A rapid, specific and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous determination of strychnine, brucine, strychnine N-oxide and brucine N-oxide in rat plasma. Plasma samples were pretreated via simple protein precipitation with methanol and ephedrine hydrochloride was used as internal standard. Chromatographic separation was carried out on an ZORBAX Eclipse XDB-C-18 column (2.1 x 150 mm, 3.5 mu m) by gradient elution with methanol and 10 mM ammonium acetate (adjusted to pH 4.0 with formic acid). The quantification of the analytes was performed by mass spectrometry with TurbolonSpray ionization (ESI) inlet in the positive ion multiple reaction monitoring (MRM) mode. The results showed that the calibration curve was linear in the concentration range of 0.510 similar to 306.3 ngmL(-1) for strychnine, brucine and 0.102 similar to 306.0 ng mL(-1) for strychnine N-oxide and brucine N-oxide, respectively. The intra- and inter-day precisions were less than 14.9%, and the accuracy ranged from 89.4 to 113% at three QC levels for the 4 analytes. The validated method was successfully applied to the pharmacokinetic study of strychnine, brucine, strychnine N-oxide and brucine N-oxide in rat plasma after oral administration of each monomer and the total alkaloids from Semen Strychni. After single oral administration of the total alkaloids from Semen Strychni at 4 dose levels, C-max, AUC(0-t) of strychnine and brucine increased and were proportional to the oral doses. In comparative pharmacokinetics studies, no significant difference was found between each monomer and the total strychnos alkaloids on the pharmacokinetic parameters such as C-max and AUC. Mean C-max and AUC of strychnine and brucine were slight increased in the monomer groups in comparison to the total strychnos alkaloids groups, which suggested that some other alkaloids in the Semen Strychni may decrease the absorption of strychnine and brucine in body. (C) 2015 Elsevier B.V. All rights reserved.