A single extraction method for the analysis by liquid chromatography/tandem mass spectrometry of fumonisins and biomarkers of disrupted sphingolipid metabolism in tissues of maize seedlings

Authors: Zitomer, Nicholas; Glenn, Anthony - Tony; Bacon, Charles; Riley, Ronald

Submitted to: Analytical and Bioanalytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/28/2008
Publication Date: 5/17/2008
Citation: Zitomer, N.C., Glenn, A.E., Bacon, C.W., Riley, R.T. 2008. A single extraction method for the analysis by liquid chromatography/tandem mass spectrometry of fumonisins and biomarkers of disrupted sphingolipid metabolism in tissues of maize seedlings. Analytical and Bioanalytical Chemistry. 391:2257-2263.

Interpretive Summary: Fusarium verticillioides, is a fungus that can cause diseases in many plants and produces toxins known as fumonisins. In addition to animal diseases, these toxins contribute to the development of disease in maize seedlings. Fumonisins interfere with the production of certain fats, called sphingolipids. This can be observed during disease by identifying some sphingolipids that are affected by fumonisins. An extraction method was developed for the simultaneous analysis of fumonisins (three naturally occurring forms, FB1, FB2, and FB3) and some of the sphingolipids in maize tissues by using a technique known as liquid chromatography/linear ion trap tandem mass spectrometry. The method involved a single extraction to detect the chemicals at low levels and with high recoveries. To test the efficacy of the method, seed of a susceptible maize line were inoculated with F. verticillioides. The seedlings were then harvested, and fumonisin content, as well as sphingolipids were measured in the leaf and root tissues. Fumonisin accumulation was significantly greater in leaf one compared to leaves two and three. While FB1, FB2, and FB3 were detected in root tissues, one of the three forms, FB1, was preferentially accumulated in leaf tissues. Accumulation of sphingolipids was evident in roots and leaves of seedlings grown from inoculated seed. The method developed was effective, fast, and sensitive for use in simultaneously measuring fumonisin in tissues and their effects on sphingolipids. The method should prove useful for screening maize cultivars for susceptibility to F. verticillioides-induced seedling diseases.

Technical Abstract: The fungus Fusarium verticillioides is a pathogen of many plants and produces fumonisins. In addition to their well-studied animal toxicoses these toxins contribute to the development of maize seedling disease in susceptible maize varieties. Fumonisin disruption of sphingolipid biosynthesis occurs during pathogenesis. An extraction method was developed for the simultaneous analysis of fumonisins B1 (FB1), B2 (FB2) and B3 (FB3), free sphingoid bases and sphingoid base 1-phosphates in maize tissues by liquid chromatography/linear ion trap tandem mass spectrometry. The method involved a single extraction using 1:1 acetonitrile:water + 5% formic acid (1 ml per 10 mg tissue). Mean recoveries ranged from approximately 50 to 99 percent, and limits of detection ranged from 10 fg µl-1 to 6900 fg µl-1. To test the efficacy of the method, seed of a susceptible maize line were inoculated with a pathogenic, fumonisin-producing strain of F. verticillioides. The seedlings were then harvested, and fumonisin content, as well as sphingoid bases and their 1-phosphates, were measured in the leaf and root tissues. Fumonisin accumulation was significantly greater in leaf one compared to leaves two and three. While FB1, FB2, and FB3 were detected in root tissues, FB1 was preferentially accumulated in leaf tissues. Accumulation of sphingoid bases and their 1-phosphates was evident in roots and leaves of seedlings grown from inoculated seed, with the level of accumulation being similar in leaves 1, 2 and 3. The method developed was effective, fast, and sensitive for use in simultaneously measuring fumonisin in tissues and their effects on sphingolipid metabolite biomarkers of disease. The method should prove useful for screening maize cultivars for susceptibility to F. verticillioides-induced seedling diseases.