Document emE7Xa4vd96B3j3j2bpLBLmkm

NorthwesBtioanalytical RSetpuodrytNNoo..NNWW''BBRS9998-=000852 ' Quantitative Determination of PFOS, PFOSA, PFOSA.A, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS in Human Serum by LC/MS/MS ! Assay Validation Report Northwest Bioanalytical (NWB) A Division of NWT Inc. 1121 East 3900 South Salt Lake City, UT 84124 t PREPARED FOR: 3M 935 Bush Avenue St. Paul, MN 55133 AUTHOR: _ David L. Vollmer, Ph.D., Project Manager APPROVED FOR RELE_Y,#_: )( :.7" _'7"::--__ " Rodger L. boltz, Ph.D., Laboratory_rector Page l DATE: I2"/'3/t_ DATE: 5"//_/:,- /_ / / Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 TABLE OF CONTENTS SIGNATURE PAGE .................................................................................................................................... 1 TABLE OF CONTENTS ............................................................................................................................. 2 LIST OF TABLES ....................................................................................................................................... 3 LIST OF FIGURES ......................................................................................................... i............................ 6 1. INTRODUCTION ................................................................................................................................. 7 2. VALIDATION SUMMARY" ................................................................................................................. 9 2.1. Range of Quantitation 9 2.2. Precision and Accuracy ................................................................................................................ 10 2.3. Repeatability and Reproducibility Test ........................................................................................ 10 2.4. System Precision Test ................................................................................................................... 11 2.5. Extraction Efficiency .................................................................................................. .................. 11 2.6. Stability Evaluation ...................................................................................................................... 12 3. DATA MANAGEMENT .................................................................................................................... 13 4. COMMENTS AND CONCLUSIONS ................................................................................................ 13 4.1. Proposed Sample Analysis Acceptance Criteria ........................................................................... 15 5. REFERENCES .................................................................................................................................... 16 6. ANALYTICAL METHOD ................................................................................................... .............. 86 6.1. Reference Materials and Matrices ......................................................................................... ,...... 86 6.2. Chemicals and Equipment ..................................................................... i...................................... 87 1 6.3. Reagents, Calibration Standard and Quality Control (QC) Solutions ............ .............................. 87 6.4. Preparation of Quality Control Samples ....................................................................................... 92 6.5. Preparation of Calibration Standards ............................................................................................ 92 6.6. Sample Preparation._ ..................................................................................................................... 93 6.7. LC/MS/MS Conditions ................................................................................................................. 95 6.8. Quantitation .............. i........................................................... ........................................................ 96 Page 2 NorthwestBioanalytical LIST OF TABLES StudyNo. NWBS98-082 ReportNo. NWBR99-005 Table 1. Summary of Rabbit Serum Calibration Curves for PFOS ......................................... :................. 18 Table 2. Summary of Rabbit Serum Calibration Curves for PFOSAA ..................................................... 19 Table 3. Summary of Rabbit Serum Calibration Curves for POAA .......................................................... 20 Table 4. Summary of Rabbit Serum Calibration Curves for PFHS ........................................................... 21 Table 5. Summary Data for Endogenous Analyte Concentrations in Human Serum Calibration Standards for Run 25 ................................................................................................................. 22 Table 6. Summary Data for Endogenous Analyte Concentrations in Human Serum Calibration 11, Standards for Runs 26, 28 and 32 ............................................................................................. 23 : Table 7. Summary Data for Endogenous Analyte Concentrations inHuman Serum Quality Control ' Samples for Runs 26, 28 and 32......................................... . ...................................................... 24 : Table 8. Summary of Human Serum Calibration Curve Parameters for PFOS ........................................ 25 Table 9. Summary of Human Serum Calibration Curve Parameters for PFOSA ..................................... 25 Table 10. Summary of Human Serum Calibration Curve Parameters for PFOSAA ................................. 26 Table 11. Summary of Human Serum Calibration Curve Parameters for N-MeFOSE-OH ...................... 26 Table 12. Summary of Human Serum Calibration Curve Parameters for N-EtFOSE-OH ........................ 27 Table 13, Summary of Human Serum Calibration Curve Parameters for POA.A ................. i................... 27 Table 14. Summary of Human Serum Calibration Curve Parameters for PFHS ....................................... 28 Table 15. Back-Calculated Concentrations ot: Human Serum Calibration Standards for PFOS .............. 29 Table 16. Back-Calculated Concentrations of Human Serum Calibration Standards for PFOSA ............ 30 Table 17. Back-Calculated Concentrations of Human Serum Calibration Standards for PFOSAA ......... 31 Table 18. Back-Calculated Concentrations of Human Serum Calibration Standards for N-MeFOSE-OH ................................................................................................................ 32 Table 19. Back-Calculated Concentrations of Human Serum Calibration Standards for N-EtFOSE-OH ............................................................................................................... 33 Page 3 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 20. Back-Calculated Concentrations of Human Serum Calibration Standards for POAA .............. 34 Table 21. Back-Calculated Concentrations of Human Serum Calibration Standards for PFHS ............... 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 ............... . ................ 42 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-EtFOSE-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 Serum Quality Control Samples .... :.-.... :.................... 49 Table 36. System Precision for PFOS ........................................................................................................ 50 Table 37. System Precision for PFOSA ............................................................................................... ...... 50 Table 38. System Precision for PFOSAA .................................................................................................. 51 Table 39. System Precision for N-MeFOSE-OH ....................................................................................... 51 Table 40. System Precision for N-EtFOSE-OH ......................................................................................... 52 Table 41. System Precislon for POAA ...................................................................................................... 52 Table 42. System Precision for PFHS ........................................................................................................ 53 Table 43. PFOS Extraction Efficiency ...................... i................................................................................ 54 Page 4 l Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 ' Table 44. PFOSA Extraction Efficiency .................................................................................................... 54 Table 45. PFOSAA Extraction Efficiency ................................................................................................. 55 Table 46. N-MeFOSE-OH Extraction Efficiency ...................................................................................... 55 Table 47. N-EtFOSE-OH Extraction Efficiency ........................................................................................ 56 Table 48. POAA Extraction Efficiency ..................................................................................................... 56 Table 49. PFHS Extraction Efficiency ....................................................................................................... 57 Table 50. Freeze/Thaw Stability for PFOS ................................................................................................ 58 Table 51. Freeze/Thaw Stability for PFOSA ............................................................................................. 59 Table 52. Freeze/Thaw Stability for PFOSAA .......................................................................................... 60 Table 53. Freeze/Thaw Stability for N-MeFOSE-OH ............................................................................... 61 Table 54. Freeze/Thaw Stability for N-EtFOSE-OH ................................................................................. 62 Table 55. Freeze/Thaw Stability for POA.A ............................................................................................... 63 Table 56. Freeze/Thaw Stability for PFHS ................................................................................................ 64 Table 57. Room Temperature Matrix Stability for PFOS .......................................................................... 65 Table 58. Room Temperature Matrix Stability for PFOSA ....................................................................... 66 Table 59. Room Temperature Matrix Stability for PFOSAA ............................................................. i...... 67 Table 60. Room Temperature Matrix Stability for N-MeFOSE-OH ......................................................... 68 'Table 61. Room Temperature Matrix Stability for N-EtFOSE-OH ........................................................... 69 1 ) J Table 62. Room Temperature Matrix Stability for POA.A ........................................................................ 70 Table 63. Room Temperature Matrix Stability for PFHS .......................................................................... 71 Table 64. Room Temperature Extract Stability for PFOS ......................... ............................................... 72 Table 65. Room Temperature Extract Stability for PFOSA ...................................................................... 73 Table 66. Room Temperature Extract Stability for PFOSAA ................................................................... 74 Table 67. Room Temperature Extract Stability for N-MeFOSE-OH ........................................................ 75 Page 5 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 68. Room Temperature Extract Stability for N-EtFOSE-OH .......................................................... 76 Table 69. Room Temperature Extract Stability for POAA ....................................................... ................. 77 Table 70. Room Temperature Extract Stability for PFHS ......................................................................... 78 Table 71. Stock Solution Stability for PFOS ............................................................................................. 79 Table 72. Stock Solution Stability for PFOSA .......................................................................................... 80 Table 73. Stock Solution Stability for PFOSAA ....................................................................................... 81 Table 74. Stock Solution Stability for N-MeFOSE-OH ............................................................................ 82 Table 75. Stock Solution Stability for N-EtFOSE-OH ............................................................................... 83 Table 76. Stock Solution Stability for POAA ............................................................................................ 84 Table 77. Stock Solution Stability for PFHS ............................................................................................. 85 LIST OF FIGURES Figure 1. Rabbit Serum Blank ........ i.................. ........................................................................................ 97 Figure 2. Human Serum Blank ...... _............................................................................... ........................... 98 Figure 3. High Standard (500 ppb) ........................................................................................................... 99 Page 6 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 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 chromatography/tandem mass spectrometry method for the measurement ofPFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS in human serum. For chemical 1 names and structures, see the Analytical Method contained in section 6 of the "report. ; This report summarizes the analytical results from the validation of the method for quantitation ofPFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-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 NWB supervisory personnel involved in the completion of this 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. 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 of a GLP regulated nonclinical study (Title 21 of the US Code of Federal Regulations Part 58). However, Northwest Bioanalytical conducts all studies within the guidelines of GLP principles. Background Early method development experiments demonstrated varying levels of endogenous PFOS, PFOSAA, POAA, and PFHS in several lots of"blank" human serum. These endogenous levels complicate tile use of human serum for the preparation of calibration standards and quality control samples. Page 7 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 To circumvent the difficulty of preparing standards and controls in the presence of endogenous levels, several animal matrices were evaluated. Rabbit serum showed the lowest endogenous levels of the 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 ofPFOSA, MeFOSE-OH, and EtFOSE-OH. A serum matrix that was pre-extracted from human serum to remove any endogenous levels of analytes prior to preparation of the curve was also evaluated. This serum matrix, however, showed greater extraction recoveries for all the analytes in the calibration curve samples than in the human serum samples that were not pre-extracted, and therefore did not demonstrate acceptable accuracy for quantitation of the analytes. 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 of human serum. Once the endogenous levels were determined for those lots of human serum, those concentrations were added to the spiked concentrations for the human serum QCs and for the human serum calibration standards. This procedure is described in detail within this report. Principles of the Method After the addition of HPLC-grade water and sodium dodecyl sulfate (SDS, internal standard) to 0.20 mL of human 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 negative-ion electrospray ionization and multiple reaction monitoring. Page S NorthwestBioanalytical 2. VALIDATION SUMMARY StudyNo. NWBS98-082 Report No. NWBR99-005 Four separate analytical runs were used in the determination of linearity, precision, and accuracy ofPFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS. The validation included determination of the stability of the analytes maintained under various storage conditions, the extraction efficiencies of the analytes and the internal standard, the system precision of the analytes, and the reproducibility of the 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 List of Tables for the location of the validation test data and summary statistics. 2.1. Range of Quantitation Rabbit Serum Calibration Curve ' Each analytical run included calibration standards in duplicate at a minimum of four different i concentrations and at least two rabbit serum blanks. Data for PFOSA, N-MeFOSE-OH and i N-EtFOSE-OH are not included because there were no endogenous amounts detected in j human serum. l ! i Range (ppb) PFOS 1.00 to 50.0 PFOSAA 1.00 to 50.0 POAA. 1.00 to 50.0 PFHS 1.00 to 50.0 Mean Correlation Coefficient 0.9961 0.9957 0.9987 0.9982 ] Human Serum Calibration Curve I ? Each analytical run included calibration standards in duplicate at a minimum of seven different concentrations, a minimum of 10 quality control samples (QCs) (two levels in replicates of five), two "blanks" and two "0-ppb QCs" (QCs containing internal standard, but no spiked analytes). Page 9 NorthwestBioanalytical Spiked Concentration Range Mean Correlation Coefficient PFOS 1.00 to 500 ppb 0.9934 PFOSA 1.00 to 500 ppb 0.9969 StudyNo. NWBS98-082 Report No. NWBR99-005 PFOSAA 0.538 to 53.8 ppb NMeFOSE- OH 1.00 to 500 ppb NEtFOSE- OH 1.00 to 500 ppb POAA 1.00 to 500 ppb PFHS 1.00 to 500 ppb 0.9966 0.9971 0.9976 0.9972 0.9978 2.2. Precision and Accuracy The precision and accuracy of the LC/MS/MS method for the quantitation of PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-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-EtFOSE-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 to 13.3% for each QC concentration. The mean percent theoretical for all levels of quality controls ranged from 95.7 to 106.7. The inter-assay %CV for N-MeFOSE-OH and N-EtFOSE-OH were less than or equal to 25.5% for each QC concentration. The mean percent theoretical for all levels of quality controls ranged from 93.5 to 99.4. 2.3. 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 run number 32 met the same acceptance criteria used for run numbers 25, 26, and 28. Page I0 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 The analytical procedure demonstrated acceptable reproducibility for all the analytes except N-MeFOSE-OH and N-EtFOSE-OH. 2.4. 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 of a normal validation run. Instrument response is defined as the peak area ratio (analyte peak area/intemal standard peak area). The intra-assay %CV for PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS were less than or l equal to 78.2% for each QC concentration. t The unextracted samples did not demonstrate acceptable precision for all the analytes (See section 4 Comments and Conclusions for discussion of system precision.). 2.5. Extraction Efficiency t The extraction efficiencies for PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-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 intemal standard added following the extraction. 2. The analyte added to serum prior to extraction and the internal standard added following extraction. 3. The internal standard added to serum prior 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%). Page 11 Northwest Bioanalytical 2.6. Stability Evaluation Study No. NWBS98-082 Report No. NWBR.99-005 For the following stability evaluations, the mean test QC sample concentrations must fall within 20% of the mean reference QC sample concentrations to demonstrate acceptable stability. Freeze-thaw Stability Stability ofPFOS, PFOSA, PFOSAA, N-MeFOSE-0H, N-EtFOSE-0H, 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 il samples subjected to one freeze/thaw cycle. ii Samples demonstrated acceptable stability after three freeze/thaw cycles for all the analytes. I Room Temperature Matrix Stability Stability of PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POA.A and PFHS in human serum stored under normal room temperature and light conditions for up to 4 hours I 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 I conditions for all the analytes except N-MeFOSE-OH and N-EtFOSE-OH. I Room Temperature ;Extract Stability 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 acceptable stability after 24 hours at normal room temperature for all the analytes except PFOS, PFOSAA, POAA and PFHS analytes (See section 4 Comments and Conclusions for discussion of room temperature extract stability). Page 12 NorthwestBioanalytical Stock Solution Stability StudyNo. NWBS98-082 ReportNo. NWBR99-005 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 solutio n stability evaluation was performed in replicates of three at the same level of unextracted standard. Stock solutions demonstrated acceptable stability after storage at -20 C for 24 days for all the analytes except N-MeFOSE-OH and N-EtFOSE-OH. Long-Term Matrix Stability and Reduced Temperature (-20 C) Extract Stability 'l These stability tests will be performed and reported at a later date in a separate report. 3. DATA MANAGEMENT The concentration values for the endogenous levels ofPFOS, PFOSAA, POAA and PFHS were calculated using PE Sciex MacQuan software (v. 1.6). All other concentration values were calculated using the Watson DMLIMS soRware. Data presented in this report may be formatted to fewer significant figures than may have been used by the computer to generate means, standard deviations and coefficients of variation. Summary statistics may be derived from unfounded data and may differ slightly from the values obtained using the rounded values. 4. COMMENTS AND CONCLUSIONS ! During the assay validation, three potential internal standards were evaluated. They were nonanesulfonic acid, octanesulfonic acid and SDS. A fourth internal standard, CsH5FI3SO3, was evaluated during method development, but was unsuitable for this assay owing to a serum interferent that demonstrated different ionization suppression from lot to lot of human serum. Of the three internal standards, SDS gave the best results for all the analytes, and thus it was used as the internal standard for each analyte. To calculate the endogenous levels of the analytes in human serum, each validation run included: (1) "blank" human serum samples, prepared in duplicate or greater, from the lot used to prepare Page 13 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 the human serum calibration curve, (2) "blank" human serum samples in five replicates from the lot 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. If endogenous 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 level of 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 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 (sum of the endogenous concentration plus the concentration spiked into each QC), respectively. These average endogenous amounts for each analyte were used over the course of the 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 ;.fieldinga 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 course of the validation. The method described in this report has been validated for the determination ofPFOS, PFOSA, PFOSAA, POAA and PFHS in human serum. The method is not considered validated for NMeFOSE-OH and N-EtFOSE-GH. The reason the results for N-MeFOSE-OH and N-EtFOSEOH did not meet the acceptance criteria for validation could be due to the following factors: (1) poor extraction efficiencies, (2) poor ionization efficiencies (i. e., 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 fragrnentation of the acetate adduct of each analyte to the acetate ion (i. e., [M + CH3CO2]"-'-> [CH3CO2]'). Page 14 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 The poor system precision for all the analytes can be attributed to a "column conditioning" affect. That is, the analytical column used for this assay yields more precise results when it is primed by injection of two or more extracted samples. When unextracted samples are injected onto the column, the condition of the column is affected and the precision decreases. This was verified by 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. Acceptable room temperature extract stability was not demonstrated for the high QCs of PFOS, PFOSAA, POAA,. and PFHS. Owing to the apparent instability of the room 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 period for an accepted validation run. 4.1. Proposed Sample Analysis Acceptance Criteria : Subject Sample Determination Subject sample concentrations for all the analytes will be determined using the human serum calibration curve, unless an analyte concentration for PFOS, PFOSAA, POAA or PFHS is less that the adjusted LLOQ concentration. If a subject sample has a concentration for any of those analytes that is below the adjusted LLOQ, then the rabbit curve will be used to quantitate that analyte in that sample. In these cases, the accuracy of the rabbit serum curve will be verified by analyzing human serum QCs containing known analyte concentrations ! between 1 and 50 ppb and calculating their concentrations using the rabbit serum curve. Page 15 Northwest Bioanalytical Calibration Curve Study No. NWBS98-082 Report No. NWBR99-005 Each run will include calibration standards in duplicate at seven or more concenu:ations covering the lower to upper limit ofquantitation. 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. Ira 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 1 The back-calculated concentrations of at least one of the duplicate lowest points in the calibration curve must be within + 20% of the target concentration to qualify as the LLOQ. If this criterion is not met, the next level is subjected to the same test and the LLOQ raised accordingly. : Quality Control Samples i Each analytical run will include low and high QC samples in triplicate. The measured concentrations of at least two-thirds of all analytical QCs must be within + 20 % of their target concentrations. If study samples require dilution, a dilution QC will be analyzed in triplicate for each dilution level. At least one dilution QC at each level must be within _+20% of the target concentration. The dilution QC acceptance is independent of the undiluted analytical QC acceptance. 5. REFERENCES [5.1 ] L. Clemen, G. Langenburg, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Blood Extracts Using HPLC-Electrospray/Mass Spectrometry," 3M Environmental Laboratory, FACT-M-4.1. [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. Page 16 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 [5.3] L. Clemen, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Sulfactants from Liver for Analysis Using HPLC-Electrospray/Mass 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 ageement. 3M will be notified concerning final disposition of records at completion of contract obligations. "1 J Page 17 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 1. Summary of Rabbit Serum Calibration Curves for PFOS Linear (weighted I/x). All concentrations are expressed as ppb. Run Date Run 1.00 5.00 25.0 50.0 Number 30-Jan-1999 25 0.896 5.42 20.0 53.1 1.03 5.61 24.6 51.4 Slope 1524 Intercept Multiple Rsquared 266 0.9950 LLOQ ULOQ (ppb) (ppb) 1.00 50.0 01-Feb-1999 26 "0.671 4.44 24.3 47.2 1407 553 0.9950 1.00 50.0 1.20 4.80 22.7 56.3 [_ 02-Feb-1999 28 0.903 6.22 26.1 47.0 1414 657 0.9977 1.00 50.0 ! 0.887 4.65 26.3 50.0 03-Feb-1999 29 0.826 4.96 21.7 51.9 1580 944 0.9966 1.00 50.0 1.02 6.07 26.8 48.7 Mean S.D. %CV, n 0.928 5.27 24.1 50.7 1481 605 0.146 0.619 2.27 2.95 74 243 15.8 11.7 9.4 5.8 7 8 8 8 4 4 * sample deactivated - poor instrument response 0.9961 0.0011 4 i Page 18 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 2. Summary of Rabbit Serum Calibration Curves for PFOSAA Linear (weighted l/x). All concentrations are expressed as ppb. Run Date Run 1.00 5.00 25.0 50.0 Number 30-Jan-1999 25 0.857 5.41 19.9 55.0 2.07 6.16 23.0 50.8 ,m Slope 212 Intercept -30 Multiple Rsquared 0.9921 LLOQ (ppb) ULOQ (ppb) 1.00 50.0 01-Feb-1999 26 * 4.80 23.2 47.6 215 1.21 4.25 25.3 54.7 103 0.9967 1.00 50.0 02-Feb-1999 28 0.724 5.29 25.3 46.8 266 0.863 6.73 26.8 49.5 188 0.9962 1.00 50.0 m. 03-Feb-1999 29 0.702 4.98 24.9 47.2 313 1.09 6.16 25.1 51.8 I59 0.9979 1.00 50.0 Mean S.D. 1.07 5.47 24.2 50.4 0.442 0.770 1.98 3.02 252 105 41 .... 84 0.9957 0.0022 %CV 41.2 14.1 8.2 6.0 n 7 8 8 8 4 4 4 * sample deactivated - poor instrument response t 1 Page 19 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 3. Summary of Rabbit Serum Calibration Curves for POAA Linear (weighted I/x). All concentrations are expressed as ppb. Run Date Run Number 1.00 5.00 25.0 50.0 Slope Intercept Multiple LLOQ ULOQ Rsquared (ppb) (ppb) 30-Jan-1999 25 1.02 5.57 21.9 52.6 2543 0.917 5.22 24.2 50.5 81 0.9976 1.00 50.0 01-Feb-1999 26 *0.643 4.51 23.8 52.2 2782 318 0.9991 1.00 50.0 1.13 5.01 24.3 50.1 1 02-Feb-1999 28 0.835 5.56 23.4 48.2 2983 859 0.9985 1.00 50.0 1.01 5.31 26.6 51.1 03-Feb-1999 29 0.857 5.26 24.0 50.3 2988 1141 0.9994 1.00 50.0 1.00 5.66 24.8 50.1 Mean S.D. -%CV n 0.925 5.26 24.1 50.6 2824 600 0.139 0.352 1.22 1.27 182 421 15.0 6.7 5.1 2.5 7 8 8 8 4 4 * sample deactivated - poor instrument response "0.9987 0.0007 4 Page 20 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 4. Summary of Rabbit Serum Calibration Curves for PFHS Linear (weighted I/x). All concentrations are expressed as ppb. Run Date Run 1.00 5.00 25.0 50.0 Slope Intercept Multiple LLOQ ULOQ Number Rsquared (ppb) (ppb) 30-Jan-1999 25 0.833 5.87 22.5 52.9 2677 567 0.9955 1.00 50.0 0.876 5.50 23.4 52.3 01-Feb-1999 26 *0.742 4.85 23.2 50.7 2824 701 0.9990 1.00 50.0 ' 1.06 5.10 24.2 51.9 02-Feb-1999 28 0.882 5.45 23.2 49.5 3013 629 0.9990 1.00 50.0 1.08 4.94 25.4 51.6 03-Feb-1999 29 0.862 5.12 23.8 49.2 3018 974 0.9994 1.00 50.0 1.03 5.54 25.4 51.0 ;I Mean 0.921 5.30 23.9 51.1 2883 718 0.9982 i S.D. 0.113 0.326 1.00 1.22 142 155 0.0016 %CV 12.3 6.2 4.2 2.4 n 7 8 8 8 4 4 4 * sample deactivated - poor instrument response 1 Page 2 i Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 5. Summary Data for Endogenous Analyte Concentrations Standards for Run 25 in Human Serum Calibration PFOS PFOSA PFOSAA N-MeFOSE-OH N-EtFOSE-OH PFHS POAA Replicate 1 16.1 ND 1.60 ND 2 15.4 ND 1.94 ND 3 16.0 ND 0_943 ND : 4 17.3 N'D 1.34 ND ND 0.497 3.08 ND 0.666 3.00 ND 0.696 3.18 ND 0.602 3.52 , Mean 16.2 N/A 1146 N/A S.D. 0.796 N/A 0.421 N/A N/A 0.615 3.20 N/A 0.088 -0.228 _ % CV 4.9 N/A 28.9 N/A N/A 14.3 7.1 ! n 4 4 4 4 4 4 4 ND = none detected N/A = not applicable I I i Page 22 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 6. Summary Data for Endogenous Standards Analyte Concentrations in Human for Runs 26, 28 and 29 Serum Calibration Run 26 1 2 3 4 PFOS PFOSA PFOSAA N-MeFOSEOH N-EtFOSEOH POAA PFHS 25.4 ND 0.646 ND 31.3 ND 1.07 ND 27.5 ND 2.94 ND 33.3 ND 1.55 biD ND 3.91 1.33 ND 5.04 1.66 ND 4.59 1.68 ND 4.79 1.68 Mean 29.4 N/A 1.55 N/A S.D. 3.61 N/A 0.998 N/A % CV 12.3 N/A 64.3 N/A Run 28 1 36.8 ND 2.88 ND 2 35.8 ND 2.30 ND 3 39.2 ND 1.25 ND 4 27.8 ND 0.953 ND N/A 4.58 1.59 N/A 0.482 0.172 N/A 10.5 10.8 ND 4.22 1.79 ND 5.05 1.94 ND 4.97 2.01 ND 4.02 1.57 Mean 34.9 N/A 1.85 N/A S.D. 4.93 N/A 0.901 N/A % CV 14.1 N/A 48.7 N/A Run 29 1 23.4 ND 2.07 ND 2 23.9 ND 1.23 ND N/A 4.56 1.82 N/A 0.521 0.195 N/A 11.4 10.7 ND 3.10 1.24 ND 2.97 1.26 Mean 23.6 N/A 1.65 N/A S.D. 0.417 N/A 0.598 N/A % CV 1.8 N/A 36.3 N/A N/A 3.04 1.25 N/A 0.091 0.017 N/A 3.0 1.4 Overall Mean 30.4 Overall S.D. 5.66 Overall % CV 18.6 N/A 1.69 N/A N/A 0.814 N/A N/A 48.2 N/A _qD= none detected q/A = not applicable N/A 4.27 1.61 N/A 0.767 0.269 N/A 18.0 16.6 Page 23 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 7. Summary Data for Endogenous Analyte Concentrations in Human Serum Quality Control Samples for Runs 26, 28 and 29 Run 26 1 2 3 4 PFOS 21.6 21.4 21.0 22.5 PFOSA PFOSAA N-MeFOSE-OH N-EtFOSE-OH ND 1.71 ND ND ND 1.69 ND ND ND 2.86 ND ND ND 3.13 ND ND POAA 1.62 1.99 1.77 2.13 PFHS 3.22 2.85 3.54 3.32 5 23.4 iNrD 1.74 ND ND 2.13 3.38 Mean 22.0 N/A 2.23 N/A S.D. 0.982 N/A 0.706 N/A % CV 4.5 N/A 31.7 N/A Run 28 1 18.2 ND 1.66 ND 2 22.3 ND 1.95 ND 3 20.7 ND 1.51 ND 4 23.0 ND 1.53 ND 5 22.1 ND 2.09 ND N/A 1.9'3 3.26 N/A 0.225 0.258 N/A 11.7 7.9 ND 1.66 3.13 ND 2.02 3.11 ND 2.04 3.18 ND 2.13 3.33 ND 2.07 3.20 Mean 21.3 N/A 1.75 N/A S.D. 1.91 N/A 0.258 N/A % CV 0.I N/A 0.1 N/A Run 29 1 21.8 ND 2.41 ND 2 23.9 ND 4.01 ND 3 22.8 ND 2.59 ND 4 23.5 ND 2.50 ND 5 26.2 ND 2.80 ND Mean 23.6 N/A 2.86 N/A S.D. 1.63 N/A 0.658 N/A % CV 6.9 N/A 23.0 N/A N/A 1.98 3.19 N/A 0.187 0.087 N/A 0.1 0.0 ND 1.73 3.41 ND 2.15 3.55 ND 1.96 2.88 ND 1.92 3.24 ND 2.31 3.42 N/A 2.01 3.30 N/A 0.224 0.258 N/A 11.1 7.8 Overall Mean 22.3 N/A 1.99 N/A N/A 1.97 3.25 Overall S.D. 1.77 N/A 0.560 N/A N/A 0.200 0.206 Overall % CV 7.9 28.2 ND = none detected N/A = not applicable 10.2 6.3 Page 24 l Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 8. Summary of Human Serum Calibration Curve Parameters for PFOS Quadratic (weighted l/x). All concentrations are expressed as ppb. Run Date 30-Jan-1999 01-Feb-1999 02-Feb-1999 10-Feb-1999 Run Number 25 26 28 32 A* B* C* -0.000002 -0.000002 -0.000001 -0.000003 0.003075 0.002977 0.001649 0.004162 0.006751 0.000357 0.007710 -0.009869 Multiple Rsquared 0.9909 0.9930 0.9937 0.9960 LLOQ (ppb) 17.2 31.4 31.4 31.4 ULOQ (ppb) 516 530 530 530 Mean S.D. n, , . -0.000002 0.002966 0.001237 0.000001 0.001029 0.008092 4 4 4 0.9934 0.0021 4 * A, B, and C are coefficients used to define the quadratic curve. Table 9. Summary of Human Serum Calibration Curve Parameters for PFOSA Quadratic (weighted l/x). All concentrations are expressed as ppb. Run Date 30-Jan-1999 01-Feb-1999 02-Feb-1999 10-Feb-1999 Run Number 25 26 28 32 A* -0.000002 -0.000002 -0.000001 -0.000002 B* 0.003444 0.003308 0.001834 0.003945 C* Multiple Rsquared 0.000622 0.000757 0.00078.9 0.001714 0.9946 0.9965 0.9982 0.9982 LLOQ (ppb) 1.00 1.00 1.00 1.00 ULOQ (ppb) 500 500 500 500 Mean S.D. n -0.000002 0.000001 4 0.003133 0.000908 4 0.000971 0.000501 4 0.9969 0.0017 4 * A, B, and C are coefficients used to define the quadratic cun'e. Page 25 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 10. Summary of Human Serum Calibration Curve Parameters for PFOSAA Quadratic (weighted I/x). All concentrations are expressed as ppb. Run Date Run Number A* 30-Jan-1999 01-Feb-1999 02-Feb-1999 10-Feb-1999 25 0.000000 26 -0.000001 28 0.000000 32 -0.000001 B* 0.000676 0.000764 0.000469 0.001170 C* -0.000208 -0.000653 -0.000089 -0.000458 Multiple Rsquared 0.9964 0.9971 0.9963 0.9965 LLOQ (ppb) 1.99 2.23 2.23 2.23 ULOQ (ppb) 270 271 271 271 I Mean S.D. n 0.000000 0.000001 4 0.000770 0.000294 4 -0.000352 0.000253 4 0.9966 0.0004 4 * A, B, and C are coefficients used to define the quadratic curve. Table 11. Summary of Human Serum Calibration Curve Parameters for N-MeFOSE-OH Quadratic (weighted I/x). All concentrations are expressed as ppb. Run Date Run Number A* B* C* Multiple LLOQ ULOQ Rsquared (ppb) (ppb) 30-3"an-1999 25 -0.000007 0.021767 -0.001921 0.9981 1.00 500 01-Feb-1999 26 -0.000004 0.012302 0.005797 0.9962 1.00 500 02-Feb-1999 28 0.000001 0.007315 0.001464 0.9970 1.00 500 10-Feb-1999 32 -0.000010 0.019016 0.003235 0.9970 1.00 500 f Mean -0.000005 0.015100 0.002144 0.9971 S.D. 0.000005 0.006538 0.003241 0.0008 n 4 4 4 4 * A, B, and C are coefficients used to define the quadratic curve. Page 26 Northwest Bioanatytical Study No. NWBS98-082 Report No. NWBR99-005 Table 12. Summary of Human Serum Calibration Curve Parameters for N-EtFOSE-OH Quadratic (weighted l/x). All concentrations are expressed as ppb. Run Date 30-Jan-1999 01-Feb-1999 02-Feb-1999 10-Feb-1999 Run Number 25 26 28 32 A* -0.000002 -0.000002 0.000000 -0.000004 B* 0.015048 0.008104 0.005031 0.011968 C* -0.003325 0.002172 -0.005001 -0.001308 Multiple Rsquared 0.9994 0.9954 0.9986 0.9969 LLOQ (ppb) 1.00 1.00 5.00** 1.00 ULOQ (ppb) 500 500 500 500 l .Mean S.D. f I ! n_ -0.000002 0.000002 4 0.010038 0.004383 4 -0.001866 0.003086 4 0.9976 0.0018 4 i * A, B, and C are coefficients used to define the quadratic curve. i **Both Standards at 1.00 ppb were statistical outliers, so the LLOQ _'as raised to 5.00 ppb. Table 13. Summary of Human Serum Calibration Curve Parameters for POAA I ' Quadratic (weighted l/x). All concentrations are expressed as ppb. i Run Date Run Number A* B* C* Multiple LLOQ ULOQ Rsquared (ppb) (ppb) 30-Jan-1999 01-Feb-1999 02-Feb-1999 10-Feb-1999 25 -0.000003 0.005601 0.000463 0.9972 4.20 503 26 -0.000004 0.006274 -0.001033 0.9963 5.27 504 28 -0.000002 0.004036 0.003976 0.9981 5.27 504 32 -0.000005 0.008652 -0.001660 0.9970 5.27 504 Mean S.D. n -0.000004 0.000001 4 0.006141 0.001919 4 0.000437 0.002522 4 0.9972 0.0007 4 * A, B, and C are coemcients used to define the quadratic cu_'e. Page 27 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 14. Summary of Human Serum Calibration Curve Parameters Quadratic (weighted l/x). All concentrations are expressed as ppb. for PFHS Run Date Run Number A* B* C* Multiple LLOQ ULOQ Rsquared (ppb) (ppb) 30-Jan-1999 01-Feb-1999 02-Feb-1999 10-Feb-1999 25 -0.000004 0.005961 0.002977 0.9979 1.62 501 26 -0.000004 0.006088 0.000767 0.9977 2.61 502 28 -0.000002 0.003814 0.003933 0.9982 2.61 502 32 -0.000006 0.007750 0.002353 0.9973 2.61 502 \ \ -[ Mean S.D. n -0.000004 0.000002 4 0.005903 0.001614 4 0.002508 0.001330 4 0.9978 0.0004 4 * A, B, and C are coefficients used to define the quadratic curve. Page 28 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 15. Back-Calculated Concentrations of Human Serum Calibration Standards for PFOS All concentrationsare expressedas ppb. Run Date 30-Jan-1999 Run 17.2 21.2 26.2 31.4 35.4 40.4 41.2 55.4 66.2 80.4 116 130 280 516 530 Number 25 16.6 19.9 27.2 16.4 18.3 29.4 44.2 65.1 103 460 45.0 69.3 120 584 01-Feb-1999 26 35.6 37.2 39.6 50.8 73.0 130 498 32.8 32.4 *32.6 53.0 *56.4 143 563 02-Feb-1999 28 28.4 36.6 39.1 34.6 35.7 42.1 53.6 76.1 62.6 68.5 126 288 566 135 290 486 10-Feb-1999 32 29.7 33.6 43.3 57.7 *65.5 140 278 501 32.4 33.8 40.5 56.7 73.6 135 262 580 Mean S.D. %CV %Bias n 16.5 19.1 28.3 32.3 34.9 40.9 44.6 55.7 67.2 72.8 112 135 280 522 532 0.141 1.13 1.56 2.77 1.90 1.75 0.566 4.20 2.97 3.17 12.0 6.24 12.8 87.7 41.6 0.9 5.9 5.5 8.6 5.4 4.3 1.3 7.5 4.4 4.4 10.7 4.6 4.6 16.8 7.8 -4. I -9.9 8.0 2.9 -1.4 1.2 8.3 0.5 1.5 -9.5 -3.4 3.8 0.0 1.2 0.4 2 2 2 6 6 5 2 6 2 4 2 6 4 2 6 * sample deactivated-statistical outlier Page 29 NorthwestBioanalytical StudyNo. NWBS98-082 ReportNo. NWBR99-005 Table 16. Back-Calculated Concentrations of Human Serum Calibration Allconcentrationsare expressedasppb. Standards for PFOSA Run Date Run Number 1.00 5.00 10.0 25.0 50.0 100 250 500 30-Jan-1999 25 0.818 4.39 10.0 25.3 51.0 86.9 456 1.14 5.02 *11.8 28.4 54.3 103 550 01-Feb-1999 26 1.19 5.06 10.1 23.2 48.3 107 454 0.929 4.93 9.82 23.4 *40.6 "120 550 ] 02-Feb-1999 28 1.00 5.01 10.2 23.9 47.7 99.9 262 525 1.05 4.80 "12.2 26.4 47.0 107 236 476 10-Feb-1999 32 0.998 4.72 "11.8 23.9 "41.4 "118 247 471 0.989 4.94 11.0 24.8 49.7 108 241 538 Mean S.D. %CV %Bias n f 1.01 4.86 10.2 24.9 49.7 102 247 503 0.116 0.222 0.456 1.77 2.69 7.99 11.3 42.2 11.5 4.6 4.5 7.1 5.4 7.8 4.6 8.4 1.0 -2.8 2.0 -0.4 -0.6 2.0 -1.2 0.6 8 8 5 8 6 6 4 8 * sample deactivated-statistical outlier Page30 Northwest Bioanalyticat Study No. NWBS98-082 Report No. NWBR99-005 Table 17. Back-Calculated Concentrations of Human Serum Calibration Standards for PFOSAA All concentrations are expressed as ppb. Run Date 30-Jan-1999 Run Number 25 1.99 2.23 4.15 4.38 6.84 7.07 14.9 15.1 28.4 28.6 55.3 55.5 136 270 271 1.74 4.03 7.29 13.7 27.3 53.2 250 2. I0 4.37 7.06 "20.1 30.6 56.7 291 01-Feb-1999 26 *3.80 4.16 6.75 14.2 26.4 54.6 264 2.47 4.53 7.95 13.3 ! "22.7 i 61.5 278 02-Feb-1999 28 2.08 4.39 "9.11 "18.3 28.1 57.0 140 294 2.35 4.29 *8.77 "18.4 29.2 57.8 126 252 10-Feb-1999 32 2.19 3.94 7.94 13.0 25.4 63.5 131 264 2.29 4.62 7.02 16.0 28.5 56.7 132 282 Mean ::: S.D. %CV %Bias n 1.92 2.28 4.20 4.32 7.18 7.42 13.7 14.1 29.0 27.5 55.0 58.5 132 271 272 0.255 0.149 0.240 0.249 0.163 0.622 1.35 2.33 1.57 2.47 3.32 5.80 29.0 15.1 13.3 6.5 5.7 5.8 2.3 8.4 9.6 8.0 5.7 4.5 5.7 4.4 10.7 5.6 -3.5 2.2 1.2 -1.4 5.0 5.0 -8.1 -6.6 2.1 -3.8 -0.5 5.4 -2.9 0.4 0.4 2 5 2 6 2 4 1 4 2 5 2 6 4 2 6 * sample deactivated-statistical outlier Page 31 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 18. Back-Calculated Concentrations of Human Serum Calibration N-MeFOSE-OH Standards for All concentrations are expressed as ppb. Run Date 30-Jan-1999 Run Number 25 1.00 5.00 10.0 25.0 50.0 100 250 500 1.18 4.54 9.73 22.8 47.7 "63.1 478 0.862 *4.04 11.3 *30.7 53.4 101 522 01-Feb-1999 26 1.10 4.69 10.1 23.6 52.8 "132 463 1.04 4.77 *7.56 24.2 *39.4 "152 538 i 02-Feb-1999 28 0.971 4.61 10.2 24.9 45.1 87.0 252 493 1.13 4.72 11.3 *29.5 *37.3 106 265 *385 ' 10-Feb-1999 32 0.945 *3.84 11.4 21.8 *40.2 103 268 460 , 1.19 4.41 10.6 21.9 *36.5 98.6 240 537 i Mean J S.D. i %CVI i %Bias _. n 1.05 4.62 10.7 23.2 49.8 99.1 256 499 0.118 0.133 0.678 1.25 4.02 7.30 12.9 33.6 11.2 2.9 6.3 5.4 8.1 7.4 5.0 6.7 5.0 -7.6 7.0 -7.2 -0.4 -0.9 2.4 -0.2 8 6 7 6 4 5 4 7 I * sample deactivated-statistical outlier Page 32 NorthwestBioanalytical StudyNo. NWBS98-082 Report No. NWBR99-005 Table 19. Back-Calculated Concentrations of Human Serum Calibration Standards for N-EtFOSE-OH All concentrationsare expressedas ppb. Run Date 30-Jan-1999 Run Number 25 1.00 1.19 0.963 5.00 10.0 25.0 50.0 100 250 500 5.01 10.1 23.4 49.8 *60.9 491 4.25 "12.0 "30.1 52.4 100 509 01-Feb-1999 26 1.11 4.65 10.0 23.7 52.5 "133 461 -] 1.03 4.75 *7.79 24.5 "38.1 "153 540 / 02-Feb-1999 28 "1.88 5.20 9.64 22.5 *40.9 *78.6 244 497 *2.06 4.98 10.9 25.5 *34.0 96.1 264 *386 10-Feb-1999 32 1.16 4.38 "11.7 22.8 42.6 107 272 477 1.14 4.75 11.3 22.2 *37.5 97.5 246 513 Mean S.D. %CV %Bias n 1.10 4.75 10.4 23.5 49.3 100 257 498 0.0861 0.321 0.687 1.17 4.65 4.84 13.7 25.7 7.8 6.8 6.6 5.0 9.4 4.8 5.3 5.2 10.0 -5.0 4.0 -6.0 -1.4 0.0 2.8 -0.4 6 8 5 7 4 4 4 7 * sample deactivated-statistical outlier Page33 Northwest Bioanalytical Table 20. Back-Calculated Concentrations of Human Serum Calibration All concentrations are expressed as ppb. Study No. NWBS98-082 Report No. NWBR99-005 Standards for POAA Run Date Run 4.2 5.27 8.2 9.27 13.2 14.3 28.2 29.3 53.2 54.3 103 104 254 503 504 Number 30-Jan-1999 25 4.49 7.26 13.4 27.6 51.7 100 461 4.30 7.69 14.0 29.6 53.3 107 549 01-Feb-1999 26 6.09 9.12 13.9 "18.9 48.7 105 500 5.14 9.55 14.1 26.7 *44.3 113 -t] 02-Feb-1999 28 4.92 9.49 14.8 27.2 53.2 104 270 528 5.05 9.39 15.6 31.0 52.1 106 239 481 10-Feb-1999 32 5.47 8.49 "16.9 27.3 *44.5 113 241 489 5.20 9.59 14.4 28.7 55.6 113 235 541 Mean 'S.D. %CV ' %Bias n 4.40 5.31 7.48 9.27 13.7 14.6 28.6 28.2 52.5 52.4 104 109 246 505 508 0.134 0.423 0.304 I0.418 0.424 0.673 1.41 1.74 1.13 2.87 4.95 4.43 16.0 62.2 25.7 3.0 8.0 4.1 4.5 3.1 4.6 4.9 6.2 2.2 5.5 4.8 4.1 6.5 12.3 5.1 4.8 0.8 -8.8 0.0 3.8 2.1 1.4 -3.8 -1.3 -3.5 1.0 4.8 -3.1 0.4 0.8 2 6 2 6 2 5 2 5 2 4 2 6 4 2 5 * sample deactivated-statistical outlier Page 34 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 21. Back-Calculated Concentrations of Human Serum Calibration Standards for PFHS All concentrations are expressed as ppb. Run Date Run Number 30-Jan-1999 25 1.62 2.61 5.62 6.61 10.6 11.6 25.6 26.6 50.6 51.6 101 102 252 501 502 1.49 5.65 11.3 26.2 49.2 98.1 458 1.50 5.31 12.1 26.1 50.5 102 554 01-Feb-1999 26 2.68 7.13 12.0 24.0 48.7 102 478 2.65 6.74 11.4 24.3 *39.0 111 525 02-Feb-1999 28 "1.82 6.86 12.2 2.16 6.55 12.4 25.3 50.3 103 250 522 29.2 48.6 111 238 494 10-Feb-1999 32 2.28 6.27 13.3 2.59 6.79 12.2 25.6 *42.5 112 243 475 25.6 50.0 106 234 566 Mean S.D. %CV %Bias n 1.50 2.47 5.48 6.72 11.7 12.3 26.2 25.7 49.9 49.4 100 108 241 506 510 0.00707 0.236 0.240 0.291 0.566 0.619 0.0707 1.86 0.919 0.876 2.76 4.42 6.90 67.9 34.7 0.5 9.6 4.4 4.3 4.8 5.0 0.3 7.2 1.8 1.8 2.8 4.1 2.9 13.4 6.8 -7.4 -5.4 -2.5 1.7 10.4 6.0 2.3 -3.4 -1.4 -4.3 -1.0 5.9 -4.4 1.0 1.6 2 5 2 6 2 6 2 6 2 '4 2 6 4 2 6 * sample deactivated-statistical outlier Page 35 NorthwestBioanalytical StudyNo. NWBS98-082 Report No. NWBR99-005 Table 22. Intra-Assay Precision for PFOS Human Serum Quality Control Samples All concentrationsareexpressedasppb. Run Number: 26 Run Date: 01-Feb-1999 ,m Replicate 1 2 3 4 5 Low QC High QC (42.3 ppb) (372 ppb) 42.0 412 44.6 * 459 45.0 324 41.4 392 43.5 443 Mean S.D. CV% %Theoretical n 43.3 1.57 3.6 102.4 5 * > 20% theoretical 406 52.8 13.0 109.1 5 Page36 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 23. Intra-Assay Precision for PFOSA Human Serum Quality Control Samples All concentrationsare expressed as ppb. Run Number: 26 Run Date: 01-Feb-1999 Low QC (20.0 ppb) High QC (350 ppb) Replicate 1 21.8 387 2 "26.1 4i0 3 23.2 324 4 21.5 374 1 5 23.2 409 Mean S.D. 23.2 1.82 .... 381 35.2 i CV% 7.8 9.2 %Theoretical 116.0 108.9 [ i n 5 5 i * > 20% theoretical Page 37 NorthwestBioanalytical StudyNo. NWBS98-082 ReportNo. NW'BR99-005 Table 24. Intra-Assay Precision for PFOSAA Human Serum Quality Control Samples All concentrationsare expressedas ppb. l r ! Run Number: 26 Run Date: 01-Feb-1999 Replicate 1 2 3 4 5 Low QC (12.7 ppb) High QC (190 ppb) 12.0 187 14.0 200 13.2 163 13.9 189 13.9 196 Mean S.D. %CV %Theoretical n 13.4 187 0.846 14.4 6.3 7.7 105.5 98.4 5 5 Page38 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 25. Intra-Assay Precision for N-MeFOSE-OH Human Serum Quality Control Samples All concentrations are expressed as ppb. Run Number: 26 Run Date: 01-Feb-1999 Replicate 1 2 3 4 5 Low QC (20.0 ppb) High QC (350 ppb) 23.2 *424 22.1 *425 23.2 336 21.7 *426 21.5 407 Mean S.D. CV% %Theoretical n 22.3 0.814 3.7 111.5 5 * > 20% theoretical 404 38.6 9.6 115.4 5 Page 39 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 26. Intra-Assay Precision for N-EtFOSE-OH Human Serum Quality Control Samples All concentrations are expressed as ppb. Run Number: 26 Run Date: 01-Feb-1999 Replicate 1 2 3 4 5 Low QC (20.0 ppb) High QC (350 ppb) 23.1 *431 22.2 *431 22.7 341 21.4 *440 22.1 403 Mean S .D. CV% %Theoretical n 22.3 0.644 2.9 111.5 5 * > 20% theoretical 409 40.6 9.9 116.9 5 Page 40 NorthwestBioanalytical SthdyNo. NWBS98-082 Report No. NWBR99-005 Table 27. Intra-Assay Precision for POAA Human Serum Quality Control Samples All concentrationsare expressedasppb. Run Number: 26 Run Date: 0I-Feb-1999 Replicate 1 2 3 4 Low QC (23.3 ppb) 21.8 24.0 22.2 22.2 High QC (353 ppb) 366 374 "281 341 I 5 18.8 365 Mean S.D. CV% %Theoretical n 21.8 345 1.88 38.1 8.6 11.0 93.6 97.7 5 5 * > 20% theoretical Page41 NorthwestBioanalytical StudyNo. NWBS98-082 ReportNo. NWBR99-005 Table 28. Intra-Assay Precision for PFHS Human Serum Quality Control Samples Allconcentrationsare expressedasppb. Run Number: 26 Run Date: 01-Feb-1999 Low QC (22.0 ppb) High QC (352 ppb) Replicate 1 22.9 343 2 25.2 384 3 23.3 *278 4 23.3 332 l 5 24.9 360 Mean S.D. CV% %Theoretical n 23.9 339 1.05 39.5 4.4 11.7 108.6 96.3 5 5 * > 20% theoretical Page42 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 29. Inter-Assay Precision for PFOS Human Serum Quality All concentrations are expressed as ppb. Control Samples Run Date Run Number 30-Jan- 1999 25 Low QC (42.3 ppb) 40.9 45.2 **33.6 **29.2 41.2 High QC (372 ppb) 414 409 408 354 398 01-Feb-1999 26 42.0 412 44.6 **459 45.0 324 41.4 392 43.5 443 " 02-Feb- 1999 28 *19.0 *'503 38.9 401 44.7 418 43.2 397 36.9 435 10-Feb-1999 32 40.7 352 43.3 340 41.9 377 38.6 344 41.5 355 Mean 40.9 S.D. 4.08 %CV 10.0 %Theoretical 96.7 %Bias -3.3 n 19 * sample deactivated - _oor instrument response **> 20% aeoretical 397 44.3 11.2 106.7 6.7 20 Page 43 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 30. Inter-Assay Precision for PFOSA Human Serum Quality All concentrations are expressed as ppb. Control Samples Run Date Run Number 30-Jan- 1999 25 Low QC (20.0 ppb) 23.9 **24.7 17.8 *'14.2 21.5 High QC (350 ppb) 386 357 410 343 381 01-Feb-1999 26 21.8 387 *'26.1 410 23.2 324 21.5 374 23.2 409 02-Feb- 1999 28 *10.5 20.5 20.9 22.2 19.2 **456 342 370 339 388 10-Feb-1999 32 20.7 338 23.4 329 20.7 347 17.0 331 20.1 317 .... ! Mean 21.2 367 , S.D. 2.81 36.5 %CV 13.3 9.9 %Theoretical 106.0 104.9 %Bias 6.0 4.9 n 19 20 * sam _ledeactivated- poor instrument res _onse **> 20% theoretical Page 44 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 31. Inter-Assay Precision for PFOSAA Human Serum All concentrations are expressed as ppb. Quality Control Samples Run Date Run Number 30-Jan-1999 25 Low QC (12.7 ppb) ** 16.6 13.6 11.1 **9.97 14.0 High QC (190 ppb) 223 225 **246 208 213 01-Feb-1999 26 12.0 187 14.0 200 13.2 163 13.9 189 13.9 196 02-Feb-1999 28 "9.11 212 12.2 186 14.8 194 13.9 177 11.8 189 1O-Feb-1999 32 13.4 202 14.3 195 12.5 205 10.9 192 12.2 175 I I Mean 13.1 199 S.D. 1.56 19.1 %CV 11.9 9.6 %Theoretical 103.1 104.7 %Bias 3.1 4.7 n 19 20 * sample deactivated- poor instrument response ** > 20% theoretical Page 45 I Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 32. Inter-Assay Precision for N-MeFOSE-OH Human Serum All concentrations are expressed as ppb. Qualib" Control Samples Run Date Run Number 30-Jan- 1999 25 Low QC (20.0 ppb) 16.4 19.0 *'15.2 *'12.2 17.4 High QC (350 ppb) 310 316 355 301 311 01-Feb- 1999 _, 26 , ,, 23.2 22.1 23.2 21.7 21.5 *'424 **425 336 **426 407 02-Feb-1999 28 " *11.6 23.6 **28.8 **26.5 22.8 **446 352 383 335 401 10-Feb- 1999 32 u...-. ** 14.8 16.0 ** 15.0 *'11.5 ,,,, ...... *'14.7 *'252 **244 328 315 , 292 Mean 19.2 348 S.D. %CV 4.90 58.9 25.5 16.9 %Theoretical %Bias 96.0 99.4 -4.0 -0.6 n 19 20 * sam:_ledeactivated- poor instrument res mnse ** > 20% theoretical Page 46 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 33. Inter-Assay Precision for N-EtFOSE-OH Human Serum All concentrations are expressed as ppb. Quality Control Samples Run Date Run Number 30-Jan-1999 25 Low QC (20.0 ppb) *'15.5 18.7 *'15.0 *'12.1 17.3 High QC (350 ppb) 316 308 340 294 316 01-Feb- 1999 26 23.1 **431 1 22.2 **4.31 I 22.7 341 21.4 *'44.0 22.1 403 02-Feb- 1999 28 *11.4 23.0 23.5 23.1 21.2 *'4.36 329 363 332 378 10-Feb-1999 32 *'15.1 16.8 *'15.4 *'12.5 *'14.9 *'251 **254 339 314 301 Mean 18.7 346 S.D. 3.97 57.6 %CV 21.2 16.6 %Theoretical 93.5 98.9 %Bias -6.5 -1.1 n 19 20 * sam _ledeactivated- _oor instrumentres _onse **> 20%theoretical Page 47 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 34. Inter-Assay Precision for POAA Human Serum Quality All concentrations are expressed as ppb. Control Samples Run Date Run Number 30-Jan-1999 25 Low QC (23.3 ppb) *'28.0 24.4 *'18.3 *'17.4 24.2 High QC (353 ppb) 349 396 377 318 379 01-Feb-1999 26 21.8 366 I [ 24.0 374 22.2 *'281 22.2 341 18.8 365 02-Feb-1999 28 * I 1.6 376 23.1 325 24.8 345 22.0 324 19.1 351 10-Feb- 1999 32 23.5 348 25.2 321 22.1 321 19.5 326 22.6 302 Mean 22.3 344 S.D. 2.70 29.5 %CV 12.1 8.6 %Theoretical 95.7 97.5 %Bias -4.3 -2.5 n 19 20 * sam 31edeactivated-poor instrument res _onse ** > 20% theoretical Page 48 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 35. Inter-Assay Precision for PFHS Human Serum Quality All concentrations are expressed as 9pb. Control Samples Run Date Run Number 30-Jan-1999 25 Low QC (22,0 ppb) **26.6 High QC (352 ppb) .o , 379 24.4 391 , 18.7 370 *'17.1 328 24.0 361 01-Feb-1999 26 22.9 343 ,,,1 25.2 384 23.3 **278 23.3 332 24.9 360 02-Feb- 1999 28 *11.9 393 23.1 359 ; 23.1 382 23.3 366 21.0 377 10-Feb-1999 32 21.7 357 22.6 344 22.6 354 20.8 3OO p 21.4 308 { Mean 22.6 353 ,,i .... S.D. 2.22 31.1 %CV 9.8 8.8 %Theoretical 102.7 100.3 %Bias ,,.., n 2.7 0.3 19 20 sample deactivated- statistical outlier ** > 20% theoretical Page 49 NorthwestBioanalytical Table 36. System Precision for PFOS StudyNo. NWBS98-082 ReportNo. NWBR.99-005 Run Date 10-Feb-1999 Run Number 32 Unextraeted Low QC (20.0 ppb) Unextracted High QC (350 ppb) Instrument Response 0.101895 1.622610 0.141723 1.870562 0.169329 1.990953 Mean 0.137649 1.828042 t S.D. 0.033901 0.187817 %CV 24.6 10.3 n 3 3 Table 37. System Precision for PFOSA Run Date lO-Feb-1999 Run Number 32 Unextraeted Unextracted Low QC (20.0 ppb) High QC (350 ppb) Instrument Response 0.006447 0.207807 0.006939 0.558348 0.018200 0.347834 Mean m, 0.010529 0.371330 S.D. 0.006648 0.176448 ) %CV 63.1 47.5 [' n 3 '3 Page50 NorthwestBioanalytical StudyNo. NWBS98-082 ReportNo. NWBR99-005 Table 38. System Precision for PFOSAA Run Date 10-Feb-1999 Run Number 32 Unextracted Low QC (12.7 ppb) Unextracted High QC (190 ppb) Instrument Response 0.017247 0.247510 0.015952 0.338792 0.029981 0.357733 Mean 0.021060 0.314678 S.D. %CV i n 0.007753 36.8 3 0.058935 18.7 3 Table 39. System Precision for N-MeFOSE-OH Run Date 10-Feb-1999 Run Number 32 Unextracted Low QC (20.0 ppb) Unextracted High QC (350 ppb) Instrument Response 0.015063 0.583610 0.010676 2.230467 0.032584 1.338583 Mean S.D. %CV n 0.019441 0.0i 1592 59.6 3 1.384220 0.824376 59.6 3 Page51 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 40. System Precision for N-EtFOSE-OH Run Date Run Number 10-Feb-1999 32 i, , Mean S.D. %CV n Unextracted Unextracted Low QC (20.0 ppb) High QC (350 ppb) Instrument Response 0.011565 0.525858 0.008637 2.211409 0.034819 1.323620 0.018340 0.014346 78.2 3 1.353629 0.843176 62.3 3 Table 41. System Precision for POAA ' Unextracted Unextracted Run Date Run Number Low QC (20.0 ppb) High QC (350 ppb) Instrument Response 10-Feb-1999 32 0.148240 2.629881 0.197618 3.363888 0.313308 3.570008 Mean S.D. %CV n 0.219722 0.084725 38.6 3 3.187926 0.494147 15.5 3 Page 52 NorthwestBioanalytical StudyNo. NWBS98-082 Report No. NWBR99-005 i Table 42. System Precision for PFHS Run Date 10-Feb-1999 Run Number 32 Unextracted Low QC (20.0 ppb) Unextracted High QC (350 ppb) Instrument Response 0.179242 2.770742 0.244403 3.162425 0.295965 3.428575 Mean 0.239870 3.120581 %SC.DV. n 0.02548.4493 3 0.31300.9607 3 Page 53 Northwest Bioanalytical Table 43. PFOS Extraction Efficiency Study No. NWBS98-082 Report No. NWBR99-005 Time of Spikinq I. Analyte and IS after extraction Mean Analyte Peak Areas 81470 86784 77797 82017 Istd Peak Areas 539547 554615 523549 539237 2. Analyte prior and IS after extraction Mean 61442 94927 54973 70447 3. IS prior and Analyte after extraction Mean 200162 461938 490278 384126 Mean extraction efficiency for the analyte = 85.9% Mean extraction efficiency for the internal standard = 71.2 % Table 44. PFOSA Extraction Efficiency Time of Spiking 1. Analyte and IS after extraction Mean Analyte Peak Areas 45280 45068 38724 43024 Istd Peak Areas 539547 554615 523549 539237 2, Analyte prior and ISafter extraction Mean 33229 47251 25711 35397 3, IS prior and Analyte after extraction Mean 200162 461938 490278 384126 Mean extraction efficiency for the analyte = 82.3% Mean extraction efficiency for the internal standard = 71.2% Page 54 Northwest Bioanalytical Table 45. PFOSAA Extraction Efficiency Study No. NWBS98-082 Report No. NWBR99-005 Time of Spiking I. Analyte and IS after extraction Mean Analyte Peak Areas 6484 6197 6383 6355 Istd Peak Areas 539547 554615 523549 539237 2. Analyte prior and IS after extraction Mean 4229 5443 3961 4544 3. ISprior and Analyte after extraction Mean 200162 449601297388 384126 Mean Mean extraction extraction efficiency efficiency for the analyte for the internal standard \ = 71.5"/, ffi 71.2 % Table 46. N-MeFOSE-OH Extraction Efficiency Time of Spiking I. Analyte and IS after extraction Mean Analvte PeakAreas 257654 262358 154951 224988 Istd Peak Areas 539547 554615 523549 539237 2. Analyte prior and IS after extraction 85560 I 226651 _j 45573 Mean 119261 3. IS prior and Analyte after extraction Mean 200162 461938 490278 384126 Mean extraction efficiency for the analyte ffi 53.0% Mean extraction efficiency for the internal standard = 71.2% Page 55 Northwest Bioanalytical Table 47. N-EtFOSE-OH Extraction Efficiency Study No. NWBS98-082 Report No. NWBR99-005 Time of Spiking I. Analyte and IS after extraction Mean Analyte Peak Areas 191529 190650 122094 168091 Istd Peak Areas 539547 554615 523549 539237 2. Analyte prior and IS after extraction Mean 52797 139842 32402 75014 3. IS prior and Analyte after extraction Mean 200162 461938 490278 384126 Mean extraction efficiency for the analyte = 44.6*/0 Mean extraction efficiency for the internal s tandard = 71.2 % Table 48. POAA Extraction Efficiency Time of Spiking 1. Analyte and IS after extraction Mean Analyte Peak Areas 87822 84427 75701 82650 Is td Peak Areas 539547 554615 523549 539237 2. Analyte prior and IS after extraction 76904 83445 62754 Mean 74368 I: 3. IS prior and Analyte after extraction 200162 461938 490278 Mean 384126 Mean extraction efficiency for the analyte = 90.0*/, Mean extraction efficiency for the internal standard -- 71.2*/, Page 56 Northwest Bioanalytical Table 49. PFHS Extraction Efficiency Study No. NWBS98-082 Report No. NWBR99-005 Time of Spiking 1. Analyte and IS after extraction Mean Analyte Peak Areas 87124 86589 82852 85522 lstd Peak Areas 539547 554615 523549 539237 2. Analyte prior and IS after extraction Mean 66595 81391 61113 69700 3. IS prior and Analyte after extraction ] Mean 200162 461938 490278 384126 Mean extraction efficiency for the analyte = 81.5% Mean extraction efficiency for the internal standard-- 71.2% Page 57 NorthwestBioanalytical Table 50. Freeze/Thaw Stability for PFOS All concentrationsare expressedas ppb. StudyNo. NWBS98-082 ReportNo. NWBR99-005 Run Number Run Date 25 30-Jan-1999 LOW QC After 3 Freeze/Thaw cycles 1 43.3 2 33.1 ! 3 40.5 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 39.0 5.27 13.5% 38.0 2.54% HIGH QC After 3 Freeze/Thaw cycles 1 330 2 315" 3 410 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 370 56.6 15.3% 397 -6.80% * sample deactivated - poor instrument response Control = mean of reference QC samples Page58 Northwest Bioanalytical Table 51. Freeze/Thaw Stability for PFOSA All concentrations are expressed as ppb. Study No. NWBS98-082 Report No. NWBR99-005 Run Number Run Date 25 30-Jan-1999 LOW QC After 3 Freeze/Thaw cycles 1 2 3 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 23.5 19.9 20.0 21.1 2.05 9.70% 20.4 3.59% I-IIGHQC After 3 FreezeThaw cycles 1 336 2 248* 3 401 Mean 369 S.D. 46.0 ! % CV 12.5% I Control (ng/mL) Mean % Dev. from Control 375 -1.73% * sample deactivated - poor instrument response Control = mean of reference QC samples Page 59 NorthwestBioanalytical Table 52. Freeze/Thaw Stability for PFOSAA All concentrationsare expressedas ppb. StudyNo. NWBS98-082 ReportNo. NWBR99-005 Run Number Run Date 25 30-Jan-1999 LOW QC After 3 Free:;e/Thaw cycles 1 13.6 2 12.5 3 12.0 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 12.7 0.819 6.45% 13.1 -3.05% HIGH QC After 3 Freege/Thaw c_cles 1 205 2 "180 3 **237 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 221 22.6 10.2% 223 -0.897% * sample deactivated - poor instrument response ** > +20% theoretical Control = mean of reference QC samples Page60 NorthwestBioanalytical Table 53. Freeze/Thaw Stability for N-MeFOSE-OH Allconcentrationsare expressedas ppb. StudyNo. NWBS98-082 ReportNo. NWBR99-005 Run Number Run Date 25 30-Jan-1999 LOW QC After 3 FreezeThaw cycles 1 18.9 2 11.8 3 Mean Mean S.D. % CV Control (ng/mL) o/0Dev. from Control 15.5 15.4 3.55 23.1% 16.0 -3.75% HIGH QC After 3 Freeze/Thaw cycles 1 320 2 213" 3 324 Mean 322 [ S.D. 2.83 I % CV 0.878% Control (ng/mL) 319 Mean % Dev. from Control 0.940% * sample deactivated - poor instrument response Control = mean of reference QC samples Page61 Northwest Bioanalytical Table 54. Freeze/Thaw Stability for N-EtFOSE-OH All concentrations are expressed as ppb. Study No. NWBS98-082 Report No. NWBR99-005 Run Number Run Date 25 30-Jan-1999 LOW QC After 3 Freeze/Thaw cycles 1 19.1 2 11.4 3 15.9 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 15.5 3.87 25.0% 15.7 -1.49% mGH QC After 3 Freeze cycles 1 302 2: 214" 3 307 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 305 3.54 1.16% 315 -3.33% * sample deactivated - poor instrument response Control = mean of reference QC samples Page 62 NorthwestBioanalytical Table 55. Freeze/Thaw Stability for POAA All concentrationsare expressedas ppb. StudyNo. NWBS98-082 ReportNo. NWBR.99-005 Run Number Run Date 25 30-Jan-1999 LOW QC After 3 Freeze cycles 1 25.3 2 23.8 3 25.5 Mean Mean S.D. % CV Control (ng/mL) %0Dev. from Control 24.9 0.929 3.74% 22.5 10.5% I-IIGI-QI C After 3 Freeze/Thaw cycles 1 330 2 144" 3 385 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 358 38.9 10.9% 364 -1.79% * sample deactivated - poor instrument response Control = mean of reference QC samples Page63 Northwest Bioanalytical Table 55. Freeze/Thaw Stability for PFHS All concentrations are expressed as ppb. Study No. NWBS98-082 Report No. NWBR99-005 Run Number Run Date 25 30-Jan-1999 LOW QC. After 3 FreezeThaw cycles 1 24.4 2 21.7 3 23.5 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 23.2 1.37 5.93% 22.2 4.50% mGl QC After 3 Freeze/Thaw cycles I 3i0 2 145" 3 365 Mean 338 I S.D. 38.9 I % CV I 1.5% Control (ng/mL) Mean % Dev. from Control 366 -7.79% * sample deactivated - poor instrument response Control = mean of reference QC samples Page 64 NorthwestBioanalytical StudyNo. NWBS98-082 ReportNo. NWBR99-005 Table 57. Room Temperature Matrix Stability for PFOS Allconcentrationasreexpressedas ppb. Run Number Run Date 32 10-Feb-1999 2 Hour LOW QC 4 Hour 1 39.5 38.4 2 41.0 36.9 -"J 3 40.8 37.5 ! Mean 40.4 37.6 S.D. 0.814 0.755 % CV 2.01% 2.01% i Control (ng/mL) 41.2 41.2 Mean % Dev. from Control -1.94% -8.74% !, HIGH Qc 2 Hour 4 Hour 1 322 343 2 271 173" 3 276 349 ! Mean 290 346 S.D. 28.1 4.24 % CV 9.69% 1.23% I Control (ng/mL) 354 354 Mean % Dev. from Control -18.1% -2.26% * sample deactivated - poor instrument response Control = mean of reference QC samples Page65 NorthwestBioanalytical StudyNo. NWBS98-082 ReportNo. NWBR99-005 Table 58. Room Temperature Matrix Stability for PFOSA All concentrationsare expressedas ppb. Run Number Run Date 32 10-Feb-I999 2 Hour LOW QC 4 Hour 1 20.0 1817 2 20.3 17.2 3 20.7 18.2 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 20.3 0.351 1.73% 20.4 -0.490% 18.0 0.764 4.24% 20.4 -11.8% " HIGI-IQC 2 Hour 4 Hour 1 310 317 2 261 174" 3 276 336 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 282 25.1 8.89% 332 -15.0% 327 13.4 4.1% 332 -1.51% * sample deactivated - poor instrument response Control --mean of reference QC samples Page66 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 59. Room Temperature Matrix Stability for PFOSA.A All concentrations are expressed as ppb. Run Number Run Date 32 10-Feb-1999 2 Hour LOW QC 4 Hour 1 2 3 Mean S.D. o/0 CV Control (ng/mL) Mean % Dev. from Control 12.8 13.3 13.3 13.1 0.289 2.21% 12.7 3.15% 11.5 11.5 10.3 11.1 0.693 6.24% 12.7 -12.6% 2 Hour HIGH QC 4 Hour 1 179 200 2 *'138 "103 1z 3 152 185 i Mean 156 193 S.D. 20.8 10.6 % CV 13.3% 5.49% Control (ng/mL) Mean % Dev. from Control 194 -19.6/0 194 -0.515% * sample deactivated - poor instrument response ** > +20% theoretical Control = mean of reference QC samples Page 67 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 60. Room Temperature Matrix Stability for N-MeFOSE-OH All concentrations are expressed as ppb. Run Number Run Date 32 10-Feb-1999 2 Hour LOW QC 4 Hour 1 2 3 Mean Mean S.D. % CV Control (ng/mL) %0Dev. from Control 14.3 14.9 13.0 14.1 0.971 6.90% 14.4 -2.31% 10.5 12.5 10.8 11.3 1.08 9.57% 14.4 -21.8% i QC 2 Hour 4 Hour 1 248 218 2, 199 131" 3 197 335 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 215 28.9 13.5% 286 -24.9% 277 82.7 29.9% 286 -3.15% * sample deactivated - poor instrument response Control = mean of reference QC samples Page 68 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 61. Room Temperature Matrix Stability for N-EtFOSE-OH All concentrations are expressed as ppb. Run Number Run Date 32 10-Feb-1999 2 Hour LOW QC 4 Hour 1 15.1 11.9 2 14.8 12.7 3 13.1 12.0 , Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 14.3 1.08 7.52% 14.9 -3.80% 12.2 0.436 3.57% 14.9 -18.12% 1 2 3 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 2 Hour HIGH QC 4 Hour 255 235 224 139" 210 339 230 23.0 10.0% 292 -21.3% 287 73.5 25.6% 292 -1.71% * sample deactivated - poor instrument response Control = mean of reference QC samples Page 69 NorthwestBioanalytical StudyNo. NWBS98-082 Report No. NWBR99-005 Table 62. Room Temperature Matrix Stability for POAA Allconcentrationsare expressedasppb. Run Number Run Date 32 10-Feb-1999 2 Hour LOW QC 4 Hour 1 22.3 20.9 2 22.8 20.1 ] 3 24.4 20.1 / Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 23.2 1.10 4.74% 22.6 2.51% 20.4 0.462 2.27% 22.6 -9.88% i ttIGtt QC 2 Hour 4 Hour i i ! 1 313 327 2 221 171" 3 280 , 308 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 271 46.6 17.2% 324 -16.3% 318 13.4 4.21% 324 -1.85% * sample deactivated - poor instrument response Control = mean of reference QC samples Page 70 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 63. Room Temperature Matrix Stability for PFHS All concentrations are expressed as ppb. Run Number Run Date 32 10-Feb-1999 2 Hour LOW QC 4 Hour 1 20.8 21.5 2 21.3 19.5 ]- 3 23.9 21.1 Mean 22.0 20.7 ' S.D. 1.66 1.06 % CV 7.57% 5.11% Control (ng/mL) Mean % Dev. from Control 21.8 0.917% 21.8 -5.05% t HIGH QC 2 Hour 4 Hour 1 333 341 2 251 172" 3 281 306 Mean Mean S.D. % CV Control (ng/mL) %0 Dev. from Control 288 41.5 14.4% 333 -13.4% 324 24.7 7.62% 333 -2.70% * sample deactivated - poor instrument response Control = mean of reference QC samples Page 71 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 64. Room Temperature Extract Stability for PFOS All concentrations are expressed as ppb. Run Number Run Date 32 10-Feb-1999 LOW QC After 24 Hours 1 35.9 2 36.5 ! 3 32.3 Mean Mean S.D. %CV Control (ng/mL) % Dev. from Control 34.9 2.27 6.51% 41.2 -15.3% ttIGI-I QC After 24 Hours 1 300 2 235 3 271 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 269 32.6 12.1% 354 -24.1% Control = mean of reference QC samples Page 72 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 65. Room Temperature Extract Stability for PFOSA All concentrations are expressed as ppb. Run Number Run Date 32 10-Feb-1999 LOW QC After 24 Hours 1 19.7 2 19.4 3 16.1 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 18.4 1.997 10.9% 20.4 -9.80% HIGH QC After 24 Hours 1 311 2 239 3 270 Mean S.D. 273 36.12 ! % CV 13.2% , Control (ng/mL) 332 Mean % Dev. from Control -17.7% Control = mean of reference QC samples Page 73 NorthwestBioanalytical StudyNo. NWBS98-082 ReportNo. NWBR99-005 Table 66. Room Temperature Extract Stability for PFOSAA Allconcentrationsare expressedas ppb. Run Number Run Date 32 10-Feb-1999 LOW QC After 24 Hours 1 12.6 2 11.7 3 10.2 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control I 1.5 1.21 10.5% 12.7 -9.45% HIGH QC After 24 Hours 1 167 2 "133 3 "147 Mean 149 S.D. 17.1 i % CV 11.5% ! Control (ng/mL) 194 Mean % Dev. from Control -23.2% * > +20% theoretical Control = mean of reference QC samples Page74 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 67. Room Temperature Extract Stability for N-MeFOSE-OH All concentrations are expressed as ppb. Run Number Run D ate 32 10-Feb-1999 LOW QC After 24 Hours 1 12.8 2 13.5 I 3 10.1 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 12.1 1.80 14.8% 14.4 -15.7% 1 2 3 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control HIGH QC After 24 Hours 276 257 242 258 17.0 6.60% 286 -9.67% Control = mean of reference QC samples Page 75 Northwest Bioanalyfical Study No. NWBS98-082 Report No. NWBR99-005 Table 68. Room Temperature Extract Stability for N-EtFOSE-OH All concentrations are expressed as ppb. Run Number Run D ate 32 10-Feb-1999 LOW QC After 24 Hours 1 12.8 2 13.5 ] 3 10.5 i Mean 12.3 S.D. 1.57 % CV 12.8% Control (ng/mL) Mean % Dev. from Control 14.9 -17.7% HIGH QC After 24 Hours 1 289 2 265 [, 3 247 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 267 21.I 7.89% 292 -8.56% Control = mean of reference QC samples Page 76 NorthwestBioanalytical StudNyo. NWBS98-082 ReportNo. NWBR99-005 Table 69. Room Temperature Extract Stability for POAA All concentrationsare expressedas ppb. Run Number Run Date 32 10-Feb-1999 LOW QC After 24 Hours 1 21.7 2 22.4 3 19.1 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 21.1 1.74 8.25% 22.6 -6.78% I-IIGH QC After 24 Hours i 1 290 2 222 3 247 Mean 253 S.D. 34.4 ! % CV 13.6% ] Control (ng/mL) 324 Mean % Dev. from Control -21.9% Control = mean of reference QC samples Page 77 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Table 70. Room Temperature Extract Stability for PFHS All concentrations are expressed as ppb. Run Number Run Date 32 10-Feb-1999 LOW QC After 24 Hours 1 20.7 2 19.9 [ 3 18.1 Mean S.D. % CV Control (ng/mL) Mean % Dev. from Control 19.6 1.33 6.81% 21.8 -10.2% nIGH QC After 24 Hours 1 283 2 216 3 241 Mean Mean S.D. % CV Control (ng/mL) % Dev. from Control 247 33.9 13.7% 333 -25.9% Control = mean of reference QC samples Page 78 NorthwestBioanalytical Table 71. Stock Solution Stability for PFOS StudyNo. NWBS98-082 ReportNo. NWBR99-005 Analyte Peak Area Prepared day of analytical run Mean SD % CV n 121195 128494 125134 124941 3653 2.92 3 Storedfor24 days Mean SD %CV n % Difference 143208 132499 122485 132731 10363 7.81 3 6.23% Page79 Northwest Bioanalytical Table 72. Stock Solution Stability for PFOSA Study No. NWBS98-082 Report No. NWBR99-005 Analyte Peak Area Prepared day of analytical run Mean SD % CV n 41058 34117 84507 53227 27310 51.3 3 t Stored for 24 days 63582 58872 46572 Mean 56342 SD 8783 % CV 15.6 n 3 % Difference 5.85% Page 80 NorthwestBioanalytical Table 73. Stock Solution Stability for PFOSAA StudyNo. NWBS98-082 ReportNo. NWBR99-005 Analyte Peak Area Prepared day of analytical run Mean SD % CV ' n 14044 15654 15150 14949 824 5.51 3 ! Stored for 24 days 17264 16136 13572 Mean 15657 SD 1892 % CV 12.1 n 3 % Difference 4.74% Page81 j NorthwestBioanalytical StudyNo. NWBS98-082 ReportNo. NWBR99-005 Table 74. Stock Solution Stability for N-MeFOSE-OH Analyte Peak Area Prepared day of analytical run Mean SD % CV n 115190 9O190 52253 85878 31689 36.9 3 Stored for 24 days Mean SD % CV n % Difference 169447 175317 125816 156860 27045 .17.2 3 82.7% el I Page82 NorthwestBioanalytical Table 75. Stock Solution Stability for N-EtFOSE-OH Study No. NWBS98-082 Report No. NWBR99-005 Analyte Peak Area Prepared day of analytical run Mean SD % CV n 113772 87610 52034 84472 30988 36.7 3 Stored for 24 days Mean SD % CV n % Difference 160189 166768 115012 147323 28175 19.1 3 74.4% Page83 NorthwestBioanalytical Table 76. Stock Solution Stability for POAA StudyNo. NWBS98-082 ReportNo. NWBR99-005 Analyte Peak Area Prepared day of analytical run Mean SD % CV n 200388 2O6992 205145 204175 3407 1.67 3 Stored for 24 days Mean SD % CV n 200970 200039 183527 194845 9813 5.04 3 % Difference -4.57% Page84 Northwest Bioanalytical Table 77. Stock Solution Stability for PFHS Study No. NWBS98-082 Report No. NWBR99-005 Analyte Peak Area Prepared day of analytical r_n Mean SD % CV n 212505 221778 218307 217530 4685 2.15 3 i Stored for 24 days 230603 224907 2O0109 Mean 218540 SD 16214 % CV 7.42 n 3 % Difference 0.464% Page 85 Northwest Bioanalytical 6. ANALYTICAL METHOD Study No. NWBS98-082 Report No. NWBR99-005 Principles of the Method After the addition of HPLC-grade water and sodium dodecyl sulfate (SDS, internal standard) to 0.20 mL of human or rabbit serum, the serum mixture is made basic with the addition of 0.5 M tetrabutylammonium hydrogen sulfate (TBA, pH I0) 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 negative-ion electrospray ionization and multiple reaction monitoring. CHEMICAL FORMULAS Abbreviation PFOS PFOSA PFOSAA PFHS P OAA N-EtFOSE-OH N-MeFOSE-OH SDS Chemical Name Perfluorooctane sulfonate Perfluorooctane sulfonylamide Perfluorooctane sulfonylamido(ethyl)aeetate Perfluorohexane sulfonate Perfluorooctanoate 2(N-ethylperfluorooctanesulfonamidoethanol 2(N-methylperfluorooctanesulfonamidoethanol Dodecyl sulfate (sodium) 6.1. Reference Materials and Matrices Formula C8F17SO3CsF 17SO2NH2 CSF17SO2N(CH2CH3)CH2CO2 C6F13SO3C7F 15CO2" C8F17 SO 2 N(CH2CH3)(CH2CH2OH ) CSF 17 SO2 N (CH3)(CH2CH2OH) CH3(CH2) 10 CH2 OSO3" Reference Material PFOS PFOSA PFOSAA N-MeFOSE-OH N-EtFOSE-OH Lot Number 193 214 617 212 936 Purity 100% 100% 53.8% 100% 100% Expiration Date Source Storage Conditions 12/31/2010 12/31/2010 12/31/2010 1213112010 12/31/2010 3M Room Temperature 3M Room Temperature 3M Room Temperature 3M Room Temperature 3M Room Temperature Page 86 NorthwestBioanalytical StudyNo. NWBS98-082 Report No. NWBR99-005 Reference Material POAA PFHS (in methanol) SDS (sodium dodecyl sulfate) Lot Number Purity 245 $398-182 17H0459 100% 100% (6200 ppm) 91.0% Expiration Date 12/31/2010 12/31/2010 1/16/2001 Source Storage Conditions 3M Room Temperature 3M - 20 C Sigma Room Temperature Matrix Human serum Rabbit serum Source Biochemed Biochemed 6.2. Chemicals and Equipment Chemicals Ammonium Acetate, 99.9 % Water, I-IPLC-_ade Methanol, HPLC-grade Methyl-tert-Butyl Ether, HPLC-grade Sodium Hydroxide, 98.4% Sodium Carbonate, 101.0% Sodium Bicarbonate, 100.3% Tetrabutylammonium Hydrogen Sulfate (TBA), approx. 97% 1 Equipment Supplies i ' Model Manufacturer Balance: Mettler Toledo AT261 Mettler-Toledo, Inc., Hightstown, NJ Balance: Mettler Toledo MT5 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, Page87 NorthwestBioanalytical StudyNo. NWBS98-082 ReportNo. NWBR99-005 Equipment/Supplies Model Ontario Manufacturer pH Meter: Orion 230A Orion, Boston, MA Pipettes: Finnpipette: Digital 1-5 mL Pipettes: Rainin Pipetman TM Volumes: fixed20; adjust.: 20, 100; 200; 1000 _L Sonicator Fisher Scientific, Pittsburgh, PA Rainin Instrument Co., Wobum, MA Branson, Danbury, CT Stir Plate: Nuova 1I Vortex: Fisher Genie 2 Thermolyne Corp., Dubuque, IA Fisher Scientific, Fair Lawn, NJ l- 6.3. 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 I-IPLC-grade water to an appropriate 1-L container. Add 26.5 g of sodium carbonate (Na2CO3) to the 1-L container. Add 21.0 g of sodium bicarbonate (NaHCO3) to the 1-L container. Fill to volume with t-IPLC-grade water. Use a stir plate to mix. : 10 N sodium hydroxide Add 400 g of sodium 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. Page 88 NorthwestBioanalytical StudyNo. NWBS98-082 ReportNo. NWBR99-005 0.5 M Tetrabuo,lammonium 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-_ade water and mix. Note: Be sure to check the pH daily when in use. 2 mM ammonium acetate in water Add 1 mL of 1 M ammonium acetate to a 500-mL container approximately half full with HPLC-grade water. QS to volume with HPLC-grade water and mix by inversion. 2 mM ammonium acetate in methanol Fill an appropriate 1-L container approximately half full with methanol. Weigh out 0.154 g of ammonium acetate (NH4 OAc) and transfer to the container. Use a stir plate to mix. Adjust the pH if desired and fill to volume with methanol. 2 mM ammonium acetate water:methanol (50:50 v/v) Fill an appropriate 1-L container with 500 mL of 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 mg of PFOS and transfer to a 10-mL volumetric flask. QS to volume with methanol. Sonicate for approximately 5 minutes. PFOSA Stock Standard (1078 ppn O 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 ppn 0 Weigh 10.310 mg of PFOSAA and transfer to a 10-mL volumetric flask. QS to volume with methanol. Sonicate for approximately 5 minutes. Page89 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 N-MeFOSE-OH Stock Standard (1210ppm) Weigh 12.103 mg of N-MeFOSE-OH and transfer to a 10-mL volumetric flask. QS to volume with methanol. Sonicate for approximately 5 minutes. N-EtFOSE-OH Stock Standard (l127ppm) 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 (1090ppm) Weigh 10.9 mg of POA.A and transfer to a 10-mL volumetric flask. QS to volume with methanol. Sonicate for approximately 5 minutes. 1 PFHS Stock Standard (lO00ppm) Add 807 _tL of PFHS (6200 ppm) to a 5-mL volumetric flask. QS to volume with methanol. Mix by inversion. L Diluted Stock Solution (10. 0 ppm) '_ Add 95 _L of PFOS Stock Solution (1053 ppm), 93 _L of PFOSA Stock Solution (1078 ppm), 97/.tL of PFOSAA Stock Solution (1031 ppm), 83 btL of N-MeFOSE-OH Stock Solution (1210 ppm), 89 _L of N-EtFOSE-OH Stock Solution (1127 ppm), 92 p.L of POAA Stock Solution (1090 ppm) and 100 _L of PFHS Stock Solution (1000 ppm) to a 10-mL volumetric flask. QS to volume with methanol. Mix by inversion. Spiking Standard 8 (5. OOppm) Add 500 p.L of methanol and 500 p.L of Diluted Stock Solution (10.0 ppm) to a 13 x 100 mm silanized glass tube. Mix by vortexing. Spiking Standard 7 (2.50ppm) Add 500 pL of methanol and 500 gL of Spiking Standard 8 (5.00 ppm) to a 13 x 100 mm silanized glass tube. Mix by vortexing. Page 90 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Spiking Standard 6 (1.OOppm) Add 800 _tL of methanol and 200 _L of Spiking Standard 7 (5.00 ppm) to a 13 x 100 mm silanized glass tube. Mix by vortexing. Spiking Standard 5 (0.500ppm) Add 900 _L of methanol and 100 _L of 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 _L of methanol and 50 p.Lof Spiking Standard 7 (5.00 ppm) to a 13 x 100 mm silanized glass tube. Mix by vortexing. Spiking Standard 3 (0.lOOppm) Add 900 _L of methanol and 100 _.L of Spiking Standard 6 (1.00 ppm) to a 13 x 100 mm silanized glass tube. Mix by vortexing ( Spiking Standard 2 (0. 0500ppm) Add 900 _L of methanol and 100 _L of Spiking Standard 5 (0.500 ppm) to a 13 x 100 mm silanized glass tube. Mix by vortexing Spiking Standard 1 (O.OlOOppm) I Add 900 ILLof methanol and 100/.tL of Spildng Standard 3 (0.100 ppm) to a 13 x 100 mm silanized glass tube. Mix by vortexing Internal Standard Solutions All internal standard solutions are transferred to 16 x 100 mm polypropylene screw-cap tubes and stored in a-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. Page 91 NorthwestBioanalytical StudyNo. NWBS98-082 ReportNo. NWBR.99-005 SDS Diluted Stock Solution (10. 0 ppm) Add 91.7 _L of SDS Stock Solution (1091 ppm) to a 10-mL volumetric flask. QS to volume with methanol and mix by inversion. SDS Working Internal Standard (1.00ppm) Add 2.50 mL of SDS DilUted Stock Solution (10 ppm) to a 25-mL class A volumetric flask. QS to volume with methanol and mix by inversion. 6.4. Preparation of Quality Control Samples l High Quality Control (350 ppb spiked) . Quantitatively transfer 1.05 mL of the 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 QualiW Control (20.0 ppb spiked) Quantitatively transfer 60 _.L of the Diluted Stock Solution (10.0 ppm) and 29.94 mL of blank human serum into a polypropylene tube. Sonicate for approximately 10 minutes and i equilibrate for approximately 10 minutes. Storage of QC Samples I I After preparation, place aliquots of the low and high QC pools into 2-mL cryogenic vials, and store in a -20 C freezer. 6.5. Preparation of Calibration Standards If endogenous 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. Page92 NorthwestBioanalytical Standard Number 5 4 2 1 Concentration of Spiking Solution (ppm) 0.500 0.250 0.0500 0.0100 Volume of Spiking Solution (p.L) 20 20 20 20 StudyNo. NWBS98-082 ReportNo. NWBR99-005 Volume of Blank Rabbit Serum (mL) 0.20 0.20 0.20 0.20 Final Spiked Concentration (ppb) 50.0 25.0 5.00 1.00 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 Standard Concentration of Volume of Spiking Volume of Blank Final Spiked .Number Spiking Solution Solution Human Serum Concentration (ppm) (_L) (mL) (ppb) 8 5.00 20 0.20 500 7 2.50 20 0.20 250 6 1.00 20 0.20 100 ' 5 0.500 20 0.20 50.0 : 4 0.250 20 0.20 25.0 i 3 0.100 20 0.20 10.0 2 0.0500 20 0.20 5.00 1 0.0100 20 I 0.20 1.00 6.6. Sample Preparation Calibration Curve Samples (prepare in duplicate) Rabbit Serum Curve Transfer 0.20 mL of HPLC-grade water and 0.20 mL rabbit serum into appropriately labeled 13 x 100 mm polypropylene tubes. Add 20 p.L of Calibration Standard Spiking Solutions 1, 2, 4 and 5 to the corresponding labeled tube. Page93 NorthwestBioanalytical Human Serum Cwwe StudyNo. NWBS98-082 ReportNo. NWBR99-005 Transfer 0.20 mL of HPLC-grade water and 0.20 mL human serum into appropriately labeled 13 x 100 mm polypropylene tubes. Add 20 _L of Calibration Standard Spiking Solutions 1-8 to the corresponding labeled tube. Quality Control Samples (prepare in duplicate) Aliquot 0.20 mL of each of the Low and High controls into appropriately labeled 13 x 100 mm polypropylene tubes. lf Aliquot 0.20 mL of blank human serum into separate 13 x 100 mm polypropylene tubes and label as QC0. Aliquot 0.20 mL of blank human serum into separate 13 x 100 mm polypropylene tubes and label as BLANK Aliquot 0.20 mL of blank rabbit serum into separate 13 x 100 mm polypropylene tubes and label as RABBIT BLANK. i Study Samples Transfer 0.20-mL aliquots of each study sample into appropriately labeled 13 x 100 mm polypropylene tubes. Extraction Procedure ! 1. Add 0.20 mL of H-PLC-grade water (this should be performed before adding the ) calibration standard spiking solutions to calibration curve samples). 2. Add 20 btLof SDS internal standard (1.00 ppm) to all samples, excluding BLANKS. 3. Vortex samples for at least 5 seconds. 4. Add 0.40 mL 0.5 M tetrabutylammonium hydrogen sulfate (TBA), pH 10, and 0.40 mL of 0.25 M carbonate buffer. NOTE: Check the pH of TBA daily. 5. Add 3.0 mL methyl-tert-butyl ether. Page94 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 6. Cap each 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 13 x 100 mm polypropylene tube with a disposable transfer pipet. 9. Dry samples under nitrogen at 45 C until dry (approximately 10 minutes). 10, Add 75 gL of 2 mM ammonium acetate water:methanol (50:50 v/v) to each tube. 11. Vortex for at least 5 seconds. ] 12. Transfer into autosampler vial insert. 1 13. Cap and store extracts at 4 C until analysis. 6.7. LC/MS/MS Conditions LC Conditions Column I.D. Mobile Phase(s) ! Betasil C-18 A: 2mM ammonium acetate in water B: 2mM ammonium acetate in methanol Flow Rate Column Temperature Injection.Volume LC Conditions 300 _tL/min 50 C 5 - 15 l.tL Gradient I The HPLC gradient required is shown below. Time (mind 0.00 4.50 11.0 12.0 13.0 % Mobile Phase A 50 50 2.7 2.7 50 % Mobile Phase B 50 50 97.3 97.3 50 Page 95 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 MS Conditions Post Column Split Source 2:1 Turbo Ion Spray Source Temp. 300 C Analysis type Compounds PFOS PFOSA Multiple reaction monitoring (MRM) Transitions monitored 499 / 80 Dwell Time (ms) 100 498 / 78 100 Collision Energy (v) 91 38 1 PFOSAA 584 / 419 200 30 I N-MeFOSE-OH 616 / 59 100 52 N-EtFOSE-OH 630 / 59 100 58 POAA 413 / 369 100 15 PFHS 399 / 80 100 48 SDS 265 / 97 100 41 The prepared standards and QCs are loaded onto the autosampler tray in a random order and injected into the instrument LC/MS/MS system. 6.8. Quantitation PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA, PFI-IS 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 regressio n analysis of calibration curves (weighted l/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 analysis of calibration curves (weighted I/x) using the peak area ratio (analyte peak area/internal standard peak area) vs. concentration. This quantitation is performed using the Watson DML12vlS software. Page 96 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 Figure 1. Total Ion Chromatogram of Rabbit Serum Blank I0.: 7.304Cl_ 6S00 _J000 ,,- f,oooo 45_ SDS-- "_ 3_ 2_ IOCQ 0.66 | .65 3.26 4.38 5.44 6.73 |. 12 Time,m _" 12.29 Page 97 Northwest Bioanalytical Study No. NWBS98-082 Report No. NWBR99-005 _ Figure 3. Total Ion Chromatogram of High Standard (500 ppb) 6.87r3 [_ I[ 60c._ '_' ,,_, ,.. I,_t _._$ i ,., t 2I ..C$e_..S i.ocs PFHS l 4.72 PFOSA/PFOSAA-- SDS-- POAA eFos [ 8.14 9.48 \ _MeFOSE-OH EtFOSE-OH 1.9,__ 2 3_,. 4 13,17 6.,7 6' ' I0 12 14 "l"u_..mi Page 99