BS EN 12823-1:2014

BSI Standards Publication

Foodstuffs — Determination of
vitamin A by high performance
liquid chromatography
Part 1: Measurement of all-E-retinol and 13-
Z-retinol
BS EN 12823-1:2014 BRITISH STANDARD

National foreword
This British Standard is the UK implementation of EN 12823-1:2014.
It supersedes BS EN 12823-1:2000 which is withdrawn.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 77938 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 31 May 2014.
Amendments/corrigenda issued since publication
Date Text affected
 BS EN 12823-1:2014

EUROPEAN STANDARD EN 12823-1

NORME EUROPÉENNE
EUROPÄISCHE NORM May 2014

ICS 67.050 Supersedes EN 12823-1:2000

English Version

Foodstuffs - Determination of vitamin A by high performance

liquid chromatography - Part 1: Measurement of all-E-retinol and
13-Z-retinol

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

vitamine A par chromatographie liquide haute performance Hochleistungs-Flüssigchromatographie - Teil 1:
- Partie 1: Dosage du tout-E-rétinol et du 13-Z-rétinol Bestimmung von all-E-Retinol und 13-Z-Retinol

This European Standard was approved by CEN on 24 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 12823-1:2014 E
worldwide for CEN national Members.
BS EN 12823-1:2014
EN 12823-1:2014 (E)

Contents Page

Foreword ..............................................................................................................................................................3
1 Scope ......................................................................................................................................................4
2 Normative references ............................................................................................................................4
3 Principle ..................................................................................................................................................4
4 Reagents .................................................................................................................................................4
5 Apparatus ...............................................................................................................................................7
6 Procedure ...............................................................................................................................................8
7 Calculation ........................................................................................................................................... 10
8 Precision .............................................................................................................................................. 11
9 Test report ........................................................................................................................................... 12
Annex A (informative) Examples of HPLC chromatograms ......................................................................... 13
Annex B (informative) Precision data............................................................................................................. 14
Annex C (informative) Alternative HPLC systems ........................................................................................ 15
Bibliography ..................................................................................................................................................... 16

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 BS EN 12823-1:2014
EN 12823-1:2014 (E)

Foreword

This document (EN 12823-1:2014) 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 November 2014, and conflicting national standards shall be withdrawn
at the latest by November 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 12823-1:2000.

This European Standard consists of two parts:

— Part 1: Measurement of all-E-retinol and 13-Z-retinol;

— Part 2: Measurements of β-carotene.

This European Standard provides the base for the analytical methods. It is intended to serve as a frame in
which the analyst can define his own analytical work in accordance to the standard procedure.

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.

3
BS EN 12823-1:2014
EN 12823-1:2014 (E)

1 Scope

This European Standard specifies a method for the determination of vitamin A in foodstuffs by high
performance liquid chromatography (HPLC). This method has been validated in an interlaboratory study with
samples of margarine and milk powder with all-E-retinol levels ranging from 653 µg/100 g to 729 µg/100 g and
with 13-Z-retinol levels ranging from 30 µg/100 g to 39 µg/100 g. The determination of vitamin A content is
carried out by the measurement of all-E-retinol, 13-Z-retinol and β-carotene. This part covers the
measurement of all-E-retinol and 13-Z-retinol.

The extract obtained after saponification in this method can be used for the determination of β-carotene, as
described in EN 12823-2:2000, Foodstuffs - Determination of vitamin A by high performance liquid
chromatography - Part 2: Measurements of β-carotene. In this case, the saponification temperature should
preferably not exceed 80 °C in order to prevent isomerisation and oxidation of β-carotene.

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

Retinol is saponified by using methanolic or ethanolic potassium hydroxide solution and extracted by an
appropriate solvent. The determination is carried out by high performance liquid chromatography (HPLC) with
either fluorometric (F) or ultraviolet (UV) detection. The substances are identified on the basis of the retention
times and determined by the external standard procedure using peak areas or heights, see [1] to [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.

4.1 Methanol.

4.2 Ethanol absolute, volume fraction, φ(C2H5OH) = 100 %.

4.3 Ethanol, φ(C2H5OH) = 96 %.

4.4 Sodium sulfate, anhydrous.

4.5 KOH solution for saponification, in suitable mass concentrations, e.g. ρ(KOH) = 50 g/100 ml or
60 g/100 ml, or alcoholic solutions, e.g. 28 g KOH in 100 ml of a mixture of 9 parts per volume of ethanol and
1 part per volume of water.

4.6 Antioxidants, such as ascorbic acid (AA), sodium ascorbate, sodium sulfide (Na2S), butylated
hydroxytoluene (BHT), pyrogallol or hydroquinone.

4.7 Solvents and extraction solvents, such as diethyl ether (peroxide-free), di-isopropylether, light
petroleum (boiling range of 40 °C to 60 °C), n-hexane, butanol, iso-octane or appropriate mixtures thereof.

4
 BS EN 12823-1:2014
EN 12823-1:2014 (E)

4.8 HPLC phases

Examples of appropriate mixtures (expressed as volume parts) include:

— n-hexane + 2-propanol (98 + 2);

— iso-octane + 2-propanol (98,5 + 1,5);

— iso-octane+ iso-butanol (98 + 2);

— n-hexane + n-butanol (98 + 2);

and gradient with 2-propanol + n-heptane, (0,5 + 99,5) to (8,5 + 91,5) in 12 min.

4.9 Standard substances

4.9.1 General

All-E-retinol (all-E vitamin A alcohol) and 13-Z-retinol can be obtained in several forms, and from different
suppliers. It is therefore necessary to determine the concentration of the calibration solution spectrometrically
(see 4.10.4). If vitamin A esters are used (e.g. retinyl palmitate or acetate), check the concentration after
saponification (see 6.3.1). Vitamin A and its derivatives are sensitive to oxygen and light. Standard
substances should be stored in the dark under nitrogen or argon at −20 °C.

Particular attention should be given to the information on the vitamin A content of the standard substances
supplied by different manufacturers.

4.9.2 All-E-retinol, vitamin A alcohol, M (C20H30O) = 286,5 g/mol, with a purity of at least 90 %.

4.9.3 Vitamin A esters.

4.9.3.1 Retinyl palmitate, vitamin A palmitate, M(C36H60O2) = 524,9 g/mol.

4.9.3.2 Retinyl acetate, vitamin A acetate, M(C22H32O2) = 328,5 g/mol, with a purity of at least 90 %.

4.9.4 13-Z-retinol, M(C20H30O) = 286,5 g/mol with a purity of at least 60 % for qualitative purposes.

4.10 Stock and standard solutions

4.10.1 All-E-retinol stock solution

Weigh out approximately 50 mg of all-E-retinol (4.9.2) to the nearest milligram into a 100 ml one-mark
volumetric flask, dissolve in n-hexane or other suitable solvents (4.7), and dilute the solution to the mark. The
stock solution contains approximately 0,5 mg/ml.

Alternatively, weigh out approximately 100 mg of retinyl palmitate (4.9.3.1), or 50 mg of retinyl acetate
(4.9.3.2) to the nearest milligram into a 100 ml one-mark volumetric flask, and dilute the solution to the mark.
The stock solution concentrations calculated as retinol are approximately 0,55 mg/ml and 0,44 mg/ml,
respectively.

Alternative masses and volumes may be used according to chromatographic separation and quantification.

Store the stock solution protected from light at approximately −20 °C. A maximum storage time should be
defined based on stability tests carried out by the user under designated conditions.

5
BS EN 12823-1:2014
EN 12823-1:2014 (E)

4.10.2 13-Z-retinol stock solution

Weigh out approximately 1 mg to 2 mg of 13-Z-retinol (4.9.4) to the nearest 0,1 mg into a 100 ml one-mark
volumetric flask, dissolve it in absolute ethanol (4.2), or other suitable solvents, and dilute the solution to the
mark. This solution contains approximately 10 µg/ml to 20 µg/ml and is used for identification purposes only.

4.10.3 All-E-retinol standard solution

Pipette 5 ml of the all-E-retinol stock solution (4.10.1) into a 100 ml one-mark volumetric flask and dilute to the
mark with n-hexane (4.7) or other suitable solvents compatible with the mobile phase. Pipette 5 ml of this
solution into a 50 ml one-mark volumetric flask, and dilute to the mark with the same solvent. The standard
solution contains approximately 2,5 µg/ml. Then carry out a concentration and purity test as described in
4.10.4.

Alternatively, retinyl palmitate or retinyl acetate stock solutions (4.10.1) may be used for the preparation of the
standard solution. In that case, saponify an aliquot of the stock solution using the conditions described in
6.3.1. After extraction and evaporation, redissolve the residue in n-hexane or other suitable solvent and carry
out a concentration test as described in 4.10.4.

Protect the standard solution from light and store at a temperature of below 4 °C. A maximum storage time
should be defined based on stability tests carried out by the user under designated conditions.

4.10.4 Concentration and purity test

Prepare a standard solution of all-E-retinol in ethanol and measure the absorbance in a quartz cell having an
optical path length of 1 cm at the maximum wavelengths of 325 nm to 326 nm with ethanol in the reference
cell. Calculate the mass concentration, ρall-E, in microgram per millilitre, of all-E-retinol using Formula (1):

A all - E ⋅ M all − E ⋅ 10 3
ρ all - E = ⋅P (1)
ε all − E

Calculate the mass concentration,ρ13-Z, in microgram per millilitre, of 13-Z-retinol using Formula (2):

A 13 - Z ⋅ M13 − Z ⋅ 10 3
ρ13 - Z = ⋅P (2)
ε 13 - Z

where

Aall-E is the absorption value at the maximum at a wavelength of 325 nm to 326 nm;
Mall-E is the molar mass (286,5 g/mol) of all-E-retinol;
εall-E is the molar extinction coefficient (52 400) for all-E-retinol dissolved in ethanol, calculated from
1%
an E1cm value of 1 830 [5], and rounded to 3 significant digits. It may change significantly with
other solvents;
A13-Z is the absorption value at the maximum at a wavelength of 328 nm;
M13-Z is the molar mass (286,5 g/mol) of 13-Z-retinol;
ε13-Z is the molar extinction coefficient (48 300) for 13-Z-retinol dissolved in ethanol, calculated from
1%
an E1cm value of 1 686 [5], and rounded to 3 significant digits. It may change significantly with
other solvents;
P is the correction factor for purity of all-E-retinol or 13-Z-retinol assessed by HPLC and calculated
using Formula (3):

6
 BS EN 12823-1:2014
EN 12823-1:2014 (E)

B
P= (3)
Btotal

where

B is the peak area or height for all-E-retinol or 13-Z-retinol obtained with the standard solution
(4.10.3);
Btotal is the sum of peak areas or heights for all-E-retinol or 13-Z-retinol obtained with the standard
solution (4.10.3).

When using newly purchased vitamin A standard substances, or ones that have been stored for a prolonged
period, check whether the absorption maximum of the all-E-retinol standard solution (4.10.3) used is between
325 nm and 326 nm using a suitable spectrometer.

For further checks on the vitamin A standards, measure the absorbance of the standard solution in quartz
cells (5.1) at wavelengths of 300 nm, 325 nm, 350 nm and 370 nm, with 2-propanol (or other suitable solvents,
see 4.7) in the reference path. Determine the following ratio at each wavelength:

E
for all-E-retinol
E 325

If the ratio does not exceed 0,602 (300 nm), 0,452 (350 nm) and 0,093 (370 nm) for vitamin A alcohol, the
standard substance is suitable for use [6], [7].

For retinyl palmitate (4.9.3.1), determine the ratio of E/E326 at wavelengths of 300 nm, 350 nm and 370 nm
with 2-propanol (or other suitable solvents) in the reference path. If the ratio does not exceed 0,593 (300 nm),
0,537 (350 nm) and 0,142 (370 nm), the standard substance is suitable for use [6], [7], [8].

5 Apparatus

Usual laboratory apparatus and, in particular, the following:

5.1 UV-VIS spectrometer, capable of measuring absorbance at defined wavelengths, with appropriate
quartz cells, e.g. of 1 cm path length.

5.2 Rotary evaporator, with water bath and vacuum unit.

The use of nitrogen is recommended for releasing of the vacuum.

5.3 HPLC system, consisting of a pump, sample injection device, a UV-VIS detector or a fluorescence
detector and data integrator/processing device.

5.4 HPLC columns

®
Suitable analytical normal phase columns are appropriate such as LiChrospher Si 60 1) (5 µm, 250 mm x
® 1)
4 mm) and LiChrosorb Si 60 (5 µm, 250 mm x 4 mm and 125 mm x 4 mm). The performance criterion for
suitable analytical columns is the baseline resolution of all-E-retinol and 13-Z-retinol.

1) ® ® ®
LiChrospher Si 60, LiChrosorb Si 60 and Extrelut are examples of suitable products available commercially. This
information is given for the convenience of users of this European Standard, and does not constitute an endorsement by
CEN of the products.

7
BS EN 12823-1:2014
EN 12823-1:2014 (E)

Column types and particle sizes other than those specified in this European standard may also be used.
Chromatographic conditions may have to be adapted for such columns to guarantee equivalent results.

5.5 Filter device

Large and small scale filter devices to filter HPLC mobile phases and sample solutions respectively, e.g. of
0,45 µm pore size is appropriate.

NOTE Filtering of the mobile phase as well as of the sample test solution through a membrane filter prior to use or
injection may increase longevity of the columns.

®1)
5.6 Extraction columns, (optional), e.g. Extrelut [1].

5.7 Phase separation filters, optional.

6 Procedure

6.1 General

Vitamin A is sensitive to UV radiation and to oxidizing agents (e.g. atmospheric oxygen). It is therefore
necessary to exclude UV light (by using amber glassware, aluminium foil, or UV-absorbing materials) and
oxygen (nitrogen flushing) when handling it. In particular, the air above the liquid shall be replaced by a
blanket of nitrogen during saponification. The solvents shall be evaporated under reduced pressure using a
rotary evaporator (5.2) at no more than 50 °C.

6.2 Preparation of the test sample

Homogenize the test sample. Grind coarse material with an appropriate mill and mix thoroughly. Precautions
such as pre-cooling shall be taken in order not to expose the sample to high temperatures for long periods of
time.

6.3 Preparation of the sample test solution

6.3.1 Saponification

Saponify 2 g to 20 g of the test sample by refluxing preferably under nitrogen using suitable amounts of
methanol (4.1) or ethanol (4.3), antioxidants (4.6) (optional) and one of the potassium hydroxide solutions
(4.5). If antioxidants such as ascorbic acid (AA), pyrogallol or butylated hydroxytoluene are used, they should
be added to the sample prior to the addition of the potassium hydroxide solution. Sodium sulfide may also be
added to obviate the oxidative catalytic effects of trace metals.

Examples of suitable ratios of reagents used by laboratories during the validation study are given in Table 1
(for more information see [3]):

Table 1 — Examples of suitable ratios of reagents

Sample mass Alcohol Antioxidants Potassium hydroxide

2 g to 5 g 50 ml methanol 0,25 g AA 5 ml of a 50 g/100 ml solution
> 5 g to 10 g 100 ml ethanol 1 g AA + 0,04 g Na2S 20 ml of a 60 g/100 ml solution
> 10 g to 20 g 150 ml ethanol 1 g AA 50 ml of a 60 g/100 ml solution

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 BS EN 12823-1:2014
EN 12823-1:2014 (E)

The usual time of saponification ranges from 15 min to 45 min with temperatures of 80 °C to 100 °C. If the
extract is to be used for determination of beta-carotene as described in EN 12823-2, the saponification should
preferably not exceed 80 °C in order to prevent isomerisation and oxidation of β-carotene.

NOTE Saponification may also be carried out at room temperature overnight with stirring (approximately 16 h) under
otherwise same conditions, or using a microwave oven [9]. These procedures were not tested in the interlaboratory study.

If after saponification and cooling, fat or oil is present on the surface of the saponification mixture, additional
ethanolic potassium hydroxide shall be added and saponification time extended.

6.3.2 Extraction

In order to avoid emulsions, an amount of water shall be added to the saponified sample solution so that the
ratio of alcohol to water in the resulting solution is 1:1.

Extract the retinol from the saponified sample solution by means of a suitable solvent or a suitable solvent
mixture (4.7) and repeat the procedure 3 times to 4 times with volumes ranging from 50 ml to 150 ml. Wash
the combined extracts to neutral with water (2 times to 4 times, 50 ml to 150 ml).
®1)
NOTE The extraction can also be performed by the solid support, liquid/liquid extraction technique (e.g. Extrelut )
when the content of retinol is not too low and the fat content of the sample is less than 10 % [1]. This procedure was not
tested in the interlaboratory study.

6.3.3 Evaporation

Remove traces of water either by drying with sodium sulfate (4.4) and/or using phase separation filter paper
(5.5) prior to evaporation or by azeotropic distillation with absolute ethanol (4.2) after evaporation. Evaporate
the extract using a rotary evaporator (5.2) under reduced pressure at not more than 50 °C. Remove solvent
residues preferably under a stream of nitrogen to avoid losses of retinol.

6.3.4 Dilution

Redissolve the residue immediately, preferably using the mobile phase (4.8), or another HPLC compatible
solvent in order to obtain an appropriate concentration for injection onto the HPLC column (5.4). This is the
sample test solution.

6.4 Preparation of the calibration solutions

Using standard solutions (4.10.3), prepare calibration solutions for HPLC using suitable solvents to cover the
analytical range required.

6.5 Identification and purity check

Identify the all-E-retinol and 13-Z-retinol by comparing the retention times from sample chromatograms with
those obtained from calibration solutions under the same chromatographic conditions. Peak identification can
also be performed by adding the standard substances to the sample test solution.

NOTE 1 The separation and quantification has proven to be satisfactory if the following chromatographic conditions are
followed (also Figure A.1). For alternative HPLC systems see Table C.1.

®
Stationary Phase: Merck LiChrosorb Si60, 5 µm
Column dimension: 250 mm x 4 mm
Mobile phase: n-hexane + n-butanol (98 + 2) (volume parts)
Flow: 2 ml/min

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BS EN 12823-1:2014
EN 12823-1:2014 (E)

Injection volume: 50 µl
Detection: Fluorescence: Excitation: 325 nm, Emission: 475 nm UV: 325 nm.

Check the purity of all-E-retinol and 13-Z-retinol standards by injecting an appropriate volume (e.g. 20 µl) of
the standard solutions (4.10.3) containing approximately 10 µg all-E-retinol or 13-Z-retinol per millilitre into the
HPLC system. Integrate the peak areas or heights of all-E-retinol and 13-Z-retinol and correct the mass
concentration of all-E-retinol standard solution if necessary.

NOTE 2 Freshly prepared all-E-retinol standards (4.10.3) usually do not contain any 13-Z-retinol or other peak
impurities. Vitamin A-ester (retinyl palmitate or –acetate) can contain some 13-Z-retinol after saponification.

6.6 Determination

Inject appropriate volumes (e.g. 20 µl) of the calibration solutions (6.4) and the sample test solution (6.3.4)
into the HPLC system (5.3). To carry out the determination by the external standard method, integrate the
peak areas or determine the peak heights obtained for sample test solutions, and compare the results with the
corresponding values for the standard substance with a similar retention time, or construct a calibration curve.
Check the linearity of the calibration curve.

NOTE Internal standard type procedures can also be used if the corresponding recovery tests have demonstrated
the same behaviour of the internal standard during analysis as the analyte itself. In addition, appropriate compounds can
be added to the test solution prior to injection onto the HPLC column for quantification purposes [7].

7 Calculation

Base the calculation either on a calibration graph over the linear range or on the following simplified
procedure.

Calculate the mass fraction, wall-E, of all-E-retinol, in mg/100 g of the sample using Formula (4):

A S ⋅ ρ all - E ⋅ VS ⋅ VSt ⋅ 100

w all - E = (4)
A all - E ⋅ m ⋅ Vis ⋅ 1000

where

AS is the peak area or height for all-E-retinol obtained with the sample test solution (6.3.4), in units
of area or height;
ρall-E is the concentration of all-E-retinol of the calibration solution (6.4), taking into account the
measurement by UV of the standard solution in 4.10.4, in micrograms per millilitre;
VS is the total volume of sample test solution (6.3.4), in millilitre;
VSt is the injection volume of the calibration solution (6.4), in microlitre;
100 is the factor to calculate the mass concentration per 100 g;
Aall-E is the peak area or height for all-E-retinol obtained with the calibration solution (6.4), in units of
area or height;
m is the mass of the test portion, in grams;
Vis is the injection volume of the sample test solution (6.3.4), in microlitre;
1 000 is the conversion factor from microgram to milligram.

Calculate the mass fraction, w13-Z, of 13-Z-retinol, in mg/100 g of the sample using Formula (5):

10
 BS EN 12823-1:2014
EN 12823-1:2014 (E)

A13-Z ⋅ ρall- E ⋅ VS ⋅ VSt ⋅100

w13-Z = (5)
A all-E ⋅ FR ⋅ m ⋅ Vis ⋅1 000

where

A13-Z is the peak area or height for 13-Z-retinol obtained with the sample test solution (6.3.4), in units
of area or height;
ρall-E is the concentration of all-E-retinol of the calibration solution (6.4), taking into account the
measurement by UV of the standard solution in 4.10.4, in micrograms per millilitre;
VS is the total volume of sample test solution (6.3.4) in millilitre;
VSt is the injection volume of the calibration solution (6.4), in microlitre;
100 is the factor to calculate the mass concentration per 100 g;
Aall-E is the peak area or height for all-E-retinol obtained with the calibration solution (6.4), in units of
area or height;
m is the mass of the test portion, in grams;
Vis is the injection volume of the sample test solution (6.3.4), in microlitre;
1000 is the conversion factor from microgram to milligram;
FR is the response factor of 13-Z-retinol to all-E-retinol assessed by HPLC using standard solution of
known concentration using Formula (6):

B13 - Z ⋅ ρ all - E
FR = (6)
B all - E ⋅ ρ13 - Z

where

B13-Z is the peak area or height obtained for the 13-Z-retinol standard solution;
Ball-E is the peak area or height obtained for the all-E-retinol standard solution;
ρ13-Z is the mass concentration of the 13-Z-retinol standard solution;
ρall-E is the mass concentration of the all-E-retinol standard solution.

If no 13-Z-retinol standard substance is available for determination of the response factor, the 13-Z-retinol
content can be calculated using a response factor of 1. The result shall be annotated accordingly.

8 Precision

8.1 General

Details of the interlaboratory test of the precision of the methods are summarized in Annex B. The values
derived from this test may not be applicable to analyte concentration ranges and matrices other than given in
Annex B.

8.2 Repeatability

The absolute difference between two single test results found on identical test material by one operator using
the same apparatus within the shortest feasible time interval will exceed the repeatability limit r in not more
than 5 % of the cases.

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BS EN 12823-1:2014
EN 12823-1:2014 (E)

The values are:

margarine all-E- x = 729 μg/100 g r = 78,4 μg/100 g

retinol:
13-Z- x = 39 μg/100 g r = 8,4 μg/100 g
retinol:
milk all-E- x = 653 μg/100 g r = 39,2 μg/100 g
powder retinol:
13-Z- x = 30 μg/100 g r = 4,2 μg/100 g
retinol:
8.3 Reproducibility

The absolute difference between two single test results found on identical test material reported by two
laboratories will exceed the reproducibility limit R in not more than 5 % of the cases.

The values are:

margarine all-E- x = 729 μg/100 g R = 204,4 μg/100 g

retinol:
13-Z- x = 39 μg/100 g R = 33,6 μg/100 g
retinol:
milk all-E- x = 653 μg/100 g R = 61,6 μg/100 g
powder retinol:
13-Z- x = 30 μg/100 g R = 20,2 μg/100 g
retinol:

9 Test report

The test report should comply with EN ISO/IEC 17025 [11] 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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 BS EN 12823-1:2014
EN 12823-1:2014 (E)

Annex A
(informative)

Examples of HPLC chromatograms

a) Standard b) Margarine c) Milk powder

Key
Y fluorescence
X time (min)
1 13-Z-retinol
2 all-E-retinol
®
Stationary phase: Merck LiChrosorb Si 60, 5 µm, 250 mm x 4,0 mm
Mobile phase: n-hexane + n-butanol (98 + 2) (volume parts)
Flow rate: 2 ml/min
Detection: fluorometric: excitation: 325 nm, emission: 475 nm

Figure A.1 — HPLC chromatograms of vitamin A standards (a), vitamin A in margarine (b) and milk
powder (c)

13
 BS EN 12823-1:2014
EN 12823-1:2014 (E)

Annex B
(informative)

Precision data

In accordance with the EU MAT Certification Study Guidelines, the parameters given in Table B.1 have been
defined in an interlaboratory test [3]. The study was organized by the Institute of Food Research, Norwich,
United Kingdom.

Table B.1 — Precision data for margarine and milk powder

a a
Parameter Margarine (CRM 122) Milk powder (CRM 421)
Sample all-E-retinol 13-Z-retinol all-E-retinol 13-Z-retinol
Year of interlaboratory test 1994 1994 1994 1994
Number of laboratories 9 5 8 5
Number of samples 2 2 2 2
Number of laboratories retained
9 5 6 5
after eliminating outliers
Number of outliers (laboratories) 0 0 2 0
Number of accepted results 45 25 40 21
Mean value x , μg/100 g 729 39 653 30
Repeatability standard deviation
28 3 14 1,5
sr, μg/100 g
Repeatability relative standard
3,8 7,7 2,1 5,0
deviation RSDr, %
Repeatability limit r [r = 2,8 × sr],
78,4 8,4 39,2 4,2
μg/100 g
Reproducibility standard deviation
73 12 22 7,2
sR, μg/100 g
Reproducibility relative standard
10,0 30,8 3,4 24,0
deviation RSDR, %
Reproducibility limit R
204,4 33,6 61,6 20,2
[R = 2,8 × sR], μg/100 g
HorRat value, according to [10] 0,85 1,68 0,28 1,26
a CRM = Certified Reference Material

14
 BS EN 12823-1:2014
EN 12823-1:2014 (E)

Annex C
(informative)

Alternative HPLC systems

The separation and quantification of all-E-retinol and 13-Z-retinol has been shown to be satisfactory if the
following chromatographic conditions given in Table C.1 are used [3]:

Table C.1 — Alternative HPLC systems and chromatographic conditions

Column Dimensions Mobile phase Detection

(particle size) (length x diameter) (V + V) (nm)
®
LiChrospher Si 60 (5 μm) 250 mm x 4 mm n-hexane + 2-propanol (98 + 2) F (325/480)

®
LiChrosorb Si 60 (5 μm) 250 mm x 4 mm n-hexane + 2-butanol (98 + 2) F (325/475)

®
LiChrosorb Si 60 (5 μm) 250 mm x 4 mm iso-propanol + n-heptane, gradient: UV 325
(0,5 + 99,5) to (8,5 + 91,5) in 12 min

15
BS EN 12823-1:2014
EN 12823-1:2014 (E)

Bibliography

[1] BOURGEOIS C.F., CIBA N. Disposal cartridge extraction of retinol and alpha-tocopherol from fatty
samples. J.A.O.A.C. 1988, 71 (1) pp. 12–15

[2] LETH T., JACOBSEN J.S. Vitamin A in Danish pig, calf and ox liver. J. Food Compos. Anal. 1993, 6
pp. 3–9

[3] FINGLAS P.M., VAN DEN BERG H., DE FROIDMONT-GORTZ I. The certification of the mass fractions of
vitamins in three reference materials: margarine (CRM 122), milk powder (CRM 421), and lyophilized
Brussels sprouts (CRM 431). EUR-Report 18039. Commission of the European Union, Luxembourg,
1997

[4] Untersuchung von Lebensmitteln: Bestimmung von Vitamin A in diätetischen Lebensmitteln L 49.00-3,
Mai 1985 (Inspection of foodstuffs; determination of the vitamin A content in dietetic foodstuffs L 49.00-
3, 1985-05) Berlin, Köln: Beuth Verlag GmbH

[5] ISLER O., BRUBACHER G. Vitamine. 1982, I p. 34 [Georg Thieme Verlag, Stuttgart]

[6] TAKASHIMA Y. et al. Stability of retinol analogs. Chem. Pharm. Bull. (Tokyo). 1979, 27 pp. 1553–1563

[7] BOLLIGER H.R. et al. The monograph of vitamin A in the European Pharmacopoeia. Pharm. Acta Helv.
1977, 52 (8) pp. 161–174

[8] Arens, M. and Gertz, Chr., 1996. Gehaltsbestimmung von Vitamin A-Standardsubstanzen – Deutsche
Einheitsmethoden zur Untersuchung von Fetten, Fettprodukten, Tensiden und verwandten Stoffen,
124. Mitt.; (Determination of vitamin A content in standard substances - German methods for analysis
of fats, fat products, tensids and related products, 124. ann) Fett/Lipid, VCH Verlag, Weinheim, 98, Nr.
5, S. 185-187

[9] HOELLER U., W OLTER D., HOFMANN P., SPITZER V. Microwave-assisted rapid determination of vitamins
A and E in beverages. J. Agric. Food Chem. 2003, 51 pp. 1539–1542

[10] HORWITZ W., ALBERT R. The Horwitz Ratio (HorRat): A useful index of method performance with
respect to precision. J. AOAC Int. 2006, 89 pp. 1095–1109

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

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