High-sensitivity workflow for LC-MS-based analysis of GalNAc-conjugated oligonucleotides: a case study

Area of Science:

• Bioanalysis
• Pharmacokinetics
• Mass Spectrometry

Background:

• Liquid chromatography-mass spectrometry (LC-MS) is crucial for bioanalysis, enabling mass-selective quantitation.
• Oligonucleotides with chemical modifications, such as N-acetylgalactosamine (GalNAc), require precise quantification to understand their behavior in vivo.
• Distinguishing between conjugated and unconjugated forms is essential for accurate pharmacokinetic profiling.

Observation:

• A sensitive LC-MS approach was developed and validated for AZD8233, a GalNAc-conjugated 16-mer oligonucleotide.
• The method utilizes liquid-liquid extraction (LLE) coupled with solid-phase extraction (SPE) and optimized LC conditions.
• Detection focused on a low-mass fragment ion for enhanced sensitivity and specificity.

Findings:

• The validated LC-MS methodology achieved high sensitivity for mass-specific measurement of AZD8233 in human plasma.
• Quantification was accurate within the range of 0.20-100 ng/ml.
• The method successfully discriminated between the GalNAc-conjugated and unconjugated forms of the oligonucleotide.

Implications:

• This validated LC-MS method provides valuable insights into the in vivo stability of the GalNAc linker for AZD8233.
• The methodology is broadly applicable to other modified oligonucleotides requiring sensitive and mass-selective quantification.
• It enables quantitative discrimination from metabolites and endogenous interferences, crucial for drug development.