The analysis of substituted cathinones. Part 2: an investigation into the phenylacetone based isomers of 4-methylmethcathinone and N-ethylcathinone

During the analysis of "seized samples", suspected of containing 4-methylmethcathinone (mephedrone) and N-ethylcathinone (ethcathinone) additional compounds were observed in the GCMS chromatogram. These compounds were suspected to be the corresponding phenylacetone isomers of mephedrone and ethcathinone respectively. These isomers are referred to as iso-mephedrone and iso-ethcathinone, respectively. The identity of these compounds was verified by synthesising the isomers from known starting materials and comparing them with the compounds found in the seized samples. Analytical data, GCMS, NMR and IR on these compounds are provided. Possible explanations for the presence of these compounds in the seized samples are explored. Contaminated starting material is one suggestion. Rearrangement of the propiophenone based product to the phenylacetone based product is also suggested. The reaction of the α-bromopropiophenone with a primary amine can also lead to the phenylacetone based product. The presence of these isomeric compounds in seized samples could be used to compare different samples and attempt to establish a common origin.     

Multiple stable isotope characterization as a forensic tool to distinguish acid scavenger samples

Acid scavengers are frequently used as stabilizer compounds in a variety of applications. When used to stabilize volatile compounds such as nerve agents, the lower volatility and higher stability of acid scavengers make them more persistent in a post-event forensic setting. Compound-specific isotope analysis of carbon, nitrogen, and hydrogen in three acid-scavenging compounds (N,N-diethylaniline, tributylamine, and triethylamine) were used as a tool for distinguishing between different samples. Combined analysis of multiple isotopes improved sample resolution, for instance differentiation between triethylamine samples improved from 80% based on carbon alone to 96% when combining with additional isotope data. The compound-specific methods developed here can be applied to instances where these compounds are not pure, such as when mixed with an agent or when found as a residue. Effective sample matching can be crucial for linking compounds at multiple event sites or linking a supply inventory to an event.     

Simultaneous separation of different types of amphetamine and piperazine designer drugs by capillary electrophoresis with a chiral selector

The recent emergence of a new class of piperazine-type compounds has brought about the need for laboratory screening methods for both seized drugs and toxicological samples. These piperazine compounds, which include 1-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), exhibit comparable physiological effects and can be substituted for the classic amphetamine-type drugs. We have optimized a chiral capillary electrophoresis (CE) separation that detects a set of 6 piperazine and 4 chiral amphetamine compounds in under 23 min using a 200 mM phosphate buffer at a pH = 2.8 with 20 mM hydroxypropyl- beta-cyclodextrin (HPbeta3CD). In addition to the above compounds, a series of "clandestine" BZP diHCl samples were also analyzed using this method to assess the ruggedness of the procedure. The novel CE separation was tailored to simultaneously detect these piperzine compounds in addition to amphetamine-type drugs. Distinct migration time and UV-spectral data were obtained for all compounds of interest.     

Neutral heroin impurities from tetrahydrobenzylisoquinoline alkaloids

Laudanosine, reticuline, codamine, and laudanine are members of the tetrahydrobenzylisoquinoline family of natural products. These alkaloids are present in the opium poppy, Papaver somniferum, and are subsequently found as impurities in clandestinely processed morphine. Morphine is then synthesized to heroin using hot acetic anhydride. During the course of this study, it was determined that these four tetrahydrobenzylisoquinolines undergo degradation to a series of 18 neutral impurities when subjected to hot acetic anhydride. Based on the degradation pathway, these new impurities were categorized into two sets of impurities called the C1-acetates compounds and the stilbene compounds. Synthesis, isolation, and structural elucidation information is provided for the tetrahydrobenzylisoquinoline alkaloids, and the new neutral impurities have been studied. Several hundred authentic heroin samples were analyzed using an established heroin signature program method. This methodology features the detection of trace neutral impurities present in heroin samples. It was determined that all 18 new impurities were detected in various quantities in four different types of heroin samples. Analytical results featuring these new impurities are reported for South American-, Southwest Asian-, Mexican-, and Southeast Asian-type heroin samples. These new impurities, coupled with other established forensic markers, enhance the ability to classify illicit heroin samples.     

Some pyridine derivatives as "route-specific markers" in 4-methoxyamphetamine (PMA) prepared by the Leuckart Method Studies on the role of the aminating agent in their distribution in the final product

The two previously unknown isomeric aryl-methylpyridines were prepared and analysed. Both 2,6-dimethyl-3,5-(4'-methoxyphenyl)pyridine and 2,4-dimethyl-3,5-di-(4'-methoxyphenyl)pyridine have been identified as a new by-product in the crude 4-methoxyamphetamine (PMA) obtained via the Leuckart method. The synthesis of 2,6-dimethyl-3,5-diphenylpyridine, which is connected to amphetamine chemistry, is also reported. It was also found that different reagents (formamide, formamide/HCOOH, ammonium formate) used in the course of the Leuckart synthesis of PMA significantly affected the impurity content. The presented results point out on the "high-boiling pyridines" as compounds especially useful in the comparative analysis, since their profile seems to be independent on the purification procedure and may be conveyed from the crude reaction mixture even into a carefully purified final product.     

Availability of target odor compounds from seized ecstasy tablets for canine detection

The aim of this study was to compare seized samples of 3,4-methylenedioxy-N-methylamphetamine (MDMA) pills, used to train law enforcement detection canine teams, to determine what differences exist in the chemical makeup and headspace odor and their effect on detectability. MDMA solutions were analyzed by liquid chromatography-mass spectrometry. Analysis of these samples showed a wide variance of MDMA (8-25%). Headspace SPME-GC/MS analysis showed that several compounds such as 3,4-methylenedioxyphenylacetone and 1-(3,4-methylenedioxyphenyl)-2-propanol are common among these MDMA samples regardless of starting compound and synthesis procedure. However, differences, such as the level of the various methylenedioxy starting compounds, were shown to affect the overall outcome of canine detection, indicating the need for more than one MDMA training aid. Combinations of compounds such as the primary odor piperonal in conjunction with a secondary compound such as MDP-2-OH or isosafrole are recommended to maximize detection of different illicit MDMA samples.     

Simultaneous determination of eight catechins and four theaflavins in bottled tea by liquid chromatography-tandem mass spectrometry for forensic analysis

Tea, and particularly bottled tea, is widely consumed worldwide and is often encountered at crime scenes in poisoning cases or used in place of urine in drug abuse monitoring. Tea is a rich source of polyphenols, such as catechins and theaflavins, and these compounds are useful for identification of trace quantities of tea samples. However, information on the contents of catechins and theaflavins in bottled tea is limited. In this study, a method was developed for simultaneous analysis of eight catechins and four theaflavins in tea using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of these polyphenols were determined in bottled black, oolong, and green teas after a simple pretreatment process by the standard addition method. The developed LC-MS/MS method was rapid and all tested polyphenol compounds were separated within ~14 min. All tea types contained all the catechins, at varying concentrations, but not all the theaflavins were present in all the tea types. This indicates that the theaflavin composition reflects the degree of the fermentation and could be used for discrimination among different types of tea. All the green tea samples contained all eight catechins; however, the concentrations of these compounds varied among the tea samples. Principal component analysis and hierarchical cluster analysis were useful for discrimination of samples. It has been unclear whether the variations of chemical components are useful for forensic discrimination. Our results demonstrate that, in addition to identification of tea varieties, catechins and theaflavins can be used for the discrimination of bottled tea samples.     

Chlorinated Opium Alkaloid Derivatives Produced by the Use of Aqueous Sodium Hypochlorite During the Clandestine Manufacture of Heroin

Abstract:  A clandestine chemist was observed producing heroin from crude morphine utilizing a solution of sodium hypochlorite during the process. Numerous chlorinated opium alkaloid derivatives were created when the morphine acetylation reaction was quenched and neutralized with a solution of sodium hypochlorite and ammonium hydroxide. Four of these compounds, 1-chloroheroin, 1-chloroacetylcodeine, 1-chloro-O⁶-monoacetylmorphine, and 2'-chloropapaverine, were characterized via preparative isolation, gas chromatography/mass spectrometry, nuclear magnetic resonance spectroscopy, and independent synthesis. These chlorinated derivatives were formed via electrophilic aromatic substitution with free chlorine during the illicit process. Although no illicit heroin exhibits containing these compounds have been observed in seizures to date, mass spectral data are provided for several of these compounds for their identification should they be seen within future seizures of illicit heroin.     

Development of a harmonised method for the profiling of amphetamines VI: Evaluation of methods for comparison of amphetamine

Amphetamine samples were analysed by gas chromatography-mass spectrometry (GC-MS), and the peak areas of 33 target compounds were transformed by applying various pretreatment techniques. The objective was to optimise the ability of a number of distance metrics to establish links between samples of amphetamine originating from the same batch (henceforth referred to as linked distances). Furthermore, partial least squares discriminant analysis (PLS-DA) was used to evaluate the effects of various pretreatment methods on separation of amphetamine batches synthesised by the Leuckart reaction, reductive amination of benzyl methyl ketone, and the nitrostyrene route. The most efficient way to pretreat GC-MS data varied for the different distance metrics, although best results were obtained when data were normalised to the sum of peak areas, and either the fourth root or a logarithm was applied to the normalised data. When pretreating normalised data by fourth root transformation, Pearson correlation was the distance metric that was most successful at finding linked samples. Normalisation and the use of fourth root also represented the best method of pretreating data when employing PLS-DA to separate samples synthesised by different routes. To achieve a faster and more user-friendly procedure for evaluating chromatograms, experiments were performed in which the number of target compounds used to compare samples was reduced. The effect of each compound that was removed was studied by applying PLS-DA and by using Pearson correlation to calculate linked distances as well as unlinked distances (between samples from different batches of amphetamine). Considering both links between samples from the same batch and separation of samples synthesised by different routes, the best results were obtained with the data set comprising 26 compounds. Finally, it was found that the profiling method developed in this work was superior to an existing technique with respect to separating linked and unlinked distances.     

Comparing the effects of weathering and microbial degradation on gasoline using principal components analysis

Ignitable liquid residues recovered from a fire scene will often show signs of weathering as a result of exposure to the heat of the fire. In addition, when the substrate is rich in organic matter, both weathering and microbial degradation may be observed. In this study, 20 μL aliquots of fresh gasoline samples were intentionally weathered and also subjected to microbial degradation in potting soil. These samples were then analyzed using a passive adsorption-elution recovery method and gas chromatography/mass spectrometry. Peak areas from compounds of interest were normalized and autoscaled and then subjected to principal components analysis. This analysis showed that while lower boiling compounds are subject to weathering, a different set of compounds are subject to microbial degradation. Of the compounds studied, heptane, octane, toluene, and ethylbenzene were the most vulnerable to both weathering and microbial degradation. In contrast, 1,3,5-trimethylbenzene and 2-ethyltoluene were the most resistant to both phenomena.     

Acid alteration of several ignitable liquids of potential use in arsons

Ignitable liquids such as fuels, alcohols and thinners can be used in criminal activities, for instance arsons. Forensic experts require to know their chemical compositions, as well as to understand how different modification effects could impact them, in order to detect, classify and identify them properly in fire debris. The acid alteration/acidification of ignitable liquids is a modification effect that sharply alters the chemical composition, for example, of gasoline and diesel fuel, interfering in the forensic analysis and result interpretation. However, to date there is little information about the consequences of this effect over other accelerants of interests. In this research paper, the alteration by sulfuric acid of several commercial thinners and other accelerants of potential use in arsons is studied in-depth. For that purpose, spectral (by ATR-FTIR) and chromatographic (by GC–MS) data were obtained from neat and acidified samples. Then, the spectral and chromatographic modifications of each studied ignitable liquid were discussed, proposing several chemical mechanisms that explain the new by-products produced and the gradual disappearance of the initial compounds. Hydrolysis, Fischer esterification and alkylation reactions are involved in the modification of esters, alcohols, ketones and aromatic compounds of the studied ignitable liquids. This information could be crucial for correctly identifying these accelerants. Additionally, an exploratory analysis revealed that some of the most altered ignitable liquid samples might be very similar with each other, which could have impact on casework.     

Australian Federal Police seizures of illicit crystalline methamphetamine ('ice') 1998-2002: impurity analysis

Nineteen crystalline methamphetamine ('ice') seizures captured by the Australian Federal Police (AFP) at the Australian border between 1998 and 2002 were analysed. Using a modified gas chromatograph-mass spectrometry (GC-MS) impurity profiling approach of these samples we have identified >30 compounds associated with methamphetamine and/or its synthetic route. Major impurities detected include 1,2-dimethyl-3-phenylaziridine 8, dimethylamphetamine 14, N-formylmethamphetamine 24, N-acetylmethamphetamine 25, 1,3-dimethyl-2-phenylnaphthalene 32, 1-benzyl-3-methylnaphthalene 33 and methamphetamine dimer 34. These data are suggestive of ephedrine/pseudoephedrine as the main precursor of the 'ice' samples seized during 1998-2002. Additionally the two naphthalenes 32 and 33 further identified that 15 items in 9 seizures were produced via the more specific ephedrine/hydriodic acid/red phosphorus method. One sample comprised 75% dimethylamphetamine and 9.7% methamphetamine, representing the first Australian seizure of imported dimethylamphetamine reported.     

Chemical fingerprinting of unevaporated automotive gasoline samples

The comparison of two or more samples of liquid gasoline (petrol) to establish a common origin is a difficult problem in the forensic investigation of arsons and suspicious fires. A total of 35 randomly collected samples of unevaporated gasoline, covering three different grades (regular unleaded, premium unleaded and lead replacement), were examined. The high-boiling fraction of the gasoline was targeted with a view to apply the techniques described herein to evaporated gasoline samples in the future.A novel micro solid phase extraction (SPE) technique using activated alumina was developed to isolate the polar compounds and the polycyclic aromatic hydrocarbons (PAHs) from a 200microl sample of gasoline. Samples were analysed using full-scan gas chromatography-mass spectrometry (GC-MS) and potential target compounds identified. Samples were then re-analysed directly, without prior treatment, using GC-MS in selected ion monitoring (SIM) mode for target compounds that exhibited variation between gasoline samples. Principal component analysis (PCA) was applied to the chromatographic data. The first two principal components (PCs) accounted for 91.5% of the variation in the data. Linear discriminant analysis (LDA) performed on the PCA results showed that the 35 samples tested could be classified into 32 different groups.     

SPME/GC-MS characterization of volatiles associated with methamphetamine: toward the development of a pseudomethamphetamine training material

The headspace profiles of eleven methamphetamine (MA) samples have been analyzed using solid-phase microextraction/gas chromatography-mass spectrometry (SPME/GC-MS). Nine of the eleven are illicit MA seizures from the Southwest U.S. border. One sample is methamphetamine base synthesized in the Drug Enforcement Administration (DEA) Southwest Laboratory, and the remaining sample is pharmaceutical-grade methamphetamine hydrochloride that is used to make training aids for drug detecting canines. In addition. volatiles associated with 1-phenyl-2-propanone (P2P), a methamphetamine precursor, have been identified for comparison with those found in methamphetamine seizure and the two reference samples. Eighty-seven different compounds were identified from all the samples, not including simple hydrocarbons and aldehydes. Only seven occur consistently in all seizure samples, and these are: acetic acid, benzaldehyde, acetophenone, P2P, 1-phenyl-1,2-propanedione (P12P), 3-phenyl-3-buten-2-one, 1-chloro-1-phenyl-2-propanone. Dimethyl sulfone, a common cutting agent in methamphetamine. was found in six of the nine seizure materials. When the reference methamphetamine and P2P samples are included, only two compounds are common to all twelve samples, and these are benzaldehyde and P2P. As such, these two compounds are likely candidates for use in a pseudomethamphetamine (PM) formulation, and their effectiveness in eliciting a canine response is being evaluated before actual deployment.     

Evaluation and classification of fentanyl-related compounds using EC-SERS and machine learning

Multiple analytical techniques for the screening of fentanyl-related compounds exist. High discriminatory methods such as GC–MS and LC–MS are expensive, time-consuming, and less amenable to onsite analysis. Raman spectroscopy provides a rapid, inexpensive alternative. Raman variants such as electrochemical surface-enhanced Raman scattering (EC-SERS) can provide signal enhancements with 10¹⁰ magnitudes, allowing for the detection of low-concentration analytes, otherwise undetected using conventional Raman. Library search algorithms embedded in instruments utilizing SERS may suffer from accuracy when multicomponent mixtures involving fentanyl derivatives are analyzed. The complexing of machine learning techniques to Raman spectra demonstrates an improvement in the discrimination of drugs even when present in multicomponent mixtures of various ratios. Additionally, these algorithms are capable of identifying spectral features difficult to detect by manual comparisons. Therefore, the goal of this study was to evaluate fentanyl-related compounds and other drugs of abuse using EC-SERS and to process the acquired data using machine learning—convolutional neural networks (CNN). The CNN was created using Keras v 2.4.0 with Tensorflow v 2.9.1 backend. In-house binary mixtures and authentic adjudicated case samples were used to evaluate the created machine-learning models. The overall accuracy of the model was 98.4 ± 0.1% after 10-fold cross-validation. The correct identification for the in-house binary mixtures was 92%, while the authentic case samples were 85%. The high accuracies achieved in this study demonstrate the advantage of using machine learning to process spectral data when screening seized drug materials comprised of multiple components.     

Metal concentrations in plants and urine from patients treated with traditional remedies

A number of traditional South African herbal remedies which are associated with morbidity and mortality were analysed for selenium, manganese, copper, lead, zinc and mercury content. Few showed high levels of toxic metals, but in these the concentrations were sufficiently high as to cause concern since there is no quality control on the production of these remedies, which are the accepted form of medication for over 80% of the population. Urine samples, obtained from 65 patients admitted to hospital following treatment with a traditional herbal remedy were also analysed for metals. Only a small number of the patients had abnormally high levels of metal excretion. These data suggest that, in contrast to experience with traditional Chinese and Indian preparations, metal contamination from plants seems not to be a problem in traditional South African remedies.     

The use of a quantitative structure-activity relationship (QSAR) model to predict GABA-A receptor binding of newly emerging benzodiazepines

The illicit market for new psychoactive substances is forever expanding. Benzodiazepines and their derivatives are one of a number of groups of these substances and thus far their number has grown year upon year. For both forensic and clinical purposes it is important to be able to rapidly understand these emerging substances. However as a consequence of the illicit nature of these compounds, there is a deficiency in the pharmacological data available for these ‘new’ benzodiazepines. In order to further understand the pharmacology of ‘new’ benzodiazepines we utilised a quantitative structure-activity relationship (QSAR) approach. A set of 69 benzodiazepine-based compounds was analysed to develop a QSAR training set with respect to published binding values to GABA_A receptors. The QSAR model returned an R² value of 0.90. The most influential factors were found to be the positioning of two H-bond acceptors, two aromatic rings and a hydrophobic group. A test set of nine random compounds was then selected for internal validation to determine the predictive ability of the model and gave an R² value of 0.86 when comparing the binding values with their experimental data. The QSAR model was then used to predict the binding for 22 benzodiazepines that are classed as new psychoactive substances. This model will allow rapid prediction of the binding activity of emerging benzodiazepines in a rapid and economic way, compared with lengthy and expensive in vitro/in vivo analysis. This will enable forensic chemists and toxicologists to better understand both recently developed compounds and prediction of substances likely to emerge in the future.     

Determining the Method Threshold of Identification via Gas Chromatography–Mass Spectrometry of Weathered Gasoline Extracted from Burnt Nylon Carpet,

The Organization of Scientific Area Committees defines threshold of identification as the minimum concentration of ignitable liquid identifiable from gas chromatographic‐mass spectrometry data using accepted pattern identification criteria. We propose a method for determining this threshold for gasoline based on base peak to qualifier ratios of six compounds. The ion ratios were established for each compound in the neat gasoline. These ratios were then compared to those obtained for gasoline and 98% weathered gasoline both spiked onto burnt nylon carpet at 20 ppt down 0.50 ppt, and recovered from the carpet using headspace extraction (ASTM 1412). Identification was confirmed if the compounds’ ion ratios fell within ±25% of that in the neat sample. We found that ion ratios for all samples were acceptable for six compounds at 1.60 and 0.80 ppt for extracted neat and extracted 98% weathered gasoline, respectively, illustrating potential for incorporating into Quality Assurance Programs.     

Preliminary evaluation of the persistence of organic gunshot residue

The organic components of gunshot residue (OGSR, also called firearms discharge residue (FDR) or cartridge discharge residue (CDR)) have been studied and discussed in the literature. These residues, consisting of particulates such as burned and unburned powder as well as molecular compounds, are rarely used in casework except for purposes such as shooting reconstructions. Molecular compounds that survive the firing event or that are created as a result of the firing event could, with focused research and development, open a new avenue for forensic gunshot residue analysis. In this study, the persistence of organic gunshot residue was evaluated using diphenylamine (DPA) as a target analyte and ion mobility spectrometry (IMS) as the detection system. Samples were collected from hands using a solvent swabbing technique and the swab was analyzed using direct thermal desorption for sample introduction into the IMS. OGSR was found to persist for at least 4h. Although DPA is a widely used industrial compound, analysis of numerous blank and background samples (n～100) did not show any significant response for DPA using this detector. Variations were noted among individuals and as such, the data set does not support estimation of a half-life as has been done for traditional primer residues. No secondary transfers were observed, suggesting the possibility of skin adhesion via interactions between the lipophilic organic compounds and skin lipids. IMS proved valuable as a means of generating patterns for forensic pattern matching and shows promise as a screening tool applied to firearms discharge.     

Spectral differentiation and gas chromatographic/mass spectrometric analysis of the lacrimators 2-chloroacetophenone and o-chlorobenzylidene malononitrile

2-Chloroacetophenone (CN) and o-chlorobenzylidene malononitrile (CS) are the most common chemical agents used as lacrimators in the United States. There is a lack of complete spectral data on these compounds in the literature. Spectral data (ultraviolet, fluorescence, proton nuclear magnetic resonance, and infrared) and a gas-liquid chromatographic/mass spectrometric method are presented that differentiate and identify CN and CS. These methods and data were used to identify a forensic science specimen from an accidental intoxication.