Document jB0jrx7yR6VyEy1N7RJewYvG2
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IDTRAITOR MYEWoLM
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Ton Spray LC/MS Determination of Perfluoro Analytical Standards Provided by 3M Medical Department
!
Prepared for 3M Medical Department, Toxicology Services,
St. Paul, MN 55133-3220
Henry Y. Wu, Ph.D. Jack D. Henion, Ph.D. Advanced BioAnalytical Services, Inc.
Date: August 30, 1995
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005902
mmmABIDOVAANNACLYETDICAL
-_-- SERVICs ES,sING. TABLE OF CONTENT
SIGNATUREPAGE oot
9SMYHWOLIM
Page
i
LINTRODUCTION +.vtiieie eee
1
!
1.1 Study OBJECHVES eee
aneas eesti
1
i
1.2Chemical Structures, Namesor 3M Identifications, and MolecularWeights ... 1
2.MATERIALSANDMETHODS .......o0oiitiitiiaiiiiene ae. 2
2.1 ChemandiMatcefiaalsls ..........................cc.c......... 2
22 LOMS INSIUMENGtON +.
3
2.3PrepofaStorckaandTtestiSoloutinons ............................
3. RESANUDDL ISCUTSSISON ........oiuiiiiiiiaiaeea iS
BET-6295 enti
eeeeeeL 6
B2T6294 oii
6
BBT-6293 titi
BAF0SE AMIE oi
3.5Perfluorooctanamide +...
8
i
4CONCLUSION ......iuuiiiiieii icici. 8
:
S.FIGURES ....iuiiitiiitiite ie eeeeee
LO)
Figure1. Total Ton Chromatogram(TIC)fortheInjection of 1 iL of DMSO ...... 9
Figure 2. TIC for the Injections of 1 uL of Top, Middle and Botiom Day Zero
Solutionof T-6295 +... uuu
10
Figure 3. TIC for the Injections of 1 jL of Top, Middle and Bottom Day Seven
Soluotf Ti-6o29n5 +...
1
Figure 4. Extracted lon Chromatograms for the Injection of 1 kLof Day Zero Top
SoloufTt62i95o..n. +... iui
12
Figur.e Full-Scan Mass Spectrumof Peak 1in Figure 4 .................. 13
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Figur6e.Full-Scan Mass SpectrumofPeak 2in Figur7e. Full-ScanMass SpectrumofPeak 3inFigured
Figured... _..........
14 15
FFiigguurr9e8e.. FFuullll--SSccaann MMaassss SSppeeccttraruomfoPfeaPkesakS4aainnFdigSubrienFigur4e ...4.
jo 17
Figure10. Full-ScanMass Figure 11. Foll-Scan Mass
SpectrumofPeak SpectrumofPeak
6 inFigure 7inFigure
4 4
................ ............ ...
1g |g
Figure 12. TIC for the Injections of 1 uL of Top, Middle and Bottom Day Zero
!
SoluoftTi62o94n ................................... 2
Figure 13. TIC for the Injections of 1 LL of Top, Middle and Bottom Day Seven
y
SolofuT-6t294ion ieee Figure 14. Extracted lon Chromatograms for the Injection of 1 uL of Day Seven
21
Top SoluotfTi6o29n4 .........cceeeii Figure 15.Full-Scan Mass SpectrumofPeak |inFigure 14...
2 3
Figure 16.Full-Scan Mass SpectrumofPeak 2in Figure Figure 17.Full-ScanMass Spectrum ofPeak 3inFigure 14
14... _................
24 25
Figure 18.Full-Scan Mass Spectrum ofPeak 4inFigure Figure 19.Full-ScanMass Spectrum ofPeak SinFigure 14
14... .................
26 27
Figure20. Figure 21.
Total TIC
Ton for
Chromatogram(TIC)fortheInjectionof 1 the Injections of 1 iL of Top, Middle and
iL of Water... Bottom Day Zero
28
SoluOftT6i29o3n ................................... Figure 22. TIC for the Injections of 1 JL of Top, Middle and Bottom Day Seven
2
SoluOtfTi6o29n3 ..............cceeeeiei Figure 23. Extracted lon Chromatograms for the Injection of 1 pL of Day Seven
3
FigureB2o4.ttFuolmlS-oSlcuatnioMnasosf TS6pe2c9t3rum o.f..Pe.a.k..1.i.n .Fi.g.ur.e..2.3 ...............................331
Figure 25. Full-Scan Mass Spectrum ofPeak 2in Figure 23... Figure26.Full-ScanMassSpectrumof Peak 3in Figure 23...
33 34
Figure
27. TIC for the Injections SoloufFtosie Aominde
of 1 L of Top, Middle and Bottom Day Zero ................................_
35
Figure
28. TIC for the Injections SoloufFtosie Aominde
of 1 L of Top, Middle and Bottom Day Seven ................................
3
Figure 2S9.oExtlraocufteFdtolsoienAComhirdnoem.atogr.a.m.s.f.o..t.h.I.n.j.ct.i.o.n.o.f.1..i.L_o.f_D.a.y..Z.e.r.o Top %
Figure 30.Full-ScanMassSpectraofPeaks 1aand IbinFigure 29...
38
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Figure31. Full-Scan MassSpectrum of Peak 2.inFigure 29 ................. 39
Figure32. Full-Scan MassSpectrumofPeak 3inFigure 29 ................. 40
Figure 33. Full-Scan Mass SpectrumofPeak 4inFigure 29 ................. 41
Figure34. Full-ScanMass SpectrumofPeak Sin Figure29 ................. 42
Figure 35. Full-Scan MassSpectrumofPeak 6inFigure 20 ................. 43
Figure36. Full-Scan Mass Spectrum ofPeak 7inFigure 29 ................. 44
Figure 37. TIC for the Injections of 1 iL of Top, Middle and Bottom Day Zero
|
SolutionofPerfluorooctanamide ............................... 45
!
Figure 38. TIC for the Injections of 1 L of Top, Middle and Bottom Day Seven
i
SoolfPuerfltuori ooctoanamnide ............................... 46
Figure 39. Extracted lon Chromatograms for the Injection of 1 pL of Day Zero Top
SolutionofPerfluorooctanamide ............................... 41
Figure40. Full-Scan Mass Spectrumof Peak 1inFigure 39 ................. 48
Figure41. Full-Scan Mass Spectrumof Peak2inFigure 39 ................. 49
Figure 42. Full-ScanMassSpectrumofPeak 3inFigure 39 ................. 50
Figure43. Full-Scan Mass SpectrumofPeak 4inFigure 39 ................. 51
Figure44. Full-ScanMass SpectrumofPeak Sin Figure 39 ................. 52
Figure 45. Full-Scan MassSpectrumofPeak 6inFigure 39 ................. 53
Figure 46.Full-ScanMassSpectrumofPeak 6inFigure 39 ................. 54
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005905
mmBAIDDVANAANLCYETDICAL i SERb VICES, ING id,
SsYHWOLIM
ADVANCED BIOANALYTICAL SERVICES
SIGNATURE PAGE
Title: Ton Spray LC/MS Determination of Perfluoro Analytical Standards Provided by 3M Medical Department
|
:
Reported by:
Henry Y. Wu, Ph.D.
Senior Research Scientist
8(3( 28
Date.
Reviewed by:
Zz
Tan 1
eft
"Mary Ririgwy s.
Date
Quatfty Assuran Manager
Authorized for
Release by:
aR Rtg
Jack/Henion, Ph.D. ABS Laboratory Director
ry 30, (975
i
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00590
AEpv2anc5en 5
-_--_-- SERVICES, INC.
SUMMARY
SSMYHWOL3M
Five perfluoro compounds including T-6293, T-6294, perfluorooctanamide were analyzed by full-scan ion spray LC/MS
T-6295, fose for composition
amide and identification
and ambient temperature stability in DMSO and water (only for T-6293). The on-line LC/MS
determination of the listed test articles was carried out under unit mass resolution conditions
following mass axis calibration using PPG reference compounds. Chromatography was
i
performed on a 2 x 100mmDeltabondTM C8 column using linear gradient elution with acetonitrile-
i
water from 5:95 to 95:5 in 15 minutes. The mass spectrometer was operated in the negative ion mode. The LC/MS experiments showed that all five perfluoro compounds contain some
impurities. In addition, all five perfluoto compounds are stable in DMSO and waterforat least a
week at ambient temperature at a concentration of 10 mM. No apparent degradation was observed
by LC/MS analyses of these samples. Attempts to obtain molecular ion information for
compounds T-6292 and butyl fose by ion spray and atmospheric pressure chemical ionization
(APCD) were unsuccessful. However,poorresponse from both ionization modes did not provide
sufficient sensitivity for the LC/MS analysis of these two compounds.
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1. INTRODUCTION
aSnedvepnerpefrlfulouroorooctcaonmapmoiudendwseriencltuhdeintgarTge-t62m9o2l,ecTu-l6e2s93f,orT-q6u2a9l4it,atTi-v6e29c5o,mpfoossietaimoindea,ndbusttyalbifloistye
ademtiedremiannatdiopne.rflAumooronogcttahneamm,idfievewepreerflaunoarloyzceodmpboyufnudlsl-isncacnluidoinngsTp-r6ay29L3C,/TM-6S29f4o,r Tc-o6m2p9o5s,itfioosne
LidCen/tMifSicadteitoenramnidnaatmiboinenotftethmepelriasttuerde tsetsatbilairttyicilnesDMwaSsOcaanrdriweadteorut(ounnldyefroruTn-i6t29m3a).ss Trheesoolnu-tliionne
|
conditions following mass axis calibration using PPG reference compounds. Chromatography
1
was performed on a 2
acetonitrile-water from
x 100 mm DeltabondTM C8
5:95 to 95:5 in 15 minutes.
column using linear gradient elution with
The mass spectrometer was operated in the
negative ion mode. some impurities. In
The LC/MS addition, all
experiments showed that all five perfluoro compounds contain five perfluoro compounds are stable in DMSO and water for at
least a week observed by
at ambient temperature ata concentration of 10 mM. LC/MS analyses of these samples. Attempts to obtain
No apparent degradation molecular ion information
was for
(cAoPmCpDo)unwdesreTu-n6s2u9cc2esasnfdulb.utyPloofrosreesbpyonisoenfsrpormayboatnhdpaostimtoisvpehaernidcnepgraetsisvuereiocnhiezmaitcioanl mioondiezsatdiiodn
not provide sufficient sensitivity for the LC/MS analysisofthese two compounds.
1.1 Study Objectives
`The objectives of this study were:
* to qualitatively determine the composition ofeach perfluoro compound.
*
to qualitatively determine the stability of each asa stock solution at 10 mM concentration.
compound
in
DMSO
or
water
(for
T-6293
only)
12 Chemical Structures, Names or 3M Identifications, and Molecular Weights
i GF; --S--o0'K* Il o
T6295
MW = 538.22
anion m/z = 499
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005908
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mmABIDOVAANNACLYETDICAL
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TT
0
F I 7 CH,CH;
CaF --SI--I N__H
TM -62=045W 27.20 anion mz = 526
CF --sTiS --~atCHaHn,o--iI --owg M T62=936W 85.29
|
2H
|
`monoanion m/z = 650
3
!
o
O'NH,*
arn 0._CHyH,
nin
i anca,on
MW =571.25
i
CoFin --5I--l Nm,
Nanwionimls=144z98
I CrFis --C--NH,
MPWerfl=u4o1ro3o.c0t9anamide anion m/z = 412
GFir --S$i--N_{~_CH;CH,CH, CH
Il HH 0H
butyl fose M=5W 99.31
2. MATERIALS AND METHODS 2.1 Chemicals and Materials SAtl.lPsaeuvl,enMpNerf5l5u1o3r3o-c3o2m2p0o.unMedtshwyelrseulpfrooxviiddeed(DbyMS3OM, MCeadtic#a2l7,D0e4p3a-r1t)mweants,oTboixaiinceodlofgryomSeSrviigcmeas., Aldrich Chemicals, Milwaukee, WI 53233. Acetonitrile (Cat #0154) was obtained from
2
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SERVICES, ING.
pBuaxrtcehra/sSecdiefnrtiofmicJ.PTr.odBuacktesr,,MDcanGvaerws,PaMrkA, I0L1962030.85.HigAhmmpuornitiyu(mNAANceOtpautree()Cawta#t7eTr0w5a9s6-m0a1d)ewabsy Bamstead Model D7331 Ultrapure Water System, Dubuque, IA 52001.
All materialsand equipment may be substituted by equivalent materials 2nd equipment,
Fisher Vortex Genie 2TM, Cat #12-812, Fisher Scientific, Springfield, NJ 07081
i
Microbalance, Model MTS, Mettler-Toledo, Inc., Hightstown, NJ 08520 Siliconized Graduated Tubes, 2 mL, Cat #M-720, Phenix Research Products, Hayward, CA
94545
i
Pipettes, Cat #P-1000, P-200, P-100, P-20, Rainin Instrument Co., Woburn, MA 01888
Siliconized Green Slick Tips, | mL, Cat #TGS-415BRS, Phenix Rescarch Products, Hayward,
CA 94545
Siliconized 94545
Clear
Tips,
250
uL,
Cat
#T-051
BRCS,
Phenix
Research
Products,
Hayward,
CA
Polypropylene autosampler vials (siliconized in house), Cat #500 102, Sun Brokers, Inc.
Wilmington, NC 28402
Snap Cap, 11 mm, Cat #69158-P, Scientific Resources Inc., Eatontown, NJ 07724
Solvent Filtration Apparatus, Cat #32-00610, Krackeler Scientific, Albany, NY 12201
Vacuum Pump, Cat #54-907, VWR Scientific, Rochester, NY 14603
0:45 pm Nylon Titan Membrane Eatontown, NJ 07724.
Filters,
Cat
#74547-NN,
Scientific
Resources
Inc.,
Liquid Nitrogen, Cat #SS180N-NIT4052, Empire Airgas, Elmira, NY 14901
U.H.P. Nitrogen, Empire Airgas, Elmira, NY 14901
U.H.P. Argon, Empire Airgas, Elmira, NY 14901
Fused-silica, 100 id. PEEK wing, 0.007"
x id.
170 pk 0.d., Polymicro Technologies, Phoenix, AZ x 1/16" 0.4., Cat. #1536, Upchurch Scientific,
85023 Inc., Oak
Harbor,
WA 98277
2.2 LC/MS Instrumentation The liquid chromatography part of the system included a Hewlett-Packard Model 1090L pump,
Hewlett-Packard, Avondale, PA 19311. DeltabondTM C8 columns, 5 um, 2 x 100 mm, and DeltabondTM C8 guard cartridges (5 jim, 2 x 10 mm) were obtained from Keystone Scientific, Inc.,
3
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Bellefonte, PA 16823. The mass spectrometer was a PE-SCIEX API III* fromPE-SCIEX, `Thomhill, Ontario.
HPLC Conditions:
Flow Rate
200 pL/min
`Column Temperature
Ambient
Injection Volume
na
|
Column
2x 100 mm DeltabondTM C8
Guard Cartridge
2x 10 mm DeltabondTM C8
]
Pressure
Eluents
70 bar
t (min) acetonitrile
Fowater
0
5
95
15
95
5
`This program was used for
For the analysis of T-6293
the
sam
analysesof
ples, water
T-6294, T-6295, fose amide and`perfluorooctanamide.
was replaced by 10 mM ammonium acetate with the
same gradient program.
Mass Spectrometer Conditions: Ion Spray Voltage
Orifice Voltage
Declustering Potential
Multiplier Voltage
Curtain Gas (UHP. N)
Nebulizer Gas TM)
Interface Heater
Turbospray Temperature
Turbospray Gas Flow Dwell Time
Mass Scan Range
Scan Rate
-4200V
"90 V (-70 V for T-6293)
60 V (-40 V for T-6293)
4000V
14 Limin
50-70 psi
60C
500C
4.0 L/min 1 msec
100-900 Da
1.69 sec/scan
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Count Controller (CC)
10
Mass calibrationof QU in the range of 100-900 was performed daily by the infusion ofa 10M%
PPG (polypropylene glycols, average MW 425, 1000 and 2000) calibration solution (PE-SCIEX
APTI" operator's manual) at a flow of 10 KL/min. Calibration was performed on m/z 59.0,
325.3, 500.38 and 906.7. The mass spectrometric parameters, MS/MS peak
sensitivity are optimized daily. Peakwidthsare approximately 0.7 amu at half-height
widths and
in thesingle
MS mode.
!
2.3 Preparation of Stock and Test Solutions
i
Stock Solutions: Accurately weigh approximately 5 mg of one of the perfluoro compounds
(assuming purity 100%) on a micro balance in a siliconized graduated tube (2 mL). Dissolve the solid sample in an appropriate amount of DMSO or water (for T-6293 only) to yield a 10 mM
solution. Store the solution at ambient temperature.
Test Solutions: Carefully take 10uL oft. he 10 mM stock solution from the top, middle and bottom parts of the solution, and dilute them with DMSO or water (for T-6293 only) by 10- to 100-fold to give test solutions with concentrations of 100-1000 HM. Vortex-mix the diluted solutions, and analyze them immediately after preparation.
`The test solutions with same concentrations for the analytes were prepared on day zero andday seven in the same way from the same stock solution. The stock solutions were stored at ambient temperature on the lab bench without disturbance.
3. RESULTS AND DISCUSSION
As requested by 3M, the polypropylene tubes and vials used in this work were siliconized. The
`Pipette tips used in
different parts of a
this work were also siliconized.
solution on both day zero and
The LC/MS conditions for
day seven were the same.
the
To
analyses of
avoid cross
contamination, solvent blanks (DMSO or water) were injected before the injection oaf different
type of test solution. No LC/MS data were obtained for compounds T-6292 and butyl fose,
`because the molecular ion signal was too weak to be used to perform LC/MS experiments.
The on-line LC/MS determination of the five compounds tested was carried out under unit mass
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{
differentiation of molecular weights differing by only one dalton, e.g. the sulfonamide sulfonic acid at m/z 498 and 499, may be reliably determined under these conditions.
vs.
the
i
31 T-6295
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shown in Figure 4. Full-scan single MS spectra of the components detected from Figure 4 are
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present is the m/z 499 anion.
32 T-6294
The and
TIC day
from the injections of top, middle and bottom seven were summarized in Figures 12-13.
parts of the T-6294 solution on Extracted ion chromatograms
day from
zero the
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14 are shown in Figures blank and different parts
15-19. of the
By carefully examining the T-6294 solutions, it appears
TIC from the injections of solvent that the bottom part of the solution
contains fewer impurities. In addition, ions at m/z 297.5, 311.5, 325.5 and 339.5 showed higher
relative responses (compared to the ion at m/z 526 for [M-HJof T-6294) for the day seven
6
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SSMYHWOLIM
solutions than 5.6-8.5 min).
those obtained Again, the m/z
for the day zero 526 anion is the
solutions (See Figures 12 and major species in the sample.
13
in
the
regions
of
33 T-6293
T-6293 was solutions for
not soluble T-6293. A
in DMSO. typical TIC
Therefore, water from the injection
was of 1
used to prepare the stock and uL of water is shown in Figure
test 20.
i
Total ion chromatograms from the injections of top, middle and bottom parts of the T6293 solution on day zero and day seven are summarized in Figures 21-22. Extracted ion
:
1
bcohtrtoommatpoagrtraomfsthfersotmoctkhesoilnujteicotnioanreofpr1eskeLntoefd dinayFisgeuvreen2t3e.stFsuolllu-tsicoann (s1inmglMe)MdSilsupteecdtrfaroomf tthhee
components detected from Figure 23 are shown in Figures and 369 were detected. Based on infusion experiments,
24-26. the ion
Components at m/z 650, at m/z 526 in Figure 25
630 is a
fragment different
of m/z 650. parts of the
By carefully examining the TIC from the injections T-6293 solutions on both day zero and day seven,
of solvent blank and there is no apparent
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somewhat higher than that for the day zero bottom solution (see Figure 21),
3.4 Fose Amide
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airnejepcrtieosnenotfed1 piLn.Foifgudraey2z9e.roFutelslt-sscoalnutsiionng(l1e0M0SMs)pecdtirluatoefd tfhreomcotmhpeotnoepnptasrtdeotfetchteedstionckFisgoulrueti2on9
are shown in Figures were detected. Peaks
30-36. marked
Components with an X are
at m/z 606, 499, 498, 398, 339.5, 325.5, and 311.5 from the chemical background of the LC eluent (see
Figures relative
34 and 36). response for
Interestingly, the top the ions at m/z 499,
portion of the day zero 339.5, 325.5 and 311.5
solution showed much higher than those obtained from the
middle showed
and the
bottom portions of the same day solution. On opposite trend for the responses of those ions.
the contrary, the day seven solution The ion at m/z 499 appeared at two
wdihfifleerepnetarket1ebntiisotnhetiimseosto(psiccefpoeramko1fatahendma1jborincFoimgpuornee2n9t).atmPezak419a8is(speoessFiibgluyrefsr3o0m aanndim3p5u).ritByy,
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carefully examining the TIC from the injections of solvent blank and different parts of the solutions of fose amide on both day zero and day seven, there is no significant difference between the three
parts of the solution and there is no apparent change in the composition of the stock solution at
ambient temperature for a week.
3.5 Perfluorooctanamide
`The TIC from the injections of top, middle and bottom parts of the solution of`perfluorooctanamide
i
on day zero and day seven are summarized in Figures 37-38. Extracted ion chromatograms from
the injection of 1 uLofday zero test solution (1 mM) diluted from the top part of the stock solution
1
are presented in Figure 39. Full-scan single MS spectraofthe components detected in Figure 39
:
are shown in Figures 40-46. Major components at m/z 412, 392, 369, 339.5, 325.5, 311.5 and
297 were detected. Peaks marked with an X are from the chemical background of the LC eluent
(see Figure 46). Again, the top portion of the day zero solution showed much higher relative
response for the ions at m/z 339.5, 325.5 and 311.5 than those obtained from the middle and
bottom portions of the same day solution. The relative responses for the ions at m/z 339.5, 325.5
and 311.5 for the day seven solution were significantly lower than those obtained for the. day zero
solution. Itis clear from Figure 39 that this sample has several `components present in addition to
the test article of interest.
4. CONCLUSION
Five perfluoro compounds were analyzed by ion spray LC/MS. In general, all five compounds are
stable in DMSO or water (for T-6293 only). No significant degradation products were detected for
any of the compounds analyzed. However, it appears that the low molecular `weight impurities are
not uniformly distributed in some solutions.
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5. FIGURES
SSMYHWOLIM
Figure 1. Total Ion Chromatogram (TIC) for the Injection of 1 uL of DMSO
TIC of al masses, 10010 500
1DMuLSoOf DLCM/SMOS,3d.et7a/b51o9n5d-C108:A45CANMHZO gradient(S% to 95% in 16min)
i
1
55,000.00
1
7
:
22
2
00T
12081 25071 assor
"01a
1sao1t
ScanTime (min)
9
-
00591
MASEpEvaAnEcRen, ee
T--
Figure 2. TIC for the Injections of 1 AL of Top, Middle and Bottom Day Zero Solution of T-6295
"nr Top
sammie
TM ge i: .
Middle
55,000,000
i i.
L | l Bottom 7
5
&2
53,500,000
-; Iwm5SonntwE am m Em w
10
=
005917
mmASIDOVAANNACLYETDICAL
SERVICES. INC.
-
TTT
Figure 3. TIC for the Injections of 1 uL of Top, Middle and Bottom Day Seven Solution of T-6295
100] Top
30,700,000
gs 7
g
)
fs
i
2=
10) Middle g 78
F 5:
22
29200000
107 Bottom g
Ex
22
010
21081
25051
8051
10113
`ScanfTime (min)
n
31,700,000
154011 -
005918
MA2DV4ANC0ED 0
---
SERVICES, ING.
SSMYHWOLIM Figure 4. Extracted lon Chromatograms for the Injection of 1 uL of Day Zero Top
Solution of T-6295
1T6L2(8150L0 UMOAOpM)T7-/6S529955-,-d1o11l:a1b1o-AndMCB ACNIHEOf(o S86%% in 15 min) 1001300 iTIC
5.400.000
|
zomase
Isa5r340
I g as8/399
z E3 wes 2
&z
499/499
1
3 4 EA
5a /
saa/s40
A
20000
0.000 130,000 100.000 2.100000 30.000
earre or
'
a0000
010 %205 5108 26775 315060 1a25 15590 Time (minyScan
12
-
005919
[| ARDVyANoCEbD i SERVICES, ING
TTT,
Figure 5. Full-Scan Mass Spectrum of Peak 1 in Figure 4
STPcr6ao2nf0sl5e40LQ-OT4MS2SCm.Ai1Nns7519355.&1415:7110A0TMT6i2m9o5n1L.O1M3mS-i1n1Scans 4042
21pUeLa(k10sD UMAop)T6285,dotabondCBACNHE0(0S65%% in 15min) wo, 1850
990
i
s 2090
zg f2 w
g
2s
2 & we oo ao
13
--
005920
MSEeSnS Sel ervoESNG
Figure 6. Full-Scan Mass Spectrum of Peak 2 in Figure 4
piotosarsA on , SI, I amniretns
1 peak
22,857
gg
Efw
2 Ez
&
-
. = on = =
"
}
005921
ADVANCED SBEIROVANIACLEYST.IICNACL
Figure 7. Full-Scan Mass Spectrum of Peak 3 in Figure 4
PSrcoafnlse 1Q413.S1C5A0 Nmis
11385
T6295LOMS- Scans 178.70 Time=t23min
143-169
T11p6UeL2a(91k500LOuMMASG.R1)T7-5529555,.do1t1a:1b1onAdMCBACNM2O(S% 0 55%n 15min)
wo
00
ssmvwoL IM 7118
es
Zz
f w
&
2
o 7% w me wo ES
1s
005922 _
15 Catherwood Road a Ithaca. New York 14850 m (607) 266-0665 m Fax (607) 266-0749
ADVANCED BIOANALYTICAL SERVICES, INC.
Figure 8. Full-Scan Mass Spectrum of Peak 4 in Figure 4
Profi QTSCAN
T6295LOMS- Scans 197-205
TSc6a2n9s51L97.2O05 m-iMn7u1s5S915-171.81:81.2111A4M.700 Time=5.64 min
21uL(0pUoMskIsop)T-6295,dolabond C8ACNHZO(S% to 95% n 15min)
100.
90
SSMYHWOLIM sam
7s
g z
fw
2
3
2. 451.0
o 20 % me oo 0
16
005923 -
15 Catherwood Road a Ithaca. New York 14850 w (607) 266-0665 m Fax (607) 266.0749
ABIDDVAANNACLYETDICAL
SERVICES, INC.
ssmvitvor sm
Figure 9. Full-Scan Mass Spectra of Peaks 5a and 5b in Figure 4
ST pcraonfs 22QO 0r.2s2c8amnuM sen1s11S 1191T8628025TLr8OeM5=S67251cma2irns20.225
TULp(10 eak MAoT205 tabardCOACNHAO(% 095% i 15min
120480
)
"we
i
gz x
|
f: a
i=
o EY
=
=
profi Qrscan
Tie205LOM wceane 28.238
ST3eUazLre1s2020O8c.Mh23So8)iT6s25155d1a181a10A8C4RAT0CN0rHZ7O.I0Em%0i1n98% i 15 mi)
peak
0
4000
= 1758800
E7
z
f2 o
2>
o EY
= we =
=
17
005924 -
15 Catherwood Road w Ithaca. New York 14850 m (607) 266-0665 w Fax (607) 266-0749
ADVANCED BIOANALY TICAL SERVICES, INC
Figure 10. Full-Scan Mass Spectrum of Peak 6 in Figure 4
Profi QTSOAN
T6295 LOIMS-1/Scans 243.245
STc6a2n9s526L3-2O45mi-Mn7u5s/S915-2-410-1:81.2114A6M.712 Time=6.85min
41pueLa(k10s0 UMAop)T-6295,deftabondC8ACNH0/(5'%0 95% in 15min)
100, 1830
SSMYHWOLIM 156,683
7s]
zg w
i
25. ais S490
was
o 200
0
0
0
mz
8
005925 -
15 Catherwood Road a Ithaca. New York 14850 a (607) 266-0665 m Fax (607) 266-0749
ADVANCED BIOANALYTICAL SERVICES, INC Figure 11. Full-Scan Mass Spectrum of Peak 7 in Figure 4
9SMYHWOL3M
Profle Q1SCAN
T6295LOMS-1/Scans 260-262
T`S6c2a8ns52L 60-2O 62 m-iM 7n1u5s/S 915-2-51- 61:&1211 6A6-M700 Time=7.33 min
1uL{0 uMop)T-6295,deliabondC8ACNH20(5%to 3peaks
95%
in
15
min)
100, 1830
101,898
|
75:
g
s
3255
25.
607.0
o: 200
400
600
800
mz
19
00592 -
15 Catherwood Road Ithaca. New York 14850 w (607) 266-0665 m Fax (607) 266-0742
ABSEIDROVVAAINNCAECLSYE.TDIICNACL
savior
Figure 12. TIC for the Injections of 1 uL of Top, Middle and Bottom Day Zero
Solution of T-6294
h Top
s
i
:Zz
| $=
50,900,000
10] Middle
igm
&3 2
162,200,000
10 Bottom
Sean0000
H i
ob; w =u mw sean mm E e) w mm@
Bl
005927 -
15Catherwood Road m haca. New York 14850 = (607) 266-0665 = Fax (607) 266.0748
ADVANCED BIOANALYTICAL SERVICES, INC.
9SMYHWOL3M
Figure 13. TIC for the Injections of 1 uL of Top, Middle and Bottom Day Seven Solution of T-6294
101 Top
43,500,000
gg 7
5
i
2
1
3
10) Middle
Ig
52
29,300,000
10 Bottom
5 g
7
z2
2
52
33,900,000
010
10218
20516
38051
4"031
1540.11
ScanTime (min)
21
005928 -
15 Catherwood Road w Ithaca. New York 14850 (607) 266-0665 m Fax (607) 266-0749
ABIDOVAANNACLEYTDICAL SERVICES, INC
ssmviwoL am
Figure 14. Extracted Ion Chromatograms for the Injection of 1 UL of Day Seven Top Solution of T-6294
0 T6294TL2OM9S.410- 7c1u29r5)-d1o0t8aPGM5 ACNHZOISKACN to SACK in 15min)
T0000
-- 10 TIC
ari0000
]:'
g
_,
wsrsrzers
|
40000
zg s.
|
mss
250000
2
: 1 sss4
150000
gt
E
2 Hoses ggn E3
soarsze g2
z
g "\ 2s
T0000 10000 soom s7esr wsoe muss msom Time (minySean
005929
22
-
15 Catherwood Road m Iinaca. New York 14850 m (607) 266-0665 Fax (607) 266.0749
ADVANCED BIOANALYTICAL SERVICES, INC.
Figure 15. Full-Scan Mass Spectrum of Peak 1 in Figure 14
ProfleQISCAN
T6204 LOMS-1/Scans 212.221
STc6a2n3s42L1O2M22S1-1mi-n7u/s1201-9251-018:.0822P5M712Time=6.08min
21pleofaTk-s6294(100ubop).detaC8ACNH2O(tSoSASACCNN in 15 in)
0, 1630
SSMYHWOLIM 36.447
7.
g
2075
fw
$
&
25.
o 200
Wo
50
BE
mz
ES G05930 15 Catherwood Road m Ithaca. New York 14850 m (607) 266-0665 m Fax (607) 266-0749
ADVANCED SBEIROVANIACLEYST,IICNAGL
ssmvwoLM
Figure 16. Full-Scan Mass Spectrum of Peak 2 in Figure 14
-6Spcr2afni4so2L3Q0IO-S2NC34EANmi1nu2s 51%218 220T46850721924TLiOmMeSx-1Scaminns230.234
apleosfT-6254(100 ido)doaG8ACNHZO(SXACN 0 SACiNn 15min
.
ans
or
i
i
[8
1550 5&
2 ew
3
2
2500 o FS 3 J ES wo
hy
005931
15 Catherwood Road w Ithaca. New York 14850 m (607) 266-0665 mw Fax (607) 266-0749
BAIDOVAANNACLEYDTICAL
SERVICES, INC.
Figure 17. Full-Scan Mass Spectrum of Peak 3 in Figure 14
SPcraonflse
2Q45I.S2C47ANminus
1232
T6204 8.256.712
LOMS-A/Scans Time=6.88 min
243.247
T101 6T2-9642L0O4M(S1.1007u1b2Ac5p5) +d1o0a8CPMBACNHEO(foS5KAACCNNin 16 min)
4pea0ks
ass
SsMYHWOLIM 150440
7s a0
g
Zz
ow
&
2
2600 a0 o E) % me ES a0
25
G05032
-
15 Catherwood Road m Ithaca. New York 14850 (607) 266-0665 wm Fax (607) 266-0749
ADVANCED BIOANALYTICAL SERVICES, INC.
Figure 18. Full-Scan Mass Spectrum of Peak 4 in Figure 14
-Q1SCAN
T6294 LCMS-1/Scans 257-260
TSc6a2n9s42L57-C260ImiM7n/u1s2S/19-5-2-510:1&082P-70M712 Time=7.26 min
p1 eulaofsT-6294(100ubtop)dolta C8ACNHZO(S%ACN to 95%ACN in 16min)
EY
9SMYHWO13M s0865
I
} 7
g
2
1830
3430
200 "0 me 00 800
15 Catherwood Road
G05933
2%
-
Ithaca. New York 14850 w (607) 266-0665 m Fax (607) 266-074
ADVANCED SBIEORAVNIACLEYST,IICNAC.L Figure 19. Full-Scan Mass Spectrum of Peak 5 in Figure 14
Profle QISCAN
T6294 LOMS-/Scans 432-435
`TS6ca2n9s44L32O-M4S3-51mi-n7us121-541-018:.084P60M712Time=12.20 min
31pleoafkTs-6294(100 uMop)l.taC8ACNIH2O(SACN to 95%ACiNn 15min)
oo
s260
9SMYHWOLIM 1.232.500
7s
z = 2
2 1690 2190
o 200
0
50
500
mz
2
0050,32
-
15 Catherwood Road w Ithaca. New York 14850 w (607) 266-0665 Fax (607) 266.0748
ADVANCED BICANALYTICAL SERVICES, INC.
oavHwOLIM
Figure 20. Total Ion Chromatogram (TIC) for the Injection of 1 iL of Water
TIC ofall masses, 10010900
H2ulOoLfHO2MO 4de-ta1G385AC-N1H1:Z5O0Agradiont(SKAGN 0 95%AON in15mir) 1
15000000
7
gz
2
2
007 208 25061 80s1 3ot 154011 ScanfTime (min)
x
0C5935
=
15 Catherwood Road m Ithaca. New York 14850 m (607) 266-0665 m Fax (607) 266.0748
aAr SASEDoRVAVAINCCEESr.Dr eIaNG T.
ssMviwoL am
Figure 21. TIC for the Injections of 1 uL of Top, Middle and Bottom Day Zero Solution of T-6293
109 Top
39800000
a
i
i.
L
g7
Middle
25 2
&) Bottom
gz
:
$
& oa
0T0 T=or
24,400,000
30700000
E&Y iPsY ScanTine (min)
a0
iso
29
-
G0593
Ll. [L]ASDoVAANNACLETDCA
SERVICES, ING.
Te
Figure
22. TIC for Solution
the Injections of T-6293
of
1
iL
of
Top,
Middle
and
Bottom
Day
Seven
10) Top
1.500.000
gm
] i.
10 Middle
]gz
32
&2
199 Bottom
15,600,000 17,900,000
i zt
22
010
2To8r 2051 3a0s1
4i0s1
156011
ScanTime (min)
30
C0593
=
mmEoSAr2DerVvrAiNv0eCieEscDi,Nevese
Sova
Figure 23. Extracted lon Chromatograms for the Injection of 1 uL of Day Seven Bottom Solution of T-6293
E TTorT-GT 2551 itn He--DIti--om dota GBACNHZO rantSHAGN to SEAN i 5
100900 10) TIC
1260000
g7
2
Zo
sesso
S000
"
1
3 | ssaeso
g7 iw FE
| aan g7
z
Z=
"| 2somso o7a womw Tins (miyean
00
s
s00000
mE e Rhe
31
oe
-
C5938
MAoE vanceE o
-_-- SERVICES, ING. Figure 24. Full-Scan Mass Spectrum of Peak 1 in Figure 23
SSMYHWOLIM
"Profle QISCAN
T6203 LOMS Scans 264.265
T`S6c2an9s32L8O4.M2S6.53m-i7n/u1s3/19.527-61&1:23469A-M712 Time=6.00 min
10101 T-6293(1 Tpoak
mM
in
H2O/bottom)
dota
C8
ACNHZO
gradent(SKACN
to
96%ACN
in
15 40000
1
EX
" zg s
2
20
0 mz 0
800
32
005939
-
WRMWeSASEovARvVaRIonCcSEES,DPNeiSnae:
Saviviwol
Figure 25. Full-Scan Mass Spectrum of Peak 2 in Figure 23
Speraontao3a1143S2o3AmNins 1254 & 45T07i122T3imLo=o8MS8Sminca3n145s25
T0l2o0f3TL-6M25S3(10mlTdinH1E31I185R4)A8adotaG8AGNHED Graon(SKAGN 10 SACNin 15min
2 pea~ks
ss00
505,068
2s
g
Zz
i3 w
&
2,
o FE
s250
& mz wo
wo
33
0C5919
-
e IA ASSEDoAvAvainccEeSR,DeIanee SOL
Figure 26. Full-Scan Mass Spectrum of Peak 3 in Figure 23
Scpearanomtso4L11N5C2Sam.oTu4rsa12n2s00.1181.440T71a2sTinLoo.S7S2cmains 412.420 2pUalsaIs.T2351 mn Hondoe C8ACNHZO Gradaeont(o SA5AOCNN i 15 mi)Er)
i
g
HIE
25
419.0
o To
% we a
ED
En
005941
-
MW ASDoVaANNaCsEDrea
ScmUmmEeZ SrERrVImCEmS,sTIsNCL.cemmarmeasmieeeesemee SEMIN
Figure 27. TIC for the Injections of 1 uL of Top, Middle and Bottom Day Zero
Solution of Fose Amide
Top
27
50400000
|
1
gTM
Middle
5 22
1097 Bottom
ioo
22
a0T
T2o8r
48,900,000
4.600000
2501
08s1
w1a
1S011
ScanfTime (min)
35
---
0035942
m ||, ERY.ew
Figure 28. TIC for the Injections of 1 pL of Top, Middle and Bottom Day Seven Solution of Fose Amide
h Top
48,500,000
2
22
.
nn
g
FLTon
_,
Bottom
es
=
&2
a
Scan/Time (min)
36
-
visoag
||. AF DVAE NCEDY SERVICEeSs. ING SsMYHWOLIM
Figure 29. Extracted lon Chromatograms for the Injection of 1 uL of Day Zero Top Solution of Fose Amide
FUosLeAofmFiodsesL Amide(M 1070/u5lS 9h5op4)3d. 2atPaMC8 ACNIHZOIG% 10.95% in 1min)
:
100500 re
4360000
sosias0
]
1a
410000
\
ssnns
120000
Lf JL A g assess
3
170000
* smsmes
4
110000
r 2 asurson N 5 50000
soniasn
B
1850000
aos/e0s 010 25
7
30000
m 0 77s wao msss msso Timo (minScan
37 005944 -
MW =E Aoovae nceRne ea,
2
EI
Figure 3PJ 0e. sc oFullA-Smciao n Mra1as0ms8Fe a8Soe4p4sec0Ate7ir1caoToofOeMPSe2a.ks 1a22a1n2d281b in Figure 29 fTlrFos 100 tno)Soe CAACH0H8E%O1L5Em) rz mo
i
fo
wp
'
&>
2975
f= proto toca
Fon Aid=e LONGmecans 3=00307
STF eoaonl FBo30Ao 1mi30178ano ra8.t)40od5n.o7s1tG2BTiAmCKM1Z0O7LSmin108% Took
18mi)
. [Peak 1b
oto
= wer
"
&5
fo Zz
2
2: >
f=
="=
=
38
-
65915
A5So2Ev5aR0c5eAn55,
Figure 31. Full-Scan Mass Spectrum of Peak 2 in Figure 29
S-- feayni5r5i0vmieit25i1Fa8ois.cAr2hid5imLa0cONS7T-omS2icnan 258245
tn 0 irr)se CoACNAZO8%G115i0
2 peaks eo
393,208
"
g
2 so
i
25:
3115
o P3
% wi =
=
39
0a 05946
-
MW SAEeorvSvainScceesnPeIaNG,
Figure 32. Full-Scan Mass Spectrum of Peak 3 in Figure 29
FSopcnraeontAsom2i5ad01-es2o5L0mCmNniSn-1s 711253955F8-o.42s17e30A27m1Pi2deTiLvoC.S61Smainns250258 21pcealksFos Anid(100 ues) et G5 AGNH2O09E5%%i 15 mi)
oo, 180
ere stog01
[s
zg 23 ow
5&2
.
=
o ES
3 me C3
wo
"0
605947
-
=e IW SASEDoRVAVAINCCESER.DeIaN,G 0
Figure 33. Full-Scan Mass Spectrum of Peak 4 in Figure 29
-pSrcoafnise2Q612S.2C7ANmis
1-250
FosoAmide LOMS-1/S2c6a2.n27s1 8 27-712 Time=7.49 min
F2opgeoaoAtfmsFiodsesLAOnNiSd-(110705u8lh6o-p4)5a2tPiCBACNMZOIS0355% i 5 mi)
oo, 1830
Ssaviwor 704
7. as
g
fi w
&
25
o 2%
& me oo
@%o
#
005945
Mr 52e5,
-T=
Figure 34. Full-Scan Mass Spectrum of Peak 5 in Figure 29
i rEEossECarsomATE00ATEFaHo,rmeuC no sS e snttsne
bE "
ano
J.
g
2
=
249.0
= - me = =
o - 005949
MW So AebrvvaN incceEsR,DeIaNG -
Figure 35. Full-Scan Mass Spectrum of Peak 6 in Figure 29
P`-oSpscraefnAisme$iQ8d10es35oL8AONmNinGus11-52000F.5o-4s2s40A.37m1i02dTmLeCx1S1.:0181mcianns 309-398 p1 eaokfFosAnid(100 ho)dataG8AGNHZO(S% 005% i 15 mi)
"80
1260025
7;
g
z
f2 w
2 =
2
2
o ES & me ES a5
43
005959
-
Ml SASEDRAVVAINNCESCSE,DReIaNG, Figure 36. Full-Scan Mass Spectrum of Peak 7 in Figure 29
SPrcoafnlse4Q251-S4C26ANminus 1-420 8.43F0o.s7o12AmTiidmaoxL1O1.M9S8:m/iSncans 425.426 Faso Amide LCM7/S505.- 132.FM 1pueLakofsFose Amide(100 uMop),delta C8ACNH20(5% to 95% in 15min)
aso
7 g
wwe 2500
2
2490
526.0
ED
wo me ES)
0
"
.
005954
I sSSeorvva2 incceeso0Rs
Figure 37. TIC for the Injections of 1 ML of Top, Middle and Bottom Day Zero Solution of Perfluorooctanamide
--.
1750000
5
Zz
i
2 os
"
gm gZz
Middle
i2
125,400,000
" Bottom
SN
5 g2
:=
== Sarin as
17,300,000
wo _
005952
||. AE DVAR NCEDY I co SEESRVTICEEm SS.N INGem e erce eee
ssmviwor
Figure 38. TIC for the Injections of 1 KL of Top, Middle and Bottom Day Seven Solution of Perfluorooctanamide
L Top
g
i
15,100,000
" Middle
gm
3
&
R Bottom
J. 5
2
ao7
5Tor
14,700,000
16,000,000
2=01 ScanimoS(aomri) wwsr bPdo
46
-
905953
|, ASr ebrvvaiy ncecsE.De ing eee eee
ssmviwoLM
Figure 39. Extracted Ion Chromatograms for the Injection of 1 pL of Day Zero
Top Solution of Perfluorooctanamide
GTULTGTFF1S1LGOoNSHSB(H2C78OA5a5p)-N2do5t2aPCMBACNHZO(S1%0 95% n 15min
Toas00
ss0000
jis
aearses
1
000
pr
2
Z000
_ sans
Toso
&
3
Zz
22 ussnss 4 S000
2z
[p--
B
"aco
anor0sz
.
12000
pr
,
ar0000
e+ 025
m so a7s wsom Time (minySean
md s omo
47
-
005954
NE, ADVANCED
SH ssU eseSS5Em7R7VeITCnFEDSe,,FIeNZC..eeeeeeeeeeeeeseeme--eme sMYHWOLIM
Figure 40. Full-Scan Mass Spectrum of Peak 1 in Figure 39
ProfleQISCAN CTFIS0ONH2LMS 2/Scans 224-226
S`cGaInFsI2S2C4O.N2H226LmOiMnSus.21272145&9252-76-72152PTMine=6.32 min
21 upLeaokfsG7FISCONH2(1 mhihop)detaC8ACNH2O0(5S%%in 15 mi)
ss2e0
oo, 1830
7.
g
i
fw z
&
2
2070 [3 2% % mz % oo
48
-
005955
mossosvaensc.e..
Figure
41. Full-Scan Mass Spectrum of Peak 2 in Figure 39
hJ er ne o Sm nr 1 uLof C7F1SCONH2(1 mMAop),deltaC8,ACNH20to(955% %in 15min)
J
"
g
i .
$
3
Le
369.0
.= = = =
49
005956 -
IW ASOovAANCEPDea, EeSERVICES. ING
Figure 42. Full-Scan Mass Spectrum of Peak 3 in Figure 39
ScParonfsie23Q91.S2C4A5mNinus
1.295
CTFILSCMSCOScaNns 2H30.2245 & 250.500Tnes6.60 min
C{UL oTfGTFFISICLONSCHZS(C21-m7O1i5o0N5).6dHo2t5a2PCMBACNHZO(S% fo 95% i 15 min)
2peaokos, 1830
ssvHwoL IM
180,925
7
g
2=
g 2
ans 2.
3 2% me ES) 0
so
005957 -
MaAosvasnc.eo.. WR servesWS
Figure 43. Full-Scan Mass Spectrum of Peak 4 in Figure 39
-- oni aman
nsil
aos asin smn wn
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52
2
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005958
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--_-- SERVICe ES.eINC.
Figure 44. Full-Scan Mass Spectrum of Peak in Figure 39 Profi Q1SCAN GIFISCONHZ LOMS2/Scans 265.271 (SCcTanFsIS2C6O5.N2H721LmiOnuMs2S1--276/25889257-46.27152PTMimo=7.53 min 2TpLoaokfsGTFISCONHR(1 mop)dotaCA8CNHZO(S% to 96% n 15 min) 00, 1830
SSMYHWOLIM 5726
7s
a
95
z
5
235
25
250
wo
3)
0
mz
52
005959
-
AEDV2AN2CE5D 0
SERVICRESS, INC. eee
ere
Figure 45. Full-Scan Mass Spectrum of Peak 6 in Figure 39
Profi Q1SCAN
GTFISCONHR LCMS2/Scans 356-357
SCcIaFnIsS3C5O6N-H3527LmOinMuSs21.-375/45/&9537-06-27512PTMi.mo=10.07 min
31 puoLaokfsGTF1SCONH(1mop),dotaC8ACNIH2009(55%i%n 15min)
100
230
9SMYHWOL3M 105,000
75
g
i 50
302.0
25] 1540
20
"0
0
0
mz
53 005960 -
ASSeoOrAvvNaiAncLceYEsT,DICmAeL
Figure 46. Full-Scan Mass Spectrum of Peak 7 in Figure 39
SaPrnofele20165.9C6AmNinus
F15CONH2 LOMS 25can 1-962 380.712 Tino=1035min
386367
STUL TFoSfOGTNFI5LGOoNMna(a1 Tthhso)6d2a5aPCi BACNHO(S% 065% 15 mi
|2peaks
4120
osvwoLM
329613
g
i z :i
2
2
2100
PA
ao me wo
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54
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