葛根素微透析体外回收率测定及其影响因素研究

Experimental factors affecting in vitro recovery of puerarin in microdialysis

目的:建立葛根素微透析体外回收率的测定方法,考察影响回收率的有关因素。方法:采用高效液相色谱法测定微透析样品中葛根素的浓度,利用增量法、减量法和零净通量法计算探针的相对回收率,并考察灌流液成分、外液浓度、灌流速度、介质温度和搅拌速率对回收率的影响。结果:对于同一探针,三种计算方法得出的回收率存在差异。采用等渗氯化钠溶液、林格液、PBS和葡萄糖溶液为灌流液时,得到的回收率分别为(71.25±2.36)%、(73.48±1.41)%、(68.50±2.43)%和(74.98±1.16)%,差异有统计学意义(P<0.01);在同一灌流速度下,探针回收率在外液葛根素浓度为0.2~25μg/m L的范围内差异无统计学意义(P>0.05);在同一浓度下,探针的回收率随灌流速度的增加呈指数下降;在25~40℃范围内,探针回收率随介质温度升高而增大;探针的回收率随着搅拌速率的增加而增大,当速率达到200转/min时,回收率达到一个相对稳定的值。结论:本研究建立了葛根素微透析体外回收率的测定方法。葛根素微透析探针体外回收率与介质中葛根素浓度无关,而受计算方法、灌流液成分、灌流速度、介质温度和搅拌速率等因素的影响。

Objective： To analyze experimental factors affecting in vitro recovery of puerarin in microdialysis. Methods： Puerarin concentration in microdialysate samples was determined by high performance liquid chromatography. The methods of direct dialysis,retrodialysis and the zero-net flux were used to calculate in vitro recovery,respectively. The effects of perfusate composition,the analyte concentration,perfusate flow rate,medium temperature and stir rates of the dialysis medium on recovery were investigated. Results： There were significant differences in the recovery values among direct dialysis,retrodialysis and zero-net flux methods. The recovery for 0. 9% Na Cl solution,Ringer＇s solution,PBS and anticoagulant dextrose solution as perfusate fluid were（71. 25 ± 2. 36）%,（73. 48 ± 1. 41）%,（68. 50 ± 2. 43）% and（74. 98 ± 1. 16）%,respectively. The composition of perfusate fluid had significant influence on the recovery（P〈0. 01）. At the same flow rate, recovery was independent of the analyte concentration. At the same concentration,the recovery was decrease with the increasing flow rate in an exponential relationship. The recovery increased with the raising temperature and stir rate of the dialysis medium,and the recovery remained stable when the stir rate reached above 200 rpm. Conclusion： A study method for in vitro recovery of puerarin in microdialysis has been established,and the recovery of puerarin is affected by calculating methods,perfusate fluids,flow rate,medium temperature and stir rate,but not affected by analyte concentrations.