Simultaneous Determination of N-Dealkylated Metabolites of Four Butyrophenone-Type Agents by HPLC with Fluorescence Detection after Pre-Column Derivatization with Dansyl Chloride

Abstract

The basic metabolites of butyrophenone-type agents are sometimes more neurotoxic than the parent compounds. Recently, we developed a simple high performance liquid chromatographic (HPLC) method coupled with dual ultraviolet detection to determine the N-dealkylated basic metabolites, i.e., 1,3-dihydro-1-(1,2,3,6-tetrahydro-4-pyridinyl)-H-benzimidazole-2-one, 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 4-(4-chloro-phenyl)-4-hydroxypiperidine, and 4-(4-bromophenyl)-4-hydroxypiperidine (BPHP), of droperidol, spiperone, haloperidol, and bromperidol, respectively, in phosphate buffered saline (pH 7.4). An HPLC analysis with fluorescence detection was also developed to quantify these metabolites in rat plasma after precolumn derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). In this study, dansyl chloride (Dns-Cl) was used for determination of these metabolites instead of NBD-F. The samples in phosphate buffered saline were mixed with borate buffer (pH 9.5) and Dns-Cl solution in acetonitrile at 60°C for 15 min. HPLC was conducted with a reversed phase (C18) column, eluted with a mixture of methanol—water—acetic acid (650:350:4, v/v/v) at a flow rate of 1.0 mL/min at 25°C. The four Dns-derivatives were well separated from each other in less than 50 min. The calibration curves were linear up to 1 µg/mL, and the lower limits of detection were 0.005 to 0.06 µg/mL. The coefficients of variation were less than 13.1%. Since Dns-Cl is a much cheaper labeling agent than NBD-F, the present method is considered to be more suitable and practical for routine determination of these metabolites.