Phosphorothioate oligonucleotide quantification by μ-liquid chromatography-mass spectrometry

Area of Science:

• Analytical Chemistry
• Pharmacology
• Biochemistry

Background:

• Phosphorothioate oligonucleotides are crucial therapeutics for modulating gene expression.
• Accurate quantification in biological matrices is essential for drug development.
• Existing analytical methods may lack the sensitivity or throughput required.

Purpose of the Study:

• To develop and validate a sensitive assay for quantifying phosphorothioate oligonucleotides in rat plasma.
• To support pharmacokinetic studies of these therapeutic agents.
• To optimize analytical parameters for enhanced detection.

Main Methods:

• Ion-pair reversed-phase chromatography on a monolithic capillary column.
• High-resolution tandem mass spectrometry for sensitive nucleic acid detection.
• Solid-phase extraction method for efficient plasma sample processing (10 μl).

Main Results:

• The assay demonstrated linearity from 100 to 1,000 nM with a limit of detection at 50 nM.
• Intra- and inter-day precision and accuracy were consistently better than 10.2%.
• The assay successfully supported pharmacokinetic analysis, detecting oligonucleotides up to 3 hours after intravenous administration.

Conclusions:

• A robust and sensitive analytical method for phosphorothioate oligonucleotides in rat plasma has been established.
• The developed assay is suitable for early-phase pharmacokinetic investigations.
• This quantitative method aids in the development and application of oligonucleotide therapeutics.