lc-msms法测定大鼠血浆中pac-1的新型抗癌衍生物的浓度及其药物动力学研究（英文）
LC-MS/MS 法测定大鼠血浆中 PAC-1 的新型抗癌衍生物的浓度及其药物动力学研究（英文） 朱刚直 易勤 范志宏 马岳慧 王丹丹 王磊 程泽能 中南大学湘雅医学院附属海口医院 湖南泰新医药科技有限公司 深圳市湘雅生物医药研究院 中南大学湘雅药学院药物代谢与药动学研究实验室 中南大学生命科学与技术学院 摘 要： 本研究建立了一个新的、灵敏的液相色谱-串联质谱法用于测定大鼠血浆中 SM-1 的浓度。在简单的蛋白沉淀处理后, 用乙腈甲醇10 mM 乙酸铵溶液 (37.5:37.5:25, v/v/v) 作为流动相, 在 C18反相色谱柱 (50 mm×4.6 mm, 3.5m) 上分离 SM-1 和内标 (吉非替尼) 。质谱检测使用三重四级杆串联质谱, 电喷雾正离子模式、质谱多反应监测技术对待测物 SM-1 和内标物分别在质核比为 407.3203.4 和 447.3128.3 处进行检测。方法学验证的线性范围为 30至 6000 ng/m L, 批内和批间的精密度都小于 4.7%。平均回收率在 98.7%104.1%之间。在样品制备和分析程序中 SM-1 都是稳定的。测定结果均符合 FDA生物分析方法指导原则的要求。该方法已成功地应用于测定单次口服50、100、200 mg/kg 剂量后, 大鼠血浆中 SM-1 的浓度。关键词： 抗肿瘤药物; LC-MS/MS; 药代动力学; 大鼠; 验证; 作者简介：Lei Wang, E-mail:wangleivvl163.com;作者简介：Zeneng Cheng, chengzncsu.edu.cn收稿日期：2017-07-20基金：National Science and Technology Major Projects (Grant No.2012ZX09103101-051) Determination of a novel derivative of the PAC-1 anticancer agent in rat plasma by LC-MS/MS and its application to a pharmacokinetics studyGangzhi Zhu Qin Yi Zhihong Fan Yuehui Ma Dandan Wang Lei Wang Zeneng Cheng Haikou Affiliated Hospital of Xiangya Medical College of Central South University; Hunan Tai Xin Medical Science and Technology Ltd; Shenzhen Research Institute of Xiangya Biomedicine; Research Institute of Drug Metabolism and Pharmacokinetics, Xiangya School of pharmacy, Central South University; Abstract： A new and sensitive liquid chromatography-tandem mass spectrometry method was developed for the determination of SM-1 in rat plasma. After a simple protein precipitation, SM-1 and internal standard (gefitinib) were separated with gradient elution on a Waters XBridge C18 (50 mm×4.6 mm, 3.5 m) using acetonitrilemethanol10 mM ammonium acetate (37.5:37.5:25, v/v/v) as mobile phase. The triple quadruple mass spectrometer was set in positive electrospray ionization mode, multiple reaction monitoring was used for quantification. The precursors to produce ion transitions monitored for SM-1 and IS were m/z 407.3203.4 and 447.3128.3, respectively. The method validation was conducted over the curve range of 306000 ng/mL. The intra-and inter-day precisions were less than 4.7%, the average extraction recoveries ranged from 98.7% to 104.1% for each analyte. SM-1 was proved to be stable during sample storage preparation and analytical procedures. All the results met the acceptance criteria in accordance with the FDA guidance for bioanalytical method. Consequently, this method was successfully applied to determine SM-1 concentrations in rats after oral administrations at the doses of 200, 100 and even 50 mg/kg.Keyword： Anti-tumor drug; LC-MS/MS; Pharmacokinetics; Rat; Validation; Received： 2017-07-201. IntroductionThe high concentration of procaspase-3 in cell contributes to the development of some types of tumors1, such as lung cancer2, hepatoma3, melanoma4, breast cancer5, colon cancer6and so on.As the first small molecule activator of procaspase-3, procaspase-activating compound 1 (PAC-1) can trigger tumor cell apoptosis by activating procaspase-3 to caspase-3.The compound of N- (3-allyl-2-hydroxy benzene methylene-2- (4-) -benzyl homopiperazine-1) acethydrazide fumarate salt, also called SM-1 (Fig.1A) , is a novel derivative of the PAC-1 that changes the piperazine of PAC-1 with the homopiperazine according to the structure-activity relationship of PAC-19.Compared with the PAC-1, SM-1shows stronger capacity for activating procaspase-3, low toxicity to nerve cells, and higher bioavailability10,11.It may be a promising anti-cancer candidate12.Pharmacokinetic (PK) analyses are essential components of the drug discovery and development process.Particularly, in early clinical trials, it is of great importance to study PK to serve itself as an“auxiliary biomarker”, which is helpful for the selection of doses in further trials and promotion of clinical rational drug use.A reliable and sensitive bioanalytical method will be essential for the PK studies of chemical candidates by providing the accurate and reliable data of the concentration.Although an HPLC-UV method has been preliminarily built by our group to assess the absolute bioavailability of SM-1 in rats, the sensitivity of the method is still high.The lower limit of quantification (LLOQ) of the method is about 0.1g/m L9.It is not able to support the determination of samples from a lower dose group.In addition, the latest results of pharmacodynamics studies in animals have shown that the antitumor activity of SM-1 is stronger than expected.The dose administered in the PK and safety studies could be further lowered to reduce the toxicity.In above cases, a more sensitive method was necessary for the sample determinations of SM-1.Figure 1.Chemical structures of SM-1 (A) and gefitinib (B) .In this work, the LC-MS/MS methodwas initially developed and validated for the determination of SM-1in rat plasma.This sensitive method was applied to the PK studies of SM-1 in rat, and the PK properties of three doses of SM-1 were investigated.2. Experim
