Chuan Zi Wan (CZW) is a traditional Chinese herbal medicine derived from Ligularia species and used as an antitussive and expectorant folk remedy. Its toxicity was recently demonstrated in rats, for which the hepatotoxic pyrrolizidine alkaloids (PAs) were detected but not fully examined. In order to further assess its safety, the entire PA profile of CZW was investigated using high-performance liquid chromatography (HPLC) coupled to electrospray ionization ion trap mass spectrometry. Twenty-six toxic PAs (25 otonecine-type and one retronecine-type PAs) and one non-toxic PA of platynecine-type were detected and/or characterized based on diagnostic ions, mass fragmentation patterns, chromatographic behaviors and biosynthetic consideration. Except clivorine, ligularine, hodgsonine and ligularizine, all other PAs were described for the first time; moreover, most otonecine-type PAs were found to contain the acetyl group, an important substructure closely linked to their toxic effects in vivo. Clivorine was unequivocally identified as a major PA in the extract, with a content of 3.94 mg g(-1) dried extractant determined by a HPLC/UV assay. The total content of the toxic PAs was estimated to be about 7.92 mg g(-1) dried extract. These results indicated that this herbal product may have a potential health threat to humans in its Chinese medical practice.