High-Sensitivity Workflow for LC–MS-Based Analysis of GALNAC-Conjugated Oligonucleotides: A Case Study

Aim: Mass-selective quantitation is a powerful attribute of LC-MS as a platform for bioanalysis. Here, a sensitive LC-MS approach has been validated for an oligonucleotide having chemical modifications (e.g., N-acetylgalactosamine [GalNAc] conjugated), to distinguish between the conjugated and unconjugated forms of the oligonucleotide, thereby enabling a nuanced view of the pharmacokinetic profile. Results: A high-sensitivity methodology for mass-specific measurement of AZD8233, a GalNAc-conjugated 16-mer oligonucleotide, using LLE-SPE with optimized LC conditions and detection of a low-mass fragment ion was successfully validated in the range of 0.20-100 ng/ml in human plasma. Conclusion: The AZD8233 LC-MS methodology adds valuable insight on the GalNAc linker's in vivo stability to the program and should be broadly applicable to oligonucleotides requiring high sensitivity and mass-selective measurement for quantitative discrimination from metabolites and endogenous interferences.