刘胜兰 唐智 陈磊 吴素芬 周玲 廖音娟 张杰

中圖分类号 R917;R593.22 文献标志码 A 文章编号 1001-0408（2021）18-2248-06

DOI 10.6039/j.issn.1001-0408.2021.18.12

摘 要 目的：建立测定人血浆中20（S）-原人参二醇（简称为“PPD”）浓度的方法。方法：血浆样品经乙腈沉淀蛋白后，以非那雄胺为内标，采用超高效液相色谱-串联质谱法测定。色谱柱为Waters ACQUITY UPLC HSS T3，流动相为5 mmol/L碳酸氢铵溶液-乙腈（梯度洗脱），流速为0.4 mL/min，柱温为40 ℃，进样量为10 μL;离子源为电喷雾离子源，以多反应监测模式（MRM）进行负离子扫描，用于定量分析的离子对分别为m/z 459.40→375.20（PPD）、m/z 371.30→315.30（内标）。同时将该法用于临床样品的测定。结果：PPD检测质量浓度的线性范围为0.25～30.00 ng/mL（r=0.999 2），定量下限为0.25 ng/mL;批内、批间RSD均小于10%，相对误差（RE）为-14.61%～12.69%;提取方法和基质效应均不影响PPD的定量测定。人参皂苷CK片100 mg组、人参皂苷CK片200 mg组、人参皂苷CK片300 mg组类风湿性关节炎患者口服相应药物第84天时的平均cmax分别为18.06、30.03、27.00 ng/mL，中位tmax分别为12.0、6.0、12.0 h，平均AUC0-t分别为622.52、668.15、1 155.97 ng·h/mL。结论：本研究成功建立了测定人血浆中PPD浓度的方法，且方法灵敏、准确、稳定，操作简便，血浆用量少，可用于临床样本的定量测定。

关键词 20（S）-原人参二醇;超高效液相色谱-串联质谱法;蛋白沉淀法;血药浓度

Determination of 20（S）-protopanaxadiol Concentration in Human Plasma by UPLC-MS/MS

LIU Shenglan1，TANG Zhi2，CHEN Lei3，WU Sufen3，ZHOU Ling2，LIAO Yinjuan1，ZHANG Jie4（1. Dept. of Pharmacy， Changsha Hospital of Hunan Normal University/the Fourth Hospital of Changsha， Changsha  410006， China; 2. Hunan Key Laboratory for Bioanalysis of Complex Matrix Samples， Changsha Duxact Biotech Co.， Ltd.， Changsha 410205， China;  3. Central Research Institute， Zhejiang Hisun Pharmaceutical Co.， Ltd.， Zhejiang Taizhou 318000， China; 4. Dept. of Pharmacy， the Third Xiangya Hospital of Central South University， Changsha 410013， China）

ABSTRACT   OBJECTIVE： To establish the method for the determination of 20（S）-protopanaxadiol （PPD） concentration in human plasma. METHODS： Plasma samples were precipitated with acetonitrile and determined by UPLC-MS/MS， using finandrogen as internal standard. The determination was performed on Waters ACQUITY UPLC HSS T3 column with mobile phase consisted of 5 mmol/L ammonium bicarbonate aqueous solution-acetonitrile （gradient elution） at the flow rate of 0.4 mL/min. The column temperature was set at 40 ℃， and sample size was 10 μL. The ion source was electrospray ion source， and negative ion scanning was carried out with multiple reaction monitoring mode. The ion pairs used for quantitative analysis were m/z 459.40→375.20 （PPD） and m/z 371.30→315.30 （internal standard）. At the same time， the method was applied to the determination of clinical samples. RESULTS： The linear range of PPD was 0.25-30.00 ng/mL （r=0.999 2）， and the limit of quantitation was 0.25 ng/mL. RSDs of intra-batch and inter-batch were all lower than 10%， and relative errors （RE） were -14.61%-12.69%. Extraction method and matrix effect did not affect the quantitative determination of PPD. In ginsenoside CK 100 mg group， ginsenoside CK 200 mg group and ginsenoside CK 300 mg group， mean cmax of patients with rheumatoid arthritis after oral administration of corresponding drugs were 18.06， 30.03， 27.00 ng/mL; median tmax were 12.0， 6.0， 12.0 h; mean AUC0-t were 622.52， 668.15，     1 155.97 ng·h/mL. CONCLUTIONS： The method for the determination of PPD concentration in human plasma is successfully established. The method is sensitive， accurate， stable， easy to operate and less plasma consumption. It can be used for the quantitative determination of clinical samples.

KEYWORDS   20（S）-protopanaxadiol; UPLC-MS/MS; Protein

precipitation method; Plasma concentration

20（S）-原人参二醇[20（S）-PPD，以下简称“PPD”]为二醇型人参皂苷的苷元，是稀有人参二醇皂苷Rd、F2、Rg3、CK、Rh2的主要活性代谢产物[1]，具有抗癌、抗抑郁、止血、激活氯离子通道和抑制钠离子通道去极化等诸多药理活性[2-5]。有报道称，人参皂苷CK在体内和体外均可被胃酸和/或肠道微生物进一步分解并转化为PPD，且PPD为二醇型人参皂苷活性最强的代谢产物[6]。对于人参皂苷CK等二醇型人参皂苷的临床药动学研究而言，准确、快速测定生物样品中PPD的浓度极为重要。……