A TD-700 Laboratory Fluorometer Suggested Method for Aflatoxin Analysis

1. SCOPE AND APPLICATION

1.1 This test method is an adaptation of the VICAM Aflatest™ Mycotoxin Testing System. It provides a fast, simple, and accurate procedure for determining low levels of aflatoxin in corn, milo, rice, peanuts, peanut butter, tree nuts, cottonseed, feed and grain samples.

1.2 This method incorporates the detection of all the major aflatoxins including, aflatoxin B1, B2, G2, M1, without the use of toxic solvents like chloroform or methylene chloride.

2. SUMMARY OF METHOD

2.1 The aflatoxin-containing materials are extracted with a 60% or 80% methanol-water extraction mixture with the aid of a high speed blender. The slurry is filtered through a gravity filter, diluted with D.I. water, and then filtered through a microfibre filter. The filtrate is then passed through an Aflatest affinity column and washed with distilled water. The aflatoxins are eluted with HPLC grade methanol into a glass cuvette. A developer solution is added to the elutant and the corresponding fluorescence is measured.

3. APPARATUS AND EQUIPMENT

• TD-700 Fluorometer with standard PMT (P/N 7000-009)
• Long Wavelength UV Filter Kit (P/N 10-032R), which includes 2 Near UV Mercury Vapor lamps (P/N 10-049), 300—400 nm excitation filter (P/N 10-069R), and 410—600 nm emission filter (P/N 10-110R-C)
• 13 mm round test tube adaptor (P/N 7000-981)
• 13 x 100 mm borosilicate glass test tubes (P/N 10-031)

3.1 Aflatest Affinity Column. Short 2 mL Aflatest-10 Affinity Column (specific for AFB1 and AFB2) or the Aflatest-P Affinity Column (specific for AFB1, AFB2, AFG1 and AFM1).

3.2 High speed blender and blender jar.

3.3 Fluted filter paper.

3.4 Microfibre filters.

3.5 Labware: All reusable labware (glass, polyethylene, Teflon, etc.) should be cleaned by soaking in laboratory grade detergent and water for 4 hours, rinsed with tap water, distilled deionized water, and methanol.

3.5.1 Assorted Class A calibrated pipettes.

3.5.2 Graduated cylinder, 100-mL.

3.5.3 Filtration and dilution cups (3).

3.5.4 Assorted volumetric flasks for preparing dilution standard.

3.5.5 Disposable 13 mm glass cuvettes.

4.0 REAGENTS AND STANDARDS

4.1 Methanol, HPLC grade.

4.2 Water, Deionized.

4.3 Salt, NaCl (5 g).

4.4 Aflatoxin Stock Standard Solution: Aflatoxin B1, B2, G1, G2, or M1 from a commercial supplier will be shipped in a sealed ampule. Weigh contents to the nearest 0.1 mg. Transfer to a volumetric flask and reweigh empty ampule. Determine by difference the mass of aflatoxin added to the flask. Dilute with determined solvent matrix. Label flask and freeze to store.

4.5 Laboratory Reagent Blank: The blank is prepared by extracting an uncontaminated portion of the material to be analyzed. It should be the last portion to be extracted in the sample set. It is used to measure any background interference due to contaminated reagent or apparatus.

4.6 Aflatoxin Dilution Standard Solutions: Make appropriate dilutions of the stock standard solution when preparing the aflatoxin standards. The dilution should be representative of the range to be analyzed.

4.7 Aflatest Developer Solution: Dilute 5.0 mL of Aflatest developer concentrate with 45.0 mL of distilled deionized water.

4.8 Salt: Sodium chloride, NaCl (5.0 g).

5.0 CALIBRATION AND STANDARDIZATION

5.1 Calibration

5.1.1 Install filter 10-069R excitation and emission filter 10-110R-C. For further assistance refer to Section III (Optical Filter Installation and Removal) on page 9 of the TD-700 Operating Manual.

5.1.2 Insert the Near U.V. Mercury Vapor lamp (P/N 10-049). Then turn on the instrument and allow a 10 minute warm-up period. For further assistance refer to Section IV (Lamp Installation and Removal) on page 11 of the TD-700 Operating Manual.

5.1.3 Calibrate instrument in the direct concentration mode with the prepared calibration standard. Be sure to shake well and allow all bubbles to rise to the surface of the cuvette before taking a reading. For further assistance refer to Section IX (Calibration: Multi-Optional Mode - Direct Concentration) on page 22 of the TD-700 manual.

5.1.4 Retain cuvette of calibration standard and blanking solution for future calibrations.

6.0 PROCEDURE

6.1 Extraction of Filter Samples

80% Methanol-Water Extraction - For corn, cottonseed, feed, and grain samples.

6.1.1 Prepare 100 mL methanol-water extraction solvent.

6.1.2 Gravity filtration setup: Fold edges of fluted filter paper over a filtration cup.

6.1.3 Diluted extract filtration setup: Place clean microfibre filter into a small funnel and place in a filtration cup.

6.1.4 Weigh 50 g of ground sample into blender jar.

6.1.5 Add 5 g of NaCl to sample.

6.1.6 Add 100 mL of methanol-water extraction solvent to jar.

6.1.7 Cover and blend for 1 minute.

6.1.8 Pour extract into fluted filter paper assembly.

6.1.9 Pipette 10.0 mL of filtered extract into a clean cup and dilute with 40.0 mL deionized water.

6.1.10 Filter diluted extract through microfibre filter. Collect filtrate in a clean filtration cup.

60% Methanol-Water Extraction - For peanuts, peanut butter, tree nuts, and nut products.

6.1.1 Prepare 125 mL methanol-water extraction solvent.

6.1.2 Gravity filtration setup: Fold edges of fluted filter paper over a filtration cup.

6.1.3 Diluted extract filtration setup: Place a clean microfibre filter into a small funnel and place in a filtration cup.

6.1.4 Weigh 25 g of ground sample into blender jar.

6.1.5 Add 5 g of NaCl to sample.

6.1.6 Add 125 mL of methanol-water extraction solvent to jar.

6.1.7 Cover and blend for 1 minute.

6.1.8 Pour extract into fluted filter paper assembly.

6.1.9 Pipette 10.0 mL of filtered extract into a clean cup and dilute with 40.0 mL deionized water.

6.1.10 Filter diluted extract through microfibre filter. Collect filtrate in a clean filtration cup.

6.2 Affinity Chromatography

6.2.1 Remove buffer solution from Aflatest affinity column.

6.2.2 Pipette 1.0 mL of filtered extract onto Aflatest column.

6.2.3 Push extract through with syringe.

6.2.4 Pipette another 1.0 mL of filtered extract onto Aflatest column.

6.2.5 Push second portion through column with syringe.

6.2.6 Fill column with deionized water.

6.2.7 Push water through with syringe.

6.2.8 Repeat wash with water. (If necessary, the column can be washed several times.)

6.2.9 Completely push out last wash portion through column.

6.2.10 Pipette 1.0 mL HPLC grade methanol through Aflatest column and collect elutant in cuvette.

6.2.11 Add 1.0 mL of diluted Aflatest Developer directly to elutant in cuvette.

6.3 Sample Analysis

6.3.1 Place cuvette in calibrated fluorometer to measure fluorescence.

6.3.2 Record concentration readout. For further assistance refer to Section X (Reading Samples) on page 27 of the TD-700 manual.

7. REFERENCES

1. Aflatest™ Mycotoxin Testing System - User's Guide, Vicam, Watertown, MA.

2. Lillehoj, E. B., T. J. Jacks, and O. H. Calvert, Aflatoxin Estimation in Corn by Measurement of Bright Greenish-Yellow Fluorescence in Aqueous Extracts, Journal of Food Protection. 49: 623-626 (1986).

3. Ferguson-Foos, Jane and J. D. Warren, Improved Cleanup for Liquid Chromatographic Analysis and Fluorescence Detection of Aflatoxins M1 and M2 in Milk Fluids, J. Assoc. Off. Anal. Chem. 67, 1111-1114 (1984).

4. Mehan, V. K., T. N. Bhavanishankar, and J. S. Bedi, Comparison of Different Methods for Extraction and Estimation of Aflatoxin B1 in Groundnut, J. of Food Science and Technology, 22, 123-125 (1985).

8. SUMMARY

1. Extraction

a. Weigh sample into blender.

b. Add 5 g NaCl (salt)

c. Add appropriate Methanol:Water extraction solvent.

d. Cover and blend for 1 minute.

e. Pour extract into fluted filter paper setup.

2. Extract Dilution

a. Pipette specified amount of filtered extract into a clean container.

b. Dilute with specified amount of D.I. water.

c. Filter through microfibre filter.

3. Affinity Chromatography

a. Setup Affinity column.

b. Pass filtered extract though column.

c. Wash twice with D.I. water.

d. Elute aflatoxins from column with HPLC grade methanol.

e. Collect in a glass cuvette.

f. Add diluted Aflatest Developer to elutant in cuvette.

g. Place cuvette in calibrated fluorometer and measure corresponding fluorescence.