α-MT is a hallucinogenic and stimulant tryptamine that was involved in several overdose fatalities in the United States and Europe. Analytical toxicology, and particularly the identification of metabolite biomarkers in biological samples, often is the only way to prove tryptamine use in clinical and forensic caseworks. We aimed to identify optimal α-MT metabolite biomarkers of consumption in humans. We identified α-MT metabolites in 10-donor-pooled human hepatocyte incubations and postmortem urine and blood from an α-MT overdose case using in silico metabolite predictions, liquid chromatography high-resolution-tandem mass spectrometry (LC-HRMS/MS), and software-assisted data mining. Nine metabolites were identified in vitro and eight additional metabolites were found in urine; five metabolites were found in blood. Metabolic transformations were hydroxylation, O-sulfation, O-glucuronidation, N-glucuronidation, and N-acetylation, consistent with the metabolism of structural analogues. The findings in hepatocyte incubations and postmortem samples were consistent, proving the in vitro model suitability. We suggest α-MT, hydroxy-α-MT glucuronide, and two hydroxy-α-MT sulfates as biomarkers of α-MT use in non-hydrolyzed urine; we suggest α-MT, two hydroxy-α-MT sulfates and N-acetyl-α-MT as biomarkers of α-MT use in blood. Further studies on α-MT clinical and forensic caseworks with different doses and routes of administration are necessary to better explore α-MT metabolism.

The current opioid overdose crisis is characterized by the presence of unknown psychoactive adulterants. Xylazine is an alpha-2 receptor agonist that is not approved for human use but is commonly used in veterinary medicine due to its sedative and muscle-relaxant properties. Cases of human intoxication due to accidental or voluntary use have been reported since the 1980s. However, reports of adulteration of illicit opioids (heroin and illicit fentanyl) with xylazine have been increasing all over Western countries. In humans, xylazine causes respiratory depression, bradycardia, and hypotension-posing individuals, using xylazine-adulterated opioids. We present a narrative review of the latest intoxication cases related to xylazine, to bring awareness to readers and also to help pathologists to detect and deal with xylazine cases.

Rapporter les difficultés d'identification de composés de type tryptamines à travers un cas d'intoxication fatale à l'alpha-méthyltryptamine (AMT). Un homme de 35 ans, présentant des antécédents psychiatriques et de toxicomanie, a été retrouvé décédé à son domicile, en état de putréfaction débutant. À proximité du corps, 6 fioles d'un produit nommé FELIZ ont été retrouvées. Les échantillons suivants ont été prélevés : sang périphérique (SP), sang cardiaque (SC), urine, bile, liquide gastrique (LG), cheveux (2 cm). Un dépistage immunologique a été effectué sur l'urine pour détecter les amphétaminiques, cocaïniques, opiacés et cannabinoïdes. La quantification des amphétaminiques a été réalisée par LC-MS/MS dans le SP. La recherche d'éthanol et de volatils a été effectuée sur le SP par HS-GC-FID. Un criblage large a été réalisé sur SP, SC, LG, urine et le produit FELIZ par GC-MS et UHPLC-UV/MS. La quantification des benzodiazépines et de l'éthylglucuronide (EtG) a été effectuée par GC-MS/MS sur le SP. L'AMT, le 5-(2-méthylaminopropyl) benzofurane (5-MAPB) et le 5-(2-aminopropyl) benzofurane (5-APB) ont été quantifiés par LC-MS/MS dans l'ensemble des matrices, avec une technique validée dans le sang en termes de linéarité, sélectivité, précision, justesse et dilution, et dans les cheveux (justesse et précision à la limite inférieure de quantification). Des études de prédictions de métabolites in silico et d'identification de métabolites dans des incubations d'hépatocytes humains ont été effectuées pour l'AMT par LC-HRMS/MS. Seul le 5-MAPB a été identifié dans les fioles. Le dépistage immunologique positif aux amphétaminiques a pu être expliqué par la présence de benzofuranes, d'éphédrine et de noréphédrine. Les concentrations dans le SP des molécules formellement identifiées lors de la phase de screening étaient les suivantes : éthanol : 0,14 g/L ; EtG < 0,10 μg/mL ; nordazépam : 170 ng/mL ; diazépam : 8,34 ng/mL ; oxazépam : 75,9 ng/mL ; éphédrine : 69,7 ng/mL ; noréphédrine : 5,63 ng/mL ; 5-MAPB : 101 ng/mL et 5-APB : 9,33 ng/mL. L'analyse du LG et de l'urine par GC-MS a révélé un composé de type X-(2-aminopropyl) indole (X-IT) dont l'identification formelle était complexifiée par l'existence d'isomères ayant des spectres de masses identiques (3-IT (autre nom de l'AMT), 5-IT et 6-IT). Dans les sangs analysés, le même pic chromatographique était coélué à une interférence matricielle en GC et particulièrement en LC avec un spectre UV, similaire à ceux d'amines de putréfaction. Suite à l'obtention du standard (délai de plusieurs mois), les spectres ont été formellement identifiés. La concentration en AMT était mesurée dans le SP (4686 ng/mL) et le SC (4653 ng/mL) ainsi que dans l'urine et la bile (> 5000 ng/mL). Les cheveux ont été analysés sans segmentation et les concentrations obtenues étaient les suivantes : AMT (24 030 pg/mg), 5-MAPB (722 pg/mg) et 5-APB (68,7 pg/mg). Toutefois, les bains de lavage présentaient également de fortes concentrations en ces composés. L'étude du métabolisme de l'AMT a mis en évidence 17 métabolites. Cinq ont été retrouvés dans le SP de la victime et les 17 dans l'urine. Les métabolites hydroxylés (OH-AMT glucuro- et sulfo-conjugués) et l'AMT-N-acétylé ont été proposés comme marqueurs de consommation d'AMT (Malaca et al., 2023 Metabolites. 13(1)). La mise en évidence de composés de type tryptamines peut être rendue complexe du fait de la présence d'interférences matricielles dans des cas d'analyse de matrices altérées, de la difficulté de la distinction des isomères en l'absence de standard et de l'absence de données sur les métabolites. Dans le cas présent, l'étude du métabolisme de l'AMT a permis d'identifier de nombreux métabolites dans deux matrices et de confirmer la consommation d'AMT. Concernant l'analyse capillaire, la présence d'AMT et de benzofuranes dans les bains de lavage suggérait une contamination externe très probablement secondaire à la sueur au moment de la phase agonique et ne permettait donc pas de documenter une habitude de consommation. Finalement, la cause de décès retenue était l'intoxication aiguë par AMT en association avec des benzofuranes et des benzodiazépines.

Tryptamine intoxications and fatalities are increasing, although these novel psychoactive substances (NPS) are not controlled in most countries. There are few data on the metabolic pathways and enzymes involved in tryptamine biotransformation. 4-acetoxy-N,N-diisopropyltryptamine (4-AcO-DiPT) is a synthetic tryptamine related to 4-hydroxy-N,N-diisopropyltryptamine (4-OH-DiPT), 4-acetyloxy-N,N-dipropyltryptamine (4-AcO-DPT), and 4-acetoxy-N,N-dimethyltryptamine (4-AcO-DMT). The aim of this study was to determine the best 4-AcO-DiPT metabolites to identify 4-AcO-DiPT consumption through human hepatocyte metabolism and high-resolution mass spectrometry. 4-AcO-DiPT metabolites were predicted in silico with GLORYx freeware to assist in metabolite identification. 4-AcO-DiPT was incubated with 10-donor-pooled human hepatocytes and sample analysis was performed with reversed-phase liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) in positive- and negative-ion modes. Software-assisted LC-HRMS/MS raw data mining was performed. A total of 47 phase I and II metabolites were predicted, and six metabolites were identified after 3 h incubation following ester hydrolysis, O-glucuronidation, O-sulfation, N-oxidation, and N-dealkylation. All second-generation metabolites were derived from the only first-generation metabolite detected after ester hydrolysis (4-OH-DiPT). The metabolite with the second-most-intense signal was 4-OH-iPT-sulfate followed by 4-OH-DiPT-glucuronide, indicating that glucuronidation and sulfation are common in this tryptamine’s metabolic pathway. 4-OH-DiPT, 4-OH-iPT, and 4-OH-DiPT-N-oxide are suggested as optimal biomarkers to identify 4-AcO-DiPT consumption.

BACKGROUND AND AIM:From few years, an emerging number of new psychoactive substances (NPS) entered the illicit market. NPS are designed to be similar to the effects of classical drugs of abuse, with increased effects and duration. Synthetic cannabinoids are cannabinoid receptor agonists (SCRAs), some of the most abused NPS.METHODS:We have herein briefly highlighted current relevant available information on the newest SCRAs generation, with relevant structural remarks as to the distinctive traits of such substances.RESULTS:Compared to the previous SCRAs generations, the structures of the last generation result in increased affinity for and efficacy at cannabinoid CB1 receptors, which are thought to be mainly responsible for the psychoactive effects of THC and its analogues. Accordingly, these more potent cannabimimetic effects may increase the number of adverse reactions such as neurological disorders, psychiatric episodes and deaths. In the last decade, more than a hundred SCRAs from different chemical classes emerged on the illicit web market. SCRAs have been thoroughly studied and the last generations include increasingly potent and toxic compounds, posing a potentially daunting health threat to consumers.CONCLUSIONS:From November 2017 to February 2021, at least 20 new "fourth-generation" SCRAs were formally reported to international drug agencies. Our understanding about the neurotoxicity of these compounds is still limited, due to the lack of global data, but their potency and their toxicity are likely higher than those of the previous generations.

Synthetic cathinones are a class of new psychoactive substances (NPS) with structural and pharmacological similarity to amphetamines. Methylone, a synthetic cathinone NPS, is the β-keto analog of MDMA. There are no controlled administration data for methylone in humans. The aim of this research was development of an LC-MS/MS method to simultaneously quantify methylone, MDMA and their metabolites 4-hydroxy-3-methoxy-N-methylcathinone (HMMC), 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymeth-amphetamine (HMMA) and 4-hydroxy-3-methoxyamphetamine (HMA) in human plasma and application of the method to the first controlled oral methylone administration study. Briefly, 10 μL 100 ng/mL internal standard mixture (methylone-d3, MDA-d5 and MDMA-d5) and 2 μL NH2 2% in H2O (pH 9) were added to 100 μL plasma, tubes were stirred in a roller mixer for 10 min and centrifuged at 3500 rpm for 5 min. Supernatants were collected into clean tubes, 100 μL acidic methanol (1% HCl) was added to prevent evaporative losses, and samples dried under nitrogen for approximately 30 min. Samples were reconstituted in 100 μL mobile phase A:B (95:5) (0.1% formic acid in water:acetonitrile) and transferred into autosampler vials for LC–MS/MS analysis. Analytes separation was obtained on a UHPLC 1290 Infinity II (Agilent Technologies Italia S.p.a.) coupled to a mass spectrometer (6470A Triple Quadrupole LC-MS) equipped with an electrospray ionization source operating in positive-ion mode (ESI+). Limits of quantification (LLOQ) were 5 (methylone) to 500 (MDMA) μg/L and 0.5 to 50 μg/L for their metabolites. A randomized double blinded study was conducted in 12 male volunteers (aged 18–45 years old; weight ranging from 50 to 90 kg) and divided into 3 sessions, with single oral doses of 50, 100, 150 or 200 mg methylone, 100 mg MDMA or placebo in each session. Plasma samples were collected before and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10 and 24 h after dosing. The analytical method was validated over five successive days in plasma following the most recent criteria for bioanalytical method development and validation. The method was linear for all analytes over the calibration range. Intra- and inter-assay accuracies were ±15%, while intra- and inter-assay precision were ±10%. Methylone plasma concentrations increased in a dose-related manner. Methylone Cmax were 138, 243, 320 and 530 μg/L following the 50, 100, 150 and 200 mg doses, respectively, while corresponding area under the curve (AUC) were 1065, 2486, 3511 and 5489 min ×μg/L. Methylone Tmax were 1 h for the lowest 50 mg dose and 2 h for the remaining doses. The last detected methylone concentrations (Clast) at 24 h were 10.5, 18.8, 36.4 and 51.8 μg/L (for 50, 100, 150 and 200 mg respectively). For methylone's HMMC metabolite, Cmax were 6.6, 16.9, 28.3, and 32.7 μg/L, while AUC were 36.9, 90.1, 129 and 173 min ×μg/L. HMMC exhibited rapid kinetics with Tmax of 1–1.25 h for all doses. As for Clast, HMMC concentrations were 0.5, 0.4, 1.3 and 0.8 μg/L (for 50, 100, 150 and 200 mg, respectively) 24 h after drug administration. Methylone is a popular drug of abuse, yet no studies addressed its metabolism and pharmacokinetics in humans. A validated LC-MS/MS method for simultaneous quantification of methylone, MDMA and their metabolites (HMMC, MDA, HMMA and HMA) in human plasma is presented. Considering the pharmacokinetic study for methylone and HMMC, concentrations following the 100 mg dose were approximately doubled compared to the 50 mg dose, and after the 200 mg dose were about four times higher than the lowest dose (within ±20%), suggesting linear pharmacokinetics for both analytes.

The need to identify and quantify a growing number of NPS represents a crucial challenge for toxicological and forensic purposes. Among biological specimens investigated for pharmaco-toxicological analyses, oral fluid (OF) provides greater advantages compared to blood to prove drug current use. OF is regarded with particular interest for drug screening at workplace and roadside testing, among others. This chapter contains an overview of analytical methodologies applied to determine the most used NPS (synthetic cannabinoids, cathinones, phenethylamines, designer piperazines, tryptamines, synthetic opioids, and designer benzodiazepines), and metabolites in OF by hyphenated techniques, reporting details of specimen extraction, separation, and detection with sensitivity, specificity, and accuracy details. The majority of the published methods used the Intercept® or the Quantisal® device for OF collection, and analytes were mostly extracted by solid-phase or liquid–liquid extraction. Liquid chromatography coupled to (tandem) mass spectrometry was the main applied technique for target analysis of NPS in OF.

Tryptamine intoxications and fatalities are increasing, although these novel psychoactive substances (NPS) are not controlled in most countries. There are few data on the metabolic pathways and enzymes involved in tryptamines biotransformation. 4-acetoxy-N,N-diisopropyltryptamine (4-AcO-DiPT) is a synthetic tryptamine related to 4-hydroxy-N,N-diisopropyltryptamine (4-OH-DiPT), 4-acetyloxy-N,N-dipropyltryptamine (4-AcO-DPT) and 4-acetoxy-N,N-dimethyltryptamine (4-AcO-DMT). The aim was to determine the best 4-AcO-DiPT metabolites to identify 4-AcO-DiPT consumption through human hepatocyte metabolism and high-resolution mass spectrometry. 4-AcO-DiPT metabolites were predicted in silico with GLORYx freeware to assist in metabolite identification. 4-AcO-DiPT was incubated with 10-donor-pooled human hepatocytes, and sample analysis performed with reversed-phase liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) in positive- and negative-ion modes. LC-HRMS/MS raw data were separately processed with targeted and nontargeted data-mining. A total of 47 phase I and II metabolites were predicted, and six metabolites were identified after 3 h incubation following ester hydrolysis, O-glucuronidation, O-sulfation, N-oxidation and N-dealkylation. All second-generation metabolites derived from the only first-generation metabolite detected after ester hydrolysis (4-OH-DiPT). Interestingly, the most intense metabolite (4-OH-DiPT) was also detected in the control samples without hepatocytes at 0 and 3 h and at 0 h incubation with hepatocytes but at much lower intensity than after 3 h incubation with hepatocytes, meaning that, although 4-OH-DiPT is formed through enzymatic reaction, it is also spontaneously formed during incubation to a lesser extent. This indicates that 4-OH-DiPT formation was overestimated in our experiments. When analyzing authentic samples, digestion (with β-glucuronidase/sulfatase) is preferred to increase the 4-OH-DiPT signal. Since the parent drug is most likely degraded and the acetyl group is eliminated quickly, toxicologists could report 4-OH-DiPT positive cases that actually were from 4-AcO-DiPT intake. The second most intense metabolite was 4-OH-iPT-sulfate followed by 4-OH-DiPT-glucuronide, indicating that glucuronidation and sulfation are common in this tryptamine's metabolic pathway. Others propose that tryptamines do not have a common metabolic pathway and that metabolism changes depending on the nature and position of their substituents, with demethylation, hydroxylation and dealkylation the most common phase I reactions, followed by glucuronidation or sulfation. 4-OH-DiPT, 4-OH-iPT and 4-OH-DiPT-N-oxide are proposed as biomarkers of 4-AcO-DiPT consumption, but the rapid enzymatic hydrolysis and lower spontaneous hydrolysis of 4-AcO-DiPT to 4-OH-DiPT might create a problem in discerning 4-AcO-DiPT from 4-OH-DiPT consumption. If the parent drug is present even in low concentrations, the ingested drug would be clear, but there is no available information regarding detection of this drug in authentic samples. These results require in vivo confirmation, which is challenging due to the small number of 4-AcO-DiPT seizures in recent years. Psychedelic tryptamine use is low but increasing, raising the importance of laboratory identification of specific metabolites to verify intake and identify potential public health NPS outbreaks. Since the parent drug is rapidly hydrolyzed and it is currently unknown whether 4-AcO-DiPT is present in authentic biological samples, identifying its metabolites is highly useful throughout, but especially late in its time course. 4-OH-DiPT, 4-OH-iPT and 4-OH-DiPT-N-oxide are optimal biomarkers to identify 4-AcO-DiPT consumption.

Cannabidiol (CBD) exhibits anti-inflammatory, anxiolytic, antiseizure, and neuroprotective proprieties without addictive or psychotropic side effects, as opposed to Δ9-tetrahydrocannabinol (THC). While recreational cannabis contains higher THC and lower CBD concentrations, medical cannabis contains THC and CBD in different ratios, along with minor phytocannabinoids, terpenes, flavonoids and other chemicals. A volumetric absorptive microsampling (VAMS) method combined with ultra-high-performance liquid chromatography coupled with mass spectrometry in tandem for quantification of CBD, THC and their respective metabolites: cannabidiol-7-oic acid (7-COOH-CBD); 7-hydroxy-cannabidiol (7-OH-CBD); 6-alpha-hydroxy-cannabidiol (6-α-OH-CBD); and 6-beta-hydroxycannabidiol (6-β-OH-CBD); 11- Hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH). After overnight enzymatic glucuronide hydrolysis at 37°C, samples underwent acidic along with basic liquid-liquid extraction with hexane: ethyl acetate (9:1, v/v). Chromatographic separation was carried out on a C18 column, with the mass spectrometer operated in multiple reaction monitoring mode and negative electrospray ionization. Seven patients with intractable epilepsy were dosed with various CBD-containing formulations and blood collected just before their daily morning administration. The method was validated following international guidelines in toxicology. Linear ranges were (ng/ml) 0.5–25 THC, 11-OH-THC, THCCOOH, 6-α-OH-CBD and 6-β-OH-CBD; 10–500 CBD and 7-OH-CBD; and 20–5000 7-COOH-CBD. 7-COOH-CBD was present in the highest concentrations, followed by 7-OH-CBD and CBD. This analytical method is useful for investigating CBD, THC and their major metabolites in epilepsy patients treated with CBD preparations employing a minimally invasive microsampling technique requiring only 30 µL blood.

Background4-Hydroxy-N,N-methylpropyltryptamine (4-OH-MPT) is a psychedelic tryptamine whose use is regulated in several countries. Due to unspecific effects, consumption can be ascertained only through toxicological analyses. However, the trace amounts of tryptamines are usually challenging to detect in biological samples. 4-OH-MPT metabolism was characterized to identify optimal metabolite markers of intake in clinical/forensic toxicology.Research design and methods4-OH-MPT was incubated with 10-donor-pooled human hepatocytes to simulate in vivo conditions; samples were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS), and data were processed with Compound Discoverer from Thermo Scientific. LC-HRMS/MS and data mining were supported by in silico metabolite predictions (GLORYx).ResultsThree phase I and four phase II metabolites were identified, including N-oxidation and N-demethylation at the alkylamine chain, and O-glucuronidation and sulfation at the hydroxylindole core.Conclusions4-OH-MPT metabolic fate was consistent with the human metabolism of tryptamine analogues: we suggest 4-OH-MPT-N-oxide and 4-hydroxy-N,N-propyltryptamine (4-OH-PT) as metabolite biomarkers of 4-OH-MPT consumption after glucuronide/sulfate hydrolysis in biological samples to improve detection of 4-OH-MPT and phase I metabolites; 4-OH-MPT-glucuronide is suggested as an additional biomarker when hydrolysis is not performed. Further research on the metabolism of structural analogues is necessary to evaluate the specificity of 4-OH-MPT metabolite biomarkers.

The aim of this study is to define, for the first time, human methylone and HMMC plasma pharmacokinetics following controlled administration of 50–200 mg methylone to 12 male volunteers. A new LC-MS/MS method was validated to quantify methylone, MDMA, and their metabolites in plasma. The study was a randomized, cross-over, double-blinded and placebo-controlled study, with a total of 468 plasma samples collected. First, 10 µL of MDMA-d5, MDA-d5 and methylone-d3 internal standards were added to 100 µL of plasma. Two mL of chloroform and ethyl acetate 9:1 (v/v) were then added, mixed well and centrifuged. The supernatant was fortified with 0.1 mL acidified methanol and evaporated under nitrogen. Samples were reconstituted with a mobile phase and injected into the LC-MS/MS instrument. The method was fully validated according to OSAC guidelines (USA). Methylone plasma concentrations increased in a dose-proportional manner, as demonstrated by the increasing maximum concentration (Cmax) and area under the curve of concentrations (AUC). Methylone Cmax values were reported as 153, 304, 355 and 604 ng/mL, AUC0–24 values were reported as 1042.8, 2441.2, 3524.4 and 5067.9 h·ng/mL and T1/2 values as 5.8, 6.4, 6.9 and 6.4 h following the 50, 100, 150 and 200 mg doses, respectively. Methylone exhibited rapid kinetics with a Tmax of 1.5 h for the 50 mg dose and 2 h approximately after all the other doses. HMMC exhibited faster kinetics compared to methylone, with a Cmax value that was 10–14-fold lower and an AUC0–24 value that was 21–29-fold lower. Methylone pharmacokinetics was linear across 50–200 mg oral doses in humans, unlike the previously described non-linear oral MDMA pharmacokinetics. An LC-MS/MS method for the quantification of methylone, MDMA and their metabolites in human plasma was achieved. Methylone exhibited linear pharmacokinetics in humans with oral doses of 50–200 mg.

BACKGROUND:Over the past few years, an emerging number of new psychoactive substances (NPSs) entered the illicit market. NPSs are designed to resemble the effects of classical drugs of abuse, reinforcing their effects and duration. Among the most abused NPS, synthetic cannabinoids are cannabinoid receptor agonists (SCRAs) that mimic the effect of the main psychotropic phytocannabinoid Δ9-tetrahydrocannabinol (THC).METHODS:We herein reviewed the international literature to provide available information on the newest SCRAs generation.RESULTS:Compared to the previous SCRAs generations, the structures of the last generation result in increased affinity for and efficacy at cannabinoid CB1 receptors, which are thought to be mainly responsible for the psychoactive effects of THC and its analogues. Accordingly, these more potent cannabimimetic effects may increase the number of adverse reactions such as neurological disorders (e.g., psychosis, agitation, irritability, paranoia, confusion, and anxiety), psychiatric episodes (e.g., hallucinations, delusions, self-harm), other physical conditions (e.g., tachycardia, hypertension, arrhythmia, chest pain, nausea, vomiting, and fever) and deaths. In the last decade, more than a hundred SCRAs from different chemical classes emerged on the illicit web market. SCRAs have been thoroughly studied: they were physico-chemically characterized, and pharmaco-toxicological characteristics were investigated. The last SCRAs generations include increasingly potent and toxic compounds, posing a potential health threat to consumers.CONCLUSION:From November 2017 to February 2021, at least 20 new "fourth-generation" SCRAs were formally reported to international drug agencies. Our understanding of the neurotoxicity of these compounds is still limited due to the lack of global data, but their potency and their toxicity are likely higher than those of the previous generations.

Variable individual responses and relationships between administered dose and CBD blood concentrations suggest therapeutic drug monitoring (TDM) as a valuable support for patient management. Currently, there is no published method for the simultaneous quantification of CBD, THC and their main active and inactive metabolites. The aim of this study was the development of an analytical method for simultaneous quantification of cannabidiol (CBD), 7-hydroxy-cannabidiol (7-OH-CBD), cannabidiol-7-oic acid (7-COOH-CBD), 6-alpha-hydroxy-cannabidiol (6-α-OH-CBD), and 6-beta-hydroxycannabidiol (6-β-OH-CBD), Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in blood using volumetric absorptive microsampling (VAMS) and LC-MS/MS. Glucuronide hydrolysis was conducted by adding 2 μL IS solution (with 11-OH-THC-d3, THC-COOH-d3, CBD-d3, THC-d3 at 100 ng/mL) and 50 μL beta-glucuronidase to 30 μL blood samples in a VAMS tip, diluting in 800 μL water and heating at 37 °C overnight. After hydrolysis, acidic and basic extractions were performed to increase the probability of isolating all metabolites. The basic extraction was performed with 4 mL hexane: ethyl acetate (9:1) and 100 μL ammonium hydroxide and the acidic extraction with 4 mL hexane: ethyl acetate (9:1) and 15 μL formic acid (≥ 99.9%). Tubes were capped, vortexed for 10 sec, mixed for 10 min and centrifuged at 5000 g for 5 min. The supernatants from both extractions were combined into a glass tube and dried under nitrogen. Samples were reconstituted with 100 μL water:methanol (1:1) and transferred into autosampler glass vials, before injection of 10 μL onto the chromatographic system. Chromatographic separation was performed on a C18 column, with multiple reaction monitoring (MRM) in both positive and negative ionization modes (ESI). Proof of concept of the method included analysis of blood from seven epileptic patients receiving controlled dosing of different CBD formulations (20 mg/kg/day Epidiolex®, 18 drops-3.86 mg/kg/day CBD oil, 3.86 mg/kg/day CBD oil Enecta and 100–250 mg/kg/day CBD crystals). Accurate small volume blood collection was achieved with VAMS, and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry to achieve low limits of quantification for CBD, THC, and their respective metabolites. Method validation followed international guidelines in toxicology, with achieved linear ranges of 0.5–25 ng/mL (6-α-OH-CBD, 6-β-OH-CBD, THC, 11-OH-THC and THC-COOH), 10–500 ng/mL (CBD and 7-OH-CBD), and 20–5000 ng/mL for 7-COOH-CBD. Limits of quantification ranged from 0.02-3.4 ng/mL. Accuracy and precision were within ±20% at LOQ for all matrices and within ±15% for all quality control samples across the linear range. Analytical recoveries ranged from 61–98.6%, and matrix effects from 88.6–97.8%. Seven patients (4 males, age 3–12 years, weight 11–28.4 kg; 3 females, age 8–20 years, weight 23.6–40 kg), with blood samples collected at trough concentration just prior to the next administration. 7-COOH-CBD was present in the highest concentrations (211–1537 ng/mL), followed by 7-OH-CBD (53.4–498 ng/mL) and CBD (19.1–2501 ng/mL). The isomers 6-α-OH-CBD and 6-β-OH-CBD were detected but in lower concentrations (0.9–28.9 and 1.0–38.7 ng/mL, respectively), in accordance with previous studies. There were no traces of THC or its metabolites in the blood samples, as expected since the prepared CBD formulations did not contain THC. This simultaneous cannabinoids analytical method is valuable for further investigation of CBD, THC and their metabolites in individuals treated with medical cannabis, THC or CBD pharmacotherapies and/or recreational cannabis use.

Tryptamines are indolealkylamines sharing their core structure with serotonin. Most tryptamines are psychoactive hallucinogens. Every year, new molecules are synthesized to circumvent the laws. To date, 53 tryptamine analogues are monitored by the EU Early Warning System, and, although the total number of intoxications and fatalities are still low, they are increasing. Tryptamines are generally potent and their concentrations in biological matrices are low, making detection challenging in analytical toxicology. Urinary metabolite biomarkers can be targeted to improve detection. However, like other novel psychoactive substances (NPS), there are no data available on the metabolism of the most recently encountered molecules (Malaca. International Journal of Molecular Sciences 2020;21:9279). 4-hydroxy-N,N-methylpropyltryptamine (4-OH-MPT), also known as meprocin, is a psychedelic tryptamine first identified in seizures in Europe in 2018. 4-OH-MPT intake was never reported in the literature or early warning systems in authentic cases, possibly due to lack of specific metabolite biomarkers. The aim of this research was to characterize 4-OH-MPT metabolism in human hepatocytes and identify optimal metabolite markers to identify 4-OH-MPT intake. Analyses were conducted following our in-house protocol (Di Trana. Talanta 2021;235:122740). To assist in metabolite identification, 4-OH-MPT metabolic fate was predicted using GLORYx freeware, generating a list of potential first- and second-generation metabolites with a probability score. 4-OH-MPT was incubated with cryopreserved ten-donor-pooled human hepatocytes for 3-h to simulate liver metabolism. Incubates were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) in full-scan and data-dependent MS/MS acquisition modes to capture the signal of all metabolites and their fragmentation pattern in two injections, i.e., in positive- and negative-ionization modes. LC-HRMS/MS data were mined with Compound Discoverer (Thermo Scientific) using a targeted/non-targeted approach to identify expected and unexpected metabolites. After excluding duplicates and putative metabolites with a molecular mass lower than 100 Da, 12 first-generation and 28 subsequent second-generation metabolites were predicted in silico; major reactions were O-sulfation, O-glucuronidation, N-dealkylation, and hydroxylation, N-oxidation, and carboxylation at the N-alkyl chain. 4-OH-MPT signal intensity decreased to 47% after 3-h incubation with hepatocytes. Seven metabolites were produced through N-demethylation and N-oxidation at the alkyl side chain, and O-glucuronidation and O-sulfation at the indole ring; 4-OH-MPT-glucuronide was the metabolite with the most intense signal. These results were consistent with the metabolic fate of structural analogues. The LC gradient was purposefully long and gradual to separate potential isomers. For this reason, O-glucuronides presented a broad shoulder peak, maybe indicating the resolution of two conformers of the same metabolites. 4-OH-MPT-N-oxide and 4-hydroxy-propyltryptamine (4-OH-PT) are proposed as metabolite biomarkers of 4-OH-MPT consumption after glucuronide and sulfate hydrolysis to increase 4-OH-MPT detection capabilities. Alternatively, 4-OH-MPT-glucuronide is suggested as an additional biomarker of 4-OH-MPT intake when hydrolysis is not performed. Unfortunately, these metabolites may not be specific to 4-OH-MPT. There are currently no data on the metabolism of structural analogues 4-hydroxy-N,N-dipropyltryptamine (4-OH-DPT), 4-acetoxy-N,N-dipropyltryptamine (4-AcO-DPT), and 4-acetoxy-N,N-methylpropyltryptamine (4-AcO-MPT) that potentially could produce similar metabolites. However, 4-OH-PT might be produced by 4-OH-DPT N-depropylation and 4-AcO-DPT ester hydrolysis and N-depropylation, making 4-OH-MPT-N-oxide crucial to document 4-OH-MPT consumption. More importantly, 4-AcO-MPT and 4-OH-MPT might produce the same major metabolites, considering the reactivity of the ester bond in 4-AcO-MPT. Therefore, the distinction between 4-OH-MPT and 4-AcO-MPT consumption may not be possible without detecting 4-AcO-MPT. The detection of tryptamine metabolites in biological matrices is crucial to document consumption in clinical and forensic settings. 4-OH-MPT-glucuronide, 4-OH-MPT-N-oxide, and 4-OH-PT are metabolite biomarkers of 4-OH-MPT consumption; however, research on the metabolism of other tryptamine structural analogues is necessary to evaluate the specificity of these metabolites to identify 4-OH-MPT only.

Cannabidiol (CBD) is a promising therapeutic agent with analgesic, myorelaxant, and anti-epileptic actions. Recently, a purified form of CBD (Epidiolex®) has been approved by the European Medicines Agency (EMA) for the treatment of two highly-refractory childhood-onset epilepsies (Dravet and Lennox-Gastaut syndrome). Given the interindividual response and the relationship between the dose administered and CBD blood levels, therapeutic drug monitoring (TDM) is a valuable support in the clinical management of patients. We herein report for the first time a newly developed and validated method using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS) to evaluate CBD and its metabolites (i.e., cannabidiol-7-oic acid (7-COOH-CBD), 7-hydroxycannabidiol (7-OH-CBD), 6-α-hydroxycannabidiol (6-α–OH–CBD) and 6-β-hydroxycannabidiol (6-β–OH–CBD)) in serum samples. The method reached the sensitivity needed to detect minimal amounts of analytes under investigation with limits of quantification ranging from 0.5 to 20 ng/mL. The validation results indicated in this method were accurate (average inter/intra-day error, <15%), precise (inter/intra-day imprecision, <15%), and fast (8 min run time). The method resulted to be linear in the range of 1–10,000 ng/mL for CBD-COOH, 1–500 ng/mL for 7-OH-CBD and CBD and 1–25 ng/mL for 6-α–OH–CBD and 6-β–OH–CBD. Serum levels of CBD (88.20–396.31 and 13.19–170.63 ng/mL) as well as of 7-OH-CBD (27.11–313.63 and 14.01–77.52 ng/mL) and 7-COOH-CBD (380.32–10,112.23 and 300.57–2851.82 ng/mL) were significantly higher (p < 0.05) in patients treated with GW pharma CBD compared to those of patients treated with galenic preparations. 6-α–OH–CBD and 6-β–OH–CBD were detected in the first group and were undetectable in the second group. 7-COOH-CBD was confirmed as the most abundant metabolite in serum (5–10 fold higher than CBD) followed by 7-OH-CBD. A significant correlation (p < 0.05) between the dose administrated and a higher bioavailability was confirmed in patients treated with a GW pharma CBD preparation.

Tanja R. Zijp, PharmD* Martha L. Toren-Wielema, PharmD* Prashant V. Nannan Panday, PharmD* Jos G. W. Kosterink, PhD* Stefan P. Berger, PhD†‡ Daan J. Touw, PhD* *Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen and University of Groningen, Groningen, the Netherlands †Department of Internal Medicine, Division of Nephrology, University Medical Center Groningen and University of Groningen, Groningen, the Netherlands ‡UMC Groningen Comprehensive Transplant Center, University Medical Center Groningen and University of Groningen, Groningen, the Netherlands

No analytical assay is currently available for the simultaneous determination of CBD major metabolites in serum or urine samples of individuals treated with medical cannabis or CBD-based pharmaceuticals. We developed and validated a method using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) for quantifying cannabidiol (CBD) and its metabolites, cannabidiol-7-oic acid (7-COOH-CBD), 7- hydroxycannabidiol (7-OH-CBD), 6-alpha-hydroxycannabidiol (6-α-OH-CBD) and 6-beta-hydroxycannabidiol (6-β-OH-CBD) in serum and urine samples of an individual treated with medical cannabis. The ionization was performed by electrospray in negative mode to reach the sensitivity required to detect trace amounts, with limits of quantification ranging from 0.05 to 0.1 ng/mL. The method is accurate (average inter/intra-day error, <15%), precise (inter/intra-day imprecision, <15%) and fast (8 min run time) and it is an essential tool to investigate CBD pharmacokinetics and pharmacodynamics in individuals treated with medical cannabis or with CBD-based medical preparations.

Our understanding of tryptamines is poor due to the lack of data globally. Tryptamines currently are not part of typical toxicology testing regimens and their contribution to drug overdoses may be underestimated. Although their prevalence was low, it is increasing. There are few published data on the many new compounds, their mechanisms of action, onset and duration of action, toxicity, signs and symptoms of intoxication and analytical methods to identify tryptamines and their metabolites. We review the published literature and worldwide databases to describe the newest tryptamines, their toxicology, chemical structures and reported overdose cases. Tryptamines are 5-HT2A receptor agonists that produce altered perceptions of reality. Currently, the most prevalent tryptamines are 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DiPT), 5-methoxy-N,N- diallyltryptamine (5-MeO-DALT) and dimethyltryptamine (DMT). From 2015 to 2020, 22 new analytical methods were developed to identify/quantify tryptamines and metabolites in biological samples, primarily by liquid chromatography tandem mass spectrometry. The morbidity accompanying tryptamine intake is considerable and it is critical for clinicians and laboratorians to be informed of the latest data on this public health threat.

Sampling and drug stability in oral fluid (OF) are crucial factors when interpreting forensic toxicological analysis, mainly because samples may not be analyzed immediately after collection, potentially altering drug concentrations. Therefore, the stability of some common drugs of abuse (morphine, codeine, 6-monoacetylmorphine, cocaine, benzoylecgonine, Delta(9)-tetrahydrocannabinol, cannabidiol, amphetamine, 3,4-methylenedioxymethamphetamine, ketamine) and the more commonly consumed new psychoactive substances in our environment (mephedrone, and N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamide 5F-AKB48 also known as 5F-APINACA) was investigated in an OF pool for the presence and absence of M3 Reagent Buffer (R) up to 1 year of storage. Fortified OF samples were stored at three different temperatures (room temperature, 4 and -20 degrees C) to determine the best storage conditions over time. Control fortified OF samples were stored at -80 degrees C for reference purposes. Compounds with concentration changes within +/- 15% of initial value were considered stable. The drugs were significantly more stable in M3 Reagent Buffer (R) than in neat OF samples in all storage conditions. All analytes were stable for 1 year at 4 degrees C and -20 degrees C in M3 Reagent Buffer (R). Drugs stability in OF varied depending on the analyte, the presence of a stabilizer, the storage duration and temperature. When immediate sample analysis is not possible, we suggest to store OF samples at 4 or -20 degrees C and test them within 2 weeks. Alternatively, OF samples may be stored at 4 or -20 degrees C with M3 Reagent Buffer (R) to be tested within 1 year.