Effects Of Ertapenem On The Pharmacokinetics Of Micafungin In Rats

Objectives: A HPLC and an UPLC method were established for the determination of micafungin and free drug in rat plasma.The influence of ertapenem on the pharmacokinetics of micafungin in rats was also investigated,consummate the pharmacokinetic study of two drugs.Methods: Chromatography conditions: HPLC,The samples were separated by Diamonsil C18?150 mm×4.6 mm,5 ?m?,with imrecoxib as the internal standard and the mobile phase of acetonitrile: 0.01 mol/L amonium acetate buffer?adjusted to pH 4.6 with glacial acetic acid??42.5: 57.5,V/V?at the detection wavelength of 269 nm.In addition the column temperature was set at 30 ?,the mobile phase was used at a flow rate of 1.0 mL·min-1.The injected volume was 10 ?L.UPLC,The samples were separated by Acquity UPLC? BEH C18?2.1mm×50 mm,1.7 ?m?,and the mobile phase of acetonitrile: 0.01 mol/L amonium acetate buffer?adjusted to p H 4.5 with glacial acetic acid??45: 55,V/V?at the detection wavelength of 269 nm.In addition the column temperature was set at 30 ?,the mobile phase was used at a flow rate of 0.2 mL·min-1.The injected volume was 2 ?L.Animals treatment: Forty rats were randomly divided into two groups with 20 rats in each.Each rat in the control group was given micafungin(15mg·kg^-1)though caudal vein injection,while each rat in the test group was given micafungin(15 mg·kg^-1)and ertapenem(100 mg·kg^-1)though caudal vein injection.Blood samples from plexus venous?approximatly 0.5 mL?at fundus oculi were collected pre-dose and at 2,10,20,30 mins and 1,2,3,4,6,12,24 hs post dose.All blood samples of rats were centrifuged at 10000 r·min-1 for5 mins.Then the plasma samples were transferred to other clean tubes and stored frozen at-40 ? for analysis.Sample preparation: For the sample preparation of plasma concentration,the 100 ?L plasma was added to a 1.5 mL tube containing 10 ?L internal standard,and added 400 ?L acetonitrile,mixed for 1min.The mixture was centrifuged for 5 min at 10000 r·min-1 and the liquid supernatant was transferred to a sample bottle,then a 10 ?L aliquot of supernatant was injected into HPLC system.For the sample preparation of plasma free drug concentration,the 100 ?L plasma was added to a glass tube containing hollow fiber,then it was centrifuged for 10 min at 6000 r·min-1.A 2 ?L aliquot of internal ultrafiltrate 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 analyze was performed on SPSS13.1 software to analysis the pharmacokinetic parameters.Differences in pharmacokinetic parameters of two groups were analyzed by using the nonparametric testor Student's t-test.The experiment was deemed significant if A P < 0.05.Results: HPLC,The retention times of micafungin and imrecoxib were 7min and 11 min,respectively.There is no interference of serum endogenous impurity.The calibration curve was Y=0.0443X-0.0025?r2=0.9994?.The relative recoveries of micafungin at the concentrations of 1.5 ?g·mL-1,25?g·mL-1 and 160 ?g·mL-1 were 99.10%,90.10% and 95.00%.The absolute recoveries of quality control samples at above three concentrations were101.7%,108.4% and 103.8%,respectively and the absolute recovery of imrecoxib was 97.70%.The RSD values of intra-day precision were 6.3%,7.9% and 7.7%.The RSD values of inter-day precision were 8.4%,8.9% and8.6%.Plasma samples of micafungin were stable when stored at-40 ? for fourteen days or under freeze-thaw cycles for three times and the processed samples had a good stability within 24 hours at 4 ?.UPLC,The retention times of micafungin was 2.33 min.There is no interference of serum endogenous impurity.The calibration curve was Y=9259X-26.721?r2=0.9994?.The relative recoveries of micafungin at the concentrations of 40 ?g·L-1,160 ?g·L-1 and 512 ?g·L-1 were 94.05%,99.52%and 96.97%.The absolute recoveries of quality control samples at above threeconcentrations were 91.81%,96.83% and 90.61%,respectively.The RSD values of intra-day precision were 2.5%,1.4% and 0.6%.The RSD values of inter-day precision were 3.8%,3.5% and 3.4%.Pharmacokinetic parameters of micafungin in experimental group and control group were as follows: AUC0-24h:?294.9±25.6?mg·h·L-1 and?301.2±41.0?mg·h·L-1;AUC0-?:?349.4±28.0?mg·h·L-1 and?359.5±53.9?mg·h·L-1;t1/2:?9.261±0.935?h and?9.295±1.133?h;V:?0.577±0.077?L·kg^-1and?0.568±0.087?L·kg^-1;CL:?0.043±0.003?L·h-1·kg^-1 and?0.043±0.007?L·h-1·kg^-1;Cmax:?65.95±9.71?mg·L-1 and?84.13±12.98?mg·L-1.Compared with the control and test group respectively,there was no significant difference in AUC0-24 h,AUC0-?,t1/2,CL and V,except Cmax.Conclusions: The HPLC and UPLC methods have best performance in terms of precision,sensitivity and accuracy.It is suitable to determine the plasma concentration of micafungin in rats.Compared with the control group and test group respectively,there was no significant difference in AUC0-24 h,AUC0-?,t1/2,CL and V,except Cmax.The experiment appears that ertapenem may have effect on the distribution of micafungin in rats.The result reminds that ertapenem have influence on pharmacokinetics of micafungin in rats.In addition the determination result of plasma free drug concentration appears that micafungin have effect on some pharmacokinetic parameters of micafugin in rats.The result reminds that ertapenem have some influence on the pharmacokinetics of micafungin in rats.