Determination Of Eptifibatide In Human Plasma And The Pharmacokinetic Study Of Eptifibatide Injection

AIM: To detect the concentration of eptifibatide in human plasma after a singleintravenous bolus administration of three doses （90μg/kg,180μg/kg and270μg/kg）to twelve healthy Chinese volunteers, a rapid and sensitive analysis method has beendeveloped and validated based on liquid chromatography-tandem mass spectrometry（LC-MS/MS）. Through the results above, we can obtain the pharmacokineticcharacteristics of eptifibatide in human plasma, so as to guide a more safe andeffective medication in clinical.METHOD: The eptifibatide plasma samples were treated with acetonitrile toprecipitate protein and then re-extracted with dichloromethane, the20μL ofextraction was injected to LC-MS/MS system. Eptifibatide and triptorelin as internalstandard （I.S.） were separated on a SB-Aq C18column （150×4.6mm I.D.,5μmparticle size） using methanol: acetonitrile:0.1%acetic acid （17.5:17.5:65，v/v/v） asmobile phase. MS/MS involved electrospray ionization in the positive ion mode andmultiple-reaction monitoring （MRM） of the transitions of eptifibatide and I.S. at m/z832.3â†'m/z159.2and m/z656.5â†'m/z249.3, respectively. The method was fullyvalidated in terms of specificity, linearity, LLOQ, precision, accuracy, extractionrecovery, matrix effect and stability studies according to relevant guidelines in foodand drug administration （FDA）.According relevant guidelines, twelve healthy Chinese volunteers were given asingle intravenous bolus administration of three doses （90μg/kg,180μg/kg and270μg/kg）, respectively. The plasma samples were collected at different time pointsafter administration. These plasma samples were determined with above establishedLC-MS/MS method. The DAS3.0software was used to calculate the correspondingpharmacokinetic parameters and the main parameters were statistically analyzedapplication of SPSS17.0software, and then describe the pharmacokineticcharacteristics of eptifibatide in the body. RESUTS: A method for the determination of eptifibatide in human plasma based onliquid chromatography-tandem mass spectrometry （LC-MS/MS） has been developedand validated. The lower limit of quantification （LLOQ） was5ng/mL and the linearover the concentration range5–3000ng/mL. The run time of this assay wasonly2.8min. The accuracy was within a range of-2.52%–2.52%in terms ofrelative error （R.E.） and the intra-and inter-day precisions in terms of relativestandard deviation （RSD） were≤6.61and≤14.2, respectively. The extraction recoveryof this method was high and stable, and there is no significant signal suppression orenhancement occurred in the ionization of eptifibatide or I.S.. In present study,eptifibatide was shown to be stable under all following storage conditions: long-termstability in plasma samples maintained at80°C for40d; three freeze/thaw cycles（20to25°C）; short-term stability in plasma samples maintained at25°C for4h; inprocessed samples in autosampler vials for4h. The rapid, selective, accurate andsensitive method was successfully applied to a pharmacokinetic study of eptifibatideinjection.After a single intravenous bolus administration of three doses （90μg/kg,180μg/kgand270μg/kg） to twelve healthy Chinese volunteers, the pharmacokinetic parameterswere followed: Cmax:485±123μg/L,1230±604μg/L and1525±611μg/L,respectively; T_1/2:1.38±1.31h,1.90±1.17h and2.47±0.46h, respectively; AUC0-t:512±112μg·h/L,936±172μg·h/L and1319±277μg·h/L, respectively; AUC0-∞:518±118μg·h/L,949±175μg·h/L and1340±277μg·h/L, respectively; CL:0.18±0.04L/h/kg,0.20±0.03L/h/kg and0.21±0.05L/h/kg, respectively; V_d:0.32±0.29L/kg,0.52±0.34L/kg and0.76±0.28L/kg, respectively; MRT:1.80±0.33h,1.80±0.29h and1.89±0.25h, respectively.The statistical results indicate that in90μg/kg-270μg/kg dose range eptifibatideshows a linear pharmacokinetic behavior; the main pharmacokinetic parameters CLand MRT among three doses are no significant difference （P>0.05）; however, there issignificant difference （P<0.05） of T_1/2and V_dbetween low and high dose. Thecompartment model fitting result shows that the pharmacokinetic behavior ofeptifibatide fits two compartment model. These results have a great significance fordetermining the route of administration, drug absorption rate and sampling time points.