1. Evolution of an Analytical Method for Brominated Flame Retardants K.A. MacPherson, T.M. Kolic and E. J. Reiner Ministry of the Environment –Toronto, Ontario

2. 2 Overview Ontario Ministry of Environment –Dioxin Laboratory BFRs Sample Preparation GC-HRMS method development (2000 – 2006) Current status/capability Future analyses / Method Development

3. 3 MOE - Laboratory Services Branch LaSB provides analytical support for the ministry's –environmental monitoring –regulatory programs –data supports standard setting –analytical method development (emerging issues) –ensures the data quality of ministry compliance enforcement and emergency analytical testing –Emergency response

4. 4 MOE – LaSB -Dioxin Laboratory Polychlorinated Dioxins and Furans(DFPCB - E3418) Dioxin-Like PCBs (DFPCB - E3418) Congener Specific PCBs by HRMS(PCBC - E3459) Brominated Flame Retardants (PBDE - E3430) Polychlorinated Naphthalenes (PCN - E3431) Perflourooctane Sulfonate (PFOS – E3457) Brominated and mixed Br/Cl dioxins and furans(PBDDc/PBDFs)* Polychlorinated Diphenylethers (PCDE - 3432)* LSB Method numbers in brackets (*) = Method under development

5. 5 BFRs in the Environment Initial estimates by Environment Canada suggest: –sewage biosolids may be a major contributor of the total PBDE loadings to the environment (excluding landfills) –Sewage sludge, a product from waste water treatment process, which is organic rich and often applied as fertilizer on agricultural land

6. 6 BFR survey in Biosolids NMA – Nutrient Management Act 2002 Legislation –The purpose of this Act is to provide for the management of materials containing nutrients in ways that will enhance protection of the natural environment and provide a sustainable future for agricultural operations and rural development. In response to increased tendency for corporate farming, diminishing concern for land stewardship and increased concern for water quality & quantity

7. 7 Sample Preparation

8. 8 Sample Cleanup Considerations Acid/Base/AgNO 3 cleanup may be repeated with difficult samples Standard dilution for injection on GC-HRMS = 1:50 Difficult samples (& “overloaded”) may require dilutions up = 1:800 (many co-extractables present)

9. 9 Ideally one injection per sample Major & Minor congener consideration –Some yet un-identified congeners present at minor concentrations –Some predominant congeners need dilution for accurate quantification

10. 10 The Era Begins….2000 4 function - SIM Experiment HP 6890+ GC, 40m.18mm x.18µm rtx500 column Ultima NT, running MassLynx (v4.0) software Monitor & quantify six congeners; Br 3 – Br 7 : BZ# 28,47,99,100,153,154

11. 11 2002 – Search for Deca 6 function - SIM Experiment Monitor & quantify; Br 1 – Br 10 (no Br 8/ Br 9 ) 13 C 12 -BDE analytical standards available in calibration standard: BZ#:3, 15, 28, 47, 99, 153, 154, 183, 209

12. 12 2004 - 2006 Non-bde analytical standards available in CVS –HBB – Hexabromobenzene –PBEB – Pentabromoethylbenzene –BB-153 – Hexabromobiphenyl –BTBPE – 1,2-Bis(2,4,6-Tribromophenoxyethane) –DBDPE - Decabromodiphenylethane

13. 13 Analytical Challenges Ensure Deca BDE efficiently introduced to GC –Injector conditions –Temperature –Liner –Carrier gas flow Ensure Deca BDE efficiently leaves GC! –Minimal Film Thickness –Minimal length –Appreciable ID

14. 14 2000-2006 Uniliner emulates on-column injection and reduces discrimination Uniliner potentially effective without sealing column and would also increase longevity Minimal injections no matter which injection port liner when high concentrations present

15. 15 Injector Maintenance ! Over time non-volatile sample residues in injector liner provide sites for absorption of critical analytes Enhanced Response With clean injector (The Reporter On-Line)

16. 16 30m RTX500.25mm x.15µm – 42 minutes

17. 17 2003 SEPARATION extract injected on each of 2 columns: 15m & 30m QUANTIFICATION - Br 3 -Br 7 30m x 0.25mm x 0.10µm DB5-HT Br 10 15m x 0.25mm x 0.10µm DB5-HT

18. 18 GC experiments 15M Injector:280  C Split/Splitless.9mL/min 110  C 40  C/min  200  C 10  C/min  330  C hold until deca elutes 30M Injector 280  C Split/Splitless 1.8mL/min 110  C 10  C/min  140  C 80  C/min  220  C 5  C/min  330  C hold until deca elutes

19. 19 30m DB-5HT (0.25mm x 0.10µm)

20. 20 15m DB5-HT (0.25mm x 0.10µm)

21. 21 15m DB-5HT (0.25mm x 0.10µm) 2005 CVS

22. 22 Wide Range in Physical Properties Mass –230 amu - mono –972 amu - deca Melting Point –300  C Boiling Point –420  C

23. 23 Analytical Challenges - HRMS Experiment Calibration Perfluorokerosene (PFK) is used as a reference calibrant Instrument calibration requires PFK masses which bracket the range of ions in the ion window (SIR experiment). Originally, modification of PFK reference file (Ultima-NT) necessary for successful SIR calibration up to ~971 Da. ( 13 C 12 BDE 209) Filename=c:\masslynx\ref\pfk.ref

24. 24 Reference Compound Mixture Perfluorokerosene -H (PFK) Boiling Point: 210 - 240  C Supplier:  1 Industrial Dr., Pelham, NH 03076 Catalogue No.: 16596

25. 25 Molecular Ion Quantification WHY 13 C 12 BDE 209 Available Analytical Standard Greater probability of positive identification No interferents in native ion channel WHY NOT 1.Better response for [M - 2Br] + ion 2.Difficult calibration at this mass range

26. 26 You never know what you’ll find 13 C 12 -BDE 209 DBDPE Decabromodiphenylethane

27. 27 Leading to production of analytical standard “ Characterization of Mass-Labeled [13C14]- Decabromodiphenylethane and its use as a Surrogate Standard in the Analysis of Sewage Sludge Samples” A.Konstantinov a *, G.Arsenault a, B.Chittim a, K.MacPherson b, A.McAlees a, R.McCrindle c, D.Potter a, E.Reiner b, C.Tashiro a and B.Yeo a. Chemosphere 2004

28. 28 2006 : Analysis for BFRs 1 Analytical Run GC: 15m DB5-HT (0.25mm x 0.10µm) HRMS: 6 function SIM Experiment CONGENERS: 46 Native + 5 non-BDE BFRs 19 13 C 12- BDEs 4 Injection Standards ~16 min. for last elutor (BDE 209) >900 samples reported

29. 29 Future Method Development Transfer method to benchtop GC-MS/MS Excellent sensitivity 1pg DecaBDE (molecular ion) Less cost per analysis Skill level for operation < sector instruments

30. 30 DBDPE 13 C 12 -BDE 209 BDE 209 13 C 12 -BDE 209 DBDPE MS/MSMS/MS HRMSHRMS

31. 31 10pg DecaBDE / 20pg DBDPE

32. 32 Acknowledgements Tereza Gobran – Sample Preparation Wellington Laboratories – Analytical Stds.