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Analytical Report: LIMS E02-1053 AnalyticalReport:LIMS E02-1053 Analytical Laboratory Report Title Comparative Analysisof Fluorochemicalsin Human Serum Samples Obtained Commercially Data Requirement Not Applicable Author Lisa Stevenson Study Completion Date 11113/02 Performing Laboratory Extractionsand Analy=_ 3M EnvironmentLaal boratory Buildin2g-3E-09,935 BushAvenue St.Paul,MN55106 Project Identification AnalyticalReport: E02-1053 Total Number of Pages 52 i. :1 3M Environmentalt _horatory 3M Environmental Laboratory Page 1 Page 1 Analytical Report: LIMS E02-1053 AnalyticaRl eport:LIMS E02-1053 i;.__ This page has been reserved for specificcountryrequirements. i 3M Environmental Laboratory 3M Environmental Laboratory Page 2 Page 2 : ,,_ J i r. _-] _;_.":_1 B 1 ::I E / Analytical Report: LIMS E02-1053 AnalyticalReport:LIMS E02-1053 Compliance Statement Analytical Laboratory Report Title: Comparative Analysis of Fluorochemicalsin Human Serum Samples Obtained Commercially Study Identification Number: E02-1053 This study was not conducted under Good Laboratory Practices. Wi Reagen, Ph.D., Laboratory Management, Sponsor Representative " Date L_a Stevenson, Principal Analytical Investigator Date r I ', !1 !,_t r F ] 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 3 Page 3 i_:ii; i _] I ,,.,,.,_ t,l _: , ! i _ ,_:;; :I !:i I , !i! >,._t Analytical Report: LIMS E02-1053 AnalyticaRl eport:LIMS E02-1053 Quality Assurance Statement Analytical Laboratory Report Title: Comparative Analysisof Fluorochemicalsin Human Serum Samples Obtained Commercially Study IdentificationNumber: E02-1053 TUhniist (sQtuAdUy) haassinbdeeicnatIendspinetchteedfboyllothwein3gMtabElnev. irTohnemfeinndtainlLgasbwoerraetorreypQourtaelditytoAtshseurance Principal Analytical Investigator(PAl) and laboratory management. "" Inspection Dates 10/17/02 10/22/02 10/30/02, 10/31/02, 11/07-08/02 11/01/02, 11/07-08/02 n Phase Sample Spiking Analysis Data Draft report Date Reported to Management PAl 10/18/02 10/18/02 10/23/02 10/23/02 11/01/02, 11/08/02 1111//0018//0022, 11101/02, 11/08/02 1111//0018//0022, QAU Representative __ 2_ Date i" F LJ 3M EnvironmentalLaboratory 3M Environmental Laboratory Page4 Page 4 ..._... _iii _::i_i 'ii,_:, :I !it ' i.:,i , _j i i I __..J Analytical Report: LIMS E02-1053 Table of Contents AnalyticalReport:LIMS E02-1053 Compliance Statement ...................................................................................................... 3 Quality Assurance Statement ............................................................................................ 4 Ust of Tables ..................................................................................................................... 6 Study Personnal and Contributors..................................................................................... 7 Location of Archives .......................................................................................................... 7 Executive Summary ........................................................................................................... 8 Introductionand Purpose .................................................................................................. 9 Speciment Receipt and Maintenance ................................................................................ 9 Chemical Characterization of the Reference Substances.................................................. 10 Sample Preparation and Analysis...................................................................................... 11 Method Summaries ....................................................................................................... 11 Preparatory and AnalyticalMethod ........................................................................ 11 Analytical Equipment ............................................................................................. 12 Data Quality Objectives and Data Integrity........................................................................ 13 Data Summary, Analyses, and Results............................................................................. 13 Summary of Data Results............................................................................................. 13 Summary of Quality ControlAnalyses Results.............................................................. 14 Statement of Data Quality............................................................................................. 14 Statistical Methods and Calculations................................................................................. 15 Statement of Conclusion ................................................................................................... 15 References ........................................................................................................................ 15 Appendix A: Characterization of the Control Matrix .......................................................... 16 Appendix B: Extraction and Analytical Method.................................................................. 17 IETS-8-231.1, Solid Phase Extractionand Analysis of FluorochemicalCompounds 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 Page ................................................................................ 52 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 5 Page 5 Analytical Report: LIMS E02-1053 AnalyticaRl eport:LIMS E02-1053 List of Tables Table 1. PFOA Data Summary of Pooled Serum.............................................................. 8 Table 2. Pooled human serum samples received from commercialvendors in July and _ _t August, 2002 ........................................................................................................ 9 Table 3. Characterization of the AnalyticalReference Substancesin Study E02-1053 ..... 10 ..... Table 4. Target Ions Monitoredin 3M LaboratoryAnalyses and Observed Retention Tim es ................................................................................................................... 12 Table 5. PFOA Data Summary of Pooled Serum.............................................................. 13 Table 6. Limit of Peak Area Threshold in the Analyses of Sere Extracts.......................... 14 Table 7. Characterization of the Control Matrix Used for Analyses in Study E02-1053 ..... 16 ; Table 8. Acceptance Criteda Summary of PFOA-NH4 QC samples Analyzed 11/01/02...37 _I Table 9. Acceptance Criteda Summary of PFOA-Acid QC samplesAnalyzed 11/01/02...37 '! r 3M Environmental Laboratory 3M Environmental Laboratory Page 6 Page 6 _. _r "_ .._.. !_i ....i :_! , :_-_:_ :__, = J i liji I_ ,_ _ _ _ -i Analytical Report: LIMS E02-1053 AnalyticaRl eport:LIMS E02-1053 Study Personneland Contributors Requestor 3M EnvironmentalTechnology and Safety Services 3M Environmental Laboratory Building2-3E-09 St. Paul, MN 55106 William Reagen, Ph.D., Laboratory Management, Sponsor Representative Analytical Chemistry Laboratory Extractionsand Analyses 3M EnvironmentaLlaboratory(3M Lab) Usa Stevenson,P/JncipalAnalytical Investigator(PAl) 3M Lab Contributing Personnel Marlene M. Heying* Ognjenka Krupljanin* Richard C. Jones* Bob W. Wynne* Contractlabprofessionasl erviceemployee Study Initiation: 10/16/02 Study Completion: 11113/02 Location of Archives All originalraw 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 archivedat the 3M EnvironmentalLaboratoryaccording to 3M Standard Operating Procedures. Remainingspecimens pertainingto the analytical phase of this study willbe archived at 3M EnvironmentalLaboratoryfor as long as the quality of the preparationaffords evaluation. 3M Environmental Laboratory 3M Environmental Laboratory Page 7 Page 7 Analytical Report: LIMS E02-1053 ,i Executive Summary AnalyticaRl eport:LIMS E02-1053 A screening study was undertaken to compare branched and linear isomersof i perfluorooctanoate (PFOA - CTFlsCOO) in 4 lots of commercial pooled human sera with ;_ concentrations ranging from 0.65 - 5.6 ng/mL PFOA. Results from this study showeda wide distributionof the percentage of branched isomers of PFOA compared to the linear isomer of PFOA in the commercialpooled populations(Table 1). Since this is a preliminaryscreeningstudy a larger sample size would be needed to determine if a significantstatisticaldifference exists between pooled human sera samples. As shown in Table 1, two of the lots of commercial pooled sera showedthe presence of branched Isomers of PFOA while the other two lots showed a much lowerpercentage (by at least 40x) of PFOA branched isomers. Table 1. PFOA Data Summary of Pooled Serum Branched UnearIsomer % Branched/ :_ Sample IdentificaUon IsomerArea Area SumBranched+ Unear ' Pooled TCR-687-Bioresource <361" 114603 <0.31 Human Lot 020821 Serum TCR-688-Lampire 8059 41466 16 Lot X324B TCR-689-Sigma 8824 55100 14 Lot 022K0965 _,ii!,Iii West LotG01406042 <ssl" lse154 <o.21 "Area threshold found as 361 in the extracted PFOA-Acid 0.534 ng/mL standard. 3M Environmenta/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 fluorochemicalin 4 lots of commercialpooled human sera with concentrationsranging ':' from 0.65 - 5.6 ng/mL PFOA. The PFOA concentrationswere determined in study ' E02-1039. Analyses of sera extractsfor determiningthe relativeisomer ratios of PFOA were completed by the 3M EnvironmentalLaboratoryunder study number E02-1053, '-i and the results of these analyses are presented in this report. The analytical portionof this study was initiated on 16 October 2002. i Specimen Receipt and Maintenance The 3M Environmental Laboratoryreceived pooled human sera samples collected from ,_ vadous commercial vendors in Julyand August,2002. All specimenswere received ....,. frozen in good conditionon dry ice. All specimens were immediatelytransferred to storage at -20C + 10C, and maintained at that temperature except when removed for extraction and analysis as describedin the method. The samples were kept isolated :i from the test materials (analyticalstandards)duringstorage. Table 2. Pooled human serum samples received from commercial vendors in July and August, 2002 i:?_i Samples Identification Lot# "' PooledHuman TCR-687-Biomsource 020821 Serum _!I TCR-688-Lampim X324B ',__ TCR-689-Sigma 022K0965 TCR-690-GoldeWn est Q01406042 _: 2.; ' The control matrix used in sera analyses performedduringE02-1053 was obtained from ..._.' a commercial source and is presented in AppendixA. Samples analyzed at the 3M Environmental Laboratorywill be stored and maintainedfollowing3M Standard _i Operating Procedures. 3M Environmental Laboratory 3M Environmental Laboratory Page 9 Page 9 iI ) ..;.i. 4t _.i:,J ) _i .... i:_._,__l i i _J '! iL_,i Analytical Report: LIMS E02-1053 AnalyticaRl eport:LIMS E02-1053 Chemical Characterization of the Reference Substances Perfluorooctanoate Ammonium Salt (PFOA-NH4) CAS Number: 3825-26-1 Chemical Formula: CTFIsCO='NH4+ Molecular Weight: 431 This chemical is a 3M electrochemicalfluorinationproductionlot and contains, as determined by NMR, approximately20% branched:80% linear isomers by weight. Perfluorooctanoate-Acid (PFOA-Acid) CAS Number: 335-67-1 Chemical Formula: CrF_sCO2H MolecularWeight: 414 This chemical is a commercialproductobtained from Oakwood Productsand contains approximately 1% branched:99% linear isomers. The molecular ion 413 was selected as the primary ionfor PFOA. This ionwas fragmented further duringanalysisto produce ions 119, 169, 219, and 369. The total ioncurrent (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 Substance 8ource 3M Lab PFOA-NH4 PFOA-Acid TCR-99131-037 TCR_17 3M OakwooPdroducts F.xpimtlon Date 12/15/2(X_ NA SCtoonrdalgffeonll Chemical Lot Numbw -20"C + 10"C 33 Room Temperature 210(X_ PDl,__y._s'idcpatlton Whitpeowder Whitcerystal Purity 95.2=/0, 99.51%*" NA--.No_t "SeeC,e_llk:atoelAnalysifsromCentreAnalytk_I.abom4ode_sAppendxF. "See _ o(,=unalymflora3M. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 10 Page 10 ?t _-,_; :,ii _: = :I ,i I _: i_!!ii!l , _._.,_ _ _!_:i! i 9 Analytical Report: LIMS E02-1053 AnalyticaRl eport:LIMS E02-1053 Sample Preparation and Analysis Human serum samples were analyzed in this study. Sera sampleswere extracted beginning on 17 October 2002 using a solid phase extraction(SPE) procedure. Sample extracts were analyzed using high-performanceliquidchromatographyelectrospray/tandem mass spectrometry (HPLC-ES/MS/MS) in the multiplereaction mode versus extracted rabbitsera standards. Qualitative analysis of branchedand linear isomersof 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 withinan approximate 0.2 minute retentiontime window from the linear PFOA isomei'. Method Summaries Followingis a brief descriptionof the method used duringthis analyticalstudy by the 3M Environmental Laboratory. A detailed description of the method used in this study is located in Appendix B. 3M Environmental Laboratory PREPARATORAYNDANALYTICALMETHOD ETS-8-231.1, =SolidPhase Extraction and Analysisof FluorochemicalCompounds from Biological Matrices" Human sera was prepared using 2.0 mL of serumthen diluted to 10 mL with reagent grade water. The diluted serum/waterwas spiked with the appropriateanalyte mixture. Acetonitrile(ACN) was added as an extractionsolvent,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 througha pre-conditionedC18SPE cartridge. The analyte(s) of interestwere eluted from the SPE cartridge with 2.0 mL of methanol and analyzed. Analyses were performed by monitoringtwo or more productions selected from a single primary ion characteristicof PFOA usingHPLC-ES/MS/MS. For example, the molecular anion 413 (C_F_sCOO'),selectedas the primaryion for analysis, was fragmented further to producecharacteristicdaughter ions 119, 169, 219, and 369. The total ion current (TIC) peak areas are the sum of the signalfor each daughter ionat specific retentionstimes for each target analyte isomer that were monitoredfor analysis. Daughter ions may also be referred to as productions. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 11 Page 11 ...!... _-+" !m::"_ +:! :.,,_ _:- !:_:!+_,_ +_ i!'iiii _ j ,j + i ,i ..:++_ Analytical Report: LIMS E02-1053 ANALY'nCALEQUIPMENT AnalyticaRl eport:LIMS E02-1053 The following is representative of the settings used duringthe analyticalphase of this study. Liquid Chromatograph: Hewlett-Packarde Series 1100 Uquid Chromatographsystem Analyticalcolumn: Keystone+ BetasilTM Cla 2x100 mnm(5 pm) Column temperature: 40C Mobile phase components: Component A: 2mM ammonium acetate Component B: methanol Flow rate: 300 pL/min Injectionvolume: 1-30 pL Solvent Gradient: 16.0 minutes Time (minutes) o.o 10.0 11.0 11.5 12.5 13.0 16.0 %B 40% 90% 90% loo% 100% 40% 40% Mass Spectrometer: MicromassAPI/Mass SpectrometerQuattro Ultima Tdple Quadrupole system Software: Mass Lynx" 3.5 Cone Voltage: 20-60 V CollisionGas Energy: 20-50 eV Mode: Electrospray Negative Source BlockTemperature: 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 Analyt8 I PFOA PrimaryIon (AMU) PI 413,0 ProductIon (_Mu) I III 119, 169, 219, 369 Isomerret_tion time Branched II Linear "=' -7.9 min. -8.1 min. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 12 Page 12 Analytical Report: LIMS E02-1053 AnalyticalReport:LIMS E02-1053 Data Quality Objectives and Data Integrity The following data qualityobjectives (DQOs) were indicatedfor this study: Calibration: Calibration curves were not a componentof this study. Isomer ratios of extracted rabbit matrix standards (labeled as RBS-date of extraction-concentration) _ were evaluated to determine instrumentresponse, Limits of Peak Area Threshold: The limitof peak area thresholdwas the lowest standard pointthat had a signal to noise ratio of at least 2 times that of the baseline noise. Acceptance Criteria" The isomer ratio of branched:linearPFOA in the extracted continuingverification(QC) sample (labeled as RBS-date of extraction-QC- _ ;_ concentration)is required to meet +30% agreement versusthe extracted initialstandard at the same concentration, Confirmatory Methods" No confirmatorymethodwill be used. ; Demonstration of Specificity: Isomer identificationwillbe substantiatedby _ chromatographic retention times of the total ioncurrent (TIC). Data Summary, Analyses,and Results Data quality objectives for the analyticalphase of this study outlined above were met with the exceptionsnoted in this report. __1_ Summary of Data Results _, _,'_ Table 5. PFOA Data Summary of Pooled Serum Sample Identification Branched Unear IsomerArea IsomerArea % Branched/ Sum Branched+ Unear Pooled TCR-687-Bioreeource <361" 114603 <0,31 ..... Human Lot020821 ! Serum TC_LroetX324B 8059 41466 16 TLCoRt0-2_29K-S0i9g5m5a 8824 55100 14 TCR-690-Golden WestLotG01406042 <361 168154 <0.21 t * Areathresholdfoundas361in theextractedPFOA-Aci0d.534ng/mLstandard ! 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 13 Page 13 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 Summary of Quality Control Analyses Results Calibration: Quantitation was not a component of this study. Comparison of branched:linearratio in the extractedrabbit matrixstandard was based ..._.. on the total ioncurrent (TIC) of peak areas at retentiontimes consistentfor the target i analyte (i.e. 7.9 and 8.1 minutesfor PFOA-NH4, and 7.9 and 8.1 minutesfor PFOA- Acid). Limits of Peak Area Threshold: The peak area thresholdwas determined based on the extracted PFOA-Acid 0.534 ng/mL standard, usingthe branchedisomer TIC peak area at -2 times the baseline noise. Table 6. Limit of Peak Area Threshold in the Analyses of Sera Extracts i i ii Analyte Concentration BranchedIsomer Instrument ' ng/mL TIC PeakArea PFOA-Acid 0.534 361" QuattroUltlma ""i ; "Be_,ed onthe extractedPFOA-Add 0.534 ng/mL standtud,_ isomer area at .. _ retention'dine7.9 minutes,wlal a TIC peak area -2 _ the baseine noise. PFOA-Add - standard sp_l with PFOA-Add starda_ mix .i ! Blanks: All blanks were belowthe limitof peak area thresholdfor the compounds of interest. Acceptance Criteria: The isomer ratioof branched:linearPFOA in the extracted continuingverification (QC) sample was within +/- 10% for all QC data. Refer to Appendix C for detailed informationregardingQC data. i_l Precision: Precisionwas not a component of this study. Matrix Spikes: Matrix spike data were not a componentof this study. S! Spike Recoveries: Spike recoverieswere not determinedfor the continuing _! verifications (QCs). Surrogates: Surrogates were not a component of this study. _:!. Statement of Data Quality The ratio of isomers observed in the standards throughoutthis 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. _L',' i 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 14 Page 14 _= i_,i, ,I ::" r i _;, ',i Analytical Report: LIMS E02-1053 AnalyticaRl eport:LIMS E02-1053 Statement of Conclusion Results from this study showeda wide distributionof 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 preliminaryscreeningstudy a larger sample size would be needed to determine if a significantstatisticaldifference exists between pooled human sera samples. As shown in Table 1, two of the lots of commercial pooled sere showed the presence of branched isomers of PFOA while the other two lotsshowed a much lower percentage (by at least 40x) of PFOA branchedisomers. References 3M EnvironmentalLaboratory Study # E02-1039, November 2002 7':: "I 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 15 Page 15 _ : i :,,,i _ :,:_'i,I ',1 i',_,l I Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 AppendixA: Characterizationof the ControlMatrix Table 7. Characterization of the Control Matrix Used for Analyses in Study E02-1053 Control Matrix Rabbit Serum TN.A-4511 Source ExpiratioDnate Storage Conditionm ChemicaLl ot # Physical Descrlp#on Sigma 09/26/2005 -20C+ 10C 99H8400 Serum ,:i)'_ : :.2 3M Environmental Laboratory 3MEnvironmentalLaborator), Page 16 Page 16 Analytical Report: LIMS E02-1053 AnalyticaRl eport:LIMS E02-1053 :,_ Appendix B: Extraction and Analytical Method This appendix includes the following method: ETS-8-231.1, SolidPhase Extractionand Analysisof FluorochemicaCl ompounds fromBiologicalMatrices,(19 pages) ::J 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 17 Page 17 , Analytical Report: LIMS E02-1053 _',,'t.t.._.... .... ; ;TI;I._, _)C II:._..,I't,. "i_" ;:T.;l'r_:ll!, l_r I1 ,!,i_, ' , i'l',_r,q If): 16 -__( ..I_-... '! ! I?,,i i"" 1 :_ ! "! ""'_ 3MEnviromnental i.abocatool _fethod ' Solid Phase ExtracUon and Analysis of Fluorochemical Compounds from Biological Matrices Method Number: ETS-8.231.1 Adoption Date: II / I_]DI Revision Date: _z// _/o 2. Effective Date: ,.9-/I ,_/OZ Approved By: lillliam K. lcagen Date LaboratorMyanager ectcopyof I_ltll Diiie i i 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Mab'ices Page I of 19 Page 18 Analytical Report: LIMS E02-1053 i D ,,.:t _i_i.':It ;t_:: :". _........ i cttrrcrt;, *, "3 l_t)t ] 4 d'.i'++_, ;ri_tn I I) . #, _..JO.. + __ !,'i_!! +"'_+ :G I i. i ill!ill ;: iJ ! ,._ ; ii c.:... ':+ i :I i "+! _ -: 1 Scope and Application Tsohliisdmpehtahsoededxetrsacrcitbioesn(tShPe Eex).traTchtiiosnmoefthtaordgemt aaayaalylsteosbferoemxtefinshd,erdatot loivtehre,rrbatioselorag,icmaolmusaetrliivceers,parnodvimdoeudstheasterthaeudsiantag quality objectives are meL 2 Method Summary An amountof biological material, determined by the anaLyst, is prepared (fluids diluted and tissues homogenized) at a 1/6 dilution, or other dilution as determined by the analyst using reagent grade water. An aliquot of the dilution/homogenate is spiked with the appropriate sun'ogate 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. Tbe supernatantis transferredto a clean tube, diluted with water, and passed through a pry- cpoernfdoirtimonanedceClilqluSidPEchcraormtraidtogger.aFpihnya-lellye,cthroesapnraalyteasndoefmintmeraessst asprecetlruotmedetfrryo(mHtPhLeCS-PEES/cMarStr/MidSge).and analyzed by high o.n.,o..i 3.1 Dilution A dilution expressed as 1:5 or 1/6 is defined as: ! mL of sample + 5 mLs of diluent for a total of 6 mLs combined, unless otherwisenoted. 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 fi-om difficult biological matrices and introduced into solvents for appropriate analysis. 3.3 Reagent grade water Waterwith no detectableconcentration(so)f the targetanalyte(s). 3.4 Quality control sample calibrationcurve (as a continuingcalibration verification). Sample used to monitor the extraction efficiency (as a matrix spike) and to verify the continued accuracy of the initial 4 Warnings and Cautions 4.1 Health and Safety Warnings Always wear appropriate gloves, eyewcar, and clothing when working with solvents, samples and/or equipment. Usecautiowniththevoltagceables forthe probe.When engagedt,heprobeemploysa voLtagoefapproximate5l0y00 volts. 4.2 Cautions Take carenot to allow the SPE column to run to drynessalter the methanoland water washes. After washingis complete, add sample then allow all oftbe liquid to pass through the SPE column to dryness. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 2 of 19 Page 19 i p !:iiii "' _-_ ! ii'i:i :} _r i I i' i _:_!'!I ,_,_ '_ " i ....: Analytical Report: LIMS E02-1053 1,_t_.:, :' I1,.._']t ',.Y_|'_:}::(' tl':Jt.!. 'I . .i_ ;.:r_t., I"Ol" {,.[Lt.l:lv-, i"l_,, " :.(ll & ._T "i}2 Do not operate solvent pumps above capacity c.f400 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 of instrumentation that comes in contact with the sample or extract. 6 Instrumentation, Supplies, and Materials The following insmmaentation, supplies, and materials are used while performing this method. i_tafion, supplies, and materials may be used in place of those listed. Equivalent 6.1 Instrumentation Vortex mixer, VWR, Vortex Genie2 Ultra-TurrTa2x5 tissuheomogenizer Vacuum Pump SPE Extraction Manifold Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR Balance (+/- 0. 1000 g) Micromass, Quattro II or Uitima triple quadrupole Mass Spectrometer equipped with an eleetrospray ionization source HP 1100 or Agilent low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 6.2 Supplies and Materials Eppendorfor disposablepipettes,plasticor glass Dissecting scalpels Polypropylene bottles, capable of holding 50 rnL to 1 L Oqalgene) Volumetric flasks, glass, type A 40 mL glass vial(sICHEIVl) PlastiscampolvvialsW,beaton,6 mL (orothearppropriastieze) Centrifuge tubes, polypropylene, 15 mL and 50 mL Labels Graduated pipettes, glass Syringes, capable ofmeusuring 5 IJ-Lto 1000 IJ.L Bottle-Top Dispenser (capable of dispensing 5*hi..ofsolven0 SPE extraction cartridge, 1 g, Sep-Pak 6 ee tri-funetional C1_(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 of Fluorochemicai Compounds from Biological Matrices Page 3 of 19 Page 20 Analytical Report: LIMS E02-1053 I)u,:tim_,'lt .._+:.vr:._:.,_ctt. f cttrr_:'_: ;,:, I+Ld:l,.'_ t..,tll '.i),:l_, 2()i;7 _, : i, I r_ t ;::, i! !._j ..... _ :_; ..... !!!._o!_ ._] i ...... i: _..:.._ _ Crimpers HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7 Reagents and Standards Reagent gradewater, Milli-Q TM,Nanopure lI, or equivalent Acetonitrile, I-IPLC grade or equivalent Methanol, HPLC grade or equivalent Ammonium acetatree,agengtrade or equivalent Biological fluids or tissues, frozen from supplier 7.1 Reagents preparation 2.0 mM ammonium acetate solution: Weigh approximately 0300 g ammonium acetate. Pour into a 2000 mL volumetric container containing reagent grade water, mix until all solids am 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 standardpreparation, 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 !00 mg of target analyte into a 100 mL volumetric flask and recordthe actuawleight in the standard logbook or oflaerappropriate location. Bring to volume with methanol for a stock standard of approximately 1000 ppm (]ag/mL). Dilute the stoek solution with methanol for a working standard ! solution of approximately 50 ppm. Example calculation: I000 It,g/rnL x 5 mL/100 mL ffi50 I.tg/mL. Dilute working standard 1 with methanol to produce a working standard 2 solution of approx. 5.0 ppm. Example calculation: 50 btg/mL x l0 mL/100mL = 5.0 blg/mL. Dilute working standard 1 with methanol to produce a working standard 3 solution of approx. 0.50 ppm. Example calculation: 50/.ls/mL x i.0 mL/100 mL = 0.5/dg/mL. 7.3 Surrogate standard preparation Prepare surrogate standard(s). The following is an example only and may or may not be appropriate for all surrogate standard preparations. Weigh approximately 90-110 mg of surrogate standard into a 100-mL volumetric flask and record the actual weight. Bring to volume with methanol fora surrogate standard stock of approximately 900 - i 100 ppm. Prepare a surrogate standard working standard. Transfer approximately 1 mL of surrogate standard stock to a 10-mL volumetric flask and bring to volume with methanol for a working standard of90-110ppm. 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-I I0 mg of internal standard into a 100-mL volumetric flask and record the actual weight. 3M Environmental Laboratory ETS-8-231. I Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 4 of 19 Page 21 Analytical Report: LIMS E02-1053 I ):lC:i;:.. !li IIJr). _:,t.'tl'-:_2t.!. 't'_..L..,.t.. __.,Ji, kt_l" .IJ .t:C,,._, I', , ,tll iiV 11. _"=.,_."_ "; Bring to volume with methanol for an internal standard stock of approximately 900 - 1100 ppm. ' Prepare an internal standard working standard. Transfer approximately 1 mL of internal standard stock to a 10-mL volumetric flask and bring to volume with methanol for a working standard of90-110pprn. Record the actual volume transferred and standard concentrations in the sumdards logbook or other appropriate location. 8 Sample Handling _53 All samples are received frozen and must be kept frozen until the extraction is performed. Allow samples to thaw to room temperature prior to extraelion. Typically fi-esh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovlals until analysis. If analysis will be delayed, extracted standards and samples may be refrigerated a1 approximately 40C indefinitely or i: ! may be stored at room texture until analysis can be perfotrned. ! 9 Quality Control _" l mR i [ _ 9.1 Blanks " 9.t.t Solvent Blank An aliquot of methanol is used as a solvent blank. Solvent blanks am not extracted. !_'_i "::i:_r ,;_ _i_ t , ':) 9.12 Method Blank 9.1.3 An aliquot of 1.0 ml., of water, or other appropriate amount, is used as a method blank. Four method blanks are extracted and analyzed with eachsetfollowintghisprocedur(etwoarcspikedwithsurrogataend two are not sp_ed). Matrix Blank An aliquot of 1.0 mL or 1.0 g ofmatrix (diluted or homogenized) is used as a matrix blank. Other amounts may be used, as appropriate. Matrix blanks are prepared from one of three sources: I) a study control matrix from a study control animal received with a sample set: 2) a commercially obtained sample of the same species as the study animals; or 3) a surrogate matrix, also obtained commercially, but of a differentspecies than the study animal. (eg. if rat isusedto ganemtestandarcdurvesandCCVsfora mousestudy).The matrixtouseis dependenotn tim matrix used for the curve. 9.1.3.1 Studycontromlatricxurve - if the stud,./conmtartorliixs used forthe 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) mal_qxcurve- if the commercially obtained matrix is used for the curve, prepare four (4) matrixblanks 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 otm'nercially available matrix of the same species as the study anhnals (twospiked with surrogate and two not sp/ked). _,_ 9.1.3.4 If limited matrix is available, the number of method and matzSxblanks may be adjusted and will be noted in -- the study protocol or in the raw data. 9.2 Sample Replicate Samplesr_licates arepreparedaccordingto eachstudyprotocolor project outline. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluoroehernical Compounds from Biological Matrices Page 5 of19 Page 22 Analytical Report: LIMS E02-1053 I)t_c',l:',,.'J]t _r,:!y be' w.,',.l. ; ,:tttrcrlt. lbr 14 dav, in:t:,, 10/! _, 2!!07_ _i i 1 : '__,'_ ;,_._ i ! ii:_,_ !:i _J f'_; _'_I ,i:__iti :.:i _ i_:_! 9.3 Surrogate standard If surrogatestandard is a componentof the study, all samples are spikedwith surrogatestandardprior to extractionto obtain a concentration in the mid-rangeof the calibrationcurve, with the exceplionof blank samples as described above. Typicallysurrogatsetandardisspikedintothe 1.0mLdiluted/homogeniszaemd pleremovedforextractionH. owever, surrogatme aybespikeddirectlyintothematrixpriorto dilutingwith water,intothediluted/homogenizseadmple priorto removingthe 1.0mL sample,or intothe 1.0mL diluted/homogenizesdampleremovedfor extraction. 9.4 Internal standard if internasl tandarids a componeontf thestudya, ll sampleasrespikedwithinternasl tandaradfterextractiotnoobtain a concentrationinthe mid-rangeof thecalibrationcurve. Typicallyinternalstandardis spikedintothe 2.0 mL of extractinthe 15 mLcentrifugerule, before_ansfetringto the autovial. 9.5 Lab Control Sample Labcontrosl amplesamnotacomponenotf thismethod. 9.6 Quality Control (QC) Sample Prepare quality control (QC) samplesto monitorextraction efficiencyand to verifythe continuedaccuracyof the initialcal_ration curve. Typically 1.0mL,or otherappropriateamount,of the samematrix used to preparethe initial calibrationcurve is used foreach QC sample. Twelve(12) qualitycontrolsamples(QC) will be preparedforeach matrixduringthe courseof a study. A minimum of 3 QC samplesmust be prepared(one at each level)on each day of sampleextraction.(e.g. lfthe study is such that sampleswill be extracted on three differentdaysthen four QC samplesmustbe preparedoneach day of extraction for atotal of twelve.) QC sampleswill consist of four samplesat each of three levels ofanalyte. The levels listedbelow may be used and may representsample concentrationsdiluted intothe rangeof the calibrationcurve: Low level: 3X to 5X the LLOQ, Mid-level:equivalentto a point nearthe middle of the calibrationcurve, Highlevel: 80%of the ULOQ Two QC sample levels are analyzedafterevery tenthsamplo injection startinga/_erthe last calibrationstandard injection, with a minimum of three QC per analysis. Solvent blanks are not considered samples but nrmy be included as suchfordeterminingwhenQC sampleswill be analyzed. QC samples extracted with a particularsample set mustbe analyzedin the same analyticalrun. Any QC samples extractdeudrintghecoursoefthestudmyaybe-ncludiendsubsequeanntalyses. If samples from multipleextractiondates are analyzedin one analyticalrun,then QC samplesfrom the same sample extractiondates mustbeincludedin thatanalysis. Each QC is expectedto show an accuracyof 75-125%of expected. A minimumof 2/3 of all QC samplesmustmeet this criteria,and a minimumof I/2 of the QC samplesat each level mustmeet this criteria. If not, the set musteither be re-analyzed or re-extracted. 9.7 Sample Dilution Any samplewithan areagreatetrhanthatof thehighestacceptabsletandarwd ill needto bedilutedintotherangeof the calibration curve. If samples are diluted into the range of the curve during analyses and enough sample remains, a post-rundilutionvalidationwill be performedto verifysamplevalues. To performthe dilution validation,one sample will be separated into two representativesamples(i.e. two 1.0 mL aliquots for fluid samplesor two 1.0gram amountsfortissue samples,orother amount as determinedby the analyst 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 6 of 19 Page 23 ,,a ;] i._:_ i"":"i _,:_ i ...._. -:' .< !i__: <j i,_71 ')A,:;_' ,_L,J Analytical Report: LIMS E02-1053 l)(:cli,)lcnt ir_<::lc ,_:,*i. i c_lircr,.l, f'or 14 tl;l'. I'r(.,1 l()ib2C)(12 and documented in a note to file) then diluted using two procedures. The first procedure consists of diluting the sample with additional matrix prior to exUaction (fluid adding fluid), while the second procedure consists of 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 of_e sample concentration. 10 Calibration and Standardization 10.1 Instrument Calibration One calibrationcurve willbe preparedfrom extractedmatrix standardsi,n the samematrix asthe samples,per study. It will consistof a minimum of nine (9) levels. Additional cah'brationcurves may he extractedon separatesample extraction dates, as determined by the analyst and docunmated in a note to file. Transfer 1.0 mL, or other appropriate amount, of diluted control fluid or homogenized control tissue to a 15 mL centrifuge tube using a disposable plastic pipette. This will be repeated while preparing aliquots for the standard curve. Be sure to mix or shakz the control matrix container between aliquots to ensure a homogenous sample is removed. Record each standardvolume on the weight/volumessheet or extraction workshoet, as appropriate. Four 1.0 mL aliquots, or other appropriate amount, ofcontroi matrix serve as matrix blanks. The standard concentrations and spiking amounts listed in Table I may be used, when appropriate, to spike one standard curve. A total of 9 standards, four matrixblanks, and four method blanks are prepared in addition to the QC samples and test samples. The number of standards and blanks may he adjusted as determined by the analyst and documented in a note to file. Us_ Attachment C, or other appropriate form. as an aid in calculating the concentrations of 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. pHrioowr etvoerre, mitomvianyg btheesp1i.k0emd Ldisraemctplyle,inotroitnhtoe mthaetr1i.x0 pmrLiorditloutdeidl/uhtoinmgowgeinthizwedatesra,minptloe trheme doivleudtedf/ohroemxtorgacetnioizne.d sample Analyze the extracted matrix standard eta're prior to each set of extracts. The curve equation will be determined by regression analysis using the peak areasof the target analyte(s) using MassLynx or other suitable soRware. Any level outside 75% - 125% of nominal mus_be deactivateda,ndregressiorne-e,alculated, except the LLOQ which must be wilhin 30./0ofnominal. All levels must show a response grealer than twice that of 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 of Fluorochemical Compounds from Biological Matrices Page 7 of 19 Page 24 Analytical Report: LIMS E02-1053 1)( "_"..."..'._. ..",It I)hlk ,',"<. _: ::':!. 'l',:lllTk_ll*,., I'f,r ',,-I (.t:l_.',_ ';;_;I_.' !l_.l(,_ ",,,t)_-3 :'-; ' .,i_ ....i. :_,,I _ I '. ...i.. __ I :-i:Ii '' !i:.__!_i!_ i_!:)_ , k;__.3 Working standard (approximate concentration) 0.500 u_.mL . 0.500 uf/ml., , 0.500 u[/mL 0.500 us/mL ' 0.500 uf/mL 5.00 uf/mL 5.00 u[/ml., 5.00 u[/mL 5.00ug/mL 5.00 u_/mL 5.00 ug/mL 50.0u_/mL 50.0 u[/mL Surrogate Std I00 u[/mL 11 Procedures 1.5 3.0 8.0 16 32 5.6 .. g.0 I6 24 32 40 5.0 6.0 10 , Approximatefinal In Matrix diluted 1:5 Blank 5.00 ng/g or ng/mL 10.0 ng/g or nB/mL 25.0 ng/g or ng/mL 50.0 ng/g orng/mL 100 n_/_ or n_/mL 175 ng/gor ng/mL 250 ng/gor ng/mL 500 n_/g or ng/mL 750n_/8orn_/mL 1000 ng/g or n_/mL 1250 ng/g or ng/mL 1500ng/gorng/rnL,., 1750 ng/g or ng/mL 6500 ng/g orng/mL conAcpenptrroaxtiiomnaotfe fainnaallyte in Final 2.0 mL volume Blank 0.375 ng/mL 0.750 n_/mL 2.00 ng/mL 4.00 ng/mL g.00 ng/mL 14.0 ng/mL 20.0 ng/mL 40.0 n[/mL 60.0n_/mL 80.0 nB/mL 100 n_/mL 125ng/mL 150 ng/mL 500 ng/mL , ,m 11.1 Tissue Sample Preparation Obtain frozen tissuesamples Cut approximately1.00(30g of tissue(+/-0.10130g), or other appropriateamount,using a dissectingscalpel. This part of the procedureis best performedquickly,not allowingthe tissueto thaw. Weigh the tissuedirectlyintoa taredplasticsampulevial. Record the weight on the weight/volumesheet,extractionworksheet,or other appropriatelocation. Returunnused'isstuoethefreezaefrterxtractaimoonunthsavebeenremoved. Add 2.5 mLof reagentwaterto sampulevial,or othervolumeas determinedby the analystanddocumentedin a note to file. Hunotmiltohgeesnaimzepthleeisahmomploeg.ePnuetotuhse.Ullra-Turraxgrinderprobein thesampleand grindfor approximately2 minutes,or Rinsethe probeintothe tubecontainingthe samplewith 2.5mLofreageotgradewater,or othervolumeas determined by the analystand documentedin a noteto file,usingapipette. Takethegrinderapartand cleanit withmethanolaRereach sample.Referto ETS-9-52formoreinformation. If an amount otherthan 1.000(3g (notwithin+/- 0.1000 g) is removedforan initialweight,adjustthe watervolume accordinglyto maintaina 1/6dilution.(e.g. if0.5 g is removedforextractiona, dda totalof 2.5 mL ofwater.),orother ratioas determinedby the analystanddocumentedin anote tofile. 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 8 of 19 Page 25 Analytical Report: LIMS E02-1053 [_,_,'.l.l.m. ..'nt :,:.: )_-. u':.'d.. :I ,:_urcnt, for I,I d:,,", l"r,)t_) I() I_, = ,".()z" ii 'i : L "i ! jI _i1 _) i:_!_i..,, '_,_ !_'_ .... : ..... L.'I ,_,'.l 11.2 Fluid Sample Preparation Obtain frozenfluid sampleandallow itto thawat roomtemperatureor inlukewarmwater. Label a 15 mL polypmpylenecentrifugetube withthe studynumber,sampleID, extractiondam and analystinitials. See attachedworksheet(AttachmentA or similarworksheet)fordocumentingthe remainingsteps. Vortexmix the fluid sample forapproximately15 seconds,thentransfer1.0mL of Huid,or otherappropriateamount to a plastic sampulevial, orotherappropriatecontainer. Returnunusedsamplesto freezerafterextractionamountshavebeen removed Add 5.0 mL of reagent waterto the 1.0mL of fluid for a I/6 dilution,or otherdilutionas determinedby the analyst anddocumentedin a note to file. If a volume other than !.0 mL is removedforan initial volume,adjustthewater volume accordinglyto maintainthe samedilutionas above. 11.3 Tissue and Fluid Sample Extraction ARertissueor fluid samples havebeenpreparedaccordingto sectionsI1.1and l 1.2,vortexmix or shake by hand the diluted/homogenizedsample for approximately15 seconds then transfer1.0mL, or other appropriatevolume,to a clean 15 mLpolypropylenecentrifugetube. Returnunused diluted/homogenizedportionsto the freezerafterextractionamountshavebeen removed. Recordthe volume removedon the extractionworksheet,(AttachmentA or similarworksheet). Spikeblanks, samples,andstandardsr, eadyforextractionwith surrogatestandardas describedin this method. Spike each cal_ration standardmatrixwith the appropriateamount of standardas describedin this method for the calibrationcurvestandardsandeachQC sample. Vortexmixthe standardcurvesamplesandQC samplesforapproximately5 seconds. To each sampleand standard,add 5.0 mLof acetonitrile,cap, and vortexmix or shake by hand approximately 15 seconds. Placeall sampleson ll_ shakerat an appropriatespeed for20 mimacsto adequatelymix (a settingof approximately 300 rpmonthe modelslistedin section6.1). Removefromthe shakerand centrifugeat an appropriatespeed for I0 minutesto adequatelypelletthe precipitate(a settingof approximately2000 rpmon the modelslistedin section6.1). Add 40.0 mL of reagent gradewaterto a clean 50 mL centrifugetube. Removesamplesfrom the centrifugeand decant the supernatantinto the waterin the 50 ml. tube,takingcarenot to introduceany of the nmtrixsolidsinto the solution. Cap and mix by invertingseveraltimes. In this step the orderof additionmay be changed (i.e. the sample maybe putinto timcentrifugetubeandthen thewateradded). Attachthe reservoirto theSPEcartridgeandattachthisreservoir/cartridguenittoa vacuummanifold. NOTE:Whenrunningthe vacuum,set the vacuumchamberatapproximately15 kPA- to givean approximatelution flow of 5-7 mL/min. Flowsmayvarythroughcartridgesand the Id)Amay be raisedfor slow tubes and dryingafter mosthave beendrawndown. Preparethe SPEcartridgeby washingtwice with approximatel5y.0 mLof methanol,followedby approximateltywo 5.0 mLaliquotsof water,taking care notto allow the columnto run to drynessaRer each wash. After washingis complete,pour the sampleinto the reservoir/cartridgeunitand allow all of the liquid to pass through the columnto dryness. Runthe vacuumon high forapproximately5 minutesto adequatelydryeach SPE cartridge. Placea collection 15 mLpolypropylenceentrifugetube undereachcartridgeandelutewith2.0 mL of methanoL Spikeextractedblanks,samples,and standardswith internalstandardas describedin this method. 3M Environmental Laboratory ETS-8-231.1 Solid PhaseExtractionand Analysis of Fluorocbemical Compounds fromBiological Matrices Page 9 of 19 Page 26 I ...i. i,, i !_:,,i i)i: , :_._ _.... Ji : _-?ii *"'" _,,'::_ !!_) _'_ '': i ii Analytical Report: LIMS E02-1053 Dolj::' .":C _rl;t',:t:.c w.,:d, 'f' ,: :rrcnl, _'.._ !.I thlv', f:,_l_, !01 i:. 2(102 Label each glass autovial, as appropriate, with the study number, vial file archive number, animal number/gender/timepoint or LIMS number, nkatrix, final solvent, analyte components (if needed), extraction type, extraction date, and analyst(s) performing the extraction. Transfer each eluant to a glass autovial and cap. 11.4 Extract Analysis 11.4.I Software set-up On the MassLynx main page, set up a sample list name. Save the list as instrument designator letter, last 2 digits of test year-month-day, and a letterthat will increase through the alphabet with each additional list for that day. Example Sample List: IYYMMDDa or A020204a I = Initialoftheinstmnentname(A-- "Amelia") YY = Test year (02) MM = Test month (02) DD = Test day (04) a = First sample list (run) of the day (the next sample listwill end with _o',the next 'c', andsoor0 Assign a filename using the instrument designator letter, the last 2 digits of the test year-month-day, and a 3-digit sequential file number that starts with I and ineteasea by one for each filename. Example filename: IYYMMD ,,Dft_.,o.r A020204001 I = Initial of instrument name Y'Y = Test year MM -- Test month DD = Test day ### = 3-digit sequential file number starting with 1 through 999 (001) Also, as part of 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 mm.gsto 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 informationon MRM. Typically the analytical batch run sequence begins with system suitability, solvent blanks, and a set of extracted matrix standards. Sample extracts are analyzed with two QC samples injected aRer every tenth sample injection. Solvent blanks should be analyzed periodically to monitor possible analym 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 of Fhorochemieal Compounds from Biological Matrices Page 10 of 19 Page 27 :_ ' "_) _ ' ! .... __ i.i_,_ zJ ii_ii' ": _ ! -_ i Analytical Report: LIMS E02-1053 _]oCl.llllc_-,, r-,t.? _,c tl:.,cf.[. 'l',.-i.w,._;i,i.. i_l I,l ifl.:,..,_ li,Jlll _l'l_ll_ 2::,1_ 11.43. 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 appmpdat location: Sample size = 10 lttI,injection Injeeffsample = 1 Cycle time = 10.0 minutes Flow rate = 300 _Jmin Mobile phase: Solvent A = 2 mM Ammonium Acetate, Solvent B = Methanol Solvent gradient program: 0.00 1.00 5.50 7.50 8.O0 10% ICY'/, 95% 95% 10% 11.4.3 Instrument set-up Refer to ETS-9-24, "Operation and Maintenance oftbe Mieromass Qunttro II Triple Quadmpole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," for details. Cheek the solvent level in HPLC reservoirs and refill ifneeessary. Cheek the stainless steel capillaryat the end oftbe 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. TOtpaenn othnethtuenneitpraoggee.n.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 !0--500uUmin or as appropriate Set HPLC pump to "On" Observe droplets or mist coming out of the tip &the probo. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed Allow to equilibrate for approximately i0 minutes. Typical instrument parameters include: .,50-4o0 ES nebul/zing gas 10-15 liters/hour 3M Environmental Laboratory ETS-8-231. I Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 11 of 19 Page 28 !. Ict.li-_,..'i;[ Jli;l_," I'.'(."tl_t_(!. !',",i'r:;L. Analytical Report: LIMS E02-1053 !_,," i._ tl;.Iv_ j_CH, ll _!')tl/" _,(),__ i ?_ i i_:' i..i .._., _i ! .. ::_ !:<: i_i HPLC constant flow mode, flow rate 10-500 pL/min Pressure <40(:1bat (this parameter is not set, it is a guide to ensure the HPLC is operating correctly.) Source block temperature approximately 150"C Desolvation temperature approximately 2500C 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 tapedinto the instrumentlog. Click on start button in the Aequisilion Control Panel (the location of the start button may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). 12 Data Analysis and Calculations 12.1 Calculations If other calculations are used than those listed, they will be documented in the raw data. Calculate the matrix amount contained in the initial dilution using the following equation: IW (g) (or IV (mL)) Matrix Amount (g/mL or mL/mL) = (lW(g) (or IV(mL))+ DV (mL) Calculate actual concentrations ofanalyte in calibration standardsusing the following equation: Spike Concentration (ug/mL) Spiked Amount (mL) x 1000 ng Concentration (ng/g or ng/mL) = SV (mL)x Matrix Amount (g/mL or mL/mL) Iug .._..i.ll i i ..... i '!'_,_::I IWV ==IInniittiiaall vwoleuigmhet (where 1.0 g = 1.0 mL ) DV = Diluent voha'ne (reagent grade water) SV = Sample volume removed for exmaction (typically 1.0 mL) AR = Analytical result from MassLynx surranaty DF = Dilution factor FV = Final volume MA = Matrix amount _ curve = MA of tissue/fluid standard curve, assumed to be I g or I mL/5 mL water _)sample = MA of tissue/fluid sample ( g or mL ofsampleY5 mL water) Calculate spike percent recoveries using the following equation: %Recovery Observed Result - Matrix Blank Result = x 100 Spiking Level 3M Environmental Laboratory ETS-g-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 12 of 19 Page 29 Analytical Report: LIMS E02-1053 l)_,,::,+t:.'aatm;.ly b..' t,'..'d, t ,:urrcrlt, ['_._1t 4 day!, I'r,_z_ilJ. Jb.:2()()2 Calculate relative standard deviation using the following equation: Relative Standard Deviation Standard Deviation = Mean x 100 i Calculate percent deviation using the following equation: '_ % Deviation = Expected Cone. - Calculated Conc. x 100 Expected Cone. ,._:_ [+i ,11.q + .... Calculate actual concentration ofanalyte in fluid 0tg/rnL): AR(ng/mL)xDFx_curve(mIJmL'lxFV(mL_inCurve x 1.0u__ = _g/g) _ sample (ml_/mL) FV (mL) in Matrix 1000 ng Calculate actual eoncenwation of analyte in tissue (llg/g): AR (ng/g) x DF x _ x FV (mL_in Curve x 1.0 ug a sample (.g/mL) FV (mL) inMatrix 1000 ng ffi (pg/g) 'i 13 Method Performance i _,_:+ ..... i"i_I ( :"+_ 13.1 System Suitability System suitability will be determined prior to the start and at the completion of each analytical run. Prior to the calibration curve and after the last sample of the run three (3) mid-level 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 of the run separately. The peak area precision must be equal to or less than 5.0*6 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 AT is the asymmetry factor. If any item of the system suitability fails, system maintenance must be completed prior to running a second set of system suitability samples and the system suitability must pass before stat_g the calibration. If system suitability fails at the completion of a run, the sample set must be reanalyzed. 13.2 Quantitation The coefficient of determination value for the calibration curve, plotted by regression using the peak areas of the analyte(s), must be 0.990 or better. All active calibration curve points must be within 25% of the theoretical value with the exception of the LOQ point, which may deviate up to 30%. Calibration standards with peak areas less than two times the curve matrix blank will be deactivated to disqualify a , data range that may be affected by background levels ofthe analyte. +..i A valid calibration curve must contain at least 6 active points above and including the LOQ. lftbe curve cannot meet these criteria, the sample set must be reanalyzed or reextracted. ::_'i_ 13.3 Accuracy Two thirds of all quality control samples and Ii2 of 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 of Fluorochemical Compounds from Biological Matrices Page 13 of 19 Page 30 Analytical Report: LIMS E02-1053 I),, _lln,:'nt _::: _:c tl_.,;('. _, ;.;trcnl, fi.:,r_.I d:lv,, fn,;_l i1!:1{, 2(R)2 i,;-i: .... i :_(i:! ' "" _r i::_'i ;..._ if 7i i .i ij _._ ':ii ::.-i _. ,'"_ Surrogates and internal standards must have a percent deviation < 50%. De,,iations outside this range will be reanalyzed to confirm. If the second analysis confirms the original, the deviation will be documented in the raw data. Iftbe second analysis is within 50%, then the second value will replace the original value. 14 Pollution Prevention and Waste Management Sample waste is disposed of in noninfectious biohazard waste containers. Flammable solvent waste is disposed of'in high BTU containers. Glass pipette waste is disposed ofin 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 Each page generated for a study must contain ",hefollowing information (if applicable): study/project or insman_nt 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 MassLyax to include with the study raw dam. Copy these pages and tape into the instrument runlog. Plot the cah'bration era-re 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 s'tom with the study raw dam. Summarize data using suitably sothgare (Excel 7.0 or LIMS) and store in the study folder. Back up electronic data to appropriate meditar_ Record in study notebook the file name and location of backup electronic data. 16 Attachments Atmc_tB: Sampte Weight/Volunm Work.sheet Attachment C, Calibration StandardConcentration Worksheet Attachment D, Dilutions Summary Worksheet 17 References ETS-9-24, "Operation and Maintenance of the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source" ETS-9-52, "Operation and Maintenance of a Tissue Grinder" 18 Affected Documents None 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 14 of 19 Page 31 I)ou,tlrlcnt Analytical Report: LIMS E02-1053 s_i3; _:4 ::_.,,t f ,'.'arrcrll. for !4 da_, _: ;'r_,t_-, it).1,.., 2 t _1" : 19 Revisions i, Revision Revision ....'i Number R,cvi_ignDescriotion Date : 1 Minorformattingchanges.Addeddetailed informationto all sections concerningthe 02/18/02 extraction procedure,analyticalprocedure,andcalculations. Added attachmentsand ,_=!2 references. !,"?:i !_/'i iI,_ 3M Environmental Laboratory ETS-8-231.1 Solid Phase Extractionand Analysisof Fluoroehemical CompoundsfromBiological Matrices Page 15 of 19 Page 32 Analytical Report: LIMS E02-1053 I),.WI.U-r_C:_r ,. ,. I;.-....t..l._.Ct!. I ,.'_I1",'t'_! i',/,I },-I L]:l'. ._- t"F,_,' !{) :,_'.,.. "{s}_/'3}_ ...... : Study Number: Prep Date: Analystsinitials: i:' 'i Box#: Attachment A - Extraction Worksheet Method Revision:ETS-8-231.1 Matrix: Sample Timepoint: Sample Number Volume of Amount and :_ or diluted sample Amount and surrogatespike Type of column Elutlonsolvent Sample Number description removed spike mix used mix used used and lot and volume Comments ::ii.i, ., !:.j _-,_:,,_ '_"i,i::_i_ _. i._ ...._ ._i_,_,,_ - -_ Blank matrix _ TN-A- ; Amountweighed/allquoted: 1. Homogenlze sampie 2. Aliquot 1 mL of diluted matrix into 15 mL polypropylenetube 3. Spike sarnptesaccorcllngly g/mL 45.. AShdadke sammpLleofof Ar 2C0Nr(aTinN-QA-_ 6. Centrifuge sample for 10 rain @ rpm )(Stohaekaecrh d,.iluted sample an) d shake or vortex mix rpm (Centrifuge .) 7. Add 40 mL of water to 50 mL polypropelanecentrifugetuba. 8. Decant extract Into centrifugetubes with water 9. Shake sample slightlyto ensure propermixing 10. Attach 6 mL C18 SPE cartridges and 75 mL reservoirsto vacuum manifold 11. Conditioncolumn with two washes of ~5 mL MeOH (TN-A- ) - do not allow columnto go to dryness 12, Wash columnwith two washes of -5 mL water - do not allowcolumnto go to dryness 13, Filter sample through conditioned column,discardingfiltrate 14, Allow column to go to dryness. After ddpplngstops,drawa high vacuumthrough columnfor at least 5 minutes. 15. Elute column with solvent( TN-A- ) intoappropriate 15 mL centrifugetube 16, Spike samples with uL of internal standard# . conc.___ 17. Transfer sample into appropriatelylabeled autovlaland cap Note: In vacuumsteps above set the vacuum chamber at approximately15 kPA - this should give approximately5-7 mL/min elutionflow Flowsmay varythrough cartridges - kPA may be raised for slowtubes and dryingafter most have been drawn down and shut off. 3M Environmental Laboratory ETS-8-231.I Solid PhaseExtraction andAnalysisof Fluorochemical Compounds from Biological Matrices Page 16 of 19 Page 33 Analytical Report: LIMS E02-1053 | ICtt:I.II'I'_Q_.l.l.. ," !.,_.' ).l<t'tt ,J' ,7!ll'r',.',)l, !i?,r I:-} (i:t'.._.. fl'_:;ll _'.l, It"_ _!(i()2 -._ Attachment B - Sample Weight/Volume Worksheet Prep Date(s): "I Analyst(s): :' Sample Matrix: Method/Revision: Study Number: Equipment Number: Final Solvent & TN Number: Sample ID Initial Wt./VoL K/mL/L Water Volume added (mL) Volume Removed (mL) Comments ,, ) ,':J ,. I ........... /!'/ ! I :i ..... Form Completion Verified By:.. ,! .... 3M Environmental Laboratory ETS-8-231.1 ', Solid Phase Extraction and Analysis of Fluorochemical Compounds from Biological Matrices Page 17 of 19 Page 34 Analytical Report: LIMS E02-1053 !)._cl,.1_i,.,,_-.:!v bc.'u_:c!,if,'_;_,...,: " ! I_..,i.1,."4,.,fr()tll ' _: ]:_ L,!()2 i,-,_ ,i ....._. _I _l L _ _ : ;:_ :i r _!!/ ._._:! J _ / ii.,_ _:<_ Prep date(s): Analyte(s): Sample matrix: Method/revision: AttachmentC: CalibrationStandardConcentrationWorksheet Standard number: Equipment number: Final solvent and TN: Blank Tissue or Fluid/identifier: Analyte Analyte Analyte mix std approx. mix std approx. mix std approx. 0.500 ug/mL: 5.00 ug/mL: 50.0 ug/mL: Surrogate std approx. I00 ug/mL: Actual concentrations ofstandards in the analyte mix Analyte Std conc ug/mL 0.5_ 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 Ain'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: rnL 2._ 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 Dilution mL/mL 0.i_7 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 _mL 0.1_0 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 of standards in relation to the final 2.0 mL solvent and initial matrix 2.0 mL Final Volume Fluid Matrix Tissue Matrix Analyte Final conc. ngmL 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 1O0 Surrogate Final conc ng/rnL 0.500 Analyte Final cone. ng/mL 5.00 10.0 25,0 50.0 ,100 175 250 500 750 I000 1250 1500 1750 Surrogate Std cone ng/mL 1O0 Surrogate, Final cone ng/mL 6500 Analyte Final conc. n_/8 5.00 10.0 25.0 50.0 100 175 250 500 750 I0OO 1250 1500 ,750 Surrogate Std conc ng/mL 1O0 Surrogate Final conc . ng/g 650,0 ) ETS-8-231.1 Solid Phase Extraction and Analysis of Fluoroehemical Compounds from Biological Matrices Page 18 of 19 3M Environmental Laboratory Page 35 Analytical Report: LIMS E02-1053 I)_,Cl;I_Y'"! 'r_;:}" bC U.....,:.-'.,.."'":3,!':i::n'loI,_r_ ': _"".l"r_,,l_i''Ir_2tlt._2 I Attachment D: Dilutions SummaryWorksheet Study: :_, Dilution Date/Analyst: '' "1,' Box Number: SolventfrN Number: Extraction Date/Analyst: Matrix/Timepoint: ....: Sample Number Dilutions , or Description 1/ 1/ 1/ 1/ 1/ 1/ 1/ Comments Verified By: ., u ,, d L'" Notes: 1/10 dilution -- of sample+ of solvent 3M Environmental Laboratory FormCompltiouVerified By: ETS-8-231.1 Solid Phase Extraction and Analysis of Fluorochemieal Compounds from Biological Matrices Page 19 of 19 Page 36 Analytical Report: LIMS E02-1053 Analytical Report: LIMS E02-1053 , AppendixC: QC Data SummaryTables '! :, Table 8. Acceptance Criteria Summary of PFOA-NH4 OC sam des Analyzed 11/01/02 ! ...i. PFOA-NH4 Sample Identification % Branched/ % Difference :, Sum Branched + Unear RBS101702.10 r_/mL-1 RBS101702-QC-10 n_mL-l-1 RBS101702-QC-10 n_)/mL-1-2 26.02 25.35 24.93 NA i 3 4 % Difference: The difference 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). !,,! !_-_ i'i_:_I_,_1 Table 9. Acceptance Criteria Summary of PFOA-Acid QC sam =les Analyzed 11/01/02 PFOA-Acid Sample Identification % Branched/ SumBranched+ Unear % Difference RBS101702-I0 ng/mL-1 2.22 NA RBS101702-QC-10 ng/mL-l-1 2.24 -1 R,. BS101702-QC-10 r_mL-1-2 2.04 8 %isoDmieffmerefonrceP:FTOhAe diniftfheereenxcterabcetetwdesetannthdeartdo(tlaalbieolnecduRrrBeSn-tdpeetaekoaf reexatrraacttiioosno-cf othnecebnratrnactihoend):alitntehaer same level as the extracted QC (labeled RBS-date of extraction-QC-concentmtlon). 31t/I Environmental Laboratory 3M Environmental Laboratory Page 37 Page 37 Appendix D: Data Spreadsheets Analytical Report: LIMS E02-1053 AnalyticaRl eport:LIMS E02-1053 i ;i 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 38 Page 38 E02-1053 Analytical Report: LIMS E02-1053 ANALYSIS 11/01/02 Branched Peak Branched Peak Linear Peak Filename Branched Ret Time 7.9/ Ret Time 7.6 Ret Time 7.9 Ret Time 8.1 D021101a Sum of Branched:Linear I i! Branched Branched Linear Identification PFOA-NH4 PFOA-NH4 PFOA-NH4 Area** Area Area * 100 Branched/l_ear !!ii!! RBS I01702-10 ng/mL-I 0 93029 264494 D021101003 26.02 RBS101702-QC-10 ng/mL-l- 1 0 81108 238850 13021101027 25.35 RBS 102802-QC-10 ng/mL-l-1 0 65454 195485 D021101029 25.08 ill!'I_ RBS 101702-QC-! 0 ng/mL- 1-2 0 59384 178832 DIY21101055 24.93 ii,_ RBS 102802-QC-10 ns/mL- 1-2 0 56858 168536 13021101057 25.23 WB 102802-H20 BIk-1 0 355 2873 13021101043 11.00 .... RBS 102802-Sara Blank- 1" 0 0 3846 D021101045 0.00 0.5 ns/mL-1 0 1566 6752 D021101063 18.83 Pooled TCR-687-Bio_* 0 361 114603 D021101007 0.314 TCR-688-Larapire 0 8059 41466 D021101009 16.27 TCR-689-Sigma 0 TCR-690-Golde.n West* 0 Below LimitofPeakAreaThreshold 8824 55100 D021101011 361 168154 D021101013 13.80 0.214 _('_ ** 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 Sara Ratios Page 39 E02-1053 Analytical Report: LIMS E02-1053 ?,_7)7 '-'-1 i i_! i ;7,_,i_i, _1'_::'i ! : -._ ._/ALYSIS PFOA-Add 11/01/02 Pooled data were not Included in the scope of the study and were not reported TIC Branched Peak Linear Peak Filename Branched Ret Tune 7.9/ RetTlme7.9 RetTime8.1 D021101a2 SumofBranehed:Linear Branched Linear * 100 Identification PFOA-Add PFOA-Add Area Area Branched/Linear Standards RBSI01702-10 ng/mL-2 RBS 101702-QC- 10 ng/mL,-2-1 RBSI02802-QC-10 ng/nff.,-2-1 RBS 101702-QC-I0 ng/mL-2-2 RBS 102802-QC-10 n_/mL-2-2 WB 102802-H20 Ilk-1 RBS102802-Sera Blank- 1 0.5 n[/ml.,-2 Pooled TCR-687-Bioresource* TCR-688-Lampire TCR-689-Sigma TCR-690-Golden West* Below Limit of Peak Area Threshold 7971 5022 2733 4416 1909 260 545 361 361 8207 8823 361 351306 219508 225367 211715 198898 2873 3787 6473 Dff21101005 D0211010311 D021101033 D021 I01058 I)021101061 DIY21101043 13021101045 D021101065 113845 41469 55095 168119 I)021101007 13021101009 I)021101011 D021101013 2.22 2.24 1.20 2.04 0.951 8.30 12.58 5.28 0.316 16.52 13.80 0,214 ETS-8-231.1 3M Environmental Laboratory Confirmation Sora Ratios Page 40 E02-1053 Analytical Report: LIMS E02-1053 ,? - _ii ii '_ ; i ; TIC 11/01/02 Analysis Branched Peak Branched Peak RedTime 7.6 Rot Time 7.9 Linear Peak Ret Time 8.1 Hlename Identification PFOA-NII4 PFOA.NII4 PFOA-NII4 RetTime7.9/ Sum of Area** Area Area Branched:Linear S_ndards&CCVs * I00 _ INff RBSI01700-10 ng/mL-1 0 93029 264494 I)021101003 26.00 NA RBS 101702-QC-10 ng/mL-l-I 0 81108 238850 D001101007 2535 3 RBS I02802-QC- 10 ng/mL- I- 1" 0 65454 195485 134321101009 25.08 4 RBS101702-QC-10 ng/mL-1-2 0 59384 178832 D021101055 24.93 4 RBS 102802-QC-I 0 n_mL-l-2* 0 56858. 168536 D021101057 25.23 3 * Data not used/reported. Not extracted on the same date as the samples and therefore not included in tables 8 and 9. **Branchedpeakat7.6minutesobservedonlyinPFOA-NHs4tandardsatconcentration>s25ng/mLN. otincludedinanycalculations. i:J . ,J I ,,,!,'\- ETS-8-231.1 3M Environmental Laboratory HumanSeraQCvs StdRatios Page 41 E02-1053 Analytical Report: LIMS E02-I053 11/01/02 Analysis Identification TIC Branched Peak Ret Time 7.9 PFOA.Acid Linear Peak Ret Time 8.1 PFOA-Acid Area Area Filename Ret Time 7.9/ Sum of Branched:Linear * 100 % Diff . _,i Standards&CCVs i RBS101702-10 rig/rob-2 7971 351306 DtY21101005 2.22 NA _?! RBS101702-QC-10 ng/mL-2-1 5022 219508 D021101031 2.24 -1 RBS 102802-QC- 10 ng/mL-2- !* 2733 225367 13021101033 1.20 46 _ RBS101702-QC- 10 ng/mL.2-2 4416 211715 i)021101059 2.04 8 i .RBS 102802-QC-10 ng/mL-2-2* 1909 198898 13021101061 0.951 57 'i * Data not used/reported. Not extracted on the san_e date as the samples and therefore not included in tables 8 and 9. .,_.J ETS-8-231.1 3M Environmental Laboratory HumanSeraQCvsStdRatios Page 42 ! [ i"_ 'i .L:J iI --:_';i_! _ Analytical Report: LIMS E02-1053 AnalyticaRl eport: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= BranchedTIC PeakAr_a * 10o (Branched TICPeak Area + LinearTIC Peak Area) SampleTCR-688 PFOA Percentage:(8059/(8059+41466)*) 100 = 16% % Differenc=e (ExpecteTdIC% Branched/SuInmitiaSl td- ObserveTdIC% Branched/SuQmC_" 100 Expected TIC % Branched/Sum InitialStd Sum= Branchedpeakarea+ Linearpeakarea % Difference of PFOA RBS102202-QC-10 ng/mL-l-1 sample analyzed 10/22/02 InitialStd= RBS101702-1n0g/mL-T1IC% Branched/Su=m22 QC = RBS102202-QC-10 ng/mL-l-1 TIC % Branched/Sum = 24 % Differenc=e 1(22-24)/ 241= 9 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 43 Page 43 Analytical Report: LIMS E02-1053 AnalyticalReport:LIMS E02-1053 i Appendix F: Interim Certificate(s) of Analysis _7,!1 '.. _j L 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 44 Page 44 :i ::i :J : !i ::::i ::_ : :,, .: ::'! -_ :__ii:!] :,:J ..... '2 i ..__,_, . .J Analytical Reoort: LIMS E02-1053 '_YO'_k'_'_kPhone: Centre AnalkjUcal tab0PatoriEs, Inc. 348 staiCnoJlePgA1e0. 1 ww.c,,,t,,f,,b.com (814)231-8032 Fax:(814)231-1253or(814)231-1580 INTERIM CERTIFICATE OF ANAL YSIS Centre Analytical Laborstorlm COA Reference #: 023-033 (Revision I) 3M Product: Ammonium Perlluorooetanoate Test Control Reference #: TCR-99131-37, Lot #: 332 Purity: 95.2% II Purity j TestName Appearance Identification NMR . Speeilleaa.o_ I White,crystallinesolid Resi ult 95.2% ii Conforms Positive Meta1.ls(CICaPlc/iMumS) 2. Magnesium 3. Sodium 4. Potassium 5. Nickel 6. Iron 7. Manganese Total% Impurity(NMR) Total% Impurity (LCJMS) Total % Impurity (GCJMS) Residual Solvents CI'GA) Purityby DSC Inorganic Anions GC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate '' ...... 1. 0.001 wtJwt.% 2. <0.001wtJwt.% 3. 0.005wtJwt.% 4. <0.001wtJwt.% 5. <0.001 wt./wt.% 6. <0.001wtJwt.% 7. <0.001wtJwt.% 0.34wtJwt.% 4.49wtJwt.% None Quantified None Detected 99.7% 1. <0.015wt./wt.% 2. <0.005wtJwt.% 3. <0.040wtJwt.% 4. <0.009wt./wt.% 56. NPhitorsitpehate 7. Sulfate Organic Acidsz (IC) 21.. TPFFAPA 3. HFBA 4. NFPA ElementaAlhalysi_: 1. Carbon 2. Hydrogen 3. Nilzugen 4. Sulfur 5. Fluorine Ammonium Analysis _ IonSelective Electrode TheoreticalValue = 22.3% TheoreticalValue = 0.935% TheoreticalValue = 3.25% TheoreticaVl alue = 0% Theoretical Value = 66.1% TheoreticalValue = 4.18% 56. <0.006wtY./wt.%% 7. <0.040 wtJwt.% 21.. <<00..11wwttJJwwt.t%,% 3. <0.1 wt./wt.% 4. <0.25wt./wt.% 1. 18.9 wtJwt.% 2. 1.31 wt./wt.% 3. 3.75 wt./wt.% 4. 4.34 wtJwt.% 5. 63.2 wtYwt.% 3.49 wtJwt. % --= COA023033-37REVl.doc 3M Environmental Laboratory Page 1 of 2 Page 45 Analytical Report: LIMS E02- IQ_5__ ' "L'I_I I Akk nmnL _vO_l=.'_k_Phone: CGnt Analgtical UaboratoHGs, Inc. 3048 ResearchDrive StateCollegeP, A16801 w'_.centrelab.com (814)231-8032 Fax:(814)231-1253or(814)231-1580 INTERIM CER TIFICA TE OF ANAL YSIS Centre Analytical Laboratories COA Reference #: 023-033 (Revision 1) 3M Product: Ammonium Perfluorooctanoate Test Control Reference #: TCR-99131-37, Lot #: 332 Date of Last A_alysis: 12/15/00 Expiration Date: 12/15/06 Storage Conditions: < -I0 *C Re-assessment Date: 12/15/06 tpttrity = 100% - (Total Metal impurities. 0.006% + Total NMR impurities, 0.34% + : ,! Total LC/MS impurities, 4.49%) , ,p i Total impurity from all tests = 4.84% Purity = 100% - 4.84% = 95.2% z TFA HFBA NFPA PFPA Trifluoroacctic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid i'_,I 3Theoretical value calculations based on the empirical formula, Cff'IsO2(')NI_ (+) (MW=431.1) _i! LC/MS Purity Profile: Peak# Retention Time (rain) Mus(s) IdelltRy Ares %Area 1 12.140 269 Ce 167596 0.73 i ,! 2 13.504 331,319 C__ologue3Ft3 860991 3.76 / 3 14.099 369 FFOA 21861700 - Total - 22890287 4.49 This work was conducted under EPA Good LaboratoryPractice Standards (40 CFR 160). Charles Sira/o/ns Date Scientist, Cenlre Analytical Laboratories !!i_i Reviewed By" //_/_/_/lf_-'_ ,,/_//S/ Flaherty / Date / Laboratory Manager, Centre Analytical Laboratories COA023033-37REVI.doc 3M Environmental Laboratory Pase 2 of 2 Page 46 'i L' "_?q; I.I ,,:"i _L,_ i i i *l 3 J Anal_ical Re_ort: LIMS E02-1053 3M ENVIRONMENTAL LABORATORY Note to File i i i i |. Project or Study Number: PACT-TCR-005 i ASSOCiated Study Number:. LIMS # E00-1762 i ml | ii ill i i i J The exlm_ '_ forPFOA_ HU 144111DaUnd332CI_Ii-_ 1314)18sadT1_-99131-037)maybe eglended5 years(12/15/06)m JiabilitywasdemomtratebdystudiesE00-18510tydmlyfis),E00-2192 Recorded By: Date 3M Environmental Laboratory Page 47 Analytical Report: LIMS E02-1053 ..... ,_::,, _:i :_ 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: Chemical Characterization of PFOA, Lot 332, TCR-99131-37 IR Spectroscopy Date: December 11, 2000 by IH-NMR, I_F-NMR, and i i i ,!i :' ,i _ i: : ! ii'_?t I_,_ _':_:'_!_ _'_'_ii_l i:i!_ ..... i !_! SAMPLE DESCRIPTION: PFOA, lot 332, TCR-99131-37; Nominal product =C7F15-CO2 (') NH4 (+) (white powder). The sample was stored in a-20 C freezer at all times except when aliquots were removed for analysis. o.BJECTIVE: This sample was subjected to SH-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. EXPERIMENTAL: FT-N P, 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 IH-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 (CFaCOzD = TFAD). An additional 400 MHz IH-NMR spectrum (#h62631.402) and a 376 MHz tgF-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 NI-L(t+)resonance that had been observed in IH-NMR spectrum of the acetone-d6 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 FI'-IR spectral analysis using the KBr disk technique. A transmission FT-IR spectrum (#A62631) was then acquired using a Digilab FTS-40 FT-IR speetrophotometer. 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+l-C02 (') NI-14 (+), 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 Page 48 December 11, 2000 Analytical Report: LIMS E02-1053 3M SMMD Analytical Lab Request # 62631 3M SMMD Analytical Lab: Bldg. 236-2B-! 1 :I RESULT_ (cont.): The qualitative and quantitative compositional results that were derived from the single trial IH-NMR and 19F-NMR spectral analyses are summarized in TABLE-I. Any water that may have been present in the sample was ignored for calculation purposes. The 19F-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 'i fluorocarbon chains contained 8 carbon atoms except where noted. In general, the tgF=NMR technique is not particularly well suited for characterizing small amounts of potential fluorochemieal 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. !_i 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 e: Lisa Clemen- ET&S- 2-3E-09 TanyaRude - QAI Rick Payfer - SA&C Analytical Lab - 236-2C-11 ii File Reference:wr6263l.studyFACITCR005.doc/78 ':.j StudyProtocol #FACT-TCR-005 3M Environmental Laboratory Page2 of 3 Page 49 Analytical Report: LIMS E02-1053 December11, 2000 3M SMMDAnalyticalLab Request# 62631 3M SMMDAnalyticalLab: Bldg. 236-2B-11 TABLE-1 ., Sample: PFOA, lot 332, TCR-99131-37 ,i Overall Compositional Results by 19F-NMR Analysis & tH-NMR Internal Standardization Analysis Structural Assignments I I_F-NMR Relative Wt. % ._ Concentrations .,. . . .: Normal chain isomer -_ CF3(CF2)x_CO2 (-)NI-14(+) (wherex assumedto be 6 forcalculationpurposes) Internal monomethyl branched isomers CF3(CF2)x-CF(CF3)-(CF2)y-CO2 (-)NH4 C+) (wherex+y assumedto be4 for calculationpurpose,andx ;_0, y ;_0) Isopropyl branch isomer =77.7% =12.5% =9.0% ',',.I (CF3)2CF'(CF_-)x'CO2(') NI_') ,. (where x assumedto be 4 forcalculationpurposes) t-butyl branch isomer -_ (CF3)zC-(CF2)x-CO2 (')NH4 (+) ' (wherex assumedto be 3 forcalculationpurposes) O =0.24% =0.19% Possible FF NI_+) Internal gem-dimethyl branch isomers i.! CF3-(CF2)x-C(CF3)2-(CF2)y-CO2 (') NH4(+) !:iI (wherex+y assumedto be 3 forcalculationpurposes:x ;e0) .(3, i J.F FI o- =0.13% _0,11% _!: _ Possible F F NH4(+) > Possible alpha branch isomer (whereCxxaFs2suxm+Ie-dCtFo(bCeF53)f-oCrOca2lc{u')laNtiIo-_np(urposes) =0.085% Probable F-SF4-Rr, =0.029% possibly as F-SF4-CnF_-CO2 (')NH4(+) (wheren assumedto be 7 forcalculationplurposes) ,i Possible CF3CF2CO2 (-) NH4c+) =0.009% CF3-O-CF2-Re, where -Rf is undefined Trace ::: ::.:I_ '.. _. / ,i ' .:.:... 1H-NMR Absolute Wt.% ._..,. ..: .. .. -.. !..:-: -.ii;-.: Concentration Total unassi_,med aliphatic materials =0.002-0.003% 1) Trace amountsof otherunidentifiedcomponentsare also detectedintheNMRspectra. StudyProtocol#FACT-TCR-005 3M Environmental Laboratory Page 3 of 3 Page 50 i imj J Analytical Report: LIMS E02-1053 [[ "' :":!i '_, i '_ _ il !_-,.] ,_:_! I,_,! _.,_'_:t _ I_ Certificate of Analysis N om|laal Product: CF3(CF2),-CO2H,where average n ,_ 6 Pentadecafluorocctanoic acid Product Code: TCR-617, Lot 210002 October28,2002 Tom KestnerandJoel/Wilier The sample of TCR-617, lot 210002 was analyzed using a combination of 19F-NMR, IH-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-I. TABLE-I Sample: TCR-617, Lot 210002 Quantitative and Qualitative Compositional Results by Combined 19F/tH-NMR and LC/MS Analyses ComponentStructures _ _H/_gF-NMR RelativWeeight% Concentrations (singlteria.lanalys)is CF3(CF2)a-CO2H where average n -- 6.02 by tgF-NI_. LC,,/MSshowedn=6 (major)n,-.-5n,=4(minors). Probable(CF3)2-CF-(CF2)a-CO2H assume n_-4for calculation purposes Probable (Cc,FlsO)-CO2H aeyclic ether acid as possible CF3CF2-O-CF(CF3)-CF2CF2-CO2H Possible CF3(CF2)x-CF(CF3)-(CF2):COaH wherex_0, _ andassume x+), = 4 for calculationpurposes Possible (CF3)3-C-(CF2)_-COaH assume nffi3for calculation purposes Possible CnH2n+s2aturated aliphatic hydroc.m'bons 1. Traceamountsof oflmrunassignedcomponeatswerealsodetec_l intheNMRspectra. <99.5 I% Purity 0.39% 0.057% 0.01904 0.013% 0.0079% ,.-'_ I C.: --.'_ ...... / ' //o i Page1of ! 3M Environmental Laboratory FileReference:Cola TCR-617Lot210002.do0 Page 51 Analytical Report: LIMS E02-1053 AnalyticaRl eport:LIMS E02-1053 .i _:_ Appendix G: Report Signature Page William Reagen, Ph.D., Laboratory Management, Sponsor Representative Date i ;if!i! ii /_isa Stevenson, Principal Analytical Investigator i Date _,__ i 3M Environmental Laboratory 3M Environmental Laboratory Page 52 Page 52