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This work first aims to investigate metabolites of 2-fluoro-deschloroketamine (2F-DCK), a new arylcyclohexylamine derivatives (a group of dissociative ketamine-based substances) using two in vitro experimental approaches, and to compare obtained results by means of molecular networking. Metabolites of 2F-DCK were investigated using both human liver microsomes (HLMs) and hepatic (HepaRG) cell line incubates using molecular networking approach: 2F-DCK pure substance was incubated with HLMs for up to 1 h at two concentrations (100 and 500 μM) and with HepaRG cells for two time periods (8 and 24 h) at one concentration (20 μM). In vitro obtained results were subsequently applied to a 2F-DCK-related fatality case. In vitro-produced metabolites were investigated using high-resolution accurate mass spectrometry using Orbitrap mass analyzer technology. Thirteen metabolites were in vitro produced and several metabolic pathways can be postulated. Seven additional metabolites were found in post-mortem samples (bile and urine) of the case, comprising three Phase II metabolites, which appear to be minor in vivo metabolites. HLMs and HepaRG cell models appear to be complementary and obtained data allowed the identification of several specific 2F-DCK metabolites in biological samples. In practical terms, observed metabolic ratios suggested that nor-2F-DCK (208.1137 m/z) and a hydrogenated metabolite (224.1443 m/z) could be proposed as reliable metabolites to be recorded in HRMS libraries in order to improve detection of 2F-DCK use., (© 2021 John Wiley & Sons, Ltd.)

Background: Histamine (HA) is a small amine playing an important role in anaphylactic reactions. In order to identify and quantify HA in plasma matrix, different methods have been developed but present several disadvantages. Here, we developed an alternative method using liquid chromatography coupled with an ultra-high resolution and accurate mass instrument, Q Exactive™ (Thermo Fisher) (LCHRMS)., Methods: The method includes a protein precipitation of plasma samples spiked with HA-d4 as internal standard (IS). LC separation was performed on a C18 Accucore column (100∗2.1mm, 2.6μm) using a mobile phase containing nonafluoropentanoic acid (3nM) and acetonitrile with 0.1% (v/v) formic acid on gradient mode. Separation of analytes was obtained within 10min. Analysis was performed from full scan mode and targeted MS2 mode using a 5ppm mass window. Ion transitions monitored for targeted MS2 mode were 112.0869>95.0607m/z for HA and 116.1120>99.0855m/z for HA-d4. Calibration curves were obtained by adding standard calibration dilution at 1 to 180nM in TrisBSA., Results: Elution of HA and IS occurred at 4.1min. The method was validated over a range of concentrations from 1nM to 100nM. The intra- and inter-run precisions were <15% for quality controls. Human plasma samples from 30 patients were analyzed by LCHRMS, and the results were highly correlated with those obtained using the gold standard radioimmunoassay (RIA) method., Conclusion: Overall, we demonstrate here that LCHRMS is a sensitive method for histamine quantification in biological human plasmas, suitable for routine use in medical laboratories. In addition, LCHRMS is less time-consuming than RIA, avoids the use of radioactivity, and could then be considered as an alternative quantitative method., (Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

The recent emergence of new synthetic opioids (NSOs) compounds in the illicit market is increasingly related to fatal cases. Identification and medical care of NSO intoxication cases are challenging, particularly due to high frequency of new products and extensive metabolism. As the study of NSO metabolism is crucial for the identification of these drugs in cases of intoxication, we aimed to investigate the metabolism of the piperazine NSO AP-237 (= bucinnazine). Two complementary approaches (in silico and in vitro) were used to identify putative AP-237 metabolites which could be used as consumption markers. In silico metabolism studies were realized by combining four open access softwares (MetaTrans, SyGMa, Glory X, Biotransformer 3.0). In vitro experiments were performed by incubating AP-237 (20 µM) in differentiated HepaRG cells during 0 h, 8 h, 24 h or 48 h. Cell supernatant were extracted and analyzed by liquid chromatography coupled to high-resolution mass spectrometry and data were reprocessed using three strategies (MetGem, GNPS or Compound Discoverer®). A total of 28 phase I and six phase II metabolites was predicted in silico. Molecular networking identified seven putative phase I metabolites (m/z 203.154, m/z 247.180, m/z 271.180, two m/z 289.191 isomers, m/z 305.186, m/z 329.222), including four previously unknown metabolites. Overall, this cross-disciplinary approach with molecular networking on data acquired in vitro and in silico prediction enabled to propose relevant candidate as AP-237 consumption markers that could be added to mass spectrometry libraries to help diagnose intoxication. [ABSTRACT FROM AUTHOR]

Consumption of mushrooms can become unsafe for the consumer in case of confusion. Some fungi of Cortinarius genus contain the nephrotoxic mycotoxin orellanine responsible for their toxicity. Related case poisoning diagnosis is a challenge for both clinicians and analysts because of a long latency period between intake and toxic syndrome, the lack of available information in literature and the numerous pitfalls of orellanine identification/quantification in biological samples. In this situation, we propose an analytical method designed for the orellanine detection and/or quantification in biological matrices such as plasma, urine and whole blood, in a context of related intoxication suspected case. Using 1 mL biological sample volume, this liquid chromatographic with high-resolution mass spectrometry detection method (i) exhibits a limit of quantification for orellanine of 0.5 µg/L in plasma and urine and (ii) enables orellanine detection in whole blood with a limit of detection of 0.5 µg/L. This validated analytical method was successfully applied to 10 suspected intoxication cases. [ABSTRACT FROM AUTHOR]

Acebutolol is a β1-selective adrenergic receptor antagonist with moderate membrane-stabilizing activity and intrinsic sympathomimetic activity; accordingly, the drug is indicated in hypertension, angina pectoris, and arrhythmia. However, acebutolol's beta-blocking properties also extend the QRS and QTc intervals, and may predispose the patient to ventricular tachydysrhythmia. Here, we report autopsy and toxicological findings on a fatal case of acebutolol self-poisoning in a 70-year-old woman. Toxicological analyses of post-mortem samples (using a liquid chromatography high-resolution mass spectrometry (LC-HR-MS) method) highlighted high concentrations of acebutolol and its metabolite diacetolol in femoral blood (92.8 mg/L and 21.2 mg/L, respectively) and other matrices (cardiac blood, urine, bile, and gastric contents). A molecular networking approach provided useful information on acebutolol's metabolism and revealed the existence of an unknown phase II metabolite of acebutolol. Molecular networking also facilitated visualization of the complex LC-HR-MS/MS datasets and the sample-to-sample comparisons that confirmed massive acebutolol intoxication by ingestion. [ABSTRACT FROM AUTHOR]

Mycotoxin intoxications can result from the consumption of amatoxins like α- and β-amanitin or of phallotoxin, present in several toxic mushrooms like Amanita phalloides. To identify and quantify amatoxins and phallotoidin in biological matrixes, we developed a method using liquid chromatography coupled with an ultra-high-resolution and accurate mass instrument (liquid chromatography–high-resolution-mass spectrometry, LC–HR-MS), Q Exactive™ (Thermo Fisher). The method includes a simple solid-phase extraction of urine samples spiked with flurazepam as internal standard (IS), using Bond Elut Agilent Certify cartridges (C18, 200 mg, 3 mL). LC separation was performed on a C18 Accucore column (100 × 2.1 mm, 2.6 µm) using a gradient of 10 mM ammonium acetate buffer containing 0.1% (v/v) formic acid and of acetonitrile with 0.1% (v/v) formic acid. Separation of analytes was obtained in 7 min, with respective retention times for α-amanitin, β-amanitin, phalloidin and IS of 1.9, 1.7, 3.5 and 3.8 min, respectively. Quantitation on the LC–HR-MS system was performed by extracting the exact mass value of each protonated species using a 5-p.p.m. mass window, which was 919.3614, 920.3455, 789.3257 and 388.1586 for α-amanitin, β-amanitin, phalloidin and IS, respectively. Calibration curves were obtained by spiking drug-free urine at 1–100 ng/mL. Mean correlation coefficients, r2, were above 0.99 for each amatoxins and phalloidin. According to currently accepted validation procedures, the method was tested for selectivity, calibration, accuracy, matrix effect, precision and recovery. Authentic urine samples from 43 patients suffering from a suspected intoxication with mushrooms were analyzed by LC–HR-MS, and the results were compared with ELISA competitive immunoassay. The LC–HR-MS presented large benefits over immunoassay of being specific, faster and more sensitive, making it suitable for daily emergency toxicological analysis. [ABSTRACT FROM PUBLISHER]

The article describes a study in which liquid chromatography-tandemmass spectrometry (LC-MS-MS) method for the simultaneous quantification of acetaminophen (APAP) and its glucuronide and sulfate metabolites (APAP-GLU and APAP-SUL) in small volumes of plasma was developed and validated and further the method was method was used to evaluate APAP metabolism in mice treated with high doses of APAP. As suggested, the validated method could be used in pathology and toxicology research.

Chlormequat is a quaternary ammonium used as plant growth regulating agent. We report here the first suicide case involving a 45 year-old farmer man who intentionally self-injected C5SUN(®), containing chlormequat and choline. An original liquid chromatography high resolution mass spectrometry method (LC-HR-MS), using a hybrid quadrupole-orbitrap mass spectrometer, was developed for qualitative and quantitative analysis of chlormequat in different biological matrices. Toxicological analyses of post-mortem samples highlighted the presence of chlormequat in the blood (2.25mg/L) and the urine (4.45mg/L), in addition to ethanol impregnation blood (1.15g/L). The route of administration (subcutaneous injection) was confirmed by the detection of chlormequat in the abdominal fat sample (chlormequat: 10.04mg/g) taken from the traumatic injury location, as well as in the syringe found at the death scene, close to the victim's body. Based on the results of these post-mortem investigations, the cause of death was determined to be consecutive to cardiac dysrhythmia and cardiac arrest following chlormequat self-injection. [ABSTRACT FROM AUTHOR]

• APAP thiomethyl metabolites are stable biomarkers to monitor therapeutic use and overdose. • The thiomethyl metabolites have delayed excretion rates. • The thiomethyl metabolites allow to monitor the elimination of the toxic fraction after overdose. • The thiomethyl metabolites are produced from the glutathione and cysteine conjugates. Paracetamol/acetaminophen (N-acetyl-p-aminophenol, APAP) is a top selling analgesic used in more than 600 prescription and non-prescription pharmaceuticals. To study efficiently some of the potential undesirable effects associated with increasing APAP consumption (e.g. , developmental disorders, drug-induced liver injury), there is a need to improve current APAP biomonitoring methods that are limited by APAP short half-life. Here, we demonstrate using high-resolution mass spectrometry (HRMS) in several human studies that APAP thiomethyl metabolite conjugates (S-methyl-3-thioacetaminophen sulfate and S-methyl-3-thioacetaminophen sulphoxide sulfate) are stable biomarkers with delayed excretion rates compared to conventional APAP metabolites, that could provide a more reliable history of APAP ingestion in epidemiological studies. We also show that these biomarkers could serve as relevant clinical markers to diagnose APAP acute intoxication in overdosed patients, when free APAP have nearly disappeared from blood. Using in vitro liver models (HepaRG cells and primary human hepatocytes), we then confirm that these thiomethyl metabolites are directly linked to the toxic N-acetyl-p-benzoquinone imine (NAPQI) elimination, and produced via an overlooked pathway called the thiomethyl shunt pathway. Further studies will be needed to determine whether the production of the reactive hepatotoxic NAPQI metabolites is currently underestimated in human. Nevertheless, these biomarkers could already serve to improve APAP human biomonitoring, and investigate, for instance, inter-individual variability in NAPQI production to study underlying causes involved in APAP-induced hepatotoxicity. Overall, our findings demonstrate the potential of exposomics-based HRMS approach to advance towards a better precision for human biomonitoring. [ABSTRACT FROM AUTHOR]

Ethanol is the most frequently psychoactive substance used in the world, leading to major public health problems with several millions of deaths attributed to alcohol consumption each year. Metabolism of ethanol occurs mainly in the liver via the predominant oxidative metabolism pathway involving phase I enzymes including alcohol dehydrogenases (ADH), cytochrome P450 (CYP) 2E1 and catalase. In a lesser extent, an alternative non-oxidative pathway also contributes to the metabolism of ethanol, which involves the uridine diphospho-glucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II enzymes. Using liquid chromatography-high resolution mass spectrometry, ethylglucuronide (EtG) and ethylsulfate (EtS) produced respectively by UGT and SULT conjugation and detected in various biological samples are direct markers of alcohol consumption. We report herein the efficient non-oxidative metabolic pathway of ethanol in human differentiated HepaRG cells compared to primary human hepatocytes (HH). We showed dose- and time-dependent production of EtS and EtG after ethanol (25 or 50 mM) treatment in culture media of differentiated HepaRG cells and HH and a significant induction of CYP2E1 mRNA expression upon acute ethanol exposure in HepaRG cells. These differentiated hepatoma cells thus represent a suitable in vitro human liver cell model to explore ethanol metabolism and more particularly EtG and EtS production. In addition, using recombinant HepG2 cells expressing different UGT1A genes, we found that UGT1A9 was the major UGT involved in ethanol glucuronidation. Unlabelled Image • Ethylglucuronide (EtG) and Ethylsulfate (EtS) are two metabolites of ethanol used as direct markers of alcohol consumption. • Using LC-HRMS, we report the dose- and time-dependent EtG and EtS production after ethanol treatment by human hepatocytes. • Using recombinant HepG2 cells, we found that UGT1A9 was the major UGT involved in ethanol glucuronidation. [ABSTRACT FROM AUTHOR]