Determination of a substance P antagonist in human plasma and urine using high-performance liquid chromatography with ultraviolet absorbance and tandem mass spectrometric detection.

A high-performance liquid chromatographic (HPLC) assay using ultraviolet (UV) detection was developed and compared with a HPLC method with tandem mass spectrometric (HPLC/MS-MS) detection for the determination of a substance P receptor antagonist 2(S)-((3,5-bis(trifluoromethyl)benzyl)-oxy)-3(S)-phenyl-4-((3-oxo-1,2,4- triazol-5-yl) methyl)morpholine (Fig. 1, Ia, L-742 694) in human plasma and urine. The drug was isolated from the biological matrix through liquid-liquid extraction. In the HPLC/UV method, the samples were initially injected onto a cyano Hypersil column, and the chromatographic region containing the peaks of interest was heart-cut onto an analytical C-18 Hypersil column via a column switching device. The analyte was quantified by monitoring absorbance at 205 nm. The limit of quantification for I extracted from 1 ml of plasma or urine was 2.5 ng ml-1, and the assays were validated in the concentration range 2.5-500 ng ml-1. The HPLC/MS-MS method were validated in the concentration range 0.2-500 ng ml-1. Both assays provided data with precision, measured as coefficient of variation, better than 10% at all points within the standard curve range and with adequate accuracy.