Project SMT-CT96-2045

1 Project SMT-CT96-2045 VALIDATION OF ANALYTICAL METHODS TO DETERMINE THE CONTENT OF AFLATOXINS, OCHRATOXIN AND PATULIN IN FOODSTUFFS OF VEGETABLE ORIGIN Partner 7 - Jörg Stroka and Elke Anklam European Commission DG JRC Ispra July 1997 Report on Workpackage 1.5 Draft of method for aflatoxin analysis in peanut butter, pistachios, fig paste and paprika powder in suitable format for intercomparison purposes

2 Section 1 Scope 4 Page 2 Normative References 5 3 Principle 5 4 Reagents 6 5 Apparatus 10 6 Procedures 13 6.1 Conditioning of immunoaffinity columns 13 6.2 Extraction 13 6.3 Immunoaffinity clean up 16 6.4 Post column derivatisation 17 6.5 Calibration curve 17 6.6 Remarks on calculation 17 6.7 Spiking procedure 18 7 Precision 18 8 Test Report 18 Bibliography 19

3 ABSTRACT This method can be applied to the determination of aflatoxins in fig paste, pistachios, peanut butter and paprika powder. The limit of quantification of the method has been demonstrated to be 0.2 µg/kg for each aflatoxin. The method involves extraction of a test portion of fig paste, pistachios or paprika powder with a solvent solution (methanol/water) or for peanut butter with the solvent mixture plus hexane (or cyclohexane). The sample extract is filtered, diluted with water to a specified solvent concentration, and applied to an immunoaffinity column containing antibodies specific to aflatoxins B 1, B 2, G 1 and G 2. The aflatoxins are removed from the immunoaffinity columns with neat methanol. Aflatoxins are quantified by reversephase liquid chromatography (RP-HPLC) with post column derivatisation (PCD) and fluorescence detection.

4 METHOD FOR THE DETERMINATION OF AFLATOXIN B 1 AND TOTAL AFLATOXINS IN FIG PASTE, PISTACHIOS, PEANUT BUTTER AND PAPRIKA POWDER 1. SCOPE This method (based on JAOAC, 1991, 74, 81-88) can be applied to the determination of aflatoxins B 1, B 2, G 1 and G 2 in fig paste, pistachios, peanut butter and paprika powder. The limit of quantification of the method has been demonstrated to be 0.2 µg/kg for each aflatoxin. CAUTION 1 - This method requires the use of solutions of aflatoxin B 1. Aflatoxins are carcinogenic to humans. Attention is drawn to the statement made by the International Agency for Research on Cancer (WHO). 2 - Aflatoxins are subject to light degradation. Protect analytical work adequately from the daylight, and keep aflatoxin standard solutions protected from light by using amber vials or aluminium foil. 3 - The use of non acid-washed glassware (e.g. vials, tubes, flasks) for aflatoxin aqueous solutions may cause a loss of aflatoxin. Special attention should be taken with new glassware. Thus, before use, soak the glassware in dilute acid (e.g. sulphuric acid, 2 mol/l) for several hours; then, rinse extensively with distilled water to remove all traces of acid (this can be checked by using a ph-paper). 4 - Decontamination procedures for laboratory wastes (1,2) were developed and validated by the International Agency for Research on Cancer (WHO).

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6 2. NORMATIVE REFERENCES This method incorporates dated or undated reference and provisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed in the hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this method only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. ISO 3696:1987: Water of analytical laboratory use - Specification and test methods ISO 5725:1986: Precision of test methods - Determination of repeatability and reproducibility for a standard test method by inter-laboratory test. 3. PRINCIPLE A test portion is either extracted with a solvent solution (methanol/water) or the solvent solution plus hexane (or cyclohexane). The sample extract is filtered, diluted with water to a specified solvent concentration, and applied to an immunoaffinity column containing antibodies specific to aflatoxins B 1, B 2, G 1 and G 2. The aflatoxins are removed from the immunoaffinity columns with neat methanol. Aflatoxins are quantified by reverse-phase high performance liquid chromatography (RP-HPLC) with post column derivatisation (PCD) involving bromination. The PCD is achieved with either electrochemically generated bromine or with pyridinum hydrobromide perbromide (PBPB) followed by fluorescence detection.

7 4. REAGENTS All reagents shall be of recognised analytical grade. Unless otherwise stated, use water complying with grade 3 of ISO 3696. 4.0 Phosphate buffer saline (PBS) [ph 7.4 ]. PBS can be prepared from potassium chloride (0.20 g), potassium dihydrogenphosphate (0.20 g), anhydrous disodium hydrogensphosphate (1.16g) [or hydrogenphosphate x 12 H 2 0 (2.92 g)] and sodium chloride (8.00 g) added to 900 ml purified water. After dissolution the ph must be adjusted to 7.4 (with 0.1 mol/l HCl or 0.1 mol/l NaOH as appropriate) and the solution made to 1.0 L. Alternatively, commercially available phosphate buffered saline tablets with equivalent properties can be used. 4.1 Sodium chloride. 4.2 Pyridine hydrobromide perbromide (PBPB) [CAS: 39416-48-3]. 4.3 Potassium bromide. 4.4 HPLC grade acetonitrile. 4.5 HPLC grade methanol. 4.6 Methanol (pure). 4.7 HPLC grade water, complying with grade 1 of ISO 3696. 4.8 Extraction solvent: Methanol (4.6) - water solution [8 + 2 (v+v)]. 4.9 Hexane (pure) or cyclohexane (pure).

8 4.10 Nitric acid, c(hno 3 ) = 4 mol/l. 4.11 Immunoaffinity column: The affinity column should contain antibodies raised against aflatoxins B 1, B 2, G 1 and G 2. The column should have a maximum capacity of not less than 100 ng of aflatoxin B l and should give a recovery of not less than 80 % for aflatoxins B 1, B 2, G 1 and not less than 60 % for aflatoxin G 2 when applied as a standard solution in methanol/water containing 5 ng of each aflatoxin. 4.12 HPLC mobile phase solvent (A): Water(4.7)/acetonitrile(4.4)/methanol (4.5) solution [6 + 2 + 3 (v+v+v)]. 4.13 HPLC mobile phase solvent (B): for use with electrochemically genearted bromine: water (4.7)/acetonitrile(4.4)/methanol(4.5) solution [6 + 2 + 3 (v + v + v)] containing 120 mg potassium bromide (4.3) and 350 µl nitric acid at 4 mol/l (4.10). NOTE 1: The mobile phase solvent (4.12/4.13) should be degassed. 4.14 Post column reagent: Dissolve 25 mg PBPB (4.2) in 500 ml H 2 O. Solution can be used for up to four days if stored in a dark place at room temperature. 4.15 Toluene/acetonitrile [98 + 2 (v + v)]. 4.16 Mixed aflatoxins stock solution for HPLC: The supplied calibrant solution contains the following aflatoxins in a toluene/acetonitrile solution (4.15): B 1, B 2, G 1 and G 2 with a concentration of 1000 ng/ml, 200 ng/ml, 1000 ng/ml and 200 ng/ml respectively.

9 Prepare a stock solution by pipetting exactly 2.0 ml of the calibrant solution into a 20 ml calibrated volumetric flask. Fill up to the mark with the toluene/acetonitrile solution (4.15) and shake well. NOTE 2: Wrap flask tightly in aluminium foil and store at less than 0 C. Before use, do not remove aluminium foil until contents have reached room temperature to avoid incorporation of water by condensation. When solution has to be stored for a longer period (e.g. 1 month), weigh flask and record any change before and after solution is to be used. 4.17 Working calibrant solutions for HPLC: Use the stock solution for pipetting the volumes as listed in Table 1 into a set of 10 ml calibrated volumetric flasks, then evaporate the toluene/acetonitrile solution just to dryness under a stream of nitrogen at room temperature. To each flask, add 4 ml of methanol, let aflatoxins dissolve, dilute to 10 ml with water, and shake well. NOTE 3: Bear in mind that methanol and water are subject to volume contraction when mixed.

10 Table 1: Preparation of working calibrant solutions Working standard Aliquot taken from stock solution in (µl) Final concentration of working calibrant in (ng/200 µl) B 1 B 2 G 1 G 2 1 40 0.080 0.016 0.080 0.016 2 120 0.240 0.048 0.240 0.048 3 200 0.400 0.080 0.400 0.080 4 280 0.560 0.112 0.560 0.112 5 360 0.720 0.144 0.720 0.144 4.18 Spiking solution: Prepare a spiking solution by pipetting exactly 2.0 ml of the supplied calibrant solution into a 10 ml calibrated volumetric flask. Then evaporate the toluene/acetonitrile solution just to dryness under a stream of nitrogen at room temperature. Fill up to the mark with methanol and shake well. The concentration of this spiking solution will be 200 ng/ml for aflatoxins B1 and G1, respectively 40 ng/ml for aflatoxins B2 and G2. NOTE 2: Wrap flask tightly in aluminium foil and store at less than 0 C. Before use, do not remove aluminium foil until contents have reached room temperature to avoid incorporation of water by condensation.

11 When solution has to be stored for a longer period (e.g. 1 month), weigh flask and record any change before and after solution is to be used. 5. APPARATUS Usual laboratory apparatus and, in particular, the following: 5.1 High speed blender/shaker High speed blender (e.g. Ultra Turrax, waring blender), explosion proof. Adjustable vertical or horizontal shaker. 5.2 Filter paper, ø 24 cm, prefolded. 5.3 Erlenmeyer flask with screw top or glass stopper. 5.4 Glass microfiber filter paper, ø 5 cm. 5.5 Reservoir, 75 ml with luer tip connector for immunoaffinity column. 5.6 Hand pump, 20 ml syringe with luer lock or rubber stopper for IAC. 5.7 Volumetric glassware of 3mL or 5 ml, 10 ml and 20 ml (shall have an accuracy of at least 0.5 %). 5.8 HPLC pump, suitable for flow rate at 1.000 ± 0.005 ml/minute. 5.9 Injection system. Capable for total loop injection. A valve with a 200 µl loop is recommended. Note 4: In the case that a different loop size than recommended is used it must be guaranteed that the limit of detection (LOD) for the

12 system is 0.2 ng/g and the limit of quantification (LOQ) is 0.5 ng/g for each aflatoxin. 5.10 RP - HPLC column (4.6 mm x 25 cm, 5 µm), e.g. LC-18 or ODS-2 (Optional but recommended: pre-column). 5.11 Post column derivatisation system with PBPB. Second HPLC pulseless pump, zero-dead volume T-piece, reaction tubing min. 45 cm x 0.5 mm internal diameter PTFE. 5.12 System for derivatisation with electrochemically generated bromine. (e.g. KOBRA cell) Note 5: In order to confirm the afltoxins B 1 and G 1 the HPLC column has to be disconnected from the bromination device and must be connected directly to the fluorescence detector (switching-off the electrical current with the bromination device still in line is no recommened due to the possibility of remaining bromine in the cell membrane of the device). 5.13 Fluorescence detector, with a wavelength of λ = 360 nm excitation filter and a wavelength of λ > 420 nm cut-off emission filter, or equivalent. (Recommended settings for adjustable detectors are Ex. = 365 nm, Em. = 435 nm, BW = 18 nm) 5.14 Disposable filter unit (0.45 µm). occur that Note 6: Prior to usage it has to be verified that no aflatoxin losses during filtration (recovery testing) since there is a possiblity various filter materials can retain aflatoxins 5.15 Single marked pipettes, 2 ml and 10 ml capacity.

13 5.16 Analytical balance, capable of weighing to 0.1 mg. 5.17 Laboratory balance, capable of weighing to 0.01 g. 5.18 Calibrated microliter syringe(s) or microliter pipette(s) (25 µl - 500 µl).

14 6. PROCEDURES 6.1. Conditioning of immunoaffinity columns: Immunoaffinity columns (4.11) should be adjusted to room temperature prior conditioning. For conditioning apply 10 ml of PBS (4.0) on the top of the column and let it pass at a speed of 2-3 ml/min through the column (gravity). Make sure that a small portion (0.5 ml) of the PBS remains on the column until the sample solution is applied. 6.2 Extraction: 6.2.1 Fig paste: Weigh, to the nearest 0.1 g, approximately 50 g of the test portion into a 500 ml Erlenmeyer flask with screw top or glass stopper. Add 5 g sodium chloride (4.1) and 300 ml methanol/water solvent (4.8). Blend for 3 min with a high speed blender (e.g. Ultra Turrax). Filter the extract using prefolded paper (5.2). Pipette 10.0 ml of the clear filtrate into a reservoir (5.5) containing 60 ml of PBS (4.0) that is placed on a conditioned immunoaffinity column. Mix with a plastic spatula and rinse residues with 1-2 ml PBS into the reservoir. Apply the solution on the column as described in section 6.3. 6.2.2 Peanut butter: Weigh, to the nearest 0.1 g, approximately 50 g of the test portion into a 500 ml Erlenmeyer flask, add 5 g sodium chloride (4.1), 200 ml methanol/water solvent (4.8) and 100 ml hexane or cyclohexane (4.9). Blend for 3 min with a high speed blender (e.g. Ultra Turrax). Note 7: Bear in mind that only high speed blending guarantees sufficient extraction results. Do not shake.

15 Filter through prefolded paper (5.2). In case of solvent layer separation (should not occur if filtration takes place immediately after blending since hexane/cyclohexane will be retained in the filter) carry on with the lower phase. Pipette 10.0 ml of the clear filtrate into a reservoir (5.5) containing 60 ml of PBS (4.0) that is placed on an immunoaffinity column. Mix with a plastic spatula and rinse residues with 1 ml - 2 ml PBS from spatula into the reservoir. Transfer solution on column as described in section 6.3. 6.2.3 Pistachios (ground): Weigh, to the nearest 0.1 g, approximately 50 g of test portion into a 500 ml Erlenmeyer flask, add 5 g sodium chloride (4.1), 200 ml methanol/water solvent (4.8) and 100 ml hexane or cyclohexane (4.9). Blend for 3 min with a high speed blender (e.g. Ultra Turrax). Note 7: Bear in mind that only high speed blending guarantees sufficient extraction results. Do not shake. Filter through prefolded paper (5.2). In case of solvent layer separation (should not occur if filtration takes place immediately after blending since hexane/cyclohexane will be retained in the filter) carry on with the lower phase. Pipette 10.0 ml of the clear filtrate into a reservoir (5.5) containing 60 ml of PBS (4.0) that is placed on an immunoaffinity column. Mix with a plastic spatula and rinse residues with 1 ml - 2 ml PBS from spatula into the reservoir. Note 8: If significant precipitation occurs during diluting with PBS, alternatively, 20 ml of the clear filtrate can be filled up with PBS to a volume of exactly 140 ml and then filtered throug a glass microfiber paper (5.4) to aviod clotting

16 of the immunoaffinity column. Use an aliquot of exactly 70 ml of this diluted filtrate for immunoaffinity column clean-up. Transfer solution on column as described in section 6.3. 6.2.4 Paprika Powder: Weigh, to the nearest 0.1 g, approximately 50 g of test portion into a 500 ml Erlenmeyer flask. Add 5 g sodium chloride (4.1) and 300 ml methanol/water solvent (4.8). Shake intensively by hand for the first 15 s - 30 s and then for 30 min. Filter the extract using prefolded filter (5.2) and pipette 10.0 ml of the clear filtrate into a reservoir (5.5) containing 60 ml of PBS (4.0) that is placed on an immunoaffinity column. Mix with a plastic spatula and rinse residues with 1 ml - 2 ml PBS from spatula into the reservoir. Apply solution on column as described in section 6.3.

17 6.3 Immunoaffinity clean up: NOTE 9: Methods for loading onto immunoaffinity columns, washing the column and elution vary slightly between column manufacturers and the specific instructions supplied with the columns should be followed precisely. In general, procedures involving sample extraction with methanol/water, filtration or centrifugation, possible sample dilution with PBS or water, loading under pressure onto (possibly pre-washed) column, washing of column with distilled water and elution of aflatoxins with methanol or acetonitrile. Pass the filtrate through the column at a flow rate of approximately 1 drop/s (approx. 3 ml/min) (gravity). Do not exceed a flow rate of 5 ml/min. Wash the column with approx. 15 ml of water (4.7) - applied in little portions of approx. 5 ml - at a flow rate of max. 5 ml/min. and dry by applying little vacuum for 5 s - 10 s or passing air through the immunoaffinity column by means of a syringe for 10 s. Elute the aflatoxins in a two step procedure: Apply 0.50 ml methanol on the column and let it pass through by gravity. Collect the elute in a calibrated volumetric flask of 3 ml or 5 ml (5.7). Wait for 1 min and apply a second portion of 0.75 ml methanol. Collect most of the applied elution solvent by pressing air through. Fill flask to the mark with water and shake well. If the solution is clear it can be used directly for HPLC analysis. If the solution is not clear, pass it through a disposal filter unit (0.45 µm) (5.14) prior HPLC injection (see Note 6, p. 12). The injection by total loop mode guarantees maximum accuracy. It is recommended (depending on the injection system, e.g. syringe or autosampler) to take a sample volume of 3 times the injection loop size and to

18 inject at least 2/3 of this volume into the valve, to ensure that the middle fraction remains in the injection loop. Thus, the loop is rinsed with the injection solvent while enough solvent remains in the valve. 6.4 Post column derivatisation: When using PBPB, mount the mixing T-piece and reaction tubing mentioned under 5.11, and then operate using the following parameters: - flow rates: 1.00 ml/min (mobile phase). 0.30 ml/min (reagent). When using electrochemically generated bromine (KOBRA cell) follow the instructions for the installation of the cell as supplied by the manufacturers and operate using the following parameters:- - flow rate: 1.00 ml/min (mobile phase) - current: 100 µa 6.5 Calibration curve: Calibration curves should be prepared using the working calibration solutions described (4.17). These solutions cover the range of 1 ng/g - 4 ng/g for aflatoxins B 1 and G 1 and the range of 0.2 ng/g - 0.8 ng/g for B 2 and G 2. Make the calibration curves prior to analysis according to Table 1 and check the plot for linearity. NOTE 10: In case that the content of aflatoxins in the sample will be outside of the calibration range, an appropriate calibration curves must be prepared. Alternatively the injection solution for HPLC analysis can be diluted to an aflatoxin content apropriate for the established calibration curve.

19 6.6 Remarks on calculation: For those sample preparation procedures that involve the use of hexane or cyclohexane, the volume of these solvents added for extraction must not be taken into account for analysis calculation. The addition of these solvents is necessary only to break possible fat layers (incapsulated aflatoxin). Aflatoxins do not dissolved in these solvents. 6.7 Spiking procedures: Add 1.0 ml of the spiking solution (section 4.18) to 50 g of the sample material (equivalent to a level 4 ng/g aflatoxins B 1 and G 1, resp. 0.8 ng/g aflatoxins B 2 and G 2 ). Let the solvent evaporate for at least 30 min. Extract the spiked material following exactly the procedure which is given in the sections 6.2.1-6.2.4. 7. PRECISION To be included after completion of the laboratory intercomparison 8. TEST REPORT The test report shall contain at least the following data: all information necessary for the identification of the sample; all information necessary for the identification of the calibrant the results and the units in which the results have been expressed; date of test; any particular points observed in the course of the test; Any operation not specified in this method or regarded as optional which might have affected the results.

20 BIBLIOGRAPHY [1] Laboratory decontamination and destruction of aflatoxins B 1, B 2, G 1 and G 2 in laboratory wastes. Castegnaro, M., Hunt D.C., Sansone E.B., Schuller P.L.Siriwardana M.G., Telling G.M., van Egmond H.P. and walker E.A. IARC Scientific publication no. 37, International Agency for Research on Cancer (WHO), Lyon (France), 1980, p. 59. [2] Laboratory decontamination and destruction of carcinogens in laboratorywastes: some mycotoxins. Castegnaro M., Barek J., Fremy J.M., Lafontaine M. Miraglia M., Sansone E.B. and Telling G.M. IARC publication no. 113, International Agency for Research on Cancer (WHO), Lyon (France), 1991, p. 63.