Drug Abuse in Sport

Introduction
• Drug abuse in sport is often called doping
• The abuse of drugs in an attempt to enhance
performance in human sporting competitions is not
new
• The effect of drugs on performance is often extremely
difficult to determine, and there is little definitive
published work for any species
• The toxic side-effects of drugs are less difficult to
ascertain, but the conclusions drawn from the available
data are often circumstantial (tdk. Langsung) (e.g. the
possible links between taking anabolic steroids and
liver cancer)
 Rule in Human sport
• Since 1999 doping issues have been taken
over by the World Anti-Doping Agency
(WADA)
• WADA provides a legal definition of doping
• WADA publishes, at least annually, a
Prohibited List as an International Standard
• This list covers substances and methods
prohibited from use by athletes
 PROHIBITED LIST (JANUARY 2020)
• SUBSTANCES & METHODS
PROHIBITED AT ALL TIMES (IN- AND OUT-OF-COMPETITION)
• PROHIBITED SUBSTANCES:
• S0: NON-APPROVED SUBSTANCES
• S1: ANABOLIC AGENTS
• S2: PEPTIDE HORMONES, GROWTH FACTORS, RELATED
SUBSTANCES, AND MIMETICS
• S3: BETA-2 AGONISTS
• S4: HORMONE AND METABOLIC MODULATORS
• S5: DIURETICS AND MASKING AGENTS
 PROHIBITED LIST (JANUARY 2020)
• SUBSTANCES & METHODS
PROHIBITED AT ALL TIMES (IN- AND OUT-OF-COMPETITION)
• PROHIBITED METHODS:
• M1: MANIPULATION OF BLOOD AND BLOOD
COMPONENTS
• M2: CHEMICAL AND PHYSICAL MANIPULATION
• M3: GENE AND CELL DOPING
 PROHIBITED LIST (JANUARY 2020)
• SUBSTANCES & METHODS
PROHIBITED IN-COMPETITION
• PROHIBITED SUBSTANCES:
• S6: STIMULANTS
• S7: NARCOTICS
• S8: CANNABINOIDS
• S9: GLUCOCORTICOIDS

• SUBSTANCES PROHIBITED IN PARTICULAR SPORTS

• P1: BETA-BLOCKERS
 S0: NON-APPROVED SUBSTANCES
• Any pharmacological
substance which is not
addressed by any of the
subsequent sections of the
List and with no current
approval by any
governmental
regulatory health authority
for human therapeutic use
(e.g. drugs under preclinical
or clinical development
or discontinued, designer
drugs, substances approved
only for veterinary use) is
prohibited at all times.
 S1: ANABOLIC AGENTS
• 1. ANABOLIC ANDROGENIC • 5-Androstenedione (androst-5-
STEROIDS (AAS) ene-3,17-dione);
when administered exogenously, 7α-hydroxy-DHEA;
including but not limited 7β-hydroxy-DHEA;
to: 7-Keto-DHEA;
1-Androstenediol (5α-androst-1- 19-Norandrostenediol (estr-4-
ene-3β,17β-diol); ene-3,17-diol);
1-Androstenedione (5α-androst- 19-Norandrostenedione (estr-4-
1-ene-3,17-dione); ene-3,17-dione);
1-Androsterone (3α-hydroxy-5α- Androstanolone (5α-
androst-1-ene-17-one); dihydrotestosterone, 17β-
1-Epiandrosterone (3β-hydroxy- hydroxy-5α-androstan-3-one);
5α-androst-1-ene-17-one); Androstenediol (androst-5-ene-
1-Testosterone (17β-hydroxy-5α- 3β,17β-diol);
androst-1-en-3-one); Androstenedione (androst-4-ene-
4-Androstenediol (androst-4-ene- 3,17-dione);
3β,17β-diol); Bolasterone;
4-Hydroxytestosterone (4,17β- Boldenone;
dihydroxyandrost-4-en-3-one); Boldione (androsta-1,4-diene-
3,17-dione);
 S1: ANABOLIC AGENTS
• 1. ANABOLIC ANDROGENIC • Epiandrosterone (3β-hydroxy-5α-
STEROIDS (AAS) androstan-17-one);
when administered exogenously, Epi-dihydrotestosterone (17β-
including but not limited hydroxy-5β-androstan-3-
to: one);
Calusterone; Epitestosterone;
Clostebol; Ethylestrenol (19-norpregna-4-en-
Danazol 17α-ol);
([1,2]oxazolo[4',5':2,3]pregna-4-en- Fluoxymesterone;
20-yn-17α-ol); Formebolone;
Dehydrochlormethyltestosterone (4- Furazabol (17α-methyl
chloro-17β-hydroxy-17α- [1,2,5]oxadiazolo[3',4':2,3]-5α-
methylandrosta-1,4-dien-3-one); androstan-17β-ol);
Desoxymethyltestosterone (17α- Gestrinone;
methyl-5α-androst-2-en-17β-ol and Mestanolone;
17α-methyl-5α-androst-3-en-17β-ol); Mesterolone;
Drostanolone; Metandienone (17β-hydroxy-17α-
methylandrosta-1,4-dien-3-one);
Metenolone;
Methandriol;
 S1: ANABOLIC AGENTS
• 1. ANABOLIC ANDROGENIC STEROIDS (AAS) • Norclostebol (4-chloro-17β-ol-estr-4-en-3-
when administered exogenously, including one);
but not limited Norethandrolone;
to: Oxabolone;
Methasterone (17β-hydroxy-2α,17α- Oxandrolone;
dimethyl-5α-androstan-3-one); Oxymesterone;
Methyl-1-testosterone (17β-hydroxy-17α- Oxymetholone;
methyl-5α-androst-1-en-3-one); Prasterone (dehydroepiandrosterone, DHEA,
Methylclostebol; 3β-hydroxyandrost-5-en-17-one);
Methyldienolone (17β-hydroxy-17α- Prostanozol (17β-[(tetrahydropyran-2-
methylestra-4,9-dien-3-one); yl)oxy]-1'Hpyrazolo[3,4:2,3]-5α-androstane);
Methylnortestosterone (17β-hydroxy-17α- Quinbolone;
methylestr-4-en-3-one); Stanozolol;
Methyltestosterone; Stenbolone;
Metribolone (methyltrienolone, 17β-hydroxy- Testosterone;
17α-methylestra-4,9,11-trien-3-one); Tetrahydrogestrinone (17-hydroxy-18a-
Mibolerone; homo-19-nor-17α-pregna-4,9,11-trien-3-
Nandrolone (19-nortestosterone); one);
Norboletone; Trenbolone (17β-hydroxyestr-4,9,11-trien-3-
one);
and other substances with a similar chemical
structure or similar biological effect(s)
 S1: ANABOLIC AGENTS
• 2. OTHER ANABOLIC AGENTS
Including, but not limited to:
Clenbuterol, selective androgen receptor
modulators
[SARMs, e.g. andarine, LGD-4033
(ligandrol), enobosarm (ostarine) and
RAD140], tibolone, zeranol and zilpaterol
 S2: PEPTIDE HORMONES, GROWTH FACTORS,
RELATED SUBSTANCES, AND MIMETICS
• The following substances, and other • 1.2 Hypoxia-inducible factor (HIF)
substances with similar chemical structure activating agents, e.g.
or similar biological effect(s), Cobalt;
are prohibited: Daprodustat (GSK1278863);
1. Erythropoietins (EPO) and agents Molidustat (BAY 85-3934);
affecting erythropoiesis, Roxadustat (FG-4592);
including, but not limited to: Vadadustat (AKB-6548);
1.1 Erythropoietin-Receptor Agonists, e.g. Xenon.
Darbepoetins (dEPO); 1.3 GATA inhibitors, e.g.
Erythropoietins (EPO); K-11706.
EPO based constructs [e.g. EPO-Fc, 1.4 TGF-beta (TGF-β) signalling inhibitors,
methoxy polyethylene glycol-epoetin beta e.g.
(CERA)]; Luspatercept;
EPO-mimetic agents and their constructs Sotatercept.
(e.g. CNTO-530, peginesatide). 1.5 Innate repair receptor agonists, e.g.
Asialo EPO;
Carbamylated EPO (CEPO).
 S2: PEPTIDE HORMONES, GROWTH FACTORS,
RELATED SUBSTANCES, AND MIMETICS
• 2. Peptide Hormones and their Releasing
Factors,
2.1 Chorionic Gonadotrophin (CG) and
Luteinizing Hormone (LH) and their
releasing factors in males, e.g. Buserelin,
deslorelin, gonadorelin, goserelin,
leuprorelin, nafarelin and triptorelin;
2.2 Corticotrophins and their releasing
factors, e.g. Corticorelin;
• 2.3 Growth Hormone (GH), its fragments and
releasing factors, including, but not limited to:
Growth Hormone fragments, e.g.
AOD-9604 and hGH 176-191;
Growth Hormone Releasing Hormone (GHRH)
and its analogues, e.g.
CJC-1293, CJC-1295, sermorelin and
tesamorelin;
Growth Hormone Secretagogues (GHS), e.g.
Lenomorelin (ghrelin) and its mimetics, e.g.
Anamorelin, ipamorelin, macimorelin and
tabimorelin; GH-Releasing Peptides (GHRPs),
e.g. Alexamorelin, GHRP-1, GHRP-2
(pralmorelin), GHRP-3, GHRP-4, GHRP-5, GHRP-
6, and examorelin (hexarelin).
 S2: PEPTIDE HORMONES, GROWTH FACTORS,
RELATED SUBSTANCES, AND MIMETICS
• 3. Growth Factors and Growth Factor
Modulators, including, but not limited to:
Fibroblast Growth Factors (FGFs);
Hepatocyte Growth Factor (HGF);
Insulin-like Growth Factor-1 (IGF-1) and its
analogues;
Mechano Growth Factors (MGFs);
Platelet-Derived Growth Factor (PDGF);
Thymosin-β4 and its derivatives e.g. TB-
500;
Vascular-Endothelial Growth Factor
(VEGF); and other growth factors or growth
factor modulators affecting muscle, tendon
or ligament protein synthesis/ degradation,
vascularisation, energy utilization,
regenerative capacity or fibre type
switching
 S3: BETA-2 AGONISTS
• All selective and non-selective beta-2 • Except:
agonists, including all optical isomers, are • Inhaled salbutamol: maximum 1600
prohibited. micrograms over
Including, but not limited to: 24 hours in divided doses not to exceed
Fenoterol; 800 micrograms over 12 hours starting
Formoterol; from any dose;
Higenamine; • Inhaled formoterol: maximum delivered
Indacaterol; dose of 54 micrograms over 24 hours;
Olodaterol; • Inhaled salmeterol: maximum 200
Procaterol; micrograms over
Reproterol; 24 hours.
Salbutamol;
Salmeterol;
Terbutaline;
Tretoquinol (trimetoquinol);
Tulobuterol;
Vilanterol.
 S3: BETA-2 AGONISTS
• The presence in urine of salbutamol in •
excess of 1000 ng/mL or formoterol in
excess of 40 ng/mL is not consistent with
therapeutic use of the substance and will
be considered as an Adverse Analytical
Finding (AAF) unless the Athlete proves,
through a controlled pharmacokinetic
study, that the abnormal result was the
consequence of a therapeutic dose
(by inhalation) up to the maximum dose
indicated above.
 S4:HORMONE AND METABOLIC MODULATORS
• The following hormone and metabolic • 2. Selective estrogen receptor modulators
modulators (SERMs)
are prohibited: including, but not limited to:
1. Aromatase inhibitors including, but not Bazedoxifene;
limited to: Ospemifene;
2-Androstenol (5α-androst-2-en-17-ol); Raloxifene;
2-Androstenone (5α-androst-2-en-17-one); Tamoxifen;
3-Androstenol (5α-androst-3-en-17-ol); Toremifene.
3-Androstenone (5α-androst-3-en-17-one); 3. Other anti-estrogenic substances
4-Androstene-3,6,17 trione (6-oxo); including, but not
Aminoglutethimide; limited to:
Anastrozole; Clomifene;
Androsta-1,4,6-triene-3,17-dione Cyclofenil;
(androstatrienedione); Fulvestrant.
Androsta-3,5-diene-7,17-dione
(arimistane);
Exemestane;
Formestane;
Letrozole;
Testolactone
 S4:HORMONE AND METABOLIC MODULATORS
• 4. Agents preventing activin receptor IIB
activation
including, but not limited, to:
Activin A-neutralizing antibodies;
Activin receptor IIB competitors such as:
Decoy activin receptors (e.g. ACE-031);
Anti-activin receptor IIB antibodies (e.g.
Bimagrumab);
Myostatin inhibitors such as:
Agents reducing or ablating myostatin
expression;
Myostatin-binding proteins (e.g. Follistatin,
myostatin
propeptide);
Myostatin-neutralizing antibodies (e.g.
Domagrozumab, landogrozumab,
stamulumab)
• 5. Metabolic modulators:
5.1 Activators of the AMP-activated
protein kinase (AMPK), e.g. AICAR,
SR9009; and Peroxisome
Proliferator Activated Receptor δ (PPARδ)
agonists, e.g. 2-(2-methyl-4-((4-methyl-2-
(4-(trifluoromethyl)
phenyl)thiazol-5-yl)methylthio)phenoxy)
acetic acid (GW1516, GW501516);
5.2 Insulins and insulin-mimetics;
5.3 Meldonium;
5.4 Trimetazidine.
 S5: DIURETICS AND MASKING AGENTS
• The following diuretics and masking agents
are prohibited, as are other substances
with a similar chemical structure or similar
biological effect(s).
Including, but not limited to:
• Desmopressin; probenecid; plasma
expanders, e.g. intravenous administration
of albumin, dextran, hydroxyethyl starch
and mannitol.
• Acetazolamide; amiloride; bumetanide;
canrenone; chlortalidone; etacrynic acid;
furosemide; indapamide; metolazone;
spironolactone; thiazides, e.g.
Bendroflumethiazide, chlorothiazide and
hydrochlorothiazide; triamterene and
vaptans, e.g. Tolvaptan.
• Except:
• Drospirenone; pamabrom; and
ophthalmic use of carbonic anhydrase
inhibitors (e.g. Dorzolamide,
brinzolamide);
• Local administration of felypressin in
dental anaesthesia.
The detection in an Athlete’s Sample at all
times or In-Competition, as applicable, of
any quantity of the following substances
subject to threshold limits: formoterol,
salbutamol, cathine, ephedrine,
methylephedrine and pseudoephedrine,
in conjunction with a diuretic or masking
agent, will be considered as an Adverse
Analytical Finding (AAF) unless the Athlete
has an approved Therapeutic Use
Exemption (TUE) for that substance in
addition to the one granted for the
diuretic or masking agent.
 S6: STIMULANTS
• All stimulants, including all optical isomers, • Fenproporex;
e.g. Fonturacetam [4-phenylpiracetam
d- and l- where relevant, are prohibited. (carphedon)];
Stimulants include: Furfenorex;
a: Non-Specified Stimulants: Lisdexamfetamine;
Adrafinil; Mefenorex;
Amfepramone; Mephentermine;
Amfetamine; Mesocarb;
Amfetaminil; Metamfetamine(d-);
Amiphenazole; p-methylamfetamine;
Benfluorex; Modafinil;
Benzylpiperazine; Norfenfluramine;
Bromantan; Phendimetrazine;
Clobenzorex; Phentermine;
Cocaine; Prenylamine;
Cropropamide; Prolintane.
Crotetamide; A stimulant not expressly listed in this
Fencamine; section
Fenetylline; is a Specified Substance.
Fenfluramine;
 S6: STIMULANTS
• b: Specified Stimulants: • Ephedrine***;
Including, but not limited to: Epinephrine**** (adrenaline);
3-Methylhexan-2-amine (1,2- Etamivan;
dimethylpentylamine); Etilamfetamine;
4-Methylhexan-2-amine Etilefrine;
(methylhexaneamine); • Famprofazone;
4-Methylpentan-2-amine (1,3- Fenbutrazate;
dimethylbutylamine); Fencamfamin;
5-Methylhexan-2-amine (1,4- Heptaminol;
dimethylpentylamine); Hydroxyamfetamine
Benzfetamine; (parahydroxyamphetamine);
Cathine**; Isometheptene;
Cathinone and its analogues, e.g. Levmetamfetamine;
mephedrone, Meclofenoxate;
methedrone, and α - Methylenedioxymethamphetamine;
pyrrolidinovalerophenone; Methylephedrine***;
Dimetamfetamine Methylphenidate;
(dimethylamphetamine);
 S6: STIMULANTS
• Nikethamide; • Except:
Norfenefrine; • Clonidine;
Octodrine (1,5-dimethylhexylamine); • Imidazole derivatives for dermatological,
Octopamine; nasal or ophthalmic use and those
Oxilofrine (methylsynephrine); stimulants included in the
• Pemoline; 2020 Monitoring Program*
Pentetrazol; * Bupropion, caffeine, nicotine, phenylephrine,
phenylpropanolamine, pipradrol, and synephrine: These
Phenethylamine and its derivatives;
substances are included in the 2020 Monitoring
Phenmetrazine; Program, and are not considered Prohibited Substances.
Phenpromethamine; ** Cathine: Prohibited when its concentration in urine is
Propylhexedrine; greater than 5 micrograms per milliliter.
Pseudoephedrine***** *** Ephedrine and methylephedrine: Prohibited when
the concentration of either in urine is greater than 10
• Selegiline; micrograms per milliliter.
Sibutramine; **** Epinephrine (adrenaline): Not prohibited in local
Strychnine; administration, e.g. nasal, ophthalmologic, or co-
Tenamfetamine administration with local anaesthetic agents.
(methylenedioxyamphetamine); ***** Pseudoephedrine: Prohibited when its
concentration in urine is greater than 150 micrograms
Tuaminoheptane; and other substances per milliliter.
with a similar chemical structure
or similar biological effect(s).
 S7: NARCOTICS
• The following narcotics, including all
optical isomers, e.g. d- and l- where
relevant, are prohibited:

• Buprenorphine;
Dextromoramide;
Diamorphine (heroin);
Fentanyl and its derivatives;
Hydromorphone;
Methadone;
Morphine;
Nicomorphine;
Oxycodone;
Oxymorphone;
Pentazocine;
Pethidine.
 S8: CANNABINOIDS
• All natural and synthetic cannabinoids are
prohibited, e.g.
•
• In cannabis (hashish, marijuana) and
cannabis products
• Natural and synthetic
tetrahydrocannabinols (THCs)
• Synthetic cannabinoids that mimic the
effects of THC
Except:
• Cannabidiol.
 S9: GLUCOCORTICOIDS
• All glucocorticoids are prohibited when
administered by oral, intravenous,
intramuscular or rectal routes.
Including but not limited to:

• Betamethasone;
Budesonide;
Cortisone;
Deflazacort;
Dexamethasone;
Fluticasone;
Hydrocortisone;
Methylprednisolone;
Prednisolone;
Prednisone;
Triamcinolone.
 M1: MANIPULATION OF BLOOD AND
BLOOD COMPONENTS
• The following are prohibited: • 3. Any form of intravascular manipulation
• of the blood or blood components by
1. The Administration or reintroduction of physical or chemical means
any quantity of autologous, allogenic
(homologous) or heterologous blood, or
red blood cell products of any origin into
the circulatory system.
2. Artificially enhancing the uptake,
transport or delivery of oxygen.
Including, but not limited to:
Perfluorochemicals; efaproxiral (RSR13)
and modified haemoglobin products, e.g.
Haemoglobin-based blood substitutes and
microencapsulated haemoglobin
products, excluding supplemental oxygen
by inhalation.
 M2: CHEMICAL AND PHYSICAL
MANIPULATION
• The following are prohibited:
•
1. Tampering, or Attempting to Tamper, to
alter the integrity and validity of Samples
collected during Doping Control.
Including, but not limited to:
Sample substitution and/or adulteration,
e.g. Addition of proteases to Sample.
2. Intravenous infusions and/or injections
of more than a total of 100 mL per 12 hour
period except for those legitimately
received in the course of hospital
treatments, surgical procedures or clinical
diagnostic investigations.
 M3: GENE AND CELL DOPING
• The following, with the potential to
enhance sport performance, are
prohibited:
•
1. The use of nucleic acids or nucleic acid
analogues that may alter genome
sequences and/or alter gene expression by
any mechanism. This includes but is not
limited to gene editing, gene silencing and
gene transfer technologies.
2. The use of normal or genetically
modified cells.
 P1: BETA-BLOCKERS
• Beta-blockers are prohibited In • Underwater sports (CMAS) in constant-
Competition only, in the following sports, weight apnoea with or without fins,
and also prohibited Out-of-Competition dynamic apnoea with and without
where indicated. fins, free immersion apnoea, Jump Blue
apnoea, spearfishing, static apnoea,
• Archery (WA)* target shooting, and variable
• Automobile (FIA) weight apnoea.
• Billiards (all disciplines) (WCBS) *Also prohibited Out-of-Competition
• Darts (WDF) Including, but not limited to:
• Golf (IGF)
• Shooting (ISSF, IPC)*
• Skiing/Snowboarding (FIS) in ski jumping,
freestyle aerials/halfpipe and snowboard
halfpipe/big air
 Therapeutic Use Exemption
• Complex rules apply to some of the
substances, not least because some have
therapeutic uses.
• The onus (tanggung jawab) is on the athlete to
obtain a ‘Therapeutic Use Exemption’ for
certain categories of substances (WADA 2020),
with the substances covered under this
possible exemption made clear in the WADA
list.
 Endogenous Anabolic Agents and
Hormones
• The WADA list also recognises that some anabolic
agents and hormones are produced
endogenously.
• Criteria regarding when an offence has been
committed are laid down for these substances
• An offence (pelanggaran) is considered to have
occurred if one (or more) of these prohibited
substances is detected in an athlete’s specimen at
a level outside of that which is considered to be
the ‘normal’ range in humans for the substance
 Particular Classes of Substances
• For some substances, offences apply both in-
or out of-competition, whereas for others an
offence applies only if the substance is
detected in-competition
• Particular classes of substances are prohibited.
• For example, betablockers are prohibited in-
competition in sports such as shooting,
billiards and gymnastics.
 Excessive Quantities of Normal
Nutrients
• The position regarding excessive quantities of
normal nutrients is much debated.
• Although not currently included in a
prohibited list, there is no doubt that certain
vitamins (notably B1, C and E) and also
creatine have been used in large doses with
the intention of affecting performance
 Substances with Threshold
• For most substances, the mere presence of
the substance or a diagnostic metabolite in
the biological fluid sampled constitutes an
offence, but for some substances (see Table)
there is a reporting threshold
• If the drug is detected but it is below the
reporting threshold, no offence is deemed to
have occurred.
 Summary of urinary concentrations above which
WADA-accredited laboratories must report findings
for specific substances
 Reported Analytical Findings
• Drugs misused in human sport include anabolic
steroids, stimulants, glucocorticoids, and peptide and
protein hormones such as erythropoietin and human
chorionic gonadotropin
• Diuretics and masking agents are also commonly found
• Of particular interest are the anabolic steroids and
glucocorticoids; not only may synthetic analogues be
misused, but also testosterone or hydrocortisone.
• This use of a pseudo-endogenous compound that is
either indistinguishable or distinguishable with
difficulty from that which is produced naturally by an
individual presents interesting analytical problems
Reported Analytical Findings
Prohibited substances most commonly reported by
IOC-accredited laboratories, in order of frequencya
Prohibited substances most commonly reported by
IOC-accredited laboratories, in order of frequencya
 Sampling
• Sample collecting procedures must take into
consideration both scientific and legal aspects as
follows:
• the health of the individual being sampled
must be safeguarded
• incorrect labelling, contamination or sample
switching must be avoided
• the rights of interested parties, generally the
owner or the individual or team in human sport,
must be safeguarded against error by the
analyst
 Sampling
• The two matrices specified for testing are urine and blood
• Urine is the primary matrix used for testing because it is
considerably less invasive in terms of sampling compared to
blood, offers relatively low health and safety risk
compared to blood in terms of sampling and analysis
• Blood may be sampled and used instead of urine provided
validated methods of analysis are applied to testing
• Blood is used as a test matrix when detecting blood
transfusion
• The collected sample is split into two: samples A and B.
• The ‘B’ sample is to be opened only after the first sample (
the ‘A’ sample) has been found to contain a banned drug
 Analytical approach
• Dope is generally administered at or near the
therapeutic dose, which results in relatively
low concentrations in biological fluids.
• Any drug used in human treatment or in
veterinary practice may be found.
• Screening procedures must be both sensitive
and of wide coverage, and they differ in detail
from other analytical schemes.
 Analytical approach
• Although the parent drug is the entity normally
described in the rules, screening procedures rely upon
the detection of either the unchanged drug or its
metabolites.
• The identification of the corresponding metabolites is
often useful supplementary evidence to support the
identification of the parent drug.
• Examples may include the detection of 3- and 4-
hydroxymepivacaine, major metabolites of the local
anaesthetic mepivacaine, or nordiazepam after
diazepam administration.
 Analytical approach
• Occasionally, the parent drug is not excreted in urine at a
detectable concentration, so knowledge of the metabolic
pathways in the particular species is essential.
• An example of this is the identification of 19-
norandrosterone and 19-noretiocholanolone in the urine
of humans as evidence of the administration of the
anabolic steroid nandrolone or a 19-norsteroid precursor
• After administration of cocaine, primarily benzoylecgonine
and ecgonine methyl ester are detected in urine unless a
huge dose of cocaine was given
 CH3 Cocaine CH3

O
O
O
O

O
O
NH
N
H3 C
O
O Cocaine
Norcocaine

HO CH3

O O

O
O

N OH
H3 C
N
O H3 C
Benzoylecgonine
HO Ecgonine-methylester

OH

Main Metabolism Pathway H3C

N
Ecgonine
 Analytical approach
• Some drugs are notable for being excreted in urine
almost entirely in conjugated form as, for instance,
morphine, apomorphine, fentanyl, nefopam and
pentazocine in the horse.
• When the presence of these drugs is suspected,
hydrolysis before extraction is essential because most
analytical methods are designed to detect drugs and
metabolites in their nonconjugated form.
• If a hydrolysis procedure is not employed, and the
analytical method used is not designed to detect
conjugates, the drug may not be detected even though
it was administered.
 Analytical approach
• No single analytical scheme will suffice to cover so many different
types of compounds
• Liquid–liquid extractions designed for group separation of drugs
followed by thin-layer chromatography (TLC) was used almost
universally for many years and is still used widely in North American
and some other racing chemistry laboratories
• Alternatively, drugs were extracted on styrene–divinylbenzene
copolymer XAD-2 resin
• The development of solid-phase extraction (SPE) in the cartridge
format in the late 1970s and the rapid advances made in the
technology associated with the technique have provided an
attractive alternative to liquid–liquid extraction in many drug-
screening programmes.
• Instrumental methods (GC using nitrogen–phosphorus detection
(GC-NPD), high-performance liquid chromatography using
ultraviolet detection (HPLC-UV), GC-MS and LC-MS(/MS)) are the
predominant analytical methods
 Solid-Phase Extraction
• In many racing chemistry laboratories throughout the world, SPE
has replaced liquid–liquid extraction for the isolation of drugs from
both urine and plasma
• The early applications of SPE to racing chemistry were in the use of
C18 bonded-phase cartridges to the isolation of anabolic steroids
and their metabolites from equine (kuda) urine
• Retention through a cation-exchange mechanism isolated the basic
drugs, whereas an anionexchange mechanism isolated acidic drugs
• Mixed-mode cartridges with retention mechanisms based upon
both hydrophobic interactions and ion-exchange processes have
found wide application in racing chemistry and many other
disciplines associated with drug analyses
Solid-phase extraction (SPE)
 Screening for Basic and Neutral Drugs and Poisons in Urine
2.5 mL Urine + 1 mL Sulphuric Acid

15 min. under reflux heated

+ 2 mL Amm. Sulphate 30% + 2,5 mL NaOH

10 N (pH 8-9) + 2,5 mL native urine
Extraction with 5 mL mixture of Et.
Ac.:MeCl:Isop. OH = 3:1:1
2 min. at 1500 g centrifugation

Aqueous Organic phase

phase
Evaporated at 60 °C

Residue: acetylation (Pyr. + Acetanhydride, 2:3) at 30 °C

Evaporated to dryness
Redue: dissolved in 50 L MeOH, 1 L injected
 Isotope ratio mass spectrometry
• Combustion isotope ratio MS (CIRMS) (see Figure) is now
used routinely by some WADA accredited laboratories
• After separation, eluted compounds are then combusted
oxidatively in a combustion reactor. Nitrogen oxides are
removed by reduction to nitrogen and water is then
removed by passing the eluent through a water separator.
• The sample is then introduced into the ion source of the
MS by an open split interface
• CIRMS provides an additional tool to help distinguish a
person whose testosterone to epitestosterone ratio may
be naturally beyond the normal range from an individual
who has administered testosterone
 Isotope ratio mass spectrometry
• The synthetic testosterone has a different proportion
of 13C to the more abundant 12C than the normal
endogenous steroid
• The extracted steroids are separated by GC and then
converted into CO2 and the relative amounts of 12C to
13C as CO2 determined for each eluting steroid in turn.
• Typically, the testosterone metabolites androsterone
and etiocholanolone or androstanediols are monitored,
or the metabolites 5a-androstanediol and 5b-
androstanediol
Combustion isotope ratio MS (CIRMS)
 Protein hormones
• Currently, in human sports drug testing, methods are
accepted for the analysis of hCG and for EPO only
• Tests for hCG are based on immunoprocedures (Cowan et
al. 1991; Laidler et al. 1994).
• A method to identify the different isoform pattern of
recombinant EPO from that of endogenous material using
isoelectric focusing (Figure 9.3) has been developed
recently (Lasne and Ceaurriz 2000; Lasne 2001)
• A method based on the perturbation (gangguan) of a
number of blood parameters (haematocrit, reticulocyte
haematocrit, percentage macrocytes, serum EPO and
soluble transferrin receptor; Parisotto et al. 2001) was used
at the Sydney Olympics in 2000.
Protein hormones
 Literature
• Clarke’s Analytical Forensic Toxicology,
Pharmaceutical Press, London, 2008, p. 263-
281