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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.

The present study relates to a new capillary electrophoresis method for the determination of N-methylpyrrolidine, an impurity considered to be toxic and also potential degradation impurity in cefepime hydrochloride drug substance. The newly developed capillary electrophoresis method for determining the content of N-methylpyrrolidine in cefepime for injection has been validated as per International Conference on Harmonization (ICH) guidelines to prove the selectivity, sensitivity, suitability, robustness, and ruggedness of the method. This simple, efficient, and rapid methodology may be used by pharmaceutical industry for routine analysis as well as during stability studies. The newly developed capillary electrophoresis method to determine the content of N-methylpyrrolidine in cefepime for injection requires 10 min for data acquisition, and uses an indirect UV photometry method to detect the analyte signal at 240 nm against the reference signal at 210 nm. The electrophoretic system is optimized to get stable base line, higher signal to noise ratio and peaks with narrow peak width. The method employs bare fused silica capillary with extended light path, effective length of capillary is 56 cm and inner diameter of capillary is 50 μm, 5 mmole of imidazole buffer adjusted to pH 5.1 with 3 molar acetic acid solution is used as background electrolyte. The sample is introduced in hydrodynamic mode employing pressure of 50 mbar for 5 s, and the desired separation is achieved with constant applied voltage of 25 kV at ambient temperature (~25°C).

A gradient reversed phase high performance liquid chromatography (RP-HPLC) method has been developed and validated for the determination for related substances of Dapagliflozin drug substance. Chromatographic separation of Dapagliflozin from its process and degradation related substances was achieved on YMC Pack Pro C18, 250mm × 4.6mm 5 i.e A stainless steel column 250 mm long, 4.6 mm internal diameter filled with Octadecyl silane chemically bonded to porous silica particles of 5 m diameter maintained column oven temperature at 25°C. Orthophosphoric acid buffer is mobile phase A and acetonitrile is mobile phase B. Wavelength for UV detection: 225nm, flow rate: 0.8 ml/min and Injection volume: 20µl. The developed method suitability was checked and validated as per ICH guidelines for specificity, linearity, accuracy, precision, limit of quantification, limit of detection robustness and ruggedness experiments. Dapagliflozin drug substance was subjected to stress conditions of thermal, hydrolysis, humidity, peroxide and photolytic to observe the degradation products. Limit of detection of each RS is less than 0.008%w/w indicating that the developed method is highly sensitive. The experiment results are given in detailed in this research article.

A new unknown impurity of cefoxitin formed during a gradient reverse phase high performance liquid chromatography (HPLC) analysis of stress stability samples of the drug substance cefoxitin, and the level of this impurity was found at up to 0.9%. This impurity was identified by LC-MS and characterized by ( 1 H NMR, 13 C NMR, LC/MS/MS, elemental analysis and FT-IR). Based on the spectral data, the impurity was named as, 3-[[(2R,3S)-[3-methoxy-3-N-[2-(thiophen-2-yl)acetamido]]-4oxoazetidin-2-ylthio]-2-[(carbamoyloxy)methyl]]-acrylic acid. The structure of this impurity was also established unambiguously, prepared by isolation and co-injected into HPLC to confirm the retention time. To the best of our knowledge, this impurity has not been reported elsewhere. Structural elucidation of the impurity by spectral data is discussed in detail.