Despite it being banned under the Chemical Warfare Convention, VX is still being used, as demonstrated in 2017 with the assassination of Kim Jong Nam in Malaysia. In addition to conducting the typical forensic investigations, incidence of the deliberate use of chemical warfare agents (CWA) such as VX could also include undertaking chemical forensics to address questions including method of production, precursor-product-matching and batch-matching. The identification of residual impurities in VX, known as chemical attribution signatures (CAS), that allow for these questions to be addressed, would be beneficial to agencies undertaking these investigations.To increase the knowledge pool regarding CAS specific to a particular production method, five synthetic routes of VX formation were chemically profiled and subjected to multivariate analysis. In total, forty-one impurities of interest were identified that could be attributed to a production method. The structures of thirty-four were confirmed through a combination of database matching, accurate mass measurement, literature analysis and comparison with authentic standards. Several of these impurities were identified as candidate CAS for method attribution. Additionally, the longevity of the CAS was assessed by analysing samples after storage for 18 months at room temperature. While samples could not be classified (via MVA) to a specific production method, nineteen candidate CAS were still able to be identified. These results are suggestive that VX method of production may still be able to be elucidated on long term storage.

Recently, the Annex on Chemicals of the Chemical Weapons Convention was amended to include four new entries to the Schedule 1 toxic chemical list. These new entries included the novichok agents A-230, A-232 and A-234. In addition to this, 17 N,N-dialkyl(alkan)imidamides precursor chemicals were added to various export control lists. These additions increased the population of chemicals that could require reporting during both the Organisation for the Prohibition of Chemical Weapons (OPCW) Official Proficiency Test (PT) and authentic sample analysis. The network of OPCW Designated Laboratories (DL) therefore needs to be aware of these newly listed chemicals, their precursor chemicals and degradation products and associated chromatographic and mass spectral properties. N,N-Dialkyl(alkan)imidamides often exhibit poor gas chromatography properties and require derivatisation prior to analysis. Complicating this is the difficulty in silylating N,N-dialkyl(alkan)imidamides in aqueous environments, in addition to the inherent instability of the subsequent silyl ester. Therefore, derivatising agents that readily react with N,N-dialkyl(alkan)imidamides to form derivatives with improved GC-MS properties and are stable for extended time periods are of importance to DLs. A possibility to address these twin concerns is derivatising with an alkyl chloroformate using modified Schotten-Baumann conditions to form the corresponding carbamate. Using these conditions, a series of N,N-dialkyl(alkan)imidamides were derivatised with methyl chloroformate and assessed for an improvement to both their GC-MS behaviour and stability. Additionally, samples from the 56th and 57th OPCW Official PT, spiked with N,N-diisopropylethanimidamide and N-methyl-N-propylethanimidamide respectively, were treated with methyl chloroformate. Significant improvements in stability and chromatographic properties were observed in all instances.

The ongoing use of chemical warfare agents (CWAs) in conflicts, assassinations, and terrorist attacks means that the detection and identification of these compounds are crucial. The forensic identification of organophosphorus nerve agents (OPNAs) and their precursors and degradation products remains challenging due to the destructive nature and extensive preparation required for conventional chromatographic methods. In this study, we characterise precursor and degradation products of Novichok analogues, including 1,1,3,3-tetramethylguanidine, N,N-diethylpentanimidamide, N,N-dipropylbutanimidamide, using 2D ¹H-¹³C heteronuclear multiple quantum coherence (HMQC) NMR. We further investigate mixtures of phosphonate compounds [dimethyl methylphosphonate (DMMP), diisopropyl methylphosphonate (DIMP), diethyl 2,2-diethoxyethylphosphonate (DEOP), and dibutyl butylphosphonate (DBBP)] and a fully degraded VX sample using ¹H diffusion-ordered spectroscopy (DOSY) and 3D ¹H-¹³C DOSY-HMQC NMR. The ¹H DOSY experiments successfully separated components of both amine and phosphonate mixtures, while analysis of the degraded VX sample revealed key degradation products, including ethyl methylphosphonic acid and bis-2-(diisopropylaminoethyl)disulfide. The 3D DOSY-HMQC method provided improved resolution of overlapping signals compared to 2D approaches, representing the first application of this experiment to CWA-related compounds. These results demonstrate that DOSY-based NMR can virtually separate complex mixtures non-destructively, providing complementary capability to GC-MS and LC-MS in forensic CWA investigations.

The host-guest chemistry of O,O '-diisopropyl fluorophosphate (DFP), a phosphonofluoridate G-series chemical warfare agent simulant, was investigated in the presence of a number of octanuclear cubic coordination cage hosts. The aim was to demonstrate cage-catalysed hydrolysis of DFP at near neutral pH: however, two octanuclear coordination cages, H-PEG (containing water-solubilising PEG groups) and H-W (containing water-solubilising hydroxymethyl groups), were actually found to increase the lifetime of DFP in aqueous buffer solution (pH 8.7). Crystallographic analysis of DFP with a structurally related host cage revealed that DFP binds to windows in the cage surface, not in the internal cavity. The phosphorus-fluorine bond is directed into the cavity rather than towards the external environment, with the cage/DFP association protecting DFP from hydrolysis. Initial studies with the chemical warfare agent (CWA) sarin (GB) with H-PEG cage in a buffered solution also showed a drastically reduced rate of hydrolysis for sarin when bound in the host cage. The ability of these cages to inhibit hydrolysis of these P-F bond containing organophosphorus guests, by encapsulation, may have applications in forensic sample preservation and analysis.

An interlaboratory evaluation of a chemical profiling method for a nerve agent precursor was performed by eight chemical analytical laboratories with a wide global distribution. A set of chemical attribution signatures (i.e. impurities) present in methylphosphonic dichloride (DC) were analysed by gas chromatography-mass spectrometry (GC/MS). The GC/MS analyses were performed with the individual instrumentation and analytical methods used by the laboratories in proficiency tests organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). Two batches of DC, produced by two different production routes, were dispatched together with one reference sample. By the use of a targeted MS-library, sixteen chemical attribution signatures were analysed in the DC samples. Retention indices, mass spectra and relative peak areas of the GC/MS data were evaluated. When the within batch-data of the eight laboratories were compared, similarity values of 0.720-0.995 were obtained. In addition, the two different batches had clearly unique chemical attribution profiles, as indicated by a large between batch-distance similarity values of 0.509-0.576. Retention indices showed less than +/- 14 retention index unit variation for the 16 chemical attribution signatures. This study showed the potential of getting consistent chemical profiling data over multiple laboratories by the use of retention indices for alignment of GC/MS-data. The results indicate that the GC/MS instrumentation and methods used at most OPCW designated laboratories are valid tools for the acquisition of chemical profiling data of importance for the attribution analysis to study suspected production or use of chemical warfare agents.

Sarin is a highly toxic nerve agent classified by the Chemical Weapon Convention as a Schedule 1 chemical with no use other than to kill or injure. Moreover, in recent times, chemical warfare agents have been deployed against both military and civilian populations. Chemical warfare agents always contain minor impurities that can provide important chemical attribution signatures (CAS) that can aid in forensic investigations. In order to understand the trace molecular composition of sarin, various analytical approaches including GC–MS, LC–MS and NMR were used to determine the chemical markers of a set of sarin samples. Precursor materials were studied and the full characterisation of a synthetic process was undertaken in order to provide new insights into potential chemical attribution signatures for this agent. Several compounds that were identified in the precursor were also found in the sarin samples linking it to its method of preparation. The identification of these CAS contributes critical information about a synthetic route to sarin, and has potential for translation to related nerve agents.

From an analytical chemistry standpoint, determining the chemical attribution signatures (CAS) of synthetic reaction mixtures is an impurity profiling exercise. Identifying and understanding the impurity profile and CAS of these chemical agents would allow them to be exploited for chemical forensic information, such as how a particular chemical agent was synthesised. Being able to determine the synthetic route used to make a chemical agent allows for the possibility of batches of the agent, and individual incidents using that agent, to be forensically linked. This information is of particular benefit to agencies investigating the nefarious and illicit use of chemical agents. One such chemical agent of interest to law enforcement and national security agencies is fentanyl. In this study two acylation methods for the final step of fentanyl production, herein termed the Janssen and Siegfried methods, were investigated by liquid chromatography- high resolution mass spectrometry (LC-HRMS) and multivariate statistical analysis (MVA). From these data, fifty-five chemical impurities were identified. Of these, ten were specific CAS for the Janssen method, and five for the Siegfried method. Additionally, analytical data from four different literature methods for production of the fentanyl precursor 4-anilino-N-phenethylpiperidine (ANPP), were compared to the results obtained from the method of production (Valdez) used in this study. Comparison of the LC-HRMS data for these five methods allowed for four Valdez specific impurities to be identified. These may be useful CAS for the Valdez method of ANPP production.

The organophosphorous nerve agent VX is classified by the Chemical Warfare Convention (CWC) as a Schedule 1 chemical; namely a substance that is highly toxic with no use that is of benefit to society. Even with this classification, the nefarious use of the Schedule 1 chemical VX has been observed, as demonstrated in 2017 in Malaysia. Therefore, undertaking chemical analysis on samples of VX to identify chemical attribution signatures (CAS) for chemical forensics is required. To further understand the chemical profile of VX, and to aid in the identification of potential CAS, three in house synthesised stocks of VX were investigated. The three VX stocks analysed were synthesised in 2014, 2017 and 2018 using the same method, allowing for a comparison of data between each of the stocks at different stages of storage. As opposed to a majority of literature reports, these agent stocks were not stabilised, nor were they subjected to forced degradation. Using NMR, high resolution (HR) LC-HRMS, GC-(EI)MS and GC-(CI)MS to gain a full insight into the CAS profile, a total of 44 compounds were identified. Of these compounds, 30 were readily identified through accurate mass measurement and NIST library matches. A further seven were identified through extensive LC-HRMS/MS studies, with seven remaining unresolved. Several compounds, identified in minor amounts, were able to be traced back to impurities in the precursor compounds used in the synthesis of VX, and hence may be useful as CAS for source attribution.

Here, samples of castor oil were extracted from Australian Ricinus communis seeds. The extracted oils were esterified and the derivatives characterised by gas chromatography-mass spectrometry (GC–MS). The fatty acid composition of the oils was profiled, enabling the trace fatty acid components of castor oil to be reported for the first time. The resulting data were also subjected to statistical analysis to test the applicability for provenance and geographic attribution purposes, but no discrimination was found.

The castor bean plant, Ricinus communis, grows wild throughout many regions of Australia. The seeds of the plant contain the schedule 1 chemical agent ricin, a type II ribosomal inhibiting protein. Currently there are limited analytical techniques that can be applied in analysis of the seeds to establish attribution. In this study, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for the analysis of seeds collected from 68 plants across 38 locations around Australia. Of the 92 elemental isotopes measured, fifteen (24Mg, 27Al, 44Ca, 53Cr, 55Mn, 57Fe, 60Ni, 65Cu, 66Zn, 75As, 85Rb, 88Sr, 98Mo, 138Ba and 202Hg) yielded data that were relevant to all collection sites. Data were further analysed using multivariate statistical analysis which facilitated the potential for the identification of unique provenance isotopes. Furthermore, this analysis indicated that 59Co was present at significant levels in Victorian and Sydney specimens only.

INTRODUCTION:Seeds of Ricinus communis contain the toxic protein ricin, a 64 kD heterodimeric type II ribosome-inactivating protein that has been used in several high-profile poisoning incidents. The ability to determine which cultivar the toxin was isolated from via an LC-MS method would be of significant use to law enforcement and forensic agencies.OBJECTIVE:To analyse via LC-MS and chemometrics (principal components analysis (PCA), orthogonal partial-least-squares discriminant analysis (OPLS-DA)) extracts of R. communis to identify compounds specific to a particular cultivar.METHODS:Seeds from eight specimens of six cultivars of R. communis ('carmencita', 'dehradun', 'gibsonii', 'impala', 'sanguineus' and 'zanzibariensis') were extracted using a standard methodology. These extracts were analysed by LC-MS then subjected to chemometric analysis (PCA and OPLS-DA). Identified compounds of importance were subjected to high-resolution Fourier transform (HRFT) MS and MS/MS to elucidate their structures.RESULTS:This analysis identified 17 ions as potential cultivar determinators. Through accurate mass measurement and MS/MS, molecular formulae for 13 ions were determined, including two known and 11 new peptides.CONCLUSION:Unique ions in extracts of 'carmencita', 'dehradun', 'gibsonii', 'impala' and 'zanzibariensis' were identified that would allow an individual cultivar to be distinguished from other cultivars in this study. Although 'sanguineus' extracts contained no unique compounds, a unique LC-MS profile would allow for cultivar assignment.

: In 2009 a National Security Science and Technology grant was awarded to the Human Protection and Performance Division for the investigation of several forensic aspects of the castor bean plant Ricinus communis. A major focus of this grant was to understand the chemical composition of the seeds, and to ascertain if these differences could be used for provenance classification. This technical report will discuss progress made during these investigations.

The methanol extract of an assemblage of Halimeda stuposa and a Dictyota sp., yielded three natural products characteristic of Dictyota sp., and one of Halimeda sp. These included the xenicane diterpene 4-hydroxydictyolactone (1), and the diterpenes dictyol E (2), 8a,11-dihydroxypachydictyol A (3) and indole-3-carboxaldehyde (4). A minor revision of 1 and new spectroscopic data for 1 and 2 are provided, along with associated anti-cancer activities of compounds.

The tetrahydro-β-carboline, callophycin A (1), was isolated from the methanol extract of the red alga Callophycus oppositifolius collected from Pugh Shoal, north east of Truant Island, Northern Territory, Australia. The structure and relative stereochemistry of 1 was determined through extensive NMR analysis. Callophycin A (1) was tested against a panel of mammalian cell lines and found to be generally cytotoxic.

While investigating the cytotoxic activity of the methanol extract of an Australian marine sponge Stelletta sp. (Demospongiae), a new diketopiperazine, cyclo-(4-S-hydroxy-R-proline-R-isoleucine) (1), was isolated together with the known bengamides; A (2), F (3), N (4), Y (5), and bengazoles; Z (6), C(4) (7) and C(6) (8). The isolation and structure elucidation of the diketopiperazine (1), together with the activity of 1-8 against a panel of human and mammalian cell lines are discussed.

Three new merosesquiterpenoids, metachromins U, V, and W (1-3), were isolated from a specimen of the marine sponge Thorecta reticulata collected off Hunter Island, Tasmania, Australia. Structures of the new compounds were elucidated through extensive NMR investigations and comparison with literature values. The cytotoxicities of 1-3 were assessed against a panel of human tumor cell lines (SF-268, H460, MCF-7, and HT-29) and a mammalian cell line (CHO-K1). All compounds were found to have 50% growth inhibition activities in the range 2.1-130 μM, with 2 being the most active (GI50 2.1-10 μM).

Bioassay-guided fractionation of extracts of the brown alga Sporochnus comosus led to the isolation of five new compounds, comosusols A-D (3-6) and comosone A (7). The structures of all isolated compounds were elucidated using standard one- and two-dimensional NMR techniques, as well as comparison with literature values. The cytotoxic activity of all compounds was investigated against a panel of human tumor and mammalian cell lines. These assays found eight of the nine compounds had GI(50) values in the 8-63 μM range.