Determination Of R-Hirudin In Urine Of Rabbits By Thrombin Time Method

Objective:To develop a bioassay,i.e.thrombin time(TT)method,for determining r-hirudin(rH)in u- rine of rabbits so as to provide a methodology for further studying rH urinary excretion pharmacokinetics.Methods: The rH level in urine was measured by means of anti-thrombin activity of rH.TT was determined in a coagulometer following successive addition of 40μL blank plasma of the rabbits ,50μL Tris-HCl buffer(pH 7.4)and 50μL 5 u· mL~(-1)thrombin solution to 10μL urine.The concentration of rH in urine was read from the logTr prolongation rate- concentration calibration curve.An appropriate dilution with blank urine was inevitable if rH level in urine samples was higher than linear range.Results:The logTT prolongation rate was found to be linearly related to the rH concen- tration in urine over the range of 0.49-2.50μg·mL~(-1)(r=0.9956).The bioassay showed a good precision of< 8.10%(within-day RSD)and <9.07 %(between-day RSD)as well as high recovery of 95.6%-97.7%(method recovery)and>90.0%(dilution recovery).rH standard urine samples were stable for at least 2 weeks at-20℃ and 8 h at room temperature.Application of the above method to the urinary excretion kinetic study of rH in rabbits dosed iv rH demonstrated an log(X_(u∞)-X_(ut))-t curve corresponding to the two-compartment model and the first-order ki- netics with elimination half life of 65.4 min.Conclusion:The TT method developed by us is well suitable for measure- ment of rH concentration in urine and for study of its urinary excretion oharmacokinetics in rabbits.