Document dQ4NjqQzkjOob1GvaYrMO4qNG

Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 A nalytical Laboratory Report Title Comparative Analysis of Fluorochemicais in Human Serum Samples Obtained Commercially Data Requirem ent Not Applicable A u th o r Lisa Stevenson Study Com pletion Date 11/13/02 Perform ing Laboratory Extractions and Analyses 3M Environmental Laboratory Building2-3E-09,935 Bush Avenue St. Paul, MN 55106 Project Identification Analytical Report: E02-1053 Total Num ber o f Pages 52 3M Environmental Laboratory 3M Environmental Laboratory Page 1 Page 1 Analytical Report: LIMSE02-1053 Analytical Report: LIMS E02-1053 This page has been reserved for specific country requirements. Il | i 3M Environmental Laboratory 3M Environmental Laboratory Page 2 Page 2 Analytical Report: LIMSE02-1053 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. William Reagen, Ph.D., Laboratory Management, Sponsor Representative Date , Principal Analytical Investigator Date 3M Environmental Laboratory 3M Environmental Laboratory Page 3 Page 3 Analytical Report: LIMSE02-1053 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 r H / fy p L Date 3M Environmental Laboratory 3M Environmental Laboratory Page 4 Page 4 Analytical Report: LIMS E02-1053 Table of Contents Analytical Report: LIMS E02-1053 Compliance Statem 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 Summaries..................................................................................................................... 11 Preparatory and Analytical Method.................................................................................. 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 Statement 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 ETS -8-231.1, Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices (19 pages).........................................................................................18 Appendix C: QC 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 ag e ...........................................................................................52 3M Environmental Laboratory 3M Environmental Laboratory 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, 2002 ..................................................................................................................... 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-Acid QC samples Analyzed 11/01/02...37 3M Environmental Laboratory 3M Environmental Laboratory Page 6 Page 6 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 Study Personnel and Contributors Requestor 3M Environmental Technology and Safety Services 3M Environmental Laboratory Building 2-3E-09 St. Paul, MN 55106 William Reagen, Ph.D., Laboratory Management, Sponsor Representative Analytical Chemistry Laboratory Extractions and Analyses 3M Environmental Laboratory (3M Lab) Usa Stevenson, PrincipalAnalytical 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 Page 7 Page 7 Analytical Report: LIMS E02-1053 Executive Summary Analytical Report: LIMS E02-1053 A screening study was undertaken to compare branched and linear isomers of perfluorooctanoate (PFOA - C7F15C O O ) 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 TC-668-Lampire Lot X324B TCR-689-Sigma Lot 022K0965 TCR-690-Golden West 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 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 TC R -689-S igm a 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 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: C7F15CO2N IV 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-Acid) CAS Number: 335-67-1 Chemical Formula: C7F|5C 0 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 19 ,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. 8 Table 3. Characterization of the Analytical Reference Substances in Study E02-1053 r Location 3M Lab PFOA-NH4 Substance TCR-99131-037 PFOA-Acid TCR-617 Source 3M Oakwood Products Expiration Date 12/15/2006 NA Storage CondMone Chemical Lot Number Physical Description Purity -20C10C 332 White powder 95.2%* Room Temperature 210002 White crystal 99.51%** NA--Not available `Sea Certificateol AnalysisIran CentreAnalytical Laboratories inAppendxF. "See Certficata ofAnalysisfrom3M. 3M Environmental Laboratory 3M Environmental Laboratory Page 10 Page 10 Analytical Report: LIMS E02-1053 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 Preparatoryand 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. T h e supernatant was transferred to a clean tube, diluted with water, and passed through a pre-conditioned C 18 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 (C7F15C O O ')I selected as the primary ion for analysis, was fragmented further to produce characteristic daughter ions 1 1 9 ,1 6 9 ,2 1 9 , 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 Page 11 Page 11 Analytical Report: LIMS E02-1053 Analytical Report: U M S E02-1053 Analytical Equipment The following is representative of the settings used during the analytical phase of this study. Liquid Chrom atograph: Hewlett-Packard Series 1100 Uquid Chromatograph system Analytical column: Keystone BetasilTM C i8 2x100 mm (5 pm) Column temperature: 40C Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 1-30 pL Solvent Gradient: 16.0 minutes Time (minutes) %B 0.0 40% 10.0 90% 11.0 90% 11.5 100% 12.5 100% 13.0 40% 16.0 40% Mass Spectrom eter: Micromass APi/Mass Spectrometer Quattro Ultima Triple Quadrupole system Software: Mass Lynx" 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 Tim es Target Primary Ion Analyta (AMU) Product Ion (amu) Isomerretention time Branched Linear PFOA 413.0 1 1 9 ,1 6 9 ,2 1 9 ,3 6 9 -7 .9 min. -8.1 min. 3M Environmental Laboratory 3M Environmental Laboratory Page 12 Page 12 Analytical Report: LIMSE02-1053 Analytical Report: LIMS E02-1053 Data Quality Objectives and Data Integrity The following data quality objectives (OQOs) were indicated for this study: C alibration: 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 A rea 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 C riteria: 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. C onfirm atory M ethods: No confirmatory method will be used. Dem onstration o f 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 Summaryf of Pooled Serum Sample Identification Branched Linear Isomer Area IsomerArea Pooled Human Serum TCR-687-Bloresource Lot 020821 TCR-688-Lampire Lot X324B TCR-689-Sgma Lot 022K0965 <361* 6059 8824 114603 41466 55100 TCR-690-Golden West Lot G01406042 <361* 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 Page 13 Page 13 Analytical Report: LIMS E02-1053 Analytical Report: LIM S E02-1053 Summary of Q uality Control Analyses Results C alibration: 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). Limits 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-Add 0.534 361* Quattro Ultima ' Based on the extracted PFOA-Add 0.534 n gM . standard, branched isomar area at retention time 7.9 minutes, with a TIC peak area -2 times the baseine noise. PFOA-Add - standard spiked with PFOA-Add 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 +/- 10% 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 3M Environmental Laboratory 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 Page 15 Page 15 Analytical Report: LIMS E02-1053 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 Bqsiration Date Storage Concfitions Chemical Lot # Physical Description Sigma 09/26/2005 -20C 10C 99H8400 Rabbit Serum 3M Environmental Laboratory 3M Environmental Laboratory Page 16 Page 16 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 Appendix B: Extraction and Analytical Method This appendix includes the following method: ETS-&-231.1, Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices, (19 pages) 3M Environmental Laboratory 3M Environmental Laboratory Page 17 Page 17 h K 'u n . . . ; n ;i\ he used, i ; ' aureus, o r ) i Analytical Report: LIMSE02-1053 r-'vi 10 16 2 !'V. 3M Environm ental lab o rato ry Method Solid Phase Extraction and Analysis o f Fluorochemlcal Compounds from Biological Matrices Method Number: ETS-8-231.1 Adoption Date: H /l3jo l Revision Date: J/'S/02- Effective Date: 3 -jt l(t>z Approved By: Willliam K. Reagen Laboratory Manager m*VYi ExactCopyofOriginal t f o - il l e s i Initial Oats 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 1 of 19 Page 18 D-'ct'.ic.-u na'* Analytical Report: LIMS E02-1053 f curron'., tor l 4 J a \ - from 10 . 2J02 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 Summ ary 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 Cig 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 + S mLs o f diluent for a total o f 6 mLs combined, unless otherwise noted. 3.2 SPE cartridge A column containing an open solvent reservoir at one end and packed with bonded silica sorbents at the other end. It is designed to retain the compounds o f interest under some solvent conditions and elute them under others. A separation is thus achieved; compounds can be removed from difficult biological matrices and introduced into appropriate solvents for analysis. 3.3 Reagent grade w ater Water with no detectable concentration^) o f the target analyte(s). 3.4 Quality control sam ple 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 Page 19 Analytical Report: LIMS E02-1053 Do not operate solvent pumps above capacity o f 400 bar (5800 psi) back pressure, [f 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 M aterials 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, VWR, Vortex Genie 2 Ultra-Turrax T25 tissue homogenizer Vacuum Pump SPE Extraction Manifold Centrifuge, Mistral 1000 or IEC 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 bolding 50 w h 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 pL Bottle-Top Dispenser (capable o f dispensing 5mL o f solvent) SPE extraction cartridge, 1 g, Sep-Pak 6 cc tri-functional C i, (Waters) 75 mL sample reservoir (or other appropriate size) Crimp cap glass autovials and caps 3M Environmental Laboratory E T S - 8 - 2 3 1.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 3 of 19 Page 20 Analytical Report: LIMS E02-1053 Document "k .v he iiscd. T' c u riv i: .'n 1l tlavs I . mu ' 0/1 <v'2{)>2 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 [I, or equivalent Acetonitrile, HPLC grade or equivalent Methanol, HPLC grade or equivalent Ammonium acetate, reagent grade o r 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(s) for the standard curve. Multicomponent analyte standards are acceptable. The following is an example only and may or may not be appropriate for all standard preparations. Weigh approximately 100 mg o f target analyte into a 100 mL volumetric flask and record the actual weight in the standard logbook or other appropriate location. Bring to volume with methanol for a stock standard o f approximately 1000 ppm (jig/mL). Dilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. Example calculation: 1000 Jlg/mL x 5 mlVl 00 mL = 50 |ig/mL. Dilute working standard 1 with methanol to produce a working standard 2 solution o f approx. 5.0 ppm. Example calculation: 50 Jig/mL x 10 mlVlOOmL = 5.0 Jig/mL. Dilute working standard 1 with methanol to produce a working standard 3 solution o f approx. 0.50 ppm. Example calculation: 5 0 JJg/m L x 1.0 m U lO O m L " 0.5 Jig/m L. 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 volumetric flask and bring to volume with methanol for a working standard o f 90-1 lOppm. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 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 o f Fluorochemical Compounds from Biological Matrices Page 4 o f 19 Page 21 \11 i i uv be used. Analytical Report: LIMSE02-1053 U 'l !-' 1:r-,s b in in'! > 2 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 lOppm. Record the actual volume transferred and standard concentrations in the standards logbook or other appropriate location. 8 Sample Handling All samples are received frozen and must be kept frozen until the extraction is performed. Allow samples to thaw to room temperature prior to extraction. Typically 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. 9.12 Method Blank An aliquot o f 1.0 mL o f 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 anim als; o r 3) a surrogate m atrix, also obtained com m ercially, 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 o f the same species as the study animals (two spiked with surrogate and two not spiked). 9.1.3.4 If limited matrix is available, the number o f 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. 3MEnvironmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 5 o f 19 Page 22 Docji'vnf irciy be u Analytical Report: LIMSE02-1053 uiTcnt. fer 14 day> coi;', 11). !rS 2n02 9.3 Surrogate standard If surrogate standard is a component o f the study, all samples are spiked with surrogate standard prior to extraction to obtain a concentration in the mid-range o f the calibration curve, with the exception o f blank samples as described above. Typically surrogate standard is spiked into the 1.0 mL diluted/homogenized sample removed for extraction. However, surrogate may be spiked directly into the matrix prior to diluting with water, into the diluted/homogenized sample prior to removing the 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 Sam ple 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 lour 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 m inim um o f three Q C per analysis. Solvent blanks are not considered sam ples but m ay 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 o f 75-125% o f expected. A minimum o f 2/3 o f all QC samples must meet this criteria, and a minimum o f 1/2 o f the QC samples at each level must meet this criteria. If not, the set must either be re-analyzed or re-extracted. 9.7 Sample Dilution Any sample with an area greater than that o f the highest acceptable standard will need to be diluted into the range o f the calibration curve. If samples 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 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 determined by the analyst 3MEnvironmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 6 o f 19 Page 23 Analytical Report: LIMS E02-1053 Document ir.uv ' *tt. i auront, for 14 tluv iVom 10 6'2002 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 Instrum ent Calibration One calibration curve will be prepared from extracted matrix standards, in the same matrix as the samples, per study. It will consist o f a minimum o f nine (9) levels. Additional calibration curves may be extracted on separate sample extraction dates, as determined by the analyst and documented in a note to file. Transfer 1.0 mL, or other appropriate amount, o f diluted control fluid or homogenized control tissue to a 15 mL centrifuge tube using a disposable plastic pipette. This will be repeated while preparing aliquots for the standard curve. Be sure to mix or shake the control matrix container between aliquots to ensure a homogenous sample is removed. Record each standard volume on the weight/volumes sheet o r 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, o r 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 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 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 o f 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 Page 24 i . i t U i i . U H C ' U i r i . l v , V U: Analytical Report: LIMSE02-1053 T tit r e n t , l o r 1-4 d:ty>- ; r u n - : ' ) !/. \ , D 2 T Mil I-: 1 A i t u o m m wi : Si'i k i m ; A t t o r s i . s h >k S i ami t u n s a s d S imkis I'SINt; I.ft Ml. oi- M ai ki x Working standard (approximate concentration) pL Approximate final concentration o f analyte in M atrix dilated 1:5 Approximate final concentration o f analyte in Final 2.0 m L volume . - Blank Blank 0.500 ug/mL 1.5 5.00 ng/g or ng/mL 0.375 ng/mL 0.500 ug/mL 3.0 10.0 ng/g or ng/mL 0.750 ng/mL 0.500 ug/mL 8.0 25.0 ng/g or ng/mL 2.00 ng/mL 0.500 ug/mL 16 50.0 ng/g or ng/mL 4.00 ng/mL 0.500 ug/mL 32 100 ng/g or ng/mL 8.00 ng/mL 5.00 ug/mL 5.6 175 ng/g or ng/mL 14.0 ng/mL 5.00 ug'm L . 8.0 250 ng/g or ng/mL 20.0 ng/mL 5.00 ug/mL 16 500 ng/g or ng/mL 40.0 ng/mL 5.00 ug/mL 24 750 ng/g or ng/mL 60.0 ng/mL 5.00 ug/mL 32 1000 ng/g or ng/mL 80.0 ng/mL 5.00 ug/mL 40 1250 ng/g or ng/mL 100 ng/mL 50.0 ug/mL 5.0 1500 ng/g or ng/mL 125 ng/mL 50.0 ug/mL 6.0 1750 ng/g or ng/mL 150 ng/mL Surrogate Std 100 ug/mL 10 6500 ng/g or ng/mL 500 ng/mL 11 Procedures 11.1 Tissue 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 die 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.S 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-S2 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 o f Fluorochemical Compounds from Biological Matrices Page 8 o f 19 Page 25 i>*eumonr / he u v il, :f current, for i l ci Analytical Report: LIMSE02-1053 from 10 |(-> 1002 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 15 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 1.0 mL o f fluid for a 1/6 dilution, or other dilution as determined by the analyst and documented in a note to file. If a volume other than 1.0 mL is removed for an initial volume, adjust the water volume accordingly to maintain the same dilution as above. . 11.3 Tissue and Fluid Sample Extraction After tissue or fluid samples have been prepared according to sections 11.1 and 11.2, vortex mix or shake by hand the diluted/homogenized sample for approximately 15 seconds then transfer 1.0 mL, or other appropriate volume, to a clean 15 mL polypropylene centrifuge tube. Return unused diluted/homogenized portions to the 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 d e c a n t th e su p e rn atan t in to th e w ater in the SO m L tu b e, taking care n o t to introduce an y o f th e m atrix so lid s into th e solution. Cap and mix by inverting several times. In this step the order o f 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/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 o f methanol, followed by approximately two 5.0 mL aliquots o f water, taking care not to allow the column to run to dryness after each wash. After washing is complete, pour the sample into the reservoir/cartridge unit and allow all of the liquid to pass through the column to dryness. Run the vacuum on high for approximately 5 minutes to adequately dry each SPE cartridge. Place a collection 15 mL polypropylene centrifuge tube under each cartridge and elute with 2.0 mL o f methanol. Spike extracted blanks, samples, and standards with internal standard as described in this method. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 9 o f 19 Page 26 ! 1 . ! -- ; ;. ' ! ' .I i . -; ' | m !$.] v*n :) - D ocij Analytical Report: LIMSE02-1053 ( iriiiv b e a- e d 'fo u iren t, for M davs Von' 1vi I o 2(K>2 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 instniment name (A = "Amelia") YY = Test year (02) MM = 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 V, and so on.) Assign a filename using the instniment designator letter, the last 2 digits o f the test year-month-day, and a 3-digit sequential file number that starts with I and 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 pan 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 M R M (M ultiple R eaction M onitoring). S et Ionization M ode as appropriate and m ass to 499 o r 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. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 10 o f 19 Page 27 i A 'CU MV Analytical Report: LIMS E02-1053 .tv be used, i vupv..l i't>r M t h v s Imi h KV Kt 2- t i 2 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: ' Time Solvent B 0.00 10% 1.00 10% 5.50 95% 7.50 95% 8.00 10% 11.4.3 Instrument set-up Refer to ETS-9-24, "Operation and Maintenance 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 fiat 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. 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-S00uL/min 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.I Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page II o f 19 Page 28 KJUTicii 111;iv be lito ti. ' i' imtc Analytical Report: LIMSE02-1053 , 4 d u rs i n im i O'] ( 2002 IIPLC 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) lOOOng SV (m L)x Matrix Amount (g/mL or mL/mL) 1ug IW = Initial weight (where 1.0 g = 1.0 mL ) IV = Initial volume DV = Diluent volume (reagent grade water) S V = Sample volume removed for extraction (typically 1.0 mL) AR = Analytical result from MassLynx summary DF = Dilution factor FV = Final volume MA = Matrix amount 3 curve = MA o f tissue/fluid standard curve, assumed to be 1 g or 1 mL/5 mL water 3 sample = MA o f tissue/fluid sample (__ g or mL o f sample/5 mL water) Calculate spike percent recoveries using the following equation: % Recovery Observed Result - Matrix Blank Result 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 Page 29 Doo.'iiv.Mir iruiv Ik- Analytical Report: LIMS E02-1053 auront, for 14 duvs Iront it/- )6/21)02 Calculate relative standard deviation using the following equation: Standard Deviation Relative Standard Deviation = x 100 Mean Calculate percent deviation using the following equation: ,,,, . . ExpectedI Cone. - Calculated Cone. __ % Deviation = -- - ------------------------------------------ x 100 Expected Cone. Calculate actual concentration o f analyte in fluid (pg/mL): AR (ng/mL) x DF * 3 curve fmL/mLl x FV (mL) 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/pf x DF x 3 curve fg/mLl x FV (mL) 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 o r less than 5.0% RSD, the precision o f 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 a t the com pletion of a run, the sam ple set m ust be reanalyzed. 13.2 Q uantitation The coefficient o f determination value for the calibration curve, plotted by regression using the peak areas o f the analyte(s), must be 0.990 or better. All active calibration curve points must be within 25% o f the theoretical value with the exception o f the LOQ point, which may deviate up to 30%. Calibration standards with peak areas less than two times the curve matrix blank will be deactivated to disqualify a data range that may be affected by background levels o f the analyte. A valid calibration curve must contain at least 6 active points above and including the LOQ. If the curve cannot meet these criteria, the sample set must be reanalyzed or reextracted. 13.3 Accuracy Two thirds o f all quality control samples and l/2 o f each quality control sample at each level are expected to show an accuracy o f 75-125%. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 13 o f 19 Page 30 IX'..uncut ;/ i:c Analytical Report: LIMS E02-1053 '' liront, for M da'- - irimi iI>1^>2(J0j 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 regressioa Print these graphs and store with the study raw data. Print data integration summary, integration method, and chromatograms horn MassLynx, and store with the study raw data. Summarize data using suitable software (Excel 1.0 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 Attachm ent B: Sam ple W eight/Volume W orksheet 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 o f Fluorochemical Compounds from Biological Matrices Page 14 o f 19 Page 31 Document m he Analytical Report: LIMS E02-1053 "4. ru rro m , for 14 d:\yv. froir. lO .'ir.'2rK)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 :i c`S 1 a 3MEnvironmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis o f Fluorochemical Compounds from Biological Matrices Page 15 o f 19 Page 32 Analytical Report: LIMS E02-1053 Document . y hc osai. 1cunvni. for 14 day'- fro'\ !() 16.-2002 Study Number: Prep Date: Analysts initials: Box#: Attachment A - Extraction Worksheet Method Revision: ETS-8-231.1 Matrix: Sample Timepoint: Sample 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/aliquoted:__________ g/mL 1. Homogenize sample 2. Aliquot 1 mL of diluted matrix into 15 ml. polypropylene tube 3. Spike sam ples 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 tubes with water 9. Shake semple 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 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, 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 mL/min elution flow Flows may vary through cartridges - kPA may be raised for slow tubes and drying after mast have been drawn down and shut off. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 16 o f 19 Page 33 Ito c i imene c Analytical Report: LIMS E02-1053 ,.'iii`r,.'|,i , for 14 eays from 1.) | s Prep Date(s): A nalyst(s): Sample M atriz: M ethod/Revision: Sam ple ID Attachment B - Sample Weight/Volume Worksheet Study Number: Equipm ent Number: Final Solvent & TN N um ber: Initial W UVol e/m L /L W ater Volume added (mL) _ Volume Removed (mL) C om m ents 1 Form Completion Verified By:. 1 3M Environmental Laboratory ETS-8-231.1 \ Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 17 o f 19 Page 34 !>ti;i:ni.v-i *.;tv be ll^ed. :f ;t>v!,1 Analytical Report: LIMS E02-1053 v . from !' ! n < i(>2 Prep date(s): A n alyte(s): Sample m atrix: M ethod/revision: Attachment C: Calibration Standard Concentration Worksheet Standard number: Equipm ent number: Final solvent and TN: B lank Tissue or Fiuid/identifier: A nalyte mix std approx. 0.500 ug/mL: A nalyte mix std ap prox. 5.00 ug/mL: A nalyte mix std approx. 50.0 ug/mL: S u rro g ate std approx. 100 ug/mL: Actual concentrations o f standards in the analyte mix A naly te 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 Am't spiked mL 0.0015 0.0030 0.0080 0.0160 0.0320 0.0056 0.0080 0.0160 0.0240 0.0320 0.0400 0.005 0.006 All Final Volume: mL 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 All Initial Fluid Dilution mL/mL 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 0.1667 All Initial Tissue Density g/mL 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 0.1600 Calculated concentrations o f standards in relation to the final 2.0 mL solvent and initial matrix 2.0 m L Final Volume Fluid M atrix Tissue M atrix A nalyte F in a l cone. ng/mL 0.375 0.750 2.00 4.00 8.00 14.0 20.0 40.0 60.0 80.0 100 125 150 Surrogate Std cone ng/mL 100 Surrogate Final cone ng/mL 0.500 A n alyte F in a l .cone. ng/mL 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 Surrogate Std cone ng/mL 100 Surrogate Final cone ng/mL 6500 A n aly te Final cone. ng/g 5.00 10.0 25.0 50.0 100 175 250 500 750 1000 1250 1500 1750 Surrogate Std cone ng/mL 100 Surrogate Final cone .ng/g 6500 3MEnvironmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 18 o f 19 Page 35 liOKin-'^r 'Hi'v be useb Analytical Report: LIMS E02-1053 lurent, foi' M b 's irm o b ' 1h ii)02 Study: Dilution Date/Analyst: Box Number: Sample Number or Description 1/ Attachment D: Dilutions Summary Worksheet Solvent/TN Number: Extraction Date/Analyst: M atrix/Tim epoint: Dilutions 1/ 1/ 11 1/ 1/ 1/ Comments Verified By: ! Notes: 1/10 dilution = _______ o f sample + ________ o f solvent 3MEnvironmental Laboratory Form Completion Verified By:________________ _______ ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 19 o f 19 Page 36 Appendix C: QC Data Summary Tables Analytical Report: LIMSE02-1053 Analytical Report: U M S E02-1053 Table 8. Acceptance Criteria Summary of PFOA-NH4 QC samples Analyzed 11/01/02 Sam ple Identification PFOA-NH4 % Branched/ Sum Branched + Linear % Difference RBS101702-10 ng/mL-1 RBS1017Q2-QC-10 ng/mL-1-1 RBS101702-QC-10 ng/mL-1-2 26.02 25.35 24.93 NA 3 4 % Difference: The cfifference between the total ion current peak area ratios of the branched:linear 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 Sam ple Identification RBS101702-10 ng/mL-1 RBS101702-QC-10 ng/mL-1-1 RBS101702-QC-10 ng/mL-1-2 PFOA-Acid % Branched/ Sum Branched + Linear 2.22 2.24 2.04 % Difference NA -1 8 % D ifferen ce : T h e d ifferen ce betw een th e to tal ion current p e a k area ratios o f th e b ran ch ed :fin ear isom ers fo r P F O A in th e extracted standard (lab eled R B S -d ate of extraction-concentration) a t th e s a m e level a s th e extracted Q C (lab eled R B S -d ate o f extraction-Q C -concentration). 3 M Environmental Laboratory 3M Environmental Laboratory Page 37 Page 37 Appendix D: Data Spreadsheets Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 3M Environmental Laboratory 3M Environmental Laboratory Page 38 Page 38 E02-1053 Analytical Report: LIMS E02-1053 ANALYSIS 11/01/02 Identification TIC Branched Peak Ret Time 7.6 Branched PFOA-NH4 Area** Branched Peak Ret Time 7.9 B ranched PFOA-NH4 Area Linear Peak Ret Time 8.1 Linear PFOA-NH4 A rea Filename D021101a Branched Ret Time 7.9/ Sum of Branched:Linear * 100 Branched/Linear Standards RBS101702-10ng/mL-l RBS101702-QC-10 ng/mL-1-1 RBS102802-QC-10 ng /m H -1 RBS101702-QC-10 ng/mL-1-2 RBS102802-OC-10 ng/mL-1-2 W B102802-H20 Blk-1 RBS 102802-Sera Blank-1* 0.5 ng/mL-1 0 93029 264494 D 0 2 1 101003 0 81108 238850 D021101027 0 65454 195485 D021101029 0 59384 178832 D 0 2 1 101055 0 56858 168536 D021101057 0 355 2873 D 0 2 1 101043 0 0 3846 D 0 2 1 101045 0 1566 6752 D021101063 26.02 25.35 25.08 24.93 25.23 11.00 0.00 18.83 Pooled TCR-687-Bioresource* 0 361 114603 D021101007 0.314 TCR-688-Lampire TCR-689-Sigma TCR-690-Golden West* 0 8059 41466 D 0 2 1 101009 0 8824 55100 D021101011 0 361 168154 D021101013 16.27 13.80 0.214 * Below Limit of Peak Area Threshold ** Branched peak at 7.6 minutes observed only in PFOA-NH4 standards at concentrations > 25 ng/mL. Not included in any calculations. ETS-8-231.1 3M Environmental Laboratory Confirmation Sera Ratios Page 39 E02-1053 Analytical Report: LIMS E02-1053 ANALYSIS 11/01/02 PFO A-A dd Pooled data w e n not included in the scope of the study and were not reported TIC Branched Peak Linear Peak Filename Branched Ret Time 7.9/ Ret Time 7.9 Ret Time 8.1 D021101a2 Sum o f Branched:Linear Branched L in ear * 100 Identification . PFOA-Add PFOA-Add Area A rea B ranched/L inear Standards RBS101702-10 ng/mL-2 RBS101702-QC-10 ng/mL-2-1 RBS102802-QC-10 ng/mL-2-1 RBS101702-QC-10 ng/mL-2-2 RBS102802-OC-10 ne/mL-2-2 W B102802-H20 Blk-1 RBS102802-Sera Blank-1 0.5 ng/mL-2 7971 5022 2733 4416 1909 260 545 361 351306 219508 225367 211715 198898 2873 3787 6473 D 0 2 1 101005 D 0 2 1 101031 D 0 2 1 101033 D 0 2 1 101059 D 0 2 1 101061 D 0 2 1 101043 D 0 2 1 101045 D 0 2 1 101065 2.22 2.24 1.20 2.04 0.951 8.30 12.58 5.28 Pooled TCR-687-B ioresource* TCR-688-Lampire TCR-689-Sigma TCR-690-Golden West* * Below Limit of Peak Area Threshold 361 8207 8823 361 113845 41469 55095 168119 D 0 2 1 101007 D 0 2 1 101009 D 0 2 1 101011 D 0 2 1 101013 0.316 16.52 13.80 0.214 ETS-8-231.1 3M Environmental Laboratory Confirmation Sera Ratios Page 40 E02-1053 Analytical Report: LIMSE02-1053 v TIC 11/01/02 Analysis Branched Peak Branched Peak Linear Peak Filename Ret Time 7.6 Ret Time 7.9 Ret Time 8.1 Identification PFOA-NH4 PFOA-NH4 PFOA-NH4 R et Time 7.9/ Area** A rea Area Sum of B ran ch ed iL in ear Standards & CCVs 100 RBS101702-10 ng/mL-1 0 93029 264494 D 0 2 1 101003 26.02 RBS101702-QC-10 ng/mL-1-1 RBS102802-QC-10 ng /m H -1 * RBS101702-QC-10 ng/mL-1-2 0 0 0 81108 65454 59384 238850 195485 178832 D 0 2 1 101027 D021101029 D 0 2 1101055 25.35 25.08 24.93 RBS102802-OC-10 ne/mL-1-2* 0 56858 168536 D 0 2 1 101057 25.23 * Data not used/reported. Not extracted on the same date as the samples and therefore not included in tables 8 and 9. ** Branched peak at 7.6 minutes observed only in PFOA-NH4 standards at concentrations > 25 ng/mL. Not included in any calculations. ETS-8-231.1 3M Environmental Laboratory Human Sera QC vs Std Ratios Page 41 E02-1053 Analytical Report: LIMS E02-1053 TIC 11/01/02 Analysis Branched Peak Linear Peak Rlename Ret Time 7.9 Ret Time 8.1 Identification PFOA-Acid PFOA-Add Ret Time 7.9/ Sum of - Area A rea Branched:Llnear *100 Standards & CCVs RBS101702-10 ng/mL-2 7971 351306 D 0 2 1 101005 2.22 RBS101702-QC-10 ng/mL-2-1 5022 219508 D021101031 2.24 RBS102802-QC-10 ng/mL-2-1* 2733 225367 D 0 2 1 101033 1.20 RBS101702-QC-10 ng/mL-2-2 4416 211715 D 0 2 1 101059 2.04 RBS102802-OC-10 n/mL-2-2* 1909 198898 D 0 2 1 101061 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 Page 42 Analytical Report: LIMS E02-1053 ' Analytical Report: LIMS E02-1053 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% % D ifferen ce = (E x p e c te d T IC % B ranched/Sum Initial S td - O bserved T IC % B ranched/S um QC1 * 1 0 0 Expected T IC % B ranched/Sum Initial Std S um = B ranched p e a k a re a + L in ear p e a k area % D ifferen ce o f P F O A R B S 1 0 2 2 0 2 -Q C -1 0 ng/m L-1-1 sam p le an alyzed 1 0 /2 2 /0 2 In itia l S td - R B S 10 1 7 0 2 -1 0 ng/m L-1 T IC % B ranched/S um = 2 2 Q C = R B S 1 0 2 2 0 2 -Q C -1 0 ng/m L-1-1 T IC % B ranched/Sum = 2 4 % D ifferen ce = l(2 2 - 2 4 ) / 2 4 I * 9 3 M Environmental Laboratory 3M Environmental Laboratory 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 Page 44 Page 44 Analytical Report: HMSE02-1053 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 Perflaorooctanoate Test Control Reference #: TCR-99131-37, Lot #: 332 _________________________ Pnrity: 95.2%_______________________ Test Name Specifications Result Purity1 95.2% Appearance White, crystalline solid Conforms Identification . NMR Positive Metals (ICP/MS) 1. Calcium 1. 0.001 wt7wt.% 2. Magnesium 2. <0.001 wt7wt% 3. Sodium 3. 0.005 wt/wt.% 4. Potassium 4. <0.001 wtVwt.% 5. Nickel 5. <0.001 wt./wt% 6. Iron 6. <0.001 wtVwt.% 7. Manganese 7. <0.001 wt/wt.% Total % Impurity (NMR) 0.34 wt/wt.% Total % Impurity (LC/MS) 4.49 wtiwt% Total % Impurity (GC/MS) None Quantified Residual Solvents (TGA) None Detected Purity by DSC 99.7% Inorganic Anions (IC) 1. Chloride 1. <0.015 wt/wt.% 2. Fluoride 2. <0.005 wt7wt% 3. Bromide 3. <0.040 wtVwt.% 4. Nitrate 4. <0.009 wt./wt% 5. Nitrite 5. <0.006 wt./wt% 6. Phosphate 7 . S u lfa te 6. <0.006 wt./wt.% 7- <0.040 wtywt.% Organic Acids1(IC) 1. TFA : I 2. PFPA 1. <0.1 wt./wt.% 2. <0.1 wtVwt.% 3. HFBA 3. <0.1 wt/wt.% 4. NFPA 4. <0.25 wt/wt.% Elemental Analysis1: 1. Carbon Theoretical Value * 22.3% 1. 18.9 wt/wt.% 2. Hydrogen Theoretical Value = 0.935% 2. 1.31 wt/wt.% 3. Nitrogen Theoretical Value = 3.25% 3. 3.75 wt/wt.% 4. Sulfur Theoretical Value = 0% 4. 4.34 wt7wt.% 5. Fluorine Theoretical Value = 66.1% 5. 63.2 wt/wt.% Ammonium Analysis' Ion Selective Electrode Theoretical Value = 4.18% 3.49 wt/w t % ...J COA023033-37REV1 .doc 3M Environmental Laboratory Page 1 o f2 Page 45 Analytical Report: LIMS E02-105^ Centre Analytical Laboratories. Inc. 3048 Research Drive State College, F*A 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: <-10*C Re-assessment Date: 12/15/06 lPurity = 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, CgFu02(")NH4(+) (MW=431.1) . LC/MS Purity Profile: Peak# 1 2 3 Total Retention Time (min) 12.140 13.504 14.099 - M a(s) 264 331, 19 369 - Identity c, C7homologue/Fu PFOA - Area 167596 860991 21861700 22890287 % Area 0.73 3.7 - 4.49 This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). Prepared By: ChlaCrlesA!Sujrfons Scientist, Centre Analytical Laboratories Date Reviewed By*: n l/ f i4 e f /* /? /* / lorn Flaherty ' Date Laboratory Manager, Centre Analytical Laboratories COA023033-37REVl.doc 3M Environmental Laboratory Page 2 o f2 Page 46 A n a l^ tic a l^ fi o rt^ IM ^ E 0 ^ 0 5 3 3M E nvironm ental Laboratory Note to File Project or Study Number: FACT-TCR-005 A ssociated Study Number; LIMS # EOO-1762 L.-J m m 3M Environmental Laboratory Page 47 Analytical Report: LIMS E02-1053 3M SPECIALTY MATERIALS & MANUFACTURING DIVISION ANALYTICAL LABORATORY Request No. 62631 Study No. FACT-TCR-005 To: William Reagen - (8-6565) - Environmental Lab- 2-3E-09 From: Tom Kestner - (3-5633) - SMMD Analytical Lab - 236-2B-11 Subject: Chemical Characterization of PFOA, Lot 332, TCR-99131-37 by `H-NMR, 19F-NMR, and IR Spectroscopy Date: December 11,2000 SAMPLE DESCRIPTION: PFOA, lot 332, TCR-99131-37; Nominal product =CyFi5-C02^ NH^+*(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 I9F-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. E X PER IM EN TA L ; FT-NMR A portion of the thawed sample was accurately weighed, spiked with a known amount of 1,4bis(trifluoromethyl)benzene (p-HFX), and then totally dissolved in acetone-d for subsequent analysis by NMR. An initial 400 MHz 1H-NMR spectrum (# h62631.401) and a 376 MHz 19F-NMR spectrum (# f62631.401) were acquired using a Varian UNTTYplus 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 MHz `H-NMR spectrum (#h62631.402) and a 376 MHz I9F-NMR spectrum (#f62631.402) were collected in the TFAD JiTi? solvent. The primary purpose of using the TFAD solvent was to help minimize the interferences associated with broad NHU<+) resonance that had been observed in 'H-NMR spectrum o f the acetone-d^ solution. The sample preparation method described above was intended to permit the use of 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-C02(') NH4<+), where V 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 Page 48 Decem ber 11, 2000 Analytical Report: LIMSE02-1053 3M SM M D Analytical Lab R equest # 62631 3M SMM D Analytical Lab: Bldg. 236-2B -11 RESULTS tcontl; The qualitative and quantitative compositional results that were derived from the single trial 'H-NMR and 19F-NMR spectral analyses are summarized in TABLE-1. Any water that may have been present in the sample was ignored for calculation purposes. The ,9F-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 of the fluorocarbon chains contained 8 carbon atoms except where noted. In general, the 19F-NMR 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 Clem en - ET&S - 2-3E-09 Tanya Rude - QAI Rick Payfer - SA&C Analytical Lab - 236-2C-11 File Reference: wrf>2631.studyFACTTCR005.doc/78 I Study Protocol #FACT-TCR-005 3M Environmental Laboratory Page 2 of 3 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 ,9F-NMR Analysis & *H-NMR Internal Standardization Analysis Structural Assignments1 19F-NM R Relative W t. % Concentrations Normal chain isomer c f 3(c f 2)x-c o 2(-) n h 4(+) (where x assumed to be 6 for calculation purposes) Internal monomethyl branched isomers C F3(CF2)x-CF(CF3)-(CF2)y-C02(`3NH4(+) (where x+y assumed to be 4 for calculation purpose, and x * 0, y * 0) Isopropyl branch isomer (CF3)2CF-(CF2)x-C 02WNH4(+) (where x assumed to be 4 for calculation purposes) t-butyl branch isomer (CF3)3C -(C F2)x-C 0 2(-) NH4(+) (where x assumed to be 3 for calculation purposes) - k 77.7% =12.5% 9.0% 0.24% =0.19% F 'l S f Possible FF NH4<+) Internal gem-dimethyl branch isomers C F3-(CF2)x-C(CF3)2-(CF2)y-C 02( ) NH4(+) (where x+y assumed to be 3 for calculation purposes: x * 0) O f P.Z lf f F^ / cp2 =0.13% =0.11% Possible F F NH4W Possible alpha branch isomer C xF2x+i-C F(C F3)-C 0 2(-) NH4(+) (where x assumed to be 5 for calculation purposes) Probable F-SF4 -R f, possibly as F-SFa-C nFin-C O ^ N H 4W (where n assumed to be 7 for calculation purposes) Possible C F3C F2C 0 2(') N I i / +) CF3-0-CF2-Rf, w here -R f is undefined Total unassigned aliphatic materials =0.085% =0.029% =0.009% Trace XH-NMR 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 Page 50 Analytical Report: LIMS E02-1053 Certificate of Analysis Nominal Product: CF^CF^n-CChH, where average n 6 Pentadecafluorooctanoic acid Product Code: TCR-617, Lot 210002 October 28,2002 Tom Kestner and Joel Miller The sample of TCR-617, lot 210002 was analyzed using a combination of 19F-NMR, 'H-NMR, and LC/MS analysis techniques. The overall qualitative and quantitative compositional results that were derived from these combined analyses are summarized below in TABLE-1. TABLE-1 Sample: TCR-617, Lot 210002 Quantitative and Qualitative Compositional Results by Combined 19F/*H-NMR and LC/MS Analyses Component Structures1 `h/ ^ f -n m r Relative Weight% Concentrations (single trial analysis) C F 3(C F 2)b-C 0 2 H where average n = 6.02 by l9F-NMR. LC/MS showed n=6 (major), n=5, n=4 (minors). Probable (CF3)2-CF-(CF2)I1-C02H assume n=4 for calculation purposes Probable (CiF^C^-Ci^H acyclic ether acid as possible CF3CF2-0 -CF(CF3)-CF2CF2-C02H Possible CF3(CF2)x-CF(CF3)-(CF2)jrCQ2H where x^O, y*0 and assume x+y > 4 for calculation purposes Possible (CF3)3-C-(CF2),,-C02H assume n=3 for calculation purposes Possible CnH2n+2 saturated aliphatic hydrocarbons 1. T race am ounts o f other unassigned components w ere also detected in the N M R spectra. 99.51% Purity 0.39% 0.057% 0.019% 0.013% 0.0079% Page 1 of 1 3M Environmental Laboratory File Reference: CofA_TCR-617_Lot 210002.doc Page 51 Appendix G: Report Signature Page Analytical Report: LIMS E02-1053 Analytical Report: LIM S E02-1053 William Reagan, Ph.D., Laboratory Management, Sponsor Representative Date A vfftn________ ____ Lisa Stevenson, Principal Analytical Investigator w i k s . Date 3M Environmental Laboratory 3M Environmental Laboratory Page 52 Page 52