Software-Assisted Automated Detection and Identification of “Unknown” Analogues: Implementation on V-Type Nerve Agents

Abstract

V-type nerve agents are among the most toxic organophosphorus chemical warfare agents, and they are under strict regulation and supervision by the OPCW (Organization for the Prohibition of Chemical Weapons). The V-type class of materials refers to a potentially large number of analogues and isomers. In order to expose instances of unfulfillment of the OPCW treaty, it is essential to have the ability to detect and identify “unknown” analogues of this family, even in the absence of an analytical standard. This work demonstrates a new automated tool for the detection and identification of V-type analogues, using high-resolution-accurate-mass LC-MS analysis, followed by “Compound Discoverer” software data processing. This software, originally developed for metabolism and metabolomics screening, is used here to automatically detect various V-type analogues by picking peaks and comparing them to “in-silico” calculated modifications made on a predefined basic V-backbone structure (according to the OPCW definitions for V-type agents). Subsequently, a complete structural elucidation for the proposed molecular formula is obtained by MS/MS data analysis of the suspected component, for both the V-type analogue (using ESI­(+) analysis) as well as its hydrolysis product (using ESI(−) analysis) for a better elucidation of the phosphonate “head” structure. This method was found to be useful for the detection and identification of several “unknown” analogues, at low ng/mL levels in soil extracts.

Discovery of Modified Metabolites, Secondary Metabolites, and Xenobiotics by Structure-Oriented LC–MS/MS

“…62,63 These approaches have been used to discover numerous drug biproducts 64−68 and nerve agents and their degradation products. 69,70 In this review, we cover the concept of structure-oriented LC−MS approaches and the analytical paradigms possible for monitoring compounds through elemental, structural moiety, or functional group similarity. We will cover five of the most common approaches, including (1) constant ion monitoring, (2) fragmentation filtering, (3) in-source fragmentation, (4) mass defect filtering, and (5) isotope pattern filtering.…”

“…Structure-oriented monitoring methods are employed for the detection of multiple classes of toxicologically or pharmacologically relevant compounds. In contrast to unbiased-shotgun methodologies, structure-oriented monitoring utilizes prior knowledge of the elemental composition, structural moiety, or functional group characteristics to develop an analytical pipeline honed for the detection of a subset of compounds. , Fragmentation-based LC–MS/MS methods are routinely used to characterize phase I and phase II metabolites such as cysteinyl, ,− sulfate, ,, or glucuronide conjugated products. ,, Additionally, discovery methods have been developed to monitor for specific classes of clinically relevant molecules by diagnostic fragmentation patterns, including steroid hormones, − covalently modified DNA and RNA nucleosides, ,− and small molecules or peptides exhibiting glycosylated, phosphorylated, or other distinct functional groups modifications. − Early stage drug discovery requires extensive characterization of drug metabolism, including description of drug-related metabolites, potential reactive intermediaries, and degradation products. ,, To characterize this array of metabolically diverse drug intermediates, structure-oriented monitoring approaches were developed to screen predictable transformations frequently occurring in vivo , such as hydroxylation, dehydrogenation, and demethylation, among other common phase I or II conjugation reactions. , These approaches have been used to discover numerous drug biproducts − and nerve agents and their degradation products. , …”

Integrating Effect-Directed Analysis and Chemically Indicative Mass Spectral Fragmentation to Screen for Toxic Organophosphorus Compounds

“…Identifying “unknown” compounds in complex matrices is one of the most difficult challenges analytical chemists face. , This challenge usually arises in environmental, security, or medical settings, where the analytical chemist has to detect and identify an unknown bioactive compound, at a trace level, out of an interfering matrix. , …”

“…1,2 This challenge usually arises in environmental, security, or medical settings, where the analytical chemist has to detect and identify an unknown bioactive compound, at a trace level, out of an interfering matrix. 3,4 To identify such compounds, one usually relies on mass spectral libraries. However, these mass spectral libraries are not always exhaustive and will miss any "unknown unknowns", compounds never previously recorded or not yet included in commercial MS databases.…”

“…We present two chromatograms: one showing the molecular ions of methomyl and tributyl phosphate obtained from a full scan experiment, and another chromatogram showing the sum of two organophosphorus-indicative ions. The two low-mass organophosphorus-indicative ions chosen, based on previous reports, , are m / z 78.9944, which corresponds to a [PO 2 CH 3 +H] + ion, and m / z 97.0050, which corresponds to a [PO 3 CH 5 +H] + ion, both with m / z resolution tolerance of 3 ppm.…”