Document 6boM8GnXV1O40gGzn11nMO9rg

INTERIM REPORT #25 - Analysis of Ground Water 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 November 22,2006 PERFORMING LABORATORY Exygen Research 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 Exygen Study Number: P0001131 Total Pages: 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen 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 Exygen Research. Weston Solutions, Inc. 3M Company Exygen Research Date Page 2 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No.: P0001131 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen 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 Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Exygen Principal Investigator and Management and to the Study Director. Phase 32. Raw Data & Interim Report review 44. Final Interim Report and Raw Data Review Date Inspected 09/01/06 Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Studv Director 09/08/06 09/12/06 09/14/06 11/21/06 11/22/06 11/22/06 11/22/06 Miwa Flaherty Senior Quality Assurance Auditor Date 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion of the study. This QA statement involves only the review of the interim report and associated raw data. Exygen Research Page 3 o f 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 CERTIFICATION OF AUTHENTICITY This interim report, for Exygen Study Number P0001131, is a true and complete representation of the raw data. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 ` (814) 272-1039 Principal Investigator, Exygen: Date Exygen Research Facility Management: Exygen Research Study Director, Weston Solutions, Inc. Jaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: IAamU A Michael A. Santoro / Director of Regulatory Affairs Exygen Research 22-/\/V Date im Date / ' Page 4 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen 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: EXYGEN STUDY NUMBER: TYPE OF STUDY: SAMPLE MATRIX: TEST SUBSTANCE: P0001131 P0001131 Residue Ground Water Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) SPONSOR: STUDY DIRECTOR: STUDY MONITOR: 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 PERFORMING LABORATORY: Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: 11/05/04 Interim Analytical Start Date: 07/19/06 Interim Analytical Termination Date: 08/04/06 Interim Report Completion Date: 11/22/06 Exygen Research Page 5 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 PROJECT PERSONNEL The Study Director for this project is Jaisimha Kesari at Weston Solutions, Inc. The following personnel from Exygen Research were associated with various phases of this interim portion of the study: Name John Flaherty Karen Risha Christine Edwards Mark Ammerman Brian McAllister Mindy Cressley Krista Gallant Cammy Graybill Ling Ling Liu Title Vice President Laboratory Supervisor Technician Sample Custodian Sample Custodian Technician Technician Technician Technician Exygen Research Page 6 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen 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.......................................................................................................11 4.0 ANALYTICAL TEST SAMPLES..............................................................................11 5.0 REFERENCE MATERIAL........................................................................................12 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................ 13 6.1. Extraction Procedure...............................................................................................13 6.2 Preparation of Standards and Fortification Solutions...............................................13 6.3 Chromatography.......................................................................................................14 6.4 Instrument Sensitivity...............................................................................................14 6.5 Description of LC/MS/MS Instrument and Operating Conditions...........................15 6.6 Quantitation and Example Calculation..................................................................... 15 7.0 EXPERIMENTAL DESIGN......................................................................................17 8.0 RESULTS...................................................................................................................17 9.0 CONCLUSIONS............................................................................................ 17 10.0 RETENTION OF DATA AND SAMPLES............................................................. 18 Exygen Research Page 7 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Table I. Table n. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Ground Water Samples.................. 20 Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples............................................................................................... 21 Exygen Research Page 8 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Figure 1. LIST OF FIGURES Page Typical Extracted Calibration Curve for PFBS in Reagent Water................ 24 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 25 Figure 3. PFBS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................... 26 Figure 4. Chromatogram Representing a Ground Water Sample Analyzed for PFBS (Exygen ID: C0192981, Data Set: 071906BR)....................... :.......... 27 Figure 5. Typical Extracted Calibration Curve for PFHS in Reagent Water................ 28 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 29 Figure 7. PFHS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................... 30 Figure 8. Chromatogram Representing a Ground Water Sample Analyzed for PFHS (Exygen ID: C0192964, Data Set: 071906A).................................... 31 Figure 9. Typical Extracted Calibration Curve for PFOS in Reagent Water................ 32 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively....................................................................................................33 Figure 11. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively..................................... 34 Figure 12. Chromatogram Representing a Ground Water Sample Analyzed for PFOS (Exygen ID: C0192964, D ata Set: 0 7 1 9 0 6 A ).......................................... 35 Exygen Research Page 9 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 LIST OF APPENDICES Page Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method V0001780 and Protocol Amendments....................... 36 Exygen Research Page 10 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No. : P0001131 1.0 SUMMARY Exygen Research extracted and analyzed ground water samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001780 (Appendix A). The limit of quantitation (LOQ) for PFBS, PFHS and PFOS in ground water was 25 ng/L. Analytical results and assessed accuracies for the analysis of PFBS, PFHS and PFOS found in ground water samples are summarized in Table I. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in ground water samples were 123 12%, 109 24%, and 124 33%, respectively. Quantitative results were obtained for PFHS is all samples and PFOS in all samples except the one sample that was not reported (NR) due to quality control failures. With the exception of the trip blank, all PFBS results are designated as NR due to quality control failures. 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 ground water according to Protocol P0001131 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFBS, PFHS, and PFOS in ground water using the analytical methods entitled, "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS." 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 July 19,2006, and the analytical termination date for this interim report was August 4, 2006. 4.0 ANALYTICAL TEST SAMPLES Thirty-five ground water samples (Exygen ID CO192964 - CO192998) representing eight ground water sampling sites (the eight LOI wells) and associated field quality control samples were received on wet ice on June 24, 2006, from Tim Frinak at Weston Solutions, Inc. The samples were logged in by Exygen personnel and placed in refrigerated storage. Exygen Research Page 11 o f 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 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 (SP0005726) and PFHS (SP0002401), were supplied by 3M. PFBS (SP0005726) was received from 3M at Exygen on May 13, 2005. PFHS (SP0002401) was received from 3M at Exygen on January 20, 2003. The analytical standard PFOS (SP0002694) was purchased from Fluka Corporation and was received at Exygen on April 23, 2003. The analytical standards, PFBS (SP0007771), PFHS (SP0007770), and PFOS (SP0007769) were supplied by 3M. PFBS (SP0007771), PFHS (SP0007770), and PFOS (SP0007769) were received from 3M at Exygen on June 13, 2006. 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 PFBS PFHS PFOS Exveen Inventory No. SP0005726 SP0002401 SP0002694 SP0007771 SP0007770 SP0007769 Lot# 101 SE036 430180-1 101 NB 120067-69 217 Puritv (% ) 96.7 98.6 101.2 96.7 98.6 86.9 Expiration Date 12/04/06 10/18/06 10/31/07 12/04/06 10/18/06 08/31/06 The molecular structures of PFBS, PFHS and PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4F9S03*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 (CFnSOaTC^) Transitions Monitored: 399 - 80 Exygen Research Page 12 o f 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Structure: FFF FFF S03 FFF FFF PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFnSCVK4) Transitions Monitored: 499 -> 80 Structure: FFFF FFFF F SO3 FFFF FFFF 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical method "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" was used for the ground water samples in this study. 6.1. Extraction Procedure A 40 mL aliquot of the water sample was used for the extraction procedure. After fortification of appropriate samples, the samples were loaded onto a Cis SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.2 Preparation of Standards and Fortification Solutions A mixed stock standard solution of PFBS, PFHS and PFOS was prepared as specified in Exygen method V0001780. The stock standard solution was prepared at a concentration of 1000 pg/mL by dissolving 100 mg of each of the standards (corrected for purity and salt content, if necessary) in methanol. From this solution, a 100 pg/mL fortification standard solution was prepared by taking 10 mL of the stock and bringing the volume up to 100 mL with methanol. By taking 10 mL of the 100 pg/mL fortification standard and Exygen Research Page 13 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 bringing the volume up to 100 mL with methanol, a 10 pg/mL fortification standard was prepared. By taking 10 mL of the 10 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 1.0 pg/mL fortification standard was prepared. By taking 10 mL of the 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 0.01 pg/mL fortification standard was prepared. A set of standards containing PFBS, PFHS and PFOS were prepared in water and processed through the extraction procedure, identical to samples. The following concentrations were prepared: Cone, of Fort Fort Solution Volume (ng/mL)1 (PL) 00 10 100 10 200 10 400 100 100 100 200 100 400 1of PFBS, PFHS and PFOS Volume of Fortified Sample (mL) 40 40 40 40 40 40 40 Final Cone, of Calibration Std. (ng/L) 0 25 50 100 250 500 1000 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.3 Chromatography Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~0.5 mins, -8.5 mins, and -11.1 mins, respectively. Peaks above the LOQ were not detected in any of the reagent blank samples corresponding to the analyte retention time. 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 25 ng/L of PFBS, PFHS and PFOS. Exygen Research Page 14 o f 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No. : P0001131 6.5 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 Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: ~30C 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 %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Total run time: ~18 min Flow Rate: 0.3 mL/min Ions monitored: Analvte PFBS PFHS PFOS Mode negative negative negative Transition Monitored 299 -> 99 399 -> 80 499 -> 80 Approximate Retention Time (min) ~0.5 min. ~8.5 min. -11.1 min. 6.6 Quantitation and Example Calculation Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations of standards. The concentration was determined from the following equations. Exygen Research Page 15 o f 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No.: P0001131 Equation 1 calculated the amount of analyte found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/L) = (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. Equation 2: Recovery (%) = (analyte found (ng/L) - analyte in control (ng/L)) xl00% amount added (ng/L) An example of a calculation using an actual sample follows (calculation is for PFHS only): Ground water sample Exygen ID: C0192984 Spk G (Set: 071906B), fortified at 500 ng/L with where: peak area intercept slope dilution factor ng/L PFHS added (fort level) amt in corresponding sample = = = = = = 260660 0.00291 507 1 500 155 (Set:071906B) From equation 1: Analyte found (ng/L) = i260660-0.002911 x 1 507 = 514 ng/L From equation 2: % Recovery = (514 ng/L - 155 ng/Ll x 100% 500 ng/L = 72 % NOTE: Numbers may differ slightly from raw data due to rounding. Exygen Research Page 16 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No. : P0001131 7.0 EXPERIMENTAL DESIGN For samples designated as field matrix spikes, PFBS, PFHS, and PFOS were added at a known concentration to the bottles in the laboratory before being shipped to the field. The samples were filled to a 200 mL volumetric fill line in the field. The samples were extracted in three sets. Each set included one reagent blank, two reagent blanks fortified at known concentrations. Two sets contained three sample sites and one set contained two sample sites along with the field blank and the field blank spikes collected for the ground water samples. For each site, a sample, a field duplicate and two-matrix field spikes were collected. For each site, a laboratory duplicate and two laboratory matrix spikes were also extracted. 8.0 RESULTS Exygen Research extracted and analyzed ground water samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001780 (Appendix A). The limit of quantitation for PFBS, PFHS and PFOS in ground water was 25 ng/L. Analytical results and assessed accuracies for the analysis of PFBS, PFHS and PFOS found in ground water samples are summarized in Table I. The primary factor used to assess the stated accuracy was based on the field matrix spikes that were in the appropriate concentration range. Primary spiking level is not appropriate if the primary sample concentration exceeds three times the concentration of the spike level. Quantitative results were obtained for PFHS in all samples and PFOS in all samples except for one sample that was not reported (NR) due to quality control failures. With the exception o f the trip blank, all PFBS results are designated as N R due to quality control failures. Fortification recoveries for PFBS, PFHS, and PFOS in ground water samples are detailed in Table II. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in ground water samples were 123 12%, 109 24%, and 124 33%, respectively. 9.0 CONCLUSIONS Except as noted above, the ground water samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001780. Exygen Research Page 17 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No.: P0001131 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 final 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. Exygen Research Page 18 o f 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 19 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Ground Water Samples Exygen ID C0192996 C0192964 CO192964 Rep C0192965 C0192968 CO192968 Rep C0192969 C0192972 C0192972 Rep C0192973 C0192976 CO192976 Rep C0192977 C0192980 CO192980 Rep C0192981 C0192984 C0192984 Rep C0192985 C0192988 C0192988 Rep C0192989 C0192992 C0192992 Rep C0192993 C lient Sam ple ID G W -TR IP-Q 2-Y06-C P0 G W -220R-Q 2-Y06-LF-0 G W -220R-Q 2-Y06-LF-0* G W -220R-Q 2-Y06-LF-D B G W -220L-Q 2-Y06-LF-0 G W -220L-Q 2-Y06-LF-0* G W -220L-Q 2-Y06-LF-D B G W -226R-Q 2-Y06-LF-0 G W -226R-Q 2-Y06-LF-0* G W -226R-Q 2-Y06-LF-D B G W -226L-Q 2-Y06-LF-0 G W -226L-Q 2-Y06-LF-0* G W -226L-Q 2-Y06-LF-D B G W -310R-Q 2-Y06-C P-0 G W -310R-Q 2-Y06-C P-0* G W -310R-Q 2-Y06-CP-DB GW -317U-Q 2-Y06-CP-0 G W -317L-Q 2-Y06-C P-0* G W -317L-Q 2-Y06-C P-DB G W -320L-Q 2-Y06-C P-0 G W -320L-Q 2-Y06-C P-0* G W -320L-Q 2-Y06-C P-DB G W -327R-Q 2-Y06-C P-0 G W -327R-Q 2-Y06-C P-0* G W -327R-Q 2-Y06-C P-DB C 4 Sulfonate P FB S C6 Sulfonate P FH S C8 Sulfonate PFO S Ptrtluorobutan--utfonato______ PTfluorohexentiulfonate_______ PerfloorooctanMulfonati Analyte Assessed Analyte Assessed Analyte Assessed Found Accuracy Found Accuracy Found Accuracy (ng/L, ppt) {+/-% ) (ng/L, ppt) < '-*> (ng/L, ppt) <+/-%) ND 30 ND 30 ND 40 NR . 55300 40 79000 40 NR - 57100 40 82200 40 NR - 54300 40 72500 40 NR . 80300 40 96000 30 NR - 81100 40 91200 30 NR " 94500 40 174000 30 NR 3150 30 17500 50 NR 3200 30 16000 50 NR 3430 30 20100 50 NR ND 30 ND 50 NR ND 30 ND 50 NR ND 30 26.4 50 NR 441000 30 1430000 30 NR 474000 30 1630000 30 NR 454000 30 988000 30 NR 155 30 222 50 NR 106 30 140 50 NR 128 30 188 50 NR ND 50 NR . NR ND 50 NR - NR ND 50 NR NR 195000 30 736000 50 NR 206000 30 748000 50 NR 177000 30 532000 50 Laboratory Duplicate ND = Not detected at or above the Lim it of Quantitation (LOQ) of 25 ng/L. NR - Not reported due to quality control failures. Exygen Research Page 20 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples Sample Description G W -T R IP -Q 2-Y 06-C P -L S (C 0192997,1.0 ppb Field S pike) G W -T R IP -Q 2 -Y 0 6 -C P -H S (C 0192999, 10 ppb Field Spike) GW-220R-Q2-Y06-LF-0 <00192904 Spk C , 10000 ng/L, Lab Spike) GW-220R-Q2-Y06-LF-0 (C01929S4 Spk D , 100000 ng/L, U b S pike) GW-220R-Q2-Y06-LF-LS (C O iesotO , 10 ppb. Field Spike) GW-220R-Q2-Y06-LF-HS (C 0192967,100 ppb, Field Spike) GW-220L-Q2-Y06-LF-0 (C01929S9 Spk E, 10000 ng/L, U b S pike) GW-220L-Q2-Y06-LF-0 <00102000 Spk F, 100000 ng/L, U b Spike) GW-220L-Q2-Y06-LF-LS <00192070,10 ppb. F ield Spike) GW-220L-Q2-Y06-LF-HS (C 0 ie 2 9 7 1 ,100 ppb, F ield Spike) GW-226R-Q2-Y06-LF-0 <00102072 Spk 0 , 5000 ng/L, U b S pike) GW-226R-Q2-Y06-LF-0 (C0192972 Spk H, 50000 ngfL, U b Spike) GW-226R-Q2-Y06-LF-LS (0 0 19 29 7 4.5 .0 ppb. Field S pike) GW-226R-Q2-Y06-LF-HS (C IH 92 97 5 ,50 ppb, F ield S pike) C4 Sulfonate PFBS___________ C6 Sulfonate PFHS___________ C8 Sulfonate PFOS Amount Amount Amount Amount Found In Amount Found In Amount Found in Amount Spiked Semple Recovered (ng/L, ppt) (ng/L, ppt) (ng/L, ppt) Ree Sample Recovered \%i (no/L, ppt) (ng/L, ppt) Ree Sample Recovered (%> (ng/L, ppt) (ng/L, ppt) Ree 1000 10000 ND ND 1310 11400 131 114 ND ND 1050 105 ND 9730 97 ND 1410 13700 141 137 10000 100000 10000 100000 NR NR NR NR NR NR 55300 75700 * 79000 106000 . NR NR 55300 207000 152 79000 268000 189 NR NR 55300 72200 79000 91400 NR NR 55300 188000 133 79000 214000 135 10000 100000 10000 100000 NR NR NR NR NR NR 80300 97700 . 96000 128000 NR NR 80300 141000 61 96000 182000 86 NR NR 80300 97200 96000 176000 * NR NR 60300 215000 135 96000 215000 119 5000 50000 5000 50000 NR NR NR NR NR NR 3150 8470 106 17500 24000 NR NR 3150 50400 95 17500 64300 94 NR NR 3150 8560 109 17500 27200 NR NR 3150 57600 109 17500 91300 148 ` Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated NO = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures. Note: Since this summery table shows rounded results, recovery values may vary slightly from the values In the raw data. Exygen Research Page 21 o f 36 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study N o.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples Continued S a m p le D e s c rip tio n GW-226L-Q2-Y06-LF-0 (C 0192976 Spk C , 100 ng/L, Lab Spike) GW-226L-Q2-Y06-CF-0 (C 0192976 Spk D, 1000 ng /L , Lab S pike) GW-226L-Q2-Y06-LF-LS (C 0192S 71,0.1 ppb. F ield Spike) GW-226L-Q2-Y06-LF-HS (C 0102070,1.0 ppb, Field S pike) C4 Sulfonate PFBS____________ C6 Sulfonate PFHS_____________C 8 Sulfonate PFOS Am ount Am ount Am ount A m o u n t F o u n d In S p ik e d S am p le (n g /L , p p t) (n o /L , p p t) Am ount R eco vered (n g /L , p p t) Ree (%| F o u n d in S am p le (n o /L , p p t) Am ount R eco vered (n s /U p p t) R ee (%) F o u n d In S a m p le (n g /L , p p t) Am ount R eco vered (n g /L , p p t) Ree (*i 100 1000 100 1000 NR NR NR NR NR NR ND NR NR ND NR NR ND NR NR ND 104 104 ND 1100 110 ND 96.2 96 ND 1360 136 ND 130 1150 149 1770 130 115 149 177 GW-310R-Q2-Y06-CP-0 (CO192OS0 Spk E , 290000 ng/L, Lab Spike) GW-310R-Q2-Y06-CP-0 (C 0192SM Spk F, 2900000 ng/L, U b S p ke ) GW-310R-Q2-Y06-CP-LS (C 01S2SS2,290 ppb. Field S pike) GW-310R-Q2-YQ6-CP-HS (C 0102093,2900 ppb. Field Spike) 250000 2500000 250000 2500000 NR NR NR NR NR NR 441000 807000 146 1430000 1390000 - NR NR 441000 3560000 125 1430000 4440000 120 NR NR 441000 699000 103 1430000 1010000 NR NR 441000 3300000 114 1430000 3410000 79 GW-317L-Q2-Y06-CP-0 (C 0192094 Spk Q , 900 ng/L, Lab Spike) GW-317L-Q2-Y06-CP-0 (C 0192094 Spk H , 9000 ng/L, Lab Spike) GW-317L-Q2-Y06-CP-LS (C 0192996,0.5 ppb, Field S pike) GW-317L-Q2-Y06-CP-HS (C 0 1 9 2 M 7 ,9.0 ppb, Field S pike) 500 5000 500 5000 NR NR NR NR NR NR 155 NR NR 155 NR NR 155 NR NR 155 515 4920 726 4740 72 222 95 222 114 222 92 222 617 5530 952 6290 79 106 146 121 GW-320L-Q2-Y06-CP-0 (C01020SB Spk C, 100 ng/L, Lab S pike) GW-320L-Q2-Y06-CP-0 (C 0102099 Spk D , 1000 ng /L , Lab Spike) GW-320L-Q2-Y06-CP-LS (C 0 1 0 2 M 0 ,0.1 ppb, Field S pike) GW-320L-Q2-Y06-CP-HS (C 0 1 0 2 M 1 ,1.0 ppb, Field S pike) 100 1000 100 1000 NR NR NR NR NR NR ND NR NR ND NR NR ND NR NR ND 99.2 1000 137 1530 99 NR 100 NR 137 NR 153 NR NR NR NR NR NR NR NR NR GW-327R-Q2-Y06-CP-0 (C0192W2 Spk E, 100000 ng/L, Lab Spike) GW-327R-Q2-Y06-CP-0 (C 0192002 Spk F, 1000000 ng/L, U b Spike) GW-327R-Q2-Y06-CP-LS (C 0102004,100 ppb, Field Spike) GW-327R-Q2-Y06-CP-HS (C 0102009,1000 ppb, Field Spike) 100000 1000000 100000 1000000 NR NR NR NR NR N R 195000 277000 62 736000 765000 NR NR 195000 1520000 133 736000 2180000 144 NR NR 195000 267000 72 736000 719000 NR NR 195000 921000 73 736000 1350000 61 A verag e: S ta n d a rd D e v ia tio n : 123 12 A verag e: S ta n d a rd D e v ia tio n : 109 24 A verag e: S ta n d a rd D e v ia tio n : 124 33 'Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated ND * Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR * Not reported due to quality control failures. Note: Since this summary table shows rounded results, recowry values may vary slightly from die values in the raw data. Exygen Research Page 22 of 36 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 23 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 1. Typical Extracted Calibration Curve for PFBS in Reagent Water 071906BR P7GO-1131 W attr.rdb (PFBS): " U n t i l" R tgrtssion p / * tatighting): y * 8 8 .4 x + 7 0 4 ( r - 0.9Q27) Area, counts Exygen Research Page 24 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study N o.: P0001131 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively X C *nt$t-$ PFBS (Standard) t>aaf aot foaad) amu -sample 1 o f4 t from UlMtA-wUt I Arj: 4400 count* Height 3.70e+002 cp* RT: 0.507 min 0.61 Exygen Research Page 25 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 3. PFBS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively I Rongont Centro/ -PFBS (Uk/tnown) 299.&9M nmn rompi* 9 o f 41 from $719$tA.w/ff ponk not fonnd) 11.36 Intensity, cps Intensity, cp s Area: 8800 oounts Haight: 6.50a+002 cps RT: 0.524 min 0.52 Afta: 80367 counts Haight: 6.04t+003 ops RT: 0.527 min Intensity, cp s Exygen Research Page 26 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Ground Water Sample Analyzed for PFBS (Exygen ID: C0192981, Data Set: 071906BR) I C0192901 PFBS {ftotaowM) 299LA 9M i n -sam pt* 14 o f 31 from 07190SBR.wfff A n a : C2*cort* H tigkb 5.42*402 cps RT: 0.543 mi 0.84 Intensity, cps Exygen Research Page 27 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No. : P0001131 Figure 5. Typical Extracted Calibration Curve for PFHS in Reagent Water 071Q06A P760-1131 W jttl.td b (PFHS): R tgresion C'1 t r f (Mighting): y = 731 x + 0.00176 ( i - 0.0084) Area, counts Exygen Research Page 28 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively I x c v n m ^ -P F H S (Stamford) 311.M0.0 p u t not found) -tampl* 1o f 41 from tm o tB .w iff 13.21 Intensity, cp s Tim, min XC072606-1 PFHS (Standard) 399.0*0.0 im u sampl 2 of 41 ffom 07190GB.wiff Ara: 13356 counts Height: 8.33+002 opt RT: 8.00 min 8.66 Intensity, cp s Exygen Research Page 29 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 7. PFHS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively I Roagomt Control - PFHS (Unknown) 399LO/8A0ama -samp/ 9 o f 41 from 0719$B.wiff 4>9J* *OtfOMH<1) Intensity, cp s Tim*, min Reagent Spk A - PFHS (QC) 399.00.0 im u sample 10 of 41 from 07190G9.mil A ita: 27567 count* Height: 1.82e+003 cp* RT: 8.68 min 8.68 Intensity, cp s Area: 270219 count* Haight 1.68e+004op* RT:8.71 min Intensity, c p s Exygen Research Page 30 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Ground Water Sample Analyzed for PFHS (Exygen ID: C0192964, Data Set: 071906A) C$192964 - PFtfS (Unknown) 3M./*& amu -u m p t* 16 o f 41 from 0719**A.wHf A n a : 2969797$ coaata Hu'gt- U S itM S cf RT: $.43 mia 8.43 Intensity, cps Exygen Research Page 31 o f 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 9. Typical Extracted Calibration Curve for PFOS in Reagent Water 07100GC R760-1131 W lttl.rdb (PFOS): " U n til* R tjiin io n f 1 / * ' wtightlng): y * 183 x + 1.81*4003 ([> 0 .0 0 8 4 ) Area, counts Exygen Research Page 32 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No. : P0001131 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively I x c m w 4 - PFOS (StMOdtrd) 4 U M L I <> -tom plr 1 o f4 1 from 0Tn06A.wrff ttoaSoot found) 1U4 Intensity, cp s ___ A rtj : 7040 count* Height: 4.17t+002 ep* RT: 11.0 min 11.04 Intensity, cp s Exygen Research Page 33 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 11. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively I ftongont Control PFOS (Unknown) 49%OTA9 nmn onmpto 9 o f 41 from 971999A.wiff &ook not fonn<0 12.72 Intensity, cp s A rti: 15503 counts Height: 0.07*+002 cps RT: 11.1 min 11.08 Intensity, cp s Arai: 151570 oounte Haight: 8.22+003 cps RT: 11.1 min 11.07 Intensity, cp s Exygen Research Page 34 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Ground Water Sample Analyzed for PFOS (Exygen ID: C0192964, Data Set: 071906A) C9192964 - PFOS (Unknown) 499.m 0.0omu . la rp /t 15 o f 41 from 0719WLwiff Atom: 16342997 com nt* H ig t 5.90**005 cp* RT: 11.1 min 11.10 5.5*5 - 5 .0 *3 - 4 .3 *5 4.0*5 3.5*5- 10.50 3.0*5 2.5*5 2.0*3 1.5*5- 1.0*5- 5.0*4- 0.0 - T im *, min Intensity, cp s Exygen Research Page 35 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods and Protocol Amendments Exygen Research Page 36 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen 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: M ichael A . Santoro Director o f Regulatory Affairs 3M Building 0236-01-B -10 St. Paul, M N 55144 Phone: (651) 733-6374 Page / oj 65 Exygen Research Page 37 o f 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 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 Exygen Research Page 2 o f65 Page 38 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0 0 0 1 131 PROTOCOL APPROVAL Study Title: Analysis o f Perfhiorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Sentm Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 APPROVALS JaisimhaKesan, Si Weston Solutions Michael Ai.. Sawitoro, Sponsor Representative 3M Comp:ady Date S Richard A. Grj Exygen Resi isident, Facility Management U .__________ ad, Quality Assurance Unit Date / m I L Date Page 3 oj 65 Exygen Research Page 39 of 104 Interim Report #25 - Analysis of Ground W ater Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 TABLE OF CONTENTS TITLE PAGE.............................................................................................................................................. 1 DISTRIBUTION......................................................................................................................................... 2 PROTOCOL APPROVAL...........................................................................................................................3 TABLE OF CONTENTS............................................................... 4 INTRODUCTION....................................................................................................................................... 5 TEST MATERIALS................................................................................................................................... 5 OBJECTIVE............................................................................................................................................... 6 TESTING FACILITY..................................................................................................................................6 STUDY DIRECTOR................................................................................................................................... 7 SPONSOR REPRESENTATIVE................................................................................................................. 7 PRINCIPAL INVESTIGATOR................................................................................................................... 7 PROPOSED EXPERIMENTAL START AND TERMINATION DATES................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM....................................................... 8 SAMPLE PROCUREMENT, RECEIPT AND RETENTION...................................................................... 8 SAMPLE IDENTIFICATION..................................................................................................................... 9 ANALYTICAL PROCEDURE SUMMARY............................................................................................... 9 VERIFICATION OF ANALYTICAL PROCEDURE...................................................................................9 METHOD FOR CONTROL OF BIAS......................................................................................................... 11 STATISTICAL METHODS........................................................................................................................ 11 GLP STATEMENT..................................................................................................................................... 11 REPORT..................................................................................................................................................... 11 SAFETY AND HEALTH............................................................................................................................ 12 AMENDMENTS TO PROTOCOL.............................................................................................................. 13 DATA RECORD KEEPING........................................................................................................................13 QUALITY ASSURANCE........................................................................................................................... 14 RETENTION OF DATA AND ARCHIVING..............................................................................................14 APPENDIX I, ANALYTICAL METHODS..................................................................................................15 Page 4 of 65 Exygen Research Page 40 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 INTRODUCTION The purpose o f 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 w ill be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit o f 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 (C4F9SO]*K'1') Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 - 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFu SOj'K*) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 3 9 9 - 8 0 Structure: S03 Page 5 o f 65 Exygen Research Page 41 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CiFpSOjTC*) Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 - 99 Structure: OBJECTIVE The purpose o f this study is to perfonn analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions o f the following Exygen analytical methods: V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f 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 Exygen Research Page 42 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number P0001131 STUDY DIRECTOR Jaisimba 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 o f 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 o f this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and term ination dates w ill b e included in the final report. Page 7o f 65 Exygen Research Page 43 of 104 Interim Report #25 - Analysis of Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOU31 IDENTIFICATION AT 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 w ill be included in the final report associated with this study. The test systems were chosen to access the environmental impact o f 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 o f samples collected will vary depending availability in the field. The total number o f samples received and analyzed for each matrix will be documented in die final report associated with this study. Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples w ill be stored refrigerated at 2C-8C. Fish, clam, vegetation and small mammal liver samples w ill be processed according to the appropriate analytical method (see Appendix I). These sam ples will b e stored frozen at -10C. Small m am m al w hole blood samples w ill be centrifuged in the field at the time o f collection and the serum fraction w ill be used for the study. Small mammal serum will be stored frozen at -10"C. The receipt and processing o f the samples will be documented in the final report and raw data associated with the study. Page 8 o f 65 Exygen Research Page 44 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 SAMPLE IDENTIFICATION Prior to analysis, each sample w ill 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 o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: `M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: `M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" The above methods use analytical conditions capable o f separating the isomers o f 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 o f fortified control samples. The test substance w ill be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen w ill 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 of 65 Exygen Research Page 45 of 104 Interim Report #25 - Analysis of Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 collection at the testing facility and have been shown to be free o f PFBS, PFHS and PFOS. Samples w ill 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 o f each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (l3C PFOA). PFOA and l3C PFOA are included in the solutions used to spike the samples. The results for PFOA and ,3C PFOA will not be reported in this study. Exygen w ill 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) w ill be extracted in duplicate and w ill 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 w ill supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. A ll 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 w ill 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 w ill 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 w ill also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. Low and high spiking levels for each matrix are defined below: M atrix Low Spiking Level High Spiking Level Water 500ng/L 5000 ng/L Soil Sediment 4ng/g 4 na/a 40ng/g 40 nu/g Fish lO n g /g lOOng/g Clams lO n g/g lOOng/g Vegetation lOng/g 100ng/g Small Mammal Liver 10ng/g lOOng/g Small Mammal Serum lOng/mL lOOng/mL Page 10 o f 65 Exygen Research Page 46 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Recoveries are anticipated to be between 70% and 130% o f the fortified levels; however, the exact precision and accuracy w ill be determined by the analysis o f the quality control samples described above. A statement o f accuracy w ill be included in the final report. METHOD FOR CONTROL OF BIAS Control o f bias will be addressed by taking representative sub-samples from a homogeneous mixture o f each matrix from untreated control samples, and by analyzing at least two levels o f fortifications. STATISTICAL METHODS Statistics w ill be limited to those specified in the subject methods and to the calculation o f average recoveries, as applicable. GLP STATEMENT A ll aspects o f 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 w ill be prepared by the principal investigator or their designee at the conclusion o f the study. The report w ill include, but w ill not be limited to, the following: The name and address o f the Study Director, Sponsor Representative, and o f the testing facility. A statement o f GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). Page II o f 65 Exygen Research Page 47 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates o f study inspections and dates findings were reported to the Study Director and Management. A description o f the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy o f 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 o f 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 o f a standard and a control sample, and a chromatogram at a fortification level. The location o f the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity o f the data w ill 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 o f an amendment signed by the Study Director. The amendment shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendment w ill be signed and dated by the Study Director and the Sponsor Representative. A ll applicable requirements for reporting o f study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel w ill practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environment to the test or reference substance(s). Page 12 o f 6S Exygen Research Page 48 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen 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 file Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation o f study plan change. However, when a change is required without sufficient time for the issue o f 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 o f the written amendment will be the date o f the documented change. Copies o f the signed amendments w ill be appended to all distributed study plan copies. The original amendment w ill be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided w ill be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description o f 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 o f study personnel, signatures and initials Chronological presentation o f all study correspondence Any other documentation necessary for the reconstruction o f the study Chromatograms- A ll chromatograms w ill contain the following: Sample identification, injection date, arrow or other indication o f the area o f interest, and injection number corresponding to the run. Additionally, fortifications w ill include the amount o f analyte added and the sample number o f the sample that was fortified. Analytical standard chromatograms w ill additionally include the concentration (e.g., pg/mL). Page 3 o f 65 Exygen Research Page 49 of 104 Interim Report #25 - Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 A s part o f the documentation the following sheets w ill 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 o f Exygen Research w ill inspect the study at intervals adequate to assure compliance with GLP's, and w ill report the findings o f 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 w ill be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period o f time specified in 40 CFR 792.19S. An exact copy o f the materials submitted to the study director will also be kept at Exygen Research. Exygen w ill obtain permission from the study director before discarding or returning samples. Exygen Research Page 14 o f 65 Page 50 of 104 Interim Report #25 Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 APPENDIX I ANALYTICAL METHODS V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781 "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782 "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783 "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" _ V0001784 `M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785 `M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786 "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" Exygen Research Page IS o f 65 Page 51 o f 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number; P0001131 ANALYTICAL METHOD Method Number V0001780 Method of Analysis for the Determinetiee of Parfleerooctaeok Acid (PFO A) fai W ater by LO M 8/M S Analytical Testing Facility; Exygen Research 3058 Research Drive Sode College, PA 16801 Approved By: C-XLi_________ Paul Connolly * Technical Leader, LC-MS, Exygen Research Date /o h n Flaherty S ~ ' Vice Preodenl, Operation, E*ygen Reeearch Date Exygen Research Total Paget: 7 Page 16 o f 65 Page 52 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 EnygtBtw iw fc Mvtbod Nunwer VOOOIHO ANALYTICAL METHOD Method o f Analysis for the Determination o fPerfluocooctsnote Acid (PFOA) in Water by LC /M S/M S 1.0 Scope T h ii method is to he employed for the isolation and quantitation o f perfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/M S) in water. 2.0 Safety 2.1 Always obecrve safe laboratory practices. 2.2 Consult foe expropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 40 mL o fted aample for extraction. 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 A ll samples must be thoroughly mixed before being sampled for extraction 3.5 Any samptea containing particles should be centrifuged at ~3000 rpm for -5 minutes and foe supernatant used for the extraction. 3.6 Sample collection procedures w ill be specified in foe sampling plan for this project 4.0 Reagents and Standards 4.1 Water- HPLC 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 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 Mesa Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balancecapable o freading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifUge tubes. 5.5 15 mL 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 Autopipettea (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg ) tC lS SPE cartridges. Page 2 of ? Page 17 o f 65 Exygen Research Page 53 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 ExypnUfMich MKbod Numb V0001780 A N A LY TIC A L m e t h o d Method o fAnalytic for foe Determination o fPerfluorooetanoic Aekl (PFOA) in Water by LC /M S/M S 5.12 SPB vacuum manifold. 5.13 Centrifbge capable o f spuming 50 mL polypropylene tubee at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Floophaae RP (Keystone Scientific), 2.1 mm x 50 mm, 5n (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase(A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Fhaae(B): Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 StA 65 65 25 25 65 SLB 35 35 75 75 35 Flow Rate fm L/m inl 0.3 0.3 0.3 0.3 0.3 6.6 Injection Volume: 15 pL (cm be increaaed to aa much as SOpL). 6.7 Quantitation: Peak Area - external standard calibration curve. The above condition are intended a a guide and may be changed in order to optimize foe HPLC system. 7.0 MS/MS System 7.1 Mode: Electroeprsy Negative M RM mode, monitoring 4 13 - 369 nv/z. The above conditions era intended ai a guide and may be changed in order to optimize foe MSMS syftcm. 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0.IS4 g of nmonium acetateto 1000 mL o f water. Alternate votaries may be prepared. Ptye J ol ' Page 18 o f65 Exygen Research Page 54 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygcaRttMich MethodNumber V0001780 ANALYTICAL METHOD Method o fAnalyst fi the Determination o f Perfiuorooctanoic Acid (PFOA) in Water 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 (conected fi purity) and dilute to 100 mL with methaool in a 125-mL LDPE bottle. 9.1.2 A 10 pg/raL fortification solution o f PFOA ia prepared by bringing 10 mL of the 100 jig/tnL solution to a final volume o f 100 with methanol in a 12S mL LOPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o fPFOA ia prepared by bringing Id mL o f the 10 pg/mL aolution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification aolution o fPFOA il prepared by bringing 10 mL o fthe 1.0 pg/mL aolution to a final volume o f 100 with methanol in a 123 mL LOPE bottle. 9.1.3 A 0.01 pg/mL fortification aolution o f PFOA ia prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol In a 123 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 fi a maximum period o f 6 months from the dateo fpreparation, 9.2 Standard Calibration Solutions 92.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are proceeaed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be Concentration o fFortification Solutionloot) 0 10 10 10 100 100 100 Fortification Volume of Volume Fortified Condri (ttU Samoli (mL) 0 40 100 40 200 40 400 40 100 40 200 40 400 40 Final Concentration of Calibration Standard foot)* 0 23 50 100 230 500 1000 Calibration Standard ID (example) XCraraddyy-O XCmroddyy*l XCmmddyy-2 XCmmddyyO XCmmddyy-4 XCmroddyy-5 XCmmddw-6 * The extracted concentration o f the calibration standard is equal to 8 * its initial concentration, due to the concentration o fthe standard during the extraction (SPE). XC extracted calibration standard. Page 4 o f ' Page 19 o f65 Exygen Research Page 55 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 BxyswXMMxdi MethodNumberVQOOI7tO J| ANALYTICAL METHOD Method o f Analysis Ihr the Determination ofPerfluorooctanoic Acid (PFOA) in Water by LC /M S/M S 9.2.3 9.2.4 9.2.5 A zero standard solutkw (reagent blank) m int be prepared with each set o fstandardsextracted Store all extracted calibration standards in 15-mL polypropylene tubes at 2*C to 6*C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as (0.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or leas) must include at least me reagent control (method blade using HPLC water) and (wo reagent controls fortified at known concentrations (lab control spike) to verify procedural rooovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in foe quality assurance plan for this project. 11.0 Sample Extraction 11.1 Measure 40 mL o f sample or a portion o f sample diluted to 40 mL with w&ter into 50 mL polypropylene centrifiige tubes (fortify as needed replace lid and mix w ell). 11.2 Condition the C u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL o fHPLC water (~ 2 dropfeec). Do not let column run dry 11.3 Load sample on conditioned C it SPE cartridge. Discard eluate. 11.4 Elute with ~5 mL 100% methanol Collect 5 mL o f eluate into graduated 1SmL polypropylene centrifiige tubes (final volume " 5 mL). !1.5 Analyze samples using electrosprmy LC/MS/MS. 12.0 Chromatography 12.1 Iqject foe 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 Standardi o f PFOA eosresponding to at least five or more concentration levels must be included in is analytical act 12.3 An entire set of extracted calibration standards must be included at the beginning and at the end o f a sample set. Extracted standards must he interspersed between every 5-10 samples. Aa an alternative, an entire set of extracted calibration standards may be injected at the beginning of a set followed by extracted calibration standards interspersed every 5*10 samples (to account for a aooond act o f extracted standards). In either case, extracted calibration standardi mustbe 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 l/x weighting of peak area Page5 of7 Page 20 o f 65 Exygen Research Page 56 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyganRmrrh MstfaodNumberV000I7M I ANALYTICAL METHOD | Method o fAoalytU forfoe Determination o fFerfluoiooctanoic Acid (PFOA) in Waterby LC/MS/MS venue calibration standard concentration using MstsLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be father diluted and reanalyzed. 13.0 AcceptanceCriteria 13.1 Chromatogram must show a peak o f t daughter ion at 369 amu from a parent of 413 amu. The 413 amu parent corresponds to the PFOA anion, while the ^'ghter ion (369 amu) represents the toss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contain PFOA at levla greater than 50 ng/L, then new blank sample roust beobtained and the entire set must be re-extracted. 13J Recoveries of control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike falls outride the acceptable limits, the entire act o f samples should be re-extracted. Any mstrix 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 ststisticaJ outlier by using (he Huge Error Test, may be excluded from the calculation o f the calibration curve. However, die total number o f extracted calibration standards that could be excluded must not exceed 20% o f the Mal number o f extracted standards injected. 13.5 The correlation coefficient (R ) for calibration curves generated must be 20.992 (R3 20.985). 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 times between standards id samples must not drift more than 4 % within an analytical run. I f retention time drift exceeds Oils lim it 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 foe standard curve (linear regression parameters) generated by foe Maas Lynx software program: PFOA found (ng/L) - (Peak area - intercept) x DF slope DF * factor by which foe final volume was diluted, if necessary. Page 6 of 7 Page 21 of 6S Exygen Research Page 57 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Hxygcn Protocol Number: POOOH31 ExygMKtMVCfc Mtdwcf Nwabrr VOOOl780 | AWALVT1CAL M IT H O P | Method ofAnalyse fcr the Determination o fPerfhiorooctmok Acid (PFOA) in Water by LC /M S/M S 14.2 Por samples fbetified w ilh known amounts o f PFOA prior to extraction, use the Allowing equation to calculate the percent recovery. Recovery (% ) - I total analytefound(ng/L) analytefound in control(ng/L)l _10Q analyteadded (ng/L) Exygen Research P tg t7 0f7 Page 22 o f 65 Page 58 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: POOOI13I ANALYTICAL METHOD Method Humber V0001781 Method o fAnalysis for the Determination o f Perfloorooctuoie A d d (PFO A) la Sell by LC /M 8/M S Analytical Testing Facility: Exygen Research 3058 Research Drive Stale College, PA 16801 Approved By: " A L-JJL____ Paul Connolly ' Technical Leader, LC-MS, Exygen Reaaarch /md/y._______ Jffhn Flaherty 7 / Vice President, Operations, BxygcnResearch Date Exygen Research Total Pages: 7 Page 23 o f65 Page 59 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygea Protocol Number: P001131 tin n U H iC M ttbodN unbcr V000178] I ANALYTICAL METHOD Method o f Analysis for foe Determination o fPerfluorooctanoic Acid (PFOA) in Soil by LC /M S/M S 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectiometric Detector (LC/MS/M S) in soil. 2.0 Safoty 2.1 Always observe safe laboratorypractices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t lead 15g o ftestsample for extraction. 3.2 No sampleproceasing is needed for soil samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 A ll tenples mustbe thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedures w ill be specified in the sampling plan for this project. 4.0 Reagenta end Standards 4.1 W ater-H PLC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Add - Sigma-Aldrich 5.0 Instrument end 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 advents equipped with t variable volume iqjector capable o f injecting 5-200 pL eonneeted to a tandem Maas Spectrometer (LC/M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o freading to 0.00001 g. 50 mL disposable polypropylene centrifuge tubes. 15 mL disposablepolypropylene centrifoge tubes. Disposable micropipets (SO-IOOiiL, !00-200uL). 125-mL LDPE narrow-mouth bottles. 2 mL clear HPLC vial kit. Disposable pipettoe. Autopipettoi (100-1000 pL and 10-100 pL), with disposable tips. Waters Sep Pak Vac 6 cc (lg ) tC I8 SPE cartridges. SPE vacuum manifold. Ultrasonic bath. Pi* 2erf' Page 24 o f 65 Exygen Research Page 60 of 104 Interim Report #25 - Analysis of Ground W ater Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 B xyya Roeireb Method NanbcrVOOO1711 ANALYTICAL M ITHOP Method o fAnalysis for the Detenmnetioo o/Perfluorooctaooic Acid (PFOA) in Soil by LC /M S/M S 5.14 Wrist-action ihaker. 5.15 Centrifuge capable ofspinning SOmL polypropylene tubes ai 5000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5m (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Pbaao (B ): Methanol 6.5 Gradient Program: X inslm io) 0.0 1.0 8.0 20.0 22.5 SLA 65 65 25 25 65 Flow Rate & fmL/min) 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 >iL). 6.7 Quantitation: Peak A rea - external standard calibration curve. 6.8 RunTime: **2 3 minutes. The above condition 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 M RM mode, monitoring 413 - 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize die 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 or ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. Pge3of? Page 25 o f 65 Exygen Research Page 61 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea tu m id i Method NumberV0001781 ANALYTICAL METHOD Method o f Analysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Soil by LC /M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a itock aoiution o f -lO O pg/raL o f PFOA by weighing 10 mg of analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 12J*mL LDPE bottle. 9.1.2 A 10 p^m L fortification solution o f PFOA is prepared by bringing 10 mL o f the 100 p^m L aoiution 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 1U mL of the 10 pg/mL aoiution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pptaL fortification solution o f PFOA ia prepared by bringing 10 m Loffoe 1.0 pg/mL aoiution to a final volume oflOO with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification aoiution o f PFOA ia prepared by bringing 10 mL o f die 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The itock 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 ire prepared in HPLC water The calibration standards are processed Enough the extraction procedure, identical to samplea. The following is a typical example: additional concentrations may be prepared asnooded. Concentration o f Fortification Solution info) 0 10 10 10 100 100 100 Fortification Volume of Volume Fortified Control (uL) SamlemL) 0 40 100 40 200 40 400 40 100 40 200 40 400 40 Final Concentration of Calibration Standard (ddO* 0 25 50 100 250 500 1000 Calibration Standard ID fexamole) XCmmddyy-0 XCmmddyy-l XCmmddyy-2 XCmmddyy-3 XCmmddyy-4 XCmmddyy*5 XCmmddW'6 * The extracted concentration o f the calibration standard is equal to 8x its initial concentration, doe to the concentration o f the standard during the extraction (SPE). XC - extracted calibration standard. Pag 4 o f 7 Page 26 o f65 Exygen Research Page 62 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 BxygBRaaaafcfc M atted NenterVOOOl7|| ANALYTICAL METHOD ] Method o fAnalysis for the Determination o f Perfluoiooctinoic Acid (PFOA) in Soil by LC /M S/M S 9.2.3 9.2.4 9.2.$ A zero ctanderd solution (reagent blank) m m be prepared with each eeto f standard* extracted. Store all m eted calibration standards in 15-mL polypropylene lubes at 2C to 6*C, up to two weeks. Alternate volumes end 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 5 mL o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural reoovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assuranceplan for this project. 11.0 Sample Extraction 11.I Weigh 5 g o f sample into 50 mL polypropylene centrifuge tubes (fortify as needed replace lid end mix well). 1U Add 5 mL o fmethanol and shake on a wrist action shaker fo r-IS minutes. 1U Transfer thetubes to an ultrasoate bath and sonicate for ~ 13 minutes. 11.4 Bring the volume up to 40 mL with water in tbe 50 mL polypropylene centrifoge tube. ^ 11.5 Centrifoge for -1 0 at -3000 rpm. 11.6 Condition the Cis SPB cartridgee (1 g, 6 m L) by passing 10 mL methanol followed by 5 mL ofHPLC water ( - 2 drop/sec). Do not let column run dry 11.7 Load (decant) the sample on the conditioned Cis SPE cartridge. Discard etoate. 11.8 Elute wife -5 mL 100% methanol. Collect 5 mL o f eluate into graduated 1S mL polypropylene centrifoge tubes (final volume 5 mL). 11.9 Analyze samples usingelectrospray LC/MS/MS. 12.0 Chromatognphy 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 (ample*. 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 set Extracted standards must be interspersed between every 5*10 samples. As an alternative, an entire set of Pag*$ of7 Page 27 o f 65 Exygen Research Page 63 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number P0001131 Exyt*a JlMttfch Method Number VOOO1781 I A N A LYTIC A L M ETH O D Method o f Analysis for theDetenninitionofPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standard! interspersed every 5-10 samples (to account for 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 MassLynx 3.3 (or equivalent! software system. 12.5 Sample response should not exeeed standard responses. Any samples that exceed standard responses should be furtherdiluted and reanalyzed. 13.0 Acceptance Critaria 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 die 1m s o fcarbon dioxide. 13.2 Method blanks muat not contain PFOA at levels greeter than the LOQ. If a blank contains PFOA at levala greater than 50 ng/L, then a new blank sample mustbe 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 foils 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 (letam ine if re-extraction is 13.4 Any calibration standard found to be a statistical outlier by using the Huge Brror 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 foe total number o f extracted standards injected. 13.5 The correlation coefficient (R ) for calibration curves generated musi be 20.992 (R2 20.985). I f calibration results fall outside these limns, then appropriate slept muat be taken to adjust instrument operation, and the standards or the relevant sat o fsamples should be reanalyzed. 13.6 Retention times between standards id samples must not drift more than 4 % within an run. I f retention time drift exceeds this lim it within an analytical ran then foe set must be reanalyzed. Pile a of7 Page 28 o f 65 Exygen Research Page 64 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E s y y Russrch M1bodNuMbarV00017tl ]_______________________ A N A LYTIC A L M ETHO D_______________________ Method o fAnalysis forth Determination o fPeriluorooctsooic Acid (PFOA) in Soil by LC /M S/M S 14.0 Calculations 14.1 Use the following equation to calculate foe amount o f PFOA found (ia ng/L, baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/L) xD F DF factor by which foe final volume waa diluted, if noccaaary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use foe following equation to calculate foe percent recovery. Recovery (%)m [ totalanalyte found(ng/L) analyte found in oontrol(ng/L)] analyteadded (ng/L) 14.3 Use the following equation to convert foe amount o f PFOA found in ng/L to ng/g(ppb). PFOA found (ppb) - fPFQA found fna/Ll x volume extracted (P.04U1 sample weight (5 g) 14.4 Use foe following equation to calculate the amount o f PFOA found in ppb based on dry weight. PFOA found (ppb) dry weight " PFOA found (ppb) x [100% / total aolida(%)] Pag* 7of7 Page 29 o f 65 Exygen Research Page 65 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOI131 ANALYTICAL METHOD Method Number V0001782 Method o f Aialysli for the Decermlnition of Perfluorooctaaoiic Acid (PFOA) In Sediment by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16601 Approved By: V --V c j L Paul Connolly ( Technical Leader, LC-MS, Exygen Research a /w ________ JohnFlaherty f Vice President, Operations, Exygen Research lO kfaA W Date A r Due Exygen Research Total Pagea: 7 Page JOof65 Page 66 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exypa Rnearck Method Number VOOO1782 I A N A LYTIC A L M ETH O D | Method o f Analysis for the Determination o fParfluorooctanoic Acid (PFOA) in Sediment by LC /M S/M S 1.0 Scope This method ia to be employed for the iaolatinn and quantitation o fperfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spcctromotric Detector (LC/MS/M S) in sediment 2.0 Safety 2.1 Always obaarvesafe laboratorypracticea. 2.2 Consult the appropriate MSDS before handling any cbemical for proper safety 3.0 Sample Requirement 3.1 At leaat 30 g ofteat sample for extraction. 32 No sample processing ia needed for aediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 A lt samples must be thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedural w ill be specified in the sampling plan for this project 4.0 4.1 W atar-H PLC grade 4.2 Methanol - HPLC grade 4.3 Acetic Add Reagent grade 4.4 Ammonium Acetate - A C .S. Reagent Grade 4.5 Parfluorooctanoic Add-Sigm a-Aldrich 5.0 Instrument and Equipment 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ife a variable volume utfedor capable o f injecting 3-200 jiL connected to a tandem Masi Spectrometer (LC/M S/M S). 3.2 A device to oolleet raw data for peak integration and quantitation. 3.3 Analytical balance capable o f reading to 0.0000) g. 3.4 50 mL disposablepolypropylene oentriAige tubes. 5.5 15 mL diyosable polypropylene oeotriftige tubes. 5.6 Disposablemicropipets (5(M 00uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposablepipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with dispocable tips. 5.11 W atenSspPakVac6cc(lg)tC18SP6cartridgee. 5.12 SPE vacuum manifold. Pap 2of7 Page 3/ o f 65 Exygen Research Page 67 of 104 Interim Report #25 - Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExjrfM Rm m rIi Method Number V0001782 I A N A LYTIC A L M ETH O D | Method o fAnalysis for the Determination o f Perfluorooctanoie Acid (PFOA) in Sediment bv LC /M S/M S ' 5.13 Vortexer. 3.14 Wrist-action shaker. 5.15 Centriflige capable ofspinning 50 mL polypropylene tubes at 3000 ipm. 6.0 Chromatographic System 6.1 Analytical Column: FluopbaaeRP (Keystone Scientific), 2.1 nun x 50 mm. 5n (P/N: 82305-052130) 6.2 Temperature: 30"C 6.3 Mobile Phase (A ): 2 mM Ammonium Acetate in Water Mobile Phase (B ) : Methanol Gradient Program: Q gim in) 0.0 1.0 8.0 20 .0 22.5 2LA 6$ 65 25 25 65 Flow Rate (m lVm inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6 .6 injection Volume: IS jiL (can be increased to as much as 50 pL). 6.7 Quantitatioa: Peak Area - external standard calibration curve. 6 .8 Run Time: - 23 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 M RM mode, monitoring 413 -* 369 m/z for PFOA. The above m diffr* ate intended u a guide sod 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 0.154 g of ammonium acetateto 1000 mL o f water. P ti'J o n Page 32 o f 65 Exygen Research Page 68 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bxyy Rwwh Method Number VOOOI7B2 A N A LYTIC A L M ETH O D Method o f Analysis for the Determinetion o f Perfluorooctanoic Acid (PFOA) in Sediment by LC /M S/M S 8.2 Extraction Solutioni 8.2.1 IS eoetic add in water ie prepared by adding 10 mL o f acetic add to 1 0 0 0 mL o fwater. Alternate volumes maybe prepared. 9.0 Standard Preparation 9.1 Stmdard Stock/Fortification Solution 9.1.1 Piepaic t etock solution o f -100 pgftnL o f PFOA by weighing 10 mg of analytical standard (corrected fix purity) and dilute to 100 mL with methanol in a 125-mL LDPfi bottle. 9.12 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 100 pg/raL solution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f tits 10 pg/mL solution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fbrtiiication solution o f PFOA is prepared by bringing 10 m Lofthe 1.0 pg/mL solution to e final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions ere to be stored in a refrigerator at approximately 4*C and are stable fix a maximum period o f 6 months from tiw date ofpreparation. 9.2 Standard CaUbration Solutioni 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o fthe 0.1 pg/mL fortification solution. 9.2J The following is a typical cample: additional concentrations may be Concentration o f Fortification Solution (nc/mL) too 100 too 10 5 2 Volume fa L ) 10 5 2 10 10 10 Diluted to (mL) 100 100 100 100 100 100 Final Concentration (na/mL) 100 5.0 2.0 1.0 0.5 0.2 Page 4 of 7 Page 33 o f 65 Exygen Research Page 69 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exypnfam iv Method N u f e r V00017? I A N A LYTIC A L M ETH O D | Method o fAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Sediment by LOMS/M S 9.2.3 Store all calibration standards in 125-raL LDPE narrow-mouth bottles al 2*C to 6 *C, 9 to six month*. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as 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 verity procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in die quality assuranceplan for tide project. 11.0 Sample Extraction 11.1 Weigh S g o f sample into 50 mL polypropylene centrifoge tubes (fortify as needed, replies lid sad mix well). 11.2 Add 35 mL o f 1% acetic acid, cap, vortex and shake on a wrist action shaker for ~60 minutes. 11.3 Centrifoge the tubes at -3000 rpm for -2 0 minutes. 114 Condition the Cu SPE cartridges (1 g, 6 m L) by passing 10 mL methanol followed by 20 mL o fHPLC water (~ 2 dmp/sec). Do not let column run dry 11.5 Load (decani) the sample on the conditioned C u SPE cartridge. Discard eluafe 11.6 Add 20 mL o f methanol lo the sediment left in the bottom o f the 50 mL centrifiigc tube. Cap, vortex and shake on a wrist action shaker for -30 11.7 Centrifoge the tubes at -3000 rpm for -2 0 minutes. 11.8 Decint the methanol onto the same SPE cartridge. Collect the eluste. 11.9 Wish the column with 4 mL o fmethanol. Collect the eluate and add it to the ehiate collected in step 11A 11.10 Condition a second Cu SPE cartridge (1 g, 6 m L) by passing 10 mL methanol followed by 20 mL o f HPLC water ( - 2 drop/eec). Do not let column run dry 11.11 Add the methanol to -200 mL o f water and load on the second conditioned SPE cartridge. 11.12 Elute with -3 mL I00H methanol. Collect 5 mL of eluate into graduated 15 mL polypropylene centrifoge tubes (final volume 5 mL). 11.13 Analyze samples using electrospny LC/MS/MS. P*g* 5 ot'7 Page 34 o f65 Exygen Research Page 70 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 S iy f Raw ct Method Number VOOOI7I2 A N A LYTIC A L M ETH O D Method o fAnalysts for the Determination o fPerfluorooctanoic Add (PFA) in Sediment by LC /M S/M S 12.0 Chromatography 12.1 12.2 12.3 12.4 12.5 Inject the same m ount o feach standard, ample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. Standards ofPFOA corresponding to it least five or more concentration levels must be included in an analytical set. An entire set o f extracted calibration standards must be included at the beginning and at foe end o f a sample id . Standards must be interspersed between every 5*10 ismplee. As an alternative, an entire set o f calibration standards may be iryected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second id of standards). In either case, calibration standards must be foe first and last iiyection in a sample set Use linear standard curvst 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 MassLynx 3.3 (or equivalent) software system. Simple 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 daughterion (369 amu) represents the loss o fcarbon dioxide. 13.2 Method blanks must not oontain PFOA at levels greater than the LOQ. If a blank *****" PFOA X levels greater thm 0.2 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 133 Recoveries o f control spikes and matrix apikea must be between 70-130% of their known values. I f a control 4 ke foils outside the acceptable limits, the entire set o f sample* should he re-extracted. Any matrix spike outside 70130H should be evaluated by foe 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 foe calculation o f foe calibration curve However, foe total number o f extracted calibration standards that could be excluded must not exceed 2 OS o f the total number o f extracted standards injected. 13.5 The correlation coefficient (R ) for calibration curves generated must be 20.992 (R* 20.945). I f calibration results foil outside these limits, then appropriate stops must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. Pae*6of? Page 35 o f 65 Exygen Research Page 71 o f 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000U3I ExygnRMMKh MeftodNuotar V000I7I2 1 ........... A N A LYTIC A L M ETH O D | Method o fAnalysis for the Determination o fPerfluorooclinoic Acid (PFOA) in Sediment by LC /M S/M S 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 lim it 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 ngftnL. bated on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ngfa&L) - (Peak area intercept) x DF slope DF 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(H )" f totalanalyte found (ng/mL) analyte found in oontrol (ng/mL)] ^ analyte added (ng/m L) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found (ppb) - fPFOA found (nahnUx final volume fS m Lll sample weight (3 g) 14.4 Use 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 [ 100% / tots) solids(%)} Pate 7 of? Page 36 of63 Exygen Research Page 72 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001783 Method o f Aaalyaia for the Determlaotloa o f Perilaorooctanolc Aetd (PPOA) in Fish end Clams by LC/M S/M S Analytical Totting Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By. y ? -I f -ll. Paul Connolly > Technical Leader, LC-MS, Exygen Research \/m d S Flaherty Vice President, Operations, Exygen Research HlVrM Date .je A k . Date Exygen Research Total Papa: 8 Page 37 o f 65 Page 73 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Eaypa Jtasmmb Msttaod Number V00017S3 I a Pia L Y T IC A L M E T H Q P I Method o f Analysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Clamsby LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mats Spectrometric Detector (LC/MS/M S) in fish and clams. 2.0 Safety 2.1 Always obaerve safe laboratory practices. 2.2 Consult tiie appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g o fteat sample for extraction. 3 2 Samplea 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. 3.3 Sample collection procedures w01 be specified in the sampling plan for this project 4.0 Reagents end Standards 4.1 W ater-H PLC 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 merit) - Reagent grade 4.6 Ploriiit (60-100 mesh) - Reagent grade 4.7 Supcrclein LC-NHj - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L-Ascorbic acid - Reagent grade 4.10 DimethyIdichloTOeiUne-Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Add - Sigma-Aldrich 5.0 bstniment 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 Mass Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 53 Analytical balance capable o f reading to 0.00001 g. Pap 2ofS Page 38 of 65 Exygen Research Page 74 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0Q01131 Exyt**RflM*ch M eted Number VOOOIW A N A LYTIC A L M ETH O D Method o f Analysis for the Determination o fPerftuorooclanoic Add (PFOA) in Fish and Claras by LC/MS/MS 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 512 5.13 5.14 5.15 5.16 Rotary evaporator. Tissumizer. 125 mL pear-shaped flasks. 50 mL disposable polypropylene centrifuge tubes. 15 mL disposable polypropylene ccntrifcge tubes. Diqxuable micropipets (50100uL, 100-200uL). 125-mL LDPE nerrow-mouth bottles. 2 mL clear HPLC vial Idt. Disposable pipettes. Aulopipettcs (100-1000 pL sod 10-100 pL), with disposable tips. SPE tubes (20mL) (Supelco cat. no. N057177). Wrist action taker. Centrifuge capable o f spuming 50 mL polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophasc RP (Keystone Scientific), 2.1 mm x 50 mm, 5*i (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A ): 2 raM Ammonium Acetate in Water Mobile Phase (B) : Methanol Gradient Program: Twna /mint 0 .0 1.0 8 .0 20 .0 22.5 &A 65 65 25 25 65 Flow Rate (mL/lmin) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6 .6 Injection Volume: IS |iL(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 may be changed in order to optimize the HPLC system. Pete 3 o ft Page 39 of 65 Exygen Research Page 75 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 BxygmKMNicfc Method Number V000I783 L AN A LYTIC A L M IT H O D 1 Method o fAnalysis for foe Determination o fPcrfluorooctanotc Acid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS Syitem 7.1 Mode: Electrocpny 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 0.154 g of ammoaiuin acetate to 1000 mL 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 m 100 mL o fmethanol. 30% Dimethyldichlorosilane in toluene is prepared by bringing 3 mL o f dimefoyldichloroatlane to a final volume o f 10 mL with toluene. Alternate vohunea maybe 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 nock solution o f-1 0 0 pg/mL o f PFOA by weighing 10 mg o f analytical standard (cocracted for purity) and dilute to 100 mL with methanol in a 12$-mL LDPE bottle. A 1.0 ufrtaL fortification solution o f PFOA is prepared by bringing 1 mL o f foe 100 pgfaiL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. A 0.1 |igfaiL fortification solution o f PFOA ia prepared by bringing 10 m Lofthe 1 .0 pgfaiL solution to a final volume o f 1 0 0 with methanol in a 125 mL LDPE bottle. A 0.01 pgfaiL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 tig faL 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 fpreparation. PafMofS Page 40 o f 65 Exygen Research Page 76 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exypa Keenreh Mnhod Number VOOOI7S3 A N A LYTIC A L M ETH O D Method ofAnalysis for die Detennination ofPcrfluorooctanoic Acid (PFOA) in Fish end Clam ibyLC/M S/M S 9.2 Standard Calibcadon Solutions 9.2.1 9.22 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 jigfrnL fortification solution. The following is a typical example: additional concentrations may be prepared aa needed. Concentration o fFortification Volume Solution(uc/mL) (mL) Diluted to (mL) Final Concentratim i (lUfalL) 1.0 5.0 100 o.os 1.0 2.5 100 0.025 10 0,03 0.023 1.0 10 10 100 100 100 0.01 0.003 0.0025 0.1 0,005 10 10 100 100 0.001 0.0003 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles it 2C to 6 *C , tv to six months. 92.4 Alternate volumes and concentrations of standards may be prepared as 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or leas) must include at least one untreated control and two imtreated controls fortified at known concentratione (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in foe quality aawmnec plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into 30 mL polypropylene centrifuge tubes (fortify aa needed, replace lid and mix well). 11.2 Add 30 mL o f acetonitrile and shake on a wrist action shaker for -13 minutes 11.3 Place the tubes in a freezer for ~1 hour. 11.4 Pack and condition foe SPE tubes and rilanize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tuba in sequence with 2 g florisil, 2 g silica gel, 2 g carbon, and 1 g LC-NHj. Condition foe columns with 20 mL o f methanol, then 20 mL o f acetonitrile. Diacard ell washes. Do not ellow the column to dry. 11.6 Sitanize foe 123 mL pew-shaped flasks by raising with the 30% dunefoyldichloroailane in toluene solution. R in a the flask with toluene once, followed by methanol (three tim a). Dry foe flasks completely before use, either by air-drying or with a stream o fnitrogen. Page 5 ofX Page 41 o f 65 Exygen Research Page 77 of 104 Interim Report #25 - Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygea Protocol Number: P0001131 Exypskawscb Method Number V000I7B3 | A N A LYTIC A L M ETH O D | Method o f Analysis for the Determination o fPerfluomoctanoie Acid (PFOA) in Fish and Clams by LC/MS/MS 11.7 Ceotrifege the 50 mL polypropylene tubes containing sample at -2000 rpm fix - 1 0 minutes. 11.8 Decant the extract on to a conditioned SPE column fitted inside the mouth of the pear-ibapod flask. Collect the eluate in the 125 mL silanized pear-shape flask. 11.9 Add 10 mL o f acetonitrile to the sample in the 50 mL centrifuge tube. Homogenize die frozen & t phase using a liaswmizer for -3 0 seconds and rinse the dssumizarwith -1 0 mL o f acetonitrile into the tube. 11.10 Shake the sample again for-1 0 minutes on s wrist-action shaker. 11.11 Place the tubes in a freezer fix - 1 hour more. 11.12 Centrifhg* die 50 mL polypropylene tubes containing sample at -2000 rpm fo r- 1 0 minutes. 11.13 Decant die extract onto die 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 aoetonitrile through the SPE column and combine the eluate in the tame pear-eluped flask. 11.15 Add 3-4 drops o f t-octanol to die extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.16 Make the final volume, by adding 2 mL o f 2% ascorbic acid in methanol to die pear-shaped flask md swiri to mix/diisoLve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze samplesusing electTOspny 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 ofPFOA corresponding to at least five or more 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 die and o f a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an Hire set o f calibration standards may be infected 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 ease, calibration standard* must be the first and last injection in a sample set. 12.4 Use linear standard curvet fix quantitation. Linear standard curves are generated for the analyte by linear regreuion using 1/x weighting o f peek area versus calibration standard concentration using MassLynx 3 3 (or equivalent) software system. P ip 6 of S Page 42 o f 65 Exygen Research Page 78 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 BaygwUmweh MethodNunbwVOOO1713 L ANALYTICA L M ETH O D Method o fAnalysis for the Detenninetiaii o fPerfluorooetinoic Acid (PFOA) in Fish end 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 i peak o f a daughter ion at 369 amu from s parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss ofcaibon dioxide. 13.2 Method blades must not contain PFOA at levels greeter then the LOQ. If a blank contains PFOA at levels peater than 0.S ppb, then a new blank sample must be obtaiaed and the entire set must be re-extracted. 13.3 Recoveries of oontroi spikes and matrix spikes must be between 70-130% of their known values. I f a oontroi spike foils outside the acceptable limits, the entire set o f singles 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, die total number o f calibration standards that could be excluded mustnot exceed 2 0 % ofthe total numbero f standards utfected. 13.5 The correlation coefficient (R ) for calibration curves generated must be 20.992 (Ra 20.985). I f calibration results foil outside these limits, then appropriate tape moat be taken to adjust instranent operation, and the standardsor the relevant eeto fsamples should be 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 lim it within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equationto cskulatatht amount of PFOA found (in ng/mL based on peek ana) using the standard curve (linear regression parameters) by die Mass Lynx software program: PFOA found (ng/mL) - (Peak trea - intercept) slope 14.2 Use the following equation to convict the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found /ppb) fPFOA found fanftnL* * final volume <mU x DF) sample weight (g) DF factor by which the final volume was diluted, if necessary. Page 7 ofU Page 43 o f65 Exygen Research Page 79 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exypq fciwreh Mathod Number VOOOI783 _______________________ A N A LYTIC A L M ETH O D Method o f Analysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For sample* fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (% ) [total analytefound (n ^ g ) analyte found in control(n g ft)l [|Q 0 analyte added (ng/g) Exygen Research PaftSofS Page 44 o f65 Page 80 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001784 Method o f Aaalyite for the Determination o f Perflooroocteaoic Acid (PFO A) io Vegetation by LC /M S/M S Analytical Totting Facility: Exygen Reteerch 3058 Reteerch Drive State College, PA 16801 Approved By. ^ c--lL Paul Connolly * Technical Leader, LC-MS, Exygen Retearch _isyuM Date >iohn Flaherty ' Vice Preeident, Operstiont, Exygen Retearch Date Exygen Research Total Paget: 7 Page 45 o f 65 Page 81 o f 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xygen Protocol Num ber: P 0 001131 Exy y a Um w eh Method Number VOOOI784 I A K A 1A TIC A L M ETH O D Method o fA n ilyiU for the Determinarian o f Perfluorooctaaoic Acid (PFOA) in Vegetation byLC /M S /M S 1.0 Scope ThU method ie to be employed for the ieoletion end quantitation o f perfluoiooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectromctric Detector (LC/MS/M S) in vegetation. 2.0 Safety 2.1 Always obeerve safe laboratory practices. 2.2 Coniult the appropriate MSDS before handling any chemical for proper safety 3.0 Sample Requirement 3.1 At leaat 20 g o ftest sample for extraction. 3.2 Saoqtles should be proewaed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Plaoe 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. 3.3 Sample collection procedures w ill be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 Water - HPLC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120*400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.3 Silica gel (00*200 mesh) - Reagent grade 4.0 Florisil (00-100 mash) - Reagent grade 4.7 Supcrclean LC-NHj - Reagent grade 4.8 1-Octtnoi - HPLC grade 4.9 L-Aacorbic a rid - Reagent grade 4.10 Dimethyldichloforilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooetflooic Acid - Sigma-Aldrich 3.0 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 3-200 jjL oomccted to a tandem Maas Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5J Analytical balance capable o fleading to 0.00001 g. Pig 2 of 7 Page 46 o f65 Exygen Research Page 82 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen P rotocol N um ber: P 0 001131 BxygmJUsesKh Method N u o ta V00017S4 A N A LYTIC A L M ETH O D ] Method ofAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 5.4 Rotary evaporator. 5.5 125 mL pear-shaped flasks. 5.6 SOmL dposablc polypropylene centri&ge tubes. 5.7 15 mL disposable polypropylene ceotriflige tub--. 5.8 Disposable micropipeta(50-100uL, !00-200uL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2m L elevK P L C via lkit 5.11 Disposable pipettes 5.12 Autopipettes (100-1000 pL and HMOOpL), with disposable tips. 5.13 SPE tubes (20mL) (Supeico cat. no. N057177). 5.14 Wrist action shaker. 5.IS Centrifoge capable o fR im ing 50 mL polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fhiophase RP (Keystone Scientific), 2.1 nun x $0 mm. 5m (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: Tim * furiti! 0 .0 1.0 8.0 20 .0 22.5 65 65 25 25 65 Flow Rate 2t fm lAninl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6 .6 Inaction Volume: 15 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 ate intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: BleetrosprayNegative MRM mode, monitoring 413 - 369 mfz for PFOA. Page 3of7 Page 47 o f 65 Exygen Research Page 83 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygoa Research Method Number V0001784 AN A LYTIC A L M ETH O D Method o f Asilyeis ihr the Determination ofPerfluorooctonoic Add (PFOA) in Vegetation by LC/MS/MS The above condition are intended u a guide and may be changed in order to optimize die MSMS eyatan. 8.0 Preparation o fSolution 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water la prepared by adding 0.154 g of ammonium acetate to 1000 mL o f water. 82 Extraction Solutions 8 .2 .1 822 2%asoorbic add in methanol i prepared by dissolving 2 g o f ascorbic add in 100 mL o fmethanol 80S DimethytdichlofOiilane b) toluene it prepared by bringing 3 mL o fdimethyldichloroiilaite to a final volume o f 10 mL with toluene. Alternate volumesmay be prepared. 9.0 Standard Preparation 9.1 Standard Slock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f-100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) end dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification aolutian o f PFOA ia prepared by bringing 1 mL o f the 100 g ^ n L aolutian to a final volume o f 100 with methanol in a 125 mL LDPE bottle. A 0.1 pgfaL fortification solution o f PFOA is prepared by bringing 10 mL o f the 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 pg/mL solution to final volume of 100 with methanol In a 125 mL LDPE bottle. The stock and fortification solution ate 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 fpreparation. 9 2 StandardCalibration Solutions 9.2.1 LC/MS/MS calibration standards ore prepared in methanol via dilution o fthe 1.0 pg/mL fortification solution. Page 4 ul ' Page 48 o f 65 Exygen Research Page 84 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 ExyiteiUttaieh Mette* Number V00017I4 ANALYTICAL METHOD Method o fAnalyse for the Deteraination o fPcrfluoiooctanotc Acid (PFOA) in Vegetation byLC /M S /M S 92.2 The following i* a typical example: additional concentrations may be prepared aa needed. Concentration Pinal ofFortification Votum Solution (unfaiL) (mL) Diluted to (mL) Concentration (I'mL) 1.0 5.0 too 0.05 1.0 2.5 100 0.025 1.0 1.0 100 0.01 0.05 0.025 10 10 100 100 0.005 0.0025 0.1 10 100 0.001 0 .0 0 $ 10 100 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles it 2*C to 6 *C , 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 or less) must include it least one untreated control and two untreated controls fortified at known concentrations (lab control q>ikc) to verify procedural recovery for the batch. 102 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 11.2 11.3 11.4 11.5 11.6 11.7 Weigh 5 g o f frozen am ple into SO mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Add 30 mL o facetonitrile and shake on a wrist action shaker for -1 5 minutes. Centrifuge foe 50 mL polypropylene tubes containing sample at -2000 rpm for- 1 0 minutes. Pack and condition foe SPB tubes and silanize the peer-shaped flasks. 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 foe columns with 20 mL o f methanol, then 20 mL o f acetonitrile. Discard all washes. Do not allow foe column to dry. Silanize foe 12S mL pear-shaped flasks by nosing with the 30% dimefoytdichloroellane in toluene solution. Rinse the flask with toluene once, followed by methanol (tim e times). Dry the flasks completely before use. eitherby air-drying or with a stream o fnitrogen. Decant the extract on to a conditioned SPE column fitted inside the mouth of the pear-shaped flask. Collect the etuate in the 12S mL silanized pear-shape Page 5 o Page 49 of 65 Exygen Research Page 85 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research anaLvticai. method Method Number V0Q0I7S4 ] Method o f Analysis for the DetennmationofPetfluotooctanwc Acid (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 mL o f acetonitrile to the ample in the SOmL centrifuge tube. 11.9 Shake the sample again for -1 0 minutes on a wriit-actioa shaker. 11.10 Centrifitge the 50 mL polypropylene tubes containing sample at -2000 rpm for -5 minutes. 11.11 Decant the extract onto foe sane SPE column. Collect the eluate into the um a pear-shaped flask and oombinc 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-octeaol to foe extract in the pea-shaped flask and evaporate a reduced pressure using a rotary evaporator (at < 40*C). 11.14 Malm the final volume, by adding 2 mL o f 2% ascorbic acid m methanol to the pear-ehaped flask and swirl to mix/disaotve. 11.15 Transfer the extracts to HPLC vials using disposable pipeU. 11.16 Analyze samples using electrospiay 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 fPFOA corresponding to at least five or more concentration levels mustbe included in an analytical set 12.3 An entire eet o f extracted calibration standards must be included at foe beginning end at foe end o f a sample set Extracted standards must be interspersed between every 5-10 sample. As an alternative, an entire set of extracted calibration standards may be injected at the beginning o f a set followed by extracted catifantion standards interspersed every 5-10 samples (to account for a second set o f extracted standards), hi either case, extracted calibration standards must be foe 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 ofpeak 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 psrern o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents foe loss o f carbon dioxide. Pegs 6 of 7 Page SOo f65 Exygen Research Page 86 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No.: P0001131 E xygen Protocol Num ber: P 0 001131 Byg-- R n c h Method Nuabar V00017S4 A N A LY TIC A L m e t h o d | Method ofAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 13.2 Mediod blanks must not oontain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greeter than 0.5 ppb, then a new blank sample mast 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 fsUs outside the acceptable limits, the entire seco f samples should be re^xtracted. 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 2 0 % of the 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 fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standardsor 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 lim it 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) slope 14.2 Use the following equation to convert the amount o f PFOA found in ngtoL to ng/g(ppb). PFOA found fppbl - [PFOA found fna/m Ll x final volume fm Ll x DF1 sample weight (g) DF - fsetor by which the final volume was diluted, if necessary. 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use die following equation to calculate the percent tooovery. Recovery (% )- [total analytefound (n^g) analytefound in control (ng/g)] ^ analyteadded (ng/g) Page 7 ol' 7 Page 5! o f 65 Exygen Research Page 87 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001785 Method o f Analysis for the Determ tietioa o f PerflnorooctenoJc Add (PF A ) io Smelt Marnassi U ver by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College. PA 101 Approved By: 'B A c j L ____ Paul Connolly I Technical Leader, LC-MS, Exygen Research ___ l lA t g - i Dale o /# J6bhn Filabherty /V'''ViciceePPreresskident, Oprations, Exygen Research Date Exygen Research Total Papa: 7 Page 52 o f 65 Page 88 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 cyg*s Rttmnk Mtbod Nunfccr V000171* | A N A LYTIC A L M ETH O D Method o fAnalysis for tbe Determination o fPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS | 1.0 Scope Thia method i t to be employed for the iaolation and quantitation o fperfluorooctanotc acid by High Performance Liquid Chromatography coupled to a tandem Maas Spectzometric Detector (LC/MS/M S) in small mammal liver. 2.0 Safety 2.1 Alwaya obearve safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety 3.0 Sample Requirement 3.1 At least Sg o fteat aample for extraction. 3.2 Sandies should be processed before extraction. Place the frozen sample in a food prooataor and homogenize with dry ice. Place the sample in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place foe samples in frozen storage until time o f analysis. Alternately, if there is an insufficient amount o f sample (-less than 3 g), then no processing is necessary and the sample can be used as supplied. 3.3 Sample collection procedural w ill be specified in the sampling plan for this project 4.0 Reagents md Standards 4.1 W ater-H PLC grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile - HPLC pads 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfruorooctanoic 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 iqjeclor capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 50 mL disposable polypropylene cantrifoge tubes. 5.5 15 mL disposable polypropylenecouriftige tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-mL LDP6 narrow-mouth bottle. 5.8 2 mL dear HPLC vial k it 2Pag* o f? Page SS of65 Exygen Research Page 89 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E x y p a ta ttid i M ttbodN unb VOOOI7IJ I ANAilV T IC a I . M ETH O D Method o fAnalysis for the Determination ofPerftuorooctmoic Acid (PFOA) in Small Manina] Liver by LC/MS/MS 3.9 Disposable pipettea. 3.10 Antopipettes (100*1000 pL and 10*100 pL), with disposable tips. 3.11 Waters Sep Pak Vac 6 cc(lg)tC 18S P E cartridges. 3.12 SPE vacuum manifold. 3.13 Tissuemizer. 3.14 Wrist-action shaker. 5.13 Centrifuge capable ofspinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x $0 mm. 5p (P/N: 82503-052130) 62 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 (min) 0.0 1.0 8.0 20 .0 22.3 &A 63 63 25 25 63 Flow Rate fmL/m in) 35 0 J 35 0.3 75 0.3 75 0.3 35 0.3 6 .6 Injection Volume: 15 pL (can be increased to at much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibratimi curve. 6 .8 RunTime: - 2 3 minutes. The above conditions are intended as a guide and may be chengod in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: ElectroeprayNegative M RM mode, monitoring 413 - 369 m/z for PFOA. The bove conditions are intended as a guide and may be changed in order to optimize the MSMS system. Page 3 o f? Page 54 o f65 Exygen Research Page 90 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001 ]31 Bxypa RMMItk MKkodNunbCT V000I7M I A?t/vLVTICAL M m tO D Method o f Analysis for the Determination o fPerfhiorooctanoic Acid (PPOA) in Small Mammal Liver by LC/MS/MS 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0154 g of ammonium acetate to 1 0 0 0 mL o f water. Alternate volumes maybe prepared. 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 (ooireeted 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 mL o f the 100 pgfaiL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortificatimi solution o f PFOA is prepared by bringing 10 m Lofthe t.O pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 Tbe stock end fottification solutions ere to be stored in a refrigerator at approximately 4*C and e stable for a maximum period of 6 months from the date o fpreparation. 9.2 Standard Calibration Solutions 92 .1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 pg/raL fortification solution. The following is a typical example: additional concentrations may be prepared aa needed. OWMeiMTWlfft o f Fortification Solution fae/mL) 100 100 100 5.0 2.0 1.0 Volume (mL) 5.0 2.0 li) 10 10 10 Dilutedto (mL) 100 100 100 100 100 100 Final Concentration tnc/mU 5.0 2.0 1.0 0.5 0.2 0.1 at 2*C to tPC, uptorix months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. Pa*e4of7 Page 55 o f 65 Exygen Research Page 91 o f 104 Interim Report #25 Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 fixyteaftMtaicb Method Nuaiber V000I7U I A N A LYTIC A L M ETH O D j Method o f Analyse fbr the Determlaatioa ofPerfhiorooctanoic Acid (PFOA) in Smell Mammal Liver by LC/MS/MS 10.0 Batch Set Up 10.1 Bach batch o f aamples extracted (typically 20 or leee) must include at least one untreated control and two uncreated 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 w ill be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 1 g o f sample into a $0 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate weights o f liver may be measured dependingon the sample rise available for use. 11.2 Add water to the sample for a final volume o f 10 mL. 11.3 Homogenize sample uring a tissuemiwr for -1 minute. 11.4 Transfer 1 mL o f the sample using a disposable pipette into a 15 mL disposable ceatrifrge tube. 11.5 Add 5 mL o f acetonitrile and shake for -2 0 minutes on a wrist-action shaker 11.6 Centrifuge the tubes at -3000 rpm for -5 minutes. 11.7 Decant fee supernatant into a 50 mL disposable centrifiige tube and add 35 m Lofwatsr. 11.8 Condition fee C SPE cartridge* (1 g, 6 mL) by pairing 10 mL methanol followed by SmL o f KPLC w tfer 2 dropfeec). Do not let column run dry 11.9 Load the sampleon conditioned Cu SPE cartridge. Discard eluate. 11.10 Elute wife *2 mL o f methanol Collect 2 mL o f eluate into a graduated 15 mL polypropylene oentriftige tube (final volume 2 mL). 11.11 Analyze aamples using electroaprty LC/MS/MS. 12.0 Chromatography 12.1 Iqject 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 analysed samples. 122 Standards o fPFOA corresponding to at least five or more 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 ai the end o f a sample set. Standards must be interspersed between every 5- 1u samples. As in alternative, an entire set o f calibration standards may be injected at fee beginning o f a set followed by calibration standards mtenpemed 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 linearregression using l/x weighting o fpeak area Plge9of7 Page 56 o f 65 Exygen Research Page 92 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExypeReieeich Msftod Number V00017I5 | A N A LYTIC A L M ETH O D ... Method o fAnalysis far the Determination ofPerfhtocooctmoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Sample impoose should not exceed standard responses. Any samples ihai exceed standard responses should be farther 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 paraot corresponds to die PFOA anion, while the U iijK if ion (369 amu) represents die 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. I f a control spike fall* outside die acceptable limits, the entire set o f samples should be re*extractod. Any matrix spike outside 70 130% should be evaluated by die 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, die total number o f calibration standards diet could be excluded must not exceed 20% ofthe total number o f standards injected. 13.5 The correlation coefficient (R ) for calibration curves generated must be 20.992 (R1 20.985). I f calibration results fall outside these limits, then appropriste steps must be takm to adjust instrument operation, and the standards 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 ran. If retention time drift exceeds this lim it within an analytical nm 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 fnf/m L) (Peek irse - intmeepf) x DF x aliquot factor slope DF - factorby which the final volume was diluted, if neoessery. Aliquot fa c to r-10 h |iio n Page 57 o f 65 Exygen Research Page 93 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocot Number: P0001131 Exypa RMMfch Method Number V00017I5 I a h a i.y u c a l m e t h o d Method o fA n lly til for li * Dcunninltioii o f Periluoroocunotc Acid (PFOA) in Smell M unnul Liver 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 (% ) - f total analyte found (ng/mL) - analytefound in control (ng/roL)] analyte added (ng/m L) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL u> n g fg (p i*). PFOA found fppbl - IPFOA found fne/m Ll x final volume (mL)} sample weight (g) Exygen Research Page 7 of7 Page 58 of65 Page 94 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001786 Method of Aaalyala for the P eterailiithH i of Perfloorooctanok Add (PFO A) in Small Mammal Seram by LC /M S/M S Analytical Testing Facility: Exyien Reaearch 3058 Reaearch Drive State Clkge, PA 16801 Approved By: C-- ___ Paul Connolly I Technical Leader, LC-MS, Exygen Reaearch S l//7 ? / / ________ John Flaherty / Vice President, Operations, Exygen Research / 'A / l Date Exygen Research Total Pager 7 Page 59 o f 65 Page 95 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number P0001131 Eaiyt--JUnMCh Method Number V000I7S6 I AWALVTICa L m e t h o d Method o f Analysis for the Detennination o f Perfluocooctanoic Acid (PFOA) in Small Mammal Seram by LC/MS/MS 1.0 Scope This method is to be employed for foe isolation and quantitation o f perfluoraoetanoic acid by High Performance Liquid Chromatography coupled to a tandem Maas Spectrametric Detector(LC/M S/M S) in small mammal scram. 2.0 Safety 2.1 Always obcervs asfc laboratorypractices. 22 Consult foe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t leest 1m Lofteet sample for extraction. 3.2 No ample proceesliig la needed for eeram samples. However, frozen serum samples must to allowed to completely thaw to room temperature before use. 3.3 Sample collection procedure w ill be specified in foe sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H PLC grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile-HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment S.t A high performance liquid chromatograph capable o f pumping up to 2 solvent equipped with a variable volume injector capable of injecting 5-200 pL connected to a tandem M a il Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capableo freading to 0.00001 g. 5.4 50 mL di^joeable polypropylenecentrifoge tubes. 5.5 15 mL disposablepolypropylenecentrifoge tubes. 5.6 Disposable micropipets (50-100uL, 100*200uL). 5.7 125-mL LDPB narrow-mouth bottlea. 5.8 2 mL dear HPLC vial kit. 5.9 p iiqrft*Mt pipirttn 5.10 Autopipettea (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters SqpPak Vac 6 ec(lg)tC 18S P E cartridges. 5.12 SPB vacuum manifold. 5.13 Voitexer. Page J o P Page 60 o f 65 Exygen Research Page 96 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen P rotocol N u iriber: POOOI131 B inalw iw fc Method Number V000I7W f A1W AT1CAL METHOD Method o fAnalysis for theDetermination o f Perfhiorooctanoic Acid (PFOA) in Small Mammal Seram by LCW S/M S 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f pinning IS mL polypropylene tubes at 3000 rpm 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x SOmm. 5p (P/N: 82505-032130) 6.2 Temperature; 30*C 6.3 Mobile Phase (A ) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase(B ) : Mediano! 6.5 Gradient Program: 0.0Time Omini 1.0 280.0.0 22.S XA 65 65 25 2S 65 Plow Rate & faiL/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 liyection Volume: 15 jtL (can be increased to a* much as SOpL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTime: - 2 3 minutes. The above conditions are jniended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative M RM modemonitoring 413 -+ 369 mil for PFOA. The above conditions ore intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o fSolutions 6.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water it prepared by adding 0.1S4 g of acetate to 1000 mL o f water. Alternate volumesmay be prepared. Page 3 a t 7 Page 61 of 65 Exygen Research Page 97 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 ExypaRMMfCfc Method Number VOOOPI& I A N A tynCA l. MKTHOD Method ofAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortificatioo Solution 9.1.1 Prepare a stock solution o f -100 |ig/m L o f PFOA by weighing 10 mg o f analytical itandard (corrected for purity) and dilute to 100 mU with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/mL fortification aolutioa o f PFOA it prepared by bringing I mL o f the 100 p^niL aolution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pf/taL fortification solution o f PFOA is prepared by bringing 10 m Loftbe 1.0 pgtaiL aolution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.4 The stock and fortification rotations 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 fpreparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o fthe 0.1 pgfaaL fortification aolutioa The following is a typical example: additional concentrations may be prepared as needed. Conftntrttiwi ofFortification Votums Diluted to Final Concentration SolutionfaahnL) (mL) (mL) (na/mL) to o 5.0 100 100 2.0 100 100 1.0 100 5.0 2.0 1.0 5.0 10 100 OS 2.0 10 100 1.0 10 100 0.2 01 9.2.3 Store ell calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six montile. 9.2.4 A tonale volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each belch o f simples extracted (typically 20 or leas) muat include at least one --t ir tiH oontrol and two untreated controls fortified at known concentrations(lab oootrol spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. Fife 4 of ? Page 62 o f65 Exygen Research Page 98 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 ExygsnRamrch Method Numtm VQ0017SC | A N A LYTIC A L M ETH O D Method o fAnalysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Seram by LC/MS/MS 11.0 Sample Extraction 11.1 Measure 1mL o f sample into aSO mL polypropylene ccntriftige tubes (tonify as needed, replace lid and mix w ell). Note dial alternate volumes o f serum maybe measured depending on die sample size available for use. 11.2 Add water to the aample for a final volume o f 20 m L Cap tightly 11.3 Vortex fo r-1 minute. 11.4 Transfor 1 mL o f the sample using a disposable pipette into a 15 mL disposable ecntrifUge tube. 11.5 Add 5 mL o f acetonitrile and shake for -2 0 minutes on t wrist-action shaker. 11.6 Centrifege the tubes a t-3000 ipm for-5 minutes. 11.7 Decant the supernatant into a 50 mL disposable oentrifoge tube and add 35 mL o f water. 11.8 Condition die Cn SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL ofHPLC water ( - 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned Cn SPE cartridge. Discard eluate. 11.10 Elute with -2 mL o f methanol. Collect 2 mL o f eluate into a graduated 15 mL polypropylene ccntriftige tube (final volume - 2 mL). 11.11 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the su m m ount 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 fPFOA corresponding to at least five or more concentration levels mustbe included in an analytical set. 12.3 An entire set o fcalibration atmdards must be included at the beginning and at the end o f t sample set Standards must be interspersed between every 5 -1 0 samples. As in alternative, an entire set o f calibration standards may be injected at the beghming o f a act followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In cither ease, 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 ofpeak area versus calibration standard concentration using MaasLynx 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 3 o f? Page 63 o f 65 Exygen Research Page 99 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000U31 Exygen Rawarcb Method Number V000)786 I A W A LYTIC A LM ^TH O D Method o f Analysis for the Determination ofPerfluoroocttnoic Acid (PFOA) in Small Mammal Seram by LC/MS/MS 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram must chow a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent conoeponds to the PFOA anion, whtle the taughiaf {on (3 6 9 amu) representa rite loaa o f carbon dioxide. Method blanks must not contain PFOA at level greater than the LOQ. if a blank contains PFOA at levels greater than 10 ng/mL, then a new blank ample must be obtained and foe entire set must be re-extracted. Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. I f a control spite falls outside the acceptable limits, the entire set o f --TMpt-- foould be re-extracted. Any matrix spike outside 70 130% should be evaluated by foe analyst to determine if re-extraction is warranted. Any calibration standard found to be a statistical outlier by using foe Huge Error Teat, may be excluded from foe calculation o f the calibration curve. However, foe total number o f calibration standards that could be excluded must not exceed 20% o ffoe total nimbar o f standards injected. 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 standardsor the relevant set o f samples should be reanalyzed. 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 ran 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 x aliquot factor slope DF fitte r by which foe final volume was diluted, if nooessary. Aliquot factor * 20 14.2 For san ies fortified with known amounts o f PFOA prior to extraction, use foe following equation to calculate the percent recovery. Recovery (% )- [total analytefound(ng/mL) - analyte found in control(ng/mL)] k|QQ analyte added(ngfrnL) PageS of 7 Page 64 o f 65 Exygen Research Page 100 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E*yt*o R jtiircii Method Number V000I7S6 I AWa L Y T IC A I. M ETH O D Method o fAnalysis for die Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ppb. PFOA found (PPM - fPFQA found fag/m Ll x final volume imL\) sample volume (m L) Exygen Research P|e7f? Page 65 o f65 Page 101 o f 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 S tate College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment Num ber 1 Effective Date: 0 1/18/05 Exygen Study Number P0001131 O ient Study N um ber. Pagel of 1 None D ES C R IPTIO N O F AM ENDED SE C TIO N 1) Analytical Procedure Sum m ary V0001780:Section 9.1 2 ) Verification o f Analytical Procedure AMENDED TO 1) Add to Section 9.1: Section 9 .1 .6 , Atterriate weights o f standards m ay b e used to prepare alternate concentrations o f stock solutions as necessary. A lternate levels of fortification solutions may also be prepared. 2 ) Low and high spiking levels o f the analytes for each m atrix m ay be altered depending on sam ple size available for extraction and/or to cover analyte concentrations expected in the sam ples. R A T IO N A L E 1 ) Higher concentrations o f standards need to be prepared In order to spike the sam ple bottles at higher levels. 2 ) T he sam ple size avalab le fo r small m am mal Iv e r and serum w as sm aller than expected. Spiking at the pre-determ ined levels in the protocol puts the spiked concentration low er than the detection lim it Also, the analyte levels in the ground w ater sam ples a re expected to greatly exceed the pre-determ ined spiking levels listed In the protocol. W hen the levels in the sam ples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the Q C sam ple. Higher spiking levels in the bottles w ill cover the analyte concentrations expected in the w ater sam ples. IMPACT ON STUDY T he LOQ is 100 ng/g fo ra 0.1 g sam ple o f small m am mal liver and is 1000 n g /rn L fo ra 0.01 mL sam ple of sm all mammal serum . Higher levels o f spiking for the w ater sam ples w ill ensure that m ore Q C recovery data can be used. LIBRARY ID: W 00012264` .. .. ' ADMINISTRATIVE FORM Exygen Research Page 102 of 104 Interim Report #25 - Analysis of Ground Water Samples Exygen Study No.: P0001131 3058 Research D rive Phone: 814-272-1039 S tate College, PA 16801 Fax: 814-231-1580 Amendment Num ber Effective Date: Exygen Study Num ber PROTOCOLAMENDMENT 2 03 /07/05 P0001131 C lient Study N u m b er Pagai oH None D ESC R IPTIO N O F AM ENDED S E C TIO N 1) Report, page 11 o f 65 2 ) T est M aterials, page 6 o f 65: PFO S transition monitored 489 -> 98. AMENDED TO 1) Instead o f one final report, interim reports will be issued. 2 ) P FO S transition monitored may also be 4 9 9 > 80. RATIO NALE 1) Due to the excessive sizes o f the data sets, interim reports w li be issued to allow the client to receive data in a tim elier m am n'>M K<* -a w 2 ) T he A P I 4 0 0 0 LC /M S/M S systems (M e e t the 4 9 9 -> 60 P FO S transition with greater sensitivity than the 4 9 9 -> 99 transition. IMPACT ON 3 T W Y 1 ) T h e d e n t will be able to receive and review the data m ore quickly. 2 ) The 4 9 9 -> 80 transition can be detected with greater sensitivity, therefore, giving better chrom atography. Sponsor Sigrupura (if required) LIBRARY ID: V000122S4- ' - Date Exygen QAU Review LjJ oJosh ADMINISTRATIVE FORM Exygen Research Page 103 of 104 Interim Report #25 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 J U L .2 5 .2 0 0 5 8 : 56Ptf1 EXYGEN RESEARCH N 0 .7 7 4 P .3 3058 Research D rive Phone: 814-272-1039 S ta i C ollege, PA 16801 Fax: 814-231-1580 Am endm ent N um ber E ffe d iv a D ate: Exygen S tudy N um ber PROTOCOL AMENDMENT 3 07 /1 8 /0 5 P1131 C lien t S tu dy N um ber: Page 1 er 1 NA : D E S C R IP T IO N O F A M E N D E D S E C fO N " V erificatio n o f A n alytical Procedure, pegs 10 o f protocol. AMEN T h e fe w d uplicate can b e used fo r the laboratory spikes and re p lic ate w hen th e prim ary sam ple volum e is lim ite d RATIONALE T h e sam ple size fo r a w a te r sam ple Is 2 0 0 m l- If a sam ple s ite req uires re-extraction for any reaso n, th ere w ould not be enough o f the prim ary sam ple to re p e a t tw o laboratory spikes and a rep licate. T h e Held duplicate Is technically th e s a m e sam p le as th e prim ary sam ple and therefore, can be used fo r laboratory spikes and rep licates as n eed ed. IM P A C T O N S T U D Y No negative impact on the study. Using the duplicate sam ple allows fo r the full QC of the sample site to be completed. Director M Exygen Maqgtfi Sponsor Slgnelure'fif required) ?8t0F / .iw k L D a te z/~ ja i - s Date 7 /is fa s D a te E x y g e n Q A U R e v ie w Y T k . f y LIBRARY ID: V000122S-S ADMINISTRATIVE FORM Exygen Research Page 104 of 104