Document LJb30d3JZ9v4EZD15Vvy2EnOd

R ep ort T itle Evaluation of the Northwest Bioanalytical Method V alidation Reports fo r PFOS and Related Analytes in Human Serum or Plasma in Meeting the Requirements o f the - _________EDA BioanalyticaLMethod Validation QAI Report No. 405-503 Data R equirem ent Full Validation o f Bioanalytical Method Author Nancy Benson Philippe Ourisson Quality Associates, Inc. 9017 Red Branch Road, Suite 102 Columbia, MD 21045 Sponsor 3M Environmental Laboratories 935 Bush Avenue Bldg 2-3E-09 St Paul, MN 55106 R eport Date December 31,2001 Report Number QAI 405-503 Number o f Pages 26 TABLE OF CONTENTS QAI Report No. 405-503 I. S U M M AR Y....................................................................................................... 3 II. INTRODUC T IO N ................................ .................. ......... ..... .......................... III. SPECIFICITY IN SERUM AND P LA S M A ......................................................5 IV. CALIBRATION IN SERUM AND P LA S M A ....................................................7 V. ACCURACY IN SERUM ANALYSIS................................................................9 VI. ACCURACY IN PLASMA A N ALYSIS........................ .......................... ....... 11 VII. ABSOLUTE RECOVERY IN S ER U M ........................................................... 12 VIII. ACCURACY AND POAA EFFECTS ON I.S. QUANTITATION............... 13 IX. ACCURACY OF SERUM QUANTITATION VS PLASMA CURVE............ 14 X. VALIDATION OF SERUM EXTRACT DILUTION PRO CEDURE............. 15 XI. PRECISION IN SERUM ANALYSES.......................................... 16 XII. PRECISION IN PLASMA A N ALYSES.......................................................... 18 XIII. SERUM SAMPLE STABILITY TO FREEZE-AND-THAW .......................... 19 XIV. SHORT-TERM AMBIENT STABILITY OF SERUM S A M PLE S ................ 20 XV. LONG-TERM FREEZER STABILITY OF SERUM SAM PLES...................21 XVI. POST-PREPARATIVE AM BIENT STABILITY OF SERUM EXTRACTS...................................................... 22 XVII. POST-PREPARATIVE 1-8C STABILITY OF SERUM EXTRACTS.........24 XVIII. POST-PREPARATIVE -20C STABILITY OF SERUM EXTRACTS.........25 XIX. STOCK SOLUTION S TA B ILITY ....................................................................26 Validation report review 2 Human Serum a t Northwest Bioanalytical QAI Report No. 405-503 I. SUMMARY The validation in human serum o r plasma was performed fo r the analytes PFOS, PFOSA, PFOSAA, POAA, PFHS, M-556, and M570. The surrogate standard IH,IH,2H,2H- perfluorooctanesulphonic acid was added before extraction, but used as an internal standard in the quantitation. -- ........................... The following parameters can be considered as com pletely meeting the FDA re quire m en ts: Specificity Calibration in serum and plasma Accuracy in serum and plasma. Precision in serum and plasma Validation of Extract Dilution Serum sample stability to: o freeze-thaw. 7 cycles o short-term am bient storage, 6.75 hr x 7 cycles o freezer storage, 42 - 55 days Serum extract stability to: o am bient storage, 7 days o refrigerated storage, 4 days o frozen storage, 5 days The following restrictions apply to these analyses: Because of poor analyte and internal standard extraction efficiencies, it is essential that samples, QCs and calibrations standards in a set be kept together fo r all analyses. Extraction efficiency w ithin a set is consistent; Samples with levels of POAA above the ULOQ w ill show LC/MS ionization suppression o f the internal standard THPFOS, resulting in a high quantitation bias on other analytes. A resolution was not demonstrated. For diluting samples with serum, the calibration curve must be prepared at the same dilution of serum as the diluted sample fo r accuracy. If the sample is at a different m atrix dilution, the results w ill not be correct. W hen a sample must be diluted, it not only must be compared to a calibration curve prepared at the same dilution of serum, but it also must be re-extracted fo r that analysis, in order to keep sam ples, QCs, and calibration standards together. The following parameters from the FDA requirements were not tested: Stock solution stability. Stability o f the surrogate standard. Quantitating serum samples against plasma calibration does not meet the FDA requirements for accuracy. Validation report review 3 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 H. INTRODUCTION Three reports w ere reviewed: NWBR00-122: Quantitative Determination o f PFOS, PFOSA, PFOSAA, POAA, PFHS, M-556 and M570 in Human Serum by LC/MS/MS: Assay Revalidation Addendum Report, 11/20/01 (study NWBS00-040) NW BR00-108: Quantitative Determination o f PFOS, PFOSA, PFOSAA, POAA, PFHS, M-556 and M570 in Human Serum by LC/MS/MS: Assay Revaluation Report, 1/24/01 (study NWBS00-040); NW BR99-005: Quantitative Determination o f PFOS, PFOSA, PFOSAA, POAA, PFHS, M-556 and M570 in Human Serum by LC/MS/MS: Assay Validation Report, 1/24/01 (study NWBS98-082); The methods used in these studies are very sim ilar with the following differences: #005 #108/122 internal SDS THPFOS standard (IS) partition basified with 0.5 M unadjusted pH ca. 6.9 tertbutylam m onium hydrogen sulfate (pH 10) + 0.25 M carbonate buffer final solvent 2mM ammonium acetate: methanol 20mM ammonium acetate in water (1:1) : 20 mM ammonium acetate in MeOH (3:7) quantitation quadratic regression, weighted 1/x, quadratic regression, weighted using area ratio vs. concentration 1/x2, using area ratio vs. concentration This review examines which o f the FDA-required tests were conducted, and which meet the standards described by FDA fo r a fu ll validation o f the method. This report did not consider w hether additional requirements, specified by protocol, were met if they did not affect the evaluation based on the FDA guideline; however, they may be discussed in the text o f this report. Validation report review 4 Human Serum at Northwest Bioanalytical QAl Report No. 405-503 n i. SPECIFICITY IN SERUM AND PLASMA This requirement is met Endogenous levels were found in ail serum and plasma samples tested, although much greater in serum. - FDA G uideline > Six sources of blank biological matrix Measure levels of each analyte NWB Report Requirem ent met. Testing was reported on 2 lots o f serum and 4 lots of plasma. R e su lts: In report #005, endogenous levels of PFOS, PFOSA, PFOSAA, POAA and PFHS in human serum were determined using rabbit serum calibration; PFOSA was not detected. Mean ng/m L in Human Serum Source: report#005 (tables 5 - 6 ) PFOS PFOSAA POAA PFHS Run 25 calibration standards (n=4) 16.2 1.46 3.20 0.615 Run 26/28/29 calibration standards (n=12) 30.4 1.69 4.27 1.61 Run 26/28/29 QC samples (n=12) 22.3 1.99 1.97 3.25 The LLOQ in rabbit serum or each ana yte was 1.00 ng/mL. In report #108, serum results indicated significant endogenous levels of ail analytes except PFOSA. These levels are higher than in report #005. Source: report #108 tables 8 -1 4 & report #122 tables 8 14 endogenous levels target LLOQ calibration LLOQ* Mean ng/m L in Human Serum PFOS PFOSA PFOSAA POAA PFHS M-556 M570 47.1 1 .0 48.1 ND 1 .0 1 .0 5.0 4.76 2.15 3.3 4.6 1 .0 1 .0 1 .0 1 .0 1 .0 6 .0 5.76 3.15 4.3 5.6 Calibration was w ith human plasma Validation report review 5 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 Pooled plasma also showed significant endogenous PFOS, PFOSAA, POAA and PFHS, but at least 5x less than in serum. The levels were determined by standards prepared in plasma a t 1 -1 0 0 ng/mL. The endogenous level is defined as the mean y-intercept of linear regressions performed on 2 separate tests. Source: report 122 tables 78 84 endogenous levels target LLOQ calibration LLOQ* PFOS 2.94 1.00 3.94 Mean ng/mL In Human Plasma PFOSA PFOSAA POAA PFHS M-556 M570 ND 1.00 1.00 0.60 1.0 1.60 0.92 0.36 ND ND 1.00 1.00 2.50 1.00 1.92 1.36 2.50 1.00 Calibration was with human plasma. Validation report review 6 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 IV . CALIBRATION IN SERUM AND PLASM A R equirem ent m e t There is how ever no ju s tific a tio n fo r th e se le ctio n o f th e co m p lica te d ca lib ra tio n procedure (q u a d ra tic w ith 1/x2 o r 1/x w e ig h tin g ). FDA G uideline Blank + 6 - 8 samples NWB R eport R equirem ent m e t 2 blanks and 2 blanks + IS were included in each run. No blank analyses were reported. Samples: 8 Levels fo r M-556; 9 levels fo r all others. Duplicate samples. 3 runs fo r serum and 2 fo r plasma. LLOQ > 5x blank response R equirem ent m e t Endogenous responses caused LLOQ approxim ately = "blank" in most cases. Summarized in table below. (A c c u ra c y ) 20% o f actual @ LLOQ R equirem ent m e t All pass, except one serum POAA. 15% of actual >LL0Q Passed, o f 550 total: 550 for PFHS 549 for PFOS 548 fo r M-556, M557 547 fo r PFOSA, PFOSAA, POAA > 4 o f 6 standards meet these A ll runs pass. criteria in each curve LLOQ: An experimental determ ination o f the analytical lim its was not performed. The lowest standard injected which met the acceptance criteria was chosen as the LLOQ. in most cases, these standard concentrations were close to the endogenous levels. Since the calibration LLOQ is only slightly ( 1 - 4 tim es) higher than the endogenous (blank) level the uncertainty o f sample measurements at the LLOQ w ill be increased by the uncertainty of measurement o f the endogenous level. Because the endogenous levels are low er in plasma than in serum, plasma blanks should be used to prepare calibration standards when an LLOQ below 5.0 ng/mL is needed. Validation report review 7 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 Range: I PFOS Serum (reports #108/122) S erum LLO Q 48 Serum ULOQ 540 Plasma (report #122 Plasma LLOQ 3.9 Plasma ULOQ 410 VAUPATEP RANGE (ng/mL) PFOSA PFOSAA | POAA T PFHS M-556 M570 10500 6.0 500 1.0 500 1.6 500 5.8 500 3.2... 4 .3 - 5.6 500 500 500 1.9 1.4 2.5 1.0 480 520 470 480 Calibration method: Quadratic regression w ith 1/x2 weighting, of peak area ratio (sam ple / surrogate standard) vs. nominal concentration. (1/x w eighting in report #005). There is no Justification for the selection o f this complicated calibration method. In all sets, there were at least fo u r out o f six calibration standard levels that met the accuracy requirement o f 15% o f actual (or 20% o f actual a t the LLOQ). Validation report review 8 Human Serum a t Northwest Bioanalyticai QAI Report No. 405-503 V . ACCURACY IN SERUM ANALYSIS The FDA requirem ents fo r th is te s t w ere m e t FDA G uideline 5 replicates @ 3 concentrations in the expected range o f concentrations. Levels: 3xLLOQ; at the m idle vel; near the upper boundary o f the standard curve (A ccuracy) Mean values 20% o f actual @ LLOQ Mean values 15% of actual >LLOQ NWB R eport 5 replicates, each on 3 separate days Levels: LLOQ (4.0 - 51.1 ng/mL; 1.1 - 4 x ) midlevel (150-200 ng/mL) high (400-450 ng/mL) R equirem ent m et A ll passed except PFOSAA (intraday) All passed. R esults: Summary tables presented below, w ith failed results bolded. In the initial test o f 3 sets, PFOSA and PFOSAA failed because the fortifications were faulty. The fortifying solution was prepared from dilute stocks by evaporation, and these two analytes were partially lost. The intra-day data below was obtained from set 17 containing ju st these 2 analytes, added in concentrated solutions. Accuracy in this set was unacceptable at the low level fo r PFOSAA, and at the high level fo r PFOSA. Interday QC data was obtained from a monitoring study analyzed in the weeks following this fourth set. A total o f fifteen sets are reported fo r PFOSA and PFOSAA, which not only validate the interday accuracy requirement, but also validate the intra-day accuracy requirement (5 out of 90 results outside the lim its for PFOSA, and 7 out o f 90 for PFOSAA). Validation report review 9 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 % Accuracy from Fortified Blank Serum Samples Source: report #108 (tables 15-30 and App. A) ________________ PFOS PFOSA PFOSAA POAA PFHS M-556 M570 Levels, ng/mL Low Mid High 51.1 4.00 197 150 446 400 9.00 155 405 8.74 154 403 6.15 152 402 5.80 "1 5 2 402 8.60 155 405 Run 13 Run 15 Run 16 Interdav Mid-level Run 13 Run 15 Run 16 Interday High level Run 13 Run 15 Run 16 Interdav 92 92 91 92 92 88 85 88 89 88 . 85 87 92 -- -- 101 99 -- -- 99 82 -- -- 98 73 103 111 108 97 -- 93 101 97 97 -- 90 104 105 104 108 95 105 103 99 108 102 110 108 104 -- 94 104 97 95 -- 93 100 97 94 100 96 105 101 98 92 101 110 103 98 -- 95 111 98 99 -- 98 105 99 96 106 98 108 100 97 PFOSA / PFOSAA intra-day results are from run 17; interday results are from study NWBS00-062. They include 15 runs o f 3 concentrations and 2 replicates each. Validation report review 10 Human Serum at Northwest Bioanafytical Q Al Report No. 405-503 V L ACCURACY IN PLASMA ANALYSIS R equirem ent m e t FDA G uideline 5 replicates @ 3 concentrations in the expected range o f concentrations: < 3x LLOQ; at the midlevel ; near the upper boundary o f the standard curve -------- N W BR eport -- 5 replicates @ 4 concentrations levels: LLOQ low (4.0 - 6.4 ng/mL) midlevel (126 -1 5 0 ng/mL) high (332 - 441 ng/mL) (A c c u ra c y ) R equirem ent m et Mean values 20% o f actual @ LLOQ All passed. Mean values 15% of actual >LLOQ A ll passed, except PFOSA and M570 at the low level. R esults: Summary tables presented below, w ith failed results bolded. W hile the low level fo r PFOSA and M570 tailed, at 116% and 118% respectively, three levels passed fo r all analytes. A ll results pass. \ % A ccu ra cy fro m F o rtifie d B la n k P lasm a S am ples Source: report #122 (tables 85-98) PFOS PFOSA PFOSAA POAA PFHS M-556 M570 Levels, ng/m L LLOQ Low 4.0 1.0 1.6 6.4 4.0 4.6 1.9 1.4 2.5 1.0 4.8 4.5 4.0 4.0 M id 126 150 151 145 156 150 150 H ig h 332 400 401 385 417 441 400 LLOQ 98 101 . 98 91 95 98 103 Low level 112 116 112 104 106 106 118 M id-level 106 107 103 106 101 99 107 H igh level 101 104 104 103 96 111 101 Validation report review 11 Human Serum at Northwest Bioanalyticai QAI Report No. 405-503 V II. ABSOLUTE RECOVERY IN SERUM Requirement met with restrictions: the design of the test was different than the guideline. The QC's and calibrations in a set must be kept together for all analyses. Extraction efficiencies are poor, but are consistent within a set FDA G uideline 3 concentrations - low, medium, high - in biological matrix Compare to unextracted standards NWB R eport 2 levels (4 & 400 ng/mL) in serum; 4 replicates, in 1 set. Compared with blank extracts fortified with known concentrations. Recovery should be consistent, precise and reproducible (lim its not specified). Accuracy not required. Extraction efficiencies are poor (15 - 70%) fo r analytes, worse (6%) for internal standard, but consistent within a set. D esign: Analytes and internal standards were added either to serum or to serum extract to determine losses during extraction. The loss calculations were based on comparing the peak area ratio o f (the one added before e xtra ctio n : the one added after extraction) to the comparable peak area ratio when both were added after extraction. The endogenous levels were included in the calculated efficiencies (levels not reported). R esults: A ll analytes showed losses during extraction. These were sim ilar at low and high levels. PFOS showed considerably higher loss at the low level, but this is probably an artifact of measurement, w ith an endogenous level on the order o f 4x the added 4 ng/ml_. Extraction losses are not a concern for this method, so long as they are consistent for samples within an extraction set. Since samples are extracted along w ith the calibration standards, as Iona as they are kept together in analysis, the relative quantitation w ill be unaffected. Losses in the internal standard are a greater concern, but, at least w ithin a set, they are consistent. There is no inter-set data. (Source: Report #122 Tables 64-70) % MEAN EXTRACTION EFFICIENCY* PFOS PFOSA PFOSAA POAA PFHS M-556 M570 Analyte low 44 69 63 17 16 38 54 high 30 70 63 14 14 40 60 overall 37 J 70 63 16 15 39 57 Internal Standard, THPFOS overall (mean 6.0%) 5.9 5.8 6.3 6.1 6.3 5.5 6.1 Validation report review 12 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 V III. ACCURACY AND POAA EFFECTS ON l.S. QUANTITATION Restriction on Accuracy: Samples with levels of POAA above the ULOQ will show a high quantitation bias for other analytes, due to LC/MS coelution and ionization suppression of the internal standard THPFOS. The scope of this effect is presented, but not completely proven. This may affect results obtained by dilution and reanalysis. FDA Guideline None NWB Report High concentration of POAA >500 ppb depresses IS response, biasing other analytes to the high side. However, the test has too many unknowns to allow many conclusions to be drawn. P lian- The internal standard THPFOS and the analyte POAA are incompletely resolved in LC/MS. Thus, POAA at high levels in samples suppresses the THPFOS response. This causes a high bias in analyte quantitation by peak area ratio. Low level QC sam ples were fortified w ith high levels o f POAA, and the analytes quantified and com pared to quantitation before fortification w ith POAA.. Results: The data show that when fortified to approximately the ULOQ level, the change in quantitation is slight (-2 to -11% ). However, at 10 - 100x this level, the bias steadily increases, in proportion to the decrease in IS peak area. (Source: Report #122 p. 26)________________ POAA Added, approx. ng/mL 592 5920 11800 59200 QC cone. PFOS PFOSA PFOSAA ng/mL 51.8 4.05 6.83 % Change from QC Mean IS Area Analytes Found +11 -2 to-11 -32 +16 to+36 -43 +28 to+56 -75 +152 to+216 PFHS M-556 M570 8.04 5.41 6.87 Validation report review 13 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 IX . ACCURACY OF SERUM QUANTITATION VS PLASMA CURVE Does n o t m eet FDA gu id e lin e fo r accuracy. FDA G uideline None NWB R eport Serum and plasma calibration standards. Serum and plasma QC's at 3 levels, 3-4 replicates, 2 sets o f each matrix Serum QC's analyzed with both calibrations Serum QC samples were analyzed against plasma calibration, because plasma has lower endogenous levels of some analytes than does serum. The results were reported as % deviation from theoretical concentration. R esults: This method of quantitating serum sam ples against plasma calibration does not meet the FDA requirements fo r accuracy. Most o f the PFOSA, PFOSAA, POAA and PFHS results are > 15% of theoretical. (Source: Report 122, Tables 141) SERUM QC vs. PLASMA CURVE PFOS PFOSA PFOSAA POAA I PFHS M-556 Accuracy, % Deviation from theoretical low 11 57 14 14 26 -19 mid 12 40 31 20 29 -7 _______ wgh 6 32 19 24 23 -11 Precision, % CV low 6 14 12 10 10 10 mid 7 3 3 5 4 4 high 6 6 5 3 6 2 # of runs 2 11 2 1 1 Levels, ng/mL low 39.3 4.00 7.20 9.54 6.41 5.30 mid 161 150 153 143 132 151 high 370 400 403 370 345 401 M570 9 15 9 14 9 8 2 6.70 153 403 All calibration sets passed the requirements, although occasional replicates failed. Accuracy and precision o f plasma and serum QC's against the same m atrix calibration met requirements, with the exception that PFOSAA / plasma precision at the low level was 16% CV and accuracy at the mid level was 17%, and POAA / serum accuracy was 16% at the low level. The low levels were above the LLOQ. Validation report review 14 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 X. VALIDATION OF SERUM EXTRACT DILUTION PROCEDURE This requirement was met, with restriction: The calibration curve must be prepared at the same dilution of serum as die diluted sample for accuracy. If the sample is at a different matrix dilution, the results will not be correct FDA G uideline T h e ability to dilute samples originally above the upper lim it o f the standard curve should be demonstrated by accuracy and precision parameters in the validation." "Any required sample dilutions should use like matrix" NWB R eport R equirem ent m et, b u t th e m ethod m u st co n ta in a re s tric tio n on d ilu tio n an alysis. 3 levels; 4 replicates; 1 set mid; high; 10x high D esign: Serum QC samples were diluted 1:10 or 1:50 with ammonium acetate buffer, and analyzed against a calibration curve prepared in 10% serum in buffer. It is known that quantitating against a whole serum curve is not accurate. R esu lts: The data show that accuracy requires that the proportion o f matrix in the sample and calibration curve extracts must be sim ilar. A ll 1:10 dilutions had acceptable (> 15%) accuracy, but the 1:50 dilution was biased unacceptably low (57 - 85%). Intra-assay precision was excellent (<7% CV) in all measurements. (Source: Report 122, Tables 113-119) PFOS PFOSA PFOSAA P recision (CV) mid high 44 5 '6 1 4 10x high 2 2 2 10x high (1:50) 2 4 4 Accuracy % recovery mid 108 96 94 high 103 96 99 10x high 107 99 100 10x high (1:50) 77 57 66 Levels, ng/m L mid 193 150 153 high 443 400 403 10x high 4040 4000 4000 POAA 3 3 1 4 106 100 105 85 157 407 4010 PFHS 2 4 2 3 105 102 107 84 154 404 4000 M-556 4 4 2 4 101 102 114 70 151 401 4000 M570 2 4 2 4 103 103 108 69 157 407 4010 Validation report review 15 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 X I. PRECISION IN SERUM ANALYSES The FDA requirem ents w ere m e t FDA G uideline (P re cisio n - in tra b a tch / in te rb a tch ) CV 20% @ LLOQ NWB Report R equirem ent m e t A ll passed, except 1 o f 4 PFOSAA sets at 1.5x LLOQ, w ith 24 %CV. CV 15% @ >LLOQ A ll passed, except 1 o f 4 PFOSA sets at 4x LLOQ, w ith 19.8 %CV. R esults: Summary tables presented below. A ll analytes passed at all concentrations, with the exception of PFOSA and PFOSAA in one set each at the low level. In the initial te st o f 3 sets, PFOSA and PFOSAA had low recoveries because the fortifications w ere faulty; a set 4 contains ju s t these 2 analytes. Both analytes have at least three sets at each levels meeting the intra-day requirements. Interday QC data was also obtained from a monitoring study analyzed later. Validation report review 16 Human Serum at Northwest Bioanaiytica! QAI Report No. 405-503 Precision (% CV) from Fortified Blank Serum Samples Source: report #108 (tables 15-30 and app. A) __________ . _______ PFOS PFOSA PFOSAA POAA PFHS M-556 M570 Levels, ng/mL Low Mid High Low level 51.1 4.00 197 150 446 400 9.00 155 405 8.74 154 403 6.15 152 402 5.80 152 402 8.60 155 405 Set 13 Set 15 Set 16 Set 17 45 6 10 4 54 2 2 2 6 3 47 4 20 11 9 5 59 6 24 Interdayl 3 14 8 6 6 67 Interday2 11 10 Mid-level Set 13 Set 15 Set 16 Set 17 Interdayl Interday2 4 2 3 4 5 5 4 9 12 8 3 5 4 9 6 8 2 4 35 5 4 44 4 1 44 6 5 66 High level Set 13 54 Set 15 36 Set 16 33 Set 17 2 Interdayl 4 7 Interday2 10 8 4 4 5 6 7 5 4 75 3 3 33 3 3 44 4 4 54 Interdayl is the mean o f sets 13,15, and 16. Interday2 PFOSA / PFOSAA data are from study NW BS00-062, a m onitoring study. This represents QC sample data from 15 runs o f 3 concentrations and 2 replicates each. Validation report review 17 Human Serum at Northwest Bioanaiyticai QAI Report No. 405-503 xn. PRECISION IN PLASMA ANALYSES The FDA requirements for this test were m et FDA Guideline (Precision - intrabatch / interbatch) CV 20% @ LLOQ CV 15% @ >LLOQ ~ NWB Report Requirement m et A ll passed. A ll passed. Results: Summary tables presented below. A ll analytes passed at all concentrations, Precision (% CV) from Fortified Blank Plasma Samples Source: report #122 (tables 8 5 -9 8 ) ______ ________ ________ PFOS PFOSA PFOSAA POAA PFHS M-556 M570 Levels, ng/mL Low 6.40 4.00 4.60 4.81 4.48 4.00 4.00 Mid 126 150 151 145 156 150 150 High 332 400 401 385 417 441 400 LLOQ 5 10 6 4 10 6 5 Low level 3 2 5 6 3 82 Mid-level 3 6 6 5 3 56 High level 2 2 3 1 2 72 Validation report review 18 Human Serum a t Northwest Bioanalytica! QAI Report No. 405-503 X III. SERUM SAM PLE STABILITY TO FREEZE-AND-THAW All requirements were m et In serum, all analytes were stable to seven freeze / thaw cycles, except PFOSAA which was stable to three cycles. Accuracy and precision were generally acceptable. FDA G uideline Two concentrations in triplicate NWB R eport R equirem ent m e t 3 concentrations in serum, approx. 1x, 4-40x and 10-100x LLOQ Analyte stability should be determined R equirem ent m e t Analyzed after three and after three freeze and thaw cycles. seven cycles. R esu lts: Serum samples fortified at 1x, 4x and 10x LLOQ were frozen and thawed seven tim es before analysis. In each thawing, the samples remained at room tem perature up to 6.75 hours. Losses are measured by comparing the sample analysis results to the corresponding control analysis. A ll analytes except PFOSA were stable to these conditions. PFOSA data indicate losses o f 11 - 20%, considerably exceeding the precision o f the sample measurement. PFOSA was stable to 3 freeze-thaw cycles in report #005. In that report, PFOSA at approxim ately 1x and 20x LLOQ showed stability, as did all the other analytes. Precision data fo r sample analyses are good. Accuracy o f controls analyses is within guidelines except fo r PFOS high le v e l, which is -15.2% . (Source: report #122 Tables 15 - 2 8 ) PFOS PFOSA % Deviation from Control low -3 -18 medium -3 -20 high -2 -11 Sample, mean ng/mL low 45.3 3.58 medium 164 120 ______________ M l 369 353 AFTER 7 FREEZE-THAW CYCLES PFOSAA POAA PFHS M-556 M570 -5 7 -6 -7 4 -15 1 -1 -5 -5 -5 0.0 0.7 0.3 -5 9.46 136 392 8.38 151 372 6.48 162 406 5.04 134 368 8.32 142 366 (Source: report #005 Table 5 1 ) PFOSA AFTER 3 FREEZE-THAW CYCLES PFOSA low high % Deviation from Control 4 -2 Sample, mean ng/mL 21.1 369 Validation report review 19 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 X IV . SHORT-TERM AM BIENT STABILITY OF SERUM SAMPLES Requirements were m et Serum samples were kept at room temperature up to 6.75 hours per cycle during seven recurring freeze-thaw cycles (discussed above). All were stable except PFOSAA, which exhibited 10 - 20%4osses. FDA Guideline 3 aliquots o f biological m atrix fortified a t low and high concentrations Store fortified samples at room tem perature 4 - 2 4 hours. NWB Report Requirement m et 3 concentrations approx. 1x, 4-40x and 10-100x LLOQ Requirement m et Fortified serum samples w ere frozen and thawed seven times. The maximum thaw tim e in a cycle was 6.75 hours; the total tim e a t room tem perature is not reported. Results: Presented in the section above. Validation report review 20 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 XV. LONG-TERM FREEZER STABILITY OF SERUM SAMPLES All requirements were m et The results demonstrate that there is no degradation over the 55-day storage period (42 days for two analytes) of extracts at freezer temperature -20 C. FDA G uideline A t least 3 aliquots o f biological matrix fortified at low and high concentrations NWB R eport R equirem ent m e t 5 replicates o f serum at approxim ately 1x, 4x and 10x LLOQ. Store fortified samples under the same conditions as the study samples, fo r a time exceeding the time between the date of first sample collection and the date of last sample analysis. R equirem ent m e t Stored at -20 C for: 42 days PFOSA and PFOSAA 55 days other analytes Stability was determined by comparison to theoretical concentration. Results: All analytes are stable under these storage conditions, within the accepted lim its o f precision of the measurements. Precision o f all measurements are <11%. (Source: report #122 Tables 57-63) SAM PLES AFTER 42 OR 55 DAYS A T -20 C PFOS PFOSA PFOSAA POAA PFHS M-556 M570 Sample, % Change from theoretical low -9 8 medium -14 0.0 '______ hiflh Sample, mean ng/mL -15 -1 low 46.7 4.34 medium 170 150 hiah 378 395 11 3 2 9.95 160 413 -10 -3 -8 7.82 149 372 12 8 0.2 6.86 164 403 -7 -7 -9 5.42 141 367 -7 -4 -5 8.02 149 386 Validation report review 21 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 X V I. POST-PREPARATIVE AM BIENT STABILITY OF SERUM EXTRACTS The requirements were met for all analytes. The results demonstrate that there is no degradation over the 7 day storage period of extracts at room temperature, .... covering at least the resident-time in die autosampler. FDA G uideline No specific requirement The stability o f the drug and the internal standard should be assessed over the anticipated run tim e NWB R eport R equirem ent m e t Serum extracts used; approx. 1x, 4-40x and 10-100x LLOQ R equirem ent m e t Serum extracts stored 3 o r 7 days at room tem perature. Stability was measured by comparison to previously analyzed or freshly prepared QC samples. R esults: Two tests w ere conducted at different times. In the first, "standards and controls" were extracted and analyzed, then reinjected on the 4th day after extraction. Three QC levels were included. In the second, low and high QC sample extracts were stored for 7 days, then analyzed against freshly extracted QC samples. A ll analytes are stable under these conditions. Recoveries were all in the range of 100% 11. W hile there is no separate data fo r the surrogate standard, the results presented at a minimum demonstrate th a t at worst, all analytes and the surrogate degrade at the same rate, if any. Sample precision is good, <10% CV in all cases. Control precision is good in the first test, but was not reported for the second. Control accuracy is good, except for PFOSA and PFOSAA. In the first test, control values were <85% o f theoretical due to a fortification error. However, the stability test is not affected by this, if both were fortified with the same solution. In the second test, accuracy could not be determined because the fortification level was not reported. Validation report review 22 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 (Source: report #122 Tables 29-35 ) EXTRACTS STORED 3 DAYS, AMBIENT PFOS PFOSA PF06AA POAA PFHS M-556 M570 % Deviation from Control low 1 -3 2 4 0.3 -2 -1 mid r . -high Sample, mean ma/MI -3 -3 -3 4 .-3.- ..... =5.. . ..... ____4 -0.6 -1 -4 .. A -0.7 --3- low 47.7 2.73 7.73 3.1 6.24 5.52 8.2 mid 168 100 110 1.6 157 145 146 high 382 286 288 4.1 427 381 390 (Source: report #122 Tables 36 - 42 ) EXTRACTS STORED 7 DAYS, AMBIENT PFOS PFOSA PF08AA POAA PFHS M-556 M570 % Deviation from Control low 3 high 3 8 11 7 12 -2 -10 0.2 11 2 0.2 3 9 Sample, mean ng/mL low high 48.0 391 3.37 8.89 334 334 7.68 378 6.14 404 5.43 379 8.91 420 Validation report review 23 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 X V II. POST-PREPARATIVE 1-8C STABILITY OF SERUM EXTRACTS A ll requirem ents w ere m e t The re s u lts de m onstrate th a t the re is no d e g ra d a tio n o ve r the fo u r day storage p e riod o f e xtra cts a t re frig e ra to r tem perature. FDA G uideline No specific requirement The stability should be assessed over the anticipated storage tim e before injection. NWB R eport R equirem ent m e t R equirem ent m e t Stored serum extracts were refrigerated at 1-8 C fo r 4 days. Stability was measured by comparison to freshly prepared QC samples. R esults: A ll analytes are stable under these conditions. Sample precision is good. Control accuracy is good; however, PFOSA and PFOSAA accuracy in the QC sam ples could not be evaluated. W hile there is no separate data fo r the surrogate standard, the results presented at a minimum demonstrate that, at worst, all analytes and the surrogate degrade at the same rate, if any. (Source: Report #122 Tables 43 - 49) PFOS PFOSA % Deviation from Control low -0.2 -5 high 2 -2 Sample Precision (CV). % . low 5 8 high 2 9 Sample, mean ng/mL low 46.6 2.96 high 384 317 EXTRACTS STORED 4 DAYS 1-8C PFOSAA POAA PFHS M-556 M570 2 4 5 1 8.22 310 6 4 6 2 8.31 387 1 2 10 3 6.95 411 16 10 11 0.8 6.29 402 4 3 9 1 8.36 397 Validation report review 24 Human Serum at Northwest Bioanalytical QAI Report No. 405-503 X V m . POST-PREPARATIVE -20C STABILITY OF SERUM EXTRACTS A ll requirem ents w ere m e t The re su lts dem onstrate th a t the re is no degradation o ve r th e fiv e day storage period o f e xtra cts a t freezer tem perature (-20 C). FDA G uideline No specific requirement N W BR eport R equirem ent m e t Two levels in triplicate. The stability should be assessed over the anticipated storage tim e before injection. R equirem ent m e t Stored serum extracts were stored at -20 C fo r 5 days. Stability was measured by comparison to freshly prepared QC samples. R esults: All analytes are stable under these conditions. W hile there is no separate data fo r the surrogate standard, the results presented at a minimum demonstrate that, at worst, all analytes and the surrogate degrade at the same rate, if any. (Source: Report #122 Tables 50-56) EXTRACTS STORED 5 PAYS a t -20 C PFOS PFOSA PFOSAA POAA PFHS M-556 M570 % Deviation from Control low -5 high -2 Sample Precision (CV), % low 7 high 2 Sample, mean ng/MI low 44.5 high 371 -2 -5 4 3 3.08 307 -1 -0.3 5 3 7.91 296 6 -0.5 21 5 8.30 370 -0.7 2 12 2 6.81 409 -0.6 0.8 14 3 5.39 370 4 -3 4 3 8.30 374 Validation report review 25 Human Serum at Northwest Bioanalytica! QAI Report No. 405-503 XIX. STOCK SOLUTION STABILITY Requirement not m et No testing reported. FDA Guideline... ........... NWB Report Store at room temperature for at least 6 hours Requirement not met Store at other temperatures and time periods as required. Requirement not met Report issued by: Philippe Ourisson Quality Associates, Inc. Manager, Scientific Support Date Validation report review I 26 Human Serum at Northwest Bioanalytical