Drugs Of Abuse Research Articles

Dietary regulation of silent synapses in the dorsolateral striatum

Obesity and drugs of abuse share overlapping neural circuits and behaviors. Silent synapses are transient synapses that are important for remodeling brain circuits. They are prevalent during early development but largely disappear by adulthood. Drugs of abuse increase silent synapses during adulthood and may facilitate reorganizing brain circuits around drug-related experience, facilitating addiction and contributing to relapse during treatment and abstinence. Whether obesity causes alterations in the expression of silent synapses in a manner similar to drugs of abuse has not been examined. Using a dietary-induced obesity paradigm, mice that chronically consumed high fat diet (HFD) exhibited increased silent synapses in both direct and indirect pathway medium spiny neurons in the dorsolateral striatum. Both the time of onset of increased silent synapses and their normalization upon discontinuation of HFD occurs on an extended time scale compared to drugs of abuse. These data demonstrate that chronic consumption of HFD, like drugs of abuse, can alter mechanisms of circuit plasticity likely facilitating neural reorganization analogous to drugs of abuse.

English

Introduction: Addressing the health problems of prison inmates is a major challenge for public administrations, which are responsible for guaranteeing the necessary services to deal with them. It is therefore essential to know which are the chronic pathologies that affect them most frequently, what impact they have on the oral cavity and what type of treatments are required. The aim of this study is to shed further light on these questions. Material and method: Articles in PubMed on chronic and oral pathologies and treatment needs were reviewed. Results: The most prevalent chronic pathologies are heart disease, chronic respiratory disease, diabetes and cancer. Anxiety and depression are also highly prevalent among the prison population, along with the consumption of tobacco, alcohol and drugs of abuse. They are responsible for the appearance of caries and periodontal diseases, the treatment of which should be carried out in the prison environment. Conclusions: The level of dental care among the prison population is very low. Therefore, it is necessary to implement preventive and therapeutic measures among inmates to improve their oral health and, consequently, their general health.

Validation of an LC-MS/MS Method for Analysis of 58 Drugs of Abuse in Oral Fluid and Method Comparison with an Established LC-HRMS Method.

Liquid chromatography-mass spectrometry (LC-MS) methods for detection of multiple drugs of abuse (DoA) in oral fluid (OF) samples are being implemented in many clinical routine laboratories. Therefore, there is a need to develop new multi-analyte methods with simple sample pre-treatment and short analysis times. The purpose of this work was to validate a method detecting 58 DoA to be used with two different OF sampling kits, the saliva collection system (SCS) from Greiner Bio-One and Quantisal from Immunalysis, using the same sample pretreatment and analytical method. A set of 110 samples collected with the SCS kit was further compared to an LC-HRMS (high resolution mass spectrometry) method in another laboratory. The method was successfully validated, with precision and accuracy of ≤15% and z-scores of <2 for external controls. Using a sensitive LC-MS/MS instrument, the detection limits were <1 µg/L in neat oral fluid. In the comparative study between the LC-MS/MS and LC-HRMS methods using SCS samples, a good agreement was observed. Discrepancies were limited to lower concentration ranges, attributable to differences in cut-off thresholds between the methods. This work contributes to the development of LC-MS multi-analyte methods for OF samples, which are suitable for clinical routine laboratories.

A Review of Drug Abuse, Misuse, and Related Laboratory Challenges.

Various definitions can be considered for drugs and substance abuse. According to the National Institute on Abuse, the use of an over-the-counter drug in a different way than that prescribed to experience or arouse emotion is a simple form of drug abuse. The World Health Organization (WHO) also defines drug abuse as the persistent or sporadic use of drugs that are incompatible or unrelated to acceptable medical practice. With the increasing non-therapeutic use of prescription drugs, serious related consequences have also increased. Therefore, there is a need to know more precisely about the types of substances and drug abuse, which is the most important part of diagnosis and recognizing the tests that cause false positive and negative results. The purpose of this review article is to collect and summarize the most important and more common types of drugs of abuse and review the drugs that cause false results in screening tests. In addition, the most common detection methods of the drug will be reviewed and the advantages and drawbacks of each method will be discussed. In this article, we aimed to point out all the facts about the emerging problems in drug abuse, the methods of screening, and the possible false results in addition to troubleshooting strategies.

Effects of oxycodone on placental lineages: Evidence from the transcriptome profile of mouse trophoblast giant cells

Pregnant women are often prescribed or abuse opioid drugs. The placenta is likely the key to understanding how opioids cause adverse pregnancy outcomes. Maternal oxycodone (OXY) exposure of pregnant mice leads to disturbances in the layer of invasive parietal trophoblast giant cells (pTGC) that forms the interface between the placenta and uterus. These cells are analogous to extravillous trophoblasts of the human placenta. They are crucial to coordinating the metabolic needs of the conceptus with those of the mother and are primary participants in the placenta-brain axis. Their large nuclear size, however, has precluded both single-cell (sc) and single-nucleus (sn) RNA-seq analyses beyond embryonic day (E) 8.5. Here, we compared the transcriptomes of placentas from pregnant mice exposed to OXY with unexposed controls at E12.5, with particular emphasis on the pTGC. The nonfluidic Parse snRNA-seq approach permitted characterization of the nuclear transcriptomes of all the major placental cell lineages and their presumed progenitors at E12.5. OXY exposure had a negligible effect on components of the placental labyrinth, including the two syncytial cell layers, but caused transcriptomic changes consistent with metabolic stress throughout the spongiotrophoblast. Most notably, there was loss of the majority of pTGC, whose normal gene expression is consistent with elevated energy demand relating to biosynthesis of multiple secretory products, especially hormones, and endoduplication of DNA. This unusual sensitivity of pTGC presumably puts the pregnancy and future health of the offspring at particular risk to OXY exposure.

Prenatal Opioid and Alcohol Exposures: Association with Altered Placental Serotonin Transporter Structure and/or Expression.

Fetal exposures to many drugs of abuse, e.g., opioids and alcohol (EtOH), are associated with adverse neurodevelopmental problems in early childhood, including abnormalities in activity of the serotonin (5HT) transporter (SERT), which transports 5HT across the placenta. Little is known about the effects of these drugs on SERT expression. Pregnant women who used EtOH or opioids were compared to gestational age-matched controls using a structured questionnaire to determine prenatal substance exposure. Following elective pregnancy termination, placental membranous vesicles and exosomes were prepared from first and second trimester human placentas. Changes in EtOH- or opioid-exposed placental SERT expression and modifications were assessed by quantitative western blot. Novel SERT isoforms were sequenced and analyzed. Opioid-exposed but not EtOH-exposed maternal placentas showed SERT cleavage and formation of new SERT fragments (isoforms). Alcohol-exposed cases showed reduced SERT levels. Antibodies to the N-terminal SERT region did not recognize either of the two cleavage products, while antibodies to the central and C-terminal regions recognized both bands. The secondary band seen in the opioid group may represent a hypophosphorylated SERT fragment. These changes in SERT modifications and expression may result in altered fetal brain serotonergic neurotransmission, which could have neurodevelopmental implications.

Ruthenium(II) N-heterocyclic carbene polymer based sensors for detection of predatory drugs like ketamine and scopolamine.

The current demand in the field of abusive drug research is to have a highly sensitive and rapid method for detecting ketamine and scopolamine, which are frequently used in drug-facilitated sexual assaults administered via beverages and food. We report here the first electrochemical sensors of N-heterocyclic carbene (NHC) coordinated ruthenium organometallic 1-dimensional polymers and their multi-walled carbon nano tube (MWCNT)-based composite for detecting ketamine and scopolamine. The preparation of ruthenium NHC organometallics and the MWCNT composite as sensors offers significant advantages for electrochemical applications, with enhanced sensitivities of 121.979 and 3.273 μA μM-1 cm-2 for ketamine and scopolamine, respectively, and selectivity in sensing applications. The complex-carbon composite sensor has a low limit of detection i.e., 0.194 and 3.18 nM for ketamine and scopolamine identification, respectively. Alongside, the selectivity of the composite sensor was evaluated in the presence of other blood constituents, and the study evidenced remarkable discernment towards the title drugs. Furthermore, real-time analyses using the composite sensor demonstrated quantitative identification of ketamine and scopolamine. Therefore, this innovation has the potential to provide valuable tools for forensic investigations and address the urgent need for real-time detection of date rape drugs. This contribution demonstrates a robust proof-of-concept that emphasizes the importance of creating non-enzymatic, environmentally friendly, and cost-effective sensors for on-site sensing applications as point-of-care devices.

NeuropsychopharmARCology: Shaping Neuroplasticity through Arc/Arg3.1 Modulation

Activity-regulated cytoskeletal associated protein (aka activity-regulated gene Arg3.1) belongs to the effector gene family of the immediate early genes. This family encodes effector proteins, which act directly on cellular homeostasis and function. Arc/Arg3.1 is localized at dendritic processes, allowing the protein local synthesis on demand, and it is considered a reliable index of activitydependent synaptic changes. Evidence also exists showing the critical role of Arc/Arg3.1 in memory processes. The high sensitivity to changes in neuronal activity, its specific localization as well as its involvement in long-term synaptic plasticity indeed make this effector gene a potential, critical target of the action of psychotropic drugs. In this review, we focus on antipsychotic and antidepressant drugs as well as on psychostimulants, which belong to the category of drugs of abuse but can also be used as drugs for specific disorders of the central nervous system (i.e., Attention Deficit Hyperactivity Disorder). It is demonstrated that psychotropic drugs with different mechanisms of action converge on Arc/Arg3.1, providing a means whereby Arc/Arg3.1 synaptic modulation may contribute to their therapeutic activity. The potential translational implications for different neuropsychiatric conditions are also discussed, recognizing that the treatment of these disorders is indeed complex and involves the simultaneous regulation of several dysfunctional mechanisms.

The Association Between Dextromethorphan/Bupropion with Alcohol and Substance Misuse: Reports to the Food and Drug Administration Adverse Event Reporting System (FAERS).

Dextromethorphan/bupropion (DXM/BUP) received Food and Drug Administration (FDA) approval for the treatment of adults with major depressive disorder (MDD) in August 2022. This combination is not known to have abuse liability and is not currently scheduled by the Drug Enforcement Administration (DEA). Notwithstanding, dextromethorphan is a drug of abuse. Herein, we sought to determine whether DXM/BUP has alcohol and/or substance misuse liability. We evaluated spontaneous reports of terms such as "alcohol problem, alcoholism, alcohol abuse, substance dependence, substance use disorder (SUD), substance abuse, drug dependence, drug use disorder and drug abuse" in the FDA Adverse Event Reporting System (FAERS). The FAERS is a spontaneous reporting database of adverse events submitted to the FDA. We performed a comparative assessment of the alcohol and/or substance misuse liability of DXM/BUP since its market authorization in August 2022, using acetaminophen as the control. Dextromethorphan served as the upper-bound reference point. Our findings showed that, since August 2022, dextromethorphan had a significant reporting odds ratio (ROR) for "drug abuse." In contrast, DXM/BUP did not have a significant ROR for any of the categories of alcohol and/or substance misuse evaluated. Limitations of our findings derive largely from the limitations of the FAERS and its data capture method. The absence of alcohol or substance misuse reported to the FAERS with DXM/BUP accords with the lack of evidence of abuse liability prior to FDA approval and its non-scheduling by the DEA.

Prior cocaine use diminishes encoding of latent information by orbitofrontal, but not medial, prefrontal ensembles.

Maladaptive decision-making is a hallmark of substance use disorders (SUDs), though how drugs of abuse alter neural representations supporting adaptive behavior remains poorly understood. Past studies show that the orbitofrontal (OFC) and prelimbic (PL) cortices are important for decision-making, tracking both task-relevant and latent information. However, previous studies have focused on how drugs of abuse impact the firing rates of individual units. More work at the ensemble level is necessary to accurately characterize potential drug-induced changes. Using single-unit recordings in rats during a multidimensional decision-making task and then applying population- and ensemble-level analyses, we show that prior use of cocaine altered the strength and structure of task-relevant and latent representations in the OFC, changes relatable to suboptimal decision-making in this and perhaps other settings. These data expand our understanding of the neuropathological underpinnings of maladaptive decision-making in SUDs, potentially enabling enhanced future treatment strategies.

Client and program-level factors associated with planned use of medications for opioid use disorder in specialty substance use treatment programs: Evidence from linked administrative data and survey data

BackgroundMost patients in specialty drug treatment programs that are not federally licensed Opioid Treatment Programs (OTPs) programs do not receive medications for opioid use disorder (MOUD). MethodsWe linked results from a survey of non-OTP treatment program directors in New Jersey (n = 81) to statewide administrative records of admissions for opioid use to those programs between July 2021–June 2022. Using multi-level regression, we examined the association of three types of factors with planned MOUD use: program survey responses, client-level factors, and program-level client characteristic mix. ResultsOf 9583 opioid treatment admissions in non-OTP settings, 41 % included treatment plans involving MOUD. Programs where directors reported staff concerns about buprenorphine's efficacy or diversion had a lower proportion of clients with planned MOUD, as did programs reporting too little physical space to prescribe. Being self-referred to treatment, unemployed and not looking for work, aged 30–49, heroin use (vs. prescription opioid use), and stimulant use in addition to opioids, were positively associated with planned MOUD; while non-Medicaid insurance, and Black and Hispanic race/ethnicity, were negatively associated with planned MOUD. Clients were more likely to have planned MOUD if their programs had a higher proportion of clients aged 30 or older, heroin as primary “drug of abuse,” stimulant use, and not working but actively looking for work. ConclusionFindings suggest addressing program staff attitudes toward buprenorphine could help increase planned MOUD. There is also a need to improve access for clients with non-Medicaid insurance, address within-program race and ethnic disparities, and address employment-related barriers to medication.

Evaluation of the efficacy of preventive therapy in chronic migraine with or without medication overuse headache: data from a prospective study

Chronic migraine (CM) affects three out of every hundred people. As the frequency of attacks increases, so does the frequency of painkillers usage, and 85% of patients with CM are also diagnosed with medication overuse headache (MOH).Objective: to investigate the efficacy of preventive therapy of CM with or without MOH.Material and methods. The study involved 117 patients with CM and MOH (group 1) and 23 patients with CM without MOH (group 2), patients were examined at the initial visit and after 3 and 9 months of follow-up. All patients in the 1st group (CM+MOH) received a 7-day "detoxification" therapy. Patients in both groups were given an educational and information session on the type of their diagnosis, treatment methods and lifestyle correction. The choice of a preventive treatment in both groups was based on the current clinical recommendations, algorithms of modern international guidelines, concomitant pathology, the presence of contraindications in a particular patient, tolerability, as well as territorial and economic factors.Results. A positive effect was found in both groups, regardless of the presence of MOH. The lack of significant differences between the groups of CM with/without MOH is probably due to the use of "detoxification" therapy and the cessation/restriction of the use of the drug of abuse in all patients with CM and MOH, although a more pronounced positive dynamics is observed in patients of the 2nd group (CM without MOH), which in turn emphasizes the severity of the disease, the greater disability and the poorer prognosis for response to preventive treatment in the presence of MOH. It was found that one third of patients in the study favored monoclonal antibodies (mAbs) due to convenience, rapid onset of action and tolerability. In the group of patients with MOH, the best results were obtained when using injection methods of treatment (anti-CGRP mAbs, botulinum therapy) or when using combined therapy compared to monotherapy with venlafaxine or topiramate.Conclusion. All first-line drugs for the CM prophylactic treatment according to the clinical guidelines of the Ministry of Health of Russia are clearly effective, including cases with both CM and MOH. The use of combined therapy in CM with/without MOH can be effective even with a low initial response to one of the monotherapies.

Cocaine taking and craving produce distinct transcriptional profiles in dopamine neurons.

Dopamine (DA) signaling plays an essential role in reward valence attribution and in encoding the reinforcing properties of natural and artificial rewards. The adaptive responses from midbrain dopamine neurons to artificial rewards such as drugs of abuse are therefore important for understanding the development of substance use disorders. Drug-induced changes in gene expression are one such adaptation that can determine the activity of dopamine signaling in projection regions of the brain reward system. One of the major challenges to obtaining this understanding involves the complex cellular makeup of the brain, where each neuron population can be defined by a distinct transcriptional profile. To bridge this gap, we have adapted a virus-based method for labeling and capture of dopamine nuclei, coupled with nuclear RNA-sequencing, to study the transcriptional adaptations, specifically, of dopamine neurons in the ventral tegmental area (VTA) during cocaine taking and cocaine craving, using a mouse model of cocaine intravenous self-administration (IVSA). Our results show significant changes in gene expression across non-drug operant training, cocaine taking, and cocaine craving, highlighted by an enrichment of repressive epigenetic modifying enzyme gene expression during cocaine craving. Immunohistochemical validation further revealed an increase of H3K9me3 deposition in DA neurons during cocaine craving. These results demonstrate that cocaine-induced transcriptional adaptations in dopamine neurons vary by phase of self-administration and underscore the utility of this approach for identifying relevant phase-specific molecular targets to study the behavioral course of substance use disorders.

Enhancing the electrochemical performance of graphite sheet electrodes for ketamine detection

Flexible graphite sheets (GS) have been a promising material for developing electrochemical sensors. In this work, we showed that the electrochemical activation protocol (+1.4 V followed by -1.0 V vs. Ag|AgCl|KCl(sat.) both at 200 s) in alkaline medium (0.5 mol l-1 NaOH) enhances the electrochemical activity of GS. Before the treatment, cyclic voltammetric profile of inner-sphere [Fe(CN)6]3-/4- redox couple at 50 mV s-1 exhibited an ill-defined voltammetric profile (ΔEp = 800 mV) while an increase in the reversibility was achieved (ΔEp = 225 mV) after the treatment. Additionally, the lower charge transfer resistance (Rct) measured by Electrochemical Impedance Spectroscopy (EIS) and a higher heterogeneous electron transfer constant (k0) corroborate with the enhancement in the electrochemical performance of treated GS electrodes. The SEM images revealed an irregular pattern with number of grooves, indicating a partially surface exfoliation and AFM analysis displayed an increase in the roughness. These findings agreed with the estimation of electroactive area which increased 3-times after the treatment. As proof of concept, ketamine (KET), an anesthetic often used as drug of abuse, was determined in synthetic saliva and veterinary pharmaceutical samples using treated GS electrodes and square wave voltammetric measurements. Linear range of 5.0–100.0 µmol l-1 with a limit of detection value of 2.1 µmol l-1 was obtained for KET. Appropriate recovery values (∼105 %) for the analysis of samples were achieved.

Post-mortem human brain analysis of the ventral pallidum in alcohol use disorder

ObjectiveAlcohol use disorder (AUD) is characterised by cycles of alcohol misuse, abstinence, and relapse. The neurobiology of AUD strongly implicates the role of the ventral pallidum (VP) in a variety of drugs of abuse, including alcohol. Pre-clinical studies have demonstrated critical role of parvalbumin and calretinin neurons in the VP in modulating relapse, mood, and motivation. However, there are a limited studies examining the VP at the cellular level in AUD in humans. MethodPost-mortem human brain tissue of AUD (n = 11), remission (n = 6), and control brains (n = 12) were processed for immunohistochemistry to examine the presence and changes in parvalbumin and calretinin neurons in the VP. ResultsSimilar to pre-clinical rodent models, parvalbumin and calretinin neurons were present in the VP, although no significant difference was found in their number or morphology across all AUD, remission, and control brains. ConclusionThe presence of parvalbumin and calretinin neurons in the VP was confirmed across all groups. This is particularly important as it supports the translatability of previous animal studies regarding the role of parvalbumin and calretinin neurons in AUD, and thus further implicates the VP in the neurobiology of AUD in humans. As there are no distinctions in the number or morphology of these neurons, their significance likely lies in their activity. The presence of both parvalbumin and calretinin in humans, particularly in both control and AUD cases supports translational capacity from preclinical findings is more feasible.

Exploring Cerebrospinal Fluid: Validation of a New Method for Quantification of 39 Drugs of Abuse by LC-MS/MS

Exploring Cerebrospinal Fluid: Validation of a New Method for Quantification of 39 Drugs of Abuse by LC-MS/MS

A five-year retrospective review of fatalities involving novel psychoactive substances in Southern Ireland

Introduction: Novel psychoactive substances (NPS) are rapidly emerging and being reformulated to evade legislative controls, creating unpredictability in drug markets and ineffective drug policies. Given that Ireland has the highest self-reported NPS use within Europe and literature regarding health risks is lacking, the National Advisory Committee on Drugs has advocated for the surveillance of regional trends. Objectives: To elucidate the demographic and autopsy findings of fatal cases involving NPS in Southern Ireland as compared to deaths involving traditional drugs of abuse (TDOA). Methods: Post-mortem reports from the Cork-Kerry region with positive toxicology for illicit substances between 2012-2016 were retrospectively analyzed to compare NPS and TDOA cases with respect to circumstances surrounding death, toxicological results, and pathological findings. Results: Between 2012-2016, there were 164 cases involving illicit substances in the Cork-Kerry region. NPS accounted for 17 (10.4%) cases, with an average annual mortality rate of 34.8 per 100,000 population. NPS contributed to the cause of death in 70.6% of cases where detected, compared to 43.5% for TDOA only. In both cohorts, fatal abusers were predominantly young males. Importantly, cases involving NPS showed a higher proportion of mono-drug intoxication and presentation to hospital prior to death. Autopsy findings were non-specific but commonly featured pulmonary congestion, aspiration, and cerebral oedema. Conclusion: NPS are detected in a small proportion of medicolegal autopsies but contribute significantly to acute intoxication deaths. This study highlights the need for effective drug monitoring and enforcement strategies, along with improved management of drug toxicities presenting to hospital.

Cell type-specific epigenetic priming of gene expression in nucleus accumbens by cocaine.

A hallmark of addiction is the ability of drugs of abuse to trigger relapse after periods of prolonged abstinence. Here, we describe an epigenetic mechanism whereby chronic cocaine exposure causes lasting chromatin and downstream transcriptional modifications in the nucleus accumbens (NAc), a critical brain region controlling motivation. We link prolonged withdrawal from cocaine to the depletion of the histone variant H2A.Z, coupled with increased genome accessibility and latent priming of gene transcription, in D1 dopamine receptor-expressing medium spiny neurons (D1 MSNs) that relate to aberrant gene expression upon drug relapse. The histone chaperone ANP32E removes H2A.Z from chromatin, and we demonstrate that D1 MSN-selective Anp32e knockdown prevents cocaine-induced H2A.Z depletion and blocks cocaine's rewarding actions. By contrast, very different effects of cocaine exposure, withdrawal, and relapse were found for D2 MSNs. These findings establish histone variant exchange as an important mechanism and clinical target engaged by drugs of abuse to corrupt brain function and behavior.

B-282 Cost and Benefits of Utilization of Ion Mobility Mass Spectrometry Testing for Drugs of Abuse Compared to Immunoassay Screening in a Large Urban Hospital

Abstract Background Urine drugs of abuse testing (DOAU) in US hospitals are mainly orders from the emergency department performed by immunoassay screening. Testing can be relatively expensive, and results may suffer from non-idealities in sensitivity, specificity, and from panel limitations. A potential approach to these issues for immunoassays is instead to employ mass spectrometry (MS), where a large and flexible analyte list can be evaluated providing needed specificity and reducing send out costs for confirmatory testing. We compared results and cost impacts of a DOAU assay on a prototype, ion mobility mass spectrometry system to those obtained from an array of immunoassays offered as routine tests at a large urban hospital. Methods Immunoassay screen-positive and screen-negative urine samples were isolated from among those submitted for a DOAU panel (amphetamines, barbiturates, benzodiazepines, cannabis, cocaine, opiates, and methadone) or for additional single drug tests (fentanyl, oxycodone, phencyclidine, buprenorphine, propoxyphene). Sample aliquots (3 mL) were retrieved after not more than 5 days of refrigerated storage of the original sample and were subsequently frozen. These samples were then subjected to MS analysis using the prototype system where identification of 62 compounds was based on mass and ion mobility-derived Collision Cross Section (CCS). An established library of m/z, SLIMCCSHe, peak intensity, and peak width for each analyte was used, with acceptable method performance characteristics as based on applicable CLIA validation guidelines. The MS list included compounds covered by the immunoassay classes as well as additional drugs not expected to be positive among the immunoassays test results but of value for routine toxicological assessments. Results comparisons, utility of results, sample-to-result turnaround times, and overall financial implications were compared between the two workflows. Results Among immunoassay screen-negative samples, MS analysis showed additional positive results for metabolites of parent drugs as well as novel psychoactive substances and known adulterants in &amp;gt;10% of cases. Among the immunoassay screen-positive results, MS analysis confirmed immunoassay results (&amp;gt;90%) but picked up an additional positive component in over 15% of cases. Cost per complete patient result for the immunoassay was approximately $2.80 in comparison to an estimated cost of $5 for the MS method. Confirmation testing for positive immunoassay screen results (16% of all samples) would cost an additional $70 per sample, whereas the need for confirmation testing is eliminated for the MS method. Thus, use of the MS testing workflow would be superior to an immunoassay with reflex testing workflow while at the same time providing a clinically superior service of more diverse positive test results and elimination of often overlooked false negatives. Turnaround time between immunoassay (1h) and MS (50m) were judged to be similar. Conclusions The MS approach confirmed &amp;gt;90% of all immunoassay positive screen results. In addition, the MS method identified additional positive results not detected by the immunoassay despite an overall similar estimated time for sample to patient. Need for development of a user friendly, MS analyzer for use in hospitals is supported based on cost-effective and analytically superior tests for DOAU compared to screens performed using immunoassay panels.

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&amp;lt;10 ng/mL) and X-POSITIVE (X&amp;gt;=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 “&amp;gt;1200 ng/mL” for X-ARK &amp;gt;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 (&amp;lt;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.