molecules Review Analysis of Organophosphorus-Based Nerve Agent Degradation Products by Gas Chromatography-Mass Spectrometry (GC-MS): Current Derivatization Reactions in the Analytical Chemist’s Toolbox Carlos A. Valdez 1,2,3,* and Roald N. Leif 1,2,3 1 Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA; [email protected] 2 Nuclear and Chemical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA 3 Forensic Science Center, Lawrence Livermore National Laboratory, Livermore, CA 94550, USA * Correspondence: [email protected] Abstract: The field of gas chromatography-mass spectrometry (GC-MS) in the analysis of chemical warfare agents (CWAs), specifically those involving the organophosphorus-based nerve agents (OPNAs), is a continually evolving and dynamic area of research. The ever-present interest in this field within analytical chemistry is driven by the constant threat posed by these lethal CWAs, highlighted by their use during the Tokyo subway attack in 1995, their deliberate use on civilians in Syria in 2013, and their use in the poisoning of Sergei and Yulia Skripal in Great Britain in 2018 and Citation: Valdez, C.A.; Leif, R.N. Alexei Navalny in 2020. These events coupled with their potential for mass destruction only serve to Analysis of Organophosphorus-Based stress the importance of developing methods for their rapid and unambiguous detection. Although Nerve Agent Degradation Products by Gas Chromatography-Mass the direct detection of OPNAs is possible by GC-MS, in most instances, the analytical chemist must Spectrometry (GC-MS): Current rely on the detection of the products arising from their degradation. To this end, derivatization Derivatization Reactions in the reactions mainly in the form of silylations and alkylations employing a vast array of reagents have Analytical Chemist’s Toolbox. played a pivotal role in the efficient detection of these products that can be used retrospectively to Molecules 2021, 26, 4631. https:// identify the original OPNA. doi.org/10.3390/molecules26154631 Keywords: nerve agents; GC-MS; derivatization; silylation; methylation; chemical warfare agents; Academic Editor: Paraskevas sarin; Novichoks D. Tzanavaras Received: 19 June 2021 Accepted: 29 July 2021 1. Introduction Published: 30 July 2021 Gas Chromatography-Mass Spectrometry (GC-MS) has been a central analytical tech- nique in the field of Chemical Warfare Agent (CWA) detection, analysis, and identification. Publisher’s Note: MDPI stays neutral The role that this form of mass spectrometric method has provided to analysts immersed with regard to jurisdictional claims in published maps and institutional affil- in this specific area is invaluable and continues to grow in importance as the CWA molec- iations. ular space rapidly expands. CWAs represent some of the most toxic chemical entities ever produced in a laboratory. Their sole purpose is to eliminate a threat or incapac- itate it [1]. Among the most notorious members in this class of toxic compounds are the organophosphorus-based nerve agents [2]. The lethal effects of nerve agents stem from their efficient inhibition of the enzyme acetylcholinesterase (AChE), which is an Copyright: © 2021 by the authors. enzyme involved in maintaining homeostasis in the nervous system and controls mus- Licensee MDPI, Basel, Switzerland. cle function [3,4]. Inhibition of AChE leads to the accumulation of the neurotransmitter This article is an open access article distributed under the terms and acetylcholine in synaptic junctions that results in complete muscle relaxation, an event that, conditions of the Creative Commons when affecting those involved in respiration, leads to the demise of the affected individual Attribution (CC BY) license (https:// by asphyxiation. creativecommons.org/licenses/by/ Traditional OPNAs fall into two main categories since their introduction in the 20th 4.0/). century, being used against military as well as civilian targets and these are the G-series Molecules 2021, 26, 4631. https://doi.org/10.3390/molecules26154631 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 18 Molecules 2021, 26, 4631 2 of 18 Traditional OPNAs fall into two main categories since their introduction in the 20th century, being used against military as well as civilian targets and these are the G-series and the V-series (Figure 1) [5–7]. The G-series were developed in Germany and hence the and the V-series (Figure1)[ 5–7]. The G-series were developed in Germany and hence G-prefix in their notation, and they share a common chemical core, a pentavalent phos- the G-prefix in their notation, and they share a common chemical core, a pentavalent phorus center containing a methyl group, a fluorine atom and an alkyl ester group that phosphorus center containing a methyl group, a fluorine atom and an alkyl ester group ultimately distinguishes each member in this class from one another. Thus, Sarin (or GB) that ultimately distinguishes each member in this class from one another. Thus, Sarin possesses the isopropoxy moiety, while Soman (or GD) possesses the chiral, 2,2-dimethyl- (or GB) possesses the isopropoxy moiety, while Soman (or GD) possesses the chiral, 2,2- 2-butoxy moiety and lastly Cyclosarin (or GF) which contains the cyclohexyloxy moiety. dimethyl-2-butoxy moiety and lastly Cyclosarin (or GF) which contains the cyclohexyloxy moiety.The OPNA The Tabun OPNA (or Tabun GA) (oris considered GA) is considered a G-series a G-seriesagent even agent though even it thoughdoes not it doesshare notmany share common many structural common structuralfeatures with features the ot withher members the other in members the group. in theGA group.possesses GA a possessescyanide moiety a cyanide instead moiety of the instead fluoride of thewhich fluoride serves which as the serves leaving as group the leaving in addition group to in a additiondimethylamino to a dimethylamino moiety instead moiety of a methyl instead grou of ap. methyl The second group. category, The second the category,V-series also the V-seriesconsists alsoof a consistspentavalent of a pentavalentphosphorus phosphorusatom featuring atom an featuring alkoxy and an alkoxya methyl and substituent a methyl substituentin addition into additiona N,N-dialkylaminoethylthiolate to a N, N-dialkylaminoethylthiolate group that serves group as that a leaving serves as group a leaving in its groupreaction in with its reaction AChE. withOne of AChE. the most One fa ofmous the mostmembers famous in this members class is in VX this (O class-ethyl- isS VX-[2- ((Diisopropylamino)ethyl]O-ethyl-S-[2-(Diisopropylamino)ethyl] methylphosphonothioa methylphosphonothioate)te) that consists thataside consists of a methyl aside ofand a methylethoxy andsubstituents, ethoxy substituents, the hallmark the N,N-diisopro hallmark N, Npylaminoethylthiol-diisopropylaminoethylthiol substituent. substituent. Addition- Additionally,ally, the other the two other members two members in this class, in this VR class, (Russian VR (Russian VX) and VX) CVX and (Chinese CVX (Chinese VX) are VX)structural are structural isomers isomersof VX and of VX of andeach of other. each In other. the Incase the of case VR, of the VR, alkoxy the alkoxy group group is the isisobutoxy the isobutoxy group group while whilethe thioalkyl the thioalkyl side chain side is chain the N is,N the-diethylaminoethylthiolN,N-diethylaminoethylthiol substit- substituent.uent. For CVX, For the CVX, alkoxy the alkoxygroup is group the n is-butoxy the n-butoxy group while group the while thioalkyl the thioalkyl side chain side is chainthe N is,N the-diethylaminoethylthiolN,N-diethylaminoethylthiol substituent. substituent. A third Acategory third category that broke that into broke the intoworld’s the world’sspotlight spotlight are the areA-series the A-series or Novichok or Novichok agents agents[8] with [8 their] with use their in usethe poisoning in the poisoning of Sergei of SergeiSkripal Skripal and his and daughter his daughter in Salisbury, in Salisbury, UK in 2018 UK [9] in 2018and the [9] poisoning and the poisoning of Alexei ofNavalny, Alexei Navalny,who became who ill became during illa flight during from a flight Tomsk from to TomskMoscow, to Moscow,Russia in Russia2020 [10]. in 2020The structures [10]. The structuresof these “newcomers” of these “newcomers” as their name as theirmeans name are very means different are very from different the G- from and the the V-series G- and theand V-series also differ and significantly also differ significantlyamong themselves among to themselves the point that to thethey point do not that share they the do same not sharestructural the same isomerism structural the V-series isomerism do. Althou the V-seriesgh the do. structural Although make-up the structural among these make-up three amongkinds of these OPNAs three is kindsdifferent, of OPNAs one common is different, factor one that common unites them factor all that is the unites presence them of all a ishighly the presence reactive, ofpentavalent a highly reactive, phosphorus pentavalent center. For phosphorus the Novichok center. agents, For thethe Novichokrole of the agents,leavingthe group role is of played the leaving by the groupfluorine is atom played just by as the in fluorinethe case of atom Sarin just or asSoman. in the One case last of Sarinreason or for Soman. the basis One of last OPNA’s reason worldwide for the basis conc ofern OPNA’s is the worldwide fact that established concern is synthetic the fact thatroutes established exist for synthetictheir large-scale routes exist production. for their large-scaleFortunately, production. a great deal Fortunately, of information a great is dealknown of informationabout these isroutes known and about the ever-evolving these routes andfield the of chemical ever-evolving forensics field is of rapidly chemical de- forensicsveloping ismethods rapidly developinginvolving chemical methods attributio involvingn signature chemical attributionanalysis to signaturehelp forensic analysis ana- tolytical help chemist forensic determine analytical chemisthow a sp determineecific OPNA how synthesized a specific OPNA [11–13]. synthesized [11–13]. Figure 1.