ORIGINAL PAPER 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 Nicholas C. Zitomer & Anthony E. Glenn & Charles W. Bacon & Ronald T. Riley Received: 15 February 2008 / Revised: 25 April 2008 / Accepted: 28 April 2008 / Published online: 17 May 2008 # Springer-Verlag 2008 Abstract The fungus Fusarium verticillioides is a patho- gen 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 B 1 (FB 1 ), B 2 (FB 2 ) and B 3 (FB 3 ), 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, seeds 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 accumu- lation was significantly greater in leaf one compared to leaves two and three. While FB 1 , FB 2 , and FB 3 were detected in root tissues, FB 1 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 be useful for screening maize cultivars for suscep- tibility to F. verticillioides-induced seedling diseases. Keywords Fumonisins . Fusarium verticillioides . Sphinganine . Phytosphingosine . Sphingoid base 1-phosphates Introduction The fungus Fusarium verticillioides is both an endophyte and a pathogen of maize. Some of the diseases caused by F. verticillioides infection include seed rot, root rot, stalk rot, kernel or ear rot, and seedling blight [1, 2]. One of the characteristics of this fungus is the production of fumoni- sins (Fig. 1), which are potent inhibitors of ceramide synthase [3]. Fumonisins are known to cause a variety of diseases when ingested by farm animals [4, 5], and have been implicated in human carcinogenesis [6] and neural tube defects [7], as well as plant diseases [8, 9]. There are many naturally produced forms of fumonisins, of which fumonisin B 1 is the most significant form based upon its toxicity and prevalence in the environment [10]. While there are many other fumonisins [11], those most common- ly found in maize are fumonisins B 1 ,B 2 and B 3 [10]. Fumonisins of the B-series are known to disrupt sphingo- lipid metabolism in both plants and animals via inhibition of ceramide synthase [8, 12, 13], resulting in accumulation of free sphingoid bases and their 1-phosphates [8, 13]. Recent advances have pointed to sphingoid bases and sphingoid base 1-phosphates as signaling molecules in plants, as well as inducers of both increased proliferation Anal Bioanal Chem (2008) 391:2257–2263 DOI 10.1007/s00216-008-2166-x N. C. Zitomer : A. E. Glenn : C. W. Bacon : R. T. Riley (*) USDA-ARS, Toxicology and Mycotoxin Research Unit, R.B. Russell Research Center, Athens, GA 30604, USA e-mail: ron.riley@ars.usda.gov