LC–MS/MS Sensitivity Enhancement Using 2D-SCX/RPLC and its Application in the Assessment of Pharmacokinetics of Clonidine in Dried Blood Spots

Abstract

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. 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 volumes, eluted to a second dimensional RP column with ammonium acetate, de-salted with highly aqueous solvent and separated in an analytical RP column. Injection of 100 µl clonidine extract exhibited essentially the same peak shape as that from 1 µl and the response of clonidine increased quantitatively in the range of 1-100 µl. The method was successfully employed to analyze clonidine DBS samples from an in-house toxicology study, where clonidine hydrochloride was administered to cynomolgus monkeys to produce hypotensive effects. Of 55 DBS samples collected post-dose, a total of 52 samples were within the curve range of 0.1-50 ng/ml, where valid clonidine PK profiles were obtained. The PK parameters agreed well with the onset of hemodynamic changes measured with implanted miniature telemetry blood pressure transmitters. In comparison, only 21 samples were within the curve range of 2 to 1000 ng/ml from a HILIC-MS/MS method, which limited useful injection volume to 5 µl.