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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.

Novel psychoactive substances (NPS) continue to represent a threat to public health and safety. The number of new drugs in the latest emergent synthetic opioid class—the 2-benzylbenzimidazole analogs—also called the nitazenes—has begun to dominate the current new synthetic opioid (NSO) subclass of NPS. We describe a liquid chromatography–tandem quadrupole mass spectrometry method for the quantification of nine analogs and/or metabolites of drugs in this series: isotonitazene, metonitazene, protonitazene, etonitazene, clonitazene, flunitazene, N-desethyl isotonitazene, 5-amino isotonitazene and 4ʹ-hydroxy nitazene in human whole blood, urine, and tissue. Samples were prepared for analysis using a basic liquid–liquid extraction. Chromatographic separation was achieved using a C-18 analytical column. Multiple reaction monitoring mode was used for detection. The calibration range for the analytes was 0.5–50 ng/mL (except for 5-amino isotonitazene, which was 1.0–50 ng/mL). The limit of detection was 0.1 ng/mL, and the limit of quantitation was 0.5 ng/mL. The method had no carryover or interferences. Ionization enhancement was observed but did not affect quantitation. All analytes passed the method validation assessment. Authentic human samples suspected of containing NSOs were obtained from a medical examiner and coroner offices, as well as partnering forensic toxicology laboratories. Isotonitazene was confirmed in 92 blood samples, and its metabolites were confirmed across various matrices. Metonitazene (n = 35), flunitazene (n = 5), protonitazene (n = 3), etodesnitazene (n = 2) and butonitazene (n = 1) were also detected in cases. These newly emerging 2-benzylbenzimidazole analogs were commonly found in combination with NPS benzodiazepines and opioids (e.g., flualprazolam, fentanyl). Nitazene analogs are potent esoteric drugs that may not be identified during routine toxicological screening, and specialized assays based on sensitive instrumentation are needed to accurately characterize these NSOs.

The purpose of this study was to examine the impact of 59 novel psychoactive compounds on common enzyme-linked immunosorbent assay (ELISA) testing kits. Concentrations above and below the individual kit reporting limits in each class were measured. Compounds that exhibited cross-reactivity were then spiked individually using a seven-point response curve to determine the level of cross-reactivity. Diclazepam, delorazepam, phenazepam, flualprazolam, bromazolam, adinazolam, 3-methoxy-PCP, 3-hydroxy-PCP (3-OH-PCP), phenylfentanyl, para-methylacetylfentanyl and para-fluorofuranylfentanyl were determined to cross-react in the respective kits below. Herein, we detail the cross-reactivity that was observed with the above novel psychoactive substances on Immunalysis Benzodiazepine (BEN), Phencyclidine (PCP), Fentanyl (FEN), Buprenorphine (BUP), Opiates (OPI) and Oxycodone (OXY) Direct ELISA kits.

Synthetic opioids constitute one of the fastest-growing groups of new psychoactive substances (NPS) worldwide. With fentanyl analogues being increasingly controlled via classwide 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 1 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.

Non-pharmaceutical fentanyl and its analogs have driven striking increases in opioid-associated overdose deaths. These highly potent opioids can be found at low concentrations in biological specimens. Little is known regarding the concentrations of these substances among survivors of non-fatal overdoses. In a locale where fentanyl is responsible for the majority of non-fatal opioid overdoses, we compared the concentration of fentanyl in blood to naloxone dosing in the presence and absence of a concurrent sedative-hypnotic exposure.In this pilot study, we enrolled adult patients presenting to the emergency department (ED) who: (1) arrived after an overdose requiring naloxone for the reversal of respiratory depression; and (2) who required venipuncture or intravenous access as part of their clinical care. Blood specimens (Nineteen of twenty participants (95%) were exposed to fentanyl prior to their overdose; the remaining participant tested positive for heroin metabolites. No participants reported pharmaceutical fentanyl use. Fentanyl analogs - acetylfentanyl or carfentanil - were present in three specimens. In 11 cases, fentanyl and its metabolites were the only opioids identified. Among the fentanyl-exposed, blood concentrations ranged from0.1-19 ng/mL with a mean of 6.2 ng/mL and a median of 3.6 ng/mL. There was no relationship between fentanyl concentration and naloxone dose administered for reversal. We detected sedative-hypnotics (including benzodiazepines, muscle relaxants, and antidepressants) in nine participants. Among the sedative-hypnotic exposed, fentanyl concentrations were lower, but naloxone dosing was similar to those without a concomitant exposure.In this study, we found that: 1) fentanyl was present in the blood of nearly all participants; 2) fentanyl concentrations were lower among study participants with concomitant sedative-hypnotic exposure; and 3) the dose of naloxone administered for overdose reversal was not associated with the measured fentanyl concentration in blood specimens. Our results underscore the role that tolerance and concomitant drug exposure play in the precipitation and resuscitation of management of opioid overdose.

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.

Isotonitazene is an illicit synthetic opioid associated with many intoxications and fatalities. Recent studies show that isotonitazene is a potent µ-opioid receptor (MOR) agonist in vitro, but little information is available about its in vivo effects.The aims of the present study were to investigate the pharmacokinetics of isotonitazene in rats, and relate pharmacokinetic parameters to pharmacodynamic effects.Isotonitazene and its metabolites were identified and quantified by liquid chromatography tandem quadrupole mass spectrometry (LC-QQQ-MS). Male Sprague-Dawley rats with jugular catheters and subcutaneous (s.c.) temperature transponders received isotonitazene (3, 10, 30 µg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, and 240 min post-injection, and plasma was assayed using LC-QQQ-MS. At each blood draw, body temperature, catalepsy scores, and hot plate latencies were recorded.Maximum plasma concentrations of isotonitazene rose in parallel with increasing dose (range 0.2-9.8 ng/mL) and half-life ranged from 23.4 to 63.3 min. The metabolites 4'-hydroxy nitazene and N-desethyl isotonitazene were detected, and plasma concentrations were below the limit of quantitation (0.5 ng/mL) but above the limit of detection (0.1 ng/mL). Isotonitazene produced antinociception (EDIn summary, isotonitazene is a potent MOR agonist whose pharmacodynamic effects are related to circulating concentrations of the parent drug. The high potency of isotonitazene portends substantial risk to users who are exposed to the drug.

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.

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.

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.

A new class of synthetic cannabinoids has emerged as new psychoactive substances (NPS). Similar in structure to JWH-022, these substances contain alkene modifications to the tail region of the synthetic cannabinoid core structure, and nomenclature denotes these new analogues as pent-4en or but-3en species. Internationally, two analogues from this new series recently emerged: MDMB-4en-PINACA and MMB-4en-PICA. Previously, data regarding activity and potential toxicity were not available.In vitroassessment of cannabinoid receptor 1 (CB1) activation via the beta-arrestin 2 recruitment was studied for three (3) pent-4en analogues, one (1) but-3en analogue, and one (1) principal metabolite. MDMB-4en-PINACA (2.47 nM, 239%), MDMB-4en-PICA (11.5 nM, 302%), and MDMB-3en-BINACA (14.3 nM, 286%) were highly potent and efficacious (comparison: JWH-018, 25.3 nM, 100%), while the potencies of MMB-4en-PICA and MDMB-4en-PINACA 3,3-dimethylbutanoic acid were markedly lower. Modifications to core and tail structural features (i.e., indole vs. indazole) led to relatively small differences in potency, while changes among the head region led to larger differences. Sample-mining and data-mining conducted on toxicology samples led to the identification of MDMB-4en-PINACA in 25 forensic toxicology cases, including postmortem and impaired driving investigations, with case details and limited histories described herein. Moderate geographical distribution of MDMB-4en-PINACA was noted in the United States with emergence in the Northeast, Midwest, South, and West regions. Results from toxicology testing paired with case history show the potential for MDMB-4en-PINACA to cause or contribute to impairment or death. Forensic scientists, public health and public safety officials, law enforcement, clinicians, medical examiners, and coroners should consider involvement of emergent synthetic cannabinoids in their work and that new analogues containing an alkene tail can retain similar or increased potency and toxicity.

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.

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.

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-β-arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC

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.

CONTEXT: Synthetic cannabinoids (SCs) are a structurally heterogenous synthetic class of drugs of abuse. The objective was to describe the incidence of acute respiratory failure in Emergency Department (ED) patients with confirmed SC exposure, and to investigate the association between SC overdose with respiratory failure compared to non-SC overdose. METHODS: This was an observational cohort of ED patients ≥18 years with suspected cannabinoid overdose between 2015 and 2020 at two tertiary-care hospitals. Patient serum was analyzed via liquid chromatography/quadrupole time-of-flight mass spectrometry using a library with >800 drugs including novel psychoactive substances. The primary outcome was acute respiratory failure. DISCUSSION: Of 83 patients with suspected cannabinoid overdose, there were 29 confirmed SC overdoses: 5 F-MDMB-PICA (n = 18) and its metabolite 5OH-MDMB-PICA (n = 16), ADB-FUBINACA (n = 4), AB-CHIMINACA (n = 4), AB-FUBINACA (n = 1), AB-PINACA (n = 1), MDMB-4en-PINACA (n = 1), and 4F-MDMB-BINACA (n = 1). Overall, incidence of acute respiratory failure was 31.3% (95%CI 21.6–42.4). Compared to non-SC overdose, confirmed SC overdose was significantly associated with respiratory failure (25.0% SC vs. 4.2% non-SC, p = 0.05). CONCLUSION: This study demonstrates that SCs are associated with respiratory failure. Since respiratory depression is a potentially lethal adverse effect of SC overdose, future research is warranted.

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.

Indazole carboxamide synthetic cannabinoids remain the most prevalent subclass of new psychoactive substances (NPS) reported internationally. However, the metabolic and pharmacological properties of many of these compounds remain unknown. Elucidating these characteristics allows members of the clinical and forensic communities to identify causative agents in patient samples, as well as render conclusions regarding their toxic effects. This work presents a detailed report on the in vitro phase I metabolism of indazole carboxamide synthetic cannabinoid APP-CHMINACA (PX-3). Incubation of APP-CHMINACA with human liver microsomes, followed by analysis of extracts via high-resolution mass spectrometry, yielded 12 metabolites, encompassing 7 different metabolite classes. Characterization of the metabolites was achieved by evaluating the product ion spectra, accurate mass and chemical formula generated for each metabolite. The predominant biotransformations observed were hydrolysis of the distal amide group and hydroxylation of the cyclohexylmethyl (CHM) substituent. Nine metabolites were amide hydrolysis products, of which five were monohydroxylated, one dihydroxylated and two were ketone products. The metabolites in greatest abundance in the study were products of amide hydrolysis with no further biotransformation (M1), followed by amide hydrolysis with monohydroxylation (M2.1). Three APP-CHMINACA-specific metabolites were generated, all of which were hydroxylated on the CHM group; one mono-, di- and tri-hydroxylated metabolite each was produced, with dihydroxylation (M6) present in the greatest abundance. The authors propose that metabolites M1, M2.1 and M6 are the most appropriate markers to determine consumption of APP-CHMINACA. The methods used in the current study have broad applicability and have been used to determine the in vitro metabolic profiles of multiple synthetic cannabinoids and other classes of NPS. This research can be used to guide analytical scientists in method development, synthesis of reference material, pharmacological testing of proposed metabolites and prediction of metabolic processes of compounds yet to be studied.

Novel psychoactive substances (NPS) are synthetic drugs that pose serious public health and safety concerns. A multitude of NPS have been identified in the United States, often implicated in forensic investigations. The most common and effective manner for identifying NPS is by use of mass spectrometry and the true utility lies within nontargeted acquisition techniques. During this study, a liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) assay was developed, validated, and implemented for forensic toxicology testing. A SCIEX TripleTOF™ 5600 + with SWATH® acquisition was used. Resulting data were compared against an extensive library database containing more than 800 compounds. The LC-QTOF-MS assay was applied to the reanalysis of biological sample extracts to discover emergent NPS. More than 3,000 sample extracts were analyzed, and more than 20 emerging NPS were detected for the first time. Among these were isopropyl-U-47700, 3,4-methylenedioxy-U-47700, fluorofuranylfentanyl, N-methyl norfentanyl, 2F-deschloroketamine, 3,4-methylenedioxy-alpha-PHP, eutylone, and N-ethyl hexedrone.

Mitragynine is the primary active alkaloid in the leaves of the tropical tree Mitragyna speciosa, and goes by the popular names “Kratom”, biak-biak and maeng da. Mitragynine is increasingly seen in forensic toxicology casework including driving under the influence of drugs and medicolegal death investigation cases. The toxicity of mitragynine continues to be debated in the scientific community as advocates highlight its long history of use in Southeast Asia and testimonials to its benefits by present-day users, while opponents point to an increasing number of adverse events tied to mitragynine use in Western societies. Quantitative reports of mitragynine in biological specimens from forensic investigations in the literature are sparse and may be influenced by poor analyte stability and inadequate resolution of mitragynine from its diastereomers, which could lead to falsely elevated concentrations and subsequently render those reported concentrations inappropriate for comparison to a reference range. Over the course of 27 months, 1,001 blood specimens submitted to our laboratory tested positive for mitragynine using a sensitive and specific quantitative LC-MS/MS method; concentrations ranged from 5.6–29,000 ng/mL, with mean and median concentrations of 410 ± 1,124 and 130 ng/mL, respectively. Mitragynine presents an analytical challenge that requires a method that appropriately separates and identifies mitragynine itself from its isomers and other related natural products. We describe a validated analytical method and present a short series of case reports that provide examples of apparent adverse events, and the associated range of mitragynine concentrations. This type of analytical specificity is required to appropriately interpret mitragynine concentrations detected in biological specimens from forensic casework and assess its potential toxicity.

The analysis of biological specimens collected at autopsy for the presence of exogenous insulin(s) is of special interest in select death investigations as they may be suspected in the cause of a death. Technical challenges include the limited stability of insulin, and the forensic requirement of differentiating endogenous insulin from pharmaceutical analogs. A novel method was developed for the detection and quantification of human insulin, Glulisine, Lispro, Aspart, Glargine and Detemir in vitreous fluid. An immunoaffinity extraction procedure is performed followed by separation of the insulin α- and β-chains. Liquid chromatography tandem mass spectrometry analysis of the β-chain allows for the unequivocal identification of each insulin analog. The analytical measurement range for each insulin was 0.5–25 ng/mL. The method was evaluated for accuracy, precision, carryover, interferences and stability. Eight vitreous fluid samples collected from cases where untoward insulin use was suspected were subjected to analysis. Positive results were obtained from three samples, and a detailed case history is provided for one of these cases. Even though insulin instability in postmortem biological fluid remains a challenge, this method allows for a reliable forensic-level analysis in vitreous fluid.

The purpose of this study was to identify the extent and types of drugs found in alleged drug facilitated sexual assaults (DFSA) in 37 states and 1 territory of the United States. In total, 1000 cases were reviewed. Between the cases that gender was provided (613), most of the victims (91.68%) were woman, mean age of 26.8 years old. Blood and/or urine samples were tested. Twenty-one point six percent of the cases were negative for intoxicating substances. A hundred and one different substances were detected. Overall, ethanol was the most prevalent substance, detected in 30.9% of the cases (309 cases), followed by cannabinoids (THC/THCCOOH/11-OH-THC) (28.8% of cases), amphetamine/methamphetamine (16.5% of cases), cocaine/metabolites (10.4% of cases), and clonazepam/metabolite (7.6% of cases). The mean, median and range concentrations of ethanol in blood (n = 309) were 98.6 mg/dL, 82.0 mg/dL and 9.2–366 mg/dL, respectively. Ethanol and cannabinoids were the most frequent combination found. The absence of alcohol and drugs in some cases may represent delay in collecting samples.

Cocaine is usually sold as a white powder and can contain several adulterants and diluents, known as cutting agents. The cutting agents play an important role in the identification of trafficking routes, and they can also modify or intensify signs and symptoms of drug intoxication increasing the risk to the health's user. The purpose of this work was to quantify cocaine and cutting agents in 116 illicit samples from NMS Labs, Willow Grove, PA, U.S. Gas chromatography - mass spectrometry (GC-MS) and handle-portable gas chromatography toroidal ion trap mass spectrometry (GC-TMS) were used as screening methods A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of cocaine, levamisole, benzocaine, phenacetin, hydroxyzine, theophylline, diltiazem, acetaminophen and caffeine. Cocaine-d3 and caffeine-d3 were used as internal standards. The method was shown to be precise, accurate and linear over a range of 50-2000ng/mL for all analytes. Cocaine was the only detected compound in 16.37% (n=19) of the samples. Between the identified cutting agents, levamisole was the most abundant substance found (79.31% of the total samples, amounts ranging from 0.2 to 74.3%), followed by phenacetin (18.96%, 0.3-46.8%), caffeine (12.06%, 0.2-32.2%), hydroxyzine (9.48%, 0.7-13.8%) and benzocaine (5.17%, 0.4-58.3%). GC-TMS was considered suitable to be used as a tool in forensic analysis as a screening method for cocaine, benzocaine, phenacetin, hydroxyzine and caffeine with restrictions to be used for levamisole, while GC-MS presented good results in screening analysis for cocaine, levamisole, benzocaine, phenacetin, hydroxyzine and caffeine.

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.

Toxic adulterants are drug or chemical agents used to add bulk volume to traditional drugs of abuse such as cocaine and heroin. These cutting agents include levamisole, metamizole, noxiptillin, phenacetin and xylazine as well as common legal drugs such as acetaminophen, caffeine, diphenhydramine, lidocaine, quinine, quetiapine and tramadol. Because they possess pharmacological activity they result in exposure of the user, but also in the case of pregnant women, the developing fetus, to potential drug toxicity. We describe the development, validation and implementation of a rapid (48 second sample-to-sample) test based on a qualitative data-dependent liquid chromatography–quadrupole time of flight mass spectrometry method for the analysis of toxic adulterating substances in umbilical cord tissue (UCT) samples. The method provides a means of studying potential in utero exposure to these agents. Library spectra comparison at three different collision energies was used in conjunction with retention time and accurate mass to identify these substances in UCT. Analytically based reporting limits were established to determine positivity rates of adulterants in UCT utilizing a standard addition approach. The method was applied to authentic cocaine and opioid positive UCT to screen for toxic adulterants. There were a total of 82 potential adulterant positives found in a 30-sample cohort of authentic UCT samples, with an average of 2.7 substances per case. Lidocaine was the predominant finding followed by caffeine, and diphenhydramine all of which could result from non-illicit drug exposure, however, there were positives for levamisole, phenacetin, noxiptillin and xylazine none of which are approved in the United States for human therapeutic use. This initial set of data established a preliminary positivity rate of potentially toxic adulterants in UCT samples positive for cocaine or opioid use.

Discovered in the 1960s, the common anthelminthic levamisole has seen widespread use in veterinary applications. Its use rapidly expanded thereafter to include human medical treatments for a variety of acute and chronic disorders. Because of reports of severe adverse effects, the US Food and Drug Administration withdrew levamisole's approval for human use in 2000; however, medical options outside the United States and illicit options worldwide allow continued accessibility to levamisole. The compound is rapidly metabolized in the body, with at least 2 known active metabolites. Levamisole has a broad range of immunomodulatory effects, including both stimulatory and inhibitory effects on immune responses. It is generally well tolerated at therapeutic concentrations, although a variety of autoimmune-related adverse effects have been reported, including agranulocytosis, leukopenia, purpura, and visible necrotized skin tissue. Individuals with levamisole-compromised immune systems are more susceptible to infections, including COVID-19. Since the early 2000's, levamisole has been frequently used as an adulterating agent in illicit street drugs, especially cocaine, fentanyl, and heroin. Although its prevalence has varied over time and geographically, levamisole has been detected in up to 79% of the street supply of cocaine at levels up to 74% by weight. Its presence in illicit drug markets also raises concern over the potential for exposure of children and neonates, although this is supported by only limited anecdotal evidence. Levamisole is not currently included in routine drug testing panels, although a variety of confirmatory testing techniques exist across a range of antemortem and postmortem specimen options. Because of its varying presence in illicit drug markets, both the medical and forensic communities need to be aware of levamisole and its potential impact on toxicological 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.

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.

FUB-AMB, an indazole carboxamide synthetic cannabinoid recreational drug, was one of the compounds most frequently reported to governmental agencies worldwide between 2016 and 2019. It has been implicated in intoxications and fatalities, posing a risk to public health. In the current study, FUB-AMB was incubated with human liver microsomes (HLM) to assess its metabolic fate and stability and to determine if its major ester hydrolysis metabolite (M1) was present in 12 authentic forensic human blood samples from driving under the influence of drug cases and postmortem investigations using UHPLC-MS/MS. FUB-AMB was rapidly metabolized in HLM, generating M1 that was stable through a 120-min incubation period, a finding that indicates a potential long detection window in human biological samples. M1 was identified in all blood samples, and no parent drug was detected. The authors propose that M1 is a reliable marker for inclusion in laboratory blood screens for FUB-AMB; this metabolite may be pharmacologically active like its precursor FUB-AMB. M1 frequently appears in samples in which the parent drug is undetectable and can point to the causative agent. The results suggest that it is imperative that synthetic cannabinoid laboratory assay panels include metabolites, especially known or potential pharmacologically active metabolites, particularly for compounds with short half-lives.

Rationale Indazole carboxamide synthetic cannabinoids, a prevalent class of recreational drugs, are a major clinical, forensic and public health challenge. One such compound, 5F-ADB, has been implicated in fatalities worldwide. Understanding its metabolism and distribution facilitates the development of laboratory assays to substantiate its consumption. Synthetic cannabinoid metabolites have been extensively studied in urine; studies identifying metabolites in blood are limited and no data on the metabolic stability (half-life, clearance and extraction ratio) of 5F-ADB have been published prior to this report. Methods The in vitro metabolism of 5F-ADB was elucidated via incubation with human liver microsomes for 2 h at 37°C. Samples were collected at multiple time points to determine its metabolic stability. Upon identification of metabolites, authentic forensic human blood samples underwent liquid-liquid extraction and were screened for metabolites. Extracts were analyzed via ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS) operated in positive electrospray ionization mode. Results Seven metabolites were identified including oxidative defluorination (M1); carboxypentyl (M2); monohydroxylation of the fluoropentyl chain (M3.1/M3.2) and indazole ring system (M4); ester hydrolysis (M5); and ester hydrolysis with oxidative defluorination (M6). The half-life (3.1 min), intrinsic clearance (256.2 mL min-1 kg-1 ), hepatic clearance (18.6 mL min-1 kg-1 ) and extraction ratio (0.93) were determined for the first time. In blood, M1 was present in each sample as the most abundant substance; two samples contained M5; one contained 5F-ADB, M1 and M5. Conclusions 5F-ADB is rapidly metabolized in HLM. 5F-ADB, M1 and M5 are pharmacologically active at the cannabinoid receptors (CB1 /CB2 ) and M1 and M5 may contribute to a user's impairment profile. The results demonstrate that it is imperative that synthetic cannabinoid assays screen for pharmacologically active metabolites, especially for drugs with short half-lives. The authors propose that M1 and M5 are appropriate markers to include in laboratory blood tests screening for 5F-ADB.

In today's environment in the field of forensic science where continual advancements in technology and analytical approaches are the norm, the need for forensic practitioners with more specialized and subject-specific knowledge is critical. An updated survey targeting the preferred educational requirements by senior practitioners, crime laboratory directors and managers for entry level applicants was conducted. Results underscored a preference for specialized coursework within specific disciplines in preparing the next generation of forensic scientists while maintaining a strong foundation in the natural sciences at the undergraduate level. Practitioners, regardless of discipline, are seeking applicants with exposure to advanced curriculum content in addition to refined professional skills and critical thinking capabilities. The results of this survey reflect a transition in the needs of crime laboratory employers from a general, broad based criminalistics curriculum as described under current accreditation guidelines, to a focused subject matter rich curriculum with additional management and professional content. Keywords: Forensic science, Undergraduate education, Graduate education, Laboratory survey, Educational requirements

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.

The synthetic opioid landscape continues to change as non-fentanyl-related substances appear in forensic toxicology casework. Among the newest synthetic opioids to emerge is isotonitazene, an analogue of a benzimidazole class of analgesic compounds. Isotonitazene is an active and potent synthetic opioid, but the extent to which this compound is causing toxicity among drug users was previously unknown. This report describes the confirmation and quantitation of isotonitazene in blood, urine and vitreous fluid through standard addition, as well as in vivo metabolic profile determination in drug users. Quantitative analysis was performed using liquid chromatography tandem mass spectrometry (LC–MS/MS), and metabolite discovery was performed using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In total, 18 cases were confirmed positive for isotonitazene, nine of which were previously negative for any opioid. The average isotonitazene concentration in blood was 2.2 ± 2.1 ng/mL (median 1.75 ng/mL, range 0.4–9.5 ng/mL), and the average isotonitazene concentration in urine was 2.4 ± 1.4 ng/mL (median 2.7 ng/mL, range 0.6–4.0 ng/mL). The lowest concentration of isotonitazene in blood was 0.4 ng/mL (two cases) with no other opioids present; findings in death investigations. Four metabolites of isotonitazene were detected in vivo. N- and O-dealkylation products were determined to be the most prominent urinary biomarkers, while 5-amino-isotonitazene was identified in most blood samples. The prevalence and popularity of isotonitazene continue to increase in the United States in early 2020. Toxicologists, medical examiners and coroners should be aware of novel opioids outside the standard scope of testing, especially in medicolegal death investigations. Forensic scientists should add isotonitazene to testing procedures, and public health officials should counsel about potent new drugs and the dangers of opioid use.

The cutting agents, classified as diluents (pharmacologically inactive) or adulterants (pharmacologically active), are substances commonly used to cut drugs of abuse to increase profits. These substances are constantly changing over time, increasing the risks to the user's health caused by the compounds' potential individual toxicities as well as their drug-drug interactions. This work aimed to develop and validate a screening method using a portable quadrupole-based gas chromatography mass spectrometer (FLIR Griffin™ G510) to identify drugs of abuse and adulterants in seized material, and compare it with a well validated standard technology, gas chromatography mass spectrometry (GC-MS). The method was validated for the identification of alprazolam, amphetamine, aminopyrine, benzocaine, caffeine, cocaine, codeine, diltiazem, ephedrine, fentanyl, fenethylline, furanylfentanyl, heroin, hydroxyzine, levamisole, lidocaine, methamphetamine, morphine, noramidopyrine (a marker of metamizole), phencyclidine, phenacetin, procaine, strychnine and xylazine. The targeted substances were chosen based on current intelligence regarding prevalent adulterants observed in multiple jurisdictions. Interference, precision, robustness and carryover were evaluated. The method was successfully validated and proved to be suitable to detect and identify the 24 target compounds proposed. The reliability of the instrument for detecting the presence of targeted compounds was analyzed by using Receiver Operating Characteristic (ROC) analysis. The portable quadrupole-based gas chromatography mass spectrometer was considered suitable for use in forensic analysis as a screening method.

Between March 2017 and November 2018, 54 prisoner fatal overdose cases submitted to the University of Florida Forensic Toxicology Laboratory involved synthetic cannabinoids including 5F-ADB, FUB-AMB, 5F-AMB, MDMB-FUBINACA and AB-CHMINACA. Analysis of blood and urine samples was performed at NMS Labs (Horsham, PA) by liquid chromatography/tandem mass spectrometry screening, confirmatory and quantitative methods validated according to Scientific Working Group for Forensic Toxicology guidelines. This work highlights the importance of effective communication between toxicologists and medical examiners/coroners, and the value of public-private partnerships to provide coverage while laboratories work to update instrumentation and validate their own new methods to keep up with the challenges of emerging substances.

The following report summarizes a study performed on seized drug exhibits collected in two U.S. states to evaluate the presence and identification of cutting agents. Aliquots of seized drug materials from Kentucky (n = 200) and Vermont (n = 315) were prepared using a dilute-and-shoot procedure. Initial analysis was performed using gas chromatography-mass spectrometry (GC-MS) followed by analysis using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF). Active compounds detected overall included caffeine (31.0%), quinine/quinidine (24.7%), levamisole (11.6%), acetaminophen, (8.2%) and procaine (8.2%). These compounds were found with several drugs of abuse, such as heroin, fentanyl, methamphetamine, and cocaine. This novel information about cutting agents used to dilute or alter drugs of abuse is important to criminal investigations and in the management of acute intoxications at health centers. However, common methodologies for analysis and standard reporting practices frequently do not include cutting agents, resulting in lacking or inadequate information regarding prevalence of these substances.

In many jurisdictions, public safety and public health entities are working together to enhance the timeliness and accuracy of the analytical characterization and toxicology testing of novel synthetic opioids. The improved sharing and early detection of these analytical data are intended to inform surveillance, interdiction efforts, patient intervention and treatment, all of which are critical to curbing the opioid epidemic. Forensic practitioners working to identify novel synthetic opioids struggle to provide timely results when encountering new or unknown substances, such as the fentanyl analogs. These compounds, which mimic heroin in pharmacologic effect but can be far more potent, are inconsistently present in chemical identification libraries, and are currently largely unavailable as reference materials for analytical comparison. Additionally, federal, state and local governments as well as nongovernmental organizations require potency, toxicity and potential-for-abuse data to evaluate the potential health risks of emerging drug threats. Subsequent scheduling efforts and criminal prosecutions also require these thorough drug characterization studies. Pilot programs have demonstrated that early communication of real-time drug toxicity and analytical data significantly impacts the successful response to emerging opioids. High-quality, real-time, national-level data on chemical composition, toxicological test data, drug toxicity and overdoses, and analysis of seized materials by law enforcement are needed to track drug trends. However, the USA still lacks a national system to coordinate and communicate toxicology, medical and medical examiner and coroner data with the broader medical and law enforcement communities. Opportunities to address these gaps as well as recent advancements collected through interagency efforts and technical workshops in the toxicology and analytical chemistry communities are presented here. Opportunities for partnership, increased communication and expanding best practices to move toward an integrated, holistic analytical response are also explored.

In recent years an increasing number of novel opioids have appeared on the illicit drug market and have been linked to the growing opioid crisis in the United States. It is suspected that synthetic-opioid-related deaths are underestimated since many laboratories either use a screening method that is not specific and sensitive enough for these compounds or testing is not performed for fentanyl and its related analogs in general. We describe a method for the analysis of 19 of the most current novel opioid drugs quantitatively in whole blood and serum, and 17 analytes qualitatively in urine using solid phase extraction with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The limit of detection was determined to be at a minimum 0.25 ng/mL for beta-hydroxythiofentanyl, 0.05 ng/mL for butyryl/isobutyrylfentanyl, AH-7921, 2-furanylfentanyl, 4-ANPP, and U-47700; 0.025 ng/mL for MT-45, para-methoxybutyrylfentanyl, 4-methylphenethyl acetyl fentanyl, U-50488, acrylfentanyl, valerylfentanyl, and carfentanil; and 0.0125 ng/mL for para-fluorofentanyl, ortho-fluorofentanyl, para-fluorobutyrylfentanyl/FIBF, and alpha-methylfentanyl. The lower limit of quantitation was determined to be 0.1 ng/mL for all analytes except AH-7921, U-47700, U-50488 which were 0.05 ng/mL and beta-hydroxythiofentanyl which was 0.5 ng/mL. The method was validated successfully according to a Scientific Working Group in Forensic Toxicology (SWGTOX) compliant approach. This method was applied to the analysis of 2758 samples between October 2016 and September 2017. It was determined that 4-ANPP, furanylfentanyl, and carfentanil were the 3 most prevalent fentanyl related compounds detected: 56.1% of cases were positive for 4-ANPP, 44.5% were positive for furanylfentanyl, and 25.2% cases were positive for carfentanil.