Foodstuffs - Determination of vitamin B6 by high performance chromatography

This European Standard specifies a method for the determination of vitamin B6 in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B6 is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives determined as pyridoxine. The β-glycosylated forms are not taken into account. These can be determined with the method given in EN 14663 [1] by which the different vitamins of vitamin B6 (pyridoxal, pyridoxamine and pyridoxine) are separated and individually quantified. A third European Standard, EN 14166 [2], determines the total vitamin B6 by microbiological assay.

Lebensmittel - Bestimmung von Vitamin B6 mit Hochleistungs-Flüssigchromatographie

Diese Europäische Norm legt ein Verfahren zur Bestimmung von Vitamin B6 in Lebensmitteln mit Hochleistungs Flüssigchromatographie (HPLC) fest. Vitamin B6 ist der Massenanteil der Gesamtmenge an Pyridoxin (PN), Pyridoxal (PL) und Pyridoxamin (PM), einschließlich ihrer phosphorylierten Derivate, die als Pyridoxin bestimmt werden. Die  glycosylierten Formen werden nicht berücksichtigt. Diese können mit dem in EN 14663 [1] festgelegten Verfahren bestimmt werden, bei dem die verschiedenen Vitamere von Vitamin B6 (Pyridoxin, Pyridoxal und Pyridoxamin) getrennt und individuell bestimmt werden. Eine dritte Europäische Norm (EN 14166) [2] bestimmt den Gesamtgehalt an Vitamin B6 mikrobiologisch.

Produits alimentaires - Détermination de la teneur en vitamine B6 par chromatographie liquide haute performance

La présente Norme européenne spécifie une méthode de dosage de la vitamine B6 présente dans les produits alimentaires par chromatographie liquide haute performance (CLHP). La vitamine B6 est la fraction massique de la somme de la pyridoxine, du pyridoxal et de la pyridoxamine, y compris leurs dérivés phosphorylés quantifiés sous la forme pyridoxine. Les formes β-glycosylées ne sont pas prises en compte. Elles peuvent être dosées selon la méthode indiquée dans l'EN 14663 [1], qui permet de séparer et de quantifier individuellement les différentes formes de vitamine B6 (pyridoxal, pyridoxamine et pyridoxine). Une troisième Norme européenne, l’EN 14166 [2], traite du dosage de la vitamine B6 totale par essai microbiologique.

Živila - Določevanje vitamina B6 s tekočinsko kromatografijo visoke ločljivosti

Standard EN 14164 navaja metodo za določanje vitamina B6 v živilih s tekočinsko kromatografijo visoke ločljivosti (HPLC). Vitamin B6 je masni delež vsote piridoksina, piridoksala in piridoksamina, vključno z njihovimi fosforiliranimi derivati, ki so določeni kot piridoksin. β-glikozilirane oblike se ne upoštevajo. Te je mogoče določiti z metodo iz standarda EN 14663, pri kateri so različni vitamini vitamina B6 (piridoksal, piridoksamin in piridoksin) ločeni in posamezno izmerjeni. Tretji evropski standard, EN 14166, določa skupno vrednost vitamina B6 z mikrobiološkim preskusom.

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Published
Publication Date
03-Jun-2014
Current Stage
9093 - Decision to confirm - Review Enquiry
Completion Date
28-Oct-2020

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Vitamin B6 mit Hochleistungs-FlüssigchromatographieProduits alimentaires - Dosage de la vitamine B6 par chromatographie liquide haute performanceFoodstuffs - Determination of vitamin B6 by high performance chromatography67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 14164:2014SIST EN 14164:2014en,fr,de01-september-2014SIST EN 14164:2014SLOVENSKI
STANDARDSIST EN 14164:20081DGRPHãþD



SIST EN 14164:2014



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 14164
June 2014 ICS 67.050 Supersedes EN 14164:2008English Version
Foodstuffs - Determination of vitamin B6 by high performance chromatography
Produits alimentaires - Détermination de la teneur en vitamine B6 par chromatographie liquide haute performance Lebensmittel - Bestimmung von Vitamin B6 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 14164:2014 ESIST EN 14164:2014



EN 14164:2014 (E) 2 Contents Page Foreword .3 1 Scope .4 2 Normative references .4 3 Principle .4 4 Reagents .4 5 Apparatus .8 6 Procedure .8 7 Calculation .9 8 Test report . 10 Annex A (informative)
Example of a chromatogram . 11 Annex B (informative)
Precision data . 12 Annex C (informative)
Sample treatment option without acid hydrolysis . 14 Annex D (informative)
Examples for molar absorption coefficients . 15 Bibliography . 16
SIST EN 14164:2014



EN 14164:2014 (E) 3 Foreword This document (EN 14164:2014) 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 14164:2008. The Annexes A, B, C and D are informative. 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 14164:2014



EN 14164:2014 (E) 4 1 Scope This European Standard specifies a method for the determination of vitamin B6 in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B6 is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives determined as pyridoxine. The -glycosylated forms are not taken into account. These can be determined with the method given in EN 14663 [1] by which the different vitamins of vitamin B6 (pyridoxal, pyridoxamine and pyridoxine) are separated and individually quantified. A third European Standard, EN 14166 [2], determines the total vitamin B6 by microbiological assay. 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 Pyridoxal, pyridoxamine and pyridoxine are extracted from food by acid hydrolysis and dephosphorylated enzymatically using acid phosphatase. By reaction with glyoxylic acid in the presence of Fe2+ as a catalyst, pyridoxamine is transformed into pyridoxal, which is then reduced to pyridoxine by the action of sodium borohydride in alkaline medium. Pyridoxine is then quantified in the sample solution by HPLC with a fluorometric detection [3], [4]. 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 Acid phosphatase, (CAS 9001-77-8), from potatoes, enzyme activity is 33 nkat/mg1) with substrate p-nitrophenyl phosphate at pH = 4,8 and T = 37 °C, for example from Boehringer or Sigma2). 33 nkat/mg corresponds to 2 U/mg. 4.1.1 Acid phosphatase solution Prepare a solution of 20 mg/ml acid phosphatase in sodium acetate solution (4.14). It is necessary to use acid phosphatase rather than Taka-diastase to obtain a complete hydrolysis of phosphorylated forms of vitamin B6. Where 300 mg of Taka-diastase is needed to obtain good dephosphorylation, only 0,5 mg of acid phosphatase is needed, see [5].
1) Katal (symbol ''kat'') is a derived SI unit of enzyme activity. One katal is that catalytic activity which will raise the rate of reaction by one mol/s in a specified assay system. 2) This information is given for the convenience of users of this European standard and does not constitute an endorsement by CEN of the supplier. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 14164:2014



EN 14164:2014 (E) 5 4.1.2 Activity check of acid phosphatase The activity of acid phosphatase can be checked as proposed below. Prepare a stock solution of approximately 0,1 mg/ml of pyridoxal phosphate (4.9) in water. Mix 3,0 ml of the PLP stock solution and 10 ml of hydrochloric acid (4.21) in a 20 ml volumetric flask and fill up to the mark with water. Check the concentration of PLP by measuring the absorbance at 293 nm in a 1 cm cell using a UV-spectrometer (5.1) against a hydrochloric acid solution (4.20) as reference. The molar absorption coefficient (0) of PLP in 0,1 mol/l HCl is 7 200. Calculate the mass concentration PLP of the stock solution, in milligram per millilitre, according to Formula (1): FMA⋅⋅=ερPLP293PLP (1) where A293 is the absorption value of the solution at 293 nm; MPLP is the molar mass of vitamin B6 standard substance, in gram per mol (MPLP = 247,14); F is the dilution factor (here F = 20/3); 0 is the molar absorption coefficient of PLP in 0,1 mol/l of hydrochloric acid at 293 nm, in l mol−1 cm−1, (here: 0 = 7 200), see [6]. Take 1,0 ml of the PLP stock solution for extraction and proceed with 6.2.1, 6.2.2, 6.2.3 and 6.2.4. Calculate the pyridoxine (PN) conversion rate from the dephosphorylated pyridoxal phosphate solution according to Formula (2): PNPLPSTPLPSHClPN0001100822,01002(%) rate ConversionMAMA⋅⋅⋅⋅⋅⋅⋅⋅⋅=⋅ρρ (2) where PN HCL is the mass concentration of pyridoxine hydrochloride in the standard test solution, in micrograms per millilitre; AS is the peak area or peak height for pyridoxine obtained with the sample test solution, in units of area or height; 2 is the factor of dilution of the reaction with sodium borohydride if acetic acid is added, otherwise the dilution factor is 1,9; 100 is the total volume of the sample test solution, in millilitre; 0,822 is the factor to convert pyridoxine hydrochloride to pyridoxine; 100 is the conversion factor for %; MPLP is the molar mass of pyridoxal phosphate (PLP), in gram per mol (MPLP = 247,14); AST is the peak area or peak height for pyridoxine obtained with the standard test solution, in units of area or height; 1 000 is the factor to convert microgram to milligram; PLP is the mass concentration of pyridoxal phosphate (PLP) in the stock solution, in milligrams per millilitre; MPN is the molar mass of pyridoxine (PN), in gram per mol (MPN = 169,1). SIST EN 14164:2014



EN 14164:2014 (E) 6 4.2 Sodium acetate, trihydrate, mass fraction w(CH3COONa · 3H2O) ≥ 99,0 %. 4.3 Glacial acetic acid, w(CH3COOH) ≥ 99,8 %. 4.4 Glyoxylic acid, w(C2H2O3 · H2O) ≥ 97,0 %. 4.5 Ferrous sulfate II, heptahydrate, w(FeSO4 · 7H2O) ≥ 99,5 %. 4.6 Sodium hydroxide, w(NaOH) ≥ 99,0 %. 4.7 Sodium borohydride, w(NaBH4) ≥ 97,0 %. 4.8 Potassium dihydrogen phosphate, w(KH2PO4) ≥ 99,0 %. 4.9 Pyridoxal phosphate (PLP), w ≥ 99,0 %. 4.10 Orthophosphoric acid, w(H3PO4) ≥ 84,0 %. 4.11 Sodium octanesulfonate, w(C8H17NaO3S) ≥ 98,0 %, or sodium heptanesulfonate, w (C7H15NaO3S) ≥ 98,0 %. 4.12 Acetonitrile (HPLC grade), w(C2H3N) ≥ 99,8 %. 4.13 Sodium acetate solution, substance concentration c(CH3COONa · 3H2O) = 2,5 mol/l. Dissolve 170,1 g of sodium acetate, trihydrate (4.2) in 500 ml of water. 4.14 Sodium acetate solution, c(CH3COONa · 3H2O) = 0,05 mol/l (pH = 4,5). Dissolve 6,8 g of sodium acetate, trihydrate (4.2) in 1 l of water. Adjust the pH to 4,5 with glacial acetic acid (4.3). 4.15 Ferrous sulfate solution, c(FeSO4 · 7H2O) = 0,0132 mol/l. Dissolve 36,6 mg of ferrous sulfate II, heptahydrate (4.5) in 10 ml of sodium acetate solution (4.14). Prepare fresh each day of use. NOTE In a study described by Mann et al., see [7], a ferrous sulfate solution of 10 g/l was used. This concentration was based on the completion of the conversion of pyridoxamine to pyridoxal at pyridoxamine levels up to 8 times the minimum level of vitamin B6 required by the infant formula Act in the US, see Mann et al. [8]. This concentration seems not to be necessary for the European situation. 4.16 Sodium hydroxide solution, c(NaOH) = 0,2 mol/l. Dissolve 800 mg of sodium hydroxide (4.6) in 100 ml of water. 4.17 Sodium hydroxide solution, c(NaOH) = 6,0 mol/l. Dissolve 24 g of sodium hydroxide (4.6) in 100 ml of water. 4.18 Sodium borohydride solution, c(NaBH4) = 0,1 mol/l. Dissolve 378 mg of sodium borohydride (4.7) in 100 ml of sodium hydroxide solution (4.16). Prepare fresh on day of use. 4.19 Glyoxylic acid solution, c(C2H2O3 · H2O) = 1 mol/l (pH = 4,5). SIST EN 14164:2014



EN 14164:2014 (E) 7 Dissolve 4,7 g of glyoxylic acid monohydrate (4.4) in 30 ml of sodium acetate solution (4.13). Adjust the pH to 4,5 with the sodium hydroxide solution (4.17) and dilute to 50 ml with water in a volumetric flask. Prepare fresh on day of use. 4.20 Hydrochloric acid, c(HCl) = 0,1 mol/l. 4.21 Hydrochloric acid, c(HCl) = 0,2 mol/l. 4.22 HPLC mobile phase In a beaker add 940 ml of water, 40 ml of acetonitrile (4.12), 160 mg of sodium octanesulfonate or sodium heptanesulfonate (4.11) and 6,8 g of potassium dihydrogen phosphate (4.8). After dissolving sodium octanesulfonate or sodium heptanesulfonate and potassium dihydrogen phosphate by stirring, adjust the pH to 2,5 with orthophosphoric acid (4.10). Transfer the solution in a 1 l volumetric flask. Adjust to the mark with water. Filter through a 0,45
4.23 Pyridoxine hydrochloride (vitamin B6 standard substance), w(C8H11NO3 HCI) ≥ 99 %. 4.24 Pyridoxine hydrochloride stock solution, mass concentration
≈ 0f5 mg/ml. Dissolve an accurately weighed amount of pyridoxine hydrochloride (4.23), e.g. 50 mg, in a defined volume, e.g. 100 ml, of water. The stock solution is stable for 4 weeks if stored at 4 °C in the dark. For the concentration test, dilute 0,5 ml of pyridoxine hydrochloride stock solution (4.24) to 20 ml with 0,1 mol/l HCI (4.20) and measure the absorbance at 290 nm in a 1 cm cell using a UV-spectrometer (5.1) against 0,1 mol/l HCl solution as reference. Calculate the mass concentration , in microgram per millilitre of the stock solution according to Formula (3): FMA⋅⋅⋅=ερ0001PNHCl290PNHCl (3) where A290 is the absorption of the value of the solution at 290 nm; MPNHCl is the molar mass of vitamin B6 standard substance, in gram per mol (MPNHCl = 205,64); F is the dilution factor (here F
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