| Research purpose: In recent years, a newly rising living organism sampling technique- microdialysis is show up. Its principle is substance can diffuse along concentration gradient and semi permeable membrane permit micro molecule compound to pass. The merits as follows: normal position sampling, real-time, on-line, non sample pretreatment and dynamic detection. But the price of on-line apparatus is high, if generalization this new analytical technique manufacture domestic equipment is imperative.At present Franz diffusion cell is often used for TDDS to determinate its drug permute velocity and to predict bioequiavailability. Unfortunately Franz diffusion cell has some shortcomings such as hydrodynamics defect,physical chemistry chemical constitution and physiological condition of ex vivo skin occur significant deviation, all these can influence experimental data.Therefore , apply self-made MD and HPLC on-line multiple analytical technique to study on pharmacokinetics and metabolism in vivo for traditional Chinese drug is a advanced,dependable and feasible way.The methods and results of research:1. Research on feasibility analysis of pharmacokinetics for puerarinEstablish a HPLC method for determining the content of puerarin in fruits of Puerraria lobata. The HPLC separation was accomplish on Hypersil-C18 column (5μm, 4.6mm×150mm) with mobile phase of methanol-water(0.1% acetic acid) (30:70) by flow rate of 1ml/min .The detection wavelength was 251nm. This method is proved to be simple, rapid, accurate and suitable for the qualty control of puerarin in fruits of Puerraria lobata. Puerarin TDDS was prepared with Azone as skin penetration enhancer and carbopol 980 as base materia1. Cumulative permeation quantity of puerarin for 32 hours was detected by improved Franz type diffusion cell with isolated rat skin and RP-HPLC was used as puerarin analytical method. The penetration equation of puerarin was Q = 11.12t -45.18. r =0.963.The cumulative permeation quantity of puerarin in 32 h was (335.18±51.45)μg/cm2. The penetration of puerarin TDDS through the rat skin followed First order kinetics. The preparation technology of puerarin TDDS is feasible and the quantities method is proved to be simple, rapid and accurate.2. Pharmacokinetics of Puerarin in Rat Skin by MD Coupled with HPLCTo investigate the in vivo pharmacokinetics of puerarin in rat skin by MD coupled with HPLC. After microdialysis probes were calibrated by Loss and Gain in vitro and in vivo, the pharmacokinetics and metabolic process of puerarin TDDS was studied. The kinetica software was adopted for analysis of the data. Concentration–time courses of unbound puerarin and main pharmacokinetics parameters were obtained.Main pharmacokinetics parameters are: (AUC) 2335.14 ug·min·ml-1, (T1/2) 230 min, (Lz) 0.003, (Cmax) 4.62277 mg/mL, (Tmax) 460 min.3. The construction of self-made MD-HPLC on-line analysis apparatusThis self-made microdialysis and high performance liquid chromatography on-line platform as its combination used 10-ports injection valve automatically. Microdialysis equipment and high-performance liquid chromatography system were integrated with the polypropylene, PEEK, flexible fused silica capillary of three pipe materials by the United States CYBERBOND (Apollo) instant adhesive compatibility and biology community. Realize the Software implementation linkage to achieve the goal of reaching 10-ports automatic injection valve injection at the same time. Apply the SPSS software to match T test on the three kinds of concentrations (0.1 ug/mL, 10 ug/mL, 100 ug/mL) of the peak time for puerarin theoretical values with measured values, statistical analysis, P 0.1 = 0.239, P 10 = 0.617, P 100 = 0.809. P values are proved to be greater than 0.05 without statistical significance, which means there is no significant difference between the theoretical values and measured values of puerarin peak time. Therefore it can be concluded that the way VCOM and CLASS-VP software separately controlled automatic 10-ports automatic injection valves and HPLC systems, which has been set up in the experiment, can meet the requirements of the experiment, and achieve implementation of microdialysis sampling with HPLC on-line linkage. The equipment is cheap, simple and practical, economic and reliable. This will be China's first self-developed on-line combined device and has been put into practical, which has laid a foundation on the promotion of this new technology in China.4. To compare MD-HPLC off-line with on-line performanceThe study is made to investigate the self-made MD and HPLC on-line technique in precision, reproducibility and stability for puerarin in order to find and tackle the existing problems so that this on-line system can be improved and consummate. The results show that this MD and HPLC on-line technique is simple, rapid, and accurate. The data was analyzed by SPSS 13.0 statistical software.1) The regression equation in the correlation coefficient (ronline = roffline = 1), regression coefficient (bonline = 31304, boffline = 29369) were inspected respectively by F tests (Fonline = 168959.6, Ponline = 0.0001, Foffline = 71114.847, Poffline = 0.0001) and t test (tonline = 411.047, Ponline = 0.0001, toffline = 266.674, Poffline = 0.0001), P <0.05. The results suggest that online and offline in the correlation coefficient of the regression equation, regression coefficient itself is statistically significant, and the self-made MD-HPLC on-line determination of concentration and peak area Puerarin relevance and MD-HPLC off-line had no significant differences. Further of the two regression equations of the regression coefficient were examined by F test to decide whether the reaction of their difference has statistical significance. General linear model was used to observe the interaction parameter, F= 208. 967, P = 0.0001, P <0.05, which proved its statistical significance. The results suggest that self-made MD-HPLC on-line with the MD-HPLC off-line determination of the same concentration of puerarin sample, on-line by the measured peak area response value is better than off-line.2) The precision (intra-day) data that is achieved by analysis compared two groups of t test shows: t 0.1 = 46.862, P 0.1 = 0.0001; t 10 = 5.035, P 10 = 0.001; t 100 = 7.090, P 100 = 0.0001, p< 0.05, and the concentration of three kinds of peak area Puerarin SD Online is smaller than SD offline. The results suggest that: when the self-made MD-HPLC and on-line with the MD-HPLC off-line are tested by determination of low, middle and high concentration of puerarin in three kinds of samples, the precision of on-line day is better than that of off-line, and it has statistical significance . Precision (inter-day) data that is achieved by the two comparative groups test analysis shows : t 0.1 = 37.222, P 0.1 = 0.0001; t 0.1 = 5.669, P 0.1 = 0.0001; t 0.1 = 12.226, P 0.1 = 0.0001, P <0.05 , and three kinds of concentrations of puerarin peak area SDOnline are smaller than SDoffline. The results suggest that: when the self-made MD-HPLC and on-line with the MD-HPLC off-line are tested by determination of low, middle and high concentration of puerarin in three kinds of samples, the precision of on-line day is better than that of off-line, and it has statistical significance .3) The reproducibility of data that is achieved by two analytic and comparative groups of t test shows: SDonline = 8352.453, SD offline = 22953.676, SD online |
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