1、BRITISH STANDARDBS EN 14152:2003Incorporating Corrigendum No. 1Foodstuffs Determination of vitamin B2 by HPLCThe European Standard EN 14152:2003 has the status of a British StandardICS 67.050g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40
2、g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN 14152:2003This British Standard, was published under the authority of the Standards Policy and Strategy Committee on 24 July 2003 BSI 2006ISBN 0 580 42316 6National forewordThis British Standard is the o
3、fficial English language version of EN 14152:2003, including Corrigendum December 2005.The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Horizontal analysis, which has the responsibility to: A list of organizations represented on this committee can be obtained on r
4、equest to its secretary.Cross-referencesThe British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI
5、Electronic Catalogue or of British Standards Online.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers t
6、o understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keepUK interests informed; monitor related international and European developments and promulgate them in the UK.Summary of pagesThis document comprise
7、s a front cover, an inside front cover, the EN title page, pages 2 to 14, an inside back cover and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date Comments16201Corrigendum No. 131 July 2006
8、Correction to clause 4.2.14, footnote 1 on page 4 and to clause 6.3.2.EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 14152July 2003ICS 67.050English versionFoodstuffs - Determination of vitamin B2 by HPLCProduits alimentaires - Dosage de la vitamine B2 par CLHP Lebensmittel - Bestimmung von Vitam
9、in B2 mit HPLCThis European Standard was approved by CEN on 2 May 2003.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographi
10、cal references concerning such nationalstandards may be obtained on application to the 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 translationunder the responsibility of a CEN membe
11、r into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Hungary, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Nor
12、way, Portugal, Slovakia, Spain, Sweden, Switzerland and UnitedKingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2003 CEN All rights of exploitation in any form and by any means reservedworl
13、dwide for CEN national Members.Ref. No. EN 14152:2003 EIncorporating Corrigendum December 2005 EN 14152:2003 (E)2ContentspageForeword. 31 Scope . 32 Normative references . 33 Principle. 34 Reagents 45 Apparatus 76 Procedure 77 Calculation. 98 Precision 99 Test report . 10Annex A (informative) Figure
14、 11Annex B (informative) Precision data. 12Annex C (informative) Alternative HPLC-Systems 13Bibliography . 14EN 14152:2003 (E)3ForewordThis document (EN 14152:2003) has been prepared by Technical Committee CEN/TC 275 “Food analysis -Horizontal methods”, the secretariat of which is held by DIN.This E
15、uropean Standard shall be given the status of a national standard, either by publication of an identicaltext or by endorsement, at the latest by January 2004, and conflicting national standards shall be withdrawn atthe latest by January 2004.Annexes A, B and C are informative.Warning The use of this
16、 standard may involve hazardous materials, operations and equipment. Thisstandard does not purport to address all the safety problems associated with its use. It is theresponsibility of the user of this standard to establish appropriate safety and health practices anddetermine the applicability of r
17、egulatory limitations prior to use.According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark,Finland, France, Germany, Greece, Hungary, Iceland, Ireland, I
18、taly, Luxembourg, Malta, Netherlands,Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and the United Kingdom.1 ScopeThis European Standard specifies a method for the determination of vitamin B2in foodstuffs by highperformance liquid chromatography (HPLC). The determination of vitamin B2content
19、 is carried out bymeasurement of riboflavin.2 Normative referencesThis European Standard incorporates by dated or undated reference, provisions from other publications.These normative references are cited at the appropriate places in the text and the publications are listedhereafter. For dated refer
20、ences, subsequent amendments to or revisions of any of these publications apply tothis European Standard only when incorporated in it by amendment or revision. For undated references thelatest edition of the publication referred to applies (including amendments).EN ISO 3696, Water for analytical lab
21、oratory use Specification and test methods (ISO 3696:1987).3 PrincipleRiboflavin in an appropriate sample solution is determined after acid hydrolysis followed by dephosphorylationusing an enzymatic treatment by high performance liquid chromatographic (HPLC) separation with afluorometric detection 1
22、 to 8.EN 14152:2003 (E)44 Reagents4.1 GeneralDuring the analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of atleast grade 1 according to EN ISO 3696, or double distilled water.4.2 Chemicals and solutions4.2.1 Methanol, mass fraction, w(CH3OH) % HPLC grade
23、4.2.2 Sodium acetate trihydrate, w(CH3COONa 3H2O) = 99 %4.2.3 Sodium acetate solution, substance concentration c(CH3COONa 3H2O) = 0,1 mol/l4.2.4 Sodium acetate solution, c(CH3COONa 3H2O) = 2,5 mol/l4.2.5 Glacial acetic acid, w(CH3COOH) = 99,8 %4.2.6 Acetic acid solution, c(CH3COOH) = 0,02 mol/l4.2.7
24、 Hydrochloric acid, w(HCl) = 36 %4.2.8 Hydrochloric acid, c(HCl) = 0,1 mol/l4.2.9 Hydrochloric acid, c(HCl) = 0,01 mol/l4.2.10 Sulfuric acid, c(H2SO4) = 0,05 mol/l4.2.11 Sodium hydroxide, w(NaOH) = 99 %4.2.12 Sodium hydroxide solution, c(NaOH) = 0,5 mol/l4.2.13 Phosphorous pentoxide, w(P2O5) = 98 %4
25、.2.15 HPLC Mobile phaseExamples of appropriate mixtures of e. g. 10 % to 50 % methanol (4.2.1) in water or using phosphate oracetate buffer are given in annex A1 and C; possibility of using ion-pairing agents is also given.4.2.14 Dephosphorylation enzyme, with the ability to hydrolyse bound riboflav
26、in from food NOTE Taka-Diastase1)has been used for the determination of the precision data. 1)Taka-Diastase Nr. T00040 is the trade name of a product supplied by Pfaltz Vitamin B2can be obtained as riboflavin from various suppliers. The purity of the riboflavin standard may vary.It is therefore nece
27、ssary to determine the concentration of the calibration solution by UV-spectrometry (seeconcentration test 4.4.3).4.3.2 Riboflavin-5-phosphateRiboflavin-5-phosphate sodium salt, w(C17H20N4NaO9P) = 95 %; for qualitative purposes only.4.4 Stock solution4.4.1 PrecautionsVitamin B2is very sensitive to l
28、ight. Measures have to be taken to protect the vitamin B2and thecorresponding solutions during the whole sample preparation procedure e. g. by using generally brownglassware.4.4.2 Riboflavin standard stock solution, mass concentration r (C17H20N4O6) 100 m g/mlDissolve an amount of riboflavin standar
29、d substance (4.3.1) previously dried and stored in dark in a desiccatorpossibly under vacuum or/and over phosphorous pentoxide (4.2.13), 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(4.2.6) using brown
30、 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 ca. 300 ml diluted acetic acid (4.2.6), on asteam bath with constant stirring until dissolved, cool and add diluted acetic acid (4.2.6) to make 500 ml.
31、Alternatively add 5 ml sodium hydroxide solution (4.2.12) to the standard substance in a 500 ml volumetricflask. Due to the instability in alkaline solutions immediately after dissolution add 1,5 ml of glacial acetic acid(4.2.5) and dilute to volume with diluted acetic acid (4.2.6), or another appro
32、priate acid. The concentration ofthe freshly prepared and if necessary also stored solution should be tested (4.4.3).4.4.3 Concentration testMix 20 ml of the riboflavin stock solution, (4.4.2) in a 200 ml volumetric flask with 3,5 ml sodium acetatesolution (4.2.3) and dilute with water to the mark.
33、For the preparation of the blind solution, mix 20 ml of aceticsolution (4.2.6) with 3,5 ml of sodium acetate solution in a 200 ml volumetric flask and dilute to the mark withwater. Take these solutions for the spectrometric measurement.Measure the absorbance of the riboflavin solution at the maximum
34、 wavelength of about 444 nm in a 1 cm cellwith a spectrometer (5.1) against the blind solution as reference. Calculate the mass concentration, r , ofriboflavin in micrograms per millilitre, of the stock solution (4.4.2) according to equation (1):32810104444=Ar (1)whereEN 14152:2003 (E)6A444is the ab
35、sorption value of the solution at the maximum wavelength of about 444 nm;328 is the 1%1cmE value of riboflavin (in acetate buffer, pH 3,8) at 444 nm 9;10 is the dilution factor.4.5 Standard solutions4.5.1 Standard solution, r (C17H20N4O6) 10 m g/mlPrepare a 1:10 dilution of the riboflavin stock solu
36、tion (4.4.2), e. g. pipette 10 ml of the riboflavin stock solution(4.4.2), into a 100 ml brown volumetric flask and add diluted acetic acid (4.2.6) or another appropriate solventto make 100 ml. Prepare this solution fresh every day.4.5.2 Standard test solutions, r (C17H20N4O6) 0,1 m g/ml to 1 m g/ml
37、Pipette corresponding volumes e. g. 1,0 ml to 10,0 ml of the standard solution (4.5.1), into brown volumetricflasks e. g. 100 ml and dilute with the mobile phase (4.2.15) to the mark. Prepare this solution fresh every day.5 ApparatusUse laboratory apparatus and, in particular, the following:5.1 UV S
38、pectrometerUV-spectrometer capable of measuring absorptions at defined wavelengths, with appropriate cells, e. g. of1 cm length.5.2 Autoclave or heating deviceAutoclave for extraction purpose, e. g. pressure cooker type, with pressure or temperature reading device;electrical heating device or water
39、bath.5.3 HPLC systemHPLC system, consisting of a pump, a sample injecting device, a fluorescence detector with an excitationwavelength set at e. g. 468 nm and an emission wavelength set at e. g. 520 nm and an evaluating systemsuch as an integrator.5.4 HPLC columnAnalytical reversed phase column, e.
40、g. of diameter 4,0 mm to 4,6 mm, length 100 mm to 250 mm, filled withparticle size 3 m m to 10 m m.Particle sizes and column dimensions other than those specified in this European Standard may be used.Separation parameters have to be adapted to such materials to guarantee equivalent results.Other sy
41、stems (see annex C) can be used providing that a satisfactory separation of riboflavin from other co-extractives is achieved.5.5 Filter deviceMembrane filter with pore size of, e. g. 0,45 m m are appropriate.EN 14152:2003 (E)7Filtering of the mobile phase as well as of the sample solution through a
42、membrane filter prior to use orinjection will increase longevity of the columns.6 Procedure6.1 PrecautionsVitamin B2is very sensitive to light. Measures have to be taken to protect the sample and the correspondingsolutions during the whole procedure e. g. by using generally brown glassware.6.2 Prepa
43、ration of the test sampleHomogenise the test sample. Grind coarse material with an appropriate mill and mix again. Measures such aspre-cooling have to be taken to avoid exposing to high temperature for long periods of time.6.3 Preparation of the sample test solution6.3.1 Sample extractionWeigh an ap
44、propriate amount of the sample to the nearest mg, e. g. 2 g to 10 g in a beaker or a conical flask.The defined volume ranging from 50 ml to 200 ml of hydrochloric acid (4.2.8), or sulfuric acid (4.2.10). The pHof the solution should be less than 2,0. Cover the container with a watch glass and either
45、 autoclave the testportion at 121 C for 30 min, or heat it at 100 C for 60 min.NOTE The data from the BCR study have shown that a wide range of conditions for the acid hydrolysis can beapplied (temperature 95 C to 130 C, time 15 min to 60 min, the higher the temperature, the shorter the time). Howev
46、er,prolonged heating of riboflavin and riboflavin-5-phosphate may cause losses. It has been shown that, notably forchocolate foods, the extraction value could drop when pH was above 2.6.3.3 Sample test solutionIf necessary, dilute an aliquot (Va) from a clear filtrate to a defined volume of the samp
47、le solution through afilter paper or use a 0,45 m m membrane filter. Centrifugation at appropriate g level may also be used to clarifythe sample solution. From the clear filtrate dilute an aliquot (VA) to a defined volume (V) with an appropriatesolvent mixture to be compatible with the HPLC system u
48、sed, or e. g. dilute 1,0 ml of the extract (6.3.2) with1,0 ml of methanol (4.2.1). This is the sample test solution for the HPLC analysis.6.3.2 Enzyme treatment For each enzyme used, optimal pH, incubation time and incubation temperature shall be checked. To ensure an optimal dephosphorylation, the
49、enzymatic step shall be checked with analysis of samples spiked with riboflavin-5-phosphate sodium salt (4.3.2), and a material similar in sample type as the test sample. This material should be a certified reference material. If Taka-Diastase has been used for dephosphorylation, the amount of riboflavin possibly brought in with the enzyme shall be considered in the calculation of the result. NOTE 1 For determination of the precision data given in this European standard, Taka-Diastase1)was used for dephosphorylation under the following cond
