Abstract: | We established a rapid and sensitive ultra high‐performance liquid chromatography tandem mass spectrometry method for the simultaneous quantification of xanthones and steroidal saponins in rat plasma. Chromatographic separation was achieved on a C18 column with a mobile phase comprising acetonitrile and 0.1% formic acid. The detection was performed by negative electrospray ionization in multiple reaction monitoring mode. The validated method showed good linearity within the tested range (r > 0.9945). The intra‐ and interday precision at high, medium, and low concentrations was less than 7.96%. The bias of accuracies ranged from −1.92 to 9.62%. The extraction recoveries of the compounds ranged from 84.78 to 88.69%, and the matrix effects ranged from 96.76 to 108.59%. This method was successfully applied to a pharmacokinetic comparison of crude and salt‐processed Anemarrhenae Rhizoma aqueous extracts after oral administration in rats. The maximum plasma concentration and area under concentration–time curve of timosaponin BIII and timosaponin AIII increased significantly (P < 0.05 or 0.01) and those of timosaponin BII decreased significantly (P < 0.05) after processing. These results could contribute to the clinical application of crude and salt‐processed Anemarrhenae Rhizoma and reveal the processing mechanism. |