Download Letter to the Editor I Ephedrine Abuse for Doping Purposes as

Journal of Analytical Toxicology, Vol. 24, July/August 2000
Letter to the Editor I
EphedrineAbusefor Doping Purposesas 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
50000Q1
Ion254.~ (253.70Io 2~.~0: Me.O
in papers dealing with the identification
of ephedrine in human hair. Only one
400~0.1
reference (2) reports the identification of
300000~
~ : 1 0 . 7 ~
the drug in human hair after
ultrasonication and overnight incubation
<C
in methanol/5N HCI (20:1, v/v).
Ephedrine was measured at an average of
2.25 ng/mg (n = 4).
Time (min)
This paper describes a GC-MS-based
k~ 2S8.00(257.) to 2~.70): MS.D
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
0.20 6.40 6.(10 8.80 7.00 720 7.40 7.80 7.80 8.00 8.20 8.40 8.80 8.80
dichloromethane at room temperature
Time (rain)
sam 2s1 (7.e~2r~n): kU2) (-)
(2 times, 2 rain each), 50 mg of hair was
,5o,0oo
hydrolyzedwith 1 mL of 1N NaOH for 15
rain at 95~ in the presence of 50 ng of
methamphetamine-d~used as internal
standard. After extraction with 5 mL of
ethyl acetate and evaporation to dryness
in the presence of 100 pL of 2-propanol/
.=
HCl (99:1, v/v), the target compounds
were derivatized with 75 pL of
<C
heptafluorobutyric acid anhydride
(HFBA) in the presence of ethyl acetate
(2:1, v/v) at 80~ for 15 rain. Then the
mixture was evaporated to dryness and
the residue resuspended in 50 pL of ethyl
acetate.
70 80 90 1(~1101201301401501eO1701801gQ2 ~ 210 220 ~ 240 ~ 21~ ~1280 2 ~ 300 310 320 ~ ~ ~
A 2-pL aliquot of the derivatized extract
m/z
was injected in splitless mode into an
apolar capillary column (Crompack Cpsil
Figure 1. Chromatogram of the hair analysis of one of the bodybuilders. Top window: ephedine (RT,
7.63 min) and internal standard (RT, 7.48 min) chromatogram. The ephedrine concentration was
8CB, 25 m x 0.25-ram i.d., 0.25-pm film
10.7 ng/mg. Bottom window: mass spectrum of ephedrine.
thickness) of a Hewlett-Packard GC (5890
series) via a Hewlett-Packard 7673
...
i ,~
0.20
. . . . . .
6.40
i ....
e.80
i ....
@80
L ....
7,00
J ....
7~0
i ....
7.40
J ....
7.00
= ....
7.80
p ....
8.00
i . . . " ~ . : , * l
-,;,,
8 ~n
8.40
8.80
",.'*
8.80
Z
~1
21o
/
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381
Journal of Analytical Toxicology, Vol. 24, July/August 2000
autosampler. The flow of carrier gas (helium N55) was i mL/min. The injector temperature was 240~ The column oven
temperature was programmed to rise from 60~ after a 1-min hold for I rain, rise to 300~ at 25~
and hold at 300~ 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-ds, 7.63 rain 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,
[3-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.
V~ronique Dumestre-Toulet1 and Pascal Kintz2
1Laboratoire BIOffice, Avenue Gay Lussac, 33370 Artigues pros Bordeaux, France and
2Institut de M~decine L~gale, 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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