ArticleACS omega2025
Enhanced Protein Precipitation with Ammonia Enables Rapid, Universal Extraction of Oligonucleotides for Bioanalysis.
Article in ACS omega, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Authors and funding
8 authors.
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Abstract
Discovery, characterization, and development of oligonucleotides (ONTs) require a universal method that is low-cost and efficient, without requiring extensive method development for each new test article. Protein precipitation using organic solvents meets these criteria for small molecules but has historically failed for ONTs due to poor recovery arising from their tendency to coprecipitate with proteins. This study introduces an effective approach utilizing small amines dissolved in organic solvents to significantly boost the extraction recovery of ONTs from biological matrices. We first present the method's development and then analytically qualify it for the bioanalysis of antisense oligonucleotides (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 acceptable recovery for ASOs and siRNAs; greater than 80% and achievable LLOQ of 1-5 ng/mL for different ONTs in plasma and tissues. We demonstrate the method is suitable for recovering multiple ONT classes from biological matrices without the 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.
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Registered trials
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