A Pilot Test Studies On The Extraction Of Taurocholate Acid From Ovine Bile And Establishment Of LC-MS/MS Method For Determination Of Taurocholate Acid

Taurocholate acid(TCA)is one type of conjugated bile acids that has been widely found in bile of sheep taking an important part of the composition of bile.TCA has great medicinal values which lead to a high market price but someway limited manufacturing technique.This paper aims to explore a pre-production process and LC-MS/MS detection method base on the pilot test.The main points are as follows:bile of sheep?deproteinization depigmentation ?deoil?concentration and recovery of ethanol?salting out?extraction?silica gel chromatographyo Result:the extractive amount of taurocholic acid in each 2L bile was 13.95±0.67 g with the average purity of 96.52±5.27g.The average recovery rate was 71.25±7.62%?A Detection method for detecting TCA with LC-MS/MS was established.Chromatographic conditions:Poroshell 120 EC-C18(3.0 mm × 100 mm,2.7 ?m)chromatographic column;sample size:1 ?L;column temperature:35?;flow rate:0.5 mL·min-1;and mobile phase:0.5%methanol solution.Mass spectrographic conditions:ion source:electrospray ionization(ESI);mass spectrographic scanning:multiple reaction monitoring(MRM),negative ionization(Negtive)detection;atomizing air pressure 4,500V;auxiliary heating air pressure 3,500V(-);scanned area(10-1000);dry gas:nitrogen;dry gas flow:12 L·min-1;and dry gas temperature 350?.External standard method was used to determine the content.The results showed the TCA,at the three concentrations,has a within-day precision RSD?2.835%,day to day precision RSD?9.42%and stability RSD?19.80%.Its average sample recovery was 94.68%-98.41%.The limit of detection was 5ng/mL-1 and limit of quantitation 20ng/mL-1.The pilot process for extracting TCA from sheep bile used in this study was compared with the pilot test.The purity,extraction and recovery of TCA are slightly lower Compared with the pilot test,the pre-production test provided more extractive of TCA higher in purity while reducing the production cost and cycle.Its simpler process is suitable for TCA extraction in industrial production.The established LC-MS/MS method showed a high degree of accuracy and simple pretreatment as well as ocular data processing.These features endow it appropriate for the qualitative and quantitative determination of taurocholic acid in bile of sheep.