HPLC-MS/MS法同时测定家兔血浆中5种皂苷成分及药动学研究

Simultaneous Determination and Pharmacokinetic Study of Five Saponins in Plasma of Rabbits by HPLCMS/MS

目的:建立快速、灵敏的基于固相萃取技术的高效液相色谱-串联质谱(HPLC-MS/MS)方法,同时测定兔血浆中人参皂苷Rg1、人参皂苷Rb1、人参皂苷Rd、人参皂苷Re和三七皂苷R1的浓度,并研究家兔肌内注射血塞通注射液后的药动学特征。方法:采用色谱柱CAPCELL CORE C18柱(150 mm×2.1 mm,2.7μm),以水-乙腈为流动相,梯度洗脱,流速:0.3ml·min^(-1)。采用多重反应监测(MRM)的扫描方式,离子源为ESI源,选择离子对m/z 823.5+→643.4+(人参皂苷Rg1)、m/z 1131.6+→789.4+(人参皂苷Rb1)、m/z 969.4+→789.5+(人参皂苷Rd)、m/z 969.4+→789.5+(人参皂苷Re)和m/z 955.6+→775.4+(三七皂苷R1)用于定量检测,进样量为1.0μl。家兔血浆样本通过固相萃取法(SPE)处理后进样。结果:5种皂苷质量浓度分别在9.43~603.40 ng·ml^(-1)(人参皂苷Rg1)、10.00~640.04 ng·ml^(-1)(人参皂苷Rb1)、4.91~157.32 ng·ml^(-1)(人参皂苷Rd)、4.84~81.89 ng·ml^(-1)(人参皂苷Re)及4.64~148.70 ng·ml^(-1)(三七皂苷R1)范围内线性良好,日内和日间精密度均小于7.80%,各成分提取回收率介于81.75%~106.83%之间,基质效应介于86.57%~97.40%之间。Rg1主要药动学数半衰期(T1/2)=(2.61±1.05)h,药时曲线下面积(AUC0→∞)=(117.41±17.43)μg·ml^(-1)·h,平均滞留时间(MRT0→∞)=(3.48±0.76) h;Rb1主要药动学参数:T1/2=(18.04±6.77) h,AUC0→∞=(2393.82±594.47)μg·ml^(-1)·h,MRT0→∞=(25.28±8.98) h;Rd主要药动学参数:T1/2=(54.52±23.33) h,AUC0→∞=(1 373.61±398.35)μg·ml^(-1)·h,MRT0→∞=(76.64±32.98) h;Re主要药动学参数:T1/2=(2.28±1.24) h,AUC0→∞=(27.27±4.94)μg·ml^(-1)·h,MRT0→∞=(3.47±0.63) h;R1主要药动学参数:T1/2=(2.61±1.05) h,AUC0→∞=(47.14±6.16)μg·ml^(-1)·h,MRT0→∞=(3.55±0.67) h。结论:该方法适用于测定家兔血浆中人参皂苷Rg1、人参皂苷Rb1、人参皂苷Rd、人参皂苷Re、三七皂苷R1的浓度,并提供药动学参数。药动学参数显示5种皂苷成分在家兔体内呈现两种不同的药动学特征。

Objective: To establish a fast and sensitive HPLC-MS/MS method based on solid phase extraction technology to simultaneously determine the concentrations of ginsenoside Rg1,ginsenoside Rb1,ginsenoside Rd,ginsenoside Re and notoginsenoside R1 in rabbit’s plasma,and to study the pharmacokinetic characteristics of rabbits after intramuscular injection of Xuesaitong injection. Methods: The chromatographic separation was performed on a CAP CELL CORE C18 column(150 mm × 2. 1 mm,2. 7 μm) with gradient elution of mobile phase consisting of water and acetonitrile at a flow rate of 0. 3 ml· min-1. Multiplereaction monitoring(MRM) was employed with m/z 823. 5+→643.4+(ginsenoside Rg1),m/z 1131.6+→789.4+(ginsenoside Rb1),m/z 969.4+→789.5+(ginsenoside Rd),m/z 969.4+→789.5+(ginsenoside Re) and m/z 955.6+→775.4+(notoginsenoside R1)as the characteristic ion masses. The injection volume was 1.0 μl. Blood samples were extracted by SPE before the analysis of LC-MS/MS. Results: Within the concentration range of 9.43-603.40 ng · ml-1 for ginsenoside Rg1,10.00-640.04 ng·ml^(-1) for ginsenoside Rb1,4.91-157.32 ng·ml^(-1) for ginsenoside Rd,4.84-81.89 ng·ml^(-1) for ginsenoside Re and 4.64-148.70 ng·ml^(-1) for notoginsenoside R1,good linearity was respectively achieved. Both inter-and intra-day precisions were below 7.80%. The extraction recoveries ranged from 81.75% to 106.83% and the matrix effect values ranged from 86.57% to 97.40% for all analytes. The main pharmacokinetic parameters of the five saponins were as follows: Rg1 T1/2=(2.61±1.05) h,AUC0→∞=(117.41±17.43) μg·ml^(-1)·h,MRT0→∞=(3.48±0.76) h;Rb1 T1/2=(18.04± 6.77) h,AUC0→∞=(2393.82± 594.47) μg·ml^(-1)·h,MRT0→∞=(25.28± 8.98) h;Rd T1/2=(54.52 ±23.33) h,AUC0→∞=(1 373.61±398.35) μg·ml^(-1)·h,MRT0→∞=(76.64±32.98) h;Re T1/2=(2.28± 1.24) h,AUC0→∞=(27.27±4.94) μg·ml^(-1)·h,MRT0→∞=(3.47±0.63) h;R1 T1/2=(2.61±1.05) h,AUC0→∞=(47.14±6.16) μg·ml^(-1)·h,MRT0→∞=(3.55±0.67) h. Conclusion: The HPLC-MS/MS method is validated for the application in simultaneous quantification of ginsenoside Rg1,ginsenoside Rb1,ginsenoside Rd,ginsenoside Re and notoginsenoside R1 in plasma of rabbits,and proved to be adaptable for following pharmacokinetic studies.According to the pharmacokinetic parameters,five saponins can be divided into two clusters which present different pharmacokinetic characteristics.