Document gDXgNjeYdX1kGmw4ZYkMQeB2V

Project / Study Archive Request Shaded areas for Archivist use only Requestor Name Kent Lindstrom Phone 651 778-5352 Date 04/05/05 Request for: E Archival Retrieval Report IE! FC LIMS number: E05-0210 Title/Summary: Interim Report #2 - Analysis of Surface 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_____________________________________________ Executive Summary__________________________________________________________________ Group: 3M Decatur_________________________________________________________ Group Project Number: Exygen Protocol Number P0001131____________________________________ Project Lead/ Study Director: Gary Hohenstein (data management), Mike Santoro (Sponsor), John Flaherty (Principal Investigator), Jaisimha Kesari P.E., DEE (Study Director, Weston Solutions, Inc.)______________________________________________________________ Contract: NA Contract Project #: NA Analyte(s): PFBS, PFHS, PFOS Test System (Sample Matrix): water Project Type 1X1 Analysis 1 1Degradation 1 1Duct Residue Analysis 1 1Ecotox 1 1M ethod V alidation na 1 1Phys/Chem Properties 1 1Research/Method Development Regulatory Status: |_| Non-GLP |Xl GLP 0 ther: Initiation Date: 11/05/04 (Protocol Signed by Study Director), Analytical Start Date for Interim Report: 02/14/05 Completion Date: Analytical Termination Date for Interim Report #2: 02/17/05 Signed by Study Director 03/23/05, Signed by Sponsor 03/30-31/05 Electronic Media: Data CD: ITI FTIR Data 1 1Instrument Data None (raw data files archived at Exygen) Page 1 o f2 Project / Study Archive Request Archive Date Scan Date Off-Site Storage Date Key Words: Decatur Memorandum of Understanding, MOU, GLP, Protocol P0001131, PFBS, PFHS, PFOS, E05-0209, E05-0210, Interim Report #2 Comments: Distribution Point Gary Hohenstein (Interim Report #2 PDF) Jai Kesari (Interim Report #2 PDF) Date 04/05/05 04/05/05 Page 2 o f2 INTERIM REPORT # 2-AnaIvsis of Surface 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 March 22, 2005 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: 100 Interim Report #2-Analysis of Surface 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 EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by Exygen Research. Jonn Eianerty ' PP rriinnrcMipnaa ll ITnn\v/#estigator Exygen Research Jaisimha Ke . Study Director Weston Solutions, Inc. 3M Company Exygen Research Date Page 2 of 100 Interim Report #2-Analysis o f Surface 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 Date Inspected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Study Director 7. Raw Data Review 03/10,11/05 03/14/05 03/18/05 03/18/05 8. Interim Analytical Report Review 03/17/05 03/18/05 03/18/05 03/18/05 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion o f the study. This QA statement involves only the review o f the interim report and associated raw data. Exygen Research Page 3 of 100 Interim Report #2-Analysis of Surface 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: 2 1 -A M rt--0 Date Study Director, Weston Solutions, Inc. Jaisimha i^esri P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: iSi/uJA & Michael A. Santoro Director of Regulatory Affairs Exygen Research 3hh5 Date 3/ 3/ / PS Date Page 4 of 100 Interim Report #2-Analysis o f Surface 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: P0001131 EXYGEN STUDY NUMBER: P0001131 TYPE OF STUDY: Residue SAMPLE MATRIX: Surface Water TEST SUBSTANCE: Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) SPONSOR: 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 STUDY DIRECTOR: Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 STUDY MONITOR: Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 PERFORMING LABORATORY: Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: 11/05/04 Interim Analytical Start Date: 02/14/05 Interim Analytical Termination Date: 02/17/05 Interim Report Completion Date: 03/22/05 Exygen Research Page 5 of 100 Interim Report #2-Analysis o f Surface 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 Paul Connolly Chrissy Edwards Mark Ammerman Amy Sheehan Title Vice President Scientist Technical Lead-LC/MS Technician Sample Custodian Associate Scientist Exygen Research Page 6 of 100 Interim Report #2-Analysis o f Surface 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..............................................................................12 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...............................................................................................15 6.5 Description of LC/MS/MS Instrument and Operating Conditions...........................15 6.6 Quantitation and Example Calculation.....................................................................16 7.0 EXPERIMENTAL DESIGN......................................................................................17 8.0 RESULTS...................................................................................................................17 9.0 CONCLUSIONS.........................................................................................................17 10.0 RETENTION OF DATA AND SAMPLES.............................................................17 Exygen Research Page 7 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Table I. Table II. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Surface Water Samples.................. 19 Matrix Spike Recovery of PFBS, PFHS and PFOS in Surface Water Samples.......................................................................................................... 20 Exygen Research Page 8 o f 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No. : P0001131 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFBS in Water............................................... 23 Figure 2. Extracted Standards of PFBS in Water, 50 ng/L and 100 ng/L, Respectively................................................................................................... 24 Figure 3. PFBS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively.......................... 25 Figure 4. Chromatogram Representing a Surface Water Sample Analyzed for PFBS (Exygen ID: C0052282, Data Set: 021405C)................................ 26 Figure 5. Typical Calibration Curve for PFHS in Water.............................................. 27 Figure 6. Extracted Standards of PFHS in Water, 50 ng/L and 100 ng/L, Respectively................................................................................................... 28 Figure 7. PFHS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively.......................... 29 Figure 8. Chromatogram Representing a Surface Water Sample Analyzed for PFHS (Exygen ID: C00522831, Data Set: 021405C).............................. 30 Figure 9. Typical Calibration Curve for PFOS in Water.............................................. 31 Figure 10. Extracted Standards of PFOS in Water, 50 ng/L and 100 ng/L, Respectively................................................................................................... 32 Figure 11. PFOS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively.......................... 33 Figure 12. Chromatogram Representing a Surface Water Sample Analyzed for PFOS (Exygen ID: C0052282, Data Set: 021405C)................................... 34 Exygen Research Page 9 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 LIST OF APPENDICES Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method: V0001780:"Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" .............................................................................. Page 35 Exygen Research Page 10 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 1.0 SUMMARY Exygen Research extracted and analyzed surface 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 water was 50 ng/L and the limit of detection was 25 ng/L. The PFBS, PFHS and PFOS residues found in surface water samples are summarized in Table 1. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in surface water samples were 139% 21%, 95% 7%, and 96% 12%, respectively. 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 surface 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 surface water using the analytical method entitled, "V0001780: Method o f 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 February 14, 2005, and the analytical termination date for this interim report was February 17,2005. Exygen Research Page 11 o f 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No. : P0001131 4.0 ANALYTICAL TEST SAMPLES Forty-four surface water samples (Exygen ID C0052282-C0052299 and C0052310C0052335) were received on wet ice on December 04, 2004 from Charles Young at Weston Solutions, Inc. The samples were logged in by Exygen personnel and placed in refrigerated storage. Sample log-in and chain of custody information will be kept at Exygen Research until the conclusion of the study; they will then be located in the raw data package associated with this study. Storage records will be kept at Exygen Research. 5.0 REFERENCE MATERIAL The analytical standards, PFBS and PFHS, were supplied by 3M. PFBS was received from 3M at Exygen on July, 06, 2000. PFHS was received from 3M at Exygen on January, 20, 2003. PFOS was purchased from Fluka Corporation and was received at Exygen on April, 23,2003. 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 Exygen Inventory No.. SP0000252 SP0002401 SP0002694 Lot# 101 SE036 430180-1 Purity (%) 96.7 98.6 101.2 Expiration Date 12/04/06 10/18/06 04/23/06 The molecular structures o f PFBS, PFHS and PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt ^FSO s'K '1') Transitions Monitored: 299 - 99 Structure: FF FF F S03 FF FF Exygen Research Page 12 of 100 Interim Report #2-Analysis o f Surface W ater Samples Exygen Study No.: P0001131 PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CeFnSCh'K*) Transitions Monitored: 399 --> 80 Structure: FFF FFF F S03 FFF FFF PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFnSCVK*) Transitions Monitored: 499 -> 80 Structure: FFFF FF F S03 FF 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 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 Cig 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. Exygen Research Page 13 o f 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No. : P0001131 6.2 Preparation of Standards and Fortification Solutions Individual stock standard solutions of PFBS, PFHS and PFOS were prepared as specified in Exygen method V0001780. The stock standard solutions were prepared at a concentration of 100 pg/mL by dissolving 10 mg of each of the standards (corrected for purity and salt content) in methanol. From these solutions, mixed 1.0 pg/mL fortification standard solutions were prepared by taking 1 mL of each of the appropriate stock solutions and bringing the volume up to 100 mL with methanol. By taking 10 mL of the mixed 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the mixed 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.01 pg/mL fortification standard was prepared. A set of standards containing PFBS, PFHS and PFOS was 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 (UL) 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 o f standard preparation will be kept at Exygen Research until the conclusion of the study; it will then be located in the raw data associated with this study. 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 ~ 3.9 mins, ~ 10.4 mins, and ~ 12.6 mins, respectively. Peaks above the LOD were not detected in any of the reagent blank samples corresponding to the analyte retention time. Exygen Research Page 14 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 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. 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.: 35 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time ('min') 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: ~18 min %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 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 ('mini ~3.9 min. ~10.4 min. ~12.6 min. Exygen Research Page 15 o f 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 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 equations below. 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 calculated the percent recovery. Equation 2: Recovery (%) = (analyte found (ng/L) - analyte in control (ng/Ll) xl00% amount added (ng/L) An example of a calculation using an actual sample follows (calculation is for PFOS only): Water sample Exygen ID: C0052282 Spk C (Set: 021405C), fortified at 500 ng/L with where: peak area = 84394 intercept = 3070 slope = 162 dilution factor =1 ng/L PFOA added (fort level) = 500 amt in corresponding sample = 0 (Not Detected) From equation 1: Analyte found (ng/L) = f84394 - 30701 x 1 162 From equation 2: % Recovery = 502.0 ng/L = (502.0 - 0 ng/L) x 100% 500 = 100 % Exygen Research Page 16 of 100 Interim Report #2-Analysis o f Surface 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 two sets. Each set consisted of one reagent blank, two reagent blanks fortified at known concentrations. One set contained five sample sites and the second set contained four sample sites, along with all the field blanks and field blank spikes collected for the surface water samples. For each site, a sample, a field duplicate and two matrix field spikes were collected. For each site, a laboratory duplicate was extracted and two laboratory matrix spikes were also extracted. 8.0 RESULTS The PFBS, PFHS and PFOS found in the surface water samples are listed in Table I. Fortification recoveries for PFBS, PFHS and PFOS in the water samples are detailed in Table II. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in water samples were 139% 21%, 95% 7%, and 96% 12%, respectively. 9.0 CONCLUSIONS The surface water samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001780. 10.0 RETENTION OF DATA AND SAMPLES When the final analytical report is complete, all original paper data generated by Exygen Research will be shipped to the sponsor. 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 17 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 18 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Surface Water Samples Client ExygenID Sample ID C0052282 DL3-SW-LOC001 -0-041201 C0052282 Rep DL3-SW-LOC001 -0-041201 * C0052283 DL3-SW-LOC001-0-041201 Dup C0052286 DL2-SW-LOC001 -0-041201 C0052286 Rep DL2-SW-LOC001 -0-041201 * C0052287 DL2-SW-LOC001-0-041201 Dup C0052290 DBC-SW-LOC001 -0-041202 C0052290 Rep DBC-SW-LOC001 -0-041202* C0052291 DBC-SW-LOC001-0-041202 Dup C0052294 DOU-SW-LOC001-0-041202 C0052294 Rep DOU-SW-LOC001 -0-041202* C0052295 DOU-SW-LOC001 -0-041202 Dup C0052298 DDO-SW-LOC001 -0-041202 C0052298 Rep DDO-SW-LOCO01-0-041202* C0052299 DDO-SW-LOC001-0-041202 Dup C0052312 DL1-SW-LOC001 -0-041202 C0052312 Rep DL1-SW-LOC001 -0-041202* C0052313 DL1-SW-LOC001-0-041202 Dup C0052316 DMC-SW-LOC001 -0-041202 C0052316 Rep DMC-SW-LOC001 -0-041202* C0052317 DMC-SW-LOC001-0-041202 Dup C0052322 DAA-SW-LOC005-0-041202 C0052322 Rep DAA-SW-LOC005-0-041202* C0052323 DAA-SW-LOC005-0-041202 Dup C0052326 DAA-SW-LOC002-0-041202 C0052326Rep DAA-SW-LOC002-0-041202* C0052327 DAA-SW-LOC002-0-041202 Dup C0052330 Field Blank C0052333 Field Blank C0052320 DMC-SW-LOC001 -2-041202 C0052321 DMC-SD-LOC001 -2-041202 Analyte Found (ppt, ng/L) C4 Sulfonate PFBS Perfluorobutanesulfonate C6 Sulfonate PFHS Perfluorohexanosu(fonate ND ND ND ND ND ND ND ND ND ND ND ND 93.8 96.2 97.5 14000 14700 12800 231 233 252 1620 1650 1600 ND ND ND ND ND ND 76.8 77.4 45.7 48.3 77.4 107 49.2 53.6 97.6 104 2460 3270 3100 379 49.5 54.0 7980 7350 7880 375 456 433 432 445 ND ND ND ND ND ND ND ND C8 Sulfonate PFOS Parfluorooctanesulfonate ND ND ND ND ND ND 3460 2880 3920 5710 5820 5640 ND ND ND 179 185 213 284 257 283 18800 18000 19600 2780 2930 3760 ND ND ND ND ` Laboratory Duplicate ND - Not detected at or above 25 ng/L. NQ - Not quantifiable = Measured concentration between 25 ng/L and the Limit of Quantitation (LOQ) which is 50 ng/L. Exygen Research Page 19 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Surface Water Samples Sample DL3-SW-LOC001-0-041201 (C00S22I2 SpkC, 500 ngli. Spike) C4 Sulfonate PFBS_____________ C6 Sulfonate PFHS_____________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery In Sample Recovered Recovery In Sample Recovered Recovery (ng/L) (ng/L) (ng/L)_____ <%) (ng/L) (ng/L)_____ (%) (ng/L) (ng/L)_____ (%> 500 ND 748 150 ND 485 97 ND 502 100 DL3-SW-LOC001-0-041201 (C00522I2 Spk D, 5000 ng/L Spike) 5000 ND 7090 142 ND 4560 91 ND 4450 89 DL3-SW-LOC001-0-041201 Low Spk (C0052254,500 ng/LSpike) 500 ND 694 139 ND 496 99 ND 508 102 DL3-SW-LOC001-0-041201 High Spk (C0052255, 5000ng/LSpike) 5000 ND 7320 146 ND 4580 92 ND 3840 77 DL2-SW-LOC001-0-041201 (C0052255 SpkE, 500 ng/L Spike) 500 ND 779 156 ND 516 103 ND 490 98 DL2-SW-LOC001-0-041201 (C00522SSSpkF. 5000ng/LSpike) 5000 ND 7200 144 ND 4630 93 ND 4680 94 DL2-SW-LOC001-0-041201 Low Spk (C00S22SS, 500ng/LSpike) 500 ND 734 147 ND 492 98 ND 515 103 DL2-SW-LOC001-0-041201 High Spk (C0052215, 5000ng/LSpike) 5000 ND 7200 144 ND 4660 93 ND 5440 109 DBC-SW-LOC001-0-041202 (C00522S0Spk0 ,500 ng/L Spike) 500 93.8 819 145 231 714 97 3460 3840 * DBC-SW-LOC001-0-041202 (C0052290SpkH, 5000 ng/L Spike) 5000 93.8 7330 145 231 5060 97 3460 7930 89 DBC-SW-LOC001-0-041202 Low Spk (C0052292,500 ng/LSpike) 500 93.8 742 130 231 667 87 3460 3800 . DBC-SW-LOC001-0-041202 High Spk (C0052295,5000 ng/L Spike) 5000 93.8 6830 135 231 4600 87 3460 7090 73 DOU-SW-LOC001-0-041202 (C0052294Spkt, 500 ng/L Spike) DOU-SW-LOC001 -0-041202 (C00522MSpkJ, 5000ngIL Spike) DOU-SW-LOC001-0-041202 Low Spk (C0052299,500 ng/LSpike) DOU-SW-LOCO01-0-041202 High Spk (C0052297,5000ng( l Spike) 500 5000 500 5000 14000 14000 14000 14000 12200 14800 15200 14100 16" * 2" 1620 2130 102 5710 5820 * 1620 6160 91 5710 10300 92 1620 2150 106 5710 6720 1620 5720 82 5710 9350 73 DOO-SW-LOCOOI-O-041202 (C005229I SpkK, 500 ng/LSpike) 500 ND 805 161 ND 482 96 ND 50 5 101 DDO-SW-LOC001-0-041202 (C005229I SpkL, 5000ng/LSpfce) 5000 ND 7240 145 ND 4590 92 ND 4600 92 DDO-SW-LOC001-0-041202 Low Spk (C00S2510,500 ng/LSpike) 500 ND 794 159 ND 517 103 ND 517 103 DDO-SW-LOC001-0-041202 High Spk (C0052311,5000ng/L Spike) 5000 ND 5310 106 ND 4680 94 ND 5000 100 ` Sam ple residue exceeds the spiking le vel sig nifican tly; therefore, an accurate recovery value cannot be calculated " P oint w as rem oved from the calculation o f average and standard deviation as p er em ail request dated 03/21/05. ND * N ot detected at o r above 25 ng/L (ppt). NQ = N ot q uantifiable * M easured concentration between 25 ng/L (ppt) and the L im it o f Q uantitation (LO Q ) w hich is 50 ng/L (ppt). Note: Since this summary table shows rounded results, recovery values may vary slightly from die values in the raw data. Exygen Research Page 20 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Surface Water Samples Continued Sample Description DL1-SW-LOC001-0-041202 (C0052312 SpkC. 500 ng/LSpike) C4 Sulfonate PFBS_____________ C6 Sulfonate PFHS_____________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery In Sample Recovered Recovery In Sample Recovered Recovery (non-) (nfl/L) ___ ( " f l - ) ________ ! * 1 ____ (no/L) w u ________ e a ____ (ng/L) w d ________________ 500 76.8 849 154 45.7 55 6 111 179 707 106 DL1-SW-LOC001-0-041202 (C0052312Spk 0.5000 ng/L Spike) 5000 76.8 7280 144 45.7 4810 96 179 4800 92 DL1-SW-LOC001-0-041202 Low Spk (C0052314.500 ng/L Spike) 500 76.8 620 149 45.7 566 113 179 726 109 DL1-SW-LOC001-0-041202 High Spk (C0Q52315,5000 ng/L Spike) 5000 76.8 7970 158 45.7 4700 94 179 4100 78 DMC-SW-LOC001 -0-041202 (C00S2315Spk E. 500 ng/L Spike) 500 107 893 157 53.6 556 100 284 764 96 DMC-SW-LOC001-0-041202 (C005231SSpk F. 5000ng/L Spike) 5000 107 7780 153 53.6 4740 94 264 5130 97 DMC-SW-LOC001-0-041202 Low Spk (C0052315,500ng/L Spike) 500 107 786 136 53.6 544 98 284 807 105 DMC-SW-LOC001-0-041202 High Spk (C0052319,5000ng/L Spike) 5000 107 6480 127 53.6 4560 90 284 3890 72 DAA-SW-LOC005-0-041202 (C0052322 Spk0 ,500 ngA Spike) DAA-SW-LOC005-0-041202 (C00S2322Spk H, 5000 ngA Spike) DAA-SW-LOC005-0-041202 Low Spk (C00S2324,500ng/L Spike) DAA-SW-LOC005-0-041202 High Spk (C005232S, 5000ng/L Spike) 500 5000 500 5000 2460 2460 2460 2460 4100 6840 3840 10600 * 88 167 7980 7980 7980 7980 9270 12200 8290 11900 * 84 78 18800 18800 18800 18800 23500 23600 20500 20200 * 96 28" DAA-SW-LOC002-0-041202 (C00S2320 SpkI, 500 ngA Spike) 500 379 1250 174 375 891 103 2780 3530 * DAA-SW-LOC002-0-041202 (C00S2320SpkJ. 5000ngA Spike) 5000 379 6430 121 375 5320 99 2780 8310 111 DAA-SW-LOC002-0-041202 Low Spk (C00S2320,500ng/L Spike) 500 379 762 77 375 860 97 2780 3440 DAA-SW-LOC002-0-041202 High Spk (C0052329.5000ngA Spike) 5000 379 7850 149 375 5180 96 2780 7740 99 Field Blank Low Spk (C0052331,500ng/L Spike) Field Blank High Spk (C0052332,5000ngA Spike) Field Blank Low Spk (C 0082334, 900 ng/L Spike) Field Blank High Spk (C0052335, 5000ngA Spike) 500 5000 500 5000 ND 594 119 ND 6070 121 N D 625 125 ND 6550 Average: Standard Deviation: 131 139 21 ND 458 92 ND 4670 93 N D 475 95 ND 4450 Average: Standard Deviation: 89 95 7 ND 574 115 ND 5470 109 N D 541 108 ND 5100 Average: Standard Deviation: 102 96 12 ` Sam ple residue exceeds the spiking le vel sig n ifica n tly; therefore, an accurate recovery value cannot be calculated " P oint w as rem oved from the calculation o f average and standard deviation as p er em ail request dated 03/21/05. ND * N ot detected a t o r above 25 ng/L (ppt). NQ * N ot q uantifiable * M easured concentration between 25 ng/L (p p t) and th e L im it of Q uantitation (LO Q ) w hich is 50 ng/L (ppt). Note: Since this summary table shows rounded results, recovery values mayvary slightly from the values in the raw date. Exygen Research Page 21 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 22 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 1. Typical Calibration Curve for PFBS in Water 021405C revl.ndb (PFBS): "Unear Regression ('1 t x" weighting): y * 52.7 x + 0.000802 (r 0.9864) Area, counts Exygen Research Page 23 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 2. Extracted Standards of PFBS in Water, 50 ng/L and 100 ng/L, Respectively I XC021105-2, 50ng/L Standard - PFBS (Standard) 299.W99.0anw -sam ple 3 o f 52 from 021405C.wiff Area: 3492counts Height: 05.2cps RJ: 3.93 min 3.83 Intensity, cps Time, min XC021105-3, 100 ng/L Standard - PFBS (Standard) 299.0/99.0 amu - sample 4 of 52 from 021405C.wiff Area: 5190 counts Height: 105. cps RT:4.00 min Intensity, cps Exygen Research Page 24 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 3. PFBS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively Reagent Control A - PFBS Jnknown) 299.9/99.9 amu -sample 9 o f 52 from 921495C.wiff (peak not found) CO Time, min Reagent Spike A, 500 ng/L - PFBS (Q0)299.0/99.0 amu - sample 10 of 52 from 021405C.wiiff Area: 26775 counts Height: 633. ops RT: 3.93 min CO Q. O 3.93 `co(f>) 500 0.83 ,,1.49 0 -- 3.18 c 5.666.437.20 8.72-9.55^10.13 8 10 Time, min 11.56^.11.95 12 14.06,14.37 >.15.64 17.36 -- I----------- I --------- 14 16 I Reagent Spike B. 5000 ng/L, DF=10 - PFBS (Q C )299.0/99.0 amu - sample 11 of 52 from 021405C.wiff Area: 25449 counts Height: 596. ops RT: 3.94 min CO Q. o 500 CO 0 -- 3.94 9.00-^9.45 11.71^12.1013.04 ^13.72 15.41,15.77 17.55 8 10 12 14 16 Time, min Exygen Research Page 25 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Surface Water Sample Analyzed for PFBS (Exygen ID: C0052282, Data Set: 021405C) C0052282 - PFBS (Unknown) 299.0/99.0 amu sample 15 o f 52 from 021405C.wiff (peak not found) 0.58 Intensity, cps Exygen Research Page 26 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 5. Typical Calibration Curve for PFHS in Water 021405C rev1 .rdb (PFHS): "Unear'' Regression ('1 / x" weighting): y = 187 x + -0.000613 (r = 0.9994) Area, counts Exygen Research Page 27 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 6. Extracted Standards of PFHS in Water, 50 ng/L and 100 ng/L, Respectively I XC021105-2, 50 ng/L Standard PFHS (Standard) 399.0/80.0 amn -sample 3 o f 52 from 021405C.wiff Area: 9721 counts Height: 695. cps RT: 10.4min Time, min I XC021105-3, 100 ng/L Standard - PFHS (Standard)399.0/80.0 amu - sample 4 of 52 from 021405C.iruiff Area: 16628 counts Height: 1380. cps RT: 10.5 min 10.46 Exygen Research Page 28 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 7. PFHS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively I Reagent Contro/ A - PFHS (Uttnown) 399.0/9M amu -sample 9 o f 52 from 021405G.mff (peak not found) (ft CL O 20 10.45 (cft -4** _0A.63-J.D.se 2.90 -3.44>1.32 6.81-.7.2_8 8.23 10.08. !a -- ^ / l -- tyS--ft 12.98^ 13.5^.13.88 15,03 ^16.69 4c-* 10 12 14 16 Time, min I Reagent Spike A, 500 ng/L - PFHS (QC) 399.0/80.0 amu sample 10 of 52 from 021405C .wifi Area: 94857 counts Height: 7770. ops RT: 10.5 min (A Q. o 10.46 (0 JLwc 10.03. 8 10 12 14 16 Time, min I Reagent Spike B, 5000 ng/L. DF=10 PFHS (QC) 399.0/80.0 amu - sample 11 of 52 from 021405C.wiff Area: 80963 counts Height: 6530. ops RT: 10.4 min co CL 5000 4<c0c-}^o 0 10.43 X10.00. 8 10 12 14 16 Time, min Exygen Research Page 29 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Surface Water Sample Analyzed for PFHS (Exygen ID: C00522831, Data Set: 021405C) I C0052282 - PFHS (Unknown) 399.0/89.0 amu -ample 15 o f 52 from 021405C.wiff A n a: 1974counts Height: 56.3 cps RT: 10.5 mitt 0.57 Intensity, cps Exygen Research Page 30 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 9. Typical Calibration Curve for PFOS in Water 021405C rev1 .rdb (PFOS): "Unear'' Regression ('1 / x" weighting): y * 162 x + 3.07e+003 (r = 0.9978) Area, counts Exygen Research Page 31 o f 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No. : P0001131 Figure 10. Extracted Standards of PFOS in Water, 50 ng/L and 100 ng/L, Respectively I XC021105-2, SOng/L Standard - PFOS (Standard) 499.M0.0 amn -sample 3 o f 52 from 02140SC.wiff A n a: 9931 counts Height: 505. cps RT: 12.6 min 12.60 Intensity, cps Time, min XC021105-3, 100 ng/L Standard - PFOS (Standard)409.0/80.0 amu - sample 4 of 52 from 021405C.wiff Area: 16106 counts Height: 925. cps RT: 12.6 min 12.62 Intensity, cps Exygen Research Page 32 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 11. PFOS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively Reagent Control A - PFOS {Unknown) 499.0/80.0 amu -sample 9 o f 52 from 021405C.wiff Area: 3107 counts Height: 5&6cp s RT: 12.6min (O Qo . &'CO c0) 50 0 5.35-1-.-17---r 2 3.55 >1.06 4 0.31 6 12.64 12.35^1 8.71 v 10.14^10.66 _ ^ 8 10 12 14.15 _ ,, 4 5 t-15.61^16.73 14 16 Time, min I Reagent Spike A, 500 ng/L - PFOS (QC) 400.0/80.0 amu-sam ple 10 of 52 from 021405C.uviff Area: 78386 counts Height: 5080. ops RT: 12.6 min o> Q. U 5000-1 &to 0- - I ------------- -------------- r 46 12.62 12.18.. I ---- -- i-,---------------------, ----------- ,------------------------ r - 8 10 12 14 16 Time, min I Reagent Spike B. 5000 ng/L, DF=10- PFOS (00 )4 6 9 .0 /8 0 .0 amu-sam ple 11 of 52 from 021405C.wiff Area: 72672 counts Height: 4660. cps RT: 12.6 min CO Q . 12.62 &<0 c0) o C. --r-- 8 10 12 14 16 Time, min Exygen Research Page 33 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Surface Water Sample Analyzed for PFOS (Exygen ID: C0052282, Data Set: 021405C) I C0052282 - PFOS (Unknown) 499.0/90.0 ama -sample 15 o f 52 from 021405C.wiff Area: 3595 counts Height: 99.5 cps RT: 12,7 min Intensity, cps Time, min 17.9K A_aJ Exygen Research Page 34 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method: V0001780:"Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" Exygen Research Page 35 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 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 36 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 o f 65 Page 37 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PROTOCOL APPROVAL Study Title: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Periluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 APPROVALS JaisimhaKesan, Study Director Weston Solutions Michael A. 3M Comparfy Sponsor Representative ohn M. Flaherty, Principal Investigator Exygen Research Richard A. Gi Exygen Resei ident. Facility Management U.Z LydfitShaffer, Technioaiead, Quality Assurance Unit Exgen Research m Date 2$ -Ccr-KZ^ Date / t Dwatj P age 3 o f 65 Exygen Research Page 38 of 100 Interim Report #2-Analysis of Surface Water 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 o f 65 Exygen Research Page 39 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study N o.: 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 will 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 (CiF^SOj'K*) Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 - 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CsFijSOjTi*) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 - 80 Structure: Page 5 o f 6} Exygen Research Page 40 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (C8F|7S03TC+) Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 - 99 Structure: OBJECTIVE The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesuifonate (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 of 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 41 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 F a x :(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 john.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 be included in the final report. Page 7o f 65 Exygen Research Page 42 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: Water (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study. The test systems were chosen to access the environmental impact 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 the final report associated with this study. Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at 2C-8C. Fish, clam, vegetation and small mammal liver samples will be processed according to the appropriate analytical method (see Appendix I). These samples will be stored frozen at -10C. Small mammal whole blood samples will be centrifuged in the field at the time o f collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at -10C. The receipt and processing o f the samples will be documented in the final report and raw data associated with the study. P age 8 o f 65 Exygen Research Page 43 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 SAMPLE IDENTIFICATION Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Sample storage conditions and locations will be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: V0001780: "Method 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" 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 will be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen will supply one bottle per sample collected. The <*** bottles will be 500 mL preclean! Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 9 o f 65 Exygen Research Page 44 of 100 Interim Report #2-Analysis of Surface Water 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 will be added to each container to a volumetric fili line at 200 mL. A field duplicate, a low field spike and a high field spike of each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (l3C PFOA). PFOA and l3C PFOA are included in the solutions used to spike the samples. The results for PFOA and l3C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containers/bags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at ^ both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. For small mammal liver, Exygen will supply a 50 mL polypropylene centrifuge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. Low and high spiking levels for each matrix are defined below: M atrix Low Spiking Level High Spiking Level Water 500ng/L 5000 ng/L Soil 4 ng/g 40 ng/g S edim ent 4 ng/g 40 ng/g Fish 10 ng/g 100 ng/g Clams 10 ng/g 100 ng/g Vegetation 10 ng/g 100 ng/g Small Mammal Liver 10 ng/g 100 ng/g Small Mammal Serum lOng/mL 100 ng/mL P age 10 o f 65 Exygen Research Page 45 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Recoveries are anticipated to be between 70% and 130% of the fortified levels; however, the exact precision and accuracy will be determined by the analysis o f the quality control samples described above. A statement of accuracy will 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 will be limited to those specified in the subject methods and to the calculation o f average recoveries, as applicable. GLP STATEMENT All 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 will be prepared by the principal investigator or their designee at the conclusion o f the study. The report will include, but will 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 l o f 65 Exygen Research Page 46 of 100 Interim Report #2-Analysis of Surface 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 will be documented in the report. Locations where raw data and the final report are to be archived. Additions or corrections to the final report shall be in the form 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 will be signed and dated by the Study Director and the Sponsor Representative. All 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 of personnel and the environment to the test or reference substance(s). Page 12 o f 62 Exygen Research Page 47 of 100 Interim Report #2-Analysis of Surface 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 the 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 will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description of any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing 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- All chromatograms will 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 will include the amount o f analyte added and the sample number o f the sample that was fortified. Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL). Page 13 o f 65 Exygen Research Page 48 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 As part of the documentation the following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITY ASSURANCE The QA Unit o f Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will 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 will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period o f time specified in 40 CFR 792.195. An exact copy o f the materials submitted to the study director will also be kept at Exygen Research. Exygen will obtain permission from the study director before discarding or returning samples. Exygen Research Page / 4 o f 65 Page 49 of 100 Interim Report #2-Analysis o f Surface 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: `M ethod 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: "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" Exygen Research P age IS o f 65 Page 50 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number; POOOt 131 ANALYTICAL METHOD Method Number: V0001780 Method o f Analysis for the Determination o f Ferflaorooctaooic A d d (PFOA) la Water by LC/MS/MS Analytical Teeting Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: JT vU c O L ______ Paul Connolly ' Technical Leader, LC-MS, Exygen Research ?l/r?/hL zf / J''Joohhnn Flahheorlty / ' VViice* fPtrrusiiitdaanitl, OrkpueMrattiinonnsi, Exygen Research ioIx-wA)/ Date Exygen Research Total Pages: 7 Page 6 o f 65 Page 51 o f 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000113) Exygta Research Method Number V0001780 I ANALYTICAL m e t h o d Method ofAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 1.0 Scope T hu method is to be employed for the isolation and quantitation o f perfluorooctanoic a d d by High Performance Liquid Chromatography coupled to a tandem Mass Spoctrometric Detector (LC/MS/MS) in water. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 40 m L o f test sample 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 All samples must be thoroughly mixed before being sampled for extraction 3.3 Any samples containing particles should be centrifuged at -3 0 0 0 rpm for -5 minutes and the supernatant used for the extraction. 3.6 Sample collection procedures will be specified in the 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 A d d - Sigma*Aldrich 3.0 Instrument and Equipment j . l A high performance liquid chromatograph capable o f pumping up to 2 solventa equipped with a variable volume injector capable o f injecting 3*200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 3.2 A device to collect raw data for peak integration and quantitation. 3.3 Analytical balance capable o f reading to 0.00001 g. 3.4 30 m L disposable polypropylene eentrifUge tubes. 3.5 15 m L disposable polypropylene centrifuge tubes. 3.6 Disposable micropipets (S0*100uL, 100*200uL). 5.7 125*mL LDPE narrow*mouth bottles. 3.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettet (100*1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (tg ) tCIS SPE cartridges. Pase2of? P age 7 o f 65 Exygen Research Page 52 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Research Method Number V0001780 ANALYTICAL m e t h o d Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Water by U V M S/M S 5.12 SPE vacuum manifold. 5.13 CentrifUge capable o f spinning 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, Sp (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in W ater 6.4 Mobile Phase <B): Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate & S fmL/mint 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can b e increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 -* 369 m/z. The above conditions are intended as guide and m ay be changed tn 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 o f ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. Page ui Page 8 o f 65 Exygen Research Page 53 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygea lUtearch Method Number VOOOI7SO ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A) in Water by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stoclc/Fortification SohUion 9.1.1 Prepare a stock solution o f ~ 100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 12S-mL LDPE bottle. 9.1.2 A 10 fig/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 100 jig/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 p ^ m L fortification solution o f PFOA is prepared by bringing to m L o f the 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o ftb e 1.0 p y faL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.5 A 0.01 pgfaiL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/m L solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LOM S/M S calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Volume o f Concentration o f o f Fortification Volume Fortified Control Calibration Calibration Standard ID Solution ioob (PL) S an ale (mL) Standard (not)* (example) 00 10 100 10 200 10 400 100 100 40 40 40 40 40 0 XCmraddyy-0 25 XCm mddyy'l 50 XCmmddyy-2 100 XCmmddyy-3 250 XCmmddyy-4 100 200 100 400 40 40 500 1000 XCmmddyy-5 XCmmddyy-6 * The extracted concentration o f the calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC extracted calibration standard. Page4 of ' P age 19 o f 65 Exygen Research Page 54 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Num b: P0001131 Exygen Rem rch Method Number V00017S0 ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctsnoic Acid (PFOA) in Water by L C /M S/M S 9.2.3 9.2.4 9.2.5 A z o o standard solution (reagent blank) m ust be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene tubes at 2*C to 6C, up to two weeks. Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f camples extracted (typically 20 o r less) must include at least one reagent control (method blank using HPLC water) and two reagent controls fortified st known concentrations (lab control spike) to verily procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in die quality assurance plan for this project. 11.0 Sample Extraction 11.1 Measure 40 mL o f sample o r a portion o f sample diluted to 40 mL with water into 50 m L polypropylene centrifiige tubes (fortify as needed, replace lid and mix well). 11.2 Condition the C u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL o f HPLC water (~ 2 drop/sec). D o not let column run dry 11.3 Load sample on conditioned C u S P E cartridge. Discard eluate. 11.4 Elute with - 5 m L 100% methanol. Colloct 5 m L o f eluate into graduated 15 mL polypropylene centrifiige tubes (final volume * 5 mL). 11.5 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or m ore concentration levels must be included in an analytical l e t 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 he interspersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards m ay be injected at the beginning o f a sei followed by extracted calibration standards interspersed every 5-10 samples (to account for a sooond 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 Pag $ of 7 P age 20 o f 65 Exygen Research Page 55 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea R m u c h Method Number V0001780 | ANALYTICAL M E T H O D ......... | Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Water by LCYMS/MS venue calibration standard concentration using M atsLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should b e Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA s! levels p e s te r than 50 ng/L, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total num ber o f extracted standards iqjected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R2 0.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevsnt set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f M ention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/L, based cm peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/L) - (Peak area - intercept) x DF slope DF factor by which the final volume was diluted, if necessary. Page 6 of 7 Page 21 o f 65 Exygen Research Page 56 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000113 1 E x y g ntow aieh Mttbod Number V0001780 | ANALYTICAL M ETHOD ~| Method o f Analysis for the Determinition o f Perfluorooctanoic Acid (PFOA) in Water by L C /M S /M S 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) - [ total analyte found (ng/L) - analyte found in control (ng/L)] analyte added (ng/L) 1 Exygen Research Page 7 of 7 Page 22 o f 65 Page 57 o f 1 0 0 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method N um ber V0001781 M ethod o f Analysis for die Determ ination o f Perfluorooctanofc Acid (PFOA) in Soil by LC /M S/M S Analytical T a tin g Facility: Exygen Research 3058 Research Drive State College. PA 16801 Approved By: "v iA C -Ji. Paul Connolly ' Technical Leader, LC-MS, Exygen Research Date Date Exygen Research Total Pages: 7 Page 23 o f 65 Page 58 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygto ReMtrcli Method Number V0001781 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 1.0 Scope T h u method ii to be employed for the isolation and quantitation o f perfluorooctanoic a d d by High Performance liq u id Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in soil. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 15 g o f test sample for extraction. 3.2 No sample processing is needed for soil samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly m ixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - 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 M ias Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifiige tubes. 5.5 15 mL disposable polypropylene centrifiige tubes. 5.6 Disposable micropipets (50-lOOuL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 jaL and 10*100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 ce (lg ) tCIS SPB cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. Pag 2 or? P a g e 24 o f 65 Exygen Research Page 59 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygea Rwcwch Method NumbnVOOO1781 ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by L C /M S/M S ] 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f apinning SOm L polypropylene tubes at 5000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophaae RP (Keystone Scientific), 2.1 mm x SO m m . 5p (P/N: 82305-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A ) : 2 mM Ammonium Acetate in Water Mobile Phase (Bj : Methanol Gradient Program: Tim e fm inl 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B fmL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as m uch as SO pL). 6.7 Quantitation: Peak Area -ex te rn a l standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, m onitoring 4 1 3 - 369 m/z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. Page 3 of 7 Page 25 o f 65 Exygen Research Page 60 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI781 ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by L C /M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f ~100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in l25*mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o fP F O A is prepared by bringing 10 mL o f the 100 pg/mL solution to a final volume oflOO with methanol in a 125 mL LOPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 10 pg/mL solution to final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0 .1 pg/mL fortification solution o fP F O A is prepared by bringing 10 m L o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f tite 0.1 pgfaiL solution to a final volume o f 100 with methanol in 125 m L LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Volume o f Concentration o f Calibration o f Fortification Volume Fortified Control Calibration Standard ID Solution (m b) (tiL) Santole (mL) Standard foot)* (examole) 0 0 40 0 XCmmddyy*0 10 100 10 200 10 400 100 100 100 200 40 40 40 40 40 25 XCmmddyy-1 50 XCmmddyy-2 100 XCmmddyy-3 250 XCmmddyy*4 500 XCmmddyy-5 100 400 40 1000 XCmmddvv6 * The extracted concentration o f the calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC extracted calibration standard. Page 4 of 7 P age 26 o f 65 Exygen Research Page 61 o f 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 ExygmfUwvcfe Method Number V00017BI | ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.2.3 9.2.4 9.2.$ A zero standard solution (reagent blank) must be prepared with each set o f standards extracted. Store all attracted calibration standards in 15-mL polypropylene lubes at 2C to 6*C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one reagent control (method blank using 5 mL o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 1l .t Weigh 5 g o f sample into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 112 Add 5 mL o f methanol and shake on a wrist action shaker for -1 5 minutes. 11.3 Transfer the tubes to in ultrasonic bath and sonicate for ~ 15 minutes. 11.4 Bring the volume up to 40 m L with water in the 50 m L polypropylene centrifiige tube. 11.5 Centrifuge fo r- 1 0 minutes at ~3000 ipm. 11.6 Condition the C it SPE cartridges (! g, 6 mL) by passing 10 mL methanol followed by 5 m L o f HPLC water ( - 2 drop/tec). Do not let column run dry 11.7 Load (decant) the sample on the conditioned C u SPE cartridge. Discard elutte. 1l.S Elute with -5 mL 100% methanol. Collect S m L o f eluate into graduated 1S mL polypropylene centrifiige tubes (final volume - S mL). 11.9 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r more concentration levels must be included in an analytical act 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample s e t Extracted standards must be interspersed between every 5*10 samples. As an alternative, an entire set o f pm * s of 7 Page 27 o f 65 Exygen Research Page 62 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VQOO1781 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS extracted calibration atandarda m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5*10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be 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 w eighting o f peak area versus calibration standard concentration using MassLynx 3 3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ton (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set must be re*extractod. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated b y the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f extracted calibration standards that could be excluded must oot exceed 20% o f the total number o f extracted 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 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 and samples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. Page 6 of 7 Page 28 o f 65 Exygen Research Page 63 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygci Rnearch Method N u n * V00017*I ANALYTICAL M ETHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by L C /M S/M S | 14.0 Calculations 14.1 U ic the following equation to calculate the amount o f PFOA found (in ng/L, baaed on peak area) uaing the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/L) * (Peak area - intercept) x DF slope DF * factor by which the final volume was diluted, i f necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (% ) [ total analyte found (ng/L) analyte found in control (ng/L)] . . . analyte added (ng/L) 14.3 Uae the following equation to convert the amount o f PFOA found in ng/L to ng/g(ppb). PFOA found (ppb) - fPFOA found (ng/L) x volume extracted (Q.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 solid$(%)] Page 7 of 7 P age 29 o f 65 Exygen Research Page 64 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001782 M ethod o f Analysis for th e D etersntioD o f Perfluerooctanoic A d d (PFOA ) in Sediment by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C JL Paul Connolly i Technical Leader, LC-MS, Exygen Research &'J/>? d U S /J oioihnn Flaherty f Viecre President, Operations, Exygen Research ___ to k b l < y t Date dr Date Exygen Research Total P a ^ a; 7 Page 30 o f 65 Page 65 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Rsaevnb Method Number VOOOI782 | ANALYTICAL M ETHOD | Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment bv LC/MS/MS 1.0 Scope This method is to be employed for foe isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in sediment. 2.0 Safety 2.1 Always observe safe laboratory practice. 2.2 Consult the appropriate MSDS before handling any ebemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 30 g o f teat sample for extraction. 3.2 No sample processing it needed for sediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly m ixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 M ethanol-H PLC grade 4.3 Acetic Acid - Reagent grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume iiyector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene ccntriftige tubes. 5.5 15 m L disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). $.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tipi. S.l I Waters Sep Pak Vac 6 ec (Ig ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. Page 2 of 7 P age 3! o f 65 Exygen Research Page 66 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOi 782 | ANALYTICAL M ETHOD ......... Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S 5.13 Vortexer. 5.14 Wrist-action shaker. 5.15 Centrifiige capable o f apinning 50 m L polypropylene tubea at 3000 rpm. 6 0 Chromatographic System 6.1 Analytical Column: FluophaseRP (Keystone Scientific) 2.1 mm x 50 mm, 5m (P/N: 825054)52130) 6.2 Temperature: 30*C 6.3 Mobile Pbaae (A) : 2 mM Ammonium Acetate in W ater 6.4 Mobile Phase (B ): Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate 2LB fmL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 p L (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external itandard calibration curve. 6.8 RunTim e: ~ 2 3 minutes. The above conditions are intended aa a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: ElectrOtpray Negative MRM mode, m onitoring 413 -> 369 m/z for PFOA. The above conditions are intended as a guide and m ay b e 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 la prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Page 3 of 7 P age 22 o f 65 Exygen Research Page 67 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exygen Reteaich Method Number VQ001782 | ANALYTICAL METHOD | Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S 8.2 Extraction Solutions 8.2.1 1% acetic acid in water is prepared by adding 10 m L o f acetic acid to 1000 mL o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 p g /cn L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LOPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 100 pg/mL solution to final volume o f 100 with methanol in a 125 mL LOPE bottle. 9.1.3 A l .0 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 10 pg/raL solution to a final volume o f 100 with methanol in a 125 m L LOPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 m LLDPB bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f tbe 0.1 pgfaiL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from tbe date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f tbe 0.1 pg/mL fortification solution. 9.2.2 The following is a typical example; additional concentrations may be Concentration o f Fortification Solutum ftix/mL) too too too 10 5 2 Volume (mL) 10 5 2 10 10 10 Diluted to (mL) 100 too 100 100 100 too Final Concentration (nn/mL) 10.0 50 20 10 05 0.2 Page 4 of 7 Page 33 o f 65 Exygen Research Page 68 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P r o to c o l N u m b e r: P 0 0 0 1 131 Exyjm Rnetrch Method Number V0001782 | ANALYTICAL M ETHOD | Method o f Analysis fbrtheDetenninationofPerfluoroocUnoic Acid (PFOA) in Sediment by L C /M S/M S 9.2.3 9.2.4 Store til cilibration etandaidt in 125-mL LOPE narrow-mouth bottles at 2*C to 6*C, up to six month*. Alternate volume* and concentration* o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f ample* extracted (typically 20 o r lest) m ust include at least one untreated control and two untreated controls fortified at known concentration* (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the qualify assurance plan fix'this project. 11.0 Sample Extraction 1l .l W eigh 5 g o f sample into 50 m L polypropylene cemriftige tubes (fortify us needed, replace lid and mix well). 11.2 Add 35 m L o f 1% acetic acid, cap, vortex and shake on a wrist action shaker for -6 0 minutes. 11.3 Centrifuge the tubes at -3000 rpm for - 2 0 minutes. 11.4 Condition the C u SPE cartridges (1 g. 6 mL) by passing 10 mL methanol followed b y 20 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.5 Load (decant) the sample on the conditioned C u SPE cartridge. Discard eluate. 11.6 Add 20 m L o f methanol to the sediment left in the bottom o f the 50 mL ccntrifUge tube. Cap, vortex and shake on a wrist action shaker for -3 0 minutes. 11.7 Centrifuge the tubes at -3 0 0 0 rpm for - 2 0 minutes. 11.8 Decant the methanol onto the same SPE cartridge. Collect the eluate. 11.9 W ash the column with 4 mL o f m ethanol Collect the eluate and add it to the eluate collected in step 11.8. 11.10 Condition a second C u SPE cartridge (1 g, 6 m L) by passing 10 mL methanol followed by 20 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.11 Add the methanol to -2 0 0 m L o f water and load on die second conditioned SPE cartridge. 11.12 Elute with - 5 m L 100% methanol. Collect 5 m L o f eluate into graduated 15 mL polypropylene centrifuge tubes (final volume 5 mL). 11.13 Analyze samples using electrospray LC/MS/MS. Pag 5 of 7 P age 34 o f 65 Exygen Research Page 69 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Rffuarcfa Method Number VOOO1782 | ANALYTICAL M ETHOD \ Method o f A nalyrii for the Determination o f Perfluorooctanoie Acid (PFOA) in Sediment by L C /M S /M S 12.0 Chromatography 12.1 Inject the Mine amount o f each standard, sample aod fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample aet. Standards must be interspersed between every 5-10 samples. A s an a lta n a n ve. an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for second set of standards). In either case, calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves arc generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be ftuther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram m ust show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 am u parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the lost o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. if a blink wpfoini PFOA at levels greater than 0.2 ngfaiL, then a new blank sample must be obtained and the entire set m ust be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f e 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 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 die calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f die total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R3 0.985). I f calibration results foil 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. Page 6 of 7 Page 35 o f 65 Exygen Research Page 70 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen ftMMieh Method Number V0001782 1 ANALYTICAL M ETHOD | Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Sediment by L C /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 tim e drift exceeds this limit within an analytical run then the set must be reanalyzed 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. baaed on peak area) uaing the standard curve (linear regression parameters) generated by foe Mass Lynx software program: PFOA found (ng/mL) - (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 (%) - [total analyte found (ng/mL) - analyte found in control (ng/mL)] analyte added (ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) - fPFOA found fn e/m U x final volume (5 tnL)l sample weight (5 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% / total solids(%)] Page 7 of 7 Page 36 o f 65 Exygen Research Page 71 o f 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method N um ber V0001783 Method o f Analysis for the Determination o f Perfluorooctaoole Acid (PFOA) lo Fish and C h u n by LC/MS/MS Analytical Testing Facility. Exygen Research 305$ Research Drive State College PA 16801 Approved By. T U . c_j L Paul Connolly 1 Technical Leader LC-MS, Exygen Research u'/# ? /f a r / ' J owhin Flaherty * VViiccee President, Operations, Exygen Research Date Date r Exygen Research Total Paget: 8 P age 37 o f 65 Page 72 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Retouch Method Number VOOO1783 AMA L Y rtC A L M ETH O D I Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooclanoic acid by High Performance Liquid Chromatography coupled to a tandem M ass Spectromtrie Detector (LC/MS/MS) in fish and clams. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g o f test sample for extraction. 3.2 Sample# should be processed before extraction. Place the frozen sample in a food procesaor and homogenize w ith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time o f analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project. Reagents and Standards 4.1 W ater-H P L C grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 M ethanol-H PLC grade 4.5 Silica gel (60*200 mesh) - Reagent grade 4.6 Florisil (60*100 mesh) - Reagent grade 4.7 Superclean LC-NHj - Reagent grade 4.8 1-O ctanol- HPLC grade 4.9 L'Ascorbic add - Reagent grade 4.10 Dimethyldichlorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium A cetate-A .C .S . Reagent Grade 4.13 Periloorooctanoic 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 Maas Spectrometer (LC/MS/MS). 5.2 A devioe to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. P.ge2 of8 Page 38 o f 65 Exygen Research Page 73 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygca Raaearch Method Number V0001783 ANALYTICAL M ETHOD Method o f Analysis for the Determinttioo ofPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 5.4 Rotary evaporator. 5.5 Tissumizer. 5.6 125 mL pear-shaped flasks. 5.7 50 mL disposable polypropylene centriftige tubes. 5.8 15 m L disposable polypropylene centrifuge tubes. 5.9 Disposable micropipets (50-1OOuL, !00-200uL). 5.10 125-mL LDPE narrow-mouth bottles. 5.11 2 mL clear HPLC vial kit. 5.12 Disposable pipettes. 5.13 Autopipettes (100*1000 pL and 10*100 pL), with disposable tips. 5.14 SPE tubes (20mL) (Sapelco cat. no. N057177). 5.15 Wrist action shaker. 5.16 Centrifuge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluopbase 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 W ater Mobile Phase (B) : Methanol Gradient Program: Tima ftnint 0.0 1.0 8.0 20.0 22.5 SU 65 65 25 25 65 Flow Rate %B fmL/minj 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to s s much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions an intended as guide and m ay be changed in order to optimize the HPLC system. Pag 3 rs Page 39 o f 65 Exygen Research Page 74 of 100 Interim Report #2-Analysis o f Surface W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001783 ANALYTICAL M ETHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: Electrospny 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 ia prepared by adding 0.IS4 g of ammonium acetate to 1000 mL o f water. 82 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol ia prepared by dissolving 2 g o f ascorbic a d d in 100 mL o f methanol. 30% Dimethyldichlorositane in toluene is prepared by bringing 3 mL o f dimethyklichloroailaneto a final volume o f 10 m L with toluene. Alternate volumes m ay b e prepared. 9.0 Standard Preparation 9.1 Standard Stock/Foitification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f 100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing I mL o f the 100 pgftnL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. A 0.1 p ^ m L fortification solution o f PFOA is prepared by bringing 10 m L o f tiw 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 a final volume o f 100 with methanol in a 125 mL LDPE bottle. The stock sod 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. Page 4 of 8 Page 40 o f 65 Exygen Research Page 75 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Retetrch Method Number V000I783 ANALYTICAL M ETHOD Method o f Analysis fi the Determination ofPerfluoroocunoic Acid (PFOA) in Fish and G am s by LC/MS/MS 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/mL fortification solution. The following is s typical example: additional concentrations may be prepared as needed. Concentration o f Fortification Volume Solution fua/raL) (mL) Diluted to (mL) Final Concentration (/mL) 1.0 1.0 1.0 0.05 0.025 0.1 0.005 5.0 2.5 1.0 10 10 10 10 100 100 100 loo 100 100 100 0.05 0.025 0.01 0.005 0.002S 0.001 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, t v to fix months. 9.2.4 Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) m ust 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 will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into 50 m L polypropylene centrifuge lubes (fortify as needed, replace lid and mix well). 11.2 Add 30 m L o f acetonitrile and shake on a wrist action shaker for '-l S minutes 11.3 Place the tubes in a freezer fo r- 1 hour. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 m L SPE tubes in sequence with 2 g florisil, 2 g silica gel, 2 g carbon, and 1 g LC-NHj. Condition the columns with 20 mL o f methanol, then 20 m L o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 125 mL pear-shaped flasks by rinsing with the 30% dimethyldichlorosilane in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use. either by air-drying or with a stream o f nitrogen. Page 5 of 8 Page 4 ! o f 65 Exygen Research Page 76 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Eitygm lUsMich Method Number VOOOI783 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctinoic Acid (PFOA) in Fish and Clams by LC/MS/MS 11.7 Centriftige the 50 mL polypropylene tubes containing sample at -2000 rpm fo r- 1 0 minutes. 11.8 Decant the extract on to a conditioned SPE column fitted inside the mouth o f the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape flask. 11.9 Add 10 mL o f acetonitrile to the sample in the 50 m L centrifuge tube. Homogenize the frozen fat phase using a riaiumizer for - 3 0 seconds and rinse the tissumizer with -1 0 mL o f acetonitrile into the tube. 11.10 Shake the sample again fo r- 1 0 minutes on a wrist-action shaker. 11.11 Place the tubes in a freezer f o r - 1 hour more. 11.12 Centrifuge the 50 mL polypropylene tubes containing sample at -2 0 0 0 rpm for -1 0 minutes. 11.13 Decant the extract onto the a n SPE column. Collect the eluate into the tam e pear-shaped flask and combine with the eluent from the initial extraction. 11.14 Pass 20 m L o f acetonitrile through the SPE colum n and combine the eluate in the same pear-shaped flask. 11.15 Add 3-4 drops o f 1-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 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze samples using eloctrosprsy LC/MS/MS. 12.0 Chromatography 12.1 Inject the tam e amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or m ore concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards m utt be included at the beginning and at foe end o f a sample set. Standards must be interspersed between every 5* 10 samples. As an alternative, an entire set o f calibration standards may be injected at foe beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards), in either case, calibration standards must be foe first end 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 peek area versus calibration standard concentration using MassLynx 3-3 (or equivalent) software system. Pag 6 of 8 P age 42 o f 65 Exygen Research Page 77 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen lUMsrch Method Number VOOOI783 ANALYTICAL M ETHOD Method o f Analysis for the Determination ofPeriluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f c a rto n dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ If a blank contains PFOA at levels greater than O.S ppb, then a new blank sample must be obtained and the entire set m ust be re-extracted. 13.3 Recoveries o f oontrol spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike Adis outside the acceptable limits, the entire set o f samples should b e re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R* 20.985). I f calibration results foil outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should b e reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) - (Pk ITPI intercept) slope 14.2 Use the folkw ing equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found (ppb) fPFQA found fng/mL) x final volume fmL) x DF1 sample weight (g) DF factor by which the final volume was diluted, if necessary. Pigs'? ofS P age 43 o f 65 Exygen Research Page 78 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOIW ANALYTIC,VL M ETH O D Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (V) - [ total analyte found (ng/g) . analyte found in control (ng/g)] analyte added (ng/g) Exygen Research Page S o f8 Page 44 o f 65 Page 79 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001784 M ethod o f Analysis fo r die D eterm ination o f Perflnorooctanoic Acid (PFO A) io Vegetation by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: caL,_ Paul Connolly Technical Leader, LC-MS, Exygen Research a /m f iU /_______ J o h n Flaherty ^ Vice President, Operations, Exygen Research __lOfc&lsi Date Exygen Research Total Pages: 7 P age 45 o f 65 Page 80 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reeetrcti Method Number V000I7W ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope ThU method ii to be employed for foe isolation and quantitation o f perfluorooctanoic a d d by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in vegetation. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place foe 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 foe samples in frozen storage until time o f analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120*400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60-200 mesh) - Reagent grade 4.6 Florisil (60-100 mesh) -R eag en t grade 4.7 Superclean LC-NH* - Reagent grade 4.8 l-Octanol - HPLC grade 4.9 L-Ascorbic a d d - Reagent grade 4.10 Dimethyidichlorosilsne - Reagent grade 4.11 Toluene-R eagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigraa-Aldrich 5.0 Instrument and Equipment 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 p L connected to a tandem M ass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 AnalyticalbalancecapablcofreiKlmgtoO.OOOOl g. Page 2 of 7 Page 46 o f 65 Exygen Research Page 81 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research Method Number V00017M ANALYTICAL M ETHOD I Method o f Analysis for the Determination o f Perfluoroocttnoic Acid (PFOA) in Vegetation by LC/MS/MS 5.4 Rotary ev^>orator. 5.5 125 mL pear-shaped flasks. 5.6 50 m L disposable polypropylene centrifuge tdbea. 5.7 15 mL disposable polypropylene centrifuge tubes. 5.S Disposable micropipets (50-1 QOuL, 100*200uL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 mL clear HPLC vial k it 5.11 Disposable pipettes. 5.12 Autopipettes (100*1000 p L and 10*100 pL), w ith disposable tips. 5.13 SPE tubes (20mL) (Supelco c a t no. N0S7177). 5.14 Wrist action shaker. 5.15 Centrifbge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 Mobile Pharo (A ) : 2 mM Ammonium Acetate in W ater 6.4 Mobile Pharo ( B ) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 2kA 65 65 25 25 65 Flow Rate Jft fmL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 p L (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak A rea-ex tern al standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions a n intended u a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 369 m/z for PFOA. Page 3 of 7 Page 47 o f 65 Exygen Research Page 82 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 Exygea Reswcb Method Number V0001784 ANALYTICAL M ETHOD Method o f Analysis for the Determination ofPerfluorooctinoic Acid (PFOA) in Vegetation by LC/MS/MS The above conditions are intended u a guide and m ay b e changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water i t prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol is prepared b y dissolving 2 g o f ascorbic acid in 100 mL o f methanol. 30% Dimethyidichlorosilane in toluene is prepared by bringing 3 mL ofdimethyklichlorosilaae to a final volume o f 10 mL with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing I mL o f die 100 pgfrnL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. A 0.1 pg/mL fortification solution o fP F O A is prepared by bringing 10 mL o f the 1.0 pg/raL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 raL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. The stock sad fortification solutions are to be stored in a refrigerator at approximately 4"C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/mL fortification solution. P**e 4 ul Page 48 o f 65 Exygen Research Page 83 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P r o to c o l N u m b e r: P 0 0 0 1 131 Exygen JUteaich Method Number V0001784 ATSLYIICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctsnoic Acid (PFOA) in Vegetation by LC/MS/MS 9.2.2 The following ic a typical example: additional concentrations may be prepared as needed. Concentration Fine! o f Fortification Volume Diluted to Concentration Solution futfm U (nO.) (mL) (llfalL) 1.0 5.0 100 0.05 1.0 2.5 100 0.025 1.0 1.0 100 0.0! 0.05 0.025 10 10 100 100 0.005 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles st 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 o r less) must include at least one untreated control end two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 3 0m L o f acetonitrile and shake on a wrist action shaker f o r- I S minuies. 11.3 Centrifuge the SO mL polypropylene tubes containing sample at -2000 rpm fo r-1 0 minutes. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped fiasks. 11.5 Pack the 20 mL SPE tubes in sequence with 2 g florisil. 2 g silica gel. 2 g carbon, and 1 g LC-NHj. Condition the columns with 20 m L o f methanol, then 20 mL o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 12$ mL pear-shaped flasks by muting with the 30% dimetbyldichloroslUne in toluene solution. Rinse the flask with toluene once, followed b y methanol (three tim et). D ry the flasks completely before use. either by sir-drying or with a stream o f nitrogen. 11.7 Decant the extract mi to a conditioned SPE column fitted inside the mouth o f the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape flask. Page 5 o f Page 49 o f 65 Exygen Research Page 84 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VQO0I7S4 A N A LYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctaaoic A d d (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 m L o f acetonitrile to the sample in the 50 m L centrifuge tube. 11.9 Shake die sample again for 10 minutes on a wrist-action shaker. 11.10 Centrifttge the 50 mL polypropylene tubes containing sample at 2000 rpm for 5 minutes. 11.11 Decant the extract onto the same SPE column. Collect the eluate into the same pear-shaped flask and combine with the eluent from the initial extraction. 11.12 R e p e a ts te p s ll.8 d tro u g h U .il again. 11.13 Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.14 M ain the final volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and tw irl to mix/diasolve. 11.15 Transfer the extracts to HPLC vials using disposable pipets. 11.16 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 122 Standards o f PFOA corresponding to st least five or m ore concentration levels must be included in an analytical set. 12.3 An entire eet o f extracted calibration standards must be included at the beginning and st the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards), b either case, extracted calibration standards must be the first and last injection in t sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using I/x weighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should b e ftirther diluted and reanalyzed. 13.0 Acceptance C riteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. Page 6 of 7 P a g e SO o f 65 Exygen Research Page 85 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exyjw lU itnh Method Number V000I7S4 a n a ly t ic a l m e t h o d Method o f Analysis for the Detennination ofPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 13.2 Method blank* must not contain PFOA at levels greater than the LOQ. If a blank contain* PFOA at levels greater than 0.5 ppb, then a new blank sample must be obtained s k i the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes m ust be between 70-130/ of their known values. I f a control spike fall* outside the acceptable limits, the entire set o f samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (RJ 0.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or die relevant set o f sample* abould be reanalyzed. 13.6 Retention time* between standards and samples must not drift more than i 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical ran then foe set must be reanalyzed. 14.0 Calculations 14.1 Use foe following equation to calculate the amount o f PFOA found (in ng/mL, baaed on peak area) using foe standard curve (linear regression parameters) generated by foe Mass Lynx software program: PFOA found (ng/mL) - ( P d ( t f t l - ifllg tro l) slope 14.2 Use foe following equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found loob) - (PFOA found In g /m D x final volume (mL) x DF1 sample weight (g) DF " factor by which foe final volume was diluted, if necessary. 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use foe following equation to calculate foe percent recovery. Recovery (%) [ total analyte found (ng/g) - analyte found in control (ng/g)] ^ ^ analyte added (ng/g) Page 7 oi 7 P a g e 51 o f 65 Exygen Research Page 86 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000! 131 ANALYTICAL METHOD Method N um ber V0001785 M ethod o f Analysis fo r th e Determ ination o f Perflnorooctanolc A d d (PFOA) in Small M amm al Liver by LG/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C O L ,____ Paul Connolly l Technical Leader, LC-MS, Exygen Research Date John Flaherty ' Vice Presiiddeenntt, Operations, Exygen Research Date Exygen Research Total Pages: 7 Page 52 o f 65 Page 87 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygM R m u c h Method Number VQ0017SJ I ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 1.0 Scope This method U to be employed for die isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in small mammal liver. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 5 g o f test sample for extraction. 3.2 Samples should he processed before extraction. Place the frozen sample in a food processo r and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. Alternately, if there is an insufficient amount o f sample (~less than 5 g), then no processing is necessary and the sample can be used as supplied. 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4 .0 Reagents and Standards 4.1 Water -H P L C 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 $.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*20<J pL connected to a tandem Maas Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f revting to 0.00001 g. 5.4 50 m L disposable polypropylene centrifrige tubes. 5.5 15 m L disposable polypropylene centriftige tubes. 5.6 D isposable m icropipets (50-100uL, 100-200uL). 5.7 125-mL LDPE narrow*mouth bottles. S.S 2 m L clear HPLC vial k it Page 2 o f ' Page 53 o f 65 Exygen Research Page 88 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygca Research Mctbod Number VOOCI78S I ANALYTICAL M ETHOD Method o f Analysis for the Determination ofPerfluorooctinoic Acid (PFOA) in Smell Mammal U v er by LC/MS/MS 5.9 Disposable pipettes. 5.10 Autopipettes (100*1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tC l 8 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 TiMuemizcr. 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 15 m L 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: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 m M Ammonium Acetate in W ater 6.4 Mobile Phase (B ): Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate Z M Im L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak A rea-ex tern al standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended a t a guide and m ay b e changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM m ode, monitoring 413 -* 369 m/z for PFOA. The above conditions ere intended as a guide and m ay be changed in order to optimize the MSMS system. - Pag3 of? P age 54 o f 65 Exygen Research Page 89 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygm Raaearch Method Number VOOOPgj I ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfhtorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 8.0 Preparation o f Solutions 8.1 Mobile Pbaae 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0 .154 g of ammonium acetate to 1000 mL o f water. Alternate volume* may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f - 1 0 0 pgftnL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/mL fortification solution o f PFOA ia prepared by bringing I m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 p g ta L fortification solution o f PFOA is prepared by bringing 10 m L o fth e t.O pg'm L solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to b e stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 922 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentration Final o f Fortification Volume Diluted to Concentration Solution fne/mL) (mL) (m L ) (ne/mL) 100 5.0 100 5.0 100 1.0 100 2.0 100 1.0 100 1.0 5X1 to 100 OS 2.0 10 100 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. Page 4 of 7 Page 55 o f 65 Exygen Research Page 90 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyjen Rcaearch M ethod Number VOOOI785 [ A N A LY TIC A L M E T H O D Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Smell Mammal Liver by LC/MS/MS 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r lees) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.) Weigh 1 g o f aample into a 50 m L polypropylene centrifuge tubes (fortify u needed, replace lid and mix well). Note that alternate weights o f liver may be measured depending on the sample size available for use. 11.2 Add water to the sample for a final volume o f 10 mL. 11.3 Homogenize sample using a tissuemizer for ~1 minute. 11.4 Transfer 1 mL o f the sample using a disposable pipette into a 15 mL disposable centriftige tube. 11.5 Add 5 mL o f acetonitrile and shake for - 2 0 minutes on a wrist-action shaker. 11.6 Centrifuge foe tubes at -3 0 0 0 rpm for - 5 minutes. 11.7 Decant the supernatant into a SO m L disposable centrifuge tube and add 35 mL o f water. 11.8 Condition foe Cia SPE cartridge* (1 g, 6 mL) by passing 10 mL methanol followed by 5 m L o f HPLC water (~ 2 drqp/soc). Do not let column run dry 11.9 Load the sample on conditioned C n SPE cartridge. Discard eluate. 11.10 Elute with - 2 mL o f m ethanol Collect 2 m L o f eluate into a graduated 15 mL polypropylene centriftige tube (final volume 2 mL). 11.11 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, a m p le and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels must be included in in analytical s e t 12.3 An entire set o f calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5-iO samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every $-10 samples (to account for a second set o f standards). In either case, calibration standards must be foe first and last injection in a sample se t 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for foe analyte by linear regression using 1/x weighting o f peak area Psge 5of? Page 56 o f 65 Exygen Research Page 91 of 100 Interim Report #2-Analysis o f Surface W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI7IS I ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.$ Sample response should not exceed standard responses. Any samples that exceed standard responses should be Amber diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 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 new blank sample must be obtained and the entire set must be re-extracted. Recoveries o f control spikes and matrix spikes m ust be between 70-130% of their known values. I f a control spike tails outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. The correlation coefficient (R) for calibration curves generated must be 20.992 (R1 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. Retention tiroes between standards and samples must not drift more than 4 % within an analytical run. I f retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program: PFOA found (ng/mL) - (Peak area - intercept! x D F x aliquot factor slope DF factor by which the final volume was diluted, i f necessary. Aliquot factor " 10 Page 6 o f7 Page 57 o f 65 Exygen Research Page 92 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOI131 BxygenResweb Method Number VOOOPIS I A N A 1.Y 1K .A L METHOD Method o f Analyaia for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 14.2 For u m p le s fortified with known amount o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (Vo) - [ total analyte found (ng/mL) - analyte found in control (ng/mL)] analyte added (ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL lu ng/g(ppb). PFOA found fppbl - fPFQA found fna/m D x final volume fm U l sample weight (g) Exygen Research Page 7 of? P age 58 o f 65 Page 93 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Num ber V0001786 Method o f Analysis for the Determination o f Perfluorooctaoofc A d d (PFOA) ia Smalt Mammal Serum by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: "v~*--V_C-- ___ Paul Connolly I Technical Leader, LC-MS, Exygen Research a /#>4 ^ / John Flaherty / Vice Presideianit, Operations, Exygen Research __iain i.? '! Date /' J r Due Exygen Research TottI Pages: 7 Page 59 o f 65 Page 94 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V00017$6 A N A LYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in small mammal serum. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 1 m L o f test sample for extraction. 3.2 No sample processing is needed for serum samples. However, frozen serum samples must to allowed to completely thaw to room temperature before use. 3J Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - 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 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume iqjector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS) 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centriftige tubes. 5.5 15 mL disposable polypropylene centriftige tubes. 5.6 Disposable micropipets (50-100uL, 100*200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial ldt. 5.9 Disposable pipettes. 5.10 Autopipettes (100*1000 |tL and 10*100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 c c (lg )tC 1 8 S P E cartridges. 5.12 SPE vacuum manifold. 5.13 Vortcxer. Page 2 o f f P age 60 o f 65 Exygen Research Page 95 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygea ReaMfcb Method Number VOOO178b I ............................A N A L Y T IC A L M E T H O D Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC^MS/MS 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 2L& 65 65 25 25 65 Flow Rate 5kB (mL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Infection Volume: 15 jiL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: ~ 23 minutes. The above conditions are intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - * 369 m/z for PFOA. The above conditions are intended as e guide and m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. Page 3 of 7 Page 6 / o f 65 Exygen Research Page 96 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POCO1131 ExygmRMMKk Method Number V000I7S6 I ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctinoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 m l unit methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing I raL o f the 100 pg/raL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o fPFO A is prepared by bringing 10 m L o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentriti! Final ofFoitification Volume Diluted Concentration Soluti! fns/mU (raL) (mL) (ni/mL) 100 5.0 100 100 2.0 too 100 1.0 100 5.0 2.0 1.0 5.0 10 100 2.0 10 100 1.0 10 100 0.5 0.2 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C 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 o r less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. Page 4 o f7 P age 62 o f 65 Exygen Research Page 97 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Nuniber: POOO1131 Exygn Rmarcfc Method Number V000l78e I ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal S o w n by LC/MS/MS 11.0 Sample Extraction 11.1 Measure 1 mL o f sample into a 50 mL polypropylene centrifuge tubes (fortify as needed , replace lid and mix well). Note that alternate volumes o f serum may b e measured depending on the sample size available for use. 11.2 Add water to the sample for a final volume o f 20 m L Cap tightly 11.3 Vortex f o r - 1 minute. 11.4 Transfer 1 m L o f the sample using a disposable pipette into a IS mL disposable centrifUge tube. 11.5 Add S m L o f acetonitrile and shake for -2 0 m inutes on a wrist-action shaker. 11.6 CentrifUge the tubes at -3 0 0 0 ipm for - 5 minutes. 11.7 Decant the supernatant into a 50 m L disposable centrifUge tube and add 35 m L o f water. 11.8 Condition the C u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL ofH PLC water (~ 2 drop/sec). D o not let column run dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eluate. 11.10 Elute with - 2 mL o f methanol. Collect 2 m L o f eluate into a graduated 1S mL polypropylene centrifUge tube (final volume - 2 mL). 11.11 Analyze samples using electroepray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to a t least five o r m ore concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5 -1u samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in a sample set 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using I/x weighting o f peak area versus calibration standard concentration using M assLynx 3 3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. PageJ of 7 Page 63 o f 65 Exygen Research Page 98 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOU31 Exygen Reaetreh______________________ Method Number VOOOI7&6 | ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Seram by LC/MS/MS 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks m ust not contain PFOA at levels greater than the LOQ. If a blank contains PFOA st levels greater than 10 ng/mL. then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes m ust be between 70-130% o f their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% foould be evalusted by the analyst to determine i f 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 foe total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.3 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 m utt be taken to adjust instrument operation, and the standards o r the relevant set o f samples should b e reanalyzed. 13.6 Retention times between standards and samples must not drift more than 1 4 % within an analytics] run. I f retention tim e drift exceeds this limit within an analytics) ran then the set must be reanalyzed. 14.0 Calculations 14.1 Use foe following equation to calculate the amount o f PFOA found (in ng/mL. based on peak ares) using the standard curve (linear regression parameters) generated by foe Mass Lynx software program: PFOA found (ng/m L) (Peak area - intercept) x DF x aliquot factor slope DF factor by which foe final volume w as diluted, if necessary. Aliquot factor 20 14.2 Foe samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) * [ total analyte found (ng/mL) - analyte found in control (ng/mL)] analyte added (ng/mL) Page 6 o f7 Page 64 o f 65 Exygen Research Page 99 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exyjaa Raearch Method Number VOOO1786 I AWa L yTICAI. METHOD Method o f A n ily iii for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Seram by LC/MS/MS 14.3 U ie the following equation to convert the amount o f PFOA found in ng/mL to ppb. PFOA found (ppb) - fPFOA found (na/mlA x final volume fm L sample volume (mL) Exygen Research Page 7 ul ? Page 65 o f 65 Page 100 of 100