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 pre-column derivatisation

This document 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 Vorsäulenderivatiserung

Dieses Dokument legt ein Verfahren zur Bestimmung von Fumonisin B1 (FB1) und Fumonisin B2 (FB2) in verarbeiteten Mais enthaltenden Lebensmitteln für Säuglinge und Kleinkinder fest. Hierzu wird die Hochleistungs¬flüssigchromatographie (HPLC) mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion (FLD) eingesetzt. 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

Le présent document 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 g/kg et 458 g/kg en FB1+FB2, entre 89 g/kg et 384 g/kg en FB1 et entre 22 g/kg et 74 g/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 predelani hrani na osnovi koruze za dojenčke in majhne otroke - Metoda HPLC s čiščenjem z imunoafinitetno kolono in fluorescenčno detekcijo po predkolonski derivatizaciji

Ta dokument določa metodo za določevanje fumonizina B1 in fumonizina B2 v predelani hrani na osnovi koruze za dojenčke in majhne otroke z metodo HPLC s čiščenjem z imunoafinitetno kolono in fluorescenčno detekcijo. Ta metoda je bila validirana v medlaboratorijski študiji z analizo naravno kontaminiranih in primešanih vzorcev od 112 µg/kg do 458 µg/kg za FB1+FB2, 89 µg/kg do 384 µg/kg za FB1 in 22 µg/kg do 74 µg/kg za FB2.
Za več informacij glede validacije glej točko 8 in dodatek B.

General Information

Status
Published
Publication Date
30-Jul-2015
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
08-Jul-2015
Due Date
12-Sep-2015
Completion Date
31-Jul-2015

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.PXQRDILQLWHWQRLebensmittel - Bestimmung von Fumonisin B1 und Fumonisin B2 in Säuglings- und Kleinkindernahrung auf Maisbasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion nach VorsäulenderivatiserungDenrées 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 précolonneFoodstuffs - 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 pre-column derivatisation67.230Predpakirana in pripravljena hranaPrepackaged and prepared foods67.060QMLKCereals, pulses and derived productsICS:Ta slovenski standard je istoveten z:EN 16187:2015SIST EN 16187:2015en,fr,de01-september-2015SIST EN 16187:2015SLOVENSKI

STANDARDSIST-TS CEN/TS 16187:20111DGRPHãþD
SIST EN 16187:2015
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16187
June 2015 ICS 67.230 Supersedes CEN/TS 16187:2011English 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 pre-column derivatisation

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

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 This European Standard was approved by CEN on 7 May 2015.

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.

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COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,

B-1000 Brussels © 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16187:2015 ESIST EN 16187:2015

EN 16187:2015 (E) 2 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 ........................................................................................................................................... 13 Annex A (informative)

Typical chromatograms ............................................................................................ 14 Annex B (informative)

Precision data ............................................................................................................. 15 Annex C (informative)

Comparison between the method in this document and EN 14352:2004 and EN 13585:2001 on fumonisins in maize ............................................................................................ 18 Bibliography ..................................................................................................................................................... 19

SIST EN 16187:2015

EN 16187:2015 (E) 3 Foreword This document (EN 16187:2015) 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 2015, and conflicting national standards shall be withdrawn at the latest by December 2015. 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 CEN/TS 16187:2011. No technical change has been introduced during the conversion of CEN/TS 16187:2011 into this final draft European Standard. 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 organisations 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. SIST EN 16187:2015

EN 16187:2015 (E) 4 1 Scope This European Standard 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. 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 3696:1995, 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 derivatisation 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 [1]. 4.1 Acetonitrile WARNING — Acetonitrile is hazardous and samples shall be shaken using an orbital shaker which is housed within a fume cupboard. After shaking, samples shall be filtered inside a fume cupboard. 4.2 Methanol 4.3 O-phthaldialdehyde (OPA) 4.4 Citric acid solution, substance concentration c(C6H8O7·H2O) = 0,1 mol/l Dissolve 21,0 g of C6H8O7·H2O in water and dilute to 1 l. 4.5 Disodium hydrogen phosphate solution, c(Na2HPO4) = 0,2 mol/l Dissolve 28,4 g of Na2HPO4 in water and dilute to 1 l. SIST EN 16187:2015

EN 16187:2015 (E) 5 4.6 2-mercaptoethanol 4.7 Citrate-phosphate buffer solution Mix 1 part per volume of citric acid solution (4.4) with 1 part per volume of disodium hydrogen phosphate solution (4.5). 4.8 Extraction solvent Mix 2 parts per volume of citrate-phosphate buffer solution (4.7) with 1 part per volume of methanol (4.2) and 1 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 1 part per volume of acetonitrile (4.1) with 1 part per volume of water. Use this solvent to prepare spiking solutions. 4.12 Mixture of acetonitrile and water B Mix 3 parts per volume of acetonitrile (4.1) with 7 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(Na2B4O7·10H2O) = 0,1 mol/l Dissolve 3,8 g of Na2B4O7·10H2O in 100 ml of water. 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 1 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 1 part per volume of glacial acetic acid (4.9). 4.16 Immunoaffinity column The immunoaffinity column shall contain antibodies raised against FB1 and FB2. The column shall have a capacity of not less than 5 µg of fumonisins and shall give a recovery of not less than 80 % for the sum of FB1 and FB2 when applied as a standard solution in PBS containing 5 µg of fumonisins. The columns shall be warmed up to room temperature before use. With these columns the working range of the method reaches SIST EN 16187:2015

EN 16187:2015 (E) 6 5 000 µg/kg of total fumonisins. The use of columns with a capacity less than 5 µg of fumonisins will reduce the working range of the method. 4.17 Certified standard solution of fumonisin B1 (FB1), mass concentration (FB1) = 50 µg/ml in a mixture of 1 part per volume of acetonitrile and 1 part per volume of water (e.g. Biopure RK 002003 1) or equivalent) 4.18 Certified standard solution of fumonisin B2 (FB2), (FB2) = 50 µg/ml in a mixture of 1 part per volume of acetonitrile and 1 part per volume of water (e.g. Biopure RK 002004 1) 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 FB1 and FB2 stock solution Prepare a mixed FB1 and FB2 stock solution by pipetting 2 000 µl of the FB1 certified standard solution (4.17) and 500 µl of the FB2 certified standard solution (4.18) into a vial. Cap the vial and shake well to obtain a stock solution containing 40,0 µg/ml of FB1 and 10,0 µg/ml of FB2. 4.20 Diluted mixed FB1 and FB2 stock solution 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 FB1 and 0,5 µg/ml of FB2. Store the solution at less than 4 °C. This solution is stable for at least 6 months at these conditions. 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 1. Make up each calibration solution to volume (5 ml) with the mixture of acetonitrile and water B (4.12) and mix well. Store the solution at less than 4 °C. This solution is stable for at least 6 months at these conditions. Table 1 — Preparation of mixed FBs calibration solutions for HPLC HPLC calibration solution Diluted mixed FBs stock solution (4.20) Final concentration of mixed FBs calibration solutions (4.21) Sample equivalent levels of

FB1 and FB2a

FB1 µg/ml FB2 µg/ml FB1 µg/kg FB2 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).

1) Biopure RK 002003 and RK 002004 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 these products. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16187:2015

EN 16187:2015 (E) 7 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 microliter pipettes or microliter 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. 5.12 Vacuum pump. 5.13 Filter paper, e.g. qualitative, strong, fast flow, 24 cm diameter, 30 equivalent. 5.14 Glass microfibre filter paper, e.g. 1,6

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 derivatisation with OPA reagent according to 6.4.1. 5.20.4 Analytical reverse-phase HPLC separating column, e.g. C18 reverse-phase column, 150 mm x 4,6 mm, 5 µm preceded by a suitable pre-column or guard filter, which provides acceptable retention and resolution for FB1 and FB2. SIST EN 16187:2015

EN 16187:2015 (E) 8 Waters C18 SymmetryShield™ 2), Agilent Zorbax SB-C18 2) or similar have been found to be suitable. 5.20.5 Column oven, capable to operate at 20 °C. NOTE C18 reverse-phase columns can also operate at ambient temperatures. 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. 5.21 Linear gradient settings The gradient conditions are given in Table 2. Table 2 — Gradient conditions Time min Flow rate ml/min Mobile phase A % Mobile phase B % 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 FB1 and FB2 by using a Waters C18 SymmetryShield™ 2) column. The use of a different column may necessitate adjustment of these conditions until acceptable resolution is achieved. 6 Procedure 6.1 Extraction The sample should be finely ground to pass through a 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 water bath (5.3) for 60 min. Put the flask on the orbital shaker (5.5) and shake for 30 min at room temperature. Transfer the sample into a centrifuge bottle (5.7) and centrifuge (5.6) at 3 000 g for 15 min. Filter through a filter paper (5.13) and collect 10 ml filtrate. Dilute the 10 ml filtrate with 40 ml of water and mix. Filter through a

2) Waters C18 SymmetryShield™ and Agilent Zorbax SB-C18 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 end

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