Document Rzaee1p9KmYVGbw1J3GX0G9B

Iif i 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 No r m a l P o o l e d H u m a n S e r u m a n d Plasma 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 CONTAIN NO CBI 000093 3M Environmental Laboratory E02-1039 This page has been reserved for specific country requirements. 000094 Page 2 of 225 3M Environmental Laboratory E02-1039 GLP Compliance 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 conducted In compliance with Toxic Substances Control Act (TSCA) Good Laboratory Practice (GLP) Standards, 40 CFR 792, with the exceptions listed below: 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. 000095 Page 3 of 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. Inspectio n D ates 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 QAU Representative !<.J u l b / 02- . Date 000096 Page 4 o f225 3M Environmental Laboratory E02-1039 Table of Contents GLP Compliance Statement............................................................................................................................3 Quality Assurance S tatem 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 a b le s ............................................................................................................................. 56 Attachment C: Chromatograms...................................................................................................................... 63 Attachment D: Prep Sheets and Traceability Information.......................................................................... 125 Attachment E: Protocol and Amendment..................................................................................................... 208 000097 Page 5 of 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: C 7 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 000038 Page 6 o f225 Study Information Sponsor 3M Environmental Laboratory E02-1039 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. 000099 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. 00014)0 Page 8 o f225 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 C 11 C i0 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] tnoresource TCR-687 (ng/mL) 17.0 [0.144] 0.295 [0.327] 0.605 2.95 [< 0.010]' Uolden 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 1.b. Endogenous levels of test substance in normal pooled human Plasma Analyte PFOS C-|2 Cn Ci0 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 Vl/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. Value < LOQ. Accurate mass measurements and elemental compositions were obtained for endogenous C7C 11, 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 O O O lO l 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. 000102 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) c8f,3coo- CtF,5C00` C8Fi7COO C9F19COO C10F21COO Ci |F23COO C8F17S03- The molecular structures are given below. Name: C7 Chemical Name: Perfluoroheptanoate Molecular Weight: 363, as shown 000103 Page 11 of 225 3M Environmental Laboratory E02-1039 Name: CC8hemical Name: Pentadecafluorooctanoate Molecular Weight: 413, as shown Name: CC9hemical Name: Heptadecafluorononanoate Molecular Weight: 463, as shown F F F FO 0 FF FFFF Name: CIO MChoelemciuclaalrNWameige:ht:No5n1a3d,eacsafslhuoowrondecanoate Name: C ll Chemical Name: Perfluoroundecanoate Molecular Weight: 563, as shown 000104 Page 12 of 225 3M Environmental Laboratory E02-1039 Name: C12 MChoelemciuclaalrNWameige:ht:Pe6r1il3u,oarsodshodoewcnanoate Name: PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 499, as shown F FFF FF F SO 3 F F F F 000105 Page 13 of 225 Reference Substance 3M Environmental Laboratory E02-1039 Table 3. Characterization of the Reference Substances Reference Substance Formula Traceability # Source Tridecafluoro heptanioc Acid Pentadecafiuoro octanoic Acid Heptadecafluoro nonanoic Add Nonadecafluoro decanoic Acid Perfluoroun decanoic Acid Perfluorodo decanoic Acid Potassium Perfluorooctane sulfonate C6F,3COOH TCR-267 C7F15COOH TCR-617 c8f,7cooh TCR-618 C9F19COOH TCR-036 c ,0f2,cooh TCR-619 C,,F23COOH TCR-037 CbFitSOs'IC TCR-018 Aldrich Oakwood Products Oakwood Products Oakwood Products Oakwood Products Oakwood Products SMM* 236-1B-10 ... ........ 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 ` Documentation of the method of synthesis is located at the source. Reference substances confirmed as linear isomers by NMR. 000106 Page 14 of 225 Test Systems 3M Environmental Laboratory E02-1039 Table 4. Description of Test Systems Used in this Study T est 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 Test System. Based on preliminary testing, normal pooled serum and plasma contain endogenous levels of the test substances. Identification of Test System. Samples were identified by the TCR number. Collection of Test Systems. 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 Matricies." 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 Method, 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 C 18 solid phase extraction 000107 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 Chromatograph: 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: Time (min.) 0.00 10.00 11.00 11.50 12.50 13.00 16.00 %B 40% 90% 90% 100% 100 % 40% 40% Mass Spectrometer: Micromass Quatro Ultima triple quadrupole mass spectrometer Software: Masslynx version 3.5. Mass Spectrometer Acquisition Parameters: (C7) Channel Parent Ion (mM Daughter Ion (m/z) Collision Cone (V) Enerav ieV) 1 363.00 119.00 20 20 2 363.00 169.00 20 20 3 363.00 219.00 20 20 4 363.00 319.00 20 20 (Ca) Channel Parent Ion (m/z) Dauahter Ion (m/z) Collision Enerav (eV) Cone (V) 1 413.00 119.00 20 20 2 413.00 169.00 20 20 3 413.00 219.00 20 20 4 413.00 369.00 20 20 000108 Page 16 of 225 (C9) Channel 1 2 3 4 Parent Ion in M 463.00 463.00 463.00 463.00 (C ,o) Channel 1 2 3 4 (C1 1 ) Channel 1 2 3 4 Parent Ion in M 513.00 513.00 513.00 513.00 Parent Ion (m/z) 563.00 563.00 563.00 563.00 (C12) Channel 1 2 3 4 Parent Ion (m/z) 613.00 613.00 613.00 613.00 3M Environmental Laboratory E02-1039 Dauahter Ion (m/z) 119.00 169.00 219.00 419.00 Collision Enerav (eV) 20 20 20 20 Cone (V) 20 20 20 20 Dauahter Ion in M 119.00 169.00 219.00 469.00 Collision Enerav (eV) 20 20 20 20 Cone (V) 20 20 20 20 Dauahter Ion in M 119.00 169.00 219.00 519.00 Collision Enerav (eV) 20 20 20 20 Cone (V) 20 20 20 20 Dauahter Ion (m/z) 119.00 169.00 219.00 569.00 Collision Enerav (eV) 20 20 20 20 Cone (V) 20 20 20 20 000109 Page 17 of 225 3M Environmental Laboratory E02-1039 PFOS Channel 1 2 3 Parent Ion ia M 499.00 499.00 499.00 Dauahter Ion (m/z) 80.00 99.00 130.00 Collision Enerav (eV) 45 45 45 Cone (VI 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 psig 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. Demonstration 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. O O O llO 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. O O O lll Page 19 o f225 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 C12 C C10 C9 Cg 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]' [0.031] [0.068] 0.650 [0.025] Bioresource TCR-687 (ng/mL) 17.0 [0.144] 0.295 [0.327] 0.605 2.95 [< 0.010]' -- GoItferTWesT 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 C-12 C11 C10 c9 C8 C7 cnmese 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] Golden West 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. Value < LOQ. Accurate Mass Determination, Accurate mass measurements and elemental compositions were obtained for endogenous C7-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. 000112 Page 20 of 225 3M Environmental Laboratory E02-1039 Table 6. Accurate Mass Determination of Endogenous Test Substances Test Substance Accurate Mass TheMoraestsical C7 362.9739 362.9691 CC98 412.9637 462.9613 412.9659 463.9627 CPFIOOS 498.9280 512.9615 498.9297 512.9595 C ll 562.9542 562.9563 De(Mpvpiaamstis)on -153..43 -3.0 -3.3 3.9 -3.8 Probable Formula cCCCC98s710o0O0O23222FFFFF,1115I7379s C 1102F21 Daughter Ions of Endogenous Test Substances, Specific daughter ions were observed at characteristic retention times for endogenous C7-C 11 and PFOS in a concentrated SPE extract of normal pooled human plasma purchased from Golden West 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 Cg c9 c 10 Cn C 12 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 - C12 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 000113 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. 000114 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 d021 106002 Sm (Mn, 1x1) MRM of 4 Channels ES TIO 3.77e5 Area 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. 000115 Page 23 of 225 Figure 2: C7 Isomer Distribution 3M Environmental Laboratory E02-1039 d021 025069 Sm (Mn, 1x1) 10th - Solvent stan dar d 30 ppb %- 7 3 <= Unear 2670"' Branched = > 0 d021 Q25091 Sm (Mn, 1x1) 100-1 262917 3 _ <= Unear Chines e plasma 10ppb 1: MRM of 4 Channels ES TIO 2.09e5 Area 1 111111111111111 1: MRM of 4 Channels ESTIC 2.66e5 Area (branched not observed) 0 i | 1 1 i i | i 1..1 i |'"i i 1 i | i-n I"l..i I "1*^1 r`i 1'1 f 11 ~M|~n 1 t | I 1"'t*1 T i'i 1 1 1 1 1 1 1 1 1 I 1 d021025098 Sm (Mn, 1x1) 1: MRM of 4 Channels ES- 100: 3 Lampire s erum %' 73 11^ <= Unear TIC 2.98e4 Area (branched not observed) 0 r T i i t i r i T T ~ r i j 'T ..1 r r [ i r i i | i " r n j i 1 1 | Yf-j I ' i i r i i i --i""'i p ni" "i t 1 i *'t i i i i d021025100 Sm (Mn, 1x1) 1: MRM of 4 Channels ES- 10Qn - Sigma serum TIC 8.32e4 Area %(branched not observed) 7 -2 jr - 0- "`TVi1*T" T f '1 1 1 11111 1 iri [ iTT.i [~~T""i t-r..i n ~ "i 1..i 1 n i I I 1 I 1 | 1 r r..1 f-T 1 i*i,~flT~T`~i*'r 1 1 d021025102 Sm (Mn, 1x1) 1: MRM of 4 Channels ES- 1 ocb Golden W est serum u< TIC 4.93e4 Area % 7 .2 <= Unear f 4M (branched not observed) 0 T ""i "", --T*"`j' "i **> i r " r "i i ; i,""i r r ; 1 i" 1 1 | 1 1 1 1 | 1 r""r 2.50 5.00 7.50 Time1t " i m| i i i i j 1 t ~r " r ]~ i t \ t j t i 1 1 | 1 r "i 10.00 12.50 15.00 Summary of C7 Acid Chromatograms: Low to non-detect levels of the branched isomers of C7 acid were observed in these lots of commercial pooled human serum. 000116 Page 24 of 225 Figure 3: Ca Isomer Distribution 3M Environmental Laboratory E02-1039 do21024012 Sm (Mn, 1x1) 10Qn Sohrent standard 30 ppb %- 8.46 44441 8.22 <= Linear 2: MRM of 4 Channels ESTIC 3.23e5 Area Branched => 484 0- "i"i i""I.i i""i i.H.i n iTi i i i I i"T-rr'i-n..i'r 'f iTT ~i.IT T ' f"i"i i" i | i' i i "i | h i i p r r T""i i.i i "i i d021024034 Sm(Mn, 1x1) 2: MRM of 4 Channels ES- 110U0Un1 Chinese plasma 10ppb 68904273 <= Linear TIC 6.80e5 Area %8.21 Branched => 534 Q- T f"r-r"1.r ... I'"'' i d021024041 Sm (Mn, 1x1) ri" i i-1 i ~^i L i i i 1i 1 11 T 2: MRM of 4 Channels ES- Summarv of C Chromatograms: Low to non-detect levels of branched isomers of C8 were observed in these lots of commercial pooled human serum. 000117 Page 25 of 225 Figure 4: C9 Isomer Distribution d021 025069 Sm (Mn, 1x1) 3M Environmental Laboratory E02-1039 2: MRM of 4 Channels ES- d021025098 Sm (Mn, 1x1) 2: MRM of 4 ChannelsES- Summary of Ca Chromatograms: Low to non-detect levels of branched isomers of Cg were observed in these lots of commercial pooled human serum. 000118 Page 26 of 225 Figure 5: C10 Isomer Distribution 3M Environmental Laboratory E02-1039 d021024012 Sm(Mn, 1x1) 100-] Solvent standard 33 ppb %- 9.91 28022 9.77 Branched => 3 9 3 f n.i"| h..i..i'| ITTI i ri i i i i rTT""f"i"rr"r"|"i i""'i d021024034 Sm (Mn, 1x1) 100: 9.89 _ 46914 Chinese plasma 10ppb 3: MRM of 4 Channels ES- TIC <= Linear 2.87e5 Area i i i 11i iT'j.i i ri""|..i"i it"|..i"i i 3: MRM of 4 Channels ES TIO <= Linear 4.97e5 Area 983 Branched => 703 0 1 1I I I I I I T I I" I I -I I" I I I I I I I I I I I I I I ""I I I I I I I I I I I* d021024041 Sm (Mn, 1x1) IOO3 Lampire serum 9.88 1745 %: 9.70 Branched => " i ....... i ... ..................... ..... 3: MRM of 4 Channels ES- TIC <= Linear 2.00e4 Area 0 - 1 - 1 ... i " i " I -i ~ i i i i r " i " i i i i i i i i i i i i i r i i I i i i 'I...I ' i i i i ...I i i i""i i i i i i i i i i i i i i i i i i i i i I d021024043 Sm (Mn, 1x1) 100 3: MRM of 4 Channels ES- 9.84 TIC 2457 i <= Linear 2.88e4 % Sigma serum I f9.69 Branched => Area 0- 1 ' I 1` ` i " 1I 1' 1' I d021024045 Sm (Mn, 1x1) 100-1 ' Golden west serum 9.84 9223 ~ ] .I........ ... . ' ' ............ 3: MRM of 4 Channels ES- <= Linear TIC 1.00e5 Area %- i i f 1 ! iTM? f 11 " i ^ r i 9.66 Branched => 497 10.86 12.30 .1+ 651 949 . i-n-i i 1 1 ^WA --|.i-- 1 T- - 11 '1' tt~p"i 1 ehTime 2.50 5.00 7.50 10.00 12.50 15.00 Summary of Cm Chromatograms: Low to non-detect levels of branched isomers of C 10 were observed in these lots of commercial pooled human serum. 000119 Page 27 of 225 Figure 6: Cn Isomer Distribution 3M Environmental Laboratory E02-1039 d021025069 Sm(Mn, 1x1) " % . Solvent standard 30 ppb d021025091 Sm(Mn, 1x1) 100: Chinese plasme 10ppb 10.31 54214 " 3: MRM of 4 Channels ES- TIC f. 72e5 <= Linear Area 10.07 ,,Branch. ed, => 1031 " " f r i 'i 1 1 p r f ri 1 1 1 1 n i r t r * 1 0 .2 8 . 432S8 i""i" 1 1 1 1 n m n r i i i t t i t 'h i i i i 3: MRM of 4 Channels ES- TIC 4.54e5 <= Linear Area 0: i..... i .....i " " i ..... i dO21 025098 Sm (Mn, 1x1) 10.05 Branched => 703 t ......i.. ...11" 11 1111" i i " " i " 111" " 11 3: MRM of 4 Channels ES- 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Summary of Cn Chromatograms: Low to non-detect levels of branched isomers of C, , were observed in these lots of commercial pooled human serum. 000120 Page 28 of 225 3M Environmental Laboratory E02-1039 Figure 7: C12 isomer Distribution d021 0251 26 Sm (Mn, 1x1) 10Cb %- Solvent standard 30 ppb 10.75 _ 02828 2: MRM of 4 Channels ESTIC <= Linear 8.0A0ree5a (branched not observed) 0- i i i i I i l i i i i i i i i i i i i i..i ....... i i 1 1 r T " i""|""i' i i i [i...i i i i i > d021025148 Sm (Mn, 1x1) 1 0 th] 10.72 4Q7SQ %' C hiese plasma 10ppb 1"7""'........ I ...... 1 I 1 1 " 2: MRM of 4 Channels ESJIG <= Linear 6.69e5 Area (branched not observed) 1i n | 111i | r i ; 1 1i n 1 f-iTT r p 1-r-r|-i n 1jr n 1 \ 1iT t | r r r r j -r i n 7r r r i~[i 1 11 d021025155 Sm (Mn, 1x1) 1OCh Lampire serum 10.75 397 (branched not observed) 2: MRM of 4 Channels ESTIC <= Linear 1 6.96e3 Area [H 1 1 1i i i i I i...i i i I i i i i-- | - r T 'l " i-- J ...1...i l i i i i i i i i i i i i i i "i " i 1 i r i ...i 1 i i i i jt r - , i i | i t-~i....r j i r i i 2.50 5.00 7.50 10.00 12.50 15.00 Summary of Ci? Chromatograms: Low to non-detect levels of branched isomers of C 12 were observed In these lots of commercial pooled human serum. 000121 Page 29 of 225 Figure 8: PFOS Isomer Distribution d021'25126 3m (Mn, 1x1) 3M Environmental Laboratory E02-1039 1 MRM cf 3 Channels ES- Summarv of PFOS Chromatograms: Branched isomers of PFOS were observed in these lots of commercial pooled human serum. O O 01Z2 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 Spike percent recoveries: % Recovery EV = Eluate volume DF = Dilution factor Relative standard deviation: Relative Standard Deviation Standard Deviation -M---e-a--n---------- x 100 Percent deviation: n%/ D_ evi.ati.on = -T--h-e--o--r-e-t-i-c-a-T-l-h-C-e-oo-r-n-ee-t-.i-c--a--lC--Ca--loc-n-u-el-.a--t-e-d---C--o-n--e-. x 100 Means and standard deviations were calculated using Excel software. 00012a 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 C7C 11, 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. 000124 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 forTHPFO S 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 amendment was written to address the removal of THPFOS and THPFDS from the study. 000125 Page 33 of 225 3M Environmental Laboratory EQ2-1039 Signature Page W e certify that this report is a true and complete representation of the data for this study: Mark E. Ellefson Study Director Date / 9 /O 2 - William K. Reagen Testing Facility Management Date 000126 Page 34 of 225 Attachment A: Method 3M Environmental Laboratory E02-1Q39 000127 Page 35 of 225 3M Environmental Laboratory E02-1039 Record of Deviation I. Identification Study/Project No. Deviation Type: (Checkone) E02-1039 SOP Ta^ethod 0 Equipment Procedure Protocol Other: Document Number. ETS-8-231.1 Date(s) of occurrence: 10 Oct 02 to 25 Oct 02 li. Description: Required Procedure/process: ETS-8-231.1 section 9.6, "Quality Control (QC) Sample" see method for details. Actual Procedure/process: The continued accuracy o f the calibration curve was monitored through the re-injection o f a curve point after no more than 10 samples and at the end o f 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 o f the calibration curve and the extraction efficiency in each matrix tested. During the course o f the study 2/3 o f the QC samples prepared in the same matrix as the calibration curve were within 25% o f expected values. All o f the 5ppb spikes passed, but only 3 of 9 low spikes passed. ___ ________ _ _ _ _ _ _ _ _ __ (such as ameHnId.mAencttiiossnusedT, aSkOePnr:evision, etc.) No additional action will be needed. Recorded By: n QunllO \ WIV. iImpact on Study / Project Date: V ' / o: This deviation does not adversely affect the quality of the data in the context of the current study. 3M Environmental Laboratory {assigned by Study DirectorDorePvroijaecttioLneadNaot..ih_e enLd of study or project) 000128 Page 36 of 225 3M Environmental Laboratory E02-1039 3MEnvironmental Laboratory Method Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Method Number: ETS-8-231.1 Adoption Date: llju jo l Revision Date: jlZ/oz. Effective Date: J .// iftz. Approved By: Willliam K. Reagen Laboratory Manager Date A CCO O) ) fp, <D a"3 ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 1 of 19 000129 Page 37 of 225 3M Environmental laboratory E02-1039 1 Scope and Application This inethod describes the extraction of target analytes from fish, rat liver, rat sera, mouse liver, and mouse sera using solid phase extraction (SPE). This inethod may also be extended to other biological matrices provided that the data quality objectives are met 2 Method Summary An amount of 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 of 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 clarity the supernatant The supernatant is transferred to a clean tube, diluted with water, and passed through a pre conditioned O n SPE cartridge. Finally, the analytes of 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 1:5 or 1/6 is defined as: 1 mL of sample + 5 mLs of diluent for a total of 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 sisepdaersaitgionnedistothruestaianchtiheevecdo;mcpoomupnodusnodfs ianmterebset urnemdeorvseodmferosmolvdeinffticcuolnt dbiitoiolongsicaanldmelautrtiecetsheamndunindterrodouthceerds. intAo appropriate solvents for analysis. 3.3 Reagent grade water Water with no detectable concentration(s) of 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 for 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 run to dryness after the methanol and water washes. After washing is complete, add sample then allow all of the liquid to pass through the SPE column to dtyness. ETS-8-23I.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds front Biological Matrices Page 2 of 19 000130 Page 38 of 225 Exact Copy of Original UTC of g)/ 3M Environmental Laboratory E02-1039 Dbaor,nthoet oHpPeLraCtewsiolllviennittiaptuemauptsomabaotviceschauptdaociwtyn.o f400 bar (5800 psi) back pressure. If the back pressure exceeds 400 Do not run solvent pumps to dryness. 5 Interferences To minimize interferences, Teflon should not be used for sample storage or any part of 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 ofthose listed. 6.1 Instrumentation Vortex mixer, VWR, Vortex Genie 2 Ultra-Turrax T25 tissue homogenizer Vacuum Pump SPE Extraction Manifold Centrifuge, Mistral 1000 or 1EC Shaker, Eberbach or VWR Balance (+/- 0.1000 g) Micromass, Quattro II or Ultima triple quadrupole Mass Spectrometer equipped with an clectrospray ionization source HP 1100 or Agilent low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 6.2 Supplies and Materials Eppcndorfor 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 sampulc vials, Wheaton, 6 mL (or otter appropriate size) Centrifuge tubes, polypropylene, 15 mL and 50 mL Labels Graduated pipettes, glass Syringes, capable of measuring 5 pL to 1000 (lL Bottlc-Top Dispenser (capable of dispensing 5mL of solvent) SPE extraction cartridge, 1g, Sep-Pak 6 cc tri-functional Cn (Waters) 75 mL sample reservoir (or other appropriate size) Crimp cap glass autovials and caps Exact Copyof Original ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 3 o f 19 O O O IC I Page 39 of 225 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 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL vgoraludme wetaritcer.coSnttoarineeartcroonotmainteinmgpereraatguernet. grade water, mix until all solids are dissolved, bring to volume using reagent Nanodtes:urWroghaetne pstraenpdaarirndgpdreifpfaerraetniot nv,olaudmjuesst athcacnortdhionsgelyli.sted in reagents preparation, target analyte standard preparation, 7.2 Target analyte standard preparation Pforlelpoawreingtairsgaent aenxaalmytpelestoannldyaarndd(sm) faoyrotrhme asytannodtabrde acpuprvroe.priMateulftoicroamllpsotannednatrdanparleyptearasttaionndsa.rds are acceptable. The Weigh approximately 100 mg of 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 ofapproximately 1000 ppm ((Ig/mL). Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. Example calculation: 1000 (ig/mL x 5 mL/100 mL = 50 jlg/mL. Dilute working standard 1 with methanol to produce a working standard 2 solution of approx. 5.0 ppm. Example calculation: 50 (Ig/mL x 10 mL/t00mL = 5.0 (Ig/mL. Dilute working standard 1 with methanol to produce a working standard 3 solution of approx. 0.50 ppm. Example calculation: 50 (ig/mL x 1.0 mL/100 mL = 0.5 (Ig/mL. 7.3 Surrogate standard preparation Prepare surrogate 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 of surrogate standard into a 100-mL voluinetric flask and record the actual weight Bring to volume with methanol for a surrogate standard stock of approximately 900 -1100 ppm. Prepare a surrogate standard working standard. Transfer approximately 1 mL of surrogate standard stock to a 10-mL' volumetric flask and bring to volume with methanol for a working standard of 90-1 lOppm. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 7.4 internal standard preparation Prepare internal standardfs) The following is an example only and may or may not be appropriate for all internal standard preparations. Weigh approximately 90-110 mg of internal standard into a 100-mL volumetric flask and record the actual weight. 4 cn o i> o. t3 S ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 4 of 19 000132 Page 40 of 225 3M Environmental Laboratory E02-1039 Bring to volume with methanol for an internal standard stock of 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 of 90-1lOppm. 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. Tcaypppiecdalalyutforveisahlsmunattirlixansatlaynsdisa.rds are prepared with each analysis. Extracted standards and/ samples are stored in 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 ofmethanol is used as a solvent blank. Solvent blanks are not extracted. 9.1.2 Method Blank An aliquot of 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 of 1.0 mL or 1.0 g of matrix (diluted or homogenized) is used as a matrix blank. Other amounts may be used, as appropriate. Matrix blanks are prepared from one of three sources: 1) a study control matrix from a study caifonnrimatrtaoillss;auonsiremd3a)tloarsegucerenrieovrgeaadtteewsmittahantdraiaxsr,daamclsuporlevoebssetata;inn2de)dCacCocmVomsmmfeorecrricaailalmyll,oybuousebt tosatfiunadeydd)i.fsfaemTrehpneltemspoaeftcrtiihexeststohaamunseethsiepsesdcteuipedseynaadsneitnmhteaols,nt(utedhgey. matrixusedforthecurve. 9.1.3.1 Study control matrix curve-ifthe study control matrix is used for the curve, using the study control matrix (two spiked with surrogate and two not spiked). prepare four (4) matrix b lanks 9.1.3.2curveC, opmrempaerrecifaolulyr obtained (same species) matrix curve - if the commercially obtained (4) matrix blanks using the same commercially available matrix (two matrix spiked is used for the with surrogate and two not spiked). 9.1.3.3 Surrogate matrix curve same commercially available ifa surrogate matrix is used for the curve, prepare four (4) matrix blanks matrix and prepare four (4) matrix blanks using a commercially available using the matrix of the same species as the study animals (two spiked with surrogate and two not spiked). 9.1.3.4the If limited matrix study protocol or is available, the in the raw data. number of method and matrix blanks may be adjusted and will be noted in Exact Copyof Original 9.2 Sample Replicate Samples replicates arc prepared according to each study protocol or project outline. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 5 of 19 000133 Page 41 of 225 3M Environmental Laboratory E02-1039 9.3 Surrogate standard If surrogate standard is a component of the sludy, all samples are spiked with surrogate standard prior to extraction to oabbotavien. a concentration in the mid-range of the calibration curve, with the exception of blank samples as described Typically surrogate standard is spiked into the l.0 mL diluted/homogenized sample removed lor extraction. However, surrogate may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the 1.0 mL sample, or into die l.0 mL diluted/homogenized sample removed for extraction. 9.4 Internal standard If internal standard is a component of the study, all samples are spiked with internal standard after extraction to obtain a concentration in the mid-range ofthe calibration curve. Typically internal standard is spiked into the 2.0 mL ofextract in the lS mL centrifuge tube, before transferring to the autovial. 9.5 Lab Control Sample Lab control samples are not a component of this method. 9.6 Quality Control (QC) Sample Prepare quality control (QC) samples to monitor extraction efficiency and to verify the continued accuracy of 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 of a study. A minimum of 3 QC samples must be prepared (one at each level) on each day of sample extraction (e.g. If the study is such that saatmotpalleosfwtwillelbvee.e)xtracted on three different days then four QC samples must be prepared on each day of extraction for QC samples will consist of four samples at each of three levels of analyte. The levels listed below may be used and may represent sample concentrations diluted into the range of the calibration curve: Low level: 3X to 5X the LLOQ, M id-level: equivalent to a point near the middle of the calibration curve, High level: 80% of the ULOQ TainswjesouctciQhonCf,orwsdaimtehtepanlenmilnienivniemglsuwmahreconfaQtnhCarleysezaeQmdCplaepfsteerwraielnlvabelyerysaisnt.eanlySthzoeldvsa.emntpblelaninkjsecatrieonnosttacrotninsgidearfetedrstahmepllaesst bcuatlimbraaytiobne isntacnluddaerdd QC samples extracted with a particular sample set must be analyzed in the same analytical run. Any QC samples extracted during the course ofthe study may he included in subsequent analyses. If samples from multiple extraction dates an: analyzed in one analytical run, then QC samples from the same sample extraction dates must be included in diat analysis. Each QC is expected to show an accuracy of 75-125% of expected. A minimum of 2/3 of all QC samples must meet 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-anulyzed or rc-cxtractcd. 9.7 Sample Dilution Any sample with an area greater than that 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 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 O..7cScr3:i o oOCL o ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 6 ofl9 000134 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 of diluting the sample with additional matrix prior to extraction (fluid adding fluid), while the second procedure consists of diluting the extract with solvent post-extraction (methanol extract adding additional methanol solvent) dIfettehremrineleawtivheicphevracleunet idsiaffceorerrneccet riespnreostenwtiatthioinn o1f5t%hefsoarmthpelesecotnwcoenstraamtipolne.s; additional testing will be required to 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 of a minimum of 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, of 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 weight/volumes sheet or extraction worksheet, as appropriate. Four 1.0 mL aliquots, or other appropriate amount, ofcontrol matrix serve as matrix blanks. The standard concentrations and spiking amounts listed in Table 1 may be used, when appropriate, to spike one sstaamnpdlaersd acnudrvete.stAsatomtaplleosf. 9Tshtaendnaurmdsb,efrooufr mstaantrdixarbdlsanaknsd, abnladnkfosumr mayetbheodadbjluasntkesdaarse dperetepramreindeidn badydtihtieonantoalythset aQnCd documented in a note to file. Use Attachment C, or other appropriate fonn, as an aid in calculating the concentrations of the working standards. Refer to section 12 to calculate the actual concentration ofanalyte 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 sample. 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 of the target analyte(s) using MassLynx or other suitable software. Any level outside 75% -125% of nominal must be deactivated, and regression re-calculatcd, except the LLOQ which tmhrueset b(3e)wleivtheilns 3m0a%y boefdneoamctiinvaal.tedAlilnlaenvyelosnme ussett,sohrotwheasreetswpoilnlsbeegrree-aatnearlythzaend.twice that of the blank. A maximum of Exact Copyof Original U K . letIt! 01 ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 7 of 19 000135 Page 43 of 225 3M Environmental Laboratory E02-1039 T able 1 A ppr o x im vte S pik in g a m o u n ts fo r Standards and S pik es Usin g l.o m l o f M atrix (approWxoimrkaitnegcsotnancednatrrdation) pL Approximate final concentration of analyte in M atrix diluted 1:5 Approximate final IcnonFciennatlra2t.i0onmoLf vaonalulymtee - - Blank Blank 0.500 ug/mL 1.5 5.00 ng/R 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 lOOng/gornR/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 nR/gor 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 l.0000 g of tissue (+/- 0.1000 g), or other appropriate amount, using a dissecting scalpel. This part of the procedure is best performed quickly, not allowing the tissue to thaw. Weigh the tissue directly into a tared plastic sampulc 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 of reagent water to sampule vial, or other volume as determined by the analyst and documented in a note to file. Homogenize tire sample. Put the Ultra-Turrax 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 2.5 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 dean it with methanol after each sample. Refer to ETS-9-52 for mote information. 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 of 2.5 mL of water.), or other ratio as determined by the analyst and documented in a note to fdc. Exact Copyof Original MC , o /i, I o x. Initial Data OTS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 8 of 19 000136 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. See attached worksheet (Attachment A or similar worksheet) for documenting the remaining steps. Vortex mix the fluid sample for approximately 15 seconds, then transfer 1.0 mL of 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 S.O mL of reagent water to the 1.0 mL of 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 dAiflutetredti/shsoume oogr efnluizidedsasmamplpelsehfaovreabpepernoxpirmepaaterelyd aISccsoercdoinngdstothseecntitorannss1fe1r.11a.0ndm1L1,.2o,rvootrhteexr ampipxrooprrsihaatekevoblyumhaen,dtothea clean 15 mL polypropylene centrifuge tube. Return unused diluted/homogenized portions to the freezer after 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 of 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 of acetonitrile, cap, and vortex mix or shake by hand approximately 15 seconds. P30la0cerpamll osnamthpelems oodneltshelisstheadkienrsaetcatinonap6p.1ro).priate speed for 20 minutes to adequately mix (a settingi of approximately 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). Adsoedlcudatni4ot0nt..h0eCmsauLppeoarnnfdaretmaangitxenibnttyogirtnahvdeeerwtwiantagetersreinvtoetrhaaelc5tli0emamensL.50tIunmbteLh,itscaeksntientprgiftcuhagereeotrnudobeter.toofRinaedtmrdoiodtviuoecnesmaamnayyplobefestchfrheoammngatethrdiex(cis.eeon.littdrhisfeuisgnaetomatpnhldee may be put into the centrifuge tube and then die 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 15 kPA - to give an approximate elution flow of 5-7 mL/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 of 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 of methanol. Spike extracted blanks, samples, and standards with internal standard as described in this method. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 9 of 19 000137 Page 45 of 225 Exact Copyof Original LdSlIc>, Initial Date 3M Environmental laboratory E02-1039 Labe! each glass autovial, as appropriate, with the study number, vial file archive number, animal enxutmrabcctiro/gncdnadtcer,/tainmdcpanoainlytsot(rs)LpIeMrfSormnuinmgbethr,e mexatrtraicxt,iofni.nal solvent, analyte components (if needed), extraction type, 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 of 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") Y Y - Test year (02) MM = Test month (02) DD = Test day (04) a = First sample list (run) ofthe 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 of 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: IYYMMDD### or A020204001 I = Initial of instrument name YY = Test year MM = Test month DD = Test day Hit#= 3-digit sequential file number starting with 1 through 999 (001) Aanldsos,aamspplaerdt eosfctrhipetisoonms.plelist, assign a method (MS) for acquiring, an inlet file, a bottle number, an injection volume, To create a method, click on Method Editor button in the MS Status Pane and select SIR (Single Ion Recording) or MmaRssM(es()M. ulAtilpsloe RseetacthtieonacMquoinsiittoiorningst)a. rtSaentdIosntiozpatitoimn eMs.odSeaavse aapcpqruoipsirtiiaotne amnedthmodas.s tIof 4M99S/MorSotihnesrtraupmpernotpsriaartee eDmatpaloAyceqdu,iasidtdioitni"onfaolr padroddituiocnt aiolninffroargmmaetinotnataionnMinRfoMrm. ation may be collected. See Micromass MassLynx "Guide to Typically the analytical batch run sequence begins with system suitability, solvent blanks, and a set of extracted matrix standards. Sample extracts are analyzed with two QC simples 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. Solid Phase ExtractioEnTaSnd-8A-2n3a1l.y1sis of Fluorochcmical Compounds from Biological Matrices Page 10 of 19 000138 Page 46 of 225 3M Environmental laboratory E02-1039 11.4.2 HPLC set-up Set up sample tray according to the sample list prepared above. RSeetcourpdtahcetuHaPl cLoCndtoititohnesfionlltohwe iinngstrcuomndeinttiolnogsbooroakt, coornodthiteiornasppthroeparniaatleyslot ccaotniosnid:ers appropriate for optimal response. Sample size = 10 fiL injection Inject/sample = 1 Cycle time =* 10.0 minutes Flow rate * 300 pL/min Mobile phase: Solvent A - 2 mM Ammonium Acetate, Solvent B - Methanol Solvent gradient program: Tune % Solvent 13 0.00 10% 1.00 10% 5.50 95% 7.50 95% 8.00 10% 11.4.3 Instrument set-up RFiettfeedr twoitEhTaSn-A9-t2m4o, s"pOhpeerircatPiorenssaunrdeMIoaniinztaetnioanncSeoourfcteh,e" Mforicdreotmaialss.s Quattro II Triple Quadtupole Mass Spectrometer Check the solvent level in HPLC reservoits and refill if necessary. Check the stainless steel capillary at the end ofthe probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillaty. Turn on the nitrogen. Open the tune page. Click on operate to initiate source block and desolvation heaters. Open 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 "OnTM Observe droplets or mist coming out of the tip of the probe. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of'he probe if no mist is observed Allow to equilibrate for approximately 10 minutes. Typical instrument parameters include: Drying gas 250-400 liters/hour ES nebulizing gas 10-15 liters/hour ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page II of 19 000139 Page 47 of 225 Exact Copyof Original & P L c / / a_ Initial Date 3M Environmental Laboratory E02-1039 HPLC constant flow mode, flow rate 10-500 (lL/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 (mL)) (IW(g) (or IV(mL))+ DV (mL) Calculate actual concentrations of analyte in calibration standards using the following equation: Concentration (ng/gor ng/mL) Spike Concentration (ug/mL)x Spiked Amount (mL) 1000 ng SV (mL)x Matrix Amount (g/mL or mL/mL) 1ug IW = Initial weight (where 1.0 g = 1.0 mL ) IV Initial volume D V = D iluent volum e (reagent grade water) 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 of tissuc/fluid standard curve, assumed to be 1 g or 1mL/5 mL water 3 sample = MA of tissuc/fluid sample (___g or mL of sample/5 mL water) Calculate spike percent recoveries using the following equation: % Recovery Observed Result - Matrix Blank Result Spiking Level 100 ETS-8-23I.I Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 12 of 19 000140 Page 48 of 225 ; act Copyof Original &>/.?!/<2.^ nitiai Data 3M Environmental Laboratory E02-1039 Calculate relative standard deviation using the following equation: Relative Standard Deviation Standard Deviation Mean x I00 Calculate percent deviation using the following equation: % Deviation Expected Cone. - Calculated Cone. Expected Cone. x 100 Calculate actual concentration of analyte in fiuid (pg/mL): AR (ng/mL) * DF x 3 curve fmL/roU * FV fmU in Curve * 1.0 ue = (pg/g) 3 sample (mL/mL) FV (mL) in Matrix 1000 ng Calculate actual concentration of analyte in tissue (pg/g): AR fnn/gt x DF x 3 curve fg/mt) * FV (mL) in Curve 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 run. Prior to the calibration curve and after the last sample of the run 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 tire 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 of the system suitability fails, system maintenance must be completed prior to running a second set of system suitability samples and the system suitability must pass before starting the calibration. If system suitability fails at the completion ofa run, the sample set must be reanalyzed. 13.2 Quantitation The coefficient of determination value for the calibration curve, plotted by regression using the peak areas of the analyte(s), must be 0.990 or better. All active calibration curve points must be within 25% of the theoretical value with the exception of 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 of 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 rcextractcd. 13.3 Accuracy Two thirds of all quality control samples and 1/2 of each quality control sample at each level are expected to show an accuracy of 75-125%. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 13 o f 19 000141 Page 49 of 225 3M Environmental Laboratory E02-1039 Surrogates and internal standards must have a percent deviation < 50%. Deviations outside this range will be reanalyzed to confirm. If the second analysis confinns 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 of in noninfectious biohazard waste containers. Flammable solvent waste is disposed of in high BTU containers. Glass pipette waste is disposed of in broken glass containers located in the laboratory. 15 Records dCaotam.plete the extraction worksheet attached to this method, or other applicable worksheet, and store with the study raw Each page generated for a study must contain the following information (if applicable): study/project or instrument nanuamlybsetr., Oacthquerisiintifoonrmmateitohnodm,aiynbteegaradtdioend imf eatphpolidc,absalemtpolethenasmtued,y.extraction date, dilution factor (if applicable), and tPhreinset pthaegetusnaendpatagpee, sinatmopthlee liinsst,traunmdenact qtuunisinitgio. n method from MassLynx to include with the study raw data. Copy 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. eBlaeccktrounpicedleactat.ronic data to appropriate medium. Record in study notebook the file name and location of backup 16 Attachments Attachment A: Extraction Worksheet Attachm ent B: Sample W cight/Volum e W oiksheet Attachment C, Calibration Standard Concentration Worksheet Attachment D, Dilutions Summary Worksheet 17 References ETS-9-24, "Operation and Maintenance of the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source" ETS-9-52, "Operation and Maintenance of a Tissue Grinder" 18 Affected Documents None ETS-8-23U Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 14 o f 19 000142 Page 50 of 225 Exact Copyof Original ?o)? , / / ) . initial Data 3M Environmental Laboratory E02-1039 19 Revisions Revision Number l Revision Description Minor formatting changes. Added detailed information to ail sections concerning the extraction procedure, analytical procedure, and calculations. Added attachments and references. Revision Date 02/18/02 A ETS-8-23U Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 15 of 19 000143 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 description removed spike mix used mix used Type of column used and lot Elution solvent and volume Comments Blank m atrix______ TN -A -____________; Amount weighedtallquoted:___________ g/mL 1. Homogenize sample 2. Aliquot 1 mL o f diluted matrix into 15 mL polypropylene tube 3. S p ike sam p les accordingly 4. A d d ____ mL of ACN (TN-A-___________ ) to each diluted sample and shake or vortex mix 5. Shake sample for 20 min @ ______ rpm (Shaker________________ ) 6. Centrifuge sample for 10 min @ _______ rpm {Centrifuge_________________ ) 7. Add 40 mL o f ____________ water to 50 m l polypropelene centrifuge tube. 8. Decant extract into centrifuge tubes with water 9. Shake sample slightly to ensure proper mixing 10. Attach 6 mL C18 S P 6 cartridges and 75 mL reservoirs to vacuum manifold 11. Condition column with two washes of - 5 mL MeOH (TN -A-__________ ) - do not allow column to go lo dryness 12. Wash column with two washes of - 5 ml____________ water * do not allow column to go to dryness 13. Filter sample through conditioned column, discarding filtrate 14. Allow column to go to dryness. After dripping stops, drew a high vacuum through column for at feast 5 minutes. 15. Elute column with solvent {___________ TN-A-___________) into appropriate 15 mL centrifuge tube 16. Spike samples w ith _________uL of internal standard t t ______________________ , cone.______________________ 17. Transfer sample into appropriately labeled autovial and cap Note: tn 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 bo raised for low tubes and drying after most have been drawn down and shut off. CJ a s 3 ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 16 of 19 000144 Page 52 of 225 3M Environmental Laboratory E02-1039 Attachment B - Sample Weight/Volume Worksheet Prep Date(s): Analyst(s): Sample Matrix: Melhod/Reviiion: StudyNumber: Equipment Number: Final Salvent Sc TNNumber: Sample ID Initial Wt/Vol. c/mL/L Water Volume added (mL) Volume Removed (mL) Comments Exact Copyof Original ( A'r i lo fe /p .................. . i 1 i____________ Form Completion Verified By. ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 17 of 19 000145 Page 53 of 225 3M Environmental Laboratory E02-1039 Prepdatc(s): Analyte(s): Sample matrix: Mcthod/revision: Attachment C: Calibration Standard Concentration Worksheet Standardnumber: Equipment number: Final solventandTN: BlankTissueor Fluid/identlfler: Analytemixstdapprox. 0.500 ug/mL: Analytemixstdapprox. 5.00 ug/mL: Analytemixstdapprox. 50.0 ug/mL: SurroAgcattuealsctdonacpenptrroatxio.n1s0o0f sutga/nmdaLr:ds in the analyte mix SAtndacloyntee ug/mL 0.500 000...555000000 055..5.000000 55..0000 5555..0000..0000 Am'tAslpliked mL 0.0015 000...000100683000 000...000300258060 00..00214600 0000...00.10348020)6500 VoFAliunlmal le: mL 2.00 222...000000 222...000000 22..0000 2222....00000000 All Initial Fluid Dilution mL/mL 0000....1111666666667777 00..11666677 000...111666666777 00..11666677 00..11666677 All Initial Tissue Density 0000g....1111/m66660000L0000 0000....1111666600000000 0.1600 0000....1111666600000000 Calculated concentrations ofstandards in relation to the final 2.0 mL solvent and initial matrix 2.0 mLFinal Volume FluidMatrix TissueMatrix Analyte cFoinnael. ng/mL 0.375 0.750 2.00 4.00 8.00 14.0 20.0 40.0 60.0 SO.O 100 125 150 Surrogate Sntdg/cmoLne 100 SFuinrarlocgoantee nn/mL 0.500 Analyte cFoinnael. nc5/.0m0L 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 SSuntrdgr/ocmgoLantee 100 Surrogate Final cone ng/mL 6500 Analyte cFoinnael. n5g.0/f0i 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 SSuntrdgr/ocmgoLnatee 100 Surrogate Final cone 6"5g0/g0 ETS-8-23I.1 Solid Phase Extraction and Analysis of Fluoroclicmical Compounds from Biological Matrices Page 18 of 19 000146 Page 54 of 225 Exact Copy of Original .c m . i o J -- Initial Date 3M Environmental Laboratory E02-1039 Study: DilutionDate/Analyst: Box Number: SoramDpeslecrNiputmiobner 1/ Attachment D: Dilutions Summary Worksheet Solvent/TNNumber: ExtractionDate/Analyst: Matrix/Timepoint: Dilutions If if 1/ 1/ V 1/ Comments Verified By: Exact Copyof Original U tfi _ to3>/)_ Initiai Date N1/o1t0esd:ilution => _______of sample + ________ of solvent Form Completion Verified By: ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochcmical Compounds from Biological Matrices Page 19 o f 19 000147 Page 55 of 225 Attachment B: Data Tables 3M Environmental Laboratory E02-1039 000148 Page 56 of 225 Human Serum Data Quantitated with Chinese Plasma Calibration Curve All data is in units nq/mL____________________________ Lam pire Serum Sigm a Serum PFOS C12 C,, c10 C9 C, C, sam ple 1 <LOQ <LOQ <LOQ <LOQ <LOQ 0.73 <LOQ sam ple 2 <LOQ <LOQ <LOQ <LOQ <LOQ 0.57 <LOQ low spike <LOQ 0.65 0.82 0.68 0.79 1.39 0.75 High spike 5.47 5.70 7.02 6.34 5.91 6.81 6.51 sam ple 1 4.74 <LOQ <LOQ <LQQ 0.19 1.60 <LOQ sam ple 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 High spike 9.06 3.74 5.45 5.17 5.91 6.64 5.78 Golden W est Serum sam ple 1 sam ple 2 low spike 25.64 <LOQ 28.41 <LOQ 24.16 0.46 0.31 0.33 <LOQ <LOQ 0.85 0.60 0.96 0.84 1.30 5.54 5.65 4.64 0.23 0.15 0.41 High spike 26.10 3.65 6.42 5.20 6.62 9.12 4.91 B ioresource Serum sam ple 1 sam ple 2 16.79 17.26 <LOQ <LOQ 0.28 0.31 <LOQ <LOQ 0.63 0.58 2.83 3.06 <LOQ <LOQ High spike 16.78 2.65 4.94 4.32 4.95 7.39 1.47 A n a ly te PFOS Ci2 Cn Cio c, C, C, C alibration Range, ng/m L 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 bystem S u ita b ility Sam ples, CCV#1 CCV #2 CCV #3 R2 %RSD recovery, % recovery, % recovery, % 0.999596 0.999621 2.48 0.99 52% 77% 3% 52% ** .. 0.999767 2.46 105% 105% 99% 0.999471 1.45 86% 90% ** 0.9997 2.94 91% 105% 102% 0.998816 6.73 94% 87% ** 0.999569 2.37 87% 95% 93% M atrix B la n k s < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ "N o n e performed Low spike 16.44 <LOQ 0.96 0.73 1.11 4.03 0.18 13 CPTQ a> 000149 i Human Plasma Data Quantitated with Chinese Plasma Calibration Curve All data is in units nq/mL_____________________________ C hinese Piasm a Lam pire Plasm a PFOS c,2 c,, C C C C7 sam ple 1 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ sam ple 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 sam ple 1 9.89 < LOQ < LOQ < LOQ 0.37 2.30 0.20 sam ple 2 12.34 < LOQ < LOQ < LOQ 0.50 2.91 < LOQ low spike 13.15 0.48 0.46 0.49 0.78 3.34 0.52 High spike 16.19 4.53 4.42 4.01 4.88 7.31 5.43 G olden W est P lasm a sam ple 1 sam ple 2 low spike 16.20 < LOQ 20.32 < LOQ 13.56 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 Plasm a sam ple 1 sam ple 2 low spike 15.61 < LOQ 15.55 < LOQ 17.32 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 A n a ly te PFOS Ct2 Cti Cto c, C, C7 C alibration Range, ng/m L 1.0 30.0 0 .1 0 -3 1 .0 0 .1 0 -3 0 .9 0.10-30.3 0.25 - 30.4 0 .5 2 -3 1 .0 0.10-30.7 Chineese Plasma, Plasma Data Cal Curves s y s te m S u ita b ility Sam ples, CCV#1 CCV #2 M atrix R 2 "A R S D r e c o v e ry , % re c o v e ry , % B la n k s 0.999721 2.20 102% 84% < LOQ 0.999759 5.87 101% 84% < LOQ 0.999585 1.21 90% 78% < LOQ 0.999695 2.76 91% 83% < LOQ 0.999847 5.07 97% 80% < LOQ 0.999736 2.85 81% 89% < LOQ 0.999695 5.85 102% 75% < LOQ 00ePnrVa>i 000150 ' i i i i i i i i 11i I !!1 Human Serum Data, 5x Sample Scale-Up with 20uL Injection Volume Quantitated with Chinese Plasma Calibration Curve All data is in units ng/mL____________________________________ L am p ire S eru m S ig m a S eru m PFOS C,, C,, C,o C C. C, S am p le 1, C o rre cted S am p le 1, o n -C o lu m n C o n cen tratio n fo r C oncen tratio n F a c to r 24.859 < LOQ 2.486 < LOQ < LOQ < LOQ 0.313 0.031 0.675 0.068 11.734 0.250 1.173 0.025 S am p le 1, onC olum n C o ncentration > ULOQ (30.2) 0.177 0.760 0.575 1.234 13.396 0.126 S a m p le 1, C o rrected for C o ncentration F a c to r > ULOQ (30.2) 0.018 0.076 0.058 0.123 1.340 0.013 G olden W e s t Serum S am ple 1, on C olum n C o ncentration > ULOQ (30.2) 0.402 2.473 2.025 3.533 26.245 < LOQ S am p le 1r C orrected fo r C o n cen tratio n F a c to r > ULOQ (30.2) 0.040 0.247 0.203 0.353 2.625 < LOQ B io reso u rce S erum S am p le 1, o n -C o lu m n C on cen tratio n S am ple 1, C orrected fo r C o n cen tratio n F a c to r > ULOQ (30.2) 1.444 > ULOQ (30.2) 0.144 2.856 3.272 2.657 26.245 < LOQ 0.286 0.327 0.266 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__________________________________ L a m p ire P lasm a G o ld e n W e s t P lasm a S am p le 1, S am p le 1, C o rrected S am ple 1, on - C orrected for S am p le 1, o n -C o lu m n fo r C o n c e n tra tio n C olum n C on cen tratio n C o n c e n tra tio n F a c to r C o n cen tratio n F a c to r In n o v a tiv e R e se a rch P lasm a S am p le 1, S am p le 1, on - C orrected fo r C olum n C o n cen tratio n C o n cen tratio n F a c to r PFOS C,, C,, C ,. C,, C, C7 > 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, 10ul_ Injection Volume A n alyte PFOS C ,2 c,, c,, c. c. C, C a lib ra tio n R an g e, n g /m L 1.01 - 30.2 0 .1 0 4 -3 1 .1 0 .1 0 3 - 30.9 0.101 - 30,3 0.254 - 30.4 0 .1 0 3 -3 1 0 0.102 - 30.7 R2 0 .9 9 9 5 3 9 0 .9 9 9 6 9 6 0 .9 9 9 5 2 6 0 .9 9 9 9 0 7 0.999812 0.999441 0 .9 9 9 9 0 3 S ys te m S u itab ility S am ples, % R S D 4 .2 1 3.94 6.34 4 .0 2 4 .1 6 5 .9 9 6.21 CCV#1 recovery, % 155% 114% 95% 94% 92% 92% 106% M atrix B lanks < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ 000151 Page 59 of 225 Matrix Spike Recovery for Human Serum Data Quantitated with Chinese Plasma Calibration Curve Analyte PFOS Ci2 C,, Co c9 Ce 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 C C,o c9 C8 C7 Golden West 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% 000152 Page 60 of 225 Matrix Spike Recovery for Human Serum Data Quantitated with Chinese Plasma Calibration Curve Analyte PFOS Ci2 C11 C10 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 Cl2 C,, Cio Cg Cs c6 Golden West 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% o o iS 3 Page 61 of 225 3M Environmental Laboratory E02-1039 3M PharmacPeFuOticSalTseAlonmaleyrtiAcanlaRlyessiseaSrucmh manadryDevelopment Ms(Lu#CEbic/m0rMo2itSm-t1eaa0dsn3sua9Qsl)yinws-Tigasostohfpfe2earfqofpuololaroodmwlreeuiddnpghouulspemiantraigamnmaense-eotArefar-gfssillailegimnshttpetld1me1iao0nbs0sTtaqsaiupbnaeleetcdetrr1fn:oramormeyteHGrP.oLldTCehnesyWssatemesmtplBeciowoulaopsgleirdcuantlosaas Table 1 Inject: PDRAecWoArandvaeCedloloegMlnuwFgamtaslhoCsnvwoeoRRTulleaRauetnnnpmmagggutpnteeeth:::::: IonizaRtieosnolMutoiodne:: 20 pL E7NB50a115,m0.09eS0A3xpt000Iba2m--m(0is86m-ei,)l00nl/mm00tCnDinlm8a,. (TmRiuminne.) HNH204CwH/ 23Cm0M2 MeOH 11132707...500 90 0 99000 10 100 100 10 10 sAucmcumraatreizmedasins mTaebasleur2e:ments and elemental compositions obtained in this analysis are Table 2 ReTteimnteion 5.8 min 6.3 min 6.5 min 6.69 min 6.71 min 6.72 min 67..18 mmiinn Accurate Mass TheMoraestsical 362.9739 412.9637 526.9693 462.9613 445291682...999762318540 562.9542 362.9691 451226..99665190 444526916382....9999566297925477 562.9563 DeMviaastsion -153..43 (ppm) -135..08 -313.49..31 -3.8 PFroorbmaubllae QCCCCCc97881,H0ooOOOH0a2232O42FFFfFO1111j37571F39sFn1S7S C11O2F21 000154 Page 62 of 225 Attachment C: Chromatograms 3M Environmental Laboratory E02-1039 000155 Page 63 of 225 i i I i i I i i 1I f Exact Copyof Original Cminitial Jo/'D3a,;tag\_ 3M Environmental Laboratory E02-1039 000156 1i I I i i I i I i f / C ^ f* U > 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1 03 9 Page 65 of 225 Exact Copyof Original JI.nWitia.l... o/D3a,ta) on 0001S7 IIIIIIi ii 3 M ENVIRONM ENTAL LABORATORY E 0 2 -1 03 9 Page 66 of 225 Exact Copyof Original & i>niXtial )o /D3at^a o . 000158 1I >*i 1I I I i 11I f 3M NVIRONMENTAL LABORATORY E02-1039 Page 67 of 225 Exact Copyof Original IP / 3 7 . Initial Date n ( )l 5 9 ! i( t I i 1i I l1I 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1039 Page 68 of 225 Exact Copyof Original Y H . >J3 < I 9 3 - inittal Date 000160 i C l 3 (l H C tI Ii i t I 3 M E n v ir o n m e n t a l L a b o r a t o r y E 0 2 -1 0 3 9 Page 69 of 225 Exact Copyof Original 0 2 2 L _ 1 1 /1 . Initial Date ____ 000161 tIIi Iii Ii iIII ( L i R C -/I 3M Environmental Laboratory E02-1039 Page 70 of 225 Exact Copyof Origina! t X L _ Z i/ > p _ Initial Date 000162 1! ( I ! I I1I! 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1 0 3 9 Page 71 of 225 Exact Copyof Original X J Z L . h / i h r .2- l1 - 000163 i iiiiIIIIIiiIII CA- A 3M Environmental Laboratory E02-1039 Page 72 of 225 Exact Copyof Original CInritniacl u ZDiaita > 000164 <i i i i i i i i i i i 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1 03 9 Page 73 of 225 Exact Copyof Original CftTH _ js I I ll & - Inltial Data 000165 000166 i 3M Environmental Laboratory E02-1039 Page 74 of 225 Exact Copyof Original Initial Dafg  I I I <i i I I i I i ! I 3M Environmental Laboratory E02-1039 Page 75 of 225 Exact Copyof Original L WIni.t[ial. .lo/Zi/6^ fiata 000167 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1 03 9 Page 76 of 225 golden west plasma 5x scale up 10/23/02 ok 3M d021030001 100n BOldeii w e st plasm a 5s scale up 10/25/02 ok )M Environm ental Lab U l/H0021030001 2M (8 383) Sm {Mn, 2x1.00); Cm (264.291) davcy 0707*1 M -Oct*200211:2l:Sa lD e u M rie /< 1 3E S - 1.39*6 %- 0J d02103C 100n 8.37 8.08 rrv } ii| "I r f 1 I 8.37 Davey070799 30-0ct-2002 11:28:58 2: MRM of 4 Channels ES413 >369 5.21e5 ' 1 [ '1* 1 ' I " 1 ^ t * r * t T ~T" 2: MRM of 4 Channels ES413 >219 9.47e5 %- 50 100 150 200 250 300 350 400 450 S0 7.798.05 d021030001 100-, 2: MRM of 4 Channels ES8.37 413>169 4.09e6 %- d021030001 100-, 8.11 \ 8.37 2: MRM of 4 Channels ES413> 119 2.26e5 %- 0 -` < 1 1 i 1 1 1 21.0r 0 i 1 >r 4.00 6.00 8.17 W 8.00 10.00 12.00 14.00 Time 16.00 Exact Copyof Original InUitial-__- l ooa/ t*s/ ^ 000168 i iIiiI I;II iiIIt C 5 A(L(TN 3 M E n v ir o n m e n t a l La b o r a t o r y 9E 0 2 - 1 0 3 Page 77 of 225 Exact Copyof Original CATC / i / ^ T Initial Data 000169 I 1i 1i i i I t I i i i 11Ii C ? /O LtiS 3M Environmental Laboratory E02-1039 Page 78 of 225 Exact Copy of Original Ca Y){ M lI iT h - Initial Date -0 0 0 1 7 0" ............ ^ 11I I i [ I i i I i i i i ( I CS-/3C <h 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1 03 9 Page 79 of 225 ExactCopy of Origina! XJC L JA /I./ W - Initial Oats 000171 1I i I 11( S11I I 1I i I1 C '? 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1 03 9 Page 80 of 225 Exact Copy of Original L ir t \W jJ b Inttfal Date 000172 i I *i I i i i i 1I 1l Ii I1 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1 03 9 Page 81 of 225 Exact Copyof Original ITniKtial l/D3at\o/o*_ 000173 i Si ii 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1 03 9 Page 82 of 225 Exact Copy of Original -WlL-- JjJh /i^-- Initial Date 000174 / C .9 M a> 3M Environmental Laboratory E02-1039 Page 83 of 225 Exact Copyof Original InitiLal_ l Dl satiai o ^ . 000175 t <i i I i I i I t i i I I Ii {i 3 M E n v ir o n m e n t a l La b o r a t o r y E02-1039 Page 84 of 225 Exact Copy of Original CM ( I Initial Date 000176 I asi I 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2-1 039 Page 85 of 225 Exact Copy of Origina! 4fH ( JJ Z i/p X -initial Date 000177 I CLQ i 3M Environmental Laboratory E02-1039 Page 86 of 225 Exact Copyof Original CJf\l _ 6 /t/fl? initial Date _ 000178 Ii 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1 03 9 Page 87 of 225 Exact Copy of Original X IJmOtiaOi i J LDZateiitfv - 000179 iI O ff A u ti 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1 0 3 9 Page 88 of 225 Exact Copy nf Orir'na! C an - initial uait 000180 f/  I I i I i i 11I i I ! i 3M Environmental Laboratory E02-1039 Page 89 of 225 Exact Copy of Original X M L. )1-- Initial Date 000181 ' 1i ' f i i i i i i i C /o golden west plasma Sx scale up 10/23/02 ok 3M Environmental Lab .d021030003 327 (9,869) Sm (Mn, 2x1.00); Cm (325:330) 100 169191 218.739 268.273 2/ 68.870 194.714 % davey 070799 30-0ct-2002 12:03:28 1: Daughters of 513ES- 6.65e4 468.288 4/ 69.303 513.307 512.651. 319.229 413.531 112.331 152.452 318.572 \ 279.504 wa/V*a*amaVv**A 382.717 349.213 422250 / 495.097 mlz 50 ' VV 100 125 150 175 200 225 ' 250 275 300 325 350 " 375 400 425 450 475 500 525 " 550 Exact Copyof Original Initial Date 000182 3 M E n v i r o n m e n t a l La b o r a t o r y E 0 2 -1 03 9 Page 90 of 225 iciiitiiiliiilllli 3M Environmental Laboratory E02-1039 Page 91 of 225 Exact Copyof Original O CQ L_ J O / ^ t [ o K . Initial Date 000183 * Siiiliitlflliili Cl i o 3 M E n v ir o n m e n t a l La b o r a t o r y E Q 2-1039 Page 92 of 225 ExactCopyof Original riYK Initial Date 000184 14i t i i : i i i ) i I I ( i C ./ f l - c i h 3M Environmental Laboratory E02-1039 Page 93 of 225 Exact Copyof Original C m 1. tnltial Date 000185 3M Environmental Laboratory E02-1039 Page 94 of 225 Exact Copy of Origina! C M C .U/JL/. Initial Datfi oooiae ____________ _______________ 02048-21-7 solvent standard 10ppb 3M d021030013 %- dip ddth Davey070799 30-0ct-2002 14:55:16 2: MRM of 4 Channels ES513 >469 1.24e5 0 !iiIT d021030013 1Q0~i %- <''"i ' r ' ~'~r 9.74 r ' T ' T- T - i - T r i - n - | . I I 2: MRM of 4 Channels ES513 >219 1,68e5 0 -W n d021030013 100n %- l 1 1 ' 1 "I 9.74 r " | t 'i ' * I ,i I 2: MRM of 4 Channels ES- 513> 169 1.22e5 0 r ' I : i -I ! I' d021030013 ..... ! ' ! I ' - I I i' i- ' -i-i i,ry - 1 - r--|--TI --KT"|'-1 ? ! T -f 1"IT T--r ^ 'T I'-p -' 'i " i - n - f T - | T 'r I I 2: MRM of 4 Channels ES- 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 95 of 225 Exact Copyof Original fA Y X 0 /1 /& -- Initial Date 000187 II I 1I t 1I : I I I I I i I: I d i o A to ilL 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 96 of 225 Exact Copyof Original U/Xl_. -- initial Data o o o iss iiti I(ii 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 97 of 225 Exact Copyof Original to /3 i/c initial Date 000189 I1i i i !Iff 3 M E n v ir o n m e n t a l La b o r a t o r y E02-1039 Page 98 of 225 Exact Copyof Original Y1C S o h il S) Initial Data 000190 It1IIi Ii 000191 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 99 of 225 Exact Copyof Original o_ !I I 000192 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 100 of 225 i Exact Copyof Original Inlai Data ` lilil 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 101 of 225 Exact Copyof Original m , ilZ iZ i> 0 _ Initial Oats 000193 d u A C -id lilil 3M Environmental Laboratory E02-1039 Page 102 of 225 n/>ExactCopyof Original . 3 L fr n hfflaJ Oatg - '........ ^ 000194 C j i m _ ,ib !III 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 103 of 225 Exact Copy of Origina! QCL. _ ( I A / oX. ` Initial Oats "*r _ . 000195. ~' i d ll 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 104 of 225 Exact Copyof Original t L m/ i I m - IrOal Date 000196 1 li SI 3 M 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 105 of 225 Exact Copyof Origina] j o / f t l l /) T initial oats 000197 G o - /v u 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 106 of 225 Exact Copyof Original C tO i_ /jjh s J js ^ Initial Dao 000198 iI 000199 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 107 of 225 Exact Copyof Original C U pL- o / z i / zn oats Iii I 000200 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 108 of 225 Exact Copyof Original Initial -- 02048-21-7 solvent standard 10ppb 3M d021030014 C L isx %- 0 - I 7- r , T -- T - T -.d0?1030014 100 % i 1! i I i I Davey070799 30-0ct-2002 15:12:19 2: MRM of 4 Channels ES- TIC 1.09e6 Y | S 1,r " r 10.75 l T -l i - P i' T " i i-- ' v t i I l' l-- '-- r - T -- i-- !-- 1 1: Daughters of 613ESTIC 8.26e7 3 M E n v ir o n m e n t a l La b o r a t o r y E02-1039 Page 109 of 225 2.00 4.00 Exact Copy of Original Initial Date 6.00 8.00 10.00 12.00 14.00 i Time 16.00 000201 i C - 1 3 P K L-'(J2> i IiII 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 110 of 225 to,jy of Original initial uOAajgt 000202 yI 0002153 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 111 o f225 r'<act Copy of Origina! CW L. Date i da Exact Copyof Origins ........ 000264 .. 3 M E n v ir o n m e n t a l La b o r a t o r y E Q 2-1039 Page 112 of 225 1I II I 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 113 of 225 Exact Copyof Original Qtm __ ) o / 3 , i^ initial Dais C00Z05 1S/, golden west plasma 5x scale up 10/23/02 ok 3M Environmental Lab d021030008 329 (8.884) Sm (Mn. 2x1.00); Cm (323:334) 100-| 79695 il davey 070799 30-0ct'2002 13:29:23 1: Daughters of499ES- 1.67e6 3 M E n v ir o n m e n t a l La b o r a t o r y E 0 2 -1 0 3 9 Page 114 of 225 129.614 %- 98.714 179.580 168.663^ 229.786 280.108 499.079 TfrrffTf " "p " TOjfprtr 140 160 180 200 |iivf|i ir 220 240 260 280 f? rrf* T JL 300 320 340 p] n iT,i'm i i |- |i i i i mTTTOT 360 380 400 420 440 460 480 (l ttoi m/z 500 520 Exact Copy of Original .C m L _ J /? < I / n Initiai Dais 000206 t ii I 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 115 of 225 Exact Copyof Original &In2it3iaLl . J oJ^h Duty / i__ 000207 ii i1 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 116 of 225 Exact Copy of Original oataCJOL. l J s U 'L Initiaf 000208 000209 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 117 of 225 Exact Copy of Original initial tate % I I 1i 000210 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 118 of 225 Exact Copyof Origins! initia! Date Itllllliiflili ~2p> f= c > i 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 119 of 225 Exact Copy of Origina! fnjtfa! oste 000211 it 3 M 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 of 225 Exact Copyof Originai L im _ u / i / t n -- Initial fjoir 000212 I i 1 i i I ! i I i ( I I i 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 121 of 225 Exact Copyof Origina! m i- -Indiai nato --------- -** 000213 ------ -- I1 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 122 of 225 Exact Copy of On; CJY \( . L lA ij a^ Initial Dsif 000214 ii I I i i 1 l i I ti |( "^ P cR 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 123 of 225 'pyof Original Cm L !n;(ai y rs.------------ -------------:*?. 000215 1II1Ii i ! i i 1IIi Ii)! 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 124 of 225 exact Copy of Original CdQ^ - i l l / r n w m 000216 3M Environmental Laboratory E02-1039 Attachment D: Prep Sheets and Traceability Information 00021*' Page 125 of 225 3M Environmental Laboratory E02-1039 SPE Columns Extraction Worksheet Prep Date: Analysts initials: 10/23/2002 OK 10 D 3 Method Revision: ETS-8-231.1 Study Number: E02-1039 Sample Number or description Blank milli-q Lampire Serum Sigma Serum Golden West Serum Bioresource Serum Lampire Plasma Golden West Plasma Innovative Resources Plasma Volume of sample Type of column filtered (ml) used 2000 ml 2000 ml 2000 ml 2000 ml Waters, 1g, 6ml Waters, 1g, 6ml Waters, 1g. 6ml Waters, 1g, 6ml 2000 ml 2000 ml 2000 ml 2000 ml Waters, 1g, 6ml Waters, 1g, 6ml Waters, 1g, 6ml Waters, 1g, 6ml hlution Amount and solvent and spike mix used volume Comments 2mlatM*OH TN NA A-6313 2mlofMOH TN NA A-6313 TCR-888 2mlofMOH TN NA A^3t3 TCR-689 2mlofMOH TN NA A-6313 TCR-600 2mlofMOH TN NA A-6313 TCR-687 2mlatM*OH TN NA A-B313 TCR-685 2mlofMeOH TN NA A-6313 TCR-684 2/nlofMeOH TN- NA A-6313 TCR-683 ___------ -- ' _....f)iL, Z ^ c r T V1 Extraction Steps (initial when/if done): _x_condition column with MeOH; _x_wash with milli-q water; _x_filter sample;; _x_vacuum dry the column; _x elute column with solvent; ' ; r . U J 2 , ? = , a , \ Additional comments: 10ml of sera diluted with 40m l of milli-q and 200ml of A C N . Sam ple w ere shaken for 20 min at JOOrpm, then centrifuged for 10 min @ 3500rpm /4C Sam ple w as transferred into 1750m l of milli-q and filtered through conditioned SP E / U J e A e ^ jlC&Ll /<'? (&^O0 P/ 00 C o U J ^ y U S / '- is j l^ a U ^ 3 aj ExactCopyof Original f K - J o /3 i / (SL . initial Data 000218 Page 126 of 225 3M Environmental Laboratory E02-1039 Prep Date: Analysts Initials: GLP SPE Columns Extraction Worksheet F7& f ? * l I j " ^ J ^ ' r 10/11/2002 ics. l l o 'J Method Revision: H OK GKJ Study Number: E 02-1039 Sample Number description or Blank milli-q-1 Blank milll-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 ppb rabbit serum curve-2.5ppb rabbit serum curve-5ppb rabbit serum curve-1 Oppb rabbit serum curve-30ppb Lampire serum blank-1 Lampire serum blank-2 Lampire serum MS-0.5ppb Lampire serum MS-5ppb Sigma serum blank-1 Siqma serum blank-2 Siqma serum MS-0.5ppb Siqma serum MS-5ppb Golden West serum blank-1 Golden West serum blank-2 Golden West serum MS-0.5ppb Golden West serum MS-5ppb Bloreseource research serum blank-1 Bioreseource research serum blank-2 Bioreseource research serum MS-0.5ppb Bioreseource research serum MS-5ppb Volume of sample Type of column filtered (ml) used 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml Waters, 1g, 6ml 400 ml 400 ml 400 ml 400 ml Waters, 1g, 6ml Waters, 1g, 6ml Waters, 1g, 6ml Waters, 1g, 6ml 400 ml 400 ml 400 ml Waters, 1g, 6ml Waters, 1q, 6ml Waters, 1g, 6ml 400 ml Waters, 1q, 6ml 400 ml Waters, 1g, 6ml Llution Amount and spike solvent and mix used volume Comments NA 2 ml of MeOH TN-A-6310 NA 2 ml of MeOH TN-A-6310 NA 2 ml of MeOH TN-A-6310 T C R -88 8 2 ml of MeOH 2ul of 02050-14 TN-A-6310 T C R -88 8 2 ml of MeOH 5ul of 02050-14 TN-A-6310 TCR-888 2 ml of MeOH 10ul of 02050-14 TN-A-6310 TCR-886 2 ml of MeOH 15ul of 02050-14 TN-A-6310 T C R -88 8 2 ml of MeOH 20ul of 02050-14 TN-A6310 T C R -88 8 2 ml of MeOH 50ul of 02050-14 TN-A-6310 T C R -60 6 2 ml of MeOH 100ul of 02050-14 TN-A-6310 T C R -88 8 2 ml of MeOH 200ul of 02050-14 TN-A-6310 TCR-8B6 2 ml of MeOH 600ul of 02050-14 TN-A-6310 T C R -88 8 NA 2 ml of MeOH 7N-A-6310 T C R -88 8 NA 2 ml of MeOH TN-A-6310 TCR-888 2 ml of MeOH 10ul Of 02050-14 TN-A-6310 T C R -88 8 2 ml of MeOH 100ul of 02050-14 TN-A-6310 T C R -88 8 NA 2 ml of MeOH TN-A-6310 T C R -68 9 NA 2 ml of MeOH TN-A-6310 T C R -88 9 2 ml of MeOH lOul0' ' 50'* TN-A-6310 v r 2 ml of MeOH lOOul TN-A6310 NA 2 ml of M e O H TN-A-6310 NA 2 ml of MeOH TN-A-6310 T C R -68 9 T C R -88 9 T C R -69 0 T C R -89 0 2 ml of MeOH 10ul of 02050-14 TN-A-6310 2 ml of MeOH 100u! of 02050-14 TN-A-6310 NA 2 ml of MeOH TN-A-6310 T C R -89 0 T C R -89 0 TCR-887 NA 2 ml of MeOH TN-A-6310 2 ml of MeOH 10ut of 02050-14 TN-A-6310 2 ml of MeOH 10Oul of 02050-14 TN-A-6310 T C R -68 7 TCR-887 T C R -88 7 Extraction Steps (initial when/if done): _x_condition column with MeOH; _x_wash with milli-q water; _x_filter sample;; _x_vacuum dry the column; _x_elute column with solvent; act CopyOf Original Additional comments: ^ ^ cK "-? 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 milll-q and filtered through conditioned SPE COL -C 'tP tfvW G -V IV .irvJi^o ^f O. ill jv 000219 Page 127 of 225 3M Environmental Laboratory E02-1039 ^flrfiGLP SPE Columns Extraction Worksheet Prep Date: Analysts initials: l ^ i i j l ^ '*'10/10/2002 l ^ OK lo iv o j C^~. Method Revision: SPE validation Study Number: E 02-1039 Sample Number description Volume or or sample Type of column filtered (ml) used Blank milli-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 cun/e-10ppb Chinese plasma curve-30ppb Chinese plasma blank-1 Chinese plasma blank-2 Chinese plasma MS-0.5ppb Chinese plasma MS-5ppb Lampire Plasma blank-1 Lampire Plasma blank-2 Lampire plasma MS-0.5ppb Lampire plasma MS-Sppb Golden West Plasma blank-1 Golden West Plasma blank-2 Golden West Plasma MS-0.5ppb Golden West Plasma MS-5ppb Inriovative Research Plasma blank-1 Innovative Research Plasma blank-2 Innovative Research Plasma MS-0.5ppb Innovative Research Plasma MS-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 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml 400 ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters. 1q. 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters. 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1q, 6ml Waters, 1g, 6ml Elution Amount and spike solvent and mix used volume Comments NA 2 ml of MOH TN-A-6310 NA 2 ml of MeOH TN-A-6310 2 ml of M*OH 2ul of 02050-14 TN-A-6310 T C R -87 4 2 ml of M*OH 5ul of 02050-14 TN-A-&310 T C R -87 4 2 ml of M*OH 10ul of 02050-14 TN-A-6310 TCR-874 2 ml of MoOH 15ul of 02050-14 TN-A-6310 T C R -87 4 2 m lo fM O H 20ul of 02050-14 TN-A-6310 TCR-874 2 ml of MeOH 50ul of 02050-14 TN-A-6310 TCR-874 2 ml of MeOH 100ul of 02050-14 TN-A-6310 T C R -87 4 2 ml of MeOH 200ul of 02050-14 TN-A-6310 TCR-674 2 ml of MeOH 600ul of 02050-14 TN-A-6310 T C R -87 4 NA 2 ml of M*OH TN-A-6310 T C R -87 4 NA 2 ml of MeOH TN-A-6310 T C R -87 4 2 ml of MeOH 10ul of 02050-14 TN-A-6310 T C R -67 4 2 ml of MeOH 100ul of 02050-14 TN-A-6310 T C R -87 4 NA NA lOul 10Oul NA NA 2 ml of MeOH TN-A-6310 2 ml of MeOH TN-A-6310 2 ml of MeOH TN-A-6310 2 ml of MeOH TN-A-8310 2 ml of MeOH TN-A-6310 2 ml of MeOH TN-A-6310 T C R -68 5 T C R -68 5 T C R -88 5 TCR-B85 T C R -68 4 T C R -68 4 2 ml of MeOH 10ul Of 02050-14 TN-A-6310 TCR-884 2 ml of MeOH 100ul of 02050-14 TN-A-6310 NA 2mfo<MeOH TN-A-6310 NA 2 ml of MeOH TN-A-6310 T C R -88 4 T C R -68 3 T C R -68 3 2 ml of MeOH 10ul Of 02050-14 TN-A-6310 2 ml of MeOH 100ul of 02050-14 TN-A-6310 TCR-683 T C R -08 3 Extraction Steps (initial when/if done): _x_condition column with MeOH; _x_wash with milli-q water; _x_filter sample;; _x_vacuum dry the column; _x_elute column with solvent; Additionalsc<o mments:ri= C '2 >\-Y* ^ .^ ,, I >1 l'y .- - ,; V tc 1^ S ( V Exact Copyof Origina) Dale 2m l of sera/plasm a diluted with 8m l of milli-q and 40m l of A C N . Samples w ere shaken for 20 min at 300rpm, then centrifuged for 10 min @ 3500rpm /4C Sam ple w as transferred into 350m l of milli-q and filtered through conditioned S P E V'M TM-A ` 000220 Page 128 of 225 3M Environmental Laboratory E02-1039 3M Environm ental Laboratory Note to File Project or Study Number: E02-1039 Associated Study Number:_________ F5poprbs,aJmabpelelssothnavtlawlesrweeprreejpcpceiddeonnta1l0ly/1s1w/0i2tc,htehda.t are B_i_o_resour_ce__R_e_se_a_rch Ser_umMS 0.5pp_b_and Recorded By: / V ---------------------------- FormETS-4 -15,0 000221 Page 129 of 225 J M C Z N V IH N M tN IA L L A tS U H 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: Retain OK 7-15 ml centrifuge tubes liquid, Biohazard TCR Substance # TCR-674 TCR # Received from: TCR-674 Northwest Bioanalytical Amount received (wt. or 7x~10ml vol): Date: 09/25/2002 Shipper: FedEx USDS (y/n) Date of Retain ; Y# N ;O Archived/Substan-ce N_o_t_A.vailab' le; Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Locatlort/Storage: Frozen F19 Molecular Formula: Comments Sera came in 7 separate tubes. Please mark which tube was used for sample, by number marked on the tube (4,5,6,7,8,11 or 12) Attachment(s) Exact Copyof Original f U / l / CT_ Initial Date 000222 Page 130 of 225 3M Environmental Laboratory E02-1039 ____________________________________ USE LOG luman plasma from China luman plasma CR-674 ________ __________________ Gross Wt./Vol. Before withdrawal Balance ID 1 Amnt. withdrawn (mass or vol) 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) 8alance Balance Initials ID ID 12 Date na na OK 10/09/2002 Exact Copyof Original C w n v / t / 0-3lnltfal Data 000223 Page 131 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 Plasma in Sodium Citrate Pooled human plasma IR02-014 09/30/2007 Initials: Number/size of containers: Condition: Retain OK 10x1 0ml plastic tubes good O 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-683 TCR # TCR-683 Received from: Innovative Research, Ml Amount received (wt. or 10x10ml vol): Date: 10/02/2002 Shipper: MSDS (y/n) Date of Retain Y N Exact Copyof Original C m c I'/ </ 0-3- Initial Data 000224 Page 132 of 225 3M Environmental Laboratory E02-1039 ___________________________________________ USE LOG 'ooled Human Plasma 'ooled human plasma CR-683 ____________________________________ Gross Wt./Vol. Amnt. Gross wt/vol Puipose (enter standard number Before withdrawn after or reason for removal) withdrawal (mass or vol) withdrawal Balance 10 1 Balance ID 2 Balance ! 1 - 8mI - E02-1039 validation Balance Balance Initials ID ID 12 Date OK 10/14/2002 Exact Copyof Original Cl'VIC HA/6X-- Initial Dais 000225 Page 133 of 225 3M Environmental Laboratory E02-1039 ______________________________________________USE LOG 'ooled Human Plasma 'ooled human plasma CR-683 ____________________________ Gross Wt.A/ol. 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, validation Balance Balance Initials ID ID 12 Date OK 10/23/2002 Exact Copyof Original Cir\ U / j V o p - Initial Data 000226 Page 134 of 225 3M Environmental Laboratory EQ2-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemical 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 1x100ml plastic bottle Frozen Q Archived/Substance Not Available I 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 It TCR-684 TCR # TCR-684 Received from: Golden West Blologlcals, Inc. Amount received (wt. or 100ml vol): Date: 10/03/2002 Shipper: UPS MSDS (y/n) Date of Retain : )Y N Exact Copyof Original C w c u /U a xInitfal Date O O O Z ii1 Page 135 of 225 3M Environmental Laboratory E02-1039 _____________________________________________ USE LOG odium Citrate Pooled Human Plasma 'ooled human plasma CR-684 Gross Wt./Vol. 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 - 8m1 - E02-1039, validation Balance Balance Initials . ID ID 12 Date na na OK 10/10/2002 Exact Copyof Original CMC h/ I / on. Initiai Oata 000228 Page 136 of 225 3M Environmental Laboratory E02-1039 _____________________________USE LOG odium Citrate Pooled Human Plasma ooled human plasma CR-684 _______________ __ ________________ Gross Wt./Vol. 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 Initials ID ID 12 Date NA NA OK 10/23/2002 ' Exact Copyof Original- t-IMniCtial u /o' a/tso j-- 000229 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 #: 3M# Expiration date: Pooled Human Plasma From Lampire Pooled Human Plasma From Lampire 22-60824A 10/08/2007 Initials: Number/size of containers: Condition: OK 1-250ml plastic bottle Frozen Retain O rchived/Substance Not Available, Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachment(s) Disease Screen Lampire Frozen F19 TCR Substance # TCR-685 TCR # TCR-685 Received from: Lampire Amount received (wt. or 100ml vol): Date: 10/10/2002 Shipper: UPS MSDS (y/n) Date of Retain i./ y # N : Exact Copy of Original rA'YIC l l / l / T - initiai Data 000230 Page 138 of 225 3M Environmental Laboratory E02-1039 _____________________________________________ USE LOG 'ooled Human Plasma From Lampire 'ooled Human Plasma From Lampire CR-685 _____ ____ Gross Wt./Vol. Before withdrawal Balance ID 1 Amnt. withdrawn (mass or voi) Balance ID 2 - 10ml Gross wt/vol Purpose (enter standard number after or reason for removal) withdrawal Balance ID 1 - E02-10:i9 Balance Balance Initials ID ID 12 Date na na OK 10/23/2002 Exact Copyof Original C fYH . i ' / i / a v Initial Dato 000231 Page 139 of 225 3M Environmental Laboratory E02-1039 _____________________________________________ USE LOG 'ooled Human Plasma From Lampire 'ooled Human Plasma From Lampire CR-685 ___________________________________ Gross Wt./Vol. Amnt. Before withdrawal withdrawn (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 Copyof Original .C m c Iy t/ pi-- Initial Data 000232 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 # Received from: TCR-687 TCR-687 Bioresource Amount received (wt. or 500ml vol): Date: 10/11/2002 Shipper: UPS MSDS (y/n) Date of Retain U Y N O A rchived/Substance Not: Available j 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 C iiZ l o X ^ Initial Date 000233 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 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 C M ( U ZlO JInttiaJ Date 000234 Page 142 of 225 3M Environmental Laboratory EQ2-1Q39 ______________ USE LOG 'ooled Human Serum From Bioresource 'ooled Human Serum CR-687 __________ _______ Gross Wt./Vol. Amnt. Gross wt/vol Purpose (enter standard number Before wilhdrawn after or reason for removal) withdrawal (mass or voi) withdrawal Balance ID 1 Balance ID 2 Balance ID 1 - 8m1 - E02-1039, validation Balance Balance Initials ID ID 12 Date OK 10/11/2002 Exact Copyof Original Q y i( u / i c ~^ iiuttal Date 00025 Page 143 of 223 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 # Pooled Human serum TCR# X324B Received from: TCR-688 TCR-688 Lampire 08/29/2007 OK 250 ml Nalgene bottle Frozen Amount received (wt. or 250ml vol): Date: 10/11/2002 Shipper: UPS MSDS (y/n) Date of Retain ;.) Y N ( ) 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 | Exact Copyof Original JZ ttC . Initial Dato 000236 Page 144 of 225 3M Environmental Laboratory E02-1039 _____________________________________________ USE LOG 'ooled Human serum from Lampire 'ooled Human serum CR-688 __________________________________ Gross Wt./Vol. Amnt. Gross wt/vol Purpose (enter standard number Before withdrawn after or reason for removal) withdrawal (mass or vol) withdrawal Balance ID 1 Balance ID 2 Balance ID 1 - 8m! - E02-1039, validation Balance Balance Initials ID ID 12 Date OK 10/11f2002 Exact Copy of Original 000237 Page 145 of 225 3M Environmental Laboratory E02-1039 _______________________________ USE LOG 'ooled Human serum from Lampire 'ooled Human serum CR-688 ________ __ _____________ Gross Wt./Vol. 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 - E02-10:i9 Balance Balance Initials ID ID 12 Date OK 10/23/2002 Exact Copyof Original 000238 Page 146 of 225 3M Environmental Laboratory EO2-1039 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 Q22K0965 07/24/2007 Initials: Number/size of containers: Condition: Retain OK 1 -1 00ml plastic bottle frozen TCR Substance # TCR-689 TCR # Received from: TCR-689 Sigma Amount received (wt. or 100ml vol): Date: 10/11/2002 Shipper: UPS MSDS (y/n) Date of Retain \ >Y V N | 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-6250 10/11/02 OK E/(3ul M / I ) OT__ Date 000253 Page 147 of 225 3M Environmental Laboratory E02-1039 _______________________________ USE LOG tale Human Serum from Sigma Hale Human serum "CR-689 Gross WtTVol. 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 Initials ID ID 12 Date OK 10/11/2002 Exact Copyof Original InitfaJ Oats 000240 Page 148 of 225 3M Environmental Laboratory E02-1039 ______________________________________________USE LOG Male Human Serum from Sigma Male Human serum rCR-689 ____________ ____ Gross Wt./Vot. Amnt. Gross wt/vol Puipose (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 Initials ID ID 12 Date - OK 10/23/2002 Exact Copyof Original UOal Date 000241 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 Golen West Pooled Human Serum G01406042 07/25/2007 Initials: Number/size of containers: Condition: Retain OK 1-500ml blastic bottle frozen TCR Substance # TCR-690 TCR # Received from: TCR-690 Golden west Amount received (wt. or 500ml vol): Date: 10/11/2002 Shipper: UPS MSDS (y/n) Date of Retain f. >Y .N ( 1Archived/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 " CiVlExactCopyof Original . C 11/ 1 / n-- Initiai oats 000242 Page 150 of 225 3M Environmental Laboratory E02-1039 ______________________________________________USE LOG 'ooled Human Serum from Golen West 'ooled Human Serum CR-690 ______________ __________ Gross WUVol. 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 Initials ID ID 12 Date OK 10/23/2002 Exact Copyof Originai 2 n c (I instai Oats 000243 Page 151 of 225 3M Environmental Laboratory E02-1039 _____________________________________________ USE LOG 'ooled Human Serum from Goien West 'doled Human Serum CR-690 ____ _________________ Gross Wt./Vol. 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 - 8ml - E02-1039, validation Balance Balance Initials ID ID 12 Date OK 10/11/2002 Exact Copyof Original < 2 V r< mu li/ l/ a i ~ sr 000244 Page 152 of 225 Standard curve tracking sheet Prep date: Analyst: 10/7/2002 OK tK ) Type of the curve: Solvent curve for SPE sera validation Target analytes: C6, C7, C8, C9, C10. C11, C12, THPFOS, THPFDS, PFOS Standard mix 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 02048-21-8 02048-21-7 02048-21-8 02048-21-9 02048-21-10 Amount of 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 2.55 511 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 Analyte concentration In every point (ppb) C9 C10 C11 C12 0.101 0.101 0.103 0.104 0.507 1.01 0505 1.01 0.515 1.03 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 15.22 20.29 30.43 10.10 15.15 20.20 30.30 10.31 15.46 20.61 30.92 10.36 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 Signature: Verified by: O l r i U A t / r L f l c O J 0 / 5 l / 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Page 153 of 225 Exact Copyof Original li22__ tlsd-' Initiai Date ^____ 000245 3M Environmental Laboratory E02-1039 Standard mix tracking sheet Sera Super mix for SPG 10X MOV Prep date: Analyst: Type of the mix: Target analytes: 10/7/2002 Exp. 11/13/02 OK sera super mix C/C / c 7 x ^ j Cfl, C7, C8, C9, C10, C11, C12, THPFOS, THPFDS, PFOS Standard number for the mix: 02002-63 Study Number: Final SolventiTN-A #: |Flnal Volume of the rnix? Analyte CC67 cCa9 C10 C11 C12 THPFOS THPFDS PFOS Analyntuemsbtaenrdard ancaomlnygct/eemniltnr(aspttipaomnnd)oarfd staanmdoaurdntuosfed (ul) coannacnleygn/ttmeralitn(iopthnpebo)mf tihxe 02040-44 02040-58 1042 1344 12 9.5 500 511 00220044004438 1230 1268 10.3 10 517 507 0022002402--6950 02040-55 0204042 1010 1356 1126 134a 12.5 191.5.5 9.5 505 515 516 512 0204049 02022-56 1002 1048 12.5 12 501 503 Sera SPE validation MeOH/6308 r~ ^ i Signature^ f e d C%/ I Verified bv: ( h j x L j s Q jt L r J L t u ' i t o / 3 i / > a t e a Copy of Original 000246 Page 154 of 225 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 SINGLE COMPONENT PREPARATION LOG Date: Ip . I O - o Q j __________ Analyst:_________ Q K J __________ Book No. 02 050 Page N o.___ 14 Description: jo *>u~7*gy. Stock Number o < 2 . o o I X - Q 3 Weight or Volume Used: 6 m l Concentration or Purity: Corrected Weight: KJA Balance ID: K )f\ Other Correction Factors: AJA HeO fSolvent and TN-A Number. JH -A Final Volume: AC Storage Location: M f Final Concentration: Expiration Date: Reviewed 000247 Exact Copyof Original <9 W I O L Initial Date /o U M Dal/ Page 155 of 225 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 Date: i o l e Analyst: ____ Q iL Description: SINGLE COMPONENT PREPARATION LOG Book No. 02 050 Page No. 17 Stock Number ( 0 ? C > O <3 Weight or Volume Used: Concentration or Purity: AJACorrected Weight: Final Volume: Storage Location: ALsSj Balance ID: Other Correction Factors: MA Solvent and TN-A Number N ? O f/ 7 k A - ( ? 3 /o Final Concentration: 6 Expiration Date: A - / 3 - O Q Exact Copyof Original fd a Z i f l - Initlai Oats -J 2. 000248 Page 156 of 225 3 M E n v ir o n m e n t a l La b o r a t o r y -------- E 02-1039 Data: /C IC 'd ^ Analyst: 0)0 Description: SINGLE COMPONENT PREPARATION LOG Book No. 02 050 Page No. 16 (X Stock Number:________ iQ D y 'S L - Weight orVolume Used: Concentration or Purity: id - cPlCc > H S y p O Corrected Weight: A b Balance ID: A Other Correction Factors: fjS \ solvent and TN Number M O / W ' A - G 3 / 0 Final Volume:__ Storage Location: c A d r^ Final C o n c e n tra tio n :__________ ( T h Expiration Date: ii-rz -o Q / Exact Copy of Original ..CJ2CX. Initial Oats *1 000249 Page 157 of 225 !I Description: i-ViWi3 Tbrj Date Prepared: -ic - EJG ~ >1^,Storage Location: u o / . Expiration Date: ~ 1 C - i i MULTI-LEVEL PREPARATION LOG Book No. 02 048 Solvent: Page No. N rO H 21 3M Trace #: /7v-4-i< r^V > V Standard ID # 02 048-21-1 02 048-21-2 02 048-21-3 02 048-21-4 02 048-21-3 02 048-21-6 02 048-21-7 02 048-21-0 Description 'Pp-'CJ oi (> tX I 2 <5 s 1 ^ IO /S ' Component Standards Description ID# Cone Expiration Date Std 1 Std 2 Amount of Component Standard Added / jJ 6 Std 3 Std 4 Std 5 Std 6 Std 7 Std 8 `S t'7Tr?jWl * f t - ^ ^ A S P e ctS5-^> \' ___ --------------1 --------- -- S 3 s o 1-3 rK io-7 < S '? S' <2 ) 0 7 `3'Cj __-_________ ---- ----- -------- " o Final Volume Final Concentration oq. - a, o. i <? 5 ___________-- - - ------- -- -3 v> l J *5 <5 1S IO 1 f in Standard ID # 02 048-21-9 02 048-21-10 02 048-21-11 02 048-21-12 02 048-21-13 02 048-21-14 02 048-21-15 02 048-21-16 >0 Description ' T r ^ ' rSo ?>o (O' ~f--X3 2 1 Component Standards Amount of Component Standard Added _____ Page 158 of 225 3M Environmental Laboratory E02-1039 MULTI-COMPONENT PREPARATION LOG Book No. 02 002 Page No. 63 Description: ' G O T j f O ' Q j Date Prepared: Prepared By: I Q- *-'3^ oK Storage Location: Expiration Data: .Iwli-oU/j r Solvent: A Ip O ^ 3M Trace #: TN-A- (p '3 q ? Description C AC*' (TV A T iI> CV O , Acv>> C* /o A f r C ' ' f^ C (~7> C r u A c- O ) I'H T r o h T h T 'F ' 5 (J f o ii I0 L 0 2 Standard ID # 02 002-63 Component Standard ID# Cone, or Purity Expiration Date Corrected Weight or Volume Balance ID Weight Final Concentration T rb cl2o ^ o _ flf IC ^S . P R Y "* a^ui O Z o ^ o (3r4 2 - 2 ^ 0 3 f puM O -Z a tfo - tl" ^ CfHM x 3 O G /jl o^c^o - i2c?y 3 -iS -z> 3 I m A U a dJA I S iO *^ 3 S '!) S IT S ol 'O 'X G Z S L - ^o frn 1 -2 0 3 /J . G CS 0 2 c A -^ ~ \^ g q r/M Q --y d 2S W * 3 te ** LSpi "O S si s t -V7 rr*-' C o < -/c> ~ 99 lo o R 7M ZR & dZ 1/5 o Z c J S -^ G rP ^ | Final Volume /lit? V -j LV) _ v Approximate Concentrationofthe mix o ( T) SO) 1 SO Reviewed by: ' ' `'Signature, 2L LJ- ! ExactCopyofOriginal C M .... n tili 1 Dato 000251 Page 159 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: TDHA Trldecafluoroheptanoic Acid PU/07219EU NA 01/01/2005 Initials: Number/size of containers: Condition: Retain SRP 1/ 30 mL plastic container clear crystals 0.3994 g TCR Substance # TCR# Received from: TCR-99131-025 TCR-267 Aldrich Amount received (wt. or 25 g, 37.1357 g gross wt. vol): Date: 12/21/1999 Shipper. Courier MSDS (y/n) ate of Retain YIJN 02/15/2000 O Archived/Substance Not Available Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments 99.5% LAC 4/24/01 MSDS KJD 3/1/00 NA F19, Frozen C6F13COOH 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 tcr99131-25msds.pdf tcr99131-25cofa.pdf Exact Copyof Original Initial uDailte"n -- OOOC52 Page 160 of 225 JfiN 26 2000 0 9 :4 8 FR ALDRICH OC 3M CERTIFICATE OF ANALYSIS E02-1039 72*-770/-34 3M e n v ir o n m e n t a l S Cs Oi TTm POsSaT9 0 po u rn - PRODUCT NUMBER: 3 4 2 0 4 -1 LOT NUMBER: 07 21 9E U PRODUCT NAME' TRIDECAFLUOROHEPTANOIC ACID, 99K FORMULA: C 7H F1302 FORMULA W EIG HT. 3 6 4 .0 4 AFPEARANCE INFRARED SPECTRUH FLUORINE NMR GAB LIQ U ID CHROMATOGRAPHY QUALITY CONTROL ACCEPTANCE DATE UHITE CRYSTALLINE SOLI CONFORMO TO STRUCTURE AND STANDARD AS ILLUSTRATES ON PACE 7980 QF ED ITIO N I , VOLUME i OF "THE ALDRICH LIBRARY OF F T -IR SPECTRA". CONFORMS TO STRUCTURE. 99.S X JUNE 1999 ExactCopyofOriginal - 'O h t / p 2 - ww oSr ALDRZCH CHEMICAL COMPANY DAVID SUESSEL JANUARY 2 6 , 2000 aldrlch chemical co.chemists Helpingchemists inresearch&industry S. K&lto393,W tm u)a.W itt#m 5XtU3A>(4i4)ZrKmtl)` FAX(*iqZ714en AUHSCNiMm>iJuiLI|iiedud9Nntomiilak4naiionMain4 h Meira har Ndroft pncttcdtani. PMctaeormuMentati* tn MMfaMtyuftheproduitforitapimculMiBe.3e*i*ei*faidefiMeio* rutaflj i(,ht Miionii MfM endonwa Mata. aif Hiv-tint K* TOTAL PAGE.04 * * 000253 Page 161 of 225 3M Environmental laboratory E02-1039 TDHA rrldecafluoroheptanoic Acid rCR-99131-025 USE LOG ___ Gross WUVol. 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 35.9819g 0.0865g 35.8954g standard 02040-58 Balance Balance Initials ID ID 12 Date 914 RWW 08/29/2002 ExactCopyofOriginal WH t o 1O^t1ta!n ~ 000254 Page 162 of 225 3M Environmental laboratory E02-1039 O ,,.: ----- r ' Analyst: Description:_ SINGLE COMPONENT PREPARATION LOG _______ _ B O O N = ._ ^ 0 Page No 58 f / s S l / ' / u a r * ) r ^ / H A l t - s C . J f r n t i - A - i -------------- Stock Number. ?/?/-XT'. Weight orVolume Used: Concentration or Purity: T2l Corrected Weight Final Volume:, SUlJ - Storage Location Balance ID: 5 ? / cZ Other Correction Factors: //A " ""S Final Concentration'^ Expiration Date: a / j - / ExactCopyofOriginal C IT Y li / 1/ p i U ttal osta (\fi* AReviewedby: J W i _ loinJox Signature, Da* 000255 Page 163 of 225 3M Environmental Laboratory E02-1039 Date: SINGLE COMPONENT PREPARATION LOG 5 ? / 2 - _________ Book No. 02 040 Page No. 58 Analyst / in J h J ____________ Description: d s S3 /^/u /is A f7 ~ /) ttrA -')________ Stock Number 9 f S t - L Weight or Volume Used: Concentration or Purity. 2 ^ 2 l Corrected Weight: FinalVolume: Storage Location: f S '' " ' T/^ Balance ID: 9 /Y . Other Correction Factors: AA Solvent and TN-A Number.' IV JJ. Final Concentration'. Expiration Date: Exact Copyof Original (MfeXdi.t_a)__lU Reviewed by. S(iVgnaftrure,A. S l r v t M * 1p | p i Jo> ate 000256 Page 163 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-99131-025 by 'H-NMR and 19F-NMR Spectroscopy Date: October 30, 2002 SAMPLE DESCRIPTION: TCR-99131-025, lot PU/07219EU from the Telomer project. Sample Spectra #'s ExperimentDescriptions TCR-99131-025 H71638.GID.401 400MHz ID 'H-NMRinaCFClj/acetone-dsolventmixture+p-HFXcross integration/internal std. TCR-99131-025 F71638.GID.401 376MHz ID l0F-NMRinaCFCIj/acetone-dssolventmixture+p-HFXcross integration/intemal std. TCR-99131-025 F71638.GID.COSY.502 470MHz2DlsF-NMRCOSY(correlatedspectroscopy) experiment ina CFCIy'acetone-dssolvent mixture. OBJECTIVE: This sample was subjected to a combination of 'H-NMR and l9F-NMR spectral analyses to determine the purity o f the nominal product. Special emphasis was also placed on attempting to identify and quantify any impurity components. EXPERIMENTAL: A portion o f the sample was accurately weighed, spiked with a known amount of l,4-bis(trifluoromethyl)benzene (p-HFX), and then totally dissolved in a CFClj/deuterated acetone (acetone-dj) solvent mixture for subsequent analysis by NMR. Initial one-dimensional (ID) 400 MHz 'H-NMR and 376 MHz ,9F-NMR spectra were acquired at room temperature using a Varian UNITYplus 400 FT-NMR spectrometer. A two-dimensional (2D) l9F-NMR correlated spectroscopy (COSY) experiment was also acquired using a Varian UNITY INOVA 500 FT-NMR spectrometer to facilitate assignment o f 19F signals uassseodciaasteedithweirth1)vaar'iHou/Is9Fim-NpMurRityinctoemrnpaolnsetanntsd.arTdhtios asallmowplethperceaplacrualtaiotinonmoetfhtohde paebrsmoliuttteedwtehieghpt-HpeFrXcetnot be concentrations o f specific components, or 2) a 'h / i9F-NMR cross integration standard to permit the cross correlation o f the relative 'H and ,9F signal intensities for evaluation o f the overall sample composition. RESULTS: The 'H-NMR and l9F-NMR spectral data indicated this sample was a relatively high purity form o f the nominal product, CFj(CF2)n-C02H, where the average value of n = 5.05. Small amounts o f a few impurity components, including probable isomers, were also assigned. A 'H/19F-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 NMR spectra, but additional work would be needed in an effort to identify or quantify some o f these other components. ExactCopyofOriginal Page 1 of2 Initial- J LDdaltxat p 2 - 000257 Page 164 of 225 3M Environmental Laboratory E02-1039 October 30, 2002 3MSMMDAnalytical Lab Request #GID:71638 TCR-99131-025, Lot PU/07219EU: Telomer Project Copies o f the NMR spectra are attached with the paper copy o f this report for your reference. If you have any questions about these results, please let me know. Tom Kestner c: Mark Ellcfson RRoicnk PPuarycfeclrl William Reagen File Reference: ls7163S.GID.TCR-'Wl3l-025_Lot PU/07219EU_Telomer Project.DOC/101 TABLE-1 Sample: TCR-99131-025, Lot PU/07219EU from the Telomer project. Overall Compositional Results by 'H/I!IF-NMR Cross Integration Analysis Component Structures 1 *H/WF-NMR Relative Weight% Concentrations (single trial analysis) CF3(CF2)n-C 02H (where average n = 5.05) Probable internal branched isomers CF3(CF2)x-CF(CF3)-(CF2)y-C 02H 98.24% 0.87% (where x*0 andassume x+y = 3.05 for calculationpurposes) Probable terminal isopropyl branched isomers (CF3)2-CF-(CF2)n-C 02H (assume n=3.05 for calculation purposes) A probable ether/acid as possible 0-rCF2CF2F-C 0F2Hl02 0.52% 0.13% 0.11% Probable F F CnH2ll+2 saturated aliphatic hydrocarbons and 0.11% functional aliphatic components, CnH2n+i-X, where -X can be possible ether and ester functional groups. Possible CF3(CF2)n-C 02CH3 0.021% 1. Trace amounts of n(wumheerroeuasvoethrearguennass=ig5n.e0d5c)omponents were also detected in the l9F-NMR spectrum. Bract Copyof Original Page 2 of2 000258 Page 165 of 225 3M Environmental laboratory E02-1039 Certificate of Analysis Nominal Product: CF3(CF2)n-C02H, where average n 5 Tridecafluoroheptanoic acid Product Code: TCR-99131-025, Lot PU/07219EU October 30,2002 The sample o f TCR-99131-025, Lot PU/07219EU was analyzed using a combination o f t9F-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 I9F/'H-NMR Spectral Analyses Component Structures 1 `h T f -n m r Relative Weight% Concentrations (single trial analysis) CF3(CF2),,-C02H (where average n = 5.05) (where x*P0rCaonFbda3(abCslseFui2mn)xete-xCr+nFya(Cl=Fb33r.)0a-5(nCcfFohr2ec)dya-licCsuo0lma2tHieorns purposes) <98.2% Purity 0.87% Probable ter(mCiFn3a)2l -iCsoFp-(rCoFp2y)l,,-bCr0a2nHched isomers (assuAmperonb=0a3-b.r0lCe5Fefto2hCreFcra/2a-lcCcui0dl2aaHtsiolp2nopssuirbpleoses) 0.52% 0.13% F FQ 0.11% Cfu,,nHc2tni+o2nPsaralotaublraiapbthleeadtiacflTifcp>o^h&maFtTpiconhFTeyndftrso, cOCaH,,rbHo2nn+s ta-Xnd, *0.11% where -X can bPeopsossibsilbelCe Fet3h(CerFa2n)nd-Ces0t2erCfHu3nctional groups. 0.021% (where average n = 5.05) 1. Trace amounts o f num erous other unassigned components were also detected in the NMR spectra. Tom Kestner Exact Copyof Original Page l of 1 File Reference: CofA_TCR-99131-025J.ot PU-07219EU.doc 000259 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: Pentadecafluoroocfanoic acid Pentadecafluorooctanoic acid 210002 Initials: Number/size of containers: Condition: Retain OK 1-10ml amber glass vial white crystals TCR Substance # TCR # TCR-617 TCR-617 Received from: Oakwood Products Amount received (wt. or 5g vol): Date: 07/19/2002 Shipper Courier MSDS {yin) Date of Retain YO N ! 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 of 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 or less per vial). LAS 10/02/02 13 TCR-617.pdf ~ (30 mL/g ExactCopyof Original Initial Oats 000260 Page 167 of 225 - >rsTi--t-in-j^; lSNm qNMENTACj LABOFpjQY Oakwood Products, Inc. 1741 Old Dunbar Road West Columbia, SC 29172 Phone (W3) 739-8800 Fax (8031 739-6957 CERTIFICATE OF ANALYSIS Date: 15-Jul*02 Material: Pentadecafluorooctanoic add Cat.No.: 1319 Cas No.: [335-67-1] Lot No.: 210002 Assay: 97+% by NaOH titration Appearance: W hite solid Melting Point: 59-61uC Exact Copyof Original v n i iU M L P -- Initial Data QC Manager 000261 Page 168 of 225 3M Environmental Laboratory E02-1039 ________________________________________ USE LOG 'entadecafluorooctanoic acid 'entadecafluorooctanoic acid CR-617 Gross Wt.A/ol. Amnt. Gross wt/vd Purpose (enter standard number Before withdrawn after or reason for removal) withdrawal (mass or voi) withdrawal Balance 10 1 Balance ID 2 Balance ID 1 15.5486g 0.0617g 15.4869g standard 02040-45 Balance Balance Initials ID ID 12 Date 914 IRWW 08/15/2002 Exact Copyof Original -OrYK. Jnitial oats 000262 Page 169 of 225 Pate: Analyst: Description: 3M Environmental Laboratory E02-1039 SINGLE COMPONENT PREPARATION LOG Book No. 02 040 Page No. 45 j> S Q C A J C /Q ft J M _____ ( 7 /~ZTP o & r f Stock Number / C A - d 7 Weight or Volume Used: > ^ / J ^ ? Balance ID: Concentration or ,.A Purity:________H .------------------------------- -- Other CoFrarecctotirosn:_______ ap ) ' A' - Corrected Weight: --------------,---/-)---/-----/----5--"-------------------------------- . Solvent aNndbTmNb-Ae r_ / / ,, , S> -S . , / ? Final Volume: fo J Final Concentration: Storage Location: $ u J ~~ T<Y* Expiration Date: jA r/? j /- EnxaicnteCopyloof Ol'riigilinOalL, initial Data Reviewed by: l\ . S k \ * n o > , Signature, D a le 000263 Page 170 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-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 = CF3(CF2)n-CO;H, where average n 6 (white powder). Sample Spectrait's ExperimentDescriptions TCR-617 TCR-617 H71638.GID.501 500MHz 'H-NMRinacetone-d6solvent+p-HFXcross integration/internal std. F71638.GID.501 470MHz *T-NMRinacetone-dsolvent+p-HFXcross integration/internal 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 o f 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 o f the impurity components. You will notice that the tentatively assigned olefin structure from the original report is now replaced with a probable (CfiF^OJ-CChH ether acid. OBJECTIVE: This sample was subjected to a combination o f *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 o f impurity components. Joel asked that I incorporate the qualitative information from his LC/MS analysis in this updated report. FT-NMR EXPERIMENTAL: A portion o f the sample was accurately weighed, spiked with a known amount o f l,4-bis(trifluoro- maneatlhyyslijsbbeynzNeMneR(.p-AHF5X00),ManHdzth'Hen-NtoMtaRllysdpeiscstorulvmedanind dae4u7te0raMteHdzacl9eFto-NneM(aRcestpoencet-rdugm) fwoerrseubacseqquuireendtat 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 'H/I9F-NMR internal standard to allow the calculation o f the absolute weight percent concentrations of specific components, or 2) a 'H/I9F-NMR cross integration standard to permit the cross correlation o f the relative 'H and l9F signal intensities for evaluation o f 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, CF3(CF2),,-C02H, where the average value of n = 6.02. Small amounts o f a few impurity components, including probable isomers, were also assigned. A 'H/19F-NMR cross integration analysis technique was then used to calculate the relative weight percent concentrations o f 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 1 of2 Exact Copyof Original 000264 rw,, Page 171 of 225 3M Environmental Laboratory E02-1039 October 28, 2002 3MSMMD Analytical LabRequest #GID:71638 UpdatedReport - LC/MS Analysis for Impurities inTCR-617, Lot 210002 RESULTS fcont.): Trace amounts o f 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 RRiocnk PPuarycfeelrl W illiam Reagen File Reference: U71638.G]D.Updatcd ReaulU_TCR-617_Lot 210002_Tdomer Project-DOC/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 Weight% Concentrations (single trial measurement) CF3(CF2)n-C 02H where average n = 6.02 by l9F-NMR. LC/MS showed n=6 (major), n=5, n=4 (minors). Probable (CF3)2-CF-(CF2),,-C02H assume n=4 for calculation purposes Probable as possible (CCF6F3Ci3F02-)0-C-C0F2H(CaFc3y)-cClicF2eCthFe2r- acid C 02H Possible CF3(CF2)x-CF(CF3)-(CF2)y-C 02H where x*0, y*0 and assume x+y = 4 for calculation purposes assPuomsseibnl=e3(CfoFr3)c3a-lCcu-(lCatFio2)nn-pCur0p2oHses Possible CnH2n+2 saturated aliphatic hydrocarbons 1. Traceamounts ofotherunassignedcomponentswerealsodetectedintheNMRspectra. 99.52% 0.39% 0.057% 0.019% 0.013% 0.0079% Page 2 of 2 000265 Exact Copyof Original Initial Oats Page 172 of 225 3M Environmental Laboratory E02-1039 Certificate of Analysis Nominal Product: CF3(CF2)n-C 02H, where average n 6 Pentadecafluorooctanoic acid Product Code: TCR-617, Lot 210002 October 28,2002 Tom fCestner and Joel Miller The sample o f TCR-617, lot 210002 was analyzed using a combination o f 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 l9F/'H-NMR and LC/MS Analyses Component Structures 1 'Htr//l`yycF-vNmM/TRD Relative Weight% Concentrations (single trial analysis) CF3(CF2)n-C 02H where average n = 6.02 by l9F-NMR. LC/MS showed n=6 (maior), n=5, n=4 (minors'). Probable (CF3)2-CF-(CF2)n-C 02H assume n=4 for calculation purposes Probable (C6Fi30 )-C 0 2H acyclic ether acid as possible CF3CF2-0-CF(CF3)-CF2CF2-C 07H Possible CF3(CF2)x-CF(CF3)-(CF2)y-C 02H where x*0, y^0 and assume x+y = 4 for calculation purposes Possible (CF3)3-C-(CF2)n-C 02H assume 0=3 for calculation purposes Possible C,,H2n+2 saturated aliphatic hydrocarbons <99.51% Purity 0.39% 0.057% 0.019% 0.013% 0.0079% 1. TraceamountsofotherunassignedcomponentswerealsodetectedintheNMRspectra. Page 1 of 1 ExactCopyofOriginal initial Date FileReference: CofA_TCR-617_Lot 210002.doc 000266 Page 173 of 225 3 M ENVIRONMENTAL LABORATORY E 02-1039 Test Control and Reference Substance Log Substance trade name or reference # Substance/chemlcal name: Lot/batch #: 3M# Expiration date: Heptadecafluorononanoic acid Heptadecafluorononanoic acid H7568 NA Initials: Number/size of containers: Condition: Retain OK 1-50ml plastic jar white crystals TCR Substance # TCR # Received from: TCR-618 TCR-618 Oakwood Products Amount received (wt. or 50 vol): Date: 07/19/2002 Shipper Courier MSDS (y/n) Date of Retain # Y C) N C Archived/Substance Not Available Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/Storage: Molecular Formula: Comments Attachment(s) 98.02% updated 10/30/02 OK, NMR analysis MSDS Certiflcat of Analysis LAS 07/25/02 Oakwood Products Room Temp, TCR-C01 C9HF1702 Shipping comments: Corrosive solid, No 3M ID# - must ship ground small quantity exception (30 mL/g or less per vial). LAS 10/02/02 7 j k- TC R-61 8.pdf 6,81:1COWofOliiml U & s -' J jz Can 000267 Page 174 of 225 Oakwood Products, Inc. 17 Old Dunbar Road FWPahxeosn(t8eC03(o8)l07u33m)97-b63i99a5-,87SSC0029172 CERTIFICATE OF ANALYSIS Date: 27~Nov-01 Material: Heptadecafluorononanoic acid Cat.No.: 2263 Cas No,: {375-95-1] Lot No.: H7568 Assay: 99+% by NaOH titration Appearance: Off-white solid Melting Point: 57-62C ..t i ExactCopyofOriginal :\ Qinnitcial ( o Df aate/g v 1 000268 Page 175 of 225 3M Environmental Laboratory E02-1039 -ieptadecafluorononanoic acid -leptadecafluorononanoic acid rCR-618 USE LOG Gross WUVol. Amnt. Before withdrawn withdrawal (mass or vol) Balance ID 1 Balance ID 2 20.8649g |0.0677g Gross wt/vol after withdrawal Balance ID 1 20.7972b Purpose (enter standard number or reason for removal) standard 02040-46 Balance Balance Initials ID ID 12 Date 914 RWW 105/18/2002 Exact Copyof Original QUi_ Ih l'ilfiL - initlaJ Data 000269 Page 176 of 225 3M Environmental Laboratory E02-1039 SINGLE COMPONENT PREPARATION LOG Date: $ f/ f / j z. Analyst: Description: /j/sn // of-0/>py)9ilr,/ -- LLcl-- Book No. 02 040 Page No. 46 </J. (LA--------- , TCA -C)%Stock Number Weight or Volume Used: Concentration or Purity: M Corrected Weight: fi* Balance ID: Other Correction Factors: AA Solvent and TN-A Number. A s # }/ T aA / .A M /I Final Volume: Final Concentration: f 2 - & ? Storage Location: A *tt J * ~ _____ Expiration Date: '>3 Exact Copy of Original Cmc tc /3 i/a i-- Initial Date Reviewed by: A* Signature, d l 'tvClAiCyt_______ _$ ! Ib i Dais i*)?. 000270 Page 177 of 225 3M Environmental Laboratory E02-1039 3M SPECIALTY MATERIALS MANUFACTURING DIVISION ANALYTICAL LABORATORY Jo: 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-618 by 'H-NMR and ,!IF-NMR Spectroscopy Date: October 28, 2002 SAMPLE DESCRIPTION: TCR-618, lot H7568 from the Telomer project. Nominal product = CF3(CF2)n-CO;H, where average n 7 (white solid). Sample Spectra #'s ExperimentDescriptions TCR-618 H71638.GID.402 400MHz `H-NMRinacetone-dsolvent+p-HFXcross integration/intemal std. TCR-618 F71638.G1D.402/.403 376MHz'T-N'MRinacetone-d,, solvent+p-HFXcross integration/internal std. OBJECTIVE: This sample was subjected to a combination of'H-NM R and 19F-NMR spectral analyses to determine the purity o f the nominal product. Special emphasis was also placed on attempting to identify and quantify any impurity components. EXPERIMENTAL: A portion o f the sample was accurately weighed, spiked with a known amount o f l,4-bis(trifluoromethyl)benzene (p-HFX), and then totally dissolved in deuterated acetone (acetone-ds) for subsequent analysis by NMR. A 400 MHz 'H-NMR spectrum and 376 MHz 19F-NMR spectra 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/I9F-NMR internal standard to allow the calculation o f the absolute weight percent concentrations o f specific components, or 2) a 'H/I9F-NMR cross integration standard to permit the cross correlation o f the relative 'H and l9F signal intensities for evaluation o f the overall sample composition. RESULTS: Tprhoedu'Hct-,NCMF3R(CanFd2),,l5-FC-0N2MH,Rwshpeercetrtahledaavteariangdeicvaatleude tohfisns=am6p.9l5e5w. aSsmaahlilgahmpouurnittys foofrma foewf thime pnuormitiynal components were also assigned. A 'H/19F-NMR cross integration analysis technique was then used to calculate the relative weight percent concentrations o f 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 o f 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 o f 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 of Original- Initial to/n3ato1( -- Page 1 of2 000271 Page 178 of 225 3M Environmental Laboratory E02-1039 October 28,2002 3MSMMD Analytical Lab Request #GID:71638 .TCR-618, Lot H7568: Telomer Project Tom Kestner c: Rick Payfer Ron Purcell William Reagen File Reference: ls7!638.GID.TCR-6lB_lol H7568_Telomer ProjeeLDOC/lOO TABLE-1 Sample: TCR-618, Lot H7568 from the Telomer project. Overall Compositional Results by 'H/i9F-NMR Cross Integration Analysis C""^ omponI ent StrTMuctures - "TM- NMR\ I Km RelativeL . 1 n l., m Weight% IC' oncentrations (single trial measurement) CF3(CF2)n-C 02H 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% 1.10% 0.65% 0.20% as possible F F or similar Total inorganic fluoride (at least 3-types), including some possible HF Possible CnH2n+2 saturated aliphatic hydrocarbons 1. Traceamounts ofotherunassignedcomponentswerealsodetectedintheNMRspectra. 0.021% 0.014% Page 2 of2 000272 ExactCopyof Original i v'K- _ - M 5 i ) 7 _ Initial Date Page 179 of 225 3M Environmental Laboratory E02-1039 Certificate of Analysis Nominal Product: CF3(CFj)n-C02H, where average n 7 Heptadecafluorononanoic acid Product Code: TCR-618, Lot H7568 October 28,2002 Tom Kestner The sample o f TCR-618, lot H7568 was analyzed using a combination of l9F-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 i9F/`H-NMR Spectral Analyses Component Structures 1 `H /'V -N M R 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. Trace amounts o f other unassigned components were also detected in the NM R spectra. f^ & W o fo -fg m , Page 1 of 1 File Reference: CofA_TCR-618_Lot H7568.doc 000273 Page 180 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: Nonadecafluorodecanolc acid Nonadecafluorodacanoic acid R11K ID#2264 12/01/2010 Initials: Number/size of containers: Condition: Retain JCP 1/120 mL plastic bottle white solid MCH 07/22/99 0.18740 TCR Substance # TCR # Received from: S036 TCR-36 Oakwood Products Inc 4/2/99 Amount received (wt. or 25g, 43.3264 g gross wt. vol): Date: 04/27/1999 Shipper: N/A MSDS (y/n) Date of Retain *YUN 10/18/1999 O Archived/Substance Not Available i 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 of 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 is , 'J L sd036msds.pdf sd036cofa.pdf Exact Copy of Original O n . _ o /Z ttcr ^ Initial Data 000274 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),,-C02H, where average n 8 (white powder). Sample Spectra#'s Experiment Descriptions SD036 SD036 1T-H71638.GID.403 400MHz 'H-NMRinacetone-d*solvent+p-HFXcross integration/intemal std. F71638.GID.404 376MHz NMRinacetone-d*solvent+p-HFXcrossintegration/intemal std. OBJECTIVE: This sample was subjected to a combination of'H-NM R and 19F-NMR spectral analyses to determine the purity o f the nominal product. Special emphasis was also placed on attempting to identify and quantify any impurity components. EXPERIMENTAL: A portion o f the sample was accurately weighed, spiked with a known amount of l,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 l9F-NMR spectrum were acquired at room temperature using a Varian UNITYpIus 400 FT-NMR spectrometer. This sample preparation method permitted the p-HFX to be used as either 1) a lH/l9F-NMR internal standard to allow the calculation o f the absolute weight percent concentrations o f specific components, or 2) a 'H/19F-NMR cross integration standard to permit the cross correlation o f the relative 'H and l9F signal intensities for evaluation o f the overall sample composition. RESULTS: Tnohme i'nHa-lNpMrodRuacnt,dCl5FFj(-CNFM2)Rn-Csp0e2cHtra, lwdhaetareintidreicaavteedratgheisvsaalumepolef wn a=s 7a.8r5e.latSimvealyll haimghoupnutrsitoyffaorfmewo f the 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 o f 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 o f these other components. Copies o f the NMR spectra are attached with the paper copy o f this report for your reference. If you have any questions about these results, please let me know. Exact Copyof Original Page t of 2 taltiai Data 000275 Page 182 of 225 3M Environmental Laboratory E02-1039 October 28, 2002 3MSMMD Analytical Lab Request # GID.71638 . SD036, Lot Rl IK: Telomer Project Tom Kestner c: R ick Payfer RcagcnRon Purcell W illiam Pile Reference: !s7l638.GID.SD036_Lol RIIIC _Telom erProjectD O C /10l TABLE-1 Sample: SD036, Lot R l IK from the Telomer project. Overall Compositional Results by `H/I9F-NMR Cross Integration Analysis Component SC tfrMulActfullMreAs I NMR\ T X i D RelativeD a ln r i.lA W eight% rC1o. ncentrations (single trial measurement) CF3(CF2)n-C 02H where average n = 7.85 Probable CF3(CF2)x-CF(CF3MCF2)y-C 02H where x*0 and assume x+y = 5.85 for calculation purposes Probable (CF3)2-CF-(CF2)n-C 02H assume n=5.85 for calculation purposes Probable I\ 98.01% 1.41% 0.36% 0.12% FF assume 11=3.85 for calculation purposes Probable L F jL 1 C F j'in 0H 0.060% FF assume n=2.85 for calculation purposes Probable CnH2n+2 saturated aliphatic hydrocarbons Total inorganic fluoride 0.026% >0.0095% 1. Traceamountsof numerous otherunassignedcomponentswerealsodetectedinthe r-NMRspectrum. Exact Copyof Original Page 2 of 2 000276 Page 183 of 225 OAKUOOD PRODUCTS Fax:803- 739-6957 SD -03U ? Oakwood Products, Inc. WP1F7ah4exo1sn(t8OeC03l(od8)l0u7D3m3)u97nb^3bi99aa5-,Sr7S6CR00o2a9d172 CERTIFICATE OF ANALYSIS Date; ll-Feb-00 Material: Nonadecafluorodecanoic add Cas No.: [335-76-2] Lot No.: R1LK Assay: PPPrerorofddluuucocttrssolmdeesocsraevoovnloiacltaiatlideledthtahnanFeFrefrlfulouroordoedceacnaoniocicadaidcd98%m<<11i%n%. Appearance: White solid Melting Point: 83-85C Exact Copyof Original J T L . io / S i Ic n Initial Dato /fumeiYantf /} ^ /Q u a lity C o n tro l v/ Page 18ST 225 3M Environmental Laboratory E02-1039 _________________________________________ USE LOG Nonadecafluorodecanoic acid Nonadecafluorodecanoic acid I5D036____________________________________ Gross Wt./Vol. Amnt. Gross wt/vol Before withdrawn after Purpose (enter standard number or reason for removal) withdrawal (mass or voi) withdrawal Balance ID 1 Balance IO 2 Balance ID 1 33.9944g 0.1196g 33.8784g standard 02022*90 Balance Balance Initials ID ID 12 Date 914 RWW 07/02/2002 Exact Copyof Original Bufai 000278 Page 186 of 225 3M Environmental Laboratory E02-1039 Date: Analyst: 0JL- Description^ SINGLE COMPONENT PREPARATION LOG ____ PBaogoek NNoo.. 0290022 Stock Number . f f ) 9 ? & Weight or Volume Used: /^ c Concentration or Purity:______ 9 % / Corrected Weight f O h O fry Balance ID :_ 9^ Other Correction Factors: a //* - Solvent and TN-A Number Final Volume: Storage Location: 2 ~ J 7 iX Final Concentration: / /O Expiration Date: / / t / > J Exact Copyof Original i'M l io/ Initial Date Reviewed by: P u X l . ( A d < W C > - l l l Sntfnalure, ~7 Data 7 1 Page L87 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 acid Perfluoroundecanoic acid U11N NA Initials: Number/size of containers: Condition: Retain OK 1-1 0ml amber glass vial white crystals TCR Substance # TCR# Received from: TCR-619 TCR-619 Oakwood products Amount received (wt. or 5g vol): Date: 07/19/2002 Shipper: Courier MSDS (y/n) Date of Retain YO N Q 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 of Analysis LAS 07/25/02 Oakwood products Room Temp, TCR-C01 C11HF2102 Shipping comments: Harmful solid, No 3M ID# - must ship ground small quantity exception (30 mL/g or less per vial). LAS 10/02/02 7 1b. TC R-61 9.Ddf Exact copy oforiginal l L Oate , 000280 Page 188 of 225 3M Environmental Laboratory E02-1039 3M SPECIALTY MATERIALS MANUFACTURING DIVISION ANALYTICAL LABORATORY Jo: 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-6I9 by *H-NMR and l9F-NMR Spectroscopy Date: October 29, 2002 SAMPLE DESCRIPTION: TCR-619, lot U 11N from the Telomer project. Nominal product = CFjfCT^n-CCkH, where average n 9 (white powder). Sample Spectra#'s Experiment Descriptions TCR-619 TCR-619 H71638.GID.404 400MHz 'H-NMRinacetone-d*solvent+p-HFXcross inteeration/intemal std. F71638.GID.405 376MHz"T-NMRinacetone-dsolvent+p-HFXcross intesration/intemal std. OBJECTIVE: This sample was subjected to a combination of 'H-NMR and l9F-NMR spectral analyses to determine the purity o f the nominal product. Special emphasis was also placed on attempting to identify and quantify any impurity components. EXPERIMENTAL: A portion o f the sample was accurately weighed, spiked with a known amount o f l,4-bis(trifluoromethyl)benzene (p-HFX), and then totally dissolved in deuterated acetone (acetone^) for subsequent analysis by NMR. A 400 MHz 'H-NMR spectrum and a 376 MHz ,9F-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/I9F-NMR internal standard to allow the calculation o f the absolute weight percent concentrations o f specific components, or 2) a 'H/I9F-NMR cross integration standard to permit the cross correlation o f the relative lH and 19F signal intensities for evaluation o f the overall sample composition. RESULTS: 9The 'H -N M R and , F -N M R spectral data indicated this sample was a relatively high purity form o f the nominal product, CF3{CF2)n-C02H, where the average value of n = 8.63. 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 o f 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 o f these other components. Copies of the NMR spectra are attached with the paper copy o f this report for your reference. If you have any questions about these results, please let me know. ExactCopyof Original Page 1 of 2 000281 Page 189 of 225 3M Environmental Laboratory E02-1039 October 29, 2002 3MSMMD Analytical Lab Request #G1D:71638 .TCR-619, LotUl IN: Telomer Project Tom Kestner c: MRiacrkkPEalylcfcfsron Ron Purcell W illiam Rcagcn Pile Reference: ls7I638.G lD .TC R -619_LotU llN _Tetom erP rojccl.D O C /l01 TABLE-1 Sample: TCR-619, Lot U1 IN from the Telomer project. Overall Compositional Results by *H/iqF-NMR Cross Integration Analysis Component Structures 1 NMR Relative Weight% Concentrations (single trial measurement) CF^CFjVCChH (where average n = 8.63) Probable internal branched isomers CF3(CF2),-CF(CF3)-(CF2)y-C 02H (where x * 0 and assume x+y = 6.63 for calculation purposes) Probable trans-olefins of general form: trans-CF3(CF2VCF=CF-(CF2)y-C 02H (assume x+y = 6.63 for calculation purposes) Possible terminal isopropyl branched isomers (CFj)2-CF-(CF2),,-C02H (assume n=6.63 for calculation purposes) Probable CnH2n+2 saturated aliphatic hydrocarbons Total inorganic fluoride 96.4% 2.6% 0.89% 0.12% 0.032% >0.0017% 1. Trace amounts ofnumerous otherunassignedcomponentswerealsodetectedinthe F-NMRspectrum. Page2 of2 Z2 $ Exact Copyof Original Y1C _ Initial -- Oats Page 190 of 225 3M Environmental Laboratory E02-1039 Certificate of Analysis Nominal Product: CF3(CF2)n-CC>2H, where average n 9 Perfluoroundecanoic acid Product Code: TCR-619, Lot U1 IN October 29, 2002 The sample o f TCR-619, lot U11N was analyzed using a combination o f ''F-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 Co o--m----p--o-n---e--n-t oStructures 1 , 'H /VF-xNr KM. rRo Relative Weight% Concentrations (single trial analysis) CF3(CF2)n-C 0 2H (where average n = 8.63) <96.4% Purity Probable internal branched isomers 2.6% CF3(CF2)x-CF(CF3)-(CF2)y-C02H (where x*0 and assume x+y = 6.63 for calculation purposes) Probable trans-olefins of general form: 0.89% '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) Probable C nH2n-2 saturated aliphatic hydrocarbons 0.032% Total inorganic fluoride >0.0017% 1. Trace amounts o f n u m erou s other unassigned components were also detected in the |4F-NM R spectrum. Tom Kestner Exact Copyof Original l o / 3 , ( 0 .2__ In itia l Oat Page I of 1 File Reference: CofA_TCR-619_LotUllN.doc 000283 Page 191 of 225 NWmNMENTAb LABOIfAV^Y Oakwood Products, Inc. 1741 Old Dunbar Road WI'heosnteC(o8l0u3m) 7b3ia9-,8S8C0029172 Fax (803) 739-6957 CERTIFICATE OF AN ALYS IS Date: 15-Jl-02 Material: Peifluoroundecanoic acid Cat.No.: 2265 Cas No.: 14234-23-5] Lot No.: UllN Assay: 99+% by NaOH titration Appearance: White solid Melting Point: 92-98cC DeadCopyof Original ad " Initial oatd m ju Yumei Yan QC Manag 000284 Page 192 of 225 3M Environmental Laboratory E02-1039 _________________________________________USE LOG erfluoroundecanoic acid 'erfluoroundecanoic acid CR-619 ____ ___________________________ Gross Wt./Vol. Amnt. Gross wt/vol Purpose (enter standard number Before withdrawn after withdrawal (mass or voi) withdrawal or reason for removal) Balance ID 1 Balance ID 2 Balance ID 1 15.5264g 0.0691g 15.4573g standard 02040-65 Balance Balance Initials ID ID 12 Date 314 RWW 08/29/2002 CopyofOriginal taitiaJ oats 000285 Page 193 of 225 3M Environmental Laboratory E02-1039 Data: Analyst: Jf /^ / o 2~- SINGLE COMPONENTPREPARATION LOG Book No. 02 040 Page No. 65 Description: f r r t L n > t j re t a i r Stock Number: 7*~f / ? Cr 9 Weight or Volume Used: --------------P----*----*- 5* Concentration or Purity: j/h Corrected Weight: 0 rO 7 ^ Balance ID: 9 / CI Other Correction Factors: /S /h Solvent and TN-A Number 7 y Final Volume: FinalConcentration: / ? TA. * Storage Location: P / 0t a ^ - /U , Expiration Data: A l / 9 ? ExactCopyofOriginal lYlC n / .' / o 3- intttal Data Reviewed by: CYiki f\x J 'lfyt/f'/lJon 1d |^ |/pX Signatum, Date 000286 Page 194 of 225 3M Environmental Laboratory E02-1039 Test Control and Reference Substance Log Substance trade name or reference ft Substance/chemical name: Lot/batch #: 3M # Expiration date: Perfluorododecanoic acid Perfiuorododecanoic acid R24K ID82266 12/01/2010 Initials: Number/size of containers: Condition: JCP 1/120 mL plastic bottle white powder MCH 07/22/99 Retain 0.2159g O Archived/Substance Not Available TCR Substance # TCR # Received from: SD037 TCR-37 Oakwood Products Inc 4/2/99 Amount received (wt. or 25g, 43.4548 g gross wt. vol): Date: 04/27/1999 Shipper: N/A MSDS (y/n) Date of Retain y'U 'n 10/18/1999 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 of 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 sd037m sds.pdf sd037cofa.pdf NM R SD037.pdf 000287 ExactCopyofOriginal -A W --\ o l - i j ) f Initial oats 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-11 Subject: Characterization of SD037 by 'H-NMR and ''F-NMR Spectroscopy Date: October 28, 2002 SAMPLE DESCRIPTION: SD-037, lot R24K from the Telomer project. SNaommpleinsal producStp=ecCtrFa 3It('C F2)n-CQ2H, where average n Ex1p0er(imwehnitteDepsocwripdtieorn)s. SSDD003377 HF7711663388..GGIIDD..440065 430706 MMHHzz `1TH--NNMMRRiinnaacceettoonnee--ddssoollvveenntt++pp--HHFFXXccrroossssiinntteegRrraattiioonn//iinntteemmaallssttdd.. OBJECTIVE: This sample was subjected to a combination of'H-NM R and ,9F-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 o f the sample was accurately weighed, spiked with a known amount o f l,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 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 o f the absolute weight percent concentrations o f 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 o f the overall sample composition. RESULTS: The ` H -NM R and l9F-N M R spectral data indicated this sample was a high purity form o f the nominal product, CF3(CF2)n-C02H, where the average value o f n = 10.042. Small amounts o f a few impurity components, including probable isomers, were also assigned. A 'H/'9F-NMR cross integration analysis technique was then used to calculate the relalive 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 me know. Page 1 of 2 ExactCopyofOriginal Cm to ls j/r ^ initial Dais 000288 Page 196 of 225 3M Environmental Laboratory E02-1039 October 28, 2002 3MSMMD Analytical Lab Request it GDD:71638 SD037, Lot R24K: Telomer Project Tom Kestner c: R ick P ayfer RWoinlliPaumr cRe lclagcn File Reference: U7l638.GID.SD037_Lot R24K_Telomer Project.DOC/]0! TABLE-1 Sample: SD037, Lot R24K from the Telomer project. Overall Compositional Results by 'H/^F-NMR Cross Integration Analysis OCon mn . p oAnM entCSttnrIu. ct1u. Are. s1 NMR RelativXTA r D O a ln t . I. A e WAA / e:iAgCh.tt)%/ /"CA--oncentrations (single trial measurement) CF3(CF2)n-C 02H where average n = 10.02 Probable (CF3)2-CF-(CF2)n-C 02H assume n=8 for calculation purposes Possible CF3(CF2)x-CF(CF3)-(CF2)y-C 02H where x*0, y*0 and assume x+y = 8 for calculation purposes Probable chlorinated impurity, Cl-CF2CF2-Rf, possibly as Cl-(CF2)n -C 02H Possible methyl ester impurity as CF3(CF2),,-C02CH3 where average n = 10.02 Probable CnH2n+2 saturated aliphatic hydrocarbons Toluene 1. Trace amounts of other unassigned components were also detected in the NMR spectra. 99.65% 0.13% 0.088% 0.049% 0.048% 0.016% 0.015% Page 2 of2 000289 ExactCopyof Original CM , |Ol Initial Dais Page 197 of 225 3M Environmental Laboratory EQ2-1039 Certificate of Analysis Nominal Product: CF3(CF2),rC02H, where average n 10 perfluorododecanoic acid Product Code: SD037, Lot R24K October 28, 2002 Tom Kestner The sample o f 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, LotR24K Quantitative Compositional Results by Combined ^F/H-NMR Spectral Analyses Component Structures 1 `H /l,>F-NMR Relative W eight"/. Concentrations (single trial analysis) CF3(CF2),,-C02H where average n = 10.02 Probable (CF3)2-CF-(CF2),,-C02H assume n=8 for calculation purposes Possible CF3(CF2),-CF(CF3)-(CF2)y-C 02H where x*0, y*0 and assume x+y = 8 for calculation purposes Probable chlorinated impurity, Cl-CF2CF2-Rf, possibly as Cl-(CF2)n -C 02H Possible methyl ester impurity as CF3(CF2)n-C 02CH3 where average n = 10.02 Probable C,,H2n+2 saturated aliphatic hydrocarbons Toluene 299.65% Purity 0.13% 0.088% 0.049% 0.048% 0.016% 0.015% 1. Trace amounts ofotherunassignedcomponentswerealsodetectedintheNMRspectra. ExactCopyofOriginal Initial Data Page 1 of 1 FileReference: CofA_SD037_LotR24K.doc 000290 Page 198 of 225 OfKWOOD PRODUCTS Fax:803-739-6957 Feb W m O R O N y ^ A L Oakwood Products, Inc. (( V // | * \\ y \J1 1WPF7ha4eox1sn(tO8eC0l(o3d8l)0u7D33m)u97nb-36bi9a9a~5,r87S8CR00o2a9d172 CERTIFICATE OF ANALYSIS Date: ll-Feb-00 Material: Perfluorododecanoic add Cat.No.: 2266 Lot No^ R22K Assay: PPreordfluucotrsomdoodreecvaonliactailde dthan Perfluorododecanoic add96%<nu2n%. Products less volatile than Perfluorododecanoic add <2% Appearance: W hite solid M elting Point: 107-109C ExactCopyofOriginal CWM 1Q / ^ l t P - ' Initial Oats 000291 Page) 199 of 225 3M Environmental Laboratory E02-1039 ________________________________________ USE LOG 'erfluorododecanoic acid 'erfluorododecanoic acid D037 ______________________________ Gross Wt./Vol. Amnt. Gross wt/vol Puipose (enter standard number Before withdrawn after or reason for removal) withdrawal (mass or voi) withdrawal Balance ID 1 Balance ID 2 Balance ID 1 42.5752a 0.0796g 42.4956a standard 02040-55 Balance Balance Initials ID ID 12 Date 914 RWW 08/29/2002 000292 ExactCopyoforiginal 1UL InitfaJ Oats Page 200 of 225 3M Environmental Laboratory E02-1039 SINGLECOMPONENT PREPARATION LOG Date: % h J ___________ Analyst: Description: _____________ P t t . ( iu a f d f u J s r j H a , i AnJ - ft, Book No. 02 040 Page No. 55 faa/J Stock Number v X O O S ~ 7 Weight or Volume Used: 2 J & S < Concentration or . Purity:________M A Corrected Weight: Balance ID: 9 /V O ther Correction Factors: A. ' T---- Solvent and TN-A Number M J r > H -- / V - A - FinalVolume:______ / Final Concentration: / / Z '-.je .jiJ * . Storage Location: JZ * r J A f o o t ' -- fi-u . T * t~ */) Expiration Date: A . / -9 /& J ? Reviewed by: ExactCopyofOriginal I huBIC j v ^c filia l UBSi> / > / . 000293 Page 201 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: PFOS Potassium Periluorooctane Sulfonate FC-95 217 98-0211-0888-5 08/31/2006 Initials: Number/size of containers: Condition: PMR 1/175 mL glass container white powder MCH-07/22/99 Retain 0.1733g O Archived/Substance Not Available ] TCR Substance # SD018 TCR # TCR-18 Received from: Jo Dickes 8/10/98 Amount received (wt. or 160.0706g gross wt. vol): Date: 04/07/1999 Shipper: Unknown MSDS (y/n) Date of Retain # YO N 10/18/1999 Purity: Records received: Location of synthesis, fabrication, or derivation records: Std Location/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 of Analysis. LAC 04/26/01 98-0211-0888-5 F19, Frozen C8F17S03-K+ Environmental Lab Traceablitity 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-O185-6(<30 g on dry Ice) LAC 06/14/01 Shipping Comments: Dangerous goods In excepted quantity of Class 6, UN2811. REGULATED-TOXIC 98-0211-0888-5 (>30 g (1 oz sample)) Not on dry ice. LAC 01/08/02 T J. sd018m sds.pdf sd018nm rreq53030.pdf sd018nm rreq61517.pc t if EOO-16 8 2 -C O A -S D 0 1 B -P F O S -R ev 3 ExactCopyofOrigina/ 000294 Page 202 of 225 3M Environmental Laboratory E02-1039 Centre Analytical Laboratories, Inc. 3 0 4 8 Research Drive State College, PA 16801 w w w .cen trelab .co m Phone: (814) 231-8032 Fax: (814) 2 3 1 -1 2 5 3 or (8 1 4 ) 2 3 1 -1 5 8 0 INTERIM CERTIFICATE OFANALYSIS C e n tre Analy tical LaboraRtoerviiessioCnO3A Reference 3M Product: PFOS,Lot217 if : 023-018A 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 Inorg1.aniCc hAlonriiodnes (IC) 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate* Organic Acids 5(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis": 1. Carbon 2. Hydrogen 3. Nifiogen 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 wt7wt.% 3. 1.439 wt,/wt.% 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 wtVwt.% 4. <0.009 wt.Avt.% 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 wt/wt.% 3. 1.74 wt/wt.% 4. 8.84 wt/wt.% 5. 54.1 wt/wt.% ExactCopyofOriginal f y \( IQ f a I ? ? - COA023-018A 000295 Page 1 o f3 Page 203 of 225 3M Environmental Laboratory E02-1039 Centre Analytical Laboratories, Inc. 3 0 4 8 Research Drive S tate College, PA 16801 w w w .ce n tre ta b .c o m Phone: (814) 231-8032 Fax: (814) 2 3 1 -1 2 5 3 or (814) 2 3 1 -1580 INTERIM CERTIFICATE OFANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 08/31/00 Expiration Date: 08/31/06 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/06 lPurity = 100% - (sum of metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+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. 4Sulfiir in the sam ple appears to be converted to S 0 4 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. ST F A HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid th e o re tic a l value calculations based on the empirical formula, CgFi7S0 3 'K +(MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). '01 M oq jagjg COA023-018A 000296 Page 2 o f3 Page 204 of 225 3M Environmental Laboratory E02-1039 Centre Analytical Laboratories, Inc. 3 0 4 8 Research Drive State College, PA 16801 w w w .ce n tre la b .c o m Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS R e v is io n 3 Centre Analytical Laboratories COA Reference #: 023-018A LC/M S Purity Profile: Im pu rity C4 C6 Cl Total wt./wt. % 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, 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. oLA ExactCopyofOriginal Prepared By: Scientist, Centre Analytical Laboratories Reviewed By: n,L mfULcj /Mf R)hn Flaherty / Date lab o ra to ry Manager, Centre Analytical Laboratories COA023-018A 000297 Page 3 of 3 Page 205 of 225 3M Environmental Laboratory EQ2-1039 I PFOS potassium Perfluorooctane Sulfonate FC-9S (5D018 USE LOG Gross Wt./Vol. 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 1 140.4480g 0.0814g 140.3666g standard 02022-56 Balance Balance Initials ID ID 12 Date 914 RWW 05/13/2002 C;;-i cpy oforiginal O t tiy vu0298 Page 206 of 225 3M environmental Laboratory EO2-1039 SINGLECOMPONENTPREPARATION LOG Date: Analyst - Description: f P S S / . J l Book No. 02 022 Page No. 56 Stock Number J J W l Weight orVolume Used:_____ > ^ Concentration or Purity: 1l Corrected Weight t -M Final Volume: Storage Location: / i t (a Balance ID: Other Correction Factors: h J 2 - Solvent and TN-A Number M s # - /T a I ' A '0 * Final Concentration: I o V f f /y - Expiration Date: Exact Copy of Original ._J o / S H lo n Initial Data Reviewed by: ft- Signatura, 000299 Page 207 of 225 Attachment E: Protocoland Amendment 3M Environmental Laboratory E02-1039 000300 Page 208 of 225 3M Environmental Laboratory E02-1Q39 STUDY PROTOCOL STUDY TITLE Analysis of Endogenous Fluorocheraicals in Normal Pooled Human Serum and Plasma SPONSOR William K. Reagen, Ph.D DATA REQUIREMENT 40 C FR 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 ExactCopyofOriginal cm ic te ljy Initial Data 000301 Page 209 of 225 Analysis of Endogenous Fluorochemicals 3 M E n v ir o n m e n t a l La b o r a t o r y E 02-1039 E02-1039 STUDY IDENTIFICATION Analysis of Endogenous Fluorochemicals in Normal Pooled Human Serum and Plasma Sponsor S tudyDirector Test Facility Proposed S tudy 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 000302 ExactCopyof Original tnltiaf Oats Page 2 of 10 Page 210 of 225 Analysis of Endogenous Fluorochemicals 3M Environmental Laboratory E02-1039 E02-1039 1.0 Introduction and Purpose/Objective 1.1 The purpose o f 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 (C l2), 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 Substance Formula Perfluorohexanoic Acid Tetradecaftuoroheptanioc Acid Pentadecafluorooctanoic Acid Heptadecafluorononanoic Acid Nonadecafluorodecanoic Acid Perfluoroundecanoic Acid Perfluorododecanoic Acid 1H,2H,3H,4H-perfluorooctanesulfonate lH,2H,3H,4H-perfluorodecane sulfonate Potassium Perfluorooctanesulfonate CsFnCOOH CflFiaCOOH C jF isC O O H c, f 17c ooh CFuCOOH C,oFj,COOH C11F23COOH C g H sF ijQ jS C ujH gFijO sS C,F,,SO,K* ExactCopyof Original CJVr -- Initiai Data 3M Environmental Laboratory Page 3 of 10 000303 Page 211 of 225 Analysis of Endogenous Fiuorocherracals 3M Environmental Laboratory E02-1039 E02-1039 4.0 Reference Substances R eference Substances Reference Substance Formula Traceability * Source Perfluorohexanoic Acid CsFuCOOH TCR-047 SMM* 236-1B-10 Tetradecafluoro beptanioc Add C^tjCOOH TCR-267 Aldrich Pentadecafluoro octanoic Add CrF.jCOOH TCR-617 Oakwood Products Heptadecafluoro nonanoic Add CaFtrCOOH TCR-618 Oakwood Products Nonadecafluoro decanoic Acid CsFibCOOH TCR-036 Oakwood Produds Perfluoroun decanoic Add C10Fj,COOH TCR-619 Oakwood Products Perfluorodo decanoic Add CiiFaCOOH TCR-037 Oakwood Produds 1H,2H,3H,4Hperfluorooctane sulfonate CJf.FnOjS TCR-343 SynQuest Labs 1H,2H,3H,4Hperfluorodecane sulfonate CioHsFitOjS TOR-627 Pace Analytical Potassium Perfluorooctane sulfonate C,F,,SOaX* TCR-018 SMM* 236-1B-10 'D o c u m e n t a t io n o f th e m e th o d o f syn th esis is lo c a te d at th e s o u rce . **A sample o f forthe reference substance has been sent characterization 5.0 Test System Test Systems 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% Storage Conditions Frozen Frozen Ambient Temperature Ambient Temperature Frozen Ambient Temperature Frozen Frozen Ambient Temperature Frozen Test System: Source T raceability Pooled Human Serum Pooled Human Serum Pooled Human Serum Pooled Human Serum Sigma-,Aldrich, Milwaukee, W l Lamplre Biological Laboratories, Pipersville, PA Bioresource Technology, Inc., Fort Lauderdale, FL Golden West Biologicals, Temecula, CA TCR-689 TCR-688 TCR-687 +CR-690 ExactCopyofOriginal initial oto 3M Environment Laboratory P age 4 of 10 000304 Page 212 of 225 Analysis of Endogenous Fluorocfiemicala 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, Pipersvllle, PA Golden West Biologicals, Temecula, CA Innovative Research, Inc., Southield, Ml Central China T raceability TCR-685 TCR-684 TCR-683 TCR-674 5.1 J u s t if ic a t io n o f th e te st sy ste m . Based on preliminary testing, normal pooled serum and plasma contain endogenous levels of the test analytes. 5.2 Iddaeten toifficina ittiioanl poref pTaersattSioyns ,t etmest. Samples substance shall be identified by the study number, 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 T raceability 1 TCR-686 6.1 J u s t if ic a t io n o f th e S u r r o g a t e M a t r ix . Based on preliminary testing, normal pooled rabbit serum contains very low endogenous levels o f the test analytes and is thereby suitable for use as a surrogate matrix. 6.2 Induemn bt iefric,adtaioten oof finS iutirarlo pgraetpeaMraatitornix, .tesStasmubpsletas nschealolrbseuirdreongatitfeiemd by the study atrix, sample number, replicate number (if applicable), analyst(s), and project leader. 7.0 Analytical Methods 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 CopyofOriginal C m ,t o l3 ll6 L - Initial Oats 3M Environmental Laboratory Page 5 of 10 000305 Page 213 of 225 Analysis of Endogenous Fluorochemicals 3M Environmental Laboratory E02-1039 E02-1039 8.0 Data Quality Objectives 8.1 Absolute Recovery: 8.1.1 The absolute recovery o f 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 o f (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 o f 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 o f the standards). 8.1.2 The accuracy (% recovery) and precision (%CV o f 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 o f 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 o f 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% o f nominal. All levels must show a response greater than twice that o f the blank. A maximum o f four (4) levels may be deactivated in any one set, or the set will be re-analyzed. 8.3 Limits o f Quantitation (LOQ): The lower limit o f 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 o f the blank. These limits will be determined during the course o f tdhee-ascttuidvaytaenddindoacpuamrteincuteladrisnett,htehreapwradcattiac.alSlhimouitldofthqeuLanLtOitaQtiolenvfeolrctahliisbrsaettiownilbl e be raised to the next acceptable level. Samples below the practical LOQ o f that set will be reanalyzed until quantitated in a set including the validated LLOQ. The upper limit o f 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 o f 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 o f the calibration curve. If samples are diluted into the range o f the curve during analyses and enough sample remains, a post-run dilution validation will be performed to verify sample values. To Exact Copyof Original 3M Environmental Laboratory Page 6 of 10 000306 Page 214 of 225 Analysis of Endogenous Fluorochemicais 3M Environmental Laboratory E02-1039 E 0 2 -1 0 3 9 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 o f diluting the sample with additional matrix 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). If the values are not within 15% o f each other additional testing will be required to determine which value is a correct representation o f 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 o f 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 o f matrix fortifications, prepared at known concentrations o f the test substance and bracketing the anticipated range o f the method will be evaluated to determine recovery and to evaluate method performance. Reagent and matrix blanks will be ran with each set to evaluate the level o f background interferences. 9.0 Statistical Methods and Calculations 9.1 Statistical methods for the analytical results will be limited to the calculations of means, standard deviations, and relative standard deviations (as appropriate). 10.0 Report A report o f 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 compl iance 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 ExactCopyofOriginal 3M Environmental Laboratory Page 7 of 10 000307 Page 215 of 225 Analysis of Endogenous Fluorochemicals 3M Environmental Laboratory E02-1039 02*1039 contain updated methods incorporating any changes or improvements. 10.1.6 A description of the test system. 10.1.7 A description o f any circumstances that may have affected the quality or the integrity o f the data. 10.1.8 The name o f the Study Director and the names o f other scientists, professionals, and supervisory personnel involved in the study. 10.1.9 A description o f the transformations, calculations, or operations performed on the data, a summary and analysis of the analytical chemistry data, and a statement o f 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 o f each o f 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. 10.1.13 A statement prepared by the quality assurance unit listing the dates that study inspections and audits were made and the dates o f 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 o f 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 cEonmviprloinanmceenwtailthLaGbooordatoLraybSortaantodrayrdPrOacpteircaetiSntganPdraorcdesduanreds.wTithhe3QMuality 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 o f 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 Copyof Original Initial oats 3M Environmental Laboratory Page 8 of 10 000308 Page 216 of 225 Analysis of Endogenous Fluorochemicals 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 o f 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 o f the reference substances used in the study will be retained in the laboratory for a period o f not less than 2 years after a report is issued. 13.2 Sample extracts will be retained and refrigerated for a period o f not less than three months from the time o f analysis. 14.0 Protocol Amendments and Deviations____________________________________ 14.1 Amendments and deviations to the protocol will be in the form o f 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 Director and filed with the raw data. 15.0 Attachments_______________________________________________________ None \ 3M Environmental Laboratory ExactCopyofOriginal U L. Initial o 5 T Page 9 of 10 000309 Page 217 of 225 Analysis of Endogenous Ruorochemlcals Protocol Signature 3M Environmental Laboratory E02-1039 E02-1039 Protocol Approval Sponsor William K. Rcagen, PhJD. ___________________________________ Date: 3M Environmental Laboratory 3M Environmental Laboratory 000310 ExactCopyofOriginal Initial Data Page 10 of 10 Page 218 of 225 3M Environmental Laboratory E02-1039 Study Title Analysis o f Endogenous Fluorochemicals in Normal Pooled Human Serum and Plasma PROTOCOL AMENDMENT NO. 1 Amendment Date: October 31,2002 Peiforming Laboratory 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55144-1000 Laboratory Project Identification 3M Environmental Laboratory Study LEMS #E02-1039 ExactCopyof Orignal 000311 Page 219 of 225 3M Environmental Laboratory E02-1039 P rotocol UEOO-1311 Amendment 1 This amendment modifies the following portion(s) of the protocol: t. P rotocol reads: Section 8.1.1: Theabsoluterecoveryofthemethodwill beevaluatedseparatelyinhuman serumandplasmaandratserum. For eachmatrix, thesampleswill befortifiedattwolevels of(500pptand5ppb). All samples will be extractedthroughthemethod, andanalyzedbycomparisonwithexternal calibrationofnonextractedstandards. Thenon-extractedstandardcalibrationcurveswill bepreparedinmethanol, andwill consistof aminimumofnine (9) levels, includingamethanolblank. Thebestappropriateregressionwill beusedtodescribe thiscurve(forbestaccuracyatall levelsofthestandards). cAommepnadrisToOnrliemaidte:dTihneitasbussoelfuutelnreescso. very of the method will not be evaluated as the matrix effects render this type of R eason: A planned change in the study's direction. 2. P rotocol reads: Section 8.2: Calibration: beCalibrationcurveswill preparedfromextractedmatrix standards, inChinese plasmaandrabbit serum. Thecurveswill consistofaminimumofnine(9) levels andamatrix bAlamnke.nd to read: Calibration: Calibrationcurves will bepreparedfromextractedmatrix standards inChinese plasma. Thecurves will consistofaminimumofnine(9) levels. Reasons fornotusingoneormoreofthese standards intheconstructionofacalibrationcuiveareuptothediscretionoftheanalystandwill bedocumentedin therawdata. uRseeadstoonc:onTshtreucstamthpelecusrwvee.reArudndiatigoanianlslty9raebxbtriatcctuerdvestsawndearrednsowt iitnhcoluutdaedblianntkh,ebduattanoftoarltlhoisfstthuedsye.standards were 3. P rotocol R eads: Section 7.1: Analysisofthetestandreferencesubstanceswill beconductedinthe3M Environmental Laboratory. Theanalyseswill beconductedas describedby3MEnvironmental LaboratoryMethod L"AESaTmobSeloi-ndr8ad-Pto2hTr3ayO1s.,eR"TESehoaxeltdirdaa:ncAPatihlnoyaanssleyeassnEiwdsxitoAlrlafnbctahetlieyocsntoiesnsadtnouadfcnFAtdelnudroaeaflryseosrcdiehsenescomcefriiFscbulaeubldosCrtbaooyncmch3peeMsomuwEincidnallvslbiCfreroooncmmompnBeodnuiutonacldltoesLgdiahcibonaorltnrmhaeBtaoit3rorMyilcoMegEsie"nct.vahlioSrmdoenlaEemtcTrteieScnde-t8asi"l-o.2n31, monitoring will be used to detect the presence of the target analytes. Tire masses scanned will be documented in the raw data and in the final report. Reason: Add clarity. 4. P r o t o c o l R e a d s : S e c t io n 823Anysamplewithanareagreaterthan110%ofthehighestacceptable standardwill needtobedilutedintotherange ofthecalibrationcurve. Ifsamples aredilutedintotherange of thecurve duringanalyses andenoughsampleremains, apost-rundilutionvalidationwill beperformedto verify samplevalues. Toperformthedilutionvalidation, onesamplewill beseparatedintotwo representativesamples (i.e. two 1 mLaliquots forfluidsamplesortwo1 gramamounts for tissuesamples) thendilutedusingtwoprocedures. The firstprocedureconsistsofdilutingthesamplewithadditional matrixpriortoextraction(seraaddingsera), whilethesecondprocedureconsists ofdilutingtheextractwith solvent post-extraction(methanol extract addingadditional methanol solvent). If thevalues arenotwithin15%ofeachotheradditional testingwill berequiredtodeterminewhichvalue is acorrect representationofthesampleconcentration. ExactCopy o 3MEnvironmental Laboratory C rV it Page 2 of 6 Initial 000312 Page 220 of 225 3M Environmental Laboratory E02-1039 Protocol # E 0 0 -1 3 1 1 Amendment 1 Amend to Reap: Any sample with an area greater than 110% of the highest acceptable standard will be reported as >ULOQ. R : studyeason Thepurposeofthis istodemonstratethepresenceofthetargetanalytes inpooledhumanserumand plasma. It is more importanttoshowthat endogenous levels aredetectablethantoshowspecificallywhatthose levels are. 5. P rotocol Reads: Section 1.1 Thepurposeofthis studyis toquantifyperfluorohexanoicacid(C6), perfluoroheptanoic acid(C7), pentadecafluorooctanoicacid(C8), heptadecafluorononanoicacid(C9), nonadecafluorodecanoicacid(CIO), perfluorodecanoicacid(Cl 1), perfluorododecanoicacid(C12), tetrahydropcrfluorooctanesulfonate(THPFOS), tetrahydroperfluorodecanesulfonate(THPFDS), and perfluorooctanesulfonate(PFOS) innormalpooledhumanserumandplasma. Thisstudydoes nothaveatypical test substance thatis dosedontoaspecific controlledtestsystemasperaconventional GLPstudy. A R :mend to ead Thepurposeofthisstudyis toquantifyperfluoroheptanoicacid(C7), pentadecafluorooctanoic acid(C8), heptadecafluorononanoicacid(C9), nonadecafluorodecanoicacid(CIO), perfluorodecanoicacid(Cl 1), perfluorododecanoic acid(Cl2), tetrahydroperfluorooctanesulfonate(THPFOS), tetrahydroperfluorodecane sulfonate(THPFDS), andperfluorooctanesulfonate(PFOS) innormal pooledhumanserumandplasma. This study does nothaveatypical testsubstancethatisdosedontoaspecificcontrolledtestsystemasperaconventional GLP study. REASQNt.The Co compoundwas eliminatedfromthestudy. 6. Pkotocol R eads: Section 3.1 Test S ubstances Test Substance Perfluorohexanoic Acid Tetradecafluoroheptanioc Acid Pentadecafluorooctanoic Acid Heptadecafluorononanoic Acid Nonadecafluorodecanoic Add Perfluoroundecanoic Acid Perfluorododecanoic Add 1H,2H,3H,4H-perfluorooctanesulfonate 1H,2H,3H,4H-perfluorodecane sulfonate Potassium Perfluorooctanesulfonate Amend to Read: T est S ubstances Test Substance Tetradecafluoroheptanioc Acid Pentadecafluorooctanoic Acid 3MEnvironmental Laboratory Formula CsFuCOOH CeFuCOOH C FisCOOH CFirCOOH C9F,jCOOH CtoF2tCOOH C,,FaCOOH CnHsFnOjS CtoHaFuOaS CiFuSOfK* Formula CsFuCOOH C7F,5COOH 000313 Page 3 of 6 ExactCopy of Or inIitXial_ I,n/,o Page 221 of 225 3M Environmental Laboratory E02-1039 Heptadecafluorononanoic Acid Nonadecafluorodecanoic Acid Perfluoroundecanoic Acid Perfluorododecanoic Acid lH,2H,3H,4H-parfluorooctanesulfonate 1H,2H,3H,4H-perfluorodacane sulfonate Potassium Perfluorooctanesulfonate Reason: The C b compound was eliminated from the study. CF,,COOH C*F,bCOOH CioFatCOOH C,,F2jCOOH c.hsf 13o3s C,oH5Fi70 3S CjF^SOj K* Protocol # E 0 0 -1 3 1 1 Amendment 1 7. Protocol Reads: Section 4.0 Reference Substances Reference Substance Formula Peril uorohexanoic Acid CsFnCOOH Telradecafluoro heptanloc Acid CeFnCOOH Pentadecafluoro octanoic Acid C7F,sCOOH Heptadecafluoro nonanoic Acid CeF,7COOH Nonadecafluoro decanoic Acid C*F,COOH Perfluoroun decanoic Acid C10F21COOH Perfluorodo decanoic Acid C,,Fj3COOH 1H,2H,3H,4Hperfluorooctane sulfonate c, hsf , 3o 3s 1H,2H,3H,4Hperfluorodecane sulfonate C.oHsFnOjS Potassium Perfluorooctane AmendsutlfoonRaetead C#Ft7 S0 3 k* Reference Substances Reference Substance Telradecafluoro heptanioc Acid Formula CaFnCOOH Pentadecafluoro octanoic Acid C7Fi5COOH Traceability It 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 SynQuest Labs Physical Description Colorless Liquid Clear Crystals White Crystals White Crystals White Solid White Crystals White Powder White Powder TCR-627 Pace Analytical White Crystals TCR-018 SMM* 236-1 B-10 White Powder Traceability # TCR-267 TCR-617 Source Aldrich Oakwood Products Physical Description Clear Crystals White Crystals Purity TBD** 99.5% > 97% > 99% 98% > 99% 96% TBD 94.7% 86.9% Purity 99.5% > 97% Storage Conditions Frozen Frozen Ambiant Temprature Ambiant Temprature Frozen Ambient Temprature Frozen Frozen Ambient Temprature Frozen Storage Conditions Frozen Ambient Temperature Exact Copy of Origina! . M A U aT- Initial Date 3MEnvironmental Laboratory Page 4 of 6 000314 Page 222 of 225 3M Environmental Laboratory E02-1039 Pentadecafluoro octanoic Acid Heptadecafluaro nonanoic Acid Nonadecafluoro decanoic Add Perfluoroun decanoic Acid C,F,sCOOH c,f17cooh CgFisCOOH CioFjiCOOH TCR-617 TCR-618 TCR-036 TCR-619 Oakwood Products Oakwood Products Oakwood Products Oakwood Products Perfluorodo decanoic Acid C11F23COOH TCR-037 Oakwood Products 1H.2H.3H.4Hperfluorooctana sulfonate C.HsFnOsS TCR-343 SynQuest Labs 1H,2H,3H,4H- perfluorodecane sulfonate C10H5F17O3S TCR-627 Pace Analytical Potassium Perfluorooctane sulfonate CaFirSOfK* TCR-018 SMM" 236-18-10 R e a s o n : T he C6compound was eliminated from the study. White Crystals White Crystals White Solid White Crystals White Powder White Powder White Crystals White Powder Protocol #EQQ-1311 Amendment 1 >97% >99% 98% Ambient Temperature Ambient Temperature Frozen > 99% Ambient Temperature 96% Frozen TBD Frozen 94.7% Ambient Temperature 86.9% Frozen 8. Photocoi, Reads; Section6.0 Surrogate Matrix ________ S urrogate Matrix: Rabbit Serum | Source Sigma-Aldrich T r a c e a b ilit y TCR-686 6.4 Justification o f the Surrogate Matrix. Basedonpreliminarytesting, normal pooledrabbitserumcontains verylowendogenouslevelsofthetestanalytesandistherebysuitableforuseas asurrogatematrix. 6 .5 Identification o f Surrogate Matrix. Samplesshall beidentifiedbythestudynumber, dateofinitial preparation, testsubstanceor surrogatematrix, samplenumber, replicatenumber(ifapplicable), analyst(s), andproject leader. A m e n d t o r e a d : None R e a s o n : It was decided not to use rabbit serum curves to generate data for this study. 9. P r o t o c o l R e a d s : S e c t io n 8.3 Limits ofQuantitation(LOQ): Thelowerlimitofquantitation(LLOQ) will be determinedforeachanalyte. The level determinedas theLLOQmustshowarecoverywithin75%- 125%forthe analytes andmust showaresponsegreaterthantwicethatoftheblank. ExactCopyofOrigin? u /,v InfflaJ oats 3MEnvironmental Laboratory Page 5 of 6 000315 Page 223 of 225 3M Environmental Laboratory E02-1039 P rotocol # E 0 0 -1 3 1 1 Am endm ent 1 A R :mend to ead Limits ofQuantitation(LOQ): Thelowerlimitofquantitation(LLOQ) will bedeterminedfor eachanalyte. Thelevel determinedastheLLOQmustshowarecoverywithin70%- 130%fortheanalytes. R e a s o n ; Since this is a screening-type study it was appropriate to relax the acceptable recovery levels in order to avoid re-running sample sets. Amendment Approval 3M Environmental Laboratory 0316 ExactCopy of Original -L iZ iU c>2_ Initial 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, IVIN 55106 Laboratory Project Identification ET&SS E02-1039 This amendment modifies the following portion(s) of the protocol: PAGE3, SECTION1.1, PROTOCOLreads: The purpose o f this study is to quantify, perfluoroheptanoic acid (C7), pentadecafluorooctanoic acid (C8), heptadecafluorononanoic acid (C9), nonadecafluorodecanoic acid (CIO), perfluorodecanoic acid (C ll), perfluorododecanoic acid (Cl2), 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. AMENDtoread: The purpose o f 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. REASON: THPFOS and THPFDS were removed from the list o f 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 o f test substances, section 3.0, page 3 and the list o f reference substances, section 4.0, page 4. Amendment Approval S ' :-' William K. Reagen, Sponsor Representative Date Page 225 of 225