Determination of fluoxetine, norfluoxetine and their enantiomers in rat plasma and brain samples by liquid chromatography with fluorescence detection

Fluoxetine (FLX) and norfluoxetine (NFLX) racemic mixtures were determined by reversed-phase liquid chromatography with fluorescence detection (λexc = 227 nm, λem = 305 nm). The calibration curves prepared from drug-free plasma and brain were linear in the range of 5–1000 ng ml−1 and 100–40,000 ng g−1 for doped samples, with detection limits of 3.2 and 2.1 ng ml−1 in plasma and 31.5 and 26.1 ng g−1 in brain tissue for FLX and NFLX, respectively. Enantiomer determination was carried out through normal phase HPLC-FD (λexc = 224 nm, λem = 336 nm) after precolumn chiral derivatization with R-1-(1-naphthyl)ethyl isocyanate. Standard curves also prepared in a drug-free matrix were linear for each enantiomer over the range of 2–1000 ng ml−1 and 20–7000 ng g−1 with detection limits for the four compounds ranging between 0.2 and 0.5 ng ml−1 in plasma and between 3.0 and 8.2 ng g−1 in brain tissue. In both methods the analytes were isolated from the biological matrix by a new solid-phase extraction procedure with recovery in plasma and brain over 90 and 87%, respectively. The repeatability of this extraction procedure was satisfactory within-day and between-day with CV < 9.1%. This study also offered the opportunity to obtain an assessment of the potential relationships between the concentration of individual enantiomers of FLX and NFLX in plasma and brain tissue after chronic treatment with racemic FLX at a dose intended to mimic the human plasma concentration of FLX in standard clinical conditions, and therefore should make for more reliable extrapolation of neurochemical findings in other species.