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INTERIM REPORT # 23 - Analysis of Sediment 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 May 2, 2006 PERFORMING LABORATORY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: 651-733-6374 PROJECT Protocol Number: P0001131 Exygen Study Number: P0001131 Total Pages: 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No. : P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number P0001131, entitled "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program," conducted for 3M Company, is being performed in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by Exygen Research, with one exception. The laboratory control and control spikes for the direct injection method were not recorded promptly at the time of extraction. Exygen Research Jaisimha . Study Director Weston Solutions, Inc. Exygen Research Date Date Page 2 o f 110 Interim Report #23 - Analysis o f Sediment 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 25. Final Raw Data Review and Interim Analytical Report Review 05/02/06 05/02/06 05/02/06 05/02/06 rtlG ^-loC Date 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion of the study. This QA statement involves only the review of the interim report and associated raw data. Exygen Research Page 3 o f 110 Interim Report #23 - Analysis o f Sediment 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: Ricnard A. Ga^zym President Exygen Resea &cirk/pi Study Director, W eston Solutions, Inc Jaisimha Kesfe'P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: Michael A Santoro Director of Regulatory Affairs | Exygen Research Date Date Page 4 o f 110 Interim Report #23 - Analysis o f Sediment 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: Sediment 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: 04/27/06 Interim Analytical Termination Date: 04/30/06 Interim Report Completion Date: 05/02/06 Exygen Research Page 5 o f 110 Interim Report #23 - Analysis o f Sediment 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 Christine Edwards Mark Ammerman Amy Sheehan Brittany Kravets Scott Crain Frances Crespi Devon Ingram Christa Gallant Title Vice President Scientist Technical Lead-LC/MS Technician Sample Custodian Associate Scientist Technician Technician Technician Technician Technician Exygen Research Page 6 o f 110 Interim Report #23 - Analysis o f Sediment 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 For Sediment......................................................................... 13 6.2 Direct Inject Extraction Procedure For Sediment..................................................... 14 6.3 Percent Solids Procedure For Sediment................................................................... 14 6.4 Preparation of Standards and Fortification Solutions............................................... 14 6.5 Chromatography.......................................................................................................15 6.6 Instrument Sensitivity...............................................................................................15 6.7 Description of LC/MS/MS Instrument and Operating Conditions...........................15 6.8 Quantitation and Example Calculations................................................................... 16 7.0 EXPERIMENTAL DESIGN......................................................................................20 8.0 RESULTS...................................................................................................................20 9.0 CONCLUSIONS......................................................................................................20 10.0 RETENTION OF DATA AND SAMPLES.............................................................21 Exygen Research Page 7 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study N o.: P0001131 Table I. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Sediment Samples............................23 Table II. Summary of PFOS in Direct Injection Sediment Samples..............................24 Table HI. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sediment Samples..... 25 Table IV. Matrix Spike Recovery of PFOS in Direct Injection Sediment Samples.......26 Table V. Total Percent Solids in Sediment Samples......................................................27 Exygen Research Page 8 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No. : P0001131 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFBS in Methanol......................................... 29 Figure 2. Non-Extracted Standards of PFBS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively................................................................................30 Figure 3. PFBS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively........................................ 31 Figure 4. Chromatogram Representing a Sediment Sample Analyzed for PFBS (Exygen ID: C0172892, Data Set: 042606F)................................................. 32 Figure 5. Typical Calibration Curve for PFHS in Methanol......................................... 33 Figure 6. Non-Extracted Standards of PFHS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively.................................................................................34 Figure 7. PFHS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively........................................ 35 Figure 8. Chromatogram Representing a Sediment Sample Analyzed for PFHS (Exygen ID: C0172892, Data Set: 042606F)................................................. 36 Figure 9. Typical Calibration Curve for PFOS in Methanol......................................... 37 Figure 10. Non-Extracted Standards of PFOS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively.................................................................................38 Figure 11. PFOS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively.........................................39 Figure 12. Chromatogram Representing a Sediment Sample Analyzed for PFOS (Exygen ID: C 0172892, Data S et:0 4 2 8 0 6 B )............................................................40 Exygen Research Page 9 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 LIST OF APPENDICES Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method V0001782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS", and Protocol Amendments............................. Page .41 Exygen Research Page 10 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 1.0 SUMMARY Exygen Research extracted and analyzed sediment samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001782 (Appendix A). The limit of quantitation for PFBS and PFHS in sediment was 0.2 ng/g. The limit of quantitation for PFOS in sediment was 2.0 ng/g. The standard sediment method (V0001782) was originally used to extract all of the samples. Use of this method yielded poor recoveries for PFOS. Because of this, a direct injection method (Section 6.2) was used to analyze for PFOS. Analytical results for the analysis of PFBS, PFHS and PFOS found in sediment samples extracted with the original method are summarized in Table I. Analytical results for the analysis of PFOS found in sediment samples by direct injection are summarized in Table II. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in sediment samples were 85 19%, 99 29%, and 93 6%, respectively. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in sediment samples extracted with the original method are summarized in Table III. Fortification recoveries for the analysis of PFOS in sediment samples by direct injection are summarized in Table IV. The total percent solids for sediment samples are detailed in Table V. 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 sediment 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 sediment using the analytical methods entitled, "V0001782: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment 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 April 27, 2006 and the analytical termination date for this interim report was April 30,2006. Exygen Research Page 11 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 4.0 ANALYTICAL TEST SAMPLES Three sediment samples (Exygen ID: CO172892-CO172894) were received on wet ice on April 22, 2006 from Tim Frinak 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 is located in the raw data package associated with this interim report. Storage records will be kept at Exygen Research. 5.0 REFERENCE MATERIAL The analytical standards, PFBS and PFHS, were supplied by 3M. PFBS was received from 3M at Exygen on May, 13, 2005. 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. SP0005726 SP0002401 SP0002694 Lot # 101 SE036 430180-1 Purity (%) 96.7 98.6 101.2 Expiration Date 12/04/06 10/18/06 10/31/07 The molecular structures of PFBS, PFHS and PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt ^FgSOsTC*) Transitions Monitored: 299 --> 99 Structure: FF FF FF FF 3 PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFnSOsTC*) Transitions Monitored: 399 -> 80 Exygen Research Page 12 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Structure: F FFF FFF 3 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CsFnSOa'K'1') Transitions Monitored: 499 - 80 Structure: FFFF FFFF F SO3 FFFF FFFF 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical method "V0001782: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" was used for the analysis of PFBS and PFHS in sediment samples. A direct injection method, detailed in Section 6.2, was used for the analysis of PFOS in sediment samples. 6.1. Extraction Procedure For Sediment Before the samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A 5 gram portion of sediment was weighed into a fifty milliliter centrifuge tube for the extraction. After fortification of appropriate samples, 35 mL of 1% acetic acid in water was added to the samples. The samples were vortexed and allowed to shake on a wrist action shaker for ~60 minutes. The samples were centrifuged for ~20 minutes at ~3000 rpm. The supernatant was then loaded onto a Cis SPE cartridge conditioned with 10 mL of methanol and 20 mL of water. The eluate was discarded. Twenty milliliters of methanol was added to the sediment samples left in the centrifuge tube. The samples were vortexed and allowed to shake on a wrist action shaker for another 30 minutes. The samples were centrifuged again for ~20 minutes at ~3000 rpm. The supernatant was then loaded onto the same Cis SPE cartridge. The eluate was collected into a 500 mL Nalgene Bottle. The column was washed with 4 mL of methanol. The wash was collected in the same bottle as the eluate. Approximately two hundred milliliters of water was added to the bottles. The samples were mixed by shaking and loaded onto another Ci8 SPE cartridge conditioned with 10 mL of methanol Exygen Research Page 13 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 and 20 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 6.2 Direct Inject Extraction Procedure For Sediment Before the samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A one-gram portion of sediment was weighed into a 15-milliliter centrifuge tube for the extraction. Ten milliliters of 1% acetic acid in methanol was added to each sample. The samples were then shaken by hand, vortexed, and sonicated for thirty minutes. The samples were then centrifuged for ~10 minutes at ~3000 rpm. Each sample was analyzed by LC/MS/MS electrospray. 6.3 Percent Solids Procedure For Sediment Percent solids were determined using the procedure indicated in Exygen method V0000427. Approximately 20 grams of sample was weighed into a pan. The weight of the sample plus the pan was recorded. The samples were then dried in an oven overnight at 104 2 C. Then the samples were transferred to a dessicator and allowed to cool for ~15 minutes. Each sample was then weighed again, including the weight of the pan. The percent solid for each sample was then calculated. See Table V for percent solids results. 6.4 Preparation of Standards and Fortification Solutions A mixed stock standard solution of PFBS, PFHS, and PFOS was prepared at a concentration of 1000 pg/mL by dissolving 100 mg of each of the standards (corrected for purity and salt content) in 100 mL of methanol. From this solution, the following fortification standards were prepared: Cone, of Fort Stock or Fort. Volume Solution (mL) (pg/mL)1 1000 10 100 10 10 10 1.0 10 0.1 10 1of PFBS, PFHS, and PFOS Final Volume (mL) 100 100 100 100 100 Final Cone, of Fortification Std. (pg/mL) 100 10 1.0 0.1 0.01 Exygen Research Page 14 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 A set of non-extracted calibration standards containing PFBS, PFHS, and PFOS was prepared in methanol, as specified in Exygen method V0001782. The following concentrations were prepared: Cone, of Fort Fort or Calibration Volume Solution (mL) (ng/mL)1 100 1.0 100 0.5 100 0.2 10 1.0 5.0 1.0 2.0 1.0 1of PFBS, PFHS, and PFOS Final Volume (mL) 10 10 10 10 10 10 Final Cone, of Calibration Std. (ng/mL) 10 5.0 2.0 1.0 0.5 0.2 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.5 Chromatography Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~0.56mins, ~9.4 mins, and -11.4 mins, respectively. Peaks above the LOD were not detected in any of the reagent blank samples corresponding to the analyte retention time. 6.6 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 0.2 ng/mL of PFBS, PFHS and PFOS. 6.7 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 Exygen Research Page 15 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: ~30 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time (mini 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: ~18 min Flow Rate: 0.3 mL/min %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Ions monitored: Analvte Mode PFBS PFHS PFOS negative negative negative 6.8 Quantitation and Example Calculations Transition Monitored 299 -99 399 -> 80 499 80 Approximate Retention Time (miri) -0.56 min. -9.4 min. -11.4 min. Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations of standards. The concentration was determined from the following equations. Equation 1 calculated the amount of analyte found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Please note that the equations in this section are presented in a different order than the equations in the method (V0001782), but this has no effect on the final calculations. Exygen Research Page 16 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Equation 1: Analyte found (ng/mL) = (Peak area - intercept) x DF Slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. For samples fortified with known amounts of PFBS and PFHS prior to extraction, Equation 2 was used to convert the amount of PFBS and PFHS found in ng/mL to ng/g (ppb). Equation 2: Analyte found (ppb) = fAnalyte found (ng/mL) x final volume (5 mDl sample weight (5 g) Equation 3 was used to calculate the percent recovery. Equation 3: Recovery (%) = (analyte found (ppb) - analyte in control (ppb)) xl00% amount added (ppb) Equation 4 was then used to calculate the amount of PFBS and PFHS found in ppb based on dry weight. Equation 4: Analyte found (ppb) dry weight = Analyte found (ppb) x [100% / total solids(%)] An example of a calculation using an actual sample follows (calculation is for PFBS only): Sediment sample Exygen ID: C0172893 Spk F (Set: 042606F), fortified at 4 ng/mL with PFBS where: peak area = 253819 intercept = -592 slope = 42300 dilution factor =1 ng/g PFBS added (fort level) = 4 amt in corresponding sample (ng/g) = 2.41 final volume (mL) =5 sample weight (g) =5 total solids (%) = 54.37 Exygen Research Page 17 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 From equation 1: Analyte found (ng/mL) = f253819 - (-592)1 x 1 42300 = 6.01 ng/mL From equation 2: Analyte found (ppb) = (6.01 ng/mL x 5 mD 5g = 6.01 ppb From equation 3: % Recovery = (6.01 ppb - 2.41 nob) x 100% 4 ppb = 90% From equation 4: Analyte found (ppb) dry weight = 6.01 ppb x (100% / 54.37%) = 11.1 ppb NOTE: This value may be slightly different than that of the raw data due to rounding. A slightly different set of calculations was used for the direct injection samples analyzed for PFOS. Equation 1: Analyte found (ng/mL) = (Peak area - intercept) x DF Slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. For samples fortified with known amounts of PFOS prior to extraction, Equation 2 was used to convert the amount of PFOS found in ng/mL to ng/g (ppb). Equation 2: Analyte found (ppb) = [Analyte found (ng/mLl x volume extracted (10 mL)l sample weight (1 g) Equation 3 was used to calculate the percent recovery. Exygen Research Page 18 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Equation 3: Recovery (%) = (analyte found (ng/g) - analyte in control (ng/g)) xl00% amount added (ng/g) Equation 4 was then used to calculate the amount of PFOS found in ppb based on dry weight. Equation 4: Analyte found (ppb) dry weight = Analyte found (ppb) x [100% / total solids(%)] An example of a calculation using an actual sample follows: Sediment sample Exygen ID: CO172892 Spk D (Set: 042806B), fortified at 400 ng/mL with PFOS where: peak area = 152371 intercept slope = 3720 = 22700 dilution factor ng/g PFOS added (fort level) amt in corresponding sample (ng/g) volume extracted (mL) = = = = 100 4000 2360 10 sample weight (g) =1 total solids (%) = 66.54 From equation 1: Analyte found (ng/L) = f!52371 - 37201 x 100 22700 = 654 ng/mL From equation 2: Analyte found (ppb) = (654 ng/mL x 10 mL) 1g = 6540 ppb From equation 3: % Recovery = (6540 ng/g - 2360 ng/g) x 100% 4000 ng/g = 105% From equation 4: Analyte found (ppb) dry weight = 6540 ppb x (100% / 66.54%) = 9830 ppb Exygen Research Page 19 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 NOTE: This value may be slightly different than that of the raw data due to rounding. 7.0 EXPERIMENTAL DESIGN For sediment samples designated as laboratory matrix spikes, PFBS, PFHS, and PFOS were added at a known concentration to the samples in the laboratory after the samples were weighed for extraction. The sediment samples were extracted in one set. The set included one reagent blank and two reagent blanks fortified at known concentrations. The set contained three sediment samples. For each sample, two laboratory matrix spikes and a duplicate were extracted. Due to poor recoveries, the three sediment samples were extracted and analyzed using a direct injection method for PFOS. This method is detailed in Section 6.2. 8.0 RESULTS Analytical results for the analysis of PFBS, PFHS and PFOS found in sediment samples extracted with the original method are summarized in Table I. Analytical results for the analysis of PFOS found in sediment samples by direct injection are summarized in Table II. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in sediment samples were 85 19%, 99 29%, and 93 6%, respectively. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in sediment samples extracted with the original method are summarized in Table III. Fortification recoveries for the analysis of PFOS in sediment samples by direct injection are summarized in Table IV. The total percent solids for sediment samples are detailed in Table V. 9.0 CONCLUSIONS The sediment samples were successfully extracted and analyzed for PFBS and PFHS according to analytical method V0001782. The sediment samples were successfully extracted and analyzed for PFOS using an alternative method, which yielded acceptable QC data. Exygen Research Page 20 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 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 study director. This does not include facility-specific raw data such as instrument or temperature logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. Exygen Research Page 21 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 22 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Sediment Samples Exyqen ID C0172892 C0172892 Rep C0172893 C0172893 Rep C0172894 C0172894 Rep Client Sam ple ID DAL-SD -B PP01 -0-0000 DA L-SD -B PP01-0-0000* D A L-SD -B PP02-0-0000 D A L-SD -B PP02-0-0000* D A L-SD -B PP03-0-0000 D A L-SD -B PP03-0-0000* C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Psrfluorobutanssulfonate______ Perfluorohsxanesulfonate______ Perfluorooctaneautfonate Analyte Found (PPb, nfl/g) Dry Welqht Assessed Accuracy (+/- %) Analyte Found (PPb, nfl/fl) Dry Weight Assessed Accuracy (+/- %) Analyte Found (PPb. ng/g) Dry Weight Assessed Accuracy (+/-% ) 3.14 3.10 4.43 4.84 9.87 8.73 40 40 30 30 30 30 84.3 86.1 74.5 64.7 139 123 30 30 30 30 50 50 NR NR NR NR NR NR NR NR NR NR NR NR ` Laboratory Duplicate NR = Not reported due to quality control result failures. See Table II for re-analysis results. Exygen Research Page 23 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Table II. Summary of PFOS in Direct Injection Sediment Samples Exygen ID CO172892 C0172892 Rep C0172893 C0172893 Rep C0172894 C0172894 Rep Client Sample ID DAL-SD-BPP01-0-0000 DAL-SD-BPP01-0-0000* DAL-SD-BPP02-0-0000 DAL-SD-BPP02-0-0000* DAL-SD-BPP03-0-0000 DAL-SD-BPP03-0-0000* ` Laboratory Duplicate C8 Sulfonate PFOS Perfluorooctanesulfonate Analyte Found (ppb, ng/g) Dry Weight Assessed Accuracy (+/-% ) 3550 3520 30 30 4160 3840 30 30 11900 12000 30 30 Exygen Research Page 24 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sediment Samples Sam ple D escription C ^ ilfo n a t^ F B S W e tW e lg h t<wi<^ BBJ 5 ^ u jio n a ^ ^ F H 8 W e tW ^ Am ount Am t Found Am ount Am t Found Am ount Am t Found Spiked In Sample Recovered Recovery in Sample Recovered Recovery in Sample (note) (ng/g) wet wt. (no/g)w etw t. (% i (ng/g) wet wt. (ng/g) w et wt. (% ) (ng/g) wet wt. Am ount Recovered Recovery (%> DAL-SD-BPP01-0-0000 (C0172892 Spk C, 4 ppb Spike) DAL-SD-BPP01-0-0000 (C0172892 Spk 0,400 ppb Spike) DAL-SD-BPP01-0-0000 (C0172892 Spk E, 4000 ppb Spike) 4 400 4000 2.09 2.09 2.09 4.47 60 262 65 56.1 2670 67 56.1 454 5190 99 128 . NR .. NR NR DAL-SD-BPP02-0-0000 (C0172893 Spk F, 4 ppb Spike) DAL-SD-BPP02-0-0000 (C0172893 Spk 6 ,400 ppb Spike) DAL-SD-BPP02-0-0000 (C0172993 Spk H, 4000 ppb Spike) 4 400 4000 2.41 2.41 2.41 6.02 90 374 93 40.5 3920 98 40.5 . 345 76 - 5090 126 NR .. .. NR NR DAL-SD-BPP03-0-0000 (C0172894 Spk 1,4 ppb Spike) DAL-SD-BPP03-0-0000 (C0172894 Spk J. 400 ppb Spike) DAL-SD-BPP03-0-0000 (C0172894 Spk K, 4000 ppb Spike) 4 400 4000 4.39 4.39 4.39 7.71 83 359 89 61.8 4840 121 61.6 283 4500 . 55 111 . NR .. .NR NR Average: Standard Deviation: 85 19 Average: Standard Deviation: 98 29 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND * Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). NR * Not reported due to quality control result failures. See Table IV for re-analysis results. Note: Since this summary table show s rounded results, recovery values may vary slig h tly from the values in the raw data. Average: Standard Deviation: NR NR Exygen Research Page 25 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFOS in Direct Injection Sediment Samples Sample Description DAL-SD-BPP01-0-0000 (C0172892 Spk D, 4000 ppb Spike) DAL-SD-BPP01-0-0000 (C01728S2 Spk E, 40000 ppb Spike) DAL-SD-BPP02-0-0000 (C0172893 Spk G, 4000 ppb Spike) DAL-SD-BPP02-0-0000 (C0172893 Spk H, 40000 ppb Spike) DAL-SD-BPP03-0-0000 (C0172894 Spk J, 4000 ppb Spike) DAL-SD-BPP03-0-0000 (C0172894 Spk K, 40000 ppb Spike) Amount Spiked (PPb) 4000 40000 4000 40000 4000 40000 C8 Sulfonate PFOS Wet Weight Amt Found Amount in Sample (ng/g) wet wt. Recovered (ng/g) wet wt. Recovery (%) 2360 2360 6540 40000 105 94 2260 2260 5950 38000 92 89 5280 5280 8770 41200 87 90 Average: Standard Deviation: 93 6 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitation (LO Q) of 0.2 ng/g (wet weight). Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 26 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Table V. Total Percent Solids in Sediment Samples Exygen ID C0172892 C0172893 C0172894 Sample Description DAL-SD-BPP01-0-0000 DAL-SD-BPP02-0-0000 DAL-SD-BPP03-0-0000 Total Solids (%) 66.54 54.37 44.46 Exygen Research Page 27 o f 110 Interim Report #23 - Analysis of Sediment Samples Exygen Study N o.: P0001131 FIGURES Exygen Research Page 28 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Figure 1. Typical Calibration Curve for PFBS in Methanol 0 4 2 6 0 6 F S td lm tn tr d b (P F B S ):"U n t i l" R t g i t s io n ('1 / x" w t !ghti ng): y 4 .2 3 1 + 0 0 4 x + - 8 0 2 ( r 0 .0 0 0 0 ) Area, counts Exygen Research Page 29 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Figure 2. Non-Extracted Standards of PFBS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively Intensity, cps 1 5500*4506 - PFBS (S tandard) 299.0/99.0 amu -sam pto 1 o f 45 from 0426QSF.wiff A rea: 8*67 co u n t* H aight: SL05+002cp# RT: 0.554m in 0.55 500 400- 300 200- 100* 0 10 Time, min SS0014595 PFBS (Standard) 200.0/90.0 amu sample 2 of 45 from 042500F.iwiff Area: 20091 counts Height: 1.40e+003 cps RT: 0.550 min 13.14 Intensity, cps Exygen Research Page 30 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Figure 3. PFBS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively Control - PFBS (Unknown) 29910/9910mir nnmpfo 0 o f 45 from $4260F.wiff p tn k not found) 13.03 Tima, min Reagent Spk A * PFBS (QC) 200.0/00.0 im u - sample 0 of 45 from 042606F.wiff Area: 15076 counts Haight: 1.08e+003 ops RT: 0.557 min 0.50 Tima, min I Reagent Spk B - PFBS (00)200.0/00.0 im u - sample 10 of 46 from 042600F.wiff Area: 108227 oounts Height: 1.11e+004 ops RT: 0.557 min 0.56 Exygen Research Page 31 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Sediment Sample Analyzed for PFBS (Exygen ID: C0172892, Data Set: 042606F) I COf72892 PFBS (UHAhowm) 2 H L H 9 1 0 M I ta m p /* 20 o f 45 from 042606F.wiff A n a : 87668 count* Heigkt: Z90*+003cp* RT: 0.570 m ia 0.57 Intensity, cps 12 3 n* 'fT~li 8 9 10 11 12 13 1 4 18 10 17 Time, min Exygen Research Page 32 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study N o.: P0001131 Figure 5. Typical Calibration Curve for PFHS in Methanol 0 4 2 0 0 8 F Sedim tnt.rdb (PFHS): ` U n t j p Rtgitscion f 1 /x " wighting): y * 7.06 + 0 0 4 x + 1.1 *+ 0 0 3 (r> 0.0 0 0 5 ) A rea, counts Exygen Research Page 33 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Figure 6. Non-Extracted Standards of PFHS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively 1 550074596 - PFHS (Staadard) 39&0/8A0 amu -sam pto 1 o f 45 from 042606P.w/ff A n a : 12774count* H a ig kt: 7 J5 4 0 2 c p f RT: 9.40 m ilt 9.40 Intensity, cps T im , min S S 0 0 1 4 5 9 5 PFHS (Standard) 3 9 9 .0 /8 0 .0 amu sample 2 of 4 0 from 042000F.w iff Ara: 3 3 9 8 3 counts Htight: 1 .73+ 003 cps RT: 9 .4 0 min 9.40 Intensity, cps Exygen Research Page 34 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Figure 7. PFHS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively Ragnt C o n tro l-P F H S (U nknow n)39%0/80.Oamu -ta m p ! 9 o f 45 fro n t 0426t6F.w iff fro n t n o t fo u nd ) 0.40 Exygen Research Page 35 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Sediment Sample Analyzed for PFHS (Exygen ID: C0172892, Data Set: 042606F) Intensity, cps COT72892 PFHS (UhAhowm) 399L0/80L0amu -ta m p ia f8 o f 45 from $42M F.w iff A rea: 39539 count* H aight: 2 23+003 cp RT: 9.41 m in 2200 i 0.41 2000- 1800- 1000- 1400- 1200 1000- 800- 000- 400 2000 .4 2 10 10 11 12 13 1 4 15 10 17 Time, min Exygen Research Page 36 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Figure 9. Typical Calibration Curve for PFOS in Methanol 0 4 2 8 0 0 B S *d . Dl.idb (PFO S): " L in t j i " R tg ru sio n /x " wnighting): y - 2 .2 7 * + 0 0 4 x + 3 .7 2 t+ 0 0 3 ( l - 0 .8 8 4 0 ) A rea, counts Exygen Research Page 37 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Figure 10. Non-Extracted Standards of PFOS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively I SSOQ14S9G -P F O S (Standard) 499.W Q.0 am a .fiw p / 1 o f 45 from 042 96B.w i1f A n a : 10759 eoaata H aight: &fif*+O02cp# R 7: 11.4 m ia Intensity, cps Intensity, cps Tim , min S S Q 0 1 4 6 9 5 - PFOS (Standard) 4 0 0 .0 /3 0 .0 j m u - s i m p l 2 of 4 6 from 0 4 2 8 0 8 B.wiff A r * j : 122 25 counts Haight: 7 .7 2 + 0 0 2 cps RT: 1 1 .4 min 700 600 500 400 300 200 100 0 J----------- - . ............ --T " . 1 I ' I 123 4 5 8 7 Tim , min 11.43 Exygen Research Page 38 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Figure 11. PFOS in Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively Raagant Coatrot - PFOS (Uakaowa) 499.0WO.Oama -sampfo 8 o f 45 from Q4290$B.wiff Araa: 338 corirtv Haight: Z49a+001 cps RT: 11.5 mia 11.46 T im e, min Reagent Spk A - PFOS (Q C )4 0 0 .0 /8 0 .0 amu * sample 0 of 46 from 042806B .w iff Area: 100 80 oounts Height: 6 .2 1 e + 0 0 2 cps RT: 11 .4 min 11.46 Tim e, min Reagent Spk B PFOS ( 0 0 )4 0 0 .0 /8 0 .0 amu sample 10 of 4 6 from 042806B.uviff Area: 037 01 counts Height: 6 .0 7 e + 0 0 3 cps RT: 1 1.4 min Exygen Research Page 39 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Sediment Sample Analyzed for PFOS (Exygen ID: C0172892, Data Set:042806B) I C0772892 - PFOS (U ataow a) 499.0/801 amu -ta m p It 16 o f 45 from 042t06B.w iff A n a : 5801 co u a tt H aight: 4 (9 > t(lll2 c p t RT: 11.5 m ia 11.<W Intensity, cps Exygen Research Page 40 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods and Protocol Amendments Exygen Research Page 41 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 13 J STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soli, 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, MN 55144 Phone: (651) 733-6374 Page / oj 65 Exygen Research Page 42 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 o f65 Page 43 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001 U l PROTOCOL APPROVAL Study Title: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 APPROVALS JaisimhalKesan, S' Weston Solutions Michael A. & 3M Comparfy Exygen Research Page 3 of 65 Page 44 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E x y g en Pro to co l N um ber: POOO1131 TABLE OF CONTENTS T IT L E P A G E ................................................................................................................................................................................1 D IS T R IB U T IO N ......................................................................................................................................................................... 2 PR O TO C O L A P P R O V A L ........................................................................................................................................................3 T A B L E O F C O N T E N T S ..........................................................................................................................................................4 IN T R O D U C T IO N ....................................................................................................................................................................... 5 T E S T M A T E R IA L S .................................................................................................................................................................. 5 O B JE C T IV E ................................................................................................................................................................................ 6 T E S T IN G F A C IL IT Y ................................................................................................................................................................ 6 ST U D Y D IR E C T O R .................................................................................................................................................................. 7 S P O N S O R R E PR E SE N T A T IV E ............................................................................................................................................ 7 P R IN C IP A L IN V E S T IG A T O R .............................................................................................................................................. 7 PR O PO SE D EX P E R IM E N T A L ST A R T A N D TE R M IN A T IO N D A T E S ............................................................... 7 ID E N T IFIC A T IO N A N D JU S T IF IC A T IO N O F T H E T E S T S Y S T E M .................................................................... 8 S A M PL E P R O C U R E M E N T , R E C E IPT A N D R E T E N T IO N ....................................................................................... 8 S A M PL E ID E N T IF IC A T IO N ................................................................................................................................................ 9 A N A L Y T IC A L PR O C E D U R E SU M M A R Y ......................................................................................................................9 V E R IF IC A T IO N O F A N A L Y T IC A L P R O C E D U R E ...................................................................................................... 9 M E T H O D F O R C O N T R O L O F B IA S ..................................................................................................................................11 ST A T IS T IC A L M E T H O D S .....................................................................................................................................................11 G LP S T A T E M E N T .................................................................................................................................................................... 11 R E P O R T ........................................................................................................................................................................................ 11 SA FE T Y A N D H E A L T H ......................................................................................................................................................... 12 A M E N D M E N T S T O P R O T O C O L ........................................................................................................................................13 D A TA R EC O R D K E E P IN G ....................................................................................................................................................13 Q U A LIT Y A S S U R A N C E ........................................................................................................................................................ 14 R E T E N T IO N O F D A TA A N D A R C H IV IN G .................................................................................................................... 14 A P P E N D IX I, A N A L Y T IC A L M E T H O D S .........................................................................................................................15 Page 4 of 65 Exygen Research Page 45 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen P rotocol N um ber: P 0 0 0 1 131 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. TESTMATERIALS 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 (C4 F9 SO3 I O Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 -* 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CF u SOsTC*) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 - * 80 Structure: Page 5 o f 65 Exygen Research Page 46 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen P rotocol N um ber: P 0 0 0 1131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFirSOjTC1') 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), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions o f the following Exygen analytical methods: V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTINGFACILITY Exygen Research 3038 Research Drive State College, PA 16801 Phone: (814) 272-1039 Page 6 o f 65 Exygen Research Page 47 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No. : P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 STUDYDIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax: (610) 701-7401 j .kesari@westonsolutions.com SPONSORREPRESENTATIVE 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 STARTAND 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 termination dates will be included in the final report. Page 7o f 65 Exygen Research Page 48 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E x y g en P ro to co l N um ber: P 0 0 0 1131 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. \ ^ SAMPLEPROCUREMENT, RECEIPTAND 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 -IC C . 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. Page 8 o f 65 Exygen Research Page 49 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 SAMPLE IDENTIFICATION E xy g en P ro to co l N um ber: P 0 0 0 1 131 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 die Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: `M ethod 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 Deteimination 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: `M ethod of 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 precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 9 o f 65 Exygen Research Page 50 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study N o.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1131 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 fill line at 200 mL. A field duplicate, a low field spike and a high field spike of each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (13C 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 4ng/g 40 ng/g Sediment Fish Clams Vegetation Small Mammal Liver 4 ng/g lOng/g 10ng/g 10ng/g 10ng/g 40 ng/g 100ng/g 100ng/g 100ng/g 100ng/g Small Mammal Serum 10 ng/mL 100 ng/mL Page 10 o f 6S Exygen Research Page 51 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E x y g en P ro to co l N um ber: POOOl 131 Recoveries axe anticipated to be between 70% and 130% o f 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 o f 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 I I o f 65 Exygen Research Page 52 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1131 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. SAFETYAND HEALTH Laboratory personnel will practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure of personnel and the environment to the test or reference substancefs). Page 12 of 65 Exygen Research Page 53 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E x y g en P ro to co l N um ber: POOOI131 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. DATARECORDKEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description o f any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence Any other documentation necessary for the reconstruction o f the study Chromatograms- 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 IS o f 65 Exygen Research Page 54 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen Pro to co l N um ber: P 0 0 0 1 131 As part o f 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. QUALITYASSURANCE 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 14 o f 65 Page 55 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000113 t APPENDIX I ANALYTICAL METHODS V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: `M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: `M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: 'M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" Exygen Research Page 15 o j 65 Page 56 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen Protoco] N um ber: POOO1131 ANALYTICAL METHOD M ethod Number: V 0001780 Method a f Analysis for the Determinati# of PerflnorooetMole Acid (PFOA) in Water by L C /M S /M S Analytic! Testing Facility: Exygen Research 3058 Research Drive State College, P A 16801 Approved By: P>aiuull CCooinMnoflllllyv ' Technical Leader, LC-M S, Exygen Reeeareh 2 J"Joohhnn Filabheerty / ' VV iicme PPrfesl iident, O perations, Exygen Reeeareh D ate D ate Exygen Research Total Pagee: 7 Page 6 o f 65 Page 57 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 ExygtaRiMfCh Mrfhod Number VOOOP80 | ANALYTICAL METHOD ................ 1 M ethod o f Analysis fcr the Determination o f Perfluorooctanoic A cid (PFO A ) in W ater by LC/M S/M S 1.0 Seope T hil m ethod is to be employed for die isolation and quantitation o f perfluorooctanoic a d d by High Performance Liquid Chrom atography coupled to a tandem M ass Spcctrometric D etector (LC/M S/M S) in water. 2 0 Safety 2.1 Alw ays observe eafe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirement 3.1 A t least 4 0 m L o f teat sam ple fo r extraction. 3.2 N o sample processing is needed for w ater sam ples. 3.3 Sam ples stored refrigerated should be allow ed to equilibrate to room tem perature. 3.4 All samples m ust be thoroughly m ixed before being sam pled for extraction 3.3 Any samples containing particles should be centrifuged at 3000 rpm for 3 minutes and the supernatant used for the extraction. 3.6 Sam ple collection procedures w ill be specified in the sam pling plan for this p ro je c t 4.0 Reagents tn d Standards 4.1 W a te r- HPLC grade 4.2 M ethanol - HPLC grade 4.3 Ammonium Acetate - A .C.S. Reagent G rade 4.4 Perfluorooctanoic Acid - Sigma-AM rieh $.0 Instrument and Equipment 3.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 A high performance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 3*200 pL connected to a tandem M ass Spectrom eter (LC/M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene centrifogo tubes. IS m L disposable polypropylene centrifoge tubes. Disposable m icropipets (50*100uL, 100*200uL). 125*mL LDPE nsrrow -m outh bottles. 2 m L clear HPLC vial kit. Disposable pipettes. Autopipcttee (100*1000 p L and 10*100 pL) w ith disposable tips. W aters Sep P ik Vac 6 c c (!g )tC 1 8 S P E cartridges. f t * ' 2 or? Page 17 o f 65 Exygen Research Page 58 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: POOO1131 ExyfnIUNwdi Method Number VOOO1780 a n a l y t ic a l m e t h o d M ethod o f Analysis for the Determ ination ofPerfluorooctanoic A cid (PFO A ) in W ater by LC/M S/M S 3.12 SPE vacuum m anifbid. 3.13 CentriAige capable o f spinning 5 0 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 A nalytical Colum n: Fluophaae R P (K eystone S cientific), 2.1 m m x 30 m m . $h (P/N: 82303-032130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ): 2 m M Am m onium Acetate in W ater M obile Phase (B ): M ethanol Gradient Program: Time (mint 0.0 1.0 8.0 20.0 22.5 &A 65 65 25 25 6$ Flow Rate 2LB fm L/m inl 3$ 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection V olum e: 13 p L (c m b e increased to a i m u ch as SO |tL ). 6.7 Quantitatioo: Peak Area - external standard calibration curve. 6.8 R unT im e: - 2 3 minutes. The abova conditions m e intended as a guide and m ay b e changed in order to optim ize the H PLC system. 7.0 M S/M S System 7.1 M ode: E lectrospray N egative M R M m ode, m o n ito rin g 4 1 3 - 3 6 9 nVz. T he above conditions a re intended as a g u id e an d m ay b e chan g ed in order to optim ize the M SM S system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w ater is prepared b y adding 0.134 g o f nm onium acetate to 1000 raL o f water. Alternate volumes m ay be prepared. Payc3 ul ' Page / 8 o f 65 Exygen Research Page 59 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen P ro to co l N um ber: POOOl 131 Ey|a R iatrch [ ANALYTICAL M ETH O D Method Number V000>780 M ethod o f A n a ly st for the Determ ination ofPerfluorooctanoic A cid (PFO A ) in W ater by LC/M S/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a etock aotution o f 10 0 pg/m L o f PFO A b y w eighing 10 m g o f analytical Sandard (oorrectod for purity) and dilute to 100 mL with m ethanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/m L fortification solution o f PF O A ia prepared b y bringing 10 m L o f th e 100 p g f o L s o lu tio n to a final v o lu m e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 1.0 pgrinL fortification aolution o f P F O A p r e p a r e d by bringing 10 m L o f foe 10 p g /m L aolution to a final v o lu m e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.4 A 0 .1 p g /m L fortification aolution o f P F O A ia prepared b y bringing 10 m L o f th e 1.0 p g ta L aolution to a final volum e o f 100 w ith methanol in a 12$ m L LDPE bottle. 9.1.5 A 0.01 pg/m L fortification aolution o f PFO A ia prepared by bringing 10 m L o f th e 0.1 p g /m L s o lu tio n to a final v o lu m e o f 100 with methanol in a 125 m L LD PE bottle. 9.1.6 T he s o c k and fortification so lu tio n s a re to b e sto re d in a refrigerator at approxim ately 4*C and are s a b le for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/M S/MS calibration standard are prepared in HPLC water. The calibration standard are proceaaed through foe extraction procedure, identical to samples. Tbs following is a typical example: additional concentrations m ay be Final Concentration Fortification Volume o f Coooentration o f Calibration o f Fortificatimi Volume Fortified Control Calibration Standard ID Solution (tab ) (MW S an ale (m L) Standard (dm )* (e xam ple) 0 0 40 10 100 40 10 200 40 0 XCmmddyy-0 2$ XCmroddyy-l 50 XCmmddyy*2 10 400 too too 100 200 100 400 40 40 40 40 100 250 500 1000 XCmmddyyO XCmmddyy-4 XCmmddyy-5 X C m m ddw -6 * The extracted concentration o f foe calibration standard is equal to 8* its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC extracted calibration standard. Ps*e 4 of ^ Page 19 o f 65 Exygen Research Page 60 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen P ro to co l N um ber: P 0 0 0 1 131 B a y y n lU --cfa Mflthed Number V0001780 ANALYTICAL. M E T H O D M ethod o f Analysis ior the Determination o f Perfluorooctanoic A cid (PFO A ) in W ater by LC/M S/M S 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent b lin k ) 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 6*C, up to tw o weeks. Alternate volum es id concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f sam ples extracted (typically 20 o r lest) m ust include at least one reagent control (method blank using HPLC water) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 11.2 11.3 11.4 11.5 M easure 40 mL o f sample or a portion o f sam ple diluted to 40 m L with water into 50 m L polypropylene ccntriflige tubes (fortify a t needed, replace lid and m ix well). Condition the C SPE cartridges (I g, 6 m L) b y passing 10 m L methanol followed b y 5 m L o f HPLC w ater (~ 2 dzop/sec). D o not let colum n run dry Load sample oo conditioned C u S P B cartridge. D iscard eluate. Elute w ith ~5 m L 100% m ethanol C ollect 5 m L o f eluate into graduated 1S m L polypropylene centrifiige tu b es (fin al v o lu m e * 5 m L). Analyze samples using electrospray LC/M S/M S. 12.0 Chrom atography 12.1 12.2 12.3 12.4 byect the sam e m o u n t o f each standard, sam ple and fortified sam ple into the L C /M S/M S system . A calib ratio n sta n d a rd m u st p reced e a n d follow all analysed samples. Standards o f PFOA corresponding to at least five o r m ore concentration levels m ust be included in an analytical sec. A n entire set o f extracted calibration standards m ust b e included at the beginning end at the end o f a sam ple set. E xtracted standards m ust be interspersed betw een every 5-10 samples. A s an alternative, an entire set of extracted calibration standards m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards m ust be the first and last injection in a sam ple set. U se linear standard curves for quantitation. L inear standard curves are generated for the analyte by linear regression using t/x w eighting o f peak area Pag 5 of 7 Page 20 o f 65 Exygen Research Page 61 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study N o.: P0001131 E xygen P ro to co l N um ber: P 0 0 0 1131 EsysuRMMfch Mbod Number V00017IQ A N A LYTIC A L M ETH O D M ethod o f Analysis forth Determ ination o f Perfluorooctanoic A cid (PFO A ) in W ater by LC/M S/M S v enue calibration etandaid concentration u ting M aseLynx 3.3 (or equivalent) software system. 12.5 Sam ple response ehould not exceed standard responses. A ny sam ples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram m ust show a peak o f t daughter ion at 369 tm u from a parent o f 413 tm u . The 413 am u parent corresponds to the PFO A anion, w hile the daughter ion (369 amu) represents the loss o f carbon dioxide. M ethod blanks m ust not contain PFO A at level greater than the LOQ. If a blank contains PFO A at level greater than 50 ng/L, then a new blank sample m ust be obtained and the entire set m ust be re-extracted. Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f their know n values. I f a control spike fitlls outside the acceptable lim its, the e n tire set o f sam ples should b e re-extracted. A ny m atrix spike ou tsid e 70* 130% should be evaluated b y th e analyst to determ ine if re-extraction is w arranted. Any calibration standard fism d to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve However, die total num ber o f extracted calibration standards that could be excluded m ust not exceed 20% o f die total num ber o f extracted standards injected. The correlation coefficient (R) for calibration curves generated m ust be 20.992 (R* 20.985). I f calibration results fall outside these lim its, then appropriate step m ust be taken to adjust instrum ent operation, and the standards o r the relevant set o f sam ples should b e reanalyzed. R etention tim es betw een standards and sam ples m ust not drift m ore than 4 % w ithin an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate th e am ount o f PF O A found (in ng/L, based on peek ares) using the standard curve (linear regression parameters) generated b y the M ass Lynx softw are program: PFOA found (ng/L) - fPeak ares - intercept) x DF slope D F A ctor by which die final volum e was diluted, if necessary. Pagf 6 of 7 Page 21 o f 65 Exygen Research Page 62 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xygen P ro to co l N um ber: P 0 0 0 1131 BiygnStMNich MMbod Nuakwr VQOO1780 I .................. ANALYTICAL METHOD | M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in W ater by LC/M S/M S 14.2 For sam ples fortified w ith known am ounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. Recovery (tt)* [ total analyte found (ng/L) - analyte found in control (ng^L)] analyte added (ng/L) Exygen Research PiS 7 of 7 Page 22 o f 65 Page 63 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 ANALYTICAL M ETHOD M ethod N um ber V0001781 M ethod o f A aaly ib (h r the D eterm taethm o f P srfluorooctanok A cid (PFO A ) le Sell by LC /M S/M S Analytical Tearing Facility: Exygen Research 3038 Research Drive S tate College, P A 16801 Approved By: T U . C J l ,____ Paul CooaoUy ' Technical Leader, L O M S , Bxygen Research ivlu .to 4 Date D ate Exygen Research Total Pages: 7 Page 23 o f 65 Page 64 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en Pro to co l N u m b e r P 0 0 0 1 131 S ty jra Rswareh Mt&od Number V000178J I ........... AN ALYTICA L M ETH O D Method o f Analyiif for the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 1.0 Scope This method ieto b e employod for tbe isolation end quantitation o f perfluorooctanoic add by High Performance Liquid Chromatograpfay coupled to a tandem Mass ' Spectromtrie Detector (LC/MS/MS) in soil. 2.0 Safety 2.1 A lw ays o bserve safe laboratory practices. 2.2 Consult tbe appropriate M SDS before handling any chem ical for proper safety precaution. 3.0 Sam ple Requirement 3.1 A t least IS g o f te s t sam ple for extraction. 3.2 N o sam ple processing is needed for soil samples. 3.3 Sam ples stored refrigerated should b e allow ed to equilibrate to room tem perature. 3.4 All samples m ust b e thoroughly m ixed before being sam pled for extraction. 3.3 Sam ple collection procedures will be specified in the sam pling plan for this project. 4.0 Reagents end Standards 4.1 W a te r-H P L C grade 4.2 M ethanol - HPLC grade 4.3 Am m onium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic A d d - Sigma*Aldrich 5.0 Instrument and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 A high perform ance liquid chrom atograph capable o f pum ping up to 2 advents equipped w ith a variable volum e injector capable o f injecting 5-200 pL connected to a tandem M ass Spectrom eter (LC/M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. SO m L disposable polypropylene centrifUge tubes. 15 m L disposable polypropylene ccntriflige tubes. Disposable m icropipets (S0-100uL, 100-200uL). 125-mL LDPB nanow -m outh bottles. 2 m L clear H PLC visl kit. Disposable pipettes. A utopipettes (100*1000 pL and 10-100 pL ), w ith disposable tips. W aters Sep Pak Vac 6 cc ( Ig) tC18 SPE cartridges. SPB vacuum manifold. Ultrasonic bath. 2 fPag* of Page 24 o f 65 Exygen Research Page 65 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xygen P ro to co l N um ber: P 0 0 0 1 131 BxygeaRmearcb Method NeubarVOOO1711 ANALYTICAL METHOD M ethod o f A nalysis for the Determ ination o f Perfiuorooctanoic A cid (PFO A ) in Soil by LC/M S/M S 5.14 W rist-action shaker. 5.15 Centrifuge capable o f qrinning 30 m L polypropylene tube al 5000 rpm. 6.0 Chromatographic System 6.1 A nalytical C olum n: Fhiophase R P (K ey sto n e S cien tific), 2.1 m m x SO nu n . 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 m M Am m onium A cetate in W ater 6.4 M obile Phase (B ): M ethanol 6.5 Gradient P ro -a m : Tim e fmiiri 0.0 1.0 8.0 20.0 22.S 65 65 25 25 65 2L fi 35 35 75 75 35 Flow Rate fmL/m m l 0.3 0.3 0.3 0.3 0.3 6 .6 Injection V olum e: IS jiL (can b e increased to a s m u c h as SO pL ). 6.7 Quantitation: Peak A n a -e x te rn a l standard calibration curve. 6.8 R unT im e: - 2 3 minutes. The above conditions t n Intended as a guide an d m ay be changed in order to optim ize the HFLC system. 7.0 M S/MS System 7.1 M ode: Electrospray N egative M R M m ode, m onitoring 413 - v 369 m /z for PFOA. T he above conditions are intended as a guide an d m ay b e ch an g ed in o rd er 10 optim ize die M SM S system. 8.0 Preparation o f Solutions 8.1 v M obile Phase 8.1.1 2 m M am m onium acetate in w a te r is p rep a re d b y ad d in g 0.1 S4 g o f amm onium acetate to 1000 m L o f water. Alternate volumes m ay b e prepared. Page 3 of 7 Page 25 o f 65 Exygen Research Page 66 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xygen P ro to co l N um ber: P 0 0 0 1 131 Exygea Research Method Number V0001781 ANALYTICAL METHOD M ethod o f Analysis for the D etenninatioii o f Perfluorooctanoic A cid (PFO A ) in Soil by LC/M S/M S 9.0 Standard Preparation 9.1 S u n d izd Stock/Fortiftcation Solution 9.1.1 P repare stock aolutioa o f *>100 p g /ra L o f P F O A b y w eig h in g 10 m g o f analytical standard (corrected for purity) and dilute to 100 mL with m ethanol in a 125-mL LD PE bottle. 9.1.2 A 10 p g /m L fortification so lution o f P F O A is prepared by bringing to m L o f th e 100 p g /m L so lution to a final v o lu m e o f 100 w ith mcihanoi in a 125 m L LOPE bottle. 9.1.3 A 1.0 pg/raL fortification so lu tio n o f P F O A is prepared b y bringing 10 m L o f t b e lO p g /m L solution to a final v o lu m e o f 100 w ith methanol in a 125 m L LOPE bottle. 9.1.4 A 0.1 p g /m L fortification s o lu tio n o f P F O A ia prepared b y bringing 10 m L o f foe 1.0 p g /m L so lution to a final v o lu m e o f 100 w ith methanol in 125 m L LD PE bottle. 9.1.5 A 0.01 pg/m L fortification solution o f PFO A ia prepared by bringing 10 m L o f the 0.1 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L L D P E bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approxim ately 4*C and are stable for a m axim um period o f 6 months fiom the date o f preparation. 9.2.1 9.2.2 LC/M S/M S calibration standard* am prepared in H PLC water The calibration standards am processed through the extraction procedure, identical to samples. The following ia a typical example: additional concentrations m ay be prepared as needed. Concentration o f Fortification Solution foob) Fortification Volume of Volume Fortified Control (uL) Semnle fmL) Final Concentration o f Calibration Standard foot)* Calibration Standard ID fexamole) 00 10 100 10 200 10 400 to o 100 100 200 too 400 40 40 40 40 40 40 40 0 25 SO 100 250 500 1000 XCmmddyy-0 XCmmddyy-l XCmmddyy-2 XCmmddyy-3 XCmmddyy>4 XCmmddyy-5 XCmroddw-6 * The extracted concentration o f the calibration standard is equal to 8* its initial concentration, due to the concentration o f the standard during th e extraction (SPE). X C extracted calibration standard. Pig4 of7 Page 26 o f 65 Exygen Research Page 67 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1131 Exy|M ItMMnh Mathod Number V000178I ANALYTICAL METHOD ] M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Soil by LC/M S/MS > 9.2.3 A zero standard solution (reag en t b lan k ) m u st b e prepared with each set o f standards extracted. 9.2.4 Store all extracted calibration standards in 15-mL polypropylene tubes at 2*C to 6*C, up to two weeks. 9.2.5 A lternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f sam ples extracted (typically 20 o r less) m ust include at least one reagent control (m ethod blank uaing S m L o f m ethanol) and two reagent controls fortified at know n concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 11.2 11J 11.4 11.5 11.6 11.7 11.8 11.9 W eigh S g o f sam ple into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and m ix well). A dd 5 m L o f m ethanol and shake on a w rist action shaker for - ] 5 minutes. Transfer the tubes to an ultrasonic bath and sonicate for -1 5 minutes. Bring the volume up to 40 m L w ith w ater in the 50 m L polypropylene centrifoge tube. Centrifuge f b r- 1 0 at -3 0 0 0 rpm. C ondition th e C is S P E cartridges (1 g, 6 m L ) b y p a s sin g 10 m L m ethanol followed by 5 m L o f HPLC water ( - 2 drop/sec). D o not let colum n run dry Load (decant) the sample on the conditioned Ci SPE cartridge. Discard chiate. Elute with - 5 m L 100% methanol. C ollect 5 m L o f eluate into graduated 15 m L polypropylene centrifoge tu b es (final v o lu m e S m L). Analyze samples using electrospray LC/M S/M S. 12.0 Chrom atography 12.1 Inject th e sam e am ount o f each standard, sam ple an d fortified sam ple into (he LC /M S/M S system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA correspond ing to at least five or m ore concentration levels m ust be included in an analytical set. 12.3 A n entire set o f extracted calibration standards m ust b e included at the beginning and at die end o f a sam ple set. Extracted standards must be interspersed betw een every 5-10 samples. Aa an alternative, an entire set o f PageS of 7 Page 27 o f 65 Exygen Research Page 68 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E xyinR M ttfcfc Method Number VOOOI7SI I A N A L Y T IC A L M E T H O D ........... | M ethod o f Analysis for the Determ ination o f Perftuorooctanolc A cid (PFO A ) in Soil by LC/M S/M S 12.4 12.5 extracted calibration standards m ay be injected at foe beginning o f a set followed b y extracted calibration standards interspersed every 5*10 samples (to account for a second set o f extracted standard). In either case, extracted calibration standards m ust be foe first and last injection in a sam ple set. U se linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using l/x weighting o f peak area versus calibration standard concentration using M aasLynx 3 3 (or equivalent) softw are system. Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram m ust show a peek o f a daughter ion at 369 am u from a parent o f 413 amu. T he 413 amu parent corresponds to the PFOA anion, while the d aughter ion (369 am u ) represents th e 1ms o f carb o n dioxide. M ethod blanks mum not contain PFO A at levels greater than the LOQ. If a blank contains PFOA a t levels greater than 50 ng/L, then a new blank ample m ust be obtained and foe entire set m ust be re-extracted. Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% of their known values. If a control spike falls outside foe acceptable lim its, the entire set o f samples should be re-extracted. A ny m atrix spike outside 70 130% sh ould b e evaluated b y th e a n aly st to d e te rm in e i f re-extraction is warranted. A ny calibration standard found to be a statistical outlier b y using the Huge E rror Test, m ay be excluded from the calculation o f the calibration curve. However, foe total num ber o f extracted calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f extracted standards injected. T he correlation coefficient (R ) for calibration curves generated m ust be 20.992 (R1 20.985). I f calibration results fall outside these limits, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or the relevant set of samples should b e reanalyzed. Retention tim es betw een standards and sam ples m ust not drift m ore than 4 % within an analytical run. I f M ention tim e drift exceeds this lim it within an analytics! run then the set m ust be reanalyzed. P*e6of7 Page 28 o f 65 Exygen Research Page 69 o f 110 Interim Report #23 - Analysis o f Sediment 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 ExygcnRamrcli Method Nuabr V0001781 I ANALYTICAL M ETH O D M ethod o f Analysis for the Determ ination o f P eriluorooctanoic A cid (PFO A ) in Soil by LC/M S/MS 14.0 Calculation 14.1 U ie th e ibiiow iiig equation to c alcu late th e am o u n t o f P F O A found (in ng/L, baaed on peak a n a ) uaing the standard curve (linear regreaaion parameters) generated by the M ass Lynx software program: PFO A found fag/L) (Peak area - intercept) x DF DF - factor by w hich the final volume waa diluted, if neceeeary. 14.2 F or eamplee fortified w ith know n amount o f PFO A p rior to extraction, use the following equation to calculate the percent recovery. Recovery (% ) [ total analyte found (n g /L ) analyte found in control (ng/L)] analyte added (ng/L) 14.3 U se the follow ing equation to convert the am ount o f P F O A found in ng/L to ng/g(ppb). PFOA found (ppb) * fPFQA found fna/L) x volum e extracted (0.04D 1 sample weight (5 g) 14.4 U ac the follow ing equation to c alcu late th e am o u n t o f P F O A found in ppb based on dry weight PFOA found (ppb) dry weight - PFO A found (ppb) x [1 0 0 % / total solids(% )] Pag# 7or7 Page 29 o f 65 Exygen Research Page 70 o f 110 Interim Report #23 - Analysis of Sediment Samples Exygen Study No.: P0001131 E x y g en Pro to co l N um ber: POO01131 ANALYTICAL METHOD M ethod N u m b er V0001782 M e th o d o f A n a ly s li fo r th e D e t e m f o id o D o f P e r fla o ro o c ta a o ic A d d (P F O A ) io Sedim ent by LC /M S/M 8 Analytical Testing Facility: Exygen Research 3031 Research Drive State College, P A 16801 Approved By: c j iL ____ Paul Connolly { Technical Leader, LC-M S, Exygen Research A loobhin Flaherty / V Vi ice P resident, O perations, Exygen Research __12imM. D ate dr Due Exygen Research Total Page,: 7 Page 30 o f 65 Page 71 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen P ro to co l N um ber: P 0 0 0 1 131 Sxyfni Rncareli Mlhod Number VOOOI7S2 | ANALYTICAL M ETH O D 1 M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Sediment by LC/M S/M S 1.0 Scope Thi* method is to he employed for the isolation and quantitation o f perfluorooctanoic a d d b y High Performance Liquid Chrom atography coupled to a tandem M ass Spectrometric D etector (LC/M S/M S) in sedim ent 2.1 A lw ays observe sa fe laboratory practioce. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirement 3.1 A t least 30 g o f tes t sam ple for extraction. 3.2 N o sam ple processing is needed for sedim ent samples. 3.3 Sem ples stored refrigerated should be allow ed to equilibrate to room tem perature. 3.4 A ll samples m ust be thoroughly m ixed before being sam pled for extraction. 3.5 Sam ple collection p ro ce d u re w ill be eperified in the sam pling plan for this p ro je c t 4.0 Reagents and Standards 4.1 W a te r-H P L C grade 4.2 M ethanol - HPLC grade 4.3 Acetic A d d - Reagent grade 4.4 Am m onium Acetate - A C .S . Reagent G rade 4.5 Perfluorooctanoic A d d - Sigma*Aldrich 5.0 Instrument and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 A high performance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5*200 jiL connected to a tandem M ass Spectrom eter (LC/M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene centrifoge tubes. 15 m L d u q m a b le p olypropylene c en trifu g e tubes. Disposable m icropipets (50-100uL, 100-200uL), 125-mL LD PE narrow -m outh bottles. 2 m L clsar H PLC v iti kit. Disposable pipettes. Autopipettes (100-1000 p L a n d 10-100 pL ), w ith disposable tips. W aters Sep Pek Vac 6 cc (lg ) tC IS SPE cartridges. SPE vacuum manifold. Page 2 of 7 Page 31 o f 65 Exygen Research Page 72 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No. : P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1131 Exygw Xeiearch Method Number VOOOI7S2 | ANALYTICAL M ETH O D 1 M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A ) in Sediment bv LC/M S/M S 5.13 5.14 5.15 V ortexer. W rist-action sh aker CentxiiUge capable o f spinning 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 A nalytical C olum n: F lu o p h w c R P (K ey sto n e S cien tific), 2.1 n u n x 50 m m . 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ): 2 m M Am m onium A cetate in W ater 6.4 M obile Phase ( B ) : Methanol 6.5 Gradient Program: Tima /m ini 0.0 1.0 8.0 20.0 22.5 SLA 65 65 25 25 65 SLfl 35 33 75 75 35 Flow Rate fm U m inl 0.3 0.3 0.3 0.3 0.3 6.6 Injection Volum e: 15 p L (can b e increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 R unT im e: ~ 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: E lectrospray N egative M R M m ode, m o n ito rin g 4 1 3 - * 3 6 9 m /z for T he above conditions ere intended u a guide and m ay b e changed in order to optim ize die M SM S system. 8.0 Preparation o f Solutions 8.1 M obile M u se 8.1.1 2 m M am m onium acetate in w a te r is p rep ared b y ad d in g 0.154 g o f ammonium acetate to 1000 m L o f water. Page 3 o f7 Page 32 o f 65 Exygen Research Page 73 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xygen P ro to co l N um ber: P 0 0 0 1 131 Bxygea llwewdi M ated Number VOOO1782 1 ANALYTICAL M ETH O D 1 M ethod o f Analysis for the Determ ination o f Perfluoroocunoic A cid (PFO A ) in Sediment by LC/M S/M S 8.2 Extraction Solution! 8.2.1 I S acetic acid in w ater ie prepared b y adding 10 m i, o f acetic acid to 1000 m L o f water. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9 1 Standard Stock/Fortification Solution 9.1.1 Prepare a etock solution o f - 1 0 0 p g /m L o f P F O A b y w eighing 10 m g o f analytical standard (corrected for purity) and dilute to 100 mL with m ethanol in a 125-mL LD PE bottle. 9 .1 2 A 10 p g fmL fortification so lu tio n o f P F O A is p rep ared b y bringing 10 m L o f the 100 p g /m L so lu tio n to a final v o lu m e o f 10 0 w ith methanol in a 125 m L LDPE bottle. 9.1 J A 1.0 p g/m L fortification so lu tio n o f P F O A ie p rep ared b y bringing 10 m L o f th e 10 pg/raL solution to a final volum e o f 100 w ith methanol in a 125 m L LD PE bottle. 9.1.4 A 0 .1 itg/m L fortification so lution o f P F O A is p rep ared b y bringing 10 m L o f the 1.0 pg /m L so lution to a final v o lu m e o f 100 w ith methanol in a 125 m L LD PE bottle. 9.1.5 A 0.01 pg/m L fortification solution o fP F O A is prepared by bringing 10 m L o f the 0.1 pg/m L aolution to a final volum e o f 100 with methanol in a 125 m L LD PE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at ^jproxim ately 4*C and are stable for a m axim um period o f 6 months from the date o f preparation. 92 Standard Calibration Solutions 9.2.1 9.2.2 LC/M S/M S calibration stn d a rd s are prepared in m ethanol via dilution o f the 0.1 pg/m L fortification solution. H ie following is a typical example: additional concentrations m ay be flm wntrttion ofFortifioatioa Solution (ru/mL) 100 too 100 10 5 2 Volume (mL) 10 5 2 10 10 10 Diluted to (mL) 100 100 100 100 100 100 Final Concentration (nurinL) 10.0 3.0 20 1.0 O.S 02 Pag 4 of 7 Page 33 o f63 Exygen Research Page 74 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study N o.: 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 E m n lM tick Mffhod Number V0001782 1 ANALYTICAL M ETH O D | M ethod o f A o ily iii for the Determ ination o f Perfluotooctanoic Acid (PFO A ) in Sediment by LC/M S/M S 9.2.3 9.2.4 Store at) calibration standard in 125-mL LD PE narrow -m outh bottles a t 2 aC to 6*C, u p to six m onths. Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set U p 10.1 E ach batch o f cam ples sxtracted (typically 20 o r l e u ) m u st include at least m e untreated oontrol and tw o untreated controls fortified at known concentrations (lib control spike) to verity procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes w ill be specified in foe quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 W eigh 5 g o f sam ple into 50 m L poly p ro p y len e c e atrifo g e tubes (fortify as needed, replace lid and m ix well). 112 A dd 35 m L o f 1% acetic acid, cap, vortex and shake on a w hat action shaker fo r *"60 m inutes. 11.3 Centrifiige foe tubes at -3 0 0 0 rpm for - 2 0 minutes. U .4 Condition foe C n SPE cartridges (1 g, 6 m L) by passing 10 m L methanol followed b y 2 0 m L o f HPLC w ater ( - 2 drop/sec). D o not let colum n run dry 11.5 Load (decant) foe sam ple on foe conditioned C u SPE cartridge. Discard eluate. 11.6 Add 20 m L o f m ethanol to the sedim ent left in the bottom o f the 50 mL centrifiige tube. C ap, vortex ro d s h ak e o n a w rist a ctio n sh a k er for *-30 m inutes. 11.7 Centrifiige the tu b a st ~3000 rpm for - 2 0 m inutes. 11.8 D e c u t foe m ethanol onto tb s tam e SPE cartridge. C ollect foe eluate. 11.9 W ash the colum n w ith 4 m L o f m e fo u o l. C ollect foe eluate and add it to foe eluate collected in step 11.8. 11.10 C ondition a second C u SPE cartridge (1 g , 6 m L ) b y passing 10 m L methanol followed by 20 m L o f HPLC w ater ( - 2 drop/eec). D o not let colum n run dry 11.11 A dd the m ethanol to -2 0 0 m L o f w ater and load o n the second conditioned SPE cartridge. 11.12 Elute w ith - 5 m L 100% m ethanol. C ollect 5 m L o f eluate into graduated 15 m L polypropylene centrifiige tubes (final volum e 5 mL). 11.13 Analyze samples using electrospny LC/M S/M S. Pa i o f1 Page 34 o f65 Exygen Research Page 75 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E x y g en P ro to co l N u m b e r P 0 0 0 1 131 Sxyga Raaunb Method Number VOOOI7K2 | ANALYTICAL M ETH O D | M ethod o f Analysis for the D otm ninatioo o f Perfluorooctanoic A d d (PFO A ) in Sediment by LC/M S/M S 12.0 Chrom atography 12.1 12.2 12.3 12.4 12.3 Iryect the tam e am ount o f each standard, sam ple and fortified sam ple into the LC /M S/M S system . A calibration standard m ust precede and follow alt analyzed samples. Standards o f PFO A corresponding to at least five o r m ore concentration levels m ust be included in an analytical set. A n entire aet o f extracted calibration standards m ust b e included at the* beginning and at die end o f a sam ple set. Standards m ust be interspersed between every 3-10 samples. A s an alternative, an entire set o f calibration standards m ay be injected at the beginning o f a set followed by calibration standards interspersed every 5*10 sam ples (to account for a second set o f standards). In either case, calibration standards m ust be the first and last injection in a sam ple se t Use linear standard curves for quantitation. Linear standard curves arc generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using M aasLynx 3.3 (or equivalent) software system. Sam ple response should not exceed standard response!. A ny sam ples that exceed standard responses should be flirther diluted end reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 Chrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. The 413 am u parent corresponds to the PFO A anion, w hile the daughter km (369 am u) represents die lo ta o f carbon dioxide. M ethod blanks m ust not oontain PFO A a t levels p e s te r than the LOQ. If a blank PFOA at levels greater than 0.2 ng/m L, th a t a new blank sam ple m u it be obtained and th e entire aet m ust be re-extracted. Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f their know n values. I f a control spike ftU s outside die acceptable lim its, the entire act o f samples should be re-extracted. A ny m atrix spike outside 70' 130% should b e evaluated b y th e analyst to determ ine i f re-extraction is warranted. A ny calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve However, the total num ber o f extracted calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f extracted standards uyected. The correlation coefficient (R ) for calibration curvet generated m ust be 20.992 (R 1 20.985). I f calibration results foil outside these lim its, then appropriate steps m ust b e taken to adjust instrum ent operation, and the standards o r the relevant set o f samples should be reanalyzed. Pag 6 of 7 Page 35 o f 65 Exygen Research Page 76 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygnRMMTch Method Numtwr V00017I2 | ANALYTICAL M ETH O D | M ethod o f A nalysis fo r th e D eterm ination o f P erfluorooctanoic A c id (P F O A ) in Sedim ent by LC/M S/M S 13.6 R etention timee betw een standards and aam plei m ust not drift m ore than 4 % w ithin an analytical nm . If retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 C a l c u l* 14.1 U se th e follow ing equation to calcu late th e am o u n t o f P F O A found (in ng/m L. baaed on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program : PFOA found fna/m L) fPeak area - intercept) x DF slope D F factor b y which the final volum e w as diluted, if necessary. 14.2 F o r sam ples fortified w ith know n am ounts o f P F O A p rior to extraction, use the follow ing equation to calcu late foe percent recovery. Recovery ( H ) - [ total analyte found (ng/m L) - analyte found in control (ng/m L)l ^ analyte added (ng/m L) 14.3 U se the following equation to convert foe am ount o f PFO A found in ng/m L to n*/g (ppb)- PF O A found (p p b ) - (P F O A found f n a / m U x fin a l vo lu m e (5 mL>1 sample weight (5 g) 14.4 U se the following equation (if necessary) to calculate the am ount o f PFOA found in ppb based on dry weight. P F O A found (ppb) d ry w eight P F O A found (p p b ) x [ 10 0 % / to tal sohdi(% >] Page 7 of? Page 36 of 65 Exygen Research Page 77 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N uniber: P 0 0 0 1 131 ANALYTICAL M ETHOD M ethod N um ber V0001783 Method of Aaafysli for the Determination of Perflaorooctaooic Acid (PFOA) in Fish a i d Clams by LC/MS/MS Analytical Testing Facility: Exygen Research 303$ Research Drive State College, P A 16801 Approved By: rj L Paul Connolly 1 Technical Leader, LC-M S, Exygen Research rt//r> __________ Vohn Flaherty 'V i c e President, Operation!, Exygen Research D ate # A tr D ate Exygen Research 8Total Pages: Page 37 of 65 Page 78 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen P ro to co l N um ber: P 0 0 0 1 131 Haygen Rt i etrch Method Number VOOO1713 a n a l y t ic a l m e t h o d I M ethod o f A nalytic for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Fish and Clara* b y LC/M S/M S 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic a d d by High Performance Liquid Chrom atography coupled to a tandem M ass Spectrom etric Detector (LC/M S/M S) in fish and clam s. 2.0 Safety 2.1 Alw ays observe safe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirement 3.1 A t least 2 0 g o f tes t sam p le fo r extraction. 3.2 Sam ples should be procem ed before extraction. P lace the frozen sam ple in a food proceaaor and hom ogaiizc w ith dry ice. Place the sam pler in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the sam ples in frozen storage until tim e o f analysis. 3.3 Sam ple collection procedures w ill be specified is d ie sam pling plan for this project 4.0 Reagents and Standards 4.1 4.2 4.3 4.4 4.3 4.6 4.7 4.6 4.9 4.10 4.11 4.12 4.13 W star-H P L C grade Acetonitrile - H PLC grade Carbon (120-400 m esh) - Reagent grade M ethanol - HPLC grade Silica gel (60-200 m eeh) - Reagent grade Plorisil (60-100 m esh) - Reagent grade Superclean LC-N Hj - Reagent grade 1-Octanol - HPLC grade L-Ascorbic acid - Reagent grade DiroethyidicM oroiilane - Reagent grade Toluene - Reagent grade Ammonium Acetate - A.C.S. Reagent G rade Perfluorooctanoic A cid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped with a variable volum e injector capable o f injecting 5-200 jiL connected to a tandem Maas Spectrom eter (LC/M S/M S). 52 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Pie* 2ofs Page 38 of 65 Exygen Research Page 79 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E x y g en P ro to co l N um ber: P 0 0 0 1 131 Exygn (tassaceli [ ANALYTICAL M ETH O D Method Number V0OO1783 M ethod o f Analysis for the Determination o f Perfloorooctanoic A d d (PFO A ) in Fish and Clam s by LC/M S/M S 9.4 9.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14 5.15 5.16 Rotary evaporator T issum izer. 129 m L pear-shaped flasks. 50 m L disposable polypropylene centrifiige tubes. 15 m L disposable polypropylene centrifuge tubes. D isposable m icropipets (SO-lOOuL, 100-200u L). 125-mL LD PE naxrow*mouth bottles. 2 m L clear HFLC vial kit. Disposable pipettes. Aulopipottes (100*1000 p L a a d 10*100 pL ), w ith disposable tips. SPE tubes (20mL) (Supelco cat. no. N 057177). W rist action baker. Centrifoge capable o f spum ing 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographie System 6 .1 Analytical Colum n: Fluopbaae R P (K eystone Scientific). 2.1 m m x 50 m m. 5m (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 M obile Phase ( A ) : 2 m M Am m onium Acetate in W ater M obile Phase (B ) : M ethanol Gradient Program: T im a (mini 0.0 1.0 8.0 20.0 22.5 2k A 65 65 25 25 65 Flow Rate & fmlVm inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: l S p L (can be increased to as m uch aa 50 pL). 6.7 Quantitation: Peak Area ~ external standard calibration curve. 6.8 R unT im e: - 2 3 minutes. The above conditions are intended as a guide and m ay b e changed in order to optim ize the HPLC system. Page 3 of8 Page 39 of 65 Exygen Research Page 80 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 Exygnt ftaaearch Method Number VOQQ17S3 ANALYTICAL M ETH O D M ethod o f Analysis for foe Determ ination ofPerfluorooctanoic A cid (PFO A ) in Fish and Clams by LC/M S/M S 7.0 M S/M S System 7.1 M ode: Electroapray N egative M R M m o d e, m o n ito rin g 4 13 - 369 m /z for PFOA. The above conditions a n intended u a guide and m ay be changed in order to optim ize the M SM S lyatem. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w a te r is p rep ared b y ad d in g 0.154 g o r ammonium acetate to 1000 m L o f water. 82 Extraction Solutions 8.2.1 82.2 2% ascorbic a d d in methanol is prepared by dissolving 2 g o f ascorbic a d d in 100 m L o f methanol. 30% Dim efoytdichlorosilJM in toluene is prepared by bringing 3 mL o f dimethyldichloroeilane to a final volum e o f l 0 m L w ith toluene. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortifieation Solution 9.1.1 P repare a stock solution o f *100 p g fa iL o f P F O A b y w e ig h in g 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with m ethanol in a 125-mL LD PE bottle. 9.1.2 A 1.0 py'hiL fortification solution o f PFO A ia prepared by bringiog 1 m L o f foe 100 p ^ n L so lution to a final v o lu m e o f 100 w ith methanol in a 125 m L LD PE bottle. 9.1.3 A 0.1 p^fcnL fortification so lution o f P F O A is p rep ared by bringing 10 m L o f foe 1.0 p ^ m L so lution to final v o lu m e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.4 A 0.01 pgrinL fortification solution o f PFO A ia prepared by bringing 10 m L o f the 0.1 pg/m L solution to a final volum e o f 100 with methanol in a 12$ m L LD PE bottle. 9.1 J T he stock and fortification so lu tio n s a re to b e sto red in a refrigerator at approxim ately 4*C and are stable fo r a m axim um period o f 6 months from the date o f preparation. Pat* * Page 40 o f 65 Exygen Research Page 81 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 ExygeaResearch Method Number V0001783 ANALYTICAL M ETH O D J M ethod o f A n a ly st far die Determ ination o f Pcrfluorooctsnoic A cid (PFO A ) in Fish end Clams by LC/M S/M S 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/M S/M S calibration standards are prepared in m ethanol via dilution o f the 1.0 pg/m L fortification solution. T he foUowing is a typical exam ple: additional concentrations m ay be prepared aa needed. Conosntietien o f Fortification Solution fuc/mL) 10 10 1.0 0.05 0.025 0.1 0.005 Votums (mL) 5.0 2.5 1.0 10 10 10 10 Diluted to (mL) 100 100 100 100 100 100 100 Final Concentration (ua/m U 0.05 0.025 0.01 0.005 0.002S 0.001 0.0005 9.2.3 Store all calibration standards in 125-mL LD PE narrow -m outh bottles at 2*C to 6*C, up to six m onths. 9.2.4 A lternate volum es and co ncentrations o f sta n d a rd s m ay b e prepared as needed. 10.0 Batch Set Up 10.1 E ach batch o f sam ples extracted (typically 20 o r leas) m ust include at least one untreated control and tw o untreated controls fortified at known concentrations (lab control spike) to v e rily procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes w ill be specified in foe quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 11.2 11.3 11.4 11.5 11.6 W eigh 5 g o f frozen sam ple info 50 m L polypropylene centrifuge tubes (fortify u needed, replace lid end m ix well). A dd 30 m L o f acetonitrile and shake o n a wrist action shaker for - 1 S minutes Place the tubes in a freezer for -1 hour. Pack and condition foe SPE tubes and ailanize the pear-shaped flasks. Pack the 20 m L SPE tubes in sequence w ith 2 g florisil, 2 g silica gel, 2 g carbon, and I g LC-N Hj. Condition the colum ns w ith 20 m L o f methanol, then 20 m L o f acetonitrile. D iscard all w ashes. D o not allow the colum n to dry. Silaniae the 125 m L prar-shaptd flasks b y raising w ith the 30% dimefoyldichlorosilane in toluene solution. R inse foe flask w ith toluene once, followed b y m ethanol (three tim es). D ry the flasks com pletely before use, either by aiw lrym g o r w ith a stream o f nitrogen. Page $ of H Page 4/ of 65 Exygen Research Page 82 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study N o.: P0001131 E xy g eu P ro to co l N um ber: P 0 0 0 1 131 Exygm Rmerch Method Number VOOOI783 ANALYTICAL M ETH O D M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFOA ) in Fish and Clam s by LC/M S/M S 11.7 C entriftge the 50 m L polypropylene tubes containing sam ple at -2 0 0 0 rpm for - 1 0 minutes. 11.8 Decant the (tract on to s conditioned SPE colum n fitted inside the m outh o f the pear-shaped flask. Collect the e lu d e in foe 125 m L silanized pear-shape flask. 11.9 A dd 10 m L o f acetonitrile to th e sam ple in foe 50 m L centrifuge tube. Homogenize the frozen fat phase using a tissum izer for -3 0 seconds and rinse the tissum izer w ith - 1 0 m L o f acetonitrile into the tube. 11.10 Shake foe sample again for -1 0 m inutes on a w rist-action shaker. 11.11 Place the tubes in e freezer for - 1 hour m ore. 11.12 Centrifuge foe 50 m L polypropylene tubes containing sam ple at -2 0 0 0 rpm fo r- 1 0 minutes. 11.13 Decant foe extract onto the sam e SPE colum n. Collect the eiuete into the sam e pear-shaped flask and com bine w ith foe eluent from the initial extraction. 11.14 P ass 20 m L o f acetonitrile through the S P E colum n and com bine the eluate in the same peaoehaped flask. 11.15 A dd 3-4 (hops o f 1-ocUno) to foe extract in foe pear-shaped flask and evaporate at reduoed pressure using a rotary evaporator (at < 40C). 11.16 M ake the final volum e, b y adding 2 m L o f 2% ascorbic acid in m ethanol to foe pear-shaped flask mid sw iri to m ix/dissolve. 11.17 Transfer the extracts to H PLC vials using disposable pipets. 11.18 Analyze sam ples using electio sp n y LC/M S/M S. 12.0 Chrom atography 12.1 Iqjeet foe sam e am ount o f each standard, sam ple a n d fortified sam ple into the LC /M S/M S system . A calibration standard m u st precede and follow all analyzed sim ples 12.2 Sttndards o f PFO A corresponding to et least five o r m ore concentration levels m ust be included in an analytical set. 12.3 A n entire se t o f calibration standards m u tt b e in clu d e d at th e beg in n in g and at the end o f a sam ple set. Standards m ust be interspersed betw een every 5-10 samples. A s an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed e v e ry 5-10 sam ples (to account fo r a seco n d s e t o f standards). In either case, ealforation standards m ust be foe first and last injection in u sample se t 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x w eighting o f peak area versus calibration standard concentration using M aasLynx 3 3 (or equivalent) software system. Pag 6 of g Page 42 o f 65 Exygen Research Page 83 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 E xygnJU m rcb Method N unbtr V0001783 ANALYTICAL m e t h o d M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Fish and C tu n i by LC/M S/M S 12.5 Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be forther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram m ust how a peak o f a daughter ion et 369 arou from a parent o f 413 amu. The 413 am u parent corresponds to foe PFO A anion, while the daughter ion (369 amu) represents foe loss o f carbon dioxide. M ethod blank* m ust not contain PFO A a t levels p e s te r than foe LOQ. If a blank contains PFO A at levels greater than 0.5 ppb, then a new blank sam ple m ust be obtained and foe entire set m ust be re-extracted. Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f their know n vshiai. I f a eontrol spike foils outside th e acceptable lim its, the entire set o f samples should b e re-extracted. A ny calibration standard found to be a statistical outlier b y using the Huge E rror Test, m ay be excluded from the calculation o f the calibration curve. However, foe total num ber o f calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f standards injected. The correlation coefficient (R ) for calibration curves generated m ust be 20.992 (RJ 20.985). I f calibration results foil outside these lim its, then appropriate steps m ust b e taken to adjust instrument operation, and the standards o r the relevant set o f sam ples should be reanalyzed. Retention tim es betw een standards and sam ples m ust not drift m ore than 4 % w ithin so analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calcula te th e am o u n t o f P F O A found (in ng/m L, baaed on peak area) using foe standard curve (linear regression parameters) generated by the M ass Lynx software program: PFO A found (ng/m L) - (Peak area intercept) slope 14.2 U se foe follow ing equation to convert foe am ount o f P F O A found in ng/m L to n$fc(P!*) P F O A found fa n b l - fP F O A found fa e /m L l x final v o lu m e (m L ) x OFT sam ple weight (g) D F foctor by w hich the final volum e w as diluted, i f necessary. P*t ? Of * Page 43 o f 65 Exygen Research Page 84 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 Exygra RMcarch Method Number VOQO1783 I ANALYTICAL M ETH O D ~| M ethod ofA nnlyei for the D etenninttion ofPerfhiorooctanoic A cid (PFO A ) in F iih nnd C lu n l by LC/M S/M S 14.3 F or sample* fortified w ith know n eroounu o f PFO A p rior to extraction, use the following equation to calculate the percent recovery. R ecovery (% ) * [ total analyte found (n ^ g ) analyte found in control (ng/g)j analyte added (ng/g) Exygen Research Page 8 of 4 Page 44 o f6S Page 85 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 ANALYTICAL METHOD M ethod N um ber V0001784 M ethod e f A nalysis fo r th e D eterm in atio n e f P e r fla o ro o e tu o lc A d d (P F O A ) in Vegetation by LC /M S/M S Analytical T otting Facility: Exygen Research 3058 Research Drive State CoUege, P A 16801 Approved By: C JtL . Paul Connolly * Technical Leader, LC-M S, Exygen Research n , / / n d / / ________ J o h n Flaherty ' ' v i c e President, O peritioni, Exygen Research _tojz&M D ate Dat Exygen Research Total Paget: 7 Page 45 of65 Page 86 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 Exygw Remrcfe Method Number VOODI7M ANALYTICAL M ETH OD M ethod o f Analyst for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Vegetation by LC/M S/M S 1.0 Scope This m ethod is to be employed for the isolation and quantitation o f perfluorooctanoic a d d b y High Perform ance Liquid Chrom atography coupled to a tandem Mass Spectrom etric Detector (LC/M S/M S) in vegetation. 2.0 Safety 2.1 A lw aya observe sa fe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirement 3.1 A t least 20 g o f test sam ple for extraction. 3.2 Sam ples should be processed before extraction. P lace the frozen sample in a food processor and hom ogenize w ith dry ice. Place the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the sam ples in frozen storage until tim e o f analysis. 3.3 Sam ple collection procedures w ill be specified in the sam pling plan for this p ro je c t 4.0 Reagents and Standards 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 W au r-H P L C grade Acetonitrile - HPLC grade Carbon (120*400 m esh) - Reagent grade M ethanol - HPLC grade Silica gel (60-200 mesh) - Reagent grade Florisil (60-100 mesh) - Reagent grade Superclean LC-N Hj - Reagent grade 1-O ctanol-H PL C grade L-Aacorbie a d d -R e a g e n t grade Dimethytdichlorosilane - R eagent grade Toluene - Reagent grade Am m onium Acetate - A.C.S. Reagent Grade Perfluorooctanoic A cid - Sigma-AJdrich 5.0 Instrument and Equipment 5.1 A hig h p erform ance liq u id chrom atograph ca p ab le o f pu m p in g up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5*200 p L connected to a tandem M ass Spectrom eter (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. S J Analytical balance capable o f reading to 0.00001 g. Pijfe 2 of 7 Page 46 of65 Exygen Research Page 87 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E x y g en Pro to co l N um ber: P 0 0 0 1 131 Exygm JUseucb MMbod Number V0Q01784 ANALYTICAL M ETH O D M ethod o f Analyst for the Detennination o f Perfluorooctanoic A cid (PFO A ) in Vegetation by LC/M S/MS 5.4 55 5.10 s .n 5.12 5.13 5.14 5.15 Rotary evaporator. 125 m L pear-ehaped flasks. SO m L disposable polypropylene centrifiige tabes. 15 m L disposable polypropylene centrifuge tubes. D isposable m icro p ip e ts (5<M 00uL, 100>200uL). 125-mL LDPE narrow -m outh bottles. 2 m L clearH P L C vial k it Disposable pipettes. A utopipettes (100-1000 p L and 10-100 pLX w ith disposable tip. S P 6 tubes (20mL) (Supelco eat. no. N057177). W rist action shaker. CsntrifUge capable o f g in n in g 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 A nalytical Colum n: Fhiophaae R P (K eystone Scientific), 2.1 m m x 50 m m, 5p (P/N: 62505-052130) 6.2 Tem perature: 30*C 6.3 M obile Phase (A ) : 2 mM Am m onium Acetate in W ater 6.4 M obile Phase (B ): M ethanol 6.5 Gradient Program: Tune fminl 0.0 1.0 8.0 20.0 22.5 2LA 65 65 25 25 65 Flow Rate & fm L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6 .6 In jection V olum e: 15 p L (ca n b e in creased to a s m u c lia s 5 0 mL). 6.7 Quantitation: Peak A re a -e x te rn a l standard calibration curve. 6.8 R un Tim e: - 23 minutes. T he above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/MS System 7.1 M ode: Electrcepray N eg ativ e M R M m ode, m o n ito rin g 41 3 369 m /z for PFOA. Page 3 of 7 Page 47 o f 65 Exygen Research Page 88 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 Exygen Rrnarcb Method Number V0001784 I ANALYTICAL M ETH O D M ethod o f Analysis for the Determination ofPerfluorooctanoic Acid (PFO A ) in Vegetation b y LC/M S/M S The above conditions are intended aa a guide and m ay b e changed in order to optim ize the M SM S system. g.O P reparation o f S olutions 5.1 M obile Phase 8.1.1 2 m M am m onium acetate in w a te r is p rep ared b y ad d in g 0.154 g o f ammonium acetate to 1000 m L o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% asoortric a d d in m ethanol is prepared b y dissolving 2 g o f ascorbic a d d in 100 a L o f m ethanol. 30% Dim ethytdichlorodiane in toluene is prepared by bringing 3 mL ofdunethyklichlofostiane to a final volum e o f 10 m L w ith toluene. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Slock/Fortificstion Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f -1 0 0 pg/m L o f PFO A by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with m ethanol in a 125-mL LD PE bottle. A 1.0 pg/m L fortification solution o f PFO A is prepared by bringing 1 m L o f th e 100 p g /ta L so lu tio n to a final v o lu m e o f 100 w ith methanol in a 123 m L LDPE bottle. A 0.1 pgfaiL fortification so lu tio n o f P F O A is prepared b y brin g in g 10 m L o f tiw 1.0 pg/m L solution to a final volum e o f 100 w ith methanol in a 12S m L LDPE bottle. A 0.01 pg/m L fortification solution o f PFO A is prepared by bringing 10 m L o f the 0.1 pg/m L solution to a final volum e o f 100 with methanol in a 123 m L LD PE bottle. The stock and fortification solutions are to be stored in a refrigerator at approxim ately 4*C and are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC /M S/M S calibration standards are prepared in m ethanol via dilution o f file 1.0 pg/m L fortification solution. Page 4 u i" Page 48 o f 65 Exygen Research Page 89 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen P ro to co l N um ber: P 0 0 0 1131 ExygtaJUmicb Method Number V00017S4 a n a l y t ic a l m e t h o d M ethod o f Analysis for the Determination o f Perfluorooctm oic A cid (PFO A ) in Vegetation by LO M S/M S 92,2 The following is a typical example: additional concentrations m ay be prepared as needed. Concentration ofFortifia tion Solution fuifaiL) 1.0 1.0 1.0 0.03 0.025 0.1 0.005 Volume ftU 5.0 15 1.0 10 10 10 10 Diluted to (mL) 100 100 100 100 100 100 100 Final Concentration (ua/m U 0.05 0.025 0.01 0.005 0.0025 0.001 0.0005 9.2.3 Store all calibration standards in 125-m L L D PE narrow -m outh bottles at 2*C to 6*C, up to six m onths. 9 2 A A lternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 Batch S et Up 10.1 E ach batch o f sam ples extracted (typically 20 o r less) m u st include at least one untreated control and tw o untreated controls fortified st known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirem ents for field end laboratory duplicates and spikes will be specified in the quality assurance plea for Out project. 11.0 Sam ple Extraction 11.1 1 1.2 11.3 11.4 11.5 11.6 11.7 W eigh 5 g o f frozen sam ple in to SO m L p o ly p ro p y len e centrifuge lubes (fortify as needed, replace lid and m ix well). A dd 30 m L o f acetonitrile end shake on a w rist action shaker fo r-1 $ minutes. Centrifuge the 30 m L polypropylene tubes containing sam ple at 2000 rpm for 10 minutes. Pack and condition the SPE tubes and ailanize foe pear-shaped flasks. Pack foe 20 m L SPE tubes in sequence w ith 2 g florisil, 2 g silica gel. 2 g carbon, and 1 g LC-NHr- Condition foe colum ns w ith 20 m L o f methanol, then 20 m L o f acetonitrile. D iscard all w ashes. D o not allow the colum n to drySilanize foe 12S m L pear-shaped flasks by rinsing w ith the 30% diraefoyldichloroeilane In toluene solution. R inse foe flask w ith toluene once, followed by m ethanol (three tim es). D ry foe flaaks com pletely before use. either by air-drying or with a stream o f nitrogen. Decant foe extract on to a conditioned SP E colum n fitted inside the m outh o f the pear-shaped flask. C ollect foe oluato in the 125 m L silanized pear-shape flask. Page S o f ' Page 49 o f 65 Exygen Research Page 90 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 Exygra RtaMrch Mttbod Number V00OI784 | A?\a LVT1CAJL METHOD ....................~ M ethod o f Analysis for the Determ ination ofPerfluorooctanoic A cid (PFO A ) in Vegetation by LC/M S/M S 11.8 A dd 20 m L o f acetonitrile to the a m p le in the 50 m L centrifuge tube. 11.9 Shake die a m p le again fo r~ 1 0 m inutes on a w rist-action shaker. 11.10 C entriftige th e SO m L polypropylene tubes c o n ta in in g sa m p le at - 2 0 0 0 rpm fo r-S minutes. 11.11 D ecant th e Detract on to th e sam e S P E colu m n . C o lle c t the elu a te into the sam e pear-ehaped flask and com bine w ith the eluent from the initial extraction. 11.12 Repeal steps 11.8 through 11.t l 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 <40*C ). 11.14 M ake the final volum e, b y adding 2 m L o f 2% ascorbic acid in m ethanol to the pear-shaped flask and swirl to mix/disaolve. 11.15 T ransfer the extracts to H P L C v ials u sin g disp o sa b le p ip e d . 11.16 Analyze samples using electrospray LC/M S/M S. 12.0 Chrom atography 12.2 12.3 12.4 12.3 Inject th e sam e am ount o f each standard, sa m p le a n d fortified sam ple into the LC /M S/M S system . A calibration stan d ard m u st p reced e and follow ull snalyzed samples. Standards o f PFO A corresponding to i t least Ave o r m ore concentration levels m ust be included in an analytical set. A n entire set o f extracted calibration standards m ust be included at the beginning and at the end o f a sam ple s e t Extracted standards must be interspersed betw een every 5-10 sam ples. A s an alternative, an entire set o r extracted calibration standards m ay be injected at the beginning o f a set followed b y extracted calibration standard interspersed every 3-10 samples (lo account for a second set o f extracted standards). In either case, extracted calibration standards m ust be tile first and last injection in a sam ple set. U se linear standard curves fo r quantitation. Linear standard curves are generated for the analyte by Unoar regression using 1/x weighting o f peak area versus calibration standard concentration using M issL ynx 3.3 (or equivalent) software system. Sam ple response should no t exceed standard responses. A ny sam ples that exceed standard m p o o w i should be Anther diluted and reanelyw d. 13.0 Acceptance Criteria 13.1 C hrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. T he 413 am u parent corresponds to the PFO A anion, w hile the daughter ion (369 amu) represents the loss o f carbon dioxide. Pag* o f 7 Page 50 o f 65 Exygen Research Page 91 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 BxjfM RflMHCh Method Number V0001754 ANALYTICAL M ETH O D M ethod o f Analysis for the Determ ination o f Periluorooctanoic A cid (PFO A ) in Vegetation byLC /M S/M S 13.2 13.3 13.4 13.5 13.6 M ethod blank* m ust not contain PFO A at level* greater than the LOQ. If a blade contain* PFO A at level* greater than 0.3 ppb, then a new blank sample m ust be obtained and the entire sot m ust be re-extracted. R ecoveries o f control spikes an d m atrix spikes m u st b e b e tw e en 70-13QV* o f their know n values. I f a control spike falls outside th e acceptable lim its, the entire set o f samples should b e re-extracted. A ny calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve. However, the total num ber o f calibration standard! that could be excluded m ust not exceed 20% o f the total num ber o f standards injected. The correlation coefficient (R ) for calibration curves generated must be 0.992 (R* 0.985). I f calibration results foil outside these lim its, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or die relevant set o f sam ples should be reanalyzed. Retention tim es betw een standards and sam ples m ust not drift m ore than 1 4 % within an analytical ran. I f retention tim e drift exceed* this lim it within an analytical run then the aet m ust be reanalyzed. 14.0 Calculations 14.1 U se th e follow ing equation to calculate the am ount o f PFO A found (in ng/m L, baaed on peak area) using the standard curve (linear regression parameters) generated b y the M ass Lynx software program: PFOA found (ng/m L) - fPcak area - intercept! slope 1 4 1 U se the following equation to convert the am ount o f PFO A found in ng'm L to ng/g(ppb). P F O A found fppM (P F O A found fn e/m lA x final v o lu m e tm U r DF1 sample w eight (g) DF A ctor by w hich foe final volum e w as diluted, if necessary. 14.3 For sam ples R atified w ith known am ounts o f P F O A p rior to extraction, use die following equation to calculate the percent recovery. Recovery (%) - [to tal analyte found (ng/g) - analyte found in control (ng/g)] analyte added (ng/g) ^ Page 7 u l ' PageSI o f 65 Exygen Research Page 92 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P00 0 1 1 31 ANALYTICAL METHOD M ethod N um ber V0001785 M ethod o f A nalysis fo r th e D eterm ination o f P erflo o ro o ctaao lc A d d (P F O A ) la Sm ell M am m al L iver by LC /M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College. PA 16801 Approved By: ____C -- Q y a ___________ Paul Connolly I Technical Leader. LC-M S, Exygen Research d - / / ________ J6 h n Flaherty / Vice President, Operations, Exygen Research ____ 1 Due D ue Exygen Research Total Pe.es: 7 Page 52 o f 6S Page 93 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No. : P0001131 E xygen Pro to co l N um ber: POOO1131 ExygMRMMicIi Method Number VOOO17S5 | ................................ .... AiSALYTICAL M T H O P ................. M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Sm all M ammal Liver b y LC/M S/M S 1.0 Scope This m ethod is to be employed for the isolation and quantitation o f perfluorooctanoic acid b y H igh Perform ance Liquid Chrom atography coupled to a tandem Maas Spectrom etric Detector (LC/M S/M S) in sm all m am m al liver. 2.0 Saftty 2.1 A lw ays o bserve sa fe laboratory p ractices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safety precaution. 3.0 Sam ple Requirement 3.1 A t least 5 g o f teat sam ple fo r extraction. 3.2 Sam ples should b e processed before extraction. P lace the frozen sam ple in a food processor and hom ogenize w ith dry ice. P lace the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the sam ples in frozen storage until tim e o f analysis. Alternately, if there is an insufficient am ount o f sam ple (-le ss then 5 g). then no processing is necessary and the sam ple can be used as supplied. 3.3 Sam ple collection procedures will be specified in the sam pling plan for this project. 4.0 Reagents and Standard 4.1 W a te r-H P L C grade 4.2 M ethanol - HPLC grade 4.3 A cetonitrile-H P L C grade 4.4 Am m onium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic A d d - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5*200 p L connected to a tandem Mass Spectrom eter (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 m L disposable polypropylene centriftige tubes. 5.5 15 m L disposable polypropylene centriftige tubes. 5.6 D isposable m icropipets (SO-lOOuL, !0 0-200uL ). 5.7 125-mL LD PE narrow -m outh bottles. 5.8 2 m L clear HPLC vial k it pgc2or>` Page 53 of 65 Exygen Research Page 94 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 ExyaxalUm tch M tbod Number V0001715 I ANALYTICAL M ETH O D ........ M ethod o f Analysis for the Determination ofPerfluorooctanoic A cid (PFO A ) in Small M ammal Liver b y LC/M S/M S 3.9 3.10 5.11 5.12 5.13 5.14 5.15 Diapoaable pipette*. Autopipottes (100-1000 pL and 10-100 pL ), w ith disposable tips. W aters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. SPE vacuum manifold. T issuem izcr. W rist-action shaker. Centrifuge capable o f spinning 15 raL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 A nalytical Colum n; Fluophase R P (K eystone Scientific), 2.1 m m x 50 m m . 5m (P/N: 82505-052130) 62 Temperature: 30*C 6.3 M obile Phase (A ) : 2 m M Am m onium A cetate in W ater 6.4 M obile P h m e (B ): M ethanol 6.5 Gradient Program; Tim e (mini 0.0 1.0 8.0 20.0 22.5 U 65 65 25 23 65 Ufi . 35 35 75 75 35 Flow Rate fm lA nin) 0.3 0.3 0.3 0.3 0.3 6.6 Injection V olum e: IS p L (can b e increased to as m uch ax 50 pL ). 6.7 Quantitation: Peak A rea - external standard calibration curve. 6.8 R unT im e: - 2 3 minutee. The above conditions are intended as a guide and m ay b e changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: E lectiocpray N egative M R M m o d e, m o n ito rin g 413 --36 9 m /z for PFOA. T h e above conditions are intended as a guide and m a y b e c h a n g ed in order to optim ize the M SM S system. Page) o f' Page 54 of65 Exygen Research Page 95 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xygen P ro to co l N um ber: P 0 0 0 1131 b y p altaN n k I M nkwl Number V000I7M 1AN/UATICA . METHOD_______________________ M ethod o f A nalysis for the D eterm ination o f Perfluorooctanoic A cid (PFO A ) in Small M ammal Liver b y LC/M S/M S 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w a te r ia p rep a re d b y ad d in g 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volume! m ay be prepared . 9.0 Standard Preparation 9.1 Standard Stock/Foctification Solution 9.1.1 P repare a lock solution o f ~ 1 0 0 )tgfoiL o f P F O A b y w eig h in g 10 m g o f analytical standard (corrected for purity) and dilute to 100 m L with m ethanol in a 125-mL LD PE bottle. 9.1.2 A 1.0 pg/raL fortification solution o f PFO A is prepared by bringing I m L o f foe 100 pgftnL so lution to a final v o lu m e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 0.1 pgAnL fortification so lution o f P F O A ia p rep ared b y b ringing 10 m L o f foe 1.0 pgfaiL solution to a final v o lu m e o f 100 w ith methanol in a 125 m L LD PE bottle. 9.1.4 Tbe stock and fortification solutions are to b e stored in a refrigerator at approxim ately 4C sod are stable for a m axim um period o f 6 months from die date o f preparation. 9.2 Standard Calibration Solution 9.2.1 9.2.2 LC/M S/M S calibration standard# are prepared in m ethanol via dilution o f the 0.1 p g fa L fortificatim i solution. T he following is s typical exam ple: additional concentrations m ay be prepared as needed. Concentration o f Fortification Solution ina/raL) 100 100 100 5.0 2.0 1JO Voltane (mL) 5.0 2.0 IA 10 10 10 Diluted to (mL) 100 100 100 100 100 100 Fine! Concentration (na/m L ) 5.0 2.0 1.0 0.5 0.2 0.1 at 2*C to 6*C, up to six m onths. 9.2.4 Alternate volum es and concentrations o f standards m ay be prepared as Page4 of 7 Page 55 o f 65 Exygen Research Page 96 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xygen P ro to co l N um ber: P 0 0 0 1131 Biyfca keataick Mwbod Number V000i785 | ANA LYTICAL M E T H O D ............... M ethod o f Analysis for the Determ ination o f P erfluorooctsnoic A cid (PFO A ) in Smell M ammal Liver by LC/M S/M S 10.0 Batch Set Up 10.1 E ach batch o f sam ples extracted (ty p ically 2 0 o r less) m ust include at least one untreated control and tw o untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 1 1.1 113 113 11.4 11.5 11.6 11.7 11.8 11.9 11.10 11.11 W eigh 1 g o f sample into a 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and m ix well). N ote that alternate w eights o f liver may be m easured depending on the sam ple size available for use. A dd w ater to the sample for a final volum e o f 10 m L. H om o g en ize sam p le u sin g a tiasu em izer Ah' - 1 m in u te. Transfer 1 m L o f foe sample using a disposable pipette into a IS mL disposable eentrifoge tube. A d d 5 m L o f acetonitrile an d sh ak e fo r <*20 m in u te s o n a w ristaction shaker. Centrifuge foe tubes st *3000 rpm for - 5 m inutes. Decant foe supernatant into a 50 m L disposable eentrifoge tube and add 35 m L o f water. Condition the C ii SPE oaitridgee ( l g, 6 m L) by passing 10 m L methanol followed by S m L o f HPLC w ater (~ 2 drop/sec). D o not let colum n run dry Load th e sam ple on conditioned C n SPE cartridge. D iscard eluate. Elute with - 2 m L o f methanol. C ollect 2 m L o f eluate into a graduated IS m L polypropylene eentrifoge tube (final volum e * 2 raL). Analyze samples using eloctrospray LC/M S/M S. 12.0 Chrom atography 12.1 Iqject th e sam e am ount o f ea ch standard, sa m p le a n d fortified s am p le into the LC /M S/M S system . A calibration standard m ust precede and follow all analyzed samples. 123 Standards o f PFO A corresponding to at least five or m ore concentration levels m ust be included in an analytical s e t 12.3 A n entire set o f calibration standards m u st b e in clu d ed at th e begin n in g and at the end o f a sam ple s e t Standards m ust be interspersed betw een every 5-IO samples. A s an alternative, an entire set o f calibration standards may be injected at the beginning o f a set follow ed by calibration standards interspersed every 5-10 sam ples (to account for a second set o f standards), in either case, calibration standards m ust be the first and last injection in a sam ple set. 12.4 U se linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x w eighting o f peak area Page 5 o f? Page 56 of 65 Exygen Research Page 97 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xygen P ro to co l N um ber: P 0 0 0 113! r^ ExysenRamrcfa Mcfcod Number VOOOI7S5 I ANALYTICAL M ETH O D ...... M etbod o f A nalysis f t r the D eterm ination ofP erfluorooctaooic A cid (PFO A ) in Smalt M am m al Liver by LC/M S/M S versus caKbrttioo standard concentration using M assLynx 3 3 (or equivalent) software system. 12.3 Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.3 13.6 Chromatogram m ust ahow a peek o f a daughter ion at 369 am u from a parent o f 413 amu. The 413 amu parent corresponds to the PFO A anion, while the daughter ion (369 amu) represents the lose o f carbon dioxide. M etbod blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFOA at levels greeter then 10 ng/g, then a new blank sample m ust be obtained and the entire set m ust be reextracted. Recoveries o f control spikes and m atrix spikes m ust be betw een 70*130% of their know n vaiuee. I f a control spike falls outside the acceptable lim its, the entire set o f samples should be re-extracted. A ny m atrix spike outside 70 130% should be evaluated b y the analyst to determ ine i f re-extraction is w arranted. A ny calibration standard found to b e a statistical outlier by using the Huge Error Test, m ay be excluded from die calculation o f the calibration curve However, the total num ber o f calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f standards injected. T he correlation coefficient (R ) for calibration curves generated m ust be 20.992 (R* 20.983). I f calibration results fall outside these limits, then appropriate step! m ust be taken to adjust instrum ent operation, and the standards or die relevant set o f samples should be reanalyzed. Retention tiroes betw een standards and sam ples m ust not drift m ore than 1 4 % w ithin an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se the following equation to calculate th e am ount o f PF O A found (in ng/mL, baaed on peak area) using the standard curve (linear regression param eters) generated by the M ass Lynx software program : PFO A found (ng/mL) slope x O F x aliquot factor O F * factor b y w hich the final volum e w as diluted, if necessary. A liquot fa c to r* 10 Pigs 6 o f 7 Page 57 o f 65 Exygen Research Page 98 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 ExygnfUMvek Method Number VOOOI785 | ................................ AN ALYTICA L M ETH O D M ethod o f Analysis for the Determ ination ofP erfluoroocU ooic A cid (PFO A ) in Smalt M amm al Liver by LC/M S/M S 14.2 F or sam pler fortified w ith know n am ount! o f PFO A prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) - [to tal analyte found (n gfaL ) - analyte found in control (ng/m L)] ^ analyte added (ng/m L) 14.3 U se the following equation to convert the am ount o f PFO A found in ng/mL io ng/g(ppb). PFOA found (p p b )-fP F O A found fna/m L) x final volum e (m ) sample weight (g) Exygen Research Page 7 of7 Page 58 o f 65 Page 99 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen Pro to co l N um ber: POOQ1131 ANALYTICAL METHOD M ethod N um ber V001786 M ethod o f A nalysis for th e D eterm leettoo o f P e rP u o ro o cto n o k A d d (PPO A ) lo Sm alt M am m al S e n n a by LC /M S/M S Analytical Teeting Facility: Exygen Research 3058 Research Drive State College, FA 16801 Approved By: Paul Connolly I Technical Leader, LC-M S, Exygen Research __ D ate Jo h n Flaherty / Vice President, Operations, Exygen Research Date Exygen Research Total Pagai: 7 Page 59 o f 65 Page 100 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study N o.: P0001131 E xygen Protocol N um ber: P 0 0 0 1131 ExypoRMMtch Method Number V00017I6 I N M /i TIC a L M ETH OD M ethod o f A nalyua for the D eterm ination o f Perfluorooctanoic A cid (PFO A ) in Small M amm al Serum by LC/M S/M S 1.0 Scope Thi* m ethod ii to be employed for the ieolation and quantitation o f perfluorooctanoic acid by High Perform ance Liquid Chrom atography coupled to a tandem Mass Spectrometric D etector (LC/M S/M S) in small m am m al serum. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirement 3.1 A t least I m L o f teat sam ple for extraction. 3.2 N o tanqrie processing is needed for eerum sam ples. H ow ever, frozen serum samples m ust to allowed to com pletely thaw to room tem perature before use. 3.3 Sam ple collection procedures will be specified in th e sam pling plan for this p ro je c t 4.0 Reagents and Standards 4.1 W a te r - H P L C f i d e 4.2 M ethanol - HPLC grade 4.3 Acetonitrile - HPLC grade ^ 4.4 Am m onium Acetate - A C .S . Reagent Grade 4.S Perfluorooctanoic A cid - Sigm s-A ldrich 5.6 Instrument and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5 .1 1 5.12 5.13 A high performance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e irqector capable o f injecting 5*200 pL connected to a tandem Maas Spectrom eter (LC/M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene centriftige tubes. 15 m L disposable polypropylene centriftige tubes. Disposable micropipets (50*100uL, 100-200uL). 12 5-m L L D P E n arrow -m outh bottles. 2 m L clear HPLC vial kit. Disposable pipettes. A utopipettes (100*1000 p L and 10-100 pL ), w ith disposable tips. W aters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. SPE vacuum manifold. Vortaxer. Pge J oH Page 60 o f65 Exygen Research Page 101 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b er: P 0 0 0 1131 Exyy a lUmrcb MethodNumber V000I7H6 I A1W .V T1C A 1. M ETH O D M ethod o f A nalysis tar th e D eterm ination o f P erfhiorooctanoic A c id (P F O A ) in Sm all M ammal Scrum by LC W S/M S 5.14 W rist-action ibiker. 5.15 C entrifuge capable o f pinning 15 raL polypropylene tubes at 3000 rpm 6.0 Chromatographic System 6.1 A nalytical Colum n: Fluopfaase R P (K eystone S cientilic), 2 .1 m m x 50 m m . 5p (P/N: 82505-052130) 6.2 Tem perature: 30*C 6.3 M obile Phase (A) : 2 m M Am m onium Acetate in W ater 6.4 M obile Phase (B ): M ethanol 6.5 Gradient Program : Time (mini 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate & fm L/m in) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Infection Volume: 15 p L (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak A re a -e x te rn a l standard calibration curve. 6.8 R unT im e: - 2 3 minutes. T he above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: E lectrospray N egative M R M m ode, m o n ito rin g 413 --369 m /z for PFOA. T h e above conditions a re intended as a guide an d m a y b e ch an g ed in order to optim ize the M SM S system. 8.0 Preparation o f Solutions 8.1 M obile P hsse 8.1.1 2 m M am m onium acetate in w ater is prepared b y adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volumes m ay b e prepared. pat3uf? Page 6 / o f 65 Exygen Research Page 102 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 B a y fo l sssaich _____________________________________ Method Number V000178& I ........ ANALYTICAL M ETH O D M ethod o f A m lyii* for the D eterm ination ofP erfluorooctanoic A cid (PFO A ) in Small M ammal Serum by LC/M S/M S 9.0 Standard Preparation 9.1 Standard Stock/F ortification Solution 9.1.1 Prepare a stock so lution o f - 1 0 0 n g /m L o f P F O A b y w eighing 10 m g o f analytical standard (corrected fo r purity) and d ilu te to 100 m L with m ethanol in a 125-mL LD PE bottle. 9.1.2 A 1.0 pgfaiL fortification solution o f PFO A ia prepared by bringing I r a L o f the 100 p g /ra L so lu tio n to a final vo lu m e o f 100 w ith methanol in a 123 m L LDPE bottle. 9.1.3 A 0.1 pgftnL fortification so lution o f P F O A is p rep ared b y bringing 10 m L o f t b e 1.0 j ig t a L so lution to a final v o lu m e o f 100 w ith methanol in a 123 m L LD PE bottle. 9.1.4 The stock and fortification aohitioni are to be stored in a refrigerator at approximately 4*C and are stable for a m axim um period o f 6 months fiora the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/M S/M S calibration standards are prepared in m ethanol via dilution o f th e 0.1 p g /m L fortification solution. The following is a typical example: additional concentrations m sy be Concentration o f Fortification Solution (nt/mL) Volume (mL) Diluted to (mL) Final Concentration (ng/mL) 100 3.0 100 2.0 100 1.0 3.0 10 100 100 100 100 3.0 2.0 1.0 0.5 2.0 10 100 1.0 10 100 0.2 0.1 9.2.3 Store sll calibration standards in 125-m L LD PE narrow -m ouih bottles at 2C to 6*C, up to six m onths. 9.2.4 A ttenute volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 E ach batch o f sam ples extracted (typically 2 0 o r leas) m ust include at least one untreated control and tw o untreated controls fortified at known concentrations (lab control apike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. Page 4 of? Page 62 of65 Exygen Research Page 103 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 E xy g en P rotocol N um ber: P 0 0 0 1 131 ExygaaRamrcb Method Number VD0017S6 I ANALYTICAL METHOD M ethod o f A nalysis for the D eterm ination o f Perftuorooctanoic A cid (PFO A ) in Small M ammal Serum by LC/M S/M S 11.0 Sam ple Extraction 11.1 M easure 1 m L o f sam ple into a SO m L poly p ro p y len e c e n trifu g e tubes (fortify as needed, replace ltd and m ix w ell). N ote that alternate volum es o f serum m ay be m easured depending on the sam ple size available for use. 11.2 Add water to the sam ple for a final volum e o f 20 m L. C ap tightly 11.3 Vortex for - 1 m inute. 11.4 T ransfor 1 m L o f the sam ple using a disposable pipette into a 15 m L disposable oentrifoge tube. 11.5 A dd 5 m L o f acetonitrile and shake for - 2 0 m inutes on a wrist-action shaker. 11.6 Centriftige the tubes at -3 0 0 0 rpm for - 5 m inutes. 11.7 Decam the supernatant into a 50 m L disposable centriftige tube and add 35 m L o f water. 11.8 C on d itio n th e C S P E cartridges <1 g, 6 m L ) b y p a s sin g 10 m L m ethanol followed b y 5 m L o f H PLC w ater ( - 2 drop/sec). D o not let colum n run dry 11.9 Load foe sam ple on conditioned C u SPE cartridge. D iscard eluate. 11.10 Elute w ith - 2 m L o f m ethanol. C ollect 2 m L o f eluate into a graduated 15 m L polypropylene centriftige tu b e (fin al v o lu m e 2 m L ). I t . 11 A nalyze sam ples using electrospray LC /M S /M S . 12.0 Chrom atography 12.1 12.2 12.3 12.4 12.5 Iqjeet the sam e am ount o f each standard, sam ple and fortified sam ple into the LC /M S/M S system. A calibration standard m ust precede and foltow all analyzed samples. Standards o f PFO A corresponding to at least five o r m ore concentration levels m ust be included in an analytical set. A n entire set o f calibration standards m ust b e included at the beginning and at the end o f a sam ple set. Standards m ust be interspersed betw een every 5'H* sam ples. A s an alternative, an entire set o f calibration standards m ay be injected at the beginning o f a set followed b y calibration standards interspersed every 5 -10 sam p le! (to account fo r a s e co n d set o f standards). In either case, calibration standards m ust be the first and last injection in u sam ple set. U se linear standard curves fo r quantitation. Linear standard curves are generated for the analyte by linear regression using l/x w eighting o f peak areo versus calibration standard concentration using M assLynx 3.3 (or equivalent) softw are system. Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be ftuther diluted and reanalyzed. Page Suf / Page 63 o f 65 Exygen Research Page 104 of 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 0 0 0 U 3 1 Exyge* Kestaicli MethodNumber V00017*6 I a n a l y t ic a l m e t h o d M ethod o f Analysis lo r the D etom ination o f Perfluorooctanoic A cid (PFO A ) in Small Mamma! Seram by LC/M S/M S I 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chrom atogram m oat show a peak o f a daughter ion at 309 em u tram a parent o f 413 amu. The 413 am u parent corresponds to the PFO A anion, while the daughter km (369 am u) represents the loss o f carbon dioxide. M ethod blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFO A at levels greeter than 10 ng/m L, then a new blank sample m ust be obtained and the entire set m ust be re-extracted. Recoveries o f control spikes and m atrix spikes m ust b e betw een 70-130% of their know n values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. A ny m atrix spike outside 70 130% should be evaluated by th e analyst to determ ine if re-extraction is warranted. A ny calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve How ever, the total num ber o f calibration standards that could be excluded m ust not exoeed 20% o f the total num ber o f standard! injected. T he correlation coefficient (R) for calibration curves generated m ust be 20.992 (R* 20.985). I f calibration results foil outside these lim its, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards o r the relevant set o f samples should be reanalyzed. R etention tim es betw een standards and sam ples m ust not drift more than 4 % w ithin in analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate th e am ount o f P F O A found (in n g /m L baaed on peak area) using the standm d curve (linear regression parameters! generated by the M ass Lynx softw are program : PFOA found (ng/mL) (Peak i r e s . intercept! x D F x aliquot factor slope DF foctor by which the final volum e w as diluted, if necessary. Aliquot factor - 2 0 14.2 For sam ples fortified w ith know n am ounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. Recovery ( % ) - [ total analyte found (ng/m L) - analyte found in control (ng/m L)] .[)Q0 analyte added (ng/mL) Pagf6 o f7 Page 64 o f65 Exygen Research Page 105 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 Exygen Pro to co l N um ber: P 0 0 0 1 131 Bayian anarch_________________________________ ________________ Method NaabtiVOOO 1716 I AWa H T I C A L M E T H O D M ethod o f A n aly aii for th e D e ta ra in a d a o o f P e ril uorooctanoic A cid (P F O A ) in Sm all M ammal Scrum by LC/M S/M S \ 14.3 U se the following equation to convert the am ount o f PFO A found in ng'm L to ppb. PFO A found (ppb) - fPFOA found fae/m L l i final volum e (m L ll sample volum e (mL) Exygen Research P aacton Page 65 o f 65 Page 106 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 S tate College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment Number 1 Effective D ate: 0 1/19/05 Exygen Study Number P0001131 Client Study N u m b er Pagel of 1 D ESC R IPTIO N O F AM ENDED SE C TIO N 1) Analytical Procedure Sum m ary V0001780:Sectk>n 9.1 2 ) Verification o f Analytical Procedure None AMENDED TO 1) Add to Section 9.1 : Section 9 .1 .6, Alternate weights o f standards m ay be used to prepare alternate concentrations o f stock solutions as necessary. A lternate levels of fortification solutions may also be prepared. 2 ) Low and high spiking levels o f the analytes for each matrix m ay be altered depending on sam ple size available for extraction and/or to cover analyte concentrations expected in the sam ples. BATIQUAU; 1 ) Higher concentrations of standards need to be prepared in order to spike the sam ple bottles at higher levels. 2 ) The sam ple size avaBable for small mammal Iv e r and serum w as sm aller than expected. Spiking at the pre-determ ined levels hi the protocol puts the spiked concentration low er than the detection lim it Also, the analyte levels in the ground w ater sam ples are expected to greatly exceed the pre-determ ined spiking levels listed In the protocol. W hen the levels hi the sam ples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the Q C sam ple. Higher spiking levels In the bottles w ill cover th e analyte concentrations expected in the w ater sam ples. IM PACT O N S TU D Y T h e LOQ is 100 ng/g fo r a 0.1 g sam ple o f small m am mal liver and is 1000 ng/m L fo r a 0.01 mL sam ple o f sm all mammal serum. Higher levels of spiking fo r the w ater sam ples w ill ensure that more Q C recovery data can be used. LIBRARY ID: W000122S= . .. '' ADMINISTRATIVE FORM Exygen Research Page 107 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No. : P0001131 3058 Research Drive Phone: 814-272-1039 S tate College, PA 16801 Fax: 814-231 -1580 Amendment Numben Effective Date: Exygen Study Number PROTOCOL AMENDAIENT 2 03/07/05 P0001131 Client Study Numben Pao i <* 1 None D ESC R IPTIO N O F AM ENDED SE C TIO N 1 ) Report, page 11 o f 65 2 ) Test M aterials, page 6 o f 65: PFO S transition monitored 499 -> 99. AMENDED TO 1) Instead o f one Anal report, interim reports will be Issued. 2 ) P FO S transition monitored m ay also be 4 9 9 -> 80. B&nQN&E 1) Due to the excessive sizes o f the data sels, interim reports wfll be issued to allow the client to receive data in a tim elierreanoFi * * * * ' otln /ir 2 ) The A P I 4 0 0 0 LC /M S/M S systems detect the 4 9 9 -> 80 PFO S transition with greater sensitivity than the 4 9 9 -> 99 transition. IMPACT Q N STU DY 1) T h e d to nt w ill be able to receive and review the data m ore quickly. 2 ) The 4 9 9 -> 80 transition can be detected with greater sensitivity, therefore, giving better chrom atography. LIBRARY ID: VD00122e-8- Exygen QAU R eview . JJ (uloslts ADMINISTRATiyE FORM Exygen Research Page 108 o f 110 Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 RESEARCH 3058 R esearch D riv e S ta te C o lle g e , PA 16801 Phone: 814-272-1039 Fax: 81 4 -2 3 1 -1 5 8 0 D EVIATIO N FORM G en eral: X Project Specific Deviation Facility Deviation ______________________ Page 1 of 2 D ate of Occurrence: 0 4 /26 /0 6 Exygen Project # : P 7 6 0 /P 1 1 3 1 Deviation # : 5 Client Project # : NA R eference # : 0 6 -0 7 6 R eo u lato rv D riv e r: D eviation T yp e: (Include V# for methods and SOPs) GMP GLP O ther None Sam ple D escrip tio n : X Protocol ______ M ethod V #:N A Notebook reference: NA _____ SO P L o g in # : L0008191 C o n ta in e r# : NA Lot#: NA S u m m ary o f D eviatio n : The three sedim ent sam ples in L8191 (C 0 1 72892 - C 0 1 7 2 8 94 ) w ere originally extracted using the sedim ent method V 0001782. Poor recoveries w ere obtained for P F O S , P FO A and ,3C PFO A. Because o f this, the study sponsor requested the use of an alternative extraction for these compounds, as follows: D ire c t In je c tio n M eth od : Before the sam ples w ere w eighed fo r the extraction, they w ere mixed thoroughly by vigorously shaking the container. A one-gram portion of sedim ent w as w eighed into a 15-m illiliter centrifuge tube for the extraction. Ten m illiliters o f 1% acetic acid in m ethanol w as added to each sam ple. T he sam ples w ere then shaken by hand, vortexed, and sonicated for thirty minutes. The sam ples w ere then centrifuged for -1 0 minutes a t -3 0 0 0 rpm . Each sam ple w as analyzed by LC /M S /M S electrospray. Using this m ethod acceptable data was obtained for PFO S , but the recoveries fo r PFO A and ,3C PFO A w ere still poor. Another alternative method w as then used for P FO A and <3C PFO A , as follows: A ltern a tiv e S P E M ethod: The sam ples that w ere prepared In 1% acetic acid fo r the direct injection method w ere used for this extraction. Five m illiliters of each sam ple w as aliquoted into a 50-m L polypropylene centrifuge tube and the volum e w as taken to 4 0 m L w ith w ater. The sam ples w ere then centrifuged for -1 0 m inutes at -3 0 0 0 rpm . The supernatant w as then loaded onto a C 1t SP E cartridge conditioned w ith 10 mL of m ethanol and 5 mL o f w ater. T he eluate w as discarded. Approxim ately five m illiliters of m ethanol w as added to the cartridge. Five m illiliters o f eluate w as collected into a graduated 15 mL polypropylene centrifuge tube. Each sam ple w as analyzed by L C /M S /M S electrospray. Cause: Preparation A n alysis Instrum ent C lient R equest X O ther Im pact: More usable data was obtained. LIBRARY ID: V0001640-6 Exygen Research ADMINISTRATIVE FORM Page iUy o HU Interim Report #23 - Analysis o f Sediment Samples Exygen Study No.: P0001131 CHEM EHSR 236 1B 651 733 1958 05-01*2901 01:01p F r O f K iT O A SOLUTIORS 05/01 '06 13:46 NO.106 03/03 T-2M F.093/993 F-319 t,7 j*,', H f M r c h D r f v f Phorir i t a H r t o l f Statt Catlap, M1HP1 FUG S IakCK - OfVtATKWFO*:. UWWIRUMIHI4M Exygen Research * . 'r* < t . v *; . /**; t \ ` /' . . m* * ' *'* . * / , . : . { . * ' ' .. ' S ' ' " *3 . * .,/ . .. . . " * i * ** .. * . . . .* / 1 '.% * : *: i .; * , 'I !** -M i ! ." ' . Page 110 o f 110
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