Department of CIinical Chemistry, Division of Ctinich Pharmacology Glostmp Huspita& DK-2600 Glosfnrp (Denmark) and Clinicat Pfrarmacoiogy Research Unit. Danish State Medic& Research Council. Copenhagen (Denmark) (Received April 6th. 1976)
A method for the determination of ethotoin snd its p-hydroxylated and dealkyhted metabolites in urine has been developed_ Etbotoin end the metabolites were extrected fkom acidified urine with ethyl acetate and silytated before injection inta a combined gzs Aromatogreph--mass spectromekx Four psrtly ic?entified metabolites were recorded, brrf their exad qua&if&ion wes not gmsihle as pure reference substances were not avzihkble. The limit of sasitioity wzs Ear below the amounts of ethotoin and of its metabolites found in tine from patients treated with therqeutic doses of ethotoin_
Recentiy, group
the a&epileptic drug etkotok-3 (Pegsnone) was shown by our dose-dependent kinetics [I]. The purpose of the preset in-
fro exkibit
pws to develop 8 method for the quantitativedetemi?zeion of echotiin and its metialites ezMed in urine in order to elari& the meckanism bekkxdtke dose-dependent kineticsof ef&oto~b_
ues@&iczn
-
(Rockford, XL, U.S.A.) was of specially purified grade. GiucurouidaselarylsuIpb.atasewas obtaiued from Eoehringer (Mannhelm, G.F.R.). Reference substances
The structures of etbotii and its metioli&s are shown in Fig-l. E&atoin and its dealkylzted met&o&e (5phenylhydantoin) were don&d by Abbott L&s_ (North Chicago, Ill., U.S.LL). The two internal skndarck, mephenytoin aud heptamd, were donated by sapldoz (Bqsle, Switzerland) and Ciba-Geigy (Bale, Switzerland), respectively. The p-hydroxylated metabolite was folated from human uriie and purified. hEetaholit.esIII, IV, V and VI were exka.&xI from urine and purified, but were not isolated in a pure state.
Metabdite
I\ 0-
H 0N\
,o
S 7’ HI C-N-CHz-CXJ c? Ethotoin __
-
2 -_
-1
Non-enzymatic
Fig.1. Metabdic
\
? f&tatsatite SS
‘C’O I C-U-CW,-CH,
P - hydmxytated eth~toin -.
--.
\
:! 0
,c’
9,
H N
H
\ --a
k?etabatite9
cacliuqated ta-hydmxytated ethotoin
Unidentified mct.&ctites
reaction possibte . pathways
of etho’ain
in rrtan.
Isoiation and ptqification. rMetabolites III, IV, V and VI ware extracted from urine samples with ethyl acetate and cleaned by column chromatography on silica geI with ethyI acetati-hexame (1:X). The uriue remainin g after this extraction was hydroLy& witfr giuCu~~tik/ asylsulpha~. The p-hydroxyl&ed mekholitfs tiesed was exkicted with ethyl acetate and cIe&ed by columm chramak~ptrJr on silica g& with ethyl acetaten-hexane (1 :Z), followed by two reay&Eations from ethyI acetaten&sane (19). The melting point of tie compound and its W spgctrum in ethand, as weII as the acid prop~9533 ax@ tie w SW of tfie eompo~& -I ,-.aad some of i% dezktives, wez determiacxt, ., :. _ _-...
163
Identi~~tion.Theidenti~oftheisola~p-hydroxyEatebethotoLnwasconf&met2 by the fallowing an&ytk.& da*& The main pe&s in the mass spectnrm
were 16 m/e higher thsn the correspondingpeaks in the msss spectrUmof ethatoin, The compound could be s&&&d z&two positions md acetylated at one. The unchanged compound was 8 we& acid, but its ac&yl&ed derivzkivewas neutral. The UV spectrum and the meKing paint were in good agreementw&h those of synthetic 3=ethyl-5-(p-hydroxyphenyl)hydantoin [2]_ No definite proof 9~~8s obtahredthat the hydrrxylation i%.ks place in the pam-position, but p-hydroxylation has been shown to be a major metabolic pathway in DWRfor phenytain [3,4] end other B-phenylhydantoinderivatives[5], the phydroxylated metabolite was found in urine from dogs treakd with ethotoin [2] and m-hydmxylation of phenytoin occurs to a slight extent ES]. The exact struck form&e of metiohtes ?Z& IV end VI have nat yet been proved. The compounds could be formed in small amounts in vitro from ethotoin, deslkylated ethotoin and p-hydroxylsted ethotoin, respectively, in alkaline solution. The mass spectm of the unchanged compounds (TIE,IV and VI) and of their silylates may point to en oxidation in position 5 in the hydantain ring system [Z] . ‘Fhe mass spectra of met&ohte V and of its silylated derivativecould be expkined by the assumption that the metabolite had arisen kom etbotoin in which the phenyl group had been converted into the 3,4_dihydroxy-l,5_cyc]ahexedien-I-yl group. This metnbalic pathway has been shown to be open for phenytoin [7]. F’uithemo~, this vvoerkd be in agreementwith the fact that the metabolite had no acidic properties. No compoundhatigtwophenoliehydrosy~groups,asshownfor phenytoin ES] wzisobserve& Mess spectiameby. A combin& gas chromatogmph--mass spectrometer (LKB 9OQO) was Used. The conditions useci for the maSSspectra were as follows: ion&&ion energy, 70 eV; kap current, 68 PA; sccele=ting voltage, 3500 V. Tables 1 end IE give the 11185s spectra of ethotoin, its met&o&es and the internal stendsrd es unchanged and as silylated compounds, =SpectlVelY. EX~TUC~~UR aRd derimfiusfiun
of
non-cunj~afed
metaimiites.
To
a 50-yl
urine sample in 8 centrifuge tube 1 rnI of 0.1 N acetic acid and 100 ,~l of mephenytoin solution (6.X gfl in ethanol) 8s intern& &nd& were tided. The szunp!e wes mixed with 3 rni of ethyl acetate for 5 min. After cent&Tug&on, the orgsniuphase ws knsferred into a taperedt&e and evapmzbd to dryness in 8 &B%%IIXof nitmgm. The residuewas derivztizd with 50 ~1 of BTSA for 15 znin and diluted with 100 ~1 of toluene. A volume of I ~1 was injectedinto the combined @s chmm&agrziphiness spectrometer. E~frcrcti~ and deh&zatimt of fatal p-izydmxykted metaboiites.To a 50 ~1 urine sample in a centrifuge tube, ZOQ@I 0.1 M s&c acid were added. After the &&ion of 5 &I.of giucmniW/~&&p~, the tube was I& for 18 h at 37”. The suhsewent pmeedure was as descr&ed far the nonconjugnted compounds* If an@ the phyc$roxykM .&&aIite vzxs to be-mexsured, gas chmrnatogmphy v&b affame-i~nizati~zkdeteCtorcotid be used. Tfie pmcedure %vasthen
‘Z&&ES SPEC=l=EtA OF IZFF’EI-EN (My)), XYDROmLATED 2TEmIN @-%-rn)
Fragmeds bdow
DEALFXLATED
AXE) M%TABom
aa3
m/e
ET0
DA-IFlY
p-H-m
(md.*.206)
i;n&ecI?6) _-_---~-_
(moI.wt.220) EL
103 104 100 36 105 119 120 121 10 132 20 133
m, lv
iIzt~~;ti~. _ _.__~.
Metehdite
100 37
m62
Metebolite
Metidite
Iv
V
100 35
89 100 87 32
86 la0
99
18
24 41 25
14s 149 174 176 191 192
29 13 40
26 30 56
i
22
p-
Tixe results
__-
135
204 202 220 238
(DA-SrO), AND v
iC0 m/c -d/or v&h intensifiesbelow 10% have heen omitted.
,gim
&tie
ETHOTO~
EX
59 19
-165
because complete isolation of the compounds was not feasible. The concentrations were estimated by comparison of the areas under the peaks in thigas
c~nm;PtogramsofthemetaboLites~~ehe~ underthepeakinagzschromatqrarn of the dealkylated metabolite of ethotoin (5-pbenylbydantoin). JL flame-ionizationdetector wss used.
Extructi~on and formztion of deriuutives Ethotoin itself could be extracted quan&&ively
with dicfrlo~methane [9]. However, it was necessary to use a more polar extraction medium such as ethyl acetate, as some of the ethotoin mekbolites are rather poh. The fact that
several of the metabolites are weak acids could not be utilized in a cleaning procedureas ethotoin and most of its metabotites a-e unstable in dk&.ne soluti0l.l.
For most of the compounds, the extraction recovery was approximately 100% (only 75% for metabolite V). Silylation of ethotoin, of the internal standard, of p-hydmxylatedethototi and of metabolite III proceeded readily at room temperature, but the dealkyk&ed ethotoin and metabotites IV and V reacted more slowly, especially in low concentrations. This effect might be due to adsorptionof the non-silylated
compounds ORthe glass walk, but a&z 20 min the derivatizationswere found to be aJmostquantitative.
Hyddysis of conjuguteed mefubolites ‘The only conjugated metabolite found in noticeable amounts was p-hydroxylated ethotoin. Enzymatic hydrolysis of conjugated p-hydroxylated ethotoin under the couditions described was found TVbe quantitative (or at Ieastmaximal) after 12-15 h. Acid hydrolysis with concentrated hydrochloric acid was not possible as it gave rise to some decomposition of the p-hydroxylated ethotoin released. Qpid mass fragmentogramsfor the determinationof etbotoin, dealkylated metabotite, metabolite IV z+nd nonconjugated p-hydroxylated ethotoin, of standardsand of urine es&ads Fran blaEk urine and from wine from a patient treated tith ethotoin are shown in Fig.2. Metabolites III, IV, V and VI could not be measured exactly as ILo reference xvi”&knows concenfxati0n.s was avail-
able. The concentrations were estirnak3 &on t&e areas of the p&s in the gas tAxomatQ~as dtibedabove. OV-1 or OV-101 co&d be used as the column material. OV-17 yielded no sqaratior, meWMite
of the sSy1 d&v&&es of etbotoin, of dealkylated e&hot&z and of IV. ARhough they vaxe measuredat different charm&, slight inter-
fe=nce from the lattq was observed at the channels measuringethotoin and
deaHq&ted ethotoirb eOnceming the co@-
metabo~~, only p-hydroxyf&ed ethotoin was found in noticeable amounts (a trace amount of conjugated metabolite V was 0-j. Ef was measured as t&d minus fqee compound (the ftee compound
I.
/‘,/
.,,,‘.
*,
00 10 ‘. ‘1;; 100 168 Iti 177 178 ii 1.88 W 180 2OB ‘G 308 a20
98 100 18 00
78 223 280 204 206 340 304
100 22 31 16” 10 15 86
78 100 106 33 147 41 162 14 234 42 ;8” 204 12 t 16 287 46 336 3: 1s* 330 11 3 348 12 364 G --l__-__--._~__-~___“M&&‘kgmentrsuaad for mm fragmentogrnphicmeeeuremonk, ‘.,,
78 I@ 162 .108 17.6 I , ,177 261 282 :I. “a,276 : ,’ ,‘I, : 73 106 Ia? 264 2G5 408
-60 21 20 13” 11 0,3
73 lQ1 202 464
__
100 1B 4* 0.4
73 147 1es 322 4G2
100 21 10 8* 2
136 176 100 201 202 283 275 200
:: 16 2
ae 13 16 loo*
Fmgmenkabelow 100 m/e (c&opt 73) and/or with intcnsiticrrbelow 10% (oxcopt molarfrngmontrl or fragmentaueod for mnmfcagm&ography) heve buanomitted. Tho reaultagivenurorelativeinteneitioe(I). _“___.._.,______ _.., ______-.._.__._- .._._ _ .I__ ___----.-.-___---T-MotobolitaIV MotnbolitoV ET0 DAJ%TO p-N*ETO Mehbolito III MutRboliteVI MPT -m/t? I m/c I m/e I m/c I m/a,,1 /m/c I m/e I m/c 1 ______-.__-I..-----_I l_..---___-l___-~ --__-._,.
~$:,’ MA86 SPECTRA OF SILYLATED COMPOUNDSOF ETHOTOIN (ETO), DEALKYLATED ETHOTOIN(DA.ETO), p.HYDROXYLA’i’j&j$;i ETHOTOIN@WETO), METABOLITESIII, IV, V AND VI AND MEPHENYTOIN(MP’I’)
;.l:,,:,;;i .“VABLEii
.;,
A method for the determination of ethotoin snd its p-hydroxylated and dealkyhted metabolites in urine has been developed_ Etbotoin end the metabolites were extrected fkom acidified urine with ethyl acetate and silytated before injection inta a combined gzs Aromatogreph--mass spectromekx Four psrtly ic?entified metabolites were recorded, brrf their exad qua&if&ion wes not gmsihle as pure reference substances were not avzihkble. The limit of sasitioity wzs Ear below the amounts of ethotoin and of its metabolites found in tine from patients treated with therqeutic doses of ethotoin_
Recentiy, group
the a&epileptic drug etkotok-3 (Pegsnone) was shown by our dose-dependent kinetics [I]. The purpose of the preset in-
fro exkibit
pws to develop 8 method for the quantitativedetemi?zeion of echotiin and its metialites ezMed in urine in order to elari& the meckanism bekkxdtke dose-dependent kineticsof ef&oto~b_
ues@&iczn
-
(Rockford, XL, U.S.A.) was of specially purified grade. GiucurouidaselarylsuIpb.atasewas obtaiued from Eoehringer (Mannhelm, G.F.R.). Reference substances
The structures of etbotii and its metioli&s are shown in Fig-l. E&atoin and its dealkylzted met&o&e (5phenylhydantoin) were don&d by Abbott L&s_ (North Chicago, Ill., U.S.LL). The two internal skndarck, mephenytoin aud heptamd, were donated by sapldoz (Bqsle, Switzerland) and Ciba-Geigy (Bale, Switzerland), respectively. The p-hydroxylated metabolite was folated from human uriie and purified. hEetaholit.esIII, IV, V and VI were exka.&xI from urine and purified, but were not isolated in a pure state.
Metabdite
I\ 0-
H 0N\
,o
S 7’ HI C-N-CHz-CXJ c? Ethotoin __
-
2 -_
-1
Non-enzymatic
Fig.1. Metabdic
\
? f&tatsatite SS
‘C’O I C-U-CW,-CH,
P - hydmxytated eth~toin -.
--.
\
:! 0
,c’
9,
H N
H
\ --a
k?etabatite9
cacliuqated ta-hydmxytated ethotoin
Unidentified mct.&ctites
reaction possibte . pathways
of etho’ain
in rrtan.
Isoiation and ptqification. rMetabolites III, IV, V and VI ware extracted from urine samples with ethyl acetate and cleaned by column chromatography on silica geI with ethyI acetati-hexame (1:X). The uriue remainin g after this extraction was hydroLy& witfr giuCu~~tik/ asylsulpha~. The p-hydroxyl&ed mekholitfs tiesed was exkicted with ethyl acetate and cIe&ed by columm chramak~ptrJr on silica g& with ethyl acetaten-hexane (1 :Z), followed by two reay&Eations from ethyI acetaten&sane (19). The melting point of tie compound and its W spgctrum in ethand, as weII as the acid prop~9533 ax@ tie w SW of tfie eompo~& -I ,-.aad some of i% dezktives, wez determiacxt, ., :. _ _-...
163
Identi~~tion.Theidenti~oftheisola~p-hydroxyEatebethotoLnwasconf&met2 by the fallowing an&ytk.& da*& The main pe&s in the mass spectnrm
were 16 m/e higher thsn the correspondingpeaks in the msss spectrUmof ethatoin, The compound could be s&&&d z&two positions md acetylated at one. The unchanged compound was 8 we& acid, but its ac&yl&ed derivzkivewas neutral. The UV spectrum and the meKing paint were in good agreementw&h those of synthetic 3=ethyl-5-(p-hydroxyphenyl)hydantoin [2]_ No definite proof 9~~8s obtahredthat the hydrrxylation i%.ks place in the pam-position, but p-hydroxylation has been shown to be a major metabolic pathway in DWRfor phenytain [3,4] end other B-phenylhydantoinderivatives[5], the phydroxylated metabolite was found in urine from dogs treakd with ethotoin [2] and m-hydmxylation of phenytoin occurs to a slight extent ES]. The exact struck form&e of metiohtes ?Z& IV end VI have nat yet been proved. The compounds could be formed in small amounts in vitro from ethotoin, deslkylated ethotoin and p-hydroxylsted ethotoin, respectively, in alkaline solution. The mass spectm of the unchanged compounds (TIE,IV and VI) and of their silylates may point to en oxidation in position 5 in the hydantain ring system [Z] . ‘Fhe mass spectra of met&ohte V and of its silylated derivativecould be expkined by the assumption that the metabolite had arisen kom etbotoin in which the phenyl group had been converted into the 3,4_dihydroxy-l,5_cyc]ahexedien-I-yl group. This metnbalic pathway has been shown to be open for phenytoin [7]. F’uithemo~, this vvoerkd be in agreementwith the fact that the metabolite had no acidic properties. No compoundhatigtwophenoliehydrosy~groups,asshownfor phenytoin ES] wzisobserve& Mess spectiameby. A combin& gas chromatogmph--mass spectrometer (LKB 9OQO) was Used. The conditions useci for the maSSspectra were as follows: ion&&ion energy, 70 eV; kap current, 68 PA; sccele=ting voltage, 3500 V. Tables 1 end IE give the 11185s spectra of ethotoin, its met&o&es and the internal stendsrd es unchanged and as silylated compounds, =SpectlVelY. EX~TUC~~UR aRd derimfiusfiun
of
non-cunj~afed
metaimiites.
To
a 50-yl
urine sample in 8 centrifuge tube 1 rnI of 0.1 N acetic acid and 100 ,~l of mephenytoin solution (6.X gfl in ethanol) 8s intern& &nd& were tided. The szunp!e wes mixed with 3 rni of ethyl acetate for 5 min. After cent&Tug&on, the orgsniuphase ws knsferred into a taperedt&e and evapmzbd to dryness in 8 &B%%IIXof nitmgm. The residuewas derivztizd with 50 ~1 of BTSA for 15 znin and diluted with 100 ~1 of toluene. A volume of I ~1 was injectedinto the combined @s chmm&agrziphiness spectrometer. E~frcrcti~ and deh&zatimt of fatal p-izydmxykted metaboiites.To a 50 ~1 urine sample in a centrifuge tube, ZOQ@I 0.1 M s&c acid were added. After the &&ion of 5 &I.of giucmniW/~&&p~, the tube was I& for 18 h at 37”. The suhsewent pmeedure was as descr&ed far the nonconjugnted compounds* If an@ the phyc$roxykM .&&aIite vzxs to be-mexsured, gas chmrnatogmphy v&b affame-i~nizati~zkdeteCtorcotid be used. Tfie pmcedure %vasthen
‘Z&&ES SPEC=l=EtA OF IZFF’EI-EN (My)), XYDROmLATED 2TEmIN @-%-rn)
Fragmeds bdow
DEALFXLATED
AXE) M%TABom
aa3
m/e
ET0
DA-IFlY
p-H-m
(md.*.206)
i;n&ecI?6) _-_---~-_
(moI.wt.220) EL
103 104 100 36 105 119 120 121 10 132 20 133
m, lv
iIzt~~;ti~. _ _.__~.
Metehdite
100 37
m62
Metebolite
Metidite
Iv
V
100 35
89 100 87 32
86 la0
99
18
24 41 25
14s 149 174 176 191 192
29 13 40
26 30 56
i
22
p-
Tixe results
__-
135
204 202 220 238
(DA-SrO), AND v
iC0 m/c -d/or v&h intensifiesbelow 10% have heen omitted.
,gim
&tie
ETHOTO~
EX
59 19
-165
because complete isolation of the compounds was not feasible. The concentrations were estimated by comparison of the areas under the peaks in thigas
c~nm;PtogramsofthemetaboLites~~ehe~ underthepeakinagzschromatqrarn of the dealkylated metabolite of ethotoin (5-pbenylbydantoin). JL flame-ionizationdetector wss used.
Extructi~on and formztion of deriuutives Ethotoin itself could be extracted quan&&ively
with dicfrlo~methane [9]. However, it was necessary to use a more polar extraction medium such as ethyl acetate, as some of the ethotoin mekbolites are rather poh. The fact that
several of the metabolites are weak acids could not be utilized in a cleaning procedureas ethotoin and most of its metabotites a-e unstable in dk&.ne soluti0l.l.
For most of the compounds, the extraction recovery was approximately 100% (only 75% for metabolite V). Silylation of ethotoin, of the internal standard, of p-hydmxylatedethototi and of metabolite III proceeded readily at room temperature, but the dealkyk&ed ethotoin and metabotites IV and V reacted more slowly, especially in low concentrations. This effect might be due to adsorptionof the non-silylated
compounds ORthe glass walk, but a&z 20 min the derivatizationswere found to be aJmostquantitative.
Hyddysis of conjuguteed mefubolites ‘The only conjugated metabolite found in noticeable amounts was p-hydroxylated ethotoin. Enzymatic hydrolysis of conjugated p-hydroxylated ethotoin under the couditions described was found TVbe quantitative (or at Ieastmaximal) after 12-15 h. Acid hydrolysis with concentrated hydrochloric acid was not possible as it gave rise to some decomposition of the p-hydroxylated ethotoin released. Qpid mass fragmentogramsfor the determinationof etbotoin, dealkylated metabotite, metabolite IV z+nd nonconjugated p-hydroxylated ethotoin, of standardsand of urine es&ads Fran blaEk urine and from wine from a patient treated tith ethotoin are shown in Fig.2. Metabolites III, IV, V and VI could not be measured exactly as ILo reference xvi”&knows concenfxati0n.s was avail-
able. The concentrations were estirnak3 &on t&e areas of the p&s in the gas tAxomatQ~as dtibedabove. OV-1 or OV-101 co&d be used as the column material. OV-17 yielded no sqaratior, meWMite
of the sSy1 d&v&&es of etbotoin, of dealkylated e&hot&z and of IV. ARhough they vaxe measuredat different charm&, slight inter-
fe=nce from the lattq was observed at the channels measuringethotoin and
deaHq&ted ethotoirb eOnceming the co@-
metabo~~, only p-hydroxyf&ed ethotoin was found in noticeable amounts (a trace amount of conjugated metabolite V was 0-j. Ef was measured as t&d minus fqee compound (the ftee compound
I.
/‘,/
.,,,‘.
*,
00 10 ‘. ‘1;; 100 168 Iti 177 178 ii 1.88 W 180 2OB ‘G 308 a20
98 100 18 00
78 223 280 204 206 340 304
100 22 31 16” 10 15 86
78 100 106 33 147 41 162 14 234 42 ;8” 204 12 t 16 287 46 336 3: 1s* 330 11 3 348 12 364 G --l__-__--._~__-~___“M&&‘kgmentrsuaad for mm fragmentogrnphicmeeeuremonk, ‘.,,
78 I@ 162 .108 17.6 I , ,177 261 282 :I. “a,276 : ,’ ,‘I, : 73 106 Ia? 264 2G5 408
-60 21 20 13” 11 0,3
73 lQ1 202 464
__
100 1B 4* 0.4
73 147 1es 322 4G2
100 21 10 8* 2
136 176 100 201 202 283 275 200
:: 16 2
ae 13 16 loo*
Fmgmenkabelow 100 m/e (c&opt 73) and/or with intcnsiticrrbelow 10% (oxcopt molarfrngmontrl or fragmentaueod for mnmfcagm&ography) heve buanomitted. Tho reaultagivenurorelativeinteneitioe(I). _“___.._.,______ _.., ______-.._.__._- .._._ _ .I__ ___----.-.-___---T-MotobolitaIV MotnbolitoV ET0 DAJ%TO p-N*ETO Mehbolito III MutRboliteVI MPT -m/t? I m/c I m/e I m/c I m/a,,1 /m/c I m/e I m/c 1 ______-.__-I..-----_I l_..---___-l___-~ --__-._,.
~$:,’ MA86 SPECTRA OF SILYLATED COMPOUNDSOF ETHOTOIN (ETO), DEALKYLATED ETHOTOIN(DA.ETO), p.HYDROXYLA’i’j&j$;i ETHOTOIN@WETO), METABOLITESIII, IV, V AND VI AND MEPHENYTOIN(MP’I’)
;.l:,,:,;;i .“VABLEii
.;,
