Doping in Athletics: A Persistent Challenge

Introduction

Doping has emerged as one of the common practices witnessed in the world of athletics. However, the practice is strongly forbidden across the athletic organizations and mostly, the World Anti-Doping Agency (WADA) which was established in the year 1999. WADA defined doping as the suspected or proven presence of the prohibited substances within the athlete’s body. WADA has further included the inventory of the substances deemed illegal across sports. More responsibilities have been assigned to the anti-doping laboratories, which guarantee the world harmonization of the guidelines linked to anti-doping. A range of the prohibited substances include pharmacological and chemical compounds. In an effort to test incidents of doping, blood and urine are regarded as the routine samples that would substantiate drug analysis. Current research has however indicated erroneous incidents which must have pointed at smaller detection window, increased exposure to bacterial growth, incidents of disease transmission, strained stability of the matrices and the invasive sample collection. For the purposes of avoiding erroneous scenarios, hair has been considered as an alternative as well as the most fundamental biological specimen profiled for drug testing. Hair testing is believed to attract a range of the sensible merits aligned to different matrices. Most of the researches have indicated that hair testing would obviously facilitate a larger detection window given its length. The new approach gives the anti-doping laboratories an extended room for detection. Hair analysis regards hair as part of the biological matrices and distinctive in the sense that it lacks active mechanism meant for drug metabolism. On the basis of this preamble, the discussion narrows down to reviewing the hair as one way of predicting doping among the athletes.

Background Information

The world of athletics is recently put under the cloud of suspicion. According to David (2015), a leaked report from the famous International Association of Athletics Federations (IAAF) noted that the blood tests of over 800 out of the 500 athletes have shown a likelihood of consistent doping. Some of the suspected cases are however still under investigation with around 146 World Championship medals being contested between the year 2001 and 2012 based on the doping data. David (2015) further noted that in the year 2009, IAAF went further in establishing the biological passport, which would consistently update the records linked to blood measurements for every athlete. The updates would further dig into traces of drugs as well as their metabolites, which is an idea that would catch the cheats who would be required to explain changes across the baseline physiology. Tom Bassindale, one of the researchers at Sheffield Hallam University, pointed out that the biological passport has been an effective tool which would help in reducing doping. However, Bassindale still argued that there are people who would try all means of getting away with doping. For instance, micro-dosing may go undetected which is something that prompted Yannis Pitsiladis to note the essence of combining biological support with newer as well as accurate tests. Bassindale further recommended that hair samples have the potential of attracting accurate results and can be aligned to the blood sample extracted from the same athlete. Testing hair samples for the purposes of detecting recreational drugs is said to have emerged as a routine among the cricketers especially after the drug related death of Tom Maynard in the year 2012. Devcic et al. (2018) further noted that doping behaviours cover the evident consumption of the prohibited performance enhancing drugs or substances, as well as application of prohibited techniques which may not be limited to use of agents who would impede regular procedures from tracking the banned substances. Doping behaviour has always been regarded is one of the most health threatening behaviour crowded with a range of negative consequences which include subdural hematomas, virilisation, acne, altered kidney and liver functions, infertility, thrombosis, peripheral edema, myocardial ischemia and cardiovascular. The global fight against doping has gained traction over the recent times. Devcic et al. (2018) further indicated that the global fight has been bolstered by a significant focus on preventive measures, identification of factors linked to doping; establish the most specific as well as targeted campaigns and strategies that would address the substance misuse.

Among many other factors, Devcic et al. (2018) believed that most of these factors can be sports related, sociopsychological, sociodemographic, coaching, motivational as well as training related factors. The anti-doping efforts are all pointed at all these factors with attempts of justifying a doping scenario. Kalliokoski et al. (2019) still believed that blood and urine samples have dominated most of the testing programs. The authors noted that most of the xenobiotics are largely detectable across the urine samples with the help of chromatography mass spectrometry (GC-MS). However, the urinary concentrations linked to classical doping agents would exceed the ones that would be found across the biological samples. In such cases, additional sample can be collected without encountering intrusion as compared to blood sampling. A number of prohormones including the androstenediol and androstenedione as well as their nor-derivatives can be synthesized across large quantities especially by nutraliments and maybe available in some of the countries. The insulin-like growth factor, the growth hormone, interleukin-3, erythropoietin and the perfluorocarbones have been highlighted as some of the new active substances that need to be traced. The compounds cannot be detected in urine based on the research conducted by Cutler et al. (2020). Due to gaps left behind by urine and blood analysis, hair analysis has consistently been the immediate alternative. Hair analysis has however been under a chain of debates with its application appearing in some of the forensic cases, which aid the establishment of a possible intake of the highly suspected anabolic steroids. Results from the hair analyses and the GC-MS have been used in the defence of most of the court cases. Kalliokoski et al. (2019) asserted that drugs which are incorporated in hair would remain in its keratin matrix for the longest period of time, which is something that has opened a wider window for doping detection. Hair analyses have become significant in the establishment of a possible use of the exogenous substances thereby confirming a possibly long term exposure. It is worth noting that the anabolic steroids are said to have been detected in hair extracted from the body builders. The nandrolone would range from 190-260 pg/mg, the testosterone ranged from 45-70 and stanazolol would range from 130-160. Additional compounds that would be detected from the hair samples include metandienone, enantate derivative, metenolone, and methyltestosterone, the ester of testosterone, salbutamol, stanazolol, clenbuterol and dehydromethyltestosterone. Therefore, hair analyses are necessary and have a role to play in an effort to support the antidoping initiatives in the world of athletics. While the use of hair analysis has been debated for long, its importance cannot be ignored by all means and therefore its use has to be embraced. This is due to the fact that both the urine and blood samples are prone to gaps that have never been addressed for a long period of time.

Anabolic androgenic steroids, (AAS)

In the course of reviewing the hair analysis, it is important to revisit the anabolic androgenic steroids. Zahnow et al. (2018) asserted that androgenic anabolic steroids are regarded as synthetic derivatives associated to male hormone testosterone. The researchers have noted that the steroids have the capacity of exerting strong effects especially on human body which turns beneficial for the performance of the athletes. The AAS are also considered as synthetically produced variants across the male sex hormone testosterone. Notable, the anabolic part of AAS denotes muscle building while the androgenic part points at the improved male sexual characteristics. Besides, steroids points at a class of drugs which are normally prescribed for treatment of certain conditions that emanate from steroid hormone deficiency. Havnes et al. (2019) pointed out that AAS was essentially utilized by the professional athletes before early 1980s. The AAS would subsequently be used by recreational athletes for the purposes of boosting the muscle strength. Estimates have indicated that AAS is dominantly used in United States, parts of Middle East and Europe as well. When used in the supra-physiological doses, AAS would have a diminishing impact especially on the commonly known hypothalamus pituitary gonad axis. This leads to a reduction in terms of the endogenous production of the testosterone. Any terminated use of AAS, lasting or temporary hypogonadism is likely to occur accompanied with such symptoms like sexual dysfunction, fatigue and even depression. Addiction may lead to physical and mental problems and body image disorder. According to the research conducted by Havnes et al. (2019), AAS has received such a huge traction over the recent times as a result of its side effects like the cardiovascular diseases, metabolic and haematological issues. Other effects include the hepatic impairment, reduced cognitive function, anatomical changes in the entire brain and disrupted sexual hormone system. Men are likely to develop gynecomastia while women are likely to experience masculinization. Some of the users have reported psychotic, maniac and hypomania symptoms. Most of these of these symptoms have strongly been associated to AAS abuse. Both the athletes and the non-athletes would abuse the drug in an effort to enhance the physical appearance and performance. Most of the users would go ahead and combine separate types of the steroids for the purposes of maximizing effectiveness, which is a practice which is denoted as stacking. Why AAS is such an important topic today as far as doping in the world of athletics is concerned? It all started with the motive of the users, which later turned into adverse effects of the abuse which can only be recognized gradually. The worst area includes the irreversible damages while athletes face a likelihood of the tendon ruptures. The largest threat revolves around cardiovascular diseases, which involve the blood vessels and the heart. Some of the diseases include cardiac arrhythmia, thromboembolism, hypertension, coronary artery diseases, and cardiomyopathy. Significant areas that are even attracting more attention include vascular calcification. Based on this concern, Liu and Wu (2019) noted that studies need to address the fundamental hypothesis associated with exogenous androgen-induced vascular calcification. It is worth noting that the immunohistochemical analysis indicates the expression of androgen receptor. The analysis touches on the calcified tissue linked to human femoral artery while the in vitro studies established that 9 days of treatment with dihydrotestosterone or testosterone would lead to increased calcification linked to phosphate-induced muscle cells. The experimental data would insinuate that androgens would increase the significant degree of the vascular calcification via AR. This would subsequently induce cell damage thereby leading to loss of the tissue elasticity as well as fibrotic hyperplasia. Another complication is atherosclerosis, which is a disease where the artery narrows as a result of lipid metabolism disorder and plague formation. AAS is known for inducing lipid metabolism disorder that leads towards decreased high density lipoprotein, as well as increased low density lipoprotein.

The two are said to increase the cerebrovascular disease and CAD. Physical exercises are however regarded as one way of increasing HDL with a remarkable effect on triglyceride and LDL levels, which is a process that impedes the harmful impact of AAS. At the same time, lipoprotein levels are likely to return back to their normal range when the AAS use is discontinued for either months or weeks. Long term use of AAS can lead to homocysteine levels, which may result into hyperhomocysteinemia. The latter is commonly classified as a risk factor for the episodes of CAD and coronary atherosclerosis. Liu and Wu (2019) also noted episodes of thromboembolism. In this case, AAS has a direct impact on fibrinolysis and coagulation system. The abusers are at the risk of intra-cardiac and arterial embolism. Hypertension is another condition associated with AAS. In case of reversible hypertension, AAS can induce water-sodium retention into the kidney, which can lead to increased blood volume as well as the blood pressure. Other risks include sudden cardiac arrest, arrhythmia, dilated cardiomyopathy, cardiac hypertrophy, coronary spasm, and myocardial apoptosis.

Methods to detect drug doping

The fight against doping has received the full support of the detection methods, which focus on the blood matrices. Faiss et al. (2019) highlighted the fact that analytical possibilities seem to have evolved over the time. While some of the bodies seem to discredit the outcomes of urinalysis, it is important to note that blood manipulation or transfusion and urinary steroidal modules have remained dominant for long. Further complementarities aligned to Athlete Biological Passport led to significant development of analytical techniques include MS/MS and UHPLC-HRMS. Most of the urine analyses would detect possible use of the Endogenous Androgenic Anabolic Steroids (EAAS). However, establishing an exogenous forbidden substance in the urine sample is likely to represent simplest means leading towards sanctions or rule violation. Despite the observation, the urinary module has gained relevance in monitoring the steroidal hormones. Faiss et al. (2019) also noted that the steroidal module would play a fundamental role in measuring concentration of a range of free urinary and glucuroconjugated compounds associated to testosterone as well as its metabolism, etiocholanolone (Etio), epitestosterone (E), androsterone (A), 5β-androstane-3α, 5α-androstane-3α, 5α-Androstane-3α, 5α-Adiol/5βAdiol, 17β-diol (5βAdiol) and 5αAdiol/E. Notably, episodes of low dosages would result into relatively short detection windows. In the blood analysis, steroid profiles play a fundamental role. The Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) approach is necessary while quantifying the 11 endogenous steroids which would be detected in serum. Concentration values which would be measured by HRMS are likely to indicate the correlation with the ones established from the mass spectrometry especially when handling the target hormones. Moreover, attention given to steroidomics gives room for untargeted simultaneous evaluation of relatively wide range of compounds. Handelsman (2020) noted that listing blood and urine analyses sounds general. It is therefore important to revisit the Prohibited List bans that would note the growth hormone, androgens, EPO and the associated substances known for stimulating the endogenous production. Across the 15 categories, S1 takes the anabolic agents; S2 captures the peptide hormones while S4 takes both the hormone as well as the metabolic modulators. S9 entails glucocorticoids. The S2 class which highlights the mimetic and growth factors equally touches on the GATA inhibitors, erythropoietin receptor agonist, TGF-β signalling inhibitors, HCG, LH, the hypoxia inducible factor and corticotrophin. P1 takes on the beta blockers and S3 involves beta2-agonists. The S4 also taps into aromatase inhibitors, estrogen receptor modulators and the anti-estrogenic substances. S5 covers the masking gents and diuretic, S6 covers stimulants and S7 takes note of the narcotics. Handelsman (2020) also pointed out that S8 takes the cannabinoids and S9 includes glucocorticoids. Significant methods that would be engaged in determining the substances include physical and chemical manipulation, blood manipulation and gene as well as cell doping. Testing methods would therefore be aligned to androgen and haemoglobin doping. Androgen doping can either be indirect or direct in which direct doping includes administration of the testosterone, synthetic and natural androgens while indirect doping involves a range of the non-androgenic drugs. Detection approach for synthetic androgens includes the L/GC-MS while natural androgens would be detected by CIRMS, T/E and L/GC-MS. In case the dopant is a designer and nutraceutical androgens, then the L/GC-MS would still be used. The hCG immunoassays are used to detect the hCG recombinant or urinary. The hLH immunoassay is applicable to hLH recombinant while L/GC-MS would detect the GnRH analogs, neurotransmitters and opioid antagonists and anti-estrogens. It is worth noting that traces associated with synthetic androgens and even their metabolites are likely to remain detectable up to several months after their time of administration.

Detection of androgen doping has further attracted the most underutilized options which cover biological matrices like nails, hair and skin. In the course of detecting androgen doping, hair carries with it the advantage of minimal invasive sample accompanied with convenient and simple storage. It is quite obvious that hair analyses for a androgens need to go through validation and rigorous standardization as well. Advanced LC-MS method is believed to be more specific when compared to the immunoassays. On the other hand, haemoglobin doping can either involve direct blood transfusion or sometimes pick on indirect methods meant for boosting haemoglobin through stimulation of erythropoiesis, which can be done via administration of erythropoietin, the mimetic or even the analogs. Direct doping can first be attained through heterologous, which can still be detected through flow cytometry, or the bimodal population of the commonly known blood group antigens. Another direct doping mechanism includes autologous, which can be tested through urine phthalate excretion, the Biological Passport Biomarkers and the Total haemoglobin mass. Indirect doping includes the use of biosimlars and rhEpo which can be detected through the urine double immunoblot. Other doping mechanism include the use of hypoxia altitude training, which is not yet banned, the hypoxia mimetic, stabilizers, hypoxia inducible factor, the DPG analogs and perfluorocarbons which can all be detected with the help of LC-MS/MS. Across these detection mechanism, the convenient test for the autologous doping include the haematological module linked to ABP, which was introduced in the year 2009. The biomarker test essentially adopts the commonly known Bayesian approach. The ABP haematological model is sometimes sensitive to both the indirect as well as direct haemoglobin doping. In addition, detection of the human EPO gene led to the growing significance of rhEPO. However, most of the studies have established that detection of EPO in the samples of urine can be complicated at some point as a result of low concentrations as well as the fundamental requirement of separating the endogenous EPO from the exogenous recombinant. For the purposes of addressing this complexity, the double immunoblot has the capacity of establishing the urinary excretion. Another observation includes the sensitivity of immune-electrophoresis test which is equally laborious but avails 1 week of post administration. Apart from blood and urine analyses, researchers are keeping an eye on the significant use of hair analysis, which is categorized as part of the new developments.

New developments

Aguilar-Navarro et al. (2019) asserted that doping cases must have been detected to around 57%, which leaves the rest of the cases untouched. This is one observation that has send out doubts over the efficiency of the urine and blood samples used in the analytical process. In other cases, the most contradicting findings have led to dismissal of some of the legal claims which question the legitimacy of the medallists. While hair analysis has strongly been doubted before, it is gradually emerging as one of the significant methods that can no longer be ignored. The analytical identification of the doping substances noted from hair analysis may not constantly be sufficient in underpinning a doping offence. However, the most underlying target includes the anabolic agents due to the fact that desired actions would relatively take longer when compared to the excretion. More sophisticated procedures might be needed for the purposes of producing positive analytical results as part of the competition control. Notably, analysis of the exogenous steroids in the hair samples may call for a significant range of the steroids across the hair matrix at trace levels. The process has been regarded to be more complicate as a result of a series of the biotransformation reactions which emanate from other precursors especially the metabolites. The precursor elements attached to the anabolic steroids, such as the esters, can sometimes be promising in terms of posing the analytical targets for the hair analysis. This is because detection can be attained after considering the exogenous intake. Apparently, quantitative analysis of the active parent compounds such as testosterone remains controversial. For the purposes of attaining accuracy, clinical applications are essentially considered for the reproducible conditions with bio-variability of the parameters remaining significant in determining the cut-off levels associated with the criterion of the abuse. Despite the complications detected across the hair analyses, the process remains necessary due to a number of reasons. First, there is need of determining the single and long term administration of the pharmaceuticals, which might be used without medical indication. Under the antidoping controls, hair analysis has the capacity of providing more information linked to urinalysis. This paves way for specific cases such as beta-2-agonists, which raises the theoretical possibilities of filtering acute administration for the purposes of attaining the stimulatory effects needed for the anabolic effect. Hair analysis is also securing a place in verification of epitestosterone and application of the testosterone esters. However, most of the blood analyses have been successful in terms of demonstrating the exogenous use of the testosterone through detection of the esters with the help of GC-MS. The same approach can still be used for hair analysis. Some of the one-time applications for the hair analysis are said to have failed in terms of influencing positive results. Research indicates that there are chances of using the GC-CIRMS hair analysis, the same it is used for the urine analyte. However, such chances are likely to be ruined by sensitivity of instrumentation with differences in terms of the concentration levels across the matrices. In addition, it is unlikely that application of the isotope ratios of the hair extracts can give substantial exploitable results. Most of the laboratories are setting their preferences on hair samples than the urine samples. This is due to the fact that hair samples are free of any form of embarrassment that is largely linked to urine sampling. Again, hair samples can easily be stored or ferried without considering the pH control, refrigeration or any other preserving agents. Several samples can still be extracted at any given time intervals for the purposes of expanding the analysis as a result of the additional analyte. Salomone et al. (2019) further noted that there are some dopants that are making hair analyses important. Some of them include clostebol, which is sometimes referred to as 4-chlorotestosterone, is commonly known for being a weak synthetic anabolic steroid and significantly used as one of the performance enhancing drugs. As a result, the drug has been banned by WADA. As one of the dopants, clostebol is essentially used in its ester form or states such as propionate, acetate and caproate. The intake is commonly through intramuscular injection or oral ingestion. Over the recent years, clostebol has attracted cases of suspension of athletes across a wide range of sports. Italy has been cited as one of the countries to have reported the highest number of cases of the usage of clostebol. The cases are believed to have escalated due to increased sell of clostebol containing medicines for the gynaecologic and dermatologic treatments.

The US, France and other European countries went ahead banning prescription of anabolic agents. Cases of incidental clostebol contamination after having sexual intercourse have also been reported among athletes. Due to rampant cases of clostebol, the Adverse Analytical Finding (AAF) found it necessary to make use of the hair analysis for the purposes of facilitating complementary information associated to the positive finding. The obvious disadvantage of urinalysis revolves around the fact that it can only avail short term information regarding the drug consumption. At the same time, the findings could not ascertain the chances of a sustained drug abuse. Based on these reasons, urinalysis has remained ineffective in terms of detecting the long term steroids. Due to such gaps in the course of testing the presence of dopants in the body of an athlete, hair analysis has gained mileage in terms of differentiating between mycotoxin zearalenone and the commonly known urinary metabolite zeranol. Across their studies, Salomone et al. (2019) developed UHPLC-MS/MS method in the course of detecting the presence of clostebol acetate and clostebol in hair. The method was applied in three cases that covered athletes who were sanctioned for using the dopant. The study was executed in the doping control laboratories as significant elements were tested elements which would lead towards positive findings.

Methods

The research focused on the significant use of the systematic literature, which touches on the critical areas of the study. This is due to the fact that the research could not conduct a real experiment but set preference on relevant case studies that would towards desirable results. While the focus is placed on hair analysis, the research further focused on comparative studies due to rarity of the experimental studies that touch in hair analysis. This section highlights the relevant experiments that have been conducted before. Due to the rare cases, the research only focused on 2-3 experimental case studies that tap into hair analyses. Based on this preamble, this section looks at the search methods, material requirements, assessed outcomes, data extraction, exclusion and inclusion criteria, instrumental conditions, sample preparation, validation and selectivity as well as data analyses.

Search Methods

The study drew its basis on the predicting factors associated with behaviour, intentions, doping and susceptibility. The search covered some of the original studies believed to have been published in some of the scientific peer-reviewed journals from the year 2014 to the year 2020. Most of the studies are believed to have been indexed in EBSCO and MEDLINE databases. Some of the search items include “drugs and athletes”, “doping”, “hair analysis” and “performance enhancing drugs”. Some of the materials extracted from the databases could essentially be determined based on the journal title, the year of publication of the material, characteristics of the involved population such as the age and size, the dependent variables such as behaviour, susceptibility and intentions, the psychological concepts and the outstanding outcomes of the given study.

Material Requirements

The research further focused on the material requirements that need to be established from the sampled case study. Some of the material requirements established in this context include the needful process meant for handling the hair samples, the chemical or any other reagents needed in the sampled tests and ay form of theoretical support. For instance, for the experiment that covers tests for the presence of clostebol, the study looked at the availability of the chemical reagents highlighted in the experiment. Some of the notable reagents and elements mentioned in the experiment include acetonitrile, methanol, testosterone-d3, the clostebol acetate and clostebol. Other considerations include the homogenizer, the metal beads and other standard solutions. Other concerns include the process of handling the samples and sample extraction while detailing the conditions and limits. In this case, the tests are pointed at the keratin matrix, which needs to be extracted, digested, hydrolysed and dissolved for the purposes of determining the dopant.

Data Extraction and assessed outcomes

The process of data extraction focused on the chances of retrieving the material from the respective database. Consideration of the findings could be aligned to the inclusion of the trials, which could essentially be extended to other case studies. The significant outcomes anticipated in any given study needed to be aligned to the predicting factors liked to the doping behaviour and the doping susceptibility. Most of the outcomes could be reported from the experimental studies established from the articles.

Exclusion and inclusion criteria

The research made the significant use of the meta-analytic approach for the purposes of reviewing limited studies before arriving at the predicting factors associated to the doping intentions, behaviour and susceptibility among the athletes. Manuscripts were excluded with studies published before the year 2015 regarded as out-dated. The study only included 2 article based on the criteria highlighted before. However, 10 more articles were considered for both experimental and theoretical support some of the supporting studies focused on the dopants, the doping behaviour banned substances and the detection mechanisms. Most of the included studies were quantitative, thereby highlighting chances of having a dopant in the body of an athlete or not. This attracted development of a scale indicating the response rates and reliability of the outcome. A total of 113 articles were excluded on the basis that they either focused more on the blood and urine samples, or ignored to consider the hair samples. Around 38 of them mentioned nothing relevant in relation to the doping relations while 12 were more of qualitative research. 52 of the articles lacked relevant experiments with most of them criticizing hair samples as credible candidates for determination of the dopants. Most of the excluded articles paid attention to blood transfusion or manipulation.

Instrumental Conditions

The study equally paid attention to the surrounding experimental conditions which would determine the outcome. One of the studies highlighted the need of a Shimadzu Nexera 30 UHPLC system, which is believed to have been interfaced with Sciex API 5500 quadruple mass spectrometer. The setup could further be supported by an electrospray turbo said to operate in positive ion mode. Waters BEH C18 Column was utilized in the course of separating the analytes. On the other hand, the MS system could be operated in SRM, which is put in the monitoring mode for the purposes of establishing the necessary SRM conditions. This can equally be attained by infusing every analyte into the necessary ESI capillary while observing the entrance potential and declustering potential. In another experiment involving the keratin matric, the instrumental analysis only needed the hair samples to be collected especially in the necessary posterior vertex zone. It is worth noting that the toxicological analysis would only require around 30-50 mg of hair. For the keratin matrix, the samples need a room temperature for storage reasons and the plastic envelopes.

Sample Preparation

Sample preparation can be such a critical process in any experimental study both in primary or secondary context. The study considered only two experiments, with the first one covering a test for clostebol across the hair sample and the second one, a toxicological analysis for the keratin matrix using hair samples. For the clostebol-based experiment, a 50 mg of hair was washed for two times using methanol and dichloromethane. The solvent wash was removed and the hair dried using gently flowing nitrogen and later pulverized with the help of a metal beads mill. Fortification of the sample was done with the help of 5µL of the testosterone-d3 thereby yielding the concentration of around 25pg/mg. In this experiment, sample extraction could be attained through addition of around 1 ml of methanol while shaking for some time. The final incubation could be done at around 550C for approximately 15 hours. The organic phase could be collected and later evaporated to dryness with the help of a Techne Sample Concentrator. In the second experiment for toxicological analysis, the hair specimens are extracted and pulverised for the purposes of yielding small fragments which gives more surface for extraction medium. Precautions have to be considered for the purposes of avoiding a possible exposure to aerosols, dust, drug powders and even smoke. Other substances that are likely to contaminate the sample include sebum and sweat. The second fundamental procedure includes isolation of drugs from the significant keratin matrix. It is worth noting that xenobiotics in hair are essentially entrapped into keratin. This means that the keratin matrix needs to be digested, extracted as well as hydrolysed. The hair analysis demands for this procedure which paves way for analysis of the metabolites. It is worth noting that metabolites can only be produced as the key products of metabolism as a process. Ultimately, the presence of metabolites would essentially confirm a possible drug intake.

Data Analyses and Synthesis

The study conforms to the psychological concepts, which are also linked to the psychological constructs which emanate from the literature. The research gives attention to comparison of the dichotomous variables. A further application of the operationalization methods tapped into significant concepts. The meta-analytical approach looks beyond the contextual findings and integrates data before reaching the necessary conclusion. While the findings from the two experiments can be dissimilar, it is important for the research to correlate them before establishing a common finding that can be applicable in almost every hair analysis.

Results and Discussion

Experimental Results

The first experiment included the clostebol-based experiment in which each of the SRM transition could be maintained in a time window of around 10 seconds. Best results could be attained with the help of source block temperature of around 6000C as well as ion spray voltage which moved close to 3800V. Gas settings included the collision gas 8 psi, a curtain gas of 35 psi, source gas 55psi and GS1 of 45 psi. The table below gives the imminent SRM transitions and the subsequent potentials.

The experiment further included linearity, LOQ and LOD. Notably, linear calibration model could subsequently be checked in the analysis of the blank hair samples. Evaluation of the calibration could be attained with the help of the least squares regression. In the experiment, the LOQ and LOD could be estimated with the help of the Hubaux-Vos’ approach, thereby pointing at the uncertainty in terms of the concentrations and signals. The table below shares the calibration data, the calibration interval, LOQ values, and the LOD values.

Notably, the matrix effect could be determined in relation to ISTD via comparison of peak area ratio with the one extracted from methanol solution. The LOD values stood at 0.3pg/mg for the clostebol as well as 1.1pg/mg for the subsequent clostebol acetate. The LOQ values stood at 0.6pg/mg as well as 2.1pg/mg respectively. One of the sampled cases for this experiment include a 33 years old professional boxer who tested positive for the clostebol metabolite that amounted to 1ng/mL in an in-competition control. He however admitted to have applied a certain ointment of clostebol acetate to his wife for medicinal reasons. The last application he did was one week to the anti-doping control. During the test, the leg hair and the arm hair were collected from him with head hair extracted from the wife. The analysis was performed in total length of the hair with full description of samples presented in the table below.

From the results, it is evident that the professional boxer tested positive for clostebol acetate both from the analysis of his leg and arm hair. He however tested negative for clostebol. The boxer’s wife tested positive for clostebol acetate from the analysis of her head hair. A further chromatogram analysis conducted yield the following graphical results as indicated.

tetrahydrocannabinol, 3, 4 methylenedioxymethylamphetamine (MDMA), codeine, morphine, 3, 4-Methylenedioxyethylamphetamine (MDEA), 3, 4 methylenedioxyamphetamine (MDA), amphetamine, cocaine and benzoylecgonine. The second approach was applied to detect 26 NPS which included the stimulants, psychedelic substituted phenethylamines such as 4-methulethcathinone, 3-methylmethcathinone, mephedrone, 4-fluoroamphetamine, alpha-pyrrolidinovalerophenone, amfepramone, methoxetamine, 4-methoxyphencyclidine, ketamine, benzofuran and burpopione. In addition, the hair samples were essentially screened for most of the synthetic cannabinoids, which happened with the help of UR-144, MAM-2201, STS-135, ABD-PINACA, ADBICA, JWH-147, JWH-016, AB-PINACA, AB-FUBINACA and AM-2233. The analytes amounted to 40. The drug in the toxicological analysis could either be a traditional drug or a new drug. Across all the samples, 36 were head hair, 3 of them were leg hair nd18 cases could not be recorded. The table below gives a summary of the drugs or toxics that could be established in different samples.

From a wide range of detection, as indicated in the experimental section, it can be established that hair analysis appears significant in doping control, clinical toxicology, occupational medicine and clinical toxicology. The practical advantage associated to hair analysis includes the larger detection window compared to the blood and urine analysis, which have relatively smaller detection window. On the grounds of hair analysis, toxics, dopants and drugs can easily be detected for even months based on the hair length or the hair shaft to be analysed. Under practical circumstances, the detection window provided for the hair and urine testing can be complementary. The analysis of urine samples would only facilitate short term information regarding drug use by the athletes. Hair analysis stands a better chance of availing the necessary and the most accurate outcomes of cocaine and amphetamines in case they are used as dopants. In the first experiment of clostebol, it could be established that synthetic anabolic steroids can be linked to a significant range of the compounds as far as doping control in the forensic contexts are put into consideration (Devcic et al. 2018). The experiment reports the very initial instance of clostebol acetate determined through hair analysis with sample extracted from a professional boxer and his wife. This is an indication that doping across the forensic cases that involve the anabolic steroids is defining a significant share of the scientific literature thereby paving way for more interpretation. Majority of the steroid administration cases are dominantly reported in powerlifting or bodybuilding. It is worth noting that drugs with relatively low pg/mg from the hair analysis are likely to reflect an occasional exposure to the substance under coverage. The hair analysis for the professional boxer and his wife showed that they were both exposed to the compound, the clostebol acetate. It is worth noting the clostebol s appears in the S1 group of the anabolic agents. Other substances that appear in this group of the prohibited list include the metandienone, drostanolone, oxandrolone, boldenone, stanozolol, metenolone, methasterone and trenbolone. Most of these anabolic steroids are regarded as synthetic derivatives of the commonly known male hormone T. They all adopt the cyclopentanoperhydrophenanthene core, which has the carbon stricture that constitutes the three cyclohexane rings.

removal of the double bonds and even replacement of halogens and heteroatoms. The question is why clostebol would be tested from the hair analysis. Clostebol being one of the exogenous AAS would extensively undergo the metabolic reactions both in phase I and Phase II for the purposes of making the drug inactive, less active or less toxic. The intermediate metabolites stand a better chance of remaining active (Esquivel López 2018). In Phase I metabolism, essential processes that would be noted include hydroxylation, reduction and oxidation, which would later lead to more polar groups that turn into steroids. In phase II reactions, also known as conjugation reactions, they cover conjugation of anabolic steroids. Sulfation and glucuronidation are regarded as key reactions in this phase. During glucuronidation, this is regarded as the predominant pathway in the course of human metabolism while the process remains irreversible. The Uridine diphosphoglucuronosyl-tranferases, denoted as UGT, is used as a catalyst while Uridine-5 –diphosphoglucuronid acid, denoted as UDPGA, is utilized as a co-substrate. The reaction would essentially lead to association of polar glucuronide acid moiety to the identified steroid structure before forming the β-glycosidic bond. It is worth noting that the liver remains the significant sire for glucuronidation. However, UGTs can still be found in the lungs, brain, kidney, breast tissue, prostate and skin at the same time. The idea that UGT can be found on the skin leads to the fact that the clostebol acetate would easily be deposited to the hair structure thereby having its space in the keratin matrix. The moiety can easily be bound to a range of the nucleophilic functional groups such as phenol, thiol, hydroxyl, the amino groups and carboxyl to the parent aglycone, which would give rise to the glucuronide isomers. Sulfation plays a focal role in terms of facilitating metabolic reactions for the exogenous compounds. The sulphotransferase enzyme (SULT) is regarded as a catalyst which be involved in transferring sulfo moiety from the 3-phosphoadenosine-5-phosphosulfate and the co-substrate to the steroid. SULT can either be cytosolic or membrane bound (Esquivel López 2018). The membrane bound SULT can be delocalized to the hair glands, intestines, adrenal gland and liver among others. For clostebol, which is an exogenous AAS, the ultimate presence of the substance itself or its metabolites across the extracted biological sample would be regarded enough to file a report with AAF. The origin of clostebol can significantly be demonstrated via the IRMS or the GC analysis, which is grounded on the basis of content of C produced in most of the synthetic compounds, or their derivatives, especially during their pharmaceutical preparations. In the second experiment, NPS could be detected across a large number of the attendees. Butylone, MXE and methylone could be detected more exclusively across the experiments. This is a pointer to the fact that synthetic cathinones can still be verified despite having encountered falsified cases. However, synthetic cannabinoids could not be established in the tested hair samples because they were out of range. It should also be noted that synthetic cathinones keeps shifting due to presence of the confiscations, which fluctuated between the years 2013 and 2015. The findings reported that methylone emerged as the dominantly confiscated compound in the year 2013 but ended decreasing in the year 2014 as well as 2015. The study further justifies the role of the multi-drug analytical approach as it leads to more sensitive and accurate outcomes as a result of analysing more than 200 metabolites across the chromatographic run (Devcic et al. 2018). The essence of this experiment is to point the fact that a range of drugs and their metabolites can be tested with the help of the hair samples. The experiment points out development as well as validation of the approach engaged in the analysis of the drugs of the toxicological nature. The approach is however sensitive, robust and selective for the purposes of confirming a range of drugs with the help of hair samples. Hair has protein in it, 65-95% of keratin, 15-35% of water, 1-9% of lipids and minerals are less than 1%. Another observation includes colour, texture and composition is likely to vary from one person to another. A range of minerals can be accumulated in hair in a range of 0.25-0.95%. It is stipulated that around 5 million hair follicles can be traced in an adult. 1 million out of the 5 million follicles can be found in head. Again, different types of hair are commonly utilized in the course of drug analysis especially when there is no scalp hair. The place where the hair comes from determines the concentration levels of cocaine, morphine, phenobarbital and methadone.

Conclusion and future plans

The study aimed at reviewing hair analysis as one way of detecting doping among athletes, the background information focused more on how urine and blood samples have commonly been used in detection of the dopants. A number of studies have equally established the limitations of urinalysis which includes a smaller window for detection. The study further established the prevailing methods that have been used to determine doping or determining the presence of a substance that appears on the prohibited list established by WADA. The second section narrowed down to establishing the methods to be used in the study. The research considered a review of the significant experiments aligned to hair analysis in support of the anti-doping process. Two experiments were established including the clostebol-based experiment and the toxicological analysis of the prohibited substances with the help of hair analysis. Both the experiments defined the requirements and the instrumental conditions needed for the process. They also specified specific to be used. Based on the findings, determination of the clostebol and its acetate in the air segments may provide proof of a regular use of a substance for the anabolic reasons. However, detection of low concentration levels especially in a single hair segment attracts chances of sustaining the occasional exposure to the prohibited substance. Further analysis has shared proof as to how clostebol and acetate would appear in the keratin matrix. This can be substantiated by the two metabolism phases both for the exogenous and endogenous AAS. Apparently, the current discussion towards handling current doping prevention prompts a research on the alternative means of testing dopants apart from making use of the blood and urine samples. Hair analysis provides the most convenient alternative for detecting the dopants, which would justify possible violation of the rules put in place by the relevant bodies. In the second experiment of toxicological analysis with the help of the hair samples, it is evident that hair samples can be used to detect a range of the prohibited substances.

These include such substances such as cocaine among others. Based on the two experiments and the findings tapped from the two processes, it can be confirmed that that the future of detecting doping among the athletes lies behind the use of hair analysis. This is due to the fact that collection of the necessary biological sample is far from harassment and hair samples provide a larger detection window. However, some of the bodies have discredited the validity of the findings deeming them less credible to justify a violation. This means that there is need to improve on the efficiency of hair analysis as one of the practices meant to detect doping instances among the athletes. In future, collection of the specimens should be protected from any form of contamination for the purposes of achieving better results. Secondly, there is need to revise the reagents for the purposes of ensuring that they do not alter the course of reaction after specimen preparation.

References

Aguilar, M., Muñoz-Guerra, J., Plata, M. and Del, J.C., 2018. Analysis of the Doping Control Test Results in Individual and Team Sports from 2003 to 2015.

Aguilar-Navarro, M., Muñoz-Guerra, J., del Mar Plata, M. and Del Coso, J., 2019. Analysis of doping control test results in individual and team sports from 2003 to 2015. Journal of Sport and Health Science.

Bird, S.R., Goebel, C., Burke, L.M. and Greaves, R.F., 2016. Doping in sport and exercise: anabolic, ergogenic, health and clinical issues. Annals of clinical biochemistry, 53(2), pp.196-221.

Cutler, C., Viljanto, M., Hincks, P., Habershon‐Butcher, J., Muir, T. and Biddle, S., 2019. Investigation of the metabolism of the selective androgen receptor modulator LGD‐4033 in equine urine, plasma and hair following oral administration. Drug testing and analysis.

Devcic, S., Bednarik, J., Maric, D., Versic, S., Sekulic, D., Kutlesa, Z., Bianco, A., Rodek, J. and Liposek, S., 2018. Identification of Factors Associated with Potential Doping Behavior in Sports: A Cross-Sectional Analysis in High-Level Competitive Swimmers. International journal of environmental research and public health, 15(8), p.1720.

Esquivel López, A., 2018. Control of anabolic steroids misuse in sport: potential of direct detection of phase II metabolites (Doctoral dissertation, Universitat Pompeu Fabra).

Fernando, P.N.J., Pigera, S., Rashani, S.A.N., Fernando, R., Weerasinghe, D.P.P., Godakumbura, K.K.D.T.D., Niriella, M.A., Jayawickreme, S. and de Silva, A.P., 2018. Do common arishta preparations manufactured in Sri Lanka contain anabolic androgenic steroids (AAS), stimulants or ethanol?.

Gerace, E., Di Corcia, D., Antidoping, C.R., Alladio, E. and di Tossicologia, C.R., 2020. Hair analysis can provide additional information in doping and forensic cases involving clostebol.

Handelsman, D.J., Hirschberg, A.L. and Bermon, S., 2018. Circulating testosterone as the hormonal basis of sex differences in athletic performance. Endocrine reviews, 39(5), pp.803-829.

Handelsman, D.J., Hirschberg, A.L. and Bermon, S., 2018. Circulating testosterone as the hormonal basis of sex differences in athletic performance. Endocrine reviews, 39(5), pp.803-829.

Handelsman, D.J., Matsumoto, A.M. and Gerrard, D.F., 2017. Doping Status of DHEA Treatment for Female Athletes with Adrenal Insufficiency. Clinical Journal of Sport Medicine, 27(1), pp.78-85.

Havnes, I.A., Jørstad, M.L. and Wisløff, C., 2019. Anabolic-androgenic steroid users receiving health-related information; health problems, motivations to quit and treatment desires. Substance abuse treatment, prevention, and policy, 14(1), p.20.

Kalliokoski, O., Jellestad, F.K. and Murison, R., 2019. A systematic review of studies utilizing hair glucocorticoids as a measure of stress suggests the marker is more appropriate for quantifying short-term stressors. Scientific reports, 9(1), pp.1-14.

Kanayama, G. and Pope Jr, H.G., 2018. History and epidemiology of anabolic androgens in athletes and non-athletes. Molecular and Cellular Endocrinology, 464, pp.4-13.

Salomone, A., Gerace, E., Di Corcia, D., Alladio, E., Vincenti, M. and Kintz, P., 2019. Hair analysis can provide additional information in doping and forensic cases involving clostebol. Drug testing and analysis, 11(1), pp.95-101.

Salomone, A., Palamar, J.J., Gerace, E., Di Corcia, D. and Vincenti, M., 2017. Hair testing for drugs of abuse and new psychoactive substances in a high-risk population. Journal of analytical toxicology, 41(5), pp.376-381.

Zahnow, R., McVeigh, J., Bates, G., Hope, V., Kean, J., Campbell, J. and Smith, J., 2018. Identifying a typology of men who use anabolic androgenic steroids (AAS). International Journal of Drug Policy, 55, pp.105-112.