A new method developed by scientists at the Office of the Texas State Chemist, the state agency that regulates the distribution of feed and fertilizer in Texas, will reduce the cost of testing, improve sensitivity to toxin levels and speed up turnaround.

The project to develop this new method was led by Dr. Wei Li, postdoctoral research associate at the Office of the Texas State Chemist, and will also help gain insight for future research into the mechanics of how fumonisin forms.

Developing a new method

Fumonisin is regulated in corn because it can cause diseases in livestock and horses when ingested. Fumonisin B1 (FB1), fumonisin B2 (FB2) and fumonisin B3 (FB3) are included in the setting of Food and Drug Administration (FDA) guidelines for industry in the United States.

The Federal Grain Inspection Service (FGIS), other grain testing bodies and industry use quick tests such as enzyme-linked immunosorbent assay (ELISA) for the purposes of assessing grain quality to facilitate commerce. These methods may be used as a screening technique by feed and food safety regulatory bodies who rely upon more sensitive methods developed by the FDA or the Association of Official Analytical Chemists International (AOACI) such as thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) for the purpose of assigning violations and taking regulatory action. The latter methods are time-consuming and expensive.

One of the most serious problems with HPLC is the cost of acetonitrile, according to the director of the Office of the Texas State Chemist, Dr. Tim Herrman. The price of this reagent has increased over 10-fold and has been rationed, causing back orders.

In 2009 Li was hired to establish a fast and reproducible quantification method. He achieved this by reducing the sample extraction, derivitization and cleanup process and replacing the $1,000 /case acetonitrile with methanol.

The method involves a single step of extraction followed by centrifugation and filtration prior to analysis with an ultra-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (UPLC/ESI/MS/MS).

“Taking advantage of the exclusion of conventional immunoaffinity cleanup procedure from the sample preparation, the time for the whole protocol is less than 30 minutes. The developed method could be satisfactorily applied as a routine procedure to identify and quantify fumonisins in laboratories of food/feed quality and safety control when a large sample load is required,” Li says.

Li’s method includes running the isotope labeled internal standard with every sample in addition to creating a calibration curve and using a control at the beginning and end of each set.

“Running the isotope with every test increases the reproducibility of measuring fumonisins FB1, FB2 and FB3,” Herrman says.

The improved extraction method and application of LC/MS/MS technology reduces the office’s turnaround time from 24 samples in two days to 24 samples in four hours, increasing productivity fourfold. They’re also now able to use this extraction for multiple analyses whereas the old method forced them to run one sample at a time.

Research community benefits

Although Li’s new method is currently being used by the Office of the Texas State Chemist for regulatory purposes, Herrman says it will improve research activities by other individuals at Texas A&M who are interested in fumonisins.

“Plant pathologists [and other grain quality researchers] will benefit from the method’s sensitivity to measuring mycotoxins,” Herrman says. “Our ability to work with very small samples, as small as a single kernel, will also be of value. For regulatory purposes, we use a 50-gram sample so it is more representative of the material, but in research applications, working with smaller samples can provide important results. Whether the research is related to trying to understand the mechanism which the fungus works to produce the toxin or it’s looking at resistance mechanisms, they will benefit from Li’s method.”