Journal of Pharmacological and Toxicological Methods
Original articleA rapid method of determining amphetamine in plasma samples using pentafluorobenzenesulfonyl chloride and electron-capture gas chromatography
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.
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