Studies On Quality Standard Of Snake Bile

Because of the complex source of snake bile and the lack of unified and perfect quality standard,the use of snake bile is disordered,adulteration occurs from time to time,which make the quality of snake bile varied.On the basis of previous studies,this experiment intends to carry out the standard research on standardizing the source of snake bile,controlling its adulteration and improving the quality of snake bile,so as to provide a basis for multi-index and specific quality control methods of snake bile.UPLC characteristic chromatograms of 9 species of 3 families of snake bile,mixed snake bile and common adulterated animal bile were established.According to similarity evaluation system,common feature peaks were matched and similarities were calculated.The characteristic chromatograms of 9 species of snake bile are different.Ten batches of mixed snake bile calibrated seven common peaks with high similarity.The similarity analysis between snake bile and adulterated animal bile showed that they had low similarities and could be distinguished clearly.In the case of adding a small amount of adulterants in snake bile,the control method was also established.Waters Xevo Ultrahigh-performance liquid chromatography with electrospray ionization quadrupole time-of-flight tandem mass spectrometry(UPLC/Qtof-MS/MS)was used to conduct a qualitative analysis on adulterated animal bile,including chook,duck,cattle,sheep,pig and rabbit.Three taurine-conjugated bile acids in chook and duck bile were identified.There were eight components in pig bile,including five glycine-conjugated bile acids,three taurine-conjugated bile acids.There were six bile acids in cattle bile,containing three glycine-conjugated bile acids,three taurine-conjugated bile acids.Eight bile acids in sheep bile were identified,consisting of four taurine-conjugated bile acids,three glycine-conjugated bile acids and cholic acid.Seven bile acids in rabbit bile were identified,involving five glycine-conjugated bile acids,one taurine-conjugated bile acid and one glycyl-taurine-conjugated bile acid.ABsciex 4500 ultrahigh-performance liquid chromatography with triple quadrupole linear iontrap mass spectrometry(UPLC-QqQ-MS/MS)was applied to determine the eight main bile acids in adulterated animal bile.According to the requirements of the Chinese Pharmacopoeia in the 2015 edition,the source,character,TLC identification,examination and determination of the main bile acid in snake bile were studied.The source and character of snake bile were clarified.In the TLC identification of snake bile,common silica gel G plate was used,reference substances,TCA and snake bile,were used as control.Snake bile and commercial snake bile showed the main spots of TCA,and there were more than three identical spots with the control medicinal material.At the same time,two spots of the same color appeared beneath the spots of TCA.TLC examinations of pig,cattle and sheep bile were established.Limit the content of GCA,and require that the spot color of GCA of the test sample should not be deeper than that of the control sample,so as to check the cattle and sheep bile.The experiment result showed that the spots of the test sample were significantly deeper than those of the control sample when 10% cattle or sheep bile was added.We used GHDCA as a reference substance to detect pig bile.The experiment result showed that when 2% pig bile was adulterated,it could be detected.Examination of smell required that snake bile should not have peculiar smell such as diesel oil.The method of HPLC-UV was used to determine the content of TCA,which had good methodology results.The contents of 26 batches of snake bile and 26 batches of commercial snake bile were determined.In our study,the differences between snake bile acids and adulterated animal bile acids were further clarified.The UPLC characteristic chromatograms of different snake bile,mixed snake bile and common adulterated animal bile were established,which can quickly identify and distinguish snake bile and adulterated animal bile.The quality standard of snake bile with strong specificity and practicability was found.The establishment of this standard plays an important role in regulating the source of snake bile,controlling the adulteration of snake bile and improving the quality of snake bile.