HPLC-MS/MS法测定大鼠血浆中的知母皂苷B-Ⅱ

Quantification of Timosaponin B-Ⅱ in rat plasma by liquid chromatography tandem mass spectrometry

目的建立快速、灵敏和准确的大鼠血浆中知母皂苷B-Ⅱ液质联用(HPLC-MS/MS)定量分析方法。方法大鼠血浆样品用乙腈沉淀蛋白,上清液经Alltima HP C18柱(2.1mm×50mm,5μm)分离,采用负离子检测多反应监测模式(MRM)、电喷雾离子化(ESI)对知母皂苷B-Ⅱ进行定量分析,内标为ParisaponinⅠ(重楼皂苷Ⅰ)。检测离子对为知母皂苷B-Ⅱ离子对(m/z919.4→757.4)与"重楼皂苷Ⅰ"离子对(m/z1033.5→901.4)。结果方法的线性范围为5～2000μg·L-1,最低定量下限5μg·L-1。日内精密度<10.9%,日间精密度<6.6%,准确度为-8.6%～7.5%;每样品分析时间为3min。结论该法准确、灵敏、特异,适用于血浆中知母皂苷B-Ⅱ的测定。

Aim To develop a rapid,sensitive and accurate high performance liquid chromatography （ HPLC） tandem mass spectrometric （ MS） method for determination of Timosaponin B-Ⅱ in rat plasma. Methods The rat plasma samples were treated with acetonitrile for protein precipitation,and the superna-tants were separated through an Alltima HP C18 column （ 2. 1 mm × 50 mm,5 μm） . The quantification was performed with electrospray ionization source in the negative multiple reaction monitoring （ MRM） mode. Internal standard was Parisaponin I. The precursor product combination ions of m/z： 919. 4→757. 4 and m/z： 1033. 5→901. 4 were used as the quantified ions for Timosaponin B-Ⅱ and IS,respectively. Results The linear calibration curves were obtained in the concentration range of 5 ～ 2000 μg·L -1. The lower limit of quantification （ LLOQ） was 5 μg·L -1. The intra-and inter-batch precisions at three quality control （ QC） levels were within 10. 9 % and 6. 6 %,respectively. The run time for each sample was 3. 0 min. The accuracy of the assay at the three QC levels was from -8. 6 % to 7. 5 % . Conclusion In the present paper a sensitive and precised LC-MS/MS method has been developed and validated for determination of Timosaponin B-Ⅱ in rat plasma.