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INTERIM REPORT # 6 - Analysis of Liver and Serum Samples STUDY TITLE Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: 610-701-3761 INTERIM REPORT COMPLETION DATE April 23, 2008 PERFORMING LABORATORY MPI Research, Inc. 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: 651-733-6374 PROJECT MPI Research Study Number: 0137.0219 MPI Project Number: P0001131 Total Pages: 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT MPI Project Number P0001131, entitled "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program," conducted for 3M Company, is being performed in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by MPI Research, Inc. MPI Research Jaisimha Kesari P.E., DEE Study Director Weston Solutions, Inc. Sponsor Representative 3M Company MPI Research, Inc. Date Date Page 2 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 QUALITY ASSURANCE STATEMENT MPI Research's Quality Assurance Unit reviewed MPI Project No. P0001131, entitled, "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program". All reviewed phases1 were inspected for conduct according to MPI Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the MPI Principal Investigator and Management and to the Study Director. Phase Date Inspected Date Reported to Date Reported Principal to MPI Date Reported to Investigator Management Studv Director Raw Data Review and Interim Analytical 05/27,31/05 Report Review 09/09/05 09/12/05 09/13/05 Final Interim Report Report and Raw Data Review 04/11/08 04/13/08 04/13/08 04/13/08 Lynann Porter Quality Assurance Group Leader, Quality Assurance Unit ^ 1 ^ 3 - Off Date 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion of the study. This QA statement involves only the review of the interim report and associated raw data. MPI Research, Inc. Page 3 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 CERTIFICATION OF AUTHENTICITY This interim report, for MPI Project Number P0001131, is a true and complete representation of the raw data. Submitted by: MPI Research, Inc. 3058 Research Drive State College, PA 16801 (814) 272-1039 Principal Investigator, MPI Research: Gw Charles Simons Director Analytical Lax> Operations MPI Research, Inc. Date MPI Research Facility Management: Kevin Lloyd General Manager, Analytical Sciences MPI Research, Inc. Study Director, Weston Solutions, Inc. Date Jaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: a< I//Iac^i^ Michael A. Sapforo Director of Regulatory Affairs Date Date / MPI Research, Inc. Page 4 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 STUDY IDENTIFICATION Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program MPI PROJECT NUMBER: P0001131 MPI STUDY NUMBER: 0137.0219 TYPE{OF STUDY: SAMPLE MATRIX: Residue Small Mammal Liver and Serum TEST SUBSTANCE: Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) SPONSOR: 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 STUDY DIRECTOR: Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 STUDY MONITOR: Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 PERFORMING LABORATORY: MPI Research, Inc. 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: 11/05/04 Interim Analytical Start Date: 11/29/04 Interim Analytical Termination Date: 02/20/08 Interim Report Completion Date: 04/23/08 MPI Research, Inc. Page 5 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 PROJECT PERSONNEL The Study Director for this project is Jaisimha Kesari at Weston Solutions, Inc. The following personnel from MPI Research were associated with various phases of this interim portion of the study: Name Charles Simons John Flaherty Karen Risha Paul Connolly Chrissy Edwards Mark Ammerman Amy Sheehan Krista Gallant Nancy Saxton Brittany Kravets Title Director Analytical Lab Operations, PI Vice President Analytical Manager Director Bioanalytical Analytical Project Leader Project Lead Sample Control Analytical Group Leader Research Chemist Associate 2 Research Chemist Associate 1 Technician MPI Research, Inc. Page 6 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: POOOl 131 TABLE OF CONTENTS Page TITLE PAGE...................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................. 2 QUALITY ASSURANCE STATEMENT..........................................................................3 CERTIFICATION OF AUTHENTICITY...........................................................................4 STUDY IDENTIFICATION................................................................................................5 PROJECT PERSONNEL.....................................................................................................6 TABLE OF CONTENTS.....................................................................................................7 LIST OF TABLES...............................................................................................................8 LIST OF FIGURES..............................................................................................................9 LIST OF APPENDICES.................................................................................................... 11 1.0 SUMMARY................................................................................................................ 12 2.0 OBJECTIVE............................................................................................................... 13 3.0 INTRODUCTION....................................................................................................... 13 4.0 ANALYTICAL TEST SAMPLES.............................................................................. 13 5.0 REFERENCE MATERIAL........................................................................................ 14 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................ 16 6.1 Extraction Procedure For Liver................................................................................ 16 6.2 Extraction Procedure For Serum by Method V0001786..........................................17 6.3 Extraction Procedure For Serum by Protocol Amendment #13...............................17 6.4 Preparation of Standards and Fortification Solutions for Initial Analysis................17 6.5 Preparation of Standards and Fortification Solutions for Re-Analysis.....................18 6.6 Chromatography.......................................................................................................19 6.7 Instrument Sensitivity...............................................................................................20 6.8 Description of LC/MS/MS Instrument and Operating Conditions.......................... 20 6.9 Quantitation and Example Calculation.................................................................... 21 7.0 EXPERIMENTAL DESIGN..................................................................................... 22 8.0 RESULTS...................................................................................................................23 9.0 CONCLUSIONS......................................................................................................25 10.0 RETENTION OF DATA AND SAMPLES.............................................................25 MPI Research, Inc. Page 7 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table I. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Hispid Cotton Rat Liver Samples ...27 Table II. Summary of PFBS, PFHS and PFOS in Re-extracted Hispid Cotton Rat Liver Samples................................................................................................ 28 Table HI. Summary of PFBS, PFHS and PFOS in Hispid Cotton Rat Serum Samples.......................................................................................................... 29 Table IV. Summary of PFBS, PFHS and PFOS in Re-extracted Hispid Cotton Rat Serum Samples.............................................................................................. 30 Table V. Matrix Spike Recovery of PFBS, PFHS and PFOS in Hispid Cotton Rat Liver Samples................................................................................................ 31 Table VI. Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Hispid Cotton Rat Liver Samples...............................................................................33 Table VII. Matrix Spike Recovery of PFBS, PFHS and PFOS in Hispid Cotton Rat Serum Samples...............................................................................................35 Table VIII. Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Hispid Cotton Rat Serum Samples.............................................................................37 Table IX. Surrogate Spike Recovery of 13C PFOS in Re-extracted Hispid Cotton Rat Liver Samples.................................................................................................38 Table X. Surrogate Spike Recovery of 13C PFOS in Re-extracted Hispid Cotton Rat Serum Samples...............................................................................................39 MPI Research, Inc Page 8 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 LIST OF FIGURES Page Figure 1. Typical Calibration Curve for PFBS in Methanol......................................... 41 Figure 2. Non-Extracted Standards of PFBS in Methanol, 0.1 ng/mL and 0.2 ng/mL, Respectively..................................................................................... 42 Figure 3. PFBS in Control Liver, 10 ng/mL Fortified Control Liver Spk A, and 50 ng/mL Fortified Control Liver Spk B, Respectively...................................... 43 Figure 4. PFBS in Control Serum, 10 ng/mL Fortified Control Serum Spk A, and 50 ng/mL Fortified Control Serum Spk B, Respectively............................... 44 Figure 5. Chromatogram Representing a Liver Sample Analyzed for PFBS (ExyLIMS ID: C0049686, Data Set: 121704B)............................................. 45 Figure 6. Chromatogram Representing a Serum Sample Analyzed for PFBS (ExyLIMS ID: C0049666, Data Set: 121304C)............................................ 46 Figure 7. Typical Calibration Curve for PFHS in Methanol......................................... 47 Figure 8. Non-Extracted Standards of PFHS in Methanol, 0.1 ng/mL and 0.2 ng/mL, Respectively.......................................................................................48 Figure 9. PFHS in Control Liver, 10 ng/mL Fortified Control Liver Spk A, and 50 ng/mL Fortified Control Liver Spk B, Respectively...................................... 49 Figure 10. PFHS in Control Serum, 10 ng/mL Fortified Control Serum Spk A, and 50 ng/mL Fortified Control Serum Spk B, Respectively............................... 50 Figure 11. Chromatogram Representing a Liver Sample Analyzed for PFHS (ExyLIMS ID: C0049686, Data Set: 121704B)............................................. 51 Figure 12. Chromatogram Representing a Serum Sample Analyzed for PFHS (ExyLIMS ID: C0049666, Data Set: 121304C)..............................................52 Figure 13. Typical Calibration Curve for PFOS in Methanol......................................... 53 Figure 14. Non-Extracted Standards of PFOS in Methanol, 0.1 ng/mL and 0.2 ng/mL, Respectively.......................................................................................54 Figure 15. PFOS in Control Liver, 10 ng/mL Fortified Control Liver Spk A, and 50 ng/mL Fortified Control Liver Spk B, Respectively................................. 55 Figure 16. PFOS in Control Serum, 10 ng/mL Fortified Control Serum Spk A, and 50 ng/mL Fortified Control Serum Spk B, Respectively............................... 56 Figure 17. Chromatogram Representing a Liver Sample Analyzed for PFOS (ExyLIMS ID: C0049686, Data Set: 121704B)..............................................57 Figure 18. Chromatogram Representing a Serum Sample Analyzed for PFOS (ExyLIMS ID: C0049666, Data Set: 121304C)..............................................58 MPI Research, Inc. Page 9 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 LIST OF FIGURES Page Figure 19. Typical Calibration Curve for 13C PFOS in Methanol.................................. 59 Figure 20. Non-Extracted Standards of 13C PFOS in Methanol, 0.1 ng/mL and 0.2 ng/mL, Respectively......................................................................................60 Figure 21. 13C PFOS in Control Liver, 0.5 ng/mL Fortified Control Liver Spk A, and 2.5 ng/mL Fortified Control Liver Spk B, Respectively......................... 61 Figure 22. 13C PFOS in Control Serum, 10 ng/mL Fortified Control Serum Spk A, and 50 ng/mL Fortified Control Serum Spk B, Respectively........................ 62 Figure 23. Chromatogram Representing a Liver Sample Analyzed for 13C PFOS (ExyLIMS ID: C0049677, Data Set: 080807B)............................................. 63 Figure 24. Chromatogram Representing a Serum Sample Analyzed for C PFOS (ExyLIMS ID: C0049668, Data Set: 080307B).............................................64 MPI Research, Inc. Page 10 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 LIST OF APPENDICES Page Appendix A MPI Project No.: P0001131 (MPI Study No.: 0137.0219) with Analytical Methods, Amendment, and Deviations................................... 65 MPI Research, Inc. Page 11 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 1.0 SUMMARY MPI Research, Inc. extracted and analyzed hispid cotton rat (Sigmodon hispidus) liver samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS)) according to MPI Method V0001785 and analyzed hispid cotton rat {Sigmodon hispidus) serum samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS)) according to MPI Method V0001786 and MPI Protocol P0001131 Amendment #13(Appendix A). The limit of quantitation for PFBS, PFHS, PFOS and 13C PFOS in liver was 20 ng/g. The limit of quantitation for PFBS, PFHS and PFOS in serum was 400 ng/mL for samples initially analyzed and being reported using MPI Method V0001786. The limit of quantitation for PFBS, PFHS, PFOS, and 13C PFOS in serum was 0.5 ng/mL for samples subsequently re-extracted and reanalyzed being reported using MPI Protocol P0001131 Amendment #13. Analytical results and assessed accuracies for the initial analysis of PFBS, PFHS and PFOS in liver samples are summarized in Table I. Analytical results and assessed accuracies for the re-extraction of PFBS, PFHS and PFOS in liver samples are summarized in Table II. Analytical results and assessed accuracies for the initial analysis of PFBS, PFHS and PFOS in serum samples are summarized in Table III. Analytical results and assessed accuracies for the re-extraction of PFBS, PFHS and PFOS in serum samples are summarized in Table IV. In several instances, the initial sample analyses did not produce quantitative results. These results were designated not reported (NR) due to quality control failures. Liver samples with adequate sample mass and serum samples with adequate volume that did not meet quality control criteria were re-extracted and reanalyzed in an attempt to obtain quantitative results. Quantitative results were obtained for all samples and analytes except for PFBS for all liver samples and three serum samples (due to inadequate sample volume), and five serum samples (three of which did not have adequate sample volume) for PFOS, which were not reported (NR) due to quality control failures. Fortification recoveries for the analysis of PFBS, PFHS and PFOS in liver samples are summarized in Table V. The overall average percent recovery standard deviation for PFHS in the liver samples was 82 11%. Fortification recoveries for the analysis of PFBS and PFHS in re-extracted liver samples are summarized in Table VI. The overall average percent recovery standard deviation for PFHS in the re-extracted liver samples was 100 32%. Fortification recoveries for the analysis of PFBS, PFHS and PFOS in serum samples using method V0001786 are summarized in Table VII. The overall average percent recovery standard deviation for PFHS in the serum samples using method V0001786 was 93 19%. Fortification recoveries for the analysis of PFBS, PFHS and PFOS in re-extracted serum samples using protocol amendment #13 are summarized in Table VIII. The overall average percent recovery standard deviation MPI Research, Inc. Page 12 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 for PFBS and PFHS in the re-extracted serum samples using protocol amendment #13 was 89 4% and 96 12%, respectively. The overall average percent recovery for PFOS in the re-extracted serum samples using protocol amendment #13 was 88%. Fortification recoveries for the analysis of 13C PFOS in re-extracted liver samples are summarized in Table IX. The overall average percent recovery standard deviation for 13C PFOS in the re-extracted liver samples was 88 21%. Fortification recoveries for the analysis of 13C PFOS in re-extracted serum samples using protocol amendment #13 are summarized in Table X. The overall average percent recovery standard deviation for 13C PFOS in the re-extracted serum samples using protocol amendment #13 was 80 12% . 2.0 OBJECTIVE The objective of the analytical part of this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) in liver and serum according to Protocol P0001131 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFBS, PFHS, and PFOS in liver and serum using the analytical methods entitled, "V0001785: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS," and "V0001786: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS," and the procedure outlined in protocol amendment #13. The study was initiated on November 05, 2004, when the study director signed protocol number P0001131. The analytical start date for this interim report was November 29, 2004, and the analytical termination date for this interim report was February 20, 2008. 4.0 ANALYTICAL TEST SAMPLES The control liver samples (ExyLIMS ID C0053650) obtained from hispid cotton rats (Sigmodon hispidus) were received on June 11, 2003 on dry ice from Harlan Bioproducts for Science, Inc. The control serum samples (ExyLIMS ID C0034001) obtained from hispid cotton rats (Sigmodon hispidus) were received on May 20, 2004 on dry ice from Lampire Biological Laboratories. The control samples were logged in by MPI Research personnel and placed in frozen storage. Fifteen liver samples (ExyLIMS ID C0049677C0049690, C0049692) and fifteen serum samples (ExyLIMS ID C0049662-C0049676) MPI Research, Inc. Page 13 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 were received on dry ice on October 23, 2004 from Tim Frinak at Weston Solutions, Inc. The preparation of the serum samples by centrifugation of the whole blood was performed in the field before the samples were shipped to MPI Research. The samples were logged in by MPI Research personnel and placed in frozen storage. Sample identification (ID) codes are of the form DFxxM01SHx00x004100x. The first four characters define the sampling area where: DF06 = Decatur Field Number 6 DF08 = Decatur Field Number 8b DF09 = Decatur Field Number 9 DF14 = Decatur Field Number 14 (control field) The fifth through the seventh characters (M01) refer to small mammal samples and the eighth through the tenth characters define the matrix where: SHL = Sigmodon hispidus liver SHB = Sigmodon hispidus serum The eleventh through the thirteenth characters define the sample number (001 through 006) for the sampling area. The fourteenth character (0) indicates the primary sample and the last six characters indicate the sample collection date in YYMMDD format. Sample login and chain of custody information is located in the raw data package associated with this interim report. Storage records will be kept at MPI Research. 5.0 REFERENCE MATERIAL The analytical standards, PFBS, PFHS and PFOS were supplied by 3M. PFBS (SP0000252) was received from 3M at MPI Research on July 06, 2000. PFBS (SP0008956) and PFHS (SP0008961) were received from 3M at MPI Research on February 19, 2007. PFHS (SP0002401) and PFOS (SP0002402) were received from 3M at MPI Research on January 20, 2003. PFOS (SP0002694) was purchased from Fluka Corporation and was received at MPI Research on April 23, 2003. 13C PFOS (SP0009538) was purchased from Wellington Laboratories and was received at MPI Research on July 24, 2007. The available information for the reference materials is listed below. PFBS and PFHS were stored frozen, PFOS and 13C PFOS were stored refrigerated. Compound PFBS PFBS PFHS ExvLIMS Inventory No. SP0000252 SP0008956 SP0002401 Lot # 101 2 SE036 Puritv (%) 96.7 97.3 98.6 Expiration Date 12/04/06 01/18/17 10/18/06 MPI Research, Inc. Page 14 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples Compound PFHS PFOS PFOS 13C PFOS ExyLIMS Inventory No. SP0008961 SP0002694 SP0002402 SP0009538 Lot #. NB0120067-69 430180-1 217 MPFOS0607 MPI Study No.: 0137.0219 MPI Project No.: P0001131 Puritv (%) 98.6 101.2 86.9 98 Expiration Date 10/18/16 10/31/07 03/31/16 06/07/10 The molecular structures of PFBS, PFHS, PFOS and 13C PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (T^FgSCVK*) Transitions Monitored: 299 -->99 Structure: FF FF F SO 3 FF FF PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFnSCVK^) Transitions Monitored: 399 -- 80 Structure: FF F F F F F SO3 F F F F FF PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFnSOs'K*) Transitions Monitored: 499 -- 80 Structure: FFFF FFFF F SO3 FFFF FFFF MPI Research, Inc. Page 15 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 13C PFOS Chemical Name: 1,2,3,4-I3C perfluorooctanesulfonate Molecular Weight: 538 supplied as the sodium salt (CgFnSCb'Na^ Transitions Monitored: 503 -- 80 Structure: 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical method "V0001785: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" was used for the liver samples in this study. The analytical method "V0001786: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" and Protocol Amendment #13 were used for the serum samples in this study. 6.1 Extraction Procedure For Liver One-gram portions of control liver were used for the liver control and spikes A and B. Due to limited sample quantity 0.1 g portions of the liver samples were used for the extraction procedure. The appropriate amount of fortification solution was then added to the samples and allowed to dry. After fortification of appropriate samples, the volume was brought up to 10 mL with water. The samples were homogenized with a tissuemizer for -1 minutes. One milliliter of sample was then transferred to a disposable centrifuge tube to which 5 mL of acetonitrile was added. The samples were placed on a wrist action shaker for ~20 minutes. The samples were then centrifuged for ~5 minutes at -3000 rpm. The supernatant was decanted into a 50 mL disposable centrifuge tube to which 35 mL of water was added. The samples were loaded onto a Cig SPE cartridge conditioned with 10 mL of methanol and 5 mL of water (25 mL of water was used after the 10 mL of methanol, to condition the column for the re-extraction). The eluate was discarded. Approximately two milliliters of methanol was added to the cartridge. Two milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. MPI Research, Inc. Page 16 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 6.2 Extraction Procedure For Serum by Method V0001786 One-milliliter portions of control serum were used for the serum control and spikes A and B. Due to limited sample quantity 0.01 mL portions of the serum samples were used for the extraction procedure. After fortification of appropriate samples, the volume was brought up to 20 mL with water. The samples were vortexed -1 minutes. One milliliter of sample was then transferred to a disposable centrifuge tube to which 5 mL of acetonitrile was added. The samples were placed on a wrist action shaker for ~20 minutes. The samples were then centrifuged for -5 minutes at -3000 rpm. The supernatant was decanted into a 50 mL disposable centrifuge tube to which 35 mL of water was added. The samples were loaded onto a Cis SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately two milliliters of methanol was added to the cartridge. Two milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.3 Extraction Procedure For Serum by Protocol Amendment #13 Fifty microliter portions of control serum were used for the extraction of the serum control and spikes A and B. Fifty microliter portions of each serum sample (volume permitting) were also used for the extraction procedure. The samples were transferred into a 1.5 mL centrifuege tube. The sample spikes were fortified with 0.5 mL of the appropriate fortification solution. All other samples not designated as spikes had 0.5 mL of acetonitrile added to them. All samples were capped and vortexed for -1 minute. The samples were then centrifueged for -10 minutes at 10,000 rpm. The sample supernatant was transferred to an autosampler vial. Each sample was analyzed by LC/MS/MS electrospray. 6.4 Preparation of Standards and Fortification Solutions for Initial Analysis Individual stock standard solutions of PFBS, PFHS and PFOS were prepared as specified in ExyLIMS methods V0001785 and V0001786. The stock standard solutions were prepared at a concentration of 100 pg/mL by dissolving 10 mg of each of the standards (corrected for purity and salt content) in 100 mL of methanol. From these solutions, mixed 1.0 pg/mL fortification standard solutions were prepared by taking 1 mL of each of the appropriate stock solutions and bringing the volume up to 100 mL with methanol. By taking 10 mL of the mixed 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.1 pg/mL fortification standard was prepared. A set of non-extracted standards containing PFBS, PFHS and PFOS was prepared in methanol. The following concentrations were prepared: MPI Research, Inc. Page 17 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples Cone, of Fort Fort Solution Volume (ng/mL)1 (mL) 100 5.0 100 2.0 100 1.0 5.0 10 2.0 10 1.0 10 1of PFBS, PFHS and PFOS Final Volume (mL) 100 100 100 100 100 100 MPI Study No.: 0137.0219 MPI Project No.: P0001131 Final Cone, of Calibration Std. (ng/mL) 5.0 2.0 1.0 0.5 0.2 0.1 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.5 Preparation of Standards and Fortification Solutions for Re-Analysis Individual stock standard solutions of PFBS, PFHS and PFOS were prepared as specified in the raw data. The stock standard solutions were prepared at a concentration of 10,000 pg/mL by dissolving 1 g of each of the standards (corrected for purity and salt content) in 100 mL of acetonitrile. From these solutions, a mixed 100 pg/mL fortification standard solution containing PFBS, PFHS and PFOS was prepared by taking 1 mL of each of the appropriate stock solutions and bringing the volume up to 100 mL with acetonitrile. By taking 1.0 mL of the mixed 100 pg/mL fortification standard and bringing the volume up to 100 mL with acetonitrile, a mixed 1.0 pg/mL fortification standard was prepared. An individual stock standard solution of 13C PFOS was prepared as specified in the raw data. The stock standard solution was prepared at 1.0 pg/mL by dissolving 1 mL of the neat standard solution (corrected for purity, concentration, and salt content) in 50 mL of acetonitrile. By taking 10 mL of the mixed 1.0 pg/mL fortification standard and 10 mL of the 1.0 pg/mL 13C PFOS fortification standard and bringing the volume up to 100 mL with acetonitrile, a mixed (containing PFBS, PFHS, PFOS and 13C PFOS) 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the mixed with13C PFOS 0.1 pg/mL fortification standard bringing the volume up to 100 mL with acetonitrile, a mixed with 13C PFOS 0.01 pg/mL fortification standard was prepared. A set of non-extracted standards containing PFBS, PFHS, PFOS and 13C PFOS were prepared in methanol for the liver re-analysis. The following concentrations were prepared: MPI Research, Inc. Page 18 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples Cone, of Fort Fort Final Solution Volume Volume (ng/mL)1 (mL) (mL) 100 5.0 100 100 2.0 100 100 1.0 100 5.0 10 100 2.0 10 100 1.0 10 100 1of PFBS, PFHS, PFOS and 13C PFOS MPI Study No.: 0137.0219 MPI Project No.: P0001131 Final Cone, of Calibration Std. (ng/mL) 5.0 2.0 1.0 0.5 0.2 0.1 A set of fortification standards containing PFBS, PFHS, PFOS and 13C PFOS to be used to fortify the control serum samples to create an extracted calibration curve, were prepared in acetonitrile for the serum re-analysis. The following concentrations were prepared: Cone, of Fort Fort Final Solution Volume Volume (ng/mL)1 (mL) (mL) 100 5.0 100 100 2.0 100 100 ' 1.0 100 5.0 10 100 1.0 10 100 0.5 10 100 1of PFBS, PFHS, PFOS and 13C PFOS Final Cone, of Calibration Std. (ng/mL) 5.0 2.0 1.0 0.5 0.1 0.05 The stock standard solutions and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.6 Chromatography Quantification of PFBS, PFHS, PFOS and 13C PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS, and PFOS in liver samples were ~ 4.7 mins, ~ 10.8 mins, and - 12.9 mins, respectively. The retention times of PFBS, PFHS, PFOS and 13C PFOS in the re-extracted liver samples were - 1.3 mins, ~ 8.4 mins, ~ 11.0 and --11.0 mins, respectively. The retention times of PFBS, PFHS and PFOS in serum samples were ~1.8 mins, - 8.2 mins, and - 10.2 mins, respectively. The retention times of PFBS, PFHS, PFOS and 13C PFOS in the re-extracted serum samples were - 1.5 mins, - 8 .9 mins, -11.4 and - 11.4 mins, respectively. Peaks above the LOQ were not detected in any of the control liver samples corresponding to the analyte retention times. Peaks above the LOQ were not detected in any of the control serum samples corresponding to MPI Research, Inc. Page 19 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 the PFBS and PFHS retention times. However, PFOS was found at ~ 10 ppb in the serum control sample. 6.7 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 0.1 ng/mL of PFBS, PFHS, PFOS and 13C PFOS. 6.8 Description of LC/MS/MS Instrument and Operating Conditions Instrument: API 4000 Biomolecular Mass Analyzer Interface: Turbo Ion Spray Liquid Introduction Interface Computer: DELL OptiPlex GX400 Software: Windows NT, Analyst 1.2 and 1.4 HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: 35 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Analyte PFBS PFHS PFOS 13C PFOS T im e ('min') %A %B 0.0 65 35 1.0 65 35 8.0 25 75 1 0 .0 25 75 1 1 .0 65 35 1 8 .0 Total run time: -18 min Flow Rate: 0.3 mL/min 65 35 Ions monitored: Approximate Retention Transition Time for Liver /Re Mode Monitored extracted Liver (min) negative 299 99 negative 399 80 negative 499 80 ~ 4.7/-1.3 min. -10.8/-8.4 min. -12.9/-11.0 min. negative 503 -> 80 -11.0 min. Approximate Retention Time for Serum/Reextracted Serum (min) ~ 1.8/-1.5 min. ~ 8.2/-S.9 min. - 10.2/~11.4min. -1 1 .4 min. MPI Research, Inc. Page 20 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples 6.9 Quantitation and Example Calculation MPI Study No.: 0137.0219 MPI Project No.: P0001131 Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations of standards. The concentration was determined from the equations below. Equation 1 calculated the amount of analyte found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/mL) = (Peak area - intercept) x DF x AF Slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. AF = Aliquot Factor, 10 for liver samples, 20 for serum samples, 1 for re extracted serum samples Equation 2 was used to convert the amount of analyte found in ng/mL of extraction solvent to ng/g (ppb) of liver or ng/mL of serum (ppb). Equation 2: Analyte found (ppb) = \Analyte found (ng/mL! x final volume (2 mL)l sample weight (g) or sample volume (mL) . For samples fortified with known amounts of PFBS, PFHS, PFOS, and 13C PFOS prior to extraction, Equation 3 was used to calculate the percent recovery. Equation 3: Recovery (%)= [Total analyte found (ppb) - analyte in control (ppb)l xl00% amount added (ppb) An example of a calculation using an actual sample follows; calculation is for PFHS only (values may differ slightly due to rounding differences): Liver sample ExyLIMS ID: C0049679 Spk E (Set: 121504B), fortified at 100 ppb with where: peak area = 16400 intercept = 2100 slope = 33800 dilution factor =1 aliquot factor = 10 ppb PFHS added (fort level) = 100 amt in corresponding sample (ng/mL)* = ND (not detected) MPI Research, Inc. Page 21 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 final volume (mL) sample weight (g) =2 = 0.1 * The primary sample result was used for all calculations. Note that the calculation uses ng/mL of extraction solvent. From equation 1: Analyte found (ng/mL) = IT6400 - 21001 x 1 xlO 33800 From equation 2: Analyte found (ppb) = 4.23 ng/mL = (4.23 ng/mL x 2 mLl 0.1 g = 84.6 ppb From equation 3: % Recovery = (84.6 ppb - 0 ppb) x 100% 100 ppb = 85 % 7.0 EXPERIMENTAL DESIGN For the initial liver and serum samples designated as laboratory matrix spikes, PFBS, PFHS and PFOS were added at known concentrations to the samples in the laboratory after the samples were weighed or measured. For the re-extraction of the liver and serum samples designated as laboratory matrix spikes, PFBS, PFHS, PFOS and 13C PFOS were added at known concentrations to the samples in the laboratory after the samples were weighed or measured. The initial liver samples were extracted in three sets. Each set included one control liver blank and two control liver blanks fortified at known concentrations and five liver samples. The initial serum samples were extracted in four sets. Each set included one control serum blank and two control serum blanks fortified at known concentrations. Two sets contained two samples, one set contained eight samples, and one set contained three samples. For each sample, two laboratory matrix spikes and a duplicate were extracted. Laboratory control liver and serum spikes in the analytical sets were prepared by adding a known concentration of the analyte to laboratoiy liver or serum. Laboratory control liver and serum spikes are used to assess method accuracy. For each sample, two laboratory matrix spikes and a duplicate were extracted. MPI Research, Inc. Page 22 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.:P0001131 The re-extracted liver samples were extracted in three sets. Each set included one control liver blank and two control liver blanks fortified at known concentrations and five liver samples. For each liver sample, two laboratory matrix spikes and a duplicate were extracted. The re-extracted serum samples were extracted in three sets. Each set included one control serum blank and two control serum blanks fortified at known concentrations. One sets contained three samples, one set contained four samples, and one set contained five samples. For each sample, one laboratory matrix spikes and a duplicate were extracted. Laboratoiy control liver and serum spikes in the analytical sets were prepared by adding a known concentration of the analyte to laboratoiy liver or serum. Laboratory control liver and serum spikes are used to assess method accuracy. Accuracies were assessed for each sample by reviewing the individual QC results obtained for each sample site. There were two laboratory spike recovery results available for each sample from the initial analysis of both liver and serum. There were two laboratory spike recovery results available for each sample from the re-analysis of liver and one laboratory spike recovery result available for each sample from the re-analysis of serum. From the laboratory matrix spikes available for each sample, the laboratory matrix spike concentration that was most appropriate for the endogenous concentration of the analyte was used to assess the accuracy. There were two individual 13C PFOS recovery results per sample for the re-analysis of the liver samples. While the lower spike of 13C PFOS failed quality control criteria for unknown reasons, the 13C PFOS high spikes met quality control criteria and were used for assessing accuracy. There was one individual 13C PFOS recovery result per sample for the re-analysis of the serum that was used to assess the accuracy. In the cases where the endogenous levels of PFOS were greater than three times the level of PFOS spiked, the 13C PFOS recoveries alone were used to access accuracy with an expanded uncertainty of +/- 50%. There were no circumstances that affected the quality or integrity of the data. 8.0 RESULTS Analytical results and assessed accuracies for the initial analysis of PFBS, PFHS and PFOS in liver samples are summarized in Table I. Analytical results and assessed accuracies for the re-extraction of PFBS, PFHS and PFOS in liver samples are summarized in Table II. Analytical results and assessed accuracies for the initial analysis of PFBS, PFHS and PFOS in serum samples are summarized in Table III. Analytical results and assessed accuracies for the re-extraction of PFBS, PFHS and PFOS in serum samples are summarized in Table IV. In several instances, the initial sample analyses did not produce quantitative results. These results were designated not reported (NR) due to quality control failures. Liver samples with adequate sample mass and serum samples with adequate volume that did not meet quality control criteria were re-extracted and reanalyzed in an attempt to obtain quantitative results. Quantitative results were obtained for all samples and analytes except for PFBS for all liver samples and three serum samples (due to inadequate sample volume), and five serum samples (three of MPI Research, Inc. Page 23 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 which did not have adequate sample volume) for PFOS, which were not reported (NR) due to quality control failures. Fortification recoveries for the analysis of PFBS, PFHS and PFOS in liver samples are summarized in Table V. The overall average percent recovery standard deviation for PFHS in the liver samples was 82 11%. Fortification recoveries for the analysis of PFBS and PFHS in re-extracted liver samples are summarized in Table VI. The overall average percent recovery standard deviation for PFHS in the re-extracted liver samples was 100 32%. Fortification recoveries for the analysis of PFBS, PFHS and PFOS in serum samples using method V0001786 are summarized in Table VII. The overall average percent recovery standard deviation for PFHS in the serum samples using method V0001786 was 93 19%. Fortification recoveries for the analysis of PFBS, PFHS and PFOS in re-extracted serum samples using protocol amendment #13 are summarized in Table VIII. The overall average percent recovery standard deviation for PFBS and PFHS in the re-extracted serum samples using protocol amendment #13 was 89 4% and 96 12%, respectively. The overall average percent recovery for PFOS in the re-extracted serum samples using protocol amendment #13 was 88%. Fortification recoveries for the analysis of 13C PFOS in re-extracted liver samples are summarized in Table IX. The overall average percent recovery standard deviation for 13C PFOS in the re-extracted liver samples was 88 21%. Fortification recoveries for the analysis of C PFOS in re-extracted serum samples using protocol amendment #13 are summarized in Table X. The overall average percent recovery standard deviation for 13C PFOS in the re-extracted serum samples using protocol amendment #13 was 80 12% . Matrix spikes that were in the appropriate concentration range (primary concentration must be less than or equal to three times the spiking level) were used to calculate the assessed accuracies. In instances of failed laboratory spike recoveries, the samples were not reported due to the quality control failure. Not all liver and serum sample results met data quality objectives. All fifteen liver samples and all fifiteen serum samples had analytes designated as not reported (NR) due to quality control failures in initial analyses. Some of the samples failed because the analyte spike levels were too low relative to the endogenous level in the sample to allow assessment of matrix interference. Some samples failed for unknown reasons. All fifteen liver samples and twelve of the serum samples were re-extracted and reanalyzed in an attempt to obtain quantitative results. Three of the serum samples did not contain sufficient remaining sample volume to re extract, and were therefore not reported. All liver and serum samples designated for re extraction and re-analysis for PFOS due to significantly high endogenous analyte concentrations were spiked with a mass labeled surrogate, 13C PFOS. The mass labeled surrogate, C PFOS, was used to assess the accuracy of PFOS. Quantitative results were obtained for all samples and analytes except for PFBS for all liver samples and three serum samples (due to insufficient volume), and five serum samples for PFOS (due to insufficient volume), which were not reported (NR) due to quality control failures. MPI Research, Inc. Page 24 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 9.0 CONCLUSIONS Except as noted above, the liver samples were successfully extracted and analyzed for PFHS and PFOS according to analytical method V0001785. Except as noted above, the serum samples were also successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001786 and protocol amendment #13. 10.0 RETENTION OF DATA AND SAMPLES When the final analytical report is complete, all original paper data generated by MPI Research will be shipped to the study director. This does not include facility-specific raw data such as instrument or temperature logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the MPI Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. MPI Research, Inc. Page 25 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 TABLES MPI Research, Inc. Page 26 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Hispid Cotton Rat Liver Samples Exygen ID C0049677 C0049677 Rep C0049678 C0049678 Rep C0049679 C0049679 Rep C0049680 C0049680 Rep C0049681 C0049681 Rep C0049692 C0049692 Rep C0049682 C0049682 Rep C0049683 C0049683Rep C0049684 C0049684 Rep C0049685 C0049685 Rep C0049686 C0049686 Rep C0049687 C0049687 Rep C0049688 C0049688Rep C0049689 C0049689 Rep C0049690 C0049690 Rep Client Sample ID DF06M01SHL0010041007 DF06M01SHL0010041007* DF06M01SHL0020041007 DF06M01SHL0020041007* DF06M01SHL0030041007 DF06M01SHL0030041007* DF06M01SHL0040041008 DF06M01SHL0040041008* DF06M01SHL0050041008 DF06M01SHL0050041008* DF06M01SHL0060041008 DF06M01SHL0060041008* DF08M01SHL0010041008 DF08M01SHL0010041008* DF08M01SHL0020041008 DF08M01SHL0020041008* D F 0 8M 01S H L 0030041008 DF08M01SHL0030041008* DF09M01SHL0010041007 DF09M01SHL0010041007* DF09M01SHL0020041007 DF09M01SHL0020041007* D F 0 9M 01S H L 0030041008 DF09M01SHL0030041008* DF14M01SHL0010041007 DF14M01SHL0010041007* DF14M01SHL0020041007 DF14M01SHL0020041007* D F14M 01SHL0030041007 DF14M01SHL0030041007* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulfonate_____Perfluorohexanesulfonate_____Perfluorooctanesulfonate Analyte Found (PPb, ng/g) Assessed Accuracy <+/- %> Analyte Found (ppb, ng/g) Assessed Accuracy <+/- %) Analyte Found (ppb, ng/g) Assessed Accuracy <+/-%) NR . 30.4 30 NR . NR - 26.2 30 NR - NR - 64.5 40 NR - NR - 45.0 40 NR . - NR . ND 30 NR . NR - ND 30 NR - NR - NR* - NR . NR - NR* - NR - NR . NR* . NR . NR - NR* - NR - NR - 39.1 30 NR . NR - 36.6 30 NR - NR . 68.9 30 NR . NR - 75.4 30 NR - NR - 20.5 30 NR . NR - ND 30 NR - NR - NR* - NR NR - NR* - NR - NR . 30.8 30 NR . NR - 31.3 30 NR - NR . 108 30 NR . NR - 113 30 NR - NR . 78.3 30 NR . NR - 80.2 30 NR - NR . ND 30 NR* . NR - ND 30 NR* - NR . NR* . NR* . NR - NR* - NR* - NR . NR* . NR* . NR - NR* - NR* - ` Laboratory Duplicate ND = Not detected above the LOQ of 20 ng/g. NR = Not reported due to quality control failures. See Table II for re-analysis results. NR* = Not reported, analytical results met quality control criteria in both initial and re-extracted analyses; see Table II for reported re-analysis results. MPI Research, Inc. Page 27 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table II. Summary of PFBS, PFHS and PFOS in Re-extracted Hispid Cotton Rat Liver Samples ExyLIMS ID C0049677 C0049677 Rep C0049678 C0049678 Rep C0049679 C0049679 Rep C0049680 C0049680 Rep C0049681 C0049681 Rep C0049692 C0049692 Rep C0049682 C0049682 Rep C0049683 C0049683 Rep C0049684 C0049684 Rep C0049685 C0049685 Rep C0049686 C0049686 Rep C0049687 C0049687 Rep C0049688 C0049688 Rep C0049689 C0049689 Rep C0049690 C0049690 Rep Client Sample ID DF06M01SHL0010041007 DF06M01SHL0010041007* DF06M01SHL0020041007 DF06M01SHL0020041007* DF06M01SHL0030041007 DF06M01SHL0030041007* DF06M01SHL0040041008 DF06M01SHL0040041008* DF06M01SHL0050041008 DF06M01SHL0050041008* DF06M01SHL0060041008 DF06M01SHL0060041008* DF08M01SHL0010041008 DF08M01SHL0010041008* DF08M01SHL0020041008 DF08M01SHL0020041008* DF08M01SHL0030041008 DF08M01SHL0030041008* DF09M01SHL0010041007 DF09M01SHL0010041007* DF09M01SHL0020041007 DF09M01SHL0020041007* DF09M01SHL0030041008 DF09M01SHL0030041008' DF14M01SHL0010041007 DF14M01SHL0010041007* D F14M01SHL0020041007 D F 14M01SH L0020041007* DF14M01SHL0030041007 DF 14M01SHL0030041007* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulfonate__________Perfluorohexanesulfonate_____Perfluorooctanesulfonate Analyte Found (PPb. ng/g) Assessed Accuracy (*/-% ) Analyte Found (PPb, ng/g) Assessed Accuracy (+/- %) Analyte Found (PPb, ng/g) Assessed Accuracy (+/-%) NR . NR . 178000 50 NR - NR - 164000 50 NR . NR . 146000 50 NR - NR - 128000 50 NR . NR . 222000 50 NR - NR - 243000 50 NR . 721 50 91500 50 NR - 665 50 92800 50 NR . 38.2 40 240000 50 NR - 97.8 40 228000 50 NR ' . NR . 499000 50 NR - NR - 408000 50 NR . NR . 402000 50 NR - NR - 394000 50 NR . NR . 301000 50 NR - NR - 138000 50 NR . NR - 262 30 433000 50 218 30 420000 50 NR . NR . 49600 50 NR - NR - 50600 50 NR . NR . 192000 50 NR - NR - 149000 50 NR . NR . 26800 50 NR - NR - 23700 50 NR . NR . 1970 50 NR - NR - 1830 50 NR . 35.9 40 1670 50 NR - ND 40 1630 50 NR . 24.2 50 822 50 NR - ND 50 834 50 'Laboratory Duplicate ND = Not detected above the LOQ of 20 ng/g NR = Not reported due to quality control failures. MPI Research, Inc. Page 28 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.:P0001131 Table III. Summary of PFBS, PFHS and PFOS in Hispid Cotton Rat Serum Samples ExygenID C0049662 C0049662 Rep CO049663 C0049663 Rep C0049664 C0049664 Rep C0049665 C0049665 Rep C0049666 C0049666 Rep C0049667 C0049667 Rep C0049668 C0049668 Rep C0049669 C0049669 Rep C0049670 C0049670 Rep C0049671 C0049671 Rep C0049672 C0049672 Rep C0049673 C0049673 Rep C0049674 C0049674 Rep C0049675 C0049675 Rep C0049676 C0049676 Rep Client Sample ID DF06MO1SHB0010041007 DF06MO1SHB0010041007* DF06M01SHB0020041007 DF06M01SHB0020041007* DF06M01SHB0030041007 DF06M01SHB0030041007* DF06M01SHB0040041008 DF06M01SHB0040041008* DF06M01SHB0050041008 DF06M01SHB0050041008* DF06M01SHB0060041008 DF06M01SHB0060041008* DF08M01SHB0010041008 DF08M01SHB0010041008* D F08M 01SHB0020041008 DF08M01SHB0020041008* DF08M01SHB0030041008 DF08M01SHB0030041008* DF09M01SHB0010041007 DF09M01SHB0010041007* DF09M01SHB0020041007 DF09M01SHB0020041007* DF09M01SHB0030041008 DF09M01SHB0030041008* DF14M01SHB0010041007 DF14M01SHB0010041007* DF14M01SHB0020041007 DF14M01SHB0020041007* DF14M01SHB0030041007 DF14M01SHB0030041007* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulfonate____ Perfluorohexanesulfonate______ Perfluorooctanesulfonate Analyte Found (ppb, ng/mL) Assessed Accuracy (+/.%) Analyte Found (ppb, ng/mL) Assessed Accuracy (+/- %) Analyte Found (ppb, ng/mL) Assessed Accuracy <+/-%) NR NR* - NR . NR - NR* - NR - NR . ND 30 NR NR - ND 30 NR - NR . ND 30 NR NR - ND 30 NR - NR . NR* . NR* . NR - NR* - NR* - NR . NR* . NR . NR - NR* - NR - NR . ND 30 NR . NR - ND 30 NR - NR . NR* - NR . NR - NR* - NR - NR . NR* . NR . NR - NR* - NR - NR . NR* - NR NR - NR* - NR - NR . NR* . NR* . NR - NR* - NR* - NR . NR* NR . NR - NR* - NR - NR . ND 30 NR . NR - ND 30 NR - NR . NR* . NR* . NR - NR* - NR* [ NR . NR* - NR NR - NR* - NR - NR . NR* . NR* . NR - NR* - NR* - 'Laboratory Duplicate ND = Not detected above the LOQ of 400 ng/mL. NR = Not reported due to quality control failures. See Table IV for re-analysis results. NR* = Not reported, analytical results met quality control criteria in both initial and re-extracted analyses; see Table IV for reported re-analysis results. MPI Research, Inc. Page 29 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table IV. Summary of PFBS, PFHS and PFOS in Re-extracted Hispid Cotton Rat Serum Samples ExyLIMS ID Client Sam ple ID C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS P e rfluo rob utan esulfonate_____ P e rfluo roh exanesulfo nate______P erfluo roo ctane sulfonate Analyte Found (ppb, ng/ml_) Assessed Accuracy (+/-% ) Analyte Found (ppb, ng/mL) Assessed Accuracy (+/- % ) Analyte Found (ppb, ng/mL) Assessed Accuracy (+/-% ) C0049662 C0049662 Rep C0049663 C0049663 Rep C0049664 C0049664 Rep C0049665 C0049665 Rep D F06M 01S H B 0010041007 DF06M01SHB0010041007' D F06M 01S H B 0020041007 D F06M 01S H B 0020041007* D F06M 01S H B 0030041007 D F06M 01S H B 0030041007* D F06M 01S H B 0040041008 D F06M 01S H B 0040041008* 8.92 9.06 A A A A 0.897 0.950 30 30 - - - - 30 30 206 181 A A A A 1610 1610 30 117000 50 30 122000 50 - A- - A- - A- - A- 30 50300 50 30 52600 50 C0049666 DF06M 01SHB0050041008 C0049666 Rep DF06M 01SHB0050041008* C0049667 D F06M 01S H B 0060041008 C0049667 Rep DF06M01SHB0060041008* C0049668 D F08M 01S H B 0010041008 C0049668 Rep DF08M 01SHB0010041008* C0049669 D F08M 01S H B 0020041008 C 0049669 Rep DF08M 01SHB0020041008* C0049670 D F08M 01S H B 0030041008 C0049670 Rep DF08M 01SHB0030041008* 1.51 1.55 A A 1.03 0.975 0.863 0 .7 3 8 0.817 0.772 30 30 30 30 30 30 30 30 100 96.4 A A 521 499 197 186 686 605 30 104000 50 30 107000 50 - A- - A- 30 NR 30 NR - - 30 NR 30 NR - - 30 292000 50 30 299000 50 C0049671 D F09M 01SH B 0010041007 1.33 30 199 C0049671 Rep DF09M01SHB0010041007* 1.24 30 221 30 56700 50 30 47400 50 C0049672 D F09M 01S H B 0020041007 1.99 30 369 30 60100 50 C0049672 Rep DF09M 01SHB0020041007* A - A - A- C0049673 C0049673 Rep C0049674 C0049674 Rep D F09M 01S H B 0030041008 D F09M 01S H B 0030041008* D F14M 01S H B 0010041007 D F14M 01S H B 0010041007* 0.585 0.555 1.73 1.57 30 30 30 30 NR NR 2.51 2.37 - 30300 50 - 32500 50 30 1490 50 30 1460 50 C0049675 D F14M 01S H B 0020041007 1.12 30 11.4 30 979 50 C 0049675 Rep DF14M 01SHB0020041007* 1.06 30 8.30 30 937 50 C0049676 D F14M 01S H B 0030041007 0.743 30 3.84 30 301 50 C0049676 Rep DF14M 01SHB0030041007* 0.756 30 3.81 30 305 50 'Laboratory Duplicate ND = Not detected above the LOQ of 0.5 ng/mL. NR = Not reported due to quality control failures. A Insufficient remaining sample volume to re-extract this sample. MPI Research, Inc. Page 30 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No. : 0137.0219 MPI Project No.: P0001131 Table V. Matrix Spike Recovery of PFBS, PFHS and PFOS in Hispid Cotton Rat Liver Samples Sample Description DF06M01SHL0010041007 (C0049877 Spk C, 100 ppb Spike) DF06M01SHL0010041007 (C0049677 Spk H, 1000 ppb Spike) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS______________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/g) (nq/fl) (ng/g) (%) (ng/g) (ng/a) (%) (ng/g) (ng/g)_____ !%!___ 100 NR NR NR 30.4 107 77 NR NR NR 1000 NR NR NR 30.4 775 74 NR NR NR DF06M01SHL0020041007 (C0049676 Spk D, 100 ppb Spike) 100 NR NR NR 64.5 131 67 NR NR NR DF06M01SHL0020041007 (C0049678 Spk 1,1000 ppb Spike) 1000 NR NR NR 64.5 816 75 NR NR NR DF06M01SHL0030041007 (C0049679 Spk E, 100 ppb Spike) 100 NR NR NR ND 84.6 85 NR NR NR DF06M01SHL0030041007 (C0049679 Spk J, 1000 ppb Spike) 1000 NR NR NR ND 769 77 NR NR NR DF06M01SHL0040041008 (C0049680 Spk F, 100 ppb Spike) DF06M01SHL0040041008 (C0049680 Spk K, 1000 ppb Spike) 100 1000 NR NR NR NR NR* NR* NR* NR NR NR NR* NR* NR* NR NR NR NR NR DF06M01SHL0050041008 (C0049681 Spk G, 100 ppb Spike) DF06M01SHL0050041008 (C0049681 Spk L, 1000 ppb Spike) 100 1000 NR NR NR NR NR* NR* NR* NR NR NR NR* NR* NR* NR NR NR NR NR DF06M01SHL0060041008 (C0049692 Spk C, 100 ppb Spike) 100 NR NR NR 39.1 126 87 NR NR NR DF06M01SHL0060041008 (C0049692 Spk H, 1000 ppb Spike) 1000 NR NR NR 39.1 871 83 NR NR NR DF08M01SHL0010041008 (C0049682 Spk D, 100 ppb Spike) 100 NR NR NR 68.9 159 90 NR NR NR DF08M01SHL0010041008 (C0049682 Spk 1,1000 ppb Spike) 1000 NR NR NR 68.9 848 78 NR NR NR DF08M01SHL0020041008 (C0049683 Spk E, 100 ppb Spike) DF08M01SHL0020041008 (C0049683 Spk J, 1000 ppb Spike) 100 1000 NR NR NR NR 20.5 NR NR 20.5 144 124 859 84 NR NR NR NR NR NR ND = Not detected above the LOQ of 20 ng/g. NR = Not reported due to quality control failures. See Table VI for re-analysis results. NR* = Not reported, analytical results met quality control criteria in both initial and re-extracted analyses; see Table VI for reported re-analysis results. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research, Inc. Page 31 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table V. Matrix Spike Recovery of PFBS, PFHS and PFOS in Hispid Cotton Rat Liver Samples Continued Sample Description DF08M01SHL0030041008 (C0049684 Spk F, 100 ppb Spike) DF08M01SHL0030041008 (C0049684 Spk K, 1000 ppb Spike) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS______________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/g) (ng/g) (ng/g) (%) (ng/g) __ (ng/g) (%) (ng/g) __ (ng/g) (%) 100 NR NR NR NR* NR* NR* NR NR NR 1000 NR NR NR NR* NR* NR* NR NR NR DF09M01SHL0010041007 (C0049665 Spk G, 100 ppb Spike) 100 NR NR NR 30.8 117 86 NR NR NR DF09M01SHL0010041007 {C0049685 Spk L, 1000 ppb Spike) 1000 NR NR NR 30.8 753 72 NR NR NR DF09M01SHL0020041007 (C0049686 Spk C, 100 ppb Spike) 100 NR NR NR 108 185 77 NR NR NR DF09M01SHL0020041007 (C0049686 Spk H, 1000 ppb Spike) 1000 NR NR NR 108 901 79 NR NR NR DF09M01SHL0030041008 (C0049687 Spk D, 100 ppb Spike) 100 NR NR NR 78.3 160 82 NR NR NR DF09M01SHL0030041008 (C0049687 Spk 1,1000 ppb Spike) 1000 NR NR NR 78.3 858 78 NR NR NR DF14M01SHL0010041007 (C0049688 Spk E, 100 ppb Spike) DF14M01SHL0010041007 (C0049688 Spk J, 1000 ppb Spike) 100 1000 NR NR NR NR ND 76.4 76 NR* NR* NR* NR NR ND 802 80 NR* NR* NR* DF14M01SHL0020041007 (C0049689 Spk F, 100 ppb Spike) DF14M01SHL0020041007 (C0049689 Spk K, 1000 ppb Spike) 100 1000 NR NR NR NR NR* NR* NR* NR* NR* NR* NR NR NR* NR* NR* NR* NR* NR* DF14M01SHL0030041007 (C0049690 Spk G, 100 ppb Spike) DF14M01SHL0030041007 (C0049690 Spk L, 1000 ppb Spike) 100 1000 NR NR NR NR NR* NR* NR* NR* NR* NR* NR NR NR* NR* NR* NR* NR* NR* Average: Standard Deviation: 82 11 ND = Not detected above the LOQ of 20 ng/g. NR = Not reported due to quality control failures. See Table VI for re-analysis results. NR* = Not reported, analytical results met quality control criteria in both initial and re-extracted analyses; see Tabte VI for reported re-analysis results. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research, Inc. Page 32 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table VI. Matrix Spike Recovery of PFBS, PFHS and PFOS in Re extracted Hispid Cotton Rat Liver Samples Sample Description DF06M01SHL0010041007 (C0049677 Spk C, 100 ng/g Spike) DF06M01SHL0010041007 (C0049677 Spk D, 1000 ng/g Spike) DF06M01SHL0020041007 (C0049678 Spk E, 100 ng/g Spike) DF06M01SHL0020041007 (C0049678 Spk F, 1000 ng/g Spike) DF06M01SHL0030041007 (C0049679 Spk G, 100 ng/g Spike) DF06M01SHL0030041007 (C0049679 Spk H, 1000 ng/g Spike) DF06M01SHL0040041008 (C0049680 Spk 1,100 ng/g Spike) DF06M01SHL0040041008 (C0049680 Spk J, 1000 ng/g Spike) DF06M01SHL0050041008 (CO049681 Spk K, 100 ng/g Spike) DF06M01SHL0050041008 (C0049681 Spk L, 1000 ng/g Spike) DF06M01SHL0060041008 (C0049692 Spk C, 100 ng/g Spike) DF06M01SHL0060041008 (C0049692 Spk D, 1000 ng/g Spike) DF08M01SHL0010041008 (C0049682 Spk E, 100 ng/g Spike) DF08M01SHL0010041008 (C0049682 Spk F, 1000 ng/g Spike) DF08M01SHL0020041008 (C0049683 Spk G, 100 ng/g Spike) DF08M01SHL0020041008 (C0049683 Spk H, 1000 ng/g Spike) DF08M01SHL0030041008 (C0049684 Spk 1,100 ng/g Spike) DF08M01SHL0030041008 (C0049684Spk J, 1000 ng/g Spike) Amount Spiked (ng/g) C4 Sulfonate PFBS C6 Sulfonate PFHS Perfluorobutanesulfonate____________ Perfluorohexanesulfonate Amt Found Amount Amt Found Amount in Sample Recovered Recovery in Sample Recovered Recovery (ng/g) (ng/g) (%> (ng/g) (ng/g) (%) 100 NR NR NR NR NR NR 1000 NR NR NR NR NR NR 100 NR NR NR NR NR NR 1000 NR NR NR NR NR NR 100 NR NR NR NR NR NR 1000 NR NR NR NR NR NR 100 NR NR NR 721 920 * 1000 NR NR NR 721 1280 56 100 NR NR NR 38.2 176 138 1000 NR NR NR 38.2 897 86 100 NR NR NR NR NR NR 1000 NR NR NR NR NR NR 100 NR NR NR NR NR NR 1000 NR NR NR NR NR NR 100 NR NR NR NR NR NR 1000 NR NR NR NR NR NR 100 NR NR NR 262 366 104 1000 NR NR NR 262 933 67 ` Sample residue exceeds the spiking level significantly (>3x spiking level); therefore, an accurate recovery value cannot be calculated. ND = Not detected above the LOQ of 20 ng/g NR = Not reported due to quality control failures. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research, Inc. Page 33 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table VI. Matrix Spike Recovery of PFBS, PFHS and PFOS in Re extracted Hispid Cotton Rat Liver Samples Continued Sample Description DF09M01SHL0010041007 (C0049685 Spk K, 100 ng/g Spike) DF09M01SHL0010041007 (C004968S Spk L, 1000 ng/g Spike) DF09M01SHL0020041007 <00049686 Spk C, 100 ng/g Spike) DF09M01SHL0020041007 (C0049686 Spk D, 1000 ng/g Spike) DF09M01SHL0030041008 (C0049687 Spk E, 100 ng/g Spike) DF09M01SHL0030041008 (C0049687 Spk F. 1000 ng/g Spike) DF14M01SHL0010041007 (C0049688 Spk G, 100 ng/g Spike) DF14M01SHL0010041007 (C0049688 Spk H, 1000 ng/g Spike) DF14M01SHL0020041007 (C0049689 Spk 1,100 ng/g Spike) DF14M01SHL0020041007 (C0049689 Spk J, 1000 ng/g Spike) DF14M01SHL0030041007 (C0049690 Spk K, 100 ng/g Spike) DF14M01SHL0030041007 (C0049690 Spk L, 1000 ng/g Spike) Amount Spiked (ng/g) C4 Sulfonate PFBS Perfluorobutanesulfonate Amt Found Amount in Sample Recovered Recovery (ng/g) (ng/g) (%) C6 Sulfonate PFHS Perfluorohexanesulfonate Amt Found Amount in Sample Recovered Recovery (ng/g) (ng/g) (%) 100 NR NR NR NR NR NR 1000 NR NR NR NR NR NR 100 NR NR NR NR NR NR 1000 NR NR NR NR NR NR 100 NR NR NR NR NR NR 1000 NR NR NR NR NR NR 100 NR NR NR NR NR NR 1000 NR NR NR NR NR NR 100 NR NR NR 35.9 169 133 1000 NR NR NR 35.9 880 84 100 1000 NR NR NR . NR NR 24.2 172 NR 24.2 902 148 88 Average: Standard Deviation: ND = Not detected above the LOQ of 20 ng/g NR = Not reported due to quality control failures. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. 100 32 MPI Research, Inc. Page 34 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table VII. Matrix Spike Recovery of PFBS, PFHS and PFOS in Hispid Cotton Rat Serum Samples Sample Description DF06M01SHB0010041007 (C0049662 Spk C, 1000 ppb Spike) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS______________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/mL) (ng/mL) (ng/mL) (%) (ng/mL) (ng/mL) (%) (ng/mL) (ng/mL) (%) 1000 NR NR NR NR* NR* NR* NR NR NR DF06M01SHB0010041007 (C0049662 Spk E, 10000 ppb Spike) 10000 NR NR NR NR* NR* NR* NR NR NR DF06M01SHB0020041007 (C0049663 Spk D, 1000 ppb Spike) DF06M01SHB0020041007 (C0049663 Spk F, 10000 ppb Spike) 1000 10000 NR NR NR NR ND 995 100 NR NR NR ND 7500 75 NR NR NR NR NR DF06M01SHB0030041007 (C0049664 Spk C, 1000 ppb Spike) 1000 NR NR NR ND 895 90 NR NR NR DF06M01SHB0030041007 (C0049664 Spk E, 10000 ppb Spike) 10000 NR NR NR ND 7470 75 NR NR NR DF06M01SHB0040041008 (C0049665 Spk D, 1000 ppb Spike) DF06M01SHB0040041008 (C0049665 Spk F, 10000 ppb Spike) 1000 10000 NR NR NR NR NR* NR* NR* NR* NR* NR* NR NR NR* NR* NR* NR* NR* NR' DF06M01SHB0050041008 (C0049666 Spk C, 1000 ppb Spike) DF06M01SHB0050041008 (C0049666 Spk K, 10000 ppb Spike) 1000 10000 NR NR NR NR NR* NR* NR* NR NR NR NR* NR* NR* NR NR NR NR NR DF06M01SHB0060041008 (C0049667 Spk D, 1000 ppb Spike) DF06M01SHB0060041008 (C0049667 Spk L, 10000 ppb Spike) 1000 10000 NR NR NR NR ND 1270 127 NR NR NR ND 7860 79 NR NR NR NR NR DF08M01SHB0010041008 (C0049668 Spk E, 1000 ppb Spike) DF08M01SHB0010041008 (C0049668 Spk M, 10000 ppb Spike) 1000 10000 NR NR NR NR NR* NR* NR* NR NR NR NR* NR* NR* NR NR NR NR NR DF08M01SHB0020041008 (C0049669 Spk F, 1000 ppb Spike) DF08M01SHB0020041008 (C0049669 Spk N, 10000 ppb Spike) 1000 10000 NR NR NR NR NR* NR* NR* NR NR NR NR* NR* NR* NR NR NR NR NR ND = Not detected above the LOQ of 400 ng/mL. NR = Not reported due to quality control failures. See Table VIII for re-analysis results. NR* = Not reported, analytical results met quality control criteria in both initial and re-extracted analyses; see Table VIII for reported re-analysis results. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research, Inc. Page 35 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table VII. Matrix Spike Recovery of PFBS, PFHS and PFOS in Hispid Cotton Rat Serum Samples Continued Sample Description DF08M01SHB0030041008 (C0049670 Spk G, 1000 ppb Spike) C4 Sulfonate PFBS ________ C6 Sulfonate PFHS______________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/mL) (ng/mL) (ng/mL) (%) (ng/mL) (ng/mL) (%) (ng/mL) (ng/mL) (%) 1000 NR NR NR NR* NR* NR* NR NR NR DF08M01SHB0030041008 (C0049670 Spk 0 , 10000 ppb Spike) 10000 NR NR NR NR* NR* NR* NR NR NR DF09M01SHB0010041007 (C0049671 Spk H, 1000 ppb Spike) DF09M01SHB0010041007 (C0049671 Spk P, 10000 ppb Spike) 1000 10000 NR NR NR NR NR* NR* NR* NR* NR* NR* NR NR NR* NR' NR* NR* NR* NR* DF09M01SHB0020041007 (C0049672 Spk I, 1000 ppb Spike) DF09M01SHB0020041007 (C0049672 Spk Q, 10000 ppb Spike) 1000 10000 NR NR NR NR NR* NR' NR' NR NR NR NR' NR* NR* NR NR NR NR NR DF09M01SHB0030041008 (C0049673 Spk J, 1000 ppb Spike) DF09M01SHB0030041008 (C0049673 Spk R, 10000 ppb Spike) 1000 10000 NR NR NR NR ND 1110 111 NR NR NR ND 8900 89 NR NR NR NR NR DF14M01SHB0010041007 (C0049674 Spk C, 1000 ppb Spike) DF14M01SHB0010041007 (C0049674 Spk F, 10000 ppb Spike) 1000 10000 DF14M01SHB0020041007 (C0049675 Spk D, 1000 ppb Spike) DF14M01SHB0020041007 (C0049675 Spk G, 10000 ppb Spike) 1000 10000 NR NR NR NR NR NR NR NR NR NR NR NR NR* NR* NR* NR* NR* NR* NR' NR* NR* NR* NR* NR* NR* NR* NR NR NR* NR* NR* NR* NR NR NR NR DF14M01SHB0030041007 (C0049676 Spk E, 1000 ppb Spike) DF14M01SHB0030041007 (C0049676 Spk H, 10000 ppb Spike) 1000 10000 NR NR NR NR NR NR NR* NR* NR* NR* NR* NR* NR* NR* NR* NR* NR* NR* Average: Standard Deviation: 93 19 ND = Not detected above the LOQ of 400 ng/mL. NR = Not reported due to quality control failures. See Table VIII for re-analysis results. NR* = Not reported, analytical results met quality control criteria in both initial and re-extracted analyses: see Table VIII for reported re-analysis results. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research, Inc. Page 36 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table VIII. Matrix Spike Recovery of PFBS, PFHS and PFOS in Re extracted Hispid Cotton Rat Serum Samples Sample Description DF06M01SHB0010041007 (C0049662 Spk C, 250 ppb Spike) Amount Spiked (ng/mL) C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulfonate________ Perfluorohexanesulfonate________ Perfluorooctanesulfonate Amt Found Amount Amt Found Amount Amt Found Amount in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/mL) (nq/mL) <%) (ng/mL) (ng/mL) (%) (ng/mL) (ng/mL) (%) 250 8.92 238 92 206 436 92 117000 117000 DF06M01SHB0020041007 (C0049663 Spk D, 250 ppb Spike) 250 A AA AA AA AA DF06M01SHB0030041007 (C0049664 Spk E, 250 ppb Spike) 250 A AA A AA A AA DF06M01SHB0040041008 (C0049665 Spk F, 1000 ppb Spike) 1000 0.897 960 96 1610 2800 119 50300 45500 * DF06M01SHB0050041008 (C0049666 Spk G, 250 ppb Spike) 250 1.51 220 87 100 328 91 104000 107000 * DF06M01SHB0060041008 (C0049667 Spk C, 250 ppb Spike) 250 A AA A AA A AA DF08M01SH B0010041008 (C0049668 Spk D, 250 ppb Spike) 250 1.03 207 82 521 724 81 NR NR NR DF08M01SHB0020041008 (C0049669 Spk E, 250 ppb Spike) 250 0.863 207 82 197 431 94 NR NR NR DF08M01SHB0030041008 (C0049670 Spk F, 1000 ppb Spike) 1000 0.817 949 95 686 1690 100 292000 283000 * DF09M01SHB0010041007 (C0049671 Spk G, 250 ppb Spike) 250 1.33 217 86 199 405 82 56700 50500 * DF09M01SHB0020041007 (C0049672 Spk C, 250 ppb Spike) 250 1.99 226 90 369 659 116 60100 53500 * DF09M01SHB0030041008 (C0049673 Spk D, 250 ppb Spike) 250 0.585 220 88 NR NR NR 30300 41800 * DF14M01SHB0010041007 (C0049674 Spk E, 250 ppb Spike) 250 1.73 222 88 2.51 224 89 1490 1660 * DF14M01SHB0020041007 (C0049675 Spk F, 250 ppb Spike) 250 1.12 225 90 11.4 246 94 979 1130 DF14M01SHB0030041007 (C0049676 Spk G, 250 ppb Spike) 250 0.743 223 89 3.84 238 94 301 521 88 Average: Standard Deviation: 89 4 Average: Standard Deviation: 96 12 Average: Standard Deviation: ND = Not detected above the LOQ of 0.5 ng/mL NR = Not reported due to quality control failures. 'Sample residue exceeds the spiking level significantly (> 3x spiking level); therefore, an accurate recovery value cannot be calculated. A Insufficient remaining sample volume to re-extract this sample. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. 88 NA MPI Research, Inc. Page 37 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table IX. Surrogate Spike Recovery of 13C PFOS in Re-extracted Hispid Cotton Rat Liver Samples ExyLIMS ID C 0049677 Spike C C0049677 Spike D C 0049678 Spike E C0049678 Spike F C 0049679 Spike G C 0049679 Spike H C 0049680 Spike 1 C0049680 Spike J C0049681 Spike K C0049681 Spike L C0049692 Spike C C0049692 Spike D C 0049682 Spike E C 0049682 Spike F C 0049683 Spike G C0049683 Spike H C 0049684 Spike I C0049684 Spike J C0049685 Spike K C 0049685 Spike L C 0049686 Spike C C 0049686 Spike D C 0049687 Spike E C0049687 Spike F C0049688 Spike G C 0049688 Spike H C 0049689 Spike I C 0049689 Spike J C 0049690 Spike K C 0049690 Spike L Sample Description D F06M 01SH L0010041007 D F06M 01SH L0010041007 DF06M01SHL0020041007 D F06M 01SH L0020041007 DF06M01SHL0030041007 DF06M01SHL0030041007 DF06M01SHL0040041008 D F06M 01SH L0040041008 DF06M01SHL0050041008 DF06M01SHL0050041008 DF06M01SHL0060041008 DF06M01SHL0060041008 D F08M 01SH L0010041008 D F08M 01SH L0010041008 DF08M01SHL0020041008 D F 08M 01SH L0020041008 D F 08M 01SH L0030041008 DF08M01SHL0030041008 D F09M 01SH L0010041007 D F 09M 01SH L0 0 10041007 DF09M01SH L0020041007 DF09M01SHL0020041007 DF09M01SHL0030041008 DF09M01SHL0030041008 D F14M 01S H L0010041007 D F14M 01SH L0010041007 DF14M01SH L0020041007 D F 14 M 0 1SH L00 20 0 4 1007 DF14M01SHL0030041007 DF 14 M 0 1SH L00 30 0 4 1007 Amount Spiked (ng/g) 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 100 1000 13C-PFOS (M+4) Amount Recovered (ng/g) NA 774 NA 830 NA 712 NA 771 NA 787 NA 1480 NA 958 NA 1130 NA 706 NA 884 NA 626 NA 804 NA 903 NA 930 NA 929 Recovery (%) NA 77 NA 83 NA 71 NA 77 NA 79 NA 148 NA 96 NA 113 NA 71 NA 88 NA 63 NA 80 NA 90 NA 93 NA 93 Average: Standard Deviation: 88 21 NA = Not applicable, high surogate spike recoveries used to assess accuracy of PFOS results. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research, Inc. Page 38 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Table X. Surrogate Spike Recovery of 13C PFOS in Re-extracted Hispid Cotton Rat Serum Samples ____________________________________________________________________ 13C-PFOS (M+4)_______________ Amount Amount ExyLIMS ID Sample Description Spiked (ng/mL) Recovered (ng/mL) Recovery (%) C0049662 SpkC D F06M 01S H B 0010041007 250 156 62 C0049663 Spk D C0049664 Spk E D F06M 01SH B 0020041007 D F06M 01S H B 0030041007 250 250 A A A A C0049665 Spk F D F06M 01S H B 0040041008 1000 934 93 C0049666 Spk G D F06M 01S H B 0050041008 250 156 62 C0049667 Spk C D F 06M 01S H B 0060041008 250 A A C0049668 Spk D DF08M01SHB0010041008 250 NR NR C0049669 Spk E D F08M 01SH B 0020041008 250 NR NR , C0049670 Spk F D F08M 01SH B 0030041008 1000 812 81 C0049671 Spk G D F 09M 01S H B 0010041007 250 180 72 C0049672 Spk C D F 09M 01S H B 0020041007 250 181 72 C0049673 Spk D D F09M 01SH B 0030041008 250 190 76 C0049674 Spk E D F14M 01S H B 0010041007 250 231 92 C0049675 Spk F D F14M 01S H B 0020041007 250 229 92 C0049676 Spk G D F14M 01S H B 0030041007 250 233 93 Average: Standard Deviation: 80 12 A Insufficient remaining sample volume to re-extract this sample. NR = Not reported due to quality control failures. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research, Inc. Page 39 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 FIGURES MPI Research, Inc. Page 40 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 1. Typical Calibration Curve for PFBS in Methanol 121704B.rdb (PFBS): "Lineai" Regression ('1 i y i ' weighting): y = 5.49e+003 x + 584 (r = 0.8986) Area, counts MPI Research, Inc. Page 41 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: POOOl 131 Figure 2. Non-Extracted Standards of PFBS in Methanol, 0.1 ng/mL and 0.2 ng/mL, Respectively 0113004-5, 0.1 ng/mL Standard - PFBS (Standard) 299,0/99.0 am a -sam ple 1 o f 36 from 121704B.wiff Area: 1150. counts Height: 50.1 cp* RT: 1.71 min 0.63 Time, min C113004-5. 0.2 ng/m L Standard - PFBS (Standard) 299.0/99.0 amu - sample 2 of 36 from 121704B.wiff A rea: 1680. counts H eight: 0 8.6 ops RT: 1.79 min 0.01 MPI Research, Inc. Page 42 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 3. PFBS in Control Liver, 10 ng/mL Fortified Control Liver Spk A, and 50 ng/mL Fortified Control Liver Spk B, Respectively CO05365O C o n tro /PFB S (Unknown) 299.0/99.0 am a -sam ple 0 ofS& from 121704B.wiff p ea k not found) Area: 2 54 0. counts H eight: 93.7 cps RT: 1.61 min 1.61 2-75 3 .2 3 4 . 7 ^ 5 ,1S 6.26 lL > w w . A M 8 -8A M r*^A A ir\/v\ 45 67 8 9 10 11 Tim e, min C0053650 Spike 0. 50 n g /m L - PFBS (Q C )2 99 .0/9 9.0 a m u -s a m p le 10 of 36 from 121 70 4 B .w iff Area: 10200. counts Height: 598. cps RT: 1.60 min 1 2 .8 4 ^ `' 14.13-0440 1530^15.58 17 44 * ` * ' -^V|- * - A 12 13 14 15 16 17 MPI Research, Inc. Page 43 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 4. PFBS in Control Serum, 10 ng/mL Fortified Control Serum Spk A, and 50 ng/mL Fortified Control Serum Spk B, Respectively CQ034001 C ontrol PFBS (Unknown) 299.0/99.0 am u sam ple 8 o f 49 from 121304C.wiff (freak n o t found) 11.31 ^-11.95,12.67 6 9 10 Tim e, min C0034001 Spike A, 10 ng/m L - PFBS (Q C )299.0/99.0 a m u- sample 9 of 48 from 121304C.wiff Area: 1770. counts Height: 87.3 cps RT: 1.65 min 1.65 1 23456789 10 11 Tim e, min C0034001 Spike B .5 0 ng/m L - PFBS (Q C )299.0/99.0 amu sample 10 of 48 from 121304C.wiff Area: 6280. counts Height: 280. cps RT: 1.62 min 1.62 MPI Research, Inc. Page 44 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project N o.: P0001131 Figure 5. Chromatogram Representing a Liver Sample Analyzed for PFBS (ExyLIMS ID: C0049686, Data Set: 121704B) C0049696 'P F B S (Unknown) 299.0/99.0 am u -sam pfe 24o f 36 from 121704B.wiff freak not found) 0 .4 9 Intensity, cps MPI Research, Inc. Page 45 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 6. Chromatogram Representing a Serum Sample Analyzed for PFBS (ExyLIMS ID: C0049666, Data Set: 121304C) C0049666 PFBS (Unknown) 299.0/99.0 am u -sam ple 30 o f 48 from 121304C.wffT (peak not found) 0.67 Intensity, cps MPI Research, Inc. Page 46 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 7. Typical Calibration Curve for PFHS in Methanol 1 2 1 7 0 4 0 .rdb (PFHS): "L in e a i'1 Regression '1 / x " w e ig htin g ): y = 3.21 e + 0 0 4 x + 2 .3 3 e+ 00 3 (r = 0.998) Area, counts MPI Research, Inc Page 47 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples y MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 8. Non-Extracted Standards of PFHS in Methanol, 0.1 ng/mL and 0.2 ng/mL, Respectively I Cf730(W-6, 0.1 ag/mL Sta n d a rd -P F H S (Standard)399.0/90.0am a sam ple 1 o f 36 from 1217Q4B.wifT Area: 5650. co u n ts Height: 1B3. cps RT: 8.20 m in 8.20 to Q_ & CO Tim e, min I C113004-5. 0.2 ng/m L Standard - PFHS (S tandard)399.0/80.0 amu - sample 2 of 36 from 121704B.wiff Area: 8470. counts H eighfc393.eps R T :8 .2 0 m in 350 300to & 250- 8.20 Tim e, min MPI Research, Inc. Page 48 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 9. PFHS in Control Liver, 10 ng/mL Fortified Control Liver Spk A, and 50 ng/mL Fortified Control Liver Spk B, Respectively C0053650 C ontrol - PFHS (Unknown) 399.9/99.9 am u -sam pte 9 o f 36 from 121794B.wiff Area: 1929. counts Height: 29.9 cps RT: 9.44m in 0.68 CO Qo. CcO 0) Tim e, min I 00053650 Spike A. 10 n g/m L - PFHS (Q C )399.0/80.0 mu sample 9 of 36 from 121704B.wiff Area: 15700. counts Height: 1060. cps RT: 8.19 min !.18 A 1-20 2 .5 4 2.92 6 .7 0 ^ 7 .3 9 ,7 .6 0 -,____^ | 1 Tim e, min I C0053650 Spike B. 50 n g /m L - PFHS (QC) 3 9 9 .0/8 0.0 m u -s a m p le 10 of 3 6 from 121 70 4 B.w iff Area: 62400. counts Height: 4970. cps RT: 8.19 min 8.19 MPI Research, Inc. Page 49 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 10. PFHS in Control Serum, 10 ng/mL Fortified Control Serum Spk A, and 50 ng/mL Fortified Control Serum Spk B, Respectively 00034007 Control -PFHS (Unknown) 399.WBQ.0 am u - sam pis 8 o f 4B from 121304C.wiff Area: 1620. co u n ts Height: 67.8 cps RT: &78 m in Qto. 40 20 12.46 ^1 3 .4 2^1 4.3 2 Tim e, min I C0034001 Spike A. 10 ng/m L - PFHS (Q 0 )3 99 .0/8 0.0 a m u - sample 9 of 48 from 121304C.wiff Area: 9420. counts Height: 720. cps RT: 8.19 min cn Qo. 6 00 400 to c0} 200 0.62 8.19 | 1 7 4 3 j.s e M 6 .5 8 ,. ,8 .5 5 10.86 1 2 3 4 5 6 7 8 9 10 11 Tim e, min I 0 0034001 Spike 0 ,5 0 n g /m L - PFHS (0 0 )3 9 9 .0 /8 0 .0 a m u -s a m p le 10 of 4 8 from 1 2 1 3 0 4 0 .iwiff Area: 39300. counts Height: 3090. cps RT: 8.20 min 8.20 12.82 13.70 12 13 14 i i 18 17 MPI Research, Inc. Page 50 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 11. Chromatogram Representing a Liver Sample Analyzed for PFHS (ExyLIMS ID: C0049686, Data Set: 121704B) I C0O49686 -PFHS (U nknow n)399.M 0.0am a .sa m p le 24o f 36 from 121704B.wiff Area: 19600. co a n ts Height: 1000. cps RT: 8.20 min 8.20 Intensity, cps MPI Research, Inc. Page 51 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 12. Chromatogram Representing a Serum Sample Analyzed for PFHS (ExyLIMS ID: C0049666, Data Set: 121304C) I C49666 PFHS (Unknown) 399.0m.Q am u -sam ple 3 0 o f 4$ from 1213C4C.wiff Area: 2370. counts Height: 123. cps RT: 8L14 mia 8.14 Intensity, cps MPI Research, Inc. Page 52 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 13. Typical Calibration Curve for PFOS in Methanol 121704B.rdb (PFOS): "L in e a r" Regression C'1 / x" w e ig htin g ): y = 2 .9 8 e + 0 0 4 x + 8.8 3 e+ 00 3 (r = 0 .9 9 4 6 ) Area, counts MPI Research, Inc Page 53 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.:P0001131 Figure 14. Non-Extracted Standards of PFOS in Methanol, 0.1 ng/mL and 0.2 ng/mL, Respectively C113004-6, f t ng/ttiL Standard - PFOS (Standard) 499.0/90.0 am a -sam ple 1 o f 36 from 121704B.wiff Area: 3200. counts Height: 457. cps RT: 10.6 m in 10.82 01 Q. cOCO) Tim e, min I C113004-5. 0.2 ng/m L Standard PFOS (S tandard)409.0/80.0 amu - sample 2 of 36 from 121704B.wiff A rea: 15200. counts H eight: 034. ops RT: 10.0 min 10.60 MPI Research, Inc. Page 54 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 15. PFOS in Control Liver, 10 ng/mL Fortified Control Liver Spk A, and 50 ng/mL Fortified Control Liver Spk B, Respectively I C0O53650 Control PFOS (Unknown) 499.0/90.0 am u -sam ple &o f 36 from 12174B.wtff Area: 9249. count Height: 260. cp RT: 10.6 min CO QO_ 4000 - CO c 2000 - QJ c 3 .6 7 s.3O Tim e, min C0053650 Spike A. 10 ng/m L - PFOS (QC) 499.0/80.0 a m u - sample 9 of 30 from 1 2 l7 0 4 B .w iff Area: 20700. counts Height: 974. cps RT: 10.6 min 6.59 10.59 9 .1 6 ^ 10-1tS _ ^ >11.24 8 9 10 11 Tim e, min I C0053650 Spike B, 50 n g/m L - PFOS (QC) 4 99 .0/8 0.0 a m u - sam ple 10 of 36 from 121704B.iAiiff Area: 61100. counts Height: 3410. cps RT: 10.6 min 12 6.58 ..... 13 14 15 16 17 MPI Research, Inc. Page 55 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 16. PFOS in Control Serum, 10 ng/mL Fortified Control Serum Spk A, and 50 ng/mL Fortified Control Serum Spk B, Respectively C0034007 C ontroi PFOS (U nknow n)499.0/M 0 am u -sam ple 9 o f 49 from 121394C.wiff Area: 79000. counts Height: 634 cps RT: 10.6 min CO 6 0 0 Q. o a r 400 CO c 200 - c 3.52.. 3.99 6.57 0 1 23456789 10 Tim e, min C0034001 Spike A, 10 n g/m L - PFOS (0 0 )4 9 9 .0 /8 0 .0 amu - sample 9 of 48 from 121304C.wiff Area: 27500. counts Height: 1080. cps RT: 10.6 min 3 .7 2 ^ 4 .0 7 6.66 1 6 8 9 10 Tim e, min C0034GQ1 Spike B, 50 n g /m L - PFOS (0 0 )4 9 9 .0 /8 0 .0 amu - sam ple 10 of 48 from 1 21304C .w iff Area: 50200. counts Height: 2630. cps RT: 10.6 min [ 13.07 18.14.15.47 ,16.21 1 12 13 14 15 16 17 2000 1000 0 10.58 10.11 19.82s. _ 11.75 12 14.07 15.43 ,,1 5 .8 3 ,,16.94 13 14 15 16 17 MPI Research, Inc. Page 56 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 17. Chromatogram Representing a Liver Sample Analyzed for PFOS (ExyLIMS ID: C0049686, Data Set: 121704B) Intensity, cps C0049696 - PFOS (Unknown) 499.0/90.0 am a -sam ple 24o f 36 from 121704B.wiff Area: 15400000. c o u n ts Height: 896000. cps RT: 10.6 m in 8.5e5 8.0e5 7.5e5 7.0e5 6.5e5 6.0e5 5.5e5 5.0e5 4.5e5 4 .0 e5 3.5e5 3.0e5 2.5e5 2.0e5 1.5 e5 1.0 e5 5 .0e4- 0.0J-------1-------.-------.-------1------ .-------.----- r 12 3 45 67 10.59 10.12^ 8 9 10 T im e, min 11 12 13 14 15 16 17 MPI Research, Inc. Page 57 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 18. Chromatogram Representing a Serum Sample Analyzed for PFOS (ExyLIMS ID: C0049666, Data Set: 121304C) C0049666 -PFO S (U nknow n)499.0/90.0am a -sam ple 3 0 o f 48 from 121304C.wiff Area: 693000. co u n ts Height: 57800. cps RT: 10.6 min 10.56 Intensity, cps MPI Research, Inc. Page 58 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 19. Typical Calibration Curve for 13C PFOS in Methanol 0 8 0 8 0 7 B P1131 Rat Liver.rdb (13C PFOS): " L in e a r Regression ("1 / * ' fre ig h tin g ): y = 3 .7 5 e + 0 0 4 x + 2 42 (r = 0 .0 9 9 8 ) Area, counts MPI Research, Inc. Page 59 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 20. Non-Extracted Standards of 13C PFOS in Methanol, 0.1 ng/mL and 0.2 ng/mL, Respectively 550023024 T3C PFOS (Standard) 5 03.W 0.0am a -sam ple 1 o f 42 from 090B07B.wiff Area: 3S12coants Height: 1.12e+002cps RT: 11.0min 11.01 <s> Q. & V) c<u Tim e, min I SS0023326 13C PFOS (S tandard)503.0/80.0 amu - sam ple 2 of 42 from 080807B.w iff Area: 8 19 5 counts H eight: 3 .6 2 e+ 00 2 cps RT: 11.0 min 350 300 250 200 150 100 50 0 A. 89 Ay -- Av MPI Research, Ine. Page 60 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 21. 13C PFOS in Control Liver, 0.5 ng/mL Fortified Control Liver Spk A, and 2.5 ng/mL Fortified Control Liver Spk B, Respectively I C0053650 C ontrol - 13CPFOS (Unknown) 503.0/80.0am u -sam ple 9 o f 42 from 080807B.w iff freak not found) CO QO. Tim e, min I C0053650 Spk A - 13C PFOS (QC)503.0>80.0 amu sa m p led of 42 from 080807B.w iff Area: 16884 counts Height: 2 .12e+003 cps RT: 11.0 min Tim e, min C0053650 Sp* B - 13C PFOS (QC) 50X0/80.0 am u - sam ple 10 o f 42 from 080807B.wiff Area: 87847connts Hbigkt: 1.10e+004cps RT: 11.0min MPI Research, Inc. Page 61 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 22. 13C PFOS in Control Serum, 10 ng/mL Fortified Control Serum Spk A, and 50 ng/mL Fortified Control Serum Spk B, Respectively MC0000263 C ontrol 13C PFOS (Unknown) S03.0l90.0amu -sam ple 0 o f 32 from 080307B.wiff p e a k not found) Tim e, min M C 0000263 Spk A - 13C PFOS (QC) 503.0/80.0 a m u - sample 9 of 32 from 0 80307B.wiff Area: 30542 counts Height: 1.45e+003 cps RT: 11.4 min IWC0000263Spk B -1 3 C PFOS (QC)S03.0J90.0amu -sam ple 10o f 32 from 080307B.w iff Area: 31076counts Height: 1.5de+003cps RT: 11.4min MPI Research, Inc. Page 62 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 23. Chromatogram Representing a Liver Sample Analyzed for 13C PFOS (ExyLIMS ID: C0049677, Data Set: 080807B) C0M9677 - 13CPFOS (Unknown) 5Q3.W9Q.0amu -sam ple 16o f 42 from Q9Q9Q7B.wiff (peak not found) Intensity, cps MPI Research, Inc. Page 63 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Figure 24. Chromatogram Representing a Serum Sample Analyzed for 13C PFOS (ExyLIMS ID: C0049668, Data Set: 080307B) C0049668 73C PFOS (Unknown) 503.0/80.0 am u -sam pfe f 5 o f 32 front 090307B.wiff freak not found) 0.39 Intensity, cps MPI Research, Inc. Page 64 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 APPENDIX A MPI Project No.: P0001131 (MPI Study No.: 0137.0219) with Analytical Methods, Amendments, and Deviations MPI Research, Inc. Page 65 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Sponsor Representative: Michael A. Santoro Director of Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone:(651) 733-6374 Page I o f 65 MPI Research, Inc. Page 66 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit MPI Research, Inc. Page 2 o f 65 Page 67 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 PROTOCOL APPROVAL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 APPROVALS JaisimhalKSn, Study Director Weston Solutions Michael A. Santoro, Sponsor Representative 3M Compaify bhn M. Flaherty, Principal Investigator Exygen Research Richard A. Grazzmi, President, Facility Management Exygen Research Lydht Shaffer, Techniy^t^ead, Quality Assurance Unit Exgen Research m 44f Date -PCI- Date ' ' Dat' Page 3 o f 65 MPI Research, Inc. Page 68 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 TABLE OF CONTENTS TITLE PA G E ..................................................................................................................................................................... 1 DISTRIBUTION............................................................................................................................................................... 2 PROTOCOL APPROVAL.............................................................................................................................................. 3 TABLE OF CONTENTS................................................................................................................................................ 4 IN T R O D U C T IO N ............................................................................................................................................................. 5 TEST M ATERIA LS........................................................................................................................................................ 5 O B JECTIV E......................................................................................................................................................................6 TESTING FACILITY...................................................................................................................................................... 6 STUDY DIRECTOR........................................................................................................................................................ 7 SPONSOR REPRESENTATIVE................................................................................................................................... 7 PRINCIPAL INVESTIGATOR.............................................................. ......................................................................7 PROPOSED EXPERIMENTAL START AND TERMINATION D A TES........................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SY ST EM ................................................................8 SAMPLE PROCUREMENT, RECEIPT AND RETEN TION................................................................................. 8 SAMPLE IDENTIFICATION........................................................................................................................................9 ANALYTICAL PROCEDURE SUMMARY.............................................................................................................. 9 VERIFICATION OF ANALYTICAL PROCEDURE................................................................................................9 METHOD FOR CONTROL OF B IA S................!....................................................................................................... 11 STATISTICAL M ETH O D S............................................................................................................................................11 GLP STA TEM ENT.......................................................................................................................................................... 11 R EPO R T.............................................................................................................................................................................11 SAFETY AND HEA LTH ................................................................................................................................................12 AMENDMENTS TO PROTO CO L............................................................................................................................... 13 DATA RECORD K EE PIN G .......................................................................................................................................... 13 QUALITY A SSURANCE...............................................................................................................................................14 RETENTION OF DATA AND A RCHIVING.............................................................................................................14 APPENDIX I, ANALYTICAL METHODS................................................................................................................. 13 Page 4 o f 65 MPI Research, Inc. Page 69 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: POOO1131 INTRODUCTION The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum using LC/MS/MS for the 3M Decatur Monitoring Program. The study will be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit of Exygen Research. TEST MATERIALS The test materials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M. PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (CFSOs'K4) Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 -> 99 Structure: FF FF FF FF PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CtFuSOflC) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 - 80 Structure: Page 5 o f 65 MPI Research, Inc. Page 70 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0Q01131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFiySCVlO) Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 -> 99 Structure: OBJECTIVE The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions of the following Exygen analytical methods: V0001780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Page 6 o f 65 MPI Research, Inc. Page 71 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax: (610)701-7401 j .kesari@westonsolutions.com SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director of Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M. Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 john.flaherty@exygen.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion of this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and termination dates will be included in the final report. Page 7 o f 65 MPI Research, Inc. Page 72 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: Water (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study. The test systems were chosen to access the environmental impact of PFBS, PFHS and PFOS in the Decatur, Alabama area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples will be received at Exygen directly from Weston Solutions. The details of sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types of samples collected will vary depending availability in the field. The total number of samples received and analyzed for each matrix will be documented in the final report associated with this study. Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at 2C-8C. Fish, clam, vegetation and small mammal liver samples will be processed according to the appropriate analytical method (see Appendix I). These samples will be stored frozen at < -10"C. Small mammal whole blood samples will be centrifuged in the field at the time of collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at < -10C. The receipt and processing of the samples will be documented in the final report and raw data associated with the study. Page 8 o f 65 MPI Research, Inc. Page 73 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 SAMPLE IDENTIFICATION Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Sample storage conditions and locations will be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: V0001780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" The above methods use analytical conditions capable of separating the isomers of PFBS, PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sample will be used for the preparation of fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen will supply one bottle per sample collected. The bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 9 o f 65 MPI Research, Inc. Page 74 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 collection at the testing facility and have been shown to be free of PFBS, PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike of each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (l3C PFOA). PFOA and UC PFOA are included in the solutions used to spike the samples. The results for PFOA and l3C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containers/bags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. For small mammal liver, Exygen will supply a 50 mL polypropylene centrifuge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. Low and high spiking levels for each matrix are defined below: Matrix Low Spiking Level High Spiking Level Water 500 ng/L 5000 ng/L Soil 4 ng/g 40 ng/g Sediment 4 ng/g 40 ng/g Fish 10 ng/g 100 ng/g Clams 10 ng/g 100 ng/g Vegetation 10 ng/g 100 ng/g Small Mammal Liver 10 ng/g 100 ng/g Small Mammal Serum 10 ng/mL 100 ng/mL Page 10 o f 65 MPI Research, Inc. Page 75 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Recoveries are anticipated to be between 70% and 130% of the fortified levels; however, the exact precision and accuracy will be determined by the analysis of the quality control samples described above. A statement of accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control of bias will be addressed by taking representative sub-samples from a homogeneous mixture of each matrix from untreated control samples, and by analyzing at least two levels of fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation of average recoveries, as applicable. GLP STATEMENT All aspects of this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative. REPORT A final report will be prepared by the principal investigator or their designee at the conclusion of the study. The report will include, but will not be limited to, the following: The name and address of the Study Director, Sponsor Representative, and of the testing facility. A statement of GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). Page / 1 o f 65 MPI Research, Inc. Page 76 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates of study inspections and dates findings were reported to the Study Director and Management. A description of the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy of the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page. Description of the instrumentation used and operating conditions. All results from all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level. Representative chromatograms for each analyte in each matrix, including chromatograms of a standard and a control sample, and a chromatogram at a fortification level. The location of the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity of the data will be documented in the report. Locations where raw data and the final report are to be archived. Additions or corrections to the final report shall be in the form of an amendment signed by the Study Director. The amendment shall clearly identify that part of the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Representative. All applicable requirements for reporting of study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel will practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure of personnel and the environment to the test or reference substance(s). Page 12 o f 65 MPI Research, Inc. Page 77 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Proj ectN o.:P0001131 Exygen Protocol Number: P0001131 AMENDMENTS TO PROTOCOL All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation of study plan change. However, when a change is required without sufficient time for the issue of a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date of the written amendment will be the date of the documented change. Copies of the signed amendments will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation of standards (stock, fortification, calibration) Description of any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing of study personnel, signatures and initials Chronological presentation of all study correspondence Any other documentation necessary for the reconstruction of the study Chromatograms- All chromatograms will contain the following: Sample identification, injection date, arrow or other indication of the area of interest, and injection number corresponding to the run. Additionally, fortifications will include the amount of analyte added and the sample number of the sample that was fortified. Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL). Page 13 o f 65 MPI Research, Inc. Page 78 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 As part of the documentation the following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITY ASSURANCE The QA Unit of Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings of audits to the Study Director, Exygen Management, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period of time specified in 40 CFR 792.195. An exact copy of the materials submitted to the study director will also be kept at Exygen Research. Exygen will obtain permission from the study director before discarding or returning samples. MPI Research, Inc. Page 14 o f 65 Page 79 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygcn Protocol Number: P0001131 APPENDIX I ANALYTICAL METHODS V0001780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" MPI Research, Inc Page J5 o f 65 Page 80 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P001131 ANALYTICAL METHOD M ethod N um ber: V 0001780 M e th o d o f A n a ly sis fo r th e D e te rm in a tio n o f P e rflu o ro o c ta n o lc A c id (P F O A ) in W a te r by L C /M S/M S A nalytical T esting Facility: Exygen R esearch 30S8 R esearch D rive S tate C ollege, P A 16801 A pproved By: __ C.-- _____________ Paul C onnolly 1 T echnical Leader, LC -M S, Exygen R esearch 10h-i-AW D ate AiUi / J o h n F laherty / ' V ice President, O perations, Exygen R esearch D ate Ap MPI Research, Inc. Total Pages: 7 Page 6 o f 65 Page 81 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOP80 [ A N A LY TIC A L M E T H O D Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by IC/MS/MS 1.0 S co p e T his m ethod is to b e em ployed for the isolation an d quantitation o f perfluorooctanoic acid by H igh Perform ance Liquid C hrom atography coupled to a tandem M ass S p ectro m cth c D etecto r (L C /M S/M S ) in w ater. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 C onsult the appropriate M SD S before h andling an y chem ical for proper safety precautions. 3.0 S am ple R equirem ent 3.1 A t least 4 0 m L o f test sam p le fo r extractio n . 3.2 N o sam ple p rocessing is n eeded for w ater sam ples. 3.3 Sam ples stored refrigerated sh o u ld b e allo w e d to equ ilib rate to room tem perature. 3.4 A ll sam ples roust be thoroughly m ixed before bein g sam pled for extraction. 3.5 A ny sam ples co n tain in g p articles should b e ce n trifu g ed at -3 0 0 0 rpm for -5 m inutes and the supernatant used for the extraction. 3.6 S am ple co llectio n p rocedures w ill b e sp ecified in the sam p lin g plan for this p ro je c t. 4 .0 R eagents and Standards 4.1 W a te r - H P L C grad e 4.2 M ethanol - H PL C grade 4.3 A m m onium A cetate - A .C .S . R eagent G rade 4.4 P erfluorooctanoic A cid - Sigm a-A ldrich 5.0 Instrum ent and Equipm ent 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5 .9 5 .1 0 5.11 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5-200 p L connected to a tandem M ass Spectrom eter (L C /M S/M S ). A device to collect raw data for peak integration and quantitation. A nalytical balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene centrifuge tubes. ' 15 m L disposable polypropylene centrifuge tubes. D isp o sab le m icro p ip ets (50-1 OOuL, 1 0 0 -2 0 0 u L ). 125-raL LD PE narrow -m outh bottles. 2 m L clear H PLC vial kit. D isposable pipettes. A utopipettes (100-1000 p L and 10-100 p L ), w ith d isposable tips. W aters S ep P ak V ac 6 cc (lg ) tC 18 S PE cartridges. Page 2 o f ? Page 7 o f 65 MPI Research, Inc. Page 82 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method N umber V000178Q A N A LY TIC A L M E T H O D M ethod o f A n aly sis for th e D eterm ination o fP erflu o ro o cta n o ic A c id (P F O A ) in W ater by LC /M S/M S 5.12 S P E v acu u m m an i fold. 5.13 C entrifugo cap ab le o f sp inning 5 0 m L p o ly p ro p y len e tu b es at 3 0 0 0 rpm . 6.0 C hrom atographic System 6.1 A naly tical C olum n: F lu o p h ase R P (K ey sto n e S cien tific), 2.1 m m x 50 m m . 5p (P/N : 82505-052130) 6 .2 T em p e ratu re : 30C 6.3 M o b ile P hase (A ) : 2 m M A m m o n iu m A cetate in W ater 6.4 M obile P hase (B ) : M ethanol 6.5 G radient Program : T im e (m in) 0.0 1.0 8.0 2 0 .0 2 2 .5 %A 65 65 25 25 65 %B 35 35 75 75 35 F low R ate (m U roin) 0.3 0.3 0.3 0.3 0.3 6.6 Injection V olum e: 15 p L (can b e increased to as m uch as 50 pL). 6.7 Q uantitation: P eak A rea - external standard calibration curve. 6.8 R u n T im e : - 2 3 m inutes. T he above conditions are intended as a g uide and m ay b e changed in order to optim ize the H PL C system . 7.0 M S /M S System 7.1 M o d e : E le c tr o s p ra y N e g a tiv e M R M m o d e , m o n ito r in g 4 1 3 -> 3 6 9 m /z . T h e ab o v e c o n d itio n s are in ten d ed as a g u id e an d m a y b e ch a n g e d in o rd e r to optim ize the M SM S system . 8.0 P reparation o f Solutions 8.1 M o b ile P h ase 8.1.1 2 m M am m o n iu m acetate in w a te r is p rep ared b y ad d in g 0.154 g o f am m onium acetate to 1000 m L o f w ater. A lternate v olum es m ay b e prepared. Page v i " Page /8 o f 65 MPI Research, Inc. Page 83 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.:P0001131 Exygen Protocol Number: P0001131 Exygen Research M ethod N umber V0001780 | A N A LY TIC A L M E T H O D j M ethod o f A nalysis fo r the D eterm ination o f Perfluorooctanoic A cid (P F O A ) in W ater by LC /M S/M S 9.0 Standard P reparation 9.1 S tan d ard S to c k /F o rtificatio n S o lu tio n 9.1.1 P rep are a stock solution o f - 1 0 0 p g /m L o f P F O A b y w eig h in g 10 nig o f analytical standard (corrected for p urity) and dilute to 100 m L with m ethanol in a 125-m L L D P E bottle. 9 .1 .2 A 10 p g /m L fo rtific a tio n s o lu tio n o f P F O A is p re p a re d b y b rin g in g 10 m L o f th e 100 p g /m L s o lu tio n to a fin al v o lu m e o f 100 w ith m ethanol in a 125 m L L D PE bottle. 9.1.3 A 1.0 p g /m L fortification so lu tio n o f P F O A is prep ared b y b rin g in g 10 m L o f th e 10 p g /m L so lu tio n to a fin al v o lu m e o f 100 w ith methanol in a 125 m L L D PE bottle. 9 .1 .4 A 0.1 p g /m L fo rtificatio n so lu tio n o fP F O A is p re p are d b y brin g in g 10 m L o f th e 1.0 p g /ra L s o lu tio n to a fin al v o lu m e o f 100 w ith m ethanol in a 125 m L L D P E bottle. 9.1.5 A 0.01 p g /m L fo rtification so lu tio n o fP F O A is p rep ared b y bringing 10 m L o f the 0 .) p g /raL solution to a final volum e o f 100 with methanol in a 125 m L L D P E b ottle. 9 .1 .6 T h e sto ck and fo rtificatio n so lu tio n s a re to b e sto red in a re frig erato r at approxim ately 4C and are stable for a m axim um period o f 6 m onths from the date o f preparation. 9.2 S tandard C alibration S olutions 9 .2 .1 9 .2 .2 L C /M S/M S ca libration stan d ard s are p re p are d in H P L C w ater. T he calibration standards are processed through the extraction procedure, identical to sam ples. T he follow ing is a typical exam ple: additional concentrations m ay be prepared as needed. C o n cen tratio n o f Fortification Solution (nob) 0 10 10 10 100 100 100 Fortification V olum e (PL) 0 100 200 400 too 200 400 V olum e o f Fortified C ontrol Sample (mL) 40 40 40 40 40 40 40 F in a l Concentration of C alib ratio n Standard (rot)* 0 25 50 100 250 500 1000 C alib ratio n Standard ID (exam ple) X C m m ddyy-0 XCmmddyy-1 X C m m ddyy-2 X C m m ddyy-3 X C m m ddyy-4 X C m m ddyy-5 X Cm m ddw -6 * T he e x tra cted co n c en tra tio n o f th e ca lib ratio n stan d ard is eq u a l to 8* its initial concentration, d u e to th e concentration o f the standard during the extraction (SPE). X C " extracted calibration standard. Page 4 o P Page 19 o f 65 MPI Research, Inc. Page 84 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VQ00I780 A N A L Y T IC A L M E T H O D M ethod o f A nalysis fo r the D eterm ination ofP erflu o ro o ctan o ic A cid (P F O A ) in W ater by LC /M S/M S 9 .2 .3 9 .2 .4 9 .2 .5 A zero standard solution (reagent blank) m u st be prepared w ith each set o f standards extracted. S tore all extracted calibration standards in 15-m L polypropylene tubes at 2C to 6C , up to tw o w eeks. A lternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 B atch S et U p 10.1 10.2 E ach b atc h o f sam p les ex tra cted (ty p ica lly 2 0 o r less) m u st in clu d e at least o n e reagent control (m ethod b lank usin g H P L C w ater) and tw o reagent co n tro ls fo rtified at k n o w n co n c en tra tio n s (lab co n tro l sp ik e) to verify procedural recovery for the batch. R equirem ents fo r field an d lab oratory d u p licates an d sp ik es w ill be specified in the q uality assurance plan for this project. 11.0 Sam ple Extraction 11.1 1 1 .2 11.3 1 1 .4 11.5 M easure 40 m L o f sam ple o r a portion o f sam ple d iluted to 4 0 m L w ith w ater in to 5 0 m L p oly p ro p y len e ce n trifiig e tu b es (fo rtify as n ee d ed , rep lace lid and m ix w ell). C o n d itio n th e C ]i S P E c a rtrid g es (1 g , 6 m L ) b y p a ssin g 10 m L m ethanol follow ed b y 5 m L o f H P L C w ater ( - 2 drop/sec). D o not let colum n run dry L o ad sam ple o n co n d itio n ed C | i S P E cartridge. D isca rd eluate. Elute w ith ~5 m L 100% m ethanol. C ollect 5 m L o f eluate into graduated 15 m L p olypropylene centrifiige tu b es (fin al v o lu m e 8 5 m L ). A nalyze sam ples using electrospray L C /M S/M S. 12.0 C hrom atography 12.1 1 2 .2 12.3 1 2 .4 Inject the sam e am ount o f each standard, sam p le an d fo rtified sam ple into the L C /M S /M S system . A c a lib ra tio n sta n d a rd m u s t p re c e d e a n d follow all analyzed sam ples. S tan d ard s o f P FO A co rresp o n d in g to at least fiv e o r m o re co n c en tra tio n levels m ust b e included in an analytical set. A n en tire set o f ex tracted ca lib ratio n sta n d a rd s m u st b e inclu d ed at the beginning and at the end o f a sam ple set. E xtracted standards m ust he interspersed betw een every 5-10 sam ples. A s an alternative, an entire set o f extracted calibration standards m ay be injected at the beginning o f a set follow ed b y extracted calibration standards interspersed every 5*10 sam ples (to ac count fo r a second set o f ex tracted stan d ard s). In eith er case, extracted calibration standards m u st b e the first an d last in jectio n in a sam p le set. U se linear standard curves for quantitation. L in ear standard curves are generated for the analyte b y linear regression u sin g 1/x w eighting o f peak area Page 5 of 7 Page 20 o f 65 MPI Research, Inc. Page 85 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research M ethod Number V0001780 ________________________________ A N A L Y T I C A L M E T H O D M ethod o f A nalysis fo r th e D eterm ination o f P erfluorooctanoic A d d (P F O A ) in W ater by LC /M S/M S 12.5 v ersus ca libration standard con cen tratio n u sin g M assL y n x 3.3 (o r equivalent) softw are system . S am ple response sh ould n o t ex ceed stan d ard resp o n ses. A n y sam ples that exceed standard responses should b e further diluted and reanalyzed. 13.0 A c cep tan ce C riteria 13.1 13.2 13.3 13.4 13.6 C hrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 am u. T h e 413 am u p aren t co rresp o n d s to the P F O A an io n , w hile the daughter ion (369 am u) represents the loss o f carbon dioxide. M ethod blanks m ust not contain P FO A at levels greater than the LOQ . If a blank contains P FO A at levels greater than 50 ng/L , then a new blank sam ple m ust be obtained and the entire set m ust be re-extracted. R ecoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f th e ir k n o w n values. I f a co n tro l sp ik e falls o u tsid e th e ac ce p ta b le lim its, the entire set o f sam ples should be re-extracted. A ny m atrix spike outside 70 13 0 % sh o u ld b e ev a lu a te d b y th e a n a ly st to d e te rm in e i f re -e x tra c tio n is w arranted. A ny calibration standard found to be a statistical outlier b y using the Huge E rror Test, m ay b e excluded from the calculation o f the calibration curve. H ow ever, the total num ber o f extracted calibration standards that could be excluded m ust not exceed 20% o f the total n um ber o f extracted standards in je c te d . T he correlation coefficient (R ) for calibration curves generated m ust be 0 .9 9 2 (R 2 0 .9 8 5 ). I f ca lib ratio n re su lts fa ll o u tsid e these lim its, then ap p ro p riate step s m ust b e taken to ad ju st in stru m en t o p eratio n , and (he standards o r the relevant set o f sam ples should b e reanalyzed. R eten tio n tim es b etw een stan d ard s an d sam p les m u st not drift m o re than 4 % w ithin an analytical run. I f reten tio n tim e d rift exceed s th is lim it w ithin an analytical run then the set m ust be reanalyzed. 1 4 .0 C alculations 14.1 U se th e fo llo w in g e q u a tio n to c a lc u la te th e a m o u n t o f P F O A fo u n d (in ng/L , based on peak area) using the standard curve (linear regression param eters] generated b y the M ass Lynx softw are program : PFO A found (ng/L) - (Peak area - intercom ) x DF slope D F = factor b y w hich the final volum e w as diluted, if necessary. Page 6 of 7 Page 21 o f 65 MPI Research, Inc. Page 86 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project N o.: P0001131 Exygen Protocol Number: P0001131 Exygen Research M ethod N umber VOO0I78O | A N A LY TIC A L M E T H O D | M ethod o f A nalysis for the D eterm ination o f P eriluorooctanoic A cid (P F O A ) in W ater by LC /M S/M S 14.2 F o r sam p les fortified w ith k n o w n am o u n ts o f P F O A p rio r to extractio n , use the follow ing equation to calculate the p ercent recovery. Recovery (%) " [ total analyte found (ng/L) - analyte found in control (ng/L )] ^ ^ analyte added (ng/L) MPI Research, Inc. Pige 7 of 7 Page 22 o f 65 Page 87 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod N u m b er V 0001781 Method of Analysia for the Determination of Perfluorooctanolc Acid (PFOA) la Soli bv LC/MS/MS ' A nalytical T esting F acility: Exygen R esearch 3058 R esearch D rive S tate C ollege, P A 16801 A pproved By: T U . C J L _____ P aul C onnolly ' Technical Leader, L C -M S, Exygen R esearch __ I D ate D ate MPI Research, Inc. Total Pages: 7 Page 23 o f 65 Page 88 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygcn Protocol Number: P0001131 Exygea Research Method Number VOOO!781 I ANALYTICAL METHOD M ethod o f A nalysis fo r th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Soil by LC /M S/M S 1.0 S cope T his m ethod is to be em ployed for the isolation and quantitation o f perfluorooctanoic acid b y H ig h P erform ance L iquid C h ro m ato g rap h y co u p le d to a tandem M ass S pectrom trie D etecto r (L C /M S/M S ) in soil. 2.0 Safety 2.1 A lw a y s o b serv e safe la b o ra to ry p ra ctices. 2.2 C o nsult the appropriate M S D S b efore h an d lin g an y ch em ical for p ro p er safety precautions. 3.0 S am ple R equirem ent 3.1 A t least 1$ g o f test sa m p le fo r ex tractio n . 3.2 N o sam ple processing is n eeded fo r soil sam ples. 3.3 S am ples sto red refrig erated should b e allo w e d to eq u ilib rate to room tem perature. 3.4 A ll sam ples m ust b e thoroughly m ix ed b efo re bein g sam p led for extraction. 3.5 S am ple co llectio n procedures w ill be sp ec ified in th e sam p lin g plan for this p ro je c t. R eagents an d Standards 4 .1 W ater - H PLC grade 4.2 M ethanol - H PL C grade 4.3 A m m onium A cetate -A .C .S . R eagent G rade 4.4 Perfluorooctanoic A cid - S ig m a-A ldrich 5.0 Instrum ent and Equipm ent 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5 .1 0 5.11 5 .1 2 5 .1 3 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5-200 pL connected to a tandem M ass S pectrom eter (L C /M S/M S). A device to collect raw d ata for peak integration and quantitation. A nalytical balance capable o fread in g to 0.00001 g. 50 m L disposable polypropylene centrifuge tubes. 15 m L disposable polypropylene centrifuge tubes. D isposable m icropipets (50-100uL, 100-200uL). 125-m L L D P E narrow -m outh bottles. 2 m L clear H PL C vial kit. D isposable pipettes. A utopipettes (100-1000 p L and 10-100 p L ), w ith disposable tips. W aters Sep P ak V ac 6 cc ( Ig ) tC 18 S P E cartridges. S PE vacuum m anifold. U ltrasonic bath. Page 2 or 7 Page 24 o f 65 MPI Research, Inc. Page 89 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method N umber V00 0 17 8 1 ]_______________________________ A N A L Y T I C A L M E T H O D ________________________________ M ethod o f A n aly sis fo r th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Soil by LC /M S/M S 5.14 W rist-action shaker. 5 .15 C entrifuge cap ab le o f sp in n in g 50 m L p o ly p ro p y len e tu b es al 5000 rpm . 6.0 C hrom atographic System 6.1 A naly tical C olum n: F lu o p h ase R P (K ey sto n e S cien tific), 2.1 m m x 50 m m . 5p (P/N : 82505-052130) 6.2 T em perature: 30C 6.3 M o b ile P hase (A ) : 2 m M A m m onium A cetate in W ater 6.4 M obile Phase (B ) : M ethanol 6.5 G radient P rogram : T im e (m in) 0.0 1.0 8.0 2 0 .0 22.5 %A 65 65 25 25 65 %B 35 35 75 75 35 F low R ate (m L /m in) 0.3 0.3 0.3 0.3 0.3 6 .6 In jectio n V olum e: 15 p L (can b e in creased to a s m u c h a s 5 0 p L ). 6.7 Q uantitation: P eak A rea - external standard calib ratio n curve. 6.8 R un Tim e: ~ 23 m inutes. T h e above cond itio n s are in tended as a g u id e a n d m ay b e c h a n g ed in o rd e r to optim ize the H P L C system . 7.0 M S/M S System 7.1 M o d e : E le c tr o s p ra y N e g a tiv e M R M m o d e , m o n ito r in g 4 1 3 - * 3 6 9 m /z for PFOA. T h e a b o v e c o n d itio n s a r e in te n d e d a s a g u id e a n d m a y b e c h a n g e d in order lo optim ize the M SM S system . 8.0 P reparation o f Solutions 8.1 M o b ile P hase 8.1.1 2 m M am m onium acetate in w a te r is p rep ared b y ad d in g 0.154 g of am m onium acetate to 1000 m L o f w ater. A lternate volum es m ay b e prepared. Page 3 o f 7 Page 25 o f 65 MPI Research, Inc. Page 90 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygeo Research Method Number VOOI78I A N A LY TIC A L M E T H O D M ethod o f A nalysis fo r th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Soil by LC /M S/M S 9.0 S tandard Preparation 9.1 S tan d ard S to c k /F o rtific atio n S olu tio n 9.1.1 P repare a stock so lu tio n o f - 1 0 0 p g /ra L o f P F O A b y w eighing 10 m g o f analytical standard (corrected fo r p u rity ) and d ilu te to 100 m L w ith m ethanol in a 125-m L L D P E bottle. 9.1 .2 A 10 p g /m L fo rtific a tio n so lu tio n o f P F O A is p re p a re d b y b rin g in g 10 m L o f th e 100 p g /m L so lu tio n to a fin al v o lu m e o f 100 w ith m ethanol in a 125 m L L D P E bottle. 9 .1 .3 A 1.0 p g /m L fo r tific a tio n s o lu tio n o f P F O A is p re p a re d b y b rin g in g 1U m L o f th e 10 p g /m L so lu tio n to a fin al v o lu m e o f 100 w ith m ethanol in a 125 m L L D P E b ottle. 9 .1 .4 A 0.1 p g /m L f o r tific a tio n s o lu tio n o f P F O A is p re p a re d b y b r in g in g 10 m L o f {he 1.0 p g /m L so lu tio n to a fin al v o lu m e o f 100 w ith m ethanol in a 125 m L L D P E bottle. 9.1 .5 A 0.01 p g /m L fo rtificatio n so lu tio n o f P F O A is p re p are d b y brin g in g 10 m L o f th e 0.1 p g /m L so lu tio n to a final v o lu m e o f 100 w ith m ethanol in a 125 m L L D P E b o ttle. 9.1 .6 T h e stock and fo rtificatio n so lu tio n s are to b e sto re d in a refrig erato r at approxim ately 4*C and are stable fo r a m axim um period o f 6 m onths from the d ate o f preparation. 9.2 S tandard C alibration Solutions 9 .2 .1 9 .2 .2 L C /M S /M S ca lib ratio n stan d ard s are p re p a re d in H P L C w ater. T he calibration standards are processed through the extraction procedure, identical to sam ples. The follow ing is a typical exam ple: additional concentrations m ay be prepared as needed. Final C o n cen tratio n o f Fortification Solution (ppb) 0 10 10 10 100 100 100 F o rtific a tio n V olum e (M 0 100 200 400 100 200 400 Volume of Fortified Control Sample (mL) 40 40 40 40 40 40 40 Concentration o f C alibration Standard (opt)* 0 25 50 100 250 500 1000 C alib ratio n Standard ID (exam ple) X C m m ddyy-0 X C m m ddyy-l X C m m ddyy-2 X C m m ddyy-3 X C m m ddyy-4 X C m m ddyy-5 X C m m ddW '6 * T h e ex tra cted co n c en tra tio n o f th e ca lib ratio n stan d ard is eq u a l to 8x its initial concentration, d u e to the concentration o f the standard during the extraction (SPE). X C " extracted calibration standard. Page 4 o f 7 Page 26 o f 65 MPI Research, Inc. Page 91 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research M ethod Number V0001781 1 ....... A N A L Y T I C A L M E T H O D M ethod o f A nalysis fo r th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Soil by LC /M S/M S 9 .2 .3 9 .2 .4 9 .2 .5 A zero standard solution (reagent blank) m ust be prepared w ith each set o f standards extracted. S tore all extracted calibration stan d ard s in 15-m L poly p ro p y len e lubes a t 2C to 6 C , u p to tw o w e e k s. A ltern ate volum es an d co n cen tratio n s o f stan d ard s m ay be prepared as needed. 10.0 B atch Set U p 10.1 10.2 E ach batch o f sam p les ex tra cted (ty p ica lly 2 0 o r less) m u st inclu d e at least o n e reagent co ntrol (m eth o d b lank usin g 5 m L o f m eth an o l) an d tw o reagent co n tro ls fortified at k now n co n cen tratio n s (lab co n tro l spike) to verify procedural recovery for the batch. R equirem ents fo r field a n d lab o rato ry d u p licates an d sp ik es w ill be specified in the quality assurance p lan for this project. 11.0 Sam ple Extraction 11.1 1 12 11.3 1 1 .4 11.5 1 1 .6 1 1 .7 11.8 11.9 W e ig h 5 g o f s a m p le in to SO m L p o ly p r o p y le n e c e n tr if u g e tu b e s (f o rtif y as n eeded, rep lace lid and m ix w ell). A d d 5 m L o f m eth an o l and sh ak e o n a w rist ac tio n sh a k e r fo r - 1 5 m inutes. T ransfer the tubes to an u ltrasonic b ath and sonicate f o r-1 5 m inutes. B ring the volum e up to 40 m L w ith w ater in th e 50 m L polypropylene centrifbge tube. C entrifuge for - 1 0 m inutes at -3 0 0 0 rpm . C o n d itio n th e C is S P E ca rtrid g es (1 g , 6 m L ) b y p a s s in g 10 m L m eth an o l follow ed by 5 m L o f H PL C w ater ( - 2 drop/sec). D o not let colum n run dry Load (decant) the sam ple on the conditioned C n SPE cartridge. D iscard eluate. E lute w ith - 5 m L 100% m ethanol. C o llect S m L o f elu ate into graduated 15 m L p oly p ro p y len e centrifU ge tu b es (final vo lu m e - 5 m L ). A nalyze sam ples using electrospray L C /M S/M S. 12.0 C h rom atography 12.1 1 2 .2 12.3 Inject th e sam e am ount o f each standard, sam ple and fo rtified sam ple into the L C /M S /M S sy stem . A c a lib ratio n sta n d ard m u s t p re c e d e a n d follow all analyzed sam ples. S tandards o f P FO A corresp o n d in g to at least five o r m o re co n cen tratio n levels m ust b e included in an analytical set. . A n entire sec o f extracted calib ratio n stan d ard s m u st b e in cluded at the beginning and at the end o f a sam ple set. E xtracted standards m ust be interspersed b etw een every 5-10 sam ples. A s an alternative, an entire set o f Page 5 of 7 Page 27 o f 65 MPI Research, Inc. Page 92 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research M ethod Number VOOO1781 A N A LY TIC A L M E T H O D M ethod o f A n aly sis fo r th e D eterm in atio n o f P erflu o ro o ctan o ic A c id (P F O A ) in Soil by LC /M S/M S ] 12.4 12.5 extracted calibration standards m ay be injected at th e beginning o f a set follow ed b y extracted calibration standards interspersed ev ery 5*10 sam ples (to account fo r a second set o f extracted standards). In either case, extracted ca libration standards m u st be the first and last in jectio n in a sam ple set. U se linear standard curves for quantitation. L inear standard curves are gen erated fo r th e an aly te b y lin ear reg ressio n u sin g 1/x w eig h tin g o f peak area versu s calibration standard co n cen tratio n u sin g M assL y n x 3.3 (o r eq u iv a le n t> softw are system . S am ple response should not exceed stan d ard resp o n ses. A n y sam p les that exceed standard responses should be further d iluted and reanalyzed. 13.0 A cceptance C riteria 13.1 13.2 13.3 13.4 13.5 13.6 C hrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 am u. T h e 4 1 3 am u p aren t co rresp o n d s to th e P F O A anion, w hile the daughter ion (369 am u) represents the loss o f carbon dioxide. M ethod blanks m ust not contain PFO A at levels greater th an the LO Q . If a blank contains P FO A at levels greater than 50 ng/L , then a new blank sam ple m ust b e obtained and the entire set m ust b e re-extracted. R ecoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f their know n values. If a control spike falls ou tsid e the acceptable lim its, the entire set o f sam ples should be re-extracted. A n y m atrix spike outside 70 130% sh o u ld b e e v a lu a te d b y th e a n a ly st to d e te rm in e i f re -e x tra c tio n is w arranted. A ny calibration standard found to be a statistical o u tlier b y using the Huge E rror T est, m ay be excluded from the calculation o f the calibration curve. H ow ever, the total n um ber o f extracted calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f extracted standards in je c te d . T h e co rrelation coefficien t (R ) fo r calib ratio n cu rv es g enerated m ust be 2 0 .9 9 2 (R J 20 .9 8 5 ). I f ca lib ratio n re su lts fall o u tsid e th ese lim its, then ap p ro p riate step s m u st b e tak e n to ad ju st in stru m en t o p eratio n , and the standards or the relevant set o f sam ples should be reanalyzed. R eten tio n tim es betw een standards an d sam p les m u st n o t drift m o re than 4 % w ithin a n analytical run. If retention tim e d rift exceeds this lim it w ithin an analytical run then the set m ust b e reanalyzed. Page 6 o f 7 Page 28 o f 65 MPI Research, Inc. Page 93 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygco Research Method Number VOOO1781 A N A LY TIC A L M E T H O D M ethod o f A nalysis fo r the D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Soil bv LC /M S/M S ' 14.0 C alcu latio n s 14.1 U se th e fo llo w in g e q u a tio n to c a lc u la te th e a m o u n t o f P F O A fo u n d (in n g/L , based on peak area) using the standard curve (linear regression param eters) generated by the M ass Lynx softw are program : PFO A found (ng/L) - (Peak m ca - intercept) * D F slope D F - factor b y w hich th e B aal volum e w as diluted, if necessary. 14.2 F o r sam p les fortified w ith k n o w n am o u n ts o f P F O A p rio r to extractio n , use the follow ing equation to calculate the percent recovery. R ecovery (% ) [ total analyte found (ng/L) - analyte found in control (ng/L )] ^ ^ analyte added (ng/L) 14.3 U se th e fo llo w in g e q u a tio n to c o n v e rt th e am o u n t o f P F O A fo u n d in ng/L to ng/g (ppb). . P F O A fo u n d (p p b ) " (P F O A fo u n d (n a /L ) x v o lu m e e x tra c te d (0.04L11 sam ple w eight (5 g) 14.4 U se th e fo llo w in g e q u a tio n to ca lc u la te th e a m o u n t o f P F O A fou n d in ppb based o n d ry w eight. P FO A fo u n d (p p b ) d ry w e ig h t P F O A fo u n d (p p b ) x [1 0 0 % / total solids(% )] Page 7 o f? Page 29 o f 65 MPI Research, Inc. Page 94 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod N u m b er V 0001782 M e th o d o f A n a ly s is f o r th e D e te r m in a tio n o f P e r f lu o r o o c ta n o ic A c id ( P F O A ) in Sedim ent by L C /M S/M S A nalytical T esting Facility: Exygen R esearch 3058 R esearch D rive S tate C ollege, P A 16801 Approved By: - A lA CJtl.___ P aul C onnolly I Technical Leader, LC -M S, Exygen R esearch 4L>jfl? / k j y / i olohhin F la h e rty f Viicc<e P r e s id e n t, O p e ra tio n s , E x y g e n R e s e a rc h __10Wf D ate D ate MPI Research, Inc Total Pages: 7 Page 30 o f 65 Page 95 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI782 I A N A LY TIC A L M E T H O D ' M e th o d o f A n a ly s ts fo r th e D e te rm in a tio n o f P e r f lu o r o o c ta n o ic A c id (PFOA) in Sediment by LC/MS/MS 1.0 S cope T his m ethod is to b e em ployed fo r the isolation and quantitation o f perfluorooctanoic acid b y H igh Perform ance L iquid C hrom atography coupled to a tandem M ass S pectrom etric D etector (L C /M S/M S) in sedim ent. 2.0 Safety 2.1 A lw a y s o b se rv e s afe la b o ra to ry p ra ctices. 2.2 C onsult th e appropriate M SD S b efore h andling an y ch em ical for p ro p er safely precautions. 3.0 S am ple R equirem ent 3.1 A t least 3 0 g o f te st sam p le fo r ex tra ctio n . 3.2 N o sam ple processing is needed for sed im en t sam ples. 3.3 S am p les sto red re frig erated sh o u ld b e allo w e d to eq u ilib ra te io room tem perature. 3.4 A ll sam ples m ust be thoroughly m ixed before bein g sam pled for extraction. 3.5 S am p le co llectio n p ro c ed u re s w ill b e sp ec ified in th e sam p lin g plan for this p ro je c t 4.0 R eagents a nd S tandards 4.1 Water - HPLC grade 4.2 M ethanol - H P L C grade 4.3 A cetic A cid - R eagent grade 4.4 A m m onium A cetate - A .C .S . R eagent G rade 4.5 P erfluorooctanoic A cid - S igm a-A ldrich 5.0 In strum ent and E quipm ent 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5 .1 0 5.11 5 .1 2 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e in jector capable o f injecting 5-200 p L connected to a tandem M ass S pectrom eter (L C /M S/M S). A device to collect raw data for peak integration and quantitation. A n a ly tic a l b a la n c e c a p a b le o f re a d in g to 0.0 0 0 0 1 g. 50 m L disposable polypropylene centriftige tubes. 15 m L d isp o sab le poly p ro p y len e ce n trifu g e tubes. D isposable m icropipets (50-100uL, 100-200uL). 125-m L L D PE narrow -m outh bottles. 2 m L clear H P L C viol kit. D isposable pipettes. A utopipettes (100-1000 p L and 10-100 p L ), w ith disposable tips. W aters Sep Pak V ac 6 cc (Ig ) tC18 S P E cartridges. S PE vacuum m anifold. Page 2 o f 7 Page 31 o f 65 MPI Research, Inc. Page 96 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method N umber VOOOI782 1 ANALYTICAL METHOD j M e th o d o f A n a ly s is fo r t h e D e te rm in a tio n o f P e r i l u o r o o c ta n o ic A c id (P F O A ) in S e d im e n t by LC /M S/M S 5 .1 3 5 .1 4 5.15 V ortcxer. W rist-action shaker. C entrifuge capable o f spinning 5 0 znL p olypropylene tubes at 3000 rpm. 6.0 C hrom atographic System 6.1 A nalytical C olum n: F lu o p h ase R P (K ey sto n e S cien tific), 2 .1 m m x 50 m m . 5p (P/N : 82505-052130) 6.2 Tem perature: 30C 6.3 M obile P hase ( A ) : 2 m M A m m onium A cetate in W ater M obile P hase (B ): M ethanol G radient Program : T im e (m in) 0.0 1.0 8.0 2 0 .0 22.5 %A 65 65 25 25 65 %B 35 35 75 75 35 F low R ate im L /m in) 0.3 0.3 0.3 0.3 0.3 6.6 Injection V olum e: 15 p L (can be increased to as m uch as 50 pL). 6.7 Q uantitation: P eak A rea - external standard calibration curve. 6.8 R u n T im e : - 2 3 m inutes. T he above conditions are intended as a guide an d m ay b e changed in order to optim ize the H PLC system . 7.0 M S /M S System 7.1 M o d e: E le c tro sp ray N e g a tiv e M R M m o d e, m o n ito rin g 4 1 3 3 6 9 m /z for PFOA. T h e a b o v e c o n d itio n s a re in ten d ed as a g u id e an d m a y b e c h a n g e d in o rd e r to optim ize the M SM S system . 8.0 Preparation o f Solutions 8.1 M o b ile P h ase 8.1.1 2 m M am m o n iu m acetate in w a te r is p re p are d b y ad d in g 0.1 5 4 g o f am m onium acetate to 1000 m L o f w ater. Page 3 of 7 Page 32 o f 65 MPI Research, Inc. Page 97 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygea Reiearch Method Number VOOI782 | ANALYTICAL METHOD | M eth o d o f A n a ly sis fo r th e D e te rm in atio n o f P erflu o ro o c ta n o ic A c id (P F O A ) in S ed im en t bv LC /M S/M S ' 8.2 E xtraction Solutions 8 .2 .1 1% a c e tic a c id in w a te r is p re p a re d b y a d d in g 1 0 m L o f a c e tic a c id to 1000 m L o f w ater. A lternate volum es m ay b e prepared. 9.0 S tandard Preparation 9.1 S tan d ard S to ck /F o rtificatio n S olu tio n 9 .1 .1 P re p a re a s to c k s o lu tio n o f --1 0 0 p g / c n L o f P F O A b y w e ig h in g 10 m g o f analytical standard (corrected for p u rity ) an d d ilu te to 100 m L w ith m ethanol in a 125-m L L D P E bottle. 9.1.2 A 10 pg/m L fortification solution o f P FO A is prepared b y bringing to m L o f th e 100 p g /m L s o lu tio n to a fin a l v o lu m e o f 100 w ith m ethanol in a 125 m L L D P E bottle. 9.1 .3 A 1.0 p g /m L fo rtific a tio n so lu tio n o f P F O A is p re p a re d b y b rin g in g 10 m L o f th e 10 p g /m L so lu tio n to a fin a l v o lu m e o f 100 w ith m ethanol in a 125 m L L D P E bottle. 9 .1 .4 A 0.1 p g /m L fo rtific a tio n s o lu tio n o f P F O A ia p re p a re d b y b rin g in g 10 m L o f th e 1.0 p g /m L s o lu tio n to a fin a l v o lu m e o f 1 0 0 w ith m ethanol in a 125 m L L D P E b ottle. 9.1.5 A 0.01 p g 'm L fortificatio n so lu tio n o f P F O A is p re p are d b y b ringing 10 m L o f th e 0.1 p g /m L so lu tio n to a fin al v o lu m e o f 100 w ith m ethanol in a 125 m L L D P E b ottle. 9.1.6 T h e stock an d fo rtification solu tio n s are to b e stored in a refrig erato r at approxim ately 4C and are stable for a m axim um period o f 6 m onths from the date o f preparation. 9.2 S tandard C alib ratio n S olutions 9.2.1 9 .2 .2 L C /M S/M S calib ratio n standards are p re p are d in m eth an o l via dilution o f th e 0.1 p g /m L fo rtification so lu tio n . T he follow ing is a typical exam ple: additional concentrations m ay be Concentration of Fortification Solution (ns/raL) 100 100 100 10 5 2 Volume (mL) 10 5 2 10 10 10 Diluted to (mL) 100 100 100 100 100 100 Final Concentration (ng/mL) 10.0 5.0 2.0 1.0 0.5 0.2 Page 4 of 7 Page 33 o f 65 MPI Research, Inc. Page 98 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001782 | A N A LY TIC A L M E T H O D ) M ethod o f A n aly sis fo r th e D etefm in atio n o f P erflu o ro o ctan o ic A c id (P F O A ) in S edim ent by LC /M S/M S 9 .2 .3 9 .2 .4 S to re all calib ratio n stan d ard s in 125-raL L D P E narro w -m o u th bottles at 2C to 6C , up to six m onths. A lternate v o lu m es and con cen tratio n s o f stan d ard s m ay b e prepared as needed. 10.0 B atch S et U p 10.1 1 0 .2 E ach b atc h o f sam p les ex tracted (ty p ica lly 2 0 o r less) m u st in clu d e at least knowno n e u n tre a te d c o n tro l a n d tw o u n tre a te d c o n tr o ls fo rtifie d a t concentrations (lab control spike) to verify p rocedural recovery for the batch. R equirem ents for field and laboratory duplicates and spikes w ill be specified in the q uality assurance p lan for thia project. 11.0 S am ple E xtraction 11.1 W e ig h 5 g o f s a m p le in to 5 0 m L p o ly p ro p y le n e c e n trifu g e tu b e s (fo rtify as 11.2 needed, A dd 35 for -6 0 mmr eipnLluaotcefesl.%l i d aancedtmiciaxcwide,lcl)a.p, vortexandshakeonawristactionshaker 11.3 by mL 6mL) by columnmruenthdarnyol11.4 C entrifuge the tubes at -3 0 0 0 rpm for - 2 0 m inutes. C o n d itio n th e C n S P E cartrid g es (1 g, passing 10 m L follow ed 20 o f H PL C w ater ( - 2 drop/sec). D o not let 11.5 L o ad (d e can t) th e sam p le o n th e co n d itio n ed C ig S P E ca rtrid g e. D iscard eluate. 11.6 A dd 20 m L o f m ethanol to th e sed im en t left in the bo tto m o f the 50 m L centrifuge tube. C ap, vortex and shake o n a w rist action shaker for -3 0 m inutes. 11.7 C entrifuge th e tubes at -3 0 0 0 rp m fo r - 2 0 m in u tes. L1.8 D e can t th e m e th an o l o n to th e sa m e S P E ca rtrid g e . C o lle c t th e eluate. 11.9 W a sh th e c o lu m n w ith 4 m L o f m eth a n o l. C o lle c t th e e lu a te a n d ad d it to the elu a te co llec ted in step 11.8. 11.1 0 C o n d itio n a sec o n d C S P E c a rtrid g e (1 g , 6 m L ) b y p a s s in g 10 m L m ethanol follow ed b y 2 0 m L o f H PL C w ater ( - 2 drop/sec). D o not let colum n run dry 11.11 A dd th e m eth an o l to - 2 0 0 m L o f w a te r an d lo ad o n th e sec o n d conditioned SPE cartridge. 11.12 E lute w ith -S m L 100% m eth an o l. C o lle ct 5 m L o f elu ate into graduated 15 m L p olypropylene centrifuge tubes (final volum e 5 m L). 11.13 A nalyze sam ples usin g electrospray L C /M S /M S . Page S o f 7 Page 34 o f 65 MPI Research, Inc. Page 99 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Rexesrch Method N um ber V 0001762 | ANALYTICAL METHOD | M ethod o f A nalysis fo rth e D e te n m n a tio n o fP e rflu o ro o c ta n o ic A cid (P F O A ) in S edim em bv LC /M S/M S ' 12.0 C hro m ato g rap h y 12.1 12.2 12.3 12.4 12.5 Inject th e sam e am ount o f each standard, sam ple an d fo rtified sam ple into the L C /M S /M S sy stem . A ca lib ra tio n sta n d ard m u st p re c e d e an d fo llo w all analyzed sam ples. Standards o f P FO A corresp o n d in g to at least fiv e o r m o re co n cen tratio n levels m ust b e included in an analytical set. A n entire set o f extracted calib ratio n stan d ard s m u st b e included at the beginning and at the end o f a sam ple set. S tandards m ust be interspersed betw een every 5 -1 0 sam ples. A s an altern ativ e, an en tire set o f calibration stan d ard s m ay b e injected at th e b eg in n in g o f a set follow ed b y calibration standards interspersed every 5-10 sam ples (to account for a second set o f standards). In eith er ca se, ca lib ratio n stan d ard s m u st b e th e first and last injection in a sam ple set. U s e lin e a r s ta n d a r d c u r v e s f o r q u a n tita tio n . Linear s ta n d a r d c u r v e s arc generated for the analyte b y linear regression u sin g 1/x w eighting o f peak area versus calibration standard co n cen tratio n usin g M assL y n x 3.3 (o r equivalent) softw are system . Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should b e A irther d iluted and reanalyzed. 1 3 .0 A cceptance C riteria 13.1 13.2 1 3 .3 1 3 .4 13.5 C hrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 am u. T h e 413 am u p aren t co rresp o n d s to the P F O A anion, w hile the daughter ion (369 am u) represents th e lo ss o f carbon dioxide. M ethod blanks m ust no t contain P FO A at levels g reater than the LOQ . If a blank contains PFO A at levels greater than 0.2 ng/m L , then a new blank sam ple m ust b e obtained and the entire set m ust be re-extracted. R ecoveries o f control spikes and m atrix spikes m ust b e betw een 70-130% o f th eir k n o w n values. I f a co n tro l sp ik e falls o u tsid e th e ac cep tab le lim its, the entire set o f sam ples should be re-extracted. A ny m atrix spike outside 70 130% sh o u ld b e e v a lu a te d b y th e a n a ly st to d e te rm in e i f rc -e x tra c tio n is w arranted. A ny calibration standard found to b e a statistical outlier b y using the Huge E rror Test, m ay be excluded from the calculation o f the calibration curve. H ow ever, the total num ber o f extracted calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f extracted standards in je c te d . T he correlation coefficient (R ) fo r calibration curves generated m ust be 0 .9 9 2 (R 3 0 .9 8 5 ). I f ca libration re su lts fall o u tsid e these lim its, then appropriate step s m u st b e tak en to ad ju st in stru m en t o p eratio n , and the standards o r the relevant set o f sam ples should be reanalyzed. P#ge 6 o f 7 Page 35 o f 65 MPI Research, Inc. Page 100 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001782 | ANALYTICAL METHOD | M ethod o f A nalysis for the D etennination o f P erfluorooctanoic A cid (P F O A ) in Sedim ent bv L C /M S/M S * 1 3 .6 R etention tim es b etw een standards an d sam p les m ust not drift m ore than 4 % w ithin an analytical run. If retention tim e d rift exceeds this lim it within an analytical run then the set m ust b e reanalyzed. 1 4 .0 C alculations 14.1 U se th e fo llo w in g e q u a tio n to c a lc u la te th e a m o u n t o f P F O A fo u n d (in ng/m L . baaed on peak area) using the standard curve (linear regression param eters) generated b y the M ass L ynx softw are program : PFO A found (ng/m L) = (Peak a re a intercept) x DF slope O F = factor b y w hich the final volum e w as diluted, i f necessary. 14.2 F o r sam p les fortified w ith k n o w n am o u n ts o f P F O A p rio r to ex tractio n , use the follow ing equation to calculate the percent recovery. R ecovery (% ) - [ total analyte found (ng/m L ) - analyte found in c ontrol (ng /m L )| ^ ^ analyte added (ng/m L) 14.3 U se th e fo llo w in g e q u a tio n to co n v e rt th e am o u n t o f P F O A fo u n d in n g/m L to ng/g (ppb). P FO A found (p p b ) " (PFO A fo u n d (n g /m L ) x fin al v o lu m e (5 m U l sam ple w eight (5 g) 14.4 U se th e follo w in g eq u atio n ( if n ecessary ) to c a lc u la te th e am o u n t o f P FO A found in p pb based o n d ry w eight. P FO A fo u n d (ppb) d ry w eight P F O A found (p p b ) x [100% / total $olids(% j] Plgc 7 of 7 Page 36 o f 65 MPI Research, Inc. Page 101 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod N um ber: V 0001783 Method of Analysis for the Dtermination of Perfluorooctanolc Acid (PFOA) in Fish and Clams by LC/MS/MS A nalytical T esting Facility: Exygen R esearch 3058 R esearch D rive State C ollege, PA 16801 Approved By: P aul C onnolly T echnical L eader, LC -M S, Exygen R esearch a Jofohhnn F la h e rty / V icee P re s id e n t, O p e ra tio n s , E x y g e n R e s e a rc h D ate D ate MPI Research, Inc. Total Pages: 8 Page 37 o f 65 Page 102 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research M ethod N umber VOOO1783 ]______________________ATNa LYTICAL m e t h o d ______________________ M ethod o f A nalysis fo r th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Fish and C lam s b y LC /M S/M S 1.0 S co p e T his m ethod is to be em ployed for the isolation and quantitation o f perfluorooctanoic a c id b y H ig h P e rf o rm a n c e L iq u id C h r o m a to g r a p h y c o u p le d to a ta n d e m Mass Spectrom etric D etector (L C /M S/M S ) in fish and clam s. 2.0 Safety 2.1 A lw a y s o b serv e safe lab o ra to ry p ractices. 2.2 C onsult the appropriate M SD S b efo re h an d lin g an y ch em ical for proper safety precautions. 3.0 S am ple R equirem ent 3.1 A t least 2 0 g o f te st sa m p le fo r ex tractio n . 3.2 S am p les sh ould b e p ro cessed b efo re ex tractio n . P la ce the frozen sam ple in a food p ro cesso r an d ho m o g en ize w ith d ry ice. P la ce the sam p les in containers and leave open in frozen storage overnight to allow for carbon dioxide sublim ation. Seal and place th e sam ples in frozen storage until tim e o f analysis. 3.3 S am ple co llectio n procedures w ill b e specified in th e sam p lin g plan fo r this p ro je c t 4 .0 R eagents an d S tandards 4.1 4.2 4.3 4 .4 4.3 4 .6 4 .7 4 .8 4 .9 4 .1 0 4.11 4 .1 2 4 .1 3 W ater - H PLC grade A cetonitrile - H PL C grade C arbon (120-400 m e sh )-R e a g e n t grade M ethanol - H PLC grade S ilica gel (60-200 m esh) - R eagent grade F loruil (60-100 m esh) - Reagent grade Superclean L C -N H j - R eagent grade 1-O ctan o l - H P L C g ra d e L-A scorbic acid - R eagent grade D im ethyldichlorosilane - R eagent grade Toluene - R eagent grade A m m onium A cetate -A .C .S . R eagent G rade Perfluorooctanoic A cid - Sigm a-A ldrich 5.0 Instrum ent and E quipm ent ' 5.1 A h ig h p erfo rm an c e liq u id ch ro m a to g ra p h c a p a b le o f p u m p in g up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5-200 p L connected to a tandem M ass S pectrom eter (L C /M S/M S ). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 A nalytical balance cap ab le o f read in g to 0.00001 g. Page 2 of 8 Page 38 o f 65 MPI Research, Inc. Page 103 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygcn Protocol Number: P0001131 Exygca Research Method Number V0001783 I A N A L Y T IC A L M E T H O D M ethod o f A nalysis fo r th e D eterm ination o f P erflu o ro o ctan o ic A cid (P F O A ) in Fish and C lam s by LC /M S/M S 5.4 5.5 5.6 5.7 5.8 5.9 5 .1 0 5.11 5.12 5 .1 3 5 .1 4 5 .1 5 5 .1 6 R otary evaporator. Tissum izer. 125 m L p ear-shaped flasks. 50 m L disposable polypropylene centrifuge tubes. 15 m L disposable polypropylene centrifuge tubes. D isposable m icropipets (50*100uL, 100-200uL). 125-m L L D PE narrow -m outh bottles, 2 m L cle ar H PL C vial kit. D isposable pipettes. A utopipettes (100*1000 p L and 10-100 p L ), w ith d isposable tips. SPE tubes (20m L) (S upelco cat. no. N 057I77). W rist action shaker. C entrifuge cap ab le o f sp in n in g 5 0 m L p o ly p ro p y len e tu b es at 2000 rpm . 6.0 C hrom atographic System 6.1 A n a ly tic a l C o lu m n : F lu o p h a s e R P ( K e y s to n e S c ie n tif ic ) , 2 .1 m m x 5 0 m m . 5(.i (P/N : 82505-052130) 6.2 Tem perature: 30C 6.3 M obile Phase (A ) : 2 raM A m m onium A cetate in W ater 6.4 M obile Phase (B ) : M ethanol 6.5 G radient Program : T im ; (min) 0 .0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 %B F low R ate (m L/m in) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection V olum e: 15 p L (can b e increased to as m uch as 5 0 nL ). 6.7 Q uantitation: P eak A r e a - e x te rn a l stan d ard ca lib ratio n curve. 6.8 R un Tim e: ~ 23 m inutes. T h e ab o v e c o n d itio n s are in ten d ed as a g u id e an d m a y b e ch a n g e d in o rd e r to optim ize the H PL C system . Page 3 o f 8 Page 39 o f 65 MPI Research, Inc. Page 104 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reiejuxh _ Method N umber VOOOI783 | A N A LY TIC A L M E T H O D | M ethod o f A nalyst* fo r th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Fish and C lam s b y LC /M S/M S 7.0 M S /M S S ystem 7.1 M o d e: E le c tro sp ray N e g ativ e M R M m o d e , m o n ito rin g 4 1 3 - 36 9 m /z for PFOA. T h e ab ove conditions are in tended as a g u id e an d m ay b e ch an g ed in o rd e r to optim ize the M SM S system . 8.0 Preparation o f Solutions 8.1 M o b ile P h ase 8.1.1 2 m M am m o n iu m ac etate in w a te r is p re p are d b y ad d in g 0 .1 5 4 g o f am m onium acetate to 1000 m L o f w ater. 8.2 E x traction S olutions 8.2.1 8 .2 .2 2% a s c o rb ic a c id in m e th a n o l is p r e p a r e d b y d i s s o lv in g 2 g o f a s c o rb ic acid in 100 m L o f m ethanol. 30% D im ethyldichlorosilane in toluene is prepared by bringing 3 mL o f dim ethyldichlorosilane to a final volum e o f 10 m L w ith toluene. A lternate volum es m ay be prepared. 9.0 Standard Preparation 9.1 S tandard S tock/F ortification S olution . 9.1.1 P rep are a stock solution o f - 1 0 0 p g /m L o f P F O A b y w eig h in g 10 mg o f analytical standard (corrected for purity) and d ilute to 100 m L with m ethanol in a 125-m L L D P E bottle. 9.1.2 A 1.0 n g /m L fo rtification so lu tio n o f P F O A is p re p are d b y bringing 1 m L o f th e 100 p g rin L so lu tio n to a fin al v o lu m e o f 100 w ith methanol in a 12S m L L D P E bottle. 9 .1 .3 A 0 .1 p g /m L f o r tific a tio n s o lu tio n o f P F O A is p re p a re d b y b r in g in g 10 m L o f th e 1.0 p g /m L s o lu tio n to a f in a l v o lu m e o f 1 0 0 w ith m ethanol in a 125 m L L D P E bottle. 9 .1 .4 A 0.01 p g /m L fortificatio n so lu tio n o f P F O A is p re p are d b y b ringing 10 m L o f th e 0.1 p g /m L so lu tio n to a fin al v o lu m e o f 100 w ith methanol in a 125 m L L D P E b ottle. 9.1.5 T h e stock a n d fo rtification so lu tio n s are to b e sto red in a refrig erato r at approxim ately 4C and are stable fo r a m axim um period o f 6 m onths from the d ate o f preparation. Page 4 of 8 Page 40 o f 65 MPI Research, Inc. Page 105 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001783 A N A LY TIC A L M E T H O D M ethod o f A n aly sis fo r th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Fish and C lam s b y LC /M S/M S 9.2 S tandard C alibration S olutions 9.2.1 9 .2 .2 L C /M S/M S ca libration standards are p rep ared in m eth an o l via dilution o f th e 1.0 p g /m L fo rtific a tio n so lu tio n . T h e follo w in g is a ty p ical ex am p le: ad d itio n al co n c en tra tio n s m ay be prepared as needed. C o n cen tratio n Final o f Fortification Volum e S o lution Cutt/raL) (m L) D iluted to tm U Concentration (ua/m L ) 1.0 1.0 1.0 0.05 0.025 5.0 2.5 1.0 10 10 100 100 100 100 100 0.05 0.025 0.01 0.005 0.0025 0.1 0.005 10 10 100 100 0.001 0.0005 9.2.3 S tore all ca libration stan d ard s in 125-m L L D P E narro w -m o u th bottles at 2C to 6C , up to six m onths. 9.2.4 A lternate volum es and concentrations o f stan d ard s m ay be prepared as needed. 1 0 .0 B atch Set Up 10.1 10.2 E ach batch o f sam ples ex tracted (ty p ica lly 2 0 o r less) m u st in clu d e at least one untreated control and tw o untreated controls fortified at know n concentrations (lab control spike) to v erify p rocedural recovery for the batch. R equirem ents fo r field an d lab oratory d u p licate s an d sp ik es w ill b e specified in the q uality assurance plan for this project. 1 1.0 Sam ple Extraction 11.1 11.2 11.3 11.4 11.5 1 1 .6 W e ig h 5 g o f f r o z e n s a m p le in to SO m L p o ly p r o p y le n e c e n tr if u g e tu b e s (fortify as needed, rep lace lid an d m ix w ell). A d d 3 0 m L o f a c e to n itrile a n d s h a k e o n a w r is t a c tio n s h a k e r fo r --15 m in u te s Place the tubes in a freezer for hour. Pack and condition the S PE tubes an d silanize the pear-shaped flasks. P ack th e 2 0 m L S PE tubes in seq u en ce w ith 2 g florisil, 2 g silica gel, 2 g carbon, and 1 g LC-NH }. C ondition the colum ns w ith 2 0 m L o f m ethanol, th e n 2 0 m L o f a c e to n itr ile . D i s c a r d a l l w a s h e s . D o n o t a llo w t h e c o lu m n to dry. Silanize the 125 m L pear-shaped flasks b y rinsing w ith the 30% dim ethyldichlorosilane in toluene solution. R inse th e flask w ith toluene once, follow ed b y m ethanol (three tim es). D ry the flasks com pletely before use, either b y air-drying o r w ith a stream o f nitrogen. Page 5 of X Page 41 o f 65 MPI Research, Inc. Page 106 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V 000P83 A N A LY TIC A L M E T H O D M ethod o f A nalysis fo r th e D eterm ination o f P erflu o ro o ctan o ic A cid (P F O A ) in Fish and C lam s by LC /M S/M S 11.7 C en trifu g e th e 5 0 m L p o ly p ro p y len e tu b es co n ta in in g sa m p le at ->2000 rpm fo r - 1 0 m inutes. 11.8 D ecant th e ex tra ct o n to a co n d itio n ed S P E co lu m n fitted in sid e th e m outh o f the p ear-shaped flask. C ollect th e eluate in th e 125 m L silan ized pear-shape flask. 11.9 A d d 10 m L o f ac eto n itrile to th e sa m p le io th e 5 0 m L cen trifu g e lube. H om ogenize the frozen fat phase using a tissum izer fo r - 3 0 seconds and rinse the tissum izer w ith - 1 0 m L o f acetonitrile into the tube. 11.10 S h ak e th e sam ple again f o r - 1 0 m in u te s o n a w rist-actio n shaker. 11.11 P la ce th e tu b es in a freezer fo r - l h o u r m ore. 11.12 C en trifu g e th e 50 m L poly p ro p y len e tu b es c o n ta in in g sam p le at -2 0 0 0 rpm f o r - 1 0 m inutes. 11.13 D ecant th e extract o n to th e sam e S P E colum n. C o lle ct th e elu ate into the sam e p ear-sh ap ed flask an d co m b in e w ith th e elu e n t fro m th e initial extraction. 1 1 .1 4 P a s s 2 0 m L o f a c e to n itr ile th ro u g h th e S P E c o lu m n a n d c o m b in e th e e lu a te in the sam e p ear-shaped flask. 11.15 A dd 3-4 d rops o f 1-octanol to the extract in th e pear-sh ap ed flask and evaporate at reduced pressure using a rotary evaporator (at < 40C ). 11.16 M a k e th e fin al v o lu m e , b y a d d in g 2 m L o f 2 % asc o rb ic a c id in m eth an o l to the pear-shaped flask and sw irl to m ix/dissolve. 11.17 T ra n sfer th e e x tra cts to H P L C v ia ls u sin g d is p o s a b le p ip ets. 11.18 A nalyze sam ples u sin g electro sp ray L C /M S /M S . 12.0 C hrom atography 12.1 12.2 12.3 1 2 .4 Inject the sam e am ount o f each standard, sam p le an d fortified sam p le into (he L C /M S /M S system . A calib ratio n stan d ard m u st p re ced e an d follow all analyzed sam ples. S tandards o f P FO A corresp o n d in g to a t least five o r m o re co n cen tratio n levels m u st b e in clu d ed in an an aly tical set. A n e n tir e s e t o f c a lib r a tio n s ta n d a r d s m u s t b e in c lu d e d a t th e b e g in n in g an d at the en d o f a sam ple set. Standards m ust be interspersed betw een every 5-10 sam ples. A s an alternative, an entire set o f calibration standards m ay be injected at the beginning o f a set follow ed b y calibration standards in tersp e rsed ev e ry 5 -1 0 sam p les (to a c c o u n t fo r a s e c o n d set o f stan d ard s). In e ith e r ca se, ca lib ratio n stan d ard s m u st b e th e first a n d last in jectio n in a sam ple set. U se linear standard curves fo r quantitation. L inear standard curves are generated fo r th e analyte b y linear reg ressio n u sin g 1/x w eig h tin g o f peak area versu s calibration stan d ard co n cen tratio n usin g M assL y n x 3.3 (o r equivalent) softw are system . Pige 6 of 8 Page 42 o f 65 MPI Research, Inc. Page 107 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research M ethod N umber V0001783 | A N A LY TIC A L M E T H O D ~ M ethod o f A nalysis for the D eterm ination o f P erfluorooctanoic A cid (P F O A ) in Fish and C lam s b y L C /M S/M S 12.5 S am p le resp o n se sh o u ld n o t exceed stan d ard resp o n ses. A n y sam p les that exceed standard responses should b e further diluted and reanalyzed. 1 3 .0 A cceptance C riteria 13.1 13.2 13.3 1 3 .4 13.5 1 3 .6 C hrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 am u. T h e 413 am u p aren t co rresp o n d s to th e P F O A anion, w hile the daughter ion (369 am u) represents the loss o f carbon dioxide. M ethod blanks m ust not contain P FO A at levels g reater than the LOQ- If a blan k contains P F O A at levels g reater th an 0.5 p pb, then a new blank sam ple m ust b e obtained and the entire set m ust b e re-extracted. R ecoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f th eir k n o w n values. I f a control sp ik e falls o u tsid e th e accep tab le lim its, the entire set o f sam ples should b e re-extracted. A ny calibration standard found to be a statistical o u tlier b y using the H uge E rror Test, m ay b e excluded from the calculation o f the calibration curve. H ow ever, the total num ber o f calibration standards that could be excluded m ust n o t exceed 2 0% o f the total num ber o f standards injected. T he correlation coefficient (R ) fo r calibration curves generated m ust be 0 .9 9 2 (R 3 0 .9 8 $ ). I f calib ratio n resu lts fall o u tsid e th ese lim its, (hen appropriate steps m ust b e taken to adjust instrum ent operation, and the standards o r the relevant set o f sam ples should b e reanalyzed. R etention tim es betw een stan d ard s an d sam p les m u st n o t drift m ore than 4 % w ithin an analytical run. I f retention tim e d rift exceeds this lim it w ithin an analytical run then the set m ust b e reanalyzed. 14.0 C alculations 14.1 U se the follow ing equation to calculate the am ount o f P FO A found (in ng/m L. based on peak area) using the standard curve (linear regression param eters) generated by the M ass Lynx softw are program : PFO A found (ng/raL) = (Peak area - intercept) slope 14.2 U se th e fo llo w in g e q u a tio n to co n v e rt th e a m o u n t o f P F O A fo u n d in n g/m L to ng/g (ppb). P F O A fo u n d (p p b ) *=I P F Q A fo u n d (n e / m L ) x f in a l v o lu m e (m U x D F ) sam ple w eight (g) D F factor b y w hich the final volum e w as diluted, if necessary. Pge 7 of 8 Page 43 o f 65 MPI Research, Inc. Page 108 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V000] 783 A N A L Y T IC A L m e t h o d M ethod o f A n aly sis fo r th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Fish and C lam s by LC /M S/M S 14.3 F o r sam p les fo rtified w ith k n o w n am o u n ts o f P F O A p rio r to extractio n , use the follow ing equation to calculate the p ercent recovery. R ecovery (% ) = [ to ta l a n a ly te fo u n d (n g /g ) a n a ly te fo u n d in c o n tr o l ( n g /g )] ]clQQ analyte added (ng/g) MPI Research, Inc. Page 8 of 8 Page 44 o f 65 Page 109 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod N um ber: V 0001784 M e th o d o f A n a ly s is f o r th e D e te rm in a tio n o f P e rf lu o r o o c ta n o ic A c id (P F O A ) in V egetation b y L C /M S/M S A nalytical T esting Facility: Exygen R esearch 3058 R esearch D rive S tate C ollege, PA 16801 A pproved By: T U . CJiL Paul C onnolly ' Technical Leader, LC -M S, Exygen R esearch a/*> / U / John F la h erty ' VV iicr e P re s id e n t, O p e ra tio n s , E x y g e n R e s e a rc h _iQfokt D ate MPI Research, Inc. T otal Pages: 7 Page 45 o f 65 Page 110 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Rweirch Method Number V000! 7M a n a l y t i c a l m e t h o d __________________________________ M ethod o f A n aly sis fo r th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in V egetation by LC /M S/M S 1.0 S cope T his m ethod is to be em ployed for the isolation and quantitation o f perfluorooctanoic acid b y H igh P erform ance L iquid C hrom atography coupled to a tandem M ass S pectrom etric D etecto r (L C /M S/M S ) in vegetation. 2.0 Safety 2.1 A lw a y s o b serv e safe lab o ra to ry p ra ctices. 2.2 C onsult th e appropriate M SD S before h andling an y chem ical for proper safety precautions. 3.0 S am ple R equirem ent 3.1 A t lea st 2 0 g o f te st s a m p le fo r ex tractio n . 3.2 Sam ples sh ould b e pro cessed b efo re ex traction. P la ce the frozen sam ple in a food processor and hom ogenize w ith d ry ice. P lace the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublim ation. Seal and place the sam ples in frozen storage until tim e of analysis. 3.3 S am p le co llec tio n p ro ced u res w ill b e sp ecified in th e sam p lin g p lan for this project. 4.0 R eagents an d Standards 4.1 4.2 4.3 4 .4 4.5 4.6 4.7 4.8 4.9 4 . 10 4.11 4 .1 2 4 .1 3 W ater - H PLC grade A cetonitrile - H PLC grade C arbon (120-400 m esh) - R eagent grade M ethanol - HPLC grade S ilica gel (60-200 m esh) - R eagent grade Florisil (60-100 m esh) -R e a g e n t grade Superclean L C -N H j - R eagent grade l -O ctanol - H PLC grade L -A scorbic acid - Reagent grade D im ethyldichlorosilane - R eagent grade Toluene - R eagent grade A m m onium A cetate -A .C .S . R eagent G rade Perfluorooctanoic A cid - Sigm a-A ldrich 5.0 Instrum ent and E quipm ent 5.1 A h ig h p erfo rm an ce liq u id ch ro m a to g rap h c a p a b le o f p u m p in g up to 2 solvents equipped w ith a variable volum e in jector capable o f injecting 5-200 p L connected to a tandem M ass S pectrom eter (L C /M S/M S). 5.2 A device to collect raw d a ta fo r p ea k integ ratio n an d q u an titatio n . 5.3 A naly tical b ala n ce cap ab le o f re ad in g to 0 .00001 g. Page 2 o f 7 Page 46 o f 65 MPI Research, Inc. Page 111 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Research Method N umber V0001784 A N A LY TIC A L M E T H O D | M ethod o f A nalysis fo r the D eterm ination o f P erflu o ro o ctan o ic A cid (P F O A ) in V egetation by LC /M S/M S 5.4 5.5 5.6 5.7 5.6 5.9 5 .1 0 5.11 5 .1 2 5 .1 3 5 .1 4 5 .1 5 R otary evaporator. 125 m L p ear-shaped flasks. 50 m L d isposable p olypropylene centrifuge tubes. 15 m L d isposable p oly p ro p y len e c e n trifu g e tubes. D isposable m icropipets (S0-100uL, 100-200uL). 125-raL L D PE narrow -m outh bottles. 2 m L clear H PLC v ial kit. D isposable pipettes. A utopipettes (100-1000 p L and 10-100 p L ), w ith disposable tips. S PE tubes (20m L ) (Supelco cat. no. N 057177). W rist action shaker. C entrifuge cap ab le o f sp in n in g 5 0 m L p o ly p ro p y len e tubes at 2 0 0 0 rpm . 6.0 C hrom atographic System 6.1 A n aly tical C o lu m n : F lu o p h a se R P (K e y sto n e S cien tific} , 2.1 m m x 5 0 m m , 5p (P/N : 82505-052130) 6.2 T em perature: 30C 6.3 M o b ile P h ase (A ) : 2 m M A m m o n iu m A cetate in W ater M obile P hase (B ) : M ethanol G radient P rogram : T im e (m in) 0 .0 1.0 8.0 2 0 .0 22.5 %A 65 65 25 25 65 F low R ate % B Im L /m in) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection V olum e: 15 p L (can be increased to as m uch as 50 pL). 6.7 Q uantitation; P eak A rea - external standard calibration curve. 6.8 R un T im e: - 23 m inutes. T h e ab ove cond itio n s are in tended as a g u id e an d m ay b e ch a n g ed in o rd e r to optim ize the H PL C system . 7.0 M S/M S System 7 .1 M ode: E lectrospray N eg ativ e M R M m o d e, m o n ito rin g 413 - t 369 m /z for PFOA. Page 3 of 7 Page 47 o f 65 MPI Research, Inc. Page 112 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method N umber VQOO17114 _________________________________ A N A L Y T I C A L m e t h o d __________________________________ M ethod o f A nalysis for the D eterm ination o f P erfluorooctanoic A cid (P F O A ) in V egetation by LC /M S/M S T h e abo v e conditions are intended as a g u ide a n d m ay b e ch an g ed in o rd er to optim ize the M SM S system . 8.0 Preparation o f S olutions 8.1 M o b ile P h ase 8.1.1 2 m M am m onium acetate in w a te r is p rep ared b y ad d in g 0.154 g o f am m onium acetate to 1000 m L o f w ater. 8.2 E x traction Solutions 8.2.1 8 .2 .2 2% a s c o rb ic a c id in m e th a n o l i s p r e p a r e d b y d is s o lv in g 2 g o f a s c o rb ic acid in 100 m L o f m ethanol. 30% D im ethyldichlorosilane in toluene is prep ared b y bringing 3 m L o f dim ethyldichloro8iIane to a final v o lu m e o f 10 m L w ith toluene. A lternate volum es m ay be prepared. 9.0 S tandard Preparation 9.1 S tandard S tock/F ortification S olution 9.1.1 P repare a stock so lu tio n o f - 1 0 0 p g /m L o f P F O A b y w eig h in g 10 m g o f analytical standard (corrected fo r p u rity ) an d d ilu te to 100 m L w iih m ethanol in a 125-m L L D P E bottle. 9.1.2 A 1.0 p g /m L fortification so lu tio n o f P F O A is p re p are d b y b ringing 1 m L o f th e 100 p g ftn L s o lu tio n to a fin al v o lu m e o f 100 w ith methanol in a 125 m L L D PE bottle. 9 .1 .3 A 0 .1 p g /ra L fo rtific a tio n s o lu tio n o f P F O A is p re p a re d b y b r in g in g 10 m L o f th e 1.0 p g /ro L s o lu tio n to a fin a l v o lu m e o f 1 0 0 w ith m ethanol in a 125 m L L D PE bottle. 9.1.4 A 0.01 p g /m L fortification so lu tio n o f P F O A is p repared by bringing 10 m L o f th e 0.1 p g /m L so lu tio n to a final v o lu m e o f 100 w ith methanol in a 125 m L L D P E bottle. . 9.1.5 T h e stock and fo rtificatio n so lu tio n s are to b e sto red in a refrig erato r at approxim ately 4C and are stable for a m axim um period o f 6 m onths from the d ate o f preparation. 9.2 S tandard C alibration Solutions 9.2.1 L C /M S /M S ca libration stan d ard s are p rep ared in m eth an o l via dilution o f th e 1.0 p g /m L fortification solution. P#ge 4 u i" Page 48 o f 65 MPI Research, Inc. Page 113 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 Exygen Retcucb Method Number V0001784 A N A L Y T IC A L m e t h o d M ethod o f A nalysis for the D eterm ination o f P erfluorooctanoic A cid (P F O A ) in V egetation by LC /M S/M S 9.2.2 T he fo llow ing is a typical exam ple: add itio n al co n cen tratio n s m ay be prepared as needed. C o n cen tratio n Final o f Fortification Volum e D iluted to C oncentration Solution fuz/m L) (mL) (m L ) (M8/mL) 1.0 5.0 100 0.05 1.0 1.0 0.05 2.5 1.0 10 100 100 100 0.025 0.01 0.005 0.025 10 100 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 S to re all ca libration stan d ard s in 125-m L L D P E narro w -m o u th bottles a t 2C to 6 C , u p to s ix m o n th s . 9 .2 .4 A ltern ate v o lum es an d con cen tratio n s o f stan d ard s m ay be prepared as needed. 10.0 B atch S et U p 10.1 10.2 E ach batch o f sam ples ex tracted (ty p ica lly 2 0 o r less) m u st include at least o ne untreated control and tw o untreated controls fortified at know n co n centrations (lab co ntrol sp ik e) to v erify p ro c ed u ra l re co v ery for the batch. R equirem ents fo r field an d lab o rato ry d u p licates and sp ik es w ill be specified in the quality assurance plan for this project. 11.0 Sam ple E xtraction 11.1 11.2 11.3 11.4 11.5 11.6 11.7 W eigh 5 g o f frozen sam ple into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and m ix w ell). A dd 3 0 m L o f acetonitrile an d shake o n a w rist ac tio n sh ak e r f o r -1 5 m inuies. C entrifuge the 50 m L polypropylene tubes co n taining sam ple at -2 0 0 0 rpm fo r- 1 0 m inutes. P ack and condition the S PE tubes an d silanize the p ear-shaped flasks. P ack the 20 m L S PE tubes in sequence w ith 2 g flonsil, 2 g silica gel. 2 g carbon, and 1 g L C -N H j. C ondition the colum ns w ith 20 m L o f m ethanol, th e n 2 0 m L o f a c e to n itrile . D is c a rd a ll w a sh e s. D o n o t a llo w th e c o lu m n to dry. S ilanize the 125 m L pear-shaped flasks by rinsing w ith the 30% dim ethyldichlorosilane in toluene solution. R inse the flask w ith toluene once, follow ed b y m ethanol (three tim es). D ry the flasks com pletely before use. either b y air-drying o r w ith a stream o f nitrogen. D ecant the extract on to a conditioned S PE colum n fitted inside the m outh o f th e p ear-shaped flask. C o llect th e elu ate in th e 125 m L silan ized pear-shape flask. Page 5 o f Page 49 o f 65 MPI Research, Inc. Page 114 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygcn Reicvcb Method Number V000I TtS4 A N A L Y T IC A L m e t h o d M ethod o f A n aly sis fo r the D eterm ination o f P erilu o ro o ctan o ic A c id (P F O A ) in V egetation by LC /M S/M S 11.8 A d d 2 0 m L o f ac eto n itrile to th e sam p le in th e 5 0 ra L c e n trifu g e tube. 11.9 S h ak e th e sam ple a g a in for ~ 1 0 m in u te s o n a w rist-a ctio n sh ak er. 11.10 C entrifuge th e 50 m L poly p ro p y len e tu b es co n ta in in g sam p le at -2 0 0 0 rpm fo r - 5 m inutes. 11.11 D ecant th e ex tra ct o n to th e sam e S P E co lu m n . C o lle c t th e elu a te into the sam e p ear-sh ap ed flask an d co m b in e w ith th e elu e n t from th e initial extraction. 11.12 R ep ea t step s 11.8 th ro u g h 1 1 .11 again. 11.13 A dd 3 -4 d rops o f 1-octanol to th e extract in th e p ear-sh ap ed flask and evaporate at reduced pressure using a rotary evaporator (at < 40C ). 1 1 .1 4 M a k e th e fin a l v o lu m e , b y a d d in g 2 m L o f 2% a s c o r b ic a c id in m e th a n o l to the pear-shaped flask and sw irl to m ix/dissolve. 11.15 T ran sfer the extracts to H P L C v ials u sin g d isp o sab le pipets. 11.16 A nalyze sam ples u sin g electrospray L C /M S /M S. 12.0 C hrom atography 12.1 12.2 12.3 12.4 12.5 In ject th e sam e am o u n t o f each stan d ard , sa m p le an d fo rtified sam p le into the L C /M S /M S sy stem . A ca lib ratio n stan d ard m u st p re ced e and follow all analyzed sam ples. S tandards o f P F O A corresp o n d in g to at least five o r m o re con cen tratio n levels m u st b e included in an analytical set. A n entire set o f extracted calib ratio n stan d ard s m u st be included at the beginning and at the end o f a sam ple set. E xtracted standards m ust be interspersed betw een every 5-10 sam ples. A s an alternative, an entire set o f extracted calibration standards m ay b e injected at the beginning o f a set follow ed b y extracted calibration standards interspersed every 5-10 sam ples (to account for a second set o f extracted standards). In either case, extracted ca libration standards m u st b e the first an d last in jectio n in a sam p le set. U se linear standard curves fo r quantitation. L inear standard curves are g enerated fo r th e an a ly te b y lin ear re g ressio n u sin g 1/x w e ig h tin g o f p eak area v ersus calibration standard co n cen tratio n usin g M assL y n x 3.3 (o r equivalent) softw are system . S am ple response should n o t exceed standard responses. A ny sam ples that exceed standard responses should be further d iluted and reanalyzed. 1 3 .0 A cceptance C riteria 1 3 .1 C hrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 4 13 am u. T h e 413 am u p aren t co rresp o n d s to th e P F O A anion, w hile the daughter ion (369 am u) represents th e loss o f carbon dioxide. Pag 6 o f 7 Page 50 o f 65 MPI Research, Inc. Page 115 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygeo Research M ethod N umber VOOO1784 a n a l y t ic a l m e t h o d M ethod o f A nalysis for th e D eterm ination o f P erfluorooctanoic A cid (P F O A ) in V egetation by LC /M S/M S 13.2 13.3 13.4 13.5 1 3 .6 M ethod blanks m ust not contain P FO A at levels g reater than the LOQ . If a b lank co n tain s P F O A at levels g reater th an 0.S p p b , th en a n ew b lank sam ple m ust be obtained and the entire set m ust be re-extracted. R ecoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f th eir know n values. I f a co ntrol sp ik e falls o u tsid e th e accep tab le lim its, the entire set o f sam ples should b e re-extracted. A ny calibration standard found to b e a statistical outlier by using the Huge E rror T est, m ay be excluded from th e calculation o f the calibration curve. H ow ever, the total num ber o f calibration standards that co u ld be excluded m ust not exceed 20% o f the total num ber o f standards injected. T he correlation coefficient (R ) for calibration curves generated m ust be 0 .9 9 2 (R 2 0 .9 8 5 ). I f ca libration resu lts fall o u tsid e these lim its, then appropriate steps m u st b e taken to a d ju st in stru m en t o p eratio n , and (he standards o r the relevant set o f sam ples should be reanalyzed. R etention tim es betw een standards an d sam p les m u st n o t drift m o re than 4 % w ithin an analytical run. I f retention tim e d rift exceeds this lim it w ithin an analytical run then the set m ust be reanalyzed. 14.0 C alcu latio n s 14.1 U se th e fo llo w in g e q u a tio n to c a lc u la te th e a m o u n t o f P F O A fo u n d (in n g /m L. based on peak area) using the standard curve (lin ear regression param eters} generated b y the M ass Lynx softw are program : PFO A found (ng/m L) (Peak area - intercept! slope 14.2 U se th e fo llo w in g eq u atio n to co n v e rt th e am o u n t o f P F O A fo u n d in n g/m L to ng/g (ppb). . P F O A fo u n d (p p b ) = fP F O A fo u n d I'n g /m L t x fin a l v o lu m e C mLt x DF1 sam ple w eight (g) D F factor b y w hich the final volum e w as diluted, if necessary. 14.3 F o r sam p les fo rtified w ith k n o w n am o u n ts o f P F O A p rio r to extractio n , use die follow ing equation to calculate the p ercent recovery. R ecovery (% ) [ total analyte found (ng/g) - analyte fo u n d in c o n tro l (ng /g )] ^ analyte added (ng/g) Page 7 ui' 7 Page 51 o f 65 MPI Research, Inc. Page 116 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod N u m b er V 0001785 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS A nalytical T estin g Facility: Exygen R esearch 3058 R esearch D rive S tate C ollege, P A 16801 A pproved By: coL Paul C onnolly I T echnical Leader, LC -M S, Exygen R esearch ff/ # d - / / . /o h n Flaherty / V ic e P re s id e n t,. O po e ra tio rn s , E x y g e n R e s e a rc h D ate D ate MPI Research, Inc. T otal Pages: 7 Page 52 o f 65 Page 117 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygea Reieirch Method Number V0001785 A N A L Y T IC A L M E T H O D M ethod o f A nalysis fo r the D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Sm all M am m al L iver b y LC /M S/M S 1.0 S cope T his m ethod is to be em ployed for the isolation and quantitation o f perfluorooctanoic acid by H igh Perform ance Liquid C hrom atography coupled to a tandem M ass S pectrom etric D etector (L C /M S/M S ) in sm all m am m al liver. 2.0 Safety 2.1 A lw ay s o b serv e safe lab o ra to ry p ractices. 2.2 C o n s u lt th e a p p r o p ria te M S D S b e f o r e h a n d l i n g a n y c h e m ic a l f o r p r o p e r s a f e ty precautions. 3.0 S am ple R equirem ent 3.1 A t least 5 g o f test sam p le fo r extraction. 3 .2 S a m p le s s h o u ld b e p r o c e s s e d b e f o re e x tra c tio n . P la c e th e f r o z e n s a m p le in u food processor and hom ogenize w ith d ry ice. P lace the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublim ation. Seal and place the sam ples in frozen storage until tim e o f a n a ly sis . A lte rn a te ly , i f th e r e is an in s u f f ic ie n t a m o u n t o f s a m p le ( - l e s s than 5 g), th en n o p ro cessing is necessary an d th e sam p le c an b e used as supplied. 3.3 S am ple collection pro ced u res w ill b e sp ecified in the sam p lin g plan fo r this project. 4 .0 R eagents an d Standards 4.1 W a ter - H P L C grad e 4.2 M ethanol - H P L C grade 4.3 A c e to n itrile -H P L C grade 4.4 A m m onium A cetate - A C S. R eagent G rade 4.5 P erfluorooctanoic A cid - S igm a-A ldrich 5.0 Instrum ent and E quipm ent 5.1 A h ig h p erfo rm a n c e liq u id ch ro m a to g ra p h c a p a b le o f p u m p in g up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5-200 p L connected to a tandem M ass S pectrom eter (L C /M S/M S). 5.2 A device to c ollect raw d a ta for peak integ ratio n an d q u an titatio n . 5.3 A naly tical b ala n ce cap ab le o f re ad in g to 0 .00001 g. 5 .4 SO m L d is p o s a b le p o ly p r o p y le n e c e n trifu g e tu b e s . 5.5 15 m L d isp o sab le p o ly p ro p y len e c e n trifu g e tu b es. 5.6 D isposable m icropipets (50-100uL , 100-200uL ). 5.7 125-m L L D P E narrow m outh bottles. 5.8 2 m L c le ar H P L C v ial kit. Page 2 o f 7 e Page 53 o f 65 MPI Research, Inc. Page 118 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 1131 ExygeoRefetrch Method Number V0G0I785 | A N A LY TIC A L M E T H O D M ethod o f A n aly sis fo r th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Sm all M am m al Liver b y LC /M S/M S 3.9 5 .1 0 5.1 1 5 .1 2 5 .1 3 5 .1 4 5.15 D isposable pipettes. A utopipettes (100*1000 p L an d 10*100 p L ), w ith d isp o sab le tips. W aters Sep Pak V ac 6 cc (lg ) tC 18 S PE cartridges. S PE vacuum m anifold. Tissuem izer. W rist*action shaker. C entrifuge capable o f sp in n in g 15 m L p o ly p ro p y len e tubes a t 3000 rpm . 6.0 C hrom atographic System 6.1 A naly tical C o lu m n : F lu o p h ase R P (K ey sto n e S cien tific), 2.1 m m x 50 m m . 5 m (P/N : 82505-052130) 6.2 T em perature: 30C 6.3 M obile P hase (A ) : 2 m M A m m onium A c etate in W ater 6.4 M obile Phase (B ) : M ethanol 6.5 G radient P rogram : T im e (m in) 0.0 1.0 8.0 2 0 .0 2 2 .5 %A 65 65 25 25 65 %B 35 35 75 75 35 F low R ate im iym ini 0.3 0.3 0.3 0.3 0.3 6.6 In je c tio n V o lu m e: 15 p L (c an b e in cre ase d to as m u c h as 50 p L ). 6.7 Q uantitation: P eak A rea - external stan d ard ca lib ratio n curve. 6.8 R u n T im e : - 2 3 m inutes. T h e ab ove co n ditions are in tended as a g u ide an d m ay b e c h a n g ed in o rd e r to optim ize the H PLC system . 7.0 M S /M S System 7.1 M o d e: E lectro sp ray N eg ativ e M R M m o d e, m o n ito rin g 413 - * 369 m /z for PFOA. T h e a b o v e c o n d itio n s a r e in te n d e d a s a g u id e a n d m a y b e c h a n g e d in o rd e r to optim ize the M SM S system . Page 3 o f ? Page 54 o f 65 MPI Research, Inc. Page 119 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Reieuch M ethod Number VOOfl 1785 | AN/vLYTICAL*MTHOP M ethod o f A nalysis for the D eteim ination o f P erfluorooctanoic A cid (P F O A ) in Sm all M am m al L iver b y LC /M S/M S 8.0 Preparation o f Solutions 8.1 M o b ile P hase 8.1.1 2 m M am m o n iu m acetate in w a te r is p re p are d b y ad d in g 0.1 5 4 g o f am m onium acetate to 1000 m L o f w ater. A lternate volum es m ay b e prepared. 9.0 Standard P reparation 9.1 S tan d ard S to c k /F o rtific atio n S o lu tio n 9.1.1 P re p a re a s to c k s o lu tio n o f --1 0 0 p g /r a L o f P F O A b y w e ig h in g 10 m g o f analytical stan d ard (corrected fo r p u rity ) an d d ilu te to 100 m L with m ethanol in a 125-m L L D P E bottle. 9.1.2 A 1.0 p g /raL fo rtification so lu tio n o f P F O A is prep ared b y b ringing I m L o f th e 100 p g /m L so lu tio n to a fin al v o lu m e o f 100 w ith methanol in a 125 m L L D P E bottle. 9 .1 .3 A 0 .1 p g /m L fo rtific a tio n s o lu tio n o f P F O A is p re p a re d b y b r in g in g 10 m L o f d ie 1.0 p g /m L so lu tio n to a fin a l v o lu m e o f 100 w ith m ethanol in a 125 m L L D P E bottle. 9.1.4 T he stock an d fo rtification so lu tio n s are to b e sto red in a refrigerator at approxim ately 4C and are stable for a m axim um period o f 6 m onths firom th e d a te o f p re p a ra tio n . 9.2 S tandard C alibration S olutions 9 .2 .1 9 .2 .2 L C /M S/M S ca libration stan d ard s are p re p are d in m ethanol via dilution o f th e 0.1 p g /m L fo rtificatio n so lu tio n . T h e follo w in g is a ty p ical ex am p le: ad d itio n al co n c en tra tio n s m ay be prepared as needed. Concentration of Fortification Solution (ng/mL) 100 100 100 5.0 2.0 1.0 Volume (mL) 5.0 2.0 1.0 10 10 10 Diluted to (mL) 100 100 100 100 100 100 Final Concentration (ng/mL) 5.0 2.0 1.0 0.5 0.2 0.1 9.2.3 S tore all calibration stan d ard s in 125-m L L D P E narro w -m o u th bottles at 2C to 6C , up to six m onths. 9.2.4 A lternate volum es and co n cen tratio n s o f stan d ard s m ay be prepared as needed. Page 4 o f 7 Page 55 o f 65 MPI Research, Inc. Page 120 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001785 A N ,U L Y T IC A L M E T H O D M e th o d o f A n a ly s is fo r th e D e te rm in a tio n o f P e r ilu o r o o c ta n o ic A c id (P F O A ) in Small M am m al Liver by L C /M S/M S j 10.0 B atch Set U p 10.1 10.2 E ach batch o f sam ples ex tracted (ty p ically 2 0 o r less) m u st in clu d e at least one untreated control and tw o untreated controls fortified at know n concentrations (lab control spike) to verify p rocedural recovery for the batch. R equirem ents for field an d laboratory d u p licates and sp ik es w ill be specified in th e quality assurance p lan fo r this project. 1 1.0 Sam ple E xtraction 11.1 1 1.2 11.3 1 1 .4 1 1 .5 1 1 .6 11.7 11.8 11.9 11.10 11.11 W eigh 1 g o f sam ple into a $0 m L p o ly p ro p y len e ce n trifu g e tu b es (fo rtify as n eeded, replace lid and m ix w ell). N o te th at altern ate w eig h ts o f liver m ay be m easured depending o n the sam ple size available fo r use. A dd w ater to the sam ple for a final volum e o f 10 m L. H om ogenize sam ple using a tissuem izer for -1 m inute. T ran sfer l m L o f the sam ple usin g a d isp o sab le p ip ette in to a IS m L disposable centrifuge tube. A dd 5 m L o f acetonitrile and shake fo r - 2 0 m inutes o n a w nst-action shaker. C entrifuge the tubes at > 3000 rpm for >5 m inutes. D ecant th e supernatant into a 5 0 m L d isp o sab le cen trifu g e tu b e and add 35 raL o f w ater. C o n d itio n th e C is S P E ca rtrid g es (1 g, 6 m L ) b y p a ssin g 10 m L m ethanol follow ed b y 5 m L o f H PL C w ater {> 2 drop/sec). D o not let colum n run dry L o ad th e sam p le o n conditioned C ii S P E cartrid g e. D iscard eluate. E lute w ith >2 m L o f m ethanol. C ollect 2 m L o f eluate into a graduated 15 m L p olypropylene centrifuge tu b e (final volum e 2 m L ). A nalyze sam ples u sing electrospray L C /M S /M S. 1 2 .0 C hrom atography 12.1 12.2 12.3 12.4 Inject the sam e am ount o f each standard, sam ple and fortified sam ple into the L C /M S /M S sy stem . A c a lib ratio n sta n d ard m u s t p re c e d e an d fo llo w all analyzed sam ples. S tan d ard s o f P F O A corresp o n d in g to a t least five o r m o re co n cen tratio n levels m u st b e included in an analytical set. A n e n tire s e t o f c a lib r a tio n s ta n d a r d s m u s t b e in c lu d e d at th e b e g in n in g an d ui the end o f a sam ple set. Standards m ust be interspersed betw een every 5 -lu sam ples. A s an alternative, an entire set o f calibration standards m ay be injected at the beginning o f a set follow ed b y calibration standards in te rs p e rs e d e v e r y 5*10 s a m p le s (to a c c o u n t fo r a s e c o n d s e t o f s ta n d a r d s ). In e ith er case, calibration standards m u st be the first and last injection in a sam ple set. U se linear standard curves fo r quantitation. L inear standard curves are generated for the analyte by linear regression using t/x w eighting o f peak area Page 5 of? Page 56 o f 65 MPI Research, Inc. Page 121 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reteareh Method Number V0001785 | A N A LY TIC A L M E T H O D M ethod o f A nalysis fo r the D eterm ination o f P erfluorooctanoic A cid (P F O A ) in Sm all M am m al Liver b y LC /M S/M S 12.5 versus calibration stan d ard co n cen tratio n u sin g M assL y n x 3.3 (o r equivalent) softw are system . S am ple response sh ould n o t exceed stan d ard resp o n ses. A ny sam ples that exceed standard responses should be further d iluted and reanalyzed. 1 3 .0 A cceptance C riteria 13.1 13.2 13.3 13.4 13.5 1 3 .6 C hrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 4 13 am u. T he 413 am u p aren t co rresp o n d s to the P FO A anion, w hile the daughter ion (369 am u) represents the loss o f carbon dioxide. M ethod blanks m ust not contain PFO A at levels greater than the LOQ . if a b lank contains P FO A at levels g re ater th an 10 n g /g , th en a n ew b lank sam ple m ust be obtained and the entire set m ust be re-extracted. R ecoveries o f control spikes and m atrix spikes m ust be betw een 70-130% of th e ir k n o w n values. I f a c o n tro l sp ik e fa lls o u tsid e th e a c c e p ta b le lim its, the entire set o f sam ples should be re-extracted. A n y m atrix spike outside 70 130% sh o u ld b e e v a lu a te d b y th e a n a ly st to d e te rm in e i f re -e x tra c tio n is w arranted. A ny calibration standard found to be a statistical o u tlier by using the Huge E rror T est, m ay be excluded from th e calculation o f the calibration curve H ow ever, the total num ber o f calibration standards that co u ld be excluded m ust not exceed 20% o f the total num ber o f standards injected. T he correlation coefficient (R ) for calibration curves generated m ust be 0 .9 9 2 (R ] 0 .9 8 5 ). I f calib ratio n re su lts fall o u tsid e th ese lim its, then appropriate steps m u st b e tak en to a d ju st in stru m en t o p eratio n , and the standards or the relevant set o f sam ples should be reanalyzed. R etention tim es b etw een stan d ard s an d sam p les m u st n o t drift m o re than 4 % w ithin an analytical ran. If retention tim e drift exceeds this lim it w ithin an analytical run then the set m ust be reanalyzed. 1 4 .0 C alcu latio n s 14.1 U se th e fo llo w in g e q u a tio n to c a lc u la te th e a m o u n t o f P F O A fo u n d (in ng/m L . based on peak area) using the standard curve (linear regression param eters) generated b y the M ass Lynx softw are program : PFO A found (n g ta L ) (Peak area - intercept) x D F x aliquot factor slope D F TMfactor b y w hich the final volum e w as diluted, if necessary, A liquot fa c to r" 10 Pige 5 of 7 Page 57 o f 65 MPI Research, Inc. Page 122 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen ReK irch Method Number V0001785 A N A L Y T IC A L M E T H O D M ethod o f A nalysis fo r th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Sm all M am m al Liver b y LC /M S/M S 14.2 F o r sam p les fo rtified w ith k n o w n am o u n ts o f P F O A p rio r to ex tractio n , use the follow ing equation to calculate the p ercent recovery. R ecovery (% ) [ total analyte found (ng/m L ) - analyte found in c ontrol (ng/m L )] analyte added (ng/m L ) 14.3 - U se th e fo llo w in g e q u a tio n to co n v e rt th e a m o u n t o f P F O A fo u n d in ng/m L to ng/g (ppb). PFO A found (ppb) = [PFO A found (ng/m L ) x final volum e fm L Il sam ple w eight (g) MPI Research, Inc. Page 7 of 7 Page 58 o f 65 Page 123 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod N um ber: V 000J 786 M ethod o f A nalysis fo r th e D eterm in atio n o f P erflu o ro o ctan o ic A cid (P F O A ) in S m all M am m al S erum by L C /M S/M S A nalytical T estin g F acility: Exygen R esearch 3058 R esearch D rive S tate C ollege, P A 16801 A pproved By: c siL/i' v - ? -- -- Paul C onnolly 1 Technical Leader, LC -M S, Exygen R esearch s if/7 ? J 6 h n Flaherty / V ice President, O perations, Exygen R esearch D ate D ate MPI Research, Inc. T otal Pages: 7 Page 59 o f 65 Page 124 of 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001786 A N A L Y T IC A L M E T H O D M ethod o f A nalysis for th e D eterm ination o f P erflu o ro o ctan o ic A c id (P F O A ) in Sm all M am m al Serum by LC /M S/M S 1.0 S cope T his m ethod is to be em ployed for the isolation an d q u antitation o f perfluorooctanoic acid b y H igh P erform ance L iquid C hrom atography coupled to a tandem M ass S pectiom etric D etector (L C /M S/M S) in sm all m am m al serum . 2.0 Safety 2.1 A lw a y s o b serv e safe lab o ra to ry p ra ctices. 2.2 C onsult th e appropriate M S D S b efore h an d lin g any ch em ical fo r proper safely precautions. 3.0 S am ple R equirem ent 3.1 A t lea st 1 m L o f te st sam p le fo r ex tra ctio n . 3.2 N o sam ple pro cessin g is n eeded fo r seru m sam p les. H o w ev er, frozen serum sam ples m ust to allow ed to com pletely thaw to room tem perature b efore use. 3.3 S am p le co llectio n p ro ced u res w ill b e sp ec ified in th e sam p lin g plan for this project. 4.0 R eagents and Standards 4.1 W a te r - H P L C g ra d e 4.2 M ethanol - H P L C grade 4.3 A cetonitrile - H P L C grade 4.4 A m m onium A cetate - A .C .S . R eagent G rade 4.5 P erfluorooctanoic A cid - S igm a-A ldrich 5.0 Instrum ent and E quipm ent 5.1 5.2 5.3 5.4 $.5 5.6 5.7 5.8 5.9 5 .1 0 5 .1 1 5 .1 2 5 .1 3 A h igh perform ance liquid chro m ato g rap h c a p ab le o f p u m p in g up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5*200 p L connected to a tandem M ass S pectrom eter (L C /M S/M S). A device to collect raw data for peak integration and quantitation. A nalytical balan ce ca p ab le o f read in g to 0.00001 g. SO m L d is p o s a b le p o ly p ro p y le n e c e n tr if u g e tu b e s . 15 m L d isp o sab le p oly p ro p y len e cen trifu g e tubes. D isposable m icropipets (50-100uL , 100*200uL). 125-m L L D PE narrow *m outh bottles. 2 m L clear H PL C vial kit. D isposable pipettes. A utopipettes (100-1000 p L and 10*100 pL ), w ith disposable tips. W aters Sep P ak V ac 6 cc (Ig ) tC18 SPE cartridges. SPE vacuum m anifold. V ortexcr, Page 2 o f? Page 60 o f 65 MPI Research, Inc. Page 125 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Research M ethod Number VOOOI7U6 | A iW V T IC A L M ETH O D M ethod o f A n aly sis for th e D eterm ination o f P erflu o ro o ctan o ic A cid (P F O A ) in Sm all M am m al S erum b y L C 'M S /M S 5.14 W rist-action shaker. 5.15 C en trifu g e cap ab le o f sp in n in g 15 m L p o ly p ro p y len e tu b es at 3000 rpm . 6.0 C hrom atographic System 6.1 A naly tical C olum n: F iu o p h a se R P (K ey sto n e S cien tific), 2.1 m m x 50 m m . 5p (P/N : 82505-052130) 6.2 T em p e ra tu re : 30C 6.3 M o b ile P hase (A ) : 2 m M A m m o n iu m A cetate in W ater 6.4 M obile Phase (B ) : M ethanol 6.5 G radient P rogram : T im e (m ini 0.0 1.0 8.0 2 0 .0 2 2 .5 %A 65 65 25 25 65 %B 35 35 75 75 35 Flow R ate im L /m inl 0.3 0.3 0.3 0.3 0.3 6.6 Injection V olum e: 15 p L (can b e increased to as m u ch as 50 pL ). 6.7 Q uantitation: P eak A rea - external standard calib ratio n curve. 6.8 R u n T im e: ~ 23 m inutes. T h e ab ove co n ditions are intended as a g u id e an d m a y b e ch a n g ed in o rd e r to optim ize the H PLC system . 7.0 M S /M S S ystem 7.1 M o d e : E le c tr o s p ra y N e g a tiv e M R M m o d e , m o n ito r in g 4 1 3 --3 6 9 mJz for PFOA. T h e a b o v e c o n d i t i o n s a r e in te n d e d a s a g u id e a n d m a y b e c h a n g e d in o r d e r io optim ize the M SM S system . 8.0 P reparation o f S olutions 8.1 M o b ile P h ase 8.1.1 2 m M am m onium acetate in w ater is prep ared b y ad d in g 0.154 g o f am m onium acetate to 1000 m L o f w ater. A lternate volum es m ay be prepared. Pag 3 o f 7 Page 61 o f 65 MPI Research, Inc. Page 126 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research M ethod Number VQ0017B6 A N A L Y T IC A L M E T H O D M ethod o f A n aly sis for the D eterm ination o fP e rflu o ro o c ta n o ic A cid (P F O A ) in Sm all M am m al Serum by LC /M S/M S 9.0 Standard Preparation 9.1 . Standard S tock/Forfk& tion Solution 9.1.1 P repare a stock so lu tio n o f " 1 0 0 p g /m L o f P F O A b y w eig h in g 10 m g o f an a ly tic a l sta n d a rd (c o rre c te d fo r p u rity ) a n d d ilu te to 100 m l. w ith m ethanol in a 125-m L L D P E bottle. 9.1.2 A 1.0 n g /m L fo rtification so lu tio n o f P F O A is prepared b y bringing 1 m L o f th e 100 y g /ra L so lu tio n to a fin al v o lu m e o f 100 w ith m ethanol in a 125 m L L D P E bottle. 9 .1 .3 A 0 . 1 p g /m L f o r tific a tio n s o lu tio n o f P F O A is p r e p a r e d b y b rin g in g IU m L o f th e 1.0 p g /m L so lu tio n to a fin al v o lu m e o f 100 w ith m ethanol in a 125 m L LD PE battle. 9 . 1.4 T h e s to c k a n d fo rtific a tio n s o lu tio n s a r e to b e sto re d in a re frig e ra to r at approxim ately 4C and are stable for a m axim um period o f 6 m onths from the date o f preparation. 9.2 S tandard C alibration S olutions 9.2.1 9 .2 .2 L C /M S/M S calibration stan d ard s are p re p are d in m eth an o l via dilution o f th e 0.1 p g /m L fo rtificatio n solution. The follow ing is a typical exam ple: additional concentrations m ay be prepared as needed. Concentration of Fortification Solution fna/mL) 100 100 100 5.0 2.0 1.0 Volume (mL) 5.0 2.0 1.0 10 10 10 Diluted to (mL) 100 100 100 100 100 100 Final Concentration (ng/mL) 5.0 2.0 1.0 0.5 0.2 0.1 9.2.3 S tore all calibration stan d ard s io 125-m L L D P E narro w -m o u th bottles at 2C to 6C , up to six m onths. 9.2.4 A lternate v o lu m es and co n cen tratio n s o f stan d ard s m ay b e prepared as needed. 1 0 .0 B atch Set U p VO.l 10.2 E ach batch o f sam p les ex tracted (ty p ica lly 2 0 o r less) m u st in clu d e at least one untreated control and tw o untreated controls fortified at know n concentrations (lab control spike) to verify p rocedural recovery for the batch. R equirem ents for field an d lab o rato ry du p licates an d sp ik es w ill b e specified in the q uality assurance plan for this project. Page 4 of 7 Page 62 o f 65 MPI Research, Inc. Page 127 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research M ethod N umber V0001786 ______________________________ A N A L Y T I C A L m e t h o d _______________________________ M ethod o f A nalysis for th e D eterm ination o f P erfluorooctanoic A cid (P F O A ) in Sm all M am m al Serum by LC /M S/M S 11.0 S am ple Extraction 11.1 11.2 11.3 11.4 1 1.5 11.6 11.7 11.8 11.9 1 1 .1 0 11.11 M e a s u re 1 m L o f s a m p le in to a SO m L p o ly p r o p y le n e c e n tr if u g e tu b e s (f o rtif y as needed, replace lid and m ix w ell). N ote that alternate volum es o f serum m ay b e m easured depending o n the sam ple size available for use. A dd w a te r to the sam p le fo r a final v o lu m e o f 2 0 m L . C ap tig h tly V ortex f o r - 1 m inute. T ransfer 1 m L o f the sam ple using a d isposable pipette into a IS m L disposable centrifuge tube. A dd 5 m L o f acetonitrile and shake for ~ 20 m inutes on a w rist-action shaker. C entrifuge the tubes at -3 0 0 0 rpm for - 5 m inutes. D e c a n t th e s u p e r n a ta n t in to a SO m L d is p o s a b le c e n tr if u g e tu b e an d a d d 35 m L o f w ater. C o ndition th e C n 5 P E cartridges (1 g, 6 m L ) b y p assin g 10 m L m ethanol follow ed b y 5 m L o f H P L C w ater ( - 2 drop/sec). D o not let colum n run dry L o ad th e sam ple o n con d itio n ed C ia SPE cartrid g e. D iscard eluate. E lute w ith ~ 2 m L o f m ethanol. C ollect 2 m L o f eluate into a graduated 15 m L poly p ro p y len e c entrifuge tu b e (fin al vo lu m e 2 m L ). A nalyze sam ples using electrospray LC /M S/M S. 12.0 C hrom atography 12.1 12.2 12.3 12.4 12.5 Inject the sam e am ount o f each standard, sam p le a n d fo rtified sam ple into the L C /M S /M S system . A ca lib ratio n stan d ard m u st p re ced e and follow alt analyzed sam ples. S tandards o f P FO A co rresponding to at least fiv e o r m o re con cen tratio n levels m ust b e included in an analytical set. A n entire set o f ca lib ratio n stan d ard s m u st b e in clu d ed at the b eg in n in g and at the end o f a sam ple set. S tandards m ust be interspersed betw een every 5 -in sam ples. A s an alternative, an entire set o f calibration standards m ay be injected at the beginning o f a set follow ed b y calibration standards in tersp e rsed ev e ry 5 -1 0 s a m p le s (to a c c o u n t fo r a s e c o n d se t o f stan d ard s). In eith er case, ca libration standards m u st b e th e first an d last injection in a sam ple set. U se linear standard curves fo r quantitation. L inear standard curves are g en erated fo r th e an aly te b y lin ear re g ressio n u sin g 1/x w e ig h tin g o f p eak area v ersus ca libration standard co n cen tratio n u sin g M assL y n x 3.3 (o r equivalent) softw are system . S am ple response sh ould not ex ceed standard resp o n ses. A n y sam ples that exceed standard responses should be further diluted and reanalyzed. Page 5 of Page 63 o f 65 MPI Research, Inc. Page 128 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project N o.: P0001131 Exygen Protocol Number: P000U 31 Exygen Research M ethod N umber VQ001786 | A N A LY TIC A L M E T H O D M ethod o f A nalysis fo r th e D eterm ination o fP e rflu o ro o c ta n o ic A c id (P F O A ) in Sm all M am m al Serum by LC /M S/M S | 13.0 A cceptance C riteria 13.1 1 3 .2 13.3 13.4 13.5 13.6 C h ro m a to g ra m m u s t s h o w a p e a k o f a d a u g h te r io n a t 3 6 9 a m u fro m a parent o f 4 1 3 a m u . T h e 4 1 3 a m u p a r e n t c o r r e s p o n d s to th e P F O A a n io n , w h ile the daughter ion (369 am u) represents the loss o f carbon dioxide. M ethod blanks m ust n o t contain P FO A at levels greater than the LOQ . if a blank contains P FO A at levels greater than 10 ng/m L , then a new blank sam ple m ust b e obtained and the entire 6et m ust be re-extracted. R ecoveries o f control spikes and m atrix spikes m ust b e betw een 70-130% o f th eir k n o w n values. I f a co ntrol sp ik e falls o u tsid e the ac cep tab le lim its, Ihe entire set o f sam ples should be re-extracted. A ny m atrix spike outside 70 130% sh o u ld b e ev a lu ated b y th e an a ly st to d ete rm in e i f re -ex tra ctio n is w arranted. A ny calibration standard found to be a statistical outlier b y using the H uge E rror T est, m ay be excluded from the calculation o f the calibration curve. H ow ever, the total num ber o f calibration standards th at could be excluded m ust not exceed 20% o f the total num ber o f standards injected. T h e correlation coefficient (R ) for calibration curves generated m ust be 0 .9 9 2 (R J 0 .9 6 5 ). I f ca lib ratio n re su lts fall o u tsid e th ese lim its, then a p p r o p ria te s te p s m u s t b e ta k e n to a d ju s t in s tr u m e n t o p e r a tio n , a n d the standards o r the relevant set o f sam ples should b e reanalyzed. R etention tim es betw een standards and sam p les m u st n o t drift m ore than 4 % w ithin an analytical run. If retention tim e d rift exceeds this lim it within an analytical run then the set m ust be reanalyzed. 14.0 C alcu latio n s 14.1 U se th e fo llo w in g e q u a tio n to ca lc u la te th e a m o u n t o f P F O A fo u n d (in ng/m L . based on peak area) using the standard curve (linear regression param eters) generated b y the M ass Lynx softw are program : PFO A found (n g /m L ) (Peak area - intercept) x D F x aliquot factor slope D F - factor b y w hich the final volum e w as diluted, i f necessary. A liquot factor - 20 14.2 F o r sam p les fo rtified w ith k n o w n am o u n ts o f P F O A p rio r to extractio n , use the follow ing equation to calculate th e percen t recovery. R ecovery (% ) = [to ta l a n aly te fo u n d (n g /m L ) - an aly te fo u n d in c o n tro l (n g /m L )] qq _______________________a n a ly te a d d e d ( n g /m L )_____________________________________ Page 6 o f? Page 64 o f 65 MPI Research, Inc. Page 129 o f 149 Interim Report #6 - Analysis of Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Research Method N umber V00Q1786 | ANa L V TIC A L M E T H O D M e th o d o f A n a ly sis fo r th e D e te rm in atio n o f P e rflu o ro o c ta n o ic A c id (P F O A ) in Sm all M am m al Serum by LC /M S/M S ! 1 4 .3 U s e th e fo llo w in g e q u a tio n to c o n v e r t th e a m o u n t o f P F O A fo u n d in ng/m L to ppb. P F O A fo u n d (ppb) ** fP F O A fo u n d C n e /m L l x fin a l v o lu m e ltnX .ll sam ple volum e (m L) MPI Research, Inc. Pge7ut"? Page 65 o f 65 Page 130 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI ProjectN o.:P0001131 E RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Am endm ent Number:_1__ Effective Date: 01/19/05 Exygen Study Num ber: P0001131 Client Study Number: Page 1 of 1 DESCRIPTION OF AMENDED SECTION 1) Analytical Procedure Sum mary V0001780:Section 9.1 2) Verification o f A n a ly tic a l P ro c e d u re None AMENDED TO 1) Add to Section 9.1: Section 9.1.6, Alternate weights o f standards m ay be used to prepare alternate concentrations of stock solutions as necessary. Alternate levels of fortification solutions may also be prepared. ' 2) Low and high spiking levels of the analytes for each matrix may be altered depending on sample size available for extraction and/or to cover analyte concentrations expected in the samples. RATIONALE 1) Higher concentrations of standards need to be prepared in order to spike the sample bottles at higher levels. 2) The sample size available for small mammal liver and serum was sm aller than expected. Spiking at the pre-determined levels in the protocol puts the spiked concentration lower than the detection limit. Also, the analyte levels in the ground water samples are expected to greatly exceed the pre-determ ined spiking levels listed in the protocol. W hen the levels in the samples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the Q C sample. Higher spiking levels in the bottles will cover the analyte concentrations expected in the water samples. IMPACT ON STUDY The LOQ is 100 ng/g for a 0.1 g sample o f small m ammal liver and is 1000 ng/m L for a 0.01 mL sam ple of sm all m ammal serum. Higher levels of spiking for the water samples will ensure that more QC recovery data can be used. LIBRARY ID'. W0001226-6 ADMINISTRATIVE FORM MPI Research, Inc. Page 131 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 3058 Research Drive Phone: 814-272-1039 S ta te C ollege, PA 16801 Fax: 814-231-1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT ______ 2______ 03/07/05 P0001131 Client Study Number: Page 1 of 1 None DESCRIPTIO N O F AMENDED SECTIO N 1) R ep o rt, p age 11 o f 65 2) Test M aterials, page 6 o f 65: PFOS transition m onitored 499 -> 99. AMENDED TO 1) Instead of one final report, interim reports will be issued. 2) PFOS transition m onitored m ay also be 499 -> 80. R A T IO N A L E 1) Due to the excessive sizes o f the data sets, interim reports will be issued to allow the client to receive da ta in a tim elie r w te e . b a n n e r c ifa /e s 2) The API 4000 LC/M S/M S system s detect the 499 -> 80 PFO S transition with greater sensitivity than the 499 -> 99 transition. IM PACT ON STUDY 1) T he client w ill be able to receive and review the data m ore quickly. 2) T he 499 -> 80 transition can be detected w ith greater sensitivity; therefore, giving better chrom atography. IM bL Study Director Signature / 7 .1 m d u L f rincipal Investigator Signature Ak Date LIBRARY IO: V0001226-8 MPI Research, Inc. ADMINISTRATIVE FORM Page 132 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 JU-.ES.2e05 8*56flM EXYGEN RESEflRCH NO. 77A P .3 3058 Research D rive Phone: 814*272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 3 07/18/05 P1131 Client Study Number: P3Be 1 of 1 NA ' DESCRIPTION OF AMENDED SECTION Verification of Analytical Procedure, page 10 of protocol. ' AMENDED TO The field duplicate can be used for the laboratory spikes and replicate when the primary sample volume is limited. r a t io n a le The sample size for a water sample is 200 m l- If a sample site requires re-extraction for any reason, there would not be enough of the primary sample to repeat two laboratory spikes and a replicate. The field duplicate is technically the same sample as the primary sample and therefore, can be used for laboratory spikes and replicates as needed. IMPACT ON STUDY No negative Impact on the study. Using the duplicate sample allows for the full QC of the sample site to be completed. LIBRARY IK VD00122S Exygen QAU Review /'T L 'l v '/ / p e - A r ADMINISTRATIVE FORM j. MPI Research, Inc. Page 133 of 149 Interim Report #6 - Analysis of Liver and Serum Samples 'r llrt^ODS 04:22pm From-WESTOF SOLUTIONS . ' NOVi22.200S 4:5BPf1 EXYOEN RESEBRCH + MPI Study No.: 0137.0219 MPI Project No.: P0001131 T-677 P.003/003 F-713 P tu .a i > r.a 3058 Research Drive Phone 814-272-1039 State College, PA 16801 Faxt 814-231-15 Amendment Number. Effective Date: Exygen Study Number PROTOCOL AMENDMENT 4 11/22 5 ' P1131 Client Study Number. Page 1 of 1 NA __ DESCRIPTION OF AMENDED SECTION Analytical Procedure Sumrrary; V000i780:"Methcd of Analysis for the Determination of Perfluoroooctaftoic Add (PFOA) in Water by LCMS/MS,' Section 11.0 ofthe method. AMENDED TO Section 11.0. Samples may be diluted before going through the extradlon procedure. RATIONALE ^ If a 40 mL portion of sample win not load onto the Cia SPE cartridge, a pre-dilution can be prepared and extracted. IMPACT-ON STUDY No negative impact on the study. Mora usable data may be obtained. Sponsor Signature pfrequired) Date Exygen QAU Review (. t I LIBRARYID: V000122S-8 RECEIVED TIME NOV.23. S:0PM ADMINISTRATIVE FORM PRINT TIME NOV.23. 5=41PM I MPI Research, Inc. Page 134 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: POOOl 131 'S CHEN EHSR 236 IB 651 733 1958 JU U gi-2003 0 5 :1 2 FROM: 3M GNU. LflB 051 778 6176 P n O V .2 Z .2 0 0 5 4 : 58PT1 EXYCEN RESEARCH 11/23 '05 14:12 NO.979 03/03 78:56517331558 no p ? ~ 3058 Research Drive Phene: 814-272-1039 State College, PA 16801 Fax: 814*231-1580 Amendment Num ber Effective Date: Exygan Study Number PROTOCOL AMENDMENT 4 11/22/06 P1131 Client Study Number: Page 1 of 1 NA ba'CRIPTIO N OF AMENDED SECTION Analytical Procedure Summary: VOOOt780r"Mefhod ofAnalysis for the Determination of Perfluoroooctanoic Add (PFOA) in Water by LC/MS/MS.' Section 11.0 of the method. am ended t 5` Section 11.0. Samples may be diluted before going through the extraction procedure. RATIONALE If a 40 m l portion of sample will not toad onto the C.a SPE cartridge, a pre-dilution can be prepared and extracted. IMPACT ON STUDY <\ No negative impact on the study. More usable data may be obtained. Study DlnsewfSignature DSfe bate ~ ~~ ~ O ita lts -n D r-o f Oita _______ /> ///. IS ,W>S Dale ExygenQAU Review */**/r I \ LIBRARY IO: V0001U6-6 ADMINISTRATIVEFORM RECEIVED TIME NOV.23. 3:32PM PRINT TIME NOV.23. 3:33PM S'" N MPI Research, Inc. Page 135 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples ' *200S 04:08pm Froo-KESTON SOUITIONS MPI Study No.: 0137.0219 MPI Project No.: P0001131 T-15Z P.oa^/ooij F-178 . . -- j- a , 11 |g r r e ; :..r c h 3BB ResearchOrive Ptione: 81*-Z7Z'1039 State Collas, PA16801 Faso 814-231*1580 ' Amendment Nu iben E__ff_e_ctive Date: Exygen Study Number T-'OCOU AMENDMENT j 12/01/05 , PO O Pm t Client Study Number Page 1 of.1 = NA l DESCRIPTION OF AMEWED SEgPQM~ Test Materials, page 5-6 of Protocol. AMENDED TO An alternate lot of p fo s m ! so-used tor this study. RATIONALE The PF08 used previously in this stutfcr (lot numDer 217 from 8M) ran out and B was necessary to use a new lot No negafive impact on study. IM FA C TO N LS T 57' study oir_____ ____ SQ-J f i Rifnclpel UtvtsSgtla: ifinalu. v O 0I oaa y - / i M Z .-U C & 'o Exygen QAU Inft/Dte T _J_3kt (ate. LIBRARY ID: VDtB""BHMr RECEIVED TIME 1RR. ;i :26PM . ADMINISTRATIVE form PRINT TIME I MOR.21. 5:2SPM MPI Research, Inc. Page 136 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 EARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number: Effective Date: Exgen Study Number PROTOCOL AMENDMENT 5 . 05/17/06 P0001131 ' Client Study Number Page 1 oM NA DESCRIPTION OF AMENDED SECTION Appendix I, Analytical Method V0001782 AMENDED TO . `4 ' As per client request, samples in login L4026 that have been tagged for re-extraction by the sponsor will be re-extracted using the following method: Direct Injection Method: Before the samples are weighed for the extraction, they are mixed thoroughly by vigorously shaking the container. A one-gram portion of sediment is weighed into a 15-milliliter centrifuge tube for the extraction. Ten milliliters of 1% acetic acid in methanol is added to each sample. The samples are then shaken by hand, vortexed, and sonicated for thirty minutes. The samples are then centrifuged for -10 minutes at -3000 rpm. Each sample is analyzed by LC/MS/MS electrospray. RATIONALE More usable data will be obtained by using an alternate method. IMPACT ON STUDY No negativeimpact on study. Studyy DirreeidrSlgnature / td d u , Dati H finclpal Investigator Signature Sponsor Signature (^required) Data Date ii-n M u i-T t/i/C Date /v /H Q -i Dai Exygen QAU lnit./Date LIBRARY ID: V0001226-9 ' ADMINISTRATIVE FORM MPI Research, Inc. Page 137 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 3058 Research Drive Phone: 814-272-1039 S ta te C ollege, PA 16801 Fax: 814-231 -1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 8 07/06/06 P0001131 Client Study Number: Page 1 of 1 ______ NA DESCRIPTION O F AMENDED SECTION A p p e n d ix I, A n a lytica l M ethod V 00 0 1 7 8 2. AMENDED TO A s per client request, sedim ent sam ples re-tagged fo r re-extractlon by the sponsor will be re-extracted using the follow ing m ethod: D ire c t In je c tio n M eth o d : Before the sam ples are weighed fo r the extraction, they are m ixed thoroughly by vigorously shaking the container. A one-gram portion o f sedim ent is w eighed into a 15-m illiliter centrifuge tube for the extraction. Ten m illiliters o f 1% acetic acid in m ethanol is added to each sam ple. T he sam p le s are then shaken by hand, vortexed, and sonicated for thirty m inutes. The sam ples are then centrifuged for - 1 0 m inutes a t -3 0 0 0 rpm. Each sam ple is analyzed by LC /M S /M S electrospray. R A T IO N A L E M ore usable data will be obtained by using an alternate m ethod. IMPACT ON STUDY No negative im pact on study. Study Director Signature ^Pfincipal Investigator Sanatre Study Director Management Signature Exygen Management Signature Sponsor Signature (if required) Date Date Date Date Date Exygen QAU Init./D ate /0 - / 'llu ln ie LIB R A R Y ID: V 0 0 0 1 2 2 6 -9 A D M IN IS T R A TIV E FO R M MPI Research, Inc. Page 138 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 CHEM EHSR 236 ife 651 733 1958 07-11-2006 03:57pm FronrWSTH SOURIONS 07/11 '06 14:09 NO.142 02/02 T--528 P .0 0 2 /0 0 2 F -5 M 3053ResearchOrWe Phone:81^27?d03? StateCollege, PA1MC1 Fa;SI4-231-1580 RESEARCH . . P k T O ' O L a m e n d m e n t ArtiendmontNumber 8 Effective Dat: m ow n ^nSftidyNunlber POOOH31~ Cileni,StpdyNumber. 7 .P a $ ii.i' fi .M L D Efe ftim N Q F AMENDED S E P T E T Appendix I, Analytical Method VQ001782. : : . wiewpRBia - i. As byp e r o t t e n t r e q u e s t s e d i m e n t s a m p l e s r a - t a g g e d f o r r p - e j c t r a c t t o n t h e s p o n s o r w lH be r e ^ x t r a d e d u s i n g t h e f o l l o w i n g r h a t h o d : . . D lrtctln jsetlo n Method: Before the samples are vrelghed.fqr the extraction, thjsy. are mixad.thoroughly by vigorously shaking tha container. A one-gram poifiofl of 8S|drrwnt la weighed Into a 15-mWTlfter centrifuge tube.fbrthe exfeactipn. te n milliliters of fyo acetic sCid In methanol Is added td.each sample. The samples are then shaken by ,. hand, vortexed, and sonicated for thirty minutes. The samples ere then cebtritoRid for -1 0 minutes at -3000 rpm. Esohsampie .is analyzed by LC/MS/MS eieetrbspray. .... ; : RATIONAL^' ' ~ Mord usable date will be obtained by using.an altemkte mettiod. a i l'. n r r a r . T ^ n r . N o negativa impact o n study. - LIBRARYID: V000122S4 RECEIVED T in e J U L . l l . 4:27P(1 ' AOMrfilSTOATWC(=ru ' PRINT TIME J U L . l l . 4:28PM MPI Research, Inc. Page 139 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: POOOl 131 01-0B -Z0O 7 03:58pm From -KSTO N SOLUTIONS T-021 P.002/005 F-058 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 81423M 580 AmendmBDt Number: Effective Date: Exygen Study Number PROTOCOL AMENDAIENT 11 1 2 /2 1 /0 6 P00Q1131 a ie n t Study Number: P a g e io fl P00Q1131 LIBRARY ID: V00012Z6-9 REOE IVED TIME J AN. 8 . 4:08PM ADMINISTRATIVE FORM PRINT TIME JAN. 8 . 4:04PM MPI Research, Inc. Page 140 of 149 Interim Report #6 - Analysis of Liver and Serum Samples SEP.2 0 .2 0 0 5 1 :52PM EXYGEN RESEARCH MPI Study No.: 0137.0219 MPI Project No.: P0001131 N O .3 7 7 P .2 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM ____________________|_____________ _________________ ___________________ ________________Pane 1 of 1 General: X Project S pecific Deviation ____ Facility Deviation Date o f Occurrence: 03/16/05 Exygen Project # : P760/P1131 Deviation # : 1/1 C lient Project # : _______ NA_______ Reference # : 05-122 Regulatory Driver: Deviation Tvds: (In c lu d e V # fo r m e th o d s a n d S O P s ) ______ X ______ _____ GMP GLP Other None Sample Description: ______ Protocol ______M ethod V#: 0001658-3 Notebook reference: NA X SOP L o g in # : ________N A_______ C o n t a i n e r # : ________ NA_______ L o t # : __________ NA Summary of Deviation: This deviation pertains to all soil and sedim ent samples analyzed fo r percent solids before 07/07/05. a. No blanks or duplicates w ere run as required b y section 9.3. b. S om e sam ple w eights exceed the allow able range (> 10g). C ause: ____ P re p a ra tio n ____ A n a ly s is _____ In s tru m e n t____ C lient R equest X O ther Impact: There has been no negative im pact on the study. All o f the percent solid values that were determined during the tim e period in question are considered valid, although the S O P w a s not follow ed. In the newly revised version o f the SO P blanks and d uplicates are no lo nger required. Also, In the new SOP, the allowable am ount o f sample to be used is < 20 g. A ll o f the sam ples in question in this deviation weighed (ess than 20 g. T he technician analyzing the sam ples fo r percent solids w as follow ing the new procedure before it was form ally approved. C o r r e c tiv e Actions: A new version o f the SOP has been issued and approved (V0000427-3). L IB R A R Y ID: V 0 0 0 1 6 4 0 -6 MPI Research, Inc. A D M IN IS T R A T IV E FO R M Page 141 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples SEP.20 .200 5 1 :52PM EXYGEN RESEARCH MPI Study No.: 0137.0219 MPI ProjectN o.:P0001131 N O .3 7 7 P .3 3058 Research Drive S ta te College, PA 16801 Phone; 8 U - 2 7 2 - 1 D 3 9 Fax: 814-231-1580 General: X Project Specific Deviation DEVIATION FORM ________ __ __________ Page 1 of 1 Facility Deviation Date o f Occurrence; 06/29/05 Exygen Project # : P760/P1131 Deviation # ; 2/2 Client Project # : NA Reference# : p ~ - V 3 3 . Regulatory Driver: Deviation Type: (Include V tt for methods a n d S O P s ) GMP GLP Other None Sample Description: X Protocol _____ Method v# :na Notebook reference: NA _____ SOP L o g in # : L4254/L4256 Container # : C00564B0-85 Lot # : NA Summary of Deviation: The protocol states that control and fortified control samples of each matrix will be analyzed; however, control dam was not obtained. Fish was used as the control for the analysis o f these six clam samples. Cause: P reparation____ A n a ly s is _____ In stru m e nt____ Client Request X Other Impact: No negative impact on this study. Corrective Actions: Deviation issued. Study Director Quality Assurance LIB R A R Y ID: V 0 0 0 1 6 4 0 - Date < Exygen Mbnagefnent- A Sponsor Representative Date A. Sponsor Management U -& -0 S Date Date Date A D M IN IS T R A TIV E FO RM MPI Research, Inc. Page 142 of 149 Interim Report #6 - Analysis of Liver and Serum Samples SEP.20 .200 5 1 :52PM EXYGEN RESERRCH MPI Study No.: 0137.0219 MPI Project No.: P0001131 NO.377 P .4 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM General: X Project Specific Deviation ____ Facility Deviation Date o f Occurrence: 12/27/04 Exygen Project # : P760/P1131 Deviation # : 3/3 Client Project # : _______ NA_______ R e fe re n ce # : / 2 1 ., . Reaulatorv Driver: GMP X GLP Other None Sample Description: D eviation Tvoe: <In c lu d e W fo r m e th o d s e n d S O P s ) Protocol Method X SOP V#: 202-20 Section 5.2.3 Notebook reference: NA L o g in # : _______ N A _______ C o n t a i n e r # ________ N A _______L o t # : ___________NA Summary of Deviation: SL2114 (30% Dimethyldichlorosilane in Toluene) was given the expiration Sate of 02/13/05, but RE544 (Toluene) used to make SL2114 expired on 03/2S/04. S L2114 was used to silanlzed glassware prepared for the fish extraction from 12/27/04 through 01/07/06. Cause: ____ P reparation____ A n a ly s is _____ In strum ent____ Client Request X Other Im pa ct: No negative impact on the study. Toluene was used only as a solvent for the glassware preparation. Dimethyldichlorosilane, which is the coating agent, was not expired. Corrective A ctions: Deviation Issued. S in n a tu re s : A P rin c ip a iin v e s tig a to r X qx/ Study Director flyC*. fk& JL tK Quality Assurance ffc jk Date m oh Batej Date S f/i ------ E xygen Mnagement Sponsor Representative k)f\ Sponsor Manegement ate Date Date LIB R A R Y ID: V 0 0 0 1 64 0 -6 A D M IN IS T R A TIV E FO RM MPI Research, Inc. Page 143 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 t RCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM General: X Project Specific Deviation Facility Deviation Exygen Project # : P760/P1131 Client Project # : NA Page 1of 2 Date of Occurrence: 04/26/08 Deviation # ; 5 Reference tt : 06-076 Reoulatorv Driver: Deviation Type: (In c lu d e V II fo r m e th o d s a n d S O P s ) GMP X GLP Olher None Sample Description.- X Protocol V#: NA Nolebdok reference: NA Method SOP Login # : L0008191 Container # : ______NA_____ Lot ft : ________ M Summary of Deviation: The three sediment samples in L8191 (C0172892 - C0172894) were originally extracted using the sediment method V0001782. Poor recoveries were obtained for PFOS, PFOA and ,5C PFOA Because of this, the study sponsor requested the use of an alternative extraction for these compounds, as follows: Direct Injection Method: Before (he samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A one-gram portion of sediment was weighed Into a 15-milliliter centrifuge tube for the extraction. Ten milliliters of 1% acetic acid in methanol was added to each sample. The samples were then shaken by hand, vortexed. and sonicated for thirty minutes. The samples were then centrifuged for -10 minutes at -3000 rpm. Each sample was analyzed by LC/MS/MS electrospray. Using this method acceptable data was obtained for PFOS, but the recoveries for PFOA and ,SC PFOA were still poor. Another alternative method was then used for PFOA and ,3C PFOA, as follows: Alternative SPE Method: The samples that were prepared in 1%acetic acid for the direct injection method were used for this extraction. Five milliliters of each sample was aliquoted into a 50-mL polypropylene centrifuge tube and the volume was taken to 40 mL with water. The samples were then centrifuged for -10 minutes at -3000 rpm. The supernatant was then loaded onto a C, SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluale was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluale was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. Cause: ___ Preparation___ Analysis_____ Instrument___ Client Request X Other Impact: More usable data was obtained. LIBRARY ID: V0001840-6 AD M IN ISTRA TIV E FO R M MPI Research, Inc. Page 144 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 CHEM EHSR 236 1B 651 733 1958 C5-GW00S GMlpo Frra-iESTCH SGlirnGRS 05/01 '06 13:46 NO.106 03/03 M M P.OOJ/OOI F--31B SEA R C H J0M Rem it!) Drive Phone: ii+ W -IW ? S titt College, PA1 fte I f ! - - d | v ia t ig n f o a m fM m w Pfp g M n ft lB e te : CSini Prefect#: V J. : FjtfHlyPwfaflbn Ib tiflia m m iiB IS tM X . ' ^ P m iP i i i 'i , ' XA ' ' ' .. b eirteB o r DttfMontnuvd- .' , * ? \1 i " , ' .' S-'. ] .* id * /* * A M v J jd n M eat t- A to r U BUdyBlrtolar OuaMyRMUmnce .W (r/< /`L ...cm -Sportier . . .c^m... . u n M triftV o s s iw M MPI Research, Inc ^ vt , _ . Page 145 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project N o.: P0001131 EHS OPNS ENVIRONMENTAL LAB 651 778 4226 07/11/06 11:46 |3 :04/07 N0:681 RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 8 1 4 -23M 580 DEVIATION FORM S55E !" X Project Specific Deviation Facility Deviation JSasMi- Data' o f Occurrence: 04/28/06 Exyijen Project # : P 7 6 0 /P 1 131 Deviation # : . f r '' ' . Client Project # : NA R efbfw ioe#: . 08-16 fftfliilntow Priyyp Deviation Type: {Include V# formafhods and SOPsi .. _____ ~W _ GMP GLP Other None _____ Protocol X Method V#: V0427-3 Notebook rference: NA SOP S A m B ltg rtP riP tia n -' Login _______ NA Container # : C00T59446 ; Lot # : :N Summary of Deviation: .: .. :. . One sod sample (CQ0159446) was weighed at ~8g for percent solid analysis, rather than at -20g which I staled In Ihe method. ' " Cause: P re p a ra tio n ____ A n a ly s is _____ instrument ___ Client Request _ X _ . Other Im pact; ' .. . . . . . ; . No negative Im pact An accurate percent solid number was obtained by slightly altering the calculation used. '' '. ' ... ' Corrective Action: Deviation Issued. StudyCDirector Quality Assuraaee Sponsor Repreq & uhJoe* ^( Date Sponsor Management C a te . MPI Research, Inc. Page 146 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 EHS OPNS ENVIRONMENTAL LAB 1? 651 778 4226 07/11/06 11:46 0 :06/07 N0:681 RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, P 16801 Fax: 814-231-1580 DEVIATION FORM O tturai: X Project Specific Deviation Facility Deviation Pm * to* Oste o f Occurrence: 68728/06 Exygan Project # : P760/P1131 : Dviation # : iff Client Project 4 : NA Reference 4 : B M W la ia o f.B ilm : GMP x_ GLP _ Other . None Saniate Description: Login- # : _______ NA Deviation Tvoa; (Include V# formethods and SOPs) Protocol ____ Method X 80P V#: 1800 Notebook reference: Container 4 : NA Lot: m. Sinhiimpt of Deviation: Peer review o f raw data w as not documented per SOP V1600 prior to 6/28/06.. Cause: ____ P re p a ra tio n _____ A n a ly s is ____ _ In strum ent____ Client Request V " Other Impact: ' ' ' ' ' " ....... . .. . / Raw data will be more thoroughly evaluated arid reviewed before QA Inspection. ' ' ' ; CartwayArtlann; A notB to fCe will be Issued to ail subsequent reports stating that overall summaries haVe been peer reviewed. ' .... ' MPI Research, Inc. Page 147 o f 149 Interim Report #6 - Analysis o f Liver and Serum Samples EHS OPNS ENVIRONMENTAL LAB 651 778 4226 MPI Study No.: 0137.0219 MPI Project No.: P0001131 0 7 /1 1 /0 6 1 1 :4 6 (3 : 0 7 /0 7 NO:681 RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM S im u li . X Project Specific Deviation _ _ Facility Deviation Exyyen Project 4 : P76/P131 J S te is L L Dele of Occurrence: 08/26/06 ' Deviation # ; ; 8/B Client Project # : _ NA ' ! Reference * : . f i t * - I l ta . Weaulatdrv Driver: DavleHan Toa: (In c lu d e V # fo r m a th d s a n d S O P s l ___ _ .X ... ___ \ GMP GLP Other None Sampte Description: X Protocol V * NA Notebook rference: LopIn * : L0008121 L00080B1 Container # : Sum mary o f Deviation: Samples were filled to 2S0 mL Instead o f 200 mL. Method coi66347 C016B354 C0171088: L o t*: . GP NA b a ite * :' X Preparation ___ ; Analysis ____ Instrument ' ' Client Reguaat htiosct: .: . . Samples could not be extracted according to the protocol. Other ' c p r a s U ta A stlm ? ; ~~ .- -. Spiking, levels were adjusted to eccommodate the alternative volume. MPI Research, Inc. Page 148 of 149 Interim Report #6 - Analysis o f Liver and Serum Samples MPI Study No.: 0137.0219 MPI Project No.: P0001131 0 1 -0 8 -2 0 0 7 0 3 :5 9 pis FronHffiSTON SOLUTIONS T-021 P .005/005 F-05B RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231 -1580 GgngaJ: X Project SpecificDeviation DEVIATION FORM Facility Deviation Pana l o f i Date of Occurrence: Nov. 2006Jan. 2007 Exygen Project#: Client Project#: POPP1131 PC001131 Deviation#: Reference#: 10 fl~CO Regulatory-Driver: DovtattonTvpe: (Inekide V#formethodsand SOPs) GMP GLP Other None Sample Description: ' Protocol X Method Vft VD001780 Notebook reference: ____ SOP Login#: L0010155 Container#: Lot#: L0010165 L0010Z54 Summary of Deviation: Stock siamterds and fomfieaficn standards used in the extraction forthese logins were prepared in acetonitrile Instead of methanol. Cause: X Preparation___ Analysis____ Instrument____ Citait Request _ Other Impact: 1 No negative Impact. Compounds are completelysoluble inacetonitrile as they are in methanol Correctiva Adlon: Deviation Issued. Preventative Actions: No actiontaken. Slanature: Date Jsfr M7 SponsorManagement UBFWRYID:VE001640-7 RECEIVED TIME JAN. 8 . 4I02PM ADMINISTRATIVE FORM PRINT TIME JAN. 8 . 4:04PM MPI Research, Inc. Page 149 of 149