Determination of rivaroxaban in patient’s plasma samples by anti-Xa chromogenic test associated to High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS/MS)
February
Determination of rivaroxaban in patient's plasma samples by anti-Xa chromogenic test associated to High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS/MS)
Priscilla Bento Matos Derogis 0 1
Livia Rentas Sanches 0 1
Valdir Fernandes de Aranda 0 1
Marjorie Paris Colombini 0 1
Cristo vão Luis Pitangueira Mangueira 0 1
Marcelo Katz 0 1
Adriana Caschera Leme Faulhaber 0 1
Claudio Ernesto Albers Mendes 0 1
Carlos Eduardo dos Santos Ferreira 0 1
Carolina Nunes FrancË a 1
João Carlos de Campos Guerra 0 1
0 Hospital Israelita Albert Einstein , São Paulo, Brazil, 2 Santo Amaro University±UNISA, São Paulo , Brazil
1 Editor: Pablo Garcia de Frutos, Institut d'Investigacions Biomediques de Barcelona , SPAIN
Rivaroxaban is an oral direct factor Xa inhibitor, therapeutically indicated in the treatment of thromboembolic diseases. As other new oral anticoagulants, routine monitoring of rivaroxaban is not necessary, but important in some clinical circumstances. In our study a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was validated to measure rivaroxaban plasmatic concentration. Our method used a simple sample preparation, protein precipitation, and a fast chromatographic run. It was developed a precise and accurate method, with a linear range from 2 to 500 ng/mL, and a lower limit of quantification of 4 pg on column. The new method was compared to a reference method (anti-factor Xa activity) and both presented a good correlation (r = 0.98, p < 0.001). In addition, we validated hemolytic, icteric or lipemic plasma samples for rivaroxaban measurement by HPLC-MS/MS without interferences. The chromogenic and HPLC-MS/MS methods were highly correlated and should be used as clinical tools for drug monitoring. The method was applied successfully in a group of 49 real-life patients, which allowed an accurate determination of rivaroxaban in peak and trough levels.
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Data Availability Statement: All relevant data are
within the paper.
Funding: The authors received no specific funding
for this work.
Competing Interests: The authors have declared
that no competing interests exist.
Introduction
Rivaroxaban (Xarelto, Janssen Pharmaceuticals, Titusville, New Jersey) is an oral anticoagulant
that directly inhibits activated factor X (FXa). It is indicated for the prevention of
thromboembolism in patients with atrial fibrillation [
1
], the treatment of deep-vein thrombosis (DVT) [
2
]
and the prevention of recurrent DVT and pulmonary embolism (PE) following an acute DVT
in adults [
3, 4
], in prevention of venous thromboembolism (VTE) in patients undergoing total
knee or hip replacement surgery [5±7].
It is postulated that rivaroxaban has predictable pharmacokinetics and pharmacodynamics
[
3, 8, 9
]. However, there is an increasing interest in rivaroxaban monitoring in special clinical
situations [
10, 11
], such as: acute renal failure, bleeding complications or thrombosis during
treatment, prior to urgent surgery, life-threatening bleeding, stroke, overdose and suspected
accumulation of drug [
9
]. In addition, a recent study showed that rivaroxaban passes into
human breast milk and its safety has not been determined [12]. Future works may recommend
the rivaroxaban monitoring in pregnancy and lactation.
According to the Scientific and Standardization Committee (SSC), through its subcommittee
on Control of Anticoagulation, of the International Society on Thrombosis and Haemostasis,
rivaroxaban can be monitored by indirect methods as activated partial thromboplastin time
(APTT) with reservation for low drug concentrations; prothrombin time (PT); and by
antiFXa assays [
13
]. However, there is a controversial about PT in the literature, some authors
pointed out PT as a good method [
13
], but others like Lindhoff-Last, Samama [
14
], explain
that PT is not a proper test for rivaroxaban measurement due to a discreet alteration in this
parameter and also the dependency of thromboplastin reagent used. Gosselin, Adcock Funk
[
15
] mentioned that drug activity measurement by chromogenic anti-Xa assay must be
preferred against PT, APTT or dilute Russell viper venom time (DRVVT) because it is a more
precise measurement and present lower bias.
High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)
methods for direct dosage of anticoagulants plasmatic concentration as rivaroxaban have been
reported [16±18]. HPLC-MS/MS is known as a standard gold method due to its high
specificity and sensitivity. The main concerns about HPLC-MS/MS use are related to method
development/validation and the usual absence of commercial calibrators and controls. Therefore, our
aim was to develop an HPLC-MS/MS assay for an accurate rivaroxaban concentration
determination in plasma samples with a simple sample preparation step.
Material and methods
Ethics statement
Signed informed consent was obtained from each patient in accordance with ethical p (...truncated)