EN 17250:2020

SLOVENSKI STANDARD
01-marec-2020
Živila - Določevanje ohratoksina A v začimbah, sladkem korenu, kakavu in
kakavovih proizvodih z IAC-čiščenjem in HPLC-FLD
Foodstuffs - Determination of ochratoxin A in spices, liquorice, cocoa and cocoa
products by IAC clean-up and HPLC-FLD
Lebensmittel - Bestimmung von Ochratoxin A in Gewürzen, Süßholz, Kakao und
Kakaoerzeugnissen nach IAC-Reinigung mit HPLC-FLD
Produits alimentaires - Dosage de l’ochratoxine A dans les épices, la réglisse, les
produits à base de réglisse, le cacao et les produits à base de cacao par purification sur
colonne d’immuno-affinité et CLHP-DFL
Ta slovenski standard je istoveten z: EN 17250:2020
ICS:
67.140.30 Kakav Cocoa
67.220.10 Začimbe Spices and condiments
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17250
EUROPEAN STANDARD
NORME EUROPÉENNE
January 2020
EUROPÄISCHE NORM
ICS 67.140.30; 67.220.10
English Version
Foodstuffs - Determination of ochratoxin A in spices,
liquorice, cocoa and cocoa products by IAC clean-up and
HPLC-FLD
Produits alimentaires - Dosage de l'ochratoxine A dans Lebensmittel - Bestimmung von Ochratoxin A in
les épices, la réglisse, les produits à base de réglisse, le Gewürzen, Süßholz, Kakao und Kakaoerzeugnissen
cacao et les produits à base de cacao par purification nach IAC-Reinigung mit HPLC-FLD
sur colonne d'immuno-affinité et CLHP-DFL
This European Standard was approved by CEN on 18 November 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 17250:2020 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 . 9
7 Procedure. 10
8 HPLC analysis . 11
9 Calculation . 13
10 Precision . 14
11 Test report . 16
Annex A (informative) Typical chromatograms . 17
Annex B (informative) Precision data . 21
Bibliography . 27

European foreword
This document (EN 17250:2020) 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
The mycotoxin ochratoxin A has a chemical structure comprising a dihydrocoumarin moiety linked to
a molecule of L-β-phenylalanine via an amide bond. Ochratoxin A is produced by several fungal species
in the Penicillium and Aspergillus genera, primarily Penicillium verrucosum, Aspergillus ochraceus and
Aspergilli of the section Nigri, especially A. carbonarius. Cereals such as wheat are especially affected, as
well as a diverse range of other foodstuffs such as dried fruit, spices, cocoa, coffee, wine, beer, liquorice
and products thereof.
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 e.g. such as in
[4].
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 is a potent nephrotoxic agent, a carcinogen and has genotoxic properties.
Ochratoxin A has been classified by IARC as Group 2B.
1 Scope
This document specifies a procedure for the determination of ochratoxin A (OTA) in chilli, paprika,
black and white pepper, nutmeg, spice mix, liquorice (root and extracts), cocoa and cocoa products by
high performance liquid chromatography (HPLC) with immunoaffinity column clean-up and
fluorescence detection (FLD).
This method has been validated in interlaboratory studies via the analysis of both naturally
contaminated and spiked samples ranging from 1,0 μg/kg to 84,9 μg/kg for spices (paprika and chili [5],
black and white pepper, nutmeg and spice mix [6]), ranging from 7,7 μg/kg to 96,8 μg/kg for liquorice
and liquorice products [7] and ranging from 2,1 μg/kg to 26,3 μg/kg for cocoa and cocoa products [6].
For further information on the validation, see Clause 10 and Annex B.
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/ui
4 Principle
Spices or liquorice and liquorice products are extracted with a mixture of methanol and aqueous
sodium hydrogen carbonate solution, whereas cocoa and cocoa products are extracted with aqueous
methanol. The extract is filtered, diluted with phosphate buffered saline (PBS), polysorbate 20 (except
for liquorice and liquorice products), and applied to an immunoaffinity column containing antibodies
specific to ochratoxin A. The ochratoxin A is isolated, purified and concentrated on the column then
released using methanol. The purified extract is quantified 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. Commercially available solutions with equivalent properties to those listed
may be used.
5.1 Nitrogen, minimum 99,95 % purity.
5.2 Methanol, technical grade.
5.3 Methanol, HPLC grade.
5.4 Acetonitrile, HPLC grade.
5.5 Glacial acetic acid, 99 % purity.
5.6 Toluene, UV grade.
5.7 Sodium hydrogen carbonate, minimum 99,5 % purity.
5.8 Sodium chloride (NaCl), minimum 99 % purity.
5.9 Disodium hydrogen phosphate dodecahydrate (Na HPO · 12 H 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 solution, volume fraction φ(HCl) = 37 % (acidimetric).
5.14 Hydrochloric acid solution, substance concentration c(HCl) = 0,1 mol/l.
Dilute 8,28 ml of hydrochloric acid solution (5.13) to 1 l with water.
5.15 Sodium hydroxide solution, c(NaOH) = 0,2 mol/l.
Dissolve 8 g of sodium hydroxide (5.12) in 1 l of water.
5.16 Acetic acid solution, mass concentration ρ(CH COOH) = 10 g/l.
Dilute 9,5 ml of glacial acetic acid (5.5) to 1 l with water.
5.17 Polysorbate 20
5.18 Polysorbate 20 solution, ρ(Tween® 20 ) = 20 g/l.
Dissolve 20 g of Polysorbate 20 (5.17) in 1 000 ml of water.
5.19 Phosphate buffered saline solution (PBS), pH = 7,4.
Dissolve 8 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) in 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 dilute to 1 l with water.
Alternatively, a PBS solution with equivalent properties can be prepared from commercially available
PBS material.
5.20 Sodium hydrogen carbonate solution, ρ(NaHCO ) = 30 g/l.
Dilute 30 g of sodium hydrogen carbonate (5.7) in 1 000 ml of water.

Tween® 20 is a trade name of a polysorbate 20-type nonionic surfactant available from different 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.
5.21 Extraction solution A (for spices, liquorice and liquorice products).
Mix methanol (5.2) with sodium hydrogen carbonate solution (5.20) (50+50, v+v). Mix well.
5.22 Extraction solution B (for cocoa and cocoa products).
Mix methanol (5.2) with water (80+20, v+v). Mix well.
5.23 Mobile phase A (for paprika and chilli).
Mix methanol (5.3) with acetonitrile (5.4), water and glacial acetic acid (5.5) (35+35+29+1, v+v+v+v).
5.24 Mobile phase B (for liquorice and liquorice products).
Mix methanol (5.3) with water and glacial acetic acid (5.5) (70+30+1, v+v+v).
5.25 Mobile phase C (for black and white pepper, nutmeg, spice mix, cocoa and cocoa products).
Mix methanol (5.3), acetonitrile (5.4), water and glacial acetic acid (5.5) (28+28+39+1, v+v+v+v).
5.26 Mobile phase D (HPLC column washing solution for liquorice and liquorice products).
100 % methanol (5.3).
5.27 Immunoaffinity column
The immunoaffinity column contains 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 (5.2) and
85 pa
...