Protonitazene detection in two cases of opioid toxicity following the use of tetrahydrocannabinol vape products in Australia.

INTRODUCTION: Protonitazene is an opioid belonging to the 2-benzylbenzimidazole structural class. We describe two cases of opioid toxicity involving the reported inhalation of a delta-9-tetrahydrocannabinol vape product in which protonitazene was detected. CASE REPORTS: Case 1 was a young male found unconscious after the reported use of a delta-9-tetrahydrocannabinol vape. He suffered two subsequent apnoeic episodes requiring bag-valve-mask ventilation before eventual recovery. Only protonitazene was detected in blood at a concentration of 0.74 µg/L. Case 2 was a young male who died shortly after being found unresponsive. The postmortem femoral blood concentrations of protonitazene and delta-9-tetrahydrocannabinol were 0.33 µg/L and 2 µg/L, respectively. Analysis of a pod vaping device found in the decedent's hand and a separate e-liquid bottle labelled as delta-9-tetrahydrocannabinol showed a mixture of protonitazene and delta-9-tetrahydrocannabinol. DISCUSSION: The opioid effects of protonitazene are mediated through ß-arrestin2 and mu opioid receptor signalling pathways. Benzimidazole opioids are lipophilic and, when mixed with a suitable solvent, can be used in a vape device. It is anticipated that naloxone would have provided effective reversal of toxicity in our cases. CONCLUSIONS: Novel routes of opioid administration, like vaping, may appear relatively innocuous in comparison to intravenous administration, but opioids may still be absorbed at high concentrations, resulting in severe opioid toxicity or death.

Characteristics of Overdose Deaths Related to Illicitly Manufactured Fentanyl - Arizona, July 2019 - June 2020.

Using the Arizona State Unintentional Drug Overdose Reporting System (AZ-SUDORS), the study aims to identify the social and drug characteristics of illicitly manufactured fentanyl (IMF)-related overdose deaths. The data include drug overdose deaths from July 1, 2019 to June 30, 2020. Decedents were categorized into four groups by types of opioids detected: (1) IMF-positive; (2) heroin-positive (negative for IMF); (3) pharmaceutical opioid-positive (negative for heroin or IMF); (4) nonopioid. Bivariate statistics were used to compare differences between IMF and other groups. Among 2,029 decedents, 77.8% tested positive for opioids. The IMF group included 57.9%, the heroin group included 9.5%, the pharmaceutical opioid group 10.5%, and the nonopioid group 22.2%. The IMF group was younger (mean age 35.0), more likely to be from a large urban area (78.2%), and with a greater proportion of ethnic/racial minorities (48.6%), compared to the other three groups. The IMF group was less likely to test positive for methamphetamine (24.9%), compared to heroin (63.7%) or pharmaceutical opioid groups (34.0%), but more likely to test positive for cannabis (31.3%), compared to the other three groups. Our data show disproportionate IMF impacts on younger persons and ethnic minorities. Interventions need to be tailored to account for distinct psychosocial profiles associated with IMF use.

Alkoxy chain length governs the potency of 2-benzylbenzimidazole 'nitazene' opioids associated with human overdose.

RATIONALE: Novel synthetic opioids (NSOs) are emerging in recreational drug markets worldwide. In particular, 2-benzylbenzimidazole 'nitazene' compounds are problematic NSOs associated with serious clinical consequences, including fatal respiratory depression. Evidence from in vitro studies shows that alkoxy chain length can influence the potency of nitazenes at the mu-opioid receptor (MOR). However, structure-activity relationships (SARs) of nitazenes for inducing opioid-like effects in animal models are not well understood compared to relevant opioids contributing to the ongoing opioid crisis (e.g., fentanyl). OBJECTIVES: Here, we examined the in vitro and in vivo effects of nitazene analogues with varying alkoxy chain lengths (i.e., metonitazene, etonitazene, isotonitazene, protonitazene, and butonitazene) as compared to reference opioids (i.e., morphine and fentanyl). METHODS AND RESULTS: Nitazene analogues displayed nanomolar affinities for MOR in rat brain membranes and picomolar potencies to activate MOR in transfected cells. All compounds induced opioid-like effects on locomotor activity, hot plate latency, and body temperature in male mice, and alkoxy chain length markedly influenced potency. Etonitazene, with an ethoxy chain, was the most potent analogue in MOR functional assays (EC50 = 30 pM, Emax = 103%) and across all in vivo endpoints (ED50 = 3-12 µg/kg). In vivo SARs revealed that ethoxy, isopropoxy, and propoxy chains engendered higher potencies than fentanyl, whereas methoxy and butoxy analogues were less potent. MOR functional potencies, but not MOR affinities, were positively correlated with in vivo potencies to induce opioid effects. CONCLUSIONS: Overall, our data show that certain nitazene NSOs are more potent than fentanyl as MOR agonists in mice, highlighting concerns regarding the high potential for overdose in humans who are exposed to these compounds.

Evaluation of fentanyl immunoassay test strips for rapid in-situ detection of fentanyl and fentanyl analogs in seized samples and alternative matrices.

BACKGROUND: Ion mobility spectrometry is used for the rapid detection of drugs at points of security but are unable to differentiate some drugs leading to the instrument alarming for a drug not present in the sample. This can be particularly problematic for samples that alarm for fentanyl. In this study, fentanyl immunoassay strips were evaluated for use as a secondary test for fentanyl, including for the testing of alternative matrices, such as powders, e-liquids, and infused papers and textiles. METHODS: The limit of detection of fentanyl immunoassay strips was examined along with their selectivity to 18 fentanyl analogsand 72 other drugs and cutting agents. The effectiveness of the test strips at the detection of fentanyl in the presence of other drugs was examined by testing a series of concentrations of fentanyl in solution in combination with other drugs. The testing of alternative matrices was explored with laboratory prepared samples through sampling with cotton buds and extraction in water. RESULTS: The fentanyl immunoassay strips detected fentanyl at concentrations of 45 ng/mL and reacted with 16 of 18 tested fentanyl analogs with carfentanil and norfentanyl being the only analogs to not react. There was no reactivity with other drugs or cutting agents. The effectiveness of the fentanyl test strips was not reduced when fentanyl was mixed with other drugs. Fentanyl was successfully detected with high sensitivity in all alternative matrices. CONCLUSION: The fentanyl immunoassay strips were found to be an effective secondary test for fentanyl and at least 16 fentanyl analogs in seized drug samples, including when mixed with other drugs. The effectiveness of the sampling methods for alternative matrices should be further evaluated using fentanyl and fentanyl analog casework samples. The use of this method by law enforcement and other agencies should be examined to assess its effectiveness and ease of use in operational settings.

Comparative neuropharmacology of structurally distinct non-fentanyl opioids that are appearing on recreational drug markets worldwide.

BACKGROUND: The emergence of novel synthetic opioids (NSOs) is contributing to the opioid overdose crisis. While fentanyl analogs have historically dominated the NSO market, a shift towards non-fentanyl compounds is now occurring. METHODS: Here, we examined the neuropharmacology of structurally distinct non-fentanyl NSOs, including U-47700, isotonitazene, brorphine, and N-desethyl isotonitazene, as compared to morphine and fentanyl. Compounds were tested in vitro using opioid receptor binding assays in rat brain tissue and by monitoring forskolin-stimulated cAMP accumulation in cells expressing the human mu-opioid receptor (MOR). Compounds were administered subcutaneously to male Sprague-Dawley rats, and hot plate antinociception, catalepsy score, and body temperature changes were measured. RESULTS: Receptor binding results revealed high MOR selectivity for all compounds, with MOR affinities comparable to those of morphine and fentanyl (i.e., nM). All drugs acted as full-efficacy MOR agonists in the cyclic AMP assay, but nitazene analogs had greater functional potencies (i.e., pM) compared to the other drugs (i.e., nM). When administered to rats, all compounds induced opioid-like antinociception, catalepsy, and body temperature changes, but nitazenes were the most potent. Similar to fentanyl, the nitazenes had faster onset and decline of in vivo effects when compared to morphine. In vivo potencies to induce antinociception and catalepsy (i.e., ED50s) correlated with in vitro functional potencies (i.e., EC50s) but not binding affinities (i.e., Kis) at MOR. CONCLUSIONS: Collectively, our findings indicate that non-fentanyl NSOs pose grave danger to those individuals who use opioids. Continued vigilance is needed to identify and characterize synthetic opioids as they emerge in clandestine drug markets.

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.

Detection of AP-237 and synthetic cannabinoids on an infused letter sent to a German prisoner.

In the past years, new psychoactive substances (NPS) started circulating in prisons, leading to health risks and challenges for the criminal justice system. Seizures of papers and cards impregnated with synthetic cannabinoid (SCs) have been reported. In November 2021, a letter suspected to be drug-infused was sent from a German prison to this laboratory. Toxicological analyses were performed by means of gas chromatography-mass spectrometry (GC-MS) for drug screening and liquid chromatography-tandem mass spectrometry (LC-MS/MS) as well as high-performance (HP) LC with diode-array detection (DAD) for semi-quantification of the compounds. The novel synthetic opioid (NSO) AP-237 was detected on the letter, with an estimated concentration of 1.2 µg/cm2 , together with the SCs MDMB-4en-PINACA (77 µg/cm2 ) and 5F-ADB (6.5 µg/cm2 ). To the best of the authors' knowledge, this is the first time an NSO was detected on a drug-infused paper seized in a prison. Highly potent NSOs could easily be dissolved in organic solvents to produce impregnated papers and textiles, and this might represent a serious threat to the health of people in prison. Given the inhomogeneity in drug concentrations, health risks might in particular arise from the consumption of highly concentrated areas of the paper-so-called "hot spots"-especially when highly potent NSOs are used for infusion. Laboratories engaged in analyzing such impregnated papers should be aware of the potential presence of NSOs and adapt the respective methods accordingly.

Toxicological and pharmacological characterization of novel cinnamylpiperazine synthetic opioids in humans and in vitro including 2-methyl AP-237 and AP-238.

The recent scheduling actions of fentanyl-related substances in both the United States and China have sparked the emergence and proliferation of other generations of "legal" opioids that are structurally distinct from fentanyl, including the recently emerged class of cinnamylpiperazines. In contrast to fentanyl, which contains a piperidine core and a phenethyl moiety, the primary structural components of cinnamylpiperazines are the piperazine core and a cinnamyl moiety. This manuscript reports on the toxicological profile for antemortem and postmortem cases where a cinnamylpiperazine was detected. Samples were quantitatively confirmed using liquid chromatography tandem mass spectrometry. The cases were received between February 2020 and April 2021. Concentrations of 2-methyl AP-237 from four postmortem cases ranged from 820 to 5800 ng/mL, and concentrations of AP-238 from two postmortem cases were 87 and 120 ng/mL. µ-Opioid receptor (MOR) activation potential for 2-methyl AP-237, AP-237, para-methyl AP-237, and AP-238 were studied using a ßarr2 recruitment assay. Efficacies (Emax, relative to hydromorphone) and potencies (EC50) were derived and of the compounds tested AP-238 was the most potent compound in the panel with an EC50 of 248 nM. 2-Methyl AP-237 was found to be the most efficacious drug (Emax = 125%) of the tested cinnamylpiperazines; however, it had substantially less efficacy than fentanyl. The in vitro MOR activation potential of the studied cinnamylpiperazines was lower than that of fentanyl and other novel synthetic opioids (NSOs), in line with the relatively higher concentrations observed in postmortem toxicology samples-an important observational link between in vitro pharmacology and in vivo toxicology.

Untargeted Metabolomics in Forensic Toxicology: A New Approach for the Detection of Fentanyl Intake in Urine Samples.

The misuse of fentanyl, and novel synthetic opioids (NSO) in general, has become a public health emergency, especially in the United States. The detection of NSO is often challenged by the limited diagnostic time frame allowed by urine sampling and the wide range of chemically modified analogues, continuously introduced to the recreational drug market. In this study, an untargeted metabolomics approach was developed to obtain a comprehensive "fingerprint" of any anomalous and specific metabolic pattern potentially related to fentanyl exposure. In recent years, in vitro models of drug metabolism have emerged as important tools to overcome the limited access to positive urine samples and uncertainties related to the substances actually taken, the possible combined drug intake, and the ingested dose. In this study, an in vivo experiment was designed by incubating HepG2 cell lines with either fentanyl or common drugs of abuse, creating a cohort of 96 samples. These samples, together with 81 urine samples including negative controls and positive samples obtained from recent users of either fentanyl or "traditional" drugs, were subjected to untargeted analysis using both UHPLC reverse phase and HILIC chromatography combined with QTOF mass spectrometry. Data independent acquisition was performed by SWATH in order to obtain a comprehensive profile of the urinary metabolome. After extensive processing, the resulting datasets were initially subjected to unsupervised exploration by principal component analysis (PCA), yielding clear separation of the fentanyl positive samples with respect to both controls and samples positive to other drugs. The urine datasets were then systematically investigated by supervised classification models based on soft independent modeling by class analogy (SIMCA) algorithms, with the end goal of identifying fentanyl users. A final single-class SIMCA model based on an RP dataset and five PCs yielded 96% sensitivity and 74% specificity. The distinguishable metabolic patterns produced by fentanyl in comparison to other opioids opens up new perspectives in the interpretation of the biological activity of fentanyl.

Quantification of fentanyl analogs in oral fluid using LC-QTOF-MS.

Oral fluid is a valuable alternative matrix for forensic toxicologists due to ease of observed collection, limited biohazardous exposure, and indications of recent drug use. Limited information is available for fentanyl analog prevalence, interpretation, or analysis in oral fluid. With increasing numbers of fentanyl-related driving under the influence of drug (DUID) cases appearing in the United States, the development of detection methods is critical. The purpose of the present study was to develop and validate a quantitative method for fentanyl analogs in oral fluid (collected via Quantisal™) using liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS). Validation resulted in limits of detection and quantification ranging from 0.5 to 1 ng/mL. Established linear range was 1-100 ng/mL for all analytes, except acetyl fentanyl at 0.5-100 ng/mL (R2  > 0.994). Within- and between-run precision and bias were considered acceptable with maximum values of ±15.2%CV and ±14.1%, respectively. Matrix effects exhibited ionization enhancement for all analytes with intensified enhancement at a low concentration (9.3-47.4%). No interferences or carryover was observed. Fentanyl analogs were stable in processed extracts stored in the autosampler (4° C) for 48h. The validated method was used to quantify fentanyl analogs in authentic oral fluid samples (n=17) from probationers/parolees. Fentanyl and 4-ANPP concentrations were 1.0-104.5 ng/mL and 1.2-5.7 ng/mL, respectively.

Availability of Illegal Drugs During the COVID-19 Pandemic in Western Germany.

Background: In response to the COVID-19-pandemic, a lockdown was established in the middle of March 2020 by the German Federal Government resulting in drastic reduction of private and professional traveling in and out of Germany with a reduction of social contacts in public areas. Research Questions: We seek evidence on whether the lockdown has led to a reduced availability of illegal drugs and whether subjects with substance-related problems tried to cope with possible drug availability issues by increasingly obtaining drugs via the internet, replacing their preferred illegal drug with novel psychoactive substances, including new synthetic opioids (NSO), and/or by seeking drug treatment. Methods: A questionnaire was anonymously filled in by subjects with substance-related disorders, typically attending low-threshold settings, drug consumption facilities, and inpatient detoxification wards from a range of locations in the Western part of Germany. Participants had to both identify their main drug of abuse and to answer questions regarding its availability, price, quality, and routes of acquisition. Results: Data were obtained from 362 participants. The most frequent main substances of abuse were cannabis (n = 109), heroin (n = 103), and cocaine (n = 75). A minority of participants reported decreased availability (8.4%), increased price (14.4%), or decreased quality (28.3%) of their main drug. About 81% reported no change in their drug consumption due to the COVID-19 pandemic and the lockdown. A shift to the use of novel psychoactive substances including NSO were reported only by single subjects. Only 1-2% of the participants obtained their main drug via the web. Discussion: Present findings may suggest that recent pandemic-related imposed restrictions may have not been able to substantially influence either acquisition or consumption of drugs within the context of polydrug users (including opiates) attending a range of addiction services in Germany.

Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry and High-Sensitivity Gas Chromatography-Mass Spectrometry Screening of Classic Drugs and New Psychoactive Substances and Metabolites in Urine of Consumers.

The use of the new psychoactive substances is continuously growing and the implementation of accurate and sensible analysis in biological matrices of users is relevant and fundamental for clinical and forensic purposes. Two different analytical technologies, high-sensitivity gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) were used for a screening analysis of classic drugs and new psychoactive substances and their metabolites in urine of formed heroin addicts under methadone maintenance therapy. Sample preparation involved a liquid-liquid extraction. The UHPLC-HRMS method included Accucore™ phenyl Hexyl (100 × 2.1 mm, 2.6 µm, Thermo, USA) column with a gradient mobile phase consisting of mobile phase A (ammonium formate 2 mM in water, 0.1% formic acid) and mobile phase B (ammonium formate 2 mM in methanol/acetonitrile 50:50 (v/v), 0.1% formic acid) and a full-scan data-dependent MS2 (ddMS2) mode for substances identification (mass range 100-1000 m/z). The GC-MS method employed an ultra-Inert Intuvo GC column (HP-5MS UI, 30 m, 250 µm i.d, film thickness 0.25 µm; Agilent Technologies, Santa Clara, CA, USA) and electron-impact (EI) mass spectra were recorded in total ion monitoring mode (scan range 40-550 m/z). Urine samples from 296 patients with a history of opioid use disorder were examined. Around 80 different psychoactive substances and/or metabolites were identified, being methadone and metabolites the most prevalent ones. The possibility to screen for a huge number of psychotropic substances can be useful in suspected drug related fatalities or acute intoxication/exposure occurring in emergency departments and drug addiction services.

A comprehensive analytical process, from NPS threat identification to systematic screening: Method validation and one-year prevalence study.

Several New Psychoactive Substances (NPS) enter the illicit drug market each year. This constant evolution of compounds to screen is challenging to law enforcement and drug chemists, and even more so to forensic toxicologists, who need to detect such compounds which might be at low concentrations in complex biological matrices. While some technological solutions are better suited than others to address such a challenge (e.g., high resolution mass spectrometry), laboratories with limited instrumental and financial resources are faced with a complex task: systematically screening for a rapidly evolving NPS panel using an accredited method run on standard equipment (e.g., liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)). This work presents a solution to this challenge: a complete workflow from the detection of a regional NPS threat to its implementation in a method accredited under the ISO 17025:2017 norm. Initial LC-MS/MS method included 55 NPS and metabolites (31 Novel Synthetic Opioids (NSO), 22 NSO metabolites and 2 designer benzodiazepines). Following their identification as relevant territorial threats, flualprazolam, then isotonitazene, were added to the contingent. By relying on development aiming for maximal integration to the current analysis workflow, systematic NPS screening using this method was easily implemented. Between March 2019 and March 2020, the 5 079 forensic cases analyzed in the province of Québec (Canada) revealed a NPS positivity rate of 3.4%. While 94% involved designer benzodiazepines, 5% involved NSO. This process, combining high efficiency, simple detection technology, ISO accreditation and rapid response to new threats resulted in a four-fold increase in NPS detection.

Novel Opioids: Systematic Web Crawling Within the e-Psychonauts' Scenario.

BACKGROUND: A wide range of novel psychoactive substances (NPSs) are regularly searched and discussed online by e-psychonauts. Among NPSs, the range of prescription/non-prescription opioids (fentanyl and non-fentanyl analogs) and herbal derivatives currently represents a challenge for governments and clinicians. METHODS: Using a web crawler (i.e., NPS.Finder®), the present study aimed at assessing psychonaut fora/platforms to better understand the online situation regarding opioids. RESULTS: The open-web crawling/navigating software identified some 426 opioids, including 234 fentanyl analogs. Of these, 176 substances (162 were very potent fentanyls, including two ohmefentanyl and seven carfentanyl analogs) were not listed in either international or European NPS databases. CONCLUSION: A web crawling approach helped in identifying a large number, indeed higher than that listed by European/international agencies, of unknown opioids likely to possess a significant misuse potential. Most of these novel/emerging substances are still relatively unknown. This is a reason of concern; each of these analogs potentially presents with different toxicodynamic profiles, and there is a lack of docking, preclinical, and clinical observations. Strengthening multidisciplinary collaboration between clinicians and bioinformatics may prove useful in better assessing public health risks associated with opioids.

Long-term stability of novel synthetic opioids in blood.

Recently, there has been an increase in overdose deaths due to novel synthetic opioids (NSO). Due to backlogs experienced by many forensic laboratories, it is important to understand drug stability in a variety of storage conditions. The objective of this study was to investigate the stability of AH-7921, U-47700, U-49900, U-50488, MT-45, W-15, and W-18 in blood at various temperatures over a 36-week period. NSO were generally stable over the 36-week period (66%-118%) at low and high concentrations when blood samples were stored in the refrigerator or freezer. Most analytes were stable for at least 2 weeks at room temperature (77%-120%). At the elevated temperature (35°C), analytes were generally stable for at least 14 days (75%-109%). This study has determined the stability of several NSO at various temperatures over a 36-week period. These results reflect the forensic significance of keeping samples stored at proper temperatures. Blood samples suspected to contain synthetic opioids should be stored refrigerated or frozen, when possible, in order to preserve analyte stability, especially at low concentrations.

Investigation of Fragmentation Pathways of Fentanyl Analogues and Novel Synthetic Opioids by Electron Ionization High-Resolution Mass Spectrometry and Electrospray Ionization High-Resolution Tandem Mass Spectrometry.

The global drug market is characterized by the fast development of new psychoactive substances such as fentanyl analogues and novel synthetic opioids, the detection of which is complicated by the lack of appropriate quality control procedures and references. Herein, we analyze the fragmentation pathways and characteristic ions of 25 novel fentanyl analogues and 5 novel synthetic opioids by electron ionization (EI) and electrospray ionization (ESI) high-resolution mass spectrometry to provide a reference for the identification of these species. In the ESI mode, fentanyl analogues mainly undergo piperidine ring degradation, phenethyl and piperidine ring dissociation, and piperidine ring and amide moiety cleavage, while piperidine ring degradation and phenethyl and piperidine ring dissociation are the major pathways in the EI mode. The five novel synthetic opioids largely undergo amide group dissociation and N-cyclohexyl bond cleavage in the ESI mode. Thus, this work facilitates the detection and quantitation of fentanyl analogues and novel synthetic opioids or other substances with similar structures in forensic laboratories.

Challenges Related to Three Cases of Fatal Intoxication to Multiple Novel Synthetic Opioids.

In the last two decades, a large increase in opioid overdose death rates has been recorded in North America. This phenomenon, related to the misuse of prescription opioids, has been dubbed an "opioids crisis". Recent years have seen the entrance of novel synthetic opioids (NSO) on the market, compounding the fatal intoxications issue. This brings several challenges for forensic toxicology laboratories: an increased number of cases, a large number of novel structurally similar compounds to include in screening analytical methods, the low concentration of drugs in biological fluids, and the challenging interpretation in the absence of sufficient literature. Three cases of fatal intoxication highlighting those challenges are presented, complete with post-mortem concentrations in cardiac blood, femoral blood and urine. Toxicological screening and quantitative analyses were performed on the biological specimens. In the first and second cases, furanylfentanyl, U-47700 and 4-anilino-N-phenethylpiperidine (4-ANPP) were detected at similar concentrations in cardiac blood. In the third case, a total of seventeen different NSO were detected. All intoxications showed a combination of NSO and other drugs. These three cases appear to be the harbinger of an increased NSO prevalence in the province of Québec, Canada.

Application of a validated UHPLC-MS/MS method for 28 fentanyl-analogue and novel synthetic opioids in whole blood in authentic forensic cases.

The abuse of fentanyl and its analogues, which are potent, short-acting, synthetic narcotic analgesics, has become a major issue. Many cases containing fentanyl-analogue and novel synthetic opioids have also been reported. Hence, we report the determination of fentanyl, fentanyl-analogue and novel synthetic opioids in whole blood by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method is characterized by the use of a simple, fast and inexpensive liquid-liquid extraction (LLE) for sample preparation, a rapid run time (8 min) and a low required volume of whole blood (100 µl). The limits of detection (LODs) ranged from 0.005 to 0.03 ng/ml, and the lower limits of quantitation (LLOQs) ranged from 0.05 to 0.2 ng/ml. The method was shown to be liner over a concentration range of 0.2-40 ng/ml for fentanyl, norfentanyl and norcarfentanil and 0.05-40 ng/ml for all other target analytes. Recoveries were within the range of 72.09%-103.22%, and the matrix effects were in the 67.95%-113.32% range. Moreover, the method was applied to the detection and quantification of fentanyl and its analogues in whole blood from real forensic cases. This methodology has great potential for use in the determination of fentanyl and its analogues in forensic cases.

In vitro metabolism of the novel synthetic opioid agonist cyclopropylfentanyl and subsequent confirmation in authentic human samples using liquid chromatography-high resolution mass spectrometry.

Novel synthetic opioids (NSOs) are a class of novel psychoactive substances (NPS) that are growing in popularity and presenting a significant public health risk. Included in this class are derivatives of the highly potent analgesic, fentanyl. Cyclopropylfentanyl (CycP-F) was first reported to the EU Early Warning System in August 2017, and was subsequently linked to more than 100 deaths in the US alone. Limited pharmacological, pharmacokinetic or toxicological data is available for many emerging NSOs; however we can expect novel fentanyl analogues to present limited detection windows, short onset, narrow therapeutic indices and the potential for very high potency. Knowledge of the metabolism of these drugs is essential for the identification of analytical targets for their detection. Therefore in vitro metabolites of CycP-F were produced using human liver microsomal incubations. Metabolites formed were elucidated using liquid chromatography-high resolution accurate mass analysis (LC-HRAM). Identified metabolites were added to our accurate mass screening database for NPS which was utilised for subsequent screening analysis. CycP-F and metabolites were identified in two human blood case samples. Eleven metabolites were identified in vitro, with the major metabolites produced via N-dealkylation, monohydroxylation and N-oxidation. Analysis of the positive case samples identified four in vivo metabolites, all of which were observed in vitro. The major metabolite identified in vitro and in vivo was the N-dealkylated nor-metabolite; two further mono-hydroxylated and one dihydroxylated metabolite were detected in vivo.

Street fentanyl use: Experiences, preferences, and concordance between self-reports and urine toxicology.

BACKGROUND: Conducted in Dayton, Ohio, the study aims to characterize user knowledge and experiences with non-pharmaceutical fentanyl-type drugs (NPFs) and compare self-reports with urine toxicology for NPFs and heroin. METHODS: Between May 2017-January 2018, 60 individuals who self-reported heroin/NPF use were interviewed using structured questionnaire on socio-demographics, NPF and other drug use practices. Unobserved urine samples were collected and analyzed using: 1) liquid-chromatography-tandem mass spectrometry (LC-MS/MS)-based method (Toxicology lab) to identify 34 fentanyl analogues, metabolites, and other synthetic opioids; 2) immunoassay-based method to screen for opiates (heroin). Sensitivity, specificity and Cohen's kappa were calculated to assess agreement between self-reports and urine toxicology. RESULTS: The sample was 52% female, and over 90% white. Almost 60% reported preference for heroin, and 40% for NPF. Participants endorsed a number of ways of distinguishing heroin from NPF, including appearance (88.3%), effects (76.7%), taste (55%), and information provided by dealers (53.3%). Almost 80% felt confident they could distinguish heroin from NPF, but knowledge about fentanyl analogues was limited. LC-MS/MS testing identified 8 types of NPFs. Over 88% tested positive for NPFs, including 86% fentanyl, 48% carfentanil, 42% acetyl fentanyl. About 47% screened positive for opiates/heroin, and all of them were also positive for NPFs. When comparing self-reported use of NPF to urine toxicology, sensitivity and specificity were relatively high (84% and 83.3%, accordingly), while Cohen's Kappa was 0.445, indicating fair agreement. Sensitivity and specificity were lower for heroin (77.8% and 50.0%, accordingly), and Cohen's Kappa was 0.296, indicating low agreement between self-reports of heroin use and urine toxicology. DISCUSSION: Nearly 90% of the study participants tested positive for NPF-type drugs. Participants were more likely to over-report heroin use and underreport NPF use. The majority had little knowledge about fentanyl analogues. Study findings will inform development of novel harm reduction approaches to reduce overdose mortality.