Li Pengfei, Liu Shanshan, Zhuang Hongyan, Niu Mengxi, Pan Fei, Wang Nan, Sha Sha, Wang Qian, Wang Jing
Beijing Anding Hospital, Capital Medical University/National Clinical Research Center for Mental Disorders/National Center for Mental Disorders/Beijing Key Laboratory of Mental Disorders, No. 5 Ankang Hutong, Dewai, Xicheng District, Beijing, 100088, China.
Advanced Innovation Center for Human Brain Protection, Capital Medical University, No.10 Xitoutiao, Youanmenwai, Beijing, 100069, China.
Sci Rep. 2025 May 13;15(1):16640. doi: 10.1038/s41598-025-01432-1.
20(S)-Protopanaxadiol (PPD) is a saponin derivative of ginsenoside, with more potent biological and pharmacological activities than Rg3 and Rh2. The lack of ionizable centers leads to low mass spectrometry reactions and internal cleavage of three hydroxyl groups, making it challenging to establish highly sensitive PPD mass spectrometry methods. The aim of this study is to establish and validate a quantitative detection method for PPD in multiple matrices using mass spectrometry. The methods used Rh2 as the internal standard and organic solvent liquid-liquid extraction under alkaline conditions for biological sample pretreatment. Isometric separation was achieved through methanol, acetonitrile, and a 10 mmol/L solution of acetic acid (45:45:10, v/v/v) at a flow rate of 0.4 mL/min. Finally, perform mass spectrometry quantification. Comprehensive method validation was conducted on rat plasma samples, and partial method validations were performed on three types of rat tissues (adipose tissue, smooth muscle, and skeletal muscle), bile, urine, fecal samples, and dog plasma samples. The results were in accordance with the requirements of NMPA for bioanalytical method validation, ensuring the accuracy and reliability of our analytical measurements. This study employed a conventional liquid-liquid extraction sample pretreatment scheme, utilizing multiple biological matrices commonly found in a single treatment protocol and liquid chromatography-tandem mass spectrometry detection parameters. The consistency of processing and detection across diverse samples eliminated the need for methodological changes, providing exceptional convenience. Up to 90% of the organic phase and a 50 mm short chromatographic column achieved rapid and effective separation of PPD. A key aspect of our work is the use of a "programmed injection" technique, which significantly reduces the analysis time from 4.2 min during method exploration to 2.4 min. These methods have achieved a relatively low quantification limit of 2.5 ng/mL. The methods established were successfully applied to the kinetic process of PPD in rats, and the pharmacokinetic characteristics of PPD in dogs were studied for the first time.
20(S)-原人参二醇（PPD）是人参皂苷的一种皂苷衍生物，其生物和药理活性比Rg3和Rh2更强。由于缺乏可电离中心，导致质谱反应较低，且三个羟基发生内部裂解，这使得建立高灵敏度的PPD质谱方法具有挑战性。本研究的目的是建立并验证一种使用质谱法对多种基质中PPD进行定量检测的方法。该方法以Rh2为内标，在碱性条件下采用有机溶剂液-液萃取法对生物样品进行预处理。通过甲醇、乙腈和10 mmol/L乙酸溶液（45:45:10，v/v/v）以0.4 mL/min的流速实现等度分离。最后进行质谱定量分析。对大鼠血浆样品进行了全面的方法验证，并对三种大鼠组织（脂肪组织、平滑肌和骨骼肌）、胆汁、尿液、粪便样品和犬血浆样品进行了部分方法验证。结果符合国家药品监督管理局对生物分析方法验证的要求，确保了我们分析测量的准确性和可靠性。本研究采用了传统的液-液萃取样品预处理方案，在单一处理方案中利用了多种常见的生物基质以及液相色谱-串联质谱检测参数。不同样品处理和检测的一致性消除了方法变更的需要，提供了极大的便利。高达90%的有机相和一根50 mm的短色谱柱实现了PPD的快速有效分离。我们工作的一个关键方面是使用了“程序进样”技术，该技术将分析时间从方法探索期间的4.2分钟显著缩短至2.4分钟。这些方法实现了相对较低的定量限，为2.5 ng/mL。所建立的方法成功应用于PPD在大鼠体内的动力学过程，并首次研究了PPD在犬体内的药代动力学特征。
