A rapid, sensitive and versatile liquid chromatography/electrospray ionization 
tandem mass spectrometry (LC/ESI-MS/MS) method was developed for the 
comprehensive analyses of the chemical constituents contained in the Chinese 
medicine-Venenum Bufonis (VB, Chan' Su in Chinese). LC analysis was carried out 
on an Agilent Eclipse plus C₁₈ RRHD column (2.1 × 150 mm, 1.8 μm) with a linear 
gradient solvent system of water (0.1% formic acid) and acetonitrile (0.1% 
formic acid) as mobile phase. Detection and quantification were performed by 
multiple reaction monitoring (MRM) transitions via electrospray ionization (ESI) 
source operating in the positive ionization mode. Through "Molecular Feature 
Extraction" (MFE), more than 900 features were detected from VB extracts. Among 
them, a total of 97 components were identified using the Agilent METLIN accurate 
mass matching database (DB) established according to those reported in the 
literatures. Further more, 30 high quality matches were obtained by comparisons 
of their accurate mass and retention times (AMRT) with those imported out in the 
developed personal database (METLIN DB with AMRT). The characteristic 
fragmentation pathways were proposed for the tentative characterization of four 
representative types of bufadienolides in the present work. The targeted MS/MS 
experiment of the 30 major compounds was performed for their quantification and 
semi-quantification. And 7 of them were quantified over the assaying 
concentration range of 5.0-500 pg/μL. The lowest limit of detection and 
quantification of them were 0.25-0.50 and 1.25-0.25 pg/μL, respectively. The 
recoveries varied from 83 to 106% depending on the chemical types and different 
extraction solvents. The remained 23 bufosteroids were simultaneously 
semi-quantified using three representative standard compounds as their standard 
references, respectively.