【作者】 刘丹；

【导师】 陈笑艳； 钟大放；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2006， 博士

【摘要】 液相色谱-串联质谱（LC/MS/MS）法因其具有选择性好、灵敏度高和测试速度快的优势,近几年来已迅速成为生物样品分析和药物动力学研究的主要方法。本文利用液相色谱-串联质谱技术,建立了4种灵敏、快速的定量方法,分别用于血浆中阿德福韦、O-去甲右美沙芬、佐米曲普坦和厄多司坦的测定。所建方法的灵敏度均高于已有方法,并已被成功应用于药物动力学研究。 一、液相色谱-串联质谱法测定血浆中的阿德福韦 目的:建立灵敏、快速的液相色谱-串联质谱法测定人血浆中阿德福韦的浓度。方法:血浆样品经沉淀蛋白后,以甲醇-水-甲酸（20:80:0.25,v/v/v）作为流动相,采用Diamonsil-C₁₈色谱柱分离,通过电喷雾离子化串联质谱,以选择反应监测（SRM）方式进行正离子检测。用于定量分析的离子反应分别为m/z 274.1→m/z（162.1+226.0）（阿德福韦）和m/z 288.1→m/z176.1（内标,PMPA）。该方法的线性范围为0.25～100ng/mL,浓度定量下限为0.25 ng/mL。结果:将此方法应用于一类新药阿德福韦第匹福酯的Ⅰ期临床药物动力学研究,为设计合理给药方案和临床安全有效用药提供理论依据。1.考察空腹口服低、中、高三个单剂量后的药物动力学,获得该药在人体内的药物动力学特征:符合二室模型类型、具有线性动力学特征。2.考察餐后口服阿德福韦第匹福酯的药物动力学,与同剂量空腹口服药物的动力学参数进行比较,确定食物不改变该药物的动力学特征。3.考察多剂量给药后阿德福韦在人体内的药物动力学,与同剂量单次口服给药的动力学参数进行比较,确定药物在体内无蓄积。结论:该方法灵敏度高,实用性强,适用于临床药物动力学研究。 二、液相色谱-串联质谱法测定血浆中的O-去甲右美沙芬 目的:建立快速、灵敏的液相色谱-串联质谱法直接测定人血浆中的O-去甲右美沙芬,用于受试者口服氢溴酸右美沙芬后的生物等效性研究。方法:血浆样品经液-液萃取后,以甲醇-水-甲酸（70:30:1,v/v/v）作为流动相,采用Zorbax Extend C₁₈色谱柱分离,通过电喷雾离子化串联质谱,以选择反应监测（SRM）方式进行正离子检测。用于定量分析的离子反应分别为m/z 258→m/z 157（O-去甲右美沙芬）和m/z更多还原

【Abstract】 Nowadays liquid chromatography-tandem mass spectrometry （LC/MS/MS）, due to its high sensitivity and selectivity, has become a valuable technique in the determination of biological samples and in the pharmacokinetic studies. Four rapid and sensitive methods for quantitative analyses of adefovir, dextrorphan, zolmitriptan and erdosteine in plasma were developed and validated by liquid chromatography-tandem mass spectrometry in this thesis. The methods have been successfully applied to pharmacokinetic studies.1. Determination of adefovir in plasma by LC/MS/MSA sensitive and selective liquid chromatography-tandem mass spectrometric （LC/MS/MS） method for the determination of adefovir in human plasma was developed. After a simple protein precipitation, the post-treatment samples were analyzed on a Diamonsil-C₁₈ column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of methanol-water-formic acid （20: 80: 0.25, v/v/v）. Selected reaction monitoring （SRM） using the precursor → product ion combination of m/z 274.1 → m/z （162.1+226.0） and m/z 288.1 → m/z 176.1 was used to quantify adefovir and PMPA, respectively. The linear calibration curves were obtained in the concentration range of 0.25-100 ng/mL for adefovir. The method has a lower limit of quantification （LLOQ） of 0.25 ng/mL for adefovir.The method was applied to a Phase I clinical trial of adefovir dipivoxil. After oral administration of increasing （low, medium, high） dose and multidose of adefovir dipivoxil, the plasma concentrations of adefovir were monitored by the developed sensitive and fast LC/MS/MS method. Mean plasma concentration-time curves of adefovir were best fitted to two-compartment models. The absorption of the drug was not significantly influenced by concomitant food intake.2. Direct determination of dextrorphan in human plasma by LC/MS/MSA sensitive and selective LC/MS/MS method for direct determination of dextrorphan in human plasma was developed and was used to study the pharmacokinetics of dextrorphan. After a single dose oral administration of dextromethorphan hydrobromide 60 mg to 18 healthy Chinese male volunteers, the plasma concentration of dextrorphan, an active metabolite of dextromethorphan, was determined. Dextrorphan and internal standard chlorpheniramine were extracted from plasma using liquid-liquid extraction, then separated on a Zorbax Extend Cig column. The mobile phase consisted of methanol-water-formic acid （70:30:1）, at a flow-rate of 0.5 mL/min. A Finnigan TSQ tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring （SRM） using the precursor to product ion combinations of m/z 258 to 157 and m/z 275 to 230 was performed to quantify dextrorphan. The pharmacokinetic parameters of dextrorphan were calculated by non-compartment model statistics. The linear calibration curves were obtained in the concentration range of 0.2-80.0 ng/mL. Each plasma sample was chromatographed within 3.0 min. The intra- and inter-day relative standard deviation （RSD） across three validation runs over the entire concentration range was less than 8%. Accuracy determined at three concentrations （0.5, 6.0 and 70 ng/mL for dextrorphan） ranged from 98.8% to 100.6%. The method was applied to study the pharmacokinetics of 18 healthy volunteers after oral administration of 60 mg of dextromethorphan hydrobromide test formulation or reference formulation. Mean peak plasma levels （Cmax） of 13.5 ± 7.6 ng/mL and 13.6 ± 7.7 ng/mL and rmax of 2.1 ± 0.7 h and 2.0 ± 0.7 h were observed. The mean tin value of 3.8 ± 1.8 h and 3.8 ± 1.5 h were obtained, AUCo* values were calculated to be 60.3 ± 37.3 ngh/mL and 65.9 ± 44.3 ng-h/mL. The method is sensitive and convenient, and is proved to be suitable for clinical investigation of dextrorphan pharmacokinetics and bioequivalence evaluation of formulations containing dextromethorphan.3. Determination of zolmitriptan in plasma by LC/MS/MSA sensitive and selective liquid chromatography-tandem spectrometry method for the determination of zolmitriptan was developed and validated over the linearity range0.05-30 ng/mL with 0.5 mL of plasma using diphenhydramine as the internal standard. Liquid-liquid extraction using a mixture of diethyl ether and dichloromethane was used to extract the drug and the internal standard from plasma. The mass spectrometer was operated under the selected reaction monitoring mode using the electrospray ionization technique. The instrument parameters were optimized to obtain 3.0 min run time. The mobile phase consisted of acetonitril-water-formic acid （70:30:0.5）, at a flow rate of 0.5 ml/min. In positive mode, zolmitriptan produced a protonated precursor ion at m/z 288 and a corresponding product ion at m/z 58. And internal standard produced a protonated precursor ion at m/z 256 and a corresponding product ion at m/z 167. The inter- and intraday precision （%RSD） were less than 8.5% and accuracy （%error） was between ±2.5%. The method had a lower limit of quantification of 0.05 ng/ml for zolmitriptan, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokinetic study of zolmitriptan after an oral administration of 5 mg zolmitriptan to 22 healthy volunteers. After oral administration of 5 mg of zolmitriptan test formulation or reference formulation, mean peak plasma levels （Cmax） of 10.3 ± 5.8 ng/mL and 10.6 ± 5.4 ng/mL and Tmm of 2.1 ± 1.3 h and 2.6 ± 1.6 h were observed. The mean t\n value of 9.7 ± 2.9 h and 9.5 ± 3.7 h were obtained, A UCo-t were calculated to be 75.5 ± 35.8 ng ? h/mL and 76.9 ± 39.4 ng ? h/mL.4. Quantification of low level erdosteine in dog plasma using a reduced sample volume with a liquid-chromatography/tandem mass spectrometryErdosteine is an acidic compound, which can be used as a functional acid radical to form new salt, ambroxol erdosteinate. When the new salt used as a mucolytic drug, it showed better mucolytic effect and the dosage was much lower than erdosteine used singly.A sensitive and selective liquid chromatography-tandem mass spectrometric （LC/MS/MS） method for the determination of erdosteine in dog plasma was developed and validated. Plasma proteins were precipitated by acetonitrile, followed by vortex mixing and centrifugation. Chromatographic separation was performed on a Cs column with a mobile phase consisting of 0.5% ammonia （aqueous）, 1% formic acid （aqueous）and methanol at a flow-rate of 0.5 ml/min. MS detection using negative electrospray ionization （-ESI） as an interface was used in selective reaction mode （SRM） to monitor transition m/z 248 -> m/z 110 （erdosteine） and m/z 404 -> m/z 114 （lisinopril, internal standard）. The method has a lower quantitation limit of down to 0.20 ng/ml and a lower consume of plasma only 25 \i\. Validation experiments have shown that the assay has good precision and accuracy over a wide concentration range （0.20²000 ng/ml）, and no interferences caused by endogenous compounds were observed by matrix effect test. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 10.0%. The accuracy determined at three concentrations （0.5,25.0 and 1800 ng/ml for erdosteine） was within ±3.0% in terms of relative error.To investigate the pharmacokinetics of erdosteine in dogs, the LC/MS/MS method was applied for the determination of erdosteine in dog plasma. The absorption of erdosteine in dogs was rapid and complete. Individual plasma-concentration data were analyzed by compartmental analysis. Mean plasma concentration curves of erdosteine were best fitted to two-compartment models. The absolute bioavailability of erdosteine in dogs was 67.5%.更多还原