吗啡游离浓度测定及人血红蛋白结合研究

目的　建立人血红蛋白溶液中吗啡游离浓度测定方法 ,了解吗啡与血红蛋白的结合率 ,计算相关参数。方法　采用超滤法分离出游离吗啡 ,高效液相色谱法测定游离吗啡浓度 ,以总浓度减去游离浓度的差值与总浓度的比值为吗啡-血红蛋白结合率。结果　吗啡的超滤平均回收率为98 5 % ,测定的精密度为 1 1%～ 1 5 %。在体外 ,吗啡总浓度在 8 5 0× 10 -5～ 1 17× 10 -2 mol·L-1、人血红蛋白浓度在1 2 9× 10 -4～ 8 5 7× 10 -4mol·L-1范围内 ,吗啡与人血红蛋白具单一类型结合部位 ,吗啡 -血红蛋白结合参数 :人血红蛋白结合部位数 (N)为 4 1,吗啡 -血红蛋白结合常数 (K)为 30 4L·mol-1。结论　超滤法结合高效液相色谱法是一种简便、快速的游离药物浓度测定方法 ,适用于药物蛋白结合率试验研究。在体外 ,吗啡与血红蛋白结合率随吗啡与血红蛋白浓度变化

Determination of free morphinen and human haemoglobin Binding in vitro

AIM To develope a reversed phase high performance liquid chromatography method to determine free concentrations of morphine (M) in the solution of human haemoglobin (Hb). To study the binding of M to haemoglobin, and evaluate the binding parameters of M to Hb. METHODS An ultrafiltration technique was used to recover morphine from the samples. Morphine was analyzed using a kromasil column (150 mm×4 6 mm) and a mobile phase of 0 1% tyiethylamine methanol (75∶25,v/v). The mobile phase pH was adjusted to 7 0 by phosphoric acid. The detection was set at 283 nm. RESULTS The ultrafiltration recovery of morphine was 98 5%. The Hb binding of M was concentration dependent of Hb and M. There were single typed binding sites for M to human Hb. The parameters determined were 4 1 for N and 340 mol·L -1 for K when the concentration of M and Hb were added from 8 50×10 -5 ～1 17×10 -2 mol·L -1 and 1 29×10 -4 ～8 57×10 -4 mol·L -1 respectively. CONCLUSION An ultrafiltration technique has proved to be simple and rapid for free drug determination. It is suitable for drug protein binding study.