Determination of eptifibatide concentration in human plasma utilizing the liquid chromatography–tandem mass spectrometry method

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to determine the concentration of eptifibatide in human plasma. Following protein precipitation, the analyte was separated on a reversed-phase C18 column. Acetonitrile:5 mM ammonium acetate:acetic acid (30:70:0.1, v/v/v) was used at a flow-rate of 0.5 mL/min with the isocratic mobile phase. An API 4000 tandem mass spectrometer equipped with a Turbo IonSpray ionization source was used as the detector and was operated in the positive ion mode. “Truncated” multiple reaction monitoring using the transition of m/z 832.6 → m/z 832.6 and m/z 931.3 → m/z 931.3 was performed to quantify eptifibatide and the internal standard (EPM-05), respectively. The method had a lower limit of quantification of 4.61 ng/mL for eptifibatide. The calibration curve was demonstrated to be linear over the concentration range of 4.61 − 2770 ng/mL. The intra- and inter-day precisions were less than 10.5% for each QC level, and the inter-day relative errors were 2.0%, 5.6%, and 2.8% for 9.22, 184, and 2490 ng/mL, respectively. The validated method was successfully applied to the quantification of eptifibatide concentration in human plasma after intravenous (i.v.) administration of a 270-μg/kg bolus of eptifibatide and i.v. administration of eptifibatide at a constant rate of infusion of 2 μg/(kg min) for 18 h in order to evaluate the pharmacokinetics.