Document 997MKbRKGGrQ8vBb96LpyQ0DR
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NW BR00-I22
Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS,
M556 and M570 in Human Serum by LC/MS/MS
Assay Revalidation Addendum Report
Northwest Bioanalytical (NWB) A Division of NWT Ine. 1121 East 3900 South Salt Lake City, UT 84124
PREPARED FOR:
3M Environmental Technology and Services 935 Bush Avenue St. Paul, MN 55133
AUTHOR:
.
Connie O. Sakashita, B.S., NWB Project Manager
APPROVED F
LEASE BY:
Patrick Bennett, M.S., M.B.A., NWB Laboratory Director
il-J-Q O
DATE:
DATE:
S '" 2 e - ct{
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Northwest Bioanalytical
Study No. NWBSOO-O40 Report No. NWBR00-122
QUALITY ASSURANCE STATEMENT
LABORATORY:
Northwest Bioanalytical (NWB) A Division o f NWT, Ine. 1121 East 3900 South Salt Lake City, UT 84124
SPONSOR:
3M Environmental Technology Services 935 Bush Avenue St Paul, MN 55133
COMPOUND(S) :
PFOS, PFOSA, PFOSAA, PFHS, M556 and M570
NWB STUDY NUMBER:
NWBS00-040
SPONSOR STUDY NUMBER:
Method Validation
NWB STUDY TITLE:
Quantitative Determination of PFOS and Metabolites in Human Serum by LC/MS/MS, Assay Validation
The method validation study described in this report is not included within the definition of a GLP regulated nonclinical study. However, Northwest Bioanalytical conducts all projects in accordance with the guidelines o f the U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 58), the OECD Principles o f Good Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety o f Drugs (Ordinance No. 21, PAB Notification No. 424.) The following inspections were performed by the NWB QAU per SOP.
Inspection and Reporting Statement.
Inspection Date 15 May 2000 15-18 May 2000 07-18 Sep 2000
24 Jan 2000
19-30 Jul 2001
05-07 Nov 2001
20 Nov 2001
Phase o f Studv Analytical Plan Assay Validation Report Draft/Raw Data (NWBR00-108) Final Report (NWBR00-108) Report Draft/Raw Data (NWBROO-122) Report Draft/Raw Data (NWBROO-122) Final Report (NWBR00-122)
Date Inspection Report Issued To
NWB Proiect Manaser NWB Manasement
15 May 2000
31 May 2000
18 May 2000
31 May 2000
20 Sep 2000
29 Sep 2000
24 Jan 2000
31 Jan 2000
30 Jul 2001
31 Jul 2001
08 Nov 2001
30 Nov 2001
20 Nov 2001
30 Nov 2001
.
`Reports to NWB Management are issued monthly.
i
As can reasonably be established, the methods and procedures described and the results incorporated into this final report accurately reflect the raw data.
Sheryl A. Mathews, A.A.S. NWB QAU Compliance Auditor
t- lv
Date
/
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Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBR00-122
COMPLIANCE STATEMENT
The method validation study described in this addendum report is not included within the definition of a GLP nonclinical regulated study. However, to the best of our knowledge, the stability, extraction efficiency, and plasma abbreviated validation experiments were conducted in accordance with the guidelines of the U.S. FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 48) and according to the methods and procedures described within this report. In addition, these experiments followed the guidelines of the OECD Principles of Good Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety of Drugs (Ordinance No. 21, PAB Notification No. 424.)
This addendum report also included results from research experiments conducted outside of the validation study. These experiments were conducted according to the procedures described within section 7 of this report.
Serum sample results obtained using plasma curves for PFOSA, PFOSAA, POAA, PFHS, M556 and M570 did not meet NWB SOP requirements for validation.
Any known circumstances that may have affected the quality or integrity of the study or data are discussed within the report. This report represents an accurate record of the raw data.
Connie O. Sakashita, B.S. NWB Project Manager
Patrick K. Bennett, M.S., M.B.A. NWB Laboratory Director
Date: /v - t - y
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Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
TABLE OF CONTENTS
SIGNATURE PAGE..........................................................................................................................1
QUALITY ASSURANCE STATEMENT...................................................................................... 2
COMPLIANCE STATEMENT....................................................................................................... 3
TABLE OF CONTENTS..................................................................................................................4
LIST OF TABLES................................................ ........................................................................... 5
1. INTRODUCTION.... ...............................................................................................................13
2. METHODOLOGY....................................................................................... .......................... 14
3. STABILITY EVALUATION.................................................................................................17
3.1. Freeze-Thaw and Room Temperature Matrix Stability..................................................... 17
3.2. Reinjection Stability............................................................................................................ 18
3.3. Extract Storage Stability..........................................................
18
3.4. Long-Term Matrix Stability in Human Serum....................................................................19
4. EXTRACTION EFFICIENCY FROM HUMAN SERUM....................................................19
5. COMMENTS AND CONCLUSIONS.................................................................................. 21
6. ABBREVIATED VALIDATION - HUMAN PLASMA...................................................... 21
6.1. Range of Quantitation.........................................................................................................23
6.2. Lower Limit of Quantitation.............................................................................................. 23
6.3. Intra-Assay Precision and Accuracy.............................................................
24
6.4. Proposed Acceptance Criteria for Human Plasma...............................................
24 .
7. ADDITIONAL POST-VALIDATION EXPERIMENTS...................................................... 25
7.1. Ion Suppression Experiment.............................................................................................. 25 7.2. Dilution Matrix Experiment............................................................................................... 27 7.3. Serum and Plasma Comparison......................................................................................... 29 8. DATA MANAGEMENT....................................................................................................... 31
9. REFERENCES.........................................................
31
10. DATA RETENTION.............................................................................................................. 31
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Study No. NWBS00-040 Report No. NWBR00-122
LIST OF TABLES
Table 1. Summary of Calibration Curve Parameters for PFOS in Human Serum....................... 32
Table 2. Summary of Calibration Curve Parameters for PFOSA in Human Serum.................... 32
Table 3. Summary of Calibration Curve Parameters for PFOSAA in Human Serum..................32
Table 4. Summary of Calibration Curve Parameters for POAA in Human Serum..................... 33
Table 5. Summary of Calibration Curve Parameters for PFHS in Human Serum....................... 33
Table 6. Summary of Calibration Curve Parameters for M556 in Human Serum....................... 33
Table 7. Summary of Calibration Curve Parameters for M570 in Human Serum....................... 33
Table 8. Back-Calculated Concentrations of Calibration Standards for PFOS in Human Serum......................................................................................
34
Table 9. Back-Calculated Concentrations of Calibration Standards for PFOSA in Human Serum........................................................................................................... 34
Table 10. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Serum........................................................................................................... 34
Table 11. Back-Calculated Concentrations of Calibration Standards for POAA in Human Serum............................................................................................................ 35
Table 12. Back-Calculated Concentrations of Calibration Standards for PFHS in
Human Serum.............................................................................................................35
Table 13. Back-Calculated Concentrations of Calibration Standards for M556 in Human Serum........................................................................................ !.................. 35
Table 14. Back-Calculated Concentrations of Calibration Standards for M570 in Human Serum.................................
36
Table 15. Quality Control Samples for PFOS in Human Serum................................................ 37
Table 16. Quality Control Samples for PFOSA in Human Serum..............................................37
Table 17. Quality Control Samples for PFOSAA in Human Serum...........................................37
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Study No. NW BS00-040 Report No. NWBROO-122
Table 18. Quality Control Samples for POAA in Human Serum................................................38
Table 19. Quality Control Samples for PFHS in Human Serum............................................ 38
Table 20. Quality Control Samples for M556 in Human Serum.............................................38
Table 21. Quality Control Samples for M570 in Human Serum.............................................38
Table 22. Freeze-Thaw and Room Temperature Matrix Stability for PFOS in Human Serum.............................................................................................................39
Table 23. Freeze-Thaw and Room Temperature Matrix Stability for PFOSA in Human Serum............................................................................................................ 40
Table 24. Freeze-Thaw Stability and Room Temperature Matrix for PFOSAA in Human Serum............................................................................................................ 41
Table 25. Freeze-Thaw and Room Temperature Matrix Stability for POAA in Human Serum............................................................................................................ 42
Table 26. Freeze-Thaw and Room Temperature Matrix Stability for PFHS in Human Serum.............................................................................................................43
Table 27. Freeze-Thaw and Room Temperature Matrix Stability for M556 in Human Serum.............................................................................................................44
Table 28. Freeze-Thaw and Room Temperature Matrix Stability for M570 in Human Serum..................................................................................................
45
Table 29. Reinjection Stability for PFOS.................................................................
46
Table 30. Reinjection Stability for PFOSA.................................... .........................:.....J..i........ 47
Table 31. Reinjection Stability for PFOSAA.................................... .........................'..................48
Table 32. Reinjection Stability for POAA.................................................................................... 49
Table 33. Reinjection Stability for PFHS..................................................................................... 50
Table 34. Reinjection Stability for M556..................................................................................... 51
Table 35. Reinjection Stability for M570..................................................................................... 52
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Study No. NWBSOO-O40 Report No. NWBR00-122
Table 36. Room Temperature Extract Stability for PFOS...........................................................53
Table 37. Room Temperature Extract Stability for PFOSA........................................................54
Table 38. Room Temperature Extract Stability for PFOSAA...................................................... 55
Table 39. Room Temperature Extract Stability for POAA..........................................................56
Table 40. Room Temperature Extract Stability for PFHS...........................................................57
Table 41. Room Temperature Extract Stability for M556...........................................................58
Table 42. Room Temperature Extract Stability for M570............................................................ 59
Table 43. Reduced Temperature (1 to 8 C) Extract Stability for PFOS.................................... 60
Table 44. Reduced Temperature (1 to 8 C) Extract Stability for PFOSA..................................61
Table 45. Reduced Temperature (1 to 8 C) Extract Stability for PFOSAA...............................62
Table 46. Reduced Temperature (1 to 8 C) Extract Stability for POAA....................................63
Table 47. Reduced Temperature (1 to 8 C) Extract Stability for PFHS.....................................64
Table 48. Reduced Temperature (1 to 8 C) Extract Stability for M556.....................................65
Table 49. Reduced Temperature (1 to 8 C) Extract Stability for M570.....................................66
Table 50. Reduced Temperature (-20 C) Extract Stability for PFOS.........................................67
Table 51. Reduced Temperature (-20 C) Extract Stability for PFOSA......................................68
Table 52. Reduced Temperature (-20 C) Extract Stability for PFOSAA...................................69
Table 53. Reduced Temperature (-20 C) Extract Stability for POAA.......................................70
Table 54. Reduced Temperature (-20 C) Extract Stability for PFHS.....................
71
Table 55. Reduced Temperature (-20 C) Extract Stability for M556.........................................72
Table 56. Reduced Temperature (-20 C) Extract Stability for M570.........................................73
Table 57. Long-Term Matrix Stability for PFOS in Human Serum............................................74
Table 58. Long-Term Matrix Stability for PFOSA in Human Serum.........................................74
Table 59. Long-Term Matrix Stability for PFOSAA in Human Serum......................................75
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Study No. NWBSOO-040 Report No. NWBR00-122
Table 60. Long-Term Matrix Stability for POAA in Human Serum.......................................... 75
Table 61. Long-Term Matrix Stability for PFHS in Human
Serum................................. 76
Table 62. Long-Term Matrix Stability for M556 in Human
Serum................................. 76
Table 63. Long-Term Matrix Stability for M570 in Human
Serum................................. 77
Table 64. PFOS Extraction Efficiency from Human Serum..................................................... ...78
Table 65. PFOSA Extraction Efficiency from Human Serum..................................................... 79
Table 66. PFOSAA Extraction Efficiency from Human Serum.................................................. 80
Table 67. POAA Extraction Efficiency from Human Serum..............................
81
Table 68. PFHS Extraction Efficiency from Human Serum........................................................ 82
Table 69. M556 Extraction Efficiency from Human Serum........................................................ 83
Table 70. M570 Extraction Efficiency from Human Serum........................................................ 84
Table 71. Summary of Calibration Curve Parameters for PFOS in Human Plasma................... 85
Table 72. Summary of Calibration Curve Parameters for PFOSA in Human Plasma................ 85
Table 73. Summary of Calibration Curve Parameters for PFOSAA in Human Plasma............. 85
Table 74. Summary of Calibration Curve Parameters for POAA in Human Plasma................. 86
Table 75. Summary of Calibration Curve Parameters for PFHS in Human Plasma................... 86
Table 76. Summary of Calibration Curve Parameters for M556 in Human Plasma................... 86
Table 77. Summary of Calibration Curve Parameters for M570 in Human Plasma................... 87
Table 78. Back-Calculated Concentrations of Calibration Standards for PFOS in Human Plasma......................................
88
Table 79. Back-Calculated Concentrations of Calibration Standards for PFOSA in Human Plasma............................................................................................................ 88
Table 80. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Plasma.............................................................................................................88
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Study No. NWBS00-040 Report No. NWBR00-122
Table 81. Back-Calculated Concentrations of Calibration Standards for POAA in Human Plasma............................................................................................................89
Table 82. Back-Calculated Concentrations of Calibration Standards for PFHS in Human Plasma............................................................................................................89
Table 83. Back-Calculated Concentrations of Calibration Standards for M556 in Human Plasma............................................................................................................89
Table 84. Back-Calculated Concentrations of Calibration Standards for M570 in Human Plasma............................................................................................................90
Table 85. Lower Limit of Quantitation for PFOS in Human Plasma.......................................... 91
Table 86. Lower Limit of Quantitation for PFOSA in Human Plasma........................................92
Table 87. Lower Limit of Quantitation for PFOSAA in Human Plasma......................................93
Table 88. Lower Limit of Quantitation for POAA in Human Plasma..........................................94
Table 89. Lower Limit of Quantitation for PFHS in Human Plasma...........................................95
Table 90. Lower Limit of Quantitation for M556 in Human Plasma...........................................96
Table 91. Lower Limit of Quantitation for M570 in Human Plasma...........................................97
Table 92. Intra-Assay Precision for PFOS Quality Controls in Human Plasma...........................98
Table 93. Intra-Assay Precision for PFOSA Quality Controls in Human Plasma........................99
Table 94. Intra-Assay Precision for PFOSAA Quality Controls in Human Plasma...................100
Table 95. Intra-Assay Precision for POAA Quality Controls in Human Plasma....... ................ 101
Table 96. Intra-Assay Precision for PFHS Quality Controls in Human Plasma....... ................. 102
Table 97. Intra-Assay Precision for M556 Quality Controls in Human Plasma.........................103
Table 98. Intra-Assay Precision for M570 Quality Controls in Human Plasma.........................104
Table 99. Summary of Calibration Curve Parameters for PFOS in Serum Diluted with Buffer................................................................................................................ 105
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Study No. NWBS00-040 Report No. NWBR00-122
%
Table 100. Summary of Calibration Curve Parameters for PFOSA in Serum Diluted
with Buffer.................................................................................................................105
Table 101. Summary of Calibration Curve Parameters for PFOSAA in Serum Diluted with Buffer................................................................................................................ 105
Table 102. Summary of Calibration Curve Parameters for POAA in Serum Diluted with Buffer.................................................................................................................106
Table 103. Summary of Calibration Curve Parameters for PFHS in Serum Diluted with Buffer.................................................................................................................106
Table 104. Summary of Calibration Curve Parameters for M556 in Serum Diluted with Buffer.................................................................................................................106
Table 105. Summary of Calibration Curve Parameters for M570 in Serum Diluted with Buffer.................................................................................................................107
Table 106. Back-Calculated Concentrations of Calibration Standards for PFOS in Serum Diluted with Buffer....................................................................................................108
Table 107. Back-Calculated Concentrations of Calibration Standards for PFOSA in Serum Diluted with Buffer....................................................................................................108
Table 108. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Serum Diluted with Buffer....................................................................................................108
Table 109. Back-Calculated Concentrations of Calibration Standards for POAA in Serum
Diluted with Buffer......................................................
109
Table 110. Back-Calculated Concentrations of Calibration Standards for PFHS in Serum Diluted with Buffer....................................................................................................109
Table 111. Back-Calculated Concentrations of Calibration Standards for M556 in Serum Diluted with Buffer....................................................................................................109
Table 112. Back-Calculated Concentrations of Calibration Standards for M570 in Serum Diluted with Buffer................... ............................................................................... 110
Table 113. PFOS Quality Controls Samples Prepared in Serum Diluted with Buffer.............. 111
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Study No. NWBS00-040 Report No. NWBR00-122
Table 114. PFOSA Quality Control Samples Prepared in Serum Diluted with Buffer..............111
Table 115. PFOSAA Quality Control Samples Prepared in Serum Diluted with Buffer...........112
Table 116. POAA Quality Control Samples Prepared in Serum Diluted with Buffer................112
Table 117. PFHS Quality Control Samples Prepared in Serum Diluted with Buffer.................113
Table 118. M556 Quality Control Samples Prepared in Serum Diluted with Buffer.................113
Table 119. M570 Quality Control Samples Prepared in Serum Diluted with Buffer................. 114
Table 120. Summary of Calibration Curve Parameters for PFOS (Serum vs. Plasma)..............115
Table 121. Summary of Calibration Curve Parameters for PFOSA (Serum vs. Plasma)...........115
Table 122. Summary of Calibration Curve Parameters for PFOSAA (Serum vs. Plasma)........115
Table 123. Summary of Calibration Curve Parameters for POAA (Serum vs. Plasma)............116
Table 124. Summary of Calibration Curve Parameters for PFHS (Serum vs. Plasma)..............116
Table 125. Summary of Calibration Curve Parameters for M556 (Serum vs. Plasma)..............116
Table 126. Summary of Calibration Curve Parameters for M570 (Serum vs. Plasma)..............117
Table 127. Back-Calculated Concentrations of Calibration Standards for PFOS (Serum vs. Plasma)................................................................................................... 118
Table 128. Back-Calculated Concentrations of Calibration Standards for PFOSA (Serum vs. Plasma)................................................................................................... 119
Table 129. Back-Calculated Concentrations of Calibration Standards for PFOSAA: . (Serum vs. Plasma)............................................................................................
120 -- -
Table 130. Back-Calculated Concentrations of Calibration Standards for POAA ; (Serum vs. Plasma)................................................................................................... 121
Table 131. Back-Calculated Concentrations of Calibration Standards for PFHS (Serum vs. Plasma)....................................................................................................122
Table 132. Back-Calculated Concentrations of Calibration Standards for M556 (Serum vs. Plasma)....................................................................................................123
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Study No. NWBSOO-040 Report No. NWBROO-122
Table 133. Back-Calculated Concentrations of Calibration Standards for M570 (Serum vs. Plasma)..................................................................................................124
Table 134. PFOS Quality Controls (Serum vs. Plasma)............................................................. 125
Table 135. PFOSA Quality Controls (Serum vs. Plasma)...........................................................127
Table 136. PFOSAA Quality Controls (Serum vs. Plasma)....................................................... 128
Table 137. POAA Quality Controls (Serum vs. Plasma)............................................................ 129
Table 138. PFHS Quality Controls (Serum vs. Plasma)....................................................... 131
Table 139. M556 Quality Controls (Serum vs. Plasma)......................................
132
Table 140. M570 Quality Controls (Serum vs. Plasma)............................................................. 133
Table 141. Serum QC Results Based on Plasma Curves............................................................ 135
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Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS
Assay Revalidation Addendum Report
1. INTRODUCTION
Northwest Bioanalytical (NWB) was contracted by 3M Environmental Technology and Services (3M) to modify and revalidate a liquid chromatography/tandem mass spectrometry method for the measurement of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in human serum. The accuracy and precision results of the method revalidation were reported in NWB report NWBR00-108 [9.1]. This report summarizes the results from the stability tests conducted as part of the method revalidation for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in human serum for 3M Environmental Technology and Services. The results from the following tests are also included: extraction efficiency from human serum; ion suppression experiment; and diluted matrix experiment. The suitability of the method for use with plasma was determined by demonstrating intra-assay and LLOQ accuracy and precision and comparing serum and plasma results.
Kris Hansen and James Lundberg at 3M Environmental Technology and Services served as the Study Monitors. The following is a list of NWB supervisory personnel involved in the completion of this work: Connie O. Sakashita, B.S. (Current NWB Project Manager); Brad I. Coopersmith, Ph.D. (NWB Project Manager until October 28, 2000); Licong . Jiang, Ph.D. (NWB Senior Scientist); Patrick Bennett, M.S., M.B.A. (NWB Laboratory Director). The following NWB laboratory personnel contributed to the completion of this work: Suzanne Newman, B.S. (NWB Scientist).
NWB SOPs were used in the conduct of this project and were available to project personnel in both electronic and hard copy formats.
Date Study Initiated: 13-Apr-2000 Date Analyses Completed: 25-Apr-2001
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Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
The method revalidation study described in this report is not included within the definition of a GLP regulated nonclinical study. However, Northwest Bioanalytical conducts all studies within the guidelines of the US FDA Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (Title 21 CFR Part 58), the OECD Principles of Good Laboratory Practice and the Japanese MHW Good Laboratory Practice Standard Ordinance for Nonclinical Laboratory Studies on the Safety of Drugs (Ordinance No. 21, PAB Notification No. 424). Any changes to or deviations from the original protocol (Analytical Plan) were documented through approved protocol amendments or deviation memos and are retained within the raw data.
2. METHODOLOGY
Reference Materials and Matrices
Analyte PFOS (FC-95)
Lot Number Purity 193 100%
Expiration Date
12/31/2010
Source 3M
PFOSA
214
100% 12/31/2010
3M
PFOSAA (FC-129)
617
53.8% 12/31/2010
3M
POAA (FC-143)
245
100%
12/31/2010
3M
PFHS* (used for serum)
PFHS (used for plasma)
M556
M570
S398-182 100%
SE-036 91.1%
NB1130478D
118506-26
99.89% 99.75%
12/31/2010 1/01/2010 12/31/2010 12/31/2010
3M 3M 3M 3M
THPFOS
59909
90% 12/31/2010 3M
* Received as a 6,200 ppm solution in methanol. ** Kept dry.
Storage Conditions
Room Temperature
Room Temperature
Room Temperature
Room
Temperature**
-20C
-20C
; Room Temperature
Room Temperature
Room Temperature
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Study No. NWBS00-040 Report No. NWBR00-122
Matrix
Human serum Human plasma (Northern Chinese)
Lot Numbers BC30399-4; BC061900-1 C5186; C4929; C5517; C5200
The reference material purity for PFOS, PFOSA, PFHS and POAA was not available prior to the conduct of this study. Therefore, all serum concentrations included in the report for these analytes are based upon an assumed purity of 100%.
After the revalidation was completed, 3M contracted with Centre Analytical Laboratories, Inc. in State College, Pennsylvania to determine the absolute concentration of PFOS, POAA and PFHS in NWB stock solutions used to prepare the analytical standards and controls used for this revalidation and subsequent analyses. All arrangements for purity determinations and transfers of NWB solutions to Centre Analytical Laboratories, Inc. were performed by 3M. Per agreement with the Sponsor, NWB quantitative results were not corrected for the purity corrections. Therefore, based on the results obtained, the serum concentrations included in this report should be corrected according to the following table (except for the serum samples included in the serum versus plasma experiments, Section 7.3):
Analyte PFOS POAA PFHS
Correction Factor 0.836 0.909 0.855
Principles of the Method
;
The analytical method consisted of a liquiddiquid extraction procedure followed by evaporation and reconstitution of the extract residue with 30:70 (v/v) 20 mM ammonium acetate in water: 20 mM ammonium acetate in methanol. The samples were analyzed by liquid chromatography/tandem mass spectrometry using a PE Sciex API 3000. The instrument was operated in the multiple reaction monitoring (MRM) mode under
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Study No. NWBS0O-O4O Report No. NWBR00-122
optimized conditions for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 detection.
A detailed description of the methodology is included in NWB report NWBR00-108
[9.1],
.
Acceptance Criteria for Human Serum
Calibration Curve
Each run will include in duplicate calibration standards at six or more concentrations covering the lower to upper limit of quantitation. For all analytes except PFOSA, at least three-fourths of the calibration standard's back-calculated concentrations must be within 15% ( 20% for LLOQ) of their individual target concentrations. For PFOSA, at least three-fourths of the calibration standard's back-calculated concentrations must be within 20% ( 25% for LLOQ) of their individual target concentrations. A calibration standard will be considered a statistical outlier if the back-calculated concentration is greater than two times the acceptance criteria for that standard.
Lower Limit o f Quantitation
The back-calculated concentrations of at least one of the duplicate lowest points in the
calibration curve must be within 25% of the target concentration for PFOSA to qualify
as the LLOQ and within 20% of the target concentration to qualify as the LLOQ for all
other analytes. If this criterion is not met, the next level is subjected to the same test and
the LLOQ raised accordingly.
' ':
Quality Control Samples
Each analytical run will include low, medium and high QC samples in duplicate. The measured concentrations of at least two-thirds of all analytical QCs must be within 20% of their target concentrations ( 25% for PFOSA), and no two QCs at the same concentration can be outside the limit. If study samples require dilution, a dilution QC will be analyzed in triplicate for each dilution level (except for PFOSA and PFOSAA
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Study No. NWBS00-040 Report No. NW BR00-I22
which should not be diluted with control matrix). At least two dilution QC at each level must be within 20% of its target concentrations in order to accept diluted study samples at that level. The dilution QC acceptance is independent of the undiluted analytical QC acceptance.
3. STABILITY EVALUATION
The revalidated method's final acceptance criteria is used as a basis for evaluating fluorochemical stability results. If the mean concentration obtained for stability test QC samples falls within 20% ( 25% for PFOSA) of the mean control QC concentrations, then stability is considered acceptable under the specified test conditions. Control QC samples are defined as samples not subjected to test conditions.
The calibration curve parameters summary and individual calibration standard results for the serum stability runs can be found in Tables 1-14. The analytical QC results for the stability runs can be found in Tables 15-21.
3.1. Freeze-Thaw and Room Temperature Matrix Stability
The stability of PFOS, PFOSA, PFOSAA, POAA, and PFHS in human serum for
three freeze-thaw cycles was demonstrated and reported under the original validation
study NWBS98-092, NWB report NWBR99-005 [9.2], Therefore, only freeze-thaw
stability for M556 and M570 was needed. Acceptable results for the three freeze-
thaw cycle stability test was obtained on Rim 4 for M556 and M570 (data not
included).
.
However, since some analyzed subject samples subsequently underwent more than three freeze-thaw cycles, the stability of PFOS, PFOSA, PFOSAA, POAA, PFHS, . M556 and M570 was evaluated after seven freeze-thaw cycles. All analytes demonstrated acceptable stability when compared against controls that underwent one freeze-thaw cycle (Tables 22 - 28).
During the seven freeze-thaw cycle test, samples were left at room temperature for up to 6.75 hour periods between each freeze cycle. Since the freeze-thaw samples
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Study No. NWBSOO-040 Report No. NWBR00-122
demonstrated acceptable stability, the stability of serum samples left at room temperature for up to 6.75 hours is also demonstrated.
3.2. Reinjection Stability
The quantitative reproducibility of standards and controls after reinjection was determined for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, and M570. The samples were extracted and analyzed on June 30,2000 (Run 15 - data shown in NWBR00-108). These samples were reinjected on July 3, 2000 after storage at room temperature (Run 23). The results from Run 23 indicate reinjection stability when compared with data obtained from the original injection [Run 15] (Tables 29 - 35).
3.3. Extract Storage Stability
Extract stability was evaluated at different storage temperatures and for various lengths of time. The evaluations were performed using QC lots prepared on June 27, 2000 for all analytes and conditions. Although the same QC lots (prepared on June 27,2000) were not used for PFOSA and PFOSAA run acceptance determination, they still provided a control value for stability evaluations.
3.3.1. Room Temperature Extract Stability
The extract stability of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 was determined for low and high serum QC extracts stored at room temperature for seven days prior to analysis. These test QC samples were compared against serum control samples that were analyzed immediately following extraction.
The extracted test QC samples demonstrated acceptable stability after seven days at normal room temperature (Tables 36 - 42).
3.3.2. Reduced Temperature fl to 8C) Extract Stability
The extract stability of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 was determined for low and high serum QC extracts stored at 1 to 8C for four
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Study No. NW BS00-040 Report No. NWBR00-122
days prior to analysis. These test QC samples were compared against serum control samples analyzed immediately following extraction.
The extracted test QC samples demonstrated acceptable stability after four days at
1 to 8 C (Tables 43 -4 9 ).
.
3.3.3. Reduced Temperature (-20 C) Extract Stability
The extract stability of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, and M570 was determined for low and high serum QC extracts stored at -20 C for five days prior to analysis. These test QC samples were compared against serum control samples analyzed immediately following extraction.
The extracted test QC samples demonstrated acceptable stability after five days at -20 C (Tables 50-56).
3.4. Long-Term Matrix Stability in Human Serum
The long-term matrix stability of PFOS, POAA, PFHS, M556 and M570 in human serum was determined by storing QC samples for approximately 55 days at -20 C. The long-term matrix stability of PFOSA and PFOSAA in human serum was determined by storing QC samples for approximately 42 days at -20 C. The mean test QC concentrations were compared against theoretical.
The test QC samples demonstrated acceptable stability after approximately 42 days for PFOSA & PFOSAA or 55 days for other analytes, at -20 C (Tables 57 - 63).
4. EXTRACTION EFFICIENCY FROM HUMAN SERUM
:
The extraction efficiencies of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 were determined by comparing the area ratios obtained for the following three cases:
1. Both the analyte and internal standard added following the extraction (post extract).
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Northwest Bioanalytical
Study No. NWBSOO-O40 Report No. NWBR00-122
2. The analyte added to serum prior to extraction and the internal standard added
following extraction (pre-extract analyte).
.
3. The internal standard added to serum prior to extraction and the analyte added following the extraction (pre-extract internal standard).
The extraction efficiencies were then determined by the area ratio of the pre-extract samples to the post-extract samples. The extraction efficiency experiments were performed at both low and high concentrations to determine that there was no concentration bias.
The total amount of analyte in the sample prior to extraction (pre-extract) is equal to the amount of analyte added plus the persistent amount in the blank matrix. The amount of analyte in the post-extract solution is the amount of analyte added plus the amount of analyte recovered (extracted) from the blank matrix. Therefore, the following formula was used to determine the extraction efficiency for PFOS, PFOSAA, POAA, PFHS, M556 and M570:
((Spk + Per)* EE) / (Spk + (Per * EE)) = Rpre / Rpost; where
Spk = the amount o f analyte added
Per = the persistent amount of analyte in the blank matrix
EE = the extraction efficiency
Rpre = the ratio o f analyte/intemal standard determined for the analyte added prior and
internal standard added after extraction
.
R post = the ratio o f analyte/intemal standard determined for the analyte and internal standard added after extraction
Mean extraction efficiency (%)
PFOS 37.3
PFOSA 69.6
PFO.SAA 62.7 : .
Mean extraction efficiency (%)
POAA 15.6
PFHS 15.3
M556 39.1
Mean extraction efficiency (%)
M570 57.1 '
The results are shown in Tables 64 - 70.
THPFOS 5.99
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Northwest Bioanalytical
5. COMMENTS AND CONCLUSIONS
Study No. NWBSO0-O4O Report No. NWBR00-122
Per agreement with the Sponsor, the regressions were not recalculated based upon the updated purity information for PFOS, POAA and PFHS. Some differences might occur due to the effect of rounding if the regressions were performed with the purity corrected concentrations, but these differences would have a negligible effect on the overall interpretation of the revalidation results.
PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in human serum appear to be stable for seven freeze-thaw cycles and demonstrated matrix stability for up to 6.75 hours at room temperature. PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 exhibit extract stability for seven days at room temperature, four days at 1 to 8 C, and five days at -20 C. The analytes also demonstrated matrix stability for approximately 55 days (for PFOS, POAA, PFHS, M556 and M570) and 42 days (for PFOSA and PFOSAA) at -20 C in human serum.
6. ABBREVIATED VALIDATION - HUMAN PLASMA
The choice of matrix for calibration curve preparation is dependent upon the desired
LLOQ. 3M supplied NWB with human plasma identified as "Northern Chinese" plasma
(referred to as Chinese plasma or CP). This matrix has very low levels of
fluorochemicals and will be used for studies with a desired LLOQ of < 5.00 ppb. The
following sections summarize the accuracy and precision of the fluorochemicals in
human plasma and the quantitative accuracy of serum sample results obtained against
plasma calibration curves using the revalidated serum method.
.
The absolute concentrations of PFOS, PFHS and POAA in the stock solution used for
plasma analyses were determined by Centre Analytical Laboratories, Inc. in State
College, Pennsylvania. Based upon the results, the concentrations of plasma standard and
quality control samples were corrected using the following factors:
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Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NWBROO-122
Analyte PFOS POAA PFHS
Correction Factor 0.821 0.961 1.043
All plasma experiment concentrations included in this report reflect adjustments based upon the correction factors above. The serum target concentrations for these experiments have also been adjusted based upon the correction factors (listed on page 15 of report).
The tests for the abbreviated human plasma validation were conducted after the serum revalidation was completed. They were performed outside of the revalidation or in sample analysis studies conducted after the serum revalidation. However, all applicable NWB SOPs were followed.
Persistent Levels of Analytes in Chinese Plasma
The persistent levels of the analytes in Chinese plasma varied in each tube of plasma
tested. Therefore, several tubes of plasma were pooled and the amounts of persistent
analytes were determined. Previous determination of persistent levels was achieved by
comparison with rabbit serum curves. For the experiments included in this report, the
persistent levels were determined as follows:
.
Known amounts of the analytes were added to Chinese plasma in the range of 1 to 50 or 1 to 100 ppb. The standards were then analyzed according to the revalidated method on at least two separate occasions. A linear regression was performed using the spiked analyte concentration as the y-value and the ratio of the analyte area to internal standard area as the x-value (Microsoft Excel 97 formulas). The mean resultant y-intercep"t determined from multiple runs was then used as an estimate of the persistent level of analyte if it was greater than 0.2 ppb.
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Northwest Bioanalytical
6.1. Range of Quantitation
Study No. NW BS00-040 Report No. NW BR00-122
The target calibrator concentrations were approximately 1.00,2.50, 10.0, 25.0, 50.0, 100, 250, 400 and 500 ppb for all analytes except M556. The target calibrator concentrations for M556 were approximately 2-50, 10.0, 25.0, 50.0, 100,250,400 and 500 ppb. The final curve range can vary based upon the persistent levels of the analyte in the human plasma pool used. For the validation study, the calibration curve ranges were as follows:
PFOS PFOSA PFOSAA POAA PFHS M556 M570
3.94 to 414 ppb 1.00 to 500 ppb 1.60 to 501 ppb 1.92 to 481 ppb 1.36 to 523 ppb 2.50 to 500 ppb 1.00 to 500 ppb
The calibration curve parameter summaries and individual calibration standard results for the plasma runs can be found in Tables 7 1 -8 4 .
6.2. Lower Limit of Quantitation
The precision at the Lower Limit of Quantitation (LLOQ) of the assay in human
plasma was determined by using six 100-pL aliquots of the pooled Chinese plasma
fortified with PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, and M570-at
concentrations equal to the LLOQ. The LLOQ concentration was dependent upon the
persistent levels found in the plasma.
;
Lower Limit of Quantitation Precision (%CV) Accuracy (%Bias)
PFOS 3.94 ppb
5.4 -1.5
PFOSA 1.00 ppb
10.1 1.0
PFOSAA 1.60 ppb
5.7 -2.5
POAA 1.92 ppb
4.4 -8.9
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Northwest Bioanalytical
Lower Limit of Quantitation Precision (%CV) Accuracy (%Bias)
PFHS 1.36 ppb
9.9 -5.1
M556 2.50 ppb
5.7 -2.0
Study No. NWBS00-040 Report No. NWBROO-122
M570 1.00 ppb
5.2 3.0
The results are shown in Tables 8 5 -9 1 .
6.3. Intra-Assay Precision and Accuracy
The precision and accuracy of the LC/MS/MS method for PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, and M570 in human plasma were determined by analyzing three levels of quality control samples in replicates of five on a single run.
Precision (%CV) Accuracy (%Theoretical)
Accuracy (%Bias)
PFOS Intra-assay 1.5 to 2.8 101.2 to 112.3 1.2 to 12.3
PFOSA Intra-assay 1.8 to 5.7 104.0 to 116.3 4.0 to 16.3
PFOSAA Intra-assay 2.6 to 5.8 102.6 to 111.7 2.6 to 11.7
POAA Intra-assay 0.8 to 5.8 103.4 to 106.2 3.4 to 6.2
PFHS Intra-assay
Precision (%CV) Accuracy (%Theoretical)
Accuracy (%Bias)
1.7 to 3.1 95.7 to 106.0
-4.3 to 6.0
M556 Intra-assay 4.7 to 7.6 98.7 to 111.3 -1.3 to 11.3
M570 Intra-assay 1.9 to 5.6 101.3 to 118.0 1.3 to 18.0
The results are shown in Tables 9 2 -9 8 .
'
6.4. Proposed Acceptance Criteria for Human Plasma
.
Calibration Curve
.
Each run will include in duplicate calibration standards at six or more concentrations covering the lower to upper limit of quantitation. For all analytes except PFOSA and PFOSAA, at least three-fourths of the calibration standard's back-calculated concentrations must be within 15% ( 20% for LLOQ) of their individual target
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Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NWBR00-122
concentrations. For PFOSA and PFOSAA, at least three-fourths of the calibration standard's back-calculated concentrations must be within 20% ( 25% for LLOQ) of their individual target concentrations. A calibration standard will be considered a statistical outlier if the back-calculated concentration is greater than two times the acceptance criteria for that standard.
Lower Limit of Quantitation
The back-calculated concentrations of at least one of the duplicate lowest points in the calibration curve must be within 25% of the target concentration for PFOSA and PFOSAA to qualify as the LLOQ and within 20% of the target concentration to qualify as the LLOQ for all other analytes. If this criterion is not met, the next level is subjected to the same test and the LLOQ raised accordingly.
Quality Control Samples
Each analytical run will include low, medium and high QC samples in duplicate. The measured concentrations of at least two-thirds of all analytical QCs must be within 20% of their target concentrations ( 25% for PFOSA and PFOSAA), and no two QCs at the same concentration can be outside the limit.
Based upon the results, the method is suitable for use with human plasma.
7. ADDITIONAL POST-REVALIDATION EXPERIMENTS
Additional experiments were conducted subsequent to completion of the revalidafion to : further elucidate the applicability of the revalidated method to sample analysis. These tests were performed outside of any revalidation or sample analysis studies.
7.1. Ion Suppression Experiment
During analysis of medical surveillance serum samples, it was noted that the internal standard response for the study samples differed from the standards and quality controls. POAA and the internal standard (THPFOS) have very similar retention
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Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
times. Therefore, THPFOS and POAA are not chromatographically resolved in extracted samples containing high concentrations of POAA. This co-elution can suppress the internal standard response and introduce a (high) bias in the measured concentration of analytes when compared against the standard curve. The suppression of the internal standard signal is proportional to the concentration of POAA in the sample and dependent on the LC resolution of the two compounds.
An experiment was performed to evaluate the magnitude of this effect. Low control samples were fortified with various (high) levels of POAA. The test samples were analyzed in triplicate in a single run (PFOSMD-41). The mean concentration of the test samples were then compared against the mean of the analytical low control samples. A summary of the results is shown in the following table:
PFOS
Control Mean Cone, (ppb) 51.8 P O A A added at -5 9 2 0 0 ppb % change from control mean 182% P O A A added at -1 1 8 0 0 ppb % change from control mean 28% P O A A added at -5 9 2 0 ppb % change from control mean 16%
P O A A added at -5 9 2 ppb
% change from control mean -2%
PFO SA PFOSAA PFH S
4.05 6.83 8.04
152% 180% 180%
39%
52% 29%
21%
30% 16%
-11% -5% -7%
M 5S6
5.41 216% 52% 36% -8%
M 570 IS A rea
6.87 108857 207% -75% 56% -43% 34% -32% -8% +11%
The experiment verified that very high levels of POAA resulted in a lower response for the internal standard, which then results in a falsely elevated concentration for the other 6 analytes. No significant bias is introduced when the extracted concentration of POAA is within the revalidated range of the analytical method (approximately 500 ppb). However, some samples can contain POAA at concentrations above the revalidated range of the analytical method, and will likely introduce a bias in the reported concentrations of PFOS, PFHS, PFOSA, PFOSAA, M556, or M570 when the concentration of these analytes are determined prior to sample dilution. The amount of bias could range up to approximately 56%. However, the amount of
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Study No. NWBS00-04O Report No. NWBR0-122
PFOS, PFHS, PFOSA, PFOSAA, M556, or M570 impurities in the POAA reference material is not known. This prevents accurate determination of the % bias. It should be noted that since the amount of ion suppression is dependent upon the amount of extracted POAA, no concentration bias is expected for an analyte result determined from a pre-diluted sample wherein the extracted POAA concentration is less than approximately 500 ppb. This bias will not significantly effect the interpretation of the analytical results.
7.2. Dilution Matrix Experiment
The amount of Chinese plasma supplied by 3M is limited. Therefore, it cannot be used on a routine basis for dilution. Human serum (control serum) is used to dilute test samples as needed to obtain measurable concentrations within the range of the calibration curve. The measured concentrations of fluorochemicals (FC) obtained for diluted samples must then be corrected for persistent levels of the analytes in the control serum. Experiments were performed to determine whether a substitute matrix that did not contain persistent levels of FC analytes could be used when performing dilutions.
Previous experiments had shown that samples diluted with buffer instead of serum did not accurately quantitate against a typical serum curve (data not reported). This indicated that the matrix to buffer ratio of the sample affected the extraction recovery. For the experiments described in this report, QC samples were diluted with buffer. The calibration curve was prepared in the same buffer to matrix ratio as'the QC samples to determine if this was a viable method for diluting samples. A different dilution ratio was prepared to determine if samples could be quantitated from a curve prepared in a matrix to buffer ratio that was different from the QC samples (e.g., if QC samples diluted with matrix by a factor of 50 (2% matrix in buffer) could be quantitated against a curve prepared in 10% matrix in buffer). For the experiments described below, ammonium acetate buffer was used to dilute the matrix for the QCs and calibration curves.
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Study No. NW BS00-040 Report No. NWBROO-122
Calibration Curve and OC Preparation
The experiment (PFOSMD run 39) used a curve prepared in 10% serum in buffer, and three levels of QC samples diluted with buffer by a factor of 10 (10% matrix to buffer ratio). The QC concentrations prior to dilution were 150,400, and 4000 ppb plus the persistent level for each analyte. QC samples at 4000 ppb (plus persistent level) were also diluted 1:50 with buffer.
The calibration curve parameter summaries and individual calibration standard results for the runs can be found in Tables 99-112. The quality control sample results can be found in Tables 113-119.
Results for 10% Serum in Buffer (PFOSMD Run 39)
The mean accuracy (% theoretical) for all analytes at all QC concentration levels
diluted with buffer by a factor of 10 (the same serum to buffer ratio as the curve)
ranged from 94.1 % to 114.3%. The intra-assay precision (%CV) for all analytes were
r'
less than or equal to 5.6% for each QC concentration.
The mean accuracy (% theoretical) for the QC samples diluted with buffer by a factor of 50 ranged from 56.5% to 84.8%. The inter-assay precision (%CV) for all analytes were less than or equal to 4.4%. All analytes were biased low, and all analytes except for POAA and PFHS were not within 20% of the target concentrations.
QC samples met the acceptance criteria of 20% of target concentration (25% for PFOSAA) when diluted with buffer if quantitated against a curve prepared in the same serum to buffer ratio. QC samples diluted with buffer at a different ratio than : the calibration curve did not meet the acceptance criteria for all analytes. .
It appears that quantitating samples diluted with buffer against a curve prepared in the same serum to buffer ratio is a viable way minimizing the corrections needed due to persistent levels of analytes in the dilution matrix. However, since most analytes cannot be accurately quantitated against a calibration curve that is prepared in a serum/buffer ratio different from that of the diluted samples, a separate curve with the same buffer to matrix ratio would need to be prepared for each dilution factor needed.
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Northwest Bioanalytical
7.3. Serum and Plasma Comparison
Study No. NWBS00-040 Report No. NWBR00-122
Because the choice of calibration curve and quality control matrix is dependent upon the desired LLOQ, it is not always possible to match the subject sample matrix with that of the calibration curve and quality control matrix. For instance, it is necessary to use Chinese plasma to prepare the calibration standards and quality control samples when analyzing serum samples where a LLOQ of < 5.00 ppb is desired. Therefore, to evaluate if, and to what extent, the use of plasma calibration standards affects the quantitative analysis of fluorochemicals in serum, a comparison of quantitative results obtained from plasma curves versus serum curves was performed.
Calibration curves were prepared in both human serum and Chinese plasma, and were analyzed along with serum and Chinese plasma quality control samples in replicates of four. The results for the calibration and quality control samples are shown in Tables 120-140.
The confidence interval around the serum QC mean was determined for serum samples quantitated against the plasma curve using Microsoft Excel 97. Alpha (a) was set to 0.05 for a 95% confidence level. The confidence interval around the mean was compared to the analytical acceptance range of the QC which is 20% ( 25% for PFOSA and PFOSAA) of the target concentration. The summary results of the experiment are shown in Table 141.
The 10/27/00 serum curve for M570 had a raised LLOQ of 5.20 ppb. Data for PFOSA and PFOSAA analyzed on 10/27/00 and data for PFHS and M556 analyzed on 4/25/01 are not used because either the serum or plasma calibration curve or the QC samples did not meet the acceptance criteria. One high QC replicate analyzed on 10/27/00 was not included in the statistical calculations because of an extraction error.
The confidence intervals around the mean serum QC results for PFOS are within the acceptance criteria at all levels.
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Study No. NWBS00-040 Report No. NWBR00-122
The confidence intervals around the mean serum QC results for PFOSA are greater than the acceptance range of 25% deviation. The confidence intervals ranged up to 78% deviation from target.
The confidence intervals around the mean serum medium QC result for PFOSAA was 33.9% deviation which is greater than the plasma acceptance range of 25% deviation. The confidence interval for the PFOSAA low and high QC samples are within the acceptance criteria.
The confidence intervals around the mean serum QC results for POAA and PFHS are greater than the acceptance range of 20% deviation. The confidence intervals ranged up to 27.4% deviation from target for POAA and up to 37.6% deviation from target for PFHS.
The confidence interval around the mean serum low QC results for M556 was -26.9% deviation. This is greater than the acceptance range of 20% deviation and may be due a low value obtained for one replicate. The confidence intervals for the M556 medium and high QC samples are within the acceptance criteria.
The confidence intervals around the mean serum medium QC result for M570 was 21.9% deviation which is slightly greater than the plasma acceptance range of 20% deviation. The confidence interval for the M570 low and high QC samples are within the acceptance criteria.
CONCLUSION
;
Serum sample results obtained using plasma curves for PFOSA, PFOSAA, POAA, PFHS, M556, and M570 did not meet NWB SOP requirements for validation. Results may vary on average up to 26% from QC results obtained using human serum calibration curves at some concentration levels for PFHS, PFOSAA, POAA, M556, and M570. For PFOSA, results may vary on average up to 43% from QC results obtained using human serum calibration curves. However, when a lower limit of
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Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBROO-122
quantitation less than 5 ppb is required by the Sponsor, it will be necessary to use plasma curves.
8. DATA MANAGEMENT
PFOS, PFOSA, PFOSAA, POAA, PFHS, M556, M570, and THPFOS chromatographic peaks were integrated using PE Sciex MacQuan software (version 1.6) with a smooth factor of one. Quantitation was based upon quadratic regression analysis of calibration curves (weighted 1/x2) using the area ratio vs. concentration calculated by the Watson DMLIMS software (versions 6.1.1.03 and 6.1.1.04).
9. REFERENCES
[9.1.] C. Sakashita. "Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M556 and M570 in Human Serum by LC/MS/MS." Assay Revalidation Report. NWB Study NWBS00-040. NWB Report NWBR00-108. January 24, 2001.
[9.2.] D. Vollmer. "Quantitative Determination of PFOS, PFOSA, PFOSAA, N-MeFOSE-OH, N-EtFOSE-OH, POAA and PFHS in Human Serum by LC/MS/MS." Assay Validation Report. NWB Study NWBS98-082. NWB Report NWBR99-005. May 13,1999.
10. DATA RETENTION
The raw data and final report for this study will be stored in the NWB Archive, 1121 East 3900 South, Salt Lake City, UT 84124 per regulations and contract agreement. 3M Environmental Technology and Services will be notified concerning final disposition of records at completion of contract obligations.
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Study No. NW BS00-040 Report No. NWBR00-122
Table 1. Summary of Calibration Curve Parameters for PFOS in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
03-M-2000 21-Aug-2000
Run A B C R-Squared LLOQ ULOQ Number
23 -0.000004 0.019854 0.112640 0.9862 48.1 547 18 0.000001 0.028989 0.257857 0.9849 48.1 547
A, B, and C are coefficients used to define the calibration curve.
Table 2. Summary of Calibration Curve Parameters for PFOSA in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
03-Jul-2000 21-Aug-2000
Run A B C R-Squared LLOQ ULOQ Number
23 -0.000017 0.054058 0.006149 0.9952 1.00 502 18 0.000005 0.082782 0.019970 0.9952 1.00 502
A, B, and C are coefficients used to define the calibration curve.
Table 3. Summary of Calibration Curve Parameters for PFOSAA in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
03-Jul-2000 21-Aug-2000
Run A B C R-Squared LLOQ ULOQ Number
23 -0.000002 0.013656 -0.020557 0.9903 6.00 505 18 0.000005 0.025511 -0.020269 0.9978 6.00/.. 505
A, B, and C are coefficients used to define the calibration curve.
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Study No. NW BS00-040 Report No. NW BR00-122
Table 4. Summary of Calibration Curve Parameters for POAA in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
03-Jul-2000 21-Aug-2000
Run A B C R-Squared LLOQ ULOQ Number
23 -0.000002 0.013683 0.010580 0.9979 5.76 505 18 0.000003 0.013704 0.017950 0.9966 5.76 505
A, B, and C are coefficients used to define the calibration curve.
Table 5. Summary of Calibration Curve Parameters for PFHS in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
03-Jul-2000 21-Aug-2000
Run A B C R-Squared LLOQ ULOQ Number
23 -0.000006 0.018323 0.012882 0.9971 3.15 502 18 -0.000001 0.022486 0.017181 0.9970 3.15 502
A, B, and C are coefficients used to define the calibration curve.
Table 6. Summary of Calibration Curve Parameters for M556 in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
03-M-2000 21-Aug-2000
Run A B C R-Squared LLOQ ULOQ Number
23 0.000000 0.002714 0.000585 0.9925 4.30 502 18 0.000001 0.011156 0.005643 0.9958 4.30 502
A, B, and C are coefficients used to define the calibration curve.
Table 7. Summary of Calibration Curve Parameters forM570 in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
03-M-2000 21-Aug-2000
Run A B C R-Squared LLOQ ULOQ Number
23 0.000000 0.003818 -0.004160 0.9963 5.60 505 18 0.000005 0.036404 -0.017720 0.9977 5.60 505
A, B, and C are coefficients used to define the calibration curve.
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Study No. NWBS00-040 Report No. N WB ROO-122
Table 8. Back-Calculated Concentrations of Calibration Standards for PFOS in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date 03-Jul-2000 21-Aug-2000
Run Number
23
;
18
48.1 49.6 57.1 72.1 97.1 147 297 447 547
50.7 52.1 64.0 77.3 98.4 158 309 472 564 45.8 45.6 53.4 68.2 86.6 138 298 435 508 51.7 54.3 60.3 80.5 101 152 304 489 566 44.1 45.6 50.5 70.5 91.1 139 286 417 521
Table 9. Back-Calculated Concentrations of Calibration Standards for PFOSA in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date 03-Jul-2000 21-Aug-2000
Run Number
23
18
1.00 2.51 10.1 25.1 50.3 100
1.10 0.918 1.01 0.990
2.54 2.38 2.73 2.30
10.6 27.3 51.6 9.36 24.6 46.2 10.4 27.9 52.7 9.62 25.1 44.7
103 95.4 106 95.8
251 402 502
259 419 534 265 398 436 253 416 530 229 378 502
Table 10. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
_
Run Date 03-Jul-2000 21-Aug-2000
Run Number
23
18
6.00 7.50 15.0 30.0 55.0 105 255 ; 405 505
6.74 7.73 16.6 31.3 57.6 111 278 446 524 5.49 6.93 13.6 27.3 52.0 99.6 249 382 451 5.83 7.71 14.5 29.8 53.7 99.3 239 397 499 6.13 7.50 14.4 32.2 57.0 108 268 414 513
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Study No. NWBSOO-040 Report No. NWBR00-122
Table 11. Back-Calculated Concentrations of Calibration Standards for POAA in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date 03-Jul-2000 21-Aug-2000
Run Number
23
18
5.76 7.26 14.8 29.8 54.8 105 255 405 505
5.93 7.11 15.2 30.6 52.0 109 265 406 524 5.74 6.93 15.6 29.4 53.1 99.6 261 404 474 5.98 7.19 13.9 28.2 52.8 102 256 401 481 6.06 6.58 15.2 32.5 55.2 108 270 412 511
Table 12. Back-Calculated Concentrations of Calibration Standards for PFHS in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date 03-Jul-2000
Run Number
23
21-Aug-2000
18
3.15 4.65 12.2 27.2 52.2 102 252 402 502
3.13 4.45 12.8 27.7 49.3 103 256 405 515 3.20 4.55 13.7 26.3 49.1 99.7 253 402 487 2.85 4.72 11.8 28.1 48.8 98.3 245 408 497 3.17 5.23 12.1 27.9 51.9 105 258 404 502
Table 13. Back-Calculated Concentrations of Calibration Standards for M556 in Human
Serum
.. <.
Quadratic weighted lIk 1. All concentrations are expressed as ppb.
:
Run Date 03-Jul-2000 21-Aug-2000
Run Number
23
18
4.30 11.8 26.8 51.8 102 252 ' 402 502
4.44 12.6 28.9 53.2 113 274 434 528 4.18 11.1 23.8 47.5 95.1 247 381 448 4.36 11.0 25.5 49.0 101 234 391 469 4.35 11.8 27.2 55.4 106 284 415 516
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Study No. NWBSQ0-040 Report No. NWBR00-122
Table 14. Back-Calculated Concentrations of Calibration Standards for M570 in Human Serum
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date 03-Jul-2000 21-Aug-2000
Run Number
23
18
5.60 7.10 14.6 29.6 54.6 105 255 405 505
6.16 6.90 15.4 29.8 54.0 107 273 414 510 5.54 6.43 14.3 28.7 52.6 104 265 404 464 5.11 7.35 13.7 30.2 53.3 101 249 401 503 5.84 7.43 14.4 31.1 54.5 106 269 406 505
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Study No. NW BS00-040 Report No. NWBR00-122
Table 15. Quality Control Samples for PFOS in Human Serum
Mean of replicate QCs reported. All concentrations are expressed as ppb.
Run Date Run Low QC Number 51.1 ppb
03-Jul-2000 23
47.7
21-Aug-2000 18
46.7
% D if. Medium QC % D if. 197 ppb
-6.65
168 -1 4 .7
-8.61
. 170
-1 3 .7
High QC 446 ppb
382
378
% D if
-14.3 -15.2
Table 16. Quality Control Samples for PFOSA in Human Serum
Mean o f replicate QCs reported. All concentrations are expressed as ppb.
Run Date Run Number
03-M-2000 23
21-Aug-2000 18
Low QC 4.00 ppb
2.73
4.34
% D if. Medium QC % D if. 150 ppb
-3 1 .8
100 -3 3 .3
8 .5 0 150 0 .0 0
High QC 400 ppb
286
395
% Dif.
-28.5 -1.25
Table 17. Quality Control Samples for PFOSAA in Human Serum
Mean o f replicate QCs reported. All concentrations are expressed as ppb.
Run Date Run Number
03-Jul-2000 23
21-Aug-2000 18
Low QC 9.00 ppb
7.73
9.95
% D if Medium QC % D if. High QC % D if.
155 ppb
405 ppb
-14.1
110 -2 9 .0 288 -2 8 .9
1 0 .6 160 3 .2 3 413 1.98
Page 37
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NWBROO-122
Table 18. Quality Control Samples for POAA in Human Serum
Mean o f replicate QCs reported. All concentrations are expressed as ppb.
Run Date Run Low QC %oDif. Medium QC % D if.
Number 8.74 ppb
154 ppb
03-Jul-2000 23
8.45 -3 .3 2
150 -2 .6 0
21-Aug-2000 18
7.82 -1 0 .5
149 -3 .2 5
High QC 403 ppb
400
372
% D if
-0 .7 4 4 -7 .6 9
Table 19. Quality Control Samples for PFHS in Human Serum
Mean o f replicate QCs reported. All concentrations are expressed as ppb.
Run Date Run Number
03-Jul-2000 23
21-Aug-2000 18
Low QC 6.15 ppb
6.24
6.86
% D if. Medium QC % D if. High QC % D if.
152 ppb
402 ppb
1 .4 6 157 3 .2 9 427 6 .2 2
1 1 .5 164 7 .8 9 403 0 .2 4 9
Table 20. Quality Control Samples for M556 in Human Serum
Mean o f replicate QCs reported. All concentrations are expressed as ppb.
Run Date Run Number
03-Jul-2000 23
21-Aug-2000 18
Low QC 5.80 ppb
5.52
5.42
% D if. Medium QC % D if. High QC % D if.
152 ppb
402 ppb
-4.83
145 -4 .6 1 381 -5 .2 2
-6.55
141 -7 .2 4 367 -8 .7 1
Table 21. Quality Control Samples for M570 in. Human Serum-::
Mean o f replicate QCs reported. All concentrations are expressed as ppb. `
Run Date Run Number
03-Jul-2000 23
21-Aug-2000 18
Low QC 8.60 ppb
8.20
8.02
% D if. Medium QC % D if. 155 ppb
-4.65
146 -5 .8 1
-6.74
149 -3 .8 7
High QC 405 ppb
390
386
% D if.
-3 .7 0 -4.69
Page 38
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 22. Freeze-Thaw and Room Temperature Matrix Stability for PFOS in Human Serum
All concentrations are expressed as ppb.
Run Number Run Date
18 21-Aug-2000
LOW QC
A fte r 7 F reeze-T h a w cycles
46.8 45.1 44.0-
M ean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
45.3 1.41 3.1 46.7 -3.0
MEDIUM QC
A fte r 7 F reeze-T h a w cycles
170 158 164
M ean S.D.
% CV Control (ng/mL) M ean % Dev. from Control
164 6.00 3.7 170 -3.5
HIGH QC
A fte r 7 F ree ze-T h a w cycles
369 376 361
M ean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
369 7.51 2.0 378 -2.4
Control = mean o f analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle
Page 39
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 23. Freeze-Thaw and Room Temperature Matrix Stability for PFOSA in Human Serum
All concentrations are expressed as ppb.
Run Number Run Date
18 21-Aug-2000
LOW QC
A fte r 7 F reeze-T h a w cycles
3.73 3.53 3.48
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
3.58 0.132
3.7 4.34 -17.5
MEDIUM QC
A fte r 7 F ree ze-T h a w cycles
108 126 125
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
120 10.1 8.4 150 -20.0
HIGH QC
A fte r 7 F reeze-T h a w cycles
348 361 350
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
353 7.00 2.0 395 -10.6
Control = mean o f analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle
Page 40
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NWBROO-122
Table 24. Freeze-Thaw Stability and Room Temperature Matrix for PFOSAA in Human Serum
All concentrations are expressed as ppb.
Run Number Run Date
18 21-Aug-2000
LOW QC
A fte r 7 F reeze-T h a w cycles
9.08 9.85 9.46
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
9.46 0.385
4.1 9.95 -4.9
MEDIUM QC
A fte r 7 F reeze-T h a w cycles
114 147 147
Mean S.D.
% CV Control (ng/mL) Mean % Dev. fro m Control
136 19.1 14.0 160 -15.0
HIGH QC
A fte r 7 F reeze-T h a w cycles
383 403 391
M ean S.D.
% CV Control (ng/mL) M ean % Dev. from Control
392 10.1 2.6 413 -5.1
Control = mean o f analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle
Page 41
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NWBR00-122
Table 25. Freeze-Thaw and Room Temperature Matrix Stability for POAA in Human Serum
All concentrations are expressed as ppb.
Run Number Run Date
18 21-Aug-2000
LOW QC A fter 7 F reeze-T h a w cycles
8.06 8.83 8.25
M ean S.D.
% CV
Control (ng/mL) Mean % Dev. from Control
8.38 0.401
4.8 7.82 7.2
M EDIUM QC
A fte r 7 F reeze-T h a w cycles
151 149 154
M ean
S.D.
% CV
Control (ng/mL) Mean % Dev. from Control
151 2.52 1.7 149 1.3
HIGH QC A fte r 7 F reeze-T h a w cycles
364 379 372
M ean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
372 7.51 2.0 372 0.0
Control = mean o f analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle
Page 42
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NW BR00-122
Table 26. Freeze-Thaw and Room Temperature Matrix Stability for PFHS in Human Serum
All concentrations are expressed as ppb.
Run Number Run Date
18 21-Aug-2000
LOW QC
A fte r 7 F reeze-T haw cycles
6.05 6.27 7.12
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
6.48 0.565
8.7 6.86 -5.5
MEDIUM QC
A fte r 7 F reeze-T h a w cycles
165 157 163
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
162 4.16 2.6 164 -1.2
HIGH QC
A fte r 7 F reeze-T h a w cycles
397 417 405
M ean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
406 10.1 2.5 403 0.7
Control = mean o f analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle
Page 43
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 27. Freeze-Thaw and Room Temperature Matrix Stability for M556 in Human Serum
All concentrations are expressed as ppb.
Run Number Run Date
18 21-Aug-2000
LOW QC A fte r 7 F reeze-T h a w cycles
4.95 5.07 5.09
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
5.04 0.0757
1.5 5.42 -7.0
MEDIUM QC
A fte r 7 F reeze-T h a w cycles
128 140 . 135
M ean S.D.
% CV Control (ng/mL) M ean % Dev. from Control
134 6.03 4.5 141 -5.0
HIGH QC
A fte r 7 F reeze-T h a w cycles
359 372 374
M ean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
368 8.14 2.2 367 0.3
Control = mean o f analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle
Page 44
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBROO-122
Table 28. Freeze-Thaw and Room Temperature Matrix Stability for M570 in Human Serum
All concentrations are expressed as ppb.
Run Number Run Date
18 21-Aug-2000
LOW QC
A fte r 7 F ree ze-T h a w cycles
7.68 8.76 8.51
M ean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
8.32 0.565
6.8 8.02 3.7
MEDIUM QC
A fte r 7 F ree ze-T h a w cycles
142 142 ' 143
M ean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
142 0.577
0.4 149 -4.7
HIGH QC
A fte r 7 F ree ze-T h a w cycles
356 371 370
M ean S.D.
% CV Control (ng/mL) M ean % Dev. from Control
366 8.39 2.3 386 -5.2
Control = mean o f analytical QC samples analyzed in the same run NOTE: Samples were left at room temperature for up to 6.75 hour periods between each freeze cycle
Page 45
Northwest Bioanalytical
Table 29. Reinjection Stability for PFOS
All concentrations are expressed as ppb.
Study No. NWBS00-040 Report No. NWBR00-122
Run Num ber R un D ate
23 3-Jul~2000
LOW QC R e in jec tio n (A fter 2.5 D ays R oom Temp.)
49.1 48.0 50.0 46.1 45.1
M ean S .D .
% CV C ontrol (ng/m L) M ean % Dev. from C ontrol
47.7 2.04 4.3 47.2 1.1
M EDIU M QC R e in je c tio n
(A fter 2.5 Days Room Temp.)
164 172 170 164 170
M ean S .D .
v .c v C ontrol (ng/m L) M ean % Dev. from C ontrol
168 3.74 2.2 173 -2.9
H IG H QC R e in je c tio n (A fter 2.5 D ays Room Temp.)
391 369 368 372 411
M ean S.D.
% CV C o n tro l (ng/m L) M ean % Dev. from C ontrol
382 18.6 4.9 393 -2 .8
Control = mean o f analytical QC samples analyzed in run 15 (original injection)
Page 46
Northwest Bioanalytical
Study No. NWBSQ0-040 Report No. NWBR00-122
Table 30. Reinjection Stability for PFOSA
All concentrations are expressed as ppb.
Run Number R un Date
23 3-Jul-2000
LOW QC R ein je c tio n (A fter 2.5 D ays R oom Temp.)
2.79 2.69 2.85 2.66 2.68
M ean S.D.
%CV C ontrol (ng/mL) M ean % Dev. from C ontrol
2.73 0.0820
3.0 2.82 -3.2
M EDIUM QC R ein jectio n
(A fter 2.5 Days R oom Temp.)
102 102 97.9 94.4 105
M ean S.D.
% CV C ontrol (ng/mL) M ean % Dev. from Control
100 4.14 4.1 103 -2.9
H IG H QC R ein jectio n (A fter 2.5 D ays Room Temp.)
ns . 273 290 284 307
M ean S.D.
%CV C ontrol (ng/mL) M ean % Dev. from Control
286 13.7 4.8 300 -4.7
Control = mean o f analytical QC samples analyzed in run 15 (original injection)
Page 47
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBR00-122
Table 31. Reinjection Stability for PFOSAA
All concentrations are expressed as ppb.
Run Number R un Date
23 3-Jul-2000
LOW QC R e in je c tio n (After 2.S Days Room Temp.)
7.78 7.58 8.15 7.41 7.74
M ean S.D.
% CV C ontrol (ng/mL) M ean % Dev. from Control
7.73 0.276
3.6 7.59 1.8
M EDIUM QC R e in jec tio n
(After 2.5 Days Room Temp.)
109 112 113 104 110
M ean S.D.
%CV C ontrol (ng/mL) M ean % Dev. from Control
.
110 3.51 3.2
H3 -2.7
HIG H QC - Reinjection (After 2.5 D ays Room Temp.)
287 285 284 278 305
M ean S.D.
%CV C ontrol (ng/mL) M ean % Dev. from Control
288 10.2 3.5 301 -4.3
Control = mean o f analytical QC samples analyzed in run 15 (original injection)
Page 48
Northwest Bioanalytical
Table 32. Reinjection Stability for POAA
All concentrations are expressed as ppb.
Study No. NW BS00-040 Report No. NWBROO-122
Run Number Run Date
23 3-Jul-2000
LOW QC R e in jec tio n (After 2.5 D ays Room Temp.)
8.68 8.43 8.31 8.41 8.41
M ean S.D.
% CV C ontrol (ng/m L) M ean % Dev. from Control
8.45 0.138
1.6 8.10 4J
M EDIU M QC R ein jectio n
(A fter 2.5 D ays Room Temp.)
150 152 156 144 147
M ean S.D.
%CV C ontrol (ng/mL) M ean % Dev. from C ontrol
150 4.60 3.1 144 4.2
H IG H QC R e in jec tio n (After 2.5 D ays Room Temp.)
417 408 386 388 401
M ean S.D.
% CV C ontrol (ng/m L) M ean % Dev. from C ontrol
to o 13.2 3.3 384 4.2
Control = mean o f analytical QC samples analyzed in run 15 (original injection)
Page 49
Northwest Bioanalytical
Table 33. Reinjection Stability for PFHS
All concentrations are expressed as ppb.
Study No. NWBS0O-040 Report No. NWBROO-122
Run Number R un D ate
23 3-Jul-2000
LOW QC R e in je c tio n (A fter 2.5 D ays R oom Tem p.)
6.30 6.32 6.48 5.98 6.11
M ean S .D .
% CV C ontrol (ng/m L) M ean % Dev. from C ontrol
6.24 0.195
3.1 6.22 0.3
M EDIUM QC R e in je c tio n
(After 2.5 D ays Room Temp.)
153 156 156 159 159
M ean S.D.
% CV C ontrol (ng/m L) M ean % Dev. from C ontrol
157 2.51 1.6 158 -0.6
H IG H QC R ein je c tio n (A fter 2.5 Days Room Temp.)
441 416 421 409 450
M ean S.D .
% CV C ontrol (ng/m L) M ean % Dev. from Control
427 17.4 4.1 445 -4.0
Control = mean o f analytical QC samples analyzed in run 15 (original injection)
Page 50
Northwest Bioanalytical
Table 34. Reinjection Stability for M556
All concentrations are expressed as ppb.
Study No. NWBS00-040 Report No. NWBR00-122
Run Number R un Date
23 3-Jul-2000
LOW QC R e in je c tio n (A fter 2.5 D ays Room Temp.)
5.78 5.73 5.45 5.29 5.33
M ean S.D.
% CV C ontrol (ng/m L) M ean % Dev. from C ontrol
5.52 0.227
4.1 5.61 -1.6
M EDIUM QC R ein jectio n
(A fter 2.5 D ays Room Temp.)
144 147 147 , 143 146
M ean S.D.
% CV C ontrol (ng/m L) M ean % Dev. from Control
145 1.82 1.3 147 -1.4
H IG H QC R e in je c tio n (A fter 2.5 D ays Room Temp.)
388 373 381 367 398
M ean S.D.
% CV C ontrol (ng/m L) M ean % Dev. from Control
381 12.2 3.2 396 -3.8
Control = mean o f analytical QC samples analyzed in run 15 (original injection)
Page 5 1
Northwest Bioanalytical
Table 35. Reinjection Stability for M570
All concentrations are expressed as ppb.
Study No. NWBS00-040 Report No. NWBR00-122
Run Num ber R un Date
23 3-Jul-2000
LOW QC R ein je c tio n (A fter 2.5 D ays Room Temp.)
8.31 8.36 . 8.52 8.10 7.71
M ean S.D .
% CV C ontrol (ng/m L) M ean % Dev. from C ontrol
8.20 0.312
3.8 8.31 -U
M EDIUM QC R ein je c tio n
(A fter 2.5 D ays Room Temp.)
145 149 146 144 145
M ean
S.D .
%cv
C ontrol (ng/m L) M ean % Dev. from C ontrol
146 1.92 1.3 147 -0.7
HIG H QC R ein je c tio n (A fter 2.5 D ays Room Temp.)
391 384 390 380 405
M ean S.D.
% CV C ontrol (ng/m L) M ean % Dev. from C ontrol
390 9.51 2.4 401 -2.7
Control = mean o f analytical QC samples analyzed in run 15 (original injection)
Page 52
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 36. Room Temperature Extract Stability for PFOS
All concentrations are expressed as ppb.
Run Number Run Date
18 21-Aug-2000
LOW QC
After 7 Days
51.7 46.0 46.2
Mean S.D.
% cv
Control (ng/mL) Mean % Dev. from Control
48.0 3.23 6.7 46.7 2.8
HIGH QC
After 7 Days
373 402 397
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
391 15.5 4.0 378 ' 3.4
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 53
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 37. Room Temperature Extract Stability for PFOSA
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC
After 7 Days
3.48 3.34 3.30
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
3.37 0.0945
2.8 3.13 7.7
HIGH QC
After 7 Days
337 362 334
Mean
S.D.
%cv
Control (ng/mL)
Mean % Dev. from Control
344 15.4 4.5 322 6.8
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 54
Northwest Bioanalytical
Study No. NWBS0O-O4O Report No. NWBR00-122
Table 38. Room Temperature Extract Stability for PFOSAA
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC
After 7 Days
8.2 9.2 9.3
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
8.89 0.607 6.8 8.02 10.8
HIGH QC
After 7 Days
321 337 343
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
334 11.4 3.4 297 12.5
Control = mean o f QC samples' analyzed in the same run immediately following extraction
Page 55
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 39. Room Tem perature Extract Stability for POAA
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC
After 7 Days
6.92 7.98 8.15
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
7.68 0.667
8.7 7.82 -1.8
HIGH QC
After 7 Days
360 395 378
Mean S.D.
% CY Control (ng/mL) Mean % Dev. from Control
378 17.5 4.6 372 1.6
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 56
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 40. Room Temperature Extract Stability for PFHS
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC
After 7 Days
6.31 6.21 5.90
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
6.14 0.214 3.5 6.86 -10.5
HIGH QC
After 7 Days
396 405 412
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
404 8.02 2.0 403 0.2
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 57
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 41. Room Temperature Extract Stability for M556
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC
After 7 Days
5.13 5.65 5.50
Mean S.D.
%CV Control (ng/mL) Mean % Dev. from Control
5.43 0.268 4.9 5.42 0.2
HIGH QC
After 7 Days
373 376 387
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
379 7.37 1.9 367 3.3
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 58
Northwest B ioanalytical
Study No. NWBSOO-040 Report No. NWBR00-122
Table 42. Room Temperature Extract Stability for M570
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC
After 7 Days
8.86 9.13 8.74 .
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
8.91 0.200 2.2 8.02 11.1
HIGH QC
After 7 Days
409 429 423
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
420 10.3 2.5 386 8.8
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 59
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBR00-122
Table 43. Reduced Temperature (1 to 8 C) Extract Stability for PFOS
All concentrations are expressed as ppb.
Run Number Run Date
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
18 21-Aug-2000
LOW QC 1 to 8C
After 4 Days
49.3 45.6 44.8
46.6 2.40 5.2 46.7 -0.2
HIGH QC 1 to 8C
After 4 Days
391 385 377
384 7.02 1.8 378 1.6
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 60
Northwest Bioanalytical
Study No. NWBS0O-O4O Report No. NWBR00-122
Table 44. Reduced Temperature (1 to 8 C) Extract Stability for PFOSA
All concentrations are expressed as ppb.
Run Number Run Date
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
18 21-Aug-2000
LOW QC 1 to 8C
After 4 Days
3.22 2.79 2.86
2.96 0.231
7.8 3.13 -5.4
HIGH QC 1 to 8C
After 4 Days
350 297 304
317 28.8 9.1 322 -1.6
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 61
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 45. Reduced Temperature (1 to 8 C) Extract Stability for PFOSAA
All concentrations are expressed as ppb.
Run Number Run Date
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
Mean S.D1
% CV Control (ng/mL) Mean % Dev. from Control
18 21-Aug-2000
LOW QC 1 to 8C
After 4 Days
7.78 8.55 8.32
8.22 0.395 4.8 8.02 2.5
HIGH QC 1 to 8C
After 4 Days
312 306 311
310 3.21 1.0 297 4.4
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 62
Northwest Bioanalytical
Study No. NWBSOO-04O Report No. NWBR00-122
Table 46. Reduced Temperature (1 to 8 C) Extract Stability for POAA
All concentrations are expressed as ppb.
Run Number Run Date
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
18 21-Aug-2000
LOW QC 1 to 8C
After 4 Days
7.78 8.75 8.39
8.31 0.490 5.9 7.82
6.3
HIGH QC 1 to 8C
After 4 Days
381 394 385
387 6.66 1.7 372 4.0
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 63
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 47. Reduced Temperature (1 to 8 C) Extract Stability for PFHS
All concentrations are expressed as ppb.
Run Number Run Date
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
18 21-Aug-2000
LOW QC 1 to 8C
After 4 Days
6.76 6.34 7.74
6.95 0.718 10.3 6.86
1.3
HIGH QC 1 to 8C
After 4 Days
400 411 423
411 11.5 2.8 403 2.0
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 64
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBROO-122
Table 48. Reduced Temperature (1 to 8 C) Extract Stability for M556
All concentrations are expressed as ppb.
Run Number Run Date
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
Mean S.D.
%CV Control (ng/mL) Mean % Dev. from Control
18 21-Aug-2000
LOW QC 1 to 8C
After 4 Days
5.47 6.57 6.82
6.29 0.718 11.4 5.42 16.1
HIGH QC 1 to 8C
After 4 Days
398 404 403
402 3.21 0.8 367 9.5
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 65
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 49. Reduced Temperature (1 to 8 C) Extract Stability for M570
All concentrations are expressed as ppb.
Run Number Run Date
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
18 21-Aug-2000
LOW QC 1 to 8C
After 4 Days
7.96 7.90 9.21
8.36 0.740
8.9 8.02 4.2
HIGH QC 1 to 8C
After 4 Days
396 394 402
397 4.1'6 1.0 386 2.8
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 66
Northwest Bioanalytical
Study No. NWBSOO-04O Report No. NWBR00-122
Table 50. Reduced Temperature (-20 C) Extract Stability for PFOS
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC -20C
After 5 Days
47.2
45.5 40.9
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
44.5
3.26 7.3 46.7 -4.7
HIGH QC -20C
After 5 Days
376 373 365
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
371 5.69 1.5 378 -1.9
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 67
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 51. Reduced Temperature (-20 C) Extract Stability for PFOSA
All concentrations are expressed as ppb.
Run Number Run Date
18 21-Aug-2000
LOW QC -20C
After 5 Days
2.95 3.16 3.14
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
3.08 0.116 3.8 3.13 -1.6
HIGH QC -20C
After 5 Days
312 312 296
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
.
307 9.24 3.0 322 -4.7
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 68
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 52. Reduced Temperature (-20 C) Extract Stability for PFOSAA
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC -20C
After 5 Days
7.52 8.32 7.89
Mean S.D.
%CV Control (ng/mL) Mean % Dev. from Control
7.91 0.400 5.1 8.02 -1.4
HIGH QC -20C
After 5 Days
294 306 287
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
296 9.61 3.2 297 -0.3
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 69
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 53. Reduced Temperature (-20 C) Extract Stability for POAA
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC -20C
After 5 Days
7.84 10.2 6.85
Mean S.D.
%CV Control (ng/mL) Mean % Dev. from Control
8.30 1.72 20.7 7.82 6.1
HIGH QC -20C
After 5 Days
351 373 386
Mean S.D.
% cv
Control (ng/mL)
Mean % Dev. from Control
370 17.7 4.8 372 -0.5
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 70
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBROO-122
Table 54. Reduced Temperature (-20 C) Extract Stability for PFHS
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC -20C
After 5 Days
6.68 6.06 7.70
Mean
S.D.
% cv
Control (ng/mL) Mean % Dev. from Control
6.81 0.828 12.2 6.86 -0.7
HIGH QC -20C
After 5 Days
403 407 417
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
409
1.2\
1.8 403 1.5
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 71
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 55. Reduced Temperature (-20 C) Extract Stability for M556
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC -20C
After 5 Days
4.51 5.69 5.97
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
5.39 0.775 14.4 5.42 -0.6
HIGH QC -20C
After 5 Days
358 373 378
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
370 10.4 2.8 367 0.8
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 72
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBR00-122
Table 56. Reduced Temperature (-20 C) Extract Stability for M570
All concentrations are expressed as ppb.
Run Number
18
Run Date 21-Aug-2000
LOW QC -20C
After 5 Days
8.43 8.52 7.94
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
8.30 0.312 3.8 8.02 3.5
HIGH QC -20C
After 5 Days
366 388 369
Mean S.D.
% CV Control (ng/mL) Mean % Dev. from Control
374 11.9 3.2 386 -3.1
Control = mean o f QC samples analyzed in the same run immediately following extraction
Page 73
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 57. Long-Term Matrix Stability for PFOS in Human Serum
All concentrations are expressed as ppb.
Run Date
Run Number
21-Aug-2000
18
Low QC 51.1 ppb 55 Days
52.2
50.1
42.3 43.8
45.0
Medium QC 197 ppb 55 Days
175
171
174
166 165
High QC 446 ppb 55 Days
381
391
361
371 388
Mean SD
%CV %Bias
N
46.7 170 378
4.26 4.55
12.4
9.1 2.7 3.3
-8.6 -13.7 -15.2
55 5
Table 58. Long-Term Matrix Stability for PFOSA in Human Serum
All concentrations are expressed as ppb.
Run Date
Run Number
21-Aug-2000
18
Low QC 4.00 ppb 42 Days
4.28
4.56
4.40 4.12
4.32
Medium QC 150 ppb 42 Days 155
158
154.
142 .
143
High QC 400 ppb 42 Days
376
420 381
397 .
400
Mean SD
%CV %Bias
N
4.34 0.161 3.7 8.5
5
150 7.37 4.9 0.0
5
395 17.4 4.4 -1.3 5
Page 74
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 59. Long-Term Matrix Stability for PFOSAA in Human Serum
All concentrations are expressed as ppb.
Run Date
Run Number
21-Aug-2000
18
Low QC 9.00 ppb 42 Days
9.64
9.11 10.5
10.2
10.3
Medium QC 155 ppb 42 Days
157
158 164
159 160
High QC 405 ppb 42 Days
403
429
406
413 416
Mean SD
%CV %Bias
N
9.95 0.568 5.7 10.6
5
160 2.70 1.7 3.2
5
413 10.2 2.5 2.0
5
Table 60. Long-Term Matrix Stability for POAA in Human Serum
All concentrations are expressed as ppb.
Run Date
Run Number
21-Aug-2000
18
Low QC 8.74 ppb 55 Days
7.74
7.54
7.72
8.05
8.03
Medium QC 154 ppb 55 Days 145
138
155 .
151 .
157
High QC 403 ppb 55 Days
364
380
354 -
371 .
389
Mean SD
%CV %Bias
N
7.82 0.219 2.8 -10.5
5
149 7.76 5.2 -3.2
5
372 13.6 3.7 -7.7 5
Page 75
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-I22
Table 61. Long-Term Matrix Stability for PFHS in Human Serum
All concentrations are expressed as ppb.
Run Date
Run
Number
21-Aug-2000
18
Low QC 6.15 ppb 55 Days
7.01
7.06
7.10 6.57
6.56
Medium QC 152 ppb 55 Days
163
158
169
161
168
High QC 402 ppb 55 Days
387 414
375 404
435
Mean SD
%CV %Bias
N
6.86 0.271 4.0 11.5
5
.164 4.66 2.8 7.9
5
, 403 23.4 5.8 0.2 5
Table 62. Long-Term Matrix Stability for M556 in Human Serum
All concentrations are expressed as ppb.
Run Date 21-Aug-2000
Run Number
18
Low QC 5.80 ppb 55 Days
6.07
5.67
*4.60
4.97
5.79
Medium QC 152 ppb 55 Days 141
137
149
137
143
High QC 402 ppb 55 Days
355
382
340
362 s .
394
Mean SD
%CV %Bias .N
5.42 141
0.612
4.98
11.3 3.5
-6.6 -7.2
55
* > 20% deviation from theoretical
367 21.5 5.9 -8.7
5
Page 76
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 63. Long-Term M atrix Stability for M570 in Human Serum
All concentrations are expressed as ppb.
Run Date
Run Number
21-Aug-2000
18
Low QC 8.60 ppb 55 Days
7.93 8.07
7.87
8.04 8.20
Medium QC 155 ppb 55 Days
147 147
158
144 150
High QC 405 ppb 55 Days
373 391
373 384
408
Mean SD
%CV %Bias
N
8.02 0.128
1.6 -6.7
5
149 5.36 3.6 -3.9
5
386 14.6 3.8 -4.7 5
Page 77
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 64. PFOS Extraction Efficiency from Human Serum
Run 22
L ow Concentration = 4.00 ng/mL
Time o f Soikine
1. A nalyte and IS after extraction
Mean
Ratio Analvte/IS 0.0679 0.0683 0.0735 0.0699
Ratio IS/Analvte 14.7 14.6 13.6 14.3
2. Analyte prior and IS after extraction
Mean
0.0656 0.0605 0.0649 0.0637
3. IS prior and Analyte after extraction
Mean
0.775 0.726 0.805 0.769
Adjusted mean extraction efficiency = 44.4% Mean extraction efficiency for the internal standard = 5.38%
H igh Concentration -- 400 ng/mL
Time o f Spikins
1. A nalyte and IS after extraction
Mean
Ratio Analvte/IS 1.65 1.55 1.65 1.62
2. A nalyte p rio r and IS after extraction
Mean
0.522 0.538 0.518 0.526
Ratio IS/Analvte 0.605 0.643 0.607 0.618
3. IS prior and A nalyte after extraction
Mean
0.0396 0.0379 0.0401 0.0392
.
i
=
Adjusted mean extraction efficiency = 30.1% Mean extraction efficiency for the internal standard = 6.34%
O verall mean extraction efficiency fo r analyte = 37.3% O verall m ean extraction efficiency fo r internal standard - 5.86%
* Due to rounding, calculations based on displayed values may differ from actual.
Page 78
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NWBR00-122
Table 65. PFOSA Extraction Efficiency from Human Serum
. Run 22
L ow Concentration = 4.00 ng/mL
Time of Soikine
1. A nalyte and IS after extraction
Mean
Ratio Analvte/IS 0.0275 0.0222 0.0214 0.0237
Ratio IS/Analvte 36.3 45.0 46.8 42.7
2. Analyte prior and IS after extraction
Mean
0.0171 0.0147 0.0172 0.0163
3. IS prior and A nalyte after extraction
Mean
2.47 2.25 2.45 2.39
Mean extraction efficiency for the internal standard = 5.60%
H igh Concentration = 400 ng/mL
Time o f Soikine
1. A nalyte and IS after extraction
Mean
Ratio Analvte/IS 2.58 2.47 2.65 2.57
Ratio IS/Analvte 0.387 0.405 0.377 0.390
2. Analyte prior and IS after extraction
Mean
1.80 1.87 1.75 1.81
3. IS prior an d Analyte after extraction
Mean
0.0243 0.0223 0.0233. ; 0.0233
:
Mean extraction efficiency for the analyte = 70.4%
Overall m ean extraction efficiencyfo r analyte = 69.6% Overall m ean extraction efficiency fo r internal standard = 5.79%
* Due to rounding, calculations based on displayed values may differ from actual.
Page 79
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBRQO-122
Table 66. PFOSAA Extraction Efficiency from Human Serum
Run 22
L o w Concentration -- 4.00 ng/mL
Time of Soikine
1. A nalyte and IS a fte r extraction
Mean
Ratio Analvte/IS 0.0246 0.0257 0.0238 0.0247
Ratio IS/Analvte 40.6 39.0 42.0 40.5
2. A nalyte prior and IS after extraction
Mean
0.0206
0.0186
0.0194
0.0195
..
3. IS prior and A nalyte after extraction
Mean
2.71 2.45 2.58 2.58
Adjusted mean extraction efficiency = 62.6% Mean extraction efficiency for the internal standard = 6.37%
H igh Concentration = 400 ng/mL
Time o f Spiking
1. A nalyte and IS a fte r extraction
Mean
Ratio Analvte/IS 1.50 1.44 1.56 1.50
2. A nalyte prior and IS after extraction
Mean
0.962 0.959 0.917 0.946
3. IS prior and Analyte after extraction
Mean
Ratio IS/Analvte 0.665 0.693 0.641 0.666
0.0419 0.0394 0.0413 0.0409
.
:
Adjusted mean extraction efficiency = 62.7% Mean extraction efficiency for the internal standard = 6.14%
O verall m ean extraction efficiency fo r analyte = 62.7% Overall m ean extraction efficiency fo r internal standard. = 6.26%
* Due to rounding, calculations based on displayed values may differ from actual.
Page 80
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBR00-122
Table 67. POAA Extraction Efficiency from Human Serum
Run 22
L ow Concentration ~ 4.00 ng/m L
Time of Soikina
1. A nalyte and IS after extraction
Mean
Ratio Analvte/IS 0.0288 0.0291 0.0302 0.0294
Ratio IS/Analvte 34.7 34.3 33.1 34.0
2. Analyte prior and IS alter extraction
Mean
0.00934 0.00760 0.0101 0.00901
3. IS prior and A nalyte after extraction
M ean
2.02 1.93 2.05 2.00
Adjusted mean extraction efficiency = 16.8% Mean extraction efficiency for the internal standard = 5.88%
H igh Concentration = 400 ng/m L
Time o f Soikina
1. A nalyte and IS after extraction
M ean
Ratio Analvte/IS 2.61 2.59 2.72 2.64
Ratio IS/Analvte 0.383 0.386 0.367 0.379
2. A nalyte prior and IS after extraction
Mean
0.377 0.385 0.383 0.382
3. IS prior and A nalyte after extraction
M ean
0.0248 0.0227 0.0241 0.0239
:
;
Adjusted mean extraction efficiency = 14.3% Mean extraction efficiency for the internal standard = 6.31%
Overall m ean extraction efficiency fo r analyte = 15.6% Overall mean extraction efficiency fo r internal standard = 6.10%
* Due to rounding, calculations based on displayed values may differ from actual.
Page 81
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBR00-122
Table 68. PFHS Extraction Efficiency from Human Serum
Run 22
Low Concentration = 4.00 ng/mL
Time o f Snikine
I . A nalyte and IS after extraction
Mean
Ratio Analvte/IS 0.0367 0.0361 0.0366 0.0365
Ratio IS/Analvte 27.3 27.7 27.3 27.4
2. A nalyte prior and IS a fte r extraction
Mean
0.00870 0.00778 0.00878 0.00842
3. IS prior and A nalyte after extraction
Mean
1.72 1.63 1.71 1.69
Adjusted mean extraction efficiency = 16.3% Mean extraction efficiency for the internal standard = 6.17%
H igh Concentration = 400 ng/m L
Time o f Spiking
I. A nalyte and IS after extraction
Mean
Ratio Analvte/IS 3.49 3.29 3.58 3.45
2. A nalyte prior an d IS after extraction
Mean
0.486 0.514 0.485 0.495
Ratio IS/Analvte 0.286 0.304 0.279 0.290
3. IS prior and A nalyte after extraction
Mean
0.0192 0.0181 0.0192 0.0188
*
:
Adjusted mean extraction efficiency = 14.3% Mean extraction efficiency for the internal standard = 6.48%
O verall m ean extraction efficiency fo r analyte = 15.3% Overall mean extraction efficiencyfo r internal standard = 6.33%
* Due to rounding, calculations based on displayed values may differ from actual.
Page 82
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBROO-122
Table 69. M556 Extraction Efficiency from Human Serum
Run 22
L o w C oncentration - 4.00 n g /m L
Time o f Soikina
I . Analyte and IS after extraction
M ean
Ratio Analvte/IS 0.00786 0.00750 0.00810 0.00782
Ratio IS/Analvte 127 133 123 128
2. A nalyte prior and IS after extraction M ean
0.00421 0.00366 0.00310 0.00366
3. IS prior and A nalyte after extraction M ean
7.54 6.18 6.78 6.83
Mean extraction efficiency for the analyte = 46.8% Adjusted mean extraction efficiency = 37.7%
H ig h C oncentration = 400 ng/m L
Time o f Soikina
1. A nalyte and IS after extraction
M ean
Ratio Analvte/IS 0.788 0.758 0.836 0.794
2. A nalyte prior an d IS after extraction M ean
0.337 0.326 0.303 0.322
Ratio IS/Analvte 1.27 1.32 1.20 1.26
3. IS p rio r and A nalyte after extraction M ean
0.0753 0.0690 0.0709 0.0717
4
:
Mean extraction efficiency for the analyte = 40.6% Adjusted mean extraction efficiency = 40.4%
O verall m ean extraction efficiency f o r analyte = 39.1% O verall m ean extraction efficiency fo r internal standard = 5.52%
* Due to rounding, calculations based on displayed values may differ from actual.
Page 83
Northwest Bioanalytical
Study No. NWBS00-O40 Report No. NWBR00-122
Table 70. M570 Extraction Efficiency from Human Serum
Run 22
L ow Concentration = 4.00 ng/mL
Time o f Sinking
I . A nalyte and IS after extraction
Mean
Ratio Analvte/IS 0.0333 0.0317 0.0335 0.0328
Ratio IS/Analvte 30.1 31.5 29.9 30.5
2. A nalyte prior and IS after extraction
Mean
0.0244 0.0220 0.0244 0.0236
3. IS prior and Analyte after extraction
Mean
1.92 1.72 1.74 1.79
Adjusted mean extraction efficiency = 54.4% Mean extraction efficiency for the internal standard = 5.87%
H igh Concentration = 400 ng/mL
Time o f Sinking
1. A nalyte and IS after extraction
Mean
Ratio Analvte/IS 2.20 2.16 2.35 2.24
2. A nalyte prior and IS after extraction
Mean
1.34 1.37 1.32 1.34
Ratio IS/Analvte 0.454 0.463 0.426 0.448
3. IS prior and Analyte after extraction
Mean
0.0294 0.0270 0.0280 0.0281
:
Adjusted mean extraction efficiency = 59.7% Mean extraction efficiency for the internal standard = 6.27%
Overall m ean extraction efficiency fo r analyte = 57.1% O verall m ean extraction efficiency f o r internal standard = 6.07%
* Due to rounding, calculations based on displayed values may differ from actual.
Page 84
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NW BR00-122
Table 71. Summary of Calibration Curve Parameters for PFOS in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number A B C R-Squared LLOQ ULOQ
12-0ct-2000 S00091-2 -0.000007 0.030663 -0.005620 27-Oct-2000 PFOSMD-40 0.000000 0.028132 0.010586
0.9986 0.9931
3.94 414 3.94 414
A, B, and C are coefficients used to define the calibration curve.
Table 72. Summary of Calibration Curve Parameters for PFOSA in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number
A
B C R-Squared LLOQ ULOQ
12-0ct-2000 S00091-2 - 0.000011 0.073810 0.006421 27-Oct-2000 PFOSMD-40 0.000001 0.052188 0.010966
0.9970 0.9912
1.00 500 1.00 500
A, B, and C are coefficients used to define the calibration curve.
Table 73. Summary of Calibration Curve Parameters for PFOSAA in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
Run
A
B
C R-Squared LLOQ ULOQ
Number
12-0ct-2000 S00091-2 -0.000003 0.032601 -0.006279 0.9988 1.60 501
27-Oct-2000 PFOSMD-40 0.000005 0.021209 -0.002450 0.9873 1.60 501
A, B, and C are coefficients used to define the calibration curve.
Page 85
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NWBROO-122
Table 74. Summary of Calibration Curve Parameters for POAA in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
Run
AB
C R-Squared LLOQ ULOQ
Number
12-0ct-2000 S00091-2 0.000000 0.017245 0.001072 0.9985 1.92 481
27-Oct-2000 PFOSMD-40 0.000002 0.014346 0.002192 0.9918 1.92 481
A, B, and C are coefficients used to define the calibration curve.
Table 75. Summary of Calibration Curve Parameters for PFHS in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
Run
A
B
C R-Squared LLOQ ULOQ
Number
12-0ct-2000 S00091-2 -0.000005 0.025818 -0.000970 0.9983 1.36 523
27-Oct-2000 PFOSMD-40 -0.000001 0.025534 0.011247 0.9953 1.36 523
A, B, and C are coefficients used to define the calibration curve.
Table 76. Summary of Calibration Curve Parameters for M556 in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
12-0ct-2000 31-Oct-2000
Run Number
S00091-2
24
A B C R-Squared L0Q ULOQ
-0.000002 0.018315 0.005561 0.9954 2.0 500 - 0.000001 0.018373 0.001923 0.9972 2.50 500
A, B, and C are coefficients used to define the calibration curve.
Page 86
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBROO-122
Table 77. Summary of Calibration Curve Parameters for M570 in Human Plasma
Quadratic weighted 1h e . All concentrations are expressed as ppb.
Run Date Run Number
A
B
C R-Squared LLOQ ULOQ
12-0ct-2000 S00091-2 -0.000006 0.048518 0.005367 27-Oct-2000 PFOSMD-40 0.000001 0.034031 0.011681
0.9978 0.9910
1.00 500 1.00 500
A, B, and C are coefficients used to define the calibration curve.
Page 87
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 78. Back-Calculated Concentrations of Calibration Standards for PFOS in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 3.94 5.17 11.3 23.6 44.2 85.4 209 332 414
12-0ct-2000 S00091-2 4.00 5.01 11.2 25.0 42.9 83.7 203 335 390 3.86 5.35 11.1 24.3 43.5 85.3 209 350 424
27-Oct-2000 PFOSMD-40 3.72 5.18 11.3 19.4 45.5 82.1 214 337 369 3.90 5.67 11.7 24.1 45.9 84.4 221 367 407
Table 79. Back-Calculated Concentrations of Calibration Standards for PFOSA in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 1.00 2.50 10.0 25.0 50.0 100 250 400 500
12-0ct-2000 S00091-2 1.04 2.60 10.4 25.1 49.1 95.9 252 380 462 0.941 2.53 8.93 25.7 50.5 102 255 444 508
27-Oct-2000 PFOSMD-40 0.961 2.59 9.81 20.2 53.4 103 259 356 443 0.999 2.71 10.4 22.1 52.3 101 267 458 515
Table 80. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 1.60 3.10 10.6 25.6 50.6 101 251 401 501 12-0ct-2000 S00091-2 1.65 3.12 10.7 25.3 50.5 98.2 255 393 469
1.54 3.10 10.4 26.3 51.0 101 245 432 513 27-Oct-2000 PFOSMD-40 1.66 3.50 11.7 21.3 53.1 104 299 406 440
1.47 2.96 10.8 23.5 47.3 90.3 261 441 475
Page 88
Northwest Bioanalytical
Study No. NWBS00-04O Report No. NWBR00-122
Table 81. Back-Calculated Concentrations of Calibration Standards for POAA in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 1.92 3.36 10.6 25.0 49.0 97.1 241 385 481
12-0ct-2000 S00091-2 2.00 3.24 10.5 26.1 49.3 97.5 241 386 477 1.96 3.11 10.1 25.2 52.1 95.0 239 392 477
27-Oct-2000 PFOSMD-40 1.73 3.01 10.5 22.9 49.7 99.1 250 367 439 2.30 3.12 10.9 25.1 50.7 96.7 262 422 474
Table 82. Back-Calculated Concentrations of Calibration Standards forPFHS in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
.
Run Date Run Number 1.36 2.92 10.7 26.5 52.6 104 262 418 523
12-0ct-2000 S00091-2 1.28 2.74 10.6 27.5. 50.2 104 253 441 518 1.44 3.08 10.8 26.9 53.2 104 259 413 524
27-Oct-2000 PFOSMD-40 1.52 2.72 10.6 24.2 54.6 107 259 466 489 1.28 2.78 10.4 28.4 54.1 102 270 425 491
Table 83. Back-Calculated Concentrations of Calibration Standards for M556 in Human
Plasma
.
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 2.50 10.0 25.0 50.0 100 250
O O
500
12-0ct-2000 31-Oct-2000
S00091-2 24
2.48 11.3 27.9 49.7 101 259 398 521 2.47 9.43 24.5 43.7 96.7 245 384 499 2.33 9.67 24.3 51.9 95.2 255 388 499 2.70 9.81 25.8 49.3 104 249 438 470
Page 89
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBROO-122
Table 84. Back-Calculated Concentrations of Calibration Standards for M570 in Human Plasma
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 1.00 2.50 10.0 25.0 50.0 100 250 400 500
12-0ct-2000 S00091-2 0.991 2.57 10.2 25.0 50.8 97.0 250 380 451 1.00 2.48 9.46 25.4 50.3 101 244 448 528
27-Oct-2000 PFOSMD-40 0.993 2.71 10.6 21.2 53.6 104 284 401 444 0.951 2.65 10.4 20.9 48.6 93.5 254 443 481
Page 90
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBROO-122
Table 85. Lower Limit of Quantitation for PFOS in Human Plasma
All concentrations are expressed as ppb.
Run Date Run Number 27-Oct-2000 PFOS MD 40
LLOQ (3.94 ppb)
3.98 3.51 3.89 4.04 4.06 3.77
Mean S.D. %CV %Theoretical %Bias
N
3.88 0.208 ' 5.4 98.5 -1.5
6
Page 9 i
Northwest Bioanalytical
Study No. NWBSOO-04O Report No. NWBR00-122
Table 86. Lower Limit of Quantitation for PFOSA in Human Plasma
All concentrations are expressed as ppb.
Run Date 27-Oct-2000
Run Number PFOS MD 40
LLOQ (1.00 ppb)
' 1.08 1.07 1.11 0.900 1.01 0.865
Mean S.D. %CV
%Theoretical %Bias N
1.01 0.102 10.1 101.0
1.0 6
Page 92
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NW BR00-122
Table 87. Lower Limit of Quantitation for PFOSAA in Human Plasma
All concentrations are expressed as ppb.
Run Date Run Number 27-Oct-2000 PFOS MD 40
LLOQ (1.60 ppb)
1.63 1.58 1.54 1.39 1.63 1.57
Mean S.D. %CV
%Theoretical %Bias N
1.56 0.0889
5.7 97.5 -2.5
6
Page 93
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 88. Lower Limit of Quantitation for POAA in Human Plasma
All concentrations are expressed as ppb.
Run Date Run Number 27-Oct-2000 PFOS MD 40
LLOQ (1.92 ppb)
1.72 1.84 1.80 1.74 1.79 1.62
Mean S.D.
%cv
%Theoretical %Bias N
1.75 0.0776
4.4 91.1 -8.9
6
Page 94
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NW BR00-122
Table 89. Lower Limit of Quantitation for PFHS in Human Plasma
All concentrations are expressed as ppb.
Run Date Run Number 27-Oct-2000 PFOS MD 40
LLOQ (1.36 ppb)
1.23 1.29 1.36 1.35 *1.06 1.42
Mean S.D. %CV
%Theoretical %Bias N
1.29 0.128 9.9 94.9 -5.1
6
* > 20% deviation from theoretical
Page 95
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NW BR00-I22
Table 90. Lower Limit of Quantitation for M556 in Human Plasma
All concentrations are expressed as ppb.
Run Date 31-Oct-2000
Run Number
24
LLOQ (2.50 ppb)
2.44 2.25 2.50 2.65 2.50 2.34
Mean S.D. %CV %Theoretical %Bias
N
2.45 0.139 5.7 98.0 -2.0
6
Page 96
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBROO-122
Table 91. Lower Limit of Quantitation for M570 in Human Plasma
All concentrations are expressed as ppb.
Run Date Run Number 27-Oct-2000 PFOS MD 40
LLOQ (1.00 ppb)
' 1.14 1.02 1.01 1.01 1.01 0.997
Mean S.D. %CV
%Theoretical %Bias N
1.03 0.0538
5.2 103.0 3.0
6
Page 97
Northwest Bioanalytical
Study No. NWBSOO-Q40 Report No. NWBROO-122
Table 92. Intra-Assay Precision for PFOS Quality Controls in Human Plasma
All concentrations are expressed as ppb.
Run Date Run Number 12-0ct-2000 S00091-2
Low QC 6.40 ppb
7.33 7.38 *7.89 7.07 6.98
Medium QC 126 ppb 132 129 138 132 137
High QC 332 ppb
343 331 334 334 340
Mean S.D. %CV
%Theoretical %Bias n
7.19 0.195 2.7 112.3 12.3
4
134 3.78 2.8 106.3 6.3
5
336 4.93 1.5 101.2 1.2
5
* Sample deactivated due to unacceptable internal standard response.
Page 98
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBROO-122
Table 93. Intra-Assay Precision for PFOSA Quality Controls in Human Plasma
All concentrations are expressed as ppb.
Run Date Run Number Low QC 4.00 ppb
12-0ct-2000 S00091-2
4.69
4.73
*4.67
4.53
4.66
Medium QC 150 ppb 148 157 172 166 163
High QC 400 ppb
423 409 425 414 409
Mean S.D. %CV
%Theoretical %Bias n
4.65 0.0866
1.9 116.3 16.3
4
161 9.15 5.7 107.3 7.3
5
416 7.62 1.8 104.0 4.0
5
* Sample deactivated due to unacceptable internal standard response.
Page 99
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBR00-122
Table 94. Intra-Assay Precision for PFOSAA Quality Controls in Human Plasma
All concentrations are expressed as ppb.
Run Date Run Number 12-0ct-2000 S00091-2
Low QC 4.60 ppb
5.20 5.50 *7.43 4.96 4.89
Medium QC 151 ppb 146 148 169 156 155
High QC 401 ppb
411 423 431 404 425
Mean S.D. %CV
%Theoretical %Bias n
5.14 0.276
5.4 111.7 11.7
4
155 9.04 5.8 102.6 2.6
5
419 11.0 2.6 104.5 4.5
5
* Sample deactivated due to unacceptable internal standard response.
Page 100
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBROO-122
Table 95. Intra-Assay Precision for POAA Quality Controls in Human Plasma
All concentrations are expressed as ppb.
'
Run Date Run Number Low QC 4.81 ppb
12-0ct-2000 S00091-2
5.33
5.17
*7.82
4.86
4.69
Medium QC 145 ppb 144 157 163 155 152
High QC 385 ppb
400 395 399 394 401
Mean S.D. %CV
%Theoretical %Bias n
5.01 0.290
5.8 104.2 4.2
4
154 6.98 4.5 106.2 6.2
5
398 3.11 0.8 103.4 3.4
5
* Sample deactivated due to unacceptable internal standard response.
Page 101
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBR00-122
Table 96. Intra-Assay Precision for PFHS Quality Controls in Human Plasma
All concentrations are expressed as ppb.
Run Date Run Number Low QC 4.48 ppb
12-Oct-2000 S00091-2
4.91
4.80
*5.41
4.72
4.58
Medium QC 156 ppb 162 158 162 150 158
High QC 417 ppb
406 388 399 403 401
Mean S.D. %CV
%Theoretical %Bias n
4.75 0.139 2.9 106.0
6.0 4
158 4.90 3.1 101.3 1.3
5
399 6.88 1.7 95.7 -4.3
5
* Sample deactivated due to unacceptable internal standard response.
Page 102
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 97. Intra-Assay Precision for M556 Quality Controls in Human Plasma
All concentrations are expressed as ppb.
'
Run Date Run Number 12-0ct-2000 S00091-2
Low QC 4.00 ppb
4.65 4.35 *4.76 4.14 3.89
Medium QC 150 ppb
147 140 150 146 159
High QC 400 ppb
441 **492 459 403 431
Mean S.D.
VoCV %Theoretical
%Bias n
4.26 148 445
0.322
6.95
33.1
7.6 4.7 7.4
106.5 98.7 111.3
6.5 -1.3 11.3
4 55
* Sample deactivated due to unacceptable internal standard response. ** > 20% deviation from theoretical
Page 103
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 98. Intra-Assay Precision for M570 Quality Controls in Human Plasma
All concentrations are expressed as ppb.
Run Date Run Number 12-0ct-2000 S00091-2
Low QC 4.00 ppb
**4.82 4.75 *5.89 4.56 4.74
Medium QC 150 ppb 146 161 170 165 158
High QC 400 ppb
407 407 414 393 403
Mean S.D. %CV
%Theoretical %Bias n
4.72 0.111 2.4 118.0 18.0
4
160 9.03 5.6 106.7 6.7
5
405 7.69 1.9 101.3 1.3
5
* Sample deactivated due to unacceptable internal standard response. ** > 20% deviation from theoretical
Page 104
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NWBR00-122
Table 99. Summary of Calibration Curve Parameters for PFOS in Serum Diluted with Buffer
Quadratic weighted 1lx 2. All concentrations are expressed as ppb.
Run Date Run Number A B C R-Squared LLOQ ULOQ 23-Oct-2000 PFOSMD-39 - 0.000001 0.009444 -0.004315 0.9954 5.30 504
A, B, and C are coefficients used to define the calibration curve.
Table 100. Summary of Calibration Curve Parameters for PFOSA in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number
A
B
C R-Squared LLOQ ULOQ
23-Oct-2000 PFOSMD-39 0.000000 0.014752 0.002265 0.9946 1.00 500
A, B, and C are coefficients used to define the calibration curve.
Table 101. Summary of Calibration Curve Parameters for PFOSAA in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
Run Number
A
23-Oct-2000 PFOSMD-39 0.000002
B 0.009880
C 0.004993
R-Squared LLOQ. ULOQ 0.9960 l'.OO 500
A, B, and C are coefficients used to define the calibration curve.
Page 105
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NW BR00-122
Table 102. Summary of Calibration Curve Param eters for POAA in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
Run
A
B
C R-Squared LLOQ ULOQ
Number
23-Oct-2000 PFOSMD-39 - 0.000001 0.011351 -0.000137 0.9967 1.70 501
A, B, and C are coefficients used to define the calibration curve.
Table 103. Summary of Calibration Curve Param eters for PFHS in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
Run Number
A
23-Oct-2000 PFOSMD-39 -0.000003
B 0.018220
C 0.007453
R-Squared LLOQ ULOQ 0.9980 1.00 500
A, B, and C are coefficients used to define the calibration curve.
Table 104. Summary of Calibration Curve Param eters for M556 in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
Run Number
23-Oct-2000 PFOSMD-39
A 0.000000
B C R-Squared LLOQ ULOQ 0.008729 -0.000146 0.9936 1.00 500
A, B, and C are coefficients used to define the calibration curve.
Page 106
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBROO-122
Table 105. Summary of Calibration Curve Parameters for M570 in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number
A
B
C R-Squared LLOQ ULOQ
23-Oct-2000 PFOSMD-39 0.000000 0.015628 0.005711 0.9969 1.00 500
A, B, and C are coefficients used to define the calibration curve.
Page 107
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 106. Back-Calculated Concentrations of Calibration Standards for PFOS in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 5.30 6.80 14.3 29.3 54.3 104 254 404 504
23-Oct-2000 PFOSMD-39 5.32 6.18 14.0 25.6 52.3 102 264 410 476 5.45 7.23 14.8 30.1 54.6 113 265 417 488
Table 107. Back-Calculated Concentrations of Calibration Standards for PFOSA in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 1.00 2.50 10.0 25.0 50.0 100 250 400 500 23-Oct-2000 PFOSMD-39 1.03 2.21 9.29 22.6 50.2 98.2 265 410 455
1.03 2.45 10.1 25.8 50.6 112 271 423 468
Table 108. Back-Calculated Concentrations of Calibration Standards for PFOSAA in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 1.00 2.50 10. 25.0 50.0 100 250 400 500 23-Oct-2000 PFOSMD-39 1.12 2.50 9.73 23.6 50.0 104 267 .408 485
0.901 2.41 9.41 24.1 50.6. 111 254 399 ; 479
Page 108
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 109. Back-Calculated Concentrations of Calibration Standards for POAA in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 1.70 3.20 10.7 25.7 50.7 101 254 401 501
23-Oct-2000 PFOSMD-39 1.56 3.22 10.4 24.7 50.9 99.8 260 401 458 1.88 3.10 10.6 25.3 50.8 109 263 432 491
Table 110. Back-Calculated Concentrations of Calibration Standards for PFHS in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 1.00 2.50 10.0 25.0 50.0 100 250 400 500 23-Oct-2000 PFOSMD-39 1.01 2.40 10.1 24.2 49.6 99.3 254 424 450
1.01 2.53 10.0 24.3 49.0 106 260 433 473
Table 111. Back-Calculated Concentrations of Calibration Standards for M556 in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 2.50 10.0 25.0 50.0 100 250 400 500 23-Oct-2000 PFOSMD-39 2.68 10.6 22.9 51.3 109 283 432 505
2.36 9.33 22.9 48.6 102 242 379 451
Page 109
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBROO-122
Table 112. Back-Calculated Concentrations of Calibration Standards for M570 in Serum Diluted with Buffer
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number 1.00 2.50 10.0 25.0 50.0 100 250 400 500
23-Oct-2000 PFOSMD-39 1.07 2.48 9.93 22.5 51.6 103 268 408 489 0.964 2.34 9.59 24.6 50.8 110 254 393 476
Page 110
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 113. PFOS Quality Controls Samples Prepared in Serum Diluted with Buffer
Run Date 23-Oct-2000
Run Number PFOSMD-39
193 ng/mL DF=10 204
219 205 202
443 ng/mL DF=10
464
423 451 477
4040 ng/mL DF=10
4240
4420 4320 4320
4040 ng/mL DF=50
*3180
*3020 *3150 *3100
Mean S.D. %CV %Theoretical %Bias
n
208 7.77 3.7 107.8 7.8
4
454 23.1 5.1 102.5 2.5
4
4330 73.7 1.7 107.2 7.2
4
3110 69.9 2.2 77.0 -23.0
4
* >20% deviation from theoretical
Table 114. PFOSA Quality Control Samples Prepared in Serum Diluted with Buffer
Run Date 23-Oct-2000
Run Number PFOSMD-39
150 ng/mL DF=10
147
147 145 136
400 ng/mL DF=10
388
360 370 409
4000 ng/mL DF=10
4020
3850 3980 3960
4000 ng/mL DF=50
*2380
*2240 *2210 *2210
Mean S.D. %CV %Theoretical %Bias
n
144 382 3950
5.25 21.5 72.7
3.6 5.6
1.8
96.0 95.5 98.8
-4.0 -4.5
-1.3
44
4
* >20% deviation from theoretical
' 2260 *81.2
3.6 56.5 -43.5
4
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBROO-122
Table 115. PFOSAA Quality Control Samples Prepared in Serum Diluted with Buffer
Run Date 23-Oct-2000
Run Number PFOSMD-39
153 ng/mL DF=10 144
146 142 142
403 ng/mL DF=10
405
379 389 417
4000 ng/mL DF=10
3920
4100 3970 4010
4000 ng/mL DF=50
*2750
*2560 *2620 *2550
Mean S.D. %CV
%Theoretical %Bias n
144 398 4000 2620
1.91 16.8 76.2
92.0
1.3 4.2 1.9 3.5
94.1 98.8 100.0 65.5
-5.9 -1.2 0.0 -34.5
44
4
4
* >20% deviation from theoretical
Table 116. POAA Quality Control Samples Prepared in Serum Diluted with Buffer
Run Date Run Number 23-Oct-2000 PFOSMD-39
Mean S.D. %CV
%Theoretical %Bias n
157 ng/mL DF=10 163 174 164 166
167 4.99 3.0 106.4 6.4
4
407 ng/mL DF=10 410 387 418 411
407 13.5 3.3 100.0 0.0 4
4010 ng/mL DF=10 4230 4190 4280
4140
4210 59.4 1.4 105.0 5.0
4
4010 ng/mL DF=50 3610 3310 3330 '3350 ' :
23400 141 4.1 84.8 -15.2 4
Page 112
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NWBR00-122
Table 117. PFHS Quality Control Samples Prepared in Serum Diluted with Buffer
Run Date 23-Oct-2000
Run Number PFOSMD-39
154 ng/mL DF=10
157
163 161 166
404 ng/mL DF=10 421
389 416 419
4000 ng/mL DF=10
4200
4210 4330 4300
4000 ng/mL DF=50 3490
3300 3410 3260
Mean S.D. %CV %Theoretical %Bias
n
162 3.77 2.3 105.2 5.2
4
411 15.0 3.6 101.7 1.7 4
4260 64.8 1.5 106.5 6.5
4
3370 105 3.1 84.3 -15.8 4
Table 118. M556 Quality Control Samples Prepared in Serum Diluted with Buffer
Run Date 23-Oct-2000
Run Number PFOSMD-39
151 ng/mL DF=10
156
157 145 148
401 ng/mL DF=10
421
385 403 417
4000 ng/mL DF=10
4480
4600 4710 4490
4000 ng/mL DF=50
*2980
*2740 *2780 *2710
Mean S.D. %CV
%Theoretical %Bias n
152 5.92 3.9 100.7 0.7
4
407 16.3 4.0 101.5 1.5 4'
4570 108 2.4 114.3 14.3 4
* >20% deviation from theoretical
. 2800: . 122
4.4 70.0 -30.0
4
Page 113
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 119. M570 Quality Control Samples Prepared in Serum Diluted with Buffer
Run Date 23-Oct-2000
Run Number PFOSMD-39
157 ng/mL DF=10
159
167 158 160
407 ng/mL DF=10
430
401 408 441
4010 ng/mL DF=10
4240
4390 4290 4330
4010 ng/mL DF=50
*2910
*2730 *2700 *2680
Mean S.D. %CV %Theoretical %Bias
n
161 4.08 2.5 102.5 2.5
4
420 18.7 4.5 103.2 3.2 4
4310 63.4 1.5 107.5 7.5
4
* >20% deviation from theoretical
2760 105 3.8 68.8 -31.2 4
Page 114
Northwest Bioanalytical
Study No. NWBSO0-O4O Report No. NWBR00-122
Table 120. Summary of Calibration Curve Parameters for PFOS (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
Run Matrix A
B
C R-Squared LLOQ ULOQ
Number
27-Oct-2000 PFOSMD-40 Plasma 0.000000 0.028132 0.010586 0.9931 3.94 414
27-Oct-2000 PFOSMD-46 Serum -0.000003 0.031081 0.084427 0.9506 36.8 454
25-Apr-2001 25 Plasma -0.000003 0.048192 0.045435 0.9841 3.28 413
25-Apr-2001
26
Serum -0.000020 0.057680 -0.301033 0.9934 36.8 454
A, B, and C are coefficients used to define the calibration curve.
Table 121. Summary of Calibration Curve Parameters for PFOSA (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
25-Apr-2001 25-Apr-2001
Run Matrix A B C R-Squared LLOQ ULOQ Number
25 Plasma -0.000005 0.120662 0.028023 0.9887 1.00 500 26 Serum -0.000032 0.166445 0.051786 0.9873 1.00 502
A, B, and C are coefficients used to define the calibration curve.
Table 122. Summary of Calibration Curve Parameters for PFOSAA (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
25-Apr-2001 25-Apr-2001
Run Matrix Number
A
B
C R-Squared LLOQ ULOQ
25 Plasma 0.000009 0.033608 0.035470 0.9938 T..30 500
26 Serum 0.000007 0.044621 -0.040847 0.9947 . 4.20 ; 503 .
A, B, and C are coefficients used to define the calibration curve.
Page 115
Northwest Bioanalytical
Study No. NWBS0O-O4O Report No. NWBROO-122
Table 123. Summary of Calibration Curve Parameters for POAA (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
Run Matrix A
B
C R-Squared LLOQ ULOQ
Number
27-Oct-2000 PFOSMD-40 Plasma 0.000002 0.014346 0.002192 0.9918 1.92 481
27-Oct-2000 PFOSMD-46 Serum 0.000003 0.016839 0.003246 0.9894 6.82 461
25-Apr-2001 25 Plasma -0.000003 0.011215 0.005552 0.9929 1.44 481
25-Apr-2001 26 Serum 0.000001 0.011414 0.000898 0.9931 6.82 461
A, B, and C are coefficients used to define the calibration curve.
Table 124. Summary of Calibration Curve Parameters for PFHS (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number Matrix A B C R-Squared LLOQ ULOQ
27-Oct-2000 PFOSMD-40 Plasma -0.000001 0.025534 0.011247 0.9953 27-Oct-2000 PFOSMD-46 Serum -0.000012 0.033256 0.018733 0.9890
A, B, and C are coefficients used to define the calibration curve.
1.36 523 3.85 431
Table 125. Summary of Calibration Curve Parameters for M556 (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
Run Matrix A
B
C R-Squared LLOQ ULOQ
Number
27-Oct-2000 PFOSMD-40 Plasma 0.000002 0.016888 0.007835 27-Oct-2000 PFOSMD-46 Serum 0.000005 0.014650 0.008960
0.9949 ` 2.50 0.9903 ' 3.8
500 501 `
A, B, and C are coefficients used to define the calibration curve.
Page 116
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NW BR00-122
Table 126. Summary of Calibration Curve Parameters for M570 (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date
Run Matrix A
B
C R-Squared LLOQ ULOQ
Number
27-Oct-2000 PFOSMD-40 Plasma 0.000001 0.034031 0.011681 0.9910 1.00 500 27-Oct-2000 PFOSMD-46 Serum 0.000003 0.034699 0.028660 0.9919 5.20 503 25-Apr-200I 25 Plasma 0.000009 0.039312 0.045599 0.9964 1.00 500 25-Apr-2001 26 Serum 0.000002 0.046817 0.021413 0.9931 3.70 503
A, B, and C are coefficients used to define the calibration curve.
Page 117
Northwest Bioanalytcal
Study No. NWBSOO-040 Report No. NWBR00-122
Table 127. Back-Calculated Concentrations of Calibration Standards for PFOS (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number Matrix 3.94 5.17 11.3 23.6 44.2 85.4 209 332 414 27-Oct-2000 PFOSMD-40 Plasma 3.72 5.18 11.3 19.4 45.5 82.1 214 337 369
3.90 5.67 11.7 24.1 45.9 84.4 221 367 407 Run Date Run Number Matrix 36.8 38.0 44.3 56.8 77.7 120 245 370 454 27-Oct-2000 PFOSMD-46 Serum 34.0 31.7 37.3 49.3 70.7 114 247 374 392
50.5 39.0 45.8 60.7 78.7 125 282 418 436 Run Date Run Number Matrix 3.28 4.52 10.7 23.0 43.5 84.6 208 331 413 25-Apr-2001 25 Plasma 2.70 '3.90 9.65 21.4 42.2 80.3 190 324 392
4.15 4.63 11.3 25.0 47.6 90.3 217 336 441 Run Date Run Number Matrix 36.8 38.0 44.3 56.8 77.7 120 245 370 454 25-Apr-2001 26 Serum 38.3 35.5 44.2 54.7 71.1 124 234 363 420
37.4 39.0 46.1 56.1 75.9 128 256 400 459
Page 118
\
Northwest Bioanalytical
Study No. NWBSO0-O4O Report No. NWBROO-122
Table 128. Back-Calculated Concentrations of Calibration Standards for PFOSA (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number Matrix 1.00 2.50 10.0 25.0 50.0 100 250 400 500 25-Apr-2001 25 Plasma 0.935 2.52 7.89 23.0 45.4 102 223 387 477
*1.77 2.99 10.3 27.3 52.5 113 254 402 546 25-Apr-2001 26 Serum 0.951 1.96 9.62 25.6 48.1 109 240 403 422
1.18 2.21 10.7 24.3 52.4 111 279 450 491
* Sample deactivated as an outlier
Page 119
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 129. Back-Calculated Concentrations of Calibration Standards for PFOSAA (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number Matrix 1.30 2.80 10.3 25.3 50.3 100 250 400 500 25-Apr-2001 25 Plasma 1.28 2.65 8.27 24.7 50.7 102 253 399 480
*2.23 3.22 9.79 24.9 54.4 110 257 391 509 R un Date Run Number Matrix 4.20 5.70 13.2 28.2 53.2 103 253 403 503 25-Apr-2001 26 Serum 4.27 5.31 12.8 26.9 50.7 119 259 391 477
4.38 5.78 13.6 25.0 51.9 111 260 407 511
* Sample deactivated as an outlier
Page 120
Northwest Bioanalytical
Study No. NW BS00'040 Report No. NWBROO-l22
Table 130. Back-Calculated Concentrations of Calibration Standards for POAA (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number Matrix 27-Oct-2000 PFOSMD-40 Plasma
Run Date Run Number Matrix 27-Oct-2000 PFOSMD-46 Serum
Run Date Run Number Matrix 25-Apr-2001 25 Plasma
Run Date Run Number Matrix
25-Apr-200l
26
Serum
1.92 1.73 2.30 6.82 7.32 *18.3 1.44 1.35 *2.14 6.82 7.98 6.23
3.36 10.6 25.0 49.0 97.1 241 385 481 3.01 10.5 22.9 49.7 99.1 250 367 439 3.12 10.9 25.1 50.7 96.7 262 422 474 8.18 15.0 28.6 51.4 97.3 234 370 461 6.86 13.9 27.2 51.9 105 252 399 421 9.23 14.9 29.4 46.2 94.9 246 392 424 2.88 10.1 24.5 48.5 96.6 241 385 481 2.65 10.5 24.1 43.4 91.1 225 396 470 3.35 11.2 26.6 46.5 90.9 241 405 498 8.18 15.0 28.6 51.4 97.3 234 370 461 8.28 15.6 28.8 50.2 103 242 383 457 7.23 15.0 26.9 48.0 100 235 378 433
* Sample deactivated as an outlier
Page 121
Northwest Bioanalytical
Study No. NWBS0O-O4O Report No. NWBR00-122
Table 131. Back-Calculated Concentrations of Calibration Standards for PFHS (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number Matrix 1.36 2.92 10.7 26.5 52.6 104 262 418 523 27-Oct-2000 PFOSMD-40 Plasma 1.52 ^ 2.72 10.6 24.2 54.6 107 259 466 489
1.28 2.78 10.4 28.4 54.1 102 270 425 491 Run Date Run Number Matrix 3.85 5.13 11.5 24.4 45.7 88.9 217 345 431 27-Oct-2000 PFOSMD-46 Serum 4.08 4.36 11.2 21.8 47.5 89.1 235 371 363
*15.4 5.80 10.6 23.5 49.0 89.7 230 388 389
* Sample deactivated as an outlier
Page 122
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBROO-122
Table 132. Back-Calculated Cohcentrations of Calibration Standards for M556 (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number Matrix 2.50 10.0 25.0 50.0 100 250 400 500 27-Oct-2000 PFOSMD-40 Plasma 2.64 10.7 23.4 52.9 101 276 396 456
2.35 9.95 23.3 48.3 94.1 260 440 481 Run Date Run Number Matrix 3.80 11.3 26.3 51.3 101 251 401 501 27-Oct-2000 PFOSMD-46 Serum 3.89 11.1 25.3 53.7 116 280 441 480
3.91 10.0 25.7 42.9 102 258 396 450''
Page 123
Northwest Bioanalytical
Study No. NWBS0O-040 Report No. NW BR00-122
Table 133. Back-Calculated Concentrations of Calibration Standards for M570 (Serum vs. Plasma)
Quadratic weighted 1/x2. All concentrations are expressed as ppb.
Run Date Run Number Matrix 1.00 2.50 10.0 25.0 50.0 100 250 400 500
27-Oct-2000 PFOSMD-40 Plasma 0.993 2.71 10.6 21.2 53.6 104 284 401 444
0.951 2.65 10.4 20.9 48.6 93.5 254 443 481
Run Date Run Number Matrix 3.70 5.20 12.7 27.7 52.7 103 253 403 503
27-Oct-2000 PFOSMD-46 Serum *5.54 4.92 11.8 27.4 51.0 116 277 432 464
*16.4 5.75 12.4 26.9 45.5 106 271 404 468
Run Date Run Number Matrix 1.00 2.50 10.0 25.0 50.0 100 250 400 500
25-Apr-2001 25 Plasma 1.01 2.43 8.50 24.6 52.8 105 246 420 483
1 *1.90 2.66 9.29 25.4 51.9 108 247 387 504
Run Date Run Number Matrix 3.70 5.20 12.7 27.7 52.7 103 253 403 503
25-Apr-2001
26
Serum 4.07 4.60 12.5 27.4 50.3 118 262 395 478
3.94 4.60 13.3 26.0 49.7 109 264 405 502
* Sample deactivated as an outlier
Page 124
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBROO-122
Table 134. PFOS Quality Controls (Serum vs. Plasma)
All concentrations are expressed as ppb.
Run Date
Run Number Low QC (Plasma) 6.40 ppb
27-Oct-2000 PFOSMD-40
6.44
5.99
5.91
Medium QC (Plasma) 126 ppb
115
136
118
High QC (Plasma) 332 ppb
316
326
298
Mean
6.11
SD 0.286
%CV
4.7
%Bias
-4.5
n3
Run Date
Run Number Low QC (Serum) 39.3 ppb
27-Oct-2000 PFOSMD-46 35.0
35.6
35.5 35.8
123 11.4 9.3 -2.4
3
Medium QC (Serum) 161 ppb 164 151 149 152
313 14.2 4.5 -5.7
3
High QC (Serum) 370 ppb
336 *199 343 346
Mean
35.5 154 342
SD
0.340
6.78
5.13
%CV
1.0 4.4 1.5
%Bias
-9.7 -4.3 -7.6
n 443
* Sample deactivated due to a sample preparation error. ** > 20% deviation from theoretical
Page 125
Northwest Bioanalytical
Study No. NWBSOO-O40 Report No. NWBR00-122
Table 134. PFOS Quality Controls (Serum vs. Plasma) Continued
All concentrations are expressed as ppb.
Run Date
Run Number Low QC (Plasma) 5.75 ppb
25-Apr-2001
25
5.44
5.62
5.59 1 5.90
Medium QC (Plasma) 126 ppb
143
139
136
132
High QC (Plasma) 331 ppb
333
325
347
333
Mean
. 5.64
SD 0.192
%CV
3.4
%Bias n
-1.9 4
Run Date
Run Number
Low QC (Serum) 39.3 ppb
25-Apr-2001
26
41.3
45.4
45.8 45.6
138 4.65 3.4 9.5
4
Medium QC (Serum) 161 ppb 174 182 172 169
335 9.15 2.7 1.2
4
High QC (Serum) 370 ppb
377 388 408 381
Mean SD
%CV %Bias
n
44.5 174 2.16 5.56 4.9 3.2 13.2 8.1
44 ** > 20% deviation from theoretical
389 13.8 3.5 5.1
4
Page 126
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NWBROO-122
Table 135. PFOSA Quality Controls (Serum vs. Plasma)
All concentrations are expressed as ppb.
Run Date
Run Number
Low QC (Plasma) 4.00 ppb
25-Apr-2001
25
4.06
3.99
4.49
4.73
Medium QC (Plasma) 150 ppb
158
175
170
159
High QC (Plasma) 400 ppb
395
412
438
424
Mean
4.32
SD 0.353
%CV
8.2
%Bias
8.0
n4
Run Date
Run Number
Low QC (Serum) 4.00 ppb
25-Apr-2001
26
3.52
4.75
4.55
**4.85
166 8.35 5.0 10.7
4
Medium QC (Serum) 150 ppb 153 163 155 151
417 18.2 4.4 4.3
4
High QC (Serum) 400 ppb
390 431 417 377
Mean
4.42 156 404
SD
0.611
5.26
24.6
%CV
13.8
3.4
6.1
%Bias n
10.5 4.0 44
1.0 4
* Sample deactivated due to a sample preparation error. ** > 20% deviation from theoretical
Page 127
Northwest Bioanalytical
Study No. NWBS00-O40 Report No. NWBR00-122
Table 136. PFOSAA Quality Controls (Serum vs. Plasma)
All concentrations are expressed as ppb.
Run Date
Run Number Low QC (Plasma) 4.30 ppb
25-Apr-2001
25
3.59
3.85 3.74
4.96
Medium QC (Plasma) 150 ppb
**187
175
172
169
High QC (Plasma) 400 ppb
429
412
440
429
Mean
4.04
SD 0.626
%CV
15.5
%Bias
-6.0
n4
Run Date
Run Number
Low QC (Serum) 7.20 ppb
25-Apr-2001
26
6.74
8.11
8.29
8.37
176 7.89 4.5 17.3
4
Medium QC (Serum) 153 ppb 150 162 159 157
428 11.6 2.7 7.0
4
High QC (Serum) 403 ppb
363 409 396 376
Mean
7.88 157 386
SD
0.766
5.10
20.5
%CV
9.7
3.2 .
5.3
%Bias
9.4 2.6 -4.2
n 444
* Sample deactivated due to a sample preparation error. ** > 20% deviation from theoretical
Page 128
Northwest Bioanalytical
Study No. NWBSOO-040 Report No. NWBR00-122
Table 137. POAA Quality Controls (Serum vs. Plasma)
All concentrations are expressed as ppb.
Run Date
Run Number
Low QC (Plasma) 4.81 ppb
27-Oct-2000 PFOSMD-40 4.57
4.31
4.52
Medium QC (Plasma) . 145 ppb
146
167
147
High QC (Plasma) 385 ppb
373
419
393
Mean
4.47
SD 0.138
%CV
3.1
%Bias
-7.1
n3
Run Date
Run Number
Low QC (Serum) 9.54 ppb
27-Oct-2000 PFOSMD-46 9.42
9.17
8.53
9.62
153 11.8 7.7 5.5
3
Medium QC (Serum) 143 ppb 158 146 148 150
395 23.1 5.8 2.6
3
High QC (Serum) 370 ppb
373 *253 375 388
Mean
9.19 151 379
SD
0.474
5.26
8.14
%CV
5.2
3.5 .
2.1
%Bias
-3.7 5.6
2.4
n
44
3
* Sample deactivated due to a sample preparation error. ** > 20% deviation from theoretical
Page 129
Northwest Bioanalytical
Study No. NWBS0O-O4O Report N o. NWBR00-122
Table 137. POAA Quality Controls (Serum vs. Plasma) Continued
All concentrations are expressed as ppb.
Run Date
Run Number
Low QC (Plasma) 4-32 ppb
25-Apr-2001
25
5.13
4.47
4.35
4.44
Medium QC (Plasma) . 145 ppb
143
145
145
137
High QC (Plasma) 385 ppb
394
383
412
404
Mean SD
%CV
4.60 0.359
7.8
143 3.79 2.7
%Bias n
6.5 -1.4 44
Run Date
Run Number
Low QC (Serum) 9.54 ppb
Medium QC (Serum) 143 ppb
25-Apr-2001
26
10.1
157
11.4 152
**13.2
149
9.82
152
Mean SD
%CV %Bias
n
11.1 153 1.54 3.32 13.9 2.2 . 16.4 7.0
44
** > 20% deviation from theoretical
398 12.6 3.2 3.4
4 High QC (Serum) 370 ppb
374 389 385 391
385 7.59 2.0 4.1
4
Page 130
Northwest Bioanalytical
Study No. NWBS00-040 Report No. NWBR00-122
Table 138. PFHS Quality Controls (Serum vs. Plasma)
All concentrations are expressed as ppb.
Run Date
Run Number
Low QC (Plasma) 4.48 ppb
27-Oct-2000 PFOSMD-40 4.62
4.35
4.39
Medium QC (Plasma) 156 ppb
157
173
151
High QC (Plasma) 417 ppb
397
407
384
Mean
4.45
SD 0.146
%CV
3.3
%Bias
-0.7
n3
Run Date
Run Number
Low QC (Serum) 6.41 ppb
27-Oct-2000 PFOSMD-46
5.65
5.70
6.87
5.71
160 11.4 7.1 2.6
3
Medium QC (Serum) 132 ppb 144 136 129 135
396 11.5 2.9 -5.0
3
High QC (Serum) 345 ppb
343 *221 395 367
Mean
5.98 136 368
SD 0.592 6.16 26.0
%CV
9.9
4-5 .
7.1
%Bias
-6.7 3.0 6.7
n 443
* Sample deactivated due to a sample preparation error. ** > 20% deviation from theoretical
Page 131
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBROO-122
Table 139. M556 Quality Controls (Serum vs. Plasma)
All concentrations are expressed as ppb.
Run Date
Run Number
Low QC (Plasma) 4.00 ppb
27-Oct-2000 PFOSMD-40
3.73
3.45
3.34
Medium QC (Plasma) 150 ppb
133
168
139
High QC (Plasma) 400 ppb
389
420
387
Mean
3.51
SD 0.201
%CV
5.7
%Bias
-12.3
n3
Run Date
Run Number
Low QC (Serum) 5 3 0 ppb
27-Oct-2000 PFOSMD-46
5.03
5.23
**4.15
5.01
147 18.7 12.7 -2.0
3
Medium QC (Serum) 151 ppb 166 152 152 155
399 18.5 4.6 -0.3
3
High QC (Serum) 401 ppb
383 **243
371 388
Mean SD
%CV %Bias
n
4.86 156
0.480
6.65
9.9 4.3 . -8.3 3.3
44
** > 20% deviation from theoretical
381 8.74 2.3 -5.0
3
Page 132
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBR00-122
Table 140. M570 Quality Controls (Serum vs. Plasma)
All concentrations are expressed as ppb.
Run Date
Run Number Low QC (Plasma) 4.00 ppb
27-Oct-2000 PFOSMD-40 3.95
3.54
3.38
Medium QC (Plasma) 150 ppb
133
161
136
High QC (Plasma) 400 ppb
379
403
360
Mean
3.62
SD 0.294
%CV
8.1
%Bias
-9.5
n3
Run Date
Run Number
Low QC (Serum) 6.70 ppb
27-Oct-2000 PFOSMD-46 6.36
6.35
**5.33
6.39
143 15.4 10.8 -4.7
3
Medium QC (Serum) 153 ppb 169 150 156 155
381 21.5 5.6 -4.8
3
High QC (Serum) 403 ppb
397 *227 367 400
Mean
6.11 158 388
SD
0.519
8.10
18.2
%CV
8.5
5.1 .
4.7
%Bias
-8.8 3.3 -3.7
n 44 3
* Sample deactivated due to a sample preparation error. ** > 20% deviation from theoretical
Page 133
Northwest Bioanalytical
Study No. NWBS0O-O4O Report No. NWBR00-122
Table 140. M570 Quality Controls (Serum vs. Plasma) Continued
All concentrations are expressed as ppb.
Run Date
Run Number Low QC (Plasma) 4.00 ppb
25-Apr-2001
25
3.24
3.49
3.58
4.05
Medium QC (Plasma) 150 ppb
180
175
164
171
High QC (Plasma) 400 ppb
420
417
440
432
Mean
3.59
SD 0.339
%CV
9.4
%Bias
-10.3
n4
Run Date
Run Number
Low QC (Serum) 6.70 ppb
25-Apr-2001
26
5.90
7.49
7.03
8.03
173 6.76 3.9 15.3
4
Medium QC (Serum) 153 ppb 166 167 167 161
427 10.7 2.5 6.8
4
High QC (Serum) 403 ppb
392 445 437 409
Mean SD
%CV %Bias
n
7.11 165
0.906
2.87
12.7 1.7 . 6.1 7.8 44
** > 20% deviation from theoretical
421 24.6 5.8 4.5
4
Page 134
Northwest Bioanalytical
Study No. NW BS00-040 Report No. NWBR00-122
I
Table 141. Serum QC Results Based on Plasma Curves
Sample ID
PFOS (ppb)
PFOSA (ppb)*
PFOSAA (ppb)*
POAA (ppb)
PFHS (ppb)*
M556 (ppb)*
M570 (ppb)
Low (analyzed 10/27/00) 41.2
11.1 7.64 4.44 6.99
Low (analyzed 10/27/00) 41.8
10.8
7.71
4.61
6.98
Low (analyzed 10/27/00) 41.8
10.1 9.22 3.67 5.93
Low (analyzed 10/27/00) 42.1
11.4 7.72 4.42 7.02
Low (analyzed 4/25/01 )
41.7
5.04
6.68
9.62
6.41
Low (analyzed 4/25/01)
46.5
6.47
8.5
9.93
7.74
Low (analyzed 4/25/01)
46.7
6.75
8.72
11.3
8.29
Low (analyzed 4/25/01)
47.0
6.88
8.83
13.0
8.94
mean *
43.6 6.29 8.18 10.9 8.07 4.29 7.29
std. dev. *
2.61 0.847 1.01
1.07 0.766 0.419 0.986
%CV *
6.0%
13.5%
12.4%
9.9%
9.5%
9.8%
13.5%
%dev *
10.9%
57.1%
13.6%
14.3%
25.9%
-19.2%
8.8%
acceptance range * 31.4 to 47.2 3.00 to 5.00 5.40 to 9.00 7.63 to 11.4 5.13 to 7.69 4.24 to 6.36 5.36 to 8.04
95% confidence range * 41.8 to 45.4 5.45 to 7.12 7.48 to 8.88 10.2 to 11.7 7.32 to 8.82 3.87 to 4.7 6.60 to 7.97
Med (analyzed 10/27/00) Med (analyzed 10/27/00) Med (analyzed 10/27/00) Med (analyzed 10/27/00) Med (analyzed 4/25/01) Med (analyzed 4/25/01) Med (analyzed 4/25/01) Med (analyzed 4/25/01)
mean * std. dev. *
%CV * %dev * acceptance range *
95% confidence range *
182 168 165 168 185 189 191 199
181 12.5 6.9% 12.3% 129 to 193
172 to 190
205 207 210 220
211 6.66 3.2% 40.3% 113 to 188
204 to 217
192 200 203 207
201 6.4 3.2% 31.0% 115 to 191
196 to 205
186 172 174 177 160 164 164 170
171 8.37 4.9% 19.5% 114 to 172
165 to 177
180 170 162 169
170 7.41 4.4% 29.0% 106 to 158
163 to 178
149 136 137 139
140 5.97 4.3% -7.1% 121 to 181
134 to 146
175 155 161 160 184 190 191 191
176
15.3 8.7% 15.0% 122 to 184
165 to 186
High (analyzed 10/27/00) 364
441 400 361 416
High (analyzed 10/27/00) 370
443 452 349 384
High (analyzed 10/27/00) 374
459 424 .366 420
High (analyzed 4/25/01)
396
493
451
453
433
High (analyzed 4/25/01)
399
.509
467
470 .
450 -
High (analyzed 4/25/01)
406
543
490
476
478
High (analyzed 4/25/01)
424
560
506
478
: 486
mean * std. dev. *
%CV * %dev * acceptance range * 95% confidence range *
390 21.8 5.6% 5.5%
296 to 444 374 to 407
526 30.7 5.8% 31.6% 300 to 500 492 to 561
479 24.3 5.1% 18.7% 302 to 504 460 to 497
460 15.2 3.3% 24.3% 296 to 444 449 to 471
425 26.0 6.1% 23.3% 276 to 414 396 to 455
359 8.74 2.4% -10.6% 321 to 481 349 to 369
438 36.0 8.2% 8.7% 322 to 484 411 to 465
* Due to rounding, calculations based on displayed values may differ from actual. NOTE: Only data from acceptable runs included in the Table (see report text).
(Data obtained on 10/27/00 from run PFOSMD-40. Data obtained on 4/25/01 from run S00040-25.)
Page 135
