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.     

Assessment of post-mortem submersion interval using total aquatic decomposition scores of drowned human cadavers

Investigating drowning-related deaths remains a significant problem for forensic personnel all over the world. The previously published decomposition scoring method like the total aquatic decomposition (TAD) score promises to estimate the correct post-mortem submersion interval (PMSI) in aquatic habitats through the assessment and calculation of the decomposition rate and accumulated degree days (ADD). The current study comprised of 53 drowned death cases belonging to various districts of Haryana from May 2016 to August 2017. The regression and Pearson's correlation indicated a significant correlation between the TAD scores and the actual ADD (calculated through water temperatures) (r² = 0.917) and between the actual and the estimated ADDs (calculated through TAD scores used by Heaton et al. [21]) (r = 0.9585). The results indicated that the estimated ADD tends to over predict the PMSI compared to the actual ADD. It is further confirmed by paired t-test, which showed the mean of actual ADD (mean = 349) to be significantly lower than the mean of estimated ADD (mean = 663). Moreover, these methods will help forensic investigators and researchers formulate region-specific regression equations for PMSI estimation.     

Validation of the InnoXtract™ DNA extraction method for bone,teeth, and rootless hair

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.     

Classification of firing pin impressions using HOG-SVM

Crimes, such as robbery and murder, often involve firearms. In order to assist with the investigation into the crime, firearm examiners are asked to determine whether cartridge cases found at a crime scene had been fired from a suspect's firearm. This examination is based on a comparison of the marks left on the surfaces of cartridge cases. Firing pin impressions can be one of the most commonly used of these marks. In this study, a total of nine Ruger model 10/22 semiautomatic rifles were used. Fifty cartridges were fired from each rifle. The cartridge cases were collected, and each firing pin impression was then cast and photographed using a comparison microscope. In this paper, we will describe how one may use a computer vision algorithm, the Histogram of Orientated Gradient (HOG), and a machine learning method, Support Vector Machines (SVMs), to classify images of firing pin impressions. Our method achieved a reasonably high accuracy at 93%. This can be used to associate a firearm with a cartridge case recovered from a scene. We also compared our method with other feature extraction algorithms. The comparison results showed that the HOG-SVM method had the highest performance in this classification task.     

Ethanol stability in unpreserved refrigerated antemortem blood

Ethanol stability in preserved antemortem blood has been widely studied since it is a common practice in cases involving suspected impaired driving to collect antemortem blood in evacuated blood tubes containing sodium fluoride. In some situations, antemortem blood is submitted to a forensic laboratory for ethanol analysis in evacuated blood tubes that contain only an anticoagulant. There has been limited research on ethanol stability in antemortem blood stored without a preservative. On two occasions, antemortem blood was collected from five ethanol-free individuals into 6-ml Vacutainer® tubes containing only 10.8 mg potassium EDTA. The blood tubes were spiked with ethanol to approximately either 0.08 or 0.15 g/dl. Dual-FID headspace gas chromatography was used to analyze 58 blood tubes, 29 from each session, for ethanol 1 day after sample collection and again after 1 year of refrigerated storage (~4°C). Statistically significant decreases in ethanol were detected at the 0.05 level of significance. Mean decreases in ethanol after 1 year of storage for the 0.08 and 0.15 g/dl samples were 0.013 and 0.010 g/dl, respectively. The mean ethanol decrease across all tubes was 0.012 g/dl. The range of decreases for the 58 blood tubes was 0.003–0.018 g/dl. The mean ethanol decreases measured in this unpreserved antemortem blood are comparable in magnitude to those previously observed in antemortem blood containing sodium fluoride after 1 year of refrigerated storage. Ethanol did not increase in the antemortem blood samples despite the absence of sodium fluoride.