This paper presents a high-throughput LC-MS/MS method that may be versatilely applied to a wide range of oligonucleotides, making it a valuable tool for rapid screening and discovery. The method is demonstrated using an in-house synthesized MALAT-1 ASO as a model. Biological samples were purified using a reversed liquid-liquid extraction process automated by Hamilton and analyzed with ion-pairing LC-MS/MS. Besides some existing considerations such as column selection, ion-pairing reagent, and sample purification, our work focused on the following four subtopics: 1) selecting the appropriate MRM transition to maximize sensitivity for trace-level ASO in biological samples; 2) utilizing a generically risk-free internal standard tenofovir to avoid the crosstalk interference from the oligo internal standard commonly shown in the LC-MS assay; 3) automating the sample preparation process to increase precision and throughput; and 4) comparing liquid-liquid extraction (LLE) and solid-phase extraction (SPE) as sample purification methods in oligo method development.
Learning Objectives:
General Objective: Participants are able to know the Novel Approaches and Troubleshooting for Developing a High-Throughput Oligonucleotide LC-MS Method beyond the Current Stage, which is beneficial to accelerate the oligo discovery research.
Specific Objective 1: How to select the appropriate Multiple Reaction Monitoring (MRM) transition to maximize sensitivity for trace-level ASO in biological samples?
Specific Objective 2: How to avoid the crosstalk interference internal standard (tenofovir) from the oligo internal standard commonly observed in the LC-MS assay?
Specific Objective 3: How to standardize the workflow by utilizing the generic internal standard and automated sample preparation?
Specific Objective 4: What are the pros and cons of using liquid-liquid extraction (LLE) and solid-phase extraction (SPE) as the sample purification method in oligo method development?