| To explore the best parts and method of embedded by the microdialysis probe, and establish a method to implant the microdialysis probe into rabbit’s knee joint capsule. To investigate the influence of different factors on recovery and loss, as a reference in vivo experiments by microdialysis.To explore the feasibility of applying microdialysis technique for pharmacokinetics study in knee joint fluid of rabbite, and provide theoretical and experimental evidence for the studies in the pharmacokinetic-pharmacodynamic(PK-PD) of sinomenine in rabbit’s knee joint fluid by microdialysis.1 The establishment of analytical method of Sinomenine in dialysateA rapid and sensitive method based on Ultra Performance Liquid Chromatography was developed for the Sinomenine in dialysate. The analytes were separated on a ACQUITY UPLC HSS Ci8(2.1mm × 50 mm,1.8(um) with the mobile phase composed of acetonitrile-water containing 0.25% ethylenediamine (30:70,v/v) at a flow rate of 0.3mL/min. The method had the injection volume was 2uL. Drawing standard curve, linear range, precision and stability testing.Under the chromatographic condition, Sinomenine had symmetrical peaks with tR 1.70min, and limit of quantification and detection limit with the experimentalrequirements. The linear range, limit of quantification and the limit of detection could met the requirement. All the validation data, such as specificity, linearity, matrix effect and stability were within the required limits. The method has been successfully applied to pharmacokinetic study of the Sinomenine in dialysate matrix.2 Establish a method to implant of the microdialysis probe into rabbit’s knee joint capsuleTo dissect rabbit’s some joints and observe the physiological structure of bone, ligament and muscle in each joint,rocord the anatomy of each join,and determine the sampling position and implant. According the characteristics of the sampling site, select the appropriate micro dialysis probe. The microdialysis probe were implanted in the lateral joint eye of rabbit’s knee capsule with the help of syringe needle and guide eatheter,and the position of the probe was verified by X-ray.To determine microdialysis after sampling site for rabbit knee, determining the probe into the knee lateral knee of the eye. The method to implant the microdialysis probe was established, and microdialysis has evident advantages in pharmacokinetic study.3 Establishment of Sinomenine microdialysis parameterTo assess the realationship of the probe relative recovery(RR) obtained by gain method and the probe relative loss(RL) obtained by loss method. To assess the feasibility of retrodialysis method by calculating the recovery of microdialysis probe in vivo. To investigate the influence of different factors(including the velocity,concentration) on recovery. To investigate the stability of Sinomenine recovery, including vitro and vivo stability of Sinomenine recovery.The probe RR obtained by gain method was similar to RL of loss method all components. The recovery of probe calculated by retrodialysis method was stable throughout all experiments, indicating that the retrodilaysis method could be applied to assessing the recovery of probe in vivo.The recovery of probe in vitro is inversely proportional to the perfusing rate. And independent of the concentrations of Sinomenine. The recovery of Sinomenine results showed that could keep stable within 14h in vitro and4 Establish a study method of joint cavity local pharmacokinetic of SinomenineThe recovery of probe calculated by retrodialysis method. Concentration-time curves was calculated. Main pharmacokinetics parameters were obtained with DASS2.1.1 software.The recovery of probe calculated by retrodialysis method was (15.82±2.12)%, The recovery good stabilities in 3 hours in vivo. Retrodialysis method is capable of monitoring the concentration of Sinomenine dynamically. The results can be used in pharmacokinetics research of Sinomenine. Providing that the relative recoveries is determined by retrodialysis, microdialysis can be used for the pharmacokinetic study of Sinomenine. |
