Drugs in Sport Flashcards
Why do athletes’ dope?
- It works!
- There are powerful incentives to dope
- Huge personal and financial rewards
- Immediate gratification versus delayed sanction (time discounting)
- “Win at all costs” mentality
- Decisions regarding doping behaviour is made by evaluating the reward vs risk
Why is there a need for dope testing?
o Effort to get clean games
o Promote fair play is sports
o Save the ethics of sport
What is a drug?
How is it abused for sport?
- A chemical substance that interacts in the body to alter its biochemical system
- It is designed for medical and therapeutic purpose for the benefit of society- this is abused for doping in sport
What is doping?
The use of prohibited substances and/or methods to enhance performance in sports
The IOC and WADA covers anti-doping activities globally, what is their definition of doping?
“Doping is defined as occurrence of one or more of anti-doping rule violations”
The anti-doping rule violations
Why do some athletes cheat?
*** Physiological **
o To get more strength
o Increase oxygen transport
o Lose weight, train harder
o To build muscle, increase energy
o Mask injury and reduce tiredness
*** Social **
o To win at all costs
o Belief that everyone is doing it
o Pressure from coaches, peers, and the media
o By winning they can earn big money
o Fear of not winning
How do athletes try and avoid detection?
- Avoid testers
- Tamper with sample- has found beer or horse urine in replacement in some cases
- Refuse to give a sample
- Give someone else’s sample
- Drink lots of water
- Masking agents
Why is there a need to ban performance enhancing drugs in sport?
- Dangerous to the health of the competitor
- Unfair- contravenes the basic characteristics of sport
- Bad example to young people
- Risk of addiction
What are some groups of prohibited drugs, their effects and example
Stimulants
Excite the CNS and relieve physical and psychological fatigue
e.g., Cocaine, Ephedrine
Cannabinoids
Improve concentration, attention and determinations
e.g., derivatives of cannabis such as hashish
Peptide hormones and similar substances
Increase performance ability, accelerate metabolism, increase resilience and stress resistance
e.g., insulin, corticotrophins, erythropoietin
Diuretics
Regulate weight, reduce body liquid
Also used in masking agents to conceal other doping substances e.g., Furosemide
Glucocorticosteroids
Have qualities similar to adrenocortical hormones and relieve inflammation
e.g., Prednisolone, dexamethasone, hydrocortisone
Aromatase inhibitors
Increase own testosterone secretion
- This is prohibited in males only
e.g., cyclofenil
Narcotics
Affect the CNS, relieve pain and discomfort which could arise from muscle strain
e.g., morphine, methadone
Beta-2 agonists
Slow down HR and reduce tremor
- generally used where precise coordination is needed such as shooting and diving
- All Beta-2-agonists are prohibited except formoterol
e.g., salbutamol is allowed to be taken in inhalations for asthma
Selective androgen receptor modulators or SARMS
Affect androgen receptors responsible for muscular fiber growth, growth of muscle bulk or strength
Myostatin inhibitors
Block myostatin effects. Myostatin is a specific protein responsible for the regulation of organic growth of muscle tissue
Anabolic steroids
Increase muscle strength, resilience, help quickly restore after strenuous physical exercise, especially protein exercise
e.g., Nandrolone, testosterone
What are the penalities for getting caught doping?
- WADA “strict liability’ policy
- Athletes are responsible for what’s in their system
- First offence- up to four-year ban
- Second offence- possible life ban
- Athletes can be stripped of medals years later
The history of doping and testing
- Ancient Greeks to modern sports
- Athletes have sought to gain a competitive advantage
- IAAF banned doping in 1928 (no tests)
- 1st IOC ‘banned list’ in 1967- there was no testing at this time only a list of 27/28 substances that were banned
- 1st Olympic tests in 1968, Mexico- Only some compounds included
- Anabolic steroids- first tests in 1976- big increase in doping violations
- Increased IOC testing after Seoul 1988
- 1999 World Anti-doping agency (WADA)
Worldwide doping control, tell me about the role of WADA
- To monitor anti-doping activities worldwide
- To ensure proper implementation of and compliance with the World Anti-doping code (code)
- Harmonizing anti-doping rules in all sports and all countries
- Accreditation and monitoring of laboratories engaged in antidoping analysis
What is the total number of WADA accrediated labs?
30
o America= 5
o Asia= 6
o Europe= 17
o Oceania= 1
o Africa= 1
They are dedicated to the analysis of sports doping control tests
WADA accreditation is based upon compliance with two international standards
**o ISO/ IEC 17025
o International standard for laboratories (ISL), WADA **
- WADA monitors the performance through continuous external quality assessment scheme (EQAS)
- Participation in the EQAS is mandatory for all WADA accredited laboratories
What is the process inside the doping control laboratory
Whats the analytical process in a doping control laboratory?
- Screening
- Confirmation
- Analytical procedures:
1. Registration and aliquoting
2. Pre-treatment: (homogenisation, centrifugation, filtration)
3. Extraction/ purification: (sample preparation methods)
4. Derivatisation
5. Instrumental analysis
Tell me about the extraction and analysis of the samples tested in doping laboratories
*** Sample extraction **
o Extraction of samples according to different screening methods
o Screening methods depends on the properties of the compounds and are not restricted to a certain class on the list
o Target specific screenings like EPO, hCG, hGH
*** Sample analysis **
o Once the samples reaches the lab, ‘A’ sample is opened and analysed
o ‘B’ sample will be securely stored
o If ‘A’ sample reveal an Adverse Analytical finding, the ‘B’ sample may be tested on request
o The seal of B sample is opened in Prescence of athlete or the independent observer
How are the results interpreted once obtained?
- Each batch contains 1 or more control samples and a blank urine
- Most of the screenings are qualitative
- Quantitative compounds
o Cannabis
o Formoterol
o Salbutamol
o Morphine
o Ephedrines
o hCG
There is a list of prohibited drugs and it is reviewd each year. Approximately how many drugs are on that list at the moment?
About 400
Tell me about the prohibited list for 2023?
- Substances and methods always prohibited
- Substances and methods prohibited in competition
- Substances prohibited in particular sports
*** Important: **athlete is responsible for everything, that enters his/her body
Whats the criteria for inclusion of prohibited substances and methods on the prohibited list?
o Enhancing sports performance
o Health risks to the athletes
o Violating the spirit of sports
o Masking use of other prohibited substances and methods
Is caffeine banned?
- Found naturally in many plants such as coffee and tea, yerba mate leaves, kola nuts, guarana seeds and cacao
- Included in the 2022 monitoring program- In competition only and is not considered prohibited substances (in case it becomes an anti-doping issue in the future
Is alcohol banned?
- WADA has not banned but sports federation may have their own ban and regulations on alcohol abuse
Is salbutamol banned?
- With threshold of 1 µg/mL
- Permitted: inhaled salbutamol: maximum 1600 micrograms over 24 hours in divided doses not to exceed 600 micrograms over 8 hours starting from any dose
In olympic testing, what is the process that actually happens with a sample?
o Collection
o Transportation
o Chain of custody
o Split into aliquots
o Analysis
Hundreds of compounds, 5 or 6 tests
o Reporting
o Involves trained personnel and is also expensive
o Half the athletes and all medallists are tested
Split into A and B bottles
o Unique number and barcode
o Up to 400 samples tested each day
o 24 hours for most results
This includes confirmations
Some specific tests may take longer
Usually, 2 weeks
o Results given to IOC
o B analysis optional
o All samples will go through “screens”
o These cover the vast majority of substances on the prohibited list
o Suspect samples will then be subjected to specific confirmation to confirm the present of a prohibited substance
**o GC-MS/MS **
o** LC-MS **
o Chromatography allows separation of a mixture of analytes
o MS provides identification (what the compound is) and quantification (the amount)
How is LC-MS used in sample testing?
Liquid chromatography mass spectrometry
Looks for around 200 plus compounds from almost all classes
Min detection limit in picograms per mL in urine (2 mL extract)
How is GC-MS/MS used in sample testing?
Gas chromatography mass spectrometry
120 compounds mainly anabolic agents
Also, some narcotics
How does chromatography work?
- Chromatography allows the separation of complicated mixtures
- Biological samples remain highly complex so further information is required for identification
How does mass spectrometry work?
- Mass spectral identification
- Fragments of analytes creates a pattern or “fingerprint
which can be used for identification - Be measuring the abundance of the ions you can work out the amount
Tell me about the use of mass spectrometry in anti-doping analysi?
- MS is applied, following separation by either LC or GC, with ionisation techniques such as EI, CI, ESI (electrospray ionisation), APCI (atmospheric pressure ionisation) and APPI (atmospheric pression photoionization)
- The only accepted technique is when a prohibited substance, its metabolite or marker is being confirmed
- Alternative detection is allowed only for substances and methods, for which mass spectrometric detection is impossible or currently impossible in the expected concentration range
- The mass spectrometer configuration ranges from low resolution quadrupole techniques, via ion-trap and sector instruments, to time-of-flight (TOF) detection
- Both target analysis and general approaches for unknown doping agents are applied
Historical timeline of dope testing at the Olympics
Tell me the potential negative side effects of anabolic steroids in men, women and men&women
Glucocorticosteroids are on the prohibited list but what are some excemptions to this?
- Potent anti-inflammatory agents
- **All GCs are banned by WADA **
- MRPL 30 ng/mL
- Adverse analytical findings is to be reported if the concentration in urine exceeds 30 ng/mL due to its therapeutic use also. Exemptions to this:
o **Betamethasone, dexamethasone- 60 ng/mL
o 6beta-hydroxy-budesonide- 45 ng/mL
o Prednisoloe- 100 ng/mL
o Prednisone- 300 ng/mL
o Triamciniolone acetonide- 45 ng/mL ** - Athlete may opt for a valid therapeutic use exemption (TUE)
- Intra-articular, inhalatory and dermal route of administration is permitted
What is the effects of beta blockers?
Block effect of adrenaline, slow down the heart and breathing and calm nerves
A few case studies
Modafinil- psychostimulant/ adrafinil- prodrug/ modafinilic acid- metabolite
* GC-MS analysis faces a major pitfall- single artifact for all three compounds- elution at same retention time with similar mass fragmentation making them non-separable
* Detected by LC-MS/MS in ESI mode. (modafinil and adrafinil, as parent compounds in +ve ionisation, modeafilil acid in -ve mode
Methylhexanamine [4-methyl-2-hexaneamine [MHA]-
* Stimulant used as a nasal decongestant but found it way into sports doping due to its stimulating properties
* An analogue to tuaminoheptane with an active chiral centre
* GC-MS analysis, derivatisation using carbon-di-sulphide gave isothiocynate derivatives of methylhexaneamine and tuaminoheptane and resolved both the compounds
Tell me about Methamphetamine
- Levo (l)- methamphetamine- over the counter decongestant- is permitted in court
- Dextra (d)-methamphetamine abused as CNS stimulant- is not permitted in court
- Difference sanctions by WADA for the levo (l) or dextro (d)- methamphetamines
- Chiral separation of d-(+) and l-(-) forms using chiral derivatising agents (s-(-)-N-trifluoroacetylprolyl chloride (TPC) and (-)-alpha-methoxy-trifluoromethylprolyl chloride (MTPA) and detection on GC-MS provides the solution
Tell me about **Erythropoietin (EPO) and human growth hormone (hGH) **
- Banned protein hormones
o Both produced naturally in the body
o Pharmaceutical versions also available, produced by DNA technology
o Difficult to tell the difference between the natural and synthetic versions
Tell me about** Erythropoietin (EPO)**
What it is, its benefits and side effecs
- A protein hormone produced naturally by the kidney which stimulates the production of red blood cells (RBCs)
- It can also be manufactured and injected into the body
- Increasing RBCs can dramatically improve and athletes’ aerobic capacity and delay fatigue. It also improves metabolism and
muscle recovery - Side effects
o Quick increases in blood pressure
o Convulsions
o Influenza-like symptoms
o Increased cancer risk
o Liver damage
o Increased viscosity of blood
Whats the Haematological module
- The haematological module collects information on markers of blood doping and contains following blood variables
o ABPS: abnormal blood profile score
o HCT: haematocrit
o HGB: haemoglobin
o IRF: immature reticulocyte fraction
o MCH: mean corpuscular haemoglobin
o MCHC: mean corpuscular haemoglobin concentration
o MCV: mean corpuscular volume
o OFFS: OFF-hr score
o PLT: platelets
o RBC: red blood cells (erythrocytes) count
o RDW-SD: red cell distribution width (standard deviation)
o RET#: reticulocyte count
o RET%: reticulocyte percentage
o WBC: white blood cells
- Indirect method to monitor the biological passport
What are the main analytical challenges in doping analysis?
- More than 300 drugs and/or metabolites
- Difference in physical and chemical properties
- No single chemical or instrumental method can screen “all in one”
- Sample matric interferences
- Requirements of low-level detection
- Limited volume of urine (60 mL)- fast reporting required
- Highest level of certainty required
- Non approved substances
Utilisation of forensics application in anti-doping
- WADA international standard for laboratories (ISL) states that the laboratory shall be competent enough to produce forensically valid results
Doping: a paradigm shift has taken place in testing
- With the advancement in techniques the approach of testing combines forensic and clinical science
- The role of anti-doping science (not “doping science”) is to protect clean athletes
Whats the role of forensic science in anti-doping?
- Anti-doping is mainly focused on direct detection, using analytical methods for the detection of doping agents in biological samples
- However, considering other ADRVs like possession, administration, trafficking of prohibited substance or methods correspond to criminal investigation,
- Hence a forensic approach to help assessing potential violation of these rules
What are some of the sophisticated techniques in place for analysis?
Recent trends in anti-doping
Recent trends in technologies
Trends in methodologies
Tell me about the current blood testing strategies
What ar the advantages and disadvantages of Dried Blood Spot tesrting?
What are some alternatives for dried blood sample testing?
Future of dope testing: DBS implementation
Why do we need biomarkers?
Athlete passport management unit (APMU)
What is gene doping and how does it work?
WADA definition- **the non-therapeutic use of genes, genetic elements and/or cells that have the capacity to enhance athletic performance **
-
Delivery
- DNA packaged into a virus and is injected into atheletes blood stream to the muscles -
Change
- The virus binds to the muscle cells and deposit the foreign gene inside. Here it integrates into the cells chromosomes. The gene then stimulates the production of the protein -
Dispersal
- EPO is produced by the altered muscle cells. this flows through the blood stream to the bone marrow and stimulates the production of RBCs. -
Enhancement
- Extra RBCs throughout the athletes body which increases the oxygen capacity and hence endurance
For gene doping, what are the gene and system targets?
Tell me about supplement testing in sports?
- Supplement testing can be done by WADA accredited lab only when asked by any anti-doping agency
- Supplement testing by non-WADA accredited lab (commercial lab)
- Advantage of supplement testing:
o This helps athlete to take the right product and avoid a positive drug test during dope testing
