| A method for purifying fusaproliferin from cultures of Fusarium subglutinans E-1583 by preparative HPLC was developed, which involved extracting culture materials with methanol, evaporating methanol extracts, transferring evaporated residues with small volume of methanol, partitioning the residues in methanol with large volumes of hexane, evaporating the hexane, and dissolving the residues in acetonitrile or methanol. The final acetonitrile solution with fusaproliferin was purified by HPLC with C18 preparative column and mobile phase of CH3CN:water (80:20, v:v, at 2.5 mL/min). The identity of the purified fusaproliferin was verified by analytical HPLC, GC-MS, 1H NMR, and LC-MS, and its purity was at least 99.86% when evaluated by analytical HPLC with the paired-sample method and LC-MS.; The newly developed GC-FID method for fusaproliferin detection included four main steps: methanol extraction, C18 cartridge cleanup, TMS-derivatization, and GC detection. This method had a detection limit (LOD) of 40 pg of fusaproliferin per injection, which was ten times lower than the analytical HPLC method (LOD of 0.5 ng per injection) and did not have the heat decomposition problem of a previously described GC method. The standard curve established by linear regression had a R2 of 0.9999 and a linear range from 0.1 to 100 ng of fusaproliferin. The fusaproliferin recovery rates from corn samples spiked with 200 ng/g, 1000 ng/g, and 5000 ng/g standard fusaproliferin were 109.1%, 85.7%, and 98.9%. When this method was used to examine corn samples, we found that most corn samples had fusaproliferin levels of less than 50 ng/g, which is below the detection limits of most published methods. Therefore, this GC-FID method could be very useful in studying the natural contamination of fusaproliferin in corn.; The results of the Ames Salmonella/microsome assay showed that fusaproliferin was a weak frameshift mutagen in tester strain TA98 in the presence of S9 and was not a mutagen in strain TA1535. Mutagenicity results in the other tester strains were equivocal. The mutagenic potency of fusaproliferin in tester strain TA98 was approximately 500 times less than that of aflatoxin B1. |
