mdch lab poster bonita3
TRANSCRIPT
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
O
O
O O
O
OO
O
O O
O
O
O
O
O
O
O
O
Individ.
cl
lcl
ucl
0.4
0.45
0.5
0.55
2 4 6 8 10 12 14 16 18 20
F11-PBDEsPBDE_047_QH
Individ.: cl : 0.609333 ucl: 0.921229 lcl: 0.297438 * Rule violationSubgrp Size 1
O
O OO O
O
O
O
O
O
OO
O
O
Individ.
cl
lcl
ucl
0.4
0.6
0.8
2 4 6 8 10 12 14 16 18 20
F11-PBDEsPBDE_100_QH
Individ.: cl : 0.335294 ucl: 0.407457 lcl: 0.263131 * Rule violationSubgrp Size 1
O
OO
O
OO
O
O
OO
OO
O
O O
O
O
Individ.
cl
lcl
ucl
0.25
0.3
0.35
0.4
2 4 6 8 10 12 14 16 18 20
F11-PBDEsPBDE_099_QH
Individ.: cl : 0.364444 ucl: 0.567712 lcl: 0.161177 * Rule violationSubgrp Size 1
O
O
O
O
O
O
O
O
O
O O
O O O
O
Individ.
cl
lcl
ucl
0.2
0.3
0.4
0.5
0.6
2 4 6 8 10 12 14 16 18 20
F11-PBDEsPBDE_154_QH
Individ.: cl : 0.268421 ucl: 0.398075 lcl: 0.138767 * Rule violationSubgrp Size 1
O
O
O
O
O
OO
O OO O
O
O O
OO
O
O
Individ.
cl
lcl
ucl
0.15
0.2
0.25
0.3
0.35
0.4
2 4 6 8 10 12 14 16 18 20
F11-PBDEsPBDE_153_QH
Individ.: cl : 0.298333 ucl: 0.413582 lcl: 0.183085 * Rule violationSubgrp Size 1
OO
O
O
O
O
O
O
O
O O O O
O
O
O
O
Individ.
cl
lcl
ucl
0.2
0.25
0.3
0.35
0.4
2 4 6 8 10 12 14 16 18 20
F11-PBDEsPBDE_183_QH
Individ.: cl : 0.38 ucl: 0.549917 lcl: 0.210083 * Rule violationSubgrp Size 1
O
O
O
O
O
OO
O
O
O
O
OO
O O
O
O
O
Individ.
cl
lcl
ucl
0.2
0.3
0.4
0.5
2 4 6 8 10 12 14 16 18 20
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
500
1000
1500
2000
2500
3000
3500
4.369
9.813
11.182
20.923 Abu
ndan
ce
FBDE-47
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
6.491
7.150 7.547
8.780
9.832
10.035
10.495 12.220
12.353
13.181
14.910 15.232 15.504
16.659 19.383
Abu
ndan
ce
Figure 8: AMAP Recovery
AMAP W0906
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
BDE-28
BDE-47
BDE-100
BDE-99
BDE-154
BDE-153
BDE-183
PPB
Inter-LabTwister
AMAP W0909
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
BDE-28
BDE-47
BDE-100
BDE-99
BDE-154
BDE-153
BDE-183
PPB
Inter-LabTwister
AMAP W1004
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
BDE-28
BDE-47
BDE-100
BDE-99
BDE-154
BDE-153
BDE-183
PPB
Inter-LabTwister
AMAP W1005
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
BDE-28
BDE-47
BDE-100
BDE-99
BDE-154
BDE-153
BDE-183
PPB
Inter-LabTwister
AMAP W1007
0.00
0.10
0.20
0.30
0.40
0.50
0.60
BDE-28
BDE-47
BDE-100
BDE-99
BDE-154
BDE-153
BDE-183
PPB
Inter-LabTwister
AMAP W1008
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
BDE-28
BDE-47
BDE-100
BDE-99
BDE-154
BDE-153
BDE-183
PPB
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
0.2000
0.4000
0.6000
0.8000
1.0000
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.