Determination Of Levamlodipine In Human Plasma And Pharmacokinetics Study

OBJECTIVE: To establish a rapid and sensitive method for the quantitation oflevamlodipine by LC-MS/MS in healthy human plasma.The method was used todetermine the concentrations of levamlodipine in plasma of24volunteers after oraladministrations of tested preparation and referred preparation, draw relevantpharmacokinetic parameters and bioequivalence evaluation through statisticalanalysis.METHOD:In accordance with the plasma samples collected method, takingdrugs before （0h） and medication after2.0h、4.0h、6.0h、8.0h、10.0h、14.0h、24.0h、34.0h、48.0h、72.0h、96.0h、120.0h in upper shallow venous blood4ml, thencentrifugated at3500×g for10min and transferred the supernatant to a polypropylenetube,stored at-20°C.Plasma samples were thawed at room temperature,then added internallevamlodipine into a certain amount of plasma sample.The mixture was extracted withdiethyl ether-dichloromethane（3:2,v/v）.And the organic phase was dried bynitrogen.Subsequently,the residue was reconstituted150μl mobile phase and30μl ofthe sample was injected into the LC-MS/MS system. Mobile phase:acetonitrile-10mMammonium acetate-formic acid（40:60:0.06，v/v/v）;flow velocity:1.0ml/minï¼›columntemperature30C;sample size：30μl. And the detection of the analyte was by thealternate of positive and negative ion mode.Multiple reaction monitoring wasperformed using the ion-pairs m/z409.2m/z238.3for levamlodipine,and theion-pairs m/z256.3m/z167.1for the internal standard respectively.The analytic methodology was evaluated by detecting the specificity, stability,linearity, accuracy, inter and intra-precision,recovery and matrix effects,lower limit ofquantization for levamlodipine in plasma according to the data to proved LC-MS/MSmethod fit for the bioequivalence study.After progressing the samples,draw the plasma concentration-time profile oflevamlodipine.Based on the concentration data, obtained the pharmacokineticparameters data.Finally,statistical analysis the pharmacokinetic parameters of the tworeagent by SPSS software to estimate whether it is or not bioequivalent.RESULT: This research established LC-MS/MS method meet the requirement to determination the concentration of levamlodipine in human plasma. And the methodwas rapid,reproducible and sensitive with a low limit of quantization of0.05ng·ml,the linear range of this method was0.05-5ng/ml，and the precision and accuracy ofthe method were all in line with the national relevant requirements,and there was nosignificant matrix effects in the plasma interfered the determination of levamlodipine.The results of the stability experiments showed that plasma samples were stableenough at room temperature for6h,at-20°C for3months or after three freeze-thawcycles.The method corresponded to the relevant norm and could be used forbioequivalence study.After24healthy volunteer oral reference product of levamlodipine, Cmax:2.84±0.72ng/ml, Tmax:6.3±4.2h, t1/2:36.44±7.96h, AUC0-Ï„:142.45±39.41ng h/ml,AUC_0-∞:159.29±45.56ng h/ml.After oral test product: Cmax:2.72±0.61ng/ml,Tmax:8.3±7.3h, t1/2:33.31±5.19h, AUC0-Ï„:137.75±36.82ng h/ml,AUC_0-∞:151.44±42.13ng h/ml，by comparison with the reference product therelative bioavailability of the test product was99.6%±21.1%.The results analyzed byBAPP2.0of the two formulations were as follows:The VOne side t-Test shows nosignificant differences.Under the90%confidence intervals,the levamlodipine of testto reference ratio were89.43%¹06.51%（Cmax）、90.94%¹04.80%(AUC_0-t), Tmaxnonparametric test results S=56.5>S0.05（17）=35, P>0.05,there was no significantdifference between reference product and test product. Statistic analysis result showsthe main pharmacokinetic parameters Cmax、Tmax、AUC_0-tall comply with relevantstandards, considered to be bioequivalent.