Determination of Huperzine A in rat plasma by high-performance liquid chromatography with a fluorescence detector.

A simple reversed-phase high-performance liquid chromatography (HPLC)-fluorescence method for the determination of Huperzine A in rat plasma was developed and validated. Separation was achieved on Kromasil C(8) column (5 microm, 150 mm x 4.6mm i.d.). The mobile phase, methanol-water-triethanol amine (45:55:0.05, v/v/v), was delivered at a flow rate of 1.0 ml/min. The eluent was monitored by a fluorescence detector with excitation wavelength at 310 nm and emission wavelength at 370 nm. No interfering peaks were observed in rat blank plasma. The relationship between Huperzine A concentration and peak-area ratio of Huperzine A to the IS was linear over the range of 2.5-250 ng/ml. The intra- and inter-day coefficients of variation were <or=10.1% and <or=14.1%, respectively. The method and extraction recovery of Huperzine A were 101.9-108.9% and 73.5-84.6%, respectively. Huperzine A was found to be stable for at least 5h at RT and 1 week at -20 degrees C. The method was applied to a pharmacokinetic study of Huperzine A in rats following intranasal administration.