SENSITIVE ENANTIOMER-SPECIFIC HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHICANALYSIS OF METHAMPHETAMINE AND AMPHETAMINE FROM SERUM USING PRECOLUMN FLUORESCENT DERIVATIZATION

In order to study the stereoselective disposition of methamphetamine ( MAP), a widely abused drug, we have developed a sensitive HPLC assay t o separate and quantitate the enantiomers of MAP and amphetamine (AP) in rat serum. Serum samples to which was added aniline sulfate (intern al standard) were alkalized with 0.02 M carbonate buffer (pH 10.6) and extracted with ethyl acetate. Following back extraction with hydrochl oric acid, neutralization, and reconstitution, the sample was derivati zed with (-)-fluorenylethyl chloroformate overnight at room temperatur e. The derivatized products were separated following injection onto a reversed-phase C,, column. The mobile phase consisted of 0.02 M acetat e buffer-acetonitrile-tetrahydrofuran (46:39:15, v/v). The fluorescent intensity of the effluent was monitored at excitation and emission wa velengths of 265 and 330 nm, respectively. The derivatized aniline, R- , S-AP, R- and S-MAP had retention times of 21.0, 22.6, 23.6, 27.7 and 29.0 min, respectively. Linear standard curves were obtained over the concentration range of 5-250 ng/ml. The inter-day and intra-day coeff icients of variation for the assay of all four compounds at 12.5, 50.0 and 250 ng/ml were in the range of 2.1-18.6%. The method was applied to quantitate the concentrations of MAP and AP enantiomers in rat seru m following a short term intravenous infusion of racemic MAP (15 mg/kg ). There were no differences in serum concentrations of MAP enantiomer s but the concentrations of S-AP were consistently greater than those of R-AP. These data suggest a stereoselective disposition for the form ation and/or elimination of amphetamine.