Submitted to: American Society for Mass Spectrometry
Publication Type: Abstract only
Publication Acceptance Date: 5/24/2012
Publication Date: 5/24/2012
Citation: Busman, M. 2012. Determination of the secondary metabolites fusarubin and 8-O-methylbostrycoidin from maize by LC-MS/MS. American Society for Mass Spectrometry. Interpretive Summary:
Technical Abstract: The quinones, fusarubin and 8-O-methylbostrycoidin, are secondary metabolites produced by fungi in grains used for human consumption. A variety of biological activities have been noted for fusarubin and 8-O-methylbostrycoidin, including antibiotic properties. Chemical characterization of fusarubin and 8-O-methylbostrycoidin frequently is based upon their deep red coloration. Here we demonstrate a technique for analysis of fusarubin and 8-O-methylbostrycoidin by HPLC coupled to ESI-MS/MS. The technique is applied to the screening of Fusarium species for fusarubin and 8-O-methylbostrycoidin production on cracked corn and to evaluation of field production in maize. Portions (10 g) of the corn were extracted with 50 mL acetonitrile for 2 hrs. Extracts were filtered with a 125 mm 2V paper filter. 10 µL of extract was applied to a MetaChem Inertsil C18 column. Chromatography was conducted utilizing a ThermoSpectraPhysics HPLC system consisting of an AS4000 autosampler coupled to a P2000 gradient pump. Elution of analyte was achieved with a gradient flow of methanol and water (0.3% acetic acid was added to the mobile phase.) The solvent program used a 35-95% gradient over 25 min. Flow was directed to a ThermoFinnigan LCQ-DECA ion trap mass spectrometer equipped with an electrospray ionization source. For quantitative analyses, abundant fragments from the [M + H]+ ions of the analytes, fusarubin and 8-O-methylbostrycoidin, were monitored in the MRM mode. To assess quantitative behavior of the LC-MS system, 10 µL injections over a wide range of fusarubin and 8-O-methylbostrycoidin concentrations were made. LC-MS/MS experiments were conducted in selected ion monitoring mode of operation. Integrations of LC-MS/MS response were recorded for the dominant fragments. Responses for the residual parent ions were also recorded. Linear response curves were obtained by using the external standard method over a range of analyte concentrations. Minimum detection limits and limits of quantitation (based on the signal-to-noise ratio of 3 and 10, respectively) were determined for fusarubin and 8-O-methylbostrycoidin spiked into corn extract. A series of 10 g portions of cracked maize were spiked with fusarubin and 8-O-methylbostrycoidin (acetonitrile) in order to achieve levels corresponding to 1 ppm fusarubin and 8-O-methylbostrycoidin. The samples were then extracted and processed according to the above described method. The analysis of the spiked samples allowed determination of recoveries of the fusarubin and 8-O-methylbostrycoidin upon quantitation utilizing prominent fragment ions for each analyte. The LC-MS/MS method was utilized to screen fusarubin and 8-O-methylbostrycoidin production by Fusarium species on cracked maize. The method was also applied to a field study of inoculations of Fusarium isolates on growing maize ears. Mature ears were harvested, shelled and extracted with acetonitrile and subjected to LC-MS/MS.