Document gDZaGyZogj32kGENjJe5ya72a

INTERIM REPORT #29 - Reanalvsis of Soil and Sludge 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 August 27, 2007 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 Protocol Number: P0001131 ExyLIMS Study Number: P0001131 Total Pages: 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT ExyLIMS Study 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. C P MPI Research, Inc. Jaisimha Kesari P.E., DEE Study Director Weston Solutions, Inc. Michael A. S Sponsor Rep 3M Company MPI Research |cH Date Page 2 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 QUALITY ASSURANCE STATEMENT MPI Research's Quality Assurance Unit reviewed ExyLIMS Study 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". 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 to Principal MPI Date Reported to Investigator Management Studv Director 51) Draft Interim Analytical Report and Raw Data Review 06/06/07 06/12/07 06/21/07 07/06/07 54) Draft Interim Analytical Report Review 08/07/07 08/13/07 08/20/07 08/24/07 55) Final Interim Analytical Report Review 08/20/07 08/21/07 08/27/07 08/27/07 L fy\A Lynannl Porter ?lanfoi Date Quality Assurance Research Group Leader, Quality Assurance Unit 'Note: All in-lab inspections and the protocol review 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 Page 3 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 CERTIFICATION OF AUTHENTICITY This interim report, for ExyLIMS Study 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: Charles Simons Director, Analytical Laboratory Operations MPI Research, Inc. MPI Research Facility Management: A-Mm_______ Kevin Lloyd General Manager, Analytical Sciences MPI Research, Inc. Study Directo/, Weston Solutions, Inc. _______ Jaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: jfy lw L // $ Michael A. Sanjdro Director of Regulatory Affairs F'Jtf<H Date mm Date MPI Research Page 4 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 PROTOCOL NUMBER: P0001131 EXYLIMS STUDY NUMBER: P0001131 TYPE OF STUDY: Residue SAMPLE MATRIX: Soil and Sludge 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: 01/25/07 Interim Analytical Termination Date: 02/07/07 Interim Report Completion Date: 08/27/07 MPI Research Page 5 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 Amy Sheehan Mark Ammerman Eric Edwards Christine Edwards Krista Gallant Mindy Cressley Cameala Graybill Natalie Cleaver Brittany Kravets Title Director, Analytical Laboratory Operations Senior Director, Analytical Laboratory Operations Manager Analytical Group Leader, Industrial Analysis Project Leader, Sample Control Sample Custodian 2 Project Leader, Industrial Analysis Research Chemist Associate 1 Research Chemist Associate 1 Research Chemist Associate 1 Research Associate 1 Technician MPI Research Page 6 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 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.................................................................................................... 10 1.0 SUMMARY................................................................................................................ 11 2.0 OBJECTIVE............................................................................................................... 11 3.0 INTRODUCTION....................................................................................................... 12 4.0 ANALYTICAL TEST SAMPLES.............................................................................. 12 5.0 REFERENCE MATERIAL........................................................................................ 12 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................ 14 6.1 Extraction Procedure For Soil/Sludge Samples........................................................ 14 6.2 Percent Solids Procedure For Soil/Sludge Samples................................................. 14 6.3 Preparation of Standards and Fortification Solutions............................................... 14 6.4 Chromatography....................................................................................................... 15 6.5 Instrument Sensitivity............................................................................................... 15 6.6 Description of LC/MS/MS Instrument and Operating Conditions...........................15 6.7 Quantitation and Example Calculation..................................................................... 16 7.0 EXPERIMENTAL DESIGN...................................................................................... 18 8.0 RESULTS................................................................................................................... 18 9.0 CONCLUSIONS......................................................................................................... 19 10.0 RETENTION OF DATA AND SAMPLES............................................................. 19 MPI Research Page 7 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Table I. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Soil/Sludge Samples.......................21 Table n. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil/Sludge Samples.......................................................................................................... 23 Table HI. Total Percent Solids in Soil/Sludge Samples.................................................27 MPI Research Page 8 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFBS in 50:50 Acetonitrile:Water................ 30 Figure 2. Non-Extracted Standards of PFBS in 50:50 Acetonitrile:Water, 0.025 ng/mL and 0.05 ng/mL, Respectively.................................................. 31 Figure 3. PFBS in Control Blank, 0.25 ng/mL Fortified Control Spike A and 2.5 ng/mL Fortified Control Spike B, Respectively...................................... 32 Figure 4. Chromatogram Representing a Soil/Sludge Sample Analyzed for PFBS (ExyLIMS ID: C0160900, Data Set: 013107E)............................................. 33 Figure 5. Typical Calibration Curve for PFHS in 50:50 Acetonitrile:Water................ 34 Figure 6. Non-Extracted Standards of PFHS in 50:50 Acetonitrile:Water, 0.025 ng/mL and 0.05 ng/mL, Respectively.................................................. 35 Figure 7. PFHS in Control Blank, 0.25 ng/mL Fortified Control Spike A and 2.5 ng/mL Fortified Control Spike B, Respectively...................................... 36 Figure 8. Chromatogram Representing a Soil/Sludge Sample Analyzed for PFHS (ExyLIMS ID: C0160882, Data Set: 013107C)............................................. 37 Figure 9. Typical Calibration Curve for PFOS in 50:50 Acetonitrile:Water................38 Figure 10. Non-Extracted Standards of PFOS in 50:50 Acetonitrile`.Water, 0.025 ng/mL and 0.05 ng/mL, Respectively.................................................. 39 Figure 11. PFOS in Control Blank, 0.25 ng/mL Fortified Control Spike A and 2.5 ng/mL Fortified Control Spike B, Respectively...................................... 40 Figure 12. Chromatogram Representing a Soil/Sludge Sample Analyzed for PFOS (ExyLIMS ID: C0153323, Data Set: 012607A )............................................41 MPI Research Page 9 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 LIST OF APPENDICES Page Appendix A Study Protocol P0001131 (ExyLIMS Study No. P0001131) with Analytical Methods, Protocol Amendments, and Deviations................... 42 MPI Research Page 10 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 1.0 SUMMARY MPI Research extracted and analyzed soil and sludge samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) using a direct injection analysis technique as specified in Protocol Amendment 12 (Appendix A). The samples in this report are re-extractions of soil and sludge samples previously designated as not reported (NR) due to quality control failures in Interim Reports #21 (Analysis of Soil Samples), #22 (Analysis of Soil Samples) and #24 (Analysis of Water, Sludge, and Sediment Samples). Re-extraction was performed in an attempt to provide quantitative values for these samples. All soil and sludge samples were initially analyzed by the SPE methods outlined in the protocol in previous reports (see Interim Reports #21, #22, and #24). When the results did not meet quality objectives, the samples were re-analyzed by the Direct Inject method outlined in Protocol Amendment 12 to eliminate the interference that was causing the SPE data to not meet quality control standards. The limit of quantitation for PFHS and PFOS in soil and sludge was 0.2 ng/g (wet weight). Due to the instrument conditions detailed in Section 6.6, the PFBS calibration curve had a raised LOQ as shown in the raw data. The LOQ provided is assuming wet weight values and the results for the actual samples are reported based on dry weight. Analytical results and assessed accuracies for the analysis of PFBS, PFHS and PFOS in soil and sludge samples based on dry weight are summarized in Table I. Fortification recoveries for the analysis of PFBS, PFHS and PFOS in soil and sludge samples based on wet weight are summarized in Table II. The overall average percent recoveries standard deviations for PFBS, PFHS and PFOS in the soil and sludge samples based on wet weight were 94 24%, 83 14%, and 107 26%, respectively. Quantitative results were obtained for 21 of the 23 samples reanalyzed for PFBS, 6 of the 11 samples reanalyzed for PFHS, and 22 of the 24 samples reanalyzed for PFOS. Those samples that did not meet matrix spike recovery criteria were not reported (NR) due to quality control failures. Percent solids for the soil and sludge samples are detailed in Table III. 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 soil and sludge according to Protocol P0001131 (Appendix A). MPI Research Page 11 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFBS, PFHS, and PFOS in soil and sludge using the Direct Inject conditions specified in Protocol Amendment 12. 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 January 25, 2007 and the analytical termination date for this interim report was February 7, 2007. 4.0 ANALYTICAL TEST SAMPLES Nine soil samples (Exygen ID: C0152909, C0152917 - C0152920, C0152924, C0152934, C0152935, and C0152939) were received on wet ice on February 17, 2006 from Tim Frinak at Weston Solutions, Inc. Nineteen soil samples (Exygen ID: C0153316 - C0153319, C0153323, C0153324, C0153327, C0153328, C0153332, C0153337 C0153339, C0153342, C0153348 - C0153350, C0153354, C0153358, and C0153359) were received on wet ice on February 22, 2006 from Tim Frinak at Weston Solutions, Inc. Thirteen soil samples (Exygen ID: C0156344, C0156356, C0156366, C0156370, C0156371, C0156375, C0156380, C0156382, C0156384, C0156388, C0156389, C0156391, and C0156394) were received on wet ice on March 2, 2006 from Tim Frinak at Weston Solutions, Inc. Three soil samples (Exygen ID: C0159457, C0159461, and C0159471) were received on wet ice on March 10, 2006 from Tim Frinak at Weston Solutions, Inc. Seven soil samples (Exygen ID: C0160871, C0160881, C0160882, C0160885, C0160897, C0160899, and C0160900) were received on wet ice on March 15, 2006 from Tim Frinak at Weston Solutions, Inc. One sludge sample (Exygen ID: C0171106) was received on wet ice on April 18, 2006 from Tim Frinak at Weston Solutions, Inc. All samples were logged in by Exygen personnel and placed in refrigerated storage. Sample log-in and chain of custody information is located in the raw data package associated with this interim report. Storage records will be kept at Exygen Research. 5.0 REFERENCE MATERIAL The analytical standards, PFBS (SP0008058) and PFHS (SP0008057), were supplied by 3M. PFBS (SP0008058) was received from 3M at Exygen on September 6, 2006. PFHS (SP0008057) was received from 3M at Exygen on September 5, 2006. The analytical standard PFOS (SP0002694) was purchased from Fluka Corporation and was received at Exygen on April 23, 2003. MPI Research Page 12 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated. Compound PFBS PFHS PFOS Exveen Inventory No. SP0008058 SP0008057 SP0002694 Lot # 2 NB-120067-69 430180-1 Puritv (% ) 97.3 98.6 101.2 Expiration Date 01/17/08 10/18/07 10/31/07 The molecular structures of PFBS, PFHS and PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (^FgSOa'K*) Transitions Monitored: 299 -> 99 Structure: FF FF F SO 3 FF FF PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFoSOaTC1") Transitions Monitored: 399 -- 80 Structure: FFF FF F F SO3 FFF FFF PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFnSOaTC1") Transitions Monitored: 499 --> 80 Structure: FFFF FF F SO3 FFFF FFFF MPI Research Page 13 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 6.0 DESCRIPTION OF ANALYTICAL METHOD The direct injection conditions outlined in Protocol Amendment 12 were used for this study. 6.1 Extraction Procedure for Soil/Sludge Samples A 0.5-gram portion of the soil/sludge sample was used for the extraction procedure. The sample was measured into a 15 mL polypropylene centrifuge tube. The appropriate samples were fortified and 4 mL of 80:20 acetonitrile:water was added. The samples were capped tightly and vortexed. The samples were placed into an ultrasonic bath at room temperature for ~2 hours. The samples were then centrifuged at ~3000 rpm for 10 minutes. A portion of the supemate was then transferred to an autosampler vial. Each sample was analyzed by LC/MS/MS electrospray. 6.2 Percent Solids Procedure for Soil/Sludge Samples Percent solids were determined using the procedure indicated in Exygen method V0000427. Approximately 20 grams of sample was weighed into a pan. The weight of the sample plus the pan was recorded. The sample was then dried in an oven overnight at 104 + 2 C. Then the sample was transferred to a dessicator and allowed to cool for ~15 minutes. Each sample was then weighed again, including the weight of the pan. The percent solid for each sample was then calculated. See Table III for percent solids results. 6.3 Preparation of Standards and Fortification Solutions A stock standard solution of each analyte was prepared as specified in the raw data. The stock standard solutions were prepared at a concentration of 10,000 pg/mL by dissolving 1.0 g of the standard (corrected for purity and salt content, if necessary) in acetonitrile. From these stock solutions, a 1000 pg/mL mixed fortification standard solution was prepared by taking 10 mL of each stock and bringing the volume up to 100 mL with acetonitrile. By taking 10 mL of the 1000 pg/mL mixed fortification standard and bringing the volume up to 100 mL with acetonitrile, a 100 pg/mL mixed fortification standard was prepared. By taking 10 mL of the 100 pg/mL mixed fortification standard and bringing the volume up to 100 mL with acetonitrile, a 10 pg/mL mixed fortification standard was prepared. By taking 10 mL of the 10 pg/mL mixed fortification standard and bringing the volume up to 100 mL with acetonitrile, a 1.0 pg/mL mixed fortification standard was prepared. By taking 10 mL of the 1.0 pg/mL mixed fortification standard and bringing the volume up to 100 mL with acetonitrile, a 0.1 pg/mL mixed fortification standard was prepared. By taking 10 mL of the 0.1 pg/mL mixed fortification standard and bringing the volume up to 100 mL with acetonitrile, a 0.01 pg/mL mixed fortification standard was prepared. MPI Research Page 14 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 A set of non-extracted calibration standards containing containing PFBS, PFHS, and PFOS were prepared in 50:50 acetonitrile:water for the direct inject method. The following concentrations were prepared: Cone, of Fort Fort Solution Volume (ng/mL)1 (mL) 100 5.0 100 2.5 100 1.0 5.0 10 2.5 10 1.0 10 0.50 10 0.25 10 1of PFBS, PFHS, and PFOS Volume of Fortified Sample (mL) 100 100 100 100 100 100 100 100 Final Cone, of Calibration Std. (ng/mL) 5.0 2.5 1.0 0.50 0.25 0.10 0.05 0.025 The stock standard solutions were stored in a freezer (-20 5C) when not in use. 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. 6.4 Chromatography Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~0.6 mins, -9.1 mins, and -11.8 mins, respectively. Peaks above the LOQ were not detected in any of the reagent blank samples corresponding to the analyte retention time. 6.5 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 0.025 ng/mL of PFBS, PFHS and PFOS. 6.6 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 1.4.1 HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Autosampler HP Vacuum Degasser HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm MPI Research Page 15 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Column Temp.: -30 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time (min') 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: -18 min Flow Rate: 0.3 mL/min Ions monitored: %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Analvte PFBS PFHS PFOS Mode negative negative negative Transition Monitored 299 99 399 -80 499 -> 80 Approximate Retention Time (min') ~0.6 min. -9.1 min. -11.8 min. 6.7 Quantitation and Example Calculation Ten 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 eight concentrations of standards. The concentration was determined from the following equations. 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 Slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. For samples fortified with known amounts of PFBS, PFHS and PFOS prior to extraction, Equation 2 was used to calculate the percent recovery. MPI Research Page 16 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Equation 2: Recovery (%) = (analyte found (ng/mL) - analyte in corresponding sample (ng/mLV) xl00% amount added (ng/mL) Equation 3 was used to convert the amount of PFBS, PFHS and PFOS found in ng/mL to ng/g (ppb). Equation 3: Analyte found (ppb) rAnalyte found (ng/mL) x volume extracted (4 mL)1 sample weight (0.5 g) Equation 4 was then used to calculate the amount of PFBS, PFHS and PFOS found in ppb based on dry weight. Equation 4: Analyte found (ppb) dry weight = Analyte found (ppb) x [100% / total solids(%)] NOTE: Total solids (%) = [dry weight (g) / wet weight (g)] x 100% An example of a calculation using an actual sample follows: Soil sample Exygen ID: C0152918 Spk G (Set: 012507A), fortified at 50 ng/mL with PFHS where: peak area 28900 intercept = 268 slope 35700 dilution factor = 100 ng/mL PFHS added (fort level) = 50 ng/mL in corresponding sample = 31.0 volume extracted (mL) 4 sample weight (g) 0.5 total solids (%) = 82.92 From equation 1: Analyte found (ng/mL) = [28900-2681 x 100 35700 From equation 2: % Recovery 80.2 ng/mL (80.2 ng/mL - 31.0 ng/mL) x 100% 50 ng/mL = 98% MPI Research Page 17 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 From equation 3: Analyte found (ppb) = (80.2ng/mLx 4mL) 0.5 g = 642 ppb From equation 4: Analyte found (ppb) dry weight = 642 ppb x (100% / 82.92%) = 774 ppb Note: Numbers may vary slightly due to rounding. 7.0 EXPERIMENTAL DESIGN For soil/sludge samples designated as laboratory matrix spikes, PFBS, PFHS, and PFOS were added at a known concentration to the samples in the laboratory after the samples were weighed for extraction. Of the 51 soil/sludge samples designated for reanalysis, four soil samples (Exygen ID: C0152934, C0153318, C0153324, and C0153327) had insufficient remaining sample mass to be re-analyzed. The remaining 47 soil/sludge samples were analyzed in eleven sets. Seven sets contained five samples, three sets contained four samples, and one set contained two samples. Each set included one matrix control blank and two matrix control blanks fortified at known concentrations. For each sample, a laboratory duplicate of the sample and two laboratory matrix spikes were also analyzed, with the exception of sample CO152917, which only had sufficient remaining sample mass for the reanalysis of the sample and one laboratory matrix spike. The laboratory spikes were fortified with known concentrations of PFBS, PFHS and PFOS. 8.0 RESULTS All soil and sludge samples were initially analyzed by the SPE methods outlined in the protocol in previous reports (see Interim Reports #21, #22, and #24). When the results did not meet quality objectives, the samples were re-analyzed by the Direct Inject method outlined in Protocol Amendment 12 to eliminate the interference that was causing the SPE data to not meet quality control standards. The limit of quantitation for PFHS and PFOS in soil and sludge was 0.2 ng/g (wet weight). Due to the instrument conditions detailed in Section 6.6, the PFBS calibration curve had a raised LOQ as shown in the raw data. The LOQ provided is assuming wet weight values and the results for the actual samples are reported based on dry weight. MPI Research Page 18 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Analytical results and assessed accuracies for the analysis of PFBS, PFHS and PFOS in soil and sludge samples based on dry weight are summarized in Table I. Fortification recoveries for the analysis of PFBS, PFHS and PFOS in soil and sludge samples based on wet weight are summarized in Table II. The overall average percent recoveries standard deviations for PFBS, PFHS and PFOS in the soil and sludge samples based on wet weight were 94 24%, 83 14%, and 107 26%, respectively. Accuracies were assessed for each sample by reviewing the individual quality control results obtained for each sample site. 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. Quantitative results were obtained for 21 of the 23 samples reanalyzed for PFBS, 6 of the 11 samples reanalyzed for PFHS, and 22 of the 24 samples reanalyzed for PFOS. Those samples that did not meet matrix spike recovery criteria were not reported (NR) due to quality control failures. Percent solids for the soil and sludge samples are detailed in Table III. 9.0 CONCLUSIONS Except as noted above, the soil and sludge samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to protocol P0001131. There were no circumstances that would have affected the quality or integrity of the data. 10.0 RETENTION OF DATA AND SAMPLES All original paper data generated by Exygen Research that pertains to this interim report 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 interim analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. MPI Research Page 19 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 TABLES MPI Research Page 20 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Soil/Sludge Samples Exvaen ID CO152909 CO152909 Rep C0152917 C0152918 C0152918 Rep C0152919 C0152919 Rep C0152920 C0152920 Rep C0152924 CO152924 Rep CO152935 C0152935 Rep CO152939 C0152939 Rep C0153316 C0153316 Rep C0153317 C0153317 Rep C0153319 CO153319 Rep C0153323 CO153323 Rep CO153328 C0153328 Rep CO153332 C0153332 Rep CO153337 C0153337 Rep CO153338 CO153338 Rep CO153339 C 0153339 Rep CO153342 C0153342 Rep C0153348 CO153348 Rep C0153349 C0153349 Rep C 0 1 53350 C0153350 Rep C0153354 C0153354 Rep C0153358 C0153358 Rep CO153359 C0153359 Rep C lie n t S a m ple ID D F 12-S B -G P 02-0-0010 D F 12-S B -G P 02-0-0010* D F 12-S B -G P 04-0-0000 D F 12-S B -G P 04-0-0005 D F 12-SB -G P 04-0-0005* D F 12-S B -G P 04-0-0010 D F 12-S B -G P 04-0-0010* D F 12-S B -G P 04-0-0020 DF 12-SB -G P 04-0-0020* DF 13-SB-G PO 1-0-0005 DF13-SB-GP01 -0-0005* D F 13-S B -G P 03-0-0005 DF 13-SB -G P 03-0-0005* D F 13-S B -G P 04-0-0000 DF 13-SB -G P 04-0-0000* D F 10-S B -G P 03-0-0000 DF 10-S B -G P 03-0-0000* D F 10-S B -G P 03-0-0005 D F 10-S B -G P 03-0-0005* D F 10-S B -G P 03-0-0020 DF 10-S B -G P 03-0-0020* D F 10-S B -G P 04-0-0010 D F 10-S B -G P 04-0-0010* D F 11-S B -G P 01-0-0005 D F 1 1-SB-G P01 -0-0005* D F 11-S B -G P 01-0-0050 D F 1 1-SB-G P01 -0-0050* D F 11-S B -G P 02-0-0050 D F 11-S B -G P 02-0-0050* DF11-SB-GPO3-0-0000 D F 11-S B -G P 03-0-0000* D F 1 1-S B -G P 03-0-0005 D F 11-S B -G P 03-0-0005* D F 1 1-S B -G P 03-0-0020 DF 11-SB-GP03-0-0020* D F 1 1-SB -G P 04-0-0020 D F 11-S B -G P 04-0-0020* D F 11-S B -G P 04-0-0050 DF 11-SB-GP04-0-0050* D F 05-S B -G P 01-0-0000 DF05-SB-GP01 -0-0000* D F 05-S B -G P 01-0-0050 DF05-SB-GP01 -0-0050* D F 05-S B -G P 02-0-0010 D F 05-S B -G P 02-0-0010* D F 05-S B -G P 02-0-0020 D F 05-S B -G P 02-0-0020* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanaaulfonate_______Parfluorohexaneaulfonate_______Perfluorooctan-- ulfonata Analyte Found (ppb, ng/g) Dry Weight 3.40 4.16 Assessed Accuracy (+/-% ) 30 30 Analyte Found (ppb, ng/g) Dry Weiaht Assessed Accuracy (+/-% ) . - Analyte Found (ppb, ng/g) Drv Weiaht Assessed Accuracy </-%) _ - - - 4.00 40 - - _ 299 30 NR . - 319 30 NR - - . 7510 40 - - 24800 40 . _ 14000 30 - - - 13600 30 8.29 7.51 50 50 - _ - . - - . 431 30 - - 931 30 9.37 8.64 . - 30 30 - . 11100 40 - 17300 40 _ 40500 30 - 34600 30 9.46 10.6 30 30 - _ 23200 50 - 29800 50 3.48 4.61 30 30 '- . -- . 62.3 30 20900 50 - 62.3 30 21000 50 . 201 30 49600 30 - 203 30 40500 30 9.70 20.7 30 30 - _ - 4310 7160 30 30 69.3 71.6 30 30 _-. .- *- ND 30 ND 30 . -. - *- NR . NR - - 8930 30 - 8120 30 - . 9470 30 - - 9860 30 _ 448 30 - - 481 30 _ . 226 30 - - 175 30 47.6 30 - 59.0 30 . - 24.7 27.1 30 30 _ - - ND 30 ND 30 ND 30 ND 30 _ 118 30 - 126 30 . 117 30 - 89.7 30 ' Laboratory Duplicate ND = Not detected at or above 0.8 ng/g (wet weight). NR = Not reported due to quality control failures. MPI Research Page 21 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Soil/Sludge Samples (Continued) E x v n e n ID C0156344 C0156344 Rep CO156356 C015 6 3 5 6 Rep C0156366 CO156366 Rep CO156370 C0156370 Rep C0156371 CO156371 Rep CO156375 CO156375 Rep CO156380 C015 6 3 8 0 Rep C 0156382 C015 6 3 8 2 Rep C0156384 C0156384 Rep C0156388 C0156388 Rep C 0156389 C0156389 Rep C 0 1 56391 C0156391 Rep C 0156394 C 0156394 Rep C0159457 C0159457 Rep C0159461 C0159461 Rep C 0 1 59471 C0159471 Rep C0160871 C 0160871 Rep C0160881 C 0160881 Rep CO160882 C0160882 Rep C0160885 C0160885 Rep C0160897 C0160897 Rep CO160899 C0160899 Rep CO160900 C0160900 Rep C0171106 C0171106 Rep Client Sample ID DF13-SS-SS03-0-0000 DF13-SS-SS03-0-0000* D F 10-S S -S S 02-0-0000 DF10-SS-SS02-0-0000* DF11-SS-SS01-0-0000 D F 1 1 -S S -S S 0 1 -0-0 000* D F 1 1-SS -S S 02-0-0003 D F 1 1-SS-S S 02-0-0003* DF 11-SS-SS03-0-0000 D F 1 1-SS-S S 03-0-0000* D F 1 1-SS -S S 04-0-0003 DF11-SS-SS04-0-0003* DF05-SS-SS02-0-0000 DF05-SS-SS02-0-0000* D F 1 2 -S S -S S 0 1-0-0000 D F 1 2 -S S -S S 0 1 -0 -0 0 0 0 * DF12-SS-SS02-0-0000 D F 12-SS -S S 02-0-0000* SS12-SS-SS04-0-0000 S S 12-S S -S S 04-0-0000* D F 12-S S -S S 04-0-0003 DF12-SS-SS04-0-0003* D F 12-S S -S S 05-0-0003 DF12-SS-SS05-0-0003* DF13-SS-SS02-0-0000 DF13-SS-SS02-0-0000* D A L -S B -6 0 4 R -0 -0 1 00 D A L -S B -6 0 4 R -0 -0 100* DAL-SB-605R-0-0010 DAL-SB-605R-0-0010* D A L -S B -1 3 8 R -D B -0 1 00 D A L -S B -1 3 8 R -D B -0 1 00* DF05-SS-SS03-0-0000 DF05-SS-SS03-0-0000* D F 0 9 -S S -0 0 0 7 -0 -0 0 0 0 DF09-SS-0007-0-0000* DF09-SS-0007-0-0003 DF09-SS-0007-0-0003* DF09-SS-0008-0-0000 DF09-SS-0008-0-0000* D F09-SS-0011-0-0010 DF09-SS-0011-0-0010* D F 09-S S -0 0 1 2 -D B -0 0 0 0 D F 0 9 -S S -0 0 1 2 -D B -0 0 0 0 * DF09-SS-0012-0-0003 DF09-SS-0012-0-0003* DPWS-SL-DCTP01 -0-0000 DPW S-SL-DCTP01 -0-0000* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutan-- ulfonate______ Perfluorolrxanasulfonate_______ Parfluorooctanaaulfonata Analyte Found (PPb, ng/g) Dry Weight Assessed Accuracy (/-% ) Analyte Found (PPb, ng/g) Dry Weight Assessed Accuracy (+/- %) Analyte Found (PPb, ng/g) Dry Weight Assessed Accuracy {*/-% ) 17.5 30 . . , 22.5 30 - - - . NR _ - _ - - NR . -- 9.24 6.80 30 30 - 5.81 5.92 - 40 40 . - - NR NR 6.26 30 7.49 30 - _ NR - NR 17.0 50 14.1 50 _ - -- NR . - -- . 23300 - 10800 _ -- -- . .. -- . -- _ - . 834000 - 857000 . 479000 - 443000 .- - 30 30 . - . - _ - . - _ - 30 30 30 30 . NR 1.06 ND 20.6 21.9 -- . -- . -- 40 40 - . -- . 15.4 - 15.6 30 30 - _ -- _ 11900 30 - 11700 30 . 803000 30 - 739000 30 __ - -- . 9.40 50 - 8.17 50 30 - 30 - - ._ -- _ NR - _ - 6.64 6.29 7.38 7.33 6.40 - - . - 30 30 30 30 30 NR NR NR NR - - - NR . -- . -- . -- _ -- - - . . - . - . - 6.96 30 _ - - -- . 1170 30 - 1630 30 'Laboratory Duplicate ND = Not detected at or above 0.4 ng/g (wet weight). NR 3 Not reported due to quality control failures. MPI Research Page 22 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil/Sludge Samples Sample Description Amount Spiked (na/g) C4 Sulfonate PFBS Wet Weight Amt Found Amount in Sample Recovered Recovery (ng/g) (ng/g) (%> C6 Sulfonate PFHS Wet Weight Am t Found Amount in Sample Recovered Recovery (ng/g) (ng/g) <%) C8 Sulfonate PFOS Wet Weight Am t Found in Sample (ng/g) Amount Recovered Recovery (ng/g) (%) DF12-SB-GP02-0-0010 (C0152909 Spk C. 8 ppb Spike) DF12-SB-GP02-0-0010 (C0152909 Spk 0 ,2 0 ppb Spike) 8 20 2.86 2.86 10.9 101 NA NA - - - DF12-SB-GP04-0-0000 (C0152917 Spk E, 20 ppb Spike) 20 - 3.27 16.0 64 . DF12-SB-GP04-0-0005 (C0152918 Spk 6 ,4 0 0 ppb Spike) DF12-SB-GP04-0-0005 (C0152918 Spk H, 400000 ppb Spike) 400 400000 . . 248 642 99 NR NR NR DF12-SB-GP04-0-0010 (C01S2919 Spk 1,20000 ppb Spike) DF12-SB-GP04-0-0010 {C0152919 Spk J, 40000 ppb Spike) 20000 40000 . . - 6300 33400 136 - 6300 NA NA DF12-SB-GP04-0-0020 (C0152920 Spk K, 8000 ppb Spike) DF12-SB-GP04-0-0020 (C0152920 Spk L, 20000 ppb Spike) 8000 20000 . .. - 11800 19800 100 - 11800 NA NA DF13-SB-GP01-0-0005 (C0152924 Spk C, 4 ppb Spike) 4 7.10 9.12 51 DF13-SB-GP01-0-0005 (C0152924 Spk D, 8 ppb Spike) 8 7.10 NA NA - - DF13-SB-GP03-0-0005 (C0152935 Spk G, 400 ppb Spike) DF13-SB-GP03-0-0005 (C0152935 Spk H, 800 ppb Spike) 400 800 . . - 357 787 108 - 357 NA NA DF13-SB-GP04-0-0000 (C0152939 Spk 1,20000 ppb Spike) DF13-SB-GP04-0-0000 (C01S2939 Spk J, 40000 ppb Spike) 20000 40000 . -- 9200 36600 137 - - 9200 NA NA DF10-SB-GP03-0-0000 (C0153316 Spk K, 20 ppb Spike) DF10-SB-GP03-0-0000 (C0153316 Spk L, 200000 ppb Spike) 20 200000 7.22 25.5 - 91 - - - 31200 224000 96 DF10-SB-GP03-0-0005 (C0153317 Spk C, 20 ppb Spike) DF10-SB-GP03-0-0005 (C0153317 Spk 0,80000 ppb Spike) 20 80000 7.26 30.2 - 115 - . . - - 17800 138000 150 DF10-SB-GP03-0-0020 (C0153319 Spk G, 8 ppb Spike) DF10-SB-GP03-0-0020 (C0153319 Spk H, 20 ppb Spike) 8 20 2.81 2.81 10.6 97 NA NA . -- -- DF10-SB-GP04-0-0010 (C0153323 Spk 1,80 ppb Spike) DF10-SB-GP04-0-0010 (C0153323 Spk J, 40000 ppb Spike) 80 40000 . . 50.2 113 79 - - - - 16900 74500 144 NR h Not reported due to quality control failure. NA = Not applicable. This matrix spike concentration was not used to assess the accuracy for this analyte. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. MPI Research Page 23 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil/Sludge Samples (Continued) Sample Description DF11-SB-GP01 -0-0005 (C0153328 sp k E, 400 ppb Spike) DF11-SB-GP01-0-0005 (C0153328 Spk F, 40000 ppb Spike) DF11-SB-GP01-0-0050 (C0153332 Spk G, 40 ppb Spike) DF11-SB-GP01-0-0050 (C0153332 Spk H, 8000 ppb Spike) DF11-SB-GP02-0-0050 (C0153337 Spk I, 200 ppb Spike) DF11-SB-GP02-0-0050 (C01S3337 Spk J, 400 ppb Spike) D F 1 1-S B -G P 0 3 -0 -0 0 0 0 (C01S3338 Spk K, 4 ppb Spike) DF11-SB-GP03-0-0000 (C0153338 Spk L, 8 ppb Spike) DF11-SB-GP03-0-0005 (C01S3339 Spk C, 8 ppb Spike) D F 1 1-S B - G P 0 3 - 0 - 0 0 0 5 (C0153339 Spk D, 6000 ppb Spike) DF11-SB-GP03-0-0020 (C0153342 Spk E, 2000 ppb Spike) D F 1 1- S B - G P 0 3 - 0 - 0 0 2 0 (C0153342 Spk F, 4000 ppb Spike) DF11-SB-GP04-0-0020 (00153348 Spk G, 200 ppb Spike) DF11-SB-GP04-0-0020 (C0153348 Spk H, 400 ppb Spike) D F 1 1-S B -G P 0 4 -D B -0 0 5 0 (C0153349 Spk 1,200 ppb Spike) D F 1 1-S B -G P 0 4 -D B -0 0 5 0 (C0153349 Spk J, 400 ppb Spike) DF05-SB-GP01-0-0000 (C0153350 Spk K, 40 ppb Spike) DF05-SB-GP01-0-0000 (C0153350 Spk L, 200 ppb Spike) DF05-SB-GP01-0-0050 (C01533S4 Spk C, 40 ppb Spike) DF05-SB-GP01-0-0050 (C0153354 Spk D, 200 ppb Spike) DF05-SB-GP02-0-0010 (C0153358 Spk E, 4 ppb Spike) DF05-SB-GP02-0-0010 (C0153358 Spk F, 400 ppb Spike) DF05-SB-GP02-0-0020 (C0153359 Spk G, 4 ppb Spike) DF05-SB-GP02-0-0020 (C01S3359 Spk H, 200 ppb Spike) Amount Spiked (no/q) C4 Sulfonate PFBS Wet Weight Amt Found Amount in Sample Recovered Recovery (nq/fl) (nq/q) (%) C6 Sulfonate PFHS Wet Weight Am t Found Amount in Sample Recovered Recovery (ng/g) (ng/g) (%> C8 Sulfonate PFOS Wet Weight Amt Found in Sample (ng/g) Amount Recovered (ng/g) Recovery (% ) 400 166 551 96 40000 40 7.68 48.6 102 - - 40900 69400 71 .. . 8000 200 56.3 -197 70 . - - 3420 10200 85 ... . 400 56.3 NA NA 4 ND 5.11 128 . -- ... - . 8 ND NA NA 8 NR NR NR -- - .. -- . 8000 2000 - -- . - - 7500 13300 73 . . 7830 NA NA 4000 - - - - 7830 11400 89 . . _200 359 502 72 400 - - - - 359 NA NA . . .200 186 364 89 400 40 - - - 186 NA NA , 40.9 69.1 71 . 200 - - 40.9 40 19.9 48.6 72 NA NA ._ . - . 200 19.9 NA NA 4 ND 3.77 94 - ._ - . 400 - -- 4 ND 4.81 120 - - - 99.2 582 121 .. . 200 - - - 104 271 84 ND * Not detected at or above 0.8 ng/g (wet weight). NR = Not reported due to quality control failure. NA * Not applicable. This matrix spike concentration was not used to assess the accuracy for this analyte. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. MPI Research Page 24 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil/Sludge Samples (Continued) Sample Description DF13-SS-SS03-0-0000 (C015S344 Spfc 1, 4 ppb Spike) DF13-SS-SS03-0-0000 (C01S6344 Spk J, 20 ppb Spike) DF10-SS-SS02-0-0000 (C0156356 Spk K, 4 ppb Spike) DF10-SS-SS02-0-0000 (C0156356 Spk L, 20 ppb Spike) DF11-SS-SS01-0-0000 (C0156366 Spk C. 8000 ppb Spike) DF11-SS-SS01 -0-0000 (C0166366 Spk 0 ,20000 ppb Spike) DF11-SS-SS02-0-0003 (C01SS370 Spk E, 8 ppb Spike) D F 1 1- S S -S S 0 2 -0 -0 0 0 3 (C0156370 Spk F, 20 ppb Spike) D F 1 1- S S -S S 0 3 -0 -0 0 0 0 (C0156371 Spk G, 4 ppb Spike) DF11-SS-SS03-0-0000 (C0156371 Spk H, 8 ppb Spike) DF11-SS-SS04-0-0003 (C0156375 Spk 1.4 ppb Spike) D F 1 1- S S -S S 04-0-0003 (00150375 Spk J, 20 ppb Spike) DF05-SS-SS02-0-0000 (00156380 Spk K, 4 ppb Spike) DF05-SS-SS02-0-0000 (C0156360 Spk L. 8 ppb Spike) DF12-SS-SS01 -0-0000 (C0156382 Spk C, 8 ppb Spike) DF12-SS-SS01-0-0000 (C0156382 Spk D, 20 ppb Spike) DF12-SS-SS02-0-0000 (C0156384 Spk E, 8 ppb Spike) D F12-SS-SS02-0-0000 (C0156364 Spk F, 200000 ppb Spike) DF12-SS-SS04-0-0000 (C0156388 Spk G, 80000 ppb Spike) DF12-SS-SS04-0-0000 (C0156388 Spk H, 400000 ppb Spike) DF12-SS-SS04-0-0003 (C0156389 Spk 1,6 ppb Spike) DF12-SS-SS04-0-0003 (C0156389 Spk J, 20 ppb Spike) DF12-SS-SS05-0-0003 (C0156391 Spk K, 6000 ppb Spike) DF 12-SS-SS05-0-0003 (C0156391 Spk L, 20000 ppb Spike) DF13-SS-SS02-0-0000 (C0156394 Spk C, 200000 ppb Spike) DF13-SS-SS02-0-0000 (C0156394 Spk D, 400000 ppb Spike) Amount Spiked (ng/g) C4 Sulfonate PFBS Wet Weight Amt Found Amount in Sample Recovered Recovery (ng/g) (ng/g) (%> C6 Sulfonate PFHS Wet Weight Amt Found Amount in Sample Recovered Recovery (ng/g) (ng/g) (%> C8 Sulfonate PFOS Wet Weight Amt Found in Sample ino/ci) Amount Recovered (ng/g) Recovery (%) 4 11.4 20 11.4 4. 20 8000 20000 8 6.54 20 6.54 4 4.63 8 4.63 4 20 4 5.15 8 5.15 8 20 8 200000 80000 400000 11.7 . 8 20 8000 20000 200000 400000 NR NR . - _ - NA NA 35.0 118 .. - . - 13.5 NA 87 NA 7.27 66 NA NA .. - 9.68 113 NA NA .. -- 16.2 56 -- .. -- NR NR NR NR .. -- .. -- NR NR . . . NR NR . NR NR . . . . . - -- - NR NR NR NR - . . 18900 NA NA - 18900 33000 71 ... ... - - . - NR NR NR NR ., . - . - NR NR NR NR . . - - - 574000 783000 105 . 356000 NA NA - - 356000 872000 129 . -- -- . . 9680 18900 115 - - 9680 NA NA . _ 607000 NA NA - - 607000 1070000 116 NR = Not reported due to quality control failure. NA Not applicable. This matrix spike concentration was not used to assess the accuracy for this analyte. Note: Since this summary table shows rounded results, recovery values may vary slightly from die values In the raw data. MPI Research Page 25 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Soil/Sludge Samples (Continued) Sample Description DAL-SB-604R-0-0100 (C0156457 Spk E. 2 ppb Spike) DAL-SB-604R-0-0100 (C0159457 Spk F, 4 ppb Spike) DAL-SB-605R-0-0010 (C0159461 Spk G, S ppb Spike) DAL-SB-605R-0-0010 (C0159461 Spk H, 20 ppb Spike) DAL-SB-138R-DB-0100 (C0159471 Spk 1,20 ppb Spike) DAL-SB-138R-DB-0100 (C0159471 Spk J, 40 ppb Spike) DF05-SS-SS03-0-0000 (C0160871 Spk K, 8 ppb Spike) DF05-SS-SS03-0-0000 (C0160871 Spk L, 20 ppb Spike) DF09-SS-0007-0-0000 (C0160881 Spk C, 2000 ppb Spike) DF09-SS-0007-0-0000 (C0160881 Spk D. 4000 ppb Spike) DF09-SS-0007-0-0003 (C0160882 Spk E, 8 ppb Spike) DF09-SS-0007-0-0003 (C0160882 Spk F, 20 ppb Spike) DF09-SS-0008-0-0000 (C016088S Spk G, 8 ppb Spike) DF09-SS-Q008-0-0000 (C0160885 Spk H, 20 ppb Spike) DF09-SS-0011-0-0010 (C0160897 Spk I, 8 ppb Spike) DF09-SS-0011-0-0010 (C0160897 Spk J, 20 ppb Spike) DF09-SS-0012-DB-0000 (00160899 Spk K, 8 ppb Spike) DF09-SS-0012-DB-0000 (C0160899 Spk L, 20 ppb Spike) DF09-SS-0012-0-0003 (C0160900 Spk C, 8 ppb Spike) DF09-SS-0012-0-0003 (C0160900 Spk D. 20 ppb Spike) DPWS-SL-DCTP01 -0-0000 (C0171106 Spk E, 400 ppb Spike) DPWS-SL-DCTP01-0-0000 (C0171106 Spk F, 800 ppb Spike) Amount Spiked (no/g) C4 Sulfonate PFBS Wet Weight Am t Found Amount in Sample Recovered Recovery (ng/g) (ng/g) (% ) C6 Sulfonate PFHS Wet Weight Am t Found in Sample (no/fl) Amount Recovered (no/Q) Recovery (% ) C8 Sulfonate PFOS Wet Weight Amt Found Amount in Sample Recovered Recovery (ng/g) (no/a) (%) 2 0.816 2.06 62 4 0.816 NA NA 8_ 20 - .20 12.4 40 - 12.4 8 16.6 23.7 89 . 20 2000 16.6 NA NA . 4000 8 - _ NR 20 - - NR 8 . . NR 20 - - - NR .8 5.72 14.7 112 20 5.72 NA NA 8 6.67 15.4 109 20 6.67 NA NA 8 5.64 15.8 127 20 5.64 .400 NA NA .. 800 - -- . . - - . 7.71 - 7.71 30.8 92 NA NA .. . - . NR - NR NR NR . NR NR NR NR . NR NR .. . - -- ... -- 268 - - 268 - 19.6 149 NA NA . - .. - NR NR NR NR .. - .. - .. - .. - .. - 737 117 NA NA A verage: S ta n da rd D e via tio n : 94 24 A verage: S ta n da rd D e via tio n : 83 14 NR = Not reported due to quality control failure. NA = Not applicable. This matrix spike concentration was not used to assess the accuracy for this analyte. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. A verage: S ta n d a rd D e via tio n : 107 26 MPI Research Page 26 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Table III. Total Percent Solids in Soil/Sludge Samples Exygen ID C0152909 C0152917 C0152918 C0152919 C0152920 C0152924 CO152934 C0152935 C0152939 C0153316 C0153317 C0153318 C0153319 C0153323 C0153324 C0153327 C0153328 C0153332 C0153337 C0153338 C0153339 C0153342 C0153348 C0153349 C0153350 C0153354 C0153358 C0153359 C0156344 C0156356 C0156366 C0156370 C0156371 C0156375 C0156380 Client Sample ID DF12-SB-GP02-0-0010 DF12-SB-GP04-0-0000 DF12-SB-GP04-0-0005 DF12-SB-GP04-0-0010 DF 12-SB-GP04-0-0020 DF13-SB-GP01-0-0005 DF 13-SB-GP03-0-0000 DF 13-SB-GP03-0-0005 DF 13-SB-GP04-0-0000 DF 10-SB-GP03-0-0000 DF 10-SB-GP03-0-0005 DF 10-SB-GP03-0-0010 DF 10-SB-GP03-0-0020 DF 10-SB-GP04-0-0010 DF10-SB-GP04-0-0020 DF11-SB-GP01-0-0000 DF11-SB-GP01-0-0005 DF11-SB-GP01-0-0050 DF11-SB-GP02-0-0050 DF11-SB-GP03-0-0000 DF11-SB-GP03-0-0005 DF11-SB-GP03-0-0020 DF 11-SB-GP04-0-0020 DF 11-SB-GP04-0-0050 DF05-SB-GP01-0-0000 DF05-SB-GP01-0-0050 DF05-SB-GP02-0-0010 DF05-SB-GP02-0-0020 DF13-SS-SS03-0-0000 DF10-SS-SS02-0-0000 DF11-SS-SS01-Q-0000 DF11-SS-SS02-0-0003 DF11-SS-SS03-0-0000 DF11-SS-SS04-0-0003 DF05-SS-SS02-0-0000 Total Percent Solids (%) 83.95 81.74 82.92 83.85 84.73 85.57 80.93 82.87 82.68 77.08 76.78 81.84 80.75 80.67 84.01 79.27 82.39 79.21 81.25 84.51 84.04 82.75 80.19 82.04 85.92 80.79 83.92 89.00 65.54 86.29 81.11 70.80 79.72 89.38 82.35 MPI Research Page 27 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Table III. Total Percent Solids in Soil/Sludge Samples (Continued) Client Exygen ID__________________Sample ID__________________ Total Percent Solids (%) C0156382 DF12-SS-SS01-0-0000 82.65 C0156384 DF12-SS-SS02-0-0000 68.74 C0156388 SS12-SS-SS04-0-0000 74.29 C0156389 DF12-SS-SS04-0-0003 78.13 C0156391 DF12-SS-SS05-0-0003 81.22 C0156394 DF13-SS-SS02-0-0000 75.65 C0159457 DAL-SB-604R-0-0100 77.17 C0159461 DAL-SB-605R-0-0010 82.01 CO159471 DAL-SB-138R-DB-0100 80.36 C0160871 DF05-SS-SS03-0-0000 80.74 C0160881 DF09-SS-0007-0-0000 86.39 C0160882 DF09-SS-0007-0-0003 89.57 C0160885 DF09-SS-0008-0-0000 86.47 C0160897 DF09-SS-0011-0-0010 86.19 C0160899 DF09-SS-0012-DB-0000 90.43 C0160900 DF09-SS-0012-0-0003 88.07 C0171106 DPWS-SL-DCTP01 -0-0000 22.83 MPI Research Page 28 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 FIGURES MPI Research Page 29 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 Figure 1. Typical Calibration Curve for PFBS in 50:50 Acetonitrile: W ater 013107E P1131 Soil.rdb (PFBS): ''Linear1' Regression C'1 / * ' w eighting): y * 9.9Se+003 x + 202 (r * 0 .9930) Concentration. ng/m L Area, counts MPI Research Page 30 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Figure 2. Non-Extracted Standards of PFBS in 50:50 Acetonitrile:Water, 0.025 ng/mL and 0.05 ng/mL, Respectively I SS0079852 - PFBS (Standard) 299UM99Lanta -sam ple 1 o f 28 from 013107E.wiff A n a : 535 counts Height: 2.14e+001 cps RT: 8.585 min Tim e, min I SS0010851 PFBS (Standard) 299.0/99.0 amu - sample 2 of 28 from 0131Q7E.wiff Area: 1165 counts H eight: 3 .69e+001 cps RT: 0.577 min 3.62 MPI Research Page 31 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Figure 3. PFBS in Control Blank, 0.25 ng/mL Fortified Control Spike A and 2.5 ng/mL Fortified Control Spike B, Respectively C9221S19 Control - PFBS (Unknown) 299.0/99.0 amu sample 19 o f 28 from 91310TF.wiff p e a k not fourni) 2 3 4 5 6 7 8 9 10 11 12 13 14 1S 1C 17 T im e, min C 0221519 S p k A - PFBS (QC) 299 .0/99.0 amu - sam ple 11 of 28 from 013 107E .w iff Area: 3271 counts Height: 1.15e+002 cps RT: 0.593 min 0.59 100 4 i 1.78 ILL, 3.41 ,3.09 _7 -51j x 7 j j _ _10i42 15J 2 / v ,,___/ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 17 Tim e, min C0221519 Spk B - PFBS (0 0 )2 9 9 .0 /9 9 .0 amu - sample 12 of 28 from 013107E.wiff Area: 200 37 counts Height: 1.01 e+003 cps R T :0 .0 0 4 m in Ll<0 Qc_>. 0.00 1000-1 1 to 5 0 0 c O) 1.75 3.45 11.03 1 2 3 4 5 7 8 9 10 11 12 13 14 15 10 17 Tim e, min MFI Research Page 32 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 Figure 4. Chromatogram Representing a Soil/Sludge Sample Analyzed for PFBS (ExyLIMS ID: C0160900, Data Set: 013107E) I CO16O900 -PFBS (Uutuown) 299. M 9.0amu -ample 1*o f 20 from 013107E w iff Area: 7213 counts HelgAt: 292i+002cps RT: 0.574m/ 0.57 Intensity, cps MPI Research Page 33 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Figure 5. Typical Calibration Curve for PFHS in 50:50 Acetonitrile: W ater 012G07C P1131 Soil.rdb (PFHS): "L in e a i" Regression C' 1 / * ' w e ightin g): y 3 .5 e + 0 0 4 x + 5.89 (r 0 .9970) Concentration, ng/mL Area, counts MPI Research Page 34 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Figure 6. Non-Extracted Standards of PFHS in 50:50 Acetonitrile:Water, 0.025 ng/mL and 0.05 ng/mL, Respectively I SS009852 - PFHS (Standard) 399.0/80.0 amu -sample 1 o f 40 from &13107C.wiff Area: 1774counts Height: 7.99e*001cps RT: 9.13min 9.13 V) Q. O & to I Area: 1869 counts Height: t.1 0 e + 0 0 2 cps RT: 9 .1 4 min 9.14 MPI Research Page 35 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Figure 7. PFHS in Control Blank, 0.25 ng/mL Fortified Control Spike A and 2.5 ng/mL Fortified Control Spike B, Respectively C0221S19 Control -PFHS (Unknown) 399.0/80.0 amu -sample 10o f 40 from 013107C.wiff (peak not found) 12.81 12.43. 13.79 14.26 2.10 A yy 84 10.52, -A- 5 6 7 8 9 10 Tim e, min C 0221519 S p k A - PFHS (QC) 399 .0/80.0 amu - sam ple 11 of 40 from 013107C .w iff Area: 9136 counts Height: 5.71 e+002 cps R T :9 .1 0 min 11.82. 9.10 U 11.73^ 12.33 8 9 10 11 12 13 14 15 Tim e, min C0221519 Spk B - PFHS (Q 0 )399.0/80.0 amu - sample 12 of 40 from 013107C.wiff Area: 866 20 counts Height: 5.85e+ 003 cps R T :9 .1 1 min 4000 2000 0 9.11 i 89 10 Tim e, min 12 13 14 15 Jl MPI Research Page 36 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Figure 8. Chromatogram Representing a Soil/Sludge Sample Analyzed for PFHS (ExyLIMS ID: C0160882, Data Set: 013107C) I C0J60882 - PFHS (Unknown) 399.0/90.0 a n a -sample 22 o f 40 from 013107C.wiff Area: 49146 count* Height: Z69e+003cps RT: 9.17 min 9.17 Intensity, cps MPI Research Page 37 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Figure 9. Typical Calibration Curve for PFOS in 50:50 Acetonitrile:Water 013107E P1131 S oil.rdb (PFOS): "L in e a i" Regression ( ' 1 / * ' w eighting): y * 1 .3 7 e + 0 0 4 x + 2.31e+Q03 ( r * 0.9959) Concentration, ng/mL Area, counts MPI Research Page 38 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Figure 10. Non-Extracted Standards of PFOS in 50:50 Acetonitrile:Water, 0.025 ng/mL and 0.05 ng/mL, Respectively I SS009852 - PFOS (Standard) 499.0/80.0 amu ample 1 o f 41 from 012607C.wiff Area: 1597 count Height: 0.44e*001 cp KT: 11.0 min 11.81 CO Q. Tim e, min I SS0019851 - PFOS (Standard) 499 .0/80.0 am u - sam ple 2 of 41 from 012607C .w iff Area: 2 4 0 4 counts Height: 1.17e+002 cps RT: 11.8 min 11.81 &'co c c0) MPI Research Page 39 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Figure 11. PFOS in Control Blank, 0.25 ng/mL Fortified Control Spike A and 2.5 ng/mL Fortified Control Spike B, Respectively C 0227579 Control - PFOS (Unknown) 499LO/84XO amu sample 1$ o f 41 from 01260?C>wiff freak not found) r>A , 1 23456789 10 Time, min C 0221519 S p k A - PFOS (QC) 409 .0/80.0 a m u -s a m p le 11 of 41 from 012607C .w iff Area: 4 6 4 4 counts Height: 3.04e+ 002 cps RT: 11.8 min 11.81 3.13 11.20 U 10.83%*. M 1 23456789 10 Tim e, min C 0221519 Spk 0 - PFOS (QC) 499.0/80.0 amu - sam ple 12 of 41 from 012607C .w iff Area: 31 8 6 2 counts Height: 2 .04e+003 cps RT: 11.8 min , ,n 1 2 7 0 ^13.20 ^.14.43 15 16 17 2000 SS 1000 0 11.81 89 A 13.48 10 11 12 13 14 15 16 17 Tim e, min MPI Research Page 40 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Figure 12. Chromatogram Representing a Soil/Sludge Sample Analyzed for PFOS (ExyLIMS ID: C0153323, Data Set: 012607A) Intensity, cps C01S3323 - PFOS (Unknown) 49918/8816 amn -sam ple 33 o f 38 front Q12607A.wiff Area: 1742515 counts Height: 1.35e+005cps RT: 11.9 min 1 .3 *6 1.2e5 - 1.1*61.0*6- 9.0e4 8.0e 4 - 7.0 e4 6.0 e4 5.0e4- 4.0*4 - 3 .0 *4 - 2 .0 *4 1.0*4- 0.0J-----.----- .-----1-----,----- ------.-----.-----1234 667 8 Tim e, min 11.81 MPI Research Page 41 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 APPENDIX A Study Protocol P0001131 (ExyLIMS Study No. P0001131) with Analytical Methods, Protocol Amendments, and Deviations MPI Research Page 42 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 Page / o f 65 MPI Research Page 43 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 Page 2 o f 65 Page 44 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 JaisimhaKesan, S' Weston Solutions Michael A. Sgfitoro, Sponsor Representative 3M Comparfy Lydft Shaffer, Technii Exgen Research L I __________ ad, Quality Assurance Unit Date 2$ -OCT- Date ftk vo '-/ Date Page 3 o f 65 MPI Research Page 45 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 TE S........................................................... 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 EN T.......................................................................................................................................................... 11 R EPO R T.............................................................................................................................................................................11 SAFETY AND H EA LTH ................................................................................................................................................12 AMENDMENTS TO PROTO CO L............................................................................................................................... 13 DATA RECORD K EE PIN G ...........................................................................................................................................13 QUALITY A SSURANCE............................................................................................................................................... 14 RETENTION OF DATA AND ARCHIVING.............................................................................................................14 APPENDIX I, ANALYTICAL METHODS................................................................................................................. 15 Page 4 o f 65 MPI Research Page 46 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 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 (C4F9SO3TU) Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 - 99 Structure: F F F F s o 3- F F PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (C6Fi3S03'K+) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 - 80 Structure: FFF FFF F S03 Page 5 o f 65 MPI Research Page 47 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CsFiySOyTC*) Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 -* 99 Structure: OBJECTIVE The purpose of this study is to perform analysis for peTfluorobutanesulfonate (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 Page 48 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: POOO1131 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 j ohn.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 Page 49 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 o f 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 S -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 Page 50 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 Page 51 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 (,3C PFOA). PFOA and l3C PFOA are included in the solutions used to spike the samples. The results for PFOA and 13C 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 foT 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 lOng/mL 100 ng/mL Page 10 o f 65 MPI Research Page 52 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 ! I o f 65 MPI Research Page 53 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 Page 54 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: 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 of 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 / J o f 65 MPI Research Page 55 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 fforn 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 Page 14 o f 65 Page 56 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: POOO1131 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 Page 15 o f 65 Page 57 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001780 M ethod o f Analysis for the D eterm ination o f Perfloorooctanoic Acid (PFOA) in W ater by LC/MS/MS Analytical Testing Facility: Exygen Research 3038 Research Drive State College, PA 16801 Approved By: TU cU L Paul Connolly 1 Technical Leader, LC-MS, Exygen Research .2 a .. ' / J 'oJohhnn Flaahh<erty / Vice DPrmensi.dent, Operations, Exygen Research o|aAW Date Date MPI Research T out Pages: 7 Page 16 o f 65 Page 58 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research Method Number VOQQpgQ ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Pcrfluorooctanoic Acid (PFOA) in Water by L C /M S/M S 1.0 Scope This method is to be employed for the isolation and quantitation o f pcrfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in water. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult foe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 40 m L o f test sample for (traction. 3.2 No sample processing is needed for water samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction. 3.3 Any samples containing particles should be centrifuged at -3 0 0 0 rpm for -5 minutes and foe supernatant used for the extraction. 3.6 Sample collection procedures will be specified in foe sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater -H P L C grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifoge tubes. 5.5 15 m L disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. Pane 2 of ? Page 7 o f 65 MPI Research Page 59 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 ExygeaJUMweb M ethod Number VQ0Q178Q A N A L Y T IC A L m e t h o d Method o f Analysis for foe Determination o f Perfluorooctanoic Acid (PFOA) in Water by L C /M S /M S 5.12 SPB vacuum manifold 5.13 CentrifUge capable o f spinning 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6 .1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 825054)52130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 m M Ammonium Acetate in W ater 6.4 Mobile Phase (B ): Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B fmL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 p L (can be increased to as much as 50 pL). 6.7 Quantitation: Peak A rea-ex tern al standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and m ay be changed in ordeT to optimize foe HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, m onitoring 413 --369 m/z. The above conditions are inteoded as a guide and may be changed in order to optimize foe MSMS system. 8-0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o f water. Alternate volumes m ay be prepared. Page 3ui ' Page 8 o f 65 MPI Research Page 60 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 ExygcnRetouch Method Number V000I780 I ..... ............. ANALYTICAL METHOD Method o f Analyse for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by L C /M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in s 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o ftb e 10 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.4 A 0.1 pgftnL fortification solution o f PFOA is prepared by bringing 10 mL o f the 1.0 pg/raL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 p g fa L fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Volume o f Concentration of o f Fortification Volume Fortified Control Calibration Solution ioob) (mU Samsle (mL) Standard (not)* Calibration Standard ID (examole) 00 10 100 10 200 10 400 100 100 100 200 100 400 40 40 40 40 40 40 40 0 XCmmddyy-0 25 XCmmddyy-1 50 XCmmddyy-2 100 XCmmddyy-3 250 XCmmddyy-4 500 XCmmddyy-5 1000 XCmmddw-6 The extracted concentration o f the calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC extracted calibration standard. Page 4 o i ' Page 19 o f 65 MPI Research Page 61 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygra Retouch M ethod N umber VOOO1780 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PPOA) in Water by L C /M S/M S 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) must be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene tubes at 2C to 6aC, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one reagent control (method blank using HPLC water) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery fewthe batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Measure 40 m L o f sample o r a portion o f sample diluted to 40 mL with water into 50 mL polypropylene centriftige tubes (fortify as needed, replace lid and mix well). 11.2 Condition the C SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 m L o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.3 Load sample on conditioned Cii SPE cartridge. Discard eluate. 11.4 Elute with - 5 m L 100% methanol. Collect 5 mL o f eluate into graduated 15 m L polypropylene centriftige tubes (final volume * 5 mL). 11.5 Analyze samples using electrospmy LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sam ple and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels must be included in an analytical set. 12.3 A n entire set o f extracted calibration standards m ust be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for t seoond set o f extracted standards). In either case, extracted calibration standards must be the first and last injection in s sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area Pag 5 of 7 Page 20 o f 65 MPI Research Page 62 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygee Research M ethod Number V 0001780 I ANALYTICAL M ETHOD | Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Water by L C /M S/M S verctu calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike falls outside the acceptable limits, (he entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total num ber o f extracted standards injected. ^ 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R* 0.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 1 4 % within an analytical ran. I f retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/L) - (Peak area - intercept) x DF slope DF - factor by which the final volume was diluted, if necessary. Page 6 o f 7 Page 21 o f 65 MPI Research Page 63 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygco Research M ethod Number VQQOl780 | ............ANALYTICAL M E T H O D | Method o f Analysts for the Determination ofPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 14 2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [total analyte found (ng/L) analyte found in control (ng/L)] analyte added (ng/L) MPI Research Page 7 o f 7 Page 22 o f 65 Page 64 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method N um ber V0001781 Method o f Analysis for the Determ ination o f P erflu o ro o ctu o ic Acid (PFOA) la Soil by L C /M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C -JL Paul Connollyv ' Technical Leader, LC-MS, Exygen Research Date Date MPI Research Total Pages: 7 Page 23 o f 65 Page 65 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Rwwreh_________________________________________________ Method Number VOOOI78I [ ... AN ALYTICA L m e t h o d Method o f Analysis for the Determination ofperfluorooctanoic Acid (PFOA) in Soil by L C /M S/M S 1.0 Scope This method is to be employed for the isolation and quantitation ofperfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spcctrometric Detector (LC/MS/MS) in soil. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 15 g o f test sample for extraction. 3.2 No sample processing is needed for soil samples. 3.3 Samples stored refrigerated should b e allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly m ixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater - HPLC grade 4.2 M ethanol-H PLC grade 4.3 Ammonium Acetate -A .C .S . Reagent Grade 4.4 Perfluorooctanoic A d d - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance Uquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5*200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifoge tubes. 5.6 Disposable micropipets (S0-l00uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100*1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. Page 24 o f 65 MPI Research Page 66 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen teeewch M ethod Num ber VOW 1781 ANALYTICAL METHOD J Method o f Analysis for the Determination ofPerfluorooctaaoic Acid (PFOA) in Soil by L C /M S/M S 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 50 m L polypropylene tubes ai 5000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: FluophaseRP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase ( A ) : 2 m M Ammonium Acetate in W ater 6.4 Mobile Phase ( B ) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B ftnUroin) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 --369 m/z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optimize die MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 raM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o f water. Alternate volumes m ay be prepared. Pg 3 of 7 Page 25 o f 65 MPI Research Page 67 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reatiicb Method Number VOOO1781 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/inL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125*mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 10 pg/raL solution to a final volume oflOO with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f )he 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f die 0.1 pgfaiL solution to a final volume o f 100 with methanol in 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards am prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Concentration of Fortification Solution (obtri Fortification Volume of Volume Fortified Control (PU Sample (mL) Final Concentration of Calibration Standard (port* Calibration Standard ID (example) 0 0 40 10 100 40 0 XCmmddyy-0 25 XCmmddyy-1 10 200 40 10 400 40 50 XCmmddyy-2 100 XCmmddyy-3 100 100 100 200 100 400 40 40 40 250 XCmmddyy-4 500 XCmmddyy-S 1000 XCmmddw-6 * The extracted concentration o f (he calibration standard is equal to 8* its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC extracted calibration standard. Page 4 o f7 Page 26 o f 65 MPI Research Page 68 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V000I781 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) 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 with each set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene lubes at 2C to 6*C, up to two weeks. Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one reagent control (method blank using 5 mL o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction I t . 1 Weigh 5 g o f sample into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 1 U Add 5 m L o f methanol and shake on a wrist action shaker fo r- 1 S minutes 11.3 Transfer the tubes to an ultrasonic bath and sonicate for ~15 minutes. 11.4 Bring the volume up to 40 m L with water in the 50 m L polypropylene centrifuge tube. ^ 11.5 Centriftige for *10 minutes at ~3Q00 rpm. 11.6 Condition the C SPE cartridges (1 g, 6 m L) by passing 10 m L methanol followed by 5 mL o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.7 Load (decant) die sample on the conditioned C h SPE cartridge. Discard cluate. 11.8 Elute with ~5 mL 100% methanol. Collect 5 m L o f eluate into graduated 15 mL polypropylene centriftige tubes (final volume - S mL). 11.9 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards m ust be included at the beginning and at die end o f s sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set of Pag S o r 7 Page 27 o f 65 MPI Research Page 69 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Rwaarch_______________________________________________________ Method N umber V0001781 1 ............ ANALYTICAL M ETHOD ~ Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by L C /M S/M S extracted calibration standards m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be die first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA i t levels greater than the LOQ- If a blank contains PFOA at levels greater than 50 ng/L. then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known vslues. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must ho 0.992 (R2 0.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention tunes between standards and samples m ust not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. Page o f 7 Page 28 o f 65 MPI Research Page 70 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygcn Protocol Number: P0001131 Exygcn Research Method Number VOOO1781 I ANALYTICAL M ETHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by foe Mass Lynx software program: PFOA found (ng/L) - (Peak area - intercept! x DF slope DF factor by which foe final volume waa diluted, if necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate foe percent recovery. Recovery (%) - [ total analyte found (ng/L) - analyte found in control (ng/L)] analyte added (ng/L) 14.3 Uae the following equation to convert the amount o f PFOA found in ng/L to ng/g (ppb). PFOA found (ppb) - fPFOA found (ng/L) x volume extracted (0.04D1 sample weight (5 g) 14.4 Use the following equation to calculate the amount o f PFOA found in pph based on dry weight. PFOA found (ppb) dry weight PFOA found (ppb) x [100% / total solids(%)] Page 7 of? Page 29 o f 65 MPI Research Page 71 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 Exygen Protocol Number: POOO1131 ANALYTICAL METHOD Method N um b: V0001782 M ethod o f Analysis fo r the D eterm ination o f Perfluorooctanoic Acid (PFOA ) ia Sedtmeot by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: vU. c jL Paul Connolly I Technical Leader, LC-MS, Exygen Research u p ? I /________ /io h n Flaherty / Vice President, Operations, Exygen Research iok b M Date Date MPI Research Total Pages; 7 Page 30 o f 65 Page 72 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 Exygen Protocol Number; P0001131 Exyftn Research Method Number V000I7B2 ANALYTICAL M ETHOD H Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LCVMS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in sediment. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 30 g o f teat sample for extraction. 3.2 No sample processing is needed for sediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly m ixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol -H P L C grade 4.3 Acetic Acid - Reagent grade 4.4 Ammonium Acetate - A C S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem M ats Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene ccntrifbge tubes. 5.5 15 m L disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL), 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (140-1000 p L and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. Page 2 of 7 Page 31 o f 65 MPI Research Page 73 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygcn R ncveh Method Number VOOO1782 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment bv L C /M S /M S ' 3.13 Vortexer. 5.14 Wrist-actioo shaker. 5.15 CentriAige capable o f spinning 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophaae RP (Keystone Scientific), 2.1 m m x 5 0 m m .5^ (P/N: 82505>052130) 6.2 Temperature: 30C 6.3 Mobile Phase ( A ): 2 m M Ammonium Acetate in W ater 6.4 Mobile Phase ( B ) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 ZA 65 65 25 25 65 Flow Rate s i n imL/min) 35 0.3 3$ 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: ~ 2 3 minutea. The above conditions are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 -* 369 m /z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 $ of ammonium acetate to 1000 mL o f water. Page 3 o f7 Page 32 o f 65 MPI Research Page 74 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 E x y g e n P r o to c o l N u m b e r: P 0 0 0 1 131 ExygcaR nm ch Method Number V0001782 I ANALYTICAL METHOD 1 Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment bv L C /M S/M S ' 8.2 Extraction Solutions 8.2.1 1% acetic acid in water is prepared by adding 10 m L o f acetic acid to 1000 mL o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 ml. with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pgfottL fortification solution o f PFOA is prepared by bringing 10 m L o f foe 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o ftb e 1.0 pg/mL solution to a final volume oflOO with methanol in a 125 m LLDPB bottle. 9.1.5 A 0.01 pg/tnL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4AC and are stable for a m aximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution offo eO .l pg/mL fortification solution. 9.2.2 The following is a typical cample: additional concentrations may be Concentration o f Fortification Solution (na/raL) too 100 too 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 20 1.0 0.5 0.2 Page 4 of 7 Page 33 o f 65 MPI Research Page 75 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygcn Protocol Number: P0001131 ExygenJUmrch Method Number V0001782 | ANALYTICAL M ETHOD 1 Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S 9.2.3 9.2.4 Store all calibration standards in 125-raL LDPE narrow-mouth bottles at 2C to 6C, tqi to six months. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f sample into SO m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 35 mLof\% aceticacid, cap, vortexand shakeona wrist action shaker for -6 0 minutes. 11.3 CentrifUge the tubes at -3 0 0 0 rpm for - 2 0 minutes. 11.4 Condition the C ti SPE cartridges (1 g, 6 mL) by passing 10 mL methanol mLfollowed by 20 ofH PLC water (~ 2 drop/sec). Do not let column nut dry 11.5 Load (decant) the sample on the conditioned C u SPE cartridge. Discard chute. 11.6 Add 20 m L o f methanol to the sediment left in the bottom o f the 50 mL centrifuge tube. Cap, vortex and shake on a wrist action shaker for *-30 minutes. 11.7 CentrifUge the tubes at -3 0 0 0 rpm for ~20 minutes. 11.8 Decant foe methanol onto the same SPE cartridge. Collect the eluate. 11.9 Wash the column with 4 mL o f methanol. Collect foe eluate and add it to the eluate collected in step 11.8. 11.10 Condition a second C u SPE cartridge (1 g, 6 m L) by passing lO m L methanol followed by 20 raL ofH PL C water (~ 2 drop/sec). Do not let column run dry 11.11 Add the methanol to -2 0 0 m L o f water and load on the second conditioned SPE cartridge. 11.12 Elute with - 5 m L 100% methanol. Collect 5 m L o f eluate into graduated 15 m L polypropylene centrifoge tubes (final volume 5 mL). 11.13 Analyze samples using electrospray LC/MS/MS. Page S o f f Page 34 o f 65 MPI Research Page 76 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygca RMMrcb M ethod Num ber V 0001782 I ANALYTICAL M ETHOD 1 Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Sediment bv L C /M S/M S 12.0 Chromatography 12.1 byoct the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5-10 sample. As an alternative, an entire set o f calibration standards may b e injected at die beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set of standards). In either case, calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves arc generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.2 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike M s outside the acceptable limits, the entire set o f sample* should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (RJ 0.985). If calibration results fall outside these limits, th en appropriate steps must b e taken to adjust instrument operation, and the standards o r the relevant set o f samples should be reanalyzed. Page 6 o f 7 Page 35 o f 65 MPI Research Page 77 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 Exygen Protocol Number: P0001131 ExygenRar c h Method Number V0001782 1 ANALYTICAL M ETHOD | Method o f Analysis for the Determination o f Pcrfluorooctanoic Acid (PFOA) in Sediment by L C /M S /M S 13.6 Retention time between ctandards and samples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) - /Peak area - intercept) x DF slope OF factor by which the final volume was diluted, if necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [total analyte found (ng/mL) analyte found in control (ng/mL)] analyte added (ng/mL) ^ 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) - (PFOA found (ng/mL) x final volume (S mL)l sample weight (5 g) 14.4 U se the following equation (if necessary) to calculate the amount o f PFOA found in ppb based on dry weight. PFOA found (ppb) dry weight - PFOA found (ppb) x (1 0 0 % /total so!tds(%)] Page 7 of 7 Page 36 o f 65 MPI Research Page 78 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001783 M ethod o f A ialyals fo r th e D eterm U atioo o f Pcrfluorooctanolc Acid (PFO A ) io Fish a id Clams by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By. vU -. f - J l. Paul Connolly 1 Technical L eads', LC-MS, Exygen Research '/# > 'ohn Flaherty Vice President, Operations, Exygen Research Date r MPI Research Total Pages: 8 Page 37 o f 65 Page 79 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 Exygen Protocol Number: P0001131 Exygea Rctttich Method Number VQ0Q1783 ANALYTICAL m e t h o d Method o f Analysis for foe Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Claras by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic a d d by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in fish and clams. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize w ith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time o f analysis. 3.3 Sample collection procedures will be specified in die sampling plan for this project Reagents and Standards 4.1 W ater-H P L C grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 M ethanol-H PLC grade 4.3 Silica gel (60-200 mesh) - Reagent grade 4.6 Floriiil (60-100 mesh) -R eagent grade 4.7 Superclean LC'N H] - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L-Ascorbic acid - Reagent grade 4.10 Dimethytdichlorositanc - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 3.0 Instrument and Equipment S.l A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5 2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Pige 2 of 8 Page 36 of 65 MPI Research Page 80 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exyga Research Method Number VOOOPgj ANALYTICAL M ETHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14 5.15 5.16 Rotary evaporator. Tisstunizer. 125 roL pear-shaped flasks. 50 mL disposable polypropylene centrifuge tubes. 15 m L disposable polypropylene centrifuge tubes. Disposable micropipets (SO-lOOuL, 100-200uL). 125-mL LDPB narrow-mouth bottles. 2 mL clear HPLC vial kit. Disposable pipettes. Autopipettes (100-1000 p L and 10-100 pL), with disposable tips. SPE tubes (20mL) (Supelco cat. no. N057177). Wrist action shaker. Centrifuge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: FluophaseRP (Keystone Scientific), 2.1 m m x 50 mm, $p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time fminl 0.0 1.0 B.O 20.0 22.5 %A 65 65 25 25 65 Flow Rate fmL/miiri 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Iqjection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optimize the HPLC system. Page 3 o f 8 Page 39 o f 65 MPI Research Page 81 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 Exygen Protocol Number: P0001131 Exygen Rateirch________ ____________________________________ Method Number VOQO1783 I ANALYTICAL M ETHOD | Method o f Analysts for the Detanmnatioo o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM m ode, monitoring 413 -* 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.IS4 g o f ammonium acetate to 1000 m L o f water, 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol is prepared by dissolving 2 g o f ascorbic acid in 100 mL o f methanol. 30% Dimethytdichlorositane in toluene is prepared by bringing 3 mL o f dimethyldichlorosilane to a final volume o f 10 m L with toluene. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Stodc/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f -1 0 0 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-raL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 1 mL o f die 100 pg/mL solution to a final volume o f 100 with methanol in a 12S m L LDPE bottle. A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o ftb e 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 jig/mL solution to a final volume o f 100 with methanol In a 123 m L LDPE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. Page 4 o f 8 Page 40 o f 65 MPI Research Page 82 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygta Research Method Number V0001783 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f die 1.0 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared aa needed. Concentration o f Fortification Volume Diluted to Final Concentration Solution (uo/mL) (mL) (mL) (ufl/mL) 1jQ 5.0 100 0.05 1.0 2.5 100 1.0 1.0 too 0.025 0.01 0.05 10 100 0.005 0.025 10 100 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for die batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 1 1.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into SO m L polypropylene centrifuge tubes (fortify as needed, replace lid and m ix well). 11.2 Add 30 mL o f acetonitrile and shake on a wrist action shaker for -1 5 minutes 11.3 Place the tubes in a freezer fo r- 1 hour. 11.4 Pack and condition the SPE tubes and ailaoize the pear-shaped flasks. 11.5 Pack the 20 m L SPE tubes in sequence w ith 2 g florisil, 2 g silica gel, 2 g carbon, and 1 g LC-NHj. Condition the columns with 20 mL o f methanol, then 20 m L o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize die 125 m L pear-shaped flasks b y rinsing with the 30% dimethyldichlorosilane in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use, either by air-drying o r with stream o f nitrogen. Page * ufX Page 41 o f 65 MPI Research Page 83 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen ReMttch Method Number VOOOI783 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 11.7 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm fo r- 1 0 minutes. 11.8 Decant the extract on to a conditioned SPE column fitted inside the mouth o f the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape flask. 11.9 Add 10 m L o f acetonitrile to the sample in the SO m L centrifuge tube. Homogenize die frozen fin phase using a tissumizer for - 3 0 seconds and nnse the ttssumizer with - 1 0 m L o f acetonitrile into the tube. 11.10 Shake the sample again fo r-1 0 minutes cm a wrist-action shaker. 11.11 Place die tubes in a freezer fo r- 1 hour more. 11.12 Centrifrige the 50 m L polypropylene tidies containing sample at -2 0 0 0 rpm fo r-1 0 minutes. 11.13 Decant the extract onto the same SPE column. Collect the eluate into the same pear-shaped flask and combine with the eluent from the initial extraction. 11.14 Pass 20 mL o f acetonitrile through the SPE column and combine the eluate in the same pear-shaped flask. 11.15 Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 409C). 11.Id M ake the final volume, by adding 2 mL o f 2% ascorbic acid in methanol to die pear-shaped flask and swirl to mix/dissolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to a t least five o r m ore concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in a sim ple set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte b y linear regression using 1/x weighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. Page 6 of it P a g e 42 o f 65 MPI Research Page 84 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number. P000U31 Exygen Research Method Number V0001783 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 am u parent corresponds to tho PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.5 ppb, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o fthe total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R2 20.985). I f calibration results foil outside these limits, (hen appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should b e reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: FFOA found (ngtaL ) - (Fcrii area interetul) slope 14.2 Uee the following equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found (ppb) - fPFQA found fng/mL) x final volume fmL) x DF1 sample weight (g) DF factor by which the final volume was diluted, if necessary. Pige 7 of 8 Page 43 o f 63 MPI Research Page 85 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study 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 Research Method N umber VOOOj 783 ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) - [ total analyte found (ng/g) analyte found in control (ng/g)] k1QQ analyte added (ng/g) MPI Research Page 8 o f 8 Page 44 o f 65 Page 86 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001784 M ethod o f Analysis for the D eterm ination o f Perfluorooetanoic A d d (PFOA ) in Vegetation by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: T tA CJiL Paul Connolly * Technical Leader, LC-MS, Exygen Research n //r> /L C /________ J o h n Flaherty / ^ Vice President, Operations, Exygen Research ___ J z & M Date Date MPI Research Total Pages: 7 Page 45 o f 65 Page 87 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research Method Number V000! 7H4 ANALYTICAL m e t h o d Method o f Analysis for the Determinadon o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope Tina method ia to be employed ft- the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectromtrie Detect (LC/MS/MS) in vegetation. 2.0 Safety 2.1 Atwaya observe safe labaratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safely precautions. 3.0 Sample Requirement 3.1 At least 20 g o fte st sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time o f analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W a te r-H P LC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120*400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60-200 mesh) - Reagent grade 4.6 Florisil (60-100 mesh) -R eagent grade 4.7 Superclean L C -N H i- Reagent grade 4.8 l -Octanol - HPLC grade 4.9 L-Ascorbic a d d - Reagent grade 4.10 Dimethyldichlorosilane - Reagent grade 4.11 Toluene - Reagent gride 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume inject capable o f injecting 5-200 pL connected to atandem Mass Spectrometer (LC/MS/MS). 5 2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g Page 2 ot' 7 Page 46 o f 65 MPI Research Page 88 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exyge# Research Method Number V0001784 ANALYTICAL METHOD .................. 1 Method o f Analysis for the Detennination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 5.4 Rotary evaporator. 5.5 125 m L pear-shaped flasks. 5.6 50 m L disposable polypropylene centrifuge tubes. 5.7 15 m L disposable polypropylene centrifUge tubes. 5.6 Disposable micropipets (SO-lOOuL, 100-200uL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 m L clear HPLC vial kit. 5.11 Disposable pipettes. 5.12 Autopipettes (100-1000 p L and 10-100 pL), w ith disposable tips. 5.13 SPE tubes (20mL) (Supelco cat. no. N057177). 5.14 Wrist action shaker. 5.15 CentrifUge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: FluophascRP (Keystone Scientific}, 2.1 m m x $ 0 m m ,5 p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase ( A ) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase ( B ) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 SLA 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: IS pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, m onitoring 413 -+ 369 m/z for PFOA. Page 3 o f7 Page 47 o f 65 MPI Research Page 89 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen R eseuch Method Number V000]784 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS The above conditions are intended as a guide and m ay b e changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.134 g o f ammonium acetate to 1000 mL o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic a d d in methanol is prepared by dissolving 2 g o f ascorbic acid in 100 m L o f methanol. 30% Dimethyldichlofosilane in toluene is prepared by bringing 3 mL o f dimethyldichloronlane to a final volume o f 10 mL with toluene. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f - l 00 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 123-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 1 mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 123 m L LDPE bottle. A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 1.0 p ^ ra L solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. A 0.01 pg/raL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4 'C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/mL fortification solution. 4 hi Page 48 o f 65 MPI Research Page 90 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygeo Asieaicb Method Number V0001784 I ANX.YI1CA L M E T H O D *~| Method o f Analysis for tbc Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 9 2 .2 The following ia a typical example: additional concentrations may be prepared as needed. C oncentration Final ofFortification Volume D iluted to C oncentration Solution fusftnL ) (m L) (m L) (ufi/m L) 1.0 5.0 100 0.05 1.0 2.5 100 0.025 1.0 1.0 100 0.01 0.05 10 100 0.005 0.02S 10 100 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 Store all calibration standards in 125-tnL LDPE narrow-mouth bottles at 2C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into 50 mL polypropylene centrifuge tubes (fortify a t needed, replace lid and mix well). 11.2 Add 30 m L o f acetonitrile and shake on a wrist action shaker for -1 5 minutes. 11.3 Centrifoge the 50 m L polypropylene tubes containing sample at -2000 rpm fo r- 1 0 minutes. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in sequence with 2 g florisil, 2 g silica gel, 2 g carbon, and 1 g LC-NHj. Condition the columns with 20 mL o f methanol, then 20 m L o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 125 mL pear-shaped flasks by rinsing with the 30% dimethyldichlorosilsne in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use, either by air-drying or with a stream o f nitrogen. 11.7 Decant the extract mi to a conditioned SPE column fitted inside the mouth o f the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape flask. Pig? 5 ol' ' Page 49 o f 65 MPI Research Page 91 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V001784 ANALYTICAL m e t h o d Method o f Analysis for the Detenunation o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 mL o f acetonitrile to the sample in the 50 raL centrifUge tube. 11.9 Shake the sample again for - 1 0 minutes on s wrist-action shaker. 11.10 Centrifuge the 50 mL polypropylene tubes containing sample at -2 0 0 0 rpm for - 5 minutes. 11.11 Decant the extract onto the same SPE column. Collect the eluate into the same pear-shaped flask and combine with the eluent from the initial extraction. 11.12 Repeat steps 11.8 through 11.11 again. 11.13 Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.14 Make the final volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. 11.15 Transfer the extracts to HPLC vials using disposable pipets. 11.16 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fbrtiflod sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample s e t Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards m ay be injected at the beginning o f a set followed b y extracted calibration standards interspersed every 5-10 samples (to account fix' a second set o f extracted standards). In either case, extracted calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while (he daughter ion (369 amu) represents the loss o f carbon dioxide. Pge 6 of 1 Page 50 o f 65 MPI Research Page 92 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 E x y g e n P r o to c o l N u m b e r: P 0 0 0 1 131 Exygoa Rcictrch Method Number V00017S4 ANALYTICAL METHOD I Method o f Analyst for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than O.S ppb, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. I f control spike fitUs outside the acceptable limits, the entire set o f samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R* 20.985). I f calibration results foil outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standard or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) - (Peril area intercept) slope 14.2 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found (ppb) - (PFOA found (ng/mLl x final volume (m D x DF1 sample weight (g) DF " factor by which the final volume was diluted, if necessary. 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use foe following equation to calculate the percent recovery. Recovery (%) - (to tal analyte found (ng/g) analyte found in control (ng/g)] analyte added (ng/g) Page 7 ol* 7 Page 51 o f 65 MPI Research Page 93 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P001131 ANALYTICAL METHOD Method Number: V0001785 M ethod o f Analysis fo r the Determ ination o f Perflnorooctaaoic Acid (PFO A ) in Smelt M am m al Liver by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly I Technical Leader, LC-MS, Exygen Research ml John Flaherty / Vice President, Operations, Exygen Research Date Date MPI Research Total Pages: 7 Page 52 o f 65 Page 94 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reteartb Method Number VOOOI785 | ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Peruorooctaooic Acid (PFOA) in Small Mammal Liver b y LC/MS/MS 1.0 Soope Thie method ia to be employed for the iaolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectroroetric Detector (LC/MS/MS) in small mammal liver. Z.O Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safely precautions. 3.0 Sample Requirement 3.1 At least 5 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. Alternately, i f there is an insufficient amount o f sample (-less than 5 g), then no processing is necessary and the sample can be used as supplied. 3.3 Sample collection procedure* will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 M ethanol-H PLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic A d d - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 m L disposable polypropylene centrifoge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-lQQuL, 100-20QuL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial k it Pag 2 u p Page S3 o f 65 MPI Research Page 95 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 E x y g en P ro to co l N um ber: P 0 0 0 1131 Exygea Research Method Number V0001 785 | ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perftuorooctanoic Acid (PFOA) in Small Mammal l iv e r by LC/MS/MS 5.9 5.10 5.11 5.12 5.13 5.14 5.15 Disposable pipettes. Autopipettee (100*1000 pL and 10-100 pL), with disposable tips. Waters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. SPE vacuum manifold. Tissuemizer. Wrist-action shaker. Centrifuge capable o f spinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5p (P/N: 8250S-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate % B (mL/tnin) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: IS pL (can b e increased to as much as 50 pL). 6.7 Quantitation: Peak A rea-e x te rn a l standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative M RM mode, m onitoring 413 - 3 6 9 m /zfoi PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. Page 3 of? Page 54 o f 65 MPI Research Page 96 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygcn Protocol Number: P0001131 Exygu Riaesrch Method Number VOO01785 I AN/vL Y TIC A L m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 8.0 Preparation o f Solutions 8.1 Mobile Phaae 8.1.1 2 mM ammonium acetate in water is prepared by adding 0 154 g o f ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 P re p are a sto c k so Iu tio n o f-IO O p g /m L o fP F O A by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/raL fortification solution o f PFOA is prepared by bringing I m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 p ^ m L fortification solution o f PFOA Is prepared by bringing 10 mL o f tiie 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may be preparod as needed. Concentration Final o f Fortification Volume Diluted to Concentration Solution fnfi/mL) im U (mL) (na /m L ) 100 5.0 100 5.0 100 2.0 100 2.0 100 1.0 100 1.0 S.0 10 100 0.5 2.0 10 100 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2"C to 6"C, up to six months. 9.2.4 Alternate volume* and concentrations o f standards m ay be prepared as needed. Page 4 o f 7 Page 55 o f 65 MPI Research Page 97 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 E x y g e n Protocol N u m b e r: P 0 0 0 1 131 Exygen Research M ethod Number V 0001785 AN.ULYTICAL M ETH O D M ethod o f Analysis for the Determination ofPerfluorooctsnoic Acid (PFOA) in Small Mammal Liver b y LC/MS/MS j 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r leas) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 1 g o f sample into a 50 mL polypropylene centrifuge tubes (fonify as needed, replace lid and mix well). Note that alternate weights o f liver may be measured depending on the sample size tvailsble for use. 11.2 Add water to the israple for a final volume o f 10 mL. 113 Homogenize sample using a tissuemizer for -1 minute. 11.4 Transfer t mL o f (he sample using a disposable pipette into a 15 mL disposable ccntrifUge tube. 11.5 Add 5 mL o f acetonitrile and shake for - 2 0 m inutes on a wrist-action shaker. 11.6 Centrifuge the tubes it ~3000 rpm for ~5 minutes. 11.7 Decant foe supernatant into a 50 m L disposable centrifoge tube and add 35 mL o f water. 11.8 Condition the C n SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 m L o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eiuate. 11.10 Elute with ~2 m L o f methanol. Collect 2 m L o f eiuate into a graduated 1S m L polypropylene centrifoge tube (final volume * 2 mL). 11.11 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or m ore concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at foe beginning and at the end o f a sample set. Standards m ust be interspersed between every 5 -1o samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area Page 5 o f 7 Page 56 o f 65 MPI Research Page 98 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen R em ich Method Number V0001785 I ANALYTICAL M ETH O D .... Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 10 ng/g, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R* 0.985). I f calibration results foil outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standard or the relevant set o f sample should b e reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) (Peak area - intercept) x DF x aliquot factor slope DF factor by which the final volume was diluted, if necessary. Aliquot factor " 10 Page 6 of 7 Page 57 o f 65 MPI Research Page 99 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygcn Protocol Number: P0001131 Exygen Re--wch____________________ ___________ Method Number VWOI785 I ........................ ANALYT ICAL M ETHOD ....... Method o f Analysis for the Determination o f Periluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 14.2 For samples fbetified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (% ) [total analyte found (ng/mL) - analyte found in coptrol(ng/mL)] ^ analyte added (ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL lo ng/g (ppb). PFOA found (ppb) - IPFOA found (ng/mL) x final volume fmL)1 sample weight (g) MPI Research Page 7 o f7 Page 58 o f 6S Page 100 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001786 M ethod o f Analysis for th e D eterm ination o f Perfluorooctaoolc Acid (PFOA ) in Small Mammal Serum by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly Technical Leader, LC-MS, Exygen Research n//Z ) / / _________ V6hn Flaherty ' / Vice President, Operations, Exygen Research Date Da MPI Research Total Feget: 7 Page 59 o f 65 Page 101 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 Exygm Retouch Method Number V0001786 1 ....ANAL YTICAL M ETH O D Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in small mammal serum. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult foe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 1 mL o f test sample for extraction. 3.2 No sample processing is needed for serum samples. However, frozen serum samples must to allowed to completely thaw to room temperature before use. 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater ~ HPLC grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic A d d - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 p i connected to a tandem Mass Spectrometer (LC/MS/MS). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 mL disposable polypropylene centrifoge tubes. 15 mL disposable polypropylene centrifoge tubes. Disposable micropipets (50-lOOuL, 100-200uL). 125-mL LDPB narrow-mouth bottles. 2 mL clear HPLC vial Idt. Disposable pipettes. Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. Waters Sep Pak Vac 6 cc (Ig ) tC18 SPE cartridges. SPE vacuum manifold. Vortexer. P*ge2oP Page 60 o f 65 MPI Research Page 102 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 E x y g e n P r o to c o l N u m b e r: P 0 0 0 1 131 Exygen Retaarch Method Number V0001786 I A IW V TIC A L METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase KP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in W ater Mobile Phase (B) : Methanol Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (roL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 |iL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 R unTim e: ~ 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.1S4 g of ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. Page 3 o f 7 Page 61 o f 65 MPI Research Page 103 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: P0001131 ExygeeRcMucfa Method Number VOOOI7B6 I ANALYTICAL METHOD M ethod o f Analysis for the Determination o f Perfluorooctonoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f "100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-raL LDPE bottle. 9.1.2 A 1.0 jig/mL fortification solution o f PFOA i$ prepared by bringing I m L o f the 100 ug/rnL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o f PFOA is prepared b y bringing 10 m L o f the 1.0 )ig/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f die 0.1 jt^ m L fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentration ofFortification Volume Diluted to Final Concentration Solution (na/mLl (mL) (mL) (ng/mL) 100 5.0 100 100 2.0 100 100 1.0 100 5.0 2.0 1.0 5.0 10 100 2.0 10 100 1.0 10 100 0.5 0.2 0.1 9.2.3 Store ail calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to tiC, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) m ust include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. Page 4 of'7 Page 62 o f65 MPI Research Page 104 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number: POOOU31 Exygec Research Method Number VG001786 I ANALYTICAL M ETHOD Method o f Analyst* for the Determination o f Perftuorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 11.0 Sample Extraction 11.1 Measure 1 mL o f sample into a SO m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate volumes o f serum may be measured depending on the sample size available for use. 11.2 Add water to the sample for a final volume o f 20 m L Cap tightly 11.3 Vortex for -1 minute. 11.4 Transfer 1 m L o f the sample using a disposable pipette into a 15 mL disposable centrifuge tube. 11.5 Add 5 mL o f acetonitrile and shake for -2 0 m inutes on a wrist-action shaker. 11.6 Centrifuge the tubes at -3 0 0 0 rpm for - 5 minutes. 11.7 Decant the supernatant into a 50 m L disposable centrifuge tube and add 3S m L o f wafer. 11.8 Condition the C tt SPE cartridges ( l g, 6 mL) by passing 10 mL methanol followed b y 5 m L ofH PL C water (~ 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned C\x SPE cartridge. Discard eluate, 11.10 Elute with - 2 mL o f methanol. Collect 2 m L o f eluate into a graduated 15 m L polypropylene centrifiige tube (final volume * 2 mL). 11.11 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed sample*. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards m ust be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in u sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12-5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. Page 5 of 7 Page 63 o f 65 MPI Research Page 105 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygen Protocol Number. P000U31 Exygen R ociitb ______________________ Method Number VQOO1786 1 ANALYTICAL M ETHOD ........ ~ Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. if a blank contains PFOA at levels greater than 10 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. I f a control spike falls outside the acceptable limits, (he entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R3 0.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should b e reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within in analytical nut. If retention tme drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) * (Peak area - intercept) x DF x aliquot factor slope DF * factor by which the final volume was diluted, i f necessary. Aliquot factor * 20 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [total analyte found (ng/mL) - analyte found in control (ng/mL)] analyte added (ng/mL) ^ Page 6 o f7 Page 64 o f 65 MPI Research Page 106 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exygcn Protocol Number: P0001131 Exy|a Research M ethod N umber VOOO1786 I ANALYTICAL METHOD ~ Method o f Analysts for the Determination o f Perfluoroocunoic Acid (PFOA) in Small Mammal Serum by 1X/MS/MS 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ppb. PFOA found (ppb) - fPFOA found fngftnL) x final volume fmLIl sample volume (mL) MPI Research Pge 7 ot f Page 65 oj 65 Page 107 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment Number 1 Effective Date: 01/19/05 Exygen Study Number P0001131 Client Study Number Page 1 of 1 DESCRIPTION OF AMENDED SECTION 1) Analytical Procedure Summary V0001780:Section 9.1 2) Verification of Analytical Procedure None ~ AMENDED TO 1) Add to Section 9.1: Section 9.1.6, Alternate weights of standards may be used to prepare alternate concentrations of stock solutions as necessary. Alternate levels of fortification solutions may also be prepared. 2) Lowand 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 smaller 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 predetermined spiking levels listed in the protocol. When the levels in the samples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the QC sample. Higher spiking levels in the bottles will cover the analyte concentrations expected in the water samples. IMPACTON STUDY The LOQ is 100 ng/g for a 0.1 g sample of small mammal liver and is 1000 ng/mL for a 0.01 mL sample of small mammal serum. Higher levels of spiking for the water samples will ensure that more QC recovery data can be used. LIBRARY ID: W000122S-6` .' MPI Research ADMINISTRATIVE FORM Page 108 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, 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 DESCRIPTION OF AMENDED SECTION 1) Report, page 11 of 65 2) Test Materials, page 6 of 65: PFOS transition monitored 469 -> 99. AMENDED TO 1) Instead of one final report, interim reports will be issued. 2) PFOS transition monitored may also be 499 > 80. RATIONALE 1) Due to the excessive sizes of the data sets, interim reports will be issued to allow the client to receive data in a timeliermanoF-. * * * nt>~ c s je tltr 2) The API 4000 LC/MS/MS systems detect the 499 > 80 PFOS transition with greater sensitivity than the 499 -> 99 transition. IMPACT ON STOP * 1) The client will be able to receive and review the data more quickly. 2) The 499 -> 80 transition can be detected with greater sensitivity; therefore, giving better chromatography. LIBRARY ID: V000122S-8- Exygen QAU Review J J O le s llS ADMINISTRATIVE FORM MPI Research Page 109 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 JU U .2 S .2005 8 :56AM EXYGEN RESEARCH H O .774 P .3 Gm & W g!nRESEARCH 3058 Research Drive Phone: 814*272-1039 State College, PA 16801 Fax:814-231-1580 Amendment Number: Effective Dale: Exygen StudyNumber PROTOCOLAMENDMENT 3 07/18/05 P1131 Client Study Number: Page1 on NA DESCRIPTION OF AMENDED SECTION! Verification ofAnalytical Procedure, page 10 Of protocol. AMENDED TO The field duplicate can be used for the laboratoiy spikes and replicate when the primary sample volume is limited. ^ ,_ BaX IQ bjA LE The sample size for a watersample Is 200 ml- If s sample site requires re-extradion 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 end 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. Sponsor Slgnsturaqtf requlrad) LIBRARYIDrV0001228 Date Exygen QAU Review f'Tk.'fv f/o a -A r ADMINISTRATIVE FORM MPI Research Page 110 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 'T 11-23-2005 04:22p Fron-WESTON SOLUTtONS NOV..22.3005 4 : 50m EXYGEN RESEARCH T-677 P.003/003 F-713 M J.S X 4 r .a E xyren 3058 Research Drive Phone: S14-27Z-1039 State College, PA16801 Fax; 814-231-1580 ^ RESEARCH Amendment Number. Effective Date: Erygen Study Number PROTOCOL AMENDMENT 4 125 Pt 131 Client Study Number. Pagai eM NA__ DESCRIPTIONOF AMENDEDSECTION Analytical Procedure Surmary: VOOOl780:*Method ofAnalysis torthe Determination of PerttuorooocteftaicAcid (PFOA) inWatarby LQMS/MS,' Section 11.0 ofthe method. AMENDED TO " Section 11.0. Samples may be diluted before going throughthe extrartion procedure. RATIONALE ; If a 40 mL portion of sample will not load onto the Cu SPE cartridge, a predilution can be prepared and extra: ' IMPACTONSTUDY " No negative impact onthe study. More usable data may be obtained. study DKenrQlgnahire RSdpel lmadgatarSaperure Study Dtaoor Man Spensersignature Qfrequired) Date Date Dale Exygen QAU Review * - l~ UBRARVID:VQ00122S4 R E V I V E D TIME NOV.23. 5:40PM ADMINISTRATIVE FORM PRINT TIME NOV.23. 5:41PM MPI Research Page 111 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 ffcHEM EHSR 236 18 651 733 1958 J U L J-2 0 0 3 0 5 :1 2 PR0M:3t1 ENU. U S 651 779 6170 P rO V .2 a .2 0 0 5 4 :58FT1 EX7SEN HESEflRW 11/23 '05 14:12 NO.979 03/03 TU: 565 1 7 3 3 1 5 5 5 k : k i NO.9 1 4 P .3 I 3058 Research Drive Phone: 814-272-1039 Stete College, PA16801 Fax: 814*23MS>0 Amendment Number. Effective Date: Exygan Study Number PROTOCOL AMENDMENT 4 11/22/OS P1131 Client Study Number: Pegel of1 NA Analytical Procedure Summery; V0001780c*Methcd ofAnalysis forth Potenninafioii of PerftuoroooctanolcAcid (PFOA) inWaterby LC/MS/MS,' SecOon 11,0 ofthe method. AMENDED T6 Section 11.0. Samples maybe diluted before going throughtne extraction procedure. RWNtSU If a 40 mL portion of samplo win not lead onto the C,, SPE cartridge, a pre-dilution can be prepared and extracted. IMPACTON STUDY i\ No negative impacton the study. M m usable data may be obtained. Study Direnarsvnaure DM r*f a BBT DM zis-M V r-a f DM Mm . U .-m s M ExvoenQAtl Review f a . tt/*~a/ e^~ UBR*RVIO:VOOOiaS6-a RECEIVED TIME NOV.23. 3:32PM ADMINISTRATIVE FORM PRINT TIME NOV.23 . 3:33PM MPI Research Page 112 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 ' -2006 04:09pm From-ffiSTON SOLUTIONS T-15J P.0oj/00^ F-US y i, . | I m 3C58 ResearchDrive Phone: M-27Z-1039' State co lle je , PA16801 FaK 4-23111580 Amendment Nu then Effective Date iicygen StudyNumber V'OCOL AMENDMENT 1 2 /p i/d ^ POObmi ClientStudyNumter DESCRIPTIONOF AMSNPEg.SESajET Teat MatofMte, page & of Protocol. paai o f1 NA AMENDEDto " AnaHsmatalotoiPFOSiTi 'y usedfariNssMy. The PFOS used previously in thfe siwtyOot numDer217 from 8M) ran out and 1 wae necessaryto ueo a newlot Na negafiveimpactonstudy. JM PA C T 'b l s j r p . Q T i S E J 'L * studyo m m oenumo^ n L m /ie J ii * M idpelli* . "S in a ia ; v DO* Dab DO M -/H d d 3 t0 6 Exygan QAU InlUDete Jjkt [e!s. U B R A R riD -V D O O P S U M RECEIVED TIME W . ; i ' 2GPM , ADMINISTRATIVE FORM PRINT TIME I MPR.21. 5:29PM MPI Research Page 113 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number. Effective Date: Exygen Study Number PROTOCOL AMENDMENT 6 05/17/06 ' P0001131 Client Study Number PageioM 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-m iIlliter 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 -1 0 minutes at -30QO rpm. Each sample is analyzed by LC/MS/MS electrospray. RATIONALE More usable data will be obtained by using an alternate method. LIBRARY ID: V0001226-8 ' ADMINISTRATIVE FORM MPI Research Page 114 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 CHEM EHSR 236 ife 651 733 1958 07-1 W O O S 03:57 FronrWESTOH S0LU7I0WS 07/11 '06 14:09 NO.142 02/02 + T-5M P.002/007 P-54S (? 3 0 5 8 f te s e s r t h D r W e StateCollege, PATD1 Fbk: 81;4-231-1580 RESEARCH . ,(; J . 1 **RQTQ(50t. AMENDMENT ' " *1 A r h e n d r i h e n t Number EfhctlvaDate: l ^ ^ d y Nurrtber .8 . P000113C Client Sfrdy Number. BE AppendixI, Analytical Method V0001782. S' . .. V ;>h . --------------------------------------- :-- '" '" i D E b t b 1 /" .. A* perdjent request. sediniem wimples re-tagged for'rp-eirtractlon by the *POnpr be reirtiacted using the follo*rfnartietfiod: .. D Ii r e c t n ] c t ) o n MetHotfc Before the samptes ere welflhed.fy t h e extraction. thiey ere mixed thoroughly byvigorously s h a k in g the container...A one-grtm Isw e i g h e d Into a 15-mlllffltor osnWfuge tube.fbrthe extradpn. Ten rrfllilltpmcf acetic ebid in methanol la added each sample. Thesamples era then sIW n o y hind, vortexed, and sonicated f o r thirtyminutes. The sample* a then centrifuged fpr -1 0 minute* a t-3000 rpm. Eachsample is analyzed by LC/MS/M8 aieetrttspray. .... -----; ' RffiftteLE .. . More usable data win be obtainedby using,an altsmate method. IMPACTBN 5TUBX~ E 3 Q sm Q A U i1 h i /D e0 s. UBRARViDi VD0012284 RECEIVED TIM E J U L . l l . 4 :2 7 P M PR IN T TIME JMWrCTjUmVf >rU ' J U L .ll. 4 :2 8 P M MPI Research Page 115 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 09-05-2008 OnOlp* Ftoi-ffiSTON SOLUTIONS T-672 ?.002/002 F-697 RESEARCH 3053 Research Drive Phone: 814-272-1039 State Collega, PA16801 Fax: 814*231-1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOLAMENDMENT to 7110/06 P i tat CfientStudy Number: P ag eloM NA ------------------------- bgSCRlPfiON o f AMENDEPSECTQQN Reference V00017B1: "Method of Analysis for the Determination of Perfluorooctanoic Add (PFOA) inSoil byLC7MS/MSwas used 1oanalyze sludge as well. ----- ----------------- flMPMO^O TO . The purpose of this study is to perform analysis for perfluorooctanoic acW (PFOA) to water, so, sediment, sludge; fish, dams, w g^ton. mammal rets, and small mammal serum using LC/MS/MSforthe 3MDecaturMonitoringProgram ------------------------ -------- - ' RATIONALE Sludge is constituted of soHwitha greaterpercentage ofwater. -------------- :--------------------- IMPACT ON STUPy Using a knownmethod produced viable data applicableto this study. I S * / / / / //4 S < X -< > .... Mitapai investigatorSignature/ -Date ' K$ . StudyDimeterMa/rMeffisnt Sigiata ' E^BnMandgoTMtStgnaftB SeoneorSanatare tifrequired) Q C jP {fa ti / Oats S ^ S 6 0 ' )& Data t 1 Exygen QAUInitiate ?/roiaL UBRARY ID: VOO122S0 RECEIVED TIME SEP. 5. 1:22PM ADMINISTRATIVE FORM PRINT TIME SEP. 5. 1:23PM MPI Research Page 116 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 01-OB-Z007 03:58pm Fron-HESTON SOLUTIONS T-021 F.002/005 F-058 Exysen {^R ESEA R C H 3098 Research Drive Phone: 814-272-1039 State College, PA 16801 Fare 814-231-1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENOMENT ____1_2_/2_1_/_0_6__ P0001131 Client Study Number: PBge1of1 P0001131 . LIBRARY ID:VD001S2M RECEIVED TIME JAN. 8 . 4:02PM ADMINISTRATIVE FORM PRINT TIME JAN. 8 . 4:04PM MPI Research Page 117 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 01-24-2007 04:34pm From-KESTON SOLUTIONS *4' T-064 P.003/004 F-103 3058 ftseanch Drive Phone: t14-Z7M039 StateCollege, PA16807 Pax: 814-231-3580 PROTOCOLAMENDMENT Amendment Number 12 effective Date: w vanr Exygsn Study Number _ W lj CfientStudy Number Page 1o f2 - N f e ____ _ DESCRiPIlONQ &mpie ExBSrton se^ons ofthe Analytical ProcedureSummaries: V0001780;'Methad T /J u Z n E t- ** P*#oodmofc Add (PFOA) tn Water by w 00!7?.1:]'ltethod AnaiysbIprthe Determination of Perfluoroooaanaic ^ d (PFOA) m_Sgll by LO M S M S : and V0001782.--M*hod of Analysis fo r the Determinationof PwfluoroooctanoicAdd (PFOA}in Sedimentby IC/MS/MS.* 2SIE53*Ia p*re*p,la"rred,*u*si*n_g the followiMnCg"mPteet!8nodi;edgnatad fer re*naiysis may be extracted and Forwatersamples: f Ten miatiters fte sample a rpaired into a 50 mL polypropylene centrifuge tube. a. ? fortitie? *i(tLa foitfleation solution. Ten maiNters of ecetortMe a J**. Wf: capped tightly, and vortatsd. The sample k m i o M for tH0 houmat mom'Tijserattire. The sampleb then mtriftiged at -3000 rpmfor 10minutes. Aportionoflhasupemate is transferredtoan autosamplecvlaL Forsoj endsedimentsampfes: ? b " S iflii 12 a IS mL potyprapytene centrifuge tube. flSSSaiC M a fcri,fca8on sofutioa Eight mMftem of 80:20 *ohlBon s to tamptes' aTM1vortex. samples am then f*#*" t,l!,P*ature-endarethen centrifuged at -3000 rpmfor ten minutes. Aportionofthesupernateistransferredto enautosarrplervial. ~ l.c)li}ffi/M$ electnosprsy against non-wdiacted standards ^' 1:'Z0, S0' :a>i n iM.OngftnLtnSO;SOacetoniWa?i*ater. The S S K 3 ffiS a i2 C 5 2 3 S r * * i* " uWnot be reported quantitatively minterim reports21. #22, and<a* due toqutftyconw sampleMures. The study directorand sponsor have requestedrwnatysfcijioengthe edoveconditions. 7 jH "'IMPACT oft STUDY ... ..... No negative impact onthestudy, M en usabledete maybe obtained. LiaMRio: WOC122E-Q noMnnsnwriveform RECEIVED TIME JAN.24. 4:?3TI PRINT TITE JAN.24. 4:40PM \ \ \ \ MPI Research Page 118 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 01-24-2007 04:35p Frra-WESTOH SOLUTIONS + T-064 P.004/004 F-103 30S8 ResearchDrive Phone: 814-272-1039 State College, PA16801 Fax: 814-231-1580 H PROTOCOLAMENDMENT Amendment Number: ___12_____ P*82 or2 . Effective Data: 01/16/07 Exygen Study Number P1131 Client Study Number: _________ _ i UfiRARY Ia V 000122W RECEIVED TIME JflN.24. A- .M BMIWSTWVnVE FORM PRINT TIME JRN.24. 4:40FM MPI Research Page 119 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 CHEM EHSR 236 IB 6517331958 07/25 ' 07 09:42 NO.254 02/03 Amendment Number Effective Date: MPI State College Project Number PROTOCOLAMENDMENT 13 ' Page 1 of 2 07/18/07 Other Study P0001131 Numbers):________ NA______ DESCRIPTION OF AMENDED SECTION ' 1) Entire Protocol 2) Sample Extraction section ofthe Analytical Procedure Summaries: VD001785: -Method of Analysts for the Determination of Perfluoroooctanolc Add (PFOA) in Small Mammal Liver by LC/MS/MS." 3) Standard Preparation and Sample Extraction sections of the Analytical Procedure Summaries: V0001786: ` Method ofAnalysis for the Determination of Perfluproooctanoic Add (PFOA) In Small Mammal Serum by LC/MS/MS.' AMENDEDTO 1) Amend all references of Bqrgen Research, Inc. (Exygen) to MPI Research, Inc. . 2) As per client request, liver samples designated for re-analysis wffl be fortified with PFBS, PFHS, and 13C labeled PFOS for the extraction. The samples will be reacted following the original method with one exception; the SPE cartridges will be conditioned with 10 mL of methanol and 25 mL of water. The '*C labeled PROS will be monitored by LCMSMS at the transition of 503 > 80. .. 3) A mixed fortification solution containing PFBS, PFHS, and MCPFOS may be prepared at 5.0 ng/mL by taking 5.0 mL of a 100 ng/mL mixed formication solution and bringing it up to volume In a 100 mL volumetric flask with acetonitrile. A mixed fortification solution may be prepared at 2.0 ng/mL by taking 2.0 mL ofa.10O ng/mL mixed fortification solution and bringing It up to vdume in 8 100 mL volumetric flask with acetonitrile. A mixed fortification solution may be prepared at 1.0 ngAnL by taking 1.0 mL of a 100 ng/mL mixed fortification solution and bringing it up to volume in a 100 mL volumtrie flaskwHh acetonitrile, a mixed fortification solution may be prepared at 0.5 ng/mL by taking 10 mL of a 5-0 ng/mL mixed fortification solution and bringing Kup to volume In a 100 mL volumetric flask with acetonitrile. A mixed fortification solution may be prepared at 0.1 ng/mL by taking 10 mL of a 1.0 ng/mL mixed fortification solution and bringing it up to volume In a 100 mL volumetric flask with acetonitrile. A mixed fortification solution may be prepared at 0.05 ng/mL by taking 10 mL ef a 0.5 ng/mLmixed forflfleafion solution and bringing Kup to volume In a 100 mL volumetric flask with acetonitrile. These fortifiestfon solutions will be used to fortify the samples and the standards for the extraction. These standards are to be atoned in tha same conditions as outlined in tha original method. As par client request, serum samples designated for re-analysis may be extracted using the following method: , - A matrix control and two matrix control spikes fortified at known levels will be extracted and analyzed with each data sat , A sample aliquot, a sample replicate aliquot and one appropriate sample aliquot spike will be extracted for each serum sample - Transfer 0.05 mL of sample Into a 1.5 mL centrifuge tube - F a ^ s p ite ^ p te s w ith 500 pL of tha appropriate spiking solution of PFBS, PFHS, - Add 500 pL of acetonitrile to unspiked samples -Vortex for-1 minute____________________________________ __________________ LIBRARY ID V0001228-12 ADMINISTRATIVE FORM MPI Research Page 120 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 CHEM EHSR 236 IB 651 733 1958 07/25 '07 09:42 NO.254 03/03 : Amendment Number: Effective Date: MPI State College Project Number PROTOCOL AMENDMENT ' 13 Page 07/18/07 Other Study P0001131 Numberfs): 2 of 2 NA_______ Centrifuge at 10,000 rpm tar -1 0 minutes * Transfersupernatant to an autosampler vial tar analysis Note: Volumes or sample may be altered depending on sample availability; also the sample replicate aliquot may not be extracted due to sample availability. Samples are analyzed against standards prepared by the same procedure outlined above. The control serum wlfl be used to make the standards by fortifying the control serum at specific levels. The standards are then processed through the same extraction as the samples. The final calibration curve will include standard concentrations at 0.5,1.0,5.0,10,20, and 50 ng/mL. The LC conditions for the analysis remate the same as outlined in the original method with the addition of the transition tor " C labeled PFOS which Is 503 - 80. RATIONALE 1) In June 2007, Exygen Research, Inc. began operating underthe name of MPI Research, Inc. 2 i 3) The requested samples to be reanalyzed could not be reported quantitatively due to quality control sample failures. The study director and sponsor have requested reanalysis using the above conditions. IMPACT ON STUDY 1) No negative impact 2 & 3) No negative Impact on the study. More usable data may be obtained. M o M ao Study Offcolor Senator y k io i: Date/ / P rn d [ li 3 fl 0 ra tu m fa le r a - Date ' StudyDirector ManaMdi1tont Signature j Ta W ^ MPI State College ManagementSjgMdya - . . Date n 2 (p"vJk0-Q i Ha x A j J . SponsorSignature (lrrequired) Date/ / ' MPI State College QAU Init/D ate J u P / jI v ^ ic H LIBRARY ID V0001226-12 ADMINISTRATIVE FORM MPI Research Page 121 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 S EP.20.2005 1:52PM EXYGEN RESERRCH NO.377 P .2 en RESEARCH 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 1 of 1 DateofOccuiTence: 03/16/05 Deviation # : 1/1 Reference # : 05-122 Regulatory Driver: Deviation Tvna: (Include Wt for m ethods and SO Ps) GMP GLP Other None Sample Description: _ _ _ __ Protocol ______ Method V * 0001658-3 Notebook reference: NA X SOP Lo gin#: NA Container#: NA L o t# : NA Summ ary o f Deviation: This deviation pertains to all soil and sediment samples analyzed for percent solids before 07/07/05. a. No blanks or duplicates were run as required by section 9.3. b. Some sample weights exceed the allowable range (> 10g). Cause: Preparation____A nalysis_____ Instrum ent_____ Client Request X Other Im pact: There has been no negative impact on the study. All of the percent solid values that were determined during the time period in question are considered valid, although the SOP was not followed. In the newly revised version of the SOP blanks and duplicates are no longer required. Also, in the new SOP, the allowable amount of sample to be used is < 20 g. All of the samples in question in this deviation weighed less than 20 g. The technician analyzing die samples for percent solids was following the new procedure before it was formally approved. Corrective Actions: A new version of the SOP has been issued and approved (V0000427-3). Signature q / i./d u b z " 'Principal Irkfistigator / ^ /1 j IQ M m . Study Direior f L ^ -- :c 1h ^ S l S L x ^ - ^ Quality Assurance / y / V , * / T /' f/r/k ' r Exygen Mariagejnfent Am 7 D ite Sponsor Representative t/i/o ff Date NA Sponsor Management _ Date Date Date LIBRARY ID: V0001640-6 ADMINISTRATIVE FORM MPI Research Page 122 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 SEP.20.2005 1:52PM EXYGEN RESEPIRCH N O .3 7 7 P .3 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM __________________________________ ______ _____________________________________________________Page 1 of 1 General: X Project Specific Deviation ____Facility Deviation Date of Occurrence: 06/29/05 Exygen Project # : P760/P11-31 D e v ia tio n # : 2/2 Client Project#: ______ NA______ R e fere n c e # : Regulatory Driver: Deviation Tvae: {Include V# for m ethods and SO Ps> _____ X _____ ____ GMP GLP Other None Sample Description: X Protocol _____ Method V#:NA Notebook reference: NA ! _____SOP Login # : L4264/L4256 Container#: C0056480-85 L o t#: 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 of these six clam LIBRARY ID: V 0 0 0 1 6 4 M MPI Research ADMINISTRATIVE FORM Page 123 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 S E P .20.2005 1:52PM EXYGEN RESEARCH NO.377 P .4 E\ygen RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 General: X Project Specific Deviation DEVIATION FORM Page 1 of 1 Facility Deviation Date of Occurrence: 12/27/04 Exygen Project # : Client Project # : P760/P1131 NA Deviation # : 3/3 Reference # : oS'-fr Regulatory Driver: Deviation Tvoe: (Include V# for m ethods end SOPs) GMP GLP Other None Sample Description: ____ Protocol V#: 202-20 Section 5.2.3 Notebook reference: NA Method X SOP Login#: _______NA Container#: NA L o t#: NA Summary of Deviation: SL2114 (30% Dimethyldichlorosilane in Toluene) was given the expiration date of 02/13/05, but RE544 (Toluene) used to makeSL2114 expired on 03/28/04. SL2114 was used to silanlzed glassware prepared for the fish extraction from 12/27/04 through 01/07/05. Cause: Preparation Analysis Instrument Client Request X Other Impact: 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 Actions: Deviation issued. Signatures; A Phncipat|nvestigat6r liO A iu j/ Study Director Quality Assurance fJ r jB ' /) ---- ' Exygen k^njgdment a te j 9 /* o /o f Date Sponsor Representative wk Sponsor Management Date Date Date LIBRARY ID: V0001640-6 ADMINISTRATIVE FORM MPI Research Page 124 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 Exfveen \\# C jresearch 3058 Research Drive Phone: 814-272-1039 5tate College, PA 16801 Fax: 814-231-1580 DEVIATION FORM ________________________________________________________________________ Pag 1 o( 2 General: X Project Specific Deviation ___ Facility Deviation Date of Occurrence: 04/26/06 Exygen Project # : P760/P1131 Deviation # : ____ 5 Client Project # : ______ NA______ Reference # : 06-076 Regulatory D river Deviation Type: (Include V# formethods end SOPs) _____ X _____ _____ GMP GLP Other None Sample Description: X Protocol ____ Method V#: NA Notebook reference: NA ____ SOP Login#: 10008191 Container#: ______ NA______Lot#: _________ NA Summary of Deviation: The three sediment samples In L8191 (C0172892 - C0172894) were originally extracted using the sediment method V0001782. Poor recoveries urere obtained for PFOS, PFOA and 1JC PFOA. Because of this, the study sponsor requested the use of an alternative extraction for these compounds, as follows: Direct Injection Method; Before the 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-miniliter 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 -1 0 minutes at -3000 ipm. Each sample was analyzed by LC/MS/MS electrospray. Using this method acceptable data was obtalnedfor PFOS, but the recoveries for PFOA and IJC PFOA were stlU poor. Another alternative method was then used for PFOA and ,5C 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 volumewas taken to 40 mL with water. The samples were then centrifuged for -1 0 minutes at -3000 rpm. The supernatant was then loaded onto a Cl( SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. Theeluale was discarded. Approximately five milliliters of methanol was added to the cartridge. Five mSiaiters of eluate 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 IO: V0001640-6 ADMINISTRATIVE FORM MPI Research Page 125 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 CHEH EHSR 236 IB 651 733 1958 OS-n-iin 01:11 Frgrnsm sourness 05/01 '06 13:A6 N0.106 03/03 M M F.on/oos M U SIA U G K I0 M R iM R h B rtvi Smt Cotton, Ftt.f14.ni.lB0 n iu rttirw e n O U ' ' > ' MPI Research Page 126 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB 6 5 1 778 4226 0 7 /1 1 /0 6 1 1 :4 6 0 :0 4 /0 7 NO:681 RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 D E V IA T IO N FO R M a n y fl: ' ' X Project Specific Deviation Facility Deviation Data' of Occurrence: 04/28X16 Exyoen Project # : P760/P31 Deviation # : ' .O " ' . Client Project rii : ___na; : R e firtn o e# : " '.'b e -m -.'" , RMMlittrifOrlvtr Devlitlon typ e : ilnclud V# h r mathda and SOR) * GMP OLP Ollier None Protocol X Method Vfc VQ0O427-3 Notebook reference: NA 3 ln B lt.P t9 9 'lP llc n : Login#: _______NA Container # : ' C00159446: Lot # : 8uniniarv of Deviation: .; , . One aoN ample (C00159446) was weighed at-B gfor percent solid analysis, rather.than at -20g which Is stated In the method. ' ' " . " . ' Preparation____Analysis____ Inslrurnsnt ^ ___ Client Request ' X- Other Impact; ; '. . .. . . , . . No negative Im ped An accurate percent solid number was obtained by slightly altering the calculation used. '. ' ' ' . SanttWiAfttorit;, Deviation Issued. ^HncIp^Wpvestlgtor / ~ Datai S M i i ______ _ 2 / Exygsn fflange/Tiert Sponsor Rapreq Quality Assures Sponsor Management ^ - jy /ir ' D ' Data '.'Date: MPI Research Page 127 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB @ 651 778 4226 0 7 /1 1 /0 6 1 1 :4 6 0 :0 6 /0 7 NO:681 RESEARCH 3058 Research Drive Pitne: 814-272-1039 State College, PA16801 Fax: 814-231-1S80 DEVIATION FORM Patiate!: X . ' Project Specific Deviation Facility Deviation Date of Occurrence: 68/28708 Exygen Project # : P760/P1131 . Dviation # : I f f ' Client Project # : NA Referenoe * : . WBUliteryPriver: DeviattOn TV0a: (Includa VUfor method* and SOPaj GMP x GLP . Olher None Semole Deaeri otlon: _____ Protocol ____ ' Wethbd V#: 1800 Notebook reference: X SP Login#: ______ NA Container # : NA Lot#:' J4L auitamarirof Deviation: 1 1 . Peer review of raw data waa not documented per SOP V100 prior t 8/28/06.. Cause: Imoict: Preparation__ ' Anryslt Instrument____Client Request V " Other . ;. Raw data will be more thoroughly evaluated arid reviewed before Q Inspection. ' ' li" Corrective Action: A note to fie will be Issued to all subsequent reports stating dial overall summaries have been peer reviewed. ' ' . . " ' <j ih Wk ia|ta it izaloc* Data m Exygeh M ^ g ii ifaju A /iJM . Sppnsr Rpreaentatlve Sponsor Mnagement Date MPI Research Page 128 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB f? 651 778 4226 0 7 /1 1 /0 6 1 1 :4 6 0 :0 7 /0 7 NO:681 I* _ RlSEARCH 3058 Research Drive S tate C ollege, PA 16801 ' Phone: 8 4 '$ fa -1 0 3 9 Fax: 814-231-1580 ' ' :v '' ' ' . PVIATION FRM ; ' ' , general; .. X ' Project Specific Deviation _ _ Facility Deviation Data of Oecrrenos: 08CMB6. ExygenProjBct#: P76/PH31 ' O'eviation # : ; 8fft Client Project # : NA. : Reference it : f . - tile : . RtnuHttry PrlYr= DavlaUenTvaa: (Includa V# for methods and SOPS) ' X ^ __ __' GMP GLP Other Norie X Protocol _____ Method V#NA. Notebook reference: _____ SOP Semate Oscrtotlon; Login# : L0008121 LOO8O01 Container # : .' aummiy:,PtWYleygni- Samples were filled to 250 mL Instead of 200 mL. C06B347 C016B354 C1718S: Lot#: NA Cause: X Preparation __ ;' Analysis Instrument Impact: .: . Samples could not be extracted acocrdlity to the protocol, llant Request _ Other SpHdng.levels were adjusted to sccommodete the alternative volume. Signatures: , Ipel Investigator ~ \u A . Study Director ue/ssur6nce J tk l&e. Date Sponsor Management Date MPI Research Page 129 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No.: P0001131 01-08-2007 03:59pm FrogHfESTON SOLUTIONS T-021 P.005/005 F-05B IT 3058 ReseanehDrive Phone: 814-272*1039 State College, PA16801 Fax: 814-231-1580 RESEARCH DEVIATION FORM X ProjectSpecificDeviation Faculty Deviation Date ofOccurrence: Nov.2006Jan. 2007 Exygen Project#: Client Project#: P0001131 P0001131 D eviation#: Reference#: 10 fl~ O Q L Doviatton ty p e: (Indude V#FOrmBthodsand SOPs) GMP GLP Other None Samle DescrlPtton: _____ Protocol X Mettio# Vil: V0001780 Notebook reference: ____ SOP Login#: L0010165 Container#: L o t# : L0010185 LODI0254 Summary of Sleek standards and fotflGation standards used In the extraction far these logins Mere prepared in acetonitrile Instead of mMhenot. Cause; X Preparation____Analysis_____Instrument Client Request ____ Other No negativeimpact. Compounds arecompletelysolubleInacetonitrileas theyareInmethanol. Corrective Action" Deviation issued. No action taken. LIBRA RY ID: VD001640-7 RECEIVED TIME JAN. 8. 4:02PM AD M INISTRATIVE FO R M PRINT TIME JAN. 8. 4:04PM MPI Research Page 130 of 131 Interim Report #29 - Reanalysis of Soil and Sludge Samples ExyLIMS Study No. : P0001131 02- 11-2007 05:04wn Fron-veSTON SOLUTIONS T-114 P .003/003 f -155 n RESEARCH 3058 Research Orive Phone: 814-272-103? State College, PA16801 Fase 814-23M580 General'. X Project Specific Deviation DEVIATION FORM Pene 1cM_ FacTity Deviation Date of Occurrence: Jen-F e b 2007 ____ Sxygen Project# : Client Project#: P0001131. P0001131 D e v ia tio n # :____11 Reference# : RMulatotvTiffver." " DwStnw Time: (Inebrie W a r m e m o s ana SOPS) GMP GLP Other None Semole DeecrfefiT X Protocol .t;. Notebocikreference: Login#: L0007S84 L0D07593 U0007667 LQ0Q7753 L0007810 L0008125 Container#: Method Lot#: SOP ry p f S p e rd fe rtre q i^ s e e correspondence in raw fetePac^ -,me. onecrem of Amendment 12 was deviated from due to sample mess avateWity. I t e ^ w g w to r sample and adding eightmilliters of,8020 scetonWienwater. only ha a gram of sample was weigpeo and four milliliters of 80:20 seetonltritewater was added. - Cause: X Preparation___ Analysis _ instrument Cfient Request. Other Itonegative impact The rado of sample to solvent in th3extraction stayed the same. Actions: Deviation issued. No action taken. Stonatura; /U u l 6rygen Management U Jul tA i Sponsor Repreamtatrve Sponsor Management ate Data LIBRARY in:VDD01W0.7 RECEIVED TIME FEB .15. 5:07PM ADMINISTRATIVE form PRINT TIME FEB.15. 5:09PM MPI Research Page 131 of 131