S.I. No. 370/1993 - European Communities (Feedingstuffs) (Methods of Analysis) (Amendment) Regulations, 1993.

1. Mobile phase

2. Pump

3. Injection valve

4. Guard column

5. HPLC analytical column

6. Confirmatory test

6.1 Further treatment of the extract (5.2.2.2).

Place the extract obtained in (5.2.2.2) into a separating funnel (4.22.1) and add 5 ml sodium chloride solution (3.15.1), and 2 ml chloroform (3.1). Shake for 2 minutes and repeat the extraction with 2 further 2 ml portions of chloroform (3.1). 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 approximately 1 g sodium sulphate).

Wash the sodium sulphate layer with a few ml of chloroform and combine with the chloroform extract. Evaporate the chloroform extract to dryness using a heating block (4.22.2). Dissolve the residue in 1 ml of chloroform and proceed to paragraph 5.6.2 of method A.

6.2 Preparation of derivative and thin layer chromatography. See Method A, point 5.6.2, Two-dimensional chromatography involving the formation of aflatoxin B

1

-hemiacetal (aflatoxin B

2a

).

7.

Calculation of results

Calculate the aflatoxin B

1

content (µg/kg) present in the sample, using the formula

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 proceeding injection of reference standard 6 (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;

Vm

=

volume of the injected sample extract in ml;

V

ext

=

final volume of sample extract in ml, allowing for any dilution that was made (5.1);

M

=

mass of sample in g;

V

f

=

volume of filtrate transferred to Florisil cartridge (5.2.1.2) 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

µ

g/kg = 20 x m.

7.1 Calculation of the results may also be done by peak height measurement.

8.

Repeatability

See under 10.1.

9.

Reproducibility

See under 10.1.

10.

Observations

10.1

Precision

A collaborative study

(1)

, carried out at 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 Commission Decision 89/610/EEC

(3)

, 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)

Level

(µg/kg)

r

R

CVr

(4)

(/)

CV

R

(%)

8 & 14

1.4

1.7

11

18

10.2.

Stabilisation of chloroform

The absorption 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.

(1)

Egmond, H.P. van, Heisterkamp, S.H. and Paulsch, W.E. (1991).

(2)

ISO 3534-1977.

(3)

OJ No. L 351, 2-12-1989, p. 39.

(4)

CV = Coefficient of Variation.

10.3

Accuracy

The correct applications of the method should 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 minus 20% to plus 10%.

10.4

Defatting

Samples containing more than 5% fats must be defatted with light petroleum (bp 40 to 60°C) after the preparation indicated in 5.1.

In such cases, the analytical result must be expressed in terms of the weight of the non-defatted sample.

GIVEN under my Official Seal, this 13th day of December, 1993.

JOE WALSH,

Minister for Agriculture, Food

and Forestry.

EXPLANATORY NOTE.

These Regulations amend the European Communities (Feedingstuffs) (Methods of Analysis) Regulations, 1978, (S.I. 250 of 1978) so as to give effect to Commission Directive 92/95/EEC which replaced the two-dimensional TLC method of analysis for aflatoxin B

1

in feedingstuffs by a HPLC method.

7. Reagent pump

8. T-joint

9. Thermostatically controlled bath

10. Spiral reaction coil

11. Fluorescence detector

12. Restrictor

13. Waste

14. Strip chart recorder/integrator

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

Figure 3 : Compensation for drift in aflatoxin B

1

respond by injecting reference standard (3.13.3) at regular intervals'

(B) HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD

1.

Purpose and Scope

To determine the level 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 portion is purified by elution through a Florisil cartridge followed by elution through a C

18

cartridge. The purified extract is subjected to reversed phase HPLC, followed by post-column derivatization with iodine in water, and fluorescence detection of the aflatoxin B

1

derivative.

3.

Reagents

Warning

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.

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).

— 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. sulphuric 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 (4.2), 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.

3.1 Chloroform, stabilized with 0.5 to 1.0% of ethanol, by mass. See observation 10.2.

3.2 Methanol, HPLC grade.

3.3 Acetone.

3.4 Acetonitrile, 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 acetonitrile (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 minutes and filter through a membrane filter (4.13). Protect the saturated solution from light to prevent photodegradation.

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

µ

g/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 (4.21). Measure the absorbency (A) at the maximum near 363 nm. Calculate the concentration of aflatoxin B

1

in micrograms per millilitre of solution from the formula.

Concentration

(

µ

g/ml) =

= 13.991 x A

3.12.1 Aflatoxin 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°C) in the dark, well sealed and wrapped in aluminium foil.

3.13 Aflatoxin B

1

calibration solutions HPLC.

NB

Use acid-washed glassware for preparation of these solutions (see Caution, third indent)

.

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

µ

l of stock standard solution (200 ng aflatoxin B

1

) into a 50 ml volumetric flask, and evaporate the solution to dryness in a current of inert 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 Calibration solution 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 (5.3).

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

µ

g/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) (4.20). 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

4.1 Mechanical shaker.

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

4.3 High performance liquid chromatograph, containing an injector with a loop suitable for the injection of 250 ml. See the manufacturers instructions for partial or complete loop filling.

4.4 HPLC analytical column: 3

µ

m or 5

µ

m C

18

packing.

4.5 Pulse-free pump for delivery of the iodine postcolumn reagent.

4.6 Valco zero dead volume Tee, stainless steel (

1

/

16

" x 0.75 mm).

4.7 Spiral reaction coil; Teflon or stainless steel. Dimensions of 3000 X0.5 mm to 5000 x 0.5 mm have been found to be appropriate in combination with 5

µ

m or 3

µ

m HPLC columns.

4.8 Thermostatically controlled water-bath adjusted to 60°C capable of temperature regulation to better than 0.1°C.

4.9 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.

4.10 Strip chart recorder.

4.11 Electronic integrator (optional).

4.12 Fluted filter paper, diameter: 24 cm, Macherey-Nagel 617 ¼ or equivalent.

4.13 Membrane filter with a pore size of 0.45

µ

m. Millipore HAWP 04700 or equivalent.

4.14 500 ml glass stoppered conical flask.

4.15 Glass column (internal diameter approximately 1 cm, length approximately 30 cm) equipped with a Luer tip.

4.16 Luer chloroform-resistant stopcock (e.g. Bio-rad 7328017, Analytichem A1 6078, J. T. Baker 4514 or equivalent).

4.17 Chemically resistant syringe, 10 ml Luer connector.

4.18 Syringe suitable for HPLC injection of 250

µ

l (see 4.3).

4.19 100

µ

l microsyringe for preparation of calibration solutions (check that the accuracy is within 2% by weighing).

4.20 10 ml glass stoppered calibrated tubes.

4.21 Spectrophotometer, suitable for making measurements in the UV region of the spectrum.

4.22 Equipment for confirmatory test (6).

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

4.22.2 Heating block, 40 to 50°C.

5.

Procedure

5.1

Extraction

Weigh 50 g of the prepared sample (see Observations 10.4) into the conical flask (4.14) and add 25 g of Celite (3.8), 250 ml of chloroform (3.1) and 25 ml of water. Stopper the flask, and shake for 30 minutes on a mechanical shaker (4.1). Filter through a fluted filter paper (4.12). 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.

5.2 Clean-up (the procedure should be carried out without significant interruptions).

5.2.1 Florisil SEP-PAK purification.

5.2.1.1 Preparation of the column-cartridge assembly.

Attach a stopcock (4.16) to the shorter stem of a Florisil cartridge (3.9) (see figure 1). Wash the cartridge and remove air by taking 10 ml chloroform (3.1) and passing 8 ml via the stopcock rapidly through the cartridge using a syringe (4.17).

Attach the longer stem of the cartridge to a glass column (4.15) and pass the remaining 2 ml chloroform through the cartridge into the column. Close the stopcock. Remove the syringe.

5.2.1.2 Purification

Add the filtrate collected in 5.1 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 elutes. 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 all of the eluate in the round bottomed flask of the rotary evaporator (4.2). Concentrate the eluate on the rotary evaporator at 40°C too 50°C until all the 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.

5.2.2 C

18

SEP-PAK Purification.

5.2.2.1 Preparation of the column-cartridge assembly.

Attach a stopcock (4.16) 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 of methanol (3,2) via the stopcock rapidly through the cartridge with a syringe (4.17). (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 (4.15) and pass the remaining 2 ml water through the cartridge in the column. Close the stopcock. Remove the syringe.

5.2.2.2 Purification

Transfer the extract collected in the 5.2.1.2 quantitatively to the glass column (4.15) and rinse 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. (If air bubbles develop in the constriction near the cartridge, stop the flow and tap the top of the glass column, to remove them). 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 all of the eluate in a 50 ml volumetric flask. Make up to the mark with water and mix. Proceed to paragraph 5.3.8.

5.3

High performance liquid chromatography

(See figure 2 for assembly 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 amount 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%). 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.

5.3.1 HPLC pump settings.

Using mobile phase (3.6), set the HPLC pump (4.3) to give a flow of 0.5 or 0.3 ml/min for a 5 µm or a 3 µm HPLC column (4.4), respectively.

5.3.2 Post-column pump settings.

Set the pump (4.5) to give a flow 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.

5.3.3 Fluorescence detector.

Set the fluorescence detector (4.9) 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

.

5.3.4 Injector.

For all solutions, inject 250

µ

l amounts following the instructions of the manufacturer of the injector.

5.3.5 Check on 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.

5.3.6 Check on the stability of the system.

Before each series of analyses, 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 (5.3.7).

5.3.7 Check for 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 references standard must not differ by more than 10% in 90 minutes). Correct the 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 proceeding to 5.3.8.

5.3.8 Chromatography of the sample extract (5.2.2.2).

Caution: It is normally not necessary to filter the final extract prior to HPLC. If it is considered necessary, cellulose filters must not be used, because they may lead to losses of aflatoxin B

1

. Teflon filters are acceptable

.

Inject the purified sample extracts, obtained in paragraph 5.2.2.2, in the following sequence: reference standard (3.13.3), extract, extract, reference standard, extract, extract, reference standard, etc.

S.I. No. 370 of 1993.

EUROPEAN COMMUNITIES (FEEDINGSTUFFS) (METHODS OF ANALYSIS) (AMENDMENT) REGULATIONS, 1993.

I, JOE WALSH, Minister for Agriculture, Food and Forestry, in exercise of the powers conferred on me by

Section 3

of the

European Communities Act, 1972

(No. 27 of 1972), and for the purposes of giving effect to Commission Directive 92/95/EEC of 9 November, 1992

(1)

, hereby make the following Regulations