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Synthetic benzimidazole opioids (BOs) are highly potent µ-opioid receptor agonists with heroin-like effects. Isotonitazene was first available in 2019 in the drug market, although new analogs have multiplied recently. The authors aimed to identify BO use trends and gather toxicological data from BO-related cases to assist in clinical and forensic investigations.A systematic literature search was conducted according to the PRISMA guidelines. PubMed and Scopus databases were accessed in October 2021 to identify scientific reports of BO-related intoxication and fatalities. Publication dates, case descriptions, symptoms, autopsy findings, and concentrations of BOs and metabolites in biological matrices were compiled.Data from 8 case reports with 93 fatalities involving isotonitazene ( n = 65), metonitazene ( n = 20), etonitazepyne ( N -pyrrolidino etonitazene) ( n = 8), flunitazene ( n = 4), and/or butonitazene ( n = 1), and 1 acute intoxication involving etonitazepyne were collected. Autopsy findings included pulmonary congestion/high lung weight (66%), cardiomegaly/high cardiac weight (39%), cerebral edema (22%), gastric contents in the airways (22%), and organ congestion (22%). Median peripheral blood concentrations were 1.7 ng/mL for isotonitazene (0.4-9.5 ng/mL, n = 13), 5.4 ng/mL for metonitazene (0.52-33 ng/mL, n = 17), 5.4 ng/mL for etonitazepyne (2.4-8.3 ng/mL, n = 2), 1.3 ng/mL for flunitazene (0.58-2.1 ng/mL, n = 2), and 3.2 ng/mL for butonitazene ( n = 1). Central nervous system depressants were almost always coadministered.Isotonitazene was predominant in cases from 2019 to mid-2020 and was replaced by metonitazene after scheduling in the United States. Typical findings on opioid overdoses have been reported. Peripheral blood concentrations were consistent with a potency similar to that of fentanyl. These results must be interpreted carefully, considering the scarcity of reports on BO-related cases and drug co-exposures.

Acetazolamide (ACZ) is a carbonic anhydrase inhibitor prescribed for the treatment of various pathologies. It is also used in doping and is prohibited in and out of sportive competitions. ACZ was reported not to undergo metabolization. However, the detection of ACZ metabolites may be critical for documenting ACZ use. We aimed to further investigate ACZ metabolic fate in humans. ACZ putative metabolites were generated in silico to assist in metabolite identification. ACZ was incubated with primary human hepatocytes to identify in vitro metabolites (10 µmol/l ACZ and 10

4-hydroxy-4-OH-MPT was incubated with 10-donor-pooled human hepatocytes to simulateThree phase I and 4 phase II metabolites were identified, including4-OH-MPT metabolic fate was consistent with the human metabolism of tryptamine analogues: we suggest 4-OH-MPT

“Light cannabis” is a product legally sold in Europe with Δ9-tetrahydrocannabinol (THC) concentration

From 2014 onwards, illicit fentanyl and analogues have caused numerous intoxications and fatalities worldwide, impacting the demographics of opioid-related overdoses. The identification of cases involving fentanyl analogues is crucial in clinical and forensic settings to treat patients, elucidate intoxications, address drug use disorders and tackle drug trends. However, in analytical toxicology, the concentration of fentanyl analogues in biological matrices is low, making their detection challenging. Therefore, the identification of specific metabolite biomarkers is often required to document consumption. β′-Phenylfentanyl (N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl]-benzenepropanamide) is a fentanyl analogue that was first detected in Sweden in 2017 and has recently reemerged onto the American illicit drug market. There is little data available on β′-phenylfentanyl effects and toxicokinetics and its metabolism is yet to be studied. We aimed to investigate β′-phenylfentanyl human metabolism to identify potential biomarkers of use. To assist in β′-phenylfentanyl metabolite identification, a list of putative reactions was generated using in silico predictions with GLORYx freeware. β′-phenylfentanyl was incubated with cryopreserved 10-donor-pooled human hepatocytes, analyses were performed by liquid chromatography–high-resolution tandem mass spectrometry (LC–HRMS-MS) and data were processed using a partially automated targeted/untargeted approach with Compound Discoverer. We identified 26 metabolites produced by N-dealkylation, oxidation, hydroxylation, O-glucuronidation, O-methylation and combinations thereof. We suggest β′-phenylnorfentanyl (N-phenyl-N-4-piperidinyl-benzenepropanamide) and further metabolites 1-oxo-N-phenyl-N-4-piperidinyl-benzenepropanamide and 1-hydroxy-N-phenyl-N-4-piperidinyl-benzenepropanamide as major biomarkers of β′-phenylfentanyl use. In silico predictions were mostly wrong, and β′-phenylfentanyl metabolic fate substantially differed from that of a closely related analogue incubated in the same conditions, highlighting the value of the experimental assessment of new psychoactive substance human metabolism. In vivo data are necessary to confirm the present results. However, the present results may be necessary to help analytical toxicologists identify β′-phenylfentanyl-positive cases to provide authentic samples.

Purpose Buprenorphine and methadone are international gold standards for managing opioid use disorders. Although they are efficacious in treating opioid dependence, buprenorphine and methadone present risks, especially during pregnancy, causing neonatal abstinence syndrome and adverse obstetrical outcomes. Buprenorphine and methadone are also abused during pregnancy, and identifying their use is important to limit unprescribed prenatal exposure. Previous studies have suggested that concentrations of buprenorphine, but not methadone markers in unconventional matrices may predict child outcomes, although currently only limited data exist. We reviewed the literature on concentrations of buprenorphine, methadone, and their metabolites in unconventional matrices to improve data interpretation. Methods A literature search was conducted using scientific databases (PubMed, Scopus, Web of Science, and reports from international institutions) to review published articles on buprenorphine and methadone monitoring during pregnancy. Results Buprenorphine and methadone and their metabolites were quantified in the meconium, umbilical cord, placenta, and maternal and neonatal hair. Methadone concentrations in the meconium and hair were typically higher than those in other matrices, although the concentrations in the placenta and umbilical cord were more suitable for predicting neonatal outcomes. Buprenorphine concentrations were lower and required sensitive instrumentation, as measuring buprenorphine glucuronidated metabolites is critical to predict neonatal outcomes. Conclusions Unconventional matrices are good alternatives to conventional ones for monitoring drug exposure during pregnancy. However, data are currently scarce on buprenorphine and methadone during pregnancy to accurately interpret their concentrations. Clinical studies should be conducted with larger cohorts, considering confounding factors such as illicit drug co-exposure.

Synthetic cathinones (SCs) constitute a heterogenous class of new psychoactive substances (NPS), structurally related to cathinone. SCs represent the widest NPS class, second to synthetic cannabinoids, accounting for approximately 160 different analogues with substitution at the phenyl group, the amine group, or the alkyl chain. In 2020, α-pyrrolidonophenone analogues were the most trafficked SCs, and were involved in many fatalities and intoxication cases. In particular, 3F-α-pyrrolidinovalerophenone (3F-α-PVP) was the cause of the highest number of SC-related fatal intoxications in Sweden in 2018. Minor structural modifications are used to avoid legal controls and analytical detection, but may also induce different toxicological profile. Therefore, the identification of specific markers of consumption is essential to discriminate SCs in clinical and forensic toxicology. In this study, we assessed 3F-α-PVP metabolic profile. 3F-α-PVP was incubated with 10-donor-pooled human hepatocytes, LC-HRMS/MS analysis, and software-assisted data mining. This well-established workflow was completed by in silico metabolite predictions using three different freeware. Ten metabolites were identified after 3 h incubation, including hydrogenated, hydroxylated, oxidated, and N-dealkylated metabolites. A total of 51 phase I and II metabolites were predicted, among which 7 were detected in the incubations. We suggest 3F-α-PVP N-butanoic acid, 3F-α-PVP pentanol, and 3F-α-PVP 2-ketopyrrolidinyl-pentanol as specific biomarkers of 3F-α-PVP consumption. This is the first time that an N-ethanoic acid is detected in the metabolic pathway of a pyrrolidine SC, demonstrating the importance of a dual targeted/untargeted data mining strategy.

For more than ten years, new synthetic cathinones (SCs) mimicking the effects of controlled cocaine-like stimulants have flooded the illegal drug market, causing numerous intoxications and fatalities. There are often no data on the pharmacokinetics of these substances when they first emerge onto the market. However, the detection of SC metabolites is often critical in order to prove consumption in clinical and forensic settings. In this research, the metabolite profile of two pyrrolidinyl SCs, &alpha, pyrrolidinohexaphenone (&alpha, PHP) and 4&rsquo, &rsquo, fluoro-&alpha, pyrrolidinovalerophenone (4F-&alpha, PVP), were characterized to identify optimal intake markers. Experiments were conducted using pooled human hepatocyte incubations followed by liquid chromatography&ndash, high-resolution tandem mass spectrometry and data-mining software. We suggest &alpha, PHP dihydroxy-pyrrolidinyl, &alpha, PHP hexanol, &alpha, PHP 2&prime, keto-pyrrolidinyl-hexanol, and &alpha, keto-pyrrolidinyl as markers of &alpha, PHP use, and 4F-&alpha, PVP dihydroxy-pyrrolidinyl, 4F-&alpha, PVP hexanol, 4F-&alpha, PVP 2&prime, keto-pyrrolidinyl-hexanol, and 4F-&alpha, keto-pyrrolidinyl as markers of 4F-&alpha, PVP use. These results represent the first data available on 4F-&alpha, PVP metabolism. The metabolic fate of &alpha, PHP was previously studied using human liver microsomes and urine samples from &alpha, PHP users. We identified an additional major metabolite (&alpha, PHP dihydroxy-pyrrolidinyl) that might be crucial for documenting exposure to &alpha, PHP. Further experiments with suitable analytical standards, which are yet to be synthesized, and authentic specimens should be conducted to confirm these results.

Sexual enhancers increase sexual potency, sexual pleasure, or libido. Substances increasing libido alter the concentrations of specific neurotransmitters or sex hormones in the central nervous system. Interestingly, the same pathways are involved in the mechanisms underlying many psychiatric and neurological disorders, and adverse reactions associated with the use of aphrodisiacs are strongly expected. However, sexual enhancers of plant origin have gained popularity over recent years, as natural substances are often regarded as a safer alternative to modern medications and are easily acquired without prescription. We reviewed the psychiatric and neurological adverse effects associated with the consumption of herbal aphrodisiacs Areca catechu L., Argemone Mexicana L., Citrus aurantium L., Eurycoma longifolia Jack., Lepidium meyenii Walp., Mitragyna speciosa Korth., Panax ginseng C. A. Mey, Panax quinquefolius L., Pausinystalia johimbe (K. Schum.) Pierre ex Beille, Piper methysticum G. Forst., Ptychopetalum olacoides Benth., Sceletium tortuosum (L.) N. E. Brown, Turnera diffusa Willd. ex. Schult., Voacanga africana Stapf ex Scott-Elliot, and Withania somnifera (L.) Dunal. A literature search was conducted on the PubMed, Scopus, and Web of Science databases with the aim of identifying all the relevant articles published on the issue up to June 2020. Most of the selected sexual enhancers appeared to be safe at therapeutic doses, although mild to severe adverse effects may occur in cases of overdosing or self-medication with unstandardized products. Drug interactions are more concerning, considering that herbal aphrodisiacs are likely used together with other plant extracts and/or pharmaceuticals. However, few data are available on the side effects of several plants included in this review, and more clinical studies with controlled administrations should be conducted to address this issue.

Purpose Drug use during pregnancy is a critical global challenge, capable of severe impacts on neonatal development. However, the consumption of cannabis and synthetic cannabinoids is on the rise in pregnant women. Obstetric complications with increased risks of miscarriage, fetal growth restriction, and brain development impairment have been associated with perinatal cannabis exposure, but data on synthetic cannabinoid use during pregnancy are limited. Methods We reviewed studies that investigated the risks associated with cannabis and synthetic cannabinoid use and those that reported the concentrations of cannabinoids and synthetic cannabinoids in maternal (breast milk) and neonatal (placenta, umbilical cord, meconium, and hair) matrices during human pregnancy. A MEDLINE and EMBASE literature search to identify all relevant articles published in English from January 1998 to April 2019 was performed. Results Cannabis use during pregnancy is associated with increased risks of adverse obstetrical outcomes, although neurobehavioral effects are still unclear. Analyses of cannabinoids in meconium are well documented, but further research on other unconventional matrices is needed. Adverse effects due to perinatal synthetic cannabinoid exposure are still unknown, and analytical data are scarce. Conclusions Awareness of the hazards of drug use during pregnancy should be improved to encourage health care providers to urge pregnant women to abstain from cannabis and, if cannabis-dependent, seek treatment. Moreover, substances used throughout pregnancy should be monitored as a deterrent to cannabis use, and potential cannabis-dependent women should be identified, so as to limit cannabis-fetal exposure during gestation, and provided appropriate treatment.

The aim of this review was to report the most recent cases of acute intoxication, fatalities and “driving under the influence” cases, involving illicit fentanyl and its newest analogs. When available, information on age, sex, circumstances of exposure, intoxication symptoms, cause of death (if applicable) and toxicology results from biological fluid testing was described. Scientific publications reporting fatalities or acute intoxications involving use of fentanyl derivatives were identified from PubMed, Scopus and institutional/governmental websites from January 2017 up to December 2019. The search terms, used alone and in combination, were as follows: fentanyl, street fentanyl, analogs, compounds, derivatives, abuse, fatality, fatalities, death, toxicity, intoxication and adverse effects. When considered relevant, reports not captured by the initial search but cited in other publications were also included. Of the 2890 sources initially found, only 44 were suitable for the review. Emergent data showed that the most common analogs detected in biological samples and seized materials are acetylfentanyl, acrylfentanyl, butyrfentanyl, carfentanil, cyclopropylfentanyl, fluorofentanyl, 4-fluorobutyrfentanyl, 4-fluoroisobutyrfentanyl, furanylfentanyl, 2-methoxyacetylfentanyl, 3-methylfentanyl and ocfentanil. These compounds were frequently administered in association with other illicit substances, medicinal drugs and/or alcohol; patients and the victims often had a previous history of drug abuse. The trend of fentanyl analogs is rapidly evolving with illicit market fluctuations. Since information about potency and lethal dosage are frequently unknown, it is important to identify the new trends for further investigation on therapeutic use, toxicity and fatal doses, and implement public health measures. Recently marketed fentanyl analogs such as crotonylfentanyl and valerylfentanyl were not involved in intoxications to date, but should be carefully monitored. Many intoxications and fatalities might have gone unnoticed, and research efforts should focus on metabolite identification studies and the implementation of updated and comprehensive analytical methods

Novel synthetic cannabinoids are appearing in recreational drug markets worldwide. Pharmacological characterization of these new drugs is needed to inform clinicians, toxicologists, and policy makers who monitor public health. [1-(5-Fluoropentyl)-1H-indol-3-yl](1-naphthyl)methanone (AM-2201) is an abused synthetic cannabinoid that was initially created as a research tool for investigating the endocannabinoid system. Here we measured the pharmacodynamic effects of AM-2201 in rats, and simultaneously determined plasma pharmacokinetics for the parent drug and its metabolites. Male Sprague-Dawley rats were fitted with surgically implanted temperature transponders and indwelling jugular catheters under pentobarbital anesthesia. One week later, rats received subcutaneous injection of AM-2201 (0.1, 0.3, and 1.0 mg/kg) or its vehicle, and serial blood specimens were withdrawn via catheters. Core temperatures and catalepsy were measured just prior to each blood withdrawal, and plasma was assayed for drug and metabolites using liquid chromatography-tandem mass spectrometry. We found that AM-2201 produced dose-related hypothermia and catalepsy that peaked at 2 hours and lasted up to 8 hours. AM-2201 plasma concentrations rose linearly with increasing dose and ranged from 0.14 to 67.9 µg/l. Concentrations of three metabolites, AM-2201 N-(4-hydroxypentyl) (≤0.17 µg/l), naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-018) N-(5-hydroxypentyl) (≤1.14 µg/l), and JWH-018 N-pentanoic acid (≤0.88 µg/l) were detectable but much lower. Peak AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid concentrations occurred at 1.3, 2.4, and 6.5 hours, respectively. Concentrations of AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid were negatively correlated with body temperature, but, given the low concentrations of metabolites detected, AM-2201 is likely the major contributor to pharmacodynamic effects under our experimental conditions.

Background The prevalence of drug use during pregnancy continues to increase despite the associated serious adverse obstetrical outcomes, including increased risk of miscarriage, fetal growth restriction, brain development impairment, neonatal abstinence syndrome, preterm delivery, and stillbirths. Monitoring drug use during pregnancy is crucial to limit prenatal exposure and provide suitable obstetrical health care. The authors reviewed published literature reporting the concentrations of common drugs of abuse and new psychoactive substances (NPS), such as synthetic cathinones and synthetic opioids, NPS, and their metabolites using unconventional matrices to identify drug use during pregnancy and improve data interpretation. Methods A literature search was performed from 2010 to July 2019 using PubMed, Scopus, Web of Science scientific databases, and reports from international institutions to review recently published articles on heroin, cocaine, amphetamine, methamphetamine, synthetic cathinone, and synthetic opioid monitoring during pregnancy. Results Meconium has been tested for decades to document prenatal exposure to drugs, but data regarding drug concentrations in amniotic fluid, the placenta, the umbilical cord, and neonatal hair are still lacking. Data on prenatal exposure to NPS are limited. Conclusions Maternal hair testing is the most sensitive alternative matrix for identifying drug use during pregnancy, while drug concentrations in the meconium, placenta, and umbilical cord offer the identification of prenatal drug exposure at birth. Adverse developmental outcomes for the infant make it critical to promptly identify maternal drug use to limit fetal exposure or, if determined at birth, to provide resources to the exposed child and family. Alternative matrices offer choices for monitoring and challenge laboratories to deliver highly sensitive and specific analytical methods for detection.

Clandestine laboratories continue producing new synthetic cannabinoids that mimic and magnify natural cannabinoids effects to circumvent drug scheduling legislation. New synthetic cannabinoids are highly potent and responsible for many acute intoxications and deaths. Characterization of metabolic pathways is critical to identify metabolite markers whose detection can prove intake. BB-22 is a new potent synthetic cannabinoid whose toxicological and metabolic properties are currently unavailable. Analytical methods require constant updating and are challenging due to extensive synthetic cannabinoid metabolism and low marker concentrations. A single non-specific BB-22 metabolite was previously identified in incubations with human liver microsomes (BB-22 3-carboxyindole). Clear characterization of BB-22’s metabolism is required to help toxicologists document BB-22 consumption in clinical and forensic cases. We incubated 10 μmol/L BB-22 with cryopreserved human hepatocytes for 3 h. Samples were analyzed by liquid chromatography on a biphenyl column and high resolution mass spectrometry. Results were processed with data mining software, identifying ten metabolites. Loss of the quinolinyl side-chain via ester hydrolysis was the main biotransformation. All other metabolites were produced by further indole or cyclohexylmethyl hydroxylation or glucuronidation. We recommend BB-22 3-carboxyindole and two BB-22 3-carboxyindole-hydroxycyclohexylmethyl isomers as metabolite targets for documenting BB-22 intake. Hydrolysis of biological samples before analysis is strongly suggested to improve detection of phase I metabolites. BB-22 3-carboxyindole is not specific for BB-22 intake, as it was previously detected as a minor MDMB-CHMICA and ADB-CHMICA metabolite. Consumption of these two synthetic cannabinoids should be ruled out to confirm BB-22 intake.

The present study aims to recommend appropriate urinary marker metabolites for documenting EG-018 consumption by investigating its metabolism in human hepatocytes. For metabolite profiling, 10 µM EG-018 was incubated in human hepatocytes for 3 h. Metabolite identification in hepatocyte samples was accomplished with high-resolution mass spectrometry via information-dependent data acquisition. EG-018 was highly metabolized in human hepatocytes. A total of eight metabolites were characterized, mainly generated from hydroxylation and carbonylation on the pentyl chain. Dihydrodiol formation, N-dealkylation, and glucuronidation of hydroxylated metabolites were the other major pathways. The primary metabolites of EG-018 in human hepatocyte incubation were pentyl hydroxylated EG-018 (M6) and pentyl carbonylated EG-018 (M8). These two metabolites are proposed as the best urinary markers for confirming EG-018 intake.

Illicit fentanyl and analogues have been involved in many fatalities and cases of intoxication across the United States over the last decade, and are becoming a health concern in Europe. New potent analogues emerge onto the drug market every year to circumvent analytical detection and legislation, and little pharmacological/toxicological data are available when the substances first appear. However, pharmacokinetic data are crucial to determine specific biomarkers of consumption in clinical and forensic settings, considering the low active doses and the rapid metabolism of fentanyl analogues. Phenylfentanyl is a novel analogue that was first detected in seized material in 2017, and little is currently known about this substance and its metabolism. We studied phenylfentanyl metabolic fate using in silico predictions with GLORYx freeware, human hepatocyte incubations, and liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). We applied a specific targeted/untargeted workflow using data-mining software to allow the rapid and partially automated screening of LC-HRMS/MS raw data. Approximately 90,000 substances were initially individuated after 3-h incubation with hepatocytes, and 115 substances were automatically selected for a manual check by the operators. Finally, 13 metabolites, mostly produced by N-dealkylation, amide hydrolysis, oxidation, and combinations thereof, were identified. We suggest phenylnorfentanyl as the main biological marker of phenylfentanyl use, and we proposed the inclusion of its fragmentation pattern in mzCloud and HighResNPS online libraries. Other major metabolites include N-Phenyl-1-(2-phenylethyl)-4-piperidinamine (4-ANPP), 1-(2-phenylethyl)-4-piperidinol, and other non-specific metabolites. Phase II transformations were infrequent, and the hydrolysis of the biological samples is not required to increase the detection capability of non-conjugated metabolites. The overall workflow is easily adaptable for the metabolite profiling of other novel psychoactive substances.

Metabolite profiling of novel psychoactive substances (NPS) is critical for documenting drug consumption. N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (ADB-FUBINACA) is an emerging synthetic cannabinoid whose toxicological and metabolic data are currently unavailable. We aimed to determine optimal markers for identifying ADB-FUBINACA intake. Metabolic stability was evaluated with human liver microsome incubations. Metabolites were identified after 1 and 3 h incubation with pooled human hepatocytes, liquid chromatography- high resolution mass spectrometry in positive-ion mode (5600+ TripleTOF®, Sciex) and several data mining approaches (MetabolitePilot™, Sciex). Metabolite separation was achieved on an Ultra Biphenyl column (Restek®); full-scan TOF-MS and information-dependent acquisition MS/MS data were acquired. ADB-FUBINACA microsomal half-life was 39.7 min, with a predicted hepatic clearance of 9.0 mL/min/kg and a 0.5 extraction ratio (intermediate-clearance drug). Twenty-three metabolites were identified. Major metabolic pathways were alkyl and indazole hydroxylation, terminal amide hydrolysis, subsequent glucuronide conjugations, and dehydrogenation. We recommend ADB-FUBINACA hydroxyalkyl, hydroxydehydroalkyl and hydroxylindazole metabolites as ADB-FUBINACA intake markers. N-dealkylated metabolites are not specific ADB-FUBINACA metabolites and should not be used as definitive markers of consumption. This is the first ADB-FUBINACA in vitro metabolism study; in vivo experiments enabling pharmacokinetic and pharmacodynamics studies or urine from authentic clinical/forensic cases are needed to confirm our results.

The drug abuse epidemic within the United States remains one of the nation’s most serious social challenges, especially among adolescents and young adults. Novel psychoactive substances continuously emerge into the illicit drugs-of-abuse market to evade legislation. In 2013, SDB-006 was detected as a novel synthetic cannabinoid (SC) with high binding affinity to CB1 (EC50 = 19 nM) and CB2 (EC50 = 134 nM). Unfortunately, no human metabolism data for SDB-006 are currently available, making it challenging to confirm intake, since all previously investigated SCs were extensively metabolized. The present study aims to recommend appropriate marker metabolites for documenting SDB-006 consumption by investigating its metabolism in human hepatocytes. For metabolite profiling, 10 µM of SDB-006 was incubated in human hepatocytes for 3 h. Metabolite identification in hepatocyte samples was accomplished with high-resolution mass spectrometry via information-dependent data acquisition. Results revealed that SDB-006 was highly metabolized in human hepatocytes. A total of 20 metabolites were characterized, generated mainly from hydroxylation and glucuronidation. Hydroxylation occurred primarily on several positions of the pentyl chain. N-Dealkylation was the other major pathway, including depentylation and debenzylation. Based on our data, we propose 4′-keto-SDB-006 (M19) and pentyl-OH-SDB-006 (M15) as optimal marker metabolites for documenting SDB-006 intake.

Recently, synthetic opioid-related overdose fatalities, led by illicitly manufactured fentanyl and analogs, increased at an alarming rate, posing a global public health threat. New synthetic fentanyl analogs have been constantly emerging onto the drug marked for the last few years, to circumvent the laws and avoid analytical detection. Analytical methods need to be regularly updated to keep up with the new trends. In this study, we aimed to develop a new method for detecting the newest fentanyl analogs with a high sensitivity, in whole blood, urine, and hair. The method is intended to provide to clinical and forensic toxicologists a tool for documenting consumption. We developed a comprehensive ultra-high-performance liquid chromatography-tandem mass spectrometry method for quantifying fentanyl and 22 analogs and metabolites. Urine samples were simply diluted before injection; a liquid-liquid extraction was performed for blood testing; and a solid phase extraction was performed in hair. The chromatographic separation was short (8 min). The method was validated with a high sensitivity; limits of quantifications ranged from 2 to 6 ng/L in blood and urine, and from 11 to 21 pg/g in hair. The suitability of the method was tested with 42 postmortem blood, urine, or hair specimens from 27 fatalities in which fentanyl analogs were involved. Average blood concentrations (±SD) were 7.84 ± 7.21 and 30.0 ± 18.0 μg/L for cyclopropylfentanyl and cyclopropyl norfentanyl, respectively (n = 8), 4.08 ± 2.30 μg/L for methoxyacetylfentanyl, (n = 4), 40.2 ± 38.6 and 44.5 ± 21.1 μg/L for acetylfentanyl and acetyl norfentanyl, respectively (n = 3), 33.7 and 7.17 μg/L for fentanyl and norfentanyl, respectively (n = 1), 3.60 and 0.90 μg/L for furanylfentanyl and furanyl norfentanyl, respectively (n = 1), 0.67 μg/L for sufentanil (n = 1), and 3.13 ± 2.37 μg/L for 4-ANPP (n = 9). Average urine concentrations were 47.7 ± 39.3 and 417 ± 296 μg/L for cyclopropylfentanyl and cyclopropyl norfentanyl, respectively (n = 11), 995 ± 908 μg/L for methoxyacetylfentanyl, (n = 3), 1,874 ± 1,710 and 6,582 ± 3,252 μg/L for acetylfentanyl and acetyl norfentanyl, respectively (n = 5), 146 ± 318 and 300 ± 710 μg/L for fentanyl (n = 5) and norfentanyl (n = 6), respectively, 84.0 and 23.0 μg/L for furanylfentanyl and furanyl norfentanyl, respectively (n = 1), and 50.5 ± 50.9 μg/L for 4-ANPP (n = 10). Average hair concentrations were 2,670 ± 184 and 82.1 ± 94.7 ng/g for fentanyl and norfentanyl, respectively (n = 2), and 10.8 ± 0.57 ng/g for 4-ANPP (n = 2).

Background: Currently, there is a great interest in the potential medical use of cannabidiol (CBD), a non-intoxicating cannabinoid. Productive pharmacological research on CBD occurred in the 1970s and intensified recently with many discoveries about the endocannabinoid system. Multiple preclinical and clinical studies led to FDA-approval of Epidiolex®, a purified CBD medicine formulated for oral administration for the treatment of infantile refractory epileptic syndromes, by the US Food and Drug Administration in 2018. The World Health Organization considers rescheduling cannabis and cannabinoids. CBD use around the world is expanding for diseases that lack scientific evidence of the drug’s efficacy. Preclinical and clinical studies also report adverse effects (AEs) and toxicity following CBD intake. Methods: Relevant studies reporting CBD’s AEs or toxicity were identified from PubMed, Cochrane Central, and EMBASE through January 2019. Studies defining CBD’s beneficial effects were included to provide balance in estimating risk/benefit. Results: CBD is not risk-free. In animals, CBD AEs included developmental toxicity, embryo-fetal mortality, central nervous system inhibition and neurotoxicity, hepatocellular injuries, spermatogenesis reduction, organ weight alterations, male reproductive system alterations, and hypotension, although at doses higher than recommended for human pharmacotherapies. Human CBD studies for epilepsy and psychiatric disorders reported CBD-induced drug-drug interactions, hepatic abnormalities, diarrhea, fatigue, vomiting, and somnolence. Conclusion: CBD has proven therapeutic efficacy for serious conditions such as Dravet and Lennox-Gastaut syndromes and is likely to be recommended off label by physicians for other conditions. However, AEs and potential drug-drug interactions must be taken into consideration by clinicians prior to recommending off-label CBD.

25C-NBOMe and 25I-NBOMe are potent hallucinogenic drugs that recently emerged as new psychoactive substances. To date, a few metabolism studies were conducted for 25I-NBOMe, whereas 25C-NBOMe metabolism data are scarce. Therefore, we investigated the metabolic profile of these compounds in human hepatocytes, an in vivo mouse model and authentic human urine samples from forensic cases. Cryopreserved human hepatocytes were incubated for 3 h with 10 μM 25C-NBOMe and 25I-NBOMe; samples were analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS) on an Accucore C18 column with a Thermo QExactive; data analysis was performed with Compound Discoverer software (Thermo Scientific). Mice were administered 1.0 mg drug/kg body weight intraperitoneally, urine was collected for 24 h and analyzed (with or without hydrolysis) by LC-HRMS on an Acquity HSS T3 column with an Agilent 6550 QTOF; data were analyzed manually and with WebMetabase software (Molecular Discovery). Human urine samples were analyzed similarly. In vitro and in vivo results matched well. 25C-NBOMe and 25I-NBOMe were predominantly metabolized by O-demethylation, followed by O-di-demethylation and hydroxylation. All methoxy groups could be demethylated; hydroxylation preferably occurred at the NBOMe ring. Phase I metabolites were extensively conjugated in human urine with glucuronic acid and sulfate. Based on these data and a comparison with synthesized reference standards for potential metabolites, specific and abundant 25C-NBOMe urine targets are 5'-desmethyl 25C-NBOMe, 25C-NBOMe and 5-hydroxy 25C-NBOMe, and for 25I-NBOMe 2' and 5'-desmethyl 25I-NBOMe and hydroxy 25I-NBOMe. These data will help clinical and forensic laboratories to develop analytical methods and to interpret results. Copyright © 2016 John Wiley & Sons, Ltd.

AM-2201 is a popular synthetic cannabinoid first synthesized in 2000. AM-2201 pharmacokinetic and pharmacodynamic data are scarce, requiring further investigation. We developed a sensitive method for quantifying AM-2201 and 13 metabolites in plasma to provide a tool to further metabolic, pharmacokinetic and pharmacodynamic studies. Analysis was performed by liquid chromatography-tandem mass spectrometry. Chromatographic separation was performed by gradient elution on a biphenyl column with 0.1% formic acid in water/0.1% formic acid in acetonitrile:methanol 50:50 (v/v) mobile phase. Sample preparation (75 μL) consisted of an enzymatic hydrolysis and a supported liquid extraction. The method was validated with human plasma with a 0.025 or 0.050 – 50 μg/L working range, and cross-validated for rat plasma. Analytical recovery was 88.8 – 110.1% of target concentration, and intra- (n = 30) and inter-day (n = 30) imprecision

Resume Compte tenu de l’interet croissant que suscitent les cannabinoides de synthese, nous proposons de faire le point sur les principales methodes utilisees a ce jour permettant leur detection et/ou leur quantification dans les milieux biologiques (sang, urine, salive et cheveu) et les produits consommes (poudres, melanges de plantes). Les principales techniques de separation et d’analyse (immuno-analyse, chromatographie sur couche mince, chromatographie en phase liquide, chromatographie en phase gazeuse, spectrometrie de masse, spectroscopie infrarouge, resonance magnetique nucleaire) sont discutees. La connaissance des methodes actuelles et des perspectives offertes par l’apport de techniques recentes doit permettre au toxicologue de faire face aux defis poses par l’analyse des cannabis de synthese.

Background Hypoglycin A has been recently identified has the causal agent of atypical myopathy (AM) in horses. Its identification and quantification in equine’s biological fluids is thus a major concern to confirm maple poisoning and to provide insight into the poorly understood mechanism of hypoglycin A intoxication. Methods Quantification of hypoglycin A has been achieved with the aTRAQ kit for amino acid analysis of physiological fluids (AB Sciex). Acquisition method on mass spectrometer has been updated to record the hypoglycin A specific MRM transition. Results Outlined accuracy profiles demonstrated very reliable data. A good linearity was observed from 0.09 to 50 μmol/L and precision was very good with coefficient of variation below 8%. Fifty-five samples collected from 25 confirmed AM horses revealed significant hypoglycin A concentrations, while toxin was not found in serum of 8 control animals. Conclusions The described aTRAQ variant method has been analytically and clinically validated. The reliability of our approach is thus demonstrated into the workup of atypical myopathy.

Plant poisonings have left their mark on history and still cause many deaths, whether intentional or accidental. The means to show toxicological evidence of such poisonings should be implemented with great care. This article presents a technique for measuring thirty-nine toxic principles of plant origin in the blood, covering a large amount of toxins from local or exotic plants: α-lobeline, α-solanine, aconitine, ajmaline, atropine, brucine, cephalomannine, colchicine, convallatoxin, cymarine, cytisine, digitoxin, digoxin, emetine, gelsemine, ibogaine, jervine, kavain, lanatoside C, lupanine, mitragynine, neriifolin, oleandrin, ouabain, paclitaxel, physostigmine, pilocarpine, podophyllotoxin, proscillaridin A, reserpine, retrorsine, ricinine, scopolamine, senecionine, sparteine, strophanthidin, strychnine, veratridine and yohimbine. Analysis was carried out using an original ultra-high performance liquid chromatography separation coupled with tandem mass spectrometry detection. Extraction was a standard solid phase extraction performed on Oasis® HLB cartridge. Thirty-four of the thirty-nine compounds were put through a validation procedure. The assay was linear in the calibration curve range from 0.5 or 5 μg/L to 1000 μg/L according to the compounds. The method is sensitive (LOD from 0.1 to 1.6 μg/L). The within-day precision of the assay was less than 22.5% at the LLOQ, and the between-day precision was less than 21.5% for 10 μg/L for all the compounds included. The assay accuracy was in the range of 87.4 to 119.8% for the LLOQ. The extraction recovery and matrix effect ranged from 30 to 106% and from −30 to 14%, respectively. It has proven useful and effective in several difficult forensic cases.

BACKGROUND In 2014, 2 novel synthetic cannabinoids, MN-18 and its 5-fluoro analog, 5F-MN-18, were first identified in an ongoing survey of novel psychoactive substances in Japan. In vitro pharmacological assays revealed that MN-18 and 5F-MN-18 displayed high binding affinities to human CB1 and CB2 receptors, with Ki being 1.65–3.86 nmol/L. MN-18 and 5F-MN-18 were scheduled in Japan and some other countries in 2014. Despite increasing prevalence, no human metabolism data are currently available, making it challenging for forensic laboratories to confirm intake of MN-18 or 5F-MN-18. METHODS We incubated 10 μmol/L of MN-18 and 5F-MN-18 in human hepatocytes for 3 h and analyzed the samples on a TripleTOF 5600+ high-resolution mass spectrometer to identify appropriate marker metabolites. Data were acquired via full scan and information-dependent acquisition-triggered product ion scans with mass defect filter. RESULTS In total, 13 MN-18 metabolites were detected, with the top 3 abundant metabolites being 1-pentyl-1H-indazole-3-carboxylic acid, pentyl-carbonylated MN-18, and naphthalene-hydroxylated MN-18. For 5F-MN-18, 20 metabolites were observed, with the top 3 abundant metabolites being 5′-OH-MN-18, MN-18 pentanoic acid, and 1-(5-fluoropentyl)-1H-indazole-3-carboxylic acid. CONCLUSIONS We have characterized MN-18 and 5F-MN-18 metabolism with human hepatocytes and high-resolution mass spectrometry, and we recommend characteristic major metabolites for clinical and forensic laboratories to identify MN-18 and 5F-MN-18 intake and link observed adverse events to these novel synthetic cannabinoids.

Atractyloside (ATR) and carboxyatractyloside (CATR) are diterpene glycosides that are responsible for the toxicity of several Asteraceae plants around the world. Mediterranean gum thistle (Atractylis gummifera L.) and Zulu impila (Callilepis laureola DC.), in particular, are notoriously poisonous and the cause of many accidental deaths, some suicides and even some murders. There is no current method for measuring the two toxins in biological samples that meet the criteria of specificity required in forensic medicine. We have endeavored to fill this analytical gap. Analysis was carried out using a solid-phase extraction and a high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry detection. The method was validated in the whole blood with quantification limits of 0.17 and 0.15 µg/L for ATR and CATR, respectively. The method was applied to a non-fatal case of intoxication with A. gummifera. To the best of the authors' knowledge, this is the first time that a concentration of ATR and CATR in blood (883.1 and 119.0 µg/L, respectively) and urine (230.4 and 140.3 µg/L, respectively) is reported. ATR and CATR were quantified in A. gummifera roots by the standard method addition (3.7 and 5.4 mg/g, respectively).

The toxicity of the sea mango (Cerbera manghas L.) is well known. The plant is ranked as one of the deadliest of the southern Asian coastline. Cardenolidic heterosides are responsible for the cardiotoxicity of trees of the Cerbera genus. We have identified and determined the concentration of the principal glycosidic steroids present in the seeds of sea mangos (Thailand). Drug screening of an extract of the seeds was carried out using ultra-high performance liquid chromatography coupled to photodiode array detection and mass spectrometry (UHPLC–PDA-MS) with quantification at 219 nm. Identification was confirmed by UHPLC–HRMS. Deacetyltanghinin (m/z 549.3055 ± 2 ppm), neriifolin (m/z 535.3259 ± 2 ppm), tanghinin (m/z 591.3169 ± 2 ppm) and cerberin (577.3375 ± 2 ppm) were the most abundant glycosidic steroids present in the sea mango seeds. A seed of the dried ripe fruit had concentrations of 1209.1, 804.2, 621.4 and 285.9 μg/g, respectively. A seed of the fresh unripe fruit had concentrations of 49.4, 47.0, 3.5 and 2.3 μg/g.

In March 2009, the body of a 51-year-old man was found in the boot of his car. The body had been frozen before being dismembered at the abdomen. The autopsy failed to determine the cause of death. Systematic toxicological analyses of the victim's peripheral blood and urine showed the presence of atropine, a powerful anticholinergic. Atropine was therefore specifically detected and quantified throughout the victim's biologic samples by HPLC-MS² in the biologic fluids and UHPLC-MS² in the hair. The atropine concentrations were 887 ng/mL in the cardiac blood, 489 ng/mL in the peripheral blood, 6693 ng/mL in the gastric contents (1.1 μg), 6753 ng/mL in the urine, and 2290 pg/mg in the hair. The blood concentrations measured in the decedent were consistent with an overdose of atropine, which was determined as the cause of death. The manner of death was a homicide with criminal intent.

BACKGROUND ADB-PINACA and its 5-fluoropentyl analog 5F-ADB-PINACA are among the most potent synthetic cannabinoids tested to date, with several severe intoxication cases. ADB-PINACA and 5F-ADB-PINACA have a different legal status, depending on the country. Synthetic cannabinoid metabolites predominate in urine, making detection of specific metabolites the most reliable way for proving intake in clinical and forensic specimens. However, there are currently no data on ADB-PINACA and 5F-PINACA metabolism. The substitution of a single fluorine atom distinguishes the 2 molecules, which may share common major metabolites. For some legal applications, distinguishing between ADB-PINACA and 5F-PINACA intake is critical. For this reason, we determined the human metabolic fate of the 2 analogs. METHODS ADB-PINACA and 5F-PINACA were incubated for 3 h with pooled cryopreserved human hepatocytes, followed by liquid chromatography—high-resolution mass spectrometry analysis. Data were processed with Compound Discoverer. RESULTS We identified 19 and 12 major ADB-PINACA and 5F-ADB-PINACA metabolites, respectively. Major metabolic reactions included pentyl hydroxylation, hydroxylation followed by oxidation (ketone formation), and glucuronidation of ADB-PINACA, and oxidative defluorination followed by carboxylation of 5F-ADB-PINACA. CONCLUSIONS We recommend ADB-PINACA ketopentyl and hydroxypentyl, and ADB-PINACA 5-hydroxypentyl and pentanoic acid, as optimal markers for ADB-PINACA and 5F-ADB-PINACA intake, respectively. Since the 2 compounds present positional isomers as the primary metabolites, monitoring unique product ions and optimized chromatographic conditions are required for a clear distinction between ADB-PINACA and 5F-ADB-PINACA intake.

In 2014, NM-2201 (CBL-2201), a novel synthetic cannabinoid (SC), was detected by scientists at Russian and US laboratories. It has been already added to the list of scheduled drugs in Japan, Sweden and Germany. Unfortunately, no human metabolism data are currently available, which makes it challenging to confirm its intake, especially given that all SCs investigated thus far have been found to be extensively metabolized. The present study aims to recommend appropriate marker metabolites by investigating NM-2201 metabolism in human hepatocytes, and to confirm the results in authentic human urine specimens. For the metabolic stability assay, 1 µM NM-2201 was incubated in human liver microsomes (HLMs) for up to 1 h; for metabolite profiling, 10 µM of NM-2201 was incubated in human hepatocytes for 3 h. Two authentic urine specimens from NM-2201-positive cases were subjected to β-glucuronidase hydrolysis prior to analysis. The identification of metabolites in hepatocyte samples and urine specimens was achieved with high-resolution mass spectrometry via information-dependent acquisition. NM-2201 was quickly metabolized in HLMs, with an 8.0-min half-life. In human hepatocyte incubation samples, a total of 13 NM-2201 metabolites were identified, generated mainly from ester hydrolysis and further hydroxylation, oxidative defluorination and subsequent glucuronidation. M13 (5-fluoro PB-22 3-carboxyindole) was found to be the major metabolite. In the urine specimens, the parent drug NM-2201 was not detected; M13 was the predominant metabolite after β-glucuronidase hydrolysis. Therefore, based on the results of our study, we recommend M13 as a suitable urinary marker metabolite for confirming NM-2201 and/or 5F-PB-22 intake.