Abstract: OBJECTIVE To establish a method for determining the content of kukoamine A hydrochloride(KuA-H) in liver microsomes and plasma of rats, and to study its metabolic stability in liver microsomes and pharmacokinetics characteristics in plasma. METHODS The rat liver microsome incubation system containing KuA-H was incubated in a water bath at 37℃ for multiple time points, and ice acetonitrile containing 76 ng·mL^-1 carbamazepine was added to terminate the reaction. The plasma samples were precipitated with acetonitrile, and the content of KuA-H in rat liver microsomes and plasma was determined by HPLC-MS/MS. The determination was performed on ACE Excel Super C₁₈ column with mobile phase consisted of water(0.1% formic acid)-acetonitrile(0.1% formic acid) for gradient elution at the flow rate of 0.2 mL·min^-1. The column temperature was set at 40℃, and the injection volume was 5 μL. Electrospray ionization source was performed in a positive electrospray ionization mode in the multiple reaction monitoring mode. The ion transitions for quantitative analysis were m/z 531.3→222.4 (KuA-H) and m/z 237.3→192.2(internal standard), respectively. Taking the content of KuA-H at 0 min of incubation as a reference, the remaining percentage of it in the rat liver microsome incubation system was calculated, and the content of KuA-H in plasma samples at different time points was determine after intragastric administration. The pharmacokinetic parameters were calculated using DAS 2.0 software. RESULTS The linear ranges of KuA-H in rat liver microsomes and plasma were 30-900 ng·mL^-1 and 12.5-2 000 ng·mL^-1, respectively. The precision and accuracy, extraction recovery rate, matrix effect and stability met the quantitative analysis requirements of biological sample. The remaining percentage of KuA-H within 60 min of incubation in rat liver microsomes ranged from 85.5% to 101.5%. The main pharmacokinetic parameters were as follows:C_max was (107.9±29.2)ng·mL^-1, t_1/2 was (4.71±1.92)h, AUC_0-t was (252.2±34.7)ng·h·mL^-1, and t_max was (0.46±0.10)h. CONCLUSION The HPLC-MS/MS established is rapid and sensitive, which is suitable for in vitro metabolic stability and in vivo pharmacokinetic studies of KuA-H.