Seized drug reporting of NPS helps to guide regional toxicological practice: A 17 month review between 2022 and 2023.

Novel psychoactive substances (NPS) are everchanging and plague forensic laboratories who must identify an unending variety of emerging substances and evolve current methodologies to detect these substances. Identifying potential regional NPS targets and timely examining trends in seized drug data could help mitigate the burden laboratories face. Over 17 months, NPS seized drug data were processed and categorized from three laboratories located across the United States to determine any NPS regional similarities and prevalent NPS drug categories: the South Carolina Law Enforcement Division (SLED), the Sedgwick County Regional Forensic Science Center (SCRFSC), and the Orange County Crime Laboratory (OCCL). Seized drug materials, including pills, powders, and plant material, were primarily analyzed for NPS via gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy. From June 2022 to October 2023, 1940 NPS seized drug identifications were reported by these laboratories with 63 different NPS reported. Novel synthetic opioids (NSO) were the most prevalent NPS class across all three laboratories (55%), with fluorofentanyl accounting for 74% of NSO identifications. This is unsurprising given the fentanyl epidemic in the United States. Furthermore, these data highlighted varying regional NPS seized drug trends: eutylone, a synthetic cathinone, was one of the most frequently identified NPS in SLED, SCRFSC observed the most diverse set of synthetic cannabinoids, and OCCL observed an increased prevalence in the designer benzodiazepine, bromazolam. NPS scope recommendations are a valuable resource for forensic laboratories; however, most focus on a national perspective. Timely analysis and reporting of NPS seized drug data may help to develop regional NPS scope recommendations laboratories may employ.

In vitro structure-activity relationships and forensic case series of emerging 2-benzylbenzimidazole 'nitazene' opioids.

2-Benzylbenzimidazole 'nitazene' opioids are presenting a growing threat to public health. Although various nitazenes were previously studied, systematic comparisons of the effects of different structural modifications to the 2-benzylbenzimidazole core structure on µ-opioid receptor (MOR) activity are limited. Here, we assessed in vitro structure-activity relationships of 9 previously uncharacterized nitazenes alongside known structural analogues. Specifically, we focused on MOR activation by 'ring' substituted analogues (i.e., N-pyrrolidino and N-piperidinyl modifications), 'desnitazene' analogues (lacking the 5-nitro group), and N-desethyl analogues. The results from two in vitro MOR activation assays (ß-arrestin 2 recruitment and inhibition of cAMP accumulation) showed that 'ring' modifications overall yield highly active drugs. With the exception of 4'-OH analogues (which are metabolites), N-pyrrolidino substitutions were generally more favorable for MOR activation than N-piperidine substitutions. Furthermore, removal of the 5-nitro group on the benzimidazole ring consistently caused a pronounced decrease in potency. The N-desethyl modifications showed important MOR activity, and generally resulted in a slightly lowered potency than comparator nitazenes. Intriguingly, N-desethyl isotonitazene was the exception and was consistently more potent than isotonitazene. Complementing the in vitro findings and demonstrating the high harm potential associated with many of these compounds, we describe 85 forensic cases from North America and the United Kingdom involving etodesnitazene, N-desethyl etonitazene, N-desethyl isotonitazene, N-pyrrolidino metonitazene, and N-pyrrolidino protonitazene. The low-to-sub ng/mL blood concentrations observed in most cases underscore the drugs' high potencies. Taken together, by bridging pharmacology and case data, this study may aid to increase awareness and guide legislative and public health efforts.

Determination of fentanyl contamination on United States paper currency by LC-QQQ-MS.

Previous research has evaluated the extent to which cocaine and other drugs were detectable on currency in the USA. The literature was in agreement that the majority of bills exhibited some degree of contamination. With the increase of fentanyl in the illicit drug supply, this study was designed to evaluate the extent that fentanyl, cocaine, methamphetamine and other substances were present on circulating currency in 2022. A quantitative assay using liquid chromatography-triple quadrupole mass spectrometry was developed and validated to detect six analytes: fentanyl, 4-anilino-N-phenethylpiperidine, acetylfentanyl, benzylfentanyl, cocaine and methamphetamine. One-dollar bills were collected from 13 cities across the country. Sample preparation consisted of soaking the bills in methanol followed by liquid-liquid extraction. Chromatographic separation was achieved using a C18 analytical column and gradient elution with ammonium formate in water (5 mM, pH 3) and 0.1% formic acid in acetonitrile. The quantitative working range for this assay was 0.1 µg to 1.0 µg per bill (equivalent to 1 ng/mL to 100 ng/mL of extract). Fentanyl was detected on the majority (63%) of samples, with 61% of samples having ≥0.1 µg of fentanyl and 4% of samples having ≥1.0 µg. Cocaine and methamphetamine were detected on 100% and 98% of bills, respectively, typically in amounts >1.0 µg. The remaining fentanyl-related substances were detected in 15% of samples in amounts no >0.69 µg per bill and exclusively in the presence of fentanyl. Unsurprisingly, areas of the country with higher incidence of fentanyl use yielded higher frequency of contaminated bills and higher concentrations. Human exposure to drugs on currency is unlikely to have any significant impacts toxicologically or pharmacologically; however, our research findings suggest that paper currency could serve as a useful substrate for surveillance of drug trends regionally, nationally and/or internationally.

Increasing prevalence of designer benzodiazepines in impaired driving: A 5-year analysis from 2017 to 2021.

Designer benzodiazepine (DBZD) use has been increasing over the past decade and poses a threat to human health and safety, particularly when involved in driving under the influence of drug (DUID) cases. Over a 5-year period between 2017 and 2021, there were 1,145 reported DBZDs in 805 blood samples submitted from law enforcement agencies for DUID testing. Eleven different DBZDs were detected, including three metabolite pairs: etizolam/alpha-hydroxyetizolam, clonazolam/8-aminoclonazolam, diclazepam/delorazepam, flualprazolam, flubromazolam, flubromazepam, bromazolam and bromazepam. Etizolam/alpha-hydroxyetizolam (n = 485) and flualprazolam (n = 149) were the most frequently detected DBZDs, at 60% and 18%, respectively. Driving behavior, standardized field sobriety test performance and physical observations of individuals suspected of DUIDs, whose blood sample was toxicologically confirmed for one or more DBZDs, were consistent with the effects caused by central nervous system depressants. Each DBZD has its own unique timeline, and toxicology testing had to be frequently updated to reflect the state of the novel psychoactive substance market. DBZDs play a role in impaired driving and can be the sole intoxicant in DUID cases.

N,N-Dimethylpentylone (dipentylone)-A new synthetic cathinone identified in a postmortem forensic toxicology case series.

Synthetic cathinones emerged on the novel psychoactive substance (NPS) drug market as alternatives to controlled stimulants and entactogens such as methamphetamine and 3,4-methylenedioxymethamphetamine. The majority of synthetic cathinones can be subclassified into two groups: beta-keto amphetamines (i.e., NPS with the suffix "drone") and beta-keto methylenedioxyamphetamines (i.e., NPS with the suffix "lone"). Although a significant number of beta-keto amphetamines have been identified, beta-keto methylenedioxyamphetamines have dominated the NPS market, including notable drugs like methylone, butylone, N-ethyl pentylone (ephylone), eutylone and now N,N-dimethylpentylone. N,N-Dimethylpentylone, also known as dipentylone or beta-keto-dimethylbenzodioxolylpentanamine, emerged into the illicit drug supply <2 months of the international control of eutylone (September 2021). A novel standard addition method was developed and validated for N,N-dimethylpentylone, pentylone and eutylone, and 18 postmortem cases were quantitated using the method described in this manuscript. The resulting blood concentration range for N,N-dimethylpentylone in this case series was 3.3 to 970 ng/mL (median: 145 ng/mL, mean: 277 ± 283 ng/mL). Pentylone, a metabolite of N,N-dimethylpentylone, was detected in all cases (range: 1.3-420 ng/mL, median: 31 ng/mL and mean: 88 ± 127 ng/mL). Due to the rise in identifications of N,N-dimethylpentylone in postmortem investigations as well as the potential misidentification of N,N-dimethylpentylone as N-ethyl pentylone, samples testing positive for pentylone should be additionally confirmed for the presence of N,N-dimethylpentylone. Based on prior trends of new synthetic cathinones, it can be theorized that N,N-dimethylpentylone may predominate the US synthetic stimulant market for the next 1-2 years; however, given the emergence of additional closely related isomeric compounds, it is important to utilize methodology capable of differentiating N,N-dimethylpentylone from its isomers (N-isopropylbutylone, N-ethyl pentylone, N-ethyl N-methyl butylone, hexylone, N-propylbutylone, diethylone and tertylone).

Detection, chemical analysis, and pharmacological characterization of dipyanone and other new synthetic opioids related to prescription drugs.

The emergence of structurally diverse new synthetic opioids (NSOs) has caused the opioid crisis to spiral to new depths. Little information is available about the pharmacology of most novel opioids when they first emerge. Here, using a ß-arrestin 2 recruitment assay, we investigated the in vitro µ-opioid receptor (MOR) activation potential of dipyanone, desmethylmoramide, and acetoxymethylketobemidone (O-AMKD) - recent NSOs that are structurally related to the prescription opioids methadone and ketobemidone. Our findings indicate that dipyanone (EC50=39.9 nM; Emax=155% vs. hydromorphone) is about equally active as methadone (EC50=50.3 nM; Emax=152%), whereas desmethylmoramide (EC50=1335 nM; Emax=126%) is considerably less active. A close structural analogue of ketobemidone (EC50=134 nM; Emax=156%) and methylketobemidone (EC50=335 nM; Emax=117%), O-AMKD showed a lower potency (EC50=1262 nM) and efficacy (Emax=109%). Evaluation of the opioid substitution product buprenorphine and its metabolite norbuprenorphine confirmed the increased in vitro efficacy of the latter. In addition to in vitro characterization, this report details the first identification and full chemical analysis of dipyanone in a seized powder, as well as a postmortem toxicology case from the USA involving the drug. Dipyanone was quantified in blood (370 ng/mL), in which it was detected alongside other NSOs (e.g., 2-methyl AP-237) and novel benzodiazepines (e.g., flualprazolam). While dipyanone is currently not commonly encountered in forensic samples worldwide, its emergence is worrisome and representative of the dynamic NSO market. Graphical Abstract.

Metabolic profile determination of 2F-viminol - A novel synthetic opioid identified in forensic investigations.

Novel synthetic opioid (NSO) continue to emerge in the United States in the midst of an opioid crisis. The NSO 2F-viminol was identified in casework at the Center for Forensic Science Research and Education through its NPS Discovery program in 2019. Little information and published literature were available for this new opioid at the time. To address this, human liver microsomes (HLMs) were used to perform in vitro metabolism studies with a drug standard. The goal was to predict in vivo metabolism. Experimental samples were prepared using HLMs, NADPH, phosphate buffer (pH 7.4), and a 2F-viminol standard. Standard samples were prepared containing only drug, control samples were prepared with drug and HLMs but no NADPH cofactor, and metabolism reaction mixtures contained drug, HLMs and NADPH. The subsequent mixtures were incubated with light shaking to allow metabolism to occur. After cleanup, metabolite mixtures were analyzed via a SCIEX TripleTOF 5600+ liquid chromatograph quadrupole-time-of-flight mass spectrometer (LC-QTOF-MS). The generated metabolic structures were elucidated using SCIEX MetabolitePilot software (version 2.0). In addition to remaining parent drug, seven metabolites of 2F-viminol were discovered, including N-dealkylated and hydroxylated species. The proposed primary metabolites of 2F-viminol were N-dealkylation (sec-butyl) + hydroxylation and N-dealkylation (sec-butyl); however, they should be confirmed in authentic samples, and forensic laboratories should consider adding 2F-viminol and its metabolites to screening protocols to help in extending the window of detection for the parent drug in toxicological samples. As NSOs continue to appear, forensic laboratories must continue metabolism experiments to generate information about pharmacokinetics.

Proliferation of Novel Synthetic Opioids in Postmortem Investigations After Core-Structure Scheduling for Fentanyl-Related Substances.

ABSTRACT: New generations of novel synthetic opioids (NSOs) have emerged to fill a void in the illicit drug markets left by the decline in popularity of fentanyl analogs subsequent to core-structure scheduling of fentanyl-related substances in the United States and China. These new opioids include members of the 2-benzyl benzimidazole (eg, isotonitazene, metonitazene, N -pyrrolidino etonitazene, protonitazene, etodesnitazene), benzimidazolone (eg, brorphine), and cinnamylpiperazine (eg, AP-238, 2-methyl AP-237) subclasses. Novel synthetic opioids continue to be detected in opioid-related fatal overdoses, demonstrating the harms associated with exposure to these drugs. Between January 2020 and December 2021, 384 casework blood samples were reported by our laboratory to contain 1 or more of the prior listed 8 NSOs. Isotonitazene (n = 144), metonitazene (n = 122), and brorphine (n = 91) were the 3 most prevalent substances, with positivity for isotonitazene and brorphine peaking just before the announcement of emergency scheduling. These NSOs have been documented as significant drivers of drug mortality, and this case series described here highlights the challenges medical examiners and coroners face in staying current with emerging drugs. Challenges include regional differences, rapid turnover, short lifecycles, variable toxicology testing, and difficulty in assessing individual drug toxicity in polydrug cases.

Pharmacological evaluation and forensic case series of N-pyrrolidino etonitazene (etonitazepyne), a newly emerging 2-benzylbenzimidazole 'nitazene' synthetic opioid.

Novel synthetic opioids continue to emerge on recreational drug markets worldwide. In response to legislative bans on fentanyl analogues, non-fentanyl structural templates, such as 2-benzylbenzimidazoles ('nitazenes'), are being exploited to create new µ-opioid receptor (MOR) agonists. Here, we pharmacologically characterize an emerging cyclic analogue of etonitazene, called N-pyrrolidino etonitazene (etonitazepyne), using in vitro and in vivo methods. A series of analytically confirmed fatalities is described to complement preclinical findings. Radioligand binding assays in rat brain tissue revealed that N-pyrrolidino etonitazene has high affinity for MOR (Ki = 4.09 nM) over δ-opioid (Ki = 959 nM) and κ-opioid (Ki = 980 nM) receptors. In a MOR-ß-arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC50 = 0.348 nM), similar to etonitazene (EC50 = 0.360 nM), and largely exceeding the potencies of fentanyl (EC50 = 14.9 nM) and morphine (EC50 = 290 nM). When administered s.c. to male Sprague Dawley rats, N-pyrrolidino etonitazene induced opioid-like antinociceptive, cataleptic, and thermic effects. Its potency in the hot plate test (ED50 = 0.0017 mg/kg) was tenfold and 2,000-fold greater than fentanyl (ED50 = 0.0209 mg/kg) and morphine (ED50 = 3.940 mg/kg), respectively. Twenty-one overdose fatalities associated with N-pyrrolidino etonitazene were found to contain low blood concentrations of the drug (median = 2.2 ng/mL), commonly in the context of polysubstance use. N-Pyrrolidino etonitazene was reported as a cause of death in at least two cases, demonstrating toxicity in humans. We demonstrate that N-pyrrolidino etonitazene is an extremely potent MOR agonist that is likely to present high risk to users. Continued vigilance is required to identify and characterize emergent 2-benzylbenzimidazoles, and other non-fentanyl opioids, as they appear in the marketplace.

Reports of Adverse Events Associated with Use of Novel Psychoactive Substances, 2017-2020: A Review.

An important role of modern forensic and clinical toxicologists is to monitor the adverse events of novel psychoactive substances (NPS). Following a prior review from 2013 to 2016, this critical literature review analyzes and evaluates published case reports for NPS from January 2017 through December 2020. The primary objective of this study is to assist in the assessment and interpretation of these cases as well as provide references for confirmation methods. Chemistry, pharmacology, adverse events and user profiles (e.g., polypharmacy) for NPS are provided including case history, clinical symptoms, autopsy findings and analytical results. Literature reviews were performed in PubMed and Google Scholar for publications using search terms such as NPS specific names, general terms (e.g., 'designer drugs' and 'novel psychoactive substances'), drug classes (e.g., 'designer stimulants') and outcome-based terms (e.g., 'overdose' and 'death'). Government and website drug surveillance databases and abstracts published by professional forensic science organizations were also searched. Toxicological data and detailed case information were extracted, tabulated, analyzed and organized by drug category. Case reports included overdose fatalities (378 cases), clinical treatment and hospitalization (771 cases) and driving under the influence of drugs (170 cases) for a total of 1,319 cases providing details of adverse events associated with NPS. Confirmed adverse events with associated toxidromes of more than 60 NPS were reported including synthetic cannabinoid, NPS stimulant, NPS hallucinogen, NPS benzodiazepine and NPS opioid cases. Fifty of these NPS were reported for the first time in January 2017 through December 2020 as compared to the previous 4 years surveyed. This study provides insight and context of case findings described in the literature and in digital government surveillance databases and websites during a recent 4-year period. This review will increase the awareness of adverse events associated with NPS use to better characterize international emerging drug threats.

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.

Analysis of the Illicit Opioid U-48800 and Related Compounds by LC-MS-MS and Case Series of Fatalities Involving U-48800.

We report a method for the detection and quantitation of 12 drugs and 2 metabolites in the same structural class as the illicit mu-opioid agonist U-47700 in human whole blood. These substances are either known or suspected to be present as potential novel opioids in illicit drug markets. The general class of these drugs was developed in pharmaceutical research programs in the 1970s, but these drugs have recently become of concern for overdoses and death in opioid users in the USA and internationally. The scope of analysis included the following compounds: methylenedioxy U-47700, ethylenedioxy U-47700, ethylenedioxy U-51754, U-69593, U-47931E (bromadoline), U-47700, U-48800, U-49900, U-51754, U-50488, propyl U-47700 and isopropyl U-47700. Additionally, two metabolites N,N-didesmethyl U-47700 and desmethyl U-47700 were also included in the scope. Drugs were extracted from human whole blood using solid-phase extraction, and the extracts were analyzed by liquid chromatography--tandem mass spectrometry. The assay was validated with respect to bias, carryover, interference, within-run and between-run precision, and accuracy. Eight medicolegal death investigation cases that had screened positive for U-48800 by liquid chromatography--time-of-flight mass spectrometry were successfully confirmed and quantified using this method. The mean and median concentrations of U-48800 in these cases were 2.5 (±2.1) and 1.8 ng/mL, respectively, with a range of concentrations of 0.27-6.2 ng/mL. Case history information including the presence of other drugs in combination are described and discussed.

Online surveillance of novel psychoactive substances (NPS): Monitoring Reddit discussions as a predictor of increased NPS-related exposures.

BACKGROUND: Novel psychoactive substances (NPS) present continuous and growing challenges for the scientific, medical, and interventional communities as emerging substances on recreational drug markets change national and international drug landscapes. NPS account for an increasing proportion of adverse events, hospitalizations, and deaths due to increasing potency and unanticipated biological effects compared to predecessors. This study evaluated the utility of drug use forums as an early indicator or predictor of impending intoxications with potentially harmful or lethal outcomes prior to their occurrences. METHODS: Eight NPS were selected for evaluation to assess the relationship between online mentions of drugs and their involvement in toxic exposures or overdoses. Mentions on Reddit drug forum discussions were tallied and toxicology testing results from forensic investigations in the US were assessed. The selected NPS covered several subclasses and a predetermined time range (2013-2020). They included carfentanil, U-47700, eutylone, flualprazolam, N-ethylpentylone, 5F-MDMB-PICA, isotonitazene, and brorphine. RESULTS: Seven NPS (excluding 5F-MDMB-PICA) appeared in discussions on Reddit prior to their implication in poisonings or intoxications. Distinct increases and decreases in number of mentions and number of exposures were observed. For most substances (n = 5, 63%), a rise in Reddit mentions was soon followed by a corresponding rise in toxicology positivity. Peak positivity for carfentanil and flualprazolam, however, preceded peak Reddit mentions. CONCLUSIONS: This study demonstrated the utility of social media sites, such as Reddit, as a predictor for future trends in NPS-related exposures. These results provide confirmation that activity on drug use forums in the virtual world can help predict changes in exposures associated with new or re-emerging NPS in the real world. The results warrant further evaluation as a strategy for inclusion in early warning systems.

The Rise and Fall of Isotonitazene and Brorphine: Two Recent Stars in the Synthetic Opioid Firmament.

Synthetic opioids constitute one of the fastest growing groups of new psychoactive substances (NPS) worldwide. With fentanyl analogues being increasingly controlled via class-wide scheduling, many non-fentanyl related opioids are now emerging on the recreational opioid market, rendering the landscape highly complex and dynamic. While new compounds are entering the supply in rapid and unpredictable manners, some recent patterns have become apparent. Many of these newly emerging opioids are being pirated from early patent literature and/or research papers, synthesized and sold online through various channels. Burdened by the identification of every newly emerging drug, many toxicology labs struggle to keep up. Moreover, by the time a "new" drug is controlled via legislative measures, illicit drug markets will have already adapted and diversified as manufacturers work to avoid the restricted product(s). Hence, the typical life-cycle of an NPS opioid is generally short (less than 6 months to one year), with only a few drugs escalating to significant numbers of detections. In this review, we summarize the key events in the emergence, rise, and subsequent decline of two non-fentanyl opioids - isotonitazene and brorphine. These two opioids sequentially dominated the NPS opioid market in 2019 and 2020. Both isotonitazene and brorphine remained in circulation for over a year, each contributing to hundreds of deaths and adverse events. By detailing the life-cycles of these opioids from their earliest synthesis as described in scientific literature to their subsequent rise and fall on recreational markets, this review illustrates the new characteristic life-cycle of synthetic opioids in the 'post-fentanyl-analogue' era.

Metonitazene in the United States-Forensic toxicology assessment of a potent new synthetic opioid using liquid chromatography mass spectrometry.

Metonitazene is considered a new psychoactive substance (NPS) and emerging potent synthetic opioid, causing increased public health concern beginning in 2020. Metonitazene joins a growing list of new synthetic opioids (NSOs) contributing to deaths among people who use drugs in the United States and other parts of the world. Metonitazene (a 2-benzylbenzimidazole analogue) first appeared in mid-2020 in the recreational drug supply and subsequently began proliferating in death investigation casework towards the end of 2020. Screening and metabolite discovery were performed by liquid chromatography quadrupole time-of-flight mass spectrometry. Quantitative confirmation was performed by liquid chromatography tandem quadrupole mass spectrometry. Metonitazene was confirmed in 20 authentic forensic postmortem cases with an average concentration in blood at 6.3 ± 7.5 ng/ml (median: 3.8 ng/ml, range: 0.5-33 ng/ml, n = 18) and in urine at 15 ± 13 ng/ml (median: 11 ng/ml, range: 0.6-46 ng/ml, n = 14). Metonitazene was the only opioid identified in 30% of cases but was also found in combination with fentanyl (55%) and NPS benzodiazepines, opioids, and hallucinogens (45%). Medical examiners included metonitazene as a drug responsible for the cause of death, and the manner of death was always ruled to be an accident. The metabolism of metonitazene was found to be similar to that of isotonitazene, a closely related analogue. Toxicology laboratories and death investigators should ensure that metonitazene is included in forensic testing protocols, all while remaining vigilant for subsequent NSOs to emerge.

The Development and Validation of a Novel Designer Benzodiazepines Panel by LC-MS-MS.

Novel illicit benzodiazepines are among the most active areas of new illicit drug manufacture and use. We describe a method for the detection and quantification of etizolam and its metabolite α-hydroxyetizolam, flubromazolam, clonazolam, diclazepam, delorazepam, bromazepam, flubromazepam, phenazepam, flualprazolam, flunitrazolam, and nitrazolam in human whole blood. After addition of internal standards, samples are buffered and extracted using a liquid-liquid extraction. Analysis is performed using positive-ion electrospray tandem mass spectrometry for detection and quantitation. Calibration ranges were established based on the method performance and differed from compound to compound. Replicates at the lowest calibration point for each compound performed within 5% of CV (Coefficient of Variation). The correlation coefficient was >0.990 for all compounds. Relative standard deviation for all compounds was ≤10% of CV and accuracy was ±10% for both within- and between-run experiments. The maximum average intra- and inter-run imprecision were 5.7%. The maximum average intra- and inter-run imprecision was -8.7%. As part of evaluating the scope for relevancy, samples testing positive in immunoassay but confirmed to be negative in traditional benzodiazepine confirmation method were re-analyzed using this method. The presence of at least one novel benzodiazepine was identified in 70% of these samples. The appearance of these novel "designer" benzodiazepines demonstrates the challenge for toxicology testing and the need for continually updated confirmation methods.

Eutylone Intoxications-An Emerging Synthetic Stimulant in Forensic Investigations.

Synthetic stimulants are the largest class of novel psychoactive substances identified each year by forensic laboratories internationally. While hundreds of these drugs appear in drug powders, only a few proliferate in use among forensically relevant populations and eventually emerge in postmortem and clinical investigations. Beta-keto-methylenedioxyamphetamines (i.e., novel psychoactive substances with names ending in "ylone") are currently the most popular subclass of synthetic stimulants. Leading up to its federal scheduling in 2018, N-ethyl pentylone was the most encountered synthetic stimulant. The popularity of N-ethyl pentylone declined once it was scheduled, but it was quickly replaced by eutylone (bk-EBDB), a structurally related analog from the same family. In cases encountered between January 2019 and April 2020, eutylone was quantitatively confirmed in 83 forensic investigations, including postmortem cases and driving under the influence of drugs cases. Matrix types included blood, urine and tissue. Eutylone was identified in cases submitted from 13 states, demonstrating proliferation around the United States; Florida accounted for 60% of the positive cases. The mean concentration of eutylone in postmortem blood was 1,020 ng/mL (standard deviation = ±2,242 ng/mL; median = 110 ng/mL, range = 1.2-11,000 ng/mL, n = 67). The mean concentration of eutylone in blood from driving under the influence of drugs cases was 942 ng/mL (standard deviation = ±1,407 ng/mL; median = 140 ng/mL, range = 17-3,600 ng/mL, n = 7). This report includes cause and manner of death data for 22 postmortem cases. Further analysis of authentic human specimens revealed the presence of three eutylone metabolites, including one unique biomarker and one metabolite in common with butylone. Laboratories should be aware that eutylone may be present in cases of suspected Ecstasy, "Molly" and/or methylenedioxymethamphetamine use, causing or contributing to impairment or death.

Flualprazolam Blood Concentrations in 197 Forensic Investigation Cases.

Flualprazolam is a designer benzodiazepine and novel psychoactive substance that is increasing in prevalence and appearing in forensic investigations. Flualprazolam was quantitatively confirmed in 197 blood samples from medicolegal death investigations and human performance cases reported between August 2019 and February 2020. Drug screening was performed using liquid chromatography-time-of-flight mass spectrometry and quantitative confirmation was performed using liquid chromatography-tandem mass spectrometry. A three-point standard addition protocol was implemented for quantitation in the absence of an available traditionally validated assay. In postmortem cases with quantitative results (n = 167), the mean (±standard deviation [SD]) flualprazolam concentration was 20 (±63) ng/mL, the median concentration was 8.2 ng/mL and the range of concentrations was 2.0-620 ng/mL. Four additional postmortem cases were reported positive (<2.0 ng/mL). In drug impaired driving cases (n = 22), the mean (±SD) flualprazolam concentration was 22 (±18) ng/mL, the median concentration was 14 ng/mL and the range of concentrations was 4.4 to 68 ng/mL. The four remaining cases were of unknown circumstances. This report details the most extensive dataset of flualprazolam intoxication cases reported to date. There was significant overlap in concentrations of flualprazolam between postmortem and DUID cases. Flualprazolam was commonly (83% of the time) found in combination with opioids (e.g. fentanyl). Toxicologists should consider quantitative flualprazolam results in the context of case history, observations, and/or other toxicological findings. Addition of flualprazolam to the scope of drug testing should be considered by all laboratories.

Brorphine-Investigation and quantitation of a new potent synthetic opioid in forensic toxicology casework using liquid chromatography-mass spectrometry.

New synthetic opioids continue to appear as novel psychoactive substances (NPS) on illicit drug markets. Isotonitazene emerged in mid-2019, becoming the most prevalent NPS opioid in the United States within a few months. Notification by the Drug Enforcement Administration of its intent to schedule isotonitazene in mid-2020 led to its decline in popularity and replacement with a new NPS opioid: brorphine. Brorphine is a potent synthetic opioid, but little information was previously available regarding its toxicity or involvement in impairment and death. Our laboratory developed an assay for the identification and quantitative confirmation of brorphine using standard addition. Quantitative analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vitro and in vivo metabolism studies were performed using pooled human liver microsomes and authentic biological specimens, respectively, with analysis by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Brorphine was confirmed in 20 authentic forensic cases, commonly found in combination with fentanyl (100%) and flualprazolam (80%). The average concentration of brorphine in blood was 2.5 ± 3.1 ng/mL (median: 1.1 ng/mL, range: 0.1-10 ng/mL). The average concentration of brorphine in urine was 4.6 ± 7.6 ng/mL (median: 1.6 ng/mL, range: 0.2-23 ng/mL). The majority of cases originated from Midwestern states. Metabolism was verified to included N-dealkylation and hydroxylation. Detailed case histories and autopsy findings are presented herein. The prevalence of brorphine continues to increase in the United States. Forensic scientists should remain aware of the ongoing emergence of new opioids, especially those outside a standard scope of toxicology testing.

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.