Simultaneous Determination of the N ?Dealkylated Metabolites of Four Butyrophenone?Type Agents in Rat Plasma by HPLC with Fluorescence Detection after Precolumn Derivatization with 4?Fluoro?7?Nitro?2,1,3?Benzoxadiazole

Abstract

The basic metabolites of butyrophenone type agents are sometimes more neurotoxic than the parent compounds. We have developed a high performance liquid chromatographic method with fluorescence detection (HPLC‐FL) to quantify the N‐dealkylated basic metabolites, i.e., 1,3‐dihydro‐1‐(1,2,3,6‐tetrahydro‐4‐pyridinyl) ‐2H‐benzimidazole‐2‐one, 1‐phenyl‐1,3,8‐triazaspiro[4.5]decan‐4‐one, 4‐(4‐chlorophenyl)‐4hydroxypiperidine, and 4‐(4‐bromophenyl)‐4‐hydroxypiperidine, of droperidol, spiperone, haloperidol, and bromperidol, respectively, in rat plasma after precolumn derivatization with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F). Mexiletine was carried through the procedure as an internal standard (IS). After liquid‐liquid extraction with benzene and evaporation, derivatization with NBD‐F was conducted in borate buffer (pH 8.5) at 60°C for 3 min. HPLC was conducted with a reversed‐phase (C₁₈) column, eluted with a mixture of methanol‐water‐trifluoroacetic acid (600:400:0.4, v/v/v) at a flow rate of 1.0 mL/min at 25°C. The derivatives of four basic metabolites and the IS were well separated from each other in less than 48 min. The calibration curves were linear up to 0.5 µg/mL, and the lower limits of detection were 0.002 to 0.03 µg/mL. The coefficients of variation were less than 13.3%. These results confirm that HPLC‐FL after precolumn derivatization with NBD‐F is sensitive and satisfactory for the simultaneous assay of basic metabolites of four butyrophenone type agents in rat plasma.