HighResolutionLCQTOFMSfortheScreeningofUnknown

ContaminantsinBiologicalandEnvironmentalSamples

M.Wang,E.Parry,E.Houtz,S.Salihovic,S.Anand,Petreas,M.,ParkJS.

BiomonitoringBranch,ECLDepartmentofToxicSubstanceControl

9/28/16

WhyQTOFScreening?Beyondthetargetedanalytes list

Agilent6550LCQTOF

The Agilent 6550 iFunnel Q-TOFsupports the Agilent Jet Stream ESI and the Dual Agilent Jet Stream ESI.

Massaccuracy:subppmlevel

Highresolution

Nolossofdataacrossentiremonitoredm/zrange(1001700,smallmolecule)

PresentationOutline

SystemandProjectsOverview TargetSuspectScreening>NontargetSuspectScreening> SemiUnknownapproach

ProjectsSummary(biologicalandenvironmentalsamples)

1.SFOwastewaterscreening2.Catserumscreening3.FoamProject

FBF(findbyformula)algorithm

MFE(molecularfeatureextraction)algorithm

1.TargetSuspectScreening:SampleswithinterestedTargetedChemicalFamily

2.NonTargetSuspectScreening:notlimitedtooneparticularlyfamilybutexpandtobroader

nontargetcontaminants

3.SemiUnknownApproach:beyondcurrentdatabases

ProjectsOverview SFOwastewater:historicalusageandcurrentannualtesting

ofAFFF(aqueousfirefightingfoam)atSFOresultinhighconcentrationofPFASsinwastewater.Whatarethoseprecursors/intermediatesintheWWinthetreatmentplantsbeforedischargetobay?TargetSuspectScreening

Catserumexposome screening:apilotprojecttosetupbiomonitoring/serumsamplemethodontheQTOF.Convenientsamplesof5HTcatsand5Normalcatsserum.compoundsprofilescomparisonbetweenHTandNormalcats.NonTargetSuspect+SemiUnknown

Participateinthesaferconsumerproductprojects(SB1019labeling):helpconfirmifthereisnopresenceofOPFRs,aswellasexpandidentificationofotherFRspresentinfabric/foams:intheprocessofsummarizeintoastandardmethod.TargetSuspectScreening

Library1. organophosphateflameretardants

(OPFR)library:922. ExpandedPFASlibrary:2443. InHouseLCMSlibrary(using

consumerproductchemicalsindex,combinewithourinhousechemicalsanduptodateliteratures):~2500

4. EPATox21library(DSSTox_ToxCastRelease_20151019):~9000

5. EOAlibrary(Environmentalorganicacids,CollaborationProjectwithUCSF):744 3.GoldSmith et.al.FoodChem.Toxicol.,2014,269279

4.Richardet.al.,Chem.Res.Toxicol.,2016,12251251

AspinoffprojectfromourLCMS/MSmethodsofPFASanalysis

0

3

6

9

12

11/19/2015 11/24/2015 11/29/2015 12/4/2015 12/9/2015 12/14/2015

Concentrationg

/L

6:2FtS

PFOS

TimelineofSampling(2015)

9

11/19FirstAFFFAddition

11/25Endof1stRoundofAFFF

12/1MoreAFFF

12/3EndofAFFF

11/1911/20

11/2311/25 11/30 12/2 12/6 12/9 12/14

12/1512/3

ErikaHoutzetal,2016ACSpresentation,SanDiego

SamplecollectedAnalyzedbyLCMS/MSforPFASSuiteAnalyzedbyTOPAssayAnalyzedbyLCQTOFMS

TotalPFASlevel(TOPassay)muchhigherthantotalconcentrationoftargetedPFASmeasuredinthewastewater

TargetedPFAS

Total TOPassay

QTOF:ExpandedPFASlibrary

workflow Full Scan TIC, triplicate data set

Inspect Data Quality, RT shift, peak intensity

MPP for compounds Grouping and Alignment,

and blank substraction

FBF (Find By Formula using appropriate parameters), search against PFAS library

Recursive Qual Analysis1. Generate Compound list2. Inspect Chromatography3. Inspect Mass spectrum4. review overall score

Further Identification and confirmation1. Literature search2. Standard comparison3. MS/MS spectra

Adatabasewascompiled,whichcontainedthemassesandelementalformulasforallpotentialAFFFfluorochemicals identifiedinpatents.

105compoundsidentifiedinAFFFsamples:C3C15

Accuratemass

Isotopicpatternmatch

WelldefinedChromatographyPeak

12/3/15CompositeInfluent

12/3/15CompositeEffluent

KatieC.HardingMarjanovic et.al.,Environ.Sci.Technol.2015,49,76667674

SFOWWScreening

Expandtheanalytes listbeyondtheoriginalLCMS/MSmethod:foundprecursors,intermediates,andnewreplacementPFASs:ADONA,EtherPFHxS,KetonePFOS

Helpconfirmationofthesourcesandtransformationpathways

WorkingonCompareWWprofilesfromdifferentsources:MPPanalysis

Previously,wehaveusedLCMS/MSserummethodtoanalyzed12PFAScompoundsincatserumsamples.

PilotprojectforbiologicalsampleQTOFscreening

NormalCatsControl(5)

HTCatsCase(5)

Questions Asked For This Study

1.Exposome ofCatSerum:NonTargetSuspectScreening:Beyondthe12commonPFCAs;

Highproductionvolume(HPV)chemicals,pharmaceuticals,pesticides,polyandperfluoroalkyl substances(PFAS)andflameretardants

2. Aretheredetectableprofiledifferencesbetweenthecontrolandcasegroups?

SerumPrep:asimpleproteinprecipitationmethod

MeOH

Vortex

Centrifuge

Evaporation

Reconstitutionandinstrumentanalysis

the MPP (Mass Profiler Professional) non targeted workflow

Full Scan TIC, triplicate injections

MFE (Molecular Feature Extraction) with appropriate parameters

Export to MPP, Grouping and Alignment: Features aligned across all data sets

Apply Quality Control Filters, Blank subtraction, Recursive analysis

PeakfilterMasstoleranceRTtoleranceIonspecies

Export for Identification: exact mass, isotope pattern, retention time1. Search against databases2. Unknowns: elemental

formula generation

Profiles Comparison MPP1. Group as control and case2. Comparison using volcanic

plot, p 2

Identify features that are up or down regulated between case and control groups

Further Identification and confirmation1. Search Chemspider/Pubmed and/or Merck Index

(search by formula, by accurate mass) 2. MS/MS spectra

Exposome Profile

CatExposome:All10samplesFeature:Mass@RT

~1300afteralignment

Cpd Label Name RT Formula Score Mass488 Cpd48benzoicacid 2.105 C7H6O2 99.14 122.036794 Cpd94allylcaproate 3.449 C9H16O2 99.03 202.121347 Cpd471octen3ol 2.593 C8H16O 99.02 174.125857 Cpd571octen3ol 2.88 C8H16O 98.94 174.1261366 Cpd362,2,6,6tetramethyl4methylideneheptane 6.813 C12H24 98.49 214.192766 Cpd66tertbutylhydroquinone 3.046 C10H14O2 98.48 166.0993763 Cpd761,2benzisothiazol3(2H)one 7.567 C7H5NOS 98.3 151.009235 Cpd352{[2,2dimethyl3(oxiran2ylmethoxy)propoxy]met 2.353 C11H20O4 98.27 216.1364302 Cpd30decene 6.194 C10H20 98.27 186.1623490 Cpd49perfluoro1butanesulfonate 4.028 C4HF9O3S 98.24 299.9508116 Cpd1 ID0273from:Env.ScienceTechnol.,2013,47(10), 3.822 C11H18O 98.21 166.1359713 Cpd7 1,2benzisothiazol3(2H)one 7.201 C7H5NOS 98.18 151.00947 Cpd7 allylcaproate 0.724 C9H16O2 98.16 202.1205

583 Cpd58dibutyladipate 4.585 C14H26O4 98.13 258.1835166 Cpd16dibutyladipate 4.511 C14H26O4 98.06 258.1832493 Cpd491,1,2,2,3,3,4,4,5,5,6,6,6Tridecafluoro1hexanesulfo 5.249 C6HF13O3S 98.06 399.94331266 Cpd1 Lufenuron 6.954 C17H8Cl2F8N2O3 97.98 509.9781130 Cpd13ethenyldecanoate 3.95 C12H22O2 97.97 244.1679139 Cpd13ethenyldecanoate 4.054 C12H22O2 97.96 244.168293 Cpd292,6,8Trimethyl4nonanoneID#204 6.104 C12H24O 97.96 244.20487 Cpd87diisopropyladipate 3.332 C12H22O4 97.89 230.1518771 Cpd77ID0270from:Env.ScienceTechnol.,2013,47(10), 8.28 C35H70O2 97.86 568.5426128 Cpd1 1,2benzisothiazol3(2H)one 3.906 C7H5NOS 97.85 151.00934 Cpd4 butyl2methylprop2enoate 0.579 C8H14O2 97.73 188.1052

191 Cpd19dibutyladipate 4.968 C14H26O4 97.69 258.1834423 Cpd4 DL12hydroxystearicacid 7.205 C18H36O3 97.64 300.2669470 Cpd47palmiticacid 7.794 C16H32O2 97.44 256.2404228 Cpd2 2,2,4trimethyl1[(2methylpropanoyl)oxy]pentan3 5.43 C16H30O4 97.41 286.214867 Cpd67isoeugenol 3.045 C10H12O2 97.4 210.0892288 Cpd28decene 6.079 C10H20 97.15 186.1616213 Cpd2 17alphaEstradiol 5.216 C18H24O2 97.05 318.183734 Cpd731H,1H,2H,2Hperfluorooctanesulfonate(6:2FTS) 5.618 C8H5F13O3S 97.04 427.9748

PFBS

PFHxS

6:2FTS

Foodpreservativeandadditives

Presentinonlyonesample

HTvs.Normal:Profilescomparison

Venndiagram,HTvs.Normal

Normal HT

Volcanoplot:UpvsDownRegulation

UpregulatedentitiesDownregulatedentities

5vs5:verylimitedsamplesize

Profilecomparison:HTvs.Normal

IDBrowserUpregulated,Score>80

Name Formula Mass RT Score commentDuloxetine C18H19NOS 343.1241 5.617 96.76 depressiondrugPFOSA C8H2F17NO2S 498.953 6.568 96.73 PFASPFOSAA C10H4F17NO4S 556.9582 6.236 95.59 PFASCatechol C6H6O2 110.0368 0.905 87.47 phenol/pesticideLleucine C6H13NO2 131.0947 0.619 86.9 dietarysupplement

acetaminophen C8H9NO2 151.0634 1.458 86.75 pharmaceuticaltexanol C12H24O3 216.1722 5.738 86.44 coalescentforlatexpaints

myristicacid C14H28O2 228.2093 7.208 85.83 commonfattyacid

acetophenone C8H8O 120.0569 5.286 85.56commonfragrantketoneused

infragance(S)hydroprene C17H30O2 266.2239 7.274 85.37 insecticide

dodecylmethacrylate C16H30O2 254.2236 7.644 82.19anesterthatmightbeusedin

PCP

Profilecomparison:HTvs.Normal

IDBrowserUpregulated,Score>80

Name Formula Mass RT Score commentDuloxetine C18H19NOS 343.1241 5.617 96.76 depressiondrugPFOSA C8H2F17NO2S 498.953 6.568 96.73 PFASPFOSAA C10H4F17NO4S 556.9582 6.236 95.59 PFASCatechol C6H6O2 110.0368 0.905 87.47 phenol/pesticideLleucine C6H13NO2 131.0947 0.619 86.9 dietarysupplement

acetaminophen C8H9NO2 151.0634 1.458 86.75 pharmaceuticaltexanol C12H24O3 216.1722 5.738 86.44 coalescentforlatexpaints

myristicacid C14H28O2 228.2093 7.208 85.83 commonfattyacid

acetophenone C8H8O 120.0569 5.286 85.56commonfragrantketoneused

infragance(S)hydroprene C17H30O2 266.2239 7.274 85.37 insecticide

dodecylmethacrylate C16H30O2 254.2236 7.644 82.19anesterthatmightbeusedin

PCP

11

3

Tentativelyidentifiedcompounds,Downregulated

Name Formula Mass RT Score commentLufenuron C17H8Cl2F8N2O3 509.9782 6.954 97.98 fleacontrolagent

(+)isostearicacid C18H36O2 284.271 7.826 93.26 fattyacid,PCP

Databasematch

MolecularFormulaGeneration

SemiUnknowns:ThosethathaveNoDatabase

match

TRUE C15H24N2O3PS 97.8 343.1243 343.1245 0.59 0.59 0.2 5.617FALSE C19H21NO3S 97.01 343.1243 343.1242 0.14 0.14 0.05 5.617

HalogenContainingFeatures

USINGMASSSEARCH

MerckIndex:searchbyformulamass,lowresolution

EPACompTox Dashboard:SearchbymonoisotopicMass,highresolution

405.9483 0.0035

CatExposome ScreeningSummary

Establishedmethodforbiologicalsampleprepandscreening.Thebiologicalexposomeincludes:variousPFASs,PCPs,pesticides,pharmaceuticals,FRs

Exploratoryprojectonprofilescomparison:procedures,criteria's

SOPinprocess

WhatsNext?SlidefromJonSobus presentation,USEPA

StandardProtocols,SuspectScreening

SampleProducts(eg.Foam)

Serum

Identification/Confirmationofnewemergingcontaminants.DevelopnewmethodsforTargeted/QuantificationAnalysis

FlameRetardants EOA/Exposome/Metabolimics

ExpandedPFAS

Wastewater

AcknowledgementsB2Group

HEMSGroupDr.SteveGarner,AlbanyAnimalHospital,Albany,

California,USAWilliamZolan,SupervisingChemist,MelLeong

TreatmentPlantDr.BruceLabelle,ECLChief

Diclazuril isusedincatstotreatIsosporaspecies

Pubmed Search

Venndiagram

Targetedvs.nontargetedSFOWW

MPPDataprocessing:nontargetedworkflow,recursiveanalysis

Profileplots,131features:76upregulated,55downregulated

SystemOverview:AnalysismethodHPLCsystem:AgilentUPLC1290 AgilentextendedC18column,1.8um,2.1X50mm ElectrosprayIonization(ESI),negative/positiveionpolarity

fullscan(m/z1001700) TwosetsofGradientsforpositive/negativeionization SystemCalibrationbeforeeachrunusingAgilentTuningmix

(ESILLowConcentrationTuningMix) Referencesusedduringthesamplerun