Quantification of l-stepholidine in rat brain and plasma by high performance liquid chromatography combined with fluorescence detection

A sensitive and reliable assay for the quantification of l-stepholidine (SPD) in rat plasma and brain was developed using high performance liquid chromatography (HPLC) combined with fluorescence detection. Brain regions (prefrontal cortex, striatum, and cerebellum) and plasma from rats treated with SPD (10 mg/kg s.c.) 20, 40, 60, or 90 min prior to euthanasia were analyzed for SPD levels. Brain samples were homogenized in ice-cold 0.1 M perchloric acid and centrifuged to remove proteins. The supernatants and diluted plasma samples, to which O-desmethylvenlafaxine was added as a process standard, were basified and extracted with ethyl acetate. The organic phase was taken to dryness and the residue taken up in mobile phase. The samples were then injected into an HPLC equipped with a fluorescence detector (excitation and emission wavelengths set at 280 and 320 nm, respectively). The mean recovery of SPD was 74.6%, and reliability studies confirmed the reproducibility of the assay (intra- and inter-assay coefficients of variation of 4.8% and 5.3%, respectively). The assay was readily applicable to the brain and plasma samples obtained from rats injected with SPD as described above; the levels and patterns of disappearance of SPD in brain regions and plasma are shown.