RESUMEN
Driving under the influence of drug (DUID) cases continue to challenge forensic toxicologists as both the volume and complexity of casework increases. Comprehensive DUID testing should also meet the drafted Academy Standards Board (ASB)/ American National Standard Institute (ANSI) standard and the National Safety Council's Alcohol, Drugs and Impairment Division (NSC-ADID) recommendations. A simple method using protein precipitation followed by filtration extraction with an 8 minute run time by liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed, and a comprehensive ASB/ANSI validation was performed. Target drugs and metabolites were quantitatively assessed in blood and qualitatively assessed in urine. Included were 127 target analytes including cannabinoids (12), amphetamines (11), cocaine and metabolites (6), benzodiazepines (36), Z-drugs (5), opioids (27), anticonvulsants (3), first-generation antihistamines (6), muscle relaxants (2), dissociatives and hallucinogens (6), barbiturates (10), and miscellaneous substances (3). Limits of detection are appropriate for DUID and other forensic casework such as drug-facilitated crime (DFC) and postmortem investigations. To demonstrate applicability, 78 proficiency test blood and urine samples and 1,645 blood and urine samples from authentic cases samples demonstrated effective detection of target analytes in forensic casework. By increasing the analytical scope of multiple drug classes via a single method, this technique detects drugs that may have previously gone undetected, such as flualprazolam, etizolam, mitragynine, gamma-hydroxybutyric acid and psilocin and improves laboratory efficiency by reducing the number of tests required. The described method is, to the authors' best knowledge, the only published single procedure to meet all drugs listed in the drafted ASB/ANSI standard and recommended Tier 1 and traditional drugs from Tier 2 for DUID screening, while also achieving many drug scope and sensitivity recommendations for DFC and postmortem testing.
Asunto(s)
Cannabinoides , Espectrometría de Masas en Tándem , Anfetaminas , Cromatografía Liquida/métodos , Toxicología Forense/métodos , Detección de Abuso de Sustancias/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Etizolam is a novel psychoactive substance and novel benzodiazepine of the thienotriazolodiazepine class, which has recently seen an increasing trend in use worldwide. We report a case series of 10 decedents with etizolam and opioids in their systems. Death investigation, expanded toxicology and medical investigation information were included for contextualization of etizolam in death. Etizolam was detected and confirmed within peripheral and cardiac blood, urine, vitreous humor and, in one case, gastric fluid, by liquid chromatography-tandem mass spectrometry and liquid chromatography-quadrupole time of flight mass spectrometry methodologies. Death investigation indicated nonmedical use of most drugs. Medical investigation commonly noted pulmonary edema, cardiomegaly and cerebral swelling. The majority of the decedents appeared to be unaware of the presence of etizolam and succumbed to the mixed drug toxicity of their routine depressant and narcotic analgesic drug of abuse in combination with etizolam. Etizolam use continues to be observed and poses as a potentially lethal contribution to multiple drug toxicity, especially in the age of the opioid crisis. Assessment of analytes like etizolam requires up-to-date methodologies and vigilance in testing to better characterize the toxicology and interpret the contribution to death.
Asunto(s)
Analgésicos Opioides , Diazepam , Cromatografía Liquida , Diazepam/análogos & derivados , Toxicología Forense , HumanosRESUMEN
Systematic toxicological approaches that employ both ideology changes and improvements in instrumentation and sample extraction allow for improved toxicology testing efficiency through lower sensitivities, higher specificity and minimized resource use. Historically, the San Francisco Office of the Chief Medical Examiner relied heavily on a gas chromatography mass spectrometry (GC-MS) testing regime, comprised of individual drug-class confirmation and quantitation assays. Traditional methods utilizing GC-MS typically require iterations of testing, exhausting sample volume, and hindering productivity and turnaround times, particularly for polypharmacy cases frequently seen in modern postmortem toxicology. The method described here consolidated the scope of seven legacy methods into a single liquid chromatography tandem mass spectrometry (LC-MS/MS) method for better sensitivity, higher throughput, minimal sample consumption for the quantitation of drugs of abuse and improved quality assurance with the incorporation of smart, automated processing. About 100 µL of blood or urine were rapidly extracted using a simple acetonitrile protein crash and subsequent in-vial filtration and injected on to an LC-MS-MS system. The developed method was fully validated to SWGTOX and international guidelines and incorporated 55 analytes along with a customized query that facilitates rapid and consistent application of acceptability criteria for data processing and review. Applicability was demonstrated with the analysis of 1,389 samples (858 blood and 531 urine) where at least 41% of positive results may have been missed due to their decreased sensitivity and 11% of results were not within the scope of the previous analytical methods estimated. On average, cases in this study would have previously required three distinct GC-MS assays, 3 mL of blood, and upwards of 30 h of active staff time. The described LC-MS-MS analytical approach has mitigated the need to perform multiple assays, utilized only 0.1 mL of sample, significantly reduced analyst work time, incorporated 10 additional analytes and allowed for a more comprehensive testing regime to better inform cause of death determinations.
Asunto(s)
Toxicología Forense/métodos , Detección de Abuso de Sustancias/métodos , Autopsia , Cromatografía Liquida , Cromatografía de Gases y Espectrometría de Masas , Humanos , Espectrometría de Masas en TándemRESUMEN
Synthetic cannabinoids (SC), are a novel class of designer drugs which emerged as a drug of abuse in the late 2000's. We report a case series of 6 patients who may have smoked a synthetic cannabinoid product in a remote wilderness setting. They presented with varying degrees of altered mental status, agitation, and seizures. Two were confirmed to have AB-PINACA, ADB-PINACA and their respective pentanoic acid metabolites in biological specimens via liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS). Both compounds had DEA Schedule I classification at the time of case presentation, and 22 SCs are currently temporary or permanent DEA Schedule I. More than 150 SCs are known to date, and new compounds are appearing at a rapid rate on darknet and surface web e-commerce websites, marketed as "research chemicals" or "legal highs." The scale and rapidity of SC evolution make legal control and analytical detection difficult. Nontargeted testing with liquid chromatography high resolution mass spectrometry (LC-HRMS), examining both parent compounds and metabolites, is the ideal method for novel SC identification and confirmation. Due to full agonism at the cannabinoid receptors CB1 and CB2, clinical effects are more severe than marijuana, which is a partial cannabinoid receptor agonist. They include agitated delirium, lethargy and coma, seizures, tachycardia, hypertension, and hallucinations, among other findings. Treatment is primarily symptomatic and aimed at airway protection and control of agitation and seizures. SCs do not appear to be abating anytime soon and require the cooperation of law enforcement, analytical scientists, and clinicians to adequately control. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'
Asunto(s)
Drogas de Diseño/envenenamiento , Indazoles/envenenamiento , Valina/análogos & derivados , Adulto , Agresión/efectos de los fármacos , Delirio/inducido químicamente , Electroencefalografía , Humanos , Masculino , Estructura Molecular , Convulsiones/inducido químicamente , Espectrometría de Masas en Tándem , Valina/envenenamientoRESUMEN
DbpA is a DEAD-box RNA helicase implicated in Escherichia coli large ribosomal subunit assembly. Previous studies have shown that when the ATPase and helicase inactive DbpA construct, R331A, is expressed in E. coli cells, a large ribosomal subunit intermediate accumulates. The large subunit intermediate migrates as a 45S particle in a sucrose gradient. Here, using a number of structural and fluorescent assays, we investigate the ribosome profiles of cells lacking wild-type DbpA and overexpressing the R331A DbpA construct. Our data show that in addition to the 45S particle previously described, 27S and 35S particles are also present in the ribosome profiles of cells overexpressing R331A DbpA. The 27S, 35S, and 45S independently convert to the 50S subunit, suggesting that ribosome assembly in the presence of R331A and the absence of wild-type DbpA occurs via multiple pathways.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ribosomas , Escherichia coli/enzimología , Escherichia coli/genética , Cinética , ARN Bacteriano/metabolismo , ARN Ribosómico/metabolismoRESUMEN
Retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that recognizes viral RNA and activates the interferon-mediated innate antiviral response. To understand the mechanism of signal activation at the receptor level, we cloned, expressed, and purified human RIG-I containing the two caspase activation and recruitment domains (CARDs) followed by the C-terminal helicase domain. We found that recombinant RIG-I is a functional protein that interacts with double-stranded RNA with substantially higher affinity as compared with single-stranded RNA structures unless they contain a 5'-triphosphate group. Viral RNA binding to RIG-I stimulates the velocity of ATP hydrolysis by 33-fold, which at the cellular level translates into a 43-fold increase of interferon-beta expression. In contrast, the isolated ATPase/helicase domain is constitutively activated while also retaining its RNA ligand binding properties. These results support the recent model by which RIG-I signaling is autoinhibited in the absence of RNA by intra-molecular interactions between the CARDs and the C terminus. Based on pH profile and metal ion dependence experiments, we propose that the active site of RIG-I cannot efficiently accommodate divalent cations under the RNA-free repressed conformation. Overall, these results show a direct correlation between RNA binding and ATPase enzymatic function leading to signal transduction and suggest that a tight control of ATPase activity by the CARDs prevents RIG-I signaling in the absence of viral RNA.