Quality Control Of Ursodeoxycholic Acid Capsule And Research On Pharmacokinetics Of UDCA In Rabbits

Ursodeoxycholic Acid (UDCA), chemically named 3α,7β-dihydroxy-5p-cholestane-24-carboxylic acid, which is a kind of hydrophilic bile acid, shows the activities of musculus sphincter ductus choledochi dilatation and promotion of bile secretion and discharge. Besides, UDCA also has the properties of cell protection and immune adjusting. It is the only one valid drug licensed by FDA for curing cholestasis hepatopathy and is widly used against liver diseases in clinic.In this thesis, the method of qualitatively controlling Ursodeoxycholic Acid Capsule was researched. According its properties and structural characteristics, we developed the methods of identification, impurity test and content determination. The content of UDCA was determinated, and the related impurities and four known impurities including cholalic acid(CA), lithocholic acid(LA), anthropodeoxycholic acid(CDCA) and 7-ketone-lithocholic acid, were also tested by HPLC. Octadecyl silicane bonded phase was used, and the mobile phase consisted of acetonitrile-0.001mol·L^-1 phosphoric buffer (45:55, adjusted to pH2.2±0.1 by phosphoric acid). The flow rate was 1.0ml·min^-1, with the detective UV wavelength at 206nm and column temperature at 30℃. Results: the calibration curve was linear (r=0.9999) within the range of 0.1～2.0mg·ml^-1 for UDCA, the limit of detection was 0.2μg(S/N=3); the calibration curve was linear(r=1.0000) within the range of 0.04～1.2mg·ml^-1 for CA, the limit of detection was 0.09μg(S/N=3); the calibration curve was linear(r=0.9998)within the range of 0.5～10mg·ml^-1 for LA, the limit of detection was 10μg(S/N=3); the calibration curve was linear (r=0.9999) within the range of 0.072～0.72mg·ml^-1 for 7-ketone-LA, the limit of detection was 5ng (S/N=3) ; the calibration curve was linear (r= 0.9998) within the range of 0.08～1.6mg·ml^-1 for CDCA, the limit of detection was 0.15μg(S/N=3). The RSD for between-day precision test was 0.16% (n=5); the RSD for repeatability test was 1.3%(n=6); the average recovery was 100.2%; according to the results assayed from batches of sample, the impurities limits were controlled of CA less than 0.5%, LA less than 0.1%, CDCA less than 1.0%, 7-ketone-LA less than 0.4%, the other single related impurity less than 0.25%, the total amount of the other related impurities less than 0.5% and the total amount of impurities less than 2.0%; the content of UDCA was controlled to 95.0%～l05.0% of labeled content. It was also examed of UDCA capsule during 6-month acceleration test under 40℃and 18-month long-term test under commom temperature based on the quality standard already established. The method was simple, accurate and suitable for the quality control of UDCA capsule.Pharmacokinetics of UDCA in rabbits was researched in the study. A sample-pretreated method of ether-extraction-precolumn-derivation was established, and the chromatographic conditions, which was used for determinating the derivant of extracted UDCA, were decided to be Hypersil C₁₈ column(250×4.6mm, 10μm), acetonitrile-methol-0.005mol·L^-1 phosphoric buffer-triethylamine(50:27:42:0.25, adjusted to pH3.0±0.1 by phosphoric acid) as mobile phase with detective UV wavelength at 247nm and column temperature at 35℃. Under these conditions, the ratio of peak areas for UDCA-derivant to internal-standard-derivant was linear(r=0.9998) with the content of UDCA in the range of 0.15～5.0μg/ml, with the LOQ of 0.15μg/ml. The extraction recovery was more than 90%, the result of recovery test was more than 95%, the RSD of within-day precision test and between-day precision test were both less than 15%. The method was confirmed to be available enough for quantitative analysis of UDCA in pharmacokinetics study in rabbits.The pharmacokinetics study of UDCA was performed with single dosage by the established analytical method in two dosage-groups respectively done in 6 healthy rabbits after intragastric administration. The experimental data showed that UDCA was very rapidly absorbed, and achieved peak concentration in plasma within 5 minutes. It manifested that UDCA abided by two-compartment model in rabbits. The main parametes of UDCA average data were as follow: Dosage-group A(20mg/kg): AUC_{0→∞} was 14.1340±6.1410mg·L^-1·h; T_1/2,ka was 0.0039±0.0042h; T_1/2,α was 0.5797±0.2907h; T_1/2,β was 45.0204±27.2369h; V₁ was 5.2149±2.7386L·kg^-1; CL was 1.7161±0.8751L·h^-1·kg^-1; Tmax was 0.0833h; Cmax was 5.9344±1.6538mg·L^-1. Dosage-group B(10mg/kg): AUC_{0→∞} was 4.3440±1.8553mg·L^-1·h; T_1/2,ka was 0.0003±0.0005h; T_1/2,α was 0.2910±0.3485h; T_1/2,β was 13.8642±25.0494h; V₁ was 0.9653±0.8329L·kg^-1; CL was 0.0183±0.0375L·h^-1·kg^-1; Tmax was 0.0833h; Cmax was 4.9410±2.8722mg·L^-1'. These experimental results could provide internal data for the clinic research of UDCA and its preparations.