Determination of a glycine/NMDA receptor antagonist in human plasma and urine using column-switching high-performance liquid chromatography with ultraviolet, fluorescence and tandem mass spectrometric detection

Abstract

High-performance liquid chromatography (HPLC) assays using ultraviolet (UV) and fluorescence (FL) detection were developed and compared with a liquid chromatography/tandem mass spectrometry (LC/MS-MS) method for determination of the glycine receptor antagonist 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1 H)-quinolone (L-701, 324, I) in human plasma and urine. The drug and internal standard (II) were isolated from the biological matrix through liquid-liquid extraction. In the HPLC-UV and HPLC-FL methods, the samples were initially injected onto a Cyano BDS Hypersil column, and the chromatographic region containing the peaks of interest was heart-cut onto an analytical C-18 BDS Hypersil column via a column-switching device. The analyte was quantified by monitoring either absorbance at 226 nm or fluorescence at 385 nm following 230 nm excitation. The limit of quantitation for I extracted from 1 ml of plasma or urine was 5 ng ml −1, and the assays were validated in the concentration range of 5–200 ng ml −1. The LC/MS-MS method also utilized a column-switching protocol and was validated in the concentration range of 1–200 ng ml −1. Both assays provided data with precision and accuracy within less than 10% for all points in the standard curve range.