Original article
A rapid method of determining amphetamine in plasma samples using pentafluorobenzenesulfonyl chloride and electron-capture gas chromatography

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Abstract

Introduction: Acute administration of (+)-amphetamine has been used as a model for mania in humans since it mimics the physiological, biochemical, and cognitive effects seen in mania. A rapid and sensitive method for the determination of amphetamine in human plasma samples using gas chromatography with electron-capture detection was developed in our laboratory to follow the time course of amphetamine levels in patients receiving this drug as part of a study using amphetamine as a model for mania. Methods: Blood samples were taken from healthy male volunteers at 30, 60, 90, 150, 210, 240, and 480 min after administration of 25 mg of (+)-amphetamine. Plasma was isolated by centrifugation and used for the analysis. This method is a modification of the procedure described by Paetsch et al. [J. Chromatogr. 573 (1992) 313] for the determination of amphetamine in rat brain tissue. Amphetamine was derivatized under basic conditions using pentafluorobenzenesulfonyl chloride (PFBSC) prior to analysis on a gas chromatograph equipped with a capillary column and an electron-capture detector. The internal standard used was benzylamine. The structure of the amphetamine derivative was confirmed using combined gas chromatography–mass spectrometry (GC-MS). Results: The limit of detection was <1 ng/ml, and the method was linear in the 1- to 100-ng range used. Mean amphetamine levels peaked at 3.5 h after drug administration, and were 40.8±1.5 ng/ml at that time. Discussion: This procedure produces a stable derivative with excellent chromatographic properties and is both simple and reproducible.

Introduction

The amphetamines and related stimulants display powerful cardiovascular, central stimulant, hyperthermic and anxiogenic properties. They have been a source of attention due to their drug abuse potential as well as therapeutic use in the treatment of narcolepsy and attention deficit hyperactivity disorder (Brust, 1993). As well, amphetamine is a useful research tool in the study of mania as it mimics the physiological, biochemical, and cognitive effects seen in mania (Jacobs & Silverstone, 1986). Symptoms of increased blood pressure and heart rate Brauer et al., 1996, Caldwell, 1996, de Wit et al., 1997, Jacobs & Silverstone, 1986, Slattum et al., 1996, decreased reaction time Rapoport et al., 1980, Servan-Schreiber et al., 1998, and elevation of mood de Wit et al., 1997, Miller & Griffith, 1983, Silverstone et al., 1983 have been demonstrated with the acute administration of oral amphetamine

Numerous techniques have previously been used to quantify amphetamine. These include: radioimmunoassay Schmidt & Ebert, 1988, Ward et al., 1994; combined gas chromatography–mass spectrometry (GC-MS) Dasgupta & Spies, 1998, Sato & Mitsui, 1997, Suzuki et al., 1989; high-resolution MS (Danielson & Boulton, 1974); GC with flame-ionization detection (Kintz, Tracqui, Mangin, Lugnier, & Chaumont, 1989), nitrogen–phosphorus detection Cheung et al., 1997, Jacob et al., 1995, Terada, 1985, or electron-capture detection (GC-ECD) Coutts et al., 1984, Paetsch et al., 1992; and high-performance liquid chromatography (HPLC) with ultraviolet detection (Farrell & Jefferies, 1983) or chemiluminescence detection (HPLC-CD) Hayakawa et al., 1989, Nakashima et al., 1992. However, some of these methods are expensive, time consuming and/or involve laborious extraction procedures.

Gas chromatography is a relatively inexpensive technique, which is accessible to many laboratories. Analysis of amphetamine generally requires derivatization of its amino group to increase sensitivity and selectivity as well as increase volatility, reduce polarity, and improve chromatographic properties. Derivatives that have been used include acetyl (Lebish, Finkle, & Brackett, 1970), n-propyl (Jacob et al., 1995), trifluoroacetyl (Suzuki et al., 1989), trichloroacetyl (Hornbeck & Czarny, 1989), trichloroethyl chloroformate (Dasgupta & Spies, 1998), heptafluorobutyric (Cheung et al., 1997), pentafluorobenzoyl (Terada, 1985), pentafluorobenzenesulfonyl (Paetsch et al., 1992), pentafluorobenzyl (Sato & Mitsui, 1997), pentafluoropropionyl (Valentine et al., 1995), perfluorooctanoyl Gjerde et al., 1993, Thompson & Dasgupta, 1994, and 4-carbethoxyhexafluorobutyl (Czarny & Hornbeck, 1989).

A relatively simple, sensitive, and reproducible assay for amphetamine levels in plasma was desirable for our clinical studies, where numerous samples were to be analyzed, and for this purpose the procedure of Paetsch et al. (1992), using pentafluorobenzenesulfonyl chloride (PFBSC) for analysis of amphetamine in rat brain, was modified as described in the present report.

Section snippets

Materials and methods

This double-blind, crossover study was part of an investigation of the physiological, cognitive, mood, and neurochemical effects of acute amphetamine administration in healthy volunteers.

Results and discussion

The procedure described here is rapid and the derivatives formed are stable and have excellent chromatographic properties. The retention times of derivatized benzylamine (the internal standard) and amphetamine were 20.1 and 21.8 min, respectively (Fig. 1). The mass spectral analysis was consistent with the structure of N-pentafluorobenzenesulfonylamphetamine. The standard curves were linear from 1 to 100 ng (r2>.99 obtained routinely) (Fig. 2). The procedure was sensitive to <1 ng/ml in plasma,

Acknowledgements

The authors are grateful to the Alberta Heritage Foundation for Medical Research and the Canadian Institutes for Health Research (CIHR) for financial support. The expert advice of Drs. Ronald Coutts, Susan Rotzinger, Veronique Tanay, and Mrs. J. van Muyden is greatly appreciated.

References (36)

  • D Servan-Schreiber et al.

    Dopamine and the mechanisms of cognition: Part II. d-amphetamine effects in human subjects performing a selective attention task

    Biological Psychiatry

    (1998)
  • M Terada

    Determination of methamphetamine and its metabolites in rat tissues by gas chromatography with a nitrogen–phosphorus detector

    Journal of Chromatography

    (1985)
  • L.J Urichuk et al.

    Determination of p-trifluoromethylphenol, a metabolite of fluoxetine, in tissues and body fluids using an electron-capture gas chromatographic procedure

    Journal of Chromatography, Biomedical Sciences and Applications

    (1997)
  • L.H Brauer et al.

    Acute tolerance to subjective but not cardiovascular effects of d-amphetamine in normal, healthy men

    Journal of Clinical Psychopharmacology

    (1996)
  • J.C.M Brust

    Neurological aspects of substance abuse

    (1993)
  • J.A Caldwell

    Effects of operationally effective doses of dextroamphetamine on heart rates and blood pressures of army aviators

    Military Medicine

    (1996)
  • R.J Czarny et al.

    Quantitation of methamphetamine and amphetamine in urine by capillary GC/MS: Part II. Derivatization with 4-carbethoxyhexafluorobutyryl chloride

    Journal of Analytical Toxicology

    (1989)
  • T.J Danielson et al.

    Detection and quantitative analysis of amphetamine

    Biomedical Mass Spectrometry

    (1974)
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