Simultaneous determination of Aprepitant and two metabolites in human plasma by high-performance liquid chromatography with tandem mass spectrometric detection

Abstract

A method for the simultaneous determination of Aprepitant, I (5-[[2( R)-[1( R)-(3,5-bistrifluoromethylphenyl)ethoxy]-3( S)-(4-fluorophenyl) morpholin-4-yl]methyl]-2,4-dihydro-[1,2,4]triazol-3-one) and two active metabolites ( II and III) in human plasma has been developed. The method was based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS-MS) detection in positive ionization mode using a heated nebulizer interface. The analytes and internal standard ( IV) (Fig. 1) were isolated from basified plasma using liquid–liquid extraction. The organic extracts were dried, reconstituted in mobile phase and injected into the HPLC-MS/MS system. The analytes were chromatographed on a narrow bore ( 50 mm×2.0 mm , 3 μm) Keystone Scientific’s Prism R.P. analytical column, with mobile phase consisting of acetonitrile (ACN):water containing trifluoroacetic acid with pH adjusted to 3 (40:60, v/v) pumped at a flow rate of 0.5 ml/min. The MS-MS detection was performed on a Sciex API 3000 tandem mass spectrometer operated in selected reaction monitoring mode. The precursor→product ion combinations of m/ z 535→277, 438→180, 452→223 and 503→259 were used to quantify I, II, III, and IV, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10–5000 ng/ml for I and II and 25–5000 ng/ml for III when 1 ml of plasma was processed. The precision of the assay (expressed as coefficient of variation, CV) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. Matrix effect experiments were performed to demonstrate the absence of any significant change in ionization of the analytes when comparing neat standards to analytes in the presence of plasma matrix. This assay was utilized to support a clinical study where multiple oral doses of I were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of Aprepitant. Concentrations of the two most active metabolites, which if present in high concentrations would increase the neurokinin-1 (NK 1) receptor occupancy level and therefore potentially contribute to the antiemetic action of Aprepitant, were determined.