Evaluating cross-reactivity of new psychoactive substances (NPS) in human whole blood by enzyme-linked immunosorbent assay (ELISA).

Due to the increase in the use of novel psychoactive substances (NPS) and their overall prevalence, it is important to have effective and reliable screening technologies to detect NPS in biological matrices. Enzyme-linked immunosorbent assays (ELISA) are among the most popular screening methods. To evaluate the effectiveness of ELISA for NPS detection, five subclasses of NPS (novel synthetic opioids, fentanyl analogs, stimulants, benzodiazepines and hallucinogens) were evaluated in whole blood for their cross-reactivity on commercially available ELISA kits. A variety of novel synthetic opioids were tested at concentrations of 1-80 ng/mL and 50-2000 ng/mL and demonstrated no cross-reactivity to a morphine ELISA plate at either concentration range. Fentanyl analogs were tested at concentrations ranging from 0.01 to 1 ng/mL and had cross-reactivities ranging from 8% to 178% on the fentanyl ELISA kit used. Both para-chloro fentanyl (178%) and acryl fentanyl (164%) showed cross-reactivities well above that of fentanyl. Novel stimulants were tested at concentrations of 0.5-40 ng/mL and 20-2,000 ng/mL. 4-Fluoroamphetamine was the only novel stimulant with cross-reactivity (3,354%) to the amphetamine ELISA plate. Novel benzodiazepines were tested at concentrations of 1-40 ng/mL on a benzodiazepine plate. Cross-reactivities ranged from 36.1% to 263%, with desalkylflurazepam having the highest cross-reactivity. Finally, novel hallucinogens were tested at concentrations of 0.5-10 ng/mL on a phencyclidine (PCP) ELISA plate, which produced no cross-reactivity and then with 10-1,000 ng/mL, which gave results from 56.6% to 151%. Both hydroxy-PCP (151%) and chloro-PCP (137%) showed cross-reactivities above that of PCP. This research has demonstrated the utility of using ELISA-based screening for novel benzodiazepines, hallucinogens and for fentanyl analogs; however, there is limited application and risk of false-negative results for the other drug classes due to low or non-existent cross-reactivities.

Xylazine: Pharmacology Review and Prevalence and Drug Combinations in Forensic Toxicology Casework.

Xylazine, an alpha-2 receptor agonist used in veterinary medicine for its sedative and muscle-relaxant effects, has been reported in forensic toxicology casework since the 1980s. It is not approved for human use, but it is used as an adulterant in heroin and illicit fentanyl. The prevalence and concentrations of xylazine in 2.5 years (January 2019-June 2021) of driving under the influence of drugs (DUID) and medico-legal death investigation (MDI) cases was investigated, including other drugs detected in combination with xylazine. Of over 170,000 cases screened for xylazine, 97% were classified as MDI. Over the course of the study period, the prevalence and geographical spread of xylazine increased. Overall, 2.8% of DUID and 2.1% of MDI cases screened positive for xylazine with concentrations of 5.1-450 ng/mL (mean = 36 ng/mL) and 5.0-11,000 ng/mL (mean = 41 ng/mL), respectively. Two MDI cases which had xylazine concentrations of 9,100 and 11,000 ng/mL were drug overdose suicides that did not involve any opioids. Opioids, primarily fentanyl and/or a fentanyl byproduct/metabolite were detected in 100% of DUID and all but two MDI cases. After opioids, stimulants, phyto-cannabinoids and benzodiazepines were the most common drug classes detected in conjunction with xylazine in both DUID and MDI casework. In summary, xylazine exposure continues to increase, mostly through the adulteration of illicit opioids. There is an extensive overlap in the concentrations between living and deceased individuals, making it difficult to interpret the role of the drug in MDI or DUID cases without other case information.

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.

Concentrations of para-Fluorofuranylfentanyl in Paired Central and Peripheral Blood Collected during Postmortem Death Investigations.

The opioid epidemic in the USA has been associated with an increasing mortality rate in large part due to the emergence and proliferation of synthetic opioids over the last 15 years. Fentanyl and its analogs have played a large part in these statistics due to their potency and toxicity. Fluorofuranylfentanyl (FFF) is a fentanyl analog that emerged in the USA in 2018 and was associated with numerous adverse events and deaths. During this study, a liquid chromatography tandem mass spectrometry workflow was developed to accurately identify the isomer of FFF present (ortho- vs. meta- vs. para-) in medicolegal death investigation cases from Pinellas County, Florida. FFF was quantified in central and peripheral blood samples collected at autopsy. In addition, the metabolism of FFF was studied using liquid chromatography quadrupole time-of-flight mass spectrometry. para-FFF was quantitatively confirmed in 29 postmortem cases; no other isomer of FFF was detected. Central blood concentrations ranged between 0.66 and 73 ng/mL (mean = 11 ± 14 ng/mL, median = 10 ng/mL) and peripheral blood concentrations ranged between 0.53 and 23 ng/mL (mean = 5.7 ± 6.4 ng/mL, median = 2.7 ng/mL). A comparison of central to peripheral blood concentrations was evaluated to determine the possibility of postmortem redistribution. The metabolism of ortho-FFF was studied and found to undergo metabolic processes similar to fentanyl, producing ortho-fluorofuranyl-norfentanyl, fluoro-4-anilino-N-phenethylpiperidine, and hydroxylated species. The results of this study demonstrate the toxicity of FFF and its implication in medicolegal death investigations. Laboratories must remain aware of new or re-emerging fentanyl analogs, as they pose significant risks to public health and public safety.

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.

Pharmacokinetics and pharmacodynamics of the synthetic cannabinoid, 5F-MDMB-PICA, in male rats.

5F-MDMB-PICA is a popular synthetic cannabinoid associated with analytically confirmed intoxications. In vitro studies show 5F-MDMB-PICA is a potent cannabinoid-1 receptor (CB1) agonist, but little information is available about in vivo pharmacokinetics and pharmacodynamics. To this end, the present study had three aims: 1) to develop a validated method for detection of 5F-MDMB-PICA and its metabolites in rat plasma, 2) to utilize the method for investigating pharmacokinetics of 5F-MDMB-PICA in rats, and 3) to relate 5F-MDMB-PICA pharmacokinetics to pharmacodynamic effects. 5F-MDMB-PICA and its metabolites were quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS) and method validation followed forensic standards. Male Sprague-Dawley rats bearing surgically implanted jugular catheters and subcutaneous (s.c.) temperature transponders received 5F-MDMB-PICA (50, 100, or 200 µg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, 240, and 480 min post-injection, and plasma was assayed using LC-MS/MS. At each blood draw, body temperature, and catalepsy scores were recorded. Maximum plasma concentrations (Cmax) of 5F-MDMB-PICA rose linearly with increasing dose (1.72-6.20 ng/mL), and plasma half-life (t1/2) ranged from 400 to 1000 min 5F-MDMB-PICA-3,3-dimethylbutanoic acid and 5OH-MDMB-PICA were the only metabolites detected, and plasma concentrations were much lower than the parent drug. 5F-MDMB-PICA induced robust hypothermia and catalepsy-like symptoms that were significantly correlated with concentrations of 5F-MDMB-PICA. Radioligand binding in rat brain membranes revealed 5F-MDMB-PICA displays high affinity for CB1 (IC50 = 2 nM) while metabolites do not. In summary, 5F-MDMB-PICA is a potent CB1 agonist in rats whose pharmacodynamic effects are related to circulating concentrations of the parent drug and not its metabolites.

Recommendations for Toxicological Investigation of Drug-Impaired Driving and Motor Vehicle Fatalities-2021 Update.

This report describes updates to the National Safety Council's Alcohol, Drugs and Impairment Division's recommendations for drug testing in driving under the influence of drug (DUID) cases and motor vehicle fatalities. The updates are based on a survey of drug testing practices in laboratories in the USA and Canada, a comprehensive review of the prior recommendations and data and research on drugs most frequently detected in DUID cases. A consensus meeting was held with representative forensic science practitioners and the authors of this report to update recommendations. No changes were made to the Tier I scope; however, there were changes to cutoffs of some analytes for blood, urine and oral fluid. Due to increased prevalence in DUID cases, trazodone and difluoroethane were added to the Tier II scope. For clarification, Tier I cutoffs reflect free concentrations, and hydrolysis is recommended but not required. The consensus panel concluded that urine is an inferior matrix to blood and oral fluid as it may represent historical use or exposure unrelated to observed impairment; therefore, future iterations of these recommendations will not include urine as a recommended matrix. Laboratories currently testing urine should work with traffic safety partners to encourage the use of blood and oral fluid as more appropriate specimens and adjust their capabilities to provide that testing.

A transnational perspective on the evolution of the synthetic cannabinoid receptor agonists market: Comparing prison and general populations.

The synthetic cannabinoid receptor agonist (SCRA) market is transnational, and the availability of individual SCRAs changes regularly in response to national and international legislative controls. This generates a cyclic pattern and near constant evolution of SCRA compounds. This study reports toxicology-based and/or seized sample-based prevalence data relating to SCRA use in prisons from Germany, the United Kingdom (UK; Scotland and Wales), and the United States (US), representing 4427 individual test results. The study examines SCRA detections in prisons from July 2018 to September 2020, and where possible, prison-based data are compared with SCRA prevalence data in the wider population. The relative influence of Chinese, other international, and national drug legislation on the prevalence of individual SCRAs in prisons is also considered. tert-Leucinate- and valinate-indole- and indazole-3-carboxamides were the most common SCRA detections, and MDMB-4en-PINACA was one of the most commonly detected SCRAs in all jurisdictions by September 2020. However, despite there being a global production and supply market, there were notable regional differences. Analog controls in German and US legislation may have led to increased compound diversity that is not reflected in the UK which has both analog controls and a blanket ban on psychoactive substances. While there were regional differences, SCRA prevalence in prisons closely aligned with the SCRAs detected on the local market, demonstrating that SCRA (and possibly other NPS) monitoring programs in prisons can act as early warning systems for the wider population in that given jurisdiction.

Evaluating Trends in Novel Psychoactive Substances Using a Sentinel Population of Electronic Dance Music Festival Attendees.

Electronic dance music (EDM) festivals have become a popular venue for recreational drug use, including the use of traditional stimulants like 3,4-methylenendioxymethamphetamine (MDMA) and novel psychoactive substances (NPS). Using this cohort of people who use drugs recreationally, this study sought to collect biological specimens and self-reported drug use data from EDM festival attendees in the United States to monitor regional and temporal trends related to NPS use and turnover between 2014 and 2017. Oral fluid samples were collected at three United States EDM festival locations, including Miami, Florida (2014 to 2017); Tampa, Florida (2017) and Atlanta, Georgia (2017). Samples were screened by liquid chromatography-quadrupole time-of-flight mass spectrometry and confirmed by liquid chromatography-tandem mass spectrometry. Over the 4 years, 1,233 oral fluid samples were collected. With respect to self-reported drug use, 63% of respondents reported medicinal and/or recreational drug use within the last week. When comparing 4 years of data from Miami (2014 to 2017), NPS trends showed the disappearance of alpha-PVP after 2014 followed by a significant increase in ethylone positivity in 2015 and rapid decrease in 2016. Dibutylone was identified for the first time in Miami 2016, and N-ethyl pentylone was identified for the first time in Miami 2017. Additionally, 3,4-methylenendioxymethamphetamine positivity steadily increased from 2014 to 2017. A comparison across study sites (Miami, Tampa and Atlanta) and specific trends with respect to novel simulant use are described within. Using this opportunistic approach of monitoring drug trends, we have found that peak positivity of novel stimulants usually is within a year of their first detection. Understanding the dynamics of NPS drug markets will allow laboratories to plan for resource allocation and scope updates within a timely fashion to assist with the detection and confirmation of these emerging substances in samples submitted for forensic analysis.

Evaluation of Synthetic Cannabinoid Metabolites in Human Blood in the Absence of Parent Compounds: A Stability Assessment.

Synthetic cannabinoids represent a chemically diverse class of novel psychoactive substances (NPS) responsible for large analytical and interpretative challenges for forensic toxicologists. Between 2016 and 2019, the three most prevalent synthetic cannabinoids in the United States were MMB-FUBINACA (FUB-AMB), 5F-MDMB-PINACA (5F-ADB) and 5F-MDMB-PICA, based on results from seized drug and toxicology testing. In 2018, accurate determination of synthetic cannabinoid positivity was brought into question as it was determined that the metabolites of these drug species were present in the absence of parent compounds in forensically relevant blood samples. During this study, the stability of MMB-FUBINACA, 5F-MDMB-PINACA and 5F-MDMB-PICA was evaluated, as well as the characterization of breakdown products. A liquid-liquid extraction method was assessed for recovery of basic parent compounds and acidic metabolites and deemed fit for use in this study. Analysis was performed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) using a SCIEX TripleTOF® 5600+. All three synthetic cannabinoids were found to be unstable when stored in blood at either room temperature or refrigerated; all analytes were considerably more stable when stored in the freezer. All three synthetic cannabinoids degraded to their respective butanoic acid metabolites: MMB-FUBINACA 3-methylbutanoic acid, 5F-MDMB-PINACA 3,3-dimethylbutanoic acid and 5F-MDMB-PICA 3,3-dimethylbutanoic acid. All three of these metabolites were studied and determined to be stable in blood at all storage conditions. Considering these results, our laboratory continued testing for synthetic cannabinoid metabolites in blood samples and found 83 positives (21%) for only a synthetic cannabinoid metabolite. A case report is presented herein where 5F-MDMB-PINACA 3,3-dimethylbutanoic acid was identified in the absence of 5F-MDMB-PINACA. Forensic toxicologists should be aware of the results of this study as they directly impact analytical consideration for test development and implementation, as well as interpretation of findings.

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.

Emerging Synthetic Cannabinoids: Development and Validation of a Novel Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry Assay for Real-Time Detection.

Synthetic cannabinoids pose significant threats to public health and safety, as their implications in overdose and adverse events continue to arise in United States and around the world. Synthetic cannabinoids have seen several generations of chemically diverse structural elements, impacting potency and effects. These factors create new analytical challenges for forensic laboratories. This report describes an efficient liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) assay for the identification of synthetic cannabinoid parent compounds and metabolites, including real-time identification of emergent compounds, using a SCIEX TripleTOF® 5600+ with non-targeted SWATH® acquisition. Method validation evaluated precision/accuracy, limits of detection, interferences, processed sample stability and carryover, for which 19 parent compounds and 19 metabolites were tested. To demonstrate feasibility, de-identified blood sample extracts were acquired from a large forensic toxicology laboratory and analyzed using the validated LC-QTOF-MS assay. In mid-2018, 200 blood extracts were analyzed, demonstrating a 19% positivity rate with > 94% agreement rate with original testing. In addition, three newly discovered synthetic cannabinoids were identified, including 5F-MDMB-PICA, 4-cyano CUMYL-BUTINACA and 5F-EDMB-PINACA. These synthetic cannabinoids were previously unreported in forensic toxicology casework in the United States. 5F-MDMB-PICA has become the most prevalent synthetic cannabinoid in United States, as of early 2019. These results demonstrate the effectiveness of this assay and workflow in the identification and characterization of synthetic cannabinoids, as well as the usefulness of sample-mining using non-targeted mass acquisition by LC-QTOF-MS for the discovery of NPS. High resolution mass spectrometry should be considered when developing new or novel assays for synthetic cannabinoids.

Detection and characterization of the new synthetic cannabinoid APP-BINACA in forensic casework.

New psychoactive substances (NPS) continue to emerge around the world. APP-BINACA (or APP-BUTINACA), a novel synthetic cannabinoid, was first reported in Europe in January 2019 and later in the United States in March 2019. APP-BINACA was identified in the United States for the first time in blood sample extracts from forensic casework by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). To date, APP-BINACA has been identified in 11 forensic toxicology cases from five states and in both medicolegal death investigations and drug impaired driving investigations. APP-BINACA was commonly found in combination with 4F-MDMB-BINACA. Subsequent to its discovery in biological samples, APP-BINACA was detected and characterized in seized drug material by gas chromatography mass spectrometry (GC-MS), LC-QTOF-MS, and nuclear magnetic resonance (NMR) spectroscopy. Further analysis of biological specimens resulted in the identification of five metabolites, including 4-HO-APP-BINACA and APP-BINACA 3-phenylpropanoic acid. The frequency of APP-BINACA detection appears to be increasing and this new synthetic cannabinoid has been identified as a possible contributory factor in adverse events, including death. This is the first literature report regarding the characterization of the new synthetic cannabinoid APP-BINACA in humans. Since it is not widely tested for, it is not yet known the extent to which APP-BINACA is contributing to morbidity and mortality, but forensic scientists, public health officials, and others should be aware of its possible presence and impact. Laboratories should incorporate APP-BINACA into testing workflows for detection and confirmation, where possible.

4F-MDMB-BINACA: A New Synthetic Cannabinoid Widely Implicated in Forensic Casework.

This is the first report regarding the characterization of the new synthetic cannabinoid 4F-MDMB-BINACA. 4F-MDMB-BINACA was first analytically confirmed in seized drug material using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF), and nuclear magnetic resonance (NMR) spectroscopy. Subsequent to this characterization, 4F-MDMB-BINACA was detected in biological specimens collected as part of forensically relevant casework, including medicolegal death investigations and drug impaired driving investigations, from a variety of regions in the United States. Further analysis of biological specimens resulted in the identification of the metabolites 4F-MDMB-BINACA 3,3-dimethylbutanoic acid and 4-OH-MDMB-BINACA. 4F-MDMB-BINACA is appearing with increasing frequency as a contributory factor in deaths, creating morbidity and mortality risks for drug users. Laboratories must be aware of its presence and impact, incorporating 4F-MDMB-BINACA into workflows for detection and confirmation.

The Detection of Novel Stimulants in Oral Fluid from Users Reporting Ecstasy, Molly and MDMA Ingestion.

"Ecstasy" and "Molly" are common drug slang terms used among club and rave cultures to denote preparations believed to contain 3,4-methylenedioxymethamphetamine (MDMA). However, users of Ecstasy and Molly have increasingly tested positive for novel psychoactive substances (NPS), notably novel stimulants. To evaluate hypothesized non-specific and interchangeable use of the terms Ecstasy, Molly and MDMA, self-reported drug use was compared against toxicological findings in biological specimens. Oral fluid specimens were collected from participants attending large multi-day electronic dance music festivals in Miami, FL; Tampa, FL; and Atlanta, GA. Participants additionally completed a structured survey about recent recreational drug use. Collected specimens were screened for therapeutic drugs, common drugs of abuse and NPS using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF). Positive screen results were confirmed by validated liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for MDMA, MDA, methylone, dimethylone, ethylone, butylone, dibutylone, eutylone, pentylone, N-ethyl pentylone (ephylone), alpha-PVP and 4-fluoroamphetamine (4-FA). During this 4-year study, 223 participants provided an oral fluid specimen and indicated recent use of Ecstasy, Molly and/or MDMA/MDA. Of these subjects, 203 (91.0%) indicated only one of these drug terms; while 20 (9.0%) participants indicated a combination of multiple terms. Of the 203 participants designating only one drug term, 123 (60.6%) reported Molly use, 55 (27.1%) reported MDMA use and 25 (12.3%) reported Ecstasy use. Seven participants reported the use of MDA, but these responses were paired with MDMA responses due to detection of MDA as a metabolite of MDMA. The results from this study indicate that there are inconsistencies between admission to drug use and toxicological findings in this population. Of the 223 participants who indicated use of Ecstasy, Molly and/or MDMA/MDA, MDMA without a novel stimulant was confirmed in 121 (54.3%) participants, while 66 (29.6%) tested positive for at least one novel stimulant.

Activity-Based Concept to Screen Biological Matrices for Opiates and (Synthetic) Opioids.

BACKGROUND: Detection of new highly potent synthetic opioids is challenging as new compounds enter the market. Here we present a novel screening method for the detection of opiates and (synthetic) opioids based on their activity. METHODS: A cell-based system was set up in which activation of the µ-opioid receptor (MOR) led to recruitment of ß-arrestin 2, resulting in functional complementation of a split NanoLuc luciferase and allowing readout via bioluminescence. Assay performance was evaluated on 107 postmortem blood samples. Blood (500 µL) was extracted via solid-phase extraction. Following evaporation and reconstitution in 100 µL of Opti-MEM® I, 20 µL was analyzed in the bioassay. RESULTS: In 8 samples containing synthetic opioids, in which no positive signal was obtained in the bioassay, quadrupole time-of-flight mass spectrometry revealed the MOR antagonist naloxone, which can prevent receptor activation. Hence, further evaluation did not include these samples. For U-47700 (74.5-547 ng/mL) and furanyl fentanyl (<1-38.8 ng/mL), detection was 100% (8/8) for U-47700 and 95% (21/22) for furanyl fentanyl. An analytical specificity of 93% (55/59) was obtained for the opioid negatives. From an additional 10 samples found to contain other opioids, 5 were correctly scored positive. Nondetection in 5 cases could be explained by very low concentrations (<1 ng/mL alfentanil/sufentanil) or presence of inactive enantiomers. CONCLUSIONS: The MOR reporter assay allows rapid identification of opioid activity in blood. Although the cooccurrence of opioid antagonists is currently a limitation, the bioassay's high detection capability, specificity, and untargeted nature may render it a useful first-line screening tool to investigate potential opioid intoxications.

N-Ethyl Pentylone (Ephylone) Intoxications: Quantitative Confirmation and Metabolite Identification in Authentic Human Biological Specimens.

N-ethyl pentylone (ephylone) has been identified as the most recent novel stimulant to emerge into the arena of evolving novel psychoactive substances (NPS). Due to its novelty, information regarding case reports with associated quantitative confirmations, biotransformation pathways, and identified unique metabolites will assist the scientific community in understanding the implications of the emergence and risks associated with N-ethyl pentylone use. Authentic blood specimens (n = 26) submitted as part of toxicological death investigations or drugged driving casework tested positive for N-ethyl pentylone, and were quantitatively analyzed by liquid chromatography tandem mass spectrometry (LC-MS-MS). N-ethyl pentylone concentrations ranged from 12 to 1,200 ng/mL, with mean (±standard deviation) and median concentrations of 313 (±366) and 125 ng/mL, respectively, excluding one case measured at 50,000 ng/mL. N-ethyl pentylone was often found in combination with other drugs of abuse and NPS, include a variety of novel opioids including fentanyl analogs. Oral fluid specimens (n = 5), collected from recreational drug users at a dance music festival, were quantitatively analyzed using LC-MS-MS. Concentrations ranged from 12.6 to 1,377 ng/mL. Additional analysis was performed to characterize the metabolic profile of N-ethyl pentylone using human liver microsomes (HLM), followed by confirmation of the presence of the proposed metabolites in a subset of the blood specimens and oral fluid specimens. Metabolomic analysis was performed using a liquid chromatograph quadrupole time-of-flight mass spectrometer (LC-QTOF), followed by data processing using MetabolitePilot™ software. In vivo verification of in vitro HLM-generated metabolites resulted in the confirmation of four metabolites. Reduction of the beta-ketone to an alcohol resulted in the most prominent metabolite found in the authentic specimens, and its uniqueness to N-ethyl pentylone leads to this metabolite being an appropriate biomarker to determine N-ethyl pentylone ingestion. This is the first study to report N-ethyl pentylone concentrations and to characterize the metabolic profile of N-ethyl pentylone.

Dibutylone (bk-DMBDB): Intoxications, Quantitative Confirmations and Metabolism in Authentic Biological Specimens.

The number of emerging novel stimulants modified based on beta-keto variations of amphetamine-like substances continues to rise. Dibutylone reports described in the medical and toxicological literature are limited, therefore little information is available in terms of quantitative confirmation or metabolism. During this study, authentic human specimens, including blood, urine, vitreous humor, oral fluid and liver were quantitatively and qualitatively analyzed for the presence of dibutylone and butylone, with paired case history and demographic information. Dibutylone concentrations were variable across all specimen types, specifically ranging from 10 to 1,400 ng/mL in postmortem blood specimens. The metabolic profile of dibutylone was mapped by in vitro incubation with human liver microsomes (HLM). Samples were analyzed using a SCIEX TripleTOF® 5600+ quadrupole time-of-flight mass spectrometer. Data processing was conducted using MetabolitePilot™. Authentic human specimens, including blood, urine, vitreous humor, oral fluid and liver, were utilized for in vivo verification of five HLM-generated metabolites in analytically confirmed cases of dibutylone use. Butylone was confirmed as a metabolite of dibutylone, but issues involving co-ingestion of these two novel stimulants or potential co-existence from synthesis lead to ineffectiveness as a true biomarker. Hydrogenation of the beta-ketone of dibutylone resulted in the most prominent metabolite found in human specimens, and its uniqueness to dibutylone over other stimulants leads to its classification as an appropriate biomarker for dibutylone ingestion. This is the first study to map the metabolic profile of dibutylone, including verification in authentic specimens, confirming metabolic conversion to butylone and identifying biomarkers more useful in forensic toxicological drug testing.

Field Detection of Drugs of Abuse in Oral Fluid Using the Alere™ DDS®2 Mobile Test System with Confirmation by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS).

The collection and analysis of drugs in oral fluid (OF) at the roadside has become more feasible with the introduction of portable testing devices such as the Alere™ DDS®2 Mobile Test System (DDS®2). The objective of this study was to compare the on-site results for the DDS®2 to laboratory-based confirmatory assays with respect to detection of drugs of abuse in human subjects. As part of a larger Institutional Review Board approved study, two OF samples were collected from each participant at a music festival in Miami, FL, USA. One OF sample was field screened using the DDS®2, and a confirmatory OF sample was collected using the Quantisal™ OF collection device and submitted to the laboratory for testing. In total, 124 subjects participated in this study providing two contemporaneous OF samples. DDS®2 field screening yielded positive results for delta-9-tetrahydrocannabinol (THC) (n = 27), cocaine (n = 12), amphetamine (n = 3), methamphetamine (n = 3) and benzodiazepine (n = 1). No opiate-positive OF samples were detected. For cocaine, amphetamine, methamphetamine and benzodiazepines, the DDS®2 displayed sensitivity, specificity and accuracy of 100%. For THC, the DDS®2 displayed sensitivity of 90%, specificity of 100% and accuracy of 97.5%, when the threshold for confirmation matched that of the manufacturers advertised cut-off. When this confirmatory threshold was lowered to the analytical limit of detection (i.e., 1 ng/mL), apparent device performance for THC was poorer due to additional samples testing positive by confirmatory assay that had tested negative on the DDS®2, demonstrating a need for correlation between manufacturer cut-off and analytical reporting limit. These results from drug-using subjects demonstrate the value of field-based OF testing, and illustrate the significance of selecting an appropriate confirmation cut-off concentration with respect to performance evaluation and detection of drug use.