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PBDE Analysis in Human Blood Serum by Gerstel Twister© Stir-bar Sorptive Extraction Technique and GC/MS-ECNI-SIM

Quality Control Materials

• Calibratorsarepreparedinpre-screenedblankfetalbovineserum• Threemethodcontrolsareaddedtoeachbatchatblank,low,andhighlevels.TheseareBlank:

consistingofprescreenedFetalbovineserum,QL:NISTHumanSerum1957,andQH:NISTHumanSerum1958.TheNIST1958alsocontainednormalhumanpopulationrangesofchlorinatedpesticidesandchlorinatedbiphenylstodemonstratetheselectivityofthemethod.

• Aserumsurrogatecontainingknownconcentrationsofanalytessimilartothecompoundsofinterest,butnotcommonlyfoundinthesamplematrixtested(PBB-155)isspikedintoeverycalibrator,unknownandQCsampletestedinordertomonitorextractionefficiencyandanalyterecovery.

• Internalstandardconsistsofdifluoro-BDE_047(FBDE-47)andBDE156arespikedintoeachsample.

Stir-bar Sorptive Extraction (SBSE)

Extraction procedure is outlined in Figure 1. 0.25 ml of serum is diluted 1:1 with water and denatured using equal volume concentrated formic acid. Twister stir bars are added and allowed to stir at room temperature for 2 hours prior to analysis. Analytes are desorbed from the stirbar in a thermo-desorption unit (Gerstel TDU) and cryo-trapped using a Gerstel CIS4, see Figure 2. A Custom BDE Mixture (1ppm for each congener, AccuStandard Inc.) was serially diluted using isopropanol to prepare the spiking standard. Standard was spiked into screened fetal bovine serum to prepare a 6-point calibration curve (0.064 ppb to 3.20 ppb). Quality control materials run with each batch consisted of blank fetal bovine serum (QB), NIST Human serum SRM-1957 (QL) and Human serum SRM-1958(QH).

Contact Information

Authors: Colin Johnson, Paul R. Loconto, Michael O’Keefe, Bonita Taffe www.michigan.gov/mdchlab

“Providing Quality Laboratory Science for Healthier People and Communities”

Analysis

Stir Bar Conditioning/Cleaning

Results Conclusion

References

Acknowledgements

Standards and Limit of Detection

Methods

Michigan Department of Community Health - Bureau of Laboratories

3350 N. Martin Luther King Blvd. P.O. Box 30035

Lansing, MI 48909Office: (517)- 335-9490

E-Mail: [email protected]: [email protected]

• ThequantificationofPBDEsinhumanserumiscarriedoutusingcapillarygaschromatographic(C-GC)separationwithelectroncapturenegativeionmassselectivedetection(ECNI-SIM,whereSIMstandsforselectiveionmonitoring).Inthismethod,tenBDEcongenersandtwoPBBcongeners(seeFigure2)arebaselineseparatedandallbutBDE-183isquantitatedagainstdifluoro-BDE_047.BDE-183isquantitatedagainstBDE-156duetocomparableretentiontime.Thismethodologydetectselutinganalytesasbromineionsatm/z79,withtheabundancem/z81servingasaqualifierion.Alltargetedanalytepeaksmustmeetthequalifierioncriteria(abundanceofm/z81100%relativetotheabundanceofquantionm/z79).

• AMulti-PurposeSamplerSingleRail,SingleInjector(MPS)wasused[GerstelGmbH&Co.KG].TheMPSismountedatoptheGC-MSD(Agilent6890GC/5973NMSD).TheMPSisintegratedwithChemStation®[AgilentTechnologies,Inc.]andisprogrammedusingMaestrosoftware[GerstelGmbH&Co.KG].

• Thestir-barisplacedinaconditionedTwisterdesorptionlinerandautomaticallytransferredfromthetrayholdertotheThermalDesorptionUnit(TDU),seeFigure3.AnalytessorbedonTwisterarethermallydesorbedintheTDUandthenarecryotrappedontoaglasslinerinsideaCooledInletSystem(CIS).AnalytesarethenthermallydesorbedintheCISintoaDB-XLB(J&WandAgilentTechnologies,Inc.)capillarycolumn.

Background levels of BDE47 and BDE99 from dust are common problems with trace analysis for these compounds due to the ubiquitous use in building materials. Steps taken to lower background in this method includes special washing of all glassware with dust free storage and the following procedure to condition the stirbars:

• SoakTwisters(50:50MeOH:MeCl,2h)• Removeanddryundernitrogen• SoakinTolueneovernight• Removeanddryundernitrogen• PlaceTwistersinglasstubes,bakeat

300oCfor2hrsunderdryN2• RemoveTwistersandallowtocoolin

holder• Increasetemperatureonconditionerto

350oC,bakefor5minutesandremove

• Storeinclean,dustfreevialuntilnextuse.

• GoodseparationofcongenerswasachievedwiththismethodandbackgroundlevelswereacceptableasshowninFigure5

• Thismethodusesa6pointcalibrationcurveinserumforanalysisofBDEcongeners,R2≥0.99.QualitycontrollimitsusingNISTSRM1958forQHandareshowninFigure6

• TheLODforeachcongenermeasuredislessthan0.2ppb• NointerferencewithextractionofBDEsbytheSBSEwasdetectedfromotheranalytesinthe

NISTSRM1958(whichcontainsPCBs,pesticidesandotherorganochlorinemetabolites)• MeasuredanalyteswereaveragedandcomparedwithNISTreportedlevels,Figure7.AllBDEs

exceptBDE85werewithin2SDofreportedvalues;BDE85waswithin3SD.MeasuredvaluesforPBB153werelowerthanNISTreportedvalues.

• AccuracyandPrecisionweremeasuredusingreplicatesoftheArcticMonitoringandProficiencyProgram(AMAP)samplesaswellastheNISTSRMandcomparedtoproficiencyprogramparticipantresults.SeeFigure8

• Arapidanalyticalmethodwhichuses0.25mlofhumanserumhasbeenvalidatedforPBDEs

• ThismethodusesGerstelTwisterSBSEextraction.AprocedureforenhancedBDEbackgroundeliminationandcleanupforstirbarre-usehasbeendeveloped.

• Themethodhasbeendemonstratedtobebothaccurateandpreciseatconcentrationlevelsfoundinhumansubpopulations

• PBDE-47and99aregenerallydemonstratedinanalysis.Thisstudydemonstrateswaystoreducehighenvironmentalbackgroundlevelswithoutextrememeasures(cleanroompreparation).

• Thelimitofdetectionforthismethodforeachcongenerislessthan0.2ppb.Thismethodcouldbeusedforpopulationscreeningtoidentifyingmorehighlyexposedsubpopulations.

1. ATSDR.ToxicologicalProfileforPolybrominatedBiphenylsandPolybrominatedDiphenylEthers(PBBsandPBDEs);AgencyforToxicSubstanceandDiseaseRegistry:Atlanta,GA,2004.

2. BettsKS2008.UnwelcomeGuest:PBDEsinIndoorDust.EnvironHealthPerspect116:A202-A208.

3. ReportonHumanBiomonitoringofenvironmentalChemicalsinCanada,MeasuresSurveyCycle1(2007),HealthCanada/LaboratoriedeToxicologie,InstitutNationaldeSantePublique,Quebec,CA,www.healthcanada.gc.ca

4. SjödinA,WongLY,JonesRS,ParkA,ZhangY,HodgeC,DiPietroE,McClureC,TurnerW,NeedhamLLandPatterson,DG,“SerumConcentrationsofPolybrominatedDiphenylEthers(PBDEs)andPolybrominatedBiphenyl(PBB)intheUnitedStatesPopulation:2003–2004”,Environ.Sci.Technol.,2008,42(4),pp1377–1384.

5. Johnson,PI,Stapleton,HM,Sjodin,A,Meeker,JD,“RelationshipsbetweenPolybrominatedDiphenylEtherConcentrationsinHouseDustandSerum”,EnvironSciTechnol,44,5627-32,2010.

6. Loconto,P.R.,“Evaluationofautomatedstirbarsorptiveextraction-thermaldesorpton-gaschromatographyelectroncapturenegativeionmassspectrometryfortheanalysisofPBDEsandPBBsinsheepandhumanserum”JournalofChromatographicScience,47(2009)656-669.

7. Loconto,P.R.“Selectivityandsensitivityimprovementsforselectedpolybrominateddiphenylethersandpolybrominatedbiphenylsusingcapillarygaschromatography/electroncaptureneagativeionmassselectivedetection:acosteffectiveapproachtobiomonitoring”LC-GCNorthAmerica26(11)(2008)1118-1130.

8. J.K.Taylor.QualityAssuranceofChemicalMeasurements.LewisPublishers,BocaRaton,FL1987

IUPAC name Abbreviation LOD1 ppb

• 2,2’,4,4’,5,5’-Hexabromobiphenyl PBB-153 • 2,2’,4,4’,6,6’-Hexabromobiphenyl PBB-155 • 2,4,4’-Tribromodiphenyl Ether BDE_028 0.122 • 2,2’,4,4’-Tetrabromodiphenyl Ether BDE_047 0.165 • 2,3’,4,4’-Tetrabromodiphenyl Ether BDE_066 0.112 • 2,2’,3,4,4’-Pentabromodiphenyl Ether BDE_085 0.160 • 2,2’,4,4’,5-Pentabromodiphenyl Ether BDE_099 0.160 • 2,2’,4,4’,6-Pentabromodiphenyl Ether BDE_100 0.190 • 2,2’,4,4’,5,5’-Hexabromodiphenyl Ether BDE_153 0.182 • 2,2’,4,4’,5,6’-Hexabromodiphenyl Ether BDE_154 0.168 • 2,2’,3,4,4’,5’,6-Heptabromodiphenyl Ether BDE_183 0.192 • 5,5’-difluoro-2,2’,4,4’-tetrabromodiphenyl ether F-BDE_047, IS • 2,3,3’,4,4’,5-Hexabromodiphenyl ether BDE_156, IS

LOD was determined by calculating the standard deviation at each standard concentration following repeated measurements (n=15-20) of the lowest 3 concentration standards in serum. The absolute values of the standard deviations were then plotted versus concentration. The intercept of the least squares fit of this line equals So, with 3So being the LOD. J.K. Taylor. Quality Assurance of Chemical Measurements. Lewis Publishers, Boca Raton, FL 1987.

Figure 2

Sample Preparation for Twister Stir-bar Sorptive Extraction in Human Serum Matrix

Rinse a 5mL headspace vial 3x with dichloromethane.

Place a previously cleaned and conditioned Twister [10mm long and 0.5mm film thickness] into the headspace vial.

To 0.25mL of unspiked or spiked human serum is added 0.25mL of HPLC Grade H2O.

Then add 0.50mL of 5% acetonitrile in concentrated formic acid.

Stir at 500 rpm for exactly two hours then removed the Twister and

place in HPLC Grade H2O to remove biological material.

Dry the Twister with a Kim-Wipe or equivalent and place the Twister in a clean desorption liner.

Carefully place the transport adapter atop the desorption liner that

contains the Twister and place in the TDU tray holder.

Figure 1

Figure 3

Thermal Desorption Unit liner with Twister stirbar in place

Thermal Desorption Unit (TDU) on Agilent GC

Figure 4

Figure 6: QC CHARTS

F11-PBDEsPBDE_028_QH

Individ.: cl : 0.467143 ucl: 0.541331 lcl: 0.392955 * Rule violationSubgrp Size 1

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Figure 5 NIST 1958 (QH) Serum Chromatogram showing baseline separation of congeners.

Time, Minutes

Blank Fetal Bovine Serum spiked with FBDE-47 and BDE-156 internal standards. This figure illustrates a stable baseline, indicating a clean solid-phase on the stir-bar as well as a clean blank serum for calibrators.

BDE/BB Congener Retention Time (min) BDE-28 8.780 BDE-47 10.035 BDE-66 10.495 BDE-100 12.353 BDE-99 13.181 BDE-85 14.190 BB-153 15.504

BDE-153 16.659 BDE-183 19.383

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

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9.813

11.182

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BDE-156

Time, Minutes 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500

3.676

3.730

3.839

3.909

3.952

4.024

4.222

4.310

4.349 4.457

4.528

5.251

5.388

5.485

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9.832

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12.353

13.181

14.910 15.232 15.504

16.659 19.383

Abu

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ce

Figure 8: AMAP Recovery

AMAP W0906

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BDE-47

BDE-100

BDE-99

BDE-154

BDE-153

BDE-183

PPB

Inter-LabTwister

AMAP W0909

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Inter-LabTwister

AMAP W1004

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AMAP W1005

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AMAP W1007

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AMAP W1008

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BDE-47

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BDE-153

BDE-183

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Inter-LabTwister

Abstract

Introduction

Polybrominated diphenyl ethers (PBDEs), widely used fire retardants, are currently under investigation as agents interfering with immunological, embryonic development and endocrine functions. Low population background levels have favored analysis by high resolution mass spectrometry (HRMS), limiting this analysis to highly specialized laboratories. This presentation describes a rapid solvent free extraction of PBDEs from a small serum sample (0.25 ml), with processing time of 2-3 hours using Twister® stirbar sorptive extraction. Gas chromatographic separation with electron capture negative ion mass selective detection (GC/ MSD-ECNI-SIM) produces quantitative results with detection limits fit for purpose using instrumentation readily available in most analytical laboratories.

Polybrominated diphenyl ethers (PBDEs) have been widely used as fire retardants in many consumer items including polyurethane foam found in furniture and carpeting, home electronics and other textiles. In 2004 Penta-BDEs and Octa-BDEs were banned by the European Union and US production was phased out at this time, however items containing these chemicals persist. The BDEs consist of 10 homolog groups (1 to 10 bromine atoms) with 209 possible congeners. BDEs are not chemically bound to products and can be mobilized by leaching or degradation of treated materials over time.

Concentrations of BDEs in indoor air and dust have been shown to be significantly higher than outdoor concentrations. A recent study of the relationship between house dust and serum concentrations of BDEs in adults shows high correlation5. Young children are considered to be at highest risk of exposure from house dust due to their full body contact with the floor and carpets and hand to mouth activity. BDEs are classed as potential endocrine disruptors and the mechanism of action is currently under investigation in animal models with respect to immunological, embryonic development and endocrine functions1,2. Further epidemiological research to evaluate human response to these agents is needed.

Low general population background levels and background environmental contamination by these analytes has favored quantitative analysis by high resolution mass spectrometry (HRMS), which is costly, time consuming and limits this analysis to highly specialized laboratories3,4. Building on previous work in this laboratory6,7 this presentation describes the characterization and validation of a rapid solvent free extraction of a small serum sample (0.25 ml), with processing time of 2-3 hours to analysis using solid phase extraction of analytes from matrix (Twister® stir bar sorptive extraction). This method involves solvent-less extraction of hydrophobic organics with a 10mm long and 0.5mm thick Twister® stir-bar coated with a non-polar polydimethylsiloxane (PDMS). The stir-bar is dropped into an aqueous matrix containing hydrophobic POPs and stirred for a period of time. Partitioning into the PDMS coating is related to the octanol-water partition coefficient for the analyte.

Analysis by capillary gas chromatographic separation with electron capture negative ion mass selective detection (GC/ MSD-ECNI-SIM), produces quantitative results with detection limits fit for purpose using instrumentation more readily available to most analytical laboratories. Validation parameters demonstrated in this method include linearity, accuracy, detection limits, and precision (evaluating intra and inter assay variation).

Figure 5

Figure 6

NIST Human Serum 1958

0.0000

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1.2000

PBDE-028

PBDE-047

PBDE-066

PBDE-100

PBDE-099

PBDE-085

PBDE-153

PBDE-154

PBDE-183

PBB-153

PPB

NIST ± SDMeasured

Figure 7

Figure 8

Thisposterwassupportedby:• TheMichiganDepartmentofCommunityHealthwithfundingfrom

theCDCPublicHealthEmergencyPreparedness(PHEP)CooperativeAgreement#2U90TP517018-11

• AnappointmenttotheEnvironmentalPublicHealth(EPH)FellowshipProgram(CJ)administeredbytheAssociationofPublicHealthLaboratories(APHL)andfundedbytheCentersforDiseaseControlandPrevention(CDC).

• GERSTELGlobalAnalyticalSolutionswithtrainingofCJandinstrumentalsupportduringthisproject.