Document 0qzOEwo74V77Kd4kqgw23LJZO

ADVANCED BIOANALYTICAL SERVICES, INC. ANALYTICAL REPORT NOV 1996 \ &A TITLE: "v^/CO'-S QUALITATIVE INVESTIGATION OF THE IN ~~ " VITRO METABOLISM OF T-6292, T-6293, T-6294 ANt) T-6295 BY RAT AND HUMAN HEPATO CYTES USING ION SPRAY LC/MS AND LC/MS/MS DATE: November 12, 1996 REPORT: 96ADEM01.3M AUTHORS: Daniel E. Mulvana, B.S. Jack Henion, Ph.D. PREPARED FOR: 3M Medical Department, Toxicology Services, St. Paul, MN 55133 NO. OF PAGES: 45 005854 15 C a th e rw o o d Road Ithaca. New York 14 85 0 (607) 2 6 6 -0 6 6 5 Fax (6 0 7 ) 2 6 6 - 0 7 4 9 2 ADVANCED BIOANALYTICAL SERVICES SIGNATURE PAGE T itle: 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 R eported by: * Daniel E. Mulvana, B.S. Senior Research Scientist 7 Date L Reviewed and Approved by: Mary Ringwood, Bt#. Quality Assurance Manager Date A uthorized for Release by: Jack Henion, P h .D .(^ & Laboratory Director Advanced BioAnalytical Services Date ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. f>S8S5 REPORT : 96ADEM01.3M 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 100 mm Betasil 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. ADVANCED BIOANALYTICAL SER V IC E S . INC. U05856REPORT : 96ADEM0; 4 TABLE OF CONTENTS SIGNATURE PAGE ABSTRACT PAGE 1. INTRODUCTION 1.1 Introduction 1.2 Study Objective 2 3 7 7 7 2. EXPERIMENTAL 2.1 Chemicals and Materials 2.2 LC/MS Instrumentation 2.3 Standard Solutions 2.4 HPLC Eluent Preparation 2.5 Sample Preparation 2.6 Sample Analysis 2.7 Data Handling 7 7 7 8 8 8 9 10 3. RESULTS AND DISCUSSION 3.1 Mass Spectrometric Results for Test Articles 3.2 Metabolism by Rat Hpatocytes 3.3 Metabolism by Human Hpatocytes 11 11 11 15 4. CONCLUSIONS 16 5. REFERENCES 16 FIGURES Figure 1. Structures of the study test articles. Figure 2. Negative ionization mass spectra obtained from the test articles T-6293 ([M- H]- = 650.1), T-6294 ([M-H]` = 526.1) and T-6295 ([M -H]' = 498.9). Figure 3. Product ion spectra obtained from the deprotonated test articles T-6293, T- 6294, and T-6295. Figure 4. Proposed CID fragmentation mechanism for T-6293. Figure 5. Proposed CID fragmentation mechanism for T-6294. Figure 6. Proposed CID fragmentation mechanism for T-6295. 17 18 19 20 21 22 ADVANCED BIDANALYTICAL SER VIC ES, INC. 005857 REPORT : 96ADEMO 1..1M 5 Figure 7. Total ion and extracted fragment ion chromatograms for T-6292 obtained from Rat Control Sample 13. Figure 8. Total ion and extracted fragment ion chromatograms for T-6292 obtained from R at 1, O-Hr sample. Figure 9. Extracted ion chromatograms of fragment peaks for T-6292 homologous series in Rat 1, O-Hr sample. Figure 10a. Proposed structure of parent compound for T-6292 homologous series from Figures 8 and 11. Figure 10b. Proposed structure of glucuronide for homologous series from Figures 12 and 13. Figure 11. Total ion and extracted fragment ion chromatograms for T-6292 obtained from R at 1, 6-Hr sample. Figure 12. Extracted ion chromatograms o f putative glucuronide peaks for T-6292 homologous series in Rat 1, 6-Hr sample. Figure 13. Extracted ion chromatograms of putative glucuronide peaks for T-6292 homologous series in Rat 1, O-Hr sample. Figure 14. Proposed structures of the sulfonamide and carboxylic acid metabolites for T6292 from Rat 1. Figure 15. Extracted ion chromatograms of sulfonamide (m/z 498) and carboxylic acid (m/z 584) metabolites for T-6292 from Rat 1, O-Hr (top) and 6-H r (bottom) samples. Figure 16. Product ion scan for carboxylic acid metabolite o f T-6292 in Rat 1, 6-H r sample and proposed fragment structures. Figure 17. Total ion and extracted parent (m/z 650) and proposed metabolite ion chromatograms for T-6293 obtained from Rat Control Sample 13. 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. Figure 19. Total ion and extracted parent (m/z 650) and proposed metabolite ion chromatograms for T-6293 obtained from Rat 1, 6-Hr sample. Figure 20. Possible structures for the observed metabolites of T-6293 with [M-H]' of 542 and 556 amu from Rat 1. 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. 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. 23 24 25 26 26 27 28 29 30 31 32 33 34 35 36 37 38 ADVANCED BIOANALYTICAL S E R V IC E S . INC. REPORT : 9 6 A D E M 0 l i k r k C C C C 6 Figure 23. Figure 24. Figure 25. Figure 26. Figure 27. Figure 28. Figure 29. Total ion and extracted parent (m/z 526) and proposed metabolite ion chromatograms for T-6294 obtained from Rat Control Sample 13. Total ion and extracted parent (m/z 526) and proposed metabolite ion chromatograms for T-6294 obtained from Rat 1, O-Hr sample. Total ion and extracted parent (m/z 526) and proposed metabolite ion chromatograms for T-6294 obtained from Rat 1, 6-Hr sample. Product ion scan for perfluorooctanesulfonamide metabolite (CF3(CF2)7S 0 2NH2, m/z 498) of T-6294 in Rat 1, 6-Hr sample and proposed fragment structures. Total ion and extracted parent (m/z 499) and homolog ion chromatograms for T-6295 obtained from Rat Control Sample 13. Total ion and extracted parent (m/z 499) and homolog ion chromatograms for T-6295 obtained from Rat 1, 0-Hr sample. Total ion and extracted parent (m/z 499) and homolog ion chromatograms for T-6295 obtained from Rat 1, 6-Hr. 39 40 41 42 43 44 45 ADVANCED B IO A N A L Y T IC A L S E R V IC E S , INC. 5S59 REPORT : 96ADEM01JM. 1. INTRODUCTION 7 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 (m/z 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 was to detect and characterize the metabolism of T-6292, T-6293, T-6294 and T-6295 by rat and human hepatocytes. 2. EXPERIMENTAL 2 .1 Chemicals and Materials T-6292, T-6293, T-6294 and T-6295 (Figure 1) were provided by 3M M edical Department, Toxicology Services, St. Paul, MN 55133. Siliconized polypropylene autosampler vial: 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 W ISP 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, ADVANCED BIOANALYTICAL S E R V IC E S . INC. 9 Q S S 6 0REPORT : 96ADEMI 8 Inc., Bellefonte, PA 16823. The mass spectrometers used were an API in + 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 o f 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 o f DMSO to yield a 10 mM solution. Working Solutions of T-6292, T-6293, T-6294 and T-6295 (10 pg/mL): Dilute an appropriate aliquot of 10 mM stock solution to 10 mL with 1:1 2 mM ammonium acetate:acetonitrile in a 16 X 101 mm siliconized polypropylene tube. 2 .4 HPLC Eluent Preparation 2 mM Ammonium 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 stirred solution was filtered under vacuum through a 0.45 pm filter. Methanol: Methanol was filtered under vacuum through a 0.45 pm 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 o f ice-cold methanol, centrifuged and shipped packed in dry ice to Advanced BioAnalytical Services (ABS). The samples were stored at ABS at -20C and were removed only for transfer of ADVANCED BIOANALYTICAL S E R V IC E S , INC. REPORT : 96ADEM0 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-H r). These were extensively studied under a variety of chromatographic and mass spectrometric conditions. HPLC Conditions: 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) % M eth an o l % W a te r (2 m M A m m .A c etate) 0 20 5 95 9 95 80 5 5 10 15 ~ 1 20 20 80 80 Mass Spectrometer Conditions: 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 ADVANCED BIDANALYTICAL S E R V IC E S , INC. REPORT : 96ADEM0I.3M r 05362 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 IIP are listed below. Similar parameters were used for the work performed on the API 300. MS Mode M S/M S M ode Ion Spray Voltage Orifice Voltage Declustering Potential Curtain Gas (U.H.P. N2) -3600 V -70 V 42V 1.2 LPM -3600 V -70 V 42V 1.2 LPM Nebulizer Gas (N2) 60 PSI 60 PSI Collision Energy 18 eV CGT 250 Scan Range 100-800 amu variable Step Size see Note below variable Dwell Time 3 msec 4 msec Note: Initial LC/MS step sizes of 1 amu were used to locate the chromatographic peaks of potential metabolites. Once located, step sizes of 0.1 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) at a flow rate of 10 |iL/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 o f a 1 fig/mL solution in 1:1 acetonitrile:2 mM ammonium acetate at a flow rate of 10 pL/min. The mass spectrometric parameters and sensitivity were optimized using this solution at 50 fiL/min. 2.7 Data Handling All raw chromatographic and mass spectrometric data were stored on the ABS file server ABS1_FS.DATA in the file hierarchy "RAWDATA: Development: 3M: 3M -------------------------------------------- -----------------------------------------------------------0 6 5 3 6 3 ADVANCED BIOANALYTICAL REPORT : 96ADEM01.3M SER VIC ES, INC. 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 articles 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 felt that extraction recoveries would be low and important metabolites could be missed. Most of the mass spectrometric information obtained in this study were from Q1 scanning experiments, followed by product ion scans. Precursor ion scans of logical CID product ions were used but resulted only in confirmation of previously found metabolites present at high levels. Neutral loss experiments were even less sensitive under the conditions used and provided little 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, since the most basic site is a tertiary nitrogen of a sulfonamide. The fact that T-6293 exists mainly in the diphosphoester form 1 (Mr =1204), comprising 82% of T-6293 solids, accounts for the reduced response seen for the monoester (Mr= 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 CED fragmentation pathways are shown in Figures 4-6. Several of these 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 IIT mass spectrometer. ADVANCED BIOANALYTICAL SER VIC ES, INC. 005864REPORT : 96ADEM01JM 3.2.1 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 1, O-Hr sample (Figure 8, top, reaction quenched at approximately time 0), several large peaks are 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 0-Hr peaks were found to contain masses in a homologous series which are compound related but which may also be impurities or degradation products. Each of the observed peaks contains masses which differ by 80 and 107 amu. An example is given in Figure 9. It is suspected that the different ions within each chromatographic peak were generated by CID in the sampling orifice region of the mass spectrometer since no collision gas was used. In the example given, the lowest mass ion, m/z 319, is a characteristic mass for perfluoroalkyl compounds, namely, C 6F I3. The second ion, m/z 426, is 107 amu higher, which has been previously described for these compounds as a loss o f E tN S 02 (Figure 5). The ion observed at m/z 506 is 80 amu higher, which correspond to S 0 3, 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 metabolites at significant levels; however, for rapid reactions, the sampling and quenching at this time point may be crucial. Summary extracted ion chromatograms of the fragment ions analogous to m/z 319, differing only by C F2 groups, are shown in Figure 8. As predicted, no corresponding peak appears for m/z 469 (C 9F I9), since this would exceed the perfluoroalkyl chain length of the parent molecule. Chromatographic peak splitting is evident in the late eluting peaks, probably due to chromatographic resolution of the branched isomers of these compounds. Corresponding spectra for the Rat 1, 6H r sample (Figure 11) indicate a decreased response for these compounds, except for the parent C 8F 17 fragment (m/z 419). The control sample contained no significant levels of these ions (Figure 7). An additional homologous series of proposed metabolites o f the parent compound and impurities has been observed which appear to be glucuronic acid conjugates. ADVANCED BIOANALYTICAL SER VIC ES. INC. REPORT : 96ADEM0UM 005S6S 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 [MH]' 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-H]' of m/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]' o f 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 CH2C 0 2 from the carboxylic acid anion would produce the ion at m/z 526. 3.2.2 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-Hr are presented at the top of Figures 17, 18 and 19, respectively. As with T-6292, a 0.5 min difference 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 chromatograms for 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 0-Hr or control samples. The parent anion at m/z 650 was found in both the 0 and 6-Hr samples, although reduced by half in the latter. Possible structures for m/z 542 and 556 are shown in Figure 20. Very little "up front" CDD 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 ADVANCED BIOANALYT1GAL S E R V IC E S , INC. REPORT : 96ADEM01.3M 005866 14 metabolite observed with an [M-H]' of m/z 584 was shown to be the carboxylic acid previously seen for T-6292 (see Figure 14) by a MS/MS product ion scan (data not shown). The glucuronide proposed for T-6292 was also seen in T-6293 (Figure 19, m/z 746). The ions 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). The likely 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 both 0 and 6-Hr samples, the later m/z 499 peak coincides with the 498 ion. This is believed to be an artifact due to the isotopic contribution of the m/z 498 ion and incomplete mass resolution between m/z 498 and m/z 499. Additionally, the observed retention time for the first peak in the m/z 499 ion chromatogram is closer to that routinely observed for perfluorooctanesulfonate than for the second (see Figure 28). Thus, it is assumed that the second m/z 499 ion is actually m/z 498 being detected as m/z 499. The m/z 499 ion was found at significantly high levels in all control samples examined (data not shown) at the same retention time 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) and an easily conceivable product of degradation and/or metabolism while m/z 327 is more difficult to rationalize and may be a product of "up front" CID with a parent ion higher than the scan range used. Further experimentation would be required. In the control sample, neither ion was detected (not shown). 3.2.3 Metabolism of T-6294 by Rat Hepatocytes The TICs and extracted ion chromatograms for the proposed metabolites of T-6294 from the control, O-Hr and 6-Hr samples for Rat 1 are presented in Figures 23-25, respectively. The parent compound ([M-H]' = 526) was observed in both 0 and 6H r samples at approximately 8.7 min. Several metabolites were found in the rat hepatocyte samples for T-6294. The main metabolite was perflurorooctane sulfonamide ([M-H]' = 498, 8.0 min), the N-dealkylation product. This metabolite was observed in both the 0 and 6-Hr samples, although it was much larger in the latter. A MS/MS product ion scan was performed on the 6-Hr sample for precursor ADVANCED BIOANALYTICAL SER VIC ES, INC. REPORT : 96ADEM01.3M 005867 ion m/z 498 (Figure 26), and all observed fragments supported the proposed structure. The structures for the radical species shown in Figure 26 are not proposed to be the most stable configurations. The metabolite observed at m/z 483 (6.6 min) is postulated to be the same as the fragment ion proposed in Figure 16. The remaining metabolites at m/z 542 and 556 are postulated to be those in Figure 20, with oxidation occurring on the terminal methyl group. 3,2.4 Metabolism of T-6295 by Rat Hepatocytes The control blank, 0-Hr and 6-Hr TICs for T-6295 in Rat 1 are presented in Figures 27-29, respectively. As previously stated (Section 3.2.2), the 499 ion was observed observed in all control samples. The remaining ions in the extracted ion chromatograms are members of the homologous series seen previously for T-6292.. It is difficult to believe that these are products of metabolism since their formation would require deletion of C F2 units from the chain. Apparently, T-6295 is metabolized minimally by rat hepatocyte incubates, as judged by the high levels of parent in both the 0 and 6-Hr samples. 3.3 Metabolism by Human Hepatocytes Metabolism of the test articles by human hepatocytes was similar to that of the rat but fewer types were seen. The human samples were examined using a Sciex API 300 mass spectrometer, however, the investigation was less extensive than that of the rat samples. Further work is certainly warranted. Since no new metabolism was discovered for the human samples, the results will only be discussed using text; no figures are presented. 3.3.1 Metabolism of T-6292 by Human Hepatocytes The homologous series presented in Figure 10a was not observed in the human. Interestingly, however, the glucuronide series presented in Figure 10b was seen in the 6-Hr samples. The sulfonamide in Figure 14 was observed at very low levels and the acid in Figure 14 was not detected at all. 3.3.2 Metabolism of T-6293 by Human Hepatocytes The parent ion was the only compound-related moiety seen in these samples; however, with optimization of the mass spectrometer focused on T-6293, further information should be attainable. Based solely on abundance the parent ion is one- ADVANCED BIOANALYTICAL S E R V IC E S , INC. REPORT : 96ADEM01JM 005868 third lower in the 6-Hr sample than in the O-Hr sample, thus some metabolism may have occurred. 3.3.3 Metabolism of T-6294 by Human Hepatocytes The parent and sulfonamide metabolite (m/z 498, see Figure 14) were the only compound-related ions observed in the samples. The abundance of the parent ion in the O-Hr sample was reduced approximately by half in the 6-Hr sample and, conversely, the abundance of the sulfonamide in the O-Hr sample was approximately doubled in the 6-Hr sample. 3.3.4 Metabolism of T-6295 by Human Hepatocytes There were no discemable differences between the 0 and 6-Hr sample for T-6295. 4 . CONCLUSIONS Several potential metabolites and impurities were determined from the LC/MS data in the study. Further MS/MS experimentation, possibly in conjunction with a sample cleanup and concentration step, would be required to further support the postulated structures in the rat samples. Additionally, for the human samples, further high sensitivity detection experimentation is required for complete preliminary investigation, followed by MS/MS experimentation for confirmation of the metabolite structures. 5 . REFERENCES 1. Gordon, Steven C., Ph.D., D.A.B.T., Health Hazard Summary of Ammonium Salts of Mono-, Di- and Tri[N-ethyl(perfluorooctane)sulfonamidoethyljphosphate (FC-807). September 14, 1994. Supplied by 3M Company. ADVANCED BIOANALYTICAL S E R VIC ES, INC, REPORT : 96ADEM01.3M 005869 Figure . Structures of the study test articles 17 T-6292 M r= 571 O II / C H 2-CH3 F3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2-S -r Q || ^ c h 2-c h 2-o h O T-6293 M r= 651 T-6294 M r= 527 T-6295 M r= 500 O <CH2-CH3 o e-OH II c h 2-c h 2-o - o OH c h 2-c h 3 II <F3 C-CF2 -CF2 -CF2 -CF2-CF2 -CF2 -C F2 -S- oH O II F3C-CF2 -CF2 -CF2-CF2 -CF2 -CF2 -C F2 -S-OH o ADVANCED BIOANALVTICAL SER VIC ES, INC. REPORT : 96ADEMOOM 005870 18 Figure 2. Negative ionization mass spectra obtained from the test articles T-6293 ([M-H]' = 650.1), T-6294 ([M-H]' = 526.1) and T6295 ([M-H]' = 498.9) ADVANCED BIOANALYTICAL S E R V IC E S , INC. REPORT : 96ADEM01JM 005871 Figure 3 Product ion spectra obtained from the deprotonated test articles T-6293, T-6294, and T-6295. T-6293 Product Ion Scan 495,000 Relative Intensity (%) Product ton Scan 405,000 Relative Intensity (%) t * 295 Product Ion Scan 1,620,000 Relative Intensity (%) rj ADVANCED BIOANALYTICAL S E R VIC ES, INC. REPORT : 96ADEM0I.3M 005872 Figure 4. Proposed CID fragmentation mechanism for T-6293. 20 T-6293 m/z = 650 O .C H 2-CH3 F3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2-S -N f II o || PCfeH (124 amu) m/z = 526 c h 2-c h 3 F3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2-S N / II o - Et -- N = S 02 (107 amu) 't m/z = 419 F3 C-CF2 -C F2 -CF2 -C F 2 -CF2 -C F2 -CF2 ADVANCED BIDANALYTICAL SER VIC ES, INC. REPORT : 96ADEM01.3M 005873 Figure 5. Proposed CID fragmentation mechanisms for T-6294. T-6294 m/z = 526 O ^11 / F 3C-CF2-CF2-CF2-CF2-CF2-CF2-CF2'-S-N c H2-c h 3 II' O O F-S-N ^ Il " O m/z = 126 (pathway not shown) - Et -- N =S02 (107 amu) m/z = 419 dc F3C -C F ^ C ^ -C F ^ -C ^ -C F 2-CF2-CF2 m/z = 119 F3 C -C F2 -C F 2 m/z = 169 F 3C -C F 2 -CF2 -C F 2 -CF2~ F3C-CF2-CF2-CF2 m/z = 269 m/z = 219 ADVANCED BIDANALYTICAL S E R V IC E S , INC. REPORT : 96ADEM01.3M 005874 Figure 6. Proposed CID fragmentation mechanisms for T-6295. O 22 C F 2(C F 2)2S 0 3 m /z = 2 3 0 C F 2C F 2S 0 3 1C F 2S 0 3 so* m/z = 180 m/z = 130 m/z = 80 O m /z = 99 (pathway not shown) ADVANCED BIOANALYTICAL SER VIC ES, INC. REPORT : 96ADEM0I .3M 005875 23 Figure 7. Total ion and extracted fragment ion chromatograms for T6292 obtained from Rat Control Sample 13. TIC of all masses, 100 to 800 control sample 13, no TA 2,805,000 Relative Intensity (%) control sample 13, no TA 169/169 Scart/Time (min) 590,000 269/269 \ _____ n , 319/319 1 All 369/369 45,000 .................................... ...........1f T W - r.A______ _ - A, 10,000 590,000 419/419 | 469/469 ] _______________________________ 0.0 2.0 4.0 1 84 165 6.0 248 Time (min)/Scan ADVANCED BIOANALYTICAL SER VIC ES, INC. 55,000 590,000 8.0 10.0 331 414 REPORT : 96ADEM01.3M 005876 Figure 8. Total ion and extracted fragment ion chromatograms for T6292 obtained from Rat 1, O-Hr sample. TIC of all masses, 100 to 800 T-6292 Rat 1, 0-Hr, 10 ul inj 5,090,000 Relative Intensity (%) 169/169 219/219 269/269 319/319 369/369 419/419 469/469 0.0 1 T- 2.0 84 Scan/Time (min) 7.25 7.54 7.75 JL 8.09 A, I 8.33 4.0 6.0 167 250 Time (min)/Scan K 8.0 333 820,000 765,000 485,000 645,000 185,000 605,000 820,000 10.0 416 ADVANCED BIQANALYTICAL S E R V IC E S , INC. REPORT : 96ADEM01.3M 005877 25 Figure 9. Extracted ion chromatograms of fragment peaks for T -6292 homologous series in Rat 1, O-Hr sample. T-6292 Rat 1. O-Hr, 10 ul inj 319/319 645,000 Rel. Int. (%) Rel. Int. (%) Rel. Int. (%) Time (min)/Scan ADVANCED BIO ANALYTICAL S E R VIC ES, INC. REPORT : 96ADEM0UM 005878 26 Figure 10a. Proposed structure of parent compound for T -6292 homologous series from Figures 8 and 11. O || .C H 2-CH3 F3 C -(C F 2 ),,-C F 2 -S -|v( so3 o Mass of Parent Anion 356 406 456 506 556 606 n 1 2 3 4 5 6 Corresponding Fragment Ion in Figures 8 and 11 169 219 269 319 369 419 Figure 10b. Proposed structure of glucuronide for homologous series from Figures 12 and 13. II / C H 2-CH3 f 3c -(c f 2)n-CF2- s - r ^ || ^ C H 2-CH2- 0 -- Glu' o Mass of Parent Anion 496 546 596 646 696 746 n 1 2 3 4 5 6 ADVANCED BIOANALYTICAL S E R V IC E S , INC. REPORT : 96ADEM0I ..1M 005879 27 Figure 11. Total ion and extracted fragment ion chromatograms for T6292 obtained from Rat 1, 6-Hr sample. TIC of all masses, 100 to 800 T-6292 Rat 1, 6-Hr, 10 ul inj 4,295,000 Relative Intensity (%) 169/169 219/219 269/269 319/319 369/369 419/419 469/469 0.0 2.0 1 84 Scan/Time (min) 7.27 /* w ..fri - A. 575,000 195,000 160,000 8.05 AA X 8.33 -rvAu. At=fy A 170,000 200,000 840,000 840,000 4.0 6.0 167 250 Time (min)/Scan 8.0 333 10.0 416 ADVANCED BIOANALYTICAL S E R V IC E S , INC. REPORT . 96ADEM0I.3M 005880 28 Figure 12. Extracted ion chromatograms of putative glucuronide peaks for T-6292 homologous series in Rat 1, 6-Hr sample. T-6292 R a ti, 6-Hr, 10 ul inj Rel. Int. (%} Rel. Int. (%) Rel. Int. (%) Rel. Int. (%) Rel. Int. (%) Rel. Int. (%) 496/496 100. 50- 01 546/546 100- 50- oh ------ 596/596 100-1 50- 0 1 -"- - t 646/646 100- 50- ol " - 11 696/696 10CH 50- Ol 1 1'" 'i 746/746 ioo- so- 01 ' 0.0 1 - - - y 2.0 84 A L J ______ a T 6.69 A 4.0 6.0 167 250 Time (min)/Scan T 8.0 333 T 90,000 10.0 416 ADVANCED BIOANALYTICAL S E R V IC E S , INC. REPORT : 96ADEM0J.3M 005881 Rei. Int. (%) Rei. Int. (%) Rei. Int. (%) Rei. Int. (%) Rei. Int. {%) Rei. Int. (%) 29 Figure 13. Extracted ion chromatograms of putative glucuronide peaks for T-6292 homologous series in Rat 1, O-Hr sample. T-6292 Rat 1,0-Hr, 10 u i Inj 496/496 10Oi 50 O' 546/546 100- 50- 596/596 10O-i 50 646/646 100- 50O' 696/696 100i 50' O' 746/746 1001 50 0.0 1 2.0 84 i " "" I-- 4.0 6.0 167 250 Time (min)/Scan 105,000 45,000 10,000 25,000 h 105,000 10,000 8.0 10.0 333 416 ADVANCED BIOANALYTICAL SER VIC ES, INC. REPORT : 96ADEM01,3M 005882 30 Figure 14. Proposed structures of the anions of the sulfonamide and carboxylic acid metabolites for T-6292 from Rat 1. Sulfonamide Metabolite (mIz 498) O |( F3C-(CF2)6-CF2-S-NH O Carboxylic Acid Metabolite (m/z 584) || / C H 2-C H 3 F3C -(C F 2)6-C F 2-S -r<^ | 'CH2-|j-0 o ADVANCED BIOANALYTICAL SERVICES, INC. REPORT : 96ADEM01.3M 005883 Figure 15. Extracted ion chromatograms of sulfonamide (m/z 498) and carboxylic acid (m/z 584) metabolites for T-6292 from Rat 1 , O-Hr (top) and 6-Hr (bottom) samples. T-6292 Rat 1, O-Hr, 10 ul inj 498/498 55,000 T-6292 Rat 1 , 6-Hr, 10 ul inj 498/498 Time (min)/Scan 425,000 Time (min)/Scan ADVANCED BIO ANALYTICAL SER VIC ES, INC. REPORT : 96ADEMOI.3M 005884 Figure 16. Product ion scan for carboxylic acid metabolite of T-6292 in Rat 1, 6-Hr sample and proposed fragment structures. Product Ion Scan , Parent =584 T-6292 Rat 1, 6-Hr, 10 ul inj 19,615 Relative Intensity (%) Carboxylic Acid Metabolite Product Ion Assignments m/z famu) 169, 219, 269, 419, 526 483 Description See Figure 5 0 1 F3C-(CF2)6-CF2-S O 83 ADVANCED BIOANALVTICAL SER VIC ES, INC. O II F-S - O REPORT : 96ADEM01.3M 00588S Figure 17. Total ion and extracted parent (m/z 650) and proposed metabolite ion chromatograms for T-6293 obtained from Rat Control Sample 13. TIC of all masses, 100 to 800 control sample 13, no TA 2,805,000 Relative Intensity (%) control sample 13, no TA 542/542 Scan/Time (min) 0 r TM -- ' .................. 556/556 'I . 1 "I 1 ................. 1 1 ' I" I .......... I I 0 1 84 165 248 331 414 Time (min)/Scan ADVANCED BIDANALYTICAL S E R V IC E S , INC, REPORT : 96ADEM0I.3M S886 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. TIC of all masses 100 to 800 Rat 1 ,0-Hr, 10 ul inj 4.89 2,680,000 Relative Intensity (%) 1 101 201 301 2.4 4.8 7.2 Scan/Time (min) 556/556 584/584 650/650 I, 746/746 0.0 Rat 1 ,0-Hr, 10 ul inj 0.0 1542/542 2.0 84 6.70 4.0 6.0 167 250 Time (min)/Scan 8.0 333 401 9.6 315,000 315,000 315,000 315,000 315,000 10.0 416 ADVANCED BIOANALYTICAL SER VIC ES, INC. REPORT : 96ADEM01.3M 005887 35 Figure 19. Total ion and extracted parent (m/z 650) and proposed metabolite ion chromatograms for T-6293 obtained from Rat 1 , 6-Hr sample. TIC of all masses 100 to 800 T-6293 Rat 1 ,6-Hr, 10u i inj 2,910,000 Relative Intensity (%) T-6293 R a ti, 6-Hr, 10 ul inj Scan/Time (min) 542/542 556/556 7.95 A 6.48 A 310,000 65,000 584/584 165,000 6.67 A 650/650 6.70 A- 746/746 6.63 L _------------- ,------------- -----------------------_-_--_-_1-_--_-_--_--_-_--_--_-_--_-_--_--_-_--_--_-_1-_--_--_-------- ---------- --- I 0.0 2.0 4.0 6.0 8.0 1 84 167 250 333 Time (min)/Scan 140,000 125,000 I 10.0 416 ADVANCED BIOANALYTICAL SER VIC ES, INC. 00S888 REPORT : 96ADEM01.3M 36 Figure 20. Possible structures for the observed metabolites of T-6293 with [M-H]" of 542 and 556 amu from Rat 1. Mr= 543 [M-H]- = 542 || y CH2-CH2OH F3C -C F 2-C F 2-C F 2-C F 2-C F2-C F 2-C F 2-S -N II O Mr= 557 [M-H]' = 556 O II f 3c -c f 2-c f 2-c f 2-c f 2-c f 2-c f 2-c f 2-s - oII c h 2-c o o h ADVANCED BIDANALYTICAL S E R VIC ES, INC. REPORT . 96ADEM01.3M 005889 37 Figure 21. Extracted ion chromatograms of sulfonamide (m/z 498) and sulfonate (m/z 499) ions for T-6293 from Rat 1, O-Hr (top) and 6-Hr (bottom) samples. Rat 1, O-Hr, 10 ul inj 498/498 260,000 T-6293 Rat 1, 6-Hr, 10u i inj 498/498 100i c| 50- cQc> 499/499 Time (min)/Scan Time (min)/Scan 645,000 265,000 ADVANCED B IO A N A L Y T IC A L S E R VIC ES, INC. REPORT : 96ADEM0UM 005890 38 Figure 22. Extracted ion chromatograms of proposed metabolites with m/z 327 and 419 for T-6293 from Rat 1, O-Hr (top) and 6-Hr (bottom) samples. Rat 1, O-Hr, 10 ul inj 327/327 85,000 T-6293 Rat 1, 6-Hr, 10 ul inj 327/327 Time (min)/Scan 215,000 Time (min)/Scan ADVANCED BIOANALYTICAL SER VIC ES, INC. REPORT : 96ADEM01JM 005891 39 Figure 23. Total ion and extracted parent (m/z 526) and proposed metabolite ion chromatograms for T-6294 obtained from Rat Control Sample 13. TIC of all masses, 100 to 800 T-6294, Control, no test article, 10 ul inj, 1090 2,905,000 Relative Intensity (%) T-6294, Control, no test article, 10 ul inj, 1090 483/483 Scan/Time (min) 498/498 526/526 542/542 556/556 0.0 2.0 1 83 ' T* -- " -- ' " I1 4.0 6.0 166 245 Time (min)/Scan --I *' T ' 8.0 328 10,000 10,000 10,000 10,000 10,000 10.0 412 ADVANCED BIOANALYTICAL S E R V IC E S , INC. REPORT . 96ADEM01.3M 005892 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. TIC of all masses, 100 to 800 T-6294, R ati, 0-hr, 10 ul Inj, 1090 5,295,000 Relative Intensity (%) T-6294, R ati, 0-hr, 10 ul inj, 1090 483/483 498/498 Scan/Time (min) 526/526 542/542 ... , 556/556 0.0 2.0 4.0 6.0 1 84 167 250 Time (min)/Scan 1,895,000 7.90 A 8.64 .A. I 180,000 1,895,000 10,000 1,895,000 8.0 10.0 333 416 ADVANCED BIOANALYTICAL SER VIC ES, INC. REPORT : 96ADEM01..1M 005593 Figure 25. Total ion and extracted parent (m/z 526) and proposed metabolite ion chromatograms for T-6294 obtained from Rat 1 , 6-Hr sample. TIC of all masses, 100 to 800 T-6294, R ati, 6-hr, 10 ul inj, 1090 7,595,000 8.1 Relative Intensity (%) T-6294, R ati, 6-hr, 10 ul inj, 1090 483/483 498/498 526/526 542/542 556/556 0.0 2.0 1 83 Scan/Time (min) 6.58 A 80,000 3,535,000 7 8.73 A 8 i 6.75 i ___________________ 4.0 6.0 8.0 165 248 331 Time (min)/Scan . 1,350,000 30,000 50,000 10.0 414 ADVANCED BIOANALYTICAL SER VIC ES, INC. REPORT : 96ADEM01.3M 005894 Figure 26. Product ion scan for perfluorooctanesulfonamide metabolite (CF3(CF2)7S 0 2NH 2, m/z 498) of T-6294 in Rat 1, 6-Hr sample and proposed fragment structures. -Profile DAUGHTER Parent =498 T-6294 Rat 1 ,6-Hr, 10 ul inj 398,750 m/z iamu) 169, 219 478 259 78 ADVANCED B O A N A L Y T IC A L SERVICES, INC. Description See Figure 5 o F3C-(CF2)6-CF-!-N " o o F2C-(CFg)2-CF-S-N O REPORT : 96ADEM0I.3M 005895 Figure 27. Total ion and extracted parent (m/z 499) and homolog ion chromatograms for T-6295 obtained from Rat Control Sample 13. TIC of all masses, 100 to 800 control media (no ta), 10 ul, gradient 7 720,000 Relative Intensity (%) Scan/Time (min) control media (no ta), 10 ul, gradient 7 499/499 440,000 6.80 L______ :___ A____ 449/449 6.58 I 15,000 399/399 6.34 i 50,000 349/349 440,000 299/299 -I--------------- ------- -------- 1 0.0 2.0 1 84 " * I I . I. I ii I 4.0 6.0 8.0 167 250 333 Time (min)/Scan 440,000 10.0 416 ADVANCED BIOANALYTICAL SER VIC ES, INC. REPORT : 96ADEM0I.3M 005896 44 Figure 28. Total ion and extracted parent (m/z 499) and homolog ion chromatograms for T-6295 obtained from Rat 1, O-Hr sample. TIC of all masses, 100 to 800 T-6295, ra ti, 0-hr, 10 ul, gradient 7 2,060,000 Relative Intensity (%) T-6295, rati, 0-hr, 10 ul, gradient 7 499/499 449/449 399/399 349/349 299/299 0.0 2.0 1 84 Scan/Time (min) 6.86 6.62 1 6.36 1 6.00 A 5.25 A 4.0 6.0 167 250 Time (min)/Scan 8.0 333 1,420,000 180,000 375,000 135,000 25,000 10.0 416 ADVANCED BIOANALYTICAL S E R V IC E S , INC. REPORT : 96ADEM01.3M 005897 Figure 29. Total ion and extracted parent (m/z 499) and homolog ion chromatograms for T-6295 obtained from Rat 1, 6-Hr. TIC of all masses^ 100 to 800 T-6295, rati, 6-hr, 10 ul, gradient 7 2,830,000 Relative Intensity (%) T-6295, ra ti, 6-hr, 10 ul, gradient 7 499/499 449/449 399/399 Scan/Time (min) ___ 6.94 A 6.66 J 6.39 349/349 1 6.03 299/299 1 5.24 __________________ k _________________________ 0.0 2.0 4.0 6.0 8.0 1 84 167 249 332 Time (min)/Scan 1,960,000 275,000 535,000 220,000 65,000 10.0 415 ADVANCED BIOANALYTICAL SER VIC ES, INC. REPORT : 96ADEM01.3M 005898