Review of analytical methods for screening and quantification of fentanyl analogs and novel synthetic opioids in biological specimens

Fentanyl, fentanyl analogs, and other novel synthetic opioids (NSO), including nitazene analogs, prevail in forensic toxicology casework. Analytical methods for identifying these drugs in biological specimens need to be robust, sensitive, and specific. Isomers, new analogs, and slight differences in structural modifications necessitate the use of high-resolution mass spectrometry (HRMS), especially as a non-targeted screening method designed to detect newly emerging drugs. Traditional forensic toxicology workflows, such as immunoassay and gas chromatography mass spectrometry (GC–MS), are generally not sensitive enough for detection of NSOs due to observed low (sub-μg/L) concentrations. For this review, the authors tabulated, reviewed, and summarized analytical methods from 2010–2022 for screening and quantification of fentanyl analogs and other NSOs in biological specimens using a variety of different instruments and sample preparation approaches. Limits of detection or quantification for 105 methods were included and compared to published standards and guidelines for suggested scope and sensitivity in forensic toxicology casework. Methods were summarized by instrument for screening and quantitative methods for fentanyl analogs and for nitazenes and other NSO. Toxicological testing for fentanyl analogs and NSOs is increasingly and most commonly being conducted using a variety of liquid chromatography mass spectrometry (LC–MS)-based techniques. Most of the recent analytical methods reviewed exhibited limits of detection well below 1 μg/L to detect low concentrations of increasingly potent drugs. In addition, it was observed that most newly developed methods are now using smaller sample volumes which is achievable due to the sensitivity increase gained by new technology and new instrumentation.     

Exploiting the post-attendee URL feature in Zoom webinar to distribute malware

The post-attendee Uniform Resource Locator (URL) feature within the video conferencing application known as Zoom is often overlooked by digital forensic experts as a potential risk for malware transmission. However, with the ability to redirect webinar participants to any URL set by the host for the webinar, the post-attendee URL can be abused by bad actors to expose webinar participants to malicious websites or, in the worst-case scenario, force participants to download a file through the use of a direct download link URL. This study aims to showcase how this exploit can be replicated by creating an experimental environment involving four Windows 10 desktops running Zoom version 5.7.5 and creating a webinar with four user accounts acting as webinar participants and setting the post-attendee URL value to the URL of a website that contained a keylogger. In another trial, the same experimental environment was utilized, with the only difference being the post-attendee URL that was set to redirect webinar participants to a download link for a .jpg file. In both instances, every user account that joined the webinar via clicking on the invitation link that was emailed to each user account after registering for the webinar was redirected to the post-attendee URL regardless of their user account role. These results not only prove that the post-attendee URL can be exploited, but also provide insight as to how this type of attack can be prevented.     

Linking ammonium nitrate-aluminum (AN-AL) post-blast residues to pre-blast explosive materials using isotope ratio and trace elemental analysis for source attribution

Forensic science practitioners are often called upon to attribute crimes using trace evidence, such as explosive remnants, with the ultimate goal of associating a crime with a suspect or suspects in order to prevent further attacks. The explosive charge is an attractive component for attribution in crimes involving explosives as there are limited pathways for acquisition. However, there is currently no capability to link an explosive charge to its source via post-blast trace residues using isotope ratios or trace elements. Here, we sought to determine if pre-blast attribution signatures are preserved after detonation and can be subsequently recovered and detected. A field study was conducted to recover samples of post-blast explosives from controlled detonations of ammonium nitrate-aluminum (AN-Al), which were then analyzed via isotope ratio mass spectrometry (IRMS) and inductively coupled plasma-mass spectrometry (ICP-MS) for quantitation and profiling of isotopes ratio and trace element signatures, respectively. Oxygen and nitrogen isotope ratios from AN-Al yielded some of the most promising results with considerable overlap within one standard deviation of the reference between the spreads of pre- and post-blast data. Trace element results from AN-Al support the findings in the isotope ratio data, with 26 elements detected in both pre- and post-blast samples, and several elements including B, Cd, Cr, Ni, Sn, V, and Zn showing considerable overlap. These preliminary results provide a proof-of-concept for the development of forensic examinations that can attribute signatures from post-blast debris to signatures in pre-blast explosive materials for use in future investigations.     

An interlaboratory study to evaluate the utility of gas chromatography-mass spectrometry and gas chromatography-infrared spectroscopy spectral libraries in the forensic analysis of fentanyl-related substances

Synthetic opioids such as fentanyl account for over 71,000 of the approximately 107,000 overdose deaths reported in the United States in 2021. Fentanyl remains the fourth most identified drug by state and local forensic laboratories, and the second most identified drug by federal laboratories. The unambiguous identification of fentanyl-related substances (FRS) is challenging due to the absence or low abundance of a molecular ion in a typical gas chromatography-mass spectrometry (GC-MS) analysis and due to a low number of fragment ions that are similar among the many potential isomers of FRS. This study describes the utility of a previously reported gas chromatography-infrared (GC-IR) library for the identification of FRS within a blind, interlaboratory study (ILS) involving seven forensic laboratories. Twenty FRS reference materials, including those with isomer pairs in the library, were selected based on either their presence in the NIST library and/or some similarity of the mass spectra information produced. The ILS participants were requested to use the Florida International University (FIU) GC-MS and GC-IR libraries supplied by FIU to search for matches to their unknown spectra generated from in-house GC-MS and GC-IR analysis. The laboratories reported improvement in the positive identification of unknown FRS from ~75% using GC-MS alone to 100% correct identification using GC-IR analysis. One laboratory participant used solid phase IR analysis, which produced spectra incompatible with the vapor phase GC-IR library to generate a good comparison spectrum. However, this improved when searched against a solid phase IR library.     

Nail analysis for the assessment of caffeine abuse in Northwest China: A population-based study

Caffeine is the most widely consumed psychoactive agent worldwide and has the potential for abuse, but studies monitoring caffeine abuse in China are scarce. This study aims to estimate the prevalence of caffeine abuse in northwest China and investigate the correlation between caffeine and other drugs in hair and nails using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Fingernail clippings were collected from 376 participants in northwest China to detect caffeine and 13 other illicit psychoactive drugs and their metabolites. Paired hair and nail samples were collected from 39 participants to investigate the correlation between caffeine and other drugs in hair and nails. The samples were decontaminated, pulverized, and extracted by a high-throughput nail sample preparation method and analyzed by UPLC-MS/MS. The results showed a risk of caffeine abuse in northwest China, with concentrations ranging from 0.43 to 10.6 ng/mg for healthy volunteers, 0.49–246 ng/mg for caffeine abusers, and 0.25–363 ng/mg for drug addicts in community rehabilitation centers. Caffeine was detected together with other illicit psychoactive drugs and their metabolites. Furthermore, positive detection correlations were found between hair and nail samples. This study provides a current perspective on caffeine abuse in northwest China and demonstrates the practical use of UPLC-MS/MS for the simultaneous detection of caffeine and 13 illicit psychoactive drugs and their metabolites in hair and nails. The results highlight the potential of nails as a supplementary matrix when hair samples are unavailable and emphasize the need for handling caffeine carefully given its potential for abuse.     

Sampling and recovery of ignitable liquid residues (ILRs) from fire debris using capillary microextraction of volatiles (CMV) for on-site analysis

A new, fast, and ultra-sensitive headspace sampling method using the Capillary Microextraction of Volatiles (CMV) device is demonstrated for the analysis of ignitable liquid residues (ILRs) in fire debris. This headspace sampling method involves the use of a heated can (60°C) to aid in the recovery of volatile organic compounds (VOCs) from medium and heavy petroleum distillates. Our group has previously reported the utility of CMV to extract gasoline at ambient temperature in less than 5 min in the field. This work evaluates the recovery and analysis of low mass loadings (tens of ng) of VOCs from charcoal lighter fluid, kerosene, and diesel fuel. Nonane, decane, undecane, tridecane, tetradecane, and pentadecane were selected for evaluation of recovery to represent these ILR classes. The face-down heated can headspace sampling technique was compared to the previously reported, non-heated, paper cup headspace sampling technique. Mass recovery improvements of 50%–200% for five of the six target compounds in diesel fuel were achieved compared to the non-heated sampling method. The average relative standard deviation (reported as % RSD) between the replicate trials decreased from an average of 28% to 6% when using the heated can method. Ignitable liquids were spiked onto burned debris in a live burn exercise and sampled using the heated can and paper cup headspace sampling techniques. The heated sampling technique reported here, for the first time, demonstrates an effective extraction method that when coupled to a portable GC–MS instrument allows for a sampling and analysis protocol in the field in less than 30 min.     

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.     

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.     

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.