Optimizing the discovery bioanalysis strategy for macrocyclic peptides

Macrocyclic peptides (MCPs) have remained a compelling modality in drug discovery and development, with many successful marketed drugs. Their unique molecular structure and ADME properties have posed bioanalytical challenges that cannot be fully addressed with conventional small molecule LC-MRM assays. In this work, we developed and optimized a high-throughput discovery bioanalytical strategy for MCPs with 16 marketed MCP drugs. By evaluating ten different sample extraction methods based on the recovery and matrix effect, we identified that the protein precipitation extraction with MeOH/ACN (1/1 v/v) with 0.5% FA outperformed the other sample extraction methods, achieving 80% recovery for 80% of the MCP drugs and 90% matrix effect for 90% of the MCP drugs. By assessing the sensitivity of the targeted-selected ion monitoring (t-SIM) and parallel reaction monitoring (PRM) on the Orbitrap HRMS and comparing with the conventional LC-MRM, we concluded that the t-SIM provided comparable sensitivity with MRM (LOQ at 1~3 ng/mL for the majority of the MCP drugs), with the extra benefits of minimal method development and high post-acquisition flexibility in data processing. The optimized bioanalytical strategy was applied to various biological matrices and displayed performance that met the quantitation requirements for discovery bioanalysis.