EN 17251:2020

Foodstuffs - Determination of ochratoxin A in pork meat and derived products by IAC clean-up and HPLC-FLD

This document describes a procedure for the determination of ochratoxin A (OTA) in pork products specifically ham, pork-based products (canned chopped pork) and pork liver using high performance liquid chromatography with fluorescence detection (HPLC-FLD).
The method has been validated for ochratoxin A in naturally contaminated ham, pork based products (canned chopped pork) and pork liver containing 0,5 μg/kg to 11 μg/kg [4], [5], [6].
Laboratory experiences have shown that this method is also applicable to pâté and kidney [4].

Lebensmittel - Bestimmung von Ochratoxin A in Schweinefleisch und Schweinefleischerzeugnissen mit Hochleistungsflüssigchromatographie und Fluoreszenzdetektion (HPLC-FLD)

Dieses Dokument beschreibt ein Verfahren zur Bestimmung von Ochratoxin A (OTA) in Schweinefleischerzeugnissen, insbesondere Schinken, Schweinefleisch-basierten Erzeugnissen (geschnittenes Schweinefleisch in Dosen) und Schweineleber mit Hochleistungsflüssigchromatographie mit Fluoreszenzdetektion (HPLC-FLD, en: high performance liquid chromatography with fluorescence detection).
Das Verfahren wurde für Ochratoxin A in natürlich kontaminiertem Schinken, Schweinefleisch-basierten Erzeugnissen (geschnittenem Schweinefleisch in Dosen) und Schweineleber im Bereich von 0,5 μg/kg bis 11 μg/kg validiert [4], [5], [6].
Laborerfahrungen haben gezeigt, dass dieses Verfahren auch auf Pastete und Nieren anwendbar ist [4].

Produits alimentaires - Dosage de l'ochratoxine A dans la viande de porc et les produits carnés issus du porc par chromatographie liquide à haute performance couplée à la détection par fluorescence (CLHP-DFL)

La présente Norme européenne décrit un mode opératoire de dosage de l’ochratoxine A (OTA) dans les produits issus du porc, notamment le jambon, les produits carnés à base de porc (porc haché en conserve) et le foie de porc, par chromatographie liquide à haute performance couplée à la détection par fluorescence (CLHP-DFL).
La méthode a été validée dans des échantillons naturellement contaminés en ochratoxine A de jambon, de produits carnés à base de porc (porc haché en conserve) et de foie de porc à des teneurs allant de 0,5 μg/kg à 11 μg/kg [4], [5], [6].
Les expériences en laboratoire ont démontré que cette méthode est également applicable aux pâtés et aux reins [4].

Živila - Določevanje ohratoksina A v svinjskem mesu in predelanih proizvodih z IAC-čiščenjem in tekočinsko kromatografijo visoke ločljivosti s fluorescenčno detekcijo (HPLC-FLD)

Ta dokument opisuje postopek za določevanje ohratoksina A (OTA) v proizvodih iz svinjskega mesa, natančneje v šunki, proizvodih, ki vsebujejo prašičje meso (sesekljana svinjina v konzervi), in svinjskih jetrih z visokozmogljivo tekočinsko kromatografijo s fluorescenčno detekcijo (HPLC-FLD).  Metoda je bila validirana za ohratoksin A z naravno kontaminirano šunko, proizvodi, ki vsebujejo prašičje meso (sesekljana svinjina v konzervi), in svinjskimi jetri z vsebnostjo 0,5 do 11 μg/kg [4, 5, 6]. Izkušnje v laboratoriju so pokazale, da je ta metoda uporabna tudi za pašteto in ledvice [4].

General Information

• Status: Published
• Publication Date: 14-Jan-2020
• Withdrawal Date: 30-Jul-2020
• ICS: 67.120.10 - Meat and meat products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 5 - Biotoxins
• Current Stage: 9060 - Closure of 2 Year Review Enquiry - Review Enquiry
• Start Date: 02-Sep-2025
• Completion Date: 02-Sep-2025
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Mandate: M/520 - Mandate for standardisation addressed to CEN for methods of analysis for mycotoxins in food
• Ref Project: SIST EN 17251:2020 - Foodstuffs - Determination of ochratoxin A in pork meat and derived products by IAC clean-up and HPLC-FLD

Overview

EN 17251:2020 (CEN) specifies a laboratory procedure for the determination of ochratoxin A (OTA) in pork meat and derived products using immunoaffinity column (IAC) clean‑up followed by high‑performance liquid chromatography with fluorescence detection (HPLC‑FLD). The scope covers ham, canned chopped pork and pork liver, with validated performance for naturally contaminated samples in the range 0.5 µg/kg to 11 µg/kg. Laboratory experience also supports applicability to pâté and kidney. The standard is intended for routine mycotoxin analysis in meat and meat products to support food safety monitoring and quality control.

Key topics and technical requirements

• Analytical principle: Extraction with a methanol / sodium hydrogen carbonate mixture, dilution with phosphate‑buffered saline (PBS) plus polysorbate 20, clean‑up on OTA‑specific immunoaffinity columns, and analysis by reversed‑phase HPLC with fluorescence detection (RP‑HPLC‑FLD).
• Immunoaffinity columns (IAC): Columns must contain antibodies specific to OTA, have a minimum capacity of 100 ng OTA, and demonstrate ≥85% recovery when tested with a standard spiked solution.
• Reagents and mobile phase: Specifies HPLC‑grade solvents (methanol, acetonitrile), acetic acid solutions, PBS, Tween‑20 (polysorbate 20), extraction solution and the HPLC mobile phase composition.
• Calibration and quantification: Preparation of calibration solutions from a defined standard (e.g., 100 ng/ml) covering a calibration range typically corresponding to 0.32 µg/kg – 14.4 µg/kg (examples provided). Injection solution and LC parameters are prescribed for reproducible results.
• Validation and performance data: Method validated for selected pork matrices; annexes include typical chromatograms and precision data. Safety warnings note OTA’s IARC Group 2B classification and the need for chemical safety procedures.
• Reference: Uses EN ISO 3696 for laboratory water quality.

Applications and who uses it

• Food testing laboratories performing regulatory monitoring, compliance testing and official controls for mycotoxins in meat products.
• Regulatory authorities and public health agencies establishing surveillance programs for OTA contamination in pork-derived foods.
• Meat processors and quality assurance teams implementing supplier testing, incoming raw material checks and shelf‑life or product safety assessments.
• Research laboratories studying mycotoxin transfer, contamination sources or exposure from pork products.

Practical benefits include standardized, validated methodology for sensitive detection of OTA in pork matrices, enabling robust inter‑laboratory comparison and regulatory conformity.

Related standards

• EN ISO 3696 - Water for analytical laboratory use (specified for reagent water quality).
• Produced by CEN/TC 275 (Food analysis – Horizontal methods); national adoptions of EN 17251:2020 are required by CEN members.

Standards Content (Sample)

SLOVENSKI STANDARD
01-marec-2020
Živila - Določevanje ohratoksina A v svinjskem mesu in predelanih proizvodih z
IAC-čiščenjem in tekočinsko kromatografijo visoke ločljivosti s fluorescenčno
detekcijo (HPLC-FLD)
Foodstuffs - Determination of ochratoxin A in pork meat and derived products by IAC
clean-up and HPLC-FLD
Lebensmittel - Bestimmung von Ochratoxin A in Schweinefleisch und
Schweinefleischerzeugnissen mit Hochleistungsflüssigchromatographie und
Fluoreszenzdetektion (HPLC-FLD)
Produits alimentaires - Dosage de l'ochratoxine A dans la viande de porc et les produits
carnés issus du porc par chromatographie liquide à haute performance couplée à la
détection par fluorescence (CLHP-DFL)
Ta slovenski standard je istoveten z: EN 17251:2020
ICS:
67.120.10 Meso in mesni proizvodi Meat and meat products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17251
EUROPEAN STANDARD
NORME EUROPÉENNE
January 2020
EUROPÄISCHE NORM
ICS 67.120.10
English Version
Foodstuffs - Determination of ochratoxin A in pork meat
and derived products by IAC clean-up and HPLC-FLD
Produits alimentaires - Dosage de l'ochratoxine A dans Lebensmittel - Bestimmung von Ochratoxin A in
la viande de porc et les produits carnés issus du porc Schweinefleisch und Schweinefleischerzeugnissen mit
par chromatographie liquide à haute performance Hochleistungsflüssigchromatographie und
couplée à la détection par fluorescence (CLHP-DFL) Fluoreszenzdetektion (HPLC-FLD)
This European Standard was approved by CEN on 6 October 2019.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17251:2020 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Reagents . 5
6 Apparatus and equipment . 8
7 Procedure. 9
8 HPLC-FLD analysis . 10
9 Calculation . 11
10 Precision . 12
11 Test report . 13
Annex A (informative) Typical chromatograms . 14
Annex B (informative) Precision data . 17
Bibliography . 19
European foreword
This document (EN 17251:2020) 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 July 2020, and conflicting national standards shall be
withdrawn at the latest by July 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN 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.
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, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.
Introduction
Ochratoxins are a class of pentaketide molecules made up of dihydroisocoumarin linked to
β-phenylalanine. Ochratoxin A (OTA) is mainly produced by Aspergillus ochraceus, A. carbonarius and
A. niger in tropical regions and by Penicillium verrucosum in temperate climates. It is found in a variety of
food products, especially cereals and their derivatives which are major contributors to exposure, but it is
also found in coffee, wine, beer, dried fruits and spices. Ochratoxin A can also be detected in pork meat
and pork based products.
WARNING 1 — Suitable precaution and protection measures need to be taken when carrying out
working steps with harmful chemicals. The latest version of the hazardous substances ordinance (EU)
1907/2006 [3] should be taken into account as well as appropriate national statements.
WARNING 2 — 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.
WARNING 3 — Ochratoxin A has been classified as substance of Group 2B by International Agency for
Research on Cancer (IARC) meaning the existence of sufficient evidence of its renal carcinogenicity to
animals and possibly to humans.
1 Scope
This document describes a procedure for the determination of ochratoxin A (OTA) in pork products
specifically ham, pork-based products (canned chopped pork) and pork liver using high performance
liquid chromatography with fluorescence detection (HPLC-FLD).
The method has been validated for ochratoxin A in naturally contaminated ham, pork based products
(canned chopped pork) and pork liver containing 0,5 μg/kg to 11 μg/kg [4], [5], [6].
Laboratory experiences have shown that this method is also applicable to pâté and kidney [4].
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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, Water for analytical laboratory use - Specification and test methods (ISO 3696)
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle
Ochratoxin A is extracted by mixing a test portion with a mixture of methanol and aqueous sodium
hydrogen carbonate solution. The extract is centrifuged, diluted with a mixture of phosphate buffered
saline (PBS) and a polysorbate 20 solution, and applied to an immunoaffinity column containing
antibodies specific to ochratoxin A.
The purified extract is analyzed by reversed-phase high performance liquid chromatography (RP-HPLC)
coupled with fluorescence detection (FLD).
5 Reagents
Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696,
unless otherwise specified. Solutions shall be of quality for LC analysis, unless otherwise specified.
Commercially available solutions with equivalent properties to those listed may also be used.
5.1 Methanol (CH OH), technical grade.
5.2 Methanol (CH OH), HPLC grade.
5.3 Acetonitrile (CH CN), HPLC grade.
5.4 Glacial acetic acid, volume fraction φ(CH COOH) approximately 99 %.
5.5 Toluene, UV grade.
5.6 Mixture of toluene and glacial acetic acid.
Mixture of toluene (5.5) and glacial acetic acid (5.4) (99+1, v+v).
5.7 Sodium hydrogen carbonate (NaHCO ), minimum 99 % purity.
5.8 Sodium chloride (NaCl), minimum 99 % purity.
5.9 Disodium hydrogen phosphate (Na HPO ∙ 12H O), minimum 99 % purity.
2 4 2
5.10 Potassium dihydrogen phosphate (KH PO ), minimum 99 % purity.
2 4
5.11 Potassium chloride (KCl), minimum 99 % purity.
5.12 Sodium hydroxide (NaOH), minimum 99 % purity.
5.13 Hydrochloric acid (HCl), φ(HCl) = 37 % (acidimetric).
5.14 Hydrochloric acid solution, substance concentration c(HCl) = 0,1 mol/l.
Dilute 8,28 ml of hydrochloric acid (5.13) to 1000 ml with water in a 1 l volumetric flask.
5.15 Sodium hydroxide solution, c(NaOH) = 0,2 mol/l.
Dissolve 8,0 g sodium hydroxide (5.12) in a 1 l volumetric flask (6.11) and fill up to the mark with water.
5.16 Acetic acid solution, mass concentration ρ(CH COOH) = 20 g/l (2 %).
Dilute 20 g of glacial acetic acid (5.4) to 1000 ml with water in a 1 l volumetric flask.
5.17 Phosphate buffered saline (PBS), pH = 7,4.
Weigh 8,0 g of sodium chloride (5.8), 2,9 g of disodium hydrogen phosphate (5.9), 0,2 g of potassium
dihydrogen phosphate (5.10) and 0,2 g of potassium chloride (5.11) and transfer into a 1 l volumetric
flask (6.11). Dissolve in water and add 900 ml of water.
After dissolution adjust the pH to 7,4 with hydrochloric acid solution (5.14) or sodium hydroxide solution
(5.15) as appropriate, then fill up to the mark with water.
Alternatively, a PBS solution with equivalent properties may be prepared from commercially available
PBS material.
® 1
5.18 Polysorbate 20, e.g. Tween 20 , lauric acid ≥ 40 %. ®
5.19 0,01 % polysorbate solution in PBS, ρ(Tween 20) = 0,1 g/l (0,01 %).
Weigh 100 mg of polysorbate 20 (5.18), transfer quantitatively into a 1 l volumetric flask (6.11) and fill
up to the mark with PBS solution (5.17).
®
Tween 20 is a trade name of a polysorbate 20-type nonionic surfactant available from various suppliers. This
information is given for the convenience of users of this European standard and does not constitute an endorsement
by CEN of this product. Equivalent products may be used if they can be shown to lead to the same results.
5.20 Sodium hydrogen carbonate solution (NaHCO ), ρ(NaHCO ) = 10,0 g/l (1 %).
3 3
Add 10 g of sodium hydrogen carbonate (5.7) into a 1 l volumetric flask (6.11) and fill up to the mark
with water.
5.21 Extraction solution.
Mix methanol (5.1) and sodium hydrogen carbonate solution (5.20) (3+2, v+v).
5.22 HPLC mobile phase.
Mix methanol (5.2), acetonitrile (5.3) and acetic acid solution (5.16) (25+35+40, v+v+v).
5.23 Injection solution.
Mix methanol (5.2) and water (1+1, v+v).
5.24 Immunoaffinity column (IAC).
The immunoaffinity column (IAC) contains antibodies raised against ochratoxin A. The IAC 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 3 volumes of methanol (5.1) and
17 volumes of PBS solution (5.17) containing 3 ng of ochratoxin A. Immunoaffinity columns shall be
stored following the instructions of the producers and shall, if necessary, be allowed to equilibrate at
room temperature before use.
5.25 Ochratoxin A (OTA), e.g. crystalline or as a film, purity greater than 98 % mass fraction, or as
certified standard solution.
5.26 Ochratoxin A stock solution, ρ = 20 µg/ml.
Dissolve ochratoxin A in crystal form (5.25) or the content of 1 ampoule (if ochratoxin A has been
obtained as a film) in mixture (5.6) to give a solution containing approximately 20 µg/ml to 30 µg/ml of
ochratoxin A.
This step may be omitted when using the certified standard solution. The certified standard solution then
serves as stock solution.
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 with an UV spectrometer (6.13) and mixture (5.6) as reference.
Identify the wavelength for maximum absorption and calculate the mass concentration of ochratoxin A, ρ,
in µg/ml, according to Formula (1):
AMmax×× 100
ρ= (1)
δε×
where
A is the maximum absorbance value determined from the absorption curve (here: 333 nm);
max
M is the molar mass of ochratoxin A, in g/mol (M = 403,8 g/mol);
δ is the path length of the quartz cell, in cm;
ε 2
is the molar absorption coefficient of ochratoxin A in mixture (5.6), in m /mol (here:
544 m /mol).
This solution can be used for 12 months if stored at −18 °C. Allow to reach room temperature before
opening. Confirm the concentration when it is used after 12 months.
5.27 Ochratoxin A standard solution, ρ = 100 ng/ml.
Dilute the stock solution (5.26) or a certified solution of ochratoxin A (5.25) with the injection solution
(5.23) to obtain a standard solution with a mass concentration of ochratoxin A of 100 ng/ml. This
solution is stable for at least one month if stored in the refrigerator at 4 °C.
5.28 Calibration solutions.
Prepare six calibration solutions from the standard solution (5.27) as follows.
With appropriate calibrated pipettes or microlitre pipettes (6.4) transfer e.g. the volumes of the
ochratoxin A standard solution (5.27) separately each into volumetric flask as listed in Table 1. Fill each
volumetric flask up to the mark with injection solution (5.23), close and mix manually. This will result in
six ochratoxin A solutions with approximately the concentrations listed in Table 1. These six solutions
cover a range from 0,32 μg/kg
...