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 for the quantitative determination of ezlopitant and its two active metabolites in human plasma using narrow-bore reversed-phase high-performance liquid chromatography (HPLC) coupled with electrospray tandem mass spectrometry (ES–MS–MS). Ezlopitant, its two pharmacologically active metabolites, an alkene analogue (CJ-12 458) and a benzyl alcohol analogue (CJ-12 764), and their corresponding trideuterated internal standards (I.S.), were extracted from plasma with methyl tert.-butyl ether (MTBE). The dried MTBE extracts were reconstituted and analyzed using a narrow-bore (2.1 mm I.D.) YMC basic HPLC column and a mobile phase of acetonitrile–20 mM ammonium acetate, pH 5 (60:40, v/v). Column effluent was monitored by pneumatically assisted electrospray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to product ions was used to quantify ezlopitant and its two active metabolites. 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 ezlopitant and its two active metabolites CJ-12 485 and CJ-12 764, respectively. The precision (RSD %) and accuracy (Deviation %) values for the method were within ±12% and ±15%, respectively, for all analytes. Sample analysis times were less than 5 min from one injection to the next. The assay proved to be suitable for pharmacokinetics studies.