Determination of Fluoxetine and Norfluoxetine in Human Serum and Urine by HPLC Using a Cholester Column with Fluorescence Detection

Abstract

Fluoxetine (FLX), a selective serotonin reuptake inhibitor, is mainly demethylated to norfluoxetine (NFLX). In this study, FLX and NFLX levels in human serum and urine (each 100 µL) were simultaneously analyzed by HPLC fluorescence detection on a Cholester column after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples into pentane, derivatization with NBD-F was conducted in borate buffer (pH 8.5) at 70°C for 2 min. Protriptyline was utilized as an internal standard. The regression equations for FLX hydrochloride and NFLX hydrochloride in human serum showed good linearity in the range of 0.01–0.5 µg/mL with the detection limit of 0.005 µg/mL, and in the range of 0.005–0.5 µg/mL with the detection limit of 0.002 µg/mL, respectively. The corresponding values for human urine were 0.1–0.5 µg/mL with the detection limit of 0.06 µg/mL, and 0.1–0.5 µg/mL with the detection limit of 0.04 µg/mL, respectively. The coefficients of variation were less than 15.7% with good recovery. Our method is useful for a simple and sensitive determination of FLX and NFLX in human serum and urine using a sample volume as small as 100 µL, and should be suitable for therapeutic drug monitoring.