Foodstuffs - Determination of ochratoxin A 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 ochratoxin A in cereal based foods for infants and young children by high performance liquid chromatography (HPLC) with immunoaffinity column 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 0,050 μg/kg to 0,217 μg/kg. For further information on the validation see Clause 8 and Annex B. Additional studies have shown that this method is applicable to cereal based baby foods containing 8 different types of cereals, honey and cocoa, at levels up to 3,540 μg/kg, see Annex C.

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

Dieser europäische Normentwurf legt ein Verfahren zur Bestimmung von Ochratoxin A in Säuglings- und Kleinkindernahrung auf Getreidebasis durch Hochleistungsflüssigkeitschromatographie (HPLC) mit Reinigung an einer Immunoaffinitätssäule fest. Dieses Verfahren wurde in einem Ringversuch durch Analyse sowohl von natürlich kontaminierten als auch von aufgestockten Proben von Säuglingsnahrung auf Weizenbasis mit Ge¬halten von 0,023 µg/kg bis 0,225 µg/kg validiert. Es wurde nachgewiesen, dass das Verfahren auf Beikost, die 8 verschiedene Getreidearten, Honig und Kakao enthält, bis zu Gehalten von 3,480 µg/kg an¬wendbar ist.Die Bestimmungsgrenze des Verfahrens liegt bei 0,007 µg/kg (aus laborinternen Versuchen abgeleitete Daten).

Produits alimentaires - Dosage de l'ochratoxine A dans les aliments à base de céréales pour nourrissons et jeunes enfants - Méthode CLHP avec purification sur colonne d'immuno-affinité et détection par fluorescence

Le présent projet de Norme Européenne spécifie une méthode pour déterminer la teneur en ochratoxine A dans les aliments à base de céréales pour nourrissons et jeunes enfants, par chromatographie liquide à haute performance (CLHP) avec purification sur colonne d’immuno-affinité. Cette méthode a été validée lors d’une étude interlaboratoires via l’analyse d’échantillons naturellement contaminés et d’échantillons dopés d’aliments à base de blé pour nourrissons, à des concentrations allant de 0,023 µg/kg à 0,225 µg/kg. Cette méthode s’est révélée applicable aux aliments à base de céréales pour nourrissons contenant 8 différents types de céréales, du miel et du cacao, à des concentrations inférieures ou égales à 3,480 µg/kg. Il a été démontré que la limite de quantification de la méthode se situe à 0,007 µg/kg (information obtenue à partir des études intralaboratoires).

Živila - Določevanje ohratoksina A v žitnih kašicah za dojenčke in majhne otroke - Metoda HPLC s čiščenjem z imunoafinitetno kolono in fluorescenčno detekcijo

Ta evropski standard opredeljuje metodo za določevanje ohratoksina A v hrani na osnovi žita 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 preko analize tako naravno kontaminiranih kot vzorcev z internimi dodatki v razponu od 0,050 μg/kg do 0,217 μg/kg. Za nadaljnje informacije o potrjevanju glej Klavzulo 8 in Dodatek B. Dodatne študije so pokazale, da je ta metoda uporabna za otroško hrano na osnovi žita, ki vsebuje 8 različnih vrst žitaric, med in kakav, v stopnjah do 3,540 μg/kg (glej Dodatek C).

General Information

Status
Published
Publication Date
02-Mar-2010
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
19-Feb-2010
Due Date
26-Apr-2010
Completion Date
03-Mar-2010

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Ochratoxin A in Säuglings- und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und FluoreszenzdetektionProduits alimentaires - Dosage de l'ochratoxine A dans les aliments à base de céréales pour nourrissons et jeunes enfants - Méthode CLHP avec purification sur colonne d'immuno-affinité et détection par fluorescenceFoodstuffs - Determination of ochratoxin A 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 15835:2010SIST EN 15835:2010en,fr,de01-maj-2010SIST EN 15835:2010SLOVENSKI

STANDARD
SIST EN 15835:2010
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15835
February 2010 ICS 67.050; 67.230 English Version

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

Produits alimentaires - Dosage de l'ochratoxine A dans les aliments à base de céréales pour nourrissons et jeunes enfants - Méthode CLHP avec purification sur colonne d'immuno-affinité et détection par fluorescence

Lebensmittel - Bestimmung von Ochratoxin A in Säuglings-und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule und Fluoreszenzdetektion This European Standard was approved by CEN on 25 December 2009.

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 15835:2010: ESIST EN 15835:2010

EN 15835:2010 (E) 2 Contents Page Foreword ..............................................................................................................................................................31Scope ......................................................................................................................................................42Normative references ............................................................................................................................43Principle ..................................................................................................................................................44Reagents .................................................................................................................................................45Apparatus ...............................................................................................................................................76Procedure ...............................................................................................................................................97HPLC analysis ..................................................................................................................................... 108Calculation ........................................................................................................................................... 119Precision .............................................................................................................................................. 1210Test report ........................................................................................................................................... 13Annex A (informative)

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

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

Data on the in-house study ....................................................................................... 16Bibliography ..................................................................................................................................................... 18 SIST EN 15835:2010

EN 15835:2010 (E) 3 Foreword This document (EN 15835:2010) 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 August 2010, and conflicting national standards shall be withdrawn at the latest by August 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. 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, 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 15835:2010

EN 15835:2010 (E) 4 1 Scope This European Standard specifies a method for the determination of ochratoxin A in cereal based foods for infants and young children by high performance liquid chromatography (HPLC) with immunoaffinity column 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 0,050 µg/kg to 0,217 µg/kg. For further information on the validation see Clause 8 and Annex B. Additional studies have shown that this method is applicable to cereal based baby foods containing 8 different types of cereals, honey and cocoa, at levels up to 3,540 µg/kg, see Annex C and [6]. 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.

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 tert-butyl methyl ether after addition of 0,5 mol/l phosphoric acid / 2 mol/l sodium chloride solution. The extract is evaporated and redissolved in methanol and phosphate buffered saline (PBS) solution. After removal of lypophilic compounds with hexane, the extract is applied to an immunoaffinity column containing antibodies specific to ochratoxin A. The toxin is eluted from the column with methanol. Ochratoxin A is determined by HPLC with enhanced fluorescence detection involving post column reaction with ammonia. NOTE Some investigations indicate that HPLC can be also performed without the use of ammonia although this results in at least a two-fold decrease of the response for ochratoxin A. In this case, the fluorescence detection conditions need to be changed (excitation wavelength = 333 nm, emission wavelength = 460 nm). 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. 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 SIST EN 15835:2010

EN 15835:2010 (E) 5 4.4 Disodium hydrogen phosphate, Na2HPO4 anhydrous or Na2HPO4·12 H2O 4.5 Potassium chloride 4.6 Potassium dihydrogen phosphate 4.7 Sodium chloride 4.8 Sodium hydroxide 4.9 Ammonium hydroxide solution, the mass fraction w(NH4OH) = 25 % in water (post column reagent) Degas the solution with a degasser (5.21.7). 4.10 Hydrochloric acid solution, w(HCl) is 37 % (acidimetric) 4.11 Phosphoric acid solution, w(H3PO4) = 85 % 4.12 Hydrochloric acid solution, c(HCl) = 0,1 mol/l Dilute 8,28 ml of hydrochloric acid solution (4.10) to 1 l of water. 4.13 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l Dissolve 4 g of sodium hydroxide (4.8) in 1 l of water. 4.14 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 (4.7), 1,2 g of anhydrous disodium hydrogen phosphate or 2,9 g of Na2HPO4·12 H2O (4.4), 0,2 g of potassium dihydrogen phosphate (4.6) and 0,2 g of potassium chloride (4.5) in 900 ml of water. After dissolution, adjust the pH to 7,4 with hydrochloric acid solution (4.12) or sodium hydroxide solution (4.13) as appropriate, then dilute to 1 l with water. Alternatively, a PBS solution with equivalent properties can be prepared from commercially available PBS material. 4.15 Mixture of phosphoric acid solution and sodium chloride solution, c(H3PO4) = 0,5 mol/l, c(NaCl) = 2 mol/l Dissolve 118 g of sodium chloride (4.7) in approximately 900 ml of water. Add 33 ml of phosphoric acid (4.11) and make up to 1,0 l with water. 4.16 Glacial acetic acid, the mass fraction ≥ 99,7 % 4.17 Acetic acid solution, the volume fraction is 9 % Add 90 ml of glacial acetic acid (4.16) and 910 ml of water. 4.18 Hexane WARNING — Hexane is highly flammable. Operations involving this solvent shall be performed in a fume cupboard. Serious health problems can be derived from prolonged exposure to this reagent. SIST EN 15835:2010

EN 15835:2010 (E) 6 4.19 Methanol, gradient grade 4.20 Toluene

4.21 Mixture of methanol and acetic acid solution Mix 72 parts per volume of methanol (4.19) with 28 parts per volume of acetic acid solution (4.17). 4.22 Tert-butyl methyl ether WARNING — Tert-butyl methyl ether is hazardous and samples shall be blended using an explosion proof blender which is housed within a fume cupboard. Centrifugation of extracts shall be performed at cool temperature (4 °C to 8 °C). 4.23 Mixture of toluene and glacial acetic acid Mix 99 parts per volume of toluene (4.20) with one part per volume of glacial acetic acid (4.16). 4.24 HPLC mobile phase A Acetic acid solution (4.17). 4.25 HPLC mobile phase B Methanol (4.19). Degas the mobile phases A and B with for example helium (4.2). Helium can be pumped into the reservoirs of both mobile phases A and B. The pumping rate should be 50 ml/min to 100 ml/min. The use of a degasser is also an acceptable option. 4.26 Immunoaffinity columns The immunoaffinity column contain antibodies raised against ochratoxin A. The column shall have a capacity of not less than 100 ng of ochratoxin A and shall give a recovery of not less than 85 % when applied as a standard solution of ochratoxin A in a mixture of 15 parts per volume of methanol (4.19) and 85 parts per volume of PBS solution (4.14) containing 3 ng of ochratoxin A. 4.27 Ochratoxin A, in crystal form or as a film in ampoules WARNING — Ochratoxin A is a potent nephrotoxin with immunotoxic, teratogenic and potential genotoxic properties. The International Agency for Research on Cancer (IARC) has classified ochratoxin A as a possible human carcinogen (group 2B). Protective clothing, gloves and safety glasses should be worn at all times, and all standard and sample preparation stages should be carried out in a fume cupboard. 4.28 Ochratoxin A stock solution Prepare a stock solution of ochratoxin A in the mixture of toluene and glacial acetic acid (4.23) with a nominal concentration of 10 µg/ml. To determine the exact concentration, record the absorption curve between a wavelength of 300 nm and 370 nm in 5 nm steps in 1 cm quartz cells (5.22) in a spectrometer with the solvent mixture (4.23) as reference. Identify the wavelength for maximum absorption and calculate the mass concentration of ochratoxin A, ota, in micrograms per millilitre, using Equation (1): bMA×××=ερ100maxota (1) SIST EN 15835:2010

EN 15835:2010 (E) 7 where Amax is the absorption determined at the maximum of the absorption curve (here: at 333 nm); M is the molar mass, in grams per mole, of ochratoxin A (M = 403,8 g/mol); 0 is the molar absorption coefficient, in square metres per mole, of ochratoxin A in the solvent mixture (4.23), (here: 544 m2/mol); b is the 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 six months. 4.29 Ochratoxin A standard solution Pipette a volume of ochratoxin A stock solution (4.28) containing exactly 400 ng ochratoxin A into a 10 ml calibrated volumetric flask (5.13) and dilute to 10 ml with the mixture of toluene and glacial acetic acid (4.23) and shake. This gives a standard solution containing 40,0 ng/ml of ochratoxin A.

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 six months. 4.30 Ochratoxin A spiking solution Pipette a volume of ochratoxin A stock solution (4.28) containing exactly 2 500 ng ochratoxin A into a 50 ml calibrated volumetric flask (5.13) and dilute to 50 ml with the mixture of toluene and glacial acetic acid (4.23) and shake. This gives a spiking solution containing 50,0 ng/ml of ochratoxin A.

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 six months. 5 Apparatus 5.1 General Usual laboratory glassware and equipment and, in particular, the following: 5.2 High speed blender 5.3 Analytical balance, capable of weighing to 0,000 1 g 5.4 Laboratory balance, capable of weighing to 0,1 g 5.5 Vacuum manifold, to accommodate immunoaffinity columns 5.6 Filter papers, suitable for qualitative analysis 5.7 pH indicator paper, for pH = 0 to pH = 14 5.8 Cooling centrifuge, capable of a centrifugal force of 15 300 g at 4 °C SIST EN 15835:2010

EN 15835:2010 (E) 8 5.9 Centrifuge bottles, of 250 ml capacity with screw cap, chemically resistant to tert-butyl methyl ether and able to work at 15 300 g without deformation 5.10 Rotary evaporator, with water bath 5.11 Disposable syringe barrels, to be used as reservoirs, of 5 ml, 20 ml and 50 ml capacity, luer locks and attachments to fit to immunoaffinity columns 5.12 Microsyringes, of 25 µl, 50 µl, 100 µl, 500 µl and 1 000 µl capacity 5.13 Calibrated volumetric flasks, e.g. of 10 ml, 50 ml and 1 000 ml capacity 5.14 Vacuum system 5.15 Round bottomed flasks, of 100 ml capacity 5.16 Calibrated volumetric pipettes 5.17 Displacement pipettes, of 100 µl and 1 000 µl capacity, with appropriate tips 5.18 Glass vials, of GC autosampler type, approximately 1,8 ml capacity and crimp caps 5.19 Separating funnel, of 250 ml capacity 5.20 Vortex mixer 5.21 HPLC apparatus, comprising the following: 5.21.1 Injection system, capable of injecting e.g. 50 µl 5.21.2 Eluent and post column reagent reservoirs 5.21.3 Mobile phase pump, gradient, capable of maintaining a volume flow rate of 1 ml/min pulse free 5.21.4 Fluorescence detector, able to provide

= 390 nm excitation and

= 440 nm emission wavelengths 5.21.5 Recorder, integrator or computer based data processing system 5.21.6 Analytical reverse-phase HPLC separating column, for example C18, base deactivated octadecyl silane (ODS), length of 25 cm, inner diameter of 4,7 mm and a particle size of 5 µm, which ensures resolution of ochratoxin A from all other peaks. The maximum overlap of peaks shall be less then 10 %. A suitable corresponding reverse-phase guard column should be used 5.21.7 Degasser, optional, for degassing HPLC mobile phases (4.24) and (4.25) a

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