DEVELOPMENT AND VALIDATION OF SEVEN PHENYL HYDRAZINE CHLORO ESTER ISOMERS (PGIs) BY RP-HPLC-UV METHOD IN ANTICOAGULANT DRUG SUBSTANCE; APIXABAN
Objective: The objective of this work was to develop and validate a simple and sensitive reverse-phase high-pressure liquid chromatography method for the determination of seven potential genotoxic impurities in Apixaban drug substance.
Methods: The optimized separation was achieved by using ACE 3 C18 PFP (150 mm×4.6 mm, 3 µm) HPLC column. The mobile phase-A was a degassed mixture of 0.01M Ammonium acetate buffer(PH adjusted 4.9±0.05 with diluted glacial acetic acid) and mobile phase-B was a degassed mixture of Acetonitrile, Isopropyl alcohol and Buffer PH 4.9 in the ratio of 60:20:20 v/v/v. The gradient program was operated at a flow rate of 1.0 ml/min and UV detection was at 330 nm.
Results: The method was superior at linearity for seven impurities and correlation coefficient values were larger than 0.999, moreover, in the separation point of view, this method further achieved no matrix interference through chromatography by better resolution of the other impurities from the Apixaban drug substance and its related impurities for the accurate analysis of seven potential genotoxic impurities. The established limits of detection (LOD), limits of quantification (LOQ) values for the seven mutagenic impurities were each of 5 ppm (0.015µg/ml) and15 ppm (0.045µg/ml) respectively. The developed method was validated as per ICH guidelines and applied as a generic method to determine these seven potential genotoxic impurities for the pharmaceutical process control and drug material release.
Conclusion: Validation of this analytical method was carried out including stability, selectivity, linearity, accuracy, system precision, method precision and intermediate precision thus proving that the described RP-HPLC method could be employed for fast and simple analysis of sevenphenyl hydrazine chloro ester isomers in Apixaban drug substance.
2. Zhang Kun, Zhao Shenghao, Kan Wusheng, Xiao Jun, Pu Feifei, Li Kun. Comparison of apixaban and rivaroxaban for anticoagulant effect after lumbar spine surgery: a single-center report. Future Sci 2018;4:297.
3. Eliquis, US FDA Label. Bristol-Myers Squibb Company; 2016.
4. International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human use, M7, Assessment and control of DNA reactive (Mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk; 2014.
5. Sawatari K, Nakanishi Y, Matsushimi T. Relationships between chemical structures and mutagen city: a preliminary survey for a database of mutagen city test results of new workplace chemicals. Ind Health 2001;39:341-5.
6. Jenny Wang, Samuel Yang, Kelly Zhang. A simple and sensitive method to analyze genotoxic impurity hydrazine in pharmaceutical materials, a simple and sensitive method to analyze genotoxic impurity hydrazine in pharmaceutical materials. J Pharm Biomed Anal 2016;126:141-7.
7. Elder DP, Snodin D, Teasdale A. Control and analysis of hydrazine, hydrazides and hydrazones-genotoxic impurities in active pharmaceutical ingredients (APIs) and drug products. J Pharm Biomed Anal 2011;54:900-10.
8. Raman NVVSS, Prasad AVSS, Reddy KR. Sensitive derivatization methods for the determination of genotoxic impurities in drug substances using hyphenated techniques. J Pharm Biomed Anal 2014;89:276-81.
9. Khan M, Kumar S, Jayasree K, Krishna Reddy KVSR, Dubey PK. Simultaneous trace level determination of potentially genotoxic hydrazine, methyl hydrazine, and alkylamines in pharmaceutical substances by CE using indirect photometric detection. Chromatographia 2013;76:801-9.
10. Jagota NK, Chetram AJ, Nair JB. Determination of trace levels of hydrazine in the penultimate intermediate of a novel anti-infective agent. J Pharm Biomed Anal 1998;16:1083-7.
11. Gorog S, Balogh G, Cschi A. Estimation of impurity profile in drugs and related materials. Part-II-the role of chromatographic and spectroscopic methods in the estimation of side-reactions in drug syntheses. J Pharm Biomed Anal 1993;11:1219-26.
12. Huybrechts T. Successfully developing and validating methods for the quantification of genotoxic impurities in APIs. Informa Genotoxic Impurities Meeting, Hilton Prague; 2007.
13. Lee CB, Hubert M, Nguyen Van Dua C. Determination of N, N-Dimethylaminoethyl chloride and the dimethyl-aziridiniumionat sub-ppm levels in diltiazem hydrochloride by LC-MS with electrospray ionization. Analyst 2000;125:1255-9.
14. Hansen SH, Sheribah ZA. Comparison of CZE, MEKC, MEEKC and non-aqueous capillary electrophoresis for the determination of impurities in bromazepam. J Pharm Biomed Anal 2005;39:322-7.
15. Ellison GK. Development of a general headspace GC-ECD method for genotoxic alkyl halides at trace levels in API-the challenge of genotoxic impurities and their analysis. Pharmaceuticals Analysis science Group (PASG) Autumn Meeting, Milton Keynes UK; 2006.
16. Liu DQ, Sun M, Korda AS. Recent advances in trace analysis of pharmaceutical genotoxic impurities. J Pharm Biomed Anal 2010;51:999-1014.
17. International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human use, Q2 (R1), Validation of Analytical Procedures: Text and Methodology; 2005.
This work is licensed under a Creative Commons Attribution 4.0 International License.