Simultaneous analysis of benzphetamine and its metabolites, and quantitation of urinary p-hydroxy-N-benzylamphetamine by micellar electrokinetic chromatography

Abstract

We developed a method for simultaneous analysis of benzphetamine (BZ) and its metabolites, p-hydroxy-N-benzylamphetamine (pHBA), p- hydroxybenzphetamine (pHBZ), amphetamine (AP), methamphetamine and p- hydroxymethamphetamine by micellar electrokinetic chromatography (MEKC). Urine samples from 0 - 15 h (3 h intervals) after oral administration of BZ (10 mg) were hydrolyzed with β-glucuronidase (EC 3.2.1.31) at 37°C overnight. The treated urine was applied to a solid phase extraction column Bond Elut Certify®. After sequentially washing the column with water, 0.1 mol/l acetic acid and methanol, the samples were eluted with dichloromethane: isopropanol: 28% ammonium hydroxide=78.4: 19.6: 2.0 (v/v %). The eluate was evaporated and the residue dissolved in running buffer was analyzed by MEKC. In urine from 0-3 h, AP, pHBZ and pHBA were detected. After that, only pHBA, which is one of the major metabolites of BZ in human urine, could be detected in the urine by the present method. A method for quantitation of pHBA by MEKC is described here. The effects of acetonitrile and sodium dodecyl sulfate in the running buffer of MEKC on the separation of BZ and its metabolites are also reported.