Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 230–235

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Simultaneous determination of rivaroxaban and dabigatran levels in human plasma by high-performance liquid chromatography–tandem mass spectrometry Marie Korostelev a , Kevin Bihan a , Lison Ferreol a , Nadine Tissot a , Jean-Sebastien Hulot a,b,c , Christian Funck-Brentano a,b,c , Noël Zahr a,∗ a

AP-HP, Pitié-Salpêtrière Hospital, Department of Pharmacology and CIC-1421, F-75013 Paris, France Sorbonne University, UPMC Univ Paris 06, Faculty of Medicine, Department of Pharmacology and UMR ICAN 1166, F-75013 Paris, France c INSERM, CIC-1421 and UMR ICAN 1166, F-75013 Paris, France b

a r t i c l e

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Article history: Received 14 July 2014 Received in revised form 31 July 2014 Accepted 6 August 2014 Available online 15 August 2014 Keywords: Rivaroxaban Dabigatran Mass spectrometry LC–MS/MS TSOAC

a b s t r a c t A sensitive and accurate liquid chromatography method with mass spectrometry detection was developed and validated for the quantification of dabigatran (Pradaxa® ) and rivaroxaban (Xarelto® ). 13 C6 -dabigatran and 13 C6 -rivaroxaban were used as the internal standard. A single-step protein precipitation was used for plasma sample preparation. This method was validated with respect to linearity, selectivity, inter- and intra-day precision and accuracy, limit of quantification and stability. The lower limit of quantification was 2.5 ng/mL for both drugs in plasma. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Since their marketing authorization, target specific oral anticoagulants (TSOACs) are widely used for prophylaxis and treatment of thromboembolic events. Indeed, these drugs directly target proteins essential for coagulation, namely thrombin (dabigatran) or factor Xa (rivaroxaban). TSOACs have demonstrated at least a similar anticoagulant effect associated with the same or a lower bleeding risk compared to vitamin K antagonist (VKA) and low molecular weight heparin (LMWH) [1,2]. Moreover, the direct mechanism of action makes these drugs particularly interesting for several reasons: short half-lives, a rapid onset of action, fewer drug and food interaction, and predictable pharmacokinetics. In contrast with VKA and LMWH, no laboratory monitoring is advised [3,4]. However, there are clinical situations, which can require monitoring of TSOACs plasma concentration to ensure the absence of

∗ Corresponding author at: Pitié-Salpêtrière Hospital, Department of Pharmacology, Assistance Publique Hôpitaux de Paris 47, Boulevard de l’hôpital, 75013 Paris, France. Tel.: +33 1 42 16 20 15; fax: +33 1 42 16 20 46. E-mail address: [email protected] (N. Zahr). 0731-7085/© 2014 Elsevier B.V. All rights reserved.

over- or underdosing (renal impairment, elderly patients, obesity) because of an increased interindividual variability and/or a modified bioavailability [5]. In these situations, the risk of a TSOAC overdose has to be minimized by a laboratory monitoring since there is currently no specific therapy that reverse the anticoagulant effect of the TSOACs in case of life-threatening bleeding. Studies were performed to examine whether the coagulation tests used for heparin monitoring could also assess the anticoagulant effect of dabigatran and rivaroxaban. It was shown that the use of Activated Partial Thromboplastin Time (aPTT) is not recommended to estimate dabigatran or rivaroxaban concentrations because of its poor sensitivity, the important inter-individual variability, and the poor correlation with LC–MS/MS [6]. In contrast, Biophen DiXaI(R) assays can be used to estimate concentrations of rivaroxaban >30 ng/mL but the quantification of low rivaroxaban levels ( 0.995. 2.6.4. Lower limit of quantification The lower limit of quantification (LLOQ) was determined in a sextuple analysis of the signal-to-noise ratio (S/N) on QC at 2.5 ng/mL on three separate days with a coefficient of variation (CV) of 1.1% for rivaroxaban and 1.5% for dabigatran. 2.6.5. Matrix effect and extraction recovery The matrix effect was assessed in triplicate by comparing the concentrations obtained with three solutions at 2.5, 50.0, and 500.0 ng/mL in blank plasma extracts with the same solutions in HCl for dabigatran and in methanol for rivaroxaban. The extraction recovery was determined in triplicate by comparing three levels of samples (2.5, 50.0 and 500.0 ng/mL) with reference solutions containing blank plasma extracts spiked with rivaroxaban and dabigatran at the same concentrations. 2.6.6. Stability Dabigatran and rivaroxaban stability in plasma samples was assessed at three concentrations (2.5, 50, and 500 ng/mL) in triplicate and each one was aliquoted on the first day (J0, baseline). Plasma samples were then stored at room temperature (20 ◦ C), +4 ◦ C and −20 ◦ C up to 24 h, 48 h, 5 days, 1 and 3 months. Both molecules were considered to be stable in plasma samples when average measured concentrations were in the limit of 85–115% of the theoretical concentrations. 3. Results

2.5. Clinical application 3.1. Optimization of LC–MS/MS conditions 65 samples were taken at trough in patients treated with dabigatran (n = 21) or rivaroxaban (n = 44) during repeated administrations. Dabigatran or rivaroxaban plasma concentrations were analyzed in blood samples taken at steady state and at trough. Fig. 3 shows an example of 2 different plasma samples from patients treated by dabigatran or rivaroxaban. Blood samples were collected using lithium heparinate as anticoagulant. All samples were centrifuged 5 min at 3000 rpm and the plasma was then stored at −20 ◦ C until analysis. 2.6. Validation The method was validated according to International Conference on Harmonization (ICH) guidelines. 2.6.1. Selectivity Six different plasma blanks were used to investigate interferences. Interferences were defined by a response above 20% of the lower limit of quantification (LLOQ).

Electrospray positive mode yielded a better spectrometer response than the negative mode. 13 C6 -dabigatran and 13 C6 rivaroxaban were judged as the most appropriate IS due to their similar structure and their lack of clinical use. Quantification was performed using multiple reactions monitoring (MRM) of following transitions: m/z 436.1 → 144.9, m/z 442.1 → 144.9, m/z 472.2 → 289.0 and m/z 478.2 → 249.9 for rivaroxaban, 13 C6 rivaroxaban, dabigatran and 13 C6 -dabigatran, respectively. The cone voltage energies were optimized at 40 V for rivaroxaban and 13 C -rivaroxaban, and 30 V for dabigatran and 13 C -dabigatran. The 6 6 collision energies were optimized at 25 V for rivaroxaban, 13 C6 rivaroxaban, dabigatran and 20 V for 13 C6 -dabigatran. To achieve symmetrical peak shapes, good resolution and a short chromatographic run time, a mobile phase consisting of (A) distilled water containing 0.1% formic acid and 0.07 g of ammonium acetate and (B) acetonitrile containing 0.1% formic acid methanol was used in the experiments. Retention times were 2.15 min for rivaroxaban and 13 C6 -rivaroxaban and 1.45 min for dabigatran and 13 C -dabigatran. 6

M. Korostelev et al. / Journal of Pharmaceutical and Biomedical Analysis 100 (2014) 230–235


Fig. 3. Rivaroxaban and dabigatran chromatograms from patients’ plasma samples.

Tandem mass spectrometry is sufficiently selective and sensitive to allow a simple and fast pre-treatment procedure as investigated in this method (protein precipitation by ZnSO4 solution). A dilution of the supernatant was then realized with a high volume of IS solution.

3.2. Method validation 3.2.1. Selectivity Sextuple analysis of blank samples showed no interfering peaks for dabigatran, rivaroxaban and IS in human plasma. Blank responses could not be distinguished from detector noise (

Simultaneous determination of rivaroxaban and dabigatran levels in human plasma by high-performance liquid chromatography-tandem mass spectrometry.

A sensitive and accurate liquid chromatography method with mass spectrometry detection was developed and validated for the quantification of dabigatra...
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