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fi.G> _ II -fi? n STUDY TITLE Analysis of Pooled Human Sera and Plasma and Monkey Sera for Fluorocarbons Using Exygen Method ExM-023-071 DATA REQUIREMENTS OECD Principles of Good Laboratory Practice, ENV/MC/CHEM(98)17, November 26,1997 STUDY DIRECTOR Emily R. Decker STUDY COMPLETED ON October 30, 2002 PERFORMING LABORATORY / TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR 3M Environmental Laboratory Building 2-3E-09 St. Paul, MN 55133-3331 Phone: 651-778-6565 PROJECT Study Plan Number: ExP-023-082 Exygen Study Number: 023-082 Sponsor Study Number: E02-1071 Total Pages: 111 CONTAIN NO CBi 000318 Exygen Study No.: 023-082 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number 023-082, entitled "Analysis of Pooled Human Sera and Plasma and Monkey Sera for Fluorocarbons Using Exygen Method ExM-023-071," conducted for 3M Environmental Laboratory, was performed in compliance with OECD Good Laboratory Practice Standards (as revised in 1997), ENV/MC/CHEM(98)17 by Exygen Research, with the following exceptions: 1. 8.3 (5): The computerized system of data generation did not provide for the retention of a full audit trail to show all changes or to associate all changes to data to a timed and dated electronic signature. 2. 6.2 (4): The stability of the test items under storage or the study test conditions was not known. Also the purity of C6 acid and THPFOS was not known. 3. E, 5.2 (3): The date of receipt of for the calf serum sample ID 0204718 was not documented. 4. t, 1.2.2 (g): The instrument used for the analysis has not been qualified. William K. Reagan, Ph.D. Sponsor Representative 3M Environmental Date Exygen Research. 000319 Page 2 of 111 Exygen Study No.: 023-082 QUALITY ASSURANCE STATEMENT The Quality Assurance Unit of Exygen Research reviewed Exygen Study Number 023082 entitled, "Analysis of Pooled Human Sera and Plasma and Monkey Sera for Fluorocarbons Using Exygen Method ExM-023-071." All phases were reviewed 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 Study Director and to management. Phase 1. Protocol Review 2. Extraction, Fortification 3. Raw Data, Draft Report Review 4. Final Report Review Date Reported to Date Date Reported to Exygen Inspected Studv Director Management 10/10/02 10/14/02 10/30/02 10/15/02 10/25/02 10/25/02 Date Reported to SDonsor 10/30/02 10/30/02 10/25-28/02 10/29/02 10/30/02 10/30/02 10/30/02 10/30/02 10/30/02 10/30/02 Naomi Lovallo Technical Lead-QA i ( 3c j<n~ Date Exygen Research. 000320 Page 3 of 111 Exygen Study No.: 023-082 CERTIFICATION OF AUTHENTICITY This report, for Exygen Study Number 023-082, is a true and complete representation of the raw data for the study. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Exygen Research Exygen Research Facility Management: ^Xlohn M. Flaherty Vice President Exygen Research Sponsor Study Monitor, 3M: William K. Reagan, Ph.D. 3M Environmental Date Date Exygen Research. 000321 Page 4 of 111 Exygen Study No.: 023-082 STUDY IDENTIFICATION Analysis of Pooled Human Sera and Plasma and Monkey Sera for Fluorocarbons Using Exygen Method ExM-023-071 STUDY PLAN NUMBER: ExP-023-082 EXYGEN STUDY NUMBER: 023-082 SPONSOR STUDY NUMBER: E02-1071 TYPE OF STUDY: Residue TEST SYSTEM: Human Serum, Human Plasma, and Monkey Serum TEST ITEMS: perfluorooctane sulfonate (PFOS), perfluorohexanoic acid (C6), perfluoroheptanoic acid (C7), pentadecafluorooctanoic acid (C8), heptadecafluorononanoic acid (C9), nonadecafluorodecanoic acid (CIO), perfluoroundecanoic acid (C ll), perfluorododecanoic acid (C12), tetrahydroperfluorooctane sulfonate (THPFOS), and tetrahydroperfluorodecane sulfonate (THPFDS) SPONSOR: William K. Reagan- Sponsor Study Monitor 3M Environmental Building 2-3E-09 St. Paul, MN 55133-3331 STUDY DIRECTOR: Emily R. Decker Exygen Research Phone: (814) 272-1039 TESTING FACILITY: Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: Experimental Start Date: Experimental Termination Date: Study Completion Date: 10/09/02 10/15/02 10/23/02 10/30/02 Exygen Research. 000322 Page 5 of 111 Exygen Study No.: 023-082 PROJECT PERSONNEL The Study Director for this project at Exygen Research was Emily R. Decker. The following personnel from Exygen Research were associated with various phases of the study: Name Paul Connolly Emily Decker Xiaoming Zhu Rickey Keller Title Technical Leader-LC/MS Scientist Technician Sample Custodian Exygen Research. 000323 Page 6 of 111 Exygen Study No.: 023-082 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.............................................................................................................. II 3.0 INTRODUCTION......................................................................................................11 4.0 TEST SYSTEM......................................................................................................... 11 5.0 TEST ITEM S............................................................................................................ 12 6.0 DESCRIPTION OF ANALYTICAL METHOD...................................................... 15 6.1 Extraction Procedure........................................................................................ 15 6.2 Preparation of Standards and Fortification Solutions.......................................15 6.3 Chromatography............................................................................................... 16 6.4 Instrument Sensitivity....................................................................................... 16 6.5 Description of Instrument and Operating Conditions...................................... 17 6.6 Quantitation and Example Calculation............................................................ 17 7.0 EXPERIMENTAL DESIGN.................................................................................... 20 8.0 RESULTS................................................................................................................. 20 9.0 CONCLUSIONS...................................................................................................... 21 10.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA.................... 21 11.0 RETENTION OF DATA AND SAMPLES.............................................................21 Exygen Research. 000324 Page 7 of 111 Exygen Study No.: 023-082 Table I LIST OF TABLES Page Summary of Recoveries for Calibration Curve in Calf Serum Compared to Standards in Methanol....................................................................................23 Table II Summary of Recoveries for Calibration Curve in Human Plasma Compared to Standards in Methanol...............................................................................23 Table HI Summary of Recoveries for Laboratory Fortified Matrix Spikes....................24 Table IV Summary of Residues for Human Serum Samples......................................... 26 Table V Summary of Residues for Human Plasma Samples........................................ 26 Table VI Summary of Residues for Monkey Serum Samples........................................ 26 Exygen Research. 000325 Page 8 of 111 Exygen Study No.: 023-082 LIST OF FIGURES Page Figure 1 Chromatogram Representing 0.1 ng/mL Calibration Standard.......................28 Figure 2 Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb (Exygen ED: 0204490 Spk J, Sponsor ID: TCR-674)..................................32 Figure 3 Chromatogram Representing a Human Plasma Sample (Exygen ID: 0204490, Sponsor ID: TCR-674)................................................................................... 36 Figure 4 Chromatogram Representing a Sample Analyzed for Three Daughters for THPFDS (Exygen ED: 0204292 Dup, Sponsor ID: X328-A)........................ 40 Exygen Research. 000326 Page 9 of 111 Exygen Study No.: 023-082 LIST OF APPENDICES Page Appendix A Study Plan ExP-023-082 (Exygen Study No. 023-082) and Deviations.................................................................................................. 41 Exygen Research. 000327 Page 10 of 111 Exygen Study No.: 023-082 1.0 SUMMARY Exygen Research conducted a quantitative screening on various human serum, human plasma, and monkey serum samples for the determination of perfluorooctane sulfonate (PFOS), perfluorohexanoate (C6), perfluoroheptanoate (C7), pentadecafluorooctanoate (C8), heptadecafluorononanoate acid (C9), nonadecafluorodecanoate (CIO), perfluoroundecanoate (Cl 1), perfluorododecanoate (C12), tetrahydroperfluorooctane sulfonate (THPFOS), and tetrahydroperfluorodecane sulfonate (THPFDS) according to protocol ExP-023-082 (Appendix A). This screening was performed on an instrument that had not been used for routine fluorochemical analysis prior to this study. The method used for this study has not been validated at the levels reported for C8 and PFOS and not validated at any level for the other anions. These levels were completely dependent on instrument sensitivity. Recoveries for fortified samples are given in Tables I-III. Residues of each anion in human serum are summarized in Table IV. Residues of each anion in human plasma are summarized in Table V. Residues of each anion in monkey serum are summarized in Table VI. 2.0 OBJECTIVE The objective of this study was to screen human serum, human plasma, and monkey serum samples and quantitate to the lowest possible level according to instrument sensitivity. 3.0 INTRODUCTION This report details the results of the analysis for perfluorooctane sulfonate (PFOS), perfluorohexanoate (C6), perfluoroheptanoate (C7), pentadecafluorooctanoate (C8), heptadecafluorononanoate (C9), nonadecafluorodecanoate (CIO), perfluoroundecanoate (Cl 1), perfluorododecanoate (C12), tetrahydroperfluorooctane sulfonate (THPFOS), and tetrahydroperfluorodecane sulfonate (THPFDS) in human serum, human plasma, and monkey serum samples. The study was initiated on October 09, 2002, when the study director signed study plan number ExP-023-082. The experimental start date was October 15, 2002, and the experimental termination date was October 23, 2002. 4.0 TEST SYSTEM Pooled human serum samples were purchased by the sponsor from Sigma-Aldrich, Milwaukee, WI, Lampire Biological Laboratories, Pipersville, PA, Bioresource Exygen Research. 000328 Page 11 of 111 Exygen Study No.: 023-082 Technology, Inc., Fort Lauderdale, FL, and Golden West Biologicals, Temecula, CA. Pooled monkey serum samples were purchased by the sponsor from Lampire Biological Laboratories, Pipersville, PA. Pooled human plasma samples were purchased by the sponsor from Lampire Biological Laboratories, Pipersville, PA, Bioresource Technology, Inc., Fort Lauderdale, FL, Golden West Biologicals, Temecula, CA, and Innovative Research, Inc. Southield, MI. In addition, blank matrix consisting of pooled human plasma collected in rural China was provided by the sponsor. Also, calf serum was purchased from Sigma-Aldrich by Exygen. Exgyen ED 0203963 0203964 0203965 0204292 0204334 0204335 0204490 0204991 0204492 0204493 0204718 0204747 Sponsor ID Lot 020821 Lot 22K0965 Lot GO140604 X328-A TCR-684 TN-A-06332 TCR-674 TN-A-6337 TN-A-06333 TN-A-06336 NA TCR-683 Matrix Human Serum Human Serum Human Serum Human Serum Human Plasma Monkey Serum Human Plasma Human Plasma Monkey Serum Monkey Serum Bovine (Calf Serum) Human Plasma Source BioResource Sigma-Aldrich Golden West Biologicals Lampire Golden West Biologicals Lampire 3M (plasma from rural China) Lampire Lampire Lampire Sigma-Aldrich Innovative Research Samples were received frozen on dry ice and then placed in frozen storage ( <-10C) until samples were logged in by Exygen personnel. All records concerning sample receipt, processing and storage can be found in the raw data package associated with this study. 5.0 TEST ITEMS The analytical standards PFOS, C6, C7, C8, C9, CIO, Cl l , C12, THPFOS, and THPFDS were received at Exygen on September 30, 2002 from 3M Environmental Technology and Services. The available information for the reference material is listed below. The reference material was stored frozen. Compound PHAA (C6) TDHA (C7) PFOA (C8) PFNA (C9) CIO C ll C12 PFOS THPFOS THPFDS Exygen Research. Exvsen Inventor/ No. SP0002086 SP0002091 SP0002087 SP0002085 SP0002090 SP0002093 SP0002089 SP0002084 SP0002088 SP0002092 Lot No. NB 117735-32 PU/07219EU 210002 H7568 R11K U11N R24K 217 Q75-91 PMR-269-83 Puritv (%) Expiration Date TBD 01/01/10 99.5 01/01/05 >97 07/19/07 >99 07/19/07 98 12/01/10 >99 07/19/07 96 12/01/10 86.9 08/31/06 Unknown 06/07/05 94.7 08/22/12 Page 12 of 111 000329 Exygen Study No.: 023-082 The molecular structures of the anions are given below. Name: PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 499, as shown Name: C6 Chemical Name: Perfluorohexanoate Molecular Weight: 313, as shown Name: Cl Chemical Name: Perfluoroheptanoate Molecular Weight: 363, as shown Name: C8 Chemical Name: Pentadecafluorooctanoate Molecular Weight: 413, as shown Exygen Research. 000330 Page 13 of 111 Exygen Study No.: 023-082 Name: C9 Chemical Name: Heptadecafluorononanoate Molecular Weight: 463, as shown Name: CIO Chemical Name: Nonadecafluorodecanoate Molecular Weight: 513, as shown '0 ` Name: C l 1 Chemical Name: Perfluoroundecanoate Molecular Weight: 563, as shown Name: C12 Chemical Name: Perfluorododecanoate Molecular Weight: 613, as shown Exygen Research. 000331 Page 14 of 111 E.xygen Study No.: 023-082 Name: THPFOS Chemical Name: Tetrahydroperfluorooctane sulfonate Molecular Weight: 427, as shown FFFH FH F S03 FH FFFH Name: THPFDS Chemical Name: Tetrahydroperfluorodecane sulfonate Molecular Weight: 527, as shown 6.0 DESCRIPTION OF ANALYTICAL METHOD Analytical method entitled "Method of Analysis for the Determination of Perfluorohexanesulfonate (PFHS), Perfluorooctanesulfonate (PFOS) and Pentadecafluorooctanoic Acid (PFOA) in Rat Liver, Serum and Urine" was used for this study. For this study, several modifications were made and have been documented in the protocol/protocol deviations. 6.1 Extraction Procedure a. Measure 2 mL of serum sample into a 15 mL disposable centrifuge tube and fortify, if appropriate. b. Add 5 mL of ACN and shake for ~20 minutes on a wrist action shaker. c. Centrifuge tubes at -3000 rpm for - 5 minutes. Carefully decant supernatant into a 50 mL disposable centrifuge tube" and add 35 mL of water. d. Load the sample onto a conditioned SPE column. Discard the eluate. Any analyte residues will be trapped on the SPE column at this point. e. Elute with 5 mL of methanol and then evaporate to less than 1 mL using a nitrogen evaporator. Bring final volume up to 1 mL with methanol. f. Analyze samples using electrospray LC/MS/MS. The volume of sample used and the volume of methanol used for elution were different than those cited in the method. This was done to allow for lower quantification limits for the anions in this study. Exygen Research. 000332 Page 15 of 111 Exygen Study No.: 023-082 6.2 Preparation of Standards and Fortification Solutions Individual stock solutions of all of the anions were prepared on October 02, 2002, as specified in method ExM-023-071. The stock standard solutions were prepared at a concentration of ~ 100 pg/mL by dissolving ~10 mg of the standard (corrected for purity and salt content when appropriate) in methanol. From these solutions, a 1.0 pg/mL mixed fortification standard solution was prepared by transferring the appropriate volume (~0.4 - 1 mL) of each of the stock solutions into a 100-mL volumetric flask and bringing the volume up to the mark with methanol. The 0.1 pg/mL mixed fortification standard was prepared by transferring 10 mL of the 1.0 pg/mL mixed fortification standard into a volumetric flask and bringing the volume up to 100 mL with methanol. A set of calibration standards were prepared by dilution in the following manner: Initial Cone. (ng/mL) 100 100 100 100 5.0 2.0 1.0 Volume (mL) 1 0.5 0.2 0.1 1 1 1 Diluted to (mL) 10 10 10 10 10 10 10 Final Cone. (ng/mL) 10.0 5.0 2.0 1.0 0.5 0.2 0.1 The stock standard solutions and all fortification and calibration standard solutions were stored in a refrigerator (6 2C) when not in use. 6.3 Chromatography Quantification was accomplished by electrospray LC/MS/MS analysis. An API 4000 Sciex system was used in this study because of its greater sensitivity and also because it had not been used for fluorochemical analysis prior to this study. Peaks were detected in the control matrices corresponding to some of the target anions, especially for C8. 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 0.1 ng/mL, which corresponds to a concentration of 0.05 ng/mL (ppb) in the extracted samples. Residues were calculated below this level where the response of the anion was approximately three times the signal to noise ratio. The results were Exygen Research. Page 16 of 111 000333 Exygen Study No.: 023-082 reported as Not Detected (ND) if the response was approximately less than three times the signal to noise ratio and Not Quantifiable (NQ) was used for negative results. All other responses were reported. 6.5 Description of Instrument and Operating Conditions Instrument: PE SCIEX API 4000 Biomolecular Mass Analyzer, (LC/MS/MS #8) SCIEX Turbo Ion Spray Liquid Introduction Interface Turbo Ion spray temperature = 350 C Auxiliary gas flow = - 7.0 L/min Harvard Infusion Pump Computer: Dell OptiPlex GX 110 Software: PE Sciex Analyst 1.2 HPLC Equipment: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Column Temperature: Mobile Phase (A) : Mobile Phase (B) : Genesis C-8, 5 cm x 2.1 mm i.d. x 4 p (Exygen ID: 71A) (JONESCHROMATOGRAPHY: Part No. FK5962E) 35C 2 mM Ammonium Acetate in Type I Water Methanol Time (min) 0.0 2.0 5.0 9.0 9.5 14.0 14.5 20.0 %A 90.0 90.0 10.0 10.0 0.0 0.0 90.0 90.0 %B 10.0 10.0 90.0 90.0 100.0 100.0 10.0 10.0 Flow Rate (mL/min) 0.3 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Injected Volume: 15 pL Ions monitored : Anion Parent ion Daughter ion C6 313 269 C7 363 319 C8 413 369 C9 463 419 CIO 513 C ll 563 C12 613 469 519 569 PFOS 499 80 THPFOS THPFDS 427 527 81 81 D w ell ("sees) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Declustering Potential -20 -20 -20 -20 -20 -20 -20 -85 -65 -75 Collision Energy -10 -10 -10 -10 -10 -10 -10 -80 -60 -66 Exygen Research. 000334 Page 17 of 111 Exygen Study No.: 023-082 6.6 Quantitation and Example Calculation Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x weighted linear regression) by Analyst software using seven concentrations of standards prepared in methanol. The residue concentration for the samples was determined from the following equations: Use Equation 1 to calculate the amount of anion found (in ng/mL, based on peak area) using the standard curve (1/x weighted linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/mL) = (peak area - intercept) slope Use Equation 2 to calculate the amount of analyte found (in ppb) Equation 2: Analyte found (ppb = ng/mL) = (analyte found (ng/mL) x FV (mL) x DF sample volume (mL) FV = final volume DF = dilution factor For samples fortified with known amounts of analyte prior to extraction, use Equation 3 to calculate the percent recovery (ppb = ng/mL) Equation 3: Recovery (%) = [ total analyte found (ppb) - analyte found in control or sample (ppb)] qq analyte added (ppb) Note: Any analyte found in the control was subtracted from analyte found. Flowever, the response for the sample duplicate was not used. Exygen Research. 000335 Page 18 of 111 Exygen Study No.: 023-082 An example of a calculation using an actual sample follows: Human Serum Sample, Exygen ID 0204491 Spk J (Data Set: I01702A), fortified with 0.5 ng/mL (calculation is using values for C6): Where: peak area = intercept = slope = dilution factor = ng/mL added (fort level) = avg. amt in controls = final volume = sample volume = 54636 632.277 59435.9 1 0.5 ng/mL 0 (Not detected) 1 mL 2 mL From equation 1: Analyte found (ng/mL) = 54636- 632.2771 59435.9 = 0.9 ng/mL From equation 2: Analyte found (ppb) = 0.9 ng/mL x 1 mL 2 mL = 0.45 ppb (ng/mL) From equation 3: % Recovery = (0.45 ng/mL - 0 ng/mL) x 100 0.5 ng/mL = 90% Note: This example calculation was done using rounded numbers, and therefore may be slightly different from the values shown in the raw data. Exygen Research. 000336 Page 19 of 111 Exygen Study No.: 023-082 7.0 EXPERIMENTAL DESIGN For the screening of each sample, duplicate extractions were performed. Also, each sample was fortified at 0.5 ng/mL and 5.0 ng/mL and then taken through the extraction procedure. Two calibration curves were also taken through the extraction procedure, one using calf serum and one using human plasma. These were treated as quality control fortifications in the data set and were not used for the calibration curve. Since there was residue detected in the samples for THPFOS and THPFDS, an additional analysis in which a three-daughter ion confirmation was performed. 8.0 RESULTS There was no significant residue detected in the reagent blank analyzed with these samples. Also, there was no carry-over present for any of the anions in the instrument blanks (methanol washes) analyzed in the analytical sets, except for C8 and C9, and this is most likely contributed to those analytes being present in the instrumental system, particularly in the mobile phase. This is especially evident with the absence of the anions (except for C8 and C9) in the methanol wash analyzed after the injection of the 10 ng/mL calibration standard. All fortifications were at a level equal to or less than the 10 ng/mL standard. Since there was no carry-over observed after the injection of this standard, the carry-over present after proceeding injections would be minimal. A representative chromatogram of a standard prepared in methanol can be found in Figure 1. Recoveries for fortified samples are given in Tables I-III. Recoveries outside the suggested range of 70% to 130% were reported, however this method has not been validated at these low levels and some of the recoveries were outside of this range because the level of residue in the sample was significantly greater than the amount fortified, especially for C8 and PFOS. Example chromatograms of fortified samples are shown in Figure 2. Residues of each anion in human serum are summarized in Table IV. Residues of each anion in human plasma are summarized in Table V. Residues of each anion in monkey serum are summarized in Table VI. Example chromatograms of a human plasma sample are given in Figure 3. The detection of THPFDS in some of the samples warranted further investigation. The presence of THPFOS and THPFDS was confirmed with a re analysis with additional daughter ion confirmation. A chromatogram detailing the three daughter ion confirmation of THPFDS is given in Figure 4. Exygen Research. 000337 Page 20 o f 111 Exygen Study No.: 023-082 9.0 CONCLUSIONS The quantitative screening of these serum and plasma samples produced levels of certain analytes at extremely low levels (< 100 ppt). These levels are based solely on the instrument sensitivity and not the method recovery. The results contained in this report should be evaluated as a quantitative screening. Contamination of these samples due to instrument conditions is very limited because the instrument used for the analyses had never been used for routine fluorochemical analysis prior to the initiation of this study. No carry-over was observed throughout the injections of the analytical sets, which was demonstrated with the absence of the target analytes in the methanol washes analyzed after the injection of the highest level of calibration standard (10 ng/mL). Two people took a set of 64 samples through the sample preparation procedure in approximately 10 hours and the analysis by LC/MS/MS took approximately 48 hours. 10.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA The method used in this study has not been validated for C8 and PFOS at the levels given in this report and at any level for the rest of the anions. Residues were reported lower than the lowest calibration standard. 11.0 RETENTION OF DATA AND SAMPLES When the final report is complete, all original paper data generated by Exygen Research will be shipped to the sponsor. This does not include facility-specific raw data such as instrument 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 archives of Exygen Research for the lifetime of the product. Sponsor permission will be obtained before discarding. Exygen Research. 000338 Page 21 of 111 Exygen Study No.: 023-082 TABLES Exygen Research. 000339 Page 22 of 111 Exygen Study No.: 023-082 Table I Summary of Recoveries for Calibration Curve in Calf Serum Compared to Standards in Methanol ________________________ %Recovery____________ Fort Level Sample ID Sponsor ID (ng/mL) C6 C7 C8 C9 CIO C ll C12 X C 101702-0 NA 0.0 - - - - - - - XC101702-1 NA 0.2 85 120 163 151 155 96 87 XC 101702-2 NA 0.5 98 111 127 132 135 110 111 XC101702-3 NA 1.0 90 113 109 124 112 111 116 XC101702-4 NA 1.5 102 113 121 127 121 120 121 XC101702-5 NA 2.0 102 108 117 122 115 109 110 XC 101702-6 NA 2.5 97 99 111 107 103 98 98 XC101702-7 NA 5.0 95 97 109 108 104 106 105 AVG: 96 109 122 124 121 107 107 STANDARD DEVIATION: 6.2 8.2 19.1 15.0 18.6 8.2 11.5 RELATIVE STANDARD DEVIATION: 6.5 7.5 15.6 12.1 15.4 7.6 10.7 PFOS ** ** ** ** ** ** ** ** ** ** THPFOS - 148 159 141 157 152 141 141 148 7.8 5.2 THPFDS - 140 144 120 138 129 122 124 131 9.6 7.3 Table II Summary of Recoveries for Calibration Curve in Human Plasma Compared to Standards in Methanol ________________________ %Recovery________________ Fort Level Sample ID Sponsor ID (ng/mL) C6 C7 C8 C9 CIO C ll C12 XC 101502-8 TCR-674 0.0 - - - - - - - XC101502-9 TCR-674 0.2 90 120 126 86 33 115 109 XC101502-10 TCR-674 0.5 87 121 146 115 84 132 114 XC101502-11 TCR-674 1.0 96 113 121 104 90 110 108 XC 101502-13 TCR-674 2.0 107 114 129 107 112 114 120 XC101502-15 TCR-674 5.0 95 97 106 97 98 101 104 AVG: 93 113 126 102 81 114 111 STANDARD DEVIATION: 10.2 9.6 14.4 10.9 29.2 11.3 6.2 RELATIVE STANDARD DEVIATION: 10.9 8.5 11.5 10.7 35.8 9.9 5.6 PFOS ** ** ** ** ** ** ** ** ** THPFOS - 142 160 151 150 127 146 12.4 8.5 THPFDS - 133 142 123 137 113 130 11.6 9.0 ** Recovery not applicable because the residues detected in sample were significantly greater than the amount fortified. Exygen Research. 000340 Page 23 of 111 Exygen Study No.: 023-082 Table III Calf Serum Summary of Recoveries for Laboratory Fortified Matrix Spikes _________________________ % Recovery_______________ Sample ID Fort Level Sponsor ID (ng/mL) C6 C l C8 C9 CIO C ll C12 PFOS 0204718 Spk A NA 0.5 98 110 99 126 115 95 0204718 SpkB NA 5.0 84 85 92 97 95 96 AVG: 91 98 96 112 105 96 STANDARD DEVIATION: 9.9 17.7 4.9 20.5 14.1 0.7 RELATIVE STANDARD DEVIATION: 10.9 18.1 5.2 18.4 13.5 0.7 105 100 103 3.5 3.4 ** ** ** ** ** THPFOS THPFDS 144 126 118 108 131 117 18.4 12.7 14.0 10.9 Human Serum _________________________ %Recovery Fort Level Sample ID Sponsor ID (ng/mL) C6 C l C8 C9 CIO C ll C12 PFOS THPFOS 0203963 Spk C Lot 020821 0.5 114 142 123 133 124 151 160 ** 0203964 Spk D Lot 22K0965 0.5 44 92 144 91 96 110 107 ** 0203965 SpkE Lot GO140604 0.5 53 120 376 153 118 139 132 ** 0204292 Spk F X328-A 0.5 94 125 380 188 129 128 131 ** 0203963 SpkM Lot 020821 5.0 106 112 164 146 131 137 134 ** 228 133 265 182 321 0203964 Spk N Lot 22K0965 5.0 76 91 106 98 97 107 113 0203965 Spk O Lot GO140604 5.0 88 109 158 119 110 117 115 0204292 SpkP X328-A 5.0 88 98 128 105 103 101 109 AVG: 83 111 197 129 114 124 125 STANDARD DEVIATION: 24.3 17.6 113.1 32.6 14.0 17.7 17.8 RELATIVE STANDARD DEVIATION: 29.3 15.9 57.3 25.3 12.4 14.3 14.2 103 ** ** ** ** ** 118 198 116 195 73.7 37.8 THPFDS 198 117 149 149 168 109 121 113 141 31.4 22.3 ** Recovery not applicable because the residues detected in sample were significantly greater than the amount fortified. Exygen Research. 000341 Page 24 of 111 Exygen Study No.: 023-082 Table III (cont') Summary of Recoveries for Laboratory Fortified Matrix Spikes Human Plasma % Recovery Sample ID Fort Level Sponsor ID (ng/mL) C6 C7 C8 C9 CIO C ll C12 PFOS THPFOS THPFDS 0204334 Spk H TCR-684 0.5 69 94 211 152 107 93 103 ** 122 0204490 Spk J TCR-674 0.5 78 78 73 84 90 91 96 ** 107 0204491 Spk J TN-A-6337 0.5 91 97 168 140 110 91 93 ** 113 0204747 Spk G TCR-683 0.5 89 124 406 182 143 132 120 ** 159 0204334 Spk R TCR-684 5.0 96 100 131 110 110 98 100 ** 114 113 102 116 146 112 0204490 SpkU TCR-674 5.0 86 85 92 90 92 92 96 0204491 Spk T TN-A-6337 5.0 97 96 109 101 95 86 88 0204747 Spk 0 TCR-683 5.0 82 85 106 92 90 83 83 AVG: 86 95 162 119 105 96 97 STANDARD DEVIATION: 9.4 13.9 108.0 35.3 17.8 15.3 11.1 RELATIVE STANDARD DEVIATION: 11.0 14.7 66.7 29.7 17.0 16.0 11.4 65 ** ** ** ** ** 109 113 111 119 17.0 14.3 102 106 94 111 15.7 14.1 Monkey Serum ________________________ %Recovery Sample ID Fort Level Sponsor ID (ng/mL) C6 C7 C8 C9 CIO C ll C12 PFOS THPFOS THPFDS 0204335 Spk I TN-A-06332 0.5 106 91 68 52 88 85 95 ** 0204492 Spk K TN-A-06333 0.5 122 112 129 134 117 129 125 ** 0204493 SpkL TN-A-06336 0.5 123 98 97 102 115 93 94 ** 0204335 Spk S TN-A-06332 5.0 100 93 97 107 108 105 109 ** 0204492 SpkU TN-A-06333 5.0 88 85 100 94 97 93 101 ** 114 162 132 107 124 129 155 120 104 116 0204493 Spk V TN-A-06336 5.0 115 100 116 114 109 106 107 AVG: 109 97 106 101 106 102 105 STANDARD DEVIATION: 13.7 9.3 20.6 27.4 11.1 15.5 11.5 RELATIVE STANDARD DEVIATION: 12.5 9.6 20.4 27.2 10.5 15.2 10.9 ** ** ** 141 130 19.8 15.3 120 124 17.2 13.9 ** Recovery not applicable because the residues detected in sample were significantly greater than the amount fortified. Exygen Research. 000342 Page 25 of 111 Exygen Study No.: 023-082 Table IV Summary of Residues for Human Serum Samples Amount Found (ng/mL) ft,, Sample ID Sponsor ID C6 C7 C8 C9 CIO C ll C12 PFOS THPFOS THPFDS 0203963 Lot 020821 0.0261 ND 2.85 0.683 0.228 0.371 0.0333 32.0 ND 0.0743 0203963 Dup Lot 020821 ND ND 2.93 0.684 0.22 0.373 0.0282 33.3 ND 0.0599 0203964 Lot 22K0965 ND 0.0940 1.45 0.307 0.107 0.0942 0.0122 8.64 ND 0.0402 0203964 Dup Lot 22K0965 ND 0.0976 1.45 0.276 0.108 0.115 0.0172 8.95 ND 0.0412 0203965 Lot GO140604 0.0687 0.145 5.93 0.508 0.214 0.207 0.0199 29.7 0.0440 0.0846 0203965 Dup Lot GO140604 0.0884 0.170 6.00 0.557 0.204 0.236 0.0184 29.3 0.0436 0.0944 0204292 X328-A ND 0.115 3.58 0.634 0.189 0.137 0.0169 16.6 0.0221 0.0791 0204292 Dup X328-A ND 0.12 4.21 0.772 0.221 0.149 0.0490 20.6 0.0394 0.0946 Table V Summary of Residues for Human Plasma Samples ________________________ Amount Found (ng/mL)__________ Sample ID Sponsor ID C6 C l C8 C9 CIO C ll C12 PFOS THPFOS THPFDS 0204334 TCR-684 ND 0.0925 2.57 0.174 0.108 0.0797 ND 14.8 0.0140 0.0539 0204334 Dup TCR-684 ND 0.0875 2.64 0.198 0.120 0.0695 ND 14.9 ND 0.0609 0204490 TCR-674 ND ND 0.0887 NQ 0.0252 0.0458 ND 4.78 0.0115 ND 0204490 Dup TCR-674 ND ND 0.130 NQ 0.0167 0.0357 ND 4.84 ND ND 0204491 TN-A-6337 ND ND 2.23 0.166 0.0840 0.0501 ND 11.4 ND 0.0252 0204491Dup TN-A-6337 ND 0.0309 2.38 0.208 0.0958 0.0346 ND 12.6 ND ND 0204747 TCR-683 ND 0.0897 2.09 0.287 0.121 0.105 ND 14.1 0.0193 0.0843 0204747 Dup TCR-683 ND 0.0876 2.92 0.416 0.163 0.178 ND 19.4 0.0292 0.109 Table VI Summary of Residues for Monkey Serum Samples ________________________ Amount Found (ng/mL) ______ Sample ID Sponsor ID C6 C l C8 C9 CIO C ll C12 PFOS THPFOS THPFDS 0204335 TN-A-06332 0204335 Dup TN-A-06332 0204492 TN-A-06333 0204492 Dup TN-A-06333 0204493 TN-A-06336 0204493 Dup TN-A-06336 ND ND ND ND ND ND ND 1.25 3.24 0.286 0.540 0.0365 17.2 ND 1.72 4.37 0.396 0.650 0.0464 22.4 ND NQ NQ 0.103 0.137 ND 16.5 ND NQ NQ 0.0880 0.114 ND 16.4 ND NQ NQ ND 0.0939 0.0227 14.8 ND NQ NQ ND 0.106 0.0321 18.1 ND ND ND ND ND ND 0.0141 ND 0.0130 ND ND ND ND = Not Detected NQ = Not Quantifiable (negative residue calculated) Exygen Research. 000343 Page 26 of 111 Exygen Study No.: 023-082 FIGURES Exygen Research. 000344 Page 27 of 111 Exygen Study No.: 023-082 Figure 1 Chromatogram Representing 0.1 ng/mL Calibration Standard Exygen Research. 000345 Page 28 of 111 Exygen Study No.: 023-082 Figure 1 (cont) Chromatogram Representing 0.1 ng/mL Calibration Standard Exygen Research, 000346 Page 29 of 111 Exygen Study No.: 023-082 Figure 1 (cont) Chromatogram Representing 0.1 ng/mL Calibration Standard Exygen Research 000347 Page 3 0 of 111 Exygen Study No.: 023-082 Figure 1 (cont) Chromatogram Representing 0.1 ng/mL Calibration Standard Exygen Research, 000348 Page 31 of 111 Exygen Study No.: 023-082 Figure 2 Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb (Exygen ID: 0204490 Spk J, Sponsor ID: TCR-674) 000349 Exygen Study No.: 023-082 Figure 2 (cont') Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb Exygen Research 000350 Page 33 of 111 Exygen Study No.: 023-082 Figure 2 (cont') Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb (Exygen ID: 0204490 Spk J, Sponsor ID: TCR-674) Exygen Research. 000351 Page 3 4 of 111 Exygen Study No.: 023-082 Figure 2 (coni') Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb (Exygen ID: 0204490 Spk J, Sponsor ID: TCR-674) Exygen Research. 000352 Page 35 o f 111 Figure 3 Exygen Study No.: 023-082 Chromatogram Representing a Human Plasma Exygen Research 000353 Page 36 of 111 Exygen Study No.: 023-082 Figure 3 (cont') Chromatogram Representing a Human Plasma Exygen Research. 3 S if Page 37 of 111 Exygen Study No.: 023-082 Figure 3 (cont') Chromatogram Representing a Human Plasma Exygen Research. 000355 Page 38 of 111 Exygen Study No.: 023-082 Figure 3 (cont') Chromatogram Representing a Human Plasma Exygen Research. 000356 Page 39 of 111 Exygen Study No.: 023-082 Figure 4 Chromatogram Representing a Sample Analyzed for Three Daughters for THPFDS Exygen Research. Page 40 of 111 Exygen Study No.: 023-082 APPENDIX A Exygen Study Plan ExP-023-082 (Exygen Study No. 023-082) and Deviations Exygen Research. .0 0 3 5 8 Page 41 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 STUDY PLAN Study Title: Analysis of Pooled Human Sera and Plasma and Monkey Sera for Fluorocarbons Using Exygen Method ExM-023-071 Study Plan Number: ExP-023-082 Exygen Study Number: 023-082 P erform ing L aboratory: E xygen R esearch 3058 R esearch D rive S tate C ollege, P A 16801 Phone: (814) 272-1039 Study Sponsor: 3M E nvironm ental L aboratory B uilding 2-3E-09 S t Paul, M N 55133-3331 Phone: (651) 778-6565 t \ tf j Exygen Research. 000359 Page J o f 59 Page 42 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 1) E m ily R. D ecker, Study D irector, E xygen Research 2) W illiam K Reagen, Sponsor Study M onitor, 3M 3) Exygen R esearch Q uality A ssurance U nit Exygen Research. 000360 Page 2 o f 59 Page 43 of 111 Exygen Study No.: 023-082 I I _ -- i Study Plan: ExP-023-082 Exygen Study No.: 023-082 S tu d y T itle: A nalysis o f Pooled H um an Sera and Plasm a and M onkey Sera for Fluorocarbons U sing Exygen M ethod ExM -023-071 Study Plan N um ber ExP-023-082 E xygen Study N um ber 023-082 This study plan w as audited by the Q uality A ssurance U nit o f E xygen Research. _________ _________________________ N aom i Lovallo Technical Lead-Q A (oQilcn-. D ate APPROVALS (JEinlly RADecker, Study D irector Exygen ^ (2 /L M / J o h n Flaherty, V ice Prsident, Facility M anagem ent Exygen icjnlT. D ate D ate r,, W illiam Reagen, Sponsor Study M onitor 3M D ate * | [ F ij 1 I i i \ i 1 y Exygen Research. 000361 Page 3 o f59 Page 44 of 111 Exygen Study No.: 023-082 10/'09/02 14:26 ftSD INF TECH 2-2E-01 -> *88142311580 NO.215 P002 Study Plan: ExP-023-082 Exygen SmdyNo.: 023-082 | Study T itle; A nalysis o f Fooled Human Sera and Plasma, and M onkey Sera for Fluorocarbons U sing Exygen M ethod ExM -023-071 ,* . I-. | Study Plan Number. ExP-023-082 Exygen Study N um ber 023-082 I. Ii ' ''i& S study plan was ancSted b y the Quality Assurance U nit o f Exygen Research. D ate |t j I | * APPROVALS ' 'Erily R . Decker, Study Director . W "' i- -I i.-:; John Flaherty, V ice President, Facility Management ilfcw * D ate D ate D ate : -I J>agt 3 of59 : __________________________ - RECEIVED TIME OCT. 9. E x y g e n 'R e s e a r c h . 4:13PM PRINT TIME OCT. 9. 000362 4:14PM Page 45 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 TITLE PAGE........................................................ 1 DISTRIBUTION... .............................................................................. 2 STUDY PLAN APPROVAL........................ ...................................................... ................................................. 3 TABLE OF CONTENTS................................................................................................................. 4 INTRODUCTION - .................... 5 TEST ITEMS.................. 5 OBJECTIVE........... ........................................... 8 TESTING FACILITY...................................................................................................................... 8 STUDY DIRECTOR................................. ...............................!...........................................................................8 SPONSOR............................................................................................................................................................... 8 SPONSOR STUDY MONITOR............................................................................................................................9 PROPOSED EXPERIMENTAL START AND TERMINATION DATES.................................. 9 C O M M U N IC A TIO N S............................... ............................................................................................................ 9 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM............................................................ 9 SAMPLE PROCUREMENT, RECEIPT AND RETENTION............................................................................ 9 SAMPLE IDENTIFICATION............................................................................................................................... 10 ANALYTICAL PROCEDURE SUMMARY...................................................................................................... 10 VERIFICATION OF ANALYTICAL PROCEDURE........................................................................................ 11 METHODS FOR CONTROL OF B IA S.............................................................................................................. 12 STATISTICAL METHODS..................................................................................................................................12 GLP STATEMENT.......................................................................................................................... 12 REPORT.................................................................................................................................................................. 12 SAFETY AND HEALTH.................................. 13 AMENDMENTS TO STUDY PLAN..................................................................................................................14 DATA RECORD KEEPING.................... 14 QUALITY ASSURANCE.................................................................................................................... ................ 15 RETENTION OF DATA AND ARCHIVING.................................................................................................... 15 APPENDIX 1......................................................................................................................................................... 16 Exygen Research. 000363 Page 4 o f 59 Page 46 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 T he purpose o f this study is to perform analysis for perfluorooctane sulfonate (PFO S), perfluorohexanoic acid (C6), perfluoroheptanoic acid (C7), pentadecafluorooctanoic acid (C8), heptadecafluorononanoic acid (C9), nonadecafluorodecanoic acid (CIO), perfluoroundecanoic acid ( C ll), perfluorododecanoic acid (C12), tetrahydroperfluorooctane sulfonate (THPFOS), and tetrahydroperfluorodecane sulfonate (THPFDS) in pooled hum an serum and plasm a and m onkey sera using Exygen m ethod ExM -023071 entitled "M ethod o f A nalysis fo r the D eterm ination o f Perfluorohexanesulfonate (PFHS), Perfluorooctanesulfonate (PFO S) and Pentadecafluorooctanoic A cid (PFO A ) in R at L iver, Serum and U rine." T he study will be audited for com pliance w ith O ECD Principles o f G ood Laboratory Practice (as revised in 1997), EN V /M C /C H EM (98)17 by the Q uality A ssurance U nit o f Exygen Research. T he test item s are perfluorooctane sulfonate (PFO S), perfluorohexanoic acid (C6), perfluoroheptanoic acid (C7), pentadecafluorooctanoic acid (C8), heptadecafluorononanoic acid (C9), nonadecafluorodecanoic acid (CIO), perfluoroundecanoic acid ( C ll), perfluorododecanoic acid (C12), tetrahydroperfluorooctane sulfonate (T H P F O S ), and tetrahydroperfluorodecane sulfonate (THPFDS). A ll test item s w ere received from the Sponsor. Name: PFOS Chem ical N am e: Perfluorooctanesulfonate M olecular W eight: 499, as show n , l. f f ! [, Exygen Research. 000364 Page 5 o f 59 Page 47 of 111 Exygen Study No.: 023-082 Name: C6 Chem ical N am e: Perfluorohexanoic acid M olecular W eight: 313, as shown Study Plan: ExP-023-082 Exygen Study No.: 023-082 Name: C7 Chem ical N am e: Perfluoroheptanoic acid M olecular W eight: 363, as shown Name: C8 Chem ical N am e: Pentadecafluorooctanoic acid M olecular W eight: 413, as shown Chem ical N am e: Heptadecafluorononanoic acid M olecular W eight: 463, as shown Exygen Research. 000365 Page 6 o f59 Page 48 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 N am e: CIO Chem ical Nam e: N onadecafluorodecanoic acid M olecular W eight: 513, as show n Name: C ll Chem ical N am e: Perfluoroundecanoic acid M olecular W eight: 563, as shown Name: C12 Chem ical N am e: Perfluorododecanoic acid M olecular W eight: 613, as shown Name: THPFOS Chem ical N am e: Tetrahydroperfluorooctane sulfonate M olecular W eight: 427, as shown Exygen Research. 000366 Page 7 o f 59 Page 49 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Name: THPFDS Chem ical N am e: Tetrahydroperfluorodecane sulfonate M olecular W eight: 527, as show n .t1: I? i T h e purpose o f this study is to perform analysis on four different lots o f pooled hum an serum , four different lots o f pooled hum an plasm a, and three lots o f pooled m onkey serum for the target fluorocom pounds using the analytical m ethod, "M ethod o f Analysis for the D eterm ination o f Perfluorohexanesulfonate (PFH S), Perfluorooctanesulfonate (PFO S) and Pentadecafluorooctanoic A cid (PFO A ) in R at Liver, Serum and U rine." Exygen Research 3058 Research D rive State College, P A 16801 Phone: (814) 272-1039 E m ily D ecker Scientist Exygen Research Phone: (814) 272-1039 em ily.decker@ exygen.com 3M Environm ental Laboratory B uilding 2-3E-09 SL Paul, M N 55133-3331 Phone: (651) 778-6565 Exygen Research. 000367 Page 8 o f 59 Page 50 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 W illiam Reagen B uilding 2-3E-09 St. Paul, M N 55133-3331 Phone: (651) 778-6565 w kreagen@ m m m .com It is proposed that the analytical portion o f this study be conducted from O ctober 14 to O ctober 21, 2002. T he actual experim ental start and term ination dates w ill be included in the final report. A ll 'o m m u n icatio n s betw een the T esting Facility, m ethod developers, a n i the Sponsor will b e directed through the Study D irector (or designate) and the Sponsor Study M onitor. C om m unications w ill be fully docum ented by the Study Director. Pooled hum an sera and plasm a and m onkey sera are used as the test system s in this study. T he m atrices w ill be provided by the sponsor. T he m atrices w ill be representative of that for w hich this analytical m ethod was designed. Pooled hum an serum sam ples were purchased by the sponsor from Sigm aA ldrich, M ilw aukee, W I, L am pire Biological Laboratories, Pipersville, PA, Bioresource Technology, Inc., Fort Lauderdale, FL, and G olden W est Biologicals, Tem ecula, CA. Pooled m onkey serum sam ples w ere purchased by the sponsor from Lam pire B iological Laboratories, Pipersville, PA . Pooled hum an plasm a sam ples w ere purchased by the sponsor from Lam pire Exygen Research. 000368 Page 9 o f 59 Page 51 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 In addition to the calibration standards described in Section 3.5.3, a set o f calibration standards will be processed through the extraction procedure identical to the sam ples, using bovine serum and also a set using hum an plasm a. T he fortification o f the standards before extraction is done according to the follow ing table: Cone. O f M ixed Fortification Solution (ng/m L) 1 10 10 10 10 100 100 Fortification V olum e (pL) 400 100 200 300 400 50 100 V olum e of C o n tro l Sam ple (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Cone, of E x tracted C alib ratio n S ta n d a rd .. (n g /m L ) 0.2 0.5 1.0 1.5 2.0 2.5 5.0 T he testing facility will establish the relationship betw een the instrum ent response and the concentration o f analyte in order to assess the linearity of the system . A standard curve should be constructed w ith at least five standards. T he testing facility should also verify the endogenous levels o f analyte in the m atrix control sam ples. This should be accom plished by analysis o f a control sam ple for each m atrix and exam ination o f the region of analyte retention. T he potential exists for interference from fluorochem icals introduced from dietary m aterial and other exogenous sources. Sam ples are fortified w ith the target analytes. T he com pounds w ill be m ade into solutions as per the m ethod, and added to the m atrices via a m icropipette. Fortified sam ples will be processed through the described procedures to ensure m ethod accuracy and to check for bias. R ecoveries should be betw een 70% and 130% of the fortified levels. T he sponsor m ay accept occasional recoveries outside of this range. T he relative standard deviation (RSD) for each fortification level as w ell as the overall R SD , should be less than or equal to 20% . A ny m odifications to the analytical m ethod w ill be docum ented in the study raw data. M odifications deem ed significant by the Sponsor or Study D irector w ill necessitate revision of the m ethod. Exygen Research. 000369 Page I I of 59 Page 53 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 C ontrol of bias w ill be addressed b y taking representative sub-sam ples from a hom ogeneous m ixture of each m atrix for untreated control sam ples, and by analyzing at least two levels o f fortifications. Statistics will be lim ited to those specified in the subject m ethod and to the calculation o f average recoveries, as applicable. A ll aspects o f this study shall be perform ed and reported in com pliance w ith O EC D Principles o f G ood L aboratory Practice (as revised in 1997), EN V /M C/CH EM (98)17. The final report or data package (supplied to the Sponsor) shall contain a statem ent that the study was conducted in com pliance w ith current and applicable G L P standards and w ill outline any deviations in the study from those standards. T his statem ent w ill be signed by the Study D irector and Sponsor. f t A final report w ill be prepared by the study director or their designee at the conclusion of the study. T he report w ill include, but w ill not be lim ited to, the follow ing: The nam e and address o f the Study D irector, Sponsor M onitor and o f the testing facility. A statem ent o f GLP com pliance (any related docum entation, such as chain-of-custody records, m ust be in the study records). The signed and dated statem ent by the Exygen Research Quality Assurance U nit regarding dates o f study inspections and dates findings w ere reported to the Study D irector and M anagem ent. Exygen Research. 000370 Page 12 o f 59 I ' Page 54 o f 1H Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 A description o f the exact analytical conditions em ployed in the study. If the subject m ethod w as follow ed exactly, it is necessary to include only a copy of the analytical m ethod. A ny m odifications to this m ethod w ill be incorporated into the report. If the m ethod is photo-reduced, the project num ber and page num ber m ust be included on each page. A ny steps considered critical, i.e. steps w here little variation is allow able o r directions m ust be follow ed precisely. D escription of the instrum entation used and operating conditions. The num ber o f w orker-hours or calendar days required to com plete one set o f samples. A ll results from all sets analyzed. Identify all control and fortified sam ples, and in the data table include sam ple num ber, fortification level, and unique identification num ber by sam ple set. Representative chrom atogram s for each analyte in each m atrix, including chrom atogram s of a standard and a control sam ple, and a chrom atogram at a fortification level. T he location o f the analyte peaks w ill be clearly identified in all chrom atogram s. A ll circum stances that m ay have affected the quality o r integrity o f the data will be docum ented in the report. Locations w here raw data and the final report are to be archived. A dditions o r corrections to the final report shall be in the form o f an am endm ent by the Study D irector. T he am endm ent shall clearly identify that part o f the report that is being altered and the reasons for the alterations. T he am endm ent w ill be signed and dated by the Study D irector and the Sponsor Study M onitor. Laboratory personnel w ill practice good sanitation and health habits. A ny health condition o f laboratory personnel that m ay be considered to adversely affect the study will be reported to the Study D irector. A ny injury to laboratory personnel occurring during the conduct o f this study w ill be reported to the study director. Exygen Research. 000371 Page 13 o f 59 Page 55 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environm ent to the test o r reference substance(s). A ll significant changes to the study plan outlined here w ill be expressed in w riting, signed and dated by the Study D irector and Sponsor Study M onitor. A m endm ents usually w ill be issued prior to initiation o f study plan change. H ow ever, w hen a change is required w ithout sufficient tim e for die issue o f a w ritten am endm ent, that change m ay be effected verbally w ith supporting docum entation signed and dated by die Study D irector and follow ed w ith a w ritten am endm ent as soon as possible. In this case, the effective date o f the w ritten am endm ent w ill be the date o f the docum ented change. C opies o f the signed am endm ents w ill be appended to all distributed study plan copies. T he original am endm ent w ill be m aintained w ith the original study plan. A ny deviations from the study plan or from the analytical m ethod as provided w ill b e docum ented and reported prom ptly to the Sponsor Study M onitor. R ecords to be m aintained include the follow ing (as appropriate): Sam ple tracking sheet(s) Sam ple receipt records, storage history, and chains of custody H istory and preparation o f standards (stock, fortification, calibration) D escription of any m odifications to the m ethod Instrum ent run sheets, bench-sheets or logs A nalytical data tables A ll chrom atographic and instrum ental conditions Sam ple extraction and analysis dates A com plete listing of study personnel, signatures and initials Chronological presentation o f all study correspondence A ny other docum entation necessary for the reconstruction of the study Chromatograms- A ll ch ro m ato g ram s w ill co n tain the follow ing: Sam ple identification, date, Exygen study num ber, arrow o r other indication o f the area of interest, and injection num ber corresponding to the run. A dditionally, fortifications w ill include the am ount of analyte added and the sam ple num ber of the sam ple that w as fortified. Exygen Research. 000372 Page 14 o f 59 Page 56 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 A nalytical standard chrom atogram s w ill additionally include the concentration (e.g., jig/m L). * A s part o f the docum entation the follow ing sheets will be included in each analytical set: a run sheet listing the sam ples to be run in the set, and an instrum ent conditions sheet describing the instrum ent type and operating conditions. T he Q A U nit o f Exygen R esearch w ill inspect the study at intervals adequate to a ssu re c o m p lia n c e w ith G L P 's , a n d w ill re p o rt th e fin d in g s o f a u d its to th e Study Director, Exygen M anagem ent, and the Sponsor Study M onitor. A ll hard copy raw data, including, but not lim ited to, the original chrom atogram s, w orksheets, correspondence, and results shall be included w ith the data package subm itted to the Sponsor. These w ill be archived w ith the original study plan, am endm ents, final report, and all pertinent inform ation from the Sponsor. T he testing facility shall keep all electronic raw data and any instrum ent, equipm ent, and storage logs for the lifetim e o f the product and shall obtain perm ission o f the sponsor before discarding. Exygen Research. 000373 Page 15 o f 59 i Page 57 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 APPENDIX I METHOD "Method of Analysis for the Determination of Perfluorohexanesulfonate (PFHS), Perfluorooctanesulfonate (PFOS) and Pentadecafluorooctanoic Acid (PFOA) in Rat Liver, Serum andUrine" Exygen Research. 000374 Page 16 o f 59 Page 58 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 I h TjE Method of Analysis for the Determination of Perfltiorohexanesulfonare (PFHS), PerfluorooctaneaulfcmatB (FFOS) and Pentadecafluoraoctandc A d d (PFOA) in Rat Liver, Serum and Urine AUTBOBS lohn Flaherty, Karen Risha, and Emily Decker B A B IS S U E P . July30,2002 SEQMSQB 3M Medical Department Corporate Toxicology 3M Center, Building 220-2E-02 S t Paul, MN 55144-1000 mEQBMMfiLLMQMTflRX Exygen Research 3058 Research Drive State College. PA 16801 METHOD NUMBER ExM-023-071 XQIAL.NUMgJR-QF.PAS.ES iI 8 t l Exygen Research. 000375 Page 17 o f 59 Page 59 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Hxygeo Method No: ExM-023-071 MANAGEMENT APPROVAL Exygen Research tyu&Q Z"/ ZWZ-. John L. Butenhoff Date Sponsor Representative 3M Medical Department Corporate Toxicology Exygea Research Exygen Research. 000376 Page 2 of 43 I Page 18 o f 59 Page 60 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygeaMethodN: ExM-02M)71 TABLE OF CONTENTS hue MANAGEMENT A PPRO V A LTABLE OF CONTENTS______ .,,3 LIST OF TABLES___________ LIST OF FIGURES________________ 1. SUMMARY__________________ -4 ...5 .7 Z EXPERIMENTAL COMPOUNDS. 8 3. CHEMICALS AND SUPPLIES-------------------------------------------------------------- 9 3.1. Chemicals_________________________________________ ___ ________9 3 3 . Standards------------------ _______________ 9 3.3. Equipment and Supplies.. 3.4. Solutions________ ____________________________1_09 3.5. Preparation of Standard Solutions... 3.5.1. Stock solution.-.-- ----------------3 3 3 . Fortification Solutions--,----------- 3.5.3. Calibration Standards---------- -- M ETHOD-___ ________________________ 4.1. FlowDiagram________ 4 Z Sample Processino____ _______________________________________________________________________________11111111101222 4 3 . Batch Set up_________ 4.4. sampleExtraction ____ ____ 13 ____ 13 4.4.1. Liver Extraction-- ____ 13 4.43. Serum and Urine Extraction.-- . -- 13 4.43. SPE Column Conditioning__ __ _________ ___________________ 14 43. Quanutation_____________ - ________________________________14 43.1. LC/MS/MS System and Operating Conditions----------------------------14 4 3 3 . Calibration Curve Procedures------ ---------------- ------------------------ 15 4 3 3 . Sample A n a l y s i s __ ____ 16 4.6. Acceptance Criteria____-- ---- ---------- ----------------------------------------- 17 4.7, Performance Criteria______ -17 4.8. Tim e Required fo r An a l y sis------------------------------------------------------ 18 5. CALCULATIONS___________________________________________________ 18 6. S A F E T Y 19 ExygenResearch Exygen Research. 000377 Pagel of43 Page 19 o f 59 Page 61 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethod No: ExM-023-071 LIST OF TABLES Table 1. Recovery Summary of PFHS in Rat Liver and Serum..__________ ,,.,, .2 0 Table 2. Recovery Summary of FFOS in R it Liver. Serum and U rine____ ___,,,,.,,,,..21 Table 3. Recovery Summary of FFOA in Rat Liver, Serum and Urine_____ 22 i i l Cs, ! Exygen Rcseirch Exygen Research. 000378 Pago 4 of43 Page 20 o f 59 Page 62 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 LIST O F FIGURES Figure 1. Calibration Curve for PFHS___ _____....____ ..23 Figure 2. Calibration Curve forPFOS____ __________ ..24 Figure3. Calibration Curve for PFOA___ ____ ....____ .25 Figure 4. Representative Chromatogram of a 0.1 ng/mL Standard Containing PFHS_______________________________ 26 Figures. Representative Chromatogram of a0.1 ng/mL Standard Containing FFOS-. 26 Figure 6. Representative Chromatogram of a 0.1 ng/mL Standard Containing PFOA.. 27 Figure 7. Representative Chromatogram of a 0.5 ng/mL Standard Containing PFHS. 27 Figure 8. Representative Chromatogram of a 0.5 ng/mL Standard Containing FFOS-..28 Figure 9. Representative Chromatogram of a 0.5 ng/mL Standard Containing PFOA., 28 Figure 10. Representative Chromatogram of a 5.0 ng/mL Standard Containing PFHS...29 Figure 11. Representative Chromatogram of a 5.0 ng/mL Standard Containing FFOS.. .29 Figure 12. Representative Chromatogram of a 5.0 ng/mL Standard Containing PFOA .30 Figure 13. Representative Chromatogram of a Reagent Blank Sample Analyzed for PFHS______________________________________________________ .30 Figure 14. Representative Chromatogram of a Reagent Blank Sample Analyzed for pros____________________________________ .31 Figure IS. Representative Chromatogram of a Reagent Blank Sample Analyzed for FTOX--------------------------------------------- ----- i ____________________ ,,31 Figure 16. Representative Chromatogram of a Control Liver Sample Analyzed for PFHS________________________ _______ ____ _______________ ,,32 Figure 17. Representative Chromatogram of s Control Liver Sample Analyzed f o r P F O S ________________________________________________ ..32 Figure 18. Representative Chromatogram of a Control Liver Sample Analyzed for PFOA________________________________________________ H gure l9. Representative Chromatogram of aControl Serum Sample Analyzed for PFHS_______________ _-- --------------------- ----------------------- Figure 20. Representative Chromatogram of aControl Serum Sample Analyzed f o r P F O S ________________________________________________ Figure 21. Representative Chromatogram of aControl Serum Sample Analyzed for PFOA---------------------------------------------- ------------- ----------- ..33 ..33 -.34 r,, -.34 Figure 22. Representative Chromatogram of a Control Urine Sample Analyzed forPFOS__________________________________ _____________ ...35 Figure 23. Representative Chromatogram of a Control Urine Sample Analyzed for PFOA___________________ :...................................................... ..35 Figure 24. Representative Chromatogram of a Control Liver Sample Fortified at 10 ng/g with PFHS............... ........................................................ ,,.36 Exygen Research Page 5 of 43 Exygen Research. 000379 Page 21 o f59 Page 63 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023*082 Exygen Method No: ExM-023-071 L IST O F FIG U R ES (continued) Figure 25. Representative Chromatogram of a Control Liver Sample Fortified at 10 ng/g with PFOS---------------- --------- ----------- --------------- ----------- 36 Figure 26. Representative Chromatogram of a Control Liver Sample Fortified at 10 ng/g with PFOA-._________...--____ __________________ ______37 Figure 27. Representative Chromatogram of a Control Liver Sample Fortified at 50 ng/g with PFHS-----------------------------------------------------------------37 Figure 28. Representative Chromatogram of a Control Liver Sample Fortified at 50 ng/g with PFOS________ ___ ___ ___ ________________ _______38 Figure 29. Representative Chromatogram of a Control Liver Sample Fortified at 50 ng/g with PFOA-- ----- ------------ ------------------------------------ ____ 38 Figure 30. Representative Chromatogram of a Control Serum Sample Fortified at 10 ng/mL with PFHS.... .............................. ..................................... Figure 31. Representative Chromatogram of a Control Serum Sample Fortified at 10 ng/mL with PFOS_______ ________ _______i __________ _ ____ 39 Figure 3Z Representative Chromatogram of a Control Serum Sample Fortified at 10 ng/mL with PFOA___________________________________ ____ 40 Figure 33. Representative Chromatogram of a Control Serum Sample Fortified at 50 ng/mL with PFHS___________ ____ _____ ____ ________ -------40 Figure 34. Representative Chromatogram of a Control Seram Sample Fortified at 50 ng/mL with PFOS____________________________________ Figure 35. Representative Chromatogram of a Control Seram Sample Fortified at 50 ng/mL with PFOA____________________ _____________..... ____ 41 Figure 36. Representative Chromatogram of a Control Urine Sample Fortified at 10 ng/mL with PFOS__ ;._______________________________ Figure 37. Representative Chromatogram of a Control Urine Sample Fortified at 10 ng/mL with PFOA_____ ______________________________ ____ 42 Figure 38. Representative Chromatogram of a Control Urine Sample Fortified at 50 ng/mL with PFOS____________ ____ ___ _____________ ____ 43 Figure 39. Representative Chromatogram of a Control Urine Sample Fortified at 50 ng/mL with PFOA .... ___ ___ _____ ______ ____ 43 i f 1 l\y \ kr l: \ \ t ! it a rri. -| i Exygen Research Exygen Research. 000380 Page 6 o f43 Page 22 o f 59 Page 64 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 L SUMMARY This report detaQr a method of analysis for residues of Peifluorohexanesulfonate (PFHS), Perfluorooctanesulfbnate (PFOS) and Pentadecafluorooctanoic Acid (PFOA) in Rat Liver, Serum and Urine. Residues of PFHS, PFOS and PFOA are extracted from each matrix with acetonitrile. The acetonitrile extract ia added to water and loaded onto a conditioned C18 solid phase extraction (SPE) cartridge. Analyte residues are eluted with 2 mL of methanol Quantification of PFHS, PFOS and PFOA is accomplished by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using multiple reaction monitoring (MRM). The proposed limit of quantitation (LOQ; the lowest fortification specified by fire method which gives adequate recovery according to EPA guidelines) for this method is 10 ng/g (parts-per-billion) each for PFHS, PFO*S and PFOA. The theoretical limit of detection (LOD) will be based on the signal to noise ratio and will be at least greater than 3 times the level of noise, based on the Instrumentation system used. For all analytes, the lowest analytical standard . corresponds to 0.1 ng/mL. This method was developed using rat liver, serum and urine. Typical percent recoveries standard deviations (at 10 and 50 ng/g) are shown below: Fortification L ev elfarfa) 10 0 PFHS Recovery in Rat Liver 115% 9.9% fa-3) 98% 3.5% fa-3) Fortification Level fax/mU 10 50 PFHS Recovery ia Rat Serum 108% 4.7% to d ) 111% 9.6% fa-3) Fortification Level fas/*) 10 50 PFOS Recovery in Rot Liver 96% 8.5% fa-3) 88% 1.5% fa-3) Fortification Level fnrfm U 10 50 PFOS Recovery in Rat Serum (8% 9.8% fa-31 120% 2.1% fa-31 PFOS Recovery In RotUrine 93% 4.7% fa-3) 79% 1.2% fa-3) Fortification Level fato/*) 50 PFOA Recovery in Rat Liver 98% 3.1% fa-3) 94% 2J% fa-3) Fortification Level fal/m L ) 10 50 PFOA Recovery in PFOA Recovery In Rat Serum Rat Urine U 7% 1.5% fa-3) 89% 2J% fa-3) 111% 4.0% fa-3) 87% 2.1% fa-3) Representative calibration curves are shown in Figures 1-3. Representative chromatograms are shown in Figures 4 to 39. i it | 1 j | Exygen Research Exygen Research. 000381 Page 7 o f43 Page 23 o f 59 Page 65 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-032 Exygen Study No.: 023-082 Exygen Method No: BxM-023-071 2 . EXPERIM ENTAL COM POUNDS The structures farFFHS, PFOS and EFOA sre given below. PFHS Chemical Name Molecular weight = = Perfluorohexanesulfonate 399, as shown s PFH3 is supplied as the potersbm salt (C.EiaSCjK*), molecular weight = 438 PFOS Chemical Name Molecular weight = = Perfluorooctanesulfcnate 499, as shown i I l t 1 F - * w * F i * PFOS is supplied as the potassium salt (CeFnSCjTC*) molecular weight = 538 FFO 'Chemical Name Molecular weight = = Pentadecafluorooctandc A dd 413, as shown fM Ft 1 F i1 Il1 ifi I1 t ii I Exygen Research Page 8 oT43 Exygen Research. 000382 P ag e 24 o f 59 ' Page 66 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: BxM-023-071 CHEM ICALS A ND SUPPLIES 3.L Chemicals C hem ical Grade Source Methanol (MeOH) HPLC EM Science Ammonium Acetate Reagent IT Baker Water Type I Exygen Acetonitrile_____________ HPLC_____ EM Science Catalog No. MX0475-1 0596-01 NA AX0145-1 Type I water= electrical resistivity, minimum of 16.67 MQ/cm at 25 *C, from a Labconco WaterproTM workstation. 3.2. Standards Standard Perfiuoruhexanesulfonate (PFHS) Perfluorooctanesulfonate (FFOS) Pentadecafluorooctanoic Acid (FFOA) TCR Number SB-036 SD-018 Lot No: 08316DO _ Purity (%) 99.99 all isomen, 8436 straight chain 86.9 96 Source 3M 3M Aldrich Chem E quipment and Supplies Equipm ent Balance, analytical (display at least 0.0001-g) 125-mL LDPE narrow mouth bottles Disposable glass micropipets (50-100 & 100-200 pL) Tissumizer Wrist action shaker Sorvall RC 5C plus Centrifuge 50 rriL disposable polypropylene centrifuge tabes ' 15 mL disposable polypropylene centrifuge tubes Visiprep vacuum manifold Sep Pak Vac 6 cc (lg) tC18 cartridges (part # WAT 036795 2-mL clear HPLC vial Kit (cat# 5181-3400) Class A pipets and volumetric flasks Standard lab equipment (graduated cylinders, disposable tubes etc.) Stand-alone drop-in guard cartridge holder (part #844017-400) Hypercarb drop-in guard column (4 mm) (part # 844017-400) HPLC Pump (LC-10AD) LC/MS/MS and HPLC systems S u p p lie r Mettler Nalgene Drummond (VWR) Tekmar Burrell Scientific _ Dupont ' VWR VWR Supelco Waten Hewlett-Packard various suppliers various suppliers Keystone Scientific Keystone Scientific Shimadzu As described in section 4.5. Exygen Research Page 9 of 43 } 11 lr i '; f i !5 j i P V '. ! Exygen Research. 000383 Page 25 0/59 Page 67 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 Note* 1. In order to avoid contamination, the use of disposable labware (containers, tubes, pipetjes, etc.) la highly recommended. 2. Teflon or Teflon-lined containers or equipment should not be need. 3. It may be necessary to check the solvents (acetonitrile, methanol) for the presence of contaminants (especially PFOA) by LC/MS/MS before use. Certain lot numbers have been found to be unsuitable for use. 4. Use disposable micropipettes or pipettes to aliquot standard solutions when preparing standards and samples for extraction. 3. Equivalent materials may be substituted for those specified in this method. 3.4. Solutions (1) 2 mM ammonium acetate in water is prepared by weighing 0.154 g of ammonium acetate and dissolving in l.L of water. (2) Hypercarb filtered type I water is prepared by filtering type I water through a Hypercarb guard column using a HPLC pump at -2-3 mTJmi-n. Before use, wash the guard cartridge with -25 mL of HPLC grade acetonitrile, then - 23 mL of type I water, then begin collecting the filtered type I water eluate for use in the extraction. Repeat the wash after filtering -2 L of water. Note: The aforementioned example is provided for guidance, alternative volumes may be prepared as long as the appropriate ratios of the solvent to solute are maintained. 3 3 . Phepasation of Standard Solutions Analytical standards are used for three purposes: 1. Calibration Standards-These standards are prepared in methanol and are used to calibrate the response of the detector used in the analysis. Z Laboratory Control Spikes - These fortifications are prepared at concentrations corresponding to the LOQ and 5x LOQ and are used, to determine analytical recovery. Laboratory control spikes are prepared in control matrix. 3. Matrix Spikes - These fortifications are prepared by spiking into the field samples at a known concentration. Matrix spikes are used to evaluate the effect of the sample matrix on analytical recovery and are prepared at the client's request. The analyst may vary the absolute volumes of the standards as long as the correct proportions of solute to solvent are maintained. Exygea Research Page 10 of 43 ! 1 l * i r : i 1` Exygen Research. 000384 Page 26 o f39 Page 68 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygea Method No: BxM-023-071 3 .5 .1. S tock s o lu tio n Prepare individual stock solutions at 100 pg/mL f ix PFHS, PFOS and PFOA by weighing out 10 mg of each analytical standard (corrected for purity and if necessary, salt content) and dilute to 100 mL with methanol in separate 100-mL volumetric flasks. The stock solutions (in 125-mL LDFE bottles) are to be stored in a refrigerator at 2*0 to 6"C and are stable for a maximum period of one year from the date of preparation. 3 J .2 . F o rtific a tio n S o lu tio n s a. Prepare a mixed fortification standard at 1.0 pg/mL (1000 ngfmL) of PFHS, PFOS and PFOA by adding 1.0 mL of each of the 100 pg/mL stock solutions into a 100-mL volumetric flask and bring up to volume with methanol. b. Prepare a mixed fortification standard at 0.1 pg/mL (100 ng/mL) of PFHS, PFOS and PFOA by diluting 10.0 mL of the 1.0 pg/mL mixed fortification solution to 100 mL with methanol in a volumetric Exanrole: one hundred microllten of the 0.1 pg/mL solution spiked into 1 g of liver or 1 mL of serum/urine is equivalent to a 10 ppb (10 ng/mL or ng/g) fortification. t r- I l Store all fortification standard solutions in a refrigerator (in 125-mL LDFB bottles) at 2C to 6C for a maximum period of one year from the date of preparation. Note also that additional concentrations may be prepared if necessary. 3 .5 J . C a lib ra tio n S tand ard s I iI LC/MS/MS calibration standards containing PFHS, PFOS and PFOA are prepared at 0.1,02., 0.5,1.0,2.0 and 5.0 ng/mL in methanol via dilution of the 0.1 pg/mL mixed fortification solution (section 3.5.2.b). The following is a typical example; additional concentrations may be prepared as needed. Initial Cone. Volume Diluted to Final Cone. (ng/mL) (mL) (mL) (ng/mL) 100 5.0 100 5.0 100 2.0 100 2.0 It 100 1.0 100 1.0 5.0 10.0 100 0.5 2.0 10.0 100 0.2 ! 1.0 10.0 100 0.1 The standards may be used for a period of one year (in 125-mL LDPE bottles) when stored refrigerated (at 2C to 6C). Exygen Research Page 11 of 43 Exygen Research. 000385 Page 27 o f59 Page 69 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 BxygeaMethodNo: ExM-<B3471 4. M ETHOD 4.L H o w Diagram The flow diagram of the method la given below, followed by a detailed description of each step. Method Plow Diagram Weigh l g of liver or measure 1 mL o f serum or urine (fortify samples designated as matrix spikes and laboratory control spikes) 4 Add 9 mL of water to liver, 19 mL of water to serum and urine, homogenize Remove 1 mL and add 5 mL of ACN, shake i Centrihige A Decant supernatant into 35 mL of water 4 Load onto conditioned SPE A ' Elute with 2 mL methanol 4. LC/MS/MS Analysis 4.2. Sample Processing Far liver samples, place frozen samples in a food processor and homogenize with dry ice. Then place .samples in containers and leave open in frozen storage overnight to allow for COj sublimation. Seal and place the samples in frozen Stonge below -10C until time of extraction. No sample processing is needed for serum and urine samples. However, frozen serum and urine samples must be allowed to completely thaw to room temperature before use. [ | Exygen Research Exygen Research. 000386 Page 12 of 43 i Page 28 o f 59 Page 70 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023471 4-3. Batch Set up a. A batch of samples should not contain more than 20 field samples. b. Each batch of samples analyzed must include at least one control (method blank using control matrix) and two matrix controls fortified at known concentrations (typically 10 and 50 a g fg for liver or ngfinL for serum and mine) to verify procedural recovery for the batch. c. At least one field sample In each batch must also be separately fortified at a known concentration and carried through the procedure to verify recovery. Additional samples in the batch may also be fortified if desired. d. All samples require duplicate injections. 4 .4 . Sa m p l e E x tr a c tio n 4 .4 .1 . L iv e r E x tra c tio n a. Weigh 1 g of liver sample into a SO mL disposable centrifuge tube and fortify, if appropriate. b. Add water to the sample for a final volume of 9 mL. Cap tightly. c. Homogenize sample using a tissuemizer for -1 minute. d. Transfer 1 mL of the sample using a disposable pipette into 15 mL disposable centrifuge tubes. Add 5 mL of ACN and shake for -20 minutes on a wrist action shaker. e. Centrifuge tubes at -3000 rpm for - 5 minutes. Carefully decant supernatant into a 50 mL disposable centrifuge tube and add 35 mL of water. f. Load the sample onto a conditioned SPE column (for conditioning details, see section 4.4.3.). Discard the eluate. Any analyte residues will be trapped on the SPE column at this point g. Elute with 2 mL of metharioL Collect 2 mL of elute into a graduated 15 mL centrifuge tube. h. Analyze samples using electrospray LC/MS/MS. 4 .4 .2 . S erum and U rin e E x tra c tio n a. Measure 1 mL of serum or urine sample into a 50 mL disposable centrifuge tube and fortify, if appropriate. b. Add 19 mL of water to sample. Cap rightly and vortex for -1 minute. Then continue with steps d-h in section 4.4.1. ; ! j j \ `i I [ 1 | { * \\ ! I ; j: I r: Exygen Research Exygen Research. 000387 Pago 13 of 43 Page 29 o f 59 } i Page 71 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygea Method N: ExM-023-071 4 .4 .3 . S P E C olum n C o n d itio n in g Place the unconditioned SPE columns on the vacuum manifold. Condition the SPE columns by pissing - 10 mL of methanol through the column followed by - 5 mL of water. The washes may be pulled through the SPE column using vacuum at a flow rate of -1 drop/sec or may be allowed to pass through the column unaided. Discard all washes. Do not allow the column to dry. 4 .5 . Qu a n tit a tio n 4 J .1 . L C /M S /M S System and O p eratin g C o n d itio n s Mass Spec: Interface: Computer Software: Micromass Quattro Ultima (Micromass) Electrospray (Micromass) Harvard infusion pump (Harvard Instruments), for tuning COMPAQ Professional Workstation AP200 Windows NT, Masslynx 3 3 HFLC: Hewlett Packard (HP) Series 1100 HP Quit Pump HP Vacuum Degasser HP Autosampler HP Column Oven Note: A 4 X10 mm hypercarb drop in guard cartridge is attached on-line after the purge valve and before the sample injector port to trap any residue contaminants that may be in the mobile phase and/or HPLC system. HPLC Column: Genesis Cs (Jones Chromatography), 2.1 mm x SO mm, 4js ' Column Temperature: 35s C Injection Volume: IS pL Mobile Phase (A): 2 mM Ammonium Acetate in Type I water Mobile Phase (B): Methanol Time 0.0 2.0 %A 90 90 %B EogJaiaCm limin) 10 0 3 10 0 3 5.0 10 90 03 9.0 10 9.5 0 90 100 03 03 14.0 0 100 03 14.5 90 10 03 20.0 90 10 03 It may be necessary to adjust the HPLC gradient in order to optimize instrument performance. Columns with different dimensions (e.g. 2.1 x 30) and also columns Exygen Research Page 14 of 43 i t * [ i1 l I Exygen Research. 000388 Page 30 o f 59 Page 72 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Bxygen Metbod No: ExU-023471 from different manufacturer* (Keystone Betaail C n etc.) could be used, provided equivalent chromatography is obtained. tons monitored: Analvte PFHS PFOS FFOA Mode Negative Negative Negative TranaitiqnMonitored 399-80 499-99 413 -* 389 Approximate Retention Tin -8.2 min. -8 .8 min --8.6 min The retention dmes may vary, on a day to day basis, depending on the batch of mobile phase etc. Drift in retention times (up to 4 96) la acceptable within an analytical run, as long as the drift continues through the entire analysis and the standards are included at the beginning and end of the analytical run. Note: An alteraadve LC/MS/MS system may be used once demonstrated to be equivalent The mass spectrometer is tuned for each analyte by infusing a - 1.0 pgfoL standard solution (at 10 pL/min, using an infusion pump) via a "T* into a abeam of mobile phase containing 50% methanol and 50% 2mM ammonium acetate in water at 0.2 ml/min flow rate. Each analyte is initially tuned for the parent ion and then tuned for the product ion. Once the instrument is tuned, the optimized parameters axe saved as a tune file. This tune file Is then used during routine analysis.. 4 .5 .2 . C a lib ra tio n C urve Proced ures a. Inject the same aliquot (between 10 to 50 pL) of each calibration standard (ranging from the lowest level standard to the highest level prepared), into the LC/MS/MS. b. Use weighted linear standard curves far quantitation. Linear standard curves are generated for each analyte by linear regression using 1/x weighting of peak area versus calibration standard concentration using Maaslynx (or equivalent) software system. Any calibration standard found to bo a statistical outlier by using an appropriate outlier test, may be excluded from. the calculation of the calibration curve. However, the total number of calibration standards that may be excluded must not exceed 20% of the total number of standards injected. c. The correlation coefficient (R) for calibration curves generated must be 0.9925 (R2 05185). If calibration results fall outside these limits, then appropriate steps must be taken'to adjust instrument operation, and the standards or the relevant set of samples should be reanalyzed i I ! Exygen Research Exygen Research. Pige 13 of 43 000389 Page 31 o f 59 Page 73 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study Mo.: 023-082 Bxyjea Method No: ExM-023-071 Typical calibration curves for FFHS, PFOS and PFOA can be found in Hgures 14 J J Sam ple A n a lysis rti./ [ ftp' a. Inject the same aliquot (between 10 to SO pL) of each standard, sample, recovery, control, etc. into the LC/MS/MS system. [ ^ b. Standards corresponding to at least five or more concentration levels (starting with the LOQ level or below) must be included in an analytical set. ; e. An entire set of calibration standards should be injected at the beginning of a set followed by calibration standards interspersed approximately every 5-10 samples (to account for a second set of extracted standards). As an alternative, an entire set of calibration standards may be included at the beginning and at die end of a sample set. In either case, calibration standards must be the first and last injection in a sample set. d. The concentration of each samplc/fortification/contxol is determined from the standard curve, based on the peak area of each analyte. The standard responses should bracket responses of the residue found in each sample se t Results may be quantitated up to 10% outside the curve by extrapolation. I t necessary, dilute the samples to give a response within the standard curve range. i i ! r : | i e. Fortification recoveries falling within 70 to 130% are considered acceptable. f. Samples must be stored refrigerated between 2C to 6C until analysis. iI g. Samples in which either no peaks are detected or peaks less than the lowest concentration of the calibration standards are detected at the corresponding i I analyte retention time will be reported as ND (not detected). Samples in ! which peaks are detected at the corresponding analyte retention time that are less than the LOQ and greater than or equal to the lowest concentration of the calibration standards will be reported as NQ (not quantifiable). I i ii The analysis performed during the method development included fortifications at j f 10 and 50 ng/g of FFHS in rat liver, 10 and 50 ng'mL of FFHS in serum, 10 and : I 50 ng/g of PFOS and FFOA in rat liver and 10 and 50 ng/mL of FFOS and FFOA I in serum and urine. Typical chromatograms can be found in Hgures 4-39. j i ti I Exygen Research Exygen Research. 000390 Page 16 of 43 } ; Page 32 o f59 j [ iis Page 74 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: BxM-033-071 4.6. Acceptance Cw teria The M ow ing criteria must be met to ensure the presence of PFHS, PFOS and FFOA: 1. The chromatogram must show a peak of a daughter ion at 80 amu from a parent of 399 amu for PFHS, a daughter ion at 99 amu from a parent of 499 amu for PFOS, and a daughter ion at 369 amu from a parent of 413 amu for FFOA. 2. Method blanks must not contain analyte at levels greater than the LOQ. If a blank contains the analyte at levels greater than 10 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 3. Recoveries o f control spikes and matrix spikes (if any) must be between 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set.of samples should be re-extracted. Any matrix spike outside 70-130% should be evaluated by the analyst to determine if re-extraction is warranted. 4. Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation of the calibration curve. However, the total number of calibration standards that could be excluded must not exceed 20% of the total number of standards injected. 3. The correladon coefficient (R) for calibration curves generated must be 0.9923 (R1 0583). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set of samples should be reanalyzed. & Retention times between standards and samples must not drift more than 4 % within an analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 4.7. Performance Criteria _ The following two criteria must be performed once as a system suitability test, before die commencement of analysis, when using an instrumentation set-up that has not been used for this method. First Criterion: Run a standard solution on LC/MS/MS corresponding to the estimated LOQ (10 ng/mL) in matrix and obtain a signal to noise ratio for the analyte transition of at least 9:1, compared to a reagent blank. 11 this criterion cannot be met, optimize and change instrument operating parameters (or increase the injection volume, if appropriate). Exygen Reseuch P ige 17 o f 43 , : [ i < ' ;\ ! j : j j j i ttr.:- t Exygen Research. 000391 Page 33 o f 59 Page 75 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: BxM-023-071 Second Criterion; Run a set of standards of five or more concentration levels, from at or below the LOQ, up to the highest concentration level to be included in die analysis. Generate a calibration curve for the analyte and obtain a linear regression with a coefficient of determination (R1) of at least 0.985 for the analyte. Once this criterion is met, samples may be analyzed with standards interspersed. i 4 A Tim e Required to r Analysis One person can take a set of 20 samples through the sample preparation procedure in approximately 4 hours. The LC/MS/MS analysis of the set (containing 20 field samples, 1 matrix blank, 2 laboratory control spikes, 1 matrix spike and 12 standard injections) will take approximately 14 hours. 5 . CALCULATIONS a. Use Equation 1 to calculate the amount of analyte found (in ng/mL, based on peak area) using the standard curve (1/x weighted linear regression parameters) generated by the Masalynx software program. Egaitton 1; Analyte found (ng/inL) = (peak area - intercept! x D F x aliquot factor slope DF a factor by which the final volume was diluted, if necessary. Aliquot factora 9 for liver, 20 for serum and urine b. For samples fortified with known amounts of analyte prior to extraction, use I Equation 2 to calculate the percent recovery. II Equation 2: Recovery (%) = _ [ total analyte found (ng/mL) - analyte found in control (ng/mL)] . 11 ......... -- -.........---- 1X lO u ! analyte added (ng/mL) Note: Subtract analyte found in control (ng/mL) from analyte found (ng/mL), if ng/mL in control is greater than LOQ. Exygen Research Page 18 of 43 i Exygen Research. 000392 Page 34 o f 59 Page 76 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Bxyiea Method No: ExM-0334171 c. Use Equation 3 to calculate the amount of analyte found (in ppb) Eqaation 3: Analyte found (ng/g or ngtaL) frnlY ta found fiw/mT.t t bv imTt nunpla weight (f} or tem ple volume (mJL) FV final volume Fox repotting purposes, samples in which either no peaks are detected or peaks less than the lowest concentration of the calibration standards am detected at the corresponding analyte retention time will be reported as ND (not detected). Samples in which peaks am detected at the corresponding analyte retention dine that are lest than the LOQ and greater than or equal to the lowest concentration of the calibration standards will be repented as NQ (not quantifiable). 6. SAFETY The analyst should read the material safety data sheets for all standards and reagents before performing this method. Use universal precautions when handling standards and reagents, including working in fume hoods and wearing laboratory coats, safety glasses, and gloves. Exygen Research Exygen Research. 000393 Page 19 of43 Page 35 o f S9 Page 77 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Bxyicn Method No: HaM -tBM Tl T ab le 1. R ecovery S um m ary o f P F H S in R a t L iv er an d Serum Recovery Summary of PFHS In Rat Liver Sample ID 0201684 SpkA 0201684 SpkB 0201684 SokC Analyte Added fnofa) 10 10 10 Average: Standard Deviation: Percent Recovery (%) 108 110 128 11S 9J9 Sample ID 0201684 SpkD 0201684 SpkE 0201684 Sldt F Analyte Added fng/g) SO SO 50 Average: Standard Deviation: Percent Recovery (%) 101 68 94 98 . 15 Recovery Summary of PFHS In Rat Serum Sample ID 0201882 SpkA 0201682 SpkB 0201682 SokC Analvte Added fno/mL) 10 10 10 Average: Standard Deviation: Perant Recovery CU 112 '103 110 108 4.7 Sample ID 0201882 SpkD 0201682 SpkE 0201682 Sok F Analvte Added fnoftnL) 50 50 50 Average: Standard Deviation: Percent Recovery <%1 107 104 122 111 9.6 Exyjeo Reaarch Exygen Research. i Pige 20 of 43 000394 Page 36 o f59 Page 78 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 E xygu MethodNo: BxM-023-071 T a b le 2 . R ecovery Sum m ary o f PFO S in R a t L iver, S eru m an d U rin e Recovery Summary ofPFOS In Rat Uver SamolelD 0201684 Spk A 0201684 8pkB 0201684 Sok C Analyte Added (natal 10 10 10 Avirag Standard Deviation: Percent Recovery mi SB 98 108 96 U Sample ID 0201684 Spk 0 0201684 Spk E 0201684 Sok F Analyte Added (natal 60 SO 60 Average: Standard Deviation: Percent Recovery Ml 90 sa 87 88 U Recovery Summary ofPFOS In Rat Serum Samla ID 0201682 Spk A 0201882 Spk S 0201682 Sok 0 Analyte Added (hgtaiL) 10 10 10 Avaraga: Standard Deviation: Percent Recovery m i 80 85 99 88 9.8 SamplalD 0201662 Spk 0201682 Spk E 0201682 Spk F Analyte Addad (nq/mL1 60 so so Average: Standard Deviation: Percent Recovery t%1 118 122 121 120 2.1 Recovery Summary ofPFOS In Rat Urine Samla ID 0201682 Spk A 0201682 Spk B 0201682 Spk O Analyte Addad (noAnL) 10 10 10 Aveng Standard Deviation: Percent Recovery ml 98 88 91 93 4.7 Sample ID 0201682 Spk D 0201882 Spk E 0201682 Spk F Analyte Addad (ngfmt.) 50 50 so Average: Standard Deviation: Pereant Recovery mi 80 78 78 79 U I? ( i II f I- [ ft i Exygen Research Pige21 of43 Exygen Research. 000395 Page 37 o f 59 Page 79 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exypm Method No: ExM-023-071 T ab le 3. R ecovery S um m ary o f PFO A in R a t L iv er, S erum an d U rine Recovery Summary of PFOA InRat Liver Sample D 0201684 SpkA 0201684 SpkB 0201884 SokC Analyte Added fndkl 10 10 10 Avenge: Standard Deviation: Percent Recovery DU 95 101 99 98 3.1 Sample ID 0201684 Spk D 0201884 Spk E 0201684 Spk F Analyte Added (hg/a) GO 50 50 Avenge: Standard Deviation: Percent Recovery (%) 87 92 94 94 25 Recovery Summary of PFOA InRat Serum Sample ID 0201682 Spk A 0201682 Spk S 0201682 Spk C * Analyte Added (noAnU 10 10 10 Avenge: Standard Deviation: Percent Recovery (%) na 117 115 117 . 1JS Sample ID 0201682 Spk D 0201682 Spk E 0201882 Sok F Analyte Added fnq/mU 50 50 50 A vm gt: Standard Deviation: Percent Recovery (%) 107 112 115 in 44) Recovery Summary ofPFOA In Rat Urine Sample ID 0201682 SpkA 0201682 SpkB 0201882 SokC Analyte Added InoAnU 10 10 10 Avenge: Standard Deviation: Percent Recovery IW 66 91 89 89 2J Sample ID 0201682 Spk D 0201682 Spk E 0201682 Spk F Analyte Added Ina/mU SO 50 50 Avenge: Standard Deviation: Percent Recovery (%) 89 88 85 87 2.1 (! t ! I i. \ L I-, iI Exygen Research Page 22 of 43 Exygen Research. 000396 Page 38 o f 59 Page 80 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 F igu re L C alibration C arve for FFH S Bxygea Method No: H*M-023-07t Canpound 1 m e PfHS OniMrll oI riMiimriilnre t m m CaM fenaiM'1l1U**X'f*.1413t . RMfMOMtype Egttnul M , Arat Cun M>KLbMi.Origin: Baft*,WdgMtog:t t t A * I n Nm Exygen Research Exygen Research. 000397 I Pegs 23 of 43 i! i I Page 39 o f 59 Page 81 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 F igu re 2 . C alibration C urve fo r PFOS ExygenMethodNo:BxM-023-071 Compound 2 nuns: PFOS Coefficient otDetaminallon: 0997009 Caferatlon curve: 3211.30 *x+409285 Heaponae type: External Std, Ana Curve type: linear, Origin: Exdude. WetohSng: 1/x. Arfa tran Nane t, V1 S! k f I ; Exygen Research Exygen Research. 000398 Ptge 24 of43 Page 40 o f 59 Page 82 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 F igu re 3. C alibration C urve fo r PFOA Exygen Method No: BxM-023-071 Compound 1 namo:PFOA CoaflUonl o( DotenrinnUon: 0.995945 Cnibfitk curw: 31270J*x+-3875.35 RoaponM typo: External Std, Aran Curve typo: Linear, Origin: ExJuda, Weighting: 1AxAlia tmn* Nona F H Exygen Research Exygen Research. 000399 Page 23 of43 f; Page 41 o f59 Page 85of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method Mo:ExM-023-071 F igu re 4. R epresentative C hrom atogram o f a 0.1 ng/m L Standard. C ontaining FFHS F igu re 5 . R epresentative C hrom atogram o f a 0.1 ng/m L Standard C ontaining PFOS Exygen Research Exygen Research. 000400 Page 26 of 43 Page 42 o f 59 Page 84 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 E tna Method No: BxM-023-071 F igu re 6. R epresentative Chrom atogram o f a 0.1 ng/m L Standard C ontaining PFOA F igu re 7 . R epresentative Chrom atogram o f a 0.5 ng/m L Standard C ontaining PFHS i Exygen Research Exygen Research. 000401 Page 27 of 43 Page 43 o f 59 Page 85 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Bxjrjen Method Noe ExM-0Z3-071 fig u r e 8. R eprsentative Chrom atogram o f a 0 5 ng/m L Standard C ontaining PFOS Ml|*M| WMdtamm a * i 7M in mai .i.m.... .l-- fig u r e 9 . R epresentative Chrom atogram o f a 0 5 ng/m L Standard C ontaining PFOA CMnoaAuxgtaLproAaMPfot aMiMmiMrsT k i Exygen Research Exygen Research. 000402 I ! Page 28 of 43 ! Page 44 o f 59 Page 86 o f 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: E*M-023-07t F igu re 10. R epresentative Chrom atogram o f a 5.0 ng/m L Standard C ontaining F F eS I r i R F igure 11. R epresentative Chrom atogram o f a 5.0 ng/m L Standard C ontaining PFOS CMHO.i.uiiaM.H'OAatfmt Iiimm>-- f* i L II Exygen Seaurcb Exygen Research. 000403 j. I I t Fige29of43 \ Page 45 o f 59 I Page 87 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 B qrjen Method No:ExM -O n^m F igure 12. R epresentative C hrom atogram o f a 5.0 ng/m L Standard C ontaining PFOA F ig u re 13. R ep resen tativ e C h ro m ato g ram o f a R eagent B lan k S am ple A nalyzed fo r P F H S ihiemeiiHA lU M niura Exyjea ReKatch Exygen Research. 000404 Page30 of43 ' Page 46 o f 59 Page 88 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 BxygeaMethodNo: ExM-023-071 F ig u re 14. R ep resen tativ e C h ro m ato g ram o f a R eagent B lank S am ple A nalyzed fo r PF O S F ig u re 15. R e p re se n ta tiv e C L ro m a to g ra m o f it R e a g e n t B la n k S am p le A nalyzed fo r PFO A Exygen Research Exygen Research. 000405 Page 31 of43 .Page 47 o f59 Page 89 of 111 Exygen Study No.: 023-082 Study Plan; ExP-023-082 Exygen Study No.: 023-082 Hxyjra MethodNo:ExM-0Z3-OTl F ig u re 16. R ep resen tativ e C h ro m ato g ram o f a C o n tro l L iv er S am ple A nalyzed fo r PF H S F ig u re 17. R ep resen tativ e C h ro m ato g ram o f a C on tro l L iv er S am ple A nalyzed fo r PF O S Exygen Resesch Exygen Research. 000406 Page32 of43 ___________ Page 4 8 o f 59 ' i ' Page 90 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 BxygeaMethodN:ExM-023-071 F ig u re 18. R ep resen tativ e C h ro m ato g ram o f a C o n tro l L iv er S am nle A nalyzed fo r P F O A F ig u re 19. R ep resen tativ e C h ro m ato g ram o f a C o n tro l S eru m S am nle A nalyzed fo r P F H S P Exygen Research. 000407 Page 49 o f 59 Page 91 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethodNo: HxM-023-071 F ig u re 20. R ep resen tativ e C h ro m ato g ram o f a C ontrol S erum S an m i. A nalyzed fo r PFO S I MHMttA N llO M a u t w i n u fM nn u t i4m ' iui F ig u re 21. R ep resen tativ e C h ro m ato g ram o f a C on tro l S eru m S am ple A nalyzed fo r P F O A Exygen Research Exygen Research. 000408 Page 34 of43 Page SO o f 59 Page 92 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 BxygeaMethodNo: HxM-023471 F ig u re 22. R ep resen tativ e C h ro m ato g ram o f a C o n tro l U rin e S am ple A nalyzed fo r P F O S l tr.: F ig u re 23. R ep resen tattv e C h ro m ato g ram o f a C o n tro l U rin e S am ple A nalyzed fo r PFO A I f it f i Exygen Reseuch Exygen Research. 000409 Page 35 of43 Page 51 o /5 9 Page 93 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethodNo: BxU-023-071 F igure 24. R epresentative Chrom atogram o f a C ontrol L iver Sam ple F ortified a t 10 ngfg w ith PFHS t I* F F igure 25. R epresentative Chrom atogram o f a Control liv e r Sam ple F ortified at 10 ng/g w ith PFOS ' amMUHrafk,iowt m w uetim m i 6 Exygen Research Exygen Research. 000410 Page 36 of43 ! Page 52 o f 59 Page 94 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Hxygen Method No:BxM-023471 fig u r e 26. R epresentative C hrom atogram o f a C ontrol L iver Sam ple F ortified a t 10 n gfe w ithP F O A fig u r e 27. R epresentative C hrom atogram o f a C ontrol L iver Sam ple F ortified a t 50 n g/g w ith PFH S u n m ih.e'kD.mpf Exygen Research Exygen Research. 000411 Page 37of 43 Ii t \ Page 53 o f 59 Page 95 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: HxM-023-071 F igure 28. R epresentative C hrom atogram o f a C ontrol L iver Sam ple F ortified at 50 ng/g w ith PFOS | F igu re 29. R epresentative C hrom atogram o f a C ontrol L iver Sam ple Fortified a t 50 ng/g w ith PFOA ' Exygen Research Exygen Research. 000412 Page 38 of43 I Page 54 o f 59 Page 96 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen MethodNo: ExM-023-071 f ig u re 30. R ep resen tativ e C h ro m ato g ram o f a C o n tro l S erum Sam p! F o rtifie d a t 10 ng/m L w ith PFH S F ig u re 31. R ep resen tativ e C h ro m ato g ram o f a C o n tro l S eru m S am ple F o rtified a t 10 ng/m L w ith PFO S maeMa MMWIifhMl t Exygen Retouch Exygen Research. Tjn u i ue um P*e 3 of Page 55 o f 59 . Page 97 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-0234J7l fig u r e 32. R epresentative Chrom atogram o f a C ontrol Serum Snm pio F ortified at 10 ng/m L w ith PFOA fig u r e 33. R epresentative Chrom atogram o f a C ontrol Serum Sam ple F ortified at 50 ng/m L w ith PFH S Exygen Research Exygen Research. 000414 Page 40 of 43 i Page 56 o f 59 Page 98 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 HxygeaM ahod No: BxH-Q23-tm F igu re 34. R epresentative Chrom atogram o f a C ontrol Serum S amp le F ortified a t 50 ng/m L w ith PFO S F igu re 35. R epresentative Chrom atogram o f a C ontrol Serum Sam ple F ortified at 50 ng/m L w ith PFOA Exygen Research Exygen Research. 000415 Page 41 of 43 Page 57 o f 59 Page 99 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethod No: ExM-C2W71 F igure 36. R epresentative C hrom atogram o f a C ontrol U rine Sam ple F ortified a t 10 ng/m L w ith PFOS F igu re 37. R epresentative C hrom atogram o f a C ontrol U rine Sam ple F ortified at 10 ng/m L w ith PFOA ' Exygen Resench Exygen Research. 000416 Page 42 of 43 Page 58 o f 59 Page 100 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Bxygen Method No; ExM-023-071 F igure 38. R epresentative Chrom atogram o f a C ontrol U rine Sam ple F ortified at 50 ng/taL w ith PFOS I%T fIfl Fi !r. . f F igu re 3 9. R epresentative Chrom atogram o f a C ontrol U rine Sam ple F ortified at 50 ng/m L w ith PFOA Exygen Research Exygen Research. 000417 Page 43 of 43 P a g e 5 9 o f 59 ! Page 101 of 111 Exygen RESEARCH ^Precise Research. Proven Results. Exygen Study No.: 023-082 Exygen Research. 000418 Page 102 of 111 Exygen Study No.: 023-082 H Exygen Study No.: 023-082 \ Proven Results. PROTOCOL DEVIATION Page 1 of 1 Deviation Num ber 3 Date ofOccurrence: throughout study Exygen Study N um ber 023-082 Protocol Number ExP-023-082 D E SC R IPT IO N O F DEVIATION 1.Analytical'Procedure Summary-Method Section 4.5.3.d-- several samples were quantified above 10% of the curve. 2.Analytical Procedure Summary-Method Section 4.5.3.e-- the acceptance critieria for QC spike recoveries could riotalways be met. A C T IO N S TAKEN forwamplB-.ttalatloalMUtNi SOPrcvtolfln. etc. 1-2.Protocol deviation issued. Recorded Bjr Date: to fa c jc O . IM PACT ON STUDY 1.No negative Impact because residue was < than 15% above the curve for C6 and C8 for sample 0204493 Spk V and for C8 for samples 0203965 and 0203965 Dup. Also, the samples that were quantified outside the curve forTHPFOS and THPFDS were only fortification recoveries and the majority of the recoveries were > 100%. 2.impact not determined only documented since thismethod has not been validated for these anions at these levels. Principal W, Signature Date Signptupe j . Date . Mpdagement Signature/ (Sponsor) U:\Forms\PROTOCOL_OEVlATION.doc Date \ Date Exygen QAU Review il tu /r/v/??9/25/2002/5 Exygen Research. 000420 3 0 5 8 Research D rive VState College, P A 16801, U S A T: 80022813219 F: 814.2 7 2 .1 0 19 exygen.com Page 104 of 111 Exygen Study No.: 023-082 \ Proven Resub. P a ge _ 1 _ of _1_ PROTOCOL DEVIATION Deviation Number: 4___ Date of Occurrence: throughout study Exygen'Study Number: 023-082 Protocol Number: ExP-023-082 D E SC R IP T IO N OF DEVIATION 1.Analytical'frocedure Summary-Method Section 4.5.3.g-- several samples were quantified ' below the aiiye and NO was used forwhen no peak was detected and NQ was used for negative results. Allother resultswere reported. 2.Analytical Procedure Summary-Method Section 4.5.3.C-- a second set ofcalibration standards was not analyzed in a set. A CTIO N S TAKEN far am ple - deviation Iwued. SOP wvMon. te. 1-2.Protocol deviation Issued. Recorded ___________ Date: IQ 3 [ Z IM PACT ON STUDY 1. No negative Impact because no claim of,quantitative accuracy has been established. 2. No negative impact because the extracted.standards were analyzed throughout the set which provide a QC check on the initialcalibration.\ t iUl Principal Investigator SiIgnaatture Date Stray D irects Signature, *. Date , jjjairniagement Signature / (Sponsor) U:\Fontit\PROTOCOk_DEVIATION.doc Date Date Exygen Q AU Review HLL- I 9/25/2002/5 Exygen Research. 000421 \jK.33005SE8 Research Drive S`tale(CCooflleege, fPA 1 6 8 0 1 , U S A ,T: 8 0 0 .2 8 1 3 2 1 9 F: 8 1 4 .2 7 2 .1 0 19 exygen.com Page 105 of 111 Exygen Study No.: 023-082 Exygen Research. 000422 Page 106 of 111 Exygen Study No.: 023-082 OCT-30-2002 12!24 P.02/06 _ 'RESEARCH kPmd Ruaweh. \ Provan Rmurs. PROTOCOL OBVIATION . OyMtfnNbmbac 1 ' = ' pat* oOccirrnk 10/13/02. 1 of ~ T ~ E*yarfetudyNum tac'023-082' -- -.V i l l.......... V i --.1... Protocof Number ExM 23*M 2 1-ix.iM. : . j -- ........ L. ' i t : Aratyjcal Pteadura' SumraryrUlCalfbr^tfe^ Standard] Did not prnvf^xuieted.standards jithvmplaima fit 1.5 and 2.3 ppb leveii.. ; .D a w t c f c i f a : . fc fritf8 T U is ~ - -- --------------- i"-- :-- r No negative impactbae^uiaonlytadtM(jtcdtunplyaflable and thattnaar'range/thecurva wasMrtabil*hed wrthout'.f^-^''--- ; J W f ' K-j t; ; igament'SIgnatur (Sponaort. U,n/m\PftOTOCOlv.D6VIATtoN.il#6 # . -:V \ ;r Pala P fjo fd L ' \ **** * // /&/ \ Date \ m J& \ Data * ' Exygen QAU Review V Ait e \ Jqx lihultL* w o im . r otypnxcm * Exygen Research. 00042a Page 107 of 111 OCT-30-2002 12:24 Exygen Study No.: 023-082 P.03/06 Exygert Research. 000424 Page 108 of 111 Exygen Study No.: 023-082 OCT-30-2002 12! 24 P .04/06 ^RESEARCH l.PnbP'ircoUveMn RHceduvs.1' V: ` *T, . ^ .' : . , Pag* -1 of .1 PROTtCpfepEVlAtlON .U^tlrcNurT!tar 3 , iOtoOOejufjenc thrcuehoutrtutiv . ' 023-081 ; i ^ :i . 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