The type and content change of alkaloids of Radix Aconiti Lateralis (Lateral root of Aconitum carmichaeli Debx, an important and popular medicinal herb in Traditional Chinese Medicine) in processing were studied using high performance liquid chromatography-electrospray ionization-multi-stage mass spectrometry (HPLC-ESI-MSn). Extract containing alkaloids, which were known to be the main bioactive components of the herb, was prepared by 1% (v/v) hydrochloric acid solution. An HPLC method which can simultaneously separate these alkaloids was established with gradient elution mode. Forty-eight compounds were structurally identified by employing LC-MSn techniques; the MSn spectra of most alkaloids displayed a characteristic behaviour of loss of CH3COOH (60 u), CH3OH (32 u), C6H5COOH (122 u), CO (28 u) and H2O (18 u). Among them, the fragmentation ion C6H5COOH (122 u) was reported for the first time. By comparison, 22 compounds were found both in the crude materials and the processed products; 17 alkaloids were only found in the processed products and 9 alkaloids,which existed in crude material, could not be detected after processing. In the process of identification, we found new kinds of alkaloids, with protonated molecules at m/z 452, 468, and 482. We called these compounds dehydra-hypaconine, dehydra-mesaconine, and dehydra-aconine, respectively. Copyright © 2012 John Wiley & Sons, Ltd.