ELISA detection of phenazepam, etizolam, pyrazolam, flubromazepam, diclazepam and delorazepam in blood using Immunalysis® benzodiazepine kit

Phenazepam and etizolam were the first uncontrolled benzodiazepines available for sale in the UK. Pyrazolam, flubromazepam and diclazepam are not used medicinally anywhere in the world; they are produced exclusively for the uncontrolled, recreational market. It is important to know whether potentially abused drugs like these can be detected in routine toxicological screening tests. The purpose of this study was to evaluate whether the Immunalysis® Benzodiazepines ELISA kit could detect phenazepam, etizolam, pyrazolam, flubromazepam, diclazepam and its metabolite delorazepam. Their cross-reactivity was assessed by comparing the absorbance of the drug with that of oxazepam, the reference standard. This study found that these uncontrolled benzodiazepines cross-react sufficiently to produce a positive result with the Immunalysis® Benzodiazepine ELISA kit. Cross-reactivity ranged from 79 to 107% for phenazepam, etizolam, pyrazolam, flubromazepam, diclazepam and delorazepam fortified into blood. The results show that it is possible to detect these newer benzodiazepines with traditional forensic toxicology laboratory tools and it is important to include these benzodiazepines in the confirmation tests

SODAS: Surveillance of Drugs of Abuse Study

Objective: Novel Psychoactive Substance (NPS) as a form of recreational drug use has become increasingly popular. There is a paucity of information with regards the prevalence and clinical sequalae of these drugs. The aim of this study was to detect NPS in patients presenting to the Emergency Department (ED) with suspected toxicological ingestion. Methods: The prospective study was performed in a large Emergency Department (ED) in the UK. During a three month period eighty patients were identified by clinicians as having potentially ingested a toxicological agent. Urine sample were analysed using liquid chromatography-high resolution mass spectrometry and basic clinical data was gathered. Results: 80 patients with a history of illicit or recreational drug consumption had urine screenings performed. 49% (39) of patients undergoing a screen had more than one illicit substance detected. 20% (16) of patients tested positive for at least one NPS. Conclusions: Almost half of patients presenting had ingestion of multiple substances which correlated poorly with self reporting of patients. Developing enhanced strategies to monitor evolving drug trends is crucial to the ability of clinicians to deliver care to this challenging group of patients

Analysis and clinical findings of cases positive for the novel synthetic cannabinoid receptor agonist MDMB-CHMICA

Context: MDMB-CHMICA is a synthetic cannabinoid receptor agonist which has caused concern due to its presence in cases of adverse reaction and death. Method: 43 cases of suspected synthetic cannabinoid ingestion were identified from patients presenting at an Emergency Department and from post-mortem casework. These were subjected to liquid-liquid extraction using tertiary-butyl methyl ether and quantitatively analysed by Electospray Ionisation Liquid Chromatography – tandem Mass Spectrometry. For positive samples, case and clinical details were sought and interrogated. Results: 11 samples were found positive for MDMB-CHMICA. Concentrations found ranged from <1 – 22 ng/mL (mean: 6 ng/mL, median: 3 ng/mL). The age range was 15 – 44 years (mean: 26 years, median: 21 years), with the majority (82%) of positive results found in males. Clinical presentations included hypothermia, hypoglycaemia, syncope, recurrent vomiting, altered mental state and serotonin toxicity, with corresponding concentrations of MDMB-CHMICA as low as <1 ng/mL. Duration of hospitalisation ranged from 3 – 24 hours (mean: 12 hours, median: 8 hours). Discussion: The concentration range presented in this case series is indicative of MDMB-CHMICA having a high potency, as is known to be the case for other synthetic cannabinoid receptor agonists. The age range and gender representation were consistent with that reported for users of other drugs of this type. The clinical presentations observed were typical of synthetic cannabinoid receptor agonists and show the difficulties in identifying reactions potentially associated with drugs of this type. Conclusion: The range of MDMB-CHMICA concentrations in Emergency Department presentations (n=9) and post-mortem cases (n=2) was reported. No correlation between the concentration of this drug and clinical presentation or cause of death was reported in this sample. However, the potential for harm associated with low concentrations of MDMB-CHMICA and the symptoms of toxicity being non-specific was highlighted

Postmortomics:The potential of untargeted metabolomics to highlight markers for time since death

The success of forensic investigations involving fatalities very often depends on the establishment of the correct timeline of events. Currently used methods for estimating the postmortem interval (PMI) are mostly dependent on the professional and tacit experience of the investigator, and often with poor reliability in the absence of robust biological markers. The aim of this study was to investigate the potential of metabolomic approaches to highlight molecular markers for PMI. Rat and human muscle tissues, collected at various times postmortem, were analyzed using an untargeted metabolomics approach. Levels of certain metabolites (skatole, xanthine, n-acetylneuraminate, 1-methylnicotinamide, choline phosphate, and uracil) as well as most proteinogenic amino acids increased steadily postmortem. Threonine, tyrosine, and lysine show the most predictable evolution over the postmortem period, and may thus have potential for possible PMI markers in the future. This study demonstrates how a biomarker discovery approach can be extended to forensic investigations using untargeted metabolomics

DISC1 regulates N-methyl-D-aspartate receptor dynamics:abnormalities induced by a Disc1 mutation modelling a translocation linked to major mental illness

Abstract The neuromodulatory gene DISC1 is disrupted by a t(1;11) translocation that is highly penetrant for schizophrenia and affective disorders, but how this translocation affects DISC1 function is incompletely understood. N-methyl-D-aspartate receptors (NMDAR) play a central role in synaptic plasticity and cognition, and are implicated in the pathophysiology of schizophrenia through genetic and functional studies. We show that the NMDAR subunit GluN2B complexes with DISC1-associated trafficking factor TRAK1, while DISC1 interacts with the GluN1 subunit and regulates dendritic NMDAR motility in cultured mouse neurons. Moreover, in the first mutant mouse that models DISC1 disruption by the translocation, the pool of NMDAR transport vesicles and surface/synaptic NMDAR expression are increased. Since NMDAR cell surface/synaptic expression is tightly regulated to ensure correct function, these changes in the mutant mouse are likely to affect NMDAR signalling and synaptic plasticity. Consistent with these observations, RNASeq analysis of the translocation carrier-derived human neurons indicates abnormalities of excitatory synapses and vesicle dynamics. RNASeq analysis of the human neurons also identifies many differentially expressed genes previously highlighted as putative schizophrenia and/or depression risk factors through large-scale genome-wide association and copy number variant studies, indicating that the translocation triggers common disease pathways that are shared with unrelated psychiatric patients. Altogether, our findings suggest that translocation-induced disease mechanisms are likely to be relevant to mental illness in general, and that such disease mechanisms include altered NMDAR dynamics and excitatory synapse function. This could contribute to the cognitive disorders displayed by translocation carriers

Bioanalytical methods for the determination of synthetic cannabinoids and metabolites in biological specimens

Recently the use of synthetic cannabinoids (SCs) has increased around the world. As a result, the importance for accurate analysis of SCs in human biological matrices is evident and continues to be especially challenging due to their chemical structures being constantly modified. Many methods have been published recently for the analysis of SCs in human biological samples. This review provides an overview of the analytical methods used for the analysis of SCs and their metabolites in biological specimens with a special focus on chromatographic analysis and sample preparation. Liquid chromatography assay is the most commonly used for confirmation purposes of SCs and their metabolites in biological matrices. In blood and oral fluid, analysis of SCs must be very sensitive. In urine, SCs have extensive metabolism pathways; therefore the main target compounds are their hydroxyl and carboxyl metabolites, which is important to recognise when establishing clinical and forensic toxicology analytical methods

A review of ethylphenidate in East and West Scotland

Ethylphenidate is a psychostimulant and analogue of methylphenidate. Interestingly it is also produced as a metabolite from the co-ingestion of methylphenidate and alcohol (ethanol). In the UK, between April and June 2015, ethylphenidate and 6 other methylphenidate based novel psychoactive substances (NPS) were subjected to a temporary class drug order under the Misuse of Drugs Act 1971. Ethylphenidate is being abused by both novel and habitual drug users, more prominently in the East of Scotland. What is unknown in the literature is the contribution of ethylphenidate in deaths. A search was conducted for an 18 month period (July 2013 to December 2014) to identify cases where ethylphenidate was detected during post-mortem toxicological analysis. Nineteen cases were identified and these cases were examined with regards to case circumstances, pathology findings, toxicology results and adverse effects. The individuals ranged in age from 20 to 54 (median 37) and the majority were male (n = 14) and from the East of Scotland (n = 16), more specifically Edinburgh and surrounding area. Current or previous heroin abuse was a common theme in these cases (n = 16) and injection was a common route of administration of “legal highs” or “burst”. The concentration of ethylphenidate in the cases ranged from 0.008 mg/L to over 2 mg/L in post-mortem femoral blood (median 0.25 mg/L, average 0.39 mg/L). Other drugs commonly detected were benzodiazepines (n = 15), followed by opiates (n = 11, 4 of which were positive for 6-monoacetylmorphine) and then methadone (n = 8). All 19 cases received a full post-mortem examination and there were 10 cases where drug toxicity was the sole or potentially contributory factor to the cause of death. Ethylphenidate was specifically mentioned in the cause of death for 5 cases, chronic intravenous (IV) drug use was named as part of the cause of death for 2 cases and in 6 cases there was evidence of complications and infections through IV drug use. As far as it is known to the authors, this is the first review of post-mortem cases involving the use of ethylphenidate in East and West Scotland. This study can be used as a guide for toxicologists and pathologists when interpreting cases which are positive for ethylphenidate

Synthetic cannabinoid receptor agonists in postmortem casework in Scotland

Synthetic cannabinoid receptor agonists (SCRAs) have been a concern to forensic toxicologists since their emergence as drugs of abuse in the mid-late 2000s. The extent of their use in Scotland appears to be low especially when compared to other drug groups such as opioids and benzodiazepines. There is a concern, however, that the use is widespread in prison populations in particular. In this work, samples of blood and urine collected during routine postmortem examination between April 2017 and March 2019 were subjected to analysis of SCRA compounds. Circumstantial and demographic information was collected on positive cases to build up a body of evidence for where SCRAs may be most likely to contribute to the cause of death. Thirteen out of 133 cases (10%) tested were positive for one or more compound in one or more matrix. Overall, the detection of 5F-MDMB-PINACA or its O-desmethyl acid metabolite was most common, followed by the metabolite shared by AB-FUBINACA and MMB-FUBINACA. SCRA-positive cases were predominantly males (92%), and the age range of all decedents was 21–49 years old (median 36 years). The majority of cases were certified as drug-related deaths (DRDs, 38%), natural/medical (31%) or suicide (23%), and two of the DRDs mentioned SCRAs specifically in the cause of death. The concentrations of SCRAs detected did not seem to be as important to the determination of the cause of death as their mere presence, but quantitative results were reported (where possible) in order to build up a body of evidence for SCRA concentrations in different case types