Observations from a decade of oligonucleotide bioanalysis by LC-MS

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• Oligonucleotide therapeutics present unique bioanalytical challenges compared to small molecules.
• Existing methods like ELISAs and PCR have limitations for comprehensive analysis.
• Liquid chromatography-mass spectrometry (LC-MS) is increasingly vital for sensitive and selective oligonucleotide quantification.

Purpose of the Study:

• To highlight common challenges encountered during oligonucleotide therapeutic bioanalysis.
• To share practical solutions and considerations derived from real-world case studies.
• To provide guidance for researchers developing LC-MS methods for oligonucleotides.

Main Methods:

• Four distinct case studies detailing specific bioanalytical issues.
• Application of liquid chromatography coupled to mass spectrometry (LC-MS).
• Analysis of challenges including analyte recovery, chromatographic resolution, and sensitivity.

Main Results:

• Identified common problems such as low/variable analyte recovery and poor resolution from related species.
• Documented chromatographic abnormalities and difficulties in achieving required sensitivity.
• Demonstrated the utility of LC-MS in addressing these complex analytical issues.

Conclusions:

• LC-MS provides a robust platform for oligonucleotide bioanalysis, balancing sensitivity and selectivity.
• Addressing challenges in recovery, resolution, and sensitivity is key for method development.
• The presented considerations can aid in optimizing bioanalytical strategies for oligonucleotide therapeutics.