SIST EN 14164:2008

SLOVENSKI STANDARD
SIST EN 14164:2008
01-september-2008
1DGRPHãþD
SIST ENV 14164:2002
äLYLOD'RORþHYDQMHYLWDPLQD%V+3/&
Foodstuffs - Determination of vitamin B6 by HPLC
Lebensmittel - Bestimmung von Vitamin B6 mit HPLC
Produits alimentaires - Dosage de la vitamine B6 par CLHP
Ta slovenski standard je istoveten z: EN 14164:2008
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN 14164:2008 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 14164:2008

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SIST EN 14164:2008


EUROPEAN STANDARD
EN 14164

NORME EUROPÉENNE

EUROPÄISCHE NORM
June 2008
ICS 67.050 Supersedes ENV 14164:2002
English Version
Foodstuffs - Determination of vitamin B by HPLC
6
Produits alimentaires - Dosage de la vitamine B par CLHP Lebensmittel - Bestimmung von Vitamin B mit HPLC
6 6
This European Standard was approved by CEN on 5 December 2007.

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 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 Management Centre has the same status as the
official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, 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
© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14164:2008: E
worldwide for CEN national Members.

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SIST EN 14164:2008
EN 14164:2008 (E)
Contents Page
Foreword.3
1 Scope .4
2 Normative references .4
3 Principle.4
4 Reagents.4
5 Apparatus .8
6 Procedure .9
7 Calculation.10
8 Test report .10
Annex A (informative) Example of a chromatogram.12
Annex B (informative) Precision data.13
Annex C (informative) Sample treatment option without acid hydrolysis.15
Annex D (informative) Examples for molar absorption coefficients.16
Bibliography .17

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SIST EN 14164:2008
EN 14164:2008 (E)
Foreword
This document (EN 14164:2008) 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 2008, and conflicting national standards shall be withdrawn
at the latest by December 2008.
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 ENV 14164:2002.
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, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
3

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SIST EN 14164:2008
EN 14164:2008 (E)
1 Scope
This European Standard specifies a method for the determination of vitamin B in foodstuffs by high
6
performance liquid chromatography (HPLC). Vitamin B is the mass fraction of the sum of pyridoxine,
6
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 vitamers of vitamin B (pyridoxal, pyridoxamine and pyridoxine) are separated and
6
1
individually quantified. A third European Standard (EN 14166 ) [2] determines the total vitamin B by
6
microbiological assay.
2 Normative references
The following referenced document is 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
enzymatically using acid phosphatase.
2+
By reaction with glyoxylic acid in the presence of Fe 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
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 Chemicals and solutions
2)
4.2.1 Acid phosphatase, (CAS 9001-77-8), from potatoes, enzyme activity is 33 nkat/mg with substrate
3)
p-nitrophenyl phosphate at pH = 4,8 and T = 37 °C, for example from Boehringer or Sigma . 33 nkat/mg
corresponds to 2 U/mg.
4.2.1.1 Acid phosphatase solution
Prepare a solution of 20 mg/ml acid phosphatase in sodium acetate solution (4.2.14).

1
Under elaboration.
2) 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.
3) 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.
4

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SIST EN 14164:2008
EN 14164:2008 (E)
It is necessary to use acid phosphatase rather than Taka-diastase to obtain a complete hydrolysis of
phosphorylated forms of vitamin B. Where 300 mg of Taka-diastase is needed to obtain good
6
dephosphorylation, only 0,5 mg of acid phosphatase is needed, see [5].
4.2.1.2 Activity check of acid phosphatase
Check the activity of each new batch of acid phosphatase as follows. Prepare a stock solution of
approximately 0,1 mg/ml of pyridoxal phosphate (4.2.9) in water. Take 5,0 ml of this solution for extraction and
procede with 6.2.1, 6.2.2, 6.2.3, 6.2.4 and 7.
Calculate the pyridoxine recovery from this solution and divide by the theoretical amount of pyridoxine
released from pyridoxal phosphate (PLP). Calculate the theoretical mass concentration ρ of PN released
PN
from PLP, in milligram per millilitre according to equation (1):
ρ × M × 2× 5,0
PLP UV PN
ρ = (1)
PN
100× M
PLP
where
ρ is the mass concentration of PLP determined by UV spectrometry;
PLP UV
M is the molecular weight of vitamin B standard substance, in gram per mol (M = 169.1);
PN 6 PN
2 is the factor of dilution of the reaction with sodium borohydride;
5,0 is the volume taken for extraction, see 4.2.1.2;
100 is the total volume of the sample test solution, in millilitres;
M is the molecular weight of PLP, in gram per mol (M = 265,16).
PLP PLP
Mix 3,0 ml of the PLP stock solution and 10 ml of hydrochloric acid (4.2.21) in a 20 ml volumetric flask and fill
up to the mark with water. Check the concentration of PLP by measuring the absorbance at 295 nm in a 1 cm
cell using a UV-spectrometer (5.2) against a hydrochloric acid solution (4.2.20) as reference. Molar absorption
coefficient (ε) of PLP in 0,1 mol/l HCl is 8 353.
Calculate the mass concentration ρ of the stock solution, in milligram per millilitre, according to equation
PLP
(2):
A × M
295 PLP
ρ = × F (2)
PLP
ε
where
A is the absorption of the value of the solution at 295 nm;
295
M is the molecular weight of vitamin B standard substance, in gram per mol (M = 265,16);
PLP 6 PLP
F is the dilution factor (here F = 20/3);
-1 -1
ε is the molar absorption coefficient of PLP in 0,1 mol/l of hydrochloric acid at 295 nm, in l mol cm
(here ε = 8 353).
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SIST EN 14164:2008
EN 14164:2008 (E)
4.2.2 Sodium acetate, trihydrate, mass fraction w(CH COONa · 3H 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) ≥ 99,0 %
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 Pyridoxal phosphate (PLP), w ≥ 99,0 %
4.2.10 Orthophosphoric acid, w(H PO ) ≥ 84,0 %
3 4
4.2.11 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
4.2.12 Acetonitrile (HPLC grade), w(C H N) ≥ 99,8 %
2 3
4.2.13 Sodium acetate solution, substance concentration 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.
4.2.14 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).
4.2.15 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 (4.2.14).
Prepare fresh each day of use.
NOTE In a study described by Mann et al., see [10], 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 B required by the infant formula Act in the US, see Mann et al. [9]. This concentration seems not
6
to be necessary for the European situation.
4.2.16 Sodium hydroxide solution, c(NaOH) = 0,2 mol/l
Dissolve 800 mg of sodium hydroxide (4.2.6) in 100 ml of water.
4.2.17 Sodium hydroxide solution, c(NaOH) = 6,0 mol/l
Dissolve 24 g of sodium hydroxide (4.2.6) in 100 ml of water.
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SIST EN 14164:2008
EN 14164:2008 (E)
4.2.18 Sodium borohydride solution, c(NaBH ) = 0,1 mol/l
4
Dissolve 378 mg of sodium borohydride (4.2.7) in 100 ml of sodium hydroxide solution (4.2.16). Prepare fresh
on day of use.
4.2.19 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 (4.2.13). Adjust the
pH to 4,5 with the sodium hydroxide solution (4.2.17) and dilute to 50 ml with water in a volumetric flask.
Prepare fresh on day of use.
4.2.20 Hydrochloric acid, c(HCl) = 0,1 mol/l
4.2.21 Hydrochloric acid, c(HCl) = 0,2 mol/l
4.2.22 HPLC mobile phase
In a beaker add 940 ml of water, 40 ml of acetonitrile (4.2.12), 160 mg of sodium octanesulfonate or sodium
heptanesulfonate (4.2.11) and 6,8 g of potassium dihydrogen phosphate (4.2.8).
After dissolving sodium octanesulfonate or sodium heptanesulfonate and potassium dihydrogen phosphate by
stirring, adjust the pH to 2,5 with orthophosphoric acid (4.2.10). Transfer the solution in a 1 l volumetric flask.
Adjust to the mark with water. Filter through a 0,45 µm filter.
4.3 Pyridoxine hydrochloride (Vitamin B standard substance), w(C H NO · HCI) ≥ 99 %
6 8 11 3
4.4 Pyridoxine hydrochloride stock solution, mass concentration ρ ≈ 0,5 mg/ml
Dissolve an accurately weighed amount of pyridoxine hydrochloride (4.3), e.g. approximately 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 (4.4) to 20 ml with 0,1 mol/l
HCI (4.2.20) and measure the absorbance at 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 ρ, in microgram per millilitre of the stock
solution according to equation (3):
A × M ×1000
290 PNHCl
ρ = × F (3)
PNHCl
ε
where
A is the absorption of the value of the solution at 290 nm;
290
M is the molecular weight of vitamin B standard substance, in gram per mol (M = 205,64);
PN 6 PN
F is the dilution factor (here F = 40);
ε is the molar absorption coefficient of pyridoxine hydrochloride in 0,1 mol/l of hydrochloric acid at
-1 -1
291 nm, in l mol cm (here ε = 8 600), see [6] .
Further information on molar absorption coefficients in other solutions than 0,1 mol/l HCl (pH ≈ 1) is given in
Annex D.
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SIST EN 14164:2008
EN 14164:2008 (E)
4.5 Standard solutions
4.5.1 Pyridoxine hydrochloride intermediate standard solution, ρ(C H NO · HCI) ≈ 10 µg/ml
8 11 3
Pipette 1 ml of the vitamin B stock solution (4.4) into a 50 ml volumetric flask and dilute to the mark with
6
water. Prepare this solution each day of analysis.
4.5.2 Pyridoxine hydrochloride standard test solutions for HPLC, ρ(C H NO · HCI) ≈ 0,1 µg/ml to 1
8 11 3
µg/ml
Prepare a series of appropriate test stand
...