Abstract
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.
Highlights
Apixaban is chemically described as ‘1-(4-methoxyphenyl)-7-oxo-6[4-(2-oxopiperidin-1-yl) phenyl]-4, 5, 6, 7-tetrahydro-1H-pyrazolo [3,4c] pyridine-3-carboxamide’, molecular formula is C25H25N5O4and molecular weight is 459.5
Apixaban has the advantage of reduced bleeding compared with rivaroxaban, it was recommended as the first choice anticoagulant after lumbar spine surgery
According to TTC concept, we have developed a new RP-HPLC method for quantification of seven identified potential genotoxic impurities in Apixaban drug substance, as these impurities having hydrazone and alkyl halide structural alerts
Summary
Apixaban is chemically described as ‘1-(4-methoxyphenyl)-7-oxo-6[4-(2-oxopiperidin-1-yl) phenyl]-4, 5, 6, 7-tetrahydro-1H-pyrazolo [3,4c] pyridine-3-carboxamide’, molecular formula is C25H25N5O4and molecular weight is 459.5. Apixaban is an anticoagulant and is routinely used in the treatment of stroke, embolism, infarct etc. Apixaban has an oral bioavailability of ~50%. It is administered as a twice-daily dose. It is confirmed that apixaban and rivaroxaban drugs were effective anticoagulation therapies that exhibited similar preventive effects against postoperative VTE after lumbar spine surgery [2]. Apixaban has the advantage of reduced bleeding compared with rivaroxaban, it was recommended as the first choice anticoagulant after lumbar spine surgery. Apixaban is formulated as oral tablets and marketed by Bristol-Myers Squibb Company with the trade name ELIQUIS. According to Less-Than-Lifetime (LTL) exposures phenomena of ICH M7 [4] for mutagenic impurities in the pharmaceutical industry, the risk assessment for carryover into the drug substance from the raw materials and intermediates to be appropriately controlled to fulfill the regulatory requirements. Genotoxic assessment (identification of genotoxic impurity, control of impurity and generated data presentation) is must w. r. t analytical approach and control of identified impurities based on TTC concept
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