Simultaneous Monitoring of Argatroban and Its Major Metabolite Using an HPLC Method: Potential Clinical Applications

Abstract

Argatroban is a peptidomimetic inhibitor of thrombin that is currently undergoing extensive clinical trials as a heparin substitute for thrombotic complications. Argatroban is readily metabolized into a major derivative, M1, that has pharmacological characteristics distinct from its parent compound. The currently available clot-based assays measure the cumulative anticoagulant effect of argatroban and its metabolite(s). Available HPLC methods do not differentiate between argatroban and M1-metabolite. A modified method was developed to simultaneouly quantitate M1-metabolite and argatroban in biological fluids. Initial validation studies for the method included clinical trials of argatroban in patients with heparin-induced thrombocytopenia, (ARG 911 Study) and coronary interventional procedures (ARG 310 Study). Plasma samples were extracted with acetonitrile and reconstituted in a mobile phase. Calibration curves were prepared by running known standards of argatroban and M1-metabolite in normal human plasma. Ultraviolet detection was made at 320 nm. The retention times for argatroban and M1-metabolite peaks were found to be 10.5 +/- 0.3 minutes and 3.9 +/- 0.1 minutes, respectively. The extraction efficiency was > 95% (r2 = 0.99). In heparin-induced thrombocytopenia patients with major bleeding complications (n = 30), the relative increase in M1-metabolite compared to argatroban varied widely (two- to eight-fold). The mean concentration of argatroban during the steady infusion period was found to be 0.7 +/- 0.35 microgram/mL, and for M1-metabolite, it was 5.5 +/- 2.8 micrograms/mL. Proportionate results were not seen when higher dosages of argatroban were administered (coronary angioplasty studies). Argatroban and M1-metabolite levels also compared well with the results in global clotting assays. Owing to the simultaneous quantitation of argatroban and M1-metabolite, this method provides a rapid assessment of the pharmacokinetics and pharmacodynamics of argatroban. The differential quantitation may be useful in the assessment of relative metabolic turnover of argatroban that can be related to the hepatic and renal functions in a given patient.

Monitoring of Argatroban in ARG310 Study: Potential Recommendations for its Use in Interventional Cardiology

“…To this end, we used a recently developed HPLC system where absolute levels of argatroban and its major Ml-metabolite can be quantitated simultaneously (19). The results of the circulating levels of argatroban based on the HPLC measurements are shown in Fig.…”

“…These calibration samples also underwent similar treatment of extraction procedure(s). The argatroban and/or Ml (if any) peak(s)' area under the curve (AUC) was determined according to the standard method of such calculations performed by computer analysis (19).…”

Use of Lepirudin in Patients with Heparin-Induced Thrombocytopenia and Renal Failure Requiring Hemodialysis

“…She was admitted to the hospital because of active gastrointestinal bleeding. Laboratory results on admission included an international normalized ratio (INR) of 4.58, hematocrit of 19.1% (normal 36-46%), hemoglobin 6.6 g/dL (12)(13)(14)(15)(16), and platelet count of 281 × 10 3 /mm 3 (130-400 ). The activated partial thromboplastin time (aPTT) was elevated at 55.3 seconds (23-32) secondary to cross-reactivity to warfarin and possible effects of rehydration from acute blood loss.…”

Prolonged elevation of plasma argatroban in a cardiac transplant patient with a suspected history of heparin‐induced thrombocytopenia with thrombosis

“…For example, no argatroban was detected in Samples A and K, and a decline of values were observed over time, both of which are consistent with the patient's clinical course. Had this not been the case, a high‐performance liquid chromatography–based measurement would have been the method of choice 17,26 …”