Jeremy Carlier

In vitro, in vivo and in silico metabolic profiling of α-pyrrolidinopentiothiophenone, a novel thiophene stimulant

BACKGROUND Little or no pharmacological or toxicological data are available for novel psychoactive substances when they first emerge, making their identification and interpretation in biological matrices challenging. MATERIALS & METHODS A new synthetic cathinone, α-pyrrolidinopentiothiophenone (α-PVT), was incubated with hepatocytes and samples were analyzed using liquid chromatography coupled to a Q Exactive™ Orbitrap mass spectrometer. Authentic urine specimens from suspected α-PVT cases were also analyzed. Scans were data mined with Compound Discoverer™ for identification and structural elucidation of metabolites. RESULTS/CONCLUSION Seven α-PVT metabolites were identified in hepatocyte incubations, and in the authentic urine samples, also with an additional monohydroxylated product and a glucuronide of low intensity. α-PVT dihydroxypyrrolidinyl, α-PVT 2-ketopyrrolidinyl, α-PVT hydroxythiophenyl and α-PVT thiophenol had the most intense in vivo signals.

Quantification of Pregabalin Using Hydrophilic Interaction HPLC-High-Resolution MS in Postmortem Human Samples: Eighteen Case Reports

Pregabalin is a drug for treating epilepsy, anxiety disorders and neuropathic pain. Cases of poisoning are rare, though some have been fatal. Concentrations of pregabalin in postmortem human samples and its distribution have very rarely been documented. As the literature is so scarce, we propose to report the concentrations in autopsy samples of 18 people who had been taking Lyrica(®), including one case of a mixed overdose involving pregabalin. Analysis was carried out using an original Hydrophilic Interaction LIquid Chromatography (HILIC) technique coupled with a high-resolution mass spectrometer (m/z 160.1334 ± 5 ppm). The sensitivity of the technique enables a quick and simple treatment of the samples by protein precipitation. The method was validated in the whole blood with detection and quantification limits of 0.025 and 0.060 µg/mL, respectively. Pregabalin was a likely factor in the cause of death in 3 of the 18 cases. In the other individuals, the concentrations ranged from 0.4 to 17.0 in the peripheral blood, 1.5 to 11.1 in the central blood, 126.6 to 2004.6 in the urine and 10.5 to 58.3 µg/mL in the bile, with median values of 5.6, 4.6, 534.6 and 17.7, respectively.

Fatal Case of a 27‐Year‐Old Male After Taking Iboga in Withdrawal Treatment: GC‐MS/MS Determination of Ibogaine and Ibogamine in Iboga Roots and Postmortem Biological Material

We report the case of a man who died twelve hours after ingesting powdered iboga root, commonly taken for its stimulant and hallucinogenic properties. Ibogaine and ibogamine were quantified in the powder ingested and the victims body fluids by GC‐MS/MS after liquid–liquid extraction (Toxi‐tubes A®). The concentrations of ibogaine measured in the blood samples taken at the scene and in the peripheral blood, urine, and gastric fluid samples taken during the autopsy were 0.65, 1.27, 1.7, and 53.5 μg/mL, while the iboga content in the powder was 7.2%. Moreover, systematic toxicological analyses of biological samples showed the presence of diazepam and methadone in therapeutic concentrations. Death was attributed to the ingestion of a substantial quantity of iboga in the context of simultaneous methadone and diazepam consumption.

The principal toxic glycosidic steroids in Cerbera manghas L. seeds: identification of cerberin, neriifolin, tanghinin and deacetyltanghinin by UHPLC-HRMS/MS, quantification by UHPLC-PDA-MS.

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 219nm. Identification was confirmed by UHPLC-HRMS. Deacetyltanghinin (m/z 549.3055±2ppm), neriifolin (m/z 535.3259±2ppm), tanghinin (m/z 591.3169±2ppm) and cerberin (577.3375±2ppm) 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 285.9μg/g, respectively. A seed of the fresh unripe fruit had concentrations of 49.4, 47.0, 3.5 and 2.3μg/g.

In Vitro Metabolite Profiling of ADB-FUBINACA, A New Synthetic Cannabinoid

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.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. .

Distinguishing Intake of New Synthetic Cannabinoids ADB-PINACA and 5F-ADB-PINACA with Human Hepatocyte Metabolites and High-Resolution Mass Spectrometry

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.

Synthetic cannabinoid BB-22 (QUCHIC): Human hepatocytes metabolism with liquid chromatography-high resolution mass spectrometry detection

HIGHLIGHTSBB‐22 metabolite profile using human hepatocytes is reported for the first time.Optimal metabolite targets for documenting BB‐22 intake are proposed.MDMB‐CHMICA and ADB‐CHMICA intake must be ruled out to confirm BB‐22 consumption. ABSTRACT 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&mgr;mol/L BB‐22 with cryopreserved human hepatocytes for 3h. 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.

Quantification of [1-(5-fluoropentyl)-1H-indol-3-yl](naphthalene-1-yl)methanone (AM-2201) and 13 metabolites in human and rat plasma by liquid chromatography-tandem mass spectrometry.

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 (75μL) consisted of an enzymatic hydrolysis and a supported liquid extraction. The method was validated with human plasma with a 0.025 or 0.050-50μg/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<11.9% coefficient of variation. Method recoveries and matrix effects ranged from 58.4-84.4% and -62.1 to -15.6%, respectively. AM-2201 and metabolites were stable (±20%) at room temperature for 24h, at 4°C for 72h, and after three freeze-thaw cycles, and for 72h in the autosampler after extraction. The method was developed for pharmacodynamic and pharmacokinetic studies with controlled administration in rats but is applicable for pre-clinical and clinical research and forensic investigations. Rat plasma specimen analysis following subcutaneous AM-2201 administration demonstrated the suitability of the method. AM-2201, JWH-018 N-(5-hydroxypentyl), and JWH-018 N-pentanoic acid concentrations were 4.8±1.0, 0.15±0.03, and 0.34±0.07μg/L, respectively, 8h after AM-2201 administration at 0.3mg/kg (n=5).

Atropine eye drops: An unusual homicidal poisoning

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 victims peripheral blood and urine showed the presence of atropine, a powerful anticholinergic. Atropine was therefore specifically detected and quantified throughout the victims 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.

Pharmacodynamic effects, pharmacokinetics, and metabolism of the synthetic cannabinoid AM-2201 in male rats

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.