医药学论文:高效液相色谱-质谱法测定人血浆中甘草次酸浓度及人体药代动力学研究
【摘要】 目的 建立人血浆中甘草次酸的高效液相色谱-质谱(LCMS)测定法,并用于健康志愿者的药代动力学研究。方法 20例健康志愿者一次口服甘草酸二铵胶囊150mg,采集肘静脉血并分离血浆,血样经乙酸乙酯提取后,以熊果酸为内标,进行LCMS分析。色谱柱:Venusil XBPC18柱(5μm, ID4.6×150mm),流动相:甲醇(5mmol/L乙酸铵)-水(85∶15,V/V),梯度洗脱;流速:0.8mL/min,柱温25℃,进样10μL。甘草次酸和内标熊果酸的检测离子和裂解电压分别为m/z 469.5、m/z 455.6和200V、100V。根据不同时间甘草次酸血浓度计算其药代动力学参数。结果 在0.5~200ng/mL范围内,甘草次酸与内标的峰面积比值与浓度线性关系良好,相关系数为0.9974,定量限为0.5ng/mL,提取回收率为76.0%~80.0%,日内、日间RSD均小于12%。甘草次酸口服给药Cmax为(95.57±43.06)ng/mL,Tmax为(10.95±1.32)h。结论 高效液相色谱-质谱测定法灵敏、准确、简便,适用于血浆甘草次酸的浓度测定及其人体药代动力学研究。
【关键词】 甘草酸二铵 甘草次酸 高效液相色谱-质谱联用法 药代动力学
glycyrrhetic acid in human plasma
ZHAO Wenjing, WANG Benjie, WEI Chunmin, YUAN Guiyan, BU Fanlong, GUO Ruichen
(Institute of Clinical Pharmacology, Qilu Hospital of Shandong University, Jinan 250012, China)
To develop a LCMS method for the determination of glycyrrhetic acid in human plasma and for evaluation of its pharmacokinetic characteristics.Methods Glycyrrhetic acid in plasma was extracted with ethyl acetate, separated on a C18 column with a mobile phase of methanol(5mmol/L ammonium acetate)water(85∶15,V/V) and analyzed by the LCMS method with ursolic acid(UA) as the internal standard.The target ions were m/z 469.5 for glycyrrhetic acid and m/z 455.6 for internal standard. The fragmentor voltages were 200V for glycyrrhetic acid and 100V for the internal standard. Results The calibration curve was linear over the range of 0.5 to 200ng/mL (r=0.9974). The quantitative limit for glycyrrhetic acid in plasma was 0.5ng/mL, the recovery rate was 76.0% to 80.0%, and the interday and intraday derivations (RSD) were less than 12%. The Cmax and Tmax of glycyrrhetic acid were (95.57±43.06)ng/mL and (10.95±1.32)h after oral administration. Conclusion The assay proved to be sensitive,accurate and convenient and can be applied in the determination of glycyrrhetic acid in human plasma and its pharmacokinetic study.
Key words: Diammoniu glycyrrhizinate; Glycyrrhetic acid; High performance liquid chromatographymass spectrometry; Pharmacokinetics
甘草次酸(glycyrrhetic acid, GA)具有较强的抗病毒、抗炎、抗过敏、抗氧自由基、解毒、防癌、抗癌、免疫调节作用[13]。甘草酸盐或甘草次酸血浓度测定方法已有文献报道[46],但灵敏度低,重现性差,不适合人体甘草酸治疗剂量的药代动力学研究。国外有采用HPLCMS/MS法测定口服给药血浆中甘草次酸浓度的报道[7],但HPLCMS/MS普及率低,且费用较高。本研究采用HPLCMS方法测定甘草二酸铵口服给药人血浆中甘草次酸浓度,旨在建立简单、可靠、灵敏的甘草次酸分析测定方法,并用于甘草二酸铵口服给药的人体药代动力学研究。
1 材料与方法
1.1 仪器与药品 Agilent 1100 Series LC/MSD高效液相色谱/质谱仪;1313A型自动进样器(美国安捷伦科学技术公司);W80A型旋涡混合器(上海精科实业有限公司);ABOTT高速离心机(美国雅培公司);PK514BP超声清洗器(德国BANDEl);梅特勒-托利多 Al 104电子天平(瑞士梅特勒公司);BHWIV型电热恒温水温箱(北京医疗设备厂)。
甘草酸二铵胶囊由济南利民制药有限责任公司生产,50mg/粒,批号070617。甘草次酸对照品由中国固体制剂制造技术国家工程研究中心提供,批号1063050327。熊果酸对照品由Sigma公司提供,批号042k1240,纯度90%。甲醇、乙酸乙酯,色谱纯,系美国J.T.Baker公司产品。乙酸铵,分析纯,系山东省化工研究院产品。
1.2 受试者选择 20名男性健康受试者,(24.55±1.36)岁,体重(64.95±5.46)kg;经体检证明,肝、肾功能正常,无急、慢性疾病及家族遗传病史,心电图正常,精神状态良好。试验前2周内无用药史,3月内未参加其他新药临床试验。志愿者试验前2周及试验期内禁烟酒,试验期间,统一饮食,试验前签署知情同意书。试验方案经山东大学齐鲁医院伦理委员会批准。
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