Separation and quantitation of the enantiomers of methamphetamine and its metabolites in urine by hplc: precolumn derivatization and fluorescence detection

Juthamas Sukbuntherng, Athiwat Hutchaleelaha, Hsiao Hui Chow, Michael Mayersohn

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21 Scopus citations


To study the disposition kinetics of methamphetamine (MAP), we have developed a sensitive high-performance liquid chromatographic (HPLC) assay to quantitate the enantiomers of MAP and its major metabolites, amphetamine (AP), p-hydroxymethamphetamine (p-OH-MAP), and p-hydroxyamphetamine (p-OH-AP), the latter two of which are hydroxylated metabolites, in rat urine. To determine conjugated hydroxylated metabolites, urine samples were treated with ²-glucuronidase. Both hydrolyzed and nonhydrolyzed p-OH-MAP and p-OH-AP were extracted into ethyl acetate and back extracted with 0.05M HCl. To determine MAP and AP, urine samples were extracted with benzene, followed by back extraction into 0.05M HCl. The acid layer was collected, and to it was added ()-1-(9-fluorenyl)ethyl chloroformate (FLEC) for the derivatization of MAP and its metabolites. Derivatization was allowed to proceed for 24 h at room temperature. The derivatized products were separated on a C18 column with a mobile phase consisting of acetate buffer (pH 3.6)-acetonitrile-tetrahydrofuran. Quantitation was achieved using a fluorescence detector at an excitation wavelength of 265 nm and an emission wavelength of 330 nm. Linear standard curves were obtained over the concentration range of 5100 ng/mL. The interday and intraday coefficients of variation for the assay for all eight enantiomers at 10 and 75 ng/mL were less than 13%. The detection limit was 5 ng/mL or 0.5 ng on-column. The method was applied to quantitate the concentration of MAP and its metabolites in rat urine following a short intravenous infusion of 7.5 mg/kg d, l-MAP. There were no significant differences in total recovery of MAP enantiomers in urine (at p of.05), but there was a greater excretion of d-AP compared with l-AP (p <.005). The urinary recovery of the l-enantiomer was consistently higher than the d-form for both p-OH-MAP and p-OH-AP (p <.02 and p <.002, respectively). These data suggest a stereoselective disposition of MAP in rats.

Original languageEnglish (US)
Pages (from-to)139-147
Number of pages9
JournalJournal of Analytical Toxicology
Issue number3
StatePublished - May 1995

ASJC Scopus subject areas

  • Analytical Chemistry
  • Environmental Chemistry
  • Toxicology
  • Health, Toxicology and Mutagenesis
  • Chemical Health and Safety


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