Determination of rivaroxaban by different factor X

Authors: Job Harenberg Roland Krämer Christina Giese Svetlana Marx Christel Weiss Martin Wehling

Publish Date: 2011/08/03

Volume: 32, Issue: 3, Pages: 267-

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Abstract

Rivaroxaban and other oral direct factor Xa inhibitors ODiXa are currently developed for prophylaxis and treatment of thromboembolic diseases using fixed doses Although routine monitoring is not required assessing the intensity of anticoagulation may be useful under certain clinical conditions ODiXa prolong coagulation times of several clotting assays and thus their concentration may be determined in factor Xa specific chromogenic substrate assays So far no standardized and validated assay is commercially available Here five methods A through E are studied and optimized to reduce interassay variability Human pooled plasma was spiked by a serial dilution of rivaroxaban 25–900 ng/ml The release of paranitroaniline from the chromogenic substrates was measured by the optical density OD at 405 nm Method B was identified to yield the lowest sum of deviations from the mean value of the OD concentration curve calculated from all assays Spline functions were developed for OD versus concentration curves for all methods The calculated OD versus concentration curves overlapped for all methods The coefficient of variation for all assays and concentrations of rivaroxaban decreased from 253 ± 114 using the original data to 38 ± 22 using the calculated data P  00001 The robustness of the chromogenic assay method B remains to be corroborated in interlaboratory comparisonsOral direct factor Xa inhibitors ODiXa are currently developed for prevention and treatment of thromboembolic diseases with the aim to overcome the limitations of the conventional anticoagulants unfractionated and lowmolecularweight heparins LMWHs fondaparinux and vitaminK antagonists 1 One of the relevant advantages of ODiXa is the alleged dispensability of laboratory monitoring for dose adjustments 2 3 However determination of the anticoagulant effect may be desirable in certain clinical situations such as acute bleeding urgent surgery analysis of compliance suspected overdose substantial decrease of renal or hepatic function in elderly or children populations 4Attempts to assess anticoagulation intensity by ODiXa showed that inhibition of factor Xa activity prolongation of prothrombin time PT and activated partial thromboplastin time aPTT correlate with plasma levels of the ODiXa rivaroxaban in this case in healthy subjects and those undergoing major orthopaedic surgery 5 6 7 8 The prolongation of PT values by rivaroxaban depends of the thromboplastin reagents used 9 Up to now all chromogenic assays use heparin for standardization and data using rivaroxaban are sparse A preliminary standardization of the reagents has been described using rivaroxaban 10 Several factor Xa amidolytic chromogenic substrate assays have been tested to specifically determine the concentration of rivaroxaban however results of those methods show a considerable interassay variability of up to 40 11 The present paper aims at reducing the interassay variability between various chromogenic substrate assays on human plasma samples spiked with rivaroxabanRivaroxaban was isolated from commercially available Xarelto® Bayer Healthcare Care Wuppertal Germany by extracting an aqueous suspension of the tablets with dichloromethane at 25°C The compound was recrystallized from dichloromethane and vacuumdried The purity and identity were checked by 1H NMR spectroscopy exact molecular mass by highresolution electrospray mass spectrometry Micromass QTOF ultima and elemental analysis Pooled plasma was spiked with increasing concentrations of rivaroxaban 25–900 ng/ml dissolved in dimethyl sulfoxide DMSOBlood was drawn from 20 healthy volunteers into 105 mmol/l sodium citrate Sarstedt Nuermbrecht Germany centrifuged at 3000×g for 15 min at 4° to obtain platelet poor plasma PPP Pooled plasma was derived from mixing PPP of 20 healthy persons Plasma samples were aliquoted transferred into plastic tubes shock frozen and stored at −70° until analysed Plasma samples were thawed only once at 37° rivaroxaban was added at various concentrations and analysed in the assays within 2 h Donors gave informed consent prior to blood sampling Volunteers gave written informed consentThe test principle is based on the inhibitory action of rivaroxaban on coagulation factor Xa which specifically cleaves paranitroaniline pNA linked to a chromogenic peptide Increasing rivaroxaban concentrations dosedependently inhibit the activity of factor Xa on the chromogenic peptide and thereby the release of pNA The concentration of rivaroxaban is plotted against the optical density OD of released pNAThe following factor Xa specific chromogenic substrates were used Coamatic Heparin assay method A S2732 chromogenic substrate SucisoleucineglutamylgammaPipglycineargininepNnitroaniline Ηaemochrom Diagnostica GmbH Essen Germany STA Rotachrom heparin method B chromogenic substrate CBS 5244 MAPAglycylargininylpnitroaniline hydrochloride Diagnostica Stago distributed by RocheDiagnostika Mannheim Germany S2222 chromogenic substrate assay method C Nbenzoyllisoleucyllglutamylglycyllargininepnitroaniline hydrochloride and its methyl ester Instrumentation Laboratory GmbH Kirchheim Germany STAheparin Liquid method D chromogenic substrate CBS0244 MAPAglycinearginylpnitroanilide Asnières sur Seine France and Technochrom antiXa method E chromogenic substrate succinylisoleucineglutamylglycylargininepnitroaniline Technoclone Vienna AustriaAll reagents were dissolved in the solvent provided by and according to the description of the manufacturers All assays were run on microtiter plates and not on the instruments proposed by the manufacturers This was decided to eliminate the variability of the experiments caused by differences of the instructions by the manufacturers and coagulation analysers Some manufacturers did not have instructions for the determination of rivaroxaban in the chromogenic assays Preliminary experiments revealed that the maximal OD at 405 nm in the absence of rivaroxaban differed substantially between the assays using the incubation procedures described below Therefore the amounts of the chromogenic substrate and of factor Xa were adjusted for every method to about 1000 OD at 405 nm in the absence of rivaroxaban The molar ratios of the substrate and factor Xa were not changed for the individual assays 25 μl human plasma containing rivaroxaban at various concentrations were diluted 15 with 25 μl normal pooled plasma followed 25 μl factor Xa and incubated at 37°C for 5 min 50 μl of synthetic chromogenic substrates were added and the samples incubated for 20 min Samples were supplemented with 25 μl antithrombin stock solution 1 unit per ml for the analysis with the technochrom antiXa assay before addition of factor Xa as recommended by the manufacturer The enzymatic activity of factor Xa was stopped by adding 50 μl 50 acetic acid OD was recorded at 405 nm and converted to rivaroxaban ng/ml plasma Pooled plasma samples were spiked with 25–900 ng/ml rivaroxaban Blank plasma was obtained by adding acetic acid prior to the chromogenic substrate to each plasma sample No dilutions of samples containing high concentrations of rivaroxaban were performed in these experiments The OD value of the plasma sample was subtracted from the OD of the test sample The assays were performed on microtiter plates in duplicates and the absorbance of pNA was read at a wavelength of 405 nm using the microtiter plate reader MR 7000 software version 32 Dynatech laboratories Denkendorf Germany

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