Document JNzO3erZ8oKjRpY5wX8OQ60M2

3M Study Title A n a l y s is o f E n d o g e n o u s F l u o r o c h e m ic a l s in N o r m a l P o o l e d H u m a n S e r u m a n d P lasma Data Requirement 40 CFR Part 792 Author Mark E. Ellefson Study Completion Date 13 November, 2002 Revised Report 9 December, 2002 Performing Laboratory 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Environmental Laboratory Study Number E02-1039 Total Number of Pages 225 3M Environmental Laboratory E02-1039 This page has been reserved for specific country requirements. Page 2 o f 225 3M Environmental Laboratory E02-1039 GLP Compliance Statement p Study Title: Analysis of Endogenous Fluorochemicals in Normal Pooled Human Serum and ; Plasma Study Identification Number: 3M Environmental Laboratory study Number E02-1039 ; j This study was conducted in compliance with Toxic Substances Control Act (TSCA) Good Laboratory Practice (GLP) Standards, 40 CFR 792, with the exceptions listed below: rq Exceptions to GLP compliance: ! 40 CFR 792.130(e): There is not an electronic audit trail of corrections. The authenticated hardcopy printouts are considered the original raw data. Page 3 o f 225 3M Environmental Laboratory E02-1039 Quality Assurance Statement Study Title: Analysis of Endogenous Fluorochemicals in Normal Pooled Human Serum and Plasma Study Identification Number: 3M Environmental Laboratory Study Number E02-1039 This study was audited by the 3M Environmental Laboratory Quality Assurance Unit (QAU), as indicated in the following table. The findings were reported to the study director and laboratory management. In s p e c tio n Da te s 10/10/02 10/11/02 10/29/02-10/30/02 10/31/02 Phase Protocol In-phase Data Final Report Date Reported to Management Study Director 10/10/02 10/10/02 10/11/02 10/11/02 10/30/02 10/30/02 11/1/02 11/1/02 u jb /0 z ~ Date Page 4 o f 225 3M Environmental Laboratory E02-1039 Table of Contents GLP Compliance Statement............................................................................................................................3 Quality Assurance Statem ent........................................................................................................................ 4 List of Tables......................................................................................................................................................6 List of Figures.................................................................................................................................................... 6 Study Information..............................................................................................................................................7 Executive Sum m ary......................................................................................................................................... 8 S u m m a ry .............................................................................................................................................................9 Introduction................................................................................................................................................. 11 Test Substance..................................................................................................................................................11 Reference Substance....................................................................................................................................... 14 Test System s.....................................................................................................................................................15 Method Summaries...........................................................................................................................................15 Preparatory and Analytical Method 15 Analytical Results.............................................................................................................................................. 18 Data Sum m ary.................................................................................................................................................. 20 Statistical Methods and Calculations............................................................................................................ 31 Statement of Conclusion................................................................................................................................. 32 References......................................................................................................................................................... 33 List of Attachments........................................................................................................................................... 33 Signature P a g e ................................................................................................................................................. 34 Attachment A: Method......................................................................................................................................35 Attachment B: Data T ab les.............................................................................................................................56 Attachment C: Chromatograms...................................................................................................................... 63 Attachment D: Prep Sheets and Traceability Information.......................................................................... 125 Attachment E: Protocol and Amendment......................................................................................................208 Pa g e 5 o f 225 3M Environmental Laboratory E02-1039 List of Tables Table 1.a. Endogenous levels of test substance in normal pooled human serum ..............................9 Table 1.b. Endogenous levels of test substance In normal pooled human Plasm a........................... 9 Table 2. Characterization of the Test Substances..................................................................................... 11 Table 3. Characterization of the Reference Substances.......................................................................... 14 Table 4. Description of Test Systems Used in this Study.........................................................................15 Table 5.a. Endogenous levels of test substance in normal pooled human serum ............................. 20 Table 5.b. Endogenous levels of test substance in normal pooled human Plasm a...........................20 Table 6. Accurate Mass Determination of Endogenous Test Substances............................................21 Table 7. Daughter Ions of Endogenous Test Substances....................................................................... 21 List of Figures Figure 1: NMR Certified 99.5% Linear Isomer PFOA and Mixed Branched and Linear Isomer PFOA Standards..............................................................................................................................22 Figure 2: C7 Isomer Distribution..................................................................................................................... 23 Figure 3: C8 Isomer Distribution.....................................................................................................................25 Figure 4: C9 Isomer Distribution.....................................................................................................................26 Figure 5: C 10 Isomer Distribution....................................................................................................................27 Figure 6: Cn Isomer Distribution....................................................................................................................28 Figure 7: C 12 Isomer Distribution....................................................................................................................29 Figure 8: PFOS Isomer Distribution.............................................................................................................. 30 P a g e 6 o f 225 3M Environmental Laboratory E02-1039 Study Information Sponsor William K. Reagen 3M Environmental Laboratory Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 651-778-6565 Study Director Mark E. Ellefson 3M Environmental Laboratory Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 651-778-5405 Study Location Testing Facility 3M Environmental Laboratory Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 William K. Reagen, Laboratory Manager Stacy R. A. Hanson, Analytical Chemist Marlene M. Heying, Analytical Chemist Cindy M. Carlson, Analytical Chemist Ognjenka Krupljanin, Analytical Chemist Study Dates Study Initiation: 10 October 2002 Experimental Initiation: 10 October 2002 Experimental Completion: 31 October 2002 Study Completion: 01 November 2002 Location of Archives All original raw data and the report have been archived at the 3M Environmental Laboratory according to 3M Standard Operating Procedures. Remaining specimens pertaining to the analytical phase of this study will be archived at 3M Environmental Laboratory for as long as the quality of the preparation affords evaluation. Page 7 of 225 3M Environmental Laboratory E02-1039 Executive Summary A screening study of three lots of commercial pooled human plasma, one lot of pooled human plasma from central China, and four lots of commercial pooled human serum was undertaken to quantify endogenous levels of perfluoroheptanoate (C7), pentadecafluorooctanoate (C8) (PFOA), heptadecafluorononanoate (C9), nonadecafluorodecanoate (C10), perfluoroundecanoate (C11), perfluorododecanoate (C12), and perfluorooctane sulfonate (PFOS). Results from this study showed quantifiable levels of the linear isomers of C7, C9-C12 ranging from < 0.010 - 0.9 ng/mL (Tables 1a and 1b). Low to non-detect levels of branched Isomers were observed for the C7 and C 9-C 12 compounds detected (Figure 1 - 1 0 ) . PFOS was present In the screened lots of commercial sera and plasma In concentrations ranging from 2.5 - 27 ng/mL and was present as branched and linear isomers. PFOA was present In the screened lots of commercial sera and plasma In concentrations ranging from 0.65 - 5.6 ng/mL and was present as branched and linear Isomers. Page 8 o f 225 3M Environmental Laboratory E02-1039 Summary Quantitative screenings were conducted on four lots of pooled human serum and four lots of pooled human plasma to determine endogenous levels of perfluoroheptanoate (C7), pentadecafluorooctanoate (C8), heptadecafluorononanoate (C9), nonadecafluorodecanoate (C10), perfluorodecanoate (C11), perfluorododecanoate (C12), and perfluorooctane sulfonate (PFOS) as described by the 3M Environmental Laboratory Study #E02-1039 protocol. The analytical screening data is summarized in tables 1.a. and 1.b. Table 1.a. Endogenous levels of test substance in normal pooled human serum Test Substance PFOS C12 Cn Cm C9 c8 C7 Sigma TCR-689 (ng/mL) 4.56 [0.018] [0.076] [0.058] 0.265 1.60 [0.013] Lampire TCR-688 (ng/mL) [2.49] [<0.010]1 [< 0.010]1 [0.031] [0.068] 0.650 [0.025] tsioresource TCR-687 (ng/mL) 17.0 [0.144] 0.295 [0.327] 0.605 2.95 [< 0.010]' Lioiden w est TCR-690 (ng/mL) 27.0 [0.040] 0.320 0.203 0.900 5.60 0.190 [ ] Denotes values determined from concentrated spe method. Value < LOQ. Table 1.b. Endogenous levels of test substance in normal pooled human Plasma Analyte PFOS C 12 C 11 c10 Cg c8 c7 umnese TCR-674 (ng/mL) < 1.01 <0.101 <0.101 <0.101 < 0.251 < 0.521 < 0.10` Lampire TCR-685 (ng/mL) 11.1 [0.036] [0.049] [0.127] 0.435 2.61 0.100 innovative Research TCR-683 (ng/mL) 15.6 [0.022] 0.135 0.170 0.585 3.07 [0.016] (ioiaen w est TCR-684 (ng/mL) 18.3 [0.024] [0.071] 0.160 0.535 3.87 0.290 [ ] Denotes values determined from concentrated spe method. V alu e < LOQ. Accurate mass measurements and elemental compositions were obtained for endogenous C7C11, and PFOS from a concentrated SPE extract of normal pooled human serum (see Table 6). Characteristic daughter ions were observed for endogenous C7-C12, and PFOS from a concentrated SPE extract of normal pooled human plasma (see Table 7). Qualitative analysis of chromatographically resolved branched and linear isomers of PFOA was accomplished using NMR certified standards of a linear isomer PFOA standard and of a mixed branched and linear isomer PFOA mixed standard. It was determined that the branched PFOA isomers elute within an approximate 0.2 minute retention time window and are baseline resolved from the linear PFOA isomer. This finding of C8 isomer retention time resolution was Page 9 of 225 3M Environmental Laboratory E02-1039 extrapolated to determine qualitatively the presence or absence of branched isomers for the higher homologues of C9-C12, and PFOS (see Figures 1 - 8). Results from this study showed quantifiable levels of the linear isomers of C7, C9-C12 ranging from < 0.010 - 0.9 ng/mL (Tables 1a and 1b). Low to non-detect levels of branched isomers were observed for the C7 and C 9-C 12 compounds detected (Figures 1 - 8). PFOS was present in the screened lots of commercial sera and plasma in concentrations ranging from 2.5 - 27 ng/mL and was present as branched and linear isomers. PFOA was present in the screened lots of commercial sera and plasma in concentrations ranging from 0.65 - 5.6 ng/mL and was present as branched and linear isomers. L: .-.1J m Page 10 of 225 3M Environmental Laboratory E02-1039 Introduction The purpose of this study is to perform quantitative screening for perfluoroheptanoate (C7), pentadecafluorooctanoate (C8), heptadecafluorononanoate (C9), nonadecafluorodecanoate (C10), perfluorodecanoate (C11), perfluorododecanoate (C12), and perfluorooctanesulfonate (PFOS) in normal pooled human serum and plasma. This study did not have a typical test substance that is dosed onto a specific controlled test system as per a conventional GLP study. The study design is in compliance with US EPA Toxic Substances Control Act (TSCA) 40 CFR Part 792. Test Substance Preliminary screening indicated that the test substances listed below are present as endogenous material in normal pooled human serum and plasma. Information pertaining to traceability, source, physical description, and storage conditions is not available for these compounds as they exist in biological matrices. Table 2. Characterization of the Test Substances T est Substance Formula Tridecafluoroheptanoate (C7) Pentadecafluorooctanoate (C8) Heptadecafluorononanoate (C9) Nonadecafluorodecanoate (C10) Perfluoroundecanoate C11) Perfluorododecanoate (C12) Perfluorooctane sulfonate (PFOS) CeF^COO C7F,5COO` C0F17COO C9F19COO C10F21COO C11F23COO C8F17S0 3 The molecular structures are given below. Name: C7 Chemical Name: Perfluoroheptanoate Molecular Weight: 363, as shown FF F FF FF F F F O O Page 11 of 225 3M Environmental Laboratory E02-1039 Name: C8 Chemical Name: Pentadecafluorooctanoate Molecular Weight: 413, as shown Name: C9 Chemical Name: Heptadecafluorononanoate Molecular Weight: 463, as shown F F F FO F FF F FF F F 0 Name: CIO Chemical Name: Nonadecafluorodecanoate Molecular Weight: 513, as shown Name: C ll Chemical Name: Perfluoroundecanoate Molecular Weight: 563, as shown FFFFFO FFFFF Page 12 of 225 3M Environmental Laboratory E02-1039 Name: C l2 Chemical Name: Perfluorododecanoate Molecular Weight: 613, as shown Name: PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 499, as shown FFFF FFFF F SO 3 FF FFFF Page 13 of 225 Reference Substance 3M Environmental Laboratory E02-1039 Table 3. Characterization of the Reference Substances Reference Substance Tridecafluoro heptanioc Acid Pentadecafluoro octanoicAcid Heptadecafluoro nonanoic Add Nonadecafluoro | decanoicAcid Perfluoroun decanoic Add Perfluorodo decanoic Acid Potassium Perfluorooctane sulfonate Formula Traceability # Source CeF,3COOH TCR-267 Aldrich C7F,5COOH TCR-617 c8f,7cooh TCR-618 C9F19COOH TCR-036 c,0f21cooh TCR-619 C11F23COOH TCR-037 Oakwood Products Oakwood Products Oakwood Products Oakwood Products Oakwood Products C8F17SO3 K* TCR-018 SMM* 236-1B-10 ` Documentation of the method of synthesis is located at the source. ` ` Reference substances confirmed as linear isomers by NMR. Physical Description Clear Crystals White Crystals White Crystals Purity 98.2%** 99.5%** 98.02%** Storage Conditions Frozen Ambient Temperature Ambient Temperature White Solid 98%** Frozen White Crystals White Powder 96.4%** 99.7%** Ambient Temperature Frozen White Powder 86.9% Frozen Page 14 of 225 3M Environmental Laboratory E02-1039 Test Systems Table 4. Description of Test Systems Used in this Study Test System: Pooled Human Serum Pooled Human Serum Pooled Human Serum Pooled Human Serum Pooled Human Plasma Pooled Human Plasma Pooled Human Plasma Pooled Human Plasma Source Sigma-Aldrich, Milwaukee, Wl Lampire Biological Laboratories, Pipersville, PA Bioresource Technology, Inc., Fort Lauderdale, FL Golden West Biologicals, Temecula, CA Lampire Biological Laboratories, Pipersville, PA Golden West Biologicals, Temecula, CA Innovative Research, Inc., Southield, Ml Central China T raceability TCR-689, Lot 022K0965 TCR-688, Lot X324B TCR-687, Lot 020821 TCR-690, Lot G01406042 TCR-685, Lot 22-60824A TCR-684, Lot G01410002 TCR-683, Lot IR02-014 . TCR-674, Lot N087P27 Justification of the T est System. Based on preliminary testing, normal pooled serum and plasma contain endogenous levels of the test substances. Identification of T est System. Samples were identified by the TCR number. Collection of Test System s. The commercial vendors indicated that three of the four lots of commercial pooled human serum and three lots of commercial pooled human plasma purchased for this study were collected from subjects residing in close proximity to the individual vendors (Sigma-Aldrich purchases blood products from blood banks located throughout the US). Hence, although limited to single lots of serum or plasma per location, analytical data from the purchased serum and plasma provide a benchmark of endogenous levels of fluorochemicals from different regions of the country. Method Summaries Preparatory and Analytical Method ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological M atricies." A 2.0 mL aliquot of serum or plasma is transferred to a 50 mL screwcapped polyethylene centrifuge tube. Spiking solution is added as appropriate, followed by the addition of 8.0 mL of ASTM Type I water. The mixture is shaken and 40.0 mL of acetonitrile are added. The sample container was capped, mixed for 20 minutes, and centrifuged at 3,500 rpm for 20 minutes to clarify the supernatant. Following centrifugation, the supernatant is transferred to a 500 mL Nalgene container, diluted with 350 mL ASTM Type I water, and passed through a pre-conditioned C 18 solid phase extraction (SPE) cartridge. The analytes of interest are eluted from the SPE cartridge using 2.0 mL of methanol. Concentrated SPE M ethod, A modified form of the SPE method described by ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matricies." used to concentrate analytes present at concentrations below the LOQ. A five fold concentration is achieved by using 5 times more serum or plasma while maintaining the final elution volume at 2.0 mL methanol. The method consists of a 10.0 mL aliquot of serum or plasma transferred to a 250 mL Nalgene container. Spiking solution was added as appropriate, followed by the addition of 40.0 mL of ASTM Type I water. The mixture is shaken and 200.0 mL of acetonitrile are added. The sample container is capped, mixed for 20 minutes, and centrifuged at 3,500 rpm for 20 minutes to clarify the supernatant. Following centrifugation, the supernatant is transferred to a 2.0 L Nalgene container, diluted with 1750 mL ASTM Type I water, and passed through a pre-conditioned C18 solid phase extraction Page 15 of 225 3M Environmental Laboratory E02-1039 (SPE) cartridge. The analytes of interest are eluted from the SPE cartridge using 2.0 mL of methanol. Further sensitivity was achieved by doubling the injection volume of the standards used to calibrate the run. Analytical Method Liquid Chrom atograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM C 18 2x100mm, 5pm particle size Column temperature: 40 C Cycle Time: 17.0 minutes Flow rate: 300pL/min Injection volume: 10 pL (20 pL for concentrated SPE method only) Mobile phase components: Solvent A: 2.0 mM ammonium acetate in water Solvent B: Methanol Solvent Gradient: Tim e (min.) 0.00 10.00 11.00 11.50 12.50 13.00 16.00 %B 40% 90% 90 % 100% 100% 40% 40 % Mass Spectrom eter: Micromass Quatro Ultima triple quadrupole mass spectrometer Software: Masslynx version 3.5. Mass Spectrometer Acquisition Parameters: (C7) Channel 1 Parent Ion (m/z) 363.00 Dauqhter Ion M 119.00 Collision Cone V1 Enerqv (eV) 20 20 2 363.00 169.00 20 20 3 363.00 219.00 20 20 4 363.00 319.00 20 20 Channel 1 2 3 4 Parent Ion in M 413.00 413.00 413.00 413.00 Dauqhter Ion (m/z) 119.00 169.00 219.00 369.00 Collision Enerqv (eV) 20 20 20 20 Cone(V) 20 20 20 20 Page 16 of 225 3M Environmental Laboratory E02-1039 (C,) Channel 1 2 3 4 Parent Ion (m/z) 463.00 463.00 463.00 463.00 Dauqhter Ion (m/z) 119.00 169.00 219.00 419.00 Collision Energy (eVl 20 20 20 20 Cone (V) 20 20 20 20 (Cio) Channel 1 2 3 4 Parent Ion in M 513.00 513.00 513.00 513.00 Daughter Ion (m /zl 119.00 169.00 219.00 469.00 Collision Energy (eV) 20 20 20 20 Cone (V) 20 20 20 20 (Cu) Channel 1 2 3 4 Parent Ion in M 563.00 563.00 563.00 563.00 Dauahter Ion Qg/zl 119.00 169.00 219.00 519.00 Collision Energy (eV) 20 20 20 20 Cone (V) 20 20 20 20 (C 12) Channel 1 2 3 4 Parent Ion in M 613.00 613.00 613.00 613.00 Daughter Ion in M 119.00 169.00 219.00 569.00 Collision Energy (eV) 20 20 20 20 Cone (V) 20 20 20 20 Page 17 of 225 3M Environmental Laboratory E02-1039 PFOS Channel 1 2 3 Parent Ion (m/z) 499.00 499.00 499.00 Dauahter Ion (m/z) 80.00 99.00 130.00 Collision Enerqv (eV) 45 45 45 Cone fV) 60 60 60 Capillary Voltage: 4000 V Gain = 1.0 EMV Mode: Electrospray Negative Gas Temperature: 250 C Drying Gas: 8.0 L /min. Nebulizer Pressure: 30 pslg Analysis Type: Multiple Reaction Monitoring (MRM) Analytical Results Data quality objectives outlined in the 3M Environmental Laboratory method were met (see Appendix A). Regressions. Quadratic curve fit weighted 1/x was applied to calibration standards and sample data to improve quantitation over the concentration range appropriate to the data. All calibration curves had a coefficient of determination of 0.998 or greater. Calibration Standards. Calibration curves were prepared from extracted matrix standards in Chinese plasma for all plasma and serum quantitations. Reported values were not corrected for endogenous levels of test substance in the Chinese plasma calibration curve (levels of all endogenous test substances in the Chinese plasma were determined to be < LOQ). The curves consisted of a minimum of nine (9) points. The equation was determined by regression analysis using the peak areas of the analyte. The accuracy of each level was verified. Any level outside 70% -1 3 0 % of nominal was deactivated, and regression re calculated. All levels showed a response greater than twice that of the blank. Continuing Calibration Verification. For quantitative determinations, a mid-level matrix calibration check was analyzed at least every ten samples to monitor instrumental drift, with a limit of 25% deviation of the target concentrations. Limit of Quantitation (LOQ). The LOQ was equal to the lowest standard in the calibration curve, with a level of accuracy within 30%. The level of analyte in the LOQ was also greater than two times the response of analyte in the blank samples. Dem onstration of Specificity. The identification of analytes was substantiated by chromatographic retention times, characteristic primary ions, characteristic daughter ions, and isomeric proportions (where applicable). Control of Bias. Two levels of matrix fortifications, prepared at known concentrations of the test substance and bracketing the anticipated range of the method were evaluated to determine recovery and to evaluate method performance. In serum samples, PFOS and C12 exhibited matrix effects identified by high (>150% recovery) or low (< 75% recovery. For all other analytes, and for all analytes in plasma samples, recoveries ranged from 75% to 105%. Reagent and matrix blanks were run with each set to evaluate the level of background interferences. Page 18 of 225 3M Environmental Laboratory E02-1039 Blanks. Method blanks and matrix blanks were evaluated in the course of this study. The method blanks showed no evidence of background contamination introduced in the sample preparation stage. The matrix blanks did show endogenous levels of the analytes. This was expected and such instances were noted in the raw data. Page 19 of 225 3M Environmental Laboratory E02-1039 Data Summary Quantitative screenings were conducted on four lots of normal pooled human serum and four lots of normal pooled human plasma for the determination of endogenous levels of perfluoroheptanoate (C7), pentadecafluorooctanoate (C8), heptadecafluorononanoate (C9), nonadecafluorodecanoate (C10), perfluorodecanoate (C11), perfluorododecanoate (C12), and perfluorooctane sulfonate (PFOS) as described by the 3M Environmental Laboratory Study #E021039 protocol. The screening data is summarized in tables 5.a. and 5.b. Matrix spike recovery information is contained in Appendix B. Table 5.a. Endogenous levels of test substance in normal pooled human serum Test Substance PFOS C 12 Cu c10 C9 C8 C7 sig m a TCR-689 (ng/mL) 4.56 [0.018] [0.076] [0.058] 0.265 1.60 [0.013] Lampire TCR-688 (ng/mL) [2.49] [< 0.010]1 [< 0.010]1 [0.031] [0.068] 0.650 [0.025] uioresource TCR-687 (ng/mL) 17.0 [0.144] 0.295 [0.327] 0.605 2.95 [< 0.010] ' uoiaen west TCR-690 (ng/mL) 27.0 [0.040] 0.320 0.203 0.900 5.60 0.190 [ ] Denotes values determined from concentrated spe method. Value < LOQ. Table 5.b. Endogenous levels of test substance in normal pooled human Plasma Analyte PFOS C12 Cu Cio Cg C8 C7 Chinese TCR-674 (ng/mL) < 1.01 < 0.101 < 0.101 <0.101 < 0.251 < 0.521 <0.10' Lampire TCR-685 (ng/mL) 11.1 [0.036] [0.049] [0.127] 0.435 2.61 0.100 innovative Research TCR-683 (ng/mL) 15.6 [0.022] 0.135 0.170 0.585 3.07 [0.016] (olden w e s t TCR-684 (ng/mL) 18.3 [0.024] [0.071] 0.160 0.535 3.87 0.290 [ ] Denotes values determined from concentrated spe method. V a lu e < LOQ. Accurate Mass Determ ination, Accurate mass measurements and elemental compositions were obtained for endogenous C 7-C 11 and PFOS in a concentrated SPE extract of normal pooled human serum purchased from Golden West Biologicals (TCR-690). Accurate mass measurements are presented in Table 6. Page 20 of 225 3M Environmental Laboratory E02-1039 Table 6. Accurate Mass Determination of Endogenous Test Substances Test Substance Accurate Mass Theoretical M ass C7 C8 C9 PFOS CIO C ll 362.9739 412.9637 462.9613 498.9280 512.9615 562.9542 362.9691 412.9659 463.9627 498.9297 512.9595 562.9563 M ass Deviation (ppm) 13.3 -5.4 -3.0 -3.3 3.9 -3.8 Probable Formula C7O2F 13 C 8 0 2F,5 C902F 17 C803F 17S C io0 2F,9 C 1102F2I Daughter Ions of Endogenous Test Substances, Specific daughter ions were observed at characteristic retention times for endogenous C 7-C 11 and PFOS in a concentrated SPE extract of normal pooled human plasma purchased from Golden W est Biologicals (TCR-684). Daughter ions verified in analytical standards of linear isomers. A summary of daughter ions observed is presented in Table 7. Table 7. Daughter Ions of Endogenous Test Substances Test Substance C7 C8 C9 c10 Cn c12 PFOS Parent Ion (m/z) 363 413 463 513 563 613 499 Daughter Ions (m/z) 319, 119 369, 219, 169, 119 419, 269, 219, 169, 119 469,319, 269,219, 169 519, 319, 219, 169, 119 569, 419, 319, 269, 219, 169, 119 130, 99, 80 Isomers, The presence or absence of distributions of branched and linear isomers of endogenous fluorochemicals were qualitatively determined in concentrated SPE extracts of commercial lots of pooled human serum and plasma (Figures 1 - 1 0 ) . Qualitative analysis of chromatographically resolved branched and linear isomers of PFOA was accomplished using NMR certified standards of a linear isomer PFOA standard and a mixed branched and linear isomer PFOA standard. It was determined that the branched PFOA isomers elute within an approximate 0.2 minute retention time window and are baseline resolved from the linear PFOA isomer. This finding of C8 retention time resolution was extrapolated to determine qualitatively the presence of branched and linear isomers for C7, the higher homologues of C9-C12, and PFOS. Low to non-detect levels of branched isomers were observed for the C7 and C 9-C 12 compounds detected. PFOS and PFOA were present in the screened lots of sera and plasma as branched and linear isomer distributions. Figure 1 contains chromatograms of NMR certified linear isomer PFOA and mixed branched and linear isomer PFOA standards. Figures 2 - 8 contain chromatograms of the corresponding standards of C7 - C 1 2 and PFOS in solvent, an extracted calibration curve point (extracted calibration curve point consists of pooled human plasma from central China spiked with 10 ng of test substance/mL of plasma), and three concentrated SPE Page 21 of 225 3M Environmental Laboratory E02-1039 extracts of commercial pooled human serum. Pooled human plasma collected in central China was used as the blank matrix to construct an extracted calibration curve because preliminary screening results indicated it did not contain quantifiable levels of endogenous test substances. Page 22 of 225 3M Environmental Laboratory E02-1039 Figure 1: NMR Certified 99.5% Linear Isomer PFOA and Mixed Branched and Linear Isomer PFOA Standards dO21 106002 S m (M n , 1x1) 100-1 Linear isomer CSstandard a.43 51249 MRM o f 4 Channels ES <= linear (99.5% by NMR) TIO ^ jjq Area %- 8.19 branched(0.5% by NMR) => 503 n *f i '| i i i i i i i i11 i i 'i i t |" f i,ii"rii y i i i i H '1 1 1 I'1 1 1 1 i 1 ' 1 1 i ' 1 1 1 I Summary of PFOA Standards: NMR certified standard of the linear isomer PFOA contains 0.5% branched and 99.5% linear isomers. NMR certified standard of mixed branched and linear isomer PFOA contains 22.3% branched and 77.7% linear isomers. Page 23 of 225 Figure 2: C7Isomer Distribution d021025069 Sm (Mn, 1x1) 3M Environmental Laboratory E02-1039 1: MRM of 4 Channels ES- d021025098 Sm (Mn, 1x1) 1: MRM of 4 Channels ES- 2.50 5.00 7.50 10.00 12.50 15.00 S u m m a ry o f C 7 A c id C h ro m a to g ra m s: Low to non-detect levels of the branched isom ers of C 7 acid were observed in these lots of com m ercial pooled human serum. Page 24 of 225 Figure 3: C8Isomer Distribution 3M Environmental Laboratory E02-1039 dO21024012 Sm (Mn, 1x1) 100- Sohrent standard 30 ppb %- 8.46 44441 <= Linear 2: MRM of 4 Channels ESTIC 3.23e5 Area 1 Branehed=> dQ21024034 Sm (Mn, 1x1) 2: MRM of 4 Channels ES- ,i 1 0 0 i 8.47 69023 <= Linear TIC 6.80e5 ; Chinese plasma 10ppb Area 1% 8.21 Branched => 534 _ i i i"i i i l i l r^pi i i i i i i i i i i i i i i i i i i i i ^ | l i i i i i i ! [i i i t i i i i i i { i i iii" r , p i i > d021024041 Sm (Mn, 1x1) 1 00 Lampire serum 8.44 _ 63605 < = Linear 2: MRM of 4 Channels ESTIC 6.30e5 Area %: 8.18 Branched => 10219 ffl 0 1i i i -i i i i i i i i i i i i 'i i t i ~| i i i i i n i i i ir-Yn^T | i i i i i i i i i*|`i `i i i i i i i i i i i i r-p i i i d021024043 Sm (Mn, 1x1) 2: MRM of 4 Channels ES- 100: 8.40 _ 7 9 3 6 0 1 <= Linear TIC 8.13e5 - Sigma serum Area %: Branched => 0.14 7251 0 i i r--i i ............. i' i i r i i l i i i i' i i' i i-M r i V _ i i | i i i i | i i i i | i i 'i i | i i r i i i d021024045 Sm(Mn, 1x1) 100: ' 8.40 _ 145120 <= Linear 2: MRM of 4 Channels ESTIC 1,28e6 j Golden west serum Area %- Branched=> 7.80 8619, 'I j i i i T 'f'T'T i i i i"r'vr"| i i i"T i i i"i1i i 2.50 5.00 7.50 11 i 1111 i 111i 11I 1I M' i 'I I I I I M I Time 10.00 12.50 15.00 S u m m a ry o f C a C h ro m a to g ra m s: Low to non-detect levels of branched isom ers of C 8 were observed in these lots of com m ercial pooled human serum. Page 25 of 225 3M Environmental Laboratory EO2-1039 Figure 4: C9Isomer Distribution d021025069 Sm (Mn, 1x1) 10Ch Solvent standard 30 ppb %- Q.10 45551 <= Linear 2: MRM of 4 Channels ESTIC 4.01 e5 Area (branched not observed) Q i i i i ji i i i i i i i i i i il,l^ i^ i i i i i i i i l i i 1 r r r y ^ d021025091 Sm (Mn, 1x1) 10th Q.10 43B0S C hiese plasma 10ppb % (branched not observed) fi i | i i i i i i i i | i i i- | i i i i | i i i i 2: MRM of 4 Channels ES- T IC <= Linear 4.81 e5 Area 0 i 11 111"fi i i 'i'i-i 11 11 y 11 11 111 11 11 111 i i i d021025098 Sm (Mn, 1x1) 2: MRM of 4 Channels ES- S u m m a ry o f C C h ro m a to g ra m s: Low to non-detect levels of branched isom ers of C9were observed In these lots of com m ercial pooled human serum. Page 26 of 225 Figure 5: Cm Isomer Distribution d021024012 Sm(Mn, 1x1) 3M Environmental Laboratory E02-1039 3: MRM of 4 Channels ES- dD21 D24041 Sm(Mn, 1x1) 3: MRM of 4 Channels ES- S u m m a rv o f C m C h ro m a to g ra m s: Low to non-detect levels of branched isom ers of C 10 were observed in these lots of com m ercial pooled human serum. Page 27 of 225 Figure 6: Cn Isomer Distribution 3M Environmental Laboratory E02-1039 d021025069 Sm (Mn, 1x1) 1QCh 10.31 54214 3: MRM of 4 Channels ESTIC 5.72e5 Solvent standard 30 ppb % 10.07 <= Linear Area 1031 Branched => Ti fi |-n 11 ) 111 r| n 1 1) 111 i pi 11 f i 111 f iTf i p-i 1 11 n n f m 'T7 1 1 1n -rm i n ri'i'rm 1 1 11 i d021025091 Sm (Mn, 1x1) 3: MRM of 4 Channels ES- d021025102 Sm(Mn, 1x1) 100-] Golden ouest serum o 1I I ' ' I I 2.00 4.00 3: MRM of 4 Channels ES- 10.23. 8903 TIC 9.71e4 <= Unear ^ re a 10.07 111141 Branched => ' I ' ' I I ' I 1` I ' ' I ' ' 1 11I` 1TIime' I 6.00 8.00 10.00 12.00 14.00 Sum m ary of Cn Chrom atogram s: Low to non-detect levels of branched isomers of Cn were observed in these lots of commercial pooled human serum. Page 28 of 225 3M Environmental Laboratory E02-1039 Figure 7: C 12 Isomer Distribution d021025126 Sm (Mn, 1x1) 1001 10.75 82828 Solvent standard 30 ppb %(branched not observed) 2: MRM of 4 Channels ES- TIC <= Linear 8.00e5 Area Q i i i i i i i 11 i i i "i1 i i r 1 i i i i i i i 1 i i i n i r n dQ21025148 Sm (Mn, 1x1) 100: 11 1 1 1 1 | 1 1 10.72 4Q7B0' C hiese plasma 10ppb y 1 1 it / i i i i | i i i i | i i i i 2: MRM of 4 Channels ES- TIC <= Linear 6.69e5 Area (branched not observed) n -T r 1 tt | r i r < 11 r 1 < f it r p 1 r r \-m i it t -i t t r i f i | i i iT yr v n p - r r r fi 1 1 1 d021025155 Sm (Mn, 1x1) 100: Lam pire serum 10.75 3Q7 ; (branched not observed) %- 2: MRM of 4 Channels ES- TIC <= Linear 1 6.96e3 Area 0 I ' ri i I i i i i i i i i i i i i i i |'t i i ~ i J i " n I T I *T J I I T T J I T ' I I 2.50 5.00 7.50 10.Q0 12.50 15.00 Summary of C Chrom atogram s: Low to non-detect levels of branched isomers of C 12 were observed in these lots of commercial pooled human serum. Page 29 of 225 Figure 8: PFOS Isomer Distribution (1021525126 3m (Mn, 1x1) 3M Environmental Laboratory E02-1039 1. mrm of 3 Channels ES- Summarv of PFOS Chromatograms: Branched isomers of PFOS were observed in these lots of commercial pooled human serum. Page 30 of 225 3M Environmental Laboratory E02-1039 Statistical Methods and Calculations Theoretical concentrations of analyte in final eluate: Concentration = (Concentration of Analytical Standard x Amount of standard added) / EV Observed result to original sample result: Original sample result = Observed result x DF f Spike percent recoveries: i>; j Ml ,,%, R,, ecovery = -O--b-s-e--r-v-e-d---R-e--s-u-l-t-----S-a--m--e--S-a--m- p--le--,-U--n--sp--ik--e-d- x 1, 00 Theoretical Concentration EV = Eluate volume DF = Dilution factor Relative standard deviation: Relative Standard Deviation Standard Deviation x 100 Mean Percent deviation: ^ .. Theoretical Cone. - Calculated Cone. I % Deviation = -------------------------------------------- x 100 Theoretical Cone. Means and standard deviations were calculated using Excel software. I 1v,'i S3 Page 31 of 225 3M Environmental Laboratory E02-1039 Statement of Conclusion Results from this study showed quantifiable levels of the linear isomers of C7, C9-C12 ranging from < 0.010 - 0.9 ng/mL (Tables 1a and 1b). Low to non-detect levels of branched isomers were observed for the C7 and C 9-C 12 compounds detected (Figures 1 -1 0 ). PFOS was present in the screened lots of commercial sera and plasma in concentrations ranging from 2.5 - 27 ng/mL and was present as branched and linear isomers. PFOA was present in the screened lots of commercial sera and plasma in concentrations ranging from 0.65 - 5.6 ng/mL and was present as branched and linear isomers. Accurate mass measurements and elemental compositions were obtained for endogenous C7C11, and PFOS that are consistent with theoretical mass and elemental composition. The presence of confirmatory daughter ions at characteristic retention times of target analytes in matrix are consistent with the linear isomer analytical standards. Page 32 of 225 3M Environmental Laboratory E02-1039 References None List of Attachments Attachment A: Extraction and Analytical Methods Attachment B: Data Summary Tables Attachment C: Sample Chromatograms Attachment D: Sample Prep Sheets, Test Substance Information and Notes to File Attachment E: Protocol, Protocol Amendments and Deviations Report Revisions Semi-quantitative estimates forTHPFOS and THPFDS were removed from tables 1a, 1b, 5a, and 5b because the data was intended as quantitative and only screening estimates could be obtained. References to THPFOS and THPFDS were removed from the list of test substances table 2 (page 11), chemical structures (page 13), reference substances table 3 (page 14), mass spectrometer acquisition parameters (page 18), and control of bias (page 19). References to THPFOS and THPFDS were also removed from the accurate mass determination discussion (page 20) and accurate mass determination data table 6 (page 21), daughter ion discussion (page 21) and daughter ion data table 7 (page 21), isomer discussion (page 21), and isomer figures 8 and 9 (pages 30 and 31). Wording in the Executive Summary (page 8), Summary (page 9), Introduction (page 11), and Statement of Conclusion (page 34) was modified to reflect the removal of the semi-quantitative estimates for THPFOS and THPFDS. Several typographical errors were also corrected, including William K. Reagen's phone number (page 7), formulas for the anionic forms of THPFOS and THPFDS in table 2 (page 11), and the units designator for mass deviation, table 6 (page 21). A protocol am endm ent was written to address the removal of TH P F O S and T H P F D S from the study. Page 33 of 225 3M Environmental Laboratory E02-1039 Signature Page W e certify that this report is a true and complete representation of the data for this study: h William K. Reagen Testing Facility Management Date Page 34 of 225 Attachment A: Method -i I li M 3M Environmental Laboratory E02-1039 S .-''I il Page 35 of 225 3M Environmental Laboratory E02-1039 Record of Deviation I. Id e n tifica tio n Study / Project No. Deviation Type: (C heck one) E0 2 -10 3 9 SOP Protocol TS^Method 0 Equipment Procedure Other: Document Number: ETS-8-231.1 Date(s) of occurrence: 10 ct 02 to 25 Oct 02 II. D e scrip tio n : Required Procedure/process: ETS-8-231.1 section 9.6, "Quality Control (QC) Sample" see method for details. Actual Procedure/process: The continued accuracy of the calibration curve was monitored through the re-injection of a curve point after no more than 10 samples and at the end of the run, not by preparing additional samples. The QC samples described in the method were prepared to monitor extraction efficiency. The QC samples were only spiked at 2 levels, 0.5 and 5ppb. In addition, only 2 QC samples were prepared for each matrix. It was possible, therefore, to distinguish between the continued accuracy of the calibration curve and the extraction efficiency in each matrix tested. _______ ___________ ___ ________ During the course of the study 2/3 of the QC samples prepared in the same matrix as the . calibration curve were within 25% of expected values. All of the 5ppb spikes passed, but only 3 of 9 low spikes passed.________________ ___ ________ __ _______ _____ _____ III. A c tio n s Taken: (such as amendment issued, SOP revision, etc.) No additional action will be needed. Recorded By: ' Date: d u 'ld IV. Im p a ct on S tu d y / P ro je c t \ U / \ / op--. This deviation does not adversely affect the quality of the data in the context of the current study. Date: 3M E nviron m en tal L ab oratory D e v ia tio n N o . (________ {assignedby Study Director or Project Lead at the end ofstudyor project) Exact Copy o f Original Page 36 of 225 3M Environmental Laboratory E02-1039 3M Environm ental Laboratory Method ~ Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Method Number: ETS-8-231.1 Adoption Date: //jl3 jd l Revision Date: J$f'o%. Effective Date: s j l ?/iz. Approved By: Willliam K. Reagen Laboratory Manager Date Exact Copy of Original / 0 / ?///)> ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page l o f t 9 Page 37 of 225 3M Environmental Laboratory E02-1039 1 Scope and Application This method describes the extraction o f target analytes from fish, iat liver, rat sera, mouse liver, and mouse sera using solid phase extraction (SPE). This method may also be extended to other biological matrices provided that the data quality objectives are m et 2 Method Summary An amount o f biological material, determined by the analyst, is prepared (fluids diluted and tissues homogenized) at a 1/6 dilution, or other dilution as determined by the analyst using reagent grade water. An aliquot o f the dilution/homogenate is spiked with the appropriate surrogate or analyte mixture. Acetonitrile (ACN) is added as an extraction solvent and also serves to precipitate the proteins. The sample is capped, mixed, and put on the centrifuge to clarify the supernatant. The supernatant is transferred to a clean tube, diluted with water, and passed through a pre conditioned C n SPE cartridge. Finally, the analytes o f interest are eluted from the SPE cartridge and analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES/MS/MS). 3 Definitions 3.1 Dilution A dilution expressed as I:S or 1/6 is defined as: 1 mL o f sample + 5 mLs o f diluent for a total o f 6 mLs combined, unless otherwise noted. - 3.2 SPE cartridge A column containing an open solvent reservoir at one end and packed with bonded silica sorbents at the other end. It is designed to retain the compounds o f interest under some solvent conditions and elute them under others. A separation is thus achieved; compounds can be removed from difficult biological matrices and introduced into appropriate solvents for analysis. 3.3 Reagent grade water W ater with no detectable coricentration(s) o f the target analyte(s). 3.4 Quality control sample Sample used to monitor the extraction efficiency (as a matrix spike) and to verify the continued accuracy o f the initial calibration curve (as a continuing calibration verification). 4 Warnings and Cautions________________________________________________ 4.1 Health and Safety Warnings Always wear appropriate gloves, eyewear, and clothing when working with solvents, samples and/or equipment Use caution with the voltage cables tor the probe. When engaged, the probe employs a voltage of approximately 5000 volts. 4.2 Cautions Take care not to allow the SPE column to tun to dryness after the methanol and water washes. After washing is complete, add sample then allow all o f the liquid to pass through the SPE column to dryness. ETS-8-231. 1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 2 of 19 Page 38 of 225 3M Environmental Laboratory E02-1039 Do not operate solvent pumps above capacity o f 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HPLC will initiate automatic shutdown. Do not tun solvent pumps to dryness. 5 Interferences____________________________________ To minimize interferences. Teflon should not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract 6 Instrumentation, Supplies, and Materials____________ The following instrumentation, supplies, and materials are used while performing this method. Equivalent instrumentation, supplies, and materials may be used in place o f those listed. 6.1 Instrumentation Vortex mixer, VWR, Vortex Genie 2 Ultra-Turrax T25 tissue homogenizer Vacuum Pump SPE Extraction Manifold Centrifuge, Mistral lOOOorlEC Shaker, Eberbach or VWR Balance (+/- 0.1000 g) Micromass, Quartm II or Ultima triple quadrupole Mass Spectrometer equipped with an clcctrospray ionization source HP 1100 or Agilent low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 6.2 Supplies and Materials Eppcndorf or disposable pipettes, plastic or glass Dissecting scalpels Polypropylene bottles, capable of holding 50 mL to 1 L (Nalgene) Volumetric flasks, glass, type A 40 mL glass vials (ICHEM) Plastic sampule vials, Wheaton, 6 mL (or other appropriate size) Centrifuge tubes, polypropylene, 15 mL and 50 mL Labels Graduated pipenes, glass Syringes, capable o f measuring 5 pL to 1000 |iL Bottlc-Top Dispenser (capable o f dispensing 5mL o f solvent) SPE extraction cartridge, 1 g, Sep-Pak 6 cc tri-functional C u (Waters) 75 mL sample reservoir (or other appropriate size) Crimp cap glass autovials and caps ETS-8-23I.I Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 3 o f 19 Page 39 of 225 Exa ct Copy o f Original j 1 ; i !-:.j I 3M Environmental Laboratory E02-1039 Crimpers HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7 Reagents and Standards Reagent grade water, Milli-QTM, Nanopure II, or equivalent Acetonitrile, HPLC grade or equivalent Methanol, HPLC grade or equivalent Ammonium acetate, reagent grade or equivalent Biological fluids or tissues, frozen from supplier 7.1 Reagents preparation 2.0 raM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing reagent grade water, mix until all solids are dissolved, bring to volume using reagent grade water. Store at room temperature. Note: When preparing different volumes than those listed in reagents preparation, target analyte standard preparation, and surrogate standard preparation, adjust accordingly. 7.2 Target analyte standard preparation Prepare target analyte standard(s) for the standard curve. Multicomponent analyte standards are acceptable. The following is an example only and may or may not be appropriate for all standard preparations. Weigh approximately 100 mg o f target analyte into a 100 mL volumetric flask and record the actual weight in the standard logbook or other appropriate location. Bring to volume with methanol for a stock standard o f approximately 1000 ppm (jlg/mL). Dilute the stock solution with methanol for a working standard I solution of approximately 50 ppm. Example calculation: 1000 jtg/mL x 5 mL/100 mL = 50 jlg/mL. Dilute working standard 1 with methanol to produce a working standard 2 solution o f approx. 5.0 ppm. Example calculation: 50 jlg/mL x 10 mL/lOOmL = 5.0 jlg/mL. Dilute working standard 1 with methanol to produce a working standard 3 solution o f approx. 0.50 ppm. Example calculation: 50 jtg/mL x 1.0 mL/100 mL = 0.5 jlg/mL. 7.3 Surrogatestandardpreparation Prepare sutrogate standard(s). The following is an example only and may or may not be appropriate for all surrogate standard preparations. Weigh approximately 90-110 mg o f surrogate standard into a 100-mL volumetric flask and record the actual weight Bring to volume with methanol for a surrogate standard stock o f approximately 900 - 1 100 ppm. Prepare a surrogate standard working standard. Transfer approximately 1 mL o f surrogate standard stock to a 10-mL volumetric flask and bring to volume with methanol for a working standard of90-l Klppm. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 7.4 Internal standard preparation Prepare internal standard(s). The following is an example only and may or may not be appropriate for all internal standard preparations. Weigh approximately 90-110 mg o f internal standard into a 100-mL volumetric flask and record the actual weight. ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochcmical Compounds from Biological Matrices Page 4 of 19 Page 40 of 225 3M Environmental Laboratory E02-1039 Bring to volume with methanol for an internal standard stock o f approximately 900 - 1100 ppm. Prepare an internal standard working standard. Transfer approximately 1 mL of internal standard stock to a 10-mL volumetric flask and bring to volume with methanol for a working standard o f 90-1 lOppm. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 8 Sample Handling All samples are received frozen and must be kept frozen until the extraction is performed. Allow samples to thaw to room temperature prior to extraction. Typically flesh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials until analysis. / If analysis will be delayed extracted standards and samples may be refrigerated at approximately 4C indefinitely or may be stored at room temperature until analysis can be performed. 9 Quality Control 9.1 Blanks 9.1.1 Solvent Blank An aliquot o f methanol is used as a solvent blank. Solvent blanks are not extracted. 9.1.2 Method Blank An aliquot o f 1.0 mL of water, or other appropriate amount, is used as a method blank. Four method blanks are extracted and analyzed with each set following this procedure (two are spiked with surrogate and two are not spiked). 9.1.3 Matrix Blank An aliquot o f 1.0 mL or 1.0 g o f matrix (diluted or homogenized) is used as a matrix blank. Other amounts may be used, as appropriate. Matrix blanks are prepared from one o f three sources: 1) a study control matrix from a study control animal received with a sample set; 2) a commercially obtained sample o f the same species as the study animals; or 3) a surrogate matrix, also obtained commercially, but of a different species than the study animal, (eg. if rat is used to generate standard curves and CCV s for a m ouse study). The m atrix to use is dependent on the matrix used for the curve. ' 9.1.3.1 Study control matrix curve - if the study control matrix is used for the curve, prepare four (4) matrix blanks using the study control matrix (two spiked with surrogate and two not spiked). 9.1.3.2 Commercially obtained (same species) matrix curve - if the commercially obtained matrix is used for the curve, prepare four (4) matrix blanks using the same commercially available matrix (two spiked with surrogate and two not spiked). 9.1.3.3 Surrogate matrix curve - if a surrogate matrix is used for the curve, prepare four (4) matrix blanks using the same commercially available matrix and prepare four (4) matrix blanks using a commercially available matrix o f the same species as the study animals (two spiked with surrogate and two not spiked). 9.1.3.4 If limited matrix is available, the number of method and matrix blanks may be adjusted and will be noted in the study protocol or in the raw data. 9.2 Sample Replicate Samples replicates arc prepared according to each study protocol or project outline. ETS-S-23U Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 5 of 19 Page 41 of 225 Exact Copy o f Original VPS !;l '.v'-'l m 3M Environmental Laboratory E02-1039 9.3 Surrogate standard If surrogate standard is a component o f the study, all samples are spiked with surrogate standard prior to extraction to obtain a concentration in the mid-range o f the calibration curve, with the exception o f blank samples as described above. Typically surrogate standard is spiked into the 1.0 mL diluted/homogenized sample removed for extraction. However, surrogate may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the l.0 mL sample, or into the l .0 mL diluted/homogenized sample removed for extraction. 9.4 Internal standard If internal standard is a component o f the study, all samples are spiked with internal standard after extraction to obtain a concentration in the mid-range o f the calibration curve. Typically internal standard is spiked into the 2.0 mL o f extract in the IS mL centrifuge tube, before transferring to the autovial. 9.5 Lab Control Sample Lab control samples are not a component o f this method. . 9.6 Quality Control (QC) Sample Prepare quality control (QC) samples to monitor extraction efficiency and to verily the continued accuracy o f the initial calibration curve. Typically 1.0 mL, cr other appropriate amount, o f the same matrix used to prepare the initial calibration curve is used for each QC sample. Twclve (12) quality control samples (QC) will be prepared for each matrix during the course o f a study. A minimum o f 3 QC samples must be prepared (one at each level) on each day of sample extraction, (e.g. If the study is such that samples will be extracted on three different days then four QC samples must be prepared on each day o f extraction for a total of twelve.) QC samples will consist o f four samples at each o f three levels o f analyte. The levels listed below may be used and may represent sample concentrations diluted into the range o f the calibration curve: Low level: 3X to 5X the LLOQ, Mid-level: equivalent to a point near the middle o f the calibration curve, High level: 80% o f the ULOQ Two QC sample levels arc analyzed after every tenth sample injection starting after the last calibration standard injection, with a minimum o f three QC per analysis. Solvent blanks are not considered samples but may be included as such for determining when Q C samples will be analyzed. QC samples extracted with a particular sample set must be analyzed in the same analytical nut. Any QC samples extracted during the course o f the study may be included in subsequent analyses. If samples from multiple extraction dates arc analyzed in one analytical run, then QC samples from the same sample extraction dates must be included in that analysis. Each QC is expected to show an accuracy o f 75-125% o f expected. A minimum of2/3 o f all QC samples must m eet this criteria, and a minimum of 1/2 of the QC samples at each level must meet this criteria. If not, the set must either be rc-anulyzcd or re-extracted. 9.7 Sample Dilution Any sample with an area greater than that o f the highest acceptable standard will need to be diluted into the range o f the calibration curve. If samples arc diluted into the range of the curve during analyses and enough sample remains, a post-run dilution validation will be perfonned to verily sample values. To perform the dilution validation, one sample will be separated into two representative samples (i.e. two 1.0 mL aliquots for fluid samples or two 1.0 gram amounts for tissue samples, or other amount as detennined by the analyst ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochctnical Compounds from Biological Matrices Page 6 o f 19 Page 42 of 225 3M Environmental Laboratory E02-1039 and documented in a note to file) then diluted using two procedures. The first procedure consists o f diluting the sample with additional matrix prior to extraction (fluid adding fluid), while the second procedure consists o f diluting the extract with solvent post-extraction (methanol extract adding additional methanol solvent) If the relative percent difference is not within 15% for these two samples; additional testing will be required to determine which value is a correct representation o f the sample concentration. 10 Calibration and Standardization 10.1 Instrument Calibration One calibration curve will be prepared from extracted matrix standards, in the same matrix as the samples, per study. It will consist o f a minimum o f nine (9) levels. Additional calibration curves may be extracted on separate sample extraction dates, as determined by the analyst and documented in a note to file. Transfer 1.0 mL, or other appropriate amount, o f diluted control fluid or homogenized control tissue to a 15 mL centrifuge tube using a disposable plastic pipette. This will be repeated while preparing aliquots for the standard curve. Be sure to mix or shake the control matrix container between aliquots to ensure a homogenous sample is removed. Record each standard volume on the weightA-olumes sheet or extraction worksheet, as appropriate. Four 1.0 mL aliquots, or other appropriate amount, o f control matrix serve as matrix blanks. The standard concentrations and spiking amounts listed in Table 1 may be used, when appropriate, to spike one standard curve. A total of 9 standards, four matrix blanks, and four method blanks are prepared in addition to the QC samples and test samples. The number o f standards and blanks may be adjusted as determined by the analyst and documented in a note to file. Use Attachment C, or other appropriate fbnn, as an aid in calculating the concentrations o f the working standards. Refer to section 12 to calculate the actual concentration o f analyte in each calibration standard and QC sample. Typically the target analyte standard is spiked into the 1.0 mL diluted/homogenized sample removed for extraction. However, it may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sam ple. prior to removing the 1.0 mL sample, or into the 1.0 mL diluted/homogenized sample removed for extraction. Analyze the extracted matrix standard curve prior to each set of extracts. The curve equation will be determined by regression analysis using the peak areas o f the target analyte(s) using MassLynx or other suitable software. Any level outside 75% - 125% o f nominal must be deactivated, and regression re-calculatcd, except the LLOQ which must be within 30% o f nominal. All levels must show a response greater than twice that o f the blank. A maximum o f three (3 ) le v els m a y b e d ea ctiv a ted in an y one set, or the set will be re-analyzed. Exact Copy of Original ETS-8-23I.I Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 7 o f 19 Page 43 of 225 3M Environmental Laboratory E02-1039 Table 1 A p p r o x im a t e S p ik in g Am o u n t s fo r Standards and Spik e s Usin g 1.0 m l o f M a trix Working standard (approximate concentration) pL Approximate final concentration of analyte in M atrix diluted 1:5 Approximate final concentration o f analyte in Final 2.0 mL volume - - Blank Blank 0.500 ug/mL 1.5 5.00 ng/g or ng/mL 0.375 ng/mL 0.500 ug/mL 3.0 10.0 ng/g or ng/mL 0.750 ng/mL 0.500 ug/mL 8.0 25.0 ng/g or ng/mL 2.00 ng/mL 0.500 ug/mL 16 50.0 ng/g or ng/mL 4.00 ng/mL 0.500 ug/mL 32 100 ng/g or ng/mL 8.00 ng/mL 5.00 ug/mL 5.6 175 ng/g or ng/mL ' 14.0 ng/mL 5.00 ug/mL 8.0 250 ng/g or ng/mL 20.0 ng/mL 5.00 ug/mL 16 500 ng/g or ng/mL 40.0 ng/mL 5.00 ug/mL 24 750 ng/g or ng/mL 60.0 ng/mL 5.00 ug/mL 32 1000 ng/g or ng/mL 80.0 ng/mL 5.00 ug/mL 40 1250 ng/g or ng/mL 100 ng/mL 50.0 ug/mL 5.0 1500 ng/g or ng/mL 125 ng/mL 50.0 ug/mL 6.0 1750 ng/g or ng/mL 150 ng/mL Surrogate Std 100 ug/mL 10 6500 ng/g or ng/mL 500 ng/mL 11 Procedures________________________________________________________ 11.1 Tissue Sample Preparation Obtain frozen tissue samples Cut approximately 1.0000 g o f tissue (+/- 0.1000 g), or other appropriate amount, using a dissecting scalpel. This part o f the procedure is best performed quickly, not allowing the tissue to thaw. Weigh the tissue directly into a fared plastic sampuic vial. Record the weight on the weight/volume sheet, extraction worksheet, or other appropriate location. Return unused tissue to the freezer after extraction amounts have been removed. Add 2.5 mL o f reagent water to sampule vial, or other volume as determined by the analyst and documented in a note to file. Homogenize the sample. Put the Ultra-Turrnx grinder probe in the sample and grind for approximately 2 minutes, or until the sample is homogeneous. Rinse the probe into the tube containing the sample with 2.S mL of reagent grade water, or other volume as determined by the analyst and documented in a note to file, using a pipette. Take the grinder apart and clean it with methttnol after each sample. Refer to ETS-9-52 for more informatioa If an amount other than 1.0000 g (not within+/- 0.1000 g) is removed for an initial weight, adjust the water volume" accordingly to maintain a 1/6 dilution, (e.g. if 0.5 g is removed for extraction, add a total o f 2.5 mL o f water.), or other ratio as determined by the analyst and documented in a note to file. Data M L. ,0 /3 ,/vx. Exact Copy of Original Initial ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 8 of 19 Page 44 of 225 3M Environmental Laboratory E02-1039 11.2 Fluid Sample Preparation Obtain frozen fluid sample and allow it to thaw at room temperature or in lukewarm water. Label a 15 mL polypropylene centrifuge tube with the study number, sample ID, extraction date and analyst initials. Sec attached worksheet (Attachment A or similar worksheet) for documenting the remaining steps. Vortex mix the fluid sample for approximately IS seconds, then transfer 1.0 mL o f fluid, or other appropriate amount to a plastic sampulc vial, or other appropriate container. Return unused samples to freezer after extraction amounts have been removed. Add 5.0 mL o f reagent water to the 1.0 mL o f fluid for a 1/6 dilution, or other dilution as determined by the analyst and documented in a note to file. If a volume other than 1.0 mL is removed for an initial volume, adjust the water volume accordingly to maintain the same dilution as above. 11.3 Tissue and Fluid Sample Extraction After tissue or fluid samples have been prepared according to sections 11.1 and 11.2, vortex mix or shake by hand the diluted/homogenized sample for approximately 15 seconds then transfer 1.0 mL, or other appropriate volume, to a clean 15 mL polypropylene centrifuge tube. Return unused diluted/homogenized portions to the freezerafter extraction amounts have been removed. Record the volume removed on the extraction worksheet, (Attachment A or similar worksheet). Spike blanks, samples, and standards, ready for extraction with surrogate standard as described in this method. Spike each calibration standard matrix with the appropriate amount o f standard as described in this method for the calibration curve standards and each QC sample. Vortex mix the standard curve samples and QC samples for approximately 5 seconds. To each sample and standard, add 5.0 mL o f acetonitrile, cap, and vortex mix or shake by hand approximately 15 seconds. Place all samples on the shaker at an appropriate speed for 20 minutes to adequately mix (a setting o f approximately .3 300rpm onthcm odclslistcdinsection6.1). ., Remove from the shaker and centrifuge at an appropriate speed for 10 minutes to adequately pellet the precipitate (a setting of approximately 2000 rpm on the models listed in section 6.1). Add 40.0 inL o f reagent grade water to a dean 50 mL centrifuge tube. Remove samples from the centrifuge and decant the supernatant into the water in the 50 mL tube, taking care not to introduce any of the matrix solids into the solution. Cup and mix by inverting several times. In this step the ortlcr of addition may be changed (i.e. the sample may be put into the centrifuge tube and then the water added). Attach the reservoir to the SPE cartridge and attach this rcservoir/cartridge unit to a vacuum manifold. NOTE: When running the vacuum, set the vacuum chamber at approximately i 5 kPA - to give an approximate elution flow o f 5-7 inL/min. Flows may vary through cartridges and the kPA may be raised for slow tubes and drying after most have been drawn down. Prepare the SPE cartridge by washing twice with approximately 5.0 mL of methanol, followed by approximately two 5.0 mL aliquots o f water, taking care not to allow the column to run to dryness after each wash. After washing is complete, pour the sample into the reservoir/cartridge unit and allow all of the liquid to pass through the column to dryness. Run the vacuum on high for approximately 5 minutes to adequately dry each SPE cartridge. Place a collection 15 mL polypropylene centrifuge tube under each cartridge and elute with 2.0 mL o f methanol. Spike extracted blanks, samples, and standards with internal standard as described in this method. ETS-8-23I.I Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 9 o f 19 Page 45 of 225 Exact Copy o f Original 3M Environmental Laboratory E02-1039 Labe! each glass autovial, as appropriate, with the study number, vial file archive number, animal mimber/gcndcr/timcpoint or LIMS number, matrix, final solvent, analyte components (if needed), extraction type, extraction date, and analyst(s) performing the extraction. Transfer each eluant to a glass autovial and cap. 11.4 Extract Analysis 11.4.1 Software set-up On the MassLynx main page, set up a sample list name. Save the list as instrument designator letter, last 2 digits o f test ycar-month-day, and a letter that will increase through the alphabet with each additional list for that day. Example Sample List: IYYMMDDa or A020204a I =Initial o fthe instrument name (A =*"Amelia") m Y Y -T e s t year (02) MM " Test month (02) ii DD = Test day (04) a " First sample list (run) o f the day (the next sample list will end with V, the next 'c', and so on.) Assign a filename using the instrument designator letter, the last 2 digits o f the test year-month-day, and a 3-digit sequential file number that starts with l and increases by one for each filename. Example filename: IYYMMDDff## or A020204001 I = Initial o f instrument name YY = Test year MM ~ Test month fa DD = Test day ### = 3-digit sequential file number starting widi 1 through 999 (001) Also, as part o f the samplelist, assign a method (MS) for acquiring, an inlet file, a bottle number, an injection volume, and sample descriptions. To create a method, click on Method Editor button in the MS Status Pane and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate mass(es). Also set the acquisition start and stop times. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx "Guide to Data Acquisition" for additional information on MRM. Typically the analytical batch ran sequence begins with system suitability, solvent blanks, and a set o f extracted matrix standards. Sample extracts are analyzed widi two QC samples injected after every tenth sample injection. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and arc not considered sample extracts but may be included as such. ETS-8-23I.I Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 10 o f 19 Page 46 of 225 m ii il m ;v,;ii 3M Environmental Laboratory E02-1039 11.4.2 HPLC set-up Set up sample tray according to the sample list prepared above. Set up the HPLC to the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook; or other appropriate location; Sample size = 10 pL injection Injcct/sample = 1 Cycle tim e3 10.0 minutes Flow rate 3 300 pLAnin Mobile phase: Solvent A 3 2 mM Ammonium Acetate, Solvent B 3 Methanol Solvent gradient program: Tune /'o Solvent B 0.00 10% 1.00 10% 5.50 95% 7.50 95% 8.00 ; 10% 11.4.3 Instrument set-up Refer to ETS-9-24, "Operation and Maintenance of the Micromass Quattro 11Triple Q uadruple Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for details. Check the solvent level in HPLC reservoirs and refill if necessary. Greek the stainless steel capillary at the end of the probe. Use an eyepiece to check die tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactoty, disassemble the probe and replace the stainless steel capillaty. Turn on the nitrogen. Open the nine page. Click on operate to initiate source block and desolvarion heaters. O pen the Inlet Editor. . Download the HPLC method and initiate solvent flow to begin system equilibrium. Set the flow to 10-500uL/min or as appropriate Set HPLC pump to "On" Observe droplets or mist coming out o f the tip of the probe. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. Readjust the tip o fthe probe if no mist is observed Allow to equilibrate for approximately 10 minutes. Typical instrument parameters include: Dtying gas 250-400 liters/hour ES nebulizing gas 10-15 liters/hour ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page II o f 19 Page 47 o f 225 Exact Copy of Original Initial Data 3M Environmental Laboratory E02-1039 1 tijiJ HPLC constant flow mode, flow rate 10-500 |iL/min Pressure <400 bar (this parameter is not set, it is a guide to ensure the HPLC is operating correctly.) Source block temperature approximately 150C Desolvation temperature approximately 250C These settings may change in order to optimize the response Print the tune page, sample list, and acquisition method from MassLynx and store it in the study binder with a copy taped into the instrument log. Click on start button in the Acquisition Control Panel (the location of the start button may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). 12 Data Analysis and Calculations_________________________________________ 12.1 Calculations If other calculations are used than those listed, they will be documented in the raw data. Calculate the matrix amount contained in the initial dilution using the following equation: Matrix Amount (g/mL or mL/mL) IW (g) (or IV (iriL)) (IW(g) (or IV(mL))+ DV (mL) Calculate actual concentrations o f analyte in calibration standards using the following equation: Concentration (ng/g or ng/mL) Spike Concentration (ug/mL)x Spiked Amount (mL) 1000 ng SV (m L)x Matrix Amount (g/mL or mL/mL) l ug 1W = Initial weight (where 1.0 g = 1.0 mL ) IV = Initial volume D V = D i l u e n t v o l u m e (re a g e n t g r a d e w a te r) SV - Sample volume removed for extraction (typically 1.0 mL) AR = Analytical result from MassLynx summary DF =>Dilution factor FV = Final volume MA = Matrix amount 3 curve **MA o f tissuc/fluid standard curve, assumed to be 1 g or 1 mL/5 mL water 3 sample = MA of tissuc/fluid sample (___g or mL of samplc/5 mL water) Calculate spike percent recoveries using the following equation: % Recovery Observed Result - Matrix Blank Result x 100 Spiking Level ETS-8-231. 1 Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 12 o f 19 Page 48 of 225 3M Environmental Laboratory E02-1039 Calculate relative standard deviation using the following equation: ,, , . _ , ,_ . . Standard Deviation ... Relative Standard Deviation ------------------------------ x 100 Mean Calculate percent deviation using the following equation: _ .. % Deviation = --Exp--e-c--t-e-d--C---o--n-e--.---C--a--l-c--u-l-a--t-e--d-C---o--n-e--. x __ 100 Expected Cone. Calculate actual concentration o f analyte in fluid Qjg/mL): AR(ng/mL) * D F * 3 curve fmL/mL) x FV fmU in Curve x I Q ug = (pg/g) 3 sample (mL/mL) FV l.'mL) in Matrix 1000 ng Calculate actual concentration o f analyte in tissue (pg/g): AR (ng/g) x DF x 3 curve ig/mLl x FV (m il in Curve x 1.0 ug = (pg/g) 3 sample (g/mL) FV (mL) in Matrix 1000 ng 13 Method Performance 13.1 System Suitability System suitability will be determined prior to the start and at the completion of each analytical tun. Prior to the calibration curve and after the last sample o f the tun three (3) mid-level unextractcd calibration standards will be analyzed. As applicable, the peak area precision, retention time precision, resolution, and peak asymmetry will be monitored at the beginning and the end of die run separately. The peak area precision must be equal to or less than 5.0% RSD, the precision of the retention time must be equal to or less than 2.5% RSD, the resolution must be > 2.0, and the peak asymmetry (fronting or tailing) must be 0.5<AF<2.0, where AF is the asymmetry factor. If any item o f the system suitability fails, system maintenance must be completed prior to running a second set o f system suitability samples and the system suitability must pass before starting the calibration. If system suitability fails at the completion o f a run, the sample set must be reanalyzed. 13.2 Quantitation The coefficient o f determination value for the calibration curve, plotted by regression using the peak areas o f the analyte(s), must be 0.990 or better. All active calibration curve points must be within 25% o f the theoretical value with the exception o f the LOQ point, which may deviate up to 30%. Calibration standards with peak areas less than two times the curve matrix blank will be deactivated to disqualify a data range that may be affected by background levels o f the analyte. A valid calibration curve must contain at least 6 active points above and including the LOQ. If the curve cannot meet these criteria, the sample set must be reanalyzed or reextractcd. 13.3 Accuracy Two thirds o f all quality control samples and 1/2 o f each quality control sample at each level ate expected to show an accuracy o f 75-125%. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 13 of 19 Page 49 of 225 Exa ct Copy o f Original 3M Environmental Laboratory E02-1039 Surrogates and internal standards must have a percent deviation < 50%. Deviations outside this range will be reanalyzed to confirm, ff the second analysis confirms the original, the deviation will be documented in the raw data. If the second analysis is within 50%, then the second value will replace the original value. 14 Pollution Prevention and Waste Management Sample waste is disposed o f in noninfectious biohazard waste containers. Flammable solvent waste is disposed o f in high BTU containers. Glass pipette waste is disposed of in broken glass containers located in the laboratoiy. 15 Records Complete the extraction worksheet attached to this method, or other applicable worksheet, and store with the study raw ' data. Each page generated for a study must contain the following information (if applicable): study/project or instrument number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. Other information may be added if applicable to the study. Print the nine page, sample list, and acquisition method from MassLynx to include with the study raw data. Copy these pages and tape into the instrument runlog. Plot the calibration curve by the appropriate regression. Print these graphs and store with the study raw data. Print data integration summary, integration method, and chromatograms from MassLynx, and store with the study raw data. Summarize data using suitable software (Excel 7.0 or LIMS) and store in the study folder. Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 16 Attachments__________________________________________________________ Attachment A: Extraction Worksheet A ttachm ent B : S am ple W eight/V alum e W oiksheet Attachment C, Calibration Standard Concentration Worksheet Attachment D, Dilutions Summary Worksheet 17 References___________________________________________________________ ETS-9-24, "Operation and Maintenance o f the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source" ETS-9-52, "Operation and Maintenance o f a Tissue Grinder" 18 Affected Documents None ___________________________________________ ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochctnical Compounds from Biological Matrices Page 14 o f 19 Page 50 of 225 Exact Copy o f Original kWL. rol?i/a. 3M Environmental Laboratory E02-1039 19 Revisions Revision Number I Revision Description M inor formatting changes. Added detailed information to all sections concerning the extraction procedure, analytical procedure, and calculations. Added attachments and references. Revision p ale 02/18/02 A ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 15 o f 19 Page 51 of 225 3M Environmental Laboratory E02-1039 Study Number: Prep Date: Analysts initials: Box#: Attachment A - Extraction Worksheet Method Revision: ETS-8-231.1 Matrix: Sample Timepoint: Sample Number Sample Number Volume of Amount and or diluted sample Amount and surrogate spike Type of column Elution solvent description removed spike mix used mix used used and lot and volume Comments Exact Copy o f Original Blank matrix_____ TN-A-_________ ; Amount weighed/aliquoted:________ g/mU 1. Homogenize sample 2. Aliquot t mL of diluted matrix into 15 mL polypropylenetube 3. Spike sam p les accordingly 4. Add___ mL of ACN (TN-A-_________ ) to each diluted sample and shake orvortex mix 5. Shake sample for 20 min @ _____ rpm(Shaker____________ ) 6. Centrifuge sample for 10 min @ ______ qxn (Centrifuge_____________ ) 7. Add 40 ml of__________water to 50 mL polypropelene centrifuge tube. 8. Decant extract into centrifuge tubes withwater 9. Shake sample slightly to ensure proper mixing 10. Altach 6 mL C18 SPE cartridges and 75 mL reservoirs to vacuum manifold 11. Condition column with two washes of -5 mL MeOH (TN-A-________ ) - do not allowcolumn togo todryness 12. Wash column with two washes of -5 ml_________ water - do not allowcolumn to go to dryness 13. Filter sample through conditioned column, discarding filtrate 14. Allowcolumn lo go to dryness. After dripping stops, drawa highvacuumthrough column far at least 5 minutes. 15. Elute column with solvent (_________TN-A-________ ) inlo appropriate 15 mL centrifuge tube 16. Spike samples with_______ uL of internal standard ff__________________ , cone._________________ 17. Transfer sample into appropriately labeled autovial and cap Note: In vacuum steps above set the vacuum chamber at approximately 15 kPA - this should give approximately 5-7 mL/mln elution flow Flows may vary through cartridges - kPA may be raised for slowtubes and drying after most have been drawn down and shut off. GTS-8-231.I Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 16 o f 19 Page 52 of 225 3M Environmental Laboratory E02-1039 Prep Date(s): A n aly st(s): Sample M atrix: M ethod/Revision: Sam p le ID Attachment B - Sample Weight/Volume Worksheet Study Number: Equipment Number: Final Solvent <!cTN N um ber: In itial W U V ol. e/m L /L W ater V olum e added (m L ) V olum e Rem oved (m L ) C om m ents Exact Copy o f Original (0 / 7L - t o f o U r . ' i1 1 Fonti Completion Verified By:. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 17 of 19 Page 53 of 225 ''il :q iI J is 3M Environmental Laboratory E02-1039 P rep date(<f): A nalyte(s): Sample m atrix: M cthad/revision: Attachment C: Calibration Standard Concentration Worksheet Standard number: Equipment number: Final solvent and TN: Blank Tissue or Fluid/identilier: A nalyte mix std approx. 0.500 ug/mL: A nalyte mix std approx. 5.00 ug/mL: A nalyte mix std approx. 50.0 ug/mL: S u rro g a te std ap p ro x . 100 ug/mL: Actual concentrations of standards in the analyte mix A n aly te Std cone ug/mL 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 All Am't spiked mL 0.0015 0.0030 0.0080 0.0160 0.0320 0.0056 0.0080 0.0160 0.0240 0.0320 0.0400 0.005 0.006 All Final Volume: mL 2 .0 0 2.00 2.00 2 .0 0 2.00 2.00 2 .0 0 2 .0 0 2.00 2.00 2.00 2.00 2.00 All Initial Fluid Dilution m l/m L 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 All Initial Tissue Density fi/mL 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 Calculated concentrations o f standards in relation to the final 2.0 mL solvent and initial matrix 2.0 m L Finn! Volume Fluid M atrix Tissue M atrix A n aly te Final cone. ne/mL 0.375 0.750 2 .0 0 4.00 8.00 14.0 2 0 .0 40.0 60.0 80.0 100 125 150 S u rro g a te Std cone ng/mL 100 Surrogate Final cone ng/mL 0.500 Analyte Final cone. nu/mL 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 Surrogate Std cone ng/mL too Surrogate Final cone ng/mL 6500 Analyte Final cone. n/ 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 Surrogate Std cone ng/mL 100 Surroeate Final cone ng/g 6500 ETS-8-231.1 Solid Phase Extraction and Analysis of Fluoroclicmical Compounds from Biological Matrices Page 18 o f 19 Page 54 of 225 Exact Copy of Original -& y\ (~ io/3 lLoxInitiai Data 3M Environmental Laboratory E02-1039 `'-i) Study: Dilution Date/Analyst: Box Num ber: S am p le N u m b e r o r D esc rip tio n 1/ Attachment D: Dilutions Summary Worksheet Solvent/TN Number: Extraction Date/Analyst: M atrix/TImepoint: D ilu tio n s 1/ 1/ 1/ 1/ 1/ 1/ C o m m en ts V e rifie d B y : 3 uJ ';.1I El ... N o tes: 1/10 dilution = of sample + ________ of solvent Date U V IL _ I p / 3>! Exact Copy of Original Initial Form Com pletion Verified By: a ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochcmical Compounds from Biological Matrices Page 19 o f 19 Page 55 of 225 Attachment B: Data Tables 3M Environmental Laboratory E02-1039 Page 56 of 225 i - Human Serum Data Quantitated with Chinese Plasma Calibration Curve All data is in units nq/mL________________________ Lampire Serum Sigma Serum PFOS ^12 C,t C,o c9 c. C7 sample 1 < LOQ < LOQ < LOQ < LOQ < LOQ 0.73 < LOQ sample 2 < LOQ < LOQ < LOQ < LOQ < LOQ 0.57 < LOQ low spike < LOQ 0.65 0.82 0 .6 8 0.79 1.39 0.75 High spike 5.47 5.70 7.02 6.34 5.91 6.81 6.51 sample 1 4.74 < LOQ < LOQ < LOQ 0.19 1.60 < LOQ sample 2 4.38 < LOQ < LOQ < LOQ 0.34 1.59 0.15 low spike 4.75 0.45 0.69 0.60 0.84 1.86 0.65 Analyte PFOS c ,2 c,, C,,, C, C, C, Calibration Range, ng/mL 1.01-30.2 0.104-31.1 0.103-30.9 0.252 - 30.3 0.101 - 30.4 0.258-31.0 0.102 - 30.7 Chineese Plasma, Serum Data Cal Curves system Suitability Samples, CCV #1 CCV #2 CCV #3 R* 0.999596 0.999621 %RSD 2.48 0.99 recovery, % recovery, % recovery, % 52% 3% .. 77% 52% .. 0.999767 2.46 105% 105% 99% 0.999471 1.45 86% 90% ** 0.9997 0.996816 2.94 6.73 91% 105% 102% 94% 87% .. 0.999569 2.37 87% 95% 93% Matrix Blanks < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ "None performed High spike 9.06 3.74 5.45 5.17 5.91 6.64 5.78 Golden West Serum sample 1 25.64 < LOQ 0.31 < LOQ 0.96 5.54 0.23 sample 2 28.41 < LOQ 0.33 < LOQ 0.84 5.65 0.15 low spike 24.16 0.46 0.85 0.60 1.30 4.64 0.41 High spike 26.10 3.65 6.42 5.20 6.62 9.12 4.91 Bioresource Serum sample 1 16.79 < LOQ 0.28 < LOQ 0.63 2.83 < LOQ sample 2 17.26 < LOQ 0.31 < LOQ 0.56 3.06 < LOQ High spike 16.78 2.65 4.94 4.32 4.95 7.39 1.47 Low 18 44 < LOQ n 96 0 73 1 11 4 03 0.18 Page 57 o f 225 Human Plasma Data Quantitated with Chinese Plasma Calibration Curve All data is in units ng/mL Chinese Plas iia Lampire Plasma PFOS c12 Cii Cio C, C, C7 sample 1 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ sample 2 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ low spike 1.30 0.52 0.65 0.40 0.59 < LOQ 0.28 High spike 4.78 5.02 5.18 4.88 4.91 4.71 5.28 sample 1 9.89 < LOQ < LOQ < LOQ 0.37 2.30 0.20 sample 2 12.34 < LOQ < LOQ < LOQ 0.50 2.91 < LOQ 13.15 0.48 0.46 0.49 0.78 3.34 0.52 High 16.19 4.53 4.42 4.01 4.88 7.31 5.43 Golden West Plasma sample 1 sample 2 low spike 16.20 20.32 13.56 < LOQ < LOQ 0.44 < LOQ 0.10 0.52 0.15 0.17 0.57 0.42 0.65 0.72 3.46 4.28 3.34 0.45 0.13 0.32 High spike 20.34 4.80 4.95 5.29 5.11 7.94 5.34 Innovative Research Plasma sample 1 sample 2 low spike 15.61 15.55 17.32 < LOQ < LOQ 0.58 0.13 0.14 0.84 0.19 0.15 0.78 0.68 0.49 0.84 3.04 3.09 3.86 < LOQ < LOQ 0.48 High spike 21.63 5.66 6.16 6.08 6.00 9.16 6.14 Analyte PFOS c12 C,, Cio C C, C, Calibration Range, ng/mL 1.0-30.0 0.10-31.0 0.10-30.9 0.10-30.3 0.25 - 30.4 0.52-31.0 0.10-30.7 Chineese Plasma. Plasma Data Cal Curves bystem Suitability Samples, CCV#1 CCV #2 Matrix R2 0.999721 0.999759 0.999585 %RSD 2.20 5.87 1.21 recovery, % recovery, % 102% 84% 101% 84% 90% 78% Blanks < LOQ < LOQ < LOQ 0.999695 2.76 91% 83% < LOQ 0.999847 5.07 97% 80% < LOQ 0.999736 0.999695 2.85 5.85 81% 102% 89% 75% < LOQ < LOQ Page 58 o f 225 WL USE Human Serum Data, 5x Sample Scale-Up with 20uL Injection Volume Quantitated with Chinese Plasma Calibration Curve All data is in units ng/mL__________________________________ Lampire Serum Sigma Serum PFOS C,2 c,, c,. c, c. C, Sample 1, Corrected Sample 1, on-Column for Concentration Concentration Factor 24.859 < LOQ < LOQ 2.486 < LOQ < LOQ 0.313 0.675 11.734 0.250 0.031 0.068 1.173 0.025 Sample 1, onColumn Concentration > ULOQ(30.2) 0.177 ' 0.760 0.575 1.234 13.396 0.126 Golden West Serum Sample 1, Corrected for Sample 1, on* Concentration Column Factor Concentration > ULOQ (30.2) > ULOQ (30.2) 0.018 0.402 0.076 2.473 0.058 2.025 0.123 3.533 1.340 26.245 0.013 < LOQ Sample 1, Corrected for Concentration Factor > ULOQ (30.2) 0.040 0.247 0.203 0.353 2.625 < LOQ Bioresource Serum Sample 1, Corrected Sample 1, on-Column for Concentration Concentration Factor > ULOQ (30.2) > ULOQ (30.2) 1.444 0.144 2.856 0.286 3.272 0.327 2.657 0.266 26.245 < LOQ 2.625 < LOQ Human Plasma Data, 5x Scale-Up with 20uL Injection Volume Quantitated with Chinese Plasma Calibration Curve All data is in units of ng/mL Lampire Plasma Golden West Plasma Innovative Research Plasma Sample 1, Sample 1, Sample 1, Corrected Sample 1, on- Corrected for Sample 1, on- Corrected for Sample 1, on-Column for Concentration Column Concentration Column Concentration Concentration Factor Concentration Factor Concentration Factor PFOS c C,, C,o c, c. C, > ULOQ (30.2) 0.364 0.486 1.270 3.799 36.207 < LOQ > ULOQ (30.2) 0.036 0.049 0.127 0.380 3.621 < LOQ > ULOQ (30.2) 0.236 0.711 2.112 3.436 > ULOQ(31.0) < LOQ > ULOQ (30.2) 0.024 0.071 0.211 0.344 > ULOQ (31.0) < LOQ > ULOQ (30.2) 0.219 1.400 2.169 4.016 37.170 0.160 > ULOQ (30.2) 0.022 0.140 0.217 0.402 3.717 0.016 Chineese Plasma Calibration Curve for Scale-Up data. IQuL Injection Volume Analyte PFOS C,, Cn Cl0 C, C. C, Calibration Range, ng/mL 1.01-30.2 0.104-31.1 0.103- 30.9 0.101 -30.3 0.254 - 30.4 0.103-31.0 0.102-30.7 R2 0.999539 0.999696 0.999526 0.999907 0.999812 0.999441 0.999903 System Suitability Samples, %RSD 4.21 3.94 6.34 4.02 4.16 5.99 6.21 CCV #1 recovery, % 155% 114% 95% 94% 92% 92% 106% Matrix Blanks < LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Page 59 o f 225 Matrix Spike Recovery for Human Serum Data Quantitated with Chinese Plasma Calibration Curve Analyte PFOS C12 Cn C10 c9 C8 C7 Lampire Serum Average Endogenous Concentration, ng/mL < LOQ < LOQ < LOQ < LOQ < LOQ 0.65 < LOQ Low Spike Recovery, % < LOQ 129% 164% 136% 158% 148% 150% High Spike Recovery, % < LOQ 114% 140% 127% 118% 123% 130% Sigma Serum Average Endogenous Concentration, ng/mL 4.56 < LOQ < LOQ < LOQ 0.27 1.60 0.15 Low Spike Recovery, % 39% 90% 138% 120% 115% 53% 100% High Spike Recovery, % 90% 75% 109% 103% 113% 101% 113% Analyte PFOS Ci2 Cii Ci0 Cg C8 c7 G olden W est Serum Bioresource Serum Average Endogenous Concentration, ng/mL 27.02 < LOQ 0.32 < LOQ 0.90 5.60 0.19 Low Spike Recovery, % -573% 92% 106% 120% 80% -191% 44% High Spike Recovery, % -19% 73% 122% 104% 114% 71% 94% Average Endogenous Concentration, ng/mL 17.02 < LOQ 0.30 < LOQ 0.61 2.95 < LOQ Low Spike Recovery, % 0% 530% 93% 864% 86% 91% 294% High Spike Recovery, % 3288% < LOQ 6% 146% 49% 624% 36% Page 60 of 225 Matrix Spike Recovery for Human Serum Data Quantitated with Chinese Plasma Calibration Curve Analyte PFOS C12 C11 Ci0 c9 c8 C7 Chinese Plasma Average Endogenous Concentration, ng/mL < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Low Spike Recovery, % 260% 104% 130% 80% < LOQ < LOQ 56% High Spike Recovery, % 96% 100% 104% 98% 98% 94% 106% Lampire Plasma Average Endogenous Concentration, ng/mL 11.12 < LOQ < LOQ < LOQ 0.44 2.61 0.20 Low Spike Recovery, % 407% 96% 92% 98% 69% 147% 64% High Spike Recovery, % 102% 91% 88% 80% 89% 94% 105% Analyte PFOS Ci2 Cii Ci0 C9 C8 C6 Golden W est Plasma Average Endogenous Concentration, Low Spike ng/mL Recovery, % 18.26 -940% < LOQ 88% 0.10 84% 0.16 82% 0.54 37% 3.87 -106% < LOQ < LOQ High Spike Recovery, % 42% 96% 97% 103% 92% 81% 125% Innovative Research Plasma Average Endogenous Concentration, Low Spike ng/mL Recovery, % 15.58 348% < LOQ 116% 0.14 141% 0.17 122% 0.59 51% 3.07 159% < LOQ 1438% High Spike Recovery, % 121% 113% 121% 118% 108% 122% 183% Page 61 of 225 3M Environmental Laboratory E02-1039 PFOS Telomer Analysis Summary 3M Pharmaceuticals Analytical Research and Development LC/MS analysis of a pooled human sera sample obtained from Golden West Biologicals (#E02-1039) was performed using an Agilent 1100 quaternary HPLC system coupled to a Micromass Q-Tof 2 quadrupole time-of-flight mass spectrometer. The sample was run as submitted using the following parameters listed in Table 1: Table 1 Inject: Analog wavelength output: PDA Wavelength Range: Recorded Mass Range: Flow Rate: Column Temp: Column: Ionization Mode: Resolution: 20 pL NA 190-600 nm 100-800 Da. 0.3 ml/min ambient Betasil C l8, 5 pm, 50x2mm ESI(-) 7,000 Run H20 w / 2mM MeOH Time N ^C H jCO z (min.) 0 90 70 12.5 0 13.0 90 17.0 90 10 100 100 10 10 Accurate mass measurements and elemental compositions obtained in this analysis are summarized in Table 2: Table 2 Retention T im e 5.8 min 6.3 min 6.5 min 6.69 min 6.71 min 6.72 min 6.8 min 7.1 min Accurate Mass Theoretical M ass 362.9739 412.9637 526.9693 462.9613 498.9280 512.9615 426.9734 562.9542 362.9691 412.9659 526.9610 463.9627 498.9297 512.9595 426.9674 562.9563 1U> b Mass D ev iatio n (ppm ) 13.3 -5.4 15.8 -3.3 3.9 14.1 -3.8 Probable Form ula C70 2F,3 Cg0 2F 15 C10H4O3F 17S C902F i7 C803F i7S C io0 2F 19 C8H4O3F 13S C n 0 2F2i Page 62 of 225 Attachment C: Chromatograms 3M Environmental Laboratory E02-1039 Page 63 of 225 C .i- f r u h > 3M Environmental La b o r a t o r y E02-1039 Page 64 o f 225 Exa c t Copy of Original Q yy Jo f S i l i s ^ Initial Data / ^0 F U 3M Environmental La b o r a t o r y E02-1039 Page 65 o f 225 Exa c t Copy o f Original JW lo lS i b -x. Initial Date 3M Environmental La b o r a to r y E02-1039 Page 66 o f 225 Data Q t >L l o l S t l e n - Exa c t Copy o f Original Initial 3M E n v ir o n m e n t a l L a b o r a t o r y E02-1039 Page 67 o f 225 Exa c t Copy o f Original 67~r>l )o l 3 i l n ~L initial O a ts 3M Environmental La b o r a to r y E02-1039 Page 68 o f 225 ---n E x a c t Copy o f Original ilL _ Initial Date 02048-21-7 solventstandard10ppb 3M d021030015 100i C l - A t / > 7.36 %- d021030015 7.15 Davey070799 30-0ct-2002 15:29:24 2: MRM of 4 Channels ES TIO 2.29e5 .............. l~'i 1: Daughters of 363ES- 3M Environmental La b o r a t o r y E02-1039 Page 69 o f 225 Exact Copy of Original Q .2 L _ H / i Inlal Date . d 3- R t l Exa ct Copy o f Original Date& p O L . i i L i l Q T - . Initial Page 70 o f 225 davey070799 30-0ct-2002 15:29:24 1: Daughters of 363ES5.29e4 319.257 T i i I j n i i | rrn p m | i rrp-i n p . 111 I I 11 [ 11 111 i 11 r'|l 111 111 11| 11 n 11 I n 11I i 11 fTl/Z 260 280 300 320 340 360 380 400 3M E n v i r o n m e n t a l La b o r a t o r y E02-1039 3M Environmental La b o r a t o r y E02-1039 Page 71 o f 225 Data Exact Copy o f Original Initial X J20L 02048-21-7 solventstandard10ppb 3M d021030015 C l- Davey070799 30-0ct-2002 15:29:24 2: MRM of 4 Channels ES- 3M Environmental LabEo0r2a-t10o3r9y Page 72 o f 225 Exact Copy of Original C W )C n A l o i . Initial Data 3M Environmental La b o r a to r y E02-1039 Page 73 o f 225 (2 ? H u b D a ta Exact Copy of Original Initial 'ce&.a e g - > < u b 3M Environmental La b o r a t o r y E02-1039 Page 74 o f 225 D a ta ; o / 3 i /o_<~ Initial V it a? Exact Copy of Original LW <- _ J o / Z i / -i^ Initial oats 3/WEnvironmental La b o r a t o r y E02-1039 Page 75 o f 225 3M E02-1039E n v ir o n m e n t a l La b o r a t o r y Page 76 o f 225 Exact Copy of Original CtY)( i laiiu Initial  S aclTtn 3M E n v i r o n m e n t a l La b o r a t o r y E02-1039 Page 77 o f 225 Exact Copy of Original 20L. initial Data I/ E02-10393MEnvironmental Laboratory Page 78 o f 225 C? Exact Copy o f Origina! > K j W -u - initial Data C Z $ Q -tb Exact Copy of Origina! DTK.. - H/t /<rt~ -- Initia] Data 3ME n v ir o n m e n t a l La bEo0r2a-t1o03r 9y Page 79 o f 225 E02-10393M Environmental Labo rato ry Page 80 o f 225 0 .*? D a te L in L i v i Exact Copy of Original Inttlal 3M E n v ir o n m e n t a l La b o r a t o r y E02-1039 Page 81 o f 225 Date Exact Copy of Original Initial 3M E02-1039E n v ir o n m e n t a l La b o r a t o r y Page 82 o f 225 Exact Copy o f Original O InintfauJ . l o lDSatiei o J - 3M Environmental La b o r a to r y E02-1039 Page 83 o f 225 D a is Q 2L- t/zifoi Exa ct Copy o f Original Initial wm / G^ p u l> 3M Environmental La b o r a t o r y E02-1039 Page 84 o f 225 Exact Copy o f Original -C-iy) ( Initial lc>/3, ! c o ^ Data C _9 Q t*ib 3M E n v ir o n m e n t a l La b o r a t o r y E02-1039 Page 85 o f 225 Exact Copy of Original Cm( -Initial n z i/o -2 ^ Date ` ............ ............................................................................................................................. ' '' Exa ct Copy o f Original I/ 3M Environmental La b o r a to r y E02-1039 Page 86 o f 225 Data Initial ( h It - 3M Environmental La b o r a t o r y E02-1039 Page 87 o f 225 (T ' Date Exact Copy of Original lm ( Initiai Q fi A U N 3M Environmental La b o r a t o r y EQ2-1039 Page 88 o f 225 Exact Copy of ORo'oa! c m i _ \v a J.p - initial Dare / d - IU> P i A ^ 3M E n v i r o n m e n t a l La b o r a t o r y E02-1039 Page 89 o f 225 Exa ct Copy o f Original c O L_ i J Z > } > - Initial Data 3M E n v i r o n m e n t a l La b o r a t o r y E02-1039 Page 90 o f 225 Exa ct Copy o f Original CQ( Initial Ql/ S i / h -- Date 3M E n v ir o n m e n t a l La b o r a t o r y E02-1039 Page 91 o f 225 Exact Copy o f Original initial Data C L;d a q , / ^> 3M Environmental La b o r a to r y E02-1039 Page 92 o f 225 Exa ct Copy of Original CJYlL i oI z i I ol- Initfal Data 3M Environmental La b o r a t o r y E02-1039 Page 93 o f 225 D a to L m i_ 1XA /& - Exact Copy of Original initial 5WEnvironmental labo rato ry E02-1039 Page 94 o f 225 Oatr C m c .u /U -x - Exact Copy of Origina! Initial d lO fo L th 3M Environmental La b o r a t o r y E02-1039 Page 95 o f 225 Exa ct Copy o f Original SAYY ( \ / \ / g -- Initial Data ' ..............-- ? ' USI d i o A d ld 3M Environmental La b o r a t o r y E02-1039 Page 96 of 225 Exact Copy o f Original UQOL_ U / 1 Initial Data -- . / Cji A^ub 3/UEnvironmental La b o rato ry E02-1039 Page 97 o f 225 Exact Copy o f Original - & > X - <0 /31 / o x. Initial Data 3M Environmental La b o r a t o r y E02-1039 Page 98 o f 225 du Data r ie io h ih S L Exact Copy of Original Initial I/' d -\ / P r tA h 3M Environmental La b o r a t o r y E 02-1039 Page 99 o f 225 Exa c t Copy o f Original 3 i_ _ WW D ata' C L 11 f i t L . / ' b 3M Environmental La b o r a to r y E02-1039 Page 100 o f 225 Exa ct Copy o f Original . - C _ I Q jz /o ^ initial Data Clu /vg^-iiN 3M Environmental La b o r a to r y EO2-1039 Page 101 o f 225 Exact Copy of Original CC L 1 1 / . 1, / P O _ ' Initial Oats Exact Copy ot Original 2 2 L H / l/ p -i - I/ 3M E n v i r o n m e n t a l La b o r a t o r y E02-1039 3M Environmental La b o r a t o r y E02-1039 Page 103 o f 225 llA / oXData Exact Copy of Original to ( initlal 3M Environmental La b o r a t o r y E02-1039 Page 104 o f 225 ;SiSr.! figS5 Exa c t Copy of Original Initial Date C o a-t rp 3MEnvironmental LabEo0r2a-t10o3r9y Page 105 o f 225 Exact Copy of Original H C Initial lo /m /on D a ta C .O . A O 3ME n v ir o n m e n t a l La bEo0r2a-t1o03r9y Page 106 o f 225 Exact Copy o f Original C J3Q U / o h i f / n Initiai D a ta C J ^ /K L^ -I f a c t Copy o f Original 3M Environmental La b o r a t o r y E02-1039 Page 107 o f 225 3M Environmental labo rato ry E02-1039 Page 108 o f 225 i, / Exa c t Copy o f Original 01--. CL)3- fiu h , Exact Copy of Origins! irdtial Date 3M Environmental La b o r a t o r y E02-1Q39 Page 109 o f 225 3M Environmental labo rato ry E02-1039 Page 110 o f 225 tlA A n o jjj t o ijy of Original tK K . filia l ( L i > P c_ -(> n 3M Environmental La b o r a t o r y E02-1039 Page 111 o f 225 r <act Copy o f Original U /y /^ A -- Data -^ .-1 * d O - tC L ib 3M Environmental La b o r a t o r y E02-1039 Page 112 o f 225 -v Dat& Exact Copy of Origina! initial 3M Environmental La b o r a to r y E02-1039 Page 113 o f 225 oata Exact Copy of Original Initial if! 3M Environmental La b o r a t o r y E02-1039 Page 114 o f 225 3M Environmental Labo r a to r y E02-1039 Page 115 o f 225 n_ Data Initial 3M Environmental La b o r a t o r y E02-1039 Page 116 o f 225 Exact Copy o f Original < ^ L J M l L -l . Initiaf oats 3M Environmental La b o r a to r y E02-1039 Page 117 o f 225 :i > f S Exact Copy o f Original 3M Environmental La b o r a to r y E02-1039 Page 118 o f 225 Exact Copy of Original - to /s d o ^ Initia! Date 3M Environmental La b o r a to r y E02-W39 Page 119 o f 225 Date -^LLU LU -- Exact Copy of Origina! inrtia! 02048-21-7 solvent standard iOppb 3M Environmental Lab davey 070799 30-0ct-2002 16:20:35 1: Daughters of 499ES- 3M E n v ir o n m e n t a l La b o r a t o r y E02-1039 Page 120 o f 225 Exact Copy of Origina1 c Imnitial . Dauu / 1/6*1-- . _______________ ^ ....... ' '.............................. ' i' ...-- 3M E n v i r o n m e n t a l La b o r a t o r y E02-1039 Page 121 o f 225 m Date i l / 1 / CS Exact Copy of Origina! - initiai 02048-21-7 solvent standard 10ppb 3M Environmental Lab davey 070799 30-0ct-2002 16:20:35 3M Environmental La b o r a to r y E02-1039 Page 122 o f 225 Exact Copy or O n :. - C A T Y \( LI A \ J . iS^-- Initial Orilf ` *--------------- " - r - . '-f-.'-r-r-........... s ' 3M Environmental La b o r a t o r y E02-1039 Page 123 o f 225 :' W of Original A*/- r - -------- 3M Environmental La b o r a t o r y E02-1039 Page 124 o f 225 Exact Copy of Original inIa! Dm 3M Environmental Laboratory E02-1039 A t t a c h m e n t D : P r e p S h e e t s a n d T r a c e a b il it y In f o r m a t io n Page 125 of 225 3M Environmental Laboratory E02-1039 SPE Columns Extraction Worksheet Prep Date: Analysts initials: 10/23/2002 OK Sample Number or description Blank milli-o Lamoire Serum Siqma Serum Golden West Serum Bioresource Serum Lamoire Plasma Golden West Plasma Innovative Resources Plasma Volume of sample Type of column filtered (ml) used 2000 ml Waters, 1q, 6ml 2000 ml W&ters, 1g, 6ml 2000 ml Waters, 1g, 6ml 2000 ml Waters, 1g, 6ml 2000 ml Waters, 1g, 6ml 2000 ml Waters, 1g, 6ml 2000 ml Waters, 1g, 6ml 2000 ml Waters, 1g, 6ml Method Revision: ETS-8-231.1 Study Number: E02-1039 Elution Amount and solvent and spike mix used volume Comments NA NA NA ZmlotMvOH A4313 2miofMOH A>6313 2mt of MiOH A4313 TN TN TCR*68B TN TCR-889 NA 2mlof MaOH TN V8313 TCR-890 NA NA NA NA A-6313 2mlofMaOH A-6313 2riilo<M*OH A-6313 Zmi o fMaOH M>313 TCR-887 TN TCR-685 TNTCR-684 TNrCR-683 ______________________J___ _ ------------------------------------ ------ \ f ~ \ ` h ^ V1 Extraction Steps (initial when/if dona): _x_condltion column with MeOH; _x_wash with milli-q water; _x_filter sample;; _X vacuum dry the column; --x_elute column with solvent; A C f^ C o u tfi -U J ' l<rT O f 2 ./ ' Additional comments: >/ 10ml of sera diluted with 40ml of milli-q and 200ml of ACN. Samples were shaken for 20 min at 300rpm. then centrifuged for 10 min @ 3500rpm/4C Sample was transferred Into 1750ml of mllll-q and filtered through conditioned SPE / " L U & A f* f ) a . a *2 " o f i n/ Co 7U S / ' - i - Y Exa c t Copy of Original Qi_ Jo/ 3 l / (Sl initial Data Page 126 of 225 3M Environmental Laboratory E02-1039 ^Npri'GLP SPE Columns Extraction Worksheet ^ ( ^ ||07 Prep DateAnalysts initials: * 10/11/2002 >'c. iio 'J H OK Ct<J Method Revision: E T S - 2 3 1 tj- J Study Number: E 02-1039 Sample Number description Volume of or sample Type of column filtered (ml) used Blank milli-a-1 Blank mllli-q-2 ._ rabbit serum blank rabbit serum curve-0.1ppb rabbit serum curve-0.25ppb rabbit serum curve-0.5ppb rabbit serum curve-0.75ppb rabbit serum curve-1 DDb rabbit serum curve-2.5ppb___________ _ rabbit serum curve-5ppb rabbit serum curve-1Oppb____________ rabbit serum curve-30ppb Lamoire serum blank-1 i ampin serum blank-2 Lampire serum MS-0.5ppb t ampire serum MS-5ppb_____ Siqma serum blank-1 Sinma serum blank-2 Rinma serum MS-0.5ppb Rinma serum MS-5DDb GoldenWest serum blank-1 Golden West serum blank-2 flolden West serum MS-0.5ppb 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 mi 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml Waters, 1g, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1g, 6ml Waters, 1q, 6ml Waters, 1g, 6ml Waters, 1g, 6ml Waters, 1g, 6ml Waters, 1g, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1g, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1g, 6ml Waters, 1q, 6ml 400 ml Waters. 1q, 6ml 400 ml Waters, 1g, 6ml Bioreseource research serum MS-5ppb 400 ml Waters, 1q, 6ml 400 ml Waters, 1g, 6ml elution Amount and spike solvent and mix used volume Comments NA 2 ml ofMiOH TNA-6310 NA 2ml ofMiOH TNA-6310 NA 2 ml of MiOH TN-A-6310 2 ml of MiOH 2ul of 02050-14 TNA-6310 2 ml of MiOH 5ul of 02050-14 TNA-6310 2mi ofMiOH 10ul of 02050-14 TNA-6310 2ml ofMiOH 15ul of 02050-14 TN-A-6310 2 ml of MiOH 20ul of 02050-14 TN-A-6310 2 ml of MiOH 50ul of 02050-14 TNA-6310 2 ml ofMiOH 100ul of 02050-14 TNA-6310 2ml ofMiOH 200ul of 02050-14 TN-A-6310 2ml of MiOH 600ul of 02050-14 TNA6310 NA 2 mi of MiOH TNA-6310 NA 2mlof MoOH TNA-6310 2ml of MiOH 10ul of 02050-14 TN-A-6310 2ml of MiOH 10Oul of 02050-14 TNA-6310 TCR-080 TCR-888 rcR-aee TCR-080 TCR-asa TCR-888 TCR-688 TCR-888 TCR-6B0 TCR-888 TCR-888 TCR-888 TCR-888 TCR-888 NA TNA-6310 NA 2ml of MiOH TNA-6310 lOuioiTso-iv 2ml of MiOH TNA-6310 o r 2miof MeOH 100ulo2osfci4 TN-A-6310 NA NA 10ul of 02050-14 TNA-6310 2 ml of MiOH TN-A-6310 2ml of MiOH TNA-6310 TCR-689 TCR-889 TCR-889 TCR-889 TCR-890 TCR-890 TCR-890 100ul of 02050-14 TN-A-6310 NA 2ml Of MiOH TNA-6310 NA 2 ml of MiOH TN-A-6310 2 ml of MiOH 1 0ui of 02050-14 TN-A-6310 TCR-890 TCR-887 * TCR-687 TCR-887 1 0 0 ul of 02050-14 TN-A-6310 TCR-887 Extraction Steps (initial when/if done): x condition column with MeOH; _x_wash with milli-q water; _xjlter sample;; _x""vacuum dry the column; _x_elute column with solvent, Exact Copy of Original O W L . l o l S \ I fSV _ Initial D ata .. . . A D O T ^ A - ? / cN c - - K / O " o ? M of ser^plasm^dTuted with 8ml of milli-q and 40ml of ACN. Samples were shaken for 20 min at 300rpm. then centrifuged for 10 min @ 3500rpm/4C Sample was transferred into 350ml of miM-g and filtered through eonAhmad SPE __ S W N ^ - wee. ^ 3 l:e' u ` Page 127 of 225 3M Environmental Laboratory E02-1039 ^flrffGLP SPE Columns Extraction Worksheet Prep Date: Analysts initials: lM ^ f ij| ,:7> 10/ 1 0 /20 0 2 OK t lQ - 3 CY-- Method Revision: SPE validation Study Num ber E 02-1039 Sample Number description Volume or or sample Type o f column filtered (ml) used Blank miili-q-1 Blank milli-q-2 Chinese plasma curve-0.1ppb Chinese plasma curve-0.25ppb Chinese plasma curve-0.5ppb Chinese plasma curve-0.75ppb Chinese plasma curve-1ppb Chinese plasma curve-2.5ppb Chinese plasma curve-5ppb Chinese plasma curve-10ppb Chinese plasma eurve-30ppb Chinese plasma blank-1 Chinese plasma blank-2 Chinese plasma MS-0.5ppb Chinese plasma MS-5ppb L a m o ire Plasma blank-1 Lamoire Plasma blank-2 Lampire plasma MS-0.5ppb Lamoire Dlasma MS-5opb Golden W est Plasma blank-1 Golden W est Plasma blank-2 Golden W est Plasma MS-0.5ppb Golden W est Plasma MS-5ppb Innovative Research Plasma blank-1 Innovative Research Plasma blank-2 Innovative Research Plasma MS-0.5ppb Innovative Research Plasma MS-5ppb 400 ml Waters, 1g, 6 m l 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml 400 ml Waters, 1g, 6 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml Waters, 1g, 6 ml 400 ml 400 ml Waters, 1g, 6 ml Waters, 1g, 6 ml Elution Amount and spike solvent and mix used volume Comments NA 2ml ofMeOH TN-A-6310 NA 2mi of MeOH TN-A-6310 2ml of MeOH 2ul o f 02050-14 TN-A-6310 2ml ofMeOH 5ul o f 02050-14 TN-A-6310 2ml ofMeOH 10ul o f 02050-14 TN-A-6310 2ml of MeOH 15ul o f 02050-14 TN-A-6310 2ml of MeOH 20ul o f 02050-14 TN-A-31Q 2ml of MeOH 50ul o f 02050-14 TN-A-6310 2ml of MeOH 10Oul o f 02050-14 TN-A-6310 2ml of MeOH 200ul of 02050-14 TN-A-6310 2ml of MeOH 600ul of 02050-14 TN-A-6310 NA 2ml of MeOH TN-A-6310 NA 2ml of MeOH TN-A-6310 2ml of MeOH 10ul of 02050-14 TN-A-6310 2mi of MeOH 10Oul of 02050-14 TN-A-6310 NA 2ml ofMeOH TN-A-6310 NA 2ml of MeOH TN-A-6310 1 0 ul 2ml of MeOH TN-A-6310 1 00ul NA 2ml of MeOH TN-A-6310 2 m l of MeOH TN-A-6310 NA 2mlof MeOH TN-A-6310 2mi of MeOH 10ul o f 02050-14 TN-A63tO 2ml of MeOH 100ul o f 02050-14 TN-A-6310 NA 2ml of MeOH TN-A-6310 NA 2mi of MeOH TN-A-6310 TCR-674 TCR-674 TCR-674 TCR-674 TCR-674 TCR-674 TCR-674 TCR-674 TCR-674 TCR-674 TCR-674 TCR-674 TCR-674 TCR-685 TCR-685 TCR-68S TCR-685 TCR-684 TCR-684 TCR-8S4 TCR-684 TCR-683 TCR-683 10ul of 02050-14 TN-A-6310 2ml of MeOH 100ul of 02050-14 TN-A-6310 TCR-683 TCR-683 Extraction Steps (initial when/if done): _x_condition column with MeOH; _x_wash with milli-q w a te r _x_filter sam ple;; Exa c t Copy o f Original x vacuum dry the column; _ x_elute column with solvent, Data A dditional comments: CVkiTT . F`-` S ( il c-ti /fc 2 IIV *' J 1.1l-v.-,: v t x j 'P 2ml of sera/plasma diluted with 8ml of milli-q and 40ml of ACN. Samples were shaken for 20 min at 300rpm, then centrifuged for 10 min @ 3500rpm/4C Sample was transferred into 350ml of milli-q and filtered through conditioned SPE fc-nJ TM-A Page 128 of 225 3M Environmental Laboratory E02-1039 3 M E n v ir o n m e n t a l L a b o r a t o r y Note to File Project or Study Number: E02-1039 Associated Study Number:_________ Page 129 of 225 J M tlN V IH V N M lzN IA L L A U K A I U K Y E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemical name: Lot/batch #: 3M# Expiration date: Human plasma from China Human plasma N087P27 Initials: Number/size of containers: Condition: OK 7-15 ml centrifuge tubes liquid, Biohazard Retain IO Archived/Substance Not Available;! TCR Substance # TCR# Received from: TCR-674 TCR-674 Northwest Bioanalytical Amount received (wt. or 7x-10ml vol): Date: 09/25/2002 Shipper: FedEx MSDS (y/n) Date of Retain J Y9 N ' P urity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachment(s) Frozen F19 Sera came in 7 separate tubes. Please mark which tube was used fo r sample, by number marked on the tube (4,5,6,7,8,11 or 12) Exact Copy o f Original n o c _ u / t / O T , Initial Data Page 130 of 225 3M Environmental Laboratory E02-1039 I_________________________________ ________ USE LOG Euman plasma from China uman plasma CR-674_______________________________________________________ Gross Wt./Vol. Before withdrawal Balance ID 1 Amnt. withdrawn (mass or voi) Balance ID 2 Gross wt/vol after withdrawal Balance ID 1 Purpose (enter standard number or reason for removal) "' 26ml * E02-1039, validation (all tubes were combined) 8 alance Balance ID ID 12 Initials Date na na OK 10/09/2002 Exa c t Copy o f Original CY\n( InitfaJ M/| ! o~x- Dato Page 131 of 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemicai name: Lot/batch #: 3M# Expiration date: Pooled Human Plasma in Sodium Citrate Pooled human plasma IR02-014 09/30/2007 Initials: Number/size of containers: Condition: Retain OK 10x 10ml plastic tubes good i 0 Archived/Substance Not Available Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachment(s) CofA Southfield, Ml Frozen, F24 TCR Substance # TCR # Received from: TCR-683 TCR-683 Innovative Research, Ml Amount received (w t or 10x 10ml vol): Date: 10/02/2002 Shipper: MSDS (y/n) Date of Retain .) Y N Exact Copy o f Original n /_ t_ /o 2 ~ Initlai Data Page 132 of 225 3M Environmental Laboratory E02-1039 ________________________________________ USE LOG 'ooled Human Plasma 'ooled human plasma CR-683 ____________________________________________ Gross Wt./Vol. Before withdrawal Balance ID 1 Amnt. withdrawn (mass or voi) Balance ID 2 - 8 ml Gross wt/vol after withdrawal Balance ID 1 - Puipose (enter standard number or reason for removal) E02-1039 validation Balance Balance ID ID 12 Initials Date OK 10/14/2002 Exact Copy o f Original .C u n e u /\/e > \- initial O a ts Page 133 of 225 3M Environmental Laboratory EO2-1039 | USE LOG pooled Human Plasma Pooled human plasma (rCR-683 ___________________________________________ Gross Wt./Vol. Amnt. Before withdrawn withdrawal (mass or voi) Balance ID 1 Balance ID 2 - 1 0 ml Gross wt/vol after withdrawal Balance ID 1 Purpose (enter standard number or reason for removal) E02-1029, validation Balance Balance ID ID 12 Initials Date OK 10/23/2002 Exa ct Copy o f Original Cmc Inittal / oO~ Date Page 134 o f 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemicaJ name: Lot/batch #: 3M# Expiration date: Sodium Citrate Pooled Human Plasma Pooled human plasma G01410002 10/03/2007 Initials: Number/size of containers: Condition: Retain OK 1x 100ml plastic bottle Frozen . O Archived/Substance Not Available ] Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachment(s) CofA Golden West Biologicals Frozen F19 TCR Substance # TCR-684 TCR # Received from: TCR-684 Golden West Biologicals, Inc. Amount received (wL or 100ml vol): Date: 10/03/2002 Shipper: UPS MSDS (y/n) Date of Retain 'Y N " Exact Copy of Original Cmc u / U g 7~ InitlaJ Data Page 135 of 225 3M Environmental Laboratory E02-1039 ________________________________________ USE LOG iodium Citrate Pooled Human Plasma 'ooled human plasma CR-684 ______________________________ Gross Wl./Vol. Amnt. Before withdrawn withdrawal (mass or voi) Balance ID 1 Balance ID 2 8mI Gross wt/vol after withdrawal Balance ID 1 - Purpose (enter standard number or reason for removal) E02-1039, validation Balance Balance Initials . ID ID 12 Date na na OK 10/10/2002 Exact Copy of Original CMC n / t/ o n -- Initial Date Page 136 of 225 3M Environmental Laboratory E02-1039 | _ ________________________________________USE LOG Sodium Citrata Pooled Human Plasma ooled human plasma CR-684 ________________________________________ Gross Wt./Vol. Amnt. Before withdrawn withdrawal (mass or vol) Balance ID 1 Balance ID 2 - 10ml Gross wt/vol after withdrawal Balance ID 1 - Purpose (enter standard number or reason for removal) E02-1039 Balance Balance Initials ID ID 12 Date NA NA OK 10/23/2002 - Exact Copy of Original- rry? ( Initial / o ')-- Data Page 137 of 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemical name: Lot/batch it: 3M # Expiration date: Pooled Human Plasma From Lamplre Pooled Human Plasma From La m p ir e 22-60824A 10/08/2007 Initials: Number/size of containers: Condition: Retain OK 1-2S0m l plastic bottle Frozen ' C) Archived/Substance Not Available-j Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachments) Disease Screen Lampire Frozen F19 TCR Substance # TCR# TCR-685 TCR-685 Received from: Lampire Amount received (wt. or 100ml vol): Date: 1 0 /1 0 /2 0 0 2 Shipper: UPS MSDS (y/n) Date of Retain u y* N : Exact Copy of Original initial Dato Page 138 of 225 3M Environmental Laboratory E02-1039 | ______________ U SE LO G Eooled Human Plasma From Lampire ooled Human Plasma From Lampire CR-685_______________________________________________________ Gross Wt./Vol. Amnt. Before withdrawal Balance 10 1 withdrawn (mass or voi) Balance ID 2 - 10ml Gross wt/vol after withdrawal Balance ID 1 - Purpose (enter standard number or reason for removal) E02-1039 Balance Balance ID ID 12 Initials Date na na OK 10/23/2002 Exa ct Copy of Original C in e Inittal n /i/a iOats Page 139 of 225 3M Environmental Laboratory E02-1039 ____________________________ _______________ U SE LOG 'ooled Human Plasma From Lampira 'ooled Human Plasma From Lampire CR-685 _____________________________________ Gross Wt./Vol. Amnt. Before withdrawn withdrawal (mass or voi) Balance ID 1 Balance ID 2 - 8ml Gross wt/vol after withdrawal Balance ID 1 - Purpose (enter standard number or reason for removal) E02-1039, validation Balance Balance ID ID 12 Initials Date na na OK 10/ 10/2002 Exact Copy o f Original C rvxc a /) / o j. Initiai Data Page 140 of 225 3M Environmental laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemical name: Lot/batch #: 3M# Expiration date: Pooled Human Serum From Bioresource Pooled Human Serum 020821 08/27/2007 Initials: Number/size of containers: Condition: Retain OK 500ml plastic bottle Frozen TCR Substance # TCR-687 TCR# TCR-887 Received from: Bioresource Amount received (wt or 500ml vol): Date: 10/11/2002 Shipper UPS MSDS (y/n) Date of Retain U Y N ! O Archived/Substance Not Available ] Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachment(s) Bioresource Frozen F19 Also assigned TN-A-6284 10/11/02 OK . Exact Copy of Original A nr Initial ftetB Page 141 of 225 3M Environmental Laboratory E02-1039 ___________________________________________ USE LOG >ooled Human Serum From Bioresource >ooled Human Serum CR-687 _________________________ _______________ Gross Wt./Vol. Amnt. Before withdrawn withdrawal (mass or vol) Balance ID 1 Balance ID 2 -' 10ml Gross wt/vol after withdrawal Balance ID 1 - Purpose (enter standard number r reason for removal) E02-1039 Balance Balance Initials ID ID 12 Date OK 10/23/2002 Exact Copy o f Original Initia) Date Page 142 of 225 3M Environmental Laboratory E02-1039 | _________________ U SE LO G pooled Human Serum From Bioresource Pooled Human Serum lTCR-687___________________________ ___________________________ Gross Wt./Vol. Amnt. Before withdrawn withdrawal (mass or vol) Balance ID 1 Balance ID 2 - 8mI Gross wt/vol after withdrawal Balance ID 1 - Purpose (enter standard number or reason for removal) E02-1039, validation Balance Balance Initials ID ID 12 Date OK 10/ 11/2002 Exact Copy of Original (> /1 / A i . initial oats Page 143 of 225 3M Environmental laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemical name: Lot/batch #: 3M # Expiration date: Initials: Number/size of containers: Condition: Retain Pooled Human serum from Lampire TCR Substance # TCR- 6 8 8 Pooled Human serum TCR# TCR- 6 8 8 X324B Received from: Lampire 08/29/2007 OK 250 m l Nalgene bottle Frozen Amount received (wt. or 250ml vol): Date: 10/ 11/2002 Shipper: UPS MSDS (y/n) Date of Retain UY N I 0 Archived/Substance Not Available; Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachment(s) Lampire Frozen F19 Also assinged TN-A-6286 10/11/02 ok " Exa c t Copy o f Original Page 144 of 225 3M Environmental Laboratory E02-1039 I ___________________ U SE LOG fooled Human serum from Lampire ooled Human serum CR-688__________________ ____________________________________ Gross Wt./Vol. Before withdrawal Balance ID 1 Amnt withdrawn (mass or voi) Balance ID 2 Gross wt/vol after withdrawal Balance ID 1 Purpose (enter standard number or reason for removal) 8ml - E02-1039, validation Balance Balance ID ID 12 Initials Date OK 10/11/2002 Exact Copy of Original -W C U / , / - , Initial Dais Page 145 of 225 3M Environmental Laboratory EQ2-1039 _________________________________ ________ U SE LOG 'ooled Human serum from Lamplre 'ooled Human serum C R - 6 8 8 _________________________________________________________________________________ Gross Wt./Vol. Amnt. Before withdrawn withdrawal (mass or voi) Balance ID 1 Balance ID 2 - 10ml Gross wt/vol after withdrawal Balance ID 1 - Purpose (enter standard number or reason for removal) E02-1039 Balance Balance Initials ID ID 12 Date OK 10/23/2002 Exact Copy of Original < 1 1/'I ! ro - Initial patg Page 146 of 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemical name: Lot/batch #: 3M# Expiration date: Male Human Serum from Sigma Male Human serum 022K0965 07/24/2007 Initials: Number/size of containers: Condition: Retain OK 1100m l plastic bottle frozen TCR Substance 1t TCR # Received from: TCR-689 TCR-689 Sigma Amount received (wt. or 100ml vol): Date: 10/ 11/2002 Shipper UPS MSDS (y/n) Date of Retain >y m N i (..) Archived/Substance Not Available Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachment(s) Sigma Frozen F19 Also assigned TN-A-625010/11/02 OK ' Exact Cu^y -M /y >o Data Page 147 of 225 3M Environmental Laboratory E02-1039 1 Male Human Serum from Sigma Male Human serum lrCR-689 USE LOG Gross WtWol. Before withdrawal Balance ID 1 Amnt withdrawn (mass or voi) Balance ID 2 - 8 m! Gross wt/vol after withdrawal Balance ID 1 - Purpose (enter standard number or reason for removal) E02-1039, validation Balance Balance ID ID 12 Initials Date OK 10/11/2002 Exact Copy of Original T.inC. ( i / i / c J i -- Initial O ats Page 148 of 225 3M Environmental Laboratory E02-1039 1 Male Human Serum from Sigma Male Human serum (rCR-689 USE LOG Gross Wt./Vol. Amnt. Before withdrawn withdrawal (mass or voi) Balance ID 1 Balance ID 2 - 10ml Gross wt/vol after withdrawal Balance ID 1 - Puipose (enter standard number or reason for removal) E02-1039 -------------------- j -- ----------------------------------------- Balance Balance ID ID 12 Initials Date OK 10/23/2002 Exact Copy o f Original V rK u / i /o-u. int&Bi Oafs Page 149 of 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemical name: Lot/batch #: 3M# Expiration date: Pooled Human Serum from Ciolen West Pooled Human Serum G01406042 07/25/2007 Initials: Number/size of containers: Condition: Retain OK 1-500ml blastic bottle frozen ITCR Substance # 1 |TCR # TCR-690 TCR-690 Received from: Golden west I lAmount received (wt. or 500ml |vol): -- (Date: jShipper: 10/ 1 1/2002 UPS |MSDS (y/n) |Date of Retain f..i y m N Ci Archived/Substance Not Available Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachment(s) Golden West Frozen F19 Also assigned TN-A-6251 10/11/02 ok Exa ct Copy o f Original - m r Initial Data Page 150 of 225 3M Environmental Laboratory E02-1039 I ______________________ U SE LOG Eooled Human Serum from Golen West ooled Human Serum CR-690_______________________________________________________ Gross WtJVol. Amnt. Gross wt/vol Purpose (enter standard number Before withdrawn after or reason for removal) withdrawal (mass or voi) withdrawal Balance ID 1 Balance ID 2 Balance ID 1 - 10ml - E02-1039 Balance Balance ID ID 12 Initials Date OK 10/23/2002 Exact Copy o f Originai Initial nata Page 151 of 225 3M Environmental Laboratory E02-1039 ____________________________ ______________ USE LOG 'ooled Human Serum from Golen West 'ooled Human Serum CR-690 ____________________________________________ Gross Wt/Vol. Amnt. Before withdrawn withdrawal (mass or voi) Balance ID 1 Balance ID 2 - 8ml Gross wt/vol after withdrawal Balance ID 1 - Purpose (enter standard number or reason for removal) E02-1039, validation Balance Balance ID ID 12 Initials Date OK 10/11/2002 Exa c t Copy o f Original <zyr^( W tM u Z l/^ rO ats Page 152 of 225 Standard curve tracking sheet Prep date: A n a lyst: Type o f the curve: 10/7/2002 OK ' C7C& J Solvent curve fo r SPE sera validation Target analytes: C6, C7, C8, C9, C10, C11. C12, THPFOS, THPFDS, PFOS Standard m ix used for preparing the curve: 02002-63 Study Number: Sera SPE validation Final Solvent&TN-A #: MeQH/6308 {Final Volume of each point: | 25| Curve point number 02048-21-1 02048-21-2 02048-21-3 > 02048-21-4 02048-21-5 02048-21-6 02048-21-7 02048-21-8 02048-21-9 02048-21-10 Amount o f mix used (ul) 5 25 50 125 250 375 500 750 1000 1500 C6 0.100 0.500 1.00 2.50 5.00 7.50 10.00 15.00 20.01 30.01 C7 0.102 0.511 1.02 2.55 5.11 7.66 10.21 15.32 20.43 30.64 C8 0.103 0.517 1.03 2.58 5.17 7.75 10.33 15.50 20.66 31.00 A nalyte con centration in every p o in t (ppb) C9 C10 C11 C12 0.101 0.507 1.01 0.101 0.505 1.01 0.103 0.515 1.03 0.104 0.518 1.04 2.54 2.53 2.58 2.59 5.07 5.05 5.15 5.18 7.61 7.58 7.73 7.77 10.14 10.10 10.31 10.36 15.22 20.29 30.43 15.15 20.20 30.30 15.46 20.61 30.92 15.54 20.72 31.08 THPFOS 0.102 0.512 1.02 2.56 5.12 7.68 10.24 15.37 20.49 30.73 THPFDS 0.100 0.501 1.00 2.51 5.01 7.52 10.02 15.03 20.04 30.06 PFOS 0.101 0.503 1.01 2.52 5.03 7.55 10.06 15.09 20.12 30.18 Signaturee:: i h u / i i * Verified by: C i r t ^ L C W ' L t O l O / ^ t / d X _ 3M E n v ir o n m e n ta l La b o r a t o r y E02-1039 Page 153 o f 225 Exact Copy of Original u r t .ic/3>/(>?- Initial Date 3M Environmental Laboratory E02-1039 Standard m ix tracking shaet Sara Super m ix fo r SPE10X MDV Prep date: Analyst: Type of the mix: 10/7/2002 OK sera super mix Exp. 11/13/02 O tC Ic - 7 x j Target analytes: C8, C 7.C 8. C9.C10, C11.C12, THPFOS, THPFDS, PFOS Standard number for the mix: 02002-63 Study Number: Final Solvant&TN-A #: I Final Volume of the mix: A nalyte C6 C7 C8 C9 CIO C11 C12 THPFOS THPFDS PFOS concentration of amount of Analyte standard analyte in standard standard used number mg/ml (ppm) (ul) 02040-44 1042 12 02040-58 1344 9.5 02040-45 1230 10.5 02040-48 . 1268 10 02022-90 1010 12.5 02040-65 1356 9.5 02040-55 1126 11.5 02040-42 1348 9.5 02040-49 02022-56 1002 1048 12.5 12 concentration o f the analyte In the mix ng/ml (ppb) 500 511 517 507 505 515 518 512 501 503 Sera SPE validation MeOH/6308 HI Signature: ^ L tiro Verified by: to /3 / 3-- Exact Copy o f Original Page 154 of 225 3M Environmental Laboratory . E02-1039 Date: tp> IvQ- O Q j A na lyst:___________^ SINGLE COMPONENT PREPARATION LOG Book No. 02 050 Page No. 14 Description: Joo y p h H S * ftp R ^ Stock Number o 3 l o o S Weight o r Volume Used: 5 uA. Concentration or Purity: to Corrected Weight: h )/\ Balance ID: K )f\ Other Correction Factors: Solvent and TN-A Number H eD V /M-A - $ '3 / O Final Volume: Storage Location: u / Final Concentration: Expiration Date: A /3 / ________ Exact Copy of Original / 4 V K <C ^ i / (fL- Initial Date Page 155 of 225 3M Environmental Laboratory E02-1039 Date: Analyst: l * /0 D 2 j ___Q iL Description: SINGLE COMPONENT PREPARATION LOG oi A-t IX " Book No. 02 050 Page No. 17 Stock Number (O J?C C -> ^ Weight or Volume Used: Concentration or Purity: Corrected Weight: m. Balance ID: UA Other Correction Factors: UA Solvent and TN-A Number. A C ( / /7 K ,^ ^ 3 i C i Final V otum : * /U Z.Q Final C oncentration: < f r7 G Storage Location: Ru d Expiration Date: // /S -O -3 / Exact Copy of Original f 'm c tc fa J o l -- Initiai Data Page 156 of 225 3M Environmental Lab o r a to r y E02-1039 Dale: Analyst: jC IC -C 'C j oo Description: SINGLE COMPONENT PREPARATION LOG Book No. 02 050 Page No. 16 IX Stock Number:_______iO ,9 r C *2 . - f c ^ Weight or Volume Used: JL@ Balance ID: AJ Concentration or Purity: Corrected Weight: A -i A t5C c(?piO Other Correction Factors: Solvent and TN-A Number M o / W - ft e ? > ic> Final V olum e: LK.'l Q Storage Location: (Ru cP i f ' Final C oncentration: <fP>.,,J3.c-. p p Expiration Date:__ n - r i- o Q i Exact Copy of Original -CYY. j o / 3 i/ o_: Initial Oats Page 157 of 225 multi-level preparation log f o /h ,4 3M Environmental Laboratory E02-1039i Page 158 o f 225 JS Exact Copy o f Original C M .... 3M Environmental Laboratory E02-1039 MULTI-COVPONENT PREPARATION LOG Book No. 02 002 P ag e No. 63 Description: X f'-Qj Date Prepared: IQ~ Ic'V -'ai. Prepared By:__ oK Storage Location^ Expiration Date:_ JjL . IIOUj r Solvent::: / "p C W 3M T race #: TN-A- Q O ? Standard ID# 02 002-63 Description Component Standard Conc. or ID# Purity Expiration Date Weight or Volume Corrected Weight Balance ID Final Concentration T fb 0 * A C *5 6 /T iD Ac 0 ,0 A frT > O 2o4o- Vt lO ^ a . / S '^ b ppv*-* 2 aJ o 2 o < -to ex O v a tto - 2 - 2 - o3> f PUM llT b o i- / o 3 \0 G y X W A (J A 0 3 c /f0 " tC ; '0 3 . G Z & ' i2 e y ff> V A |o K ' f f 'M \-2 o 3 l jJ L fi/J . S c>> S -I) S \T Sol SO S A u. <c c O i ;ei C f A r f 7> CS" cam A c i 2 j c. .` - S ' ^ n2 J M -# si *5 \Y iH T r o S s Th 5 0 @ o tf* -* 0 ^ 0 7 c<_ } V O % 99 nn ii g j J (M A U l s 0^ o fc J K -'s C r f^ Final Volume /[LC^ `b l 3 j SO 1 1& j A . V L 50 0 $5 Approximate Concentration of the mix s o o j f Reviewed by: l ll/L l Data ' Page 159 of 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemicat name: Lot/batch #: 3M# Expiration date: TDHA Trldecafluoroheptanoic Acid PU/07219EU NA 01/01/2005 initials: Number/size of containers: Condition: Retain SRP 1/ 30 m L plastic container clear crystals 0.3994 a TCR Substance # TCR-99131-025 TCR# Received from: TCR-267 Aldrich Amount received (wt or 25 g, 37.1357 g gross wt. vol): Date: 12/21/1999 Shipped Courier MSDS (y/n) Date of Retain YO N 02/15/2000 i-O Archived/Substance Not Available j Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments 99.5% LAC 4/24/01 MSOS KJD 3/1/00 NA F19, Frozen C5F13COOH Standard has been moved to Freezer 19 in room 347 KJD 06/07/00 Standard was stored at room temperature prior to 06/07/00 LAC 12/22/00 Attachment(s) Shipping comments: Corrosive, No 3M ID# - must ship ground small quantity exception (30 mL/g or less per vial). LAS 10/02/02 A* A* tcr99131-25msds.pdf tcr99131-25cofa.pdf B e a d Copy o f Original initial Data Page 160 of 225 JAN 26 2000 0 9 :4 8 FR ALDRICH GC JM CERTIFICATE OF ANALYSIS E02-1039 14/04 7Z /.-7 V 3 I-M 3H ENVIRONMENTAL SCOTT POST S t 77S 3890 PO NWR: PRODUCT NUMBERS 34204-1 LOT NUMBER: 07219EU PRODUCT NAME TRIDECAFLUCROHEPTANOIC ACID 99X FORMULA; C7HF1302 FORMULA WEIGHT: 3 6 4 .0 * APPEARANCE INFRARED SPECTRUM FLUORINE NMR GAS LIQUID CHROMATOGRAPHY QUALITY CONTROL ACCEPTANCE DATE WHITE CRYSTALLINE SOLID CONFORMS TO STRUCTURE AND STANDARD AS ILLUSTRATED ON PAGE 79BB OF EDITION 2 , VOLUME 1 OF "THE ALDRICH LIBRARY OF FT-IR SPECTRA". CONFORMS TO STRUCTURE. 99.3 X JUNE 1999 Exact Copy o f Original L - L o / f r l a ~> H tW Data ALDRICH CHEMICAL COMPANY DAVID SUESSEL JANUARY 2 6 I 2000 churrusts helping chemists in research & Industry , a ld r lc h c h e m ic a l c o . A U m C M w vm ra d M tiU p itd ic a o m m io tia iA m i vic e n ta lm d In nd ehar AHndi ptioUotUuns. Pwdwnr m et dam m i* ft H--tUtyiJthiin&ixtleritipimaMttima.iltrnimiitooikNai t r p utta; tip Mr M ia u l (m m end n o u s nrfufc, K Q . B w 333 , A U tm tu tW , W kM M M O ttf USA > (4 W Z F H M Q ` F A 3 tt* U ) Z f* 4 m M H n.ii ** TOTAL PAGE.04 ** Page 161 of 225 3M Environmental Laboratory E02-1039 TDHA rridecafluoroheptanoic Add rCR-99131-02S USE LOG _______________ Gross Wt-A/ol. Amnt. Gross wt/vol Before withdrawn after Purpose (enter standard number or reason for removal) withdrawal (mass or voi) withdrawal Balance ID 1 Balance ID 2 Balance ID 1 35.9819g 0.0865g 35.8954g standard 02040-58 Balance Balance Initials ID ID 12 Date 914 RWW 08/29/2002 B a c t Copy o f Original t o / / f 3-- u h otta Page 162 of 225 3M Environmental Laboratory E02-1Q39 Dale: Analyst SINGLE COMPONENT PREPARATION LOG ___________ Book No. 02 040 Page No.___ 58 Description: / Z 1/ e / s S l / J w a r * } IL O -L J r J f T f i X - A - i Stock Number____ 9 ^ / / / - X S ' Weight orVolume Used:_____ As 9A Concentration or Purity: 99. S?. Corrected Weight Balance ID:_____ * -/ Other Collection Factors: AA Solvent and TN-A Number ' A l s o / / - / A /" ~ 2.V *7 Final Volume: >,______J */HFinalConcenlraBon: ) Storage Location: f/t> 0 f tA L Expiration Date: Exact Copy of Original C Z Q L ^ tiZ ilc n - M tU O ats Reviewed by: ( V k A 'I- W n /o n Signature, ip /p j/o X Date Page 163 of 225 3M Environmental Laboratory E02-1039 Data:_Analyst: F SINGLE COMPONENT PREPARATION LOG ________ __________ Book No. 02 040 Page No. 58 Description: d M if f T J J X -A -') \; Stock Number Cij Weight or Volume Used: Balance ID: ^ / * / Concentrationor Purity: 9 9 .5 ? * OtherCorrection Factors: a SY S ' & \! CorrectedWeight ...... , i Solvent and TN-A Number ' 6 2 -V * / Final Volume: : r/? _ / Final Concentration: 3/ W f i n , Storage Location: F /e f if A f s * * / ) - Expiration Date: -J * Exact Copy of Original Cmc waai B r Reviewed by: ( V k A S lfJ rtA lO fl. Signature, Ip /p i/o l Date Page 163 of 225 3M Environmental Laboratory E02-1039 3M S P E C IA L T Y M A T E R IA L S M A N U FA C T U R IN G D IV ISIO N A N A LY TIC A L L A B O R A T O R Y To: Lisa Stevenson - (8-5568) - ET&SS - 2-3E-09 Request # GID:71638 From: Tom Kestner - (3-5633) - SMMD Analytical Lab - 236-2B-11 Subject: Characterization of TCR-99131-025 by 'H-NMR and l9F-NMR Spectroscopy Date: October 30, 2002 SAMPLE DESCRIPTION: TCR-99131-025, lot PU/07219EU from the Telomer project Nominal product = CF3(CF2),,-C02H, where average n 5 (white solid). Sam ple Spectra it' s Experiment Descriptions TCR-99131-025 TCR-99131-025 TCR-99131-025 H 7 1 6 3 8 .G ID .4 0 1 F 71 6 3 8 .G ID .4 0 1 F 7 1 6 3 8 .G ID .C O S Y .5 0 2 400 M H z ID `H -N M R in a C FC lj/acetone-dj solvent m ixture + p -H F X cross integration/intemal std. 376 M H z ID 1VF-N M R in a C F C V acetone-dj solvent m ixture + p-H F X cross integration/intem al std. 470 M H z 2D '"F-N M R C O SY (correlated spectroscopy) experim ent in a CFCly'acetone-d* solvent mixture. OBJECTIVE: This sample was subjected to a combination of 'H-NMR and l9F-NMR spectral analyses to determine the purity of the nominal product Special emphasis was also placed on attempting to identify and quantify any impurity components. EXPERIMENTAL: A portion of the sample was accurately weighed, spiked with a known amount of l,4-bis(trifluoro- methyl)benzene (p-HFX), and then totally dissolved in a CFCb/deuterated acetone (acetone-de) solvent mixture for subsequent analysis by NMR. Initial one-dimensional (ID) 400 MHz 'H-NMR and 376 MHz l9F-NMR spectra were acquired at room temperature using a Varian UNITYplus 400 FT-NMR spectrometer. A two-dimensional (2D) ,9F-NMR correlated spectroscopy (COSY) experiment was also acquired using a Varian UNITY INOVA 500 FT-NMR spectrometer to facilitate assignment of 19F signals p-HFX to bea s s o c i a t e d w i t h v a r i o u s i m p u r i t y c o m p o n e n t s . T h i s s a m p l e p r e p a r a t i o n m e t h o d p e r m i t t e d t h e u s e d a s e ith e r 1 ) a 'H / 'T - N M R in te rn a l sta n d a rd to a llo w th e c a lc u la tio n o f th e a b so lu te w e ig h t p e rc e n t *H/19F-NMR thec o n c e n t r a t i o n s o f s p e c i f i c c o m p o n e n t s , o r 2 ) a c r o ss in te g ra tio n sta n d a rd to p e r m it cro ss correlation of the relative *H and l9F signal intensities for evaluation of the overall sample composition. ' RESULTS: The 'H-NMR and l0F-NMR spectral data indicated this sample was a relatively high purity form o f the nominal product, CFj(CF2),,-C02H, where the average value of n = 5.05. Small amounts of a few impurity components, including probable isomers, were also assigned. A `H/I9F-NMR cross integration analysis technique was then used to calculate the relative weight percent concentrations of the identified components. The qualitative and quantitative compositional results that were derived from the single trial 'H/|ijF-NMR cross integration analysis are summarized below in TABLE-1. The relative weight percent concentrations shown in TABLE-1 should be very close to their respective absolute weight percent values assuming no water was present in the sample. Trace amounts of numerous other unassigned components were also detected in the NMR spectra, but additional work would be needed in an effort to identify or quantify some of these other components. Exact Copy of Original Page i of2 ijr t- , infflal Data ' Page 164 of 225 3M Environmental Laboratory E02-1039 O ctober 30, 2002 3M S M M D A nalytical Lab R equest if G ID :7 1 6 3 8 . T C R -99131-025, Lot PU /07219EU : T elo m er P roject Copies of the NMR spectra are attached with the paper co p y o f this report for your reference. If you have any questions about these results, please let me know. Tom Kestner c: Mark Ellcfson Rick Payfcr Ron Purcell William Rcagen . File Reference: ls71638.GlD.TCR-'W13i-023_Lot PU/07219EU_Telomer Project.DOC/101 T A B LE -1 Sample: TCR-99131-025, Lot PU/07219EU from the Telomer project. Overall Compositional Results by 'H/19F-NMR Cross Integration Analysis Component Structures 1 `H/,yF-NMR Relative Weight% Concentrations (single trial analysis) CF3(CF2)n-C02H (where average n = 5.05) Probable internal branched isomers CF3(CF2)x-CF(CF3)-(CF2)y-C02H . x*0 x+y = 3.05( w h e r e and assu m e fo r calcu latio n p u rp o se s) Probable terminal isopropyl branched isomers (CF3)2-CF-(CF2),,-C02H (assume n=3.05 for calculation purposes) A probable ether/acid as possible 0-rCF2CF2-C02Hl2 F F0 98.24% 0.87% 0.52% 0.13% 0.11% ' V ^ 0H P ^ l f'F Probable F F CnH2n+2 saturated aliphatic hydrocarbons and functional aliphatic components, CnH2n+rX, 0.11% where -X can be possible ether and ester functional groups. Possible CF3(CF2)n-C02CH3 (where average n = 5.05) 0.021% 1. T race am ounts o f n u m e ro u s other unassigned com ponents w ere also detected in the l9F-N M R spectrum . Exact Copy o f Original Wai Data Page 2 o f 2 Page 165 of 225 3M Environmental Laboratory E02-1039 Certificate o f Analysis Nominal Product; CF3(CF2),,-C02H, where averagen5 Tridecafluoroheptanoic acid Prodnct Code: TCR-99131-025, Lot PU/07219EU October 30,2002 The sample of TCR-99131-025, Lot PU/07219EU was analyzed using a combination of I9F-NMR and 'H-NMR spectral analysis techniques. The overall qualitative and quantitative compositional results that were derived from these combined analyses are summarized below in TABLE-1. TABLE-1 Sample: TCR-99131-025, Lot PU/07219EU Overall Compositional Results by Combined '^F/1H-NMR Spectral Analyses Component Structures1 lH/`yF-NMR Relative Weight% Concentrations (single trial analysis) CF3(CF2)n-C02H (where averagen = 5.05) Probable internal branched isomers CF3(CF2)x-CF(CF3)-(CF2V C 0 2H (w h e re x?rf) a n d a s s u m e x + y = 3 .0 5 fo r c a lc u la tio n p u r p o s e s ) Probable terminal isopropyl branched isomers (CF3)2-CF-(CF2)n-C02H (assume n=3.05 for calculation purposes) A probable ether/acid as possible O-rCF2CF2-C02Hl2 F F9 <98.2% Purity 0.87% 0.52% 0.13% 0.11% F" i Probable FF CnH2n+2saturated aliphatic hydrocarbons and functional aliphatic components, C,,H2n+i-X, 0.11% where -X can be possible ether and ester functional groups. Possible CF3(CF2),,-C02CH3 0.021% (where average n = 5.05) 1. T race am ounts o f n u m e ro u s other unassigned com ponents were also detected in the N M R spectra. Tom Kestner Exa c t Copy o f Original Page 1 o f 1 File Reference: CofA_TCR-99131-025_Lot PU-07219EU.doc Page 166 of 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemical name: Lot/batch #: 3M# Expiration date: Pentadecafluorooctanoic acid Pentadecafluorooctanoic acid 210002 Initials: Number/size of containers: Condition: Retain OK 1-10 ml amber glass vial jwhite crystals |TCR Substance # TCR-617 jTC R i TCR-617 |Received from: Oakwood Products I Amount received (wt. or 5g (voi): {Date: 07/19/2002" jShippen Courier |MSDS (y/n) Date of Retain yO n I O Archived/Substance Not Available" Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments 99.51%, updated 10/30/02 OK, NMR analysis MSDS Certificate o f Analysis LAS 07/25/02 Oakwood products Room Temp, TCR-C01 C8HF1502 Corrosive Solid LAS 07/19/02 Attachment(s) Shipping comments: No 3M ID# - must ship ground small quantity exception o r less per vial). LAS 10/02/02 \ TCR-617.pdf (30 mL/g Exact Copy of Original O H L - Lo / Z ) / f x > . Initial Oats Page 167 of 225 :*7V***? ' \l-^ l N V m Q N M E N T A L j LABOFfAJlQY k O akw ood Products/ Inc. 1741 Old Dunbar Road West Colombi*, S C 29172 Phone Ilit) 739-3800 Fax (803) 739-6957 CERTIFICATE OF ANALYSIS D ate: 15-JuI-02 M a te ria l: P e n ta d e c a flu o ro o c ta n o ic a rid C a t.N o .: 13 2 9 C a s N o .: [3 3 5 -6 7-1] t o t N o .: 2 10 0 0 2 Assay: 9 7 + % b y N a O H titra tio n A p p earan ce: W K ite o lid Melting Point: 59-62C -".r.. '* .*..' V * ... Exa ct Copy o f Original .'?S -x < A i Q L - (C /3J L - ' j Initial Data i W M \& i umei Yan QC Manager Page 168 of 225 3M Environmental Laboratory E02-1039 __________________________________________ USE LOG `entadecafluorooctanoic acid 'entadecafluorooctanoic acid C R - 6 1 7 _____________________________ _______________ _ Gross Wt./Vol. Before withdrawal Balance ID 1 Amnt. withdrawn (mass or voi) Balance ID 2 15.5486g 0.0617g Gross wt/vol after Purpose (enter standard number or reason for removal) withdrawal Balance ID 1 15.4869g standard 02040-45 Balance Balance ID ID 12 Initials Date 914 RWW 08/15/2002 Exact Copy o f Original Initial Oats Page 169 of 225 3M Environmental laboratory E02-1039 Date: ih A , A n aly st: / j J y / SINGLE COMPONENT PREPARATION LOG Book No. 02 040 Page No. 45 Description: A s a / j J s r j . J /ls s a c / - > *>>/ e / b / J Stock Number ! C A - U 7 Weight or Volume Used: 22- ) Concentration or Purity:________ n A Corrected Weight: Balance ID:______f <-f Other Correction Factors: jjS L Solvent and TN-A i Number: / - / / O / ? / T A /> /) . `/ S Final Volume: 5~a ~ i Storage Location: $ \ J f- / f, ,, r ~~ $./> _ Final Concentration: Expiration Date: J. f t S '/ J Exact Copy o f Original ~ rm . (o l3ilO L, Initial Dais Reviewed Signaturo, Dalo Page 170 of 225 3M Environmental Laboratory E02-1039 3M S P E C IA L T Y M A T E R IA L S M A N U FA C T U R IN G D IV ISIO N A N A LY TIC A L L A B O R A T O R Y To: Lisa Stevenson - (8-5568) - ET&SS - 2-3E-09 Request # GID:71638 From: Tom Kestner - (3-5633) - SMMD Analytical Lab - 236-2B-11 Subject: Characterization of TCR-6I7 by 'H-NMR, l9F-NMR, and LC/MS Analyses Date: October 28, 2002: Updated Report - LC/MS Analysis for Impurities SAMPLE DESCRIPTION: TCR-617, lot 210002 from the Telomer project. Nominal product = CFjfCFzln-COzH, where average n 6 (white powder). Sample Spectra it's Experiment Descriptions TCR-617 TCR-617 H71638.GID.501 500 MHz 'H-NMR in acetone-d solvent + p-HFX cross intepration/interaal std. F71638.GID.501 470 MHz r>F-NMR in acetone-d solvent + p-HFX cross intesration/intemal std. UPDATE: After the initial NMR compositional analysis report was issued to you on 10-24-02, Joel Miller performed a qualitative LC/MS analysis to assist in the assignment of some of the impurity components in the sample of TCR-617. This updated reported summarizes the new information regarding the identities and concentrations for some of the impurity components. You will notice that the tentatively assigned olefin structure from the original report is now replaced with a probable (CFuOj-COzH ether acid. OBJECTIVE: This sample was subjected to a combination of 'H-NMR and l9F-NMR spectral analyses to determine the purity of the nominal product. Special emphasis was also placed on attempting to identify and quantify any impurity components. The sample was also given to Joel Miller for a qualitative LC/MS analysis to assist in the assignment of impurity components. Joel asked that I incorporate the qualitative information from his LC/MS analysis in this updated report. FT-NMR EXPERIMENTAL: A portion of the sample was accurately weighed, spiked with a known amount of l,4-bis(trifluoro- m eth yl)benzen e (p -H F X ), and then totally d issolved in deuterated acetone (acetone-do) for su bsequent analysis by NMR. A 500 MHz 'H-NMR spectrum and a 470 MHz l9F-NMR spectrum were acquired at room temperature using a Varian UNITY INOVA 500 FT-NMR spectrometer. This sample preparation method permitted the p-HFX to be used as either 1) a lH/l,F-NMR internal standard to allow the calculation of the absolute weight percent concentrations of specific components, or 2) a 'h/19F-NMR cross integration standard to permit the cross correlation of the relative 'H and l9F signal intensities for evaluation of the overall sample composition. RESULTS: The combined 'H-NMR, l9F-NMR, and LC/MS analyses indicated this sample was a high purity form of the nominal product, C F j(CF{),,-C02K, where the average value of n = 6.02. Small amounts o f a few impurity components, including probable isomers, were also assigned. A 'H/I9F-NMR cross integration analysis technique was then used to calculate the relative weight percent concentrations of the identified components. The qualitative and quantitative compositional results that were derived from the combined single trial 'H/I9F-NMR cross integration analysis, and the qualitative LC/MS analysis, are summarized below in TABLE-1. The relative weight percent concentrations shown in TABLE-1 should be very close to their respective absolute weight percent values assuming no water was present in the sample. ' Page l o f 2 Exact Copy o f Original Page 171 of 225 3M Environmental Laboratory E02-1039 O ctober 28, 2002 3 M S M M D A n a ly tic a l L ab R e q u e st It G ID :7 1 6 3 8 U pdated R eport - LC/M S A nalysis for Im purities in T C R -617, L ot 210002 RESULTS (cont.): Trace amounts of a few other unassigned components were also detected in the NMR spectra, but additional work would be needed in an effort to identify or quantify these other components. Copies of the NMR spectra are attached with the paper copy of this report for your reference. If you have any questions about these updated results, please let me know. Tom Kestner c: Joel Miller Rick Payfer Ron Purcell William Reagen File Reference: U7l638.GID.Updited Reaultj_7CR-6l7_Lot 210002_Telomer ProjecLDOC/101 TABLE-1 Sample: TCR-617, Lot 210002 from the Telomer project. Overall Compositional Results by *H/19F-NMR Cross Integration and LC/MS Analyses Component Structures 1 NMR Relative Welght% Concentrations (single trial measurement) CF3(CF2),,-C02H where average n = 6.02 by 19F-NMR. LC/MS showed n=6 (maior), n=5, n=4 (minors). Probable (CF3)2-CF-(CF2)n-C02H assume n=4 for calculation purposes Probable (C6Fi3 0 )-C0 2H acyclic ether acid as possible CF3CF2-0-CF(CF3)-CF2CF2-C02H Possible CF3(CF2)x-CF(CF3)-(CF2)y-C02H w h ere x*0, y*0 and assume x+y = 4 for calculation purposes Possible (CF3)3-C-(CF2)n-C02H a s s u m e n--3 for ca lcu la tio n pu rp oses Possible CnH2n+2saturated aliphatic hydrocarbons 1. T race am ounts o f o th er unassigned com ponents w ere also detected in the N M R spectra. 99.52% 0.39% 0.057% 0.019% 0.013% 0.0079% Page2 of2 Exact Copy o f Original _ ic R i(r/u - Inffial Date Page 172 of 225 3M Environmental Laboratory E02-1039 Certificate o f Analysis Nominal Product: CF3(CF2),,-C02H, where average n 6 Pentadecafluorooctanoic acid Product Code: TCR-617, Lot 210002 October 28,2002 Tom Kestner and Joel Miller The sample of TCR-617, lot 210002 was analyzed using a combination of 19F-NMR, 'H-NMR, and LC/MS analysis techniques. The overall qualitative and quantitative compositional results that were derived from these combined analyses are summarized below in TABLE-1. TABLE-1 Sample: TCR-617, Lot 210002 Quantitative and Qualitative Compositional Results by Combined ^F/1H-NMR and LC/MS Analyses Component Structures 1 1H/li'F-NMR Relative Weight% Concentrations (single trial analysis) CF3(CF2)n-CC>2H where average n = 6.02 by 19F-NMR. LC/MS showed n=6 (major), n=5, n=4 (minors). Probable (CF3)rCF-(CF2),,-C02H assume n=4 for calculation purposes Probable (C6F]30)-C02H acyclic ether acid as possible CF3CF2-0-CF(CF3)-CF2CF2-C02H Possible CF3(CF2)x-CF(CF3)-(CF2VC02H where x*0, y*0 and assume x+y = 4 for calculation purposes Possible (CF3)3-C-(CF2),,-C02H a ss u m e n --3 fo r c a lc u la tio n p u rp oses Possible C,,H2n+2 saturated aliphatic hydrocarbons 1. T race am ounts o f o ther unassigned com ponents w ere also detected in the N M R spectra. 99.51% Purity 0.39% 0.057% 0.019% 0.013% 0.0079% Page 1 of 1 File Reference: C ofA _TCR-6l7_Lot 210002.doc Exact Copy o f Original initial -)I3i1n t-- o ata~ Page 173 of 225 3 M Environmental Laboratory E02-1039 Test Control and Reference Substance Log r. Substance trade name Heptadecafluorononanoic acid or reference # TCR Substance # TCR-618 Substance/chemical name: Heptadecafluorononanoic acid TCR # TCR-618 Lot/batch it: H7S68 Received from: Oakwood Products 3M # Expiration date: NA Amount received (wt or 5g vol): initials: Number/size of containers: Condition: OK 1-50ml plastic jar white crystals . Date: Shipper MSDS (y/n) 7/19/2002 Courier y Ij n ' Retain Date of Retain : C) Archived/Substarice Not Available! I r"i c t Copy o f Original Page 174 of 225 WT May 1? -001 'm m m m 2:20 EG & 1039 .'jprar O a l w o o d ipx o d n c i s f l n c 1741 O ld D u n b a r R oad West Columbia, SC 2 9 2 7 /> Phone (803) 739-8S00 " Fax- (8 0 3 ) 7 3 9 - 6 9 5 7 CERTIFICATE OF ANALYSIS D ate: 27-Nov-Ol Material: Heptadecafluanmonanoic add Cat.No.: 2263 C a s N o . : [375-95-1] t o t N o .: U7568 Assay: 99+% b y N a O H titr a tio n Appearance: Off-white solid Melting Point: 57-62C Exact Copy of Original Si2 L _ 1 Initial paia -k m J lA nm ei Van Q C M ana Page 175 of 225 3M Environmental Laboratory E02-1039 1 Heptadecafluorononanoic acid Heptadecafluorononanolc acid |rCR-618 USE LOG Gross Wt.A/ol. Amnt. Before withdrawn withdrawal (mass or voi) Balance ID 1 Balance ID 2 20.8649g 0.0677g Gross wt/vol after withdrawal Balance ID 1 20.7972g Purpose (enter standard number or reason for removal) standard 02040-46 Balance Balance Initials ID ID 12 Date 914 RWW 05/18/2002 Exa ct Copy of Original ( M .1 , /f ^/3 1 (52-- Initial Data Page 176 of 225 3M Environmental Laboratory E02-1039 Date: f//fA SINGLE COMPONENT PREPARATION LOG 2.___________ Analyst: tciS * 3 _____________ Book No. 02 040 Page No. 46 Description: J , n / A H / C- / , , J eM f x Stock Number T C - 6 / 9 , Weight or Volume Used: (?e 3 4 Concentration or Purity:_________ / y / T Corrected Weight: Balance ID: Iff Other Correction Factors: /V Solvent and TN-A Number / i s * ; -/ / T a / - Final Volume: Storage Location: Final Concentration: 2 - ? . . / />. Expiration Date: 3 Exact Copy of Original CMC ' ic/3i/6i-- Initial Date Reviewed Signature, V) w i i s l h ?. Dais Page 177 of 225 3M Environmental Laboratory E02-1039 3 VI S P E C I A L T Y M A T E R I A L S M A N U F A C T U R I N G D I V I S I O N A N A L Y T I C A L L A B O R A T O R Y To: Lisa Stevenson - (8-5568) - ET&SS - 2-3E-09 Request #GID:71638 From: Tom Kestner - (3-5633) - SMMD Analytical Lab - 236-2B-11 Subject: Characterization of T C R -6 1 8 b y 'H-NMR and 19F-NMR Spectroscopy Date: October 28,2002 SAMPLE DESCRIPTION: TCR-618, lot H7568 from the Telomer project. Nominal product = CFi(CF2)n-C02H, where average n 7 (white solid). S am p le Spectra It's Experiment Descriptions TCR-618 TCR-618 H 7 1 6 3 8 .G ID .4 0 2 F 7 1638.G ID .402/.403 400 M H z `H -N M R in acetone-d< solvent + p-H FX cross intecration/intem al std. 376 M H z "TF-NMR in acetone-d solvent + p-HFX cross integration/intem al std. OBJECTIVE: This sample was subjected to a combination of 'H-NMR and I9F-NMR spectral analyses to determine the purity of the nominal product. Special emphasis was also placed on attempting to identify and quantify any impurity components. EXPERIMENTAL: A portion of the sample was accurately weighed, spiked with a known amount of 1,4-bis(trifluoromethyl)benzene (p-HFX), and then totally dissolved in deuterated acetone (acetone-di) for subsequent analysis by NMR. A 400 MHz 'H-NMR spectrum and 376 MHz l9F-NMR spectra were acquired at room temperature using a Varian UNTTYplus 400 FT-NMR spectrometer. This sample preparation method permitted the p-HFX to be used as either 1) a 'H/i9F-NMR internal standard to allow the calculation of the absolute weight percent concentrations of specific components, or 2) a 'H/I9F-NMR cross integration standard to permit the cross correlation of the relative 'H and l9F signal intensities for evaluation of the overall sample composition. RESULTS: T h e 'H -N M R and ,9F -N M R spectral data ind icated th is sam p le w a s a h igh purity form o f the nom inal product, CF3(CF2),,-C02H, where the average value of n = 6.955. Small amounts of a few impurity components were also assigned. A 'H/,9F-NMR cross integration analysis technique was then used to calculate the relative weight percent concentrations of the identified components. The qualitative and quantitative compositional results that were derived from the single trial 'H/I9F-NMR cross integration analysis are summarized below in TABLE-1. The relative weight percent concentrations shown in TAB LE-1 should be very close to their respective absolute weight percent values assuming no water was present in the sample. Trace amounts of a few other unassigned components were also detected in the NMR spectra, but additional work would be needed in an effort to identify or quantify these other components. Copies of the NMR spectra are attached with the paper copy of this report for your reference. If you have any questions about these results, please let me know. B ract Copy o f Original- Page 1 o f2 Inflfai D a ta Page 178 of 225 O ctober 28, 2002 3M Environmental Laboratory E02-1039 3M SM M D A nalytical L ab R equest # GED:71638 . TC R -618, L otH 7568: T elom er P roject Tom Kestner Rick Payfer Ron Purcell William Rcagen File Reference: Is71638.G1D.TCR-618_Lot H7568_TeIomer ProjectDOC/lOO TABLE-1 Sample: TCR-618, Lot H7568 from the Telomer project. Component Structures 1 CF3(CF2),,-C02H where average n = 6.955 Probable H-(CF2),,-C02H assume n = 8 for calculation purposes Probable CF3(CF2),,-C02CH3 where average n = 6.955 A probable cyclic ether NMR Relative Weight% Concentrations (single trial measurement) 98.02% 1.10% 0.65% 0.20% . as possible F F . or similar Total inorganic fluoride (at least 3-types), including some possible HF Possible C,,H2n+2 saturated aliphatic hydrocarbons 1. T race am ounts o f o th er unassigned com ponents w ere also detected in the N M R spectra. 0.021% 0.014% Page 2 o f2 Exact Copy o f Original 'm c Inlal M 3i I Data Page 179 of 225 3M Environmental Laboratory E02-1039 Certificate o f Analysis Nominal Product: CF3(CF2)n-C02H, where average n a 7 Heptadecafluorononanoic acid Product Code: TCR-618, Lot H7568 October 28,2002 Tom Kestner The sample of TCR-618, lot H7568 was analyzed using a combination of 19F-NMR and 'H-NMR spectral analysis techniques. The overall qualitative and quantitative compositional results that were derived from these combined analyses are summarized below in TABLE-1. TABLE-1 Sample: TCR-618, Lot H7568 Quantitative Compositional Results by Combined '^F/'H-NMR Spectral Analyses /C->o__m__p_o__n_e__ntt Structures 11 i'Hu //i'VVF-xNnMu oR Relative Weight% Concentrations (single trial analysis) CF3(CF2),,-C02H where average n = 6.955 Probable H-(CF2),,-C02H assume n = 8 for calculation purposes Probable CF3(CF2),,-C02CH3 where average n = 6.955 A probable cyclic ether 98.02% Purity 1.10% 0.65% 0.20% as possible F F or similar Total inorganic fluoride (at least 3-types), 0.021% including some possible HF Probable C,,H2n+2 saturated aliphatic hydrocarbons 0.014% 1. T race am ounts o f o th er unassigned com ponents were also detected in the N M R spectra. Page 1 o f 1 File Reference: CofA_TCR-618_LotH7568.doc Page 180 of 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference it Substance/chemical name: Lot/batch #: 3M# Expiration date: Nonadecafluorodecanoic acid Nonadecafluorodecanolc acid R11K ID#2264 12/01/2010 Initials: Number/size of containers: Condition: Retain JCP 1/120 mL plastic bottle w hite solid MICH 07/22/99 0.1874g TCR Substance # TCR# Received from: SD036 TCR-36 Oakwood Products Inc 4/2/99 Amount received (wt. or 25g, 43.3264 g gross w t vol): Date: 04/27/1999 Shipper: N/A MSDS (y/n) Date of Retain WTTTn 10/18/1999 : ! O Archived/Substance Not Available j Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachment(s) 98.01%, updated 10/30/02 OK NMR analysis MSDS, Certificate o f Analysis Unknown F19, Frozen C9F19COOH TN-A-2451 prior to 4/27/99 JCP 04/27/99 Standard has been moved to Freezer 19 in room 347 KJD 06/06/00 Standard was stored at room temperature prior to 06/06/00. LAC 12/19/00 A fa ' sd036msds.pdf sd02 6cofa.pdf Exact C o p y o f O rig in al CM. _ IntUal Date Page 181 of 225 3M Environmental Laboratory E02-1039 3M SPECIALTY MATERIALS MANUFACTURING DIVISION ANALYTICAL LABORATORY To: Lisa Stevenson - (8-5568) - ET&SS - 2-3E-09 Request# GID:71638 From: Tom Kestner - (3-5633) - SMMD Analytical Lab - 236-2B-11 Subject: Characterization of SD036 by 'H-NMR and I9F-NMR Spectroscopy Date: October 28,2002 SAMPLE DESCRIPTION: SD036, lot RI IK from the Telomer project. Nominal product = CF3(CF2)n-C02H, where average n 8 (white powder). Sam ple S p ectra # 's Experiment Descriptions SD036 SD036 H 7 1638.G ID .403 F 7 1 6 3 8 .G ID .4 0 4 40 0 M H z 'H -N M R in acetone-<L solvent + p-HFX cross integration/intem a] std. 376 M H z |,'F -N M R in acetone-d solvent + p-H FX cross integration/intem al std. OBJECTIVE: This sample was subjected to a combination of 'H-NMR and l9F-NMR spectral analyses to determine the purity of the nominal product Special emphasis was also placed on attempting to identify and quantify any impurity components. EXPERIMENTAL: A portion of the sample was accurately weighed, spiked with a known amount of 1,4-bis(trifluoromethyl)benzene (p-HFX), and then totally dissolved in deuterated acetone (acetone-d^) for subsequent analysis by NMR. A 400 MHz 'H-NMR spectrum and a 376 MHz 19F-NMR spectrum were acquired at room temperature using a Varian UNITYplus 400 FT-NMR spectrometer. This sample preparation method permitted the p-HFX to be used as either 1) a 'H/'^-NMR internal standard to allow the calculation of the absolute weight percent concentrations of specific components, or 2) a 'H/19F-NMR cross integration standard to permit the cross correlation of the relative 'H and 19F signal intensities for evaluation of the overall sample composition. RESULTS: T h e 'H -N M R and l5F -N M R spectral data ind icated this sam p le w a s a relatively h igh pu rity form o f the nominal product, CF3(CF2)n-C02H, where the average value of n = 7.85. Small amounts of a few impurity components, including probable isomers, were also assigned. A `H/I9F-NMR cross integration analysis technique was then used to calculate the relative weight percent concentrations of the identified components. The qualitative and quantitative compositional results that were derived from the single trial `h/ i9F-NMR cross integration analysis are summarized below in TABLE-1. The relative weight percent concentrations shown in TABLE-1 should be very close to their respective absolute weight percent values assuming no water was present in the sample. Trace amounts of a numerous other unassigned components were also detected in the l9F-NMR spectrum, but additional work would be needed in an effort to identify or quantify some of these other components. Copies of the NMR spectra are attached with the paper copy of this report for your reference. If you have any questions about these results, please let me know. Exact Copy o f Original UYY Initial Data Page t of2 Page 182 of 225 3M Environmental Laboratory E02-1039 O ctober 28, 2002 3M SM M D A nalytical Lab R equest # G ID :71638 . SD 036, Lot R l IK : T elom er Project Tom Kestner c: Rick Payfcr Ron Purcell William Rcagcn Pile Reference: Ij71638.GID.SD036_Lol RIIK_Telomer ProjectDOC/IOl TABLE-1 Sample: SD036, Lot Rl IK from the Telomer project. Overall Compositional Results by 'H/^F-NMR Cross Integration Analysis Component Structures 1 NMR Relative Weight% Concentrations (single trial measurement) CF3(CF2)n-C02H where average n = 7.85 Probable CFjiCFzlx-CFiCFjHCFOy-CCbH where x*0 and assume x+y = 5.85 for calculation purposes Probable (CF3)2-CF-(CF2)n-C02H assume n=5.85 for calculation purposes Probable 98.01% 1.41% 0.36% 0.12% - y 5 r '^ 0H IsF FF assume n=3.85 for calculation purposes Probable L fL A 0.060% FF assume n=2.85 for calculation purposes Probable CnH2n+z saturated aliphatic hydrocarbons Total inorganic fluoride 0.026% >0.0095% 1. T race am ounts o f n u m e ro u s other unassigned com ponents w ere also detected in the l0F -N M R spectrum . Exact Copy of Original Page 2 of 2 Page 183 of225 OfKUGOD PRODUCTS F ax :8 0 3 -7 3 9 -6 9 5 7 Feb W M f 0Nt ^ l AL LA^ ir^ / <O- R Y 039 'S b-oeio Oalovood Products, Old Dunbar Kn^ Ine. West Columbia, SC 29172 s* Wwwe (803) 739-8800 F a x (803) 73 9 ^9 5 7 c e r t ific a t e OF ANALYSIS Date; ll-Feb-00 M a " riaI- N o " a < .d anoicaci(i C a s N o - 1335-76-2] L o i N o .; U lU C Perfluoxodecaaoic^acid ^ Fnfl" oto<l""'< 'ic aied <5./, Prodarts less volatile than ^erflaorodecanoic add Appearance; White solid M elting Point: 8385*c ExactCopyof Original 4 r f4 M * L jl 7Utnei y a /Q u ality thtrol t I ( j i Page 185 o f 225 3M Environmental Laboratory E02-1039 ____________________________________________________ U S E L O G lonadecafluorodecanoic acid lonadecafluorodecanoic acid :D036 ____________________ ________________ Gross Wt/Vol. Before withdrawal Balance ID 1 Amnt. withdrawn (mass or vol) Balance ID 2 33.9944g 0.1196g Gross wt/vol after withdrawal Balance ID 1 33.8784g Purpose (enter standard number or reason for removal) standard 02022-90 Balance Balance ID ID 12 Initials Date 914 RWW 07/02/2002 )i Exa c t Copy o f Original X L- j I ^ / ^ Dato tM Page 186 of 225 3M Environmental Laboratory E02-1039 Date: Analyst: SINGLE COMPONENT PREPARATION LOG Book No. 02 022 Page No. 90 Description: A A r Stock N um ber_ a > Ji j Weight or Volume Used: Concentration or Purity: w ! O hCorrected Weight 0 My Balance ID:_ Other Correction Factors: Solvent and TN-A Number Final Volume: /O 0 ^ ) Storage Location: 2 * J / % > * / Final Concentration: / O t Q jr> p ^ Expiration Date: //x /e J Page 187 of 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemical name: Lot/batch #: 3M# Expiration date: Perfluoroundecanoic add Perfluoroundecanoic acid U11N NA Initials: Number/size of containers: Condition: Retain OK 1 -1Oml amber glass vial white crystals. TCR Substance # TCR # Received from: TCR-619 TCR-619 Oakwood products Amount received (wt. or Sg vol): Date: 07/19/2002 Shipper Courier MSDS (y/n) Date of Retain * y (.; N ! O Archived/Substance Not Available Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachment(s) >99% LAS 07/25/02 MSDS Certificate o f Analysis LAS 07/25/02 Oakwood products Room Temp, TCR-C01 C11HF2102 Shipping comments: Harmful solid, No 3M ID# - must ship ground sm all quantity exception (30 mL/g or less per vial). LAS 10/02/02 T C R -6 1 9 .p d f Exact c o p y o f Original l K t^ . [ c / - ? i l inftlal Dtay Page 188 of 225 3M Environmental Laboratory E02-1039 3M SPECIALTY MATERIALS MANUFACTURING DIVISION ANALYTICAL LABORATORY To: Lisa Stevenson - (8-5568) - ET&SS - 2-3E-09 Request # GID:71638 From: Tom Kestner - (3-5633) - SMMD Analytical Lab - 236-2B-11 Subject: Characterization of TCR-619 by 'H-NMR and 19F-NMR Spectroscopy Date: October 29, 2002 SAMPLE DESCRIPTION: TCR-619, lot U1 IN from the Telomer project. Nominal product = CFjfCFzVCOiH, where average n 9 (white powder). Sam ple Spectra it's Experiment Descriptions TCR-619 TCR-619 H 7 1 6 3 8 .G ID .4 0 4 F 7 1 6 3 8 .G ID .4 0 5 400 M H z 'H-NM R in acetone-d* solvent + p-HFX cross integration/internal std. 376 M H z i!T -N M R in acetone-d solvent + p-H FX cross integration/internal std. OBJECTIVE: This sample was subjected to a combination of'H-NMR and l9F-NMR spectral analyses to determine the purity of the nominal product. Special emphasis was also placed on attempting to identify and quantify any impurity components. EXPERIMENTAL: A portion of the sample was accurately weighed, spiked with a known amount of 1,4-bis(trifluoromethyl)benzene (p-HFX), and then totally dissolved in deuterated acetone (acetone-da) for subsequent analysis by NMR. A 400 MHz 'H-NMR spectrum and a 376 MHz l9F-NMR spectrum were acquired at room temperature using a Varian UNITYplus 400 FT-NMR spectrometer. This sample preparation method permitted the p-HFX to be used as either 1) a 'H/19F-NMR internal standard to allow the calculation of the absolute weight percent concentrations of specific components, or 2) a 'H/i9F-NMR cross integration standard to permit the cross correlation of the relative 'H and 19F signal intensities for evaluation of the overall sample composition. RESULTS: T h e 'H-NTTvIR. a n d 10F->JTvR sp ectral data in d ica ted this sa m p le w a s a r ela tiv e ly h ig h p u rity fo r m o f th e nominal product, CF3(CF2)n-C02H, where the average value of n = 8.63. Small amounts of a few impurity components, including probable isomers, were also assigned. A 'H/,9F-NMR cross integration analysis technique was then used to calculate the relative weight percent concentrations of the identified components. The qualitative and quantitative compositional results that were derived from the single trial !H/I9F-NMR cross integration analysis are summarized below in TABLE-1. The relative weight percent concentrations shown in TABLE-1 should be very close to their respective absolute weight percent values assuming no water was present in the sample. Trace amounts of numerous other unassigned components were also detected in the l9F-NMR spectrum, but additional work would be needed in an effort to identify or quantify some of these other components. ' Copies of the NMR spectra are attached with the paper copy of this report for your reference. If you have any questions about these results, please let me know. Copy o f Original O H _ io f Initial Data Page 1 o f2 Page 189 of 225 3M Environmental Laboratory E02-1039 O ctober 29, 2002 3M SM M D A nalytical Lab R equest # G 1D :71638 .T C R -619, L o tU l IN: T elom er Project Tom Kestner c: Mark Ellcfson Rick Payfer Ron Purcell William Rcagcn Pile Reference: ls7I638.GID.TCR-619_LotUllN_TelomerPiojcct.DOC/101 TABLE-1 Sample: TCR-619, Lot U1 IN from the Telomer project. Overall Compositional Results by 'H/I9F-NMR Cross Integration Analysis Component Structures 1 NMR Relative Weight% Concentrations (single trial measurement) CF3(CF2),,-C02H (where average n = 8.63) 96.4% Probable internal branched isomers CF3(CF2))t-CF(CF3HCF2)y-C01H 2.6% (where x*0 and assume x+y = 6.63 for calculation purposes) Probable trans-olefins of general form: trans-CF3(CF2)x-CF=CF-(CF2)y-C02H (assume x+y = 6.63 for calculation purposes) 0.89% Possible terminal isopropyl branched isomers (CF3)2-CF-(CF2),,-C02H 0.12% (assume n=6.63 for calculation purposes) Probable CnH2,,+2 saturated aliphatic hydrocarbons 0.032% Total inorganic fluoride >0.0017% 1. T race am ounts o f n u m e ro u s other unassigned com ponents w ere also detected in the l9F-N M R spectrum . . Page 2 o f2 Exact Copy of Original -- . U ^ I/T L , Initial Oats Page 190 of 225 3M Environmental Laboratory E02-1039 I C ertifica te o f A n aly sis V.'-'-i Nominal Product: CF3(CF2)n-C02H, where average n 9 Perfluoroundecanoic acid Product Code: TCR-619, LotUllN October 29,2002 The sample of TCR-619, lot U11N was analyzed using a combination of l!lF-NMR and 'H-NMR spectral analysis techniques. The overall qualitative and quantitative compositional results that were derived from these combined analyses are summarized below in TABLE-1. TABLE-1 Sample: TCR-619, Lot U1 IN Overall Compositional Results by Combined 19F/'H-NMR Spectral Analyses Component SCtfrnu.nchtuirraecs. 11 ,H/,WF-XNTMH/TRD Relative Weight% Concentrations ''> 13 (single trial analysis) )) J CF3(CF2)n-C02H <96.4% (where average n = 8.63) Purity Probable internal branched isomers 2.6% CF3(CF2)x-CF(CF3HCF2)y-C02H (where x*0 and assume x+y = 6.63 for calculation purposes) Probable trans-olefins of general form: 0.89% U trans-CF3(CF2)x-CF=CF-(CF2)y-C02H (assume x+y = 6.63 for calculation purposes) Possible terminal isopropyl branched isomers 0.12% (CF3)2-CF-(CF2),,-C02H (assume n=6.63 for calculation purposes) P ro b a b le C ,,H 2,, satu rated a lip h atic hyd rocarbons 0.032% Total inorganic fluoride >0.0017% 1. T race am ounts o f n u m e ro u s other unassigned com ponents w ere also detected in the |,JF -N M R spectrum . S TomKestner Exact Copy o f Original lo /Si / 02-- Data Page I of 1 File Reference: CofA_TCR-619_LotUlIN.doc Page 191 of 225 E m m N M E N M LABOffATWY OaJcwood Products, Inc. 1741 Old Dunbar Road West Columbia, SC 29172 I'hone (803) 739-8800 Fax (803) 739-6957 CERTIFICATE OF ANALYSIS Date: 15-JuI-02 M aterial: Perfluoroundec anoic acid Cat.No.: 2265 Cas No.: [4 2 3 4 -2 3 -5 ] Lot No.: U11N Assay: 99+% by NaOH titration Appearance; W hite solid M eltin g Point: 92-98C v V \ >' i ; . i T- . : Exact Copyof Original Initial Daw; ^Yurnei Yaii; QC Manager Page 192 of 225 3M Environmental Laboratory E02-1039 __________________________________________ USE LOG 'erfluoroundecanoic acid 'erfluoroundecanoic acid CR-619 ___________ _________________ Gross WL/Vol. Amnt. Before withdrawal withdrawn (mass or voi) 8 alance ID 1 Balance ID 2 15.5264g 0.0691 g Gross wt/vol after withdrawal Balance ID 1 15.4573g Purpose (enter standard number or re a s o n fo r removal) standard 02040-65 Balance Balance ID ID 12 Initials Date 914 RWW 08/29/2002 iffsst CopyofOriginal Ciltt -- Initial Data Page 193 of 225 31WEnvironmental Laboratory E02-1039 Data: Analyst: Description: SINGLE COMPONENT PREPARATION LOG Book No. 02 040 Page No. 65 / L . e r b -M iA 's r v t o i e Stock Num ber 7~f f t O' / 9 Weight orVolume Used: ----2-------------P---- -^5*. Concentration or Purity: rfA ~ Corrected Weight: 0 'O i7 i } 7 Balance ID:_____ ^ / 6/ Other Correction Factors: Solvent and TN-A Number Final Volume: J. FinalConcanlration: J ? f / , Storage Location: A /o , a ^ - / L - y / < * Expiration Data: /u > / > ? Exa ct Copy o f Original . ('/ / o 3Wtfel Hatff Reviewed by: Data Page 194 of 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemical name: Lot/batch # : 3M * Expiration date: Perfluorododecanoic acid Perfluorododecanoic acid R24K 10*2266 12/ 0 1 /2 0 1 0 Initials: Number/slze of containers: Condition: Retain JCP 1/120 mL plastic bottle w hite powder MCH 07/22/99 0.2159a TCR Substance * SD037 TCR# TCR-37 Received from: Oakwood Products Inc 4/2/99 Amount received (wt. or 25g, 43.4548 g gross w t vol): Date: 04/27/1999 Shipper N/A MSDS (y/n) Date of Retain * Y (J N 10/18/1999 I D Archived/Substance Not Available : Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage; Molecular Formula: Comments 99.65% updated 10/30/02 OK, NMR analysis MSDS, Certificate o f Analysis Unknown '' F19, Frozen C11F23COOH TN-A-2453 prior to 4/27/99 JCP 04/27/99 Standard has been moved to Freezer 19 in room 347 KJD 06/06/00 Standard was stored at room temperature prior to 06/06/00. LAC 12/19/00 Attachment(s) Shipping comments: Irritant, no shipping information - ship as small quantity exception. LAS 10/02/02 > - i sd037msds.pdf sd037cofa.pdf NMR SD037.pdf ' Exact Copy o f Original Initial Data Page 195 of 225 3M Environmental Laboratory E02-1039 3M SPECIALTY MATERIALS MANUFACTURING DIVISION ANALYTICAL LABORATORY To: Lisa Stevenson - (8-5568) - ET&SS - 2-3E-09 Request # GID:71638 From: Tom Kestner - (3-5633) - SMMD Analytical Lab - 236-2B-II Subject: Characterization of SD037 by 'H-NMR and 19F-NMR Spectroscopy Date: October 28,2002 SAMPLE DESCRIPTION: SD-037, lot R24K from the Telomer project. Nominal product = CFjfCFzVCOzH, where average n 10 (white powder). S am ples Spectra tt'i Experiment Descriptions SD037 SD037 H 71 6 3 8 .G ID .4 0 5 F 7 1 6 3 8 .G ID .4 0 6 400 M H z `H-N M R in acetone-d solvent + p-H FX cross integration/intem al std. 376 M H z ''F -N M R in acetone-dfi solvent + p-H FX cross integration/internal std. OBJECTIVE: This sample was subjected to a combination of'H-NMR and 19F-NMR spectral analyses to determine the purity of the nominal product. Special emphasis was also placed on attempting to identify and quantify any impurity components. EXPERIMENTAL: A portion of the sample was accurately weighed, spiked with a known amount of 1,4-bis(trifluoromethyl)benzene (p-HFX), and then totally dissolved in deuterated acetone (acetone-d6) for subsequent analysis by NMR. A 400 MHz 'H-NMR spectrum and a 376 MHz 19F-NMR spectrum were acquired at room temperature using a Varian UNITYplus 400 FT-NMR spectrometer. This sample preparation method permitted the p-HFX to be used as either 1) a 'H/19F-NMR internal standard to allow the calculation of the absolute weight percent concentrations of specific components, or 2) a 'H/^F-NMR cross integration standard to permit the cross correlation of the relative 'H and 19F signal intensities for evaluation o f the overall sample composition. RESULTS: T h e 'H -N M R an d l5F -N M R sp ectral data ind icated th is sa m p le w a s a h igh p u rity form o f th e n o m in a l product, CF3(CF2)n-C02H, where the average value of n = 10.042. Small amounts of a few impurity components, including probable isomers, were also assigned. A 'H/I9F-NMR cross integration analysis technique was then used to calculate the relative weight percent concentrations of the identified components. The qualitative and quantitative compositional results that were derived from the single trial 'H/I9F-NMR cross integration analysis are summarized below in TABLE-1. The relative weight percent concentrations shown in TABLE-1 should be very close to their respective absolute weight percent values assuming no water was present in the sample. Trace amounts of a few other unassigned components were also detected in the NMR spectra, but additional work would be needed in an effort to identify or quantify these other components. . Copies of the NMR spectra are attached with the paper copy of this report for your reference. If you have any questions about these results, please let rne know. Exact Copy o f Original CVXM ip|3>|/T t^ Initial Data Page 1 o f2 Page 196 of 225 3M Environmental Laboratory E02-1039 O ctober 28, 2002 3M SM M D A nalytical Lab R equest U G ID :71638 SD 037, Lot R24K: T elom er Project Tom Kestner c: Rick Puyfer Ron Purcell William Rcagcn File Reference: U7163*.GID.SD037_Lol R24K_Tclomer PrajectDOC/IOI TABLE-1 Sample: SD037, Lot R24K from the Telomer project. Overall Compositional Results by `H7I9F-NMR Cross Integration Analysis Component Structures 1 NMR Relative Weight% Concentrations (single trial measurement) ' CF3(CF2),,-C02H where average n = 10.02 Probable (CF3)2-CF-(CF2),,-C02H assume n=8 for calculation purposes Possible CF3(CF2)x-CF(CF3)-(CF2)y-C02H where x*0, y*0 and assume x+y = 8 for calculation purposes Probable chlorinated impurity, Cl-CF2CF2-Rf, possibly as Cl-(CF2)n-C02H Possible methyl ester impurity as CF3(CF2)n-C02CH3 where average n = 10.02 Probable CnH2n+2saturated aliphatic hydrocarbons Toluene 1. T race am ounts o f o th e r unassigned com ponents w ere also detected in the N M R spectra. 99.65% 0.13% 0.088% 0.049% 0.048% 0.016% 0.015% Page 2 o f 2 Exact Copy of Original t'm i Initial Oats Page 197 of 225 3M Environmental Laboratory E02-1039 Certificate o f Analysis Nominal Product: CF3(CF2),,-C02H, where average n 10 perfluorododecanoic acid Product Code: SD037, Lot R24K. October 28,2002 Tom Kestner The sample of SD037, lot R24K was analyzed using a combination of 19F-NMR and 'H-NMR spectral analysis techniques. The overall qualitative and quantitative compositional results that were derived from these combined analyses are summarized below in TABLE-1. TABLE-1 Sample: SD037, Lot R24K Quantitative Compositional Results by Combined i9F/'H-NMR Spectral Analyses Component Structures 1 1'H/lI59F--]NMR Relative Weight% Concentrations ______ (single trial analysis)______ CF3(CF2)n-C02H where average n = 10.02 Probable (CF3)2-CF-(CF2)n-C02H assume n=8 for calculation purposes Possible CF3(CF2)*-CF(CF3)-(CF2)y-C02H where x*0, y^0 and assume x+y = 8 for calculation purposes Probable chlorinated impurity, Cl-CF2CF2-Rf, possibly as Cl-(CF2)n-CQ2H Possible methyl ester impurity as C F3(C F 2),,-C 02C H 3 99.65% Purity 0.13% 0.088% 0.049% 0.048% w h e re a v e r a g e n = 1 0 .0 2 Probable Ci,H2n+2 saturated aliphatic hydrocarbons 0.016% Toluene 0.015% 1. Trace amounts of other unassigned components were also detected in the NMR spectra. Exact Copy of Original lo fli/c n - banal Oats Page 1 of 1 File Reference: CofA_SD037_Lot R24K.doc Page 198 of 225 OflKUOOD PRODUCTS Fax:803-739-6957 F e b SM W W RO iw qtTTAL La ^ q ^ to r y . a fiW O akw o o d Products, Inc. 1741 Old Dunbar Road West Columbia. SC29172 Phone (803) 739-8800 Fax (803) 739-4957 CERTIFICATE OF ANALYSIS Date: ll-Peb-00 M aterial: Perfluorododecanoic add Cat.No.: 2266 LotN o^ R22K Assay: Products more volatile than Perfluorododecanoic a d d < 2% Perfluorododecanic ad d 96% nun. Products less volatile than Perfluorododecanoic a d d <2% Appearance: W hite solid M eltin g Point: 107-109C Exact Copy of Original CWIC \2 lM 2 y ' Initial O a ts % Page* 199 of 225 3M Environmental Laboratory E02-1039 __________________________________________ USE LOG 'erfluorododecanoic acid 'erfluorododecanoic acid ID037 ________________ ___________ __ Gross Wt./Vol. Amnt. Before withdrawn withdrawal (mass or vol) Balance ID 1 Balance ID 2 42.5752g 0.0796g Gross wt/voi after withdrawal Balance ID 1 42.4956g Purpose (enter standard number or reason for removal) standard 02040-55 Balance Balance Initials ID ID 12 Date 914 RWW 08/29/2002 Exact Copy o f Original toltlsl Dits Page 200 of 225 3M Environmental Laboratory E02-1039 Date: 2- Analyst: jfc lrftO SINGLE COMPONENT PREPARATION LOG Book No. 02 040 Page No. 55 Description: P t t ( l u a / ' P t ) a J s r j a , e - A f i J - f 90/J Stock Number v_S~D 0 Weight orVolume Used: Concentration or Purity: Ml Corrected Weight: > J Z - J c j Balance ID:______ ^ / V Other Correction Factors: A / Solvent and TN-A ___ Number /* J f e > -- / V - A - W Final Volum e: T ^J- Flnal Concentration: ! ) 2 . , j * * Storage Location: f r o e Y * - /?<. T * a Expiraon Date: A . /) - < } / o S Reviewed by: Exact Copy o f Original C ivic InttU Oat Page 201 of 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substanca trade name or reference # Substance/chemical name: Lot/batch #: 3M# Expiration date: PFOS Potassium Perfluorooctane Sulfonate FC-95 217 98-0211-0888-5 08/31/2006 Initials: Number/size of containers: Condition: Retain PMR 1/175 mL glass container w hite powder MCH-07/22/99 0.1733g TCR Substance # SD018 TCR # TCR-18 Received from: Jo Dickes 8/10/98 Amount received (wt. or 160.0706g gross w t vol): Date: 04/07/1999 Shipper: Unknown MSDS (y/n) Date of Retain * YU N 10/18/1999 : O Archived/Substance Not Available Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Locatlon/Storage: Molecular Formula: Comments Attachment(s) 86.9% LAC 09/19/00 NMR Results/Characterization #53030, MSDS KJD 02/29/00 Interim final report from Centre and Certificate o f Analysis. LAC 04/26/01 98-0211-0888-5 . F19, Frozen C8F17S03-K+ Environmental Lab Traceablltity number was TN-A-2130 PMR 04/07/99 Moved to cold storage (< 0C) on 05/16/00 LAC 05/16/00 Standard has been moved to Freezer 19 in room 347 KJD 06/06/00 Standard was stored at room temperature prior to 05/16/00. LAC 12/19/00 Shipping Codes: FC-S000-0185-6(<30 g on dry ice) LAC 06/14/01 Shipping Comments: Dangerous goods in excepted quantity o f Class 6, UN2811. REGULATED-TOXIC 98-0211-0888-5 (>30 g (1 oz sample)) Not on dry ice. LAC 01/08/02 - sd018msds.pdf sd018nmrreq53030.pdf sd018nmrreq61517.pt ih E 0 0 -16 82 -C O A -S D 01 8-P F O S -R ev3 Exact Copy of Original Page 202 of 225 3M Environmental Laboratory E02-1039 Centre Analytical Laboratories. Inc. 3048 Research Drive State Collage, PA 16801 www.centrelab.com Phone:(814)231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 C entre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFOS, Lot 217 Reference#: SD-018 ___________ Purity: 86.9%__________ Test Name Specifications Purity1 Result 86.9% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds - POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic Acids0(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis0: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine White Crystalline Powder 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Conforms Positive 1. 0.005 wt./wt.% 2. 0.001 wt./wt.% 3. 1.439 wtVwt.% 4. 6.849 wt./wt% 5. <0.001 wt./wt.% 6. 0.005 wt./wt% 7. <0.001 wt./wt.% 1.91 wt./wt.% 8.41 wt./wt.% None Detected 0.33 wt./wt.% None Detected Not Applicable'' 1. <0.015 wt./wt.% 2. 0.59 wt./wt.% 3. <0.040 wt7wt.% 4. <0.009 wt7wt.% 5. <0.006 wt./wt.% 6. <0.007 wt./wt.% 7. 8.76 wt./wt.% 1. <0.1 wt./wt.% 2. <0.1 wt./wt.% 3. 0.10wt./wt.% 4. 0.28 wt./wt.% 1. 12.48 wt/wt.% 2. 0.244 wt7wt.% 3. 1.74wt./wt.% 4. 8.84 wt.Avt.% 5. 54.1 wtVwt.% COA023-018A Page 1 o f3 Exact Copy of Original Page 203 of 225 3M Environmental Laboratory E02-1039 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/06 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/06 'Purity = 100% - (sum o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0 .5 9 %+NMR impurities, 1.905%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.07% Purity = 100% -13.07% = 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials of low purity. No endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. STFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CgFi7SC>3'K+(MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). .1 # O dS COA023-018A Page 2 o f3 Page 204 of 225 3M Environmental Laboratory E02-1039 Centre Analytical Laboratories, Inc 3048 Research Drive State College, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023'018A LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total wt./wt. % 1 .2 2 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curvto, respectively. The C5 value was calculated using the average result from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average result from the C6 and C8 standard curves. [' i t! Exact Copy of Original Scientist, Centre Analytical Laboratories Reviewed By: nLm fLbii }6hn Flaherty / Date / tLa.1b_o_r_a*t_o_ryxManager, Centre Analytical Laboratories COA023-018A Page 3 o f3 Page 205 o f 225 3M Environmental Laboratory E02-1039 USE LOG 'FOS 'otasslum P e r ilu o r a o c t a n e S u lfo n a te FC-95 0018 ____________________________ Gross Wt.A/ol. Before withdrawal Balance 10 1 AmnL withdrawn (mass or voi) Balance ID 2 140.448Og 0.0814g Gross wt/vol after withdrawal Balance IO 1 140.3666g Purpose (enter standard number or reason for removal) standard 02022-56 Balance Balance Initials ID ID 12 Date 914 RWW 05/13/2002 L ;-;{ ^ 1 -- <o lP t//t? -- initial g y Page 206 of 225 3M Environmental laboratory E02-1039 Date: A n a ly s t Description: SINGLE COMPONENT PREPARATION LOG Book No. 02 022 Page No. 56 S h ilL Stock Number Weight orVolume Used: X Concentration or Purity: ^ f/s ,9 V* S~ Z . -!$CorrectedWeight Balance ID: </// 9Z 7 S 'Other Correction Factors: />, Solvent and TN-A Number M * . c J 1/ Final Volume:_____ . 5~D*~ ! Storage Location: lilt, Final Concentration: / WZ/yt-to. Expiration Date: Exact Copy of Original l (3 iU > L _ Initial Data Reviewed by: Data Page 207 of 225 A ttac h m ent E : Pr o to c o la n d A m endm ent 3M Environmental Laboratory E02-1039 Page 208 of 225 3M Environmental Laboratory E02-1039 3M STUDY PROTOCOL STUDY TITLE Analysis of Endogenous Fluorochemicals inNormal Pooled Human Serumand Plasma SPONSOR William K. Reagen, Ph.D DATA REQUIREMENT 40 CFR Part 792 TESTING FACILITY 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St Paul, MN 55106 LABORATORY STUDY IDENTIFICATION 3M Environmental Study Number E02-1039 NUMBER OF PAGES 10 Exact Copy o f Original r m c 10 ( 3 1 Initial Data Page 209 of 225 Analysis of Endogenous Fluorochemicals . 3M Environmental La b o r a t o r y E02-1039 ' E02-1039 STUDY IDENTIFICATION Analysis o f Endogenous Fluorochemicals in Norm al Pooled H um an Serum and Plasm a Sponsor StudyDirector Test Facility Proposed Study Timetable Experimental Start Date Experimental Termination Date 'William Reagen, Ph.D. 3M Environmental Laboratory 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 (651)778-6565 Mark E. Ellefson 3M Environmental Laboratory 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 (651)778-5405 3M Environmental Laboratory 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106 10 October 2002 01 November 2002 3M Environmental Laboratory Exact Copy of Original ... j o l B i / -- Initial oats Page 2 of 10 Page 210 of 225 Analysis of Endogenous Fluorochemioils 3M Environmental Laboratory E02-1039 E 02-1039 1.0 Introduction and Purpose/Objective 1.1 The purpose of this study is to quantify perfluorohexanoic acid (C6), perfluoroheptanoic acid (C7), pentadecafluorooctanoic acid (C8), heptadecafluorononanoic acid (C9), nonadecafluorodecanoic acid (CIO), perfluorodecanoic acid (Cl 1), perfluorododecanoic acid (C12), tetrahydroperfluorooctane sulfonate (THPFOS), tetrahydroperfluorodecane sulfonate (THPFDS), and perfluorooctanesulfonate (PFOS) in normal pooled human serum and plasma. This study does not have a typical test substance that is dosed onto a specific controlled test system as per a conventional GLP study. 2.0 Regulatory Compliance 2.1 This study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practice Regulations for the Toxic Substances Control Act, 40 CFR 792. 3.0 Test Substances 3.1 Preliminary screening indicates that the test compounds listed below are present as endogenous material in normal pooled human serum and plasma. Information . pertaining to traceability, source, physical description, and storage conditions is not available for these compounds as they exist in biological matrices. Test Substances Test S u b s ta n c e Perfluorohexanoic Acid Tetradecafluaroheptanioc Acid Pentadecafluorooctanoic Acid Heptadecafluorononanoic Acid Nonadecafluorodecanoic Acid Perfiuoroundecanoic Acid Perfluorododecanoic Acid 1 H ,2 H ,3 H ,4 H -p e rfIu o ro o c fa n e s u lfo n a te 1H,2H.3H.4H-perfluorodecane sulfonate Potassium Perfluorooctanesulfonate Formula C sF,,C O O H C .F,jC O O H C j F isC O O H C ,F,,C O O H CtFiaC O O H CioFjtC O O H C ,,FaC O O H CH sF ,j O j S C ioH jFi/O aS C .F.rSO j-K ' Exa ct Copy o f Original C (V r /o jJ tltn -- Initial Data 3M Environmental Laboratory Page 3 of 10 Page 211 of 225 Analysis of Endogenous Fluorochemicals 3M Environmental Laboratory E02-1039 E02-1039 4 .0 R eference Substances Reference Substances Reference Substance Formula Traceability Source Perfluorohexanoic Acid CsFiiCOOH TCR-047 SMM* 236-1B-10 Tetradecafluoro heptanioc Add CFCOOH TCR-267 Aldrich Pentadecafluoro octanoic Add C/FhCOOH TCR-617 Oakwood Products Heptadecafluoro nonanoic Add CsFnCOOH TCR-618 Oakwood Products Nonadecafluora decanoic Acid CF,,COOH TCR-038 Oakwood Products Perfluoroun decanoic Add CioFjiCOOH TCR-619 Oakwood Products Perfluorodo decanoic Add CuFaCOOH TCR-037 Oakwood Products 1H,2H,3H,4Hperfluorooctane sulfonate CsHiFnOjS TCR-343 SynQuest Labs 1H,2H,3H,4Hperfluorodecane sulfonate CioHjFuOjS TCR-627 Pace Analytical Potassium Perfiuonooctane sulfonate CsFuSOt'K* TCR-018 SMM* 236-1 B-10 "D o cu m en tatio n o f th e m ethod o f synthesis is located at the source. Physical Description Colorless Liquid Clear Crystals White Crystals White Crystals White Solid White Crystals White Powder White Powder White Crystals White Powder Purity TBD** 99.5% >97% > 99% 98% > 99% 96% TBD 94.7% 86.9% **A sample o f the reference substance has been sent for characterization Storage Conditions Frozen Frozen Amblent Temprature Amblent Temprature Frozen Ambient Temprature Frozen Frozen Amblent Temprature Frozen 5.0 T est System T est System s v. Test System: Pooled Human Serum Pooled Human Serum Pooled Human Serum Pooled Human Serum Source Sigma-Aldrich, Milwaukee. W l Lampire Biological Laboratories, Plpersviile, PA Bioresource Technology, Inc., Fort Lauderdale, FL Golden West Blologicals, Temecula, CA Traceability TCR-689 TCR-688 TCR-687 TCR-690 Exact Copy of Original initial C a ia 3M Environmental Laboratory Page 4 of 10 Page 212 of 225 Analysis of Endogenous Fluorochemicala 3M Environmental Laboratory E02-1039 E02-1039 Test System: Pooled Human Plasma Pooled Human Plasma Pooled Human Plasma Pooled Human Plasma Source Lampire Biological Laboratories, Pipersville, PA Golden West Biofogicals, Temecula, CA Innovative Research, Inc., Southield, Ml Central China T raceability TCR-685 TCR-684 TCR-683 TCR-674 5.1 J u s tific a tio n o f th e te st system . Based on preliminary testing, normal pooled . serum and plasma contain endogenous levels of the test analytes. 5.2 Id e n tific a tio n o f T est S ystem . Samples shall be identified by the study number, date of initial preparation, test substance or test system, sample number, replicate number (if applicable), analyst(s), and project leader. 6.0 Surrogate Matrix Surrogate Matrix Surrogate Matrix: Rabbit Serum Source Sigma-Aldrich Traceability | TCR-686 | 6.1 J u s tific a tio n o f th e S u rro g a te M a trix . Based on preliminary testing, normal pooled rabbit serum contains very low endogenous levels of the test analytes and is thereby suitable for use as a surrogate matrix. 6.2 Id e n tific a tio n o f S u rro g a te M a trix . Samples shall be identified by the study number, date of initial preparation, test substance or surrogate matrix, sample number, replicate number (if applicable), analyst(s), and project leader. 7.0 A nalytical M ethods 7.1 Analysis of the test and reference substances will be conducted in the 3M Environmental Laboratory. The analyses will be conducted as described by 3M Environmental Laboratory Method ETS-8-231, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological matrices". Exact Copy of Original C J K . fo /3 t) $2-- Initial D atoy 3M Environmental Laboratory Page 3 of 10 Page 213 of 225 Analysis of Endoenous Fluorochemicals 3M Environmental Laboratory E02-1039 E02-1039 8.0 Data Quality Objectives 8.1 Absolute Recovery: 8.1.1 The absolute recovery of the method will be evaluated separately in human serum and plasma and rat serum. For each matrix, the samples will be fortified at two levels of (500ppt and 5ppb). All samples will be extracted through the method, and analyzed by comparison with external calibration of non-extracted standards. The non-extracted standard calibration curves will be prepared in methanol, and will consist of a minimum of nine (9) levels, including a methanol blank. The best appropriate regression will be used to describe this curve (for best accuracy at all levels of the standards). 8.1.2 The accuracy (% recovery) and precision (%CV of the recoveries) will be determined at each level, and for all levels combined. There is no control limit for accuracy; precision must be better than 15% at each level. 8.2 Calibration: Calibration curves will be prepared from extracted matrix standards, in Chinese plasma and rabbit serum. The curves will consist of a minimum of nine (9) levels and a matrix blank. The equation will be determined by regression analysis using the peak areas of the analyte. The accuracy of each level will be verified. Any level outside 75% -125% of nominal must be deactivated, and regression re-calculated, except the LLOQ which must be within 30% of nominal. All levels must show a response greater than twice that of the blank. A maximum of four (4) levels may be deactivated in any one set, or the set will be re-analyzed. 8.3 Limits of Quantitation (LOQ): The lower limit of quantitation (LLOQ) will be determined for each analyte. The level determined as the LLOQ must show a recovery within 75% -125% for the analytes and must show a response greater than twice that of the blank. These limits will be determined during the course of in rawt h e s t u d y a n d d o c u m e n t e d th e d ata. Should t h e LLOQ level calibration be de-activated in a particular set, the practical limit of quantitation for this set will be raised to the next acceptable level. Samples below the practical LOQ of that set will be reanalyzed until quantitated in a set including the validated LLOQ. The upper limit of quantitation (ULOQ) will be determined in serum and plasma. The level determined as the ULOQ must show a recovery within 75% - 125% for the analytes. These limits will be determined during the course of the study and documented in the raw data. Should the ULOQ level calibration standard be de activated in a particular set, the practical limit of quantitation for this set will be lowered to the next highest acceptable level. Any sample with an area greater than 110% of the highest acceptable standard will need to be diluted into the range of the calibration curve. If samples are diluted into the range of the curve during analyses and enough sample remains, a post-run dilution validation will be performed to verify sample values. To Bract Copy of Original 3M Environmental Laboratory Page 6 of 10 Analysis of Endogenous Fluorochemicals 3M Environmental Laboratory E02-1039 E02-1039 perform the dilution validation, one sample will be separated into two representative samples (i.e. two 1 mL aliquots for fluid samples or two 1 gram amounts for tissue samples) then diluted using two procedures. The first procedure consists of diluting the sample with additional matrix prior to extraction (sera adding sera), while the second procedure consists of diluting the extract with solvent post-extraction (methanol extract adding additional methanol solvent). If the values are not within 15% of each other additional testing will be required to determine which value is a correct representation of the sample concentration. 8.4 Use o f Confirmatory Methods Confirmatory methods are typically not needed with LC/MS/MS analysis. 8.5 Demonstration of Specificity 8.5.1 The identification of analytes will be substantiated by chromatographic retention time, by the characteristic primary ion, the characteristic product ion, and isomeric proportions (where applicable). 7.6 Control of Bias Two levels of matrix fortifications, prepared at known concentrations of the test substance and bracketing the anticipated range of the method will be evaluated to determine recovery and to evaluate method performance. P.eagent and matrix blanks will be run with each set to evaluate the level of background interferences. 9.0 Statistical Methods and Calculations 9.1 Statistical methods for the analytical results will be limited to the calculations of m eans, standard deviations, and relative standard deviations (as appropriate). 10.0 Report________________________________________________________ A report of the results of the study will be prepared by 3M Environmental Laboratory. The report will include, but not be limited to, the following, when applicable: 10.1.1 Name and address of the facilities performing the study, 10.1.2 Dates upon which the study was initiated and completed. 10.1.3 A statement of compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 10.1.4 A copy of the protocol, and any amendments and deviations. 10.1.5 A description of the methods used to conduct the test(s). The report will Exa c t Copy o f Original Initial Data 3M Environmental Laboratory Page 7 of 10 Analysis of Endogenous Fluorochemicals 3M Environmental Laboratory E02-1039 E02-1039 contain updated methods incorporating any changes or improvements. 10.1.6 A description of the test system. 10.1.7 A description of any circumstances that may have affected the quality or the integrity of the data. 10.1.8 The name of the Study Director and the names of other scientists, professionals, and supervisory personnel involved in the study. 10.1.9 A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the analytical chemistry data, and a statement of the conclusions drawn from the analyses. 10.1.1 OStatistical methods used to evaluate the data, if applicable. 10.1.1 IThe signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable. 10.1.12The location where raw data and the final report are to be stored. 10.1.13A statement prepared by the quality assurance unit listing the dates that study inspections and audits were made and the dates of any findings reported to the Study Director and Management. 10.2 If it is necessary to make corrections or additions to a final report after it has been accepted, the changes will be made in the form of an amendment issued by the Study Director. The amendment will clearly identify the part of the final report that is being amended, provide the reasons for the amendment, and will be signed by the Study Director. 11.0 Quality Assurance 11.1 The 3M Environmental Laboratory Quality Assurance Unit will review the protocol and audit study conduct, data, and the final report to determine co m p lian ce w ith G ood Laboratory Practice Standards and w ith 3M Environmental Laboratory Standard Operating Procedures. The Quality Assurance Unit will report all findings to the Sponsor Representative and the Study Director. 12.0 Location of Raw Data, Records, and Final Report_____________________ 12.1 Original data or copies thereof, will be available at 3M Environmental Laboratory. When the final report is completed all original paper data, including those items listed below, will be retained in the archives of 3M Environmental Laboratory following signing of the final report. 12.2 The following raw data and records will be retained in the study folder in the archives according to 3M Environmental Laboratory SOPs. 12.2.1 Approved protocol and amendments Exact Copy of Original Initial Oats 3M Environmental Laboratory Page 8 oMO Page 216 of 225 Analysis of Endogenous Fluorochemlcals 3M Environmental Laboratory E02-1039 E02-1039 12.2.2 Study correspondence 12.2.3 Shipping records . 12.2.4 Raw data 12.2.5 Approved final report (original signed copy) 12.2.6 Electronic copies of data 12.3 The following supporting records will be retained separately from the study folder in the archives according to 3M Environmental Laboratory SOPs: 12.3.1 Training records 12.3.2 Calibration records 12.3.3 Instrument maintenance logs 12.3.4 Standard operating procedures, equipment procedures, and methods 13.0 Sample Retention__________________________________________________ 13.1 A portion of the reference substances used in the study will be retained in the laboratory for a period of not less than 2 years after a report is issued. 13.2 Sample extracts will be retained and refrigerated for a period of not less than three months from the time of analysis. 14.0 Protocol Amendments and Deviations 14.1 Amendments and deviations to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. All changes to the protocol will be indicated in the final report. Any other changes will be in the form of written deviations, signed by the Study D i r e c t o r a n d filed with the r a w data. 15.0 Attachments None 3M Environmental Laboratory Exa ct Copy of Original initial Datg Page 9 of 10 Page 217 of 225 Analysis of Endogenous Fluorochemicals Protocol Signature 3M Environmental Laboratory E02-1039 EQ2-103g Protocol Approval Sponsor . William K. Rcagen, PhD. 3M Environmental Laboratory Date: 3M Environmental Laboratory Exa c t Copy o f Original *)< IP/Z\/6 L - Initial Data Page 10 o f 10 Page 218 of 225 3M Environmental Laboratory E02-1039 Study Title Analysis of Endogenous Fluorochemicals in Normal Pooled Human Serum and Plasma PROTOCOL AMENDMENT NO. 1 Am endm ent Date: October 31,2002 Perform ing Laboratory 3M Environmental Laboratory' Building 2-3E-09 ' 935 Bush Avenue St. Paul, MN 55144-1000 Laboratory Project Identification 3M Environmental Laboratory Study LIMS #E02-1039 Exact Copy o f Orig'nal Page 219 of 225 3M Environmental Laboratory E02-1039 Protocol UEOO-1311 Amendment 1 This amendment modifies thefollowing portion(s) of the protocol: 1. P rotocol r eads: S ection 8.1.1: T he absolute recovery o f the m ethod will b e evaluated sep arately in h u m an serum and plasm a and rat serum. For each matrix, the samples will be fortified at two levels o f (500ppt and 5ppb). All samples will be extracted through the method, and analyzed by comparison with external calibration o f nonextracted standards. The non-extracted standard calibration curves will be prepared in methanol, and will consist o f a minim um o f nine (9) levels, including a methanol blank. The best appropriate regression will be used to describe (his curve (fo r best accuracy at all levels o f the standards). Amend to read: The absolute recovery o f the method will not be evaluated as the matrix effects render this type o f comparison limited in its usefulness. R eason: A planned change in the study's direction. 2. Protocol reads: Section 8.2: Calibration: Calibration curves will be prepared from extracted m atrix standards, in C hinese plasm a and rabbit serum. Tire curves will consist o f a minimum o f nine (9) levels and a m atrix blank. A m end t o r ea d : Calibration: Calibration curves will be prepared from extracted matrix standards in Chinese plasma. T he curves will consist o f a minimum o f nine (9) levels. Reasons for not using one or m ore o f these standards in the construction o f a calibration curve are up to the discretion o f the analyst and will be docum ented in the raw data. Reason: The sam ples were run against 9 extracted standards without a blank, but not all o f these standards were used to construct the curve. Additionally rabbit curves were not included in the data for this study. 3. Protocol R eads: S ection 7.1: Analysis o f the test and reference substances will be conducted in the 3M Environm ental Laboratory. The analyses will be conducted as described by 3M Environmental Laboratory M ethod E T S -8 -2 3 1, "S olid P hase E xtraction and Analysis o f Fluorochem ical Com pounds from B iological m atrices" . Amend to R ead: Analysis o f the test and reference substances will be conducted in the 3M Environmental L aboratory. T h e analyses will be conducted as described b y 3M E nvironm ental Laboratory M ethod E T S -8 -2 3 1, "Solid Phase E xtraction and Analysis o f Fluorochemical Compounds from Biological matrices". Selected ion m onitoring w ill be u sed to detect the presence o f the target analytes. The masses scanned will be docum ented in the raw data and in the final report. R eason: Add clarity. 4. P rotocol R eads: Section 8J Anv sam ple w ith an area greater than 110% o f the highest acceptable standard will need to be diluted into the range o f the calibration curve. I f sam ples are diluted into the range o f the curve during analyses and enough sample remains, a post-run dilution validation will be perform ed to verify sam ple values. To perform the dilution validation, one sample will be separated into two representative samples (i.e. two 1 mL aliquots for fluid samples or two 1 gram amounts for tissue sam ples) then diluted using two procedures. The first procedure consists o f diluting the sample with additional m atrix prior to extraction (sera adding sera), while the second procedure consists o f diluting the extract with solvent post-extraction (methanol extract adding additional methanol solvent). I f the values are not w ithin 15% o f each other additional testing w ill be required to determ ine w hich value is a correct representation o f the sample concentration. Exact Copy o 3MEnvironmental Laboratory Page 2 of 6 rV l( Initial Page 220 of 225 3M Environmental Laboratory E02-1039 Protocol UE00-1311 Amendment 1 Amend to Read; Any sample with an area greater than 110% o f the highest acceptable standard will be reported as >ULOQ. Reason: The purpose o f this study is to demonstrate the presence o f the target analytes in pooled human serum and plasma. It is more important to show that endogenous levels are detectable than to show specifically what those levels are. S. P n o f o c o i . Re a ps: Section 1.1 T he purpose o f this study is to quantify perfluorohexanoic acid (C 6), perfluoroheptanoic acid (C7), pentadecafluorooctanoic acid (C8), heptadecafluorononanoic acid (C9), nonadecafluorodecanoic acid (CIO ), perfluorodecanoic acid ( C l 1), perfluorododecanoic acid (C12), tetrahydroperfluorooctane sulfonate (THPFOS), tetrahydroperfluorodecane sulfonate (THPFDS), and perfluorooctanesulfonate (PFO S) in normal pooled hum an serum and plasma. This study does not have a typical test substance that is dosed onto a specific controlled test system as per a conventional GLP study. Amend to Reap: T he purpose o f this study is to quantify perfluoroheptanoic acid (C7), pentadecafluorooctanoic acid (C8), heptadecafluorononanoic acid (C9), nonadecafluorodecanoic acid (CIO), perfluorodecanoic acid (C l 1), perfluorododecanoic acid (C l 2), tetrahydroperfluorooctane sulfonate (THPFOS), tetrahydroperfluorodecane sulfonate (THPFDS), and perfluorooctanesulfonate (PFOS) in normal pooled human serum and plasma. T his study does not have a typical test substance that is dosed onto a specific controlled test system as per a conventional GLP study R e a s o n : .T h e Q , com p o u n d was elim inated fro m the study. 6. Protocol R e a p s : Section 3.1 Test Substances Test Substance Perfluorohexanoic Acid Tetradecafluoroheptanioc Acid Pentadecafluorooctanoic Acid H eptadecafluorononanoic Acid Nonadecafluorodecanoic Acid Perfluoroundecanoic Acid Perfluorododecanoic Acid 1 H ,2 H ,3 H .4 H -p e rflu o ro o c ta n e s u lfo n a te 1H,2H,3H,4H-perfluorodecane sulfonate Potassium Perfluorooctanesulfonate Amend to Read: Test Substances Test Substance T etradecafluoroheptanioc Acid P entadecafluorooctanoic Acid 3MEnvironmental Laboratory Formula CsFnCOOH CsFuCOOH CrFisCOOH C.F,7COOH CsFuCOOH C ,oF21COOH C11F23COOH CaHsFtaOsS CtoHsFoOjS CjFijSOj'K ' . Formula C4F,jCOOH CrFtsCOOH Page 3 of 6 Exact Copy of Origino! V W / O. Data Page 221 of 225 3M Environmental Laboratory E02-1039 Heptadecafluorononanoic Acid Nonadecatluorodecanoic Acid Perfluoroundacanoic Acid Perfluorododecanoic Acid t H,2H,3H,4H-perfluofooctanesulfonate 1H,2H,3H,4H-perfluorodecane sulfonate Potassium Perfluorooctanesulfonate R e a s o n : T he C 6 com poun d was elim in ated fro m the study. CaF,,COOH CaFtttCOOH CioFjtCOOH C11F23C0 0 H CiHsFtjOjS CioHsFtjOjS CiFnSOj'K* Protocol MOO-1311 Amendment 1 7, P r o t o c o l R e a d s : Se c t io n 4.0 Reference Substances Reference Substance Perflut >rohexanoic Acid Tetradecafluoro heptanloc Acid Pentadecafluoro octanoic Acid Heptadecafluoro nonanoic Acid Nonadecafluoro decanoic Acid Perfluoroun decanoic Acid Perfluorodo decanoic Acid 1H.2H.3H.4Hperfluorooctane sulfonate 1H,2H,3H,4Hperfluorodecane sulfonate Potassium Perfluorooctane sulfonate Amend to Reap Formula CsFnCOOH C.F,3COOH C,F,sCOOH CoF^COOH CsFnCOOH C,0F2i COOH Ci ,F23COOH CaHsFijOjS C10H5F17O3S C,F,7SOj K* Traceability tt TCR-047 TCR-267 TCR-617 TCR-618 TCR-036 TCR-619 TCR-037 TCR-343 Source SMM* 236-1B-10 Aldrich Oakwood Products Oakwood Products Oakwood Products Oakwood Products Oakwood Products Physical Description Colorless Liquid Clear Crystals White Crystals White Crystals White Solid White Crystals White Powder SynQ uest Labs W hite Powder Purity TBD** 99.5% > 97% > 99% 98% > 99% 96% TBD TCR-627 Pace Analytical White Crystals 94.7% TCR-018 SMM* 236-1B-10 White Powder 86.9% Storage Conditions Frozen Frozen Ambient Temperature Ambient Temperature Frozen Ambient Temperature Frozen Frozen Ambient Temperature Frozen Reference Substances Reference Substance Tetradecafluoro heptanioc Acid Formula CiFnCOOH [ Pentadecafluoro octanoic Acid C ,F ,sCOOH Traceability # TCR-267 TCR-617 Source Aldrich Oakwood Products Physical Description Clear C ry s ta ls White Crystals Purity 99.5% > 97% Storage Conditions Frozen Ambient Temperature Exa c t Copy o f Origina! . LL/l lJ.qT - Initial Date 3MEnvironmental Laboratory Page 4 of 6 Page 222 of 225 3M Environmental Laboratory E02-1039 Protocol #E00-1311 Amendment 1 Pentadecafluoro octanoic Add c , f 15c o o h TCR-617 Oakwood Products White Crystals > 97% Ambient Temperature Heptadecafluoro nonanoic Acid c , f ,7c o o h TCR-618 Oakwood Products White Crystals >99% Ambient Temperatura Nonadecafluoro decanoic Acid CgFioCOOH TCR-036 Oakwood Products White Solid 98% Frozen Perfluoroun decanoic Add CioFrtCOOH TCR-619 Oakwood Products While Crystals >99% Ambient Temperature Perfluorodo decanoic Add C,,Fn COOH TCR-037 Oakwood Products White Powder 96% Frozen 1H.2H.3H.4Hperfluorooctane sulfonate CeMsFisOaS TCR-343 SynQuest Labs While Powder TBD Frozen 1H,2H,3H,4Hperfluorodecane sulfonate C10H5F17O3S TCR-627 Pace Analytical White Crystals 94.7% Ambient Temperature Potassium Perfluorooctane sulfonate CsFpSOjK* TCR-018 SMM* 236-1B-10 White Powder 86.9% Frozen j; Reason: T he C6 com p o u n d was elim inated from the study. 8 . P r o t o c o l R e a d s : Se c t io n 6.0 Surrogate Matrix ______ . ; -, Surrogate Matrix: Source Traceability Rabbit Serum | Slgma-Aldrich TCR-686 m 6.4 Justification o f the Surrogate Matrix. B ased on prelim inary testing, norm al pooled rab b it serum contains very low endogenous levels o f the test analytes and is thereby suitable for use as a surrogate matrix. E 6. 5 Identification o f Surrogate Matrix. Sam ples shall be identified by the study num ber, date o f initial preparation, test substance or surrogate m atrix, sample number, replicate num ber (if applicable), analyses), and project leader. A m e n d TO r e a d : N one REASON: It w as d ecid ed n ot to use rabbit serum curves to generate data for this study. 9, Protocol R e a d s : SECTION 8.3 Lim its o f Q uantitation (LOQ): T he low er limit o f quantitation (L L O Q ) w ill be determ ined for each analyte. T he level determined as the LLOQ must show a recovery within 75% - 125% for the analytes and m ust show a response greater than twice that o f the blank. Exa ct Copy o f OriginsI 'n ( Initial 3MEnvironmental Laboratory Page 5 of 6 Page 223 of 225 3M Environmental Laboratory E02-1039 Protocol UE00-1311 Amendment 1 A m e n d TO R e a d : Limits o f Quantitation (LOQ): The lower limit o f quantitation (LLOQ) will be determ ined for each analyte. T he level determined as the LLOQ m ust show a recovery within 70% - 130% for the analytes. R e a s o n : Since this is a screening-type study it was appropriate to relax the acceptable recovery levels in ord e r to avo id re -running sam ple sets. Amendment Approval W illiam K. R ea g en , Sponsor Representative '/j Date 3MEnvironmental Laboratory Exa c t Copy o f Original Initial - l i / 1 !_ 0 7 _ Data Page 6 of 6 Page 224 of 225 Protocol E02-1039 Amendment #2 Study Title Analysis of Endogenous Fluorochemicals in Normal Pooled Human Serum and Plasma PROTOCOL AMENDMENT NO. 2 Amendment Date: 6 December, 2002 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS E02-1039 This amendment modifies the following portion(s) of the protocol: P A G E 3 , S e c t i o n 1 .1 , P R O T O C O L r e a d s : The purpose of this study is to quantify, perfluoroheptanoic acid (C7), pentadecafluorooctanoic acid (C8), heptadecafluorononanoic acid (C9), nonadecafluorodecanoic acid (CIO), perfluorodecanoic acid (Cl 1), perfluorododecanoic acid (C12), tetrahydroperfluorooctane sulfonate (THPFOS), tetrahydroperfluorodecane sulfonate (THPFDS), and perfluorooctanesulfonate (PFOS) in normal pooled human serum and plasma. This study does not have a typical test substance that is dosed onto a specific controlled test system as per a conventional GLP study. A M E N D TO r e a d : The purpose of this study is to quantify perfluoroheptanoic acid (C7), pentadecafluorooctanoic acid (C8), heptadecafluorononanoic acid (C9), nonadecafluorodecanoic acid (CIO), perfluorodecanoic acid (Cl 1), perfluorododecanoic acid (C l2), and perfluorooctanesulfonate (PFOS) in normal pooled human serum and plasma. This study does not have a typical test substance that is dosed onto a specific controlled test system as per a conventional GLP study. REASO N: THPFOS and THPFDS were removed from the list of analytes in the revised report because the data was intended as quantitative and only screening estimates could be obtained. In addition, THPFOS and THPFDS were removed from the list of test substances, section 3.0, page 3 and the list of reference substances, section 4.0, page 4. Amendment Approval -- William K. Reagen, Sponsor Representative Date Page 225 of 225