Published: Journal of Analytical Toxicology, ISSN 0146-4760, Volume 24, Number 5, July/August, pp.381-382

Here is where the title stuff goes

Ephedrine Abuse for Doping Purposes as Demonstrated by Hair Analysis

To the Editor:

Ephedrine is a sympathomimetic amine that mimics the stimulant action of amphetamine. For this reason, ephedrine is included in the list of banned drugs of the International Olympic Committee.

The standard of testing for sympathomimetic drugs and derivatives in case of doping control is gas chromatography coupled to mass spectrometry (GC–MS) conducted on a urine sample by accredited laboratories. In urine, the elimination of ephedrine occurs within a few days. To increase the window of detection and to document long-term abuse, hair has been recently proposed in sports (1). This specimen has found numerous applications for 20 years in both forensic and clinical toxicology.

The international literature is very poor in papers dealing with the identification of ephedrine in human hair. Only one reference (2) reports the identification of the drug in human hair after ultrasonication and overnight incubation in methanol/5N HCl (20:1, v/v). Ephedrine was measured at an average of 2.25 ng/mg (n = 4).

This paper describes a GC–MS-based procedure for the identification and quantitation of ephedrine in hair and its application in doping control. The procedure is based on a modification of the screening method proposed by Sachs and Kintz (3).

After decontamination with 5 mL of dichloromethane at room temperature (2 times, 2 min each), 50 mg of hair was hydrolyzed with 1 mL of 1N NaOH for 15 min at 95°C, in the presence of 50 ng of methamphetamine-d5 used as internal standard. After extraction with 5 mL of ethyl acetate and evaporation to dryness in the presence of 100 µL of 2-propanol/ HCl (99:1, v/v), the target compounds were derivatized with 75 µL of heptafluorobutyric acid anhydride (HFBA) in the presence of ethyl acetate (2:1, v/v) at 80°C for 15 min. Then the mixture was evaporated to dryness and the residue resuspended in 50 µL of ethyl acetate.

A 2-µL aliquot of the derivatized extract was injected in splitless mode into an apolar capillary column (Crompack Cpsil 8CB, 25 m x 0.25-mm i.d., 0.25-µm film thickness) of a Hewlett-Packard GC (5890 series) via a Hewlett-Packard 7673 autosampler. The flow of carrier gas (helium N55) was 1 mL/min. The injector temperature was 240°C. The column oven temperature was programmed to rise from 60°C after a 1-min hold for 1 min, rise to 300°C at 25°C/min, and hold at 300°C for 5 min. The detector was a Hewlett-Packard 5971 operated in the electron impact mode. The electron multiplier voltage was set at +200V above the EI tune voltage. Acquisition was realized in scan mode from 80 to 400 amu. Analytes were identified and quantitated on the basis of retention time and a comparison of the abundance of specific ions (7.48 min and m/z 258 for methamphetamine-d5, 7.63 min and m/z 254, 169, 210 for ephedrine).

The assay was linear from 0.1 to 50 ng/mg (r = 0.998) with a recovery of 87% at 4 ng/mg. The limit of detection for ephedrine was 0.04 ng/mg with a signal-to-noise ratio of 3.

This procedure was used for the unique identification of ephedrine abuse by two bodybuilders and one cyclist.

The first case concerns two bodybuilders in possession of ephedrine tablets and ampoules of various anabolic steroids, b-agonists, and growth hormone who were arrested by the French Police at the Spanish border (Hendaye, France). The older athlete (45 years of age) was on the French team for 10 years; the other (28 years of age) was a French winner in his category. Ephedrine was identified and quantitated in hair from both bodybuilders at concentrations of 10.7 (Figure 1) and 0.67 ng/mg, respectively. No other stimulants were identified.

The second case concerns an internationally competing 26-year-old cyclist with blond hair. Ephedrine was identified and quantitated in the cyclist’s hair at a concentration of 0.66 ng/mg. Amphetamine was also measured at a concentration of 0.37 ng/mg.

In conclusion, this is the first report where ephedrine was identified as a doping agent through hair analysis. This technique may be a useful adjunct to conventional drug testing in sports. Methods for evading urine analysis do not affect the drug concentrations in hair. Specimens can be more easily obtained with less embarrassment, and hair can provide a more accurate history of drug use. Costs are too high for routine use, but the data generated are extremely helpful in documenting positive urine cases. This new technology may find useful applications in doping control, if accepted by the International Olympic Committee.

Some issues have to be discussed before considering hair as a valid specimen by the International Olympic Committee and the International Sport Federations. The relationship between urine and hair results is not yet established, and a negative hair test result does not mean that no doping has taken place.

Véronique Dumestre-Toulet1 and Pascal Kintz2

1Laboratoire BIOffice, Avenue Gay Lussac, 33370 Artigues pr`es Bordeaux, France and 2Institut de Médecine Légale, 11, rue Humann, 67000 Strasbourg, France

References

1. P. Kintz. Hair testing and doping control in sport. Toxicol. Lett. 102-103: 109–113 (1998).
2. Y. Nakahara and R. Kikura. Hair analysis for drugs of abuse—determination of ephedrine and its homologs in rat hair and human hair. J. Chromatogr. B 700: 83–91 (1997).
3. H. Sachs and P. Kintz. Testing for drugs in hair. Critical review of chromatographic procedures since 1992. J. Chromatogr. B 713: 147–161 (1998).

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