Methods of Analysis for the Official Control of Feedingstuffs

L.N. 285 of 2005

VETERINARY SERVICES ACT

(CAP. 437)

Methods of Analysis for the Official Control of Feedingstuffs

Rules, 2005

IN exercise of the powers conferred by article 25 of the Veterinary

Services Act, 2001, the Minister for Rural Affairs and the Environment

has made the following rules>–

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

Official Control of Feedingstuffs Rules, 2005.

(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.

For the purposes of these rules -

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.

Acetone.

Chloroform, stabilized with 0·5 to 1·0 % of 96 % ethanol (v/v).

N-hexane.

Methanol.

Anhydrous diethyl ether, free from peroxides.

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

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

Silica gel, for column chromatography, particle size 0·05 to 0·20 mm.

Absorbent cotton wool, previously defatted with chloroform, or glass wool.

Sodium sulphate, anhydrous, granular.

Inert gas, e.g. nitrogen.

1 N Hydrochloric acid.

50 % (v/v) sulphuric acid.

Kieselguhr (hyflosupercel), washed in acid.

Silica gel G-HR or equivalent, for TLC.

Standard solution with about 0·1 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 0·1 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 tank;

Diethyl ether (3.5)/methanol (3.4)/water: 94/4·5/1·5 (v/v/v), saturated tank;

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

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

“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 B1, be

carried out in accordance with the methods described in the Schedule

to these rules.

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 obtaina uniform layer 0·25 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 110 ºC 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.

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 50 ºC 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 benzene-

acetonitrile 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).

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.

Quantitative determinations

Determine either visually or by fluorodensitometry as indicated below.

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.

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.

Confirmation of the identity of aflatoxin B 1

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

below.

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.

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 2·5 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 from the tank and allow to dry in the dark at ambient

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

2·5 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

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.

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)

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 0·1 µg/ml. If kept in a

refrigerator at 4 ºC, this solution is stable for two weeks.

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

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

observation10.2.

Methanol, HPLC grade for preatation of 3.6.

Acetone.

Acetonitril, HPLC grade.

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

ultrasonically.

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

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

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

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.

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

solutionfrom light to prevent photodegradation.

Acid washed Celite 545, or equivalent.

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

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

Inert gas e.g. nitrogen.

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 (4 ºC) in the dark, well sealed and wrapped in

aluminium foil.

AflatoxinB 1 calibraltion solutions HPLC.

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

Apparatus).

3.13.1. Calibration solution 4ng/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.

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/ml.

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.

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.

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).

Reagents for confirmatory test (6).

Sodium chloride saturated solution.

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.

Grinder-mixer.

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

Mechanical shaker.

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

flask.

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.

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 60 ºC, capable of

temperature regulation to better than 0,1 ºC.

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 617 1/4 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 1 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).

4.24.1. Acid-rinsed 100 ml separating funnel with Teflon stopcock.

4.24.2. Heating block, 40 to 50 ºC.

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.

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).

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 in5.3 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 40 ºC to 50 ºC 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.

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.

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.

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).

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.

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 .

Injector

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

manufacturer of the injector.

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.

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).

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.

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

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.

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) =

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.

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

Precision

A collaborative study ( 1 ), 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 ( 2 ) 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, 17-29

ISO 3534-1977.

Level

r

R

CV r (*)

CV R

(µg/kg)

(%)

(%)

8 & 14

1,4

1,7

(*) CV = coefficient of variation

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.

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 ºC) after the preparations indicated in 5.1.

Insuch 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 meanvalue for meanvalues of aflatoxin B, from 10 and up to 20

µg/kg;

± 10 µg/kg on the meanvalue for meanvalues greater than 20 and up to 50

µg/kg;

± 20 % of the meanvalue for meanvalues above 50 µg/kg.

Appendix to Annex

Figure 1

Figure 2

Figure 3

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