Enhanced protein precipitation with ammonia enables rapid, universal extraction of oligonucleotides for bioanalysis

Extracting oligonucleotides from biological matrices for mass spectrometry (MS) using current methodologies is time-consuming and costly. Accelerating the discovery, characterization, and development of oligonucleotides (ONTs) requires a universal method that is low-cost, efficient, and that does not need extensive method development for each new test article. Protein precipitation using organic solvents meets these criteria for small molecules but has historically failed for oligonucleotide therapeutics due to poor recovery arising from their tendency to co-precipitate with proteins. This study introduces a novel approach utilizing small amines dissolved in organic solvents to significantly boost extraction recovery of ONTs from biological matrices. We first present the method’s development and then analytically qualify it for the bioanalysis of ASOs and small interfering RNAs (siRNAs) extracted from various tissues, using ion-pairing reverse phase (IPRP) liquid chromatography coupled to tandem MS and high resolution MS (HRMS) detection. This method, Enhanced Protein Precipitation (EPP), achieves near-complete recovery across multiple ONT classes from biological matrices without need of sample digestion, costly solid phase extraction plates, or custom-designed hybridization probes. The method has proven to be more versatile and sustainable than conventional approaches.