Determination of flumazenil in human plasma by liquid chromatography–electrospray ionisation tandem mass spectrometry

A liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MS/MS) method was developed to determine unlabelled flumazenil (Ro 15-1788) in human plasma in [11C]flumazenil positron emission tomography (PET) studies. N-Methyl tri-deuterated flumazenil was used as an internal standard. The analyte and internal standard were extracted from plasma samples using solid-phase extraction, with a recovery of 78%. This was determined through the convenience of radioactivity measurements of 11C-labelled flumazenil. The evaporated and reconstituted eluate was analysed by LC–ESI–MS/MS. The calibration curve was linear over the tested concentration range of 0.05–0.5 nM (15–150 pg/ml) with a correlation coefficient, R2, of 0.998±0.001. A high precision was achieved, with mean intra-assay and inter-assay relative standard deviations of at most 6 and 7%, respectively. The accuracy of the method ranged from 95 to 104%. As a proof of concept, the validated method was applied in the determination of flumazenil in plasma from two healthy volunteers participating in a PET study with three repeated investigations. A bolus-infusion protocol was used to achieve a constant concentration level of flumazenil. The average plasma concentrations ranged from 0.11 and 0.19 nM and all measurements were within the calibration standard range. The flumazenil concentrations were relatively constant within each scan and the average intra-scan precision was 15%.