Development of a Versatile High-through-put Oligonucleotide LC-MS Method to Accelerate Drug Discovery

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• Oligonucleotide quantification is vital for biological understanding and diagnostics.
• High-throughput methods are needed for rapid screening and discovery.
• Existing liquid chromatography-mass spectrometry (LC-MS) methods require optimization for diverse oligonucleotides.

Purpose of the Study:

• To develop and validate a versatile, high-throughput LC-MS/MS method for oligonucleotide quantification.
• To optimize key assay parameters including Multiple Reaction Monitoring (MRM) transitions and internal standards.
• To automate sample preparation for enhanced precision and throughput.

Main Methods:

• Developed an automated reversed liquid-liquid extraction (LLE) process using a liquid handling workstation.
• Employed ion-pairing LC-MS/MS for analysis of MALAT-1 Antisense Oligonucleotide (ASO).
• Utilized a generic internal standard (tenofovir) to prevent interference and compared LLE with solid-phase extraction (SPE).

Main Results:

• Achieved a lower limit of quantification (LLOQ) of 2 nM in rat cerebrospinal fluid (CSF) and plasma.
• Demonstrated assay sensitivity, specificity, precision, accuracy, recovery, matrix effect, and stability.
• Successfully quantified MALAT-1 ASO in rat CSF and plasma post-injection, evaluating matrix differences.

Conclusions:

• The developed high-throughput LC-MS/MS method is sensitive and versatile for oligonucleotide quantification.
• Automated sample preparation and generic internal standards improve assay robustness and throughput.
• This method provides a foundation for oligonucleotide discovery and the development of diagnostic tools.