本文選題：石杉堿甲 + 緩釋微球��； 參考：《吉林大學》2017年碩士論文

【摘要】：目的:建立快速、專屬、準確、靈敏度高的定量分析石杉堿甲的LC-MS/MS方法;觀察注射用石杉堿甲緩釋微球的血藥濃度經(jīng)時變化過程,計算人體藥代動力學參數(shù),評價石杉堿甲緩釋微球在健康人體的藥代動力學特征及緩釋效果;探索石杉堿甲的代謝途徑,尋找可能的代謝產(chǎn)物,為石杉堿甲新藥研發(fā)和臨床使用后毒副作用的分析提供理論基礎。研究方法:本研究采用液液萃取的方法將石杉堿甲從血漿中提取出來,進入LC-MS/MS系統(tǒng)進行定量分析。以10 m M醋酸銨溶液為水相,甲醇為有機相,經(jīng)Ascentis-C18柱(10 cm×2.1 mm,5mm)梯度洗脫將待測物石杉堿甲分離后,進入質(zhì)譜檢測系統(tǒng)。選用電噴霧離子化源(ESI),多重反應檢測(MRM)的正離子檢測模式,定量分析的線性范圍是10-3000 pg/m L,用于定量分析的離子對為m/z 243.1?m/z 210.1(石杉堿甲)和m/z 285.2?m/z 193.1(內(nèi)標地西泮)。試驗以健康成年志愿者為對象,采用隨機、雙盲、安慰劑對照的試驗設計,單次給予志愿者肌肉注射0.75 mg、1.5 mg、3.0 mg、4.5 mg和6.0 mg五個劑量的石杉堿甲,用以上所述LC-MS/MS定量方法對各時間點人血漿中石杉堿甲的濃度進行測定,用DAS 3.0軟件計算藥代動力學參數(shù),根據(jù)數(shù)據(jù)評價其藥代動力學特征;收集受試者服用石杉堿甲后的尿液樣本,經(jīng)Oasis HLB固相萃取柱處理后進行質(zhì)譜分析,結(jié)合石杉堿甲的結(jié)構(gòu)和采集得到的色譜、一級質(zhì)譜、二級質(zhì)譜數(shù)據(jù),推測其在人體內(nèi)可能發(fā)生的代謝反應。結(jié)果:本研究建立的人血漿樣本中石杉堿甲的LC-MS/MS定量分析方法專屬性強、準確度好、靈敏度高,適用于注射用石杉堿甲緩釋微球的人體藥代動力學研究。達到了很高的靈敏度(LLOQ為10 pg/m L),樣品的分析時間為8.5 min,準確度在85.33%到112.33%之間,日內(nèi)精密度小于9.73%,日間精密度小于9.63%,提取回收率高,基質(zhì)效應對石杉堿甲和內(nèi)標地西泮的測定無影響。穩(wěn)定性研究結(jié)果表明:石杉堿甲血漿樣品-80°C冰凍放置360天、-80°C反復凍融三次、樣品處理前室溫放置4 h及提取后進樣小瓶中放置4 h均穩(wěn)定。本研究發(fā)現(xiàn)石杉堿甲緩釋微球在人體內(nèi)的AUC0-t、AUC0-∞及Cmax與給藥劑量呈線性相關(guān),消除速率常數(shù)與劑量沒有依賴關(guān)系,在0.75 mg~6.0 mg給藥劑量范圍內(nèi),石杉堿甲具有線性藥代動力學特征。其達峰時間和半衰期明顯延長,實現(xiàn)了緩釋的目的。結(jié)合石杉堿甲代謝實驗所采集的數(shù)據(jù),推測其可能發(fā)生了去飽和、氧化、脫氨基同時去飽和的三種反應。
[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 擄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，

本文編號：1887645