1、August 2014Translation 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!%:;“2232404www.din.deDDIN EN 14152Foodstuffs Determination of vitamin B2 by high performance liquidchromatography;English version EN 14152:2014,English translation of DIN EN 14152:2014-08Lebensmittel Bestimmung von Vitamin B2 mit Hochleistungs-Flssigchromatographie;Englische Fassung EN 14152:
3、2014,Englische bersetzung von DIN EN 14152:2014-08Produits alimentaires Dtermination de la teneur en vitamine B2 par chromatographie liquide hauteperformance;Version anglaise EN 14152:2014,Traduction anglaise de DIN EN 14152:2014-08SupersedesDIN EN 14152:2003-10 andDIN EN 14152 Corrigendum 1:2006-03
4、www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 17 pages07.14 DIN EN 14152:2014-08 2 A comma is used as the decimal marker. National foreword This document (EN 14152:2014) has been prepared by Technical Committee CEN/TC 275 “Food analys
5、is 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
6、DIN 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 14152:2003-10 and DIN EN 14152 Corrigendum 1:2006-03 as follows: a) the scope of the standard has been extended; b) Annex B has been supplemented to include Tables B.2 and B.3; c) the Bibliography has been updated; d) the standard has been editorially re
8、vised. Previous editions DIN EN 14152: 2003-10 DIN EN Corrigendum 1: 2006-03 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
9、 14152June 2014 ICS 67.050 Supersedes EN 14152:2003English Version Foodstuffs - Determination of vitamin B2 by high performance liquid chromatography Produits alimentaires - Dtermination de la teneur en vitamine B2 par chromatographie liquide haute performanceLebensmittel - Bestimmung von Vitamin B2
10、 mit Hochleistungs-Flssigchromatographie This European 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. U
11、p-to-date lists and bibliographical references concerning 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 tran
12、slation under the responsibility of a CEN member 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, Finla
13、nd, Former Yugoslav Republic of Macedonia, France, 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
14、 Marnix 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 14152:2014 EEUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGEUROPEAN STANDARDNORME EUROPENNEEUROPISC
15、HE NORMEN 14152:2014 (E) 2 Contents PageForeword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus .6 6 Procedure .7 7 Calculation 8 8 Precision .9 9 Test report . 10 Annex A (informative) Examples of HPLC chromatograms . 11 Annex B (informative) Precision data . 12 Annex
16、C (informative) Alternatives HPLC systems 14 Bibliography . 15 DIN EN 14152:2014-08EN 14152:2014 (E) 3 Foreword This document (EN 14152:2014) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shal
17、l 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 the elements of this doc
18、ument 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 14152:2003. Annexes A, B and C are informative. According to the CEN-CENELEC Internal Regulations, the national standards organizatio
19、ns 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, Lithuania, Luxembourg, Malta,
20、 Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. WARNING The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated wit
21、h its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. DIN EN 14152:2014-08EN 14152:2014 (E) 4 1 Scope This European Standard specifies a method for the determinatio
22、n of vitamin B2in food by high performance liquid chromatography (HPLC) and fluorescence detection. This method has been validated in two interlaboratory studies. The first study was for the analysis of samples of milk powder and pigs liver ranging from 1,45 mg/100 g to 10,68 mg/100 g. The second st
23、udy was for the analysis of samples of tube feeding solution, baby food, powdered milk, meal with fruits, yeast, cereal and chocolate powder ranging from 0,21 mg/100 g to 87,1 mg/100 g. Vitamin B2is the mass fraction of total riboflavin including its phosphorylated derivatives. For further informati
24、on 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 application. For dated references, only the edition cited applies. For undated references, the latest edition
25、 of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696) 3 Principle Riboflavin is extracted from food after acid hydrolysis followed by dephosphorylation using an enzymatic treatment, and separated b
26、y HPLC, and detected by fluorometric detection. An external standard is used for quantification. For further information see 1 to 11. 4 Reagents During 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 d
27、ouble distilled water. 4.1 Methanol, mass fraction w(CH3OH) 99,8 %, HPLC grade. 4.2 Sodium acetate trihydrate, w(CH3COONa 3H2O) = 99 %. 4.3 Sodium acetate solution, substance concentration c(CH3COONa 3H2O) = 0,1 mol/l. 4.4 Sodium acetate solution, c(CH3COONa 3H2O) = 2,5 mol/l. 4.5 Glacial acetic aci
28、d, w(CH3COOH) = 99,8 %. 4.6 Acetic acid solution, c(CH3COOH) = 0,02 mol/l. 4.7 Hydrochloric acid, w(HCl) = 36 %. 4.8 Hydrochloric acid, c(HCl) = 0,1 mol/l. 4.9 Hydrochloric acid, c(HCl) = 0,01 mol/l. 4.10 Sulfuric acid, c(H2SO4) = 0,05 mol/l. 4.11 Sodium hydroxide, w(NaOH) 99 %. DIN EN 14152:2014-08
29、EN 14152:2014 (E) 5 4.12 Sodium hydroxide solution, c(NaOH) = 0,5 mol/l. 4.13 Phosphorous pentoxide, w(P2O5) = 98 %. 4.14 Enzyme or enzyme mixture, with the ability to liberate vitamin B2from foods as free riboflavin. NOTE For the precision data in Table B.1, Taka-Diastase from Pfaltz and Bauer1)has
30、 been used. 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. 4.15 HPLC Mobile phase Examples of appropriate mixtures of e.g. 10 % to 50 % methanol (4.1) in water or using phosphate or acetate buffer are given in
31、 Annex A and Annex C. The possibility of using ion-pairing agents is also given. 4.16 Phosphate buffer (pH = 3,5), c(KH2PO4) = 9,0 mmol/l. 4.17 Tetraethylammoniumchloride, w(C8H20ONCl) 98 %. 4.18 Sodium heptanesulfonate, w(C7H15NaO3S) 98 %. 4.19 Standard substances 4.19.1 Riboflavin, w(C17H20N4O6) =
32、 98 %. Vitamin B2can be obtained as riboflavin from various suppliers. The purity of the riboflavin standard may vary. It is therefore necessary to determine the concentration of the calibration solution by UV-spectrometry (see concentration test in 4.20.3). 4.19.2 Riboflavin-5-phosphate, w(C17H20N4
33、NaO9P) = 95 %. Riboflavin-5-phosphate sodium salt (for check of enzyme and retention time in chromatogram). 4.20 Stock solution 4.20.1 Precautions Vitamin B2is very sensitive to light. Measures shall be taken to protect the vitamin B2and the corresponding solutions during the whole sample preparatio
34、n procedure e.g. by using generally brown glassware. 4.20.2 Riboflavin stock solution, M = 376,36, (C17H20N4O6) 100 g/ml. Dissolve an amount of riboflavin standard substance (4.19.1) previously dried and stored in dark in a desiccator possibly under vacuum and/or over phosphorous pentoxide (4.13), w
35、eighed to the nearest milligram, e.g. approximately 50 mg in a defined volume, e.g. 500 ml in an appropriate solvent e.g. diluted acetic acid (4.6) using brown volumetric flasks. This solution can be stored at 4 C in the dark for 2 months. Riboflavin is sparingly soluble. To facilitate dissolution w
36、arm with approximately 300 ml diluted acetic acid (4.6), on a steam bath with constant stirring until dissolved, cool and add diluted acetic acid (4.6) to make 500 ml. Alternatively add 5 ml of sodium hydroxide solution (4.12) to the standard substance in a 500 ml volumetric flask. Due to the instab
37、ility in alkaline solutions immediately after dissolution add 1,5 ml of glacial acetic acid (4.5) and dilute to volume with diluted acetic acid (4.6), or another appropriate acid. The concentration of the freshly prepared and if necessary also stored solution should be tested (4.20.3). 1) The inform
38、ation of the suppliers of Taka-Diastase, Pfaltz M is the molar mass, in grams per mol. The value is 376,36; A444is the absorption value of the riboflavin solution. 4.21 Standard solutions 4.21.1 Riboflavin standard solution, (C17H20N4O6) 10 g/ml. Prepare a 1:10 dilution of the riboflavin stock solut
39、ion (4.20.2), e.g. pipette 10 ml of the riboflavin stock solution, into a 100 ml brown volumetric flask and add diluted acetic acid (4.6) or another appropriate solvent to make 100 ml. Prepare this solution fresh every day. 4.21.2 Riboflavin standard test solution, (C17H20N4O6) 0,1 g/ml to 1 g/ml. P
40、ipette corresponding volumes e.g. 1,0 ml to 10,0 ml of the standard solution (4.21.1), into brown volumetric flasks e.g. 100 ml and dilute with the mobile phase (4.15) to the mark. Prepare this solution fresh every day. 5 Apparatus Use laboratory apparatus, glassware, and, in particular, the followi
41、ng: 5.1 UV spectrometer, UV spectrometer capable of measuring absorption at defined wavelengths (444 nm), with appropriate cells, e.g. of 1 cm length. 5.2 Autoclave or heating device, autoclave for extraction purpose, e.g. pressure cooker type, with pressure or temperature reading device, electrical
42、 heating device or water bath. 5.3 HPLC system, consisting of a pump, a sample injecting device, a fluorescence detector with an excitation and emission wavelength set at e.g. 468 nm and 520 nm, respectively (see Annex C), and an evaluation system such as an integrator. 5.4 HPLC column Analytical re
43、versed phase column, e.g. of diameter 4,0 mm to 4,6 mm, length 100 mm to 250 mm, filled with particle size 3 m to 10 m. Other systems (see Annex A) can be used providing that a satisfactory separation of riboflavin from other co-extractives is achieved. DIN EN 14152:2014-08EN 14152:2014 (E) 7 Other
44、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 equivalent results. 5.5 Filter device Filtering of the mobile phase as well as of the sample solution through a membrane filter, e.g.
45、a pore size of 0,45 m, prior to use or injection will increase longevity of the columns. 6 Procedure 6.1 Precautions Vitamin B2is very sensitive to light. Measures shall be taken to protect the sample and the corresponding solutions during the whole procedure e.g. by using generally brown glassware.
46、 6.2 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. 6.3 Preparation of the sample test solution 6.3.1 Extraction Weigh
47、 an appropriate amount of the sample to the nearest mg, e.g. 2 g to 10 g in a beaker or a conical flask. Add a defined volume ranging from 50 ml to 200 ml of hydrochloric acid (4.8), or sulfuric acid (4.10). The pH of the solution should not be more than 2,0. Cover the container with a watch glass a
48、nd 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, time 15 min to 60 min). The higher the temperature is, the shorter the t
49、ime should be. However, prolonged heating of riboflavin and riboflavin-5-phosphate can cause losses. It has been shown that, notably for chocolate foods, the extraction efficiency could drop when pH was above 2. 6.3.2 Enzyme treatment After cooling to room temperature adjust the extract to the optimal pH for the enzyme used with sodium acetate solution (4.4) and add a suitable amount of dephosphorylating enzyme (4.14) to the sample. Incubate the
