Studies On The Optimization Of Extraction Process Of Taurocholate Acid In Ovine Bile And The Application Of HPLC For Taurocholate Acid

Taurocholic acid (TCA), the essential components in animal bile, is a type of conjugated bile acid. It is secreted most in the snake gall, also in ovine bile and ox-gall. This study was to optimize the extraction and purification processes of ovine bile taurocholic acid based on estibalished processes of extraction and purification.The effects of the ratio of mobile phase, pH, salting out, the amount of NaCl, and column chromatography velocity on the extraction of taurocholic acid extraction and its purity were investigated by orthogonal experimental design (orthogonal table L9 (34)) from ovine bile, and the best method of extraction of taurocholic acid was selected. Orthogonal experiment results shown that the mobile phase and the proportion of the velocity were the main reasons determining the effiency of ovine bile extraction, and the amount of NaCl and the proportion of mobile phase were the main reasons determining the purity of taurocholic acid.The optimized taurocholate acid extraction process in Ovine Bile as follows:the proportion of mobile phase condition of 1, velocity condition of 3, pH condition of 2, salting out to join the amount of NaCl condition of 3. Using thin layer chromatography, thin layer scanning method, and high performance liquid chromatography (HPLC) method for qualitative and quantitative analysis, the results shown that the purity of taurocholic acid reached 98%, extraction amount reached 0.7509g, and the recovery rate can reach 75%.Using high performance liquid chromatography (HPLC) method for determining the content of the taurocholic acid in the extraction, within the scope of 12.5 200μg/mL taurocholic acid concentration had better linear relationship duing the peak area, the regression equation for Y=1267.5 X+68025, R2=0.9984; The average recovery rate exceeded 92.89%; CV value of within-day was 0.34-2.08%; CV value of inter-day was 1.88 to 4.95%. Testing conditions were confirmated as follows: mobile phase with 30% acetonitrile solution; The flow rate was 0.5 mL/min; Detection wavelength was setted at 210 nm; Column temperature at 30℃; Sample quantity was 0.8μL. Determine the retention time of taurocholic acid was 3.268 min. Results shown the similarity between taurocholic acid that extracted from ovine bile and the standard taurocholic acid using this testing conditions. Therefore, this optimized method is sensitive, accurate, convenient, practical, and applicable to taurocholic acid detection.