udarbejdet på internationalt og/eller europæisk niveau, og som ikke har fået status som standard, fx en teknisk rapport, eller europæisk præstandard DS-håndbog samling af standarder, eventuelt suppleret med informativt materiale DS-hæfte publikation med informativt materiale Til disse publikationstyper kan endvidere udgives tillæg og rettelsesblade DS-publikationsform Publikationstyperne udgives i forskellig form som henholdsvis fuldtekstpublikation (publikationen er trykt i sin helhed) godkendelsesblad (publikationen leveres i kopi med et trykt DS-omslag) elektronisk (publikationen leveres på et elektronisk medie) DS-betegnelse Alle DS-publikationers betegnelse begynder med DS efterfulgt af et eller flere præfikser og et nr., fx DS 383, DS/EN 5414 osv. Hvis der efter nr. er angivet et A eller Cor, betyder det, enten at det er et tillæg eller et rettelsesblad til hovedstandarden, eller at det er indført i hovedstandarden. DS-betegnelse angives på forsiden. Overensstemmelse med anden publikation: Overensstemmelse kan enten være IDT, EQV, NEQ eller MOD IDT: Når publikationen er identisk med en given publikation. EQV: Når publikationen teknisk er i overensstemmelse med en given publikation, men præsentationen er ændret. NEQ: Når publikationen teknisk eller præsentationsmæssigt ikke er i overensstemmelse med en given standard, men udarbejdet på baggrund af denne. MOD: Når publikationen er modificeret i forhold til en given publikation.
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM EN 14663 December 2005 ICS 67.050 English Version Foodstuffs - Determination of vitamin B6 (including its glycosylated forms) by HPLC Produits alimentaires - Dosage de la vitamine B6 (y compris ses formes glycosylées) par CLHP This European Standard was approved by CEN on 26 October 2005. Lebensmittel - Bestimmung von Vitamin B6 (einschließlich glucosidisch gebundener Verbindungen) mit HPLC 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 14663:2005: E
EN 14663:2005 (E) Contents Page Foreword...3 1 Scope...4 2 Normative references...4 3 Principle...4 4 Reagents...4 5 Apparatus...9 6 Procedure...9 7 Calculation...11 8 Precision...12 9 Test report...14 Annex A (informative) Precision data...15 Annex B (informative) Examples for suitable HPLC-conditions for the determination of vitamin B 6 compounds...19 Annex C (informative) Examples for molar extinction coefficients...20 Annex D (informative) Figures...21 Bibliography...22 Figure Figure D.1 Standard substances and sample potato puree...21 Tables Table 1 Examples for molecular extinction coefficients of vitamin B6 compounds...7 Table A.1 Precision data for Semolina with milk, powder...15 Table A.2 Precision data for Potato puree,powder...16 Table A.3 Precision data for vegetables with ham (baby food)...17 Table A.4 Precision data for multi vitamin drink...18 Table B.1 Examples for suitable HPLC-conditions for the determination of vitamin B6 compounds...19 Table C.1 Examples for molar extinction coefficients (E) of vitamin B6 compounds [3], [4]...20 2
EN 14663:2005 (E) Foreword This document (EN 14663:2005) 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 June 2006, and conflicting national standards shall be withdrawn at the latest by June 2006. 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. 3
EN 14663:2005 (E) 1 Scope This document specifies a method for the determination of vitamin B 6 in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B 6 is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives as well as the β-glycosylated forms, calculated as pyridoxine. This method has been successfully validated with semolina with milk (infant food), potato puree, vegetables with ham (convenient products) and a multi vitamin drink at levels from 0,034 mg/100 g to 1,21 mg/100 g. 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 (ISO 3696:1987). 3 Principle Pyridoxal, pyridoxamine and pyridoxine are extracted from food by acid hydrolysis and dephosphorylated and deglycosilated enzymatically using acid phosphatase and β-glucosidase. The different derivatives of vitamin B 6 (pyridoxal, pyridoxamine and pyridoxine) are separated by HPLC and quantified by fluorometric detection [1], [2]. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at least grade 1 according to EN ISO 3696, or double distilled water. 4.2 Di-potassium hydrogen phosphate, mass fraction w(k 2 HPO 4 3 H 2 O) 99,9 % 4.3 Sodium acetate, without crystal water, w(ch 3 COONa) 99,0 % 4.4 Trichloroacetic acid (TCA), w(cl 3 CCOOH) 99,0 % 4.5 Sodium acetate solution, substance concentration c(ch 3 COONa) = 2,5 mol/l Dissolve 205 g of sodium acetate (4.3) in 1 l of water. 4.6 Post-column reagent (optional), K 2 HPO 4 solution c(k 2 HPO 4 ) = 0,15 mol/l Dissolve 34,2 g of di-potassium hydrogen phosphate (4.2) in water, dilute to 1 000 ml, mix and degas. 4