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Vervangt NEN-EN 14122:2001 Ontw.
Nederlandse norm
NEN-EN 14122 (en)
Foodstuffs - Determination of vitamin B1 by HPLC
ICS 67.050
juli 2003
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NEN-EN 14122
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Als Nederlandse norm is aanvaard: - EN 14122:2003,IDT
Normcommissie 370 275 "Voedingsmiddelenanalyse - Horizontale methoden"
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Apart from exceptions provided by the law, nothing from this publication may be duplicated and/or published by means of photocopy, microfilm, storage in computer files or otherwise, which also applies to full or partial processing, without the written consent of the Netherlands Standardization Institute.
Although the utmost care has been taken with this publication, errors and omissions cannot be entirely excluded. The Netherlands Standardization Institute and/or the members of the committees therefore accept no liability, not even for direct or indirect damage, occurring due to or in relation with the application of publications issued by the Netherlands Standardization Institute.
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NEN-EN 14122
Nederlands voorwoord Voor de in deze norm vermelde normatieve verwijzingen bestaan in Nederland de volgende equivalenten: vermelde norm
Nederlandse norm
titel
EN ISO 3696:1995
NEN-EN-ISO 3696:1995
Water voor analytische laboratoriumdoeleinden Eisen en beproevingsmethoden (en)
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EUROPEAN STANDARD
EN 14122
NORME EUROPÉENNE EUROPÄISCHE NORM
May 2003
ICS 67.050
English version
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Foodstuffs - Determination of vitamin B1 by HPLC
Produits alimentaires - Dosage de la vitamine B1 par CLHP
Lebensmittel - Bestimmung von Vitamin B1 mit HPLC
This European Standard was approved by CEN on 17 March 2003. 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 Management Centre or to any CEN member.
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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 Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and United Kingdom.
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EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36
© 2003 CEN
B-1050 Brussels
All rights of exploitation in any form and by any means reserved worldwide for CEN national Members.
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Ref. No. EN 14122:2003 E
EN 14122:2003 (E)
Contents Page Foreword......................................................................................................................................................................3 Scope ..............................................................................................................................................................4
2
Normative references ....................................................................................................................................4
3
Principle ..........................................................................................................................................................4
4
Reagents .........................................................................................................................................................4
5
Apparatus .......................................................................................................................................................7
6
Procedure .......................................................................................................................................................8
7
Calculation....................................................................................................................................................11
8
Precision .......................................................................................................................................................11
9
Test report ....................................................................................................................................................12
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Annex A (informative) Examples of HPLC chromatograms ..................................................................................13 Annex B (informative) Precision data......................................................................................................................15 Annex C (informative) Alternative HPLC systems .................................................................................................17
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Bibliography ..............................................................................................................................................................18
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EN 14122:2003 (E)
Foreword This document (EN 14122:2003) has been prepared by Technical Committee CEN/TC 275 "Food analysis Horizontal methods", the secretariat of which is held by DIN.
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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 2003, and conflicting national standards shall be withdrawn at the latest by November 2003. Annexes A, B and C are informative. WARNING — The use of this European Standard can involve hazardous materials, operations and equipment. This European Standard does not purport to 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.
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According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and the United Kingdom.
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EN 14122:2003 (E)
1
Scope
This European Standard specifies a method for the determination of vitamin B1 in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B1 is the mass fraction of total thiamin including its phosphorylated derivatives.
2
Normative references
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This European Standard incorporates by dated or undated references, provisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies (including amendments). EN ISO 3696:1995, Water for analytical laboratory use – Specification and test methods (ISO 3696:1987).
Principle
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Thiamin is extracted from food after acid hydrolysis followed by dephosphorylation using an enzymatic treatment and quantified by HPLC with pre- or post-column derivatization to thiochrome [1] to [6].
4
Reagents
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4.1 General
4.2
Chemicals and solutions
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During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at least grade 1 according to EN ISO 3696:1995, or double distilled water.
Methanol, HPLC grade mass fraction w(CH3OH) 99,8 %
4.2.2
Acetic acid solution, substance concentration c(CH3COOH) = 0,02 mol/l
4.2.3
Isobutanol, w(C4H10O) 98 %
4.2.4
Sodium dihydrogen phosphate, w(NaH2PO4) 99,8 %
4.2.5
Hydrochloric acid, w(HCl) = 36 %
4.2.6
Hydrochloric acid, c(HCl) = 0,1 mol/l
4.2.7
Sulfuric acid, c(H2SO4) = 0,05 mol/l
4.2.8
Sodium hydroxide, w(NaOH) 99 %
4.2.9
Sodium hydroxide solution, mass concentration (NaOH) = 150 g/l
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4.2.1
4.2.10 Sodium hydroxide solution, (NaOH) = 200 g/l 4.2.11 Potassium hexacyanoferrate III, w{K3[Fe(CN)6]} 99 % 4
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EN 14122:2003 (E)
4.2.12 Potassium hexacyanoferrate III solution, {K3[Fe(CN)6]} = 10 g/l 4.2.13 Alkaline potassium hexacyanoferrate III solution (pre-column derivatization), {K3[Fe(CN)6]} = 0,4 g/l Dilute 2,0 ml of the hexacyanoferrate solution (4.2.12) to 50 ml with sodium hydroxide solution (4.2.9). Prepare fresh each day of analysis. 4.2.14 Alkaline potassium hexacyanoferrate III solution (post-column derivatization), {K3[Fe(CN)6]} = 0,5 g/l Dilute 2,5 ml of the hexacyanoferrate solution (4.2.12) to 50 ml with sodium hydroxide solution (4.2.10).
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4.2.15 Taka diastase or suitable alternative 1)
4.2.16 Sodium acetate solution, c(CH3COONa · 3H2O) = 2,5 mol/l
4.2.17 Sodium acetate solution, c(CH3COONa · 3H2O) = 0,5 mol/l 4.2.18 HPLC mobile phases
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Examples of appropriate mixtures with volume fractions of e.g. 10 % to 50 % methanol (4.2.1) in water or using phosphate or acetate buffer are given in annex C. The possibility of using ion pairing agents is also given. 4.2.19 Phosphate buffer (pH 3,5), c(KH2PO4) = 9,0 mmol/l 4.2.20 Tetraethylammoniumchloride, w(C8H20NCl ) 98 %
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4.2.21 Sodium heptanesulfonate, w(C7H15NaO3S) 98 % 4.2.22 Acetate buffer, (pH 4,0), c(CH3COOH) = 50 mmol/l
4.3.1
Standard substances General
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Thiamin chloride hydrochloride can be obtained from various suppliers. The purity of the thiamin standard substances can vary and it is therefore necessary to determine the concentration of the calibration solution by UVspectrometry (see 4.4.4). 4.3.2
Thiamin chloride hydrochloride, w(C12H17ClN4OS · HCl) 99 %
4.3.3
Thiamin monophosphate chloride, w(C12H17ClN4O4PS) 98 %
4.3.4
Thiamin pyrophosphate chloride (cocarboxylase), w(C12H19ClN4O7P2S) 98 %
4.4 4.4.1
Stock solutions
Thiamin chloride hydrochloride, (C12H17ClN4OS · HCl) 0,1 mg/ml
Dissolve an accurately weighed amount of the thiamin chloride hydrochloride standard substance (4.3.2) in a defined volume of an appropriate solvent, for example 10 mg of vitamin B1 standard substance in 100 ml of hydrochloric acid (4.2.6). This solution can be stored for four weeks at + 4 °C.
1) e.g Taka-Diastase Nr T00040, Pfalz & Bauer, Waterbury, CT 06708, USA. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results.
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