National Institute on Drug Abuse Clinical Trials Network Meeting Report: Managing Patients Exposed to Xylazine-Adulterated Opioids in Emergency, Hospital and Addiction Care Settings.

Used as a veterinary sedative and not approved for human use, xylazine has been increasingly linked with opioid overdose deaths in the United States. A growing number of people have been exposed to xylazine in the illicit opioid supply (especially fentanyl) or in other drugs, particularly in some areas of the Northeast. Xylazine is an α-2 adrenergic agonist that decreases sympathetic nervous system activity. When combined with fentanyl or heroin, it is purported to extend the duration of the opioid's sedative effect and to cause dependence and an associated withdrawal syndrome; however, data to support these concerns are limited. Despite the escalating frequency of detection of xylazine in people with nonfatal and fatal opioid overdose, direct links to these outcomes have not been identified. Because the strongest causal link is to fentanyl coexposure, ventilatory support and naloxone remain the cornerstones of overdose management. Xylazine is also associated with severe tissue injury, including skin ulcers and tissue loss, but little is known about the underlying mechanisms. Nonetheless, strategies for prevention and treatment are emerging. The significance and clinical effects of xylazine as an adulterant is focused on 4 domains that merit further evaluation: fentanyl-xylazine overdose, xylazine dependence and withdrawal, xylazine-associated dermal manifestations, and xylazine surveillance and detection in clinical and nonclinical settings. This report reflects the Proceedings of the National Institute on Drug Abuse Center for the Clinical Trials Network convening of clinical and scientific experts, federal staff, and other stakeholders to describe emerging best practices for treating people exposed to xylazine-adulterated opioids. Participants identified scientific gaps and opportunities for research to inform clinical practice in emergency departments, hospitals, and addiction medicine settings.

Understanding sensitivity and cross-reactivity of xylazine lateral flow immunoassay test strips for drug checking applications.

The continued prevalence of xylazine in the illicit drug market has necessitated development of quick and simple methods for identification, including lateral flow immunoassays (also known as "test strips"), like those frequently used to detect fentanyl. This study explored the drug checking applicability of the first publicly available xylazine test strips (XTS) using four sub-studies: reproducibility (i.e., consistency of positive results in a highly-concentrated xylazine solution); limit of detection on a calibration curve of xylazine concentrations; cross-reactivity against 77 commonly encountered drugs, cutting agents, and other structurally similar compounds; and applicability for analyzing community-acquired samples-where 100 drug residue samples were analyzed using XTS, direct analysis in real time mass spectrometry (DART-MS), and gas chromatography tandem mass spectrometry (GC-MS/MS). XTS consistently detected xylazine at concentrations ≥2.5 µg/ml, and XTS results were reproducible. Sensitivity and specificity of XTS were calculated by comparing expected versus obtained results based on xylazine concentration of community-acquired samples measured by GC-MS/MS. XTS consistently detected xylazine in samples with concentration >2 µg/ml and yielded a sensitivity of 0.974, specificity of 1.00, and overall accuracy of 0.986. Cross-reactivity with lidocaine, a common cutting agent, and lack of XTS reactivity with other α2 -agonists found in the illicit drug supply highlight the need to offer consumers comprehensive drug checking services that identify a range of substances and better inform them about drug contents.

On the challenge of unambiguous identification of fentanyl analogs: Exploring measurement diversity using standard reference mass spectral libraries.

Fentanyl analogs are a class of designer drugs that are particularly challenging to unambiguously identify due to the mass spectral and retention time similarities of unique compounds. In this paper, we use agglomerative hierarchical clustering to explore the measurement diversity of fentanyl analogs and better understand the challenge of unambiguous identifications using analytical techniques traditionally available to drug chemists. We consider four measurements in particular: gas chromatography retention indices, electron ionization mass spectra, electrospray ionization tandem mass spectra, and direct analysis in real time mass spectra. Our analysis demonstrates how simultaneously considering data from multiple measurement techniques increases the observable measurement diversity of fentanyl analogs, which can reduce identification ambiguity. This paper further supports the use of multiple analytical techniques to identify fentanyl analogs (among other substances), as is recommended by the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG).

Rapid Analysis of Drugs: A Pilot Surveillance System To Detect Changes in the Illicit Drug Supply To Guide Timely Harm Reduction Responses - Eight Syringe Services Programs, Maryland, November 2021-August 2022.

A record number of 2,912 drug overdose deaths occurred in Maryland during the 12-month period July 1, 2020-June 30, 2021. Illicitly manufactured fentanyl, fentanyl analogs, or both* were involved in 84% of these deaths.† Timely identification of illicit drug market changes (e.g., fentanyl rapidly replacing heroin) could improve the public health response, specifically communications about risks for novel psychoactive substances. During November 19, 2021-August 31, 2022, the National Institute of Standards and Technology (NIST)§ tested 496 deidentified drug paraphernalia samples that staff members collected at eight Maryland syringe services programs (SSPs), also known as needle exchange programs,¶ in partnership with the Maryland Department of Health Center for Harm Reduction Services (CHRS).** All test results were available within 48 hours. Among the 496 paraphernalia samples collected, 367 (74.0%) tested positive for an opioid, and 364 (99.2%) of these samples contained fentanyl or fentanyl analogs. Approximately four fifths of fentanyl-positive samples also tested positive for the veterinary medicine xylazine, a sedative that when combined with opioids might increase the potential for fatal respiratory depression and soft tissue infections when injected (1). For 248 of the 496 samples, SSP participants also completed a questionnaire about the drugs they had intended to purchase. Among the 212 participants who had intended to buy an opioid, 87.7% were exposed to fentanyl, fentanyl analogs, or both, and 85.8% were unknowingly exposed to xylazine. Results improved awareness of fentanyl and xylazine among SSP staff members and galvanized efforts to enhance SSPs' wound care services for participants experiencing soft tissue injuries possibly associated with injecting xylazine. Rapid analysis of drug paraphernalia can provide timely data on changing illicit drug markets that can be used to mitigate the harms of drug use more effectively.

Identification of the veterinary sedative medetomidine in combination with opioids and xylazine in Maryland.

Public health, public safety, and forensic science personnel continue to face the emergence of new compounds into the drug market. While focus is often put on the detection of new analogs of known illicit drugs, monitoring the changes in cutting agents and other compounds can be equally as important. Over the last year, near real-time monitoring of the drug supply in Maryland has been completed through a public health-public safety partnership whereby residue from suspected drug packaging or used paraphernalia is collected and analyzed. Through this project, we have recently detected the presence of the veterinary sedative medetomidine in a small number of samples. The presence of medetomidine has been identified in both public health and law enforcement samples and in the presence of fentanyl and xylazine-another veterinary sedative that has been widely observed over the last year. While the rate at which medetomidine has been detected remains low, it is concerning and worthy of continued monitoring.

A Collaborative Platform for Novel Compound Identification - Characterization of Designer Phencyclidines (PCPs) POXP, PTHP, and P2AP.

With the sustained prevalence and introduction of new emerging drugs throughout the world there is a need for continued development and maintenance of platforms that enable rapid identification and characterization of unknown compounds. To complement existing efforts, a collaborative platform between the National Institute of Standards and Technology (NIST) and practicing forensic agencies is being deployed which enables laboratories to leverage techniques and expertise that may not exist at their facilities. Using this approach, unknown compounds are identified and characterized using a suite of analytical tools to obtain (1) a rapid preliminary identification followed by (2) a more complete characterization and confirmation of the preliminary identification. To demonstrate this platform, the characterization of three previously unreported analogs of phencyclidine (PCP) - POXP, PTHP, and P2AP - are described. A preliminary identification of the three substances was obtained using direct analysis in real time mass spectrometry (DART-MS) with confirmation by nuclear magnetic resonance (NMR) spectroscopy, gas chromatography mass spectrometry (GC-MS) and gas chromatography flame ionization detection (GC-FID).

Incorporating measurement variability when comparing sets of high-resolution mass spectra.

Mass spectra are an important signature by which compounds can be identified. We recently formulated a mathematical approach for incorporating measurement variability when comparing sets of high-resolution mass spectra. Leveraging replicate mass spectra, we construct high-dimensional consensus mass spectra-representing each of the compared analytes-and compute the similarity between these data structures. In this paper, we present this approach and discuss its applications and limitations when trying to discriminate methamphetamine and phentermine using in-source collision induced dissociation mass spectra collected with direct analysis in real time mass spectrometry.

Qualitative Analysis of Real Drug Evidence Using DART-MS and the Inverted Library Search Algorithm.

Chromatographic-less mass spectrometry techniques like direct analysis in real-time mass spectrometry (DART-MS) are steadily being employed as seized drug screening tools. However, these newer analytical platforms require new computational methods to best make use of the collected data. The inverted library search algorithm (ILSA) is a recently developed method designed specifically for working with mass spectra of mixtures collected with DART-MS and has been implemented as a function in the NIST/NIJ DART-MS data interpretation tool (DIT). This paper demonstrates how DART-MS and the ILSA/DIT can be used to analyze seized drug evidence, while discussing insights gathered during the evaluation of 92 adjudicated case samples. The evaluation verified that the combination of DART-MS and the ILSA/DIT can be used as an informative tool to help analysts screen seized drug evidence but also revealed several factors─such as the influence of incorporating multiple in-source fragmentation spectra and the effect of scoring thresholds─an analyst must consider while employing these methods. Use cases demonstrating the benefit of the nonscoring metrics provided by the ILSA/DIT and demonstrating how the ILSA/DIT can be used to identify novel substances are also presented. A summary of considerations for using the ILSA/DIT for drug screening concludes this paper.

Updates to the Inverted Library Search Algorithm for Mixture Analysis.

Identifying mixture components is a well-known challenge in analytical chemistry. The Inverted Library Search Algorithm is a recently proposed method for identifying mixture components using in-source collision induced dissociation (is-CID) mass spectra of a query mixture and a reference library of pure compound is-CID mass spectra ( J. Am. Soc. Mass Spectrom. 2021, 32 (7), 1725-1734). This article presents several subtle but important advances to the algorithm, including updated compound matching strategies that improve result explainability and spectral filtering to better handle noisy mass spectra as is often observed with real-world samples such as seized drug evidence.

Rapid, presumptive identification of seed-based toxins using direct analysis in real time mass spectrometry (DART-MS) and its variants.

Detection of seed-based toxins is a need for forensic chemists when suspected poisonings occur. The evidence that is found is often physically unidentifiable, as the seeds are mashed to extract the toxin. This work investigates potential strategies for rapid detection of seed-based toxins and seed mashes containing these toxins using chemical signatures obtained by direct analysis in real time mass spectrometry (DART-MS). Seven toxins (digoxin, digitoxin, hypaconitine, hyoscyamine, lanatoside, oleandrin, and scopolamine) and six seeds containing these toxins were studied. While detection of four of the toxins was readily attainable, detection of digoxin, digitoxin, and lanatoside was hindered by the inability to thermally desorb these larger compounds under normal operating conditions. The use of DART-MS variants capable of higher desorption temperatures (thermal desorption (TD)-DART-MS and infrared thermal desorption (IRTD)-DART-MS) enabled detection of these compounds. Detection of toxins from direct analysis of seed mashes and methanolic seed mash extracts was found to be compound and technique dependent. Principal component analysis (PCA) of generated mass spectra enabled differentiation of seed species, even in cases where the toxins were undetectable.

Improving particle collection efficiency of sampling wipes used for trace chemical detection.

Improvement of the particle collection efficiency of sampling wipes is desirable for optimizing the performance of many wipe-based chemical analysis techniques used for trace chemical screening applications. In this note, commercially available Teflon coated fiberglass and calendered Nomex sampling wipes were modified by mechanically scoring the wipe surface to produce topography that promoted enhanced and localized particle collection. Wipe surface modifications improved particle collection efficiency, relative to unmodified wipes, by factors of 3 to 13 depending on sampling conditions, wipe type, and surface sampled. Improvements were demonstrated for both model polystyrene latex microspheres and inkjet printed explosive particles. The modifications also concentrated particles into pre-defined locations on the wipe which can be engineered to ensure maximum overlap with the thermal desorber of a trace contraband detection system allowing for more effective analysis of collected trace residues.

Comparing two seized drug workflows for the analysis of synthetic cannabinoids, cathinones, and opioids.

As the challenges faced by drug chemists persist, due to the presence of emerging drugs, laboratories continue to look for new solutions, ranging from existing methods to implementation of entirely new technology. A common barrier for making workflow changes is a lack of pre-existing data demonstrating the potential impact of these changes. In this study, we compare, qualitatively and quantitatively, an existing workflow for seized drug analysis to an experimental workflow. Four chemists were asked to analyze a total of 50 mock case samples across the two workflows. The existing workflow employed color tests for screening alongside general purpose GC-FID and GC-MS analyses for confirmation. The experimental workflow combined DART-MS screening with class-specific (targeted) GC-MS analysis for confirmation. Comparison of the workflows showed that screening by DART-MS required the same amount of time as color tests but yielded more accurate and specific information. Confirmation using the existing workflow required more than twice the amount of instrument time and data interpretation time while also presenting other analytical challenges that prevented compound confirmation in select samples. Targeted GC-MS methods simplified data interpretation, reduced consumption of reference materials, and addressed almost all limitations of general-purpose methods. While the experimental workflow requires modifications and answering of additional research questions, this study shows how rethinking analytical workflows for seized drug analysis could reduce turnaround times, backlogs, and standards consumption. It also demonstrates the potential impact of being able to investigate workflow changes prior to implementation.

Rapid GC-MS as a Screening Tool for Forensic Fire Debris Analysis.

Techniques developed for the screening of forensic samples can be useful for increasing sample throughput and decreasing backlog in forensic laboratories. One such technique, rapid gas chromatography mass spectrometry (GC-MS), allows for fast sample screening (≈1 min) and has gained interest in recent years for forensic applications. This work focuses on the development of a method for ignitable liquid analysis using rapid GC-MS. A sampling protocol and temperature program were developed for the analysis of these volatile samples. Using the optimized method for analysis, the limits of detection for compounds commonly found in ignitable liquids ranged from 0.012 mg/mL to 0.018 mg/mL. Once the method was developed, neat ignitable liquids (i.e., gasoline and diesel fuel) were analyzed, and major components in each liquid were identified. The identification of major compounds in gasoline and diesel fuel in the presence of substrate interferences was then assessed through the analysis of simulated fire debris samples. Three different substrates were spiked with each ignitable liquid, burned, and analyzed. Major compounds in both liquids were identified using the total ion chromatograms, relevant extracted ion profiles, and deconvolution methods.

The Min-Max Test: An Objective Method for Discriminating Mass Spectra.

Deciding whether the mass spectra of seized drug evidence and a reference standard are measurements of two different compounds is a central challenge in forensic chemistry. Normally, an analyst will collect mass spectra from the sample and a reference standard under identical conditions, compute a mass spectral similarity score, and make a judgment about the sample using both the similarity score and their visual interpretation of the spectra. This approach is inherently subjective and not ideal when a rapid assessment of several samples is necessary. Making decisions using only the score and a threshold value greatly improves analysis throughput and removes analyst-to-analyst subjectivity, but selecting an appropriate threshold is itself a nontrivial task. In this paper, we describe and evaluate the min-max test-a simple and objective method for classifying mass spectra that leverages replicate measurements from each sample to remove analyst subjectivity. We demonstrate that the min-max test has an intuitive interpretation for decision-making, and its performance exceeds thresholding with similarity scores even when the best performing threshold for a fixed dataset is prescribed. Determining whether the underlying framework of the min-max test can incorporate retention indices for objectively deciding whether spectra are measurements of the same compound is an ongoing work.

Development and evaluation of a synthetic opioid targeted gas chromatography mass spectrometry (GC-MS) method.

As seized drug casework becomes increasingly complex due to the continued prevalence of emerging drugs, laboratories are often looking for new analytical approaches including developing methods for the analysis of specific compounds classes. Recent efforts have focused on the development of targeted gas chromatography mass spectrometry (GC-MS) confirmation methods to compliment the information-rich screening results produced by techniques like direct analysis in real time mass spectrometry (DART-MS). In this work, a method for the confirmation of synthetic opioids and related compounds was developed and evaluated. An 11-component test solution was used to develop a method that focused on minimizing overlapping retention time acceptance windows and understanding the influence of instrument parameters on reproducibility and sensitivity. Investigated settings included column type, flow rate, temperature program, inlet temperature, source temperature, and tune type. Using a DB-200 column, a 35-min temperature ramped method was created. It was evaluated against a suite of 222 synthetic opioids and related compounds, and successfully differentiated all but four compound pairs based on nonoverlapping retention time acceptance windows or objectively different mass spectra. Compared to a general confirmatory method used in casework, the targeted method was up to 25 times more sensitive and provided at least a two-fold increase in retention time differences. Analysis of extracts from actual case samples successfully demonstrated utility of the method and showed no instance of carryover, although the high polarity column required wider retention time windows than other columns.

A New Library-Search Algorithm for Mixture Analysis Using DART-MS.

Forensic analysis of seized drug evidence often involves determining whether the components of an unknown mixture are illicit compounds. One approach to this task is to screen the evidence using direct analysis in real time mass spectrometry (DART-MS) to make presumptive identifications. This manuscript introduces a new library-search algorithm that enhances presumptive identifications of mixture components using a series of in-source collision-induced dissociation mass spectra collected through DART-MS. The multistage search, titled the Inverted Library-Search Algorithm (ILSA), identifies potential components in a mixture by first searching the lowest fragmentation mass spectrum for target peaks, assuming these peaks are protonated molecules, and then scoring each target peak with possible library matches. As a proof of concept, the ILSA is demonstrated through several example searches of model seized drug mixtures of acetyl fentanyl, benzyl fentanyl, amphetamine, and methamphetamine searched against a small library of select compounds and the freely available NIST DART-MS Forensics Database. Discussion of the search results and several open areas of research to further extend the method are provided. This new approach for presumptive identification provides analysts with refined information about mixture components and will be of immediate importance in forensic analysis using DART-MS. A prototype implementation of the ILSA is available at https://github.com/asm3-nist/DART-MS-DST.

Design, Synthesis, and Biological Evaluation of Novel 1,3-Oxazole Sulfonamides as Tubulin Polymerization Inhibitors.

A series of novel 1,3-oxazole sulfonamides were constructed and screened for their potential to inhibit cancer cell growth. These compounds were evaluated against the full NCI-60 human tumor cell lines, with the majority exhibiting promising overall growth inhibitory properties. They displayed high specificity within the panel of leukemia cell lines versus all other lines tested. When examined in the dose-response assay, GI50 values fell within the low micromolar to nanomolar ranges. 1,3-Oxazole sulfonamide 16 displayed the best average growth inhibition, whereas the 2-chloro-5-methylphenyl and 1-naphthyl substituents on the sulfonamide nitrogen proved to be the most potent leukemia inhibitors with mean GI50 values of 48.8 and 44.7 nM, respectively. In vitro tubulin polymerization experiments revealed that this class of compounds effectively binds to tubulin and induces the depolymerization of microtubules within cells.

A framework for the development of targeted gas chromatography mass spectrometry (GC-MS) methods: Synthetic cannabinoids.

With the increased presence of novel psychoactive substances (NPSs) in casework, drug analysis has become more challenging. To address these challenges, new screening technologies with improved specificity are being implemented, allowing for the creation and adoption of targeted confirmatory analyses that produce more conclusive results. This paper outlines a six-step, data-driven, framework to develop and evaluate gas chromatography mass spectrometry (GC-MS) methods for targeted classes of drugs. The process emphasizes maximizing retention time differences (to minimize the potential for retention time acceptance windows to overlap) and understanding the trade-offs between sensitivity and reproducibility using a test solution containing pairs of compounds that are difficult to distinguish. The method is then evaluated by expanding the panel of compounds analyzed, identifying limitations in compound discrimination, comparing to current methods, and analyzing representative casework to establish usability. To demonstrate this framework, a method for synthetic cannabinoids was created. The developed method utilizes a DB-200 column and an isothermal temperature program. It was found that sensitivity could be adjusted, without compromising reproducibility, by altering the split ratio and injection volume. The targeted method successfully differentiated 50 cannabinoids based on either retention time differences or mass spectral dissimilarity - determined using a newly developed spectral comparison test. Compared to a general method used for casework, the targeted method was an order of magnitude more sensitive, a minute shorter, and provided major increases in retention time differences. This framework can be implemented and adapted to develop targeted methods for other applications or compound classes.

Development and evaluation of a synthetic cathinone targeted gas chromatography mass spectrometry (GC-MS) method.

To address challenges associated with the increased prevalence of novel psychoactive substances (NPSs), laboratories often adopt new techniques or new methods with the goal of obtaining more detailed chemical information with a higher level of confidence. To demonstrate how new methods applied to existing techniques can be a viable approach, a targeted gas chromatography mass spectrometry (GC-MS) method for synthetic cathinones was developed. To create the method, a range of GC-MS parameters were first investigated using a seven-component test solution with the goal of minimizing compounds with overlapping acceptance windows by maximizing retention time differences within a reasonable runtime. Once developed, the targeted method was evaluated through several studies and was compared to a general GC-MS confirmatory method. The method produced a twofold increase in retention time differences of the test solution compounds with a 3.83-min shorter runtime than the general method. Limitations of the method were also studied by analyzing an additional forty-eight cathinones to identify instances where definitive compound identification may not be possible due to overlapping acceptance windows and mass spectra. Thirty-eight pairs of compounds had retention times differences of less than 2% and, of those thirty-eight, one pair had indistinguishable mass spectra. A set of case samples were also analyzed using the method to evaluate suitability for casework. An increase in split ratio was required to obtain acceptable sensitivity. The development of this method is part of a larger project to measure benefits and drawbacks of different drug chemistry workflows.

Creation and Release of an Updated NIST DART-MS Forensics Database.

Facing increasing caseloads and an everchanging drug landscape, forensic laboratories have been implementing new analytical tools. Direct analysis in real time mass spectrometry (DART-MS) is often one of these tools because it provides a wealth of information from a rapid, simple analysis. The data produced by these systems, while extremely useful, can be difficult to interpret, especially in the case of complex mixtures, and therefore, mass spectral databases are often used to assist in interpretation of data. Development of these databases can be expensive and time-consuming and often relies on manual evaluation of the underlying data. The National Institute of Standards and Technology (NIST) released an initial DART-MS in-source collisional-induced dissociation mass spectral database for seized drugs in the early 2010s but it has not been updated to reflect the increasing prevalence of novel psychoactive substances. Recently, efforts to update the database have been undertaken. To assist in development of the database, an automated data evaluation process was also created. This manuscript describes the new NIST DART-MS Forensics Database and the steps taken to automate the data evaluation process.