Strategies for effective development of ultra-sensitive LC-MS/MS assays: application to a novel STING agonist.

Background: The continual need for the development and validation of ultra-sensitive (low pg/ml) LC-MS/MS assays in the pharmaceutical industry is largely driven by the ultra-low analyte exposure or very low sample volume. Methodology: Strategies and systematic approaches for sensitivity enhancement are provided which cover all aspects of a LC-MS/MS bioanalysis. A case study where such strategies were applied for the validation of a 5.0 pg/ml assay for a STING agonist is discussed. Conclusion: Analytical protocols were developed to extract analytes from large volume of plasma samples (600 and 400 µl) with high throughput. The guidance provided in this publication can serve as a resource to influence LC-MS/MS method development activities.