EN 17424:2020

SLOVENSKI STANDARD
oSIST prEN 17424:2019
01-oktober-2019
Živila - Določevanje aflatoksina v začimbah, razen v papriki, z IAC-čiščenjem in
HPLC-FLD s postkolonsko derivatizacijo
Foodstuffs - Determination of aflatoxins in spices other than paprika by IAC clean-up and
HPLC-FLD with post-column derivatization
Lebensmittel - Bestimmung von Aflatoxinen in Gewürzen außer Paprika mit IAC
Reinigung und HPLC-FLD mit Nachsäulenderivatisierung
Produits alimentaires - Détermination des aflatoxines dans les épices (pour lesquelles un
niveau maximal a été établi par l'UE) autres que le paprika
Ta slovenski standard je istoveten z: prEN 17424
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.220.10 Začimbe Spices and condiments
oSIST prEN 17424:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN 17424:2019
oSIST prEN 17424:2019
DRAFT
EUROPEAN STANDARD
prEN 17424
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2019
ICS 67.050
English Version
Foodstuffs - Determination of aflatoxins in spices other
than paprika by IAC clean-up and HPLC-FLD with post-
column derivatization
Produits alimentaires - Détermination des aflatoxines Lebensmittel - Bestimmung von Aflatoxinen in
dans les épices (pour lesquelles un niveau maximal a Gewürzen außer Paprika mit IAC Reinigung und HPLC-
été établi par l'UE) autres que le paprika FLD mit Nachsäulenderivatisierung
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 275.
If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17424:2019 E
worldwide for CEN national Members.

oSIST prEN 17424:2019
prEN 00275281:2019 (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 . 11
7.1 Extraction of aflatoxins from the sample . 11
7.2 Determination of recovery . 11
7.3 Immunoaffinity clean-up . 11
7.4 HPLC-FLD analysis . 12
7.4.1 General. 12
7.4.2 Confirmation . 12
7.4.3 Post-column derivatization . 12
7.4.4 Identification . 13
7.4.5 Calibration . 13
8 Calculation . 13
9 Precision . 14
9.1 General. 14
9.2 Repeatability . 14
9.3 Reproducibility . 14
10 Test report . 16
Annex A (informative) Example conditions for suitable HPLC-FLD systems . 17
A.1 General. 17
A.2 Example HPLC Conditions . 17
A.3 Other examples of suitable columns and mobile phase conditions . 17
Annex B (informative) Typical chromatograms . 19
Annex C (informative) Precision data . 24
Annex D (informative) Supplementary information . 28

oSIST prEN 17424:2019
prEN 17424:2019 (E)
European foreword
This document (prEN 17424:2019) has been prepared by Technical Committee CEN/TC 275 “Food
analysis — Horizontal methods”, the secretariat of which is held by DIN.
The document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
oSIST prEN 17424:2019
prEN 00275281:2019 (E)
Introduction
Aflatoxins consist of a group of approximately twenty related fungal metabolites, although only
aflatoxins B , B , G and G are normally found in foods. Aflatoxins B and G are the dihydro derivatives
1 2 1 2 2 2
of the parent compounds. Aflatoxins are produced by at least three species of Aspergillus, A. flavus,
A. parasiticus and A. nominus, and can occur in a wide range of important raw food commodities,
including cereals, nuts, spices, figs and dried fruit.
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, Regulation (EC) No 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 — Aflatoxins are known to have carcinogenic effects and to be both acutely and
chronically toxic.
oSIST prEN 17424:2019
prEN 17424:2019 (E)
1 Scope
This document describes a procedure for the determination of aflatoxins B , B , G and G and total
1 2 1 2
aflatoxins (sum of B , B , G and G ) in spices for which EU maximum levels are established, other than
1 2 1 2
paprika, by high performance liquid chromatography (HPLC) with post-column derivatization (PCD)
and fluorescence detection (FLD) after immunoaffinity column clean-up.
The method is applicable to the spices capsicum, pepper, nutmeg, ginger, turmeric and mixtures
thereof.
The method has been validated for aflatoxins B , B , G and G and total aflatoxins in a range of test
1 2 1 2
samples that comprised: ginger, pepper, nutmeg, chilli, turmeric as individual spices and mixed
pepper+chilli+nutmeg (90+5+5, m+m+m), mixed spice+ginger (6+4, m+m) mixed spice, mixed
turmeric+ginger (2+8, m+m).
The validation was carried out over the following concentration ranges: aflatoxin B = 1 µg/kg to
16 µg/kg and total aflatoxins = 2,46 µg/kg to 36,1 µg/kg.
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 http://www.iso.org/obp
4 Principle
Aflatoxins are extracted from the spices with a mixture of methanol, acetonitrile and water. The sample
extract is filtered, diluted with phosphate buffered saline (PBS) and applied to an immunoaffinity
column (IAC) containing antibodies specific to aflatoxins B , B , G and G . The aflatoxins are eluted from
1 2 1 2
the immunoaffinity column. Aflatoxins are quantified by reversed-phase high performance liquid
chromatography (HPLC) with PCD followed by FLD.
5 Reagents
Use only reagents of recognized analytical grade, p.a. (pro analysi) and water complying with grade 1 of
EN ISO 3696, unless otherwise specified. Solvents shall be of quality for HPLC analysis, unless otherwise
specified.
WARNING 1 — Decontamination procedures for laboratory wastes of aflatoxins were developed by the
International Agency for Research on Cancer (IARC) [5], [6].
WARNING 2 — Aflatoxins are subject to light degradation. Protect the laboratory, where the analyses
are done, adequately from daylight. This can be achieved effectively by using Ultraviolet (UV) absorbing
foil on the windows in combination with subdued light (no direct sunlight) or curtains or blinds in
combination with artificial light (fluorescent tubes are acceptable). Protect aflatoxin containing
oSIST prEN 17424:2019
prEN 00275281:2019 (E)
solutions from light as much as possible (keep in the dark, use aluminium foil or amber-coloured
glassware) and store at the temperature recommended by the manufacturer (e.g. −18 °C).
5.1 Methanol, (CH OH).
5.2 Acetonitrile, (CH CN).
5.3 Potassium bromide (KBr).
5.4 Sodium hydroxide (NaOH).
5.5 Sodium hydroxide solution, substance concentration c(NaOH) = 2 mol/l.
Dissolve 8,0 g of sodium hydroxide (5.4) in 100 ml of water.
5.6 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l.
Dissolve 0,4 g of sodium hydroxide (5.4) in a 100 ml of water.
5.7 Hydrochloric acid solution (HCl), volume fraction φ(HCl) = 37 % (acidimetric).
5.8 Hydrochloric acid solution, c(HCl) = 0,1 mol/l.
Dilute 8,28 ml of hydrochloric acid solution (5.7) to 1 l with water.
5.9 Sulfuric acid solution, for rinsing glassware, c(H SO ) = 2 mol/l.
2 4
5.10 Sodium chloride (NaCl).
5.11 Potassium chloride (KCl).
5.12 Potassium dihydrogen phosphate (KH PO ).
2 4
5.13 Disodium hydrogen orthophosphate (Na HPO ).
2 4
5.14 Phosphate buffered saline (PBS), pH = 7.4.
Weigh 0,20 g of potassium chloride (5.11), 0,20 g of potassium dihydrogen phosphate (5.12), 1,16 g of
disodium hydrogen orthophosphate (5.13) and 8,00 g of sodium chloride (5.10) to the nearest 0,01 g
and transfer into a 1 l volumetric flask. Dissolve in water and add 900 ml of
...