SLOVENSKI STANDARD
SIST EN 14152:2014
01-september-2014
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SIST EN 14152:2003
SIST EN 14152:2003/AC:2006
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Foodstuffs - Determination of vitamin B2 by high performance liquid chromatography
Lebensmittel - Bestimmung von Vitamin B2 mit Hochleistungs-Flüssigchromatographie
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Produits alimentaires - Détermination de la teneur en vitamine B2 par chromatographie
liquide haute performance
SIST EN 14152:2014
[Link]
Ta slovenski standard je istoveten cef28da3673e/sist-en-14152-2014
z: EN 14152:2014
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN 14152:2014 en,fr,de
[Link] inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
SIST EN 14152:2014
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SIST EN 14152:2014
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cef28da3673e/sist-en-14152-2014
SIST EN 14152:2014
EUROPEAN STANDARD EN 14152
NORME EUROPÉENNE
EUROPÄISCHE NORM June 2014
ICS 67.050 Supersedes EN 14152:2003
English Version
Foodstuffs - Determination of vitamin B2 by high performance
liquid chromatography
Produits alimentaires - Détermination de la teneur en Lebensmittel - Bestimmung von Vitamin B2 mit
vitamine B2 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.
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CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
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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.
SIST EN 14152:2014
[Link]
cef28da3673e/sist-en-14152-2014
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 14152:2014 E
worldwide for CEN national Members.
SIST EN 14152:2014
EN 14152:2014 (E)
Contents Page
Foreword ..............................................................................................................................................................3
1 Scope ......................................................................................................................................................4
2 Normative references ............................................................................................................................4
3 Principle ..................................................................................................................................................4
4 Reagents .................................................................................................................................................4
5 Apparatus ...............................................................................................................................................6
6 Procedure ...............................................................................................................................................7
7 Calculation ..............................................................................................................................................8
8 Precision .................................................................................................................................................9
9 Test report ........................................................................................................................................... 10
Annex A (informative) Examples of HPLC chromatograms ......................................................................... 11
Annex B (informative) Precision data............................................................................................................. 12
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Annex C (informative) Alternatives HPLC systems ...................................................................................... 14
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Bibliography ..................................................................................................................................................... 15
SIST EN 14152:2014
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SIST EN 14152:2014
EN 14152:2014 (E)
Foreword
This document (EN 14152:2014) 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 14152:2003.
Annexes A, B and C 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 standard can involve hazardous materials, operations and equipment.
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This standard does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this standard to establish appropriate safety and health practices and
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determine the applicability of regulatory limitations prior to use.
SIST EN 14152:2014
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SIST EN 14152:2014
EN 14152:2014 (E)
1 Scope
This European Standard specifies a method for the determination of vitamin B2 in food by high performance
liquid chromatography (HPLC) and fluorescence detection. This method has been validated in two
interlaboratory studies. The first study was for the analysis of samples of milk powder and pig's liver ranging
from 1,45 mg/100 g to 10,68 mg/100 g. The second study was for the analysis of samples of tube feeding
solution, baby food, powdered milk, meal with fruits, yeast, cereal and chocolate powder ranging from
0,21 mg/100 g to 87,1 mg/100 g. Vitamin B2 is the mass fraction of total riboflavin including its phosphorylated
derivatives.
For further information on the validation, see Clause 8 and Annex B.
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
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Riboflavin is extracted from food after acid hydrolysis followed by dephosphorylation using an enzymatic
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treatment, and separated by HPLC, and detected by fluorometric detection. An external standard is used for
quantification. For further information see [1] to [11].
SIST EN 14152:2014
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4 Reagents cef28da3673e/sist-en-14152-2014
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 Methanol, mass fraction w(CH3OH) ≥ 99,8 %, HPLC grade.
4.2 Sodium acetate trihydrate, w(CH3COONa · 3H2O) = 99 %.
4.3 Sodium acetate solution, substance concentration c(CH3COONa · 3H2O) = 0,1 mol/l.
4.4 Sodium acetate solution, c(CH3COONa · 3H2O) = 2,5 mol/l.
4.5 Glacial acetic acid, w(CH3COOH) = 99,8 %.
4.6 Acetic acid solution, c(CH3COOH) = 0,02 mol/l.
4.7 Hydrochloric acid, w(HCl) = 36 %.
4.8 Hydrochloric acid, c(HCl) = 0,1 mol/l.
4.9 Hydrochloric acid, c(HCl) = 0,01 mol/l.
4.10 Sulfuric acid, c(H2SO4) = 0,05 mol/l.
4.11 Sodium hydroxide, w(NaOH) ≥ 99 %.
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EN 14152:2014 (E)
4.12 Sodium hydroxide solution, c(NaOH) = 0,5 mol/l.
4.13 Phosphorous pentoxide, w(P2O5) = 98 %.
4.14 Enzyme or enzyme mixture, with the ability to liberate vitamin B2 from foods as free riboflavin.
For the precision data in Table B.1, Taka-Diastase from Pfaltz and Bauer 1 has been used. For the precision
)
NOTE
1)
data in Table B.2 and Table B.3 an enzyme mixture of β-amylase from barley and Taka-Diastase from Serva have been
used.
4.15 HPLC Mobile phase
Examples of appropriate mixtures of e.g. 10 % to 50 % methanol (4.1) in water or using phosphate or acetate
buffer are given in Annex A and Annex C. The possibility of using ion-pairing agents is also given.
4.16 Phosphate buffer (pH = 3,5), c(KH2PO4) = 9,0 mmol/l.
4.17 Tetraethylammoniumchloride, w(C8H20ONCl) ≥ 98 %.
4.18 Sodium heptanesulfonate, w(C7H15NaO3S) ≥ 98 %.
4.19 Standard substances
4.19.1 Riboflavin, w(C17H20N4O6) = 98 %.
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Vitamin B2 can be obtained as riboflavin from various suppliers. The purity of the riboflavin standard may vary.
It is therefore necessary to determine the concentration of the calibration solution by UV-spectrometry (see
concentration test in 4.20.3).
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4.19.2 Riboflavin-5’-phosphate, w(C17HSIST EN 14152:2014
20N4NaO9P) = 95 %.
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Riboflavin-5’-phosphate sodium salt (for cef28da3673e/sist-en-14152-2014
check of enzyme and retention time in chromatogram).
4.20 Stock solution
4.20.1 Precautions
Vitamin B2 is very sensitive to light. Measures shall be taken to protect the vitamin B2 and the corresponding
solutions during the whole sample preparation procedure e.g. by using generally brown glassware.
4.20.2 Riboflavin stock solution, M = 376,36, ρ(C17H20N4O6) ≈ 100 μg/ml.
Dissolve an amount of riboflavin standard substance (4.19.1) previously dried and stored in dark in a
desiccator possibly under vacuum and/or over phosphorous pentoxide (4.13), weighed to the nearest
milligram, e.g. approximately 50 mg in a defined volume, e.g. 500 ml in an appropriate solvent e.g. diluted
acetic acid (4.6) using brown volumetric flasks. This solution can be stored at 4 °C in the dark for 2 months.
Riboflavin is sparingly soluble. To facilitate dissolution warm with approximately 300 ml diluted acetic acid
(4.6), on a steam bath with constant stirring until dissolved, cool and add diluted acetic acid (4.6) to make
500 ml. Alternatively add 5 ml of sodium hydroxide solution (4.12) to the standard substance in a 500 ml
volumetric flask. Due to the instability in alkaline solutions immediately after dissolution add 1,5 ml of glacial
acetic acid (4.5) and dilute to volume with diluted acetic acid (4.6), or another appropriate acid. The
concentration of the freshly prepared and if necessary also stored solution should be tested (4.20.3).
1) The information of the suppliers of Taka-Diastase, Pfaltz & Bauer, Waterbury, CT 06708, USA (No T00040), and
Serva is given for the convenience of users of this European standard 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.
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EN 14152:2014 (E)
4.20.3 Concentration test
Mix 20 ml of the riboflavin stock solution, (4.20.2) in a 200 ml volumetric flask with 3,5 ml sodium acetate
solution (4.3) and dilute with water to the mark. For the preparation of the blank solution, mix 20 ml of acetic
acid solution (4.6) with 3,5 ml of sodium acetate solution (4.3) in a 200 ml volumetric flask and dilute to the
mark with water. Take these solutions for the spectrometric measurement.
Measure the absorbance of the riboflavin solution at the maximum wavelength of about 444 nm (A444) in a
1 cm cell with a spectrometer (5.1) against the blank solution as reference. Calculate the mass concentration,
ρ, of riboflavin in micrograms per millilitre, of the stock solution (4.20.2) according to Formula (1):
A 444 ⋅ M ⋅1 000
ρ=
ε (1)
where
ε is the molar absorption coefficient of riboflavin at the maximum wavelength of about
−1 −1
444 nm. The value is 12 340 l · mol · cm . This value is calculated from the extinction
1%
coefficient, E 1cm = 328, in acetate buffer (pH = 3,8) at 444 nm [9] and the molar mass,
M = 376,36. The value is given with four significant figures;
M is the molar mass, in grams per mol. The value is 376,36;
A444 is the absorption value of the riboflavin solution.
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4.21 Standard solutions
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4.21.1 Riboflavin standard solution, ρ(C17H20N4O6) ≈ 10 μg/ml.
SIST EN 14152:2014
Prepare a 1:10 dilution of [Link]
the riboflavin stock solution (4.20.2), e.g. pipette 10 ml of the riboflavin stock
solution, into a 100 ml brown volumetric flask and add diluted acetic acid (4.6) or another appropriate solvent
cef28da3673e/sist-en-14152-2014
to make 100 ml. Prepare this solution fresh every day.
4.21.2 Riboflavin standard test solution, ρ(C17H20N4O6) ≈ 0,1 μg/ml to 1 μg/ml.
Pipette corresponding volumes e.g. 1,0 ml to 10,0 ml of the standard solution (4.21.1), into brown volumetric
flasks e.g. 100 ml and dilute with the mobile phase (4.15) to the mark. Prepare this solution fresh every day.
5 Apparatus
Use laboratory apparatus, glassware, and, in particular, the following:
5.1 UV spectrometer, UV spectrometer capable of measuring absorption at defined wavelengths (444 nm),
with appropriate cells, e.g. of 1 cm length.
5.2 Autoclave or heating device, autoclave for extraction purpose, e.g. pressure cooker type, with
pressure or temperature reading device, electrical heating device or water bath.
5.3 HPLC system, consisting of a pump, a sample injecting device, a fluorescence detector with an
excitation and emission wavelength set at e.g. 468 nm and 520 nm, respectively (see Annex C), and an
evaluation system such as an integrator.
5.4 HPLC column
Analytical reversed phase column, e.g. of diameter 4,0 mm to 4,6 mm, length 100 mm to 250 mm, filled with
particle size 3 μm to 10 μm. Other systems (see Annex A) can be used providing that a satisfactory separation
of riboflavin from other co-extractives is achieved.
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EN 14152:2014 (E)
Other particle sizes or column dimensions than those specified in this European Standard may be used.
Separation parameters shall be adapted to such materials to guarantee equivalent results.
5.5 Filter device
Filtering of the mobile phase as well as of the sample solution through a membrane filter, e.g. a pore size of
0,45 µm, prior to use or injection will increase longevity of the columns.
6 Procedure
6.1 Precautions
Vitamin B2 is very sensitive to light. Measures shall be taken to protect the sample and the corresponding
solutions during the whole procedure e.g. by using generally brown glassware.
6.2 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.
6.3 Preparation of the sample test solution
6.3.1 Extraction iTeh STANDARD PREVIEW
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Weigh an appropriate amount of the sample to the nearest mg, e.g. 2 g to 10 g in a beaker or a conical flask.
Add a defined volume ranging from 50 ml to 200 ml of hydrochloric acid (4.8), or sulfuric acid (4.10). The pH of
the solution should not be more than 2,0. SIST Cover ENthe container with a watch glass and either autoclave the test
14152:2014
portion at 121 °C for [Link]
30 min, or heat it at 100 °C for 60 min.
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The data from the BCR study have shown that a wide range of conditions for the acid hydrolysis can be
applied (temperature 95 °C to 130 °C, time 15 min to 60 min). The higher the temperature is, the shorter the
time should be. However, prolonged heating of riboflavin and riboflavin-5’-phosphate can cause losses. It has
been shown that, notably for chocolate foods, the extraction efficiency could drop when pH was above 2.
6.3.2 Enzyme treatment
After cooling to room temperature adjust the extract to the optimal pH for the enzyme used with sodium
acetate solution (4.4) and add a suitable amount of dephosphorylating enzyme (4.14) to the sample. Incubate
the mixture at the optimal time and temperature for the enzyme(s) used. After cooling to room temperature
transfer to a light protected volumetric flask using diluted acetic acid (4.6) or another appropriate solvent and
dilute to a defined volume (VE).
For each enzyme used, optimal pH, incubation time and incubation temperature shall be checked.
To ensure an optimal dephosphorylation, the enzymatic step shall be checked, for example by analysis of
samples spiked with riboflavin-5'-phosphate sodium salt (4.19.2), or a material similar in sample type as the
test sample. This material should be a reference material.
The amount of riboflavin possibly brought in with the enzyme shall be considered in the calculation of the
result.
NOTE For determination of the precision data given in Table B.1, Table B.2 and Table B.3, Taka-Diastase
(Table B.1) or Taka-Diastase combined with β-amylase from barley (Table B.2 and Table B.3) were used for
dephosphorylation under the following conditions. The extract was adjusted to pH = 4,0 and pH = 4,5, respectively, with
sodium acetate solution (4.4) and 100 mg of Taka-Diastase and 10 mg β-amylase per gram of sample was added. The
mixture was incubated at 37 °C to 45 °C for 4 h to 24 h, see [9], [10] and [13].
