FluoroQuant® Afla Plus Test Kit for Aflatoxin Testing Granted Performance TestedSM Status by AOAC

The Aflatoxins are a chemically related group of compounds that are important hepatotoxic mycotoxins. They are produced by fungi, and can contaminate several important crops including corn, peanuts, and tree nuts under favorable environmental conditions.

These toxins can cause liver disease in humans and animals, a decrease in milk and egg production, and are immunosuppressive, carcinogenic and mutagenic. The most commonly occurring and most potent of the aflatoxins is Aflatoxin B1. The major fungi associated with production of the aflatoxins are Aspergillus flavus and Aspergillus parasiticus, with the latter being particularly important in contamination of peanuts.

Warm temperatures with drought-like conditions cause grain to become more susceptible to aflatoxin formation in the field. In addition, insect damage can allow the organism to access individual kernels, thereby increasing the chance of aflatoxin contamination in an infested crop. The toxins may also be formed under poor storage conditions in which the grains are kept in a high temperature, high moisture environment or are inadequately dried.




Figure 1:Structure of Aflatoxin B1


Aflatoxin concentrations are typically measured quantitatively using ELISA technology, HPLC, or fluorometric test kits. ELISA technology often requires long incubation times, skilled analysts, and is more conducive to analysis of batches of samples rather than single samples. HPLC requires expensive equipment and highly trained analysts. In addition, many approved HPLC methods or HPLC methods used in collaborative studies require long sample cleanup or analysis times.

Fluorometric test kits have the advantage of speed, ease of use, and are applicable to single-sample testing. These are highly desirable qualities in an environment where single samples or small numbers of samples must be tested rapidly.





FluoroQuant® Afla Plus is an improved fluorometric test kit which has been granted Performance TestedTM status by AOAC.

This kit employs a solid-phase, single-step clean-up column followed by fluorometeric analysis to determine aflatoxins in corn, almonds, and peanuts. Extraction uses acetonitrile/water or methanol/water (optional for peanuts). This quantitative method is rapid, accurate, inexpensive, and can be applied to individual samples. It is simple enough for use in grain processing facilities, feed and food handling facilities, and export terminals. It is also suitable for use in analytical laboratories.

The method is straight forward and user friendly. A sample is first collected, ensuring that a representative sample of the batch to be tested is taken. The sample is ground, and a 50g portion is placed in a blender jar.

Corn, peanuts, and almonds are extracted with 86/14 acetonitrile/water. Optionally for peanuts, 80/20 methanol/water may also be used in place of acetonitrile/ water. The samples are blended for 30 seconds (for corn) or 1 minute (for almonds and peanuts) and filtered into a glass or polypropylene container. From this extract, 2mL of sample is placed on a SolSep® 2001 cleanup column, included with the FluoroQuant® Afla Plus test kit. If the methanol extraction option has been used for peanuts, the samples are blended for 3 minutes.

Following extraction, 1 mL of sample and 1 mL of diluent are added to the column and mixed with the pipette tips.

The samples are then pushed through the column, and a 1mL portion of the purified extract is placed into a clean cuvette.

1mL of developer is added to the purified sample, the sample is mixed using a vortex, and is placed in a calibrated fluorometer.

The fluorometer displays the result in ppb following a preprogrammed delay. The method is very rapid and can be completed in five minutes or less including extraction.





AOAC Performance TestedSM Status

Performance testing by an independent, international organization provides consumers with confidence that an independent third party has evaluated a product and found that it complies with its performance claims.

The AOAC Research Institute is a non-profit, scientific organization which provides internationally recognized performance testing of analytical methods. They serve as an independent third party, and grant Performance TestedTM status to test kits that are evaluated and found to be in compliance with their claims.

To achieve Performance TestedTM status, Romer Labs® performed an internal validation study and submitted a validation report supporting the FluoroQuant® Afla Plus test kit’s performance claims to a General Referee, who is an expert in the field of mycotoxin analysis. The General Referee distributed the report to an internationally-based panel of experts for review.

Kits were also sent to an independent analytical laboratory, approved by AOAC-RI, for testing. The General Referee and experts then reviewed the validation report and results of the independent laboratory’s testing before granting the kit“Performance TestedTM” Status.

FluoroQuant® Afla Plus Validation

To verify kit performance, Romer Labs® conducted a validation study in which naturally contaminated and spiked samples of corn, and spiked samples of raw almonds and peanuts were analyzed on several kit lots over a period of several days.

The results were compared to an established HPLC reference method. In addition, stability and ruggedness of the test kit method was examined.

To determine the LOD, ten samples of each non-detect commodity were extracted and analyzed according to the test kit method. The LOD was determined to be the mean plus 2 times the standard deviation. The LOD of the kit varied by commodity and was found to be 1.9 or less for all tested commodities, as shown in Table 1.





To test accuracy and precision of the test kit, naturally contaminated samples of corn and fortified samples of corn, peanuts, and almonds were tested according to the kit method.

Twenty-one individual corn samples of naturally contaminated material at each aflatoxin level were extracted and analyzed by three analysts who did not know the level of analyte.

In addition, five replicates of each fortified level of corn, peanuts, and almonds were analyzed by three analysts using the kit. The kit was shown to perform very well on corn naturally contaminated with aflatoxins within the range of non-detect to 100ppb. All average results fell within the acceptable ranges, and at least 95% of the individual results fell within range.

The test kit results agreed well with the HPLC method results.

Recoveries from fortified samples ranged from 84 – 92%. The kit also performed very well on peanuts and almonds fortified with aflatoxin. All average results fell within the acceptable ranges, and 100% of the individual results also fell within range.

Recoveries from fortified samples ranged from 85 – 115%. The measurement uncertainty for corn was found to be ±2ppb at 5ppb, ±5ppb at 10ppb, ±6ppb at 20ppb, and ±21ppb at 100ppb at a 95% confidence interval.

The kits were also tested for ruggedness and stability. The kit showed good results over changes in environmental temperature, variations in extraction time, extract age, cleanup timing, and developer age.

Table 1:LODs of Validated Commodities

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Table 2:Naturally Contaminated Corn – Independent Laboratory Testing

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Table 3:Spiked Raw Unblanched Peanuts, Raw Blanched Peanuts, and Raw Almonds

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Table 4:Recovery Data

ABOUT THE AUTHOR
	Name: Donna Houchins, BSc. Position: Analytical Chemist Education: Truman State University Since 2000: Analytical Chemist R&D - Romer Labs Inc. 1999-2000: R&D Laboratory Assistant, Truman State University