UGANDA STANDARD  
      US ISO 17375
First Edition
   2009 -09-04
 
Reference numbe r 
   US ISO 17375:2006
 © UNBS 200 9 
  
Animal feeding stuffs — Determination of aflatoxin B 1
 US ISO 17375:2006  
 
© 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 17375: 2006  
© 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 17375:2006,  Animal feeding stuffs — Determination of aflatoxin B1, is identical 
B1, is identical with and  has been reproduced from an International Standard ISO 17375:2006,  Animal feeding 
stuffs — Determination of aflatoxin B 1, and has been adopted as a Uganda Standard . 
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard."   
 
  
 Reference number
ISO 17375:2006(E)
© ISO 2006
  
 
 INTERNATIONAL 
STANDARD  ISO
17375
First edition
2006-06-01
Animal feeding stuffs — Determination 
of aflatoxin B 1 
Aliments des animaux — Dosage de l'aflatoxine B1 
 ISO 17375:2006(E) 
PDF disclaimer 
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed b ut 
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editin g. In 
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Se cretariat 
accepts no liability in this area. 
Adobe is a trademark of Adobe Systems Incorporated. 
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-cr eation 
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bod ies. In 
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below. 
 
©   ISO 2006 
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 either ISO at the address be low or 
ISO's member body in the country of the requester. 
ISO copyright office 
Case postale 56 • CH-1211 Geneva 20 
Tel.  + 41 22 749 01 11 
Fax  + 41 22 749 09 47 
E-mail  copyright@iso.org Web  www.iso.org 
Published in Switzerland 
 
ii © ISO 2006 – All rights reserved
  ISO 17375:2006(E) 
© ISO 2006 – All rights reserved  iiiContents Page 
Foreword....................................................................................................................... ..................................... iv  
1 Scope ........................................................................................................................ ............................. 1  
2 Normative references ......................................................................................................... .................. 1  
3 Principle.................................................................................................................... ............................. 1  
4 Reagents..................................................................................................................... ........................... 1  
5 Apparatus .................................................................................................................... .......................... 4  
6 Procedures ................................................................................................................... ......................... 6  
6.1 Conditioni ng of IACs ....................................................................................................... ..................... 6  
6.2 Extraction ................................................................................................................. ............................. 6  
6.3 Immunoaffini ty clean up .................................................................................................... .................. 6  
6.4 Post-column derivatization................................................................................................. ................. 7  
6.5 Calibrati on curve.......................................................................................................... ......................... 7  
6.6 Calculation................................................................................................................ ............................. 7  
6.7 Spiking procedures for recovery determination .............................................................................. .8 
7 Precision.................................................................................................................... ............................ 8  
7.1 Interlabor atory test ....................................................................................................... ........................ 8  
7.2 Repeatability.............................................................................................................. ............................ 8  
7.3 Reproduc ibility............................................................................................................ .......................... 9  
8 Test report .................................................................................................................. ........................... 9  
Annex A  (informative)  Results of an inte rlaboratory test............................................................................. 10  
Bibliography ................................................................................................................... .................................. 11  
 ISO 17375:2006(E) 
iv © ISO 2006 – All rights reserved
 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 . 
 INTERNATIONAL STANDARD ISO 17375:2006(E)
 
© ISO 2006 – All rights reserved  1Animal feeding stuffs — Determination of aflatoxin B 1 
1 Scope 
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 µg/kg for aflatoxin B 1 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
1. The aflatoxin B1 is removed from 
the IAC with neat methanol, and then quantified by reverse-phase high-performance liquid chromatography (RP-HPLC) with post-column derivatization (PCD) involv ing 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. 
NOTE Decontamination procedures for laboratory wastes [2],[3] 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 17375:2006(E) 
2 © ISO 2006 – All rights reserved
 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
3) = 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 B1. The IAC should have a capacity of not less 
than 40 ng of aflatoxin B1 and should give a recovery of not less than 80 % for aflatoxin B 1 when applied as a 
standard solution in acetone/water containing 0,25 ng of aflatoxin B 1. 
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). ISO 17375:2006(E) 
© ISO 2006 – All rights reserved  34.16 Aflatoxin B1 standard material , in form of crystals or a dry film for analytical purposes. 
WARNING 1 — This method requires the use of solutions of aflatoxin B1. Aflatoxins are carcinogenic 
to humans. Attention is drawn to the statement made by the International Agency for Research on 
Cancer (WHO) [2]. 
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
1 (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
a, in micrograms per millilitre, using 
Equation (1): 
a
am a x
a100 McAdε×=×× (1) 
where  
Amax is the absorbance determined at the maximum of the absorption curve;  
Ma is the molar mass of aflatoxin B1, in grams per mole (312 g/mol);  
εa is the molar absorptivity of aflatoxin B1, in square metres per mole (1930 m2/mol for 
toluene/acetonitrile and 2150 m2/mol for methanol solutions); 
d is the optical path length of the cell, in centimetres. 
4.17.2 Calibration stock solution  
Prepare an aflatoxin B1 (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 17375:2006(E) 
4 © ISO 2006 – All rights reserved
 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 
least 5 different 20 ml volumetric flasks. Evaporate the toluene/acetonitrile solution just to dryness under a stream of nitrogen at room temperature. If methanol is used in preparation of the stock solution, evaporation is not required. To each flask, add approximately 10 ml of methanol. Allow the aflatoxins to dissolve, then dilute further with neat methanol (not with methanol/water) to the mark and shake well. Then transfer exactly 1 ml of 
this calibration working solution to an acid-washed glass vial (see Warning in 5.7), evaporated to dryness 
according to Option B (6.3.3) and than redissolved in exactly the same volume that will be used to redissolve the samples prior to injection (6.3). Calculate the concentration of aflatoxin 
B1 in the evaporated and 
redissolved solution in nanograms per millilitre. Use these concentration values for the calculation according 
to 6.6. In this case the calibration range will remain unchanged. 
Table 1 — Preparation of calibration working solutions 
 Option A Option B 
Working 
standard Calibration stock 
solution Concentration 
of aflatoxin B1 Calibration stock 
solution  Concentration 
of aflatoxin B1 
 µl ng/ml µl ng/ml 
1 20 0,050 100 0,250 
2 70 0,175 350 0,875 
3 120 0,300 600 1,500 
4 170 0,425 850 2,125 
5 220 0,550 1100 2,750 
5 Apparatus 
Usual laboratory apparatus and, in particular, the following. 
5.1 Vertical or horizontal shaker , adjustable . 
5.2 Filter paper , of diameter 24 cm, prefolded (e.g. cellulose for fine precipitates). 
5.3 Erlenmeyer flask , with screw top or glass stopper. 
5.4 Glass microfibre filter paper , of diameter 5 cm (e.g. 1,6 µm retention). 
5.5 Reservoir , 75 ml with Luer tip connector for IAC. 
5.6 Hand pump , 20 ml syringe with Luer lock or rubber stopper for IAC. 
5.7 Volumetric flasks , of 5 ml, 10 ml and 20 ml capacity, with an accuracy of at least 0,5 %. 
WARNING — The use of non acid-washed glassware (e.g . vials, tubes, flasks) for aflatoxin aqueous 
solutions may cause a loss of aflatoxin. Special at tention should be taken with new glassware. Thus, 
before use, soak the glassware in dilute acid (e.g. su lfuric acid, 2 mol/l) for several hours, then rinse 
extensively with distilled water to remove all traces of acid (this can be checked by using a pH paper). 
5.8 HPLC pump , suitable for flow rate at (1,000 ± 0,005) ml/min. ISO 17375:2006(E) 
© ISO 2006 – All rights reserved  55.9 Injection system . 
Suitable for total loop injection (a valve with a loop of at least 100 µl is recommended). It shall be guaranteed 
that the relative standard deviation (RSD) of the integrator signal for a multiple injection ( n = 10) of a standard 
solution of aflatoxin B1 (concentration equivalent to a contamination level of 1 µg/kg) results in a maximum 
value of 10 %. These data shall be reported. 
5.10 RP-HPLC column , e.g. LC-18 or ODS-2, with optional but recommended pre-column. 
5.11 Post-column derivatization system with PBPB  (alternative to 5.12), comprising. 
⎯ second HPLC pulse-less pump, 
⎯ zero-dead volume T-piece, and 
⎯ reaction tubing with minimum internal diameter of 45 cm × 0,5 mm, of PTFE. 
The reaction time shall be at least 4 s before detection. 
5.12 System for HPLC post-column derivatization with electrochemically generated bromine . 
The device shall be installed according to the manufacturer's instructions. In order to confirm the aflatoxin B1 
content, the HPLC column shall be disconnected from the bromination device and shall be connected directly 
to the fluorescence detector. 
Switching-off the electrical current with the bromination device still in line is not recommended due to the 
possibility of bromine remaining in the cell membrane of the device. 
5.13 Fluorescence detector , with a 360 nm excitation filter and a > 420 nm cut-off emission filter, or 
equivalent. 
Recommended settings for adjustable detectors are Ex = 365 nm, Em = 435 nm, BW = 18 nm. 
5.14 Disposable filter unit (0,45 µm), prior to usage verify that no aflatoxin losses occur during filtration 
(recovery testing) since there is a possibility that various filter materials can retain aflatoxin B1. 
5.15 One-mark pipettes , of 1 ml, 2 ml, 5 ml and 10 ml capacity. 
5.16 Analytical balance , capable of weighing to the nearest 0,1 mg. 
5.17 Laboratory balance , capable of weighing to the nearest 0,01 g. 
5.18 Calibrated microlitre syringe(s) or microlitre pipette(s) , 20 µl to 500 µl. 
5.19 Evaporator , optional, only needed for Option B (6.3.3). 
5.20 Spectrophotometer . 
5.21 Quartz glass cell , 1 cm optical path length. ISO 17375:2006(E) 
6 © ISO 2006 – All rights reserved
 6 Procedures 
6.1 Conditioning of IACs 
IACs (4.11) should be at room temperature prior to conditioning. For conditioning, follow the manufacturer's 
instructions. If not stated, apply 10 ml of PBS (4.2) on the top of the IAC and let it pass at a speed of 2 ml/min to 3 ml/min through the IAC (by gravity). Make sure that a small portion (0,5 ml) of the PBS remains on the IAC until the sample solution is applied. 
6.2 Extraction 
Weigh, to the nearest 0,1 g, approximately 50 g of the test portion into a 500 ml Erlenmeyer flask with screw 
top or glass stopper. Add 250 ml of acetone/water extraction solvent (4.9). Shake intensively by hand for the 
first 15 s to 30 s and then for 30 min with a shaker (5.1). Filter the extract using prefolded paper (5.2). Pipette (5.15) 5,0 ml of the clear filtrate into a 100 ml volumetric flask (5.7) and dilute to the mark with PBS or water. 
The dilution solvent (PBS or water) shall be selected according the specifications of the IAC manufacturer. If 
not stated, the dilution shall be made with PBS. If the solution is not clear, refilter through a glass fibre filter (5.4) and apply exactly 50 ml of the clear filtrate in a reservoir that is placed on a conditioned IAC. (If the 
solution is clear, the diluted solution may be directly applied onto the IAC.) Apply the solution onto the IAC as 
described in 6.3.1. 
6.3 Immunoaffinity clean up 
6.3.1 General 
Methods for conditioning, loading, washing and eluting vary slightly between IAC manufacturers and the 
specific instructions supplied with the IACs should be followed precisely. In general, procedures involving sample extraction with methanol/water, filtration or centrifugation, possible sample dilution with PBS or water, loading under pressure onto (possibly prewashed) IAC, washing of IAC with distilled water and elution of 
aflatoxin B
1 with methanol or acetonitrile. 
Pass the filtrate through the IAC at a flow rate of approximately 1 drop per second (approx. 3 ml/min) (gravity). 
Do not exceed a flow rate of 5 ml/min. Wash the IAC with approx. 20 ml of water (4.8), applied in two portions of approx. 10 ml at a flow rate of 3 ml/min. Dry by applying a light vacuum for 5 s to 10 s or passing air through the IAC by means of a syringe for 10 s.  
Elute the aflatoxin B
1 in a two-step procedure. 
a) Apply 0,50 ml of methanol on the IAC and allow it to pass through by gravity. Collect the eluate in a 
volumetric flask of 5 ml (5.7). 
b) Wait for 1 min and apply a second portion of 1,25 ml of methanol. Collect most of the applied elution 
solvent by pressing air through, after most has passed through by gravity. 
6.3.2 Option A (recommended) 
NOTE  This option is recommended, but requires an appropriate fluorescence detector and injection system (see also 
5.9). Option B only applies if the detector signal is insufficient for the analysis according to Option A. 
Collect the eluate in a 5 ml volumetric flask (5.7). Fill the flask to the mark with water and shake well (final 
volume according to 6.6). If the solution is clear, it may be used directly for HPLC analysis. If the solution is 
not clear, pass it through a disposal filter unit (0,45 µm) (5.14) prior HPLC injection. The injection by total loop mode guarantees maximum accuracy. It is recommended, depending on the injection system (e.g. syringe or 
autosampler), to take a sample volume of three times the injection loop size and to inject at least two-thirds of 
this volume into the valve, to ensure that the middle fraction remains in the injection loop. Thus, the loop is rinsed with the injection solvent while enough solvent remains in the valve. ISO 17375:2006(E) 
© ISO 2006 – All rights reserved  76.3.3 Option B (only if applicable) 
If the detector signal is not sufficient to guarantee the required relative standard deviation (see 5.9), an 
additional evaporation step may be included to meet the required relative standard deviation. 
Collect the aflatoxin-containing methanol eluate from the IAC in an acid-washed glass vial (see Warning in 
5.7). Evaporate the eluate to dryness under a gentle stream of nitrogen at 40 °C ± 5 °C. Redissolve the 
aflatoxin in an aqueous methanol solution (35 % MeOH). Use exactly the same volume for the evaporated 
sample residues as that used for the evaporated calibration solutions (4.17). The volume for redissolving (final 
volume according to 6.6) will depend on the size of the injection loop. Use the total loop mode for injection as 
described in Option A. 
6.4 Post-column derivatization 
When  using PBPB, mount the mixing T-piece and reaction tubing (see 5.11), and then operate using the 
following parameters: 
a) flow rates: 
⎯ 1,00 ml/min (mobile phase 4.12); 
⎯ 0,30 ml/min (reagent 4.14). 
When using electrochemically generated bromine, follow the manufacturer's instructions for the installation of 
the cell and operate using the following parameters: 
a) flow rate: 
⎯ 1,00 ml/min (mobile phase 4.13); 
b) current: 
⎯ 100 µA. 
6.5 Calibration curve   
Linear regression should be performed using a scientific calculator or statistical program.  
A calibration curve should be prepared using the calibration working solutions described in 4.17. These 
solutions cover the range of 0,5 µg/kg to 5,5 µg/kg for aflatoxin B1. Make the calibration curve prior to analysis 
according to Table 1 and check the plot for linearity. If the content of aflatoxin B 1 in the sample is outside the 
calibration range, an appropriate calibration curve shall be prepared. Alternatively, the injection solution for HPLC analysis may be diluted to an aflatoxin B
1 content appropriate for the established calibration curve. 
6.6 Calculation 
Plot the signals as x-axis (height or area) against the concentration of aflatoxin B1 (ng/ml) as the y-axis. 
Determine the calibration curve from these data using linear regression. Use the resulting function to calculate the concentration of aflatoxin B
1 in the measured solution (ng/ml) from the test samples: 
ya x b=+  
Calculate the aflatoxin concentration of the injected solutions from the calibration curve (function) obtained by linear regression: 
aAbρ=×+  ISO 17375:2006(E) 
8 © ISO 2006 – All rights reserved
 SED
c
ext cleanVVVwmV Vρ×××=×× 
E
c100 Vwmρ××=  
where 
m is the mass of sample material taken for analysis, in grams (50 g); 
VS is the volume of solvent taken for extraction, in millilitres (250 ml); 
Vext is the volume of aliquot taken from the extract, in millilitres (5 ml); 
VD is the volume achieved after dilution with PBS (water), in millilitres (100 ml); 
Vclean is the volume of aliquot taken for the immunoaffinity clean-up, in millilitres (50 ml); 
VE is the final volume achieved after elution from the IAC, in millilitres; 
ρ is the concentration of aflatoxin calculated from linear regression, in nanograms per millilitre; 
wc is the contamination of sample material with aflatoxin B1, in micrograms per kilogram; 
A is the area or height of the aflatoxin peak obtained from the measured solution (units). 
It is stressed that for sample and standard solutions, the same volume shall be injected to comply with the 
formula. 
6.7 Spiking procedures for recovery determination 
For determination of the recovery, the spiking procedure shall be carried out using a methanol stock solution 
of aflatoxin B1. The spiking level shall be within the calibration range (preferably the mean value). Take care 
that not more than 2 ml of the spiking solvent are added (solution must have an adequate concentration of aflatoxin B
1) and that the subsequent evaporation takes place in the dark and should last 0,5 h to 2 h. 
7 Precision 
7.1 Interlaboratory test 
Details of an interlaboratory study test on the precision of the method are summarized in Annex A. The values derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than 
those given. 
7.2 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: 
⎯ x= 1,33 µg/kg r = 0,22 µg/kg  (fortified); 
⎯ x= 3,89 µg/kg r = 0,69 µg/kg  (fortified); 
⎯ x= 0,54 µg/kg r = 0,11 µg/kg  (naturally contaminated); ISO 17375:2006(E) 
© ISO 2006 – All rights reserved  9⎯ x= 0,87 µg/kg r = 0,21 µg/kg  (naturally contaminated); 
⎯ x= 4,19 µg/kg r = 0,72 µg/kg (naturally contaminated). 
7.3 Reproducibility 
The absolute difference between two single test results, obtained using the same method on identical test 
material in different laboratories with different operators using different equipment, will in not more than 5 % of 
cases exceed the reproducibility limit R: 
⎯ x= 1,33 µg/kg R = 0,72 µg/kg  (fortified); 
⎯ x= 3,89 µg/kg R = 1,87 µg/kg  (fortified); 
⎯ x= 0,54 µg/kg R = 0,27 µg/kg  (naturally contaminated); 
⎯ x= 0,87 µg/kg R = 0,47 µg/kg  (naturally contaminated); 
⎯ x= 4,19 µg/kg R = 2,30 µg/kg (naturally contaminated). 
8 Test report 
The test report shall contain at least the following data: 
a) all information necessary for identification of the sample; 
b) all information necessary for identification of the calibrant; 
c) the test results and the units in which the results have been expressed; d) date of test; 
e) any particular points observed in the course of the test; f) any operation not specified in this method or regarded as optional which might have affected the results.  ISO 17375:2006(E) 
10 © ISO 2006 – All rights reserved
 Annex A  
(informative) 
 
Results of an interlaboratory test 
An international collaborative test involving 21 laboratories in 14  countries was carried out on 5 cattle feed 
samples composed out of the following components in descending quantities: corn gluten, soy bean peels, 
grain (wheat, barley, rye), extracted sugar cane and beet, citrus pulp, extracted sunflower, soy, corn, grass, 
molasses, calcium chloride, sodium chloride, vitamin premix and trace element premix. The blank material was prepared by mixing the above feed ingredients (without wheat and corn) with wheat and corn containing aflatoxin B
1 at level of approximately 0,15 µg/kg. 
The test was organized by Joint Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium, in 1999 (see Reference [5]) and the results obtained were subjected to statistical analysis in 
accordance with the IUPAC harmonized protocol. 
Table A.1 — Precision data 
Blank Spiked with 
aflatoxin B1 Naturally contaminated 
with aflatoxin B1 
µg/kg  µg/kg    
1,2a 3,6a 0,5b 1,0b 5b 
Number of laboratories participating 21 21 21 21 21 21 
Number remaining after eliminating outliers 0 1 1 3 2 3 
Number of test results from remaining laboratories 21 20 20 18 19 18 
Mean value, ,x µg/kg < 0,02 1,33 3,89 0,54 0,87 4,19 
Repeatability standard deviation, sr, µg/kg — 0,08 0,25 0,04 0,08 0,26 
Repeatability relative standard deviation, % — 5,9 6,4 7,2 8,7 6,2 
Repeatability limit, r (= 2,8 sr), µg/kg — 0,22 0,69 0,11 0,21 0,72 
Reproducibility standard deviation, sR, µg/kg — 0,26 0,67 0,10 0,17 0,82 
Reproducibility relative standard deviation, % — 19,4 17,5 17,9 19,4 19,6 
Reproducibility limit, R (= 2,8 sR), µg/kg — 0,72 1,87 0,27 0,47 2,30 
Horrat valuec   — 0,45 0,47 0,36 0,42 0,54 
Recovery — 110,8 108,1 n.a. n.a. n.a. 
a These levels were obtained by fortification of blank material with unknown (coded) standard solutions. 
b These levels are target levels (best estimates) during the production of the test materials for the trial. 
c Since these values are below 1,5, there is no real effect on the applicability or validity of the method. 
 ISO 17375:2006(E) 
© ISO 2006 – All rights reserved  11Bibliography 
[1] W ERNER , G., Bestimmung der Aflatoxine in Futtermitteln nach selektiver Reinigung an einer 
Immunoaffinitaetssaeule. Agribiological Research , 44 (4), 1991, pp. 289-298 
[2] Laboratory decontamination and destruction of aflatoxins B1, B2, G1 and G2 in laboratory wastes. 
CASTEGNARO , M., H UNT, D.C., S ANSONE , E.B., S CHULLER , P.L. S IRIWARDANA , M.G., T ELLING , G.M., 
van E GMOND , H.P. and W ALKER , E.A. IARC Scientific publication No. 37 , International Agency for 
Research on Cancer (WHO), Lyon (France), 1980, p. 59. 
[3] Laboratory decontamination and destruction of carcinogens in laboratory-wastes: some mycotoxins. 
CASTEGNARO , M., B AREK , J., F REMY , J.M., L AFONTAINE , M. M IRAGLIA , M., S ANSONE , E.B. and 
TELLING , G.M. IARC Scientific publication No. 113 , International Agency for Research on Cancer 
(WHO), Lyon (France), 1991, p. 63 
[4] Methodenbuch Band III , Erg. 1997, Sec. 16.1.4. VDLUFA-Verlag Darmstadt, Germany 
[5] S TROKA , J., R EUTTER , M., VON HOLST , C. and A NKLAM , E. Immunoaffinity column clean-up with liquid 
chromatography using post-column bromination for the determination of aflatoxin B1 in cattle feed. 
JAOAC , 86 (6), 2003, pp. 1179-1186 
[6] N ESHEIM , S., T RUCKSESS , M.W. and P AGE, S.W.  Molar Absorptivities of Aflatoxins B1, B2, G1 and G2 in 
Acetonitrile, Methanol and Toluene-Acetonitrile (9+1) (Modification of AOAC Official Method 971.22): 
Collaborative Study. JAOAC , 82 (2), 1991, pp. 251-258 ISO 17375:2006(E) 
ICS  65.120 
Price based on 11 pages 
© ISO 2006 – All rights reserved