Plastic minicolumn for aflatoxin detection

Contents - Previous - Next

by Srisit Karunyavanij

 

A method for determination of aflatoxin in food and agricultural products suitable for a rural setting is needed for quality testing of the products. The method should require inexpensive equipment, easy to operate by inexperienced persons, and be sensitive enough to detect aflatoxin down to the tolerated limit of 20 ppb. (microgram/ kilogram). To fulfill this goal, a technique using plastic minicolumn, a kind of small chromatographic tube, was developed by the Department of Medical Sciences of Thailand. With this method, aflatoxin could be measured within two hours with an accuracy of lower than 10 ppb. The method is described in this paper.

The development of this method started since 1976 based on some minicolumn procedures of Holyday, dip column, Velasco et al, Romer etc. by using 5 ml. pipette.

In 1979, glass minicolumn with conical shape on the top part, 4 mm. diameter and 16 mm. length was developed. This minicolumn can contain 1 ml. of the extraction. Alumina, silica gel and florisil were packed in the column. Sodium sulfate anhydrous was used to absorb moisture, Aflatoxin was examined as a blue fluorescent band at the top of the florisil layer under high intensity long weave UV lamp (365 µm.). This procedure can be completed in 1 hour starting from extraction and has a limit of detection of 5 µg/kg.

After this modified glass minicolumn was developed and used at the Regional Center of Medical Sciences for sometime, there was a problem of the changes of the packing materials in the glass column which led to more time consumed to finish the procedure.

In 1981, plastic minicolumn was investigated using clear polyethylene straw (without any UV fluoresing substances) instead of glass column. The diameter of this plastic minicolumn is 6 mm. and 25 cm. in length. Same kinds of materials were packed in the column. Moisture and water traces were absorped by calcium sulfate anhydrous. The precipitating solution was a mixture of zinc acetate and saturated ammonium sulfate which will increase the precipitation property in the interaction to reduce interfering substances in the extraction. Saturated NaCI was used as precipitating solvent that will precipitate more in higher acidity while aflatoxin will be extracted more easily.

This procedure can be completed within 15 minutes and has a limit of detection of less than 10 ppb. Final extract from this procedure can also be used for TLC.

 

MATERIALS AND METHODS

Solid samples such as grain or feed should be finely ground and mixed very thoroughly.

Apparatus

  1. High speed blender(1 It-explosion-proof feature not required)
  2. Ultraviolet light - Longwave UV with intensity of 430 watt/cm² at 15 cm. at 365 µm, or Chromatovue cabinet.
  3. Plastic minicolumn - clear polyethylene straw (non fluorescent materials) 6.0 mm. in diameter and 250 mm. in length.
  4. Glassware
    4.1 beaker (250 ml.)
    4.2 cylinder 25,50 and 100 ml.
    4.3 funnel (75 mm.)
    4.4 filter paper (Whatman No.1) 125 ml.
    4.5 Test tube 15 x 150 mm.
    4.6 Pipette pasteur or graduated pipes (5 cm³)
    4.7 Glass rod 150-170 mm.
  5. Reagents
    5.1 Organic solvents: Methanol, Acetone, Chloroform, Benzene, Ispropanol (Analytical Grade)
    5.2 Extracting solvents: Methanol: 4% KCI or 5% NaCI (60:40), Burning alcohol + 1% Salt solution 60+40
    5.3 Precipitating solution:
  1. Zinc acetate solution: dissolve 125 9 Zn (OAc)2 and 62.5 9 (NH4)2SO4 in distilled water, add 1 ml. glacial acetic acid, add water and make up to 1000 ml.
  2. 0.1 MH3PO4: dissolve 5.6 ml H3PO4 spgr. 1.750 in distilled water, make up to 1000 ml.

5.4 Diatomaceous earth: Celite 545 or Hyflo-Super Cel
5.5 Eluting solution: Chloroform: Acetone (90:10)
5.6 Packing materials:

  1. Alumina Neutral: E. Merck 70-230 mesh Astm Brockman Activity I, Art 1077.
  2. Calcium Sulfate anhydrous fine.
  3. Florisil: E. Merck 60-100 mesh Astm Art 12518.
  4. Silica gel 60: for c.c.F. Merck No. 7734.
  5. Sodium sulfate anhydrous.
  6. Defatted cotton wool. (The packing materials a, c and d should be dried at 110°C for 1-2 hours twice a week and stored in a desiccator over silica-gel).

 

COLUMN PREPARATION

Tamp a small plug of cotton wool on the bottom of the column and then add the packing materials: calcium sulfate anhydrous (10 mm), florisil (10 mm), silica gel (20 mm), alumina neutral (15 mm) and calcium sulfate anhydrous (10 mm) in a consecutive order. Tamp a small piece of cotton wool on the top (as shown in fig.1). Tap the columm between addition of the materials to ensure even packing. Then seal the column with alcohol lamp.

 

PURIFICATION OF THE EXTRACT

  1. Weigh 50 9 of the prepared sample and put into a conical flask. Add 200 ml of extracting solvent and shake contents of the flask well by a shaker for 30 min. or blend with a medium speed blender for 3 min.
  2. Filter contents through a folded paper filter into a graduated cylinder.
  3. Take 40 ml of the filtrate and pour into a 250 ml beaker. Add 20 ml of zinc acetate solution, stir, and mix well. Leave for 1 min. Then add 40 ml of 0.1 MH3PO4, stir and mix well, and stand for 5 min. Add 5 9 of celite. Mix well.
  4. Filter through a folded paper filter into a graduated cylinder.
  5. Take 50 ml of filtrate and place the solution into a separating funnel. Add 4 ml of benzene, tighten the glass stopper and shake vigorously for 1 min. Leave until the solutions are separated. Collect benzene from the upper layer and discard the lower layer. Use 1-2 9 Na2 SO4 anhydrous to clear or lessen emulsion in benzene solution.

 

COLUMN CHROMATOGRAPHY BY A MINICOLUMN

This minicolumn screening technique falls in the category of semi-quantitative methods used in testing foodstuffs for aflatoxin content. It is very useful in screening and grading food products. The procedures are as follow:

Cut both ends of the column and hold the column in place with a clamp. Place a test tube under the column to collect eluate. Add 1 ml of benzene final extract to the column. Let the solvent run down to the surface of the sorbant layer. Add eluting solution 4 times at 1 ml each time. Let all the solvent run down into the test tube. To hasten the flow, use a small rubber bulb with gentle pressure.

Inspect the minicolumn in the dark with a high intensity, long wave UV light (365 µm). Compare results with a standard minicolumn of 20, 50, and 100 ppb. If a bluish-green fluorescent band is detected at the proper height (as shown in Fig.1), the sample is judged to be positive to aflatoxin. The higher intensity shows high concentration of aflatoxin.

Good estimation by this method depends on the following factors: 1) skills of analyst, 2) UV light intensity, and 3) darkness of the place used in viewing the chromatogram.

When the sample's column shows higher intensity when compared with standard 20 ppb minicolumn, this means that the sample has aflatoxin higher than 4 ppb but less than 20 ppb. If the sample's column has a higher intensity than the 20 ppb standard but lower than the 100 ppb standard minicolumn, the sample has an aflatoxin concentration from 20-100 ppb.

When the sample's column has higher intensity than the 100 ppb standard, the sample has more than 100 ppb aflatoxin. Aflatoxin concentration in this sample can be estimated by diluting the final extract (1 ml of the extract eluded with 4 ml eluting solvent in another column) and comparing again with 100 ppb standard. If the diluted column has about the same intensity as the 100 ppb standard, the sample has an aflatoxin content of around 500 ppb.

 

REFERENCES

  1. Barabolak, R., Colburn, C.R. and Smith, R.J.J. Assoc. Off. Anal. Chem. 1974, 57: 764-766.
  2. Goldblatt, A.F., Pons, W.A. Jr., and Goldblatt. L.A.J. Assoc. Off. Anal. Chem. 1972, 55: 1114-1119.
  3. Danteman, J. and Stoloff, L.J. Assoc. Off. Anal. Chem. 1972, 55: 139-141.
  4. Hesseltine, C.W.C. and Shorwell, Q.L.Pure Appl. Chem. 1983, 35: 259-266.
  5. Holaday, GE. and Lansden, J. Agric. Food Chem. 1975, 23: 1134-1136.
  6. Holaday, C.E. J. Am. Oil Chem. Soc. 1976, 53: 603-605.
  7. Jemmali, M.J.Assoc. Off. Anal. Chem. 1973, 56: 1512-1513.
  8. Knake, R.P., Rao, C.S. and Deyoe, C.W. Feedstuffs. 1972, 44:32.
  9. Mc Kinney, J.D.J. Am. Oil Chem. Soc. 1975, 52 : 213
  10. Romer, T.R.J. Assoc. Off. Anal, Chem. 1975, 58: 500-506.
  11. Romer, T.R. and Campbel, A.D.J. Assoc. Off. Anal. Chem. 1976, 59: 110-117.
  12. Romer, T.R., Ghouri, N. and Boling, T.M. J.Am. Oil Chem. Soc. 1979, 56: 795-797.
  13. Shannon, G.M., Stubblefield, S.D. and Shotwell, O.L.J. Assoc. Off Anal. Chem. 1973, 56: 1024-1025.
  14. Velasco, J.J. Am. Oil Chem. Soc. 1972, 49: 141-142.
  15. Velasco, J. Assoc. Off. Anal, Chem. 1972, 55: 1357-1360.
  16. Velasco, J. Assoc. Off. Anal. Chem. 1975, 58: 757-763.
  17. Official Methods of Analysis. 1980. 3rd Ed., AOAC, Washington, DC.

Contents - Previous - Next