Anti Doping Testing Procedures

LECTURES OUTLINE : 1. Definition of doping and inadvertent doping 2. World anti-doping code 3. Prohibited substances and prohibited methods 4. Examples of prohibited substances and prohibited methods 5. Anti-doping testing procedures. 6. Urine and blood samples analysis.

ANALYSIS OF SAMPLE

ANTIDOPING TESTING PROCEDURE




The use of chemical, synthetic or naturally occurring, substances which artificially improve the physical or psychological condition of an athlete before or during an event. ⇒ Use of substances and/or methods prohibited under a sport’s anti-doping rule.
Doping contravenes the ethics of sport and medical science.

DOPING




Occurs when an athlete takes a medication to treat an injury or illness without realizing that it contains a prohibited substance.
Anti-doping policies - the principle of strict liability ⇒ athletes are responsible for any banned substances found in their body. ⇒ athletes are responsible for checking the status of all substances and medications they consumed. ∴ ignorance is no excuse.

Inadvertent doping


The World Anti-Doping Code
World Anti-doping Agency (WADA) → Prohibited substances and prohibited methods
List reviewed and updated annually
Latest – The 2008 Prohibited List (effective 1 January 2008)

Prohibited substances and methods

Prohibited substances : 1. Anabolic agents 2. Diuretics 3. Narcotics 4. Peptide hormones, mimetics and analogues 5. Stimulants 6. Agents with anti-oestrogenic activity 7. Masking agents Prohibited methods : 1. Enhancement of oxygen transfer 2. Gene doping 3. Pharmacological, physical and physical manipulaton

Examples

Two basic steps : 1. The athlete selection and the sample collection (urine only or combined blood and urine). 2. The sample analysis and the reporting of the analysis results.

Anti-doping Testing Procedures









Athletes can be selected for a drug test, any time, anywhere. Notified in person, by telephone or by written notice. During competition (In-Competition tests) During the training periods (Out-Of-Competition tests)

Athlete Selection




Procedure for sample collection is the same for the in- or out-of- competition tests. 1. After the competition or during the training, the selected athlete is notified and accompanied to the Doping Control Station by the responsible antidoping officer or chaperone. 2. The officer will record the athlete’s details on a notification form. The athlete will sign the form and be provided a copy. 3. The athlete can drink from sealed containers non-alcoholic, caffeine-free drinks.

Urine sample collection

4. When he/she is ready to give a sample, the athlete will be asked to select a sample collection vessels. 5. The athlete will be asked to provide a urine sample in the presence and direct observation of the chaperone who is the same gender as the athlete. 6. After having provided the required amount of urine (usually 80 ml), the athlete selects a pair of sealed sample containers, pours two thirds of the urine (not < 60 ml) into bottle A and one third into bottle B, closes the two bottles and reseal with new security seal tags.

7. The anti-doping officer measures the pH (5 – 7) and the specific gravity (≥ 1.010) of the urine left in the collection vessel to check the suitability of the sample for analysis. 8. The officer records to the drug testing form any medication and nutritional supplements the athlete declares that has been consumed in the preceding 3 – 7 days. 9. After the check of the information recorded, the codes and the proper sealing of the urine bottles, the athlete certifies the entire procedure by signing the drug testing form. The athlete is also given a copy of the form.

1. The athlete will be asked to select and check the blood testing equipment. 2. The athlete will be asked to provide a blood sample, collected in two tubes (labelled Part “1” and Part “2”) by a phlebotomist. 3. After collecting the blood sample, the phlebotomist will remove the blood test collection equipment from the athlete’s body, thereby sealing the collection equipment containers. 4. The athlete is then responsible for controlling his/her sample until it is sealed in a sample collection kit.

Blood sample collection
















The collected samples are transported to an IOCaccredited laboratory (e.g. Doping Control Centre, USM) by antidoping officers for analysis. Both the A and B bottles are transferred to the laboratory, where after their reception, the laboratory staff becoming responsible for their security. The laboratory will analyse sample A for the presence of prohibited substances or methods. Sample B is stored in a refrigerator, locked and guarded. If sample A returns a positive test result the athlete has the right to have sample B analysed to confirm the positive test result. Results of the analysis will be communicated by the laboratory to the authority for action.

Urine sample analysis







The laboratory will analyse Part “1” and Part “2” of a blood sample for indicators of prohibited substances or methods. If a blood sample returns a positive result, sample A of the urine sample will be analysed. If sample A returns a positive test result, the athlete can elect to have sample B analysed to confirm the positive test result. Even if the blood analysis is negative, the urine sample will undergo analysis in accordance with the procedure.

Blood sample analysis

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Sample preparation involving hydrolysis of urine sample and extraction of the hydrolysed sample. Analysis of the extracted urine sample by gas chromatography/mass spectrometry (GC-MS) procedure. Substances such as hCG, LH, ACTH can be measured in the urine samples using immuno-assay. No reliable tests yet for hGH, IGF-1 and EPO. Synthetic EPO has been shown to produce red blood cells which is smaller and bind more iron than natural EPO. So method being developed to analyse the size and iron content of red blood cells from a blood sample to determine whether an athlete has used EPO.

Analytical procedures

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Urine sample with an added internal standard is passed through an SPE column and eluted with methanol. The eluent obtained is evaporated to dryness under nitrogen at 60ºC. The dried residue is redissolved in acetate buffer, β glucuronidase is added and the mixture allowed to incubate overnight at 37ºC. The hydrolysed sample is next extracted into diethylether at pH 9.0-9.5. Ether extract then evaporated to dryness. A derivatised agent (MSTFA) is added to the dried extract heated at 60ºC for 30 minutes. The resultant product is dissolved into hexane before injection into a GC-MS system.

A typical analytical procedure










Most common methods of chemical analysis of urine and blood samples. In GC, retention time of each substance is reported and plotted to create a chromatogram. Standard samples of drugs are also run ⇒ identification and quantification. In MS, samples are blown apart by an electron beam ⇒ fragments ⇒ accelerated down magnetic tube to a detector ⇒ mass spectrum “fingerprint”. Confirmed by mass spectrum of standard.

Gas Chromatography/Mass Spectrometry