Sensitive enantiomer-specific high-performance liquid chromatographic analysis 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 to separate and quantitate the enantiomers of MAP and amphetamine (AP) in rat serum. Serum samples to which was added aniline sulfate (internal standard) were alkalized with 0.02 M carbonate buffer (pH 10.6) and extracted with ethyl acetate. Following back extraction with hydrochloric acid, neutralization, and reconstitution, the sample was derivatized with (-)-fluorenylethyl chloroformate overnight at room temperature. The derivatized products were separated following injection onto a reversed-phase C₁₈ column. The mobile phase consisted of 0.02 M acetate buffer-acetonitrile-tetrahydrofuran (46:39:15, v/v). The fluorescent intensity of the effluent was monitored at excitation and emission wavelengths 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 coefficients 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 serum following a short term intravenous infusion of racemic MAP (15 mg/kg). There were no differences in serum concentrations of MAP enantiomers but the concentrations of S-AP were consistently greater than those of R-AP. These data suggest a stereoselective disposition for the formation and/or elimination of amphetamine.