| Glycocholic Acid(GCA)is one kind of conjugated bile acid which has been widely found in bovine bile as the principal constituent.It has high medicinal value.Based on the laboratory test,this study aims to establish a pilot process route for extracting and purifying GCA from bovine bile as well as a LC-MS/MS method for the detection of GCA.The main research contents are as follows;(1)By the methods of protein removal,concentration,salting out,extraction and chromatography,the technological route for extracting and purifying GCA from bovine bile was established under the condition of pilot scale test.Results:The average extraction amount of GCA was 5.98±0.33g per 2L bovine bile,while the average purity was 94.42±0.05%and the average recovery was 79.42±0.05%.(2)The detection method for GCA by LC-MS/MS has been established with the chromatographic conditions and mass spectrometry scanning mode are as follows.Chromatographic conditions were Poroshell 120 EC-C18(3.0 mm×100 mm,2.7?m)column;column temperature of 42?;injection volume of 1 pL;flow rate of 0.5 mL·min-1 and chromatographic grade methanol solution as the mobile phase.Mass spectrometry conditions were electrospray ion source(ESI)as ion source;multiple reaction monitoring mode(MRM)as mass spectrometry scanning mode,negative ionization mode(Negtive)detection;atomization gas pressure of 4,500V;auxiliary heating gas pressure of 3,500V(-);scanning range with 10-1000;dry gas nitrogen;dry gas flow rate of 12 L·min-1;dry gas temperature of 350? and external standard method as the method of content determination.The results of GCA showed that at three concentrations,the RSD of intra-day precision was below 6.54%,the RSD of three days of inter-day precision was below 3.35%,and the RSD of stability of glycocholic acid at three different temperatures was less than 7.82%;meanwhile,the average recovery rate was 103.24%,the detection limit was 2 ng/mL and the quantitative limit was 10 ng/mL.Conclusion:Compared with the pilot test of the same method,the extraction amount,purity and average recovery rate of GCA extracted from bovine bile in this study were slightly lower than the lab test.The extraction process has the characteristics of short production cycle,low cost and stability which qualified for industrial production.The established LC-MS/MS method can meet the requirements of qualitative and quantitative detection of GCA in terms of accuracy,sensitivity and stability. |
PDF Full Text Request