Plastic minicolumn for aflatoxin detection
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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.
- 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.
- 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:
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
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.
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