Butyryl Fentanyl Research Articles

In vitro and in vivo study of butyrylfentanyl and 4-fluorobutyrylfentanyl in female and male mice: Role of the CRF1 receptor in cardiorespiratory impairment.

Fentanyl analogues have been implicated in many cases of intoxication and death with overdose worldwide. The aim of this study is to investigate the pharmaco-toxicology of two fentanyl analogues: butyrylfentanyl (BUF) and 4-fluorobutyrylfentanyl (4F-BUF). In vitro, we measured agonist opioid receptor efficacy, potency, and selectivity and ability to promote interaction of the μ receptor with G protein and β-arrestin 2. In vivo, we evaluated thermal antinociception, stimulated motor activity and cardiorespiratory changes in female and male CD-1 mice injected with BUF or 4F-BUF (0.1-6 mg·kg-1). Opioid receptor specificity was investigated using naloxone (6 mg·kg-1). We investigated the possible role of stress in increasing cardiorespiratory toxicity using the corticotropin-releasing factor 1 (CRF1) antagonist antalarmin (10 mg·kg-1). Agonists displayed the following rank of potency at μ receptors: fentanyl > 4F-BUF > BUF. Fentanyl and BUF behaved as partial agonists for the β-arrestin 2 pathway, whereas 4F-BUF did not promote β-arrestin 2 recruitment. In vivo, we revealed sex differences in motor and cardiorespiratory impairments but not antinociception induced by BUF and 4F-BUF. Antalarmin alone was effective in blocking respiratory impairment induced by BUF in both sexes but not 4F-BUF. The combination of naloxone and antalarmin significantly enhanced naloxone reversal of the cardiorespiratory impairments induced by BUF and 4F-BUF in mice. In this study, we have uncovered a novel mechanism by which synthetic opioids induce respiratory depression, shedding new light on the role of CRF1 receptors in cardiorespiratory impairments by μ agonists.

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.

Old and new synthetic and semi-synthetic opioids analysis in hair: A review

New Psychoactive Substances (NPS), and particularly synthetic opioids, are constantly growing in the illicit market, and represent a particular danger due to their high potency and the fact that they're often not covered by legislation yet. Thus, there is the constant need for sensitive, reliable and reproducible techniques to detect and identify these substances in a variety of different matrices, both for clinical and forensic purposes. In particular, hair analysis can provide important information on past and/or continuative use of different substances such as psychoactive drugs.In this study, we report a review of the existing literature on synthetic opioids analysis in hair, in order to summarize the analytical methods currently used for synthetic and semi-synthetic opioids and NPS hair testing. Methanol with addition of diluted acidic solution represents the most frequently extraction solution. Purification procedures, either carried out through solid phase or liquid/liquid are still needed in order to achieve an adequate sensitivity; in particular, several methods provided a limit of detection lower than 1 pg/mg for most of the monitored substances. Chromatographic techniques coupled with mass spectrometric systems confirmed to be the best choice among the available analytical techniques for monitoring psychoactive substances and metabolites in keratin matrices. Though high-resolution mass spectrometric instruments, such as Time of Flight, are gaining interest, triple quad still guarantees better sensitivity. Beside common synthetic and semi-synthetic opioids, such as hydromorphone, hydrocodone and tramadol, the most frequently detected compounds in hair samples, among fentanyl derivatives, are furanyl fentanyl, sufentanil and butyryl fentanyl.

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.

Affinity, potency, efficacy, selectivity, and molecular modeling of substituted fentanyls at opioid receptors

Substituted fentanyls are abused and cause rapid fatal overdose. As their pharmacology is not well characterized, we examined in vitro pharmacology and structure–activity relationships of 22 substituted fentanyls with modifications of the fentanyl propyl group, and conducted in silico receptor/ligand modeling. Affinities for mu, kappa, and delta opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in mammalian cells were assessed in agonist radioligand binding assays. At MOR, furanyl fentanyl had higher affinity than fentanyl, while acryl, isobutyryl and cyclopropyl fentanyls had similar affinities. Comparing affinities, thiophene and methoxyacetyl fentanyls had highest selectivity for MOR (2520- and 2730-fold compared to KOR and DOR, respectively). Functional activities were assessed using [35S]GTPγS binding assays. At MOR, furanyl fentanyl had higher potency and 11 substituted fentanyls had similar high potencies compared to fentanyl. Eight compounds were full agonists of MOR and twelve compounds were partial agonists, with efficacies from 8.8% (phenyl fentanyl) to 60.2% (butyryl fentanyl). All efficacious compounds had selective functional potency for MOR. The predicted binding poses of flexible fentanyl and rigid morphine against MOR show partially overlapping binding pockets, with fentanyl maintaining additional interaction with the transmembrane (TM) 2 helix. Subsequent molecular dynamics simulations revealed a predominant fentanyl binding pose involving various TM interactions. The piperidine nitrogen of substituted fentanyls establishes a salt-bridge with the conserved D-1473.32 residue and the propanamide carbonyl group establishes a hydrogen bond with the indole side-chain (–NH) of W-3187.35. The simulation suggests theN-linked phenethyl group may regulate the rotameric switch of W-2936.48. The predicted binding pose, in conjunction with in vitro binding affinity, clarified the molecular basis of the binding/selectivity profile of furanyl fentanyl and other derivatives at the sequence level. In summary, substituted fentanyls with high MOR potencies, selectivities, and efficacies are likely to have abuse and overdose potential. The work presented here is a prototype to investigate fentanyl derivatives and their abuse potential.

Determination of fentanyl and 19 derivatives in hair: Application to an Italian population

Nowadays fentanyl and its analogs represent the most numerous group among synthetic opioid and, due to their higher potency in comparison to traditionl opioids, the main cause of the critical increase of fatal intoxications opioids-intake related in the USA. We developed an LC-MS/MS method for the detection and quantification of fentanyl and its analogs in hair, then applied to 117 real samples, 97 collected from drugs users and 20 from postmortem cases of drugs addicts. The ionization and MRM parameters have been optimized for 27 molecules: 20 reached the acceptance criteria for identification and quantification. LODs and LOQs of 0.2 and 0.5 pg/mg, respectively, were reached for most of the substances, except for five compounds for which were set at 0.5 and 1.0 pg/mg. 2 out of the 97 samples collected from drug users tested positive; one for carfentanil, butyryl fentanyl, THFF and ocfentanil; the other one for 3-methyl norfentanyl. 2 out of the 20 postmortem samples show positive results: one only for fentanyl, the other for furanyl fentanyl, acetyl fentanyl, methoxyacetyl fentanyl, methoxyacetyl norfentanyl, ocfentanil and 4-ANPP. Despite the relatively small number of samples, the results suggest that the method should be included in routine hair analyses for monitoring the new synthetic opioids potential intake by drug users.

Determination of 21 synthetic cathinones, phenethylamines, amphetamines and opioids in influent wastewater using liquid chromatography coupled to tandem mass spectrometry

Human consumption of illicit novel psychoactive substances (NPS) is a continuing problem. New derivatives are constantly appearing, circumventing national and international laws. The use of these compounds tend to be sporadic and many are consumed as mixtures, meaning very low amounts of each are detectable at any one time. The analysis of excreted NPS in wastewater provides information on community prevalence. A wastewater-based epidemiology approach has been applied in the current study for the quantification of 21 NPS. These include three phenethylamines (25B–NBOMe, 25C–NBOMe, 25I–NBOMe), ten synthetic cathinones (3-ethylmethcathinone (3-EMC), 3-methylbuphedrone, 3-methylmethcathinone (3-MMC), 4-fluoromethcathinone (4-FMC), 4-methylbuphedrone, 4-methylethcathinone (4-MEC), buphedrone, butylone, N-ethylpentylone and pentylone), five synthetic opioid analgesics (AH-7921, butyryl fentanyl, furanyl fentanyl, U-47700 and valeryl fentanyl) as well as the synthetic amphetamine 4-fluoroamphemtaine (4-FA), ketamine analogue methoxetamine and methiopropamine. Limits of detection were between 0.01 and 0.5 ng/L and limits of quantification were between 0.05 and 1 ng/L. The method was applied to wastewater samples from South Australia collected over the Christmas-New Year period when recreational drug use tends to be high. Seven NPS (butylone, butyryl fentanyl, furanyl fentanyl, methoxetamine, N-ethylpentylone, pentylone and valeryl fentanyl) were found, with N-ethylpentylone showing the highest mass loads at 36 mg/day/1000 inhabitants.

On-site detection of fentanyl and its derivatives by field portable nano-liquid chromatography-electron lonization-mass spectrometry (nLC-EI-MS)

There is a need to develop a field-portable analytical technique for on-site confirmatory analysis of illicit drugs that are flooding illegal markets. GC-EI-MS and LC-ESI-MS/MS are two commonly used analytical techniques in forensic analyses of drugs and explosives. For years, portable GC–MS has been used for on-site identification of chemical compounds such as illicit drugs and explosives. The primary advantage of the GC–MS is its extensive library searching capability, which allows identification of unknown compounds. LC-ESI-MS has been useful in addressing the issue of thermal degradation; however, minimal fragmentation is observed under ESI-MS. By using a tandem mass spectrometer (MS/MS), compounds can be fragmented; however, no wide-ranging library for LC-MS/MS currently exists. Previously, LC electron ionization MS (LC-EI-MS) was developed to combine the advantages of the LC for sample introduction at ambient temperature with the EI-MS ionization, fragmentation, and library searching capability; however, currently no full functionality portable LC-EI-MS is available. Here for the first time we report development of a field-portable nLC-EI-MS by using an Easy nanoLC in conjunction with a full-functionality field-portable Viking 573 mass spectrometer. For proof of concept, the nLC-EI-MS was transported to the field and tested using a low power (1500 Watt) portable generator. A mixture containing heroin and synthetic opioids including fentanyl, carfentanil (oxalate salt), acetyl fentanyl, and butyryl fentanyl was successfully analyzed and identified via library match in less than 10 min. The results were compared with a laboratory nLC-ESI-MS as well as a laboratory GC–MS. Excellent reproducibility of retention times was achieved.

Unique Structural/Stereo-Isomer and Isobar Analysis of Novel Fentanyl Analogues in Postmortem and DUID Whole Blood by UHPLC-MS-MS.

The presented analytical method enabled the Toxicology Department at the Cuyahoga County Medical Examiner's Office to identify 26 and quantitatively report 24 compounds in 500μL of whole blood, including fentanyl analogues (fentalogues) such as methoxyacetyl fentanyl (MeOAF) and cyclopropyl fentanyl (CPF). This second-generation method (FG2) was developed with the objective to improve the existing analysis (FG1) by decreasing sample size, lowering limits of detection (LOD) and lower limit of quantitation, minimizing ion suppression and resolving chromatographic interferences. Interferences may occur in the analysis of fentanyl, MeOAF, CPF, 3-methylfentanyl (3MF), butyryl fentanyl and isobutyryl fentanyl due to isobars and structural or geometric isomerism with another analogue or metabolite. The isomeric and isobaric fentalogues were grouped into three sets. The LOD established for Set 1 [MeOAF, para-methoxyacetyl fentanyl, para-fluoro acryl fentanyl (isobar), fentanyl carbamate], 2-furanyl fentanyl, Set 2 [CPF, (E)-crotonyl fentanyl] and carfentanil was 0.0125ng/mL. The LOD established for N-methyl norfentanyl, norfentanyl, norcarfentanil, despropionyl fentanyl (4-ANPP), acetyl fentanyl, β-hydroxy fentanyl, benzyl fentanyl, acryl fentanyl, alfentanil, fentanyl, para-fluoro fentanyl, Set 3 [(±)-trans-3MF, (±)-cis-3MF, isobutyryl and butyryl fentanyl], para-fluoroisobutyryl fentanyl, sufentanil, phenyl fentanyl and cyclopentenyl fentanyl was 0.0625ng/mL. Seven-point linear calibration curves were established between 0.025 and 4.0ng/mL for the 8 analytes with the lower LOD and 0.125 and 20ng/mL for the 18 analytes with the higher LOD. 4-ANPP and cyclopentenyl fentanyl met qualitative reporting criteria only. The results for five postmortem and two driving under the influence of drugs authentic case samples are presented. To the authors' knowledge, FG2 is the first published method that achieved baseline resolution of the nine structural/stereo isomers and one isobar by ultra-high performance liquid chromatography-MS-MS and provided quantitative validation data for nine compounds. FG2 may be used as the new baseline for future isomers that need to be chromatographically separated.

Postmortem Toxicology of New Synthetic Opioids.

One hundred fifteen Americans die every day from opioid overdose. These overdose fatalities have been augmented by the increased availability of potent synthetic opioids, such as fentanyl and its derivatives. The death rate of synthetic opioids, other than methadone, increased by 72.2% from 2014 to 2015, and doubled from 2015 to 2016, situating the USA in the midst of an opioid overdose epidemic. The analytical identification of these opioids in postmortem samples and the correct toxicological data interpretation is critical to identify and implement preventive strategies. This article reviews the current knowledge of postmortem toxicology of synthetic opioids and the chemical and pharmacological factors that may affect drug concentrations in the different postmortem matrices and therefore, their interpretation. These factors include key chemical properties, essential pharmacokinetics parameters (metabolism), postmortem redistribution and stability data in postmortem samples. Range and ratios of concentrations reported in traditional and non-traditional postmortem specimens, blood, urine, vitreous humor, liver and brain, are summarized in tables. The review is focused on fentanyl and derivatives (e.g., acetyl fentanyl, butyryl fentanyl, carfentanil, furanyl fentanyl, 4-methoxybutyrylfentanyl, 4-fluorobutyrylfentanyl, ocfentanil) and non-traditional opioid agonists (e.g., AH-7921, MT-45, U-47700). All of these data are critically compared to postmortem data, and chemical and pharmacological properties of natural opioids (morphine), semi-synthetic (oxycodone, hydrocodone, hydromorphone, and oxymorphone), and synthetic opioids (methadone and buprenorphine). The interpretation of drug intoxication in death investigation is based on the available published literature. This review serves to facilitate the evaluation of cases where synthetic opioids may be implicated in a fatality through the critical review of peer reviewed published case reports and research articles.

Quantitative Analysis of Fentanyl and Six Fentanyl Analogs in Postmortem Specimens by UHPLC-MS-MS.

Since 2013, opioid-related deaths have increased in Miami-Dade County, FL due to fentanyl and fentanyl analogs in the local heroin supply. From 2014 to 2015, a near 600% increase in fentanyl and fentanyl analog-related deaths was observed, followed by another 200% increase in 2016. In addition to fentanyl, six fentanyl analogs were identified in this time period: β-hydroxythiofentanyl, acetyl fentanyl, furanyl fentanyl, carfentanil, butyryl fentanyl and para-fluoroisobutyryl fentanyl. As a result, an analytical method to quantify these compounds in postmortem biological fluids and tissues using solid-phase extraction with analysis by ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS) was developed and validated. All analytes except carfentanil were linear from 1 to 50 ng/mL with a limit of detection (LOD) of 0.5 ng/mL; carfentanil was linear from 0.2 to 10 ng/mL with a LOD of 0.1 ng/mL. For all analytes, bias was within ±14%, intra- and inter-day precision were ≤5% and extraction efficiency was >87%. Dilution integrity was assessed at 1:1, 1:4 and 1:9 (v/v) ratios; percent error from target was <12% for all analytes. No exogenous interferences were observed. Analytes were stable in preserved whole blood stored at 4°C for 9 months; extracted samples were stable in the refrigerated autosampler (15°C) for up to 72 h. The ability to distinguish the isomer pairs isobutyryl/butyryl fentanyl and p-fluoroisobutyryl/p-fluorobutyryl fentanyl is achieved by this method. The method was applied to 312 cases in which fentanyl and/or fentanyl analogs were previously identified. Postmortem concentrations in blood ranged from <0.2 to 0.73 ng/mL for carfentanil and from 30.6 to 91.7 ng/mL for p-fluoroisobutyryl fentanyl in cases in which these analytes were listed as the sole cause of death.

Carfentanil in Impaired Driving Cases and the Importance of Drug Seizure Data

Drug seizures containing carfentanil continue to increase in Palm Beach County, FL, USA despite international efforts to control the distribution of the drug. The analysis of drug seizures from the county in 2016 and 2017 demonstrated that carfentanil was the most commonly identified fentanyl analog and was most often detected in combination with heroin, fentanyl, furanyl fentanyl and/or other fentanyl analogs. Carfentanil is an ultra-potent opioid requiring a method with adequate sensitivity for detection in blood specimens from impairment cases. Previous research indicated that carfentanil could not be identified in biological specimens by routine drug testing protocols and that detection requires targeted analysis with greater sensitivity. Due to the prevalence of carfentanil in drug seizures, a sensitive targeted qualitative method by liquid chromatography tandem mass spectrometry in antemortem blood samples was evaluated, validated and implemented. The method included identification of carfentanil, acetyl fentanyl, beta-hydroxythiofentanyl, butyryl fentanyl, fentanyl, furanyl fentanyl, kavain, mitragynine, MT-45 and U-47700. In 2017 carfentanil was the second most frequently detected drug, after ethanol, in driving under the influence blood cases. Of all blood cases in which drug testing was performed (n = 145), carfentanil was detected in 38% followed by alprazolam (29%), fentanyl (27%), delta-9-tetrahydrocannabinol (24%) and morphine (23%). In toxicology cases carfentanil was rarely identified alone (only four cases) and was most commonly identified with other opioids (73% of cases), benzodiazepines (43%) and stimulants (29%). The high incidence of carfentanil-positive cases detected by this method underscores the importance of continually monitoring regional drug seizure trends, and evaluating the adequacy of toxicology testing panels based on these trends.

LC-MS/MS-Based Method for the Multiplex Detection of 24 Fentanyl Analogues and Metabolites in Whole Blood at Sub ng mL-1 Concentrations.

The United States and numerous other countries worldwide are currently experiencing a public health crisis due to the abuse of illicitly manufactured fentanyl (IMF) and its analogues. This manuscript describes the development of a liquid chromatography-tandem mass spectrometry-based method for the multiplex detection of N = 24 IMF analogues and metabolites in whole blood at concentrations as low as 0.1–0.5 ng mL–1. These available IMFs were fentanyl, norfentanyl, furanyl norfentanyl, remifentanil acid, butyryl norfentanyl, remifentanil, acetyl fentanyl, alfentanil, AH-7921, U-47700, acetyl fentanyl 4-methylphenethyl, acrylfentanyl, para-methoxyfentanyl, despropionyl fentanyl (4-ANPP), furanyl fentanyl, despropionyl para-fluorofentanyl, carfentanil, (±)-cis-3-methyl fentanyl, butyryl fentanyl, isobutyryl fentanyl, sufentanil, valeryl fentanyl, para-fluorobutyryl fentanyl, and para-fluoroisobutyryl fentanyl. Most IMF analogues (N = 22) could be easily distinguished from one another; the isomeric forms butyryl/isobutyryl fentanyl and para-fluorobutyryl/para-fluoroisobutyryl fentanyl could not be differentiated. N = 13 of these IMF analogues were quantified for illustrative purposes, and their forensic quality control standards were also validated for limit of detection (0.017–0.056 ng mL–1), limit of quantitation (0.100–0.500 ng mL–1), selectivity/sensitivity, ionization suppression/enhancement (87–118%), process efficiency (60–95%), recovery (64–97%), bias (<20%), and precision (>80%). This flexible, time- and cost-efficient method was successfully implemented at the Montgomery County Coroner’s Office/Miami Valley Regional Crime Laboratory in Dayton, Ohio, where it aided in the analysis of N = 725 postmortem blood samples collected from February 2015 to November 2016.

Multiple fatalities in the North of England associated with synthetic fentanyl analogue exposure: Detection and quantitation a case series from early 2017

BackgroundSynthetic fentanyl analogues are highly potent opioid drugs which have no pharmaceutical use in humans. We detected the synthetic fentanyl analogues; carfentanil, butyryl fentanyl, fluorobutyrylfentanyl, furanylfentanyl, and alfentanil as well as fentanyl itself in 25 cases in early 2017. There have been no previous reports of synthetic fentanyl deaths in the United Kingdom (UK). MethodsCases in which the history clearly stated drug use but where a post mortem blood morphine concentration was lower than would be expected to explain the sudden death, were referred for further analysis by high resolution accurate mass (HRAM) mass spectrometry. Results25 post mortem cases in which synthetic fentanyl analogues were implicated in the cause of death were reported from January to May 2017. No cases were seen in June 2017. The age range was 21–54 years and 22 were male. There was a history of heroin use, or markers of heroin use on toxicology screening in 21/25 cases. Carfentanil and fentanyl were detected in 7 cases. Multiple synthetic fentanyl analogues were present in 13 cases, with the remaining 5 cases having only carfentanil present. Synthetic fentanyl analogues were detected in combination with other drugs in all cases. Significant concentrations of ethanol were detected in only 2 cases. The concentration range of carfentanil in blood was 90–4004pg/mL. Of note, the 3 cases in which ante mortem carfentanil was quantified ranged from 21 to 98pg/mL. In all cases, death was attributed to combined central nervous system depression. ConclusionsThis paper highlights a new and rapid emergence of these drugs into the UK illicit drug arena. Synthetic fentanyl analogues represent a significant challenge both analytically and clinically within the groups who misuse drugs. It is worthwhile considering the possibility of the presence of these drugs in cases in which a toxicological cause of death is not apparent analytically but there is a history of drug use and circumstantial evidence exists to support a drug-related death as the most likely cause. It may be that synthetic fentanyl analogues should be screened for routinely to avoid reporting any false negative results, but the cost implications and viability of this have not been fully evaluated.

Development of a quantitative approach in blood plasma for low-dosed hallucinogens and opioids using LC-high resolution mass spectrometry

The WHO annually reports an increasing abuse of new psychoactive substances (NPS), which are a heterogeneous group of synthetic drugs and are consumed as substitute for controlled drugs of abuse. In this work, we focused on highly potent derivatives such those of phenethylamine (2C), N-2-methoxybenzyl phenethylamine (NBOMes), lysergic acid diethylamide (LSD), and fentanyl. Severe to fatal intoxications were described due to their high potency. Therefore, they have to be taken at very low doses resulting in low blood concentration in the low ng/mL range, which is a challenge for reliable analytical detection and quantification. The aim of this work was therefore to design a simple, robust, and fast method for simultaneous detection and quantification of multiple substances of the different classes in human blood plasma using liquid chromatography (LC) high resolution (HR) mass spectrometry (MS) with alternating HR full-scan (HRFS) MS and “All-ions fragmentation” (AIF) MS. The paper contains results of the method validation according to the EMA guideline, including intra-/interday accuracy and precision, matrix effects, storage and benchtop analyte stability as well as selectivity and carryover. All validation criteria were fulfilled for most tested compounds except for the NBOMe derivatives, one out of ten 2C-derivatives and butyryl fentanyl, which failed at accuracy and/or precision or at the acceptance criteria for matrix effect. Reasons for this are discussed and solutions presented. Despite some limitations, the HRFS + AIFMS analysis allowed detection of most of the analytes down to 0.1ng/mL, seamless integration of new or unexpected analytes, identification and quantification with no limitations on the number of monitored compounds, and reevaluation of the acquired data also concerning metabolism studies using group-indicating fragment ions.

Qualitative Identification of Fentanyl Analogs and Other Opioids in Postmortem Cases by UHPLC-Ion Trap-MSn.

Since 2013, the Miami-Dade County Medical Examiner Department has experienced an increase in the number of opioid-related deaths. The majority of cases coincided with the introduction of fentanyl into the local heroin supply. From 2014 to 2015, Miami-Dade County experienced a near 600% increase in fentanyl-related deaths, followed by an additional 200% increase in 2016. In 2015, two novel fentanyl analogs were identified in medical examiner cases: beta-hydroxythiofentanyl and acetyl fentanyl. In 2016, four additional fentanyl analogs emerged: para-fluoroisobutyryl fentanyl, butyryl fentanyl, furanyl fentanyl and carfentanil, as well as the synthetic opioid U-47700. In order to address this epidemic, a method was developed and validated to identify 44 opioid-related and analgesic compounds in postmortem samples using ultra high performance liquid chromatography ion trap mass spectrometry with MSn capabilities. The limit of detection for all compounds ranged from 0.1 to 5 ng/mL, with a majority having MS3 spectral fragmentation. Blood, urine, liver or brain specimens from ~500 postmortem cases were submitted for analysis based on case history and/or initial screening results. Of those cases, 375 were positive for illicit fentanyl and/or one or more fentanyl analogs. Due to the potency of these compounds, they were almost always included in the cause of death. Worth emphasizing and extremely alarming is the detection of carfentanil in 134 cases, 104 of which were initially missed by gas chromatography mass spectrometry. By incorporating this sensitive, highly specific, and evolving screening procedure into the workflow, the toxicology laboratory continues to effectively assist the medical examiners in determining the cause and manner of death of decedents in Miami-Dade County.

Validation of the Neogen® Fentanyl ELISA Kit for Blood and Urine.

The Neogen® Fentanyl ready-to-use enzyme-linked immunosorbent assay kit was validated following the Scientific Working Group for Forensic Toxicology Standard Practices for Method Validation in Forensic Toxicology Laboratory Guidelines. Two decision points, 0.5 and 1 ng/mL, were successfully validated for whole blood. For urine, two decision points, 1 and 5 ng/mL, were also successfully validated. The validation included the evaluation of sensitivity, precision, specificity, carryover, plate drift, ruggedness/robustness and a case sample evaluation. The empirically determined limit of detection was 0.25 ng/mL for blood and 0.5 ng/mL for urine. Precision was determined at five different concentrations ranging from 0.25 to 1.5 ng/mL with 15 replicates at each level for whole blood and demonstrated a <2.4% coefficient of variation (CV). In urine, the CV was <5.6% at six different concentrations from 0.5 to 7.5 ng/mL with 15 replicates at each level. Cross-reactivity was evaluated for norfentanyl, acetyl fentanyl, 4-anilino-N-phenethylpiperidine, beta-hydroxythiofentanyl, butyryl fentanyl and furanyl fentanyl.

Forced degradation of fentanyl: Identification and analysis of impurities and degradants

Fentanyl, N-(1-phenethylpiperidin-4-yl)-N-phenylpropionamide is a rapid-acting, powerful opioid analgesic used extensively for anesthesia and chronic pain management. A forced degradation study of fentanyl active pharmaceutical ingredient (API) was performed using light, acid, base, heat and oxidation. Under acidic conditions, fentanyl was shown to degrade to N-phenyl-1-(2-phenylethyl)-piperidin-4-amine (PPA 1 1 N-Phenyl-1-(2-phenylethyl)-piperidin-4-amine. ). Fentanyl was stable to light exposure and base treatment with no degradation observed. Oxidation with hydrogen peroxide produced fentanyl N-oxide by rapidly oxidizing the nitrogen on the piperidine ring. Five degradants were formed during thermal degradation of fentanyl. The two known degradants included propionanilide (PRP 2 2 Propionanilide. ) and norfentanyl (NRF 3 3 Norfentanyl. ). The three unknown degradants were first identified by mass using LC/MS, and postulated compounds were synthesized and confirmed by LC/MS and 1H NMR. These degradants were identified as 1-phenethylpyridinium salt (1-PEP 4 4 1-Phenethylpyridinium salt. ), 1-phenethyl-1H-pyridin-2-one (1-PPO 5 5 1-Phenethyl-1H-pyridin-2-one. ), and 1-styryl-1H-pyridin-2-one (1-SPO 6 6 1-Styryl-1H-pyridin-2-one. ). In addition to the seven degradants, three known process impurities, acetyl fentanyl, pyruvyl fentanyl and butyryl fentanyl were also detected by reverse-phase high performance liquid chromatography (HPLC) with UV detection. All degradants and impurities were identified and confirmed using authentic materials. Method validation was performed for the assay of fentanyl and its related compounds in accordance to ICH guideline Q2(R1), and the method was demonstrated to be specific, linear ( r > 0.999 for fentanyl assay and r > 0.996 for related compounds), accurate (recovery > 99.6% for fentanyl assay and recovery > 91.0 for related compounds), precise (%RSD < 0.8% for fentanyl assay and <4.8% for related compounds), sensitive (limit of detection = 0.08 μg/mL or 0.016% of nominal concentration), robust and suitable for its intended use. The chemical structures for the degradants and impurities were submitted to three in silico toxicity programs to identify any structural alerts.