Investigation Of The Influence Of Four Traditional Chinese Medicine Compound Preparations On The Pharmacokinetics Of The Active Metabolite Of Clopidogrel In Rats

Objectives: To develop an LC-MS/MS method for the determination of clopidogrel and its active metabolite.By comparing the pharmacokinetic parameters of the active metabolite of clopidogrel in rats after a single dose or pretreatment with four traditional chinese medicine compound preparations(Rongshuan capsules,Xuezhikang capsules,Xinyuan capsules,Songling Xuemaikang capsules)to investigate whether the four traditional chinese medicine compound preparations induce or inhibit the pharmacokinetic of the active metabolite of clopidogrel.Methods: Chromatography conditions: The samples were separated on a chromatographic column of Diamonsil C18(150 mm × 4.6 mm,5 ?m)and the column temperature was set at 40 °C.A mobile phase consisting of acetonitrile-1 mmol/L ammonium acetate-formic acid(80:20:0.01,V/V/V)was used at a flow rate of 1.0 mL/min.The injected volume was 10 ?L and ticlopidine was selected as the internal standard.Mass spectrometry conditions: The mass spectrometer was operated in the positive electrospray ionization mode with an electrospray ionization interface(ESI);the source voltage was maintained at 5500 V and the ion source temperature was set at 550 °C;The curtain gas pressure was fixed at 25 psi and the collision gas pressure was at 6 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 were 68 V and21 eV,respectively;the DP and CE values of its derivatized active metabolite(CAMD)were 114 V and 26 eV,respectively;the DP and CE values of ticlopidine were 60 V and 23 eV,respectively.Quantification was performed using multiple reaction monitoring(MRM)of the transitions of m/z: 322.2?212.0 for clopidogrel,m/z: 504.4 ? 354.1 for CAMD;m/z: 264.2 ?154.1 for ticlopidine,respectively.Animals treatment: Forty rats were randomly divided into five groups(n=8),control group and four experimental groups.Rats of Experimrntal groups were pretreated with Rongshuan capsules(75 mg/kg,tid),Xuezhikang capsules(60 mg/kg,bid),Xinyuan capsules(120 mg/kg,tid)and Songling Xuemaikang capsules(150 mg/kg,tid)for 7 days,respectively;rats of control group were given same volume distilled water.On day 8,after administration of Rongshuan capsules(75 mg/kg),Xuezhikang capsules(60 mg/kg),Xinyuan capsules(120 mg/kg),Songling Xuemaikang capsules(150 mg/kg)and same volume distilled water respectively,clopidogrel was administered orally at a dose of 7.5 mg/kg to all rats.Blood samples(approximate 0.5 mL)from plexus venous at fundus oculi were collected at 5,10,20,30,45 min,1,1.5,2,4,6,8,10 h post dose.Blood samples were collected in tubes containing Heparin sodium and 20 ?L of 125 mM 2-Bromo-3'-methoxyacetophenone(MPB)in acetonitrile.Immediately after drawing,the blood samples were mixed for 30 s and centrifuged at 6500×g for 5 min and the plasma samples stored at-40 °C until analysis.Sample preparation: For the sample preparation,the plasma(200 ?L)were combined with 600 ?L of acetonitrile(containing 10.5 ng/mL IS)for protein precipitation.The mixture was vortex-mixed for 1 min and centrifuged at 6500×g for 5 min.Then 10 ?L of the supernatant was injected into the LC-MS/MS system.Data analysis and statistical analysisAll the data was analyzed by using DAS 2.1.1 software and standard methods were used to calculate the pharmacokinetic parameters.Statistical analysis was performed on SPSS 17.0 software.Difference in parameters of two groups was analyzed by using the Student's t-test(normality)or nonparametric test(one or two annormality).A P value less than 0.05 was deemed significant.Results: The retention times of CAMD,clopidogrel and ticlopidine were3.21 min,5.10 min and 5.68 min,respectively.There was no endogenous interference from plasma or at any other point in the chromatogram.The calibration curve was Y = 0.02075 X + 0.00588(r=0.9992),and the linear range was 0.53~52.50 ng/mL for clopidogrel.The calibration curve was Y =0.00800 X-0.0005(r=0.9990),and the linear range was 2.00~200.00 ng/mL for CAMD.The relative recoveries of quality control samples at above three concentrations for clopidogrel were 99.89%,100.29% and 98.97%,respectively.The relative recoveries of quality control samples at above three concentrations for CAMD were 98.96%,97.98% and 99.63%,respectively.The absolute recoveries of clopidogrel at the concentrations of 1.64 ng/mL,6.56 ng/mL and 42.00.00 ng/mL were 85.02%,85.47% and 86.93%,respectively;the absolute recoveries of CAMD at the concentrations of 6.25ng/mL,25.00 ng/mL and 140.00 ng/mL were 81.54%,82.50% and 83.11%,respectively,and the absolute recovery of ticlopidine was 85.50%.The RSD values of intra-day and inter-day precision were all less than 8%.Plasma samples were stable when stored at-40 °C for a month or under freeze-thaw cycles for three times and the processed samples had a good stability within 8hours at room temperature.Pharmacokinetic parameters of CAMD in rats treated with distilled water were as follows: AUC0-t: 20574.45 ± 5451.02 ng/mL·min,AUC0-?: 21532.80 ±6223.07 ng/mL·min,Cmax: 207.50 ± 79.33 ng/mL,Tmax: 45.00 ± 8.02 min,t1/2:147.11 ± 70.92 min,CL: 0.38 ± 0.16 L/min/kg,V: 76.02 ± 27.96 L/kg;Pharmacokinetic parameters of CAMD in rats treated with Rongshuan capsules were as follows: AUC0-t: 22584.72 ± 8754.93 ng/mL·min,AUC0-?:23705.66 ± 9089.06 ng/mL·min,Cmax: 184.00 ± 58.91 ng/mL,Tmax: 50.62 ±11.16 min,t1/2: 150.69 ± 35.81 min,CL: 0.36 ± 0.15 L/min/kg,V: 79.29 ±37.18 L/kg;Pharmacokinetic parameters of CAMD in rats treated with Xuezhikang capsules were as follows: AUC0-t: 15338.44 ± 2958.96ng/mL·min,AUC0-?: 17989.99 ± 5221.61 ng/mL·min,Cmax: 181.04 ± 57.84ng/mL,Tmax: 28.12 ± 9.98 min,t1/2: 211.27 ± 66.40 min,CL: 0.44 ± 0.10L/min/kg,V: 129.78 ± 30.34 L/kg;Pharmacokinetic parameters of CAMD inrats treated with Xinyuan capsules were as follows: AUC0-t: 19585.22 ±8037.93 ng/mL·min,AUC0-?: 21615.22 ± 10084.68 ng/mL·min,Cmax: 225.25± 50.81 ng/mL,Tmax: 38.12 ± 9.98 min,t1/2: 181.96 ± 73.68 min,CL: 0.40 ±0.14 L/min/kg,V: 98.69 ± 44.23 L/kg;Pharmacokinetic parameters of CAMD in rats treated with Songling Xuemaikang capsules were as follows: AUC0-t:17804.36 ± 6443.45 ng/mL·min,AUC0-?: 19812.59 ± 6111.02 ng/mL·min,Cmax: 143.12 ± 53.63 ng/mL,Tmax: 43.75 ± 13.56 min,t1/2: 217.56 ± 132.35 min,CL: 0.41 ± 0.14 L/min/kg,V: 140.13 ± 118.15 L/kg.Compared with the control group,there was a significant decrease in AUC0-t and Tmax and a marked increase in t1/2 and V of the group treated with Xuezhikang capsules(P<0.05).On the contrary,no significant difference were observed on the pharmacokinetic parameters of CAMD between groups treated with Rongshuan capsules,Xinyuan capsules,Songling Xuemaikang capsules and control group(P>0.05).Conclusions: The LC-MS/MS method has a good performance in terms of accuracy,sensitivity and precision.It is suitable to determine the plasma concentration of the active metabolite of clopidogrel in rats.Compared with control group,it was observed that there was an obvious decrease in the exposure,decrease in excretion and increase in retention time of CAMD in the group pretreated with Xuezhikang capsules.It was also observed that the pharmacokinetic parameters have altered in groups pretreated with Rongshuan capsules,Xinyuan capsules and Songling Xuemaikang capsules compared with control group,but not significant.In conclusion,when clopidogrel was coadministration with Xuezhikang capsules,risk of clopidogrel resistant and clopidogrel active metabolite accumulation should be focused on.Coadministration of clopidogrel and Xuezhikang capsules should avoid.