Sensitive determination of a pharmaceutical compound and its metabolites in human plasma by ultra-high performance liquid chromatography–tandem mass spectrometry with on-line solid-phase extraction

This paper describes the determination of a drug candidate and two metabolites in human plasma by column-switching LC–MS/MS after protein precipitation. Starting from a standard method with a quantitation limit of 0.5ng/mL, a highly sensitive assay was developed, employing UHPLC separation and detection on an API 5000 mass spectrometer. The injected plasma equivalent was increased from 6 to 20μL; conventional column trapping for compound enrichment and removal of matrix constituents was combined with high-pressure analytical separation using small particle columns to improve resolution and signal-to-noise ratio. Quantitation limits were thus lowered to between 5 and 20pg/mL, offering the possibility to provide bioanalytical support for microdosing studies in humans. Excellent assay quality and robustness were achieved by both methods.