FprEN 17424

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

Produits alimentaires - Détermination des aflatoxines dans les épices (pour lesquelles un

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

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-

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

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

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

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

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

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17424:2019 E

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

This document (prEN 17424:2019) has been prepared by Technical Committee CEN/TC 275 “Food

This document has been prepared under a mandate given to CEN by the European Commission and the

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

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,

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

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

This document describes a procedure for the determination of aflatoxins B , B , G and G and total

aflatoxins (sum of B , B , G and G ) in spices for which EU maximum levels are established, other than

paprika, by high performance liquid chromatography (HPLC) with post-column derivatization (PCD)

The method is applicable to the spices capsicum, pepper, nutmeg, ginger, turmeric and mixtures

The method has been validated for aflatoxins B , B , G and G 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

The validation was carried out over the following concentration ranges: aflatoxin B = 1 µg/kg to

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)

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

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

the immunoaffinity column. Aflatoxins are quantified by reversed-phase high performance liquid

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

WARNING 1 — Decontamination procedures for laboratory wastes of aflatoxins were developed by the

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).

5.7 Hydrochloric acid solution (HCl), volume fraction φ(HCl) = 37 % (acidimetric).

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

Alternatively, a PBS solution with equivalent properties may be prepared from commercially available

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

Slowly add 25,5 ml of concentrated HNO (70 %, mass concentration ρ(HNO ) = 1,413 g/ml at 25 °C) to

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.

Dissolve aflatoxin B (5.20), B (5.21), G (5.22) and G (5.23) separately in acetonitrile (5.2) to give

separate solutions with a mass concentration of 10 µg/ml for each aflatoxin. Transfer the stock

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,

ε is the molar absorption coefficient of each aflatoxin in acetonitrile (5.2), in m /mol.

Table 1 — Molar mass and molar absorption coefficient of aflatoxins B , B , G and G in

Prepare a mixed stock solution containing 1 000 ng/ml of aflatoxin B and G , 500 ng/ml of aflatoxin B

and G in acetonitrile (5.2) by appropriate dilution of aflatoxins (B , B , G and G ) stock solutions (5.24).

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

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

Protect the solution from light and store below 4 °C when not in use. This solution can be stored under

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 ,

Protect the solution from light and store below 4 °C when not in use. This solution can be stored under

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

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

These calibration solutions cover the range from 0,48 µg/kg to 9,6 µg/kg for aflatoxins B and G and

Mix water, acetonitrile (5.2) and methanol (5.1) (56+30+14, v+v+v). Degas the solution before use if an

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

The affinity column contains antibodies raised against aflatoxins B , B , G and G . The column shall

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.

6.9 Filter paper, e.g. 190 mm diameter, pre-folded, wet strengthened, nominal particle retention

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

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

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

6.13.7 Alternative System for derivatization by photochemical reaction, e.g. Photochemical Reactor

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

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).

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.

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.

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.

Recommended settings for adjustable detectors are wavelengths of λ = 364 nm (excitation), λ = 440 nm

All samples shall be extracted and cleaned-up with the immunoaffinity columns on the same day as

NOTE During method development and validation lower recovery values were observed for sample extracts

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

of sodium chloride (5.10). A larger sample weight may be used provided the sample or extraction

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

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-

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

and 2,5 µg/kg aflatoxin B and G in the sample) and leave for approximately 30 min at room

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.

The use of immunoaffinity columns can vary slightly from one manufacturer to another. Follow the

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

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

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

directly for HPLC analysis. If the solution is not clear, filter through a syringe filter (6.12) prior to HPLC

It is recommended (depending on the injection system) to take a sample volume of three tim