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目的:建立用生色底物法(chromogenic substrate assay,CSA)测定大鼠血浆中重组水蛭素(re-combinant hirudin,rH)含量的方法,并用该法研究rH在大鼠血浆中的药动学。方法:以TGPApNA(tosyl-gly-cyl-polyl-arginine-para-nitraniline)为底物,凝血酶(thrombin,Th)能水解TGPApNA释放出对-硝基苯胺(para-nitraniline,pNA),pNA在405nm处有特异性吸收,通过测定反应体系中未被rH中和的Th量,间接测定rH的活性。结果:CSA法能有效地测定大鼠血浆中rH的含量;rH的浓度在3.125~40 ng.mL-1范围内呈线性关系;日间和日内精密度相对标准偏差均符合要求。应用上述方法测定大鼠iv 2.0 mg.kg-1的rH后不同时间的血药浓度,结果表明该药符合二室模型一级动力学过程。结论:用生色底物法测定大鼠血浆中rH具有准确、可靠、重现性好的特点,可用于rH药动学的研究。
OBJECTIVE: To establish a method for determination of recombinant human hirudin (rH) in rat plasma by chromogenic substrate assay (CSA) and to study the pharmacokinetics of rH in rat plasma . Methods: The pNA was released by hydrolysis of TGPApNA by thrombin (Th) with TGPApNA (tosyl-gly-cyl-polyl-arginine-para-nitraniline) 405nm at a specific absorption, by measuring the reaction system is not neutralized rH Th, indirect determination of rH activity. Results: CSA method could effectively determine the content of rH in rat plasma. The concentration of rH was linear in the range of 3.125 ~ 40 ng.mL-1. The relative standard deviations (RSDs) of intra-day and intra-day were satisfactory. The above method was used to determine the plasma concentrations of rat 2.0 mg.kg-1 iv at different times after rH. The results showed that the drug conformed to the first-order kinetics of two-compartment model. Conclusion: The determination of rH in rat plasma with chromogenic substrate method is accurate, reliable and reproducible. It can be used for rH pharmacokinetic study.