10. Development and Validation of an Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS) Method for Direct Monitoring of Argatroban Anticoagulation

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Marion L Snyder Brigham and Womens Hospital

*Marion L. Snyder, #Anne M. Winkler, #Corinne R. Fantz, #Ross J. Molinaro. *Brigham and Women's Hospital, Boston, MA #Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA.

Background: Argatroban, a direct thrombin inhibitor (DTI) approved for prophylaxis and treatment of venous thromboembolism in patients with heparin-induced thrombocytopenia (HIT), is most commonly monitored using the clotting-based activated partial thromboplastin time (aPTT). Unfortunately, the aPTT can overestimate the degree of anticoagulation in patients with clotting factor deficiencies or inhibitors, other nonspecific inhibitors (eg, lupus anticoagulants), and patients being bridged to oral anticoagulants. The aPTT response to argatroban also varies with different manufacturers’ reagents and argatroban concentrations. For these reasons, a direct measure of plasma argatroban concentrations is preferred and may also be used to establish concentration-based therapeutic ranges. Objectives: To evaluate a novel, highly sensitive and specific ultra-performance liquid chromatography-coupled electrospray positive ionization tandem mass spectrometry (UPLC-MS/MS) method for direct quantification of plasma argatroban concentration and determination of argatroban therapeutic ranges, in comparison with established (aPTT) and newly developed (Hemoclot Thrombin Inhibitors [HTI], HYPHEN BioMed) automated clotting-based methods for argatroban monitoring. Method: Argatroban was isolated from 100 μL of plasma after internal standard–spiked methanol extraction and centrifugation. The supernatant was injected into the UPLC-MS/MS (Waters ACQUITY UPLC TQD) and quantified using a 7-point standard curve. The aPTT, prothrombin time (PT), and HTI clotting-based assays were all performed on the Dade Behring BCS automated analyzer. Validation/Results: The UPLC-MS/MS method is linear from 0.04 to 2.0 μg/mL, with inter- and intra-assay precisions (CVs) of <10% over the entire linear range. Correlation with the HTI method revealed a slope of 0.98, an intercept of 0.1, and r2 of 0.99 for spiked drug-free patient plasma. Plasma argatroban levels also correlated well with PT and aPTT, although the responses differed for patients receiving concurrent warfarin therapy and patients with baseline prolonged aPTTs. Conclusions: The UPLC-MS/MS method demonstrates good correlation between plasma argatroban mass- and activity-based assays. Although the aPTT and HTI methods offer quick alternatives, our UPLC-MS/MS argatroban assay is preferred in patients with underlying factors leading to inaccurate estimations of argatroban anticoagulation using clotting-based assays. This novel assay is also useful for determining argatroban concentration–based therapeutic ranges.

Email: mlsnyder@partners.org

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