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Wayman Turner, Wanda Whitfield, Troy Cash, Emily DiPietro, Cheryl McClure and Andreas Sjodin

Using the FMS Power-Prep6 for Same Sample Cleanup for Multiple Classes of POPs and Quantification by GC/ID-HRMS on the Thermo Fisher DFS. Wayman Turner, Wanda Whitfield, Troy Cash, Emily DiPietro, Cheryl McClure and Andreas Sjodin.

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Wayman Turner, Wanda Whitfield, Troy Cash, Emily DiPietro, Cheryl McClure and Andreas Sjodin

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  1. Using the FMS Power-Prep6 for Same Sample Cleanup for Multiple Classes of POPs and Quantification by GC/ID-HRMS on the Thermo Fisher DFS Wayman Turner, Wanda Whitfield, Troy Cash, Emily DiPietro, Cheryl McClure and Andreas Sjodin 8th International Symposium on Recent Advances in POPs Analysis April 11, 2013 – San Francisco, CA National Center for Environmental Health Division of Laboratory Science

  2. Each step is Important and has its own unique challenges • Sample collection, storage & handling • Spiking with 13C-internal standards • Pre-cleanup (extraction, SPE etc…) • Sample Cleanup and Enrichment • Sample Evaporation • Analysis & Quantitation (GC/HRMS) • QA/QC • Data Handling

  3. Median serum dioxin levels in selected populations Ground troop veterans in AOVS Controls Low Medium High Ranch Hand veterans Controls Officers - non-flying Officers - flying (navigators) Officers - flying (pilots) Enlisted men - flying Enlisted men - non-flying Occupational exposures NIOSH workers - quintile 1 quintile 2 quintile 3 quintile 4 157 quintile 5 355 German plant workers New Zealand sprayers Seveso, Italy population 4540 Without chloracne 16,600 With chloracne 0 20 40 60 80 100 1000 10,000 Serum dioxin (ppt)

  4. Time tends for TCDD levels “Birth Cohort Effect” - observed level related to birth year. Source: DioxinFacts.org

  5. The Ranch Hand Dioxin Exposure Index was not correlated with serum TCDD levels 400 300 200 Serum TCDD level (ppt) 100 0 0 10 10 20 30 40 50 60 70 80 Ranch Hand exposure index (N = 147)

  6. Anal Chem 56 (1984) 1830-1842

  7. Smith, Stalling & JohnsonSchematic

  8. Late-1980’s Automated System

  9. First Generation FMS Prototype Cleanup System with Programmable Peristaltic Pump

  10. Current FMS Power-Prep6 System

  11. Close-up of Modules

  12. Packaging – ABN-Silica, Alumina & Carbon

  13. Serum vs. Adipose 2,3,7,8-TCDD Levels

  14. Total 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 Organohalogen Toxicant Measurements in Human Samples. Chemosphere 68: 824-831 (2007) Short Formula = 2.27*TCHOL + TRIG + 62.3 Long Formula = 1.667*(TCHOL-FCHOL) + FCHOL +TRIG + PLIPID • Ryan J.J. and Mills P. Lipid Extraction from Blood and Biological Samples and Concentrations of Dioxin-Like Compounds. Chemosphere 34 (1997) 999-1009.

  15. Plot of SF_TOTLIP vs. LF_TOTLIP for the 228 Pools by Age Group (R2=0.995).

  16. GilsonMicroman Pipet

  17. Gilson 215 Liquid Handler

  18. Rotary Evaporator

  19. Biotage [Zymark] Turbovap II

  20. Separtory Funnel

  21. Selected References • Chang R.R., Jarman W.M. and Hennings J.A. Sample Cleanup by Solid-Phase Extraction for the Ultatrace Determination of Polychlorinated Dibenzo-p-dioxins and Dibenzoburans in Biological Samples. Anal. Chem. 65: 2420-2427 (1993). • Turner W., DiPietro E., Cash T.P., McClure P.C., Patterson, D.G Jr., and Shirkhan H. An Improved SPE Extraction and Automated Sample Cleanup Method for Serum PCDDs, PCDFs and Coplanar PCBs. Organohalogen Compounds 19: 31-35 (1994). • Brock J.W., Burse V.W., Ashley D.L., Najam A.R., Green V.E., Korver M.P., Powell M.K., Hodge C.C. and Needham L.L. An Improved Analysis for Chlorinated Pesticides and Polychlorinated Biphenyls (PCBs) in Human and Bovine Sera Using Solid-Phase Extraction. J Anal. Toxicol . Nov/Dec: 528-536 (1996). • Barr J.B., Maggio V.L., Barr D.B., Turner W.E., Sjodin A., Sandau C.D., Pirkle J.L., Needham L.L., and Patterson D.G. Jr. New High-Resolution Mass Spectrometric Approach for the Measurement of Polychlorinated Biphenyls and Organochlorine Pesticides in Human Serum. J. Chromatography B, 794: 137-148 (2003). • Sjodin A., Jones, R.S., Lapeza C.R., Focant J-F, McGahee E.E. and Patterson D.G. Jr. Semiautomated High-Throughput Extraction and Cleanup Method for the Measurement of Polybrominated Diphenyl Ethers, Polybrominated Biphenlys and Polychlorinated Biphenlys [& Organohalogen Pesticides] in Human Serum. Anal. Chem. 76: 1921-1927 (2004).

  22. Automated C18-SPE

  23. Needle Evaporator

  24. Biotage [Zymark] Turbovap LVII

  25. FMS PowerVap

  26. High Resolution Mass Spectrometers • Micromass 70E (Adipose) • Micromass 70SE (Serum) • MicromassAutospec • MicromassAutospecUltima • Thermo Electron MAT95 XL (Apr ‘00) • Thermo Electron MAT95 XP • Thermo Fisher DFS (Since May ‘06) • Cryogenic Zone Compression HRMS?

  27. Thermo Fisher DFS

  28. LOD (pg/g Lipid) for TCDD MAT95 vs. DFSAssumptions: 70% Recovery & Total Lipid 0.6 %

  29. NHANES (www.cdc.gov/nhanes) National Health and Nutrition Examination Survey (administered by NCHS, CDC) Stratified, multistage, national probability sample of the civilian, non-institutionalized population Data cycles every 2 years starting 1999-2000 30 localities via mobile trailers; about 10,000 people every 2 year cycle Environmental components are 1/3 subsets Data collected Extensive questionnaire on demographics and health behaviors Extensive physical exam Medical and nutritional laboratory tests

  30. Current NHANES Analyte List

  31. Selected References

  32. Polychlorinated Dibenzo Dioxins and Furans Native Dioxins OCDD 33445P 3344P 3445P 234678F 123478F 123678D 123678F 123478D 2378D 123789D 1234678D 1234679D 23478F 2378F 1234678F OCDF 1234789F 12378F 12378D 334455P 123789F 234678F 123678F 33445P 13C Labeled Dioxins 3445P 12378D 3355P 334455P 123678D 3344P OCDF 123478D 2378D 123478F 123789D 1234678F 123789F 1234678D 23478F 2378F OCDD 1234789F 12378F 1234679D 1234D Figure 1

  33. PolychlorinatedNaphthalenes Native PCN N66/67 N52/60 N27 Relative Abundance N64/68 N73 N75 Time (min) 13C Labeled PCN N66/67 N52/60 N27 Relative Abundance N64/68 1234D N73 N75 Time (min) Figure 2

  34. Polybrominated Dibenzo Dioxins and Furans Native Brominated Dioxins 1234678BF 2378BF 123478/678BD 2468BF 23478BF 1234678BD 123478BF 2378BD 123789BD OBDD 12378BD 12378BF OBDF 13C Labeled Brominated Dioxins 1234678BF 123478/678BD 2378BF 2468BF 23478BF 123478BF 2378BD 1234678BD 123789BD OBDD 12378BF 12378BD OBDF Figure 3

  35. PCDD/PCDF/cPCBs: Unadjusted Geometric Means and Selected Percentiles (95% Confidence Intervals) of Serum Concentrations (pg/g lipid) by Age, Sex, Race/Ethnicity for the U.S. Population, NHANES 2003-2004

  36. PCDD/PCDF/cPCBs: Unadjusted Geometric Means and Selected Percentiles (95% Confidence Intervals) of Serum Concentrations (pg/g lipid) by Age, Sex, Race/Ethnicity for the U.S. Population, NHANES 2003-2004

  37. Is it time to get into the pool(ing)?

  38. “Any data is better than no data!”Larry Needham

  39. NHANES 2001-2002 Pools

  40. Serum Pools: NHANES 2001-2002 • 34 People per pool (Total 1,734 people; 51 pools) • Unweighted: used for estimates of the “means” • 0.75mL Serum per person • 25.5 g Serum per pool • 2 g BFRs/PCBs/Persistent Pesticides • 22 g PCDDs/PCDFs/cPCBs; PCNs • 0.5 g Total Lipids • 0.4 g PFCs

  41. Geometric mean TEQ NHANES 2001-2002 Pools Patterson et al. Chemosphere 73 (2008) S262-S277 Caudill et al. Chemosphere 69 (2007) 371-380 (Geometric mean estimation from pooled samples)

  42. Pooled-Sample Design for Dioxins and PCBsUsing NHANES 2005-2006 Samples8 Samples per Pool

  43. Pooling NHANES 2005-2006 SamplesWas it worth all the trouble? • The samples in the NHANES 2005-2006 survey were pooled with 8 samples per pool and analyzed for 74 PCDDs/PCDFs/cPCBs, PBDDs/PBDFs, PCNs, PCBs, PBDEs and OC pesticides. The number of samples analyzed was reduced from 2041 to only 247 at a savings of ~$2.8 million. • Pooling samples also resulted in lower limits of detection (LODs) because of the larger sample volumes are available for analysis.

  44. PCDDs/PCDFs/cPCBs (pg/g Lipid)

  45. PCNs (pg/g Lipid)

  46. PBDDs/PBDFs (pg/g Lipid)

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