Detection and identification of chemical warfare agents in environmental samples. Martin Köhler, Mario Francesco Mirabelli

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1 Detection and identification of chemical warfare agents in environmental samples Martin Köhler, Mario Francesco Mirabelli

2 What are chemical weapons? Chemical weapons (CW) belong to the weapons of mass destruction Munitions or devices that can use toxic chemicals to inflict deaths or harm on human beings The toxic chemicals can be classified in nerve -, blister -, blood - and choking agents Martin Köhler, Mario Francesco Mirabelli

3 Historical facts 400 B.C. Spartans have used chemical weapons to release sulfur dioxide (destructive effect on lungs) or to poison rivers 400 A.C. Chinese have intoxicated their enemies via smoke which particularly contains sulfur mustard WWI: Genesis of modern chemical warfare; first use of chlorine, phosgene, sulfur mustard; chemical weapons caused deaths and 1.2 million injuries Martin Köhler, Mario Francesco Mirabelli

Martin Köhler, Mario Francesco Mirabelli 16.12.2014 4 http://de.wikipedia.

4 Martin Köhler, Mario Francesco Mirabelli

5 Historical facts Geneva Protocol: Protocol for the Prohibition of the Use in War of asphyxiating, Poisonous or other Gases, and of Bacteriological Methods of Warfare (1928) Ratification by 65 states WWII: development of sarin, tabun and soman but never used Zyklon B and carbon monoxide in gas chambers during holocaust Sarin attack in Tokyo (1995) and Syria (2013) Martin Köhler, Mario Francesco Mirabelli

The chemical weapon convention (CWC) CWC has been in effect since 1997 Its aim is to eliminate chemical weapons by prohibiting their development, production, acquisition, retention transfer or use by

6 The chemical weapon convention (CWC) CWC has been in effect since 1997 Its aim is to eliminate chemical weapons by prohibiting their development, production, acquisition, retention transfer or use by States Parties Membership is not universal, 190 states parties Intergovernmental organization OPCW was founded by the CWC-states Covers wide number of chemicals, listed in schedules in the appendix Martin Köhler, Mario Francesco Mirabelli

7 Overview about chemical warfare agents Martin Köhler, Mario Francesco Mirabelli

Biological effect of Sarin - As a nerve agent sarin attacks the nervous system - interfering with the re-absorption of neurotransmitters at neuromuscular junctions or synaptic gaps - Death will occur

8 Biological effect of Sarin - As a nerve agent sarin attacks the nervous system - interfering with the re-absorption of neurotransmitters at neuromuscular junctions or synaptic gaps - Death will occur as a result of asphyxia due to the inability to control the muscles involved in breathing function - Inactivates acetylcholinesterase by binding covalently to the active site Martin Köhler, Mario Francesco Mirabelli

9 Properties of Chemical Warfare Agents LD 50 (i.v.) mg/kg Tabun Sarin Soman VX mouse rat Substance Sunny, breeze, about 15 C Windy and rainy, about 10 C Windless, snow on the ground, about -10 C S-Lost 2-7 d d 2-8 weeks Tabun 1-4 d h 1-14 d Sarin h h 1-2 d Soman d 3-36 h 1-6 weeks VX 3-21d 1-12 h 1-16 weeks Martin Köhler, Mario Francesco Mirabelli

10 Off-site Analysis of Chemical Warfare Agents Sample preparation dependent on the type of sample (air, organic, wipe, aqueous liquid ) Extraction Recommended operating procedure (ROP) Screening Analysis, data evaluation and identification Martin Köhler, Mario Francesco Mirabelli

11 Screening-techniques First selection Search for specific fragment or signal Decrease the number of chemical candidates Targeted / non-targeted screening methods Targeted: only a defined set of chemicals is being searched for (GC/MS, LC/MS) Non-targeted: more universal property, such as an element or a common part of the molecule is being searched for (NMR) Martin Köhler, Mario Francesco Mirabelli

12 Screening-techniques GC P, N and S compound NPD: Nitrogen phosphorus detector FDP: Flame photometric detector AED: Atomic-emission detector (all elements) EI/MS for non-containing P,N and S compounds LC/MS Degradation products Polar non-volatile analytes NMR 1 H, 31 P and 19 F Enzyme-based methods Martin Köhler, Mario Francesco Mirabelli

13 Analysis, data evaluation and identification Confirmation Synthesis of the suspected chemical Three methods of interpretation (OPCW) Interpretation of spectrum (slow) Comparison with reference spectrum (faster) Comparison with a spectrum from an authentic standard (safest) Techniques: GC, GC/MS, GC/MS/MS, GC/CI/MS LC/MS, LC/MS/MS GC/FTIR NMR LC/MS/NMR Martin Köhler, Mario Francesco Mirabelli

14 Martin Köhler, Mario Francesco Mirabelli

15 Comparison of different techniques Advantage Disadvantage NMR Non-destructive Expensive Structure resolution fast Sensitivity (mg) Complexity of the spectra GC Sensitivity (pg-fg) Need a volatile but heatresisting sample Cheap Small amount of analyte MW < 700 Da Need different instruments with different detectors Martin Köhler, Mario Francesco Mirabelli

16 Comparison GC/MS vs. LC/MS GC/MS MW < 700 Da Highly volatile samples Non-polar compounds Sensitive Investigation of intact chemical warfare agent Heat-resisting sample LC/MS 50 < MW < 50kDa Non-volatile Highly polar compounds Sensitive Investigation of degration products Martin Köhler, Mario Francesco Mirabelli

17 On- site analysis Use of SOP approved by OPCW - Preparation, transport to POE and inspection site, mobile lab setup, on- site sampling and analysis, (off- site analysis), return to HQ and reporting Sampling - Liquids or solids are preferred. Wipe samples can also be collected - Splitting - Background samples (matrix effects) Samples should preferably analyzed on site - Off- site analysis if results are ambiguous/unclear or lack of equipment Martin Köhler, Mario Francesco Mirabelli

18 On- site analysis Ideal case: bring the whole lab to test site (impractical) Portable devices are a compromise: false- positives and negatives Detection kits based on chemical reactions: paper, tubes - Yes/No response or qualitative information Point detection instruments - IMS: transit time of ions as they pass through a drift region under the inqluence of an electric Qield at ambient pressure High repetition rates. Low separation (false positives) - GC: Air pre- concentration; sample injection and separation (5min cycle) Different detectors, e.g. ECD, NPD, AED and MS - blinded mode (AMDIS): spectra and chromatogram not shown - GC- HRIMS- MS Martin Köhler, Mario Francesco Mirabelli

19 M 908 -High pressure mass spectrometer (micro ion trap) -Extremely light: 2Kg -Multi phase detection (solids, liquids and gas) -Explosives, CWAs, drugs, toxic industrial chemicals Martin Köhler, Mario Francesco Mirabelli

Analyst, 2008,133, 1064 107 Low duty-cycle and throughput

20 Mini 10.5 portable MS Reduced weight: 10Kg 35W power consumption Analyst, 2008,133, Low duty-cycle and throughput Low sensitivity and resolution Martin Köhler, Mario Francesco Mirabelli

21 Analytical strategy Physical state of sample(s) Nature of analytes (a large number of CWA compounds have to be considered) Concentration range Matrix effects Instrumentation Time Personnel Design of a proper analytical strategy Use of ROP, but Qlexibility in decision Optimization of available time Martin Köhler, Mario Francesco Mirabelli

22 Analytical strategy Aqueous liquid samples Water samples are usually screened for polar non- volatile degradation/precursor products without sample cleanup Original sample is divided into 4 or more parts Extractions with different solvents are performed - CH 2 Cl 2 ; H 2 O Liquid matrices can cause suppression, e.g. presence of hydrocarbons - When analytes are known, speciqic sample cleanup can be performed Martin Köhler, Mario Francesco Mirabelli

23 Analytical strategy MPA (methylphosphonic acid): detection is problematic if a proper extraction procedure is not followed - Cation exchange - Evaporation of aqueous residue - Derivatization - Analysis Martin Köhler, Mario Francesco Mirabelli

24 Analytical strategy Solid matrices can be very diverse Soil: multiple extraction (aqueous extraction without sonication) Concrete: evaluation of the degree of penetration of chemicals (superqicial?) and size of sample Hair: usually chemicals are adsorbed on the exterior part, so that a regular solvent extraction can be used Martin Köhler, Mario Francesco Mirabelli

25 Analytical strategy 50 samples for sarin and its degradation products 2 days of time Two independent methods for unambiguous identiqication Samples can be screened by IMS (or direct injection ESI- MS, if clean) - Screening by GC is not practical due to lack of time Alternatively, screening/identiqication for can be performed by 31 P, 1 H- NMR - Low amounts of liquid samples: NMR is non destructive (used Qirst) - Best for medium/high concentration samples - Faster results can obtained if samples can be grouped (e.g. 5 by 5) Martin Köhler, Mario Francesco Mirabelli

26 Analytical strategy GC- EIMS for unambiguous identiqication (derivatization of degradation products) LC- MS/MS for conqirmation and for degradation products Because the compounds are known, automated LC- MS/MS method with speciqic MRM transitions can be used. ConQirmation by a second method can be performed with GC- MS or NMR for medium/high concentration samples Use of duplicates instrumentation to double the number of runs per time unit Martin Köhler, Mario Francesco Mirabelli

27 Analytical strategy Diisopropyl methylphosphonate- d 14 DIMP is a schedule 2 chemical, degradation or precursor of schedule 1 chemicals Diisopropyl methylphosphonate (DIMP) Martin Köhler, Mario Francesco Mirabelli

28 Diisopropyl methylphosphonate- d 14 Analytical strategy Compared to the non- deuterated version: - Different mass - Only 3 hydrogens GC with element speciqic detector (NPD) for screening (small shift in r.t.) 31 P- NMR 1 H- NMR does not give enough information 13 C- NMR cannot be used if concentration is low Detectable by GC- MS (different fragmentation and small shift in r.t.) Detectable by LC- MS (different m/z and small shift in r.t.) LC- MS/MS only with the correct MRM transition, after parent ion is found with LC- MS Synthesis of a standard is fundamental Used in ProQiciency test to evaluate labs Qlexibility in Qind uncommon (but still classiqiable as) CWAs Martin Köhler, Mario Francesco Mirabelli

29 References Mesilaakso, Markku. "Chemical Weapons Convention Chemicals Analysis." MesilaaksoM (ed). John Wiley: Chichester 151 (2005). Martin Köhler, Mario Francesco Mirabelli

30 Thank you for your attention! Martin Köhler, Mario Francesco Mirabelli

Chemical Warfare Agents. Hanna Hunziker, Céline Prange Analy:cal Strategy, 13. October 2015

Chemical Warfare Agents. Hanna Hunziker, Céline Prange Analy:cal Strategy, 13. October 2015 Chemical Warfare Agents Hanna Hunziker, Céline Prange Analy:cal Strategy, 13. October 2015 World War I 124 000 tons of gas Aerosols or vapours Ethylbromoacetate Chloroacetone Xylylbromide Chlorine Gas