Structure Characterization and Analysis of Drugs Relevant to Sports Drug Testing by GC-Q/TOF

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Mario Thevis

Agilent eSeminar, September 11, 2013 Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

1771: Testicular implantation to capons

Castrated roosters recovered some of their loss of maleness after testicular implantation

John Hunter (1728 – 1793)

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

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…

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

1889: Misinterpretation and placebo effects…

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

1935: The synthesis of Testosterone

Leopold Ruzicka (1935)

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Anabolic-Androgenic Steroids – unlimited

ANDROGENS AND ANABOLIC AGENTS

CHEMISTRY AND PHARMACOLOGY

JULIUS A. VIDA

1969 ACADEMIC PRESS New York and London

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Anabolic-Androgenic Steroids – unlimited

For Forensic Use. Franke & Berrendonk Clin Chem, 1997, 1262 Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Anabolic-Androgenic Steroids – unlimited

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. Franke & Berrendonk Clin Chem, 1997, 1262 Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Anabolic-Androgenic Steroids – Stanozolol

For Forensic Use. Hoberman & Yesalin, Sci Am 1995, 76 Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

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. Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

For Forensic Use. www.wada-ama.org Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

For Forensic Use. Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

For Forensic Use. Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry …SARMs structures

R1 O O R2 Arylpropionamide R3 N H OH CH3

OH O

N R3 Bicyclic hydantoins R1 R2 N O R1 R2

R3 Quinolines O N N H H R4 R3 R1 X

Tetrahydroquinolines z N Y R4 H R2 For Forensic Use. Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

O2N O H O N

CF3 N O H HO CH3 H3C

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

NC O O CN

CF3 N H HO CH3 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.

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Enobosarm seizure

- confiscated in 2012 at German customs - 1 kg pure Enobosarm (MK-2866)

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry …SARMs structures

Tropanol derivatives (ACP-105)

Phenyloxadiazole derivative (RAD140)

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Tropanol derivative ACP-105

100 217.0532

(%) 80

70 + 60 M abundance 50 216.0445 362.1582 131.0886 198.1305 347.1345 40

Relative Relative 231.0677 30 73.0467 170.0992 20 273.1145 108.0803 185.1227 10 257.0835

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

For Forensic Use. Thevis et al. Rapid Commun. Mass Spectrom. 2013, 27: 1173 Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Tropanol derivative ACP-105

73.0472 100

(%) 80 MS/MS m/z 347

60 abundance 50

40 257.0850 347.1351 Relative Relative 30 217.0534 20 93.0569 183.1073 10 142.0543 273.1166

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

Tropanol derivative ACP-105 – monohydroxylated metabolite

-103 Da M+ 100 -130 Da 450.1935

347.1361 -130 Da

217.0507 50 73.0482 131.0890 305.0902 198.1308 257.0842 277.0591 320.1110 Relative Abundance (%) Abundance Relative 361.1518 435.1697 113.1323

0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 m/z

For Forensic Use. Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Tropanol derivative ACP-105 – monohydroxylated metabolite

For Forensic Use. Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Tropanol derivative ACP-105 – monohydroxylated metabolite

For Forensic Use. Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Phenyloxadiazole derivative RAD140

117.0737 421.1134 100

(%) 80 73.0464 70

60 abundance 50

Relative Relative 450.1146 30 279.0721 + 20 M 10 172.0505 291.0833 349.0733 465.1392

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

Phenyloxadiazole derivative RAD140

For Forensic Use. Park et al. Cell, 2012, 148: 421 Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Natural and synthetic PDE4-inhibitors

Roflumilast

Cilomilast

For Forensic Use. Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Roflumilast

100 241.0686

439.1072

M+

50 474.0756 314.1033 73.0475 419.0992

Relative Abundance (%) Abundance Relative 147.0434187.0220 385.0463 290.0521 121.0289 167.0232 213.0373 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 m/z

For Forensic Use. Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

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.

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

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)

Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

GW1516 m/z 256 O CH3 O Si(CH3)3 O S S CF3 100 256.0402 CH N 2CH S 3 + CF

3 N 3CH

50 Relative Abundance (%) Abundance Relative 510.0842 M+ 85.0109 237.0410 525.1056 139.0207 191.1792 362.0776 439.0531 0 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 m/z

For Forensic Use. Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Established substances – new long-term metabolites?

Exploiting the sniffer-dog quality of GC/C/IRMS and identification capability of GC-Q/TOF

For Forensic Use. Thevis et al. Rapid Commun. Mass Spectrom. 2013, 27: 1904 Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Established substances – new long-term metabolites?

Blank urine

Established substances – new long-term metabolites?

8 hours

Established substances – new long-term metabolites?

141 hours

Established substances – new long-term metabolites?

477 hours

Established substances – new long-term metabolites?

-> structure characterization by GC-Q/TOF

Established substances – new long-term metabolites?

-> structure characterization by GC-Q/TOF

Established substances – new long-term metabolites?

-> structure characterization by GC-Q/TOF

18-nor-17α-trideuteromethyl-17β- methyl-5β-androst-1,13-diene-3α-ol (3-acetate) 256.2138 [C H D ]+ 1.0 19 22 3

0.8

0.6 138.1336 166.1645 271.2364 [C10H12D3]+ [C12H16D3]+ 188.1502 331.2563 0.4 [C20H25D3]+ [C H D O ]+ [C14H14D3]+ 22 29 3 2

0.2 110.1009 84.0864 316.0379 0 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 m/z

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. Zentrum für Präventive Dopingforschung / Institut für Biochemie Center for Preventive Doping Research / Institute of Biochemistry

Center for Preventive Doping Research Dr. Thomas Piper Dr. Andreas Thomas Oliver Krug

CER Groupe Dr. Philippe Delahaut

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

Ministry of the Interior, Federal Republic of Germany (BMI)

Manfred-Donike Institute for Doping Analysis