BS en 13585-2002

BRITISH STANDARD BS EN
13585:2002
Foodstuffs —
Determination of
fumonisins B1 and B2 in
maize — HPLC method
with solid phase
extraction clean-up
The European Standard EN 13585:2001 has the status of a

British Standard
ICS 67.060
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BS EN 13585:2002
National foreword
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EN 13585:2001.
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Amendments issued since publication
Amd. No. Date Comments
© BSI 04 March 2002
ISBN 0 580 39204 X

EUROPEAN STANDARD EN 13585
NORME EUROPÉENNE
EUROPÄISCHE NORM December 2001
ICS 67.060
English version
Foodstuffs - Determination of fumonisins B1 and B2 in maize -

HPLC method with solid phase extraction clean-up
Produits alimentaires - Dosage des fumonisines B1 et B2 Lebensmittel - Bestimmung von Fumonisin B1 und B2 in
dans le maïs - Méthode CLHP avec purification par Mais - HPLC-Verfahren mit Reinigung durch
extraction en phase solide Festphasenextraktion
This European Standard was approved by CEN on 2 November 2001.
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 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 Management Centre has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION

COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2001 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 13585:2001 E
worldwide for CEN national Members.
EN 13585:2001 (E)
Contents
page
Foreword......................................................................................................................................................................2
1 Scope ..............................................................................................................................................................3
2 Normative references ....................................................................................................................................3
3 Principle ..........................................................................................................................................................3
4 Reagents and materials.................................................................................................................................3
5 Apparatus .......................................................................................................................................................4
6 Sampling .........................................................................................................................................................5
7 Preparation of the test sample .....................................................................................................................5
8 Procedure .......................................................................................................................................................6
9 Calculation......................................................................................................................................................6
10 Precision .........................................................................................................................................................7
11 Test report ......................................................................................................................................................8
Annex A (informative) Typical chromatogram........................................................................................................10
Annex B (informative) Recovery and relative standard deviation........................................................................11
Annex C (informative) Precision data......................................................................................................................12
Bibliography ..............................................................................................................................................................15
Foreword
This European Standard 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 June 2002, and conflicting national standards shall be withdrawn at the latest by
June 2002.
The 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, Czech Republic, Denmark, Finland,
France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden,
Switzerland and the United Kingdom.
2
EN 13585:2001 (E)
1 Scope
This European Standard specifies a method for the determination of fumonisin B1 (FB1) and fumonisin B2 (FB2) in
maize using high performance liquid chromatography (HPLC).
The method has been successfully validated in an interlaboratory study according to AOAC Guidelines for
Collaborative Studies [1] on maize containing 405 µg/kg to 6732 g/kg fumonisin B1 and 152 g/kg to 2619 g/kg
fumonisin B2. The method works well with maize or minimally processed maize (e.g. fresh, dried and milled maize),
but does not provide reliable results with most maize-based processed products.
2 Normative references
This European Standard incorporates by dated or undated reference, provisions from other publications. These
normative references are cited at the appropriate places in the text, and the publications are listed hereafter. For
dated references, subsequent amendments to or revisions of any of these publications apply to this European
Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the
publication referred to applies (including amendments).
EN ISO 3696 Water for analytical laboratory use - Specification and test methods (ISO 3696:1987).
3 Principle
Fumonisins are extracted from the sample of maize with a mixture of methanol and water. The filtered extract is
purified on a strong-anion-exchange (SAX) solid-phase extraction (SPE) cartridge, and the fumonisins are eluted
with a mixture of acetic acid and methanol. The extract is evaporated and the residue is redissolved in methanol
and o-phthaldialdehyde/2-mercaptoethanol (OPA/MCE) is added to form fluorescent fumonisin derivatives. The
derivatives are analysed by reverse-phase high performance liquid chromatography (HPLC) with fluorescence
detection.
WARNING - Fumonisins are hepatoxic, nephrotoxic and carcinogenic to rats and mice. Effects on humans
are not fully known. Observe appropriate safety precautions for handling fumonisins. Any laboratory spills
should be washed with a 5 % solution of sodium hypochlorite.
4 Reagents and materials
4.1 General
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only distilled
water or water of grade 1 as defined in EN ISO 3696. Solvent shall be of quality for HPLC analysis.
4.2 Methanol, abs.
4.3 Methanol solution, volume fraction (CH3OH) = 75 %
Mix 75 parts per volume of methanol (4.2) with 25 parts per volume of water.
4.4 Acetonitrile solution, (CH3CN) = 50 %
Mix 50 parts per volume of acetonitrile with 50 parts per volume of water.
4.5 o-phosphoric acid, (H3PO4) = 85 %
4.6 Acetic acid-methanol solution, (CH3COOH) = 1 % for eluting the SPE column.
Mix 1 part per volume of glacial acetic acid with 99 parts per volume of methanol (4.2).
3
EN 13585:2001 (E)
4.7 o-phthaldialdehyde (OPA)
4.8 2-mercaptoethanol (MCE)
4.9 Sodium dihydrogen phosphate solution, substance concentration c(NaH2PO4·2H2O) = 0,1 mol/l
Dissolve 15,6 g of NaH2PO4·2H2O in 1 l of water.
4.10 Disodium tetraborate solution, c(Na2B4O7·10 H2O) = 0,1 mol/l
Dissolve 3,8 g of Na2B4O7·10H2O in 100 ml of water.
4.11 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l
Dissolve 0,4 g of NaOH in 100 ml of water.
4.12 Mobile phase
Mix 77 volume parts of methanol (4.2) with 23 volume parts of sodium dihydrogen phosphate solution (4.9). Adjust
to pH 3,35 with o-phosphoric acid (4.5). Filter the solution through a 0,45 m membrane (5.7) filter.
The mobile phase composition may have to be adjusted to conform with individual HPLC column characteristics.
4.13 Derivatization reagent
Dissolve 40 mg of OPA (4.7) in 1 ml of methanol (4.2) and dilute with 5 ml of disodium tetraborate solution (4.10).
Add 50 l of MCE (4.8) and mix. The reagent solution is stable for up to one week at room temperature in a dark
and capped amber vial.
4.14 FB1 and FB2 standard solution
Prepare individual stock solutions of FB1 and FB2 at mass concentrations of 250 µg/ml in acetonitrile solution (4.4).
Commercially available standard solutions may be used. Transfer 100 l aliquots of each stock solution to a glass
vial and add 300 l of acetonitrile solution to yield 500 l of a standard solution containing both fumonisins at
individual mass concentration of 50 g/ml. If a calibration curve is made, mix different aliquots of standard solutions
(i.e. 20 l, 50 l, 100 l and 200 l) of individual fumonisins and dilute to 500 l with acetonitrile solution to obtain
the relevant calibration solutions.
°
Fumonisin standard solutions are stable up to at least six months when stored at approximately 4 C.
5 Apparatus
Usual laboratory equipment and, in particular, the following:
5.1 HPLC apparatus, comprising the following
5.1.1 High performance liquid chromatograph, isocratic pump set to deliver e.g. 1 ml/min constant flow rate,
equipped with an injection system capable to deliver e.g. 10 l.
5.1.2 Analytical reverse-phase separating column, e.g. octyldecylsilane (ODS), which ensures a baseline
resolution of the fumonisin peaks from all other peaks, with the following characteristics:
stainless steel;
4
EN 13585:2001 (E)
a length of 150 mm;
an inner diameter of 4,6 mm;
a stationary phase with particle size of 5 m;
a suitable corresponding reverse-phase guard column.
Columns of other dimensions can also be used.
5.1.3 Fluorescence detector with the capability of using excitation wavelength of = 335 nm and emission
wavelength of = 440 nm.
5.1.4 Data system
5.2 Blender, homogenizer, or mixer.
5.3 Fluted filter paper
5.4 Strong-anion-exchanging solid phase extraction column, e.g. Bond-Elut 1) SAX-cartridges,

containing 500 mg of sorbent, or similar has been found to be suitable.
5.5 SPE extraction manifold
5.6 Solvent evaporator, with heating module, or similar.
5.7 Membrane filter, for aqueous solutions, with a pore size of 0,45 m.
6 Sampling
It is important that the laboratory receives a sample which is truly representative and has not been damaged or
changed during transport or storage.
7 Preparation of the test sample

Grind the sample to pass through a 1,0 mm sieve and homogenize the sample.
8 Procedure
8.1 Extraction
Place 50 g of the test sample into a suitable glass or plastic container (e.g. a 250 ml plastic centrifuge bottle).
-1
Extract for 3 min with 100 ml of methanol solution (4.3) using the blender (5.2) at approximately 10 000 min . The
time needed for complete extraction can vary with the type of equipment used.
Centrifuge the blended extract for 10 min at 500 g and filter the supernatant through a fluted filter paper (5.3).
Check the pH of the eluate and adjust, if necessary, with sodium hydroxide solution (4.11) to between pH 5,8 and
pH 6,5.
1 Bond-Elut is an example of a suitable product available commercially. This information is given for the convenience of users
of this European Standard and does not constitute an endorsement by CEN of this product.
5
EN 13585:2001 (E)
8.2 Clean up
Attach the SPE cartridge (5.4) to the SPE manifold (5.5) and condition by washing successively with 5 ml of
methanol (4.2) and then with 5 ml of methanol solution (4.3). While maintaining a flow rate of no more than
2 ml/min, apply a 10 ml aliquot of the filtered sample extract to the SPE cartridge. Wash the SPE cartridge with 8 ml
of methanol-water solution (4.3), followed by 3 ml of methanol (4.2). Do not allow the cartridge to run dry. Discard
the washings. Elute the fumonisins with 10 ml of methanolic acetic acid 1 % (4.6) at a flow rate not more than
1 ml/min. Collect the eluate in a suitable vial.
Transfer the eluate in the collection vial to a suitable vial and evaporate the eluate to dryness by using the solvent
evaporator (5.6) under nitrogen at approximately 60 °C. Wash the collection vial with 1 ml of methanol (4.2) and
add the washing to the suitable vial. Evaporate the additional methanol to dryness under nitrogen, ensure that all
the acetic acid has evapored, and cap the vial. Retain the dried residue at approximately 4 °C until HPLC analysis,
that should be performed within two weeks.
8.3 Derivatization and determination
8.3.1 Standard derivative solution
Transfer 25 l of the fumonisin standard solution (4.14) to the base of a small test tube. Add 225 l derivatization
reagent (4.13), mix the solutions vigorously, and inject an aliquot (e.g. 10 l = 0,050 µg FB1 and FB2) of the
derivatized solution onto the HPLC column (5.1.2) at a reproducible time within 3 min after addition of the
derivatization reagent. If a single point calibration is used, adjust the sensitivity of the fluorescence detector (5.1.3)
to at least an 80 % recorder response.
8.3.2 Maize test solution
Redissolve the dried residue (see 8.2) in 200 l of methanol (4.2). Acetonitrile solution (4.4) may also be used.
Transfer 25 l of this solution to the base of a small test tube and add 225 l of the derivatization reagent (4.13).
Mix the solutions and inject an aliquot, e.g. 10 l of the derivatized solution onto the HPLC column (5.1.2) at a
reproducible time within 3 min after addition of the derivatization reagent (4.13). If fumonisin chromatographic
peaks exceed those of fumonisin standard solution or exceed the highest point of the calibration curve (4.14), make
additional dilutions of the sample extracts with methanol (4.2) and repeat derivatization.
NOTE 1 It is critical to adhere to reproducible times between the addition of the reagent (4.13) and the injection onto the HPLC
column, because of progressive decay in fluorescence of the fumonisin-derivatives that occurs after periods in excess to 4 min.
NOTE 2 Limits of detection and quantification vary considerably according to the sensitivity of the detector used. Limits of
detection (signal/noise = 3) as low as 5 µg/kg can be reached with the new generation of fluorometric detector.
NOTE 3 A typical chromatogram is given in annex A.
9 Calculation
Calculate the mass of each fumonisin mti in micrograms, injected onto the HPLC column using equation (1):
Pt
m ti = m si (1)
Ps
where
Pt is the fumonisin peak of the test sample, in units of height or area;
Ps is the fumonisin peak of the standard solution, in units of height or area;
6
EN 13585:2001 (E)
msi is the mass of each fumonisin FBi standard in micrograms, injected onto the HPLC column, based on the
concentration in the fumonisin standard solution (here: 0,050 µg of each fumonisin);
Calculate the mass fraction of each fumonisin, wi, in micrograms per kilogram, in the sample using equation (2):
mti V t D
wi = (2)
W Vi
where
Vt is the volume of the derivatized solution (8.3), in microlitres (Vt = 250 l);
D is the dilution factor that may have been used;
W is the sample equivalent mass derivatized, in grams (W = 0,625 g in 25 l of sample test solution);
Vi is the injected volume (8.3), in microlitres (Vi = 10 l).
If a calibration curve is performed, calculations should be made accordingly.
10 Precision
10.1 General
Details of the interlaboratory test on the precision of the method are summarised in annex B and annex C. The
values derived from the interlaboratory tests may not be applicable to analyte concentration ranges and
matrices other than given in annex B and annex C.
10.2 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 interval will exceed the repeatability limit r in not more than 5 %
of the cases.
The values for fumonisin B1 are:
x = 405 µg/kg r = 80,6 µg/kg

x = 813 µg/kg r = 132,7 µg/kg
x = 1621 µg/kg r = 349,2 µg/kg
x = 3245 µg/kg r = 559,2 µg/kg
x = 6732 µg/kg r = 2062,5 µg/kg
x = 4246 µg/kg r = 1572,8 µg/kg (naturally contaminated)
x = 152 g/kg r = 35,6 µg/kg

x = 313 µg/kg r = 74,5 µg/kg
x = 618 µg/kg r = 205,5 µg/kg
x = 1294 µg/kg r = 260,7 µg/kg
7
EN 13585:2001 (E)
x = 2619 µg/kg r = 891,5 µg/kg

x = 1234 µg/kg r = 603,1 µg/kg (naturally contaminated)
10.3 Reproducibility
The absolute differences between two single test results on identical test material reported by two laboratories will
exceed the reproducibility limit R in not more than 5 % of the cases.
x = 405 µg/kg R = 157,4 µg/kg

x = 813 µg/kg R = 356,7 µg/kg
x = 1621 µg/kg R = 731,1 µg/kg
x = 3245 µg/kg R = 1383,5 µg/kg
x = 6732 µg/kg R = 3079,2 µg/kg
x = 4246 µg/kg R = 2643,5 µg/kg (naturally contaminated)
x = 152 µg/kg R = 69,2 µg/kg

x = 313 µg/kg R = 138,3 µg/kg
x = 618 µg/kg R = 335,2 µg/kg
x = 1294 µg/kg R = 622,4 µg/kg
x = 2619 µg/kg R = 1309,6 µg/kg
x = 1234 µg/kg R = 922,3 µg/kg (naturally contaminated)
11 Test report
The test report shall contain the following data:
all information necessary for the identification of the sample (kind of sample, origin of sample, designation);
a reference to this European Standard;
the date and type of sampling procedure (if known);
the date of receipt;
the date of test;
the test results and the units in which they have been expressed;
any particular points observed in the course of the test;
any operations not specified in the method or regarded as optional, which might have affected the results.
8
EN 13585:2001 (E)
Annex A
(informative)
Typical chromatogram
Key
a Time (min)
b Fluorescence response (mV)
Figure A.1
Operating conditions:
Pre-column derivatisation 25

Injection volume 20
Column Discovery C18 (150 mm x 4,6 mm)
Mobile phase Methanol-phosphate buffer (77 + 23, v + v), pH 3,35
Flow rate 1 ml/min
Detection ex = 335em = 440 nm)
9
EN 13585:2001 (E)
Annex B
(informative)
Recovery and relative standard deviation
Table B.1 shows recovery and relative standard deviation of HPLC determination of fumonisins B1 and B2 at
different spiked levels in maize. In IUPAC/AOAC Interlaboratory Collaborative Study with 12 participating
laboratories, nine laboratory data were elaborated. This method was used in the IUPAC/AOAC Interlaboratory
Study for the HPLC determination of fumonisins B1, B2 and B3 in maize [2]. It has been adopted by AOAC
International as official first action method [3].
Table B.1 - Recovery and relative standard deviation data

a) b)
Spiking level (g/kg) Mean recovery x (%) RSDr (%) RSDR (%)
Fumonisin B1
500 81,1 7,1 13,9
1000 81,3 5,8 15,7
2000 81,1 7,7 16,1
4000 81,1 6,2 15,2
8000 84,2 10,9 16,3

c)
4246
- 13,2 22,2
Fumonisin B2
200 75,9 8,4 16,3
400 78,3 8,5 15,8
800 77,3 11,9 19,3
1600 80,9 7,2 17,2
3200 81,9 12,2 17,9

d)
1234
- 17,5 26,7
a) RSDr is the repeatability relative standard deviation.

b) RSDR is the reproducibility relative standard deviation.
c) Maize naturally contaminated with fumonisin B1 at a mean concentration of 4246 µg/kg.
d) Maize naturally contaminated with fumonisin B2 at a mean concentration of 1234 µg/kg.
10
EN 13585:2001 (E)
Annex C
(informative)
Precision data
The following data were obtained in interlaboratory tests according to AOAC Guidelines for Collaborative Studies
[1] conducted by IUPAC/AOAC [2].
Table C.1 - Precision data

Sample Fumonisin B1 Fumonisin B2
Year of inter-laboratory test 1994 1994
a) a)
Number of laboratories 9 9
Number of laboratories retained after eliminating outliers 8 8
Number of outliers (laboratories) 1 1
Number of accepted results 16 16
Mean value x , µg/kg 405 152
Repeatability standard deviation sr, µg/kg 28,8 12,7

Repeatability relative standard deviation RSDr, % 7,1 8,4
Repeatability limit r [r = 2,8 sr], µg/kg 80,6 35,6
Reproducibility standard deviation sR, µg/kg 56,2 24,7
Reproducibility relative standard deviation RSDR, % 13,9 16,3
Reproducibility limit R [R = 2,8 sR], µg/kg 157,4 69,2
Recovery, % 81,1 75,9
a) Three laboratories out of the original 12 participants were removed because judged invalid.

a) a)
Number of outliers (laboratories) -

Recovery % 81,3 78,3
11
EN 13585:2001 (E)

a) a)
Number of outliers (laboratories) - -

Recovery, % 81,1 77,3

a) a)

Recovery, % 81,1 80,9
12
EN 13585:2001 (E)

a) a)

Recovery, % 84,2 81,9

a) a)
b) b)

a) a)
Recovery, % - -
a) naturally contaminated maize
b) Three laboratories out of the original 12 participants were removed because judged invalid.
13
EN 13585:2001 (E)
Bibliography
[1] AOAC Official Methods Program 1995, Associate Referee’s Manual on development, Study,
Review, and Approval Process, Part IV AOAC Guidelines for Collaborative Studies pp.23-51.
[2] E.W. Sydenham, G.S. Shepard, P.G. Thiel, S. Stockenstrom, P.W. Snijman, D.J. Van Schalkwyk.
Liquid Chromatographic Determination of Fumonisins B1, B2 and B3 in corn: AOAC-IUPAC
Collaborative Study. Journal of AOAC International 1996, Vol. 79, No. 3, pp. 688-696.
th
[3] AOAC INTERNATIONAL Official Methods of Analysis, 17 Ed, 2000, Volume II, method 49.5.01
(995.15)
14
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13585:2002
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BRITISH STANDARD BS EN

The European Standard EN 13585:2001 has the status of a

NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW

This British Standard is the official English language version of

— aid enquirers to understand the text;

This British Standard, having

Amendments issued since publication

Amd. No. Date Comments

© BSI 04 March 2002

ISBN 0 580 39204 X

Foodstuffs - Determination of fumonisins B1 and B2 in maize -

This European Standard was approved by CEN on 2 November 2001.

EUROPEAN COMMITTEE FOR STANDARDIZATION

Management Centre: rue de Stassart, 36 B-1050 Brussels

The annexes A, B and C are informative.

4 Reagents and materials

4.2 Methanol, abs.

4.3 Methanol solution, volume fraction (CH3OH) = 75 %

4.4 Acetonitrile solution, (CH3CN) = 50 %

4.5 o-phosphoric acid, (H3PO4) = 85 %

4.7 o-phthaldialdehyde (OPA)

4.8 2-mercaptoethanol (MCE)

Dissolve 15,6 g of NaH2PO4·2H2O in 1 l of water.

4.10 Disodium tetraborate solution, c(Na2B4O7·10 H2O) = 0,1 mol/l

Dissolve 3,8 g of Na2B4O7·10H2O in 100 ml of water.

4.11 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l

Dissolve 0,4 g of NaOH in 100 ml of water.

4.12 Mobile phase

4.13 Derivatization reagent

4.14 FB1 and FB2 standard solution

Usual laboratory equipment and, in particular, the following:

5.1 HPLC apparatus, comprising the following

a length of 150 mm;

an inner diameter of 4,6 mm;

a stationary phase with particle size of 5 m;

a suitable corresponding reverse-phase guard column.

Columns of other dimensions can also be used.

5.1.4 Data system

5.2 Blender, homogenizer, or mixer.

5.3 Fluted filter paper

5.4 Strong-anion-exchanging solid phase extraction column, e.g. Bond-Elut 1) SAX-cartridges,

5.5 SPE extraction manifold

5.6 Solvent evaporator, with heating module, or similar.

7 Preparation of the test sample

8.3 Derivatization and determination

8.3.1 Standard derivative solution

8.3.2 Maize test solution

NOTE 3 A typical chromatogram is given in annex A.

Pt is the fumonisin peak of the test sample, in units of height or area;

Ps is the fumonisin peak of the standard solution, in units of height or area;

If a calibration curve is performed, calculations should be made accordingly.

The values for fumonisin B1 are:

x = 405 µg/kg r = 80,6 µg/kg

The values for fumonisin B2 are:

x = 152 g/kg r = 35,6 µg/kg

x = 2619 µg/kg r = 891,5 µg/kg

x = 405 µg/kg R = 157,4 µg/kg

The values for fumonisin B2 are:

x = 152 µg/kg R = 69,2 µg/kg

The test report shall contain the following data:

b Fluorescence response (mV)

Pre-column derivatisation 25

Injection volume 20

Pre-column derivatisation 25

Detection ex = 335em = 440 nm)