Presentation on theme: "D ETECTION AND I DENTIFICATION OF C HEMICAL W ARFARE A GENTS IN E NVIRONMENTAL S AMPLES Sandra Luginbühl María de Lourdes Aja Montes."— Presentation transcript:
D ETECTION AND I DENTIFICATION OF C HEMICAL W ARFARE A GENTS IN E NVIRONMENTAL S AMPLES Sandra Luginbühl María de Lourdes Aja Montes
O UTLINE 1. Historical overview of chemical weapons/chemical warfare agents (CWA) regulations. 2. Controlled chemicals A. Toxicity B. Characteristics 3. Strategies for the analysis of CWA A. Early Warning B. On-site C. Off-site 4. Case Study: Ricin 1. Characteristics 2. Detection methods (unambiguous identification)
H ISTORICAL O VERVIEW 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 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 Partiesbecoming binding international law Every 5 years there is a review of the CWC implementation process.
C HEMICAL W EAPONS C ONVENTION (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)
T OXICITY I NDICES 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/cm 3 ] ICt50 = concentration * time at which half of the population is incapacitated. [min*mg/cm 3 ]
C LASSES OF CONTROLLED CHEMICALS : S CHEDULE 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)
C LASSES OF CONTROLLED CHEMICALS : S CHEDULE 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)
C LASSES OF CONTROLLED CHEMICALS : S CHEDULE 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)
D EGRADATION P RODUCTS (H YDROLYSIS P ATHWAYS )
C HALLENGES FOR A NALYSIS 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 shouldnt 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
E ARLY W ARNING S YSTEMS Detection paper Detection tickets SAW Sensor IMS https://www.ecbc.army.mil/ps/products_detection.htm Hill, HH and SJ Martin. Pure Appl. Chem. 2002, 74 (1 2),
E ARLY W ARNING S YSTEM : D ETECTION P APER (M8) Distinguish between three different types of liquid CW agents. LOD = 5 mg/m 2 Disadvantages: Other substances can dissolve the pigments (fat, oil, fuel, etc.) Paper: Celulose Dyes (2 or 3) pH indicator Nerve Agent Mustard Agent Vx (blue + yellow)
E ARLY W ARNING S YSTEM : D ETECTION T ICKETS 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.
E ARLY W ARNING S YSTEM : SAW S ENSOR 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 phase when a substance is adsorbed onto the crystal Piezoelectric Crystal Detector Thin film which selectively adsorbs a specific CW H. H Hill & S. J Martin, Pure Appl. Chem. 2002, 74 (12),
E ARLY W ARNING S YSTEM : I ON M OBILITY S PECTROMETRY (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, H. H Hill & S. J Martin, Pure Appl. Chem. 2002, 74 (12),
O N -S ITE A NALYSIS (OPCW S TANDARD M ETHOD ) 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.
O N -S ITE 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 (arent routinely taken) Schep LJ et al. Chemical Weapons Convention Chemicals Analysis. John Wiley&Sons, 2005.
S AMPLE S PLITTING 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 O N -S ITE ANALYSIS : S AMPLE P REPARATION BSTFA DMT Derivatizing agents Schep LJ et al. Chemical Weapons Convention Chemicals Analysis. John Wiley&Sons, 2005.
O N -S ITE ANALYSIS : GC-MS BY B RUKER 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: Hexachlorobenzene Schep LJ et al. Chemical Weapons Convention Chemicals Analysis. John Wiley&Sons, 2005.
S PECTRUM : D ERIVATIZED L EWISITE -1 Hooijschuur EWJ, et al. Journal of Chromatography A, December 2002, 982 (2),
O N -S ITE ANALYSIS : OTHER S EPERATION T ECHNIQUES 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
O FF -S ITE A NALYSIS M ETHODS MethodAdvantagesDisatvantage 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 -MS n 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
R ICIN 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 LD 50 (human/inhalation) = 55 μg/kg (optimal AED ~ 1μm) LD 50 (human/ingestion) = 20 mg/kg LD 50 (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, Schep LJ, et al. Environ Int. 2009, 35 (8),
R ICIN : I DENTIFICATION Immunocapture HPLC (C18 column) MALDI-ToF Surface Enhanced Raman Spectroscopy Adenin Becher F, et al. Anal. Chem. 2007, 79, He L, et al. J. Food Sci. 2011, 76 (5), Duriez E, et al. J. Proteome Research. 2008, 7, RNA depurination Ricine Elution Enzimatic Proteolysis ZipTip C18 ESI-MS/MS 3 Ricin Peptides Trypsine (Digestion) + ACN 80% (Protein denaturalization) LOD Ricin = 0.1 ng/mL Analyte: Adenin t analysis 6 hrs (3LOD) 24 hrs LOD Ricin = 50 ng/mL Analytes: 3 Ricin A-Chain Peptides ( m/z , m/z , m/z ) t analysis 5 hrs 0.1% TFA Peptide fractionation LOD Ricin = 4 µg/mL Analyte: Ricin t analysis 20 min Nanosubstrate addition Silver dendrites + Ricin
A DDITIONAL SLIDES
S TATES THAT HAVE NEITHER SIGNED NOR ACCEDED TO THE C HEMICAL W EAPONS C ONVENTION Angola Egypt North Korea Somalia South Sudan Syria
N ERVE A GENTS
B LISTER A GENTS
B LOOD A GENTS CWC LCt50 Vapor inhalation min*mg/m³ LD50 liquid injection mg ICt 50 Vapor inhalation min*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 concentrat ions, instaneno us 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 immediate15 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) ~3000~500030s-15minfastColorless liquid InhalationDestroys Cyt C Oxidase. You cant breath. Developed by IG Farben: Zyklon B.
P ULMONARY A GENTS CWC LCt50 Vapor inhalation min*mg/m³ LD50 liquid injection mg ICt 50 Vapor inhalation min*mg/m Time to symptom Time to death State at RT Main mode of entering body Typical symptoms after inhalationStructure Pulmonary Agents ~10 hours 1-2 days Impedes persons 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) 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 rapidColorless, oily liquid InhalationNose and throat irritation, coughing, vomiting, long damage, skin burns
E ARLY W ARNING S YSTEM : D ETECTION T ICKETS 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.
SAW S ENSOR (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
S AMPLING 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
S AMPLE P REPARATION 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, HPLC a 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
S UPPORT E QUIPMENT 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 S PECTRA OF S ARIN Schep LJ et al. Chemical Weapons Convention Chemicals Analysis. John Wiley&Sons, Spectra from Spiez lab
NMR S PECTRA OF S ARIN
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. 7
Duriez E, et al. J. Proteome Research. 2008, 7, C OMPLETE MALDI-T O F R ICIN S PECTRUM