Abstract

A gradient reversed phase HPLC method was developed and validated for the analysis of related substances in zonisamide (1,2-benzisoxazole-3-methanesulfonamide), using a Waters Symmetry C8 (150*3.9 mm) column with a flow rate of 1.0 ml/min and detection at 280 nm. The mobile phase component A consisted of a mixture of 0.02 M aqueous potassium dihydrogen phosphate-acetonitrile-methanol (75:10:15 v/v/v), pH adjusted to 4.0 with orthophosphoric acid. The mobile phase component B consisted of a mixture of 0.02 M aqueous potassium dihydrogen phosphate-acetonitrile-methanol (15:40:45 v/v/v), pH 2.0 with orthophosphoric acid. The limit of detection and limit of quantitation were in the range of 0.001-0.007% and 0.0035-0.25% respectively with respect to sample concentration of 2 mg/ml. The method was linear in the range of LOQ level to 200% of specified limits for II-VIII (< 0.10%, r ² = 0.9958-0.9999). The method is sensitive, specific, linear, accurate, precise and stability-indicating for the detection and quantitation of precursors (viz., 4-hydroxycoumarin, 1,2-benzisoxazole-3-acetic acid, 1,2-benzisoxazole-3-bromoacetic acid, 1,2-benzisoxazole-3-methylbromide, sodium 1,2-benzisoxazole-3-methanesulfonate), process impurities (viz., 2-hydroxyacetophenone oxime and 3,3,3-tribromomethyl-1,2-benzisoxazole) and drug degradation products formed under stress conditions.