Analysis of human insulin and its analogs by liquid chromatography coupled with high-resolution mass spectrometry in blood and tissue samples in a forensic context : interests and limitations in postmortem data interpretation

Le but de ce travail était de développer et de valider des techniques analytiques basées sur la chromatographie liquide couplée à la spectrométrie de masse haute résolution pour l’analyse de l’insuline humaine et de ses analogues dans des milieux biologiques. Une partie bibliographique décrit les matrices biologiques d’intérêt et rassemble les connaissances actuelles sur l’analyse des insulines dans les milieux biologiques. Une partie expérimentale présente le développement de méthodes basées sur l’immunopurification et la spectrométrie de masse, à travers 2 articles analytiques. L’un rapporte la première validation d’une méthode d’analyse quantitative des insulines dans le sérum post-mortem par LC-MS/HRMS. Le second article présente la mise au point d’une analyse qualitative dans les tissus, validée en termes de limite de détection et de spécificité. L’application à des cas réels a permis d’illustrer les performances des 2 méthodes. D’après les données obtenues, un protocole pré-analytique a été établi avec des recommandations sur les matrices d’intérêt et les modalités de prélèvement en cas de suspicion d’intoxication aux insulines. Enfin, une aide à l’interprétation des résultats a été proposée sous la forme d’un logigramme à travers l’étude de la littérature et les différents cas rapportés et analysés. La mise en œuvre et l’interprétation des résultats des analyses des insulines dans un cadre médico-légal et en particulier en post-mortem sont complexes. Les travaux réalisés proposent des méthodes analytiques validées et fiables. Celles-ci devraient contribuer à acquérir de nouvelles données pour améliorer les connaissances de la toxicologie médico-légale des insulines.The aim of this study was to develop and to validate analytical techniques based on liquid chromatography coupled with high-resolution mass spectrometry for analysis of human insulin and its analogs in biological samples. A literature section describes the biologic matrices of interest and gathers current knowledge on insulins analysis in biological media. An experimental section presents the development of methods based on immunopurification and mass spectrometry, through 2 analytical papers. The first one reports the first validation of a quantitative method for insulins analysis in postmortem serum by LC-MS/HRMS. The second reports the development of a qualitative analysis of tissue samples, validated in terms of limit of detection and specificity. The application to authentic cases illustrated the performances of the both methods. Based on the data obtained, a pre-analytical process has been established with recommendations on matrices of interest and sampling methods in case of suspected insulin poisoning. Finally, a tool to help in interpreting results was proposed in the form of a logic diagram through the study of the literature and the various cases reported and analyzed. Performing and interpreting results of insulins analyses in a forensic context and particularly in postmortem are complex. The present work proposes validated and reliable analytic methods. These should contribute to the acquisition of new data to improve knowledge on the forensic toxicology of insulins

Analyse par chromatographie liquide couplée à la spectrométrie de masse haute résolution de l’insuline humaine et de ses analogues dans des prélèvements sanguins et tissulaires dans un cadre médico-légal : intérêts et limites pour l’interprétation des données post-mortem

The aim of this study was to develop and to validate analytical techniques based on liquid chromatography coupled with high-resolution mass spectrometry for analysis of human insulin and its analogs in biological samples. A literature section describes the biologic matrices of interest and gathers current knowledge on insulins analysis in biological media. An experimental section presents the development of methods based on immunopurification and mass spectrometry, through 2 analytical papers. The first one reports the first validation of a quantitative method for insulins analysis in postmortem serum by LC-MS/HRMS. The second reports the development of a qualitative analysis of tissue samples, validated in terms of limit of detection and specificity. The application to authentic cases illustrated the performances of the both methods. Based on the data obtained, a pre-analytical process has been established with recommendations on matrices of interest and sampling methods in case of suspected insulin poisoning. Finally, a tool to help in interpreting results was proposed in the form of a logic diagram through the study of the literature and the various cases reported and analyzed. Performing and interpreting results of insulins analyses in a forensic context and particularly in postmortem are complex. The present work proposes validated and reliable analytic methods. These should contribute to the acquisition of new data to improve knowledge on the forensic toxicology of insulins.Le but de ce travail était de développer et de valider des techniques analytiques basées sur la chromatographie liquide couplée à la spectrométrie de masse haute résolution pour l’analyse de l’insuline humaine et de ses analogues dans des milieux biologiques. Une partie bibliographique décrit les matrices biologiques d’intérêt et rassemble les connaissances actuelles sur l’analyse des insulines dans les milieux biologiques. Une partie expérimentale présente le développement de méthodes basées sur l’immunopurification et la spectrométrie de masse, à travers 2 articles analytiques. L’un rapporte la première validation d’une méthode d’analyse quantitative des insulines dans le sérum post-mortem par LC-MS/HRMS. Le second article présente la mise au point d’une analyse qualitative dans les tissus, validée en termes de limite de détection et de spécificité. L’application à des cas réels a permis d’illustrer les performances des 2 méthodes. D’après les données obtenues, un protocole pré-analytique a été établi avec des recommandations sur les matrices d’intérêt et les modalités de prélèvement en cas de suspicion d’intoxication aux insulines. Enfin, une aide à l’interprétation des résultats a été proposée sous la forme d’un logigramme à travers l’étude de la littérature et les différents cas rapportés et analysés. La mise en œuvre et l’interprétation des résultats des analyses des insulines dans un cadre médico-légal et en particulier en post-mortem sont complexes. Les travaux réalisés proposent des méthodes analytiques validées et fiables. Celles-ci devraient contribuer à acquérir de nouvelles données pour améliorer les connaissances de la toxicologie médico-légale des insulines

GC-MS, GC-QTOF and NMR analyses to elucidate composition of 41 powders from an NPS collector

International audienc

α-Methyltryptamine (α-MT) Metabolite Profiling in Human Hepatocyte Incubations and Postmortem Urine and Blood

: 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

α-Methyltryptamine (α-MT) Metabolite Profiling in Human Hepatocyte Incubations and Postmortem Urine and Blood

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

Severe 25E-NBOH intoxication associated with MDPHP intake: a case report, metabolism study, and literature review

International audienceN-Benzylphenethylamine derivatives are 5-HT2A receptor agonists with hallucinogenic properties, including NBOMe (N-(2-methoxybenzyl)-2-(3,4,5-trimethoxyphenyl)ethan-1-amine) and NBOH (2-(((2,5-dimethoxyphenethyl)amino)methyl)phenol). We reported here the case of a 23-year-old man who presented a serotoninergic syndrome and a loss of consciousness following the consumption of a powder labelled as 25I-NBOH. Toxicological analyses of biological samples were carried out using a liquid chromatography high-resolution mass spectrometry. Two new psychoactive substances were identified and confirmed with certified reference materials: 25E-NBOH (2-(((4-ethyl-2,5-dimethoxyphenethyl)amino)methyl)phenol) and MDPHP (1-(benzo[d][1,3]dioxol-5-yl)-2-(pyrrolidin-1-yl)hexan-1-one). Pharmaceuticals administered to the patient during his medical care were found in plasma and urine. 25E-NBOH and MDPHP concentrations were respectively at 2.3 ng/mL and 3.4 ng/mL in plasma, and 25.7 ng/mL and 30.5 ng/mL in urine. 25I-NBOH (2-(((4-iodo-2,5-dimethoxyphenethyl)amino)methyl)phenol) was specifically searched in both samples and was not detected. These results are discussed along with a literature review on human cases of exposure to N-benzylphenethylamine derivatives. Using molecular networking approach, we propose the first 25E-NBOH metabolism study using authentic biological samples (plasma and urine). We described seven metabolites (M1 to M7), including two phase I (m/z 330.172; m/z 288.160) and five phase II metabolites (m/z 464.191, m/z 478.207, m/z 492.223, m/z 508.218; m/z 396.156). The M6 (m/z 492.223) was the most intense ion detected in plasma and urine and could be proposed as a relevant 25E-NBOH consumption marker. Overall, we described an original case of 25E-NBOH poisoning and identified metabolites that could potentially be used as consumption markers to detect 25E-NBOH intoxications with a higher confidence level and probably a longer detection window