Narrow-bore high-performance liquid chromatography in combination with ionspray tandem mass spectrometry for the determination of the substance P receptor antagonist ezlopitant and its two active metabolites in plasma

A simple, but highly sensitive and specific, assay was developed fur the qu antitative determination of ezlopitant and its two active metabolites in hu man plasma using narrow-bore reversed-phase high-performance liquid chromat ography (HPLC) coupled with electrospray tandem mass spectrometry (ES-MS-MS ). Ezlopitant, its two pharmacologically active metabolites, an alkene anal ogue (CJ-12 458) and a benzyl alcohol analogue (CJ-12 764), and their corre sponding trideuterated internal standards (I.S.), were extracted from plasm a with methyl tert.-butyl ether (MTBE). The dried MTBE extracts were recons tituted and analyzed using a narrow-bore (2.1 mm I.D.) YMC basic HPLC colum n and a mobile phase of acetonitrile-20 mM ammonium acetate, pH 5 (60:40, v /v). Column effluent was monitored by pneumatically assisted electrospray t andem mass spectrometry. Multiple reaction monitoring (MRM) using the paren t to product ions was used to quantify ezlopitant and its two active metabo lites. The assay exhibited a linear dynamic range of 0.1-100 ng/ml. Average absolute recoveries from plasma were approximately 71, 80 and 99% for ezlo pitant and its two active metabolites CT-12 485 and CJ-12 764, respectively . The precision (RSD %) and accuracy (Deviation %) values for the method we re within +/-12% and +/-15%, respectively, for all analytes. Sample analysi s times were less than 5 min from one injection to the next. The assay prov ed to be suitable for pharmacokinetics studies. (C) 2000 Elsevier Science B .V. All rights reserved.