Compared interest between hair analysis and urinalysis in doping controls: Results for amphetamines, corticosteroids and anabolic steroids in racing cyclists

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Abstract

In France during a famous bicycle race, the newspapers documented the degree in which doping seemed to be supervised in some teams by managers and doctors. Use of anabolic steroids and other substances was officially banned in the mid-seventies by sports authorities. This policy has been enforced through urine testing before competition. It is well known, however, that a latency period is all that is necessary to defeat these tests. Nevertheless, hair analysis could be a promising tool when testing for periods that are not accessible to urinalysis any more. We have developed different sensitive methods for testing hair for amphetamines, anabolic steroids and their esters and corticosteroids. For amphetamines, 50 mg of hair were digested with 1 M NaOH, extracted with ethyl acetate, derivatized with TFA and analyzed by gas chromatography positive chemical-ionization mass spectrometry. For corticosteroids, 50 mg of powdered hair were treated with methanol in an ultrasonic bath and subsequently purified using a C18 solid phase extraction column. Analysis was realized by high performance liquid chromatography coupled to electrospray-ionization tandem mass spectrometry. For anabolic steroids and their esters, 100 mg of powdered hair were treated with methanol in an ultrasonic bath for extraction of esters, then alkaline digested with 1 M NaOH for an optimum recovery of other drugs. The two liquid preparations were subsequently extracted with ethyl acetate, pooled, then finally highly purified using a twin solid phase extraction on aminopropyl and silica cartridges. Residue was derivatized with MSTFA prior to injection. Analysis was conducted by gas chromatography coupled to a triple quadrupole mass spectrometer. Thirty cyclists were sampled and tested both in hair and in urine. Amphetamine was detected 10 times in hair (out of 19 analyses) compared to 6 times in urine (out of 30 analyses). Corticosteroids were detected 5 times in hair (methylprednisolone 1 case, triamcinolone acetonide 3 cases and hydrocortisone acetate 1 case) in hair (out of 12 analyses) compared to 12 times (triamcinolone acetonide 10 cases and betamethasone 2 cases) in urine (out of 30 analyses). Anabolic steroids were detected twice (nandrolone 1 case, and testosterone undecanoate 1 case) in hair (out of 25 analyses) compared to none in urine (out of 30 analyses).

Introduction

Doping is understood to be the utilization of one or more substances or procedures likely to produce an artificial enhancement of the mental or physical conditioning of an athlete [1], [2]. One of the most famous cases is the death of Tom Simpson during the Tour de France 1967 (13th stage, ascent of Mont Ventoux). The tour’s doctor refused the burial certificate and Public Justice appointed a toxicologist expert who, after the autopsy, revealed a high level of methyl amphetamine detected in urine, blood, gastric content and viscera [2]. In October 1968, the death of an 18-year-old amateur football player, J.L.Q., led to a court case [2]. Following an autopsy a judicial expert toxicologist revealed amphetamines in all viscera. For more than 30 years judicial experts from different European countries have been designated by the law courts to analyze biological specimens of blood and urine from deceased sportsmen and other cases. For the past two years in France both head and pubic hair have been analyzed. Since 1998 we have been appointed on more than thirty occasions by prosecutors in cases concerning doping. We were asked to find the cause of suspicious deaths of young athletes during sporting events; after complaints of sportsmen (positive finding by the French IOC Laboratory) for substance administration without their knowledge; to reveal doping substance administration by 61 indicted sportsmen; and to analyze products seized by Customs, Judicial Police, from sportsmen or found in cars or even along roads and in fields. All of these cases thoroughly illustrate, how forensic laboratories could be deeply involved in detection of doping agents. In relation to specimens from dead or living sportsmen, analysis of hair plays a major role in such cases when documenting doping practice in the general process of the research of the causes of death.

With regard to seizures of doping products, such as narcotics, it is important for prosecutors to know if the indicted person is a user, a dealer, or both. In this case hair analysis plays an important part. It allows distinction between repeated use or not, and gives an indication of quantities consumed.

Use of anabolic steroids was officially banned in the mid-seventies by sports authorities. In 1974 testosterone esters attracted a great deal of interest when it became known that they were undetectable as esters in urine where there is physiological testosterone. Thus, athletes became interested in using testosterone esters as an alternative several weeks before competition. The past situation in former East Germany shown by Franke and Berendonk [3], although well documented [4], [5], [6], [7], [8], is not an exception. A Californian doctor, Robert Kerr, admitted prescribing anabolic steroids to over 20 Olympic medallists of the 1984 Games in Los Angeles [9]. During the 1998 Tour de France bicycle race, newspapers and afterwards two books [15], [16] showed that doctors, trainers and the cyclists themselves [10], [17], [18] almost always organized doping in some teams. The quantity and the type of seizures of doping products (anabolic steroids, EPO, corticosteroids and stimulating agents) in cars, rooms or from athletes confirm data of the press about this subject.

Another approach to determine the extent of traffic is by observing official seizures by Customs or Police. In 1997 Customs carried out 61 seizures of doping products totaling 105,280 doses (tablets, ampoules and capsules). The Judicial Police service responsible for narcotics and doping products estimate they seize 5–10% of the black market. We can easily conclude that doping was and still is widely practiced at least for some sports.

The official statistics of 1997 indicate that weight lifting and cycling federations have the most significant number of positive cases (38 cases for weight lifting and 15 for cycling).

Random testing of cyclists in the Flanders region of Belgium from 1987–1994 revealed a positive rate of 7.8% for 4374 analyses [11]. Heading the list of banned substances were amphetamines, ephedrine, nandrolone and testosterone. During anti-doping tests performed in 1988 by the 20 laboratories accredited by the International Olympic Committee (IOC), 47,069 samples were tested and 1153 positive cases were found: nandrolone had the most occurrences (304), followed by 155 for testosterone [12].

We think that the relatively small number of positive cases is directly related to the matrix used. It is well known that a latency period is all that is necessary to invalidate these urine tests, which explains the small percentage of positive results from the IOC Laboratory.

More advanced techniques of detection such as tandem mass spectrometry (MS–MS) or high-resolution mass spectrometry (HRMS) should allow, in theory, to investigate further back in the past [13], [14]. In practice however, the number of positive cases found by the French IOC Laboratory is still approximately the same.

Sport doping practice is illegal, so trainers or competent doctors do not publish the techniques used on their athletes in medical or scientific reviews. However, data, gathered during interviews of athletes who came to our laboratory to have specimens taken, confirm declarations of anonymous sportsmen published in the press and may explain the small percentage of positive cases of anabolic steroids found by the IOC Laboratory. For these cyclists, the treatment was given during winter months and stopped 1–2 months before trials.

In relation to corticosteroids, we must remember that the IOC Laboratory does not look for them, which is the reason for a lack of results regarding these substances which are nevertheless used a lot.

Our experience of more than 1000 analyses of cow hair looking for anabolic steroids, whose use is forbidden in Europe for cattle breeding, showed us how to detect 24 anabolic steroids in hair, seven of which are testosterone esters.

Daily forensic science analyses on biological specimens from cadavers has allowed us to master analytical techniques for different drugs and narcotics in all biological specimens from autopsies including blood, hair, urine and even viscera.

Performing analyses on blood, hair and urine and having been appointed by prosecutors many times to analyze these specimens, from either autopsies or indicted sportsmen, it seemed interesting to compare results from these different biological environments, even though IOC Laboratory only has the authorization to work with urine.

Section snippets

Experimental

According to the recommendations of the Society of Hair Testing, our washing procedure included a wash with methanol for 2 min followed by two washes with hot water for 3 min and then by two 3-min washes with dichloromethane [19], [20].

The hair samples were then finely cut with scissors and processed for 2 min in a ball mill (model EI 4000 from the KLECO manufacturer located in Visalia, CA, USA).

Amphetamines

In Table 3 the results for amphetamines are presented. What is very remarkable is that the only identified drug was amphetamine itself. Urine was tested positive 6 times out of thirty analyses (range 2.2–906.0 ng/ml), that was 20% of positives. Hair analysis was positive 10 times out of 19 analyses (range 0.08–1.22 ng/mg), that was 52% of positives.

Fig. 1 displays a urine sample tested positive for amphetamine at a measured concentration of 906.0 ng/ml.

Corticosteroids

Table 4 summarizes the results for

Conclusion

We have developed simple PCI–GC–MS, HPLC–MS–MS and GC–MS–MS methods for the measurement of amphetamines, corticosteroids and anabolic steroids in hair. This matrix appeared as the sample of choice for testing testosterone and hydrocortisone abuse under their ester forms, as it is the sole matrix that could document without ambiguity such a treatment. Detection of other doping agents has also been demonstrated. Hair appears as a sensitive matrix for the detection of amphetamines and anabolic

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