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 Class3 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 degrees C, respectively. The optimized chromatographic separation achieved on Waters Acquity HSS T3 C18 column (100 mm x 2.1 mm, 1.8 & mu;m) in a gradient elution mode with 0.13% formic acid in water and acetonitrile as mobile phases, column temperature kept at 35 degrees 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.

Automated summary

Two potential genotoxic impurities were identified in the Molnupiravir synthetic routes. (Q)SAR methods were used to assess their genotoxicity and MRM technique was utilized for quantification. A validated UPLC-MS/MS method demonstrated excellent linearity and accuracy for the simultaneous quantification of the impurities and MOPR in drug substance and formulation dosage form.