1、BSI Standards PublicationBS EN 14122:2014Foodstuffs Determinationof vitamin B1 by highperformance liquidchromatographyBS EN 14122:2014 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 14122:2014. It supersedes BS EN 14122:2003 which is withdrawn.The UK participat
2、ion 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.This publication does not purport to include all the necessary provisions of a contract. Users are
3、responsible for its correct application. The British Standards Institution 2014.Published by BSI Standards Limited 2014ISBN 978 0 580 77939 8ICS 67.050Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Stand
4、ards Policy and Strategy Committee on 30 June 2014.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS EN 14122:2014EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 14122 June 2014 ICS 67.050 Supersedes EN 14122:2003English Version Foodstuffs - Determination of vitamin B
5、1 by high performance liquid 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
6、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 appli
7、cation 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 into its own language and notified to the CEN-CENELEC Man
8、agement 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, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latv
9、ia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marn
10、ix 17, B-1000 Brussels 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14122:2014 EBS EN 14122:2014EN 14122:2014 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents . 4 5 Apparatus . 7 6
11、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
12、st-column derivatization 19 Bibliography 21 BS EN 14122:2014EN 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 status o
13、f 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 subjec
14、t 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 this E
15、uropean 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, Slovakia,
16、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 of th
17、e user of this European Standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. BS EN 14122:2014EN 14122:2014 (E) 4 1 Scope This European Standard specifies a method for the determination of vitamin B1in food by high perfo
18、rmance 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 and fre
19、eze-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/100 g. V
20、itamin 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 for its
21、 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 extracted
22、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 otherwi
23、se 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(C4H10O)
24、 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 concentration (NaOH
25、) = 150 g/l. 4.10 Sodium hydroxide solution, (NaOH) = 200 g/l. BS EN 14122:2014EN 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 derivatization)
26、, 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 2,5 ml of the
27、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. For the pre
28、cision 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 solution, c(CH3C
29、OONa 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 possibility of us
30、ing 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 Thiamin chlorid
31、e 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 suppliers of Taka-Di
32、astase, 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 ml of the t
33、hiamin 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, (C12H17ClN4O4PS) 1 g/
34、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. BS EN 14122:2014EN 14122:2014 (E)
35、 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 solution can be
36、 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 device, autoclave
37、 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 set at e.g. 366 n
38、m 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 guarantee equivale
39、nt 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 250 mm. 5.4.3 Post
40、-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, prior to use or inje
41、ction 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 LichrosorbSi 60, Sph
42、erisorbSi, 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 CEN of these prod
43、ucts. BS EN 14122:2014EN 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 time. 6.2 Preparati
44、on 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
45、 not be higher than pH = 2,0. Cover the container with a watch glass and either autoclave the test portion at 121 C for 30 min, or heat it at 100 C for 60 min. The data from the BCR study have shown that a wide range of conditions for the acid hydrolysis can be applied (temperature 95 C to 130 C, ti
46、me 15 min to 60 min). The higher the temperature is, the shorter the time should be. 6.2.2 Enzyme treatment After cooling to room temperature adjust the extract to the optimal pH for the enzyme used with sodium acetate solution (4.16) or (4.17) and add a suitable amount of enzyme or enzyme mixture (
47、4.15) to the sample. Incubate the mixture at the optimal time and temperature for the enzyme(s) used. After cooling to room temperature transfer the solution to a volumetric flask using distilled water, or another appropriate solvent and dilute the sample test solution to a defined volume (Vts). For
48、 each enzyme used, optimal pH, incubation time and incubation temperature shall be checked. To ensure an optimal dephosphorylation, the enzymatic step shall be checked with analysis of samples spiked with thiamin monophosphate chloride (4.23.2) or thiamin pyrophosphate chloride (4.23.3), and a mater
49、ial similar in sample type as the test sample. This material should be a certified reference material. The amount of thiamin possibly brought in with the enzyme(s) (4.15) shall be considered in the calculation of the result. NOTE For determination of the precision data given in this European Standard in Table B.1, Table B.2 and Table B.3, Taka-Diastase and Taka-Diastase combined with -amylase from barley was used for dephosphorylation under the following conditions. The extract was ad
