| Part one Analysis the factors of influencing on the concentrations of clopidogrel active metabolite in patients with acute coronary syndromeObjective: To discuss the factors influencing the concentrations of clopidogrel active metabolite in plasma of patients with acute coronary syndrome(ACS).Methods: One hundred and five patients with ACS were administrated aspirin(100 mg·d-1)and clopidogrel bisulfate(75 mg·d-1)for 4 days.On day 5,blood samples(approximate 4 m L)were collected at 1 h after administration with empty stomach.Half of the blood was added 20 ?L 2-Bromo-3'-methoxyacetophenone(MPB,500 mmol·L-1)for determination clopidogrel bisulfate active metabolite immediately.And the other was used for determination the cytochrome P450 2C19*2(CYP2C19*2),*3,and paraoxonase-1(PON1).By single factor analysis,the binary variable and ranked related to the concentration of clopidogrel bisulfate active metabolites were analyzed.And the measurement data related to the blood drug concentration of clopidogrel bisulfate active metabolites were analyzed using Spearman analysis.Results: The concentration of clopidogrel bisulfate active metabolites had a significant negative correlation with diabetes,hyperlipidemia,PCI,PON1 carrier(P<0.05);but positive correlation with ?-blockers,statins and serum albumin(ALB)(P<0.05).Conclusion: The diabetes,hyperlipidemia,PCI can reduce the concentration of clopidogrel bisulfate active metabolites in blood;while the ALB,?-blockers and statins can increase the concentration of clopidogrel bisulfate active metabolites in blood.Part two Effect of the intestinal flora changes on the pharmacokinetics of clopidogrel bisulfate and its active metabolite in ratsObjective: To develop an LC-MS/MS method for the determination of clopidogrel bisulfate and its active metabolite,and study the effect of changes in intestinal flora on pharmacokinetics of clopidogrel bisulfate and its active metabolite in rats.Methods: Chromatography and mass spectrometry conditions: The concentration of clopidogrel bisulfate and its active metabolite were determined by Diamonsil C18 chromatographic column(150 mm × 4.6 mm,5 ?m),and the temperature was set at 40?.The mobile phase was consisted of acetonitrile-1 mmol/L ammonium acetate(1‰ formic acid containing)(80:20,V/V)with a flow rate of 1 m L·min-1.Ticlopidine hydrochloride was selected as the internal standard.The mass spectrometer was operated in the positive electrospray ionization mode with an electrospray ionization interface(ESI);the source voltage was maintained at 5.5 k V and the ion source temperature was set at 550?;The curtain gas pressure was fixed at 25 psi and the collision gas pressure was at 4 psi;Nitrogen was used as GS1(pressure was 55 psi)and GS2(pressure was 50 psi);The declustering potential(DP)and collision energy(CE)values of clopidogrel bisulfate were 68 V and 21 e V,respectively;the DP and CE values of its derivatized active metabolite(CAMD)were 114 V and 26 e V,respectively;the DP and CE values of ticlopidine hydrochloride were 60 V and 23 e V,respectively.Quantification was performed using multiple reaction monitoring(MRM)of the transitions of m/z: 322.2?212.0 for clopidogrel bisulfate,m/z: 504.4?354.1 for CAMD,m/z: 264.2?154.1 for ticlopidine hydrochloride,respectively.Animals experiment: Twenty-four SD rats were randomly divided into probiotic group,antibiotics group and control group.Rats of test group were administered Live Combined Bifidobacterium and Lactobacillus Tablets(0.8 g·kg-1)or Amoxicillin and Clavulanate Potassium Tablets(125 mg·kg-1)for 7 days,respectively;rats of control group were given same volume distilled water.On day 8,clopidogrel bisulfate(10 mg·kg-1)was administered orally to all rats.Blood samples were obtained for pharmacokinetic study 0.083,0.17,0.33,0.5,0.75,1,1.5,2,4,6,8,10 h after clopidogrel bisulfate administration and collected in tubes containing Heparin sodium and 20 ?L 125 m M 2-Bromo-3'-methoxyacetophenone(MPB),and vortexed for 30 s,immediately.After centrifugation(10900 r·min-1,5 min),200 ?L plasma sample was added into 600 ?L acetonitrile containing 2 ng·m L-1 ticlopidine hydrochloride,then vortexed 1 min and centrifugated 5 min(10900 r·min-1),10 ?L sample was injected to LC-MS/MS analysis.The pharmacokinetic parameters were calculated with DAS 2.1.1 software and the statistic analysis was processed with SPSS 21.0 software.The P value less than 0.05 were deemed significant.Results: Pharmacokinetic parameters of clopidogrel bisulfate in probiotic group,antibiotics group and control group were as follows: AUC0-t(6.83±1.81),(6.80±1.75)and(6.50±1.87)ng·h·m L-1;AUC0-?(12.11±4.90),(11.67±5.75)and(13.94±6.68)ng·h·m L-1;Cmax(3.25±1.73),(2.87±0.73)and(2.76±1.15)ng·m L-1;t1/2(9.07±4.57),(7.75±4.64)and(12.5±10.95)h;Tmax(0.30±0.19),(0.33±0.08)and(0.43±0.23)h;CL(966.61±418.40),(1000.34±366.34)and(836.05±303.94)L·(h·kg)-1;V(11261.73±4270.92),(9437.67±2945.94)and(11655.10±5789.74)L·kg-1,respectively.Pharmacokinetic parameters of CAMD in probiotic group,antibiotics group and control group rats were as follows: AUC0-t(362.26±126.43),(491.68±169.72)and(404.85±92.71)ng·h·m L-1;AUC0-?(404.85±92.71),(1524.19±176.40)and(422.45±91.08)ng·h·m L-1;Cmax(258.12±121.92),(272.00±72.04)and(258.55±112.03)ng·m L-1;t1/2(2.19±0.76),(3.59±1.95)and(2.42±0.83)h;Tmax(0.52±0.16),(0.84±0.18)and(0.78±0.16)h;CL(30.45±13.39),(20.76±5.95)and(24.67±5.40)L·(h·kg)-1;V(100.37±60.28),(110.66±73.57)and(87.10±35.88)L·kg-1,respectively.There was no significant difference in pharmacokinetic parameters of clopidogrel bisulfate and its active metabolite between control group and probiotic group(P>0.05)or antibiotics group.Conclusions: This study developed an LC-MS/MS method for the determination of clopidogrel bisulfate and its active metabolite in rat plasma.There is no significant influence on pharmacokinetics of clopidogrel bisulfate and its active metabolite when the intestinal flora was changed in rats.Part three Effects of the intestinal flora on the pharmacokinetics of ticagrelor in ratsObjectives: To develop an LC-MS/MS method for the determination of ticagrelor,and study the effect of changes in intestinal flora on pharmacokinetics of ticagrelor in rats.Methods: Chromatography and mass spectrometry conditions: The concentration of ticagrelor were determined by Diamonsil C18 chromatographic column(150 mm × 4.6 mm,5 ?m),and the temperature was set at 35?.The mobile phase was consisted of acetonitrile-1‰ formic acid(80:20,V/V)with a flow rate of 1 m L·min-1.Ibuprofen was selected as the internal standard.The mass spectrometer was operated in the negative electrospray ionization mode with an electrospray ionization interface(ESI);the source voltage was maintained at 5.5 k V and the ion source temperature was set at 600?;The curtain gas pressure was fixed at 40 psi and the collision gas pressure was at 10 psi;Nitrogen was used as GS1(pressure was 55 psi)and GS2(pressure was 50 psi);The declustering potential(DP)and collision energy(CE)values of ticagrelor were-111.36 V and-32.43 e V,respectively;the DP and CE values of ibuprofen were-49.61 V and-10.27 e V,respectively.Quantification was performed using multiple reaction monitoring(MRM)of the transitions of m/z: 521.2?361.3 for ticagrelor,m/z: 205.1?161.1 for ibuprofen,respectively.Animals experiment: Forty-five SD rats were randomly divided into probiotic group,antibiotics group and control group.Rats of test group were administered Live Combined Bifidobacterium and Lactobacillus Tablets(0.8 g·kg-1)or Amoxicillin and Clavulanate Potassium Tablets(125 mg·kg-1)for 7 days,respectively;rats of control group were given same volume distilled water.On day 8,ticagrelor(18 mg·kg-1)was administered orally to all rats.Blood samples were obtained for pharmacokinetic study at 0.083,0.25,0.5,1,1.5,2,3,4,6,8,12,24 h after ticagrelor administration and collected in tubes containing Heparin sodium.After centrifugation(10900 r·min-1,5 min),200 ?L plasma sample was added into 600 ?L acetonitrile containing 150 ng·m L-1 ibuprofen,then 1 min vortexed and 5 min centrifugated(10900 r·min-1),10 ?L sample was injected to LC-MS/MS analysis.The pharmacokinetic parameters were calculated with DAS 2.1.1 software and the statistical analysis was processed with SPSS 21.0 software.The P value less than 0.05 were deemed significant.Results: Pharmacokinetic parameters of ticagrelor in probiotic group,antibiotics group and control group rats were as follows: AUC0-t(6336.24±1840.46),(4444.05±1033.43)and(4469.32±928.47)ng·h·m L-1;AUC0-?(6841.98±1975.95),(4656.66±1083.78)and(4736.47±897.42)ng·h·m L-1;Cmax(858.65±275.98),648.81±215.59)and(617.49±168.95)ng·m L-1;t1/2(6.40±2.18),(5.25±1.39)and(5.68±2.08)h;Tmax(0.88±0.23),(0.90±0.21)and(1.30±0.59)h;CL(2.82±0.72),(4.07±0.99)and(3.95±0.91)L·(h·kg)-1;V(26.07±12.00),(31.45±14.65)and(32.95±14.17)L·kg-1.Compared with the control group,there was a significant increase in AUC0-t,AUC0-?and Cmax and decrease in Tmax and CL of the probiotic group(P<0.05).On the contrary,no significant difference were observed on the pharmacokinetic parameters of ticagrelor between antibiotics group and control group(P>0.05).Conclusions: This study developed an LC-MS/MS method for the determination of ticagrelor in rat plasma.Intestinal weight increase in the number of probiotic bacteria can increase the AUC0-t,AUC0-? and Cmax of ticagrelor,at the same time decreased the CL of ticagrelor. |
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