ANTID

OPING TESTIN

G PROCEDURE

ANALYSIS O

F SAMPLE

LECTURES O

UTLINE :

1.

Definition of doping and inadvertent doping

2.

World anti-doping code

3.

Prohibited substances and prohibited m

ethods

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

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 m

edication to

treat an injury or illness without realizing that it

contains a prohibited substance.

�Anti-doping policies -the principle of strict lia

bility

⇒athletes are responsible for any banned

substances found in their body.

⇒athletes are responsible for checking the status of

all substances and m

edications they consumed.

∴ignorance is no excuse.

Prohibited substances and m

ethods

�The W

orld Anti-D

oping 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 horm

ones, mim

eticsand analogues

5.

Stimulants

5.

Stimulants

6.

Agents with anti-oestrogenic

activity

7.

Masking agents

Prohibited m

ethods :

1.

Enhancement of oxygen transfer

2.

Gene doping

3.

Pharm

acological, 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 tim

e,

anywhere.

�Notified in person, by telephone or by written notice.

�During competition (In-C

ompetition tests)

�During the training periods (Out-Of-Competition

tests)

tests)

Urine sample colle

ction

�Procedure for sample colle

ction 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 C

ontrol Station by the responsible

antidoping officer or chaperone.

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

colle

ction vessels.

5.

The athlete will be asked to provide a urine

sample in the presence and direct observation of

the chaperone w

ho is the same gender as the

athlete.

athlete.

6.

After having provided the required amount of

urine (usually 80 m

l), the athlete selects a pair of

sealed sample containers, pours two thirds of

the urine (not < 60 m

l) 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 colle

ction 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

athlete declares that has been consumed in the

preceding 3 –

7 days.

9.

After the check of the inform

ation recorded, the

codes and the proper sealin

g 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, colle

cted in two tubes (labelle

dPart “1”

and Part “2”) by a phlebotomist.

3.

After colle

cting the blood sample, the

phlebotomist will remove the blood test colle

ction

phlebotomist will remove the blood test colle

ction

equipment from the athlete’s body, thereby

sealin

g the colle

ction equipment containers.

4.

The athlete is then responsible for controlling

his/her sample until it is sealed in a sample

colle

ction kit.

Urine sample analysis

�The collected samples are transported to an IOC-

accredited laboratory (e.g. Doping C

ontrol 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 w

ill analyse sample A for the presence of

�The laboratory w

ill 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 w

ill 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.

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/m

ass spectrometry (GC-M

S) procedure.

3.

Substances such as hCG, LH, ACTH can be m

easured in

the urine samples using immuno-assay.

4.

No reliable tests yet for hGH, IG

F-1 and EPO. Synthetic

4.

No reliable tests yet for hGH, IG

F-1 and EPO. Synthetic

EPO has been shown to produce red blood cells w

hich is

smaller and bind m

ore iron than natural EPO. So m

ethod

being developed to analyse the size and iron content of

red blood cells from a blood sample to determ

ine whether

an athlete has used EPO.

A typical analytical procedure

1.

Urine sample w

ith an added internal standard is passed

through an SPE column and eluted w

ith m

ethanol.

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 m

ixture allowed to

incubate overnight at 37ºC

.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 (M

STFA) is added to the dried

extract heated at 60ºC

for 30 m

inutes.

6.

The resultant product is dissolved into hexane before

injection into a G

C-M

S system.

Gas Chromatography/M

ass Spectrometry

�Most common m

ethods of chemical analysis of

urine and blood samples.

�In G

C, retention tim

e of each substance is

reported and plotted to create a chromatogram.

Standard samples of drugs are also run ⇒

identification and quantification.

�In M

S, samples are blown apart by an electron

beam ⇒

fragments ⇒

accelerated down

magnetic tube to a detector ⇒

mass spectrum

“fingerprint”. Confirm

ed by m

ass spectrum of

standard.