Common Drugs Of Abuse Research Articles

B-281 Performance Characteristics Evaluation of the ARK Diagnostics Immunoassay for Xylazine/4-HydroxyXylazine in Urine

Abstract Background Xylazine exposure is currently common in some US cities, but routine laboratory testing is not available. We evaluated the ARK Diagnostics immunoassay for qualitative detection of xylazine/4-hydroxy Xylazine in urine, prior to its anticipated release as a Forensic Use Only assay. Evaluation of performance characteristics of the ARK assay were conducted at two university hospitals, using either the semi-quantitative application of the assay, as performed on the Roche Cobas 503 analyzer (Institution A), or the qualitative application of the assay, as performed on the Beckman Coulter AU480 analyzer (Institution B). Methods Xylazine (X) and 4-hydroxy Xylazine (4-OHX) were obtained from Cayman Chemical (Ann Arbor, MI). Pooled xylazine-free urine was spiked either with X or 4-OHX in the range of 0-2000 ng/mL. De-identified, to-be-discarded patient urine samples were obtained from among those submitted for routine drugs-of-abuse panel testing. X measurements on all samples were performed by LC-MS/MS; samples were segregated into X-NEGATIVE (X<10 ng/mL) and X-POSITIVE (X>=10 ng/mL). The ARK assay was conducted according to manufacturer’s instructions, either as a semi-quantitative assay (Institution A, Roche analyzer) or as a qualitative assay (Institution B, Beckman Coulter analyzer), with 10 ng/mL as the manufacturer-specified cutoff for ARK POSITIVE, using manufacturer-supplied calibration standards ranging from 0-500 ng/mL X. Results The ARK semi-quantitative analysis (Roche analyzer) showed essentially equivalent curvilinear response curves for either X or 4-OHX at concentrations below 1000 ng/mL; for the semi-quantitative ARK assay, we use “>1200 ng/mL” for X-ARK >1200 ng/mL. By mixing studies (Roche), there were neither positive nor negative interferences observed as assessed for common drugs of abuse or for common therapeutic drugs. The presence of hemoglobin, bilirubin or moderate lipemia were devoid of assay interference. Precision studies for manufacturer-supplied controls were as follows: intra-assay CVs were 6.9% for the NEGATIVE control (5.1±0.35 ng/mL) and 5.9% for the POSITIVE control (15.7±0.53 ng/mL) (n=20); interassay CVs were 7.8% for the NEGATIVE control (5.1±0.40 ng/mL) and 5.7% for the POSITIVE control (15.9±0.90 ng/mL) (n=20). For the qualitative ARK assay as performed on the Beckman analyzer, POSITIVE and NEGATIVE controls ran invariably according to their designations. For the semi-quantitative assay application (Roche analyzer): among 78 X-NEGATIVE samples by LC-MS/MS, there were 0% ARK-POSITIVE results (false-positive rate=0%); among 74 X-POSITIVE samples by LC-MS/MS, there was one ARK-NEGATIVE result (false-negative rate=1.4%); this sample (X=65 ng/mL) was found to have a high amorphous crystalline content, and was unable to be reanalyzed. For the qualitative assay application (Beckman Coulter analyzer): among 78 X-NEGATIVE samples there was one ARK-POSITIVE sample (false-positive rate=1.3%) for a sample with xylazine=8.8 ng/mL by LC-MS/MS, with undetectable 4-OHX (<10 ng/mL); among 74 X-POSITIVE samples, there were no ARK-NEGATIVE results (false-negative rate=0%). Conclusions The ARK xylazine immunoassay demonstrated acceptable analytical performance with respect to detection of xylazine in urine at 10 ng/mL. With additional consideration of the assay’s cross reactivity with the major xylazine metabolite, 4-OH-xylazine, the assay was judged to be highly suitable for routine use as a screening test for detection of xylazine exposure via urine testing.

Quantitative determination by UHPLC-MS/MS of 18 common drugs of abuse and metabolites, including THC and OH-THC, in volumetric dried blood spots: a sustainable method with minimally invasive sampling

The increased use of drugs of abuse urges forensic toxicologists to create quick, simple, minimally invasive sampling techniques for biological fluids combined with analytical methods assuring accurate results. To this purpose, a method was developed aimed at quantifying 18 drugs of abuse and metabolites in DBS. Validation of the method was conducted by spiking blank whole blood with the analytes on Capitainer® B cards. The extracts were analyzed by a targeted UHPLC-MS/MS method. Linear calibration was achieved in the range of 1–100 ng/mL for: amphetamine, MDA, MDMA, methamphetamine, cocaine, codeine, benzoylecgonine, cocaethylene, morphine, 6-MAM, buprenorphine, methadone, EDDP, ketamine, norbuprenorphine norketamine, THC, and OH-THC. Experimental LOD was found at 0.5 ng/mL for all analytes except for norbuprenorphine, THC and THC-OH which yielded a LOD of 1 ng/mL. Intra- and inter-day accuracy was satisfactory with bias% resulting within 5%. Evaluation of intra- and inter-day precision yielded CV% values within 20%, for all compounds except EDDP. Average extraction recovery calculated at low (2 ng/mL) and high (75 ng/mL) concentration levels was 63% while average matrix effect determined at the same levels was found to be within 85% − 115% for all analytes except from codeine (70%) and MDMA (131%). The method was applied to authentic blood samples spotted onto the DBS card and the minimum value detected was 1.3 ng/mL. HPLC-MS/MS proved capable to identify all the targeted analytes at low concentrations in the small blood volumes obtainable from DBS cards, which in turn confirmed to be effective and sustainable micro-sampling devices.

Comment on ‘Comprehensive toxicological screening of common drugs of abuse, new psychoactive substances, and cannabinoids in blood using supported liquid extraction and liquid chromatography–quadrupole time-of-flight mass spectrometry’

Comment on ‘Comprehensive toxicological screening of common drugs of abuse, new psychoactive substances, and cannabinoids in blood using supported liquid extraction and liquid chromatography–quadrupole time-of-flight mass spectrometry’

Fluorodeschloroketamine found as a street drug in drug seizures and drug driving cases in Hong Kong

BackgroundWith the decline of the use of ketamine, one of the common drugs of abuse in Hong Kong, detection of ketamine-related analogues in local laboratories has been encountered. Aim: A brief account of the occurrence of fluorodeschloroketamine (FDCK) in forensic cases is reported through a retrospective study of all drug seizures and driving under the influence of drugs (DUID) cases since its first appearance. Methods: Identification of FDCK in drug seizures was achieved through gas chromatography – mass spectrometry (GC-MS) and/or liquid chromatography – diode array detection (LC-DAD) methods while its quantification was performed using gas chromatography – flame ionization detection (GC-FID). For the analysis of blood samples in DUID cases, identification and quantification were performed using LC-MS/MS by monitoring the respective transitions of FDCK and fluorodeschloronorketamine (FDCNK) using ketamine-d4 and norketamine-d4 respectively as internal standards. Results: Since its first submission in November 2018, a total of 74 drug seizure cases (151 items) and 6 drug driving cases were encountered till December 2019. Drug seizures found with FDCK were physically similar to those of ketamine seizures. The majority of items were detected with FDCK only (103 items, ∼67%) or as a mixture of FDCK with ketamine (42 items, ∼28%). The drug purity detected with either FDCK only or FDCK mixed with ketamine was high which was similar to those purity found in ketamine seizures. The blood drug concentrations of FDCK of the 6 drug driving cases were in the range of <0.002–1.1 μg/mL and other psychoactive drug(s)/metabolite(s) were also identified. Except for one case where the analysis of the metabolite, fluorodeschloronorketamine (FDCNK), was not conducted due to insufficient sample, the FDCK (FDCNK) concentrations in blood found in the 6 cases were <0.002 (0.005), 0.002 (0.002), 0.002 (0.003), 0.02 (0.035), 0.87 (0.44) and 1.1 (not determined) μg/mL. Conclusions: With the drug seizures found with FDCK resembled in physical appearance with ketamine seizures, users might likely misuse it as ketamine. Though complicated by other drugs found, it is speculated that the two cases with higher concentration of FDCK found in blood (1.1 and 0.87 μg/mL) might have contributed to the impairment observed.

Colorimetric Detection of Fentanyl Powder on Surfaces Using a Supramolecular Displacement Assay.

Fentanyl is a potent synthetic opioid with an alarmingly low lethal dosage of 2 mg. The equipment necessary to detect fentanyl in field settings (e.g., hand-held spectrometers) is restricted to highly trained, well-funded, and specialized personnel. Established point-of-need technologies, such as lateral flow immunochromatographic strips, are available; however, they often involve multiple contact-based steps (e.g., collection, mixing) that pose a higher risk to users handling unknown substances. Herein, we developed a colorimetric displacement assay capable of contactless detection of fentanyl in liquid or solid samples. The basis of our assay relies on the presence of fentanyl to displace a redox mediator, ferrocene carboxylic acid, inclusively bound in the cavity of a supramolecular host, CB[7]. The displacement is only possible in the presence of high affinity binding guests, like fentanyl (KA ∼ 106 M-1). The liberated redox guest can then react with indicator reagents that are free in solution, producing either: (i) a distinct blue color to indicate the presence of fentanyl or (ii) a pale blue tint in the absence of fentanyl. We demonstrate rapid and specific detection of fentanyl free base and fentanyl derivatives (e.g., acetyl fentanyl and furanyl fentanyl) against a panel of 9 other common drugs of abuse (e.g., morphine, cocaine, and heroin). Furthermore, we highlight the intended use of this assay by testing grains of fentanyl derivatives on a surface with a drop (i.e., 25 μL) of the assay reagent. We anticipate that this approach can be applied broadly to identify the presence of fentanyl at the point of need.

Clinical laboratory techniques for detecting drugs of abuse: Feasibility in developing nations

Drugs of abuse are prescription, over-the-counter, or other forms of drugs that are often used for purposes other than those for which they are meant to be used, or in excessive amounts. The abuse of illicit drugs (for example cocaine, heroin, and codeine) poses a serious threat to public health, not to mention a great challenge to the health care system. It is important to review the techniques used in detecting these drugs of abuse to stay ahead of this growing global drug problem. This review aims to give an overview of known clinical laboratory techniques used in detecting some common drugs of abuse that could serve as an integral part of determining the presence of these drugs in bodily fluids and samples. Relevant literature was reviewed in various search engines (Google Scholar, PubMed, ScienceDirect, Bing). Findings showed that some of the current clinical laboratory techniques for detecting and quantifying drugs of abuse include Microcrystalline tests, thin-layer chromatography, colourimetric tests, immunoassays, urine dipstick tests, and ultraviolet spectroscopy. Apart from these known techniques, new and emerging techniques are being validated to serve as additions to these already existing techniques and be used in clinical settings. In conclusion, the burden of drug abuse is on the rise and becoming a public health concern. There should be an ever-increasing interest in developing new analytical methodologies not only to detect but also to quantify drugs of abuse which may be applied in a plethora of areas including clinical settings.Some of the outlined techniques are highly priced and for cost ineffectiveness, they may be difficult to afford and sustain in resource-restraint settings like developing nations. Hence, grants and laboratory infrastructural support may be needed from international donor agencies.

Monocyte chemoattractant protein-1 and cyclooxygenase enzymes in rats treated with drugs of abuse

The CC chemokine monocyte chemoattractant protein (MCP)-1/CCL2 and cyclo-oxygenase-2 enzyme are potent mediators of neuroinflammation. We studied the effect of treatment with Cannabis sativaresin extract and/or tramadol, two common drugs of abuse on the level of MCP-1 in brain and serum of rats. In addition, serum levels of cyclo-oxygenase 1 (COX-1) and cyclo-oxygenase 2 (COX-2) enzymes in rats given cannabis werealso determined. Cannabis sativaresin at 5, 10 or 20 mg/kg (expressed as delta-9-tetrahydrocannabinol), tramadol at 5, 10 or 20 mg/kg or tramadol at 10 mg/kg combined with cannabis at 5-20 mg/kg were given once a day, subcutaneously, for six weeks. Resultsshowed that, as compared to corresponding controls, Cannabis sativa resin produced a significant increase in MCP-1 increased inbrain tissue (29.8-56.4%) and serum (14.8-31.0%). MCP-1 showed also significant increments in brain (34.6%) and serum (22.1%) by 20 mg/kg tramadol. After the concomitant treatment with both cannabis and tramadol, MCP-1 increased in brainby 88.5-145.7%and in serum by 32.6-64.8%. Moreover, cannabis given at 20 mg/kg significantly increasedserum COX-1 and COX-2 by 362% and 58.8% compared to corresponding control values. The histopathological study of the striatum of cannabis-treated animals revealed the presence of apoptotic cells and pyknotic nuclei and congestion of blood vessels. The striatum from animals given tramadol showed neuronal perinuclear cytoplasmic vacuolations and apoptotic cells. Moreover, the combined administration of both cannabis and tramadol resulted in neuronal necrosis, vacuolation, apoptosis and pyknosis. Collectively, these results indicate that treatment with cannabis resin and/or tramadol is associated with a proinflammatory response that involves MCP-1 and cyclooxygenases and which could result in the development of brain injury.

Characterization of Emergency Department Poisoning Epidemiology in Belize

Objective: Little is known about the epidemiology of poisonings in Belize. The purpose of this study was to characterize poisonings presenting to Karl Heusner Memorial Hospital, Department of Accident and Emergency in Belize City, Belize. Methods: Charts were reviewed for patients with possible toxicologic exposures, presenting between May 31, 2016 – December 31, 2018. Variables of interest included patient age and sex, type and route of poisoning, reason for exposure, and disposition. Results: Of the 60,310 patient visits during the study period, 768 patients, 1.3%, presented after a toxicologic exposure. The age distribution of poisoned patients varied with the largest proportion being between 19-30 years of age, 29.3%. There was a male predominance of patients, 60.4%. Approximately 44% of all poisonings involved ethanol only. Marijuana was the most common drug of abuse. Other common inhalation and ingested exposures included cleaning agents, household agents, acetaminophen, NSAIDs, pesticides, organophosphates, sedating medications, antibiotics, and ciguatera poisoning. Nearly 48 % of intoxication cases were due to intentional abuse, while about eight percent were from suicide attempts. Of identified poisoned patients, 84.4% were ultimately discharged, 13.8% were admitted, 1.6% were “self-discharged,” 0.13% were transferred, and 0.13% died. Conclusions: This study illustrates that poisoning is an important cause of Accident and Emergency visits in Belize, whose patterns may differ from those in other countries. Understanding the current state of poisonings in Belize can aid in the development of treatment protocols, antidote stocking, and educational activities for poisoning prevention.

Comprehensive Toxicological Screening of Common Drugs of Abuse, New Psychoactive Substances, and Cannabinoids in Blood Using Supported Liquid Extraction and Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF-MS).

Immunoassay (IA) is currently the most widely used technique for toxicological screening in drug impaired driving investigations. However, practical limitations in the scope of testing, and the emergence of new psychoactive substances (NPS), have highlighted the need for alternative approaches, particularly mass spectrometry-based screening. High resolution mass spectrometry (HRMS) broadens the scope of testing to include NPS, and increases analyte specificity compared to IA. In addition, it provides a platform with increased flexibility and adaptability to incorporate emerging drugs of interest due to the transient drug market. In this study, a comprehensive screening procedure was developed to identify more than two hundred drugs of interest, including cannabinoids and NPS in whole blood. Supported liquid extraction (SLE) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOF-MS) using All Ions data acquisition was employed. The method was validated in accordance with published recommendations, and all compounds of interest were identified at recommended cutoffs for driving under the influence of drugs (DUID) investigations. Cannabinoids, including 11-nor-9-carboxy-Δ9-THC (THC-COOH), fentanyl analogs, buprenorphine, novel synthetic opioids (NSOs), and synthetic cannabinoids were identified at low to sub-ng/mL concentrations in whole blood using both positive and negative electrospray ionization (ESI) acquisition methods.

Experimental Evidence on Age-related Differential Outcomes Associated With Substance Abuse.

Growing evidence indicates that adolescent substance abuse is now an alarming concern that imposes a considerable socio-economic burden on societies. On the other hand, numerous studies have shown that due to specific neurophysiological features, the brain is more vulnerable to the adverse effects of psychoactive drugs at an early age. Unfortunately, these negative effects are not limited to the period of drug use, but can persistently affect the brain's responsiveness to future exposures to the same or other types of drug. For researchers to develop pharmacological strategies for managing substance abuse disorders, they need to gain a deep understanding of the differences in behavioral outcomes associated with each type of drug across different age groups. The present study was conducted to review the experimental evidence revealing the mentioned differential effects with an emphasis on common drugs of abuse, including cocaine, nicotine, cannabis, and opioids. Although the cellular mechanisms underlying age-related effects have not been exclusively addressed for each drug, the most recent results are presented and discussed. Future studies are required to focus on these mechanisms and reveal how molecular changes during brain development can result in differential responses to drugs at the behavioral level.

Evaluating the Reliability of Dipstick Drug Screens on Vitreous and Postmortem Blood as a Triage Modality in Forensic Pathology.

Dipstick drug screens are cheap, easy to use, and quick presumptive tests to detect common drugs of abuse. Dipsticks are designed for drug detection in urine. There is no literature regarding their potential use on fluids different from urine. The study aimed to determine the performance of dipstick screening tests on postmortem vitreous and blood specimens compared to urine dipsticks and final confirmatory toxicology analyses on blood. The study population included cases in which a complete toxicology analysis was performed. Each subject was screened for three substances: cocaine, fentanyl, and opiates. Dipstick results were checked by visual inspection. Results were compared with urine screening tests and quantitative, confirmatory toxicological analyses by gas chromatography/mass spectrometry on postmortem blood samples as the gold standards for screening and confirmatory analysis, respectively. There was a high number of false-negative results for opiates. Cocaine dipsticks in blood showed the highest reliability. Fentanyl dipsticks in vitreous showed a high number of false-negative results. Both vitreous and blood dipstick screening tests for all substances performed well on negative cases. When both blood and vitreous screening tests are negative, the chance that the confirmatory toxicology analysis will be positive is very low.

Frequency of new psychoactive substances in hair and urine samples of individuals subject to drug testing in driving license regranting-A toxicological perspective.

In recent years, numerous new psychoactive substances (NPS) have emerged on the illicit drug market. The assumed non-detectability of these drugs is often a key motivation for individuals subject to drug testing, such as those in driving license regranting programs. In these programs, NPS are not routinely tested for, and thus, subjects who have to prove abstinence from common drugs of abuse might switch to NPS to avoid positive drug tests. The aim of the study was to determine the frequency of these substances in the hair and urine samples of individuals undergoing drug testing in driving license regranting. A total of 1037 samples (577 hair and 460 urine samples) collected from 949 subjects between February 2017 and December 2018 were retrospectively analyzed for designer drugs and synthetic cannabinoids by liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS). For a more sensitive analysis of synthetic cannabinoids and their metabolites, additional testing was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Overall, 42 hair and two urine samples, which were obtained from 40 subjects, tested positive for NPS resulting in a frequency of 4.2%. While synthetic cannabinoids were detected in all cases, designer drugs were only found in three of these cases. With regard to the 577 hair samples analyzed, 7.3% screened positive, whereas only 0.4% of the 460 tested urine samples contained NPS. The results of this study indicate that synthetic cannabinoid use seems to be popular among this population, and therefore, testing for synthetic cannabinoids should be requested more often preferably using hair analysis.

Qualitative Confirmation of 94 New Psychoactive Substances and Metabolites in Urine Using Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry.

Numerous methods and techniques have been published for the identification of new psychoactive substances (NPS) and their metabolites in urine. However, there lacks a holistic approach to analyze different groups of NPS and their metabolites with decision points for reporting their use. In this study, data-dependent acquisition workflow using liquid chromatography--quadrupole time-of-flight mass spectrometry was developed and validated for the identification of a total of 94 NPS and metabolites in urine using the established decision points. The limit of identification for all analytes was determined at 25% below their respective decision points. The method was demonstrated to be accurate and precise at their respective decision points with extraction recoveries and ion suppression/enhancement ranging from 51.0% to 103.5% and -81.6% to 159.1%, respectively. There was no observed carryover up to 200 ng/mL for all analytes and no interferences from urine matrixes, internal standards and other common drugs of abuse. The extracted drug analytes were stable at 4 and 15°C for up to 3 days. The validated method was successfully evaluated and applied in the testing of urine samples from NPS users. In conclusion, this validated method can analyze a wide range of NPS and their metabolites with the use of decision points for consistency in reporting.

Acute Intoxication by Bisoprolol and Drowning: Toxicological Analysis in Complex Suicides

In complex suicides, more than one suicide method is applied at the same time or one after the other. The most common complex suicide includes the ingestion of drugs combined with drowning. A case of acute intoxication by Bisoprolol and drowning is reported. The dead body of a 40-year-old woman was discovered on a river side, soon after her husband found a suicide note at home. In the woman’s vehicle four empty boxes of Bisoprolol, a widely used beta blocker, were also found. Main autopsy findings were consistent with drowning and represented by plume of froth at the mouth and nostrils with frothy fluid also in the airways, water into the stomach, and a remarkable pulmonary edema as a result of fluid aspiration. Toxicological analyses were performed on peripheral blood, urine and gastric content samples using liquid and gas chromatography, coupled with mass spectrometry. Toxicological results were negative for ethanol and other common drugs of abuse. High levels of bisoprolol were found in blood (7.54 mg/L), far exceeding the therapeutic range, in the urine (1.14 mg/L), and gastric content (13.12 mg/L). Bisoprolol intoxication was assessed as a relevant contributing condition to the immediate cause of death represented by drowning. Although Bisoprolol would certainly have a heart-depressing effect, it is not possible to determine if the victim fell unconscious or if she simply collapsed into the water with a secondary drowning.

Recent advances in the detection of drugs of abuse by dried blood spots.

The purpose of this review study was to sum up the main recent advances reported in the scientific literature about the detection of common drugs of abuse in biological samples upon their collection by dried blood spot devices. The most recent, innovative and fully validated methods for the qualitative and/or quantitative detection of common drugs of abuse are reported, including alprazolam, clonazepam, diazepam, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methyl-enedioxyethylamphetamine, amphetamine, methamphetamine, cocaine, tetrahydrocannabinol, 6-monoacetylmorphine, morphine, codeine, hydromorphone, hydrocodone, oxycodone, noroxycodone, tramadol, methadone, buprenorphine, fentanyl, ketamine and their respective metabolites and γ-hydroxybutyric acid. Dried blood spot proved to be extremely promising for routine analysis of forensic cases, although large-scale experiments on real samples need to be performed to confirm the emerging advantages of the technique and remove the potential limitations still affecting its widespread application.

Phospholipid metabolites of GHB as potential biomarkers in whole blood: Synthesis, analytics, and in vitro formation of homolog 16:0/18:1.

Gamma-hydroxybutyric acid (GHB) is a common drug of abuse, and the detection of a consumption or administration is a longstanding research objective in clinical and forensic toxicology. However, until now, the short detection window of GHB could not be enlarged by the use of GHB metabolites. Therefore, new biomarkers for the detection of a GHB intake are needed. In analogy to phosphatidylethanols as long-time biomarkers of ethanol, phospholipids with GHB might represent a promising compound class. While the availability of reference compounds often represents a bottleneck in clinical and forensic toxicological research, two phospholipids-phosphatidyl-GHB (16:0/18:1) and its isomer phosphatidyl beta-hydroxybutyric acid (16:0/18:1)-were successfully synthesized by a new highly versatile synthetic route. Structural characterization data, together with 1 H-, 13 C-, and 31 P-NMR and high-resolution mass spectrometry (HRMS) spectra, are reported. Subsequently, a HPLC-MS/MS method was established for the determination of both compounds (limits of detection [LOD] ≤ 2ng/ml), and the formation of these metabolites was investigated in two in vitro experiments. The formation of phosphatidyl-GHB (16:0/18:1) was observed in an incubation experiment by converting phosphatidylcholine (16:0/18:1) and GHB with phospholipase D and in whole blood samples spiked with 50 mM GHB, respectively. Therefore, phosphatidyl-GHB (16:0/18:1) might represent a valuable new metabolite of GHB with the potential for an extension of the detection window as GHB biomarker.

An overview of the SAMPL8 host–guest binding challenge

The SAMPL series of challenges aim to focus the community on specific modeling challenges, while testing and hopefully driving progress of computational methods to help guide pharmaceutical drug discovery. In this study, we report on the results of the SAMPL8 host–guest blind challenge for predicting absolute binding affinities. SAMPL8 focused on two host–guest datasets, one involving the cucurbituril CB8 (with a series of common drugs of abuse) and another involving two different Gibb deep-cavity cavitands. The latter dataset involved a previously featured deep cavity cavitand (TEMOA) as well as a new variant (TEETOA), both binding to a series of relatively rigid fragment-like guests. Challenge participants employed a reasonably wide variety of methods, though many of these were based on molecular simulations, and predictive accuracy was mixed. As in some previous SAMPL iterations (SAMPL6 and SAMPL7), we found that one approach to achieve greater accuracy was to apply empirical corrections to the binding free energy predictions, taking advantage of prior data on binding to these hosts. Another approach which performed well was a hybrid MD-based approach with reweighting to a force matched QM potential. In the cavitand challenge, an alchemical method using the AMOEBA-polarizable force field achieved the best success with RMSE less than 1 kcal/mol, while another alchemical approach (ATM/GAFF2-AM1BCC/TIP3P/HREM) had RMSE less than 1.75 kcal/mol. The work discussed here also highlights several important lessons; for example, retrospective studies of reference calculations demonstrate the sensitivity of predicted binding free energies to ethyl group sampling and/or guest starting pose, providing guidance to help improve future studies on these systems.

Cardiac Complications of Common Drugs of Abuse: Pharmacology, Toxicology, and Management.

Cardiovascular complications from drugs of abuse are becoming more apparent because of increased usage worldwide. Substance abuse can cause acute and chronic cardiovascular complications and is increasing in prevalence especially in young adults. These substances contribute to the development of acute coronary syndrome, type 2 myocardial injury, arrhythmias, and cardiomyopathies, and have numerous other cardiovascular complications. Although no screening guidelines exist, clinical awareness of these potential complications and their prevention, clinical presentation, diagnosis, and treatment are critically important. Management of cardiovascular disease should be coupled with appropriate social and mental health interventions to provide sustained clinical benefit. The higher the number of substances used recreationally, the greater the risk of premature heart disease. Epidemiological studies showed that 1 in 5 young adults misuse several substances and often start using at younger ages with a greater risk for adverse health outcomes over the long term. The aim of this review is to highlight the basic epidemiology, cardiac complications, and disease-specific treatment options of commonly abused substances including methamphetamine, cocaine, alcohol, anabolic-androgenic steroids, cannabis, and tobacco.

Detected drugs in homicides: A tale of two cities – Chicago and Detroit

To compare the drugs observed in homicides in two cities in the American Mid-West. We looked at drugs detected in two major American cities in the Mid-West in 2020 to determine which drugs were most commonly associated with homicides in recent years. All cases that were reported as homicides (by whatever means) in 2020 in Cook County (Chicago, Illinois) and Wayne County (Detroit, Michigan) were included. For purpose of this study, only common drugs of abuse were included since frequently comprehensive toxicology testing is not performed in homicide cases. Only drugs with a reported incidence of > 1.0% were included. Toxicology testing for drugs consisted of immunoassay screening followed confirmation testing by either LC-MS/MS or GC-MS. Ethanol was screened using dual-column headspace GC-FID with confirmation by single column headspace GC-FID on a second aliquot. For the 986 homicide cases (rate = 19K/100K) in Cook County the most common drugs were cannabinoids (60%), ethanol (28%), methylenedioxymethamphetamine (MDMA, 12%), cocaine (benzoylecgonine 7.7%, cocaine, 4.8%, cocaethylene 2.0%), fentanyl (6.8%), methamphetamine (5.1%), morphine (3.2%), midazolam (2.3%), phencyclidine (2.3%), alprazolam (1.7%), oxycodone (1.4%), 6-acetylmrophine (1.4%) and methadone (1.2%). For the 441 homicide cases (rate = 25K/100K) in Wayne County the most common drugs were cannabinoids (52%), ethanol (29%), cocaine (benzoylecgonine 9.1%, cocaine 5.7%, cocaethylene 2.0%), fentanyl (8.8%), oxycodone (7.2%), methamphetamine (5.0%), morphine (4.8%), midazolam (2.3%), 6-acetylmorphine (1.8%), alprazolam and hydrocodone (1.6% each), and lorazepam and nordiazepam (1.4%, each). A review of the results demonstrated some similarities as well as some distinct differences between the Cook and Wayne County cohorts. Notably, the most common stimulant drug observed in Cook County homicides cases was MDMA ( n = 122) compared to only a single observation in Wayne County homicides. Other stimulant drugs – cocaine and methamphetamine – were similar in both counties. Historically CNS depressants were less commonly observed in homicides than stimulants, but with the ubiquitous abuse of fentanyl it was not surprising to see a considerable number of homicide cases positive for that drug. The hallucinogen, Phencyclidine, was also observed in 23 Cook County homicides and with no observations in Wayne County. It has been noted that the use of a stimulant drug with ethanol can cause behavioral effects of both the stimulant drug and the disinhibitive effects of ethanol leading to behavior that may put the individual at greater risk for homicide. Stimulants combined with ethanol were detected in 101 (10%) of Cook County homicides, but only 16 (3.6%) of Wayne County homicides. Multiple stimulant drugs were detected in 30 Cook County and 6 Wayne County homicides. While the homicide rate in Cook County was lower than in Wayne County, the number of homicides was more than double, possibly associated with the greater use of stimulant drugs. Drugs of abuse are frequently associated with homicide cases, but the drugs may vary regionally. Notably in the Chicago the presence of MDMA and PCP was frequently observed while this was not the case in Detroit. Historically, cocaine was observed in around 30% of homicides in urban areas. While cocaine is still frequently associated with homicides, other stimulant drugs such as methamphetamine and, regionally, MDMA and PCP are now often detected.

Automating DUID methods using robotics for rapid high throughput sample preparation – Section 4 and Section 5A UK Road Traffic Act Compounds in Whole Blood

To fully automate a 96-well plate sample preparation technique using liquid handling robotics for the purpose of significantly increasing sample throughput for whole blood samples taken under the UK Road Traffic Act (RTA). Introduction: demand for analysis of whole blood samples taken under the Road Traffic Act 1988 has increased in recent years due to UK Policing and Government policy on tackling driving under the influence of drugs (DUID). Previous extraction techniques utilised by our laboratory include manual or semi-automated approaches that are lengthy and labour intensive. Both approaches require time consuming human input, to precipitate blood and prepare calibrators and quality controls. Recent developments have sped up chromatographic methods through use of techniques such as ultra-high performance liquid chromatography. However, there has been little progress with speeding up sample preparation and extraction processes, particularly for whole blood to compliment increased LC-MS/MS capacity. Use of 96-well plate formats for protein precipitation and extraction provide a platform that can be readily coupled to automated liquid handling (ALH) instruments. This provides a solution to the increase in demand by allowing for extraction of more samples, with minimal human input, in a much shorter amount of time. Here we present development, validation and implementation of an automated robotic sample preparation method for whole blood for analysis of drugs in RTA cases. Aliquots (200 μL) of blank blood and casework samples were dispensed into a 96-well plate using a robotic pipetting system before being loaded onto a Hamilton ALH. Casework samples and blank blood were spiked with internal standard. Additional blank blood samples were further spiked to produce calibrants and quality controls. The plate was mixed with the addition of a cell lysis solution and the aliquots transferred to a protein/lipid depletion (PPT) plate. A positive pressure manifold was used to draw the solution through the PPT plate into a collection plate where the precipitate was dried. The precipitate was reconstituted and loaded by the Hamilton onto a microelution SPE plate. Following extraction, the samples were reconstituted and the collection plate placed in the LC-MS/MS autosampler for analysis. The LC-MS/MS method consists of a 2.1 mm × 30 mm, 1.7 μm, C18 column running a 4 minute gradient of water and methanol with 0.1% formic acid. Quantitation of 34 compounds (including common drugs of abuse and their metabolites) was successfully validated with CVs ranging from 3.0%–9.0% and accuracies of 85.8%–111.7%. Within batch precision had CVs of < 10.0%. The robotic platform was able to undertake sample pre-treatment and extraction of 40 case samples in 3.5 hours. The method was implemented into casework following UKAS accreditation and in 2021, 12,400 samples were analysed using the method. Introduction of this method to our laboratory has resulted in a significant increase in speed for a sample to be processed and results obtained. Each ALH is capable of extracting two batches per day; equating to 160 case sample versus 28 cases on semi-automated methods. This, along with a reduced sample volume requirement and reduced solvent consumption has had a very beneficial impact in terms of cost, turnaround times and ability to use low sample volume cases. Full automation of sample preparation of whole blood samples coupled with high speed chromatographic techniques can significantly increase throughput, decrease turnaround times and reduce solvent consumption.