微透析法测定秋水仙碱体外血浆蛋白结合率及与超滤法比较

Determination in vitro of plasma protein binding rate of colchicine by microdialysis and comparison with ultrafiltration

目的:采用微透析技术研究秋水仙碱的体外血浆蛋白结合率,并与超滤法比较。方法:微透析探针分别浸于含125μg/mL、250μg/mL、500μg/mL秋水仙碱的兔、大鼠血浆中。灌注器以2.5μL/min的流速依次灌注40 min不同浓度的血浆平衡,每60 min间隔采样1次,高效液相色谱法(HPLC)分析秋水仙碱浓度,计算兔、大鼠血浆蛋白结合率,并与超滤法进行比较。结果:秋水仙碱在0.09992~99.9200μg/mL呈良好线性关系,标准方程为A=25052 C-6613.6,r=0.9997;日内精密度和日间精密度相对标准差(RSD)分别为0.36%、0.85%、0.92%和2.53%、1.74%、1.19%;秋水仙碱浓度在13.96μg/mL、52.10μg/mL、71.44μg/mL的回收率分别为(99.53±0.04)%、(99.99±0.13)%、(99.92±0.22)%;秋水仙碱浓度在120μg/mL、250μg/mL、500μg/mL的兔、大鼠血浆体外平均回收率分别为46.43%、35.46%,秋水仙碱浓度为125μg/mL时,兔、大鼠体外血浆8 h内稳定性考察平均值分别为41.22%、30.23%,RSD分别为1.36%、1.01%。在相同浓度相同种属下,低、中浓度秋水仙碱在大鼠血浆中的蛋白结合率差异无统计学意义(P>0.05),在兔血浆中的蛋白结合率差异有统计学意义(P<0.05)。结论:秋水仙碱与兔、大鼠血浆有较高的蛋白结合率,微透析法和超滤法结果差异不大。

Objective:To determine in vitro the plasma protein binding rate of colchicine using the microdialysis technique and compare it with the ultrafiltration method.Methods:Microdialysis probes were immersed in rabbit and rat plasma containing colchicine at 125μg/mL,250μg/mL and 500μg/mL,respectively.Different concentrations of plasma equilibrium were perfused at a flow rate of 2.5μL/min for 40 min and sampled every 60 min.Colchicine concentrations were analyzed by HPLC,and the binding rates of rabbit and rat plasma protein were calculated and compared with the ultrafiltration method.Results:Colchicine showed good linearity from 0.09992 to 99.9200μg/mL with the standard equation of A=25,052C-6,613.6,r=0.9997.The intra-day precision and interday precision RSDs were 0.36%,0.85%,0.92%and 2.53%,1.74%,1.19%,respectively.The recoveries were(99.53±0.04)%,(99.99±0.13)%and(99.92±0.22)%for colchicine concentrations of 13.96μg/mL,52.10μg/mL and 71.44μg/mL,respectively;the average in vitro recoveries of rabbit and rat plasma with colchicine concentrations of 120μg/mL,250μg/mL and 500μg/mL were 46.43%and 35.46%,and the mean values of the rabbit and rat plasma stability examination within 8 h at a colchicine concentration of 125μg/mL were 41.22%and 30.23%with RSDs of 1.36%and 1.01%,respectively.The differences in protein binding rates in rat plasma of low and medium concentration of colchicine at the same concentration and in the same species were not statistically significant(P>0.05),and the differences in protein binding rates in rabbit plasma were statistically significant(P<0.05).Conclu-sion:Colchicine has a high protein binding rate with rabbit and rat plasma,and the difference between the results of microdialysis and ultrafiltration methods is not significant.