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One unknown impurity in azoxystrobin bulk material at a level 0.15% was detected by a gradient reverse phase high performance liquid chromatography (HPLC). This impurity was isolated from a crude sample of azoxystrobin using reverse phase preparative LC. The impurity isolated by preparative LC was characterized by NMR, MS experimental data. Based on the results obtained from different spectroscopic experiments, these impurity was characterized as methyl 2-(2- (6- (2-cyanophenoxy)-2-((4-(2-cyanophenoxy)-6- (2- (1, 3-dimethoxy-3-oxoprop-l-en-2-yl)phenoxy) pyrimidin-5-yl) methyl)pyrimidin-4-yloxy)phenyl)-3-methoxyacrylate. Formation of this impurity is also discussed.