CEN/TS 16187:2011
(Main)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
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
This Technical Specification specifies a method for the determination of fumonisin B1 (FB1) and fumonisin B2 (FB2) in processed maize-containing foods for infants and young children by high performance liquid 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 FB1+FB2, 89 µg/kg to 384 µg/kg for FB1 and 22 µg/kg to 74 µg/kg for FB2.
For further information on the validation see Clause 8 and Annex B.
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
Diese Technische Spezifikation legt ein Verfahren zur Bestimmung von Fumonisin B1 (FB1) und Fumonisin B2
(FB2) in verarbeiteten Mais enthaltenden Lebensmitteln für Säuglinge und Kleinkinder durch Hochleistungsflüssigchromatographie
(HPLC) mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion
(FLD) fest. Dieses Verfahren wurde in einem Ringversuch durch die Untersuchung sowohl von natürlich
kontaminierten Proben als auch von aufgestockten Proben mit Gehalten von 112 µg/kg bis 458 µg/kg für
FB1 + FB2, 89 µg/kg bis 384 µg/kg für FB1 und 22 µg/kg bis 74 µg/kg für FB2 validiert.
Weitere Informationen zur Validierung befinden sich in Abschnitt 8 und Anhang B.
Denrées alimentaires - Dosage de la fumonisine B1 et de la fumonisine B2 dans les aliments pour nourrissons et jeunes enfants contenant du maïs transformé - Méthode par CLHP avec purification sur colonne d'immunoaffinité et détection de fluorescence après dérivation précolonne
La présente Spécification technique spécifie une méthode de dosage de la fumonisine B1 (FB1) et de la
fumonisine B2 (FB2) dans les aliments pour nourrissons et jeunes enfants contenant du maïs transformé par
chromatographie liquide haute performance (CLHP) avec purification sur colonne d’immunoaffinité et
détection par fluorescence (DFL). Cette méthode a été validée lors d’une étude interlaboratoires en procédant
à l’analyse d’échantillons naturellement contaminés et supplémentés à des teneurs comprises entre
112 µg/kg et 458 µg/kg en FB1+FB2, entre 89 µg/kg et 384 µg/kg en FB1 et entre 22 µg/kg et 74 µg/kg en FB2.
Pour de plus amples informations sur la validation, voir l’Article 8 et l’Annexe B.
Živila - Določevanje fumonizina B1 in fumonizina B2 v predelanih hrani na osnovi koruze za dojenčke in majhne otroke - Metoda HPLC s čiščenjem z imunoafinitetno kolono in fluorescenčno detekcijo po predkolonski derivatizaciji
Ta tehnična specifikacija opredeljuje metodo za določevanje fumonizina B1 in fumonizina B2 v predelani hrani na podlagi koruze za dojenčke in majhne otroke z metodo s tekočinsko kromatografijo visoke ločljivosti (HPLC) s čiščenjem z imunoafinitetno kolono in fluorescenčno detekcijo. Ta metoda je bila potrjena v medlaboratorijski študiji z analizami naravno kontaminiranih vzorcev in vzorcev z internimi dodatki v razponu od 111,6 μg/kg do 458,0 μg/kg za FB1+FB2, 89,1 µg/kg do 384,4 µg/kg za FB1 in 22,5 µg/kg do 73,6 µg/kg za FB2.
Za dodatne informacije o potrjevanju glej točko 8 in dodatek B.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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.
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
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.
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
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 .
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
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).
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
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
and FB when applied as a standard solution in PBS containing 5 µg of fumonisins. The columns shall be
warmed up to room temperature before use.
4.17 Certified standard solution of fumonisin B (FB
...
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