本文選題：急性冠脈綜合征 + LC-MS/MS�。� 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：第一部分分析急性冠脈綜合征患者體內(nèi)氯吡格雷活性代謝產(chǎn)物的影響因素目的:分析影響急性冠脈綜合征(ACS)患者體內(nèi)硫酸氫氯吡格雷活性代謝產(chǎn)物血漿濃度的主要因素。方法:105例ACS患者均給予阿司匹林100 mg·d-1及硫酸氫氯吡格雷75 mg·d-1,連續(xù)口服4 d;第5天晨起空腹給藥1 h后,抽取靜脈血4 m L,用乙二胺四乙酸抗凝。其中2 m L立即加入500 mmol·L-1的衍生化試劑2-溴-3’-甲氧基苯乙酮(MPB)溶液20μL,用于檢測硫酸氫氯吡格雷活性代謝產(chǎn)物的濃度;另2 m L用于熒光檢測細(xì)胞色素P450 2C19*2(CYP2C19*2)、*3和氧磷酶1(PON1)基因型。用單因素分析探索二分類變量及等級資料對硫酸氫氯吡格雷活性代謝產(chǎn)物血藥濃度的影響,用Spearman分析探索硫酸氫氯吡格雷活性代謝產(chǎn)物血藥濃度與連續(xù)性變量的相關(guān)性。結(jié)果:糖尿病、高脂血癥、PON1攜帶、進(jìn)行冠狀動脈介入手術(shù)后與硫酸氫氯吡格雷活性代謝產(chǎn)物血藥濃度呈負(fù)相關(guān)(P0.05),β受體阻滯藥、他汀類藥物、血清白蛋白(ALB)與硫酸氫氯吡格雷活性代謝產(chǎn)物的濃度呈正相關(guān)(P0.05)。結(jié)論:糖尿病、高脂血癥、PON1攜帶及進(jìn)行冠狀動脈介入手術(shù)后等可減少硫酸氫氯吡格雷活性代謝產(chǎn)物血藥濃度,而ALB以及合用β受體阻滯藥或他汀類藥物可增加其血藥濃度。第二部分腸道菌群對硫酸氫氯吡格雷及其活性代謝產(chǎn)物在大鼠體內(nèi)藥動學(xué)的影響目的:建立大鼠血漿中硫酸氫氯吡格雷及其活性代謝產(chǎn)物的LC-MS/MS測定方法,并考察大鼠腸道菌群變化對其藥代動力學(xué)的影響。方法:色譜及質(zhì)譜條件:Diamonsil C18柱(150 mm×4.6 mm,5μm),流動相為乙腈-1 m M乙酸銨(含0.1‰的甲酸)(80∶20,V/V),流速1m L·min-1,柱溫40℃,進(jìn)樣量10μL,內(nèi)標(biāo)為鹽酸噻氯匹定。離子化方式:電噴霧離子化源(ESI);檢測方式:正離子模式;掃描方式:多反應(yīng)監(jiān)測(MRM);離子源噴射電壓:5500 V;離子源溫度:550℃;氣簾氣(CUR)壓力:25 psi;霧化氣NEB(GS1,N2)壓力:55 psi;輔助氣AUX(GS2,N2)壓力:50 psi;碰撞氣(CAD)壓力:4 psi;硫酸氫氯吡格雷的去簇電壓(DP)和碰撞能(CE)值分別為68 V和21 e V,活性代謝產(chǎn)物衍生物(CAMD)的DP和CE值分別為114 V和26 e V,鹽酸噻氯匹定的DP和CE值分別為60 V和23 e V;用于定量分析的離子對分別為m/z 322.2→212.0(硫酸氫氯吡格雷),504.4→354.1(CAMD),264.2→154.1(鹽酸噻氯匹定)。動物實(shí)驗(yàn):24只SD大鼠隨機(jī)分為益生菌組、抗生素組和對照組,每組8只。分別灌胃雙歧桿菌乳桿菌三聯(lián)活菌(0.8 g·kg-1)、阿莫西林克拉維酸鉀(125 mg·kg-1)和蒸餾水,連續(xù)7天,每天一次。第8天每只大鼠給予硫酸氫氯吡格雷片(10 mg·kg-1),并于給藥前和給藥后5、10、20、30、45 min、1、1.5、2、4、6、8、10 h眼內(nèi)眥取血,于肝素抗凝離心管(含20μL 125 m M 2-溴-3’-甲氧基苯乙酮(MPB)衍生化試劑),立即渦旋30 s混勻,離心取上層血漿200μL,加入600μL含1.5 ng·m L-1內(nèi)標(biāo)的乙腈溶液,渦旋1 min,10900 r·min-1離心5 min,取上清液10μL進(jìn)樣。采用DAS 2.1.1軟件擬合大鼠的藥動學(xué)參數(shù),使用SPSS 21.0軟件對三組藥動學(xué)參數(shù)進(jìn)行統(tǒng)計(jì)學(xué)分析,P值小于0.05則認(rèn)為有統(tǒng)計(jì)學(xué)差異。結(jié)果:益生菌組、抗生素組和對照組硫酸氫氯吡格雷的主要藥動學(xué)參數(shù)如下:AUC0-t分別為(6.83±1.81)、(6.80±1.75)和(6.50±1.87)ng·h·m L-1;AUC0-∞分別為(12.11±4.90)、(11.67±5.75)和(13.94±6.68)ng·h·m L-1;Cmax分別為(3.25±1.73)、(2.87±0.73)和(2.76±1.15)ng·m L-1;t1/2分別為(9.07±4.57)、(7.75±4.64)和(12.5±10.95)h;Tmax分別為(0.30±0.19)、(0.33±0.08)和(0.43±0.23)h;CL分別為(966.61±418.40)、(1000.34±366.34)和(836.05±303.94)L·(h·kg)-1;V分別為(11261.73±4270.92)、(9437.67±2945.94)和(11655.10±5789.74)L·kg-1。益生菌組、抗生素組和對照組CAMD的主要藥動學(xué)參數(shù)如下:AUC0-t分別為(362.26±126.43)、(491.68±169.72)和(404.85±92.71)ng·h·m L-1;AUC0-∞分別為(404.85±92.71)、(1524.19±176.40)和(422.45±91.08)ng·h·m L-1;Cmax分別為(258.12±121.92)、(272.00±72.04)和(258.55±112.03)ng·m L-1;t1/2分別為(2.19±0.76)、(3.59±1.95)和(2.42±0.83)h;Tmax分別為(0.52±0.16)、(0.84±0.18)和(0.78±0.16)h;CL分別為(30.45±13.39)、(20.76±5.95)和(24.67±5.40)L·(h·kg)-1;V分別為(100.37±60.28)、(110.66±73.57)和(87.10±35.88)L·kg-1。使用SPSS 21.0進(jìn)行統(tǒng)計(jì)學(xué)分析發(fā)現(xiàn),與對照組相比益生菌組和抗生素組各藥動學(xué)參數(shù)均沒有統(tǒng)計(jì)學(xué)差異。結(jié)論:本實(shí)驗(yàn)建立了大鼠血漿中硫酸氫氯吡格雷及其活性代謝產(chǎn)物的LC-MS/MS測定方法。藥動學(xué)實(shí)驗(yàn)結(jié)果表明腸道菌群的變化對硫酸氫氯吡格雷及其代謝產(chǎn)物的藥代動力學(xué)無影響。第三部分腸道菌群對替格瑞洛在大鼠體內(nèi)藥動學(xué)的影響目的:建立大鼠血漿中替格瑞洛的LC-MS/MS測定方法,并考察大鼠腸道菌群變化對替格瑞洛藥代動力學(xué)的影響。方法:色譜及質(zhì)譜條件:Diamonsil C18柱(150 mm×4.6 mm,5μm),流動相為乙腈-1‰的甲酸(80∶20,V/V),流速1 m L·min-1,柱溫35℃,進(jìn)樣量10μL,內(nèi)標(biāo)為布洛芬。離子化方式:電噴霧離子化源(ESI);檢測方式:負(fù)離子模式;掃描方式:多反應(yīng)監(jiān)測(MRM);離子源噴射電壓:5500 V;離子源溫度:600℃;氣簾氣(CUR)壓力:40 psi;霧化氣NEB(GS1,N2)壓力:55 psi;輔助氣AUX(GS2,N2)壓力:50 psi;碰撞氣(CAD)壓力:10 psi;替格瑞洛的解離電壓(DP)和碰撞能(CE)值分別為-111.36 V和-32.43 e V,布洛芬的DP和CE值分別為-49.61 V和-10.27 e V;用于定量分析的離子對分別為m/z 521.2→361.3(替格瑞洛),205.1→161.1(布洛芬)。動物實(shí)驗(yàn):45只SD大鼠隨機(jī)分為益生菌組、抗生素組和對照組,每組15只。分別灌胃雙歧桿菌乳桿菌三聯(lián)活菌(0.8 g·kg-1)、阿莫西林克拉維酸鉀(125 mg·kg-1)和蒸餾水,連續(xù)7天,每天一次。第8天每只大鼠給予替格瑞洛(18 mg·kg-1),并于給藥前和給藥后5、15、30 min、1、1.5、2、3、4、6、8、12、24 h眼內(nèi)眥取血,于肝素抗凝離心管,離心取上層血漿200μL,加入600μL含150 ng·m L-1內(nèi)標(biāo)的乙腈溶液,渦旋1 min,10900 r·min-1離心10 min,取上清液10μL進(jìn)樣。采用DAS 2.1.1軟件擬合大鼠的藥動學(xué)參數(shù),使用SPSS 21.0軟件對三組藥動學(xué)參數(shù)進(jìn)行統(tǒng)計(jì)學(xué)分析,P值小于0.05則認(rèn)為有統(tǒng)計(jì)學(xué)差異。。結(jié)果:益生菌組、抗生素組和對照組替格瑞洛的主要藥動學(xué)參數(shù)如下:AUC0-t分別為(6336.24±1840.46)、(4444.05±1033.43)和(4469.32±928.47)ng·h·m L-1;AUC0-∞分別為(6841.98±1975.95)、(4656.66±1083.78)和(4736.47±897.42)ng·h·m L-1;Cmax分別為(858.65±275.98)、(648.81±215.59)和(617.49±168.95)ng·m L-1;t1/2分別為(6.40±2.18)、(5.25±1.39)和(5.68±2.08)h;Tmax分別為(0.88±0.23)、(0.90±0.21)和(1.30±0.59)h;CL分別為(2.82±0.72)、(4.07±0.99)和(3.95±0.91)L·(h·kg)-1;V分別為(26.07±12.00)、(31.45±14.65)和(32.95±14.17)L·kg-1。使用SPSS 21.0進(jìn)行統(tǒng)計(jì)學(xué)分析發(fā)現(xiàn),與對照組比較益生菌組的AUC0-t、AUC0-∞和Cmax增大,Tmax和CL減小(P值均小于0.05);抗生素組與對照組相比,藥動學(xué)參數(shù)均沒有統(tǒng)計(jì)學(xué)差異。結(jié)論:本研究建立了大鼠血漿中替格瑞洛的LC-MS/MS測定方法。藥動學(xué)實(shí)驗(yàn)結(jié)果顯示,口服益生菌使大鼠腸道菌群發(fā)生變化后,可增大替格瑞洛在大鼠體內(nèi)的峰濃度,降低清除速率,增加替格瑞洛在大鼠體內(nèi)的暴露量。
[Abstract]:The first part analyzed the influence factors of clopidogrel active metabolites in patients with acute coronary syndrome: the main factors affecting the plasma concentration of clopidogrel sulfate active metabolites in patients with acute coronary syndrome (ACS) were analyzed. Methods: 105 patients with ACS were given aspirin 100 mg. D-1 and 7 of clopidogrel sulfate. 5 mg. D-1, continuous oral 4 D, and 1 h on the fifth day morning, 4 m L and anticoagulant with ethylenediamine tetra acetic acid. 2 m L immediately added 500 mmol L-1 derivative reagent 2- bromide -3 '- methoxy acetophenone (20) to detect the concentration of the active metabolites of the hydrogen sulfate clopidogrel; and 2 for fluorescence detection. Cytochrome P450 2C19*2 (CYP2C19*2), *3 and oxyphosphatase 1 (PON1) genotypes. The effects of two classified variables and grade data on the blood drug concentration of the clopidogrel sulfate active metabolites were investigated by single factor analysis. The correlation between the blood concentration of the clopidogrel sulfate metabolites and the continuous variables was investigated by Spearman analysis. Disease, hyperlipidemia, PON1 carrying, after coronary intervention, was negatively correlated with the blood concentration of the clopidogrel sulfate active metabolites (P0.05). Beta blockers, statins, serum albumin (ALB) were positively correlated with the concentration of the active metabolites of hydrogen sulfate clopidogrel (P0.05). Conclusion: diabetes, hyperlipidemia, PON1 The blood drug concentration of the clopidogrel sulfate active metabolites can be reduced with and after coronary intervention, while ALB and the combination of beta blockers or statins can increase the concentration of blood drugs. The effect of the second part of the intestinal flora on the pharmacokinetics of clopidogrel and its active metabolites in rats LC-MS/MS method for the determination of clopidogrel and its active metabolites in rat plasma and the effect of intestinal microflora change on its pharmacokinetics in rats. Methods: chromatographic and mass spectrometry conditions: Diamonsil C18 column (150 mm x 4.6 mm, 5 u m), mobile phase of acetonitrile -1 m M acetate (including 0.1% formic acid) (80: 20, V/V), flow rate 1m L. The column temperature is 40 C, the injection amount is 10 L, the internal standard is tilopidine hydrochloride. Ionization mode: electrospray ionization source (ESI); detection mode: positive ion mode; scanning mode: multi reaction monitoring (MRM); ion source injection voltage: 5500 V; ion source temperature: 550; CUR pressure: 25 psi; atomization gas NEB (GS1, N2) pressure: 55 psi; auxiliary gas AUX (GS2,) pressure Force: 50 psi; collision gas (CAD) pressure: 4 psi; the de cluster voltage (DP) and collision energy (CE) value of clopidogrel sulfate (CE) are 68 V and 21 e V respectively. The DP and CE values of the active metabolite derivatives (CAMD) are 114 V and 26 respectively, respectively, 60 and 23 respectively, respectively. The ion pairs for quantitative analysis are 322.2 to 212., respectively. 0 (clopidogrel sulfate), 504.4 to 354.1 (CAMD), 264.2 to 154.1 (ticlopidine hydrochloride). Animal experiment: 24 SD rats were randomly divided into probiotics group, antibiotic group and control group, 8 rats in each group. They were fed with Lactobacillus Bifidobacterium triad (0.8 g. Kg-1), amoxicillin clavulanate potassium (125 mg. Kg-1) and distilled water for 7 days, one day for 7 days. At the next eighth days, each rat was given Clopidogrel Bisulfate Tablets (10 mg. Kg-1), and blood was taken from the eye canthus in 5,10,20,30,45 min and 1,1.5,2,4,6,8,10 h before and after administration, and the heparin anticoagulant centrifuge tube (including 20 mu L 125 m M 2- bromine -3 'methoxy acetophenone (MPB) derivatization reagent), and immediately the vortex 30 was mixed, and the upper plasma was centrifuged to 200 mu, and 600 was added to 600. L containing 1.5 ng. M L-1 internal standard acetonitrile solution, vortex 1 min, 10900 r min-1 centrifuge 5 min, taking the supernatant 10 micron samples. The pharmacokinetic parameters of rats were fitted with DAS 2.1.1 software. Three groups of pharmacokinetic parameters were statistically analyzed using SPSS 21 software. The values were less than 0.05. Results: probiotics group, antibiotic group and The main pharmacokinetic parameters of the control group were as follows: AUC0-t was (6.83 + 1.81), (6.80 + 1.75) and (6.50 + 1.87) ng. H. M L-1, AUC0- infinity (12.11 + 4.90), (11.67 + 5.75) and (13.94 + 6.68) ng. H. M L-1 respectively, Cmax (3.25 + 1.73) and ng. .64) and (12.5 + 10.95) H; Tmax (0.30 + 0.19), (0.33 + 0.08) and (0.43 + 0.23) h, CL respectively (966.61 + 418.40), (1000.34 + 366.34) and (836.05 + 0.30) L. (H. Kg) -1; V respectively, the main pharmacokinetic parameters of the antibiotic and the control group. As follows: AUC0-t (362.26 + 126.43), (491.68 + 169.72) and (404.85 + 92.71) ng. H. M L-1, AUC0- infinity respectively (404.85 + 92.71), (1524.19 + 176.40) and (422.45 + 91.08) ng. H. M L-1 respectively, Cmax are respectively (258.12 + 121.92) and ng. X (0.52 + 0.16), (0.84 + 0.18) and (0.78 + 0.16) h, CL respectively (30.45 + 13.39), (20.76 + 5.95) and (24.67 + 5.40) L. (H. Kg) -1, V respectively (100.37 + 60.28), and L kg-1. using SPSS statistical analysis, found that the pharmacokinetic parameters of probiotics group and antibiotic group were not compared with the control group. There were statistical differences. Conclusion: this experiment established a LC-MS/MS method for the determination of clopidogrel and its active metabolites in rat plasma. The results of pharmacokinetic experiment showed that the changes in intestinal flora had no effect on the pharmacokinetics of clopidogrel and its metabolites. The third part of intestinal microflora was in the rat body of tigreloo. The objective of internal pharmacokinetic study: to establish a method for the determination of Grillo's LC-MS/MS in rat plasma and to investigate the effect of the change of intestinal flora on the pharmacokinetics of tegloro. Methods: chromatographic and mass spectrometry conditions: Diamonsil C18 column (150 mm x 4.6 mm, 5 u m), mobile phase (80: 20, V/V), 1 m L. Min-1, and 35 column temperature. Centigrade is 10 mu L, internal standard is ibuprofen. Ionization mode: electrospray ionization source (ESI); detection mode: negative ion mode; scanning mode: multi reaction monitoring (MRM); ion source injection voltage: 5500 V; ion source temperature: 600; gas curtain gas (CUR) pressure: 40 psi; NEB (GS1, N2) pressure of atomization gas: 55 psi; auxiliary gas AUX (GS2, N2) pressure: 50; collision The gas (CAD) pressure: 10 psi; the dissociation voltage (DP) and the collision energy (CE) value of the DP and -32.43 e V respectively. The DP and CE values for the quantitative analysis are respectively 521.2 to 361.3 (Ti Gray Lo) and 205.1 to 161.1 (Bloven). Animal experiment: 45 rats were randomly divided into probiotics. The bacteria group, the antibiotic group and the control group, 15 in each group, were fed with Lactobacillus Bifidobacterium triad (0.8 g. Kg-1), amoxicillin potassium clavulanate potassium (125 mg. Kg-1) and distilled water for 7 days, once a day. Each rat was given to gilligillo (18 mg. Kg-1) for eighth days, and 5,15,30 min and 1,1.5,2,3,4,6,8,12,24 h eyes were given before and after administration. When the inner canthus was taken, the heparin anticoagulant centrifuge tube was centrifuged and the upper plasma plasma was centrifuged to 200 mu L, and the acetonitrile solution containing 150 ng. M L-1 was added to 600 mu. The vortex 1 min and 10900 R. Min-1 were centrifuged 10 min and 10 micron L of the supernatant. The pharmacokinetic parameters of the rats were fitted with DAS 2.1.1 software. Three groups of pharmacokinetic parameters were statistically analyzed with 21 software. The values were less than 0.05. Results: the main pharmacokinetic parameters of the probiotics group, the antibiotic group and the control group for Grillo were as follows: AUC0-t (6336.24 + 1840.46), (4444.05 + 1033.43) and (4469.32 + 928.47) ng. H. M L-1, AUC0- infinity (6841.98 + 1975.95), (4656.66 + 1083.78) and (4736.47 + 897.42) ng. H. M L- respectively 1; Cmax (858.65 + 275.98), (648.81 + 215.59) and (617.49 + 168.95) ng. M L-1, t1/2 respectively (6.40 + 2.18), (5.25 + 1.39) and (5.68 + 2.08) h, Tmax respectively (6.40 + 617.49) and h, respectively, CL and L. + 14.17) L. Kg-1. used SPSS 21 to make statistical analysis. Compared with the control group, the AUC0-t, AUC0- and Cmax increased, Tmax and CL decreased (P less than 0.05), and the pharmacokinetic parameters were not statistically different between the antibiotic group and the control group. Conclusion: This study established the LC-MS/MS determination of Grillo in the rat plasma. The results of pharmacokinetic study showed that after oral probiotics changed the intestinal flora of rats, the peak concentration of Grillo in rats could be increased, the clearance rate was reduced and the exposure of tiprolol in rats was increased.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R969.1

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