METHODS OF ANALYSIS FOR THE OFFICIAL CONTROL OF FEEDINGSTUFFS RULES *

METHODS OF ANALYSIS FOR THE

OFFICIAL CONTROL OF FEEDINGSTUFFS

SUBSIDIARY LEGISLATION 437.77

METHODS OF ANALYSIS FOR THE OFFICIAL

CONTROL OF FEEDINGSTUFFS RULES *

12th August, 2005

LEGAL NOTICE 285 of 2005, as amended by Legal Notice 62 of 2006.

(1) The title of these rules is the Methods of Analysis for

the Official Control of Feedingstuffs Rules.

(2) The scope of these rules is to implement the rules found

under European Union Council Directive 76/372/EEC establishing

methods of analysis for the official control of feedingstuffs.

Amended by

L.N. 62 of 2006.

DETERMINATION OF AFLATOXIN B 1

A.

ONE-DIMENSIONAL THIN LAYER CHROMATOGRAPHIC METHOD

1. Purpose and scope

The method makes it possible to determine the level of aflatoxin B 1 in raw

materials and straight feedingstuffs. This method can not be applied in the presence

of citrus pulp. The lower limit of determination is 0,01 mg/kg (10 ppb).

In the presence of interfering substances it is necessary to repeat the analysis using

method B (high performance liquid chromatography).

2. Principle

The sample is subjected to extraction with chloroform. The extract is filtered, and

an aliquot portion taken and purified by column chromatography on silica gel. The

eluate is evaporated and the residue redissolved in a specific volume of chloroform

or of a mixture of benzene and acetonitrile. An aliquot portion of this solution is

subjected to thin-layer chromatography (TLC). The quantity of aflatoxin B 1 is

determined under UV irradiation of the chromatogram, either visually or by

fluorodensitometry, by comparison with known quantities of standard aflatoxin B 1 .

The identity of the aflatoxin B 1 extracted from the feedingstuff must be confirmed

by the procedure indicated.

3. Reagents

NB: All the reagents must be of ‘analytical reagent’ quality unless otherwise

stated.

3.1.

Acetone.

3.2.

Chloroform, stabilized with 0ꞏ5 to 1ꞏ0% of 96% ethanol (v/v).

3.3.

N-hexane.

3.4.

Methanol.

3.5.

Anhydrous diethyl ether, free from peroxides.

3.6.

Mixture of benzene and acetonitrile: 98/2 (v/v).

3.7.

Mixture of chloroform (3.2) and methanol (3.4): 97/3 (v/v).

3.8.

Silica gel, for column chromatography, particle size 0ꞏ05 to 0ꞏ20 mm.

3.9.

Absorbent cotton wool, previously defatted with chloroform, or glass

wool.

3.10.

Sodium sulphate, anhydrous, granular.

3.11.

Inert gas, e.g. nitrogen.

3.12.

1 N Hydrochloric acid.

3.13.

50% (v/v) sulphuric acid.

3.14.

Kieselguhr (hyflosupercel), washed in acid.

3.15.

Silica gel G-HR or equivalent, for TLC.

3.16.

Standard solution with about 0ꞏ1 µg of aflatoxin B 1 per ml in chloroform

(3.2) or the benzene/acetonitrile mixture (3.6), prepared and checked as indicated in

Section 7.

Standard solution for qualitative testing purposes containing about 0ꞏ1

µg of aflatoxin B 1 and B 2 per ml in chloroform (3.2) or the benzene/acetonitrile

mixture (3.6). These concentrations are given as a guide. They must be adjusted so

as to obtain the same intensity of fluorescence for both aflatoxins.

Developing solvents

Chloroform (3.2)/acetone (3.1): 9/1 (v/v), unsaturated tank;

Diethyl ether (3.5)/methanol (3.4)/water: 96/3/1 (v/v/v), unsaturated

Diethyl ether (3.5)/methanol (3.4)/water: 94/4ꞏ5/1ꞏ5 (v/v/v), saturated

Chloroform (3.2)/methanol (3.4): 94/6 (v/v), saturated tank;

Chloroform (3.2)/methanol (3.4): 97/3 (v/v), saturated tank.

For the purposes of these rules -

"the competent authority" means the Veterinary

Services within Malta as provided under article 2 of

the Veterinary Services Act, or any other authority to

which such responsibility has been delegated by the

Veterinary Services.

The competent authority shall require that analyses for

official controls of feedingstuffs, as regards their content of

aflatoxin B 1 , be carried out in accordance with the methods

described in the Schedule.

*These rules have been repealed by Legal Notice 37 of 2025.

Apparatus

Grinder-mixer.

Shaking apparatus or magnetic stirrer.

Fluted filter papers, Schleicher and Schüll No 588 or equivalent,

diameter: 24 cm

Glass tube for chromatography (internal diameter: 22 mm, length: 300

mm), with a PTFE cock and a 250-ml reservoir.

Rotary vacuum evaporator, with a 500-ml round-bottom flask.

500-ml conical flasks, with ground-glass stoppers.

TLC apparatus.

Glass plates for TLC, 200 × 200 mm, prepared as follows (the quantities

indicated are sufficient to cover five plates). Put 30 g of silica gel G-HR (3.15) into a

conical flask. Add 60 ml water, stopper and shake for a minute. Spread the

suspension on the plates so as to obtain a uniform layer 0ꞏ25 mm thick. Leave to dry

in the air and then store in a desiccator containing silica gel. At the time of use,

activate the plates by keeping them in the oven at 110ºC for one hour.

Ready-to-use plates are suitable if they give results similar to those obtained with

the plates prepared as indicated above.

Long-wavelength (360 nm) UV lamp. The intensity of irradiation must

make it possible for a spot of 1 ng of aflatoxin B 1 to be still clearly distinguished on

a TLC plate at a distance of 10 cm from the lamp.

10 ml graduated tubes with polyethylene stoppers.

UV spectrophotometer.

Fluorodensitometer (optional).

Procedure

Preparation of the sample (see under ‘Observations’, Part C, point 1).

Grind the sample so that the whole of it will pass through a sieve with a 1-mm

mesh (in accordance with recommendation ISO R 565).

Extraction

Put 50 g of ground, homogenized sample into a 500-ml conical flask (4.6). Add 25

g of Kieselguhr (3.14), 25 ml of water and 250 ml of chloroform (3.2). Stopper the

flask, shake or stir for 30 minutes with the apparatus (4.2) and filter through a fluted

filter paper (4.3). Discard the first 10 ml of the filtrate and then collect 50 ml.

5.3.

Column clean-up

Insert into the lower end of a chromatography tube (4.4) a cotton or glass wool

plug (3.9), fill two-thirds of the tube with chloroform (3.2) and add 5 g of sodium

sulphate (3.10).

Check that the upper surface of the sodium sulphate layer is flat, thenadd 10 g of

silica gel (3.8) in small portions. Stir carefully after each addition to eliminate air

bubbles. Leave to stand for 15 minutes and then carefully add 15 g of sodium

sulphate (3.10). Let the liquid fall until it is just above the upper surface of the

sodium sulphate layer.

Mix the 50 ml of extract collected in 5.2 with 100 ml of n-hexane (3.3) and

quantitatively transfer the mixture to the column. Let the liquid fall until it is just

above the upper surface of the sodium sulphate layer. Discard this washing. Then

add 100 ml of diethylether (3.5) and again allow it to fall to the upper surface of the

sodium sulphate layer. During these operations see that the rate of flow is 8 to 12 ml

per minute and that the column does not run dry. Discard the liquid that comes out.

Then elute with 150 ml of the chloroform/methanol mixture (3.7) and collect the

whole of the eluate.

Evaporate the latter almost to dryness at a temperature not exceeding 50ºC and

under a stream of inert gas (3.11) with the rotary evaporator (4.5). Quantitatively

transfer the residue, using chloroform (3.2) or the benzeneacetonitrile mixture (3.6),

to a 10 ml graduated tube (4.10). Concentrate the solution under a stream of inert gas

(3.11) and then adjust the volume to 2 ml with chloroform (3.2) or the benzene/

acetonitrile mixture (3.6).

5.4.

Thin-layer chromatography

Spot on a TLC plate (4.8), 2 cm from the lower edge and at intervals of 2 cm, the

volumes indicated below of the standard solution and the extract

- 10, 15, 20, 30 and 40 µl of the standard aflatoxin B 1 solution (3.16);

- 10 µl of the extract obtained in 5.3 and, superimposed on the same point, 20 µl of

the standard solution (3.16);

- 10 and 20 µl of the extract obtained in 5.3.

Develop the chromatogram in the dark with one of the developing solvents (3.18).

The choice of the solvent must be made beforehand, by depositing 25 µl of the

qualitative standard solution (3.17) on the plate and checking that, when developed,

aflatoxin B 1 and B 2 are completely separated. Let the solvents evaporate in the dark

and then irradiate with UV light, placing the plate 10 cm from the lamp (4.9). The

spots of aflatoxin B 1 give a blue fluorescence.

5.5.

Quantitative determinations

Determine either visually or by fluorodensitometry as indicated below.

5.5.1.

Visual measurements

Determine the quantity of aflatoxin B 1 in the extract by matching the fluorescence

intensity of the extract spots with that of one of the standard solution spots.

Interpolate if necessary. The fluorescence obtained by the superimposition of the

extract on the standard solution must be more intense than that of the 10 µl of extract

and there must not be more than one visible spot. If the fluorescence intensity given

by the 10 µl of extract is greater than that of the 40 µl of standard solution, dilute the

extract 10 or 100 times with chloroform (3.2) or the benzene/acetonitrile mixture

(3.6) before subjecting it again to thin-layer chromatography.

5.5.2.

Measurements by fluorodensitometry

Measure the fluorescence intensity of the aflatoxin B 1 spots with the

fluorodensitometer (4.12) at an excitation wavelength of 365 nm and an emission

wavelength of 443 nm. Determine the quantity of aflatoxin B 1 in the extract spots by

comparison of their fluorescence intensities with that of the standard aflatoxin B 1

spots.

5.6.

Confirmation of the identity of aflatoxin B 1

Confirm the identity of the aflatoxin B 1 in the extract by the processes indicated

below.

5.6.1.

Treatment with sulphuric acid Spray sulphuric acid (3.13) onto the

chromatogram obtained in 5.4. The fluorescence of the aflatoxin B 1 spots must turn

from blue to yellow under UV irradiation.

5.6.2.

Two-dimensional chromatography involving the formation of aflatoxin

B 1 - hemiacetal (aflatoxin B 2a )

NB: The operations described below must be carried out following carefully the

diagram in figure 3.

5.6.2.1.

Application of the solutions

Score two straight lines on the plate (4.8) parallel to two contiguous sides (6 cm in

from each side) to limit migration of the solvent fronts. Spot the following solutions

on the plate using capillary pipettes or microsyringes

-

on point A: a volume of purified extract of the sample, obtained in 5.3,

containing about 2ꞏ5 nm of aflatoxin B 1 ;

-

on points B and C: 25 µl of the standard solution (3.16).

5.6.2.2.

Development

Develop the chromatogram indirection I, in the dark, using the developing solvent

(3.18.1) (1 cm layer in an unsaturated tank) until the solvent front reaches the

solvent limit line.

Remove the plate f rom the tank and allow to dry in the dark at ambient

temperature for five minutes. Then spray hydrochloric acid (3.12) along a band 2ꞏ5

cm high, covering points A and B (indicated by the hatched area in figure 3) until it

darkens, protecting the rest of the plate with a glass sheet. Allow to react for 10

minutes in the dark and dry with a stream of air at ambient temperature.

Next, develop the chromatogram indirection II, in the dark, using the developing

solvent (3.18.1) (1 cm layer in an unsaturated tank) until the solvent front reaches

the solvent limit line. Remove the plate from the tank and allow to dry at ambient

temperature.

5.6.2.3. Interpretation of the chromatogram Examine the chromatogram under

UV light (4.9) and check for the following features.

(a) Appearance of a blue fluorescent spot of aflatoxin B 1 originating from

the standard solution applied at C (migration in the direction I).

(b) Appearance of a blue fluorescent spot of unreacted (with the

hydrochloric acid) aflatoxin B 1 and a more intense blue fluorescent spot

of aflatoxin B 1 - hemiacetal, both originating from the standard solution

applied at B (migration in direction II).

(c) Appearance of spots matching those described in (b), originating from

the sample extract applied at A. The position of these spots is defined

first by the migration distance of the aflatoxin B 1 from point A in

direction I (the same as that traveled by the standard applied at C), and

then by the migration distances from there in direction II of the

aflatoxin B 1 - hemiacetal (same as those travelled by the standard

applied at B). The fluorescence intensities of hemiacetal spots

originating from the extract and from the standard applied at B should

match.

Calculation of the results

6.1.

From the visual measurements

The content in micrograms of aflatoxin B 1 per kg of sample (ppb) is given by the

formula

W • X

in which

Y and X are respectively the volumes in microlitres of the standard solution of

aflatoxin B 1 (3.16) and of the extract having a similar intensity of fluorescence;

S = concentration in micrograms of aflatoxin B 1 per ml in the standard solution

(3.16);

V = final volume of the extract in microlitres, allowing for any dilution that was

necessary;

W = weight in grammes of the sample corresponding to the volume of extract

subjected to column clean-up.

6.2.

From the fluorodensitometric measurements

The content in micrograms of aflatoxin B 1 per kg of sample is given by the

formula

S • V

W • Y

in which

Y = volume in microlitres of the extract spotted on the plate (10 or 20 µl);

S = quantity in nanograms of aflatoxin B 1 in the extract spot (proportional to the

value of Y taken), deduced from the measurements;

V = final volume of the extract in microlitres, allowing for any dilution that was

necessary;

W = weight in grammes of the sample corresponding to the volume of extract

subjected to column clean-up.

Preparation and testing of the standard solution (3.16)

7.1.

Determination of the concentration of aflatoxin B 1

Prepare a standard solution of aflatoxin B 1 in the chloroform (3.2) or the benzene/

acetonitrile mixture (3.6) with a concentration of 8 to 10 µg/ml. Determine the

absorption spectrum between 330 and 370 nm with the aid of the spectrophotometer

(4.11).

Measure the optical density (A) at 363 nm in the case of the chloroform solution[

or at 348 nm in the case of the solution in the benzene/acetonitrile mixture.

Calculate the concentration in micrograms of aflatoxin B 1 per ml of solution from

the formulae below

312 • A • 1000

for the chloroform solution;

20600

312 • A • 1000

for the solution in the benzene/acetonitrile mixture.

19800

Dilute as appropriate, away from day light, to obtain a working standard solution

with a concentration of aflatoxin B 1 of about 0ꞏ1 µg/ml. If kept in a refrigerator at

4ºC, this solution is stable for two weeks.

7.2.

Testing of chromatographic purity

Spot on a plate (4.8) 5 µl of the standard solution of aflatoxin B 1 containing 8 to

10 µg/ml (7.1). Develop the chromatogram as indicated in 5.4. In UV light the

chromatogram should show only one spot and no fluorescence must be perceptible in

the original deposit zone.

Repeatability

The difference between the results of two parallel determinations carried out on

the same sample by the same analyst should not exceed

-

25% related to the highest result for contents of aflatoxin B 1 from 10

and up to 20 µg/kg;

-

5 µg, in absolute value, for contents greater than 20 and up to 50 µg/kg;

-

10% related to the highest value for contents above 50 µg/kg.

Reproducibility

See under ‘Observations’, Part C, point 2.

B.

DETERMINATION OF AFLATOXIN B 1 . HIGH PERFORMANCE LIQUID

CHROMATOGRAPHIC METHOD

1. Purpose and scope

This method is for the determination of aflatoxin B 1 in animal feedingstuffs

including those containing citrus pulp. The lower limit of determination is 0,001 mg/

kg (1 ppb).

2. Principle

The sample is extracted with chloroform. The extract is filtered and an aliquot

portionis purified on a Florisil cartridge followed by a C 18 cartridge. The final

separation and determination is achieved by high performance liquid

chromatography (HPLC) using a reversed phase C 18 column, followed by post-

column derivatization with iodine in water, and fluorescence detection.

Note: mycotoxins are extremely toxic substances. Manipulations should be

performed in a designated fume cupboard. Special precautions should be taken when

toxins are in a dry form because of their electrostatic nature and resulting tendency

to disperse in working areas.

3. Reagents

3.1.

Chloroform, stabilized with 0,5 to 1,0% of ethanol, by mass. See

observation10.2.

3.2.

Methanol, HPLC grade for preatation of 3.6.

3.3.

Acetone.

3.4.

Acetonitril, HPLC grade.

3.5.

Eluting solvents{ Prepare one day before use, or remove air in the

solvents ultrasonically.

3.5.1.

Mixture of acetone (3.3) and water, 98 + 2 (v/v).

3.5.2.

Mixture of water and methanol (3.2), 80 + 20 (v/v).

3.5.3.

Mixture of water and acetone (3.3), 85 + 15 (v/v).

3.6.

Mobile phase for HPLC

Mixture of water, methanol (3.2) and acetonitril (3.4), 130 + 70 + 40 (v+v+v).

NB: The composition of the mobile phase solvent may need to be adjusted,

depending on the characteristics of the HPLC column used.

3.7.

Saturated iodine solution: add 2 g of iodine to 400 ml of water. Mix for

at least 90 min and filter through a membrane filter (4.15). Protect the saturated

solution from light to prevent photo degradation.

3.8.

Acid washed Celite 545, or equivalent.

3.9.

Florisil cartridge (Waters SEP-PAK), or equivalent.

3.10.

C 18 cartridge (Waters SEP-PAK), or equivalent.

3.11.

Inert gas e.g. nitrogen.

3.12.

Aflatoxin B 1 standard solution in chloroform, concentration 10 µg/ml.

Check the concentration of the solution as follows: determine the absorption

spectrum of the solution between 330 and 370 nm by means of the

spectrophotometer (4.23). Measure the absorbance (A) at the maximum near 363 nm.

Calculate the concentration of aflatoxin B 1 in micrograms per millilitre of solution

from the formula

Concentration (èmg/ml) = 312 x A x 1000 = 13,991 x A

22 300

3.12.1.

Aflatoxine B 1 stock standard solution in chloroform.

Transfer quantitatively 2,5 ml of the aflatoxin B 1 standard solution (3.12) to a 50

ml volumetric flask and adjust to the mark with chloroform (3.1). Store this solution

in a cool place (4ºC) in the dark, well sealed and wrapped in aluminium foil.

3.13.

Aflatoxin B 1 calibraltion solutions HPLC.

NB: Use alcid-washed glassware for preparation of these solutions (see 4,

Apparatus).

3.13.1.

Calibration solution 4 ng/ml.

Allow the volumetric flask with stock standard solution (3.12.1) to warm up to

room temperature in the aluminium foil (a few hours). Transfer 400 µl of the stock

standard solution (200 ng aflatoxin B 1 ) into a 50 ml volumetric flask, and evaporate

the solution to dryness in a current of inet gas (3.11).

Dissolve the residue obtained in approximately 20 ml of water/acetone mixture

(3.5.3), make up to the mark with the water/acetone mixture and mix well.

3.13.2.

Calibrationsolution 3 ng/ml.

Transfer quantitatively 7,5 ml of the calibration solution (3.13.1) into a 10 ml

volumetric flask, make up to the mark with the water/acetone mixture (3.5.3), and

mix well.

3.13.3.

Calibration solution, 2 ng]/l.

Transfer quantitatively 25 ml of the calibration solution (3.13.1) to a 50 ml

volumetric flask, make up to the mark with the water/acetone mixture (3.5.3) and

mix well.

This solution is also referred to as the reference standard, to be used for repetitive

injection during HPLC (5.5).

3.13.4.

Calibration solution 1 ng/ml.

Transfer quantitatively 2,5 ml of the calibration solution (3.13.1) to a 10 ml

volumetric flask, make up to the mark with the water/acetone mixture (3.5.3) and

mix well.

3.14.

Ampoule containing a mixture of aflatoxins B 1 , B 2 , G 1 and G 2

concentrations approximately 1, 0,5, 1 and 0,5 µg/ml respectively, in 1 ml

chloroform.

3.14.1.

Chromatographic test solution.

Transfer the content of the ampoule (3.14) into a glass-stoppered test-tube or

screw-capped vial. Transfer 40 µl of this solution into a glass-stoppered test tube

(acid-rinsed) (4.22). Evaporate the chloroform in a stream of inert gas (3.11) and

redissolve into 10 ml of the water/acetone mixture (3.5.3).

3.15.

Reagents for confirmatory test (6).

3.15.1.

Sodium chloride saturated solution.

3.15.2.

Sodium sulphate, anhydrous, granular.

4. Apparatus

Caution: Use of non acid-washed glassware for aqueous aflatoxin solutions may

cause losses. Particular care should be taken with new glassware and disposable

glassware such as autosampler vials and Pasteur pipettes. Therefore laboratory

glassware coming into contact with aqueous solutions of aflatoxins should be soaked

in dilute acid (e.g. sulfuric acid = 2 mol/l) for several hours, then rinsed well with

distilled water to remove all traces of acid (e.g. three times, check with pH-paper). In

practice, this treatment is necessary for the round bottomed flask (4.4), the

volumetric flasks, measuring cylinders, vials or tubes used for calibration solutions

and final extracts (particularly vials for autosamplers), and Pasteur pipettes, if these

are used to transfer calibration solutions or extracts.

4.1.

Grinder-mixer.

4.2.

Sieve of aperture size 1,0 mm, (ISO R 565).

4.3.

Mechanical shaker.

4.4.

Rotary vacuum evaporator, equipped with a 150 to 250 ml round

bottomed flask.

4.5.

High performance liquid chromatograph, injector with a loop suitable

for the injection of 250 ml. See the manufacturers instructions for partial or complete

loop filling.

METHODS OF ANALYSIS FOR THE

OFFICIAL CONTROL OF FEEDINGSTUFFS

HPLC analytical column: 3 µm or 5 µm C 18 packing.

Pulse-free pump for delivery of the iodine post-column reagent.

Valco zero dead volume Tee, stainless steel (1/16" × 0,75 mm).

Spiral reaction coil[ Teflon or stainless steel. Dimensions of 3 000 × 0,5

mm to 5 000 × 0,5 mm have been found to be appropriate in combination with 5 µm

or 3µm HPLC columns.

Thermostatically controlled water-bath adjusted to 60ºC, capable of

temperature regulation to better than 0,1ºC.

Fluorescence detector, with excitation at 365 nm and emission at 435 nm

wavelengths. (For filter instrument: emission wavelength : 400 nm). Detection of at

least 0,05 ng aflatoxin B 1 shall be possible. Some back pressure may be advisable

(e.g. restrictor, Teflon or stainless steel coil connected to the outlet of the detector),

to suppress air bubbles in the flow-cell.

Strip chart recorder.

Electronic integrator (optional).

Fluted filter paper diameter: 24 cm, Macherey-Nagel 617¼ or

equivalent.

Membrane filter with a pore size of 0,45 µm, Millipore HAWP 04700 or

equivalent.

500 ml glass stoppered conical flask.

Glass column (internal diameter

approximately

cm,

length

approximately 30 cm) equipped with a Luer tip.

Luer chloroform-resistant stopcock (e.g. Bio-rad 7328017, Analytichem

A1 6078, J.T. Baker 4514 or equivalent).

Chemically resistant syringe, 10 ml Luer connector.

Syringe suitable for HPLC injection of 250 µl (see 4.5).

100 µl microsyringe for preparation of calibration solutions (check that

the accuracy is within 2% by weighing).

10 ml glass stoppered calibrated tubes.

Spectrophotometer, suitable for making measurements in the UV region

of the spectrum.

Equipment for confirmatory test (6).

Acid-rinsed 100 ml separating funnel with Teflon stopcock.

Heating block, 40 to 50ºC.

5. Procedure

Preparation of the sample.

Grind the sample so that it passes through the sieve (4.2).

Test portion.

Weigh 50 g of the prepared test sample into the conical flask (4.16).

Extraction

Add 25 g of Celite (3.8), 250 ml of chloroform (3.1) and 25 ml of water to the test

portion (5.2). Stopper the flask, and shake for 30 minutes on a mechanical shaker

(4.3). Filter through a fluted filter paper (4.14). Collect 50 ml of the filtrate. If

necessary, take an aliquot of the filtrate and dilute to 50 ml with chloroform so that

the concentration of aflatoxin B 1 is not greater than 4 ng/ml.

5.4.

Clean-up (the procedure should be carried out without significant

interruptions).

Caution

-

protect the laboratory, where the analyses are done, adequately from

daylight. This can be achieved effectively by using

(i)

UV absorbing foil on the windows in combination with subdued

light (no direct sunlight);

(ii) Curtains or blinds in combination with artificial light (fluorescent

tubes are acceptable);

-

Aflatoxin containing solutions must be protected from light as much as

possible (keep in the dark, use aluminium foil).

5.4.1.

Florisil SEP-PAK purification

5.4.1.1.

Preparation of the column-cartridge assembly

Attach a stopcock (4.18) to the shorter stem of a Florisil cartridge (3.9) (see figure

1). Wash the cartridge and remove aid by taking 10 ml chloroform (3.1) and passing

8 ml via the stopcock rapidly through the cartridge using a syringe (4.19). Attach the

longer stem of the cartridge to a glass column (4.17) and pass the remaining 2 ml

chloroform through the cartridge into the column. Close the stopcock. Remove the

syringe.

5.4.1.2.

Purification

Add the filtrate collected in 5.3 to the column-cartridge assembly and drain by

gravity. Rinse with 5 ml of chloroform (3.1), followed by 20 ml of methanol (3.2).

Discard the eluates. During these operations, ensure that the column cartridge

assembly does not run dry.

Elute aflatoxin B 1 with 40 ml of the acetone-water mixture (3.5.1) and collect the

whole of the eluate in the round bottomed flask of the rotary evaporator (4.4).

Concentrate the eluate on the rotary evaporator at 40ºC to 50ºC until no more

acetone is distilled.

(NB: approximately 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 then no significant amount of acetone. Residues of acetone might

lead to losses of aflatoxin B 1 on the C 18 cartridge).

Add 1 ml of methanol (3.2), swirl the flask to dissolve aflatoxin B 1 on the sides of

the flask, add 4 ml water, and mix. Disconnect and discard the cartridge. Rinse the

glass column with water and retain for the C 18 purification step.

5.4.2.

C 18 SEP-PAK purification

5.4.2.1.

Preparation of the column-cartridge assembly.

Attach a stopcock (4.18) to the shorter stem of a C 18 -cartridge (3.10) (see figure

1). Prime the cartridge and remove any air by passing 10 ml methanol (3.2) via the

stopcock rapidly through the cartridge with a syringe (4.19) (Air bubbles in the

cartridge are visible as light spots on the otherwise greyish background).

Take 10 ml water, and pass 8 ml through 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 (4.17) and pass the remaining 2 ml water through

the cartridge in the column. Close the stopcock. Remove the syringe.

5.4.2.2.

Purification

Transfer the extract collected in 5.4.1.2 quantitatively to the glass column (4.17),

rinsing the flask twice with 5 ml water/methanol mixture (3.5.2) and drain by

gravity. During these operations, ensure that the column-cartridge assembly does not

run dry. (When 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. Then continue).

Elute with 25 ml water/methanol mixture. Discard the eluate. Elute the aflatoxin B 1

with 50 ml water/acetone mixture (3.5.3), and collect the whole of the eluate in a 50

ml volumetric flask. Make up to the mark with water and mix: the resulting test

solution is used for chromatography (5.5).

Caution: Filtration of the final extract prior to HPLC is normally not necessary. If

considered necessary, cellulose filters are not to be used, because they may lead to

losses of aflatoxin B 1 . Teflon filters are acceptable.

5.5.

High performance liquid chromatography

(See Figure 2 for setting-up of the equipment). Allow sufficient time for

conditioning and stabilizing the instruments.

Note 1

The flow-rates given for the mobile phase and the post-column reagent are

indicative only. They may need to be adjusted depending on the characteristics of the

HPLC column.

Note 2

The detector response to aflatoxin B 1 depends on the temperature, therefore

compensation should be made for drift (see Figure 3). By injecting a fixed amoung

of aflatoxin B 1 reference standard (3.13.3) at regular intervals (i.e. every third

injection), the aflatoxin B 1 peak values between these reference standards can be

corrected using the mean response, provided that the difference between responses

of consecutive reference standards is very small (< 10%). Therefore injections must

be made without interruptions. If interruption is necessary, the last injection before

interruption and the first injection after interruption must be the reference standard

(3.13.3). Because the calibration curve is linear and passes through the origin, the

amounts of aflatoxin B 1 in the sample extracts are determined directly by reference

to the adjacent standards.

5.5.1.

HPLC pump settings

Set the HPLC pump (4.5) to give a flow of 0,5 or 0,3 ml/min for a 5 µm or a 3 µm

HPLC column (4.6) respectively using the mobile phase (3.6).

5.5.2.

Post-column pump settings

Set the pump (4.7) to give a flow of 0,2 to 0,4 ml/min of the iodine-saturated water

solution (3.7). As a rough guide: Flows of approximately 0,4 or 0,2 ml/min are

advisable in combination with flows of 0,5 and 0,3 ml/min of the mobile phase (3.6)

respectively.

5.5.3.

Fluorescence detector

Set the fluorescence detector (4,11) to exc. = 365 nm and em = 435 nm (filter

instrument; > 400 nm). Adjust the detector attenuator to obtain approximately 80%

full scale deflection of the recorder pen for 1 ng of aflatoxin B 1 .

5.5.4.

Injector

For all solutions, inject 250 µl amounts following the instructions of the

manufacturer of the injector.

5.5.5.

Check of chromatographic separation

Inject the chromatographic test solution (3.14.1). Valleys should be less than 5%

of the sum of peak heights of the adjacent peaks.

5.5.6.

Check of the stability of the system

Before each series of analyses, respectively inject the reference standard (3.13.3),

until stable peak areas are achieved (NB Peak responses for aflatoxin B 1 between

consecutive injections should not differ by more than 6%). Proceed without delay

with the check of linearity (5.5.7).

5.5.7.

Check of linearity

Inject the aflatoxin B 1 calibration solutions (3.13.1 to 3.13.4). Every third

injection use the reference standard (3.13.3), for correction of drift in response (NB

Peak responses for this reference standard must not differ by more than 10% in 90

minutes). Correct for drift according to the formula in 7. The calibration graph

should be linear and pass through the origin, within twice standard error of Y-

estimate. Values found must not differ by more than 3% from the nominal values. If

these requirements are fulfilled, continue without delay. If not, identify and correct

the sources of the problem before continuing.

5.5.8.

Injection of sample extracts

Inject the purified sample extracts (5.4.2.2). After every two sample extracts

repeat the injection of the reference standard (3.13.3) according to the following

sequence: reference standard, extract, extract, reference standard, extract, extract,

reference standard etc.

6. Confirmatory test

6.1.

Further treatment of the extract (5.4.2.2)

Add 5 ml sodium chloride solution (3.15.1) to the final extract obtained at 5.4.2.2.

Extract three times each with 2 ml chloroform (3.1) for one minute, in the separating

funnel (4.24.1). Pour the combined chloroform extracts over approximately 1 g

sodium sulphate (3.15.2) into a 10 ml test tube. A small funnel (diameter: 4 cm) can

be used with a piece of cottonwool in the constriction, covered with a approximately

1 g sodium sulphate.

Wash the sodium sulphate layer with a few ml of chloroform and collect the

washing in the same test tube. Evaporate the chloroform extract to dryness in the

same test-tube using the heating block (4.24.2) and redissolve in 1 ml of chloroform.

6.2.

Preparation of derivative and thin layer chromatography

See Annex to European Council Directive 76/372/EEC method A, point 5.6.2.

7. Calculation of results

Calculate the aflatoxin B 1 content (µm/kg) present in the sample, using the

formula

aflatoxin B 1 content in èmg/kg =

m x V e x t

V m x M x

V f

V c

where

m = amount of aflatoxin B 1 in ng represented by the B 1 peak of the sample,

calculated as follows

P(sample)

m -

x 2 r(st)

P(st 1 ) + P(st 2 )

P(sample) = peak area of aflatoxin B 1 for the sample

P(st 1 ) = peak area of aflatoxin B 1 resulting from the preceeding injection of

reference standard (3.13.3)

P(st 2 ) = peak area of aflatoxin B 1 resulting from the following injection of

reference standard (3.13.3)

r(st) = injected amount of aflatoxin B 1 in the reference standard (3.13.3) in ng

V m = volume of the injected sample extract in ml

V e x t = final volume of sample extract in ml, allowing for any dilution that was

made (5.3)

M = mass of sample in g

V f = volume of filtrate transferred to Florisil cartridge (5.4.1.2) in ml

V c = volume of chloroform, used for the extraction of the sample in ml

If the procedure is followed as in this protocol, the formula reduces to

aflatoxin B 1 content in µg/kg = 20 × m.

7.1.

Calculations of the results may also be done by peak height

measurement.

8. Repeatability

see under 10.1

9. Reproducibility

see under 10.1

Observations

10.1.

Precision

A collaborative study * , carried out at the international level on mixed feeding

stuffs gave the results for repeatability and reproducibility indicated in Table 1. The

term repeatability (r) used here is defined as the largest ratio which is not significant

at the 95% probability level for comparison of two readings of the same sample in

the same laboratory under similar conditions. The term reproducibility (R) is

similarly defined for comparing two different laboratories. In accordance to ISO

3534 - 1977, 2.35 † and European Union Commission Decision 89/610/EEC r and R

are also given in Table 1 in terms of coefficients of variation.

Table 1

Repeatability (r) and reproducibility (R) expressed as ratios

and corresponding coefficients of variation

(15 laboratories)

*Egmond, H.P. van, Heisterkamp, S.H. and Paulsch, W.E. (1991). Food Additives and Contaminants 8, 17-

†ISO 3534-1977.

Level

r

R

CV r (*)

CV R

(µg/kg)

(%)

(%)

8 & 14

1,4

1,7

(*) CV = coefficient of variation

10.2.

Stabilization of chloroform (3.1)

The adsorption characteristics of the florisil cartridge may be changed if

stabilizers other than ethanol are used. This should be verified in accordance with

10.3 when the chloroform described is not available.

10.3.

Accuracy

The correct applications of the method shall be verified by making replicate

measurements on certified reference materials. If these are not available, the

performance of the method should be verified by recovery experiments made on the

fortified blank samples. The deviation of the mean from the actual value, expressed

as a percentage of the actual value, shall not lie outside the limits -20 to +10%.

Figure 1: Column-cartridge assembly

Figure 2: Flow diagram of the LC system with iodine post-column derivatization

Figure 3: Compensation for drift in aflatoxin B 1 response by injecting reference

standard (3.13.3) at regular intervals

C.

OBSERVATIONS CONCERNING METHODS A AND B

1. Defatting

Samples containing more than 5% fats must be defatted with light petroleum (bp

40 to 60 degrees C) after the preparations indicated in 5.1.

In such cases, the analytical results must be expressed in terms of the weight of the

non-defatted sample.

2. Reproducibility of the results for Method A

The reproducibility of the results, i.e. the variation between the results obtained by

two or more laboratories on the same sample has been estimated at

± 50% of the mean value for mean values of aflatoxin B, from 10 and up

to 20 mg/kg;

± 10 mg/kg of the mean value for mean values greater than 20 and up to

50 mg/kg;

± 20% of the mean value for mean values above 50 mg/kg.

Appendix to Annex

Figure 1

Figure 2

Figure 3