Studies on Comparison of Metabolites in Urine between Deprenyl and Methamphetamine. III. Enantiomeric Composition Analysis of Metabolites in Mouse Urine by HPLC with GITC Chiral Reagent.

Abstract

Enantiomeric compositions of methamphetamine (MA) and its major metabolite, amphetamine (AP), in the urine of mice administered with deprenyl (DPN) and MA were analysed by high-performance liquid chromatography using the tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate (GITC) derivatization method for the discrimination between the use of l-DPN and MA enantiomers. l-DPN, d-MA, l-MA and dl-MA were intraperitoneally administered to mice at 1, 5 and 10 mg/kg. Urine samples were collected at 0-12, 12-24, 24-48 and 48-72 h after administration, Urine added with N-methylphenethylamine (internal standard) was applied to BOND ELUT CERTIFY (1 ml), washed with 1 ml of 0.1 M acetic acid and 6 ml of methanol, and eluted with 2 ml of conc. HCl-methanol (9 : 91). The eluent was evaporated, redissolved in 1 ml of water, basified with 1 N NaOH and extracted with chloroform. The amphetamines in chloroform layer was derivatized with GITC at room temperature for 30 min. The GITC-derivatives were analysed by HPLC with a UV detector (254 nm). As a result, administration of l-DPN could be clearly discriminated from that of d-MA and dl-MA by comparisons of the enantiomeric compositions of MA excreted in the urine. Discrimination between administration of l-DPN and l-MA could be carried out by the apparent difference of ratio of AP/MA in the urine. The AP/MA ratio of the former was 10 times that of the latter.