Simultaneous monitoring of argatroban and its major metabolite using an HPLC method: Potential clinical applications

Argatroban is a peptidomimetic inhibitor of thrombin that is currently unde rgoing extensive clinical trials as a heparin substitute for thrombotic com plications. Argatroban is readily metabolized into a major derivative, MI, that has pharmacological characteristics distinct from its parent compound. The currently available clot-based assays measure the cumulative anticoagu lant effect of argatroban and its metabolite(s). Available HPLC methods do not differentiate between argatroban and MI-metabolite. A modified method w as developed to simultaneously quantitate MI-metabolite and argatroban in b iological fluids. Initial validation studies for the method included clinic al trials of argatroban in patients with heparin-induced thrombocytopenia, (ARC 911 Study) and coronary interventional procedures (ARG 310 Study). Pla sma samples were extracted with acetonitrile and reconstituted in a mobile phase. Calibration curves were prepared by running known standards of: arga troban and MI-metabolite in normal human plasma. Ultraviolet detection was made at 320 nm. The retention times for argatroban and MI-metabolite peaks were found to be 10.5 +/- 0.3 minutes and 3.9 +/- 0.1 minutes, respectively . The extraction efficiency was > 95% (r(2) = 0.99). In heparin-induced thr ombocytopenia patients with major bleeding complications (n = 30), the rela tive increase in M1-metabolite compared to argatroban varied widely (two- t o eight-fold). The mean concentration of argatroban during the steady infus ion period was found to be 0.7 +/- 0.35 mu g/mL, and for M1-metabolite, it was 5.5 +/- 2.8 mu g/mL. Proportionate results were not seen when higher do sages of argatroban were administered (coronary angioplasty studies). Argat roban and M1-metabolite levels also compared well with the results in globa l clotting assays. Owing to the simultaneous quantitation of argatroban and Mi-metabolite, this method provides a rapid assessment of the pharmacokine tics and pharmacodynamics of argatroban. The differential quantitation may be useful in the assessment of relative metabolic turnover of argatroban th at can be related to the hepatic and renal functions in a given patient.