The identification of firearms is of paramount importance for investigating crimes involving firearms, as it establishes the link between a particular firearm and firearm-related elements found at a ...crime scene, such as projectiles and cartridge cases. This identification relies on the visual comparison of such elements against reference samples from suspect firearms or those existing in databases. Whenever this approach is not possible, the chemical analysis of the gunpowder and gunshot residue can provide additional information that may assist in establishing a link between samples retrieved at a crime scene and those from a suspect or in the identification of the corresponding model and manufacturer of the ammunition used. The most commonly used method for the chemical analysis of gunshot residue is scanning electron microscopy with energy dispersive X-ray, which focuses on the inorganic elements present in ammunition formulation, particularly heavy metals. However, a change in the legal paradigm is pushing changes in these formulations to remove heavy metals due to their potential for environmental contamination and the health hazards they represent. For this reason, the importance of the analysis of organic compounds is leading to the adoption of a different set of analytical methodologies, mostly based on spectroscopy and chromatography. This manuscript reviews the constitution of primer and gunpowder formulations and the analytical methods currently used for detecting, characterising, and identifying their compounds. In addition, this contribution also explores how the information provided by these methodologies can be used in ammunition identification and how it is driving the development of novel applications within forensic ballistics.
Novel psychoactive substances (NPS) represent a rising public health threat due to the associated side effects and the lack of success on their control policies. NPS are freely sold, mostly on the ...internet, as legal and safe replacements of controlled drugs of abuse. Synthetic cannabinoids (SCs) represent one of the largest groups of NPS and are designed as mimetics of Δ9-tetrahydrocannabinol from cannabis
1
. After the identification of JWH-018, the first SC appearing on the market, many countries took measures to control the free circulation of these products
2
. Nevertheless, efforts to control them are rapidly contoured, since clandestine manufacturers are constantly changing their chemical structures leading to previously unknown SCs
3
. The aim of this study is to show how chemical changes on recent generations of SCs to evade the law lead to the production of more harmful compounds, with potential application in forensic context. With that purpose, the toxicity of three SCs, representatives of the first, second and third generations
4
, was assessed.
The SC JWH-018 was obtained as >98.5% pure powders from Lipomed AG Switzerland. SCs THJ-018 and EG-018 were bought through an internet website (
www.chem.eu
). THJ-018 and EG-018 were purified through HPLC/DAD and confirmed by GC/MS. Pure SCs stock solutions were prepared in 100% DMSO accordingly to the stipulated final concentrations and diluted into the culture medium prior addition to cells. The neuroblastoma human cell line SH-SY5Y was exposed, for 24 h, to several concentrations of each SC. Cell toxicity was evaluated through MTT assays.
Figure 1
shows the MTT results for SH-SY5Y viability in the presence of JWH-018, THJ-018 and EG-018. SH-SY5Y cells viability does not decrease in the presence of JWH-018 at the range of concentrations used. However, when the same cell line is exposed to THJ-018 or EG-018 there is a decrease in cell viability with the increase in the concentration of these substances. Discussion and conclusions: The results from the present study points THJ-018 and EG-018, JWH-018 analogues of 2nd and 3rd generations, as more toxic to the neuroblastoma cells than JWH-018, the first SC found on the street market. These results suggest that emerging modified SCs, to avoid the law, are becoming more toxic and dangerous. Given the emergence of this situation, it is time to rethink current legislation to prevent rise on public health issues derived from consumption of molecules of unknown toxicological profile.
Synthetic cannabinoids (SCs) are novel psychoactive substances that mimic the effects of cannabis
1-3
. These products were sold via online shops and consisted of herbal mixtures sprayed with SCs
3
.... Since then, a deluge of chemical variations of SCs has been occurring worldwide due to their synthesis in clandestine laboratories, often based on pharmaceutical research and patents, posing a growing challenge for authorities regarding regulation of these substances
1
,
3
. More recently, several highly potent compounds have emerged on the drug market, synthesised as stated in the patent application of Bowden and Williamson ("SGT-compounds"). These drugs are characterised by a cumyl substituent, which is attached to an indole, indazole or azaindole structure. One of the first cumyl-derivatives, CUMYL-PEGACLONE, was found in 2016 on the German drug market, being sold under the street name SGT-151
1
,
2
. The present study aims to understand what is inside of a SGT-151 package sold in the internet as a 'research chemical'.
The cannabinoid identification was based on its mass spectra using the Cayman database (Chemical C. Cayman Spectral Library, vol. v08302018). GC/MS was the technique used to analyse the compound using a MEGA-5 MS capillary column (0.25 µm, 0.32 mm, 30m). Chromatographic analysis was carried out under the following conditions: injection volume 1 µL and splitless injection at 280 °c. The initial oven temperature was 100 °C for 3 min, ramped to 310 °C at a rate of 30 °C/min and held at 310 °C for 10 min. The MS conditions were as follows: ionisation energy was set at 70 eV; acquisition was carried out in a scan mode range of m/z 30-450. Helium was used as the carrier gas. For purification, a HPLC/DAD, operated by Clarity software, was used with a reversed-phase column. The mobile phase was a solvent gradient system consisting of (A) 5% 10 mM ammonia format and (B) 95% acetonitrile, optimised to achieve the best resolution. The results were recorded at λ = 252 nm.
Figure 1(A)
shows the GC/MS chromatogram of SGT-151. It is clear the presence of a peak corresponding to SGT-151 and several other peaks from other compounds. It was not possible to identify the remaining peaks.
Figure 1(B)
shows a GC/MS chromatogram after purification of the package content. Discussion and conclusions: The data gathered in this study shows that SGT-151 contains a significant amount of impurities. This working progress data is the initial step of a bigger project aiming to understand whether there are differences in the toxicity of pure and non-pure SGT-151 on human cell lines, once it is important to understand if the impurities may have any influence on the cytotoxicity, compared to substances in their pure form, since most of the times the substances that are sold on the streets are not 100% pure.
One of the major challenges in forensic document analysis is estimating the age of ink deposition on a manually written document. The present work aims to develop and optimise a methodology, based on ...the evaporation of 2-phenoxyethanol (PE) over time, that can contribute to ink age estimation. A black BIC
Crystal Ballpoint Pen was purchased in a commercial area, and ink deposition began in September 2016 over 1095 days. For each ink sample, 20 microdiscs were subjected to n-hexane extraction in the presence of an internal standard (ethyl benzoate) followed by derivatisation with a silylation reagent. A gas chromatography-mass spectrometry (GC/MS) method was optimised for PE-trimethylsilyl (PE-TMS) to characterise the ageing curve. The developed method presented good linearity between 0.5 and 50.0 μg mL
, as well as limits of detection and quantification of 0.026 and 0.104 μg mL
, respectively. It was possible to characterise PE-TMS concentration over time, which reveals a two-phase decay behaviour. First, there was a substantial decline between the 1st and the 33rd day of deposition, followed a by a stabilisation of the signal, which allowed to detect the presence of PE-TMS up to 3 years. Two unknown compounds were also present and allowed to identify three dating time frames for the same ink stroke: (i) between time 0 and 33 days, (ii) between time 34 and 109 days, and (iii) more than 109 days. The developed methodology allowed to characterise the behaviour of PE over time and to establish a relative dating of three-time frames.
Understanding which peptides and proteins have the potential to undergo amyloid formation and what driving forces are responsible for amyloid-like fiber formation and stabilization remains limited. ...This is mainly because proteins that can undergo structural changes, which lead to amyloid formation, are quite diverse and share no obvious sequence or structural homology, despite the structural similarity found in the fibrils. To address these issues, a novel approach based on recursive feature selection and feed-forward neural networks was undertaken to identify key features highly correlated with the self-assembly problem. This approach allowed the identification of seven physicochemical and biochemical properties of the amino acids highly associated with the self-assembly of peptides and proteins into amyloid-like fibrils (normalized frequency of β-sheet, normalized frequency of β-sheet from LG, weights for β-sheet at the window position of 1, isoelectric point, atom-based hydrophobic moment, helix termination parameter at position j+1 and ΔG° values for peptides extrapolated in 0 M urea). Moreover, these features enabled the development of a new predictor (available at http://cran.r-project.org/web/packages/appnn/index.html) capable of accurately and reliably predicting the amyloidogenic propensity from the polypeptide sequence alone with a prediction accuracy of 84.9 % against an external validation dataset of sequences with experimental in vitro, evidence of amyloid formation.
2-Phenoxyethanol (PE) is a volatile compound present in the composition of inks. After the deposition in a document, it starts to evaporate over time. This ageing process potentially allow to ...estimate the date of an ink in a document
1
, however this is a complex system where the PE derivatization with different derivatization agents and methods can contribute to improve ink dating validation
2
. The aim of this work is to determine if PE derivatization with MSTFA:TMCS, will increase the sensitivity of the method contributing to the estimation of the ink age for a longer period of time.
In order to compare peak resolution between PE and derivatized PE, a solution of 50 µg/ml of PE was prepared in hexane. The derivatized sample was prepared using chemical derivatization with MSTFA:TMCS (95:5) at a 80ºC during 30 min. The samples were analysed using GC/MS with an Agilent Technologies 5973 − 6890 N. GC MEGA-5 MS; 0.25µm, 0.32mm, 30 m capillary column was used. Chromatographic analysis was carried out under the following conditions: injection volume 2 µl, split ratio of 2:1 and injection temperature at 280 ºC. The oven temperature program starting at 90 ºC during 8 min, then ramped at 10 ºC min
−1
to 100 ºC, and increased to 240 ºC at a rate of 30 ºC min
−1
during 4.67 min. The MS analysis was carried out in SIM mode, which m/z of PE was 77, 94, 138 and PE-TMS was 151, 195, 210.
The chromatogram results (
Figure 1
) showed that PE-TMS has a longer retention time (23.2 min) and a better resolution than PE (retention time of 13.2 min). Discussion and conclusions: This preliminary study showed an increase of the sensitivity of the PE-TMS. Our results revealed that PE derivatization with MSTFA:TMCS, might be useful in ink dating determination and can contribute to the decreasing of LOQ. However, more work has to be done. In conclusion, derivatization proves to be promising in the field of ink ageing, allowing getting sensitivity to estimate the age of an ink, in a long period of time.
Parkinson's disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of α-synuclein (a-syn) in various brain regions is ...the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.
Phytocannabinoids are psychotropic substances found in cannabis that bind to the endocannabinoid receptors regulating a variety of physiological processes in human body, including synaptic activity ...in the central nervous system and metabolic effects in the peripheric nervous system among many others
1
,
2
. Synthetic cannabinoids emerged as popular alternative to cannabis. Most of these substances are synthetic analogues of Δ
9
-THC, the psychotropic compound of cannabis, binding with higher affinity to the endocannabinoid receptor CB
1
and eliciting a stronger and long-lasting effect on brain cells. Molecular structure of synthetic cannabinoids is always changing escaping the control by authorities and increasing the hazard for general population. The popularity of cannabis and its derivatives may lead, and often does, to child's exposure to cannabinoids both in utero and through breastfeeding by a drug-consuming mother. Prenatal exposure to cannabis has been associated with higher risk of newborn morbidity
2
,
3
, altered rate of mental development and significant changes in nervous system functioning
4
,
5
. However, direct evidence that these effects are mediated through the binding of cannabinoids to endocannabinoid receptors is still lacking. Thus, it is paramount to better understand the psychoactive effects of natural and synthetic cannabinoids on the developing human brain.
We conveyed a pilot study in which human induced pluripotent stem cells (hiPSCs) were induced into neural differentiation and treated with a non-psychotropic component of cannabis, cannabidiol, known to bind the CB
2
receptor, and two synthetic Δ
9
-THC analogues, THJ-018 and EG-018. Neuronal differentiation and functional maturation were assessed by immunofluorescence, qRT-PCR and single cell calcium imaging. Our results indicate that all three substances have profound impact on the differentiation, maturation and functioning of developing CNS neurons, providing a new evidence for the importance of thorough research of the impact of pre-natal exposure to natural and synthetic cannabinoids.
Nowadays, there is a trend to enrich food and food supplements (FS) with Vitamin D
3
. Economic operators who places FS on the market does not have to perform running safety trials, but only to ...comply with the food safety regulations applicable in the European Union
1
. This scenario may present public health and legal issues. To overcome these challenges, it is mandatory to know accurately the amount of vitamin D
3
in FS and if it corresponds to the label value. HPLC methods are a preferential approach to determine accurately the content of vitamin D
3
in pharmaceuticals and supplements. Actually, HPLC/MS has become the technique of choice for vitamin D
3
determination in complex matrixes. Notwithstanding, this analysis must be preceded by time-consuming sample preparation. In fact, the success to measure accurately vitamin D
3
depends heavily in a reliable sample preparation. Moreover, it becomes even more critic when dealing with different formulations of vitamin D
3
, such has gel pills, solid pills, liquid or cutaneous applications. There are literature validating methodology to determinate vitamin D
3
in various matrixes according to the International Conference on Harmonisation (ICH) guidelines by HPLC-UV
2
. However, this published study does not use an internal standard (IS). The internal standard is useful to improve the precision of quantitative analysis, removing the error of losing sample during sample preparation. Our aim is to test o-cresol as an internal standard to future validation the methodology.
Vit D
3
present in FS was analysed by HPLC/DAD. Sample was spiked with internal standard (o-cresol) and liquid extraction coupled to an ultrasound bath was used has an extraction procedure. After a centrifuged step the supernatant was filtered. The separation was performed using a C18 column with a solvent gradient consisted in (A) 0.1% formic acid and (B) acetonitrile. The acquisition was performed on DAD-detector at 265 nm.
Figure 1
shows a chromatogram of a FS containing Vit D
3
. Two major peaks are present, corresponding to o-cresol (RT 5.45 min) and Vit D
3
(RT 11.20 min). Discussion and conclusions: The results show the capacity of the chromatographic method to separate and resolve the two chromatographic peaks. These preliminary results show that o-cresol can be used as an internal standard to determine Vit D3 in FS, normalising the loss of Vit D3 during preparation step. Since in Europe and USA, the association of vitD toxicity with the use of FS has been described, this methodology will be useful to confirm the VitD composition stated on the label.
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