Detecting Vx Metabolites in a Human Victim

DETECTING VX METABOLITES IN A HUMAN VICTIM

KEL DANIEL MARSHALL UNIVERSITY FORENSIC SCIENCE PROGRAM APRIL 11, 2014 OUTLINE

• VX Background - Toxicity • Banning - Chemical Weapons Convention • Analysis of serum from a confirmed victim • Proof of VX use • New preparation method for analysis to increase sensitivity • Conclusions

2 VX BACKGROUND

Chemical Name: • O-ethyl S-2-diisopropylaminoethyl methylphosphonothioate

• Irreversible Acetylcholinesterase inhibitor • Injection, Inhalation, Ingestion, Absorption th • LD50,intravenous = 0.0201 mg/kg ≈ 3/100 of a drop • 4,400 tons ≈ 3 trillion deaths 3 CHEMICAL WEAPONS CONVENTION

International treaty banning the possession and use of chemical weapons

As of October 14, 2013: • State parties signed and ratified: 190 • State parties signed but not ratified: 2 • State Parties neither signed nor acceded to the CWC: 4 North Korea South Sudan Egypt Angola 4 CHEMICAL WEAPONS CONVENTION

The US has destroyed 90% of its Category 1 chemical weapons. Category 1: VX, Sarin, Soman, Tabun, Sulfur Mustards, Saxitoxin, Ricin and precursors

5 CHEMICAL WEAPONS CONVENTION

Blue Grass Army Depot in Kentucky still houses 523 tons of VX, Sarin, and Mustard Gas

6 7 VICTIM OF VX

• Aum Shinrikyo – Japanese cult

• Created 100-200 g of VX and 20-30 kg of Sarin

• Sarin used in: Matsumoto City: 8 killed and hundreds injured Tokyo subway: 13 killed and thousands injured

• VX used to eliminate a former member

8 VICTIM OF VX

9 VICTIM OF VX

Biomarker: unchanged VX? EMPA? MPA? 10

VICTIM OF VX

VX Derivatized!

EMPA 11 AQUEOUS LAYER

t-BDMS

GC-CI-MS-MS product ion spectrum of [M+H]+ confirming ID of t-BDMS derivatization of EMPA 12 ORGANIC LAYER

DAEMS

GC-CI-MS-MS product ion spectrum of showing DAEMS 13 PROOF OF VX IN HUMAN BODY

14 PROOF OF VX IN HUMAN BODY

15 PROOF OF VX IN THE HUMAN BODY

To be used for forensic purposes, a biomarker must: 1. Lifetime • Alleged military use – several days, possibly longer • Terrorism – much less 2. Chemical stability in biological sample 3. No non-chemical warfare sources 4. Detectable at trace levels • Organization for the Prohibition of Chemical Weapons • Diagnosis of victims • Monitor health of employees at destruction facilities

16 NEW DETECTION METHOD

17 NEW DETECTION METHOD

Na2SO4

SO -2 EMPA 4 Na+ Na+ EMPA Na+ EMPA + -2 Na SO4 Na+ Na+ Na+ EMPA Na+ -2 SO4 EMPA

-2 Urine impurities SO4 18 NEW DETECTION METHOD PFBBr = detectable at low concentrations using negative ion chemical ionization

PFBBr

PFBBr PFBBr EMPA

EMPA EMPA

PFBBr PFBBr

EMPA EMPA Urine impurities 19 NEW DETECTION METHOD Phosphate Buffer and Hexane

PFBBr

PFBBr PFBBr EMPA

EMPA EMPA

PFBBr PFBBr

EMPA EMPA Urine impurities 20 NEW DETECTION METHOD Phosphate Buffer and Hexane

SO -2 Urine impurities 4 Na+ -2 -2 + SO4 SO4 Na Na+ + Na+ Na Na+ + + Na -2 Na SO4 Phosphate Buffer Hexane PFBBr PFBBr PFBBr PFBBr EMPA PFBBr EMPA EMPA EMPA EMPA 21 NEW DETECTION METHOD

• Direct derivatization with pentafluorobenzyl bromide on solid support • Liquid-liquid extraction • Reduced analyte loss and improved recovery • Isotope-dilution gas chromatography-negative ion chemical ionization tandem mass spectrometry in selected reaction monitoring mode • Quantitative calibration linear range 0.1 – 50 ng/mL with R2 = 0.998 • LOD = 0.02 ng/mL

22 CONCLUSIONS

• VX is very bad. • Thankfully, 98% of the world has agreed to the CWC • Research is still necessary due to terrorism and clean-up contamination • Methods are being developed that have high sensitivity for the metabolites present in biological samples • These methods can be used to determine if VX is used in a deliberate attack or if destruction workers are accidentally exposed

23 ACKNOWLEDGEMENTS

• Topic Advisor: Dr. Lauren Waugh • Dr. Pamela Staton

Practice Audience members: • Nickel B. Daniel • Penelope A. Daniel

24 REFERENCES

1. Coleman, K. A History of Chemical Warfare; Palgrave Macmillan: New York, 2005. 2. Hoenig, S. L. Handbook of Chemical Warfare and Terrorism; Greenwood Press: Westport,Conn., 2002. 3. Masson, P.; Lockridge, O. Butyrylcholinesterase for Protection from Organophosphorus Poisons: Catalytic Complexities and hysteretic Behavior. Archives of Biochemistry and Biophysics. 2010, 494, 107-120. 4. Benschop, H. P.; De Jong, L. P. A. Nerve agent stereoisomers: analysis, isolation and toxicology. Acc. Chem. Res. 1988, 21, 368-374. 5. Conference on Disarmament. The Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on their Destruction. CD/1170, Geneva, August 1992. 6. Organization for the Prohibition of Chemical Weapons. Note by the Technical Secretariat. Status of Participation in the Chemical Weapons Convention as at 14 October 2013. S/1131/2013. 14 October 2013. http://www.opcw.org/about-opcw/member-states/status-of-participation-in- the-cwc/ 7. http://www.opcw.org/chemical-weapons-convention/annex-on-chemicals/b- schedules-of-chemicals/schedule-1/

25 REFERENCES CONT.

8. Organization for the Prohibition of Chemical Weapons. Report of the OPCW on the Implementation of the Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on their Destruction in 2011. C-17/4, 27 November 2012. http://www.opcw.org/documents-reports/annual-reports/ 9. Tsuchihashi, H.; Katagi, M.; Nishikawa, M.; Tatsuno, M. Identification of Metabolites of Nerve Agent VX in Serum Collected from a Victim. J. Anal. Toxic. 1998, 22, 383-388. 10. Black, R. An Overview of Biological Markers of Exposure to Chemical Warfare Agents. J. Anal. Tox. 2008, 32, 2-9. 11. Subramaniam, R.; Ostin, A.; Nilsson, C.; Astot, C. Direct Derivatization and Gas Chromatrography – Tandem Mass Spectrometry Identification of Nerve Agent Biomarkers in Urine Samples. J. Chromat. B. 2013, 928, 98-105. 12. Lin, Y.; Chen, J.; Yan, L.; Guo, L.; Bidong, W.; Li, C.; Feng, J.; Liu, Q.; Xie, J. Determination of Nerve Agent Metabolites in Human Urine by Isotope-Dilution Gas Chromatography-Tandem Mass Spectrometry After Solid Phase Supported Derivatization. Anal. Bioanal. Chem. 2014. 26 PICTURE REFERENCES 1. http://www.filminamerica.com/Movies/TheRock/ 2. http://www.fastcocreate.com/3016518/better-chemistry- through-research-how-writers-make-breaking-bad-so- uncomfortably-real 3. http://www.cma.army.mil/bluegrass.aspx 4. http://www.nytimes.com/learning/general/onthisday/big/032 0.html 5. http://www.globalpost.com/dispatch/news/afp/130506/syria n-rebels-used-sarin-gas-un-human-rights-investigator-0 6. http://www.nti.org/gsn/article/former-cult-member-charged- vx-attacks/ 7. http://www.sudantribune.com/spip.php?article43175 8. http://theathleticnerd.com/category/all-time-best/page/11

27 QUESTIONS?