The Feedingstuffs (Sampling and Analysis) (Amendment) Regulations 1994

Regulation 3(f)

SCHEDULE 3 PROVISIONS TO BE SUBSTITUTED FOR METHOD 26b IN SCHEDULE 2 TO THE PRINCIPAL REGULATIONS

“26b. AFLATOXIN B₁ — HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD

1.    Scope and Field of Application

This method is for the determination of aflatoxin B₁ in feedingstuffs whether or not 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 on a Florisil cartridge followed by a C₁₈cartridge. The final separation and determination is achieved by high performance liquid chromatography (HPLC) using a reversed phase C₁₈column, followed by post-column derivatization with iodine in water, and fluorescence detection.

Note:

mycotoxins are extremely toxic sustances. 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, with the danger of ingestion. Care should be taken to decontaminate all equipment after use.

3.    Reagents

3.

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

Methanol, HPLC grade.

Acetone.

Acetonitrile, HPLC grade.

Eluting solvents: Prepare one day before use, remove air 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 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.

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 photodegradation.

Acid washed Celite 545, or equivalent.

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

C₁₈ cartridge (Waters SEP—PAK), or equivalent.

Inert gas, e.g. nitrogen.

Aflatoxin B₁ standard solution in chloroform, concentration 10ng/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 B₁in micrograms per millilitre of solution from the formula:

Aflatoxin B₁ stock standard solution in chloroform.

Transfer quantitatively 2.5 ml of the aflatoxin B₁ 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.

Aflatoxin B₁ calibration solutions for HPLC.

NB Use acid-washed glassware for preparation of these solutions (see 4, Apparatus).

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 400μl of the stock standard solution (200 ng aflatoxin B₁) 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.

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.

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 repeated injection during HPLC (5.5.).

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 alflatoxins B₁, B₂, G₁ and G₂, 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 sulfate, anhydrous, granular.

Light petroleum, boiling range 40 to 60° C.

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 μl. See the manufacturers instructions for partial or complete loop-filling.

HPLC analytical column: 3 μm or 5 μm C₁₈packing.

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 3 μm HPLC columns.

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

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 B₁ should 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 ¼ 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 250μl (see 4.5.).

100μl 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 50° C.

5.    Procedure

5.

Defatting.

5.0.   Samples containing more than 5% oil or fat must be defatted with light petroleum (3.16.) after the material is ground and sieved (5.1.). In such cases the analytical results may be expressed in terms of the weight of the non-defatted sample.

Preparation of the sample.

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

Test portion.

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

Extraction.

5.3   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 portion of the filtrate and dilute to 50 ml with chloroform so that the concentration of aflatoxin B₁ 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);

• solutions containing aflatoxin 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 air by taking 10 ml of 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 of 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 B₁ 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° C to 50° C 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 remaining. Residues of acetone might lead to losses of aflatoxin B₁ on the C₁₈ cartridge). Add 1 ml of methanol (3.2.), swirl the flask to dissolve aflatoxin B₁ 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 C₁₈ purification step.

5.4.2.   C₁₈ SEP—PAK purification.

5.4.2.1.   Preparation of the column-cartridge assembly.~

Attach a stopcock (4.18.) to the shorter stem of a C₁₈-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 of 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 of 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 B₁ 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 B₁. Teflon filters are acceptable.

High performance liquid chromatography.

5.5.   (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 B₁ depends on the temperature, therefore compensation should be made for drift (see Figure 3). By injecting a fixed amount of aflatoxin B₁ reference standard (3.13.3.) at regular intervals (i.e. every third injection), the aflatoxin B₁ 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 the1in 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 B₁.

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, repeatedly inject the reference standard (3.13.3.), until stable peak areas are achieved (NB Peak responses for aflatoxin B₁ 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 B₁ calibration solutions (3.13.1. to 3.13.4.). For 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 the standard error of the 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 source 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.

Further treatment of the extract (5.4.2.2.).

6.1.   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 of chloroform (3.1.) for one minute, in the separate funnel (4.24.1.). Pour the combined chloroform extracts over approximately 1 g sodium sulfate (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 sulfate.

Wash the sodium sulfate 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.

6.2.   See Annex to Council Directive 76/372/EEC(1) method A, point 5.6.2.

7.    Calculation of results

Calculate the aflatoxin B₁ content (μg/kg) present in the sample, using the formula:

where:

• m = amount of aflatoxin B₁ in ng represented by the B₁ peak of the sample, calculated as follows:

  

  • P(sample) = peak area of aflatoxin B₁ for the sample

  • P(st₁) = peak area of aflatoxin B₁ resulting from the preceding injection of reference standard (3.13.3.).

  • P(st₂) = peak area of aflatoxin B₁ resulting from the following injection of reference standard (3.13.3.).

  • r(st) = injected amount of aflatoxin B₁ in the reference standard (3.13.3.) in ng.

  • V_m = volume of the injected sample extract in ml.

  • V_cxt = final volume of the 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 the 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 B₁ 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.

10.    Observations

10.

Precision.

10.1.   A collaborative study(2), carried out at the international level on mixed feedingstuffs gave the results for repeatability and reproducability indicated in Table 1. The term repeatability (r) used here is defined as te 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 reproducability (R) is similarly defined for comparing two different laboratories. In accordance with ISO 3534 — 1977, 2.35(3) and Commission Decision, 89/610/EEC(4) 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	r	R	CVr(1)	CVR
(1) CV = coefficient of variation.
(μg/kg)			(%)	(%)
8 & 14	1.4	1.7	11	18

Stabilization of chloroform (3.1.).

10.2.   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.

10.3.   The correct application 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%.

”

(1)

OJ No. L 102, 15.4.1976, p. 8.

(2)

Egmond, H.P. van, Heisterkamp, S.H. and Paulsch, W.E. (1991). Food Additives and Contaminants 8, 17-29.

(3)

ISO 3534-1977.

(4)

OJ No. L 351, 2.12.1989, p. 39.