BS EN 14164:2014

BSI Standards Publication

Foodstuffs — Determination
of vitamin B6 by high
performance chromatography
BS EN 14164:2014 BRITISH STANDARD

National foreword
This British Standard is the UK implementation of EN 14164:2014. It
supersedes BS EN 14164:2008 which is withdrawn.
The UK participation in its preparation was entrusted to Technical
Committee AW/275, Food analysis - Horizontal methods.
A list of organizations represented on this committee can be
obtained on request to its secretary.
This publication does not purport to include all the necessary
provisions of a contract. Users are responsible for its correct
application.
© The British Standards Institution 2014.
Published by BSI Standards Limited 2014

ISBN 978 0 580 77941 1

ICS 67.050

Compliance with a British Standard cannot confer immunity from

legal obligations.
This British Standard was published under the authority of the
Standards Policy and Strategy Committee on 30 June 2014.
Amendments/corrigenda issued since publication
Date Text affected
 BS EN 14164:2014

EUROPEAN STANDARD EN 14164

NORME EUROPÉENNE
EUROPÄISCHE NORM June 2014

ICS 67.050 Supersedes EN 14164:2008

English Version

Foodstuffs - Determination of vitamin B6 by high performance

chromatography

Produits alimentaires - Détermination de la teneur en Lebensmittel - Bestimmung von Vitamin B6 mit

vitamine B6 par chromatographie liquide haute performance 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 Ref. No. EN 14164:2014 E
worldwide for CEN national Members.
BS EN 14164:2014
EN 14164:2014 (E)

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

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 BS EN 14164:2014
EN 14164:2014 (E)

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.

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EN 14164:2014 (E)

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 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.

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.
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

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/mg 1) with substrate p-
nitrophenyl phosphate at pH = 4,8 and T = 37 °C, for example from Boehringer or Sigma 2 ). 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.

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EN 14164:2014 (E)

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 (ε) of PLP in 0,1 mol/l HCl is 7 200.

Calculate the mass concentration ρPLP of the stock solution, in milligram per millilitre, according to Formula (1):

A293 ⋅ M PLP
ρPLP = ⋅F (1)
ε

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);
ε is the molar absorption coefficient of PLP in 0,1 mol/l of hydrochloric acid at 293 nm, in
−1 −1
l mol cm , (here: ε = 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):

ρ PN⋅HCl ⋅ AS ⋅ 2 ⋅100 ⋅ 0,822 ⋅100 ⋅ M PLP

Conversion rate (%) = (2)
AST ⋅1 000 ⋅ ρ PLP ⋅ M PN

where

ρPN 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;
ρPLP 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).

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EN 14164:2014 (E)

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).

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EN 14164:2014 (E)

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 μm filter.

4.23 Pyridoxine hydrochloride (vitamin B6 standard substance), w(C8H11NO3 HCI) ≥ 99 %.

4.24 Pyridoxine hydrochloride stock solution, mass concentration ρ ≈ 0,5 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 ρ, in microgram per millilitre of the stock
solution according to Formula (3):

A290 ⋅ M PNHCl ⋅ 1 000

ρ PNHCl = ⋅F (3)
ε

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 = 40);
ε is the molar absorption coefficient of pyridoxine hydrochloride in 0,1 mol/l of hydrochloric acid
−1 −1
at 291 nm, in l mol cm (here ε = 8 600), see [9].

Further information on molar absorption coefficients in other solutions than 0,1 mol/l HCl (pH ≈ 1) is given in
Annex D.

4.25 Standard solutions

4.25.1 Pyridoxine hydrochloride intermediate standard solution, ρ(C8H11NO3 · HCI) ≈ 10 μg/ml.

Pipette 1 ml of the vitamin B6 stock solution (4.24) into a 50 ml volumetric flask and dilute to the mark with
water. Prepare this solution each day of analysis.

4.25.2 Pyridoxine hydrochloride standard test solution for HPLC, ρ(C8H11NO3 · HCI) ≈ 0,1 μg/ml to
1 μg/ml.

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EN 14164:2014 (E)

Prepare a series of appropriate test standard solutions of concentrations ranging from e.g. 0,1 μg/ml to
1 μg/ml of pyridoxine hydrochloride by using the pyridoxine intermediate standard solution (4.25.1). Prepare
these solutions fresh on day of use.

Perform a system suitability test by injecting a mixed standard test solution for HPLC of pyridoxine (PN) and
pyridoxal (PL). PL and PN should have about the concentration in the standard test solution for HPLC. PL
elutes before PN. The PN/PL pair shall have baseline separation. Add pyridoxamine (PM) as spike recovery in
one sample run to ensure complete deamination and oxidation.

5 Apparatus

Usual laboratory apparatus, glassware, and, the following:

5.1 UV Spectrometer, capable of measurement of absorbance at defined wavelengths.

5.2 Heating device, oven or water bath, with shaking facilities.

5.3 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.
®
5.4 HPLC column, e.g. reversed phase column, such as LiChrospher 60 RP C8 Select B 3), particle size
of 5 μm, diameter 4,0 mm, length 250 mm.

Other particle sizes or column dimensions than specified in this European Standard may be used. Separation
parameters shall be adapted to such materials to guarantee equivalent results. The performance criterion for
suitable analytical columns is the baseline resolution of the analytes concerned.

5.5 Filter device, filtering of the mobile phase as well as of the test sample solution through a membrane
filter, with e.g. a pore size of 0,45 μm, prior to use or injection will increase longevity of the columns.

6 Procedure

6.1 Preparation of the test sample

Homogenize the test sample. Grind coarse material with an appropriate mill and mix again. Measures such as
pre-cooling shall be taken to avoid exposing to high temperature for long periods of time.

6.2 Preparation of the sample test solution

6.2.1 Extraction

Weigh an appropriate amount of the sample to the nearest milligram, e.g. 2,5 g (if the vitamin B6 content
exceeds 2 μg/g) or 5 g (if the vitamin content is less than 2 μg/g) in a conical flask. Add 50 ml of hydrochloric
acid (4.20). Heat for 30 min in a boiling water bath.

NOTE 1 For samples, with a high water content or low contents of vitamin B6, it can be useful to increase the sample
weight, e.g. 20 g, to add an adapted volume of water, e.g. 25 ml, and to add directly 5 ml of hydrochloric acid
c (HCl) = 1 mol/l.

3) LiChrospher® 60 RP C8 Select B is an example of a suitable product available commercially. This information is given
for the convenience of users and does not constitute an endorsement by CEN of the product named. Equivalent products
can be used if they lead to equivalent results.

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EN 14164:2014 (E)

NOTE 2 For samples with a high fat content it can be useful to remove fat with e.g. light petroleum before the acid
hydrolysis.

NOTE 3 The main advantage of a preliminary acid hydrolysis is to improve the filtration step for samples with starch.
The existing data in Annex B have been mainly obtained without any acid hydrolysis. A modification of the procedure
(without acid hydrolysis) is described in Annex C.

6.2.2 Enzyme treatment and transformation steps

After cooling to room temperature, adjust the extract to pH 4,5 with sodium acetate solution (4.13) and add
2,5 ml of 1 mol/l glyoxylic acid solution (4.19), 400 μl of ferrous sulfate solution (4.15) and 1 ml of acid
phosphatase solution (4.1.1). Incubate the solution overnight at 37 °C with continuous agitating.

After cooling to room temperature, dilute to 100 ml with water in a volumetric flask. Shake and filter. Mix 5,0 ml
of filtrate and 4,5 ml of 0,1 mol/l sodium borohydride solution (4.18). Shake at least for 3 min. In order to
ensure the complete destruction of the excess of sodium borohydride, it is possible to add 0,5 ml of glacial
acetic acid (4.3). Take care for the dilution factor. Shake for 1 min. Filter through 0,45 μm filter. Use this filtrate
for chromatography.

6.2.3 Identification

Identify the pyridoxine by comparison of the retention time of the individual peaks in the chromatograms
obtained with the test sample solution, and with the standard test solution. Peak identification can also be
performed by adding the pyridoxine standard substance to the sample test solution.

The separation and the quantification have proven to be satisfactory if following experimental conditions are
followed (see also Figure A.1).

®
Stationary phase: LiChrospher 60 RP C8 Select B, 5 μm, 250 mm x 4,0 mm;
Mobile phase: according to 4.22;
Flow rate: 1 ml/min;
Injection volume: 30 μl;
Detection: Fluorometric: Excitation: 290 nm; Emission: 395 nm.

6.2.4 Determination by HPLC

Inject the same appropriate volumes (up to 50 μl) of the standard solution as well as of the sample test
solution into the HPLC-system. To carry out a determination by external calibration, integrate the peak areas
or peak heights and compare the results with the corresponding values for the standard substance.

7 Calculation

Base the calculation on a calibration graph, or use the corresponding programs of the integrator, or use the
following simplified procedure.

Calculate the mass fraction, w, of vitamin B6 as pyridoxine in mg/100 g of the sample using Formula (4):

A s ⋅ ρ ⋅ 100 ⋅ 2
w= ⋅ 100 ⋅ 0,822 (4)
A st ⋅ m ⋅ 1000

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EN 14164:2014 (E)

where

As is the peak area or peak height for pyridoxine obtained with the sample test solution, in units of
area or height;
Ast is the peak area or peak height for pyridoxine obtained with the standard test solution, in units of
area or height;
ρ is the mass concentration of pyridoxine hydrochloride in the standard test solution, in
micrograms per millilitre;
m is the sample mass, in gram;
100 is the total volume of the sample test solution, in millilitre;
2 is the factor of dilution of the reaction with sodium borohydride if acetic acid is added, otherwise
the dilution factor is 1,9;
1 000 is the factor to convert microgram to milligram;
100 is the factor to calculate the content per 100 g;
0,822 is the factor to convert pyridoxine hydrochloride to pyridoxine.

Report the result for vitamin B6 calculated as pyridoxine in mg/100 g.

8 Test report

The test report should comply with EN ISO/IEC 17025 [14] and shall contain at least the following data:

a) all information necessary for the identification of the sample;

b) a reference to this European Standard or to the method used;

c) the date and time of sampling procedure (if known);

d) the date of receipt;

e) the date of test;

f) the results and the units in which the results have been expressed;

g) any particular points observed in the course of the test;

h) any operations not specified in the method or regarded as optional which might have affected the results.

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Annex A
(informative)

Example of a chromatogram

Key
Y fluorescence
X time (min)
1 pyridoxine
®
Stationary phase: LiChrospher 60 RP C8 Select B, 5 μm, 250 mm x 4,0 mm
Mobile phase: according to 4.22
Flow rate: 1 ml/min
Injection volume: 30 μl
Detection: fluorometric: excitation: 290 nm; emission: 395 nm

Figure A.1 — Example for an HPLC quantification of pyridoxine in milk powder

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EN 14164:2014 (E)

Annex B
(informative)

Precision data

The existing data have been mainly obtained by using the method as outlined in Annex C without any acid
hydrolysis. The results are not modified by the acid hydrolysis.

The precision data for the determination of vitamin B6 in Table B.1 were established in an interlaboratory test
according to ISO 5725 [10] carried out by DGCCRF (Direction Génerale de la Concurrence, de la
Consommation et de le Repression des Fraudes). The collaborative study has been carried out by using the
®
following analytical column: LiChrospher 60 RP 8 Select B, particle size of 5 μm, diameter of 4,0 mm and
length of 250 mm.

The precision data for the determination of vitamin B6 in Table B.2 were established in an interlaboratory test
according to AOAC Guidelines for collaborative study procedures to validate characteristics of a method of
analysis, see [11] carried out by Mann et al., see [7] and [8] and based on Bergaentzlé's method, see [3]. The
®
collaborative study has been carried out using a Luna 5 μm phenyl-hexyl HPLC column 4).

Table B.1 — Precision data for the determination of vitamin B6

a
Sample 1 2 3 4 5 6 7 8
Year of interlaboratory test 1993 1993 1993 1993 1993 1993 1993 1993
Number of laboratories 12 12 12 12 12 12 12 12
Number of laboratories retained after eliminating
11 10 11 12 11 11 12 11
outliers
Number of results retained 32 29 31 36 33 33 35 33

Mean value, x , mg/100 g 0,06 0,14 0,22 0,53 0,55 0,67 1,50 3,28
Repeatability standard deviation, sr, mg/100 g 0,01 0,02 0,02 0,04 0,02 0,03 0,10 0,09
Repeatability relative standard deviation, RSDr, % 18 13 10 8 4 4 6 3
Repeatability limit, r, [r = 2,8 × sr ], mg/100 g 0,03 0,05 0,06 0,12 0,06 0,08 0,27 0,26
Reproducibility standard deviation, sR, mg/100 g 0,02 0,05 0,07 0,14 0,07 0,08 0,18 0,43
Reproducibility relative standard deviation, RSDR,
30 35 30 26 13 12 12 13
%
Reproducibility limit, R, [R = 2,8 × sR], mg/100 g 0,05 0,14 0,19 0,39 0,20 0,23 0,51 1,22
Horrat values, according to [12] 1,7 2,3 2,1 2,1 1,1 1,1 1,1 1,4
a
1 Baby food, 2 Biscuit, 3 Cereal B, 4 Yeast, 5 Tube-feeding solution, 6 Chocolate powder, 7 Cereal A, 8 Powdered milk

4) Luna ® 5 μm phenyl-hexyl HPLC column is an example of a suitable product available commercially. This information
is given for the convenience of users and does not constitute an endorsement by CEN of the product named. Equivalent
products can be used if they lead to equivalent results.

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EN 14164:2014 (E)

Table B.2 — Precision data for the determination of vitamin B6 in reconstituted infant formula
a
Sample 1 2 3 4 5 6 7 8
Year of interlaboratory test 2003 2003 2003 2003 2003 2003 2003 2003
Number of laboratories 11 11 11 11 11 11 11 11
Number of laboratories retained
9 9 8 9 7 9 9 9
after eliminating outliers
Number of results retained 18 18 16 18 14 18 18 18

Mean value, x , mg/100 g 0,013 0,036 0,057 0,101 0,005 0,028 0,056 0,106
Repeatability standard deviation
0,001 0,001 0,001 0,004 0,001 0,002 0,003 0,004
sr, mg/100 g
Repeatability relative standard
10 3,3 2,0 4,0 16,4 5,9 4,5 3,5
deviation, RSDr, %
Repeatability limit r
0,0028 0,0028 0,0028 0,0112 0,0028 0,0056 0,0084 0,0112
[r = 2,8 × sr], mg/100 g
Reproducibility standard deviation
0,002 0,003 0,005 0,006 0,002 0,003 0,004 0,007
sR, mg/100 g
Reproducibility relative standard
17,5 8,2 8,4 8,4 52,1 11,2 7,4 6,4
deviation, RSDR, %
Reproducibility limit R
0,0056 0,0084 0,014 0,0168 0,0056 0,0084 0,0112 0,0196
[R = 2,8 × sR], mg/100 g
Horrat values [12] 0,81 0,44 0,48 0,53 2,06 0,58 0,42 0,43
a
1 Non-fortified milk based infant formula, 2 to 4 fortified milk based infant formula, 5 non-fortified soy based infant
formula, 6 to 8 fortified soy based infant formula.

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EN 14164:2014 (E)

Annex C
(informative)

Sample treatment option without acid hydrolysis

Replace 6.2.1 to 6.2.2 of the main text by the following procedure:

Weigh an appropriate amount of the sample to the nearest mg, e.g. 2,5 g (if the vitamin B6 content exceeds
2 μg/g) or 5,0 g (if the vitamin content is less than 2 μg/g) in a conical flask. Add 25 ml of sodium acetate
solution 0,05 mol/l (4.14), 2,5 ml of glyoxylic acid 1 mol/l (4.19), 400 μl of ferrous sulfate solution (4.15) and
20 mg of acid phosphatase (4.1). Incubate the solution overnight at 37 °C with continuous agitating.

After cooling to room temperature dilute to 50 ml with water in a volumetric flask. Shake and filter. Mix 5,0 ml
of filtrate and 4,5 ml of 0,1 mol/l sodium borohydride solution (4.18). Shake for at least for 3 min. Add 0,5 ml of
glacial acetic acid (4.3). Shake for 1 min. Filter through a 0,45 μm filter. Use this filtrate for chromatography. At
the calculation step in Formula (4), replace 100 by 50 (total volume of the sample test solution in ml).

14
 BS EN 14164:2014
EN 14164:2014 (E)

Annex D
(informative)

Examples for molar absorption coefficients

Table D.1 — Examples for molar absorption coefficients (ε) of vitamin B6 compounds [6], [13]

λmax ε M
Compounds Solvent −1 −1 −1
nm l mol cm g mol
Pyridoxine hydrochloride 0,1 mol/l HCl, pH ≈ 1 290 8 600 205,6
Pyridoxine hydrochloride 0,1 mol/l phosphate buffer, pH = 7 323,8 7 300 205,6
Pyridoxal hydrochloride 0,1 mol/l HCl, pH ≈ 1 288 8 960 (9 000) 203,6
Pyridoxal-5’-phosphate 0,1 mol/l phosphate buffer, pH = 7 388 5 020 247,1
Pyridoxamine dihydrochloride 0,1 mol/l HCl, pH ≈ 1 292 8 200 241,1
Pyridoxamine dihydrochloride 0,1 mol/l phosphate buffer, pH = 7 253 4 600 241,1
Pyridoxamine-5’-phosphate 0,1 mol/l phosphate buffer, pH = 7 326 8 370 241,1
hydrochloride
Pyridoxal-5'-phosphate 0,1 mol/l HCl, pH ≈ 1 293 7 200 247,1

15
BS EN 14164:2014
EN 14164:2014 (E)

Bibliography

[1] EN 14663, Foodstuffs - Determination of vitamin B6 (including its glycosylated forms) by HPLC

[2] EN 14166, Foodstuffs - Determination of vitamin B6 by microbiological assay

[3] BERGAENTZLÉ M., ARELLA F., BOURGUIGNON J.B., HASSELMANN C. Determination of vitamin B6 in foods
by HPLC: a collaborative study. Food Chem. 1995, 52 pp. 81–86

[4] REITZER-BERGAENTZLÉ M., MARCHIONI E., HASSELMANN C. HPLC determination of vitamin B6 in foods
after pre-column derivatization of free and phosphorylated vitamers into pyridoxol. Food Chem. 1993,
48 pp. 321–324

[5] NDAW S., BERGAENTZLÉ M., AOUDÉ-W ERNER D., HASSELMANN C. Extraction procedures for the Liquid
Chromatographic determination of thiamin, riboflavin and vitamin B6 in foodstuffs. Food Chem. 2000,
71 pp. 129–138

[6] MACHLIN L.J., ed. Handbook of Vitamins. Second Edition

[7] MANN D.L., W ARE G.W., BONNIN E. Liquid Chromatographic analysis of vitamin B6 in reconstituted
infant formula: Collaborative Study. JAOAC. 2005, 88 (1) pp. 30–37

[8] Mann D.L., Chase G.W., Eitenmiller R.R., Liquid Chromatographic analysis of vitamin B6 in soy-based
infant formula. JAOAC (2001), 84, 5:1596

[9] METZLER AND SNELL. Spectra and ionisation constants of the vitamin B6 group and related 3-
hydroxypyridine derivates. J. Am. Chem. Soc. 1955, 77 pp. 2431–2437

[10] ISO 5725, Precision of test methods — Determination of repeatability and reproducibility for a standard
test method by inter-laboratory tests

[11] AOAC INTERNATIONAL. AOAC Official Methods Program, Associate Referee's Manual on development,
Study, Review, and Approval Process. Part IV AOAC Guidelines for Collaborative Studies, 1995,
pp. 23–51.

[12]. Evaluation of Analytical Methods used for Regulation of Foods and Drugs, W. Horwitz. Anal. Chem.
1982, 54 (1) pp. 67A–76A

[13] OLLILAINEN V. HPLC analysis of vitamin B6 in foods. Agricultural and Food Science in Finland. 1999, 8
p. 559

[14] EN ISO/IEC 17025:2005, General requirements for the competence of testing and calibration
laboratories (ISO/IEC 17025:2005)

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