Document O1zK5pKQ693Ooq75Lvb2mGbRv
ADVANCED BIOANALYTICAL SERVICES, ING.
ARDE-0l63 #14 &
ANALYTICAL REPORT
TITLE: Additional Characterization of Metabolites of T-6292, T-6293
and T-6294 from Rat and Human Hepatocytes by
TurbolonSpray LC/MS and LC/MS/MS. Semi-Quantitative
Analysis of T-6295 in Rat and Human Hepatocytes Incubated
|
with T-6292, T-6293 and T-6294 by LC/MS/MS.
DATE: REPORT:
27 January, 1998 98AGKPO1.3M
AUTHOR:
Grace K. Poon, PhD. Steve Lowes, PhD
PREPARED 3M Medical Department,
i
FOR:
Toxicology Services,
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St. Paul, MN $5133
FB-2~ i
NO.OF PAGES: 69
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SIGNATURE PAGE
Title:
Additional Characterizationof Metabolitesof T-6292, T-6293 and T-
6294 from Rat and Human Hepatocytes by `TurbolonSpray LC/MS
and LC/MS/MS. Semi-Quantitative Analysis of T-6295 in Rat and
3
Human Hepatocytes Incubated with T-6292, T-6293 and T-6294,by
|
LC/MS/MS.
Reported by: pace
:
fefnaicoer KR. esPeoaornc,hPShcDie.ntist
977 Jan. 1998 Date
ARvepvrioevweeddbayn:d it L. 1 A|D~ 0z }
J2e7.0_ 958
i
MAuadriktoLrentham, B.A.
Date
'
]1
Authorized for
Release by: Steve Lowed, Ph.
2Du1c :Too-d%
i
-
AScdiveantnicfeicd
Director
BioAnalytical
Services
|
04059
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REPORT: 98AGKP01.3M
I2SSEIoR0VAeINCAsELSYe,TIICNAGL
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Te T mecwmw
SIGNATUREPAGE...
3
TABLE OF CONTENTS ...mssssmmsssenso,3
LISTOF TABLES cosmo.
|
LIST OFFIGURES
sss
6
PART A: Characterization of Metabolites ofT-6292, T-6293 and T-6294 from Rat and
Human Hepatocytes by TurbolonSpray LC/MS and LOMSAMS............ 10
1 INTRODUCTION sss
10
1112 SBTAUCDKYGORBOEUNCDTVE
eo
10
2 EXPERIMENTAL
A
seems
14
:
21. 22. 23:
SCTAHNDAERDMSAOINLUDTCMIAOTNAESR.I.LA.LSS HPLC ELUENT PREPARATION...
c ssnco o.com
14 19
24: 23. 26.
DLSAACMT/PAMLSEHPAARNENDPDLAIRLNACGTI.IMOSN./.M.S..I.N.S.T.R.U..M.sE.N.TeATI7 sONsvo ero
13 16
]
3. RESULTSAND DISCUSSION...
18 18
, :I
3312: CMEOTNATBROOULSEMXOPFETR-I6ME0N.T.S............. 321 Metabolism ofT-6292 by Rat Hepatocytes at 0
hornee19
:
332232.. 3Ja.
MeMMteettaaabbbooollliiisssmmmooofffTTT6--66222999222 bbinyyHHRuaumtnaHaennpaHHteoepcaypttoeacsyttaetosa6caty60hthoehwso ouu rrr .r.."3 " 33109
333333323.(MMEMeMeteTttaaabAbboooBlliOiOssFLmmTooI-ff6S2TT-9M-63269c 3b2byy9RRa3att HHeepao ptoacytteosacatty60o htheoso uru .n .r.""222
34,Ja(MEMeTtaAboBlliOiOssFmLmToIo6fTf2S-T06-M60229933bbyy HHuummaaonnoHHoeepaptoacytteosacatty60thoeusr nhrour"" 2332
342.ede MMeettaabboolliissmmooffTT--66229944bbyy RRaatt HHeeppaattooccyytteess aatt 06
horn" 2200 hour." 24
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3.53..44.PRODUMCetTa-bIoOlNisMmAoSfSTS-PE6C2TR9A4..b..y..H.u..m. anr Hepatocytes at 6hour ......... --------}.
3.6.
POSITIVE
IONIZATION
LC/MS
ANALYSIS
OF
ess HUMAN HEPATOCYTESINCUBATED
25
WITH T-6292 AND T-6293.........occoo.... rmsesnsn------------------------------.--------. 2]
PART B: Semi-Quantitative Analysis of T-6295 in Rat and HumanHepatocytes
Incubated with T-6292, T-6293 and T-6294 by LCMS/MS.................. 28
]
8,
EXPERIMENTAL......coooroerroreo.
TSI3
1 I
5.1. STANDARD SOLUTIONS............... ee ---------- 5.2. PREPARATION OF QUENCHING SOLUTION
nn --------------------------------. 28
... 53 HPLC ELUENT PREPARATION. ........... es------------
TETSU. nn -------------------------------- 29
5.4. 5.5.
SAMPLEPREPARATION................. er PREPARATION OF RAT HEPATOCYTES
------------------ STANDARD`CURVE SA
sn
29
5.6.
LC/MS/MS INSTRUMENTATION...
MPLES .................... 29 rss ssssssseses 30
5.6.1. HPLCconditions....................... rss 5.6.2. MS CONItiONS..........ccooomereeereeesosrrsoo resins
sss
ess JO
5.63. Data Handling...........coooeeceoeeeerooooo rere
sss 30. sssssssssssssass 31
6.
Results and Discussion: ........................
sissies
31
7. RECIENTES toss
35
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LIST OF TABLES
Table I:
S0ummHaOryMEofHProEpoP sedAStrOuctCureYs oIf MeEtabSoli.tes.ofoT-i629e2 Isncubated
with Rat
oo 12
Table II: S3u00mmHaUrMyANofHPErPoUpOosCeHdESStrr uctures of Metabolites ofo T-6293 Incubated with Ra13t
]
Table
III:
ASnudmHmUaMrAyNofHEPDrUoOpoCsYeIdESS.tructuc res ofo Metabos lites m of T-6o 294 Ins cubated
in
Rat
14
-
Table IV: Proposed `product-ion fragmentation for Metabolite VI..... En ----)
!
Table V: Proposed product-ion fragmentationfor Metabolite VL................... 27
Table VI: QIunacnutbiattaitoinvewitAhnaTl-y6s2i9s2,oTf-6T2-96329a5nd Ti-n62R9a4t. and HumansHs epastocytes a3f2te.r
i
Table VII: Quantitative Analysis of T-6295 in T-6292, T-6293 and T-6294. Ee --
1
!
ii
t 004082
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ean
SABEIoFOv.LaAnCLcESYe.DTINCAeL
5
Figure 1.
LIST OF FIGURES aRnecdoLnsCtIrMucSt/eMdSjopnrocdhurcotmiaotnomgarsasmsp(ei)c,tnregative ionization mass spectrum (ii)
um (i) ofauthentic T-g2og. 36
Figur2e.
RSePcEonCsTt,ru6ct)edOF iAoUnNTcIhrEoTm-at6o2g0r4a.m...(.)
c and Lr C/MS/MS
production ne
mass
Figure 3.
aRnedcoLnsCtIrMucStIeMdSjopnrocdhurcotmiaotnomgarsasm s(pie),ctnreugmat(iive) offonaiuztahteinotnicmTa-s6s20s5p.ectrum
(ii) on
5!
Figur4e. IFnulClUBAsLcEaDnFr6eOcoHnOsFUtLrSucWtietdh 1i0onESLchArCoEmaSt.o.gr.ams of control rat hepatocytes
a
'
Figure S. FCuUllBBsIcaEn Or7e6conDsOtUructWeidthi1on0 (c8hrAomCatEogSr.am.s.of control o humanhe epatocytes
Figure 6. WFullOscaBnCPrUecOoCnYsItErSuctc ed ion chromatogro ams of T-6292 inco ubated for 6hours
. i
Figure 7. FSuRlOl sBeEanPrOeCcoYnsItrEucSted ion chromatos grams of T-6e 293 incubated for 6 hours
i'
Figure 8. WFulHl OscaHnErPeAcOoCnYsItErSucted fon chromatr ograms of T6s 294 incubated d for hours
Figured.
mFeultlaboslciatnesafretcroinnstcruubcatteidng
ion ra
hecphartoomcyatteosgrwaitmhs
T-s6h2o9wi2nagt.
0
hthoe uprre.se.nc.e
of 44
| =
Figure
I0.Full scan metabolites
arfetceornisntcruubcatreidng
ion rat
hecphartoocmyatteosgrwaitmhs T-s6h2o9w2infgor
6thheouprrs.en.ce.
of 4
1
:
Figure
1LFmueltlaboslciatne
arfetceornisntcruubcatteidng ihounmacnhrhoempaattoocgyrtaemsswitshhoTw-i6n2g52
the
at 0
hpreesen.ce
of
42
{
Figure
12.mFueltlaboslcitaens
arfetceornisntcruubcatteidng
ihounmacnhrhoepmaattoocgyrtaemsswitshhTo-wi6n2g52
the
for
6
phroeusre.nc.e.
of
41
{
:
Figure 13.(i(S)iiRSi)MRSMcRhcMrhormocamhtarotogomrgaartmaosmgsrofaomfshruaotmfahenhpuahtmeopacanyttoechsyetpieanstcouicbnyacttueebsdatwieindtchuwbinattohedtneostwtiaetrshtticanlroe.si;le(riis)
FEIES
ste
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'
Figure
14.SRM_ chromatograms hepatocytes incubated
wsihtohwTi-n6g29t2heatab0sehnocure;
of (ii)
metabolite IV rat hepatocytes
in (i) rat incubated
hwoiuthr aTn-d62(9iv2) hfourm6anhohuerp;at(oiciiy)tehsuminacnubhaetpeadtowciyttheTs-i6n2c9u2baftoerd6whioturh...T.-..6.2..9.2.. at490
Figure
15.
SRM 6292
tsottaanldiaornd cshorloutmiaotno;g(riai)msinschuobwaitinogn thoef pTr-e6se2n9c2e
woifthmetraatbohleipteatVoIcIytiens(ia)tT0-
HOhFoUuMTrA-,6N(2iR9iiE2)PRwiIniOctuChbYhaItEuiSmoaBnnL6ofhHeTOp-UaE6t.2o9cr 2ytweistaht r0ae thohuerpaatnodcs y(tve)siantcu6s bahtoiuorn; o(fivT)-i6n2c9u2bawtiitohn
, $0
Figure
16. SRM 6292
total ion standard
cshorluotmiaotn;og(ria)msinschuobawtiinogn
the of
pTr-e6s2e9n2cewoifthmertaatbohleiptaetoIcXytiens(ia)tT0-
HOhfoUuMTrA-;6N(2iR9iE2)PRiwIniOctuChbYahItEuiSmoBannL6ofhHeTOp-Ua6tT2o9c2ytweistahtt r0athohuerpaatnodcs y(tve)siantcu6bahe toiuorn; o(fivT)-i6n2c9u2bawtiitohn
oe S1
Figure
17.
SRM 6292
total ion standard
scohlurtoimoant;oigir)aimnscusbhaotiwoinngoftThe-6p2r9esweintche
of rat
metabolite X in hepatocytes at 0
iD) incubation of T-6292 with rat hepatocytes at 6 hour; (iv) incubation
(i) Thour:
D62U9A2NwiRtEhPAhIOuCmYaInEShAep6atHoOcUEy:ter s at 0 hours and (v) incs ubation of T-6292 owfi5Tt2-h
Figure
18.Fmueltlaboslciatnes arfetceornisntcruubcatteidngiroatn hecphartoomcyatteosgrwaitmhsT-s6h2o9w3infg 0othhoerur,pres..e..n...c.e
of 53
Figure
19.Fmueltlaboslciatnes arfetceronisntcruubcatteidngiroatn hecphartoocmyatteosgwriatmhsT-s6h2o9w3infgo6rthheour.pres.e..n.c. e
of 54
1
Figure
20.Full scan metabolites
arfetceornisntcruubcatteidng
ihounmacnhrheopmaattoocgyrtaemsswitshhoTw-i6n2g93
the for
0
presence hour......
of 55
Figure
21.Full scan metabolites
arfetceornisntcruubcatteidng ihounmacnhrhoempaattoocgyrtaemsswitshhoTw-i6n2g93
the for
6
presence hour.......
of 56
Figure 22.wSiinRtchMubarttaoittoanlheoipfoanTt-occ6hy2rt9oe3smawtiaottghr6raamthsohuesrph;aotwo(iciyin)tgestihnaectu0bparhteoisuoenrn;ce(oifio)fTi-nm6ceut2ba9ab3toiloiwntietohfIVTh-ui6nm2a9(in3) OhepHatOocytes at 0 hour and (iv) incubation ofs T-6293 wis th humano hepatocyte$s 7at
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6293 hour;
standard solution; (ii) incubation (iii) incubation of T-6293 with rat
of T-6293 with hepatocytes at 6
rat hepatocytes hour;
at
0
ohfumT-a6n2h9e3pawtiotchyhteusmaatn6hhoeupra..t.o.c.y..t.e.s...at..0 hour -- and--(v)--in-- cuba-- tio-- n o(-- fivT)--- i6n2c-- 9u3bawtii3to8hn
Figure 24. SRM total ion chromatograms showing the presence of `metabolite IX in (i) T-
6293 `hour:
standard solution; (ii) incubation (iii) incubation of T-6293 with rat
of T-6293 with hepatocytes at 6
rat hepatocytes hour; (iv)
at
0
iI hofumT-a6n29he3pawtiotchyhteusmaatn6hheopuat.oc.y..t.e..s...a.t..0...h..o.ur arndr(v) incuba--ti--on--o--f T-----i6n2--c9u3.bawt3iito9hn
T
Figure 25. SRM total ion chromatograms showing the presence of metabolite Xin (i) T-
6293 hour;
standard solution; (ii) incubation (iii) incubation of T-6293 with rat
of T-6293 with hepatocytes at 6
rat hepatocytes hour; (iv) incu
at
0
ohfumTa-6n2h9e3pawtiotchyhteusmaat n6hheopaut.ocy..t.e..s...a.t...0...h.ourrsasnd (v) incubatio--n--o----f--T-----6ss2s93bawtii0to0hn
Figure 26.Reconstructed ion chromatograms showing the presence of `metabolites after
incubating rat hepatocytes with T-6294 FOF ONOUL. ccm 61
Figure
27.
iRneccuobnasttirnugctraetdheiopnatcohcyrtoemsatwoitghraTm-s62s9h4owfoirng6
the presence of `metabolites ROUT. ............ouv...........
after 63
Figure 28. iRneccuobnasttirnugctheudmaionn hcehpartoomcayttoegsrwaimtsh sT-h6o2w9i4ngftOh0re hpOrUe.s.e.n.c.e..o.f..m.e.t.a.b.o.l.i.t.e.s..a.f.t6e3r
Figure
29.
iRneccuobnasttirnugctheudmaionn hcehpartoomcayttoegsrwaimtsh
showing the
T-6294 fo6r
hporUe.s.en.c.e..o.f..m.e.t.a.b.o.l.i.t.e.s..af.t6e4r
]
Figure 30.SRM total ion chromatograms showing the presence of metabolite Vil in (i) T-
'
6294 hour;
standard solution; (iii) incubation of
(ii) incubationof T-6294 with T-6294 with rat hepatocytes at
rat hepatocytes at 0 6 hour; (iv) incubation
: ohfumT-a6n29he4pawtiotchyhteusmaatn6hheopautorcytes....a.t....0....h..o...u..r...and (v) incubation os f T.6s 294wit6h3
Figure
31.SRM
6294
total ion
standard
chromatograms showing
solution; (ii) incubation
the
of
Tp-re6s2e9n4cewiotfhmerattabhoelpiatteoXcytiens(ia)tT0-
hour, (iii)
Of T-6294
iwnictuhbahtuimoannofheTp-a6t2o9cy4tweistaht
r0athohuerpaatnodcy(tve)siantcu6bahtoiuorn:
(iv) incubation
of T-6294 with
human hepatocytes at 6 hour...................... mn
-------------- 08
Figure 32. Negative ionization product-ion mass spectra of (i) metabolite I; (if)
`metabolite VI and (iii) metabolite VIII. ................. wenmssssrsssssssssssssssssanans 67
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Figure 33. Production mass spectra of (i) Metabolite XIII and (i) metabolite XL....... 6
Figure34SCealleicbtraetdionReSatcatnidoanrd HMeopnaittoocryitnegExtCrharcotm(a2tonggr/ammLsT-6f2r95o)m....L..C./..M..S./..MS
of a go
i
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1. INTRODUCTION
|
11. BACKGROUND
:
`The metabolismofT-6292, T-6293 and T-6294 in rat hepatocytes was investigated at
Advanced BioAnalytical Services, Inc. using liquid chromatography onspray tandem
mass spectrometry (Reference 1). Several metabolites had been identified, namely the glucuronide acid conjugate, the carboxylic acid the sulfonamido alcohol, the N-dealkyl
sulfonamido alcohol and the sulphonamide of T-6293,
12. Stupy OBECTIVE
`The principle aim of this work was
(To complete the characterization of the metabolic products of T-6292, T-6293 and T-6294 which were formed by incubating each compound with rat and human
|
hepatocytes;
@) tstarnudcteumramlacshsarsapcetcetrriozmaettiroyn;of the carboxylic acid and alcohol analogues using
3) iiodnenstpirfaiyc;ation of additional metabolites by LC/MS, using positive ionization
4) identification of the aldehyde and N-dealkyl aldehyde analogues of T-6293; (9) semi-quantitative analysis to determine the concentration of T-6295 in the rat and
human hepatocytes after incubating with T-6292, T-6293 and T-6294,
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(TChheroqmuaetnocghreadphiinccusbeaptaersatwieonreofditrehctvlayriionujsecctoendsaintduesnetpsarwaatsedaocncoamnplHiPshLeCd cuosliunmgng.radient
elution. Metabolites were characterized by LC/MS and LC/MS/MS, and the results are
sMuSmmmaordieze(dm/izn T1a5b0l-8e0s0)[.- 1T11a. nTdheem mmaassss ssppeeccttrroommeetterrywwaass oupseerdatfeord fiunrtthheerfusltlruscctaunraslingle
elucidation and confirmation.
1
!
i '
ii
i
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Table I: SRuamtmaanrdyHoufmParnopHoespeadtoSctyrtucetsures of Metabolites of T6292 Incubated with
Proposed Structure" [Abbreviation| (MAH I
IONSPaomon | 629T2) 570 [|rONefsopooMunSrse|T|TMr6NeshopoMounSrse|| | r0NehsopoMounssre| 6our|
Fn TO Sm enpan
3
7
7
I :
J, =i)
v 368
a EO Sew, coon v 5 7
7
rmeld [ Ep Te T arms.an--cnon on, --cio Vi EC
o [ crsr -- omse .wrcim coon | = x [2 7 1 7 [77 [77]
TON en, | (T6299)
Crrieso0,x "=e, X
Trace
j
J
[SArSom
CFrsof
XI [ee | 7 7 ] 7
! PR OT an--noson p6a2y95) 50
7
7
v X
Observed Not observed
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Table
II:
Summary of Proposed Structuresof
Rat and HumanHepatocytes
Metabolitesof T-6293
Incubated
with
Frese er
Asko .
|
BH|Ram] [ Chou T Ghour |O
hour| hour]
|i
SO een,
or LT
T
570 | NoMS | NoMS
T enon | (T6292)
Cl T PR ies
response | response
No MS response
T carson.eonan vs
z
ml et
[roan|ET] -- Gmso--Now
VI
540
a--rs--on>oicncoon
VII
.
556
tm
}
Tx ncn, | (1-6294)
trace
v
7
--------
Sou
,
[Shrsoi[Caw
CF SoH
Xo
2
v
Vv
7
(T-6295)
/
Observed
X Not observed
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Table
III:
SRautmamnadryHuofmaPrnopHoespeadtoSctyrtuecstures
of
Metabolites of T-6294
rr Incubated in
Proposed Structure | Abbreviation| [M[ -HJ Ra | Human
TIT--Nou enon
VI
542
[0hour[6hour[0
v
our | 6hour|
v
J
GrTsOo--noox an Vir 530
x
E
rsa?"
Vill 536
TT owcoon
v
Rill >
xX
526
(T-6294)
vvvv
CFiTsO0,-- onmn, X
[Fso-- 1 XT ww |v
CF SOH
XI
(16295)
777 7
Observed X Notobserved
2. EXPERIMENTAL
|
21. CHEMICALS AND MATERIALS
T-6292, T-6293, T-6294 and T-6295 were provided by 3M Medical Department,
:
Toxicology Services, St. Paul, MN 55133.
Polypropylene autosampler vial: Polypropylene screw-cap tubes: Ethanol
Cat# 6008-014, WR Scientific Inc, Bridgeport, NJ 08014 16x 100 mm, Cat# S00 765, Sun Brokers Inc. Wilmington, NC 28402 Cat # 112000200CSPP, Pharmaco Products, Brookefield, CT 06804
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HPLC Grade Methanol Dimethyl Sulfoxide Ammonium Acetate
Cat # 230-4, Baxter/Scientific Products, McGaw Park, IL 60085 Cat #27043-1, Sigma-Aldrich Inc., Milwaukee, WI53233 Cat #24019-2, Sigma-Aldrich Inc., Milwaukee, WIS3233
.
22. STANDARD SOLUTIONS
Stock solutions of T-6292, T6293, T-6294 and T-6295 were prepared at a concentration
$
of 100 pg/mL in dimethyl sulfoxide (DMSO). Working solution (10 pg/mL) of each
standard were prepared by diluting ImL of the stock solution into 9mLethanol, All
0storcokosmoltuetmipoenrsaatnudrewboerfkoirnegusseo.lutions were stored at 4 C and were allowed to equilibrate
Preparation of Analytical Standard Stock Solutions
Stock Solutions of T-6292, T-6293, T-6294 and T-6295 (100 pg/mL): Each test article was accurately weighed on a micro balance and transferred to.a 16 x 100 mm polypropylene tube. The solid samples were dissolved in appropriate amount of DMSO to yield a 100 pg/mL solution.
1
Working Solutions of T-6292, T-6293, T-6294 and T-6295 (10 pg/mL): .
!
eEtahcahnoslta(n9damrLd)wionraki1n6gxso1l0u0timonmwaposlpyrperpoapryeldenbeytduibleut.ing the stock solution (mL) with
:
23. HPLC ELUENT PREPARATION
.
1M Ammonium Acetate Solution: Ammonium acetate (77) was added to NANOpure
}
water (IL) in a volumetric flask. The solution was filtered through a 0.45 pm filter and
stored at 4 C,
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2mM Ammonium Acetate Solution: 1 M ammonium acetate Solution (2 mL) was diluted to IL with NANOpure water. The solution was filtered through a0.45 mm filter and stored at ambient temperature. A fresh solution was prepared every month.
Methanol : 2 mM Ammonium Acetate (90:10 v/v), Eluent: Methanol (450 mL) was mixed with 2 mM ammonium acetate solution (50 mL). The solution was stored at ambient temperature. A fresh solution was prepared every month.
Methanol : 2 mM Ammonium Acetate (10:90 v/v), Eluent: Methanol (50 mL) was mixed with 2 mM ammonium acetate solution (450 mL). The solution was stored at `ambient temperature. A fresh solution was prepared every month,
24. SAMPLE PREPARATION
The metabolism study was carried out at SRI International, Menlo Park, CA. The incubates were quenched with an equal volume of ice-cold methanol, centrifuged and shipped in dry ice to Advanced BioAnalytical Services (ABS). The samples were stored aL ABS (20 C). During sample analysis, the supernatant was transferred to an eaxucteopstamdpulreirngviailn.jecTthioen.autosampler vials were maintained in an ice-water bath at all times
25. LC/MS AND LC/MS/MS INSTRUMENTATION
i
Liquid chromatography was performed on Shimadzu LC-10AD pumps and Shimadzu
.
SCLIOAgradientcontroller (Shimadzu Scientific Instruments, Inc., Columbia MD
21046). Samples were introduced with a Perkin-Elmer 200 series autosampler. The
HPLC column (Betasil C18, 2 x 100 mm) was obtained from Keystone Scientific, Inc.,
Bellefonte, PA 16823. A Sciex APLIII" triple quadrupole mass spectrometer was used for
sample analysis.
REPORT: 98AGKPO1.3M
04073
.
NI
---
cSEoRVsICeES,.ING
}
LLCC CGroanddiietnito:ns:
}
Time
A
7
%B
] [r CF we w wr-- 1
I
Flow rate:
200 plmin
:
Typical Mass Spectrometer Conditions:
5
Source:
MS Mode TurbolonSpray
!
Ton Spray Voltage
3800 V
.
Orifice:
78V
:
Declustering Potential: "48v
:
Tonization Mode:
Negative
:
CurtainGas (UHP.N) 1.21PM
;
Nebulizer Gas (Na)
60 PSI
!
Collision Gas Thickness: N/A
]
Collision Energy:
NA
Dwell Time:
Smsee
i
Step Size: -
Lamu
Scan Range:
150-800 amu
MS/MS Mode TurbolonSpray 3900 v 60V
30v
Negative 1.20PM 60 PSI 280 x 10 atoms/square cm sev 200 msec
variable
I I
Metabolite IV Transitions: mz 568 -> 269; 526 -> 169; 526 -> 119; 52683; 526 -> 65
: i
Metabolite VII Transitions: m/z 540 -> 269; 540 -> 169; 540 -> 119; 540 > 83; 540 -> 65
:
Metabolite IX Transitions: m/z 526 -> 419; 526 -> 269; 526 -> 169:
|
REPORT: 98AGKPO1.3M
] 04074
}
am ASIDOVAANNACLEYDTAL
.
SERVICES, ING.
1
!
:
526-> 123; 526 > 97
-
Metabolite X Transitions: m/z 606 -> 269; 606 -> 169; 606 -> 119;
606 -> 83; 606 > 65
Data System:
RMuAlDtivVieerwsiVoenrs2.i6o;nL1C.2;TuMnaecSVpeercsiVoenr2s.i4o;n 3.3; MacQuan Version 1.4
i
PPG Calibration
Instrument mass axis calibration was performed by infusion of PPG-425 calibration
i
solution (polypropylene glycols, averagemolecular weight 425, dissolved in 1:1
methanol : 2 mM ammonium acetate containing 0.1 9% formic acid and 0.1 % acetonitrile)
ata flow rate of 10 uL/min in the positive ion mode of detection. Peak widths were
approximately 0.6 amu at half-height in the single MS mode. A calibration check was
performed for the analytes on QI by infusion of a I pg/mL solution in ethanol at a flow
.
ate of 10 L/min. The mass spectrometric parameters and sensitivity were optimized
:
using 60%B of the HPLC mobile phase at 200 uL/min.
-
26. DATA HANDLING
'
All raw chromatographic and mass spectrometric data were stored on the ABS file server
]
ABSI_FS.DATA in the file hierarchy "RAWDATA:Validation:3MSRI:3MSRI
Qualitative". All experimental information was stored in ABS notebook No: 2070.
3. RESULTS AND DISCUSSION
T-6293, T-6294 and T-6295 displayed [M-H] ions at m/z 650, 526 and 499respectively,
i
The reconstructed ion chromatograms and negative ionization product ion mass spectra
of compounds T-6293, T-6294 and T-6295 are shown in Figures 1 -3. Their
1
fragmentation pattems were described in details in previous study [reference 1). As
observed before, T-6292 did not show any signal in positive or negative ionization modes
REPORT: 98AGKPOI.3M
|
04075
i
ADVANCED
SERVICES. ING.
[reference 1). Therefore, no attempt was made to characterize T-6292 as a parent drug or
4
a metabolite in this study.
Rat hepatocytes (reference # 4) and human hepatocytes (Female 1, reference #HH3S7)
were used for the study.
3.1. CONTROL EXPERIMENTS
y
`The full scan reconstructed ion chromatograms of the incubation media which contained
J
rat and human hepatocytes but no test articles are displayed in Figures 4 and 5
1
respectively. These samples were analysed as control samples. The blank samples did
i
;
not show any
metabolites.
signal
at
the
retention
times
expected
for
the
T-6292,
T-6293
and
T-6294
3
The full scan reconstructed ion chromatograms of the incubation media which contained
'
T-6292, T-6293 and T-6294 but no hepatocytes are displayed in Figures 6,7 and 8
1
respectively. These samples were analysed as control samples. However, compounds IX,
i
and XI were present in the T-6292 sample (Figure 6). In the T-6293 sample, apart from
|
the parent drug,
from the parent
compound XII was
drug, compound XT
observed (Figure 7).
was detected (Figure
In
8),
the
T-6294
sample,
apart
|
32. METABOLISM OF T-6292
J
321. Metabolism of T-6292 by Rat Hepatocytes at0 hour
1
"The full scan LC/MS reconstructed ion chromatograms of the rat hepatocytes sample
El
incubated with T-6292 for 0 hour are shown in Figures 9. Figure 9 (i - x) displays the
|
ion chromatograms for the following: the parent compound (m/z 570), metabolite III
| (m/z 746), metabolite V (m/z 584), metabolite VI (m/z 542), metabolite VIII (m/z 556),
i
`metabolite IX (m/z 526), metabolite X (m/z 606), metabolite XI (m/z 498), metabolite XII
:
(m/z 499) and metabolite XIII (m/z 650). Even though compound IX, XI and XII were
detected in this sample, compound IX and XI were observed in the control sample
containing T-6292 and no hepatocytes (Figure 6).
REPORT: 98AGKP01.3M
|
04076
ae. SERVICES. Ne.
.
322. Metabolism of T-6292 by Rat Hepatocytes at 6 hour
iTnhceubfaultledscwainthLCT-/6M2S92refcoorn6sthroucutreadreiosnhcohwrnominatFoiggruraemss 1o0f.thFeigautrehe1p0atiocyt)esdsiasmppllaey the (fonn/7c4h6r)om,amteotgarbaomlistfeoVrt(henzfo5l8l4o)w,inmge;tatbholpiatreenVtIc(onmepo54u2n)d, (mneut:ab5o7l0i)t,emVeItIaIb(onlieeI556), ] V(menIt.La4b9oIlX9i,)tXeaIn,IdXXm(Ientavanb5od2l6Xi)t,Ie mXeItIaIb(olnizte65X0).(mTzhi6s06s)a,mmpelteabcoolnittaeinXeId (mentzab4o9l8i)t,esmeIt,aboVl,itVeI,XI1
!
Full scan mass spectra were
metabolite IX (m/z 526) and
not obtained
X (m/z 606).
for
As
metabolites
an altemativ
IV
e,
(yz 568), VII (m/z
a selected reaction
540
),
m1o69n,it1o1r9i.n8g3(aSnRdMD65aniaolnyssfisrowmasthpeeprrfeocrumresdo.r iFoonramtemt/azbo5l6i8tewIeVr,etmhoniftoorrmeadt,ioFnoorf mz 269,
i '
metabolite ak $40
VIL, were
the formation of mz 269, 169, monitored. For metabolite IX,
119, 83 and 65 ion from the formation of nz 269,
the precursor fon 169, 119, 123
i :
faonrdma9t7ioionnosffmr/ozm2t6h9e,pr1e6c9,ur1s1o9r,i9o7n,a8t3maznd52665 wieonrsefmroonimtotrheedp.recFuorrsomretiaobnolaittmezX,60th6e
}
"wTeernteatmiovneitiodreendi.ficSaitnicoen othfemseetparboodluicttesiIonVsawnedreVIcIhawraacstearcihsiteivceodftihnitshcelsatsudoyfscaommpploeusnds.
i
when signals were observed at their appropriate transitions.
J
MNeotmaebtaobloliVitIteeIIwVaswapsredseetnetctinedthien rtahteh0epoartochyotuers rtarteastteuddywistahmTpl-e6s29(2Fiogurr6e h1o4urasn(dFiifg)u.re
|
|
h1e5paiit)o,cybtuetsabcosnetnrtolinstahmep0lehsou(rFisgaumrpesle13(Fiagnurde1155)i,), and the no test article or no
} I
hFeipgautroecy1t6e(si)inacnudba(tie)d rweiptrhesTe-n6t2t9h2e fSorR0Matnodtal6 ihoonurchrresopmeacttoivgerlaym,ssh(ToIwCi)ngofthreatpresence
coofnmtertoalbsoalmitpeleIcXonitnatihneisnagmpTl-e6s2.92Haonwdevneorh,etphaitsoccoytmepson(Feingturceou1l6d1)a,lso be observed in the
|
REPORT: 98AGKPOL3M
|
04077
ADVANCED
}
--- SERVICES, ING
!
:
M1e7tiaib)olbiutteaXbsewnatsinprtehseenOthionutrhsearmaptlheep(aFtiogcuyrtees17trief)ataenddwtihteh nTo-6h2e9pa2tfooeryt6ehsocuorns(trFoilgure
sample (Figure 17 i)
A potential metabolite (XII) was observed at 6.1 min and displayed an [M-H]" ion at
m/z 650 (Figure 10 x). An addition of 80 mass units relative to T6292 could correspond
0 the additionof a sulphate moiety. The product-ion mass spectrum of metabolite XIII
1
in Figure 33(i) showed fragment ions at m/z 526, 419 (C4Fy7), 319 (CoF1y), 269 (CsFyy),
219 (C4F5). 169 (C3Fy), 97 (HSO, or FNSO;) and 80 (S03). The fragment ionsatmz
1
80s consistent with the presence of the sulphate moiety.
,
323. Metabolism of T-6292 in Human Hepatocytes at 0 hour
The full scan LC/MS reconstructed ion chromatograms of the human hepatoeytes sample
i
incubated with T-6292 for0 hour are shown in Figures 11. Figure 11 (i =x) displays the
ion chromatograms for the following: the parent compound (nu 570), metabolite ITI
1
(2 746), metabolite V (m/z 584), metabolite VI (mz 542), metabolite VIII (nz 556),
|
(memtuab4o9l9i)teaIndX (me/tzab5o2l6i)t,e mXeItIaIb(oml/izte65X0)(.vzCo6m06p)o,umnedtsabIoXliatendXXII(mw/ezre49d8e)t,emcetteadbionlitthiesXII
| sheapmaptloec,ytasesth(eyFiwegru6e rvoibeisearnvdedixi)n. the control sample containing T-6292 and no.
3
324. Metabolism of T-6292 by Human Hepatocytes at 6 hour
]
The full scan LC/MS reconstructed ion chromatograms of the human hepatocytes sample incubated with T-6292 for6 hour are shown in Figures 12. Figure 12 (i - x) displays the
I
on chromatograms for the following: the parent compound (m/z 570), metabolite ITT (2 746), metabolite V (nu 584), metabolite VI (mz 542), metabolite VIII (mv 556),
i !
metabolite XI (m/z 498), metabolite IX (vz 526), metabolite X (m/z 606), metabolite XII (m/z 499) and metabolite XIII (m/z 650). This sample contained the metabolites IIL, V,
VI VIL IX, XI, XII and XIII.
i
REPORT: 98AGKPO1.3M
i
04078
em ASoOvAaNnAcLePnCAC
SERVICES. ING.
TwehreeSdRisMpltaoyteadl iinonFicghurroemsat14o,gr1a5,ms16foarnmdet17a.bolNiotemseItaVbo(lrivte56I8V),waVsIIde(tme/czt5e4d0i)n atnhedsXtudy samples (Figure 14 iii and iv). Metabolite VII was present in the human hepatocytes (sFaimgpulree r1eSative)d, awnitdhtThe-6n2o9t2esft oarr6tihcloeurosr n(Foihgeupraeto1c5y+t)e,sbuctonatbrsolenstaimnptlhees0(Fhioguurressam1p3liei and 151),
The SRM total ion chromatograms of human hepatocytes incubated with T-6292 for0
!
maentdab6olhiotuer IarXeidnibsoptlhayseadmpilneFsi.gurHeow1e6ve(irv,atnhdsv)c,omrepsopneectnitvecloyu,lsdhaolwsionbgetohbesperrevseedncien tohfe
control sample containing T-6292 and no hepatocytes (Figure 16).
Metabolite X was observed in the T6292 (Figure 17), but absent
human in the O
hepatocytes hour sample
sample incubated for 6 (Figure 17 iv), and the
hours no
with
hepatocytes control sample (Figure 17 i).
3.3. METABOLISM OF T-6293
33.1. Metabolism of T-6293 by Rat Hepatocytes at 0 hour
The full scan LC/MS reconstructed ion chromatograms of the rat hepatocytes sample incubated with T-6293 for 0 hour are shown in Figure 18. Figure 18 (i -x) displays the
fon chromatograms for the following; the parent compound (m/z 650), metabolite II
|
(m/e 570), metabolite 1 (mz 746), metabolite V (mz 584), metabolite VI (nz 542), metabolite VII (my 556), metabolite IX (m/z 526), metabolite X (nz 606), metabolite
XL 498)and metabolite XII (nz 499). Apart rom the parent drug, compound XII
as detected in this sample. However compound XII was sample containing T-6293 and no hepatocytes (Figure 7x).
also
observed
in
the
control
332. Metabolism of T-6293 by Rat Hepatocytes at 6 hour
iTnhceubfaultledscwainthLCT-/6M2S93rfeocron6sthrouucrteadreiosnhcohwrnominatFoiggruarems19o.f tFhiegurrateh1e9pa(ito~cxy)tesdissapmlpaylsethe fon chromatograms for the following; the parent compound (nz 650), metabolite If
REPORT: 98AGKPO1.3M
04079
ee SERVICES. ING
Y
m(remtab5o7l0i)t.e mVeItIaIbo(lmi/tze5156),(7me4ta6b)ol,itmeetIaXbo(lvizte5V26)(,rmzet5a8b4o)l,imteetXabo(lmiyte6V06I),(vmezta5b4o2)l,ite
XI VIL
498) and metabolite IX, XT and XIL
XII
(mz 499).
This
sample
contained
metabolites
II,
V,
VI,
SRM analyses were performed for metabolites IV (nz 568), VII (nv 540) and IX
_
(m2 221
526) andX (mv 606). and ii). Metabolite VII
No metabolite was present in
IV the
was detected in rat hepatocytes
the study samples (Figure treated with T-6293 for
i ,
horounrosh(eFpiagtuorcey2t3esic)o,ntbruotlasbasmepnlteisn (tFhieg0urheosu1r3siamapnlde2(3Fi)g.urCeo2m3piofuanndd ItXhewnaostfesotunardtiicnle
t6h2e93T-(6F2i9g3urset2a4n1d,aridvsaonldutvi)o.nNaondmettheab6olhiotuerXrawtahsepoabtsoecryvteesd sinamtpheleraitnhceupbaattoecdytweisthsTtu-dy
samples (Figure 25 if and ii).
333. Metabolism of T-6293 by Human Hepatocytes at 0 hour
;
The full scan LC/MS reconstructed ion chromatograms of the human hepatocytes sample
incubated with T-6293 ion chromatograms for
for hour are the following:
shown in Figures 20, the parent compound
Figure 20 (nv 650),
i -x) displays metabolite I
the
(mz 570), metabolite TI (nz 746), metabolite V (nz 584), metabolite V1 (nz 542),
mXe1t(anb/ozli4t9e8V)IIaIndnmzeta5b5o6l)i,tmeeXtIaIbo(lintze 4I9X9)(.nvAp5a2r6t),frmoemtatbhoeliptaereXnt(dmrvug6,0c6)o,mpmeotuanbdolXiIteI
] ,
was detected in 0 hepatocytes
this sample, (Figure 7 x).
as
was
observed
inthe
control
sample
containing
T-6293
and
i
i
334. Metabolism of T-6293 by Human Hepatocytes at hour
.
he full scan LC/MS reconstructed ion chromatograms of the human heptoesytes sample
i
ifonncucbhartoemdawtiotghrTa-m6s2f9o3r ftohre 6fohloluorwianrge:sthheowpnarienntFicgoumrpesou21n.d (Firgvur6e502)1,(mietxa)boldiitseplIays the
i
(m/z 570), metabolite I (nz 746), metabolite V (m/z 584), metabolite VI (vz 542),
|
metabolite VIII nz 556), metabolite IX (vz 526), metabolite RXE(PrOuRT6:06)9,8mAeGtaKbPoOliIte3M
ii
04050
mmm ASIoCvAaNnAcLYeToICAL
SERVICES, ING
2
XI 498) and metabolite XIT (vz 499). VIL IX, XI and XII
This sample contained metabolites I, V, v1,
aSnRdMX a(nma/lzys6e0s6)w.erNeopemretfaobromleidtefoIrVmewtaasbodleitteecsteIdVi(n nthveSs6t8u)d,yVsIaImp(lnevs 5(4F0i)g,urIeX2(2miz a52n6d)
iv). Metabolite VII was present in the human hepatocytes treated with T-6293 for 6.
]
cFoonurtsol(Fsiagmuprlee23(Fvi)gubruet2a3b)s.entMeitnatbhoeli0tehoIuXrwsaasmpfloeu(nFdiginurtehe230 aivn)da6ndhotuhresntoudhyepsaatmocpylteess
:
(Figure samples
24 iv and v). (Figure 25 iv
No metabolite and v).
X
was
observed
in
the
human
hepatocytes
study
34. METABOLISM OF T-6294
34.1. Metabolismof T-6294 by Rat Hepatocytes at 0 hour
.
The full scan LC/MS reconstructed ion chromatograms of the ra hepatocytes sample incubated with T-6294 for hour are shown in Figure 26. Figure 26 (i vi) displays the
moentacbhorliotmeatVoIgIIra(mnszfSor56t)h,etfhoellpoawrienngt:cmoemtpaoboulnidte(VmIz(5m26z),54m2e)t,abmoeltiabtoelXite(uVzII6(06m)z, 540),
metabolite XI (n/z 498) and metabolite XII (vz 499). Apart from the parent drug, compounds XI and XII were detected in this sample. However compound XI was also
observed inthe control sample containing T-6294 and no hepatocytes (Figure 8v).
]
342. MetabooflTi-6s29m4 by Rat Hepatocytes at 6 hour
!
`The full scan LC/MS reconstructed jon chromatograms of the rat hepatocytes sample
:
:
incubated with T-6294 for 6 hour are shown in Figure 27. Figure 27 (i=vii) displays the
fon chromatograms for the following: metabolite VI (mz 542), metabolite VII (mz 540),
i
mmeettaabboolliittee XVIII(In(znz49385)6)a,ndthemeptaarbeonltitceoXmIpIou(nvdz(49m9z).52T6h),ismestaambpolleitceoXnta(imneyd606),
'
metabolites VI, VIII, IX, XI and XII
i
REPORT: 98AGKPO1.3M
i
04031
mmSSERRVICAES,ReINaG
-
metabolite
VIIewaes pretsenot ienthoer0more6 htoures rVaTheTp7at5o7cy0t)esasramples
607-No-- (Figure 30ii and
fi). No metaboliteX (Figure 31 ii and ii).
was observed in the 0 or 6 hours rat hepatocytes study samples
343. Metabolism of T-6294 by Human Hepatocytes at 0 hour
1 :
TinhceubfaultledscwainthLTC-/6M2S94refcoorn0sthrouuctreadreiosnhcohwrnomiantFoiggruarems28o.f tFhieguhruem2a8nihe-pavtiocyditsepslsayasmple
fon
chromatograms
for
the
following:
metabolite
VI
(ne
542),
metabolite
VII
(m/z
the 540),
3 cmmeoettmaapbboooullinittdeesXVXIIII(an(n/d2nz4X9I58I5)6w)ae,nrdtehmdeeepttaaerbceotnletidtcieonXtmhIpiIosu(smna/dmzp(4l9en9.z).H5o2A6wp)ea,vrmetertfarboomlitthee Xpar(en2t d6r0u6g),,
observed
inthe
control
sample
containing
T-6294
and
no
compound XI hepatocytes (Figure
was 84),
also
344. MetabolismofT-6294 by Human Hepatocytes at 6 hour
i
iTnhceubfaultledscwainthLCT-/6M2S94refcoorn6sthrouuctreadreiosnhcohwrnomiantFoiggruarems29o.f tFhieguhruem2a9nihe-pavtiocytes sample
fon chromatograms fo th metabolite VII (n= 56),
following: metabolite VI the parent compound (1
(nz $42), 526),
metabolite
y displays the VII (nz 540),
memteatabboolliitteesXVII,(V4II9I,8I)X,aXnId amnedtaXb1ol1ite XII (499).
metaboliteX (ns606), This sample contained
J
mSeRtaMboalniatleysVeIsI wwearseppreersefnotrminedthfeor0moertabholoiutresshVuImIa(nm/hzep5a4t0o)cyatnedsXsa(mmp/z
606).
.
No
]
and). samples
No metabolite X was (Figure 31 iv and v),
observed
in
the
0
or
6
hours
human
les (Figure 30 hepatocytes study
iv
'
35. PRODUCT-10N MASS SPECTRA
(The product-ion Figure 32. The
mas spectrum base peak is at
of metabolite 1 (MH] at vz 746) is displayed mv 526 corresponds to the loss of "CH:CH:0Glu
in
fragment. This negative ion mass spectrum contained a fragment ion atm;1 13, due to
REPORT: 98AGKPO3MI
04082
ee
ImADVANCED 26
eraTrRAERTaUONofThe gTuCaronideSeid ahdTTTaCates The Presence oF----------
[em Tw Table IV: Proposed product-ion fragmentation for Metabolite VI
]
SFS0, NTi, --caon
: | Freee |e]
CsFiy
419
J
`The product-ion mass spectrum of metabolite VI is displayed in Figure 32 ii and the
]
corresponding fragment ions are illustrated in Table IV.
REPORT: 98AGKP01.3M
04083
amBAIDCVAANNACLEYDTICAL
SERVICES. ING.
2
Table V: Proposed product-ion fragmentation for Metabolite VIII
[ CF 50T , /ei-co oo_ m556a
[sree
The product-ion mass spectrum of metabolite VIII is displayed in Figure 32 iii, The fragmentation pattern at m/z 498, 219, 169, 83 and 65 is comparable to that observed for Metabolite VI (Figure 32 ii, and the corresponding fragment ions are shown in Table V. `fTohnespartomd/uzct4-7i8onamnads7s8srpeepcrtesreunmtoaflmoestsaboofliHtFe XanIdisCysFhioswHn mioniFeitgiuersef3r3omii.thTehdeepfrroatgomneantted `molecule, respectively. 36. WPIOSTIHTTI-V6E2I9O2NIAZNADTITO-N62L9C3/MS ANALYSIS OF HUMAN HEPATOCYTES INCUBATED The human hepatocytes incubated with T-6292 and T-6293 fo6r hours were also examined by full scan LC/MS using positive ionization detection. However, the compoundsofinterest could not be detected in the positive ionization mode, and there twheerpeaernendtogdernuogunsormattheerimaeltsabporleisteentwianstohbessearmvpeldeisnatthihsigahnacloynsicse.ntration; therefore neither
REPORT: 98AGKPO1.3M
04084
;
ms SI0AeNALsYTICAL SERVICES. ING
2%
PARTE Se QUATIave ATaIySi of T529 Tr Ra am Ruma HEpaocytes------
.
Incubated with T-6292, T-6293 and T-6294 by LC/MS/MS
4. INTRODUCTION
T-6292, T-6293 and T-6294 undergo metabolic biotransformation in rat and human
hepatocytes to form T-6295. The purpose of this study was to determine the
concentration of T-6295 in human and rat hepatocytes after incubating with T-6292,
1
T-6293 and T-6295, using LC/MS/MS analysis.
i
S. EXPERIMENTAL
:
S51. STANDARD SOLUTIONS
Preparation of Analytical Standard Solutions for T-6292, T-6293 and T-6294: See Experimental Section 2.2.
:: pPerrefplauroartoioonctoafnIenstuelrfnoanlicStaacnidda-rd Stock Solution (1 mg/mL): 1H,1H.2H,2H
.
Accurately weigh 2 mg of the 1H, IH,2H,2H perfluoro octane sulfonic acid on amicro
balance and transfer the chemical to a polypropylene tube. Dilute with anappropriate
volume of methanol to yield a | mg/mL solution. Store the solution at 4 C and bring to
ambient temperature before use.
]
Preparation of Internal Standard Working Solution (8 g/mL): 1H,1H,2H;281
perfluoro octane sulfonic acid -
i temperature before use. i
Dilute 0.8 mL of analytical standard stock solution withwater in a 100 mLvolumetric
flask to yield a 8 pg/mL solution. Store the solution at 4 C and bring to ambient
5
Preparation ofInternal Standard Spiking Solution (800 ng/mL):
|
1H, 1H,2H.2H perfluoro octane sulfonic acid (10 mL at 8 pg/mL. concentration) was
.
:
sdoilluuttieodnwwitahs NaAddNeOdptuoreeacwhatsetrud(y90sammLpl)e,in a 100 mL volumetric flask. 25MLof this
REPORT: 98AGKPO1.3M
04085
rmSSEoRAVNIACELS.CIANLG
SMZethanPoRlEwPaAsRmAiTxIeOdNwOitFhQhUepEaNtCocHyIteNsGcSoOnLtrUoTlImOeNdi,a~ solution for BOI~ 1-95 (supplied by----
3M)in the ratio of 2:1 (viv).
5.3. HPLC ELUENT PREPARATION: See Experimental Section 2.3.
54. SAMPLE PREPARATION
`The metabolism study was carried out at SRI International, Menlo Park, CA. The
|
incubates were quenched with an equal volume of ice-cold methanol, centrifuged and
shipped in dry ice to ABS. The samples were stored at -20 C.
Hepatocytes isolated from Male Rat #2, Female Rat #5, Male Human #341 and Female Human #2, each incubated with 0.1 mMof T-6292, T-6293 and T-6294, respectively,
were analysed for T-6295,
aRsatcoanntdroHl usmamapnleHsepatocytes incubated with no test articles for 0 and 6 hours were used.
Five stock solutions prepared in DMSO with no hepatocytes (supplied by SRI Tnternational): T-6292 from rat 2 and rat studies; T-6293 from rat 2 study; T-6294 from
rat 2 and rat studies (10 UL each) were also used as control samples. Each sample
(10 HL) containing the analytical standard was diluted with the quenching solution
(90 WL), then spiked with the internal standard solution (25 pL).
The intemal standard solution (25 L of 1H,1H,2H.2H perfluoro octane sulfonic acid, at
|
800 ng/mL concentration) was added per study sample (100 uL).
5:5. PREPARATION OF RAT HEPATOCYTES STANDARD CURVE SAMPLES aAcpcporrodpirnigatteo vtohelusmcehsemoef To-ut6l2i9n5edwboerlkoiwn:g solution was spiked to control at hepatocytes
REPORT: 98AGKPO1.3M
C04086
moSBEIsROVAINeCAELSsY,TIICNAGL.
30
.
Plasma Standard Prep.
| |
T6295 Conc.
mL added
| HepaCtoonctyrtoelsra(tmL)|
Volume (mL)
61250
Soin saw |oso
om
53
I
100 "sun [oo [ ' 0009 Y54]
1
20TsSaumi[Toooo
| I
I
o3s 01%
054 |
Lo T7 TXo 0 73To1 m To0 ss]|
!1
One standard curve was injected at the beginning of the analytical run and asecond
standard curve was injected at the end of the analytical run
5.6. LC/MS/MS INSTRUMENTATION
5.61. HPLC conditions
:
HPLC Column BetasilC18 (2x 100mm)
Mobile Phase
10:90 90:10
MMeeOOHH::22mmMM
NH,OAc NH,0Ac
(A) (B)
LC Gradient
[Mme0t02 min Tag 30%
J
42110074mmiinn
30110001%00%
7t08 min
10010 30%
i
Flowrate ~~ 200 umin
i
562. MS conditions
i
Sciex APL IIT TurbolonSprayTM Interface
'
Ton Spray Voltage:
3900 v
Orifice:
-60V
Declustering Potential 30v.
REPORT: 98AGKPOI.3M 04087
mSo BEIROVAoINACLEsYST.IICNAG.L
Ell
i
Tomato mote
Nga
-
Collision Gas Thickness 280 x 10" atoms/square cm
Collision Energy
35ev
Dwell Time
200 msec
Transitions Monitored: m/z 499 -> 99 for T-6295
m/z 499 > 80 for T-6295
m/z.427 +> 81 for internal standard
1
The production mass spectrum of T-6295 (IM-H at nz 499) produced two fragment
|
fons at m/z 80 and 99 (see Figure 3 i), which are likely due to (SO) and (FSO5)
respectively. Both transitions were monitored.
Data System
RAD Version 2.6; LC Tune Version 2.4;
i
Multiview Version 1.2; MacQuan Version 1.4
.
5.63. Data Handling
i
All raw chromatographic and mass spectrometric data were stored on the ABS file server
:
QAuBaSn"I._FASl.DeAxpTeAriimnentthaelfiilenfhoiremraatricohnyw"aRsAsWtoDrAedTiAn:AVBaSlindoatteibooonk:3NMo:SR2E037M0S,RI Semi.
:
6. RESULTS AND DISCUSSION:
]
One set of male rat hepatocytes, one set of female rat hepatocytes, one set of mile human
]
LheCp/aMtoSc/yMtSes.aTnhdeorneesuslettsoafrefesmhaolwenhiunmTaanblheepVaIt.ocyte samples were analysed by
i sTohleutisoanms.e Tsehteorfesstualtnsdaarrde csahloibwrnatiinonTacbulreveVIsIa.mples was used to analyse the five stock
{ REPORT: 98AGKP01.3M
|
04088
mamABIDOVAANNACLEYTDICAL
32
SERVICES. ING.
Im Table VI
Vege
IQunacnutbiattaitoinvewiAtnhalTy-s6i2s92o,f TT--66229935 ainndRaTt-6a2n9d4Human Hepatocytes after
Snipcen= =
oon ooo
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oss
Cone Cale Gone
our oz
[[ooobiwBBanek
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ses
aw n |ewin | a
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sai926
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lhssaaiissiiso
[Hse ssaarmiezssscioe
wa | ain
wn | [aos |
an we
wa | wn | an
ssicon0 sicns
23461 NNootHeepTT22922000
--
34 No epTema 0
wwan | [awns || aona
ssicnss aicoso
336411 NNooipTT622903410..
=,
wo | wa |
as | son |
am wa
wa | om | an
:
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334411NNooTeesptATerzair1oc.0 34 NoTs vice 16.
as | wa |
om | am |
aa a
wa | ws |oa
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HHoammaannFReenmsi 176622592210160. HamanFest 276293 0
wa | ann |
am | ison |
ow wa
wa | ess | an
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an orn |
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| |
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awne | w[ewsa || oawen wa | me | an
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04089
er Lm lee mmmABIDDVAANNACLEYTDICAL
3
!
TIEVT
SERVICES. ING.
QUAI Incubation
ATaIySISof with T-6292,
T-629' T-6293
TarndRAT-T6A29H4u(mcaonntsinHuEepalocs
fesaer------
Cone. [ Cale Con. d)
Fo [Racou0 [Racoss
Fore fs Mle
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To e294
120
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faRattfmmlse
55NNooHHeeppTTeezw
6. 0.
Rofra No Hep7629 6.
wa | wa |
am | am |
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wa | eos |
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1.0.
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| |
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| |
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www|| swoms || ssoms
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| |
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: 1
Tno62te9st5awrtaisclensot(TdaebtleectVeId)in the control samples which contained only the hepatocytes and
:
For the rat and human hepatocytes, T-6295 was detected in the control samples which
:
were incubated with T-6293 and T-6294 but with no hepatocytes (Table VI)
REPORT: 98AGKPO1.3M
04090
mamSAIoDvAaNnAcLYeToICAL
34
SERVICES, ING
FsoarmpTlheeTs ianecuFbauterdawihtehpTa-t6o2e9ye2,sSTt6u2d9y3SaamnpdIeTs-,62T954.29T5-W6a2s95pwreassenntot ohbese0rhvae rin --the------
hour samples.
sFaomrptlhee ifnecmuablaethedumwiatnh hTe-p6a2t9o2c.y0tesansdtu6dyhosuarmpslaemsp,lTes-6i2n9c5ubwaatesdprweistehnTt-i6n2t9h3e 0anhdouT-r6294,
and For
was not the male
observed in the at hepatocytes
6hour study
samples samples,
incubated with T-6292 (Table VI) T-6295 was detected inthe and 6
hour
samples incubated with T6292, T-6293 and T-6294 (Table VI)
3
Finocrutbhaetefdemwailteh rTa6t2h9e2pa,t0ocyatneds6shtuoduyr ssaammpplleess,iTn-c6u2b9at5ewdawsitphreTs-e6n2t9i3n athned6T-h6o2u9r4s,aamnpdle
Was not observed in the O hour sample incubated with T-6292 (Table VI),
LC/MS/MS analyses of the stock solutions showed T-6295 was presenta high dcuornicenngtsraatmipolneiannaalllystihse,stahimsplafefsec(tTsabtlhee vVIaIl)u.esSoifnctheecsaerrcyoonvderseftoorfTs-t6a2n9da5rwdacsurovbes,erved particularly the calibration standards at low concentrations.
REPORT: 98AGKPO1.3M
04091
-- mm _sSBEoO-- RvAVaNInACcLEeYS-- .nTEIANGL --ueeswe 35 Table VII Quantitative Analysisof T-6295 in T-6292, T-6293 and T-6294
Fewer Stock Solutions A hsprem = oooooinso
i
Frenne Bar
conTTe. | CaoterCo)ne.|| Apcemusey
.
oasr2assos Retmso
| aoa || oasnm|| aann | ara | see | ara
rsatsessesso Rotessec
|| aana || nwsose || a | ia | 20s |e
Istitz saz
wIa | sIsEs | a 2 | ise | mas
ssaazt sa:
wwa || dswm || swoer wa | es | wm
oshe sus1
o || sssesn || oome 0 | sas | nar
Jus
Do | uss | om
Isstas61 reo
I
Basoo || o|
asmwsanrrs||
w9w8e.ae1s7
fers
o | swe | an
!
7. REFERENCES :
Report No. S6ADEMO1 3M 1
D.E. Mulvana and J. Henion
QaunadliTt-a6t2i9ve5 ibnyversattiagantdiohnuomfatnheheipnavtiotcryotmesetuasbionlgiisomnosfpTr-a6y2L9C2,/MT-S62a9n3d, T-6294
LC/MS/MS.
November 12, 1996.
2
ABS Laboratory Notebook No. 2070
REPORT: 98AGKPO1.3M
04092
ADVANCED 36
i
Figure I. Reconstructed
ion chromatogram(1); negative Tomzafion mass --------
S... hem rn asuptehcetnrtuimc T(i-i)6a29n3d .LC/MS/MS product-ion`mass spectrum (iii) of
Somosmt a i
23
56 Th aime
rt | % Intensity
650 [M-H]"
j ... |
605
i
Wom aw wo se eo oom
.
d
.w
-
169
|
ee=
319
|;i REPORT: 98AGKP01.3M
|
04093
MW oSSOEAReNVAIeLCYEeTSICIAeL
37
=
Figure2. sRpeecocntsrturmuc(it)eodfiaountchehnrtoimcaTt-o6g2r0a4.m () and LCIMSIMS production mass
TICirnonms3M T6293 & T6294 Ss Prod (1
q| o ;f o
Ta 07560e5 cps on
i Splencetrnusmyfrom 9.P4Y9 min (9 scans$ ) frome 3M T6293 & Te2m 94 sudsR Prod (1), centroidelde A3.T54es53c6ps 50
o
J
Gy
Id
|
2
|
sus
I)
|
Cm
me
-,,
6 am
em
REPORT: 98AGKPOI 3M
---
04094
1 i 1
J ] i: :
--
y a BIOAoNALsYTICAL
38
SERVICES. ING.
Figure 3. (Riei)coannsdtrLuCc/teMdS/i3 oMnScphrroodmuactt-oigornamma(s3;)s, nsepgeacttirvuemf--o(--niiiiz)aotfioanutmhaesnstiscpTe-c6t2r9u5m,
XIC (period1) % Innsity
from
499.0-49a9m.5
rom
T-6295
Sud
(1)
605 T6295
3.556 cps
80
0 60 "0
2
1 234567 e
Spectum from % Inensity
6.07
min
(2
sane)
rom
T-6295
Sd
(1), 499
subtracted, (HY
subucte,cenroided
50!
Gi 60: "0
2
9 "Time, min 149eeps
`Spectrum
20 from
6.03
m0 min (5 scans)
aw from T6295
a0 Dav 499(1)
so centeoided
io
Intensit&y
80 %
720 800 mz 4924 cps
Gi)
0
20
MH 499
) 120 150 20 300 360 a0 480 we
REPORT: 98AGKPO1.3M
04095
SO0vAaNnAcLEYDTCAL
3
SERVICES. ING
.
gre 3. FOISCA FECTS Uetedfom ChromATOEFATS oT Control FathepaTocyes ne
incubated for 6 hours with no test articles
XIC (ero nemsiey
1)
fom 1g
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a
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41
Figure 6. Fnuollhespcaatnocryetceosnstructed ion chromatogramsofT-6292 incubated for 6 hours with
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2
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;
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oi 9
1
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30264e0s pe oss
3Stes cps 7\ 100 Toes asso
2499.0499.5 ForCompound Xi
ro 352scps
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J
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Ei
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9.56
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REPORT: 98AGKPO1.3M
04103
SASeoorAvvNaiAncLceYse,nICNASL
a
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Fig[uXrIeCLp1e2r.aiodmaFeu1)tlltafbosomclaSint1ers0ec0aof:nes55rta7rimu0ncactuoebdmatii1onn1gchh1urom7ma6an2t9ohe2gp6raa2tm)oscystheoswwiintghtThe opressenece osfouJr.E.
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Fw
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m0353[aP0o 92ess
Mi on sss 510m " 6 190 on
'
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w w
610 cn
1.765 cps
ETT rT
i 04104 REPORT: 98AGKPO1.3M
_--
I2SSEoIROnVAIeNCEAsSLe.TCIANGL rr
8
Figore 13. a((r)itiSSclRResMM; (ccihh)rroSommRaatMtoogcgrhraramomsmsaootfforgahrtuahmmeaspnaotfohhceypuatmteaoscniynthceeuspbaiatntocecudybtwaeitstehdinnwcoiutbthaetnseqdartitesit;cles
no test articles.
h
IICmefrnosmiRy No TA 6h MRM 0b
1o0c2eps
3
i
0
@
"0 "
450
IC fom BOLI FI No TA 6h sample 0 MRAZ
"0
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I
i
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| di
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DOEnaE y fen
i
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Lis
1 50c2.cps
!
0
5
aw "
os TTTime, min eT a
REPORT: 98AGKPO3M1
04105
MoBIsDANeALsYTICAL SERVICES. ING
49
Figure 14. hiSenRpcauMtboaccthyertdeoswmiaittnhocTug-bra6at2me9sd2swfhioortwh6iTnh-go6ut2rh9;e2(aaibits0enhcoeuro;f m(ei)tarbaothleiptaetIoYcyinte(si) rat
T6292 hour.
at0
Hour
and
(iv)
human
hepa)tohcuymteasnihnecpuabtaotceydtewsitmhoTwp-a3t5e3fdwoirth
T%ICInrenosmitTy6292 Rs Oh vz 568
25061 cps.
.
El
IC fromBOI Ra T62926hmie 568 (1) o i) : a
100e2 ps
TIC fiom T6292 F1 Oh rv 568
Gin
ss
x
238
22002 cps
h
TICfom BOLF1 T6292 6h iz 68 (1) [)
a i 2 as
10062 cps Th Swen
REPORT: 98AGKPO1.3M
04106
mn sEeaee, ADVANCED
50
Figure
15.
SRM total ion chromatograms showing the (i) T-6292 standard solution; (ii) incubatio
presence
of
metabolite
VII
in
at 0 hour; (iii) incubation incubation of T-6292 w
of
T-6292
with
n of T-6292 with rat hepatocytes at
rat hepatocytes 6 `hour; (iv)
wonTt."ommTTa2nt8o 4c50oewnAPRA w--on of
T-6292
with
human
ith human hepatocytes hepatocytes at 6 hour.
at
0
hour
and
(v)
incubation
tom Taz vse wn"
-- crn
| waE"lT -- Fie
-- Io
Hc ton an avi
,
ssn
5.
. ye
7.80 (VID)
C4e 1s In e ee
REPORT: 98AGKPO01.3M
04107
MW 2BIoOAnNsALeYeTICAL
st
SERVICES. INC
Figure
16.
ST-R62M92tostatlaBsniodnarcdhrsoolmuatitoong;r(aim)sisnchuobwaitnigontohfeTp-r6es2e9n2cewiotfhmreattabhoelpiatteocXyitens
(i) at
--
oifnhcTou-ub6ra2;t9ii2oinw)ioiftnhTch6ubu2am9ta2ionwnihotefhpTaht-uo6cm2y9at2neswhiaettpha6trohacotuyhrte.epsaattoc0ytheosurata6ndho(vu)rin(icvu)bation
T%ICIntfernosmitTy6292 STD mz 526.
i
5of
7.2062 cps 931 @0
TIC from T6292 Rs Oh mz 526 Gy b6y0 098
L194 cps 931 (1X)
TIC from T6292 Re 6h mz 526 Gi) :
30 oss
896e3 cps 933 1%) ols
TIC from T6292FI Ohr mz 526
4
60!
1
) 30,
1.4804 cps 931%)
' i
TIC trom T6252 F1 6h mz 526
9.4803 cps
931 (0)
i
60
*
614
47
Torr 4 5 eT 8 Steen
REPORT: 98AGKPO1.3M
04108
y
m SABEDORAVVNAIANCLCEYEST.DIICNAEL
52
1
:
Figure 17.
SRM
in ()
Tto6t2al92iosntcahnrdoamrdatsooglrutaimosn.;`(sih)owiinncgubtahteipornoesfeTn-c6e2o9f
`wmietthabroaltite
X
`hepatocytes at at 6 hour; (iv)
0 hour; (iii) incubation of incubationof T-6292 with
T-6292 hu
with
rat
hepatocytes
and
(v)
incubation
ofT-6292
with
human
man hepatocytes at 0hour hepatocytes at 6 hour.
TICIefnrsoimtTy6292 STD mz 606
30061 cps
iSE
Pa loll A
A h A tTA
,:
TIC from T6292 R4 Ohr m/z 606
| @ So 3|% :
60
3.50e2 cps.
660
{
TIC from T6292 R8 6he mz 606
1]:
ai S0o
:
TIC from T6292 F1 Ohe yz 606.
105
J
46
68600
5.70e2 cps
ssseren
|
TIC from T6292 FI 6h mz 606
636 0)
S00e2cps
I
"0 1 0.96
2s
|
Cor
Ese
Thee
|
REPORT: 98AGKPO1.3M
| 04109
SASeDOAvAvaNinAeceEdDPewas
5
Figure 18. Full scan reconstructed ionchromatograms showing the presence of
KIC (u1i0m 65006505 ama rm 5011 14Tope `metabolites after incubating rat hepatocytes with T-6293 for 0 hour.
%I
o
ntensity
oe
6.15 (T-6293)
A
Waieseps
0 $700.5703Ffior Compornt
mn
rr
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4
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win9g us
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ws
0 4350490orCcoompmo pound
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To TT,
REPORT: 98AGKPO1.3M
04110
ii
_--
SBAEIDROVVAAINNCAECLSYE,TDIICNAGL
54
4
Figure 19. FafutlelrsicnacnubraetcoinnsgtrrautctheedpaitooncycthersowmiatthoTg-r6a2m9s3sfhoorw6inhgoutrh.e presenceofmetabolites
wen65i0n0e6505
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0 g8l
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Ton |
58 vim
184e5 con oa Arsen
18665 cps
2 sn A
s6 1 HOD
TT
320kcps
[2
M1
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7.0004 sens
AN
20 om S38 cos
70
16tes os
TTT em REPORT: 98AGKPO1.3M
04111
SBAEIDR0VVAAINNACCLEYEST,DIICNAG.L
55
Figure
20.
Full scan reconstructed
metabolites after inc
ion
chromatograms. showing
the
presence
of
XItCe(mpeiriyod
1)
rom
650.650am.u
urboamtBiOLnIgFhIuT6m2a9n3
0hhep2a)tocytes
with
T-6293
for
0 hour.
33065 ps
0
a1 am J
2u70.0.57051F0or Compounad s1
|
W8
20004 cps om
! cs cmp so |
2 746.0746.5 For Compound
20068 cps
Lis
64
0
55;
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5Istescos
imel ]
2 $420F.o[r5SCo4mp2ou3nd VI
22ctens
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ins 556.Fo5rC5omp6ou5nd VII
PO
ws
n
xr ~
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25260-5265 FoFYr Compound IX
256008 cos
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rater) v
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-
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ow0 8
24;5
sn
72088 cos
)
se
m/z 499.0499.5For Compound XII
w
aa
ors
5.72 (xm
ET TTT
se 8.004 cos TS.
REPORT: 98AGKP01.3M
04112
ABIOCVAANNACLYETDICAL
56
metaEbolT itesSaEfFRteUVrIiSCnCEcAuSTb,atEIiCNnGOg.humaen hTeopamtCochyOtWesRwIiotrhaTm-6s2S9h3owing represenceof
for 6 how.
IlCnc(npeirtiyod1)fom 650.0-650.5 amu rom BOI FI T6293 6 (2) 615
ses cps
0}
J
me5700-5705 11s
|
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220c4 cps
|
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605 am
B00et cps
.
me 5840-5845
wa
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205cps
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we sa20.5025 0@]
wIege3n5s6it0y.55 0 @50
TM 5260-5265ForCompound IX.
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30
1'!
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5
: i'
Giw mgl6|498.0-498.5For Compound XI
S88 vm
S80 sep A
880c4 cps
0 83526008cps
"0c 89 586
709 on \
Sides cps
i
i 499.0.499.5 For Compound Xi
w@ "
sna 10
11065 cps
Poors eT
Tt TT
REPORT: 98AGKPO1.3M
04113
I 2SSIe2OrsAvNniAccLesVsTe,INCAGL
57
! =
ER in2c9u5bawitatiholnOroatfCTheH6p2aOt9o3caywtietrshartsat6 Shheoopuarwt;oc(yift)ehsienacptr0eshoeunrcse o(fi) minecuablationToYff1r.()--
hheeppaattooccyytteess aatt 60 hhoouurr.and (iv) incubation ofuTb-a6t2i9o3nwoiftTh-h6a2g9a3sw,ith haan
T%ICInfternosmitTy6293 R Or rz S63
ps
132
-
@
]
p
Q
19 105e20ps
!
IC rom BOI Ra T6293 6hnz 568 (1) 90
.
iy
:
0
15062.cps
!
IC from T6293 F1 one vz S68 %
;
iy 60
i
0
766 510 900kicps
J
T-ICfrom BOLI FI T62936hmv 568 (1)
500 cps
]
)
30
1
Cy rae,
|
REPORT: 98AGKPO1.3M
|
04114
mea SosaensTs oa
=
Figure
23.
SRM total ionchromatograms (i) T-6293 standar
showing
the
presence of `metabolite
VII
in
at
0
hour;
(iii)
d solution; (if) incubationof T-6293 incubation of T-6293 `with rat `hepato
with
rathepatocytes
oifncTu-b6a2t9i3onwoifthT-h6u2m9a3nwihtehpahtuocmyatnes`hatep6athoocuyrt.es at 0 hcoyutresaantd6(hv)ouirn;cu(biav)tion
[ToEgoT
ssttens
i
0"* aisle hi HR| at AA AOA
TICfom 625 Reteusst
asses
w"
se
TC om T2980
?
i) 30.
it
TIC tomToe1hrs
;i
6300
Jase site risers
TIC fom T53 1 si ol iw
on Tasco sz
30
CE eeT
REPORT: 98AGKPO1.3M
04115
asBIsOANoAL,YTICAL
59
--- SERVICES. ING.
:
Figure
24.
TS6R2M93totsatlanidonarcdhrsoolmuatitoong;r(aim)sisnchuobwaitnigonthoef hours (i) incubation
Tpr-e6s2e9n3cewiotfh`rmaettahbeoplaitteocIyXteisn
(i) at
OifncTu-b6a2t9i3onwiotfhTh6u2m9a3nwiohtefhpTaht-uo6cm2y9at3neswhiaettpha6trhoaoctuyhtree.psaattoc0yhteosuratandho(uvr),inavc)ubation
TICIntfernosmitTy6293 STD mz 526
i
3
T9423e14%cp)s
TIC from T6293 R Oh mz 526
098
2.08e3 cps.
0}
:
TIC from T6293 Re 6he vz 526
098
927.1%) 20263 cps
3
576
875 931 00)
|
TIC from T6293 F1 Ohr mvz 526 098
1
=? 2 /
S852 cps
.
6K20
sem
: TIC from T6293 F1 hr mz 526
ii
P 30 T
12963 cps 03300)
&10
2s 4s 6 7% sme
REPORT: 95AGKPO1.3M
04116
;
Im SSAEODRAVVNAIANCLCEYESDCIAnLe
60
7
Figure
25.
SRM T-62
totail on
chromatograms
showing
the
presence of meta
93
standard
solution;
(ii)
incubation
of
T-6293
with
rat
bolitXe in (i) hepatocytes at
0 hour; (iii) incubation of T-6293 incubation of T-6293 with hu
with
rat
hepatocytes
at
6hour;(iv)
TIICnefnrsomtyT6293 STD mza6. 45001 cps Of T-6293 with human hepatocmyatneshaetpa6thoocuyrt.es at 0 hour and (v) incubation
i
@ ol
Alito carPL| O bay
:
TIC from T6293 R4 Ohr m/z606
of To 3.752 cps.
hd 30
938
IC from T6T29o3 Rs he vs 06
S02
ainoof [
ois
TIC fom T0502493 1 hie06.
I i
601
1 =
TIC fomT6020983 1 6h vs 06 af
2502 eps rs s0e2cps
tora EET
REPORT: 98AGKPO1. 3M
04117
ABSDEoVRAAVNINACLCEYEST.DICINAGL
6
-
Figure 26. aRfetceornisntcruubcatteidngiornatchhrepoamtaotcoygtersamwsitshhoTw-6i2n9g4tfheop0rrehsoeunrc,eof metabolites
XInC riyod 1 fom 5620-5am42ro.m BOLI R4T629 he 1ForCompoun v1
26008
0 la
Nn An semetnA
a ,
i
5400-F5orC4om0pou5nd VI
2a0ctps
|
w
MLiser
ast
: 25560-5565 For Compouna VI Sat~es
Lio
(i)
saa on
bd
RL
5260-5265 For Compound X(T6296)
sacs
! w sotMN
26060-6065ForCompound X
Sonecps
115
ses
a1 /\
J
224980.4984098-.9585FFoor ComCopmpouundnd X1
som aassess teoss
oo
1
y
2494999:5Fo.rCo0mpo.und Xi
M 0c ps
f
:
I f
som 7s A Ma
os rT oa,
i
REPORT: 98AGKPOI.3M
04118
SSAEOoRAvVNaIAnCLcEYeST,nIICNAGL
6
:
Figure 27. aRfetceornisntcruubcatteidngiornatchhrepoamtaotcoygtersamwsitshhTo-w6i2n5g4tfhoerprensoeenc:eof metabolites
%XInCte(npesriitoyd 1) fom $420542.5 amu rom BOI R T294 6 hr 1
6200s cps
o of 13
sal
28407 Fi
2 540.0-5405 For Compound VII
|
eo) 126
,
24008 ps 929
me 556.0-556:5 For Compound VII i
5I99 (vim
1865 cps
:
2 5260-5265 For Compound IX
Wg
Seassops m9 9A.40
;
V2606.0606. For Compoundx.
)
6 Lis
J
980-Fe4rCo9mp8oun5d
i1
w | o
ii
2499.0 499.5 For Compound X11
1
) 60
:
LE
i
M731m) 731 sn am
1126s cps 5
AN
sorers
2246p
|
REPORT: 98AGKPOI.3M
i
|
04119
m BAIDOVAANNACLYETDICAL
63
SERVICES, INC,
RT28 RaEftCeOrMincubatinTgohmCuhmraonmhaetpoagtroacymtseSshwoiwtihnTg-6Th2e9p4rfesoe0rncheoourf.metabolites --
XIC (period % Intensity
1)
from
542.0-542.5
amu
from
BOI
FI
T6294Oh(1)
137
@ 60:
a 192
5.18
3.00e4 cps: 9A3
m/z 540.0-540.5 For Compound VII
ii) 60:
p\
m/z 556.0 - 556.5 For Compound VIII
Lio
Gil) 60:
J
m/z 526.0 - 526.5For Compound IX (iv) 60:
m/z606.0-606.5For CompounXd ) 60.
MA A 54s A
2.404 cps 7.76 831
A~
3.40c4 cps
6m
bid
/
4.526 cps 8.86 T-6294 /TM
9.00c4 cps 9.19 /\
'
2498.0 ~498.5 For Compound XI
wi) of T
in
7.32 (Xn 1.8265 cps
A
2.49.0 - 499.5 For Compound XII iy af
1 234
$)73 (xm) 2.32 5678
1.065 cps 8.86
Time, min
REPORT: 98AGKPO1.3M
04120
I SsBEIoROsVAINnCAeELsSY,ToIICNACL
6
Figure 29. aRfetceornisntcruubcatteidngiohnucmharnomhaetpoatgorcaymtsesshwoitwhinTg-6t2h9e4prfeosre6ncheouorf.metabolites
IC (period1 % Intensity
rom 542.0-542.5am
rom BOI1 F1 T6294ahr(1)
380c8 cps
@ of A
|
m2 5400-5405ForCompound VII
|
Gi) 60:
N
761 v1) 678 A
584 240kcps
N
~
TM2 556.0 -55556.5FoForr CompCoumnpodundVIIVIII
i 60
N
2.5260 526.5 For Compound IX
[2 |
Sos vm, A / 6i m
2.4064ct cpcpss ~
331e6 cps 892 (T290) [I
2606.0 606.5For CompounXd
"|
i
:
2498.0. 498.5>For Compound XI
i 60:
7.60cd ps
914
568
/\
73301) M
3886s
2499.0.499.5 For Compound XIT
73
6
5730
Tr
as 6
9.6865 cps seen
REPORT: 98AGKPO1.3M
04121
mesSEasvamsreTo,y o t
igure 30.
lon chromatograms showing 'presenceof mel
in (i) T-6294 standard hepatocytes at 0 hour;
solution; (ii) incubation (iii) incubation
of
T-6294
with
rat
at 6 and
hour; (iv) (v) incub
incubationof
T-6294
of with
T-6294 human
with rathepatocytes hepatocytes at 0 hour
CThionmsise TD 50 350 ation of T-6294 with human hepatocytes at 6 hour.
)
o% bua FY
Mec, AAl
NT
:
oo
sen
@"
Thom Tasers senso
@
.
Ww
Chom Tewo e msi
|
i" 5
a
1
-
-TIC from T6294 FI 6he yz 540
3
233
2 s78
--
1.1063 cps
REPORT: 98AGKPO1.3M
04122
m ADVANCED 66
Figure 31.
SRM total jonchromatogramsshowing T-6294 standard s
the
presence
of
`metabolitXe
in
(i)
0
hour;
(iii)
olution; (ii) incubationof T-6294 incubation of T-6294 with rathepato
with
rat
hepatocytes
at
incubation
of
T-6294
with
human
hepatocytes
cytes at 6 hour; (iv) at 0 hour and (v) incubation
of T-6294 with human hepatocytes at 6 hour.
i 0?` 10 duc Ae A
TIC from T6294 R4 Ohr m/z 606
o
EE .
3.20e2 cps.
-
|
0
[)30
'
-
o
REPORT: 98AGKPO1.3M
04123
i
I 2BSIEoORaAVNInACeLEsYST,oIICNAG.L
67
3
mFeitgaubroel3i2t.e VI Naengdat(iiivl)emieotnaibzoaltiitoen pVrIoId.uct-ion mass spectra of () metabolite 0; Gi)
`lSnpieencsttryom 24min (6 cans)from3MT5292Rs 6 PROD
i 50
Metabolite II
]
of
0 4)
5
" woe 2
w
S0stcpe HT 745
! i:
SFphetcetnnsunmyfiom%8.48mii n(319%scans)fromBOoLInFIT6293 0r (4) ES) 0 & 720 mz
80
Metabolite VI
Hei
:
of 2
DH) 582
. i
"| ES
wo
3
a9
F`iSnpgercatyrum2i0rmo1imn
0
((112c2a5s)fom
BOI
WW F1T629G3e
x
an
ap Es as
)
-
0
Metabolite VI
a
t
iiy ] 6i. 1%
2
o 8,
a8
f
.
]
Om RT hw @ wom
iI
i
|
1 REPORT: 98AGKPO1.3M
i
04124
MoSIsOANeALsY TIAL
68
Figure 33. Product-ion mass spectra of () Metabolite XITT and (i) metabolite XI
`FSipnonneyfrom 21min (25s= cans) roanIM T292RGPRODE5458
25 12965
Metabolite X11
]
7
i
,:
20 110
aE
a
VHT 650
.
Spermfro0m 31min (3T6wsca)fom IMETD6292Raio6hPER)OD6504F88n(1) "0 reise wn
:
:
|!
@
MetaboliXteI
ET 8
}
ny 3 E) 0 70 % EF
ms me
4
|
REPORT: 98AGKPOI 3M
i 041x5
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