Testosterone Misuse

Abstract

Testosterone is a key compound of the anabolic androgenic steroids (AAS) family. It has largely been misused in human and animal doping targeting a muscle tissue growth and an enhancement of performances. Such practices constitute a violation against ethical values, food safety, and animal welfare. Consequently, the use of such substance is regulated by WADA and International committees for some animal species such as equine and bovine. Although efficient, the detection of testosterone misuse remains challenging in some cases due to its endogenous origin and its inter- and intra-individual level fluctuation in biological fluids. Novel analytical strategies have been developed and are continuously evolving in order to tackle this issue and to provide a better control of testosterone misuse.

1. Introduction

“Doping” as a term has usually been correlated to fraudulent practices undertaken by dishonest athletes to improve their sporting results. However, the use of this term overpasses the boundaries of human sport field to cover the field of sports and food-producing animals such as horses and bovine. Androgenic anabolic steroids (AAS) misuse, in particular, represents one of the hottest and most challenging topics in this context.

Testosterone belongs to androgenic anabolic steroids (AAS) family, which is a group of derivatives originating from the cholesterol biosynthesis in mammals or issued from synthetic chemical reactions. These compounds are reported to be masculinizing agents (androgenic) and promoting skeletal muscle building (anabolic) [1, 2]. The interesting anabolic properties were demonstrated for the first time by Brown-Sequard [3], who reported an improvement in strength and force upon an injection of an extract of dog and guinea pig testicles. Based on its beneficial effects, testosterone has originally been used as therapeutical agent for clinical treatments such as the treatment of hypogonadism or anemia. Consequently, the availability of testosterone increased, and its use was diverted from their original purpose.

In addition to their unethical use to enhance performances and produce higher volume of muscle carcasses, these doping practices pop out as a major concern and a threat for human and animal health and welfare.

In food-producing animals, testosterone is often used by unscrupulous cattle breeders in order to stimulate bovine muscle growth and make a higher profit with regard to the consequent gain in weight over a short period of time [4]. The worldwide debate about the use of such practices has existed since the discovery of their misuse. Several international projects such as the “Transatlantic Trade and Investment Partnership” tend to harmonize the laws regarding the use of AAS in the world. These projects are mainly bringing together for negotiating process the European Union countries, which are imposing a strict regulation system regarding the AAS misuse and the USA, regulations of which offer some permissions for the use of some AAS such as estradiol and stanozolol.

The strict regulation toward AAS misuse in food-producing animals adopted by the European Union countries originates from the various scandals that stroke the region due to the misuse of these substances. One of the biggest high-profile scandals occurred in the 1980s in Italy with the discovery of diethylstilbestrol, a growth hormone promoter, in baby food. Consequently, several measures have been taken by the European Union [5, 6]. According to these directives, adequate analytical methods had to be developed and implemented in the different accredited control laboratories in order to guarantee the consumer’s security toward these active chemicals [7]. The analytical methods are mainly based on chromatography coupled to mass spectrometry techniques permitting the detection of residues in biological matrices. The system revealed to be efficient for detecting a large spectrum of anabolic strategies. However, some particular cases remain challenging with regard to the current methodologies adopted by reference laboratories. The use of “hormone cocktails” is among these particular issues [8] and consists of the administration of different anabolic agents such as testosterone, each occurring at low concentration levels. These low quantities of anabolic agents cannot be detected with regard to the detectability limitations of the analytical methods. The use of “Designer Drugs” constitutes another issue for control laboratories. Indeed, “Designers” are exclusively xenobiotic substances not listed among prohibited substances. Considering the lack of knowledge on their exact chemical structure, they cannot be detected by the classical targeted analytical strategies. Finally, the use of natural steroids such as testosterone constitutes a challenge for control laboratories with regard to the similar structures exhibited by these administered substances and their corresponding endogenous analogs.

In the equestrian world, “doping” has been in existence since the ancient Roman era where horses were fed with a “magic potion” called “Hydromel,” a mixture of milk and honey, in order to improve their physical performances in the Arena. In the twentieth century, administering alkaloids such as cocaine or strytchine to horses emerged as a new doping treatment permitting to minimize the feeling of physical tiredness. The fraudulent use of these exciting substances increased simultaneously with the appearance of horserace bets, which generated important financial gains. In addition, the use of AAS spread considering their power to enhance physical performances. In order to tackle this issue threatening the equestrian world as a whole in terms of ethical values and animal welfare and with the hope to avoid a tarnished image of horse racing competitions, the International Federation of Horseracing Authorities (IFHA) put into practice a strict control system similar to the one regulating athletes doping implemented by the World Anti-Doping Agency (WADA). This control system, “the racing code,” forbids the use of any medication during horseracing competitions and bans in particular the use of AAS in horses during training sessions, pre-competition or competition periods. Targeting an efficient application of this control system, IFHA reference laboratories are committed to developing analytical methods mainly based on chromatography coupled to mass spectrometry techniques allowing the identification and quantification of residues in biological matrices. Although being highly efficient, these methods highlighted, similarly to the official analytical methods adopted in food-producing animal field, some limitations for detecting naturally occurring steroids misuse.

2. Structure and pharmacology of steroid hormones

Anabolic-androgenic steroid hormones share the same substructure based on a sterane nucleus (Figure 1). The different families of steroid hormones result from the existence of minor modifications occurring at the sterane nucleus. Progestagens such as progesterone, corticosteroids such as cortisone and aldosterone, estrogens such as estradiol, and androgens such as testosterone are some example of these families.

Figure 1.

Structure of a cyclopentanoperhydrophenantrene nucleus or sterane.

All of these steroids are derived from cholesterol, which is mainly biosynthesized after dietary intake. This steroid biosynthetic pathway begins with an oxidation of the side chain of the cholesterol to form the androgens prior to the formation of estrogens. This oxidation reaction, catalyzed by the Cytochrome P450, generates pregnenolone, which whether can be biotransformed into progesterone through the enzyme 3β-HSD initiating the creation of the progestagen family or into 17α-hydroxypregnenolone through an oxidation by CP_17α. The latter compound is subsequently metabolized into DHEA and then to 4-androstenedione, precursor of testosterone.

Estrogens are formed during the reaction of aromatization occurring under the action of CP450. The aromatization consists of the oxidation of the methyl group at position C19 and the consecutive elimination of this newly formed to form estrogens such as estrone and estradiol. This reaction results in the creation of secondary products such as nandrolone. All these transformations are summarized in Figure 2.

Figure 2.

Global overview of the metabolization products deriving from cholesterol and the occurring biotransformations generating the different steroid families.

The secretion of naturally occurring hormones is enhanced by the Luteinizing Hormone (LH), which activates the biotransformation of cholesterol into pregnenolone and the Follicle-Stimulating Hormone (FSH), which participates in the aromatization of testosterone into estradiols. The secretion of two latter hormones is regulated by the Gonadotropin-Releasing Hormone (GnRH) according to the occurring plasmatic concentration levels of steroid hormones.

The steroid hormones are subsequently submitted to phase I biotransformations (oxidations, reductions, epimerizations, etc.) and phase II biostransformations (mainly sulfo and glucuruno conjugation) in order to be eliminated predominantly through urine.

The nature of these biotransformations is essentially correlated to the steroid nature and the occurring enzymatic system in the organism. Therefore, the steroids biotransformations present some differences depending on the species. For instance, phase II steroid biotransformations consist mainly of sulfoconjugation reactions in equine species while they consist of glucurunoconjugation reactions in humans and bovine species.

While the previously described steroid hormones such as testosterone occur naturally in the organism, some other steroids are exclusively synthetic. They usually exhibit minor modifications to their endogenous analogues such as methyltestosterone. They can also be part of a new category of steroids called “Designers.” These steroids are unmarketed compounds exhibiting a chemical structure based on marketed substances presenting minor modifications. They are usually synthesized as part of the pharmaceutical industry for medical use. With regard to their inexistence in the list of WADA banned substances, their detection using classical targeted analytical methods becomes problematic. Estra-4,9-diene-3,17-dione [9] and tetrahydrogestrinone [10] are some examples of this category of synthetic compounds.

3. Use of steroid hormones

The use of anabolic steroids in food-producing and sport animals has been flourishing with regard to their capacity to increase zootechnic performances [11, 12]. Testosterone has been one of the main steroids used in this context. Typical quantities of administered steroids to human, bovine, and equine for doping purposes range between hundreds of micrograms and few milligrams per kilogram. The administration of these steroids may be performed orally, through parenteral or alternative pathways:

Oral pathway: minor chemical modifications to the structure of natural anabolic steroids have to be performed prior to an oral administration. These modifications protect the steroid from a submission to a quick metabolization through the hepatic pathway. These modifications consist mainly of an alkylation or an esterification. One example of such steroids is the methyl testosterone, efficiency of which is four times higher than testosterone when administered orally.

Parenteral pathway: this relates to any administration occurring on a different way than the digestive one. When the steroid is administered under its free form through this pathway, it undergoes a quick metabolization leading to its degradation and elimination. Therefore, a step of esterification of the steroid is necessary to provide the desired effect on an extended period of time. The intramuscular administration of the steroid ester is immediately followed by an enzymatic hydrolysis in blood [13]. Prior to hydrolysis, the steroid ester migrates gradually from its lipophilic excipient toward blood stream [14]. This gradual liberation results in a longer effect of the doping substance. The effect is even longer when the steroid ester presents a longer lipophilic side chain [15]. Once the active steroid is formed, it is transferred through the blood stream to the corresponding hormonal receptor.

The administration of the anabolic steroid could be also performed through an intravenous pathway or through solid implants.

Alternative pathways: the transdermal application of steroids is also a possible way for administration with regard to the lipophilic nature of the skin. Anabolic steroids are usually administered through the application of spray or “pour-on” [16]. These pathways have the advantage not to submit the steroids to the first hepatic transition caused by the liver. However, a frequent application is necessary with regard to the low biodisponibility of the steroids using this pathway.

3.1 Transport of steroid hormones

Hormone steroids are rarely present in the blood under their free form. Instead, they are carried in the bloodstream bound to serum carrier proteins [17]. There are two categories of binding proteins:

Non-specific binding carrier proteins: albumin is one example of these binding proteins, and it is characterized by a high capacity of transport but a low affinity with steroids. It is found at a relatively high concentration in blood. Its main function is to ensure the transport of steroids to their corresponding hormonal receptors.

Specific binding carrier proteins: SHBG (Sex Hormones Binding Globulin) is an example of this class of proteins [18]. They are characterized by a high affinity with steroids but a low capacity of transport.

The bioavailability of the endogenous steroids is ensured by these proteins. These proteins play indeed the role of a reservoir of steroids regulating the concentration levels of steroid hormones in the metabolism. Moreover, these proteins protect the transported steroids by preventing their biotransformation and subsequent diffusion and elimination [19].

3.2 Protein production activation

When the steroids reach the target cell, they penetrate through the phospholipidic membrane of the cell by passive diffusion. This passage is guaranteed with regard to the fat-soluble nature of the hormonal steroids. Once inside the cell, the steroids bind to their specific androgenic receptor located in cytoplasm [20]. This binding results in the formation of a steroid-receptor complex, which infiltrates the nucleus to bind to a specific receptor located on chromatine. Consequently, the production of RNA messenger (mRNA) is initiated [21]. These mRNAs are then transported to the cytoplasm for further proteins synthesis.

3.3 Biological effects

The AAS are well-known to enhance muscle growth (anabolic effect) and to promote masculinization (androgenic effect). In addition, the use of these substances may lead to harmful effects such as hypogonadism and cardiovascular risks [22]. These effects are mainly caused by the disturbance occurring through the administration of exogenous steroids, which results in a negative feedback on the secretion of natural steroid hormones. Furthermore, the increase in steroids concentration leads to a higher synthesis of proteins and subsequent higher amount of muscular tissue, which needs, as a consequence, a higher volume of blood irrigating it and finally causing cardiovascular problems. High blood pressure could be observed also due to a regular administration of testosterone. In fact, such an administration causes a reduction in the secretion of HDL (High-Density Lipoproteins) resulting in an imbalance between HDL and LDL amounts leading to hypertension troubles. Other effects could be observed for female subjects submitted to a regular intake of AAS on hormonal cycle (ovulation and menstruation). The abuse affects also the psychological aspect and could lead to an aggressive attitude and even to depression.

4. Regulation

4.1 Bovine

The use of anabolic steroids as growth promoters in food-producing animals is prohibited in the European Union according to the EU Directive [5]. Different EU Directives have been issued to date in order to avoid any misuse of these substances in food-producing animals. The main purpose of these directives is to guarantee the security of the consumer toward these active chemicals [7].

The harmonization of the regulation concerning the use of these growth promoters in bovine, mainly between the European Union and the United States, has been problematic. The problem became even greater with the increase in the trade flows of bovine meat all over the world. While the use of anabolic steroids in the European Union is strictly banned in food-producing animals, the use of implants of 17β-testosterone, la 17β-estradiol, progesterone, trenbolone, or zeranol in cattle is still legal outside the European Union. As a consequence of the important position of the European Union in the bovine meat trade market, the bovine field in the United States witnessed a gradual decrease in the use of growth promoters. Although several arrangements have recently been taken in order to meet the different expectations of the different parties (MOU) (WT/DS26/28, September 30, 2009, 74 Federal Register), the problem is still persisting. In June 2013, talks between the European Union and the United States have been taken over in order to find a solution to this recurring issue. In July 2013, the TTIP “Transatlantic Trade and Investment Partnership,” a project aiming to set a transatlantic market based on a liberal model ruled by a free circulation of goods and investments, was created. This project will also cover the agriculture field and would affect the regulation put into practice for the control of agricultural products such as bovine meat quality.

The recognized reference laboratories are submitted to the identification criteria established by the European Decisions 2002/657, which defines mainly the tolerated relative retention time and abundances percentual errors depending on the performed analytical technique.

4.2 Equine

The use of anabolic steroids is strictly banned in equestrian sports. Unlike WADA, where the list of prohibited substances for athletes is classified according to their respective pharmacological families, the IFHA has classified the doping substances for horses with regard to their respective target system of the organism. For instance, article 6 of IFHA “International Agreement on Breeding, Racing and Wagering” highlights the ban of substances that affect the nervous, the cardiovascular, the respiratory, the digestive, the urinary, the reproductive, the musculoskeletal, the blood, the immune, or the endocrine system.

The control of the use of these substances protects the integrity of horse races. This control can be conducted at any time of the year on a large panel of biological matrices.

With regard to the natural occurrence of some anabolic steroids such as nandrolone, the International Equestrian Federation put into practice in March 1988 an advisory committee formed of analysts and veterinarians, whose duty is to establish concentration or ratio thresholds of certain prohibited substances when it is necessary. Consequently, different confirmation thresholds have been approved by IFHA for natural occurring anabolic steroids misuse. For instance, boldenone urinary concentration should not overpass 15 ng/mL in entire male horses, testosterone urinary concentration should be inferior to 20 ng/mL in gelded horses and less than 55 ng/mL in mares and fillies. Finally, 5α-estrane-3β,17α-diol, one of the main recognized biomarkers of nandrolone administration, urinary concentration should be lower than 45 ng/mL in entire male horses [23]. In addition to the latter criterion, a complementary threshold consisting of a ratio of the urinary concentration of 5α-estrane-3β,17α-diol and 5(10)-estrene-3β,17α-diol of 1 has been supplemented [23].

The reference IFHA laboratories are submitted to the identification criteria established by AORC (Association of Official Racing Chemists), which defines mainly the tolerated retention time and relative abundances percentual errors depending on the analytical technique adopted [24].

5. The detection of steroid abuse in bovine and equine species

With regard to the high number of analysis performed by control laboratories, high-throughput screening analytical methods are required in order to offer a first discrimination between negative and suspicious samples. Once the sample is considered suspicious, it is subsequently submitted to confirmatory methods.

5.1 Screening

Screening methods require a dual compromising aspect by being high throughput and multi-residue but at the same time showing a high efficiency and low costs. These methods allow the detection or the concentration level estimation of a certain group of prohibited doping agents. Consequently, they affect a suspicious or a compliant status to the biological sample. These methods should present an as low as possible of “false positive” results and especially minimize the rate of “false negative” results (Decision 2002/657/EEC). These analytical techniques can either be immunological or based on chromatography coupled to mass spectrometry. The following synthesis review will highlight additionally novel analytical techniques, “omics” techniques, aiming at an indirect screening of prohibited substances, in particular natural occurring steroids.

5.1.1 Immunochemical techniques

Immunochemical techniques are based on an antigen-antibody interaction. There are two main techniques used: Radioimmunoassay (RIA), which is based on a radio element, and enzyme-linked immunosorbent assay (ELISA), which is based on an enzymatic system. These techniques permit the quantification of steroid hormones through the bindings established between their corresponding tracers, an analog of antigen, and the antibodies occurring in the biological sample. They classify hence the sample as compliant or suspicious. RIA needs a prior purification of the biological sample and offers several advantages, mainly the possibility to analyze different substances at once and to denature the sex hormone binding globulin (SHBG), which is transporting and bound to these substances. However, this tool requires large sample volumes especially when the substance is present at low concentration levels. It could also lead to erroneous results due to the reactivity of the antibody [25]. As for ELISA, it permits to measure the antigen through an enzymatic pathway. ELISA represented the reference tool for screening steroids abuse before the emergence of mass spectrometry tools. The main advantage of ELISA, in comparison with RIA, lies in the absence of a radio element tracer. However, ELISA exhibits lower specificity as compared with what can offer mass spectrometry.

5.1.2 Chromatography coupled to mass spectrometry

The analytical methods based on this technique allow identifying the administered substances or the markers resulting from such an administration. As far as endogenous AAS are concerned, this technique permits to give an idea about their concentration levels in the biological matrix in order to compare it with the settled thresholds. Two main branches of chromatography have been widely used for screening purposes: Gas Chromatography and Liquid Chromatography coupled to mass spectrometry.

5.1.2.1 Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS remains the most widespread technique used for screening analysis of steroid hormones in the anti-doping control laboratories [26, 27]. A sample preparation step consisting of a hydrolysis, extraction, concentration, and purification of the analytes is necessary in order to detect trace levels of substances of interest in the biological matrix. This technique usually requires a prior derivatization of the compounds with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), PentaFluoroBenzaldehyde (PFB), or PentaFluoroPropionic Anhydride (PFPA) to enable their analysis on GC-MS. 5MS columns (5% phenyl and 95% of methylpolysiloxane) are usually used for chromatographic separation. Negative Chemical Ionization (NCI) and Electron Impact (EI) ionization modes are frequently used for analyses. Different analyzers have been used: ion trap [28], simple and triple quadripole [27, 29]. High-Resolution Mass spectrometry (HRMS) analyzers have also been used such as Time of Flight and Electromagnetic sector [29, 30].

The GC-MS techniques permit to obtain a better chromatographic separation than when operating with a Liquid Chromatography-Mass Spectrometry (LC-MS), even with the emergence of Ultra Performance Liquid Chromatography (UPLC). The main advantage of GC-MS, in comparison with LC-MS, is the absence of ion suppression effects.

GC-MS was in particular used for testosterone screening in equine species [31]. In bovine, GC-MS was used to figure out some potential biomarkers and thresholds for natural occurring steroids misuse. For instance, 5α-pregnane-3β,17α-diol was suggested as a biomarker for progesterone misuse, 5β-androstane-3α,17β-diol for testosterone misuse, 17β-androst-1-ene-3-one for boldenone misuse, and 17α-estradiol for estradiol misuse [32, 33].

New bidimensional separative techniques (GC × GC) have recently been reported in the anti-doping field [34]. The chromatographic separation on these systems is performed through two capillary columns placed in series permitting to obtain a higher separation power in comparison with unidimensional GC technique. In addition, the bidimensional gas chromatography results in a simplification of the sample preparation step by eliminating the purification process [35].

5.1.2.2 Liquid Chromatography-Mass Spectrometry (LC-MS)

LC-MS use in the anti-doping field as a screening technique has greatly increased with regard to the performance allowed [36]. LC-MS process results in a drastic gain of time in comparison with GC-MS technique regarding the lack of a derivatization step. Moreover, it enables the study of the conjugated form of the steroid in addition to its free form and therefore requires no hydrolysis step. This capacity allows a better understanding of the global metabolism of the steroids.

The ionization modes frequently used when dealing with AAS analyses in LC-MS are Atmospheric Pressure Chemical Ionization (APCI), Atmospheric Pressure Photo-Ionization (APPI), and Electrospray Ionization (ESI) [37]. The ionization type and mode depend strongly on the nature of the steroid. For instance, conjugated steroids are usually ionized in ESI⁺ or ESI⁻ modes [38] and free steroid uniquely in ESI⁺ mode.

In addition to the mass analyzers described previously for GC-MS analyses, LC offers the possibility to be coupled with other types of high-resolution mass analyzers such as Orbitrap or Fourier Transform Ion Cyclotron Resonance (FT-ICR) [39]. All of these analyzers offer the possibility to work with a high throughput, which is extremely important criterion for screening analyses [40, 41].

LC-MS was performed for screening purposes of more than 50 doping agents including testosterone, estradiol, boldenone, and norandrostenedione in porcine and bovine species [42]. It also detects fraudulent use of boldenone in bovine through the detection of the sulfoconjugated form [43]. In addition, it was performed in equine to detect testosterone, nandrolone, and boldenone misuse in entire male horses through the quantification of sulfoconjugated forms [44].

The emergence of UPLC permitted to improve the separation power of steroids and considerably reduced the analysis time. Different studies used this technique for AAS analysis and screening [45].

5.1.3 “Omic” techniques

“Omic” strategies have been applied in different scientific research fields mainly for biological applications. The “omic” techniques aim to study the recorded variations at different biological levels ranging from gene sequencing to metabolites expression [46]. It generates consequently a large amount of data permitting to understand the biological functioning of the organism as a whole. In the present context, the main objective of these approaches would be to determine biomarkers capable of characterizing the administration of a particular anabolic agent or a particular family of those agents [47]. These techniques have already shown their relevance in the anti-doping field, particularly, dealing with the issue of natural occurring steroids misuse. “Genomics” and “transcriptomics,” “proteomics,” and “metabolomics” have in particular been investigated to reveal candidate biomarkers that would serve screening purposes. Most of reported transcriptomic studies in the anti-doping field dealt with the influence of peptide hormones such as human Growth Hormone (hGH), Erythropoietin EPO and Insulin-like growth factor (IGF), and anabolic steroid hormones on the Deoxyribonucleic Acid (DNA) transcription [48, 49, 50]. These studies permitted to highlight some gene biomarkers.

The second strategy of interest in the cascade of “omics” (Figure 3) is “proteomics,” which sheds the light on the proteome investigation. This approach is of high importance in the anti-doping field since permitting a profiling of the proteins concentration variations following the administration of anabolic substances [51]. Proteomics study may either be performed in an untargeted way or more targeted, focusing on a limited number of proteins of interest, a-priori selected. The untargeted proteomic study is usually performed in three main steps: first, the proteins are separated based on two-dimensional electrophoresis (2-DE) or multidimensional chromatography, then the analysis of the separated proteins is made by Matrix-Assisted Laser Desorption Ionization-Time Of Flight (MALDI-TOF) or MALDI-TOF/TOF, and finally, the proteins are identified. Very few untargeted proteomics works are reported in the field. However, several studies reported the use of targeted proteomics. For instance, Mooney confirmed the decrease of binding capacity of SHBG globulins in bovine following an administration of AAS [52]. In another study, Cacciatore demonstrated the variations occurring in concentration levels of IGF1, inhibins, and osteocalcin after administration of estradiol and nandrolone [53]. In equine, Nagata confirmed a decrease in Luteinizing Hormone (LH) and inhibins concentration levels after an administration of nandrolone in stallions [54].

Figure 3.

“ Omics ” cascade.

“Metabolomics” is the third family of interest in this “omics” family cascade. It focuses on the metabolome that is formed by the whole group of low-molecular-weight molecules. Sugar, organic acids, amino acids, vitamins, and steroids are part of the molecules included in metabolomic studies. Metabolomics consists of the study of the disturbance resulting from a genetic tampering of the organism or from a toxicological exposition or a treatment. The main objective of metabolomics is to shed light on compounds of interest potentially used to detect biological disturbance encountered by the organism. Many studies in the anti-doping field have evaluated this strategy, namely in human [55, 56], equine [57], or bovine [58, 59] species. These studies confirmed the feasibility and the efficiency of such an approach in this field. The different steps encountered for a metabolomic study are represented in Figure 4.

Figure 4.

The different steps of a metabolomic study.

“Steroidomics,” a subfamily of “Metabolomics,” focuses on the study of the steroidome, the whole group of steroids in the organism. Steroidomics exhibits a similar analytical workflow as the one performed in metabolomics except that it focuses, through sample preparation and acquisition mode, on steroids monitoring [60]. It may be of interest in the anti-doping field, to study the variations of steroid concentration. This monitoring enables a better understanding of the consequences of anabolic steroids administration on AAS secretions in the organism. Consequently, a more accurate identification of doping cases with natural occurring steroids and an identification of potential candidate biomarkers are realized. This multidimensional approach mixing analytical and statistical tools requires an adequate sample preparation step and a specific detection method. Once the steroid profiles are obtained, they are submitted to a multivariate statistical processing to offer a first general view of the data distribution and the generated discrimination. Principal Components Analysis (PCA) and Orthogonal Partial Least Squares (OPLS) are two examples of, respectively, non-supervised and supervised statistical multivariate analysis tools. While PCA models permit to obtain a first overview of the sample distribution without any a priori instruction regarding their status, OPLS models are characterized by an incremented Y variable indicating the status of the sample and affecting a supervised nature to the model. Before proceeding to the statistical process of the data, the latter is normalized in order to decrease the statistical dominance of very intense peaks over less intense but interesting peaks. Finally, quality control samples, consisting of pools of the different samples of the study, should be injected all along the analyzed batches to assess the analytical robustness of the whole process and guarantee accurate analytical findings. Steroidomics can be divided in three main categories: non-targeted steroidomics, semi-targeted steroidomics, and targeted steroidomics. Non-targeted steroidomics requires a minimal sample preparation to avoid the loss of any steroid of interest. Rijk tested this technique to evidence many phase I and II steroid hormones biomarkers following testosterone precursors, DeHydroEpiAndrosterone (DHEA) and pregnenolone, administration in bovine [61]. Semi-targeted steroidomics consist of the assessment and monitoring of a specific fragment characterizing a group or a family of steroids. This approach has been used by Thevis, who discovered new synthetic steroids based on fragments at m/z 77, 91, and 105 corresponding respectively to fragments of steroids A ring and phenol group of estrogens [62]. In another study, Anizan selected fragment ions at m/z 97 and 113 to identify new sulfo and glucuruno-conjugated steroids attesting of an illegal administration of testosterone precursor, androstenedione, to bovine [63]. Finally, targeted steroidomics consists of the quantitative profiling of a selected set of steroids in order to study their concentration levels occurring variations following an anabolic treatment. Such a strategy was for example performed in order to establish suspicion thresholds and identify new biomarkers for DHEA, DiHydroTestosterone (DHT), and testosterone administration in men [64], to discriminate estradiol-treated bovine from the control group [65] and nandrolone-treated equine from control group [66] and served to evidence the evolution of steroid concentrations with regard to the seasonality [67].

5.2 Confirmatory strategies

The confirmation step is performed once the sample is declared suspicious following the screening process. The following part will review two main analytical approaches implemented to confirm natural AAS misuse.

5.2.1 Steroid esters detection

Steroid esters are synthetic compounds composed of a steroid moiety (the active part of the substance) and a side chain, which can be located on carbon 3 or more often on carbon 17 of the steroid. Since steroid esters are widely used in bovine and equine, several analytical strategies have already been developed to detect their misuse.

With regard to their lipophilic nature, the steroid esters cannot be detected in hydrophilic matrices such as urine. However, they can be detected in tissues [68], fat [69], hair [70], and blood [71].

While tissues and fat can only be collected at the slaughterhouse, hair and blood may be collected unambiguously from bovine and equine. Regarding hair, substances are actually transmitted to the hair through the hair follicle from blood stream or from sweat to the hair shaft. Hair can consequently provide a historical background on the steroid esters misuse. However, hair is a relatively complex matrix, and the analytical process adopted to extract substances from it is quite challenging. In fact, using harsh analytical conditions such as strong acidic or basic digestion of the hair can lead to the degradation of the ester. Instead, the use of soft analytical conditions such as methanolic extraction will result in a very low extraction recovery of the ester.

Taking into account these drawbacks, blood appears to be an interesting matrix with regard to its containment in steroid esters and the relatively easy analytical process to perform for extraction. The esters are transported through this matrix to the target cells and are usually bound to some blood proteins such as albumin [72] and SHBG [73]. These esters are gradually hydrolyzed in the blood stream by enzymatic esterase and are consequently found at trace levels (dozens to hundreds of pg/mL) [74]. Therefore, very sensitive analytical methods have been developed in order to detect them in blood [75] using some specific derivatization reactions in order to optimize the sensitivity [76, 77].

5.2.2 Isotopic approach

This approach consists of calculating isotopic ratios of different chemical elements such as ¹³C/¹²C. This ratio can be expressed as an isotopic deviation following the application of the equation below:

$${\delta }^{13}C[‰]=\left(\frac{{\left({}^{13}C{/}^{12}C\right)}_{sample}-{\left({}^{13}C{/}^{12}C\right)}_{standard}}{{\left({}^{13}C{/}^{12}C\right)}_{standard}}\right)\times {10}^3$$E1

This ratio is measured based on a reference compound, the isotopic deviation of which is fixed at zero may be used for the purpose (e.g., Vienna Pee Dee Belemite (VPDB)).

In the anti-doping field, the purpose of this technique is to differentiate endogenous and exogenous anabolic steroids according to their enrichment in ¹³C. The first applications involving the use of a GC-C-IRMS permitted to different anti-doping laboratories to identify exogenous testosterone in human [78, 79, 80]. This technique covered thereafter the animal anti-doping field and different studies using GC-C-IRMS focused on problematic issues related to the administration of natural steroids to equine [81] and bovine [82].

The small differences between endogenous and exogenous anabolic steroids isotopic deviations are one of the main difficulties encountered when applying the GC-Isotope Ratio Mass Spectrometry (IRMS) technique. In fact, the endogenous isotopic deviation of anabolic steroids depends mainly on the diet [83, 84]. Depending on the type of food consumed, the isotopic deviation of ingested cholesterol, and hence deriving anabolic steroids, will not be the same. Some plants called “C3 plants” such as wheat, rice, and soya exhibit low endogenous isotopic deviations in comparable ranges with those exhibited by synthetic steroids, mainly synthesized starting from C3 plants. However, the “C4 plants” such as maize and sugar cane generate a high endogenous isotopic deviation, which in this case of food-based diet permits to identify unambiguously exogenous administration of anabolic steroids.

6. Conclusion and perspectives

While the classical analytical methods, targeting a single or a large panel of anabolic substances, as currently implemented in the anti-doping and control laboratories are efficient to detect abuse with a large set of prohibited compounds, they have been showing some limits to detect abuse in some specific cases related to androgenic anabolic steroids misuse in racing and food producing animals. The difficulty has become even greater with the application of new doping methodologies aiming at bypassing the current analytical methods detection limits. The administrations of anabolic steroids cocktails, designer steroids, or natural occurring steroids are some examples of these new doping methodologies, which permit to escape control.

The use of natural occurring steroids such as testosterone appears to be one of the most challenging and problematic issues for doping control laboratories. With regard to their natural occurrence in the metabolism, the differentiation between their endogenous and exogenous origin becomes problematic. Moreover, the concentration levels of these steroid hormones vary between the different subjects part of the same species introducing inter-variability factors. Furthermore, these levels vary in the same subject at different time points due to different parameters such as seasonality, mating, and diet, which define the intra-variability factors. These two factors render the settling of a decision limit for a natural occurring steroid misuse difficult.

Testosterone in particular is among these steroids a problematic one for control laboratories since it has been shown to occur naturally in equine and bovine species while its administration under synthetic form is known to be very potent. Therefore, analytical strategies allowing to point out the exogenous administration are more than expected by the scientific community.

While confirmatory strategies seem to enable tackling the natural hormones issue and fulfill corresponding requirements, an additional effort appears necessary to validate the developed screening approach based on steroidomic profiling.

With regard to the general issue of natural steroids misuse in equine, more research work is needed in order to investigate minor metabolites of such administrations, which can reinforce the current international criteria. A stronger collaboration between the different reference laboratories worldwide is essential in order to settle population reference ranges of endogenous AAS and hence obtain a repository for future steroid doping challenges. A deeper investigation of the biosynthetic and physiological system of horses is also very important in order to have a better understanding of the biosynthetic pathway of endogenous steroids and hence generating a higher efficiency in tackling the issue.

Furthermore, other “upstream” omics techniques such as “genomic” and “transcriptomic” have been reported to be efficient for detecting natural steroids misuse and collaboration between these different fields would offer a mass global approach. For instance, Cannizzo developed a method based on DNA microarray data analysis [85] showing a large set of differentially expressed genes between controls and prednisolone treated bovine. Moreover, genomic instability and DNA damage were proven after nandrolone decanoate administration to rats [86]. However, such genotyping is costly in addition to the uneasiness of its application in the context of doping control. Many studies reported the possibility of generating information about the genotype starting from the phenotype and specifically steroid profiles [87, 88, 89]. These reported findings would offer a general overview of different organism levels and would be of a great help to evidence several challenging doping cases.