Hair analysis and detectability of single dose administration of androgenic steroid esters

Abstract

Detection of anabolic steroids in hair samples has been possible only in fatal cases or in cases of high-continuous dosages. In order to verify the possibility of detecting an acute administration, a sensitive and specific assay has been developed for the simultaneous determination of testosterone, nandrolone and some of their esters in hair. The analytes were extracted from finely cut hair with methanol–trifluoroacetic acid overnight. After the incubation, the mixture was evaporated to dryness, redissolved and extracted with hexane. The dried organic layer was silanised and analysed by GC–MS and GC–MS–MS. A sensitivity of at least 20 pg injected was obtained for all the analytes. In guinea pigs treated with a single intramuscular dose of 10 mg/kg nandrolone decanoate, neither nandrolone decanoate nor nandrolone were found in hair collected after 13 days, while both compounds were clearly detectable after four repeated doses (each dose every 3–4 days) of 20 mg/kg nandrolone decanoate. Neither nandrolone decanoate nor nandrolone could be detected in hair from a male healthy volunteer 1 month after treatment with 50 mg nandrolone decanoate, while his urine still tested highly positive for the main nandrolone metabolite (>100 ng/ml). Testosterone esters could not be detected in hair of healthy subjects collected respectively 3, 2 and 1 month after a single intramuscular administration of 250 mg testosterone enanthate (five subjects), a single intramuscular coadministration of 25 mg testosterone propionate plus 110 mg testosterone enanthate (one subject), or a single oral administration of 120 mg testosterone undecanoate (three subjects). Otherwise, hair analysis revealed an increase of testosterone concentration corresponding to the period of treatment. Analysis of blood and urine samples confirmed the absorption of those compounds. At the sensitivity achieved by the present method, no detection of nandrolone, nandrolone decanoate nor testosterone esters in hair seems to be obvious after a single dose administration.

Introduction

The detection of anabolic steroids misuse in sports is based on the analysis of urine samples [1]. In particular, the detection of testosterone misuse (usually administered in esterified forms) is currently based on the urinary testosterone to epitestosterone ratio, although in some rare instances physiological or pathological conditions could compromise the application of this general criterion [2]. In the case of nandrolone esters, nandrolone metabolites (norandrosterone and noretiocholanolone) are commonly investigated in urine. Recent works suggest that naturally produced norandrosterone can be detected with especially sensitive methods (gas chromatography–high resolution mass spectrometry GC–HRMS or gas chromatography–tandem mass spectrometry GC–MS–MS) at low concentrations (e.g. below 1 ng/ml in men) [3]. The detection in the body of traces of the unchanged esters would offer unequivocal confirmation of their consumption. Indeed, some authors proposed the determination of testosterone esters in plasma as a definitive proof of the administration of exogenous testosterone [4]. However, a problem still remains as blood collection cannot be executed without violating individual privacy and its collection is not permitted at the moment for doping controls.

The measurement of anabolic steroids in keratin matrix could be interesting since it offers an increase of time-window in retrospective detection of doping with these substances and the advantage of noninvasive collection. In the last decade, hair analysis has been regarded as an useful tool to detect some drugs (stimulants, β-adrenergic drugs, narcotics) which are also misused in sport [5], [6], [7], [8]. However, in the case of anabolic steroids, detection in hair samples has been possible only in fatal cases or in cases of high-continuous dosages [9].

The aim of the study was to verify the possibility of detecting an acute administration of some anabolic steroids, using sensitive and specific assays for the simultaneous determination of testosterone, nandrolone and some of their esters in hair. The entire procedure was tested by analysing hair samples obtained from guinea pigs chronically treated with nandrolone decanoate and was applied to healthy subjects receiving these drugs as single-dose administration.