Document gaKbDxZ5yz5vK65agJEvO4e2J
ST-508 Final Report
ToxDocs 14-192
TOXICOLOGY ASSESSMENT AND COMPLIANCE ASSURANCE GROUP 3M COMPANY
14-192: ORAL ADME STUDY OF MTDID 37312 IN RATS
FINAL REPORT
ToxDocs Study Number
14-192
Test Material Lot Number(s) Strategic Tox Lab Study Number
MTDID 37312
FM-3923, mixture of lots 30035, 30037, and 30039 ST-508
Other Identification Number(s) N-EtFOSE Alcohol, FC-10
19F/1H-NMR Analytical Report SMD Analytical Report 816 GID Number
Study Director
Jill Hart, AAS, LATg Senior Laboratory Technologist
Principle Investigator: (Study Monitor)
Sue Chang, Ph.D. Senior Toxicology Specialist
Testing Facility In-Life Start Date
3M Strategic Toxicology Laboratory 3M Center, Bldg 270, room SB324 Saint Paul, MN 55084
August 6, 2014
In-Life Termination Date
August 27, 2014
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`ToxDoes 14-192
"TOXICOLOGY ASSESSMENT AND COMPLIANCE ASSURANCE GROUP 3M COMPANY
14-192: ORAL ADME STUDY OF MTDID 37312 IN
RATS
Compliance Statement and Signature Page
`rTehgiuslasttourdyy gwuaisdecloinnedsu.cted for research and development purposes and does not conform to
`This finalreporthas been reviewed and approved by:
<Tipll Haert,ADASo, rLA,Tg SSetnuidoyrDLiarbeocrtaotrory Technologist
9 JAN 20/5" Date
I
== S
`Sue Chang, Ph.D.
Senior Toxicology Specialist
Study Monitor
[9 Jan, 2015
Date
ST-508 Final Report
ToxDocs 14-192
1 Summary
This study was to evaluate the serum uptake and metabolism of MTDID 37312 (white solid, 99.9%pure, N-ethyl-heptadecafluoro-N-(2-hydroxyethyl)-1-octanesulfonamide, N-EtFOSE) in male Sprague Dawley rats after a single oral dose. The formation and presence of its metabolites in serum, perfluorooctane sulfonate (PFOS) and perfluorooctane sulfonamide (PFOSA), were measured by LC-MS/MS.
Clinical observations were normal throughout the study period. All animals appeared normal during the study and all of them survived to the scheduled necropsy. There were no significant findings or gross lesions observed during the necropsy.
All of the animals had quantifiable PFOS and PFOSA in the serum after receiving an oral dose of N-EtFOSE, demonstrating that N-EtFOSE can metabolize to PFOS and PFOSA. While serum PFOS concentrations continued to increase up to Day 21 post-dose (study termination), PFOSA peaked around 30 - 48 hours post-dose.
Within the limit of the study design, data obtained here demonstrated that N-EtFOSE can metabolize to PFOS and PFOSA in the blood upon oral administration. Percent conversion from N-EtFOSE to PFOSA was not available due to limited pharmacokinetic information available. Approximately 10 - 15 % of the oral administered N-EtFOSE dose was converted to PFOSequivalent dose.
2 Study Objective
This study was to evaluate the serum uptake and metabolism of MTDID 37312 (white solid, 99.9%pure, N-ethyl-heptadecafluoro-N-(2-hydroxyethyl)-1-octanesulfonamide, N-EtFOSE) in male Sprague Dawley rats after a single oral dose. The formation and presence of its metabolites in serum, perfluorooctane sulfonate (PFOS) and perfluorooctane sulfonamide (PFOSA), were measured by LC-MS/MS.
3 Test Material Information
3.1 Test Substance
Test Material ID
Lot
Other Identification
19F/1H-NMR Analytical Report LIMS Number Purity Physical State of Test Material Chemical Name
MTDID 37312 FM-3923, mixture of lots 30035, 30037, and 30039
N-EtFOSE Alcohol, FC-10 M13-0520 & M13-0647 (see Appendix 1: Certificate of Analysis) 98.24% White solid at room temperature
N-ethyl-heptadecafluoro-N-(2-hydroxyethyl)-1octanesulfonamide
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Chemical Structure 3.2 Dose Preparation
CF3(CF2)7-SO2-N(CH2CH3)-CH2CH2-OH
Dose Preparation
Physical State of administered material Route of Administration
The test material was mixed in 2% Tween 20 to a final concentration of 10 mg/mL. The mixture was sonicated in a water bath (with heat) to achieve maximum solubility. Solution
Oral
4 Test System
4.1 Test System Information 4
Species Strain Source Age at Initiation of Treatment Weight at Initiation of Treatment Number and sex Identification IACUC Animal Usage Application Number
Rat Sprague Dawley Charles River 6-8 weeks 285 - 353 g 6 males Unique tail mark in indelible ink 2011-0001
4.2 Animal Husbandry
Housing Diet/Water
Housing Environment
All animals were single housed in wired bottom cages during the entire study until study termination. Harlan Teklad Rat/Mouse 2018 Diet (Harlan Teklad, Madison, WI) and tap water were available ad libitum throughout the study. Temperature Range 72 3F Humidity Range 30 - 70% Minimum of 10 exchanges of room air per hour 12 hour light/dark cycle
5 Study Design
On Study Day 1, all animals (fasted overnight) received a single dose of N-EtFOSE via oral gavage at 50 mg/kg body weight. The dose group designation is presented below:
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Group Dose Sex N (mg/kg)
Interim tail vein bleeding (for sera)
Euthanasia
1
50
M 3 Pre-dose; also 2 hrs, 8 hrs, 21 days PD
30 hrs, day 6 and day 14
post-dose (PD)
2
50
M 3 Pre-dose; also 2 hrs, 18 21 days PD
hrs, 48 hrs, day 6 and day
6
14 post-dose (PD)
Parameters Evaluated
6.1 Clinical Observations
Each animal was observed immediately following dosing and throughout the study for mortality and morbidity.
6.2 Body Weights
Each animal was weighed prior to dose administration (for dosing volume calculation purpose) and daily thereafter.
6.3 Serum Collection
For all rats, interim blood samples including pre-dose (approximately 200 uL) were collected via tail vein to labelled collection tubes without anticoagulant at designated time as outlined in Study Design. On Day 21 post-dose, all animals were euthanized via CO2 asphyxiation and for each animal, blood was collected ( 6 mL) via the abdominal aorta and transferred to labeled collection tubes without anticoagulant.
The blood samples were allowed to clot for 15 to 30 minutes at room temperature and then
centrifuged at 2000 x g for 15 minutes. The resulting supernatant (serum) was transferred to labelled polypropylene microcentrifuge tubes. Serum samples were stored frozen at -70oC
pending LC-MS/MS analyses.
6.4 Gross Necropsy, Organ Weights and Tissue Collection
On Day 21 post-dose, all rats were euthanized by CO2. Following euthanasia, a gross necropsy was performed on all animals. Livers were excised and the weights were recorded. Liver samples were stored in a -70C pending LC-MS/MS analyses.
6.5 Analytical
Serum samples collected were analyzed for PFOS and PFOSA by LC-MS/MS.
7 Statistical Analysis
Mean values were calculated using Microsoft Excel software.
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8 Raw Data
The raw data for body weights and dosing volumes are recorded in 3M Notebook #166568, pages 19 - 22.
9 Results
9.1 Clinical Observations & Gross Necropsy
Clinical observations were normal throughout the study period. All animals appeared normal during the study and all of them survived to the scheduled necropsy. There were no significant findings or gross lesions observed during the necropsy.
9.2 Body Weight and Liver Weight
Animal Tail # Mark
4R270 4R271 4R295 4R299 4R312 4R313
Black I Black II Black III Black IV Black V Black VI
Tube Code
M-1 M-2 M-3 M-4 M-5 M-6
Body Weight (g) Pre- D 1 D2 D5 D6 D7 D8 D9 D12 D13 D14 D15 D16 D19 D20 D21 dose 303 313 315 324 327 334 333 337 342 348 349 349 348 360 359 365 288 298 297 308 309 314 317 323 327 329 330 332 337 343 345 348 353 352 350 358 362 361 365 365 371 376 377 376 379 386 385 391 314 316 322 328 336 340 35 350 359 360 361 367 367 381 380 385 353 358 353 359 363 362 365 369 374 374 376 381 379 385 388 391 285 294 297 307 314 317 318 320 330 333 334 338 336 352 352 358
Liver Weight
(g) 15.57 14.20 15.05 16.15 15.73 14.70
9.3 Analytical Results
Concentrations of serum PFOS and PFOSA were determined using LC-MS/MS and the corresponding data are presented blow.
Animal # Tube Code pre-dose
4R270 4R271 4R295 4R299 4R312 4R313
M-1 M-2 M-3 M-4 M-5 M-6
<LLOQ <LLOQ <LLOQ <LLOQ <LLOQ <LLOQ
2h
743 787 880 427 405 585
Serum
8hr
5420 6290 5720
PFOS 18h
8130 7080 9060
concentration,
30h 48h
15400 13800 14000
15800 10900 16250
ng/mL 144h (D6) 21500 20700 22300 19300 15100 20300
336h (D14) 20800 19600 19900 18200 12700 17000
504h (D21) 23400 28500 23500 22300 14300 19600
Animal #
4R270 4R271 4R295 4R299 4R312 4R313
Tube Code pre-dose
M-1 M-2 M-3 M-4 M-5 M-6
<LLOQ <LLOQ <LLOQ <LLOQ <LLOQ <LLOQ
2h
97.4 117 96.7 91.0 70.6 29.8
Serum PFOSA concentration, ng/mL
8hr
18h
30h
48h
144h (D6)
542
1890
228
785
1570
232
499
1400
234
931
1290 244
701
743
222
917
1130 224
336h (D14) 75.4 55.8 57.1 67.8 78.0 66.8
504h (D21) 29.2 35.0 28.6 37.1 28.4 30.1
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9.4 Theoretical PFOS-equivalent Dose derived from Serum PFOS Concentration
Animal #
4R270 4R271 4R295 4R299 4R312 4R313
Tube Code
M-1 M-2 M-3 M-4 M-5 M-6
pre-dose
<LLOQ <LLOQ <LLOQ <LLOQ <LLOQ <LLOQ
PFOS-equivalent dose, mmol/kg
Assuming each time point is Cmax and Dose = Cmax x Vd; Vd = 300 mL/kg
2h
8hr
18h
30h
48h
144h 336h
(D6)
(D14)
0.00045 0.00326
0.00926
0.01293 0.01251
0.00047 0.00378
0.00830
0.01244 0.01178
0.00053 0.00344
0.00842
0.01341 0.01196
0.00026
0.0049
0.00950 0.01160 0.01094
0.00024
0.0043
0.00655 0.00908 0.00764
0.00035
0.0054
0.00977 0.01220 0.01022
504h (D21) 0.01407 0.01713 0.01413 0.01341 0.00860 0.01178
9.5 Percent Dose Converted from Inhaled N-EtFOSE Dose to Serum PFOS Dose
Animal #
4R270 4R271 4R295 4R299 4R312 4R313
N-EtFOSE dose
administered, mmol/kg 0.088 0.088 0.088 0.088 0.088 0.088
PFOS-equivalent dose relative to oral N-EtFOSE dose, %
pre-dose 2h 8hr
--
0.508 3.70
--
0.538 4.30
--
0.601 3.91
--
0.292
--
0.277
--
0.400
18h 30h 48h 144h (D6)
10.52
14.69
9.43
14.14
9.56
15.24
5.55
10.79 13.19
4.84
7.45 10.32
6.19
11.10 13.87
336h (D14) 14.21 13.39 13.60 12.43 8.68 11.61
504h (D21) 15.99 19.47 16.05 15.24 9.77 13.39
Mean
0.088
--
0.436 3.969 5.527 9.84 9.78 13.57 12.32 14.98
S.D.
0
--
0.134 0.302 0.677 0.60 2.03 1.74 2.01 3.23
10 Conclusion
All of the animals had quantifiable PFOS and PFOSA in the serum after receiving an oral dose of N-EtFOSE, demonstrating that N-EtFOSE can metabolize to PFOS and PFOSA. While serum PFOS concentrations continued to increase up to Day 21 post-dose (study termination), PFOSA peaked around 30 - 48 hours post-dose.
Within the limit of the study design, data obtained here demonstrated that N-EtFOSE can metabolize to PFOS and PFOSA in the blood upon oral administration. Percent conversion from N-EtFOSE to PFOSA was not available due to limited pharmacokinetic information available. Approximately 10 - 15 % of the oral administered N-EtFOSE dose was converted to PFOSequivalent dose.
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Appendix 1: Certificate of Analysis
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Analytical Characterization of narrow range N-EtFOSE FM-3923 Mixture of Lot 30035, Lot 30037, and Lot 30039
Document Prepared by:
David Ehresman Alan Eveland Sue Chang
3M Medical Department
August 5, 2014
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Introduction:
N-EtFOSE (CF3(CF2)7-SO2-N(CH2CH3)-CH2CH2-OH, N-ethyl-heptadecafluoro-N-(2hydroxyethyl)-1-octanesulfonamide, CASRN 08-7781-1) is a major precursor of perfluorooctanesulfonyl fluoride (POSF)-based compounds. Both in vitro and in vivo studies have shown that N-EtFOSE can undergo biotransformation resulting in the formation of perfluorooctanesulfonate (PFOS) as ultimate metabolite (Butenhoff and Seacat, 2001; Xie et al., 2009; Xu et al., 2004). Due to the inherent nature of electrochemical fluorination process used in producing N-EtFOSE, there often were homologous perfluoroalkyl species (both lower and higher molecular weights) present in N-EtFOSE. N-EtFOSE FM-3923 is a mixture from three different lots (Lot 30035, Lot 30037, and Lot 30039) and it was used in a number of mammalian toxicology studies conducted by 3M Company, such as a 2-year chronic feeding study in rats (Thomford et al., 2002), tetratology studies in rats and rabbits (Case et al., 2001), and metabolism study in non-human primates (AR226-0532).
N-EtFOSE FM-3923 was characterized in 1997 to be 98.24% pure (3M Specialty Material Division Analytical Report 816, see Appendix 1-1); however, in a more recent inhalation work with rats exposed to N-EtFOSE at low atmospheric concentration (3M Strategic Toxicology Lab Study # ST-460), a different lot of N-EtFOSE (TCR-1085) was used. N-EtFOSE Lot TCR-1085 was analyzed in 2013 to be 99.1% pure (3M MRD Analytical Laboratory LIMS Request # M131896, see Appendix 1-2). However, because there is a very limited amount of N-EtFOSE lot TCR-1085, N-EtFOSE FM-3923 is being considered to be used in a follow-up work with rats exposed to N-EtFOSE via oral gavage (3M Strategic Toxicology Lab Study # ST-508, ToxDocs 14-192).
To verify its strength, N-EtFOSE FM-3923 was analyzed by LC-MS/MS and the resulting chromatogram was compared to N-EtFOSE lot TCR-1085. Appendix 1-3 included in this report are the resulting chromatograms for both samples.
Briefly, both samples were prepared in methanol at 100 ng/mL. Ten (10) L of the sample was analyzed by LC-MS/MS (Sciex API 5000 mass spectrometer from Applied Biosystems / MDSSciex Instrument Corporation) with turbo ion spray (pneumatically assisted electrospray ionization) in negative ion mode. Separations of the compounds were completed using an ACE C-18, 5 m, 75 x 2.1 mm i.d. HPLC column (dual column method). The column flow rate was set at 0.25 mL/min with the following gradient condition:
100% Acetonitrile 10% 95% 95% 10%
2 mM ammonium acetate 90% 5% 5% 90%
Time (min) 0 4 7 11
For both analyses, the transition ion monitored was 629.902 59.029 amu, with a retention time at 6.29 minutes.
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Based on the information obtained, it appeared that N-EtFOSE FM-3923 has similar strength as N-EtFOSE lot TCR-1085, which is consistent with the respective analytical reports. Therefore, N-EtFOSE FM-3923 is deemed acceptable to be used as the test material for ST-508 (ToxDocs 14-192).
References:
Butenhoff, J., Seacat, A.M., 2001. Comparative sub-chronic toxicity of perfluoroctane sulfonate (PFOS) and N-ethyl perfluorooctanesulfonamidoethanol (N-ETFOSE) in the rat (abstract 1655). Toxicol Sci 60 Supplement 1, 348.
Case, M.T., York, R.G., Christian, M.S., 2001. Rat and rabbit oral developmental toxicology studies with two perfluorinated compounds. Int J Toxicol 20, 101-109.
Thomford, P.J., Seacat, A.M., Butenhoff, J.L., 2002. Terminal observations in Sprague Dawley rats after lifetime dietary exposure to N-ethylperfluorooctanesulfonamido ethanol (abstract 907). The Toxicologist 66 185-186.
Xie, W., Wu, Q., Kania-Korwel, I., Tharappel, J.C., Telu, S., Coleman, M.C., Glauert, H.P., Kannan, K., Mariappan, S.V., Spitz, D.R., Weydert, J., Lehmler, H.J., 2009. Subacute exposure to N-ethyl perfluorooctanesulfonamidoethanol results in the formation of perfluorooctanesulfonate and alters superoxide dismutase activity in female rats. Arch Toxicol 83, 909-924.
Xu, L., Krenitsky, D.M., Seacat, A.M., Butenhoff, J.L., Anders, M.W., 2004. Biotransformation of N-ethyl-N-(2-hydroxyethyl)perfluorooctanesulfonamide by rat liver microsomes, cytosol, and slices and by expressed rat and human cytochromes P450. Chem Res Toxicol 17, 767-775.
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Appendix 1-1 Specialty Materials Division Analytical Report # 816 for N-EtFOSE FM-3923
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Appendix 1-2 3M MRD Analytical Laboratory LIMS Request # M13-1896 for N-EtFOSE TCR-1085
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3M MATERIALS RESOURCE DIVISION ANALYTICAL LABORATORY
LIMS Request No. M13-1896
To:
Sue Chang - (733-9073) -Toxicology Assessment and Compliance Assurance - 220-
6W-08
Dave Ehresman - (733-5070) - Toxicology Assessment and Compliance Assurance
236-1B-22
Kent Lindstrom - (733-9882) - 3M Environmental Lab- 260-5N-17
From: Tom Kestner - (733-5633) - 3M MRD Analytical Laboratory - 236-2B-11
Subject: 1H/19F-NMR Characterization of TCR-1085: Purified N-Et FOSE Alcohol for Tox Study
Date: November 24, 2013 ______________________________________________________________________________
SAMPLE DESCRIPTION: TCR-1085 is purified N-Et FOSE alcohol.
The nominal product is C8F17-SO2N(CH2CH3)CH2CH2OH (white powder).
NMR Spectra #'s
Summary of NMR Spectra Acquired for TCR-1085
Experiments Descriptions
H131896.401 F131896.401
400 MHz 1H-NMR spectrum for a portion of the sample dissolved in acetone-d6 and spiked with small amounts of CFCl3 and p-HFX internal standard. 376.3 MHz 19F-NMR spectrum for a portion of the sample dissolved in acetone-d6 and spiked with small amounts of CFCl3 and p-HFX internal standard.
OBJECTIVE:
The sample of TCR-1085 was subjected to 1H-NMR and 19F-NMR spectral analyses to determine the purity of the nominal product and to characterize as many impurity components as possible. Joel Miller also performed an LCMS analysis to assist in the characterization of impurity components and his results were reported previously. In addition, you requested that I prepare and issue a certificate of analysis as a separate document in support of an upcoming 3M toxicology study.
EXPERIMENTAL:
A 62 mg portion of the TCR-1085 sample was totally dissolved in ~ 0.7 mL of deuterated acetone (acetone-d6) and then the solution was spiked with small amounts of CFCl3 and 1,4bis(trifluoromethyl)benzene (p-HFX) for NMR analyses. A 400 MHz 1H-NMR spectrum and a 376.3 MHz 19F-NMR spectrum were acquired using an Agilent VNMRS 400 FT-NMR
spectrometer that was operating with a 5mm inverse-detection gradient probe at an analysis
temperature of 22-23 C.
RESULTS:
The combined 19F-NMR and 1H-NMR spectral data indicate the sample of TCR-1085 consists of ~ 99.9 relative wt.% of the nominal product, CnF2n+1-SO2-N(CH2CH3)-CH2CH2OH, in at least six isomeric forms, where n is mainly 8. Trace amounts of two impurities were also assigned. The qualitative and quantitative compositional results that were derived from a single trial
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19F/1H-NMR cross integration spectral analysis are summarized in TABLE-1. The relative weight percent concentrations shown in TABLE-1 should be very close to their respective absolute wt.% values. Trace amounts of unassigned components are also detected in the NMR spectra but additional work would be needed in an effort to assign or quantify the other impurities.
Small amounts of longer or shorter chain fluorochemical homolog impurities could have easily gone undetected because the 19F-NMR technique is not particularly well suited for characterizing small amounts of homologs unless the chains are very short (i.e., C1-C3). Characterization of fluorochemical homolog impurities was performed by Joel Miller using LCMS.
If you have any questions about these NMR results, or if any further work is needed, please let me know. .
Tom Kestner
c: Joel Miller Scott Schmick
TABLE-1
Sample: TCR-1085, Purified N-Et FOSE alcohol
Compositional Results by 19F/1H-NMR Cross Integration Spectral Analysis
Structural Assignments 1
19F/1H-NMR Relative Wt.%
Concentrations
Sum of at least 6 desired product isomers: CnF2n+1-SO2-N(CH2CH3)CH2CH2-OH
where n assumed to be 8 for calculation purposes
Purity = 99.9%
CF3(CF2)x-SO2-N(CH2CH3)-CH2CH2-OH
(Normal chain, where x assumed to 7 for calculation purposes)
CF3(CF2)x-CF(CF3)-(CF2)y-SO2-N(CH2CH3)-CH2CH2-OH
(Internal monomethyl branch, where x+y assumed to be 5, and x 0, y 0, for calculation purposes)
(CF3)2CF-(CF2)x-SO2-N(CH2CH3)-CH2CH2-OH
(Isopropyl branch, where x assumed to be 5 for calculation purposes)
CxF2x+1-CF(CF3)-SO2-N(CH2CH3)-CH2CH2-OH
(Alpha branch, where x assumed to be 6 for calculation purposes)
(CF3)3C-(CF2)x-SO2-N(CH2CH3)-CH2CH2-OH
(t-butyl branch, where x assumed to be 4 for calculation purposes)
CF3-(CF2)x-C(CF3)2-(CF2)y-SO2-N(CH2CH3)-CH2CH2-OH
(Internal gem-dimethyl branch, where x+y assumed to be 4, and x 0, for calculation purposes)
Probable carboxamide CmF2m+1-CO-NH-CH2CH3
where m assumed to be 7 for calculation purposes
Probable N-Me FOSE alcohol CnF2n+1-SO2-N(CH3)-CH2CH2-OH
where n assumed to be 8 for calculation purposes
70.55% 16.93%
10.36% 1.67% 0.24% 0.12%
0.12% 0.018%
1. Trace amounts of unassigned impurity components are also detected in the NMR spectra.
File Ref.: sc.de.kl131896.LIMS_FT-NMR compositional results_TCR-1085_Purified N-Et FOSE Alcohol.docx
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Appendix 1-3 LC-MS/MS Chromatograms N-EtFOSE FM-3923 vs. N-EtFOSE TCR-1085
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Intensity, cps
XIC of -MRM (3 pairs): 629.902/59.029 Da from Sample 104 (EhtFOSE New) of DataSET1.wiff (Turbo Spray)
Max. 2.2e6 cps.
2.2e6 2.1e6 2.0e6 1.9e6 1.8e6 1.7e6 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5
0.0
6.29
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
Time, min
Figure 1 N-EtFOSE FM-3923 100 ng/ml std.
XIC of -MRM (3 pairs): 629.902/59.029 Da from Sample 105 (EthFOSE Old) of DataSET1.wiff (Turbo Spray)
2.1e6 2.0e6 1.9e6 1.8e6 1.7e6 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5
0.0
6.29
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
Time, min
Figure 2 N-EtFOSE TCR-1085 100 ng/ml std.
Max. 2.1e6 cps.
11.0
12.0
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Intensity, cps