Simultaneous Determination of Three Bioactive Constituents from by UPLC-MS/MS and Application of the Technique to Pharmacokinetic Analyses.

has been widely used as a valuable hemostatic agent for thousands of years due to the high levels of bioactive constituents it contains. Here, we used a sensitive ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) method for the simultaneous determination of three major active ingredients of the extract, namely, -isobutylmalic acid, gymnoside I, and militarine in rat plasma. The three major active ingredients were determined using the multiple reaction monitoring (MRM) mode at 189 ⟶ 129 for -isobutylmalic acid, 457.2 ⟶ 285.1 for gymnoside I, 725.3 ⟶ 457.2 for militarine, and 417.0 ⟶ 267.0 for the IS puerarin. All calibration curves showed good linearity (  ≥ 0.999) over the concentration range with the lower limit of quantification between 0.015 and 0.029 g/mL. The relative standard deviations of intraday and interday measurements were less than 15%, and the method was accurate within 93.3-100.4%. The extraction recovery was 92.65-100.98%, and no matrix effect was observed. The results indicated that after oral administration of in rats, the of -isobutylmalic acid was significantly longer than that of gymnoside I and militarine and the mean residence time and area under the curve of -isobutylmalic acid and gymnoside I in rats were significantly higher than those of militarine. Moreover, the blood concentration-time curve of -isobutylmalic acid showed double peaks, suggesting that -isobutylmalic acid could exhibit the phenomenon of enterohepatic circulation or metabolic conversion. We also explored some of the pharmacokinetic characteristics of three ingredients from extract , and the data obtained may provide a basis for the further investigation of .