Validation of a high-sensitivity assay for zatebradine in dried blood spots of human blood at pg/ml concentrations using HILIC–MS/MS

Abstract

Background: One of the main reasons for the increased popularity of dried blood spots (DBS) is related to the 3Rs principles (replacement, refinement and reduction) for animal use in drug development. The small blood volume collected using this technique may have a significant impact on the assay sensitivity. An approach that made use of hydrophilic interaction chromatography (HILIC) to enhance the LC–MS assay sensitivity was explored and optimized for a tool compound. Results: A very high-sensitivity assay in dried spots of human blood was validated in the range of 5 to 5000 pg/ml. The use of HILIC increased LC–MS sensitivity up to fivefold compared with other reversed phase chromatographic methods. Conclusion: The good compatibility of the DBS extracts with HILIC and the results of the assay validation for zatebradine at a very low LLOQ demonstrate the high potential and the high performance of this approach.