Development of a chiral assay for a novel, nonfluorinated quinolone, PGE-9509924, in dog plasma using high performance liquid chromatography with electrospray tandem mass spectrometry or fluorescence detection

Abstract

PGE-9509924 is a nonfluorinated quinolone and is active against a variety of susceptible and drug resistant bacteria in vitro and in animal infection models. A method for determining both enantiomers of PGE-9509924 in dog plasma has been developed. The enantiomers are derivatized with a chiral derivatizing agent, (−)-1-(9-fluorenyl)ethyl chloroformate (FLEC) and the resulting diastereomers are separated by reverse phase chromatography. Plasma samples are prepared via solid phase extraction (SPE) in a 96-well format prior to being derivatized. Samples are then analyzed by electrospray-LC/MS/MS with multiple reaction monitoring or by HPLC with fluorescence detection. Results of a side-by-side validation of the method with LC/MS/MS and HPLC/Fl detection are presented. Over the range selected for validation (0.025–10 μg/ml), both methods give similar results with identical limits of quantitation. Due to the selectivity of LC/MS/MS and the use of a stable-isotopically labeled internal standard, significantly shorter chromatographic runtimes are achieved with LC/MS/MS, making it the method of choice for sample analysis.