Chromatographic analysis of chemical warfare agents and their metabolites in biological samples

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Highlights

  • The state of the art of chromatographic analysis of CWA and their metabolites in biological material is presented.

  • The analysis of metabolites and adducts of CWA in blood, urine, plasma, serum, tissues and hairs is described.

  • The identification of metabolites and adducts of CWA in biological material is more important than identification of CWA.

Abstract

The objective of this study is to present and critically analyze the current state of knowledge on the chromatographic analysis of chemical warfare agents (CWAs) and their metabolites in biological and biomedical samples originating from humans and animals. Fast and reliable analysis of these chemicals is extremely important at a time, when more and more cases of CWA intoxication take place by terrorist groups and radical non-state organizations use or plan to use chemical weapons to achieve their own psychological, political and military goals. The review builds on original scientific publications and reports published from 2010 onwards, including some valuable papers released before that year. The authors stated that during last ten years increased number of laboratories are focused not only on trace analysis of CWA (mostly nerve and blister agents) in environmental and biological samples, but the growing number of research are devoted to instrumental analysis of metabolites, adducts and degradation products of these substances. The identification of very low-level concentrations of CWA's metabolites and adducts in biological material is often more important and difficult than the identification of original CWA, because of lower level of concentration and a very large number of compounds are present in biological samples. Many of them are hydrolysis products, metabolites and adducts, and are present in the samples in the ionic and hydrophilic form. For this reason, different chromatographic methods, in most cases liquid chromatography, are used to perform an unambiguous analysis of biological samples: blood, urine, plasma, serum, tissues, hairs.

Introduction

When the Chemical Weapons Convention (CWC) [1] entered into force in 1997, it seemed that interest in chemical weapons and analysis of chemical warfare agents would be less intense as compared to time before the Convention. However, this was not the case, and laboratories dealing with the analysis of chemical agents have increased in number. This is primarily due to the need to improve procedures for analytical verification of the Convention's provisions and, secondly, to the fact that chemical weapons are still in use. They were employed on numerous occasions during armed conflicts, e.g. in Syria [2], and terrorist acts, e.g. in the assassination of Kim Jong-nam on 13 February 2017 at the Kuala Lumpur airport [3] and the near-fatal poisoning of the Skripals on 4 March 2018 in Salisbury, UK [4]. The development of new and more reliable procedures for analyzing chemical agents in environmental and biological samples and the improvement of the existing procedures is the principal focus of the Organization for the Prohibition of Chemical Weapons (OPCW), an international agency which conducts, inter alia, proficiency tests of laboratories designated for the analysis of chemical agents. This has led to studies into new testing procedures for chemical agents. Some of them are referred to in review studies on the analysis of chemical warfare agents, their precursors and degradation products [5,6].

The analysis of chemical agents covered by the CWC [1] includes analysis of toxic agents, substrates for their production and their decomposition products. Most tests are carried out for chemicals which tend to be present in the environment – water, soil, air or food – following the use of chemical warfare agents. Of special importance, however, is the analysis of chemical agents and their metabolites in living human and animal organisms. On the one hand the analysis of biological material can provide evidence that an organism has been poisoned with a specific type of chemical agent, and on the other, it allows insight into how chemical agents affect living organisms and promotes the search for effective methods for poisoning treatment.

The knowledge on the analysis of chemical agents in biological material as well as the historical background and development prospects for this area of research have been outlined in the review studies by Black et al. published between 2008 and 2013 [7,8]. Numerous original research papers have been published since then, which demonstrates the substantial progress that has been made in analytical testing of the presence of chemical agents and their transformations in living organisms. A point of interest is to what extent the prospects envisaged by Black have become a reality. There are also other review papers which, however, address the issues of this study only to a limited extent [9,10].

The objective of this study was to present and critically analyze the current state of knowledge on the chromatographic analysis of chemical warfare agents (CWAs) and their metabolites in biological and biomedical samples originating from humans and animals. The study builds on original scientific publications and reports published from 2010 onwards, including some valuable papers released before that year.

CWAs can be analyzed in vitro, in model samples, using biological material of human and animal origin, as well as in vivo when humans or animals come into direct contact with toxic substances. In the latter case, the analysis of CWAs is combined with the assessment of the level of human exposure to organophosphorus pesticides. In the recent years, studies on the analysis of CWAs have focused essentially on organophosphorus compounds and sulfur mustard. There are few studies concerning other substances [11,12].

The analysis of biological material for CWAs is a very responsible and a difficult task. In accordance with the OPCW's guidelines, results of chemical tests performed for the purpose of collecting evidence must be indisputable and obtained using at least two different analytical methods for different markers.

Some laboratories are approved by the OPCW for testing biological material for CWAs, e.g. Dutch and German laboratories which have been designated laboratories since 2016 [13].

The OPCW organizes training sessions aimed to improve the methodology of testing biological material. The procedures described in the studies [[14], [15], [16]] have been used during the OPCW's confidence-building exercises for analysis of biomedical samples.

Section snippets

Preparation of samples for analysis of CWA metabolites

In most chemical analysis, including analysis of biological material, of critical importance is the preparation of samples. The procedure used for preparing samples determines whether it will be at all possible to perform analysis and with what uncertainty. In many cases it is a highly time-consuming step, which generally requires more time than the analysis itself. The procedure of sample preparation varies according to what substance is to be detected and identified, and whether it is to be

General problems in the analysis of CWAs in biological material

Tests for toxic agents and their markers in biological material are mostly performed by liquid chromatography, including ionic chromatography [71]. This includes increasingly rapid liquid chromatography and micro-chromatography. The analysis is usually performed using reverse phase chromatography with mobile phase gradient. The HILIC method [44,45] is rarely used.

In some procedures, the analysis for markers of chemical agents can be completed within a short period of time, e.g. the separation

Conclusions

Based on a review of literature on chromatographic analysis of CWA's metabolites in the last decade, it can be concluded that the related studies coincided to a substantive extent with the prospects outlined by Black et al. [7,8] and by van der Schans [96] regarding the chemical analysis of biomedical samples. Chromatographic analysis is currently the most common method of testing for markers of CWA poisoning. Of greatest importance among chromatographic methods is high-performance and

Acknowledgments

This research work was founded by the statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Advanced Technologies and Chemistry of Military University of Technology (projects PBS 23/900/WAT and UGB 23/762/WAT).

Abbreviations - Acronyms

[S-HETE]-CPF
sulfur-hydroxyethylthioethyl cysteine-proline-phenylalanine
AChE
acetylcholinesterase
ACN
acetonitryle
APA
alkylphosphonic acid
APH
Acylpeptide hydrolase
BChE
butyrylcholinesterase
Bis-G
bis(2-ethyl-N7-guanine) thioether
2-CEES
2-chloroethylethyl sulfide (CEES)
ChE
cholinesterase
CHMPA
o-cyclohexyl methylphosphonic acid (cyclosarin)
CMPA
cyclohexyl methylphosphonic acid
C-VX
Chinese VX
CWA
chemical warfare agent
CWA
Chemical Weapons Convention
DAP
dialkyl phosphate
DEDTP
diethyl dithiophosphate
DEP
diethyl

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