Food Contaminants - American Chemical Society

Chapter 17

Ethanol Extraction Method for a Rapid Test for Aflatoxin in Corn Nancy Zabe, Kedist Ayalew, and Stephen Powers

Downloaded by CORNELL UNIV on December 18, 2014 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch017

VICAM, 313 Pleasant Street, Watertown, MA 02472

Methanol-water mixtures or acetonitrile-water mixtures have been used for years for the extraction of aflatoxin from corn, grains, and nuts. Many labs are concerned about the health hazard of acetonitrile and the hazardous waste disposal charges for both acetonitrile and methanol. The data in this presentation show that ethanol can be used as a substitute for methanol in a rapid method for total aflatoxin determination using immunoaffinity column chromatography with fluor escence detection (AflaTest). The ethanol extraction method meets the same performance specifications for precision, accuracy and limit of detection as the methanol extraction method. The results from the rapid method using ethanol and water extraction also correlate with an HPLC method using methanol extraction. Ethanol can then be used rather than methanol as a solvent for the rapid determination of aflatoxin thus reducing hazardous waste generation.

© 2008 American Chemical Society

In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

297


298 Aflatoxins are naturally occurring toxins produced by Aspergillusflavus,A. parasiticus, and other Aspergillus fungi growing on corn, grains, nuts, spices, cottonseed, and other food products. Aflatoxin is classified by the International Agency for Research on Cancer (IARC) as a group one (proven) carcinogen along with being hepatotoxic and immunosuppressive (7). Aflatoxins are regulated worldwide at levelsfromzero to 35 ppb (jig/kg) (2). VICAM's aflatoxin test kit using methanol-water (80:20, v/v) extraction has received United States Department of Agriculture - Grain Inspection, Packers and Stockyards Administration (USDA-GIPSA) Certificate of Conformance no. FGIS 2006-101 as a quantitative test kit for the determination of total aflatoxins in corn, condensed distillers solubles, corn bran, corn flour, corn germ meal, corn gluten feed, corn gluten meal, corn meal, corn soy blend, distillers dried grains, distillers dried grains with solubles, flaking corn grits, milled rice, popcorn, rough rice, sorghum, soybeans, and wheat. The objective of this study was to generate supporting data for VICAM's AflaTest® procedure to detect aflatoxin in corn for food or animal feed using immunoaffinity column chromatography with fluorescence detection while substituting ethanol for the methanol used in extraction and elution. Results generated were evaluated using performance criteria set by the USDA-GIPSA (3).

Materials and Methods Materials Immunoaffinity columns (AflaTest®), filter papers, bromine developer solution, and fluorometer calibration standards were obtained from VICAM (Watertown, MA). Deionized water was from a MilliQpius system (Millipore Corp). Ethanol was not denatured: HPLC/spectrophotometric Grade Ethanol, 200 proof (example: Sigma cat # 459828). Methanol was HPLC grade. A l l other reagents were ACS grade or better. A VICAM Series 4fluorometerwas used for quantitation.

Procedure This study determined the limit of detection, accuracy, precision, and correlation with LC values. The limit of detection was determined by multiple measurements on aflatoxin-free corn. Accuracy and precision were determined using aflatoxin-free corn samples spiked at five and 20 ppb total aflatoxins (in the ratio of 10 aflatoxin B,: 1 aflatoxin B : 1 aflatoxin G : 1 aflatoxin G ). Correlation with HPLC values was determined by assaying four well-ground 2

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299 and mixed naturally aflatoxin contaminated corn samples that had previously been quantified by HPLC. The ethanol extraction and elution method for aflatoxin quantitation is as follows. Fifty g of ground corn sample was blended for one min with 5 g of sodium chloride and 100 mL ethanol-water (80:20, v/v). The mixture was passed through a medium filter paper (VICAM part #31240) and the extract collected in a clean vessel. Ten mL of the extract was diluted with 20 mL of water and the extract was filtered again through a microfiber filter paper (VICAM part #31955) to remove particles. One mL of the extract was passed through an AflaTest® immunoaffinity column (VICAM part #12022), containing immobilized antibodies to aflatoxin, at a rate of about one drop per second. The column was washed with two one mL portions of water at a rate of about one drop per second. The aflatoxin was eluted from the immunoaffinity column by one mL of ethanol at a rate of about one drop per second and the one mL eluate collected in a glass cuvette. A developer solution (VICAM part #32010) consisting of a dilute bromine solution was added to the cuvette to enhance thefluorescenceof aflatoxin B\ and The cuvette was then read after 60 sec in a VICAM series 4fluorometercalibrated withfluorescentcalibration standards (VICAM part #33030) setting the red calibration vial to 180 and the green calibration vial to -3.0. Thefluorometerreadout was in parts per billion (ppb, jig/kg). The methanol extraction and elution method was the same as above with the following exceptions. The sample was blended with 100 mL methanol-water (80:20, v/v) rather than ethanol:-water. The immunoaffinity column was eluted with 1 mL methanol rather than ethanol. The fluorometer was calibrated by setting the red vial to 160 rather than 180. The change in calibration compensates for a slight difference influorescenceof aflatoxin in methanol compared to ethanol. The green vial setting remained the same at -3.0. The methanol extraction and elution method is a modification of the AOAC International Official Method 991.31 {4).

Results and Discussion Limit of Detection The performance evaluation standards from the USDA-GIPSA for aflatoxin kits define the Limit of Detection (LOD) as equal to the mean plus twice the standard deviation (SD) for an aflatoxin-free ground corn sample. An aflatoxinfree corn sample (less than 0.2 ppb aflatoxin) was spiked with one ppb of aflatoxin. Both the aflatoxin-free and one ppb sample were extracted and the extract assayed ten times. Results are presented in Table I. The mean plus twice the SD of an aflatoxin-free corn sample is zero ppb. The USDA-GIPSA


300 evaluation standard for LOD is less than or equal to 3.0 ppb. The ethanol-water extraction method meets the USDA-GIPSA standard for LOD. In order to obtain an actual value for limit of detection, we have tested a corn sample spiked at one ppb aflatoxin as well. Defining LOD as the minimum level at which aflatoxin can be reliably detected, the ethanol-water extraction method can detect aflatoxin at one ppb.

Table I. Limit of Detection


Sample Aflatoxin-free com/ ppb Spiked Corn 1 2 3 4 5 6 7 8 9 10 Mean SD %RSD

0 0 0 0 0 0 0 0 0 0 0 0 0

1.6 1.9 1.3 1.2 0.78 0.66 0.45 0.34 1.2 1.1 1.1 0.50 47%

Accuracy and Precision Five aflatoxin-free ground corn samples were spiked at 5 ppb and five were spiked at 20 ppb. Total aflatoxin was spiked in a ratio of 10 aflatoxin B : 1 aflatoxin B : 1 aflatoxin G : 1 aflatoxin G . Each sample extract was analyzed once. Results are presented in Table II. The USDA-GIPSA performance evaluation standards for accuracy and precision are listed in Table II. The ethanol extraction and elution method is accurate and gives 18% RSD at 5 ppb and 4.6% RSD at 20 ppb. The method meets USDA-GIPSA performance standards for both accuracy and precision. x

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Correlation with HPLC and Methanol Extraction Four ground corn samples naturally contaminated with aflatoxins at approximately 5, 10, 20, and 100 ppb were extracted in duplicate using the


301 Table II. Accuracy and Precision ~ I ] Corn Sample

.. . ] Spike Level

USDA-GIPSA Standards

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Mean SD % RSD

5 ppb spike 6.5 4.4 4.8 4.4 4.5 4.9 0.90 18

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Food Contaminants - American Chemical Society

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