EN 14663:2005

Foodstuffs - Determination of vitamin B6 (including its glycosylated forms) by HPLC

This document 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 as well as the b-glycosylated forms that have been added to foodstuffs, calculated as pyridoxine.
This method has been successfully validated with semolina with milk (infant food), potato puree, vegetables with ham (convenient products) and a multi vitamin drink at levels from 0,034 mg/100 g to 1,21 mg/100 g.

Lebensmittel - Bestimmung von Vitamin B6 (einschließlich glucosidisch gebundener Verbindungen) mit HPLC

Dieses Dokument legt ein Verfahren für die Bestimmung von Vitamin B6 in Lebensmitteln mit Hochleistungs-Flüssigchromatographie
(HPLC) fest.
Vitamin B6 ist der Massenanteil der Summe von Pyridoxin, Pyridoxal und Pyridoxamin einschließlich ihrer
phosphorylierten Verbindungen, wie auch von ß-glycosylierten Verbindungen, berechnet als Pyridoxin.
Dieses Verfahren wurde im Ringversuch erfolgreich für Massenanteile von 0,034 mg/100 g bis 1,21 mg/100 g
in Milch-Griesbrei (Kleinkindernahrung), Kartoffelpüree, Schinken mit Gemüse (Fertignahrung) und einem
Multivitamingetränk validiert.

Produits alimentaires - Dosage de la vitamine B6 (y compris ses formes glycosylées) par CLHP

Le présent document spécifie une méthode de dosage de la vitamine B 6 présente dans les produits
alimentaires, par chromatographie liquide à haute performance (CLHP).
La vitamine B 6 est la fraction massique de la somme de la pyridoxine, du pyridoxal et de la pyridoxamine, y
compris leurs dérivés phosphorylés ainsi que les formes b-glycosylées calculés en tant que pyridoxine.
La présente méthode a été validée avec succès avec de la semoule au lait (aliment pour bébé), de la purée
de pommes de terre, des légumes au jambon (produits tout préparés) et avec une boisson multivitaminée, à
des niveaux de concentration de 0,034 mg/100 g à 1,21 mg/100 g.

Živila – Določevanje vitamina B6 (vključno z glikosilirano obliko) s HPLC

General Information

• Status: Published
• Publication Date: 06-Dec-2005
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 9 - Vitamins and Carotenoids
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Completion Date: 03-Sep-2021
• Ref Project: SIST EN 14663:2006 - Foodstuffs - Determination of vitamin B6 (including its glycosylated forms) by HPLC

Standards Content (Sample)

SLOVENSKI STANDARD
01-marec-2006
äLYLOD±'RORþHYDQMHYLWDPLQD%YNOMXþQR]JOLNRVLOLUDQRREOLNRV+3/&
Foodstuffs - Determination of vitamin B6 (including its glycosylated forms) by HPLC
Lebensmittel - Bestimmung von Vitamin B6 (einschließlich glucosidisch gebundener
Verbindungen) mit HPLC
Produits alimentaires - Dosage de la vitamine B6 (y compris ses formes glycosylées) par
CLHP
Ta slovenski standard je istoveten z: EN 14663:2005
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN 14663
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2005
ICS 67.050
English Version
Foodstuffs - Determination of vitamin B6 (including its
glycosylated forms) by HPLC
Produits alimentaires - Dosage de la vitamine B6 (y Lebensmittel - Bestimmung von Vitamin B6 (einschließlich
compris ses formes glycosylées) par CLHP glucosidisch gebundener Verbindungen) mit HPLC
This European Standard was approved by CEN on 26 October 2005.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2005 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14663:2005: E
worldwide for CEN national Members.

Contents Page
Foreword .3
1 Scope.4
2 Normative references.4
3 Principle.4
4 Reagents.4
5 Apparatus.9
6 Procedure.9
7 Calculation.11
8 Precision.12
9 Test report.14
Annex A (informative)  Precision data .15
Annex B (informative) Examples for suitable HPLC-conditions for the determination of vitamin B
compounds.19
Annex C (informative)  Examples for molar extinction coefficients .20
Annex D (informative) Figures.21
Bibliography.22

Figure
Figure D.1 — Standard substances and sample potato puree.21

Tables
Table 1 — Examples for molecular extinction coefficients of vitamin B6 compounds.7
Table A.1 — Precision data for Semolina with milk, powder.15
Table A.2 — Precision data for Potato puree,powder.16
Table A.3 — Precision data for vegetables with ham (baby food) .17
Table A.4 — Precision data for multi vitamin drink.18
Table B.1 — Examples for suitable HPLC-conditions for the determination of vitamin B6
compounds .19
Table C.1 — Examples for molar extinction coefficients (E) of vitamin B6 compounds [3], [4] .20

Foreword
This document (EN 14663:2005) 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 June 2006, and conflicting national standards shall be withdrawn at
the latest by June 2006.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic,
Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland
and United Kingdom.
1 Scope
This document specifies a method for the determination of vitamin B in foodstuffs by high performance liquid
chromatography (HPLC).
Vitamin B is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their
phosphorylated derivatives as well as the β-glycosylated forms, calculated as pyridoxine.
This method has been successfully validated with semolina with milk (infant food), potato puree, vegetables
with ham (convenient products) and a multi vitamin drink at levels from 0,034 mg/100 g to 1,21 mg/100 g.
2 Normative references
The following referenced documents are indispensable for the application of this document. 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 3696:1987).
3 Principle
Pyridoxal, pyridoxamine and pyridoxine are extracted from food by acid hydrolysis and dephosphorylated and
deglycosilated enzymatically using acid phosphatase and β-glucosidase.
The different derivatives of vitamin B (pyridoxal, pyridoxamine and pyridoxine) are separated by HPLC and
quantified by fluorometric detection [1], [2].
4 Reagents
4.1 General
During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at
least grade 1 according to EN ISO 3696, or double distilled water.
4.2 Di-potassium hydrogen phosphate, mass fraction w(K HPO · 3 H O) ≥ 99,9 %
2 4 2
4.3 Sodium acetate, without crystal water, w(CH COONa) ≥ 99,0 %
4.4 Trichloroacetic acid (TCA), w(Cl CCOOH) ≥ 99,0 %
4.5 Sodium acetate solution, substance concentration c(CH COONa) = 2,5 mol/l
Dissolve 205 g of sodium acetate (4.3) in 1 l of water.
4.6 Post-column reagent (optional), K HPO solution c(K HPO ) = 0,15 mol/l
2 4 2 4
Dissolve 34,2 g of di-potassium hydrogen phosphate (4.2) in water, dilute to 1 000 ml, mix and degas.
4.7 Hydrochloric acid, c(HCl) = 1 mol/l
c(HCl) = 0,1 mol/l
4.8 Hydrochloric acid,
4.9 Hydrochloric acid, c(HCl) = 0,2 mol/l
4.10 Sulfuric acid, c(H SO )= 1 mol/l
2 4
4.11 Light petroleum, boiling range of 40 °C to 60 °C
1)
4.12 Acid phosphatase, from potatoes. Enzymatic activity approximately 5,3 U/mg .
It is important that the enzyme used complies with the activity check 4.13.2, for further information see [2], [7].
4.13 Acid phosphatase solution
4.13.1 General
Dissolve/solubilise 60 mg of acidic phosphatase (4.12) in 10 ml of water in a beaker by stirring for 2 min.
Prepare this solution on the day of analysis.
4.13.2 Activity check of Acid Phosphatase
Weigh 10 g of pork, 5 g of potato puree or 5 g of whole meal into a beaker, and extract with acid as described
in 6.2.1. Add 1 ml of acid phosphatase solution (4.13.1) and optional 1 ml of β-glucosidase solution (4.15) to
12,5 ml of the extracted sample solution and mix. Incubate the solution at least 12 h or overnight at 37 °C with
continuous stirring. Repeat this step with the double amount of acid phosphatase solution.
Determine the mass concentration of vitamins according to 6.6. The activity of the enzyme used is sufficient, if
the resulting mass concentrations of vitamin B compounds in both sample solutions are equivalent. The
chromatogram shall not show a peak arising from pyridoxamin phosphate.
)
NOTE For the interlaboratory test, the acid phosphatase from Sigma Nr P 3752 has been used.
4.14 ββ-Glucosidase, from almonds. Enzymatic activity of approximately 3,2 U/mg.
ββ
It is important that the enzyme used complies with the activity check 4.15.2, for further information see [2], [7].
4.15 ββββ-Glucosidase solution
4.15.1 General
Dissolve/solubilise 100 mg of β-glucosidase (4.14) in 10 ml of water in a beaker by stirring for 2 min. Prepare
this solution on the day of analysis.

1)
U, this unit (often called the International unit or standard unit) is defined as the amount of enzyme which catalyses
the transformation of 1 µmol substrate per minute under standard conditions.
) This information is given for the convenience of users of this document 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.
4.15.2 Activity check of ββ-glucosidase
ββ
Weigh 10 g of pork, 5 g of potato puree or 5 g of whole meal into a beaker, and extract with acid as described
in 6.2.1. Add 1 ml of acid phosphatase solution (4.13.1) and 1 ml of β-glucosidase solution (4.15.1) to 12,5 ml
of the extracted sample solution and mix. Incubate the solution at least 12 h or overnight at 37 °C with
continuous stirring. Repeat this step with the double amount β-glucosidase solution.
Determine the mass concentration of vitamin B compounds according to 6.6. The activity of the enzyme used
is sufficient, if the resulting mass concentrations of vitamin B compounds in both sample solutions are
equivalent. The chromatogram shall not show a peak arising from pyridoxamin phosphate.
)
NOTE For the interlaboratory test, the β-glucosidase from Sigma Nr G-0395 has been used.
4.16 Mobile phase for HPLC (Sulfuric acid, c(H SO )= 0,015 mol/l containing 0,005 mol/l TCA)
2 4
Dissolve 817 mg ± 5 mg of trichloroacetic acid (4.4) in 15 ml of 1 mol/l sulfuric acid (4.10), transfer into a 1 000 ml
volumetric flask, dilute to the mark with water, mix and degas.
4.17 Silicon oil, for defoaming
4.18 Standard substances
4.18.1 General
Pyridoxamine (PM), Pyridoxal (PL) and pyridoxine (PN) can be obtained from various suppliers. The purity of
the standards may vary, and it is therefore necessary to determine the concentration and purity (see 4.19.4
and 4.20.7).
4.18.2 Pyridoxamine (PM) dihydrochloride, w(C H N O · 2HCl) ≥ 98 %
8 12 2 2
4.18.3 Pyridoxal (PL) hydrochloride, w(C H NO · HCl) ≥ 98 %
8 9 3
4.18.4 Pyridoxine (PN) hydrochloride, w(C H NO · HCl ) ≥ 98 %
8 11 3
4.19 Stock solutions
4.19.1 Pyridoxamine (PM) stock solution, mass concentration ρ(PM) approximately 500 µg/ml
Dissolve 71,7 mg of pyridoxamine dihydrochloride (4.18.2) in a 100 ml volumetric flask in 0,1 mol/l HCl (4.8) and
dilute to the mark with 0,1 mol/l HCl. The solution can be stored without any losses for up to one week at 4 °C or
up to two months at -18 °C.
4.19.2 Pyridoxal (PL) stock solution, ρ(PL) approximately 500 µg/ml
Dissolve 60,9 mg of pyridoxal hydrochloride (4.18.3) in a 100 ml volumetric flask in 0,1 mol/l HCl (4.8) and dilute
to the mark with 0,1 mol/l HCl. The solution can be stored without any losses for up to one week at 4 °C or up to
two months at -18 °C.
1)
This information is given for the convenience of users of this document 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.
4.19.3 Pyridoxine (PN) stock solution, ρ(PN) approximately 500 µg/ml
Dissolve 60,8 mg of pyridoxine hydrochloride (4.18.4) in a 100 ml volumetric flask in 0,1 mol/l HCl (4.8) and dilute
to the mark with 0,1 mol/l HCl. The solution can be stored without any losses for up to one week at 4 °C or up to
two months at – 18 °C.
4.19.4 Concen
...