Determination of BRL 46470 in human plasma by high performance liquid chromatography with ultraviolet absorbance detection followed by post-column photochemical reaction and fluorescence detection.

Abstract

A very sensitive and specific quantitative assay for BRL 46470, a selective 5-HT3 receptor antagonist, in human plasma was developed. The method uses HPLC with serial UV absorbance detection followed by post-column photochemical reaction and fluorescence detection to provide an ultra-sensitive and specific method with a wide quantitative range. The post-column photochemical reaction enhances the very weak native fluorescence of BRL 46470 by a factor of approximately 150. The quantification ranges were determined to be 0.1-1.5 ng ml-1 (fluorescence detection) and 1.5-200 ng ml-1 (UV absorbance detection) for BRL 46470. Results from a 3 d validation at nominal BRL 46470 concentrations of 0.1, 0.4, 1.0 and 1.5 ng ml-1, using post-column photochemical reaction and fluorescence detection, demonstrated precision ranges of 3.4-5.8% (average within-day) and 1.6-5.6% (between-day). The average accuracy ranged from 93.4 to 114.5%. Results from a 3 d validation at nominal BRL 46470 concentrations of 1.5, 4.0, 25 and 200 ng ml-1, using UV absorbance detection, demonstrated precision ranges of 2.0-8.2% (average within-day) and 1.0-3.4% (between-day). The average accuracy ranged from 86.3 to 103.7%. The recovery of BRL 46470 from human plasma was approximately 64%. Assay specificity was confirmed by HPLC-MS.