LC-MS/MS sensitivity enhancement using 2D-SCX/RPLC and its application in the assessment of pharmacokinetics of clonidine in dried blood spots

Background: Dried blood spot (DBS) technology offers distinctive preclinical and clinical advantages primarily ascribed to microscale sampling (e.g., 40–80 µl per time point), and the nature of solid-state samples in filter papers. Logistic benefits in sample collection, storage and shipping also result. However, the effective DBS samples available for bioanalysis are finite, that is, in the order of approximately 1 µl equivalent of plasma (3-mm punch) from a DBS of approximately 15–20 µl whole blood samples. This represents 20- to 100-times fewer samples for bioanalysis compared with a typical plasma assay. It is critical to increase LC–MS/MS sensitivity to accommodate DBS bioanalysis. Results: We developed a 2D strong cation exchange reversed-phase LC–MS/MS (2D-SCX/RPLC–MS/MS) for online enrichment, separation and detection of basic polar compounds, using clonidine hydrochloride as a model compound. Positively charged clonidine was retained and enriched in the first dimensional SCX column even in large ...