Document MMnoBOwQ81MQ8JLGBmY5y4jka

ADVANCED Mm BIOANALYTICAL SERVICES, INC. PRELIMINARY ANALYTICAL REPORT TITLE: QUALITATIVE INVESTIGATION ARII6-0313 CECE N NOV 17 1005} So, "ear OF THE IN VITRO METABOLISM OF T-6292, T-6293, T-6294 AND T-6295 BY RAT AND HUMAN HEPATO- CYTES USING ION SPRAY LC/MS AND LC/MS/MS DATE: November 12, 1996 REPORT: 96ADEMO01.3M . AUTHORS: Daniel E. Mulvana, B.S. Jack Henion, Ph.D. PREPARED FOR: NO. OF PAGES: 3M Medical Department, Toxicology Services, St. Paul, MN 55133 45 005854 15Cathorwood Road = ithaca, New York 14850 = (607) 266-0665 =Fax (607) 266-0749 2 ADVANCED BIOANALYTICAL SERVICES SIGNATURE PAGE Title: QUALITATIVE INVESTIGATION OF THE IN VITRO METABOLISM OF T6292, T-6293, T-6294 AND T-6295 BY RAT AND HUMAN HEPATOCYTES USING ION SPRAY LC/MS AND LC/MS/MS Reported by: wd Yisberc Daniel E. Mulvana, B.S. Senior Research Scientist wfnf4t Date Reviewed and Approved by: Nace Authori byzed for nif fa 4 Mary Ringwood, Quality Assurance Manager fizfan Date: BEegT Jack Henion, PhD.(%16 46-2) Laboratory Director Advanced BioAnalytical Services fn Dac mm ASIDOVAANNACLYETDICAL SERVICES. INC 905855 Reroxr eave 3 QUALITATIVE INVESTIGATION OF THE IN VITRO METABOLISM OF T-6292, T-6293, T-6294 AND T-6295 BY RAT AND HUMAN HEPATOCYTES USING ION SPRAY LC/MS AND LC/MS/MS ABSTRACT The in vitro metabolism of T-6292, T-6293, T-6294 and T-6295 was investigated using a sensitive and highly specific analytical method developed by Advanced BioAnalytical Services, Inc., Ithaca, NY. This method involves a simple direct injection LC/MS analysis of processed hepatocyte incubate. Siliconized polyethylene transfer pipets and polypropylene autosampler vials were used for all samples. LC/MS and LC/MS/MS experiments were performed in the negative ion mode using an ion spray interface. Chromatography was performed on a 2 mm x 100mmBetasil ODS column using gradient elution with either aqueous ammonium acetate with methanol or water with methanol. Using a direct injection method, rather than one using extraction, enabled detection of a wide variety of metabolites in a single injection. Included in this report are metabolism data from individual rat and human samples. Several metabolites were found in the rat and human samples and differences were observed between the species. [| Eee SERVICES, INC. rrr 005856 ` `TABLE OF CONTENTS SIGNATURE PAGE 2 ABSTRACT PAGE 3 1. INTRODUCTION 7 Ll Introduction 7 12 Study Objective 7 2. EXPERIMENTAL 7 2.1 Chemicals and Materials 7 22 LC/MS Instrumentation 7 23 Standard Solutions 8 24 HPLC Eluent Preparation 8 25 Sample Preparation 8 2.6 Sample Analysis 9 27 Data Handling 10 3. RESULTS AND DISCUSSION 1 3.1 Mass Spectrometric ResultsforTest Articles n 32 Metabolism by Rat Hepatocytes n 33 Metabolism by Human Hepatocytes 15 4. CONCLUSIONS 16 5. REFERENCES 16 FIGURES Figure I. Structures of the study test articles. 17 Figure 2. Negative ionization mass spectra obtained from the test articles T-6293 (M- HJ = 650.1), T-6294 ([M-H] = 526.1) and T-6295 ([M-H] = 498.9). 18 Figure 3. Product ion spectra obtained from the deprotonated test articles T-6293, T- 6294, and T-6295. 19 Figure 4. Proposed CID fragmentation mechanismfor T-6293. 20 Figure 5. Proposed CID fragmentation mechanism for T-6294. 21 HoTo oMo o ccms.mmmmm----ser--somIPnSEeS? Figure 6. Proposed CID fragmentation mechanism for T-6295. 2 5 Figure 7. Total ion and extracted fragment ion chromatograms for T-6292 obtained from Rat Control Sample 13. 2 Figure 8. Toul ion and extracted fragment ion chromatograms for T-6292 obtained from Rat 1, 0-Hr sample. 2% Figure 9. Extracted ion chromatograms of fragment peaks for T-6292 homologous series in Rat 1, 0-Hr sample. 25 Figure 10a. Proposed structure of parent compound for T-6292 homologous series from Figures 8 and 11. 26 Figure 10b. Proposed structure of glucuronide for homologous series from Figures 12 and 13. 2 Figure 11. Total fon and extracted fragment ion chromatograms for T-6292 obtained i from Rat 1, 6-Hr sample. 27 Figure 12. Extracted ion chromatograms of putative glucuronide peaks for T-6292 homologous series in Rat 1, 6-Hr sample. 23 Figure 13. Extracted ion chromatograms of putative glucuronide peaks for T-6292 homologous series in Rat 1, 0-Hr sample. 29 Figure 14. Proposed structures of the sulfonamide and carboxylic acid metabolites for T- 6292 from Rat 1 30 Figure 15. Extracted ion chromatograms of sulfonamide (m/z 498) and carboxylic acid (m/z 584) metabolites for T-6292 from Rat 1, 0-Hr (top) and 6-Hr (bottom) samples. 31 Figure 16. Product ion scan for carboxylic acid metabolite of T-6292 in Rat 1, 6-Hr sample and proposed fragment structures. 32 Figure 17. Total ion and extracted parent (m/z 650) and proposed metabolite ion chromatograms for T-6293 obtained from Rat Control Sample 13. 3 Figure 18. Total ion and extracted parent (mz 650) and proposed metabolite ion chromatograms for T-6293 obtained from Rat 1, 0-Hr sample. 34 Figure 19. Total ion and extracted parent (m/z 650) and proposed metabolite fon chromatograms for T-6293 obtained from Rat 1, 6-Hr sample. 35 Figure 20. Possible structures for the observed metabolites of T-6293 with [M-HJ of 542 and 556 amu from Rat 1. 36 Figure 21. Extracted ion chromatograms of sulfonamide (m/z 498) and sulfonate (m/z 499) ions for T-6293 from Rat 1, 0-Hr (top) and 6-Hr (bottom) samples. 37 Figure 22. Extracted ion chromatograms of proposed metabolites with m/z 327 and 419 for T-6293 from Rat 1, 0-Hr (top) and 6-Hr (bottom) samples. 38 once, mam BSIEDRAVNIACLEYST.IICNACL REPORT: 60HO8 SB0 S8 6 Figure 23. Total ion and extracted parent (m/z 526) and proposed metabolite ion chromatograms for T-6294 obtained from Rat Control Sample 13. 39 Figure 24. Total ion and extracted parent (m/z 526) and proposed metabolite ion chromatograms for T-6294 obtained from Rat 1, 0-Hr sample. Figure 25. Total ion and extracted parent (m/z 526) and proposed metabolite ion 4 chromatograms for T-6294 obtained from Rat 1, 6-Hr sample. Figure 26. Product ion scan for perfluorooctanesulfonamide metabolite 4 (CF,(CF,),SO,NH,, m/z 498) of T-6294 in Rat 1, 6-Hr sample and proposed fragment structures. 2 Figure 27. Total ion and extracted parent (m/z 499) and homolog ion chromatograms for i `T-6295 obtained from Rat Control Sample 13. 43 Figure 28. Total ion and extracted parent (m/z 499) and homolog ion chromatograms for T-6295 obtained from Rat 1, 0-Hr sample. a" Figure 29. Total ion and extracted parent (m/z 499) and homolog ion chromatograms for T-6295 obtained from Rat 1, 6-Hr. 45 MW 2oncee SERVICES. INC 5859 7 1. INTRODUCTION 1.1 Introduction The in vitro metabolism of T-6292, T-6293, T-6294 and T-6295 in rat and human hepatocyte incubates was investigated using ion spray LC/MS and LC/MS/MS. `The quenched incubates were directly injected and separated on an HPLC column. Chromatographic resolution of the various constituents was accomplished using gradient elution with methanol and either water or 2 mM ammonium acetate in water. The mass spectrometer was operated in the full scan (mz 100 to 800) single MS mode and peaks of interest were then studied by MS/MS techniques for confirmation of selected metabolite peaks. 1.2 Study Objective The objective of this study wastodetect and characterize the metabolismofT-6292, T-6293, T-6294 and T-6295 by rat and human hepatocytes. 2. EXPERIMENTAL 2.1 Chemicals and Materials T6292, T-6293, T-6294 and T-6295 (Figure 1) were provided by 3M Medical Department, Toxicology Services, St. Paul, MN 55133. Siliconized polypropylene autosampler via: cat#500 102, Sun Brokers, Inc., `Wilmington NC 28402. Siliconized by Eagle Picher Industries, Inc., Miami, OK 74354 Siliconized polypropylene screw-cap tubes: 16 X 101 mm, Cat# 60.541, Sarstedt Inc., Newton, NC 28658, siliconized in-house. 2.2 LC/MS Instrumentation Liquid chromatography was performed using either two Shimadzu LC-10AD pumps (Shimadzu Scientific Instruments, Inc., Columbia, MD 21046) with a Waters WISP 717 plus autosampler (Waters, Milford MA 01757) or a HewlettPackard 1090L liquid chromatograph (Hewlett-Packard, Avondale, PA 19311). A Betasil C18 column, 2 mm x 100 mm, was obtained from Keystone Scientific, Me ADVANCED SERVICES. INC. a 8 Inc., Bellefonte, PA 16823. The mass spectrometers used were an API III* and API 300 from PE-SCIEX, Concord, Ontario. 2.3 Standard Solutions Since certificates of analysis were not available for the test compounds, 100% purity was assumed, although in the case of T-6293, the purity was known to differ significantly from this value. Stock solutions of T-6292, T-6293, T-6294 and T6295 were prepared at a concentration of 10 mM in dimethyl sulfoxide. `Working solutions (10 pg/mL) of each standard were prepared in 1:1 acetonitrile:2 mM `ammonium acetate. All stock solutions and working solutions were stored at 4C and were allowed to equilibrate to room temperature before use. Preparation of Analytical Standard Stock Solutions Stock Solutions of T-6292, T-6293, T-6294 and T-6295 (10 mM): Accurately weigh each test article on a micro balance and transfer it to a 16 X 101 mm siliconized polypropylene tube. Dissolve the solid sample in an `appropriate. amount of DMSO to yield a 10 mM solution. Working Solutions of T-6292, T-6293, T-6294 and T-6295 (10 Hg/mL): Dilute an appropriate aliquot of 10mM stock solution to 10 mL with 1:1 2 mM ammonium acetate:acetonitrile in a 16 X 101 mm siliconizedpolypropylene tube. 2.4 HPLC Eluent Preparation L 2mMAmmonium acetate: Ammonium acetate (154.2 mg) was weighed and | transferred into a 1 liter graduated cylinder. After adding water to the 1-L `mark, the i stirred solution was filtered under vacuum through a 0.45 pm filter. Methanol: Methanol was filtered under vacuum through 2 0.45 um filter. 2.5 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 packed in dry ice to Advanced BioAnalytical Services (ABS). The m--e-- eer-- mmm-------------------- samples were stored at ABS at -20C and were removed only for transfer of _ mm AA ovanD cEDot SERvices, inc xeroer senon BOREL 9 supernatant to an autosampler vial. After sampling, the autosampler vials were `maintained in an ice-water bath at all times except during injection. 2.6 Sample Analysis The samples analyzed included Rat 1 (all test articles, 0 and 6-Hr and control sample 13 (no test article)) and Human #H-116 (all test articles, 0 and 6-Hr). These were extensively studied under a variety of chromatographic and mass spectrometric conditions. HELCConditions: Initial HPLC analyses were successfully performed using gradient elution with water and methanol only. Later in the study, it was found that ammonium acetate was required for elution of T-6293. Thus, water was replaced by 2 mM ammonium acetate for further analytical work. Because two different gradient pumps were used for this work, it was difficult to reproduce the gradient obtained using the HP-1090L with the Shimadzu LC-10AD, thus for consistency within gradient systems, the same gradient program was used for both systems. Although this resulted in some variability in retention times between days, depending on the system used, all comparable samples were analyzed with the same system. Gradient Program: (Time (Min) | %eMethanol| %Water (2mM AmmAcetate] = 5 E 95 e-- 55 = 10 20 80 SEi== MassSpectrometerConditions: The mass spectrometer Was operated in the ion spray mode with negative ion detection. The curtain gas was ultra-high purity nitrogen. Although MS and MS/MS conditions were varied for different analytes, the main differences were in ion spray voltage, orifice voltage and collision energy. No positive ionization work was performed, as the analytes of interest did not ionize under these conditions. It mamaovancen SERVICES, INC rons senor ~o5362 10 is conceivable that additional metabolites could be detected using positive ionization, and this should be a part of further experimentation. `The approximate MS and MS/MS acquisition parameters used for the API TI" are listed below. Similar parameters were used for the work performed on the API 300. MS Mode MS/MS Mode Ton Spray Voltage 3600 V 3600 V Orifice Voltage: 0V 0v Declustering Potential av av Curtain Gas (UH.P. Ng) 120LPM 12LPM Nebulizer Gas (N2) 60 PSI 60 PSI Collision Energy 18ev cer 250 Scan Range 100-800 amu variable Step Size: see Note below variable Dwell Time Imsec 4 msec Note: Initial LC/MS step sizes of 1 amu were used to locate the chromatographic peaks ofpotential metabolites. Once located, step sizes of0.1 amu were used for `mass confirmation. Instrument mass axis calibration was performed by infusion of PPG-425 calibration solution (polypropylene glycols, average molecular weight 425, dissolved in 1:1 methanol:2 mM ammonium acetate containing 0.1% formic acid and 0.1% acetonitrile) ata flow rate of 10 WL/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 Q1 and Q3 by infusion of a 1 ng/mL solution in 1:1 acetonitile:2 mM ammonium acetate at a flow rate of 10 L/min. The mass spectrometric parameters and sensitivity were optimized using this solution at 50 L/min. 27 Data Handling All raw chromatographic and mass spectrometric data were stored on the ABS file server ABSI_FS.DATA in the file hierarchy "RAWDATA: Development: 3M: 3M ADVANCED SERVICES. INC. 665863 1 Metabolism. All pertinent experimental information was stored in ABS notebook No. 1067. 3. RESULTS AND DISCUSSION Initial investigations of the in vitro metabolism of the test artcles were performed. using direct injection of the quenched hepatocyte incubates rather than sample extraction. The reason for this is that, due to the polar nature of these compounds. and suspected metabolites, it was fel that extraction recoveries would be low and important metabolites could be missed. Most of the mass spectrometric information obtained in this study were fromQI scanning experiments, followed by product ion scans. Precursor ion scans of logical CID product ions were used but resulted only i in confirmation of previously found metabolites present at high levels. Neutral loss `experiments were even less sensitive under the conditionsusedand provided litle information. 3.1 Mass Spectrometric Analysis Results for Test Articles Structures of T-6292, T-6293, T-6294 and T-6295 are shown in Figure 1. The negative ionization mass spectra obtained from infusion of the individual analytes are shown in Figure 2. It should be noted that T-6292 could not be ionized in either the negative ionization mode, since it has only a very weakly acidic alkyl hydroxyl group or the positive ionization mode, ince the most basic site is a tertiary nitrogen ofa sulfonamide. The fact that T-6293 exists mainly in the diphosphoester form! (M, =1204), `comprising 82% of T-6293 solids, accounts for the reduced response seen for the 3 `monoester (M,= 651), when compared to T-6294 and T-6295. The diester was not detectable under the conditions used in this study. The negative ionization collision - induced dissociation (CID) full-scan spectra of the compounds are presented in Figure 3. T-6295 was found to be extremely stable and required both a high orifice voltage and high collision energy for fragmentation. A summary description of the proposed plausible CID fragmentation pathways are shown in Figures 4-6. Several ofthese fragments were diagnostic for metabolite identification. 3.2 Metabolism by Rat Hepatocytes Metabolism of the test articles by rat hepatocytes was examined using a Sciex API TII* mass spectrometer. _ --ee---------------------- Li.[L] On ror. woo 5864 SERVICES, INC. 12 32.1 Metabolism of T-6292 by Rat Hepatocytes Although T-6292 did not ionize under the conditions used, and was thus not detectable in these experiments, several potential metabolites were nonetheless found. When comparing the total ion chromatogram (TIC) obtained from the control rat sample (Figure 7, top; no test article added) with that obtained from the Rat I, O-Hr sample (Figure 8, top, reaction quenched at approximately time 0), several large peaks arc observed in the latter over the range of 7.2 to 8.5 min which are absent in the control sample. It should be noted that there is a 0.5 min difference between the TICs, due to operator error; the control TIC peaks are 0.5 `min earlier. The O-Hrpeaks were found to contain masses in a homologous series which are compound related but which may also be impurities or degradation products. Eachofthe observed peaks contains masses which differ by 80 and 107 amu. An example is given in Figure 9. Iti suspected that the different ions within ach chromatographic peak were generated byCID in the sampling orifice region of the mass spectrometer since no collision gas was used. In the example given, the lowest mass ion, mz 319, is a characteristic mass for perfluoroalkyl compounds, namely, C,F,;. The second ion, m/z 426, is 107 amu higher, which has been previously described for these compounds as a loss of EXNSO, (Figure 5). The ion observed at m/z 506 is 80 amu higher, which correspond to SOy, a common loss under these conditions. Thus, the proposed structures of this and the other analogs are shown in Figure 10a. It may be somewhat surprising that 0-Hr sample would contain metabolitesat significant levels; however, for rapid reactions, the sampling and quenching at this time point maybecrucial. Summary extracted ion chromatogramsofthe fragment ions analogous to mz 319, differing only by CF, groups, are shown in Figure 8. As predicted, no corresponding peak appears for m/z 469 (CF), since this would exceed the perfluoroalkyl chain lengthoftheparent molecule. Chromatographic peak splitting is evident in the lat eluting peaks, probably due to chromatographic resolution of the branched isomers of these compounds. Corresponding spectra for the Rat 1, 6Hr sample (Figure 11) indicate a decreased response for these compounds, except for the parent C,F,, fragment (m/z 419). The control sample contained no significant levels of these ions (Figure 7). An additional homologous series of proposed metabolites of the parent compound and impurities has been observed which appear to be glucuronic acid conjugates. ADVANCED SERVICES. INC. 005865 13 `The masses of these compounds match those of the O-glucuronides (Figure 10b) of these compounds. Figure 12 shows the extracted ion chromatograms for the [MHJ of the suspected intact glucuronides in the series for Rat 1, 6-Hr. The `glucuronides of the C3 analog through the parent compound, the C8 analog, are presented. By comparison, the peaks are greatly diminished in the 0-Hr sample (Figure 13) and absent in the control sample (data not shown). No evidence was found for any of the possible N-glucuronides. An additional major metabolite was the N-di-dealkylated perfluoroalkyl sulfonamide (Figure 14) with an [M-HJ" ofm/z 498. For this compound, a homologous series was also observed, but only for the C7 and C6 homologs (data not shown). A carboxylic acid metabolite was also found (Figure 14) with an [M-H]' of m/z 584. A homologous series was found for this metabolite which included the C4-C8 homologs (data not shown). Extracted ion chromatograms of the C8 homologs of these metabolites from Rat 1, 0 and 6-Hr are presented in Figure 15. To support the proposed structure of the carboxylic acid metabolite, an MS/MS product ion scan was performed for precursor ion m/z 584 (Figure 16). The fragment ions formed were consistent with the structure. The parent ion is not observed but a conceivable loss of the three-membered lactone CH,CO, from the carboxylic acid anion would produce the ion at m/z 526. 32.2 Metabolism of T-6293 by Rat Hepatocytes T-6293 also underwent significant metabolism by rat hepatocytes. The TICs obtained from the control sample, Rat 1, 0-Hr and Rat 1, 6-H are presented at the topofFigures 17, 18 and 19, respectively. As with T-6292, a 0.5 min difference k exists between the TICs, due to operator error; the control TIC peaks are 0.5 min earlier. Several possible metabolites were found in the rat samples. The extracted ion chromatogramsfor the anions of these metabolites are also shown in Figures 17-19. None of the metabolites found in the 6-Hr sample were found in either the O-orcHontrrol samples. The parent anion at m/z 650 was found in both the 0 and 6-Hr samples, although reduced byhalfin the latter. Possible structures for m/z 542 and 556 are shown in Figure 20. Very little "up front" CID occurred for these peaks, thus MS/MS experiments would be required to support the proposed structures. The observed retention times are consistent with the proposed structures, with the acid eluting earlier than the alcohol. The Imteeereeteemeeeeeeeeroemereeee----e LL] tee. SERVICES, INC. REPORT: S6ADEMOL 0 005866 { 14 19, m/z 746). ! metabolite observed with an [M-HJof mz 584 was shown to be the carboxylic acid previously seen for T-6292 (see Figure 14) by a MS/MS product jon scan (data not shown). The glucuronide proposed for T-6292 was also seen in T-6293 (Figure `The fons 498 and 499 were present at significant levels in both 0 and 6-Hr samples, although they both had higher responses in the 6-Hr sample (Figure 21), Thelikely structures of these compounds are perfluorooctanesulfonate (m/z 499, T-6295) and `perfluorooctanesulfonamide (m/z 498). From Figure 21, it can be seen that for i both 0 and 6-Hr samples, the laterm/z 499peak coincides with the 498 ion. This is | believed to incomplete be an mass artifact due to the isotopic contribution resolution between mz 498 and mz of the 499. m/z 498 jonand Additionally, the observed retention time for the first peak in the m/z 499 jon chromatogram is closer to that routinely observed for `perfluorooctanesulfonate than for the second (see Figure 28). Thus, it is assumed that the secondm/z 499 ion is actually m/z 498 being in all detected as m/z control samples 499. The examined mz 499 jon was (data not shown) found at the at significantly same retention high time levels as in these samples. Thus, while it may also be a product of metabolism, there is an endogenous background level as well. `Two additional ions were found in the TIC, m/z 327 and m/z 419 (Figure 22). The ion at m/z 419 is a common fragment ion (see Figure 4) product of degradation and/or metabolism while m/z and 327 an is easily more conceivable difficult to rationalize scan range and may used. be a product of "up front" Further experimentation CID with a. would be parent ion required. higher In the than the control sample, neither ion was detected (not shown). 3.2.3 Metabolism of T-6294 by Rat Hepatocytes mam aovaneen ---- The TICs and extracted ion chromatograms for the from the control, 0-Hr and 6-Hr samples for Rat 1 Proposed metabolites of are presented in Figures T-6294 23-25, respectively. The parent compound ([M-HJ' = 526) was observed in both 0 and 6. Hr samples at approximately 8.7 hepatocyte samples for T-6294. min. Several The main `metabolites metabolite were was found in the rat perflurorooctane sulfonamide ([M-H] = 498, 8.0 min), the N-dealkylation product. This metabolite was observed in ater. A MS/MS both the product 0 and 6-Hr ion scan was samples, although performed on the it was much larger in the 6-Hr sample forprecursor _ a---- SERVICES. INC 005867 i : 15 fon m/z 498 (Figure 26), structure. The structures and all for the observed fragments supported the. proposed radical species shown in Figure 26 are not Proposed (6.6 min) to is be the most stable configurations. postulated to be the same as the The metabolite observed fragment ion proposed in at m/z Figure 483 16. The remaining metabolites at 20, with oxidation occurring mvz 542 and 536 are postulated on the terminal methyl group. to be those in Figure 3.2.4 Metabolism of T-6295 by Rat Hepatocytes The control blank, O-Hr and 6-Hr TICs for T-6295 in Rat 1 are presented in | Figures 27-29, respectively. As observed observed in all control previously stated (Section 3.2.2), samples. The remaining fons in the the 499 jonwas. extracted ion i chromatograms are members of the homologous series seen previously for T-6292, tis difficult to would require believe deletion that of these CF, are products of units from the metabolism since their formation chain. Apparently, T-6295 is metabolized minimally by rat parent in both the 0 and 6-Hr hepatocyte samples incubates, as judged by the high levels of 3.3 Metabolism by Human Hepatocytes bMuettafbeowleristmypoefstwheerteessteaernt.iclTehsebhyuhmuamnansahmepplaetsocwyetrees ewxaasmisnimeidlaursitongthaatScoifetxheAPraIt 300 mass spectrometer, however, the rat samples. Further work is the investigation was certainly warranted. less extensive Since no new than that of metabolism was ext; discovered for the human no figures are presented. samples, the results wil only be discussed using 33.1 Metabolism of T-6292 by Human Hepatocytes ITnhteerehsotminoglloyg,ohuoswesveerrie,stphreegsleunctuerdoinnidFeigsuerriees1p0raesweanstendoitnoFbisgeurrveed10ibn wtahes hsueemnani.n the `and 6-Hr samples. The sulfonamide in Figure 14 the acid in Figure 14 was not detected a al, was observed at very low levels 3.3.2 Metabolism of T-6293 by Human Hepatocytes hTohweevpearr,entwitihonopwtaismiztahteioonnloyf tchoemmpaosusndsrpeelcattreomdetmeorieftoycusseeednonin Tt-h6e2s9e3,safmuprltehse;r information should be attainable. Based solely on abundance the parent ion is one. LL] BIOANgAmLY:TICAL SERVICES, INC. REPORT s6ADEMOILM 005868  16 third have lower in occurred. the 6-Hr sample than in the 0-Hr sample, thus some metabolism may 3.3.3 Metabolism of T-6294 by Human Hepatocytes Tcohmepopuanrde-nrtealnadtesduilofnosnaombisdeervmeedtaibnoltihteesa(mmplzes4.98,ThseeeabFuignudraenc1e4)ofwtehreepatrheentonilony in the 0-Hr conversely, sample was reduced the abundance of approximately by the sulfonamide half in in the 6-Hr the O-Hr sample sample and, was approximately doubled in the 6-H sample. i T3h3e.r4e wMeerteanbooldiisscmemoafbleT-d6if2f9e5renbcyes bHeutwmeaenn thHeep0aatnodc6y-tHerssample for T-6295, 4. CONCLUSIONS Sinevtehrealsptuodtye.ntiaFlumretthaebrolMitSe/sMaSndeixmppeurriimteinetsatwieorne, dpeotsesrimbilnyedinfrcoomnjtuhnectLiCo/nMwSitdhataa psoasmtpulleatceldeastnruupctaunrdescoinnctehnetrraattiosnamsptleeps,.w~ouAlddditbieonarlelqyu,irefdortothfeurhtuhemransupspaomrptletsh,e further high sensitivity detection experimentation is required for complete preliminary investigation, followed by MS/MS experimentation for confirmation of the metabolite structures S. REFERENCES 1. Gordon, Steven C., Ph.D., D.A.B.T., Health Hazard Summary of Ammonium Salts of Mono-, Di- and Tri(N-cthyl(perfluorooctane)sulfonamidoethyl}phosphate (FC-807). September 14, 1994, `Supplied by 3M Company. Lc LL] SBIvOAyNcALeYpTICAL SERVICES. INC. REPORT soADEMOL 005869 | 17 Figure 1. Structures of the study test articles o T-6292 FiC-OF OFCy OF OF Il Cry OF 31 orzo . M,=571 | SeHzcHoon o | 1{ o T-6293 Fi0-CFy OF OFF OF OF Fy1 I cree, o [I] M,=651 I CH,-CH,-0-P-OH | OH T-6204 M,=527 F007 07, oI rrcree II o creche wn TM-6,=295500 i F4C-CF,-CF,-CF-CF,-CF,-CF,-CFp-S-OH I o -_ IM 2ocee, BSEIROVAINCAELSY,TIICNACL REP0ORT0: e5an8EM7OL0 I 18 i Figure 2. Negative ionization T-6293 (IM-HJ = mass spectra obtained from 650.1), T-6294 ((M-H] = the test 526.1) articles and T. 6295 (IM-H]" = 498.9) Spectrum from 1.6263 q1.01 & 180s] 220.0 1.9507 cps 650.1 | z 1.e2e06 255.2 = 6.005 200 300 400 mz,50a0mu 600 Spectrum from T.6204 q1.02 4 9.005 526.1 5 co = a0 700 1.9607 cps 200 300 400 mz5a00mu 600 Spectrum from T6295 q1.08 9.006 438.9 5 6.006 E 3.008 220-0 309.1 200 300 400 mz,50a0mu 600 700 1.937 cps 700 _ ADVANCED SERVVIICCESE. S, iINC. 005871 | 19 Figure 3. Product ion spectra obtained from the deprotonated test articles T-6293, T-6294, and T-6295. 628p3Product onScan g TM 2% ss 495000 850 2 | 3 123 "18 1 100 20 0 mewo 50 0 700 T6294 Product on Scan oo 28 405.000 g5 3 = 100 219 i les ms 29 wo 0 20 %0 me 50 0 7.6295Product onScan 2zoo7s 2i sw 3 oo We 20 an gy 100 20 meED 0 1P.6S20000 0 _-- MW 2BIoOAcNAeLYeTICAL SERVICEISN,C REPORT: seADEMO1 0 005872 | 20 Figure 4. Proposed CID fragmentation mechanism for T-6293. T-6203 m/z = 650 oI cHecH o Fi0-OF OF OF OF OF OF Oa_ I I~ (iret -OH o- - GVCa " " (124 amu) miz = 526 FSC CR Re o wl CHaCH, creer Or aN, Oo - Et--N=S0, (107 amu) miz=419 F5C-CFC,FOFpy-OF--CFCCFF, -- ee e --e -- e--ee-------- am ABSEIcROaVANINCAcELESY,TDIICNAGL REPORT -senDEMwm 005873 | i 21 Figure 5. Proposed CID fragmentation mechanisms for T-6294. T-6294 m/z=526 F4C-CF5yCF-y-COFFp-CyF-CCFlFlSP-voNdpoa I ha ~~ 0 | I7N I g onea CH : F1 = ~ Et--N=S0, (107 amu) miz=126 (pathway not shown) mE=419 pr FiOCCFOR,CyR,CCF,E OF, a c FAC-CF,~ mz=119 FiC-CF;CF;"_ mz =169 a b FAC-CF,C-FCCF,,~ FaC-CFo-CFo-CF;_ mz=260 miz=219 mamABIDDAvNaALcYmTICAL Services, ING Rerony saver m| 005874 22 | Figure 6. Proposed CID fragmentation mechanisms for T-6295. i miz=499 i Pathway A - 50, (80 amu) i aPlaotnhgwpaeyrBi:uoHroomaollyyltcihcacilneavages rN } medi) FiCOFCQRECRQCFEF a< FsC-OF," miz= 119 FsCCFyOF; mz= 169 ba FaC-CF,-CF,C-FC,F" FoC-CFyOFy-OFTM mlz = 269 miz=219 "CFACRNSO; "CF2A(CCFFsS05 "CF(CF,S0; "CF,CF,S0; +CF,80; -so5" miz=3%0 miz=280 miz=200 miz=180 miz= 130 mz =80 0 AIls I miz=99 (pathway not shown) -- ar-- te --e eatee r s-------- ADVANCED SERVICES. INC. 005875 23 TC amasse 1001080 Figure 7. Total ion and extracted fragment ion chromatograms for T6292 obtained from Rat Control Sample 13. cont sa1n5, m0i 5 wo 2ens000 f5 i w i Fie 1! cgoxdno t5onolTA 0= Se2iis ri) %1 W&w ones sooan0 zioare Tao one som swore "oo sores m0 ron sso oes soo0m -- ! EFE i E=EBE E EB wl cee Time ys eet te-- _ SAISDEOVRAAVNINACCLEESY.DTIANLG 005876 REPORT: S6ADEMOI IM 24 Figure 8. Total ion and extracted fragment ion chromatograms for T6292 obtained from Rat 1, 0-Hr sample. TIC of all massa, 0010800 252 Rat 1, 04, 10u -z 0 8 5090000 zn 5- 51 7 | 22 a8 30 30 ii 2 00 Tor 201 24 pr) on 72 on 96 16ar1s0 Scan (mie) 725 20000 219219 75 7e5000 257260 7s 85000 store 800 a45000 asrs69 ] 185.000 020 rare a05000 aoveen ao a="o -_ ADVANCED SERVICES. INC iwoo =Pe) Time (minyScan 20000 =0 oaso out expos 0058777 ! 25 : Figure 9. Extracted ion chromatograms of fragment peaks for T-6292 homologous series in Rat 1, 0-Hr sample. T6202Rat 1,0104ul, strat 645.000 oo Z2 | FE ! "esizs 125000 ogo 2 g so1508 I 280000 og o zs g [11 2a0 "607 Time (rioyScan2i5)0 58303 1h0t0 _ ADVANCED SERVICES. INC 005878 26 Figure 10a. Proposed structure of parent compound for T-6292 homologous series from Figures 8 and 11. oII Ficoranors $a o| CHacHy "sos MassofParentAnion a ) 356 1 ' 406 2 456 3 506 4 556 5 606 6 Corresponding Fragment Ion inFigures Sand11 169 219 269 319 369 419 Figure 10b. Proposed structure of glucuronide for `homologous series from Figures 12 and 13. 9 [CARAIIII ScSaccHehc,H0--ciu_ o `Massof ParentAnion 496 546 596 646 696 746 --e ADVANCED SERVICES, INC 1 1 2 3 4 5 6 005879 ! 27 ! Figure 11. Total 6292 fon and obtained extracted from Rat fragment ion chromatograms 1, 6-Hr sample. for T. : TIC ofallmasses, 10010800 Tie252 Ra1t,6+, 10 uf 81 4.205.000 z fw | Fs 1 i7 00 01 20 wr or 24 Scan4T8ime (min) 72 56 tearten 75.000 727 w- 210219 280289 A s1arst 195000 AE A PA. A zo A 80s 0000 360/369 200000 82 soto 840.000 "asr40 40.000 010 280 6wo 2<0 Time (rinyScan _ ABIDOvAaNnAcLYeTnICAL SERVICES, INC. =50 0i0e OO REPORT: 6AD-- GO1 0 005880 28 Figure 12. Extracted ion chromatograms of putative glucuronide peaks for T-6292 homologous series in Rat 1, 6-Hr sample. T6202Rat, 64 100i 0 soerass Ew | =Fa sewrsis i EZw I E sous g1 Ez i so asain sss 155000 sar 80000 S000 kd ao 00000 g r 696/696 ,Ei oo Taerrie + i _ 000 20 ost ae Kk T4o0r imo(riyscan=60 =80 10,000 "0000 1G0e0 -_-- ADVANCED SBEIROVAINCAELSY.TIICNACL 005881 REVERY: S4000194 29 Figure 13. Extracted jon chromatograms of putative glucuronide peaks for T-6292 homologous series in Rat 1, 0-Hr sample. 252 Rat1,0410, nf souvase g7 105000 i E 546/546. Z sw 45,000 ! " 596/596. g Zw 10,000 & wersss g 25000 Ew g eourese 2 105000 | 0& 746/746 10,000 z py & 0.0 2a0 i4o0rTime (minysc2an5600 =80 1p0i0s ESr--o-- e ------ Ls.1] ABIaOAnNALgYTICAL SERVICES. INC Heron seapesin w 005882 30 Figure 14. Proposed structures of the anions of the sulfonamide and carboxylic acid metabolites for T-6292 from Rat 1. SuMleftaobnoalmiitede(m/z 498) o FeieRscry1NH | o CMaertbaobxoylliiptceA(cmi/dz 584) i F45CC-(-CF(CF,Js CFSI Oo CH-CHy one o- o remem ---------------------------------------- ADVANCED SERVICES. INC 005883 31 Figure 15. Extracted ion chromatograms of sulfonamide (m/z 498) and carboxylic acid (m/z 584) metabolites for T-6292 from Rat 1, 0-H (top) and 6-Hr (bottom) samples. T6262Rat1, 0+10 u,lin 1 sseasn S500 g searses 0000 g E E 010 zao pr16y7Tine(ninyScan2s5o0 580 1i0e0 T6252 Rat 1,6+, 10ulif asiase "2s000 1 ; sues 90000 Ek z 1 01 2a0 w6o7"Time (minyScan26500 Ed50 1"0ie0 c--e ----t-------------------------------------------- am sBIoOvAeNnAcLYeTnICAL SERVICES, INC REPORT: senpevoL 005884 32 Figure 16. san pron Product ion Rat 1, 6-Hr scan for carboxylic acid metabolite of T-6292 sample and proposed fragment structures, in 100) ae % ; i i .o ea - 169, 219, 269, 419, 526 483 . iSee Figure 5 foerecray = o i 83 F-S- __ Oo 005885 Figure 17. Total ion and extracted parent metabolite ion chromatograms for Control Sample 13. (m/z 650) and T-6293 obtained 33 proposed from Rat cToInCtoof asammpal 1s3,17s00 1TeA08,00 70 ws o 2e05000 z 9g7 i E 5 a Ln se wo T37 ai ) d =o i A ee d i a0 =101 Sea2np0T1in (rin =301 4p0d1 contosampl 13,00 suas ssasas o Sauses sso Teas 0 w 3 a n ioTima ms iysean2a n = mfe sree I sBOoAvNAeLeYTeICAL eee Heros E-- aon SeAvices, ING 005856 34 Figure 18. Total ion and extracted parent (m/z 650) and proposed metabolite ion chromatograms for T-6293 obtained from Rat 1, 0-Hr sample. TICofalmasses 10010800 Ratt, 0, 10ulin g_ 0 99 a8 2880000 : 2izow 140 ve 2 . an 74 := & on9 2o4r Ratt, 0410u,n searsa 20"18 w2r ScanTime (ni) 9w%r 15000 sseisss s15000 Er) 315.000 esoveso asia 0 315.000 a15000 0v0 2a0 iwe 200 Time (mica =0 p10H0 -_-- Apvancen SERVICES, INC. 0058877 i 35 Figure 19. Total ion and extracted parent (m/z 650) and proposed metabolite ion 6-Hr sample. chromatograms for T-6293 obtained from Rat 1, TTI6C28of3lRlamta1s, s6e+s,10100u1l0800 8 z 2810000 450 850 1, i i on $s uf Bo ay !! i1 00 12041 2i0s1 721 w96r T6Rat2 1,6+0,103 uly ScanTime (ni) sisi 795 310000 ssesss aan 65000 souses 67 165.000 esaies 70 140000 Tasi7ie 653 125000 010 2a0 1p8y7 200 Time (minyScan 38%0 1u0s0 _ ABIDDVAANNACLYETDICAL SERVICES. INC. REPORT: seDENDL IM 005858 Figure 20. Possible structures for the observed metabolites with [M-H]" of 542 and 556 amu from Rat 1. 36 of T-6293 M[M,-=H5T4=3 542 Pon CH,0H F43CC--CFCyF-C,F-CF,-CCFF,-CF,C-FCoF-,CF-,C-FC,Fy-CF,"L5 eu ` o | 1 M[,Mv=A5T5=7556 oIl, cHzcoon F"340-CF C-CF-COFFy-CF,CF-5-CCFF-CF-CF,-CCF,-CF,J<x" o -_ am ADVNANCCEDD te SERVICES, INC Reson savior 005859 | 37 | Figure 21. Extracted jon chromatograms of sulfonamide (m/z 498) and asunldfo6n-atHer ((mb/oztto4m9)9)saimopnlsesf.or T-6293 from Rat 1, 0-Hr (top) Rat,040, 10uo asaase . 20000 Eg w & 1 i sontase 100000 : zg w g o1o Zao w6o7Time (minysc2a5nP0Y 2Pys 0"r0e T6253Rat,64, 10 souise 65000 g E50 assase 285.000 2 sof 0 Z5o i"0e 250 Time (minySean 250 0f0e _ Lc. [1]BSAIEdORvAVaNInCAEcLSeY,oTCINACL. RE.PORT:s6ADEMOL 30 005890 38 Figure 22. (bottom) samples. Extracted ion chromatograms of proposed metabolites with m/z 327 and 419 for T-6293 from Rat 1, 0-Hr (top) and 6-Hr Rat, 04 10 ulin zr E om gEs i g } 1 aioe asm Zg w g 0V pZio iw Tina (rican2w20o =50 1"080 T0293Rat, 6 100 senszr 21500 Z2 w g rorero 1 s70000 gZs 9\) FyZo wiooTime (niyse2an20 =20 1p0s0 LL. LL Ee I SBeIrOvAiNcAeLsY,TIICNAGL Rer0oR0T 5sen8op9uor1 39 Figure 23. Total ion and extracted parent metabolite ion chromatograms for Control Sample 13. TCofamassa, 10010800 (m/z 526) and T-6294 obtained proposed from Rat 6254, onl,notes ari, 10 in, 1090 2 7s 2005000 5 so t Zs 22 x 32 42 A ! = 01 170)1 Sca2on0S1Tn rie) =301 40p1r T6254, Consol, notes ari, 00 1050 prs T0000 "enim 000 Sauezs T0000 same 10000 Soares 100% a0i F2S0 oioTine (riseFnoAo E20S poi o Im ADVANCED SERVICES, iNCe 0 xoeon sno6p0e5s892 I 40 Figure 24. Total ion and extracted parent (m/z 526) and proposed metabolite ion chromatograms for T-6294 obtained from Rat 1, 0-Hr sample. aetatmsan sucrose Tazo, har, 0, 10 wi, 030 0 He | : 7a we sso so : = oo1 =101 Sca2n=0t1 rn) =201 Ta,Pat oh 10 1000 avis 5401 sesso on wm "woo sao ae Tams sae Tow po Tams00 --; =z =wTime rnin= Pr2 ="o8 Im ABSIeDDrVAvANiNAcCLeYEsTDIRCAeL OOOO 0REP0ORT5: 8sA9DE3MDL 0 41 Figure 25. Total fon and extracted parent (m/z 526) and proposed metabolite fon chromatograms for T-6294 obtained from Rat 1, 6-Hr sample. TIC of altmasses, 10010800 T6284, Rat, 6, 10 i, 1090 i g1 a 7.685.000 i z 7s x 87 ]! HE te 30 "o . eo00 0241 2i0s1 703 o9r7 ScanTime (min) T6204, Rat, 6, 10 ln, 1030 saae os 0000 "sase 1 3535000 sasrsas ars 1389000 sass 1 sseisss 010 2&0 812 ars wesoTime (minyScan248 =50 30000 50000 1"0a0 _ ABIDOVAANNACLYETDICAL SERVICES. INC REP0ORT0: 5Se8n09EM4OLIM i 42 Figure 26. Product fon scan for perfluorooctanesulfonamide metabolite (CFy(CF,),SO,NH,, m/z 498) of T-6294 in Rat 1, 6-Hr sample and proposed fragment structures. loDAUGHPaTrtE4R50 Tzert 1,044 oui . PSw0750 I " i _ 3 i w = ww 219 259 EQ we 3 a" ES 169, 219 Desert See Figure 5 i po. erniie 5 7 o aI nn 1 mn -- ee BSIEORAVNIACLEYS,TIICNAC.L 005J8CA9DE5MOI 43 Figure 27. Total fon and extracted parent (m/z 499) and homolog ion chromatograms for T-6295 obtained from Rat Control Sample 13. TIC of all masses, 100 10800. contol media (nota), 10, gradient7 10 109 7 650 720000 Ld ans i f o= 288 i] i7 00 1201 03 072 0o1s ScanfTime (min) `control madia (10a),10ul,racent 7 "osasn 60 waists es couraos 6% saorsao 2081299 o1o 2B0 p16y7 200 =50 Tie (minySean "0000 15.000 50.000 "0000 "000 1"0i0e -- BAIDOVANAANLCYETDICAL SERVICES, ING REPORT: 3enDEMOLM 005896 1 a4 ! Figure 28. Total ion and chromatograms extracted parent (m/z 499) and for T-6295 obtained from Rat 1, homolog ion 0-Hr sample. TIC of all masses, 10010 800 T6295, at, 0, 10, gradien7t gn 63 2080000 gz 7 i 22 108 ast x : L3 E) ] 00 101 201 01 or 24 Scan4T8ime (nin) 72 96 T6295, at, Ov, 10, racen7t assaz0 686 1.420000 "asad 602 180000 soora09 63 375.000 saorzag 600 135.000 200/299 525 25000 010 o2a0 067 200 Time (mean =50 1w0e0 ------ ABIDOVAANNACLYETDICAL SERVICES, INC. ------------------ RERORT SADE 3 005897 45 Figure 29. Total ion and extracted parent (m/z 499) and homolog ion chromatograms for T-6295 obtained from Rat 1, 6-Hr. ICofall masses, 10010800 T6295, Fatt, 6-1, 10 ul, gradien7t 7 z 7 2 = 1.98 cy 00 10214 T6295, att, 6-hr, 10ul, gradient 7 499/499 634 6 ago 603, 24081 7031 `ScanTime (min) 634 2830000 40071 1.960.000 49/449 68s 275.000 3997399 638 535,000 s4sra9 608 220,000 2909299 524 65,000 010 2a0 14607 24609 3820 1a0ts0 "Time (minyScan _-- mADVANCED BIOANALYTICAL SERVICES. INC. S-- e---- -- EE---------- i-- 0 REORE MADEMRY 005898