Acetyl Fentanyl Research Articles

Dependency of fentanyl analogue protomer ratios on solvent conditions as measured by ion mobility-mass spectrometry.

Recently, our group has shown that fentanyl and many of its analogues form prototropic isomers ("protomers") during electrospray ionization. These different protomers can be resolved using ion mobility spectrometry and annotated using mobility-aligned tandem mass spectrometry fragmentation. However, their formation and the extent to which experimental variables contribute to their relative ratio remain poorly understood. In the present study, we systematically investigated the effects of mixtures of common chromatographic solvents (water, methanol, and acetonitrile) and pH on the ratio of previously observed protomers for 23 fentanyl analogues. Interestingly, these ratios (N-piperidine protonation vs. secondary amine/O = protonation) decreased significantly for many analogues (e.g., despropionyl ortho-, meta-, and para-methyl fentanyl), increased significantly for others (e.g., cis-isofentanyl), and remained relatively constant for the others as solvent conditions changed from 100% organic solvent (methanol or acetonitrile) to 100% water. Interestingly, pH also had significant effects on this ratio, causing the change in ratio to switch in many cases. Lastly, increasing conditions to pH ≥ 4.0 also prompted the appearance of new mobility peaks for ortho- and para-methyl acetyl fentanyl, where all previous studies had only showed one single distribution. Because these ratios have promise to be used qualitatively for identification of these (and emerging) fentanyl analogues, understanding how various conditions (i.e., mobile phase selection and/or chromatographic gradient) affect their ratios is critically important to the development of advanced ion mobility and mass spectrometry methodologies to identify fentanyl analogues.

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

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

Differentiating Structurally Similar Fentanyl Analogs by Comparing Density Functional Theory (DFT) Calculations and Surface-Enhanced Raman Spectroscopy (SERS) Results.

Fentanyl and fentanyl analogs are the main cause of recent overdose deaths in the United States. The presence of fentanyl analogs in illicit drugs makes it difficult to estimate their potencies. This makes the detection and differentiation of fentanyl analogs critically significant. Surface-enhanced Raman spectroscopy (SERS) can differentiate structurally similar fentanyl analogs by yielding spectroscopic fingerprints for the detected molecules. In previous years, five fentanyl analogs, carfentanil, furanyl fentanyl, acetyl fentanyl, 4-fluoroisobutyryl fentanyl (4-FIBF), and cyclopropyl fentanyl (CPrF), gained popularity and were found in 76.4% of the fentanyl analogs trafficked. In this study, we focused on 4-FIBF, CPrF, and structurally similar fentanyl analogs. We developed methods to differentiate these fentanyl analogs using theoretical and experimental methods. To do this, a set of fentanyl analogs were examined using density functional theory (DFT) calculations. The DFT results obtained in this project permitted the assignment of spectral bands. These results were then compared with normal Raman and SERS techniques. Structurally similar fentanyl analogs show important differences in their spectra, and they have been visually differentiated from each other both theoretically and experimentally. Additional results using principal component analysis and soft independent modeling of class analogy show they can be distinguished using this technique. The limit of detection values for FIBF and CPrF were determined to be 0.35 ng/mL and 4.4 ng/mL, respectively, using SERS. Experimental results obtained in this project can be readily implemented in field applications and smaller laboratories, where inexpensive portable Raman spectrometers are often present and used in drug analysis.

A quantitative LC-MS/MS analysis of Xylazine, p-Fluorofentanyl, Fentanyl and Fentanyl-Related compounds in postmortem blood

The purpose of this study was to develop and validate a method to quantitate the veterinary sedative xylazine as well as 4-anilino-N-phenethylpiperidine (4-ANPP), acetyl fentanyl, fentanyl, norfentanyl, and p-fluorofentanyl in blood utilizing liquid chromatography tandem mass spectrometry. This method also qualitatively monitors for the presence of o-fluorofentanyl and m-fluorofentanyl isomers. UCT Clean Screen® DAU extraction columns were utilized to isolate the analytes in postmortem blood samples. The extracts were eluted, evaporated, reconstituted, and then analyzed using a Waters Acquity™ UPLC coupled a triple quadrupole mass spectrometer. The lower limit of quantitation was determined to be 0.1 ng/mL for all analytes, except for xylazine (0.2 ng/mL). The upper limit of quantitation for all analytes was 100 ng/mL. No interferences from matrix, internal standard, or common drug analytes were observed. Bias (-13.1–4.6 %) and precision (-13.14–10.3 %) fell within the acceptable ± 20 % criteria range. Dilution integrity at x2, x10, and x100 was evaluated and all results were within ± 20 % of the target concentration. Processed extract stability was evaluated after 72 h and all results were within ± 20 % of the analyte initial concentration. Matrix effects were the most prominent with xylazine, but deemed acceptable as the deuterated internal standard also observed comparable enhancement. Analysis of 89 postmortem blood samples by this method resulted in positive results for fentanyl (0.27–66 ng/mL, n = 82), xylazine (0.24–958 ng/mL, n = 21), 4-ANPP (0.10–38 ng/mL, n = 72), acetyl fentanyl (0.18–1.5 ng/mL, n = 3), p-fluorofentanyl (0.11–33 ng/mL, n = 30), and norfentanyl (0.10–98 ng/mL, n = 73).

Assessment of two brands of fentanyl test strips with 251 synthetic opioids reveals “blind spots” in detection capabilities

BackgroundFentanyl test strips (FTS) are a commonly deployed tool in drug checking, used to test for the presence of fentanyl in street drug samples prior to consumption. Previous reports indicate that in addition to fentanyl, FTS can also detect fentanyl analogs like acetyl fentanyl and butyryl fentanyl, with conflicting reports on their ability to detect fentanyl analogs like Carfentanil and furanyl fentanyl. Yet with hundreds of known fentanyl analogs, there has been no large-scale study rationalizing FTS reactivity to different fentanyl analogs.MethodsIn this study, 251 synthetic opioids—including 214 fentanyl analogs—were screened on two brands of fentanyl test strips to (1) assess the differences in the ability of two brands of fentanyl test strips to detect fentanyl-related compounds and (2) determine which moieties in fentanyl analog chemical structures are most crucial for FTS detection. Two FTS brands were assessed in this study: BTNX Rapid Response and WHPM DanceSafe.ResultsOf 251 screened compounds assessed, 121 compounds were detectable at or below 20,000 ng/mL by both BTNX and DanceSafe FTS, 50 were not detectable by either brand, and 80 were detectable by one brand but not the other (n = 52 BTNX, n = 28 DanceSafe). A structural analysis of fentanyl analogs screened revealed that in general, bulky modifications to the phenethyl moiety inhibit detection by BTNX FTS while bulky modifications to the carbonyl moiety inhibit detection by DanceSafe FTS. ConclusionsThe different “blind spots” are caused by different haptens used to elicit the antibodies for these different strips. By utilizing both brands of FTS in routine drug checking, users could increase the chances of detecting fentanyl analogs in the “blind spot” of one brand.

Attitudes and experiences with fentanyl contamination of methamphetamine: exploring self-reports and urine toxicology among persons who use methamphetamine and other drugs

BackgroundThere are growing concerns about illicitly manufactured fentanyl (IMF) contamination of methamphetamine. This study aims to characterize the lay views and experiences with IMF-contaminated methamphetamine (IMF/meth) and identify participants with unknown IMF exposures through urine toxicology analysis.MethodsBetween December-2019 and November-2021, structured interviews were conducted with 91 individuals who reported past 30-day use of methamphetamine and resided in Dayton, Ohio, USA. Lab-based urine toxicology analyses were conducted to identify fentanyl/analogs, methamphetamine, and other drugs. Bivariate analyses were conducted to identify characteristics associated with attitudes and experiences with IMF/meth, and unknown IMF exposures.ResultsThe majority (95.6%) of the study participants were non-Hispanic white, and 52.7% were female. Past 30-day use of methamphetamine was reported on a mean of 18.7 (SD 9.1) days, and 62.6% also reported past 30-day use of heroin/IMF. Most (76.9%) had a history of an unintentional drug-related overdose, but 38.5% rated their current risk for an opioid overdose as none. Besides fentanyl (71.9%), toxicology analysis identified nine fentanyl analogs/metabolites (e.g., 42.7% acetyl fentanyl, 19.0% fluorofentanyl, 5.6% carfentanil), and 12.4% tested positive for Xylazine. The majority (71.4%) believed that IMF/meth was common, and 59.3% reported prior exposures to IMF/meth. 11.2% tested positive for IMF but reported no past 30-day heroin/IMF use (unknown exposure to IMF). Views that IMF/meth was common showed association with homelessness (p = 0.04), prior overdose (p = 0.028), and greater perceived risk of opioid overdose (p = 0.019). Self-reported exposure to IMF/meth was associated with homelessness (p = 0.007) and obtaining take-home naloxone (p = 0.025). Individuals with unknown IMF exposure (test positive for IMF, no reported past 30-day heroin/IMF use) were older (49.9 vs. 41.1 years, p < 0.01), and reported more frequent past 30-day use of methamphetamine (24.4 vs. 18.0 days, p < 0.05). They indicated lower perceived risk of opioid overdose (0.1 vs. 1.9, scale from 0 = “none” to 4 = “high,” p < 0.001).DiscussionThis study suggests a need for targeted interventions for people who use methamphetamine and expansion of drug checking and other harm reduction services.

Examining fentanyl and its analogues in the unregulated drug supply of British Columbia, Canada using drug checking technologies.

The emergence of fentanyl and its analogues have contributed to a drastic rise in overdose-related mortality in recent years. The objective of this study was to determine the number of drug checking samples containing fentanyl and fentanyl analogues using both point of care and confirmatory drug checking technologies. Point-of-care drug checking data, using a combination of fentanyl immunoassay strips and Fourier-transform infrared spectroscopy (FTIR), were collected at harm reduction sites in Vancouver and Surrey, British Columbia. Based on current recommendations from the British Columbia Centre on Substance Use Drug Checking Project, a subset of these samples was sent for confirmatory analysis using quantitative nuclear resonance spectroscopy, gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry. A total of 22,916 samples were tested using FTIR and fentanyl immunoassay strips, of which 6125 (29%) were positive for fentanyl and/or fentanyl analogues. FTIR identified a fentanyl analogue in five samples (all carfentanil). Of the 1467 samples sent for confirmatory analysis, fentanyl was identified in 855 (58%) and fentanyl analogues in 85 (6%), including: carfentanil (n=56), acetyl fentanyl (n=15), furanyl fentanyl (n=9) and cyclopropyl fentanyl (n=5). Our research found that FTIR does not consistently distinguish between fentanyl and its analogues at point of care and that highly sensitive confirmatory drug checking technologies are needed to identify fentanyl analogues. These findings underscore the limitations of current drug checking technologies and the importance of using both point of care and confirmatory drug checking initiatives for monitoring changes in the drug supply.

Reaching new sensitivity levels for the detection of fentanyl analogs and highly potent novel synthetic opioids in blood

The aim of this presentation is to demonstrate a sensitive quantitative workflow for the analysis of 32 of potent substances in poly-drug, discarded postmortem case samples at low (pg/mL) levels. The presentation will demonstrate that the unparalleled quantification performance of the method enabled accurate detection of potent substances in poly-drug, discarded postmortem case samples at trace levels that were not previously achievable, providing toxicologists a valuable insight into the causation of accidental overdoses. A total of 32 NSO including 17 fentanyl analogs and 15 newly emerging non-fentanyl opioids were selected for this panel. NSO were extracted from the 200 μL of spiked whole blood mixtures using a liquid-liquid extraction (LLE) procedure. Analytes were chromatographically separated using a Phenomenex Kinetex C18 column (50 × 3.0 mm, 2.6 μm, 00B-4462-Y0). Mobile phase A (MPA) and mobile phase B (MPB) were ammonium formate with formic acid and formic acid in methanol and acetonitrile, respectively. The injection volume was 10 μL and the total LC runtime was 15.5 minutes. A single acquisition method consisting of 68 MRM transitions (64 for the NSO and 4 for the internal standards) was created using the Scheduled MRM Algorithm. Control human whole blood samples spiked with the 32 target analytes were prepared at concentrations ranging from 1 pg/mL to 100 ng/mL. Excellent linearity was observed across the concentration ranges analyzed with R2 values greater than 0.99 for all of the NPS in the panel. Reported LLOQ values ranged between 10 to 50 pg/mL for the 32 analytes in the panel. The accuracy and precision of measurements ranged from 80.50–116.64% and from 0.42–17.80%, respectively. The excellent accuracy and precision were observed over the entire concentration range, including at the LLOQ. Overall, the developed method showed excellent reproducibility and linearity, proving the robustness of the developed method and quantitative performance of the SCIEX 7500 System even at low concentration levels for each of the targeted NSO in this study. The applicability of the method to analyze discarded postmortem case samples from subjects suspected of NPS ingestion resulting in accidental overdoses was investigated. For example, the results from the analysis of one of these case samples showed the successful detection of one NSO and its metabolite: isotonitazene and 5-aminoisotonitazene, as well as fentanyl and four of its analogs/metabolites: beta-hydroxy-fentanyl, norfentanyl, 4-ANPP and acetyl fentanyl at concentrations of 1434.33, 7.93, 599.10, 9756.67, 147.69 and 1465.00 pg/mL, respectively. The results demonstrate that the developed method enabled accurate detection of potent substances in poly-drug, authentic case samples at trace levels that were not previously achievable. A robust and sensitive drug screening workflow for the analysis of 32 potent NSO was successfully developed using the SCIEX 7500 System. Overall, the remarkable quantification performance of the SCIEX 7500 System enabled accurate detection of potent NSO at concentrations that were not previously achievable, providing a means to monitor ultra-potent NSO in overdose scenarios. The sensitivity levels afforded by the SCIEX 7500 system provided the ability to detect low levels of NSO in postmortem case samples that would normally go undetected, providing a clearer picture for help in determining the cause of death.

Fentanyl analog trends in Washington D.C. observed in needle-exchange syringes

Fentanyl has played a significant role in the opioid crisis, proving to be a persistent problem due to its analgesic effects and addictive nature. Consequently, fentanyl analogs have been identified as a rising threat in the illicit drug market, contributing to thousands of both fatal and nonfatal overdoses in the United States. A profile of the fentanyl and fentanyl analogs present in Washington D.C. was developed by analyzing syringe extracts obtained from needle-exchange programs in the city. Gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM) acquisition provided a targeted analysis scheme to detect trace amounts of nine compounds: acetyl fentanyl, para-fluorofentanyl, fentanyl, methoxyacetyl fentanyl, meta-chlorofentanyl, carfentanil, valeryl fentanyl, beta-hydroxythiofentanyl, and furanyl fentanyl. In total, 332 syringe extracts were analyzed. The detected analytes and their percent prevalences were fentanyl (72.29 %), acetyl fentanyl (11.44 %), para-fluorofentanyl (6.63 %), and furanyl fentanyl (3.61 %). Tandem use was detected with combinations of fentanyl with acetyl fentanyl (12.08 %), fentanyl with para-fluorofentanyl (7.08 %), fentanyl with furanyl fentanyl (4.58 %), and fentanyl with acetyl fentanyl and para-fluorofentanyl (2.08 %). The identities of the analogs present, their relative potencies, and tandem use trends provides valuable information, especially for medical personnel who respond to opioid-related overdoses and deaths. Based on the fentanyl analog trends and tandem use of these compounds revealed in this study, it is recommended that Congress passes the permanent classification of fentanyl analogs as Schedule I drugs.

Determination of Nine Fentanyl Drugs in Hair Samples by GC-MS/MS and LC-MS/MS.

We established GC-MS/MS and LC-MS/MS analysis methods for nine fentanyl drugs in hair samples. Human hairs were prepared by soaking in a solution of water-dimethyl sulfoxide with target analytes. The drugs were norfentanyl, acetyl fentanyl, para-fluorofentanyl, isobutyryl fentanyl, fentanyl, thiofentanyl, 4-fluoroisobutyr fentanyl, ocfentanil, and tetrahydrofuran fentanyl. For a single-factor experiment, a Box–Behnken design-response surface was used to optimize the pretreatment conditions of samples. The prepared samples were quantitatively analyzed by GC-MS/MS and LC-MS/MS. The working curve method was used for quantitative analysis with fentanyl-D5 as the internal standard. The concentrations of the nine fentanyl drugs in the samples were 1.488–6.494 ng mg–1, RSDs < 5.0%. For GC-MS/MS, the linear range of the nine fentanyl drugs was 0.5–5.0 ng mg–1, r2 > 0.999. The detection limits were 0.02–0.05 ng mg–1, and the recovery rates were >86%. For LC-MS/MS, the nine fentanyl drugs had an excellent linear relationship within the concentration range of 3.0–220.0 pg mg–1, r2 > 0.999. The detection limits were 0.05 pg mg–1 and the recovery rates were >84%. The established methods were used for the detection of fentanyl drugs in human hairs, with high sensitivity, accuracy, and specificity. These two methods can be used for the certification of fentanyl certified reference substances (CRMs). In the experiment, the developed hair CRMs, which will continue to be studied in the future, are expected to be used in forensic drug abuse detection.

Toxicological Analysis of Fluorofentanyl Isomers in Postmortem Blood.

The opioid epidemic continues to evolve in the USA, with fentanyl the most prevalent synthetic opioid in fatal drug overdoses. Following the scheduling of fentanyl's core structure in 2018, there was a notable decline in the prevalence of fentanyl analogs in decedents; however, fluorofentanyl began being reported in casework in the winter of 2020. Fluorofentanyl has three positional isomers (para-fluorofentanyl (p-FF), ortho-fluorofentanyl (o-FF) and meta-fluorofentanyl (m-FF)), with the most predominant isomer that has recently emerged in the USA being p-FF. The goal of this study was to identify p-FF in postmortem cases between October 2020 and April 2021. Urine and blood were extracted using UCT Clean Screen® extraction columns and then screened using an Agilent 1290 Infinity liquid chromatograph (LC) coupled to an Agilent 6545 accurate mass time-of-flight mass spectrometer (TOF-MS) and quantified using an Agilent 6890N GC system coupled with an Agilent 5973 MS. The limit of quantitation (LOQ) for fentanyl, acetyl fentanyl, butyryl fentanyl, p-FF, o-FF and m-FF was 2.5 ng/mL. The screening method could not differentiate the three positional isomers of fluorofentanyl. Suspected overdose cases (n = 370) received from October 2020 through April 2021 from four Medical Examiner Districts in the state of Florida were analyzed for the presence of fluorofentanyl. The LC-QTOF-MS screen yielded 27 decedents positive for fluorofentanyl, with a majority being Caucasian (93%) and male (70%) with ages ranging from 27 to 63 years. Analysis of the blood and urine by GC-MS yielded 14 decedents positive for p-FF, nine of which were positive in the blood. The blood concentrations (n = 9) for p-FF ranged from <LOQ to 30 ng/mL, with an average and median of 9.9 ng/mL and 5.5 ng/mL, respectively. p-FF was identified in the blood of 33% of the cases, and the concentration of p-FF was generally higher than previously reported.

Pathological Lung Lesions of COVID-19 and Opioid Use Disorder: A Post-Mortem Lung and Toxicology Examination of 43 Decedents

The COVID-19 pandemic has continued from 2019 to current times, exacerbating existing epidemics since its beginning. Pathologic lung lesions in acute cases have consisted of hyaline membrane disease and microthrombi. More severe cases have pathologic lesions of alveolar and bronchiolar epithelial necrosis, lymphocytic interstitial infiltrate, and peribronchiolar and perivascular lymphocytic infiltrate. The opioid epidemic is one epidemic exacerbated by COVID-19. Those with opioid use disorder (OUD) are more likely to have compromised lung function, of which infection with the SARS-CoV-2 virus can worsen. The combined symptoms lead to an increased risk of overdose. In addition to anatomical risks, those with OUD require person-to-person contact to obtain opioids. This enhances the spread of COVID-19 and increases the risk of acquiring the disease. Pathological findings in those with chronic OUD can contain fluorescing talc powder with multinucleated giant cells and granulomas in the lung tissue. Twenty-one positive cases and twenty-two negative cases were collected from COVID-19 suspicious decedents in Marion County Coroner’s Office. Toxicology screens and COVID-19 RT-PCR tests were run on each of the 43 decedents. Microscopic evaluation was done by hematoxylin and eosin (H & E) and Masson’s Trichrome stains. The age range of those collected was 3 to 81 years. The ethnicities were 1 Asian, 8 African American, 1 Cuban, 3 Hispanic, 29 Caucasian, and 1 unknown. The male to female ratio was 3.8:1. The histological features found in this study were alveolar and bronchiolar epithelial necrosis, lymphocytic interstitial infiltrates, perivascular and peribronchiolar lymphocytic infiltrate, fibrosis, microthrombi, and hyaline membrane. Fluorescing talc was seen in 5 cases with granulomas and multinucleated giant cells. Toxicology reports showed 11 drug-related deaths, with cocaine, methamphetamine, alcohol, fentanyl, morphine, acetyl fentanyl, and sertraline as the causative agents.

Lay knowledge and practices of methamphetamine use to manage opioid-related overdose risks.

Methamphetamine use has increased among individuals with opioid use disorder. The key aims of this study are to detail and contextualise lay knowledge, attitudes, and behaviours related to methamphetamine use in relation to opioid overdose risks in an area dominated by non-pharmaceutical fentanyl-type drugs (NPF). The study recruited 41 individuals in Dayton, Ohio, who reported past 30-day use of methamphetamine and heroin/fentanyl. Interviews included structured and qualitative questions. Urine toxicology analysis was conducted to identify NPFs and other drugs. Open-ended interview sections were audio-recorded, transcribed, and analysed qualitatively using NVivo. The mean age was 38.3 years, 51% were female, and 100% non-Hispanic white. Participants described an exceedingly unpredictable local opioid market that became saturated with NPFs. The sample tested positive for 10 NPFs, including fentanyl (100%), acetyl fentanyl (61%), tetrahydrofuran fentanyl (29%), and carfentanil (12%). Most participants believed that methamphetamine could help prevent and/or reverse an opioid-related overdose. Nearly half had personally used it to help manage overdose risks related to NPF. These beliefs were embedded in a lay understanding of how methamphetamine works to stimulate the cardiovascular system. They were acted upon in the context of last resort situations that were determined by a lack of immediate access to naloxone, ambiguities surrounding overdose symptomatology, and easy access to plentiful and inexpensive methamphetamine. Lay efforts to rely on methamphetamine to manage NPF-related overdose risks highlight the need for a continuing expansion of take-home-naloxone programs and implementation of other novel harm reduction approaches in communities affected by NPFs.

Pressure-Sensitive Adhesive Combined with Paper Spray Mass Spectrometry for Low-Cost Collection and Analysis of Drug Residues.

Illicit drug use causes over half a million deaths worldwide every year. Drugs of abuse are commonly smuggled through customs and border checkpoints and, increasingly, through parcel delivery services. Improved methods for detection of trace drug residues from surfaces are needed. Such methods should be robust, fieldable, sensitive, and capable of detecting a wide range of drugs. In this work, commercially produced paper with a pressure-sensitive adhesive coating was utilized for the collection and analysis of trace drug residues by paper spray mass spectrometry (MS). This modified substrate was used to combine sample collection of drug residues from surfaces with rapid detection using a single paper spray ticket. The all-in-one ticket was used to probe different surfaces commonly encountered in forensic work including clothing, cardboard, glass, concrete, asphalt, and aluminum. A total of 10 drugs (acetyl fentanyl, fentanyl, clonazolam, cocaine, heroin, ketamine, methamphetamine, methylone, U-47700, and XLR-11) were evaluated and found to be detectable in the picogram range using a benchtop mass spectrometer and in the low nanogram range using a portable ion trap MS. The novel approach demonstrates a simple yet effective sampling strategy, allowing for rapid identification from difficult surfaces via paper spray mass spectrometry.

Quantification of fentanyl analogs in oral fluid using LC-QTOF-MS.

Oral fluid is a valuable alternative matrix for forensic toxicologists due to ease of observed collection, limited biohazardous exposure, and indications of recent drug use. Limited information is available for fentanyl analog prevalence, interpretation, or analysis in oral fluid. With increasing numbers of fentanyl-related driving under the influence of drug (DUID) cases appearing in the United States, the development of detection methods is critical. The purpose of the present study was to develop and validate a quantitative method for fentanyl analogs in oral fluid (collected via Quantisal™) using liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-QTOF-MS). Validation resulted in limits of detection and quantification ranging from 0.5 to 1ng/mL. Established linear range was 1-100ng/mL for all analytes, except acetyl fentanyl at 0.5-100ng/mL (R2 >0.994). Within- and between-run precision and bias were considered acceptable with maximum values of ±15.2%CV and ±14.1%, respectively. Matrix effects exhibited ionization enhancement for all analytes with intensified enhancement at a low concentration (9.3-47.4%). No interferences or carryover was observed. Fentanyl analogs were stable in processed extracts stored in the autosampler (4⁰ C) for 48h. The validated method was used to quantify fentanyl analogs in authentic oral fluid samples (n=17) from probationers/parolees. Fentanyl and 4-ANPP concentrations were 1.0-104.5ng/mL and 1.2-5.7ng/mL, respectively.

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.

Longitudinal Opioid Surveillance Project Involving Toxicologic Analysis of Postmortem Specimens from 9 Counties in Michigan Suggests the Discovery of New High-Intensity Drug Trafficking Areas.

Acetyl fentanyl (AF) is a Schedule I fentanyl analog that has been increasingly seen in heroin and fentanyl polydrug toxicity overdoses in Michigan (MI). Drug users are often unaware of the presence of AF in their drugs because it is often sold mixed into or disguised as heroin. High levels of AF in heroin drug products can cause increased incidence of overdose. This article describes data from a longitudinal opioid surveillance program and details 102 decedents in MI who were found to have evidence of heroin in their postmortem blood. A large portion of these decedents were also found to have evidence of fentanyl and AF. Our data further show significant overlap in incidence rates of AF and heroin-related overdose deaths in several MI counties, suggesting that AF is becoming enmeshed in heroin trafficking. Furthermore, we report unprecedented high incidence rates of AF and heroin-related overdose deaths in Calhoun county, and we propose that it is a high-intensity drug trafficking area. Highways US-131 and US-31 are likely used to transport these drugs. More study is needed into the drug trafficking trends in MI to ascertain drug sources and monitor the ever developing and dangerous polydrug heroin combinations.

Chemical attribution of fentanyl: The effect of human metabolism

Chemical attribution typically aims to establish a link between material found at a crime scene and a person, location or other evidence. In the field of illicit drugs, chemical attribution signatures are usually impurity profiles. Extending these to metabolized samples would create new possibilities in forensic investigations. The present study explores the effect of human metabolism on the impurity profile of fentanyl, as representative of synthetic opioids. Two different methods (Gupta and Siegfried) were used to synthesize fentanyl, after which the samples were incubated with liver microsomes to mimic human metabolism. The impurity profiles have been characterized with gas chromatography-mass spectrometry (GC-MS), gas chromatography with flame ionization detector (GC-FID), liquid chromatography quadrupole-time of flight mass spectrometry (LC-Q-TOF-MS) and liquid chromatography orbitrap mass spectrometry (LC-Orbitrap-MS). It was found that GC-FID and LC-Orbitrap-MS can both be used to discriminate between the Gupta and Siegfried synthesis method. This holds both for the analyses performed before and after metabolism. In addition, principal component analysis (PCA) identified acetyl fentanyl as the most important marker compound. Associated detection limits are in the range of concentrations expected in case work. While acetyl fentanyl is not stable during metabolism, its discriminating potential is transferred to its metabolic product acetyl norfentanyl. In addition, the stable impurities phenylacetamide and 1-phenylethylpiperidin-4-ol were found to be significant classifiers. To implement the results in a forensic framework, linear discriminant analysis (LDA) was applied and used to establish likelihood ratios. To our knowledge, the present work demonstrates for the first time the possibility of chemical attribution of drugs through the analysis of metabolic trace levels in biological samples.

Fentanyl analogs on the Swedish webforum flashback: Interest and impact of scheduling

BackgroundSweden regulates new psychoactive substances, including fentanyl analogs, individually. This reactive scheduling procedure enabled the existence of a recreational market for unscheduled fentanyl analogs sold from surface webshops. We measure the interest in 24 named fentanyl analogs and the impact of scheduling. MethodsWe scraped posts in threads on named fentanyl analogs from the Swedish internet forum Flashback.org, 2012–2019. The sample consists of 24 threads with a total of 8761 posts. We construct five measures of interest based on duration of threads, number of posts, and number of distinct posters, and fit a non-seasonal ARMA model to test if there was a change in mean activity after scheduling. ResultsAcross the five measures, there was most interest in acryl fentanyl, butyr fentanyl, and acetyl fentanyl. The number of daily posts was significantly reduced in nine out of 13 threads after scheduling. ConclusionThe scheduling of fentanyl analogs impacted interest on Flashback.org. The biggest effect sizes were from the narcotics scheduling of 2-Me-MAF, acryl, and acetyl fentanyl, while furanyl fentanyl saw the biggest reduction after health scheduling. The reductions were bigger for narcotics scheduling compared to health scheduling.

Analysis of 17 fentanyls in plasma and blood by UPLC-MS/MS with interpretation of findings in surgical and postmortem casework

The opioid crisis is linked to an increased misuse of fentanyl as well as fentanyl analogs that originate from the illicit drug market. Much of our current understanding of fentanyl and fentanyl analog use in our communities comes from postmortem toxicology findings. In the clinical settings of addiction medicine and pain management, where the opioid abuse potential is high, the use of fentanyl, as well as specific fentanyl analogs, may be underestimated due to limited plasma testing and limited availability of assays with suitable analytical sensitivity and selectivity to detect misuse of fentanyls. We report plasma and blood assays for 17 fentanyls (these include fentanyl, fentanyl analogs, fentanyl metabolites and synthetic precursors) in clinical, and medical examiner, casework. A mixed-mode solid phase extraction of diluted plasma or precipitated blood was optimized for maximum recovery of the fentanyls with minimized matrix effects. Analysis was performed using a Waters ACQUITY UPLC I-Class interfaced with a Waters Xevo TQ-S micro tandem quadrupole mass spectrometer. Method parameters were optimized and validated for precision, accuracy, carryover, linearity and matrix effects. Application studies were performed in postmortem blood obtained in 44 fentanyl-related fatalities and in serial plasma samples from 18 surgical patients receiving intravenous fentanyl therapy while undergoing parathyroidectomy. Fentanyls found in postmortem cases included fentanyl, norfentanyl, despropionyl-fentanyl (4-ANPP), beta-hydroxy fentanyl (β-OH fentanyl), acetyl fentanyl, acetyl norfentanyl, methoxyacetyl fentanyl, furanyl fentanyl, cyclopropyl fentanyl, and para-fluorobutyryl fentanyl, with fentanyl, norfentanyl, 4-ANPP and β-OH fentanyl predominating in frequency. Fentanyl concentrations ranged from 0.2 to 56 ng/mL and fentanyl was nearly always found with 4-ANPP, norfentanyl and β-OH fentanyl. Concentrations of other fentalogs ranged from <1 to 84 ng/mL (extrapolated). In the surgical cases, fentanyl was detected and quantified along with norfentanyl and β-OH fentanyl, but without detection of 4-ANPP in any of the samples. The association and relative concentrations of β-OH fentanyl, fentanyl and norfentanyl in the postmortem and clinical studies indicated a metabolic, rather than an illicit, source of β-OH fentanyl.