Determination of lonazolac and its hydroxy and O-sulfated metabolites by on-line sample preparation liquid chromatography with fluorescence detection

A reliable method, which can be used for the determination of lonazolac and its hydroxylated and O-sulfated metabolites in cell culture media with methyllonazolac as the internal standard is described. The procedure employs on-line sample enrichment using a BioTrap 500 MS™ (20×4 mm I.D.) extraction pre-column and subsequent gradient separation on an Xterra MS C18-H™ (100×3 mm I.D., 3.5 μm particles) analytical column in the back-flush mode. Signal monitoring was done by measurement of fluorescence responses at 273 nm for excitation and 385 nm for emission. Structural identity of analyte peaks was confirmed by liquid chromatography coupled to mass spectroscopy (LC–MS–MS) using an electrospray ionization (ESI) source in the selected reaction monitoring (SRM) mode. Mean recoveries of lonazolac, hydroxylonazolac and lonazolac sulfate, respectively, from the biological matrix were 104.2±3.5, 96.7±2.2, and 100.9±3.5%. The limit of detection (LOD) for the three compounds was about 5 ng/ml using a total sample volume of only 50 μl. Linearity of signal responses versus concentration for all three analytes was accomplished in the range 10–600 ng/ml. The mean values of the coefficients of variation (C.V.) for quality control samples measured in duplicate at three different days at the 10, 40, 100, and 400 ng/ml level were 4.46±1.15, 3.94±2.13 and 4.79±2.07% for lonazolac, hydroxylonazolac and lonazolac sulfate. The target analytes were sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.