SIST-TS CEN/TS 16187:2011

SLOVENSKI STANDARD
SIST-TS CEN/TS 16187:2011
01-julij-2011
äLYLOD'RORþHYDQMHIXPRQL]LQD%LQIXPRQL]LQD%YSUHGHODQLKKUDQLQDRVQRYL
NRUX]H]DGRMHQþNHLQPDMKQHRWURNH0HWRGD+3/&VþLãþHQMHP]LPXQRDILQLWHWQR
NRORQRLQIOXRUHVFHQþQRGHWHNFLMRSRSUHGNRORQVNLGHULYDWL]DFLML
Foodstuffs - Determination of fumonisin B1 and fumonisin B2 in processed maize
containing foods for infants and young children - HPLC method with immunoaffinity
column cleanup and fluorescence detection after precolumn derivatization
Lebensmittel - Bestimmung von Fumonisin B1 und Fumonisin B2 in Säuglings- und
Kleinkindernahrung auf Maisbasis - HPLC-Verfahren mit Reinigung an einer
Immunoaffinitätssäule und Fluoreszenzdetektion nach Vorsäulenderivatisierung
Produits alimentaires - Fumonisine B1 et B2 dans les aliments à base de maïs pour
bébés et jeunes enfants
Ta slovenski standard je istoveten z: CEN/TS 16187:2011
ICS:
67.060 äLWDVWURþQLFHLQSURL]YRGLL] Cereals, pulses and derived
QMLK products
67.230 Predpakirana in pripravljena Prepackaged and prepared
hrana foods
SIST-TS CEN/TS 16187:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 16187:2011

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SIST-TS CEN/TS 16187:2011


TECHNICAL SPECIFICATION
CEN/TS 16187

SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION
April 2011
ICS 67.230
English Version
Foodstuffs - Determination of fumonisin B1 and fumonisin B2 in
processed maize containing foods for infants and young children
- HPLC method with immunoaffinity column cleanup and
fluorescence detection after precolumn derivatization
Denrées alimentaires - Dosage de la fumonisine B1 et de la Lebensmittel - Bestimmung von Fumonisin B1 und
fumonisine B2 dans les aliments pour nourrissons et
Fumonisin B2 in Säuglings- und Kleinkindernahrung auf
jeunes enfants contenant du maïs transformé - Méthode Maisbasis - HPLC-Verfahren mit Reinigung an einer
par CLHP avec purification sur colonne d'immunoaffinité et Immunoaffinitätssäule und Fluoreszenzdetektion nach
détection de fluorescence après dérivation précolonne Vorsäulenderivatisierung
This Technical Specification (CEN/TS) was approved by CEN on 22 February 2011 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2011 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16187:2011: E
worldwide for CEN national Members.

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SIST-TS CEN/TS 16187:2011
CEN/TS 16187:2011 (E)
Contents Page
Foreword .3
1 Scope .4
2 Normative references .4
3 Principle .4
4 Reagents .4
5 Apparatus .7
6 Procedure .8
6.1 Extraction .8
6.2 Immunoaffinity column cleanup .9
6.3 Spiking procedure .9
6.4 Derivatization and HPLC determination .9
6.4.1 Automated pre-column derivatization programme .9
6.4.2 HPLC injections . 10
6.4.3 Peak identification . 10
6.4.4 Determination . 10
7 Calculation . 10
8 Precision . 10
8.1 General . 10
8.2 Repeatability . 11
8.3 Reproducibility . 11
9 Test report . 12
Annex A (informative) Typical chromatograms . 13
Annex B (informative) Precision data . 14
Annex C (informative) Comparison between the method in this document and EN 14352:2004 and
EN 13585:2001 on fumonisins in maize . 17
Bibliography . 18

2

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SIST-TS CEN/TS 16187:2011
CEN/TS 16187:2011 (E)
Foreword
This document (CEN/TS 16187:2011) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
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 has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
Annexes A, B and C are informative.
WARNING — The use of this document can involve hazardous materials, operations and equipment.
This document does not purport to address all the safety problems associated with its use. It is the
responsibility of the user of this document to establish appropriate safety and health practices and
determine the applicability of regulatory limitations prior to use.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus,
Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy,
Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia,
Spain, Sweden, Switzerland and the United Kingdom.
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SIST-TS CEN/TS 16187:2011
CEN/TS 16187:2011 (E)
1 Scope
This Technical Specification specifies a method for the determination of fumonisin B (FB ) and fumonisin B
1 1 2
(FB ) in processed maize-containing foods for infants and young children by high performance liquid
2
chromatography (HPLC) with immunoaffinity cleanup and fluorescence detection (FLD). This method has
been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples
ranging from 112 µg/kg to 458 µg/kg for FB +FB , 89 µg/kg to 384 µg/kg for FB and 22 µg/kg to 74 µg/kg for
1 2 1
FB .
2
For further information on the validation see Clause 8 and Annex B.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
EN ISO 3696:1995, Water for analytical laboratory use ― Specification and test methods (ISO 3696:1987)
3 Principle
Fumonisins are extracted from the sample with a mixture of citrate-phosphate buffer with methanol and
acetonitrile. The filtered extract is diluted with water and applied to an immunoaffinity column containing
antibodies specific to fumonisins. Fumonisins are eluted from the column with methanol and water and
quantified by HPLC/FLD with pre-column derivatization with o-phthaldialdehyde (OPA) reagent.
4 Reagents
Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995,
unless otherwise specified. Solvents shall be of quality for HPLC analysis, unless otherwise specified.
Commercially available solutions with equivalent properties to those listed may be used.
WARNING — Dispose of waste solvents according to applicable environmental rules and regulations.
Decontamination procedures for laboratory wastes have been reported by the International Agency for
Research on Cancer (IARC), see [3].
4.1 Acetonitrile.
WARNING — Acetonitrile is hazardous and samples shall be blended using an explosion proof
blender which is housed within a fume cupboard. After blending, samples shall be filtered inside a
fume cupboard.
4.2 Methanol.
4.3 O-phthaldialdehyde (OPA).
4.4 Citric acid solution, substance concentration c(C H O ·H 0) = 0,1 mol/l.
6 8 7 2
Dissolve 21,0 g of C H O ·H 0 in water and dilute to 1 l.
6 8 7 2
4.5 Disodium hydrogen phosphate solution, c(Na HPO ) = 0,2 mol/l.
2 4
Dissolve 28,4 g of Na HPO in water and dilute to 1 l.
2 4
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SIST-TS CEN/TS 16187:2011
CEN/TS 16187:2011 (E)
4.6 2-mercaptoethanol.
4.7 Citrate-phosphate buffer solution.
Mix one part per volume of citric acid solution (4.4) with one part per volume of disodium hydrogen phosphate
solution (4.5).
4.8 Extraction solvent.
Mix two parts per volume of citrate buffer solution (4.7) with one part per volume of methanol (4.2) and one
part per volume of acetonitrile (4.1).
4.9 Glacial acetic acid.
4.10 Phosphate buffered saline (PBS) solution, c(NaCl) = 137 mmol/l, c(KCl) = 2,7 mmol/l, c(phosphate
buffer) = 10 mmol/l, pH = 7,4 at T = 25 °C.
Dissolve one tablet of commercially available PBS material in 200 ml of water.
4.11 Mixture of acetonitrile and water A.
Mix one part per volume of acetonitrile (4.1) with one part per volume of water. Use this solvent to prepare
spiking solutions.
4.12 Mixture of acetonitrile and water B.
Mix three parts per volume of acetonitrile (4.1) with seven parts per volume of water. Use this solvent to
prepare calibration solutions and to redissolve dried extracts from immunoaffinity cleanup.
4.13 Sodium tetraborate solution, c(Na B O ·10H O) = 0,1 mol/l.
2 4 7 2
Dissolve 3,8 g of Na B O ·10H O in 100 ml of water.
2 4 7 2
4.14 OPA reagent solution.
Dissolve 40 mg of OPA (4.3) in 1 ml of methanol (4.2) and dilute with 5 ml of sodium tetraborate solution
(4.13). Add 50 µl of 2-mercaptoethanol (4.6) and mix for 1 min. This reagent solution is stable for up to one
week at room temperature in the dark in a capped amber vial.
4.15 HPLC mobile phase.
4.15.1 HPLC mobile phase A.
Mix 30 parts per volume of acetonitrile (4.1) with 69 parts per volume of water and one part per volume of
glacial acetic acid (4.9).
4.15.2 HPLC mobile phase B.
Mix 60 parts per volume of acetonitrile (4.1) with 39 parts per volume of water and one part per volume of
glacial acetic acid (4.9).
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4.15.3 Linear gradient settings.
Table 1 — Gradient conditions
Time Flow rate Mobile phase A Mobile phase B
min ml/min % %
0,00 1,00 60 40
5,00 1,00 60 40
26,00 1,00 12 88
29,00 1,00 12 88
29,10 1,00 - 100
30,00 1,00 - 100
30,10 1,00 60 40
38,00 1,00 60 40

The gradient programme above has proven to give acceptable resolution for FB and FB by using a Waters
1 2
1)
C SymmetryShield™ column. The use of a different column may necessitate adjustment of these
18
conditions until acceptable resolution is achieved.
4.16 Immunoaffinity column.
The immunoaffinity column shall contain antibodies raised against FB and FB . The column shall have a
1 2
capacity of not less than 5 µg of fumonisins and shall give a recovery of not less than 80 % for the sum of FB
1
and FB when applied as a standard solution in PBS containing 5 µg of fumonisins. The columns shall be
2
warmed up to room temperature before use.
4.17 Certified standard solution of fumonisin B (FB ), mass concentration ρ(FB ) = 50 µg/ml in a mixture
1 1 1
1)
of one part per volume of acetonitrile and one part per volume of water (e.g. Biopure RK 002003 or
equivalent).
4.18 Certified standard solution of fumonisin B (FB ), ρ(FB ) = 50 µg/ml in a mixture of one part per
2 2 2

1)
volume of acetonitrile and one part per volume of water (e.g. Biopure RK 002004 or equivalent).
WARNING — Fumonisins are nephrotoxic, hepatotoxic and carcinogenic to rats and mice and
classified as possible human carcinogen by IARC. These compounds should be treated with extreme
caution. Gloves and safety glasses shall be worn at all times and all standard and sample preparation
stages shall be carried out in a fume cupboard.
4.19 Mixed FB and FB stock solution.
1 2
Prepare a mixed FB and FB stock solution by pipetting 2 000 µl of the FB certified standard solution (4.17)
1 2 1
and 500 µl of the FB certified standard solution (4.18) into a vial. Cap the vial and shake well to obtain a stock
2
solution containing 40,0 µg/ml of FB and 10,0 µg/ml of FB .
1 2

1) Waters C SymmetryShield™, Biopure RK 002003 and RK 002004 are examples of suitable products available
18
commercially. This information is given for the convenience of users of this Technical Specification and does not constitute
an endorsement by CEN of these products. Equivalent products may be used if they can be shown to lead to the same
results.
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4.20 Diluted mixed FB and FB stock solution.
1 2
Pipette 500 µl of the mixed stock solution (4.19) into a 10 ml calibrated volumetric flask. Fill up to the mark
with the mixture of acetonitrile water B (4.12) and shake well to obtain a diluted mixed stock solution
containing 2,0 µg/ml of FB and 0,5 µg/ml of FB .
1 2
4.21 Mixed FBs calibration solutions for HPLC.
Prepare five HPLC mixed calibration solutions in 5 ml calibrated volumetric flasks by further diluting the diluted
mixed FBs stock solution (4.20) according to Table 2. Make up each calibration solution to volume (5 ml) with
the mixture of acetonitrile and water B (4.12) and mix well.
Table 2 — Preparation of mixed FBs calibration solutions for HPLC
HPLC calibration Diluted mixed FBs Final concentration of mixed Sample equivalent
solution stock solution FBs calibration solutions (4.21) levels of
a
(4.20) FB and FB
1 2
µl FB FB FB FB
1 2 1 2
µg/ml µg/ml µg/kg µg/kg
1 100 0,04 0,01 20,0 5,0
2 200 0,08 0,02 40,0 10,0
3 400 0,16 0,04 80,0 20,0
4 1 000 0,40 0,10 200,0 50,0
5 2 400 0,96 0,24 480,0 120,0
a
For a sample extract reconstituted in 500 µl of the mixture of acetonitrile and water B (4.12).

5 Apparatus
Usual laboratory glassware and equipment and, in particular, the following:
5.1 Analytical balance, capable of weighing to 0,000 1 g.
5.2 Laboratory balance, capable of weighing to 0,1 g.
5.3 Thermostated water bath.
5.4 Conical flasks, of 250 ml capacity with screw caps.
5.5 Orbital shaker.
5.6 Centrifuge, capable of a centrifugal force up to 3 000 g.
5.7 Centrifuge bottles, of 250 ml capacity with screw caps.
5.8 Calibrated microlitre pipettes or microlitre syringes, of 100 µl, 200 µl or 1 000 µl capacity.
5.9 Displacement pipettes, of 5 ml, 10 ml or 25 ml capacity.
5.10 Vacuum manifold, to accommodate immunoaffinity columns (4.16).
5.11 Reservoirs (of 25 ml capacity) and attachments to fit to columns.
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5.12 Vacuum pump.
5.13 Filter paper, e.g. qualitative, strong, fast flow, 24 cm diameter, 30 µm pore size, prefolded or
equivalent.
5.14 Glass microfibre filter paper, e.g. 1,6 µm pore size or equivalent.
5.15 Heating block with nitrogen or air gas supply.
5.16 Vials, of 4 ml to 12 ml capacity with screw caps.
5.17 HPLC autosampler vials, of 1,8 ml capacity with caps.
5.18 Glass flat bottom vial insert, of 250 µl volume capacity.
5.19 Vortex mixer, or equivalent.
5.20 HPLC apparatus, comprising the following:
5.20.1 Injection system.
5.20.2 Mobile phase pump (binary, ternary or quaternary pump), capable of generating a binary gradient at
1 ml/min.
5.20.3 Autosampler, capable of performing automated pre-column derivatization with OPA reagent
according to 6.4.1.
5.20.4 Analytical reverse-phase HPLC separating column, e.g. C reverse-phase column,
18
150 mm × 4,6 mm, 5 µm preceded by a suitable pre-column or guard filter, which provides acceptable
retention and resolution for FB and FB .
1 2
2) 2)
Waters C SymmetryShield™ , Agilent Zorbax SB-C or similar have been found to be suitable.
18 18
5.20.5 Column oven, capable to operate at 20 °C.
NOTE C reverse-phase column can also operate at ambient temperature.
18
5.20.6 Fluorescence detector, fitted with a flow cell and suitable for measurements with excitation
wavelength of 335 nm and emission wavelength of 440 nm.
5.20.7 Recorder, integrator or computer based data processing system.
6 Procedure
6.1 Extraction
The sample should be finely ground to pass through 0,5 mm sieve and thoroughly mixed to ensure complete
homogenization.
Weigh, to the nearest 0,1 g, a 20 g test portion of the ground sample into a conical flask (5.4). Add 100 ml of
extraction solvent (4.8) and close the flask with screw cap. Heat at 55 °C (static condition) in the thermostated

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