EN 14122:2003
(Main)Foodstuffs - Determination of vitamin B1 by HPLC
Foodstuffs - Determination of vitamin B1 by HPLC
This European Standard specifies a method for the determination of vitamin B1 in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B1 is the mass fraction of total thiamin including its phosphorylated derivatives.
Lebensmittel - Bestimmung von Vitamin B1 mit HPLC
Produits alimentaires - Détermination de la vitamine B1 par CLHP
La présente Norme européenne spécifie une méthode de dosage de la vitamine B1 présente dans les produits alimentaires, par chromatographie liquide haute performance (CLHP). La vitamine B1 correspond à la fraction massique de la thiamine totale, y compris ses dérivés phosphorylés.
Živila – Določevanje vitamina B1 s HPLC
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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
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
Examples of HPLC chromatograms.13Annex B (informative)
Precision data.15Annex C (informative)
Alternative HPLC systems.17Bibliography.18SIST EN 14122:2003
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
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
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
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
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