(T0930-06-40) Optimizing Discovery Bioanalytical Strategy for Macrocyclic Peptides and Degraders with Orbitrap High Resolution Mass Spectrometry

Purpose: LC-MRM is considered the gold standard for quantitative analysis of small-molecule drugs during bioanalysis for its excellent sensitivity, good selectivity and wide dynamic range, however, when targeting new modality drugs such as macrocyclic peptides (MCPs) and degraders, such an approach might have its limitations. Using the MCP target as an example, after electrospray ionization, the peptide signal intensities spread over different adduct forms and multiple-charge ions, and poor fragmentation efficiency is often observed, making high resolution mass spectrometer (HRMS) a better available option for the quantitative analysis. In this work, we optimize the bioanalysis procedure with a focus on evaluating the performance of LC-HRMS with various types of peptides and degrader drugs. The investigation includes optimizing sample extraction and comparing LC-HRMS analysis under different acquisition modes with the traditional LC-MRM approach.

Methods: QC samples were prepared in triplicates by spiking the selected drug compounds in different biological matrices including mouse plasma, diluted whole blood, liver, lung, and brain. Various types of sample extraction procedures, such as protein precipitation with ACN, ACN/MeOH mix, acidified ACN, acidified ACN/MeOH mix, silica-based and polymeric based reverse phase SPE plates, were evaluated by comparing the recovery of the selected drug compounds. Once the sample extraction procedure was optimized, calibration standard curves with a concentration range from 0.33 ~ 20000 ng/mL were prepared in different biological matrices as listed above with selected drug compounds. The standard curves will be analyzed under different Orbitrap HRMS acquisition modes including full scan MS, targeted SIM, and targeted MS/MS; the same curves will also be analyzed with the traditional LC-MRM approach.

Results: With the QC samples extracted from different extraction methods, we optimize the sample extraction based on the overall best recovery in different biological matrices. With the calibration curve analyzed under different acquisition modes, we can evaluate the sensitivity from the lower limit of quantitation levels, the selectivity from the matrix interference, the dynamic range from the calibration curve regression fit.

Conclusion: In the end, we conclude that with LC-HRMS, we can achieve either better or comparable sensitivity, selectivity, and dynamic range when compared with the traditional LC-MRM method for quantitative analysis. When we perform a parallel full scan MS and targeted SIM acquisition, we can obtain valuable preliminary biotransformation information without sacrificing the performance of the quantitative analysis.

The figure illustrates the optimized LC methods, and the examples of LC spectrum for selected drug compounds.

The table contains preliminary LLOQ results from mouse plasma calibration curves for selected drug compounds.