Detection of misuse of anabolic androgenic steroid hormones

24.5.1 Organization of doping tests

Doping control is organized by national and international sport federations and by the WADA for the different types of sports. Increasingly national anti-doping programs are organizing dope control by one overall organization. This strategy seems to be the most effective testing action as any possible intention by individual sport federations to hide positive cases and to protect their athletes can be excluded. The IOC only performed doping tests during the Olympic winter and summer games and it has no "out of competition testing program". This lack has now been compensated by WADA.

For a doping test athletes are selected according to the rules of the responsible sports federation. The doping test is carried out in two steps. The first step includes the sample-taking procedure and transportation of the urine specimens to an IOC accredited laboratory. In the following step the laboratory analyzes the sample for banned drugs. The sample-taking procedure is an important step. To avoid any manipulation athletes have to deliver a urine sample under visual inspection by an accredited supervisor. The urine is divided into an A- and a B-sample, both samples are sealed and then transported to the laboratory. All steps during this procedure are documented and the athlete has to sign a protocol of the sample-taking procedure and sealing of samples. All handling of a urine specimen (sample-taking, transportation containers and laboratory tests) must be documented and is designated as "chain of custody". The laboratory is not in possession of the athlete's name corresponding to the urine sample. For this reason all samples have code numbers. The reason for dividing the urine specimen into A- and B-samples is to guarantee the best chain of custody: if the A-sample is tested positive, the B-sample will be analyzed in the presence of the athlete and his advisers. If the B-analysis confirms the A-result, the samples are considered as positive. Based on this result the federation can impose sanctions on the athlete.

Doping test samples are analyzed by WADA accredited laboratories. Laboratories seeking accreditation have to comply with the requirements for doping drug testing set by the WADA World-Anti-Doping Code. Additionally the laboratory has to be accredited by a national accreditation body following the standard of ISO 17025. The laboratory must show that it has the capability to analyze all banned substances below the specified concentration limits within a controlled quality system.

The prerequisite for this accreditation system is a standardization of analytical techniques and detection limits of banned substances among the different laboratories. Information concerning new doping drugs and doping techniques is rapidly distributed in order to deal with new problems in a co-ordinated manner. Especially for the detection of synthetic AAS, which are misused mainly during training periods, the laboratory has to use highly sensitive methods.

At the present time 30 laboratories all over the world (18 in Europe, 2 in North America, 2 in South America, 5 in Asia, 1 in Australia and 2 in Africa) are accredited.

24.5.2 Detection and identification of misused anabolic androgenic steroids

Synthetic AAS were first banned in 1974. As no comprehensive analytical method for the detection of AAS in human urine was available at the beginning of the seventies, new methods had to be developed. The first methods were based on radioimmunoassay (RIA) techniques, e.g. Brooks et al. (1975) developed an antiserum for metandienone with some cross-reactivity to other 17a-methyl steroids. The RIA techniques were discouraging for several reasons: the method did not consider the high degree of metabolism of AAS (therefore screening for the parent steroidwas less successful), the antisera had only limited sensitivity for other steroids and the possibility of false positives, which was not acceptable for routine analysis. As early as 1977 Ward et al. presented a gas chromatography / mass spectrometry (GC-MS) method for the detection of the AAS metandienone, nortestosterone, norethandrolone and stanozolol. Nevertheless, the RIA technique was used as a screening method during the 1976 Olympic Games in Montreal and in Moscow in 1980, but confirmation of suspicious samples was performed by GC-MS. After

1981 all IOC accredited laboratories used GC-MS as the main analytical tool for AAS identification (Donike et al. 1984; Masse et al. 1989; Schanzer and Donike 1993).

Analysis of AAS can be divided and described in two steps: first a sample preparation is performedwith the aim to separate the banned substances from the biological matrix (urine) and to reduce biological interference (biological background). Sample preparation for AAS also includes a chemical modification (derivatization) of the isolated substances to improve their analytical detectability. The second step covers the analytical measurement, which is based on a physical principle, mainly on gas chromatography in combination with mass spectrometry (GC-MS). Additionally liquid chromatography and mass spectrometry (LC/MS) can be used for substances which show poor gas chromatographic properties or are sensitive to temperature.

The main advantage ofchromatographic techniques such as GC-MS is the possibility of analyzing a high number of substances within one run. This minimizes costs and allows a high throughput of samples. In fact it is possible to run a maximum of 50 samples per day and per GC-MS instrument. Metabolism

To a large extent anabolic androgenic steroids are metabolized by phase I and phase II reactions and only a few AAS are excreted unchanged in urine for a short period of time after administration. To detect the misuse of AAS which are not excreted in urine or only to a small extent, the analytical method cannot rely on monitoring only the parent steroid but must identify its metabolites. Detection of an AAS metabolite in urine is proof for the misuse of a banned anabolic androgenic doping substance. This presumes that the metabolite cannot be generated from endogenous steroids in the body compartment.

Metabolites of the most frequently misused anabolic steroids have been investigated by different working groups in recent years (Schanzer 1996; Schanzer and Donike 1993). Basically the metabolism of AAS follows the metabolic pathways of the principal androgen testosterone. This includes reduction of the double bond at C4-C5 to form 5a- and 56-isomers, the reduction of the 3-keto group to a 3a-hydroxy function and, in case of 176-hydroxy steroids with a secondary hydroxy group, the oxidation yielding a 17-keto function. Additionally many AAS are metabolized by cytochrom P-450 hydroxylation reactions, and steroids with hydroxy groups mainly at C-6S, C-16a and C-16S are produced. In the metabolism of stanozolol, a synthetic steroid with a condensed pyrazol ring on the steroid A-ring, further hydroxylation occurs at C-4S and C-3' of the heterocyclic ring (Fig. 24.5). In general in the course of phase I metabolism steroids are enzymatically transformed

Steroid Metabolites
Fig. 24.5 The main metabolites in the metabolism of stanozolol (1), 3'-hydroxy-stanozolol (2), 16B-hydroxystanozolol (3), and 4B-hydroxystanozolol (4).

to more polar but pharmacologically inactive compounds. Phase I reactions are often followed by phase II processes, also known as phase II conjugation. In the case of AAS and their metabolites the reaction creates steroid conjugates with sulfate or glucuronic acid (Thevis etal. 2001). These highly polar compounds are then rapidly eliminated in the urine.

In the last ten years excretion studies performed with 17a-methyl steroids demonstrated that the metabolism of AAS is highly complex and the detection of more than 20 metabolites after administration of one single AAS is not unusual. Similar results regarding the high number of metabolites are already known for the metabolism of testosterone (Kochakian 1990). Pharmacokinetics

A further important factor which has to be considered for detection of AAS is the pharmacokinetics of the parent compound and its excreted metabolites. As AAS are misused during training and the number of checks are limited, it is desirable to detect AAS as long as possible after their last administration. Analysis of the parent steroids and/or their metabolites, which are excreted very rapidly, is less effective for screening analysis than the detection of metabolites excreted long-term: these are steroids detectable for the longest possible period of time after administration (Schanzer 1996). The main differences between the pharmacokinetics of AAS are caused by their pharmaceutical preparation and the kind of application. Depot preparations, e.g. 19-nortestosterone injected intramuscularly as its undecanoate ester (Deca-Duraboline®), are detectable in urine for several weeks, whereas most oral preparations are completely eliminated within a few days after intake. Once they became aware of these scientific data, athletes switched their doping activities to AAS with short elimination times and to steroids which were believed to be undetectable. Sample preparation

For sample preparation of anabolic steroids it has to be considered that most of the AAS and their metabolites are excreted in conjugated form. Following sample preparation unconjugated steroids can be separated by extracting an aliquot of urine (e.g. 2 ml) with a polar organic non-water miscible solvent. Based on their polar and acidic character, conjugated steroids are not extractable and remain in the aqueous layer. These conjugates (mainly glucuronides) can be liberated by enzymatic hydrolysis of the urine specimen. The enzyme used can be added directly to the urine or to an isolate obtained via an adsorber resin. Enzymatic hydrolysis is achieved completely using enzyme preparations with 6-glucuronidase from E.coli or 6-glucuronidase/arylsulfatase from Helix pomatia. The "free" steroids (conjugated fraction) are then extracted from the aqueous phase via a simple liquid extraction with tert-butyl methyl ether, or in case of less polar steroids, with an alkane (e.g. n-pentane).

The first analysis is a screening procedure by which all banned AAS are detected in one single analytical run. Suspicious samples are confirmed by a second aliquot of the same urine specimen, which is isolated using a substance-specific isolation technique. Derivatization

Based on the polar groups of AAS (hydroxy and keto groups) high interactions with polar functions of the GC-column phase reduce the detectability of AAS at low concentrations. Derivatization of polar functions of AAS can lead to a distinct improvement in peak intensity and detection limit of the analytical method. The most frequently used derivatization methods are acylation (e.g. trifluoroacety-lation) and silylation (e.g. trimethylsilylation). For doping analysis of AAS sily-lation is the method of choice and the introduction of a trimethylsilyl group to an AAS is the most common derivatization reaction, converting polar groups such as hydroxy and keto functions to less polar trimethylsilyl ethers with excellent GC behaviour. For this kind of derivatization a respectable reagent MSTFA (N-methyl-N-trimethylsilyltrifluoro-acetamide) was developed (Donike 1969). Additionally, the mass spectrum is generally changed to higher and more abundant molecular and fragment ions, which also improves the signal-to-noise ratio of the substance to be identified compared to the analytical and biological background. Therefore derivatization for GC-MS detection ofsubstances isolated from biological fluids unequivocally yields a more accurate analytical result, which is an absolute requirement in view of the complex matrix and large number of possible interferences.

24.5.3 Detection of synthetic anabolic androgenic steroids

In some instances AAS are differentiated into endogenous and exogenous AAS according to their route of administration. The term "synthetic" should amplify the fact that these AAS are not produced in the body, they are chemically synthesized and can only enter the circulating blood system by exogenous application. AAS which are naturally synthesized in the glands of mammalian cells are called endogenous steroids, even though their application can be exogenous. As synthetic AAS and/or their metabolites are not present in the human organism, their identification in a urine sample of an athlete constitutes the misuse of a banned steroid. The criteria for identification of a substance are based on the analytical method applied.

In GC-MS identification of synthetic AAS obtained from a urine specimen it is mandatory to register a full mass spectrum or a selected ion monitoring (SIM) profile of the main abundant fragment ions. The mass spectrometrical data (MS spectrum or SIM profile) of the isolated substance should be in accordance with an authentic synthesized reference substance or, in the event that a synthesized reference metabolite is not available, with a well-characterized metabolite from an excretion study with the corresponding AAS. In addition to the MS data, the GC retention time of the isolated steroid has to agree with the GC retention time of the reference substance. For this purpose reference metabolites of frequently misused AAS but not commercially available were synthesized (Schanzer and Donike, 1993).

As an example Fig. 24.6 shows the criteria for a positive sample for a long-term excreted metabolite of metandienone:

• registration of a full mass spectrum which can be compared with the reference spectrum or

• in case of low concentrations, a selected ion monitoring (SIM) profile with the main intense fragment ions of the metandienone metabolites 17,17-dimethyl-18-nor-5S-androst-1,13-dien-3a-ol.

To increase the efficiency of AAS misuse testing and to detect AAS for a longer period of time after administration more selective and sensitive MS techniques were used during the last decade. The main improvements were first, installation of more sensitive and selective mass spectrometers, and secondly, by substance-specific sample preparation (Schanzer et al. 1996). The use of high resolution mass spectrometry (HRMS) was announced to the public at the Olympic Games 1996 in Atlanta. This technique was established after 1992 in a few IOC-accredited laboratories. The advantage of HRMS became apparent before Atlanta when, during doping testing by the International Weightlifting Federation, more than forty athletes were confirmed positive only by HRMS and not by the conventional MS technique. Following these results the IOC decided that it was neccessary that accredited laboratories use more sophisticated equipment, such as HRMS or MS/MS.

100 n 80 60 -40200


B Positive sample m/z: 216.1878

E+04 7.239

E+04 1.155

B Positive sample m/z: 216.1878

E+04 7.239

E+04 1.155

E+03 3.856


100 50 0


38c c Reference standard m/z: 216.1878





rn~r 17

E+03 3.856

100 50 0


E+05 1.236

" '' | I I I I | I I I I | I I I I | M I I | I I

Fig. 24.6 Criteria for a positive confirmation: 1. The registrated EI mass spectrum (e.g. mass spectrum of an isolated metabolite of metandienone: 17,17-dimethyl-18-nor-5B-androsta-1,13-dien-3a-ol TMS (A) has to be in accordance with the mass spectrum of an authentic reference substance, or 2. The main abundant fragment ions of the isolated substance show similar intensities (B) when selected ion monitoring (SIM) registration is applied in comparison to the intensities of the same fragments of the reference compound (C).

The basic principle of HRMS is that elements do not have an integral number of atomic weight but a decimal form. Only carbon, as the reference element, has an integral number of 12 as its atomic weight. Thus hydrogen does not have an atomic weight of 1 but 1.00783, nitrogen the weight of 14.00307, and oxygen the weight of 15.99491. Molecular fragments with the same integral number of mass, e.g. the fragment ions C3H6O+ and C3H8N+ both have the rounded mass 58 but the exact calculated mass of 58.04186 for C3H6O+ and 58.06567 for C3H8N+. Neither mass fragments can be separated by conventional (low resolution) mass spectrometry but only by using high resolution MS with a resolution of2500. Thus in practical terms, in this example the instrument (HRMS) can be set to detect only the signal of the mass of58.04186 for C3H6O+, and all masses differing by more than 0.0024 masses, such as 58.06567 for C3H8N+, will be discriminated. Based on this fundamental physical principle HRMS analysis of AAS steroids and their metabolites isolated from urine reduces the biological background and increases the signal-to-noise ratio, yielding a much higher selectivity in screening and confirmation.

24.5.4 Detection of endogenous anabolic androgenic steroids Indirect detection methods

The misuse of testosterone by athletes is also tested by GC-MS analysis of urinary extracts. However, the method reveals only the presence of testosterone and its ratio to epitestosterone. The mass spectrometrical data alone indicate whether testosterone originates exogenously (doping) or whether it was produced endoge-nously. In 1983 Donike et al. developed a method to calculate urinary excreted testosterone by a ratio to 17-epitestos-terone. Both isomeric steroid hormones are excreted mainly as glucuronides which are enzymatically hydrolyzed before GC-MS analysis. The urinary testosterone/epitestosterone ratio (T/E ratio) represents a relatively constant factor within an individual and alterations under physical excercise have not been noted. Exogenous application of testosterone results in an increase in the urinary concentration of testosterone glucuronide, whereas epitestosterone glucuronide is not influenced. Based on measurements of large reference groups Donike proposed a T/E ratio of 6:1 as a marker to handle a urine specimen suspicious for testosterone misuse. An increased T/E value (T/E > 6) is not immediately considered as a positive sample. Following the WADA rule the athlete has to be further investigated and it has to be determined that the increased value is not caused by physical or pathological conditions. In practice this requires several test samples of the athlete and evaluation of previous tests in order to establish the athlete's individual T/E reference values (subject-based reference values). The test sample is considered positive when the tested T/E ratio clearly exceeds the subject-based reference values (> mean + 3 standard deviations) of the athlete. In addition to the T/E ratio the testosterone and epitestosterone concentrations as well as the concentrations of the main testosterone metabolites are assessed.

Doping with dihydrotestoserone (DHT) became public knowledge after the Asian Games in 1994 when seven athletes were tested positive for DHT misuse. The criteria for DHT doping are also based on statistical methods and population-based reference values with limits for the ratios of DHT/epitestosterone, epitestosterone, DHT/etiocholanolone, 5a-androstane-3a, 178-diol/58-androstane-3a,178-diol, and androsterone/etiocholanolone established (Kicman et al. 1995; Donike et al. 1995).

The main weakness of all the methods confirming doping with endogenous AAS is the application of statistical parameters. These methods are therefore indirect methods and they only confirm that an increased value varies from the normal values of the athlete. These methods do not identify any physical characteristics of the exogenous steroid differing from the steroid produced endogenously as direct proof of doping. Direct detection method: gas chromatography - combustion - isotope ratio mass spectrometry (GC-C-IRMS)

The T/E ratio results can be supported by gas chromatography-combustion-isotope ratio mass spectropmetry (GC-C-IRMS). This method was first introduced by Bec-chi et al. in 1994 and has been adopted by other research groups (Aguilera et al. 1996; Horning et al. 1997; Shackleton et al. 1997) with distinct modifications. The principle of IRMS is the precise measurement of the 13C/12C isotope ratio of organic compounds. This method became practical for trace analysis in doping control when instruments combining gas chromatography and isotope ratio mass spectrometry were developed. Isotopes are elements with the same number of protons but different numbers of neutrons. Carbon occurs in three kinds of isotopes: 12C (6 protons and 6 neutrons) with a frequncy of approximately 98.9%, 13C (6 protons and 7 neutrons) at a rate of 1.1% and 14C (6 protons and 8 neutrons), a radioactive isotope with a half-life of 5760 years (used in determination of age), in traces. In the course of synthesizing organic compounds 12C atoms react slightly fasterthan 13C atoms. This effect results in a reduction ofthe 13C amount compared to 12C. The 13C/12C ratio is calculated in promill [813C(%o)] relative to a reference gas with a standardized 13C/12C ratio. The 8-value becomes more negative when the 13C portion is reduced, as was explained during synthetic pathways. For isotope measurement urinary excreted steroids have to be isolated to high purity. Most research groups use HPLC separation of steroids, or isolation of steroidal diols is performed via the Girard reagent (Shackleton etal. 1997). For gas chromatographic separation derivatization is applied using acetylation of steroids with the aim to improve GC peak shape or analysis refers to the underivatized steroids.

Steroids are separated by gas chromatography followed by complete oxidation to carbon dioxide in a combustion chamber. The carbon dioxide is then introduced to the mass spectrometer where the exact masses m/e 44 for 12CO2 and m/e 45 for 13CO2 are independently registered. For this kind of isotope ratio measurement a minimum of 5-10 ng of a steroid has to be used to obtain precise data. The 13C/12C

Fig. 24.7 Testosterone determined in urine after oral application of 40 mg of Andriol® (testosterone undecanoate): T/E ratio (line) and carbon isotope ratio mass spectrometry (column).

Fig. 24.7 Testosterone determined in urine after oral application of 40 mg of Andriol® (testosterone undecanoate): T/E ratio (line) and carbon isotope ratio mass spectrometry (column).

ratio can be estimated with an accuracy of ± 0.0002% (± 0.2 permill to the 13C/12C ratio of the reference gas).

Fig. 24.7 presents data of GC/C/IRMS and T/E ratio analysis after oral administration of 40 mg of testosterone undecanoate (Andriol®) to a single male volunteer. A direct proof of exogenous testosterone application is possible as the 8-values are decreased to -28 ppm after administration, in comparison to -24 ppm before intake and at the end of the elimination curve. It is also obvious that the 8-values are still decreased when the T/E ratio drops below six and is close to the normal value. This method can therefore also be used when ethnic differences influence testosterone metabolism, e.g. in Asians who have low T/E ratios and when a testosterone application will not necessarily exceed the T/E ratio of six (de la Torre etal. 1997). Exogenous testosterone also influences the 13C/12C ratio of the metabolites of testosterone. Based on these data it was proved that precursors within the synthetic pathway of testosterone, such as pregnanediol, pregnanetriol (metabolites of progesterone and 17a-hydroxyprogesterone) and cholesterol are not influenced by exogenous testosterone, whereas testosterone and its metabolites have decreased 8-values indicating exogenous application. The results of a positive testosterone finding are presented in Fig. 24.8. The T/E-ratio of the positive urine sample was 14.7. Following the rules, the athlete was further investigated and 10 urine samples were collected over a period of two days and analyzed. The T/E ratio during this study was 1.0 ± 0.1 and confirmed that the sample with a T/E ratio of 14.7 was not in accordance with endogenous production of testosterone and was considered as an offence against the doping regulations. The IRMS data of the corresponding positive urine sample (Fig. 24.8) show the decreased values for testosterone and the metabolites androsterone and etiocholanolone, whereas the higher 8-values of

Indirect method

Direct method

Positive sample

Endocrinological study

- T/E-ratio



13C/12C Isotope

ratio in

S [%o]
















* T/E-ratio 1.0 ± 0.10, n = 10 samples

Fig. 24.8 T/E-ratio and IRMS results of a testosterone positive urine sample and urine sample of the same athlete obtained during an endocrinological study.

the precursors and the values obtained from the endocrinological study were in the same range.

It was also suggested to detect testosterone misuse by analysis of testosterone esters in blood (de la Torre 1995), but this method is limited to the application of testosterone esters in the form of injectable preparations and is not applicable to the analysis of urine samples.

The isotope ratio mass spectrometry method can additionally be applied to detect and identify doping with other endogenous AAS such as dihydrotestos-terone and dehydroepiandrosterone, where reliable methods are less efficient or not available.

24.6 Key messages

• Misuse of androgens in competitive sport has been banned since 1974 and is tested by IOC and WADA accredited laboratories.

• Androgens are used by athletes during training to improve muscle strength. For this reason doping tests have been extended to out-of-competition tests.

• Non-therapeutical hormones such as prohormones of testosterone and nortestosterone have been marketed as nutritional supplements since 1999 in many countries e.g. United States.

• Positive doping cases have been proved to originate from the use of nutritional supplements "contaminated" with prohormones of nortestosterone.

• Testosterone, nortestosterone, stanozolol and metandienone represent the most frequently misused AAS in controlled sports.

• Androgens are detected and identified by gas chromatographic / mass spectrometric analysis of urinary extracts.

• Derivatization methods for steroid analysis improve detection limits for anabolic steroids.

• Synthetic androgens are extensively metabolized and doping tests are focused on urinary excreted metabolites.

• Doping with endogenous steroids is controlled by indirect methods, e.g. testosterone misuse is tested by a ratio of testosterone to epitestosterone (6:1). Positive findings are followed by additional studies to exclude physiological and pathological influences.

• Recently direct methods, such as gas chromatography-combustion-carbon isotope ratio mass spectrometry have become available to identify doping with endogenous steroids unambiguously.


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