Analysis of 4‐Bromo‐2,5‐DimethoxyphenethylamineAbuser's Urine: Identification and Quantitation of Urinary Metabolites

The metabolites of 4‐bromo‐2,5‐dimethoxyphenethylamine (2C‐B), a psychoactive drug with hallucinogenic activity, were investigated in a urine sample from a user of 2C‐B. The urine sample was deconjugated enzymatically and the metabolites were recovered by liquid–liquid extraction. The extract was analyzed by gas chromatography/mass spectrometry after derivatization, and the results were used to identify and quantitate the metabolites. 4‐Bromo‐2,5‐dimethoxyphenylacetic acid was the most abundant metabolite of 2C‐B in human urine and accounted for 73% of the total amount of detected metabolites, followed by 4‐bromo‐2‐hydroxy‐5‐methoxyphenylacetic acid (13%) and 4‐bromo‐2,5‐dimethoxyphenylethyl alcohol (4.5%). According to the literature, the main metabolites of 2C‐B in rat urine are N‐(4‐bromo‐2‐methoxy‐5‐hydroxyphenylethyl)acetamide and N‐(4‐bromo‐2‐hydroxy‐5‐methoxyphenylethyl)acetamide. However, these metabolites accounted for only a small proportion of the total amount of detected metabolites in human urine, which indicates that there are significant species‐specific differences in the metabolism of 2C‐B. 4‐Bromo‐2,5‐dimethoxyphenylacetic acid, which was the most abundant metabolite in human urine, is thought to be generated by deamination of 2C‐B by monoamine oxidase (MAO) followed by oxidation by aldehyde dehydrogenase. Our results suggest that MAO plays a crucial role in the metabolism of 2C‐B in humans.     

Unexpected Serotonin Syndrome,Epileptic Seizures,and Cerebral Edema Following 2,5‐dimethoxy‐4‐bromophenethylamine Ingestion

4‐bromo‐2,5‐dimethoxyphenethylamine (2C‐B) is a designer drug. In Europe, 2C‐B is easily obtained and used for recreational purposes. It is known for its stimulating effects similar to those of 3,4‐methylenedioxymethamphetamine, although in higher doses it has more hallucinogenic effects. Here, we report a case of 2C‐B ingestion, confirmed by liquid chromatography‐tandem mass spectrometry, in an 18‐year‐old man. The neurological consequences were severe, including the development of serotonin syndrome and severe brain edema. Supportive therapy resulted in a stable condition, although, after several months, the patient still suffered from severe neurological impairment due to the drug‐induced toxicity. This case showed that 2C‐B could not be identified with the drugs of abuse screening routinely used in Dutch hospitals. The use of 2C‐B carries many risks, with potentially profound neurological damage, that both consumers and healthcare physicians are unaware of.     

Novel O‐propyl,S‐propyl,and N‐propyl substituted 1,4‐naphthoquinones and 1,4‐anthraquinones as potential fingermark development reagents for porous surfaces

Lawsone is a 2‐substituted‐1,4‐naphthoquinone derivative, which has been proposed as an alternative to the reagents currently used for fingermark detection on porous surfaces. 2‐substituted‐anthraquinones, which contain an additional conjugated benzene ring, have a similar chemical structure to that of lawsone. In this study, a new series of 2‐substituted‐1,4‐naphthoquinones and 2‐substituted‐1,4‐anthraquinones were synthesized and completely characterized by¹H NMR,¹³C NMR, IR, and HPLC‐TOF/MS analyses. All newly synthesized 2‐substituted‐1,4‐quinones were investigated for their ability to develop latent fingermarks on porous surfaces, and this ability was compared with that of lawsone. Each fingermark developed was graded using an established method; thus, quantitative data were attributed to each fingermark. It has been demonstrated that the 1,4 ‐ quinones react with amino acids present in latent fingermarks on selected paper surfaces to produce faint yellow‐orange impressions, which exhibit strong photoluminescence when illuminated with a forensic light source at 440 nm and observed through a red filter. None of the compounds caused background darkening. The results obtained were generally similar to those of lawsone, however, 8‐dibromo‐2‐(propylamino)naphthalene‐1,4‐dione and 5,8‐dibromo‐2‐(propylthio)naphthalene‐1,4‐dione yielded better results for copier paper and colored (blue) copier paper used in this analysis. To the best of our knowledge, this is the first study to examine the role of 1,4‐anthraquinone derivatives as potential fingermark development reagents. The results indicate that 1,4‐quinones have a potential to be used as reagents for enhancement of latent fingermarks.     

Designer Psychostimulants in Urine by Liquid Chromatography–Tandem Mass Spectrometry,

Designer psychostimulants are known by recreational drug users to produce a complex array of adrenergic and hallucinogenic effects. Many of these drugs are not targeted during routine toxicology testing and as a consequence, they are rarely reported. The purpose of this study was to develop a procedure for the detection of 15 psychostimulants in urine using liquid chromatography–tandem mass spectrometry (LC‐MS/MS), specifically 2,5‐dimethoxy‐4‐bromophenethylamine (2C‐B), 2,5‐dimethoxy‐4‐chlorophenethylamine (2C‐C), 2,5‐dimethoxy‐4‐methylphenethylamine (2C‐D), 2,5‐dimethoxy‐4‐ethylphenethylamine (2C‐E), 2,5‐dimethoxyphenethylamine (2C‐H), 2,5‐dimethoxy‐4‐iodophenethylamine (2C‐I), 2,5‐dimethoxy‐4‐ethylthiophenethylamine (2C‐T‐2), 2,5‐dimethoxy‐4‐isopropylthiophenethylamine (2C‐T‐4), 2,5‐dimethoxy‐4‐propylthiophenethylamine (2C‐T‐7), 2,5‐dimethoxy‐4‐bromoamphetamine (DOB), 2,5‐dimethoxy‐4‐chloroamphetamine (DOC), 2,5‐dimethoxy‐4‐ethylamphetamine (DOET), 2,5‐dimethoxy‐4‐iodoamphetamine (DOI), 2,5‐dimethoxy‐4‐methylamphetamine (DOM), and 4‐methylthioamphetamine (4‐MTA). Analytical recoveries using solid‐phase extraction were 64–92% and the limit of detection was 0.5 ng/mL for all drugs except 2C‐B (1 ng/mL). The assay was evaluated in terms of analytical recovery, precision, accuracy, linearity, matrix effect, and interferences. The technique allows for the simultaneous detection of 15 psychostimulants at sub‐ng/mL concentrations.     

Near‐fatal Intoxication with the “New” Synthetic Opioid U‐47700: The First Reported Case in the Czech Republic

Recreational use of the potent synthetic opioid 3,4‐ dichloro‐N‐(2‐(dimethylamino)cyclohexyl)‐N‐methylbenzamide (U‐47700) is rising, accompanied by increasingly frequent cases of serious intoxication. This article reports a case of near‐fatal U‐47700 intoxication. A man was found unconscious (with drug powder residues). After 40 h in hospital (including 12 h of supported ventilation), he recovered and was discharged. Liquid chromatography/high‐resolution mass spectrometry (LC/HRMS) or gas chromatography/mass spectrometry (GC/MS) were used to detect and quantify substances in powders, serum and urine. Powders contained U‐47700 and two synthetic cannabinoids. Serum and urine were positive for U‐47700 (351.0 ng/mL), citalopram (<LOQ), tetrahydrocannabinol (THC: 3.3 ng/mL), midazolam (<LOQ) and a novel benzodiazepine, clonazolam (6.8 ng/mL) and their metabolites but negative for synthetic cannabinoids. If potent synthetic opioids become cheaper and more easily obtainable than their classical counterparts (e.g., heroin), they will inevitably replace them and users may be exposed to elevated risks of addiction and overdose.     

Identification of 2‐(ethylamino)‐1‐(4‐methylphenyl)‐1‐pentanone (4‐MEAP), a New “Legal High” Sold by an Internet Vendor as 4‐Methyl Pentedrone

Online vendors are offering a new legal high, 4‐methylpentedrone (4‐MPD). Information for potential users provided by internet vendors of 4‐MPD includes incorrect structures and nonexistent CAS numbers. A sample of 4‐MPD was obtained and analyzed using GC‐MS, NMR, and LC‐EIS. The fragmentation data from the GC‐MS and LC‐EIS produced an M‐1 ion that suggested the molecular mass was 219 amu, rather than 205 amu as calculated for 4‐methylpentedrone. The difference in molecular mass corresponded to the addition of a methyl group. Based on the mass and fragmentation pattern, two standards were synthesized, 2‐(ethylamino)‐1‐(4‐methylphenyl)‐1‐pentanone and 1‐(4‐methylphenyl)‐2‐(propylamino)‐1‐butanone. The synthesis involved bromination of the appropriate ketone followed by the reaction with ethylamine or propylamine. Based on the NMR data and unique fragmentation patterns produced by these molecules, the sample was identified as 2‐(ethylamino)‐1‐(4‐methylphenyl)‐1‐pentanone, not 4‐methylpentedrone.     

Drug Recognition Evaluation and Chemical Confirmation of a 25C‐NBOMe‐Impaired Driver

This case report details an individual arrested for drug‐impaired driving after leaving the scene of multiple motor vehicle collisions and evading police. The driver was examined by a drug recognition expert and failed the drug recognition evaluation. The driver admitted to using cocaine, marijuana, an antidepressant medication and “N‐bomb,” a novel psychoactive substance that possesses hallucinogenic properties. Toxicological analyses at the Centre of Forensic Sciences’ Toronto laboratory revealed only the substance 2‐[4‐chloro‐2,5‐dimethoxyphenyl]‐N‐[(2‐methoxyphenyl)methyl]ethanamine (25C‐NBOMe) in the accused's urine. This is the first report in which 25C‐NBOMe was identified through DRE and toxicological analyses in a drug‐impaired driver.     

Isolation and Identification of Three By‐products Found in Methylamphetamine Synthesized by the Emde Route

This article describes the isolation and structural elucidation of three compounds produced during the synthesis of methylamphetamine by the so‐called “Emde” procedure. The “Emde” procedure involves the preparation of the intermediate chloropseudoephedrine or chloroephedrine from ephedrine or pseudoephedrine, respectively. The intermediates are then reduced to methylamphetamine with hydrogen under pressure in the presence of a catalyst. The by‐product compounds were isolated from methylamphetamine by column chromatography and liquid chromatography (LC). Proton nuclear magnetic resonance spectroscopy (¹H NMR), carbon nuclear magnetic resonance spectroscopy (¹³C NMR), and nanospray quadrupole‐time of flight‐mass spectrometry (Q‐TOF‐MS) were used to identify them as two stereoisomers of the compound N, N′‐dimethyl‐3,4‐diphenylhexane‐2,5‐diamine and N‐methyl‐1‐{4‐[2‐(methylamino)propyl]phenyl}‐1‐phenylpropan‐2‐amine.     

Profenofos metabolites in human poisoning

Profenofos and its metabolites were determined in a case of fatal poisoning. Little profenofos and large amounts of metabolites were detected by gas chromatography/flame photometric detection in the acid extracts of blood and urine after methylation with diazomethane. Four major metabolites containing phosphorus were identified with the synthesized metabolites, namely, despropylated profenofos, desethylated profenofos and des-S-propylated profenofos, respectively. 4-Bromo-2-chlorophenol (BCP), an aryl moiety of profenofos, was also determined in blood and urine with high performance liquid chromatograph (HPLC) as free or conjugated metabolites.     

Synthesis and identification of urinary metabolites of 4-iodo-2,5-dimethoxyphenethylamine

This article describes the synthesis and identification of urinary metabolites of 4-iodo-2,5-dimethoxyphenethylamine (2C-I), a new psychoactive drug. 2C-I hydrochloride was administered orally to male Sprague-Dawley rats, and the urinary extracts were analyzed by gas chromatography/mass spectrometry (GC/MS), then five putative 2C-I metabolites were synthesized in our laboratory. In the synthetic process of the 2C-I metabolites, iodination of the aromatic ring was successfully carried out using iodine and orthoperiodic acid as the iodination reagent, and selective debenzylation of aryl benzyl ether was accomplished by the acid hydrolysis method using trifluoroacetic acid and thioanisole. The synthesized metabolites were well separated and detected by GC/MS after valeryl derivatization. The results showed that 2C-I underwent O-demethylation, N-acetylation, and deamination, followed by oxidation to the corresponding carboxylic acid in rats. The data presented in this study will be very useful for the analysis of 2C-I and its metabolites in forensic samples.     

Experimental Study on the Postmortem Redistribution of the Substituted Phenethylamine, 25B‐NBOMe

2‐(4‐Bromo‐2,5‐dimethoxyphenyl)‐N‐(2‐methoxybenzyl)ethanamine (25B‐NBOMe) is a substituted phenethylamine, which has become highly prevalent worldwide since 2014. Recently, in an autopsy case involving fatal 25B‐NBOMe intoxication, we found the postmortem increase of 25B‐NBOMe concentration in the cardiac blood approximately 2 days after death. The aim of this study was to investigate the distribution of 25B‐NBOMe and reproduce the postmortem redistribution using a rat model. Sprague‐Dawley rats were killed 30 min after intraperitoneal injection of 25B‐NBOMe (0.5 mg/kg) and left for 0, 3, 6, 9, 15, or 24 h (six rats at each time point). Postmortem 25B‐NBOMe concentrations in the cardiac blood increased by more than 10‐fold at 6‐h postmortem. 25B‐NBOMe accumulated primarily in the lung. Moreover, this postmortem redistribution occurred even in rats that had died 1 week following the 25B‐NBOMe administration. These findings indicate that attention should be paid to sample collection and data interpretation in the toxicological analysis of 25B‐NBOMe.     

Fatal Intoxication with α‐PVP,a Synthetic Cathinone Derivative

This study presents the fatal case of a young man who was admitted to the ICAU due to sudden cardiac arrest. An interview revealed that the patient had taken some unspecified crystals. From the moment of admission, his condition deteriorated dramatically as a result of increasing circulatory insufficiency. After a few hours, sudden cardiac arrest occurred again and the patient was pronounced dead. In the course of a medicolegal autopsy, samples of biological material were preserved for toxicology tests and histopathological examination. The analysis of samples using the LC‐MS/MS technique revealed the presence of α‐PVP in the following concentrations: blood—174 ng/mL, urine—401 ng/mL, brain—292 ng/g, liver—190 ng/g, kidney—122 ng/g, gastric contents—606 ng/g. The study also presents findings from the parallel histopathological examination. Based on these findings, cardiac arrest secondary to intoxication with alpha‐PVP was determined as the direct cause of the patient's death.     

Characterization and Differentiation of Geometric Isomers of 3‐methylfentanyl Analogs by Gas Chromatography/Mass Spectrometry,Liquid Chromatography/Mass Spectrometry,and Nuclear Magnetic Resonance Spectroscopy

The cis and trans isomers of 3‐methylfentanyl and its three analogs were chemically synthesized, and these compounds were characterized and differentiated by gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), and nuclear magnetic resonance (NMR) spectroscopy. The cis and trans isomers of the 3‐methylfentanyl analogs were completely separated by GC/MS. Although the high temperature of the GC injection port caused thermal degradation of β‐hydroxy‐3‐methylfentanyl, the degradation was completely suppressed by trimethylsilyl derivatization. The isomers were also well separated by LC/MS on an octadecylsilyl column with 10 mM ammonium acetate and methanol as the mobile phase. The proton NMR signals were split when the hydrochloride salts of the 3‐methylfentanyl analogs were dissolved in deuterated chloroform because stereoisomers were formed by the coordination of the hydrochloride proton to the nitrogen of the piperidine ring of the 3‐methylfentanyl analogs.     

Blunt Craniofacial Trauma as a Manifestation of Excited Delirium Caused by New Psychoactive Substances

The body of a 19‐year‐old male was found apparently concealed underneath bushes with recent head and facial trauma, and multiple superficial abrasions. Subsequently, it was discovered that the decedent had been running into objects and buildings following the ingestion the evening before of what was thought to be lysergic acid diethylamide (LSD). Blood staining of a nearby wall close to where the body was lying was in keeping with the described behavior. Toxicology revealed 3,4‐methylenedioxymethamphetamine (Ecstasy), in addition to two only recently available drugs 2‐(4‐bromo‐2,5‐dimethoxyphenyl)‐N‐[(2‐methoxyphenyl)methyl]ethanamine, (25B‐NBOMe), and 1‐(3,4‐methylenedioxyphenyl)‐2‐(1‐pyrrolidinyl)‐1‐butanone, (MDPBP). At autopsy, the skull was fractured with cerebral swelling, contusions, and subarachnoid hemorrhage. Death was due to blunt cranial trauma against a background of mixed drug toxicity. The case demonstrates a rare cause of death in a drug‐induced acute delirium, as well as highlighting two new designer street drugs that may result in significant aberrant behavior.     

Stability of Morphine,Codeine, and 6‐Acetylmorphine in Blood at Different Sampling and Storage Conditions

The stability of drugs in biological specimens is a major concern during the evaluation of the toxicological results. The stability of morphine, codeine, and 6‐acetyl‐morphine in blood was studied after different sampling conditions: (i) in glass, polypropylene or polystyrene tubes, (ii) with addition of dipotassium ethylene diamine tetraacetic acid (K₂EDTA) or sodium oxalate (Na₂C₂O₄), and (iii) with or without the addition of sodium fluoride (NaF). Spiked blood samples were stored at two different temperatures (4 and ?20^°C), analyzed after different storage times and after three freeze–thaw cycles. Opiate concentrations were decreased in all conditions, but the most unstable was 6‐acetyl‐morphine. The addition of NaF as preservative improved the stability of opiates at all conditions studied, whereas the type of anticoagulant did not affect the stability of opiates. It was concluded that blood samples should be stored at ?20^°C in glass tubes containing oxalate and NaF for maximum stability.     

Development of Rapid and Economical Colorimetric Screening Method for p‐Phenylenediamine in Variety of Biological Matrices and its Application to Eleven Fatal Cases of p‐Phenylenediamine Poisoning

A rapid colorimetric method for detection of p‐phenylenediamine (PPD) in various biological samples is developed. The o‐cresol test for acetaminophen detection has been modified to detect PPD in blood, urine, gastric contents, and liver. After precipitating protein with trichloroacetic acid solution (2 mL, 10% w/v), biological specimens were required to convert PPD metabolites to PPD by acid hydrolysis. Finally, o‐cresol solution (1 mL, 1% w/v), hydrogen peroxide (200 μL, 3%v/v), and concentrated ammonium hydroxide (0.5 mL) were added in the biological samples. The presence of PPD was indicated by formation of violet color which was turned to bluish green color within 10–15 min. The limit of detection was found to be 2 mg/L in blood, urine, and gastric contents and 2 mg/Kg in liver. This method is also free from any potential interference by p‐aminophenol, acetaminophen, and other amine drugs under test conditions. This method was successfully employed to thirteen fatal cases of PPD poisoning.     

Individual Identification of Racehorses from Urine Samples Using a 26‐Plex Single‐Nucleotide Polymorphism Assay

To construct a system for identifying individual horses from urine samples that are submitted for postracing doping tests, we developed a genotyping assay based on 26‐plex single‐nucleotide polymorphisms (SNPs). DNA was isolated from urine using a commercially available DNA/RNA extraction kit, and SNP genotyping was achieved with a SNaPshot^? technique. DNA profiles including 26 SNPs were acquired from urine samples and blood/hair samples. Within the studied Thoroughbred population, the 26‐plex assay showed a probability of identity of 5.80 × 10^?11. Compared to the conventional short tandem repeat assay, the SNP assay used less DNA, and the rate of successful genotyping was improved to 97% using aliquots of horse urine as small as 140 μL. The urinary DNA could be successfully genotyped under proper storage concerning refrigeration or freeze–thawing. This SNP assay can be used for individual identification when suspicious results are obtained from horse doping tests.     

Identification of 1‐(2,3‐dihydro‐1H‐inden‐5‐yl)‐2‐(ethylamino)pentan‐1‐one (bk‐IVP) in a Seized Drug Exhibit

To circumvent the law by evading regulation and obscuring their identities in routine analyses, numerous substituted cathinones have entered the illicit drug market. These compounds have been coined “bath salts” by users. In the described case, the laboratory received an unknown white powder for controlled substances identification. The sample could not be immediately identified using standard methods and procedures. Ultimately, the structure was elucidated using GC‐MS, NMR, FTIR, GC‐SPIR, UV, and color tests to be 1‐(2,3‐dihydro‐1H‐inden‐5‐yl)‐2‐(ethylamino)pentan‐1‐one (bk‐IVP), a cathinone analog with a rarely observed nonoxygenated bicyclic ring system. Features of spectra and chemical tests are reported that distinguish this class of cathinones from heterocyclic analogs.     

Determination of Acetamiprid and IM‐1‐2 in PostMortem Human Blood,Liver, Stomach Contents by HPLC‐DAD

An HPLC‐DAD method was developed to detect and quantify a neonicotinoid insecticide acetamiprid (ATP) and its metabolite IM‐1‐2 in autopsy samples of a fatal intoxication case. The postmortem blood and tissue distribution of ATP and IM‐1‐2 was determined for the first time. The method showed acceptable precisions and recoveries with relative standard deviations of <10% for ATP level and 1.38 % for IM‐1‐2. The detection and quantification limits for ATP were 0.015 μg/mL and 0.030 μg/mL for blood and were 0.035 μg/g and 0.050 μg/g for liver samples, respectively. The mean contents of ATP were 0.79 μg/g in the liver, 47.35 μg/g in the stomach contents and 2.7 μg/mL in the blood. IM‐1‐2 content was 17.0 μg/g in the stomach contents. ATP and IM‐1‐2 were not detected in the urine. The presence of ATP and IM‐1‐2 in the samples was confirmed by GC‐MS. The method can be exploited in future forensic casework.     

Fatal Overdose of Gamma‐hydroxybutyrate Acid After Ingestion of 1,4‐Butanediol

We report a case of fatal intoxication from 1,4‐butanediol (1,4‐BD), which was ingested by a young and “naïve” gamma‐hydroxybutyrate (GHB) consumer during a party with the co‐ingestion of alcohol, cannabis, and methylene‐dioxy‐methamphetamine. The following drug concentrations were found using gas chromatography coupled with mass spectrometry on autopsy samples and on a cup and a glass found at the scene: 20,350 mg/L (bottle) for 1,4‐BD; 1020 mg/L (femoral blood), 3380 mg/L (cardiac blood), 47,280 mg/L (gastric content), and 570 mg/L (vitreous humor) for GHB. The concentration of GHB is difficult to interpret in forensic cases due to the possibility of an endogenous production of GHB. The variable tolerance of the user may also modify the peri‐ and postmortem GHB concentrations. This case underscores the need to have many different sources of toxicology samples analyzed to avoid the hypothesis of endogenous production of GHB.