微透析液相色谱联用的构建及在经皮药动学研究的应用
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摘要
研究目的:微透析(Microdialysis,MD)技术是近年来新兴的一种生物活体取样技术,它是利用物质沿浓度梯度扩散和半透膜对小分子化合物具有通透性的原理设计的,具有原位取样、实时、在线、无需样品预处理、动态检测等特点。其与色谱分析仪器联合应用为体内药物分析开辟了高效、精确、简便的新途径。目前国外微透析技术已经与多种色谱分析仪器进行了在线联用,形成了标准化商品,但其设备价格昂贵,国内均依靠进口,欲在我国推广这一新的分析技术,设备的国产化势在必行。
中药经皮给药系统在我国历史悠久,目前评价中药及其有效成分经皮给药的研究系统最常用的方法是应用Franz扩散池测定药物的透皮速率,以此来预测药物的生物等效性。但Franz扩散池存在一些不足,如不能满足溶液流体动力学、离体皮肤的物理化学结构和生理条件发生显著差异等,都影响实验数据的准确性与科学性。
因此将自行研制与构建微透析与液相色谱在线联用技术应用到中药经皮给药的体内药动学与代谢研究中,克服了以往研究方法的不足,为开展这一新技术创造了条件,提供了可靠和全新的手段。同时也为中药现代化研究与中药的质量控制提供了的先进、可行的新技术。
研究方法与结果:
1葛根素高效液相色谱条件的建立与离体大鼠皮肤体外渗透性实验研究本研究建立了测定葛根素的高效液相色谱方法。色谱柱:Hypersil C18柱(5μm, 4.6 mm×150 mm);紫外检测波长:251 nm;流动相:甲醇:水(含0.1%醋酸)(30:70);流速:1min·mL-1;柱温:室温:25±0.1oC。药物浓度与峰面积之间成良好线性关系。方法精密度好、准确度高,符合分析要求。
采用改良Franz竖直扩散池,以卡波姆、氮酮为辅料制备葛根素凝胶剂,离体大鼠皮肤为透皮屏障,用HPLC法测定葛根素的累积渗透量。实验结果表明,葛根素凝胶剂体外透皮释药时间累积透过量拟合方程:Q = 11.12t -45.18 r =0.9635,32 h累积渗透量为335.18±51.45μg/cm2。葛根素以一级动力学透过皮肤,所研制的葛根素经皮给药系统体外渗透性能良好,质量控制方法简便、快捷、准确。
2微透析与高效液相色谱技术(离线)联用对葛根素凝胶剂型经皮给药系统的药代动力学研究
本研究首次以中药单体葛根素模型药物,采用微透析取样与高效液相色谱分析(离线)联用技术(MD-HPLC off-line),考察了其微透析探针体内外回收率,继而进行了葛根素凝胶剂经皮给药系统的皮肤药代动力学和代谢过程的研究。所得数据经kinetica软件处理,得到葛根素经皮给药后皮下葛根素浓度-时间曲线图。其中药时曲线下面积为(AUC) 2335.14 ug·min·mL-1,半衰期为(T1/2) 230.515 min,消除速率常数为(Lz) 0.003,最大血药浓度为(Cmax) 4.62277 mg/mL与其所对应的时间为(Tmax) 460 min。
3微透析与高效液相色谱在线联用技术平台的构建
本研究在国内率先自行研制与构建微透析与高效液相色谱在线联用平台(MD- HPLC on-line),采用10孔自动进样阀将微透析设备与高效液相色谱系统,通过聚丙烯、聚醚醚酮、弹性熔融石英毛细管三种材质的管路,经美国CYBERBOND (Apollo)瞬间生物兼容性粘合剂粘合而联为一体。实现软件联动,最终达到10孔自动进样阀进样的同时,高效液相色谱系统检测器对样品同时进行监测,仪器性能数据应用SPSS软件,配对T检验对3种浓度(0.1 ug/mL,10 ug/mL,100 ug/mL)的葛根素出峰时间理论值与实测值,进行统计学分析,P 0.1=0.239,P 10=0.617, P 100=0.809,P值均大于0.05,无统计学意义,既葛根素的理论值和实测值没有显著性差异。因此得出结论,本实验建立的VCOM和CLASS-VP软件分别控制10孔自动进样阀和HPLC系统,可以做到进样和检测同步完成,仪器精密、准确,完全可以达到实验的要求,实现了微透析取样与高效液相色谱检测在线联动。仪器设备价格低廉、简便实用,经济可靠。这将成为国内第一台自行构建并投入实用的在线联用装置,为推动这一新技术在我国的广泛应用奠定了基础。
4微透析与高效液相色谱离线与自制在线联用平台性能的考察与比较
本研究以葛根素为模型药物,测定MD-HPLC off-line与自制的MD-HPLC on-line的仪器精密度、重现性、稳定性等相关参数,考察MD-HPLC on-line仪器性能,发现与解决存在的问题,进一步改进与完善该系统,数据经SPSS 13.0统计软件分析,具体结果如下所示:
1)回归方程中相关系数(r在线=r离线=1),回归系数(b在线=31304,b离线=29369)分别进行F检验(F在线=168959.6,P在线=0.0001,F离线=71114.847,P离线=0.0001)和t检验(t在线=411.047,P在线=0.0001,t离线=266.674,P离线=0.0001),P值均小于0.05。结果提示,在线和离线回归方程中相关系数、回归系数本身具有统计学意义,且自行研制的MD-HPLC on-line测定浓度和葛根素峰面积的相关性与MD-HPLC off-line无明显差异。进一步对两回归方程中的回归系数进行F检验,反应其差值是否具有统计学意义。采用General linear model观察其交互性参数,F=208.967,P=0.0001,P值均小于0.05,有统计学意义。结果提示,自行研制的MD-HPLC on-line与MD-HPLC off-line测定同一浓度葛根素样品时,MD-HPLC on-line所测得的峰面积响应值优于MD-HPLC off-line。
2)精密度(日内)数据经两组比较t检验分析: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值均小于0.05,且3种浓度葛根素峰面积SD在线 3)重现性数据经两组比较t检验分析:SD在线=8352.453,SD离线= 22953.676,SD在线 4)稳定性数据经两组配对t检验分析:SD在线= 1741.153,SD离线= 2007.673,SD在线 5微透析与高效液相色谱在线联用技术对阿魏酸线性探针的校正本研究首次采用自行研制与构建微透析与高效液相色谱在线联用平台,对见光或受热后极易发生异构化进而降解的中药单体成分阿魏酸进行了线性微透析探针的体内外回收率校正。体外回收率结果:增量法为37.88±1.56 %,减量法为37.11±0.50%;体内回收率结果:减量法为20.69±1.87 %。再次考察和验证MD-HPLC on-line联用平台,同时避免了这类不稳定,易降解的化合物存放中发生的变化,解决了自动分析的问题。为阿魏酸的研究,提供了更加精确、快速、简便的分析测定方法。
6微透析与高效液相色谱在线联用技术在中药经皮给药系统体内药物代谢动力学中的应用
本研究首次采用自行研制与构建的微透析与高效液相色谱在线联用平台与技术,对大鼠皮下的线性微透析探针与颈静脉中的血管微透析探针同时取样,实现同步、实时、在线监测,以全面反映葛根素凝胶制剂给药后在大鼠皮肤与血液中的葛根素药代动力学代谢过程,数据经Kinetica软件处理分析,得到葛根素凝胶经皮给药后皮下和血管内葛根素浓度-时间曲线图。线性微透析探针:药时曲线下面积为(AUC)3662 ug·min·mL-1,半衰期为(T1/2) 519 min,消除速率常数为(Lz) 0.0018,最大血药浓度为(Cmax) 9.6483 ug/mL与其所对应的时间为(Tmax) 381 min;血管微透析探针:药时曲线下面积为(AUC) 2391.11 ug·min·ml-1,半衰期为(T1/2) 250 min,消除速率常数为(Lz)0.0027,最大血药浓度为(Cmax) 14.6563 ug/mL与其所对应的时间为(Tmax) 48 min。提示葛根素在血液中消除快,而在皮下组织中消除较慢,生物利用度皮下组织稍高于血液。
结论:本项目自行研制微透析取样与高效液相色谱在线联用(MD-HPLC on-line)设备,价格低廉、简便实用,把这一新技术的开展引入国内。同时应用这一技术进行具有我国特色的传统中药及其有效成分的经皮给药系统研究,为中药质量可控性、标准化、以及药代动力学与代谢研究提供可靠的科学依据与分析方法。
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 puerarin
Establish 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 HPLC
To 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 apparatus
This 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 performance
The 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 4) The stability of the data that is achieved by two analytic and comparative groups of t test shows: SDonline = 1741.153, SDoffline = 2007.673, SDonline 5、Calibration of linear probe for ferulic acid by self-made MD-HPLC on-line multiple analytical technique.
In this study, the self-made MD-HPLC on-line has been used for the first time to correct the in vitro and in vivo probe recovery of ferulic acid which can easily change when it meets light or heat. The MD-HPLC on-line was re-inspected and validated, while avoiding changes of such instable, easily degradable compounds and resolving the problem of automatic analysis. It has also provided a more accurate, rapid and simple method of analysis for the research of ferulic acid. In vitro recovery rate results:gain method for the 37.88±1.56%, loss method for the 37.11±0.50%; recovery results in vivo: loss method for the 20.69±1.87%.
6、The application of in vivo pharmacokinetics TDDS for puerarin by MD-HPLC on-line multiple analytical technique.
To monitor the in vivo pharmacokinetics of puerarin in rat skin and in jugular vein by self-made MD and HPLC on-line multiple analytical technique, 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 : Linear-(AUC) 2391.11 ug·min·ml-1, (T1/2) 519 min,(Lz) 0.0018,(Cmax) 9.6483 ug/ml, (Tmax) 381 min.;Vascular-(AUC) 2391 ug·min·ml-1,(T1/2) 250 min,(Lz) 0.0027,(Cmax) 14.6563 ug/ ml, (Tmax) 48 min. It has indicated that Puerarin eliminates slower at subcutaneous tissue than in the blood, which means the bioavailability of subcutaneous at subcutaneous tissue is higher.
Conclusion: In this paper, we established self-made MD and HPLC on-line multiple analytical technique. It has cut down the high price of this apparatus,and met the domestic's requirement. Meanwhile the application of this technique to TDDS provides an quality control standards for the traditional Chinese drug .It can be the guarantee of controllability and standardization of the pharmacokinetics and also be a base for the further qualitative or quantitative analytical research.
中药经皮给药系统在我国历史悠久,目前评价中药及其有效成分经皮给药的研究系统最常用的方法是应用Franz扩散池测定药物的透皮速率,以此来预测药物的生物等效性。但Franz扩散池存在一些不足,如不能满足溶液流体动力学、离体皮肤的物理化学结构和生理条件发生显著差异等,都影响实验数据的准确性与科学性。
因此将自行研制与构建微透析与液相色谱在线联用技术应用到中药经皮给药的体内药动学与代谢研究中,克服了以往研究方法的不足,为开展这一新技术创造了条件,提供了可靠和全新的手段。同时也为中药现代化研究与中药的质量控制提供了的先进、可行的新技术。
研究方法与结果:
1葛根素高效液相色谱条件的建立与离体大鼠皮肤体外渗透性实验研究本研究建立了测定葛根素的高效液相色谱方法。色谱柱:Hypersil C18柱(5μm, 4.6 mm×150 mm);紫外检测波长:251 nm;流动相:甲醇:水(含0.1%醋酸)(30:70);流速:1min·mL-1;柱温:室温:25±0.1oC。药物浓度与峰面积之间成良好线性关系。方法精密度好、准确度高,符合分析要求。
采用改良Franz竖直扩散池,以卡波姆、氮酮为辅料制备葛根素凝胶剂,离体大鼠皮肤为透皮屏障,用HPLC法测定葛根素的累积渗透量。实验结果表明,葛根素凝胶剂体外透皮释药时间累积透过量拟合方程:Q = 11.12t -45.18 r =0.9635,32 h累积渗透量为335.18±51.45μg/cm2。葛根素以一级动力学透过皮肤,所研制的葛根素经皮给药系统体外渗透性能良好,质量控制方法简便、快捷、准确。
2微透析与高效液相色谱技术(离线)联用对葛根素凝胶剂型经皮给药系统的药代动力学研究
本研究首次以中药单体葛根素模型药物,采用微透析取样与高效液相色谱分析(离线)联用技术(MD-HPLC off-line),考察了其微透析探针体内外回收率,继而进行了葛根素凝胶剂经皮给药系统的皮肤药代动力学和代谢过程的研究。所得数据经kinetica软件处理,得到葛根素经皮给药后皮下葛根素浓度-时间曲线图。其中药时曲线下面积为(AUC) 2335.14 ug·min·mL-1,半衰期为(T1/2) 230.515 min,消除速率常数为(Lz) 0.003,最大血药浓度为(Cmax) 4.62277 mg/mL与其所对应的时间为(Tmax) 460 min。
3微透析与高效液相色谱在线联用技术平台的构建
本研究在国内率先自行研制与构建微透析与高效液相色谱在线联用平台(MD- HPLC on-line),采用10孔自动进样阀将微透析设备与高效液相色谱系统,通过聚丙烯、聚醚醚酮、弹性熔融石英毛细管三种材质的管路,经美国CYBERBOND (Apollo)瞬间生物兼容性粘合剂粘合而联为一体。实现软件联动,最终达到10孔自动进样阀进样的同时,高效液相色谱系统检测器对样品同时进行监测,仪器性能数据应用SPSS软件,配对T检验对3种浓度(0.1 ug/mL,10 ug/mL,100 ug/mL)的葛根素出峰时间理论值与实测值,进行统计学分析,P 0.1=0.239,P 10=0.617, P 100=0.809,P值均大于0.05,无统计学意义,既葛根素的理论值和实测值没有显著性差异。因此得出结论,本实验建立的VCOM和CLASS-VP软件分别控制10孔自动进样阀和HPLC系统,可以做到进样和检测同步完成,仪器精密、准确,完全可以达到实验的要求,实现了微透析取样与高效液相色谱检测在线联动。仪器设备价格低廉、简便实用,经济可靠。这将成为国内第一台自行构建并投入实用的在线联用装置,为推动这一新技术在我国的广泛应用奠定了基础。
4微透析与高效液相色谱离线与自制在线联用平台性能的考察与比较
本研究以葛根素为模型药物,测定MD-HPLC off-line与自制的MD-HPLC on-line的仪器精密度、重现性、稳定性等相关参数,考察MD-HPLC on-line仪器性能,发现与解决存在的问题,进一步改进与完善该系统,数据经SPSS 13.0统计软件分析,具体结果如下所示:
1)回归方程中相关系数(r在线=r离线=1),回归系数(b在线=31304,b离线=29369)分别进行F检验(F在线=168959.6,P在线=0.0001,F离线=71114.847,P离线=0.0001)和t检验(t在线=411.047,P在线=0.0001,t离线=266.674,P离线=0.0001),P值均小于0.05。结果提示,在线和离线回归方程中相关系数、回归系数本身具有统计学意义,且自行研制的MD-HPLC on-line测定浓度和葛根素峰面积的相关性与MD-HPLC off-line无明显差异。进一步对两回归方程中的回归系数进行F检验,反应其差值是否具有统计学意义。采用General linear model观察其交互性参数,F=208.967,P=0.0001,P值均小于0.05,有统计学意义。结果提示,自行研制的MD-HPLC on-line与MD-HPLC off-line测定同一浓度葛根素样品时,MD-HPLC on-line所测得的峰面积响应值优于MD-HPLC off-line。
2)精密度(日内)数据经两组比较t检验分析: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值均小于0.05,且3种浓度葛根素峰面积SD在线
6微透析与高效液相色谱在线联用技术在中药经皮给药系统体内药物代谢动力学中的应用
本研究首次采用自行研制与构建的微透析与高效液相色谱在线联用平台与技术,对大鼠皮下的线性微透析探针与颈静脉中的血管微透析探针同时取样,实现同步、实时、在线监测,以全面反映葛根素凝胶制剂给药后在大鼠皮肤与血液中的葛根素药代动力学代谢过程,数据经Kinetica软件处理分析,得到葛根素凝胶经皮给药后皮下和血管内葛根素浓度-时间曲线图。线性微透析探针:药时曲线下面积为(AUC)3662 ug·min·mL-1,半衰期为(T1/2) 519 min,消除速率常数为(Lz) 0.0018,最大血药浓度为(Cmax) 9.6483 ug/mL与其所对应的时间为(Tmax) 381 min;血管微透析探针:药时曲线下面积为(AUC) 2391.11 ug·min·ml-1,半衰期为(T1/2) 250 min,消除速率常数为(Lz)0.0027,最大血药浓度为(Cmax) 14.6563 ug/mL与其所对应的时间为(Tmax) 48 min。提示葛根素在血液中消除快,而在皮下组织中消除较慢,生物利用度皮下组织稍高于血液。
结论:本项目自行研制微透析取样与高效液相色谱在线联用(MD-HPLC on-line)设备,价格低廉、简便实用,把这一新技术的开展引入国内。同时应用这一技术进行具有我国特色的传统中药及其有效成分的经皮给药系统研究,为中药质量可控性、标准化、以及药代动力学与代谢研究提供可靠的科学依据与分析方法。
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 puerarin
Establish 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 HPLC
To 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 apparatus
This 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 performance
The 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
In this study, the self-made MD-HPLC on-line has been used for the first time to correct the in vitro and in vivo probe recovery of ferulic acid which can easily change when it meets light or heat. The MD-HPLC on-line was re-inspected and validated, while avoiding changes of such instable, easily degradable compounds and resolving the problem of automatic analysis. It has also provided a more accurate, rapid and simple method of analysis for the research of ferulic acid. In vitro recovery rate results:gain method for the 37.88±1.56%, loss method for the 37.11±0.50%; recovery results in vivo: loss method for the 20.69±1.87%.
6、The application of in vivo pharmacokinetics TDDS for puerarin by MD-HPLC on-line multiple analytical technique.
To monitor the in vivo pharmacokinetics of puerarin in rat skin and in jugular vein by self-made MD and HPLC on-line multiple analytical technique, 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 : Linear-(AUC) 2391.11 ug·min·ml-1, (T1/2) 519 min,(Lz) 0.0018,(Cmax) 9.6483 ug/ml, (Tmax) 381 min.;Vascular-(AUC) 2391 ug·min·ml-1,(T1/2) 250 min,(Lz) 0.0027,(Cmax) 14.6563 ug/ ml, (Tmax) 48 min. It has indicated that Puerarin eliminates slower at subcutaneous tissue than in the blood, which means the bioavailability of subcutaneous at subcutaneous tissue is higher.
Conclusion: In this paper, we established self-made MD and HPLC on-line multiple analytical technique. It has cut down the high price of this apparatus,and met the domestic's requirement. Meanwhile the application of this technique to TDDS provides an quality control standards for the traditional Chinese drug .It can be the guarantee of controllability and standardization of the pharmacokinetics and also be a base for the further qualitative or quantitative analytical research.
引文
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[10]Amal Kaddoumi,Mihoko N,Nakashima,Toshihide Maki,et a1. Liquid chromatography studies on the pharmacokinetics of phenterm ine and fennuramine in brain and blood microdialysates after intraperitoneal administration to rats.Journal of Chromatography B,2003,791:291-303
[11]Amal Kaddoumi, Toyomi Mori, Mihoko N, Nakashima,et a1. High performance liquidchromatography with fluorescence detection for the determination of phenylpro-panolamine in human plasma and rat’s blood and brainmicrodialysates using DIB-CI as a labe1.Journal of Pharmaceutical and Biomedical Analysis,2004,34:643-650
[12]Tung Hu Tsai,Mei Chun Liu.Determination of unbound theophyllinein rat blood and brain by microdialysisand liquidchromatography.Journal of Chromatography A,2004,10321:97-101
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