Animal biles and
gallstones are popularly used in traditional Chinese medicines, and
bile acids are their major bioactive constituents. Some of these medicines, like cow-
bezoar, are very expensive, and may be adulterated or even replaced by less expensive but similar species. Due to poor ultraviolet absorbance and structural similarity of
bile acids, effective technology for species differentiation and quality control of bile-based Chinese medicines is still lacking. In this study, a rapid and reliable method was established for the simultaneous qualitative and quantitative analysis of 18
bile acids, including 6 free
steroids (
cholic acid,
chenodeoxycholic acid,
deoxycholic acid,
lithocholic acid,
hyodeoxycholic acid, and
ursodeoxycholic acid) and their corresponding
glycine conjugates and
taurine conjugates, by using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This method was used to analyze six bile-based Chinese medicines: bear bile, cattle bile, pig bile, snake bile, cow-
bezoar, and artificial cow-
bezoar. Samples were separated on an Atlantis dC₁₈ column and were eluted with
methanol-
acetonitrile-water containing
ammonium acetate. The mass spectrometer was monitored in the negative electrospray ionization mode. Total ion currents of the samples were compared for species differentiation, and the contents of
bile acids were determined by monitoring specific ion pairs in a selected reaction monitoring program. All 18
bile acids showed good linearity (r² > 0.993) in a wide dynamic range of up to 2000-fold, using
dehydrocholic acid as the internal standard. Different animal biles could be explicitly distinguished by their major characteristic
bile acids:
tauroursodeoxycholic acid and
taurochenodeoxycholic acid for bear bile,
glycocholic acid,
cholic acid and
taurocholic acid for cattle bile,
glycohyodeoxycholic acid and
glycochenodeoxycholic acid for pig bile, and
taurocholic acid for snake bile. Furthermore, cattle bile, cow-
bezoar, and artificial cow-
bezoar could be differentiated by the existence of
hyodeoxycholic acid and the ratio of
cholic acid to
deoxycholic acid. This study provided
bile acid profiles of bile-based Chinese medicines for the first time, which could be used for their quality control.