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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 PROTECT Study Plan Number: ExP-023-082 Exygen Study Number: 023-082 Sponsor Study Number: E02-1071 Total Pages: 111 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. 5.2 (3): The date of receipt of for the calf serum sample ED 0204718 was not documented. 4. 4 1-2-2 (g): The instrument used for the analysis has not been qualified. Exygen Research William K. Reagan, Ph.D. Sponsor Representative 3M Environmental Date Exygen Research. Page 2 of 111 Exygen Study No.: 023-082 QUALITY ASSURANCE STATEMENT The Quality Assurance Unit of Exygen Research reviewed Exygen Study Number 023 082 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 InsDected Date Reported to Date Reported to Exygen Studv Director Manaeement Date Reported to SDonsor 10/10/02 10/14/02 10/30/02 10/30/02 10/15/02 10/25/02 10/25/02 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 iU > - Naomi Lovallo Technical Lead-QA iff)JfSt (y** to / & te n - Date Exygen Research. 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: / John M. Flaherty Vice President Exygen Research Sponsor Study Monitor, 3M: William K. Reagan, Ph.D. 3M Environmental tohobi Date Date Date Exygen Research. 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 (C l2), 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. 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. 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..............................................................................................................11 3.0 INTRODUCTION............................................................................... 11 4.0 TEST SYSTEM.........................................................................................................11 5.0 TEST ITEMS............................................................................................................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. 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 III 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. Page 8 of 111 Exygen Study No.: 023-082 Figure 1 Figure 2 Figure 3 Figure 4 LIST OF FIGURES Chromatogram Representing 0.1 ng/mL Calibration Standard Page ....28 Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb (Exygen ID: 0204490 Spk J, Sponsor ID: TCR-674)................................. 32 Chromatogram Representing a Human Plasma Sample (Exygen ID: 0204490, Sponsor ID: TCR-674).................................................................................. 36 Chromatogram Representing a Sample Analyzed for Three Daughters for THPFDS (Exygen ID: 0204292 Dup, Sponsor ID: X328-A)........................ 40 Exygen Research. Page 9 of 111 Exygen Study No.: 023-082 LIST OF APPENDICES Page A ppendix A Study Plan ExP-023-082 (Exygen Study No. 023-082) and Deviations................................................................................................. 41 Exygen Research. 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 (C ll), 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 o f the analysis for perfluorooctane sulfonate (PFOS), perfluorohexanoate (C6), perfluoroheptanoate (C7), pentadecafluorooctanoate (C8), heptadecafluorononanoate (C9), nonadecafluorodecanoate (CIO), perfluoroundecanoate (C ll), 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. 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, Cl, C8, C9, CIO, C il, 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. Exveen Inventory 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 (%) Exoiration 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 Exygen Study No.: 023-082 The molecular structures of the anions are given below. Ii Name: PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 499, as shown FFFF FF F S03 FF FFFF Name: C6 Chemical Name: Perfluorohexanoate 1 Molecular Weight: 313, as shown j Name: C7 Chemical Name: Perfluoroheptanoate Molecular Weight: 363, as shown Name: C8 Chemical Name: Pentadecafluorooctanoate Molecular Weight: 413, as shown 'O' Exygen Research. 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 Name: C ll Chemical Name: Perfluoroundecanoate Molecular Weight: 563, as shown Name: C12 Chemical Name: Perfluorododecanoate Molecular Weight: 613, as shown Exygen Research. Page 14 of 111 Exygen Study No.: 023-082 Name: THPFOS Chemical Name: Tetrahydroperfluorooctane sulfonate Molecular Weight: 427, as shown FFFH FH F S03 FFFH 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. 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 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 otherresponses were reported. 6.5 Description of Instrum ent 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 temprature = 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 finin') 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 fmL/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 C7 363 C8 413 269 319 369 C9 CIO C ll C12 PFOS THPFOS 463 513 563 613 499 427 419 469 519 569 80 81 THPFDS 527 81 Dwell fsecs') 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. 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 (TnL) 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)] xjqq analyte added (ppb) Note: Any analyte found in the control was subtracted from analyte found. However, the response for the sample duplicate was not used. Exygen Research. 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: 101702A), 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) = f54636- 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. 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. PS 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 pm 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 som e 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. Page 20 of 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 o f all raw data, as well as a signed copy o f 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. Page 21 of 111 Exygen Study No.: 023-082 TABLES Exygen Research. 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 XC101702-0 NA 0.0 -- - XC101702-1 NA 0.2 85 120 163 XC101702-2 NA 0.5 98 111 127 XC101702-3 NA 1.0 90 113 109 XC101702-4 NA 1.5 102 113 121 XC101702-5 NA 2.0 102 108 117 XC101702-6 NA 2.5 97 99 111 XC101702-7 NA 5.0 95 97 109 AVG: 96 109 122 STANDARD DEVIATION: 6.2 8.2 19.1 RELATIVE STANDARD DEVIATION: 6.5 7.5 15.6 C9 CIO C ll C12 ---- 151 155 96 87 132 135 110 111 124 112 111 116 127 121 120 121 122 115 109 110 107 103 98 98 108 104 106 105 124 121 107 107 15.0 18.6 8.2 11.5 12.1 15.4 7.6 10.7 PFOS ** ** ** ** ** ** ** 9ft ** ** ** 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 XC101502-8 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 XC101502-13 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. Page 23 of 111 Exygen Study No.: 023-082 Table III . Calf Serum Summary of Recoveries for Laboratory Fortified Matrix Spikes ________________________%Recovery______________ Fort Level Sample ID Sponsor ID (ng/mL) C6 C7 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 144 118 131 18.4 14.0 THPFDS 126 108 117 12.7 10.9 Human Serum _______________________ %Recovery Sample ID Fort Level Sponsor ID (ng/mL) C6 Cl C8 C9 CIO C ll C12 PFOS THPFOS 0203963 SpkC Lot 020821 0203964 SpkD Lot 22K0965 0203965 SpkE Lot GO140604 0204292 Spk F X328-A 0203963 SpkM Lot 020821 0.5 0.5 0.5 0.5 5.0 114 142 123 133 124 151 160 44 92 144 91 96 110 107 53 120 376 153 118 139 132 94 125 380 188 129 128 131 106 112 164 146 131 137 134 ** ** ** ** ** 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 PEVIATION: 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. 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 SpkH TCR-684 0.5 69 94 211 152 107 93 103 ** 122 0204490 SpkJ TCR-674 0.5 78 78 73 84 90 91 96 ** 107 0204491 SpkJ 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 SpkR 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 O 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 0 2 0 4 4 9 2 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 Spk U TN-A-06333 5.0 88 85 100 94 97 93 101 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 ** ** ** * ** ** ** ** ** ** 114 162 132 107 124 141 130 19.8 15.3 129 155 120 104 116 120 124 17.2 13.9 ** Recovery not applicable because the residues detected in sample were significantly greater than the amount fortified. Exygen Research. Page 25 of 111 Exygen Study No.: 023-082 Table IV Summary of Residues for Human Serum Samples Amount Found (ng/mL) Sample ID Sponsor ED 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 0203963 Dup Lot 020821 ND ND 2.93 0.684 0.22 0.373 0.0282 33.3 0203964 Lot 22K0965 ND 0.0940 1.45 0.307 0.107 0.0942 0.0122 8.64 0203964 Dup Lot 22K0965 ND 0.0976 1.45 0.276 0.108 0.115 0.0172 8.95 0203965 Lot G0140604 0.0687 0.145 5.93 0.508 0.214 0.207 0.0199 29.7 0203965 Dup Lot G0140604 0.0884 0.170 6.00 0.557 0.204 0.236 0.0184 29.3 0204292 X328-A ND 0.115 3.58 0.634 0.189 0.137 0.0169 16.6 0204292 Dup X328-A ND 0.12 4.21 0.772 0.221 0.149 0.0490 20.6 ND ND ND ND 0.0440 0.0436 0.0221 0.0394 0.0743 0.0599 0.0402 0.0412 0.0846 0.0944 0.0791 0.0946 Table V Summary of Residues for Human Plasma Samples _______________________ Amount Found (ng/mL)__________ Sample ID Sponsor ID C6 C7 C8 C9 CIO C ll C12 PFOS THPFOS THPFDS 0204334 TCR-684 0204334 Dup TCR-684 0204490 TCR-674 0204490 Dup TCR-674 0204491 TN-A-6337 0204491Dup TN-A-6337 0204747 TCR-683 0204747 Dup TCR-683 ND 0.0925 2.57 0.174 ND 0.0875 2.64 0.198 ND ND 0.0887 NQ ND ND 0.130 NQ ND ND 2.23 0.166 ND 0.0309 2.38 0.208 ND 0.0897 2.09 0.287 ND 0.0876 2.92 0.416 0.108 0.0797 ND 0.120 0.0695 ND 0.0252 0.0458 ND 0.0167 0.0357 ND 0.0840 0.0501 ND 0.0958 0.0346 ND 0.121 0.105 ND 0.163 0.178 ND 14.8 0.0140 14.9 ND 4.78 0.0115 4.84 ND 11.4 ND 12.6 ND 14.1 0.0193 19.4 0.0292 0.0539 0.0609 ND ND 0.0252 ND 0.0843 0.109 Table VI Summary of Residues for Monkey Serum Samples _______________________Amount Found (ng/mL)__________ Sample ID Sponsor ED C6 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 C7 C8 C9 CIO C ll C12 PFOS THPFOS THPFDS 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. Page 26 of 111 Exygen Study No.: 023-082 FIGURES Exygen Research. Page 27 of 111 Exygen Study No.: 023-082 Figure 1 Chromatogram Representing 0.1 ng/mL Calibration Standard Exygen Research. Page 28 of 111 Exygen Study No.: 023-082 Figure 1 (cont) Chromatogram Representing 0.1 ng/mL Calibration Standard Exygen Research. Page 29 of 111 Exygen Study No.: 023-082 Figure 1 (cont) Chromatogram Representing 0.1 ng/mL Calibration Standard Exygen Research. Page 30 of 111 Exygen Study No.: 023-082 Figure 1 (cont) Chromatogram Representing 0.1 ng/mL Calibration Standard 'vi - v,n I i \ Exygen Research, 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) Exygen Research. Page 32 of 111 Exygen Study No.: 023-082 Figure 2 (cont') Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb Exygen Research. 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 SpkJ, Sponsor ID: TCR-674) Exygen Research. Page 34 of 111 Exygen Study No.: 023-082 Figure 2 (cont') Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb (Exygen ID: 0204490 SpkJ, Sponsor ID: TCR-674) Exygen Research. Page 35 of 111 Figure 3 Exygen Study No.: 023-082 Chromatogram Representing a Human Plasma Exygen Research. Page 36 of 111 Exygen Study No.: 023-082 Figure 3 (conf) Chromatogram Representing a Human Plasma Exygen Research. Page 37 of 111 Exygen Study No.: 023-082 Figure 3 (cont') Chromatogram Representing a Human Plasma m ! Exygen Research. Page 38 of 111 Exygen Study No.: 023-082 Figure 3 (cont') Chromatogram Representing a Human Plasma Sample (Exygen ID: 0204490, Sponsor ID: TCR-674) i 'i 1., ri P Exygen Research. 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. 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 Performing Laboratory: 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 Exygen Research. Page 1 of39 Page 42 of 111 Exygeij Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 1) Emily R. Decker, Study Director, Exygen Research 2) William K Reagen, Sponsor Study Monitor, 3M 3) Exygen Research Quality Assurance Unit i: 3' C [ Exygen Research. 1 Page 2 o f59 Page 43 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study N o.: 023-082 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 This study plan was audited by the Quality Assurance Unit of Exygen Research. ___________ ______________________________ Naomi Lovallo Technical Lead-QA (ill>HlCflDate APPROVALS (J2mlly R/lDecker, Study Director Exygen'1 { ? . fiO /Joh n Flaherty, Vice Prsident, Facility Management Exygen m i z Date Date William Reagen, Sponsor Study Monitor 3M Date i 5 [' t$ Exygen Research. Page 3 o f 59 Page 44 of 111 Exygen Study No.: 023-082 10/09/02 14:26 1:I ` -!I!.'. ! . RSD INF TECH 2-2E-01 + *86142311580 `. ' . '' HO.215 P002 ' Study Plan: ExP-023-082 ' Exygen Smdy No.: 23-082 ! . i. : ' !Study T ide: A nalysis o f Pooled Human S e n and Plasm a and M onkey Sera for [ Fluorocarbons U sing Exygen M ethod ExM -023-071 ! . h | Study Plan Number. ExP-023-082 ' Exygen Study Num ber 023-082 ' tI '!i& s study plan was audited b y the Quality AssuranceU nit ofE xygen Research. gfennii Lovallo , 3&&nical Lead-QA I I APPROVALS .I E M ly R. Decker. Study D irectes D ate - Date :t 1 John Flaherty, V ice President, Facility Management D ate tr jl | -RECEIVED TINE OCT. 9. 4 :13PM Exygeri'Research. .Paga 3 o f59 PRINT TIME OCT. 9 . 4 : 14PM ^ ^ Page 45 o f 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 8aaaaaaaaaaaaaHaaaaaaaaaaaaaaaaaHaHaaaaaaaaaaaaaaaaaaaaaaaaaaaaaakaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaHa STUDY DIRECTOR_______________________________ !........................................................................... 88 SPONSOR 8 SPONSOR STUDY MONITOR 9a aMaaaaaaaaaaaaaaaaaaaaMaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa*aaaaaa#iaaaaaaaaaaaaaaaaaa<aaaaaaaaaaaaaaaa PROPOSED EXPERIMENTAL START AND TERMINATION DATES 9 COMMUNICATIONS 9 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM ,9 ,9SAMPLE PROCUREMENT, RECEIPT AND RETENTION1 aaaa<Maaaataaaaaaaaaaaaaaaa*aaaaaaaMaaaaaMaaaaaaaaaaaiaaaaaaaaaakk*aaa SAMPLE IDENTIFICATION.................. 10 ANALYTICAL PROCEDURE SUMMARY____________ _____- ................................................................10 VERIFICATION OF ANALYTICAL PROCEDURE................................................................................. -- 11 METHODS FOR CONTROL OF B IA S.............................................................................................................. 12 STATISTICAL METHODS 12ataaaaaaaataaH aaatM aaaaannM M km kH M HM HM ktaaaataM aatM M aiaatiiaaaaataaaaaaatM kttaM aaaiaiaaaaaaaaaaaaaaHaaaaaa GLP STA TEl^El^r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 REPORT________________________________________________________________________________ 12 SAFETY AND HEALTH.........___ ........................-- --------......--------------- ----- -------- --- .................... 13 AMENDMENTS TO STUDY PLAN 14aaaaaaaaaaaaaaaaaafaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaMaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaiaaaaaaaaa aaaaaaaaaaaaaaa DATA RECORD KEEPING_______________________________________________ 14 QUALITY ASSURANCE 15aaaaaiaaaaaHaHaaaaaakaaaaaaiaaaaaataiaaaaaakaaaaaaaaaaaaaakaataaaaaaaaaaaaiaaaaaaaaaaaaaaaaiaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa RETENTION OF DATA AND ARCHIVING................................ 15 APPENDIX 1 .......................................................................................................................................................... 16 J ii R t } It }; i l i, 5 ir. i. h 1 Exygen Research. Page 4 o/59 ^ae 46 o f 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 The purpose of this study is to perform analysis for 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 CTHPFOS), and tetrahydroperfluorodecane sulfonate (THPFDS) in pooled human serum and plasma and monkey sera using Exygen method ExM-023071 entitled "Method of Analysis for the Determination of Perfluorohexanesulfonate (PFHS), Perfluorooctanesulfonate (PFOS) and Pentadecafluorooctanoic Acid (PFOA) in Rat Liver, Serum andUrine." The study will be audited for compliance with OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17 by the Quality Assurance Unit of Exygen Research. !. [ r : ; ; i The test items are 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). All test items were received from tire Sponsor. Name: PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 499, as shown ` ; I l(1. I ' ! \ Exygen Research. Page 5 o f 59 Page 47 of 111 Exygen Study No.: 023-082 Name: C6 Chemical Name: Perfluorohexanoic acid Molecular Weight: 313, as shown Study Plan: ExP-023-082 Exygen Study No.: 023-082 Name: C7 Chemical Name: Perfluoroheptanoic acid Molecular Weight: 363, as shown Name: C8 Chemical Name: Pentadecafluorooctanoic acid Molecular Weight: 413, as shown Name: C9 - Chemical Name: Heptadecafluorononanoic acid Molecular Weight: 463, as shown Exygen Research. Page 6 o f 59 Page 48 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Name: CIO Chemical Name: Nonadecafluorodecanoic acid Molecular Weight: 513, as shown Name: C ll Chemical Name: Perfluoroundecanoic acid Molecular Weight: 563, as shown Name: C12 Chemical Name: Perfluorododecanoic acid Molecular Weight: 613, as shown Name: THPFOS Chemical Name: Tetrahydroperfluorooctane sulfonate Molecular Weight: 427, as shown Exygen Research. Page 7 o f59 Page 49 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Name: THPFDS Chemical Name: Tetrahydroperfluorodecane sulfonate Molecular Weight: 527, as shown The purpose of this study is to perform analysis on four different lots of pooled human serum, four different lots of pooled human plasma, and three lots of pooled monkey serum for the target fluorocompounds using the analytical method, "Method of Analysis for the Determination of Perfluorohexanesulfonate (PFHS), Perfluorooctanesulfonate (PFOS) and Pentadecafluorooctanoic Acid (PFOA) in Rat Liver, Serum and Urine." Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Emily Decker Scientist Exygen Research Phone: (814) 272-1039 emily.decker@exygen.com 3M Environmental Laboratory Building 2-3E-09 S t Paul, MN 55133-3331 Phone:(651)778-6565 Exygen Research. Page 8 o f 59 Page 50 of 111 Exygen Study No.: 023-082 Study Plan; ExP-023-082 Exygen Study No.: 023-082 William Reagen Building 2-3E-09 S t Paul, MN 55133-3331 Phone: (651) 778-6565 wkreagen@mnun.com It is proposed that the analytical portion of this study be conducted from October 14 to October 21, 2002. The actual experimental start and termination dates will be included in the final report. All communications between the Testing Facility, method developers, and the Sponsor will be directed through the Study Director (or designate) and the Sponsor Study Monitor. Communications will be fully documented by the Study Director. Pooled human sera and plasma and monkey sera are used as the test systems in this study. The matrices will be provided by the sponsor. The matrices will be representative of that for which this analytical method was designed. - | | Pooled human serum samples were purchased by the sponsor from SigmaAldrich, Milwaukee, WI, Lampire Biological Laboratories, Pipersville, PA, Bioresource 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 Exygen Research. Page 9 o f 59 Page 51 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Biological Laboratories, Pipersville, PA, Bioresource Technology, Lie., Fort Lauderdale, FL, Golden West Biologicals, Temecula, CA, and Innovative Research, Inc. Southield, ML In addition, blank matrix consisting of pooled human plasm a collected in rural C hina w as provided by the sponsor. T he samples will be used as received. The receipt and processing of the matrices will be documented in the final report and raw data associated with the study. The samples will be stored frozen at ^ -10C. j I | | Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Sample storage conditions and locations will be documented throughout the validation. . > j ;r ; [ Reference: Exygen Method ExM-023-071 "Method of Analysis for the Determination of Perfluorohexanesulfonate (PFHS), Perfluorooctanesulfonate (PFOS) and Pentadecafluorooctanoic Acid (PFOA) in Rat Liver, Serum and Urine." A copy of the method can be found in Appendix L The method will be modified as follows: M ethod Summary L Serum a. Measure 2 mL of serum sample into a IS 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 SOmL disposable centrifuge tube and add 3S 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 1 mL of methanol. Collect 1 mL of elute into a graduated 15 mL centrifuge tube. f. Analyze samples using electrospray LC/MS/MS. n. Calibration Standards ; ' i : [ -\ ! | f ,! ! ; j ! | 1 Exygen Research. Page 10 o f 59 ! Page 52 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 of calibration standards will be processed through the extraction procedure identical to the samples, using bovine serum and also a set using human plasma. The fortification of the standards before extraction is done according to the following table: Cone. Of Mixed Fortification Solution (ng/mL) 1 10 10 10 10 100 100 Fortification Volume (pL) 400 100 200 300 400 50 100 Volume of Control Sample (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Cone, of Extracted Calibration Standard (ng/mL) 0.2 0.5 1.0 1.5 2.0 2.5 5.0 The testing facility will establish the relationship between the instrument response and the concentration of analyte in order to assess the linearity of the system. A standard curve should be constructed with at least five standards. The testing facility should also verify the endogenous levels of analyte in the matrix control samples. This should be accomplished by analysis of a control sample for each matrix and examination of the region of analyte retention. The potential exists for interference from fluorochemicals introduced from dietary material and other exogenous sources. Samples are fortified with the target analytes. The compounds will be made into solutions as per the method, and added to the matrices via a micropipette. Fortified samples will be processed through the described procedures to ensure method accuracy and to check for bias. Recoveries should be between 70% and 130% of the fortified levels. The sponsor may accept occasional recoveries outside of this range. The relative standard deviation (RSD) for each fortification level as well as the overall RSD, should be less than or equal to 20%. Any modifications to the analytical method will be documented in the study raw data. Modifications deemed significant by the Sponsor or Study Director will necessitate revision of the method. Exygen Research. Page 11 o f 59 Page 53 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082* Control of bias will be addressed by taking representative sub-samples from a homogeneous mixture of each matrix for untreated control samples, and by analyzing at least two levels of fortifications. Statistics will be limited to those specified in the subject method and to the calculation of average recoveries, as applicable. All aspects of this study shall be performed and reported in compliance with OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor. A final report will be prepared by the study director or their designee at the conclusion of the study. The report will include, but will not be limited to, the following: The name and address of the Study Director, Sponsor Monitor and of the testing facility. A statement of GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates of study inspections and dates findings were reported to the Study Director and Management Exygen Research. Page 12 o f 59 Page 54 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 A description of the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy of the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page. Any steps considered critical, i.e. steps where little variation is allowable or directions must be followed precisely. Description of the instrumentation used and operating conditions. The number of worker-hours or calendar days required to complete one set of samples. All results horn all sets analyzed. Identify all control and fortified samples, and in the data table include sample number, fortification level, and unique identification number by sample set. Representative chromatograms for each analyte in each matrix, including chromatograms of a standard and a control sample, and a chromatogram at a fortification level. The location of the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity of the data will be documented in the report. Locations where raw data and the final report are to be archived. Additions or corrections to the final report shall be in the form of an amendment by the Study Director. The amendment shall clearly identify that part of the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Study Monitor. : ; . 1 I [ f : ; I ' ti 1 : : t . i i [ ; 'i Laboratory personnel will practice good sanitation and health habits. Any health condition of laboratory personnel that may be considered to adversely affect the study will be reported to the Study Director. Any injury to laboratory personnel occurring during the conduct of this study will be reported to the study director. Exygen Research. 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 of personnel and the environment to the test or reference substance(s). All significant changes to the study plan outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Study Monitor. Amendments usually will be issued prior to initiation of study plan change. However, when a change is required without sufficient time for die issue of a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date of the written amendment will be the date of the documented change. Copies of the signed amendments will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations horn the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Study Monitor. Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains of custody History and preparation of standards (stock, fortification, calibration) Description of any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing of study personnel, signatures and initials Chronological presentation of all study correspondence Any other documentation necessary for the reconstruction of the study Chrom atogram s- All chromatograms will contain the following: Sample identification, date, Exygen study number, arrow or other indication of the area of interest, and injection number corresponding to the run. Additionally, fortifications will include the amount of analyte added and the sample number of the sample that was fortified. Exygen Research. Page 14 o f 59 Page 56 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Analytical standard chromatograms will additionally include the concentration (e.g., |ig/mL). As part of the documentation the following sheets will be included in each analytical set a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. The QA Unit of Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings of audits to the Study Director, Exygen Management, and the Sponsor Study Monitor. All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Sponsor. These will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the lifetime of the product and shall obtain permission of the sponsor before discarding. l [ | {ile-f t [ 1 ; ! . ; Exygen Research. IlI j. Page 15ofS9 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. Page 16 o f59 Page 58 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 T IT L E M ethod of Analysis for the Determination ofPerfluorohexanesulfonate (PFHS), Perfluorooctanesulfonato (PFOS) and Pentadecafluorooctanoic A dd (PFOA) in Eat Liver, Sentm and Urine AUTHORS John Flaherty, Karen Risha, and Emily Decker DATE ISSUED July 30,2002 SPONSOR 3M Medical Department Corporate Toxicology ' 3M Center, Building 220-2E-02 S t Paul, MN 55144-1000 PERFORM ING LABORATORY Exygen Research 3058 Research Drive State College, PA 16801 METHOD NUMBER ' ExM-023-071 TOTAL NUMBER OF PAGES 43 Exygen Research. Page 17 o f 59 Page 59 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 MANAGEMENT APPROVAL .o L m r d f Jdfin Flaherty / ^Laboratory Manager Exygen Research Date Exygen Research I .e S -2i>J ZW 7L. John L. Butenhoff Date Sponsor Representative 3M Medical Department Corporate Toxicology tL \ Exygen Research Exygen Research. Page 2 o f43 t ! tI 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 Exygen Method No: ExM-023-071 TABLE OF CONTENTS T ITLE--------------------------------------- ;__________________ MANAGEMENT APPROVAL_________________________ TABLE OF CONTENTS______________________________ LIST OF TABLES_____________________ ______________ LIST OF FIGURES______ ,________________________ 3 1. SUMMARY_____________________________________ 2. EXPERIMENTAL COMPOUNDS_________________ _ 3. CHEMICALS AND SUPPLIES_____________________ 3.1. Chemicals_________________________________ 3.2. Standards..................... ........................................... 3 2 . EQUIPMENTANDSTTPPTms ___ . 3.4. Solutions__________________________________ 3-5. Preparation o f Standard Solutions__________ 32.1. Stock solution................ .................................. 3 2 2 . Fortification Solutions--,_________________ 3 2 2 . Calibration Standards-----r.................... ,,,,...... 4. METHOD............. ................ ........... ......... ......................... 4.1. Flow Diagram_________ __________ ____ __ ___ 4.2. Sample Processing__ _____ __________________ 4.3. Batch Setu p_______________________________ 4.4. SampleExtraction_________________________ 4.4.1. Liver Extraction --___________ ______ --,, 4.42. Scrum and Urine Extraction.--_--_______ _ 4.4 2. SPE Column Conditioning___________ _ 4 2 . Quantitation_____________ _______ _________ 42.1. LC/MS/MS System and Operating Conditions 4 2 2 . Calibration Curve Procedures 4 2 2 . Sample Analysis________ ____ _____ ____ 4.6. Acceptance Criteria________________________ _ 4.7. Performance Criteria_______________________ 4.8. Time Required for Analysis __________ ________ 5. CALCULATIONS______________________________ 6. SAFETY________________________________ Exygen Research Page} of 43 Ii j lI I I t I j i Exygen Research. Page 19 o f 59 Page 61 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 LIST OF TABLES Table 1. Recovery Summary ofPFHS in Rat Liver and Serum...____ ____________ 20 Table Z Recovery Summary o f PFOS in Rat Liver, Serum and Urine ___ ,, .2 1 Table 3. Recovery Summary of PFOA in Rat liv e r. Serum and Urine_____________ 22 Exygen Research Exygen Research. i Page 4 of43 . Page 20 o/59 Page 62 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 BxyganMethod No: ExM-023-071 LIST OF FIGURES Figure 1. Calibration Curve forPFHS_______________ ---------------- 23 FigureZ Calibration Curve for PFOS_____ ... ---------------- 24 Figure 3. Calibration Curve for FFOA _____ _______ ---------------- 25 Figure 4. Representative Chromatogram of a 0.1 ng/mL Standard Containing PFHS,,,,__ ____ ____ ----------------26 F igures. Representative Chromatogram of a 0.1 ng/mL Standard Containing PFOS...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 FFHS...27 Figure 8. Representative Chromatogram of a 0-5 ng/mL Standard Containing PFO S...2S F ig u red Representative Chromatogram of a 0.S 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 PFOS...29 Figure 1Z Representative Chromatogram of a 5.0 ng/mL Standard Containing PFOA _30 Figure 13. Representative Chromatogram of aReagent Blank Sample Analyzed far FFH S________________________________________________________3o Figure 14. Representative Chromatogram of a Reagent Blank Sample Analyzed for PFO S--------------------------------------------------------------------- ,, ------------31 F ig u ris. Representative Chromatogram of a Reagent Blanlc Sample Analyzed for PFOX--------------------------------------------------------- 1_31 Figure 16. Representadve Chromatogram of a Control Liver Sample Analyzed for FFHS__ ._________________________________ _____ ___________32 F ig u re n . Representadve Chromatogram of a Control Liver Sample Analyzed for PFOS________________________________________________ ,.32 Figure 18. Representative Chromatogram of a Control Liver Sample Analyzed for PFOA_________________________________________ _____ ,.33 Ftgure-19. Representative Chromatogram of a Control Serum Sample Analyzed for FFHS_____ ___ ____ ________ ___ _______ ______ ___________ m33 Figure 20. Representative Chromatogram of a Control Serum Sample Analyzed for PFOS_____________________________________________________ _ E gure21. Representative Chromatogram of a Control Serum Sample Analyzed for PFOA_____ _____________________________________________ ___34 Figure 2Z Representative Chromatogram of a Control Urine Sample Analyzed for PFOS________________________________________________ ..35 Figure 23. Representative Chromatogram of a Control Urine Sample Analyzed for PFOA__ _______________..................... .......... ................................35 E gure24. Representative Chromatogram of a Control Liver Sample Fortified at 10 ng/g with PFHS__ _____________________ ___________ ____ ___36 Exygen Research Page 5 of 43 Ii tI i Exygen Research. ______ . Page 21 o f 59 , i .: 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 LIST OF FIGURES (continued) Figure 25. Representative Chromatogram of a Control Liver Sample Fortified at 10 ng/g with FFOS______________________ ___________________ 35 Figure 26. Representative Chromatogram of a Control Liver Sample Fortified at 10 ng/g with PFOA___________________ ___ ________ _______ 37 Figure 27. Representative Chromatogram of a Control liv e r Sample Fortified at 50 ng/g with PFH5 ...________ ___ _____ _____ ____ ______________37 Figure 23. Representative Chromatogram o f a Control Liver Sample Fortified at 50 ng/g with FFOS _________ ________ __________ ___ _ __ ..33 Figure 29. Representative Chromatogram of a Controlliv e r Sample Fortified at 50 ng/g with PFOA________ __ ______ ___ ____________ __ ______ 33 Figure 30. Representative Chromatogram of a Control Seram Sample Fortified at 10 ng/mL with PFHS___ __________________ _____ ___ ___ ____ 30 Figure 31. Representative Chromatogram of a Control Serum Sample Fortified at 10 ng/mL with FFOS_____ .....______ ........ .......;_______ ....________39 Figure 32. Representative Chromatogram of a Control Seram Sample Fortified at 10 ng/mL with FFOA________________________ __ _____________ 40 Figure 33. Representative Chromatogram of a Control Seram Sample Fortified at 50 ng/mL with FFHS_________________________________________ 40 Figure 34. Representative Chromatogram of a Control Serum Sample Fortified at 50 ng/mL with FFOS___ ________________ .......____ ____ ,, ___ 41 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 FFOS ___________ __._ _________________ 42 Figure 37. Representative Chromatogram of a Control Urine Sample Fortified . at 10 ng/mL with PFOA____ __ ___________ ____ ________________ 42 Figure 38. Representative Chromatogram o f a Control Urine Sample Fortified ` . at 50 ng/rnL with PFOS____________________________ _____________43 Figure 39. Representative Chromatogram of a Control Urine Sample Fortified at 50 ng/mL with PFOA.. .................................................. ....................... 43 l i i 1 I Exygen Research Exygen Research. Page 6 of43 . Page 22 o f59 Page 64 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethod No: ExM-023-071 L SUMMARY This report details a method of analysis for residues of Perfluorohexanesulfonate (PFHS), Perfluoroactanesulfonate (PFOS) andPentadecafluoiooctanoic Acid (PFOA) in Rat Liver, Serum and Urine. Residues of PFHS, PFOS and PFOA are extracted from each matrix with acetonitrile. The acetonitrile extract is 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 die method which gives adequate recovery according to EPA guidelines) for this method is 10 ng/g (parts-per-billion) each for PFHS, PFOS and PFOA. The theoretical limit of detection (LOD) will be based on die 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 30 ng/g) are shown below: Fortification Level (ng/g) to 30 FFHS Recovery in Rat liv e r 115% 9.9% fa-31 98% 33% fa-31 Fortification Level (ng/mL) 10 so FFHS Recovery in Rat Serum 108% 4.7% fa-31 111% 9.6% fa-31 Fortification Level in/*) 10 so PFOS Recovery in Rat Liver 96% 83% fa-31 88% 13% fa-31 Fortification Level faa/mLl 10 SO PFOS Recovery in Rat Serum 88% 9.8% fa-31 120% 2.1% fa-31 PFOS Recovery in R at U rine 93% 4.7% fa-31 79% 1.2% fa-31 Fortification Level fan/e) 10 50 PFOA Recovery in Rat Liver 98% 3.1% fa-31 94% 23% (n=31 Fortification Level (ng/mL) 10 50 PFOA Recovery in PFOA Recovery in Rat Senun R at U rine 117% 13% fa-31 89% 23% fa-31 111% 4.0% fa-31 87% 2.1% fa-31 Representative calibration curves are shown in Figures 1-3. Representative chromatograms are shown in Figures 4 to 39. i ; ; . . : . : ! I t j ! j | : Exygen Research Exygen Research. Page 7 of43 Page 23 o f 59 Page 65 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method NotExM-023-071 2 . EXPERIM ENTAL COM POUNDS The structures forPFHS, PFOS and PFOA are given below. PFHS Chemical Name Molecular weight = = Perfluorohexanesulfonate 399, as shown 0j - FFHS is supplied as the potassium salt (CeFuSOjX*), molecular weight = 438 PFOS . Chemical Name Molecular weight Perfluorooctanesulfonate 499, as shown PFOS is supplied as the potassium salt (CeFiTSOa'K*) molecular weight = 538 PFOA 'Chemical Name Molecular weight = - . Pentadecafluorooctanoic Acid 413, as shown Exygen Reseuch Exygen Research. Page 8 of 43 . I Page 24 o f59 Page 66 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 3 . CHEM ICALS AND SUPPLIES 3 .1 . Chemicals Chemical Methanol (MeOH) Ammonium Acetate Water Acetonitrile Grade HPLC Reagent Type I HPLC Source EM Science JT Baker Exygen EM Science Catalog No. MX0475-1 0596-01 NA AX0145-1 Type I water= electrical resistivity, minimum of 16.67 Mil/cm at 25 *C, from a Labconco WaterproTM workstation. 3.2. Standards____________________________________________________ Standard______________ TCR Number Perfluorohexanesulfonate SE-036 (PEHS) Perfluorooctanesulfonate SD-018 (FFOS) Pentadecafluorooctanoic Lot No: Acid (PFOA) 08316DO Purity (%) Source 9939 all isomers. 3M 84.36 straight chain 86.9 3M 96 Aldrich Chem 3.3. E quipment and Supplies . Equipment Balance, analytical (display at least 0.0001-g) 125-mL LDPE narrow mouth bottles Disposable glass micropipets (50-100 & 100-200 pL) Tlssumiznr Wrist action shalcer Sorvall RC 5C plus Centrifuge 50 mL disposable polypropylene centrifuge tabes ' IS mL disposable polypropylene centrifuge tubes ` Visiprep vacuum manifold Sep Pak Vac 6 cc (lg) tC18 cartridges (part# WAT 036795 2-mL clear HPLC vial S t (cat # 5181-3400) Class A pipets and volumetric flasks Standard lab equipment (graduated cylinders, disposable tubes etc.) Stand-alone drop-in guard cartridge bolder (part #844017-400) Hypercaxb drop-in guard column (4 mm) (part # 844017-400) ' HPLC Pump (LC-10AD) LC/MS/MS and HPLC systems Supplier Mettler Nalgene Drummond (VWR) Tekmar Burrell Scientific Dupont VWR VWR Supelco Waters Hewlett-Packard various suppliers various suppliers Keystone Scientific Keystone Scientific Shimadzu As described in section 4.5. \ l 1 Exygen Research Page 9 of43 . Exygen Research. Page 25 o f59 Page 67 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethodNo: ExM-023-071 Notes: 1. Ei order to avoid contamination, die ose of disposable labware (containers, tubes, pipettes, etc.) is highly recommended. 2. Teflon or Teflon-lined containers or equipment should not be used. 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. 5. 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 1 water through a Hypercarb guard column using a HPLC pump at -2-3 mL/min. Before use, wash the guard cartridge with -25 mL of HPLC grade acetonitrile, then - 25 mL of type I water, then begin collecting the filtered type I water eluate for use in the extraction. Repeat the wash after filtering - 2L of water. 't 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. 3.5. Preparation o f 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. 2. 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. . ! ! I * 1: f ; t j j < [ j ! : ! t j : .1 '| > 1 : ' ; | , ; ExygenResearch Page 10of43 . Exygen Research. P age 2 6 o f 59 Page 68 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method Mb: ExM-023-071 3 J J . Stock solution Prepare individual stock solutions at 100 |ig/mL for PFHS, PFOS and FFOA 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 2aC to 6C and are stable for a maximum period of one year from the date of preparation. 3 3 3 . Fortification Solutions a. Prepare a mixed fortification standard at 1.0 pg/mL (1Q00 ng/mL) of PPHS, PFOS and FFOA by adding 1.0 mL of each of the 100 jig/mL stock solutions into a 100-mL voluinetric flask and bring np to volume with methanol b. Prepare a mixed fortification standard at 0.1 (ig/mL (100 ng/mL) of PFHS, PFOS and PFOA by diluting 10.0 ml. of the 1.0 ig/mT. mixed fortification solution to 100 mL with methanol in a volumetric flask. Example: one hundred microliters of the 0.1 pg/raL soludon spiked into 1 g of liver or 1 mL of serum/unne is equivalent to a 10 ppb (10 ng/mL or ng/g) fortification. Store all fortification standard solutions in a refrigerator (in 125-mL LDPE 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 3 3 . Calibration Standards LC/MS/MS calibration standards containing PFHS, PFOS and PFOA are prepared at 0.1,0.2,0.5 ,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. (ng/mL) 100 100 100 5.0 2.0 1.0 Volume Diluted to (mL) (mL) 5.0 100 ZO 100 1.0 100 10.0 100 10.0 100 10.0 100 Final Cone. (ng/mL) 5.0 ZO 1.0 0.5 0.2 0.1 The standards may be used for a period of one year (in 125-mL LDPE bottles) when stored refrigerated (at 2C to 6Q . Exygen Research Page 11 of 43 . ! t i I [ ' : ; ; ; j ! , > | l j f j: j ; ; . i |: | j. : ! : Exygen Research. Page 27 o f 59 Page 69 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 4. METHOD 4.1. Flow Diagram The flow diagram of the method ia given below, followed by a detailed description of each step. ' Method Flow Diagram Weigh 1 g of liver or measure 1 mL of 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 S mL of ACN, shake 1 Centrifuge 4. Decant supernatant into 33 mL of water 4 . Load onto conditioned SFE l ' Elute with 2 mL methanol .i LC/MS/MS Analysis 4.2. Sam ple P rocessing For 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 COi sublimation. Seal and place the samples in frozen storage 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. !i r t f f ! i Exygen Research Exygen Research. Page 12 of 43 . I I Page 28 o f59 Page 70 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen MethodNo: ExM-023-071 4.3. Batch Set dp a. A batch of samples should not contain more than 20 field samples. b. Each batch o f samples analyzed must include at least one control (method blank using control matrix) and two matrix controls fortified at known concentrations (typically 10 and SO ng/g for liver or ng/mL for serum and urine) 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. Sam ple Extraction . 4.4.1. liver Extraction a. 'Weigh 1 g of liver sample into a 50 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 IS mL disposable centrifuge tubes. Add S mL of ACN and shake for -20 minutes on a wrist action shaker. c. Centrifuge tubes at -3000 rpm for - S minutes. Carefully decant supernatant into a SO mL disposable centrifuge tube and add 3S 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 methanoL Collect 2 mL of elute Into a graduated IS mL centrifuge tube. h. Analyze samples using electrospray LC/MS/MS. . 4.4.2. Serum and Urine Extraction a. Measure 1 mL of serum or urine sample into a SO mL disposable centrifuge tube and fortify, if appropriate. b. Add 19 mL of water to sample. Cap tightly and vortex for -1 minute. Then continue with steps d-h in section 4.4.1. ; ! * ' ; _ ' ;i j I : : I1 ExygenResearch Page 13 of43 . Exygen Research. Page 29 o f59 Page 71 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 4.43. SPE Column Conditioning Place the unconditioned SPE columns on the vacuum manifold Condition the SPE columns by passing - 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. ik. P iI. I 1h- f t i 4 5 . Quantitation 43.1. LCJMS/MS System and Operating Conditions Mass Spec: Interface: Computer Software: Micromass Quattro Ultima (Micromass) Electrospray (Micromass) ' Harvard inhision pump (Harvard Instruments), for tuning COMPAQ Professional Workstation AP200 Windows NT, Masslynx 3 3 HPLC: Hewlett Packard (HP) Series 1100 HP QuatPump HP Vacuum Degasser ' HP Autosampler . , * HP Column Oven Note: A 4 x 10 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 Ci (Jones Chromatography), 2.1 mm x SOmm, 4)1 ` Column Temperature: 35* C Injection Volume: 15 pL . Mobile Phase (A): 2 mM Ammonium Acetate in Type I water ' Mobile Phase (B): Methanol - Time 0.0 2.0 5.0 9.0 95 14.0 145 20.0 S lA 90 90 10 10 0 0 90 90 % B Flow Rate CmL/min'i 10 0 3 10 0 3 90 03 90 0 3 100 03 100 0 3 10 0 3 10 0 3 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 11 of43 i f f 1 I l I Exygen Research. Page 30 o f59 Page 72 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethod No: ExM-023-071 from different manufacturers (Keystone Betasil C u etc.) could be used, provided equivalent chromatography is obtained. rk Ions monitored: Analyte PEHS PFOS PFOA Mode Negative Negative Negative Transition Monitored 399 -- 80 4 9 9 -- 99 413 - 369 Approximate Retention Time - 8.2 min. - 8.8 min - 8.6 min The retention times may vary, on a day to day basis, depending on the batch of mobile phase etc. Drift in retention times (up to 4 %) is 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 alternative 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 pg/mL standard solution (at 10 |iL/min. using an infusion pump) via a "T* into a stream i tI i Iir. ! 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 1 i and then tuned for the product ion. Once the instrument is tuned, the optimized parameters are saved as a tune file. This tune file is then used during routine analysis, _ 4.5.2. Calibration CurveProcedures 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 for quantitation, lin ear standard curves are generated for each analyte by linear regression using 1/x weighting of peak area versus calibration standard concentration using Masslynx (or equivalent) software system. Any calibration standard found to be a statistical outlier by using an appropriate outlier test, may be excluded from the calculation of the calibration curve. However, die 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 20.9925 (R5 20.985). 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- Exygen Research Page 15 of 43 Exygen Research. Page 31 o f 59 Page 73 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethodNo: BxM-023-071 Typical calibratioti curves for PEHS, PFOS and PFOA can be found in Figures 1 3. it K. j 4 S 3 SampleAnafyjit a. Inject the same aliquot (between 10 to SO pL) of each standard, sample, recovery, control, etc. into the LCIMS/MS system. b. Standards corresponding to at least Eve or more concentration levels (starting with the LOQ level or below) must be included in an analytical se t c. An entire set of calibration standards should be injected at the beginning of a set followed by calibration standards interspersed approximately every S-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 the end of a sample set In either case, calibration standards must be the first and last injection in a sample se t d. The concentration of each sample/fortification/contiol 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 s e t Results may be quantitated up to 10% outside the curve by extrapolation. If necessary, dilute the samples to give a response within the standard curve rangp. _ e. Fortification recoveries falling within 70 to 130% are considered acceptable. f. Samples must be stored refrigerated between 2C to 6C until analysis. g. Samples in which either no peaks are detected or peaks leas than the lowest concentration of the calibration standards are detected at the corresponding . 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 titan or equal to the lowest concentration of the calibration standards will be reported as NQ (not quantifiable). The analysis performed during the method development included fortifications at 10 and 50 ng/g of FFHS in rat liver, 10 and 50 ng/mL of PFHS in serum, 10 and 50ng/gofPFOS and PFOA in rat liver and 10 and 50 ng/mL of PFOS andPFOA in serum and urine. Typical chromatograms can be found in Figures 4-39. fI I 1 1 t Exygen Research Exygen Research. Page 16 of 43 . i Page 32 o f 59 Page 74 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen MethodNo: E*M-023-071 4.6. Acceptance Crttexia The following criteria must be met to ensure the present of PFHS, PFOS and PFOA: 1. The chromatogram most show a peak of a daughter ion at 80 amu from a parent of 399 amu far 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 PFOA. - 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 of 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 o f samples should be re-extracted. Any matrix spike outside 70-130% should be evaluated by the analyst to determine if re-extraction is warranted. 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 correlation coefficient (R) for calibration curves generated must be 20.9925 (R1 20.985). 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. & Hetendon times between standards and samples must not drift mote 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. P e r fo r m a n c e C r it e r ia . The following two criteria must be performed once as a system suitability test, before the 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 o f at least 9:1, compared to a reagent blank. If this criterion cannot be met, optimize and change instrument operating parameters (or increase the injection volume, if appropriate). Exygen Reseirch Pigo 17 of 43 , ; | [ l , ' . ' ;\ ' , '< ; . 1 j 1> ; 1 j j ` , j 1 I' i Exygen Research. Page 33 o f 59 Page 75 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethodNo: ExM-Q23-071 SgsmJCriterisac 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 the 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. 4.8. TimeREQtuREDrORAnalysis 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 Masslynx software program. Equation 1: Analyte found (ng/mL) = (peak area - intercept! xD P x aliquot factor slope UP a factor by which the final volume was diluted, if necessary. Aliquot factors 9 for liver, 20 for serum and urine ' b. For samples fortified with known amounts of analyte prior to extraction, use Equation 2 to calculate the percent recovery. Equation 2: Recovery (96) = ; ; I' fjj | l ' - I | _! f ! j l ; 1 l [total analyte found (ng/mL) - analyte found in control (ng/mL)] analyteadded (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 Exygen Research. Pago 18 of 43 . Page 34 o f59 Page 76 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 c. Use Equation 3 to calculate the amount of analyte found (in ppb) Equation 3: Analyte found (og/g orng/mL) /analyte faunrf ftizAnLl x FV fmX ^ ample weight (g) or sample volume (mi.) . FV = final volume For reporting 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 are detected at the corresponding analyte retention time that are less than the LOQ and greater than or equal to the lowest concentration o f the calibration standards will be reported 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. 1 ' f \ I : ! . Exygen Research Exygen Research. !I Page 19 of 43 Page 35 o f59 Page 7.7 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 T able 1 . R ecovery Sum m ary o f FFH S in R at L iver and Serum Recovery Summary of PFHS In Rat Liver Sample ID 0201684SpkA 0201684 SpkB 0201684 SpkC Analyte Added (nq/ql 10 10 10 Average: Standard Deviation: Percent Recoven 108 110 126 115 9.9 0201654 SpkD 0201684 SpkE 0201684 SpkF Average: Standard Deviation: 98. 3.5 Recovery Summary of PFHS In Rat Serum Sample ID 0201682 Spk A 0201682 SpkB 0201682 SpkC Analvte Added fng/mLl 10 10 10 Average: Standard Deviation: Percent Recovery CM 112 . 103 110 108 4.7 Sample ID 0201682 SpkD 0201682 SpkE 0201682 Spk F Analvte Added (ng/mLl 50 50 so Average: Standard Deviation: Percent Recovery CM 107 104 122 111 9.8 . - : : ! ' ` : ' Exygen Research Exygen Research. i 1 t ti Pago 20 of43 . Page 36 o f59 Page 78 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethodN:ExM-023-071 T able 2 . R ecovery Sum m ary o f FIFOS in R at L iver, Serum and U rine Recovery Summary ot PFOS In Rat Liver 0201634 Spk A 0201634 Spie B 0201684 Spk C Sample ID 0201684 Spk O 0201684 Spk E 0201684 Spk F 10 10 10 ' Average: Standard Deviation Analyte Added fnq/q) 60 60 60 Average: Standard DavlaUom 88 88 10S 88 6.8 Percent Recoveryf%1 90 88 87 <8 M Recovery Summary of PFOS In Rat Serum Sampla ID 0201882 Spk A 0201682 Spk B 0201682 Spk C Sample ID 0201682 Spk O 0201682 Spk E 0201682 Spk F Analyte Added (nq/mU 10 10 10 Average: Standard Deviation Percent Recovery 80 85 99 88 9.8 Analyte Added (nq/mU 60 60 50 Average: Standard Deviation Percent Recovery(7.1 118 122 121 120 2.1 Recovery Summary of PFOS In Rat Urine Sample ID 0201682 Spk A 0201682 Spk B 0201682 Spk C Analyte Added fnq/mU 10 10 10 Average: Standard Deviation Percent Recovery f%) 98 89 91 93 4.7 Sampla ID 0201682 Spk D 0201682 Spk E 0201682 Spk F Analyte Added fnq/mL) 60 60 60 Average: Standard Deviation Percent Recovery 80 78 . 78 79 1.2 ExygenResearch Page21 of43 '! ! ( i i 1 [ i ! : y i P iii i Exygen Research. Page 37 o f 59 Page 79 of 111 Exygen Study No.: 023-082 Study Plan; ExP-023-082 Exygen Study No.: 023-082 . Exygen MethodNo: ExM-023-071 Table 3. R ecovery Sum m ary o f PFO A in R at L iver, Serum and U rine Recovery Summary of PFOA In Rat Uver Semola ID 0201884 Spk A 0201684Spk 3 0201684 SokC Anelvte Added (ngfa) 10 10 10 Average: - Stenderd Devletlom Percent Recovery(%1 96 101 99 91 3.1 Semole ID 0201684 Spk D 0201684 Spk E 0201684 Spk F Anelvte Added (ng/g) 50 so so Average: Stenderd Devletlom Percent Recovery(%1 97 92 94 94 2S Recovery Summary of PFOA In Rat Serum Sample ID 0201682 Spk A 0201682 Spk B 0201882 SokC * Anelvte Added (nq/mU 10 10 10 Average: Standard Deviation: Percent Recovery(% ) 118 117 115 117 . 1J Semple ID 0201682 Spk D 0201682 Spk E 0201682 Spk F Anelvte Added fnq/mU 50 50 50 Average: Standard Deviation: Percent Recovery f%1 107 112 IIS 111 AJQ Recovery Summary o f PFOA In Rat Urine Sample ID 0201682 SpkA 0201882 SpkB 0201682 SokC Analyte Added {nq/mU 10 10 10 Average: Standard Deviation: Percent Recovery(%) 66 91 89 89 2JS Sample ID 0201682 Spk 0201682 SpkE 0201882 SpkF Analyte Added fnq/mO so 50 50 ' Average: Standard Deviation: Percent Recoveryf%1 89 88 85 87 2.1 ExygenResearch Page 22 of43 Exygen Research. Page 38 o f 59 Page 80 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 lig u r e L C alibration Curve for PFHS ExygenMethodNckExM-023-071 Cenpound 1 nan: PFHS Coaflldenl a l Detemilnallaie 11998463 CeUwtkincurv: 18112 ` x-5.14134 . Reeponse type: External Slrf. A m C um type: Linear, Origin: Esclude, Weighting: MeA * ta n None ExygenResearch Exygen Research. ! 1 1 i. I l ! Page23 of43 ---- -- Page 39 o f 59 .! ' Page 81 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 lig u r e 2 . C alibration C urve for PFOS ExygenMethod No: ExM-023-071 Compound2 nuns: PFOS Coefficient ofDetermination: 0997009 CaUbratkjncurve: 3211.30*X+ 48.9285 Response type: External Std, Area Curve type: Unear, Origin: Exclude, Weighting: Vx, Atie trans: None I f ! ; ExygenResearch Exygen Research. I Page24 of43 Page 40 o f59 Page 82 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Figure 3. C alibration C urve for PFO A Exygen Method No: ExM-023-071 Compound 1 naira: PFOA Coaffldont of Determination: 0.995945 Calibration curva: 31270.9 *x+-3675.36 Raaponsa type: External Std, Area Curvetypo: Unoar, Origin: Esclude, Weighting: 1/x, Aidstrans: Nona Exygen Research Exygen Research. Fags 25 of 43 . Page 41 o f 59 Page 83 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMe&odNo:ExM-0234)71 Figure 4 . R epresentative Chrom atogram o f a 0.1 ng/m L Standard C ontaining FFHS ^ *`isr ** -i s.-1 Figure 5 . R epresentative Chrom atogram o f a 0.1 ng/m L Standard C ontaining PFOS COOMM.a.1 niM LPRw uri prat ExygenResearch Exygen Research. Page26 of43 Page 42 o f59 ' Page 84 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethodNa ExM-023-071 F ig u r e s. R epresentative Chrom atogram o f a 0.1 ng/m L Standard C ontaining PFO F igure 7 . R epresentative Chrom atogram o f a 0 5 ng/m L Standard C ontaining PFHS CQtt1 M ,U n |M L P n a al JalW IM M ExygenResearch Exygen Research. Page27 of43 ! t t Page 43 o f 59 Page 85 of 111 Exygen Study No.: 023-082 Study Flan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 F igure 8. R epresentative Chrom atogram o f a 0 5 ng/m L Standard C ontaining PFOS CaBS0M ,UngM LPf0AMdPf01 0*dutW 1fcSW 7 MMaJaLOaUmMmMslilit u t i n u i...4.....N.....i...s....>....u.....i..i, a, i_M___a_ M tom iin F igure 9 . R epresentative Chrom atogram of a 0 5 ng/m L Standard C ontaining PFOA . coasoa-s&jMoM.proAaadm wuMLoCoMxtS*/Mi9aIr7 i t Exygen Reseirch Exygen Research. I l Pigs 23 of43 . Page 44 o f59 Page 86 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethodNo:ExM-G23-07t Figure 10. R epresentative C hrom atogram o f a 5.0 ug/inL Standard C ontaining PFHS o o u n ,U n i< i.im ' n toiwiiraiat j i h i figure 11. Representative Chromatogram of a 5.0 ng/mL Standard Containing PFOS . co*uo3>i,tjieM.m>A*fidm>3 wjuiaoicc/auaaf/tMwsarr Ii Exygen Research Exygen Research. l l Page29 of43 i f t i Page 45 o f59 i Page 87 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMe*od No: ExM-023-071 Figure 12. R epresentative C hrom atogram o f a 5.0 ng/m L Standard C ontaining PFOA eeas8H,ianew.pfoa pros MiWiotmo.it.ji Figure 13. R epresentative Chrom atogram o f a R eagent B lank Sam ple A nalyzed fo r PFHS ` UnMiMi* >*jnanna4!<a ExygenResearch Exygen Research. Pago30 of43 ' . \ f Page 46 o f 59 Page 88 of 111 Exygen Study No.: 023-082 ... j <TOJ ; .i Study Plan: ExP-023-082 Exygen Study No.: 023-082 .1 BxjgenMethodNo: E*M-023-07I F igure 14. R epresentative Chrom atogram o f a Reagent Blank Sam ple A nalyzed for PFOS R a g IHinkA . M MlfHHia.n.H t I i. [ F igu re 15. R epresentative Chrom atogram o f a R eagent Blank Rampi A nalyzed for FFOA . t l t I j Exygen Research. .i .Page 47 o f59 ' 1 Page 89 of 111 Exygen Study No.: 023-082 :: . Study Plan; ExP-023-082 Exygen Study No.: 023-082 ExygenMethodNo: ExM-023^>71 Figure 16. R epresentative Chrom atogram o f a C ontrol L iver SnmpTp A nalyzed for PFHS ' Mownanitis *1 Figure 17. R epresentative C hrom atogram o f a Control L iver Sam ple ilK,j A nalyzed for PFOS *` mtMUwrltMkA m jui aB w aiasjT IX M lM tS ty tIV':-'}M L'ii ExygenResearch Exygen Research. Page32 of43 Page 48 o f59 Page 90 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethod NoeExM-023-071 Figure 18. R epresentative C hrom atogram o f a C ontrol L iver Sam ple A nalyzed fo r PFOA ' U M uum t f: ! Figure 19. R epresentative Chrom atogram o f a C ontrol Serum Sam ple A nalyzed fo r PROS * 1* ! I. VtI. ExygenResearch Exygen Research. P ige33of43 I i Page 49 o f59 i '' ( Page 91 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethodNo: BxM-023-071 Figure 20. R epresentative Chrom atogram o f a Control Serum Sam ple A nalyzed for FFOS m uiM iM A - Mumtiaw Figure 21. Representative Chromatogram of a Control Serum Sample Analyzed forPFOA ' I Exygen Research Exygen Research. I ii ir II t !I I Page 34 of43 Page 50 o f59 .* *' Page 92 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: ExM-023-071 Figure 22. R epresentative Chrom atogram o f a C ontrol U rine Snmpi A nalyzed for PFOS - n !' I Figure 23. R epresentative Chrom atogram o f a C ontrol U rine Sam ple A nalyzed for FFOA i .9 I { Exygen Research Exygen Research. Page35 of43 \ i iI r- i ?a f \ Page 51 o f 59 Page 93 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethodNo: ExM-G23-071 fig u r e 24. R epresentative Chromatogram o f a Control L iver Sam ple F ortified a t 10 ngfgw ith PFHS flmmuMrapkMBppk * n iwintn iinium ic/timuir fig u r e 2 5. R epresentative Chromatogram o f a C ontrol L iver Sam ple F ortified a t 10 ng/g w ith PFOS * B 81M 4U w rS pkA ,10ppfc M uH O Q taxabS t LCAUMSO ExygenResearch Exygen Research. i * S Page36 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 ExygenMethodNo:ExM-023-071 fig u r e 26. R epresentative Chrom atogram o f a Control L iver Sam ple F ortified a t 10 ng/g w ith PFO A F igure 27. R epresentative C hrom atogram o f a C ontrol liv e r Sam ple F ortified a t 50 n g/g w ith PFH S ' OxruM Lhm ra?icX ta p p i I I jK -- BilM t \k it f11 [ I1 '* t1ji ExygenResearch Pago37 of43 . \ !i Exygen Research. Page 53 o f 59 Page 95 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygea Method No: ExM-023-071 Figure 28. R epresentative Chrom atogram o f a C ontrol L iver Sam ple F ortified a t 50 ng/g w ith PFOS Figure 29. R epresentative Chrom atogram o f a C ontrol L iver Sampii F ortified a t 50 ng/g-with PFOA ` a o iw u m rsp n a n p i* oa*mwoasz%43rli I ExygenResearch Exygen Research. Page38 of43 il t iI l l Page 54 o f59 Page 96 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethod No:ExM-023-071 Figure 30. R epresentative Chrom atogram o f a Control Serum Sampi F ortified a t 10 ng/m L w ith PFHS Figure 31. R epresentative Chrom atogram o f a C ontrol Serum Sampi F ortified a t 10 ng/m L w ith PFOS MIMIlawn m M iooiBK iaar aMM#L<OchUM4SMM7Ak>MUeMt.r * \ UM 7J* tfi* Itm ' iua '"no* ' *** ExygenResearch Exygen Research. Page39 of43 '! Page 55 o f 59 . Page 97 of 111 Exygen Study No.: 023-082 Study Plan; ExP-023-082 Exygen Study No.: 023-082 ExygenMethodNo: ExM-023471 Figure 32. R epresentative Chrom atogram o f a C ontrol Serum Snmpl F ortified at 10 ng/m L w ith PFOA Figure 33. R epresentative Chrom atogram o f a C ontrol Serum Sam ple F ortified at 50 ng/m L w ith PFH S ' ExygenResearch Exygen Research. r Pago40 of43 Page 56 o f 59 Page 98 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 Exygen Method No: &M4&3-071 Figure 34. R epresentative Chrom atogram o f a C ontrol Serum Snm py F ortified at 50 ng/m L w ith PFOS eauntauaapta.Hpfk oejuMna Figure 3 5 . R epresentative Chrom atogram o f a C ontrol Serum Sam ple F ortified at 50 ng/m L w ith PFOA mra>watpta,Mpi eejuom miioi ExygenResearch Exygen Research. Page41 of43 I Page 57 o f 59 Page 99 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygeaMeihod Nix BxM-023-071 lig u r e 36. R epresentative Chrom atogram o f a Control U rine S n Pv F ortified a t 10 ng/m L w ith PFOS 10Cttim UrtM *kA ppfc -- UiMOI i Figure 37. R epresentative Chrom atogram o f a Control U rine Sampi F ortified a t 10 ng/m L w ith PFOA ' Mauta%k4Wwt ^*3 . ExygenResearch Exygen Research. It ! Pmgo42of43 . ( 1 \ ! I Page 58 o f 59 Page 100 of 111 Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygen Study No.: 023-082 ExygenMethod No: ExM-023-071 fig u r e 38. R epresentative Chrom atogram o f a Control U rine Sam ple F ortified a t 50 ng/m L w ith PFOS uimiMMatkn,iQp0i tCM M lSfr k Kt h F ( i Y \ Figure 39. R epresentative Chrom atogram o f a Control U rine Sam ple F ortified at 50 ng/m L w ith PFO A ` aauntkiMSHiDtnpk wjuUmiomw L cusnun t ExygenResearch Exygen Research. Page43 of43 Page 59 o f 59 Page 101 of 111 Exygen Study No.: 023-082 H .I ; ;Vj Exygen Research. Page 102 of 111 _ RESEARCH Precise Research. Exygen Study No.: 023-082 Exygen Research. Page 103 of 111 Exygen Study No.: 023-082 EWg:enRESEARCH ..P..rPerecciisse Research. Proven Results. Page _1_ of PROTOCOL DEVIATION Deviation Number 3____ Date of Occurrence: throughout study 1 Exygen Study Number 023-082 Protocol Number ExP-023-082 \ DESCRIPTION OF DEVIATION 1. Analytical Procedure Summary-Method Section 4.5.3.d--several samples w ere quantified above 10%of th e curve. 2. Analytical Procedure Summary-Method Section 4.5.3.e --th e acceptance critierfa for QC spike recoveries could tiqt always be m et. \ ACTIONS TAKEN \ far example - deviation Issued. SOP revision, etc. 1 -2.Protocol deviation issued. Recorded Bye i t)e c U / Datei:: IcfacjtfL IMPACT ON STUDY 1.No negative im pact because residue was. < than 15% above the curve for C6 and C8 for sample 0204493 Spk V and for C8 for samples 02039.65 and 0203965 Dup. Also, the samples th at w ere quantified outside th e curve for THPFOS and-.THPFDS w ere only fortification recoveries and the majority of the recoveries w ere 100%. \ 2.Impact not determined only documented sin ceth is method has not been validated for these anions a t these levels. \ U:\Forms\PROTOCOL DEVIAT10N.doc Exygen Research. \ Date Exygen QAU Review \ i f I t , 9/25/2002/5 3058 Research Drive State College, PA 16801, USA J : 800.281.3219 F: 814.272.1019 exygen.com Page 104 of 111 Exygen Study No.: 023-082 E\ygen RESEARCH ^PPrreecciisse Research. ' Proven Results. Page 1 of 1 PROTOCOL DEVIATION Deviation Number: 4 Date of Occurrence: throughout study ExygenStudy Number: 023-082 Protocol Number ExP-023-082 \ DESCRIPTION OF DEVIATION 1.Analytical Procedure Summary-Method Section 4 .5 .3 .g--several samples w ere quantified below the curve and ND was used for when no peak was detected and NQwas used for negative results. All other results w ere reported. \ 2.Analytical Procedure Summary-Method Section 4.5.3.C--a second se t of calibration standards was not analyzed In a set. ACTIONS TAKEN far example - deviation Issued. SOP revision, ate. 1-2-Protocol deviation issued. IMPACT ON STUDY 1. No negative im pact because no claim of..quantitative accuracy has been established. 2. No negative im pact because the extractectstandards w ere analyzed throughout the s e t which provide a QC check on the initial calibration. '--. U:\Foirrt\PROTOCQU_DEVlATION.doc Exygen Research. D ate ic h o ltfi. Date D ate D ate Exygen QAU Review hJLL i 6Ij Ien9/25/2002/5 3058 Research Drive State College, B \ 16801, USA J: 800.281 .3219 F: 814.272.1019 exygen.com Page 105 of 111 Exygen Study No.: 023-082 Exygen Research. Page 106 of 111 Exygen Study No.: 023-082 Received Fax OCT 30 300? 13:13 OCT-30-2802 12:24 Fax Station : FXYGFM RFSFARCH P.02/05 ___ R E S E A R C H kP\rePcrsotvReenseRaernchilo.. . \ Pas*_1_ of : PRO. OTOvCttOinL'NPbErnVbjeAcT'1fON OatpofOecurwie: 10/15/02 ExygerrStudyNumber 023-082 ProtocolNumtier BtE-a23-C82 AnalyticalP\ro,cedureSumrnaryrlOl^CEa3lCjbRrSIPtloTAIOSNtaOndFaDrdEVIATION ^ Didn.o1tprepaNre^extnicted.st:andardsj`it' h;ym! ;aplaima 1.5a'nd.2.3'ppblevels.. " !: V\ :* * : * *. '` V'*' ' . ' : V /!ACTIONSTAKEN " i -1 -terrninilr.dtyllai>^udjgOPWrimnn.ale.: . . Protocol deviation issued.V 1 T'. . T i " A; ; : - : wNaosn"se.tg.ilaltEivsteaib.m;l!isph.aecdVt:bweiathuoieut;oi:tn;tiloy'sfbipatwdotillMmIleyBj;j.eb!A%CECrd*-.t'f.!aOm"NptSleT'iaUy:DaUYabl.e.andt"h'elinea'r'range1Ief.the! curv~e * `* V i- v ` .\ Principal.Iny^tjqatof'Sjjpalure; ... ; Dato * . P f a l d L ! Fate. , (A&nagemntSignatun.-'r-, . O.'W onnilPftTOGOU.DeVIA'bN.SSG Date - Z c M / o x _ Date . E*xygenQAUReviewt V Site ithaln.* . $j' ` ` ! msooluis JlM(L281'J21'9 ^ P.814272.1019 '; exygan.com 1 ; Exygen Research. Page 107 of 111 OCT-30-2002 12=24 Exygen Study No.: 023-082 P .0 3 '0 6 Exygen Research. Page 108 of 111 Exygen Study No.: 023-082 Received Fax MMtfwha OCT-30-2002 12!24 ___ R E S E A R C H ' ertsrResearch. ' . ^Proven Rauls. m na P.04/06 >. . \ '! : " P R Q T C D pE V lA flO N ' ? -4 ' :Xi^JtNurpibar. 3 . . : : ofKUfjtric' throuahouf itu'v . ' ' : B y s e r.;^ ,Numb.fr 023-M2 ! ProtocotNumber. ExMod ^ l . ! above10?oftheairvei '* * severalsampleswerequantified i recoveries could noti*#'tiemet:1 \ r :;. V 'i. spike-; . : V A C T I O N S T A K E N . . 1 rsnmale - a r ^ in ^ u .^ so p wvltinn. m 1-2.Protocol deviation tsstiadj' " "* ` I ' 1 :.s h' 1 *; .. I . `I * : *"' ! : :-r :------- . \ \ Date M to , ^ a e n Q U R e v i e w > -7 - ia ffa ? t 1 ' i u ^ o in w v rrtU T u C O l^ _ D E V |A T lO N jd c , ; " :'"-r y : ' ;, \ . ' W . . ; s a M O o i/S ': C " *< ' , .* t ' ^ e iQ 5 a 'R e e w H D riv e :` ; ^ * < M ^ I ,A ; 1 8 0 1 , U S A - . . V' '' V, - r i - i./.i '1 " S 'r 1 1 ! . : .: 1 ' ' . . ^ s w i n . - . / . . ' ' * r* * ' I.;: ' : Exygen Research. Page 109 of 111 Rpcg-ivert Fax ___OCT M 201)3 OCT-30-2002 12:25 ' fat Statinrv r FXVGFM RFSFARCH Exygen Study No.: 023-082 P .06/06 Exygen Research. Page 111 of 111 Exygen Study No.: 023-082 Recpivert Fax OCT 30 ?002 13:13 OCT-30-2002 12:24 Fax Station : FXYfiEM RESEARCH ^tarsSk,s e a r c h red Research. 1 : P.05^06 ' , | \ i i ' : II \ 1 'L lt'H ^ ^ irtA iid N ; ' ' -rv A .f : ` '! - DjPyjtv*isptciqaajr^r>nnihc'aeK':. 4tnro- uanoutet.u..dv. . 77" ..1 ' . [ . :. ; , * * J * * ' *. *. .XI.* rkrd, :j[ :.:"; '.TTl . ; ; I ' . i " *I1 . "* .1 ; j : .! ; "; f r*-' ` : 1 : ' p. . . . S H1'\^ <: . i I ,] i , * : . ** . . 1 *K* J '-h -t.; :. ! : . -.f.-j . h i : -.i ' I ` ' ' :I Mar a n a h ^ `tibmMhM^ k l . u : : L ^ . I " .\ ' 7> : : ; ;. ' i.v'^BhbAU.iRevlBW A C C l o f o l .l: . :' '.u^or.rM^RaT"Qca.i^a.E. V.l-AT. io.Nt.cl . 1- -l--lr--. - 1T *, . i . .1 .. ~ 1 v . . "". " rs/`js"aw*s/s- '1;' ) ' j ; . ' 'v' v \ . vr: f: :| -i ' : ! ' ,; V '.,.-!-: v i :/ ! ; ' > : T: V: ",: Exygen Research. Page 110 of 111