Simultaneous Determination of Triterpenoidal Saponins in Rat Urine After Oral Administration of Pulsatilla chinensis Extract Using HPLC-MS

The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous quantitative determination of the following triterpenoidal saponins: anemoside A3, anemoside B4, 23-hydroxybetulinic acid, pulsatilloside B, pulsatilloside C, and cirenshenoside S in rat urine. The chromatographic separation was performed using a Sapphire C18 column (250 mm x 4.6 mm, 5 mu m) and gradient elution was used during the analysis. Anemoside A3, anemoside B4, and 23-hydroxybetulinic acid were detected with the mass spectrometer in negative ion mode monitoring at m/z 749.6/471.2, 1,219.7/749.4, 471.4/471.4 and pulsatilloside B, pulsatilloside C, and cirenshenoside S were in positive ion mode monitoring at m/z 819.4/347.2, 965.5/493.2, and 1,097.9/493.1, respectively. The calibration curves were indicative of good linearity (r (2) a parts per thousand yen 0.9965) in the range of interest for each analyte. Intra-day and inter-day precision (CV %) was less than 9.1 and 8.7 %, respectively, and accuracy was between -8.4 and 8.3 %. Recovery was 81.02-102.9 %. The method is very rapid, simple, and reliable, and suitable for excretion studies. It can be routinely used for simultaneous determination of six triterpenoidal saponins in rat urine. These results indicated that the newly developed method can also be applied to studies after administration of extraction of saponins from Pulsatilla chinensis to rats.