Evaluation of New Degradation Products Formed Under Stress Conditions of Betrixaban by LCMS/MS: Establishment of HPLC Method for Quantification of Genotoxic Impurities of Betrixaban
Abstract
This study focused to optimize an accurate HPLC method for evaluation of genotoxic impurities of Betrixaban and further structural identification of forced degradation products (DPs) of betrixaban. The analytes were resolved on Zorbax SB C18 (4.6×250mm, 5μm, Agilent) column at 35°C temperature using 75 % aqueous ammonium formate (5 mM) and 50 % ammonium formate in acetonitrile in 60:40 (v/v) in isocratic elution at 1.0 mL/min and 245 nm as detection wavelength. In the optimized experimental conditions, the retention times of the analytes were precisely determined, resulting in retention times of 10.30 min for betrixaban, 3.77 min for the 2-amino impurity, 7.79 min for the 2-nitro impurity, and 13.55 min for the 4-cyanobenzamido impurity, all exhibiting acceptable system suitability and specificity. The method demonstrated excellent sensitivity, capable of detecting impurities up to 0.009 µg/mL, with 25-150 µg/mL calibration range for betrixaban and 0.025-0.15 µg/mL for impurities. Notably, all parameters studied during the validation process exhibited results within permissible limits for both betrixaban and its impurities, affirming the robustness and reliability of the analytical method. The drug underwent exposure to various stress-inducing conditions following the guidelines outlined in ICH Q1A (R2).The stress-induced DPs were identified using LCMS/MS in ESI positive mode. By comparing their collision-induced dissociation mass spectral data with that of betrixaban, potential structures for four of DPs were proposed. The results of other validation studies were also agreeable, confirming their adequacy for the routine analysis of betrixaban and its genotoxic impurities in both bulk drug and formulations. Additionally, these validated methods can be utilized to investigate the mechanisms underlying the stress-induced degradation of betrixaban.
Keywords:
Betrixaban, characterization through LCMS, genotoxicimpurities, stress degradation products
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References
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Bhupatiraju, R. V., Kumar, B. S., Tangeti, V. S., Rekha, K., & Sayed, F. (2024) LC and LC-MS/MS Studies for Identification and Characterisation of Related Substances and Degradation Products of Abrocitinib, Toxicology International, 31(2), 321-334.https://doi.org/10.18311/ti/2024/v31i2/36370
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Bikshal, B. K., Useni, R. M., Venkateswara, R. A., &Maheshwara, R. L. (2018). Intended high-performance liquid chromatography procedure for the quantification of norfloxacin and its potential impurities in active pharmaceutical ingredient and tablet dosage forms.Thai Journal of Pharmaceutical Sciences. 42(1), 27-36.
Chen, G., Warrack, B. M., & Goodenough, A. K. (2010). LC-MS: Analysis of Impurities and Degradants in Pharmaceuticals by High Resolution Tandem Mass Spectrometry and On-line H/D Exchange LC/MS. American Pharmaceutical Review, 13(3), 20.
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Davis, J. M., & Gidding, J. C. (1983). Impurity profile: A review. J. Anal. Chem, 55, 418.
De Spiegeleer, B., Vergote, V., Pezeshki, A., Peremans, K., &Burvenich, C. (2008). Impurity profiling quality control testing of synthetic peptides using liquid chromatography-photodiode array-fluorescence and liquid chromatography-electrospray ionization-mass spectrometry: the obestatin case. Analytical biochemistry, 376(2), 229-234. https://doi.org/10.1016/j.ab.2008.02.005
El-Masry, A. A., El-Wasseef, D. R., Eid, M., Shehata, I. A., &Zeid, A. M. (2021).Optimization and validation of a facile RP-HPLC method for determination of betrixaban and lercanidipine in pharmaceutical and biological matrices. Journal of Chromatographic Science, 59(8), 785-794. https://doi.org/10.1093/chromsci/bmab082
El-Masry, A. A., El-Wasseef, D. R., Eid, M., Shehata, I. A., &Zeid, A. M. (2022). Development of three ecological spectroscopic methods for analysis of betrixaban either alone or in mixture with lercanidipine: greenness assessment. Royal Society Open Science, 9(2). https://doi.org/10.1098/rsos.211457
El-Masry, A. A., Zeid, A. M., El-Wasseef, D. R., Eid, M., & Shehata, I. A. (2020). A Validated Quantitative 1H Nuclear Magnetic Resonance (1H-qNMR) Method for Quantification of a Novel Anti-Coagulant Drug (Betrixaban Maleate) with Assessing Its Stability by Application to Degradation Study. Analytical Chemistry Letters, 10(6), 768-783. https://doi.org/10.1080/22297928.2020.1779774
Eriksson, B. I., Quinlan, D. J., & Weitz, J. I. (2009). Comparative pharmacodynamics and pharmacokinetics of oral direct thrombin and factor Xa inhibitors in development. Clinical pharmacokinetics, 48, 1-22. https://doi.org/10.2165/0003088-200948010-00001
Feng, W., Liu, H., Chen, G., Malchow, R., Bennett, F., Lin, E., ... & Chan, T. M. (2001). Structural characterization of the oxidative degradation products of an antifungal agent SCH 56592 by LC–NMR and LC–MS. Journal of pharmaceutical and biomedical analysis, 25(3-4), 545-557. https://doi.org/10.1016/S0731-7085(01)00377-7
Ghante M. R., Vaidya S., &Bhusari V. K. (2021). Development and validation of stability indicating method for estimation of Betrixaban in bulk and pharmaceutical formulation. International Journal of Science, Engineering and Management (IJSEM), 6(5), 139-145. https://doi.org/10.26452/ijsem.v6i5.139-145
ICH, D. R. G. (2003, February). Stability testing of new drug substances and products Q1A (R2). In Proceedings of the International Conference on Harmonization. Geneva, Switzerland.
ICH, H. T. G. (1996). Photostability Testing of New Drug Substance and Products. Federal Register, 62, 27115-27122.
International Conference of Harmonisation.ICH.Q2(R1). (1994). Validation of analytical procedures: text and methodology. International Conference on Harmonization, Geneva.
Jasemizad, T., & Padhye, L. P. (2019). Simultaneous analysis of betrixaban and hexazinone using liquid chromatography/tandem mass spectrometry in aqueous solutions. MethodsX, 6, 1863-1870. https://doi.org/10.1016/j.mex.2019.07.018
Kasimala, B. B., Anna, V. R., & Mallu, U. R. (2018). Stability-Indicating Reversed-Phase Hplc Method for the Separation and Estimation of Related Impurities of Cilnidipine in Pharmaceutical Formulations. Indian Drugs, 55(12).https://doi.org/10.53879/id.55.12.11185
Li, J., Chen, L., Yan, X., Li, Y., Wei, D., & Wang, D. (2015). A facile method for the synthesis of Betrixaban. Journal of Chemical Research, 39(9), 524-526.
Mallu, U. R., Anna, V. R., & Kasimala, B. B. (2019). Rapid stability indicating HPLC method for the analysis of leflunomide and its related impurities in bulk drug and formulations. Turkish Journal of Pharmaceutical Sciences, 16(4), 457-465. https://doi.org/10.4274/tjps.galenos.2018.0004
Mastannamma, S. K., Navya, P., Annapurna, U., & Varalaxmi, M. (2018). Development and validation of a stability-indicating RP-HPLC method for the determination of Betrixaban in bulk and its laboratory synthetic mixer. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 9(1), 71-79.
Surukonti, S. R., Surya, S. B. M., Katari, N. K., &Yerla, R. R. (2023). Investigating Betrixaban Maleate drug degradation profiles, isolation and characterization of unknown degradation products by mass-triggered preparative HPLC, HRMS, and NMR. Journal of Pharmaceutical and Biomedical Analysis, 235, 115643. https://doi.org/10.1016/j.jpba.2023.115643
Turpie, A. G., Bauer, K. A., Davidson, B. L., Fisher, W. D., Gent, M., Huo, M. H., ... & EXPERT Study Group. (2009). A randomized evaluation of betrixaban, an oral factor Xa inhibitor, for prevention of thromboembolic events after total knee replacement (EXPERT). Thrombosis and haemostasis, 101(01), 68-76.https://doi.org/10.1160/TH08-09-0609
Varma, B. H. R., & Rao, B. S. (2023). Gas chromatography-head space-mass spectrometry sensor based quality control of dobutamine hydrochloride bulk material for a mutagenic impurity, 2-bromopropane. Res J Chem Environ, 27, 54-61. https://doi.org/10.25303/2702rjce054061
Varma, R. B., & Rao, B. S. (2022). Gas Chromatography-Head Space-Flame Ionization Sensor based assessment of four residuary solvents in rivaroxaban bulk medication. Research Journal of Pharmacy and Technology, 15(11), 5158-5163. https://doi.org/10.52711/0974-360X.2022.00868
Vergote, V., Burvenich, C., Van de Wiele, C., & De Spiegeleer, B. (2009). Quality specifications for peptide drugs: a regulatory‐pharmaceutical approach. Journal of peptide science: an official publication of the European Peptide Society, 15(11), 697-710.https://doi.org/10.1002/psc.1164
Bari, S. B., Kadam, B. R., Jaiswal, Y. S., &Shirkhedkar, A. A. (2007). Impurity profile: significance in active pharmaceutical ingredient. Eurasian J Anal Chem, 2(1), 32-53.
Bhupatiraju, R. V., Battula, S. R., Kapavarapu, M. V. N. R., &Mandapati, V. R. (2023, January). Assessment of gas chromatography methodology approach for the trace evaluation of carcinogenic impurity, methyl chloride, in trimetazidine dihydrochloride. In Annales PharmaceutiquesFrancaises, 81(1), 64-73. https://doi.org/10.1016/j.pharma.2022.06.012
Bhupatiraju, R. V., Kasimala, B. B., Nagamalla, L., & Sayed, F. (2024). Structural evaluation of degradation products of Loteprednol using LC-MS/MS: Development of an HPLC method for analyzing process-related impurities of Loteprednol. 37(2), 98-113. https://doi.org/10.5806/AST.2024.37.2.98
Bhupatiraju, R. V., Kumar, B. S., Peddi, P., &Tangeti, V. S. (2023). An effective HPLC method for evaluation of process related impurities of Letermovir and LC-MS/MS characterization of forced degradation compounds. Journal of Chemical Metrology, 17(2), 1-19. http://doi.org/10.25135/jcm.98.2311.2975
Bhupatiraju, R. V., Kumar, B. S., Tangeti, V. S., Rekha, K., & Sayed, F. (2024) LC and LC-MS/MS Studies for Identification and Characterisation of Related Substances and Degradation Products of Abrocitinib, Toxicology International, 31(2), 321-334.https://doi.org/10.18311/ti/2024/v31i2/36370
Bhupatiraju, R. V., Rao, B. S., Venkata Narayana Rao, K. M., & Reddy, M. V. (2022). A Novel Rivaroxaban Degradation Impurity Detection by RP-HPLC, Extraction by Preparatory Chromatography, and Characterization by Lcms, Nmr, and FT-IR: Analysis of Novel Impurity in Batch Samples and Tablets of Rivaroxaban. Rasayan Journal of Chemistry, 15(4).2373-2381.https://doi.org/10.31788/RJC.2022.1547008
Bikshal, B. K., Useni, R. M., Venkateswara, R. A., &Maheshwara, R. L. (2018). Intended high-performance liquid chromatography procedure for the quantification of norfloxacin and its potential impurities in active pharmaceutical ingredient and tablet dosage forms.Thai Journal of Pharmaceutical Sciences. 42(1), 27-36.
Chen, G., Warrack, B. M., & Goodenough, A. K. (2010). LC-MS: Analysis of Impurities and Degradants in Pharmaceuticals by High Resolution Tandem Mass Spectrometry and On-line H/D Exchange LC/MS. American Pharmaceutical Review, 13(3), 20.
Cohen, A. T., Spiro, T. E., Büller, H. R., Haskell, L., Hu, D., Hull, R., ... &Tapson, V. (2013). Rivaroxaban for thromboprophylaxis in acutely ill medical patients. New England Journal of Medicine, 368(6), 513-523.https://doi.org/10.1056/NEJMoa1111096
Davis, J. M., & Gidding, J. C. (1983). Impurity profile: A review. J. Anal. Chem, 55, 418.
De Spiegeleer, B., Vergote, V., Pezeshki, A., Peremans, K., &Burvenich, C. (2008). Impurity profiling quality control testing of synthetic peptides using liquid chromatography-photodiode array-fluorescence and liquid chromatography-electrospray ionization-mass spectrometry: the obestatin case. Analytical biochemistry, 376(2), 229-234. https://doi.org/10.1016/j.ab.2008.02.005
El-Masry, A. A., El-Wasseef, D. R., Eid, M., Shehata, I. A., &Zeid, A. M. (2021).Optimization and validation of a facile RP-HPLC method for determination of betrixaban and lercanidipine in pharmaceutical and biological matrices. Journal of Chromatographic Science, 59(8), 785-794. https://doi.org/10.1093/chromsci/bmab082
El-Masry, A. A., El-Wasseef, D. R., Eid, M., Shehata, I. A., &Zeid, A. M. (2022). Development of three ecological spectroscopic methods for analysis of betrixaban either alone or in mixture with lercanidipine: greenness assessment. Royal Society Open Science, 9(2). https://doi.org/10.1098/rsos.211457
El-Masry, A. A., Zeid, A. M., El-Wasseef, D. R., Eid, M., & Shehata, I. A. (2020). A Validated Quantitative 1H Nuclear Magnetic Resonance (1H-qNMR) Method for Quantification of a Novel Anti-Coagulant Drug (Betrixaban Maleate) with Assessing Its Stability by Application to Degradation Study. Analytical Chemistry Letters, 10(6), 768-783. https://doi.org/10.1080/22297928.2020.1779774
Eriksson, B. I., Quinlan, D. J., & Weitz, J. I. (2009). Comparative pharmacodynamics and pharmacokinetics of oral direct thrombin and factor Xa inhibitors in development. Clinical pharmacokinetics, 48, 1-22. https://doi.org/10.2165/0003088-200948010-00001
Feng, W., Liu, H., Chen, G., Malchow, R., Bennett, F., Lin, E., ... & Chan, T. M. (2001). Structural characterization of the oxidative degradation products of an antifungal agent SCH 56592 by LC–NMR and LC–MS. Journal of pharmaceutical and biomedical analysis, 25(3-4), 545-557. https://doi.org/10.1016/S0731-7085(01)00377-7
Ghante M. R., Vaidya S., &Bhusari V. K. (2021). Development and validation of stability indicating method for estimation of Betrixaban in bulk and pharmaceutical formulation. International Journal of Science, Engineering and Management (IJSEM), 6(5), 139-145. https://doi.org/10.26452/ijsem.v6i5.139-145
ICH, D. R. G. (2003, February). Stability testing of new drug substances and products Q1A (R2). In Proceedings of the International Conference on Harmonization. Geneva, Switzerland.
ICH, H. T. G. (1996). Photostability Testing of New Drug Substance and Products. Federal Register, 62, 27115-27122.
International Conference of Harmonisation.ICH.Q2(R1). (1994). Validation of analytical procedures: text and methodology. International Conference on Harmonization, Geneva.
Jasemizad, T., & Padhye, L. P. (2019). Simultaneous analysis of betrixaban and hexazinone using liquid chromatography/tandem mass spectrometry in aqueous solutions. MethodsX, 6, 1863-1870. https://doi.org/10.1016/j.mex.2019.07.018
Kasimala, B. B., Anna, V. R., & Mallu, U. R. (2018). Stability-Indicating Reversed-Phase Hplc Method for the Separation and Estimation of Related Impurities of Cilnidipine in Pharmaceutical Formulations. Indian Drugs, 55(12).https://doi.org/10.53879/id.55.12.11185
Li, J., Chen, L., Yan, X., Li, Y., Wei, D., & Wang, D. (2015). A facile method for the synthesis of Betrixaban. Journal of Chemical Research, 39(9), 524-526.
Mallu, U. R., Anna, V. R., & Kasimala, B. B. (2019). Rapid stability indicating HPLC method for the analysis of leflunomide and its related impurities in bulk drug and formulations. Turkish Journal of Pharmaceutical Sciences, 16(4), 457-465. https://doi.org/10.4274/tjps.galenos.2018.0004
Mastannamma, S. K., Navya, P., Annapurna, U., & Varalaxmi, M. (2018). Development and validation of a stability-indicating RP-HPLC method for the determination of Betrixaban in bulk and its laboratory synthetic mixer. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 9(1), 71-79.
Surukonti, S. R., Surya, S. B. M., Katari, N. K., &Yerla, R. R. (2023). Investigating Betrixaban Maleate drug degradation profiles, isolation and characterization of unknown degradation products by mass-triggered preparative HPLC, HRMS, and NMR. Journal of Pharmaceutical and Biomedical Analysis, 235, 115643. https://doi.org/10.1016/j.jpba.2023.115643
Turpie, A. G., Bauer, K. A., Davidson, B. L., Fisher, W. D., Gent, M., Huo, M. H., ... & EXPERT Study Group. (2009). A randomized evaluation of betrixaban, an oral factor Xa inhibitor, for prevention of thromboembolic events after total knee replacement (EXPERT). Thrombosis and haemostasis, 101(01), 68-76.https://doi.org/10.1160/TH08-09-0609
Varma, B. H. R., & Rao, B. S. (2023). Gas chromatography-head space-mass spectrometry sensor based quality control of dobutamine hydrochloride bulk material for a mutagenic impurity, 2-bromopropane. Res J Chem Environ, 27, 54-61. https://doi.org/10.25303/2702rjce054061
Varma, R. B., & Rao, B. S. (2022). Gas Chromatography-Head Space-Flame Ionization Sensor based assessment of four residuary solvents in rivaroxaban bulk medication. Research Journal of Pharmacy and Technology, 15(11), 5158-5163. https://doi.org/10.52711/0974-360X.2022.00868
Vergote, V., Burvenich, C., Van de Wiele, C., & De Spiegeleer, B. (2009). Quality specifications for peptide drugs: a regulatory‐pharmaceutical approach. Journal of peptide science: an official publication of the European Peptide Society, 15(11), 697-710.https://doi.org/10.1002/psc.1164
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Anna, V., Rohini, V., Hemabala, K., Tatavarti, B., & N., V. (2024). Evaluation of New Degradation Products Formed Under Stress Conditions of Betrixaban by LCMS/MS: Establishment of HPLC Method for Quantification of Genotoxic Impurities of Betrixaban. International Journal of Advancement in Life Sciences Research, 7(4), 64-78. https://doi.org/https://doi.org/10.31632/ijalsr.2024.v07i04.006
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