1、August 2014Translation by DIN-Sprachendienst.English price group 12No 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!%:;(“2232405www.din.deDDIN EN 14122Foodstuffs Determination of vitamin B1 by high performance liquidchromatography;English version EN 14122:2014,English translation of DIN EN 14122:2014-08Lebensmittel Bestimmung von Vitamin B1 mit Hochleistungs-Flssigchromatographie;Englische Fassung EN 14122
3、:2014,Englische bersetzung von DIN EN 14122:2014-08Produits alimentaires Dtermination de la teneur en vitamine B1 par chromatographie liquide hauteperformance;Version anglaise EN 14122:2014,Traduction anglaise de DIN EN 14122:2014-08SupersedesDIN EN 14122:2003-09 andDIN EN 14122 Corrigendum 1:2006-0
4、3www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 24 pages07.14 DIN EN 14122:2014-082 A comma is used as the decimal marker. National forewordThis document (EN 14122:2014) has been prepared by Technical Committee CEN/TC 275 “Food analysi
5、s Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-01-13 AA Vitamine und Carotinoide. The D
6、IN Standards corresponding to the International Standards referred to in this document are as follows, whereby EN ISO Standards are only listed below 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)
7、Amendments This standard differs from DIN EN 14122:2003-09 and DIN EN 14122 Corrigendum 1:2006-03 as follows: a) the scope of the standard has been extended; b) Subclause 8.2 “Repeatability” has been extended; c) Subclause 8.3 “Reproducibility” has been extended; d) Annex D “Vitamin B1 compound: 2-(
8、1-hydroxyethyl)thiamin (HET) performing post-column derivatization” has been added; e) the Bibliography has been updated; f) the standard has been editorially revised. Previous editions DIN EN 14122: 2003-09 DIN EN 14122 Corrigendum 1: 2006-03 National Annex NA (informative) Bibliography DIN ISO 369
9、6, Water for analytical laboratory use Specification and test methods DIN ISO 5725 (all parts), Accuracy (trueness and precision) of measurement methods and results EN 14122June 2014 ICS 67.050 Supersedes EN 14122:2003English Version Foodstuffs - Determination of vitamin B1 by high performance liqui
10、d chromatography Produits alimentaires - Dtermination de la teneur en vitamine B1 par chromatographie liquide haute performanceLebensmittel - Bestimmung von Vitamin B1 mit Hochleistungs-Flssigchromatographie This European Standard was approved by CEN on 17 April 2014. CEN members are bound to comply
11、 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 CEN-CENELEC M
12、anagement 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 into its own language and notified to the CEN-CENELEC Management Centre has the same
13、 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, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg,
14、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 of exploitation in any form and by any means reserved worldwide for CEN national Memb
15、ers. Ref. No. EN 14122:2014 EEUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 14122:2014 (E) 2 Contents PageForeword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents . 4 5 Apparatus . 7 6
16、Procedure . 8 7 Calculation 10 8 Precision . 11 9 Test report 12 Annex A (informative) Examples of HPLC chromatograms 13 Annex B (informative) Precision data 15 Annex C (informative) Alternative HPLC systems . 18 Annex D (informative) Vitamin B1compound: 2-(1-hydroxyethyl)thiamin (HET) performing po
17、st-column derivatization 19 Bibliography 21 DIN EN 14122:2014-08EN 14122:2014 (E) 3 Foreword This document (EN 14122:2014) 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 stat
18、us 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 the elements of this document may be the su
19、bject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 14122:2003. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement th
20、is 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, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovak
21、ia, 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 to address all the safety problems associated with its use. It is the responsibility o
22、f 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 14122:2014-08EN 14122:2014 (E) 4 1 Scope This European Standard specifies a method for the determination of vitamin B1in food by hi
23、gh performance liquid chromatography (HPLC) with enzymatic treatment and pre- or post-column derivatization. This method has been validated in two interlaboratory studies. The first study was for the analysis of samples of whole meal flour, milk powder/spray dried milk, freeze-dried mixed vegetables
24、 and freeze-dried pigs liver ranging from 0,295 mg/100 g to 0,807 mg/100 g. The second study was for the analysis of samples of tube feeding solution, baby food with vegetables, powdered milk, meal with fruits, yeast, cereal, chocolate powder and food supplement ranging from 0,11 mg/100 g to 486 mg/
25、100 g. Vitamin B1is the mass fraction of total thiamin including its phosphorylated derivatives. For further information on the validation, see Clause 8 and Annex B. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable
26、 for its application. 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) 3 Principle Thiamin is ex
27、tracted from food after acid hydrolysis followed by dephosphorylation using an enzymatic treatment and quantified by HPLC with pre- or post-column derivatization to thiochrome. An external standard is used for quantification. For further information see 1 to 7. 4 Reagents During the analysis, unless
28、 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 Methanol, mass fraction w(CH3OH) 99,8 %, HPLC grade. 4.2 Acetic acid solution, substance concentration c(CH3COOH) = 0,02 mol/l. 4.3 Isobutanol, w
29、(C4H10O) 98 %. 4.4 Sodium dihydrogen phosphate, w(NaH2PO4) 99,8 %. 4.5 Hydrochloric acid solution, w(HCl) = 36 %. 4.6 Hydrochloric acid solution, c(HCl) = 0,1 mol/l. 4.7 Sulfuric acid solution, c(H2SO4) = 0,05 mol/l. 4.8 Sodium hydroxide, w(NaOH) 99 %. 4.9 Sodium hydroxide solution, mass concentrati
30、on (NaOH) = 150 g/l. 4.10 Sodium hydroxide solution, (NaOH) = 200 g/l. DIN EN 14122:2014-08EN 14122:2014 (E) 5 4.11 Potassium hexacyanoferrate III, wK3Fe(CN)6 99 %. 4.12 Potassium hexacyanoferrate III solution, K3Fe(CN)6 = 10 g/l. 4.13 Alkaline potassium hexacyanoferrate III solution (pre-column der
31、ivatization), K3Fe(CN)6 = 0,4 g/l. Dilute 2,0 ml of the potassium hexacyanoferrate III solution (4.12) to 50 ml with sodium hydroxide solution (4.9). Prepare fresh each day of analysis. 4.14 Alkaline potassium hexacyanoferrate III solution (post-column derivatization), K3Fe(CN)6 = 0,5 g/l. Dilute 2,
32、5 ml of the potassium hexacyanoferrate III solution (4.12) to 50 ml with sodium hydroxide solution (4.10). 4.15 Enzyme or enzyme mixture, with the ability to liberate vitamin B1from foods as free thiamin. NOTE 1 For the precision data in Table B.1, Taka-Diastase from Pfaltz and Bauer1)has been used.
33、 For the precision data in Table B.2 and Table B.3 an enzyme mixture of -amylase from barley and Taka-Diastase from Serva1)have been used. NOTE 2 If incomplete dephosphorylation occurs, this can be solved by the separate quantification of TMP (Thiamin Mono Phosphate), see 7. 4.16 Sodium acetate solu
34、tion, c(CH3COONa 3H2O) = 2,5 mol/l. 4.17 Sodium acetate solution, c(CH3COONa 3H2O) = 0,5 mol/l. 4.18 HPLC mobile phases Examples of appropriate mixtures with volume fractions of e.g. 10 % to 50 % methanol (4.1) in water or using phosphate or acetate buffer are given in Annex A and Annex C. The possi
35、bility of using ion pairing agents is also given. 4.19 Phosphate buffer (pH = 3,5), c(KH2PO4) = 9,0 mmol/l. 4.20 Tetraethylammoniumchloride, w(C8H20NCl) 98 %. 4.21 Sodium heptanesulfonate, w(C7H15NaO3S) 98 %. 4.22 Acetate buffer (pH = 4,0), c(CH3COOH) = 50 mmol/l. 4.23 Standard substances 4.23.1 Thi
36、amin chloride hydrochloride, w(C12H17ClN4OS HCl) 99 %. For external calibration, see 6.3. 4.23.2 Thiamin monophosphate chloride, w(C12H17ClN4O4PS) 98 %. For check of enzymes, see 6.2.2. 4.23.3 Thiamin pyrophosphate chloride (cocarboxylase), w(C12H19ClN4O7P2S) 98 %. 1) The information of the supplier
37、s of Taka-Diastase, Pfaltz M is the molar mass, in grams per mol. The value is 337,21; A247is the absorption value of the thiamin chloride hydrochloride solution. 4.25 Standard solutions 4.25.1 Thiamin chloride hydrochloride standard solution, (C12H17ClN4OS HCl) 1 g/ml to 10 g/ml. Pipette 1 ml to 10
38、 ml of the thiamin chloride hydrochloride solution (4.24.1) into a 100 ml volumetric flask and dilute to the mark with the appropriate solvent, e.g. hydrochloric acid solution (4.6). This solution can be stored at 4 C in the dark for 1 month. 4.25.2 Thiamin monophosphate standard solution, (C12H17Cl
39、N4O4PS) 1 g/ml to 10 g/ml. Pipette 1 ml to 10 ml of the thiamin monophosphate solution (4.24.2) into a 100 ml volumetric flask and dilute to the mark with the appropriate solvent, e.g. hydrochloric acid solution (4.6). This solution can be stored at 4 C in the dark for 1 month. DIN EN 14122:2014-08E
40、N 14122:2014 (E) 7 4.25.3 Thiamin pyrophosphate standard solution, (C12H19ClN4O7P2S) 1 g/ml to 10 g/ml. Pipette 1 ml to 10 ml of the thiamin pyrophosphate solution (4.24.3) into a 100 ml volumetric flask and dilute to the mark with the appropriate solvent, e.g. hydrochloric acid solution (4.6). This
41、 solution can be stored at 4 C in the dark for 1 month. 5 Apparatus Usual laboratory apparatus, glassware, and the following: 5.1 UV spectrometer, UV spectrometer, capable of measuring absorption at defined wavelengths (247 nm), with appropriate cells, e.g. of 1 cm length. 5.2 Autoclave or heating d
42、evice, autoclave for extraction purpose, e.g. pressure cooker type, with pressure or temperature reading device, electrical heating device or water bath. 5.3 HPLC system HPLC system, consisting of a pump, a sample injecting device, a fluorescence detector with an excitation and emission wavelength s
43、et at e.g. 366 nm and 435 nm, respectively (see Annex C), and an evaluation system such as an integrator. 5.4 HPLC column 5.4.1 General Other particle sizes or column dimensions than those specified in this European Standard may be used. Separation parameters shall be adapted to such materials to gu
44、arantee equivalent results. The performance criterion for suitable analytical columns is the baseline resolution of the thiamin from interferences2). 5.4.2 Pre-column oxidation Analytical columns, e.g. Lichrospher60 RP Select B 2), particle size of 5 m, diameter 4,0 mm to 4,6 mm, length 100 mm to 25
45、0 mm. 5.4.3 Post-column oxidation Analytical columns, e.g. SupelcoLC-18- DB 2), particle size of 5 m, diameter 4,0 mm to 4,6 mm, length 100 mm to 250 mm. 5.5 Filter device Filtering of the mobile phase as well as of the sample solution through a membrane filter with, e.g. a pore size of 0,45 m, prio
46、r to use or injection will increase longevity of the columns. 5.6 Post-column reactor pump and derivatization tube, a suitable reagent delivery system, a T-type connecting tube and a derivatization tube (e.g. 10 m x 0,33 mm). 2)Suitable silica column packing materials available commercially are Lich
47、rosorbSi 60, SpherisorbSi, HypersilSi and Lichrospher100 DIOL. Suitable RP column packing materials are SpherisorbODS, -Bondapakradial C18, Supelco. LC-18- DB and HypersilODS. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by C
48、EN of these products. DIN EN 14122:2014-08EN 14122:2014 (E) 8 6 Procedure 6.1 Preparation of the test sample Homogenize the test sample. Grind coarse material with an appropriate mill and mix again. Measures such as pre-cooling shall be taken to avoid exposing to high temperature for long periods of
49、 time. 6.2 Preparation of the sample test solution 6.2.1 Extraction Weigh an appropriate amount of the test sample to the nearest mg, e.g. 2 g to 10 g in a conical flask. Add a defined volume ranging from 60 ml to 200 ml of hydrochloric acid solution (4.6), or sulfuric acid solution (4.7). The pH of the solution should not be higher than pH = 2,0. Cover the container with a watch glass and either autoc
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