Studies On The Composition And Quality Control Of Bile Acids In Snake Bile

Snake bile(Shedan),which has been used as a precious TCM for hundred years,is commonly used for the treatment of fever,cough,eclampsia and gastrointestinal diseases.Due to the disordered headstream and lacking of quality standard,the quality of preparation of snake bile is not effectively controlled and it is common to see snake bile adulteration from other animal bile in market,which greatly reduced the efficacy of snake bile.To solve the adulterartion problem and control the quality of snake bile,the comprehensive study of components in snake bile is necessary.Bile acids(BAs)are the main active ingredients of snake bile.However duo to the minor contents and low UV absorbance of trace BAs in snake bile(except TCA),routine detective methods,such as thin layer chromatography(TLC),and high-performance liquid chromatography(HPLC)could not sensitive and selective enough to accurately determine the BAs in snake bile.Therefore,the study of the bile acid composition in snake bile and establishing the analytical method of BAs in snake bile to control its quality is significant.Waters Xevo Ultrahigh-performance liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry(UPLC/ESI-QTOF-MS/MS)has been applied to conduct a qualitative analysis on snake bile acids.Totally 15 compounds,including 4 pairs of isomers,were identified by comparing the retention time,exact molecular mass and fragmentation behaviors with reference standards,respectively.And three new bile acids were proposed for the first time.Waters TQD ultra high performance liquid chromatography with triple quadrupole linear iontrap mass spectrometry(UPLC-QqQ-MS/MS)in multiple-reaction monitoring mode has been developed to conduct adulterartion control of snake bile.The common adulterations in Chinese market were cattle bile,sheep bile,swine bile,chook bile and duck bile,and this study aimed at finding the characteristic components of the adultering bile and controlling their highest contents in snake bile.The chromatographic separation techniques of gel and semi-preparative HPLC were used to separate the methanol extraction of snake bile.The purified monomers were identified by MS and NMR spectroscopy.Total of 5 monomers were obtained,including two new compounds.ABSciex 4500 ultrahigh-performance liquid chromatography with triple quadrupole linear iontrap mass spectrometry(UPLC-QqQ-MS/MS)was applied to conduct a quantitative analysis on snake bile acids in this study.Seven components,taurocholic acid(TCA),taurochenodeoxycholic acid(TCDCA),taurodeoxycholic acid(TDCA),glycocholic acid(GCA),cholic acid(CA),tauro-3α,7α,12α,23R-tetrahydroxy-5β-cholenoic acid and tauro-3α,12α-dihydroxy-7-oxo-5β-cholenoic acid were simultaneously determined,among which,the latter two were first reported concerning their quantitative determination.In present study,ultra high-performance liquid chromatography/electrospray ionization tandem mass spectrometry(UPLC/ESI-MS/MS),due to the great separation effect of UPLC and the high sensitivity of MS/MS,has been successfully used to conduct qualitative analysis,quantitative anlaysis and adulteration control on snake bile acids.This experiment provided a strong basis for the quality control of snake bile.