|SOFT - TIAFT 1998||Poster Session 3||Thursday October 8, 1998|
SEPARATION OF AMPHETAMINE AND METHAMPHETAMINE STEREOISOMERS IN URINE BY CHIRAL HIGH PRESSURE LIQUID CHROMATOGRAPHY|
Veeravan Lekskulchai and Alphonse Poklis
Department of Pathology, Medical College of Virginia Campus at Virginia Commonwealth University, Richmond, VA. 23298-0165, USA
|An enantiomeric separation of amphetamine and methamphetamine in urine by chiral high pressure liquid chromatography (HPLC) column is described. The method involved liquid/liquid extraction of 2 mL of urine and derivatization of the amphetamines with benzoyl chloride. The analytes were separated on a (S)-indole carboxylic acid and (R)-1-napthyl-ethylamine (Phenomenex, CA) chiral HPLC column and detected at a wavelength of 220 nm with a diode array detector. Hexane:2-propanol (90:10) was used as the mobile phase with a flow rate of 1.0 mL/min. Under these conditions, the retention times of d-amphetamine, l-amphetamine, d-methamphetamine, l-methamphetamine, and N-methylphenethyl amine (internal standard) were 13.3, 14.0, 8.6, 8.9, and 9.8 min., respectively. Quantitation was performed with by the method of peak height ratios.
Calibration curves were linear from 50 to 5,000 ng/mL with a LOD of 20 ng/mL and a LOQ of 50 ng/mL. The overall within run precision of the method yielded CVs of 6% (d-amphetamine), 8% (l-amphetamine), 4% (d-methamphetamine), and 6% (l-methamphetamine) at 500 ng/mL (n=5) and 2% (d-amphetamine), 5% (l-amphetamine, 3% (d-methamphetamine), and 3% (l-methamphetamine) at 2500 ng/mL (n = 5). The overall recovery of the method was 97% at analyte concentrations of 500 ng/mL and 102% at analyte concentrations of 2500 ng/mL. Popular phenylisopropylamine drugs were found not to interfere with the method. The results of enantiomeric analysis of 30 urines from methamphetamine abusers by the presented method and chiral GC/MS were compared by linear regression analysis which yielded good correlation for each enantiomer: r2 s ranged from 0.952 to 0.974. An advantage of the chiral HPLC method over the chiral GC/MS method was the greater accuracy in determining the ratio of the amphetamine optical isomers as there was no contribution to apparent isomer content due to contamination of d-TPC in l-TPC GC/MS derivatizing reagent.