structure characterization and analysis of drugs · pdf filestructure characterization and...

Zentrum für Präventive Dopingforschung / Institut für Biochemie

Center for Preventive Doping Research / Institute of Biochemistry

Structure Characterization and Analysis of Drugs Relevant

to Sports Drug Testing by GC-Q/TOF

Mario Thevis

Agilent eSeminar, September 11, 2013



1771: Testicular implantation to capons

John Hunter (1728 – 1793)

Castrated roosters recovered

some of their loss of maleness

after testicular implantation



1849: On the importance of the testes

On August 2 I caponized six cockerels…The

dewlaps and combs were left intact. Two

cockerels had both testes removed; these

cockerels later exhibited the typical nature of

capons, behaved cowardly, agreed only rarely

to shiftless fights with other cockerels…



1889: Misinterpretation and placebo effects…



1935: The synthesis of Testosterone

Leopold Ruzicka (1935)



Anabolic-Androgenic Steroids – unlimited

ANDROGENS

AND ANABOLIC AGENTS

CHEMISTRY AND PHARMACOLOGY

JULIUS A. VIDA

1969 ACADEMIC PRESS New York and London




Franke & Berrendonk Clin Chem, 1997, 1262 For Forensic Use.




Franke & Berrendonk Clin Chem, 1997, 1262

Fig. 1. Effects of an androgenic-anabolic steroid,

Oral-Turinabol, on the shot-put performance

(in meters, y -axis) of a female athlete (code

identification 1/68.

For Forensic Use.



Hoberman & Yesalin, Sci Am 1995, 76

Anabolic-Androgenic Steroids – Stanozolol

For Forensic Use.



Numerous challenges for sports drug testing laboratories

- utmost comprehensiveness

- speed / turn-around times

- broad spectrum of physico-chemical properties of analytes

- etc.

For Forensic Use.



www.wada-ama.org For Forensic Use.



For Forensic Use.



…SARMs structures

R3 NH

O

OH CH3

O R2

R1

Arylpropionamide

Bicyclic hydantoins

NH

NH

O

R1 R2

R3

R4

Quinolines

NH

XR3

Y R4z

R1

R2

Tetrahydroquinolines

NN

O

O

OH

R1 R2

R3

For Forensic Use.



CF3 N

H

O

OH CH3

O NH

O2N

O

CH3

http://www.jamaicaobserver.com/



Canadian bobsledder Chris Korol suspended for doping infraction Nets 15-month ban for presence of androgen The Canadian Press, Posted: May 14, 2013 2:23 PM ET

Canadian bobsled pilot Chris Korol has been given a 15-month

suspension by the Canadian Centre for Ethics in Sport after testing

positive for a prohibited anabolic agent.

The substance was detected in a urine sample collected during in-

competition doping control on Oct. 21 at the Canadian bobsled

championships in Calgary. The sample was found to contain SARM S-

22, which the CCES classifies as a new drug with presumed anabolic

properties.

In its decision, the CCES said "the mere presence of SARM S-22 in an

athlete's sample is an anti-doping rule violation."

Korol, from Hamilton, piloted sleds in both the two-men and four-men

competitions at the Canadian championships, finishing third in both

races.

CF3 N

H

O

OH CH3

O

NC

CN



Enobosarm

seizure - confiscated in 2012 at German customs

- 1 kg pure Enobosarm (MK-2866)



…SARMs structures

Tropanol derivatives

(ACP-105)

Phenyloxadiazole

derivative

(RAD140)



80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

m/z

10

20

30

40

50

60

70

80

90

100

Rela

tive a

bundance (%

)

362.1582 347.1345

273.1145 257.0835

231.0677

217.0532

216.0445198.1305

170.0992

185.1227

131.0886

108.0803

73.0467

Tropanol derivative ACP-105

M+

Thevis et al. Rapid Commun. Mass Spectrom. 2013, 27: 1173 For Forensic Use.



80 100 120 140 160 180 200 220 240 260 280 300 320 340 360

73.0472

257.0850 347.1351

217.0534 93.0569

183.1073 142.0543 273.1166 10

20

30

40

50

60

70

80

90

100

Rela

tive a

bundance (%

)

MS/MS m/z 347

Tropanol derivative ACP-105

m/z




0

50

100

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460

450.1935

347.1361

217.0507

305.0902 131.0890 73.0482

257.0842 198.1308 277.0591

113.1323

Rela

tive A

bundance

(%

)

m/z

Tropanol derivative ACP-105 – monohydroxylated metabolite

435.1697 320.1110

-103 Da

361.1518

-130 Da

-130 Da

M+

For Forensic Use.



Tropanol derivative ACP-105 – monohydroxylated metabolite

For Forensic Use.



450.1146

80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480

117.0737 421.1134

279.0721

349.0733 172.0505 465.1392 291.0833

73.0464

m/z

10

20

30

40

50

60

70

80

90

100

Rela

tive a

bundance (%

)

Phenyloxadiazole derivative RAD140

M+




Phenyloxadiazole derivative RAD140




Park et al. Cell, 2012, 148: 421 For Forensic Use.



Roflumilast

Cilomilast

Natural and synthetic PDE4-inhibitors

For Forensic Use.



0

50

100

Rela

tive A

bundance

(%

)

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480

241.0686

439.1072

474.0756

314.1033 73.0475 419.0992

385.0463 187.0220 147.0434

290.0521 167.0232 213.0373 121.0289

m/z

Roflumilast

M+

For Forensic Use.



First cycling positive for GW501516, Rusvelo’s Valery Kaykov provisionally suspended By Shane Stokes @ 9:07 AM, April 11, 2013

Following a recent WADA warning concerning the substance GW501516, banned under its rules since

2009 and linked to potentially lethal health issues, the first case in cycling has been announced today by

the UCI.

The governing body has stated that the Rusvelo rider Valery Kaykov is provisional suspended in relation to

the black market substance, which was never cleared for human use.

“The decision to provisionally suspend this rider was made in response to a report from the WADA

accredited laboratory in Köln indicating an Adverse Analytical Finding of metabolite GW1516 sulfone –

Metabolic Modulator in a urine sample collected from him in an out of competition test on 17th March

2013,” stated the UCI in an announcement.

It added that Kaykov would remain suspended until such time as a hearing panel of the Russian Cycling Federation ruled on his

case, and that the rider could request and attend the analysis of his B sample.

He has been racing with the Rusvelo team for the past two seasons. Last year he finished third in the Russian time trial

championships, one minute and ten seconds behind Denis Menchov (Katusha). He was also 22nd in the 1.1-ranked Coppa

Bernocchi.

Responding today, the Rusvelo said that it had terminated his contract and insisted that its main ideology “is zero tolerance to

doping in cycling. We do support clean cycling and we will stick firmly to our policy,” it said.

A little over two weeks ago WADA issued a rare warning to all athletes, speaking about severe consequences of using

GW501516 [also known as GW1516 - ed.].

“The side effect of this chemical compound is so serious that WADA is taking the rare step of warning “cheats” to ensure that

there is complete awareness of the possible health risks to athletes who succumb to the temptation of using GW501516 for

performance enhancement,” it stated then.

“GW501516 was a developmental drug that was withdrawn from research by the pharmaceutical company and terminated when

serious toxicities were discovered in pre-clinical studies. Clinical approval has not, and will not be given for this substance.”

It is understood that the substance has been linked to the spread of tumours. According to New Scientist, the original

manufacturers GlaxoSmithKline abandoned its development in 2006 after serious effects were detected in rats used as the first

test subjects. It was determined that in all doses, the drug quickly caused tumours in a number of organs, including the

liver, bladder, stomach, skin, thyroid, tongue, testes, ovaries and womb.

http://www.velonation.com/News/ID/14196/WADA-issues-urgent-warning-about-black-market-substance-GW501516.aspx



GW501516 positives confirmed, three out of 4 riders are from the same BCR Pizza Hut team By Shane Stokes @ 3:10 PM Monday, April 15, 2013

UCI confirms Vargas Barrantes, Mudarra Segura, Morales Castillo,

Villalobos Azofeifa, and Miguel Ubeto provisionally suspended

Sergej Lisin arguably first athlete tested positive with GW1516 (2012)



60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540

256.0402

85.0109 525.1056 237.0410 139.0207 191.1792 439.0531 362.0776

0

50

100

Rela

tive A

bundance

(%

)

m/z

GW1516

M+ 510.0842

N

S

CF3

CH3

S

CH3

O

O

OSi(CH3)3

m/z 256

N

S

CF3

CH3

CH2

+

For Forensic Use.



Established substances – new long-term metabolites?

Exploiting the sniffer-dog quality of GC/C/IRMS and

identification capability of GC-Q/TOF





Blank urine

PD





8 hours

PD




141 hours

PD






477 hours

PD





-> structure characterization by GC-Q/TOF




0

0.2

0.4

0.6

0.8

1.0

m/z

60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330

256.2138

138.1336 166.1645 271.2364

188.1502

110.1009

84.0864 316.0379

[C19H22D3]+

[C20H25D3]+ [C14H14D3]+

[C12H16D3]+ [C10H12D3]+ 331.2563

[C22H29D3O2]+

18-nor-17α-trideuteromethyl-17β-methyl-5β-androst-1,13-diene-3α-ol (3-acetate)


-> structure characterization by GC-Q/TOF




Summary

- Utmost comprehensiveness of detection assays in sports drug

testing desirable – necessitates targeted AND non-targeted

approaches

- GC-HRMS(/MS) complements existing LC-HRMS/(MS)

methods

- Metabolism studies and structure characterization

enabled/supported by GC-HRMS/MS with MS/MS feature being

essential

- Extensive analytical data allow for retrospective data mining,

which facilitates estimation of prevalences of substances and/or

their abuse in sport

For Forensic Use.



World Anti-Doping Agency (WADA Grant #10A10MT)

Ministry of the Interior, Federal Republic of Germany (BMI) Manfred-Donike Institute for Doping Analysis

Center for Preventive Doping Research

Dr. Thomas Piper

Dr. Andreas Thomas

Oliver Krug

CER Groupe

Dr. Philippe Delahaut

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