Foodstuffs - Determination of ochratoxin A in spices, liquorice, cocoa and cocoa products by IAC clean-up and HPLC-FLD

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.

Lebensmittel - Bestimmung von Ochratoxin A in Gewürzen, Süßholz, Kakao und Kakaoerzeugnissen nach IAC-Reinigung mit HPLC-FLD

Dieses Dokument legt ein Verfahren zur Bestimmung von Ochratoxin A (OTA) in Chili, Paprika, schwarzem und weißem Pfeffer, Muskatnuss, einer Gewürzmischung, Süßholzwurzel (Wurzel und Extrakte), Kakao und Kakaoerzeugnissen fest, wobei die Hochleistungsflüssigchromatographie (HPLC, en: high performance liquid chromatography) und Fluoreszenzdetektion (FLD) nach Reinigung an einer Immunoaffinitätssäule angewendet wird.
Das Verfahren wurde in Ringversuchen an sowohl natürlich kontaminierten als auch an aufgestockten Proben von 1,0 μg/kg bis 84,9 μg/kg für Gewürze (Paprika und Chili [5], schwarzem und weißem Pfeffer, Muskatnuss und einer Gewürzmischung [6]), von 7,7 μg/kg bis 96,8 μg/kg für Süßholzwurzel und Süßholzwurzelerzeugnisse [7] und von 2,1 μg/kg bis 26,3 μg/kg für Kakao und Kakaoerzeugnisse [6] validiert.
Weitere Informationen zur Validierung siehe Abschnitt 10 und Anhang B.

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

Le présent document spécifie un mode opératoire de dosage de l’ochratoxine A (OTA) dans le piment, le paprika, le poivre noir et le poivre blanc, la noix de muscade, les mélanges d’épices, la réglisse (racine et extraits), le cacao et les produits à base de cacao par chromatographie liquide à haute performance (CLHP) avec purification sur colonne d’immuno-affinité et détection par fluorescence (DFL).
Cette méthode a été validée lors d’études interlaboratoires par l’analyse d’échantillons naturellement contaminés et d’échantillons dopés de 1,0 μg/kg à 84,9 μg/kg pour les épices (paprika et piment [5], poivre noir et poivre blanc, noix de muscade et mélanges d’épices [6]), de 7,7 μg/kg à 96,8 μg/kg pour la réglisse et les produits à base de réglisse [7] et de 2,1 μg/kg à 26,3 μg/kg pour le cacao et les produits à base de cacao [6].
Pour plus d’informations sur la validation, voir l’Article 10 et l’Annexe B.

Živila - Določevanje ohratoksina A v začimbah, sladkem korenu, kakavu in kakavovih proizvodih z IAC-čiščenjem in HPLC-FLD

Ta dokument opisuje postopek za določevanje ohratoksina A (OTA) v čiliju, papriki, črnem in belem popru, muškatnem oreščku, mešanici začimb, sladkem korenu (in njegovem izvlečku), kakavu in kakavovih proizvodih z visokozmogljivo tekočinsko kromatografijo (HPLC) s čiščenjem z imunoafinitetno kolono in fluorescenčno detekcijo.  Ta metoda je bila validirana v medlaboratorijskih študijah z analizo naravno kontaminiranih in primešanih vzorcev v razponu od 1,0 do 84,9 μg/kg za začimbe (paprika in čili [5], črni in beli poper, muškatni orešček in mešanica začimb [6]), od 7,7 do 96,8 μg/kg za sladki koren [7] ter od 2,1 do 26,3 μg/kg za kakav in kakavove proizvode [6].
Za več informacij glede validacije glej točko 9 in dodatek B.

General Information

Status
Withdrawn
Publication Date
29-Apr-2020
Withdrawal Date
30-Jul-2020
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
22-Jan-2020
Due Date
20-Apr-2019
Completion Date
22-Jan-2020

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SLOVENSKI STANDARD
SIST EN 17250:2020
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
SIST EN 17250:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 17250:2020
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SIST EN 17250:2020
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.
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SIST EN 17250:2020
EN 17250:2020 (E)
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

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SIST EN 17250:2020
EN 17250:2020 (E)
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.
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EN 17250:2020 (E)
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.
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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.
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EN 17250:2020 (E)
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.
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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 parts per volume of PBS solution (5.19) containing 3 ng of ochratoxin A.

5.28 Ochratoxin A, in crystal form or as a film in ampoules or as a certified standard solution.

5.29 Ochratoxin A stock solution, ρ(ochratoxin A) = 10 µg/ml.

Prepare a stock solution of ochratoxin A (5.28) in a mixture of toluene (5.6) and glacial acetic acid (5.5)

in ratio 99 + 1 (v+v) 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 in a spectrometer with the solvent mixture (toluene +

glacial acetic acid, 99 + 1, v+v) as reference. Identify the wavelength for maximum absorption and

calculate the mass concentration of ochratoxin A, ρ, in µg/ml, using Formula (1):

AM×× 100
max
ρ= (1)
ε ×b
where

A is the maximum absorbance value determined from the absorption curve (here: at 333 nm);

max
M is the molar mass of ochratoxin A, in g/mol (here: M = 403,8 g/mol);

ε is the molar absorption coefficient of ochratoxin A in the solvent mixture (toluene + glacial

2 2
acetic acid: 99+1, v+v), in m /mol (here: 544 m /mol);
b is the path length of the quartz cell, in cm.

This solution can be used for 6 months if stored at approximately −18 °C. Allow to reach room

temperature before opening. Confirm the mass concentration of the solution if it is older than 6 months.

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This step may be omitted when using a certified standard solution, provided that the product comes

with a certificate, giving sufficient evidence on the correctness of the stated mass fraction. The certified

standard solution then serves as stock solution.

The exact mass concentrations of ochratoxin A in the standard, spiking and calibration solutions are

calculated from the initial concentration of this stock solution and the subsequent volumes used.

5.30 Ochratoxin A standard solution, ρ(ochratoxin A) = 1 µg/ml.

Pipette (6.4) 100 µl of ochratoxin A stock solution (5.29) into a 1 ml volumetric flask (6.10), dry by a

gentle flow of nitrogen (5.1), then dilute to 1 ml (up to the mark) with the appropriate mobile phase

(5.23, 5.24 or 5.25) and shake it vigorously. This gives a standard solution containing 1 µg/ml of

ochratoxin A. This solution can be used for 6 months if stored at approximately −18 °C. Allow to reach

room temperature before opening. Confirm the mass concentration of the solution if it is older than

6 months.
5.31 Ochratoxin A spiking solution, ρ(ochratoxin A) = 400 ng/ml.

Pipette 1 ml of ochratoxin A stock solution (5.29) into a 25 ml volumetric flask (6.10) and dilute to the

mark with a mixture of acetonitrile (5.4) and glacial acetic acid (5.5) in a ratio of 99 + 1, v+v, and shake.

This gives a spiking solution containing 400 ng/ml of ochratoxin A.

This solution can be used for 6 months if stored at approximately −18 °C. Allow to reach room

temperature before opening. Confirm the mass concentration of the solution if it is older than 6 months.

5.32 Calibration solutions
Prepare six calibration solutions from the standard solution (5.30) as follows:

With appropriate pipettes (6.4) transfer e.g. the volumes of the ochratoxin A standard solution (5.30)

separately each into volumetric flasks as specified in Table 1. Fill each volumetric flask up to the mark

with the appropriate mobile phase (5.23, 5.24 or 5.25), close and shake manually. This results in six

ochratoxin A calibration solutions with approximately the concentrations as listed in Table 1.

These six calibration solutions cover a range from approximately 1,2 µg/kg to approximately 100 µg/kg

for ochratoxin A for all spices, cocoa and cocoa products and from approximately 2,4 µg/kg to

approximately 200 µg/kg for liquorice and liquorice products.

Protect calibration solutions from light. These solutions can be used for 1 month if stored at

approximately −18 °C.
Table 1 — Preparation of calibration solutions
Calibration Standard Final Mass concentration of
solution solution (5.30) volume calibration solution
ml ng/ml
1 15 50 0,3
2 15 25 0,6
3 25 25 1
4 50 10 5
5 150 10 15
6 250 10 25
Transfer these calibration solutions into LC vials (6.9) before injection.
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6 Apparatus and equipment
Usual laboratory glassware and equipment, in particular, the following:
6.1 Laboratory balance, accuracy: 0,01 g.
6.2 Analytical balance, accuracy: 0,1 mg.
6.3 Laboratory shaker, and shaker for centrifuge tubes

6.4 Pipettes, e.g. 100 μl to 2 000 µl and 4 ml volumetric pipettes, suitable for organic solvents.

6.5 Disposable syringe reservoir, of 100 ml capacity, and attachments to fit to immunoaffinity

columns.

6.6 Glass microfibre filter paper, 1,6 µm retention size, 150 mm diameter, or equivalent. As an

alternative, filter paper (6.7) may be used when they have been proven to give equivalent results.

6.7 Cellulose filter paper, 11 µm pore size, 150 mm diameter.
6.8 SPE vacuum manifold/elution station.

6.9 LC vials, approximately 2 ml capacity, or 2 ml LC vial with insert, with crimp caps or equivalent.

6.10 Volumetric flasks, of various capacities (e.g. 1 ml, 2 ml, 5 ml, 10 ml, 25 ml, 50 ml).

6.11 Conical flasks, 100 ml or 500 ml with screw cap, or similar recipient.
6.12 HPLC system, comprising the following:

6.12.1 HPLC pump, gradient, capable of maintaining a volume flow rate of 0,8 ml/min pulse free and

1,0 ml/min pulse free.
6.12.2 Injection system.

6.12.3 Pre-column, of suitable dimensions, with stationary phase material the same or similar to the

analytical column.
6.12.4 Reversed-phase HPLC column.

A suitable column and appropriate HPLC conditions (isocratic or gradient programme) with a retention

factor of at least two that ensures base line separation to distinguish peaks of ochratoxin A from all

other signals.

Some examples of columns which have been found to be suitable are given in Annex A.

6.12.5 Degasser, optional, for degassing mobile phases (5.23; 5.24; 5.25; 5.26).
6.12.6 Column oven, capable of maintaining a constant temperature.
6.12.7 Fluorescence detector.
6.12.8 Data evaluation system.
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7 Procedure
7.1 Preparation of the test sample

Mix or stir the laboratory sample thoroughly before removing the test portion. Weigh the test portion

into a conical flask or similar recipient of 500 ml (6.11).
7.2 Extraction

For spices, cocoa and cocoa products weigh a test portion of 12,5 g (m) to the nearest 0,1 g, and for

liquorice and liquorice products weigh a test portion of 10 g (m) to the nearest 0,1 g.

The test portion solvent ratio shall be 1 to 8 except for liquorice and liquorice products where the

solvent ratio shall be 1 to 20.

For spices add 100 ml (V ) of extraction solution A (5.21). For cocoa and cocoa products, add 100 ml

(V ) of extraction solution B (5.22). For liquorice and liquorice products, add 200 ml (V ) of extraction

1 1

solution A (5.21). Shake by hand for a few seconds to obtain a homogeneous suspension, and then shake

for 40 min with a laboratory shaker (6.3).

Filter at least 10 ml of the extract through the 150 mm glass fibre (or cellulose) filter paper (6.6, 6.7),

conically folded. Collect the filtered extract in a screw cap conical flask (6.11) for further analysis.

Proceed immediately with the immunoaffinity column clean-up procedure (7.3).
7.3 Immunoaffinity column clean-up

Connect the immunoaffinity column (5.27) to the vacuum manifold (6.8) and attach a syringe reservoir

(6.5) to the immunoaffinity column.

The immunoaffinity column shall be allowed to reach room temperature prior to using.

For spices, cocoa and cocoa products transfer in the reservoir (6.5) 50 ml of PBS (5.19) and 1 ml of 2 %

Polysorbate 20 solution (5.18) and pipette(6.4) 4 ml [V ] of the filtered extract as obtained in 7.2 and

mix.

For liquorice and liquorice products transfer in the reservoir (6.5) 15 ml of PBS (5.19), and pipette

0,5 ml [V ] of the filtered extract as obtained in 7.2 and mix.

Draw the mixture (extract and PBS) through the column by gravity at a steady flow rate (the flow rate

shall result in a dropping speed of 1 drop/s, which is about 3 ml/min) until all extract has passed the

column and the last solvent portion reaches the frit of the column.

If necessary, the process may be accelerated by applying slight pressure to the immunoaffinity column

by a syringe or by applying little vacuum (e.g. by using the vacuum system described in 6.8). In both

cases, attention shall be paid not to exceed the flow rate of 3 ml/min (= 1 drop/s).

CAUTION — If using a vacuum manifold, extra care is necessary to avoid increasing the flow rate

through the column as recovery can adversely be affected.
7.4 Preparation of sample test solutions

For paprika and chilli, wash the column with 10 ml of water at a rate not exceeding 3 ml/min. Dry the

column by pushing 50 ml air through it with a syringe. For spices, cocoa and cocoa products and

liquorice and liquorice products wash the column with 1 ml of 2 % Polysorbate 20 solution (5.18)

followed by 10 ml of water at a rate not exceeding 3 ml/min. For liquorice and liquorice products repeat

the washing steps. Dry the column by passing nitrogen or air through it for about 1 s to 2 s, and discard

the eluate from this stage of the clean-up procedure.
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For spices, cocoa and cocoa products place a 2 ml volumetric flask (6.10) under the column and pass

0,5 ml of methanol (5.3) through the column, collecting the eluate. After the last drops of methanol have

passed through the column, wait approximately 1 min. Then add a further 0,5 ml of methanol (5.3) in

the same volumetric flask and collect the eluate. Finally add 0,5 ml of methanol (5.3) and continue to

collect the eluate. Carefully pass air through the column in order to collect any final drops. Add acetic

acid solution (5.16) into the flask up to the mark and shake. This results in a final volume of exactly

2,0 ml [V ]. Transfer a sufficient portion into an LC vial (6.9) and cap.

For liquorice and liquorice products, place a 2 ml vial (6.9) under the column and pass 0,5 ml of

methanol (5.3) through the column, collecting the eluate. After the last drops of methanol have passed

through the column, wait approximately 1 min. Then add a further 0,5 ml of methanol (5.3) and collect

the eluate. Finally add 0,5 ml of methanol (5.3) and continue to collect the eluate. Carefully pass air

through the column in order to collect any final drops.

Evaporate the methanolic eluate to dryness applying a gentle stream of nitrogen (5.1) at about 30 °C to

35 °C. Re-dissolve the purified sample residues in 200 µl [V ] of mobile phase B (5.24), cap vial and

shake on a vortex mixer (6.3) for at least 15 s, making sure the lower part of the vial is thoroughly

rinsed by the solvent. Transfer the test solution into an LC vial (6.9) and analyse.

CAUTION — Since all the immunoaffinity column eluate is used for the quantitative analysis it is

very important to dry the immunoaffinity column effectively by air after the washing step and

after the elution by methanol. Shaking the vials before injection is also critical to ensure

homogeneity of the solution prior injection.

7.5 Spiking procedure (optional, if no certified reference material (CRM) is used)

From the blank samples of spices, cocoa and cocoa products weigh a test portion of 12,5 g to the nearest

0,1 g and from the blank samples of liquorice and liquorice products weigh a 10 g test portion to the

nearest 0,1 g into a conical flask or similar recipient of 500 ml (6.11). Pipette 40 μl of ochratoxin A

spiking solution (5.31) onto the blank matrix. After addition of the spiking solution, let the solvent

evaporate in a fume cupboard for at least 2 h prior to extraction. Proceed as in 7.2 starting with the

addition of the extraction solution.
8 HPLC analysis
8.1 HPLC operating conditions
8.1.1 General

Inject equal volumes of the sample test solution and of each calibration solution into the HPLC-FLD-

system.
8.1.2 Paprika and chilli (mobile phase A)

When using an RP-C18 type column as specified under 6.12.4 and the mobile phase A as specified in

5.23, the following settings have been shown to be applicable:
Flow: 0,8 ml/min
Injection volume: 20 μl
Column oven temperature (including the guard column): 22 °C ± 1 °C
Autosampler (optional) temperature: 15 °C to 20 °C
Excitation wavelength: 332 nm
Emission wavelength: 476 nm
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SIST EN 17250:2020
EN 17250:2020 (E)

Ochratoxin A has a retention time of approximately 6,6 min. Other column dimensions may be used,

provided that the required resolution is achieved. This shall be demonstrated (maximum overlap of

ochratoxin A with any other peaks if present shall be less than 10 %). The flow rate may be adjusted

according to the column dimension. A typical chromatogram for paprika is shown in Annex A,

Figure A.1.
8.1.3 Liquorice and liquorice products (mobile phase B)

When using an RP-C18 type column as specified under 6.12.4 and the mobile phase B specified in 5.24,

the following settings have been shown to be applicable:
Flow: 1,0 ml/min
Injection volume: 20 μl
Column oven temperature (including the guard column): 22 °C ± 1 °C
Autosampler (optional) temperature: 15 °C to 20 °C
Excitation wavelength: 332 nm
Emission wavelength: 476 nm

Ochratoxin A has a retention time of approximately 10 min. A typical chromatogram for liquorice

extract powder is shown in Annex A, Figure A.2.

After immunoaffinity column clean-up the purified extract may still contain substances that can elute

later or in subsequent chromatographic runs appearing as broad peaks. To prevent this, wash the

column for 10 min with mobile phase D as washing solution (5.26) followed by 10 min re-equilibration

with mobile phase B (5.24) prior to the next run. The necessity of the washing step varies with different

sample types and may be skipped if no interferences are noted from late elution peaks.

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SIST EN 17250:2020
EN 17250:2020 (E)
8.1.4 Spices, cocoa and cocoa products (mobile phase C)

When using a phenyl-hexyl column as specified under 6.12.4 and the mobile phase C specified in 5.25,

the following settings have been shown to be applicable:
Flow: 1,0 ml/min
Injection volume: 20 μl
Column oven temperature (including the guard column): 50 °C ± 1 °C
Autosampler (optional) temperature: 15 °C to 20 °C
Excitation wavelength: 332 nm
Emission wavelength: 476 nm
Ochratox
...

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