A Brief 25-year History and Recent Developments in...
Transcript of A Brief 25-year History and Recent Developments in...
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A Brief 25-year History and Recent
Developments in Automated High-
Throughput Sample Preparation of
Human Specimens for the
Measurement of PCDDs, PCDFs,
cPCBs, PCNs, PBDDs, PBDFs,
PCBs, PBDEs and OC Pesticides
by GC/ID-HRMS.
A Brief 25-year History and Recent
Developments in Automated High-
Throughput Sample Preparation of
Human Specimens for the
Measurement of PCDDs, PCDFs,
cPCBs, PCNs, PBDDs, PBDFs,
PCBs, PBDEs and OC Pesticides
by GC/ID-HRMS.
Wayman Turner - Senior Laboratory Scientist – Centers
for Disease Control and Prevention Foundation
4th International Seminar on Total Solutions for POPs Analysis
Workshop at Dioxin 2011, Brussels, Belgium
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Each step is Importantand has its own unique challenges
Each step is Importantand has its own unique challenges
• Sample collection, storage & handling
• Spiking with 13C-internal standards
• Pre-cleanup (extraction, SPE etc…)
• Sample Cleanup and Enrichment Procedure
• Sample Evaporation
• Analysis & Quantitation (HRGC/HRMS)
• Data Handling & QA/QC
• Sample collection, storage & handling
• Spiking with 13C-internal standards
• Pre-cleanup (extraction, SPE etc…)
• Sample Cleanup and Enrichment Procedure
• Sample Evaporation
• Analysis & Quantitation (HRGC/HRMS)
• Data Handling & QA/QC
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Smith, Stalling & Johnson
Schematic
Smith, Stalling & Johnson
Schematic
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“Mid-1980’s” First Attempt At “Hand” Automation
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Late-1980’s Automated SystemLate-1980’s Automated System
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Eldex ControllerEldex Controller
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RUNAWAY (Tom Selleck) – killer spider robots
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FMS Programmable PumpFMS Programmable Pump
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Early FMS Power-Prep Early FMS Power-Prep
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Six-way & Two-way ValvesSix-way & Two-way Valves
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Early 5-Carbon Column SystemEarly 5-Carbon Column System
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Current FMS Power-Prep
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FMS Six-way Valve
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Caliper (Zymark) TurboVap II & LVCaliper (Zymark) TurboVap II & LV
TurboVap II TurboVap LVTurboVap II TurboVap LV
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Gilson 215 Liquid Handler
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1,015,472 Analyses(PCDDs/PCDFs/cPCBs, PCBs and OCPs)
1,015,472 Analyses(PCDDs/PCDFs/cPCBs, PCBs and OCPs)
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Rotary EvaporatorRotary Evaporator
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Separtory FunnelSepartory Funnel
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Automated C18-SPEAutomated C18-SPE
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FMS PowerVapFMS PowerVap
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Needle EvaporatorNeedle Evaporator
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Gilson Microman PipetGilson Microman Pipet
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Biotage [Zymark] Turbovap II Biotage [Zymark] Turbovap II
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Biotage [Zymark] Turbovap LVIIBiotage [Zymark] Turbovap LVII
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Total Lipid ReferencesTotal Lipid References
• Akins J.R., Waldrep K., and Bernert J.T. Jr. The Estimation of Total Serum Lipids by a Completely Enzymatic 'Summation' Method. Clin. Chim. Acta. 184: 219-226 (1989).
• Phillips D.D., Pirkle J.L., Burse V.W., Bernert J.T., Henderson L.O. and Needham L.L. Chlorinated Hydrocarbon Levels in Human Serum: Effects of Fasting and Feeding. Arch. Environ. Contam. Toxicol. 18: 495-500 (1989).
• Bernert J.T., Turner W.E., Patterson D.G. Jr. and Needham L.L. Calculation of Serum “Total Lipid” Concentrations for the Adjustment of Persistent OrganohalogenToxicant Measurements in Human Samples. Chemosphere 68: 824-831 (2007)
Short Formula = (2.27*TCHOL + TRIG + 62.3Long Formula = 1.667*((TCHOL-FCHOL) + FCHOL +TRIG + PLIPID
• Akins J.R., Waldrep K., and Bernert J.T. Jr. The Estimation of Total Serum Lipids by a Completely Enzymatic 'Summation' Method. Clin. Chim. Acta. 184: 219-226 (1989).
• Phillips D.D., Pirkle J.L., Burse V.W., Bernert J.T., Henderson L.O. and Needham L.L. Chlorinated Hydrocarbon Levels in Human Serum: Effects of Fasting and Feeding. Arch. Environ. Contam. Toxicol. 18: 495-500 (1989).
• Bernert J.T., Turner W.E., Patterson D.G. Jr. and Needham L.L. Calculation of Serum “Total Lipid” Concentrations for the Adjustment of Persistent OrganohalogenToxicant Measurements in Human Samples. Chemosphere 68: 824-831 (2007)
Short Formula = (2.27*TCHOL + TRIG + 62.3Long Formula = 1.667*((TCHOL-FCHOL) + FCHOL +TRIG + PLIPID
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NOTHING IS IMPOSSIBLEFor those who don’t have to do the work!
NOTHING IS IMPOSSIBLEFor those who don’t have to do the work!
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Analyte List
Polychlorinated Dibenzo-p-dioxins and Dibenzofurans Polybrominated Dibenzo-p-dioxins and Dibenzofurans1,2,3,4,6,7,8,9-Octachlorodibenzo-p-dioxin (OCDD) 2,3,7,8-Tetrabromorodienzo-p-dioxin (TBDD)
1,2,3,4,6,7,8-Heptachlorodibenzo-p-dioxin (HpCDD) 1,2,3,7,8-Pentabromodibenzo-p-dioxin (PeBDD)
1,2,3,4,7,8-Hexachlorodibenzo-p-dioxin (HxCDD) 1,2,3,4,7,8-Hexabromorodibenzo-p-dioxin (HxBDD)
1,2,3,6,7,8-Hexachlorodibenzo-p-dioxin (HxCDD) 1,2,3,6,7,8-Hexabromodibenzo-p-dioxin (HxBDD)
1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin (HxCDD) 1,2,3,7,8,9-Hexabromodibenzo-p-dioxin (HxBDD)
1,2,3,7,8-Pentachlorodibenzo-p-dioxin (PeCDD) 1,2,3,4,6,7,8-Heptabromoodibenzo-p-dioxin (HpBDD)
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) 1,2,3,4,6,7,8,9-Octabromodibenzo-p-dioxin (OBDD)
1,2,3,4,6,7,8,9-Octachlorodibenzofuran (OCDF) 2,3,7,8,-Tetrabromodibenzofuran (TBDF)
1,2,3,4,6,7,8-Heptachlorodibenzofuran (HpCDF) 1,2,3,7,8-Pentabromodibenzofuran (PeBDF)
1,2,3,4,7,8,9-Heptachlorodibenzofuran (HpCDF) 2,3,4,7,8-Pentabromodibenzofuran (PeBDF)
1,2,3,4,7,8-Hexachlorodibenzofuran (HxCDF) 1,2,3,4,7,8-Hexabromodibenzofuran (HxBDF)
1,2,3,6,7,8-Hexachlorodibenzofuran (HxCDF) 1,2,3,6,7,8-Hexabromodibenzofuran (HxBDF)
1,2,3,7,8,9-Hexachlorodibenzofuran (HxCDF) 1,2,3,7,8,9-Hexabromodibenzofuran (HxBDF)
1,2,3,7,8-Pentachlorodibenzofuran (PeCDF) 2,3,4,6,7,8,-Hexabromodibenzofuran (HxBDF)
2,3,4,6,7,8-Hexachlorodibenzofuran (HxCDF) 1,2,3,4,6,7,8-Heptabromodibenzofuran (HpBDF)
2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) 1,2,3,4,7,8,9-Heptabromodibenzofuran (HpBDF)
2,3,7,8-Tetrachlorodibenzofuran (TCDF) 1,2,3,4,6,7,8,9-Octabromodibenzofuran (OBDF)
Dioxin-like Polychlorinated Biphenyls - cPCBs Polychlorinated Naphthalenes3,3',4,4'-Tetrachlorobiphenyl (PCB 77) 1,2,3,4-Tetrachlorinated naphthalene (PCN 27)
3,4,4',5-Tetrachlorobiphenyl (PCB 81) 1,2,3,5,7- and 1,2,4,6,7-Pentachlorinated naphthalene (PNC 52 & 60)
3,3',4,4',5-Pentachlorobiphenyl (PCB 126) 1,2,3,4,5,7- and 1,2,3,5,6,8-Hexachlorinated naphthalene (PNC 64 & 68)
3,3',4,4',5,5'-Hexachlorobiphenyl (PCB 169) 1,2,3,4,6,7- and 1,2,3,5,6,7-Hexachlorinated naphthalene (PNC 66 & 67)
1,2,3,5,7,8-Hexachlorinated naphthalene (PCN 69)
1,2,3,4,5,6,7-Heptachlorinated naphthalene (PCN 73)
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We Have Come a Long WayAutomation has significantly changed our throughput
• In 1987 one person (original LLE & semi-automated cleanup method) could process 10 samples for TCDD only in 5 days.
• Today one person (using SPE & PowerPrep) can process 60 samples in 5 days for PCDDs/PCDFs/cPCBs + PCNs and PBDDs/PBDFs.
• In addition today one person (using SPE & Rapid Trace) can process 150 samples in 5 days for PCBs (including mPCBs for TEQ), OC pesticides and PBDEs.
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Polychlorinated Dibenzo Dioxins and Furans
Figure 3
Native Dioxins
13C Labeled Dioxins3355P
3445P3344P
3344P
3445P
2378F
2378F
2378D
2378D
33445P
33445P
1234D
12378F
23478F
12378D334455P
12378F
23478F
334455P12378D
123478F
123678F234678F
123478D123678D
123789D
123789F
123478F
123678F
234678F
123478D
123678D
123789D
123789F
OCDF
OCDD
OCDD
OCDF
1234678F
1234679D
1234678D
1234789F
1234678F1234789F
1234679D
1234678D
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Polychlorinated Naphthalens
Figure 4
Native PCNs
13C Labeled PCNs
N42
N42
N28
N27
N27
N48
N52/60
N50N54
N53
N52/60
N66/67
N71/72N64/68
N69
N73
N66/67 N64/68
N73
1234D
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Native Pesticides
13C Labeled Pesticides
Pesticides 2010
Figure 2
a-HCCHHCB
PCAnisol
b-HCCH
g-HCCH
HCB PCAnisol
a-HCCH
b-HCCH
g-HCCH
Aldrin
Aldrin
OCS
OCS
Isodrin
Isodrin
Oxychlor
Oxychlor
H.Epox
H.Epox
g-Chlrdn
g-Chlrdn
a-Chlrdn
a-Chlrdn
t-Nona
t-Nona
op-DDE
op-DDE
pp-DDE
pp-DDE
Dieldrin
Dieldrin
Endrin
Endrin
op-DDT
op-DDT
pp-DDT
pp-DDT
Par50
Par50
Par62
Par62
Par2
6P
ar2
6
Mirex
Mirex
c-N
ona
c-N
ona
pp-Moxyc
pp-Moxyc
1234D
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Observation from the ‘real world’ by an analytical
chemist who retired after 57 years from NIST ...
It has been estimated that concentrations of 1 in 1018 of almost any
substance can be expected to be present in almost any sample. This
translates to approximately a thousand molecules. In: Quality Assurance of
Chemical Measurements by John K. Taylor (1987, p48).
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Top : Native PCBs
Bottom: 13C-labeled PCBs
Figure 118
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19
4
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Figure 2
Top : Native Pesticides
Bottom: 13C-labeled Pesticides
HCB
HCCHOXYCHLOR
H. EPOX
T-NONA
ppDDE
DIELDRIN
opDDTppDDT
MIREX
HCB
OXYCHLOR
H. EPOX
T-NONA
ppDDE
DIELDRIN
opDDT
ppDDT
MIREX
HCCH
HCCH
HCCH
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LOD (pg/g Lipid) for TCDD MAT95 vs. DFSAssumptions: 70% Recovery & Total Lipid 0.6 %
LOD (pg/g Lipid) for TCDD MAT95 vs. DFSAssumptions: 70% Recovery & Total Lipid 0.6 %
SWEIGHT MAT95 DFS
5 1.786 0.595
10 0.893 0.298
15 0.595 0.198
20 0.446 0.149
25 0.357 0.119
30 0.298 0.990
35 0.255 0.085
40 0.223 0.074
45 0.198 0.066
50 0.179 0.060
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High Resolution Mass SpectrometersHigh Resolution Mass Spectrometers
• Micromass 70E (Missouri Adipose)
• Micromass 70SE (Missouri Serum)
• Micromass Autospec
• Micromass Autospec Ultima
• Thermo Electron MAT95 XL (Apr „00)
• Thermo Electron MAT95 XP
• Thermo Fisher DFS (May „06)
• 2-D GC (GCxGC)/HRMS (Future?)
• Micromass 70E (Missouri Adipose)
• Micromass 70SE (Missouri Serum)
• Micromass Autospec
• Micromass Autospec Ultima
• Thermo Electron MAT95 XL (Apr „00)
• Thermo Electron MAT95 XP
• Thermo Fisher DFS (May „06)
• 2-D GC (GCxGC)/HRMS (Future?)
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Thermo Fisher DFS Thermo Fisher DFS
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Extra SlidesExtra Slides
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