Journal of Chromatography B, 893–894 (2012) 168–172 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B jo u r n al hom epage: www.elsevier.com/locate/chromb Short communication Development of a fast and simple liquid chromatography–tandem mass spectrometry method for the quantitation of argatroban in patient plasma samples Jeanne M. Rhea a,1 , Marion L. Snyder b,1 , Anne M. Winkler a , Charbel Abou-Diwan c , Corinne R. Fantz a , James C. Ritchie a , Fania Szlam d , Kenichi A. Tanaka d , Ross J. Molinaro a,∗ a Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA b Greenville Hospital System University Medical Center, Greenville, SC, USA c Banner Good Samaritan Medical Center, Phoenix, AZ, USA d Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA, USA a r t i c l e i n f o Article history: Received 16 November 2011 Accepted 23 February 2012 Available online 3 March 2012 Keywords: Mass spectrometry Argatroban monitoring Hemoclot Thrombin Inhibitors [HTI] Plasma Method comparison Hospitalized patient samples Heparin-Induced Thrombocytopenia (HIT) a b s t r a c t An ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the direct measurement of argatroban in human plasma was developed and compared with the activity-based Hemoclot Thrombin Inhibitors assay. UPLC–MS/MS was performed using diclofenac as an internal stan- dard. In summary, argatroban and diclofenac were extracted from 100 L of plasma using a methanol precipitation protocol, and chromatographic separation was performed on an ACQUITY TM TQD mass spectrometer using a UPLC C18 BEH 1.7 m column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation were performed using positive ion electrospray ionization and multiple reaction monitoring (MRM) mode. The UPLC–MS/MS method was linear over the concen- tration range of 0.003–3.0 g/mL, with a lower limit of quantitation for argatroban of 0.003 g/mL. The intra- and inter-assay imprecision was less than 12% at the plasma argatroban concentrations tested. Good correlation was demonstrated between the UPLC–MS/MS method and the indirect activity-based assay for determination of argatroban. However, increased plasma fibrinogen levels caused underestima- tion of argatroban levels using the indirect activity-based assay, whereas the UPLC–MS/MS method was unaffected. UPLC–MS/MS provides a relatively simple, sensitive, and rapid means of argatroban monitor- ing. It has successfully been applied to assess plasma argatroban concentrations in hospitalized patients and may provide a more accurate determination of argatroban concentrations than an activity-based assay in certain clinical conditions. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Heparin-induced thrombocytopenia (HIT) is a complication occurring in approximately 1–5% of patients treated with hep- arin [1,2], and requires the immediate replacement of heparin therapy with an alternative, rapidly active anticoagulant such as argatroban [3]. In contrast to heparins, which require formation of heparin–antithrombin–thrombin complexes to block throm- bin activity [4], argatroban is a direct thrombin inhibitor (DTI) which reversibly binds to the thrombin active site. Derived from ∗ Corresponding author at: Pathology and Laboratory Medicine, Emory University School of Medicine, Emory University Hospital Midtown, 550 Peachtree Avenue NE, Davis Fischer Building, Room 1239, Atlanta, GA 30308, USA. Tel.: +1 404 686 1913; fax: +1 404 727 9656. E-mail address: rjmolin@emory.edu (R.J. Molinaro). 1 These authors contributed equally to this work. l-arginine, argatroban was licensed by the Food and Drug Adminis- tration for prophylaxis or treatment of thrombosis in patients with HIT and, in 2002, was approved for use during percutaneous coro- nary interventions in patients who have or are at risk for developing HIT. Argatroban is administered intravenously, with steady-state blood levels and anticoagulant effect of argatroban usually obtained 1–3 h after initiation of therapy. Pharmacokinetic and pharmaco- dynamic studies have revealed that renal function, age, and sex do not have a clinical effect on metabolism, distribution, elimination, or anticoagulation of argatroban [5,6]. The main route of argatroban metabolism is hydroxylation and aromatization in the liver. This metabolism results in four known metabolites: M1, M2, M3, and M4 [7]. Of these metabolites, M1 is known to possess pharmaco- logical activity, but is a significantly less potent thrombin inhibitor compared to argatroban [7]. Argatroban and other DTIs are frequently monitored using clotting-based methods such as the activated partial 1570-0232/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2012.02.041