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Over the next decade NP-based AIT will be largely used to treat allergic disorders.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 STI