1、BSI Standards PublicationBS EN 14152:2014Foodstuffs Determinationof vitamin B2 by highperformance liquidchromatographyBS EN 14152:2014 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 14152:2014. It supersedes BS EN 14152:2003 which is withdrawn.The UK participat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 77940 4ICS 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 14152:2014EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 14152 June 2014 ICS 67.050 Supersedes EN 14152:2003English Version Foodstuffs - Determination of vitamin B
5、2 by high performance liquid chromatography Produits alimentaires - Dtermination de la teneur en vitamine B2 par chromatographie liquide haute performanceLebensmittel - Bestimmung von Vitamin B2 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 14152:2014 EBS EN 14152:2014EN 14152:2014 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus .6 6 Pr
11、ocedure .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 C (informative) Alternatives HPLC systems 14 Bibliography . 15 BS EN 14152:2014EN 14152:2014 (E) 3 Foreword This document (EN 14152:2
12、014) 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 December 201
13、4 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 subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights
14、. This document supersedes EN 14152:2003. Annexes A, B and C are informative. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic,
15、 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, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. WARNING T
16、he use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the a
17、pplicability of regulatory limitations prior to use. BS EN 14152:2014EN 14152:2014 (E) 4 1 Scope This European Standard specifies a method for the determination of vitamin B2in food by high performance liquid chromatography (HPLC) and fluorescence detection. This method has been validated in two int
18、erlaboratory 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 study was for the analysis of samples of tube feeding solution, baby food, powdered milk, meal with fruits, yeast, cereal and chocolate powder
19、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 information on the validation, see Clause 8 and Annex B. 2 Normative references The following documents, in whole or in part, are normatively referenc
20、ed in this document and are indispensable 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 met
21、hods (ISO 3696) 3 Principle Riboflavin is extracted from food after acid hydrolysis followed by dephosphorylation using an enzymatic treatment, and separated by HPLC, and detected by fluorometric detection. An external standard is used for quantification. For further information see 1 to 11. 4 Reage
22、nts 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 double distilled water. 4.1 Methanol, mass fraction w(CH3OH) 99,8 %, HPLC grade. 4.2 Sodium acetate trihydrate, w(CH3COONa 3H2O) = 99 %. 4.3 S
23、odium 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 acid, 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
24、) = 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 %. BS EN 14152:2014EN 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 mixtur
25、e, 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 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 u
26、sed. 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 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 T
27、etraethylammoniumchloride, w(C8H20ONCl) 98 %. 4.18 Sodium heptanesulfonate, w(C7H15NaO3S) 98 %. 4.19 Standard substances 4.19.1 Riboflavin, w(C17H20N4O6) = 98 %. Vitamin B2can be obtained as riboflavin from various suppliers. The purity of the riboflavin standard may vary. It is therefore necessary
28、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(C17H20N4NaO9P) = 95 %. Riboflavin-5-phosphate sodium salt (for check of enzyme and retention time in chromatogram). 4.20 Stock solution 4.20.1 Precaution
29、s Vitamin B2is very sensitive to light. Measures shall be taken to protect the vitamin B2and the corresponding solutions during the whole sample preparation 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 r
30、iboflavin standard substance (4.19.1) previously dried and stored in dark in a desiccator possibly under vacuum and/or over phosphorous pentoxide (4.13), weighed 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.
31、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 warm with approximately 300 ml diluted acetic acid (4.6), on a steam bath with constant stirring until dissolved, cool and add diluted acetic acid
32、 (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 instability in alkaline solutions immediately after dissolution add 1,5 ml of glacial acetic acid (4.5) and dilute to volume with diluted acetic acid (
33、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 information 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 ri
34、boflavin solution. 4.21 Standard solutions 4.21.1 Riboflavin standard solution, (C17H20N4O6) 10 g/ml. Prepare a 1:10 dilution of the riboflavin stock solution (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 anot
35、her 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. Pipette 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 t
36、he mobile phase (4.15) to the mark. Prepare this solution fresh every day. 5 Apparatus Use laboratory apparatus, glassware, and, in particular, the following: 5.1 UV spectrometer, UV spectrometer capable of measuring absorption at defined wavelengths (444 nm), with appropriate cells, e.g. of 1 cm le
37、ngth. 5.2 Autoclave or heating device, 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, consisting of a pump, a sample injecting device, a fluorescence detector with an excitation and e
38、mission 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 reversed 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
39、A) can be used providing that a satisfactory separation of riboflavin from other co-extractives is achieved. BS EN 14152:2014EN 14152:2014 (E) 7 Other particle sizes or column dimensions than those specified in this European Standard may be used. Separation parameters shall be adapted to such materi
40、als 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. 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 l
41、ight. Measures shall be taken to protect the sample and the corresponding solutions during the whole procedure e.g. by using generally brown glassware. 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-co
42、oling 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 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 2
43、00 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 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
44、 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 time should be. However, prolonged heating of riboflavin and riboflavin-5-phosphate can cause losses. It has been shown that, notably for chocolate fo
45、ods, 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
46、 mixture at the optimal time and temperature for the enzyme(s) used. After cooling to room temperature transfer to a light protected volumetric flask using diluted acetic acid (4.6) or another appropriate solvent and dilute to a defined volume (VE). For each enzyme used, optimal pH, incubation time
47、and incubation temperature shall be checked. To ensure an optimal dephosphorylation, the enzymatic step shall be checked, for example by analysis of samples spiked with riboflavin-5-phosphate sodium salt (4.19.2), or a material similar in sample type as the test sample. This material should be a ref
48、erence material. The amount of riboflavin possibly brought in with the enzyme shall be considered in the calculation of the result. NOTE For determination of the precision data given in Table B.1, Table B.2 and Table B.3, Taka-Diastase (Table B.1) or Taka-Diastase combined with -amylase from barley
49、(Table B.2 and Table B.3) were used for dephosphorylation under the following conditions. The extract was adjusted to pH = 4,0 and pH = 4,5, respectively, with sodium acetate solution (4.4) and 100 mg of Taka-Diastase and 10 mg -amylase per gram of sample was added. The mixture was incubated at 37 C to 45 C for 4 h to 24 h, see 9, 10 and 13. BS EN 14152:2014EN 14152:2014 (E) 8 6.3.3 Sample test solution If necessary, filter the sample solution (6.3.2) through a filter paper or a 0,45 m membrane filter. Centrifugation at a
