Metabolism of anabolic steroids in man: synthesis and use of reference substances for ...

26 Pages·2001·1.98 MB·English

Anaiytzca Chumca Acta, 275 (1993) 23-48 32
Elsevler hence Publishers B V , Amsterdam
msilobateM fo anabolic sdiorets in man: sisehtnys and esu
fo ecnerefer substances rof identification fo anabolic
diorets setilobatem
1lliW rexnahcS and derfnaM ekmoD
ehcstukL S’rthochschuk ,nloK tuthrnI f?u Bwchemre, Carl-L&m-Weg 4 5ooO Cologne 41 (Germuny)
(Recewed 20th May 1992)
AhStl-&
The use of anabohc steroids was banned by the International Olympic Comnuttee for the first tune at the Olympic
Games m Montreal m 1976 Since that tie the nususe of anabohc steroxls by athletes has been controlled by
analysis of urme extracts by gas chromatography-mass spectrometry @C-MS) The excreted steroids or their
metabohtes, or both, are isolated from urme by XAD-2 adsorption, enzymatx hydrolyss of coqlugated excreted
metabobtes anth /?-glucuromdase from Es&en&u co& bqmd-bqmd extractlon mth diethyl ether, and courted
mto tnmethylsdyl (‘INiS) derwatwes The confirmation of an anabobc stenxd nususe ts based on comparison of the
electron nnpact lomzation (EI) mass spectrum and GC retention tie of the isolated steroid and/or its metabohte
with the mEla ss spectrum and GC retention time of authentic reference substances For this purpose excretion
studies with the most common anabobc steroids were performed and the mam excreted metabobtes were synthesued
for bolasterone, boldenone, 4-chiorodehydromethyltestosterone, clostebol, drostanolone, tluoxymesterone, forme-
bolone, me&a&one, mesterolone, metandlenone, methandnol, metenolone, methyltestosterone, nandrolone,
norethandrolone, oxandrolone and stanozolol The metabobsm of anabohc Hero& the synthesis of theu mam
metabGoCht erse,t ethnetmu n and EI mass spectra as TMS dewames are dIscussed
&ywor& Gas chromatography, Gas chromatography-mass spectrometv, Anabobc steroids, Metabohsm
ehT esusim fo enoretsotset and chobana choba sdiorets and neht sethobatem era detalosi
sdiorets yb setelhta ot evorpmi rieht ecnamrofrep morf ,emru dezrtavrred htrw -htemnt-N-lyhtem-N
has del ot a ban no chobana sdiorets yb eht edmratecaoroulfntlyltsly )APTSM( and desylana
lanoitanretnI cipmylO eettnnmoC )COI( and -an yb gas yhpargotamorhc )CG( and mass -mortceps
lanoit and lanortametm strops snoitaredef -nA yrte )SM( usmg nortcele tcapmi DE( nortaxmor at
choba sdiorets erew dennab rof eht tsrif emit at 70 Ve esuaceB tsom chobana sdiorets era -moc
eht cipmylO semaG 1976 m laertnoM and -sotset yletelp dexhobatem and on tnerap diorets is -xe
enoret at eht cipmylO semaG 1984 m soL -egnA ,deterc eht msilobatem fo eht chobana sdiorets
sel oT lortnoc eht esusim fo an chobana diorets must eb nwonk srepaP no eht msilobatem fo
eht semru fo setelhta era detcelloc yltcerid retfa chobana sdrorets m man erew elbaliava rof -edlob
nortrtepmoc ro tuo fo nortrtepmoc and evah ot eb enon enoretsotsetlyhtemordyhedorolhc-4,]1[ ,]3,2[
desylana yb IOC detidercca seirotarobal ehT -na enonerdnatem ,]5,4[ enoretsotsetlyhtem ,]6[ -nan
enolord 17[ and enolordnahteron ]8[ ehT -ifitnedi
nortac fo eseht sethobatem was desab no eht
Comzvpondence to W Schanzer, Deutsche Sporthochschule
noitalosi and nortacifrrup fo yramru detercxe
Koln, Instltut fir Bmchenue, Carl-Diem-Weg 6,5ooO Cologne
sethobatem retfa noitacilppa fo egral stnuoma fo
41 (Germany)
0003~2670/93/$06 00 Q 1993 - Elsevler Science Publishers B V All nghts reserved
24 W Schanzer and M Lkwukz /Am! Chm Acta 275 (1993) 23-48
eht chobana diorets and noltazlretcarahc yb -tlem mmmm ,edndyh nunmorhc ,edlxont mumtalp
gm stniop and derarfm ,)RI( H‘ raelcun citengam edlxold and %01 palladium no laocrahc erew
ecnanoser (NMR) and mass yrtemortceps At desahcrup morf hcirdlA )mlehmetS( enoretsaloB
tsael eht desoporp erutcurts fo eht ethobatem was desahcrup morf ,nhoJpU -ordyhedorolhcC
was detadicule yb its sisehtnys enoretsotsetlyhtem was a tfig morf mrahpaneJ
CG htiw a&s-desuf yraldpac snmuloc -nroc ,)aneJ( enoretsemyxoulf was a tfig morf Cuba-
demb htiw MS htiw IE noltazmol swolla yrev tsaf ,ygieG lobazaruf was a &g morf lhcnaD ,)oykoT(
and ylevitaler elpmis elpmas noitaraperp ot -osi enolordnaxo was a itg morf ,elraeS enoretsemyxo
etal small stnuoma fo crlobana sdiorets and rieht was a tf@ morf olraC ,abrE ,enoloretsem
sethobatem morf eniru 311-9[ ehT detalosi enolonetem and enolonetem etateca erew g&s
sethobatem era deziretcarahc yb neht CG -neter morf gmrehcS ,)nilreB( enolobemrof was a tfig
noit semit and IE mass artceps A ethobatem is morf A H ttekceB ,)nodnoL( enolonatsord -lporp
deifitnedi nehw eht nolsnapmoc wth an -nehtua etano was a tfig morf lahtnenurG ,)nehcaA(
cit ecnerefer ecnatsbus shows eht emas CG -neter ,enonedlob lobetsolc ,etateca ,enolonatsem
noit emit and IE mass murtceps ,enoneldnatem ,londnahtem lolrdnahtem -orpld
As tsom fo eht chobana diorets sethobatem ,etanolp ,enoretsotsetlyhtem enolordnan and
erew ton yllaicremmoc elbalava m reilrae ,semit lolozonats erew desahcrup morf amgiS -nesleD(
dellac-os ecnerefer“ substances” erew deniatbo ,)frod ,enolordnahteron enoretsotsetyxordyh&6
morf emru m noitercxe seiduts htlvv chobana and enoldenetsordnayxordyh-3/6 erew desahcrup
sdiorets A mslcltnc fo this dohtem was eht wol morf sdlolaretS aiv leseaP )trufknarF(
ycarucca fo a yraniru ecnerefer“ ”ecnatsbus rof edlmatecaoroulflrtlyllslyhtemlrt-N-lyhtem-N
hcihw eht erutcurts 1s ton yltcaxe nwonk and eht )AFTSM( was deusehtnys m eht yrotarobal llA
elbissop yt?rupnn fo eht yramru tcartxe rehto stnegaer and stnevlos erew fo -er-lacltylana
oT tnevmucric this egatnavdasid ew detrats m tnega edarg
1987 eht sisehtnys fo eht mam sethobatem fo
esoht chobana sdiorets hcihw erew ylniam -slm
Metabohsm studws
desu m strops ehT sisehtnys fo sethobatem fo
mshobateM seiduts erew demrofrep yb laro
enonedlob [ 121, enoneldnatem [ 131, lolozonats [ 141
noitacilppa fo eht chobana sdiorets XX-I ot elam
and eht sisehtnys fo cnenupe-71 sdiorets 151[
sreetnulov and yb noltartslmmda-fles yllausU a
evah neeb dehsilbup rehtruF snoltachbup era m
elgms esod fo 20 gm fo diorets was deilppa and
noitaraperp
eht gmwollof eniru samples erew detcelloc and
sihT repap sevig an weMevo fo eht mam
derots at 4°C
cilobatem ,syawhtap eht sisehtnys fo eht mam
setrlobatem fo chobana sdiorets and rieht
Isolatwn fo c&&na sdiorets dna ueht metabo-
lyllslyhtemnt )SMT( noltazltavlred rof SM-CG
htes
and stneserp eht CG noitneter secldm and IE
ehT sdiorets ro/dna neht sethobatem erew
mass artceps fo eht chobana sdiorets detercxe
detalosi as debircsed yb ekmoD te al ]9[ wth
degnahcnu and rieht mam ,sethobatem hcihw era
emos snoitacifidom ehT lautca elpmas -araperp
ylemtuor desu rof gnmeercs and noitamrifnoc
noit 1s debircsed
ehT scitemkocamrahp fo eht detercxe sdiorets
and sethobatem lliw ton eb dessucsid
Isolatwn fo detaguJnoca?u excreted stero&
oT 5 lm fo emru era dedda 0 5 g fo muidos
muissatop-etanobracnegordyh etanobrac 2( 1,
EXPERIMENTAL
w/w> dilos reffub and 50 ng fo -yxordyh-a4
Steroids dna chermcals lolozonats as lametm standard and eht -u~nocnu
edlrtkeleS-K 1( M mmssatop tes-nt -lytub- detag sdiorets era detcartxe htiw 5 lm fo lyhteld
edndyhorob m ,)narufordyhartet mmhth -ula rehte dellltsld( revo muiclac )edirdyh ehT rehte
W SchamerandM Donke/Anal Chm Acta275(1993)23-48 25
reyal SI derrefsnart retfa noltagufrrtnec and -pave nmuloC C was desu ees( DIF-CG )sretemarap
detaro ot ssenyrd rednu muucav ehT nmuloc erutarepmet was demmargorp morf
eeS osla eht noltalosl fo detagulnoc sdiorets COoo2 at 4O”C/mm ot 320°C dleh( rof 3 )mm ehT
rotcejm erutarepmet was 300°C and eht ecafretni
Isolatwn fo detagulnoc excreted sterouii dna erutarepmet 3u)“C
metabohtes detaguJnoC fractwn The stnemurtsni and -rac
oT 2 ml fo emru 1s dedda 1 pg fo -lyhtem reir gas erew eht emas as evoba nmuloC A was
enoretsotset lanretm( standard) and eht sdiorets desu ees( DIF-CG )sretemarap ehT nmuloc
era debrosda no ethrebmA XAD-2 enerytsylop erutarepmet was demmargorp morf 180°C at
niser ehT XAD-2 snmuloc ruetsaP( ,ettepip CC/mm ot 240°C and at 15”C/mm ot 320°C
desolc htiw a glass ,lraep deb thgieh 2 )mc era ehT rotcejni erutarepmet was 300°C and eht -m
washed htiw 2 lm fo ylbuod dellltsld retaw and ecafret erutarepmet 320°C
detule wth 2 lm fo lonahtem ehT chonahtem
etaule 1s detaropave ot ssenyrd and eht eudiser 1s GC-FID parameters
devlossid m 1 lm fo 0 2 M muidos etahpsohp An PH 5880 gas hpargotamorhc was desu ehT
reffub Hp( )7 reirrac gas was muheh at 15 mm/lm at ,C”o81
At this egats a noitarapes fo detagujnoc and gnittilps oitar :1 10, erusserp 27 ISP ehT nmuloc
detagujnocnu detercxe sdiorets 1s elbissop ehT erutarepmet was demmargorp morf 180°C at
detaguJno.cnu sdiorets nac eb detcartxe htiw 5 lm 5”C/mm ot 320°C nmuloC A was -ttelweH
fo lyhteld rehte ehT gmmamer rehte must eb drakcaP l-artlU desuf s&a, deknil-ssorc -lyhtem
devomer yb noitaropave fo eht reffub noitulos rof enocilis ,11-VO( 17 m X 0 2 mm 1 d , mlif -kcrht
a trohs emit oT eht reffub noitulos 50 ~1 fo ness 0 11 mp umuloC B was drakcaP-ttelweH
esadmoroculg-P morf E cob ,21K( ,regmrheoB 1-PH desuf ,achls deknil-ssorc enoc&slyhtem
)nuehnnaM era dedda and retfa 1 h at 50°C eht ,0-VO( 17 m x 0 25 mm I d , mlif ssenkciht 0 33
reffub noitulos 1s edam emlakla Hp( )01-9 htiw mp nmuloC C was kcapmorhC TOCW desuf
250 ~1 fo %5 mmssatop etanobrac noitulos and achls LIS-PC ,BC8 dekml-ssorc %5 -lynehp
eht sdiorets era detcartxe htm 5 lm fo lyhteld eno&slyhtem ,)45-ES( 17 m x 0 25 mm 1 d , mlif
rehte no a orblv rexim rof 30 s retfA -agitrtnec ssenkciht 0 25 mp nmuloC D was -yerehcaM
nolt eht rehte reyal 1s derrefsnart and detaropave legaN ,dnobamreP desuf ,acilis dekml-ssorc %5
ot ssenyrd rednu muucav enocldslyhtem-lynehp ,145-ES( 17 m x 0 25 mm
1 d, mlif ssenkciht 025 mp nmuloC E was
Denvatlzatwn for GC-MS analyszs drakcaP-ttelweH ,5-PH desuf ,acllls %5 -lynehp
detaguJnocnU fractzon ehT yrd eudiser SI enoc&lyhtem ,)45-E@ 17 m X 0 2 mm 1 d , mlif
dezltavlred htiw 50 ~1 fo -lyhtem&-N-lyhtem-N sseukclht 0 33 mp
elozadum-edlmatecaoroulffrtlyds )unI-AFTSM(
001( 2, )w/v and detaeh rof 30 mm at 80°C 19[ usehtnyS fo reference substances (general -ed
detaguJnoC fractwn ehT yrd eudiser is -avired scnptwn)
dezLt htiw 100 ~1 fo mmnomma-AFTSM nwtad& fo secondary hydroxy groups mth
lotnhtyreolhtld-edidoI 0001( 2 4, )w/w/v and chromrum trwxuk ehT ecnatsbus hcihw was ot eb
detaeh rof 15 mm at 60°C sihT noitcaer erutrum dezldlxo was devlossid m %69 citeca dica and
1s tnelavmqe ot a erutxun fo -lyhtemnt-AFTSM htiw gmrrlts a noitulos fo %01 mmmorhc edlxolrt
enallsodol )SIMT( 0001( 2, )v/v 161[ m %69 citeca dica was dedda mhtw 10 mm nI
lareneg an ralonuuqe noitidda fo mmmorhc -xolrt
CC-MS parameters edl was ,tneiciffus gmdlely evltatltnauq noltadlxo
detaguJnocnU fractwn A drakcaP-ttelweH PH retfA 30 mm fo noitcaer eht noitcaer erutxmr was
5890 gas hpargotamorhc and an PH 5970 mass dezhartuen and neht edam emlakla Hp( )41-21
retemortceps erew .desu ehT remat gas was -eh wth a delooc suoeuqa noitulos fo %02 mmssatop
muh at 1 mm/lm at 18O”C, sphttmg ortar 1 10 edixordyh ehT noitcaer stcudorp erew detcartxe
W Schanzer ad M Dontkc /Ad Chm Acta 275 (1993) 23-48
htrw a dlofnet emulov fo lyhtetd rehte ehT rehte devlossid m lonahte ro .lonaporpos~ ehT -onahte
reyal saw dehsaw htrw ,retaw detrd revo mmdos ch noitulos saw desopxe ot thghnus elrhw gmruts
etahplus dna detaropave ot ssenyrd rednu -cav rof tuoba 4-8 h ehT lone-3-nerd-5,3-atsordna
dna lone-3-nerrt-5,3,1-atsordna srehte-SMT erew
UUIU
Reductwn fo eht elbuod-5,4 dnob nortcudeR fo dexrdrxootua rednu eseht snortrdnoc at C-6, -dlery
eht elbuod-5,4 dnob m ene-4-otek-3 sdiorets saw gm a eno-3-ne-4-tsordnayxordyh-3/6 ro -yh-3/6
demrofrep httw negordyh gmsu lonahtem ro eno-3-nerd-4,1-atsordnayxord erutcurts gmruD
6-lonahtem M mmssatop ro muidos edtxordyh rehtruf elpmas nortaraperp gmmamer SMT
(10 1, >v/v as tnevlos dna mumtalp edtxotd ro ,spuorg e g , ,SMT-0-3/71 erew desylordyh A -ed
10% mmdallap no laocrahc as tsylatac deltat nortprrcsed fo stht sesehtnys st m -araperp
Reductwn fo o&k-3 groups wzth nuuhtb -ula .noit
muuum hydrzde nortcudeR htiw mmhth -ula
munum edirdyh saw demrofrep 111 etulosba yrd
lyhtetd rehte ro m narufordyhartet htob( ylhserf
STaJUSER DNA NOISSUCSID
dellrtstd revo mmclac )edrrdyh gmdneped no eht
yttlrbulos fo eht ecnatsbus hcihw saw ot eb re-
Syntheszs fo reference substances
decud ehT noitcaer erutxtm saw dleh rednu ar-
A lareneg nortprrcsed fo eht netfo desu reac-
nog dna elthw gmrrtts dilos mmhth mmmmula
noit semehcs rof eht sisehtnys fo chobana diorets
edirdyh saw dedda (1 ralonnuqe )ssecxe retfA a
sethobatem lliw eb debrrcsed ehT sesehtnys fo
rehtruf 15 mm eht noitcaer erlctxmr saw deruop
enonedlob [ 121, detagumoc enonetdnatem [13],
otm eht emas emulov fo retaw dna eht noitcaer
lolozonats [14] sethobatem dna nortaxtrempe-71
stcudorp erew detcartxe htrw a dlofnet emulov fo
fo yxordyh&71-lyhtem-a71 sdrorets ]U[ evah al-
lyhterd rehte ehT rehte reyal saw dehsaw htrw
ydaer neeb dehshbup ehT sisehtnys fo rehto
,retaw derrd revo mmdos etahplus dna neht -pave
sethobatem llrw eb dehshbup .erehwesle
detaro ot ssenyrd rednu muucav
llA eht dezisehtnys sethobatem erew dematbo
Reductwn fo otek-3 groups wrth K-Select&e
as erup slatsyrc httw a dlety neewteb 60 dna 2000
ehT nortcaer dna elpmas nortaraperp erew -rep
gm
demrof m eht emas yaw as eht noitcaer htrw
mmhth mmmnuha .edrrdyh ehT edrrtkeleS-K saw
dedda m na ralonnuqe tnuoma ot eht derrtts nwtaiu& fo secondary hydmxy groups huw
nortulos dleh rednu nogra mhttw 10 mm chnmuum trwxuie
A evrtceles nortadrxo fo ylno eht yxordyh-3
Reductwn fo o&k-3 groups httw hydrogen ehT
noitcuder fo otek-3 spuorg htrw negordyh gmsu puorg ro wordyh&71 puorg nehw htob yxordyh
munitalp edtxotd ro 10% mmdallap no laocrahc spuorg erew tneserp saw ton elbtssop sA all
dexrsehtnys sethobatem evah a -otek-71 ro -yh-71
saw demrofrep m eht emas yaw as eht noitcuder
fo eht elbuod-5,4 ,dnob tub eht noitcaer saw lyhtem-71-yxord ,puorg srht cIttcepsnu nortadrxo
degnolorp ot 3 syad saw nesohc dewollof yb a evitceles-puorg -cuder
nort fo eht otek-3 puorg ylno
6/3-Hydroxylatwn. crfrcepsoeretS -lyxordyh-3/6
norta fo seno-3ne4-tsordna dna -rdC,l-atsordna
seno-3ene saw demrofrep httw eht gmwollof -orp Reductwn fo eht elbuodP,4 dnob
erudec ehT ecnatsbus saw devlossid m lyhte ac- ehT nottcuder demrofrep 111 lonahtem dedlery
mumomma-AFTSM-etate edidoi (100 10 0 5, m lareneg a erutxrm fo 5a- dna sremosi-?jS m
)w/v/v dna dexulfer rof 15 mm ehT dematbo elbarapmoc stnuoma saerehw eht noitcaer m
lone-3-nerd-5,3-atsordna dna -5,3,1-atsordna suoeuqa-lonahtem mmssatop edrxordyh ro -rdos
lone-3nerrt srehte-SMT erew detcartxe htiw -n nru edrxordyh sruovaf eht nortcudorp fo eht -p5
enatnep retfa noitidda fo mmssatop etanobrac remosi oT matbo a remosi-rcS lonahtem saw -ohc
dna retaw ot eht noitcaer erutxrm ehT enatnep-n nes as ,tnevlos gmdlery dna ,sremosi-3/S hcrhw
5a-
esahp saw detaropave ot ssenyrd dna eht eudtser erew neht detarapes yb nmuloc yhpargotamorhc
W Schanzera nd M Do&e/Ad Chim. Acta 275 (1993) 23-48 27
no s&a leg dewollof yb detaeper noltazlllatsyrc SI ,tneserp e g , m ,enold-71,3-enartse-a5 htob
ehT ylnommoc desu hcriB noitcaer hcihw sdleiy otek spuorg lliw tcaer yletaldenum wth eht
ylmam eht remosI-aS was ton dehppa tnegaer ehT nortcuder SI cifcepsoerets ot otek-3
spuorg hcihw lit eb decuder ylmam ot -yh&3
Reductwn fo otek-3 groups wrth nuuhth -ula yxord elbaT( )1 nehw eht gmr-A has a 5a ro
muuum hydnde ene-4 noltarugifnoc tub eht otek-3 puorg ~11 eb
noitcudeR wth mmhth mmmmula edndyh 1s decuder m revo %08 dleiy ot yxordyh-a3 nehw
ton puorg evitceles fI erom than eno otek puorg eht gnir-A has a 3/S noitarugifnoc elbaT( )1
ELBAT 1
noitcudeR sdleiy fo otek-3 spuorg htnu 4WAL dna edntkeleS-K
ecnatsbuS ,m edntkeleS-K
3a-OH 3&OH 3a-OH 3/3-OH
)%( b )%( b (46) b ($6) b
Sa-ConfZguratum
eno-3&?/71-natsordnA-aS 12 88 88 12
enotd-71,3-enatsordnA-aS 11 89 93 7
enold-71,3-enartsE-aS a 13 87 93 7
enold71,3_enatsordna-a5-enelyhteM-1 ’ 7 93 95 5
eno-3-lo&71-natsordna-rc5-lyhteM-al 49 51 >99 <l
enold-71,3-enatsordna-aS-lyhteM-al ’ 49 51 >99 <l
eno-3-lo-~7l-natsordna-a5-lyhteM-a2 13 87 > 99 <l
enold-71,3-enatsordna-zAlyhteM-rc2 a 8 92 99 1
eno-3-lo-3/7l-natsordna-a5-lyhteM-a71 11 89 88 12
eno-3-lo-a7l-natsordna-a5-lyhteM-3/71 ’ 11 89 93 7
eno-3-l+p71-nartse-a5-lyhtE-a71 B 14 86 93 7
eno-3-lop7l-natsordna-aS-lyhtennD-a7l,a7 ’ 10 90 87 13
nwtaul&noC-3/s
eno-3-1o-p71-natsordnA-pS 89 11 96
enotdFl,3-enartsE-ifS ’ 90 10 94
eno-3-lo-3/7l-natsordna-~5-lyhteM-a71 87 13 91
eno-3-lo-a7l-natsordna-~5-lyhteM-~71 ’ 88 12 94
eno-3-lop71-nartse-3jS-lyhtE-a71 ’ 90 10 95
eno-3-lo-3/7l-natsordna-3/S-lyhtenuD-a7l,a7 ’ 80 20 96
noltmugFnoc-eneu&jnoc-ene&-tqi?.&+,-a5
eno-3-io-~7l-n.e-l-tsordna-a5-lyhteM-l 9 91 10 90
enold-71,3-ene-1-tsordna-a5-lyhteM-1 ’ 10 90 10 90
mkta~noc-eno-3-nee-tsvtdnA
eno-3-lo-3/71-ne-4-tsordnA 13 87 30 70
eno-3-1o-a71-ne-4-tsordnA 14 86 38 62
eno-3-io-p71-ne-4-rtsE 19 81 42 58
eno-3-lop71-ne_4-tsordnalyhteM-a71 10 90 28 72
eno-3-lo-a71-ne-4-tsordnalyhteM&71 a 14 86 36 64
eno-3-lo~d-p71&6-ene-4-tsordnA 14 86 15 85
cnold-71,3-lo-3/6-ne-4-tsordnA 19 81 16 84
eno-3-lo~d-I#7l,~ll-ene-4tsordnalyhtem-a71-orou~-u9 28 72 90 10
eno-3-1ont-~71,3/11,3/6-ene4tsordna1yhtem-a71-orouiF-a9 ’ 27 73 72 28
eno-3-lo&7l-ne4tsordnaorolhC-4 11 89 14 86
eno&71,3-ene4tsordnaorolhC-4 ’ 13 87 19 81
’ dezisehtnyS ecnerefer ecnatsbus b egatnecreP seulav erew detaluclac morf DIF-CG sisylana retfa noitcuder ees( )latnenurepxE
fo 3 gm fo hcae ecnerefer dnuopmoc
“free”
and
c Sd
urme EIJU@@ Free”
Excretron m ConJugated ConJugated ConJugated “Free” ~&nJugated “Free” “ ConJugated cosrusa~ Comugated “Free” “Free” CouJugated ConJugated Comugated fzoqagated Comugated Conprgated co4sueated ckmJug&ted coslwmted Cornugated ‘“Free” “Free” CwJuBated CouJugated Comugated ConJugated “Free” Ck.mJugated ConJugated
b
d un~nJugat~
used study study ue =
s
of ex ex he
confirmatton yut
S “Free”
Ollgm substance for Synthesued Parent Synthesued S~thes~d Synthesued Syuthesmed Syuthesrxed Syuthesmed Unne Synthesmed Synthestzed Syuthesmed Synthesmed Synthesmed S~thes~ Synthestxed Parent Synthesmed Synthesrxed Synthestxed Synthesmed Synthesmed Synthesmed Parent Synthesrxed Parent Synthesixed Unne Synthestxed Synthesrxed Synthesmed c
study
excretron
an
(XVI
BWD
@I) (X) (XI) (XIV) (X) (XIV) (X) from
(VI
1992) &iD (XX)
(III)
(TX) (XXVI obtamed
March a (XIII)
1st (XII) (XVHD (XIX) CCXIW OCXW fXXVH)
OfXU~
wll~ metabohte
substance metabohte Q WUII~ =
WI) (XXI)
Laboratory, (II) study
excreted and/or ex
Cologne testosterone androsta-4,13-dlen-l~~~l-3~ne 11~,17/3-tetrol drene-lla,l7~-dml-3-one 3z,z,l7j%trtoJ
Mam parent 7a,l7a-Dnnethyl-5/3-androstane-3a,l7~-dtol(1) Boldenone 5/3-Androst-l-en-17El_ol3-one 6~-Hydro~-4~hlor~deby~ometbyl- 4-~lofo-androst-4~en-3a~I-l7_one 2a-MethyI-5a-androstan-3a-ol-17-one 9a-~uo~l~nor-l7,17-d~ethyl- 9a-Fluoro-l7a-methyl-aadrost-4-ene-3a,6~, 2-Hy~~ethyl-l7a-me~l-andr~ta-l,4- 16z-Hydro~r~a~l 17a-Methyl-!5a-androstane-3a,t7~-~ol la-Methyl-Sa-androstan-3a-ol-17-one 17-Epunetandrenone 6~-Hydro~et~dienone 17a-Methyl-5~-sndrostane-3a,l7~-drol(XIV) 17~-Methyl-S~-~~t-l-ene-3a,l7a~tol 17a-Methyl-5#?-androstane-3a,l7~-drol Metenolone I-Methylene-5a-androstan-3a-al-17one 17a-Metbyl-5a-audrostane-3a,l7~-dtol 17a-Methyl-5jJ-androstane-3a,17/3-drol Sa-E&run-3a-o1-17-one SjJ-Estran-3a-oL17-one 17a-~t~l-5~~s~ne-3a,l7~-dlol Oxundroione 17-Eptoxandrolone Oxymesterone 17a-Meth~-Sa-~~~e-3a,l7~-dlol 2z-Hydroxymethyl-17a-methyl-5a-androstane- 3’-Hydroxystanoxolol 3‘-Hydr~-l7~p~~~iol 4B-Hydroxystanozoiol 16&Hydroxystauozolol Urme
b
(Status
rdenhfied
sterotds
not
2 for anabohc steroid sterone drolone ~nfi~rat~n
Eola methyltestosterone Nan z =
TAFJLE Screenmg Auabobc Boldenone cl-Cblorodehydro- ClosteboJ Drostsnolone ~uo~terone Formebolone Ftlrazabol Mestanolone Mesterolone Metandrenone Metlumdrtol Metenolone Nore~~drolone Oxandroione Oxymesterone Oxymetholone a
W Schanzer and M Lbuke/Anal Chm~. Acta 275 (1993) 2348 29
Reductwn fo otek-3 groups htiw K-Sekctnde nolt elbaT( )1 and ti SI decuder m erom than %58
ehT use fo edntceleS-K ]81,71[ daetsni fo dleiy ot yxordyh-3/3 nehw eht gmr-A has a 3/5
mmhth mmmmula edndyh shows puorg -vrtceles noitarugifnoc elbaT( 11, m htob secnatsm eht
,ytl e g , eht otek-3 puorg m -ld-71,3-enartse-a5 noitcaer sdleiy eht etisoppo noitarugifnoc ot -er
eno lllrH eb decuder tsrif and neht htnH ssecxe fo noitcud htm mmhth nunmmula edndyh nehW a
tnegaer eht otek-71 puorg stcaer ,revoeroM eht ene-4-otek-3 noitarugifnoc SI decuder htm -K
noitcuder fo otek-3 spuorg 1s ,evltcelesoerets tub edntkeleS eht otek-3 puorg stcaer ylmam ot evig
tnereffid ot mmhth mmmmula edndyh noitcuder wordyh-I/3 ylralnms ot eht noitcaer wth mmhth
ehT otek-3 puorg stcaer m revo %58 dleiy ot ev@ nunmmula edndyh elbaT( 11, tub eht dleiy fo eht
yxordyh-a3 nehw eht gnir-A has a 5a -aruqfnoc remosi-yc3 SI hcum rehgih than m eht noitcuder
ELBAT 3
erutarepmeT emmargorp stiivoK secldm fo chobana sdiorets dna ueht sethobatem ’
dioretS llmUl0C
A B C D E
1-vo 1-vo 45-ES 45-ES 45-ES
mpllO 033pm 025pm 025jkm 033pm
eno-71-1o-a3-nar&E-aS SMT-nb )IIIV@ 2440 2453 2451 2454 2468
eno-3-lo-3/71-ne-1-tsordnA-3/S SMT:Bb <III> 2452 2467 2469 2471 8842
loid-a7l,a3-ene-l-tsordna&5-lyhteM-3/71 SMT-slb )vx( 2454 2468 2472 2474 2492
e-71-10-a3-nartsE3/5 SMT-slb )XIX( 2490 2505 2502 2504 2520
eno-71-la-a3-natsordna-aS-lyhteM-a2 SMT-slb )IW 2555 2574 2563 2565 5686
eno-71-lo-a3-natsordna-a5-nelyhteM-1 SMT-slb )IIVX( 2583 2604 2.599 2602 2623
-lyhtemld-71,71-ron-81-oroulF-a9
eno-3-lo-3/11-neld-31,4 SMT-slb )IIV( 2600 2621 2619 2621 2641
loid-3/7l,a3-ene-l-tsordna-~5-lyhteM-a71 SMT-slb )VX-lpe( 2607 2628 2626 2630 2649
eno-71-lo-a3-natsordna-a5-lyhteM-al SMT-slb )IX( 2607 2630 2619 2623 2644
lold-3/7l,a3-enatsordna-a5-lyhteM-a71 SMT-sb 1X( 2611 2632 2625 2628 2650
loid-~7l,a3-enatsordna-I#5-lyhteM-a71 SMT-slb )VIX( 2617 2636 2628 2631 2654
enoneldnatenupE-71 TMS )IIX( 2625 2657 2674 2680 2713
enonedloB SMT-slb (II) 2648 2671 2671 2674 2698
enolordnaxolpE-71 TINS )IIXX( 2673 2707 2735 2741 2777
lold3/7l,a3-enatsordna-3/S-lyhtemlD-a7l,a7 SMT-slb (1) 2692 2713 2706 2710 2730
eno-71-lo-Yc3-ne4-tsordnaorolhC-4 bu+TMS 0 2693 2712 2720 2724 2746
enoloneteM SMT-slb )IVX( 2694 2718 2716 2721 2743
lold-3/7l,a3-enartse-3/5-lyhtE-a71 SMT-slb )XX( 2695 2715 2710 2714 2733
enoretsotsetlyhteM SMT-slb lanretnI( )dradnats 2754 2778 2775 2779 2803
enolordnaxO TMS )IXX( 2778 2816 2845 2851 2893
enoneldnatemyxordyH&6 SMT-srb )IIIX( 2846 2873 2877 2882 2911
,3/6,a3-ene-4-tsordnalyhtem-a7l-oroulF-a9
lortet-3/71&11 SMT-artet )IIIV( 2854 2855 2855 2854 2860
enoretsemyxO SMT-snt )IIIXX( 2952 2977 2968 2972 2993
-lyhtemordyhedorolhc-4-yxordyH-3/6
enoretsotset SMT-slb )VI( 3007 3039 3044 3048 3081
lolozonatslpe-71-yxordyH-’3 SMT-snt VXX( 3100 3113 3119 3122 3137
-4,1-atsordna-lyhtem-a71-lyhtemyxordyH-z2
eno-3-lold-@71,-all-eneld 9.MT-snt )XI( 3163 3187 3203 3206 3232
lolozonatsyxordyH-’3 SMT-snt )VIXX( 3219 3238 3242 3245 32456
lolozonatsyxordyH&4 SMT-snt )IVXX( 3219 3245 3244 b 3277
lolozonatsyxordyH-a4 SMT-snt lametm( )dradnats 3238 3262 3268 b 32%
lolozonatsyxordyH&61 SMT-snt )IIVXX( 3334 3360 3368 3402 3397
a secidnI erew demmreted no snmuloc E-A ees( )latnemnepxZJ erutarepmeT p emmargor morf 180°C at mm/C”S ot 324FC
b ecnatsbuS dewohs gnorts gnhtat
03 W Schanzer and M Dontke/AnaL Chun Acta 275 (1993) 23-48
htiw mmhth mumumha ,edirdyh m ,ralucitrap elpnus dohtem rof eht sisehtnys fo -natempe-71
enoretsemyxoulf and enoretsemyxoulfordyh@6 enoneld yehT devlossid &s-71-enoneldnatem
tcaer ot ewg eht yxordyh-a3 sremosi htiw 90 and etahp m retaw ehT etahplus desopmoced m wa-
%27 sdleiy elbaT( )1 ,ret gmdlely lareves noitardyhed stcudorp and
enoneldnatenupe-71 nI this ,krow setahplus-71
Reabctwn fo otek-3 groups htiw hydrogen fo yxordyh@71-lyhtem-a71 sdiorets erew -ehtnys
ehT citylatac noitcuder fo otek-3 spuorg was dezis usmg sulphur emdlryp-ednont xelpmoc m
puorg ewtceles esuaceb eht otek-3 puorg stcaer ,edmramroflyhtenud and neht devlossid m ,retaw
hcum retsaf than eht otek-71 puorg A -esoerets gmdlely eht sremtpe-71
ewtcel noitcuder gmdlely yllaitnereferp a -a3 ro
yxordyh-3/3 nottarugifnoc was ton deniatbo Screerwg of cilobana stemds
ylemtuoR 20 chobana sdiorets erew dellortnoc
6/3-Hydroxyiatwn yb a gmneercs dohtem rof 29 sdiorets 4( tnerap
noltalyxordyH&6 fo sdiorets was tsrif -ed sdiorets and 25 ,sethobatem ees elbaT 21, 27 fo
debltcs rof l~troc m 1954 yb metsruB te al ]91[ hcihw )IIVXX-I( erew elbaluiva as erup ecnerefer
and 111 1956 yb ledaN te al ]02[ rof losltroc nI secnatsbus 4( tnerap sdnuopmoc and 23 -ehtnys
1967 Card1 and manglsuL ]12[ dehshbup a yrev dens )sethobatem ow‘? sethobatem lobazaruf(
elpmis erudecorp rof eht snehtnys fo yxordyh&6 and )enolohtemyxo erew dematbo morf yramru
sdiorets yehT detrevnoc seno-3-ene-4-tsordna noitercxe seiduts elbaT 2 shows rehtruf hcihw
otm rieht gmdnopserroc eneld-5,3lykla-n lone chobana sdiorets and sethobatem era detercxe
,srehte yllmcepse lyhte lone ,srehte and desopxe detagujnocnu ro detaguqoc ehT sisylordyh fo
,meht devlit m ,lonahte ot tcerid sunlight ehT setagujnoc was demrofrep htiw esadmoruculg-B
noltadlxootua dedleiy -3-ne-4-tsordnayxordyh-3/6 morf E co6 ehT gnineercs samples erew anal-
eno sdiorets m hgih dleiy WOT noitcaer was -ohc desy yb SM-CG ees( )latnenurepxE htiw detceles
nes ereh ot matbo yxordyh&6 sevltavlred fo 1011 gmrotmom )MIS( usmg owt ro eerht fo eht
,enoretsotset ,enoneldnatem -ordyhedorolhc-4 tsom abundant snoi rof hcae ecnatsbus
enoretsotsetlyhtem and enoretsemyxoulf As eht
noitamrof fo lone-3 lykla srehte fo -lyhtem-rc71 Kova’ts mndaces (methylene warts)
yxordyh&71 sdiorets yb ylnommoc debircsed demmargorp-erutarepmeT sthvoK secidni
sdohtem del ot noitardyhed fo eht cidica elbal enelyhtem( )stinu erew denimreted rof eht
yxordyh-3/71 ,puorg lyllslyhtennrt lone srehte erew detalyllslyhtenurt chobana sdiorets and -ehtnys
desu and eseht snoitcaer erew ylisae demrofrep dezis sethobatem ot eb deneercs elbaT( )3 A
usmg mumomma-AFTSM edidoi ehT -atsordna nmuloc erutarepmet emmargorp morf 180°C at
lone-3-neld-5,3 and lone-3-nent-5,3,1-atsordna mm/C”S ot 320°C was desu s~lT emmargorp was
srehte-SMT erew elbats m lonahte and erew -ua nesohc ot enimreted all eht sdiorets mhtw a
dezrdlxoot nehw desopxe ot sunhght, gnidleiy mm-52 emmargorp eviF tnereitd snmuloc erew
eno-3-ne-4-tsordna-yxordyh-3/6 and -yxordyh-3/6 ,detset yleman 1-VO and 45-ES htiw tnereffid
eno-3neld-4,1-atsordna sdiorets enoretsotseT mlif sessenkciht and morf tnereitd seinapmoc
was desu as a ecnerefer and was .dezldlxootua ot llA eht snmuloc had a htgnel fo 17 m ehT
,enoretsotsetyxordyh@6 hchw was demrifnoc us- chobana sdiorets and neht sethobatem erew -ed
gm citnehtua ecnerefer ecnatsbus denimret as SMT sevrtavrred eht( emas -avned
sevlt as desu ni enituor ,)lortnoc eht detaguJnoC
nwtaznenupE-71 fo 17mnethyl-17/3-hydroxy detercxe sdiorets and enoretsemyxoulf -obatem
sterords seth erew detalydslyhtenurt ot SMT-rep -avned
srenupE-71 erew deasehtnys rof eht tsrif emit sevlt saerehw eht detagujnocnu sdiorets erew
yb dlanodcaM te al ]5[ ni 1971, gmmrifnoc -71 dezltavlred htiw ,unI-AFTSM hcihw seod ton
enoneldnatenupe as a ethobatem fo -natem ezltavrred seno-3eneld-4,1-atsordna ot lone SMT
enonerd dnuldE te al 1221 m 1989 dehshbup a sevltavrred
W Schanzer and M Lkxuke/AnaL Chun Acta 275 (1993) 23-48 31
ehT stluser rof nmuloc B (OV-1, 0 33 )mp and
nmuloc C ,45-ES( 0 25 )mp wohs rof tsomla all
eht sdiorets tsomla lacitnedI secldm secnereitd(
less than 10 enelyhtem )stmu enolordnaxO )IXX(
and enolordnaxolpe )IIXX( htob wohs a -reitd
ecne fo tuoba 30 enelyhtem units and eht
&oi&oH
ethobatem fo enolobemrof a ecnereitd fo 16
enelyhtem umts
Mass spectrometry
llA chobana sdiorets and rieht sethobatem
erew desylana as SMT sevifavned yb mass -ceps
yrtemort htiw IE nortazlnol at 70 Ve lareneG IE H H
noitatnemgarf snrettap erew deretsrger rof -uc71 Rg 1 Metabohsm of methyltestosterone (1) to 17a-methyl-
yxordyh@71-lyhtem sdiorets and rieht sremlpe-71 S~-androstane-3a,l7&dlol (X) and 17rr-methyl-Sp-andros-
tane-3a,l7&dlol (XIV)
hcihw wohs as SMT sevltavrred an abundant -D
gmr tnemgarf at z/m 143 and htlrH less ytlsnetm
an noi at m/z 130 ]32,5[ and ffolageS m 1962 ,16[ gnlyfttnedl -lyhtem-a71
detalyxordyH-61 yxordyh-3/71-lyhtem-a71 -ets lold_B7l,uc3-enatsordna-aS )X( and -lyhtem-a71
sdior yalpsid eht gmdnopserroc tnemgarf snoi lold_B7l,a3-enatsordna-3/5 )VIX( as eht mam
(+ 88 )u nehw yeht era ,detalyllslyhtennrt at m/z sethobatem GF( 0, a sun&u gmr-A mshobatem
231 and 218, ylevitcepser ot that rof enoretsotset .]62[
oteK-71 sdiorets wohs a lacipyt gnir-D/C -garf derapmoC htlrH eht mshobatem fo -sotset
tnem at z/m 169 nehw eht otek-71 puorg 1s ,enoret eht oitar fo eht yramru detercxe -a71
detalyllslyhtenurt ot an lone SMT evitavrred ehT lyhtem sremosl&5/a5 1s tnerelfd ot eht oltar fo
tnemgarf at m/z 169 SI IIQS demalpxenu and eht enoretsordna ot enolonalohcmte ,@5/a5( eht
ylno noltamrofm 1s that noltaretued at C-16 evag mam sethobatem fo enoretsotset noitercxE stud-
a tnemgarf at z/m 170 stluser( ton )nwohs sei htlru a elam reetnulov dewohs a 5a/5/3
)XIX/X( oitar fo 1 6 4 retfa laro noltachppa fo
Metabohsm fo anabol~c sterordr dna synthesu fo 100 gm fo enoretsotset 06-O( h retfa )noltachppa
metabohtes and a oitar fo 1 5 1 nehw 10 gm fo -sotsetlyhtem
ehT mshobatem fo 20 chobana sdiorets retfa enoret erew deilppa yllaro ehT emas tcejbus
laro noitacilppa was detagitsevni elbaT( )2 roF dewohs m htob noitercxe seiduts an -sordna
eht gmwollof nolssucsld eht chobana sdiorets erew enolonalohcmte/enoret oltar fo 1 1
1 *
dedlvld gmdrocca ot rieht erutcurts otm evif htoB X and VIX erew dezisehtnys yb a -caer
sessalc ,seno=3neC-tsordna -nerd&l-atsordna noit fo eht drangnG tnegaer mmsenganhyhtem
,seno-3 ,seno-3-natsordna-aS senatsordna-aS htrw edrdm htlru eno-71-1o-Yc3-natsordna-YcS -sordna(
laiceps erutcurts and londnahtem )enoret and eno&71-lo-a3-natsordna-@5 -olte(
roF eht detagltsevm chobana sdiorets a feirb )enolonalohc ehT otek-71 puorg stcaer 111 tuoba
wesrevo fo eht mam detercxe sethobatem and %59 dleiy ot evig a yxordyh&71-lyhtem-a71
ueht sisehtnys SI detneserp erutcurts
ehT IE mass murtceps fo X SMT-sb 1s nwohs
Androst-4-en-3-on4zs III giF 2 and that fo VIX SMT-slb m giF 3 htoB
Methyltestosterone enoretsotsetlyhteM -u71( sevltavrred wohs an abundant gmr-D tnemgarf at
)eno-3-lo_B71-ne-4-tsordnalyhtem was tsrif -nys m/z 143 htrw %001 evitaler ytlsnetm esab( )kaep
dezlseht 539lm yb akelzuR te al 1241 and m 1937 enoddnaVi enolordnaN -3&o&71-ne-4-rtse(
yb reuahneppO 152E ehT mshobatem fo -lyhtem )eno was tsrif dezisehtnys m 1950 yb hcuB ]72[
enoretsotset m man was detagitsevm yb enognoR and yb W&is and nosleN m 1953 ]82[ ehT
32 W Schanzer and M Lhmke /Ad Chm Acta 275 11993) 23-48
Pd
OH
1
&
O-TM
CHa
80' ’
0
no&Ho&
341
B so- 0+!MT
6
H
-2
H H
Fig 4 Metabohsm of emkordnan (1) to -71-la-a3-nartse-aS
one )IIIVX( dna Sp-estran-3a-oL17-one (XIX)
00I
200 300 400
egrahc/ssaM mmmorhc edlxont ot dleiy enoid-71,3-ene+rtse
Fig 2 EI ssam spectrum of -enatsmdna-aS-lyhtem-a71
tneuqesbuS noitcuder htiw negordyh m lonahtem
3a,17/3-d101 bls-TMS (XI, ralucelom noi ta z/m 450
gnisu mumtalp edixoid as tsylatac dedleiy 35% fo
enoid-71,3-enartse-yl5 dna 65% fo -enartse-3/S
msilobatem saw detagitsevm yb legnE te al m enoid-71,3 htoB sremosi erew detarapes yb re-
1958 [71, ohw detalosi owt sethobatem -nartse-aS detaep nortazhlatsyrc dna dluoc eb ylisae -mtsrd
eno-71-lo=a3 )IIIVX( dna eno-71-lo-a3-nartse-~5 dehsutg yb rieht gmtlem ,stmop hcihw are 73-
(KIK) giF( 4) htoB sethobatem erew dezisehtnys C°57 rof enoenold-71,3-enartse-a5 [30] dna 179-
m 1960 yb refpuK te al [291 dna m 1961 yb C°181 rof d-71,3-enartse-3/5 [31] noitcudeR
IIesnuoC [301 fo enoid-71,3-enatsordna-a5 htiw edrrtkeleS-K
htoB sethobatem erew dexisehtnys ereh start- dedleiy IIIVX m tuoba 93% dleiy elbaT( 1) -eR
gm htiw enoretsotsetron hcihw saw dezldrxo htiw noitcud fo enold-71,3-enartse-I/5 htiw negordyh
gnisu mumtalp edixoid as tsylatac dedleiy tuoba
64% fo KIK dna 36% fo eht ,remos@S saerehw
eht noitcuder htiw edirtkeleS-K dedleiy KIK 111
143 tuoba 6% dna eht remosi-3/3 m 94% dleiy elbaT(
/ /
& 1)
0-TMS
ehT IE ssam murtceps fo IIIVK SMT-sih si
c&l
nwohs m giF 5 dna taht fo KIK SMT-sih ni grF
up 6
3& enolordnahteroN -lyhte-a71(
Norethundrohe
0-?!MT
H )eno-3-lo-3/71-ne-4-rtse saw tsrif dezisehtnys m
\ 1957 yb notloC te Ia [32]
loid-3/7l,a3-enartse-a5-lyhtE-a71 dna 17a-
loid&7l,a3-enartse-3/5-lyhte 0IX( erew detroper
as mam sethobatem yb skoorB te al 181 m 1971
nehW na noitercxe yduts saw edam fo
,enolordnahteron ylno XX dluoc eb deifitnedi dna
on remosi-aS fo KX saw detceted giF( 7) htoB
300 004 sremosi erew dezisehtnys dna are llew detarapes
egrahc/%h
m CG as SMT-sih sevitavired ecnereffid[ fo 40
Fig 3 EI ssam spectrum of -enatsordna-BS-lyhtem-a71
3a,17/3dlol bls-TMS 0, ralucelom ton ta z/m 450 enelyhtem stmu no na 1-VO yu&pac nmuloc