Analysis of munitions constituents in IMX formulations by HPLC and HPLC–MS

doi:10.1016/j.talanta.2014.02.013

Talanta

Volume 128, 1 October 2014, Pages 524-530

https://doi.org/10.1016/j.talanta.2014.02.013 Get rights and content

Highlights

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The single column method for detection of new and legacy munitions constituents.
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UV and MS detection for quantitation and identification of NQ, NTO, RDX and DNAN.
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Optimized gradient elution profile for optimal compound separation.

Abstract

The use of Insensitive Munitions eXplosives (IMX) is increasing as the Army seeks to replace certain conventional munitions constituents, such as 2,4,6-trinitrotolene (TNT), for improved safety. The IMX formulations are more stable and therefore less prone to accidental detonation while designed to match the performance of legacy materials. Two formulations, IMX 101 and 104 are being investigated as a replacement for TNT in artillery rounds and composition B Army mortars, respectively. The chemical formulations of IMX-101 and 104 are comprised of four constituents;2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), 1-nitroguanidine (NQ), and Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) which are mixed in various ratios to achieve the desired performance. The current work details the analysis of the IMX constituents by single column HPLC–UV–ESI-MS. Detection limits determined are in agreement with similar HPLC analysis of compounds, ranging from 7 to 9 μg/L. Gradient mobile phases are used to allow separation of the 4 target compounds in more complex mixture of other concomitant compounds. Mass spectra are used to confirm analyte identity with chromatographic retention time.

Graphical abstract

Introduction

The use of Insensitive Munitions eXplosives (IMX) is increasing as the Army seeks to replace certain conventional munitions constituents (MCs) for improved soldier safety. The IMX formulations are more stable and less prone to accidental detonation while designed to match the performance of legacy materials [1]. Time Magazine named, the BAE Systems developed IMX 101 as one of the top 50 inventions of 2010 [2]. Two formulations of IMX are currently being produced; IMX 101 is qualified as a replacement for trinitrotoluene (TNT) in artillery rounds while IMX 104 is a replacement for composition B (Comp B) [3], [4].

The increase in potential IMX use results in the need for a simple detection method for the four constituents of IMX-101 and 104; 2,4-dinitroanisole (DNAN, C₇H₆N₂O₅), 3-nitro-1,2,4-triazol-5-one (NTO, C₂H₂N₄O₃), 1-nitroguanidine (NQ, C₁H₄N₄O₂), and Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX, C₃H₆N₆O₆). The standard environmental test method, U.S. EPA method 8330, for nitroaromatic, nitramine, and nitroester analysis uses high performance liquid chromatography (HPLC) separation and detection by ultra-violet light absorption [5]. The target analyte list for the U.S. EPA method 8330 contains 17 components: 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 3,5-dinitroaniline, 1,3-dinitrobenzene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), nitrobenzene, nitroglycerin, 2-nitrotoluene, 3-nitrotoluene, 4-nitrotoluene, [3-Nitrooxy-2,2-bis(nitrooxymethyl)propyl] nitrate (PETN), RDX, N-methyl-N,2,4,6-tetranitroaniline (tetryl), 1,3,5-trinitrobenzene, 2,4,6-trinitrotoluene. Variations of this method can use electrospray ionization mass spectrometry (ESI-MS) or tandem mass spectrometry (MS–MS) for detection and quantitation of these constituents. An alternative U.S. EPA method, 8095 [6], uses GC-ECD to quantify all of the target compounds in method 8330. However, three of the IMX constituents, NTO, NQ and DNAN, are not currently on the target analyte list of either U.S. EPA method 8330 or 8095. Of the three, DNAN has been shown previously to be separated from concomitant compounds under the conditions of the US EPA method 8330 by Chow et al. [7].

At present, there is not a simple method which efficiently separates and quantifies insensitive munitions constituents and legacy compounds on a single HPLC column [8]. Previous work has focused on either the separation of individual IMX munitions constituents or the separation of one component and its derivatives [7], [8], [9], [10], [11]. Consequently, the detection methods presently available in the literature utilize multiple columns for the analysis of IMX constituents, extending analysis time and cost.

Previous studies have utilized a two column approach in order to quantify the components of IMX formulations. The two column approach is documented in dissolution studies of NTO from IMX compositions [9]. The researchers used a Thermo Scientific Hypercarb column with an acidified eluent mixture for the analysis of the highly water soluble components, NQ and NTO, and a Dionex Acclaim^® E1 column under the U.S. EPA method 8330 conditions for the analysis of RDX and DNAN. The separation of NQ and DNAN in the presence of RDX has been demonstrated by ultrafast liquid chromatography [10]. NTO and its derivatives have been analyzed by HPLC and capillary electrophoresis [11]. Recently Can and coworkers have studied NTO in complex nitro-explosive mixtures [12].

The objective of the present work was to develop a streamlined HPLC–UV–ESI-MS technique for detection and quantitation of IMX constituents in aqueous matrixes, such as ground and surface water. Modifications to the U.S. EPA method 8330 resulted in a single chromatographic separation and subsequent quantification of NQ, NTO, DNAN and RDX simultaneously, and in the presence of other explosive compounds. The results of water samples analyzed by HPLC–UV–ESI-MS are quantitatively compared to demonstrate the utility and evaluate any limitations of the single column method. The developed HPLC gradient was also used to analyze the compounds of interest on a second chromatographic column which can then provide for dual column analyte confirmation when MS analyte confirmation is not available.

The HPLC method presented allows for the separation of NQ, NTO, DNAN and RDX in a single analysis. The developed method uses an acidified mobile phase gradient, detailed below in Table 1. The gradient steps from an aqueous composition of 86% to 51% before returning to 86% over 45 mins. The mobile phase composition returns to 86% aqueous to allow the column to re-equilibrate prior to the injection of the following sample. The eluent composition consists of trifluoroacetic Acid (TFA), acetonitrile (ACN), methanol (MeOH), and water to separate the constituents on a Synergi 4 m Hydro-RP 80A column. The presented method significantly reduces analysis time, solvent use, and costs for simultaneous detection of the analytes of interest and traditional explosive compounds. This method advances the field of analytical chemistry for detection of insensitive munitions and has the potential to be useful in the analysis of IMX munitions constituents from complex matrixes, such as ground water, and ultimately, soil and tissue.

Section snippets

Reagents and supplies

All commercially available chemicals were of analytical grade or higher purity and were used without further purification. MeOH and ACN were purchased from JT Baker (Phillipsburg NJ). DNAN was purchased from Alfa Aesar(Ward Hill, MA). RDX, NQ, EPA mix A, and EPA mix B were purchased from SigmaAldrich (St. Louis, MO). Military grade crystalline NTO, IMX 101 and IMX 104 were supplied by BAE systems (Holston Army Ammunition Plant, TN) and used without further purification. 18.3 MΩ cm resistivity

Standard chromatograms and detection limits

HPLC–UV–ESI-MS analysis showed excellent separation between DNAN, RDX, NTO, and NQ on the single column method described. The retention times for the IMX munitions constituents are given in Table 1, Table 2 for the Phenomenex Synergi column. Fig. 1 shows the separation of IMX components NQ, NTO, RDX and DNAN from the analysis of a 10 mg/L standard. Two wavelengths are used for optimal detection of all analytes. The insensitive munitions components are easily separated from the solvent void

Conclusions

An HPLC method is described for the single column analysis of IMX munitions constituents. The developed method utilizes an acidified mobile phase gradient to separate the more hydrophilic compounds from the void volume, while maintaining the separation of the more hydrophobic compounds. The method provides quantitative results with acceptable quality control sample results for all four of the IMX munitions constituents. With no preparation changes, the method also allows for simultaneous

Acknowledgments

The use of trade, product, or firm names in this report is for descriptive purposes only and does not imply endorsement by the U.S. Government. The tests described and the resulting data presented herein, unless otherwise noted, were obtained from research conducted under the Environmental Quality and Installations program by the US Army Engineer Research and Development Center. Permission was granted by the Chief of Engineers to publish this information. The findings of this report are not to

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There have been few attempts to consolidate legacy and insensitive munitions analyses. Furthermore, there are no standard methods for insensitive munitions (IM) in tissues, resulting in overlapping methods and supplementary analyses. The goal of the present study was to validate extraction and instrumental methods previously developed and address analytical methodology gaps (missing tissue matrices, combined legacy and IM analysis, and IM compounds). The method encompasses analytes in waters, soils, and tissues. The primary and secondary instrumental methodologies use high performance liquid chromatography-ultraviolet (HPLC-UV) and an alternate LC-mass spectrometry (MS) method, which includes 26 compounds of interest (legacy munitions, IM, IM degradation products, and other munitions compounds). The methods were formally evaluated during a series of double-blind round robin studies including a broad variety of laboratories (Government Department of Defense (DoD), Government non-DoD, ammunition manufacturing, commercial, and academic). The results of these round robin studies were gathered to generate recovery ranges for each of the 26 compounds in each of the matrices. The recovery ranges were subsequently compared with existing recovery ranges for standard explosives analysis, United States Environmental Protection Agency (USEPA) 8330B. The validation study reveals a capable method, which reduces analysis time for IM and legacy munitions analyses.
Genomic investigations of acute munitions exposures on the health and skin microbiome composition of leopard frog (Rana pipiens) tadpoles
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Natural communities of microbes inhabiting amphibian skin, the skin microbiome, are critical to supporting amphibian health and disease resistance. To enable the pro-active health assessment and management of amphibians on Army installations and beyond, we investigated the effects of acute (96h) munitions exposures to Rana pipiens (leopard frog) tadpoles and the associated skin microbiome, integrated with RNAseq-based transcriptomic responses in the tadpole host. Tadpoles were exposed to the legacy munition 2,4,6-trinitrotoluene (TNT), the new insensitive munition (IM) formulation, IMX-101, and the IM constituents nitroguinidine (NQ) and 1-methyl-3-nitroguanidine (MeNQ). The 96h LC50 values and 95% confidence intervals were 2.6 (2.4, 2.8) for ΣTNT and 68.2 (62.9, 73.9) for IMX-101, respectively. The NQ and MeNQ exposures caused no significant impacts on survival in 96h exposures even at maximum exposure levels of 3560 and 5285 mg/L, respectively. However, NQ and MeNQ, as well as TNT and IMX-101 exposures, all elicited changes in the tadpole skin microbiome profile, as evidenced by significantly increased relative proportions of the Proteobacteria with increasing exposure concentrations, and significantly decreased alpha-diversity in the NQ exposure. The potential for direct effects of munitions exposure on the skin microbiome were observed including increased abundance of munitions-tolerant phylogenetic groups, in addition to possible indirect effects on microbial flora where transcriptional responses suggestive of changes in skin mucus-layer properties, antimicrobial peptide production, and innate immune factors were observed in the tadpole host. Additional insights into the tadpole host's transcriptional response to munitions exposures indicated that TNT and IMX-101 exposures significantly enriched transcriptional expression within type-I and type-II xenobiotic metabolism pathways, where dose-responsive increases in expression were observed. Significant enrichment and increased transcriptional expression of heme and iron binding functions in the TNT exposures served as likely indicators of known mechanisms of TNT toxicity including hemolytic anemia and methemoglobinemia. The significant enrichment and dose-responsive decrease in transcriptional expression of cell cycle pathways in the IMX-101 exposures was consistent with previous observations in fish, while significant enrichment of immune-related function in response to NQ exposure were consistent with potential immune suppression at the highest NQ exposure concentration. Finally, the MeNQ exposures elicited significantly decreased transcriptional expression of keratin 16, type I, a gene likely involved in keratinization processes in amphibian skin. Overall, munitions showed the potential to alter tadpole skin microbiome composition and affect transcriptional profiles in the amphibian host, some suggestive of potential impacts on host health and immune status relevant to disease susceptibility.
Methods for simultaneous determination of legacy and insensitive munition (IM) constituents in aqueous, soil/sediment, and tissue matrices
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Currently, no standard method exists for analyzing insensitive munition (IM) compounds in environmental matrices, with or without concurrent legacy munition compounds, resulting in potentially inaccurate determinations. The primary objective of this work was to develop new methods of extraction, pre-concentration, and analytical separation/quantitation of 17 legacy munition compounds along with several additional IM compounds, IM breakdown products, and other munition compounds that are not currently included in U. S. Environmental Protection Agency (EPA) Method 8330B. The eight additional compounds included were nitroguanidine, 3-nitro-1,2,4-triazol-5-one, picric acid, 2,4-dinitroanisole, 2,4-dinitrophenol, 2-nitrophenol, 4-nitrophenol, and new surrogate ortho-nitrobenzoic acid (o-NBA). Analytical methods were developed to enable sensitive, simultaneous detection and quantitation of the 24 IM and legacy compounds, including two orthogonal high-performance liquid chromatography (HPLC) column separations with either ultraviolet (UV) or mass spectrometric (MS) detection. Procedures were developed for simultaneous extraction of all 24 analytes and two surrogates (1,2-dinitrobenzene, 1,2-DNB; o-NBA) from high- and low-level aqueous matrices and solid matrices, using acidification, solid phase extraction (SPE), or solvent extraction (SE), respectively. For low-level aqueous samples extracted by SPE, all compounds were recovered within current Department of Defense Quality Systems Manual (DoD QSM) Ver5.3 accepted limits for aqueous samples analyzed by EPA Method 8330B (57–135%), except NQ, which was consistently recovered at approximately 50%. Likewise, all compounds were recovered from six geographically/geochemically unique soil types within current QSM accepted limits for solid samples analyzed by EPA Method 8330B (64–135%). Further, the majority of compounds were recovered from four tissue types within current limits for solids, with generally low recovery only for Tetryl (from 4 to 62%). A preparatory chromatographic interference removal procedure was adapted for tissue extracts, as various analytical interferences were observed for all studied tissue types.
Identifying degradation products responsible for increased toxicity of UV-Degraded insensitive munitions
2020, Chemosphere
Citation Excerpt :
UV-degradation solutions were mixed well prior to determination of degradation by high performance liquid chromatography (HPLC, Agilent 1200 series, Agilent Technologies Santa Clara, CA), and transported in 1L amber bottles to the toxicity lab to avoid further UV-degradation. Degradation for all IMs were measured using HPLC (Agilent 1200 series, Santa Clara, CA Bruker Hystar software Billerica, MA) using a C18 column (Phenomenex, Synergi™ 4 μm Hydro-RP 80 Å liquid chromatography column (LC), 250 × 4.6 mm, Torrance, CA) at a flow rate of 1.1 mL/min, a column temperature of 25 °C, and diode array detection (Russell et al., 2014). NQ was analyzed using a 100% water mobile phase, NTO was analyzed using a 0.005% aqueous trifluoroacetic acid mobile phase, and DNAN was analyzed using an isocratic mobile phase of 40% methanol in water.
Degradation of insensitive munitions (IMs) by ultraviolet (UV) light has become a topic of concern following observations that some UV-degradation products have increased toxicity relative to parent compounds in aquatic organisms. The present investigation focused on the Army's IM formulation, IMX-101, which is composed of three IM constituents: 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), and nitroguanidine (NQ). The IM constituents and IMX-101 were irradiated in a UV photo-reactor and then administered to Daphnia pulex in acute (48 h) exposures comparing toxicities relative to the parent materials. UV-degradation of DNAN had little effect on mortality whereas mortality for UV-degraded NTO and NQ (and associated degradation products) increased by factors of 40.3 and 1240, respectively, making UV-degraded NQ the principle driver of toxicity when IMX-101 is UV-degraded. Toxicity investigations for specific products formed during UV-degradation of NQ, confirmed greater toxicity than the parent NQ for degradation products including guanidine, nitrite, ammonia, nitrosoguanidine, and cyanide. Summation of the individual toxic units for the complete set of individually measured UV-degradation products identified for NQ only accounted for 25% of the overall toxicity measured in the exposures to the UV-degraded NQ product mixture. From these toxic unit calculations, nitrite followed by CN⁻ were the principal degradation products contributing to toxicity. Given the underestimation of toxicity using the sum toxic units for the individually measured UV-degradation products of NQ, we conclude that: (1) other unidentified NQ degradation products contributed principally to toxicity and/or (2) synergistic toxicological interactions occurred among the NQ degradation product mixture that exacerbated toxicity.
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After the first shaking period, aliquots of the DNAN stock solution were added to the suspensions to give initial concentrations ranging from 0 to 20 mg L−1 in solution. Afterwards, the tubes were bought to the same 20 mL volume with water, recapped and shaken for another 24 h, centrifuged at 2988 xg for 10 min, and the supernatant sampled and analyzed for DNAN by HPLC (Russell et al., 2014). DNAN sorption was calculated by difference between the initial and final concentrations.
Soil heterogeneity is a major contributor to the uncertainty in predicting the environmental fate of data-scare contaminants. For this paper, we focused on research designed to predict the soil environmental fate of the new munition compound, called 2,4-dinitroanisole (DNAN) -a compound increasingly employed by the U.S. and international militaries in the next-generation, insensitive explosive formulations. Here, we employed multivariate statistical correlation models to predict DNAN sorption among different soil “types” seeking to reduce the uncertainty common in all contaminant sorption models by using soil taxonomic designation as a calibrant. We collected composite soils classified under the Ultisols taxonomic Order in the U.S. National Resource Conservation Service soil classification system and quantified their properties via physical and chemical characterizations. Using multivariate statistical modeling modified for compositional data analysis (CoDa), we developed quantitative analogies of the Ultisols by partitioning the characterization data up into four different compositions: Water-extracted, Mehlich-III (referring to the weak acid) extracted, particle-size distribution, and solid-phase carbon‑nitrogen‑sulfur compositions. DNAN sorption was measured in batch soil suspensions and distribution coefficients (K_D) were calculated using linear regression modeling. Prediction models testing the correlation of the DNAN K_D values to the centered logratio -transformed compositions were calculated using CoDa-modified multilinear regression. Results showed that DNAN sorption was only predictable by dissolved organic carbon, pH, and the exchangeable cations Ca and K within the water-extracted composition. Analogies for DNAN sorption were the most discriminating at the Suborder level because of the inherent ambiguity in the Hapludults class at the Great Group level.
The effect of soil type on the extraction of insensitive high explosive constituents using four conventional methods
2019, Science of the Total Environment
Citation Excerpt :
This research found that maintaining the temperature at 100 °C was sufficient to promote fast and efficient extraction (20 min) while also preserving the explosives (Campbell et al., 2003). Although the extraction of traditional explosives is well established (Lapointe et al., 2016; Russell et al., 2014; Thomas et al., 2018; Walsh et al., 2012; Pascoe et al., 2010; Holmgren et al., 2012), the current methods may not be suitable to extract new generation Insensitive High Explosives (IHE) from soil (Taylor et al., 2013; Felt et al., 2016; Mark et al., 2017). The IHE currently being brought into service can consist of mixtures of up to three of the following energetic materials: RDX, 2,4‑dinitroanisole (DNAN), 3‑nitro‑1,2,4‑triazolin‑5‑one (NTO) and 1‑nitroguanidine (NQ), which are likely to be deposited in combination on soil (Richard and Weidhaas, 2014a).
Explosive contamination is commonly found at military and manufacturing sites (Hewitt et al., 2005; Clausen et al., 2004; Walsh et al., 2013). Under current environmental legislation the extent of the contamination must be characterized by soil sampling and subsequent separation of the explosive contaminants from the soil matrix by extraction to enable chemical analysis and quantification (Dean, 2009). It is essential that the extraction method can consistently recover explosive residue from a variety of soil types i.e. all materials that have not degraded or irreversibly bound to the matrix, so that any resultant risk is not underestimated. In this study, five different soil types with a range of organic content, particle size and pH, were spiked with a mixture of RDX, DNAN, NQ and NTO at 50 mg/kg and were extracted using one of four one-step extraction methods: stirring, shaking, sonication, and accelerated solvent extraction (ASE). Analysis of the extraction efficiencies of the four methods found that they were broadly successful for the extraction of all IHE constituents from all five soils (an average of 84% ± 14% recovery across 80 extractions). However, soils with high organic content (Total Organic Content (TOC) ≥ 2%) were found to significantly affect extraction efficiency and reproducibility. NTO and DNAN were the least consistent in extraction efficiency with poorest recovery of NTO as low as 37% ± 2%. Of the four tested methods shaking was found to be the most reproducible, though less efficient than stirring (64%–91%). ASE was found to have the most variable results for extraction of IHE constituents suggesting that ASE was the most affected by the different soil types. Therefore, it is recommended that the efficiency and reproducibility of the selected extraction method should be validated by extracting known concentrations of the IHE from the soil of interest and that any required correction factors are reported.

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