Document NNNoOE435jXq1Jmpzr3Z2kBkp
A 2 2 2 -lclort
Report Title Evaluation of the Northwest Bioanalytical Method Validation Reports for
P F O S and Related Analytes in Human Serum or Plasm a in Meeting the Requirements of the
_________ EDABioanalyticaLM ethod Validation Q A I Report No. 405-503
Data Requirement Full Validation of Bioanalytical Method
Author Nancy Benson Philippe O urisson Quality Associates, Inc. 9017 Red Branch Road, Suite 102 Columbia, M D 21045
Sponsor 3M Environmental Laboratories
935 Bush Avenue Bldg 2-3E-09
St Paul, M N 55106 Report Date
December 31, 2001 Report Number QAI 405-503
Number of Pages 26
337
TABLE OF CONTENTS
I. S U M M A R Y .............................
3
II. INTR O D U C T IO N ........ ................
...,
III. S P E C IF IC IT Y IN S E R U M A N D P L A S M A .......................................... 5
IV. C A L IB R A T IO N IN S E R U M A N D P L A S M A ........................................ 7
V. A C C U R A C Y IN S E R U M A N A L Y S IS ................................................ 9
VI. A C C U R A C Y IN P L A S M A A N A L Y S IS ...................
11
VII. A B S O L U T E R E C O V E R Y IN S E R U M ............................................. 12 V ili. A C C U R A C Y A N D P O A A E F F E C T S O N I.S. Q U A N T IT A T IO N .......... . 13
IX. A C C U R A C Y O F S E R U M Q U A N T IT A T IO N V S P L A S M A C U R V E ......... 14
X. V A LID A T IO N O F S E R U M E X T R A C T D ILU T IO N P R O C E D U R E ......... 15
XI. P R E C IS IO N IN S E R U M A N A L Y S E S ................................
16
XII. P R E C IS IO N IN P L A S M A A N A L Y S E S .............................................18
XIII. S E R U M S A M P L E ST A B IL IT Y T O F R E E Z E -A N D -T H A W .............
19
XIV. S H O R T -T E R M A M B IE N T S T A B IL IT Y O F S E R U M S A M P L E S ............ 20
XV. LO N G -T E R M F R E E Z E R S T A B IL IT Y O F S E R U M S A M P L E S .............. 21
XVI. P O ST -P R E P A R A T IV E A M B IE N T S T A B IL IT Y O F S E R U M
E X T R A C T S .........................................
22
XVII. P O S T -P R E P A R A T IV E 1-88C S T A B IL IT Y O F S E R U M E X T R A C T S ...... 24
XVIII. P O S T -P R E P A R A T IV E -20C S T A B IL IT Y O F S E R U M E X T R A C T S ......25
XIX. S T O C K SO LU T IO N S T A B IL IT Y ....................................................26
Validation report review
2 Hum an Serum at Northwest Bioanalytical
QAi Report No. 405-503
I. S U M M A R Y
The validation in human serum or plasm a w as performed for the analytes PFO S,
PFO SA , PFO SAA, POAA, PFH S, M-556, and M570. The surrogate standard IH,IH,2H,2H-
perfluorooctanesulphonic acid w as added before extraction, but used as an internal
standard in the quantitation.
-- ........................
The following parameters can be considered a s completely meeting the F D A requirements: Specificity Calibration in serum and plasm a Accuracy in serum and plasma. Precision in serum and plasm a Validation of Extract Dilution Serum sample stability to:
o freeze-thaw. 7 cycles o short-term ambient storage, 6.75 hr x 7 cycles o freezer storage, 42 - 55 days Serum extract stability to: o ambient storage, 7 days o refrigerated storage, 4 days o frozen storage, 5 d ays
The following restrictions apply to these analyses: Because of poor analyte and internal standard extraction efficiencies, it is essential
that samples, Q C s and calibrations standards in a set be kept together for all analyses. Extraction efficiency within a set is consistent;
Sam ples with levels of P O A A above the U LO Q will show LC/M S ionization suppression of the internal standard T H P F O S, resulting in a high quantitation bias on other analytes. A resolution w as not demonstrated.
For diluting sam ples with serum, the calibration curve must be prepared at the sam e dilution of serum a s the diluted sam ple for accuracy. If the sam ple is at a different matrix dilution, the results will not be correct.
W hen a sample must be diluted, it not only m ust be compared to a calibration curve prepared at the sam e dilution of serum, but it also must be re-extracted for that analysis, in order to keep sam ples, Q C s, and calibration standards together.
The following parameters from the F D A requirements were not tested: Stock solution stability. Stability of the surrogate standard.
Quantitating serum sam ples against plasm a calibration does not meet the F D A requirements for accuracy.
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H. IN TRO D U C TIO N
Three reports were reviewed:
N W BR00-122: Quantitative Determination of P FO S, P FO SA , P F O SA A , PO AA, P F H S, M -556 and M 570 in Hum an Serum by LC/MS/MS: A ssa y Revalidation Addendum Report, 11/20/01 (study N W BS00-040)
N W BR00-108: Quantitative Determination of P FO S, PFO SA , P F O SA A , PO AA, P FH S, M -556 and M 570 in Hum an Serum by LC/MS/MS: A ssa y Revaluation Report, 1/24/01 (study NW BS0O-04);
NW BR99-005: Quantitative Determination of P F O S, P FO SA , P FO SA A , PO AA, P FH S, M -556 and M 570 in Hum an Serum by LC/MS/MS: A ssa y Validation Report, 1/24/01 (study NW BS98-082);
The methods used in these studies are very similar with the following differences:
#005
#108/122
internal
SDS
THPFOS
standard
(IS)
partition
basified with 0.5 M
unadjusted pH ca. 6.9
tertbutylammonium hydrogen
sulfate (pH 10) + 0.25 M carbonate
buffer
final solvent 2m M ammonium acetate: methanol 20m M ammonium acetate in water
(1:1)
: 20 m M ammonium acetate in
M eO H (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 exam ines which of the FDA-required tests were conducted, and which meet the standards described by F D A for a full validation of the method. This report did not consider whether additional requirements, specified by protocol, were met if they did not affect the evaluation based on the F D A guideline; however, they m ay be discussed in the text of this report.
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IH . S P E C IF IC IT Y IN S E R U M A N D P L A S M A
This requirement is m et Endogenous levels were found in ail serum and plasm a
sam ples tested, although much greater in serum.
----- -
F D A Guideline > S ix sources of blank biological matrix M easure levels of each analyte
N W B Report
Requirem ent met. Testing w as reported on 2 lots of serum and 4 lots of plasma.
Results:
In report #005, endogenous levels of PFO S, PFO SA, PFO SAA, PO AA and P F H S in human serum were determined using rabbit serum calibration; P F O S A was not detected.
Mean ng/mL in Hum an 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 Q C samples (n=12)
22.3 1.99
1.97 3.25
The LLOQ in rabbit serum or each ana yte w as 1.00 ng/mL.
In report #108, serum results indicated significant endogenous levels of ail analytes except PFO SA. 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 LLO Q *
Mean ng/mL in Human Serum P FO S P F O SA P F O SA A P O A A PFH S M-556 M570
47.1 ND 1.0 1.0 48.1 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 w as with human plasm a
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Pooled plasma also showed significant endogenous PFO S, PFO SA A , PO AA and PFHS, but at least 5x less than in serum. The levels were determined by standards prepared in plasma at 1 - 1 0 0 ng/mL. The endogenous level is defined a s the mean y-intercept of linear regressions performed on 2 separate tests.
Source: report# 122 tables 78 84
endogenous levels
target LLO Q
calibration LLO Q *
PFOS
2.94 1.00 3.94
Mean ng/mL In Human Plasm a P F O SA P F O SA A P O A A P FH S 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 w as with human plasma.
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IV . C A L IB R A T IO N IN S E R U M A N D P L A S M A
Requirem ent m et There is how ever no justification for the selection o f the com plicated calibration procedure (quadratic w ith 1/x2 o r 1/x w eighting).
F D A Guideline Blank + 6 - 8 sam ples
LLO Q > 5x blank response
NW B Report Requirem ent m e t
2 blanks and 2 blanks + IS were included in each run. No blank analyses were reported.
Sam ples: 8 Levels for M-556; 9 levels for all others. Duplicate sam ples. 3 runs for serum and 2 for plasma.
Requirem ent m e t Endogenous responses caused LLO Q approximately = "blank" in most cases. Sum m arized in table below.
(Accuracy) 2 0 % of actual @ LLO Q
1 5 % of actual >LL Q
Requirem ent m et
All pass, except one serum PO AA.
Passed, of 550 total: 550 for P F H S 549 for P F O S 548 for M-556, M 557 547 for P FO SA , P FO SA A , P O A A
> 4 of 6 standards meet these All runs pass. criteria in each curve
LLO Q : A n experimental determination of the analytical limits w as not performed. The lowest standard injected which met the acceptance criteria w as chosen a s the LLOQ. In most cases, these standard concentrations were close to file endogenous levels. Since the calibration LLO Q is only slightly ( 1 - 4 times) higher than the endogenous (blank) level the uncertainty of sam ple m easurem ents at the LLO Q will be increased by the uncertainty of measurement of the endogenous level.
Because the endogenous levels are lower in plasm a than in serum, plasm a blanks should be used to prepare calibration standards when an LLO Q below 5.0 ng/mL is needed.
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Range:
I PFOS Serum (reports #108/122)
Serum LLOQ
48
Serum ULOQ
540
Plasm a (report #122)
Plasma LLOQ
3.9
Plasma ULOQ 410
V A LID A T E D R A N G E (ng/m U P F O S A P F O S A A P O A A P FH S M-556 M570
6.0 500 500
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 with 1/x2 weighting, of peak area ratio (sam ple / surrogate standard) vs. nominal concentration. (1/x weighting in report #005). There is no justification for the selection of this complicated calibration method.
In all sets, there were at least four out of six calibration standard levels that met the accuracy requirement of 1 5 % of actual (or 2 0 % of actual at the LLOQ).
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V. A C C U R A C Y IN S E R U M A N A L Y S IS The FD A requirements for th is test were m et
F D A G uideline
5 replicates @ 3 concentrations in the expected range of concentrations. Levels: : 3x LLOQ; at the m idlevel; near the upper boundary of the standard curve
(Accuracy)
M ean values 2 0 % of actual @ LLO Q
M ean values + 1 5 % of actual >LLO Q
NW B Report 5 replicates, each on 3 separate days
L@vq j * LLO Q (4.0 - 51.1 ng/mL; 1.1 - 4 x ) midlevel (150-200 ng/mL) high (400-450 ng/mL)
Requirem ent met All passed except P F O S A A (intraday)
Ail passed.
R e su lts:
Sum m ary tables presented below, with failed results bolded.
In the initial test of 3 sets, P F O S A and P F O S A A failed because the fortifications were faulty. The fortifying solution w as prepared from dilute stocks by evaporation, and these two analytes were partially lost. The intra-day data below w as obtained from set 17 containing just these 2 analytes, added in concentrated solutions. Accuracy in this set w as unacceptable at the low level for P FO SA A , and at the high level for P FO SA .
interday Q C data w as obtained from a monitoring study analyzed in the w eeks following this fourth set. A total of fifteen sets are reported for P F O S A and P FO SA A , which not only validate the interday accuracy requirement, but also validate the intra-day accuracy requirement (5 out of 90 results outside the limits for P FO SA , and 7 out of 90 for P FO SA A ).
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% Accuracy from Fortified Blank Serum Sam ples
Levels, ng/mL
PFOS PFOSA PFOSAA POAA
PFHS
M-556 M570
Low M id H ig h
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
Run 13 Run 15 Run 16 Interday
M id -le v e l
92 92 92 -- 91 -- 92 101
73 103 111 108 97
_
93 101
97 97
-- 90 104 105 104
108 95 105 103 99
Run 13 Run 15 Run 16 Interday
H igh level
92 99 88 -- 85 -- 88 99
108 102 110 108 104
--
94 104
97 95
--
93 100
97 94
100 96 105 101 98
Run 13 Run 15 Run 16 interday
89 88 . 85 87
82
--
--
98
92 101 110 103 98
--
95 111
98 99
_
98 105
99 96
106 98 108 100 97
P F O S A / P F O S A A intra-day results are from run 17; interday results are from study MW BSOO-062. They include 15 runs of 3 concentrations and 2 replicates each.
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V L A C C U R A C Y IN P L A S M A A N A L Y S IS
Requirem ent m et
P D A Guideline
5 replicates @ 3 concentrations in the expected range of concentrations:
< 3x LLOQ; at the m idievel; near the upper boundary of the standard curve
-------
NW B-Report
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)
(Accuracy) M ean values 2 0 % of actual @ LLO Q
Requirem ent met All passed.
M ean values 1 5 % of actual >LLO Q
All passed, except P F O S A and M 570 at the low level.
R e su lts:
( Sum m ary tables presented below, with failed results bolded. W hile the low level for
P F O S A and M 570 failed, at 1 1 6 % and 1 1 8 % respectively, three levels passed for all analytes. Ail results pass.
\
% A ccu ra cy from Fortified B la n k P lasm a Sa m p le s
Source: report #122 (tables 85-98)
P FO S P F O SA P FO SA A PO AA P FH S M-556 M570
Levels, ng/mL
LLOQ
4.0 1.0 1.6
1.9 1.4 2.5 1.0
Low
6.4 4.0 4.6
4.8 4.5 4.0 4.0
M id
126 150
151
145 156 150 150
Hiflh
332 400 401
385 417 441 400
LLOQ Low level
98 101 . 98 112 116 112
91 95 98 103 104 106 106 118
M id -le v e l
106 107 103
106 101
99 107
H igh level
101 104 104 103 96 111 101
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V II. A B S O L U T E R E C O V E R Y IN S E R U M
Requirem ent met w ith restrictions: the d e sig n o f the test w a s different than the guideline. The Q C 's and calibrations in a se t m u st be kept together for all analyses. Extraction efficiencies are poor, but are co n siste n t w ithin a s e t
F D A G uideline
3 concentrations - low, medium, high - in biological matrix
NW B Report
2 levels (4 & 400 ng/mL) in serum; 4 replicates, in 1 set.
Com pare to unextracted standards
Com pared with blank extracts fortified with known concentrations.
Recovery should be consistent, precise and reproducible (limits not specified). Accuracy not required.
Extraction efficiencies are poor (15 - 7 0 % ) for analytes, w orse (6 % ) for internal standard, but consistent within a set.
D e sign : Analytes and internal standards were added either to serum or to serum extract to determine losses during extraction. The lo ss calculations were based on comparing the peak area ratio of (the one added before extraction: 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 e su lts: All analytes showed lo sse s during extraction. These were similar at low and high levels. P F O S showed considerably higher loss at the low level, but this is probably an artifact of measurement, with an endogenous level on the order of 4x the added 4ng/mL.
Extraction losse s are not a concern for this method, so long a s they are consistent for sam ples within an extraction set. Since sam ples are extracted along with the calibration standards, a s Iona a s they are kept together in analysis, the relative quantitation will be unaffected. L o sse s in the internal standard are a greater concern, but, at least within a set, they are consistent. There is no inter-set data.
(Source: Report #122 Tables 64-70)
% M EA N EXT R A C T IO N E FF IC IE N C Y *
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
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
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VTTI. A C C U R A C Y A N D P O A A E F F E C T S O N I.S. Q UAN TITATIO N
Restriction on Accuracy: S a m p le s w ith levels of P O A A above the U L O Q w ill s h o w s high quantitation b ia s fo r other analytes, due to LC /M S coelution and ionization su p p re ssio n of the internal standard T H PFO S. The sco p e of th is effect is presented, but not com pletely proven. T h is m ay affect results obtained b y dilution and reanalysis.
F D A G uideline None
N W B Report
High concentration of P O A A >500 ppb depresses IS response, biasing other analytes to the high side. However, the test has too many unknowns to allow m any conclusions to be drawn.
D e sig n : The internal standard T H P F O S and the analyte P O A A are incompletely resolved in LC/MS. Thus, P O A A at high levels in sam ples suppresses the T H P F O S response. This causes a high bias in analyte quantitation by peak area ratio. Low level Q C sam ples were fortified with high levels of PO AA, and the analytes quantified and com pared to quantitation before fortification with POAA..
R e su lts:
The data show that when fortified to approximately the U LO Q 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)__________________
PO A A Added, approx. ng/mL
592
5920
11800
59200
QC cone.
PFOS
PFOSA PFOSAA
ng/mL
51.8 4.05 6.83
% Change from Q C Mean
IS Area
Analytes Found
+11 -2 to-11
CM CO
i
+16 to +36
-43 +28 to+56
-75 +152 to+216
PFHS 8.04
M-556 5.41
M570 6.87
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IX . A C C U R A C Y O F S E R U M Q U AN TITATIO N V S P L A S M A C U R V E
D o e s not meet F D A guideline for accuracy.
F D A Guideline None
NW B Report
Serum and plasm a calibration standards. Serum and plasm a Q C 's at 3 levels, 3-4 replicates, 2 sets of each matrix Serum Q C 's analyzed with both calibrations
Serum Q C sam ples were analyzed against plasm a calibration, because plasm a has lower endogenous levels of som e analytes than does serum. The results were reported a s % deviation from theoretical concentration.
R e su lts:
This method of quantitating serum sam ples against plasm a calibration does not meet the F D A requirements for accuracy. M ost of the P FO SA , P FO SA A , P O A A and P F H S results are > 1 5 % of theoretical.
(Source: Report 122, Tables 141)
SE R U M Q C vs. P L A S M A C U R V E
P F O S P F O S A PFOSAA P O A A P F H S M -556
A ccuracy, % Deviation from theore deal
low 11
57 14
14 26 -19
mid 12 40 31
20 29
-7
high 6 32 19
24 23 -11
Precision, % CV
low 6 14 12 10 10 10
mid 7
33
54
4
high 6 6 5
362
# of runs
2
11
2
1
1
Levels, ngftnL
low 39.3
4.00 7.20 9.54 6.41 5.30
mid 161 150 153 143 132 151
_________ hiatL 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 of plasm a and serum Q C 's against the sam e matrix calibration met requirements, with the exception that P F O S A A / plasm a precision at the low level w as 1 6 % C V and accuracy at the mid level w as 17% , and P O A A /serum accuracy w as 1 6 % at the low level. The low levels were above the LLOQ.
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X. VALIDATIO N OF SE R U M EX T R A C T DILUTIO N P R O C E D U R E
T his requirement w as met, with restriction: The calibration curve m ust be prepared at the sam e dilution of serum a s the diluted sam ple for accuracy. If the sam ple is at a different matrix dilution, the
results will not be correct
F D A G uideline
"The ability to dilute sam ples originally above the upper limit of the standard curve should be demonstrated by accuracy and precision parameters in the validation." "A ny required sample dilutions should use like matrix"
NW B Report
Requirem ent met, but the m ethod m ust contain a restriction on dilution a n alysis. 3 levels: 4 replicates; 1 set mid; high; 10x high
D e sign : Serum Q C sam ples were diluted 1:10 or 1:50 with ammonium acetate buffer, and analyzed against a calibration curve prepared in 1 0 % serum in buffer. It is known that quantitating against a whole serum curve is not accurate.
R esults:
The data show that accuracy requires that the proportion of matrix in the sam ple and calibration curve extracts must be similar. All 1:10 dilutions had acceptable (> 1 5 % ) accuracy, but the 1:50 dilution w as biased unacceptably low (57 - 85%). Intra-assay precision w as excellent (< 7 % C V) in ail measurements.
(Source: Report 122, Tables 113-119)
PFO S PFO SA PFOSAA
Precision (CV)
mid 4 4 1
high 5 ' 6
4
10x high
2
22
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/mL
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
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X L P R E C IS IO N IN S E R U M A N A L Y S E S
The FD A requirements were m et
F D A Guideline
(P re cisio n - intrabatch 1interbatch)
CV 20% @ LLOQ
N W B Report
Requirem ent met
All passed, except 1 of 4 P F O S A A sets at 1.5x LLO Q , with 24 % C V .
CV 15% @ >LLOQ
All passed, except 1 of 4 P F O S A sets at 4x LLOQ, with 19.8 % C V .
R e su lts:
Sum m ary tables presented below. All analytes passed at all concentrations, with the exception of P F O S A and P F O S A A in one set each at the low level.
In the initial test of 3 sets, P F O S A and P F O S A A had low recoveries because the fortifications were faulty; a set 4 contains just these 2 analytes. Both analytes have at least three sets at each levels meeting the intra-day requirements.
Interday Q C data w as also obtained from a monitoring study analyzed later.
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P re cisio n (% C V) from Fortified B lan k Se ru m Sa m p le s Source: report #108 (tables 15-30 and app. A )
P FO S P F O SA P F O SA A P O A A P FH S M-556 M570
Levels, ng/mL
Low
51.1 4.00
9.00
8.74
6.15
5.80 8.60
M id
197 150
155 154 152 152 155
H igh
446 400
405 403 402 402 405
Low level
Set 13
45
6
10 4
54
Set 15 Set 16 Set 17
2 2 2 6 3 47
4 20
11
9
5
59
6 24
Interdayl 3 14
8
6 6 67
Interday2
11 10
M id-level
Set 13 Set 15 Set 16 Set 17
4 5 3 2 4 35 2 5 5 5 4 44 3 4 4 4 1 44
99
In t e rd a y l In t e rd a y 2
4
12 8
6 8
6 5 66
H igh level
Set 13 Set 15 Set 16 Set 17
5 4 8 5 4 75
3 6 4 3 3 33 3 3 4 3 3 44
25
In te rd a y l
4
7
Interday2
10
6 7
4 4 54
<
linterdayl is the mean of sets 13,15, and 16.
Interday2 P F O S A / P F O S A A data are from study N W BS00-062, a monitoring study.
This represents Q C sample data from 15 runs of 3 concentrations and 2 replicates each.
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x n . P R E C IS IO N IN P L A S M A A N A L Y S E S
The FD A requirem ents for th is test w ere m et
F D A Guideline
(Precision - intrabatch / interbatch)
C V 20% @ LLOQ
N W B Report Requirem ent m et
All passed.
GV 15% @ >LLOQ
All passed.
R e su its:
Sum m ary tables presented below. All analytes passed at all concentrations,
P re cision (% CV) from Fortified B la n k P lasm a Sam p le s
Source: report #122 (tables 85 - 98)
______ _________ _________ _____
P FO S P F O SA P FO SA A PO A A P F H S M-556 M570
Levels, ng/mL
Low
6.40
4.00
4.60
4.81
4.48
4.00 4.00
M id
126 150 151
145 156 150 150
H ig h
332 400 401
385 417 441 400
LLOQ
5 10
6
4 10 6 5
Lo w level
3 2 5 6 3 82
M id-level
3 6 6 5 3 56
H igh level
2 2 3 1 2 72
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Q AI Report No. 405-503
XIII. S E R U M S A M P L E S T A B IL IT Y TO F R E E Z E -A N P -T H A W
A ll requirem ents were m et In serum, all analytes were stable to seven freeze / thaw cycles, except P F O S A A w hich w as stable to three cycles. Accuracy and precision were generally acceptable.
F D A Guideline Tw o concentrations in triplicate
N W B Report Requirem ent met. 3 concentrations in serum, approx. 1x, 4-40x and 10-100x LLO Q
Analyte stability should be determined Requirem ent m e t Analyzed after three and
after three freeze and thaw cycles.
seven cycles.
R e su lts: Serum sam ples fortified at 1x, 4x and 10x LLO Q were frozen and thawed seven times before analysis. In each thawing, the sam ples remained at room temperature up to 6.75 hours. Lo sse s are measured by comparing the sam ple analysis results to the corresponding control analysis.
All analytes except P F O S A were stable to these conditions. P F O S A data indicate losse s of 11 - 2 0 % , considerably exceeding the precision of the sample measurement.
P F O S A w as stable to 3 freeze-thaw cycles in report #005. In that report, P F O S A at approximately 1x and 20x LLO Q showed stability, a s did all the other analytes.
Precision data for sample analyses are good. Accuracy of controls analyses is within guidelines except for P F O S high le ve l, which is -1 5 .2 % .
(Source: report #122 Tables 15 - 2 8 ) PFOS PFOSA
% Deviation from Control
low -3
-18
medium -3
-20
_______ :______ high Sample, mean ng/mL
-2
-11
low 45.3 3.58
medium 164 120
______________ high. 369
353
A FT ER 7 FREEZE-THAW C Y C LES 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 51 ) P F O S A A F T E R 3 F R E E Z E -T H A W C Y C L E S
P F O S A low
high
% Deviation from Control
4 -2
Sample, mean ng/mL
21.1 369
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QAI Report No. 405-503
X IV . SH O R T -TER M A M B IEN T ST A B ILIT Y O F SE R U M S A M P L E S
Requirem ents were m et Serum sam p le s were kept at room temperature up to 6.75 hours per cycle during seven recurring freeze-thaw cycles (discussed above). AH were stable except P F O SA A , w hich exhibited 10 - 2 0 % 4 o sse s.-- - -
F D A G uideline
3 aliquots of biological matrix fortified at low and high concentrations
Store fortified sam ples at room temperature 4 - 2 4 hours.
N W B Report
Requirem ent m e t 3 concentrations approx. 1x, 4-40x and 10-100x LLO Q
Requirem ent m e t Fortified serum sam ples were frozen and thawed seven times. The maximum thaw time in a cycle w as 6.75 hours; the total time at room temperature is not reported.
R e su lts: Presented in the section above.
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XV. LO NG -TERM FR EE Z E R ST A B ILIT Y O F SE R U M S A M P L E S
A ll requirem ents w ere met. The re su lts dem onstrate that there is no degradation over the 55-day storage period (42 d a y s for two analytes) o f extracts at freezer tem perature -20 C.
F D A Guideline
NW B Report
At least 3 aliquots of biological matrix R equirem ent m e t 5 replicates of serum at fortified at low and high concentrations approximately 1x, 4x and 10x LLOQ .
Store fortified sam ples under the sam e conditions a s the study samples, for a time exceeding the time between the date of first sample collection and the date of last sample analysis.
Requirem ent m e t Stored at -20 C for:
42 days P F O SA and P F O SA A
55 days other analytes Stability w as determined by com parison to theoretical concentration.
R e su lts:
All analytes are stable under these storage conditions, within the accepted limits of precision of the measurements. Precision of all m easurem ents are <11% .
(Source: report #122 Tables 5 7 -6 3 ) S A M P L E S A F T E R 42 O R 55 D A Y S A T -20 C PFOS PFOSA PFOSAA POAA PFHS M-556 M570
Sample, % Change from theoretical
low -9
8
medium -14
0.0
high -15
-1
Sample, mean ng/mL
low 46.7 4.34
medium 170 150
high 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
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Q AI Report No. 405-503
XVT. PO ST-PREPA RATIV E A M B IEN T ST A B ILIT Y O F SE R U M EX T R A C T S
The requirements were met for ail analytes. The results dem onstrate that there is no degradation over the 7 day storage period of extracts at room temperature, - covering at least the resident-time in the autosam pler.
F D A Guideline No specific requirement
N W B Report
Requirem ent met. Serum extracts used; approx. 1x, 4-40x and 10-100x LLO Q
The stability of the drug and the internal standard should be a sse sse d over the anticipated run time
Requirem ent m e t Serum extracts stored 3 or 7 d ays at room temperature. Stability w as measured by com parison to previously analyzed or freshly prepared Q C samples.
R e su lts:
Two tests were conducted at different times. In the first, "standards and controls"were extracted and analyzed, then reinjected on the 4th day after extraction. Three Q C levels were included. In the second, low and high Q C sample extracts were stored for 7 days, then analyzed against freshly extracted Q C samples.
All analytes are stable under these conditions. Recoveries were all in the range of 1 0 0 % 11. W hile there is no separate data for the surrogate standard, the results presented at a minimum demonstrate that at worst, aH analytes and the surrogate degrade at the sam e rate, if any.
Sam ple precision is good, < 1 0 % C V in all cases. Control precision is good in the first test, but w as not reported for the second. Control accuracy is good, except for P F O S A and P F O SA A . In the first test, control values were < 8 5 % of theoretical due to a fortification error. However, the stability test is not affected by this, if both were fortified with the sam e solution. In the second test, accuracy could not be determined because the fortification level w as not reported.
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Q AI Report No. 405-503
(Source: report#122 Tables 29-35 ) E X T R A C T S S T O R E D 3 D A Y S, A M B IE N T PFOS PFOSA PFOSAA POAA PFHS M-556 M570
% Deviation from Control
low 1 -3 2
4
0.3 -2
-1
mid ........ .......-_..-hioh Sample, mean ng/MI
low mid high
-3 -3 -3
4
... - =5- . ... *4-- ____ 4
47.7 168 382
2.73 100 286
7.73 110 288
3.1 1.6 4.1
-0.6 -4
6.24 157 427
-1 -0.7
.. A ..... --3 -
5.52 145 381
8.2 146 390
(Source: report #122 Tables 36 - 42 ) E X T R A C T S S T O R E D 7 D A Y S , A M B IE N T PFOS PFOSA PFOSAA POAA PFHS M-556 M570
% Deviation from Control
moX1
low 3
8 11
-2
0.2 11
high Sample, mean ng/mL
3
7 12
2 0.2 3
9
low 48.0 3.37 8.89 7.68 6.14 5.43 8.91
high 391
334 334
378 404
379 420
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Q AI Report No. 405-503
X V IL P O S T -P R E P A R A T IV E 1-8C S T A B IL IT Y O F S E R U M E X T R A C T S
All requirements were m et The results dem onstrate that there is no degradation over the four day storage period of extracts at refrigerator temperature.
F D A Guideline
No specific requirement
The stability should be asse sse d over the anticipated storage time before injection.
NW B Report
Requirem ent m et
Requirem ent m e t Stored serum extracts were refrigerated at 1-8 C for 4 days. Stability w as m easured by com parison to freshly prepared Q C samples.
R e su lts:
All analytes are stable under these conditions. Sam ple precision is good. Control accuracy is good; however, P F O S A and P F O S A A accuracy in the Q C sam ples could not be evaluated. W hile there is no separate data for the surrogate standard, the results presented at a minimum demonstrate that, at worst, all analytes and the surrogate degrade at the sam e rate, if any.
(Source: Report #122 Tables 4 3 - 4 9 ) PFOS PFOSA
% Deviation from Control
low -0.2 -5
high 2
-2
Sample Precision (CV). % . low 5
8
________ high Sample, mean ng/mL
2
9
low 46.6 2.96
high 384
317
E X T R A C T S S T O R E D 4 D A Y S 1-8C PFOSAA POAA PFHS M-556 M570
2 6 1 16 4 4 4 2 10 3
5 1
8.22 310
6 2
8.31 387
10 3
6.96 411
11 0.8
6.29 402
9 1
8.36 397
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Q AI Report No. 405-503
X V m . P O S T -P R E P A R A T IV E -20C S T A B IL IT Y O F S E R U M E X T R A C T S
All requirem ents w ere m et The results dem onstrate that there is no degradation ove r the five day storage period o f extracts at freezer tem perature (-20 C).
F D A Guideline
No specific requirement
The stability should be asse sse d over the anticipated storage time before injection.
NW B Report
Requirem ent met. Two levels in triplicate.
Requirem ent m e t Stored serum extracts were stored at -20 C for 5 days. Stability w as measured by comparison to freshly prepared Q C samples.
R e su lts:
Ail analytes are stable under these conditions. While there is no separate data for the surrogate standard, the results presented at a minimum demonstrate that, at worst, all analytes and the surrogate degrade at the sam e rate, if any.
(Source: Report #122 Tables 50-56) E X T R A C T S S T O R E D 5 D A Y S at -20 C PFOS PFOSA PFOSAA POAA PFHS M-556 M570
% Deviation from Control
low -5 -2 -1
6
-0.7 -0.6
4
hiah -2
-5
-0.3 -0.5
2
0.8 -3
Sample Precision (CV)> %
low 7 4 5 21 12 14 4
____________ h!9h
2
33
5
2
33
Sample, mean ng/MI
low 44.5 3.08 7.91 8.30 6.81 5.39 8.30
high 371
307 296
370 409 370 374
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Q AI Report No. 406-503
X IX . ST O C K SO LU TIO N ST A BILIT Y
Requirem ent not m e t No testing reported.
F D A G uideline-------------
NW B Report
Store at room temperature for at least 6 hours
Requirement not met
Store at other temperatures and time periods as required.
Requirem ent not met
Report issued by:
Philippe Ourisson Q uality A sso cia tes, Inc. Manager, Scientific Support
Date
Validation report review
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3CZ
Hum an Serum at Northwest Bioanalytical
