A Roadmap guide on bioanalysis challenges and practical solutions for accurate quantification of...

Therapeutic oligonucleotides have emerged as a transformative drug class, yet their physicochemical complexity poses unique analytical challenges in bioanalysis. Liquid chromatography mass spectrometry (LC-MS) has become a powerful platform for their quantification, offering high specificity and structural insight. However, accurate measurement requires addressing challenges such as nonspecific binding, matrix effects, nuclease degradation, and ion-pairing interferences from sample preparation to LC-MS analysis. This review provides a practical roadmap for establishing robust LC-MS workflows for oligonucleotides bioanalysis, emphasizing optimized sample preparation, column and mobile phase selection, ionization control, and fragmentation tuning. Key strategies to minimize analytical artifacts, improve recovery, and ensure regulatory compliance are discussed in the context of current FDA or EMA bioanalytical validation guidelines. Collectively, this work outlines the critical considerations and systematic optimizations needed to achieve reliable, reproducible, and sensitive quantification of therapeutic oligonucleotides in complex biological matrices, supporting their successful clinical translation by informing pharmacokinetics, therapeutic potential, and safety profiles.