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.

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.

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.

Optimization of confined direct analysis in real time mass spectrometry (DART-MS).

Direct analysis in real time mass spectrometry (DART-MS) is seeing increased use in many fields, including forensic science, environmental monitoring, food safety, and healthcare. With increased use, novel configurations of the system have been created to either aid in detection of traditionally difficult compounds or surfaces, provide a more reproducible analysis, and/or chemically image surfaces. This work focuses on increasing the fundamental understanding of one configuration, where the DART ionization gas is confined in a junction, such as with thermal desorption (TD) DART-MS. Using five representative compounds and a suite of visualization tools, the role of the DART ionization gas, Vapur flow rate, gas back pressure, and exit grid voltage were examined to better understand both the chemical and physical processes occurring inside the confined configuration. The use of nitrogen as a DART ionization gas was found to be more beneficial than helium because of enhanced mixing with the analyte vapors, providing a more reproducible response. Lower Vapur flow rates were also found to be advantageous as they increased the analyte residence time in the junction, thus increasing the probability of its ionization. Operation at even lower Vapur flow rates was achieved by modifying the junction to restrict the DART gas flow. The DART exit grid voltage and gas back pressure had little observed impact on analyte response. These results provide the foundation to better understand and identify best practices for using a confined DART-MS configuration.

Detection of Nonvolatile Inorganic Oxidizer-Based Explosives from Wipe Collections by Infrared Thermal Desorption-Direct Analysis in Real Time Mass Spectrometry.

Infrared thermal desorption (IRTD) was coupled with direct analysis in real time mass spectrometry (DART-MS) for the detection of both inorganic and organic explosives from wipe collected samples. This platform generated discrete and rapid heating rates that allowed volatile and semivolatile organic explosives to thermally desorb at relatively lower temperatures, while still achieving elevated temperatures required to desorb nonvolatile inorganic oxidizer-based explosives. IRTD-DART-MS demonstrated the thermal desorption and detection of refractory potassium chlorate and potassium perchlorate oxidizers, compounds difficult to desorb with traditional moderate-temperature resistance-based thermal desorbers. Nanogram to sub-nanogram sensitivities were established for analysis of a range of organic and inorganic oxidizer-based explosive compounds, with further enhancement limited by the thermal properties of the most common commercial wipe materials. Detailed investigations and high-speed visualization revealed conduction from the heated glass-mica base plate as the dominant process for heating of the wipe and analyte materials, resulting in thermal desorption through boiling, aerosolization, and vaporization of samples. The thermal desorption and ionization characteristics of the IRTD-DART technique resulted in optimal sensitivity for the formation of nitrate adducts with both organic and inorganic species. The IRTD-DART-MS coupling and IRTD in general offer promising explosive detection capabilities to the defense, security, and law enforcement arenas.

Recent advances in ambient mass spectrometry of trace explosives.

Ambient mass spectrometry has evolved rapidly over the past decade, yielding a plethora of platforms and demonstrating scientific advancements across a range of fields from biological imaging to rapid quality control. These techniques have enabled real-time detection of target analytes in an open environment with no sample preparation and can be coupled to any mass analyzer with an atmospheric pressure interface; capabilities of clear interest to the defense, customs and border control, transportation security, and forensic science communities. This review aims to showcase and critically discuss advances in ambient mass spectrometry for the trace detection of explosives.

Broad spectrum infrared thermal desorption of wipe-based explosive and narcotic samples for trace mass spectrometric detection.

Wipe collected analytes were thermally desorbed using broad spectrum near infrared heating for mass spectrometric detection. Employing a twin tube filament-based infrared emitter, rapid and efficiently powered thermal desorption and detection of nanogram levels of explosives and narcotics was demonstrated. The infrared thermal desorption (IRTD) platform developed here used multi-mode heating (direct radiation and secondary conduction from substrate and subsequent convection from air) and a temperature ramp to efficiently desorb analytes with vapor pressures across eight orders of magnitude. The wipe substrate experienced heating rates up to (85 ± 2) °C s-1 with a time constant of (3.9 ± 0.2) s for 100% power emission. The detection of trace analytes was also demonstrated from complex mixtures, including plastic-bonded explosives and exogenous narcotics, explosives, and metabolites from collected artificial latent fingerprints. Manipulation of the emission power and duration directly controlled the heating rate and maximum temperature, enabling differential thermal desorption and a level of upstream separation for enhanced specificity. Transitioning from 100% power and 5 s emission duration to 25% power and 30 s emission enabled an order of magnitude increase in the temporal separation (single seconds to tens of seconds) of the desorption of volatile and semi-volatile species within a collected fingerprint. This mode of operation reduced local gas-phase concentrations, reducing matrix effects experienced with high concentration mixtures. IRTD provides a unique platform for the desorption of trace analytes from wipe collections, an area of importance to the security sector, transportation agencies, and customs and border protection.

Strategies for potential age dating of fingerprints through the diffusion of sebum molecules on a nonporous surface analyzed using time-of-flight secondary ion mass spectrometry.

Age dating of fingerprints could have a significant impact in forensic science, as it has the potential to facilitate the judicial process by assessing the relevance of a fingerprint found at a crime scene. However, no method currently exists that can reliably predict the age of a latent fingerprint. In this manuscript, time-of-flight secondary ion imaging mass spectrometry (TOF-SIMS) was used to measure the diffusivity of saturated fatty acid molecules from a fingerprint on a silicon wafer. It was found that their diffusion from relatively fresh fingerprints (t ≤ 96 h) could be modeled using an error function, with diffusivities (mm(2)/h) that followed a power function when plotted against molecular weight. The equation x = 0.02t(0.5) was obtained for palmitic acid that could be used to find its position in millimeters (where the concentration is 50% of its initial value or c0/2) as a function of time in hours. The results show that on a clean silicon substrate, the age of a fingerprint (t ≤ 96 h) could reliably be obtained through the extent of diffusion of palmitic acid.

Rapid detection of sugar alcohol precursors and corresponding nitrate ester explosives using direct analysis in real time mass spectrometry.

This work highlights the rapid detection of nitrate ester explosives and their sugar alcohol precursors by direct analysis in real time mass spectrometry (DART-MS) using an off-axis geometry. Demonstration of the effect of various parameters, such as ion polarity and in-source collision induced dissociation (CID) on the detection of these compounds is presented. Sensitivity of sugar alcohols and nitrate ester explosives was found to be greatest in negative ion mode with sensitivities ranging from hundreds of picograms to hundreds of nanograms, depending on the characteristics of the particular molecule. Altering the in-source CID potential allowed for acquisition of characteristic molecular ion spectra as well as fragmentation spectra. Additional studies were completed to identify the role of different experimental parameters on the sensitivity for these compounds. Variables that were examined included the DART gas stream temperature, the presence of a related compound (i.e., the effect of a precursor on the detection of a nitrate ester explosive), incorporation of dopant species and the role of the analysis surface. It was determined that each variable affected the response and detection of both sugar alcohols and the corresponding nitrate ester explosives. From this work, a rapid and sensitive method for the detection of individual sugar alcohols and corresponding nitrate ester explosives, or mixtures of the two, has been developed, providing a useful tool in the real-world identification of homemade explosives.

Mass spectrometry detection and imaging of inorganic and organic explosive device signatures using desorption electro-flow focusing ionization.

We demonstrate the coupling of desorption electro-flow focusing ionization (DEFFI) with in-source collision induced dissociation (CID) for the mass spectrometric (MS) detection and imaging of explosive device components, including both inorganic and organic explosives and energetic materials. We utilize in-source CID to enhance ion collisions with atmospheric gas, thereby reducing adducts and minimizing organic contaminants. Optimization of the MS signal response as a function of in-source CID potential demonstrated contrasting trends for the detection of inorganic and organic explosive device components. DEFFI-MS and in-source CID enabled isotopic and molecular speciation of inorganic components, providing further physicochemical information. The developed system facilitated the direct detection and chemical mapping of trace analytes collected with Nomex swabs and spatially resolved distributions within artificial fingerprints from forensic lift tape. The results presented here provide the forensic and security sectors a powerful tool for the detection, chemical imaging, and inorganic speciation of explosives device signatures.

Chemical imaging of artificial fingerprints by desorption electro-flow focusing ionization mass spectrometry.

Desorption electro-flow focusing ionization (DEFFI) mass spectrometry was used to image chemical distributions of endogenous, e.g., fatty acids, and trace exogenous compounds, e.g., explosives, narcotics and lotions, in deposited and lifted artificial fingerprints, directly from forensic lift tape. An artificial fingerprint mold and synthetic fingerprint material were incorporated for the controlled deposition of material for technique demonstration and evaluation.

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).

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).

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.

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.

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.

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.

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.

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.