Determination of the benztropine analog AHN-1055, a dopamine uptake inhibitor, in rat plasma and brain by high-performance liquid chromatography with ultraviolet absorbance detection

Abstract

N-Substituted 3α-[bis(4′-fluorophenyl)methoxy] tropanes represent a series of novel potential cocaine abuse therapeutics. AHN-1055, a member of this series, has been assessed to be the most suitable analog for pharmacokinetic studies. A sensitive and specific high-performance liquid chromatography method was developed to quantitate AHN-1055 in rat plasma and brain tissue. Reversed-phase chromatography with ultraviolet detection ( λ=220 nm) was utilized to quantitate the eluate. Plasma or brain tissue samples were prepared by liquid–liquid extraction using hexane, followed by evaporation, reconstitution in mobile phase, and injection onto an ABZ+plus column. AHN-1055 and oxprenolol (internal standard) eluted at ∼9.9 and 5.01 min, respectively, without any interfering peaks. The calibration curves were found to be linear in the range of 25–10 000 ng/ml for plasma and 50–5000 ng/g for brain ( r 2≥0.999). The intra- and inter-day variabilities were ≤10% whereas the intra- and inter-day errors were ≤8.5%. Plasma and brain recoveries of AHN-1055 were 95 and 79%, respectively. Stability studies showed plasma quality control samples to be stable through at least three freeze–thaw cycles (error<3.5%), for at least 24 h when subjected to room temperature (error<3%) and for at least 30 h after loading the processed samples onto the autosampler (error<3%). AHN-1055 stock solution was found to be stable for at least 4 months when stored at 4 °C (error<6%). The validated method accurately quantified AHN-1055 in plasma and brain samples collected from a pharmacokinetic study consisting of an intravenous bolus in the tail vein of adult male Sprague–Dawley rats.