石杉碱甲缓释微球体内药代动力学研究

发布时间：2018-05-14 11:18

  本文选题：石杉碱甲 + 缓释微球　； 参考：《吉林大学》2017年硕士论文

【摘要】：目的:建立快速、专属、准确、灵敏度高的定量分析石杉碱甲的LC-MS/MS方法;观察注射用石杉碱甲缓释微球的血药浓度经时变化过程,计算人体药代动力学参数,评价石杉碱甲缓释微球在健康人体的药代动力学特征及缓释效果;探索石杉碱甲的代谢途径,寻找可能的代谢产物,为石杉碱甲新药研发和临床使用后毒副作用的分析提供理论基础。研究方法:本研究采用液液萃取的方法将石杉碱甲从血浆中提取出来,进入LC-MS/MS系统进行定量分析。以10 m M醋酸铵溶液为水相,甲醇为有机相,经Ascentis-C18柱(10 cm×2.1 mm,5mm)梯度洗脱将待测物石杉碱甲分离后,进入质谱检测系统。选用电喷雾离子化源(ESI),多重反应检测(MRM)的正离子检测模式,定量分析的线性范围是10-3000 pg/m L,用于定量分析的离子对为m/z 243.1?m/z 210.1(石杉碱甲)和m/z 285.2?m/z 193.1(内标地西泮)。试验以健康成年志愿者为对象,采用随机、双盲、安慰剂对照的试验设计,单次给予志愿者肌肉注射0.75 mg、1.5 mg、3.0 mg、4.5 mg和6.0 mg五个剂量的石杉碱甲,用以上所述LC-MS/MS定量方法对各时间点人血浆中石杉碱甲的浓度进行测定,用DAS 3.0软件计算药代动力学参数,根据数据评价其药代动力学特征;收集受试者服用石杉碱甲后的尿液样本,经Oasis HLB固相萃取柱处理后进行质谱分析,结合石杉碱甲的结构和采集得到的色谱、一级质谱、二级质谱数据,推测其在人体内可能发生的代谢反应。结果:本研究建立的人血浆样本中石杉碱甲的LC-MS/MS定量分析方法专属性强、准确度好、灵敏度高,适用于注射用石杉碱甲缓释微球的人体药代动力学研究。达到了很高的灵敏度(LLOQ为10 pg/m L),样品的分析时间为8.5 min,准确度在85.33%到112.33%之间,日内精密度小于9.73%,日间精密度小于9.63%,提取回收率高,基质效应对石杉碱甲和内标地西泮的测定无影响。稳定性研究结果表明:石杉碱甲血浆样品-80°C冰冻放置360天、-80°C反复冻融三次、样品处理前室温放置4 h及提取后进样小瓶中放置4 h均稳定。本研究发现石杉碱甲缓释微球在人体内的AUC0-t、AUC0-∞及Cmax与给药剂量呈线性相关,消除速率常数与剂量没有依赖关系,在0.75 mg~6.0 mg给药剂量范围内,石杉碱甲具有线性药代动力学特征。其达峰时间和半衰期明显延长,实现了缓释的目的。结合石杉碱甲代谢实验所采集的数据,推测其可能发生了去饱和、氧化、脱氨基同时去饱和的三种反应。
[Abstract]:Objective: to establish a rapid, exclusive, accurate and sensitive LC-MS/MS method for quantitative analysis of Huperzine A, observe the time-varying process of blood drug concentration of Huperzine A sustained release microspheres for injection, and calculate the pharmacokinetic parameters of human body. To evaluate the pharmacokinetic characteristics and sustained release effect of Huperzine A sustained release microspheres in healthy volunteers, to explore the metabolic pathway of Huperzine A, and to search for possible metabolites. To provide a theoretical basis for the development of new drug Huperzine A and the analysis of side effects after clinical use. Methods: Huperzine A was extracted from plasma by liquid-liquid extraction and entered LC-MS/MS system for quantitative analysis. Using 10 mm ammonium acetate solution as water phase and methanol as organic phase, Huperzine A was separated by gradient elution with Ascentis-C18 column (10 cm 脳 2. 1 mm ~ 5 mm), and then entered the mass spectrometric detection system. The positive ion detection model of electrospray ionization source (ESI) was used. The linear range of quantitative analysis was 10-3000 pg/m / L, and the ion pairs used for quantitative analysis were m / z 243.1?m/z 210.1 (Huperzine A) and m / z 285.2?m/z 193.1 (internal standard diazepam). A randomized, double-blind, placebo-controlled trial was conducted in healthy adult volunteers. The volunteers were given a single intramuscular injection of 0.75 mg / L 1.5 mg / g 3.0 mg / L Huperzine A (4.5 mg and 6.0 mg / kg) respectively. The concentration of Huperzine A in human plasma was determined by the LC-MS/MS quantitative method mentioned above. The pharmacokinetic parameters were calculated by DAS 3.0 software, and the pharmacokinetic characteristics were evaluated according to the data. The urine samples were collected and analyzed by Oasis HLB solid phase extraction column. The structure of Huperzine A was combined with the collected data of chromatography, primary mass spectrometry and secondary mass spectrometry. The possible metabolic response in the human body is inferred. Results: the LC-MS/MS quantitative analysis method of Huperzine A in human plasma samples established in this study has strong specificity, good accuracy and high sensitivity. It is suitable for the pharmacokinetic study of sustained release microspheres of Huperzine A for injection. The sensitivity is 10 pg/m / L ~ (-1), the analytical time is 8.5 min, the accuracy is between 85.33% and 112.33%, the intra-day precision is less than 9.73 and the daytime precision is less than 9.63, and the recovery rate is high. The matrix effect had no effect on the determination of Huperzine A and diazepam. The results of stability study showed that the plasma samples of Huperzine A were frozen at -80 掳C for 360 days and thawed repeatedly for three times. The samples were stored at room temperature for 4 hours before treatment and in the sample bottles after extraction. In this study, we found that the sustained release microspheres of Huperzine A in human body showed linear correlation with the dosage of AUC0- 鈭，

本文编号：1887645