EN 17250:2020
(Main)Foodstuffs - Determination of ochratoxin A in spices, liquorice, cocoa and cocoa products by IAC clean-up and HPLC-FLD
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
Overview
EN 17250:2020 (CEN) specifies a validated laboratory procedure for the determination of ochratoxin A (OTA) in spices, liquorice and liquorice products, and cocoa and cocoa products. The method uses immunoaffinity column (IAC) clean‑up followed by reversed‑phase high performance liquid chromatography with fluorescence detection (HPLC‑FLD). The standard defines scope, reagents, apparatus, extraction procedures, HPLC conditions, calibration and quality checks, and reports on method precision validated in interlaboratory studies.
Key topics and requirements
- Scope and matrices: chilli, paprika, black/white pepper, nutmeg, spice mixes, liquorice (root & extracts), cocoa and cocoa products.
- Analytical principle: matrix extraction (methanol/aqueous buffers), filtration, dilution with phosphate buffered saline (PBS), IAC clean‑up specific to OTA, elution with methanol and quantification by RP‑HPLC‑FLD.
- Reagents & mobile phases: defined extraction solutions (A and B) and mobile phases for different matrices (paprika/chilli, liquorice, pepper/nutmeg/cocoa).
- Immunoaffinity column performance: column capacity ≥ 100 ng OTA and recovery ≥ 85 % under specified test conditions.
- Standards and calibration: preparation of OTA stock, standard, spiking and six calibration solutions covering approximately 1.2 µg/kg to 100 µg/kg (spice/cocoa) and wider range for liquorice.
- Validation and precision: method performance validated by interlaboratory studies across naturally contaminated and spiked samples (reported ranges for spices: 1.0–84.9 µg/kg; liquorice: 7.7–96.8 µg/kg; cocoa: 2.1–26.3 µg/kg). See Clause 10 and Annex B for precision data.
- Safety notes: OTA is a toxic mycotoxin (IARC Group 2B). The standard cautions about hazardous chemicals and laboratory safety measures.
Practical applications
- Routine testing for OTA contamination in spices, liquorice and cocoa supply chains.
- Food safety laboratories performing regulatory monitoring and compliance testing.
- Quality control in spice processing, confectionery, cocoa and liquorice product manufacturers.
- Research and method validation labs assessing mycotoxin extraction, IAC performance and HPLC‑FLD quantitation.
Keywords: ochratoxin A (OTA), HPLC‑FLD, immunoaffinity column, spices testing, liquorice analysis, cocoa products, mycotoxin analysis, EN 17250:2020, CEN standard.
Who should use this standard
- National and commercial food testing laboratories
- Food safety authorities and regulators monitoring mycotoxins
- Manufacturers and QA teams in spice, liquorice and cocoa industries
- Method developers and interlaboratory validation teams
Related standards
- EN ISO 3696 – Water for analytical laboratory use (normative reference used in EN 17250:2020)
- Prepared under CEN/TC 275 “Food analysis – Horizontal methods”
This standard provides a robust, traceable procedure for laboratories needing reliable OTA measurement in high‑risk food matrices using IAC clean‑up and HPLC‑FLD.
Standards Content (Sample)
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 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.
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
µl
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.
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.
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).
CAUTIO
...
Frequently Asked Questions
EN 17250:2020 is a standard published by the European Committee for Standardization (CEN). Its full title is "Foodstuffs - Determination of ochratoxin A in spices, liquorice, cocoa and cocoa products by IAC clean-up and HPLC-FLD". This standard covers: 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.
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.
EN 17250:2020 is classified under the following ICS (International Classification for Standards) categories: 67.140.30 - Cocoa; 67.220.10 - Spices and condiments. The ICS classification helps identify the subject area and facilitates finding related standards.
EN 17250:2020 is associated with the following European legislation: EU Directives/Regulations: 882/2004; Standardization Mandates: M/520. When a standard is cited in the Official Journal of the European Union, products manufactured in conformity with it benefit from a presumption of conformity with the essential requirements of the corresponding EU directive or regulation.
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제목: EN 17250:2020 - 식품 - 어커토신 A의 칠리, 파프리카, 후추, 겨자, 향신료 혼합물, 관자통(뿌리 및 추출물), 코코아 및 코코아 제품에서의 결정에 관한 IAC 정화 및 HPLC-FLD 분석 방법 내용: 본 문서는 어커토신 A(OTA)를 칠리, 파프리카, 후추, 겨자, 향신료 혼합물, 관자통(뿌리 및 추출물), 코코아 및 코코아 제품에서 고성능 액체 크로마토그래피(HPLC)와 면역친화력 칼럼 정화 및 형광 검출(FLD)을 통해 확인하는 방법을 정의합니다. 이 방법은 스파이스(파프리카와 칠리 [5], 후추, 겨자 및 향신료 혼합물 [6])의 경우 1.0 μg/kg에서 84.9 μg/kg, 관자통 및 관자통 제품 [7]의 경우 7.7 μg/kg에서 96.8 μg/kg, 그리고 코코아 및 코코아 제품 [6]의 경우 2.1 μg/kg에서 26.3 μg/kg 범위의 자연오염 및 도핑된 샘플을 분석함으로써 국제 연구소 간 연구를 통해 검증되었습니다. 검증에 대한 자세한 정보는 10조와 부록 B를 참조하십시오.
記事のタイトル: EN 17250:2020 - 食品 - スパイス、甘草、ココアおよびココア製品中のオクラトキシンAの決定法：IAC清浄化およびHPLC-FLD 記事の内容: この文書は、ハイパフォーマンス液体クロマトグラフィー（HPLC）と免疫親和性カラムのクリーンアップと蛍光検出（FLD）を用いて、スパイス（チリ、パプリカ、黒胡椒、白胡椒、ナツメグ、スパイスミックス）、甘草（根とエキス）、ココア、およびココア製品中のオクラトキシンA（OTA）の検出法を定めています。 この方法は、天然に汚染されたおよび添加されたサンプルを使用して、スパイス（パプリカとチリ[5]、黒胡椒、白胡椒、ナツメグ、スパイスミックス[6]）では1.0 μg/kgから84.9 μg/kg、甘草および甘草製品では7.7 μg/kgから96.8 μg/kg、ココアおよびココア製品では2.1 μg/kgから26.3 μg/kgまでの範囲の間で分析されました。 詳細な検証情報については、10条と付録Bを参照してください。
記事のタイトル：EN 17250:2020 - 食品 - 香辛料、甘草、ココアおよびココア製品におけるオクラトキシンAの決定に関するIACクリーンアップおよびHPLC-FLD 記事の内容：この文書は、高性能液体クロマトグラフィー（HPLC）と蛍光検出（FLD）を使用して、香辛料、甘草、ココア、およびココア製品におけるオクラトキシンA（OTA）の存在を決定する手順を定めています。この方法は、スパイス（パプリカとチリ [5]）、黒コショウと白コショウ、ナツメグ、スパイスミックス [6]などの食品から天然に汚染されたおよびスパイクされたサンプルを使用して、1.0μg/kgから84.9μg/kgの範囲で検証されています。甘草および甘草製品 [7]の場合は、約7.7μg/kgから96.8μg/kgの範囲のサンプルの分析が行われ、ココアおよびココア製品 [6]の場合は、約2.1μg/kgから26.3μg/kgの範囲でサンプルの分析が行われました。検証に関する詳細な情報については、10節および付録Bを参照してください。
기사 제목 : EN 17250:2020 - 식품 - 향신료, 감초, 코코아 및 코코아 제품에서 오크라토신 A의 결정을 위한 IAC 정화 및 HPLC-FLD 기사 내용 : 이 문서는 인공광쇄검출장치 (HPLC)와 형광 검출 (FLD)을 사용한 향신료, 감초, 코코아 및 코코아 제품에서 오크라토신 A (OTA)의 결정을 위한 절차를 명시하고 있습니다. 이 방법은 스파이스 (파프리카와 칠리 [5]), 검정 후추와 흰 후추, 겨자, 스파이스 혼합물 [6] 등의 식품부터 약 1.0 μg/kg에서 84.9 μg/kg의 범위로 오염된 샘플 및 스파이크된 샘플을 통해 검증되었습니다. 감초 및 감초 제품 [7]의 경우, 약 7.7 μg/kg에서 96.8 μg/kg의 범위로 샘플을 분석하였으며, 코코아 및 코코아 제품 [6]의 경우, 약 2.1 μg/kg에서 26.3 μg/kg의 범위로 샘플을 분석하였습니다. 검증에 대한 자세한 내용은 10 절 및 부록 B를 참조하십시오.
The article discusses the EN 17250:2020 standard, which outlines a procedure for determining the presence of ochratoxin A (OTA) in various food items including spices (chilli, paprika, black and white pepper, nutmeg, spice mix), liquorice (root and extracts), cocoa, and cocoa products. The method involves high performance liquid chromatography (HPLC) with immunoaffinity column clean-up and fluorescence detection (FLD). The method has been validated through interlaboratory studies using naturally contaminated and spiked samples, with concentration ranges varying depending on the type of food item analyzed. Further details on the validation process can be found in Clause 10 and Annex B of the standard.
The article discusses the EN 17250:2020 standard, which outlines a procedure for determining the presence of ochratoxin A (OTA) in various food items including spices, liquorice, cocoa, and cocoa products. The method utilizes high-performance liquid chromatography (HPLC) with immunoaffinity column clean-up and fluorescence detection (FLD). The procedure has been validated through interlaboratory studies using naturally contaminated and spiked samples in different concentration ranges for each food item. Additional information on the validation can be found in Clause 10 and Annex B.
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