Foodstuffs - Determination of vitamin B2 by HPLC

This draft European Standard specifies a method for the determination of vitamin B2 in foodstuffs by high performance liquid chromatography (HPLC). The determination of vitamin B2 content is carried out by measurement of riboflavin.

Lebensmittel - Bestimmung von Vitamin B2 mit HPLC

Diese Europäische Norm legt ein Verfahren zur Bestimmung von Vitamin B2 in Lebensmitteln mit Hochleistungsflüssigchromatographie  (HPLC) fest. Der Gehalt an Vitamin B2 wird durch die Messung von Riboflavin bestimmt.

Produits alimentaires - Détermination de la teneur en vitamine B2 par CLHP

La présente Norme européenne spécifie une méthode de dosage de la vitamine B2 présente dans les produits alimentaires par Chromatographie Liquide à Haute performance (CLHP). La détermination de la teneur en vitamine B2 est réalisée par dosage de la riboflavine.

Živila - Določevanje vitamina B2 s HPLC

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Vitamin B2 mit HPLCProduits alimentaires - Détermination de la teneur en vitamine B2 par CLHPFoodstuffs - Determination of vitamin B2 by HPLC67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 14152:2003SIST EN 14152:2003en01-november-2003SIST EN 14152:2003SLOVENSKI

SIST EN 14152:2003

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14152July 2003ICS 67.050English versionFoodstuffs - Determination of vitamin B2 by HPLCProduits alimentaires - Dosage de la vitamine B2 par CLHPLebensmittel - Bestimmung von Vitamin 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 bibliographical 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 member 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, Norway, Portugal, Slovakia, Spain, Sweden, Switzerland and UnitedKingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2003 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14152:2003 ESIST EN 14152:2003

EN 14152:2003 (E)2ContentspageForeword.31Scope.32Normative references.33Principle.34Reagents.45Apparatus.76Procedure.77Calculation.98Precision.99Test report.10Annex A (informative)
Figure.11Annex B (informative)
Precision data.12Annex C (informative)
Alternative HPLC-Systems.13Bibliography.14SIST EN 14152:2003

EN 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 European 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 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 regulatory 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, Italy, 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 B2 in foodstuffs by highperformance liquid chromatography (HPLC). The determination of vitamin B2 content 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 references, 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 laboratory 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] to [8].SIST EN 14152:2003

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 grade4.2.2 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 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 %4.2.14 Dephosphorylating enzyme,
with the ability to hydrolyse bound riboflavin from food1)4.2.15 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.
1)e.g. Taka-Diastase Nr. T00040, Pfalz & Bauer, Waterbury, CT 06708, USA was used for the collaborative study. Thisinformation is given for the convenience of users of this standard and does not constitute endorsement by CEN of theproduct named. Equivalent products may be used if they can be shown to lead to the same results [7, 10, 11].SIST EN 14152:2003

EN 14152:2003 (E)54.2.16 Phosphate buffer (pH 3,5), c(KH2PO4) = 9,0 mmol/l4.2.17 Tetraethylammoniumchloride, w(C8H20NCl) = 98 %4.2.18 Sodium heptanesulfonate, w(C7H15NaO3S) = 98 %4.3 Standard substances4.3.1 Riboflavin, w(C17H20N4O6) = 98 %;Vitamin B2 can 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 (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 B2 is very sensitive to light. Measures have to be taken to protect the vitamin B2 and 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 mg/mlDissolve an amount of riboflavin standard 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 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.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 appropriate 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. 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 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)whereSIST EN 14152:2003

EN 14152:2003 (E)6A444is the absorption value of the solution at the maximum wavelength of about 444 nm;328is the 1%1cmEvalue of riboflavin (in acetate buffer, pH 3,8) at 444 nm [9];10is the dilution factor.4.5 Standard solutions4.5.1 Standard solution, r(C17H20N4O6) » 10 mg/mlPrepare a 1:10 dilution of the riboflavin stock solution (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 mg/ml to 1 mg/mlPipette 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 SpectrometerUV-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 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. g. of diameter 4,0 mm to 4,6 mm, length 100 mm to 250 mm, filled withparticle size 3 mm to 10 mm.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 systems (see annex C) can be used providing that a satisfactory separation of riboflavin from other co-extractives is achieved.5.5 Fi

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