Structure elucidation of dyes that are formed in the colorimetric detection of the improvised explosive urea nitrate

Urea nitrate (uronium nitrate, UN) is a powerful, improvised explosive that can be easily made from urea and nitric acid. It is considered the most frequently used, illegal explosive in the Israeli arena, which is responsible for the loss of more than a hundred lives in terrorist incidents. Urea nitrate is a colorless, crystalline substance that looks very much like sugar. A sensitive color test for UN was developed recently. It is based on the formation of a red dye in the reaction between p-dimethylaminocinnamaldehyde and UN under neutral conditions. A similar reaction with p-dimethylaminobezaldehyde produces a yellow dye. The two dyes have been synthesized, and their structures determined by X-ray crystallography. Both dyes are protonated Schiff bases, prevailing in the crystal in a quinoid form. They are identical to the compounds, which are obtained in the colorimetric detection of urea with the same reagents, under strong acidic conditions, whose structures have been postulated in the literature, but never fully proved experimentally.     

Trace analysis of urea nitrate in post-blast debris by GC/MS

Urea nitrate (uronium nitrate, UN) is an improvised explosive that looks very much like sugar and is easily made from accessible starting materials, urea and nitric acid. During the last decade it has been frequently used by terrorists in the Israeli arena and in other countries as well. It is difficult to identify urea nitrate in post-explosion debris, since only a very small fraction survives the blast. Also, in the presence of water, it readily decomposes to its original components, urea and nitric acid, two ubiquitous substances with relatively low evidential value. By further modifying McCord's recent version of Clark's method for the detection of minute amounts of urea, we were able to identify with high degree of certainty traces of uronium ion, the main characteristic factor of urea nitrate, in post-blast residues by GC/MS. The analytical process is based on the initial formation of xanthenyl urea by the reaction of uronium cation with xanthydrol, followed by reaction with alcohol to form xanthylurethane, which is readily identified by GC/MS. The reaction mechanism was corroborated by the use of labeled ¹⁵N-urea. By applying the technique to residues collected from scenes of controlled firing experiments, 4 out of 16 samples showed the presence of uronium cation as indicated by the formation of the corresponding xanthylurethane. Potential interferences such as urea and ammonium nitrate did not respond under standard conditions. However, under strongly acidic conditions (pH < 2), urea is converted into uronium ion, which is a nuisance, since it behaves as an authentic uronium cation. Such conditions, however, do not prevail at common crime scenes.     

Trace analysis of urea nitrate by liquid chromatography-UV/fluorescence

In this paper we have adapted a technique, previously used to determine the presence of urea in aqueous samples of wine and urine, to detect trace levels of urea nitrate explosives. The procedure involves the reaction of the uronium ion (protonated urea) with a fluorophore, xanthydrol. By modification of the procedure to utilize non-aqueous reagents, in neutral conditions, it can be made specific to the presence of the urea nitrate ion pair. The procedure includes selective detection of derivatization products by UV and fluorescence following separation by High-Performance Liquid Chromatography (HPLC). Analytical method development included optimization of HPLC conditions (solvent, gradient), UV and fluorescence wavelengths, and derivatization parameters (xanthydrol amount, reaction times, temperature). The extraction of urea nitrate from surfaces was also investigated and optimized. For best quantification, it was shown that an internal standard was required; this resulted in a quantification limit around 0.17mM (21mg/L). The entire procedure could be performed in less than 30min per sample and potential interferences such as ammonium, nitrate, and urea did not produce a response under standard conditions.     

Recovery and detection of urea nitrate in traces

Urea nitrate (uronium nitrate, UN) is a powerful improvised explosive, frequently used by terrorists in the Israeli arena. It was also used in the first World Trade Center bombing in New York in February 1993. It is difficult to identify UN in postexplosion debris, because in the presence of water it readily decomposes to its original components, urea and nitric acid. A method for the recovery and detection of urea nitrate in traces is described. The residues are extracted with hot acetone, and the extract chromatographed on chromosorb G-HP. The eluent is analyzed by liquid chromatography mass spectrometry using atmospheric pressure chemical ionization (APCI). By applying this technique, we were able to identify urea nitrate in actual exhibits. It was found that UN can be also formed during the analytical procedure, by certain combinations of urea, nitrate ions, and a source of protons and, hence, the presence of the characteristic adduct ion does not necessarily indicate an "authentic" urea nitrate. Several solutions are suggested to overcome this ambiguity.     

A simple histochemical technique for the identification of gunshot residue

Alizarin red S (ARS) is a commonly used organic dye useful in the histologic identification of calcium deposits. ARS also forms colored reaction products with other metal ions, including barium and lead, which are present in primer residue. In histochemical studies, ARS is shown to identify primer residues from several manufacturers as well as primer residue deposited in tissue, either experimentally or in close-range gunshot wounds. This can be easily accomplished with routine histologic techniques. ARS does not stain gunpowder residue, tattoo pigment, melanin, graphite, india ink, or anthracotic pigment.     

Chemicals reacting with various forms of hemoglobin: biological significance, mechanisms, and determination.

The clinical chemistry of various forms of hemoglobin occupies a large fraction of the total activities of hospital and forensic science laboratories. Some of the potential pitfalls are reviewed here along with an account of the accidental discovery of two novel chemical forms. Human or mouse red blood cells were exposed to excess sodium nitrite to convert the intracellular pigment to methemoglobin. When these were subsequently incubated in Krebs-Ringer-phosphate-glucose medium, pH 7.4 at 37 degrees C under nitrogen and in the presence of various concentrations of methylene blue, a blood pigment was generated in high yield which had unique properties. In lysates, the pigment was stable in air, and it could be maintained in liquid nitrogen for as long as a year without deterioration. The pigment had properties different from those of oxyhemoglobin, deoxyhemoglobin, methemoglobin, or carboxyhemoglobin. After separation by isoelectric focusing, the pigment gave a strong signal on electron paramagnetic resonance (EPR) spectroscopy. The other forms of hemoglobin given above are EPR-silent. The pigment was eventually identified as the nitrosylated valency hybrid species, (alpha 2+ beta 3+)2(NO)2. The corresponding species, (alpha 3+ beta 2+)2(NO)2, has similar properties. These species apparently owe their unusual stability in air to the presence of the oxidized subunits in the same tetramer.     

Improved enhancement of ninhydrin developed fingerprints by cadmium complexation using low temperature photoluminescence techniques

Fingerprints developed with ninhydrin form stable, colored complexes when treated with various metal salts. Many of these colored complexes can be used to increase the sensitivity of detection of latent prints because of photoluminescent properties. The intensity of this photoluminescence is increased at low temperature (77K), and this is a common characteristic of each of the complexes formed with salts of the IIb group of the Periodic Table. Spectral characteristics of these Group IIb metal complexes and the influence of environmental factors on their formation are reported. These data have helped determine optimal conditions for the enhancement of ninhydrin developed fingerprints. Taking into account spectral characteristics, solubility, versatility, stability, and reproducibility, the use of the cadmium nitrate tetrahydrate complex is advocated for general use for fingerprint enhancement. The use of zinc nitrate is favored if toxicological considerations are paramount, but ninhydrin development has to be carefully controlled if optimal results are to be obtained. Limited applications for mercuric complexes are found when a red shift is desired to remove background effects.     

Discriminating red spray paints by optical microscopy, Fourier transform infrared spectroscopy and X-ray fluorescence

Red spray paints from different European suppliers were characterised to determine the discriminating power of a sequence of analysing techniques. A total of 51 red spray paints were analysed with the help of three techniques: (1) optical microscopy, (2) Fourier transform infrared spectrometry and (3) X-ray fluorescence. Infrared spectra were classified according to binder type, filler and pigment composition and a searchable spectral library was created. Due to the difference in the elemental composition of spray paints, a further discrimination was possible. The microscopic analysis was not taken into consideration for classification purposes. The structure of the substrate under a paint coating strongly affects the surface characteristics of this spray paint. Together with the spectral library, a database of information of spray paints was build.     

Identification of colorants in pigmented pen inks by laser desorption mass spectrometry

Pigments are rapidly replacing dyes as colorants in pen and printer inks, due to their superior colors and stability. Unfortunately, tools commonly used in questioned document examination for analyzing pen inks, such as TLC, cannot be used for the analysis of insoluble pigments on paper. Laser desorption mass spectrometry is demonstrated here as a tool for analyzing pigment-based pen inks. A pulsed nitrogen laser can be focused onto a pen stroke from a pigmented ink pen on paper, and positive and negative ions representative of the pigment can be generated for subsequent mass spectrometric analysis. Targeted pens for this work were a set of Uni-ball 207 pigmented ink pens containing blue, light blue, orange, green, violet, red, pink, and black inks. Copper phthalocyanine was identified as the pigment used to make both blue inks. A mixture of halogenated copper phthalocyanines were identified in the green ink. Unexpectedly, the pink ink was found to contain a red pigment, Pigment Red 12, treated with a mixture of water-soluble dyes. Each sample yielded ions representative of the pigments present.     

The impact of force,time, and rotation on the transfer of ammonium nitrate: A reductionist approach to understanding evidence dynamics

Empirical studies evaluating the conditions under which the transfer of forensic materials occurs can provide contextual information and offer insight into how that material may have been transferred in a given scenario. Here, a reductionist approach was taken to assess the impact of force, time, and rotation on the transfer of an explosive compound. An Instron ElectroPuls E3000 material testing instrument was used to bring porous and non-porous surfaces adulterated with an ammonium nitrate into direct contact with a human skin analogue, controlling for the force of contact, duration of contact, and rotation applied during contact. Quantifiable amounts of ammonium nitrate were recovered from all of the recipient surfaces demonstrating that ammonium nitrate is readily transferred from one surface to another, even when contact occurs for a short duration with a relatively low force. More particulates were transferred from non-porous surfaces onto the human skin analogue, but the amount of ammonium nitrate transferred did not depend upon the force of contact, duration of contact, or the amount of rotation applied. However, when contact occurred and involved rotation, a greater transfer of ammonium nitrate was observed, compared to those contacts occurring without rotation being applied. This approach complements more commonly-used holistic experiments that test multiple interacting variables in a realistic setting by isolating these variables, allowing them to be examined individually. This can be utilised to better understand the individual impact that specific variables have on the transfer of trace evidence in relevant crime reconstruction contexts.     

On the autofluorescence of fingermarks

The autofluorescence of fingermarks is used for their detection. The components responsible for this autofluorescence are largely unknown. Thin layer chromatography and fluorescence spectroscopy were used to identify autofluorescent components and evaluate their forensic value. Based on our results, tryptophan is hypothesized to be a major contributor to the autofluorescence when part of peptides or proteins, id est, not in its free form. Part of the autofluorescence could be assigned to a kynurenine derivative. Pheophorbide A, a metabolite of chlorophyll, is inferred as a red fluorescent fingermark component. Chlorophyll is a plant pigment which implies that dietary information can potentially be retrieved from fingermarks.     

Evaluation of canine training aids containment for homemade explosive and components by headspace analysis and canine testing

While canines are most commonly trained to detect traditional explosives, such as nitroaromatics and smokeless powders, homemade explosives (HMEs), such as fuel–oxidizer mixtures, are arguably a greater threat. As such, it is imperative that canines are sufficiently trained in the detection of such HMEs. The training aid delivery device (TADD) is a primary containment device that has been used to house HMEs and HME components for canine detection training purposes. This research assesses the odor release from HME components, ammonium nitrate (AN), urea nitrate (UN), and potassium chlorate (PC), housed in TADDs. Canine odor recognition tests (ORTs) were used with analytical data to determine the detectability of TADDs containing AN, UN, or PC. Headspace analysis by gas chromatography/mass spectrometry (GC/MS) with solid-phase microextraction (SPME) or online cryotrapping were used to measure ammonia or chlorine, as well as other unwanted odorants, emanating from bulk AN, UN, and PC in TADDs over 28 weeks. The analytical data showed variation in the amount of ammonia and chlorine over time, with ammonia from AN and UN decreasing slowly over time and the abundance of chlorine from PC TADDs dependent on the frequency of exposure to ambient air. Even with these variations in odor abundance, canines previously trained to detect bulk explosive HME components were able to detect all three targets in glass and plastic TADDs for at least 18 months after loading. Detection proficiency ranged from 64% to 100% and was not found to be dependent on either age of material.     

Evaluation of antisera for bloodstain grouping. I. ABH, MN, and Rh

Sixty-eight different commercially available blood grouping antisera and lectins with ABH, MN, and Rh D, C, E, c, and e specificities were serologically evaluated for their applicability to bloodstain antigen determination. The characteristics of the antisera were determined with red cells, with fresh bloodstains, and with series of aging bloodstains. The Rh antisera were tested under a variety of serological conditions and with bloodstains on various substrata. Additionally, studies on optimization of absorption-elution procedure variables were carried out, and some data on the storage characteristics of red cells and blood grouping antisera were gathered.     

Postmortem red coloration of teeth. A retrospective investigation of 26 cases

Postmortem red coloration of teeth (pink teeth) was found in 26 of 119 consecutive autopsy or forensic dental identification cases during 1973-1984. The present study confirms that red coloration of teeth may occur some time after death under conditions that promote putrefaction and adipocere formation. The red coloration is due to hemolysis after exudation of hemoglobin derivatives through the dentine tubules in cases in which blood has accumulated in the head, either due to congestion or by a postmortem head-down position, as after submersion in water.     

Surface Modification for the Collection and Identification of Fingerprints and Colorimetric Detection of Urea Nitrate

Glass surfaces were modified with a combination of dyes and reagents to allow for the potential simultaneous recording of a detailed fingerprint and the detection of the explosive urea nitrate (UN), as a proof of principle of surface modification for simultaneous linking of identity to manipulation of explosives. By coating microscope slides with 9,10‐diphenylanthracene (DPA), p‐dimethylaminobenzaldehyde (p‐DMAB) and p‐dimethylaminocinnamaldehyde (p‐DMAC), a colorimetric change was observed in the presence of UN, while revealing a fingerprint with enough resolution to isolate at least 10 minutiae. This is the first step in creating point‐of‐care devices capable of detecting low concentrations of explosives and drug metabolites and connecting them to a fingerprint.     

Identification and dating of the fountain pen ink entries on documents by ion-pairing high-performance liquid chromatography

A novel approach for the identification and dating of the fountain pen ink entries on paper has been established by ion-pairing high-performance liquid chromatography (IP-HPLC). Twelve black and six red fountain inks have been collected, and their ink entries have been prepared by drawing lines on paper. The chromatographic conditions for separation of their dye components after extraction with solvents were optimized. Under the optimized conditions, the 18 fountain pen inks were differentiated individually by comparing the number of detectable main or minor dye components, and the relative peak intensities of each component. The ink entries were artificially and naturally aged, and the analysis results showed that the ink dye components were significantly decomposed when exposed to UV or fluorescent light compare to those of inks stored under natural condition. The changes of the relative peak height for the dye components were linearly related to the aging time, especially under natural aging conditions. The degradation characteristics of the dye components under different aging conditions provide scientific evidences for dating of the suspicious fountain pen ink entries on document.     

Source Determination of Red Gel Pen Inks using Raman Spectroscopy and Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy combined with Pearson's Product Moment Correlation Coefficients and Principal Component Analysis

The potential combination of two nondestructive techniques, that is, Raman spectroscopy (RS) and attenuated total reflectance–fourier transform infrared (ATR‐FTIR) spectroscopy with Pearson's product moment correlation (PPMC) coefficient (r) and principal component analysis (PCA) to determine the actual source of red gel pen ink used to write a simulated threatening note, was examined. Eighteen (18) red gel pens purchased from Japan and Malaysia from November to December 2014 where one of the pens was used to write a simulated threatening note were analyzed using RS and ATR‐FTIR spectroscopy, respectively. The spectra of all the red gel pen inks including the ink deposited on the simulated threatening note gathered from the RS and ATR‐FTIR analyses were subjected to PPMC coefficient (r) calculation and principal component analysis (PCA). The coefficients r = 0.9985 and r = 0.9912 for pairwise combination of RS and ATR‐FTIR spectra respectively and similarities in terms of PC1 and PC2 scores of one of the inks to the ink deposited on the simulated threatening note substantiated the feasibility of combining RS and ATR‐FTIR spectroscopy with PPMC coefficient (r) and PCA for successful source determination of red gel pen inks. The development of pigment spectral library had allowed the ink deposited on the threatening note to be identified as XSL Poppy Red (CI Pigment Red 112).     

An Autopsy Case of Sudden Unexplained Death Caused by Malaria*

Abstract: Sudden unexplained deaths, especially those unwitnessed can lead to forensic issues and would necessitate the need for a meticulous and complete postmortem examination including ancillary investigations to discover the cause of death. We herein report a case of sudden unexplained death caused by malaria in an apparently healthy individual. This fatal case is presented to remind the forensic pathologist of the possibility of malaria as a cause of sudden unexplained death in malaria‐endemic regions. In the present case, histopathological examination demonstrated the presence of parasitized red blood cells with malarial pigment in the blood capillaries in the brain, myocardium, pericardium, lungs, kidneys, liver, and the spleen. Cerebral malaria with acute renal insufficiency or pulmonary edema with an acute respiratory distress syndrome might have been the cause of death.     

Why is methenamine detected in Goma-2 dynamites originally methenamine free? An interpretation of relevant forensic results

After the train bombing in Madrid (Spain) on 11 March 2004, methenamine was detected in some of the specimens of Goma-2 ECO dynamite submitted to the forensic laboratories when analyzed by gas chromatography coupled with mass spectrometry detection (GC-MS). Methenamine is synthesized from formaldehyde and ammonia through a condensation reaction. However, neither methenamine nor any of these compounds were used to manufacture Goma-2 ECO dynamite. Four different experiments were designed in order to explain the presence of methenamine detected in the dynamite samples analyzed. In the first one, GC-MS was used to analyze the individual components of Goma-2 ECO provided by the manufacturer and the components mixed in a raw paste. Methenamine was detected in the manufacturer's ammonium nitrate and in the raw paste. The other experiments were designed to find the precursors sources for methenamine generation in Goma-2 ECO. Results revealed that these sources could be ammonium nitrate for ammonia and sawdust for formaldehyde. Under heating conditions, dynamite could produce these precursors, which could condense in the injection port of the GC-MS system and generate methenamine. However, methenamine was not always detected in these dynamites. This was explained by the existence of two opposite effects: (a) dynamite stability makes difficult that ammonium nitrate releases ammonia and (b) there is a gradual loss of formaldehyde in sawdust along the time. Both effects can prevent the formation of an amount of methenamine large enough to be detected.     

Enhancement of microcrystalline identification of gamma-hydroxybutyrate

An enhancement of the microcrystalline test for the detection of gamma-hydroxybutyrate (GHB) is described. The original test used a silver/copper reagent which consisted of 0.1 g of silver nitrate and 0.1 g of copper nitrate in 10 mL water. The enhanced test utilizes lanthanum nitrate in place of copper nitrate. A detection limit of 0.5 mg/mL was achieved and the visual discrimination was improved because of larger sized crystals. Transient crystals were observed between 0.1 and 0.4 mg/mL. Silver nitrate alone appeared to be suitable for GHB detection but was not specific as other hydroxyl acids, such as glycolic acid, produced a similar crystal pattern. Tests conducted on chemical precursors of GHB and substances with similar biological activity highlight the specificity of the enhanced test. The reagent is therefore selective and sensitive for GHB in aqueous solutions. However, in beverage testing, crystal formation appeared to be inhibited for some drinks. Citric acid was identified as a possible interference depending on its concentration relative to GHB.