SIST EN 14122:2003

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Vitamin B1 mit HPLCProduits alimentaires - Détermination de la vitamine B1 par CLHPFoodstuffs - Determination of vitamin B1 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 14122:2003SIST EN 14122:2003en01-november-2003SIST EN 14122:2003SLOVENSKI
STANDARD



SIST EN 14122:2003



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14122May 2003ICS 67.050English versionFoodstuffs - Determination of vitamin B1 by HPLCProduits alimentaires - Dosage de la vitamine B1 par CLHPLebensmittel - Bestimmung von Vitamin B1 mit HPLCThis European Standard was approved by CEN on 17 March 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 14122:2003 ESIST EN 14122:2003



EN 14122:2003 (E)2ContentsPageForeword.31Scope.42Normative references.43Principle.44Reagents.45Apparatus.76Procedure.87Calculation.118Precision.119Test report.12Annex A (informative)
Examples of HPLC chromatograms.13Annex B (informative)
Precision data.15Annex C (informative)
Alternative HPLC systems.17Bibliography.18SIST EN 14122:2003



EN 14122:2003 (E)3ForewordThis document (EN 14122:2003) 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 identical text orby endorsement, at the latest by November 2003, and conflicting national standards shall be withdrawn at the latestby November 2003.Annexes A, B and C are informative.WARNING — The use of this European Standard can involve hazardous materials, operations andequipment. This European Standard does not purport to address all the safety problems associated withits use. It is the responsibility of the user of this European Standard to establish appropriate safety andhealth practices and determine 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.SIST EN 14122:2003



EN 14122:2003 (E)41 ScopeThis European Standard specifies a method for the determination of vitamin B1 in foodstuffs by high performanceliquid chromatography (HPLC). Vitamin B1 is the mass fraction of total thiamin including its phosphorylatedderivatives.2 Normative referencesThis European Standard incorporates by dated or undated references, provisions from other publications. Thesenormative references are cited at the appropriate places in the text and the publications are listed hereafter. Fordated references, subsequent amendments to or revisions of any of these publications apply to this
EuropeanStandard only when incorporated in it by amendment or revision. For undated references the latest edition of thepublication referred to applies (including amendments).EN ISO 3696:1995, Water for analytical laboratory use – Specification and test methods (ISO 3696:1987).3 PrincipleThiamin is extracted from food after acid hydrolysis followed by dephosphorylation using an enzymatic treatmentand quantified by HPLC with pre- or post-column derivatization to thiochrome [1] to [6].4 Reagents4.1 GeneralDuring the analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at leastgrade 1 according to EN ISO 3696:1995, or double distilled water.4.2 Chemicals and solutions4.2.1 Methanol, HPLC grade mass fraction w(CH3OH)
99,8 %4.2.2 Acetic acid solution, substance concentration c(CH3COOH) = 0,02 mol/l4.2.3 Isobutanol, w(C4H10O)
98 %4.2.4 Sodium dihydrogen phosphate, w(NaH2PO4)
99,8 %4.2.5 Hydrochloric acid, w(HCl) = 36 %4.2.6 Hydrochloric acid, c(HCl) = 0,1 mol/l4.2.7 Sulfuric acid, c(H2SO4) = 0,05 mol/l4.2.8 Sodium hydroxide, w(NaOH)
99 %4.2.9 Sodium hydroxide solution, mass concentration (NaOH) = 150 g/l4.2.10 Sodium hydroxide solution, (NaOH) = 200 g/l4.2.11 Potassium hexacyanoferrate III, w{K3[Fe(CN)6]}
99 %SIST EN 14122:2003



EN 14122:2003 (E)54.2.12 Potassium hexacyanoferrate III solution, {K3[Fe(CN)6]} = 10 g/l4.2.13 Alkaline potassium hexacyanoferrate III solution (pre-column derivatization), {K3[Fe(CN)6]} = 0,4 g/lDilute 2,0 ml of the hexacyanoferrate solution (4.2.12) to 50 ml with sodium hydroxide solution (4.2.9). Preparefresh each day of analysis.4.2.14 Alkaline potassium hexacyanoferrate III solution (post-column derivatization), {K3[Fe(CN)6]} = 0,5 g/lDilute 2,5 ml of the hexacyanoferrate solution (4.2.12) to 50 ml with sodium hydroxide solution (4.2.10).4.2.15 Taka diastase or suitable alternative 1)4.2.16 Sodium acetate solution, c(CH3COONa · 3H2O) = 2,5 mol/l4.2.17 Sodium acetate solution, c(CH3COONa · 3H2O) = 0,5 mol/l4.2.18 HPLC mobile phasesExamples of appropriate mixtures with volume fractions of e.g. 10 % to 50 % methanol (4.2.1) in water or usingphosphate or acetate buffer are given in annex C. The possibility of using ion pairing agents is also given.4.2.19 Phosphate buffer (pH 3,5), c(KH2PO4) = 9,0 mmol/l4.2.20 Tetraethylammoniumchloride, w(C8H20NCl )
98 %4.2.21 Sodium heptanesulfonate, w(C7H15NaO3S)
98 %4.2.22 Acetate buffer, (pH 4,0), c(CH3COOH) = 50 mmol/l4.3 Standard substances4.3.1 GeneralThiamin chloride hydrochloride can be obtained from various suppliers. The purity of the thiamin standardsubstances can vary and it is therefore necessary to determine the concentration of the calibration solution by UV-spectrometry (see 4.4.4).4.3.2 Thiamin chloride hydrochloride, w(C12H17ClN4OS · HCl)
99 %4.3.3 Thiamin monophosphate chloride, w(C12H17ClN4O4PS)
98 %4.3.4 Thiamin pyrophosphate chloride (cocarboxylase), w(C12H19ClN4O7P2S)
98 %4.4 Stock solutions4.4.1 Thiamin chloride hydrochloride, (C12H17ClN4OS · HCl)
0,1 mg/mlDissolve an accurately weighed amount of the thiamin chloride hydrochloride standard substance (4.3.2) in adefined volume of an appropriate solvent, for example 10 mg of vitamin B1 standard substance in 100 ml ofhydrochloric acid (4.2.6). This solution can be stored for four weeks at + 4 °C.
1)e.g Taka-Diastase Nr T00040, Pfalz & Bauer, Waterbury, CT 06708, USA. This information is given for the convenience of users of thisEuropean Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can beshown to lead to the same results.SIST EN 14122:2003



EN 14122:2003 (E)64.4.2 Thiamin monophosphate, (C12H17ClN4O4PS)
0,1 mg/mlDissolve an accurately weighed amount of the monophosphate standard substance (4.3.3) in a defined volume ofan appropriate solvent, for example 10 mg of monophosphate standard substance in 100 ml of hydrochloric acid(4.2.6). This solution can be stored for four weeks at - 20 °C.4.4.3 Thiamin pyrophosphate, (C12H19ClN4O7P2S)
0,1 mg/mlDissolve an accurately weighed amount of the pyrophosphate standard substance (4.3.4) in a defined volume of anappropriate solvent, for example 10 mg of the pyrophosphate standard substance in 100 ml of hydrochloric acid(4.2.6).4.4.4 Concentration testsDilute 10 ml of the thiamin chloride hydrochloride stock solution (4.4.1) with hydrochloric acid solution (4.2.6) in a100 ml volumetric flask to the mark. Measure the absorbance of this solution at the maximum of about 247 nm in a1 cm cell against hydrochloric acid solution (4.2.6) in the reference cell using an UV spectrometer (5.2). Calculatethe mass concentration, , in microgram per millilitre of the stock solution using equation (1):42110×10×4247=(1)where247is the absorption value of the solution at the maximum wavelength of about 247 nm;421 is the absorption coefficient 1%1cmAof thiamin chloride hydrochloride in 0,1 mol/l hydrochloride acid (see [7])10is the dilution factor.4.5 Standard solutions4.5.1 Thiamin chloride hydrochloride, (C12H17ClN4OS · HCl)
1 g/ml to 10 g/mlPipette 1 ml to 10 ml of the thiamin chloride hydrochloride stock solution (4.4.1) into a 100 ml volumetric flask anddilute to the mark with the appropriate solvent, e.g. hydrochloric acid (4.2.6). This solution can be stored at 4 °C inthe dark for 1 month.4.5.2 Thiamin monophosphate, (C12H17ClN4O4PS)
1 g/ml to 10 g/mlPipette 1 ml to 10 ml of the monophosphate stock solution (4.4.2) into a 100 ml volumetric flask and dilute to themark with the appropriate solvent, e.g. hydrochloric acid (4.2.6). This solution can be stored at 4 °C in the dark for 1month.4.5.3 Thiamin pyrophosphate, (C12H19ClN4O7P2S)
1 g/ml to 10 g/mlPipette 1 ml to 10 ml of the pyrophosphate stock solution (4.4.3) into a 100 ml volumetric flask and dilute to themark with the appropriate solvent, e.g. hydrochloric acid (4.2.6). This solution can be stored at 4 °C in the dark for 1month.5 Apparatus5.1 GeneralUsual laboratory apparatus, glassware, and the following:SIST EN 14122:2003



EN 14122:2003 (E)75.2 UV spectrometerCapable of measurement of absorbance at defined wavelengths.5.3 Autoclave or heating deviceAutoclave for extraction purpose, e.g. pressure cooker type, with pressure or temperature reading device, electricalheating device or water bath.5.4 High performance liquid chromatographic systemConsisting of a pump, sample injecting device, fluorescence detector with excitation and emission wavelengths sete.g. at 366 nm and 420 nm, respectively (see annex C), and an evaluation system such as an integrator.5.5 HPLC columns5.5.1 GeneralOther particle sizes or column dimensions than specified in this European Standard may be used. Separationparameters have to be adapted to such materials to guarantee equivalent results. The performance criterion forsuitable analytical columns is the baseline resolution of the analytes concerned 2).5.5.2 Pre-column oxidationAnalytical columns, e.g. Lichrospher 60 RP Select B 2), particle size of 5 µm, diameter 4,0 mm to 4,6 mm, length100 mm to 250 mm.5.5.3 Post-column oxidationAnalytical columns, e.g. Supelco LC-18-DB 2), particle size of 5 µm, diameter 4,0 mm to 4,6 mm, length 100 mm to250 mm.5.6 Filter deviceFiltering of the mobile phase as well as of the test sample solution through a membrane filter with, e.g. a pore sizeof 0,45 µm, prior to use or injection will increase longevity of the columns.5.7 Post-column reactor pump and derivatization tubeA suitable reagent delivery system, a T-type connecting tube and a derivatization tube (e.g. 10 m x 0,33 mm).6 Procedure6.1
Preparing of the sample solutionHomogenise the test sample. Grind coarse material with an appropriate mill and mix again. Measures such as pre-cooling have to be taken to avoid exposing to high temperature for long periods of time.6.2 Preparation of the sample test solution6.2.1 Extraction
2)Suitable silica column packing materials available commercially are Lichrosorb® Si 60, Spherisorb® Si, Hypersil® Si andLichrospher® 100 DIOL. Suitable RP column packing materials are Spherisorb® ODS, -Bondapak radial C18, SupelcoLC-18-DB and Hypersil® ODS. This information is given for the convenience of users of this European Standard and does notconstitute an endorsement by CEN of these products.SIST EN 14122:2003



EN 14122:2003 (E)8Weigh an appropriate amount of the test sample to the nearest mg, e.g. 2 g to 10 g in a conical flask. Add a definedvolume ranging from 60 ml to 200 ml of hydrochloric acid (4.2.6), or sulfuric acid (4.2.7). The pH of the solutionshould not be higher than pH = 3,0. Cover the container with a watch glass and either autoclave the test portion at121 °C for 30 min, or heat it at 100 °C for 60 min.NOTEThe 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 time should be.6.2.2 Enzyme treatmentAfter cooling to room temperature adjust the extract to pH = 4,0 with sodium acetate solution (4.2.16) or (4.2.17)and add 100 mg of taka diastase (4.2.15) per gram of sample. Incubate the mixture at 37 °C to 46 °C for 16 h to24 h. After cooling to room temperature transfer the solution to a light protected volumetric flask using distilledwater, or an other appropriate solvent and dilute to a defined volume (Ve).NOTE 1The dephosphorylation can depend on the sample matrix and on the enzyme used. Complete dephosphorylationcan be reached within shorter time, see [8].NOTE 2If other suitable enzymes are use
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