| Summary: | <i>Lycopodii Herba</i> is a widely used traditional medicinal herb, and contains diverse fascinating alkaloids. In this study, a fast and sensitive LC-MS/MS method for the simultaneous determination of lycodoline, α-obscurine, and <i>N</i>-demethyl-α-obscurine from <i>Lycopodii Herba</i> in rat plasma and brain tissue was developed and validated. Biological samples were extracted via a protein precipitation procedure using methanol as the extraction solvent and Huperzine B as the internal standard. Chromatographic separation was carried out using a Thermo Syncronis-C18 column (50 mm × 2.1 mm, 5 μm) and a gradient mobile phase containing methanol and water with 0.05% formic acid. The three alkaloids were detected by positive electrospray ionization in selective reaction monitoring mode. The selectivity, crosstalk, carryover effect, linearity, accuracy, precision, extraction recovery, matrix effect, and stability of the current method were validated. Then, using the validated method, the plasma pharmacokinetics and brain tissue distribution of the alkaloids in rats were investigated after intragastrical administration of <i>Lycopodii Herba</i> extract. The three alkaloids were shown to be rapidly absorbed into the blood (<i>T</i><sub>max</sub>, 0.79−1.58 h), and then also eliminated rapidly (<i>t</i><sub>1/2</sub>, 1.27−2.24 h). All of them could pass through the blood−brain barrier. The method provides a new research approach to expand preclinical studies of <i>Lycopodii Herba</i>.
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