Novel Synthetic Opioids Research Articles

Novel Synthetic Opioids (NSO) Use in Opioid Dependents Entering Detoxification Treatment.

IntroductionOver the last decade, the use of New/Novel Synthetic Opioids (NSO) has emerged as an increasing problem, and especially so in the USA. However, only little is known about the prevalence and history of NSO use in European heroin dependents.MethodA cross-sectional multicenter study, carried out with the means of both standardized interviews and urine toxicology enhanced screening, in a sample of opioid addicted patients referred for an in-patient detoxification treatment.ResultsSample size included here n = 256 patients; prior to admission, 63.7% were prescribed with an opioid maintenance treatment. Lifetime use of heroin and opioid analgesics was reported by 99.2 and 30.4%, respectively. Lifetime NSO/fentanyl use was reported by 8.7% (n = 22); a regular use was reported by 1.6% (n = 4), and ingestion over the 30 days prior to admission by 0.8% (n = 2). Most typically, patients had started with a regular consumption of heroin, followed by maintenance opioids; opioid analgesics; and by NSO. Self-reported data were corroborated by the toxicology screenings carried out; no evidence was here identified for the presence of heroin being contaminated by fentanyl/derivatives.DiscussionNSO and also opioid analgesics did not play a relevant role in the development and the course of opioid/opioid use disorders in German patients referred for an inpatient detoxification treatment.

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 5800ng/mL, and concentrations of AP-238 from two postmortem cases were 87 and 120ng/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 βarr2 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 248nM. 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.

Characteristics of fatal ‘novel’ synthetic opioid toxicity in Australia

BackgroundAims: To determine 1. The characteristics of all recorded cases of fatal drug poisoning involving novel synthetic opioids (NSOs) in Australia; 2. The toxicology of cases; and 3. The major autopsy findings. MethodsReview of all fatal poisonings related to NSOs in Australia 2000–2021 identified in the National Coronial Information System. ResultsThirty-one cases were identified, 96.8% due to unintentional drug toxicity. The mean age was 31.9 years and 87.1% were male. Only six were aged over forty. A history of substance use problems was documented in 80.6% and 58.1% had a history of injecting drug use. In 32.3% the final route of administration of a NSO was by non-injecting routes of administration. Ten NSOs were identified. Fentanyl analogues were present in 67.2%, most commonly furanylfenatyl (19.4%). Other NSO types were present in 39.7%, most commonly U-47700 (35.5%). Substances other than NSOs were present in 90.3%, most commonly benzodiazepines (67.7%) and other opioids (51.6%). A CNS depressant in addition to NSOs was present in 90.3%, and a new psychoactive substance other than a NSO in 25.8%. Pulmonary oedema was diagnosed in 82.6%, aspiration of vomitus in 30.4%, and acute bronchopneumonia in 17.4%. ConclusionsTen NSOs were identified. Case characteristics suggest a younger cohort whose profile is more typical of use of other NPS than of the established opioids. A large proportion used NSOs by non-injecting routes of administration.

A comprehensive evaluation of the potential binding poses of fentanyl and its analogs at the µ-opioid receptor

Fentanyl and its analogs are selective agonists of the µ-opioid receptor (MOR). Among novel synthetic opioids (NSOs), they dominate the recreational drug market and are the main culprits for the opioid crisis, which has been exacerbated by the COVID-19 pandemic. By taking advantage of the crystal structures of the MOR, several groups have investigated the binding mechanism of fentanyl, but have not reached a consensus, in terms of both the binding orientation and the fentanyl conformation. Thus, the binding mechanism of fentanyl at the MOR remains an unsolved and challenging question. Here, we carried out a systematic computational study to investigate the preferred fentanyl conformations, and how these conformations are being accommodated in the MOR binding pocket. We characterized the free energy landscape of fentanyl conformations with metadynamics simulations, and compared and evaluated several possible fentanyl binding conditions in the MOR with long-timescale molecular dynamics simulations. Our results indicate that the most preferred binding pose in the MOR binding pocket corresponds well with the global minimum on the energy landscape of fentanyl in the absence of the receptor, while the energy landscape can be reconfigured by modifying the fentanyl scaffold. The interactions with the receptor may stabilize a slightly unfavored fentanyl conformation in an alternative binding pose. By extending similar investigations to fentanyl analogs, our findings establish a structure–activity relationship of fentanyl binding at the MOR. In addition to providing a structural basis to understand the potential toxicity of the emerging NSOs, such insights will contribute to developing new, safer analgesics.

Pharmacological and metabolic characterization of the novel synthetic opioid brorphine and its detection in routine casework

After their first emergence in 2009, Novel synthetic opioids (NSO) have become an emerging class of New Psychoactive Substances (NPS) on the market for these new drugs. So far, 67 NSO have been reported to the Early Warning system of the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). It is presumed that NSO mainly target the four known opioid receptors, i.e. the μ-opioid (MOR), the δ-opioid (DOR), the κ-opioid (KOR) and nociceptin receptors and that their consumption can result in serious adverse effects such as massive respiratory depression or death. In the present study we investigated the in vivo and in vitro metabolism of brorphine, a NSO that was first identified on the NPS market in August 2019 in the United States, using both a pooled human liver microsome assay and real forensic case samples. For the detection of metabolites LC-HR-MS/MS was used and quantification of brorphine was performed using an LC-MS/MS method. Additionally, we pharmacologically characterized brorphine regarding its activation of the MOR and KOR via G protein recruitment using the [35S]-GTPγS assay. In forensic urine samples, 14 distinct metabolites were identified, whereas in blood only four metabolites could be found. The pooled human liver microsome assay generated six distinct in vitro phase I metabolites. The most prominent in vivo metabolite was formed by N-oxydation, whereas the main in vitro metabolite was formed by hydroxylation. The pharmacological characterization at the MOR and KOR revealed brorphine to be a potent MOR agonist and a weak, partial KOR agonist in the [35S]-GTPγS assay.

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.

PNS102 Knowledge about NOVEL Opioids and Nalaxone Use Attitudes Among Pharmd Students

Since 2010, the synthetic opioid related overdose deaths have increased. Educating PharmD students who will become licensed pharmacists in the future can aid in reducing synthetic opioids misuse and fighting the opioid crisis in the United States (US). This study assessed the difference in knowledge about novel synthetic opioids (NSOs) and Kratom and difference in attitudes about naloxone use among PharmD students upon delivering an educational intervention in a public pharmacy school in Huntington, West Virginia.

Detection of two potent synthetic opioids carfentanil and 3-methylfentanyl in forensic investigations during a four-year period 2017‒2020

There has been an alarming increase of novel synthetic opioids (NSOs) in the illegal drug market since 2016. These substances are still detected in forensic case investigations in biological samples as well as in seized materials. Although NSOs are steadily emerging, other potent “old” synthetic opioids are still circulating and involved in fatal overdose cases. This is the case of the potent designer drugs carfentanil and 3-methylfentanyl. These drugs were associated with several deaths since at least 20 years ago, and they are still a public health threat.In this study, we presented postmortem (PM) forensic cases that were positive for carfentanil or 3-methylfentanyl over a four-year period 2017–2020. Quantitative confirmation was via a quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method that targets 21 designer opioids, able to differentiate isomers such as cis- and trans-3-methylfentanyl, with a limit of quantification of 0.05 ng/mL.A total of 1136 and 153 PM cases were received in the laboratory from different regions of the United States positive for carfentanil and 3-methylfentanyl, respectively. PM peripheral blood ranged from 0.050‒32 ng/mL for carfentanil, and 0.057‒7.2 and 0.057‒2.2 ng/mL for cis- and trans-3-methylfentanil, respectively. During this period, fentanyl was the most common concomitant drug detected within these cases. Heroin and cocaine were also highly prevalent in those cases positive for carfentanil. Other NSOs such as 2-furanylfentanyl, cyclopropylfentanyl and para-fluorobutyrylfentanyl were also significant findings in positive carfentanil cases. For 3-methylfentanyl, cocaine was the second most prevalent co-occurrence drug, while heroin decreased over the years in those cases where 3-methylfentanyl was confirmed.

Use and knowledge of novel synthetic opioids: An Italian survey

The recreational use of novel synthetic opioids (NSOs) is increasing, especially among the youngest part of the population. We conducted a naturalistic observational survey. The main outcome was to assess the use and the knowledge of NSOs among the youngest population in Italy. A sociodemographic questionnaire was also administrated. Nine hundred twenty questionnaires were completed. Fentanyl was the most used NSO (1.63% of the subjects). In all, 14.3% of the sample revealed awareness of NSOs. Although the diffusion in Italy is small, the large knowledge of NSOs requires future monitoring of this phenomenon.

U-47700 and Its Analogs: Non-Fentanyl Synthetic Opioids Impacting the Recreational Drug Market.

The recreational use of opioid drugs is a global threat to public health and safety. In particular, an epidemic of opioid overdose fatalities is being driven by illicitly manufactured fentanyl, while novel synthetic opioids (NSOs) are appearing on recreational drug markets as standalone products, adulterants in heroin, or ingredients in counterfeit drug preparations. Trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide (U-47700) is a prime example of a non-fentanyl NSO that is associated with numerous intoxications and fatalities. Here, we review the medicinal chemistry, preclinical pharmacology, clandestine availability, methods for detection, and forensic toxicology of U-47700 and its analogs. An up-to-date summary of the human cases involving U-47700 intoxication and death are described. The evidence demonstrates that U-47700 is a potent μ-opioid receptor agonist, which poses a serious risk for overdosing and death. However, most analogs of U-47700 appear to be less potent and have been detected infrequently in forensic specimens. U-47700 represents a classic example of how chemical entities from the medicinal chemistry or patent literature can be diverted for use in recreational drug markets. Lessons learned from the experiences with U-47700 can inform scientists, clinicians, and policymakers who are involved with responding to the spread and impact of NSOs.

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.

In vitro and in vivo pharmacological characterization of the synthetic opioid MT-45

MT-45 is a synthetic opioid that was developed in the 1970s as an analgesic compound. However, in recent years MT-45 has been associated with multiple deaths in Europe and has been included in the class of novel psychoactive substances known as novel synthetic opioids (NSOs). Little is known about the pharmaco-toxicological effects of MT-45. Therefore, we used a dynamic mass redistribution (DMR) assay to investigate the pharmacodynamic profile of this NSO in vitro compared with morphine. We then used in vivo studies to investigate the effect of the acute systemic administration of MT-45 (0.01–15 mg/kg i.p.) on motor and sensorimotor (visual, acoustic and tactile) responses, mechanical and thermal analgesia, muscle strength and body temperature in CD-1 male mice. Higher doses of MT-45 (6–30 mg/kg i.p.) were used to investigate cardiorespiratory changes (heart rate, respiratory rate, SpO2 saturation and pulse distention). All effects of MT-45 were compared with those of morphine. In vitro DMR assay results demonstrated that at human recombinant opioid receptors MT-45 behaves as a potent selective mu agonist with a slightly higher efficacy than morphine. In vivo results showed that MT-45 progressively induces tail elevation at the lowest dose tested (0.01 mg/kg), increased mechanical and thermal antinociception (starting from 1 to 6 mg/kg), decreased visual sensorimotor responses (starting from 3 to 6 mg/kg) and reduced tactile responses, modulated motor performance and induced muscle rigidity at higher doses (15 mg/kg). In addition, at higher doses (15–30 mg/kg) MT-45 impaired the cardiorespiratory functions. All effects were prevented by the administration of the opioid receptor antagonist naloxone. These findings reveal the risks associated with the ingestion of opioids and the importance of studying these drugs and undertaking more clinical studies of the current molecules to better understand possible therapeutic interventions in the case of toxicity.

Use of new psychoactive substances to mimic prescription drugs: The trend in France

Among the expanding world of New Psychoactive Substances (NPS), Designer Medicines (DM) are designed to mimic psychoactive drugs and might lead to adverse events of various severity. The DM category includes designer benzodiazepines (DB), phenmetrazine, modafinil, methylphenidate analogs, and novel synthetic opioids (NSO).To investigate DM-related complications in France, all data on NPS collected in the French Addictovigilance network database through spontaneous reports (SRs) and the annual survey on deaths related to the abuse of licit and illicit psychoactive substances (DRAMES survey) between 2009 and 2017 were analyzed. From 2009–2017, about 800 cases of NPS-related abuse or somatic complications were reported to the French Addictovigilance Network, including 71 fatal cases (9%). DM use progressively increased over the years, particularly after 2013 (4% of all SRs on NPS in 2011 versus 14 % in 2017). Moreover, DM were implicated in 17 % of NPS-related deaths in France, just after cathinones (43 %) and dissociative drugs (22 %). NSO, DB and phenidate analogs were identified in 42 %, 25 % and 25 % of all DM-related death reports, respectively.DM seem to interest a new target group of users that includes mainly patients and healthy people rather than substance users. The availability on the Internet of compounds mimicking therapeutic drugs is a worrying phenomenon that could lead to their uncontrolled use.

Assessment of structure-activity relationships and biased agonism at the Mu opioid receptor of novel synthetic opioids using a novel, stable bio-assay platform

Fentanyl and morphine are agonists of the Mu opioid receptor (MOR), which is a member of the GPCR family. Their analgesic effects are associated with unwanted side effects. On a signaling level downstream from MOR, it has been hypothesized that analgesia may be mediated through the G protein pathway, whereas the undesirable effects of opioids have been linked to the β-arrestin (βarr) pathway. Despite being an increasingly debated subject, little is known about a potential ‘bias’ (i.e. the preferential activation of one pathway over the other) of the novel synthetic opioids (NSO) – including fentanyl analogs – that have emerged on the illegal drug market. We have therefore developed and applied a novel, robust bio-assay platform to study the activity of 21 NSO, to evaluate to what extent these MOR agonists show biased agonism and to investigate the potential correlation with their structure. In addition, we evaluated the functional selectivity of TRV130, a purported G protein-biased agonist. We applied newly established stable bio-assays in HEK293T cells, based on the principle of functional complementation of a split nanoluciferase, to assess MOR activation via recruitment of a mini-Gi protein (GTPase domain of Gαi subunit) or βarr2. All but two of the tested NSO demonstrated a concentration-dependent response at MOR in both bio-assays. The developed bio-assays allow to gain insight into the βarr2 or G protein recruitment potential of NSO, which may eventually help to better understand why certain opioids are associated with higher toxicity. Adding to the recent discussion about the relevance of the biased agonism concept for opioids, we did not observe a significant bias for any of the evaluated compounds, including TRV130.

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.

Toward the Interpretation of Positive Testing for Fentanyl and Its Analogs in Real Hair Samples: Preliminary Considerations.

The detection of new psychoactive substances (NPS) in hair has become extensively researched in recent years. Although most NPS fall into the classes of synthetic cannabinoids and designer cathinones, novel synthetic opioids (NSO) have appeared with increasing frequency in the illicit drug supply. While the detection of NSO in hair is now well documented, interpretation of results presents several controversial issues, as is quite common in hair analysis. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry method able to detect 13 synthetic opioids (including fentanyl analogs) and metabolites in hair was applied to 293 real samples. Samples were collected in the USA between November 2016 and August 2018 from subjects who had reported heroin use in the past year or had already tested positive to hair testing for common opiates. The range, mean and median concentrations were calculated for each analyte, in order to draw a preliminary direction for a possible cut-off to discriminate between exposure to either low or high quantities of the drug. Over two-thirds (68%) of samples tested positive for fentanyl at concentrations between LOQ and 8600pg/mg. The mean value was 382pg/mg and the median was 95pg/mg. The metabolites norfentanyl and 4-ANPP were also quantified and were found between LOQ and 320pg/mg and between LOQ and 1400pg/mg, respectively. The concentration ratios norfentanyl/fentanyl, 4-ANPP/fentanyl and norfentanyl/4-ANPP were also tested as potential markers of active use and to discriminate the intake of fentanyl from other analogs. The common occurrence of samples positive for multiple drugs may suggest that use is equally prevalent among consumers, which is not the case, as correlations based on quantitative results demonstrated. We believe this set of experimental observations provides a useful starting point for a wide discussion aimed to better understand positive hair testing for fentanyl and its analogs in hair samples.

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.

Interpretation of hair analysis for fentanyl and analogues

Nowadays, the detection of new psychoactive substances (NPS) in hair has been extensively described. Although most NPS fall into the classes of synthetic cannabinoids and designer cathinones, novel synthetic opioids (NSO) have also appeared recently on the drug market with a progressive increase in their consumption. In turn, also the detection of NSO in hair has already been presented [1] , but their results interpretation still presents several controversial issues, as is quite common in hair analysis. Lack of knowledge about both effective dosage of NSO and their keratin binding make any attempt to establish a reasonable cut-off value for each drug questionable. In this presentation, preliminary results from a large dataset of real samples positive to fentanyl or analogues will be discussed. A simple, fast, specific and sensitive UHPLC-MS/MS method able to detect 13 synthetic opioids (including fentanyl analogues) and metabolites in 25 mg of hair was applied to 293 real samples. LOD values were in the range 0.1–0.3 pg/mg for all analytes, with the exception of oxycodone which was 1.5 pg/mg. Samples were collected in the United States between November 2016 and August 2018 from subjects who had reported heroin use in the last year or had already tested positive to common opiates. The range of concentrations, mean and median were calculated for each analyte. In particular, 68% of samples resulted positive for fentanyl at concentrations between LOQ and 8600 pg/mg. The mean value was 382 pg/mg and the median was 95 pg/mg. Furthermore, the metabolites norfentanyl and 4-ANPP were also monitored. Ranges of values lied between LOQ and 320 pg/mg and between 1 and 1400 pg/mg, respectively. The ratio norfentanyl/fentanyl, 4-ANPP/fentanyl and norfentanyl/4-ANPP were also evaluated. Fentanyl and analogues were measured in a wide range of concentrations. Median and box-plot calculations were used to draw a preliminary direction for a possible cut-off to discriminate between exposure to either low or high quantities of the drug. The main metabolites could also be detected in the majority of hair samples, thus the active use could be confirmed. Finally, several ratios proved promising as possible markers of active use and to discriminate the intake of fentanyl from other analogues. Like other classes of NPS, more data about the detection of NSO in hair are needed before any clear interpretation of analytical findings can be given. However, our preliminary results provide a useful starting point for a wide discussion aimed to a better understanding of fentanyl (and analogues) positive testing from hair analysis.

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.

Quantification of U-47700 and its metabolites in plasma by LC-MS/MS

Novel Synthetic Opioids (NSO) have caused a recent epidemic both nationally and globally. NSO have gained popularity in the illicit drug market and have brought about an increase in fentanyl and its derivatives, as well as other chemically unrelated opioid agonists. U-47700, a non-fentanyl analog analgesic opioid, was first developed by The Upjohn Company and has a reported potency of 7.5 times that of morphine. Like many NSO, U-47700 is usually sold as a research chemical that can be purchased online but can also be found in “Gray Death” which is a mixture of fentanyl(s), heroin, and U-47700. With the emergence of these NSO, there is a need for laboratories to be able to detect these drugs in various matrices. In this study, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was optimized and validated to detect and quantify U-47700 and its metabolites, N-desmethyl-U-47700 and N,N-didesmethyl-U-47700, in 100 μL human plasma using an optimized solid phase extraction procedure. A small sample size (100 μL) was utilized for a future pharmacokinetic study in rats. The method was validated according to SWGTOX guidelines, including: precision and bias, linearity, carryover, interferences, matrix effects, limit of detection (LOD), limit of quantification (LOQ), dilution integrity, and stability. The LOD were 0.05 ng/mL for U-47700 and N-desmethyl-U-47700 and 0.1 ng/mL for N,N-didesmethyl-U-47700. Linear ranges for U-47700 and N-desmethyl-U-47700 were 0.1–100 ng/mL and 0.5–100 ng/mL for N,N-didesmethyl-U-47700. Matrix effects were analyzed following the post-extraction addition approach and were <5%, indicating little ion suppression or enhancement. Extraction recovery was >79%. Analytes were stable in all conditions and no endogenous or exogenous interferences were detected. This method was cross-validated in rat plasma with acceptable bias (2.1–6.2%) and precision (−14.7–15.7%) within acceptable limits. Matrix effects and extraction efficiency was comparable to human plasma validation. Postmortem whole blood samples (n = 15) were analyzed with the validated method. U-47700, N-desmethyl-U-47700 and N,N-didesmethyl-U-47700 concentration ranges were 1.1–1367 ng/mL, 4.0–1400 ng/mL and 0.7–658 ng/mL, respectively.