Effects Of Sarpogrelate Hydrochloride On The Pharmacokinetics Of Metoprolol Tartrate In Rats

Objectives:An UPLC method was established for the determination of metoprolol tartrate in rat plasma.The influence of sarpogrelate hydrochloride on the pharmacokinetics of metoprolol tartrate in rats was also investigated,consummate the pharmacokinetic study of combination of two drug.Methods:Chromatography conditions:The samples were separated on an Acquity UPLC^? BEH C₁₈ Diamonsil C₁₈（2.1mm x 50 mm,1.7 μm）,with lamotrigine as the internal standard and the mobile phase of methanol:0.01 mol/L potassium dihydrogen phosphate buffer（adjusted to pH 3.6 with phosphoric acid）（31:69,V/V）at the detection wavelength of 225 nm.In addition the column temperature was set at 30℃,the mobile phase was used at a flow rate of 3.6 mL·min^-1.The injected volume was 2 μL.Animals treatment:Forty rats were randomly divided into two groups with 20 rats in each.Each rat in the control group was given metoprolol tartrate(27 mg·kg^-1)by gavage,while each rat in the test group was given metoprolol tartrate(27 mg·kg^-1)and sarpogrelate hydrochloride(10 mg·kg^-1)by gavage.Blood samples from plexus venous（approximate 0.5 mL）at fundus oculiwere collected pre-dose and at 0 h,0.17 h,0.33 h,0.5h,0.75 h,1 h,1.5 h,2 h,2.5 h,3 h,4 h and 5 h post dose.All blood samples of rats were centrifuged at 16600×g for 2 min.Then the plasma samples were transferred to another clean tube and stored frozen at-40℃ for UPLC analysis.The other part of rats which were not given drug once.All rats were randomly divided into control group and test group with 20 rats in each group.The experimental group was intragastric administration with metoprolol tartrate(27mg·kg^-1)once a day and sarpogrelate hydrochloride(10mg·kg^-1)three time a day;the control group was intragastric administration with metoprolol tartrate(27mg·kg^-1)once a day,continue to lavage in six days.Immediately take blood from the inner canthus in five hours.All blood samples of rats were centrifuged at 16600×g for 2 min.Then the plasma samples were transferred to another clean tube and stored frozen at-40℃ for UPLC analysis.Sample preparation:For the sample preparation,the 200 μL plasma was added to a 5 mL tube containing 20 μL internal standard,vortexed for 1min.1 mL ethyl acetate was added,and then mixed for 1 min.The mixture was centrifuged for 2 min at 16600×g and the upper organic layer was transferred and evaporated to dryness under the stream of nitrogen at-40℃.The dry residue was reconstituted with 100 μL mobile phase.After mixing for 2 min,the mixture was centrifuged for 3 min at 16600×g,then a 2 μL aliquot of supernatant was injected into UPLC system.Data analysis and statistical analysis: All the data was analyzed by using DAS 2.1.1 software to calculate.Statistical analysis was performed on SPSS 13.0 software to analysis the pharmacokinetic parameters.Difference in pharmacokinetic parameters of two groups was analyzed by using the nonparametric testor Student’s t-test.The experiment was deemed significant,if A P value less than 0.05.Results:The retention times of metoprolol tartrate and lamotrigine were 1.45 min and 1.75 min,respectively.There is no interference of serum endogenous impurity.The calibration curve was Y = 1.358 X + 0.006（r=0.9995）.The relative recoveries of metoprolol tartrate at the concentrations of 0.0625μg·mL^-1,0.5μg·mL^-1 and 2μg·mL^-1 were 96.69%,93.51% and 95.33%.The absolute recoveries of quality control samples at above three concentrations were 67.12%,69.48% and 70.57%,respectively and the absolute recovery of lamotrigine was 86.84%.The RSD values of intra-day precision were 9.45%,5.20% and 5.68%.The RSD values of inter-day precision were 8.75%,1.05% and 7.87%.Plasma samples of metoprolol tartrate were stable when stored at-40℃ for five days or under freeze-thaw cycles for three times and the processed samples had a good stability within 4 hours at 4℃.Pharmacokinetic parameters of metoprolol tartrate in rats who were given metoprolol tartrate and sarpogrelate hydrochloride for once were as follows:AUC0-5h:0.82± 0.37mg·h·L^-1,AUC0-∞:0.93 ± 0.44mg·h·L^-1,t1/2:1.45 ± 0.58 h,Tmax:0.89 ± 0.27 h,CL:35.1 ± 16.1 L·h^-1·kg^-1,V:66.3 ± 22.9 L·kg^-1,Cmax:0.49 ± 0.24 mg·L^-1;Pharmacokinetic parameters of metoprolol tartrate in rats who were given metoprolol tartrate for once were as follows:AUC0-5h:0.96 ± 0.46 mg·h·L^-1,AUC0-∞:1.05 ± 0.49 mg·h·L^-1,t1/2:1.42 ± 0.41 h,Tmax:0.85 ± 0.28 h,CL:30.1 ± 11.2 L·h^-1·kg^-1,V:61.5 ± 30.6 L·kg^-1,Cmax:0.64 ± 0.35 mg·L^-1;Pharmacokinetic parameters of metoprolol tartrate in rats who were given metoprolol tartrate and sarpogrelate hydrochloride for seven days were as follows:AUC0-5h:0.98 ± 0.41 mg·h·L^-1,AUC0-∞:1.08 ± 0.42 mg·h·L^-1,t1/2:1.40 ± 0.36 h,Tmax:0.66 ± 0.31 h,CL:28.60 ± 10.89 L·h^-1·kg^-1,V : 57.02 ± 23.46 L·kg^-1,Cmax: 0.62 ± 0.39 mg·L^-1;Pharmacokinetic parameters of metoprolol tartrate in rats who were given metoprolol tartrate and sarpogrelate hydrochloride for seven days were as follows:AUC0-5h:0.84 ± 0.40 mg·h·L^-1,AUC0-∞:0.91 ± 0.41 mg·h·L^-1,t1/2:1.23 ± 0.36 h,Tmax:0.51 ± 0.10 h,CL:33.94 ± 13.59 L·h^-1·kg^-1,V:58.19 ± 22.61 L·kg^-1,Cmax:0.71 ± 0.40 mg·L^-1.Compared with the metoprolol tartrate group respectively,there was no significant difference in AUC0-24 h,AUC0-∞,t1/2,Tmax,CL,V and Cmax of the group treated with metoprolol tartrate and sarpogrelate.Conclusions:Compared with HPLC method and LC-MS/MS method,the UPLC method has best performance in terms of precision,sensitivity and accuracy.It is suitable to determine the plasma concentration of metoprolol tartrate in rats.Compared with the metoprolol tartrate group respectively,there was no significant difference in AUC0-24 h,AUC0-∞,t1/2,Tmax,CL,V and Cmax of the group treated with metoprolol tartrate and sarpogrelate.The experiment in which rats were given drugs for once appears that sarpogrelate hydrochloride have no effect on the metabolism of metoprolol tartrate in rats.The result reminder that sarpogrelate hydrochloride have no competitive inhibition on metoprolol tartrate in rats.In addition The experiment in which rats were given drugs for seven day appears that sarpogrelate hydrochloride have no effect on the metabolism of metoprolol tartrate in rats.The result reminder that sarpogrelate hydrochloride have no suicide inhibition of metoprolol tartrate in rats.