<jats:title>Abstract</jats:title><jats:p>The fragmentation mechanism of six alkaloids, namely: dihydronitidine, dihydrochelerythrine, 8‐acetonyldihydronitidine, 8‐acetonyldrochelerythrine, nitidine and 1,3‐bis(8‐dihydronitidinyl)acetone, was investigated by electrospray ionization multi‐stage tandem mass spectrometry (ESI‐MS<jats:sup>n</jats:sup>). Tandem mass spectrometry experiments indicated that different substitution sites of the methoxyl groups at C‐9 and C‐10 or at C‐10 and C‐11 determined the different abundances of the MS<jats:sup>2</jats:sup> fragmentation ions using the same collision energy. According to the different abundances of MS<jats:sup>2</jats:sup> product ions, positional isomeric benzo[<jats:italic>c</jats:italic>]phenanthridine alkaloids can be differentiated. Moreover, ten constituents in the crude alkaloidol extract from the roots of <jats:italic>Zanthoxylum nitidium</jats:italic> were rapidly identified by high‐performance liquid chromatography coupled with tandem mass spectrometry (HPLC/MS<jats:sup>n</jats:sup>), through comparing the retention times and ESI‐MS<jats:sup>n</jats:sup> spectra with the authentic standards. This work demonstrates that not only the characteristic fragments but also the characteristic abundances of the fragment ions can be used for detailed structural characterization. Copyright © 2007 John Wiley & Sons, Ltd.