Detection and identification of chemical warfare agents using HPTLC

Introduction

Chemical warfare agents present a considerable threat to human health, inducing a spectrum of symptoms ranging from irritation to fatality. It is imperative for law enforcement agencies and military personnel to possess the knowledge and tools required to detect and prevent exposure to these hazardous substances. There are various methods to categorize chemical warfare agents, one common approach is to categorize them based on the primary symptoms they cause. Nerve agents, for instance, are organic chemicals that disrupt the mechanisms through which nerves convey messages to organs. This disruption arises from the inhibition of acetylcholinesterase (AChE), an enzyme facilitating the breakdown of acetylcholine.

Blistering agents, also known as vesicants, are chemical warfare agents that induce skin blisters, eye damage, and respiratory harm. Typically, these agents manifest as oily liquids that can persist on surfaces for extended durations. Exposure to blistering agents can lead to severe burns, lung damage, and even death. In contrast, irritant agents elicit irritation on the skin, eyes, and respiratory system. Although less lethal than nerve agents and blistering agents, irritant agents can still inflict significant harm on exposed individuals. Examples of irritant agents include substances like chlorine gas, phosgene gas, and tear gas.

Arsenic agents represent another category of chemical warfare agents capable of causing substantial harm to human health. Exposure to arsenic agents can result in symptoms ranging from irritation to death.

HPTLC is a reliable and widely used analytical technique for the identification of chemical warfare. HPTLC separates the individual components of a mixture, making it possible to identify specific nerve agents such as Russian VX (RVX), O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothioate (VX), Soman (GD), Tabun (GA), cyclosarin (GF), and sarin (GB) based on their characteristic retention factor (RF) values [1]. TLC methods were transferred to HPTLC.

For six blistering agents and irritants, namely sulfur mustard (HD), HN-3 (TTA), 2-chlorobenzylidenemalononitrile (CS), 2-chloroacetophenone (CN), bromobenzyl cyanide (CA), and benzyl bromide (CB) [2], as well as three arsenic agents Lewisite (L), Clark 1 (DA), and Adamsite (DM) [3], their initial TLC methods were successfully transferred to HPTLC. This underscores the adaptability and efficacy of HPTLC in extending the capabilities of traditional TLC methods for the comprehensive analysis of chemical warfare agents.

Standard solutions

Individual standard solutions were prepared according to the table below, and for quantification purposes each solution was applied at different application volumes to generate a calibration curve.

System Suitability Test (SST): the ready-to-use solution of Universal HPTLC mix (UHM) was prepared in house according to [4] and applied on track 8 of each plate.

Chromatogram layer

HPTLC plates silica gel 60 F₂₅₄ (Supelco), 20 × 10 cm are used.

Sample application

Samples are applied as bands with the Automatic TLC Sampler (ATS 4), 15 tracks, band length 8.0 mm, distance from left edge 20.0 mm, distance from lower edge 8.0 mm.

Chromatography

Plates were developed with the following three developing solvents in ADC 2 with activation of the plate at 33 % relative humidity for 10 min using a saturated solution of magnesium chloride. For nerve agents, acetone – cyclohexane – ethyl acetate – methanol 1:5:3:0.2 (V/V), for blistering agents and irritants, toluene, and for arsenic agents, cyclohexane – dichloromethane – methanol 7:2:1 (V/V), are used as developing solvents with 20 min chamber saturation (with saturation pad). The developing distance for all three methods was 70 mm (from the lower edge). Plates were dried for 5 min.

Post-chromatographic derivatization

For nerve agents:
 

1. Spraying solution A: Acetylcholinesterase

Reagent preparation:
Dissolve 55.0 mg of acetylcholinesterase (55 mg = 150 U) in 100.0 mL of buffer solution (dissolve 19.0 g of Na₂HPO₄ x 12 H₂O and 1.8 g of KH₂PO₄ in 1.0 L of de-ionized water (pH approx. 7.4)).

Reagent use:
Spray the plate with 4.0 mL of spraying solution A with the Derivatizer, yellow nozzle, spraying level 4, and leave the plate (horizontal; outside of the Derivatizer) for 15 min at room temperature.

[Note]: with 4.0 mL, the plate should not dry out.

2. Spraying solution B: Fast blue salt

Reagent preparation:
Mix 40.0 mL of fast blue solution (100.0 mg of fast blue salt B in 40.0 mL of de-ionized water) with 10.0 mL of 1-naphthyl acetate solution (25.0 mg of 1-naphthylacetate in 10.0 mL of ethanol).

Reagent use:
Spray the plate with 2.0 mL of praying solution B with the Derivatizer, yellow nozzle, spraying level 4, and record the images after 30 min.

For blistering agents and irritants (optional):
 

1. Spraying solution C: 4-(4’-Nitroenzyl)-pyridine solution

Reagent preparation:
Dissolve 5.0 g of 4-(4’-nitrobenzyl)-pyridine in 100.0 mL of ethanol.

2. Spraying solution D: Benzofurazan-(1)-oxide solution

Reagent preparation:
Dissolve 1.0 g of benzofurazan-(1)-oxide in 100.0 mL of ethanol.

Reagent use:
Spray the plate with 2.0 mL of spraying solution B with the Derivatizer, yellow nozzle, spraying level 4, and record the images after 30 min.

3. Spraying solution E: NaOH solution

Reagent preparation:
Dissolve 4.0 g of NaOH in a mixture of 50.0 mL of de-ionized water and 50.0 mL of methanol.

Reagent use:
Spray the plate with spraying solution C with the Derivatizer (yellow nozzle, 3.0 mL, spraying level 4), heat at 150 °C for 30 s, and immediately record images. Spray the plate with spraying solution D with the Derivatizer (yellow nozzle, 3.0 mL, spraying level 3), and then with spraying solution E with the Derivatizer (yellow nozzle, 3.0 mL, spraying level 6), and record the images within the next 2 min.

Documentation

TLC Visualizer in UV 254 nm, UV 366 nm, and white light prior to derivatization, and UV 366 nm, and white light after derivatization (as needed).

Densitometry

For the UHM, TLC Scanner 4 and visionCATS, absorbance measurement at 254 nm, slit dimension 5.00 mm x 0.20 mm, scanning speed 50 mm/s, and in fluorescence mode at 366>/400 nm. For the other substances, each standard is detected at their maximum of absorption as described in the following table.

Results and discussion

For each method, the UHM was used as SST and the R_F values to obtain for each method are described as follows:

For nerve agents, a large-scale untargeted screening of samples was developed, involving the detection of toxic substances without specific identification

In this approach, each sample (utilizing reference substances in our example) was applied at different Y positions, forming a zone equivalent to a 1.0 mm band, with varying application volumes. In our example, the screening was applied on a 20 x 10 cm plate, but the screening could also be applied to a 20 x 20 cm plate.

Following the application, no development was conducted, but the entire plate underwent derivatization. Positive zones were observed as yellow against a pink/violet background. This test revealed that each nerve agent was still detectable at very low absolute quantities (amount on the plate):

• GA, VX, RVX < 0.25 ng
• GB < 0.125 ng
• GD < 0.025 ng
• GF < 0.01 ng

Conclusion

The examples above show that HPTLC is a valuable tool for identifying nerve agents, blistering agents and irritants, as well as arsenic agents which are important for law enforcement and military personnel in preventing chemical warfare. HPTLC’s format preserves the separated zones, allowing for further investigation including bioassays like acetylcholinesterase inhibition. Additionally, the use of HPTLC instruments reduces the need for analysts to physically interact with toxic samples, enhancing safety.

Literature

[1] CAMAG Application note A-142.1: Identification and quantification of arsenics agents L, DA and DM by HPTLC.

[2] CAMAG Application note A-143.1: Identification and quantification of blistering agents and irritants HD, TTA, CS, CN, CA and CB by HPTLC.

[3] CAMAG Application note A-144.1: Identification and quantification of nerve agents RVX, VX, GD, GA, GF and GB by HPTLC, and methodology for a large-scale untargeted screening.

[4] T. K. T. Do et al., J Chromatogr A (2021) 1638

Further information is available on request from the authors.

Contact

Dr. Tiên Do, Sonnenmattstrasse 11, 4132 Muttenz, Switzerland, tien.do@camag.com