Document O3OdBobMNjw1bxV437m62zGBp

| Northwest Bioanalytical ' ReSptourdty NNoo.. NNWWBBRSS988.-000852 ARR26- 1208 : Quantitative Determination of PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS in Human Serum by LC/MS/MS | Assay Validaatcion Report y Northwest Bioanalytical (NWB) ADivisionof NWTInc. 1121East 3900 South i Salt Lake City, UT 84124 | prepareDFOR: 3M ! 935 Bush Avenue St Paul, MN 55133 35 gx = = 57 oe o =~ ! AUTHOR: AR David L. Vollmer, Ph.D. Project Manager APPROVED FORRELEASEBY cott-- Rodger L. Foltz, Ph.D, Laboratory Blrector DATE: _/3/4 DATE _ S/)2 J3 PagCe ONTAIN NO CBI 000003 Northwest Bioanalytical IST OF TABLES LIST OF FIGURES... TABLE OF CONTENTS RepSoturdty NNoo.. NNWWBBRS9998..000552 messmo 3 | 21. RANGEOF QUARIANON 22 PIEGISION 80 ACKICY sesso srs smn : 23. Repeatabilaintdy ReproducibilTietsyt.urermsosssns 10 smensiss ld 2 SPE PRECISION Terns 11 2.5. 2 SUD EVAN tee Extraction EFCIENCY cooroerssrersnssseressssssesesmssnnns sss snes |1 12 #1. Proposed Sample Analysis AGSEPIANCE CHET... 15 6 ANALYTICAL METHOD. 86 6.1. Reference Materials and MARHICES ..coersssorssenersssssosses ms ------------------. 86 ! 6.2. 63. RCehaegmeinctasl,sCaalnidbrEaQtUiIoPnMSEtNatn.d.a.r.dcoarnrdsQruraelrietrysCronterosl (sQCe)sS--O--I----i--o--n--s----r--r----.----.--.--sss787 6.4. Preparationof Quality Control Samples... rss ----------------------. 92 6.5. Preparation of Calibration SHANGBIS.covrreooersersesesssesnssene ssn 92. 6.6. Sample Preparation................... --------------------------------------------.I3 6.7. LC/MS/MS Conditions................. Hr --------------) 6.8. QUANLALION ..cvoee ren --------------------------------------------------------I0 Page 000004 [I -- | LIST OF TABLES ReSptourdtyNNoo.. NNWwBBRS9998..08025 Table 1. SummaryofRabbit Serum Calibration Curves for PFOS..............ccoco... TR -- `Table 2. SummaryofRabbit Serum Calibration Curves for PFOSAA ........................ SE -- `Table 3. Summaryof Rabbit Serum Calibration Curvesfor POAA................... Ee ------ | Table 4. SummofaRarbby it Serum Calibration Curvesfor PFHS...................... wissen 2] Table 5. Summary Data for Endogenous Analyte Concentrations in Human SerumCalibration Standards for Run 25......ccvvvrervrrssne esas sess dd. Table 6. Summary Data for Endogenous Analyte Concentrations in Human Serum Calibration I Standards for Runs 26, 28 and 32...............oc... mr --------------------------------.--) Table 7. Summary Data for `Endogenous Analyte Concentrations in Human Serum Quality Control Samplesfor Runs 26, 28 and 32.........oc..eoe.. rss sans. 2d. Table 8. Summary of Human Serum Calibration Curve Parameters for PFOS.......... SE --. Table 9. Summary of Human Serum Calibration Curve Parameters for PFOSA............ ------------1 `Table 10. Summaryof Human Serum Calibration Curve Parameters for PFOSAA. a-- `Table 11. Summary of Human Serum Calibration Curve Parameters for N-MeFOSE-OH..........coocco....26. Table 12. Summaryof Human Serum Calibration Curve Parameters for N-EtFOSE-OH.............. 2? Table 13, Summaryof Human Serum Calibration Curve Parameters for POAA mess 27 ! `Table 14. Summaryof Human Serum Calibration Curve Parameters for PFHS............... S----] | Table1S. BackCalculated Concentrations of Human Serum Calibration Standards for PFOS.........20 ! Table 16. Back-Calculated Concentrationsof Human Serum Calibration Standards for PFOSA ...........30 Table 17. Back-Calculated Concentrationsof Human Serum Calibration Standards for PFOSAA........31 Table 18. Back-Calculated Concentrations of Human Serum CalibrationStandards for N-MeFOSE-OH ...ccconsmvrrrrrmn erases sr 32 Table 19. Back-Calculated Concentrationsof Human Serum Calibration Standards for N-E{FOSE-OH HE ---------------------------- 1 ssssaeasesesesseseeenee 33 pe 000005 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS9998..000552 Table 20. Back-Calculated Concentrations ofHuman Serum Calibration Standards for POAA...........34 `Table 21. Back-Calculated Concentrations ofHuman Serum Calibration Standards for PFE .........35 Table 22. Intra-Assay Precision for PFOS Human Serum Quality Control Samples ...............36 `Table 23. Intra-Assay Precision for PFOSA Human Serum Quality Control Samples... 37 Table 24. Intra-Assay Precision for PFOSAA Human Serum Quality Control Samples.................. 38 `Table 25. Intra-Assay Precision for N-MeFOSE-OH Human Serum Quality Control Samples ..........39 Table 26. Intra-Assay Precision for N-EtFOSE-OH Human Serum Quality Control Samples.............40 ] Table 27. Intra-Assay Precision for POAA Human Serum Quality Control Samples ......................41 Table 28. Intra-Assay Precision for PFHS Human Serum Quality Control Samples ................ud2 Table 29. Inter-Assay Precision for PFOS Human Serum Quality Control Samples.......................43 Table 30. Inter-Assay Precision for PFOSA Human Serum Quality Control Samples ................44 Table 31. Inter-Assay Precision for PFOSAA Human Serum Quality Control Samples ...............45 Table 32. Inter-Assay Precision for N-MeFOSE-OH Human Serum Quality Control Samples ........46 Table 33. Inter-Assay Precision for N-EFOSE-OH Human Serum Quality Control Samples.............47 Table 34. Inter-Assay Precision for POAA Human Serum Quality Control Samples.....................48 Table 35. Inter-Assay Precision for PFHS Human SerumQuality Control Samples .....o....r....49 Table 36. System Precision for PROS... 50 Table 37. System Precision for PFOSA wrest 50 ! Table 38. System Precision for PFOSAA... wrmem-------------------- Table 39. System Precisionfor N-MeFOSE-OH.......cmsnsssmnsssmssssmmssssnS] `Table 40. System Precision for N-EtFOSE-OH...cvomritsssesssssssmnn $2 Table 41. System Precision for POAA wnt 52 Table 42. SYSIEM PIECISION for PHS coven $3 Table 43. PFOS EXTACHON EMfICIoNCY crore54 -- 000006 | ---- ReSpuodryt NNoo..NNWWBBRS9S98..000552 i Table 44. PFOSAExtractionEfficiency............ ------------------h `Table45. PFOSAA EXITACUON EMCIENCY rrr 58 Table 46. N-MeFOSE-OH EXIFaCON EfCIONCY rss smmnnnanSS Table 47. N-EFOSE-OH EX{Taction EFiCiency........mmmmmsssssinnsnnne $6 Table 45.POAA EXUTRGHON FICIENCY cvs 56 `Table49. PFHS EXURCHON EFCIENCY cvvsrnssssssssssssnmnnS] Table 50. FreeSzbileit/y10T8PhOSaw ccc S8 | `Table 51. Freeze/Thaw Sability fr PROSA ors 39 Table 52. Freeze/Thaw Stability for PROSAA v.80 `Table 53. Freeze/Thaw Stability for N-MeFOSE-OH rnin61 Table 54. Freeze/Thaw Stabilityfor N-EFOSE-OH orcs 61 Table55. Freeze/Thaw Stability for POAA covers 63 ! Table 56. Freeze/Thaw Stability for PEHS ......cvoursmsssumssssessnsssesssssssssmesmssmesssssssssssssss64. ; Table 57. Room Temperature Matrix Stility for PFOS....rcmesrnssinn 65 Table 58. Room Temperature Matrix Stability for PFOSA eros 66 {Table 59 Room Temperature Matix Sahil or BEOSAA re 7 Table 60. Room Temperature Matrix Stability for N-MeFOSE-OH cvs68. Table 61. Room Temperature Matrix Stability for N-EEFOSE-OH...verrrrrssrrorrs69 I raee2 room `Temperature Matrix Stability for POAA ersten 10 `Table 63. Room Temperature Matrix Stability for FHS... 1 Table 64. Room Temperature EXract Stability f0r PROS rss 2 `Table 65. Room Temperature Extract Stability for PFOSA ..ccvccnttcsnnssnronencT3 Table66. Room Temperature Extract Sability for PROSAA cece Td Table 67. Room Temperature Extract Stability for N-MeFOSE-OH vrai TS Pages 000007 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS9998..000852 `Table 68. Room Temperature Extract Stability for N-EtFOSE-OH.....ccc.covmuvrrrrreresssossssrs, eerie 76 Table 69. Room Temperature Extract Stability for POAA cocci 77 Table 70. Room Temperature Extract Stability for PFHS ....oocevcvveerssnssssseossssssos sss S--1 Table 71. Stock Solution Stability fr PFOS .....c.occoevrcmsssnssssssssersnsss senses eseessssssererernsnsnsnseeres79 Table 72. Stock Solution Stability for PFOSA...ccvcvomersomsssssssssssesomemsomssssns wnssssssssssssenenensn 80 `Table 73. Stock Solution Stabilityfor PFOSAA ..c.vcvevsvvserersomermesssesssn resmsnssssssmmmsmennsnsenn8] `Table 74. Stock Solution Stability for N-MeFOSE-OH -------------- nn 82 | `Table 75. Stock Solution Stability for N-EFOSE-OH.....ccccerrvrrrrrrne TSS. x `Table 76. Stock Solution Stability Or POAA ..o..cvrmerressssssssrssssssmemessssesesesn -------------- | `Table 77. Stock Solution `Stability Or PFHS ......covsvevsereeeosesomessssesessesen esseressssssmsrerssssssssssise 83 LIST OF FIGURES i Figure 1. Rabbit Serum Blank................ooooooeren ssn 97 Figured, Human Serum BI ceo mons 53 I Figure 3. High $1a0dard (S00 PP) vss 99 ) | Pages 000008 Northwest Biosnaytcal ReSptourdtyNNoo.. NNWWBBRS9998..000852 Quantitative Determination of PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS in Human Serum by LC/MS/MS 1. INTRODUCTION Assay Validation Report Northwest Bioanalytical (NWB) was contracted by 3M to develop and validate a liquid chromatographytandem mass spectrometry method for the measurementof PEOS, PFOSA, PFOSAA, N-McFOSE-OH, N-EFOSE-OH, POAA and PFHS in human serum. For chemical ] names and structures, see the Analytical Method contained in section 6 of the report. : This report summarizes the analytical results from the validation ofthe method for quantitation Of PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EFOSE-OH, POAA and PFHS in human : serum for 3M. Kris Hansen, Ph.D. at 3M served as the Study Monitor. The following is a list of NW supervisory personnel involved in the completionofthis work: David L. Vollmer, Ph.D. ; (Project Manager); Brad Coopersmith, Ph.D. (Senior Scientist; Rodger L. Foltz, Ph.D., (NWB ! Laboratory Director). ! 3M SOP AMDT-S-12 (effective 12/12/95) and NWB SOPs were used in the conduct of this . project and were available to project personnel in electronic or hard copy formats. 1 Date Study Initiated: January 15,1999 Date Analyses Completed: _February 10, 1999 `The method validation study described in this report is not included within the definition ofa | `GLP regulated nonclinical study (Title 21 of the US Codeof Federal Regulations Part 58). However, Northwest Bioanalytical conducts al studies within the guidelines of GLP principles. Background Early method development experiments demonstrated varying levels of endogenous PFOS, PFOSAA, POAA, and PFHS in several lotsof "blank" human serum. These endogenous levels complicate the useofhuman serum for the preparationofcalibration standards and quality control samples. Page? 000009 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS9998..000852 To circumvent the difficultyofpreparing standards and controls in the presenceof endogenous levels, several animal matrices were evaluated. Rabbit serum showed the lowest endogenous levelsofthe aforementioned analytes when directly compared to rat, dog, and monkey serum Utilizing a calibration curve prepared in rabbit serum, however, did not demonstrate acceptable precision and accuracy for the determination of PFOSA, MeFOSE-OH, and E{FOSE-OH A serum matrix that was pre-extracied from human serum to remove any endogenous levels of analytes prior to preparationofthe curve was also evaluated. This serum matrix, however, showed greater extraction recoveries for all the analytes in the calibration curve samples than in | `tahcecheputmabalne ascecruurmacsyamfporleqsuatnhtaittwateiroenonfotthperea-neaxltyrtaecst.ed, and therefore did not demonstrate : Because the four analytes (PFOS, PFOSAA, POAA, and PFHS) that did show acceptable precision and accuracy with the rabbit serum curve were the same four analytes that showed measurable endogenous concentrations in "blank" human serum, an abbreviated rabbit curve was used to calculate the endogenous levels in specific lots ofhuman serum. Once the endogenous : levels were determined for those lots of human serum, those concentrations were added to the. `sTphiiksepdrcoocnecdeunrteraitsidoensscrfiobretdheinhduemtaainl wsietrhuimn QthCissraepnodrtf.or the human serum calibration standards. Principles of the Method , After the additionofHPLC-grade water and sodium dodecyl sulfate (SDS, internal standard) to 0.20mLofhuman or rabbit serum, the serum mixture is made basic with the addition of0.5 M | tetrabutylammonium hydrogen sulfate (TBA, pH 10) and 0.25 M carbonate buffer. This mixture is then extracted with methyl-tert-butyl ether. After sufficient mixing, the sample is centrifuged. The organic layer is transferred via pipette into a clean test tube. The organic layer is then evaporated to dryness and the sample is reconstituted into 2 mM ammonium acetate water:methanol (50:50 v/v). The extracts are then analyzed by liquid chromatographytandem mass spectrometry using negative-ion electrospray ionization and multiple reaction monitoring, Pages 000010 Northwest Bioanalytical 2. VALIDATION SUMMARY ReSptourdtyNNoo.. NNWWBBRS9998..-008025 Four separate analytical runs were used in the determinationoflinearity, precision, and accuracy of PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-E(FOSE-OH, POAA and PFESS. The validation included determination of the stability of the analytes maintained under various storage conditions, the extraction efficienciesofthe analytes and the intemal standard, the system precisionofthe analytes, and the reproducibilityofthe analytes for this assay. All values reported in the validation summary and tables are based upon the actual concentrations of solutions utilized during this validation. Please refer to the ListofTables for ] the locationof the validation test data and summary statistics. 21. Rangeof Quantitation Rabbit Serum Calibration Curve : Each analytical run included calibration standards in duplicate at a minimumoffour different | concentrations and at least two rabbit serum blanks. Data for PFOSA, N-McFOSE-OH and ! N-E(FOSE-OH are not included because there were no endogenous amounts detected in i human serum. 3 FFOS PFOSAA POAA FEES i Range (ppb) 10010300 10010500 1.000500 100%0500 , cMecan Coorrelation CoefficieB nt O0.9O 961 N 03to957 05987 00 9952A ! Human Serum Calibration Curve Each analytical run included calibration standards in duplicate at a minimumofseven different concentrations, a minimum of 10 quality control samples (QC) (two levels in replicatesoffive), two "blanks" and two "0-ppb QC" (QC containing intemal standard, but no spiked analytes), Page 000011 I Norwest Bioanalytical ReSptourdtyNNoo.. NNWWEBRS9998..000852 TT Fos OSA Thkd RCoanncgeentration Mean Corelation _Co--efficient Ww 10w S00ppb 500ppb 09934 09969 ROSA MN FOSE- W EtFOSE- Tom OH OH FES oS3m8ppbeLS00pe pb TSoO0pwpbTS0w 0ppb T50H0 pepb 09966 09971 09976 09972 09978 22. Precision and Accuracy The precision and accuracy of the LC/MS/MS method for the quantitationofPFOS, PFOSA, | PFOSAA, N-MeFOSE-OH, N-E{FOSE-OH, POAA and PFHS in human serum were determined by analyzing two levels of quality controls in replicates of five on four separate days. The intra-assay %CV for PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-E{FOSE-OH, POAA and PFHS were less than or equal to 13.0% for each QC concentration. The inter-assay %CV for PFOS, PFOSA, PFOSAA, POAA and PFHS were less than or equal i 013.3% for each QC concentration. The mean percent theoretical for all levelsofquality controls ranged from 95.7 to 106.7. The inter-assay %CV for N-MeFOSE-OH and N-E(FOSE-OH were less than or equal to 25.5% for each QC concentration. The mean percent theoretical for all levelsofquality controls ranged from 93.5 to 99.4. | 1 23. Repeatability and Reproducibility Test A different extractionist prepared samples for run number 32 than the extractionist who had prepared the samples for run numbers 25, 26, and 28. The samples were analyzed under the same operating conditions and with the same instrument. The calibration standards and QCs from runnumber 32 met the same acceptance criteria used for run numbers 25, 26, and 28. _ 000012 Northwest Bioaslyicsl ReSptourdtyNNoo.. NNWWEBRS9998..000552 The analytical procedure demonsisated acceptable reproducibility for al the analytes except 'N-MeFOSE-OH and N-EtFOSE-OH. 24. System Precision Test `The system precision for PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS was determined by injecting unextracted low and high QC samples at the beginning and end ofa normal validation run. Instrument response is defined as the peak area ratio (analyte peak area/internal standard peak area). The intra-assay %CV for PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS were less than or ] equal to 78.2% for each QC concentration. The unextracted samples did not demonstrate acceptable precision for all the analytes (See section 4 Comments and Conclusions for discussionofsystem precision). ! 25. Extraction Efficiency The extraction efficiencies for PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-E(FOSE-OH, ' POAA, PFHS and SDS (intemal standard) from human serum were determined by comparing the peak areas obtained for the following three cases: 1. Both the analyte and internal standard added following the extraction. t 2. eTxhteraacntailoyn.te added to serum prior to extraction and the internal standard added following ! 3. The internal standard added to serum prio to extraction and the analyte added following the extraction. | `The extraction efficiencies were then determined by the changes in peak area for the various cases. The mean extraction efficiencies were PFOS (85.9%), PFOSA (82.3%), PFOSAA (71.5%), N-MeFOSE-OH (53.0%), N-EtFOSE-OH (44.6%), POAA (90.0%), PFHS (81.5%) and SDS (71.2%). Pagel s 000013 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS9998..000852 2.6. Stability Evaluation For the following stability evaluations, the mean test QC sample concentrations must all `within 20% of the mean reference QC sample concentrations to demonstrate acceptable stability. Freeze-thaw Stability Stability of PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS in `human serum subjected to freezing and thawing for three cycles before processing was determined for low and high QC samples. Test QCs were compared against reference QC ] `samples subjected to one freeze/thaw cycle. i `Samples demonstrated acceptable stability after three freeze/thaw cycles for all the analytes. ' Room Temperature Matrix Stability StabilityofPFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EFOSE-OH, POAA and PFHS in { human serum stored under normal room temperature and light conditions for up to 4 hours `was determined for low and high QC samples. Test QCs were compared against reference | QC samples prepared immediately upon thawing. i Samples demonstrated acceptable stability after 4 hours at normal room temperature and light 1 conditions for all the analytes except N-MeFOSE-OHand N-EtFOSE-OH. Room Temperature Extract Stability 1 i Stability of PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS extracts stored at room temperature for approximately 24 hours prior to analysis was determined for low and high QC extracts. Test QCs were compared against reference QC samples analyzed immediately following extraction. Extracted samples demonstrated acceptablestabilityafter 24 hours at normal room temperature for all the analytes except PFOS, PFOSAA, POAA and PFHS analytes (See section 4 Comments and Conclusionsfordiscussion ofroom temperature extract stability) Page 12 000014 Northwest Bioanalytical Stock Solution Stability ReSptourdtyNNoo., NNWWBBRS9998..000552 The stock solution stability was evaluated by comparing the concentrations for a set of unextracted standards made from a stock solution prepared on January 12, 1999 with a set made from a stock solution prepared February 5, 1999. The stock solution stability evaluation was performed in replicatesofthree at the same levelof unextracted standard, tShteocaknasloyltuetsioenxscedpetmoNn-sMtreaFtOedSEac-cOepHtaabnldeNs-taEbtilFiOtySaEf-eOrHs.torage at -20 C for 24 days for all 1 Long-Term Matrix Stability and Reduced Temperature (-20 C) Extract Stability These stabiliy tests will be performed and reported at later date in a separate report. ' 3. DATAMANAGEMENT The concentration values for the endogenous levels ofPFOS, PFOSAA, POAA and PFHS were calculated using PE Sciex MacQuan software (v. 1.6). Al other concentration values were ! calculated using the Watson DMLIMS software. Data presented in this report `may be formatted 0 fewer significant figures than may have been usedbythe computer to generate means, : standard deviations and coefficientsof variation. Summary statistics may be derived from unrounded data and may differ slightly from the values obtained using the rounded values. 4. COMMENTS AND CONCLUSIONS During the assay validation, three potential intemal standards were evaluated, They were | nonanesulfonic acid, octanesulfonic acid and SDS. A fourth intemal standard, C4HsF13S0s, was evaluated during method development, but was unsuitable for this assay owing to a serum interferent that demonstrated different ionization suppression from lot to lotofhuman serum. OF the three intemal standards, SDS gave the best results for al the analytes, and thus it was used as the intemal standard for each analyte. To calculate the endogenous levelsofthe analytes in human serum, each validation run included: (1) "blank" human serum samples, prepared in duplicate or greater, from the lot used to prepare Pgez 000015 [R -- ReSptourdty NNoo.. NNWWBBRS9998..00085 the human serum calibration curve, (2) "blank" human serum samples in five replicates from the ot used to prepare the QCs, and (3) a calibration curve in duplicate prepared using rabbit serum with four different concentrations covering the range 1 to 50 ppb for each analyte. See Tables 5-7 for human serum endogenous concentration data. Ifendogenous analyte was detected in the "blank" human serum samples, that concentration was calculated using the rabbit serum calibration curve. The calibration standard and the analytical QC target concentrations were then adjusted to include those endogenous levels. For example, PFOS was determined to have an average endogenous levelof 30.4 ppb in the ] human serum lot used for the calibration standards on run numbers 26, 28, and 32. The curve range was then adjusted to be 31.4 to 530 ppb; that is, the sum of the endogenous concentration i plus the concentration spiked into each calibration standard. The average endogenous PFOS level for the human serum lot used for the analytical QCs was determined to be 22.3 ppb. Thus, the analytical low and high QC target levels were adjusted to 42.3 and 372 ppb (sumofthe endogenous concentration plus the concentration spiked into each QC), respectively. : `These average endogenous amounts for each analyte were used over the courseofthe validation except for run number 25. A different lot of human serum was used for the calibration curve on ! run number 25 than on run numbers 26, 28, and 29, thus yielding a different adjusted human ! calibration curve range for run number 25. The same human serum lot for the analytical and stability QCs was utilized over the courseofthe validation. ) The method described in this reporthasbeen validated for the determinationof PFOS, PFOSA, PFOSAA, POAA and PFHS in human serum. The method is not considered validated for N- MeFOSE-OH and N-E(FOSE-OH. The reason the results for N-MeFOSE-OH and N-E{FOSE- OH did not mest the acceptance criteria for validation could be due to the following factors: (1) poor extraction efficiencies, (2) poor ionization efficiencies (i. ., no efficiently ionizable functional groups), and (3) because the only useable MRM transition monitored for these two analytes is not derived from a molecular fragmentation, but rather fragmentationofthe acetate `adductofeach analyte to the acetate ion (i. e., [M + CHyCOs]' => [CH;COLT'). Page 14 000016 Northwest Bioanalytical ReSptourdty NNoo,. NNWWBBRS9s98..000852 The poor system precision for all the analytes can be attributed to a "column conditioning" affect. hati, the analytical column used for this assay yields more precise results when it is primed by injectionoftwo or more extracted samples. When unextracted samples are injected onto the column, the conditionofthe column is affected and the precision decreases. This was verifiedby injecting two validation runs, with and without unextracted samples. The run without the unextracted samples gave more accurate and precise results than the run that included the unextracted samples. v Acceptable room temperature PFOSAA, POAA, and PEHS. extract Owing stability was not demonstrated for the to the apparent instabiolfitthye room high QCs of PFOS, temperature extracts, | the samples must be extracted and analyzed on the same day and the analytical run times must be | less than 24 hours, which is the longest time periodfor an accepted validation run. 4.1. Proposed Sample Analysis Acceptance Criteria Subject Sample Determination : Subject sample co calibration curve, ncentrations for a unless an analyte ll the analytes concentration will be for PFO determined using the S, PFOSAA, POAA human s or PFHS er is u m : less that the adjusted those analytes that is LLOQ below concentration.If a subject sample has a concentration the adjusted LLOQ, then the rabbit curve will be used for fo any of ' quantitate that analyte in that sample. In these cases, the accuracy ofthe rabbit serum curve. will be verified by analyzing human serum QCs containing known analyte concentrations 1 between 1 and 50 ppb and calculating their concentrations using the rabbit serum curve, | ret 000017 Northwest Biossalyticsl ReSptourdtyNNoo.. NNWWBBRS9998..000852 Calibration Curve Each run will include calibration standards in duplicate at seven or more concentrations covering the lower to upper limitof quantitation. At least two thirds of the non-LLOQ calibration standard's back-calculated concentrations must be within + 15% (LLOQ must be within + 20%)of their individual target concentrations. If a standard is not within the acceptance criteria, it is deactivated. This process starts from the highest standard down to the lowest standard until all the active standards are within the acceptance criteria. Lower Limit of Quantitation ] `The back-calculated concentrations ofat least one ofthe duplicate lowest points in the ' calibration curve must be within & 20% of the target concentration toqualifyas the LLOQ. If : this criterion is not met, the next level is subjected to the same test and the LLOQ raised accordingly. i Quality Control Samples ! Each analytical run will include low and high QC samplesin triplicate. The measured i concentrationsofat least two-thirds of all analytical QCs must be within + 20 %of their target concentrations. If study samples require dilution, adilution QC will be analyzed in | triplicate for each dilution level. At least one dilution QC at each level must be within + 20% ofthe target concentration. The dilution QC acceptance is independentofthe undiluted ! analytical QC acceptance. | 5. REFERENCES [5.1] L. Clemen, G. Langenburg, "Analysis of Potassium Perfluorooctanesulfonate or Other SFpleucotrroocmheetmriyc,a"ls3iMn ESnevriurmonomreBnltoaoldLaEbxotrraatcotsryU,sFiAnCgTH-PML-C4-.E1l.ectrospray/Mass [5.2] L.Clemen, G. Langenburg, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals Compounds from Serum or Other Fluids for Analysis Using HPLCElectrospray/Mass Spectrometry," 3M Environmental Laboratory, FACT-M-3.1 ete 000018 Northwest Bioanalytical Study No. NWBS98.082 Report No. NWBR99.005 [53] LF.luColreomcehne,mi"cEaxltrSaucltfaicotnoanftPsoftraosmsiLuimvePrefrofrluAonraoloycstiasnUessuilnfgonHaPtLeCo-rElOetchterroAsnpiroanyi/cMass Spectrometry," 3M Environmental Laboratory, FACT-M-1.0. The raw data and final report for this study will be stored in the NWB Archives, 1121 East 3900 South, Salt Lake City, UT 84124 per regulations and contract agreement. 3M will be notified concerning final dispositionofrecords at completionof contract obligations. ] i i | 000019 Page 17 ! Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS9998..000852 Lm[or To oe Toe | Table 1. Summary of Rabbit Serum Calibration Curves for PFOS Liner (weighted 14). All concentrationsar expressed as ppb, oamims| as TOE[5 [00[Tn| Rsquared| (ppb) | (ppb) | ow | oo[ou| [ [Twsemese 1 1 Fr [e a [e ors e[2a3ye [9[a2a [Thor1 [ss |ooo[|roo[soo] ] [rheoe iomr[eTntToOO[eGr8[r T1900[aw|rT |ovr |1m| a0] } [ Lw T es IT 1mT se 1 T 11 1 ] [orm| [056[456|307[519 | [Tepe mer 6|sa |ove |10 | | I TTT TT I CC a [e sol s Tow Twas[s ao[s a5 [7[3m own |] ! Lo is[as [e+ T= [ 1T1 * sample deactivated ~ poor instrument response. i | Page 1 000020 Senne Table 2. SA Summary of Rabbit Serum Calibration Curves for PFOSAA EEREEEE [CsCmonT[T N5um5T be[r 8T 7[e 5T[55T[5m 50T5T[T e 0oRT sqwuares d|v(p0T pb)[|u(pnpb)] | [ reo s | e h [wo[mrhes r wro1 o we 7Fwonr1ho]] C [[ ariI ]I wr[m om si o[ae [T wrr |a| m wT a| y e]e T T 1 s C [mre s Jo]r [wo [|5e 0|ww s w |e] [eEElfwC m er f ctmc]co w1 o lm e--cc1l-- CET Te T e i * sample deactivated ~ poor instrument response 1 | 000021 ---- -- Table 3. Summaryof Rabbit Serum Calibration Curves for POAA iTr Tee | Commis |Naumber[TorwE [ma m[wse|e| owe we| Rsquared| (ppb)| (ppb) C [Fr ome e rm ] EN 0.643| 451 | 238| 522 2782 318 0.9991 [ too| 500| C emo o mret mr Totop wa] FmteJowe |T oe]m TT=]Ty Tt] r] C C o fasC e t eereree T e or eT * sample deactivated ~ poor instrument response | 000022 ae Table 4. Summary of Rabbit Serum Calibration Curves for PFHS [a om [oe To oe Foe | [oC e rm | [owTe vT[ws[T 0T [2T 07| s 5RsT qu5areLd|T(mppb)[|](0ppb)| [aren| I[wmlwewm[pr e o]oo CI c e T m T c m cT a Ry ialEC [ET [T eres|v[[oww[eT wpwper|T ow wo| ST [ Y e ecr cfn c Ln Co O] E Ce Lr TS emeC ee * sample deactivated -- poor instrument response | | _ 000023 Sy Table 5. Summary Data for Endogenous Analyte Concentrations in Human SerumCalibration Standards for Run 25 C[oJe rrox oroseTrrosm [rarestonl emoseon]vrs| vous| S oe ow[[ [ ow o | o w |e ewefe w oeoemwr owne]] | Cr emmT w TwT T[ wTT ew |e wo1w ww 1or] ECe E C T2Tc ewm Tewm T Tw e S 7 T oe T LwB] r ! ND = none detected N/A = not applicable i | Page 2 Gooo24 Ft tibiia ld Table 6. Summary Data for Endogenous Analyte Concentrations in Human SerumCalibration Standards for Runs 26, 28 and 29 OH TTo oofotIw2m:r]T[e| [ w 0.6w 46 w e w EE |[Cowwoe [ [ow39w 1 rew 1m]33 O LCee[ [nl >oe[ [7 a[= S 5 C o a wTN[[EoTowwwSew [| w |we IewL]]C CoCwm=o [ ER[1 w7aS T CE n 7 1 7 wa | 5I[oLeL] T we ww m Tw [ow] = C [ ms e weZ [e w e Www ee e [TTw w t[[aeweeo |Fea|]r] LI ovraoll]5Wa5T[[own7aaaS[T RS 71w 7 a wa [ [w7 aaa|[Iomewe[[|1o0er7|] [C eyE TTweT [ TF v wT e [w eT e Do= e ICmaeTw7enmSE TCoEoTe 7 a[7 Twae[ fIo| m[oo] [OvmwvYio=n]R25E%[|w7an[| C19E0TxaS|7 wa[ |EC|01]] [I omer] 56[wa #7 T7 wa 7 a C0a | ee] ND = none detected N/A = nol applicable Page 23 000025 Sn Table 7. Summary Data for Endogenous Analyte Concentrations in Human SerumQuality [_Teros erCoosntTrorl Soavmpales[vfoirrRounsss.26o,n2[8waendwo2s9eron| roms | vrs| C CC fr ooe o o w w w1 [Te oow [ w ww ep w w|[e meowowee]]] C eIlPTTww[[C2o770v[T Tw we ewoTw wwe [ ||oom wv|| we www] | orseoloL[aa[L om wa Tw wa a |om7 om] [C Ro = eT e we e [ [www w wf w w w[w e eonow]] EC r [w Zw[ [w mCe w w[w ww o[ m w|| ov Ceesweolm llL 5o eawamn[[TwO05aTT |n a w e To a wm [o w ooe [[rw ooww]]] [C CRo oo rmp7 m m oo7 TTewoT1 TTIp oa p p onp TT w p p ] ] 5] Cel I C = wLrwewe TpEw 3w9CT T Y w we|e w w L [o oe |e e CSovSeTsono]]l7 T3ea5[Tuana[[[035m0|T[ ua7aw S 7 w wa [[ oLomEr7o3] m3]] [Coowwmmsnna 577[--a[[m018 |awowa mm]]oome]] ND = none detected N/A = not applicable Pages BHHAIR [I -- Repodryt NNoo..NNWWBBRS9I9R.008052 ! `Table 8. Summary of Human Serum Calibration Curve Parameters for PFOS Quadratic (weighted 1). All concenmtions are expressed s pb. Run Number| A* c | Murtiple |LLOQ|ULOQ Rsquared| (ppb)| (ppb) 30Jan1999[25 | 0.000002| 0.003075| 0006751 | 09909 OLF|e-2619|09 0009 002| 0002977| 0.000357| 0.9930 02Fe| b2-8 1|90.90009001 | 0.001649| 0007710| 09937 10Fe| b3-2 1|90.90009003| 0.004162| 0.009869| 0960 [31a| s30 | Le TeTe e A,B,35d Care cociients used to dethefquadrnatic curve Table 9. Summary of Human Serum Calibration Curve Parameters for PFOSA Quadratic (weighted 1). All concentrations are expresseasd ppb. ar c |Rmsuqtuiaprteed|[L(LppOb)Q|[ U(LppOb)Q ' 30-Jan-1999 0003444 [0000622| 0.9946|10 | s0 00| OL-F|eo26-1|9000900902| 0.003308| 0.000757| 0.9965 02-Feb-1999[28 |-0.000001| 0.001834| 0000785| 0.9982[C100|s00| 10Feb-3219[09.00900|02| 0.003945| 0001714| 09982[100[500| [sp[ Toooooor [0000508| ooo0sor | voor Lo TeTeToTe * A.B. and Care coefiiens used to defn the quadratic curve. 1 1] 000027 Page28 -- ReSptourdtyNNoo,. NNWWBBRSS998..000852 Table 10. Summary of Human Serum Calibration Curve Parameters for PFOSAA Quadratic (weighted x). Al concentrations ce expressed s pb. 30-Jan-1999 01-Feb-1999 02-Feb-1999 10-Feb-1999 Run Number| A* c+ |muttipie [LLOQ| ULOQ _ [Rquarea| (ppb)| (ppb) 0.000000 | 0000676 [0.000208| 0.9964|19 |219 0| -0.000001| 0.000764| 0.000653| 0.9971 0.000000[ 0.000469 0.000085| 0.9963 0.000001 [0.001170| 0.000458| 0.9965 [01 Tomom Tomo [soon Tome |] * A,B, and Care coefiiens uedtodefn he quadratic curve Table 11. Summary of Human Serum Calibration Curve Parameters for N-MeFOSE-OH Quadratic (weighted 1x). Al concentrations are expressed a py Run Number| A* ct | muiple [LLogQ| ULOQ Rsquared| (ppb) | (ppb) ! 30-Jan-1999| 25 | 0.000007| 0021767| 0.001921| 09981 [1|0s00 0| 01-Feb-1999 0012302| 0.005797| 0.9962 |10 |s00 0_| | 02Feb-1999|28| 0.000001 | 0.007315| 0.001464 | 0.9970 [1|0500 0 | 10-Feb-1999 0.000010 | 0.019016| 0003235| 09970 |10 |s00 0| TT L TeT ToT 1] A.B. 3nd Care coefficients usetdo define the quadrtic cure _ 000028 Nortwes Bioanalytical ReSptourdtyNNoo.. NNWWBBRSS998..000852 Table 12. Summary of Human Serum Calibration Curve Parameters for N-E{FOSE-OH Quadiatic (weghied 1). All concenraronsar xpresed as ppb. 30-Jan-1999 01-Feb-1999 02-Feb-1999 10-Feb-1999 ar ct | mupie [LLog[ uLog Rsquared| (ppb) | (ppb) -0.000002| 0015048 [0.003325| 0.9994 |1.|0 so0| -0.000002 | 0.008104| 0.002172| 09956 |1.|0 s000| 0000000[ 0.005031 [0.005001| 0.9986 |50|0s0 0_| -0.000004 | 0.011968 [0.001308| 0.9969 | [Mem] -oo00002| 0010038|001866| 03576 1] ** Bol Stand*arA,dBs, afa1n.d00Cpapbewceoerfefisciiienstisculseadu0td,efsontthheeqLuLadOrQatwiacscurraviese0d 5.00 pb. i, `Table 13. Summary of Human Serum Calibration Curve Parameters for POAA : Quadratic (weighted 1). All concentraions ae expressed as ppb. i Run Number] A* ct [ Munipie [LLOQ[ ULOQ Rsquared| (ppb)| (ppb) i 30-Jan-1999 -0.000003| 0.005601| 0.000463 | 0.9972 |42 | 500 3 | 01-Feb-1999 0.000004| 0.006274[ -0.001033| 0.9963 02-Feb-1999 0000002| 0.004036| 0.003976| 0.9981 |52 | 57 04| | 10-Feb-1999 -0.000005 | 0.008652 [0.001660| 0.9970|52 |s07 4_| * A,B, and Care coefficients us0defdineth quadratic cure. Page 27 609029 Norwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS9398..000532 Table 14. Summary of Human Serum Calibration Curve Parameters for PFHS Quad (weghicd 1x). All concentrations ae expresseda pp. Run Number| A c [ mutipte [LLOQ| ULOQ Rsquared| (ppb) | (ppb) 30-Jan-1999 0.000004| 0.005961 | 0.002977| 0.9979 02-Feb-1999 10-Feb-1999 0.000002[ 0.003814| 0003933| 0.9982 0.000006| 0.007750| 0.002353| 0.9973 Co TeTT TT * A,B, and Care coufcents sedto defn the quadratic curve, | Page 2s 000030 Sn Table 15. Back-Calculated Concentrations of Human Serum Calibration Standards for PFOS [[mirposJ|Eua[rwes [Pwse[oaP]|T|TJeas]eToer]Teaoln| ae Tw] | FI oes|p5| ms| TsalH salwseO |[ea]on[tmslfoTm w]f| rTo] J CI rEo F2533E 3 0 E ECeY EE EYA ABEEY EE 1 TTDwlholail Tas Tas]Tw] Tu [er1 s| EY |T| TTTmwlolwwlsoloso]] T[wor]] J[sme]l TJwwa]]TTowr]) "| V_eson] [[voesw[liosr[[5r5s5s[5a2m5 iesoo [svros[[sovsee[]a570[oavar[[520evmo[e0ae[zso[ror[ore [oe oT wl el [TosoTe ssasT[aal o[[ooea[e uoar[T[ool ooa[[voosse [Taeonsl[[sol aolsrJaaaTree ]eaolsaLTel osnl[JauaTss o]e5rs]] * sample deactivated-statistical outlier page 20 000031 I Northwest Bioanalytical Pantie `Study No. NWBS98-082 Table 16. Back-Calculated A cocemions se xpd 5b Concentrations of Human Serum Calibration Standards for PROSA [xen raw [soserioon| vem 2s 0Ton ss[550 Toms|aw|tea[33 503 [30s | 510[ses| |] SL E ETN eTH so T ns aF lso[mn| Jo] fovremo| 26 TToioo[[5em6[[oa1m[23a2[|mwoos[[o0r]] Ja] [sw] | ! [oL zreviom | T 2s |v0T |sor[102T |259| 77[39T 5| 302|53] [ L ThI os[asT o]T maT les1T wo]T wr[aT ]a] [oL ses99| 52 TToasswos[aasrms[[T 11110s[[226358[|5va7ns[wovwe[|22aa |o53t5| [e L n TTvo1 r[assT [T 102T|205T| wo1 r| a1 n|371 50 [so [ev To0uss[oam6 osass|]o1a77[]s26a0[[1a39[|a11s3|] aaez| [wie Le |TT T Troo [29[20[s Toss[0e6f2os 0le1al]sos]] | * sample deactivated-statistical outlier Fags 30 000032 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSS998..000852 `Table 17. Back-Calculated Concentrations of Human Serum Calibration Standards for PFOSAA All concentrations are expressed ss ppb. [eo [og [Te [ex[feeoso [m[ im | 55 TT oe] TTeooo|l"o T[ooow]l TJooor]o T[ooas]] [[msear]ll | | [Joano]]|| __ [orton[3 [Two TT rrr 11 Jars] Jors| [wa] [seal [ses] | [ow] [TTTeal Tes Tos[ ws] [mo] Jas] | [an | [orm Zo rT TT] Too Tow] [sul Jowa[ [ar] [sol mo| [mi] [ [ 1 1Tesr lr Tr ool Tenr [wd T [T wal [salu]1o]n] [omioss[32| Taw [TT Tel sos] Tal [roa][wo] [sal Jovs|mi| Jost] [re Two][ws] [sel] [a] [ven] F [soe lToe awl fowlm oaemlT[eo1esa[asn]Df1o5r7o[eaws3r5l2[oo53e0ai] rne5|]1] [ CoT w Tse Taa[l l va[als so |a50e looi[ewl ol|aar ase s[a0]sesl al[ea0l]aa 0a][sos]] * sample deactivated-statistical outlier Paget 000033 ne Table 18. Back-Calculated Concentrations of Human Serum Calibration Standards for N-MeFOSE-OH [[oSnwipemtesT|un5omber[v"1s00[[5o0s0e[[1o0m0[335203[so7o[[1a0r0[]2 J|o50] T ToweTlT rmiT lafT w| ] fs] VT [orem T| veo[ewo[wsorm[5aa[[mrre[mime] e[0]] |b[ or E errwr em n|r er[m ve[m5] Cr TrooT [enTir w[rmarls]rwsfras ors] C [ [wm]woC v wsTT[ener[wierffaaT e sr[[eersr|w[er||T]] Lo [[sCehell TToev[owma owmrn]maas[eoasr[[7o3n0[[usf|o] | [[ Ce e Twa[se[[e aeawn[woes ols[uaeo[or]] CA IeTel retette] | * sample deactivated:statistical outlier 000034 ese Table 19. Back-Calculated Concentrations of Human Serum Calibration Standards for N-EtFOSE-OH [o[ rm T Soo [[so soorT[m rorem [meo[m one [os]e r [or] [r orem| T[ovwnT[[wseT ms[[T 0m0o[l5m7i[[Tasse[Tw[|TT] o]] | [T I rm e owTe [520T [T va n T 7[iaorsl[ooms eT 2J[o7] [rT I am]5[Tevaioss[aT ows[[T 711I 097][5as3sT[[raaesT a|]sonT [[2m0] [ro]u] H esl TovowJa[Y uosa([omsr[[oonwwso[[ow[|os] i [ soall TTooe ma[[[eo0osrT[fe4os0e[[]os12ow7[|[T osaaes|osaoosr[|oa3so7T5d5o]7]] C eT reTete | * sample deactivated-statistical outlier Page 33 000035 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRSS998..000852 Table 20. Back-Calculated Concentrations of Human Serum Calibration Standards for POAA. All concentrations are expresed as pb. [> pa [ToT = PRT wT) Number [esosTeo| Trae] [wal [os [sr] Two]| [ai] | [T TelTw]Te w Tm] [wll ] |Tw]| EE A AHH [orem] se | Tew] Ton] [ws[ [ws[ Twa] [ws] | wo] S [f E Tes rTT are T[T wr JT w l Jw] | | | | [ozs]5 [Ton] [ool [ws] [mal Twa][wm] Im] [TT Tel Tew Tse [ool Tar Jules] [wi] I rT] [orm]mTTso] Tow] Tues] [on] [was] [won|Jw] C ee Twa a] Tama] [se] [usm] sa TrrT rrrrrri : Vel [a0]s31]748[27| 57] 1a [286 |282[525[sae[100[109|306 | 05[sos| sol Tonslosssosufoaslocasoer] ar[174[113[207[ass[43|160[cz 257 jel TsoTao arTas on[a6[45[6a [2a [55[as a1|es[13]sa| ew] TasTos[es oo[5a[21|oa[9s[43[as|to[as[31]04[os| JL TET a ela sa sleslalelela]s] | * sample deactivated-statistical outlier | res 000036 ES -- ReSptourdtyNNoo.. NNWWBBRS9998..000552 `Table 21. Back-Calculated Concentrations of Human Serum Calibration Standards for PFHS All concentrations are expressead ppb. prom 5Two| Tse us| Jaa| [wa] Tour] | wa]| pee Je I rT pe] fe Tel Tel] rrr [orem] 26 Toa | Tool [we] uo] [wr] Tw]| [wn] [ NE I T Tewe A Taal Tae n [we]Jwl ]I | Jos) Co [TT Tow 73 EY EYEWE ERET Tes]Tas[ [wal Teel Twila] Jo] T TrT [[ osm] a [aT a[TTeerw|] [[5wsae ]l [waesl] [[wsaoso]]l [[wwiesa0]] [[sass] rr rT TT T1711 [so ooo loasefoass[ozosee[osisoor135 [oomoe]276 asa [aso[ero[547] [e [em v] T[oesTTooe[easaTehs[Twaoss[5eo0|[205[[7o2a[[1asa[[1asr[2oa]|a5x0[[2a0a[]30a]]a1s| [Tels elelelelz elalalelela]0] * sample deactivated-statistical outlier | pages 000037 Northwest Bioanalytical ReSpteurdtyNNoo,. NNWWBBRS9998..000552 Table 22. Intra-Assay Precision for PFOS Human Serum Quality Control Samples All concentrations se expressed as pb. Run Number: 26 | Low QC| High QC Run Date: 01-Feb-1999 | (42:3 PPO)| (372 ppb) Tera 2Tas[es | [ TT ea rw e[w]=] w o Te i w we T] e Theoretical |1024_| 109.0 LLT5175] . * > 20% theoretical | Page 36 000038 Norwest Bioanalytical ReSptourdtyNNoo.. NNWWBBSR998..000552 Table 23. Intra-Assay Precision for PFOSA Human Serum Quality Control Samples Al concentrationssr expressed as pp . Run Number: 26 | Low QC| High QC Run Date: 01-Feb-1999|(30-0 PPD)| (350 ppb) TeTw I [ T I 2To a[ao] TerTe : emiaTeiesT|rr]] ! LT 5 1757 | *>20% theoretical ! Page? 000039 Northwest Bioanalytical ReSptourdty NNao,. NNWWBBRS9998..000852 Table 24. Intra-Assay Precision for PFOSAA Human Serum Quality Control Samples All concentrationsare expressed as ppb. eT] Run Number: 26 | Low QC| High QC Run Date: 01-Feb-1999|(127 PPb)| (190 ppb) [5Tw[we 1 [5 Tos[ we] N ee T TreT LLT5157] Page 36 600040 NortowestBioanalyical RSetoudnyNNoo, NNWWBBRS9958..000852 `Table 25. Intra-Assay Precision for N-MeFOSE-OH Human Serum Quality Control Samples All concentrations are expressed as ph. e: e TT Run Number: 26 | Low QC| High QC Run Date: 01-Feb-1999|(20-0 ppb)| (350 ppb) I I a meTe I *> 20% theoretical 1 Page 39 000041 Northwest Bioanalyical ReSptourdtyNNoo.. NNWWBBRS9998..000852 `Table 26. Intra-Assay Precision for N-EtFOSE-OH Human Serum Quality Control Samples All concenttions ae expressed as pb Run Number: 26 | Low QC| High QC Run Date: 01-Feb-1999| (30-0 PPb)| (350 ppb) TwTe ] Tee TT e TT wTw] : Co ToTw T T Ee eT ] ] i *>20% theoretical i | Pagedo 000042 [-- ReSptoryt NNoo,. NNWWBBRSS955..000852 Table 27. Intra-Assay Precision for POAA Human Serum Quality Control Samples All concentrations ae expressed a yb. Run Number: 26 | Low QC| High QC Run Date: 01-Feb-1999| (233 Pb)| (353 ppb) ITT wTT w] onTo] / [T= [sa | [5 ws |se | ee imTwi ow oeTio] : L515] *>20% theoretical Paget 000043 Norwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS9998..000852 `Table 28. Intra-Assay Precision for PFHS Human Serum Quality Control Samples Al concentrationssr expresasspebd. Run Number: 26 | Low QC | High QC Run Date: 01-Feb-1999| (#20 PPD)| (352 ppb) TTTw woTTToawrr] ] [ [ Tm a 5 [[wswe] oowwT m T T iaw a r] T CT T CL = 1515] *>20% theoretical | Page a2 000044 ne aan Table 29.Inter-Assay Precision for PFOS Human Serum Quality Control Samples CC [ omin[ T TTT fe w ewmg5 seeT e T Tao we e ee | (423ppb) | (372 ppb) TaTe eeeeT Tee e e CT ew C e TTw w a] a I-- ] T 1 Te a eTe T fw eI] [ e a Cit tT se r sww] re]| CeeT s T e ] Page 43 000045 Rinhducie: `Table 30. Inter-Assay Precision for PFOSA Human Serum Quality Control Samples Run Number Low QC High QC (20.0 ppb) (350 ppb) T Tv w[oeww]] T LTT T T a Tosw w Toww]] T EE Ee T ETe we T TT w ws oewe]] [T L ormT [ o Toww| s |[|wwowe]|| TT ws a]| i E TTC ew s [ooww]| T T w T [weew]] 1: _ eTlT T T 1e %= |e wn] esollT Twoo ws || eveeslT ] too e0T| oious|] CAT --T--=] 000046 -- a saaTn Table 31. Inter-Assay Precision for PEOSAA Human Serum Quality Control Samples T Comin| 51w vies|ow | Run Number (L2o7wppQbC)| (H1i9g0hppQbC) T Tnw e] wn ] [we [ew T II e EoTw [ CTe T Tww] T Tw w Toww]| [ofa |To|i wn | T T Te wwow] ! I o _-- l EE TI o2[|o1 w | [me ie] TT o| oere]] esme] Page ds 000047 Table 32. Inter-Assay Precision for N-MeFOSE-OH Human Serum Quality Control Samples Run Number| LowQC | High QC (20.0ppb)| (350 ppb) I T I m EwTe] T[vw e Toaii]] I TaTa a I[ E 1 T EETRo osT|s oww]] TT wwwe |wwar E TT T |L w =me|Tw| T CTu T=ems oewe|]| I Tn ! --_-- eel[| [te Tioa]l T TTi w |a 0: | 000048 I le s--s-- Table 33.Inter-Assay Precision for N-EtFOSE-OH Human Serum Quality Control Samples Run Number| Low QC High QC (20.0 ppb) (350 ppb) T Tw wT Tw e Te Te es T Te e | [T C orm T |[Twam o rTror]] T ee ae TTww] . . T T TT m m TmT TTewe]] Tw [ms] [a] T TT w we me] L mlTT TTT w =woTTeo]] Cesm olln we e meT ] ] 000049 `Table 34. Inter-Assay Precision for POAA Human Serum Quality ControlSamples LowQC | High QC (233 ppb)| (353 ppb) T I I o we] e L T Tw [ee s] Tw| | T Tm w eown]] T EL TT T TT =wo Teswae|]] T T a T wer owwe ] T E TT T e T o Towwen]] [T orm| |T os|wom|] T Ts e wwme] | [ wlT m Two e [T |ww wwe || esollT T a a |w awse ]| [ [t miee3l 57n | aa 5] s] 000050 [I -- Study No. NWESIS.082 Report No. NWBRS9.005 ! Table 35. Inter-Assay Precision for PFHS Human Serum Quality Control Samples All concentrations see expressed as ppb. LowQC | (2.0ppb) | HighQC (352 ppb) T Tw T ei T Tn T e a] | [oerme Tm mrT |SLwe Lo | T C T TTT o o we]| ores] aw [n | s aw]| : mw [Ta|r wm [Tm 5a 1 a0 | 7 || TT me a [Tm ww ] ! T E nlT a L w e C [me Te e 0i 7T | ] ie oos | C esT T m 1%0]| Sample dea>ct2i0v%attehdeosraettitciacla] ule Page 49 000051 Northwest Bioanalytical Table 36. System Precision for PFOS ReSptourdtyNNoo.. NNWWBBRS3998..000552 RunNumber| U(nLex0otwr2aQc5Cte||d| UneHeixntgrohaQcotCea ! [ven obn|| ama | [sol Tromor | orm [L we 51 m6T5 o Table 37. System Precision for PFOSA Run Number| U(nLe0xo0twr0aQ0cCt,e|d|| HUniegmxmthroQaccCted W0Feo139299| 00067 | 0207807 ,! [ [evsleonl ] wr[Town|| 7 camo| omes| ois Page0 000052 `Table 38. System Precision for PROSAA =[erEEE Low QC High QC (12.7ppb) | (190 ppb) T T T o eo m oo m o [[oom] a wen|| [ elw so w |w ows| 51] Table 39. System Precision for N-MeFOSE-OH Unextracted| Unextracted ne T E To oon w n[|in aoe wues|| : C [ els T [o wwoem] [] owmee|| Page st 000053 Northwest Bioanalytical Table 40. System Precision for N-E(FOSE-OH ReSptourdtyNNoo..NNWWBBRS9998..000852 UnLexotwraQcCted|| UnHeixgthraQcCted (20.0ppb)| (350 ppb) 10Fe0-193299]| o0onises | osasess | 0| I oT 5 5] Table 41. System Precision for POAA Reales UnLexotwraQcCte|d| HUinegsthraQcCted 20.0ppb) | (350 ppb) 10Feo-193299]| o4s200 | 2.629881 _-- [Men 0aT m | Sian | ! [[es v of1 Toms me | ome ws | LT5] Page 2 000054 [-- ` Table 42. System Precision for PFHS RStuedyNNopo.,NNoWWEBRSSr398..0t00552 Run Daze UnLexotwraQcCted|| UnHeixgthraQcCted (200ppb) | (350 ppb) F199 32 ]| ome | 270mm | Mew] [Tomo | OY smn | [sol [Tommi | [we Tw awww we | LT 5 T5 | Pages 000055 I Norwest Bioanalytical `Table 43. PFOS Extraction Efficiency ReSptourdtyNNoo.. NNWWBBSR998..000852 1. AnalyTeimaenod fSpakfienegxtraction AnsiviePesk Areas 81470 ses Mean me 82017 IstdPeak Areas S30547 sssls ss49 539257 2 Anaiye priorsad 5 serexiacion 61a 94927 Mean 7si0o8773 ] 5.1Sprior and Ansiye fer extraction 20162 4s6s10923788 Mean 384126 Mean extraMcetaionn eexftfriacciteinocny effofritchieenicnytefronratlhsetaannadlayrted = = 85.9% 71.2% `Table 44. POSA Extraction Efficiency ' 1. AnalyTtiemaenodfSSapfiekienxgtraction Anslvte Peak Areas 45280 Istd Peak Areas 539547 45068 3724 ss6ls ust Mean Ey 59237 2 Analyte prior and Sate extraction 329 ast Mean 25711 35397 :| 3.1 prior andAnsyte airextraction 20162 a4o601297388 Mean 384126 Mean extraMcetaionnexetfrfiacciteinocnyeffofrictiheenicnytefronratlhsetaanndaalrydt=e= 8721..32%% 000056 Page 54 Northwest Bioanalytical Table 45. PFOSAA Extraction Efficiency ReSptourdtyNNoo.. NNWWBBRS9998..000852 1. AnaTlimyaentodf1Spekirngex.traction Mean 2 Analy prior snd 1afer extraction Mean Ansi6vePseakAreas 6197 a8 655 as su3 = 391 [sid Peak Areas 39547 sags s23549 39237 | 3. ISprior andAnsiyteafter extraction 20162 46a010923788 ean 384126 Mean extraMcetaionn extraction efficiency feofrftihcieenicnytefronratlhestaannadlayrt==de 71.5% 712% `Table 46. N-MeFOSE-OH Extraction Efficiency 1. AnaTliymaeaodfSSapfiekrienxgtraction Analy2t5e7P6e5a4k Areas [std Peak Areas 39547 26358 15451 s54615 sass Mean 220588 539237 J| 2. Anayte prior sad IS ateextraction 85560 26651 Mean 45573 Tis261 ! 3.15prir sdArtraxrciion 20102 54601297388 Mean 38126 Mean extraMcetaionn eexftfriacciteinocny feofrfitchieenicnytefrornatlhestaannadlayrt==de 53.0% 71.2% Page 55 000057 Northwest Bioanalytical Table 47. N-EtFOSE-OH ExtractionEfficiency ReSptourdty NNoo.. NNWWBBRS9995..000852 1. AmaTliieneanodfSaStoetkrienxg.rcion Mean 2. Analye price snd 1 afer extraction Mean AnslvePeskAress 191901655209 1T2e2so0o9r 1237589872 7352001024 sid 3Pe9a5k47Aress ssi1s sSa3i0s23e70 1 5. 1Sprior andAnsiyte stir extraction 200162 Mean s46o1u93s8 Saiz Mean extraMcetaionneexftlrcaicetniocny feofrfitchieenicnytfeornatlhsetaananldyater=d= 7414..265%, Table 48. POAA ExtractionEfficiency 1. AntTeinaenodf1Saptlekrinegxvacion AmsiviLePee)dkArens IstdP53e95a4k7 Areas: 7854740217 5s2s3i5s4i9s Mean 52650 30237 2. Aly prior snd1Sater extraction 76904 assuas Mean Tra 1 3.15 prior and Anite air extraction 200162 asio0n Mean 384126 `Mean extraMcetaionneexitrcaicetniconyeffofritchieenIcnytefronratlhestaannadlayrt=de 79102.90%% -- Page 56 000058 Northwest Bioanalytical Table 49. PFHS ExtractionEfficiency ReSptourdtyNNoo.. NNWWBBRS9998..00058 1. AmaliTeimanedofSaSholekriensgscrion Analvi1eP2e4skArcas [sid 5P3e9a54A7re: s8s655829 Mean 85322 sss3issat9s Ss5237 2 Anayeprio snd Stier exten 6659s ss1i39s1 Mean 65700 | 31S pror sd Ave ser extraction 20162 Mean 446510923788 64126 Mean extraMcetaionneexftircaicetniocny effoirctihenicnytfeornatlhestanaanldytaer=d= 7811..35%% Pages? 000059 || Norwest Bioanalytical Table 50. Freeze/Thaw Stability for PFOS All concentration ae expressed a ppb. ReSptourdtyNNoo.. NNWWBBRSS998..00025 Run Number Run Date 25 30-Jan-1999 LOW QC After 3 Freeze/Thaw cycles 1 433 | 2 33.1 3 405 Mean S.D. % CV Mean % Control Dev. from (ng/mL) Control 39.0 527 13.5% 380 254% HIGH QC After 3 Freeze/Thaw cycles 1 330 2 315% 3 410 Mean 370 1 i S.D. % CV 56.6 153% Control (ng/mL) 397 Mea%n Dev. from Control 6.80% * sample deactivated ~ poor instrument response Control = mean ofreference QC samples 000060 Page 58 Northwest Bioanalytical Table 51. Freeze/Thaw Stability for PFOSA All concentations are expresseads pp. ReSptourdtyNNoo.. NNWWBBRS9998..000852 Run Number Run Date 25 30-Jan-1999 LOWQC Afte3r Freeze/Thaw cycles 1 235 | 2 19.9 3 20.0 Mean% Mean S.D. Control (n%g/mCLV) Dev. from Control 211 2.05 92.07.04% 3.59% HIGH QC After 3 Freeze/Thaw cycles 1 336 2 248% 3 401 Mean 369 I ! %SC.DV. 1426..50% Control (ng/mL) 375 Me%a Den v. from Control -1.73% * saCmopnlterodlea=ctmievaatneodf~repfoeorrenicnsetQruCmesnatrmeplsepsonse Page 59 000061 Norwest Bioanalytcsl `Table 52. Freeze/Thaw Stability for PFOSAA All concentrations are expressed 1s ppb, ReSptourdty NNoo.. NNWWBBRSS998..000852 Run Number Run Date 25 30-Jan-1999 LOW QC After3 Freeze/Thaw cycles 1 136 2 125 ] 3 120 Mean 127 SD. 0819 % CV 6.45% Control (ng/mL) 131 Mean % Dev. from Control 3.05% HIGH QC After3 Freeze/Thaw cycles 1 205 . 2 *180 } 3 +237 Mean 21 | S.D. 26 % CY 102% Control (ng/mL) 23 Mean % Dev. from Control 0.897% * sample deactivated - poor instrument response: **>20% theoretical Control = manofreference QC samples 000062 Page 60 Norwes Bioamytal ReSptoarty NNoo., NNWWBBRS3998..000852 `Table 53. Freeze/Thaw Stability for N-MeFOSE-OH All concentations ar expressed a ppb. Run Number Run Date 25 30-Jan-1999 Lowoe After 3 Freeze/Thaw cycles 1 18.9 2 11.8 1 3 15.5 MeSaD.n Control (n%g/mCLV). Mean % Dev. from Control 3155.54 2136.10% 3.75% HIGH QC After 3 Freeze/Thaw cycles 1 320 i 2 M13 3 324 Mean 2 | %ScDv. 0.288738% Control (ng/mL) 319 Mean % Dev. from Control 0.940% * sample deactivated -- poor instrument response: Control = meanofreference QC samples Faget 000063 Northwest Biosmlyical ReSptourdtyNNoo.. NNWWEBRS9998..000852 `Table 54. Freeze/Thaw Stability for N-EtFOSE-OH All concentration are expressed as pp, Run Number Run Date 25 30-Jan-1999 LOW QC After 3 Freeze/Thaw cycles 1 19.1 ) 2 11.4 I 3 15.9 Mean SD. % CV Control (ng/mL) Mean % Dev. from Control 15.5 387 25.0% 15.7 -1.49% HIGH QC After 3 Freeze/Thaw cycles 1 302 2 214% 3 307 Mean 305 S.D. 3.54 ! % CV 1.16% Control (ng/mL) 31s Mean % Dev. from Control -3.33% * sample deactivated ~poor instrument response Control = meanofreference QC samples Page 62 000064 Norwest Bioanalytical Table 55. Freeze/Thaw Stability for POAA All concentration are expressed as ppb. ReSptourdtyNNoo.. NNWWBBRS9S98..000852 Run Number Run Date 25 30-Jan-1999 LOW QC After 3 Freeze/Thaw cycles 1 25.3 2 238 i 3 255 Mean 24.9 SD. 0.929 % CV 3.74% Control (ng/mL) 25 Mea%n Dev.from Control 10.5% HIGH QC Afte3r Freeze/Thaw cycles 1 330 ! 2 144% 3 385 Mean 358 || SD. 389 % CV 109% Control (ng/mL) 364 Mean% Dev. from Control 179% * sample deactivated ~ poor instrument response Control = meanofreference QC samples 000065 Page 3 Northwest Biossalyticsl Table 56. Freeze/Thaw Stability for PHS Al concentaions sr expresseda pp. ReSptourdty NNoo.. NNWWBBRS9998..000552 Run Number Run Date 25 30-Jan-1999 LOW Qc After 3 Freeze/Thaw cycles 1 244 I 2 217 3 25 . Mean 232 SD. 137 % Cv 5.93% Control (ng/mL) 22 Mean % Dey. from Control 450% i HIGH QC Afte3r Freeze/Thaw cycles i 1 310 2 145% 3 365 | Mean 338 sD. 389 % Cv 115% Control (ng/mL) 366 Mean% Dev. from Control 1.79% * saCmopnlterodlea=ctmievaatneodf--repfoeorreinncsetQruCmesnatmrpelsepsonse 000066 Page 6s Northwest Bioaralyical ReSptourdty NNoo,. NNWWBBRSS998..000852 Table 57. Room Temperature Matrix Stability for PFOS All concentrations ae expressed a pb. Run Number Run Date 3 10-Feb-1999 2 Hour Low Qc 4 Hour 1 39.5 384 2 410 369 1 3 4038 375 . Mean 404 37.6 SD. 0314 0.755 % cv 201% Control (ng/mL) 412 201% 412 Mean % Dev. from Control ~~ -1.94% 8.74% i 2 Hour HIGH QC 4 Hour ;' 1 22 2 271 343 173% i . 3 276 349 , Mean 29% 346 sD. 281 424 / Control % cv (ng/mL) 9.69% 354 123% 354 Mean % Dev. from Control ~~ -18.1% 226% * saCmopnlterodlea=ctmievaatneodf--rpeofoerrenicnsetQruCmesnatmrpelsepsonse 000067 Pages Norwest Bioanalytical ReSptourdty NNoo,. NNWWBBSRS98..000852 `Table 58. Room Temperature Matrix Stability for PFOSA All concenaions are expressed ss ppb. Run Number Run Date 32 10-Feb-1999 LOW QC 2 Hour 4 Hour 1 200 187 2 203 172 | 3 207 182 Mean SD. % CV Control (ng/mL) Mean % Dev. from Control 203 0.351 1.73% 204 0.490% 18.0 0.764 4.24% 204 11.8% HIGH QC 2 Hour 4 Hour 1 310 317 2 261 174% 3 276 336 Mean 282 327 SD. 25.1 13.4 | % CV 8.89% 4.1% Control (ng/mL) 332 332 Me%aDenv. from Control "15.0% 151% * sample deactivated - poor instrument response `Control = meanofreference QC samples 000068 Page 66 Norwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRSS998..000852 Table 59. Room Temperature Matrix Stability for PFOSAA All concentrations are expressed 1s pb. Run Number Run Date 32 10-Feb-1999 LOW QC Ho Hour 1 128 ns ] --2 133 0ns Mean 13.1 sD. 0289 % Cy 221% Control (ng/mL) 127 Mean% Dev. from Control 315% 1.1 0.693 624% 127 12.6% )i four HIGH QC Hour : 1 179 200 i 2 138 *103 i 3 85 : Mean 156 193 sD. 208 106 | % Cv 133% 5.49% ! Control (ng/mL) 194 194 Mean % Dev. from Control "19.6% 0.515% * sample deactivated ~ poor instrument response **> 20% theoretical Control = mean of reference QC samples . Page 67 00069 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS9S95..000552 `Table 60. Room Temperature Matrix Stability for N-MeFOSE-OH All concentrationsar expresseda pp. Run Number Run Date 32 10-Feb-1999 2 Hour Low QC 4 Hour 1 143 10.5 ] 2 14.9 3 13.0 125 108 : Mean 14.1 13 ! sD. 0.971 1.08 % CV Control (ng/mL) 6.90% 14.4 9.57% 14.4 Mean % Dev. from Control 231% -21.8% ! HIGH QC 2 Hour 4 Hour ! 1 248 218 ' 2 199 31+ i 3 197 335 Mean 215 2717 S.D. 289 82.7 | % CV 13.5% 29.9% i Control (ng/mL) 286 286 Mean % Dev. from Control "24.9% 3.15% * sample deactivated ~ poor instrument response Control =meanofreference QC samples Page 68 000070 Norwest iosealyscal ReSptourdtyNNoo.. NNWWBBRS9998-.000852 Table 61. Room Temperature Matrix Stability for N-EtFOSE-OH All concentrations are expresseda ppb. Run Number Run Date 32 10-Feb-1999 Hour Low Qc our 1 15.1 11.9 ] 23 5148 0127 . : Mean 143 122 sD. 1.08 0.436 % CV Control (ng/mL) Mean % Dev. from Control 7.52% 149 -3.80% 3.57% 149 -18.12% ! HIGH QC our. 4 Hour 1 255 235 z 2 224 139% 3 210 9 Mean 230 287 S.D. 23.0 735 | % CV. 10.0% 25.6% Control (ng/mL) 292 292 Mean % Dev. from Control "21.3% "171% * sample deactivated - poor instrument response Control = meanofreference QC samples 000071 Pages Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS9998..000852 `Table 62. Room Temperature Matrix Stability for POAA All concentrations are expressed as ppb. Run Number Run Date 32 10-Feb-1999 Low QC 2 Hour 4 Hour 1 23 209 | 2 28 201 3 244 20.1 Mean 232 204 ! S.D. 1.10 0.462 % CV 4.74% Control (ng/mL) 26 227% 226 Mean % Dev. fromControl 251% 9.88% ! HIGH QC 2 Hour 4 Hour , 1 313 327 2 21 171% ! 3 280 308 Mean 271 318 | sD. 46.6 134 % CV 17.2% 421% i Control (ng/mL) 324 324 Mean% Dev. from Control 163% "1.85% * saCmopnlterodlea=ctmievaatnedof~rpeofoerreinncsetQruCmesnatmrpelsepsonse Page 70 000072 1 Northwest Bioanalytical ReSptourdtyNNoo., NNWWEBRS9998..000852 Table 63. Room Temperature Matrix Stability for PFHS All concentrations are expresseda ppb. Run Number Run Date 32 10-Feb-1999 Low QC 2 Hour 4 Hour 1 208 21s 2 213 19.5 ] 3 239 211 : Mean 20 207 SD. 1.66 1.06 % Cv 7.57% 5.11% Control (ng/mL) 218 218 Mean% Dev. from Control 0.917% 5.05% } 2 Hour HIGH QC 4 Hour | 1 333 341 , 2 251 172% i 3 281 306 i Mean 288 324 SD. 4s 247 % CV 14.4% 7.62% | Control (ng/mL) 333 333 Mean % Dev. from Control "13.4% 2.70% * sample deactivated - poor instrument response Control = meanofreference QC samples Pagel 000073 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRSI9S8.-000852 Table 64. Room Temperature Extract Stability for PFOS Al concentaions se expeesed as pb. Run Number Run Date 32 10-Feb-1999 LOW QC After 24 Hours 1 35.9 2 36.5 | 3 323 Mean S.D. % CV. Control (ng/mL) Mea%n Dev. from Control 349 227 6.51% 412 153% . HIGH QC After 24 Hours 1 300 23 227315 ! Mean 269 \ %S.CDv. 132216% ! Control (ng/mL) 354 Mean % Dev. from Control 24.1% Control = mean ofreference QC samples Page? 000074 Northwest Bioanalytical ReSptourdty NNoo., NNWWBBRS9998..000552 `Table 65. Room Temperature Extract Stability for PFOSA All concentrations ae expresse1ds ppb. Run Number Run Date 32 10-Feb-1999 LOW QC After 24 Hours 1 19.7 2 19.4 ] 3 16.1 Mean SD. % CV Control (ng/mL) Mean% Dev. from Control 184 1.997 109% 204 9.80% ! HIGH QC After 24 Hours 1 31 2 239 ' 3 270 Mean 273 S.D. 36.12 , % Cv 13.2% Control (ng/mL) 332 Mea%n Dev. from Control 17.7% Control = meanofreference QC samples Page 73 000075 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS9998..000852 `Table 66. Room Temperature Extract Stability for PFOSAA All concentrations are expresseda ppb. Run Number Run Date 32 10-Feb-1999 LOW QC fier 24 Hours 1 126 | 2 17 3 102 Mean 1s sD. 121 % CV 10.5% Control (ng/mL) 127 Mean % Dev. from Control 9.45% HIGH QC 1 167 2 133 3 147 Mean 149 sD. 17.1 ' % Cv 115% Control (ng/mL) 194 Mean % Dev. from Control 232% * > 20% theoretical Control = mean of reference QC samples Page 74 000076 Northwest Bioamalytcal ReSptourdtyNNoo.. NNWWBBRSSS98..000852 Table 67. Room Temperature Extract Stability for N-MeFOSE-OH Al concentrations se expressed as pp. Run Number Run Date 32 10-Feb-1999 LOW QC After 24 Hours 1 128 2 135 | 3 10.1 Mean SD. % CV Control (ng/mL) Mean % Dev. from Control 121 180 14.8% 144 15.7% ! HIGH QC After 24 Hours 1 276 . 2 257 : 3 202 Mean 258 S.D. 17.0 | % CV 6.60% i Control (ng/mL) 286 Mean % Dev. from Control 9.67% Control = meanofreference QC samples Page 75 000077 Northwest Bioanalytical ReSptourdtyNNoo., NNWWEBRS9998..000852 `Table 68. Room Temperature Extract Stability for N-EtFOSE-OH All concentrations are expressed 1s ppb. Run Number Run Date 32 10-Feb-1999 LOW QC After 24 Hours 1 128 2 135 1 3 10.5 . Mean 123 S.D. 157 % CV 12.8% Control (ng/mL) 149 Mean% Dev. from Control 7.7% HIGH QC After 24 Hours 1 289 2 265 ! 3 247 Mean 267 SD. 211 | % CV 7.89% } Control (ng/mL) 292 Mea%n Dev. from Control 8.56% Control = meanofreference QC samples Page 76 000078 Northwest Bioanalytical ReSptourdtyNNoo., NNWWBBRS9998..000852 `Table 69. Room Temperature Extract Stability for POAA All concentrations are expressed as ppb. Run Number Run Date 2 10-Feb-1999 LOW QC After 24 Hours 1 217 2 2.4 ] 3 19.1 . Mean 211 SD. 174 % CV 825% Control (ng/mL) 226 Mean% Dev. from Control 6.78% ! HIGH QC After 24 Hours 1 290 . 2 222 i 3 247 Mean 253 S.D. 344 % CV 13.6% | Control (ng/mL) 324 Mean % Dev. from Control 219% Control = mean of reference QC samples Page 77 000079 Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBSR998.-000852 `Table 70. Room Temperature Extract Stability for PFHS Al concentrations ae expressed as pp. Run Number Run Date 2 10-Feb-1999 LOW QC After 24 Hours 1 207 2 199 i 3 18.1 Mean SD. % CV Control (ng/mL) Mean % Dev. from Control 196 133 681% 218 "10.2% HIGH QC After 24 Hours 1 283 2 216 3 241 Mean 247 SD. 339 , % CV 13.7% I Control (ng/mL) 333 Mean % Dev. from Control 259% Control = meanofreference QC samples Page 78 000080 Northwest Bioanalytical Table 71. Stock Solution Stability for PFOS ReSptourdtyNNoo.. NNWWBBRS9998-.000852 Analyte Peak Area Prepared dayof analytical run 121195 128494 5134 Mean 124941 sD 3653 %CV 292 n 3 ] Storedfor 24 days 143208 132499 122485 Mean 132731 SD 10363 %CV 7.81 n 3 : % Difference 623% | Page 79 000081 Northwest Bioanalytical `Table 72. Stock Solution Stability for PFOSA ReSptourdtyNNoo..NNWWBBSR9S8..000552 Analyte Peak Area Prepared day of analytical run 41058 34117 84507 Mean 53227 sp 27310 %Cv 513 n 3 ] Storedfor 24 days 63582 58872 46572 Mean 56342 sD 8783 %CV 15.6 n 3 : % Difference 585% { i 000082 Page 80 Norwest Bioanalytical Table 73. Stock Solution Stability for PFOSAA ReSptourdty NNoo.. NNWWBBRSSI58..000552 Analyte Peak Area Prepared dayof analytical run 14044 15654 15150 Mean 14949 SD 824 %CV 5.51 n 3 !i Storedfor 24 days 17264 16136 13572 Mean 15657 %eSvD 1182912 n 3 ' % Difference 474% | Pages) 000083 | Norte Baayen ReSptoryt NNao., NNWWBBRS9998..000852 Table 74. Stock Solution Stability for N-MeFOSE-OH Analyte Peak Area Prepared dayofanalytical run Mean weSyD n 115190 90190 253 85878 33166899 3 | Storedfor 24 days 169447 175317 125816 Mean 156860 SD 27045 % CV 172 n 3 Difference 827% i 000084 Pgesz Northwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS99S8..000852 `Table 75. Stock Solution Stability for N-EtFOSE-OH Analyte Peak Area Prepared dayofanalytical run 13772 87610 52034 Mean 84472 sD 30988 %CV 36.7 n 3 ! Storedfor 24 days 160189 166768 115012 Mean 147323 sD 28175 %Cv 19.1 n 3 % Difference 74.4% 1 Page 83 000085 Northwest Bioanalytical `Table 76. Stock Solution Stability for POAA ReSptourdtyNNoo.. NNWWBBRSSS58..000552 Analyte Peak Area Prepared dayofanalytical run 200388 206992 208145 Mean 204175 sD 3407 %Cv 167 n 3 I Storedfor 24 days 200970 200039 : 183527 Mean 194845 sD 9813 %CvV 5.04 n 3 : % Difference 4.57% i || 000088 Pages Northwest Bioanalytical `Table 77. Stock Solution Stability for PFHS ReSptourdtyNNoo,. NNWWBBRS9998..000852 Analyte Peak Area Prepared dayof analytical run 212505 21778 18307 Mean 217530 sD 4685 %wov 215 n 3 | Storedfor 24 days 230603 224507 200109 Mean 218540 sD 16214 wey 742 n 3 %Difference 0.464% ) Page ss 000087 Northwest Bioanalytical 6. ANALYTICAL METHOD ReSptourdty NNoo.. NNWWBBRS9998..000652 Principles of the Method After the additionofHPLC-grade water and sodium dodecyl sulfate (SDS, internal standard) to 0.20 mLofhuman or rabbit serum, the serum mixture is made basic with the addition of 0.5 M tetrabutylammonium hydrogen sulfate (TBA, pH 10) and 0.25 M carbonate buffer. This mixture is then extracted with methyl-tert-butyl ether. After sufficient mixing, the sample is centrifuged. The organic layer is transferred via pipette into a clean test tube. The organic layer is then evaporated to dryness and the sample is reconstituted into 2 mM ammonium acetate ] water:methanol (50:50 v/v). The extracts are then analyzed by liquid chromatography/tandem mass spectrometry using negafive-ion electrospray ionization and multiple reaction monitoring, CHEMICAL FORMULAS APbFbOrSeviation Chemical Name Perfluorooctane sulfonate: Formula CgF17505 PFOSA Perfluorooctane sulfonylamide CgF1750;NH, PFOSAA Perfluoroostane sufonylamido(ethylacetate CaF1S0,N(CHyCHy)CH2C05- PHS Perfluorohexane sulfonate CeF13503" PoAA Perfluorooctanoate C7F15C0y" N-EFOSE-OH 2(N-ethylperfluorooctanesulfonamidoethanol - CgF7 S03 N(CH,CH3)(CH,CH,0H) : N-MeFOSE-OH 2(N-methylperfluorooctanesulfonamidoethanol CgF7 SO N (CH3)(CHyCHOH) Ds Dodecyl sulfate (sodium) CH3(CHy) 1g CH 0503 6.1. Reference Materials and Matrices 1 _-- Reference Material Lot -_-- Number Purity Espiration Date Source Storage Conditions PFOS. 193 100% 12312010 3M Room Temperature PFOSA 214 100% 12312010 3M Room Temperature PFOSAA a7 538% 12312010 3M Room Temperature N-MeFOSE-OH a2 100% 1231/2010 3M Room Temperawre N-BFOSE-OH 96 100% 12312010 3M Room Temperaure Page S6 000083 Norwest Bioanalytical Reference Material TT roaA PFHS (in methanol) DS (sodium dodecyl sulfate) Lot Number 285 $398-182 17HO4S9 Purity 100% (6210000p%pm) 91.0% Expiration Date 12512010 2312000 1162001 ReSptourdty NNoo.. NNWWBBRS9998..000852 Source Storage Conditions 3M RoomTemperate 3M 20C Sigma Room Temperature Matrix Human serum ] Rabbit serum 62. Chemicals and Equipment Source Biochemed Biochemed Ammonium Acetate, 99.9% Water, HPLC-grade Chemicals ! Methanol, HPLC-grade , Methyl-terr-Butyl Ether, HPLC-grade ! `Sodium Hydroxide, 98.4% : Sodium Carbonate, 101.0% Sodium Bicarbonate, 100.3% `Tetrabutylammonium Hydrogen Sulfate (TBA), approx. 97% | Equip/mSueppnlites : Model Manufacturer Balance: Mettler Toledo AT261 Mettler-Toledo, Inc., Hightstown, NJ Balance: Mettler Toledo MTS Mettler-Toledo, Inc., Hightstown, NJ Centrifuge: Beckman GS-6R Beckman Instruments, Fullerton, CA Evaporator: Turbo Vap LV, Model 43750 Zymark Corp., Hopkinton, MA Liquid Chromatograph: Hewlett Packard 1100 Hewlett Packard, Palo Alto, CA Mass Spectrometer: Perkin Elmer Sciex API 3000 Perkin Elmer Sciex, Thomhill, Page 87 030089 Norwest Bioanalytical ReSptourdtyNNoo.. NNWWBBRS9998..000852 Equip/mSueppnlites Model Manufacturer Ontario - PH Meter: Orion 230A Orion, Boston, MA Pipettes: Finnpipette: Digital 1-5 mL Pipettes: Rainin PipetmanTM Volumes: fixed20; adjust.: 20, 100; 200; 1000 uL Sonicator Stir Plate: Nuova II Fisher Scientific, Pittsburgh, PA Rainin Instrument Co., Wobum, MA Branson, Danbury, CT `Thermolyne Corp., Dubuque, IA `Vortex: Fisher Genie 2 Fisher Scientific, Fair Lawn, NJ ] 63. Reagents, Calibration Standard and Quality Control (QC) Solutions `The calibrator, quality control and stock solution concentrations listed below are reported as they were prepared for the validation study and serve as a general guideline for future preparations. Reagents | All reagent solutions are stored at room temperature. + 0.25 M carbonate buffer : Add approximately 500 mL HPLC-grade waterto an appropriate 1-L container. Add 26.5 gofsodium carbonate (N2:CO3) to the 1-L container. Add 21.0 gofsodium bicarbonate (NeHCO3) to the 1-L container. Fill to volume with HPLC-grade water. Use a tir plate | to mix. + 10N sodium hydroxide Add 400 g ofsodium hydroxide (NaOH) to an appropriate 1-L container. Slowly add approximately 900 mL of HPLC-grade water while mixing. After the NaOH is completely dissolved, allow the solution to cool to room temperature. Fill to volume with HPLC-grade water and mix. Caution: The solution will become very hot as water is added! Prepare in the hood. 000090 Page 8 Northwest Biossalytcal ReSptourdty NNoo,. NNWWBBRS9998.-000832 + 0.5 M Tetrabutylammonium Hydrogen Sulfate (TBA, pH 10) `Add approximately 50 mL of HPLC-grade water to an appropriate 100-mL container. Add 16.9 g of TBA to the container. Adjust the pH with 10 N sodium `hydroxide. Fill to volume with HPLC-grade water and mix. Note: Be sure to check the pH daily when in use. + 2mM ammonium acetate in water Add 1 mL of | M ammonium acetate to a 500-mL container approximatelyhalffull with HPLC-grade water. QS to volume with HPLC-grade water and mix by inversion. + 2mM ammonium acetate in methanol | Fill an appropriate 1-L container approximatelyhalffull with methanol. Weigh out 0.154 g ofammonium acetate (NH OAc) andtransferto the container. Usea stir plate to mix. Adjust the pHifdesired and fill to volume with methanol. + 2mM ammonium acetate water:methanol (50:50 vv) Fill an appropriate 1-L container with 500mLof 2 mM ammonium acetate in water and 2 mM ammonium acetate in methanol. Calibration Standard and Quality Control (QC) Solutions All calibration standard solutions are transferred to 16 x 100 mm polypropylene screw-cap tubes and stored in a -20 C freezer. + PFOS Stock Standard (1053 ppm) Weigh 10.527 mgof PFOS and transfer to a 10-mL volumetric flask. QS to volume with ! `methanol. Sonicate for approximately 5 minutes. + PFOSA Stock Standard(1078 ppm) Weigh 10.778 mg of PFOSA and transfer to a 10-mL volumetric flask. QS to volume `with methanol. Sonicate for approximately 5 minutes. + PFOSAA Stock Standard (1031 ppm) Weigh 10.310 mg of PFOSAA and transfer to a 10-mL volumetric flask. QS to volume with methanol. Sonicale for approximately minutes. Pages 000094 Norwest Bioanalytical ReSptourdtyNNoo..NNWWBBSR998.-000852 + N-MeFOSE-OH Stock Standard(1210 ppm) `Weigh 12.103 mg of N-MeFOSE-OH and transfer to a 10-mL volumetric flask. QS to `volume with methanol. Sonicate for approximately $ minutes. + N-EtFOSE-OH Stock Standard(1127 ppm) Weigh 11.266 mg of N-EtFOSE-OH and transfer to a 10-mL volumetric flask. QS to volume with methanol. Sonicate for approximately 5 minutes. + POAA Stock Standard (1090 ppm) Weigh 10.9mgofPOAA and transfer to a 10-mL volumetric flask. QS to volume with 1 `methanol. Sonicate for approximately 5 minutes. , + PFHS Stock Standard (1000 ppm) i `Add 807 pL of PFHS (6200 ppm) to a S-mL volumetric flask. QS to volume with `methanol. Mix by inversion. + Diluted Stock Solution (10.0 ppm) { Add 95 pL of PFOS Stock Solution (1053 ppm), 93 pLof PFOSA Stock Solution (1078 | ppm), 97 uL of PFOSAA Stock Solution (1031 ppm), 83 uLof N-MeFOSE-OH Stock | Solution (1210 ppm), 89 L ofN-EFOSE-OH Stock Solution (1127 ppm), 92pLof | PPOAA Stock Solution (1090 ppm) and 100 uLof PFHS Stock Solution (1000 ppm) to a 10-mL volumetric flask. QS to volume with methanol. Mix by inversion. ; + Spiking Standard 8 (5.00ppm) Add 500 uL of methanol and 500 pLofDiluted Stock Solution (10.0 ppm) to a 13 x 100 | `mm silanized glass tube. Mix by vortexing. | - Spiking Standard7(2.50 ppm) Add 500 pLofmethanol and 500 uLof Spiking Standard 8 (5.00 ppm) to a 13 x 100 mm silanized glass tbe. Mix by vortexing. 000092. Page 90 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS9998..000852 + Spiking Standard 6 (1.00ppm) Add 800 kLof methanol and 200 LofSpiking Standard 7 (5.00 ppm) to.2 13 x 100 mm silanized glass tube. Mix by vortexing. + Spiking Standard 5 (0.500ppm) Add 900 kL ofmethanol and 100 Lof Spiking Standard 7 (5.00 ppm) to a 13 x 100 mm silanized glass tube. Mix by vortexing, + Spiking Standard 4 (0.250ppm) Add 950 HLof methanol and 50 uLofSpiking Standard 7 (5.00 ppm) to a 13 x 100 mm | silanized glass tube. Mix by vortexing. + Spiking Standard 3 (0.100ppm) Add 900 kLof methanol and 100 kLof Spiking Standard 6 (1.00 ppm) to a 13x 100 mm silanized glass tube. Mix by vortexing + Spiking Standard 2 (0.0500ppm) Add 900 iL ofmethanol and 100 kL of Spiking Standard $ (0.500 ppm) to 2 13 x 100 3 mm silanized glass tube. Mix by vortexing + Spiking Standard 1 (0.0100ppm) | Add 900 kLof methanol and 100 Lof Spiking Standard 3 (0.100 ppm) to a 13 x 100 mn silanized glass tube. Mix by vortexing Internal Standard Solutions : All intemal standard solutions are transferred to 16 x 100 mm polypropylene screw-cap tubes and stored in 2-20 C freezer. - SDS Stock Solution (1091 ppm) Weigh 5.986 mg of SDS and transfer to a 5-mL volumetric flask. QS to volume with methanol. Sonicate for 10 minutes. 000093 Paget Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS9S98.-000852 SDS Diluted Stock Solution (10.0 ppm) Add 91.7 pLof SDS Stock Solution (1091 ppm) to 2 10-mL volumetric flask. QS to volume with methanol and mix by inversion. + SDS Working Internal Standard (1.00 ppm) Add 2.50 mLof SDS Diluted Stock Solution (10 ppm) to a 25-mL class A volumetric flask. QS to volume with methanol and mix by inversion. 64. Preparation of Quality Control Samples High Quality Control (350 ppb spiked) | Quantitatively transfer 1.05mLofthe Diluted Stock Solution (10.0 ppm) and 28.95 mL of | `blank human serum into a polypropylene tube. Sonicate for approximately 10 minutes and equilibrate for approximately 10 minutes. Low Quality Control (20.0 ppb spiked) Quantitatively transfer 60 uLofthe Diluted Stock Solution (10.0 ppm) and 29.94 mL of : `blank human serum into a polypropylene tube. Sonicate for approximately 10 minutes and ! equilibrate for approximately 10 minutes. Storage ofOC Samples { After preparation, place aliquotsof the low and high QC pools into 2-mL cryogenic vials, and store in a -20 C freezer. 6.5. Preparation of Calibration Standards | Ifendogenous analyte concentrations in human serum need to be calculated, a rabbit serum | calibration curve is prepared by spiking standards as shown in the table below. The calibration curve range is 1.00 ppb to 50.0 ppb. Page 92 00 0094 Northwest Bioanalytical Study No. NWBS98.082 Report No. NWBR99.005 Standard| Concentration of| Volume of Spiking| Volume of Blank | Final Spiked Number| Spiking Solution Solution Rabbit Serum | Concentration (ppm) (HL) (mL) (ppb) [ses Tm[oa [so| [eo [om TT [ eo TT m0 | [2[ ooemm T= T [ ooT xw Tsiem]| `The human serum calibration curve is prepared on the day of each run by spiking standards as "shown in the table below. The unadjusted calibration curve range is 1.00 ppb to 500 ppb. 1 ! SNtuamnbdaerrd|| SCpoinkcienngtrSaotliuotnioonf | VolSuomleuotfioSpniking| VHoulummaenoSfeBrluamnk || CFoinncaelntSrpaitkieodn (ppm) (HL) (mL) (pb) [o &[w sw | 12 | ooxw [ | swom|] ; [o 5[ew [m 20 | 0 a w |[ smme|] ; oToowm1 [ o 1 oowwn T ||swwe|] : Co eow 1 20 | on [ ww | 6.6. Sample Preparation Calibration Curve Samples (prepare in duplicate) Rabbit Serum Curve Transfer 0.20 mLofHPLC-grade water and 0.20 mL rabbit serum into appropriately labeled 13x 100 mm polypropylene tubes. Add 20 kL of Calibration Standard Spiking Solutions 1, 2,4 and 5 to the corresponding labeled tube. Page 03 000095 Northwest Bioanalytical Human Serum Curve ReSptourdtyNNoo,. NNWWBBRSS998..000852 Transfer 0.20 mLofHPLC-grade water and 0.20 mL human serum into appropriately labeled 13x 100 mm polypropylene tubes. Add 20 kL of Calibration Standard Spiking Solutions 1-8 10 the corresponding labeled tube. Quality Control Samples (prepare in duplicate) Aliquot 0.20 mLofeachofthe Low and High controls into appropriately labeled 13 x 100 `mm polypropylene tubes. ] Aliquot 0.20 mLofblank human serum into separate 13 x 100 mm polypropylene tubes and label as QCO. ! Aliquot 0.20 mLofblank human serum into separate 13 x 100 mm polypropylene tubes and . label as BLANK Aliquot 0.20 mLofblank rabbit serum into separate 13 x 100 mm polypropylene tubes and | label as RABBIT BLANK. | Study Samples | Transfer 0.20-mL aliquotsofeach study sample into appropriately labeled 13 x 100 mm : polypropylene tubes. ) Extraction Procedure | } 1. cAadldibr0a.t2i0onmsLtaonfdHaPrdLCs-pigkriangdesowlauttieorn(sthtioscsahlioburladtiboen performed before curve samples). adding the 2. Add 20 uL of SDS intemal standard (1.00 ppm) to all samples, excluding BLANKS. 3. Vortex samples for at least 5 seconds. 4. Add 0.25 0.40 mL 0.5 M carbonate Mbuftfeert.raNbOutTyEl:amCmhoenciukmthheydprHogoefnTsBulAfadtaeil(yT.BA), pH 10, and 0.40 mL of 5. Add 3.0 mL methyl-tert-butyl ether. Poge 94 000096 Northwest Bioanalysis! ReSptourdtyNNoo.. NNWWBBRS9995..000852 6. Cap cach sample, vortex and rotate for approximately 10 minutes. 7. Centrifuge at approximately 3500 rpm for 10 minutes (or until the layers are well separated). 8. Transfer the organic layer to a clean 13x 100 mm polypropylene tube with adisposable transfer pipet. 9. Dry samples under nitrogen at 45 C until dry (approximately 10 minutes) 10. Add 75 pL of2mM ammonium acetate water:methanol (50:50 v/v) o each tube. 11. Vortex for at least 5 seconds. 1 12. Transfer into autosampler vial insert. ! 13. Cap and store extracts at 4 C until analysis. 67. LC/MS/MS Conditions LC Conditions i Column LD. Betasil C-18 Mobile Phases) | A: 2mM ammonium acetate in water B: 2mM ammonium acetate in methanol Flow Rate 300 pLmin | Column Temperature 50C Injection Volume S-15pL LC Conditions Gradient | `The HPLC gradient required is shown below. : Time % Mobile PhasAe % Mobile PhasBe (min.) 0.00 EY 50 450 50 50 110 27 97.3 120 27 973 130 50 50 Page 0s 000097 Norwest Bioanalytical ReSptourdtyNNoo., NNWWBBRSS9S8..000852 MS Conditions Post Column Split 21 Source Turbo lon Spray Source Temp. 300C Analysis type Multiple reaction monitoring (MRM) Compounds PFOS Transitions monitored 499/80 Dwell Time Collision (ms) Energy (v) 100 El PFOSA 498/78 100 38 | PFOSAA 584/419 200 30 N-MeFOSE-OH 616/59 100 52 N-EtFOSE-OH 630/59 100 E POAA 4137369 100 1s PFHS 399/80 100 48 SDs 265/97 100 a `The prepared standards and QC are loaded onto the autosampler tray in a random order and injected into the instrument LC/MS/MS system. 68. Quantitation PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA, PFHS and SDS chromatographic peaks are integrated using the instrument manufacturer supplied software with a smooth factor of 1. Quantitation of the rabbit calibration curve is based upon linear ! regression analysisof calibration curves (weighted 1/x) using the analyte peak areas. This quantitation is performed using the instrument manufacturer supplied software. Quantitation of the human calibration curve is based upon quadratic regression analysisofcalibration curves (weighted 1/x) using the peak area ratio (analyte peak area/internal standard peak area) vs. concentration. This quantitation is performed using the Watson DMLIMS software. -- 000038 Northwest Bioanalytical ReSptourdty NNoo.. NNWWBBRS9S98..000852 Figure 1. Total Ion Chromatogram of Rabbit Serum Blank - - : -- | - SDS-- ) Page97 C50099 Noriwes Boas RSepuodnyNNoo.. NNWWBBRS9998..000552 Figure 3. Total Ton Chromatogram of High Standard (500 ppb) " i" fo- :i a | we I PFOSA/PFOSAA SDS - | Hi POAA pris | PFOS a -- MeFOSE-OH | -- EtFOSE-OH | || \ re 000100