With the lack of standardized validation protocols across the forensic chemistry community, validation of instrumentation can be a challenging and time-consuming task. However, this process is crucial to understanding the associated capabilities and limitations, especially for nascent technologies. Rapid GC-MS is one such emerging analytical technique being increasingly implemented in forensic laboratories due to its fast and informative screening capabilities. However, a full validation for forensic samples has yet to be published since its debut. This work presents the results of a comprehensive validation of a rapid GC-MS system for seized drug screening through the assessment of nine components: selectivity, matrix effects, precision, accuracy, range, carryover/contamination, robustness, ruggedness, and stability. Single- and/or multi-compound test solutions of commonly encountered seized drug compounds were used to assess method and system performance. Results met the designated acceptance criteria for a majority of components. For example, retention time and mass spectral search score % RSDs were ≤ 10 % for precision and robustness studies. Limitations were identified for components that did not meet the acceptance criteria (., inability to differentiate some isomers). The study design is part of a larger validation package developed for rapid GC-MS that includes a validation plan and automated workbook. The template, available for adoption by laboratories, ultimately aims to reduce the barrier of implementation for rapid GC-MS technology.

Use of immunoassay test strips for the detection of fentanyl in drug samples has become commonplace in harm reduction, law enforcement, public health, customs, and forensic science settings for testing drug product. With the increase of xylazine in the drug supply in recent years, use of xylazine test strips has also increased. As use of test strips expands, a desire to implement them for other drugs may emerge. However, since these strips are designed for urine testing, it is important to understand their applicability to testing drug product. In this work, we investigate the utility of seven types of urine immunoassay test strips - amphetamine, benzodiazepine, cocaine, methamphetamine, nitazene, opiate, and xylazine - for drug checking applications. Reproducibility, sensitivity, cross-reactivity, and the effect of prolonged exposure to elevated temperatures were studied. Generally, the tests were found to be reproducible, able to detect trace (μg/mL) levels of the analyte of interest, and minimally affected by prolonged storage at elevated temperatures. Nearly all tests showed cross-reactivity with compounds other than the analyte of interest, highlighting the need to better understand these limitations prior to implementation in a drug checking scenario. The viability of expired cocaine, fentanyl, and methamphetamine test strips was also interrogated, and little to no change in sensitivity was found even though the tests were multiple years expired.

Remediation of clandestine laboratory and processing operations for fentanyl and its analogs (i.e., fentalogs) may involve application of oxidants, most commonly household hypochlorite bleach, to chemically degrade the fentanyl to enable disposition of the site. While the chemical degradation of fentanyl has been reported, the degradation of immediate precursors has not been. Chemical degradation of fentanyl and fentalogs can cause these immediate precursors to reform. The immediate precursors of fentanyl investigated here are norfentanyl and 4-ANNP, which are controlled substances, along with norcarfentanil, precursor for carfentanil and remifentanil. The 4-ANNP was degraded within minutes in chlorine bleach, somewhat faster than fentanyl itself, whereas norfentanyl and norcarfentanil showed apparent degradation half lives up to days. Also, degradation rates were reduced in the presence of common diluents, like cutting agents, with apparent half-lives of several weeks for fully dissolved solutions. When the fentalog was added as a powder, globules were observed which could indefinitely prevent contact between the chlorine and the contents of the globules, prolonging persistence unless the globules are mechanically or otherwise broken. Because of shared structural characteristics among fentalogs, such as presence of reactive nitrogen groups, the results of this study may be applicable to a wide array of fentalogs. This study may also help inform remediation and waste management strategies to provide not only effective remediation of contamination, but also reduce the possibility that wastes of controlled substances are handled inappropriately, e.g., leading to continued personnel exposure and/or reprocessing of controlled substances.

Analysis of ignitable liquids in fire debris samples can be a time-consuming process, from extraction of volatile compounds to instrumental analysis. Rapid gas chromatography-mass spectrometry (GC-MS) is a screening technique that can be utilized prior to confirmatory GC-MS analysis to provide an informative screening approach and possibly reduce the need to further analyze negative samples. Though rapid GC-MS is fast (less than two minutes), extraction techniques such as passive headspace extraction remain a bottleneck for decreasing overall workflow times. In this work, solid phase microextraction (SPME) was implemented with rapid GC-MS for ignitable liquid analysis for a faster, more sensitive screening approach compared to extraction with passive headspace. Using optimized inlet conditions, limits of detection as low as 27 ng/mL per compound were achieved. Gasoline and diesel fuel were extracted and analyzed, and major compounds in each liquid were identified in the resulting chromatograms. Extracted ion profiles (EIPs) and deconvolution methods were useful for additional compound identifications. Lastly, the SPME-rapid GC-MS workflow was extended to the analysis of gasoline and diesel fuel in mock burn samples using carpet and wood substrates. From SPME sample extraction to rapid GC-MS instrumental analysis and data processing, the total workflow for a single sample was reduced to under 20 min. These results indicate that SPME is a suitable injection technique for rapid GC-MS to provide a fast and sensitive screening approach for fire debris applications.

An LC-MS/MS method for the analysis of 53 benzodiazepines, including various designer benzodiazepines, was developed. The developed method was applied to a total of 79 illicit street drug samples collected in Chicago, IL. Of these samples, 68 (84%) had detectable amounts of at least one benzodiazepine. Further, of the 53 benzodiazepines included in the developed method just 14 were measured in samples. Clonazolam, a potent designer benzodiazepine and derivative of clonazepam, was the most frequently measured benzodiazepine in 63% of samples and was measured in the highest concentrations. Other benzodiazepines measured in more than 10% of samples included clonazepam, alprazolam, flualprazolam, and oxazepam. Mixtures of benzodiazepines were frequently measured in samples, with just 24% of samples containing just one benzodiazepine. To determine the response of benzodiazepines on a rapid, point-of-use drug checking tool, all 53 benzodiazepine standards were screened on a lateral flow immunoassay benzodiazepine test strip. Sixty eight percent of standards gave a positive BTS response at a concentration of 20 μg/mL, demonstrating BTS have response to a wide variety of benzodiazepines, including many designer benzodiazepines. A comparison of this data to previous data reported for the same samples demonstrated all samples containing a benzodiazepine also had an opioid present, with fentanyl being present in 94% of benzodiazepine samples. These results highlight high rates of polysubstance drug presence in Chicago, IL illicit drug samples, posing an increased risk of drug overdoses in people who use drugs.

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).

Drug checking services are being utilized worldwide to provide people who use drugs information on the composition and contents of their drugs as a tool for harm reduction and accidental overdose prevention. Existing drug checking services use a variety of techniques including immunoassay strips and spectroscopic techniques like FTIR and Raman. Few services utilize LC-MS based methods for primary or secondary analysis and few methods exist for direct analysis of illicit drugs. To address this, an LC-MS/MS method was developed for 22 illicit drugs and cutting agents using LC-MS/MS with application to 124 illicit drug samples that were collected from Chicago, IL. Samples were also analyzed using fentanyl and benzodiazepine immunoassay test strips. Fentanyl test strips gave a positive result for 86% of samples with only one sample showing a positive result on a benzodiazepine test strip. LC-MS/MS analysis of samples show that opioids were the most commonly quantified in 96% of samples, followed by stimulants at 12% and benzodiazepines at 1%. Fentanyl was measured in 91% of samples, co-occurring with heroin in 58% of opioid-containing samples. A comparison of the gold-standard LC-MS/MS results to fentanyl test strips shows a high level of accuracy for the fentanyl test strips, with just 5% of samples being classified as false negatives and no false positives. These results demonstrate the strengths and benefits of LC-MS/MS when incorporated as a secondary analysis tool for drug checking.

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 (, 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 need for rapid chemical analyses and new analytical tools in forensic laboratories continues to grow due to case backlogs, difficult-to-analyze cases, and identification of previously unseen materials such as new psychoactive substances. To adapt to these needs, the forensics community has been pursuing the use of ambient ionization mass spectrometry, and more specifically direct analysis in real time mass spectrometry (DART-MS), for a wide range of applications. From the inception of DART-MS forensic applications have been researched with demonstrations ranging from drugs of abuse to inorganic gunshot residue to printer inks to insect identification. This article presents a review of research demonstrating the use of DART-MS for forensically relevant samples over the past five years. To provide more context, background on the technique, sampling approaches, and data analysis methods are presented along with a discussion on the potential future and research needs of the technology.

Seventeen laboratories participated in three interlaboratory exercises to assess the performance of refractive index, micro X-ray Fluorescence Spectroscopy (μXRF), and Laser Induced Breakdown Spectroscopy (LIBS) data for the forensic comparison of glass samples. Glass fragments from automotive windshields were distributed to the participating labs as blind samples and participants were asked to compare the glass samples (known vs. questioned) and report their findings as they would in casework. For samples that originated from the same source, the overall correct rate was greater than 92% for each of the three techniques (refractive index, μXRF, and LIBS). For samples that originated from different vehicles, an overall correct rate of 82%, 96%, and 87% was observed for refractive index, μXRF, and LIBS, respectively. Special attention was given to the reporting language used by practitioners as well as the use of verbal scales and/or databases to assign a significance to the evidence. Wide variations in the reported conclusions exist between different laboratories, demonstrating a need for the standardization of the reporting language used by practitioners. Moreover, few labs used a verbal scale and/or a database to provide a weight to the evidence. It is recommended that forensic practitioners strive to incorporate the use of a verbal scale and/or a background database, if available, to provide a measure of significance to glass forensic evidence (i.e., the strength of an association or exclusion).

Canine training aids based on vapor capture-and-release into a flexible polymer, polydimethylsiloxane (PDMS), have been used for in canine detection of explosives that have volatile or semi-volatile odorants. To enhance the rate of odor capture for less volatile targets, two temperatures are used for aid preparation. By using an elevated temperature for the target explosive, the amount of vapor is enhanced, increasing the production of the characteristic odor profile. The polymeric adsorbent is maintained at a cool temperature, favoring vapor capture. The success of this two-temperature approach is demonstrated for training aids targeting the low volatility explosive TNT using SPME (solid-phase microextraction) headspace analysis. In addition, the effect of using two temperatures on preparing training aids based on TNT and its more volatile impurities 2,4-DNT and 2,6-DNT are evaluated in canine trials. A thermal pretreatment to minimize the non-target odors in the PDMS polymer is presented.

Rapid identification of new or emerging psychoactive substances remains a critical challenge in forensic drug chemistry laboratories. Current analytical protocols are well-designed for confirmation of known substances yet struggle when new compounds are encountered. Many laboratories initially attempt to classify new compounds using gas chromatography-electron ionization-mass spectrometry (GC-EI-MS). Though there is a large body of research focused on the analysis of illicit substances with GC-EI-MS, there is little high-level discussion of mass spectral trends for different classes of drugs. This manuscript compiles literature information and performs simple exploratory analyses on evaluated GC-EI-MS data to investigate mass spectral trends for illicit substance classes. Additionally, this work offers other important aspects: brief discussions of how each class of drugs is used; illustrations of EI mass spectra with proposed structures of commonly observed ions; and summaries of mass spectral trends that can help an analyst classify new illicit compounds.

While the drug background in forensic laboratories has been quantified, the processes that most contribute to the background have not been extensively researched. This work presents both qualitative visualization and quantitative analysis of the spread of simulant drug particulate during the process of taking net weights. The process was modeled using three masses of powder (0.2 g, 2 g, and 100 g). The net weight process, in which the mixture was poured onto weighing paper, was mimicked and the resulting aerosolized particulate was allowed to settle. Wetted cotton swabs were then used to sample 6.45 cm (1 in) squares extending up to 61 cm (24 in) away from the weigh paper. The swabs were then extracted and quantified using LC-MS/MS and two-dimensional color plots were created to visualize the magnitude of particulate spread. Qualitative flow visualization of the process, accomplished using laser light sheet videography, was also completed to support the quantitative extraction experiments and provide a visual representation of the mechanism of particulate spread. Surface concentrations were found to be highest in the area immediately surrounding the weigh paper, though transport as far as 61 cm (24 in) was observed with all mass loadings. The amount of the material aerosolized and transported on the bench surrounding the weigh paper was dependent upon the mass of material being poured. These results highlight that weighing activities encountered in forensic labs may be a primary contributor to drug background and may be a potential source of inhalation exposure for chemists.

Microspectrophotometry (MSP) is a rapid, nondestructive technique for the analysis of color in textile fibers. This technique combines microscopy and ultraviolet (UV)/visible (Vis) spectroscopy, allowing for very small colored samples, like dyed textile fibers, to be analyzed directly and thereby eliminates the need for time consuming and destructive extractions. While MSP is generally accepted to be a nondestructive evaluation method, a loss of color during analysis, or photofading can occur. In this work, cotton fabric dyed with blue, yellow, and red direct dyes at different concentrations. Dye photofading during MSP examination was investigated by measuring the absorbance at a specific position on the fibers from these fabrics, periodically over the course of 30 minutes. Visible color loss and a reduction in absorbance was observed for all three colors, but was most pronounced for the fibers dyed red. A major goal of this study is to increase awareness of the photofading phenomenon when analyzing cotton fibers using MSP.

The quest for a reliable means to detect cannabis intoxication with a breathalyzer is ongoing. To design such a device, it is important to understand the fundamental thermodynamics of the compounds of interest. The vapor pressures of two important cannabinoids, cannabidiol (CBD) and Δ-tetrahydrocannabinol (Δ-THC), are presented, as well as the predicted normal boiling temperature (NBT) and the predicted critical constants (these predictions are dependent on the vapor pressure data). The critical constants are typically necessary to develop an equation of state (EOS). EOS-based models can provide estimations of thermophysical properties for compounds to aid in designing processes and devices. An ultra-sensitive, quantitative, trace dynamic headspace analysis sampling called porous layered open tubular-cryoadsorption (PLOT-cryo) was used to measure vapor pressures of these compounds. PLOT-cryo affords short experiment durations compared to more traditional techniques for vapor pressure determination (minutes versus days). Additionally, PLOT-cryo has the inherent ability to stabilize labile solutes because collection is done at reduced temperature. The measured vapor pressures are approximately 2 orders of magnitude lower than those measured for n-eicosane, which has a similar molecular mass. Thus, the difference in polarity of these molecules must be impacting the vapor pressure dramatically. The vapor pressure measurements are presented in the form of Clausius-Clapeyron (or van't Hoff) equation plots. The predicted vapor pressures that would be expected at near ambient conditions (25 °C) are also presented.

Fentanyl and fentanyl analogues represent a current and emerging threat in the United States as pure illicit narcotics and in mixtures with heroin. Because of their extreme potency, methods to safely and rapidly detect these compounds are of high interest. This work investigates the use of thermal desorption direct analysis in real time mass spectrometry (TD-DART-MS) and ion mobility spectrometry (IMS) as tools for the rapid and sensitive (nanogram to picograms) detection of fentanyl, 16 fentanyl analogues, and five additional opioids. Competitive ionization studies highlight that detection of these compounds in the presence of heroin is readily achievable, down to 0.1% fentanyl by mass with TD-DART-MS. With IMS, detection of nanogram levels of fentanyl in a binary fentanyl and heroin mixture is possible but can be complicated by decreased resolution in certain commercial instrument models. Modifications to the alarm windows can be used to ensure detection of fentanyl in binary mixtures. Additionally, three complex background matrices (fingerprint residue, dirt, and plasticizers) are shown to have a minimal effect of the detection of these compounds. Wipe sampling of the exterior of bags of questioned powders is shown to be a safe alternative method for field screening and identification, removing the need to handle potentially lethal amounts of material.

While background studies have been commonplace in many occupational fields for a long time, attempts to understand the chemical background in forensics labs has been largely understudied. Such studies can help define the efficiency of cleaning procedures and the integrity of collected data, which is becoming increasingly important due to improving sensitivity of instrumentation and the prevalence with which potent drugs of abuse, such as the opioids, are being seen. The results from this study provide a snapshot of the drug background levels on surfaces in a laboratory system comprised of a central laboratory and two satellite laboratories. Samples were collected from work surfaces by swiping with meta-aramid wipes, and extracted for analysis by LC/MS/MS, for quantitation, and TD-DART-MS, for non-targeted screening. Surfaces were sampled from within the drug unit (where drug evidence is processed) and the evidence receiving unit (where drug cases are handled) in all laboratories as well as the report writing area, the toxicology unit and the crime scene unit in the central laboratory. Results showed that the background was restricted primarily to the benches, balances, and instrumentation within the drug unit - with approximately an order of magnitude higher concentrations observed on the balances, compared to the benches. Higher levels were also observed in analyst specific surfaces when compared to general use surfaces within the drug unit - which corresponded to where bulk evidence handling was completed. Background in the evidence receiving and report writing sections was minimal. Comparison of the main laboratory to the satellite laboratories showed similarities amongst frequently encountered drugs like cocaine, but noticeable differences in opioids which could be attributed to differences in the make-up of exhibits each laboratory receives. Understanding the background levels of drugs in a forensic laboratory environment is crucial to improving cleaning protocols, helping define detection limits for highly sensitive analyses, and providing additional results to the broader community that has been establishing background levels in other environments.

Identifying and quantifying the drug background in operational environments such as forensic laboratories is an emerging body of research. Knowing these levels is crucial to addressing issues like occupational exposure risk - due to the emergence of potent novel psychoactive substances and synthetic opioids - and data integrity - due to improvements in instrument sensitivity. The work presented here builds upon a prior study to provide a broader representation of the average drug background levels found on surfaces in forensic laboratories. Over 700 samples from 20 laboratories were collected, extracted, and analyzed quantitatively using LC-MS/MS, and qualitatively using TD-DART-MS. Quantitative analysis by LC-MS/MS included a panel of 18 drugs while the non-targeted qualitative analysis by TD-DART-MS screened for over three hundred drugs and excipients. The study focused primarily on surfaces within the drug unit and evidence receiving area of the laboratories, but also investigated other operational units (crime scene, drug interdiction, latent prints, and toxicology) as well as report writing. Background levels were highest within the drug unit of the laboratory, though detectable (tens of nanograms) levels were observed in nearly all sampled areas. The data from this expanded study plays a critical role in addressing laboratory concerns such as establishing drug identification reporting limits for new instrumentation and establishing new workflow or cleaning protocols while also providing a more comprehensive dataset for general environmental background studies.

Recent work has shown that detectable levels of drugs exists on nearly all surfaces within a forensic laboratory - especially within the drug chemistry unit. This is an expected occurrence due to the handling and opening of drug evidence that contains powder material. The process of opening evidence, which produces aerosolized particulate that can settle on surfaces throughout the lab, has never been visualized. This work presents the first attempt to visualize the spread of particulate throughout the laboratory during the analysis of drug evidence and introduces an easy to implement approach laboratories can use to evaluate their specific protocols. By creating two simulated bricks of drugs that contained fluorescent particles, the spread of particulate was able to be monitored throughout the evidence handling process up to and including cleaning of surfaces after analysis. The protocols in this work, showed the spread of particulate, prior to cleaning, to be quite extensive, with transfer onto surfaces and items that were handled. In this study, cleaning with methanol after processing the evidence was shown to be effective at removing nearly all particulate that was released in the process. The use of visualization techniques such as this demonstrate promise for helping laboratories identify processes in their own protocols that may contribute to drug background levels and educate forensic chemists how trace residues spread.

This manuscript outlines a straight-forward procedure for generating a of similarity between spectra of a set. When applied to a reference set of spectra for Type I fentanyl analogs (molecules differing from fentanyl by a single modification), the map illuminates clustering that is applicable to automated structure assignment of unidentified molecules. An open-source software implementation that generates mass spectral similarity mappings of unknowns against a library of Type I fentanyl analog spectra is available at http://github.com/asm3-nist/FentanylClassifier.

Determination of the ethanol concentration in beverages remains an occasional request to forensic laboratories. While traditionally determined using headspace gas chromatography (HS-GC), this process involves lengthy run times and the need for specialized headspace sampling equipment for the GC system. The work presented here highlights a potential alternative to HS-GC analysis, using direct analysis in real time mass spectrometry (DART-MS). By incorporating a simple T-junction to the orifice of the mass spectrometer, analysis of the headspace of a beverage can be completed, resulting in measurement of the ethanol concentration in a matter of seconds with greater than 99% accuracy. This work presents the development of a method for ethanol quantitation as well as results from the analysis of both ethanol reference material standards and in-house created ethanol-containing solutions to evaluate the precision and accuracy of the technique. Analysis of ethanolic beverages via DART-MS is shown to be a rapid and reliable alternative to traditional analysis by HS-GC.