Enantiomeric Determination of Amphetamine and Methamphetamine in Urine by Precolumn Derivatization with Marfey's Reagent and HPLC

• Published in: Journal of analytical toxicology 1998-07, Vol.22 (4), p.265-269
• Main Authors: Foster, Bryan S., Gilbert, Don D., Hutchaleelaha, Athiwat, Mayersohn, Michael
• Format: Article
• Language: English
• Subjects: Alanine - analogs & derivatives; Amphetamine - urine; Amphetamines - urine; Biological and medical sciences; Central Nervous System Stimulants - urine; Chromatography, High Pressure Liquid - methods; Dinitrobenzenes; Drug addictions; Drug Monitoring - methods; Female; Humans; Male; Medical sciences; Methamphetamine - urine; Reference Values; Sensitivity and Specificity; Toxicology
• ISSN: 0146-4760; 1945-2403
• DOI: 10.1093/jat/22.4.265

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.