Animal feeding stuffs - Determination of aflatoxin B1 (ISO 17375:2006)

This International Standard specifies a method for the determination of aflatoxin B1 in animal feeding stuffs using high-performance liquid chromatography with post-column derivatization. It is applicable to animal feeding stuffs with a fat content of up to 50 %. The limit of quantification of this method has been demonstrated to be better than 0,5 ug/kg for aflatoxin B1 for a signal-to-noise ratio of 6.

Futtermittel - Bestimmung von Aflatoxin B1 (ISO 17375:2006)

Diese Internationale Norm legt ein Verfahren zur Bestimmung von Aflatoxin B1 in Futtermitteln mittels Hochleistungs-Flüssigchromatographie mit Nachsäulenderivatisierung fest.
Das Verfahren ist anwendbar für Futtermittel mit einem Fettgehalt bis zu 50 %.
Die quantitative Nachweisgrenze dieses Verfahrens wurde bei einem Signal-Rausch-Verhältnis von 6 für Aflatoxin B1 als besser als 0,5 µg/kg nachgewiesen.
ANMERKUNG   Das Verfahren basiert auf dem in der Literaturstelle [1] angegebenen Verfahren.

Aliments des animaux - Dosage de l'aflatoxine B1 (ISO 17375:2006)

L'ISO 17375:2006 spécifie une méthode de dosage de l'aflatoxine B1 dans les aliments des animaux en utilisant la chromatographie liquide haute performance avec dérivation post-colonne.
Elle est applicable aux aliments pour animaux dont la teneur en graisse est inférieure ou égale à 50 %.
Il a été démontré que la limite de quantification de l'aflatoxine B1 par cette méthode est inférieure à 0,5 g/kg avec un rapport signal/bruit de 6.

Krma – Določevanje aflatoksina B1 (ISO 17375:2006)

General Information

Status
Published
Publication Date
31-Aug-2006
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
01-Sep-2006
Due Date
01-Sep-2006
Completion Date
01-Sep-2006

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SLOVENSKI STANDARD
SIST EN ISO 17375:2006
01-september-2006
.UPD±'RORþHYDQMHDIODWRNVLQD% ,62
Animal feeding stuffs - Determination of aflatoxin B1 (ISO 17375:2006)
Futtermittel - Bestimmung von Aflatoxin B1 (ISO 17375:2006)
Aliments des animaux - Dosage de l'aflatoxine B1 (ISO 17375:2006)
Ta slovenski standard je istoveten z: EN ISO 17375:2006
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN ISO 17375:2006 en

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

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SIST EN ISO 17375:2006
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SIST EN ISO 17375:2006
EUROPEAN STANDARD
EN ISO 17375
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2006
ICS 65.120
English Version
Animal feeding stuffs - Determination of aflatoxin B1 (ISO
17375:2006)

Aliments des animaux - Dosage de l'aflatoxine B1 (ISO Futtermittel - Bestimmung von Aflatoxin B1 (ISO

17375:2006) 17375:2006)
This European Standard was approved by CEN on 12 May 2006.

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 Central Secretariat 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 Central Secretariat has the same status as the official

versions.

CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,

Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,

Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels

© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17375:2006: E

worldwide for CEN national Members.
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SIST EN ISO 17375:2006
EN ISO 17375:2006 (E)
Foreword

This document (EN ISO 17375:2006) has been prepared by Technical Committee ISO/TC 34

"Agricultural food products" in collaboration with Technical Committee CEN/TC 327 "Animal

feeding stuffs - Methods of sampling and analysis", the secretariat of which is held by NEN.

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 December 2006, and conflicting national

standards shall be withdrawn at the latest by December 2006.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of

the following countries are bound to implement this European Standard: Austria, Belgium,

Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,

Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,

Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

Endorsement notice

The text of ISO 17375:2006 has been approved by CEN as EN ISO 17375:2006 without any

modifications.
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SIST EN ISO 17375:2006
INTERNATIONAL ISO
STANDARD 17375
First edition
2006-06-01
Animal feeding stuffs — Determination
of aflatoxin B
Aliments des animaux — Dosage de l'aflatoxine B
Reference number
ISO 17375:2006(E)
ISO 2006
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SIST EN ISO 17375:2006
ISO 17375:2006(E)
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© ISO 2006

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ii © ISO 2006 – All rights reserved
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SIST EN ISO 17375:2006
ISO 17375:2006(E)
Contents Page

Foreword............................................................................................................................................................ iv

1 Scope..................................................................................................................................................... 1

2 Normative references........................................................................................................................... 1

3 Principle................................................................................................................................................. 1

4 Reagents................................................................................................................................................ 1

5 Apparatus.............................................................................................................................................. 4

6 Procedures............................................................................................................................................ 6

6.1 Conditioning of IACs ............................................................................................................................ 6

6.2 Extraction.............................................................................................................................................. 6

6.3 Immunoaffinity clean up ...................................................................................................................... 6

6.4 Post-column derivatization.................................................................................................................. 7

6.5 Calibration curve................................................................................................................................... 7

6.6 Calculation............................................................................................................................................. 7

6.7 Spiking procedures for recovery determination ...............................................................................8

7 Precision................................................................................................................................................ 8

7.1 Interlaboratory test ............................................................................................................................... 8

7.2 Repeatability.......................................................................................................................................... 8

7.3 Reproducibility...................................................................................................................................... 9

8 Test report............................................................................................................................................. 9

Annex A (informative) Results of an interlaboratory test............................................................................. 10

Bibliography ..................................................................................................................................................... 11

© ISO 2006 – All rights reserved iii
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SIST EN ISO 17375:2006
ISO 17375:2006(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies

(ISO member bodies). The work of preparing International Standards is normally carried out through ISO

technical committees. Each member body interested in a subject for which a technical committee has been

established has the right to be represented on that committee. International organizations, governmental and

non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the

International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. ISO shall not be held responsible for identifying any or all such patent rights.

ISO 17375 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal

feeding stuffs.
iv © ISO 2006 – All rights reserved
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SIST EN ISO 17375:2006
INTERNATIONAL STANDARD ISO 17375:2006(E)
Animal feeding stuffs — Determination of aflatoxin B
1 Scope

This International Standard specifies a method for the determination of aflatoxin B in animal feeding stuffs

using high-performance liquid chromatography with post-column derivatization.
It is applicable to animal feeding stuffs with a fat content of up to 50 %.

The limit of quantification of this method has been demonstrated to be better than 0,5 µg/kg for aflatoxin B for

a signal-to-noise ratio of 6.
NOTE The method is based on that given in Reference [1].
2 Normative references

The following referenced documents are indispensable for the application 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.

ISO 3696:1987, Water for analytical laboratory use — Specification and test methods

3 Principle

A test portion is extracted with a solvent solution (acetone/water). The sample extract is filtered, diluted with

water or phosphate-buffered saline to a specified solvent concentration. A test portion is applied on an

immunoaffinity column (IAC) containing antibodies specific to aflatoxin B . The aflatoxin B is removed from

1 1

the IAC with neat methanol, and then quantified by reverse-phase high-performance liquid chromatography

(RP-HPLC) with post-column derivatization (PCD) involving bromination. The PCD is achieved with either

electrochemically generated bromine or with pyridinium hydrobromide perbromide (PBPB) followed by

fluorescence detection.
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.

WARNING — This method requires the use of toxic inflammable liquids such as acetone, methanol

and acetonitrile. Avoid contact and keep away from heat, sparks or open flames.
[2],[3]

NOTE Decontamination procedures for laboratory wastes have been developed and validated by the

International Agency for Research on Cancer (WHO).
4.1 Water, complying with grade 3 in accordance with ISO 3696:1987.
4.2 Phosphate buffer saline (PBS), pH 7,4.
© ISO 2006 – All rights reserved 1
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SIST EN ISO 17375:2006
ISO 17375:2006(E)

PBS may be prepared from potassium chloride (0,20 g), potassium dihydrogen phosphate (0,20 g), anhydrous

disodium hydrogen phosphate (1,16 g) [or disodium hydrogen phosphate dodecahydrate (2,92 g)] and sodium

chloride (8,00 g) added to 900 ml purified water. Adjust the pH to pH 7,4 (with 0,1 mol/l HCl or 0,1 mol/l NaOH

as appropriate) and dilute the solution to 1,0 l.

Alternatively, commercially available phosphate-buffered saline tablets with equivalent properties may be used.

PBS is not microbiologically stable and should be prepared fresh at least once a week.

4.3 Pyridinium hydrobromide perbromide (PBPB, CAS: 39416-48-3).

This reagent is not required in the case of using electrochemically generated bromine.

4.4 Potassium bromide.
This reagent is not required in the case of using the PBPB reagent.
4.5 HPLC-grade acetonitrile.
4.6 HPLC-grade methanol.
4.7 Acetone, pure.
4.8 HPLC grade water, complying with grade 1 of ISO 3696:1987.

4.9 Extraction solvent, solution of acetone (4.7) and water (4.8) [85+15 (by volume)].

4.10 Nitric acid, c(HNO ) = 4 mol/l.
This reagent is not required in the case of using the PBPB reagent.
4.11 Immunoaffinity column (IAC)

The IAC should contain antibodies raised against aflatoxin B . The IAC should have a capacity of not less

than 40 ng of aflatoxin B and should give a recovery of not less than 80 % for aflatoxin B when applied as a

standard solution in acetone/water containing 0,25 ng of aflatoxin B .
4.12 HPLC mobile phase solvent A, for use with PBPB post column reagent only.

Use a solution of water (4.8)/acetonitrile (4.5)/methanol (4.6) [6+2+3 (by volume)]. The ratio of solvents may

be adjusted to give optimum separation parameters.

4.13 HPLC mobile phase solvent B, for use with electrochemically generated bromine only.

Use a solution of water (4.8)/acetonitrile (4.5)/methanol (4.6) [6+2+3 (by volume)] containing 120 mg

potassium bromide (4.4) and 350 µl nitric acid at 4 mol/l (4.10) per litre of mobile phase. The ratio of solvents

may be adjusted to give optimum separation parameters.
The mobile phase solvents (4.12 and 4.13) should be degassed.
4.14 Post-column reagent, for use with PBPB post column reagent only.

Dissolve 25 mg of PBPB (4.3) in 500 ml of water. This solution may be used for up to 4 days if stored in a dark

place at room temperature. This post-column reagent shall be used only in combination with HPLC mobile

phase solvent A (4.12) but not with HPLC mobile phase solvent B (4.13).
NOTE Post column reagent is only stable for 3 days.
4.15 Toluene/acetonitrile, 98 + 2 (by volume).
2 © ISO 2006 – All rights reserved
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SIST EN ISO 17375:2006
ISO 17375:2006(E)

4.16 Aflatoxin B standard material, in form of crystals or a dry film for analytical purposes.

WARNING 1 — This method requires the use of solutions of aflatoxin B . Aflatoxins are carcinogenic

to humans. Attention is drawn to the statement made by the International Agency for Research on

[2]
Cancer (WHO) .

WARNING 2 — Aflatoxins are subject to light degradation. Protect analytical work adequately from the

daylight, and keep aflatoxin standard solutions protected from light by using amber vials or

aluminium foil.
4.17 Calibration stock solutions for HPLC
4.17.1 General stock solution

Prepare an aflatoxin B (4.16) stock solution containing 10,0 µg/ml in toluene/acetonitrile (4.15).

NOTE The toluene/acetonitrile stock solution is stable for at least one year provided it is stored in acid-washed

glassware and kept for storage at –18 °C in the dark. If stock solutions are used up in a much shorter period (maximum of

3 months), methanol (4.6) might be used as an alternative. Note that methanolic solutions are more sensitive to an

alkaline ambient of the glass surface and to day light than toluene/acetonitrile solutions.

Wrap the flasks tightly in aluminium foil and store them at less than 4 °C. To determine the exact

concentration of aflatoxins in this stock solution, record the absorption curve between a wavelength of 330 nm

and 370 nm in 1 cm quartz glass cells (5.21) in a spectrometer (5.20), with the stock solution solvent in the

reference cell. Calculate the mass concentration of each aflatoxin, c , in micrograms per millilitre, using

Equation (1):
M ×100
cA=× (1)
amax
ε × d
where
A is the absorbance determined at the maximum of the absorption curve;
max
M is the molar mass of aflatoxin B , in grams per mole (312 g/mol);
a 1

ε is the molar absorptivity of aflatoxin B , in square metres per mole (1930 m /mol for

a 1
toluene/acetonitrile and 2150 m /mol for methanol solutions);
d is the optical path length of the cell, in centimetres.
4.17.2 Calibration stock solution

Prepare an aflatoxin B (4.16) calibration solution containing 50,0 ng/ml in either toluene/acetonitrile (4.15) or

methanol (4.6) from the stock solution (4.17.1).
4.17.3 Option A (see 6.3)

Pipette from the calibration stock solution (4.17.2) the volumes as listed in Table 1 (Option A) into a set of

20 ml calibrated volumetric flasks. Evaporate the toluene/acetonitrile solution just to dryness under a stream of

nitrogen at room temperature. If methanol is used in the preparation of the stock solution, evaporation is not

required. To each flask, add 7 ml of methanol. Allow the aflatoxins to dissolve, then dilute to the mark with

water, and shake well.

NOTE Remember that methanol and water are subject to volume contraction when mixed.

© ISO 2006 – All rights reserved 3
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SIST EN ISO 17375:2006
ISO 17375:2006(E)
4.17.4 Option B (see 6.3)

Pipette from the calibration stock solution (4.17.2) the volumes as listed in Table 1 (Option B) into a set of at

lea
...

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