Foodstuffs - Determination of aflatoxin B1 in cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection

This European Standard specifies a method for the determination of aflatoxin B1 in baby food by high performance liquid chromatography (HPLC) with immunoaffinity cleanup and fluorescence detection. This method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples ranging from 0,07 µg/kg to 0,18 µg/kg. For further information on the validation, see Clause 9 and Annex B.

Lebensmittel - Bestimmung von Aflatoxin B1 in Säuglings- und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion

Diese Europäische Norm legt ein Verfahren zur Bestimmung von Aflatoxin B1 in Säuglingsnahrung durch
Hochleistungsflüssigchromatographie (HPLC) mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion
fest. Dieses Verfahren wurde in einem Ringversuch durch die Untersuchung von natürlich kontaminierten
Proben und aufgestockten Proben mit Gehalten von 0,07 µg/kg bis 0,18 µg/kg validiert.
Weitere Informationen zur Validierung befinden sich in Abschnitt 9 und Anhang B.

Produits alimentaires - Dosage de l'aflatoxine B1 dans les produits pour nourrissons et jeunes enfants à base de céréales - Méthode de chromatographie liquide haute performance avec purification sur colonne d'immunoaffinité et détection par fluorescence

Le présent projet de norme européenne spécifie une méthode pour le dosage de l’aflatoxine B1 dans les aliments pour nourrissons par chromatographie liquide haute performance (CLHP) avec purification par immunoaffinité. Cette méthode a été validée suite à une étude interlaboratoire via l’analyse d’échantillons naturellement contaminés compris entre 0,07 μg/kg et 0,17 μg/kg, et des échantillons supplémentés compris entre 0,10 μg/kg et 0,18 μg/kg. La limite de quantification de la méthode est de 0,05 μg/kg (valeur obtenue à partir d’une étude interne et d’une étude interlaboratoire), selon l’équipement utilisé.

Živila - Določevanje aflatoksina B1 v žitnih kašicah za dojenčke in majhne otroke - Metoda HPLC z imunoafinitetnim kolonskim čiščenjem in fluorescenčno detekcijo

Ta evropski standard določa metodo za določevanje aflatoksina B1 v hrani za dojenčke z tekočinsko kromatografijo visoke ločljivosti (HPLC) z imunoafinitetnim čiščenjem in fluorescenčno detekcijo. Ta metoda je bila potrjena v medlaboratorijski študiji preko analiz  naravno kontaminiranih vzorcev in vzorcev z internimi dodatki v razponu od 0,07 μg/kg do 0,18 μg/kg. Za nadaljnje informacije o potrjevanju, glej Klavzulo 9 in Dodatek B.

General Information

Status
Published
Public Enquiry End Date
13-Jan-2009
Publication Date
15-Jul-2010
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
08-Jun-2010
Due Date
13-Aug-2010
Completion Date
16-Jul-2010

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Aflatoxin B1 in Säuglings- und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und FluoreszenzdetektionProduits alimentaires - Dosage de l'aflatoxine B1 dans les produits pour nourrissons et jeunes enfants à base de céréales - Méthode de chromatographie liquide haute performance avec purification sur colonne d'immunoaffinité et détection par fluorescenceFoodstuffs - Determination of aflatoxin B1 in cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection67.230Predpakirana in pripravljena hranaPrepackaged and prepared foods67.060QMLKCereals, pulses and derived productsICS:Ta slovenski standard je istoveten z:EN 15851:2010SIST EN 15851:2010en,fr,de01-september-2010SIST EN 15851:2010SLOVENSKI

STANDARD
SIST EN 15851:2010
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15851
April 2010 ICS 67.060 English Version

Foodstuffs - Determination of aflatoxin B1 in cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection

Produits alimentaires - Dosage de l'aflatoxine B1 dans les produits pour nourrissons et jeunes enfants à base de céréales - Méthode de chromatographie liquide haute performance avec purification sur colonne d'immunoaffinité et détection par fluorescence

Lebensmittel - Bestimmung von Aflatoxin B1 in Säuglings- und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion This European Standard was approved by CEN on 27 February 2010.

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 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 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, 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 © 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15851:2010: ESIST EN 15851:2010

EN 15851:2010 (E) 2 Contents Page Foreword ..............................................................................................................................................................31Scope ......................................................................................................................................................42Normative references ............................................................................................................................43Principle ..................................................................................................................................................44Reagents .................................................................................................................................................45Apparatus ...............................................................................................................................................76Procedure ...............................................................................................................................................87HPLC analysis ........................................................................................................................................98Calculation ........................................................................................................................................... 119Precision .............................................................................................................................................. 1110Test report ........................................................................................................................................... 12Annex A (informative)

Typical chromatogram .............................................................................................. 13Annex B (informative)

Precision data ............................................................................................................. 14Bibliography ..................................................................................................................................................... 15

SIST EN 15851:2010

EN 15851:2010 (E) 3 Foreword This document (EN 15851:2010) 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 October 2010, and conflicting national standards shall be withdrawn at the latest by October 2010. 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. WARNING — The use of this standard can involve hazardous materials, operations and equipment. 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 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 implement this European Standard: 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. SIST EN 15851:2010

EN 15851:2010 (E) 4 1 Scope This European Standard specifies a method for the determination of aflatoxin B1 in baby food by high performance liquid chromatography (HPLC) with immunoaffinity cleanup and fluorescence detection. This method has been validated in an interlaboratory study via the analysis of both naturally contaminated and spiked samples ranging from 0,07 µg/kg to 0,18 µg/kg. For further information on the validation, see Clause 9 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 A test portion is extracted with a mixture of methanol and water. The extract is filtered, diluted with phosphate buffered saline (PBS) to a specified solvent concentration, and applied to an immunoaffinity column containing antibodies specific to aflatoxin B1. Aflatoxin B1 is purified and concentrated on the column and removed from the antibodies using methanol as eluent. Aflatoxin B1 is quantified by reverse-phase high performance liquid chromatography (RP-HPLC) with post column derivatization (PCD) involving bromination followed by fluorescence detection. The post column derivatization is achieved with either electrochemically generated bromine or with pyridinium hydrobromide perbromide (PBPB). 4 Reagents 4.1 General 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 [4]. 4.2 Helium purified compressed gas. 4.3 Nitrogen. 4.4 Disodium hydrogen phosphate, Na2HPO4 anhydrous or Na2HPO4·12 H2O. 4.5 Potassium bromide. 4.6 Potassium chloride. 4.7 Potassium dihydrogen phosphate, KH2PO4. 4.8 Sodium chloride. SIST EN 15851:2010

EN 15851:2010 (E) 5 4.9 Sodium hydroxide. 4.10 Hydrochloric acid solution, mass fraction w(HCl) =

37 % in water. 4.11 Hydrochloric acid solution, substance concentration c(HCl) = 0,1 mol/l. Dilute 8,28 ml of hydrochloric acid solution (4.10) to 1 l with water. 4.12 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l. Dissolve 4 g of sodium hydroxide (4.9) in 1 l of water. 4.13 Phosphate buffered saline (PBS) solution, c(NaCl) = 120 mmol/l, c(KCl) = 2,7 mmol/l, c(phosphate buffer) = 10 mmol/l, pH = 7,4. Dissolve 8,0 g of sodium chloride (4.8), 1,2 g of anhydrous disodium hydrogen phosphate or 2,9 g of Na2HPO4·12 H2O (4.2), 0,2 g of potassium dihydrogen phosphate (4.7) and 0,2 g of potassium chloride (4.6) in 900 ml of water. After dissolution, adjust the pH to 7,4 with hydrochloric acid solution (4.11) or sodium hydroxide solution (4.12) as appropriate, then dilute to 1 l with water. Alternatively, a PBS solution with equivalent properties can be prepared from commercially available PBS material. 4.14 Pyridinium hydrobromide perbromide (PBPB), [CAS: 39416-48-3]. 4.15 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. 4.16 Methanol, HPLC grade. 4.17 Methanol,

technical grade. 4.18 Toluene. 4.19 Extraction solvent. Mix eight parts per volume of methanol (4.17) with two parts per volume of water. 4.20 Nitric acid, c(HNO3) = 4 mol/l. 4.21 HPLC mobile phase A, for use with PBPB. Mix six parts per volume of water with two parts per volume of acetonitrile (4.15) and three parts per volume of methanol (4.16). Degas mobile phase A with for example helium (4.2). 4.22 HPLC mobile phase B, for use with electrochemically generated bromine. Mix six parts per volume of water with two parts per volume of acetonitrile (4.15) and three parts per volume of methanol (4.16). Add 120 mg of potassium bromide (4.5) and 350 µl of nitric acid (4.20) per litre of mobile phase. Degas mobile phase B with for example helium (4.2). 4.23 Post-column reagent. Dissolve 50 mg of PBPB (4.14) in 1 l of water. To be used with mobile phase solvent A (4.21). The solution may be used up to four days if stored in a dark place at room temperature. SIST EN 15851:2010

EN 15851:2010 (E) 6 4.24 Mixture of toluene and acetonitrile. Mix nine parts per volume of toluene (4.18) with one part per volume of acetonitrile (4.15). 4.25 Immunoaffinity column. The immunoaffinity column shall contain antibodies raised against aflatoxin B1. The column shall have a capacity of not less than 100 ng of aflatoxin B1 and shall give a recovery of not less than 80 % when 5 ng of

aflatoxin B1 are applied as a standard solution in a mixture of ten parts per volume of methanol and 90 parts per volume of water. 4.26 Aflatoxin B1, in crystal form or as a film in ampoules or in form of commercially available aflatoxin B1 solution. WARNING — Aflatoxins are subject to light degradation. Protect the laboratory, where the analyses are done, adequately from daylight. This can be achieved effectively by using ultraviolet (UV) absorbing foil on the windows in combination with subdued light (no direct sunlight) or curtains or blinds in combination with artificial light (fluorescent tubes are acceptable). Protect aflatoxin containing solutions from light as much as possible (keep in the dark, use aluminium foil or amber-coloured glassware). 4.27 Aflatoxin B1 stock solution, c ≈ 10 µg/ml. Prepare a solution of aflatoxin B1 in the mixture of toluene and acetonitrile (4.24) to give a solution with a mass concentration of approximately 10 µg/ml. To determine the exact mass concentration, record the absorption curve between 330 nm and 370 nm in 1 cm quartz cells in a spectrometer (5.14) with the mixture of toluene and acetonitrile (4.24) as reference. Identify the wavelength for maximum absorption (between 330 nm and 370 nm). Calculate the mass concentration of aflatoxin B1, afl, in µg/ml, using Equation (1):

bMA×××=ερ100maxafl (1) where

Amax is the absorption determined at the maximum of the absorption curve (between 330 nm and 370 nm);

M is the molar mass, in g/mol, of aflatoxin B1 (M = 312 g/mol);

0 is the molar absorption coefficient, in square metres per mole, of aflatoxin B1 in the mixture of toluene and acetonitrile (4.24) (1 930 m2/mol, see [5]);

b is the optical path length, in centimetres, of the quartz cell. Store this solution in a freezer at approximately - 18 °C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the concentration of the solution if it is older than 12 months. 4.28 Aflatoxin B1 standard solution,

= 5,00 ng/ml. Pipette a volume of aflatoxin B1 stock solution (4.27) containing exactly 1,00 µg aflatoxin B1 into a 200 ml calibrated volumetric flask and dilute to the mark with the mixture of toluene and acetonitrile (4.24). This solution contains 5,00 ng/ml aflatoxin B1. SIST EN 15851:2010

EN 15851:2010 (E) 7 Wrap the flask tightly in aluminium foil and store it at less than 4 °C. Before use, do not remove the aluminium foil until the contents have reached room temperature to avoid incorporation of water by condensation. A solution stored in this way is stable for at least four weeks. 4.29 Aflatoxin B1 spiking solution,

= 2 µg/ml. Pipette a volume of aflatoxin B1 stock solution (4.27) containing exactly 20 µg aflatoxin B1 into a 10 ml calibrated volumetric flask.

Evaporate the mixture of toluene and acetonitrile solution just to dryness under a stream of nitrogen at room temperature. Make up to volume with methanol (4.16) and shake well. The concentration of this spiking solution is 2 µg/ml for aflatoxin B1. Wrap the flask tightly in aluminium foil and store it at less than 4 °C. Before use, do not remove the aluminium foil until the contents have reached room temperature to avoid incorporation of water by condensation. A solution stored in this way is stable for at least three months. 5 Apparatus WARNING — All glassware coming into contact with aqueous solutions of aflatoxins shall be washed with acid solution before use. Many laboratory washing machines do this as part of the washing programme. Otherwise soak laboratory glassware coming into contact with aqueous solutions of aflatoxins in sulfuric acid (c = 2 mol/l) for several hours (e.g. 15 h overnight), then rinse well (e.g. at least three times) with water to remove all traces of acid. Check the absence of acid with pH paper.

This treatment is necessary, because the use of non-acid washed glassware can cause losses of aflatoxins. In practice, the treatment is necessary for round bottomed flasks, volumetric flasks, measuring cylinders, vials or tubes used for calibration solutions and final extracts (particularly autosampler vials), and Pasteur pipettes, if these are used to transfer calibration solutions or extracts. 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 0,01 g. 5.3 Adjustable vertical or horizontal shaker. 5.4 Filter paper, e.g. 24 cm diameter, prefolded. 5.5 Conical flask, with screw top or glass stopper of 500 ml capacity. 5.6 Glass microfibre filter, retention size 1,6 µm or smaller. 5.7 Reservoir, of 75 ml capacity with luer tip connector and attachments for immunoaffinity column (IAC). 5.8 Hand pump, 20 ml syringe with luer lock or rubber stopper for IAC. 5.9 Volumetric flasks, of 5 ml, 10 ml, 20 ml, 150 ml, and 200 ml capacity with an accuracy of at least 0,5 %. 5.10 Disposable syringe filter unit, with pore size of 0,45 µm. Prior to usage, verify that no aflatoxin losses occur during filtrat

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