Abstract: AimTo elucidate the metabolic pathway of glufosfamide in rats. MethodsIn this study, a liquid chromatography-tandem mass spectrometric method was developed and applied to characterize the metabolites of glufosfamide in rat urine, after an iv administration of 50 mg·kg^-1. The analysis was performed under two ionization modes in two different chromatographic systems, separately. To make sure that the compounds detected in rat urine were metabolites or degradation products, the stability of glufosfamide, isophosphoramide mustard (M1), and the degradation products of M1 in urine were investigated. ResultsIn positive ionization mode, besides glufosfamide, two metabolites, isophosphoramide mustard and monoaziridinyl derivative of isophosphoramide mustard, were detected. In negative ionization mode, only glufosfamide itself was detected, while derivatives of isophosphoramide mustard have no response in such condition. ConclusionGlufosfamide was mainly unchanged excreted in urine, and two metabolites were detected as isophosphoramide mustard and monoaziridinyl derivative of isophosphoramide mustard.