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, postdoctorial 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. The mold by-product is also a threat to human health. The International Agency for Research on Cancers has declared fumonisin B1, the most abundant form of the fumonisins, to be a group 2B carcinogen, or possibly carcinogenic to humans.
Considering their widespread prevalence and potential toxicities, fumonisin B1 (FB1), fumonisin B2 (FB2) and fumonisin B3 (FB3), the major fumonisins found in naturally contaminated corn, 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 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. These later methods are time-consuming and expensive.
One of the most serious problems with HPLC involved the cost of acetonitrile according to the director of the Office of the Texas State Chemist, Dr. Tim Herrman. The cost of this single reagent has increased in price over 10-fold and has been rationed, where back orders are common place for the past few years. The reagent is a by-product of the plastic manufacturing industry that has been impacted by a number of factors including the global recession.
In 2009 Li was hired to research and establish a fast and reproducible quantification method that would solve the office’s two main problems. He achieved this by reducing the sample extraction, derivitization and cleanup process and replacing the nearly $1,000 per case reagent with methanol, a more economical reagent which runs about $50 per case.
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). The LC/MS/MS method developed here represents the fastest and simplest procedure that takes less than 30 minutes, with the higher throughput analysis than both the conventional HPLC methods and other LC/MS methods involved with solid phase extraction (SPE) cleanup.
Herrman explains another advantage of Li’s new method.
“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..”
The improved extraction method and application of LC/MS/MS technology reduces the office’s turnaround time from 24 samples in two days with their previous testing method to 24 samples in 4 hours, increasing productivity four-fold. 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.”
Future grain research will benefit from the improved sensitivity and level of detection (LOD) offered by mass spectrometry. Both the LOD and level of quantitation (LOQ) has improved 50 fold compared to HPLC. The improved sensitivity, the ability to test FB1, FB2 and FB3 simultaneously and quick turnaround of results will cut down on the time and expense researchers encounter when exploring new methods to control fumonisins.
Universities such as Iowa State University and Purdue with grain quality research initiatives may invest in the type of system Li invented in the years to come.
Grain companies benefit
Using the methanol as a reagent in their new testing method, the Office of the Texas State Chemist has significantly reduced its operating costs for this test. The adoption of new technology enables the Office to provide improved service without adding cost to the regulated community.
“By operating on or near the technology frontier through the adoption of new instrumentation, we can provide much better service at a lower cost,” says Herrman. “We have experienced a significant return on capital investment through the adoption of a technology strategy that ultimately reduces cost and enables the office to not raise tonnage fees that pay for the regulatory service we provide for consumers and agribusiness. We’ve been able to hold our tonnage rates steady for the past six years and I don’t anticipate that we will need to raise fees in the foreseeable future.”
They’ve also been able to reduce their staff while managing to increase oversight and the reliability of their results, thus improving the regulatory process.
“Good regulation is good for everyone,” says Herrman. “I like to refer to it as the currency of confidence. As long as consumers have confidence in the marketplace and agribusiness has confidence that they are competing on a level playing field, commerce will happen. When people lose confidence in the safety or reliability of products, it impedes commerce. Poor regulatory oversight increases risk for industry, which in turn, may result in a firm’s decision not to invest or expand in a particular market.”
Although more regulation is never welcome in our industry, improving the methods by which regulation is done can positively affect the feed and grain stakeholder.
Through research, not only has the Office of the Texas State Chemist stopped using a very expensive reagent and cut costs, it has increased the reproducibility of results as well as level of detection and is an example of a regulatory body moving forward and making progress.
