Document 2NKKDGqo2RDNk0BG3raNq9Rdg
AR QR G-- 138K
Protein Binding of
Perfluorohexane Sulfonate,
(Human, RatP,erafnlduoMroonokcteayn)e,SaunldfoVnaartieoaunsdHPuemrafnl-uDoerroiovcetdanPolaattsoemaPPlraostmeain Fractions
STUDY ID: 9921.7
Southern Research Institute 2000 Ninth Avenue South
P.O. Box 55537 Birmingham, AL 35255.5537
000090
StPuadgye92902f177 SUMMARY opTnlhaiesbpmoeaurtpcredonsttewpiirnosftrmeaiecnatsbiouinnrsdeiwdnaigsnofrdaeftt,oeumrrmiponeerdfn.lauonAkrddodhinetauitmoenyadancllopym,lpaposrumonatdisnibisndeivnegfodirftfhereenstahmuemapno-uderived `oZehe ipreottesipwbaosuinndcutbesatteardtifcolre 1wSamsisneuptaersataetd3f7roCm itnheeancohnp-lbaosuamntadfiptvreeottdeaiirftnficeflrreaecnbtycioounncloesrntpprllaatmsinomnaasss.aumsEpialncegh. sChteanentedrlaiurfedr.neteffrioltmratthieonudlervaiiciersatwiiotnh awansomainnaallyzmeodlfeoecuslatr `awretiigchlet c(oNncMeWnt)ractioun troelfoa3t0ifv,e0ft00sdacltaonss,
000081
RCeosrceanrncahKAesrssitstnaenrt-WIlolod, BS Bioanalytical Chemistry Group Lori Coward, BS Sr. Research Associate Bioanalytical Chemistry Group Greg Gorman, PD. Manager Bioanalytical Chemistry Group
KEY PERSONNEL
Sudy99217
Page3 of7
000082
StPuadgye9d9o2f1?7
1. OBJECTIVE
hTuhmeaonb,jercatt,ivaendofmtohniskesytupdlyawsamas
to as
determine the well as various
pheurmcaennt-doefriprvoetdeipnlabsimnadipnrgotfeoirn
each test fractions
article
in
2. SAFETY
ttAhhlielsMnsaettcuedeyrs.isaalrPySrapofrceoetcdyeudrauenrsdeosDuttaoltiaenneSsdhuerieenttSshOe(PMsaS2fDe-St2)y9,-o3ftphfrooerviahdnaaenlddylsbitynsgtwhheeuremspabonanssaeondrdofpnorriintmfhaeotretmeasttiaornticcolenstauisneedd ia
the human-derived plasma protein fractions were also followed.
plasma a6 well so
3. COMPLIANCE
`T3ph5ei4rs8f,owr3om6re0dk6,iwn3at6sh0e7p)se.pirrfWiothorifmlteehdetuhrisseignwugolarvtkairowinoasusussniponrtgevaciuaodluiisbtlreaydteivdanlacindodamtpveladliiaadnnacatleeydtwiiicntashltrGmuLemtePnhtoradetgsiuoln(a,tions, it was 4. EXPERIMENTAL
4.1 Analytical Procedures
aItndtiwvoidcuoanlceanqtureaotuisonssolasutiinodniscoafteedaicnhthheutmaablne
derived below:
plasma
protein
fraction
were
prepared
=Plasma
ProteinFraction GAaliphmaa--GGlloobbuluilnin FAilbphrai-n2o-gMeancroglobulin BTertaan-sLfieprorpinroteins
El Physiological
`Plasma Protein 956057--21501357mmgg//110000mmLL. 210500--640200 mmgg//110000mmLL 228000--434200 mmgg//110000mmLL
Prepared Plasma Protein
Prepared Plasma Protein
{10Co%ncePnhtysriaotlioognical) (100Co%nPcheynstiroaltioognical)
113966mmgg//110000mmLL 3687 mmgg//110000mmLL 2402mmgg//110000mmLL
11276182mmgg//1010m0mL0L 240008 mmgg//110000mmLL 229070mmg/g1/010m0m0lL
moSritxo2tcukcrosenotwlruaotsliovpnolsratosefmxaeeadmcafhotrrtei|sxtmtiaorntuiptcrleoedawunecdreienasceufpbianaratlaetdceolanytc3se7pnitrkCaetdfiioonrnt1oo5ftmh1ien0putlpeagssh.mmal.proTtehien
factions resulting
ouflttrhaefilitnzcautbioantidoenv,iecaec(h NsaMmpWlceutwoafsfa=gai3n0,v0o0r0tedxaeldtofnosr)1amnidnucetnetarinfdupgeldacaetdU2ip0not0on0acxoCemgnptlfreoirtfii3oe0ne
minutes. The ultrafiltrate was analyzed to determine the amountofunbound test article
000033
SuPdayge99S2o1f7?
42 Results
`The the
resuolftalslofthe endofthe report.
analysesarepresaseperncetntbeoudndtestartiincTalblee
II
at
5.0 Conclusion Tdehreivperdotpeliansbmiandpirnogtoeifn fforuarctpieornfslautotrownoatdiefdfceroemnpt opurontdesinwfarsacetviaolnucaotnecdenitnrsaetvieonnsse(pTaarbatlee1haunmdanI-. `mAoddnikteioynpallalys,mparoatte5indbififnedrienngtcfoonrctehnetrsaatmieonfso(uTracbolmepIoLuI.ndTshweads eavianltuTaatbealdeinI raant,dIhTusmhaonw,satnhdat baceoltnbacu-elmniitpnroiapstriobotyenisfn.asr.TthhTeehlfeaorupgrretoshttesciionnmgbplioenupdnridong,teePixFnpOebSri,inmdweeanrftssofrfotouhrnrtdeheetoorfabtte,hhheiufgmohaulnry,cbooamnudpndomuobnnydkbseoytthpelaalssbaumttmabieosnthdahonwd veryhigh levelsofbinding at all concentrations tested and no evidenceofsaturation.
000034
SuPadgyeomo1f77
Table I Percent Binding to Human Plasma Protein Fractions (~10% Physiological Conc.)
Albumin| Gamma-| Alpha- | Fibrinogen| Alpha2- | Transferin| Beta:
Fess
Globulin| Globulin
50
Te
Macroglobulin
or--
[BEES[75005 | 150 | 336 | 156 | 50 |
Lipoproteins.
7] -
[PROS1990 1765 [PROC | 964 | 35
| 499| 01 | 285| 54
| |
125 75
75] | To |
25 90.1 Tos
Table I Percent Binding to Human Plasma Protein Fractions (100% Physiological Conc)
Albumin| GGlaombmulai-n|| GAllopbhual-in Fibrinogen Ma`cArlopghlao-b2u-lin Transferrin LipBoeptroat-eins
el [BEES 1 5%5
TT [7261 | 157| <o1 |
1 <0 | 6a |
@i
[EROS1998 [PROC 957
T7241 1750
| 504 | 711.0 |
<o1| <01 <o0 <1
| =of| 21
55% 396
Table I % Protein Binding to Rat, Human and Monkey Plasma
1 Ol dl cl dl [NominalTT peruorabeuanesuifonsie|
fPecliuorooctanecarboxalate
FT joo{=o 100 | =io6 | ou |=o =o|=os~]
F Le e 2s TTTosoero| 5w0s0[|919090[][999575[15799999
995 | "swe | 994 | 995] 7909 | 576 | 995 | 06|
%
Bi
n
di
n
g
Vales
re
ported
1
as 10"
r
ona 05
eflec nonquaniale
99a [900 1565 {995
amount ofst etl nh pla
| 375
waterBQ
L
<
s95[30
6.25 gf.
4]
000095
StaPayge99T2a1l.?7
60 References M(1o).sbKoyplPaurb,liLs.h,iPnegscCeo,. A., "Clinical Chemistry (Theory, Analysis andCorrelation) 1984, pg. 1416,C.V.
7.0 Review and Approvals
fin Li Sond,
renna Kerstner-Wood, BS Research Assistant ITI Bioanalytical Chemistry Group.
Ze Gort Lori Coward, BS,
Sr. Research Associate
Bioanalytical Chemistry Group
orman, Ph.D. Manager
Bioanalytical Chemistry Group
06-03-03
Date
6363 Date
&-oj-03
Date
000096
Method No.: BACG-3548 ANALYTICAL METHOD
Page lof 13
Title: PDrepearattioen aornfdnPAeruifluyosariosoacbtaytneHciParLbCooalMnaatessin SMpoenckteryomSeetrryu/mMaasnsd USrpienec:trSoammeptrlye (HPLC/MS/MS)
--_------e 10 PRINCIPLE
SP(eePrrRfuOlmCuoo)rroisocuftoraritnniefeciaesrdabwmoiprtalhleiastnaeri(nePtFeorObnCta)al.isnteaTndhdfearrsdoe(mr1ucSm)y,noproemruforlilungoeuros(hcme.xgo.an,nke0s.eu5ylfsmotLnr)aetaceto(entdPaFwiHniiStnhg Tbhyeesxatmrpaclteisoanrweitthheentmhyilxaecdetwaitte.haTnhieone-tphayilriancegtraetaegelnaty,erbuifs freermaonvedd,wateevra,pfoorlatleodwe1)d0 HdarPmymLneoCsnsMi,aursmecsaoScnpesettcaittteur,toemfdieilttrneyre/9dM5,aa%snmsdSepttehrcaatnnrsooflemrecrtoerndyta(tiHonaPiuLntgCo/s1Ma.mS5p/lM%ef Sr)v.iaTolhse,ararcanidda,mn5al%i yzSedc mbMy rensgu/ltmsLeinxuritnfe.reoSmaanmbpoludets20csotnota1i0n0i,n0g0P0nFgO/CmLatocfoPncFeOntCriantisoenrsugmraeantdefrnrgtohemaon1f01re0tl0oi,z50b00l00e nwgil/lmbLemwiatyhibnetdhielurtaendgweiotfhrceolnitarbloelbrelsaunlktsmpartiroirxtso oantahlaystitsh,econcentroaftPiFoOnC TTrhheedeufmcoaetnshssseppfreacytosrooumrrectermovyfooolafttPhaetRgreOpiCoistseanenottdiaaPltnFlyH-iS2n0ite0sr0afvceocrloitmnsgpilWoihnsihscehedxiitsnrltaochtewendeegafaotuigvehitoongmreoadtel.y `Ionftohredenretgoatmiavexifmriazgmeesnetnsiiotinvi(tMy,-tChOiOsHm)e'thfoordiPsRbOaCs.edonmixedreactior ntmhoenomiattormriixn.g iCItAhUnTuIniOcvNbelr:solaoSlidn,pu cprelepcard asiummtaaitiaoennssd.mnsaeyRreg ucfmaer,rraytroeaSntuoOmbPbeencruomnosbfzieodroenrSoeRsdIaes2s,-b5a-li5 ufonorrheasaerazvnedadhartinsddslueessd, safety procedures to be used when handling unpreserved primate tissued.escription of 20 REAGENTS AND SOLUTIONS
`The listed reagents or their equivalents may be used. 21 Neat Reagents
000097
ANALYTICAL METHOD
Page20f 13
Method No.: BACG-3548
Title:
PDreetpearrmaitniaotnionaonfdPeArnfalluyosriosoctbayneHcaPrLboCralMaatsesin
MonkeySerum and Spectrometry/Mass
Urine: Sample Spectrometry
(HPLC/MS/MS)
-_--m
211 ar, deionized and organic free (from in-house purification system; e.g, Ingalls
212 Methanol, HPLC grade
2.13 Perfluorooctanccarboxalate (analyte), as provided by the client 214 Perfluorohexanesulfonate (internal standard), 97% 2.15 Ammonium acetate, HPLC grade
21.6 Blank control monkey serum
.
2.17 Sodium Carbonate, Certified ACS Grade or equivalent
2.18 Sodium Bicarbonate, Certified ACSGradeor equivalent
2.19 Ethyl Acetate, HPLC grade
2.110 Tetrabutylammonium Hydrogen Sulfate
2.111 Sodium Hydroxide 50% solution, Certified grade or equivalent 2.12 Blank control monkey urine
2113 Formic Acid
22 Prepared Solutions
Appropriate changes in the solutions may be made at the discretionofthe analyst 221 5mMAmmaceo tatein norgi anicfu reewam ter
000038
Method No.: BACG-3548 ANALYTICAL METHOD
Page3of 13
Title: DPerteepramriantaiotnionaonfdPAenalrysfislbuy oHPrLCooMacsistnMSapoeunckteeryomSceetraruyrm/MaabasdsoUrSripneaect:lrSoamameptelrye (HPLC/MS/MS)
--_--mmm------ 22.1.1 2F4odrienxoamrplge,a0npriweaptcaer-e(4felgir.tere4s,Lem)e.aMsuirxe woeultlaamndmointieumtharcoeutgatheH(Pe.L5C.,mo1b.5i4l2egp)haasned
filtration apparatus.
222 TBA lon-Pairing Solution (0.5 M tetrabutylammonium hydroxide)
222.1
Fsuolrfateexaimnpdleei,ontiozepdrewpaatreer
2a5ndmLad,judsittsshoelpvHe
4.24g to 10
woifthte5t0r%abuNtayOlaHmmsoonluituimonh.ydrogen
Naodtjeu:stamemnotrs.e dilute solution of NaOiHn water may be used toeffect smaller pH
223 Carbonate/Bicarbonate Buffer Solution
223.1 wFaeonlrdleatpxopaermonpxsliuemr,taetocepolrmyep2pl.ae1rt0eegd1oi0sf0ssomoluLdt,iioudnmi.sbsioclavrebaopnparteoxiinma1t0e0lmyL2.6o5f gdoeifosnoizdeiduwmatcearr,bonMaixt.e
3.0 INSTRUMENTS, MATERIALS, AND APPARATUS
The following or their equivalents may be used.
31 HPLC pump(s), autosampler, and triple quadrupole mass spectrometer 32 Auosampler vials with inserts
33 Vortex mixers (e.g. touch mixer and IKA-Vibirax platform mixer) 34 Solveaconceatration apparatus (.g., Zymark Turbo-Vap with source of nitrogen) 3.5 HPLC mobile phase filtration apparatus
36 Filters for HPLC mobile phase filtration apparatus (.g.. Nylon-66, 0.20 uz)
000099
MethodNo.: BACG-3548 ANALYTICAL METHOD
Page dof 13
Title: (DPreHetPpeLarrCmai/tnMiaoStni/oMnaSno)df PAeurafluyosriosoctbaynscHaPrbLoCxalMaatseisnMSpoenckteroymSeetrryu/mMaasnsd USrpineec:trSoammeptlrey _ 37 Analy-- tical balance _--me
38 Volumetricflasks (e.g., 10an25mdL)
39 Disposable Pasteur pipets
310 Micropipettor(s) with tips
3.41 Culture tubes with teflon-lined caps
312 Centrifuge
313 Assorted glassware and syringes
3.14 Culture tubes (vials) for use with solvent-concentration apparatus 3.15 1m Plastic syringes with 0.2 um PVDF syringefilters 3.16 Variable speed horizontal platform shaker 317 pHmeter
40 PREPARATION OF STOCKS AND WORKING STOCKS
Adipspcrroeptriioantoef sheets.
changes in the analyst.
theActcuonaclendtilruattiioonnss
of will
the be
solutions may documented on
be made at the the preparation
41 Main Stock Solution of PFOC ~1000 g/mL,
411 acoPcrrugerapanatirecelfyraenwee~wia1gth0ea0rb0ooduuigts/s1mo0llmv.eg.solDPuFtiiOoeCntoionftthoPeRamOaC1r0k-i.mnLdvAeloitloeurnmnieaztteirdvieoclryfg,laanswikc)-.ferAteedhidewcadtoeemiropno(ic.a5e.d, guhnd
000100
ANALYTICAL METHOD
Page Sof 13
Method No.: BACG-3548
Title:
`PDreetpearrmaitniaotnioanondfPeArufalluyosriosocbtayneHcaPrLboCralMaatsesin
Monkey Serum and Spectrometry/Mass
Urine: Sample Spectrometry
(HPLC/MS/MS)
_----mm----
intoan water.
aMpixpwrelolp. vrTeirsasanestlfeer
(te.hg.e, csuolltuutrieotnubtoe)aacnldeaanddves1s0eml iLf doefsdierieodn.izedorganic-free
42 Stock Solution of Internal Standard (PFE), ~200 g/mL
421 afPcrrceeuepraawtraeetleayrwneti~og2hd0i0assbooplugvt/em1la0.nmdsgoidlinulttuiotoenatSoof0t-PhmeFLHvmoSalruikmnedwtiertiihocnifdlzeaeisdokn)o.irzAgeaddndiocdr-egfiarnoeineci-zwfeardteoeerrgwa(aact.gs,. 2Aldtder5n0atmivLeolyf,dweeiiognihztehdeocrogmpaonuincdi-nwftaortearae.Mapipxrowperlila.teTvessrel a(teh.ge.ns,ocluuslttiuorfnettoubeae)ctalrenerad,n. vesselif desired.
43 Spiking solutionofInternal Standard (PFHS), ~ S0ug/mL 43.1 Pr2empLaorefatnhe~25000 gg//mmL.ssoloutlionuiotnftioPoaFcEnulitunrdeetiuobneiazneddoargdadni6cm-fLreoefwadetieornbiyzepidpweattteirn.g
Mix well
44 Working Stock Solutions of PROC
44.1 ITffoodlelspoirwreiepndagrateambowldeoi.rfkiPierndedgpialsrutetoicioknns1sco0hl-uemtmiLoencvsoa,lnumbmaeetkuresiectdhfaleanspdkrdsooopcreourtmhedenilrtuaetpdipoirnnsotpharesisatstuehdogywlranescsiwnarteh.e ords.
`Working Stock Level (WSL)
Approximate Concentration
(ng/mL)
Sloe |] Volume of PFOC solution | Final Volume in deionized organic free water (mL)
smiofswugml| 10 |
000101
ANALYTICAL METHOD
Page 6 of 13
Method No.: BACG-3548
_ Title:
Determination of
Preparation and
PAenrafllyusoriosocbtyaneHcaPrLboCxalMaatsesin
Monkey Serum and
Spectrometry/Mass
Urine:Sample
Spectrometry
(HPLC/MS/MS)
*
om Tommi
hoem [ow
Tmoarammmoam|ereeo]] orosoma0ws]]
oioom owwo oes vorn i omo e sowo s 0 o e0ep ] ] a]||
4.42 Summary of concentrationsofserum standards:
`Standard Level
--
Volume and Spike Conc.
_
`Approximate Concentration of PFOC in serum
(ng/mL)
[To [s,pm m | mo oo]
[T=o1ow[wutsmmomoomnn]|usyoao mr]|
000102
Method No.: BACG-3548
ANALYTICAL METHOD
Page 7 of 13
Title:
_--
DP(reHetPpeLarrmCai/tnMiaoStni/oMnaSno)dfPeArnfalluyosriosoctbayneHcaPrLboCxalMaatseisn MSpoenckteroymSeetrryu/mMaasnsd SUpriencet:rSoammeptlrey
TEER [oe [owosooosmmom]| [+ [asiomn|10| [o[wmosa| ssmw a|
[x [oaosoomwer |aw| [+[omoso|omwwe| Lv[odozs |o0me| [~[omor|m2 mn|
[0[wmosmw|om0wm| Lr [Tomoasomm |5]
5.0
PREPARATION OF SPIKED STANDARDS AND BLANKS
dAipspcrroeptriionatoef cthheanagnaelsysti.n the concentrations of the solutions may be made at the
5.1
Multiple (e.g.,aboutthree) setsofmatrix standards and amatrixblank (blank +15)
are analyzedwitheachsetofunknown samples. Amatrix double `blank (blank-1S)`may
also be analyzed if desired.
000103 52 Ianptprooipnrdiiavtiedvuoallu~m2e0a-smdLecsuclrtiubreedtiunbtehse,ptiabpleetabbloavnekfmoarteraicxh(set.agn.da,r0d..5mFLo)r.tPihpeetblianntkhs,e pipet 10 uL of organic-free water insteadof theworkingstocksolution. Add the 10 ul
ANALYTICAL METHOD
Page8of 13
Method No.: BACG-3548
Title: PDreetpearrmaitniaotnionanodf PAenrafllyusoirsoocbtyaneHcaPrLboCxalMaatsesinSMpoenckteroymSeterryu/mMaasnsd USrpience:trSoammeptrlye (HPLC/MS/MS)
_--_----
ooffionrtgearnniacl-fsrteaendwaartdesrtoicnkst(ea~d)50anpdg/vmorLt)extofcorac~h5tusbeeecxoncdesp.tthe blank-IS (pipet 10 pL 53 cTaorbocnaacthe/btiwcbareboanadtde b5uf0fe0r,uaLndo1fmtLhoefdTeiBonAizieodno-rpgaainriicnfgrseoelwuattieorn.,Vo1rmteLx eoacfh
tube for about 5 seconds.
54
Add 2.5 setting.
ml.ofethylacetateandextracton
horizontal
mixerfor
1
hourat
a
lowspeed
55 R5emmoinvuetetsh.etubefromtheshakerandplacein acentrifuge(e.g.,2500rpm)forabout
56 d`Traykneeosfsf(te.hge.t, o~p 5e0tmhiynluatceestiantethleaTyeurrabnod-pVaupti)twiinttho aagcelnetalnestturbeeaamnodfenviatproorgaentaentdo `moderate heat (e.g., 50C).
57 cRoenctoanisntiintgut1e.t5h%eforersmiidcuaeciind5a0n0duvoLrtofex5b%rie5flmytMomaimxm.oFinliteurmthaecestaatmep:le9s5t%hrmoeutghhan0o.2l 4am PVDF or Nylon syringe filters into autosampler vials.
60 PREPARATION OF SAMPLES
61 bAultloviwgeoraocuhslsye.rumPispaetmpalneatloiqtuhoatwotfoearcohomsatmemppleera(tc.ugr.e,.0.5VomrLte)xienatochinsdaimvpidlueablri~e2fl0y-,
mL that
culture tubes. the expected
If necessary, concentration
dilute of the
an aliquotofany test article being
samplewithblank analyzed will fall
matrix so within the
5co0ngce/nmtrLa)tiotno eraancghetuobfethaendstvaonrdtaerxdfcourrvae.couApdledof10suecLoonfdisn.ternal standard stock (~
62
To cach tube add carbonate/bicarbonate
500 buffer,
uL and
1ofmLtohfe deTioBnAizeidono-rpgeainriicngfreesowlautteiro.n,
1 mL of Vortex each
tube for about 5 seconds.
000104
ANALYTICAL METHOD
Page9of 13
Method No.: BACG-3548
Title: PDreetpearrmaitniaotnioannodf PAenrafllyuscirsoocbtyanHecParLbCoraMlaasesin SMpoenckteryomSeetrryu/mMaasnds USrpience:trSoammeptlrey (HPLC/MS/MS)
--_--
63
Add2.5 setting.
mLofethyl
acetateandextractonhorizontal
mixerfor
1hourat
a
low
speed.
64 R5emmionvuteets.hetubefromtheshakerandplacein acentrifuge (e.g,,2500rpm)forabout
65 dTraykneesosff(et.gh.e,t~op50etmhiynlutaecestianttehleaTyeurrbaon-dVapupt%)itwiinttho aagcelaetalnestturbeeamaonfdnietvrapoogreantaentdo `moderate heat (e.g. 50 C).
66 Rcoenctoanisntiinugt1e.t5h%efrormeicaiscind5i0an0dduvoLruotefx5ebr%ief5lmytMomaimxm.oFniiltuemrtahceetsaatmep:le9s5t%hrmoeutghhan0o.l2 4am PVDF or Nylon syringe filers into autosampler vials. Cap vials for analysis,
7.0 AMNAASLSYSSPIESCTBYROHMIEGTHRYP/EMRAFSOSRSMPAENCCTEROMLEITQURIYD(CHPHLRCO/MMSA/TMOS)GRAPHY
71 Conditions are to be optimizedifnecessary.
7.1.1 HPLCConditions
Analytical Column:
Keystone Scientific equivalent
Aquasil
C18,
150
mm
x
2
mm
ID,
or
Guard Column: Elution Flow rate: Injection volume Mobile phase:
Keystone Aquasil C18 400 uL/min.
10 mm x 2 mm
3uL
A: B:
SmM 1.5%
ammonium formic acid
acetate buffer in methanol
GradientProfile:
00.-50-.75.m5imni.n. 510%0A:%:5900A%%BBlineargradient 7.58min. 10%A : 90% Bstepgradient
000105
ANALYTICAL METHOD
Page 100f 13
Method No.: BACG-3548
Tide: DPreetpearrmaitniaotnionanodf PAenraflluyosriosocbtyancHcaPrLboCxalMaatsesinSMpoenckteroymSeterryu/mMaasnsd USrpineec:trSoammeptlrey (HPLC/MS/MS)
--_--m
ee
`Temperature:
81lmin. Ambient
S0%A:S0%B
7.12 PSoEftSwcairee:x APPIE 3S0c0ie0xTTruiprlbeoQQuuaadnrupole Mass Spectrometer Conditions
TuNrotbeo:ioVnaSlpuersayliSsoteudrucneder "MS/MS Acquisition Conditions" override parameters in
this table.
.
Auxiliary Gas
Air (e.g,Grade0.1) at 85popuersnquadreis nch
`Parameter
Value
s
a0
xe
o
=
"w
on
x
mo
2
s
El
`
st
0
so
`
0
0
sn
"
so
2
oe
0
ca
1
000106
ANALYTICAL METHOD
Page 11of 13
Method No.: BACG-3548
Title: DPreetpearrmaitniaotnionanodf PAenrafllyusoirsoocbtyancHcaPrLboCxalMaatsesinSMpoenckteroymSeterryu/mMaasnsd USrpineec:trSoammeptlrey (HPLC/MS/MS)
_--_--mmm--
`Patameter a an oo a wo vou ve
---- Sean type: Polarity Acquisition mode: Pause time:
Value i ` s o ' o o
i MRM Negative Profile 5 milliseconds
Masses requested:
Qsaiosca: n) QEnuien
Qruea ssaslim) Q0u5is
iom ksy o@a 2
D=aiTie DEeS Tine
000107
ANALYTICAL METHOD
Page 120f 13
Method No.: BACG-3548
Title: DPreetpearrmaitniaotnionanodf PAenrafllyusoirsoocbtyaneHcParLboCxalMaatseisnMSpoenckteryomSeterryu/mMaasnsd USrpineec:trSoammeptrlye (HPLC/MS/MS)
---- e m ee 80 CALCULATIONS
81 ttAihtmeeIt,Shpaeereaenkadcschoeafpptteah,belaaenn.daTlphyeteiacdkaalhtearimugnah,ytrabenevdispemweoaoektaahcrheedcaahdsreaotpmeparrtmoiporngiaratatimeo.ntooFfeotnrhsqeutureeasnttthaeirttriaecttleieaauotnsinode,n the ion profilesatthe following mass-to-charge ratios:
AnPaFlOyCte PFHS
I4o1n2Pr9o1f0i3l6e89 3989103989
82 P(lPoFtHSt)hefpreoamkalalrsetaanrdeasrpdosnsveerosfusPtFheOcCondcievnitdreadtiobnyoftthehepetaekstaarretiaclreesinptohnessetaonfdtahredsI,S Alfitteomfatthievdealtya,t(hce.pg.e,aqkuhaedirgahtticsmfiat ywbeieguhsteeddiwnistthea1d/ocfonpceeanktarraetiaosn.ofOthbe ttetshtebaaretsictilceuornrvae cquuradvreataincdiitt).maNoyteb:e Tnheecbeessstarcyutrvoehfaitvmeamyobreedtehpaennodennetsotnatnhdearradncguerovfetfhoerstvaanrdiaoruds concentration ranges using the following:
y=ad+bx+c
whete y= PethaekIhSe(iPghFtHrSe)spionnssteanodfarPdFs.OCdividedbypeakheightresponseof
x= a,b,c
Concentration of the = Constants derived
PFOCin from the
standards. regression
analysis.
8:3
CUosrirnegctthtehesrteasnudltasrdofcusravmep,lecsalfcourlaatney
the level dilutions.
of
PFHC
in
each
unknown
sample.
000108
Page 13 of 13
ANALYTICAL METHOD
Method No.: BACG-3548
Title:
-_
Determination of Perfluorooctanecarboxalate in Monkey Serum and Urine: Sample
Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry
(HPLC/MS/MS)
NOTE:
Due to unresolvable interferences with the test article and/orinternal
stdaisncdraertidofnrooftmthheesmuapterrivxi,seixntgemrnaaslsstspaencdtarrodmqeutarnitsitt.ation may be usedatthe
9.0
9.1
10.0
10.1
ACCEPTANCE AND REJECTIONCRITERIA
Refer to SOP SRI 91-3 for acceptance/rejection criteria except acceptable accuracy for
standards is 80-120% of theoretical.
REPORTING
Results of reports are
all to
analyses are tabulated, and the be filed in the appropriate study
raw file.
data,
original
chromatograms,
and
Greg6ryS. Gorman, Ph.D.,StaffChemist
Bioanalytical Chemistry Group
Approved by:
sD. Jol lytical
IS, MBA, Manager `mistry Group
Date
A daopee
Date
000109
Method No: BACG-3606 ANALYTICAL METHOD
Page lof14
Tite:
DPreetpearrmaitniaotnionaondfPeArnfalluyosrioshebxyaneHsPulLfConaMtaesisn MSoprekcetyroSmeertruymMaansds UrSipneec:trSoammeptlrey
(HPLC/MS/MS)
--------
10 PRINCIPLE
SPeerrfulmuroohrexuarnienseulfsoanmaptlee(sPFarEe).obTthaeinsedefrroofmuurcimynneo(mcogl,g0u.s5mmLon)kceoysnttraeaite(ndPiFwniStg)h sisamfporlteisfiaedrewitthhenamniixnetderwnailthsatanndaiornd-p(IaSi)r,inPgerrefalgueonrto,ocbiuafnfcecraarbnodxwaaltaeter,(fPoFOlCl).owTbehdye acreeetcxaontsett,iftirultwteiaedrtehicdne,ta9htny5dl%iatcrmeoaetnatsthnfeae.nrTorelhdetceootnhatayuiltnaoicsneagtmap1t.le5elra%yveirfaolirsm,riaecnmaodcvaiednd,a,l5eyvz%aepdoSrbamytMeHdPatmoLdmCroynnMeiasussm,s f`rSopmecatbrooumettr5yt/0Ma2s0s,0S0p0ecntgr/ommeLtorfy (PHFPHLSCi/nMsS/MeS).ranTdhuferrmoamng1e0otfroe5li0a0blnegre/sumlitLsnexurtienned.s `SwaitmhpclesoconnbtltaainnrkinmgoaPtrFliHxSsoattchoanctehnetcroantcieonntsragrteiaotnerotfhPaFnH2S0,w0i0l0l nbegw/imtLhimnatyhebeadnigleuotefd reliable results prior to analysis.
TThheemiaosnsspsrpaeycstoruormceetvroylotfagPeFiHssSeatnadt -P2F0O0Civoslatcscwohmipclhiisshleodwienntoheugnhetgaotgirveeatiloynrmeodduec.e the formation ofother potentially interfering ionsextractedfromthematrix. inCcAluUdTiIngObNl:oodS,ipncleapsrmiamaantedsmsearuymc,ararryetoa nbuecmobnesriodferzeodoansobsieosh,aazlalrudnsparnedshearnveddletdiswsiuets, uprnoicveedrusraelsptroecbaeuutsieodnsw.heRnefhearndtloiSngOuPnpnruemsbeerrveSdRpIri2m-a5t-e5tifsosruea. description ofsafety 20 REAGENTS AND SOLUTIONS
Thelistedreagentsortheir equivalents maybeused. 21 Neat Reagents
211 Water, deioannidozrgeandi fre (fromin-house purificasytsiteonns;e.g, Ingalls 210N) 212 Methanol, HPLC grade
000110
ANALYTICAL METHOD
Page2of14
Method No.: BACG-3606
Title: PDreetpearrmaitniaotnioanondfPeArnfalluyosrioshebxyaneHsPulLfConaMtaesisn MSopnekcetryoSmeerturymMaansds UrSipneec:trSoammeptlrey (HPLC/MS/MS)
_--m--m------------------------------
213 Perfluorohexanesulfonate (analyte), as providedbythe client 214 Perfluorooctanccarboxalate (intemal standard), 97% 215 Ammonium acetate, HPLC grade 216 Blankcontrolmonkeyserum 217 Sodium Carbonate, Certified ACS Grade or equivalent 218 Sodium Bicarbonate,Certified ACSGradeorequivalent 219 Ethyl Acetate, HPLC grade 2110 Tetrabutylammonium Hydrogen Sulfate, Aldrich 97% 2111 Sodium Hydroxide 50% solution, Certified grade or equivalent 2112 Blankcontrolmonkeyurine 2113 Formic Acid, 88%
22 Prepared Solutions
Appropriste changes in the solutions may be made at th discretionofthe analyst 221 5mMAmmaceto atein norgai nicfu reewm ater 2211 Fionroerxgaamnipcl-ef,rteeopwraetpearre(e4.lgi,te4rs1,)m.easMuirxeowuetllamamnodnfiiulemratchertoautgeh(eH.Pg,LC1.5m4o2bgi)laenpdhaadsde
filtration apparatus.
222 TBA lon-Pairing Solution (0.5 M tetrabutylammonium hydro.xide) 000111
Page3of 14 ANALYTICAL METHOD Method No.: BACG-3606
Title: PDreetpearrmaitniaotnioanondfPeArnfalluyosrioshebxyaneHsPulLfConaMtaesisn MSopnekcetryoSmeerturymMaansds UrSipneec:trSoammeptlrey (HPLC/MS/MS)
_--mmm--
222.1 hFoyrdreoxgaemnplsuel,fattoepirnepdaerieon2i5zemdLw,adtiesrsaolnvdeaadpjpursotxtihmeapteHlyto4.1204wgiotfht5e0t%raNbuatOyHlasmomloutniiounm.
Note: a more adjustments.
dilate
solution
of
NaOH
in
water
may
be
used
toeffect
smaller
pH
223 Carbonate/Bicarbonate Buffer Solution for Serum (0.25M/0.25M)
223.1 aForpexapmplre, t2oo.p1rx0egpaoirfes1om0d0imuamL,btidciasersbolovnleataeypiprnox1i0m0amteLloyf2d.6e5 igoofsnoidwziauteemdrc.aMriboxnwateellantdo ensure complete dissolution.
224 Carbonate/Bicarbonate Buffer Solution for Urine (1.0M/1.0M)
2241 Faoprperxoamxpilmea,tte8ol.py4regpoarfeso1d0i0ummLb,idciasrsboolnvaetaepipnro1x0i0mmatLeloyf1d0e.6igoofnsiowdzaiteuemrd.caMrbioxnawteellantdo ensure complete dissolution.
30 INSTRUMENTS, MATERIALS, AND APPARATUS
The following or their equivalentsmaybe used.
31 HPLC pump(s), autosampler, and triple quadrupole mass spectrometer 32 Autosampler vials with inserts
33 Vortex mixers (e.g, touch mixer and IKA-Vibrax platform mixer) 34 Solvent-concentration apparatus (e.g., Zymark Turbo-Vap with sourceofnitrogen) 35 HPLCmobile phasefiltrationapparatus
36 Filters for HPLC mobile phase filtration apparatus (c.g, Nylon-66, 0.20 um)
000112
ANALYTICAL METHOD
Pagedof 14
Method No.: BACG-3606
Title: `PDreetpearrmaitniaotnioannodf PAenrafllyusoirsohebxyanHesPuLlfConaMtaesisn MSopnekcteryomSeetrruym/Maansds UrSipneec:trSoammeptlrey (HPLC/MS/MS)
--_----
37 Analytical balance
38 Volumetric flasks (.g., 10 and 25 mL)
39 Disposable Pasteur pipettes
310 Micropipettor(s) with tips
3.1 Culturetubeswithteflon-linedcaps
.
312 Cemifuge
313 Assorted glassware and syringes
3.14 Culture tubes (vials) for use with solvent-concentration apparatus 315 1m Plasticsyringeswith 0.2 pm PVDFsyringe filters 316 Variablespeedhorizontal platform shaker
317 pHumeter
40 PREPARATION OF STOCKS AND WORKING STOCKS oApfptrhoeprainaaltyescth.anAgcetusialntdihleuctoinocnsenwtirlatbieondsoocfutmheenstoleudtoionntshmeapyrebpearmaatdioenasthteheetsd,iscretion
41 Main Stock SolutionofPFHS ~1000 pg/mL.
#411 Pacrceupraarteelaynwe~i1g0h00abpougt/m1L0msgoluPtFiHonSoifntPoFaHS10i-nmLdevioolnuimzeetdriocrgfalnasikc)-.freAeddwadteeiron(iez.egd, organic-free watertodissolve.Dilutetothemark. Altemativly, weigh the compound
000113
PageSof 14
ANALYTICAL METHOD
Method No: BACG-3606
Title:
DPreetpearrmaitniaotnioanondfPAenrafllyusoirsohebxyaneHsPulLfConaMtaesisn MSopnekcteryomSeetrruym/Maansds UrSipneec:trSoammeptlrey
(HPLC/MS/MS)
--mmm
iwnattoear.naMpiprxowperlila.teTrveasnssefler(et.hge.scoulluttuiroenttuobae)claenadnavdedss1el0idmfeLsoifrdeedi.onized organic-free
42 Stock SolutionofInternal Standard (PROC), ~200 pg/mL,
421 `Pwreeipgahreaabonu~t21000mpgg/imnLtosoaluSt0io-nmoLfvPoFluOmCetirnidcefiloansikz)e.dAordgdandiec-iforneiezewdatoerrgaen.igc,.farceceurwaatteelry
ttohdeiscsoomlpvoeaunndddiilnuttoetaotahneampaprrokpwriitathedevieosnsiezled(eo.rgg.a,niccu-ltfurreeewtautbeer).Aalntderaatdidve5l0y,mweLigohf
deionized organic-free water. desired.
Mix well.
Transfer the solution to a clean vessel if
43 `Spiking solution of Internal Standard (PFOC), ~ S0pg/mL
43.1 2Pm repaoreftaL hne~25000gp/g/mmLLssoluotiloniounfttoPaFiOcuClotiunnrdeetiuobneizaenddoardgadni6cm-Lfroeefwdaetieornibzyepdipweattteirn.g Mix well.
44 `Working Stock Solutionsof PFHS
44.1 Ttoabpler.epParreepwaorrekiinn1gs0t-omcLksvoolluutmieotnrsi,cmfalkaesktshoerprotohpeerradpiplurtoipornisataessghlaoswsnwairnet.hIeffdoelsliorweidnga `modifieddilutionschemecanbe usedand documentedinthestudyrecords.
000114
ANALYTICAL METHOD
Page6of 14
Method No.: BACG-3606
Title:
mee DePtreepramriantaiotnioanonfdPeArnfalluyosrioshebxyaneHsPulLfConaMtaesisn (HPLC/MS/MS)
`SMpoenckteryomSeetrruym/Maansds
Urine: Sample
Spectrometry
FE ome E [ey `Approximate Concentration
[ [c s ooa oesosmooowwolsm wcsm m mrwmomoammwa rmommmoooammomwo o mmoansommage|| | | | || 0 m0 ow 0 s g eoe] ] ] ] w] ] a ]]]n] (ng/mL)
`Volume of PFHS solution
Final Volume in
deionizeodrganic free water (mL)
000115
Method No.: BACG-3606
ANALYTICAL METHOD
Page Tof 14
Title:
mes Determination of Preparation and
`APnearlfylsuiosrohbeyxanHesPuLlfConaMtaesisn
(HPLC/MS/MS)
Monkey Serum and
SpectrometryMass
Urine:Sample
Spectrometry
442 Summaryofconcentrationsofserum standards:
Standard Level
Volume and Spike Conc.
Approximate
`Concentration of 'PFHS in serum
(ng/mL)
[To otowtmoosom||rsrnooamgr|]
:
| [ |orein m|| gaaw m0e]] Tr [omosa|smsgmm]
To| oirossorsomngn| | 0] ]
iomo0 rioiwomngns] |20]
CToowmeasn | |0] ]
000116
Method No: BACG-3606 ANALYTICAL METHOD
Page8of14
Title:
Determination of Preparation and
PAenrafllyusoirsohebxyanHesPulLfConaMtaesisn
Monkey Serum and Spectrometry/Mass
Urine: Sample Spectromenry
(HPLC/MS/MS)
_--_--mnmm
50 PREPARATION OF SPIKED STANDARDS AND BLANKS oApfptrhoeprainaaltyescth.angesintheconcentrations ofthesolutionsmaybemadea thediscretion
51 bMaeunlaatlniyapzlleyedzw(eieditgfh.deeaasbciohruetsdte.htroefeu)nsketnsoowfmmsaatmrpilxesst.aAndamradtsraixnddouabmlaetbrliaxabkl(abnlkan(kIbS)l+maaIySn)aalkrseo
52 pIainpptpoertoip1nrd0iiakvtLie.duovafololr~ugma2ne0iac-s.mfdrecesueclwrtaiubtreeedtiiunnbsettshe,eapdtioapfbelttehbaelbaownvokermkfaiotnrrgiexsatc(ohec.ksgt,saon0ld.ua5tridmo.nL.)F.orPtihpeetbliannktsh,e
oorfgianntiecm-aflrseteawnadtaerrdisntsotcekad()~a5n0duvgo/rmtle)xftoore~a5chsteucbonedse.xceptthe blankIS
Addthe 10 I. (pipet 10 of
53 osForolgruastneiiorcnu,fmr1eemsawLamotpelfre.0s.aV2do5drMtt/eh0xo.fe2oa5lcMlhoctwauirbnbegotnfaootreea/abbcoihuctatrubboesn:eacto5en0bd0usf.Lfero,fatnhde1TmBLAoifodne-ipoaniirzienyg. `foclalrowbiongnatto ee/abchictaubbruebffo1enrmaLtnedo1fmTLBoAfdieoino-npiazireidnwgatseorl.utViFooonr,rteu1xrimenaeLcshtaomufpble1ef.so0rMaa/db1do.tu0htMe 5 seconds.
54 Asdettdin2g..5mLofethylacetateandextractonhorizontalmixerfor |hourat a low speed
55
Remtohevtubee `minutes.
fomtheshakerandplacein
acentrifuge
(e.g,
2500
rpm)
for
abou5t
56 Re(m8o,v~e5t0hemtionputeetshiynlatcheetTautrebloay-eVraapndp)uwtitthiantgoenatclleesatnrteuabmeoafnnditervoagpeonraatnedtomdordyenreastse heat (e.g., 50C).
000117
ANALYTICAL METHOD
Page9of 14
Method No.: BACG-3606
Title:
Determination of Preparation and
PAenrafllyusoirschebxyancHsPulLfConaMtaesisn
Monkey Serum and Spectrometry/Mass
Urine: Sample Spectrometry
(HPLC/MS/MS)
----
57 Rceocnotnasitniitnugt1e.t5h%e froersmidiuceacinid5a0n0dkvLorotfex5br%ie5flmytMo amimxm.oFnilituermthaecetsaatmep:l9es5%thmroeutghhan0o.l2 umPVDForNylonsyrifnilgteres intoautosamplervials.
60 PREPARATION OF SAMPLES
61 Avlilgoorwoeusalcy.hsPeruimaspnaamlepilqeuttototohfaewacthosraomopmlete(mep.egr,a0t.u5rmeL.)iVnotrtoeixnedaivcihdsuaalm~p2l0eb-rmiLefcluyl,tbuurte `teuxbpeesc.teIdcfonnceecnetsrsaatriyo,ndiolfuttheeatnestaartlicleiobefiqanngauynsaalomyzpteldewwiillthfalbllwaintkhimntahtreicxonscoentthraattitohne ratunbgeeaonfdthvoerstteaxfndoarrdaccuoruvpel.eoAfddsec1o0npdLs.ofinternalstandardstock (~ 50pg/mL)toeach
62
For serum solution, 1
smaLmoplfes0.a2d5dM/t0he.2f5oMllcoawribnognattoe/cbaicchartbuobnea:te50b0ufifeLro, fatnhde1
TBA mLof
fon-pairing deionized
organicfrocwater. following to each
tVuobrete1xemacLhtoufbTefBoAraiboonu-ptai5rsiencgosnodlsu.tiFoon,r
ur1inmeLsaomfpl1e.s0aMd/d1t.h0eM
Scasercbonodns.ate/bicabrubfofenraatned
1mL ofdeionized :
water.
Vortex
each
tube
for
about
63
Add2.5 setting.
mLofethyl
acetateandextract
on
horizontal
mixer
for1
hour
at
a
low
speed
64 R`mineutesm.theotubvefreomtheshaankdpelarce in acentrifuge (e.g. 2500 rpm)for about
65 Re(moev~e5.t0hmeigtnoupt,eetshyilnatcheetaTtuerlbaoy-eVraapndp)uwtitithiatgoenatclleesanttubreoafnednietavraopgmeonraatnetdmooddreyrnaetses heat (e.g, 50C).
66 Rcoenctoanisntitnugt1e.t5h%e froersimdiuceaciind5a0n0dvuoLrtoefx5b%ri5efmltMyo maimx.moFniiltuermtahceetsaatmep:l9es5%thrmoeutghhan0c2l 1m PVDForNylonsyringe filters into autosampler vials. Capvials for analysis.
000118
Method No.: BACG-3606 ANALYTICAL METHOD
Page 100f14
Title:
DPreetpearrmaitniaotnioanondfPeAnrafllyusoirsohebxyaneHsPulLfConaMtaesisn MSopnekcetyroSmeertruymMaansds UrSipneec:trSoammeptlrey
(HPLC/MS/MS)
_--_--m
70 ASNPAELCYTSRIOSMBEYTHRIYG/HMAPSESRFSOPREMCATNRCOEMELTIQRUYI(DHCPHLCR/OMMSA/TMSO)GRAPHY MASS 71 Conditionsaretobeoptimized ifnecessary.
7.11 HPLCConditions
Analytical Column: Keystone ScientificAquasil C18, 150mm x 2mmID,orequivalent
Guard Column: Elution Flow rate: Injection volume: Mobile phase:
Keystone Aquasil C18 600 pL/min.
10mx 2m mm
suL
AB::1.S5m%Mfoarmmmiocnaciiudmiancmeteattheabnuoflfer
Gradient Profile: Temperature:
0-3min. 3-5min. S-8min. 8-10min. Ambient
S50%A:S50%B 2800%B l% ineaA rgrad: ient
10%A:ste9pg0ra%dieBnt SO%A : SO%Bstep gradient
7.12 SPoEfStcwaireex:APPIE3S0c0i0exTTruiprlbeoQQuuaadnrupoleMass SpectrometerConditions
TNotue: rVablSupeorsayliisStooeudruncnede table.
r
"MS
/MS
Acquisition
Conditions"
overri
de
p
a
r
a
m
e
t
e
r
s
in
this
AuxiliaryGas: Air (e.g., Grade 0.1)at 85popuersnquadreisnch
Parameter
Value
3
2000
000119
ANALYTICAL METHOD
Page 11 of 14
Method No.: BACG-3606
_--_--m(HPLCMSIMS) Title:
Determination ofPerfluorohexanesulfonate in Monkey Serum and Urine:Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry
Parameter
Value
Ne
0
TEM
450
OR
-20
RNG
"120
Qo
10
Q1
1
ST
15
RO1
11
Q2
20
RrOZ
50
ST3
60
RO3
52
DF
250
EM
1800
NEB
15
cur
6
CAD
5
QE
0
POL
1
vem
'
:
000120
Page 120f 14 ANALYTICAL METHOD Method No: BACG-3606 Title: DPreetpearrmaitniaotnioannodf PAenrafllyusoirsohebxyaneHsPulLfConaMtaesisn MSopnekcteryomSeetrruym/Maansds UrSipneec:trSoammeptlrey (HPLC/MS/MS) --_--mnmm
Parameter
Value
PE
0
Scan type: MRM Polarity: Acquisition mode: Pause time: Masses requested: prs: O9L8M9ass(unm)
Negative Profile S milliseconds
M398e9 s)
PFOC(S) OL2M9as (unm)
RDOa2me R0133
1 RsOtI
1
3Q6M8a9ss(ems) 3Ssar S35m I7 r3 1I8] 118 221 221
Duell2T0ieng) D2u0 Time(s)
000121
ANALYTICAL METHOD
Page 130f 14
Method No. BACG-3606
Title:
PDreetpearrmaitniaotnioannodfPeArnfalluyosrioshebxyaneHsPulLfConaMtaesisn
Monkey Serum and Spectrometry/Mass
Urine: Sample Spectrometry
(HPLC/MS/MS)
_--_--m
80 CALCULATIONS
81 Aptaectaochkpseiheaabnpldeeo.,faTnthdhepeaednaaatklayhtmeiiacgyahblteaunnsd,mrpeoevoaitkehwaeredeaaacdsheactpehprrrmooimpnraaittaiotogenr.aomfFttohoreetqneussautnratreitttiahctlireoeant,nednuttsiheoentIhtSeimfaoerne,. profiles at thefollowingmass-to-charge ratios:
PAnFaHlySte PFOC
I3o9n8Pr9o1f0i3l9e89 4129103689
82 PflrootmthaelpleasltaanrdeaarrdsesvpeornssuesofthPeFHcSondcievnitdreatdiboyntohfeptehaekafersetaraertsipcolensienoftthehesItSan(dParFdOs,C) Aqlfiuttaedomrfaattthiievcedfliatyt),a.t(hNeeo.pgt.ee,:aqTkuhhaederibagethsittcscmfuiatrywvebeiefiguthstmeeaddyiwnbisetthedae1dp/ocefonnpdceeenanttkraoarntetiahosne.orfaOtnhgebeotfettsththeabearetssitctialencduonarrrvdea cucornvceenatnradtiitonmraayngbeseunseicnegstshaeryfotlolohwianvge: morethanonestandardcurve forvarious
y=al+bx+c
. where y= PethaekIhSe(iPgFhtOCr)einsstpanoodafrndPssF.HoSdividedbypeakheightresponosef
x= b,c
=CCoonstnancts edeorfnitvehtdefPrrFoHmaSthietnrsetgiarnedosasridnosn.analysis.
83 Uthseinregsutlhtesostfasnadmarpdlecsurfvoer,acanlyoudlialutteitonhse.level ofPFE in each unkuown sample. Correct NOTE: Dfruoemttohuenmraetsroilxv,aebxlteienrtnearlsfetraenndcaersdwiqtuahntthietatteisotnarmtaicylbeaenuds/eordiantttehrenadlissctraentdiaornd ofthe supervising mass spectrometrist.
000122
Page 140f 14
ANALYTICAL METHOD
Method No: BACG-3606
Title:
DPreetpearrmaitniaotnionaonfdPeArnfalluyosrioshebxayneHsuPlLfConaMtaesisn MSopnekcetryoSmeertruymMaansds UrSipneec:trSoammeptlrey
(HPLC/MS/MS)
--_--mm
90 ACCEPTANCE AND REJECTION CRITERIA
91
Refer to SOP SRI 91-3 for acoeptancelrejection standards is 80-120%oftheoretical.
criteria
except
acceptable
accuracy
for
100 REPORTING 10.1 Reasreulttosbeoffalleldanianltyhseeaspaprreoptrabiualtaetesdt,uadnydftilhee. rawdata,originalchromatograms, and reports
Author(s): on - l St. Research Associste Bioanalytical Chemistry Group
Approved by: Greg PhD. `MBainoaangaelyrtical Chemistry Group
Date"2/72/63 9/5/03
Date
000123
ANALYTICAL METHOD
Page1of13
Method No.: BACG-3607
Title: PDreetpearrmaitniaotnioannodf PAemrafllyusoirsoocbtyaneHsPulLfConaMteasisn MSopnekcetyroSmeerturmyMaansdsUrSipneec:trSoammeptlrey (FHPLC/MS/MS)
_--_--mnm
10 PRINCIPLE
PeiSrseflrfouuromtriofiooecrdtawunireitsnhuelafnsoanimanpttleee(msPalFOasrSte)a.nodTbahtradeisn(1eSde), fPrroeorrmfuulrucimoynrneo(oocmetogal.ngeu0cs.a5rbmmooLxna)lkcaetoyesnttraeait(endPiFwOniStgh) esamxpltes rawrieatthhceetnthymliiaxceeotdatnwei.tThhaenetihoynl-paacieitantgelraeaygeernits,rbeumfofveerda,nedvawpaotreart,(ePdfOotCold)lro.ywneedTshbse,y Sracepecetocanttsret,oimtfeuittletrdeyr/eiMdn,a9sas5nSd%ptemrceattnrhsofamenerotrlredyco(tnoHtPaaLiuCntio/nsMgaSm/1p.Ml5Se)r%.vifaolrsm,iacnadciadn,a5ly%zedSbmyMHaPmLmConMiaass, fSraommpalbeosucto2nt0ationi1n0g,P0F00OSnga/tmcL.oofnPcFeOSnitnrsgearretautimearoTtnhhndeafsnrroa1nm0g,e0o100f0rtn oel5ia0bg 0lnemrae/ gsyubl/tiem sndemixultrL ueiLtnnededs. iretlihabcle oresnubltltsapnrrkimoraottroliaxnasloystihsa.ttheconcentration ofPROSwillbewithintherange of
tTh`ThehfeeomriaomnsastsipsorpaneyocsftoruoortmcheeetrvrpoyolottfeangPteiFiaOsllSsyeiatnanttder-Pf2Fe0rO0i0Cnvgoilisotanscswecxhotmirpcalhcitssehdelodfwinetnrthoheuegnohemgattaortgmiirxve.eatiloynrmeodduec.e
inCucnAliuUvdeTirInsgaOlNblp:oroeSdci,anupctleaisoptnrnsia.maantRedessfeemrrauytmo,cSaarrOrePy 9ambnueumcmbobenersriSdoRefIrzeo2do-an5s-ob5sieofsoh,raazalaldruednsspcarrniedpstheiarovnneddletidswsiuets,,
procedurestobeusedwhenhandlingunpreserved primatetissue,
of safety
20 REAGENTS AND SOLUTIONS
The listed reagents or their equivalents may be used. 21 Neat Reagents
211 Water, deionizedandorgani fre (fromin-housepurificationsyste;e.g, Ingalls210N) 212 Methanol, HPLC grade
000124
ANALYTICAL METHOD
Page2of13
Method No.: BACG-3607
Title:
PDreetpearrmaitniaotnioannodf (HPLCMS/MS)
PAenrafllyusoirsoo
cbtyan
eHsPulLfCon
aMteasisn
MSopnekcetryomSeetrruym/
Maansds
UrSipneec:tSraommeptlrey
_--_--m
213
214
215 2.16 217 218
219
2.1.10 211 2112 21.13
22
221 2211
222
Perfluorooctanesulfonate (analyte),asprovided by the client
Perfluorooctanecarboxalate (internal standard), 97%
Ammonium acetate, HPLC grade
`Blank control monkey serum
Sodium Carbonate, Certified ACSGradeorequivalent
SodiumBicarbonate, Certified ACS Grade orequivalent
Ethyl Acetate, HPLC grade
`Tetrabutylammonium Hydrogen Sulfate, 97% Sodium Hydroxide 50% solution, Certified grade or equivalent `Blankcontrol monkeyurine
Formic Acid, 88%
Prepared Solutions
Appropriatechangesinthesolutionsmaybemadeatthediscretion ofth analyst
5 mMAmmoniumacetatein organic free water
Siltration apparatus. For example, toprepare 4liters, measureout ammonium acetate (e.g.1, .542g)andadd in organic-free water (e.g., 4 L). Mix well and filter through HPLC mobilephase
`TBA lon-Pairing Solution (0.5 M tetrabutylammonium hydroxide)
000125
Method No: Title:
_--_--
ANALYTICAL METHOD
Page3of 13
BACG-3607
DPreetpearrmaitniaotnioannodf PAenrafllyusoirsoocbtyaneHsPulLfConaMteasisn MSopnekcetryomSeetrruym/Maansds UrSipneec:trSoammeptlrey (HPLCMS/MS)
222.1
223 223.1
224 224.1
30
31 32 33 34 35 36
iFnordeexioanmipzleed,watotperreapnadraed2j5usmtLt,hdeispsHolvtoe 41.02w4igtohft5e0t%rsNbautOyHlasmolmuotnioinu.m hydrogen sulfite
Note: 2 more adjustments.
dilute
solutionof
NaOH
in
water
maybe
used
to
effect
smaller
pH
Cabonate/Bicarbonate Buffer SolutionforSerum (0.25M/0.25M) F`oraexapmplpe, rt2o.p1or0epgxaorefis1o0dm0imuLam,bidtciasrseboolvnleataepyiprnox1i0m0amteLlyo2f.d6e5iognoifzsedodwiatuemr,caMrbioxnawteellantdo ensure complete dissolution.
Carbonate/Bicarbonate Buffer Solution for Urine (1.0M/1.0M) Faoprpreoxximaatemltypop8.l4reepgoar,feso1d0i0ummL,bidicsasroblvoenaapitprenox1i0m0amtLeloyf1d0.e6igoofnsoidwziautemedrc.aMrbioxnwaetellantdo ensure complete dissolution.
INSTRUMENTS, MATERIALS, AND APPARATUS
The following or their equivalentsmaybe used.
"HPLC pump(s), autosampler, and triple quadrupole mass spectrometer Autosampler vials with inserts
Vortex mixers (e.g. touch mixer and IKA-Vibrax platform mixer)
Solvent-concentration apparatus (e.g., Zymark Turbo-Vap with sourceofnitrogen) HPLC mobile phase filtration apparatus
Filters for HPLC mobile phase filtration apparatus (e.g, Nylon-66, 0.20 um)
000126
Page dof 13
ANALYTICAL METHOD
Method No.: BACG-3607
Title:
Determination of (PrHePpaLrCatMiSon/MaSn)d
Perfluorooctanesulfonate in
Analysis by HPLC Mass
Monkey Serum and
Spectrometry/Mass
Urine: Sample
Spectrometry
_--mmmm----------------
37
Analytical balance
38
Volumetric flasks (.g., 10 and 25mL)
39 Disposable Pasteur pipettes
3.10
Micropipettor(s) with tips
311
Culture tubes with teflon-lined caps
312
Centrifuge
3.13
Assorted glassware and syringes
3.14
Culture tubes (vials) for use with solvent-concentration apparatus
315
1 mL Plasticsyringeswith 0.2 pm PVDFsyrifnilgteers
3.16 Variable speed horizontalplatformshaker
37
PH meter
4.0
PREPARATION OF STOCKS AND "WORKING STOCKS
a1 ApoMpfatrihoneprSaintaaoltcyekscthS.aolnAugctteiusoainlnodtiflhuPetcFiooOnncSsew~nit1lr0la0tb0ieodnposgoc/fumtmLhe.entsoeldutoinotnshmeapryebpearmaatidoensahteehtesd,iscretion
411 oParcrgceaupnraiarcte-eflaryenewe~wi1ag0the0r0atbopoudtigss1mo0lmvsego.lDPuitFliOuotnSeotifonttPohReaOmSa1r0ki-.nmLdAelvitooelrnuuimazeietvdreilocyr,gflawanseikic).g-fhrAteddwadteeiron(iea.egg, he compovad
000127
Page Sof 13
ANALYTICAL METHOD
Method No.: BACG-3607
Title:
PDreetpearrmaitniaotnioanondf PAenrafllyusoirsoocbtyaneHsPulLfConaMteasisn MSopnekcetryoSmeetrruym/MaansdsUrSipneec:trSoammeptlrey
(HPLC/MS/MS)
_-----
iwnattoera.n Mapipxrowperlila.teTrvaensssefler(ct.hge.scoulluttuiroenttuobae)claenadnavdedss1el0mifdLeosfirdeedi.onized organic-ree
42 Stock SolutionofInternal Standard (FOC), ~200 pg/mL
421 P`wreeipgahroabaonu~t21000mpggfiunltosoaluSt0io-nmoLfvPolOumCetirnidcefiloansikz).edoArdgadnidce.iforneiezweadotregra(nei.cg..farceceurwaatteelsy
tthoedcisosmoplvoeuannddidnittoetaoatnheampaprroiprwiiatthedevieosnseilze(dco.rgg,acnuicl-tfurreeetwautbeer).aAnltdeamadtdive5l0y,mweLioghf
deionized desired.
organic-free
water.
Mix well.
Transfer the solution to a clean vessel if
43 Spiking solutionofInternal Standard (PFOC), ~ S0pig/mL 431 P2rmepLaorfetahne~20500ggi/lm.LssoloutliounoitfntPioFoaOcnCulitnurdeetiuonbiezaenddoragdadn6cm-Lfroeefdweioanbizytepdiwpeaettterirng
Mix well.
44 `Working Stock Solutions ofPFOS
44. Ttoablper.ePpraerpeawroerikinng10s-tomcLkvsoolluumtieotnrsi,cmfalkasektshoeroptrhoepreradpiplruotpiroinasatesgslhaosswwnairnet.hIeffdoelsloiwrien5dg `modified dilution scheme can be used and documented in the study records.
AWpoprrkoixnigmSattoecCkoLnecveenltr(aWtiSoLn)| (ug/ml)
Volume ofPFOS solution| Final Volume in deionized organic free water (mL)
salotiooougnt |10| sulotstoooorgmt | 10 |
000128
Page6 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
meesosomosonswonollllsssmmrmmowmmumeateomrooomsmmmmrmnwmoeamesoatom|[ | | ||[mm| n0 mm0 0 e r5us] e] m] e] ] ] ] ]eaa `Title:
PDreetpearrmaitniaotnioannodfPeArvfalluyosriosocbtyaneHsuPlLfConaMteasisn
Monkey Serum and
Spectrometry/Mass
Urine:Sample
Spectrometry
(HPLC/MS/MS)
442 Summaryofconcentrationsofserum standards:
Standard Level
Volume and Spike Conc.| Approximate
Concentration of PFOS in serum
(ng/mL)
o Tiowumomgn [=5]Lio[onwovamassaes||| wo| o o ||ozo
Page 7 of 13
ANALYTICAL METHOD
Method No.: BACG-3607
Title:
mes DeterminationofPerfluorooctanesulfonate in Monkey Serum and Urine:Sample Preparation and Analysis by HPLC Mass Spectrometry/Mass Spectrometry (HPLC/MS/MS)
[(F_ oom | sm]
TooTo[ouonssommero||0nwn] ws] CTioowoprommmm|| 0] ] CcTom momgnr 5]
5.0
PREPARATION OF SPIKED STANDARDS AND BLANKS
Appropriate changesinthe concentratoifotnhse solutionsmaybemadeatthediscretion of the analyst.
5.1
Multiple(e.g.,aboutthree)sets ofmatrix standardsand amatrixblank(blank +IS)are
analyzed with each set ofunknown samples. A matrix double blank(blank-IS) `may also
be analyzed ifdesired.
52
Into individual ~20-mL culture tubes, pipet blank matrix (eg, 05mL). Pipet in the
appropriate volume as described in the table above foreachstandard. For `the blanks,
o`pfipientte1r0naklLsotafnodragradnisct-ofcrke(ew~a50teprgi/nmsLte)atdooef
atchhetwuobrekeixncgespttotchkesbollauntki-oInS.(Apdipdetth1e0
10p
iL
uL
of
organic-free`waterinstead)andvortexfor ~5 seconds.
53 For serum samples add the following to each tube: 500 Lofthe TBA ion-pairing solution, 1mLof0.25M/0.25M carbonate/bicarbonate `buffer, and 1 mLofdeionized `organicfreewater. Vortex eachtube for about 5 seconds. Forurine samples add the following to each tube 1 mL of TBA ion-pairing solution, 1 mL of 1.0M/1.0M carbonate/bicabrufbfoernaantd1e mLof deionizedwater. Vortexeachtu fob rabe out 5 seconds.
000130
Page8of 13 ANALYTICAL METHOD Method No: BACG-3607
Title: DPreetpearrmaitniaotnioannodf PAenrafliyusoirsoocbtyancHsPulLfConaMtaesisn MSopnekcetyroSmeerturmyMaansds UrSipneec:trSoammeptlrey (HPLC/MS/MS) _--mmmm--m--m--m--
54 sAedttdin2g.,5mLofethylacetateandextractonhorizontalmixerfor | hourat lowspeed
55
Remove thetubefrom theshakerandplacein `minutes.
acentrifuge
(e.g.,
2500 rpm) forabout
56
`Takeoffthetop ethylacetatelayerand putitinto acleantubeandevaporatetodryness
(e.8,~50. `minutes in the Turbo-Vap ) with a. `gentle streamofnitrogen and moderate
heat (e.g., 50 C).
57
Reconstitute the residue in 500 uL of 5% 5 mM ammonium acetate: 95% methanol
`containing 1.5% formic acid and vortex briefly to mix. Filter um PVDF or Nylon syringe filters into `autosampler vials.
the
`samples
through
0.2
6.0
PREPARATION OFSAMPLES
61 Avlilgoorwouesalcy.hsPeirpuemtsaanmaplilqeutoototfhaewacthosaromopmlete(mep.ge.r,a0t.u5rme.L)Voirnttoeixnediavcihdsuaalmp~l2e0b-rmieLflcyuf,btuurte
etxupbeecs.tedIcfonnceecenstsraartyi,ondoifluttheeatnetaalritqiucolteboeifnagnaynaslaymzpeldewiwlitfhalbllwainthkimnatthreicxonscoenttrhaatttiohne
`rangeof the standardcurve.Add10 WL ofinternalstandardstock tubeandvortex for acoupleof seconds.
(~
50
pg/mL)to each
62
For serum samples add the following to each tube: 500 uLofthe TBA ion-pairing
solution, 1 mL of 0.25M/0.25M carbonate/bicarbonate `buffer, and 1 mLofdeionized
cfoarorglbalononiwacitnfegr/ebteiocwaaertabecorhn.attVeuobbreutfef1xermeaaLncdho1tfumbTeLBofAfordeiaiobnoo-nuptiazi5eridsnewgcaotsneordl.su.tiVFooonr,rtue1rximenaeLcshaotmfupbl1ee.sf0oaMrd/a1db.ot0uhMte
5 seconds.
63
Asdetdtin2g..5ml. ofethylacetateandextracton horizontalmixerfor 1 hourat low speed
000131
ANALYTICAL METHOD
Page9of 13
Method No.: BACG-3607
Title:
DPreetpearrmaitniaotnio
n of
and.
Perfluoro
Analysis
octa
by
neHsPulLfConaMteasisn
Monkey Serum and
SpectrometryMass
Urine: Sample
Spectrometry
(HPLC/MS/MS)
_--_--mnmmm
64 R`emminouvtees.thetubefromtheshakerandplacein a centrifuge (e.8., 2500rp form abou) t5
heat eg. 50C). 65
`Takeoffthetopethylacetatelayerandput itinto aclean tube and evaporatetodryness
(e.g, ~50.`minutes in the Turbo-Vap ) with a gentle streamofnitrogen and moderate
6.6
7.0 7.1
711
Recconatnasitniitnugte1t.h5e%rfeosrimdiuce iacnid5a0n0duvoLrotfex5b%rie5fmlyMtoammixm.onFiilutemratcheetsataem:pl9e5s%thmreotuhgahn0o.l2 + wm PVDF or Nylon syringe filters into `autosampler vials. Cap vials for `analysis.
ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRY/MASS SPECTROMETRY(HPLC/MS/MS) Conditionsaretobe optimizedifnecessary.
HPLCConditions
Analytical Columa: KeystoneScieutificAquasil C18, 150mm x 2mmID,orequivalent
Guard Column: Elution Flow rate: Injection volume: Mobile phase:
Gradient Profile:
Temperature:
Keystone Aquasil C18 10mx2m mm 400 pl/min.
Sul
BA:: 1S.m5M%foarmmmiocnaiciudm iancemteattheabnuoflfer
0-3min
3 - Smin. 5-8min.
8-10min. Ambient
50%A:SO%B
20% A : 80% Blineargradient 10%A : 90% Bstepgradient
50%A. : 50%Bstepgradient
712 SPoEftSwcaireex:APPEI 3S0ci0e0xTTruiprlbeoQQuuaadnrupole Mass Spectrometer Conditions 000132
Page 10013 ANALYTICAL METHOD Method No: BACG-3607
Title: DPreetpearrmaitniaotnioannodf PAenrafllyusoirsoocbtyaneHsPulLfConatMeasisn MSopnekcetyroSmeerturmyMaansds UrSipneec:trSoammeptlrey
(HPLC/MS/MS)
------------------------eeee
TNuortbeo:ioVnalSupersayliSsoteudrucned:er "MS/MS Acqision Conditions" override parameters in is
Auxiliary Gas:
Parameter I] NC TEM Or RNG Q@ Q1 sT ROI Q2 RO2 sT3 RO3 DF CEM
Air (8, Grade0.1)at 85popuersnquadreisnch
Value 2000 0 450 20 -120 10 1 15 1 20 50 60 52 250 1800
000133
MethodNo: Tite:
_--_--
Page 110f13 ANALYTICAL METHOD BACG-3607
DP(reHetPpeLarrCmai/tnMiaoStn/ioManSn)odf PAenrafllyusoirsoocbtyaneHsPulLfConaMteasisn MSopnekcetyroSmeerturmyMaansds UrSipnee:ctrSoammepilrey
PNaEraBmeter
V1aslue
Cur
6
cap
5
QE
0
,
POL
1
vem
0
PE
0
s SPoclaamts.: MRM Negative APcqausisttiimoe:nmo$dmiel:liseconPdrosfile
Massesrequested:
OP9Lr8oM9sa:s arm) Q989M) Du2e0l Time(s)
OPL9RMOaCsS)am) Q36M89wsem) DRoZoce 3San 3Ssop
sROm} nsousw
(R Q3t) Z B 1 o1s
De0llTime(s)
000134
Page 120f 13 ANALYTICAL METHOD Method No. BACG-3607
Title: PDreetpearrmatiinoant.ioanndo.f PAenrafllyusoirsoocbtyaneHsPulLfConaMteasisn MSopnekcetryoSmeetrruym/MaansdsUrSipneec:trSoammeptlrey (HPLC/MS/MS)
_--mmm ROi)I 21 21
80 CALCULATIONS 81 Apaetcactekhpsetheaabnpldeeo.,faTnthdhepeeadanatakalhymetaiicgyahbltearunnsd,mrpoeevoaitkehawerdceaaacdshecatpheprrrmooipmnraaiatttioeog.nroafmFttohoreetqneussautnartrietttiahctelieroaent,nednuttsiehoenthtISeeaior,ne,
profiles at the following mass-to-charge ratios:
AnPaFlOySte PFOC
I4o9n8P.r9o1f0il4e989 4129103689
82 PfAlrltooemtrntaahlteilpvesetalakyn,atdrhaereapdrseveaeksrhespiugsoohttfhnsPemRsaOcyoeSnbdceeinvutisrdeaedtdiibnoysntotehfaedtophefaepketaeaskrtaeaarrreteaissc.ploeOnisnebotfhttethhseetbIaaSensd(aPrFdsO,C) qiutaodfrtahtiecdfaitl)a. (Neo.tge,:qTuhaedrbaetsitccfuitrvweeifgithtmeadywbiethde1p/econndceennttoanttiohneroftahenotfgesttheaertsitctialencodunrarrvd5e cu`crovnceeantnrdatiitonmraanygbeseunseicnegstshaeryftololohwainvge:morethan one standard curve forvarious
y=altbrte
where y= PtheeakISh(eiPgFhOtCr)esipnosntsaendoafrPdsR.OS divided by peak height response of
x= b,c
=CoCnocnesnttarnattsiodneorfivtehdefPrFoOmSthienrsetgarnedsasridso.n analysis.
000135
ANALYTICAL METHOD
Page 130f 13
Method No.: BACG-3607
-------- Title:
DPreetpearrmaitniaotnioannodf PAenrafllyusoirsoocbtyaneHsPulLfConaMteasisn MSopnekcetyroSmeerturmyMaansds UrSipneec:tSraommeptlrey
(HPLC/MS/MS)
83 Utshienrgetsuhletssotfansdaamrpdlceusrvfeo,rcaalncyudlialtuettihonesl.evel ofPFHCineachunknown sample. Correct
NOTE:
`Dueto unresolvableinterferenceswiththe testarticleand/orinternalstandard
offrotmhtehseumpaetrvriisxi,negxmtearsnsalsspteacntrdoamredtqruiasntt.itationmaybe used atthediscretion
92.0
ACCEPTANCE AND REJECTION CRITERIA
9.1 R`setfaenrdatrodsSiOs P80S-R1I209%1o-f3tfhoeroraectciecpatla.nce/rejection criteria except acceptable accuracy for
10.0 REPORTING
10.1 Reasreultotbseoffialeldainnaltyhseeasapreptarboulpatrsetdiu,adanydtfitlehee. rawdata, original chromatograms,andreports
rubor:
C br nAn Lori Coward, BS
BSir.oaRneasleytairccahlACshseomciisattery Group
Approve:
8
FAD.
Manager
"Lolo
Date
5/5/60)
Date
000136