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

BACKGROUND: Fentanyl 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. METHODS: In 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. RESULTS: Of 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. CONCLUSIONS: The 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.

BACKGROUND: There 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. METHODS: Between 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. RESULTS: The 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). DISCUSSION: This study suggests a need for targeted interventions for people who use methamphetamine and expansion of drug checking and other harm reduction services.

Fentanyl and other New Psychoactive Synthetic Opioids. Challenges to Prevention and Treatment.

Synthetic opioids have played a significant role in the current opioid crisis in the United States (U.S.) and Canada and are a matter of concern worldwide. New psychoactive opioids (NPOs) are classified in the internationally recognized new psychoactive substances (NPSs) category. This group comprises compounds that may have been synthesized decades ago but appeared only recently in the illicit drug market. Such is the case of fentanyl, fentanyl analogs, and non-fentanyl opioids. Most NPOs have effects similar to morphine, including euphoria and analgesia, and can produce fatal respiratory depression. Here, we present an overview of the systemic and molecular effects of main NPOs, their classification, and their pharmacological properties. We first review the fentanyl group of NPOs, including the four compounds of clinical use (fentanyl, alfentanil, sufentanil, and remifentanil) and the veterinary drug carfentanil. We also provide essential information on non-medical fentanyl analogs and other synthetic opioids such as brorphine, etonitazene, and MT-45, used as adulterants in commonly misused drugs. This paper also summarizes the scarce literature on the use of NPOs in Mexico. It concludes with a brief review of the challenges to prevention and treatment posed by NPOs and some recommendations to face them.

Comparative analysis of vapor profiles of fentalogs and illicit fentanyl.

Availability of fentanyl is at a record high with 3138 kg of fentanyl and related substances being seized in 2019. Fentanyl's high toxicity makes a lethal dose for most mere milligrams. With such a high potency and a consistent rise of abuse, the chances of injury or death of frontline workers increase with every interaction. Development of a non-contact detection method for fentanyl would decrease the chances of a workplace mishap. To aid in the development of a non-contact detection method, target analytes in the vapor profile of fentanyl need to be identified. In order to achieve this goal, semi-quantitative headspace analysis of fentanyl analogs and confiscated fentanyl exhibits was accomplished using solid-phase microextraction and gas chromatography coupled with mass spectrometry (SPME-GC-MS). The vapor signatures of these samples were compared to a previously reported reference-grade fentanyl vapor signature to determine the target analyte(s) for fentanyl detection in the vapor phase. A total of 20 fentalogs and confiscated exhibits, with masses ranging from 2 to 19 mg, were sampled. N-Phenylpropanamide(NPPA) or N-phenethyl-4-piperidone(NPP) was identified as target analytes in 75% of these samples. This is a crucial component for the development of a non-contact detection method for fentanyl.

Wooden Chest syndrome: The atypical pharmacology of fentanyl overdose.

WHAT IS KNOWN AND OBJECTIVE: A large percentage of opioid overdose fatalities involve fentanyl or one of its legal or illegal analogs (F/FAs). Is there something about the pharmacology of these drugs that make them unusually dangerous in an overdose? COMMENT: Some of the reasons for the dangers of overdose of F/FAs is their high potency and low cost (that leads to wide distribution). But it is rarely asked if the basic pharmacology of F/FAs differ in some fundamental way from conventional opioids such as morphine and heroin. In addition to centrally mediated respiratory depression via opioid receptors, F/FAs cause rigidity in the key respiratory muscles of the chest, upper airway and diaphragm ("wooden chest syndrome," WCS) by a non-opioid mechanism. WHAT IS NEW AND CONCLUSION: WCS is an atypical pharmacology of F/FAs. Because of its rapid onset and non-opioid mechanism, WCS makes F/FA overdose particularly dangerous.

First Polish case of fatal single-substance poisoning with cyclopropylfentanyl, a new synthetic opioid.

The article presents the first Polish case of fatal single-substance poisoning with cyclopropylfentanyl, a representative of fentanyl derivatives, whose victim was a 37-year-old man. This opioid was detected in biological material collected during medicolegal autopsy and in the syringe found near the deceased. Blood and urine samples were analyzed using liquid chromatography with mass spectrometry. The concentration of cyclopropylfentanyl was 24 ng/mL in blood and 73 ng/mL in urine.

Correlations between metabolism and structural elements of the alicyclic fentanyl analogs cyclopropyl fentanyl, cyclobutyl fentanyl, cyclopentyl fentanyl, cyclohexyl fentanyl and 2,2,3,3-tetramethylcyclopropyl fentanyl studied by human hepatocytes and LC-QTOF-MS.

Recently, a number of fentanyl analogs have been implicated in overdose deaths in Europe and in the US. So far, little is known of the molecular behavior of the structurally related subgroup; the alicyclic fentanyls. In this study, reference standards of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and 2,2,3,3-tetramethylcyclopropyl fentanyl (TMCPF) at a final concentration of 5 µM were incubated with cryopreserved human hepatocytes (1 × 106 cells/mL) for 0, 1, 3 and 5 h. The metabolites formed were identified by liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. The most abundant biotransformation found was N-dealkylation (formation of normetabolites) and oxidation of the alicyclic rings. As ring size increased, the significance of N-dealkylation decreased in favor of alicyclic ring oxidation. An example of this was cyclopropyl fentanyl, with a three-carbon ring, whose normetabolite covered 82% of the total metabolic peak area and no oxidation of the alicyclic ring was observed. In contrast, TMCPF, with a seven-carbon ring structure, rendered as much as 85% of its metabolites oxidized on the alicyclic ring. Other biotransformations found included oxidation of the piperidine ethyl moiety and/or the phenethyl substructure, glucuronidation as well as amide hydrolysis to form metabolites identical to despropionyl fentanyl. Taken together, this study provides a base for understanding the metabolism of a number of structurally related fentanyl analogs formed upon intake.

Global trends, local harms: availability of fentanyl-type drugs on the dark web and accidental overdoses in Ohio.

As America's opioid crisis has become an "epidemic of epidemics," Ohio has been identified as one of the high burden states regarding fentanyl-related overdose mortality. This study aims to examine changes in the availability of fentanyl, fentanyl analogs, and other non-pharmaceutical opioids on cryptomarkets and assess relationship with the trends in unintentional overdoses in Ohio to provide timely information for epidemiologic surveillance. Cryptomarket data were collected at two distinct periods of time: (1) Agora data covered June 2014-September 2015 and were obtained from Grams archive; (2) Dream Market data from March-April 2018 were extracted using a dedicated crawler. A Named Entity Recognition algorithm was developed to identify and categorize the type of fentanyl and other synthetic opioids advertised on cryptomarkets. Time-lagged correlations were used to assess the relationship between the fentanyl, fentanyl analog and other synthetic opioid-related ads from cryptomarkets and overdose data from the Cincinnati Fire Department Emergency Responses and Montgomery County Coroner's Office. Analysis from the cryptomarket data reveals increases in fentanyl-like drugs and changes in the types of fentanyl analogues and other synthetic opioids advertised in 2015 and 2018 with potent substances like carfentanil available during the second period. The time-lagged correlation was the largest when comparing Agora data to Cincinnati Emergency Responses 1 month later 0.84 (95% CI 0.45, 0.96). The time-lagged correlation between Agora data and Montgomery County drug overdoses was the largest when comparing synthetic opioid-related Agora ads to Montgomery County overdose deaths 7 months later 0.78 (95% CI 0.47, 0.92). Further investigations are required to establish the relationship between cryptomarket availability and unintentional overdose trends related to specific fentanyl analogs and/or other illicit synthetic opioids.

Assessment of the antinociceptive, respiratory-depressant, and reinforcing effects of the low pKa fluorinated fentanyl analogs, FF3 and NFEPP.

RATIONALE: Recent studies report that fentanyl analogs with relatively low pKa values produce antinociception in rodents without other mu opioid-typical side effects due to the restriction of their activity to injured tissue with relatively low pH values. However, it is unclear if and to what degree these compounds may produce mu opioid-typical side effects (respiratory depression, reinforcing effects) at doses higher than those required to produce antinociception. OBJECTIVES: The present study compared the inflammatory antinociceptive, respiratory-depressant, and reinforcing effects of fentanyl and two analogs of intermediate (FF3) and low (NFEPP) pKa values in terms of potency and efficacy in male and female Sprague-Dawley rats. METHODS: Nociception was produced by administration of Complete Freund's Adjuvant into the hind paw of subjects, and antinociception was measured using an electronic Von Frey test. Respiratory depression was measured using whole-body plethysmography. Reinforcing effects were measured in self-administration using a progressive-ratio schedule of reinforcement. The dose ranges tested for each drug encompassed no effect to maximal effects. RESULTS: All compounds produced full effects in all measures but varied in potency. FF3 and fentanyl were equipotent in antinociception and self-administration, but FF3 was less potent than fentanyl in respiratory depression. NFEPP was less potent than fentanyl in every measure. The magnitude of potency difference between antinociception and other effects was greater for FF3 than for NFEPP or fentanyl, indicating that FF3 had the widest margin of safety when relating antinociception to respiratory-depressant and reinforcing effects. CONCLUSIONS: Low pKa fentanyl analogs possess potential as safer analgesics, but determining the optimal degree of difference for pKa relative to fentanyl will require further study due to some differences between the current results and findings from prior work with these analogs.

Psychostimulant drug co-ingestion in non-fatal opioid overdose.

Introduction: In 2019, there were over 16,000 deaths from psychostimulant overdose with 53.5% also involving an opioid. Given the substantial mortality stemming from opioid and psychostimulant co-exposure, evaluation of clinical management in this population is critical but remains understudied. This study aims to characterize and compare clinical management and outcomes in emergency department (ED) overdose patients with analytically confirmed exposure to both opioids and psychostimulants with those exposed to opioids alone. Methods: This was a secondary analysis of a prospective consecutive cohort of ED patients age 18+ with opioid overdose at 9 hospital sites from September 21, 2020 to August 17, 2021. Toxicologic analysis was performed using liquid chromatography quadrupole time-of-flight mass spectrometry. Patients were divided into opioid-only (OO) and opioid plus psychostimulants (OS) groups. The primary outcome was total naloxone bolus dose administered. Secondary outcomes included endotracheal intubation, cardiac arrest, troponin elevation, and abnormal presenting vital signs. We employed t-tests, chi-squared analyses and multivariable regression models to compare outcomes between OO and OS groups. Results: Of 378 enrollees with confirmed opioid overdose, 207 (54.8%) had psychostimulants present. OO patients were significantly older (mean 45.2 versus 40.6 years, p < 0.01). OS patients had significantly higher total naloxone requirements (mean total dose 2.79 mg versus 2.12 mg, p = 0.009). There were no significant differences in secondary outcomes. Conclusion: Approximately half of ED patients with confirmed opioid exposures were also positive for psychostimulants. Patients in the OS group required significantly higher naloxone doses, suggesting potential greater overdose severity.

Combining surface-enhanced Raman spectroscopy (SERS) and paper spray mass spectrometry (PS-MS) for illicit drug detection.

There is an ongoing effort in the US illicit drug market to make new psychoactive compounds more potent and addictive. Due to continuous chemical modifications, many fentanyl analogs are developed and mixed with more traditional illicit drugs, such as cocaine and heroin. Detecting fentanyl and fentanyl analogs in these illicit drug mixtures has become more crucial because of the increased potency and associated health risks. Most confirmatory procedures require time-consuming and expensive, highly sophisticated laboratory equipment and experimental procedures, which can delay critical information that might save a victim or find a suspect. In this study, we propose miniaturizing and accelerating this process by combining surface-enhanced Raman spectroscopy (SERS) analysis and paper spray mass spectrometry (PS-MS). For this aim, dual-purposed paper substrates were developed through soaking in Au/Ag nanostars suspensions. These novel, in-house prepared paper SERS substrates showed stability for up to four weeks with and without the presence of drug compounds. Fentanyl analogs with similar SERS spectra were differentiated by coupling with PS-MS. The limit of detection (LOD) for fentanyl on the paper substrates is 34 µg/mL and 0.32 µg/mL for SERS and PS-MS, respectively. Fentanyl and fentanyl analogs show selective SERS enhancement that helped to detect trace amounts of these opioids in heroin and cocaine street samples. In short, we propose the combination of SERS/PS-MS by using modified paper substrates to develop cost-effective, sensitive, rapid, portable, reliable, and reproducible methods to detect illicit drugs, especially trace amounts of fentanyl and fentanyl analogs in illicit drug mixtures. The combination of these two category A techniques allows for the identification of illicit drugs according to the SWGDRUG guidelines.

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.

Does occupational exposure to fentanyl cause illness? A systematic review.

INTRODUCTION: The opioid epidemic in the United States continues to result in an increasing number of deaths and is increasingly dominated by fentanyl and fentanyl analogs. As a result, first responders are likely to come into contact with fentanyl-containing substances daily. Concerns persist regarding occupational exposure resulting in intoxication. We performed a systematic review to describe occupational illnesses from fentanyl and its analogs. METHODS: We conducted a systematic review of the literature following the 2020 Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines to assess the danger of occupational exposure to fentanyl. The PubMed, EMBASE, Google Scholar, SCOPUS, CINAHL, and National Institute for Occupational Safety and Health databases were queried to identify occupational fentanyl exposures. Studies included were single case reports, case series, observational studies, controlled studies, and abstracts from scientific presentations. We reviewed articles meeting the eligibility criteria and abstracted outcome data. Outcomes included study design, number of study subjects and study demographics, description of exposure, personal protective equipment used, duration of symptoms, illness developed, medical evaluation performed, treatment provided, hospitalizations, deaths, drug testing performed, and any situation review performed to prevent illness, analytical confirmation of the identity of culprit agent, and concentrations of drug in serum/blood. RESULTS: Our search yielded 454 citations after deduplication. After abstract and text review, 12 unique reports met the inclusion criteria. All identified studies were observational studies. Ten of the 12 were Health Hazard Evaluation reports from the National Institute for Occupational Safety and Health; two reports describe the same exposure case. There were no reported instances of comprehensive drug testing using liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry in exposed first responders. Among first responders possibly exposed to fentanyl or fentanyl analogs, none were admitted to the hospital, and only three first responders received naloxone. The three officers who received naloxone lacked recommended personal protective equipment and had subjective improvement of symptoms following naloxone. There were no instances of severe respiratory depression requiring assisted ventilation or hospital admission. Among forensic laboratory technicians, only one instance of detectable concentrations of fentanyl in urine was reported, and there were no instances of symptomatic cases. CONCLUSIONS: Among published reports of 27 first responders with symptoms after possible ambient fentanyl exposure, symptoms, recorded physical findings, and vital signs were inconsistent with acute opioid toxicity. Breaches in the recommended use of personal protective equipment appeared common. Only three persons received naloxone, although none had plausible effects of fentanyl. No suspected exposure to fentanyl led to hospitalization or death. Based on these low-quality data, there were no plausible opioid effects from ambient exposure to suspected fentanyl.

In vitro metabolite identification of acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl using LC-QTOF-HRMS together with synthesized references.

Acetylbenzylfentanyl, benzoylbenzylfentanyl, 3-fluoro-methoxyacetylfentanyl, and 3-phenylpropanoylfentanyl are fentanyl analogs that have been reported to the European Monitoring Centre for Drugs and Drug Addiction in recent years. The aim of this study was to identify metabolic pathways and potential biomarker metabolites of these fentanyl analogs. The compounds were incubated (5 µM) with cryopreserved hepatocytes for up to 5 h in vitro. Metabolites were analyzed with liquid chromatography-quadrupole time of flight-high-resolution mass spectrometry (LC-QTOF-HRMS). The experiments showed that acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-phenylpropanoylfentanyl were mainly metabolized through N-dealkylation (forming nor-metabolites) and 3-fluoro-methoxyacetylfentanyl mainly through demethylation. Other observed metabolites were formed by mono-/dihydroxylation, dihydrodiol formation, demethylation, dehydrogenation, amide hydrolysis, and/or glucuronidation. The experiments showed that a large number of metabolites of 3-phenylpropanoylfentanyl were formed. The exact position of hydroxy groups in formed monohydroxy metabolites could not be established solely based upon recorded MSMS spectra of hepatocyte samples. Therefore, potential monohydroxy metabolites of 3-phenylpropanoylfentanyl, with the hydroxy group in different positions, were synthesized and analyzed together with the hepatocyte samples. This approach could reveal that the ß position of the phenylpropanoyl moiety was highly favored; ß-OH-phenylpropanoylfentanyl was the most abundant metabolite after the nor-metabolite. Both metabolites have the potential to serve as biomarkers for 3-phenylpropanoylfentanyl. The nor-metabolites of acetylbenzylfentanyl, benzoylbenzylfentanyl, and 3-fluoro-methoxyacetylfentanyl do also seem to be suitable biomarker metabolites, as do the demethylated metabolite of 3-fluoro-methoxyacetylfentanyl. Identified metabolic pathways and formed metabolites were in agreement with findings in previous studies of similar fentanyl analogs.

Influence of carbon side chain length on the in vivo pharmacokinetic and pharmacodynamic characteristics of illicitly manufactured fentanyls.

Since 2016, illicitly manufactured fentanyls and fentanyl analogs (referred to as IMFs) have contributed to an increase in drug overdoses. Although fentanyl has been characterized and evaluated extensively in animals and humans, many of the clandestinely synthesized analogs of fentanyl have not and users may unknowingly ingest these IMFs leading to overdose and potentially death. The pharmacodynamic (PD) and pharmacokinetic (PK) properties of four IMFs and fentanyl were evaluated in Sprague-Dawley rats. A 300-µg/kg subcutaneous dose of each compound (fentanyl, acetylfentanyl, cyclopropylfentanyl, butyrylfentanyl, and valerylfentanyl) was given. PD parameters were measured using a tail flick meter and core body temperature. Blood was drawn to evaluate PK parameters utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS). Fentanyl displayed the greatest and longest lasting analgesia with a tail flick response of 10 s (the maximum cutoff). Additionally, fentanyl produced an average -4.9°C in core body temperature resulting in the greatest decrease in core body temperature. Acetylfentanyl, with the shortest carbon side chain, displayed the shortest T½, and lowest AUC and Cmax and resulted in an increase in body temperature. There were no other PK differences among the IMFs assessed. As IMFs are commonly seen on the streets and can pose significant risks to users (although these risks do depend on other factors such as dose and route of administration), there is a benefit to having the pharmacological properties of these compounds characterized to better understand the potential harm to humans.

The rise and rise of fentanyl in postmortem casework.

Forensic toxicology laboratories are navigating a period of time with increasing drug overdose deaths, an opioid epidemic, the impact of the COVID-19 pandemic, and the illicit drug market flooded with novel psychoactive substances. In New York City, the Department of Forensic Toxicology has experienced a 56% increase in postmortem casework in the past decade with fentanyl detected in 80% of all overdose deaths. Over a period of 2.5 years, 15,638 postmortem cases were tested for the presence of fentanyl and fentanyl analogs using liquid-chromatography tandem mass spectrometry (LCMSMS). Fentanyl was detected in approximately one third of cases and of these 4447 cases with femoral blood. A twofold increase in cases with high concentrations of fentanyl (>100 ng/mL) was observed between 2021 and 2022. The minor metabolite and precursor chemical, 4-ANPP (4-anilino-N-phenethylpiperidine) may help differentiate between illicit and licit fentanyl. 4-ANPP blood concentrations were <10 ng/mL in 98% of the cases and the 4-ANPP:fentanyl ratio was <0.67 for 99.1% of blood specimens. Only six cases had 4-ANPP concentrations higher than the corresponding fentanyl blood concentration. This study also highlights, the changing fentanyl analogs found in postmortem cases since 2016 in NYC with the emergence of fluorofentanyl initially identified in 2020 and continuing to dominate in comparison with the prevalence of other analogs, many of which are no longer detected in casework. The detection of one of the latest drugs to be mixed with fentanyl, namely xylazine, has also increased in prevalence by 36.7% in 2022 compared with 2021.

Review of analytical methods for screening and quantification of fentanyl analogs and novel synthetic opioids in biological specimens.

Fentanyl, fentanyl analogs, and other novel synthetic opioids (NSO), including nitazene analogs, prevail in forensic toxicology casework. Analytical methods for identifying these drugs in biological specimens need to be robust, sensitive, and specific. Isomers, new analogs, and slight differences in structural modifications necessitate the use of high-resolution mass spectrometry (HRMS), especially as a non-targeted screening method designed to detect newly emerging drugs. Traditional forensic toxicology workflows, such as immunoassay and gas chromatography mass spectrometry (GC-MS), are generally not sensitive enough for detection of NSOs due to observed low (sub-µg/L) concentrations. For this review, the authors tabulated, reviewed, and summarized analytical methods from 2010-2022 for screening and quantification of fentanyl analogs and other NSOs in biological specimens using a variety of different instruments and sample preparation approaches. Limits of detection or quantification for 105 methods were included and compared to published standards and guidelines for suggested scope and sensitivity in forensic toxicology casework. Methods were summarized by instrument for screening and quantitative methods for fentanyl analogs and for nitazenes and other NSO. Toxicological testing for fentanyl analogs and NSOs is increasingly and most commonly being conducted using a variety of liquid chromatography mass spectrometry (LC-MS)-based techniques. Most of the recent analytical methods reviewed exhibited limits of detection well below 1 µg/L to detect low concentrations of increasingly potent drugs. In addition, it was observed that most newly developed methods are now using smaller sample volumes which is achievable due to the sensitivity increase gained by new technology and new instrumentation.

Identifying reliable ions for the statistical differentiation of structurally similar fentanyl analogs.

Definitive identification of fentanyl analogs based on mass spectral comparison is challenging given the high degree of structural and, hence, spectral similarity. To address this, a statistical method was previously developed in which two electron-ionization (EI) mass spectra are compared using the unequal variance t-test. Normalized intensities of corresponding ions are compared, testing the null hypothesis (H0 ) that the difference in intensity is equal to zero. If H0 is accepted at all m/z values, the two spectra are statistically equivalent at the specified confidence level. If H0 is not accepted at any m/z value, then there is a significant difference in intensity at that m/z value between the two spectra. In this work, the statistical comparison method is applied to distinguish EI spectra of valeryl fentanyl, isovaleryl fentanyl, and pivaloyl fentanyl. Spectra of the three analogs were collected over a 9-month period and at different concentrations. At the 99.9% confidence level, the spectra of corresponding isomers were statistically associated. Spectra of different isomers were statistically distinct, and ions responsible for discrimination were identified in each comparison. To account for inherent instrument variations, discriminating ions for each pairwise comparison were ranked based on the magnitude of the calculated t-statistic (tcalc ) value. For a given comparison, ions with higher tcalc values are those with the greatest difference in intensity between the two spectra and, therefore, are considered more reliable for discrimination. Using these methods, objective discrimination among the spectra was achieved and ions considered most reliable for discrimination of these isomers were identified.

Field-portable detection of fentanyl and its analogs: A review.

The need to detect fentanyl and its analogs in the field is an important capability to help prevent unintentional exposure or overdose on these substances, which may result in death. Many portable methods historically used in the field by first responders and other field users to detect and identify other chemical substances, such as hazardous materials, have been applied to the detection and identification of these synthetic opioids. This paper describes field portable spectroscopic methods used for the detection and identification of fentanyl and its analogs. The methods described are automated colorimetric tests including lateral flow assays; vibrational spectroscopy (mid-infrared and Raman); gas chromatography-mass spectrometry; ion mobility spectrometry, and high-pressure mass spectrometry. In each case the background and key details of these technologies are outlined, followed by a discussion of the application of the technology in the field. Attention is paid to the analysis of complex mixtures and limits of detection, including the required spectral databases and algorithms used to interrogate these types of samples. There is also an emphasis on providing actionable information to the (likely) non-scientist operators of these instruments in the field.

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).