School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China; Beijing Key Lab of TCM Collateral Disease Theory Research, Beijing 100069, China.
School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China; Beijing Key Lab of TCM Collateral Disease Theory Research, Beijing 100069, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Aug 15;1092:199-206. doi: 10.1016/j.jchromb.2018.06.016. Epub 2018 Jun 7.
A specific, sensitive and rapid ultra high performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method was developed and validated for simultaneous determination of six major bioactive constituents in Rhizoma Panacis Japonici (RPJ), including oleanolic acid-type chikusetsusaponin V, IV, hemsgiganoside B, damarane-type ginsenoside Rb1, Rg1 and Re in rat plasma, using estazolam as the internal standard (IS). Plasma samples were pretreated with methanol/acetonitrile (1:1, V/V) for protein precipitation. Chromatographic separation was performed on an Agilent Eclipse Plus C column, using a gradient mobile phase consisting of methanol and 0.1% formic acid aqueous solution. A tandem mass spectrometric detection with an electrospray ionization (ESI) interface was conducted via multiple reaction monitoring (MRM) under positive ionization mode. For all the six analytes of interest, the calibration curves were linear in the concentration range of 2.00-500 ng/mL with r ≥ 0.9956. The intra- and inter-day precisions (in terms of relative standard deviation, RSD) were all below 10.2% and the accuracies (in terms of relative error, RE) were within -5.0% to 6.3% for all six analytes. Extraction recovery, matrix effect and stability data all met the acceptance criteria of FDA guideline for bioanalytical method validation. The developed method was applied to the pharmacokinetic study in rat. After oral administration of the total saponins from RPJ, six analytes were quickly absorbed into the blood and presented the phenomenon of double peaks. Among the six analytes, ginsenoside Rb1 showed slowest elimination from plasma with a t of 16.00 h, while that of the others were between 1.72 and 5.62 h. In conclusion, the developed method was successfully used to simultaneously analyze major oleanolic acid-type and damarane-type saponins of RPJ in rat plasma after oral administration.
建立并验证了一种专用于同时测定大鼠血浆中六种主要生物活性成分的超高效液相色谱-串联质谱（UHPLC-MS/MS）方法，这六种成分包括齐墩果酸型柴胡皂苷 V、IV、次黄嘌呤苷 B、达玛烷型人参皂苷 Rb1、Rg1 和 Re，以依唑仑为内标（IS）。用甲醇/乙腈（1:1，V/V）沉淀蛋白预处理血浆样品。采用 Agilent Eclipse Plus C 柱，以甲醇和 0.1%甲酸水溶液为梯度流动相进行色谱分离。采用电喷雾电离（ESI）接口，以正离子化模式进行多反应监测（MRM）串联质谱检测。对于所有六种感兴趣的分析物，校准曲线在 2.00-500ng/mL 浓度范围内呈线性，相关系数（r）≥0.9956。所有六种分析物的日内和日间精密度（以相对标准偏差，RSD 表示）均低于 10.2%，准确度（以相对误差，RE 表示）均在-5.0%至 6.3%范围内。提取回收率、基质效应和稳定性数据均符合 FDA 生物分析方法验证指南的验收标准。所建立的方法应用于大鼠的药代动力学研究。口服 RPJ 总皂苷后，六种分析物迅速被血液吸收并呈现双峰现象。在这六种分析物中，人参皂苷 Rb1 从血浆中的消除最慢，t1/2 为 16.00 h，而其他分析物的 t1/2 在 1.72-5.62 h 之间。总之，该方法成功地用于口服给药后大鼠血浆中 RPJ 的主要齐墩果酸型和达玛烷型皂苷的同时分析。
