SIST EN 14164:2014

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 - Dosage de la 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.

General Information

• Status: Published
• Public Enquiry End Date: 19-Jun-2012
• Publication Date: 10-Aug-2014
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 05-Aug-2014
• Due Date: 10-Oct-2014
• Completion Date: 11-Aug-2014
• Ref Project: EN 14164:2014 - Foodstuffs - Determination of vitamin B6 by high performance chromatography

Relations

• Replaces: SIST EN 14164:2008 - Foodstuffs - Determination of vitamin B6 by HPLC
• Effective Date: 01-Sep-2014

Overview

EN 14164:2014 - Foodstuffs: Determination of vitamin B6 by high performance liquid chromatography (HPLC) - is a CEN standard that specifies an HPLC method for measuring the mass fraction of vitamin B6 in food. Vitamin B6 is reported as the sum of pyridoxine, pyridoxal and pyridoxamine (including their phosphorylated derivatives) expressed as pyridoxine. The procedure combines acid hydrolysis, enzymatic dephosphorylation and chemical conversion steps, followed by quantification by HPLC with fluorometric detection.

Key topics and technical requirements

• Analytes covered: pyridoxine (PN), pyridoxal (PL) and pyridoxamine (PM) and their phosphorylated forms; β‑glycosylated forms are excluded (see EN 14663 for these).
• Principle: acid hydrolysis → dephosphorylation with acid phosphatase → conversion of PM → PL with glyoxylic acid/Fe2+ → reduction to PN with sodium borohydride → HPLC quantification as PN.
• Reagents & solutions: sodium acetate buffers, glyoxylic acid solution, ferrous sulfate, sodium borohydride, acetonitrile (HPLC grade), sodium octanesulfonate or heptanesulfonate, pyridoxine hydrochloride standard.
• HPLC conditions & detection:
  • Reversed‑phase column (example: C8 type), mobile phase containing water, acetonitrile, ion‑pairing agent and KH2PO4 adjusted to pH 2.5, filtered (0.45 µm).
  • Fluorescence detection: excitation ~290 nm, emission ~395 nm.
  • Performance criterion: baseline separation of PL and PN; system suitability and spike recovery (PM) recommended.
• Apparatus: UV spectrometer (for standard checks), oven/water bath, HPLC system with fluorescence detector, appropriate filters.
• Sample preparation: homogenization, acid hydrolysis (HCl), enzymatic treatment (acid phosphatase), conversion/reduction steps, filtration and injection.
• Quality & traceability: calibration with pyridoxine hydrochloride standards; activity check procedure for acid phosphatase; annexes provide chromatogram examples, precision data and an alternative procedure without acid hydrolysis.

Applications and users

• Food analysis laboratories performing routine or regulatory testing of vitamin B6 in foodstuffs (cereals, dairy, nutritional products, fortified foods).
• Quality control teams in food manufacturing and ingredient suppliers to verify label claims and formulation.
• Research labs studying vitamin stability, biofortification, processing effects or nutritional composition.
• Regulatory agencies and accreditation bodies implementing standardized, comparable results across Europe.

Related standards

• EN 14663 - method for β‑glycosylated forms of vitamin B6 (separate quantification of PL, PM, PN).
• EN 14166 - total vitamin B6 determined by microbiological assay.
• EN ISO 3696 - water for analytical laboratory use (normative reference in EN 14164).

Keywords: EN 14164:2014, vitamin B6, HPLC, high performance liquid chromatography, pyridoxine, pyridoxal, pyridoxamine, food analysis, fluorometric detection, CEN standard.

Standards Content (Sample)

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

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
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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

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
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

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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