EN 15835:2010

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 and [6].

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

Dieses Dokument legt ein Verfahren zur Bestimmung von Ochratoxin A in Säuglings- und Kleinkindernahrung auf Getreidebasis durch Hochleistungsflüssigchromatographie (HPLC) mit Reinigung an einer Immuno¬affinitätssäule und Fluoreszenzdetektion fest. Dieses Verfahren wurde in einem Ringversuch an sowohl natürlich kontaminierten als auch aufgestockten Proben mit Gehalten von 0,050 µg/kg bis 0,217 µg/kg validiert. Weitere Informationen zur Validierung, siehe Abschnitt 8 und Anhang B. In weiteren Untersuchungen wurde nachgewiesen, dass das Verfahren auf Beikost, die 8 verschiedene Getreidearten, Honig und Kakao enthält, bis zu Gehalten von 3,540 µg/kg anwendbar ist, siehe Anhang C und [6].
WARNUNG - Bei der Anwendung dieser Norm ist es möglich, dass gefährliche Substanzen, Arbeits¬gänge und Geräte angewendet werden. Diese Norm erhebt nicht den Anspruch, dass alle mit ihrer Anwendung verbundenen Sicherheitsprobleme angesprochen werden. Es liegt in der Verantwortung des Anwenders dieser Norm, geeignete Vorkehrungen für den Arbeits- und Gesundheitsschutz zu treffen und vor der Anwendung die Anwendbarkeit einschränkender Vorschriften zu bestimmen.

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 document 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 et détection fluorimétrique. La présente méthode a été
validée dans une étude interlaboratoires via l’analyse d’échantillons naturellement contaminés et
d’échantillons dopés à des concentrations allant de 0,050 μg/kg à 0,217 μg/kg. Pour de plus amples
informations sur la validation voir l’Article 8 et l’Annexe B. Des études supplémentaires ont démontré que
cette méthode est applicable aux aliments à base de céréales pour nourrissons contenant 8 types différents
de céréales, miel et noix de coco, à des niveaux jusqu’à 3,540 μg/kg, voir l’Annexe C et [6].
AVERTISSEMENT — L’utilisation de la présente norme peut impliquer l’utilisation de produits et la
mise en oeuvre de modes opératoires et d’appareillages à caractère dangereux. La présente norme n’a
pas pour but d’aborder tous les problèmes de sécurité liés à son utilisation. Il incombe à l’utilisateur
de la présente norme d’établir, avant de l’utiliser, des pratiques d’hygiène et de sécurité appropriées
et de déterminer l’applicabilité des restrictions réglementaires.

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-Feb-2010
• Withdrawal Date: 30-Aug-2010
• ICS: 67.050 - General methods of tests and analysis for food products
• ICS: 67.060 - Cereals, pulses and derived products
• ICS: 67.230 - Prepackaged and prepared foods
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 5 - Biotoxins
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 28-Oct-2020
• Completion Date: 23-Sep-2025
• Mandate: M/383 - Mandate for standardisation addressed to CEN in the field of Methods of analysis for Mycotoxins in food
• Ref Project: SIST EN 15835:2010 - Foodstuffs - Determination of ochratoxin A in cereal based foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection

Overview

EN 15835:2010 is a CEN European Standard for the determination of ochratoxin A in cereal‑based foods for infants and young children. The method uses high performance liquid chromatography (HPLC) combined with immunoaffinity column (IAC) cleanup and fluorescence detection, including a post‑column derivatization step (ammonia) to enhance signal. The standard was validated by an interlaboratory study on naturally contaminated and spiked samples (0.050 µg/kg to 0.217 µg/kg) and shown applicable to complex baby‑food matrices (multiple cereals, honey, cocoa) up to 3 540 µg/kg.

Key topics and requirements

• Analytical principle: extraction with tert‑butyl methyl ether after acid/salt treatment, solvent evaporation, reconstitution in methanol/PBS, hexane defatting, IAC cleanup and HPLC‑fluorescence detection (post‑column ammonia derivatization).
• Critical reagents and solvents: tert‑butyl methyl ether, methanol (HPLC grade), hexane, phosphate buffered saline (PBS), ammonium hydroxide (post‑column reagent).
• Immunoaffinity columns: antibodies specific to ochratoxin A; column capacity ≥ 100 ng ochratoxin A and recovery ≥ 85 % in the prescribed test solution.
• HPLC conditions: reverse‑phase column (e.g., C18), gradient-capable pump, fluorescence detector (standard conditions and an alternative excitation/emission pair if ammonia derivatization is omitted).
• Validation and performance: interlaboratory precision data and annexed precision/in‑house study results (see Annexes B and C). The standard cites EN ISO 3696 for laboratory water quality.
• Safety: handling ochratoxin A and solvents requires fume hood, protective clothing and appropriate waste disposal; ochratoxin A is a possible human carcinogen (IARC group 2B).

Applications and users

EN 15835:2010 is intended for use by:

• Food safety and testing laboratories performing routine and confirmatory analyses of baby foods and infant cereals.
• Manufacturers and quality assurance teams in baby‑food production for compliance testing and vendor/raw‑material control.
• Regulatory and public health authorities assessing compliance with ochratoxin A limits and conducting official control programs.
• Research and reference laboratories validating methods, performing interlaboratory comparisons or method development for mycotoxin analysis.

Practical uses include monitoring ochratoxin A in finished cereal‑based infant foods, investigating contamination sources, and generating data for risk assessment and regulatory enforcement.

Related standards and references

• EN ISO 3696:1995 - Water for analytical laboratory use (referenced for water quality).
• CEN/TC 275 publications and informative annexes (A, B, C) in EN 15835 provide chromatograms, precision data and in‑house applicability studies.

Keywords: EN 15835, ochratoxin A, HPLC method, immunoaffinity column, fluorescence detection, baby food testing, cereal based foods, mycotoxin analysis, CEN standard.

Standards Content (Sample)

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
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

Typical chromatograms . 14Annex B (informative)
Precision data . 15Annex C (informative)
Data on the in-house study . 16Bibliography . 18 SIST EN 15835:2010

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.
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

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

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

= 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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