Foodstuffs - Determination of vitamin B1 by high performance liquid chromatography

This European Standard specifies a method for the determination of vitamin B1 in food by high performance liquid chromatography (HPLC) with enzymatic treatment and pre- or post-column derivatization. This method has been validated in two interlaboratory studies. The first study was for the analysis of samples of whole meal flour, milk powder/spray dried milk, freeze-dried mixed vegetables and freeze-dried pig's liver ranging from 0,295 mg/100 g to 0,807 mg/100 g. The second study was for the analysis of samples of tube feeding solution, baby food with vegetables, powdered milk, meal with fruits, yeast, cereal, chocolate powder and food supplement ranging from 0,11 mg/100 g to 486 mg/100 g. Vitamin B1 is the mass fraction of total thiamin including its phosphorylated derivatives.
For further information on the validation, see Clause 8 and Annex B.

Lebensmittel - Bestimmung von Vitamin B1 mit Hochleistungs-Flüssigchromatographie

Diese Europäische Norm legt ein Verfahren zur Bestimmung von Vitamin B1 in Lebensmitteln mit Hochleistungsflüssigchromatographie (HPLC) mit Enzymbehandlung und Vor- oder Nachsäulenderivati¬sierung fest. Diese Bestimmung wurde in zwei Ringversuchen validiert. In dem einen Ringversuch wurde Vollkornmehl, Milchpulver/sprühgetrocknete Milch, gefriergetrocknetes Mischgemüse und gefrier¬getrocknete Schweineleber mit Gehalten von 0,295 mg/100 g bis 0,807 mg/100 g analysiert. Im zweiten Ringversuch wurde Krankenhausnahrung, Babynahrung mit Gemüse, Milchpulver, Fertignahrungsmittel mit Früchten, Hefe, Getreide, Schokoladenpulver und Nahrungsergänzungsmittel von 0,11 mg/100 g bis 468 mg/100 g untersucht. Vitamin B1 ist der Massenanteil an Gesamtthiamin einschließlich der phosphorylierten Derivate.

Produits alimentaires - Détermination de la teneur en vitamine B1 par chromatographie liquide haute performance

La présente Norme européenne spécifie une méthode de dosage de la vitamine B1 présente dans les produits alimentaires par chromatographie liquide haute performance (CLHP) avec traitement enzymatique et dérivation pré- ou post-colonne. Cette méthode a été validée lors de deux essais interlaboratoires. Le premier essai concernait l’analyse d’échantillons de farine complète, de lait en poudre/lait séché par atomisation, d’assortiments de légumes lyophilisés et de foie de porc lyophilisé allant de 0,295 mg/100 g à 0,807 mg/100 g. Le deuxième essai s’appliquait à l’analyse d’échantillons de solution pour alimentation entérale, d’aliment pour bébé aux légumes, de lait en poudre, de substitut de repas aux fruits, de levure, de céréale, de chocolat en poudre et de complément alimentaire allant de 0,11 mg/100 g à 486 mg/100 g. La vitamine B1 correspond à la fraction massique de thiamine totale, y compris ses dérivés phosphorylés.
Pour plus d'informations sur la validation, voir l'Article 8 et l'Annexe B.

Živila - Določevanje vitamina B1 s tekočinsko kromatografijo visoke ločljivosti

Standard EN 14122 navaja metodo za določanje vitamina B1 v živilih s tekočinsko kromatografijo visoke ločljivosti (HPLC) z encimsko obdelavo ter pred- ali post-kolonsko derivatizacijo. Ta metoda je bila validirana z dvema medlaboratorijskima študijama. Prva študija je bila namenjena analizi vzorcev polnozrnate moke, mleka v prahu/atomiziranega mleka, zmrznjene mešane zelenjave in zmrznjenih prašičjih jeter, ki vsebujejo od 0,295 mg/100 g do 0,807 mg/100 g vitamina B1. Druga študija je bila namenjena analizi vzorcev raztopin za krmljenje, zelenjavnih formul za dojenčke, mleka v prahu, grobe moke s sadjem, kvasa, žitaric, čokolade v prahu in prehranskih nadomestil, ki vsebujejo od 0,11 mg/100 g do 486 mg/100 g vitamina B1. Vitamin B1 je masni delež celotne vrednosti tiamina, vključno z njegovimi fosforiliranimi derivati. Za več informacij glede validacije glejte točko 8 in dodatek B.

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Vitamin B1 mit Hochleistungs-FlüssigchromatographieProduits alimentaires - Dosage de la vitamine B1 par chromatographie liquide haute performanceFoodstuffs - Determination of vitamin B1 by high performance liquid chromatography67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 14122:2014SIST EN 14122:2014en,fr,de01-september-2014SIST EN 14122:2014SLOVENSKI

STANDARDSIST EN 14122:2003/AC:2006SIST EN 14122:20031DGRPHãþD
SIST EN 14122:2014
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 14122
June 2014 ICS 67.050 Supersedes EN 14122:2003English Version

Foodstuffs - Determination of vitamin B1 by high performance liquid chromatography

Produits alimentaires - Détermination de la teneur en vitamine B1 par chromatographie liquide haute performance Lebensmittel - Bestimmung von Vitamin B1 mit Hochleistungs-Flüssigchromatographie This European Standard was approved by CEN on 17 April 2014.

CEN 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 application 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 Management 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, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 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 14122:2014 ESIST EN 14122:2014

EN 14122:2014 (E) 2 Contents Page Foreword ...................................................................................................................................................................... 3 1 Scope .............................................................................................................................................................. 4 2 Normative references .................................................................................................................................... 4 3 Principle .......................................................................................................................................................... 4 4 Reagents ......................................................................................................................................................... 4 5 Apparatus ....................................................................................................................................................... 7 6 Procedure ....................................................................................................................................................... 8 7 Calculation .................................................................................................................................................... 10 8 Precision ....................................................................................................................................................... 11 9 Test report .................................................................................................................................................... 12 Annex A (informative)

Examples of HPLC chromatograms .................................................................................. 13 Annex B (informative)

Precision data ...................................................................................................................... 15 Annex C (informative)

Alternative HPLC systems ................................................................................................. 18 Annex D (informative)

Vitamin B1 compound: 2-(1-hydroxyethyl)thiamin (HET) performing post-column derivatization ................................................................................................................................................ 19 Bibliography .............................................................................................................................................................. 21

SIST EN 14122:2014

EN 14122:2014 (E) 3 Foreword This document (EN 14122:2014) 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 2014 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. This document supersedes EN 14122:2003. 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, 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 — The use of this European Standard can involve hazardous materials, operations and equipment. This European Standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this European Standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. SIST EN 14122:2014

EN 14122:2014 (E) 4 1 Scope This European Standard specifies a method for the determination of vitamin B1 in food by high performance liquid chromatography (HPLC) with enzymatic treatment and pre- or post-column derivatization. This method has been validated in two interlaboratory studies. The first study was for the analysis of samples of whole meal flour, milk powder/spray dried milk, freeze-dried mixed vegetables and freeze-dried pig's liver ranging from 0,295 mg/100 g to 0,807 mg/100 g. The second study was for the analysis of samples of tube feeding solution, baby food with vegetables, powdered milk, meal with fruits, yeast, cereal, chocolate powder and food supplement ranging from 0,11 mg/100 g to 486 mg/100 g. Vitamin B1 is the mass fraction of total thiamin 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 referenced 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 methods (ISO 3696) 3 Principle Thiamin is extracted from food after acid hydrolysis followed by dephosphorylation using an enzymatic treatment and quantified by HPLC with pre- or post-column derivatization to thiochrome. An external standard is used for quantification. For further information see [1] to [7]. 4 Reagents 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 Acetic acid solution, substance concentration c(CH3COOH) = 0,02 mol/l. 4.3 Isobutanol, w(C4H10O) ≥ 98 %. 4.4 Sodium dihydrogen phosphate, w(NaH2PO4) ≥ 99,8 %. 4.5 Hydrochloric acid solution, w(HCl) = 36 %. 4.6 Hydrochloric acid solution, c(HCl) = 0,1 mol/l. 4.7 Sulfuric acid solution, c(H2SO4) = 0,05 mol/l. 4.8 Sodium hydroxide, w(NaOH) ≥ 99 %. 4.9 Sodium hydroxide solution, mass concentration (NaOH) = 150 g/l. 4.10 Sodium hydroxide solution, (NaOH) = 200 g/l. SIST EN 14122:2014

EN 14122:2014 (E) 5 4.11 Potassium hexacyanoferrate III, w{K3[Fe(CN)6]} ≥ 99 %. 4.12 Potassium hexacyanoferrate III solution, {K3[Fe(CN)6]} = 10 g/l. 4.13 Alkaline potassium hexacyanoferrate III solution (pre-column derivatization), {K3[Fe(CN)6]} = 0,4 g/l. Dilute 2,0 ml of the potassium hexacyanoferrate III solution (4.12) to 50 ml with sodium hydroxide solution (4.9). Prepare fresh each day of analysis. 4.14 Alkaline potassium hexacyanoferrate III solution (post-column derivatization), {K3[Fe(CN)6]} = 0,5 g/l. Dilute 2,5 ml of the potassium hexacyanoferrate III solution (4.12) to 50 ml with sodium hydroxide solution (4.10). 4.15 Enzyme or enzyme mixture, with the ability to liberate vitamin B1 from foods as free thiamin. NOTE 1 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 used. NOTE 2 If incomplete dephosphorylation occurs, this can be solved by the separate quantification of TMP (Thiamin Mono Phosphate), see [7]. 4.16 Sodium acetate solution, c(CH3COONa · 3H2O) = 2,5 mol/l. 4.17 Sodium acetate solution, c(CH3COONa · 3H2O) = 0,5 mol/l. 4.18 HPLC mobile phases Examples of appropriate mixtures with volume fractions 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. 4.19 Phosphate buffer (pH = 3,5), c(KH2PO4) = 9,0 mmol/l. 4.20 Tetraethylammoniumchloride, w(C8H20NCl) ≥ 98 %. 4.21 Sodium heptanesulfonate, w(C7H15NaO3S) ≥ 98 %. 4.22 Acetate buffer (pH = 4,0), c(CH3COOH) = 50 mmol/l. 4.23 Standard substances 4.23.1 Thiamin chloride hydrochloride, w(C12H17ClN4OS · HCl) ≥ 99 %. For external calibration, see 6.3. 4.23.2 Thiamin monophosphate chloride, w(C12H17ClN4O4PS) ≥ 98 %. For check of enzymes, see 6.2.2. 4.23.3 Thiamin pyrophosphate chloride (cocarboxylase), w(C12H19ClN4O7P2S) ≥ 98 %.

1) The information of the suppliers of Taka-Diastase, Pfaltz & Bauer, Waterbury, CT 06708, USA (No T00040), and Serva is given for the convenience of users of this European standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 14122:2014

EN 14122:2014 (E) 6 For check of enzymes, see 6.2.2. 4.24 Stock solutions 4.24.1 Thiamin chloride hydrochloride stock solution, (C12H17ClN4lS

HCl) ≈ 0,1 mg/ml. Dissolve an accurately weighed amount of the thiamin chloride hydrochloride standard substance (4.23.1) in a defined volume of an appropriate solvent, for example 10 mg of vitamin B1 standard substance in 100 ml of hydrochloric acid solution (4.6). This solution can be stored for four weeks at + 4 °C. 4.24.2 Thiamin monophosphate stock solution, (C12H17ClN4O4PS) ≈ 0,1 mg/ml. Dissolve an accurately weighed amount of the thiamin monophosphate chloride (4.23.2) in a defined volume of an appropriate solvent, for example 10 mg of thiamin monophosphate chloride in 100 ml of hydrochloric acid solution (4.6). This solution can be stored for four weeks at - 20 °C. 4.24.3 Thiamin pyrophosphate stock solution, (C12H19ClN4O7P2S) ≈ 0,1 mg/ml. Dissolve an accurately weighed amount of the thiamin pyrophosphate chloride (4.23.3) in a defined volume of an appropriate solvent, for example 10 mg of the thiamin pyrophosphate chloride in 100 ml of hydrochloric acid solution (4.6). 4.24.4 Concentration tests - thiamin chloride hydrochloride Dilute 10 ml of the thiamin chloride hydrochloride solution (4.24.1) with hydrochloric acid solution (4.6) in a 100 ml volumetric flask to the mark. Measure the absorbance of this solution at the maximum of about 247 nm (A247) in a 1 cm cell against hydrochloric solution (4.6) in the reference cell using an UV spectrometer (5.1). Calculate the mass concentration, , in microgram per millilitre thiamin chloride hydrochloride solution (4.24.1) using Formula (1): ερ0001247⋅⋅=MA (1) where 0 is the molar absorption coefficient of thiamin chloride hydrochloride at the maximum wavelength of about 247 nm. The value is 14 200 l · mol−1 · cm−1. This value is calculated from the extinction coefficient, 1%1cmE = 421, in 0,1 mol/l HCl [8][7] and the molar mass, M = 337,21. The value is given with four significant figures; M is the molar mass, in grams per mol. The value is 337,21; A247 is the absorption value of the thiamin chloride hydrochloride solution. 4.25 Standard solutions 4.25.1 Thiamin chloride hydrochloride standard solution, (C12H17ClN4lS

HCl) ≈ 1

Pipette 1 ml to 10 ml of the thiamin chloride hydrochloride solution (4.24.1) into a 100 ml volumetric flask and dilute to the mark with the appropriate solvent, e.g. hydrochloric acid solution (4.6). This solution can be stored at 4 °C in the dark for 1 month. 4.25.2 Thiamin monophosphate standard solution, (C12H17ClN4O4mS) ≈ 1

Pipette 1 ml to 10 ml of the thiamin monophosphate solution (4.24.2) into a 100 ml volumetric flask and dilute to the mark with the appropriate solvent, e.g. hydrochloric acid solution (4.6). This solution can be stored at 4 °C in the dark for 1 month. SIST EN 14122:2014

EN 14122:2014 (E) 7 4.25.3 Thiamin pyrophosphate standard solution, (C12H19ClN4O7P2S) ≈ 1

Pipette 1 ml to 10 ml of the thiamin pyrophosphate solution (4.24.3) into a 100 ml volumetric flask and dilute to the mark with the appropriate solvent, e.g. hydrochloric acid solution (4.6). This solution can be stored at 4 °C in the dark for 1 month. 5 Apparatus Usual laboratory apparatus, glassware, and the following: 5.1 UV spectrometer, UV spectrometer, capable of measuring absorption at defined wavelengths (247 nm), with appropriate cells, e.g. of 1 cm length. 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 HPLC system, consisting of a pump, a sample injecting device, a fluorescence detector with an excitation and emission wavelength set at e.g. 366 nm and 435 nm, respectively (see Annex C), and an evaluation system such as an integrator. 5.4 HPLC column 5.4.1 General Other particle sizes or column dimensions than those specified in this European Standard may be used. Separation parameters shall be adapted to such materials to guarantee equivalent results. The performance criterion for suitable analytical columns is the baseline resolution of the thiamin from interferences2). 5.4.2 Pre-column oxidation Analytical columns, e.g. Lichrospher® 60 RP Select B 2), particle size of 5 µm, diameter 4,0 mm to 4,6 mm, length 100 mm to 250 mm. 5.4.3 Post-column oxidation Analytical columns, e.g. Supelco® LC-18- DB 2), particle size of 5 µm, diameter 4,0 mm to 4,6 mm, length 100 mm to 250 mm. 5.5 Filter device Filtering of the mobile phase as well as of the sample solution through a membrane filter with, e.g. a pore size of 0,45 µm, prior to use or injection will increase longevity of the columns. 5.6 Post-column reactor pump and derivatization tube, a suitable reagent delivery system, a T-type connecting tube and a derivatization tube (e.g. 10 m x 0,33 mm).

2) Suitable silica column packing materials available commercially are Lichrosorb® Si 60, Spherisorb® Si, Hypersil® Si and Lichrospher® 100 DIOL. Suitable RP column packing materials are Spherisorb® ODS, µ-Bondapak radial C18, Supelco. LC-18- DB and Hypersil® ODS. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of these products. SIST EN 14122:2014

EN 14122:2014 (E) 8 6 Procedure 6.1

Preparation of the test sample Homogenize the test sample. Grind coarse material with an appropriate mill and mix again. Measures such as pre-cooling shall be taken to avoid exposing to high temperature for long periods of time. 6.2 Preparation of the sample test solution 6.2.1 Extraction Weigh an appropriate amount of the test sample to the nearest mg, e.g. 2 g to 10 g in a conical flask. Add a defined volume ranging from 60 ml to 200 ml of hydrochloric acid solution (4.6), or sulfuric acid solution (4.7). The pH of the solution should not be higher than pH = 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 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 treatment After cooling to room temperature adjust the extract to the optimal pH for the enzyme used with sodium acetate solution (4.16) or (4.17) and add a suitable amount of enzyme or enzyme mixture (4.15) to the sample. Incubate the mixture at the optimal time and temperature for the enzyme(s) used. After cooling to room temperature transfer the solution to a volumetric flask using distilled water, or another appropriate solvent and dilute the sample test solution to a defined volume (Vts). For each enzyme used, optimal pH, incubation time and incubation temperature shall be checked. To ensure an optimal dephosphorylation, the enzymatic step shall be checked with analysis of samples spiked with thiamin monophosphate chloride (4.23.2) or thiamin pyrophosphate chloride (4.23.3), and a material similar in sample type as the test sample. This material should be a certified reference material. The amount of thiamin possibly brought in with the enzyme(s) (4.15) shall be considered in the calculation of the result. NOTE For determination of the precision data given in this European Standard in Table B.1, Table B.2 and Table B.3, Taka-Diastase and Taka-Diastase combined with -amylase from barley was used for dephosphorylation under the following conditions. The extract was adjusted to pH = 4,0 and pH = 4,5, respectively, with sodium acetate solution (4.16) or (4.17) and 100 mg of Taka-Diastase and 10 -amylase per gram of sample was added. The mixture was incubated at 37 °C to 45 °C for 4 h to 24 h, see [5], [10] and [16]. 6.2.3 Sample test solution If necessary, filter the sample solution (6.2.2) through a filter paper or a 0,45 µm membrane filter. Centrifugation may also be used. This is the sample test solution for oxidation (6.3.2 or 6.3.3). 6.3 Oxidation of thiamin to thiochrome 6.3.1 General The oxidation may be performed either pre-column (6.3.2) or post-column (6.3.3). SIST EN 14122:2014

EN 14122:2014 (E) 9 6.3.2 Pre-column oxidation 6.3.2.1 Procedure for oxidation step Pipette 1 ml of the enzymatically treated sample (6.2.3), standard (4.25.1) or blank i.e. hydrochloric acid solution (4.6) or sulfuric acid solution (4.7) depending which was used in 6.2.1 into suitable vials or flasks, add 1 ml of alkaline hexacyanoferrate III solution (4.13). Shake the sample test solution for a fixed amount of time (e.g. 10 s), leave to stand for a specified time (e.g. 1 min). In order to remove interfering compounds and to protect the HPLC column it is recommended to neutralize the sample test solution (e.g. with H3PO4) or to perform a clean-up using solid phase extraction (for more information see [5]). Filter through a 0,45 µm membrane filter. This is the sample test solution to inject into the reverse phase HPLC system (6.3.2.2). Alternatively, the oxidized solution can be extracted into 1,5 ml isobutanol (4.3) and the extract can be injected. NOTE The oxidative conversion of thiamin to thiochrome can be inhibited in

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