Separation and quantitation of the enantiomers of methamphetamine and its metabolites in urine by HPLC

Precolumn derivatization and fluorescence detection

J. Sukbuntherng, A. Hutchaleelaha, Hsiao-Hui Chow, Michael Mayersohn

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21 Citations (Scopus)

Abstract

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-hydroxy-methamphetamine (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 β-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 5-100 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 I-AP (p < .005). The urinary recovery of the I-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
Volume19
Issue number3
StatePublished - 1995

Fingerprint

Methamphetamine
Enantiomers
Metabolites
urine
metabolite
fluorescence
Fluorescence
Urine
liquid
Liquids
Rats
Amphetamine
Assays
acetate
assay
p-Hydroxyamphetamine
wavelength
Recovery
Wavelength
Acetonitrile

ASJC Scopus subject areas

  • Analytical Chemistry
  • Health, Toxicology and Mutagenesis
  • Toxicology

Cite this

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title = "Separation and quantitation of the enantiomers of methamphetamine and its metabolites in urine by HPLC: Precolumn derivatization and fluorescence detection",
abstract = "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-hydroxy-methamphetamine (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 β-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 5-100 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 I-AP (p < .005). The urinary recovery of the I-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.",
author = "J. Sukbuntherng and A. Hutchaleelaha and Hsiao-Hui Chow and Michael Mayersohn",
year = "1995",
language = "English (US)",
volume = "19",
pages = "139--147",
journal = "Journal of Analytical Toxicology",
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TY - JOUR

T1 - Separation and quantitation of the enantiomers of methamphetamine and its metabolites in urine by HPLC

T2 - Precolumn derivatization and fluorescence detection

AU - Sukbuntherng, J.

AU - Hutchaleelaha, A.

AU - Chow, Hsiao-Hui

AU - Mayersohn, Michael

PY - 1995

Y1 - 1995

N2 - 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-hydroxy-methamphetamine (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 β-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 5-100 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 I-AP (p < .005). The urinary recovery of the I-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.

AB - 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-hydroxy-methamphetamine (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 β-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 5-100 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 I-AP (p < .005). The urinary recovery of the I-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.

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