(284) Characterization of the Enantiomeric Purity of Molnupiravir in Presence of its Active Metabolite

Objective : A novel HPLC chiral separation technique was developed for the determination of enantiomeric purity of R,S‐Molnupiravir in the presence of its active metabolite β-D-N4-hydroxycytidine (NHC) and subsequent quantification in different dosage forms. In the present study, we used a reversed‐phase high‐performance liquid chromatography with a chiral stationary phase consisting of Bonded Macrocylic Glycopeptide Teicoplanin Aglycone coated on silica‐gel (Astec® CHIROBIOTIC® TAG).

Methods: All separations were performed using Astec CHIROBIOTIC™ TAG, 10 cm x 2.1 mm column. Temperature was varied from 25 to 50 °C. Samples were dissolved in methanol and were injected automatically by an autosampler (5 μl) and the flow rate was 1 ml/min. The mobile phases were composed of either water 0.1% triethylamine acetate (TEAA) buffer, pH 4.0, with different amounts of methanol or acetonitrile as organic modifiers. The buffer was adjusted with acetic acid to the appropriate pH prior to the addition of an organic modifier.

Results: Our initial findings indicated minimal separation of the enantiomers under reversed phase conditions. Given this observation and the lack of success with normal phase chiral columns, we opted to refine chromatographic conditions utilizing the Astec CHIROBIOTIC™ TAG column. Although slower flow rates usually generate broad peaks and longer analysis time, the decrease of flow rate from 1.0 to 0.8/ 0.6 ml/min resulted in a satisfactory resolution and reasonable analysis time, in which complete baseline separation of the enantiomers was achieved.

Conclusions: This study assesses the enantiomeric purity of MOL in different tablet dosage forms. Multiple chromatographic parameters were found to influence these separations, with particular emphasis on the composition and concentration of salt in the mobile phase, as well as the pH level.