Risdiplam retention studies and development of extraction from serum

The primary objective was to investigate the retention of risdiplam under various conditions in reversed-phase ultra-high performance liquid chromatography. Such a study proved to be crucial to better understand the retention mechanism of risdiplam. The study was conducted using five stationary phases: octadecyl, pentafluorophenyl, diphenyl, phenyl and phenyl-hexyl. The π-π interactions were shown to be dominant for risdiplam, resulting in higher retention for phenyl-based stationary phases compared to octadecyl phases. The effect of the mobile phase composition (water, acidified water, salt addition, salt concentration and pH) was evaluated, with particular attention paid to the effect of the pH, as this parameter appears to have an impact on the risdiplam molecule. The pentafluorophenyl column was chosen because of its short retention time regardless of the mobile phase used and relatively symmetrical peaks. The developed method allowed for precise, repeatable, and sensitive analysis of risdiplam in just 3 minutes. It was applied for the analysis of risdiplam in serum samples. The protein precipitation procedure was optimized (type and volume of solvent) and used during extraction. Finally, 10% TFA was selected as a solvent for precipitation, since it allowed for the recovery of 90%. This is the first such complex study on the chromatographic behaviour of risdiplam, as well as its extraction from serum samples.