DETECTION AND IDENTIFICATION OF CHEMICAL WARFARE

AGENTS IN ENVIRONMENTAL

SAMPLES

Sandra Luginbühl

María de Lourdes Aja Montes

OUTLINE

1. Historical overview of chemical weapons/chemical warfare agents (CWA) regulations .

2. Controlled chemicalsA. Toxicity B. Characteristics

3. Strategies for the analysis of CWA A. Early Warning B. On-siteC. Off-site

4. Case Study: Ricin1. Characteristics2. Detection methods (unambiguous identification)

HISTORICAL OVERVIEW

1899 Hague Peace Conference Abstain from the “use of projectiles, the sole object of which is the diffusion

of asphyxiating or deleterious gases”

1925 Geneva Protocol for the Prohibition of the Use of Asphyxiating, Poisonous or Other Gases, and Bacteriological Methods of Warfare.

Only bans the use of chemical and biological weapons in war. Does not prohibit the development, production or possession of CW.

1970 US–Soviet initiative on Chemical Weapons (CW) Need to:

Control the precursors of CW Establish mechanisms to oversee the implementation of the treaty Routine inspections

29 April 1997 Chemical Weapons Convention (CWC) Entered into force with 87 States Parties—becoming binding

international law Every 5 years there is a review of the CWC implementation process.http://www.opcw.org

CHEMICAL WEAPONS CONVENTION (CWC)

Arms control agreement which outlaws the production, stockpiling, and use of chemical weapons.

188 Member states 

Main Previsions: Prohibition of production and use of chemical weapons. Destruction of CW and their production facilities. Investigations of allegations of use and production of chemical

weapons based on intelligence of other state parties. Systematic inspection for the production of chemicals which

might be converted to chemical weapons (evaluation of chemical plants).

Organization for the Prohibition of Chemical Weapons (OPCW)

http://www.opcw.org

TOXICITY INDICES

LD50= median lethal dose Dose required to kill half the members of a tested population

after a specified test duration. [mg per 70 kg male person]

LCt50 = Lethal concentration * time, for which and after which half of the population is killed. [min*mg/cm3]

ICt50 = concentration * time at which half of the population is incapacitated. [min*mg/cm3]

CLASSES OF CONTROLLED CHEMICALS:SCHEDULE 1

Chemicals which have few, or no uses outside of CW. 8 Toxic chemicals and 4 Precursors

Use and production mainly for research, medical, pharmaceutical or chemical weapon defence testing purposes. Production >100 g must be reported to the OPWC

Dorn, A.W. and D. Scott. The Compliance Provisions in the Chemical Weapons Convention. PSIS, Laussane: 1995

Ricin(Ribosome-inactivating Protein)

Saxitoxin (Neurotoxin)

CLASSES OF CONTROLLED CHEMICALS:SCHEDULE 2 Chemicals which have legitimate small-scale applications.

3 Toxic chemicals and 9 Precursors Similar prescriptions for production as for Schedule 1 Chemicals.

Dorn, A.W. and D. Scott. The Compliance Provisions in the Chemical Weapons Convention. PSIS, Laussane: 1995

Amiton or VG (nerve agent)

PFIB

BZ(Incapacitating

Agent)

CLASSES OF CONTROLLED CHEMICALS:SCHEDULE 3

Chemicals which have large-scale industrial uses. 4 Toxic chemicals and 13 Precursors.

Production above 30 Tons must be reported to the OPWC.

Dorn, A.W. and D. Scott. The Compliance Provisions in the Chemical Weapons Convention. PSIS, Laussane: 1995

Common functional groups of CWA Precursors Amines Phosphore (phorous, phosphines) Sulfur (Thiols,sulfur chlorides) Organo-Arsenic

Phosgene(Pulmonary Agent)

Cyanogen chloride(Blood agent)

Hydrogen cyanide or Prussic Acid(Blood agent)

Chloropicrin(Pulmonary Agent)

DEGRADATION PRODUCTS (HYDROLYSIS PATHWAYS)

CHALLENGES FOR ANALYSIS

Complicated and diverse matrices: air, soil, vegetation, water and snow.

Decomposition products and precursors

LOD < toxicity (range: ng/mL)

CWC Unambiguous identification of a compound: 2 methods are needed (one must be a spectrometric method)

3 different situations call for 3 separate methods of analysis: Early warning system: immediate response

On-site analysis: analysis shouldn’t take longer than a day, should be a non-specific method to screen for all compounds

Off-site analysis: structure determination, higher resolution and sensitivity. Time not as critical

EARLY WARNING SYSTEMS

Detection paper Detection tickets SAW Sensor IMS

https://www.ecbc.army.mil/ps/products_detection.htmhttp://www.sandia.gov/mission/homeland/chembio/assets/images/IMS_0427-t.jpgHill, HH and SJ Martin. Pure Appl. Chem. 2002, 74 (12), 2281-2291

EARLY WARNING SYSTEM:DETECTION PAPER (M8)

Distinguish between three different types of liquid CW agents.

LOD = 5 mg/m2

Disadvantages: Other substances can dissolve the pigments (fat,

oil, fuel, etc.)

http://www.opcw.org/our-work/assistance-and-protection/protection-against-chemical-weapons/detection/

Paper:• Celulose • Dyes (2 or 3)• pH indicator

Nerve Agent

Mustard Agent

Vx (blue + yellow)

EARLY WARNING SYSTEM: DETECTION TICKETS Distinguish between different types of aerosolized CW

agents.

Enzymatic substrate-based reactions (15 min). Presence of the CWA is indicated by a specific color change.

Blister agent test

Blood agent

Nerve agent

LOD: 20 – 50 ng/mL

Disadvantages: petroleum products and high temperatures may produce false readings.

http://www.crcnetbase.com/doi/pdf/10.1201/9780203485705.ch10http://www.opcw.org/our-work/assistance-and-protection/protection-against-chemical-weapons/detection/http://www.sc-ems.com/ems/NuclearBiologicalChemical/MedicalAspectsofNBC/chapters/chapter_16.htm

EARLY WARNING SYSTEM: SAW SENSOR

Results within 2 minutes LOD: 0.01 mg/m3 Can be made small and portable An array of different sensors can be put into

one hand-held device

E(t) Acoustic Wave

Change in amplitude and/or phasewhen a substance is adsorbed ontothe crystal

Piezoelectric Crystal

DetectorThin film which selectively adsorbs a specific CW

H. H Hill & S. J Martin, Pure Appl. Chem. 2002, 74 (12), 2281-2291

EARLY WARNING SYSTEM: ION MOBILITY SPECTROMETRY (IMS)

Gas phase is ionized and separated according to their differences in velocities through a gas in a weak electric field.

Splitting m/z Exposure time 1 min

For detection of Blister, V and G type nerve agents.Westhoff M, et al. Thorax 2009, 64, 744-748H. H Hill & S. J Martin, Pure Appl. Chem. 2002, 74 (12), 2281-2291

ON-SITE ANALYSIS (OPCW STANDARD METHOD)

The inspection team has: Sampling kit Sample transport kit

For Off-site Analysis Sample preparation kit Portable GC-MS Portable laboratory

Schep LJ et al. Chemical Weapons Convention Chemicals Analysis. John Wiley&Sons, 2005.

ON-SITE ANALYSIS: SAMPLING Correct documentation

Appropriate protection (gas masks, biohazard suits, etc.)

Types of Samples taken: Soil Liquid

Water (from surface up to 5 m) Surface wipes Air (aren’t routinely taken)

Schep LJ et al. Chemical Weapons Convention Chemicals Analysis. John Wiley&Sons, 2005.

SAMPLE SPLITTING

OPCW procedures require sample splitting into eight portions, which are distributed as follows:

2 on-site analysis by the inspection team

1 Inspected State Party (ISP)

1 kept as a reference on site under joint OPCW

and ISP seal

4 to be sent for off-site analysis to a minimum

of 2 different authorized laboratories.

30 min to 1 hr

6 hours

ON-SITE ANALYSIS:SAMPLE PREPARATION

BSTFA

DMT

Derivatizing agents

Schep LJ et al. Chemical Weapons Convention Chemicals Analysis. John Wiley&Sons, 2005.

ON-SITE ANALYSIS:GC-MS BY BRUKER EM 640S

Especially developed for analysis of CWA

Ready for use after 30 min

High Sensitivity (ng)

Performance test before and after each work-day with: OPCW GC/MS test mixture in dichloromethane 10µg/mL, containing:

n-Alkanes C8 –C24 even numbers Trimethylphosphate 2,6-Dimethylphenol 5-Chloro-2-methylaniline Tri-n-butylphosphate Dibenzothiophene Malathion Methylstearate

Internal Standard: HexachlorobenzeneSchep LJ et al. Chemical Weapons Convention Chemicals Analysis. John Wiley&Sons, 2005.

SPECTRUM: DERIVATIZED LEWISITE-1

Hooijschuur EWJ, et al. Journal of Chromatography A, December 2002, 982 (2),177-200

ON-SITE ANALYSIS: OTHER SEPERATION TECHNIQUES LC

Minimum sample preparation Able to analyze non-volatile

degradation products which are difficult for GC.

In combination with ESI very low LOD

Capillary Electrophoresis Miniaturization possible

„Lab-on-a-chip“ Small sample volume

http://www.labor-spiez.ch/en/dok/po/pdf/LC-MS_Techniques_for_Screening_and_Identification_od_Degradation_Products_of_Nerve_Agents_1998.pdfhttp://biodevices.et.tudelft.nl/Bio-projects/BIOMAS/project.php

OFF-SITE ANALYSIS METHODSMethod Advantages Disatvantage

Q-MS (GC-MS / LC-MS)

-Standardized-Good for routine analysis-Fast and easy to operate-Make reference spectra for use at on-site analysis

-Interface needed between seperation method and MS-Difficult to identify new compounds

FTICR -MSn capabilities-Study reactions and dynamic processes-Very high sensitivity-Very large mass range -> protein analysis-Good for complex mixtures

-Difficult to operate- Very Expensive-Slow-Cooling needed

Orbitrap -Similar capabilities as ICR-Cheaper than ICR

- Expensive

NMR -Structure elucidation-Confirm that reference compound has been synthesized-Analyze unknown substances

-Cooling needed-Low senstivity

IR/ GC-IR -Fast and cheap-GC-IR: ‚functional group monitoring‘-Fingerprint information

-Highly ambiguous results-Coupling is difficult-Derivatizing for GC changes IR pattern

RICIN Highly toxic glycoprotein readily isolated from the seeds of the castor

bean plant (Ricinus communis). Composition:

B Chain Binds to the surface of the cell and transports A into the cytosol. A Chain Possesses N-glycosidase activity which irreversibly inactivates ribosomal RNA,

inhibiting the synthesis of proteins. (2000 ribosomes per minute)

Types of ricin: Functional (only one that presents a threat to human health) Nonfunctional

Toxicity LD50 (human/inhalation) = 55 μg/kg (optimal AED ~ 1μm) LD50 (human/ingestion) = 20 mg/kg LD50 (human/injection)= 5-10 μg/kg

Schep LJ et al. concluded that:

“Ricin as a toxin is deadly but as an agent of bioterror is unsuitable”Becher F, et al. Anal. Chem. 2007, 79, 659-665Schep LJ, et al. Environ Int. 2009, 35 (8), 1267-71

RICIN: IDENTIFICATIONImmunocapture

HPLC (C18

column)

MALDI-ToFSurface Enhanced

Raman Spectroscopy

Adenin

Becher F, et al. Anal. Chem. 2007, 79, 659-665 He L, et al. J. Food Sci. 2011, 76 (5), 49-53 Duriez E, et al. J. Proteome Research. 2008, 7, 4154-4163

RNA depurination

Ricine Elution Ricine Elution

Enzimatic Proteolysis

ZipTip C18

ESI-MS/MS

3 Ricin Peptides

Trypsine (Digestion)+ ACN 80% (Protein denaturalization)

LODRicin= 0.1 ng/mL

Analyte: Adenin

tanalysis ≈ 6 hrs (3LOD) 24 hrs

LODRicin= 50 ng/mL

Analytes: 3 Ricin A-Chain Peptides

( m/z 1013.6, m/z 1310.6, m/z 1728.9)

tanalysis ≈ 5 hrs

0.1% TFA

Peptide fractionation

LODRicin= 4 µg/mL

Analyte: Ricin

tanalysis ≈ 20 min

Nanosubstrate addition

Silver dendrites + Ricin

IMMUNOCAPTURE

Any Questions?

ADDITIONAL SLIDES

STATES THAT HAVE NEITHER SIGNED NOR ACCEDED TO THE CHEMICAL WEAPONS CONVENTION

Angola Egypt North Korea Somalia South Sudan Syria

NERVE AGENTS

BLISTER AGENTS

BLOOD AGENTSCWC LCt50

Vapor inhalationmin*mg/m³

LD50 liquidinjectionmg

ICt50

Vapor inhalationmin*mg/m

Time to symptom

Time to death

State at RT

Main mode of entering body

Typical symptoms after inhalation Structure

Blood Agents

At high enough concentrations, instanenous

At high enough concentrations, instanenous

liquid Inhalation of an aerosol

Prevent the exchange of O2 and CO2 between the blood and the cells.Suffocation, violent convulsions.

Cyanogen Chloride(CK)

~10000 ~10000 immediate 15 min Colorless gas

Inhalation. Can penetrate gas masks

drowsiness, rhinorrhea (runny nose), sore throat, coughing, confusion, nausea, vomiting, edema, loss of consciousness, convulsions, paralysis,

Hydrogen Cyanide(AC)

~3000 ~5000 30s-15min fast Colorless liquid

Inhalation Destroys Cyt C Oxidase. You can’t breath.

Developed by IG Farben: Zyklon B.

PULMONARY AGENTSCWC LCt50

Vapor inhalationmin*mg/m³

LD50 liquidinjectionmg

ICt50

Vapor inhalationmin*mg/m

Time to symptom

Time to death

State at RT Main mode of entering body

Typical symptoms after inhalation Structure

Pulmonary Agents

~10 hours 1-2 days

Impedes person’s ability to breath, leads to a build-up of fluids in the lungs. Also: corrosive to eyes and skin, blurred vision and severe deep burns.

Phosgene(CG)

3200 1600 Slow, 4-8 hours

~10 hrs Colorless liquid

inhalation Corrosive to lung tissue: causes lesions and fluid from blood goes into lungs.

Used in WW1

Chloropictrin ? Very rapid Colorless, oily liquid

Inhalation Nose and throat irritation, coughing, vomiting, long damage, skin burns

OTHER

EARLY WARNING SYSTEM: DETECTION TICKETS Distinguish between different types of aerosolized CW agents.

Enzymatic substrate-based reactions (15 min). Presence of the CWA is indicated by a specific color change.

Nerve agent Filter paper

Blood agent Glass fiber filter

Blister agent test Chromatographic media

LOD: 20 – 50 ng/mL

Disadvantages: petroleum products and high temperatures may produce false readings.

http://www.crcnetbase.com/doi/pdf/10.1201/9780203485705.ch10http://www.opcw.org/our-work/assistance-and-protection/protection-against-chemical-weapons/detection/http://www.sc-ems.com/ems/NuclearBiologicalChemical/MedicalAspectsofNBC/chapters/chapter_16.htm

SAW SENSOR (KSPS 4200) Ultimate Vapor Tracer & Analyzer is the only Portable Real-Time Ultra-Fast Analyzer

on the market that can Sniff-out, Detect and Analyze all types of vapors and identify traces of organic, biological & chemical compounds accurately and very rapidly.

It can accurately detect and identify: All types of military, commercial and homemade explosives including: RDX, PETN, Tetryl,

TNT, NG, DNT, Ammonium Nitrate, Black Powder* and others. All types of drugs & illicit narcotics including: Heroin, Cocaine, Marijuana, PCP,

Methamphetamines, LSD, THT and others. All types of biological, nerve & chemical agents including: Sarin, Soman, Anthrax, Mustard

Gas and others.

The Electronic Nose and Vapor Analysis Technology is based upon Surface Acoustic Wave (SAW) sensors and flash chromatography. For the very first time, the fragrance, odor, or smell associated with virtually any chemical process, manufacturing operation, or product can be chemically analyzed and quantified with part per billion accuracy within seconds.

The 4200 comes complete with Notebook Controller configured and integrated with the following: MicroSense proprietary software provides Vaporprints in 2D high-resolution visual olfactory images that can be used as a pattern recognition method for continuous

product monitoring. GPS compatible for linking location data with chemical data. Chromatograms

http://ksps-bh.com/Explosives-detectors.html

SAMPLING KIT Preassembled kits for sampling of water or organic liquid, separately

packed and sealed.

Scoops, bowl, spatulas for collection of soil samples

Separately packed wipes, stiff wires, alligator clips, clamps to take wipe samples

Vials and glass bottles, different sizes, separately packed and sealed, to hold the samples

Spare items for sampling of water or liquid samples, i.e. spare syringes, vacutainers, tubing

Pipettes and pipette balls used for sample splitting

Bags, tape, and seals to package sample vials or bottles

‘Peli’ cases to transport the sampling equipment to the sampling point

General items: gloves, goggles, pens, knife, scissors, labels, markers, ground sheets, garbage bags

SAMPLE PREPARATION KIT Centrifugal evaporator with vacuum pump Heater/evaporator to be used with pure nitrogen from a nitrogen generator (optional

item) or a nitrogen cylinder Basic laboratory equipment, i.e. balance, centrifuge, ultrasonic bath Laboratory glassware, i.e. vials, tubes, funnels, beakers, pipettes, syringes, measuring

cylinders Supporting equipment, i.e. trays, filter paper, pH paper, SPE cartridges, adjustable

pipettes and pipette tips, labels, markers, scissors, bench coat paper, containers and bags for waste collection, laboratory coats, gloves, stationary

Laboratory fume hood Portable refrigerator, on-site sample transport container Chemicals and solvents for sample preparation and GC/MS analysis:

Dichloromethane, GC grade Methanol, GC grade Hexane, GC grade Water, HPLCa grade Tetrahydrofurane 99%, stabilized with 0.025% butylated hydroxy toluene (THF)b 2% (v/v) Hydrochloric acid in methanol 0.1N Hydrochloric acid, reagent grade 2N Hydrochloric acid, reagent grade 0.1N Ammonium hydroxide, reagent grade 1% (v/v) Triethylamine in methanol Sodium sulfate, anhydrous, reagent grade N,O-bis-(Trimethylsilyl)trifluoroacetamide (BSTFA) 3,4-Dimercaptotoluene in acetone, 5 mg/mL freshly prepared (DMT) OPCW GC/MS TEST MIXTURE OPCW GC/MS HCB mixture containing 50μg/mL of hexachlorobenzene in dichloromethane

SUPPORT EQUIPMENT individual protective equipment (e.g. gloves, protective goggles, coverall, mask with

canister, air permeable suit, boots, chemical resistant suit, SCBA (self containing breathing apparatus) team decontamination equipment detectors (i.e. hand held detectors, military detector kit); power generator (for laboratory power

supply).

NMR SPECTRA OF SARIN

Schep LJ et al. Chemical Weapons Convention Chemicals Analysis. John Wiley&Sons, 2005. Spectra from Spiez lab

NMR SPECTRA OF SARIN

NMR SPECTRA OF SARIN

NMR SPECTRA OF SARIN

NMR SPECTRA OF SARIN

ZIPTIP C18 C-18 Tips are ready-to-use pipette-tip columns of C18 resin

that enable fast and efficient capture, concentration, desalting and elution of peptides for MALDI mass spectrometry and other methods.

http://www.millipore.com/userguides.nsf/a73664f9f981af8c852569b9005b4eee/f9c60ff6912cf3bf852575de006399f5/$FILE/TN226.pdf

Duriez E, et al. J. Proteome Research. 2008, 7, 4154-4163

COMPLETE MALDI-TOF RICIN SPECTRUM