ANTIDOPING TESTING PROCEDURE

ANALYSIS OF SAMPLE
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.
DOPING

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.
 Inadvertent 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.
Prohibited substances and methods

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)
Examples
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
Anti-doping Testing Procedures

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.
Athlete Selection

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)
Urine sample collection

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.
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.
Blood sample collection
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.
Urine sample analysis

The collected samples are transported to an IOC-
accredited 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.
Blood 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.
Analytical procedures
1. Sample preparation involving hydrolysis of urine sample
and extraction of the hydrolysed sample.
2. Analysis of the extracted urine sample by gas
chromatography/mass spectrometry (GC-MS) procedure.
3. Substances such as hCG, LH, ACTH can be measured in
the urine samples using immuno-assay.
4. 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.
A typical analytical procedure
1. Urine sample with an added internal standard is passed
through an SPE column and eluted with methanol.
2. The eluent obtained is evaporated to dryness under
nitrogen at 60ºC.
3. The dried residue is redissolved in acetate buffer, β
glucuronidase is added and the mixture allowed to
incubate overnight at 37ºC.
4. The hydrolysed sample is next extracted into diethylether
at pH 9.0-9.5. Ether extract then evaporated to dryness.
5. A derivatised agent (MSTFA) is added to the dried
extract heated at 60ºC for 30 minutes.
6. The resultant product is dissolved into hexane before
injection into a GC-MS system.
Gas Chromatography/Mass Spectrometry

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.