Shi Mengge, Han Han, Yang Li, Wang Zhengtao, Chen Kaixian
The MOE Key Laboratory of Standardization of Chinese Medicines, Shanghai Key Laboratory of Compound Chinese Medicines, SATCM Key Laboratory of New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
J Ethnopharmacol. 2024 Jul 15;329:118136. doi: 10.1016/j.jep.2024.118136. Epub 2024 Apr 6.
Panax ginseng C.A. Meyer., a famous and valuable traditional Chinese medicine with thousand years of history for its healthcare and therapeutic effects. It is necessary and meaningful to study the pharmacokinetic behavior of ginsenosides in vivo as they are the most active components. Dried blood spots (DBS) are a mature and advanced blood collection method with meet the needs for the measurement of numerous analytes.
This study aimed to explore the feasibility on DBS in the metabolic profile analysis of complex herbal products.
An ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of ginsenosides. The preparation of DBS samples was conducted by spiking the whole blood with analytes to obtain 20 μL of blood spots on Whatman 903 collection card. A punched dish of 10 mm in diameter was extracted with 70 % methanol aqueous solution, digoxin was used as an internal standard. Target compounds were separated on a Waters T3 column (2.1 × 100 mm, 1.8 μm) with acetonitrile and water (0.1 % formic acid) at a flow rate of 0.4 mL/min.
The various ginsenosides showed good linearity in the range of 1-2000 ng/mL. The extraction recoveries and matrix effects of the target analytes were above 82.2%. The intra- and inter-batch accuracy and precision were within the limits of ≤15% for all tested concentrations. Moreover, the collected dried blood spot samples could be stably stored at room temperature for 14 days and 4 °C for 1 month without being affected. And it is delightful that the DBS-based analysis is compatible or even superior to the conventional protein precipitation in terms of sensitivity, linearity, and stability. In particular, the target analytes are stable in the DBS sampling under normal storing condition and the sensitivity for some trace metabolites of ginsenosides, such as 20(S)-Rg3, 20(R)-Rg3, F1, Rk1, Rg5, etc. increases 3-4 folds as evaluated by LLOQ.
The established method was successfully applied to pharmacokinetic studies of ginseng extract in mice, this suggests a more feasible strategy for pharmacokinetic study of traditional and natural medicines both in animal tests and clinical trials.
人参（Panax ginseng C.A. Meyer.）是一种著名且珍贵的传统中药，具有数千年的保健和治疗历史。人参皂苷作为其最具活性的成分，研究其体内药代动力学行为具有必要性和重要意义。干血斑（DBS）是一种成熟且先进的采血方法，能够满足多种分析物的检测需求。
本研究旨在探讨DBS用于复杂草药产品代谢谱分析的可行性。
建立并验证了一种超高效液相色谱 - 质谱联用（UHPLC - MS/MS）法用于测定人参皂苷。DBS样品的制备是通过向全血中加入分析物，在Whatman 903采集卡上获得20 μL血斑。用直径10 mm的冲孔圆盘，以70%甲醇水溶液进行提取，地高辛用作内标。目标化合物在Waters T3柱（2.1×100 mm，1.8 μm）上分离，流动相为乙腈和水（含0.1%甲酸），流速为0.4 mL/min。
各种人参皂苷在1 - 2000 ng/mL范围内呈现良好的线性关系。目标分析物的提取回收率和基质效应均高于82.2%。所有测试浓度下的批内和批间准确度和精密度均在≤15%的范围内。此外，采集的干血斑样品在室温下可稳定保存14天，在4℃下可稳定保存1个月不受影响。令人欣喜的是，基于DBS的分析在灵敏度、线性和稳定性方面与传统的蛋白沉淀法相当甚至更优。特别是，目标分析物在正常储存条件下的DBS采样中稳定，并且通过定量下限（LLOQ）评估，人参皂苷一些痕量代谢物如20(S)-Rg3、20(R)-Rg3、F1、Rk1、Rg5等的灵敏度提高了3 - 4倍。
所建立的方法成功应用于小鼠人参提取物的药代动力学研究，这为传统天然药物在动物试验和临床试验中的药代动力学研究提供了一种更可行的策略。
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