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Letters |
Institut de Médecine Légale, 11 rue Humann, F-67000 Strasbourg, France
a Author for correspondence. Fax 33-3-88-24-00-85; e-mail pascal.kintz{at}wanadoo fr.
To the Editor:
Nandrolone (19-nortestosterone or 17ß-hydroxy-19-nor-4-androsten-3-one) has been one of the most abused anabolic steroids, and doping practices are increasing, as revealed by numerous positive cases during recent years. Athletes use nandrolone because it has been claimed to increase lean body mass, strength, and aggressiveness, and shorten the recovery time between workouts.
Nandrolone is metabolized to norandrosterone (NA) and noretiocholanolone (1). Other 19-norsteroids, such as norandrostenedione or norandrostenediol, classified as anabolic androgenic steroids by the Olympic Movement Anti-doping Code of December 1999 (2), are available over the counter or through the Internet and have the same metabolites as nandrolone (1)(3)(4).
Although norandrostenediol and norandrostenedione are banned by the International Olympic Committee, there is a great need in forensic and sports sciences to discriminate nandrolone from other 19-norsteroids. This is obviously not possible in urine because the metabolites are common. The ratio between NA and noretiocholanolone has been recently proposed to differentiate the nature of the doping agent, but mixtures can be ingested (4).
Because forensic laboratories can be involved in testimony dealing with doping agents, the idea of using hair for doping control has emerged because hair analysis has been accepted in other court cases. Hair can both confirm repetitive abuse and identify the exact nature of the parent compound (e.g., nandrolone, norandrostenediol, or norandrostenedione in cases of positive urine for NA) because it has been accepted by the scientific community that the parent compound is the major analyte that is incorporated in hair (5)(6). Thus, hair analysis would discriminate nandrolone abuse from over-the-counter preparations containing 19-norsteroids.
Recently, our laboratory was requested by an attorney to evaluate potential doping practices of an athlete. The 30-year-old subject had tested positive for NA in urine at 230 µg/L. The analysis was done in an accredited laboratory, but the athlete denied the result. Hair samples were then tested for nandrolone, norandrostenediol, and norandrostenedione by a modification of our previous procedure for testosterone (7).
Full-length hair samples (12 cm long) were taken at the surface of the skin from the vertex of the athlete and stored in plastic tubes at room temperature. Only the first 6 cm from the root were analyzed. The hair color was brown.
We incubated 100 mg of hair decontaminated with dichloromethane in 1 mL of 1 mol/L NaOH for 15 min at 95 °C in the presence of 10 ng of nandrolone-d3 used as internal standard. After cooling, the homogenate was neutralized with 1 mL of 1 mol/L HCl, and 2 mL of 0.2 mol/L phosphate buffer (pH 7.0) was added. The drugs were extracted by solid-phase extraction. Isolute C18 columns were conditioned with 3 mL of methanol, followed by 2 mL of deionized water. After sample addition, the columns were washed twice with 1 mL of deionized water. After the columns were dried, analytes were eluted by the addition of three 0.5-mL aliquots of methanol. The eluant was evaporated to dryness under nitrogen, and the residue was reconstituted in 1 mL of 0.2 mol/L phosphate buffer (pH 7.0). The sample was further purified by the addition of 100 mg of Na2CO3-NaHCO3 (1:10 by volume) and 2 mL of pentane. After agitation and centrifugation, the organic phase was removed and evaporated to dryness. The residue was derivatized by the addition of 50 µL of MSTFA-NH4I-2-mercaptoethanol (1000:2:5 by volume), and then incubated for 20 min at 60 °C.
A 4-µL aliquot of the derivatized extract was injected into the column of a Hewlett Packard gas chromatograph (6890 Series) via a Hewlett Packard (model 7673) autosampler. The flow of carrier gas (helium, purity grade N 55) through the column [HP5-MS capillary column; 5% phenyl-95% methylsiloxane; 30 m x 0.25 mm (i.d.); 0.25 mm film thickness] was 1.0 mL/min. The injector temperature was 270 °C, and splitless injection was used, with a split valve off-time of 1.0 min, using the pulsed mode. The column oven temperature was programmed to increase from an initial temperature of 150 °C, maintained for 1 min, to 295 °C at 30 °C/min, and was maintained at 295 °C for the final 8 min. The detector was a Hewlett Packard 5973 operated in the electron impact (EI) mode. The electron multiplier voltage was set at 600 V above the EI-tune voltage.
Under the chromatographic conditions used, there was no interference
with the analytes by any extractable endogenous materials present in
hair. The three drugs were stable during alkaline treatment. Selected
ions, retention times, and analytical parameters of the drugs are
reported in Table 1
. Analytes were identified and quantified on the basis of their
retention times (± 1% from that of the calibrators) and the abundance
of three diagnostic ions (with ions ratios within ± 20% of the
calibrators).
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Responses for the three norsteroids were linear at 1–50 pg/mg, with correlation coefficients of 0.992–0.997. The within-run imprecisions (as CVs) were 11–18%, as determined by analysis of six replicates of 100 mg of drug-free hair obtained from the same subject with a final concentration of 10 pg of norsteroid added per mg of hair. The extraction recovery (n = 3) was 76.1–86.4%. The limits of detection (signal-to-noise ratio >3) of the doping agents were 0.5–1 pg/mg and 4–8 pg absolute.
The analysis of a strand of hair obtained from the athlete revealed the presence of 19-norandrostenedione at the concentration of 7 pg/mg. This result was not challenged by the athlete. Nandrolone has also been identified by this laboratory in the hair of bodybuilders, in concentrations of 1–7 pg/mg (8). NA and noretiocholanolone were not detected in any hair.
The sensitive, specific, and reproducible method developed appears to be suitable for the detection and quantification of 19-norsteroids in human hair. The determination of one 19-norsteroid in hair may allow unambiguous confirmation of the exact nature of the abused agent.
Hair analysis may be a useful adjunct to conventional drug testing in sports because hair can provide a more accurate history of drug use than urine (9). This technology may find useful applications in doping control if accepted by the International Olympic Committee.
References
5 anabolic steroids 5-androsen-3,17-dione; 5-androsten-3ß, 17ß-diol; dehydroepiandrosterone; and 19-nor-5-androsten-3,17-dione: excretion studies in men. J Anal Toxicol 2000;24:188-193.[Medline]
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