Development of a Method for Measuring Benzphetamine Metabolites in Urine Using HPLC after Solid Phase Extraction

Abstract

A method for quantitative analysis of p-hydroxy-N-benzylamphetamine which is the main urinary metabolite of benzphetamine in rats was developed using HPLC. The rat urine sample was adjusted to pH 6 with 0.1 M acetic acid. Each sample was applied to a solid phase extraction column, Bond Elut Certify which contains the octyl silyl group and cation exchange functional group. After subsequently washing the column with water, 0.01 M acetic acid and methanol, p-hydroxy-N-benzylamphetamine was eluted with dichloromethane: isopropanol: 28% ammonium hydroxide =96.04: 1.96: 2.00 (v/v%). The eluate was evaporated and the residue dissolved in acetonitrile: PIC B-5 (5 mM l-pentane sulfonic acid) =5: 95 was analysed by HPLC. The calibration curves showed good linearity between the following two concentration ranges: 0.138-27.5 nmol/ml and 6.90-1,380 nmol/ml. The detection limit of p-hydroxy-Nbenzylamphetamine was 0.021 nmol. The mean recovery was 103.3%. The amount of urinary phydroxy-N-benzylamphetamine obtained from rats after subcutaneous injection of 5mg/kg or 50mg/kg of benzphetamine hydrochloride was determined by the present method. As a result, about 8% of benzphetamine was excreted as p-hydroxy-N-benzylamphetamine and almost all of p-hydroxy-N-benzylamphetamine was excreted within 24 h.