Document peOvDDenxKGXwoyM3dbE0qRra
So
AR224-1907
Report Title
Evaluationofthe Northwest Bioanalytical Method Validation Reports for PFOS and Related Analytes in Human Serum or Plasma in Meeting the Requirementsofthe EDABioanalyticalMethod Validation
QAI Report No. 405-503
------
Data Requirement Full Validation of Bioanalytical Method
Author
Nancy Benson Philippe Ourisson
`QUALITYASSOCIATES, 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
Report Date
December 31, 2001
Report Number
QAI 405-503
Number of Pages
26
337
!
.
TABLE OF CONTENTS QAI Report No. 405-503
Me INTRODUCTION cosmos
Ul. SPECIFICITY IN SERUM AND PLASMA...
IV. CALIBRATION IN SERUM AND PLASMA...
V. ACCURACY IN SERUM ANALYSIS ....ccovrrssmsnssrirens
VI. ACCURACY IN PLASMAANALYSIS....cccorrrrrrsssrssns1s1
VII. ABSOLUTE RECOVERY IN SERUM...
2
Vill. ACCURACYAND POAA EFFECTS ON LS. QUANTITATION...........1.3
IX. ACCURACY OF SERUM QUANTITATION VS PLASMA CURVE......... 14
X. VALIDATION OF SERUM EXTRACT DILUTION PROCEDURE .......... 16
XL. PRECISION IN SERUM ANALYSES. ....cccvermrrsesrsnsinsises 16
XIl. PRECISION IN PLASMAANALYSES...
18
XIll. SERUM SAMPLE STABILITY TO FREEZE-AND-THAW......ccrcecc. 19
(
XIV. SHORT-TERM AMBIENT STABILITY OF SERUM SAMPLES ............20
XV. LONG-TERM FREEZER STABILITY OF SERUM SAMPLES...............21
XVI. POST-PREPARATIVE AMBIENT STABILITY OF SERUM
XVII. POST-PREPARATIVE 1-8C STABILITY OF SERUM EXTRACTS........24
XVIll. POST-PREPARATIVE -20C STABILITY OF SERUM EXTRACTS.......25.
XIX. STOCK SOLUTION STABILITY...
28
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L SUMMARY
"The validation in human serum or plasma was performedfor the analytes PFOS,
PpeFrOfSluAo,roPoFctOaSnAesAu,ipPhOonAiAc,acPiFdHwSa,sMa-5d5d6e,d abenfdorMe57e0xt.racTthieon,subrurtoguasteedsatsanadnaridntIeHr,naIlH,2H,2H-
standard in the quantitation-.
=
ere?
The following parameters can be considered ascompletelymeeting the FDA requirements:
+ Specificity
Calibration in serum and plasma
APrceccuirsaicoyn iinn sseerruumm aanndd ppllaassmmaa.
VSaelirduamtisoanmopflEextstraabcitliDtiyltuot:ion
o freeze-thaw. 7 cycles
shortterm ambient storage, 6.75 hrx 7 cycles
o freezer storage, 42 - 55 days
Serum extract stabilty to:
o o
aremfbriiegenrtatsetdorsatgoer,ag7e,da4ydsays
o frozen storage, 5 days
ThBeefcoalluoswienogfrepsotorirctainoanlsaypteplayntdointtheemsaelasntaalnydsaersd: extraction efficiencies, it is essential that samples, QCs and calibrations standards in asetbe kept together for all analyses. Extraction efficiency within a set is consistent;
Samples with levels of POAA above the ULOQ wil show LC/MS ionization suppression of 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
dmialturtiixonodiflustieorn,utmhaesretshueltdsilwuitllednostambpelceorfroercta,ccuracy.Ifthe sample is aat different When a sample must be diluted, itnot only must be comparedto a calibration curve
prepared at the same dilution of serum, but it also must be re-extracted for that analysis, inorderto keep samples, QCs, and calibration standards together.
The following parameters from the FDA requirements werenottested: SSttaboiclkitsyoolfuttihoen sstuarbirloigtya.te standard.
Quantiating serum samples against plasma calibration does not meet the FDA requirements for accuracy.
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QAI Report No. 405-503
! I. INTRODUCTION
Three reports were reviewed: + NWBR00-122: Quantitative Determination of POS, PFOSA, PFOSAA, POAA,
APdFHdSe,ndMu-m55R6epaonrdt,M1517/200i/n01Hu(smtaundySNeWrBuSm00b-y0L4C0/)MS/MS: Assay Revalidation + NPWFBHSR,OOM--150586:anQduaMnt5i7ta0tiinveHDuemtearnmiSneartuiomnboyf PLCFIOMSS,/PMFS:OSAAs,saPyFORSevAaAl,idaPtOiAonA,
Report, 1/24/01 (study NWBS00-040); NPFWHBSR,9SM--050556:anQduanMt5i7ta0tiivneHDuemtaernmiSneartuiomnobyf PLCF/OMSS,/PMFS:OSAAs,saPyFOVaSlAiAda,tiPoOnARAep,ort,
1124/01 (study NWBS88-082);
formal THPFOS The methods used in these studies are very similar with the following differences:
[Tw -- o -- s -- T#o0sA2
15)standard basfed wih 05 M
Unadjusted pH ca. 6.9
tertbutylammonium hydrogen
sulfate (pH 10) + 0.25 M carbonate
buffer
2mM ammonium acetate: methanol|20mM ammonium acetate In water
(1:1)
+20 mM ammonium acetate in
MeOH(3:
quantitation | quadratic regression, weighed Tv, | quadratic regression, weightd
using area ratio vs. concentration | c1o%n,cenutsirnagtiaornea ratio vs.
TthhiesstraenvideawrdesxdaemsicnreisbewdhibcyhoFfDtAhfeorFaDAf-urlevqauliirdaetdiotnesotfstwheermeectohnoddu.ctTehdi,sarnedpowrhtidcihd nmoetet consider whether additional requirements, specified by protocol, were metif they did not tahffeetctexttheoefvtahilsuaretpioornt.based on the FDA guideline; however,they may be discussed in
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4 Human Serumat Northwest Bioanalytical 340
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QAI Report No. 405-503
IL SPECIE
This requiremeinst met. Endogenous levelswere found in all serum and plasma
samples tested, although much greater in serum.
--- we
[_ FDAGuidelne ~~ [ ~~ NWBReport
|
> Six sourcesofblank biological matrix|Requirement met. Testing was reported on 2
Measure levels of each analyte
a
Results:
sInerreupmorwte#r0e05d,eteenrdmoigneednouussinlgervaeblbsiotfsPeFrOuSm,caPliFbOraStAi,on;PFPOFSOASAA,wPaOsAnAotadnedtePctFeHdS. in human
[Sees pot#osbr=5
[PFSMe|anPnFgO/SmALAin|HPumOaAn|SePruFmHS |
[Run25calibrationstandards (n=4)
| 162| 146 | 320] 0615]
Run 26/28/29 calibration standards (n=1 [304 1.69| 427 161|
i [Run26/28/29QCsamples(n="1T2h)e LLOQ inrabb|its2er2u3mf|or e1a.ch8a0n|alytew1a.s917.]00325ng/|mL.
Inreport#108, serum results indicated significant endogenous levels of all analytes except
PFOSA. These levels are higher than in report #005.
[evo vs | 77| Wo | 50 | wre 2s| 93 | 40| Sowa: report#108tables 8-14 3eport 22tables 8 -
hid
Mean ng/mL in Human Serum
[iaettioa | 10 70| 10 | wo[46[10[0]
caltration LLOQ" |481|10[ 60 |576[315|43 |56|
Calibration was with human plasma
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QAI Report No. 405-503
, Pooled plasma also showed significant endogenous PFOS, PFOSA, POA and PFHS, bpultasatmaleaatst1 5-xl1e0s0stnhgainmL.in sTehreuem.ndTohgeenloeuveslslewveelreisddeetfeirnmeidnaesdbtyhestmaenadnaryd-snptreerpcaeroepdtfin
linear regressions performed on2 separate tests.
[edrommrines| 231| | om |om|ow| | 0] --
i
Moan ng/mL In HumanPlasma
a Sp ze ll ed el ll ed il
[aoetiioa |100| foo[ 100 | 1.00]1.00|250] 100] [catbrationLLOQ"|304[1.00 160|Cal1ib9rat2io]n w1a.s3w6i|th h2um5a0n]pl1a0sm0a.]
(
|
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IV. CALIBRATIONINS!
MA
cRoemqpuliirceamteendtcmaelti.braTthieorneprisocheodwuervee(rqnuaodjruasttiicfiwciatthio1nf4orotrhe1/sxewleeicgthitoinnogf).the
[FDA| GuidNWe BRepl ort ine|
Blank + 6 8 samples
Requirement met.
2 blanks and 2 blanks + 1S wers included in each un.
No blank analyseswere reported.
Samples: 8 Levels for M-556; 9 levels for all others.
Duplicate samples. 3 runs for serum and 2 for plasma.
LLOQ 5x blank response | Requirement met. Endogenous responses caused SLuLmOmQaraipzpreodxiinmattaeblley b=e"lbolwa.nk" in most cases.
+20%ofactual @LLOQ | All pass, exceptone serum POAA.
+ 16% of actual >LLOQ Passed,of 550total:
:
550 for PFHS
549 for PFOS
554478 ffoorr MP-F5O5S6A,,MP5F5O7SAA, POAA
24 of6 standards met these| All runs pass.
criteria in each curve
LloLwOeQst:stAanndeaxrpderiinmjeenctteadl wdheitcehrmmienatttihoenoafctcheeptaannaclyeticcriatlerliiamiwtsawsacshonsotenpearsfotrhmeeLdL.OQT.he SInimnocestthceascaelsi,brtahteiosne LstLaOnQdairsdocnolnycselniighrtaltyi(o1ns-4wetriemecsl)osheigthoertthheaenndtohgeeennoduosgelenvoeluss. t(hbleaunnkc)elretvaeilnttyhoefumneacseurretmoeafnsitoanmfpttlhyee emnedaosguerneomuesntlsevaelt.the LLOQ will be increased by sBheocualudsbeethueseedndtoogpernepoaurselceavleilbsraatrieonloswtearndianrpdlsawshmaenthaann LinLsOeQrubme,lopwla5s.m0anbgl/amnLksis
needed.
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7 Human Serum at Northwest Bioanalytical
:
+
QAI Report No. 405-503
Range:
'
[CC
VALIDATED RANGE (ng/mL)
TPFOS [PFOSA|PFOSAA|POAA|PFHS|M-566 [M670 |
`Serum (reports #108/122)
|Sewmitoa T48] 40 [60 | 58] 32]43] 56] SeumuL0Q [540[800 [500 [500 [500 [500 [500|
Plasma (report #122)
[Pasmalioa|39 10| 16 |19]14] 25] 10] PlasmaUlOQ [410 [500 [500 [480 [520[470 [480|
CaQluaidbrraattiicornemgerteshsoidon:with 11x weighting,ofpeak area ratio (sa/msuprrlogaete jstuasntdairfdi)cvsfa.otrniotohmeninsaelleccotnicoennotfrtahtiisonc.om(p1l/icwaetiegdhtcianlgibrinatrieopnormtet#h0o05d).. There is no
`Inacaclul rsaectys,rethqeurierewmeernet aotf le1as6t%fooufraocuttuaolf(soirx 2ca0li%braotfioanctsutaalnadtatrhdeleLvLeOlsQ)t.hatmetthe
(
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:
V. ACCIU N SER RUMA ANAC LYSY IS
The FDA requirements for thistestwere met.
[ FDAGuideline
[|
NWBRepot
|
5 replicates @ 3 concentrations inthe | 2 "ePicates, each on 3 separate days
expected rangeofconcentrations.
Levels:
Levels:
<3 LLOQ;
LLOQ (4.0- 51.1 ngimL; 1.1 - 4)
anteatrhethmeiduipepveelr ;boundoaftrhye standard | mhiidglhe(v4e0l0(-145500-2n0g0/mnlg)/ml)
curve
Mean values + 20% of actual @ LLOQ Mean values + 15%of actual >LLOQ
| All passed except PFOSAA (intraday) All passed.
Results:
`Summary tables presented below, with failed results bolded. fIanultlhye. inTithiealftoersttfoyifn3g sseotlsu,tiPoFn OwaSsAparnedpaPrFedOSfrAoAm fdaiilluetde bsetoccakussbeytheevfaoprtoirfaitciatoino,nsanwdetrheese tcownotaainnailnygtejsuswtetrheepsa2retiaalnlaylylotsets., Tahdedeidntirna-cdoanycednaitrabateeldowswolautsionosb.taAicnceudrfarcoymisnetthi1s7set `was unacceptable at the low level for PFOSAA, and at the high level for PFOSA. tIhnitsefroduarytQh Csetd.atAatwoatasloofbftiafitneeedn fsertosmaaremorneiptoorrtiendgfsotruPdFy OanSaAlyaznedd PinFtOhSeAwAe,ekwhsifcohllnoowting ornelqyuivraelimdeantte(t5hoeuitntoefr9d0ayraescuclutrsaocuytsriedqeuitrheemelinmti,tsbfuotr aPlFsOo SvaAl,idaanted t7heouitntorfa-8d0ayfoarccuracy
PFOSAA).
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;
Source: report #108 (tables 15-30%anAdccAuppr.acAy from Fortified Blank Serum Samples
[___ols_, n_g_/mTLPFOS[PFOSA[PFOSAATPOAA|PFHS|M556 |W670|
Low
511 | 400 900 | 874
5.80 8.60
Mid
197 | 150 155 154
High
446 | 400 405 403
"152 | 155| 402 | 405
ow level
Run 13
2
103 | 111 | 108 | o7
Run 15
92
93 | 101 97 | 97
Run 16 91 Interday 92
9| 104 | 105 | 104 95| 105 | 103 | 99
id-level
Run 13 Run 15
92 9 88 -
108 102 | 110 | 108 | 104 - 94 | 104 97 | 95
Run 16 Interd
85 ~ 88 9
-~ 100
93| 100 o7| 94 %| 105 | 101 | 98
[Figh level
Run 13 Run 15
89 82 88 -
02 101 | 110 | 103 | 98 - 9%| 1m 98| 99
(
Run16 | 85
Interd
87
-
~-
1] 108
9%| 105 99 | 96 98| 108 | 100 | 7
INWBS00-062. They include 15 runs of3 concentrations and 2 replicates each.
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10 Human Serum at Northwest Bioanalytical
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QAI Report No. 406-503
! VI. ACCINUPLARSMA AANC ALYY SIS
Requirement met.
replicates @ 3 concentrations in the | 5 replicates @ 4 concentrations levels:
`expected rangeofconcentrations:
Loa
<3LLOQ;
low (4.06.4 ngimL)
standarddocwuervleboundaryofthe
Ve2r05 na59
Mean values + 15%ofactual >LLOQ
igh 822 miigdlevel (-14264-1 ) 1n5g/0mnLg)/mL.)
All passed, except PFOSAand M570
atthe low level.
Results:
(
Summary tables presented below, with failed results bolded. While thelow level for
-
PPFOSA and M570 failed, at
analytes. All results pass.
116%
and
118%
respectively,
three
levels
passed
for
all
Source: report#122 (tables
[ ovels, n_ g_ imL[P_ FOS
Loa
4.0
Low
6.4
Mid
126
High
332
LoQ
98
ow level
112
Mid-level
106
High level
101
% Accuracy from Fortified Blank Plasma Samples 85-98)
[PFOSA| PFOSAA|POAA|PFHS|N-556 |M570|
1.0 4.0 150 400
101 116
107 104
16 4.6 151 401
98 12 103 104
19 48 145 385
o1 104 106 103
14 4.5 156 417
95 106 101 9%
25 40 150 441
98 108 99 111
1.0 4.0 150 400
103 118 107 101
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`QAI Report No. 405-503
VIL ABSOLUTE RECOVERY IN SERUM
Requirementmetwith 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 consistentwithin a set.
[ FoAGuideline
| NWBRepori |
3 concentrations -- low, medium, high --in | 2 levels (4 & 400 ng/mL) in serum;
biological matrix
4 replicates, in 1 set.
`Compare to unextracted standards
Recovery should be consistent, precise
and reproducible (limits not specified). `Accuracy not required.
Compared with blank extracts fortified with known concentrations.
| Extraction efficiencies are poor (15
-- 70%) for analytes, worse (6%) for internal standard, but consistent `within a set.
Design: Analytes and intemal standards were addedeitherto serumorto serum
extractot d
comparing
etermine
the peak
losses during extraction. The loss
area ratioof(the one added before
calculations
extraction :
were ba
the one
sed on
added
afte
r
extraction) to the comparable peak arearatiowhenbothwere added after extraction.
i
"The endogenous levels were includedinthecalculated efficiencies (levels not reported).
Results: All analytes showedlosses during extraction. These weresimilar at low and high levels. PFOS showed considerablyhigherloss at the low level, but this is probably an artifactof measurement, with an endogenous level on the orderof4x the added 4 ng/mL.
Extraction losses are not a concerforthis method, so long as they areconsistentfor samples within an extraction set. Since samples are extracted along with the calibration standards, as long as they are kept together in analysis, the relative quantitation will be unaffected. Losses in the internal standard are a greater concern, but, at least within a set, they are consistent. There is no inter-set data.
(F -- [ m(Souarce-- :sRepor-- t #1--22 T---- arb@loe--ssT[64-- P-- -7r0 )os -- [v%& rm | oMsE-- aA]N PEoXwaT-- vRaA[CTw PIr-- OiNs|EFFmm I-- sCIsEN|CYw-- *ewun) I TensA[700 | T01404O0T| 50
[over [ a 59l ] 5i 8](6m 3]e6a 4] n 636 ] 0 55% ] 6) 1]
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12 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
i
VIII. ACCURACYANDPOAAEFFECTS ON .S.QUANTITATION
Restriction on Accuracy: fSoarmoptlheesr waniatlhyltoevse,lsduoef PtoOLACA/aMbSocvoeeltuhteioUnLaOnQdwiiolnliszahtoiwonashuipgphreqsusanitointaotfitohnebias cinotmeprnlaeltsltyanpdraorvdenT.HPThFiOsSm.aTyhaeffseccotpreeosfuitthsiosbtefafiencetdisbyprdeisleunttieond,anbudtrneoantalysis.
[_FDA|Guid NWSe Repol rt ine|
High concentrationof POAA >500 ppb depresses IS Hreoswpeovnesre,,tbhieatseisntg hotahsertoaonamlaynteysutnoktnhoewhnisghtosiadlel.ow many conclusions to be drawn.
Design: resolved
iTnhLeC/inMtSe.maTlhsutsa,ndPaOrdAATHatPFhiOgSh
and the levels in
asnaamlpylteesPsOuApApraerseseisnctohmeplTeHtePlFyOS
QreCspsoanmsep.leTshwiesrceaufosretisfiaedhwiigthhbhiiagshilneavnealslyotfePqOuaAnAt,itaatnidontbhey apneaalkytaerseqauaranttiiof,ieLdoawndlevel
`compared to quantitation before fortification with POAA...
i
Results:
qTuhaentdiattataisonhoiswstlihgahttw(h-2etnof-or1t1i%f)ie.d tHooawpepvreorx,imaatte1l0y- t1h0e0UxLthOisQlleevveell,,tthheebcihasansgteeadiinly increases, in proportion to the decrease in IS peak area.
(Source: Report #1p2.226
%ChafronmQCgMeean
POAA Added, approx. ng/mL] 1SArea |AnalyFotuneds|
sel wii sti |
MisiT ee0o[ 3 43 [|etsetorvse ||
CL [QCconc. TPFOS
so00| 75 | is2tov2te | PFOSA |PFOSAA| PFAS |mss | wero |
lngml T7518 | 405 | 685 | sos | sa1 | 687 ]
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13 Human Serum at Northwest Bioanalytical
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QAI Report No. 405-503
} IX. ACCURA ERUM [TATION VS PLASMA CURVI
Does not meet FDA guidsline for accuracy.
[Fo| AGuWWE BRms er ne|
Serum and plasma calibration standards. Serum and plasma QC's at 3 levels, 3-4 replicates,
2 sets of each matrix
Serum QC's analyzed vith both calbrations
SloewreurmenQdCogseanmopulseslewveerlse oafnsaloymzeedanaaglayitnesst tphlaansmdaoecsalsiberrautmio.n,Thbeecraesuusletspwlearsemarehpaosrted as % deviation from theoretical concentration.
Results;
"tThheisFmDeAthroedqouifrequmaennttistfaotirnagcsceurraucmy.saMmopslteosfatghaeinPstFOpSlAas,mPaFcOalSiAbrAa,tioPnOdAoAesannodtPmFeHetS results are > 16%oftheoretical.
(Source: Report 122, Tables 141) SERUM QC vs. PLASMA CURVE
[ Accuracy, 1 % DeviaPtFioOnSf|roPmFtOheSorAe[fiPcFalOSAA|POAA|PFHS|M-556 |M570|
[low] 11T &7 | 1a[94 [ 26 T 10 | 9|
[mid[ 92 | 40 | 81 [20[2 | 7| 15|
[hgh 6| 32 | 49 | 2a|2 [ 41 | 9|
[Precision,%cv
1]
[ ow[ 8[7[4 [ 0 [ 10 T 0 [ 4 |
md 7 | 83 [3[5[41419|
[high 6| &| 5| 3T7828}
[ #ofmns| 2[7[1[2| 7| 1[2|
[C~ evels,ngmt ~~|
[____ fow[ 393] 400] 720] 954] B41] 530] 6.70]
[mid 61 | 450 [ 65 | 14s | 32 | Asi | 53 |
[CC high| 370 | 400| 4035 | 370 | 345 | of | 403 |
A`Alcccaulriabcryatainodn spertescipsaiosnseodf ptlheasrmeaquainrdemsenetrsu,maQlCt'hosuagghaioncsctatsihoenaslarmeeplmiactatreisx fcaaillbedr.ation met requirements, iththe exception that PFOSAA/ plasma precision at the low level
w1a6s%a1t6t%heCVloawnldevaelc.cuTrhaecyalotwthleevemlisdwelerveelawbaovset17h%e,LaLnOdQ.POAA / serumaccuracywas
Validation report review
14 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
X. VALIDATION OF SER
DILUTION PROCEDURE
TThhiescraelqiburiarteimoennctuwrvaesmmuest,twbiethprreesptarriectdiaont:the same dilutionofserum as the diluted sample for accuracy. If the sample is ata different matrix dilution, the results will not be correct.
[[ FDAGuidene ____[
NWBReport
|
"abTohveeatbihleityutpopedirlultiemitsoafmptlheesstoaringdianradlly|| Rcoenqtuaiirneamernetstrmiectt,ibonuotnthdeilmuteitohnoadnamlyussits.
`caucrcvueraschyoualnddbperedceimsoionnstpraartaemdetbeyrs in | 360i:" 4 replicates; 1 set tdhileutviaolnisdasthioonu.l"d*uAsney lreiqeumiarterdixs"ample | mid; high; 10x high
aDensdiagnna:lySzeerduamgaQiCnssatamcpalliebsrwaetiroendciulruvteedpr1e:p1a0reord 1i:n5100wi%tshearmummoinn ibuufmfear.cetIatitsekbnufofwern, that quantitating against a whole serum curve is not accurate.
Results:
{
The datashowthat accuracy requires that the proportion of matrix in the sample and
calibration curve extracts must be similar. All 1:10 dilutions had acceptable (> +15%)
accuracy, but the 1:50 dilution was biased unacceptably low (--585%7). Intra-assay
precision was excellent (<7% CV) in all measurements.
Source: Report 122, Tables 113-119)
[____T PFOS|PFOSA|PFOSAATPOAA|PFHS|M586|M5670 |
[P~ recisionicy) ~|
[m T&T d T52a 412
[CC hgh| 5 176[4[3T 4[4 4|
[_Aoxhigh| 2| 2 | 2[7 2[2 2]
foxhigh(i60)| 2 | 4 | 4|4 35 | 414]
[Accuracy%recovery
|
[md] 108 [66 T 94 [ 06 | 105 | 701 | 103|
[ high| {03 | 66 | 65 | foo | {02 | {fo [ 103 |
[___A0xhigh| 07 | 86 [400 | #05 | dor | fia | q08 |
Kamae] wv| @ | 8 |8|#0 8]
[-- mid[ 163 T 7480 [ 63 T 157 [ 54 | 151[ ds7| [ high| 443 | 400|405 | 407 |404 |" dof | dor | 10x high| 4040 |4000 |4000 |4010 | 4000 |4000 |4070 |
Validation report review
15 Human Serum at Northwest Bioanalytical 3s5/
QAI Report No. 405-503
{
XI. PREICN SI ERUSM AI NAO LYSN ES
The FDArequirementswere met.
[ FoAGudeine
| NWBReprt |
Procision - ntrabatch /Intorbatch) |Requirementmet|
CV20% @LLOQ
ALlLOpQa,ssweidt,he2x4ce%pCtV1.of 4 PFOSAAsetsat 1.5x
cv 15% @>LL0Q
ALlLlOpQa,ssweidt,he1x9c.e8pt%C1Vo.f 4 PFOSAsetsat4x
Results;
SexucmemptairoynotfabPleFsOpSrAesaenndtePdFbOelSoAw.A iAnlloannealsyetteesapcahsastetdheatlaollwcloenvceel.ntrations, with the
fIonrtthifeiciantitiioanlswtereoeff3asusletttys;,aPsFeO4tSAconatnadinPsFjOusSttAhAeshead2laonwalryetceosv.erBioetshbaencaaluysteesthhea.ve at ( least three setsateach levels meeting the inra-day requirements.
Interday QC datawas also obtained from a monitoring study analyzed later.
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QAI Report No. 405-503
.t
Pracision (% CV) from Fortified Blank Serum Samples. Source:report #108 (tables 15-30 and app. A)
[|Pros[PFOSA[PFOSAATPOAA|PFHS|M-556 |M570|
[Levels, ng/mL
Low
51.1
900| 874 615| 580] 860
Mid
197
155 154 | 152 | 152 | 155
-
High
448 00 405 403 | 402| 402| 405
"ow level Set13
5
6
10
4
Set 15 Set 16
2
2
6
20
11
9
7 9
Set 17
8
24
Interday1
14
8
6
70
Intorday2
11
10
id-level Set 13
3
4
3
Set15 Set 16
5
4
4
4
1
4
Set 17 Interday1
9
6
5
6
Interday
8
{
igh level
-
Set 13
4
8
75
SSeett 1156
6
4
3
4
3
3
44
Set17
2
5
Interday1
7
6
54
Interda
10
7
:
linterday1isthe meanofsets 13,15, and 16. lInterday2 PFOSA/ PFOSAA data are from study NWBS00-062, a monitoring study.
istopresents GC sampledatafrom 15 runsof 3 concentrations and 2 replicates
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!
XI. CISION IN
IALY:
EiThe FDA requirements for thistestwere met.
interbatch)
QAI Report No. 405-503
Results:
`Summary tables presented below. All analytes passed at all concentrations,
Precision (% CV) from Fortified Blank Plasma Samples
Source:report #122 (tab85l-e9s8
[P| FOS[PFOSATPFOSAA
PFHS
M570
640| 400| 460 | 481| 448
4.00
(
g
132362 || 410500 || 410511
145 | 158 385 | 417
150 400
.0Q
5
6
4
8
ow level
3
id-level
3
5
6
65
85
igh level
2
3
1
7
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; XIIISERUMSAMPLESTABILITYTOFREEZE-AND-THAW A`ltlharwecqyucilreesm,eenxtcsewpetrPeFmOeSt.AAInwsheircuhmw,aasllsatnaabllyetetso wtherreoesctyacbllees.toAscecvuernacfyreaezned| precision were generally acceptable.
[__"DA| GudNWe BRel pori t ne|
Two concentrations in triplicate.
Requirement met. 3 concentrations in serum,
`approx. 1x, 4-40x and 10-100x LLOQ
Aafntaerlyttheresetafbrileietzyeshaonudldthbaewdceytcelremsi.ned|| Rseevqeunirceycmleens.t met. Analyzed after three and
RSeesvuelntst:imeSserbeufmorseamanpalleyssifso.rtiIfnieedaacth1txh,a4wxinagn,dth1e0xsaLmLpOleQswreermeaifrnoezdenatarnodotmhawed temperature up to 6.75 hours. Losses are measured by comparing the sample analysis
results to the corresponding control analysis.
Allolsasneasloyfte1s1 e--xc2e0p%t,PcFonOsSidAewraebrleysetxacbeleedtiontghetshee pcroencdiistiioonnos.ftPheFOsSamApdlaetameiansdiucraetmeent. { aPpFpOroSxAimwaatselsyt1axblaentdo 230fxreLeLzeO-Qthsahwocwyecdlesstainbirletyp,orats#0di0d5.alItnhtehaotthreerpoarnta,lyPtFesO.SA at
Pguriedceilsiinoensdeaxtcaefpotr sfoarmPpFleOSanhailgyhselsevaerle, wgohoidc.h iAsc~c1u5r.a2c%y.ofcontrols analyses is within
(Source: report #122 Tables 15 - 28
AFTER 7 FREEZE-THAW CYCLES
[ PFOS[PFOSA[PF| OSAA] POAA|PFHS |M356| Mero|
fw|[ 1 8[5[71 ST 714] [ medum| 8| 20 | 5| 1[4 5[51] [_-- hgn]l 2[ -11| -5 | oo] off 03] 51
[l 453] o 358] 046] w 838] 648]] 50d] B32]
[ [CC
medum|164 | 120| 13 | high] 369| 353 | 302 |
51| 312|
diez| 406|
a4 | 368 |
42 | 366|
(Source: report#005 Table 51) PFOSA AFTER 3 FREEZE-THAW CYCLES
[__
PFOSATfw T Wah |
[% Deviation fromControl | 4| 2 |
(Sample, meanngiml | 21.1| 369 |
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!
XIV. SHORT-TERM AMBIENT STABILITY OF SERUM SAMPLES
Requirements were met. Serum samples were kept at room temperature up to 6a.b7o5veh)o.urAsllpwearrecysctlaebdluereixncgospetvPeFnOrSeAcuAr,riwnhgifcrheeezxeh-itbhitaewdc1y0cl-e2s0(%dilsocsussesse.d ~~ ~
3 aliquotsofbiological matrix fortified at low and high concentrations Store fortified samples at room
temperature 4 --24 hours.
| Requirement met. 3 concentrations -- `approx. 1x, 4-40x and 10-100x LLOQ Requirement met. Fortified serum samples wmearxeifmruomzetnhaawndtitmehawienda sceycvleenwtaisme6s..75Thheours; the total time at room temperature is not reported.
Results: Presented inthesection above.
-
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20 Human Serum at Northwest Bioanalytical 356
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i
XV. LONG-]
'REEZER STABILITY OF S|
PLES
Aolvlerretqhueir5e5m-ednatysstwoerraegmeepte.rioTdh(e42redsauylstsfodretmwoonsatnraaltyetetsh)aotftehxetrreaicstnsaotdfergoreazdeartion temperature -20 C.
`fAotrltefiaesdt a3talloiwquaontsdohfibgiholcoognicceanltmraattriioxns||aRpepqruoixrimeamteenltym1ex,t.4x5 arnedpl1i0caxteLsLoOfQs.erum at sStaomree fcoorntidfiiteidosnsamapsltehseusntduedrythe. Requirement met. Storedat-20 C for: sbaemtpwleeesn,tfhoer adattieomefefxrcsetesdaimnpgltehe time|4525 d4a2yy8s PotFhOerSaAnaalnydtePsFOSAA canoalllyescitsi.on and the date of last sample |tshaepoirlettiycwalacsodnceetnerramtiinoend.by comparison to
Results: Aplrlecainsailoyntoefstahree msteaabslueruenmdeenrtsth.ePsreecsitsoiroangoefcaolnldimteiaonssu,rweimtehinntsthaeraec<c1e1pt%e.d limits of
{
(Source: report #122 Tables 57-63) _ SAMPLES AFTER 42 OR 65 DAYS AT 20C
[ PrOS ]PFOSA[PFI OSAA] POAA |PFHS |M856 |Wsm |
(Sample,meanagml_ [o oT"8 w ai [0 4l [71771
[ medum[ -14 | 00 3[ 318 7[ 4| [h 5| 1[g 2 [| 8 0h 2 015]
[
low[ 467] 434] 095]782] 686] 542] 802]
[ medum| 170 | 150 | 160| 140|164| 141| 149|
[C
high]378| 395 |413| 372| 403 | 367 | 388|
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Human Serum at Northwest Bioanalytical
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QAI Report No. 405-503
XVI. T-PREPARATIVE AMBIE!
ILITY.
RACTS
Tnhoedregerqaudiarteimoennotvsewretrheem7edtafyorstalolraangaelpyetresi.odTohfeexrtesrualcttssdaetmroonosmtrtaetmeptehraatttuhreer,e- is-
.
coveringat least the residenttime in the autosampler.
[[___F| OAGuiNWd BRe epol rtine|
No specific requirement
Rapepqruoixr.e1mxe,n4t-4m0ext.anSde1r0u-m10e0xtxraLcLtOs Qused;
Tinhteemstaalbisltitaynodfartdheshdoruulgdabned the R3e0qru7idraeymseanttrmoeot.m tSeemrpuermatexutrrea.ctSstasbtiolriteyd
assessedoverthe anticipated run time
|wpraesvimoeusalsyuraneadlbyzyecdoomrpafrreissholny ptorepared
QC samples.
Results:
Texwtoratcetsetdsawnedreancaolnydzuecdt,etdheatndriefifnejreecnttetdiomnest.heIn4t"hedafiyrastf,t"ersteaxntdraarctdisona.ndTchorneteroQlsC" wleevreels
{
twheerneainnacllyudzeedd.agIanitnhsetfsreecsohnldy,elxotwraacnteddhQigCh sQaCmpslaems.ple extracts were stored fo7r days,
All analytes are stable under these conditions. Recoveries were allintherangeof p1r0e0s%ent1e1d.atWhaimlientihmeruemisdenmoosnesptarraatteethdaatt,aatfowrotrhset,suarlraongaaltyetsetsanadnadrdt,hethseurrreosgualttes. degrade at the same rate,if any.
`teSsatm,pbluet pwraescisniootnirsepgoortoedd,fo<r1t0h%e CseVcoinnda.ll cCaosnetsr.olCaocncturroalcpyreicsigsoioodn,iesxgcoeopdtifnotrhePfFirOsStA faonrtdifPicFaOtiSoAn Aer.rorI.n tHhoewfeivrsetrt,estt,hceonsttraobllvyaltueesst iwsenroet <af8f5ec%toefdtbheyotrheitsiicfablodthuewteoreafortified with the same solution. In the second test, accuracy could not be determined because
the fortification level was not reported.
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355
Human Serum at Northwest Bioanalytical
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QAI Report No. 405-503
{ [Co(Source: "1 report#122 TabPlresos20|-3P5FoSA E|XPTFROSAACAT|SPSOTAOARE|DP3HDSAYS|,MaAMsBeI|ENwTeno |
pe
mWaT wTTa3T5z[ T4 a eesela 11 ior]]
: [memere nen Tae rr] ziL]a77l] aaiT] a emLosamL[osaa f
[ honm ! desesp|| 102m0 || 21d 1s0 || aiiel146577] | [ 14s5t| [ 37406| ]
[Co(Source: report #122T1 arbos3l6[eP-r4s2o)SAE|XPTrRoAsCT|SPS OATA |OPR 7FDE SAYD |S,WaAMsBsI|ENwTera|
WT els 3T1s7[w7l[221[of|l o5as ]7ov]]
[Sample,meanngmt
|
[l 480] o 337] 889] w 768] 6.14]] 543] 881]
[CC
high| 391 | 334|334 | 378 | 404| 379| 420|
(
i
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QAI Report No. 405-503
' XVIL POST-P1:8RCSETABPILIATYORFSAERUTMEIXTVRACETS
Aolvlerretqhueifroeumrendtasywsetroeramgeet.periTohdeofreesxutlrtascdtesmaotnrsetfrraigteertathoartttehmepreeriastnuroed.egradation
oe en [poproent| FDA Guidelin[ e NwBReport
|
The stability should be assessed over the anticipated storage time
before injection.
Requirement met. Stored serum extracts
were refrigerated at 1-8 C fo4r days.
Stability was measured by comparison to
freshly prepared QC samples.
Results:
All analytes are stable under these conditions. Sample precision is good. Control accuracy is good; however, PFOSA and PFOSAA accuracy in the QC samples could notbe evaluated. While there is no separate dataforthe surrogate standard, the results presented at a minimum demonstrate that, at worst, all analytes and the
sumogate degrade at the same rate, if any.
!
(Source: Report #122 Tables 43 - 49)
EXTRACTS STORED 4 DAYS 1-8C
[ PFoS [FFOSATPRO1 SAA[POAA|PFHS |W556|W&T0|
[ Cm o 2021[2S1T7 a w |412il ] ] l0 4 3 [S( ampleprecision 3 H| B J |
ml 21 el11 213| os 1]
C heno s#e8s][s127s[]3B1w 0m|]3s8a7i[] aens] s|[ 640m2 |3873|
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24 Human Serum at Northwest Bioanalytical
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QAI Report No. 405-503
XVITLPOST-P-2R0CSETABPILIATY ORFSAERT UMEIXTV RACETS
All requirements were met. The results demonstrate that there is no degradation over the five day storage periodofextracts at freezer temperature (-20 C).
[_FDAGudeline
No specific requirement
| ___NWBRepor
|
Requirement met. Two levels in triplicate.
The stabllty should be assessed | Requirement met. Stored serum extracts
over the anticipated storage time | were stored at -20 C for 5 days. Stabilty
before injection.
was measured by comparison to freshly
prepared QC samples.
Results:
All analytes are stable under these conditions. While there is no separate dataforthe asnuarlryotgeasteasntdatnhdearsdu,rrtohgearteesudletgsrpardeesaetnttehdeastaammeirnaitem,uifmadneym.onstrate that,atworst, all
(Source: Report#122 Tables 50-56) EXTRACTS STORED 5 DAYS at 20 C
[_____ TPFOS[PFOSA[PFOSAA]POAA|PFHS |M555|Wer |
i
CC
ew[ 5T 2TT 6 07] 06] 41
[ [ Samplh e2 P[ r5 ec | i g 0s 3]i0o5]n2(h ] cv0) 8],3%]
[
dew[7 aT 5Ta a[ 4 4]
Sample,meannhgglh 213137 5]2]313]
[
low[ #457 308]76 830] 681] 539] 830]
high] 371 | 307 | 206 | 370 |400 | 370| 374|
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fo
)
QAI Report No. 405-503
f XIX. ST
u
Lr
}
Requirement not met. No testing reported.
FDAGuideline ------
oe
lSetaosrte6athoruorosm temperature for at tSitmoerepeatriootdhserastermepqeuirraetdu.res and
|Requirement not met
Report issued by:
(
QPhUiAlLiIpTpYeAOSuSrOiCsIsAoTnES, ING.
Date
Manager, Scientific Support
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B