Chromatographic purification and identification of polar metabolites of benazolin-ethyl from soybean

The metabolism of benazolin-ethyl (4-chloro-2-oxobenzothiazolin-3-ylacetic acid ethyl ester), a post emergence herbicide, has been studied in soybean using (14C)-phenyl labelled compound. Preliminary studies were performed on excised soybean leaves. Following hydrolysis of the ethyl ester to benazolin acid (4-chloro-2-oxobenzothiazolin-3-ylacetic acid), extensive metabolism to polar conjugates was observed. The polar fraction from a Bligh-Dyer extraction was purified by solvent partitioning, preparative TLC and reverse phase HPLC with ion suppression. The two major metabolites were characterised by fast atom bombardment mass spectrometry with accurate mass determination as an aspartate conjugate and a malonyl-beta-glucose ester of benazolin acid. Subsequent experiments were performed by spraying intact plants at growth stage V4. The major polar metabolite isolated one month after treatment was identified as the aspartate conjugate by mass spectrometry and high resolution nuclear magnetic resonance spectroscopy.