ENV 14164:2002
(Main)Foodstuffs - Determination of vitamin B6 by HPLC
Foodstuffs - Determination of vitamin B6 by HPLC
This European Prestandard specifies a method for the determination of vitamin B6 in foodstuffs by HPLC.
Vitamin B6 is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives determined as pyridoxin. The b-glycosylated forms are not taken into account.
Lebensmittel - Bestimmung von Vitamin B6 mit HPLC
Diese Europäische Vornorm legt ein Verfahren zur Bestimmung von Vitamin B6 in Lebensmitteln mit Hochleistungs-Flüssigchromatographie (HPLC) fest.
Vitamin B6 ist der Massenanteil der Gesamtmenge an Pyridoxin, Pyridoxal und Pyridoxamin einschließlich ihrer phosphorylierten Derivate, die als Pyridoxin bestimmt werden. Die b-glycosilierten Formen werden nicht berücksichtigt.
Produits alimentaires - Détermination de la vitamine B6 par CLHP
Cette Prénorme européenne spécifie une méthode de dosage de la vitamine B6 présente dans les produits alimentaires par chromatographie en phase liquide à haute performance (CLHP).
La vitamine B6 est la fraction massique de la somme de pyridoxine, pyridoxal, pyridoxamine y compris leurs dérivés phosphorylés identifiés comme étant de la pyridoxine. Les dérivés b-glycosilés ne sont pas pris en considération.
Živila - Določevanje vitamina B6 s HPLC
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST ENV 14164:2002
01-junij-2002
äLYLOD'RORþHYDQMHYLWDPLQD%V+3/&
Foodstuffs - Determination of vitamin B6 by HPLC
Lebensmittel - Bestimmung von Vitamin B6 mit HPLC
Produits alimentaires - Détermination de la vitamine B6 par CLHP
Ta slovenski standard je istoveten z: ENV 14164:2002
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST ENV 14164:2002 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST ENV 14164:2002
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SIST ENV 14164:2002
EUROPEAN PRESTANDARD
ENV 14164
PRÉNORME EUROPÉENNE
EUROPÄISCHE VORNORM
February 2002
ICS 67.050
English version
Foodstuffs - Determination of vitamin B6 by HPLC
Produits alimentaires - Détermination de la vitamine B6 par Lebensmittel - Bestimmung von Vitamin B6 mit HPLC
CLHP
This European Prestandard (ENV) was approved by CEN on 15 November 2001 as a prospective standard for provisional application.
The period of validity of this ENV is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the ENV can be converted into a European Standard.
CEN members are required to announce the existence of this ENV in the same way as for an EN and to make the ENV available promptly
at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the ENV) until the final
decision about the possible conversion of the ENV into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, 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
© 2002 CEN All rights of exploitation in any form and by any means reserved Ref. No. ENV 14164:2002 E
worldwide for CEN national Members.
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SIST ENV 14164:2002
ENV 14164:2002 (E)
Foreword
This European Prestandard has been prepared by Technical Committee CEN/TC 275, "Food analysis - Horizontal
methods", the secretariat of which is held by DIN.
The annexes A, B, C and D are informative.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following coun-
tries are bound to announce this European Prestandard: Austria, Belgium, Czech Republic, Denmark, Finland,
France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Malta, Netherlands, Norway, Portugal, Spain, Swe-
den, Switzerland and the United Kingdom.
1 Scope
This European Prestandard specifies a method for the determination of vitamin B in foodstuffs by HPLC.
6
Vitamin B is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated
6
derivatives determined as pyridoxine. The -glycosylated forms are not taken into account.
2 Normative references
This European Prestandard incorporates by dated or undated reference, provisions from other publications. These
normative references are cited at the appropriate places in the text, and the publications are listed hereafter. For
dated references, subsequent amendments to or revisions of any of these publications apply to this European
Prestandard only when incorporated in it by amendment or revision. For undated references the latest edition of the
publication referred to applies (including amendments).
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
enzymatically using acid phosphatase.
By reaction with glyoxylic acid in presence of Fe++ as 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 [1], [2].
It is also possible to quantify pyridoxine, pyridoxal and pyridoxamine separately and to sum the different contents
obtained. In that case all risk of interference with the matrix will have to be verified and the analytical conditions
adapted. The procedure is not described in this European Prestandard because it has not been validated. Adapted
conditions can be found in the bibliography [1] to [4].
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.
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SIST ENV 14164:2002
ENV 14164:2002 (E)
4.2 Chemicals and solutions
1)
4.2.1 Acid phosphatase, from potatoes (it can be obtained from Boehringer) Enzymatic activity: 2 U/mg.
4.2.2 Sodium acetate, trihydrate, w (CH COONa 3 H O) 99,0 %
3 2
4.2.3 Glacial acetic acid, w (CH COOH) 99,8 %
3
4.2.4 Glyoxylic acid, w (C H O H O) 97,0 %
2 2 3 2
4.2.5 Ferrous sulfate II, heptahydrate, w (FeSO 7H O) 99,5 %
4 2
4.2.6 Sodium hydroxide, w (NaOH) 98,0 %
4.2.7 Sodium borohydride, w (NaBH ) 97,0 %
4
4.2.8 Potassium dihydrogen phosphate, w (KH PO ) 99,0 %
2 4
4.2.9 Orthophosphoric acid, w (H PO ) 84,0 %
3 4
4.2.10 Sodium octanesulfonate, w (C H NaO S) 98,0 %, or sodium heptanesulfonate,
8 17 3
w (C H NaO S) 98,0 %
7 15 3
4.2.11 Acetonitrile (HPLC grade), w (C H N) 99,8 %
2 3
4.2.12 Sodium acetate solution, c (CH COONa · 3H O) = 2,5 mol/l
3 2
Dissolve 170,1 g of sodium acetate, trihydrate (4.2.2) in 500 ml of water.
4.2.13 Sodium acetate solution,c (CH COONa · 3H O) = 0,05 mol/l (pH 4,5):
3 2
Dissolve 6,8 g of sodium acetate, trihydrate (4.2.2) in 1 l of water. Adjust the pH to 4,5 with glacial acetic acid
(4.2.3).
4.2.14 Ferrous sulfate solution,c (FeSO · 7H O) = 0,0132 mol/l
4 2
Dissolve 36,6 mg of ferrous sulfate II, heptahydrate (4.2.5) in 10 ml of sodium acetate solution (4.2.13).
4.2.15 Sodium hydroxide solution,c (NaOH) = 0,2 mol/l
Dissolve 800 mg of sodium hydroxide (4.2.6) in 100 ml of water.
c (NaBH ) = 0,1 mol/l
4.2.16 Sodium borohydride solution,
4
Dissolve 378 mg of sodium borohydride (4.2.7) in 100 ml of sodium hydroxide solution (4.2.15).
4.2.17 Glyoxylic acid solution,c (C H O · H O) = 1 mol/l (pH 4.5):
2 2 3 2
Dissolve 4,7 g of glyoxylic acid monohydrate (4.2.4) in 30 ml of sodium acetate solution (4.2.12). Adjust the pH to
4,5 with the sodium acetate solution (4.2.12) and dilute to 50 ml with water in a volumetric flask.
1) This information is given for the convenience of users of this standard method 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.
3
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SIST ENV 14164:2002
ENV 14164:2002 (E)
4.2.18 Hydrochloric acid,c (HCl) = 0,1 mol/l
4.2.19 HPLC mobile phase
In a beaker add 940 ml of water, 40 ml of acetonitrile (4.2.11), 160 mg of sodium octanesulfonate or sodium hep-
tanesulfonate (4.2.10) and 6,8 g of potassium dihydrogen phosphate (4.2.8).
After shaking, adjust the pH to 2,5 with orthophosphoric acid (4.2.9). Transfer the solution in a 1 l volumetric flask.
Adjust to the mark with water.
Filter through a 0,45 μm filter.
4.3 Vitamin B standard substance
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Pyridoxine hydrochloride, w (C H NO · HCl) 99 %
8 11 3
4.4 Stock solution
4.4.1 Vitamin B stock solution, (C H NO · HCl) 0,5 mg/ml
6 8 11 3
Dissolve an accurately weighed amount of the vitamin B standard substance (4.3), e.g. approximately 50 mg in a
6
defined volume, e.g. 100 ml, of water. The stock solution is stable for 4 weeks if stored at 4 °C in the dark.
4.4.2 Concentration test
Dilute 0,5 ml of vitamin B stock solution (4.4.1) to 20 ml with 0,1 mol/l HCI (4.2.18) and measure the absorbance at
6
290 nm in a 1 cm cell using a UV-spectrometer (5.2) against 0,1 mol/l HCl solution as reference.
Calculate the mass concentration , in microgram per millilitre of the stock solution according to equation (1):
A M
w
F (1)
8,6
where
A is the absorbance value of the solution at 290 nm;
M is the molecular weight of vitamin B standard substance, in gram per mol;
w 6
F is the dilution factor, i.e. 40.
8,6 is the molar extinction coefficient of pyridoxine hydrochloride in 0,1 mol/l hydrochloric acid at 290 nm [4],
-1 -1
[5], in mmol cm ;
Further information on molar extinction coefficients in other solutions than 0,1 mol/l HCl (pH ~ 1) can be seen in
annex D.
4.5 Standard solutions
4.5.1 Vitamin B intermediate standard solution, (C H NO · HCl) 10 μg/ml
6 8 11 3
Pipette 1 ml of the vitamin B stock solution (4.4.1) into a 50 ml volumetric flask and dilute to the mark with water.
6
Prepare this solution each day of analysis.
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SIST ENV 14164:2002
ENV 14164:2002 (E)
4.5.2 Vitamin B standard test solutions for HPLC
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H NO · HCl)~0,1μg/ml to 1μg/ml
8 11 3
Prepare a series of appropriate standard solutions of concentrations ranging from e.g. 0,1 μg/ml to 1 μg/ml of pyri-
doxine hydrochloride by using the pyridoxine intermediate standard solution (4.5.1).
Prepare these solutions each day of analysis.
5 Apparatus
5.1 General
Usual laboratory apparatus, glassware, and, the following:
5.2 UV Spectrometer
Capable of measurement of absorbance at defined wavelengths.
5.3 Heating device
Oven or water bath, with shaking facilities.
5.4 High performance liquid chromatographic system
Consisting of a pump, sample injecting device, fluorescence detector with excitation and emission wavelengths set
at 290 nm and 395 nm, respectively and an evaluation system such as an integrator.
®
2)
5.5 HPLC-Column, e.g. reversed phase column, such as LiChrospher 60 RP C8 Select B , particle size of
5 μm, diameter 4,0 mm, length 250 mm.
Other particle sizes or column dimensions than specified in this European Prestandard may be used. Separation
parameters have to be adapted to such materials to guarantee equivalent results. The performance criterion for
suit
...
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