Applications of liquid chromatography-mass spectrometry in metabolic studies of explosives

Liquid chromatography-mass spectrometry was used for the detection and identification of metabolites of 2,4,6-trinitrotoluene (TNT) in urine and blood. The metabolites were found in the urine of rats and in the blood of rabbits fed with TNT, in the urine of rats exposed to TNT by skin absorption and in the urine of TNT munition workers. The detected metabolites, formed by reduction processes, included 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2,6-diamino-4-nitrotoluene, in addition to untransformed TNT.     

一种新的分离三硝基甲苯及其代谢产物的高效液相色谱方法

An HPLC method is described for separation of TNT and its main reduction metabolites. Two columns(LC-C(18) and LC-CN)are connected in series and operated isocratically at 1.0mL/min with water-methanol-tetrahydrofuran(60+ 35+ 5 ). The baseline separation of 4-amino-2,6-dinitrotoluene(4A) and 2-amino-4,6-dinitrotoluene(2A) ,which are main metabolites of TNT,is obtained(Rs=2.1).     

Liquid chromatographic separation of 2,4,6-trinitrotoluene and its principal reduction products

A liquid chromatographic method is described for the baseline separation of 2,4,6-trinitrotoluene (TNT) and its main reduction products. Two analytical columns (LC-18 and LC-CN) are connected in series and eluted isocratically at 1.5 ml min^?1 with water-methanol-tetrahydrofuran (60.5 + 25 + 14.5). The capacity factors (k′) are 1.4, 1.6, 5.1, 6.4 and 7.0 for 2,6-diamino-4-nitroluene, 2,4-diamino-6-nitrotoluene, TNT, 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene, respectively.     

Use of reversed-phase high-performance liquid chromatography-diode array detection for complete separation of 2,4,6-trinitrotoluene metabolites and EPA Method 8330 explosives: influence of temperature and an ion-pair reagent

Explosives such as 2,4,6-trinitrotoluene (TNT), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) are widely distributed environmental contaminants. Complete chromatographic separation is necessary in order to accurately determine and quantify explosives and their degradation products in environmental samples and in (bio)transformation studies. The present study describes a RP-HPLC method with diode array detection using a LC-8 guard column, a Supelcosil LC-8 chromatographic column, and a gradient elution system. This gradient method is capable of baseline separating the most commonly observed explosives and TNT transformation metabolites including 2,4,6-triaminotoluene (TAT) in a single run. The TNT metabolites separated were 2-hydroxylamino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, 2,4-dihydroxylamino-6-nitrotoluene, 4,4',6,6'-tetranitro-2,2'-azoxytoluene, 2,2',6,6'-tetranitro-4,4'-azoxytoluene, 4,4',6,6'-tetranitro-2,2'-azotoluene, 2,2',6,6'-tetranitro-4,4'-azotoluene, 2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,6-diamino-4-nitrotoluene, 2,4-diamino-6-nitrotoluene, and TAT. The same gradient method at a different column temperature can also be used to baseline separate the explosives targeted in the Environmental Protection Agency (EPA) Method 8330 with approximately 22% reduction in total run time and 48% decrease in solvent consumption compared to previously published methods. Good separation was also obtained when all TNT metabolites and EPA Method 8330 compounds (a total of 23 compounds) were analyzed together; only 2,6-DANT and HMX co-eluted in this case. The influence of temperature (35-55 degrees C) and the use of an ion-pair reagent on the chromatographic resolution and retention were investigated. Temperature was identified as the key parameter for optimal baseline separation. Increased temperature resulted in shorter retention times and better peak resolution especially for the aminoaromatics investigated. The use of an ion-pair reagent (octanesulfonic acid) generally resulted in longer retention times for compounds containing amine functional groups, more baseline noise, and decreased peak resolution.     

Synthesis of 5,6-Dihydrophenanthridine (DHPA) Sulfonamides and Subsequent Acid-Catalyzed Rearrangement to Diaryl Sulfones

Selective method of reduction of the ortho-nitro groups in 2,4,6-trinitrotoluene by hydrazine hydrate in the presence of FeCl₃ and charcoal has been elaborated. This method allows obtaining either 2-amino-4,6-dinitrotoluene or 2,6-diamino-4-nitrotoluene as well as 2,4,6-triaminotoluene from TNT.     

Simultaneous determination of organic and cationic species in explosives residues with column-switching liquid chromatography-ion chromatography system

A new column-switching method has been proposed for the determination of 14 organic explosives (1,3,5,7-tetranitro-N-methylaniline, 1,3,5-trinitro-1,3,5-triazacyclohexane, 1,3,5-trinitrobenzene, 1,3-dinitrobenzene, nitrobenzene, 2,4,6-N-tetranitro-N-methylaniline, Trinitrotoluene, 4-amino-2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene, 2,6-dinitrotoluene, 2,4-dinitrotoluene, 2-nitrotoluene, 4-nitrotoluene, and 3-nitrotoluene) and/or five inorganic cations (Na(+), NH(4)(+), K(+), Mg(2+), and Ca(2+)) using liquid chromatography linked to ion chromatography by a switching valve. The mobile phase was methanol-water (40/60, v/v) for a C18 reversed-phase column and 3 mM of methanesulfonic acid (pH 2.5) for a cation-exchange column, respectively. Under the optimal conditions, the 14 organic explosives and the five inorganic cations were separated and detected simultaneously within 45 min. The limits of detection (S/N = 3) of the 14 organic explosives and the five inorganic cations were in the range of 0.0048-0.0333 mg/L and 0.0116-0.1851 mg/L, respectively. The linear correlation coefficients were 0.9971-0.9999, and the relative standard deviation of the retention time and the peak area were 0.02-0.31% and 0.51-3.64%, respectively. The method was successfully applied to the determination of organic explosives and inorganic cations in dust samples.     

A Simple Synthesis of 2,6-Diamino-4-nitrotoluene

A two-step facile synthesis of 2,6-diamino-4-nitrotoluene (1) is described via a sequential selective reduction of nitro groups from trinitrotoluene (TNT).     

Rapid voltammetric determination of nitroaromatic explosives at electrochemically activated carbon-fibre electrodes

The electrochemical behaviour of some nitroaromatic explosives (2,4,6-trinitrotoluene, TNT; 2,6-dinitrotoluene, 2,6-DNT; 2-nitrotoluene, 2-NT; 2-amino-4,6-dinitrotoluene, 2-A-4,6-DNT; 3,5-dinitroaniline, 3,5-DNA; and nitrobenzene, NB) at electrochemically activated carbon-fibre microelectrodes is reported. Electrochemical activation of such electrode material by repeated square-wave (SW) voltammetric scans between 0.0 and +2.6 V versus Ag/AgCl, produced a dramatic increase in the cathodic response from these compounds. This is attributed to the increase of the carbon-fibre surface area, because of its fracture, and the appearance of deep fissures along the main fibre axis into which the nitroaromatic compounds penetrate. Based on the important contribution of adsorption and/or thin layer electrolysis to the total voltammetric response, a SW voltammetric method for rapid detection of nitroaromatic explosives was developed. No interference was found from compounds such as hydrazine, phenolic compounds, carbamates, triazines or surfactants. The limits of detection obtained are approximately 0.03 g mL^–1 for all the nitroaromatic compounds tested. The method was applied for the determination of TNT in water and soil spiked samples; recoveries were higher than 95% in all cases.     

Sorption of nitroaromatic compounds to synthesized organoclays

This project quantifies the ability of seven engineered organoclays to sorb TNT and two of its reduction products: 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) and 4-amino-2,6-dinitrotoluene (4-A-2,6-DNT). The organoclays used in the TNT sorption studies were synthesized in the laboratory by combining bentonite with benzyltriethylammonium chloride (BTEA) at 50, 75, and 100% of the bentonite's cation exchange capacity and with hexadecyltrimethylammonium bromide (HDTMA) at 25, 50, 75, and 100% of the bentonite's cation exchange capacity. For sorption of 2-A-4,6-DNT and 4-A-2,6-DNT, two organoclays were tested: BTEA at 50% CEC and HDTMA at 75% CEC. Sorption data with HDTMA organoclay and TNT were fit to linear isotherms and demonstrated that the clay's sorptive capacity increased as the amount of total organic carbon exchanged onto the clay increased. Sorption data with BTEA organoclay and TNT were fit to Langmuir isotherms; however, the clay's sorptive capacity increased as the amount of total organic carbon sorbed to the clay's surface was decreased. Sorption behavior for TNT reduction products 2-A-4,6-DNT and 4-A-2,6-DNT to one HDTMA organoclay and one BTEA organoclay demonstrated that HDTMA organoclay at 10.3% total organic carbon was a more effective sorbent than BTEA organoclay at 5.2% total organic carbon.     

Selection of hapten structures for indirect immunosensor arrays

A multianalyte immunosensor array can be implemented by immobilization of different haptens in distinct areas of a single cavity or flow cell. In this case a mixture of different antibodies for different analytes is used in an indirect ELISA-format. The selection of the right hapten structures is very important to build up an array successfully. A system of independent hapten/antibody combinations is needed, with one immobilized hapten (coating antigen) reacting only with one antibody. If more than one antibody binds to a coating antigen no ideal calibration curves are obtained. This phenomenon is known as shared-reactivity and can lead to double-sigmoidal curves. To use monoclonal antibodies to 2,4,6-trinitrotoluene (TNT) and 2,4-dichlorophenoxyacetic acid (2,4-D), two different haptens had to be found, one only reacting with the TNT-antibody, the other only binding to the 2,4-D-antibody. 2,4-Dichlorophenoxybutyric acid was used for the 2,4-D antibody and 2,4,6-trinitrophenyl-8-aminooctanoic acid for the TNT antibody. Although 4-nitrotoluene, 2,4-dinitrotoluene and 4-amino-2,6-dinitrotoluene showed only very low cross-reactivities to the 2,4-D antibody the corresponding haptens 4-nitrophenylacetic acid, 2,4-dinitrophenyl-6-aminohexanonic acid, and 4-amino-2,6-dinitrotoluyl-(N)-glutarate are useful coating antigens for this antibody. The structure of the coating antigens had no significant influence on the midpoints (IC50) of the test for 2,4-D and even haptens with very low cross-reactivities could be used. With all haptens a test midpoint of about 0.2 μg/L for 2,4-D was achieved. For the direct assay format with immobilized antibodies the same test midpoint of 0.2 μg/L for 2,4-D was obtained. As a conclusion, the selectivity of a monoclonal antibody should not be influenced by the used tracer or coating antigen as well. It could be shown that the affinity constants of an antibody to the analytes are the main sensitivity and selectivity determining parameters for competitive immunoassays. A two-dimensional microtiter plate array was used to determine the analytes 2,4-D and TNT in parallel with a mixture of antibodies. Received: 29 July 1998 / Revised: 21 October 1998 / Accepted: 10 November 1998     

Pathways for Degrading TNT by Thu-Z: a Pantoea sp. Strain

2,4,6-Trinitrotoluene (TNT), an extensively used and versatile explosive, is harmful in soil and water. In the present study, four bacterial strains capable of degrading TNT have been isolated from contaminated sites and named as Thu-A, Thu-B, Thu-C, and Thu-Z. Thu-Z, which gave the highest degradation efficiency compared to the others, was assigned to the genus Pantoea according to its 16S rRNA gene. Similarities in both biochemical properties and morphology suggested that Thu-Z was a Pantoea sp. strain. Thu-Z was proved to be capable of using TNT as a sole nitrogen source by cleaving NO₂ from the nitroaromatic ring by direct aromatic ring reduction. Under nitrogen-limited conditions, 96.6?%?N of TNT was consumed by Thu-Z for growth, which was determined in terms of NaNO₂. Trace nitro reduction metabolites such as 2,4-diamino-6-nitrotoluene (24Dam) and 2,6-diamino-4-nitrotoluene (26Dam) were identified in the presence of (NH₄)₂SO₄. On the other hand, 4,4??,6,6??-tetranitro-2,2??-azoxytoluene (22Azo) and 2,2??,6,6??-tetranitro-4,4??-azoxytoluene (44Azo) were detected in the absence of (NH₄)₂SO₄. These indicated the existence of a dual pathway for Thu-Z, while the direct aromatic ring reduction was predominant. Addition of a nitrogen source ((NH₄)₂SO₄) after inoculation stimulated the growth of Thu-Z and accelerated TNT degradation.     

Determination of nitroaromatic, nitramine, and nitrate ester explosives in soil by gas chromatography and an electron capture detector

Hazardous waste site characterization, forensic investigations, and land mine detection are scenarios where soils may be collected and analyzed for traces of nitroaromatic, nitramine, and nitrate ester explosives. These thermally labile analytes are traditionally determined by high-performance liquid chromatography (HPLC); however, commercially available deactivated injection port liners and wide-bore capillary columns have made routine analysis by gas chromatography (GC) possible. The electron-withdrawing nitro group common to each of these explosives makes the electron capture detector (ECD) suitable for determination of low concentrations of explosives in soil, water, and air. GC-ECD and HPLC-UV concentration estimates of explosives residues in field-contaminated soils from hazardous waste sites were compared, and correlation (r>0.97) was excellent between the two methods of analysis for each of the compounds most frequently detected: 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4-dinitrotoluene (2,4-DNT), 1,3-dinitrobenzene (1,3-DNB), 1,3,5-trinitrobenzene (TNB), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). The analytes were extracted from soils with acetonitrile by 18 h of sonication in a cooled ultrasonic bath. Two soil-to-solvent ratios were evaluated: 2.00 g:10.00 ml and 25.0 g:50.0 ml. GC-ECD method detection limits were similar for the two soil-to-solvent ratios and were about 1 mug kg(-1) for the di- and trinitroaromatics, about 10 mug kg(-1) for the mono-nitroaromatics, 3 mug kg(-1) for RDX, 25 mug kg(-1) for HMX, and between 10 and 40 mug kg(-1) for the nitrate esters (nitroglycerine [NG] and pentaerythritol tetranitrate [PETN]). Spike recovery studies revealed artifacts introduced by the spiking procedure. Recoveries were low in some soils if the amount of soil spiked was large (25.0 g) compared to the volume of spike solution added (1.00 ml). Recoveries were close to 100% when 2.00-g soil samples were spiked with 1.00 ml of solution. Analytes most frequently found in soils collected near buried land mines were the microbial transformation products of TNT (2-amino-4,6-dinitrotoluene [2-Am-DNT] and 4-amino-2,6-dinitrotoluene [4-Am-DNT]), manufacturing impurities of TNT (2,4-DNT, 2,6-DNT, and 1,3-DNB), and TNT. The microbial reduction products of the isomers of DNT and of 1,3-DNB were also detected, but the ECD response to these compounds is poor.     

One-step isolation and identification of hydroxylamino-dinitrotoluenes, unstable products from 2,4,6-trinitrotoluene metabolites, with thin-layer chromatography and laser time-of-flight mass spectrometry

Two kinds of hydroxylamino-dinitrotoluenes (HADNTs), 2-hydroxylamino-4,6-dinitrotoluene (2HADNT) and 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), are known to be major metabolites produced from 2,4,6-trinitrotoluene (TNT) by bacteria. These chemicals could not be identified as TNT metabolites produced by Pseudomonas sp. strain TM15 because the mass spectra of these chemicals could not be obtained by liquid chromatography-mass spectrometry (MS) or gas chromatography-MS, which are the classic methods for identifying the metabolites of xenobiotics. However, these problems are overcome by isolating 2HADNT and 4HADNT from TNT metabolites with one-step thin-layer chromatography using dichloromethane as the developing solvent, and individually extracting them into acetonitrile by collecting spots of 2HADNT and 4HADNT. The purity of each HADNT was approximately 98%, based on the results of high-performance liquid chromatographic analyses. 2HADNT and 4HADNT are identified by obtaining their mass spectra with laser time-of-flight MS. 2HADNT and 4HADNT dissolve in distilled water and are spontaneously broken down with time. Also, heat treatment (increasing temperatures) and dissolved oxygen accelerate the destruction of HADNTs. This technique may be applicable for the identification and exact quantitative analysis of unstable and fragile compounds such as HADNTs.     

Heterophase nitration of <Emphasis Type="Italic">o</Emphasis>-nitrotoluene with sulfuric-nitric acid mixtures of low nitrating activity in gas-containing emulsions

Heterophase nitration of o-nitrotoluene, in which an increase in the contribution of the reaction in the boundary adsorption monolayer under conditions of surface aeration leads to a higher yield of 2,4-dinitrotoluene and larger 2,4-/2,6-dinitrotoluene isomer ratio, was studied.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C18 and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5-21.5 nmol/ml for NEF and 0.4-9.5 nmol/ml for metabolites in serum and 4-86 nmol/ml for NEF and 8-190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Analysis of explosives using electrospray ionization/ion mobility spectrometry (ESI/IMS)

The analysis of explosives with ion mobility spectrometry (IMS) directly from aqueous solutions was shown for the first time using an electrospray ionization technique. The IMS was operated in the negative mode at 250°C and coupled with a quadrupole mass spectrometer to identify the observed IMS peaks. The IMS response characteristics of trinitrotoluene (TNT), 2,4-dinitrotoluene (2,4-DNT), 2-amino-4,6-dinitrotoluene (2-ADNT), 4-nitrotoluene (4-NT), trinitrobenzene (TNB), cyclo-1,3,5-trimethylene-2,4,6-trinitramine (RDX), cyclo-tetramethylene-tetranitramine (HMX), dinitro-ethyleneglycol (EGDN) and nitroglycerine (NG) were investigated. Several breakdown products, predominantly NO₂⁻ and NO₃⁻, were observed in the low-mass region. Nevertheless, all compounds with the exception of NG produced at least one ion related to the intact molecule and could therefore be selectively detected. For RDX and HMX the [M+Cl⁻]⁻ cluster ion was the main peak and the signal intensities could be greatly enhanced by the addition of small amounts of sodium chloride to the sprayed solutions. The reduced mobility constants (K₀) were in good agreement with literature data obtained from experiments where the explosives were introduced into the IMS from the vapor phase. The detection limits were in the range of 15–190 μg l⁻¹ and all calibration curves showed good linearity. A mixture of TNT, RDX and HMX was used to demonstrate the high separation potential of the IMS system. Baseline separation of the three compounds was attained within a total analysis time of 6.4 s.     

Determination of nitrazepam and its main metabolites in urine by thin-layer chromatography and direct densitometry

A method for the direct quantitative densitometry of nitrazepam and its main metabolites (7-aminonitrazepam, 7-acetamidonitrazepam and 2-amino-5-nitrobenzophenone) in urine was developed. The unchanged drug and its metabolites were extracted with benzene-dichloromethane (4:1), subjected to thin-layer chromatography, and determined by direct ultraviolet densitometry. Recovery experiments showed that the method was quantitative. The limit of detection was 5 ng/ml for 2-amino-5-nitrobenzophenone and 10 ng/ml for other compounds. The method was applied to the determination of nitrazepam and its metabolites excreted in human urine after administration of 10 mg of the drug.     

超声-气相联合扩散法快速合成ZIF-8用于硝基芳香化合物炸药的传感

三维微孔沸石咪唑基骨架(ZIF-8)纳米晶通过超声-气相联合扩散法快速合成.对该纳米晶进行荧光研究表明,纳米晶对硝基芳香化合物炸药具有良好的荧光淬灭能力.通过建立的Stern-Volmer方程,在1×10-4～8×10-4 mol/L范围内,每种炸药的浓度与纳米晶的荧光淬灭程度呈线性关系.对于2,4,6-三硝基苯酚(T...     

Regioselective preparation of 5-amino- and 6-amino-1,3-benzoxazole-4,7-diones from symmetrical diaminophenol and aminoresorcinol

Regioisomeric 5-amino- and 6-amino- 1,3-benzoxazole-4,7-diones were prepared from 2,6-diamino-4-methoxyphenol and 2-amino-5-methoxyresorcinol, respectively. These symmetrical precursors have the property to be antisymmetrical to each other with respect to their amino and hydroxy substituents.     

The determination of nitroaromatics and nitramines in ground and drinking water by wide-bore capillary gas chromatography

A method has been developed to determine the concentration of nitroaromatics and nitramines in drinking water at levels below those previously achieved by gas chromatography. The nitroaromatics and nitramines are extracted from water using toluene and isoamyl acetate, respectively. The extracts are analyzed via a gas chromatograph equipped with a DB-1301 widebore fused-silica capillary column and an electron capture detector. Method detection limits of 0.003 micrograms/L for 2,6-dinitrotoluene (2,6-DNT), 0.04 micrograms/L for 2,4-dinitrotoluene (2,4-DNT), 0.06 micrograms/L for 2,4,6-trinitrotoluene (TNT), 0.3 micrograms/L for cyclotrimethylenetrinitramine (RDX), and 6.0 micrograms/L for cyclotetramethylenetetranitramine (HMX) have been obtained using this method.