Document qkk4wen2pby6z8NY0rpa7nKrx
Report Title Evaluationofthe Northwest BioanalyticalMethod Validation Reports for
PFOSandRinelMaeteetdiAnngatlhyeteRseiqnuiHruemmeanntSseorfutmheorPlasma EDABioanalyticalMethod Validation
QAI Report No. 405-503
_
Data Requirement
Full Validation of Bioanalytical Method
Author
-
Nancy Benson Philippe Ourisson QUALITY ASSOCIATES, INC. 9017 Red Branch Road, Suits 102 Columbia, MD 21045
(
Sponsor
3M Environmental Laboratories 935 Bush Avenue Bldg 2-36-08 St Paul, MN 55106
Report Date
December 31, 2001
Report Number
QAI 405-503
Number of Pages 2
.
QAIRepNo.o40r5-5t03
TAOFBCONLTENETS
cM
W.
IV.
V.
VI.
VIL.
Vill.
IX.
X.
XI.
XIl.
Xill.
(
XIV.
XV.
XVI.
INTRODUCTION css
SPECIFICITY IN SERUM AND PLASMA... oreo
.
CALIBRATION IN SERUMAND PLASMA...
T
ACC INSU ERUMR ANAA LYSC IS..Y .
ACCURACY INPLASMAANALYSIS... 11
ABSOLUTE RECOVERY IN SERUM...
12.
ACCURACYAND POAA EFFECTS ON L.S. QUANTITATION............13
ACCURACY OF SERUM QUANTITATION VS PLASMA CURVE......... 14
VALIDATION OF SERUM EXTRACT DILUTION PROCEDURE .......... 15
PRECISION INSERUMANALYSES...
16
PRECISION INPLASMAANALYSES... 18
SERUM SAMPLE STABILITY TO FREEZE-AND-THAW..........co. 19
SHORT-TERM AMBIENT STABILITY OF SERUM SAMPLES ............20
LONG-TERM FREEZER STABILITY OF SERUMSAMPLES..............21
POST-PREPARATIVE AMBIENT STABILITY OF SERUM
XVII. POST-PREPARATIVE 1-8C STABILITY OF SERUM EXTRACTS........24 XVII. POST-PREPARATIVE -20C STABILITY OF SERUM EXTRACTS....... 25
XIX. STOCKSOLUTIONSTABILITY .....ocooocccvrrrrriseensrrsssssssssssssssssssssssss2e6e8s.
Validationreportreview
2 Human SeatrNoruthwmest Bioanalytical
QAI Report No. 405-503
LI SUMMARY
`PThFeOvSaAl,idPatFiOoSnAiAn,huPmOaAAn,sePrHuSm,orM-p5l5a6s,maanwdaMs5p7e0r.foTrhmeedsfuorrftohgaeteansatlayntdeasrdPFIHOISH2,H,2H-
perfluorooctanesulphonic acid was added before extraction, but used as an internal
standard in the quantitation.
-----
ne
`Thefollowing parameters can be considered ascompletelymeetingthe FDA
re+quiSrpeecmiefnictist:y
-
Calibration in serum and plasma
*PrAeccciusriaocny iinn sseerruumm aanndd ppllaassmmaa.
ValidationofExtract Dilution
+ Serum osamfprleeezset-atbhilaiwt.y 7to:cycles o fsrheoerzetrtestmoraamgbei,e4nt2.st5or5adgea,ys6.75 hr x 7 cycles
Serumoextaramcbtisetnatbislittoyrtaog:e, 7 days o refigerated storage, 4 days
o frozen storage, 5 days
| T+heBfeoclalouwsienogrfepsotroirctaionnaslyatpeplayndtointtheemsaelasntaalnydsaersd: extraction efficiencies, it is essential tahnaatlyssaemsp.leEsx,trQaCctsioannedffciacliiebnrcaytiwointshisntaansdeatrdisscionnsaissteetnbt;e kept together for all
+ SSaumppplreessswiionthofltevheelsinotfePmaOlAsAtaanbdoavrde TthHePUFOLSO,QrweislullsthinogwiLnaC/hMigShiqounaiznattiiaotnion bias
on other analytes. A resolution was not demonstrated.
Fodirlutdiiolnutoifngsesraummpalessthweithdisleurteudm,satmhpelcealfiobrraatcicounrcaucry.ve Imfuthset sbaempprleepiasaretdaatdtihfeersenatme
matrix dilution, the results will not be correct.
* When a sample must be diluted, it not only must be compared to a calibration curve
apnraelpyasrise,daitnotrhdeerstaomekedeiplutsiaomnoplfesse,rQumC,,buatndit aclalsiobrmautsitonbsetarned-eaxrtdrsacttoegdetfhoerrt.hat
T+hSetStfoaocblilklotwsyionloguftptiahorneassmtuaerbtireloirgtsya.tferosmtatnhdearFdD.A requirements werenottested: Qrueaqnutiirteamteinntgssfeorruamccsuarmapcyl.es against plasma calibration does not meet the FDA
Validation report review
3 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
! I INTRODUCTION
`Three reports were reviewed:
+ NWBR00-122: Quantitative Determinationof 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)
+ NWBRO00-108: Quantitative Determination of PFOS, PFOSA, PFOSAA, POAA, PFHS, M-556 and M570 in Human Serum by LC/MS/MS: Assay Revalidation Report, 1/24/01 (study NWBS00-040);
+ NWBR99-005: Quantitative Determinationof 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 studiesarevery similarwiththe following differences:
[Twos Tw#oenz
internal
SDS
standard
THPFOS
(1S)
partition
basified with 0.5 M
unadjusted pH ca. 6.9
tertbutylammonium hydrogen
sulfate (pH 10) + 0.25 M carbonate
buffer
final solvent|2mM ammonium acetate: methanol|20mM ammonium acetate in water
1)
20 mMammoniumacetate in
MeOH (3:
`quadratic regression, weighted 1/x, | quadratic regression, weighted usingarearatiovs. concentration | 142,using arearatiovs.
concentration
Thisreviewexamineswhichofthe FDA-requiredtests were conducted, and which meet the standards described by FDAfor a full validationofthe method. This report did not considerwhether additional requirements, specified by protocol, were metifthey did not
affect the evaluation based on the FDA guideline; however, they may be discussed in
the text of this report.
Validation report review
4 Human Serum at Northwest Bioanalytical
`QAI Report No. 405-503
I. SPECIIN SEFRUIMACNDIPLTASYMA
FDA Guideline[ WWeRepor | saThmipslreesqtueisrteemde,natlitshmoeutg.hEmnudcohggerneoauteslreivneslesrwuemr.e
found
in
all
serum
andplasma
-
2 Six sourcesofblank biological matrix |Requirement met. Testing was reported on 2.
Me relevelsof analyte
lotsofserum and 4lotsof plasma.
Results:
In report #005, endogenous levels of PFOS, PFOSA, PFOSAA, POAA and PFHS in human `serumwere determined using rabbit serum calibration; PFOSAwasnotdetected.
[RSuvns75mcaAbrasfo@niskteasnd=a6rds _ (5=4 --[P feMeza|]nP1F nFgO4/SmL6AAi|n[HO PuOm3Aa2n0|Se1S rPouFemHiS5]] ( [RRuunn22067228872200QcaClbsraamtipolnesst(an=d1a2rd)s (1=12) || 320243|1 119609 || 4f2o7r]] 317651 ||
Inreport#108, serum results indicated significant endogenous levelsofall analytes except
PFOSA. These levels are higher than in report #005.
i report#108 tables 8 - 14
4[oportH2tables 8-
Pros Mean ng/mL in Human Serum A
[endogenous eves| 474| wo | so | 476[215|ss | as |
[ettioa |10|10| 10 [710[10|10[10| [calibrationLLOQ" | 48.1 | 10| 60 |576|315|43|56|
Calibrationwaswith human plasma
Validation report review
5 Human Serum at Northwest Bioanalytical
(QAIReport No. 405-503
oo ea |
Pooledplasmaalsoshowedsignificantendogenous PFOS, PFOSAA, POAAand PFHS,
butatleast5xlessthaninserum.Thelevels weredeterminedbystandardspreparedin
plasmaat 1 -- 100ng/mL. The endogenouslevelis definedasthemeany-intercept of
linearregressionsperformedon 2separatetests.
Fm llvlllidil |[e[nadomoegoernniccusae6v"|es|| v20o4u||| 7wN00o0|o] ovvsm0o{||eon5z2[||1ov3a8ms||| w2so5aa|]]iWo]||
(
:
Validation report review
6 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
IV. CALIIBNSRERUAMATNDIPLOASNMA
Rceoqmupilriecmateendtmcaelti.braTthieorneprisohcoewdeurveer(qnuoajdruatisc wtithi1ff5oroitrhe1csxewlaeeictgthitoiinnogof)t.nhe.
Blank + 6 -- 8 samples LLOQ: 5x blank response
Requirement met. 2 blanks and 2 blanks + IS were included in each run. No blank analyses were reported. `Samples: 8 Levels for M-656; 9 levels for all others. Duplicate samples. 3 runsforserum and 2 for plasma.
| Requirement met. Endogenous responses caused LLOQ approximately = "blank" in most cases. Summarized in table below.
+1of5act%ual >LLOQ
Pasosf 5e50 tdota,l: 550 for PFHS 549 for PFOS 548 for M-556, M557 547 for PFOSA, PFOSAA, POAA
24of6 standards meet these| All runs pass. criteria in each curve
LLOQ: An experimental determinationofthe analytical limits was not performed. The fowest standard injected which metthe acceptancecriteriawas chosen as the LLOQ. SiInncmeostthecacsaelsi,brtahteisone LstLaOnQdairsdcoonlnycesnltigrhattliyo(n1s-w4etriemcelso)sheitgohetrhteheanndtohgeeennoduosgelenvoeluss. (blank) level the uncertaintyofsample measurementsatthe LLOQ will be increased by the uncertainty of measurement of the endogenous level. Becausethe endogenous levels are lower 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;
'
VALIDATED RANGE (ng/mL)
[
Serum
[ProsTPFOSA
(reports #1081122)
[PFOSAA|POAA| PFHS
[W658[W&70
|
Somioa [46 [40 |60
SmUloa_]540
[500 [50
| 58] 52] as]66] [50 [500[500[00|
Plasma (report #122)
[Pasmatioa T 59]10 | 16 | te] 14]26] 10] PlasmaULOQ [410[500 [50 [480 [520 [470 [480|
Calibrationmethod:
sQtuaanddraartdi)crvesg.rneosmsiinoanwlictonhce1/nxt*rawteiiognh.tin(g1,/owfepigehatkinagreianrreaptioor(t#s0a0m5p)l.e /Tshuerrreogiastneo justificationforthe selectionofthis complicated calibration method.
`Inaaclclusreatcsy,rtehqeurierweemrenotaotfl1ea5st%fooufroacuttuoafl (ioxrca2li0b%raotfioancsttuaanldaatrtdhleevLeLlOsQt)h.atmetthe
(
Validation report review
8 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
V. ACCIU N SER RUMA ANAC LYSY IS
The FDA requirementsforthis test were met.
5 rep@l3iconccentarattionseintshe expected range of concentrations.
| 9repleaichocn3asetpareatseda,ys
Le<v3eLlsL:OQ;
LLeLvOelQs:(4.0- 51.1 ng/mL; 1.1 --4x)
at the midievel ;
`midlevel (150-200 ng/ml)
necuarrvethe upper boundoaftrhye standard | high (400-450 ng/mL)
Meanvalues + 20% of actual @ LLOQ All passed except PFOSAA (intraday)
Results:
Summary tables presented below, with failed results bolded.
In the iniia test of 3 sets, PFOSA and PFOSAA failed because the fortifications were faulty.Thefortifying solutionwas preparedfromdilutestocksbyevaporation,andthese tcownotaainnailnygtejsuswttehreespear2tiaalnlaylylotsets., Tahdedeidntirna-cdoanycednattarabteeldoswowluatsioonsb.taiAncecdurfarcoymisnetthi1s7set was unacceptableatthe low level for PFOSAA, and at the highlevelfor PFOSA.
Interday QC datawas obtained from a monitoring study analyzed in theweeksfollowing thisfourthset. Atotaloffifteensetsarereportedfor PFOSAandPFOSAA,whichnot only validate the interday accuracy requirement,butalso validate the intra-day accuracy PreFqOuSiArRem)e.nt (5 outof 90 results outside the limits for PFOSA, and 7outof 90 for
Validation report review
9 Human Serum at Northwest Bioanalytical
QAIRepNo.o40r5-5t03
i
`Source: report #108 (tables 15-30a%nAdccAuprpa.cyA from FortifiedBlankSerumSamples
eve_r- ]|PFOS|PFOSA |PFOSAA|PORA|PFHS| Mi656 |M570|
Low Mid
g! Cow level
Run 13 Run 15
Run 1d 6
514 | 400 197| 150 446 | 400
92 92
a21
900 | 185 |
874| 154 |
615 152
580| 860 |~152 | 155
f=
405 | 403 | 402 | 402| 405
73
103
"1
108 87
-
3
101
97 7
108-
290| 110045 | 110035 | 10094
Run 13
92 | 9 108 | 102 | 110 | 108 | 104
RRuunn1158
888
--
--
8943 | 110040 | 9977 | 8945
I!
88 |
100 9| 105 | 101 | 8
igh level
Run 13
8
92 | 101 | 110 | 103 | o8
RRuunn 1165
888
--
995 |
11m05 |
08
9|
99
%
(
Interd
87
106 9| 108 | 100 | o7
PFOBSSA00/-P06F2O.STAhAeyintirnac-lduadyere1s5urlutnssaorfef3rocomnrcuenntr1a7t;iionntseradnadyre2rseuplltiscaarteesferaomchs.tudy
Validationreportreview
10 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
! VL ACCINUPLARSMA AANACLYSYIS
Requirementmet.
replicates @ 3 concentrations inthe
expectedrangeofconcentrations:
us
snteaanrdtahreducpuprevreboundaryofthe
5replicates @ 4 concentrations levels:
Loa
low (4.06.4 ng/mL)
hmiigdhl(ev3e3l2(1- 24641- 1n5g0/nmgL)/mL)
Mor fm gia [Mpes|
Mean value+s 15%ofactual >LLOQ | All passed, except PFOSAand M570 atthelowlevel.
Results:
{
`Summary tablespresentedbelow,withfailedresuitsbolded. Wh thei lowl levele for
-
PPFOSA andM570 failed, at 116% and 118%respectively, threelevelspassedforall
analytes. All results pass.
% Accuracy from FortifiedBlank Plasma Samples Source: report #122(tables 85-98)
[______ TPFOS[PFOS|APOPAAF | PO FHSS|MA55A 6 | M570 sols, ng/mL
LLoQ Low Mid
40 6.4 126
10 4.0 150
18 46 151
19 48 145
14 45 156
1.0 4.0 150
lig
332 400
401
385
497
400
oQ
id g!
98
101
28
112 116
12
106
107
103
101
104
104
91
95
98 103
104
106
108 118
106
101
29 107
103
2%
nm
101
Validation report review
1" Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
VIL ABSOLUTE RECOVERY IN SERUM
gRuiedeqliunei.rTehmemeQteCw'nistthanrdesctarliicbtiroantsi:ontsheindaessiegtnomufstthebteeksetpwtatsogdeitfhfeerrefnotrtahlaln the analyses. Extraction efficiencies are poor, but areconsistentwithin a set.
[ *DAGudeine
| KWReporr |
3 concentrations biological matrix
--
low,
medium,
hig--h
in
|
2 4
lreepvleilcsa(t4es&, 4in010snegt./mL)
in
serum;
Compare to unextracted standards
Compared with blank extracts fortified with known concentrations.
Recovery should and reproducible
be consistent, precise (limits not specified).
Accuracy not required.
| |Ex7tr0a%c)tifoonreafnfailcyiteensc,iewsoarrseep(o6o%r)(f1o5r internal standard, but consistent within a set.
Design: Analytes and intemal standards were added either to serum or to serum
extractot determine losses during extraction. The loss calculations were based on
`comparing the peak area raotf(ithoe one added before extraction:the one added after
extraction)tothe comparable peak area ratio when both were added after extraction.
`The endogenous levels were included in the calculated efficiencies (levels not reported).
Results: All analytes showed losses during extraction.Theseweresimilar at low and high levels. PFOS showed considerably higher lossatthe low level, but this is probably an artifactofmeasurement, with an endogenous level on theorderof 4x the added 4nghmL.
Extraction losses are not a concem for this method, so longasthey are consistent for samples within an extraction set. Since samples are extracted alongwiththe calibration suntaafnfdeacrtdesd,.asLloosnsgeassintthheey ainrteekrneapltsttoagnedtahredriarneanaalgyrseiast,ertcheonrceleamt,ivebuqtu,anattilteaatsitonwiwtihllinbae set, they are consistent. There is no inter-set data.
(Source: Report #122 Tables 64-70) __% MEAN EXTRACTION EFFICIENCY"
[P TPrr OSA[PFOSAA] Po OAA|PFHS |s M55 |W&ro|
[Analyte-- --------
1
[o 4 [ eo|e w [ 7 | 16] | 38| 54|
[h 30[70[ g 63 | 14 | 14h [ 40[60|
[o 3 | v 70| 63 |e 16 | 15|] 30| 57|
[overall(meane0%)[ 56] 58] 63] 61] 63] 55] 61]
Validation report review
12 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
; VILACCURACYANDPOAAEFFECTSON1S.QUANTITATION Restriction on Accuracy: Sfaomrpoltheesrwaintahlylteevse,lsduoef PtoOLACA/aMbSocvoeeltuhteioUnLaOnQdwliolnliszahtioonawshuipgphreqsusanitointaotfitohnebias icnotmeprlneatlasltyanpdraorvednT.HPThFiOsSm.aTyhaeffseccotpreeosufltthsiosbetfafeicnteidsbyprdeisleunttieond,anbudtrneoantalysis.
High concentrationof POAA >500 ppb depresses IS
response, biasing other analytestothe high side.
Hmoawneyvecro,nctlhuesitoesnts htaosbetodormawann.y unknowns to allow
Design: resolved
The internal standard THPFOS in LC/MS. Thus, POA at high
and the levels in
asnaamlpylteesPsOuApApraerseseisntchomeplTeHtePlFyOS
rQeCspsoanmspe.leTshwiesrceafuosretsfiaedhwiigthhbhiiagshinleavneallsoytfePqOuaAnAt,itaatnidonthbey apneaalkytaerseqauaranttiiof.ieLdoawndlevel
comparteod quantitationbeforefortificationwithPOAA..
`The data showthatwhenfortified quantitation is slight (-2 to -11%).
to approximattehley ULOQ level, However, at 10 - 100xthis level,
the change in the bias steadily
increases, in proportion to the decrease in IS peak area.
(Source: Report P#O1AAp2.A22d6)ded, approx. ng/mL %1CShAarnegea f|roAmQnCaMelaynFoluneds|
sel wi 2t-ti
|
50200] 75 | +is2to+216 | [QC conc.T"PFOSTM | PFOSA|PFOSAA| PFHS |Wss6 | Mero | [ngmt "T7518 | 405 | 683 | 80a | 541 |eer |
Validation report review
13 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
}
IX. ACCURACY OF SERUM
Ti
c
Does not meet FDA guideline for accuracy.
[_FO| AGuMd WWBRe epol rn ine
`Serum and plasma calibration standards.
Serum and plasma QC's a3t levels, 3-4 replicates,
2 sets of each matrix
Serum QC's analyzed with both calibrations
lSoewreurmenQdCogseanmopluesslweeverlesoafnsaolmyzeedanaaglayitnessttphlaansdmaoecsalsiebrrautmi.on,Thbeerceasuusltespwlearsemarehpaosrted 2s % deviation from theoretical concentration.
Results:
tThhiesFmDeAthrodeofqquuaintritfaeotrimnagcecsunerratucmys.saMmploeossfatgthaeinPstFOpSlaAs,mPaFcOalSiAbrAa,tioPnOdAoAesannodtPmeFeHtS results are > +15%oftheoretical.
(Source: Report 122, Tables 141 SERUM QC vs. PLASMA CURVE
[_Ac_cura_cy,___%_De[viPatFiOonSf|roPmFtOheSoAreTtPicFaOl SAA|POAA|PFHS|M-556 |M70|
[owl 41 [ 67 [ 7a [ 1a [ 26 | to | 0|
[md 92 | 40 | a1 | 20 [| 20 | 7| 5 |
[_____hgh|"& | 32 |1 | 2a | 25| a1| o|
[Precilontec -- ---- ---- ---- _--
|
erorerere
[md 7 5 5 5T 41 4|
[hgh 6T 6[5|3 | 2| &|
[__#ofmns[ 2 4[7[2| 1[ 11 2|
[L[ovells,ongiwmi.]-- 3--]~ 4m~ ] 72--0--em] ea] sm] 670]
[I mid[761 [450 [ 63 | 143| ta | 761 | 753 |
AAclccuarlaibcryatainondspertescipsaisonsoefd ptlheasrmeaquainredmsenetrsu,maQlCt'hosuagghaioncsctastihoenaslarmeeplmiactartiexs fcaailliebdr.ation met requirements, with the exception that PFOS/plAasmAa precisionatthe low level 1w6a%sa1t6t%hCe lVoawnledvael.cTcheulaortwltaehvecemliy sdwleerveelawbaosvet1h7%e,LLanOdQ.POAA /serum accurawcays
Validation report review
4 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
X. VALIDATIONOFSERUMEXTRACTDILUTIONPROCEDURE TThhiescraleiqbruaitiroen mcuweranvstemmuestt, bwiethprreepstarriecdtaiotn:the same dilutionofserum as the diluted sample for accuracy. If the sample is ata different matrix dilution, the
results will not be correct.
a"bTohveeatbhieltyutpopedirlulteisomafmitphlteesstoaringdianraldly|| Rcoenqtuaiirneamernesttrmiectt,iobnuotnthdeilmuteitohnoadnamluyssits. caucrcvueraschyoualnddbepredceimsoionnstpraartaemdetbeyrs in | > 12V1S: 4 repli-- cates; 1 set dtihleutviaolnidsastihoon.u"d"uAsney lriekqeumiarterdixs"ample |mid; high; 10x high
Daensdiagnna:lySzeerduamgaQinCstsaacmaplliebsrawtieornecduirlvuteedpr1e:p1a0reord 1in:5100w%itshearmummoinnibuufmfear.cetaitsekbnufofwern,
that quantitating against a whole serum curve is not accurate.
Results:
i
Tcaachlceiubrdraaacttyiao,nsbuhcutortwvhetehea1xt:ta5rca0ccdtuisrlaumtcuiysontrwebaqeusisribemisialtsahrea.dt
tAlhle1p:r1o0podritliutoinoonfs mhaatdr unacceptably low (57
iaxccinetphtaebslaem(p>l+e1a5n%d) - 85%). Intra-assay
precisionwas excellent (<7% CV) in all measurements.
(Source: Report 122, Tables 113-119)
[|PFOS |PFOSA PFOSAA]POAA|PFHS|M-556|M570|
[Precision(Cv)--
~~~~ |
[ md[ 4T"4 7 + TT 31271 471 2]
[high 5T76[4[3 4| 4| 4|
[LT_Oxdhoigxh(hi5i0g)|h|
2[2| 2[7[2[2[2| 2[4|4[4| 3| 4[ 4|
[Accuracyrecovery~~--
|
[mid] 108T9 [ 94 | 706 | 105 | foi|103|
[high] 703 6 [| 8 | 100| 702 | 102| 103|
[C__foxhigh| 707| 99 | 00 |105 | for | 114 | 108 |
[Toxhigh(1:50) 77| 67 | 66 | | e | 70| es |
[Lovels,ngiml -- ---- ---- ---- ----
|
[mid]183 T 960 [ 158[ 167 | 54 | 61 | 157|
[high] 443 "400 | 403 | 407 | 404| 401| 4o7|
[fOxhigh|4040 | 4000 [4000 | 4010 |4000 | 4000 |4010 |
Validation report review
16 Human Serum at Northwest Bioanalytical
`QAI Rep Noo .40r 5-5t 03
{ XI. PRECINSIERUSMAINAOLYSNES
The FDArequirem weerenmtets.
Pre- cntriabatseh/iinteorbantch)|Requirementmet|
CV120% @LLOQ
Allpassed,except 1 of 4 PFOSAAsetsat 1.5x
LLwiO th2Q 4%C, V.
CV+15% @>LLOQ
All passed,except 1of 4 PFsO etS satA 4x LL witO h 19Q .8%, CV.
Results:
`Summary tables presented below. All analytes passedatall concentrations, with the `exceptionof PFOSAandPFOSAAinoneseteachatthelowlevel.
Inthe initialtestof 3sets, PFOSA and PFOSAAhadlowrecoveriesbecausethe fortificationswere faulty; a set 4 contains justthese 2analytes. Both analhyatveeast
{
leastthreesetsateach levelsmeetingtheintra-dayrequirements.
Interday QCdatawasalso obtained from a monitoring study analyzed later.
Validationreportreview
16 HumanSerumat NorthwestBioanalytical
QAIReport No. 405-503
.
Precision(%CV)from FortifiedBlankSerumSamples
S[ ource:_ report_ #108 _ (tables [1_ P5F-O30S_ Aan[dPapFp_ O.SA)AA_ |POA[ A|PP FHS |FNi6O 66|MS 570
Low,ng/mL. 51.1
9.00| 874
5.80 8.60
Mid
197
155 | 154
152| 155
g
ow level
446
00
405
403
402 405
Set13
4
Set 15
2
6
10
2
6
4 7
Set 18
4
1
9
9
Sot17
2%
Interday! 3
8
6
70
Interday2
10
devel
Sot 13
4
3
Set 15
2
5
SSeett1176
3
4
9
Intorday1 4
6
Interday2
8
{
ah level
Set 13
4
8
Set 16
6
4
Set 16
3
4
75 33 44
SIentta1r7day1
72
65
54
Interd
10 7
:
Interday1isthemeoafsents 13,15, and 16.
[nterday2 PFOSA/PFOSAAdataarefromstudyNWBS00-062, a monitoring study.
isrepresents QCsample datafrom 15runsof 3concentrationsand 2replicates
Validation report review
17 HumanSerumat Northwest Bioanalytical
QA! Report No. 405-503
!
XII. PREIC NPLIASS MAAINAO LYSN ES
`The FDA requirements for this test were met.
[__FoA|G~~ud WWes Repi ot ne|
oovresaomnn gu0can (iPnrteecribsaitocnh -- intrabatch /
Results:
Summary tables presented below. All analytes passedatall concentrations,
Precision(%CV) from Fortified Blank Plasma Samples
`Source:report #122 (ta8b5l-e9s8)
Ese
Cro |PFOSATPFOSAA |PORA T PFHS [WHEt8TWETo|
Low
(
Mid
g
o 3
Hig
4.00
150
400
53 3
2
448
00
156
417
6
.
8
5
z
Validation report review
18 Human Serum at Northwest Bioanalytical
`QAI Report No. 405-503
i
Xm.
S!
TO FREEZE-ANI
All requirementswere met. In serum, all ana werl esty ablt etoe sevs en freez/e thaw cycles, except PFOSAAwhichwasstable to three cycles. Accuracy and precision were generally acceptable.
[_*oAGudeine| WWeRepor
"Two concentrations in triplicate
Requirement met. 3 concentrations in serum,
approx. 1x, 4-40x and 10-100x LLOQ
Analyte stability should be determined | Requirement met. Analyzed after three and
afterthree freeze and thaw cycles.
`seven cycles.
Results: Serum samples fortified at 1x, 4x and 10x LLOQwerefrozen and thawed seventimesbeforeanalysis. In eachthawing,thesamplesremainedatroom
temperature up to 6.75 hours. Losses are measured by comparing the sample analysis resutlotthse corresponding control analysis.
All analytes except PFOSA were stabletothese conditions. PFOSA data indicate losses of 11 -- 20%,considerablyexceeding the precisionofthe sample measurement.
{
PFOSA was stablteo 3freeze-thawcycles in report #005. In that report, PFOSA at
approximately 1x and 20x LLOQ showed stability,asdidallthe other analytes.
Precisiondata for sample analyses are good. Accuor fcoantcroy ls analysesiswithin guidelinesexceptfor PFOS high level , whichis ~15.2%.
[ (Source:report #122TablPersos1 [-P5 2r8oSA [P| rOASFMT|EPROR7KFR|EPEZrEH-sT|HAWWaCsYsC|LEwSero | -- CL ew Y sw 3 7T ] a]71+ (S[ ample,meanngmi. 2m 11s[ool l or] os] 5]| C [me | e ies | s2md 0[]7s ssae|i ]75a1m|] u eeaT []sssum |l 5a32|] Chgn| 300| ss [302 | 372| ob| 300| 506|
[-- %(DSoeuvricaet:ionreproortm#0Co0n5trToalbl| Ee 51) C4PF|OSA__AF2TE|R 3 FREEZE-THAW CYCLES (Sample,moangmt. | 217| 360 |
Validation report review
19 Human Serum at Northwest Bioanalytical
QAIRepNoo .40r5-5t03
! XIV. SHORT-TERMAMBIENTSTABILITYOFSERUM SAMPLES
Re6q.7u5irhoeumresnptesrwcyecrleemedutr.iSnegrsuemvesnarmecpulrersiwnegrfreekeezpet-tahtarwocoymclteesmp(edriastcuursesuepd to
above).All were stableexcept PFOSAA,which exhibited 10 -
20%-oses.-- - -....- --
3aliquotsof biologicalmatrix fortified atlow andhighconcentrations
Store fortified samplesatroom temperature 4 --24hours.
Requirementmet. 3concentration--s approx. 1x, 4-40xand 10-1L0L0OxQ
Requirementmet.Fortifiedserum samples `werefrozenandthawedseventimes. The
tmhaextoitmaulmttimheaawttriomoeimnteacmypcleerwaasti6us.nr7o5ethours;
reported.
Results: Presentedinthesection above.
{
Validation report review
20 Human SerumatNorthwest Bioanalytical
QAI Report No. 405-503
i
XV. LONG-TERMFREEZER STABILITYOF SERUMSAMPLES
oAvllerrtehquei5r5e-mdeanytsstwoerraegemepte.rio~dTh(e42redsauylstfsodretmwoonsatnraaltyteetsh)aotftheexrteraicstsnaotdfergereazdeartion temperature -20 C.
Atfolretiafsietda3tallioqwuoatsndofhbigihocolnocegntimracattraiiolxns||Raeppqruoixriemamteenltym1ext, .4x5arnde1p0lx iLoLcOfaQs.tereusm at SSstaaommrpeelfeocsrot,nidffioitreidaiosnamsmaepsleetsxhecuensedtdueidrnytghtehe tine |R4e2qudiarsemPeFnOtSmAeta.ndSPFtOSaoAtA2r0eCfdor: between the dateoffirst sample 55 days other analytes ancaolllyescitsio.n and the date of last sample | tshteaobrieittiycawlacsondceetnetrrmaitnioend.by comparison to
Results:
Aplrlecainsailoyntoefstahreemsetaasbluereunmdeenrttsh.esPerescitsoiroangoefcaolnldimteiaosnsu,rweimtehinntsthaeraec<c1e1pt%e.d limits of
{
(Source: report #122 Tables 57-63) _ SAMPLESAFTER42 OR 55 DAYS AT 20C
le 00 |PFm OSA|PROSAR]Pm OAA |PFS|Wa ises|W670|
-- Jowl STs[AT[ 40 [ 42 7 7| medum|[ a| o0[ 3[318 7[41 [h 51 e 11 2 |8 h o2[] 5 5]
[o "4677 434] 0w 85] 762] 68l 6] 542] 6.02]
C[Cmedi"unimn| o4r7e01|753005|| 1#6103||134729|| 146043||3a617|| 134896||
Validation report review
21 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
XVL
\RATIVE AMBIE
i
TS
Tnhoedergerqaudiarteimoennotvsewretrheeme7 tdafyosrtaollraangaelypteersi.odTohfeexrtesrualcttss dateromomotenmpstehrtaattrtuhraeer,te.ies
-
covering at least the resident.time in the autosampler.
[ FDAGudeline| WWBRepon
|
No specific requirement
Requirement met. Serum extracts used;
approx. 1x, 4-40x and 10-100x LLOQ
The stabilityofthe drug and the intemal standard should be
|
Requirement 3or7 days at
met. room
tSeemrpeurmateuxirrea.ctSstasbtiolrietyd
`assessedoverthe anticipated run | was measured by comparison to
time
pQrCevisoaumspllyesa.nalyzedor freshly prepared
Results:
``eTxwtortaectsetdswanedraenacloynzdeudc,tetdheantrdeiifnfjereecnttetdiomnest.heInt4h"defaiyrastf,te"rsetxatnrdaacrtdisona.ndcTonhtroQrlCs"lweeevreeels ( wtehreen ainnaclluydzeedd. aIgnatihnestsfercesohnldy,elxotwraacntdedhiQgCh sQaCmsplaemsp.leextractswerestoredfor 7days,
All analytes are stableunderthese conditions. Recoverieswere all in therange of 1p0r0e%sent1e1d.aWthailmeitnhiemruemisdneomosnesptarraatteetdhaatt,aaftorwtorhset,suarlrl oagnaatleystteasnadnadrdt,hethseurrreosgualttse degrade at the same rate, If any. tSeastm,pbluetwpraescinsoitorneipsogrotoedd,fo<r1t0he%sCeVcoinnda.ll cCaosnetsr.olCaontcrolcpriescuigsoiorodn,ieasxgcoecopdtifynotrhPefFiOrsStA faonrtdifPicFaOtiSoAn Aer.rorI.n tHhoewfeivrsetrt,estt,hceosnttarboillivtayltueests iwsenroeta<f8fe5c%teodf bthyeothriest.iicablotdhuewetroeafortified Wtihtehftorhteifsicaamtieonsloelvuetliown.asInnottherespeocrtoendd. test, accuracy couldnotbe determined because
Validation report review
22 Human Serum at Northwest Bioanalytical
v
QAI Report No. 405-503
{ [oo(Source:report #122 T|aPbrleoss20|-35vrooEA[XpTrRoAmCT|S FSToOkREIDo3FDAY[S,esAMeBI|ENTero
.
* emoa ew]m A TlTT3 s T 7 a ]a T a ss r ea ] |
Ee miagnl[3e8e2To06T o198rA i8TT4s 11es7as |eaom ra 13] ] 0]
(Source:report #122 Tab3l6e- 4s2) EXTRACTSSTORED 7DAYS,AMBIENT
Ee onemalSTT 7TT T 7T1ooTi 51s dw]3]
ET -------- [l 480] o 337] ee]7w 6s]ea]] 543] 691]
LC
high| 301| 33 |334| ars | 40a | 370| 420|
(
.
Validation report review
23 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
!
XVII. POST-PREPARATIVE 1-8C STABILITY OF SERUM EXTRACTS
oAvlelrrtehqueifroeumrendtasywsetroeramgeet.periTohdeofreesxutlrtascdtseatmreofrnigserttahtraotratthetemrpeserIastnuroed.egradation
Fm ee est| [_*DAGudeline| WWBRepon
|
"The stabilty should be assessed obveefrorteheiannjetcitciionp.atedstoragetime
|Requirementmet. Stored serum extracts wSetrabeirietfyrwiagesramteedaastur1e-d8byCfcoormp4adraiysso.n to freshly prepared QC samples.
Results:
A`lalccaunraalcyyteiss gaoroeds;tahbolweeuvnedre,rPtFheOsSeAcoanndditPioFnOs.SASAamapclceurparceyciinstiohniesQgCoosda.mpCloenstrcooluld nreostubletsevparleusaetnetde.d aWthailmeitnhiemruemisdneomsoenpsatrraattee tdhaatta,faotrtwohrests,uarlrloagnaatleytsteasnadnarddt,htehe sumogate degradeatthe same rate,if any.
!
(Source: Report #122 Tab4l3e-4s9)
EXTRACTS STO4DR AYSE1-6D8C
[ PFOS[PFOSA[PF1 OSAA]POAA|PFHS |Wsss| Mero|
[o 02]5 2 w 11 ] 1 46 4]
[S[ amplePrcisionh (Cv), % 2[ 2 g 4]h 4121] 10] 3) |
wl 518] 71 ] 10 4] 9]
l [Sae mplemeannghhntg.h]"2|e| 1 213] os 1]|
[
lw[ 468] 206] 822] 831] 695] 620] 8636]
["hign| 384| 317 |310 | 387 |411|402 | 307 |
Validation report review
24 Human Serum at Northwest Bioanalytical
`QAI Report No. 405-503
XVIILPOST-P-2R0CE STAP BILIATYORFSAERT UMEIXTRVACETS
All requirements
over the five day
wsetorreamgeetp.erTihoedorfeesxulttrsacdtesmaotnfsrtereazteerttheamtptehrearteuirsen(o-d2e0g
rad
C).
a
t
i
on
The stability should be assessed
overtheanticipatedstoragetime before injection.
Sento mot
Requirementmet. Stored serum extracts `werestoredat-20 Cfor 5days. Stability `was measured by comparisontofreshly prepared QC samples.
Results: All analytes are stable under these conditions. While there is no separatedata forthe
`asnuarlryotgeasteasntdatnhdearsdu,rrtohgeatreesudletgsrpardeesaetnttehdeastaammeirnaitem,uifmadneym.onstrate that, at worst, all
[ (Source: Report #122TarbrloesL s5[0P-r5o6)sAE[XPrToRsAaCaT]SPoS aAT T | P5O rDiAsR Y|SWaE at-s2sD 0|Cwero| i CBw T2 Ts] wr] os] a]
[ [ CC S_ a_ mo_ p wl7l --_ 27e [ _ 5 a P_ rs0o_ 3a]ci_ 0o 8]]h n2 Bm ( g | e 0wh 8m, ]]al 3%]] [(--Sa--mp--l--e,--mengFhg_h[--2 -- 8]3] 5]2]3] 3] C[ C wd on[ 47415[]530o 078[]270061|]3w 8030|] 6do8o1|]5] 5039[]78a30|]
Validation report review
25 Human Serum at Northwest Bioanalytical
QAI Report No. 405-503
!
XIX. STOCK SOLUTIONSTABILITY
Requirement not met. No testing reported.
[_FOAGuideline -------] __-- NWBReport ___ ]
toe
Slteoarseta8throouorsmtemperatureforat Requirenmoetmnett
Stitmoerepaetriootdhserastermepqeuriraetdu.res and
Report issued by:
(
Philippe Ourisson QUALITYASSOGIATES, INC.
Date
Manager, Scientific Support
Validationreportreview
26 Human Serum at Northwest Bioanalytical