UGANDA STANDARD  
       US ISO 14718
First Edition
   2009 -09-04
 
Reference numbe r
  US ISO 14718:1998
 © UNBS 200 9 
  
Animal feeding stuffs — Determination of aflatoxin B 1 content 
of mixed feeding stuffs — Method using high-performance 
liquid chromatography 
 US ISO 14718:1998  
 
© UNBS  2009 – All rights reserved Compliance with this standard does not, of itself confer immunity from legal obligations 
A Uganda Standard does not purport to include all necessary provisions of a contract. Users are 
responsible for its correct application  
© UNBS 2009  
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilised in any 
form or by any means, electronic or mechanical, in cluding photocopying and microfilm, without prior written 
permission from UNBS. 
Requests for permission to reproduce this document should be addressed to 
The Executive Director 
Uganda National Bureau of Standards 
P.O. Box 6329 
Kampala  
Uganda 
Tel: 256 41 505 995 
Fax: 256 41 286 123 
E-mail: unbs@infocom.co.ug
    US ISO 14718: 1998  
© UNBS  2009 - All rights reserved National foreword 
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry  of Tourism, Trade and 
Industry established by the Act of Parliament of 198 3, of the Laws of Uganda.  UNBS is mandated to co-
ordinate the elaboration of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex A limentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT/SPS Agr eements of the World Trade Organisation (WTO). 
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a gi ven field or area and consists of representatives of 
consumers, traders, academician s, manufacturers, governme nt and other stakeholders. 
Draft Uganda Standards adopted by the Technical Committ ee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council. 
This Uganda Standard, US ISO 14718:1998, Animal feeding stuffs —  Determination of aflatoxin B1
content of mixed feeding stuffs — Method using high-performance liquid chromatography, is identical with and 
has been reproduced from an Inte rnational Standard ISO 14718:1998,  Animal feeding stuffs — 
Determination of aflatoxin B 1 content of mixed feeding stuffs — Method using high-performance liquid 
chromatography, and has been adopted as a Uganda Standard . 
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." A Reference numbe r
ISO 14718:1998 (E)INTERNATIONAL
STANDARDISO
14718
First edition
1998-12-15
Animal feeding stuffs — Determination of
aflatoxin B 1 content of mixed feeding
stuffs — Method using high-performanceliquid chromatography
Aliments des animaux — Détermination de la teneur en aflatoxine B 1 dans
les aliments composés — Méthode par chromatographie liquide à haute
performanceISO 14718:1998(E)
©   ISO 1998
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by an y means, electronic
or mechanical, including photocopying and microfilm, without permission in writing from the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet iso@iso.ch
Printed in Switzerland
iiForeword
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 technicalcommittees. Each member body interested in a subject for which a technical committee has been established hasthe right to be represented on that committee. International organizations, governmental and non-governmental, inliaison with ISO, also take part in the work. ISO collaborates closely with the International ElectrotechnicalCommission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.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.
International Standard ISO 14718 was prepared by Technical Committee ISO/TC 34, 
Agricultural food products ,
Subcommittee SC 10, Animal feeding stuffs .
Annexes A and B of this International Standard are for information only.INTERNATIONAL STANDARD    © ISO ISO 14718:1998(E)
1Animal feeding stuffs — Determination of aflatoxin B 1 content of
mixed feeding stuffs — Method using high-performance liquidchromatography
1 Scope
This International Standard specifies a high-performance liquid chromatographic (HPLC) method for the
determination of aflatoxin B1 content of animal feeding stuffs including those containing citrus pulp.
The lower limit of determination is 1 mg/kg.
NOTE 1 This International Standard may be applicable for the determination of the aflatoxin B1 content of a number of raw
materials and straight feeding stuffs such as corn gluten, groundnut, palm kernel, copra, citrus pulp, tapioca, soya bean, ricebran, pollard, rape seed, niger seed and cotton seed (see references [1] and [2]). These materials were, however, not includedin the collaborative testing of the method.
NOTE 2 This International Standard may also be applicable for the determination of the content of the sum of the
aflatoxins B
1, B2, G1 and G2. However, the method has not been validated for this parameter by collaborative testing.
2 Normative reference
The following normative document contains provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subseqent amendments to, or revisions of, this publication do notapply. However, parties to agreements based on this International Standard are encouraged to investigate thepossibility of applying the most recent edition of the normative document indicated below. For undated references,the latest edition of the normative document referred to applies. Members of IEC and ISO editions maintainregisters of currently valid International Standards.
ISO 6498:1998, 
Animal feeding stuffs — Preparation of test sample .
3 Principle
The sample is extracted with chloroform. The extract is filtered and an aliquot portion is purified on a Florisil®1)
cartridge and a C18 cartridge. The final separation and determination is achieved by high-performance liquid
chromatography (HPLC) using a reverse-phase C18 column, followed by post-column derivatization with iodine or
bromine, and fluorescence detection.
4 Reagents and materials
Use only reagents of recognized analytical grade.
                                                     
1)  Florisil® is the trade-name of a commercially available product. This information is given for the convenience of users of this
International Standard and does not constitute an endorsement by ISO of this product. Equivalent products may be used if they
can be shown to lead to the same results .ISO 14718:1998(E) © ISO
24.1   Water, demineralized or deionized, with resistivity of at least 10 M W◊cm, or water of at least equivalent purity.
4.2   Concentrated sulfuric acid , c(H2SO4) = 18 mol/l, r(H2SO4) = 1,84 g/ml.
4.3   Sulfuric acid , c(H2SO4) = 2 mol/l.
Carefully add 105 ml of concentrated sulfuric acid (4.2) to 895 ml of water and mix well. Avoid excessive heating of
the solution.
4.4   Control sample .
Prepare a control sample of about 2 kg of compound feed with an aflatoxin B1 content of about 5 mg/kg by
combining samples of previous determinations with an aflatoxin B1 content of about 5 mg/kg. Mix thoroughly.
The aflatoxin B1 content of the control sample should be determined five times by two analysts following the
procedure described in clause 8. From the results the mean aflatoxin B1 content, the standard deviation and the
coefficient of variation should be calculated.
4.5   Acid-washed Celite® 545 , or product of equivalent quality2).
4.6   Florisil® Sep-Pak style cartridge, Waters No. 51960 , or product of equivalent qualty3).
4.7   C18 Sep-Pak style cartridge, Waters No. 51910 , or product of equivalent quality3).
4.8   Acetone.
4.9   Methanol .
4.10   Acetonitrile .
4.11   Chloroform , stabilized with ethanol (mass fraction 0,5 % to 1,0 %).
WARNING: Chloroform is a toxic substance. Avoid inhalation of and exposure to chloroform. Work in a
fumehood when handling the solvent and solutions thereof.
The adsorption characteristics of the Florisil® cartridge (4.6) may change if stabilizers other than ethanol are used.
When chloroform as described is not available, the adsorption characteristics should be verified in accordance withclause 8.
4.12   Mixture of acetone and water , 98 + 2 (by volume).
Combine 980 ml of acetone (4.8) and 20 ml of water (4.1). Mix well.
4.13   Mixture of acetone and water , 15 + 85 (by volume).
Combine 150 ml of acetone (4.8) and 850 ml of water (4.1). Mix well.
4.14   Mixture of acetone and water,  5 + 95 (by volume).
                              
                       
2)  Celite® is the trade-name of a commercially available product.
3)  Florisil® is the trade-name of a commercially available product. Florisil® Sep-Pak style cartridge article number 51960 and
C18 Sep-Pak style catridge article number 51910, from Waters Associates (Milwaukee, USA), are examples of suitable
products available commercially.
This information is given for the convenience of users of this International Standard and does not constitute an endorsement by
ISO of these products. Equivalent products may be used if they can be shown to lead to the  same results .© ISO ISO 14718:1998(E)
3Combine 50 ml of acetone (4.8) and 950 ml of water (4.1). Mix well.
4.15   Mixture of methanol and water , 20 + 80 (by volume).
Combine 200 ml of methanol (4.9) and 800 ml of water (4.1). Mix well.
4.16   Concentrated nitric acid , c(HNO3) = 14 mol/l, r(HNO3) = 1,40 g/ml, for HPLC with bromine derivatization.
4.17   Potassium bromide  (KBr), for HPLC with bromine derivatization.
4.18   Mobile phase for HPLC .
4.18.1   Mobile phase for HPLC with iodine derivatization .
Combine 120 ml of acetonitrile (4.10), 210 ml of methanol (4.9) and 390 ml of water (4.1) and mix. Filter the eluent
through a 0,45 mm PTFE membrane filter using the solvent filtration system (5.1) and degas for 10 min in the
ultrasonic bath (5.2) before use.
NOTE  The composition of the mobile phase solvent may need adjustment depending on the characteristics of the HPLC
column used.
4.18.2   Mobile phase for HPLC with bromine derivatization .
Combine 400 ml of acetonitrile (4.10), 700 ml of methanol (4.9) and 1 300 ml of water (4.1) and mix. Add to the
mixture 286 mg of potassium bromide (4.17) and 152 ml of concentrated nitric acid (4.16). Mix well and degas with a
stream of inert gas for 15 min.
4.19   Saturated iodine solution for HPLC with iodine derivatization .
Add 2 g of iodine to 400 ml of water. Mix for at least 90 min and filter through a 0,45 mm PTFE membrane filter
(see 5.1). Prepare the solution fresh on the day of use.
Protect the saturated solution from light to prevent photodegradation.
4.20   Sodium hypochlorite solution  (household quality), r(active chlorine) = 100 g/l.
4.21   Sodium hypochlorite solution , volume fraction 1 %.
Dilute 10 ml of sodium hypochlorite solution (4.20) with 990 ml of water-acetone mixture (4.14).
4.22   Inert gas, e.g. nitrogen.
4.23   Aflatoxin B1 standard material  (C17H12O6), 2,3,6aa,9aa-tetrahydro-4-methoxycyclopenta[ c]furo[3 ¢,2¢:4,5]-
furo[2,3-h][1]benzopyran-1,11-dione; Chemical Abstracts Service Registry (CAS) number 1162-65-8.
WARNINGS
1  Mycotoxins are extremely toxic substances. Perform all manipulations in a designated fume cupboard.
Take special precautions when toxins are in a dry form because of their electrostatic nature and resultingtendency to disperse in working areas.
2  Aflatoxins are sensitive to UV radiation. Therefore, conduct all operations in the absence of sunlight or
artificial white light. Provide sufficient, but not excessive, illumination with tungsten filament lamps. Low-energy lamps and fluorescent tubes may be used, but the use of amber glassware (vials, volumetric flasks)is recommended.
3  Glassware that has been in contact with solutions of aflatoxin B
1 has to be soaked overnight in a
hypochlorite solution (4.21), before cleaning, in order to remove traces of aflatoxin B1.ISO 14718:1998(E) © ISO
44.24   Aflatoxin B1 standard solution , r(aflatoxin B1) ≈ 10 mg/ml.
Transfer the content of an ampoule containing aflatoxin B1 (4.23) to a flask and dissolve in chloroform (4.11).
Transfer the solution to a convenient size volumetric flask and dilute to the mark with chloroform so as to obtain asolution with an aflatoxin B
1 content of about 10 mg/ml. Mix.
Transfer the solution to amber vials or an airtight screw-cap bottle and store in a cool place (4 °C) in the dark, well
sealed and wrapped in aluminium foil.
4.25   Aflatoxin B1 stock standard solution .
Transfer quantitatively 2,5 ml of the aflatoxin B1 standard solution (4.24) to a 50 ml volumetric flask and dilute to the
mark with chloroform (4.11).
Transfer the solution to amber vials or an airtight screw-cap bottle and store in a cool place (4 °C) in the dark, well
sealed and wrapped in aluminium foil.
4.26   Aflatoxin B1 calibration solutions for HPLC .
4.26.1   Calibration solution I , r(aflatoxin B1) ≈ 4 ng/ml.
Allow the volumetric flask with stock standard solution (4.25) to reach room temperature in the aluminium foil (a few
hours).
Transfer 400 ml of the stock standard solution (equivalent to about 200 ng of aflatoxin B1) to an acid-washed 50 ml
volumetric flask, and evaporate the solution to dryness in a stream of inert gas (4.22). Dissolve the residue in 20 mlof the water-acetone mixture (4.13). Dilute to the mark with the water-acetone mixture and mix well.
4.26.2   Calibration solution II , r(aflatoxin B
1) ≈ 3 ng/ml.
Transfer quantitatively 7,5 ml of the calibration solution I (4.26.1) to an acid-washed 10 ml volumetric flask. Dilute to
the mark with the water-acetone mixture (4.13) and mix well.
4.26.3   Reference calibration solution , r(aflatoxin B1) ≈ 2 ng/ml.
Transfer quantitatively 25 ml of the calibration solution I (4.26.1) to an acid-washed 50 ml volumetric flask. Dilute to
the mark with the water-acetone mixture (4.13) and mix well.
This solution is used for repetitive injection during HPLC (8.5).
4.26.4   Calibration solution III , r(aflatoxin B1) ≈ 1 ng/ml.
Transfer quantitatively 2,5 ml of the calibration solution I (4.26.1) to an acid-washed 10 ml volumetric flask. Dilute to
the mark with the water-acetone mixture (4.13) and mix well.
4.27   Chromatographic test solution .
Prepare an ampoule containing a mixture of aflatoxins B1, B2, G1 and G2 in 1 ml of chloroform with concentrations
of approximately 1,0 mg/ml, 0,5 mg/ml, 1,0 mg/ml and 0,5 mg/ml respectively.
Transfer the contents of the ampoule to a glass-stoppered test tube or screw-capped vial. Transfer 40 ml of this
solution to an acid-washed glass-stoppered test tube (5.4). Evaporate the chloroform in a stream of inert gas (4.22)and dissolve the residue into 10 ml of the water-acetone mixture (4.13).© ISO ISO 14718:1998(E)
55 Apparatus
Before use, laboratory glassware coming into contact with aqueous solutions of aflatoxins shall be soaked in sulfuric
acid (4.3) for several hours, then rinsed well (e.g. three times) with water to remove all traces of acid. Check theabsence of acid with pH paper.
In practice, this treatment is necessary for the round-bottomed flask of the rotary evaporator (5.12), the volumetric
flasks, measuring cylinders, vials or tubes used for calibration solutions and final extracts (particularly autosamplersvials), and Pasteur pipettes, if these are used to transfer calibration solutions or extracts.
NOTE  Laboratory glassware coming into contact with aqueous solutions of aflatoxins has to be soaked in dilute acid
because the use of non-acid-washed glassware may cause losses of aflatoxin B1. Particular care should be taken with new
glassware and disposable glassware such as autosampler vials and Pasteur pipettes.
Usual laboratory apparatus and, in particular, the following.
5.1   Solvent filtration system , suitable for PTFE membrane filters with a pore size of 0,45 mm.
5.2   Ultrasonic bath .
5.3   Microsyringe , of capacity 100 ml, for preparation of calibration solutions.
Check by weighing that the inaccuracy does not exceed 2 % of the mass.
5.4   Glass-stoppered calibrated tubes , of capacity 10 ml.
5.5   Spectrometer , suitable for measurements in the UV region of the spectrum, provided with quartz cuvettes of
optical path length 10 mm ± 0,1 mm.
5.6   Conical flask , of capacity 500 ml, made of borosilicate glass, with a wide neck and a glass stopper or a screw
cap fitted with a PTFE liner.
5.7   Mechanical shaker , horizontal rotation or reciprocating, with frequency 250 min-1 to 300 min-1.
5.8   Fluted filter paper , of diameter 24 cm.
5.9   Luer® chloroform-resistant threeway stopcock  4).
5.10   Chemically resistant syringe , 10 ml, with Luer® connector  4).
5.11   Glass column , with internal diameter 10 mm to 15 mm, length about 30 cm to 50 cm, equipped with a Luer®
tip4).
NOTE  When a glass column of internal diameter about 10 mm and length about 30 cm is used, it is advisable to use a
plastics reservoir (chemically resistant syringe barrel) of at least 70 ml capacity.
5.12   Rotary vacuum evaporator , equipped with a 150 ml to 250 ml round-bottomed flask.
5.13   General HPLC system .
See Figures 1 and 2 for a diagrammatic representation of the HPLC system for derivatization with iodine and
bromine respectively.
5.13.1   Pump, pulse free, capable of maintaining a volume flow rate of 0,1 ml/min to 1,0 ml/min.
                                                     
4)  Luer® is the trade-name of a commercially available product. This information is given for the convenience of users of this
International Standard and does not constitute an endorsement by ISO of this product. Equivalent products may be used if they
can be shown to lead to the  same results .ISO 14718:1998(E) © ISO
6Key
1 HPLC mobile phase 10 Reaction coil
2 HPLC pump 11 Fluorescence detector3 Injector 12 Restrictor4 Guard column 13 Waste5 Analytical column 14 Recorder/integrator6 Saturated iodine solution 15 Derivatization cell (KOBRA®)7 Reagent pump 16 Tension meter8 Tee joint 17 Power supply, 10 V d.c.
9 Water bath (60 °C) 18 Resistor, 100 k W
Figure 1 — Diagrammatic representation of the HPLC system for derivatization with iodine
NOTE  See Figure 1 for Key.
Figure 2 — Diagrammatic representation of the HPLC system for derivatization with bromine© ISO ISO 14718:1998(E)
75.13.2   Injection system , with loop suitable for the injection of 250 ml.
5.13.3   Fluorescence detector , with excitation at a wavelength of 365 nm and emission at wavelength of 435 nm
(for filter instruments: emission wavelength > 400 nm). Detection of at least 0,05 ng aflatoxin B1 shall be possible.
Some back pressure may be advisable [e.g. by applying a restrictor or a coil of stainless steel orpolytetrafluorethylene (PTFE) connected to the outlet of the detector] to suppress air bubbles in the flow cell.
5.13.4   Recorder .
5.13.5   Guard column : C
18 packing, particle size 37 mm to 50 mm, length 10 mm to 20 mm, internal diameter
3,9 mm; or a guard column of equivalent quality.
5.13.6   Analytical column : C18 packing, particle size 3 mm or 5 mm, length 200 mm, internal diameter 3,0 mm; or
an analytical column of equivalent quality.
5.13.7   Electronic integrator  (optional).
5.14   HPLC system for HPLC with iodine derivatization .
5.14.1   Pump, pulse free, for delivery of the iodine post-column reagent.
5.14.2   Zero dead volume Tee , stainless steel, 1,59 mm ¥ 0,75 mm.
5.14.3   Spiral reaction coil , polytetrafluorethylene (PTFE) or stainless steel.
Dimensions of 3 000 mm ¥ 0,5 mm to 5 000 mm ¥ 0,5 mm have been found to be appropriate in combination with
5 mm or 3 mm HPLC columns.
5.14.4   Thermostatically controlled water bath  or solid-state heating device , adjusted to 60 °C, capable of
temperature regulation to the nearest 0,1 °C.
5.15   HPLC system for HPLC with bromine derivatization .
5.15.1   Electrochemical derivatization cell : Kok’s Bromine Apparatus (KOBRA®5)).
5.15.2   Power supply , 0 V to 20 V d.c.
5.15.3   Tension meter , range 0 V to 10 V d.c., impedance > 50 k W.
5.15.4   Resistor, 100 k W.
5.16   Syringe, suitable for HPLC injection of 250 ml.
6 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling method is
given in ISO 6497 [7].
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changed during transport or storage.
                                                     
5) KOBRA® is the trade-name of a commercially available apparatus. This information is given for the convenience of users of
this International Standard and does not constitute an endorsement by ISO of this product. Equivalent apparatus may be usedif it can be shown to lead to the same results.ISO 14718:1998(E) © ISO
87 Preparation of the test sample
Prepare the test sample in accordance with ISO 6498.
Grind the laboratory sample (usually 500 g) so that it passes completely through a sieve with 1 mm apertures. Mix
thoroughly.
8 Procedure
8.1 General
To each series, add a spiked blank sample with an aflatoxin B1 content of 10 mg/kg and a certified reference
material or a control sample (4.4). Addition of a blank sample to each series is strongly recommended to check forcontamination from the glassware.
The results shall comply with the criteria in clause 10.
8.2 Determination of the absorption spectrum of the aflatoxin B1 standard solution
In cuvettes, determine the absorption spectrum of the aflatoxin B1 standard solution (4.24) between wavelengths of
330 nm and 370 nm by means of the spectrometer (5.5), using chloroform as blank. Measure the absorbance ( A) at
the maximum near a wavelength of 363 nm.
8.3 Extraction
Weigh, to the nearest 0,1 g, 50,0 g of the prepared test sample (see clause 7) into the conical flask (5.6).Consecutively add 25 g of Celite® (4.5), 250 ml of chloroform (4.11) and 25 ml of water. Stopper the flask, swirl andrelease the pressure. Restopper the flask and shake it for 30 min on the mechanical shaker (5.7).
NOTE  In order to reduce the use of chloroform, one-half of the specified quantities may be used; i.e. 25,0 g of the prepared
test sample (see clause 7), 12,5 g of Celite® (4.5), 125 ml of chloroform (4.11) and 12,5 ml of water.
Filter through a fluted filter paper (5.8). If filtration is proceeding slowly, cover the funnel in order to prevent
evaporation of chloroform. Collect 50 ml of the filtrate ( Vs).
If necessary, take an aliquot portion of the filtrate and dilute to 50 ml ( Vf) with chloroform so that the aflatoxin B1
content does not exceed 4 ng/ml.
Use the filtrate for sample clean-up in accordance with 8.4.
8.4 Clean-up
Carry out the procedure without significant interruptions.
8.4.1 Florisil® purification
8.4.1.1 Preparation of the column-cartridge assembly
Attach a stopcock (5.9) to the shorter stem of a Florisil® cartridge (4.6). Wash the cartridge and remove air by
taking 10 ml of chloroform (4.11) and passing 8 ml of it via the stopcock rapidly through the cartridge using a syringe(5.10).
Attach the longer stem of the cartridge to a glass column (5.11) and pass the remaining 2 ml of chloroform through
the cartridge into the column. Close the stopcock. Remove the syringe.© ISO ISO 14718:1998(E)
98.4.1.2 Purification
Add the filtrate ( Vs or Vf) collected in step 8.3 to the column-cartridge assembly and drain by gravity. Rinse with 5 ml
of chloroform (4.11), followed by 20 ml of methanol (4.9). Discard the eluates.
During these operations, ensure that the column-cartridge assembly does not run dry.
Elute aflatoxin B1 with 50 ml of the acetone-water mixture (4.12) and collect the eluate in the round-bottomed flask
of the rotary evaporator (5.12).
NOTE 1 The quality of Florisil® varies per batch. Depending on this quality, 50 ml of acetone-water mixture (4.12) may not
be sufficient for elution. If so, the use of 60 ml to 70 ml of the acetone-water mixture (4.12) is recommended.
Concentrate the eluate on the rotary evaporator at a temperature of between 40 °C and 50 °C until no more acetone
is distilled.
NOTE 2 About 0,5 ml of liquid remains in the flask at this point. Experiments have shown that further evaporation is not
harmful and that when 0,5 ml of liquid remains, there is no significant amount of acetone. Residues of acetone may lead tolosses of aflatoxin B
1 on the C18 cartridge.
Add 1 ml of methanol (4.9), swirl the flask to dissolve aflatoxin B1 on the sides of the flask, add 4 ml of water and
mix. Disconnect and discard the cartridge. Rinse the glass column with water and retain for the C18 purification step
(8.4.2).
8.4.2 C18 purification
8.4.2.1 Preparation of the column-cartridge assembly
Attach a stopcock (5.9) to the shorter stem of a C18 cartridge (4.7). Prime the cartridge and remove any air by
passing 10 ml of methanol (4.9) via the stopcock rapidly through the cartridge with a syringe (5.10). Air bubbles inthe cartridge are visible as light spots on the otherwise greyish background. Take 10 ml of water and pass 8 ml of itthrough the cartridge. Avoid introduction of air into the cartridge when switching from methanol to water.
Attach the longer stem of the cartridge to a glass column (5.11) and pass the remaining 2 ml of water through the
cartridge in the column. Close the stopcock. Remove the syringe.
8.4.2.2 Purification
Transfer the extract obtained in 8.4.1.2 quantitatively to the glass column (5.11), rinsing the flask twice with 5 ml of
the water-methanol mixture (4.15) and drain by gravity.
During these operations, ensure that the column-cartridge assembly does not run dry. If air bubbles develop in the
constriction near the cartridge, stop the flow and tap the top of the glass column to remove the air bubbles. Thencontinue.
Elute with 25 ml of the water-methanol mixture (4.15). Discard the eluate. Elute the aflatoxin B
1 with 25 ml of the
water-acetone mixture (4.13) and collect the eluate in a 50 ml volumetric flask. Dilute to the mark with water andmix. Use the resulting solution for chromatography (8.5).
NOTE  Filtration of the final extract prior to HPLC is normally not necessary. If considered necessary, cellulose filters
should not be used because they may lead to losses of aflatoxin B1. PTFE filters are acceptable.
8.5 High-performance liquid chromatography
8.5.1 General
Allow sufficient time for conditioning and stabilizing the apparatus.
The volume flow rates given for the mobile phase and the post-column reagent are indicative only. They need to be
adjusted depending on the characteristics of the HPLC column.ISO 14718:1998(E) © ISO
10The detector response to aflatoxin B1 depends on the temperature, therefore compensation should be made for
drift.
By injecting a fixed amount of reference calibration solution (4.26.3) at regular intervals (e.g. every third injection),
the aflatoxin B1 peak values between the results for the reference calibration solution can be corrected for using the
mean response, provided that the difference between responses of consecutive measurements of referencecalibration solution is very small (< 10 %). Therefore injections shall be made without interruptions whereverpossible. If interruption is necessary, the last injection before interruption and the first injection after interruptionshall be the reference calibration solution (4.26.3).
Because the calibration curve is linear and passes through the origin, the mass of aflatoxin B
1 in the sample
extracts is determined directly by reference to the adjacent results for the reference calibration solution.
8.5.2 HPLC pump settings
Set the pump (5.13.1) to give a volume flow rate of the mobile phase (4.18) of 0,5 ml/min or 0,3 ml/min for a 5 mm or
a 3 mm HPLC column (5.13.6) respectively.
If iodine derivatization is used, proceed in accordance with 8.5.3.If bromine derivatization is used, proceed in accordance with 8.5.4.
8.5.3 Post-column pump settings for HPLC with iodine derivatization
Set the pump (5.14.1) to give a volume flow rate of the saturated iodine solution (4.19) of between 0,2 ml/min and
0,4 ml/min. As a rough guide, volume flow rates of about 0,4 ml/min or 0,2 ml/min are advised in combination withvolume flow rates of the mobile phase (4.18) of 0,5 ml/min and 0,3 ml/min respectively.
8.5.4 Fluorescence detector
Set the fluorescence detector (5.13.3) to an excitation wavelength of 365 nm and an emission wavelength of
435 nm (filter instrument: > 400 nm). Adjust the detector attenuator to obtain about 80 % full-scale deflection for1 ng of aflatoxin B
1.
8.5.5 Injector
For all solutions, inject volumes of 250 ml following the instructions of the manufacturer of the injector.
8.5.6 Check of chromatographic separation
Inject the chromatographic test solution (4.27). Valleys should be less than 5 % of the sum of the peak heights of
the adjacent peaks.
8.5.7 Check of the stability of the system
Before each series of analyses, repetitively inject the reference calibration solution (4.26.3) until stable peak heights
are achieved. Peak responses for aflatoxin B1 between consecutive injections should not differ by more than 6 %.
Proceed without delay with the check of linearity (8.5.8).
8.5.8 Check of linearity
Inject the aflatoxin B1 calibration solutions (4.26.1 to 4.26.4). Every third injection use the reference calibration
solution (4.26.3) for correction of drift in response. Peak responses for this reference calibration solution shall notdiffer by more than 10 % in 90 min. Correct for drift with the formula given in 9.3.
The calibration graph shall be linear and pass through the origin within twice the standard error of the estimate of
the response. Values found shall not differ by more than 3 % from the nominal values.© ISO ISO 14718:1998(E)
11If these requirements are fulfilled, continue without delay. If not, identify and correct the sources of any problem
before continuing.
8.5.9 Injection of sample extracts
Consecutively inject the reference calibration solution (4.26.3), the blank feed extract, the spiked blank feed extract,
the reference calibration solution (4.26.3), the extract of a certified reference material or the control sample (4.4)and again the reference calibration solution (4.26.3).
Inject the purified sample extracts (8.4.2.2). After every two sample extracts, repeat the injection of the reference
calibration solution (4.26.3). When the series contains more than 10 samples, the last injections should be theaflatoxin B
1 calibration solutions (4.26.1 to 4.26.4).
9 Calculation of results
9.1 Calculation of the aflatoxin B1 content of the aflatoxin B1 standard solution
Calculate the aflatoxin B1 content of the aflatoxin B1 standard solution (4.24) using the equation:
rk =   
  MA
d¥
¥
where
ris the aflatoxin B1 content of the aflatoxin B1 standard solution (4.24), in mg/ml;
Mis the molar mass of aflatoxin B1, in g/mol ( M = 312 g/mol);
Ais the absorbance measured in 8.2, corrected for the blank;
dis the optical path length of the cuvette, in cm ( d = 1 cm);
kis the molar absorption coefficient of aflatoxin B1 in chloroform at 363 nm, in l ◊mol–1◊cm–1
(k = 22 300 l ◊mol–1◊cm–1).
9.2 Calculation of the mass of aflatoxin B1 in the injected reference calibration solution
Calculate the mass of aflatoxin B1 in the injected reference calibration solution (4.26.3) using the equation:
cmfV =     ic ¥¥r
where
mcis the mass of aflatoxin B1 in the reference calibration solution (4.26.3) injected, in ng;
fis the dilution and units correction factor, in ng/mg ( f = 200 ng/mg);
ris the aflatoxin B1 content of the aflatoxin B1 standard solution (4.24), calculated in 9.1, in mg/ml;
Vicis the volume of reference calibration solution (4.26.3) injected, in ml ( Vic = 0,25 ml).
9.3 Calculation of the mass of aflatoxin B1 in the test solution
Calculate the mass of aflatoxin B1 in the test solution using the equation:ISO 14718:1998(E) © ISO
12mAm
AAa =   2
 + sc
cc¥
12
where
mais the mass of aflatoxin B1 in the test solution, in ng;
Asis the peak area corresponding to the aflatoxin B1 in the test solution, in area units;
mcis the mass of aflatoxin B1 in the reference calibration solution (4.26.3) injected, calculated in 9.2, in ng;
Ac1is the peak area corresponding to aflatoxin B1 resulting from the preceding injection of reference
calibration solution (4.26.3), in area units;
Ac2is the peak area corresponding to aflatoxin B1 resulting from the subsequent injection of reference
calibration solution (4.26.3), in area units.
NOTE  The peak height (in length units) may be used instead of the peak area.
9.4 Calculation of the aflatoxin B1 content of the sample
Calculate the aflatoxin B1 content of the test sample by the equation:
aas c
si sfwmV V
mVV =   
    ××
××
where
wais the aflatoxin B1 content of the test sample, in µg/kg;
mais the mass of aflatoxin B1 in the test solution, in ng;
Vsis the volume of the undiluted sample extract obtained in 8.3 used in the subsequent procedure, in ml
(Vs = 50 ml if it is not necessary to dilute the filtrate obtained in 8.3);
msis the mass of the test sample, in g ( ms = 50,0 g);
Visis the volume of sample extract injected (8.5.8), in ml ( Vis = 0,25 ml);
Vfis the volume of filtrate used for the clean-up (8.4), in ml ( Vf = 50 ml);
Vcis the volume of chloroform used for extraction of the sample (see 8.3), in ml ( Vc = 250 ml).
If ms = 50,0 g, Vs = 50 ml, Vis = 0,25 ml, Vf = 50 ml and Vc = 250 ml, the equation becomes:
wa = 20 g–1 × ma
10 Accuracy
10.1 Systematic error
Verify the correct application of the method by making replicate measurements on certified reference materials or
control samples. If these are not available, verify the performance of the method by recovery experiments made onfortified blank samples. The deviation of the mean from the actual value, expressed as a percentage of the actualvalue, shall not lie outside the limits of –20 % to +10 %.© ISO ISO 14718:1998(E)
1310.2 Precision
10.2.1 Interlaboratory test
Details of an interlaboratory test on the precision of the method are given in annex A. The values derived from this
test may not be applicable to concentration ranges and matrices other than those given.
Annex B gives a summary of statistical results of interlaboratory tests of the equivalence of iodine and bromine
derivatization.
10.2.2 Repeatability
The absolute difference between two independent single test results, obtained using the same method on identical
test material in the same laboratory by the same operator using the same equipment within a short interval of time,
will in not more than 5 % of cases exceed the repeatability limit r derived from the equation:
r w= 0,37 g/kg+0,19 m a (wa < 15 mg/kg)
where
wais the mean of the two test results, in mg/kg;
ris the repeatability limit, in mg/kg.
10.2.3 Reproducibility
The absolute difference between two single test results, obtained using the same method on identical test material
in different laboratories by different operators using different equipment, will in not more than 5 % of cases exceed
the reproducibility limit R derived from the equation:
Rw = 0,67 g/kg+0,33 m a (wa < 15 mg/kg)
where
wais the mean of the two test results, in mg/kg;
Ris the reproducibility limit, in mg/kg.
11 Test report
The test report shall specify:
 all information necessary for the complete identification of the sample;
 the sampling method used, if known;
 the test method used, with reference to this International Standard;
 all operating details not specified in this International Standard, or regarded as optional, together with details of
any incidents which may have influenced the test result(s);
 the test result obtained; or
 if the repeatability has been checked, the final quoted result obtained.ISO 14718:1998(E) © ISO
14Annex A
(informative)
Results of interlaboratory test
The precision of the method was established by an interlaboratory test carried out in accordance with
ISO 5725-2:1994 [6]. In this test, 22 laboratories from 11 European countries participated. The study involved sixunknown samples, consisting of blind duplicate samples of compounded feeding stuffs with target aflatoxin B
1
contents of < 2 mg/kg, 8 mg/kg and 14 mg/kg.
Table A.1 — Statistical results of the interlaboratory test of the method applying derivatization with iodine
Parameter Sample a
123
Number of laboratories retained after eliminating outliers 15 15 15
Mean aflatoxin B1 content, mg/kg 0,8 7,0 12,6
Repeatability standard deviation ( sr), mg/kg 0,20 0,44 1,20
Repeatability coefficient of variation, % 25,0 6,3 9,5
Repeatability limit, r (r = 2,8 ¥ sr), mg/kg 0,55 1,24 3,37
Reproducibility standard deviation, sR, mg/kg 0,34 1,02 1,80
Reproducibility coefficient of variation, % 42,5 14,6 14,3
Reproducibility limit, R (R = 2,8 ¥ sR), mg/kg 0,95 2,86 5,05
a) Sample 1:  blank; target aflatoxin B1 content < 2 mg/kg; composition: 10 % barley, 20 % manioc, 15 % citrus
pulp, 20 % "Hi-pro" soya, 10 % liquid molasses, 20 % pelleted cattle feed, 1,7 % fat, 1,5 % dicalciumphosphate and 0,7 % salt.
Sample 2:  target aflatoxin B
1 content of 8 mg/kg; other components as sample 1.
Sample 3:   target aflatoxin B1 content of 14 mg/kg; other components as sample 1.
NOTE  In the report on the interlaboratory test (see reference [3]), values of r and R are mentioned (see Table A.2) which
deviate from those in Table A.1. The differences result from calculations having been performed on the natural logarithms ofthe measured results and from the application of deviating definitions of r and R. In Table A.2 r is the largest ratio of two
readings of the same sample in the same laboratory under similar conditions which is not yet significant at the 95 % probabilit y
level. R is similarly defined for comparing two different laboratories, always using single determinations.
Table A.2 — Summary of the published statistical results of the interlaboratory test
Aflatoxin B1 content
mg/kgr
mg/kgR
mg/kgRepeatability
coefficient of
variation
%Reproducibility
coefficient of
variation
%
8 and 14 1,4 1,7 11 18© ISO ISO 14718:1998(E)
15Annex B
(informative)
Equivalence of iodine and bromine derivatization
The equivalence of the iodine and bromine derivatization can be deduced from published data (see references [3]
and [4]). Furthermore, equivalence is apparent from data collected by KDLL (Kwaliteitsdienst LandbouwkundigeLaboratoria: Quality Service for Agricultural laboratories, The Netherlands) in the period between October 1992 andJuly 1994. In this period a number of interlaboratory tests were performed in which both derivatization methodswere tested for a number of different compound and straight feeding stuffs with aflatoxin B
1 contents between
2 mg/kg and 90 mg/kg. The results of these interlaboratory tests are included in Table B.1.
Table B.1 — Statistical results of interlaboratory tests of the equivalence of iodine
and bromine derivatization
Sample Iodine derivatization aBromine derivatization aSignificance b
wa ns wa ns
Cattle feed (pelleted)  2,1
 2,2 2,2 2,3 4
 4 6 6 0,4
 0,3 0,5 0,5 2,1
 2,2 1,9 2,0 8
 8
1111 0,3
 0,3 0,3 0,4n.s.
n.s.n.s.n.s.
 4,5 4,7 5,0 5,1 3
 3 6 6 0,2
 0,2 0,8 0,7 4,8
 4,7 5,4 5,4 8
 8
1111 0,4
 0,5 0,9 0,8n.s.
n.s.n.s.n.s.
 8,3 9,6
10,812,1 5
 5 6 6 1,6
 1,1 1,6 1,4 9,0
10,1
 9,5
11,810
10
 9 9 1,0
 1,7 1,6 1,8n.s.
n.s.n.s.n.s.
Corn gluten 16,3
17,6 6
 6 2,3
 2,416,4
18,911
11 2,0
 1,5n.s.
n.s.
Copra 19,7
20,227,929,0 6
 6 4 4 4,4
 3,1 3,4 3,117,7
18,629,333,2 8
 8 8 8 2,7
 2,3 1,6 1,7n.s.
n.s.n.s.
p < -0,01
Palm kernel and
groundnut38,1
53,7 6
 6 7,4
10,735,3
48,711
11 5,3
 7,3n.s.
n.s.
Groundnut 82,0
83,0 4
 4 4,1
 9,984,4
86,0 7
 7 7,1
10,7n.s.
n.s.
Groundnut and copra 80,0
85,5 4
 4 5,5
 9,286,1
94,1 9
 9 7,3
 9,3n.s.
n.s.
a) wa    is the mean aflatoxin B1 content, in mg/kg;
    n       is the number of results;
    s       is the standard deviation.
b) n.s. means not significant in Student's  t-test, iodine tested against bromine.ISO 14718:1998(E) © ISO
16Bibliography
[1] Traag W.A., van Trijp J.M.P., Tuinstra L.G.M.Th. and Kok W.Th.  Sample clean-up and post-column
derivatization for the determination of Aflatoxin B1 in feedstuffs by liquid chromatography.  J. Chrom ., 396,
pp. 389 - 394 (1987).
[2] RIKILT-DLO, SOP A0446, State Institute for the Control of Agricultural Products, Agricultural Research
Department (DLO), Grondstoffen voor veevoeder. Screening op het gehalte aan aflatoxine B1 — High
Performance Liquid Chromatography — Post-column derivatisering — Fluorescentiedetectie (Screening of
straight feedingstuffs on aflatoxin B1 by HPLC and fluorescence detection).
[3] van Egmond H.P., Heisterkamp S.H. and Paulsch W.E. EC-collaborative study on the determination of
aflatoxin B1 in animal feeding stuffs. Food Additives and Contaminants , 8, pp.17 - 29 (1991).
[4] van Egmond H.P., Patel S., Paulsch W.E., Sizoo E.A. Tuinstra L.G.M.Th., Wood G., Boenke A., Schurer
B. and Wagstaffe P.J.  The development of five animal feed reference materials, certified for theiraflatoxin B
1 content. Food Additives and Contaminants,  11, pp. 449 - 477 (1994).
[5] ISO 5725-1:1994, Accuracy (trueness and precision) of measurement methods and results — Part 1:
General principles and definitions.
[6] ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results — Part 2:
Basic method for the determination of repeatability and reproducibility of a standard measurement
method.
[7] ISO 6497,  Animal feeding stuffs — Sampling.
[8] ISO 6651,  Animal feeding stuffs — Determination of aflatoxin B1 content (TLC methods).ISO 14718:1998(E) © ISO
ICS  65.120
Descriptors:  agricultural products,  food,  animal feeding products,  mixtures,  chemical analysis,  determination of content,  aflatoxins,   high
performance liquid chromatography.
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