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.     

Occupational exposure to traumatic evidence and posttraumatic stress symptoms in forensic science professionals: Prevalence and patterns

Forensic science professionals are routinely exposed to potentially traumatizing evidence. This study aimed to estimate the prevalence of occupational posttraumatic stress disorder (PTSD) among forensic science professionals, identify job-related correlates of PTSD symptoms, and examine the role of social support in mitigating PTSD symptomology. In response to recruitment through the American Academy of Forensic Sciences, the American Society of Crime Laboratory Directors, and Evidence Technology Magazine, 449 forensic science professionals participated in the current study. Results found that 73.5% (n = 330) of the overall sample experienced at least one work-related traumatic event consistent with meeting Criterion A for PTSD, and these rates were higher for field-based respondents (n = 203, 87.9%). The prevalence of past-month provisional PTSD was 21.6% for the full sample. Disaggregated PTSD rates were 29.0% and 14.5% for field-based and non-field-based respondents, respectively. These rates were 6- to 8-fold higher than the past-year prevalence of PTSD in the general US population, estimated to be 3.5%, and were found to be at least as high as those observed in prior epidemiological research with non-treatment seeking members of the US military deployed to war zones in Iraq and Afghanistan. Results further showed that social support was protective against PTSD symptomology. The high rates of occupational exposure to trauma and consequent PTSD symptomology observed in this large sample of forensic science professionals highlight the under-acknowledged psychological risks of these occupations and the need for enhanced attention to mental health resources for these professionals.     

Geoforensic search to crime scene: Remote sensing,geophysics, and dogs

In the absence of surface indications of burial sites, law enforcement or humanitarian organizations are faced with the difficult task of focusing large-scale ground searches to a manageable excavation area. A geoforensic-based survey may exclude parts of the landscape for reasons such as diggability or viewshed analysis but leave areas still too large for invasive exploration. This work examines how drone-based remote sensing, geophysics, and search dogs may be combined to narrow such searches. Here, we ask the reader to consider two examples where forensic geomorphology and land use provided a range of possible burial locations. Following this is a multi-proxy approach to similar dilemma, with a search-to-scene case study using remote sensing (drone photography), geophysics, ground probes, and search dogs. This approach is not presented as a definitive guide, but serves as an example of the conjunctive use of well-studied methods to approach a common problem in geoforensics.     

Fatal intoxication with 1,4-butanediol: Case report and comprehensive review of the literature

A fatal case of 1,4-butanediol (1,4-BD) oral ingestion is reported here, in which a 51-year-old man was found dead in his bed. According to the police report, the deceased was a known drug user. A glass bottle labeled (and later confirmed to be) “Butandiol 1,4” (1,4-BD) was found in the kitchen. Furthermore, the deceased's friend stated that he consumed 1,4-BD on a regular basis. The autopsy and histological examination of postmortem parenchymatous organ specimens did not revealed a clear cause of death. Chemical-toxicological investigations revealed gammahydroxybutyrat (GHB) in body fluids and tissues in the following quantities: femoral blood 390 mg/L, heart blood 420 mg/L, cerebrospinal fluid 420 mg/L, vitreous humor 640 mg/L, urine 1600 mg/L, and head hair 26.7 ng/mg. In addition, 1,4-BD was qualitatively detected in the head hair, urine, stomach contents, and the bottle. No other substances, including alcohol, were detected at pharmacologically relevant concentrations. 1,4-BD is known as precursor substance that is converted in vivo into GHB. In the synoptic assessment of toxicological findings, the police investigations and having excluded other causes of death, a lethal GHB-intoxication following ingestion of 1,4-BD, can be assumed in this case. Fatal intoxications with 1,4-BD have seldom been reported due to a very rapid conversion to GHB and, among other things, non-specific symptoms after ingestion. This case report aims to give an overview to the published of fatal 1,4-BD-intoxications and to discuss the problems associated with detection of 1,4-BD in (postmortem) specimens.     

GC–MS analysis of eight aminoindanes using three derivatization reagents

Aminoindanes are a class of novel psychoactive substances (NPSs) that have become more prevalent over the past decade. GC–MS is often utilized for identifying seized drugs and is well regarded for its ability to separate mixtures. However, certain aminoindanes have similar mass spectral data and require specific gas chromatographic stationary phases for separation. Derivatization is an alternative method that can be applied to GC–MS to enhance chromatographic results, providing more selective analysis in seized-drug identification. This study investigates derivatization techniques to provide options for forensic science laboratories in accurately identifying aminoindanes. Three derivatization reagents, N-methyl-bis(trifluoroacetamide) (MBTFA), heptafluorobutyric anhydride (HFBA), and ethyl chloroformate (ECF) were evaluated for the analysis of eight aminoindanes by GC–MS using two common gas chromatographic stationary phases, Rxi®-5Sil MS and Rxi®-1Sil MS. All three derivatization methods successfully isolated eight aminoindanes, including the isomers 4,5-methylenedioxy-2-aminoindane (4,5-MDAI), and 5,6-methylenedioxy-2-aminoindane (5,6-MDAI) that could not be differentiated prior to derivatization. Reduced peak tailing and increased abundance were observed after derivatization for all the compounds, and mass spectra of the derivatives contained individualizing fragment ions that allowed for further characterization of the aminoindanes. This excluded 4,5-MDAI and 5,6-MDAI as they shared the same characteristic ions and were only distinguishable by their retention times. All three derivatization techniques used in this study allow for successful characterization of the aminoindanes and give forensic science laboratories flexibility in their analysis approach when they encounter these compounds.     

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.     

Development and validation of a LC–MS/MS method for analysis of vortioxetine in postmortem specimens. First data from an authentic case

Vortioxetine is an antidepressant recently licensed in USA and EU for the treatment of major depressive disorder. Neither fatal case due to overdose nor data about postmortem concentrations on blood or other specimens have been reported. The aims of this study were the development and validation of a method for vortioxetine analysis by Liquid Chromatography Tandem Mass Spectrometry (LC–MS/MS) in postmortem samples and its application in an authentic case. The method was validated and applied on blood, vitreous humor, bile, brain, liver, kidney, and gastric content. After protein precipitation, the supernatant was directly injected into LC–MS/MS. Analysis was carried out by Multiple Reaction Monitoring (MRM) mode. The authentic case concerned a 38 years-old woman, affected by depression, who was found hanged at home. The method determined an acceptable sensitivity, selectivity, linearity, precision, and accuracy for all matrices. No interference was shown for all matrices. The matrices do not significantly reduce the peak intensity of vortioxetine. No carryover was shown. Toxicological analysis of the authentic case showed vortioxetine in blood (234 ng/ml), vitreous humor (10.5 ng/ml), brain (490 ng/g), lung (479 ng/g), liver (3751 ng/g), kidney (798 ng/g), bile (2267 ng/ml) and gastric content (253 ng/ml). Our case suggests that even at blood concentrations of vortioxetine equal to 234 ng/ml, the subject was able to stage and carry out the hanging. Vortioxetine concentrations found in the other cadaveric samples (biological fluids, organs, and gastric content) may be helpful to evaluate further similar comparable cases.     

Developing a new scoring method to evaluate human decomposition in a humid,continental (Dfb) climate in Quebec

The published literature shows a lack of methods to evaluate the patterns and extent of decomposition of human remains and to estimate the post-mortem interval (PMI) in humid, continental (Dfb) climates such as Quebec. The aim of this study was to address this gap in the current knowledge base by providing the first observations from human corpses studied under controlled conditions in Quebec. A 12-month study was conducted at the site for Research in Experimental and Social Thanatology; the first human taphonomy facility in Canada. Six human donors with known time of death were deposited across spring (n = 1), summer (n = 3), and autumn (n = 2) 2021. The lack of suitability of the total body score method to evaluate the extent of decomposition at the facility prompted the development of a new scoring system based on the macromorphoscopic changes observed. The scoring system was applied to the donors to evaluate decomposition throughout seasons. All donors followed comparable decomposition trajectories, regardless of the season of deposition. Eighty-five percent of taphonomic patterns appeared in the first 25 experimental days or 5000 Kelvin accumulated degree days (350 ADD). Extensive desiccation of tissues was observed at a median of 21 experimental days across donors, resulting in a plateau within decomposition with no extensive skeletonization. To the authors' knowledge, this is the first published report of experimentally observed desiccation in such a form in a Dfb climate. This study provides new data on the types of decomposition patterns to expect in forensic investigations in southern Quebec and comparable climates.