Stability studies of ALD-52 and its homologue 1P-LSD

With the emergence of new psychoactive substances (NPSs) over the years, the substances detected on stamps (also known as blotter papers) have also evolved from the traditional drug—lysergic acid diethylamide (LSD) to the multiple variants of lysergamides such as ALD-52 and 1P-LSD. The analysis of such blotter papers is usually done by solvent extraction followed by identification using gas chromatography–mass spectrometry (GC-MS). This study has shown that hydrolysis to form LSD was observed in GC-MS analysis when ALD-52 was extracted with methanol. The extraction of ALD-52 using other solvents such as acetonitrile, ethanol, isopropyl alcohol, ethyl acetate, and acetone, followed by GC-MS analysis, was investigated. It is shown that alcoholic solvents such as methanol and ethanol will result in the conversion of ALD-52 to LSD during GC-MS analysis, whereas the sterically hindered isopropyl alcohol will prevent this conversion. Investigation also shows that the hydrolysis of ALD-52 to LSD occurs at the GC injector port. It was also observed that the degree of hydrolysis was more pronounced at a lower concentration (0.1 mg/mL). The study was extended to a close analog—1P-LSD, and the results showed that 1P-LSD similarly hydrolyzes to LSD. However, 1P-LSD was observed to be more stable than ALD-52 due to steric hindrance because of the propanoyl group.     

Deep learning for sex determination: Analyzing over 200,000 panoramic radiographs

The objective of this study is to assess the performance of an innovative AI-powered tool for sex determination using panoramic radiographs (PR) and to explore factors affecting the performance of the convolutional neural network (CNN). The study involved 207,946 panoramic dental X-rays and their corresponding reports from 15 clinical centers in São Paulo, Brazil. The PRs were acquired with four different devices, and 58% of the patients were female. Data preprocessing included anonymizing the exams, extracting pertinent information from the reports, such as sex, age, type of dentition, and number of missing teeth, and organizing the data into a PostgreSQL database. Two neural network architectures, a standard CNN and a ResNet, were utilized for sex classification, with both undergoing hyperparameter tuning and cross-validation to ensure optimal performance. The CNN model achieved 95.02% accuracy in sex estimation, with image resolution being a significant influencing factor. The ResNet model attained over 86% accuracy in subjects older than 6 years and over 96% in those over 16 years. The algorithm performed better on female images, and the area under the curve (AUC) exceeded 96% for most age groups, except the youngest. Accuracy values were also assessed for different dentition types (deciduous, mixed, and permanent) and missing teeth. This study demonstrates the effectiveness of an AI-driven tool for sex determination using PR and emphasizes the role of image resolution, age, and sex in determining the algorithm's performance.     

Quantitative PCR overestimation of DNA in samples contaminated with tin

Metals can pose challenges while conducting forensic DNA analysis. The presence of metal ions in evidence-related DNA extracts can degrade DNA or inhibit PCR as applied to DNA quantification (real-time PCR or qPCR) and/or STR amplification, leading to low success in STR profiling. Different metal ions were spiked into 0.2 and 0.5 ng of human genomic DNA in an “inhibition study” and the impact was evaluated by qPCR using the Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) and an in-house SYBR Green assay. This study reports on a contradictory finding specific to tin (Sn) ions, which caused at least a 38,000-fold overestimation of DNA concentration when utilizing Quantifiler Trio. This was explained by the raw and multicomponent spectral plots, which indicated that Sn suppresses the Quantifiler Trio passive reference dye (Mustang Purple™, MP) at ion concentrations above 0.1 mM. This effect was not observed when DNA was quantified using SYBR Green with ROX™ as the passive reference, nor when DNA was extracted and purified prior to Quantifiler Trio. The results show that metal contaminants can interfere with qPCR-based DNA quantification in unexpected ways and may be assay dependent. The results also highlight the importance of qPCR as a quality check to determine steps for sample cleanup prior to STR amplification that may be similarly impacted by metal ions. Forensic workflows should recognize the risk of inaccurate DNA quantification of samples that are collected from substrates containing tin.     

Effects of obesity on talar micro- and macro-morphology

The addition of information regarding obesity status to the forensic anthropological biological profile could significantly contribute to the identification of human skeletal remains since over 40% of the U.S. adult population is currently obese. This study examines the differences in talar shape and trabecular bone structure between obese and non-obese individuals. A sample of 20 obese and 20 non-obese divided evenly by sex was selected from the Texas State University Donated Skeletal Collection. Tali were imaged using x-ray computed tomography (voxel size: 28–38.7 μm). Image stacks were processed to produce binary images as well as trabecular thickness and spacing maps. Landmark-based geometric morphometric analyses were conducted to quantify shape variation. Shape coordinates were used to locate 100 geometrically homologous volumes of interest within each talus. Bone volume fraction, trabecular thickness, and trabecular spacing were extracted at each volume of interest. Within each sex, a one-way ANCOVA was used to determine if significant differences exist between obese and non-obese individuals in trabecular bone after controlling for age. The size of the talus as well as subtle aspects of shape were found to distinguish the sexes. The results further indicate that bone volume fraction significantly differs between obese and non-obese males. In females, bone volume fraction is correlated with age but does not differ between obese and non-obese. The study demonstrates that bone microstructure is a promising approach to estimating body mass or body mass index category but age effects diminish the potential for the talus to be used alone.     

Evaluation of portable gas chromatography–mass spectrometry (GC–MS) for the analysis of fentanyl,fentanyl analogs,and other synthetic opioids

Potent synthetic opioids including fentanyl and its analogs are frequently encountered in the field and require detection and identification by first responders to maintain the safety of drug abusers, first responders, health-care providers, and the public at large. Due to the low concentration at which these substances may be encountered and the complicating matrices within which they may be dispersed, the use of portable gas chromatography–mass spectrometry (GC–MS) for their identification in the field offers great potential value. This research established that portable GC–MS is a useful method for the detection and identification of a large number of synthetic opioids, especially fentanyl and its analogs. In this study, 250 synthetic opioids and related substances including 210 fentanyl analogs were analyzed using portable GC–MS. It was concluded that 225 of the 250 (90.0%) opioids analyzed were successfully detected onboard at the time of analysis and identified as either the substance (55.2%) or an analog (34.8%). These outcomes have equivalent benefit for the field analysis of illicit drugs due to both initiating the same subsequent actions by first responders.     

Decoding hidden darknet networks: What we learned about the illicit fentanyl trade on AlphaBay

The opioid epidemic, impacted from the proliferation of fentanyl, has added impetus to the need to detect fentanyl, sources of fentanyl, and places where fentanyl and drugs adulterated with fentanyl are available. Many darknet marketplaces (DNMs) have rules that ban fentanyl. However, it is unclear how these affect the fentanyl market. Using the AlphaBay DNM as a case study, we conducted mixed methods qualitative research. We scraped and analyzed data from the AlphaBay I2P website using, among other methods, content and social network analysis, to uncover hidden fentanyl networks. Our research highlights the next evolution of darknet marketplaces – the migration of DNMs from Tor to I2P and the methods that can be used identify fentanyl networks, irrespective of where sites are: I2P, Tor, or multihomed on I2P and Tor. Despite its ban in the Global AlphaBay Rules, our research revealed the sale of fentanyl on the AlphaBay DNM. Unlike previous studies, our findings predominantly revealed the covert sale of fentanyl on AlphaBay and predatory vendors selling illicit drugs, which unbeknownst to buyers, contained fentanyl. To a lesser extent, our findings identified the overt sale of fentanyl patches on AlphaBay. Although we examined only one DNM, the prevalence of the covert sale of fentanyl and the presence of predatory vendors underscores the importance of research that decodes the language of vendors who surreptitiously sell fentanyl or drugs adulterated with fentanyl or other illicit substances. The results of our research can inform strategies aimed at disrupting and dismantling DNM fentanyl networks.     

The impact of lipid degradation on fingerprint quality on fired firearm cartridges

The recovery of identifiable fingerprints from fired cartridge cases is challenging. Therefore, the characterization of chemical modifications and their effects on fingerprint integrity post-firing is essential. In this study, the primary fingerprint lipids, including myristic acid, pentadecanoic acid, palmitoleic acid, palmitic acid, stearic acid, squalene, and cholesterol in fired and unfired cartridges, were extracted with acetonitrile, followed by derivatization using N,O-Bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (BSTFA/1%TMCS). Squalane was used as the internal standard, and all quantifications were performed using gas chromatography coupled with mass spectrometry using a triple-quadrupole mass filter. All lipids identified in the unfired cartridges were also detected in the fired cartridges, and statistical analysis using Student's t-test and F tests was performed with a 95% confidence level. The concentration of lipids in the unfired cartridges was found to be similar to that detected in the fired cartridges, except for squalene, the recovery of which was 28% lower in the fired cartridges.     

Persistence of cellular material after exposure to water

Disposing of items of forensic relevance in bodies of water is one countermeasure offenders can use to avoid detection. The impact of immersion in water has been explored for blood, saliva, and semen; however, few studies have assessed touch DNA. Here we report on the effect of exposure to water on the persistence of touch DNA over prolonged periods of time. To evaluate the persistence of cells from touch DNA, after water exposure, three substrates and two water types were tested: plastic, metal, and ceramic, submerged into seawater or tap water. Diamond™ Nucleic Acid Dye was used to stain cells deposited by touch. Cell counts before and after water exposure were compared to investigate cell loss over time, ranging from 6 hours to 5 days. A logarithmic increase in the percent of cells lost was observed over time when the data for substrate and water type conditions were combined. Substrate type influenced the persistence of cells, with the metal substrate retaining cells longer than plastic or ceramic. The influence of water type appeared dependent on the substrate, with varied cell persistence on metal whereas plastic and ceramic recorded similar cell loss over time between water types. The ability to visualize cells after exposure to water could assist in triaging evidence within operational forensic laboratories and allow for targeted sampling. This proof-of-concept study demonstrated that greater than 50% of cells can persist on various items submerged in aqueous environments for at least 5 days, highlighting the possibility for downstream DNA testing.     

Repeatability and reproducibility of comparison decisions by firearms examiners

In a comprehensive study to assess various aspects of the performance of qualified forensic firearms examiners, volunteer examiners compared both bullets and cartridge cases fired from three different types of firearms. They rendered opinions on each comparison according to the Association of Firearm & Tool Mark Examiners (AFTE) Range of Conclusions, as Identification, Inconclusive (A, B, or C), Elimination, or Unsuitable. In this part of the study, comparison sets used previously to characterize the overall accuracy of examiners were blindly resubmitted to examiners to assess the repeatability (105 examiners; 5700 comparisons of bullets and cartridge cases) and reproducibility (191 examiners of bullets, 193 of cartridge cases; 5790 comparisons) of firearms examinations. Data gathered using the prevailing AFTE Range were also recategorized into two hypothetical scoring systems. Consistently positive differences between observed agreement and expected agreement indicate that the repeatability and reproducibility of examiners exceed chance agreement. When averaged over bullets and cartridge cases, the repeatability of comparison decisions (involving all five levels of the AFTE Range) was 78.3% for known matches and 64.5% for known nonmatches. Similarly averaged reproducibility was 67.3%% for known matches and 36.5% for known nonmatches. For both repeatability and reproducibility, many of the observed disagreements were between a definitive and inconclusive category. Examiner decisions are reliable and trustworthy in the sense that identifications are unlikely when examiners are comparing non-matching items, and eliminations are unlikely when they are comparing matching items.     

Pressure sensitive adhesives and paper spray-mass spectrometry for the collection and analysis of fentanyl-related compounds from shipping materials

The rise of fentanyl and fentanyl analogs in the drug supply pose serious threats to public health. Much of these compounds enter the United States through shipping routes. Here we provide a method for fentanyl screening and analysis that utilizes pressure-sensitive adhesive (PSA) lined paper to recover drug residues from parcel-related surfaces. The paper used is commercially available repositionable notes (also called post-it or sticky notes). From this paper, mass spectra were obtained by paper spray-mass spectrometry (PS-MS), where PSA paper served as both a sampling and analysis substrate. Seven fentanyl-related compounds were analyzed: fentanyl, 4-anilino-N-phenethylpiperidine (4-ANPP), N,1-diphenethyl-N-phenylpiperidin-4-amine (phenethyl-4-ANPP), valerylfentanyl, 4-fluoroisobutyrylfentanyl (4-FIBF), carfentanil, and p-fluorofentanyl. These compounds were recovered by PSA paper and identified by PS-MS from packaging tape and plastic at 50 ng and from cardboard and shipping labels at 100 ng. The impact of cutting agents on PS-MS analysis of fentanyl analogs was explored. No trends of analyte suppression were found at high concentrations of the cutting agents caffeine, diphenhydramine, and lidocaine when recovered from surfaces. A cartridge that required no precise cutting of PSA paper prior to sampling or analysis was evaluated for use in PS-MS for fentanyl screening. Recovery and detection of fentanyl from plastic sheeting was demonstrated with this cut-free cartridge. The cut-free cartridge showed somewhat less consistency and lower analyte signal than the standard cartridge, but performance was suitable for potential screening applications. In combining PSA surface sampling with PS-MS for drug screening, both sampling and detection of fentanyl-related compounds is simple, rapid, and low-cost.