Effect Of Sarpogrelate Hydrochloride On The Pharmacokinetics Of Mexiletine Hydrochloride In Rabbits

Objective:A HPLC method was established for the determination of the concentration of mexiletine hydrochloride in rabbit plasma.And the effects of sarpogrelate hydrochloride on the pharmacokinetics of mexiletine hydroch-loride in rabbits were investigated and consummated the pharmacokinetic study of combination of these two drugs by analyzing the blood concentration of mexiletine hydrochloride in rabbits in the test group of sarpogrelate?administered mexiletine hydrochloride and sarpogrelate hydrochloride?and the control group?administered distilled water and mexiletine hydrochloride?.Methods:Administration method and plasma sample collection:Twelve healthy male rabbits were randomly divided into the sarpogrelate group?hereinafter referred to as"test group"?and the control group,with 6 rabbits in each group.The test group was given sarpogrelate hydrochloride 16.67mg·kg^-1 by gavage and the control group was given the same amount of water by gavage administration three times one day.These two groups were continuously administered for 6 days.On the 7th day,the test group was given16.67 mg·kg^-1 of sarpogrelate hydrochloride and 65 mg·kg^-1 of mexiletine hydrochloride by gavage and the control group was given 65 mg·kg^-1 of mexiletine hydrochloride by gavage.Then blood samples from plexus venous collected at different times after administration and stored frozen at-40?before HPLC analysis.Sample preparation:For the sample preparation,the 200?L plasma was added to a 10 mL tube containing 10?L internal standard(40?g·mL^-1dioxpromazine hydrochloride solution),vortexed for 30 s and 200?L of 4mol·L^-1 NaOH was added,then mixed for 1 min.The mixture was added 3mL extractant n-heptane,vortexed for 120 s,then centrifuged for 10 min at2253×g and 2400?L of the upper organic layer was transferred to a 10 mL tube,added 40?L of 0.0025 mol·L^-1 phosphoric acid solution,vortexed for 30s and evaporated to dryness under the stream of nitrogen at 50?.The dry residue was reconstituted with 100?L mobile phase.Then a 10?L aliquot of supernatant was injected into HPLC system.Chromatography conditions:The samples were separated on an Diam-onsil C₁₈?150 mm×4.6 mm,5 um?,Waters 486 UV detector,with dioxetazine hydrochloride as the internal standard and the mobile phase of acetonitrile:0.01 mol·L^-1 phosphate buffer?adjusted to pH 4.3 with phosphoric acid,22:78?at the detection wavelength of 210 nm.In addition the column temperature was set at 30?,the mobile phase was used at a flow rate of 1 mL.min^-1.The injected volume was 10?L.Pharmacokinetic analysis:The plasma samples of the test group and the control group were treated according to the treatment method and determined,and the blood concentration of mexiletine hydrochloride was calculated according to the standard curve equation.All the data was analyzed by using DAS 2.1.1 software to calculate.Statistical analysis was performed on SPSS13.0 software to analyse the pharmacokinetic parameters.If the two groups of data to be compared are subjected to a normal distribution,the two independent samples are analyzed by using the nonparametric tester Students t-test.If any one of the two groups does not obey the normal distribution,the nonparametric test is used for analysis.The experiment was deemed significant,if a P<0.05.Results:The retention times of mexiletine hydrochloride and dioxpro-mazine hydrochloride were 8.5 min and 11.9 min,respectively and plasma endogenous impurities did not interfere with the determination of the concentration of mexiletine hydrochloride and dioxpromazine hydrochloride.The assay had good specificity.The calibration curve was Y=0.481X-0.0155?r=0.9998?.The relative recoveries of mexiletine hydrochloride at the concentrations of low-concentration(0.25?g·mL^-1),medium(1?g·mL^-1),high(4?g·mL^-1)were 101.11%,99.76%and 104.53%respectively.The absolute recoveries of quality control samples at above three concentrations were 75.32%,75.61%and 80.37%respectively and the absolute recovery of dioxpromazine hydrochloride was 84.76%.The RSD values of intra-day precision of mexiletine hydrochloride at the concentrations of low-concentration(0.25?g·mL^-1),medium(1?g·mL^-1),high(4?g·mL^-1)were0.7%,1.36%and 1%.The RSD values of inter-day precision at above three concentrations were 1.26%,1.08%and 1.86%.Plasma samples of mexil-etine hydrochloride were stable when stored at-40?for seven days or under freeze-thaw cycles for three times and the processed samples had a good stability within 4 hours at room temperature.In the test group,the AUC_0-12h of mexiletine hydrochloride after adminis-tration of sarpogrelate hydrochloride was compared with the control group AUC_0-12h(2.06±0.24 mg.h.L^-1 vs 4.52±1.09 mg.h.L^-1,P<0.05)and AUC_0-?(2.11±0.26 mg.h.L^-1 vs 4.26±1.09 mg.h.L^-1,P<0.05)significantly increased.The t_1/2 of mexiletine hydrochloride with no significance?1.62±0.29 h vs1.74±0.23 h,P>0.05?;C_max was significantly increased(0.77±0.09 mg.L^-1 vs1.38±0.21 mg.L^-1,P<0.05);T_max was significantly earlier?1.15±0.4 h vs0.84±0.11 h,P<0.05?;V was significantly decreased(157.65±38.48 L.kg^-1 vs91.91±28.27 L.kg^-1,P<0.05);CL was significantly accelerated(62.6±8.56 L.h^-1.kg^-1 vs 29.53±7.18 L.h^-1.kg^-1,P<0.05).Conclusion:Compared with LC-MS/MS method,the HPLC method has best performance in terms of precision,sensitivity and accuracy,also with simple sample pre-treatment,low cost and easily to operate.After a comprehensive methodological verification,the established method is simple,specific,sensitive,precise,reproducible.It can be used for exploring the effect of sarpogrelate hydrochloride on the pharmacokinetics of mexiletine hydrochloride in rabbits.After the combination of sarpogrelate and mexiletine,the AUC_0-12,AUC_0-?,t_1/2 and C_max of mexiletine increased significantly,T_max was significantly advanced,V and CL were significantly decreased,suggesting that sarpogrelate greatly increased the exposure of the mexiletine in rabbits by inhibiting the metabolism of mexiletine,which had a significant effect on the absorption and elimination of mexiletine.