Liquid chromatographic–tandem mass spectrometric method for the quantitation of huperzine A in dog plasma

A rapid and sensitive LC–MS–MS method for the determination of huperzine A in dog plasma using huperzine B as internal standard has been developed and validated. The analyte and internal standard were extracted from plasma using n-hexane–dichloromethane–2-propanol (300:150:15, v/v/v), chromatographed on a C18 column (5 μm, 50 mm×4.6 mm i.d.) with a mobile phase consisting of acetonitrile–methanol–10 mM ammonium acetate (35:40:25, v/v/v), and detected using a tandem mass spectrometer with a TurboIonSpray ionization interface. The run time was only 2 min. The assay was linear over the concentration range 0.05–20 ng/ml and intra- and inter-day precision over this range were <5.3% with good accuracy. The limit of detection in plasma was 0.01 ng/ml. The method was successfully applied to define plasma concentration–time curves of huperzine A in dogs after the last dose of an intramuscular injection (10 μg/kg per day for 15 days) of a sustained-release formulation of huperzine A.