SIST EN 17424:2021
(Main)Foodstuffs - Determination of aflatoxins in spices other than paprika by IAC clean-up and HPLC-FLD with post-column derivatization
Foodstuffs - Determination of aflatoxins in spices other than paprika by IAC clean-up and HPLC-FLD with post-column derivatization
This document describes a procedure for the determination of aflatoxins B1, B2, G1 and G2 and total aflatoxins (sum of B1, B2, G1 and G2) in spices for which EU maximum levels are established, other than 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 B1, B2, G1 and G2 and total aflatoxins in a range of test 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 B1 = 1 µg/kg to 16 µg/kg and total aflatoxins = 2,46 µg/kg to 36,1 µg/kg.
Lebensmittel - Bestimmung von Aflatoxinen in Gewürzen außer Paprika mit IAC Reinigung und HPLC-FLD mit Nachsäulenderivatisierung
Dieses Dokument beschreibt ein Verfahren zur Bestimmung der Aflatoxine B1, B2, G1 und G2 und des Gesamtgehalts an Aflatoxinen (Summe von B1, B2, G1 und G2) in Gewürzen außer Paprika, für die EU Höchstgehalte festgelegt wurden, mittels Hochleistungsflüssigchromatographie (en: High Performance Liquid Chromatography, HPLC) mit Nachsäulenderivatisierung (en: post column derivatization, PCD) und Fluoreszenz¬detektion (FLD) nach Reinigung an der Immunoaffinitätssäule (en: immunoaffinity column, IAC).
Das Verfahren ist anwendbar auf die Gewürze Capsicum (außer Paprika), Pfeffer, Muskatnuss, Ingwer, Kurkuma und Mischungen daraus.
Dieses Verfahren wurde für die Aflatoxine B1, B2, G1 und G2 und die Gesamtaflatoxine in einer Reihe von Untersuchungsproben validiert. Diese umfassten: Ingwer, Pfeffer, Muskatnuss, Chili, Kurkuma als einzelne Gewürze und gemischten Pfeffer + Chili + Muskatnuss (90 + 5+ 5, m + m + m), gemischte Gewürze + Ingwer (6 + 4, m + m) gemischtes Gewürz, gemischtes Kurkuma + Ingwer (2 + 8, m + m).
Die Validierung wurde für die folgenden Konzentrationsbereiche durchgeführt: Aflatoxin B1 = 1 µg/kg bis 16 µg/kg und Gesamtaflatoxine = 2,46 µg/kg bis 36,1 µg/kg.
Produits alimentaires - Détermination des aflatoxines dans les épices (pour lesquelles un niveau maximal a été établi par l'UE) autres que le paprika
Le présent document décrit un mode opératoire pour le dosage des aflatoxines B1, B2, G1 et G2, ainsi que des aflatoxines totales (somme des aflatoxines B1, B2, G1 et G2) dans les épices pour lesquelles des teneurs maximales européennes sont établies, autres que le paprika, par chromatographie liquide à haute performance (CLHP) avec dérivation post-colonne (PCD) et détection par fluorescence (FLD) après purification sur colonne d’immunoaffinité (CIA).
La méthode est applicable au piment (à l’exclusion du paprika), au poivre, à la muscade, au gingembre, au curcuma, ainsi qu’à leurs mélanges.
La méthode a été validée pour les aflatoxines B1, B2, G1 et G2, ainsi que pour les aflatoxines totales dans un éventail d’échantillons d’essai comprenant : du gingembre, du poivre, de la muscade, du piment, du curcuma seuls, et un mélange poivre + piment + muscade (90 + 5 + 5, m + m + m), un mélange d’épices + gingembre (6 + 4, m + m), un mélange d’épices, un mélange curcuma + gingembre (2 + 8, m + m).
La validation a été effectuée dans les gammes de concentration suivantes : aflatoxine B1 = 1 µg/kg à 16 µg/kg et aflatoxines totales = 2,46 µg/kg à 36,1 µg/kg.
Živila - Določevanje aflatoksina v začimbah, razen v papriki, z IAC-čiščenjem in HPLC-FLD s postkolonsko derivatizacijo
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN 17424:2021
01-januar-2021
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: EN 17424:2020
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
SIST EN 17424:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN 17424:2021
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SIST EN 17424:2021
EN 17424
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2020
EUROPÄISCHE NORM
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 - Dosage des aflatoxines dans les Lebensmittel - Bestimmung von Aflatoxinen in
épices autres que le paprika par purification sur Gewürzen außer Paprika mit IAC Reinigung und HPLC-
colonne d'immunoaffinité et CLHP-FLD avec dérivation FLD mit Nachsäulenderivatisierung
post-colonne
This European Standard was approved by CEN on 14 September 2020.
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 17424:2020 E
worldwide for CEN national Members.
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SIST EN 17424:2021
EN 17424:2020 (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 . 6
6 Apparatus and equipment . 10
7 Procedure . 11
7.1 Extraction of aflatoxins from the sample . 11
7.2 Determination of recovery . 12
7.3 IAC clean-up . 12
7.4 HPLC-FLD analysis . 12
7.4.1 General. 12
7.4.2 Confirmation . 12
7.4.3 Post-column derivatization . 13
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 . 15
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 . 27
Bibliography . 31
2
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SIST EN 17424:2021
EN 17424:2020 (E)
European foreword
This document (EN 17424:2020) 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 May 2021, and conflicting national standards shall be
withdrawn at the latest by May 2021.
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.
3
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SIST EN 17424:2021
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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.
4
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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 (IAC) clean-up.
The method is applicable to the spices capsicum (excluding paprika), 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
1
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 https://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 containing antibodies specific to aflatoxins B , B , G and G . The aflatoxins are eluted from the
1 2 1 2
IAC and are quantified by reversed-phase HPLC with PCD followed by FLD.
5
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5 Reagents
Use only reagents of recognized analytical grade, p.a. (pro analysis) 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
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)
3
5.2 Acetonitrile (CH CN)
3
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 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
6
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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 water.
After dissolution adjust the pH to 7,4 with hydrochloric acid solution (5.8) or sodium hydroxide
solution (5.6) 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
5.15 Polysorbate 20, e.g. Tween 20 , lauric acid ≥ 40 %
5.16 PBS/Polysorbate 20 solution
Mix Polysorbate 20 (5.15) with PBS (5.14) (1 + 9, V + V).
5.17 Extraction solution
Mix acetonitrile (5.2), methanol (5.1) and water (40 + 35 + 25, V + V + V).
5.18 Nitric acid solution (HNO ), c(HNO ) = 4 mol/l
3 3
Slowly add 25,5 ml of concentrated HNO (70 %, mass concentration ρ(HNO ) = 1,413 g/ml at 25 °C) to
3 3
25 ml water, adjust the final volume to 100 ml with water.
5.19 Pyridinium bromide perbromide solution (PBPB), ρ(PBPB) = 50 mg/l
Weigh 50 mg ± 10 mg of PBPB into a 20 ml glass vial. Dissolve in approximately 20 ml water, use an
ultra-sonic bath if required, and transfer quantitatively into a 1 l amber glass bottle. Fill up to 1 l with
water. The solution can be stored in a dark place at room temperature for four days.
5.20 Aflatoxin B e.g. crystalline, as a film or as certified standard solution
1
5.21 Aflatoxin B e.g. crystalline, as a film or as certified standard solution
2
5.22 Aflatoxin G e.g. crystalline, as a film or as certified standard solution
1
5.23 Aflatoxin G e.g. crystalline, as a film or as certified standard solution
2
5.24 Aflatoxins (B , B , G and G ) stock solutions
1 2 1 2
Dissolve aflatoxin B (5.20), B (5.21), G (5.22) and G (5.23) separately in acetonitrile (5.2) to give
1 2 1 2
separate solutions with a mass concentration of 10 µg/ml for each aflatoxin. Transfer the stock
solutions to amber vials and store them below 4 °C when not in use.
1
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.
7
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To determine the exact mass concentration of aflatoxins in each stock solution, record the absorption
curve between a wavelength of 330 nm and 370 nm in 1 cm quartz glass cells using a UV-spectrometer
(6.5) with acetonitrile (5.2) as reference. Calculate the mass concentration of each aflatoxin, ρ, in µg/ml,
according to Formula (1):
A × M × 100
max
(1)
ρ =
δε×
where
A is the maximum extinction value determined from the absorption curve (here determined at
max
a wavelength of 350 nm);
M is the molar mass of each aflatoxin, in g/mol;
δ is the path length of the quartz cell, in cm;
2
ε is the molar absorption coefficient of each aflatoxin in acetonitrile (5.2), in m /mol.
M and ε of aflatoxins B , B , G and G are given in Table 1 [7].
1 2 1 2
Table 1 — Molar mass and molar absorption coefficient of aflatoxins B , B , G and G in
1 2 1 2
acetonitrile (5.2)
Aflatoxin M ε
2
g/mol m /mol
B 312 2 070
1
B 314 2 250
2
G 328 1 760
1
G 330 1 890
2
5.25 Mixed stock solution
Prepare a mixed stock solution containing 1 000 ng/ml of aflatoxin B and G , 500 ng/ml of aflatoxin B
1 1 2
and G in acetonitrile (5.2) by appropriate dilution of aflatoxins (B , B , G and G ) stock solutions (5.24).
2 1 2 1 2
A certified mixed aflatoxins stock standard solution which is ready to use may be used as an alternative.
Protect the solution from light and store below 4 °C when not in use. This solution can be stored under
these conditions for two months.
5.26 Mixed intermediate solution
Prepare a mixed intermediate solution by pipetting 2,0 ml of the mixed stock solution (5.25) into a
10 ml volumetric flask. Dilute to the mark with acetonitrile (5.2) and shake well. The concentration of
this mixed intermediate solution is 200 ng/ml of aflatoxin B and G , and 100 ng/ml of aflatoxin B and
1 1 2
G .
2
Protect the solution from light and store below 4 °C when not in use. This solution can be stored under
these conditions for two months.
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5.27 Mixed standard solution
Pipette 1,0 ml of the mixed intermediate solution (5.26) into a 10 ml volumetric flask, fill to the mark
with the acetonitrile (5.2) and mix well to give a solution containing 20 ng/ml of aflatoxin B and G ,
1 1
10 ng/ml of aflatoxin B and G for preparation of calibration solutions.
2 2
Protect the solution from light and store below 4 °C when not in use. This solution can be stored under
these conditions for two months.
5.28 Calibration solutions
Add different volumes of the mixed standard solution (5.27) to five 10 ml volumetric flasks to obtain
five calibration levels across the calibration range. The values in Table 2 were used in the validation
study.
Evaporate the acetonitrile until dryness under a stream of nitrogen at room temperature. To each flask,
add 4 ml of methanol (5.1), dissolve the aflatoxins by mixing, then dilute to 10 ml with water, and shake
well. Methanol and water are subject to volume contraction when mixed, so adjust the volume with
water again to the given volume.
These calibration solutions cover the range from 0,48 µg/kg to 9,6 µg/kg for aflatoxins B and G and
1 1
and G .
the range from 0,24 µg/kg to 4,8 µg/kg for B2 2
Table 2 — Example preparation of HPLC calibration solutions
Mixed Targeted mass concentration per analyte
Calibration standard
solution solution
B B G G
1 2 1 2
(5.27)
µl ng/ml ng/ml ng/ml ng/ml
1 10 0,02 0,01 0,02 0,01
2 20 0,04 0,02 0,04 0,02
3 50 0,10 0,05 0,10 0,05
4 100 0,20 0,10 0,20 0,10
5 200 0,40 0,20 0,40 0,20
These solutions should be freshly made on each day of analysis.
5.29 HPLC mobile phase solvent A
Mix water, acetonitrile (5.2) and methanol (5.1) (56 + 30 + 14, V + V + V). Degas the solution before use
if an online system is not available on the HPLC (6.13).
5.30 HPLC mobile phase solvent B
Mix water, acetonitrile (5.2) and methanol (5.1) (56 + 30 + 14, V + V + V). Add 350 µl of nitric acid
solution (5.18) and 119 mg of potassium bromide (5.3) per litre of mobile phase. Degas the solution
before use if an online system is not available on the HPLC (6.13).
5.31 Immunoaffinity column
The IAC contains antibodies raised against aflatoxins B , B , G and G . The column shall have a capacity
1 2 1 2
of not less than 100 ng of aflatoxin B .
1
9
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6 Apparatus and equipment
The use of non-acid washed glassware may cause losses of aflatoxins. It is recommended that all
glassware coming into contact with aqueous solutions of aflatoxins should be washed with acid solution
before use. Many laboratory washing machines do this as part of the washing program. Otherwise soak
such laboratory glassware in sulfuric acid (5.9) for several h (e.g. 15 h overnight), then rinse well (e.g.
three times) with water to remove all traces of acid. Check the absence of acid with pH paper.
In practice, the treatment is necessary for round bottomed flasks, volumetric flasks, measuring
cylinders, vials or tubes used for calibration solutions and final extracts (particularly autosampler
vials), and Pasteur pipettes, if these are used to transfer calibration solutions or extracts.
Usual laboratory apparatus and, in particular, the following:
6.1 Laboratory balance, accuracy of 0,01 g.
6.2 Analytical balance, accuracy of 0,1 mg.
6.3 Centrifugation bottle, e.g. 250 ml.
6.4 Centrifuge, suitable for relative centrifugal force of 2 000 g to 2 500 g.
−2
NOTE g = 9,81 m ⋅ s .
6.5 UV-spectrometer with quartz cuvettes.
®2
6.6 Laboratory blender, e.g. Ultra Turrax .
6.7 Laboratory shaker, adjustable.
6.8 pH meter.
6.9 Filter paper, e.g. 190 mm diameter, pre-folded, wet strengthened, nominal particle retention
rating of 30 µm.
6.10 Pipettes, 25 µl, 50 µl, 250 µl, 1 ml, 10 ml capacity.
6.11 Syringe, plastic disposable, 5 ml capacity.
6.12 Syringe filter, 0,45 µm polytetrafluoroethylene (PTFE).
6.13 HPLC-FLD system, with the following components:
6.13.1 HPLC pump, suitable for flow rate at 1,0 ml/min.
6.13.2 Injection system, capable for total loop injection (a 100 µl loop is recommended).
6.13.3 HPLC column, e.g. C18 or ODS-1 (length of 250 mm, inner diameter of 4,6 mm and particle size
of 5 µm), which ensures a baseline resolution of the aflatoxin B , B , G and G peaks from all other
1 2 1 2
peaks. A suitable pre-column shall be used.
2 ®
Ultra Turrax is a trade name of a product commercially 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
the product named. Equivalent products may be used if they can be shown to lead to the same results.
10
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6.13.4 Column oven.
6.13.5 Post-column derivatization system, with PBPB (only to be used with mobile phase A (5.29)).
Consisting of an HPLC pulseless pump, zero-dead volume T-piece, reaction PTFE tubing of
34 cm minimum and 0,5 mm internal diameter.
® 3
6.13.6 System for derivatization with electrochemically generated bromine, e.g. KOBRA CELL (only
to be used with mobile phase B (5.30)) and PTFE reaction tubing of 34 cm minimum and 0,5 mm
internal diameter.
6.13.7 Alternative System for derivatization by photochemical reaction, e.g. Photochemical Reactor
TM4
for Enhanced Detection (PHRED ), only to be used with mobile phase A (5.29). The photochemical
reactor is inserted between the HPLC column and the detector inlet. The knitted reactor consists of
PTFE tubing of 20 m and 0,25 mm internal diameter.
6.13.8 Fluorescence detector.
Recommended settings for adjustable detectors are wavelengths of λ = 364 nm (excitation), λ = 440 nm
(emission) and a bandwidth of 18 nm.
6.13.9 Data evaluation system.
7 Procedure
7.1 Extraction of aflatoxins from the sample
All samples shall be extracted and cleaned-up with the IAC on the same day as analysis due to lack of
stability of the analytes in extracted samples.
NOTE During method development and validation lower recovery values were observed for sample extracts
that were not cleaned-up on the same day.
Weigh a test portion of 25 g [m ] to the nearest 0,1 g into a 250 ml centrifugation bottle (6.3). Add 5,0 g
s
of sodium chloride (5.10). A larger sample weight may be used provided the sample or extraction
solvent volume or sodium chloride weight ratio is maintained.
Add 100 ml [V ] of the extraction solution (5.17) and blend for approximately 3 min to 5 min with a
e
blender (6.6). Alternatively, shake vigorously by hand for approximately 15 s to 30 s and then for
approximately 30 min with a shaker (6.7). For various types of shakers (e.g. horizontal platform shaker
or vertical wrist shaker) the motion speed shall be adjusted to obtain maximum agitation of the
extraction mixture.
After extraction, place the bottles in a centrifuge (6.4) and centrifuge for approximately 10 min at
approximately 2 000 g to 2 500 g, and then filter the supernatant through filter paper (6.9).
Add 59 ml of PBS/polysorbate 20 solution (5.16) and 1,0 ml of the filtrate into a conical flask (or
similar). Shake well by hand. Check the pH of the diluted extract and adjust if necessary to pH 7,4 with
sodium hydroxide solution (5.5). Use an aliquot of the diluted sample extract for IAC clean-up (7.3).
3 ®
KOBRA CELL is a trade name of a product commercially available. This information is given for the convenience
of users of this European Standard and does not constitute an endorsement by CEN of the product named.
Equivalent products may be used if they can be shown to lead to the same results.
4 TM
PHRED is a trade name of a product commercially available. This information is given for the convenience of
users of this European Standard and does not constitute an endorsement by CEN of the product named.
Equivalent products may be used if they can be shown to lead to the same results.
11
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SIST EN 17424:2021
EN 17424:2020 (E)
7.2 Determination of recovery
To determine the recovery rate by surrogate spike, add 125 µl of the mixed stock solution (5.25) to a
known blank sample prior to extraction (corresponds to a mass fraction of 5 µg/kg aflatoxin B and G
1 1
and 2,5 µg/kg aflatoxin B and G in the sample) and leave for approximately 30 min at room
2 2
temperature.
Follow the procedure according to 7.1 and 7.3 for the spiked and unspiked samples.
Where available, a suitable reference material may be used to determine the recovery rate.
7.3 IAC clean-up
The use of IAC can vary slightly from one manufacturer to another. Follow the manufacturers'
instructions.
Pass 30 ml of the diluted filtrate, (this contains 0,5 ml of the original extract, V ) through the IAC (5.31)
c
at a flow rate of approximately 3 ml/min (i.e. a dropping speed of approximately 1 drop/s) or by
gravity. Do not exceed a flow rate of 5 ml/min.
After the filtrate has passed through the column, wash the column with approximately 20 ml of PBS
(5.14) at a flow rate of maximum 5 ml/min and dry by applying little vacuum for approximately 5 s to
10 s or passing air through the IAC by means of a syringe (6.11) for approximately 10 s.
Elute the aflatoxins in a two-step procedure. Apply 0,50 ml of methanol (5.1) on the column and let it
pass through by gravity. Collect the eluate in a 4 ml vial that can be capped. Apply a second portion of
1,0 ml of methanol (5.1). Use a syringe to pass air through the column to collect the last few drops.
Pass 1,5 ml of water through the column and collect this in the sa
...
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.
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oSIST prEN 17424:2019
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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.
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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
2
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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.
3
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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.
4
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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
1
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
5
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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).
3
5.2 Acetonitrile, (CH CN).
3
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 water.
After dissolution adjust the pH to 7,4 with hydrochloric acid solution (5.8) or sodium hydroxide
solution (5.6) 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
5.15 Polysorbate 20, e.g. Tween® 20 , lauric acid ≥ 40 %.
5.16 PBS/Polysorbate 20 solution.
Mix Polysorbate 20 (5.15) with PBS (5.14) (1+9, v+v).
1)
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.
6
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5.17 Extraction solution.
Mix acetonitrile (5.2), methanol (5.1) and water (40+35+25, v+v+v).
5.18 Nitric acid solution (HNO ), c(HNO ) = 4 mol/l.
3 3
Slowly add 25,5 ml of concentrated HNO (70 %, mass concentration ρ(HNO ) = 1,413 g/ml at 25 °C) to
3 3
25 ml water, adjust the final volume to 100 ml with water.
5.19 Pyridinium bromide perbromide solution (PBPB), ρ(PBPB) = 50 mg/l.
Weigh 50 mg ± 10 mg of PBPB into a 20 ml glass vial. Dissolve in approximately 20 ml water, use an
ultra-sonic bath if required, and transfer quantitatively into a 1 l amber glass bottle. Fill up to 1 l with
water. The solution can be stored in a dark place at room temperature for four days.
5.20 Aflatoxin B e.g. crystalline, as a film or as certified standard solution.
1
5.21 Aflatoxin B e.g. crystalline, as a film or as certified standard solution.
2
5.22 Aflatoxin G e.g. crystalline, as a film or as certified standard solution.
1
5.23 Aflatoxin G e.g. crystalline, as a film or as certified standard solution.
2
5.24 Aflatoxins (B , B , G and G ) stock solutions.
1 2 1 2
Dissolve aflatoxin B (5.20), B (5.21), G (5.22) and G (5.23) separately in acetonitrile (5.2) to give
1 2 1 2
separate solutions with a mass concentration of 10 µg/ml for each aflatoxin. Transfer the stock
solutions to amber vials and store them below 4 °C when not in use.
To determine the exact mass concentration of aflatoxins in each stock solution, record the absorption
curve between a wavelength of 330 nm and 370 nm in 1 cm quartz glass cells using a spectrometer
(6.5) with acetonitrile (5.2) as reference. Calculate the mass concentration of each aflatoxin, ρ, in µg/ml,
according to Formula (1):
A × M × 100
max
ρ = (1)
δε×
where
A is the maximum extinction value determined from the absorption curve (here:
max
350 nm);
M is the molar mass of each aflatoxin, in g/mol;
δ is the path length of the quartz cell, in cm;
2
ε is the molar absorption coefficient of each aflatoxin in acetonitrile (5.2), in m /mol.
M and ε of aflatoxins B , B , G and G are given in Table 1 [7].
1 2 1 2
7
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Table 1 — Molar mass and molar absorption coefficient of aflatoxins B , B , G and G in
1 2 1 2
acetonitrile (5.2)
M ε
Aflatoxin
g/mol m2/mol
B
312 2 070
1
B
314 2 250
2
G
328 1 760
1
G
330 1 890
2
5.25 Mixed stock solution.
Prepare a mixed stock solution containing 1 000 ng/ml of aflatoxin B and G , 500 ng/ml of aflatoxin B
1 1 2
and G in acetonitrile (5.2) by appropriate dilution of aflatoxins (B , B , G and G ) stock solutions (5.24).
2 1 2 1 2
A certified mixed aflatoxins stock standard solution which is ready to use may be used as an alternative.
Protect the solution from light and store below 4 °C when not in use. This solution can be stored under
these conditions for two months.
5.26 Mixed intermediate solution.
Prepare a mixed intermediate solution by pipetting 2,0 ml of the mixed stock solution (5.25) into a
10 ml volumetric flask. Dilute to the mark with acetonitrile (5.2) and shake well. The concentration of
this mixed intermediate solution is 200 ng/ml of aflatoxin B and G , and 100 ng/ml of aflatoxin B and
1 1 2
G .
2
Protect the solution from light and store below 4 °C when not in use. This solution can be stored under
these conditions for two months.
5.27 Mixed standard solution.
Pipette 1,0 ml of the mixed intermediate solution (5.26) into a 10 ml volumetric flask, fill to the mark
with the acetonitrile (5.2) and mix well to give a solution containing 20 ng/ml of aflatoxin B and G ,
1 1
10 ng/ml of aflatoxin B and G for preparation of calibration solutions.
2 2
Protect the solution from light and store below 4 °C when not in use. This solution can be stored under
these conditions for two months.
5.28 Calibration solutions.
Add different volumes of the mixed standard solution (5.27) to five 10 ml volumetric flasks as listed in
Table 2 to obtain five calibration levels across the calibration range. The values in Table 2 were used in
the validation study.
Evaporate the acetonitrile just to dryness under a stream of nitrogen at room temperature. To each
flask, add 4 ml of methanol (5.1), dissolve the aflatoxins by mixing, then dilute to 10 ml with water, and
shake well. Methanol and water are subject to volume contraction when mixed, so adjust the volume
with water again to the given volume.
These calibration solutions cover the range from 0,48 µg/kg to 9,6 µg/kg for aflatoxins B and G and
1 1
the range from 0,24 µg/kg to 4,8 µg/kg for B and G .
2 2
8
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Table 2 —Preparation of HPLC calibration solutions
Targeted mass concentration per analyte
Mixed standard
Calibration
solution (5.27)
B B G G
1 2 1 2
solution
µl ng/ml ng/ml ng/ml ng/ml
1 10 0,02 0,01 0,02 0,01
2 20 0,04 0,02 0,04 0,02
3 50 0,10 0,05 0,10 0,05
4 100 0,20 0,10 0,20 0,10
5 200 0,40 0,20 0,40 0,20
These solutions should be freshly made on each day of analysis.
5.29 HPLC mobile phase solvent A.
Mix water, acetonitrile (5.2) and methanol (5.1) (56+30+14, v+v+v). Degas the solution before use if an
online system is not available on the HPLC (6.13).
5.30 HPLC mobile phase solvent B.
Mix water, acetonitrile (5.2) and methanol (5.1) (56+30+14, v+v+v). Add 350 µl of nitric acid solution
(5.18) and 119 mg of potassium bromide (5.3) per litre of mobile phase. Degas the solution before use if
an online system is not available on the HPLC (6.13).
5.31 Immunoaffinity column (IAC).
The affinity column contains antibodies raised against aflatoxins B , B , G and G . The column shall
1 2 1 2
have a capacity of not less than 100 ng of aflatoxin B .
1
6 Apparatus and equipment
The use of non-acid washed glassware may cause losses of aflatoxins. It is recommended that all
glassware coming into contact with aqueous solutions of aflatoxins should be washed with acid solution
before use. Many laboratory washing machines do this as part of the washing program. Otherwise soak
such laboratory glassware in sulfuric acid (5.9) for several h (e.g. 15 h overnight), then rinse well (e.g.
three times) with water to remove all traces of acid. Check the absence of acid with pH paper.
In practice, the treatment is necessary for round bottomed flasks, volumetric flasks, measuring
cylinders, vials or tubes used for calibration solutions and final extracts (particularly autosampler
vials), and Pasteur pipettes, if these are used to transfer calibration solutions or extracts.
Usual laboratory apparatus and, in particular, the following:
6.1 Laboratory balance, accuracy of 0,01 g.
6.2 Analytical balance, accuracy of 0,1 mg.
6.3 Centrifugation bottle, e.g. 250 ml.
6.4 Centrifuge, suitable for relative centrifugal force of 2 000 g to 2 500 g.
−2
NOTE g = 9,81 m ⋅ s .
9
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6.5 UV-spectrometer with quartz cuvettes.
®2
6.6 Laboratory blender, e.g. Ultra Turrax .
6.7 Laboratory shaker, adjustable.
6.8 pH meter.
6.9 Filter paper, e.g. 190 mm diameter, pre-folded, wet strengthened, nominal particle retention
rating of 30 µm.
6.10 Pipettes, 25 µl, 50 µl, 250 µl, 1 ml, 10 ml capacity.
6.11 Syringe, plastic disposable, 5 ml capacity.
6.12 Syringe filter, 0,45 µm polytetrafluoroethylene (PTFE).
6.13 HPLC-FLD system, with the following components:
6.13.1 HPLC pump, suitable for flow rate at 1,0 ml/min.
6.13.2 Injection system, capable for total loop injection (a 100 µl loop is recommended).
6.13.3 HPLC column, e.g. C18 or ODS-1 (length of 250 mm, inner diameter of 4,6 mm and particle size
of 5 µm), which ensures a baseline resolution of the aflatoxin B , B , G and G peaks from all other
1 2 1 2
peaks. A suitable pre-column shall be used.
6.13.4 Column oven.
6.13.5 Post-column derivatization system, with PBPB (only to be used with mobile phase A (5.29)).
Consisting of an HPLC pulseless pump, zero-dead volume T-piece, reaction tubing minimum
34 cm × 0,5 mm internal diameter PTFE.
® 3
6.13.6 System for derivatization with electrochemically generated bromine, e.g. KOBRA CELL (only
to be used with mobile phase B (5.30)) and reaction tubing minimum 34 cm × 0,5 mm internal diameter
PTFE.
6.13.7 Alternative System for derivatization by photochemical reaction, e.g. Photochemical Reactor
TM4
for Enhanced Detection (PHRED ), only to be used with mobile phase A (5.29). The photochemical
reactor is inserted between the HPLC column and the detector inlet. The knitted reactor consists of
25 m of PTFE, 1/16 inch OD × 0,25 mm ID tubing.
6.13.8 Fluorescence detector, with a wavelength of λ = 360 nm excitation filter and a wavelength of
λ > 420 nm cut-off emission filter, or equivalent (e.g. a detector with an adjustable monochromator).
2 ®
Ultra Turrax is a trade name of a product commercially 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
the product named. Equivalent products may be used if they can be shown to lead to the same results.
3 ®
KOBRA CELL is a trade name of a product commercially available. This information is given for the convenience
of users of this European Standard and does not constitute an endorsement by CEN of the product named.
Equivalent products may be used if they can be shown to lead to the same results.
4 TM
PHRED is a trade name of a product commercially available. This information is given for the convenience of
users of this European Standard and does not constitute an endorsement by CEN of the product named.
Equivalent products may be used if they can be shown to lead to the same results.
10
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Recommended settings for adjustable detectors are wavelengths of λ = 364 nm (excitation), λ = 440 nm
(emission) and a bandwidth of 18 nm.
6.13.9 Data evaluation system.
7 Procedure
7.1 Extraction of aflatoxins from the sample
All samples shall be extracted and cleaned-up with the immunoaffinity columns on the same day as
analysis due to lack of stability of the analytes in extracted samples.
NOTE During method development and validation lower recovery values were observed for sample extracts
that were not cleaned-up on the same day.
Weigh a test portion of 25 g [m ] to the nearest 0,1 g into a 250 ml centrifugation bottle (6.3). Add 5,0 g
s
of sodium chloride (5.10). A larger sample weight may be used provided the sample or extraction
solvent volume or sodium chloride weight ratio is maintained.
Add 100 ml [Ve] of the extraction solution (5.17) and blend for approximately 3 min to 5 min with a
blender (6.6). Alternatively, shake vigorously by hand for approximately 15 s to 30 s and then for
approximately 30 min with a shaker (6.7). For various types of shakers (e.g. horizontal platform shaker
or vertical wrist shaker) the motion speed shall be adjusted to obtain maximum agitation of the
extraction mixture.
After extraction, place the bottles in a centrifuge (6.4) and centrifuge for approximately 10 min at
approximately 2 000 g to 2 500 g, and then filter the supernatant through filter paper (6.9).
Add 59 ml of PBS/polysorbate 20 solution (5.16) and 1,0 ml of the filtrate into a conical flask (or
similar). Shake well by hand. Check the pH of the diluted extract and adjust if necessary to pH 7,4 with
sodium hydroxide solution (5.5). Use an aliquot of the diluted sample extract for immunoaffinity clean-
up (7.3).
7.2 Determination of recovery
To determine the recovery rate by surrogate spike, add 125 µl of the mixed stock solution (5.25) to a
known blank sample prior to extraction (corresponds to a mass fraction of 5 µg/kg aflatoxin B and G
1 1
and 2,5 µg/kg aflatoxin B and G in the sample) and leave for approximately 30 min at room
2 2
temperature.
Follow the procedure according to 7.1 and 7.3 for the spiked and unspiked samples.
Where available, a suitable reference material may be used to determine the recovery rate.
7.3 Immunoaffinity clean-up
The use of immunoaffinity columns can vary slightly from one manufacturer to another. Follow the
manufacturers' instructions.
Pass 30 ml of the diluted filtrate, (this contains 0,5 ml of the original extract, Vc) through the
immunoaffinity column (IAC) (5.31) at a flow rate of approximately 3 ml/min (i.e. a dropping speed of
approximately 1 drop/s) or by gravity. Do not exceed a flow rate of 5 ml/min.
After the filtrate has passed through the column, wash the column with approximately 20 ml of PBS
(5.14) at a flow rate of maximum 5 ml/min and dry by applying little vacuum for approximately 5 s to
10 s or passing air through the immunoaffinity column by means of a syringe (6.11) for approximately
10 s.
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Elute the aflatoxins in a two-step procedure. Apply 0,50 ml of methanol (5.1) on the column and let it
pass through by gravity. Collect the eluate in a 4 ml vial that can be capped. Apply a second portion of
1,0 ml of methanol (5.1). Use a syringe to pass air through the column to collect the last few drops.
Pass 1,5 ml of water through the column and collect this in the same glass vial, to give a final volume of
3,0 ml (V ). Cap the 4 ml vial and mix for approximately 5 s. If the solution is clear it can be used
final
directly for HPLC analysis. If the solution is not clear, filter through a syringe filter (6.12) prior to HPLC
injection.
7.4 HPLC-FLD analysis
7.4.1 General
The injection by total loop mode ensures maximum accuracy.
It is recommended (depending on the injection system) to take a sample volume of three tim
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Questions, Comments and Discussion
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