【作者】 谭志荣；

【导师】 周宏灏；

【作者基本信息】 中南大学 ， 临床药理， 2009， 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10年来已迅速成为生物样品分析和药物代谢动力学研究的主要方法。Waters公司首次推出的超高效液相色谱(UPLC)具有超高的分离度、超高的灵敏度和超高的速度,他“把分离科学推向一个新领域”。本文利用LC-MS/MS和UPLC技术,建立了7种简单、灵敏、快速的定量方法,分别用于血浆中巴替非班、美普他酚、尼可地尔、伪麻黄碱和西替利嗪、水飞蓟素、艾普拉唑和舒林酸代谢产物的测定。所建立的方法的灵敏度均优于已有方法,并成功用于药物动力学研究。一、LC-MS/MS法测定血浆中新药巴替非班的浓度目的:建立灵敏、快速的液相色谱-串联质谱法测定人血浆中巴替非班的浓度。方法:血浆经固相萃取后,以水:乙腈:甲酸(40:60:0.1,v/v/v)作为流动相,采用Thermo Hypurity 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/z820.2→m/z(632.9+159.4)(巴替非班)和m/z834.4→m/z(646.7+159.3)(依替非巴肽)。该方法的线性范围为2.45-5000 ng/mL,LLOQ(定量下限)为2.45 ng/mL。结果:将此方法应用于一类新药巴替非班的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.考察空腹静脉滴注低、中和高三个单剂量后的药物动力学,获得该药在体内的药物动力学特征:符合二室模型、具有线性动力学特征。2.考察多剂量给药后巴替非班在人体内的药物动力学,与同剂量单次滴注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美普他酚的浓度目的:建立快速、灵敏的液相色谱-串联质谱法测定人血浆中的美普他酚,用于受试者口服美普他酚后的药代动力学研究。方法:血浆样品经MTBE(叔丁基甲基醚)萃取后,以乙腈:50mM甲酸胺(70/30,v/v)作为流动相,采用Hypurity 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。用于定量的离子反应分别为m/z 234→m/z 234(美普他酚)和m/z152→m/z110(对乙酰氨基酚)。该方法的线性范围为0.29-292.5ng/mL,LLOQ为0.2925ng/mL。结果:美普他酚在血浆中分析方法的线性范围为0.2925-292.5 ng/mL,定量下限可达0.2925 ng/mL。在美普他酚血浆质控(QC)样品浓度分别为5.85,11.7和117 ng/mL下的分析方法日内精密度(RSD)<8.54%,日间精密度(RSD)<8.61%,准确度(RE)在-4.2%-5.44%之间。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。三、液相色谱-串联质谱法测定血浆中的尼可地尔的浓度目的:建立快速、灵敏的液相色谱-串联质谱法测定人血浆中的尼可地尔,用于受试者口服尼可地尔后的药代动力学研究。方法:血浆样品经MTBE(叔丁基甲基醚)萃取后,以乙腈:0.1%甲酸(50:50,v/v)作为流动相,采用Hypurity 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。用于定量的离子反应分别为m/z 212→m/z(79+136)(尼可地尔)和m/z308→m/z(140+237)(丁洛地尔)。该方法的线性范围为0.51-520 ng/mL,LLOQ为0.51ng/mL。结果:尼可地尔在血浆中分析方法的线性范围为0.51-520 ng/mL,定量下限可达0.51 ng/mL。在尼可地尔血浆质控(QC)样品浓度分别为1.02,16.25和260 ng/mL下的分析方法日内精密度(RSD)<8.54%,日间精密度(RSD)<8.61%,准确度(RE)在-4.2%-5.44%之间。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。四、液相色谱-串联质谱法同时测定血浆中的西替利嗪和伪麻黄碱的浓度目的:建立快速、灵敏的液相色谱-串联质谱法同时测定人血浆中的西替利嗪和伪麻黄碱,用于受试者口服复方西嗪伪麻的药代动力学研究。方法:血浆样品经乙腈直接沉淀后,以乙腈:0.1%甲酸(50:50,v/v)作为流动相,采用Hypurity 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。用于定量的离子反应分别为m/z 166→m/z148(伪麻黄碱)、m/z 389→m/z201(西替利嗪)和m/z264→m/z246(曲马多)。结果:将该方法的线性范围西替利嗪和伪麻黄碱均为5.0-1000 ng/mL,LLOQ均为5.0ng/mL。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。五、液相色谱-串联质谱法测定血浆中的水飞蓟素的浓度目的:建立快速、灵敏的液相色谱-串联质谱法测定人血浆中的水飞蓟素,用于研究体内肝药酶和转运体基因多态性对水飞蓟素体内浓度的影响。方法:包含CYP3A5*3、CYP2C9*3和MDRl C3435T三种基因型22名健康受试者,同时服用1片利加隆(140 mg/片)后,血浆样品经MTBE(叔丁基甲基醚)萃取后,以乙腈:10mM甲酸胺(含0.1%甲酸)(50:50,v/v)作为流动相,采用Hypurity C18色谱柱分离,通过电喷雾离子化串联质谱,以多反应监测(MRM)方式进行负离子监测。用于定量的离子反应分别为m/z 481.3→m/z124.9(水飞蓟素)和m/z355.2→m/z 339.9(VBE-1)。该方法的线性范围为0.64-325ng/mL,LLOQ为0.64 ng/mL。结果:利用建立的LC-MS/MS方法测定0-48 h水飞蓟素的血药浓度。CYP3A5*3和CYP2C9*3多态性对水飞蓟素的血药浓度没有明显影响,MDR1 C3435T多态性对水飞蓟素的药代参数有明显影响。结论:该方法灵敏度高,实用性强,适用于水飞蓟素的临床药物动力学研究。水飞蓟素可能是MDR1的一个底物。六、液相色谱-串联质谱法测定血浆中艾普拉唑及其代谢产物及在药代动力学上的应用目的:建立快速、灵敏的液相色谱-串联质谱法测定人血浆中的艾普拉唑及其代谢产物,用于受试者口服艾普拉唑后的药代动力学研究。方法:选用Thermo HyPUR/TY C18柱,流动相为10mM甲酸胺水溶液:乙腈(50:50),柱温30度,三重四极杆质谱以多反应监测方式进行检测,检测的离子:艾普拉唑m/z367.2→184.0;艾普拉唑砜:m/z:383.3→184.1;艾普拉唑硫醚:m/z:351.2→168.1;内标奥美拉唑:m/z:346.2→198.0。12名健康受试者分别口服5mg的艾普拉唑肠溶片在0-48h采血,用建立的LC-MS/MS方法检测其体内艾普拉唑及其两个代谢产物的血药浓度。结果:其线性范围艾普拉唑为0.23-2400.00 ng/mL;艾普拉唑砜为0.05-105.00 ng/mL;艾普拉唑硫醚为0.06-45.00 ng/nL。LLOQ分别为0.23、0.05和0.06ng/mL。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。七、超高效液相色谱法测定血浆中舒林酸及其代谢产物及在药代动力学上的应用目的:建立快速、灵敏的超高效液相色谱法测定人血浆中的舒林酸及其代谢产物,用于受试者口服舒林酸后的药代动力学研究。方法:血浆样品经二氯甲烷萃取后,以乙腈:20mM甲酸胺(含1%醋酸)作为流动相梯度洗脱,采用Waters AcQUITY BEH C18(2.1*50mm,1.7um)色谱柱分离,检测波长为325nm,柱温为30℃。该方法的线性范围舒林酸为0.16-20.82μg/mL,舒林酸砜为0.16-20.88μg/mL,舒林酸硫化物为0.18-23.52μg/mL,LLOQ分别为0.16、0.16、0.18μg/mL。结果:将此方法应用于舒林酸及其代谢产物的体内药代动力学研究,并根据FM03中E308G和E158K位点突变,发现E308G和E158K野生型和突变性之间,舒林酸药代参数发生明显改变,而两个代谢产物却没有明显改变。结论:本方法简单、快速和灵敏,能用于舒林酸及其代谢产物的药代动力学研究。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. Ultra Performance Liquid Chromatography (UPLC) which was first made by Waters corporation(USA), this new category of analytical separation science retains the practicality and principles of HPLC while increasing the overall interlaced attributes of speed, sensitivity, and resolution. Eight drugs including batifiban, ilaprazole, mepta, nicorandil, sulindac, silibin, pseudoephedrine and cetirizine in human plasma were developed and validated by LC-MS/MS and UPLC in these thesis. The methods have been successfully applied to pharmacokinetic studies.1. Determination of batifiban in plasma by LC-MS/MSA sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of batifiban in human plasma was developed. After a solid phase extraction, the post-treated samples were analyzed on a Thermo HyPURITY C18 column(150*2.1mm, 5μm) interfaced with a triple quadruple tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitril: water: formic acid(60:40:0.1, v/v/v). Selected reaction monitoring (SRM) using the precusor→product ion combination of m/z 819→m/z (623+159 ) and m/z 833→m/z(645+159) was used to quantify batifiban and IS (eptifibatide), respectively. The linear calibration curves were abtained in the concentration range of . The method has a lower limit of quantification (LLOQ)of for batifiban.The method was applied to a PhaseⅠclinical trial of batifiban.After oral administratin of increasing(low, medium, high) dose and multidose of batifiban, the plasma concentrations of batifiban were monitored by the developed sensitive and fast LC-MS/MS method.2. Direct determination of nicorandil in human plasma by LC-MS/MS A sensitive and selective LC-MS/MS method for direct determination of nicordil in human plasma was developed and was used to study the pharmacokinetics of nicorandil. After a single dose intravenous injection aminstration of nicorandil 1mg to 12 healthy Chinese volunteers, the plasma concentration of nicorandil was determined. Nicorandil and internal standard buflomedil were extracted from plasma using liquid-liquid extraction, then separated on a Hypurity C18 column. The mobile phase consisted of acetonitrile-water-formic acid(60:40:0.1,v/v/v), at a flow-rate of 0.25mL/min. A Waters QuattroMicro API 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 212 to (79+136) and m/z 308 to (140+237) was performed to quantify nicorandil and internal standard . The pharmacokinetic parameters of nicorandil were calculated by non-compartment model statistics. The linear calibration curves were obtained in the concentration range of 0.51-520 ng/mL. Each plasma sample was chromatographed within3.0min. The intra- and inter-day relative standard deviation(RSD) across three validation runs over the entire concentration range was less than 15%. Accuracy determined at three concentrations (1.02, 16.25 and 260 ng/mL for nicorandil) ranged from 98.8% and 100.6%. The method was applied to study the pharmacokinetics of 12 healthy volunteers after intravenous injection adminstration 1 mg of nicorandil. The method is sensitive and conventient, and is proved to be suitable for clinical investigation of nicorandil pharmacokinetics.3. Determination of metazinol in plasma by LC/MS/MSA sensitive and selective liquid chromatography-tandem spectrometry method for the determination of metazinol was developed and validated over the linearity range 0.29-292.5ng/mL with 0.5 mL of plasma using acetaminophen as the internal standard. Liquid-liquid extraction using MTBE 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-10mM ammonium formate water solution(70:30, v/v), at a flow rate of 0.25mL/min. In positive mode, metazinol produced a protonated precursor at m/z 234 and a corresponding product ion at m/z 234. And internal standard produced a protonated precursor ion at m/z 152 and a corresponding product ion at m/z 110. The inter- and intra-day precision(%RSD) were less than 15% and accuracy (%error) was between±4.5. The method has a lower limit of quantification of 0.29 ng/mL for metazinol, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokintiec study of metazinol after an oral administration of 100 mg metazinol tablet to 12 healthy volunteers.4. Simulantance quantification of pseudoephedrine and cetirizine in plasma by LC-MS/MSA sensitive and selective liquid chromatography-tandem spectrometry method for the simulantance determination of pseudoephedrine and cetirizine was developed and validated over the linearity range 5.0-1000 ng/mL with 0.2 mL of plasma using tramadol as the internal standard. Direct prodein precipitation using acetronitril 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 5.0 min run time. The mobile phase consisted of 65% methanol and 35% water (contained 0.1% formic acid, 10 mM ammonium format, at a flow rate of 0.2 mL/min. In positive mode, pseudoephedrin produced a protonated precursor at m/z 166 and a corresponding product ion at m/z 148. cetirizine produced a protonated precursor at m/z 389 and a corresponding product ion at m/z 201. And internal standard produced a protonated precursor ion at m/z 264 and a corresponding product ion at m/z 246. The inter- and intra-day precision(%RSD) were less than 15%. The method has a lower limit of quantification of 5.0 ng/mL for pseudoephedrine and cetirizine, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokintiec study of pseudoephedrine and cetirizine after an oral administration of a cetirizine and pseudoephedrine sustained-release capsules (including 120mg pseudoephedrine and 5 mg cetirizine) to 12 healthy volunteers.5. Direct determination of silibin in human plasma by LC-MS/MSA sensitive and selective liquid chromatography-tandem spectrometry method for the determination of silibin was developed and validated over the linearity range 0.64-325 ng/mL with 0.5 mL of plasma using VBE-1 as the internal standard. Liquid-liquid extraction using MTBE 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-10mM Ammonium Formate water solution (contained 0.1%formic acid) (50:50, v/v), at a flow rate of 0.3 mL/min. In negative mode, silibin produced a protonated precursor at m/z 481 and a corresponding product ion at m/z 125. And internal standard produced a protonated precursor ion at m/z 355 and a corresponding product ion at m/z 339. The inter- and intra-day precision(%RSD) were less than 15%. The method has a lower limit of quantification of 0.64 ng/mL for silibin, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokintiec study of silibin after an oral administration of a 140 mg silibin capsules to 22 healthy volunteers.6. Determination of ilapraozle and its two metabolites in human plasma by LC-MS/MSAn analytical method based on liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been improved and validated for the quantitative measurement of ilaprazole and it’s two metablites in human plasma. Separation of analytes and the internal standard (IS) omeprazole was performed on a Thermo HyPURITY C18 column (150×2.1mm, 5μm) with a mobile phase consisting of 10mM ammonium formate water solution- acetonitrile (50:50,v/v) at a flow rate of 0.25 mL/min. The API4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode via positive electrospray ionization interface using the transition m/z 367.2→m/z184.0 for ilaprazole, m/z 383.3→m/z184.1 for ilaprazole sulfone, m/z 351.2→m/z168.1 for ilaprazole thiol ether and m/z 346.2→m/z198.0 for omeprazole. The method was linear over the concentration range of 0.23 -2400.00 ng/mL for ilaprzole, 0.05-105.00 ng/mL for ilaprazole thiol ether and 0.06 - 45.00 ng/mL for ilaprazole sulfone, respectively. The intra- and inter- day prcisions were all less than 15% in terms of relativestandard deviation (R.S.D), and the accuracy was within 15% in terms ofrelative error (R.E) for ilaprazole, ilaprazole sulfone and ilaprazole thiolether. The lower limit of quantification (LLOQ) was identifiable andreproducible at 0.23, 0.05 and 0.06 ng/mL with acceptable precision andaccuracy for ilaprazole, ilaprazole sulfone and ilaprazole thiol ether,respectively. The validated method offered sensitivity and wide linearconcentration range. This method was successfully applied for theevaluation of pharmacokinetics of ilaprazole and it’s two metablites aftersingle oral doses of 5 mg ilaprazole to 12 Chinese healthy volunteers.7. Using UPLC to detect sulindac and its two metabolites in plasmaA Waters AcQURITY BEH C18 (50*2.1 mm, 1.7μm) column wasselected. The mobile phase was 20 mM ammonium acetate water solution(including 1%Acetic Acid). The column temperature was 30℃, thedetection wavelength was 325 nm. The subjects received 200 mgSulindac capsules. Sulindac capsules on first day morning after fastingover night. Then blood samples were collected at 0-36 hours after takingthe drug. Sulindac and it’s two metabolites pharmacokinetics wasmeasured by UPLC with ultraviolet detection. Sulindac and it’s twometabolites was retented within 5min, the peaks was in a good separate,and this method was very stable. In the FMO3 E308G and E158K linkagemutation, sulindac pharmacokintics parameter has significant difference,but not in sulidac sulfone and sulidac sulfide.更多还原