Determination of free-form amphetamine in rat's brain by in vivo microdialysis and liquid chromatography with fluorescence detection

The determination of free amphetamine in rat's brain by in vivo microdialysis technique and HPLC/Fluorescence derivatization method is described. In vivo microdialysis was used to sample the extracellular amphetamine and its contents were analyzed by HPLC/Fluorescence derivatization assay. The effects of pH, temperature and reaction time on the derivatization were examined. The detection limit, linearity and precision associated with this assay were also evaluated. The stability of amphetamine-dansyl chloride in different storage conditions was determined. A single 3.0 mg/kg i.p. amphetamine administration in rats resulted in a maximum concentration of 654 +/- 201 mu M in the medial prefrontal cortex. By using a non-linear curve fitting of two-compartment open model, the first-order rate constant for the appearance of amhetamine (k(1)) was 0.0280 and the first-order rate constant for the disappearance of amhetamine (k(2)) was 0.0281. These results document the utility of our method in pharmacokinetics studies.