Document 8VDBNRXVBBoOppZ7Op9zDm3dZ

f1 r Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 J.A&_ uro A nalytical Laboratory Report Title Comparative Analysis of Fluorochemicats in Human Serum Samples Obtained Commercially Data Requirem ent Not Applicable A u th o r Lisa Stevenson Study Com pletion Date 1 1 /1 3 /0 2 Perform ing Laboratory Extractions and Analyses 3M Environmental Laboratory Building 2-3E-09,935 Bush Avenue St. Paul, MN 55106 Project Identification Analytical Report: E 02-1053 Total Num ber o f Pages 52 3 M Environmental Laboratory 3M Environmental Laboratory CONTAIN NO CBI 000041 Page 1 Page 1 Analytical Report: LIMS E02-1053 Analytical R eport: L IM S E 0 2 -1 0 5 3 This page has been reserved for specific country requirem ents. 3M Environmental Laboratory 3M Environmental Laboratory 000042 Page 2 Page 2 Analytical Report: LIMS E02-1053 Compliance Statement Analytical Report: LIMS E02-1053 Analytical Laboratory Report Title: Comparative Analysis of Fluorochemicals in Human Serum Samples Obtained Commercially Study Identification Number: E02-1053 This study was not conducted under Good Laboratory Practices. Wi ,, . m tory Management, Sponsor Representative Date Date 3M Environmental Laboratory 3MEnvironmental Laboratory 000043 Page 3 Page 3 Analytical Report: LIMS E02-1053 Quality Assurance Statement Analytical Report: LIMS E02-1053 Analytical Laboratory Report Title: Comparative Analysis of Fluorochemicals in Human Serum Samples Obtained Commercially Study Identification Number: E02-1053 This study has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the Principal Analytical Investigator (PAI) and laboratory management. Inspection Dates Phase 10/17/02 Sample Spiking 10/22/02 Analysis 10/30/02,10/31/02,11/07-08/02 Data 11/01/02,11/07-08/02 Draft report Date Reported to Management PAI 10/18/02 10/18/02 10/23/02 11/01/02, 11/08/02 11/01/02, 11/08/02 10/23/02 11/01/02, 11/08/02 11/01/02, 11/08/02 QAU Representative H/tlfOL. Date 3M Environmental Laboratory 3M Environmental Laboratory 000044 Page 4 Page 4 Analytical Report: LIMSE02-1053 Table of Contents Analytical Report: LIMS E02-1053 Compliance S tatem ent................................................................................................................... 3 Quality Assurance Statem ent........................................................................................................4 List of Tables.....................................................................................................................................6 Study Personnal and Contributors................................................................................................7 Location of Archives........................................................................................................................7 Executive Summary.........................................................................................................................8 Introduction and Purpose.............................................................................................................. 9 Speciment Receipt and Maintenance..........................................................................................9 Chemical Characterization of the Reference Substances........................................................10 Sample Preparation and Analysis.................................................................................................11 Method Sum m aries.................................................................................................................... 11 Preparatory and Analytical M ethod.................................................................................11 Analytical Equipm ent........................................................................................................ 12 Data Quality Objectives and Data Integrity.................................... ............................................ 13 Data Summary, Analyses, and Results...................................................................................... 13 Summary of Data Results........................................................................................................ 13 Summary of Quality Control Analyses Results..................................................................... 14 Statem ent of Data Quality........................................................................................................ 14 Statistical Methods and Calculations...........................................................................................15 Statement of Conclusion............................................................................................................... 15 References....................................................................................................................................... 15 Appendix A: Characterization of the Control M atrix................................................................ 16 Appendix B: Extraction and Analytical Method......................................................................... 17 E TS -8-231.1, Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices (19 pages)....................................................................................... 18 Appendix C: Q C Data Summary Tables................................................................................... 37 Appendix D: Data Spreadsheets (5 pages)..............................................................................38 Appendix E: Example Calculations.............................................................................................43 Appendix F: Interim Certificate(s) of Analysis (8 pages)........................................................ 44 Appendix G: Report Signature P a g e ......................................................................................... 52 3M Environmental Laboratory 3M Environmental Laboratory 000045 Page 5 Page 5 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 List of Tables Table 1. PFOA Data Summary of Pooled Serum...................................................................... 8 Table 2. Pooled human serum samples received from commercial vendors in July and August, 2 0 0 2 ...................................................................................................................... 9 Table 3. Characterization of the Analytical Reference Substances in Study E 02-1053......10 Table 4. Target Ions Monitored in 3M Laboratory Analyses and Observed Retention T im es................................................................................................................................... 12 Table 5. PFOA Data Summary of Pooled Serum.......................................................................13 Table 6. Limit of Peak Area Threshold in the Analyses of Sera Extracts..............................14 Table 7. Characterization of the Control Matrix Used for Analyses in Study E 02-1053..... 16 Table 8. Acceptance Criteria Summary of PFOA-NH4 QC samples Analyzed 11/01/02...37 Table 9. Acceptance Criteria Summary of PFOA-Add QC samples Analyzed 11/01/02...37 3M Environmental Laboratory 3M Environmental Laboratory 000046 Page 6 Page 6 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-10S3 Study Personnel and Contributors Requestor 3M Environmental Technology and Safety Services 3M Environmental Laboratory Building 2-3E-09 St. Paul, MN 55106 W illiam Reagen, Ph.D., Laboratory Management, Sponsor Representative Analytical Chemistry Laboratory Extractions and Analyses 3M Environmental Laboratory (3M Lab) Lisa Stevenson, Principal Analytical Investigator (PAI) 3M Lab Contributing Personnel Marlene M. Heying* Ognjenka Krupljanin* Richard C. Jones* Bob W . Wynne* ` Contract lab professional service employee Study Initiation: 10/16/02 Study Completion: 11/13/02 Location of Archives All original raw data and analytical report have been archived at the 3M Environmental Laboratory according to 3M Standard Operating Procedures. The analytical reference standard reserve samples are 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. 3M Environmental Laboratory 3M Environmental Laboratory 000047 Page 7 Page 7 Analytical Report: LIMS E02-1053 Executive Summary Analytical Report: LI MS E02-1053 A screening study was undertaken to compare branched and linear isomers of perfluorooctanoate (PFOA - C7F,sCOO') in 4 lots of commercial pooled human sera with concentrations ranging from 0.65 - 5.6 ng/mL PFOA. Results from this study showed a wide distribution of the percentage of branched isomers of PFOA compared to the linear isomer of PFOA in the commercial pooled populations (Table 1). Since this is a preliminary screening study a larger sample size would be needed to determine if a significant statistical difference exists between pooled human sera samples. As shown in Table 1, two of the lots of commercial pooled sera showed the presence of branched Isomers of PFOA while the other two lots showed a much lower percentage (by at least 40x) of PFOA branched isomers. Table 1. PFOA Data Summary of Pooled Serum Sample Pooled Human Serum Identification TCR-687-Bioresource Lot 020821 TCR>688-Lampire LotX324B TCR-689-Sgma Lot 022K0965 TCR-690-Golden W est Lot G01406042 Branched Isomer Area <361* 8059 8824 <361* Linear Isomer Area 114603 41466 55100 168154 % Branched/ Sum Branched + Linear <0.31 16 14 <0.21 * Area threshold found as 361 in the extracted PFOA-Acid 0.534 ng/mL standard. 3M Environmental Laboratory 3M Environmental Laboratory 000048 Page 8 Page 8 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 Introduction and Purpose The purpose of the study is to determine the relative isomer ratios of PFOA fluorochemical in 4 lots of commercial pooled human sera with concentrations ranging from 0.65 - 5.6 ng/mL PFOA. The PFOA concentrations were determined in study E02-1039. Analyses of sera extracts for determining the relative isomer ratios of PFOA were completed by the 3M Environmental Laboratory under study number E02-1053, and the results of these analyses are presented in this report. The analytical portion of this study was initiated on 16 October 2002. Specimen Receipt and Maintenance The 3M Environmental Laboratory received pooled human sera samples collected from various commercial vendors in July and August, 2002. All specimens were received frozen in good condition on dry ice. All specimens were immediately transferred to storage at -20C 10C, and maintained at that temperature except when removed for extraction and analysis as described in the method. The samples were kept isolated from the test materials (analytical standards) during storage. Table 2. Pooled human serum samples received from commercial vendors in July and August, 2002 Samples Pooled Human Serum Identification TCR-687'Bioresource TCR-688-Lampire T CR-689-Sigma TCR-690-Golden West Lot# 020821 X324B 022K0965 G01406042 The control matrix used in sera analyses performed during E02-1053 was obtained from a commercial source and is presented in Appendix A. Samples analyzed at the 3M Environmental Laboratory will be stored and maintained following 3M Standard Operating Procedures. 3M Environmental Laboratory 3M Environmental Laboratory 000049 Page 9 Page 9 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 Chemical Characterization of the Reference Substances Perfluorooctanoate Ammonium Salt (PFOA-NH4) CAS Number: 3825-26-1 Chemical Formula: C rF^C C k'N fV Molecular W eight: 431 This chemical is a 3M electrochemical fluorination production lot and contains, as determined by NMR, approximately 20% branched:80% linear isomers by weight. Perfluorooctanoate-Acid (PFOA-Acld) CAS Number: 335-67-1 Chemical Formula: C7Ft5CC>2H Molecular Weight: 414 This chemical is a commercial product obtained from Oakwood Products and contains approximately 1% branched:99% linear isomers. The molecular ion 413 was selected as the primary ion for PFOA. This ion was fragmented further during analysis to produce ions 1 1 9 ,1 6 9 ,2 1 9 , and 369. The total ion current (TIC ) was monitored for analysis. Chemical characterization information on the reference substances used in this study is presented in tabular form below. Table 3. Characterization of the Analytical Reference Substances in Study E02-1053 Location 3M Lab Substance PFOA-NH4 TCR-99131-037 PFOA-Add TCR-617 S o u rea 3M Oakwood Products E xpiration Data 12/15/2006 NA S torage C onditions -20C 10C Room Temperature Cham leal Lot N um ber 332 210002 P h y sic a l D escription White powder White crystal P u rity 95.2%* 99.51% ** NA--NotavalaUa 'See CertificatoofAnalysisIramCentre Analytical Laboratories inAppendx F. "See CertificateofAnalysisfrom3M. 3M Environmental Laboratory 3M Environmental Laboratory 000050 Page 10 Page 10 Analytical Report: LIMSE02-1053 Sample Preparation and Analysis Analytical Report: LIMS E02-1053 Human serum samples were analyzed in this study. Sera samples were extracted beginning on 17 October 2002 using a solid phase extraction (SPE) procedure. Sample extracts were analyzed using high-performance liquid chromatographyelectrospray/tandem mass spectrometry (HPLC-ES/MS/MS) in the multiple reaction mode versus extracted rabbit sera standards. Qualitative analysis of branched and linear isomers of PFOA was accomplished using NMR certified standards of a linear isomer PFOA standard (PFOA-Acid) and a mixed branched and linear isomer PFOA standard (PFOA-NH4). It was determined that the branched PFOA isomers elute within an approximate 0.2 minute retention time window from the linear PFOA isomer. Method Summaries Following is a brief description of the method used during this analytical study by the 3M Environmental Laboratory. A detailed description of the method used in this study is located in Appendix B. 3M Environmental Laboratory Preparatory and Analytical Method ETS-8-231.1, "Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices" Human sera was prepared using 2.0 mL of serum then diluted to 10 mL with reagent grade water. The diluted serum/water was spiked with the appropriate analyte mixture. Acetonitrile (ACN) was added as an extraction solvent, which also served to precipitate the proteins. The sample was capped, mixed, and put on the centrifuge to clarify the supernatant. The supernatant was transferred to a clean tube, diluted with water, and passed through a pre-conditioned C i8 SPE cartridge. The analyte(s) of interest were eluted from the SPE cartridge with 2.0 mL of methanol and analyzed. Analyses were performed by monitoring two or more product ions selected from a single primary ion characteristic of PFOA using HPLC-ES/MS/MS. For example, the molecular anion 413 (C7F15COO ), selected as the primary ion for analysis, was fragmented further to produce characteristic daughter ions 119,169, 219, and 369. The total ion current (TIC) peak areas are the sum of the signal for each daughter ion at specific retentions times for each target analyte isomer that were monitored for analysis. Daughter ions may also be referred to as product ions. 3M Environmental Laboratory 3M Environmental Laboratory 000051 Page 11 Page 11 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 Analytical Equipment The following Is representative of the settings used during the analytical phase of this study. Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM C18 2x100 mm (5 pm) Column temperature: 40C Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pUmin Injection volume: 1-30 pL Solvent Gradient: 16.0 minutes (minutes) %B 0.0 40% 10.0 90% 11.0 90% 11.5 100% 12.5 100% 13.0 40% 16.0 40% Mass Spectrometer: Micromass API/Mass Spectrometer Quattro Ultima Triple Quadrupole system Software: Mass LynxTM 3.5 Cone Voltage: 2 0 -6 0 V Collision Gas Energy: 2 0 -5 0 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Table 4. Target Ions Monitored in 3M Laboratory Analyses and Observed Retention Times Target Primary Ion Analyte (AMU) Product Ion (a m u ) Isomer retention time Branched Linear PFOA 413.0 119,169, 219, 369 ~7.9 min. -8.1 min. 3M Environmental Laboratory 3M Environmental Laboratory 000052 Page 12 Page 12 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) were indicated for this study: Calibration: Calibration curves were not a component of this study. Isomer ratios of extracted rabbit matrix standards (labeled as RBS-date of extraction-concentration) were evaluated to determine instrument response. Lim its of Peak Area Threshold: The limit of peak area threshold was the lowest standard point that had a signal to noise ratio of at least 2 times that of the baseline noise. Acceptance Criteria: The isomer ratio of branched:linear PFOA in the extracted continuing verification (QC) sample (labeled as RBS-date of extraction-QCconcentration) is required to meet 30% agreement versus the extracted initial standard at the same concentration. Confirm atory Methods: No confirmatory method will be used. Demonstration of Specificity: Isomer identification will be substantiated by chromatographic retention times of the total ion current (TIC). Data Summary, Analyses, and Results Data quality objectives for the analytical phase of this study outlined above were met with the exceptions noted in this report. Summary of Data Results Table 5. PFOA Data Summanf of Pooled Serum Sample Identification Branched Linear IsomerArea IsomerArea Pooled Human Serum TCR-687-Bioresource Lot 020621 TCR-688-Lampire Lot X324B TCR-689-Sigma Lot 022K0965 TCR-690-Golden West Lot G01406042 <361* 8059 8824 <361* 114603 41466 55100 168154 % Branched/ Sum Branched + Linear <0.31 16 14 <0.21 * Area threshold found as 361 in the extracted PFOA-Acid 0.534 ng/mL standard 3M Environmental Laboratory 3M Environmental Laboratory 000053 Page 13 Page 13 Analytical Report: LIMSE02-1053 Analytical Report: LIM S E 0 2-1053 Summary of Q uality Control Analyses Results Calibration: Quantitation was not a component of this study. Comparison of branched:linear ratio in the extracted rabbit matrix standard was based on the total ion current (TIC) of peak areas at retention times consistent for the target analyte (i.e. 7.9 and 8.1 minutes for PFOA-NH4, and 7.9 and 8.1 minutes for PFOAAcid). Lim its of Peak Area Threshold: The peak area threshold was determined based on the extracted PFOA-Acid 0.534 ng/mL standard, using the branched isomer TIC peak area at ~2 times the baseline noise. Table 6. Limit of Peak Area Threshold in the Analyses of Sera Extracts Analyte Concentration Branched Isomer Instrument ng/mL TIC PeakArea PFOA-Acid 0.534 361* Quattro Ultima * Based on the extracted PFOA-Add 0.534 n^m L standard, branched isomer area at retention time 7.9 minutes, with a TIC peak area -2 times the baseine noise. PFOA-Acid - standard spited with PFOA-Acid standard mix Blanks: All blanks were below the limit of peak area threshold for the compounds of interest. Acceptance Criteria: The isomer ratio of branched:linear PFOA in the extracted continuing verification (QC) sample was within + /-1 0% for all QC data. Refer to Appendix C for detailed information regarding QC data. Precision: Precision was not a component of this study. Matrix Spikes: Matrix spike data were not a component of this study. Spike Recoveries: Spike recoveries were not determined for the continuing verifications (QCs). Surrogates: Surrogates were not a component of this study. Statem ent o f Data Quality The ratio of isomers observed in the standards throughout this study were stable and a reliable identifier of the source product. Statistical Methods and Calculations Statistical methods were limited to the calculation of means, standard deviations, and percent difference. See Appendix E for example calculations used in E02-1053. 3M Environmental Laboratory 3MEnvironmental Laboratory 000054 Page 14 Page 14 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 Statement of Conclusion Results from this study showed a wide distribution of the percentage of branched isomers of PFOA compared to the linear isomer of PFOA in the commercial pooled populations (Table 1). Since this is a preliminary screening study a larger sample size would be needed to determine if a significant statistical difference exists between pooled human sera samples. As shown in Table 1, two of the lots of commercial pooled sera showed the presence of branched isomers of PFOA while the other two lots showed a much lower percentage (by at least 40x) of PFOA branched isomers. References 3M Environmental Laboratory Study # E02-1039, November 2002 3M Environmental Laboratory 3M Environmental Laboratory 000055 Page 15 Page 15 Analytical Report: LIMSE02-1053 Analytical Report: LIMS E02-1053 Appendix A: Characterization of the Control Matrix Table 7. Characterization of the Control Matrix Used for Analyses in Study E02-1053 Control Matrix Rabbit Serum TN-A-4511 Source Expiration Oate Storage Conditions Chemical Lot # Physical Description Sigma 09/26/2005 -20C 10C 99H8400 Rabbit Serum 3M Environmental Laboratory 3M Environmental Laboratory 000056 Page 16 Page 16 Analytical Report: LIMSE02-1053 Analytical Report: LIMS E02-1053 Appendix B: Extraction and Analytical Method This appendix Includes the following method: ETS-8-231.1, Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices, (19 pages) 3M Environmental Laboratory 3M Environmental Laboratory 000057 Page 17 Page 17 I locum..1 ;ri,j\ he um'd. i 'curren!, l'or Analytical Report: LIMS E02-1053 my. in .u 10.16 2CV: 3M Environmental Laboratory Method Solid Phase Extraction and Analysis of Fluomchemlcal Compounds from Biological Matrices Method Number: ETS-8-231.1 Adoption Date: IIf lif o i Revision Date: J^/S/oz. Effective Date: -2-//?/oZ- Approved By: Willliam K. Reagen Laboratory Manager Date Exact Copy of Original IA ; , j i k i i s i Initial Data 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis ofFluorochemical Compounds from Biological Matrices Page 1 o f 19 000058 Page 18 O' ",:i'.i!!c;U ri;!'- Analytical Report: LIMS E02-1053 .. -.1!. I curivi 1:. for I 4 d u \ - ;mm 10 . 0 2 j02 1 Scope and Application This method describes the extraction o f target analytes from fish, rat liver, rat sera, mouse liver, and mouse sera using solid phase extraction (SPE). This method may also be extended to other biological matrices provided that the data quality objectives are m et 2 Method Summary An amount o f biological material, determined by the analyst is prepared (fluids diluted and tissues homogenized) at a 1/6 dilution, or other dilution as determined by the analyst using reagent grade water. An aliquot o f the dilution/homogenate is spiked with the appropriate surrogate or analyte mixture. Acetonitrile (ACN) is added as an extraction solvent and also serves to precipitate the proteins. The sample is capped, mixed, and put on the centrifuge to clarify the supernatant The supernatant is transferred to a clean tube, diluted with water, and passed through a pre conditioned C u SPE cartridge. Finally, the analytes o f interest are eluted from the SPE cartridge and analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES/MS/MS). 3 Definitions 3.1 Dilution A dilution expressed as 1:5 or 1/6 is defined as: 1 mL o f sample + 5 mLs o f diluent for a total o f 6 mLs combined, unless otherwise noted. 3.2 SPE cartridge A column containing an open solvent reservoir at one end and packed with bonded silica sorbents at the other end. It is designed to retain the compounds of interest under some solvent conditions and elute them under others. A separation is thus achieved; compounds can be removed from difficult biological matrices and introduced into appropriate solvents for analysis. 3.3 Reagent grade water Water with no detectable concentration(s) o f the target analyte(s). 3.4 Quality control sample Sample used to monitor the extraction efficiency (as a matrix spike) and to verify the continued accuracy o f the initial calibration curve (as a continuing calibration verification). 4 W arnings and Cautions _________ ______________________________ 4.1 Health and Safety W arnings 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 o f 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 o f the liquid to pass through the SPE column to dryness. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 2 o f 19 000059 Page 19 Analytical Report: LIMS E02-1053 IX'c.!,|1cnt iruiv ii'-oil. : ..a,..;r:L toi' 14 oav- ; r < Jfv.il 102 Do not operate solvent pumps above capacity o f 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HPLC will initiate automatic shutdown. Do not run solvent pumps to dryness. 5 Interferences To minimize interferences, Teflon should not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract 6 Instrum entation, Supplies, and Materials The following instrumentation, supplies, and materials are used while performing this method. Equivalent instrumentation, supplies, and materials may be used in place o f those listed. 6.1 Instrum entation Vortex mixer, VW R, 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 electrospray ionization source HP 1100 or Agilent low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 6.2 Supplies and Materials Eppendorf or disposable pipettes, plastic or glass Dissecting scalpels Polypropylene bottles, capable o f holding 50 ml. to 1 L (Nalgene) Volumetric flasks, glass, type A 40 mL glass vials (ICHEM) Plastic sampule vials, Wheaton, 6 mL (or other appropriate size) Centrifuge tubes, polypropylene, 15 mL and 50 mL Labels Graduated pipettes, glass Syringes, capable o f measuring 5 pL to 1000 piL Bottle-Top Dispenser (capable o f dispensing 5mL o f solvent) SPE extraction cartridge, 1 g, Sep-Pak 6 cc tri-functional C i8 (Waters) 75 mL sample reservoir (or other appropriate size) Crimp cap glass autovials and caps 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 3 o f 19 000060 Page 20 Analytical Report: LIMSE02-1053 D o c u n v n t ' K . v he used. I c u r i v i ; ..u ! t ilavv i . >m ' 0 / ] n 2!)<k: 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 volumetric container containing reagent grade water, mix until all solids are dissolved, bring to volume using reagent grade water. Store at room temperature. Note: When preparing different volumes than those listed in reagents preparation, target analyte standard preparation, and surrogate standard preparation, adjust accordingly. 7.2 Target analyte standard preparation Prepare target analyte standard^) for the standard curve. Multicomponent analyte standards are acceptable. The following is an example only and may or may not be appropriate for all standard preparations. Weigh approximately 100 mg o f target analyte into a 100 mL volumetric flask and record the actual weight in the standard logbook or other appropriate location. Bring to volume with methanol for a stock standard o f approximately 1000 ppm ((ig/mL). Dilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. Example calculation: 1000 jtg/mL x 5 mL/100 mL = 50 f.lg/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/100mL = 5.0 |lg/mL. Dilute working standard 1 with methanol to produce a working standard 3 solution o f approx. 0.50 ppm. Example calculation: 50 |Xg/mL x 1.0 mL/100 mL " 0.5 |Tg/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 o f surrogate standard into a 100-mL volumetric flask and record the actual weight Bring to volume with methanol for a surrogate standard stock o f approximately 900 - 1100 ppm. Prepare a surrogate standard working standard. Transfer approximately 1 mL o f surrogate standard stock to a 10-mL volnteme flask and bring to volume with methanol for a working standard o f 90-1 lOppm. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 7.4 Internal standard preparation Prepare internal standard(s). The following is an example only and may or may not be appropriate for all internal standard preparations. Weigh approximately 90-110 mg o f internal standard into a 100-mL volumetric flask and record the actual weight. 3MEnvironmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 4 o f 19 000061 Page 21 1V h: n i li u v PC IP CC. Analytical Report: LIMS E02-1053 ii']' ! ' v s ii >en i i >' ! Bring to volume with methanol for an internal standard stock o f approximately 900 - 1100 ppm. Prepare an internal standard working standard. Transfer approximately 1 mL o f internal standard stock to a 10-mL volumetric flask and bring to volume with methanol for a working standard o f 90-1 IOppm. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 8 Sample Handling All samples are received frozen and must be kept frozen until the extraction is performed. Allow samples to thaw to room temperature prior to extraction. Typically fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials until analysis. If analysis will be delayed, extracted standards and samples may be refrigerated at approximately 4C indefinitely or may be stored at room temperature until analysis can be performed. 9 Q uality Control 9.1 Blanks 9.1.1 Solvent Blank An aliquot o f methanol is used as a solvent blank. Solvent blanks are not extracted. 9.1.2 Method Blank An aliquot o f 1.0 mL of water, or other appropriate amount, is used as a method blank. Four method blanks are extracted and analyzed with each set following this procedure (two are spiked with surrogate and two are not spiked). 9.1.3 Matrix Blank An aliquot o f 1.0 mL or 1.0 g o f matrix (diluted or homogenized) is used as a matrix blank. Other amounts may be used, as appropriate. Matrix blanks are prepared from one o f three sources: 1) a study control matrix from a study control animal received with a sample set; 2) a commercially obtained sample o f the same species as the study animals; or 3) a surrogate matrix, also obtained commercially, but o f a different species than the study animal, (eg. if rat is used to generate standard curves and CCVs for a mouse study). The matrix to use is dependent on the matrix used for the curve. 9.1.3.1 Study control matrix curve - if the study control matrix is used for the curve, prepare four (4) matrix blanks using the study control matrix (two spiked with surrogate and two not spiked). 9.1.3.2 Commercially obtained (same species) matrix curve - if the commercially obtained matrix is used for the curve, prepare four (4) matrix blanks using the same commercially available matrix (two spiked with surrogate and two not spiked). 9.1.3.3 Surrogate matrix curve - if a surrogate matrix is used for the curve, prepare four (4) matrix blanks using the same commercially available matrix and prepare four (4) matrix blanks using a commercially available matrix of the same species as the study animals (two spiked with surrogate and two not spiked). 9.1.3.4 If limited matrix is available, the number of method and matrix blanks may be adjusted and will be noted in the study protocol or in the raw data. 9.2 Sample Replicate Samples replicates are prepared according to each study protocol or project outline. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 5 o f 19 000062 Page 22 D o a ii'v n t m:iy he : auront, lor !4 Analytical Report: LIMS E02-1053 uni;: -o.'io ;v '02 9.3 Surrogate standard If surrogate standard is a component o f the study, all samples are spiked with surrogate standard prior to extraction to obtain a concentration in the mid-range of the calibration curve, with the exception o f blank samples as described above. Typically surrogate standard is spiked into the 1.0 mL diluted/homogenized sample removed for extraction. However, surrogate may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the 1.0 mL sample, or into the 1.0 mL diluted/homogenized sample removed for extraction. 9.4 Internal standard If internal standard is a component o f the study, all samples are spiked with internal standard after extraction to obtain a concentration in the mid-range o f the calibration curve. Typically internal standard is spiked into the 2.0 mL o f extract in the 15 mL centrifuge tube, before transferring to the autovial. 9.5 Lab Control Sample Lab control samples are not a component o f this method. 9.6 Q uality Control (QC) Sample Prepare quality control (QC) samples to monitor extraction efficiency and to verify the continued accuracy o f the initial calibration curve. Typically 1.0 mL, or other appropriate amount, o f the same matrix used to prepare the initial calibration curve is used for each QC sample. Twelve (12) quality control samples (QC) will be prepared for each matrix during the course o f a study. A minimum o f 3 QC samples must be prepared (one at each level) on each day o f sample extraction, (e.g. If the study is such that samples will be extracted on three different days then four QC samples must be prepared on each day o f extraction for a total o f twelve.) QC samples will consist o f four samples at each o f three levels o f analyte. The levels listed below may be used and may represent sample concentrations diluted into the range o f the calibration curve: Low level: 3X to 5X the LLOQ, Mid-level: equivalent to a point near the middle o f the calibration curve, High level: 80% o f the ULOQ Two QC sample levels are analyzed after every tenth sample injection starting after the last calibration standard injection, with a minimum o f three QC per analysis. Solvent blanks are not considered samples but may be included as such for determining when QC samples will be analyzed. QC samples extracted with a particular sample set must be analyzed in the same analytical run. Any QC samples extracted during the course o f the study may be included in subsequent analyses. I f samples from multiple extraction dates are analyzed in one analytical run, then QC samples from the same sample extraction dates must be included in that analysis. Each QC is expected to show an accuracy of 75-125% o f expected. A minimum o f 2/3 o f all QC samples must meet this criteria, and a minimum of 1/2 o f the QC samples at each level must meet this criteria. I f not, the set must either be re-analyzed or re-extracted. 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 o f 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 Mil 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 determined by the analyst 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 6 o f 19 000063 Page 23 Document iru:y !' C Analytical Report: LIMS E02-1053 :i a u ro n t, for 14 ilav from lu i b '2002 and documented in a note to file) then diluted using two procedures. The first procedure consists o f diluting the sample with additional matrix prior to extraction (fluid adding fluid), while the second procedure consists o f diluting the extract with solvent post-extraction (methanol extract adding additional methanol solvent.) If the relative percent difference is not within 15% for these two samples; additional testing will be required to determine which value is a correct representation o f the sample concentration. 10 Calibration and Standardization 10.1 Instrument Calibration One calibration curve will be prepared from extracted matrix standards, in the same matrix as the samples, per study. It will consist o f a minimum 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, o f diluted control fluid or homogenized control tissue to a 15 mL centrifuge tube using a disposable plastic pipette. This will be repeated while preparing aliquots for the standard curve. Be sure to mix or shake the control matrix container between aliquots to ensure a homogenous sample is removed. Record each standard volume on the weight/volumes sheet or extraction worksheet, as appropriate. Four 1.0 mL aliquots, o r other appropriate amount, o f control matrix serve as matrix blanks. The standard concentrations and spiking amounts listed in Table 1 may be used, when appropriate, to spike one standard curve. A total o f 9 standards, four matrix blanks, and four method blanks are prepared in addition to the QC samples and test samples. The number o f standards and blanks may be adjusted as determined by the analyst and documented in a note to file. Use Attachment C, or other appropriate form, as an aid in calculating the concentrations o f the working standards. Refer to section 12 to calculate the actual concentration of analyte in each calibration standard and QC sample. Typically the target analyte standard is spiked into the 1.0 mL diluted/homogenized sample removed for extractioa 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 o f extracts. The curve equation will be determined by regression analysis using the peak areas o f the target analyte(s) using MassLynx or other suitable software. Any level outside 75% -125% o f nominal must be deactivated, and regression re-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 of three (3) levels may be deactivated in any one set, or the set will be re-analyzed. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 7 o f 19 000064 Page 24 in. tv L ./ i ! Analytical Report: LIMS E02-1053 T u i r a i t , l ' or M d a y s \ ,"<n>: i n '! h \ , 0 2 1 Mil 1 1 VlTKOMM \ 11 SI'lklM. A MO I M S 1OK M \ MI Mills \ \ 1>Srikl s 1 SIM. l.U Ml. <11 M \ 1Ul\ Working standard (approximate concentration) - pL - Approximate final concentration o f analyte in M atrix dilated 1:5 Blank Approximate final concentration o f analyte in Final 2.0 m L volume Blank 0.500 ug/mL 1.5 5.00 ng/g or ng/mL 0.375 ng/mL 0.500 ug/mL 3.0 10.0 ng/g or ng/mL 0.750 ng/mL 0.500 ug/mL 8.0 25.0 ng/g or ng/mL 2.00 ng/mL 0.500 ug/mL 16 50.0 ng/g or ng/mL 4.00 ng/mL 0.500 ug/mL 32 100 ng/g or ng/mL 8.00 ng/mL 5.00 ug/mL 5.6 175 ng/g or ng/mL 14.0 ng/mL 5.00 ug'mL 8.0 250 ng/g or ng/mL 20.0 ng/mL 5.00 ug/mL 5.00 ug/mL 16 500 ng/g or ng/mL 24 750 ng/g or ng/mL 40.0 ng/mL 60.0 ng/mL 5.00 ug/mL 32 1000 ng/g or ng/mL 80.0 ng/mL 5.00 ug/mL 40 1250 ng/g or ng/mL 100 ng/mL 50.0 ug/mL 5.0 1500 ng/g or ng/mL 125 ng/mL 50.0 ug/mL 6.0 1750 ng/g or ng/mL 150 ng/mL Surrogate Std 100 ug/mL 10 6500 ng/g or ng/mL 500 ng/mL 11 Procedures_________________ 11.1 Tissue Sam ple Preparation Obtain frozen tissue samples Cut approximately 1.0000 g o f tissue (+/- 0.1000 g), or other appropriate amount, using a dissecting scalpel. This part o f the procedure is best performed quickly, not allowing the tissue to thaw. Weigh the tissue directly into a tared plastic sampule vial. Record the weight on the weight/volume sheet, extraction worksheet, or other appropriate location. Return unused tissue to the freezer after extraction amounts have been removed. Add 2.5 mL o f reagent water to sampule vial, or other volume as determined by the analyst and documented in a note to file. Homogenize the sample. Put the Ultra-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 o f reagent grade water, or other volume as determined by the analyst and documented in a note to file, using a pipette. Take the grinder apart and clean it with methanol after each sample. Refer to ETS-9-52 for more 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 o f 2.5 mL o f water.), or other ratio as determined by the analyst and documented in a note to file. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 8 o f 19 00006S Page 25 Hi xjimKMir Analytical Report: LIMS E02-1053 !v irvi.!. :I current, tor II d., , l'ntm HL io 1/102 11.2 Fluid Sam ple 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 l S seconds, then transfer 1.0 mL o f fluid, or other appropriate amount to a plastic sampule vial, or other appropriate container. Return unused samples to freezer after extraction amounts have been removed. Add 5.0 mL o f reagent water to the l.O mL o f fluid for a l/6 dilution, or other dilution as determined by the analyst and documented in a note to hie. If a volume other than l .0 mL is removed for an initial volume, adjust the water volume accordingly to maintain the same dilution as above. 11.3 Tissue and Fluid Sample Extraction After tissue or fluid samples have been prepared according to sections 11.1 and 11.2, vortex mix or shake by hand the diluted/homogenized sample for approximately 15 seconds then transfer 1.0 mL, or other appropriate volume, to a clean 15 mL polypropylene centrifuge tube. Return unused diluted/homogenized portions to the 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 o f standard as described in this method for the calibration curve standards and each QC sample. Vortex mix the standard curve samples and QC samples for approximately 5 seconds. To each sample and standard, add 5.0 mL o f acetonitrile, cap, and vortex mix or shake by hand approximately 15 seconds. Place all samples on the shaker at an appropriate speed for 20 minutes to adequately mix (a setting o f approximately 300 rpm on the models listed in section 6.1). Remove from the shaker and centrifuge at an appropriate speed for 10 minutes to adequately pellet the precipitate (a setting o f approximately 2000 rpm on the models listed in section 6.1). Add 40.0 mL o f reagent grade water to a clean 50 mL centrifuge tube. Remove samples from the centrifuge and decant the supernatant into the water in the 50 mL tube, taking care not to introduce any o f the matrix solids into the solution. Cap and mix by inverting several times. In this step the order of addition may be changed (i.e. the sample may be put into the centrifuge tube and then the water added). Attach the reservoir to the SPE cartridge and attach this reservoir/caitridge unit to a vacuum manifold. NOTE: When running the vacuum, set the vacuum chamber at approximately 15 kPA - to give an approximate elution flow o f 5-7 mL/tnin. 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 o f methanol, followed by approximately two 5.0 mL aliquots o f water, taking care not to allow the column to ru n to dryness after each wash. After washing is complete, pour the sample into the reservoir/cartridge unit and allow all of the liquid to pass through the column to dryness. Run the vacuum on high for approximately 5 minutes to adequately dry each SPE cartridge. Place a collection 15 mL polypropylene centrifuge tube under each cartridge and elute with 2.0 mL o f methanol. Spike extracted blanks, samples, and standards with internal standard as described in this method. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 9 o f 19 000066 Page 26 | ) o c u : r . -f:[ m;iv be Analytical Report: LIMS E02-1053 'I 'a u r o n t, for !4 J:ivs Iro n' [O lo 2(102 Label each glass autovial, as appropriate, with the study number, vial file archive number, animal number/gender/timepoint or LIMS number, matrix, final solvent, analyte components (if needed), extraction type, extraction date, and analyses) performing the extraction. Transfer each eluant to a glass autovial and cap. 11.4 Extract Analysis 11.4.1 Software set-up On the MassLynx main page, set up a sample list name. Save the list as instrument designator letter, last 2 digits o f test year-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 f the instrument name (A = "Amelia") YY = Test year (02) M M - Test month (02) D D = Test day (04) a - First sample list (run) o f the day (the next sample list will end with V, the next 'c1, and so on.) Assign a filename using the instrument designator letter, the last 2 digits o f the test year-month-day, and a 3-digit sequential file number that starts with 1and increases by one for each filename. Example filename: IYYMMDD### or A020204001 I **Initial o f instrument name YY = Test year MM = Test month DD = Test day ### = 3-digit sequential file number starting with 1 through 999 (001) Also, as part o f the samplelist, assign a method (MS) for acquiring, an inlet file, a bottle number, an injection volume, and sample descriptions. To create a method, click on Method Editor button in the MS Status Pane and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate mass(es). Also set the acquisition start and stop times. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx "Guide to Data Acquisition" for additional information on MRM. Typically the analytical batch run sequence begins with system suitability, solvent blanks, and a set o f extracted matrix standards. Sample extracts are analyzed with two QC samples injected after every tenth sample injection. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered sample extracts but may be included as such. 3MEnvironmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 10 of 19 000067 Page 27 Analytical Report: LIMS E02-1053 L 'o ci.m v;1' t m -. oc u >cd. T..ruP'ct.L 'or M d'l'.'s io un 10-'1 *' 2Lii2 11.4.2 HPLC set-up Set up sample tray according to the sample list prepared above. Set up the HPLC to the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook, or other appropriate location: Sample size = 10 pL injection 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: 1Ilk- S n lu 'll! It 0.00 10% 1.00 10% 5.50 95% 7.50 95% 8.00 10% 11.4.3 Instrument set-up Refer to ETS-9-24, "Operation and Maintenance o f the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for details. Check the solvent level in HPLC reservoirs and refill if necessary. Check the stainless steel capillary at the end o f the 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 capillary. Turn on the nitrogen. Open the tune page. G ick 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/ntin or as appropriate Set HPLC pump to "On" Observe droplets or mist coming out o f the tip o f the probe. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. Readjust the tip o f the 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 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 11 o f 19 000068 Page 28 : njui''.'i'ii in,-tv be JVv it c :;;.. Analytical Report: LIMSE02-1053 4 ilavs fm in 1(VI f 2002 I IPLC constant flow mode, flow rate 10-500 pL/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 o f 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 o f analyte in calibration standards using the following equation: Concentration (ng/g or ng/mL) Spike Concentration (ug/mL) x Spiked Amount (mL) 1000 ng SV (mL)X Matrix Amount (g/mL or mL/mL) 1ug IW = Initial weight (where 1.0 g = 1.0 m L ) IV = Initial volume DV = Diluent volume (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 9 curve = MA o f tissue/fluid standard curve, assumed to be 1 g or 1 mL/5 mL water 9 sample = MA o f tissue/fluid sample (__ g or mL o f sample/5 mL water) Calculate spike percent recoveries using the following equation: ,, Observed Result - Matrix Blank Result % Recovery = ------------------------------------------------------ x 100 Spiking Level 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 12 o f 19 000069 Page 29 )<v iir. -nf may bo Analytical Report: LIMS E02-1053 i current, for 14 davs fmi,: il) I(v 2002 Calculate relative standard deviation using the following equation: Relative Standard Deviation Standard Deviation Calculate percent deviation using the following equation: % Deviation Calculate actual concentration o f analyte in fluid (pg/mL): AR (ne/m U * DF * 3 curve ImL/ntLI * FV (m D in Curve x 1,0 ug = (pg/g) 3 sample (mlVmL) FV (mL) in Matrix 1000 ng Calculate actual concentration o f analyte in tissue (pg/g): AR (ng/g) x DF x 3curve(g/m L) x FV (m L) in Curve x 1,0 ug 3 sample (g/mL) FV (mL) in Matrix 1000 ng - (pg/g) 13 Method Perform ance 13.1 System Suitability System suitability will be determined prior to the start and at the completion o f each analytical run. Prior to the calibration curve and after the last sample o f the run three (3) mid-level unextracted 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 o f the run separately. The peak area precision must be equal to or less than 5.0% RSD, the precision of the retention time must be equal to or less than 2.5% RSD, the resolution must be > 2.0, and the peak asymmetry (fronting or tailing) must be 0.5<AF<2.0, where AF is the asymmetry factor. If any item o f the system suitability fails, system maintenance must be completed prior to running a second set o f system suitability samples and the system suitability must pass before starting the calibration. If system suitability fails at the completion o f a run, the sample set must be reanalyzed. 13.2 Quantitation The coefficient o f determination value for the calibration curve, plotted by regression using the peak areas o f the analyte(s), must be 0.990 or better. All active calibration curve points must be within 25% o f the theoretical value with the exception o f the LOQ point, which may deviate up to 30%. Calibration standards with peak areas less than two times the curve matrix blank will be deactivated to disqualify a data range that may be affected by background levels o f the analyte. A valid calibration curve must contain at least 6 active points above and including the LOQ. If the curve cannot meet these criteria, the samp le set must be reanalyzed or reextracted. 13.3 Accuracy Two thirds o f all quality control samples and 1/2 o f each quality control sample at each level are expected to show an accuracy o f 75-125%. 3M Environmental Laboratory E T S - 8 - 2 3 1 .1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 13 o f 19 000070 Page 30 Analytical Report: LIMS E02-1053 Dr. ament !. re iivjt' .rent, for M day. frnn il' D' 2(K)J Surrogates and internal standards must have a percent deviation < 50%. Deviations outside this range will be reanalyzed to confirm. If the second analysis confirms the original, the deviation will be documented in the raw data. If the second analysis is within 50%, then the second value will replace the original value. 14 Pollution Prevention and Waste Management Sample waste is disposed o f in noninfectious biohazard waste containers. Flammable solvent waste is disposed o f in high BTU containers. Glass pipette waste is disposed o f in broken glass containers located in the laboratory. 15 Records Complete the extraction worksheet attached to this method, or other applicable worksheet, and store with the study raw data. Each page generated for a study must contain the following information (if applicable): study/project or instrument number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. Other information may be added if applicable to the study. Print the tune page, sample list, and acquisition method from MassLynx to include with the study raw data. Copy these pages and tape into the instrument runlog. Plot the calibration curve by the appropriate regression. Print these graphs and store with the study raw data. Print data integration summary, integration method, and chromatograms from MassLynx, and store with the study raw data. Summarize data using suitable software (Excel 7.0 or LIMS) and store in the study folder. Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 16 Attachments Attachment A: Extraction Worksheet Attachment B: Sample Weight/Volume Worksheet Attachment C, Calibration Standard Concentration Worksheet Attachment D, Dilutions Summary Worksheet 17 References ETS-9-24, "Operation and Maintenance o f the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source" ETS-9-52, "Operation and Maintenance o f a Tissue Grinder" 18 Affected Documents None 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fiuorocbemical Compounds from Biological Matrices Page 14 o f 19 000071 Page 31 Analytical Report: LIMS E02-1053 Docili nom may k ' :: -. T current, for !4 day* iron, 2m.)2 19 Revisions Revision Number 1 Revision Description M inor formatting changes. Added detailed information to all sections concerning the extraction procedure, analytical procedure, and calculations. Added attachments and references. Revision Date 02/18/02 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 15 o f 19 000072 Page 32 Analytical Report: LIMS E02-1053 1 . !':c 1'in vpt 4\j:ty^ fro>\ : I; i 6 2002 Study Number: Prep Date: Analysts initials: Box#: Attachment A - Extraction Worksheet Method Revision: ETS-8-231.1 Matrix: Sample Timepoint: Samle Number Sample Number or description Volume of Amount and diluted sample Amount and surrogate spike removed spike mix used mix used Type of column used and lot Elution solvent and volume Comments Blank matrix______ TN-A-___________; Amount weighed/allquoted:__________ g/mL 1. Homogenize sample 2. Aliquot 1 mL of diluted matrix into 15 mL polypropylene tube 3. Spike samples accordingly 4. Add____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 mL polypropelene centrifuge tube. 8. Decant extract into centrifuge lubes with water 9. Shake sample slightly to ensure proper mixing 10. Attach 6 mL C18 SPE cartridges and 75 mL reservoirs to vacuum manifold 11. Condition column with two washes of -5 mL MeOH (TN-A-__________ ) - do not allow column to go to dryness 12. Wash column with two washes of -5 m L__________ 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, draw a high vacuum through column for at least 5 minutes. 15. Elute column with solvent (__________ TN-A-__________ ) into appropriate 15 mL centrifuge tube 16. Spike samples w ith________ uL of internal standard # _____________________ _ cone.____________________ 17. Transfer sample into appropriately labeled autovial and cap Note: In vacuum steps above set the vacuum chamber at approximately 15 kPA - this should give approximately 5-7 mUmin elution flow Flows may vary through cartridges - kPA may be raised for slow tubes and drying after most have been drawn down and shut off. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 16 of 19 000073 Page 33 f Moumcnr " ; Analytical Report: LIMS E02-1053 r ..Mirv<>t. o r 1 4 i i \ f i v . i u 1 ) I 6 - 1 0 0 2 P rep D ate(s): A nalyses): Sam ple M atrix: M ethod/R evision: Sam ple ID Attachment B - Sample Weight/Volume Worksheet Study N um ber: E quipm ent N um ber: Final Solvent & TN N um ber: Initial W U Vol. g/m L/L W ater Volume added (m L) V olum e R em oved (m L) Com m ents Form Completion Verified By:. ! 1 1 3MEnvironmental Laboratory ETS-8-231.1 \ Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 17 o f 19 000074 Page 34 Analytical Report: LIMS E02-1053 I>k:i :i'i'*.; *.nv be irvd. : f ,! . r i-i Invs from ! !o *-i>2 P rep date(s): A nalyte(s): Sam ple m atrix: M ethod/re vision: Attachment C: Calibration Standard Concentration Worksheet Standard num ber: E quipm ent num ber: Final solvent and TN: B lank Tissue o r Fluid/ldentlfier: A n aly te m ix std a p p ro x . 0.500 ug/mL: A n aly te m ix std a p p ro x . 5.00 ug/mL: A n aly te m ix std a p p ro x . 50.0 ug/mL: S u rro g a te std a p p ro x . 100 ug/mL: Actual concentrations o f standards in the analyte mix A nalyte Std cone ug/m L 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 All A in't spiked mL 0.0015 0.0030 0.0080 0.0160 0.0320 0.0056 0.0080 0.0160 0.0240 0.0320 0.0400 0.005 0.006 All Final V o lu m e: mL 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 AH Initial Fluid D ilu tio n mL/rnL 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 All Initial T issu e D en sity g/m L 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 Calculated concentrations o f standards in relation to the final 2.0 mL solvent and initial matrix 2.0 m L F in al V olum e Fluid M atrix Tissue M atrix A nalyte Final cone. ng/m L 0.375 0.750 2.00 4.00 8.00 14.0 20.0 40.0 60.0 80.0 100 125 150 S u rro g a te Std cone ng/m L 100 Surrogate Final cone ng/mL 0.500 A nalyte Final cone. n g/m L 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 S u rro g a te Std cone ng/m L too Surrogate Final cone ng/mL 6500 A nalyte Final cone. ng/g 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 S u rro g a te Std cone ng/m L 100 Surrogate Final cone ng/g 6500 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 18 o f 19 000075 Page 35 Analytical Report: LIMS E02-1053 lieiniv --r 'f'y be u s e d '."Mirent, for ! 4 ' ' '-s trn.i f' b ) u 2 S tu d y : D ilution D ate/A nalyst: Box N um ber: Sample Number or Description 1 / Attachment D: Dilutions Summary Worksheet Solvent/TN N um ber: E xtraction D ate/A nalyst: M atrix/Tim epoint: Dilutions 1/ 1 / 1/ 1/ 1 / 1/ Comments Verified By: 1 1 Notes: 1 /1 0 dilution = ______ of sample + ________of solvent 3M Environmental Laboratory Form Completion Verified By: ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 19 o f 19 000076 Page 36 Appendix C: QC Data Summary Tables Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 Table 8. Acceptance Criteria Summary of PF0A-NH4 QC samples Analyzed 11/01/02 Sample Identification PFOA-NH4 % Branched/ Sum Branched + Unear % Difference RBS1Of702-10 ng/mL-1 RBS101702-QC-10 ngfaiL-1-1 RBS101702-QC-10 ng/mL-1-2 26.02 25.35 24.93 NA 3 4 % Difference: The difference between the total ion current peak area ratios of the branched:iinear isomers for PFOA in the extracted standard (labeled RBS-date of extraction-concentration) at the same level as the extracted QC (labeled RBS-date of extraction-QC-concentration). Table 9. Acceptance Criteria Summary of PFOA-Acid QC samples Analyzed 11/01/02 Sample Identification RBS1017Q2-10 ng/mL-1 RBS101702-QC-10 ng/ml_-1-1 RBS101702-QC-10 ng/mL-1-2 PFOA-Acid % Branched/ Sum Branched + Unear 2.22 2.24 2.04 % Difference NA -1 8 % Difference: The difference between the total ion current peak area ratios of the branched'.linear isom ers fo r P F O A in the extracted standard (labeled R B S -d ate of extraction-concentration) at the same level as the extracted QC (labeled RBS-date of extraction-QC-concentration). 3M Environmental Laboratory 3MEnvironmental Laboratory 000077 Page 37 Page 37 Appendix D: Data Spreadsheets Analytical Report: LIMS E02-I053 Analytical Report: LIMS E02-1053 3 M Environmental Laboratory 3M Environmental Laboratory 000078 Page 38 Page 38 E02-1053 Analytical Report: LIMS E02-1053 A NALYSIS 11/01/02 Identification TIC Branched Peak Ret Tim e 7.6 B ranched P F O A -N H 4 A rea** Branched Peak Ret Time 7.9 B ranched PF O A -N H 4 A rea Linear Peak Ret T im e 8.1 L in e a r P F O A -N H 4 A rea Filenam e D02110U Branched Ret Time 7.9/ Sum o f Branched:Linear * 100 B ranched/Linear S tan d ard s RBS101702-10 ng/m L-1 RBS101702-QC-10 ng/mL-1-1 RBS102802-QC-10 ng/mL-1-1 RBS101702-QC-10 ng/mL-1-2 RBS102802-OC-10 mt/mL-1-2 W B 102802-H 20 Blk-1 RBS 102802-Sera Blank-1* 0.5 ng/m L-1 0 93029 264494 D 021101003 0 81108 238850 D 0 2 1 101027 0 65454 195485 D 021101029 0 59384 178832 D 021101055 0 56858 168536 DQ21101057 0 355 2873 D 021101043 0 0 3846 DQ21101045 0 1566 6752 D 021101063 26.02 25.35 25.08 24.93 25.23 11.00 0.00 18.83 Pooled T C R -6 8 7 -B io teso u rce* 0 361 114603 D 021101007 0.314 T C R -688-L am pire 0 8059 41466 D 021101009 16.27 T C R -689-S igm a 0 8824 55100 D 021101011 13.80 TCR-690-Golden West* 0 361 168154 D 021101013 0.214 * Below Limit o f Peak A rea Threshold ** Branched peak at 7.6 m inutes observed only in PFO A-N H4 standards at concentrations > 25 ng/mL. N ot included in any calculations. ETS-8-231.1 3M Environmental Laboratory Confirmation Sera Ratios 000079 Page 39 E02-1053 Analytical Report: LIMSE02-1053 ANA LY SIS 11/01/02 PFO A -A dd Pooled d ata w ere n ot Induded in the scope o f the study and w ere not reported TIC Branched Peak Linear Peak Filename Branched Ret Tim e 7.9/ Ret Tim e 7.9 Ret T im e 8.1 D021101a2 Sum o f B ranched:Linear B ran ch ed L in e a r * 100 Identification PFOA-Add PFO A-A dd A rea A rea B ranched/Linear S ta n d a rd s RBS101702-10 ng/mL-2 RBS101702-QC-10 ng/m l^2-l R B S 102802-Q C -10 ng/m L-2-1 R B S101702-QC-10 ng/mL-2-2 RBS102802-OC-10 n/mL-2-2 W B 102802-H 20 Blk-1 RBS102802-Sera B lank-1 0.5 ng/mL-2 7971 5022 2733 4416 1909 260 545 361 351306 219508 225367 211715 198898 2873 3787 6473 D 021101005 D 021101031 D 021101033 D 0 2 1 101059 D 021101061 D 021101043 D 021101045 D 0 2 1 101065 2.22 2.24 1.20 2.04 0.951 8.30 12.58 5.28 Pooled T C R -6 8 7 -B io teso u rce* TCR-688-Lampire TCR-689-Sigma TCR-690-Colden W est* * Below Lim it of Peak Area Threshold 361 8207 8823 361 113845 41469 55095 168119 D 021101007 D 0 2 1 101009 D 021101011 D 0 2 1 101013 0.316 16.52 13.80 0.214 ETS-8-231.1 3M Environmental Laboratory Confirmation Sera Ratios 000080 Page 40 E 0 2-1053 Analytical Report: LIMS E02-1053 TIC 11/01/02 A nalysis Branched Peak Branched Peak Linear Peak Filenam e R et T im e 7.6 R et T im e 7.9 R et T im e 8.1 Identification PFOA-NH4 PFOA-NH4 PFOA-NH4 R et Tim e 7.9/ Sum of A rea** A rea A rea B ranchedilinear *100 Standards & CCVs RBS101702-10 ng/m L-1 0 93029 264494 D 021101003 26.02 RBS101702-QC-10 ng/mL-1-1 0 81108 238850 D 0 2 1 101027 25.35 RBS102802-QC-10 ng/mL-1-1* 0 65454 195485 D 0 2 1 101029 25.08 RBS101702-QC-10 ng/mL-1-2 0 59384 178832 D 0 2 1 101055 24.93 RBS102802-OC-10 ne/mL-1-2* 0 56858 168536 D 0 2 1 101057 25.23 * D ata not used/reported. N ot extracted on the sam e date as the samples and therefore not included in tables 8 and 9. ** B ranched peak at 7.6 m inutes observed only in PFO A-N H4 standards at concentrations > 25 ng/m L. N ot included in any calculations. ETS-8-231.1 3M Environmental Laboratory Human Sera QC vs Std Ratios oooosi Page 41 E02-1053 Analytical Report: LIMS E02-1053 ne 11/01/02 A nalysis Branched Peak Linear Peak Blenam e Ret Tim e 7.9 Ret T im e 8.1 Identification PFOA-Add PFO A -A dd R et T im e 7.9/ Sum of A rea A rea B ranchedtLlnear 100 Standards & CCVs RBS101702-10 ng/mL-2 7971 351306 D 021101005 2.22 RBS101702-QC-10 ng/mL-2-1 5022 219508 D 021101031 2.24 R B S 102802-Q C -l 0 n g /m L -2 -1* 2733 225367 D 021101033 1.20 RBS10I702-QC-10 ng/mL-2-2 4416 211715 D 021101059 2.04 RBS102802-OC-10 na/mL-2-2* 1909 198898 D02U01061 0.951 * Data not used/reported Not extracted on the same date as the samples and therefore not included in tables 8 and 9. ETS-8-231.1 3M Environmental Laboratory Human Sera QC vs Std Ratios 000082 Page 42 Analytical Report: LIMSE02-1053 Analytical Report: LIM S E 02 -1 0 5 3 Appendix E: Example Calculations Calculations used for Analyses In Study E02-1053 Percentage of Branched.Linear Isomer (also referred to as ratio in the report) Percentage* Branched TIC Peak Area * 100 (Branched TIC Peak Area + Linear TIC Peak Area) Sample TCR-688 PFOA Percentage: (8059/(8059+41466)) * 100 = 16% % Difference = (Expected TIC % Branched/Sum Initial Std - Observed TIC % Branched/Sum QC) * 100 Expected TIC % Branched/Sum Initial Std Sum = Branched peak area + Linear peak area % Difference of PFOA RBS102202-QC-10 ng/mL-1-1 sample analyzed 10/22/02 Initial Std = RBS101702-10 ng/mL-1 TIC % Branched/Sum = 22 QC = RBS102202-QC-10 ng/mL-1-1 TIC % Branched/Sum = 24 % Difference = l(22 - 24) / 24I = 9 3M Environmental Laboratory 3M Environmental Laboratory 000083 Page 43 Page 43 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 Appendix F: Interim Certificate(s) of Analysis 3M Environmental Laboratory 3M Environmental Laboratory 000084 Page 44 Page 44 Analytical Report: LIMS E02-1053 Ientre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICA IE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-033 (Revision 1) 3M Product: Ammonium Perfluorooctanoate T est Control Reference #: TCR-99131-37, Lot #: 332 _________________________ Purity: 95.2% _________ Test Nun* Specifications Result Purity1 95.2% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6 . Iron 7. Manganese Total % Impurity (19MR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Residual Solvents CTGA) Purity by DSC Inorganic Anions (IQ 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6 . Phosphate 7. Sulfate Organic Acids'1 (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis^ 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine Ammonium Analysis"* Ion Selective Electrode White, crystalline solid Theoretical Value - 22.3% Theoretical Value = 0.935% Theoretical Value =*3.25% Theoretical Value = 0% Theoretical Value = 66.1% Theoretical Value = 4.18% Conforms Positive 1 . 0 . 0 0 1 wt./wt% 2 . <0 .0 0 1 wtVwt.% 3. 0.005 wt./wt.% 4. <0.001 wt7wt.% 5. <0.001 wt./wt.% 6 . <0 .0 0 1 wt/wt.% 7. <0.001 wt./wt% 0.34 wtVwt.% 4.49 wt7wt.% None Quantified None Detected 99.7% 1. <0.015 wt./wt.% 2. <0.005 wt./wt.% 3. <0.040 wt7wt.% 4. <0.009 wt./wt% 5. <0.006 wt./wt% 6 . <0.006 wtywt.% 7. <0.040 wtywt.% 1 . <0 .1 wtiwt.% 2 . <0 .1 wtywt.% 3. <0.1 wt./wt.% 4. <0.25 wt./wt.% 1. 18.9 wtywt.% 2 . 1.31 wt./wt.% 3. 3.75 wt./wt.% 4. 4.34 wt./wt.% 5. 63.2 wtywt.% 3.49 wt./wt % C O A 023033-37R E V I .doc 3M Environmental Laboratory 000085 Page 1 o f2 Page 45 Analytical Report: LIMS E02-1053 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 www.centrelab.com Phone: (814) 231*8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference # : 023-033 (Revision 1) 3M Product: Ammonium Perfluorooctanoate Test Control Reference #: TCR-99131-37, Lot #: 332 Date o f Last Analysis: 12/15/00 Expiration Date: 12/15/06 Storage Conditions: <-10C Re-assessment Date: 12/15/06 `Purity = 100% - (Total Metal impurities, 0.006% + Total NMR impurities, 0.34% + Total LC/MS impurities, 4.49%) Total impurity from all tests = 4.84% Purity = 100% - 4.84% - 95.2% 2 TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid 3Theoretical value calculations based on the empirical formula, CgFijC>2(`)NH4 (+) (MW==431.1) LC/MS Purity Profile: TikF RetentionTime(min) Masi(s) Identity Area %Area 1 12.140 265 c, 167596 0.73 2 0154 331,319 Cy homoIogiie/Fi) 860991 3.76 3 14.099 369 PFOA 21861700 - T o ta l - - - "525552fr 4.49 This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). cA J LPrepared By: Charles !Sinians Date Scientist, Centre Analytical Laboratories Reviewed By: /*/ M / John Flaherty ' Date F TLaobKonrraattortrny/ XMifaanriaflgorewr, fCentre Analytical Laboratories C O A 023033-37R E V 1 .doc 3M Environmental Laboratory 000086 Page 2 o f2 Page 46 Ajialj^tca^Rfijgiort^^IM^E02^^53 3M Environmental Laboratory Note to File Project or Study Number: FACT-TCR-005 A ssociated Study Number. UM S # EOO-1762 3M Environmental Laboratory 000087 Page 47 Analytical Report: LIMS E02-1053 3M SPECIALTY MATERIALS & MANUFACTURING DIVISION ANALYTICAL LABORATORY To: William Reagen - (8-6565) - Environmental Lab- 2-3E-09 Request No. 62631 Study No. FACT-TCR-005 From: Tom Kestner - (3-5633) - SMMD Analytical Lab - 236-2B-11 Subject: Chem ical C haracterization of PFOA, Lot 332, TCR-99131-37 by `H-NM R, 19F-NM R, and IR Spectroscopy Date: December 11,2000 SAMPLE DESCRIPTION: PFOA, lot 332, TCR-99131-37; Nominal product =C7 FiS-C 0 2(-) NH4{+) (white powder). The sample was stored in a -2 0 C freezer at all times except when aliquots were removed for analysis. OBJECTIVE; This sample was subjected to 'H-NMR and 19F-NMR spectral analyses to determine the purity of the nominal product and to characterize as many impurity components as possible. An FT-IR spectrum was also acquired for the purpose of confirming the nominal perfluorinated carboxylate salt functional group. EX PER IM EN TA L: FT-NM R A portion o f the thawed sample was accurately weighed, spiked with a known amount of 1,4bis(trifluoromethyl)benzene (p-HFX), and then totally dissolved in acetone-de for subsequent analysis by NMR. An initial 400 M Hz `H-NMR spectrum (# h62631.401) and a 376 MHz 19F-NMR spectrum (# f62631.401) were acquired using a Varian UNITYplus 400 FT-NMR spectrometer. A second portion of the sample was also accurately weighed, spiked with a known amount of p-HFX, and then totally dissolved in deuterated trifluoroacetic acid (CF3CO2D = TFAD). An additional 400 M Hz 1H-NMR spectrum (#h62631.402) and a 376 MHz 19F-NMR spectrum (#f62631.402) were collected in the TFAD solvent. The primary purpose of using the TFAD solvent was to help minimize the interferences associated with broad NH4 (+) resonance that had been observed in 1H-NMR spectrum of the acetone-d6 solution. The sam ple preparation method described above was intended to permit the use o f the p-HFX as an internal standard for absolute weight percent measurements in either solution. FT-IR A portion of the sample was prepared for FT-IR spectral analysis using the KBr disk technique. A transmission FT-IR spectrum (#A62631) was then acquired using a Digilab FTS-40 FT-IR spectrophotometer. RESULTS: The combined NMR spectral data indicated the sample of PFOA, lot 332, TCR-99131-37 consisted of a high purity form of the nominal isomeric product mixture, CnF2n+i-COi(`) NHU(+), where `n ' was mainly 7. A few trace-level impurity components were also observed, tentatively assigned, and quantified from the NMR spectral data. 3M Environmental Laboratory Page 1 of 3 ooooss Page 48 December 11, 2000 Analytical Report: LIMS E02-1053 3M SMMD Analytical Lab Request # 62631 3M SMMD Analytical Lab: Bldg. 236-2B-11 RESULTS (cont): The qualitative and quantitative compositional results that were derived from the single trial 'H-NMR and ,5F-NM R spectral analyses are summarized in TABLE-1. Any water that may have been present in the sample was ignored for calculation purposes. The l9F-NMR relative weight percent concentrations shown in TABLE-1 should be very close to their respective absolute weight percent values with the stated assumptions. In order to perform the relative weight percent calculations, I assumed all o f the fluorocarbon chains contained 8 carbon atoms except where noted. In general, the l9F-NM R technique is not particularly well suited for characterizing small amounts of potential fluorochemical homolog impurity components unless the chains are very short. A more complete characterization of any other impurity homologs would require analysis by electrospray MS or a similar technique. Trace amounts of other unidentified impurities are also detected in the NMR spectra, but additional work would be required in an effort to identify or quantify these other materials. The FT-IR spectrum was used to verify the nominal perfluorinated carboxylate salt functional group. Copies of the NMR and IR spectra are attached for your reference. If you have any questions about these results, or if any further work is needed, please let me know. Tom Kestner c: Lisa Clemen - ET&S - 2-3E-09 Tanya Rude - QAI Rick Payfer - SA&C Analytical Lab - 236-2C-11 Hie Reference: wr62631,studyFACTrCR005.doc/78 Study Protocol #FACT-TCR-005 3M Environmental Laboratory Page 2 of 3 000089 Page 49 Analytical Report: LIMS E02-1053 December 11, 2000 3M SMMD Analytical Lab Request # 62631 3M SMMD Analytical Lab: Bldg. 236-2B-11 TABLE-1 Sample: PFOA, lot 332, TCR-99131-37 Overall Compositional Results by l9F-NMR Analysis & 'H-NM R Internal Standardization Analysis Structural Assignm ents1 19F-N M R R elative W t % Concentrations Normal chain isomer CF3(CF2)x-C 0 2(') NH4<+) (where x assumed to be 6 for calculation purposes) Internal monomethyl branched isomers CF3(CF2)ll-CF(CF3)-(CF2VC02t') NH4(+) (where x+y assumed to be 4 for calculation purpose, and x Z 0, y * 0) Isopropyl branch isomer (CF3)2CF-(CF2)x-C02(') NH4(+> (where x assumed to be 4 for calculation purposes) t-butyl branch isomer (CF3)3C-(CF2)x-C 0 2(-) NH4(+) (where x assumed to be 3 for calculation purposes) 77.7% 12.5% -9.0% 0.24% =0.19% f H fF Possible FF NH*(+) Internal gem-dimethyl branch isomers CF3-(CF2)x-C(CF3)2-(CF2)y-C 0 2(-) NH4(+) (where x+y assumed to be 3 for calculation purposes: x * 0 ) o f -J-f f Vl f l y^o - =0.13% 0.11% Possible F F NR,W Possible alpha branch isomer CxF2x+1-CF(CF3)-C 0 2(-) NH4(+) (where x assumed to be 5 for calculation purposes) Probable F-SF4-Rf , possibly as F-SF4-CnF2n-C 0 2(-) NH4(+) (where n assumed to be 7 for calculation purposes) Possible CF3CF2C 0 2(') Nk / +) CF3-0-C F 2-Rf, where -R f is undefined Total unassigned aliphatic materials 0.085% 0.029% 0.009% Trace ^ -N M R Absolute W t.% Concentration =0.002-0.003% 1) Trace amounts of other unidentified components are also detected in the NMR spectra. Study Protocol #FACT-TCR-005 3M Environmental Laboratory Page 3 of 3 000090 Page 50 Analytical Report: LIMSE02-1053 Certificate of Analysis Nominal Product: CF3(CF2)n-C02H, where average n * 6 PentadecaQuorooctanoic acid Product Code: TCR-617, Lot 210002 October 28,2002 Tom Kestner and Joel Miller The sample o f TCR-617, lot 210002 was analyzed using a combination of 19F-NMR, 'H-NMR, and LC/MS analysis techniques. The overall qualitative and quantitative compositional results that were derived from these combined analyses are summarized below in TABLE-1. TABLE-1 Sample: TCR-617, Lot 210002 Quantitative and Qualitative Compositional Results by Combined 19F/lH-NMR and LC/MS Analyses PCrot mt n pn on nn emntt SO ftrrtuincHtuivraeas*1 ^'Uh//i ^^CfA-nT Vml Dr Relative W eight% Concentrations (single trial analysis) CFaiCFzVCOiH where average n * 6.02 by l9F-NMR LC/MS showed n=6 (major), n=5, n=4 (minors). Probable (CF3>2-CF-(CF2)n-C02H assume n=4 for calculation purposes Probable (C6F i30 )-C02H acyclic ether acid as possible CF3CF2-0-CF(CF3)-CF2CF2-C02H Possible CF3(CF2)*-CF(CF3)-(CF2)yC02H where x*0, y*0 and assume x+y - 4 for calculation purposes Possible (CF3)3-C-(CF2),,-C02H assume n=3 for calculation purposes Possible CnH2iH-zsaturated aliphatic hydrocarbons 1. Trace amounts o f other unassigned components were also detected in the NMR spectra. ^99.51% Purity 0.39% 0.057% 0.019% 0.013% 0.0079% V, /c '- / Page 1 of 1 3MEnvironmental Laboratory File Reference: CofA_TCR-<517_Lot 210002.doc 000091 Page 51 Appendix G: Report Signature Page Analytical Report: LIMS E02-1053 Analytical Report: UMS E02-1053 22 W illiam Reagan, Ph.D., Laboratory Management, Sponsor Representative Date vft>h______________________ Lisa Stevenson, Principal Analytical Investigator n/13/oa Date 3M Environmental Laboratory 3M Environmental Laboratory 000092 Page 52 Page 52