Showing papers in "Forensic Toxicology in 2020"
TL;DR: This is the first work presenting a GC–MS/MS method for the determination of NPS in blood samples, demonstrating its utility in the quantification of a wide number of psychoactive substances in routine toxicological analyses.
Abstract: Psychoactive compounds that contain a phenylethylamine structure (such as amphetamine-type stimulants and synthetic cathinones) are one of the major classes of stimulants on the recreational drug market. Approximately 670 new psychoactive substances (NPS) are monitored only in Europe; however, new psychoactive compounds are being developed for illicit trade each year. In this context, the development of new analytical procedures for the determination of such compounds in biological specimens for forensic toxicology is of great importance. Gas chromatography–tandem mass spectrometry (GC–MS/MS) technique was applied for analysis of amphetamines and synthetic cathinones. The volumes of 200 µL of each whole blood sample and 1 mL of liquid-liquid extraction solvent were used for extraction, followed by pentafluoropropionyl derivatization. A high-throughput, robust, rapid, and sensitive procedure involving a simple liquid-liquid extraction for the simultaneous determination of 45 amphetamine-type stimulants and synthetic cathinones in whole blood was developed. The assay was validated based on its recovery (83.2–106%), interday accuracy (89.0–108%), and interday precision (≤ 8.1%). In view of the low limits of detection (ranged between 0.02 and 0.72 ng/mL) and limits of quantification (1 and 2.5 ng/mL), the developed method can serve as a less expensive and more ecologically friendly alternative to the liquid chromatography–tandem mass spectrometric methods. To the best of our knowledge, this is the first work presenting a GC–MS/MS method for the determination of NPS in blood samples. The presented procedure was applied to authentic samples from forensic cases, demonstrating its utility in the quantification of a wide number of psychoactive substances in routine toxicological analyses. The developed procedure can also be easily expanded to additional compounds.
52 citations
TL;DR: Five cases of death involving its fluorinated analog, 5F-Cumyl-PEGACLONE, a recently emerged γ-carbolinone derived SC, are reported and the Toxicological Significance Score (TSS) was assigned to the compound.
Abstract: Cumyl-PEGACLONE was the first synthetic cannabinoid (SC) with a γ-carbolinone core structure detected in forensic casework and, since then, it has dominated the German SC-market. Here the first four cases of death involving its fluorinated analog, 5F-Cumyl-PEGACLONE, a recently emerged γ-carbolinone derived SC, are reported. Complete postmortem examinations were performed. Postmortem samples were screened by immunoassay, gas chromatography mass spectrometry (GC–MS) or liquid chromatography tandem mass spectrometry. For quantification of SCs, the standard addition method was employed. Herbal blends were analyzed by GC–MS. In each case of death, the Toxicological Significance Score (TSS) was assigned to the compound. 5F-Cumyl-PEGACLONE was identified at concentrations ranging 0.09–0.45 ng/mL in postmortem femoral blood. In case 1, signs of hypothermia and kidney bleedings were noted. Despite a possible tolerance due to long term SC use, a TSS of 3 was assigned. In case 2, an acute heroin intoxication occurred and a contributory role (TSS = 1) of 5F-Cumyl-PEGACLONE was suggested. In case 3, a prisoner was found dead. GC–MS analysis of herbal blends, retrieved in his cell together with paraphernalia, confirmed the presence of 5F-Cumyl-PEGACLONE and a causative role was deemed probable (TSS = 2). In case 4, the aspiration of gastric content due to a SC-induced coma was observed (TSS = 3). 5F-Cumyl-PEGACLONE is an emerging and extremely potent SC which raises serious public health concerns. A comprehensive analysis of circumstantial, clinical, and postmortem findings, as well as an in-depth toxicological analysis is necessary for a valid interpretation and for the assessment of the toxicological significance.
21 citations
TL;DR: The developed method enables the determination of N-ethylpentylone with high sensitivity and selectivity and was used to make determinations in biological material in the case of fatal intoxication with N- methylpentylones.
Abstract: We present a fully validated method for determination of N-ethylpentylone in biological material and a case report of fatal intoxication with N-ethylpentylone. Blood and urine samples were extracted with ethyl acetate from alkaline medium (pH 9). The analysis was carried out using ultra-high-performance liquid chromatography–tandem mass spectrometry. MDMA-d5 was used as the internal standard. Validation criteria were evaluated for blank blood and urine at concentrations of 10 and 100 ng/mL. The validation parameters were as follows: lower limit of quantification: 1 ng/mL for blood and urine, coefficient of determination: blood > 0.9996, urine > 0.9975, precision for 10 and 100 ng/mL, respectively: blood 4.87% and 4.47%, urine 1.93% and 2.43%, accuracy for 10 and 100 ng/mL, respectively: blood 14.7% and −2.95%, urine 19.1% and 2.10%, recovery for 10 and 100 ng/mL, respectively: blood 91.5% and 100.2%, urine 97.4% and 96.7%, matrix effect in blood was 127% and 117% for 10 and 100 ng/mL, respectively, in urine 124% and 117% for 10 and 100 ng/mL, respectively. In the present case of fatal intoxication with N-ethylpentylone, the determined concentration of this substance was 10.6 µg/mL in peripheral blood and 17.6 µg/mL in urine. In both materials, four metabolites of N-ethylpentylone were determined qualitatively. The developed method enables the determination of N-ethylpentylone with high sensitivity and selectivity. The method was used to make determinations in biological material in the case of fatal intoxication with N-ethylpentylone.
21 citations
TL;DR: Angel’s trumpets have high toxicological significance both in their native and non-native regions, mainly due to their hallucinogenic property and accidental ingestion.
Abstract: The toxicological significance of human exposures to angel's trumpet plants (Brugmansia and Datura species) in their native American and non-native regions (Asia, Africa, and Europe) was highlighted, and the poisoning potential of various plant parts was discussed. Nearly 2500 cases of human plant exposures, reported to the Hungarian Toxicological Information Service between 2005 and 2017, were analyzed and compared to data of other toxicology centers in America, Asia and Europe, focusing on exposures to tropane alkaloid-containing plants. In America, Brugmansia and Datura were not among the 15 most common plant ingestions, but were responsible for 20% of the fatal outcomes in a 26-year period. In Asia, the anticholinergic toxidrome, related to Brugmansia and Datura, was among the most frequent plant-related intoxications, which included accidental ingestion, improper use of herbal medicines and plant abuse. In Europe, Brugmansia and Datura were among the top four plant taxa causing intoxications with major outcomes, being the leading plants ingested for their hallucinogenic properties, and accounted for 60% of abuse cases in Hungary in a 13-year period. Use of Brugmansia and Datura for criminal purposes has been reported from America, Asia and Europe. The concentrations of tropane alkaloids vary with the species, seasons and plant parts. Ingestion of the seeds and flowers has the highest toxicological significance, but exposure to the leaves and floral nectar can also cause intoxication. Angel’s trumpets have high toxicological significance both in their native and non-native regions, mainly due to their hallucinogenic property and accidental ingestion.
21 citations
TL;DR: The results are expected to help toxicologists to reliably identify these substances in case of suspected abuse and allow them a thorough risk assessment.
Abstract: The presented study aimed to elucidate the toxicokinetics of the four synthetic cathinones 4-chloroethcathinone (4-CEC), N-ethylnorpentylone (N-ethylpentylone, ephylone), N-ethylhexedrone (NEH), and 4-fluoro-alpha-pyrrolidinohexiophenone (4-fluoro-alpha-pyrrolidinohexanophenone, 4-F-α-PHP, 4F-alpha-PHP, 4F-PHP). First, their metabolism was studied using human urine and blood samples. Analysis of specimens was performed by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and gas chromatography–mass spectrometry (GC–MS). LC-HRMS/MS was also used to analyze in vitro incubations of the new psychoactive substances using pooled human liver S9 fraction (pS9), to identify the monooxygenases involved in the initial metabolic steps, and determination of plasma concentrations after a standard addition method. Metabolic stability was tested in pooled human liver microsomes incubations analyzed by LC-ion trap MS. Using LC-HRMS/MS, 47 metabolites in total were found in patient samples and pS9 incubations. Using GC–MS, 4-CEC, ephylone, NEH, and five of their metabolites were detectable in urine. The following main phase I reactions were observed: carbonyl group reduction, N-deethylation, hydroxylation, lactam formation (4F-PHP), and demethylenation (ephylone). Mainly glucuronidations were observed as phase II reactions besides conjugates with the dicarboxylic acids malonic, succinic, and glutaric acid (4-CEC), sulfation, methylation (both ephylone), and N-acetylation (NEH). A broad range of monooxygenases was involved in the initial steps with exception of NEH (only CYP1A2 and CYP2C19). 4F-PHP had the shortest in vitro half-life (38 min) and highest intrinsic clearance (15.7 mL/min/kg). Plasma concentrations ranged from 0.8 to 8.5 ng/mL. Our results are expected to help toxicologists to reliably identify these substances in case of suspected abuse and allow them a thorough risk assessment.
20 citations
TL;DR: The phytosteroid ecdysterone was recently reported to enhance performance in sports and may thus be considered as a substance of relevance in anti-doping control to assess its integration into initial testing procedures (ITP) in doping control laboratories.
Abstract: The phytosteroid ecdysterone was recently reported to enhance performance in sports and may thus be considered as a substance of relevance in anti-doping control. To trace back an administration of ecdysterone from urine samples, analytical properties have been investigated to assess its integration into initial testing procedures (ITP) in doping control laboratories. Analytical properties of ecdysterone were evaluated using gas chromatography-quadrupole/time-of-flight-mass spectrometry (GC–QTOF–MS) and liquid chromatography (LC)–QTOF–MS. Its metabolism and elimination in human were studied using urines collected after administration. The detectability of ecdysterone by GC–MS (after derivatization) and/or LC–MS(/MS) has been demonstrated and sample preparation methods were evaluated. Dilute-and-inject for LC–MS(/MS) or solid phase extraction using Oasis HLB for GC–MS or LC–MS were found most suitable, while liquid–liquid extraction was hampered by the high polarity of ecdysteroids. Most abundantly, ecdysterone was detected in the post administration urines as parent compound besides the metabolite desoxy-ecdysterone. Additionally, desoxy-poststerone was tentatively assigned as minor metabolite, however, further investigations are needed. An administration of ecdysterone can be targeted using existing procedures of anti-doping laboratories. Ecdysterone and desoxy-ecdysterone appeared as suitable candidates for integration in ITP. Using dilute-and-inject a detection of the parent compound was possible for more than 2 days after the administration of a single dose of ~ 50 mg.
20 citations
TL;DR: This manuscript reports useful analytical data about MPHP, one of its metabolites (4′-carboxy-PHP) and 4-MEAP, and the number of fatalities related to cathinones use is expected to increase in the coming years.
Abstract: The correlation between the rising consumption of new psychoactive drugs (NPS), including that of cathinones, and the occurrence of death has not been sufficiently backed up with published analytic data. In fact, the identification of cathinones in human biological samples remains difficult mainly due to the diversity of these substances and their high turnover. In this context, this manuscript aims at documenting a fatal case of a 39-year-old man: autopsy findings consisted in unspecific asphyxic syndrome. Blood ethanol concentration determination and toxicological screenings were performed using gas chromatography with flame ionization detection, liquid chromatography with diode array detection and gas chromatography with mass spectrometry detection, respectively. Liquid chromatography with high-resolution mass spectrometry detection allowed the confirmation of the presence of NPS and the subsequent metabolic study. The analyses have shown the presence of ethanol, tetrahydrocannabinol and two cathinones, 4′-methyl-α-pyrrolidinohexanophenone (MPHP) and N-ethyl-4′-methylnorpentedrone (4-MEAP). MPHP/4-MEAP concentrations were 47/1.6, 97/3.5 and 2380/49,700 µg/L in femoral blood, cardiac blood and urine, respectively. The in vitro metabolic study has highlighted the presence of five metabolites derived from MPHP and three from 4-MEAP but only two metabolites of these products have been detected in biological samples. The 4′-carboxy-PHP, one of the metabolites of MPHP, was detected in every biological sample with higher chromatographic signals than MPHP itself. The number of fatalities related to cathinones use is expected to increase in the coming years. This manuscript reports useful analytical data about MPHP, one of its metabolites (4′-carboxy-PHP) and 4-MEAP.
19 citations
TL;DR: Analytical properties of the designer benzodiazepine 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]; flualprazolam are reported to assist forensic laboratories that encounter these newly emerging compounds in casework.
Abstract: This publication reports analytical properties of the designer benzodiazepine 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine (flualprazolam) seized in an anesthesia robbery case. The target compound was identified by liquid chromatography–quadrupole time-of-flight-mass spectrometry (LC–QTOF-MS), gas chromatography–mass spectrometry (GC–MS), and nuclear magnetic resonance (NMR) spectroscopy. We could obtain detailed analytical data of flualprazolam—a new designer benzodiazepine available on the designer drug market. More designer benzodiazepines have been detected and seized on the illegal drug scene as new psychoactive substances during the last 5 years. In this study, we presented analytical data of flualprazolam to assist forensic laboratories that encounter these newly emerging compounds in casework. This is the first report on this compound in illegal products.
15 citations
TL;DR: The first demonstration of 3-fluorofentanyl and methoxyacetylfentanyl in hair samples collected from an authentic abuser is reported, despite the patient claimed not consuming three out of the six detected fentanyls.
Abstract: To demonstrate the usefulness of hair testing to determine exposure pattern to fentanyls. A 43-year-old male was found unconscious with respiratory depression 15 min after snorting 3 mg of a powder labeled as butyrylfentanyl. He was discharged from hospital within 2 days without blood or urine testing. Two locks of hair were sampled 1 month (M1 A: 0–2 cm (overdose time frame); B: 2–4 cm; C: 4–6 cm) and 1 year (Y1: A: 0–2 cm; B: 2–4 cm) later to monitor his exposure to drugs of abuse by liquid chromatography–tandem mass spectrometry after liquid-liquid extraction. Hair analysis at M1 showed a repetitive exposure to 3-fluorofentanyl (A/B/C: 150/80/60 pg/mg) with higher concentration in segment A reflecting the overdose period. The non-detection of butyrylfentanyl was consistent with the analysis of the recovered powder identified as 3-fluorofentanyl. Furanylfentanyl (40/20/15 pg/mg) and fentanyl (37/25/3 pg/mg) were also detected in hair. The second hair analysis at Y1 showed the use of three new fentanyls, with probably repetitive exposures to methoxyacetylfentanyl (A/B: 500/600 pg/mg), and single or few exposures to carfentanil (2.5/3 pg/mg) and acetyl fentanyl (1/1 pg/mg). A decreasing exposure to 3-fluorofentanyl (25/80 pg/mg), and increasing consumption of furanylfentanyl (310/500 pg/mg) and fentanyl (620/760 pg/mg) were also observed despite methadone treatment initiation. The patient claimed not consuming three out of the six detected fentanyls. We report single or repetitive exposure to several fentanyls using hair testing. To our knowledge, this is the first demonstration of 3-fluorofentanyl and methoxyacetylfentanyl in hair samples collected from an authentic abuser.
15 citations
TL;DR: In this article, two mixedmode cation-exchange sorbents in solid phase extraction (SPE) and their efficacy in pretreating urine samples for the determination of synthetic cathinones by liquid chromatography high-resolution mass spectrometry (LC-HRMS) were evaluated.
Abstract: Cathinone derivates have become very popular as legal highs and are easily obtained on the market as “bath salts”, getting around the legislation due to their modified structure based on banned substances. The aim of this project is to evaluate two mixed-mode cation-exchange sorbents in solid-phase extraction (SPE) and their efficacy in pretreating urine samples for the determination of synthetic cathinones by liquid chromatography high-resolution mass spectrometry (LC-HRMS). A method for determining a group of eleven trending synthetic cathinones in urine using SPE followed by LC-HRMS using Orbitrap as analyser was developed and validated. In the extraction step, two different cation-exchange sorbents were evaluated and compared, a weak one (Oasis WCX) and a strong one (Oasis MCX). Better results were obtained for Oasis MCX in terms of recoveries and matrix effects (lower, if a clean-up step was applied). Method quantification limits were set at 0.2 ng mL−1 and method detection limits were between 0.04 and 0.16 ng mL−1. The results obtained enabled the studied compounds to be quantified at the usual levels at which they are present in urine samples. Moreover, these limits were lower than the ones found in the literature using similar extraction strategies, which shows that the reported strategy can be a useful tool in forensic and toxicological analyses.
14 citations
TL;DR: The background of nerve agents (NAs) and their hydrolysis products is provided and more analysis methods are expected to be developed and may involve the development of sensors and lab-on-chip devices that should address specificity, sensitivity, stability, response time, and repeatability/reproducibility.
Abstract: This review article provides an overview of the background of nerve agents (NAs) and their hydrolysis products. The analytical techniques that are used for the analysis of hydrolysis products are also discussed. The analysis methods include the chromatographic, mass spectrometric, electrophoretic, and sensing technologies that are currently available for the detection of exposure to a number of hydrolysis products of NAs. All reviewed information was gathered through a detailed search of Scopus, PubMed, and the World Wide Web using relevant keywords, e.g., NA, hydrolysis products, and methylphosphonic acid. Most analyses of NA hydrolysis products still rely on chromatographic techniques such as gas and liquid chromatography. Analyses based on mass spectrometry are now fairly routine and can be further improved by providing analyses with precise and accurate masses and faster scan speeds that provide further gains in sensitivity. Although these techniques have high selectivity and sensitivity, most of them are rarely applied to real samples or suffer from destructive real sample analysis. In addition, there is still a lack of studies on the development of portable sensors for the detection of NA hydrolysis products. Although the analysis of NA hydrolysis products has experienced a transition from qualitative analysis toward rapid and quantitative identification, portable, and fast sensing technologies have not been well established or investigated extensively. In the future, more analysis methods are expected to be developed and may involve the development of sensors and lab-on-chip devices that should address specificity, sensitivity, stability, response time, and repeatability/reproducibility.
TL;DR: In this article, the authors reported the identification and structural elucidation of three new NBOHs detected in seized blotter papers: 25B-NBOH, 25C-nboH, and 25E-NboH.
Abstract: The recreational drug market remains dynamic. After the introduction of 25I-NBOH, an N-benzylphenethylamine and new psychoactive substance, as option for LSD and NBOMe drugs, new NBOH substances have been identified in recent years. Herein, we report our efforts for the identification and structural elucidation of three new NBOHs detected in seized blotter papers: 25B-NBOH, 25C-NBOH, and 25E-NBOH. Blotter papers seized between 2017 and 2018 by local police force in Brazil were submitted to extraction, purification, identification and characterization using attenuated total reflectance-Fourier transform infrared spectroscopy, gas chromatography—mass spectrometry, liquid chromatography—tandem mass spectrometry, and one- and two-dimensional nuclear magnetic resonance spectroscopy. Three new NBOHs were characterized: 2-(((4-ethyl-2,5-dimethoxyphenethyl)amino)methyl)phenol (25E-NBOH, 2C-E-NBOH), 2-(((4-chloro-2,5-dimethoxyphenethyl)amino)methyl)phenol (25C-NBOH, 2C-C-NBOH), and 2-(((4-bromo-2,5-dimethoxyphenethyl)amino)methyl)phenol (25B-NBOH, 2C-B-NBOH). To our knowledge, this is the first report for identification and detailed characterization of 25B-NBOH, 25C-NBOH, and 25E-NBOH in seized samples. NBOH substances are not under United Nations Conventions control. The identification of seized blotter papers between 2014 and beginning of 2019 showed that NBOH substances have become the main hallucinogenic drug in the region. These group are thermolabile under gas chromatographic conditions, demanding other analytical approaches of analysis to avoid misidentifications. Unfortunately, the knowledge about toxicology of NBOHs are limited.
TL;DR: Xylazine was the subject of extensive metabolism in zebrafish and 4-Hydroxylated and oxygenated derivatives were the most abundant phase I metabolites as the main targets for doping control.
Abstract: This study aims to investigate a zebrafish (Danio Rerio) water tank (ZWT) as an alternative model for the study of the metabolism of xylazine. The ZWT approach for the study of metabolism consisted of two aquariums, where 18 fish and xylazine were added into the first tank. The second one, with 18 fish without drug, served as a negative control. The samples were submitted to a comprehensive analytical method developed for doping control purposes by liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) operating in five different acquisition modes. Glycoconjugate metabolites were evaluated indirectly by extracting the samples with and without the enzymatic hydrolysis step using β-glucuronidase. In total, 11 phase I and II metabolites were detected and characterized, of which four were previously described for humans and two for horses, and five metabolites were described for the first time. The main metabolites were 4-hydroxylated (M2) and oxygenated (M1) derivatives. Both metabolites were suggested as analytical targets for xylazine misuse. Around 79% of para- and meta-hydroxylated derivatives were in glycoconjugate form, whereas for oxo-hydroxylated and sulfone-hydroxylated derivatives of xylazine, around 83% and 70% were metabolized to the glycoconjugate form, respectively. Xylazine was the subject of extensive metabolism in zebrafish. 4-Hydroxylated (M2) and oxygenated (M1) derivatives were the most abundant phase I metabolites as the main targets for doping control.
TL;DR: The toxicokinetic data provided by this study will help forensic and clinical toxicologists to reliably identify these substances in case of abuse and/or intoxication and will allow them a thorough risk assessment.
Abstract: Toxicokinetic studies are essential in clinical and forensic toxicology to understand drug–drug interactions, influence of individual polymorphisms, and elimination routes, as well as to evaluate targets for toxicological screening procedures. N-(2-Methoxybenzyl)-substituted phenethylamines (NBOMe analogues) intake has been associated with severe adverse reactions including deaths. 1-(1-Benzofuran-5-yl)-N-[(2-methoxyphenyl)methyl]propan-2-amine (5-APB-NBOMe), 2-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b′]difuran-4-yl)-N-[(5-chloro-2-ethoxyphenyl)methyl]ethan-1-amine (2C-B-FLY-NB2EtO5Cl), and 2-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b′]difuran-4-yl)-N-[(2-methoxyphenyl)methyl]ethan-1-amine (2C-B-FLY-NBOMe) are three emerging NBOMe analogues, which have encountered on the drugs of abuse market. So far, their toxicokinetic data are completely unexplored. The study included mass spectrometry-based identification of phase I and II metabolites following exposure to the terminally differentiated human hepatocellular carcinoma cells (HepaRG). The determination of enzymes involved in the major phase I/II metabolic steps and determination of plasma protein binding (PPB) were done. Finally, the evaluation of the toxicological detectability by different hyphenated mass spectrometry techniques in standard urine screening approaches (SUSAs) was investigated. The compounds were extensively metabolized in HepaRG cells mainly via O-dealkylation, hydroxylation, glucuronidation, and combinations thereof. CYP1A2, 2D6, 2C8, 2C19, and 3A4, were involved in the initial reactions of all investigated compounds. Glucuronidation of the phase I metabolites—when observed—was mainly catalyzed by UGT1A9. The PPB of all compounds was determined to be > 85%. Only the high-resolution mass spectrometry-based SUSA allowed detection of all compounds in rat urine, but only via metabolites. The toxicokinetic data provided by this study will help forensic and clinical toxicologists to reliably identify these substances in case of abuse and/or intoxication and will allow them a thorough risk assessment.
TL;DR: In this article, lowvoltage paper spray ionization coupled with quadrupole time-of-flight mass spectrometry (QTOF-MS) method was developed and employed for the qualitative analysis of NPS in street drug blotter samples.
Abstract: Analysis of new psychoactive substances (NPS) requires high selective analytical methodologies, including high-resolution mass spectrometry. In addition, the development/application of a suitable sample preparation step can provide methods in which low amounts of solvents and samples are used, as well as minimal sample manipulation is required. Herein, the use of ambient ionization techniques such as paper spray emerges as a powerful tool to enhance the performance of methodologies, particularly in forensic sciences. In this study, low-voltage paper spray ionization coupled with quadrupole time-of-flight-mass spectrometry (QTOF-MS) method was developed and employed for the qualitative analysis of NPS in street drug blotter samples. Analysis revealed the presence of LSD (m/z 324.2058); 25C-NBOH (m/z 322.1212); 25E-NBOH (m/z 316.1917); 25I-NBOMe (m/z 428.0699); DOB (m/z 274.0432); and 4′-fluoro-α-pyrrolidinohexanophenone (m/z 264.1768) in the samples analyzed. This technique was successfully applied for the analysis of NPS and allowed for fast and accurate analytical method that can be highly exploited in forensic screening purposes.
TL;DR: The largest number (247 cases) of FIBF levels in blood samples of its users is given to aid in the interpretation and analysis of significance of this compound.
Abstract: 4-Fluoroisobutyrylfentanyl (FIBF) was first identified at the New York City Office of Chief Medical Examiner (OCME) in May 2017 and was reported qualitatively due to the constant changes in “fentalogs” (analogs of fentanyl) identified. However, by the year’s end, FIBF was the fifth most common non-methadone synthetic opioid detected and a quantitative method was developed to better understand the significance of this compound. A full quantitative validation was performed utilizing liquid chromatography–tandem mass spectrometry (LC–MS/MS). All cases submitted between May 2017 and December 2018 that tested positive for FIBF qualitatively were chosen for quantitation. In addition to compiling FIBF concentrations, user demographics and concurrent drug use were also investigated. There were 247 FIBF-positive cases that were tested with blood concentrations ranging from < 0.1 to 331 ng/mL. The most commonly detected drugs in conjunction with FIBF were other opioids, benzodiazepines, cocaine, cannabinoids and ethanol. The most frequent users were male, White, and between 35 and 54 years of age. The concentrations of FIBF varied widely and showed no clear distinction when considering various case types. Although most often used in tandem with other drugs of abuse, the danger of this compound was demonstrated by the fact that FIBF was also reported as the sole intoxicant responsible for death. The development of a quantitative method for FIBF has been beneficial at the OCME due to the significant number of positive cases reported and the current lack of sufficient published information on this analog. To our knowledge, there are only two papers that include FIBF concentrations in four cases; this article gives the largest number (247 cases) of FIBF levels in blood samples of its users to aid in the interpretation and analysis of significance of this compound.
TL;DR: A case of fatal poisoning by ingestion of an unknown powder (labelled as Tabernanthe iboga) is reported and molecular networking is applied to tentatively identify the plant involved in a woman’s death and suggests an alternate Apocynaceae species.
Abstract: Identifying xenobiotics involved in deaths remains a challenge in toxicology, especially when they are missing from the usual suspect databases. The tool development for the rapid processing of untargeted screening data is a valuable asset. In recent years, the molecular networking has been developed in various fields, including toxicology, because of its ability to graphically display and compare complex data acquired from tandem mass spectrometry. Here, we report a case of fatal poisoning by ingestion of an unknown powder (labelled as Tabernanthe iboga) and apply molecular networking to tentatively identify the plant involved in a woman’s death. The supposedly ingested powder, an authentic Tabernanthe iboga root powder and postmortemed biological samples (peripheral blood and bile) were extracted and analyzed according to a screening method using high resolution liquid chromatography hyphenated to tandem mass spectrometry (Q-Exactive®). Analysis of these four matrices by molecular networking revealed a cluster of nodes composed of indolomonoterpenic alkaloids, a characteristic of the Apocynaceae family (ibogaine, ibogamine, ajmaline, reserpiline, yohimbine). Analysis of the spectral annotations of the supposedly ingested powder also shows a majority of occurrences related to the genus Rauwolfia, which differed from the authentic Tabernanthe iboga root powder. Molecular networking allowed us to discard the Tabernanthe iboga identification hypothesis and suggest an alternate Apocynaceae species, most likely belonging to the Rauwolfia genus. In addition, eleven putative compounds could be detected by the molecular networking, many of which seemed to be metabolites of the major components in the Rauwolfia genus.
TL;DR: The cannabinoid intoxication is characterized by three main conditions: neurological (drowsiness, seizures, hypotonia, and behavioral disorder), cardiovascular (tachycardia, hypertension), and general (bradypnea, pupil dilation) symptoms.
Abstract: The use of Cannabis sativa oils has been growing in the last years as an emerging therapy for non-responsive clinical conditions. Some benefits have been noticed, mainly in neurological diseases such as multiple sclerosis, epilepsy, psychosis, depression, and anxiety [1, 2]. Its applications also include the reduction of adverse effects in cancer therapy and the treatment of chronic pain [3]. Moreover, the pathways related to the cannabis benefits are still being discovered [4]. The therapeutic properties can be correlated to the presence of cannabidiol (CBD), although the pharmacological activity can be influenced by other phytocannabinoids found in the plant. However, high concentrations of Δ9-tetrahydrocannabinol (THC) and cannabinol (CBN) can be associated with several side effects [5]. THC is considered the main psychoactive substance present in cannabis and it has already been related to neurocognitive impairments [6]. These effects are correlated to the binding ability as partial agonist with the endocannabinoid receptors CB1 and CB2 [7]. Both CBD and THC are produced in the plant glandular trichomes as acids. They are derived from the same precursor: cannabigerolic acid, being CBD biosynthesis a limiting factor for THC biosynthesis and vice versa [8, 9]. Moreover, CBN is produced by the oxidation of THC and it can be used as a characterization of the procedure, storage, and administration of cannabis as a quality control parameter [7]. Due to the presence of this compound in cannabis products, intoxication cases can occur from undue utilization of oils [10, 11]. The cannabinoid intoxication is characterized by three main conditions: neurological (drowsiness, seizures, hypotonia, and behavioral disorder), cardiovascular (tachycardia, hypertension), and general (bradypnea, pupil dilation) symptoms [11, 12]. Additionally, cannabis legislation is one of the most discussed topics nowadays on the scenario of recreational use as well as for medical purposes [13, 14]. In Brazil, the legislation concerning the regulation of cannabis-based products has been performed by the Brazilian National Health and Surveillance Agency (ANVISA). Recently, this agency has approved the requirements for the regulation of the distribution/sale of cannabis products for medical use in the country. However, the cultivation of cannabis plants is prohibited [15]. As a result, along with the expansion in the use of cannabis products to achieve its medicinal benefits, the requirements for methodologies able to perform quality control in these samples have also been increased. Analytical approaches consist of the best option for this type of analysis due to its high sensitivity and specificity. Most of the publications describe the use of high-performance liquid chromatography (HPLC) coupled to diode array and/or mass spectrometry (MS) [16-19], although other techniques can also be applied such as gas chromatography (GC) [20, 21] and nuclear magnetic resonance [22]. These analytical systems can increase the cost of the analysis, being a challenge for small laboratories without continuous financial support. In this scenario, GC coupled to the conventional flame ionization detector (FID) is an affordable alternative since it presents sensitivity and robustness with a reduced price Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1141 9-020-00522 -1) contains supplementary material, which is available to authorized users.
TL;DR: The present study proved that n-propanol was not a reliable marker for either putrefaction or ethanol source distinction by showing considerable false rate.
Abstract: Concentration ratio of ethanol/n-propanol has been employed to distinguish the source of ethanol in postmortem blood, though its reliability remains controversial. Forty-two postmortem human blood samples with ethanol levels in the range of 0.07–4.64 mg/mL were investigated. Ethanol and n-propanol were determined by head-space gas chromatography coupled with flame ionization detection, while ethyl glucuronide (EtG) and ethyl sulfate (EtS) were determined by liquid chromatography–tandem mass spectrometry. EtG and EtS were both negative in 26% of the investigated postmortem blood samples and 11% of n-propanol-negative postmortem samples, indicating that n-propanol was not a reliable marker of putrefaction. It was also found that the ratio of ethanol/n-propanol (supposed to be < 20 without antemortem ethanol consumption) was unreliable by showing great individual differences and was opposite with the result of EtG and EtS in at least 17% of n-propanol-positive postmortem blood samples. Meanwhile, 140 antemortem blood samples were investigated, as an aid to estimate the blood alcohol concentration (BAC) at the time of death for postmortem samples with ethanol both from postmortem formation and antemortem consumption. By comparing with the maximum or minimum value of EtG and EtS concentration under certain BAC in antemortem samples, the BAC range at the moment of death could be estimated in 93% of postmortem samples. The present study proved that n-propanol was not a reliable marker for either putrefaction or ethanol source distinction by showing considerable false rate.
TL;DR: There is a wide scope to be explored on the effects of 3,4-DMMC, and Clinicians and pathologists should be encouraged to report their findings in scientific literature, as this will aid in evaluating potential detrimental effects of the drug in further clinical and forensic investigations.
Abstract: To provide a review of the available data on 3,4-dimethylmethcathinone (3,4-DMMC), its physicochemical properties, detection methods, patterns of abuse and prevalence, biological effects, pharmacodynamics, pharmacokinetics, metabolism, and mechanisms of toxicity have been presented and discussed. An exhaustive literature search was carried out with PubMed, drug use forums and blogs, and sites from governmental agencies. 3,4-DMMC is a synthetic cathinone that was first detected in Europe in 2010. Its recreational use and trade in retail outlets have been prohibited in several countries, but the drug remains readily available for purchase on the Internet. 3,4-DMMC has been considered a very appealing drug to be used in combination with other abuse substances. The drug combines the features of stimulants and psychoactive phenethylamines, displays high affinity for 5-HT2 and adrenergic receptors, inhibits monoamine transporters, specially serotonin transporter, but does not promote a significant 5-HT efflux. Recently, anecdotal reports on its abuse sprang up, and 3,4-DMMC has been detected in several seized products and in blood and urine of abusers. 3,4-DMMC metabolites in the urine of habitual users showed N-demethylation, β-ketoreduction, hydroxylation, and oxidation as the major reactions involved in its biotransformation. There is a wide scope to be explored on the effects of 3,4-DMMC. Clinicians and pathologists should be encouraged to report their findings in scientific literature, as this will aid in evaluating potential detrimental effects of the drug in further clinical and forensic investigations. Further studies on pharmacokinetic properties of 3,4-DMMC are also necessary to improve the methods of drug detection.
TL;DR: This case is, to the authors' knowledge, the first described case of inhaled scopolamine robbery attempt, with quantitative toxicological findings, and blood and urine should be collected as soon as possible for toxicological analysis.
Abstract: Scopolamine, an alkaloid found in certain plants, has become a drug of use for recreational and predatory purposes. We present here the case of an attempted scopolamine-facilitated robbery, via inhalation, with full recovery of the victim. We also performed a review of toxicological findings for scopolamine poisoning cases in available scientific literature. Whole blood and urine samples were collected 4 h after the assumed poisoning. Concentrations of scopolamine were determined in both samples by a validated and accredited method using liquid chromatography–tandem mass spectrometry. Scopolamine was the only substance found in both whole blood and urine samples, at 7 and 510 ng/mL, respectively. Scopolamine poisoning remains rare, as its half-life is short; blood and urine should be collected as soon as possible for toxicological analysis. This case is, to our knowledge, the first described case of inhaled scopolamine robbery attempt, with quantitative toxicological findings.
TL;DR: In this article, the authors used QuEChERS extraction coupled with gas chromatography-tandem mass spectrometry (GC-MS/MS) to establish the concentration of furanylfentanyl (Fu-F) in whole blood samples.
Abstract: The purpose of this work is the determination of the probable cause of a person’s death on the basis of analytical results obtained from the deceased’s blood sample. To establish the concentration of furanylfentanyl (Fu-F) in whole blood samples, QuEChERS extraction coupled with gas chromatography–tandem mass spectrometry (GC–MS/MS) was applied. The following QuEChERS parameters have been established as optimal for Fu-F analysis: NaCl 80 mg; MgSO4 150 mg; acetonitrile 650 µL, and C-18 sorbents 12.5 mg for a 350 mg blood sample. The following validation parameters characterized the analytical utility of the described method: linearity (R2 = 0.9975), limit of detection = 0.42 ng/mL and limit of quantification = 1.39 ng/mL, and satisfactory inter- and intraday precisions (% relative standard deviation ≤ 5.73). The postmortem analysis of whole blood samples disclosed a high concentration of Fu-F at 11.2 ng/mL. The present paper describes a fatal case of Fu-F overdose. The described analytical method involving QuEChERS sample preparation procedure followed by multiple reaction monitoring of GC–MS/MS was very convenient to find the presence of Fu-F in postmortem whole blood samples and to establish its concentration. The known circumstances of death in combination with the obtained analytical data indicated that the deceased young male probably got fatally poisoned by Fu-F. The high concentration of Fu-F (11.2 ng/mL) in his postmortem whole blood sample was several times higher than that described in the forensic literature. In our knowledge, this is the first trial to analyze Fu-F by GC–MS/MS.
TL;DR: Yohimbine acute intoxication appeared to be the most likely cause of death in the current case, and the blood concentration highlighted here is the highest ever reported to date.
Abstract: Yohimbine is an indole alkaloid extracted from the bark of the Pausinystalia yohimbe tree, which has found a clinical application through the treatment of erectile dysfunction. Yohimbine is also widely used by the bodybuilding community for its sympathomimetic effects and its presumed lipolytic effects. This substance is, however, associated with adverse effects, including cardiac and neurologic side-effects, but only few fatal cases have been described to date. In this context, this manuscript aims at documenting a fatal case of a 27-year-old male. Ethanol concentration determination and toxicological screening were performed using gas chromatography with flame ionization detection, liquid chromatography with diode array detection and gas chromatography with mass spectrometry detection. Liquid chromatography with diode array detection allowed the quantification of yohimbine. Toxicological analysis of subclavian blood, vitreous humor, and urine samples highlighted the presence of yohimbine and tramadol. Yohimbine was quantified at the concentration of 8000 µg/L in the peripheral blood and 2600 µg/L in the vitreous humor. Tramadol blood concentration was below the toxic threshold. Only a very few number of yohimbine fatalities has been reported so far. In the current case, yohimbine acute intoxication appeared to be the most likely cause of death. In fact, the blood concentration highlighted here is the highest ever reported to date.
TL;DR: In this paper, a forensic toxicological library was developed to identify 56 natural toxic substances by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) with positive and negative ionization.
Abstract: The present study aims to develop a forensic toxicological library to identify 56 natural toxic substances by liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (LC–QTOF-MS/MS). For setting up the library of product ion spectra, individual substances (31 plant toxins, 7 mushroom toxins, 5 marine toxins, 5 frog venoms, 4 mycotoxins, and 4 substances derived from plants) were analyzed by LC–QTOF-MS/MS with positive and negative ionization. The product ion spectra were acquired at the collision energies (CEs) of 20, 35, and 50 eV in single enhanced product ion mode and then in collision energy spread mode in which the CE ramp range was set to 35 ± 15 eV. To test the performance of the library, human blood plasma samples were spiked with a mixture of lycorine and domoic acid, extracted by acetonitrile deproteinization and analyzed by LC–QTOF-MS/MS. Identification by our library search could be achieved for these toxins at the purity scores of 79.1 and 67.2, respectively. The method was also applied to postmortem blood from a death case with an aconite intake, and showed that four toxins in an aconite could be identified in the blood sample at the purity scores of 54.6–60.3. This library will be more effective for the screening of natural toxic substances in routine forensic toxicological analysis. To our knowledge, there are no reports dealing with development of library for natural toxic substances by LC–QTOF-MS/MS.
TL;DR: The method was successfully applied to the analyses of plasma midazolam and propofol concentrations in a brain-injured patient subjected to hypothermia, which validated the present methodology.
Abstract: A sensitive method was developed for the simultaneous determination of midazolam and propofol in human plasma samples via modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction for their analysis by gas chromatography–tandem mass spectrometry (GC–MS/MS). Detailed optimization and validation experiments were conducted; plasma quality control samples spiked with two drugs and two internal standards (ISs) were extracted using the QuEChERS technique and determined by GC–MS/MS. The present method was applied to the measurements in an actual clinical case. The regression equations for midazolam and propofol in plasma displayed good linearity (r > 0.9978) from 10 to 1000 ng/mL with a limit of quantification of 10 ng/mL. The intra- and interday precisions for both drugs were not greater than 8.6%. The accuracies of quantitation ranged from 98 to 110%. The extraction efficiencies of midazolam and propofol for plasma were 93 to 117%. The method was successfully applied to the analyses of plasma midazolam and propofol concentrations in a brain-injured patient subjected to hypothermia, which validated the present methodology. In this study, the authors have successfully established the detailed procedure for GC–MS/MS analysis of midazolam and propofol in human plasma. The sensitivity and specificity of the present method for the drugs were almost comparable to those by the standard liquid chromatography–tandem mass spectrometry. To their knowledge, this is the first report for analysis of midazolam/propofol by GC–MS/MS.
TL;DR: The identified metabolites may provide useful markers for possible forensic and clinical determination of the mode of ingestion but the use of dog and rat animal models was not indicated.
Abstract: The metabolism of carfentanil was assessed using human, dog, and rat pulmonary microsomes. Mass spectrometry based analysis allowed for metabolite identification and species differentiation. Participation of different metabolic enzymes in carfentanil biotransformation was also assessed. Metabolite profiling was accomplished by incubating 10 µM carfentanil in human, dog, and rat lung microsomes. The metabolites were separated and analyzed by ultra-high performance liquid chromatography/high-resolution mass spectrometry. In total, 18 metabolites were detected. Nine metabolites were authentically identified through comparison to synthesized reference standards. In human lung microsomes, nine metabolites were identified. In dog lung microsomes, 15 metabolites were identified with three being dog specific. In rat lung microsomes, 15 metabolites were identified and two were rat specific. Proposed metabolic pathways included N-dealkylation, monohydroxylation, dihydroxylation, N-oxidation of piperidine ring nitrogen, and ketone formation. Participation of enzymes CYP2B6, CYP2E1, CYP2J2, and CYP3A4/5 to carfentanil metabolism was suggested by selective enzymatic inhibition. The pulmonary clearance in human lung microsomes was lower than the previously reported hepatic metabolism suggesting organ specific metabolic rates. The contribution of multiple cytochrome P450 enzymes to human, dog, and rat pulmonary microsomal carfentanil biotransformation varied between species. The identified metabolites may provide useful markers for possible forensic and clinical determination of the mode of ingestion but the use of dog and rat animal models was not indicated. To our knowledge, this is the first reported use of chemically synthesized reference standards for the unequivocal identification of lung carfentanil metabolites.
TL;DR: These novel ethyl esters may be useful additional markers of cannabinoid administration, and especially so if they prove to have longer half-lives than their parent compounds.
Abstract: Synthetic cannabinoids (SCs) represent a large proportion of novel psychoactive substances on the black market and have caused a number of deaths. Polydrug use including combination of SCs and ethanol could further complicate the toxicological impact. To the best of our knowledge, there have been no reports presenting evidence of transesterification between SCs and ethanol in vitro. The in vitro metabolism of the four carboxylate SCs PB-22, NPB-22, 5-fluoro-PB-22 (5F-PB-22), and 5-fluoro-NPB-22 (5F-NPB-22) in the presence of ethanol using human liver microsomes with and without appropriate enzyme inhibitors was studied. Newly identified SC ethyl esters were chemically synthesised and fully characterised. The activity of these SCs and their ethanol transesterification products were assessed using cannabinoid receptor (CB1 and CB2) activation assays. SCs/ethanol transesterification products were detected and studied using liquid chromatography–high-resolution mass spectrometry. We have shown that the SC ethyl ester formation is mediated by human carboxyl esterase enzymes. The ethyl esters exhibited a reduced activity for the CB receptors compared with their parent compounds. These novel ethyl esters may be useful additional markers of cannabinoid administration, and especially so if they prove to have longer half-lives than their parent compounds.
TL;DR: A potentiometric sensor based on calix[4]arene derivative provided a rapid and perspective technique for the detection of cathinone derivatives and shows that potentiometry sensors can become promising future tools for NPSs detection.
Abstract: Cathinone derivatives are new psychoactive substance(s) (NPSs) with amphetamine-like effects and currently represent a great problem for public health. For this reason it is necessary to develop new sensitive, selective fast and inexpensive methods for large-scale screening of NPSs. The purpose of this study was to evaluate the capabilities of 4-tert-butylcalix[4]arene tetra-acetate as a receptor of potentiometric sensor to recognize NPSs based on cathinone derivatives and one aminoindane for comparison. Potentiometry was used to determine the selectivity of a 4-tert-butylcalix[4]arene tetra-acetate towards cathinone derivatives. Density functional theory (DFT) computations and nuclear magnetic resonance (NMR) titration were used to explain the binding modes of the calix[4]arene derivative towards selected NPSs. The basic binding mode of the calix[4]arene derivative for NPSs, namely a hydrogen bonding towards the NH group, was confirmed using DFT calculations and NMR titration. Importantly, the calix[4]arene derivative as the receptor of the potentiometric sensor provides (1) discrimination between cathinone derivatives themselves and cathinones from aminoindane as well as the (2) possibility of detection of buphedrone in samples containing the mixture of the different NPSs including cathinones (Sr ≤ 0.11). A potentiometric sensor based on calix[4]arene derivative provided a rapid and perspective technique for the detection of cathinone derivatives. These results show that potentiometric sensors can become promising future tools for NPSs detection.
TL;DR: In this article, a matrix-assisted laser-desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometric imaging (MALDI-FT ICR MSI) was developed for direct identification and imaging of synthetic indole-3-carboxamide cannabinoids in hair samples using the positive ion mode.
Abstract: Different kinds of new synthetic cannabinoids (SCs) have been continuously developed to evade drug monitoring. Segmental hair analysis offers a longer period for retrospective drug detection compared with blood or urine. In this study, matrix-assisted laser-desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometric imaging (MALDI-FT ICR MSI) was developed for direct identification and imaging of synthetic indole-3-carboxamide cannabinoids in hair samples using the positive ion mode. The target SCs include N-(adamantan-1-yl)-1-pentyl-1H-indole-3-carboxamide (APICA), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (5F-AB-PICA), N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (ADB-PINACA) and N-(1-amino -3,3-dimethyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (5F-ADBICA). The MALDI-MS and MS/MS were first performed on the scraped hair soaked in a mixture of the four SCs after matrix sublimation. This method may provide a detection power for SCs to the 0.1 ng level per 2 cm hair. Target cannabinoids were identified by MS1 and MS2. Matrix deposition methods including airbrush sprayer and sublimation were compared. The method was then applied in revealing the spatial distribution of APICA and 5F-ADBICA in real hair samples from two drug abusers by comparing MS1 and MS2 spectra. The metabolites of APICA and 5F-ADBICA were also presumed to be present in the positive hair samples. Furthermore, a comprehensive comparison between a MALDI-FT ICR MS and a MALDI time-of-flight–MS instrument was performed in detection-sensitivity and specificity for positive real samples. The proposed method provides a powerful tool for drug supervision and forensic medicine analysis in a wide time window, and the sample amount required was also decreased.
TL;DR: Hydroxy metabolites for norflurazepam, N-(hydroxyethyl), desethyl and didesethyl for flurazapam, hydroxy for fludiazepam and glucuronides and N-(Hydroxyethyl) for cinolazep am are recommended for monitoring.
Abstract: Norflurazepam, also a metabolite of other prescription benzodiazepines, appeared on the new psychoactive substances (NPS) drug market recently, complicating the interpretation of NPS findings. The aims of the study were to tentatively identify potential metabolites of norflurazepam and structural analogues (flurazepam, fludiazepam and cinolazepam) produced by hepatocytes and in authentic human samples and to discuss the possibility to differentiate drug consumption. Each drug (5 µmol/L) was incubated with pooled human hepatocytes, and metabolites were identified using liquid chromatography–high-resolution mass spectrometry (LC–HRMS). Similarly, urine with/without hydrolysis and blood/serum from three flurazepam and seven presumptive norflurazepam cases were analysed by LC–HRMS. No metabolites were detected for norflurazepam in hepatocytes, but six metabolites for flurazepam, two for fludiazepam and three for cinolazepam were found. In human specimens collected after flurazepam ingestion, a total of eight metabolites, in good agreement with hepatocyte metabolites, were detected. In specimens of presumptive norflurazepam intake, norflurazepam and its metabolites (four hydroxy metabolites and one glucuronide of a hydroxy metabolite) were found. Based on the results, hydroxy metabolites for norflurazepam, N-(hydroxyethyl), desethyl and didesethyl for flurazepam, hydroxy for fludiazepam and glucuronides and N-(hydroxyethyl) for cinolazepam are recommended for monitoring. While flurazepam, fludiazepam and cinolazepam were metabolised by hepatocytes at side chain, norflurazepam was not, which seems to indicate that hepatocytes have difficulty in modifying the benzene/diazepine rings of some 1,4-benzodiazepines. As for confirming the intake of norflurazepam, the urine ratio of 3-hydroxy-norflurazepam/norflurazepam might be the key; a high ratio might be correlated to norflurazepam intake, thereby enabling the differentiation.