Enantiomeric determination of amphetamine and methamphetamine in urine by precolumn derivatization with Marfey's reagent and HPLC

An analytical method was developed for enantiomeric determination of amphetamine and methamphetamine in human urine. The enantiomers were isolated from urine by solid-phase extraction, and diastereomers were formed by derivatization with the chiral Marfey's reagent (1-fluoro-2,4-dinitrophenyl- 5-l-aniline amide). The diastereomers were separated by reversed-phase high- performance liquid chromatography in a water/methanol mobile phase and detected by absorbance spectrophotometry at 340 nm. Linear standard curves were obtained for all four enantiomers over a concentration range of 0.16- 1.00 mg/L in urine. The detection limit was 0.16 mg/L urine for each enantiomer, and the limit of quantitation was 0.40 mg/L. The urine of 10 decedents was analyzed by this method and by a previously published precolumn derivatization procedure using (-)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as the derivatizing agent and fluorescence detection. Comparison of the results of the two methods by linear regression showed comparable results for both d-amphetamine and d-methamphetamine. Neither method detected the presence of the l-enantiomers in the urine samples.