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.
Keywords:
- carryover
- LC–MS/MS
- LLQ
- nonspecific binding
- pharmacokinetic
- sensitivity enhancement