Detection and Analysis of the New “Designer Steroids”

Victor Uralets

Quest Diagnostics, 4230 Burnham Avenue, Las Vegas, Nevada 89119

Winter 2007

Redondo Beach, CA Androgenic Anabolic Steroids• Provide enhancement in muscle

strength and endurance far beyond that, which could be achieved without steroids even in rigorous training.

• Performance cannot remain at the same level without continued steroid use

• Abused for the performance enhancement and cosmetic purposes

Milestones1939 Nobel prize was awarded for testosterone discovery

1950-1965 Development and introduction of synthetic anabolic steroid drugs

1974 Synthetic AAS are banned by the IOC

1976 First (limited) testing at Montreal Olympics (8 positives)

1988 Positive test Ben Johnson, Seoul Olympics

1990 AAS banned in the US - Schedule III Controlled Substances

1997 Dietary supplements: androstendione, androstendiol

1997-2004 Prohormones, designer anabolic steroids.

2003-2004 US doping scandal, THG, BALCO, Baseball

2005 Amendment to the Controlled Substances Act

All Endogenous Steroids are metabolites of CHOLESTEROL

CH3

CH3

CH3

CH3

H3C

HO

A B

C D1

2

34

56

7

89

10

1112

13

14 1516

17

18

19

20

2122 23

24

25

26

27

Cholest-5-en-3b-ol (Cholesterin)

Steroid Classes

Testosterone

Androgens

Estradiol

Estrogens Progestogens Corticosteroids

Progesterone Cortisone

Secreted by the testis and is responsible for the development of the male sexual characteristics

Secreted by the ovaries, maintain the female reproductive function.

Progestogens are produced in females and serve as protectors of pregnancy

Corticosteroids regulate multiple functions of the human body:electrolyte balance, maintenance of homeostasis, glycogen, carbohydrate synthesis, suppress inflammatory reactions

Testosterone

• The principle androgenic steroid

• In the 1930s the Nobel prize was awarded for its discovery

• Stimulates anabolic activities: nitrogen retention, muscle growth

• Testosterone is not effective when administered orally or by injection

• Rapidly absorbed and metabolized by the liver

Testosterone Metabolism

Hydrogenation at C-4,55a- and 5ß-Reductase

Testosterone Metabolism5α-DHT

A B

Hydrogenation at C-4,55α-Reductase

5α-Dihydrotestosterone

5α- Androstane-17βol-3-one

Testosterone Metabolism

Hydrogenation at C-4,55ß-Reductase

5β- Androstane-17βol-3-oneEtiocholane-17βol-3-one

Testosterone Metabolism

Reduction of 3-Keto group3a-Hydroxysteroid-Dehydrogenase

5α- Androstane-3α,17β-diol

Testosterone Metabolism

Androsterone

Oxidation of 17ß-Hydroxy group17ß-Hydroxysteroid-Dehydrogenase

5α- Androstane-3α-ol-17-one

Phase II Steroid Metabolism

HOOOH

OH

OH

COOH

Glucuronide ester

3a-hydroxy steroids

HO

S

O

OHO

Sulfate ester

3b-hydroxy steroids

Synthetic Anabolic Steroidsdesigned to slow down metabolism

• Reduce androgenic activity

• Enhance anabolic potency

• Improve oral bioavailability

Synthetic Anabolic SteroidsChemical Alterations of Testosterone

OH

O

123 4

171-Methylation

1,2-Dehydrogenation

2-Methylation

4-Chlorination4-hydroxylation

17-Methylation

Synthetic Anabolic Steroids (examples)C17-OH Esterification

• Pharmacologically Efficient IM Form• Slowly Hydrolyses into Active Free C17-OH steroid• May be detected in urine for long periods of time

O CO

O 19-nortestosterone17-Decanoate

(Nandrolone)

Detection and Analysis

• Immunoassays are not used• Basic Screen by GC/MS• LC/MS/MS screen is applied

for labile steroids• Isotope Ratio Mass Spec.

(IRMS): endogenous steroids• High Resolution Mass Spec.

(HRMS) – High Sensitivity

Sample Preparation for GC/MS Screen

• Enzymatic Hydrolysis of Steroid Conjugates– Deconjugation by β-Glucuronidase from E.coli– E.coli is fast (1 hour), specific, has no sulfatase

activity– Helix Pomatia juice is slower (3 hours), but it

cleaves additional steroid sulfates– H.pomatia enzyme produce side reactions, such

as conversion of androst-5-en-3β,17β-diol into testosterone

Sample Preparation for GC/MS Screen

• Solvolysis of Steroid Sulfates– May be performed after enzymatic hydrolysis to

recover steroid sulfate fraction– Ethyl Acetate + 4N H2SO4 1 hour incubation– Yields useful long-term 3β -hydroxy metabolites

• Acidic Hydrolysis was found Destructive and has been abandoned

Sample Preparation for GC/MS Screen

• Liquid-Liquid Extraction– is used after hydrolysis to isolate deconjugated

steroids– pH 9 with tert.butyl methyl ether, yields ~ 90%

• Solid Phase Extraction– C18 cartridges are washed with methanol and

water and then hydrolyzed urine is applied– The column is rinsed with 30% aqueous CH3CN

and then the steroids are eluted with methanol

Sample Preparation for GC/MS Screen

• Derivatization of Steroids for GC/MS

Prohormones, precursors of testosterone

Prohormones, precursors of nandrolone

1

10

100

1000

10000

100000

0 50 100 150 200 250

Norandrosterone ng/ml

Time [h] after administration2000 4000

2 ng/ml Norandrosterone

Positive Urine Test for Norandrosterone

10 ng/ml

NandroloneDepo-form

Prohormone19-Norsteroid

Contaminated Product

Urinary Norandrosterone Excretion after different Applications of 19-Norsteroids

Urinary Norandrosterone Excretion after different Applications of 19-Norsteroids

Designer Anabolic Steroids

β α

Designer Anabolic Steroids

Potent androgenic-anabolic steroid

Orally efficient

Anti-Estrogen agents

2003 Doping Scandal in the US

• BALCO was charged with supplying athletes with “undetectable“ designer steroid THG

• High profile cases involving Olympic star athletes

• Large scale abuse in baseball

Tetrahydrogestrinone (THG)

2004 Amendment to the Controlled Substances Act

• Ban on designer steroids and prohormones– classified as Schedule III Controlled

Substances– no longer available over-the-counter

The End …?

2005 New Designer Anabolic Steroids, “Dietary Supplements”

not included in 2004 Controlled Substances Act

3,2-c]pyrazole-5alpha-etioallocholane-17beta-tetrahydropyranol

Methasterone Desoxymethyltestosterone Prostanozol5α-Androstan-17β-ol-[2,3c]pyrazol, 17-tetrahydropyranyl

2005 New Designer Anabolic Steroids, “Dietary Supplements”

not included in 2004 Controlled Substances Act

5α-Androstan-2α,17α-dimethyl-17β-ol-3-one

5α-Androst-2-en-17α-methyl-17β-ol 5α-Androstan-17β-ol-[2,3c]pyrazol, 17-tetrahydropyranyl

1-Androstendiol 1-AD1-AndrostendioneAndrostendiol (Δ4- and Δ5-androsten-3β,17β-diols) AdiolAndrostendione (Δ4- and Δ5-androsten-3,17-diones) AndrostenBolasterone (7α,17α-dimethyltestosterone) MyagenBoldenone (1-dehydrotestosterone) Equipose, ParenabolBoldione (androsta-1,4-dien-3,17-dione) Equi-BolanCalusterone (androst-4-en-7β,17α-dimethyl-17β-ol-3-one) MethosarbClenbuterol SpiropentClostebol (4-chlorotestosterone) Turinabol, SteranabolDanazol (17-ethynyl-17β-ol-4-androsteno[2,3-d]isoxazol) Cyclomen, DanatrolDehydrochloromethyltestosterone Oral-Turinabol, DHCMTDehydroepiandrosterone DHEADesoxymethyltestosterone (5α-androst-2-en-17β-ol) Ergo Max LMGDihydrotestosterone 5α-DHTDrostanolone (2α-methyl-5α-dihydrotestosterone) Masterid, MasteroneEpitestosterone (4-androsten-17α-ol-3-one) > 200 ng/mLEstra-4,9-dien-3,17-dione FiniGenX MagnumFluoxymesterone (9α-fluoro-17α-Me-11β-OH-testosterone) Halotestin, UltadrenFormebolone (formyldienolone) EsicleneFormestane (4-hydroxyandrostendione) LentaronFurazabol Miotolon4-Hydroxytestosterone Formadrol6α-Methylandrostendione M-1-PMestanolone (17α-methylandrostan-17β-ol-3-one) AndrostanoloneMesterolone (1α-Methyl-5α-dihydrotestosterone) ProvironMethandienone (Methandrostenolone) DianabolMethasterone (2α,17α-dimethyl-5α-androstan-17β-ol-3-one) Methyl Masterdrol, SuperdrolMethenolone (1-Methyl-5α-androst-1-en-17β-ol-3-one) PrimobolanMethandriol (17α-Methyl-5-androsten-3β,17β-diol) AndrodiolMethylnortestosteroneMethyl-1-testosterone (17α-Methyl-5α-androst-1-en-17β-ol-3-one) M-1-TMethyltestosterone (17α-Methyltestosterone) MetandrenMibolerone (1α,17α-Dimethyltestosterone) Cheque, MatenonNandrolone (19-Nortestosterone) Deca-Durabolin19-Norandrostendiol (Δ4- and Δ5-androsten-3β,17β-diols) Bolandiol, Nandrodiol19-Norandrostendione (Δ4- and Δ5-androsten-3,17-diones) NorandroNorclostebol (19-nor-4-chlorotestosterone)Norethandrolone (17α-Ethyl-19-nortestosterone) NilevarOxandrolone (2-Oxa-17α-methyldihydrotestosterone) AnavarOxymesterone (4-Hydroxy-17α-methyltestosterone) OranabolOxymetholone (17b-OH-2-hydroxymethylen-5α-DHT) AnandrolProbenecid, masking agent Proben, BenemidProstanozol (5α-androstano-17β-ol-3-one-[2,3-c]pyrazol) Orastan-EQuinbolone (1-dehydrotestosterone 17-cyclopent-1’-enyl ether) Anabolicum VisterStanozolol (5α-androstano-17α-methyl-17β-ol-3-one-[2,3-c]pyrazol) Stromba, WinstrolTestolactone (D-homo-17α-oxaandrosta-1,4-dien-3,17-dione) Teslac1-Testosterone (5α-androst-1-en-17β-ol-3-one) 1-Test; 1-TTestosterone (T/E ratio > 6) Depotest, TestinonT b l (A d t 4 9 11 t i 17β l 3 ) P b l Fi j t

T/E Ratio

Epitestosterone Testosterone

Normal

T/E ~ 1

High

T/E > 6

Androsterone/ Etiocholanolone (A/E)

NormalHigh Low

A A

AE

E E

Possible use of 5α-DHT (5α-dihydrotestosterone)

Possible use of 5α-reductase inhibitors, masking agents

A & E are main urinary androgen metabolites

Androsterone - 5α metabolite

Etiocholanolone - 5β metabolite

Fast GC - ~ 1 second separationStanozolol Metabolites Confirmation

Minutes

2.49 2.51

Differentiation of Natural and Synthetic Endogenous Steroids by IRMS –Isotope Ratio Mass Spectrometry

• The ratio of two stable carbon isotopes 13C and 12C allow to discriminate endogenous from chemically identical, but synthetic steroids in urine.

• Synthetic substances usually exhibit different composition of 13C and 12C isotopes than material of exclusive biological origin

IRMS for carbon isotope measurement

MS analysis of CO2

GC separation

On-line combustion to CO2 and HO2

• Ion abundances are measured at• M/Z = 44 represents 12C

• M/Z = 45 represents 13C

•M/Z = 46 correction for the Oxygen isotopes 17O and 18O

Carbon isotope method limitations

• Only ~ 1.11% of all carbon consists of 13C

• Large amount of substance is required

• Complete baseline separation is needed

• Sufficiently large signal-to-noise ratio

• Low and constant background condition

Isotope ratio 13C/12C

Steroid Synthesis in the Body Metabolites (Urine)

Cholesterol

Pregnenolone Progesterone Pregnandiol

17α-Hydroxy-pregnenolone

17α-Hydroxy-progesterone

Pregnantriol

DHEA Androstendione

Testosterone

Androsterone

Etiocholanolone5α-Androstandiol

5β-Androstandiol

Anabolic Steroid Liver Toxicity

O

OHCH3

Methandienone TestosteroneOH

O

H

Toxicity

>

17-alkylated Steroids are toxic (Metandienone and Stanozolol),

Nonalkylated Steroids (Testosterone and 19-Nortestosterone) are less toxic at comparable Doses

Estrogenic Side EffectsGinecomastia and Fluid Retention

Female Type Brest Growth in Males

O

OHOH

HOaromatizable

Testosterone Estradiol

O

OH

H3C

OH

HO

H3C

aromatizable

Drostanolone

Non

Aromatizing compounds are used in bulking phases of training to increase body mass including fat and fluids

Non-aromatizing steroids increase the look of definition and muscularity

Drugs Used to Counteract Steroid Side Effects

• Anti-Acne – Accutane• Accutane - Potent oral acne medication very effective for its

intended use, numerous side effects have been reported

• Anti-Estrogen• Anastrozol – blocks enzyme aromatase• 1,4,6-Androstentrien-3,17dione – aromatase inhibitor• Formestane (4-Hydroxy-4-androsten-3,17-dione)• Clomiphene – used also as fertility aid• Tamoxifen – estrogen antagonist• Testolactone

Drugs Used to Counteract Steroid Side Effects

• 5α- Reductase inhibitors• Dinasterid (Avodart)

– one of the latest drugs to treat benign prostate enlargement

• Finastride (Propecia, Proscar)– blocks 5α-DHT formation, may be used to slow down

anabolic steroid metabolism, anti-baldening agent

• Tanning Agents• Trioxsalen (Trisoralen), Methoxalen (Oxoralen) –

Synthetic melanizing and repigmenting agents.– The dark tan makes the physique look much more

appealing. Brings out muscle separation. Increases skin sensitivity to light, makes one prone to skin damage

Auxiliary Drugs

• Masking agents– Diuretics– Epitestosterone– Probenecid– 5α- Reductase inhibitors

• Anti-Estrogens• Other (non steroid) anabolic agents

– Clenbuterol, tibolone, zeranol, zilpaterol• Peptide Hormones: EPO, hGH, IGF, LH, hCG

• Beta-2 Agonists: salbutamol, terbutalin, etc.

Assistance of Stacy Sweeney and Ben Salazar of Quest Diagnostics in Las Vegas is gratefully acknowledged