Foodstuffs - Determination of ochratoxin A in wine and beer - HPLC method with immunoaffinity column clean-up

This European Standard specifies a method for the determination of ochratoxin A content in wine and beer using immunoaffinity column clean up and high performance liquid chromatography (HPLC), see [2] and [3]. This method has been validated in an interlaboratory study according to AOAC International Guidelines [4] for collaborative study procedures to validate characteristics of a method of analysis for the determination of ochratoxin A in wine and beer via the analysis of naturally contaminated and spiked samples of wine and beer at
levels ranging from 0,1 ng/ml to 3 ng/ml.

Lebensmittel - Bestimmung von Ochratoxin A in Wein und Bier - HPLC-Verfahren mit Reinigung an einer Immunoaffinitätssäule

Diese Europäische Norm legt ein hochleistungs flüssigchromatographisches Verfahren (HPLC) mit Reinigung an einer Immunoaffinitätssäule (IAS) zur Bestimmung von Ochratoxin A in Wein und Bier fest, siehe [2], [3].
Dieses Verfahren wurde in einem Ringversuch nach den AOAC "Guidelines for collaborative study procedures to validate characteristics of a method of analysis" [4] mit natürlich kontaminierten und aufgestockten Bier- und Weinproben mit Ochratoxin A Gehalten von 0,1 ng/ml bis 3 ng/ml validiert.

Produits alimentaires - Dosage de l'ochratoxine A dans le vin et la bière - Méthode par purification sur colonne d'immuno-affinité suivie d'une analyse par chromatographie liquide haute performance (CLHP)

La présente Norme européenne spécifie une méthode de dosage de l’ochratoxine A dans le vin et dans la
bière par chromatographie liquide à haute performance (CLHP) avec purification sur colonne
d’immuno-affinité, voir [2] et [3].
Cette méthode a été validée dans le cadre d’une étude interlaboratoires réalisée conformément aux directives
internationales de l’AOAC [4] relatives aux modes opératoires d’études comparatives visant à valider les
caractéristiques d’une méthode d’analyse, pour le dosage de l’ochratoxine A dans le vin et la bière, via
l’analyse d’échantillons naturellement contaminés et dopés, dans les limites d’une gamme de concentrations
comprise entre 0,1 ng/ml et 3 ng/ml.

Živila - Določevanje ohratoksina A v vinu in pivu - Metoda HPLC z imunoafinitetnim kolonskim čiščenjem

General Information

Status
Published
Publication Date
26-May-2009
Withdrawal Date
29-Nov-2009
ICS
67.160.10 - Alcoholic beverages
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
Ref Project
SIST EN 14133:2009 - Foodstuffs - Determination of ochratoxin A in wine and beer - HPLC method with immunoaffinity column clean-up

Relations

Replaces
EN 14133:2003 - Foodstuffs - Determination of ochratoxin A in wine and beer - HPLC method with immunoaffinity column clean-up
Effective Date
08-Jun-2022
Replaces
EN 14133:2003/AC:2006 - Foodstuffs - Determination of ochratoxin A in wine and beer - HPLC method with immunoaffinity column clean-up
Effective Date
30-May-2009

Overview

EN 14133:2009 (CEN) specifies a validated laboratory method for the determination of ochratoxin A in wine and beer using immunoaffinity column clean‑up followed by HPLC (high performance liquid chromatography) with fluorescence detection. The method was validated in an interlaboratory study following AOAC collaborative study guidelines and is intended for routine control of ochratoxin A at low concentration levels (validated range ~0.1 ng/ml to 3 ng/ml).

Key topics and requirements

  • Scope and validation: Method validated for naturally contaminated and spiked wine and beer; precision and reproducibility data are provided (see Annex A).
  • Sample preparation:
    • Dilution with a solution containing polyethylene glycol (PEG) and sodium hydrogen carbonate to improve recoveries and chromatographic cleanliness.
    • Degassing of sparkling wines and beers; filtration when necessary.
  • Clean-up: Use of immunoaffinity columns with antibodies specific for ochratoxin A; columns must meet minimum capacity and recovery criteria (≥85% recovery for specified challenge).
  • HPLC analysis:
    • Reversed‑phase column (e.g., C18) and isocratic mobile phase (acetonitrile/water/acetic acid).
    • Fluorescence detection (excitation ~333 nm, emission ~460 nm); method sensitivity allows detection at low nanogram levels.
    • Daily calibration with multiple calibrants; calibration curve may be forced through zero for accurate low-level quantification.
  • Quantification and calculation: External standard calibration and formulae provided to convert HPLC response to ng/ml in the original sample.
  • Safety and reagents: Warnings about ochratoxin A toxicity (IARC Group 2B), and hazards of solvents such as acetonitrile and toluene; work in fume hood with appropriate PPE.
  • Apparatus: Typical analytical lab equipment plus HPLC system, fluorescence detector, evaporation apparatus, microbalance and immunoaffinity column manifold.

Applications and users

This standard is intended for:

  • Food control and public health laboratories performing regulatory monitoring of ochratoxin A in alcoholic beverages.
  • Quality assurance teams in wineries and breweries integrating mycotoxin testing into their HACCP/QA programs.
  • Contract analytical laboratories offering mycotoxin testing services.
  • Regulatory agencies and compliance auditors requiring a harmonized, validated method for enforcement and surveillance.

Practical benefits include reliable low‑level detection, standardized clean‑up to reduce matrix interference, and interlaboratory‑validated precision suitable for compliance testing.

Related standards

  • EN ISO 3696 (water for analytical laboratory use) - referenced for reagent water quality.
  • AOAC International guidelines - used for collaborative validation procedures.

Keywords: EN 14133:2009, ochratoxin A, HPLC method, immunoaffinity column, wine testing, beer testing, mycotoxin analysis, food safety, CEN standard.

Frequently Asked Questions

EN 14133:2009 is a standard published by the European Committee for Standardization (CEN). Its full title is "Foodstuffs - Determination of ochratoxin A in wine and beer - HPLC method with immunoaffinity column clean-up". This standard covers: This European Standard specifies a method for the determination of ochratoxin A content in wine and beer using immunoaffinity column clean up and high performance liquid chromatography (HPLC), see [2] and [3]. This method has been validated in an interlaboratory study according to AOAC International Guidelines [4] for collaborative study procedures to validate characteristics of a method of analysis for the determination of ochratoxin A in wine and beer via the analysis of naturally contaminated and spiked samples of wine and beer at levels ranging from 0,1 ng/ml to 3 ng/ml.

This European Standard specifies a method for the determination of ochratoxin A content in wine and beer using immunoaffinity column clean up and high performance liquid chromatography (HPLC), see [2] and [3]. This method has been validated in an interlaboratory study according to AOAC International Guidelines [4] for collaborative study procedures to validate characteristics of a method of analysis for the determination of ochratoxin A in wine and beer via the analysis of naturally contaminated and spiked samples of wine and beer at levels ranging from 0,1 ng/ml to 3 ng/ml.

EN 14133:2009 is classified under the following ICS (International Classification for Standards) categories: 67.160.10 - Alcoholic beverages. The ICS classification helps identify the subject area and facilitates finding related standards.

EN 14133:2009 has the following relationships with other standards: It is inter standard links to EN 14133:2003, EN 14133:2003/AC:2006. Understanding these relationships helps ensure you are using the most current and applicable version of the standard.

You can purchase EN 14133:2009 directly from iTeh Standards. The document is available in PDF format and is delivered instantly after payment. Add the standard to your cart and complete the secure checkout process. iTeh Standards is an authorized distributor of CEN standards.

Standards Content (Sample)


2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Ochratoxin A in Wein und Bier - HPLC-Verfahren mit Reinigung an einer ImmunoaffinitätssäuleProduits alimentaires - Dosage de l'ochratoxine A dans le vin et la bière - Méthode par purification sur colonne d'immuno-affinité suivie d'une analyse par chromatographie liquide haute performance (CLHP)Foodstuffs - Determination of ochratoxin A in wine and beer - HPLC method with immunoaffinity column clean-up67.160.10Alcoholic beveragesICS:Ta slovenski standard je istoveten z:EN 14133:2009SIST EN 14133:2009en,fr,de01-december-2009SIST EN 14133:2009SLOVENSKI
STANDARDSIST EN 14133:2003/AC:2007SIST EN 14133:20031DGRPHãþD

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14133May 2009ICS 67.160.10Supersedes EN 14133:2003
English VersionFoodstuffs - Determination of ochratoxin A in wine and beer -HPLC method with immunoaffinity column clean-upProduits alimentaires - Dosage de l'ochratoxine A dans levin et la bière - Méthode par purification sur colonned'immuno-affinité suivie d'une analyse par chromatographieliquide haute performance (CLHP)Lebensmittel - Bestimmung von Ochratoxin A in Wein undBier - HPLC-Verfahren mit Reinigung an einerImmunoaffinitätssäuleThis European Standard was approved by CEN on 24 May 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards 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 translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, 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 STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre:
Avenue Marnix 17,
B-1000 Brussels© 2009 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14133:2009: ESIST EN 14133:2009

Precision data . 11 Bibliography . 13
Pipette 0,5 ml of the 2 µg/ml ochratoxin A standard solution (4.15) into a 10 ml volumetric flask (5.3) and evaporate the solvent under a stream of nitrogen. Redissolve in 10 ml of mobile phase (4.11). This gives a mass concentration of 100 ng/ml ochratoxin A.
Prepare six HPLC calibrants in separate 5 ml volumetric flasks (5.3) according to Table 1. Dilute each solution to 5 ml with HPLC mobile phase (4.11). SIST EN 14133:2009

Table 1 — Preparation of calibration solutions
Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 µl of filtered mobile phase (4.11) 4970 4900 4700 4000 3000 2000 µl of 100 ng/ml OTA solution
30 100 300 1000 2000 3000 OTA mass concentration (ng/ml) 0,6 2,0 6,0 20 40 60 injected ng OTA
0,06 0,20 0,60 2,00 4,00 6,00
Prepare the calibration solutions at the beginning of every day of the analysis.
4.17 Immunoaffinity columns The immunoaffinity column shall contain antibodies raised against ochratoxin A. The column shall have a total capacity of not less than 100 ng of ochratoxin A and shall give a recovery of not less than 85 % when a diluted wine solution containing 100 ng of ochratoxin A is applied.
5 Apparatus Usual laboratory equipment and, in particular, the following: 5.1 Microbalance, capable to measure 0,01 mg 5.2 Glass vials, approximately 4 ml, with polytetrafluoroethylene (PTFE)-lined screw cap, or appropriate sealable screw cap. Certain types of vials can lead to losses of ochratoxin A during evaporation. To avoid this, silanization can be applied. Prepare vials by filling them with silanizing reagent and leave this reagent in the vial for 1 min. Rinse the vial twice with appropriate solvent (toluene, acetone or hexane) followed by water (twice) and dry the vial. 5.3 Volumetric flasks, 5 ml and 10 ml volume 5.4 Vacuum manifold, to accommodate immunoaffinity columns 5.5 Reservoirs and attachments, to fi
...

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SIST EN 14133:2009 표준 문서는 와인과 맥주에서 오크라톡신 A를 측정하기 위한 HPLC 방법을 상세히 설명하고 있습니다. 이 표준의 주요 범위는 면역친화성 열(column) 청소를 이용하여 오크라톡신 A의 함량을 정밀하게 분석할 수 있는 방법을 제시하는 것입니다. 본 표준은 AOAC International Guidelines에 따라 다기관 연구에서 검증되었으며, 이는 신뢰성과 일관성 있는 결과를 제공하는 데 중요한 요소입니다. 이 표준의 강점 중 하나는 자연적으로 오염된 샘플과 인위적으로 오염된 샘플을 분석하여 0.1 ng/ml에서 3 ng/ml의 수준에서 오크라톡신 A를 정확히 검사할 수 있는 능력입니다. 이러한 광범위한 검출 범위는 다양한 종류의 와인과 맥주 샘플에서의 오염 여부를 판단하는 데 매우 유용합니다. 또한, SIST EN 14133:2009은 품질 보증과 관련된 요구 사항을 충족하는 방법론을 제시함으로써, 식품 안전 분야에서 오크라톡신 A의 존재를 모니터링하는 데 필수적인 기준을 마련하고 있습니다. 이 표준은 업계 전문가와 연구자들에게 신뢰할 수 있는 분석 방법을 제공하며, 와인과 맥주의 안전성을 확보하는 데 기여합니다. 결론적으로, SIST EN 14133:2009 표준은 오크라톡신 A 분석 방법론에 대한 깊이 있는 이해를 제공하며, 식품 안전성과 관련된 중요한 내용을 포함하고 있어, 업계에서의 활용 가치가 매우 높습니다.

Die Norm EN 14133:2009 behandelt die Bestimmung des Gehalts an Ochratoxin A in Wein und Bier und bietet eine präzise und zuverlässige Methode zur Analyse dieser kontaminierenden Substanz. Der Fokus liegt auf der Anwendung einer Hochleistungsflüssigkeitschromatographie (HPLC) in Verbindung mit einer Immunaffinitäts-Säulentechnologie zur Säuberung der Proben. Der Anwendungsbereich der Norm ist klar definiert und spricht die Notwendigkeit an, Ochratoxin A in Wein und Bier sowohl in natürlich kontaminierten als auch in angereicherten Proben zu quantifizieren. Die Validierung dieses Verfahrens erfolgte gemäß den AOAC International Richtlinien, was die wissenschaftliche Robustheit und die Akzeptanz der Methode in der Gemeinschaft unterstreicht. Zu den Stärken der Norm zählt die präzise Detektion von Ochratoxin A in Konzentrationen von 0,1 ng/ml bis 3 ng/ml. Diese hohe Empfindlichkeit ist entscheidend, um die Lebensmittelsicherheit zu gewährleisten, da Ochratoxin A ein bekanntes Mykotoxin ist, das gesundheitsgefährdend sein kann. Darüber hinaus unterstützt die Norm die Harmonisierung von Analyseverfahren in der Europäischen Union, was insbesondere für den internationalen Handel von Lebensmitteln, insbesondere von Wein und Bier, von Bedeutung ist. Die Relevanz der EN 14133:2009 ist unbestreitbar, da sie nicht nur Standards für die Qualitätskontrolle in der Lebensmittelindustrie setzt, sondern auch dazu beiträgt, die Verbraucher vor gesundheitlichen Risiken zu schützen, die durch den Verzehr von kontaminiertem Wein und Bier entstehen können. Somit ist die Norm ein unverzichtbares Instrument für Hersteller und Prüflabore, die sich mit der Überwachung von Mykotoxinen in Lebensmitteln befassen.

Le document standard SIST EN 14133:2009 constitue une référence importante pour l'analyse des aliments, spécifiquement en ce qui concerne la détermination de l'ochratoxine A dans le vin et la bière. Cette norme européenne définit une méthode rigoureuse utilisant la chromatographie liquide haute performance (HPLC) en combinaison avec une purification sur colonne d'immunoaffinité, ce qui assure une précision et une fiabilité optimales pour l'évaluation du contenu d'ochratoxine A. L'un des points forts de cette norme est son approche méthodologique validée par une étude interlaboratoire, conforme aux directives d'AOAC International pour les procédures d'études collaboratives. Cela garantit que la méthode proposée est non seulement robuste, mais également reconnue au niveau européen, ce qui est crucial pour les laboratoires d'analyse et les producteurs de vin et de bière qui doivent se conformer à des normes de sécurité alimentaire strictes. La pertinence de la norme SIST EN 14133:2009 repose également sur sa capacité à analyser des échantillons de vin et de bière, qu'ils soient naturellement contaminés ou spiked, avec une sensibilité permettant de détecter des niveaux d'ochratoxine A allant de 0,1 ng/ml à 3 ng/ml. Cela ouvre la voie à une détection précoce et à un contrôle de qualité efficace dans la chaîne de production des boissons alcoolisées, offrant ainsi une protection supplémentaire aux consommateurs. Cette norme s’inscrit dans une logique de sécurité alimentaire et de conformité réglementaire qui est essentielle dans l'industrie agroalimentaire. En fournissant une méthode standardisée pour la détermination de l'ochratoxine A dans le vin et la bière, elle permet d'harmoniser les pratiques d'analyse et de maintenir la confiance des consommateurs envers les produits concernés. En somme, le SIST EN 14133:2009 représente un outil indispensable pour garantir la qualité et la sécurité des aliments, tout en contribuant à l'amélioration continue des méthodes d'analyse dans le domaine des boissons alcoolisées.

SIST EN 14133:2009は、ワインおよびビール中のオクラトキシンAの Determinationに関する欧州規格であり、高性能液体クロマトグラフィー (HPLC) を用いた免疫親和性カラム清浄化法を指定しています。この規格は、ワインとビールに含まれるオクラトキシンAの量を正確に測定するための信頼性の高い方法を提供し、その適用範囲は特に重要です。 この標準化文書の強みは、その明確な手法の規定と、国際的なガイドラインに基づくバリデーションの実施にあります。AOAC国際ガイドラインに沿ったインターレボラトリー研究により、自然感染およびスパイクサンプルを用いた分析が行われ、オクラトキシンAの定量的評価が0.1 ng/mlから3 ng/mlの範囲で確認されています。このような精密な標準化は、食品業界における安全性基準の強化に貢献します。 さらに、SIST EN 14133:2009の関連性は、ワインやビールの監視と規制において、オクラトキシンAの影響を軽減するための基盤を提供する点にあります。この規格の採用は、製造業者が消費者に対して高品質で安全な食品を提供するために不可欠であり、業界全体の信頼性向上にも寄与します。 以上のように、SIST EN 14133:2009は、食品業界におけるオクラトキシンAの分析に対して、精度と信頼性を兼ね備えた重要な標準であり、その採用は今後の食品安全向上に寄与するでしょう。

EN 14133:2009 is a highly relevant and essential standard in the food safety sector, particularly concerning the quality assurance of beverages such as wine and beer. The scope of this standard focuses on the determination of ochratoxin A, a mycotoxin that poses potential health risks, thereby underlining its critical role in ensuring consumer safety. One of the significant strengths of EN 14133:2009 lies in its methodology. The standard employs a sophisticated high-performance liquid chromatography (HPLC) technique in conjunction with an immunoaffinity column clean-up process, which enhances the accuracy and reliability of ochratoxin A quantification. This method is particularly noteworthy due to its validation through a rigorous interlaboratory study, aligning with AOAC International Guidelines for collaborative studies. Such validation not only bolsters confidence in the method's precision but also ensures its applicability across various laboratories, thus standardizing testing procedures for ochratoxin A detection in different wine and beer samples. Furthermore, the standard specifies a testing range from 0.1 ng/ml to 3 ng/ml, accommodating varying levels of contamination. This range enhances the practical relevance of the method, allowing for both the detection of naturally contaminated samples and the evaluation of spiked samples, which is especially beneficial for quality control processes in the beverage industry. By providing a clear, validated method for the determination of ochratoxin A, EN 14133:2009 plays a pivotal role in safeguarding public health and contributing to the overall quality assurance practices within the food and beverage sectors. Its adoption can significantly support efforts to monitor and reduce ochratoxin A levels, thereby ensuring compliance with food safety regulations and consumer safety standards.