A sensitive UPLC-MS/MS method for the simultaneous assay and trace level genotoxic impurities quantification of SARS-CoV-2 inhibitor-Molnupiravir in its pure and formulation dosage forms using fractional factorial design

Two potential genotoxic impurities were identified (PGTIs)-viz. 4-amino-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (PGTI-1), and 1-(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2,4(1H,3H)-one (PGTI-II) in the Molnupiravir (MOPR) synthetic routes. COVID-19 disease was treated with MOPR when mild to moderate symptoms occurred. Two (Q)-SAR methods were used to assess the genotoxicity, and projected results were positive and categorized into Class-3 for both PGTIs. A simple, accurate and highly sensitive ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was optimized for the simultaneous quantification of the assay, and these impurities in MOPR drug substance and formulation dosage form. The multiple reaction monitoring (MRM) technique was utilized for the quantification. Prior to the validation study, the UPLC-MS method conditions were optimised using fractional factorial design (FrFD). The optimized Critical Method Parameters (CMPs) include the percentage of Acetonitrile in MP B, Concentration of Formic acid in MP A, Cone Voltage, Capillary Voltage, Collision gas flow and Desolvation temperature were determined from the numerical optimization to be 12.50 %, 0.13 %, 13.6 V, 2.6 kV, 850 L/hr and 375 °C, respectively. The optimized chromatographic separation achieved on Waters Acquity HSS T3 C18 column (100 mm × 2.1 mm, 1.8 µm) in a gradient elution mode with 0.13% formic acid in water and acetonitrile as mobile phases, column temperature kept at 35 °C and flow rate at 0.5 mL/min. The method was successfully validated as per ICH guidelines, and demonstrated excellent linearity over the concentration range of 0.5–10 ppm for both PGTIs. The Pearson correlation coefficient of each impurity and MOPR was found to be higher than 0.999, and the recoveries were in between the range of 94.62 to 104.05% for both PGTIs and 99.10 to 100.25% for MOPR. It is also feasible to utilise this rapid method to quantify MOPR accurately in biological samples

Isolation, identification, structural elucidation, and toxicity prediction using (Q)-SAR models of two degradants: AQbD-driven LC method to determine the Roxadustat impurities

ABSTRACT: Roxadustat (RDT) is the first orally administrated HIF-prolyl hydroxylase inhibitor drug used to treat anemia caused by chronic kidney disease. Two unknown degradants were detected in photolytic and oxidative stress conditions during the forced degradation study of RDT and isolated them using the preparative HPLC. The structural characterization of these impurities was confirmed using ESI-LC-MS, NMR, and FT-IR spectroscopic analysis. To evaluate the toxicity of degradants, (Q)-SAR models such as Derek and Sarah model were utilized. The current study aims to develop a stability-indicating related substances quantification method in RDT along with degradant impurities by implementing AQbD principles. The Box-Behnken Design (BBD) was utilized to optimize the final analytical method conditions. p-values for the model and lack of fit were 0.05, respectively. The optimized CMPs are 30:70 (v/v) ACN: Methanol in mobile phase-B, 50:50 mobile phase-A & B in the initial gradient program, 0.95 mL/min of flow rate, and 40 °C as column oven temperature. Agilent Zorbax XDB-C8, (250 × 4.6) mm, 5 μm analytical column was used to separate the desired components from the sample matrix peaks and themselves. The optimized method was validated in compliance with regulatory requirements. The recoveries for all the impurities ranged from 96.8% to 99.7%, with an%RSD 0.998. The current method can be used in quality-control laboratories to quantify RDT impurities without any developmental trials