背景:人参C.A.Meyer.,一种著名而宝贵的中药,具有数千年的医疗保健和治疗作用。人参皂苷是最具有活性的成分,因此研究人参皂苷在体内的药动学行为是必要和有意义的。干血斑点(DBS)是一种成熟先进的血液采集方法,可满足多种分析物的测量需求。
目的:本研究旨在探讨DBS在复杂草药产品代谢谱分析中的可行性。
方法:建立了超高效液相色谱-质谱(UHPLC-MS/MS)方法,并对其进行了验证,以测定人参皂苷。DBS样品的制备通过用分析物掺入全血来进行,以在Whatman903收集卡上获得20μL血斑。用70%甲醇水溶液提取直径10mm的冲孔盘,地高辛被用作内标。目标化合物在WatersT3色谱柱(2.1×100mm,1.8μm)与乙腈和水(0.1%甲酸),流速为0.4mL/min。
结果:各种人参皂苷在1-2000ng/mL范围内呈良好的线性关系。目标分析物的提取回收率和基体效应均在82.2%以上。对于所有测试浓度,批次内和批次间的准确性和精密度均在≤15%的范围内。此外,采集的干血斑样品可以在室温下稳定储存14天,在4°C下稳定储存1个月,不受影响。令人愉快的是,基于DBS的分析在灵敏度方面兼容甚至优于常规蛋白沉淀,线性度和稳定性。特别是,目标分析物在正常保存条件下在DBS采样中是稳定的,对人参皂苷某些微量代谢产物的敏感性,例如20(S)-Rg3、20(R)-Rg3、F1、Rk1、Rg5等。根据LLOQ评估,增加3-4倍。
结论:所建立的方法成功应用于人参提取物在小鼠体内的药代动力学研究。这为传统药物和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更可行的策略。
BACKGROUND: 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.
OBJECTIVE: This study aimed to explore the feasibility on DBS in the metabolic profile analysis of complex herbal products.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.