A Validated Chiral-RP-UPLC-MS/MS Method for the Enantiomeric Detection of Rivaroxaban In vitro

Author(s): Fuxin Chen, Xiaoxian Ma, Chuangqian Chen, Kanshe Li, Suying Chen, He Wen, Pin Gong*

Journal Name: Current Pharmaceutical Analysis

Volume 15 , Issue 4 , 2019

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Background: Rivaroxaban is the first oral, selective direct FXa inhibitor with rapid onset of action and its biological toxicity may be related to the enantiomer.

Objective: The aim of the current study was to develop and validate a precise, accurate, and specific direct Chiral-RP-UPLC-MS/MS method for the enantiomeric separation and detection of rivaroxaban and its enantiomer.

Methods: The present study screened various conditions of chromatographic and mass spectra, including chromatographic column model, flow velocity, phase ratio, column temperature, and collision energy, parent/daughter ion pairs, etc. Try to match the chromatographic and mass spectrometric conditions.

Results: Good Rs (Rs>2.5) was achieved on a Chiralpak IC column (4.6 × 250 mm, 5µm) using H2O:acetonitrile (10:90) as mobile phase at 25 oC column temperature. The rate of flow was set at 0.4 ml/min and enantiomers were detected by triple-quadruple tandem mass spectrometry using positive electrospray ionization (ESI) with MRM transitions of m/z 436.07>144.95. The cone voltage and collision energy were kept at 48 V and 28 eV, respectively. The limit of detection and quantification of (S)- rivaroxaban were 0.39 and 1.30 ng/ml, respectively. This method was validated and found to be selective, precise, accurate, linear and robust for the quantitative determination of chiral impurities. It is also a good application for the blood samples analysis in vitro.

Conclusion: Chiral-RP-UPLC-MS/MS method has entirely detected (S)-rivaroxaban and its (R)- enantiomer in very low concentration and complex matrix directly, especially for blood samples.

Keywords: UPLC-MS/MS, enantiomers, rivaroxaban, chiral separation, in vitro, chiral inversion.

Liu, W.P.; Gan, J.Y.; Schlenk, D.; Jury, W.A. Enantioselectivity in environmental safety of current chiral insecticides. PNAS, 2005, 102, 701-706.
Tverdislov, V.A.; Yakovenko, L.V.; Zhavoronkov, A.A. Chirality as a problem of biochemical physics. Russ. J. Gen. Chem., 2007, 77, 1994-2005.
Rauws, A.G.; Groen, K. Current regulatory (draft) guidance on chiral medicinal products: Canada, EEC, Japan, United States. Chirality, 1994, 6, 72-75.
Chu, B.L.; Guo, B.G.; Zuo, H.J.; Wang, Z.Y.; Lin, J.M. Simultaneous enantioseparation of antiparkinsonian medication Rotigotine and related chiral impurities by capillary zone electrophoresis using dual cyclodextrin system. J. Pharmaceut. Biomed., 2008, 46, 854-859.
Roehrig, S.; Straub, A.; Pohlmann, J.; Lampe, T.; Pernerstorfer, J.; Schlemmer, K.H.; Reinemer, P.; Perzborn, E. Discovery of the novel antithrombotic agent 5-chloro-N-((5S)-2-oxo-3-[4-(3-oxom-orpholin-4-yl)phenyl]-1,3-oxazolidin-5-ylmethyl) thiophene-2-car-boxamide (BAY 59-7939): an oral, direct factor Xa inhibitor. J. Med. Chem., 2005, 48, 5900-5908.
Favaloro, E.J.; Pasalic, L.; Curnow, J.; Lippi, G. Laboratory monitoring or measurement of direct oral anticoagulants (DOACs): Advantages, limitations and future challenges. Curr. Drug Metab., 2017, 18, 598-608.
Samos, M.; Stanciakova, L.; Skornova, I.; Bolek, T.; Kovar, F.; Stasko, J.; Galajda, P.; Mokan, M.; Kubisz, P. Review of the pharmacology of the emerging possibilities of the direct oral anticoagulants’ reversal. Curr. Drug Metab., 2017, 18, 643-650.
Antonio, G.O.; M Luisa, S.G Gonzalo, C.R.; Ramon, L.; Eduardo, R.; Carmen, P.H.; Ana Isabel, T.F.; Emilio, V.C. Discovery of anticoagulant drugs: A historical perspective. Curr. Drug Discov. Technol., 2012, 9, 83-104.
Kvasnicka, T.; Malikova, I.; Zenahlikova, Z.; Kettnerova, K.; Brzezkova, R.; Zima, T.; Ulrych, J.; Briza, J.; Netuka, I.; Kvasnicka, J. Rivaroxaban - metabolism, pharmacologic properties and drug interactions. Curr. Drug Metab., 2017, 18, 636-642.
Kreutz, R. Pharmacokinetics and pharmacodynamics of rivaroxaban - An oral, eirect factor Xa inhibitor. Curr. Clin. Pharmacol., 2014, 9, 75-83.
Wingert, N.R.; Nunes, M.A.G.; Barden, A.T.; Gomes, P.; Muller, E.I.; Flores, E.M.M.; Steppe, M. Ultra-performance LC-ESI/Q-TOF MS for the rapid analysis of rivaroxaban: Method validation using experimental design for robustness Evaluation. Curr. Anal. Chem., 2015, 11, 124-129.
Bebawy, L.I.; Mostafa, A.A.; Girge, M.A. High performance liquid chromatography, TLC densitometry, first derivative and first-derivative ratio spectrophotometry for determination of rivaroxaban and its alkalinedegradates in bulk powder and its tablets. Anal. Chem. Indian J., 2013, 13(5), 172-181.
Sekaran, C.B.; Bind, V.H.; Damayanthi, M.R.; Sireesha, A. Development and validation of UV spectrophotometric method for the determination of rivaroxaban. Der Pharma Chem., 2013, 5, 1-5.
Prabhune, S.S.; Dighe, V.; Pradhan, N.S. Enantiomeric separation of Rivaroxaban by a chiral liquid chromatographic method. Int. J. Pharm. Pharm. Sci., 2015, 7, 399-402.
Önder, Ö.; Shao, W.G.; Lam, H.; Brisson, D. Tracking the sources of blood meals of parasitic arthropods using shotgun proteomics and unidentified tandem mass spectral libraries. Nat. Protoc., 2014, 9, 842-850.
Baldelli, S.; Cattaneo, D.; Pignatelli, P. Validation of an LC-MS/MS method for the simultaneous quantification of dabigatran, rivaroxaban and apixaban in human plasma. Bioanalysis, 2016, 8, 275-283.
Sadutto, D.; Ferretti, R.; Zanitti, L.; Casullib, A.; Cirillia, R. Analytical and semipreparative high performance liquid chromatography enantioseparation of bicalutamide and its chiral impurities on an immobilized polysaccharide-based chiral stationary phase. J. Chromatogr. A, 2016, 1445, 166-171.
Yan, J.; Zhang, R.; Wang, X.; Wang, D.Z.; Zhou, Z.Q.; Zhu, W.T. Enantiomeric separation of chiralpesticides by permethylated β-cyclodextrin stationary phase in reversed phase liquid chromatography. Chirality, 2016, 28, 409-514.
Barhate, C.L.; Wahab, M.F.; Breitbach, Z.S.; Bellb, D.S.; Armstrong, D.W. High efficiency, narrow particle size distribution, sub-2 μm based macrocyclic glycopeptide chiral stationary phases in HPLC and SFC. Anal. Chim. Acta, 2015, 898, 128-137.
Grecsó, N.; Ilisz, I.; Gecse, Z.; Schönstein, L.; Fülöp, F.; Péter, A. High-performance liquid chromatographic enantioseparation of amino alcohol analogues possessing 1,2,3,4-tetrahydroisoquinoline skeleton on polysaccharide-based chiral stationary phases. Biomed. Chromatogr., 2015, 29, 788-796.
Korostelev, M.; Bihan, K.; Ferreol, L.; Tissota, N.; Hulot, J.S.; Brentano, C.F. Simultaneous determination of rivaroxaban and dabigatran levels in human plasma by high-performance liquid chromatography-tandem mass spectrometry. J. Pharm. Biomed. Anal., 2014, 100, 230-235.
Cao, M.; Fraser, K.; Huege, J.; Featonby, T.; Rasmussen, S.; Jones, C. Predicting retention time in hydrophilic interaction liquid chromatography mass spectrometry and its use for peak annotation in metabolomics. Metabolomics, 2015, 11, 696-706.
Schmitz, E.M.; Boonen, K.; van den Heuvel, D.J.; van Dongen, J.L.J.; Schellings, M.W.M.; Emmen, J.M.A. van der Graaf, F.; et al. Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants. J. Thromb. Haemost., 2014, 12, 1636-1646.
Jiao, Z.; Zhang, S.; Chen, H. Determination of tetracycline antibiotics in fatty food samples by selective pressurized liquid extraction coupled with high-performance liquid chromatography and tandem mass spectrometry. J. Sep. Sci., 2015, 38, 115-120.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Published on: 19 March, 2019
Page: [305 - 311]
Pages: 7
DOI: 10.2174/1573412914666180409145403
Price: $65

Article Metrics

PDF: 58