Abstract

Following series of synthetic cannabinoid and synthetic cathinone derivatives, the illicit drug market has begun to see increased incidence of synthetic opioids including fentanyl and its derivatives, and other chemically unrelated opioid agonists including AH-7921 and MT-45. Among the most frequently encountered compounds in postmortem casework have been furanyl fentanyl (N-(1-(2-phenylethyl)-4-piperidinyl)-N-phenylfuran-2-carboxamide, Fu-F) and U-47700 (trans-3,4-dichloro-N-(2-(dimethylamino)cyclohexyl)-N-methylbenzamide). Both drugs have been reported to be present in the heroin supply and to be gaining popularity among recreational opioid users, but were initially developed by pharmaceutical companies in the 1970s as candidates for development as potential analgesic therapeutic agents. A method was developed and validated for the analysis of U-47700, U-50488 and furanyl fentanyl in blood specimens. A total of 20 postmortem cases, initially believed to be heroin or other opioid-related drug overdoses, were submitted for quantitative analysis. The analytical range for U-47770 and U-50488 was 1–500 and 1–100 ng/mL for furanyl fentanyl. The limit of detection was 0.5 ng/mL for all compounds. Within the scope of the method, U-47700 was the only confirmed drug in 11 of the cases, 5 cases were confirmed for both U-47700 and furanyl fentanyl, and 3 cases were confirmed only for furanyl fentanyl. The mean and median blood concentrations for U-47700 were 253 ng/mL (±150) and 247 ng/mL, respectively, range 17–490 ng/mL. The mean and median blood concentrations for furanyl fentanyl were 26 ng/mL (±28) and 12.9 ng/mL, respectively, range 2.5–76 ng/mL. Given the widespread geographical distribution and increase in prevalence in postmortem casework, toxicology testing should be expanded to include testing for “designer opioids” in cases with histories consistent with opioid overdose but with no traditional opioids present or insufficient quantities to account for death.

Introduction

Opiate drugs are commonly abused in the USA and are common toxicological findings in death investigation cases. The substances involved have included natural products (e.g., morphine, codeine), semi-synthetic opioids (e.g., hydrocodone, oxycodone, hydromorphone and diacetylmorphine) and synthetic opioids (e.g., fentanyl, sufentanil). Within the last 2 years, we have observed a sharp increase in novel non-prescribed opioids in our death investigation casework. These include many fentanyl analogs such as acetyl fentanyl (1, 2) and butyryl fentanyl (3), and other novel compounds such as MT-45 (4, 5) and AH-7921 (6, 7). Structures of these and related compounds are shown in Figure 1. These substances are increasingly encountered on their own or in combination with other opioids in what appear to be routine intravenous drug deaths. Traditional laboratory approaches to drug screening may fail to detect them due to little or no cross-reactivity on traditional immunoassay tests (8), or failure to include fentanyl analogs in the mass spectral libraries used in drug screening.

Figure 1.

Chemical structure of (a) acetyl fentanyl, (b) butyryl fentanyl, (c) furanyl fentanyl, (d) U-47700, (e) U-50488, (f) MT-45, (g) AH-7921 and (h) AH-7921 d3 (ISTD).

Figure 1.

Chemical structure of (a) acetyl fentanyl, (b) butyryl fentanyl, (c) furanyl fentanyl, (d) U-47700, (e) U-50488, (f) MT-45, (g) AH-7921 and (h) AH-7921 d3 (ISTD).

Beginning in late 2015, we began to encounter two novel opioids, furanyl fentanyl and U-47700 (Figure 1) in postmortem toxicology casework.

U-47700 (trans-3,4-dichloro-N-(2-(dimethylamino)cyclohexyl)-N-methylbenzamide) (Figure 1) is an opioid analgesic drug developed by the pharmaceutical company Upjohn in the 1970s and is structurally related to AH-7921, an earlier experimental opioid agonist developed by the drug company Allen and Hanbury (9). U-47700 is a selective µ-opioid receptor agonist, and in animal models has been demonstrated to have ~7.5 times the potency of morphine (10, 11), while fentanyl has a potency of 50–100 times that of morphine. U-47700 was never studied in humans, but based on animal models is expected to produce effects similar to those of other potent opioid agonists, such as analgesia, sedation, euphoria, constipation, itching and respiratory depression. U-47700 is currently uncontrolled in the USA; however, in March of 2016, its analog AH-7921 was placed into the USA Federal Schedule as a schedule I compound (12). Recently Elliott et al. reported a single case in the UK of a fatality involving U-47700 (13).

U-50488 (trans-3,4-dichloro-N-methyl-N-[​2-(1-pyrrolidinyl) cyclohexyl]-benzeneacetamide), also developed by Upjohn at the same time (9), is a κ-opioid receptor (KOR) agonist, with some reported µ-opioid receptor respiratory antagonist effects (14). U-50488 has been studied in animal models for its analgesic, diuretic, antitussive and anticonvulsant properties (15). KOR agonists also mediate the dysphoria and hallucinations associated with drugs such as levorphanol, pentazocine and salvinorin A. U-50488 is currently uncontrolled and available from online research chemical supply companies; however, its abuse potential is unknown. KOR agonists have, however, been investigated as possible pharmacotherapeutic agents for treatment of addiction, and other KOR agonists are generally considered to be addictive (16).

Furanyl fentanyl (N-(1-(2-phenylethyl)-4-piperidinyl)-N-phenylfuran-2-carboxamide, “Fu-F”) is a fentanyl derivative, which was synthesized and patented in 1986, and has a furanyl ring in place of the methyl group adjacent to the carbonyl bridge (Figure 1). Furanyl fentanyl has an ED-50 in mice of 0.02 mg/kg (17), which is comparable to that of fentanyl (0.016 mg/kg) (18). Although never studied in humans, furanyl fentanyl would be expected to produce effects similar to those of fentanyl. One death in Sweden has been reported involving administration of furanyl fentanyl (7).

Methods

Chemicals and reagents

U-47700 was purchased from Cayman Chemical (Ann Arbor, Michigan), U-50488 hydrochloride was purchased from Tocris Bioscience (Bristol, UK), furanyl fentanyl was purchased from Finechems.net (Poland) and AH-7921-d3 hydrochloride (internal standard) was obtained from Cerilliant Corporation (Round Rock, Texas). The furanyl fentanyl standard was subsequently verified by liquid chromatography tandem mass spectrometry (LCMSMS), gas chromatography mass spectrometry (GCMS) and liquid chromatography quadrupole time of flight mass spectrometry (LCQTOF), analysis against a certified reference standard from Cayman Chemical, and was determined a purity of 75%. Calibration curves for quantitative analysis were adjusted accordingly.

Liquid chromatography mass spectrometry  grade water and methanol for liquid chromatography mobile phases were purchased from Honeywell (Muskegon, MI). Formic acid was purchased from Sigma Aldrich (St. Louis, MO).

Standards, calibrators and control preparation

Stock solutions of U-47700, U-50488 and furanyl fentanyl were prepared at a concentration of 1000 ng/µL in methanol and stored at 20°C in amber glass vials. A stock solution of the internal standard, AH-7921-d3, was purchased at a concentration of 100 ng/µL. A working solution of the internal standard was prepared at a concentration of 1 ng/µL.

Calibrators were prepared by spiking from dilutions created from the stock at concentrations of 10, 1 and 0.1 ng/µL into defribrinated sheep's blood. Only a single source of the reference material was available at the time of analysis, so controls were spiked by a different individual from the same stock. Calibrators for U-47700 and U-50488 were prepared at concentrations of 1, 3, 10, 30, 50, 100, 250 and 500 ng/mL. Calibrators for furanyl fentanyl were prepared at concentrations of 1, 3, 10, 30, 50 and 100 ng/mL. For optimal quantitative analysis of U-47700 and U-50488, calibrators below the lowest quantitative value in the samples were dropped to process all samples within a range within two orders of magnitude or less. Controls were prepared at values of 5, 80 and 400 ng/mL to allow the curve to be processed using an appropriate range for the amount of drug present in the samples. For example, if the appropriate range for quantitation was 1–100 ng/mL, the 5  and 80 ng/mL controls were used as low and high controls; if the appropriate range was 30–500 ng/mL, the 80 and 400 ng/mL controls were used as low and high controls. The controls for furanyl fentanyl were prepared at 5 and 80 ng/mL.

Instrumentation

Analysis was performed by LCMSMS using an Agilent 1100 series high-performance liquid chromatograph coupled to an Agilent 6430 triple quadrupole tandem mass spectrometer (Santa Clara, CA). Chromatographic separation was achieved on an Agilent ZORBAX Eclipse Plus C18 column (4.6 × 100 mm, 3.5 µm) with 0.1% formic acid in water (MPA) and 0.1% formic acid in methanol (MPB) using a gradient elution with initial conditions of 60:40 (MPA:MPB) and adjusting to 90:10 over 5 min  and returning to 60:40 at 5.5 min with a total run time of 10 min using 10 µL injection and flow rate of 0.5 mL/min (Figure 2). The instrument was operated in positive electrospray, multiple reaction monitoring (MRM) mode (Table I).

Figure 2.

Total ion chromatogram in MRM mode for all compounds within the scope of the method.

Figure 2.

Total ion chromatogram in MRM mode for all compounds within the scope of the method.

Table I.

MS/MS parameters

Analyte Fragmentor (V) Collision (eV) Transitions 
U-47700 120 20 329 > 284 
120 25 329 > 204 
U-50488 115 20 369 > 298 
115 35 369 > 218 
Furanyl fentanyl 125 25 375.1 > 188.2 
125 40 375.1 > 105 
AH-7921 d3 120 14 332.4 > 287.1 
120 28 332.4 > 175.9 
Analyte Fragmentor (V) Collision (eV) Transitions 
U-47700 120 20 329 > 284 
120 25 329 > 204 
U-50488 115 20 369 > 298 
115 35 369 > 218 
Furanyl fentanyl 125 25 375.1 > 188.2 
125 40 375.1 > 105 
AH-7921 d3 120 14 332.4 > 287.1 
120 28 332.4 > 175.9 

Sample preparation

Samples were extracted by solid phase extraction using 130 mg Clean Screen® DAU extraction columns (UCT, Bristol, PA). After aliquoting 500 µL of blood, 50 µL of internal standard solution was added. Samples were pretreated with 2 mL of phosphate buffer (pH 6), mixed and centrifuged for 5 min. The columns were conditioned with 3 mL of methanol, followed by 3 mL of deionized water and 1 mL of phosphate buffer. After application of the sample, the columns were washed with 1.5 mL deionized water, 0.5 mL 0.1 M acetic acid and 1.5 mL methanol and dried under full vacuum for 5 min. Samples were eluted with 2 mL of ethyl acetate/acetonitrile/ammonium hydroxide (78:20:2). Finally, the eluent was evaporated to dryness at 40°C and reconstitute in 60:40 mobile phase (MPA:MPB).

Method validation

A fit-for-purpose method validation was performed by assessing the limit of detection, linear range, bias, accuracy and precision over the course of 3 days. Stability was assessed at low (5 ng/mL) and high (80 ng/mL) control values in duplicate at temperatures of 4°C and −20°C on Days 2, 3 and 10. Criteria for acceptable performance were as follows: a signal of three times that of the noise at the limit of detection, mean bias for the controls had to be <20%, within and between run precision had to have a coefficient of variation of <15%, accuracy had to be within 20% of the target concentration of for limit of quantitation and 15% for all control values. Analytes were determined to be stable if the percent coefficient of variation for the response ratio was <15%.

Results

Method validation

The limit of detection for all three compounds was 0.5 ng/mL. The calibration performance over 3 days of replicates resulted in average correlation coefficients of 0.9995 and 0.9999 for U-47700 and U-50488, respectively. The calibration range for furanyl fentanyl was 1–100 ng/mL and the calibration performance for 3 days of replicates resulted in an average correlation coefficient of 0.9990. All calibration curves were quadratic using 1/x weighting regression analysis of the peak area of the analyte to the peak area of the internal standard. For U-47700, the bias was 5% for the low controls (80 ng/mL) and 7% for the high controls (400 ng/mL), when processed using a calibration range of 30–500 ng/mL. When using the calibration range of 1–100 ng/mL for U-47700, the bias was 5% for the low controls (5 ng/mL) and 6% for the high controls (80 ng/mL). For U-50488, the bias was 5% for the low controls (80 ng/mL) and 8% for the high controls (400 ng/mL), when processed using a calibration range of 30–500 ng/mL, respectively. When using the calibration range of 1–100 ng/mL for U-50488, the bias was 2% for the low controls (5 ng/mL) and 6% for the high controls (80 ng/mL). For furanyl fentanyl, the bias was 7% for the low control (5 ng/mL) and 5% for the high control (80 ng/mL). Within and between run precision met acceptability criteria for percent coefficient of variation and accuracy for all three compounds. All compounds were stable at temperatures of 4°C and −20°C for up to 10 days, with no significant change in concentration over 10 days.

The method described was used to analyze blood samples from postmortem casework in which the presence of U-47700 or furanyl fentanyl was indicated through unconfirmed peaks in GCMS analysis from casework in other laboratories, data mining of NMS Labs LCTOF data, or an artifact in an LC–MS/MS method looking for AH-7921. AH-7921 and U-47700 both have the same molecular weight, and although U-47700 appeared in the scan window for AH-7921, it did not meet acceptance criteria for retention time. This was verified with the U-47700 standard reference material.

Since no deuterated internal standard was available, to account for possible matrix effects the method was also validated using standard addition, with a blank and two spikes (10 and 100 ng/mL). A subset of case samples (n  =  16) was also analyzed according to both calibration methods. No significant difference in performance of the controls was seen using the direct calibration curves versus the standard addition approach, and the quantitative values of the casework samples agreed within ±30%, suggesting no significant contributions to inaccuracy from matrix effect.

Case samples

Samples from 20 cases involving deaths occurring between October 2015 and March 2016, and suspected of involving furanyl fentanyl and/or U-47700 were submitted for analysis. Of the 3 compounds in the scope, 11 cases were positive for U-47700 only, 3 cases were positive for furanyl fentanyl alone and 5 cases contained both compounds. One case was presumptively positive for furanyl fentanyl, but failed to confirm. U-50488 was not detected in any cases. The mean and median (N = 7) concentrations for furanyl fentanyl in whole blood were 26 ng/mL (±28) and 12.9 ng/mL, and were all detected within a range of 2.5–76 ng/mL. For U-47700, the mean and median concentrations (N = 16) were 253 ng/mL (±150) and 247 ng/mL within a range of 17–490 ng/mL. Within the above cases, all subjects were male with the exception of 1 female, and the mean age was 28. A summary of the case history and findings can be found in Table II .

Table II.

Summary of cases submitted for analysis including demographic information, case history and analytical findings

Case number Age Sex State Case history Additional toxicology findings Source of blood U-47700 concentration (ng/mL) Furanyl fentanyl concentration (ng/mL) 
20 CO Emergency personnel were called to the scene of an at home death. The decedent had a history of substance abuse and had recently taken prescription drugs from relatives, including hydrocodone, hydromorphone and gabapentin. Several recent cellular messages referenced acquiring heroin. Drug paraphernalia was located in proximity to the decedent and the decedent was found with a syringe still clutched in his hand. The decedent had a prescription for dextroamphetamine. Blood: Amphetamine 12 ng/mL, caffeine Peripheral blood 382 ND 
18 FL The decedent, who had a history of attention deficit hyperactivity disorder and drug abuse, was found unresponsive in bed. Syringes and two white powders were found at the scene. The white powders were chemically analyzed and determined to be butyryl fentanyl and U-47700 (presumptively determined). Blood: Ethanol 0.030 g/dL, butyryl fentanyl 26 ng/mL Peripheral blood 17 ND 
Urine: carbamazepine, ibuprofen, iminostilbene, oxcarbazepine and suspected butyryl fentanyl 
Vitreous: Ethanol 0.030 mg/dL 
39 NM A male was found by his wife unresponsive and slumped on a sofa; a syringe was found on the floor. The decedent had a history of ordering supplements and designer drugs of the internet. Blood: Mephedrone 22 ng/mL, Etizolam Femoral blood 217 ND 
25 AK The decedent, who had a history of poly-substance abuse, was released from a halfway house. His roommate found him unresponsive, with evidence of pulmonary edema. A white powder was found at the scene with a rolled-up dollar bill; the powder was chemically confirmed to be U-47700. Blood: No positive findings Peripheral blood 334 ND 
23 MN The decedent was in a rehabilitation center and found lying on the bathroom floor with a ligature around his arm and a needle/syringe on the floor near the decedent. A packet containing a powdery substance labeled “U-47700” was also found in the bathroom. Blood: Citalopram 43 ng/mL Peripheral blood 252 ND 
Urine: Cotinine, Caffeine, Nicotine, Citalopram 
 
29 NY The decedent had no past medical history and was complaining of a headache to his girlfriend the day of his death and suddenly collapsed; emergency services responded and attempted resuscitation. The decedent was known to use tobacco and ethanol. A glass pipe, a container labeled “Chiesel 60% Sativa”, capsules labeled “1G322 300 mg” and an empty vial labeled amoxicillin were noted at the scene. An autopsy was performed later the same morning; cerebral and pulmonary edema was noted. Initial toxicology testing showed the presence of blood cannabinoids as well as the presence of mitragynine and gabapentin. However, confirmatory analysis with an RL of 1.0 mcg/mL did not confirm gabapentin. blood 453 ND 
29 OH The decedent, with a family history of prescription drug abuse, was recently released from a drug rehabilitation facility. After his release while he was house sitting for his parents, he was found unresponsive with evidence of pulmonary edema. A rolled-up 10 dollar bill with a residue of white powder was collected from the scene, along with a series of packets of white powders. Blood: Delta-9 Carboxy THC 5.3 ng/mL Peripheral blood 242 ND 
26 FL The decedent, with a history of drug abuse, was found deceased at home. Benadryl and etizolam pills were reportedly found at the scene, along with 5 syringes, 3 blue glass dropper bottles with liquid and 15 diphenhydramine tablets. Blood: Diphenhydramine 694 ng/mL Unknown 103 ND 
Urine: Diphenhydramine and metabolites, 3-methoxy phencyclidine 
Gastric contents: Diphenhydramine and metabolites 
21 NC The decedent, who had a history of drug abuse, with drug convictions. He was found with an injection site in the right arm containing a needle. A cooking apparatus was also reportedly found at the scene. Blood: Tramadol <250 ng/mL, alprazolam 47 ng/mL, lorazepam 11 ng/mL, 3-methoxy-PCP 180 ng/mL Aorta blood 299 ND 
Urine: Alprazolam, amphetamine, tramadol 
10 32 NC The decedent was found dead sitting up in a chair and was originally suspected to be shooting up oxycodone. Blood: Oxycodone 11 ng/mL, venlafaxine 2,600 ng/mL, O-Desmethylvenlafaxine 380 ng/mL Aorta blood 311 ND 
Liver: Venlafaxine 7.5 mg/kg, O-Desmethylvenlafaxine 0.87 mg/kg 
11 24 NC The decedent was reported to be using a synthetic opioid “U-47700.” He had obtained “U-47700” over the internet and was using it when last known alive. He was found unconscious and unresponsive at home the next day by a friend. The substance was secured by family in the packages it had arrived in and delivered to medical examiner (ME) for shipment to toxicology. Blood: Etizolam 86 ng/mL, chlorpheniramine <250 ng/mL, diphenhydramine 250 ng/mL Aorta blood 487 ND 
12 24 NC The decedent, who had a history of substance abuse, was found unresponsive with a syringe in arm. Heroin was originally suspected. Blood: 4-ANPP and quinine Aorta blood 59 Positive 
13 36 NC The decedent was found unresponsive in a bathroom with a syringe cap in his mouth. A heroin overdose was suspected. Blood: Ethanol, 4-ANPP and quinine Aorta blood 135 26 
14 33 NC The decedent was found deceased as a result of a probable drug overdose. The subject had a history of heroin and cocaine use. Blood: morphine 48 ng/mL, 4-ANPP, 6-monoacetylmorphine and quinine Aorta blood 167 56 
15 29 NC The decedent was found unresponsive. A heroin overdose was suspected. Blood: 4-ANPP and quinine Aorta blood 490 76 
16 40 NC The decedent had a history of heroin/opioid abuse and was found deceased. Blood: 4-ANPP and quinine Aorta blood 105 2.5 
17 30 AK The decedent, who had a history of heroin abuse, was found unresponsive on his bedroom floor, on his elbows and knees. Syringes and foil with a dark residue were found at the scene. Autopsy findings included pulmonary edema, gastritis and chronic hepatitis. Blood: Delta-9 THC 1 ng/mL, tramadol 75 ng/mL, O-Desmethyltramadol 40 ng/mL and butyryl fentanyl 0.33 ng/mL Peripheral blood ND 6.1 
18 32 ME The decedent, who had a history of drug abuse, was found unresponsive face down at home. Death was called due to acute intoxication of combined effects of acetyl fentanyl and diphenhydramine. Blood: Delta-9 THC 9.6 ng/mL, delta-9 carboxy THC 13 ng/mL, diphenhydramine 140 ng/mL, acetyl fentanyl 0.65 ng/mL and naloxone Femoral blood ND 12.9 
19 26 IL The decedent had a history of heroin and prescription drug abuse. The subject was found unresponsive laying on his right side, back against the wall of the bathroom by family. Two days prior to this incident, he had been admitted to the hospital after passing out, due to a possible overdose but was also reporting to family he did not feel well. Drug paraphernalia was found on the sink and suspected heroin packets were located in the decedent's wallet. Upon autopsy, severe pulmonary and cerebral edema was noted. Individual was prescribed Vicodin, Xanax and Effexor. Blood: Alprazolam 25 ng/mL, hydrocodone 120 ng/mL, hydromorphone 1.8 ng/mL, dihydrocodeine 21 ng/mL, venlafaxine 83, O-Desmethylvenlafaxine 400 ng/mL, delta-9 carboxy THC 7.2, diphenhydramine 90 ng/mL, caffeine and nicotine Peripheral blood ND 6.2 
Urine: Presumptively positive for opiates, benzodiazepines, cannabinoids and fentanyl/metabolites 
Mean 28      253 26 
Standard deviation      150 28 
Range 18–40      17–490 2.5–76 
Median 27.5      247 12.9 
Case number Age Sex State Case history Additional toxicology findings Source of blood U-47700 concentration (ng/mL) Furanyl fentanyl concentration (ng/mL) 
20 CO Emergency personnel were called to the scene of an at home death. The decedent had a history of substance abuse and had recently taken prescription drugs from relatives, including hydrocodone, hydromorphone and gabapentin. Several recent cellular messages referenced acquiring heroin. Drug paraphernalia was located in proximity to the decedent and the decedent was found with a syringe still clutched in his hand. The decedent had a prescription for dextroamphetamine. Blood: Amphetamine 12 ng/mL, caffeine Peripheral blood 382 ND 
18 FL The decedent, who had a history of attention deficit hyperactivity disorder and drug abuse, was found unresponsive in bed. Syringes and two white powders were found at the scene. The white powders were chemically analyzed and determined to be butyryl fentanyl and U-47700 (presumptively determined). Blood: Ethanol 0.030 g/dL, butyryl fentanyl 26 ng/mL Peripheral blood 17 ND 
Urine: carbamazepine, ibuprofen, iminostilbene, oxcarbazepine and suspected butyryl fentanyl 
Vitreous: Ethanol 0.030 mg/dL 
39 NM A male was found by his wife unresponsive and slumped on a sofa; a syringe was found on the floor. The decedent had a history of ordering supplements and designer drugs of the internet. Blood: Mephedrone 22 ng/mL, Etizolam Femoral blood 217 ND 
25 AK The decedent, who had a history of poly-substance abuse, was released from a halfway house. His roommate found him unresponsive, with evidence of pulmonary edema. A white powder was found at the scene with a rolled-up dollar bill; the powder was chemically confirmed to be U-47700. Blood: No positive findings Peripheral blood 334 ND 
23 MN The decedent was in a rehabilitation center and found lying on the bathroom floor with a ligature around his arm and a needle/syringe on the floor near the decedent. A packet containing a powdery substance labeled “U-47700” was also found in the bathroom. Blood: Citalopram 43 ng/mL Peripheral blood 252 ND 
Urine: Cotinine, Caffeine, Nicotine, Citalopram 
 
29 NY The decedent had no past medical history and was complaining of a headache to his girlfriend the day of his death and suddenly collapsed; emergency services responded and attempted resuscitation. The decedent was known to use tobacco and ethanol. A glass pipe, a container labeled “Chiesel 60% Sativa”, capsules labeled “1G322 300 mg” and an empty vial labeled amoxicillin were noted at the scene. An autopsy was performed later the same morning; cerebral and pulmonary edema was noted. Initial toxicology testing showed the presence of blood cannabinoids as well as the presence of mitragynine and gabapentin. However, confirmatory analysis with an RL of 1.0 mcg/mL did not confirm gabapentin. blood 453 ND 
29 OH The decedent, with a family history of prescription drug abuse, was recently released from a drug rehabilitation facility. After his release while he was house sitting for his parents, he was found unresponsive with evidence of pulmonary edema. A rolled-up 10 dollar bill with a residue of white powder was collected from the scene, along with a series of packets of white powders. Blood: Delta-9 Carboxy THC 5.3 ng/mL Peripheral blood 242 ND 
26 FL The decedent, with a history of drug abuse, was found deceased at home. Benadryl and etizolam pills were reportedly found at the scene, along with 5 syringes, 3 blue glass dropper bottles with liquid and 15 diphenhydramine tablets. Blood: Diphenhydramine 694 ng/mL Unknown 103 ND 
Urine: Diphenhydramine and metabolites, 3-methoxy phencyclidine 
Gastric contents: Diphenhydramine and metabolites 
21 NC The decedent, who had a history of drug abuse, with drug convictions. He was found with an injection site in the right arm containing a needle. A cooking apparatus was also reportedly found at the scene. Blood: Tramadol <250 ng/mL, alprazolam 47 ng/mL, lorazepam 11 ng/mL, 3-methoxy-PCP 180 ng/mL Aorta blood 299 ND 
Urine: Alprazolam, amphetamine, tramadol 
10 32 NC The decedent was found dead sitting up in a chair and was originally suspected to be shooting up oxycodone. Blood: Oxycodone 11 ng/mL, venlafaxine 2,600 ng/mL, O-Desmethylvenlafaxine 380 ng/mL Aorta blood 311 ND 
Liver: Venlafaxine 7.5 mg/kg, O-Desmethylvenlafaxine 0.87 mg/kg 
11 24 NC The decedent was reported to be using a synthetic opioid “U-47700.” He had obtained “U-47700” over the internet and was using it when last known alive. He was found unconscious and unresponsive at home the next day by a friend. The substance was secured by family in the packages it had arrived in and delivered to medical examiner (ME) for shipment to toxicology. Blood: Etizolam 86 ng/mL, chlorpheniramine <250 ng/mL, diphenhydramine 250 ng/mL Aorta blood 487 ND 
12 24 NC The decedent, who had a history of substance abuse, was found unresponsive with a syringe in arm. Heroin was originally suspected. Blood: 4-ANPP and quinine Aorta blood 59 Positive 
13 36 NC The decedent was found unresponsive in a bathroom with a syringe cap in his mouth. A heroin overdose was suspected. Blood: Ethanol, 4-ANPP and quinine Aorta blood 135 26 
14 33 NC The decedent was found deceased as a result of a probable drug overdose. The subject had a history of heroin and cocaine use. Blood: morphine 48 ng/mL, 4-ANPP, 6-monoacetylmorphine and quinine Aorta blood 167 56 
15 29 NC The decedent was found unresponsive. A heroin overdose was suspected. Blood: 4-ANPP and quinine Aorta blood 490 76 
16 40 NC The decedent had a history of heroin/opioid abuse and was found deceased. Blood: 4-ANPP and quinine Aorta blood 105 2.5 
17 30 AK The decedent, who had a history of heroin abuse, was found unresponsive on his bedroom floor, on his elbows and knees. Syringes and foil with a dark residue were found at the scene. Autopsy findings included pulmonary edema, gastritis and chronic hepatitis. Blood: Delta-9 THC 1 ng/mL, tramadol 75 ng/mL, O-Desmethyltramadol 40 ng/mL and butyryl fentanyl 0.33 ng/mL Peripheral blood ND 6.1 
18 32 ME The decedent, who had a history of drug abuse, was found unresponsive face down at home. Death was called due to acute intoxication of combined effects of acetyl fentanyl and diphenhydramine. Blood: Delta-9 THC 9.6 ng/mL, delta-9 carboxy THC 13 ng/mL, diphenhydramine 140 ng/mL, acetyl fentanyl 0.65 ng/mL and naloxone Femoral blood ND 12.9 
19 26 IL The decedent had a history of heroin and prescription drug abuse. The subject was found unresponsive laying on his right side, back against the wall of the bathroom by family. Two days prior to this incident, he had been admitted to the hospital after passing out, due to a possible overdose but was also reporting to family he did not feel well. Drug paraphernalia was found on the sink and suspected heroin packets were located in the decedent's wallet. Upon autopsy, severe pulmonary and cerebral edema was noted. Individual was prescribed Vicodin, Xanax and Effexor. Blood: Alprazolam 25 ng/mL, hydrocodone 120 ng/mL, hydromorphone 1.8 ng/mL, dihydrocodeine 21 ng/mL, venlafaxine 83, O-Desmethylvenlafaxine 400 ng/mL, delta-9 carboxy THC 7.2, diphenhydramine 90 ng/mL, caffeine and nicotine Peripheral blood ND 6.2 
Urine: Presumptively positive for opiates, benzodiazepines, cannabinoids and fentanyl/metabolites 
Mean 28      253 26 
Standard deviation      150 28 
Range 18–40      17–490 2.5–76 
Median 27.5      247 12.9 

Discussion and conclusions

Opiates are among the most frequently encountered drugs in drug-related deaths (1923). In addition to the commonly encountered opiates, there has been increasing reports of opioid derivatives and “designer opioids” in postmortem casework. Between June 2013 and December 2015, NMS Labs has reported 582 cases of acetyl fentanyl use, mostly in death investigation casework, with blood concentrations ranging from 0.11 to 3,900 ng/mL, and mean and median concentrations of 133 and 12 ng/mL, respectively. Acetyl fentanyl is reported to be 10–15 times more potent than heroin, and 80 times more potent than morphine (24). Since June 2015, butyryl fentanyl, a fentanyl analog with a potency of approximately a quarter that of fentanyl (24, 25), has been detected by our laboratory (NMS Labs) in 20 cases in a 10-month period between July 2015 and May 2016, with concentrations ranging from 0.33 to 270 ng/mL.

Beginning in October 2015, furanyl fentanyl, another analog, has been detected by us in over 30 cases, often accompanied by the novel compound U-47700. Aside from one reported death in Sweden related to furanyl fentanyl with a reported serum concentration of 148ng/mL (7), there are no published concentrations of these compounds in the scientific literature. In 19 of the 20 cases we describe, U-47700, furanyl fentanyl, or a combination of both drugs was confirmed. One case initially screened positive for furanyl fentanyl, but was present below the laboratory's reporting limit on confirmation. Five cases, all from North Carolina had both drugs present.

Reported fentanyl concentrations in deaths attributed to fentanyl following intravenous administration (as opposed to use of fentanyl patches) are 27.5 (26), 4.8 (27) and 4.0 ng/mL (28). Fentanyl concentrations in 5 decedents, who injected fentanyl extracted from patches, had concentrations between 5 and 27 ng/mL (29). Furanyl fentanyl, which has an ED-50 that is comparable to fentanyl (17), had mean and median concentrations of 26 and 12.9 ng/mL in our case series, respectively, similar to the range of fentanyl concentrations cited above.

The potency of U-47700 is reported to be ~7.5 times that of morphine in animal studies (10), making it about 10 times less potent than fentanyl. In the cases reported here, mean and median concentrations of U-47700 were 253–247 ng/mL respectively (range 17–490ng/mL), ~10-fold higher than the concentrations in the reported intravenous fentanyl deaths cited above. The single case reported by Elliott et al. (13) involved the death of a 27-year-old male, who subsequently tested positive in urine for quetiapine, amphetamine, amitriptyline, mexedrone and ketamine; quetiapine (<0.05 mg/L), amphetamine (<0.1 mg/L) and naproxen (<0.8 mg/L). U-47700 was subsequently measured at a concentration of 1.46 mg/L (1,460 ng/mL) in postmortem femoral blood.

Novel compounds continue to appear on a monthly basis, including the appearance in the USA of W-18 and W-15. These compounds are two of a series of drugs with analgesic properties of unknown origin synthesized in Canada in 1981 (30). The researchers evaluated these compounds using the mouse phenylquinone writhing tests, which are a measure of a compounds ability to induce analgesia. In this study, a dose of 3.7 × 10−2 mg/kg morphine resulted in 50% inhibition of writhing. The doses of W-15 and W-18 required for the same degree of inhibition were 0.7 × 10−3 and 3.7 × 10−6 mg/kg, respectively.

Deaths with histories consistent with opioid overdose, but negative toxicology, should be subjected to further testing for these novel opioid-like compounds. Additionally, positive fentanyl immunoassay screens that fail to confirm for traditional opioids should be investigated for other fentanyl analogs. The geographical distribution of these findings indicates that “designer opioids” are widespread across the country and their prevalence in postmortem casework should be continually monitored.

Acknowledgements

The authors of this report would like to acknowledge the following parties for their contribution: Ms Marilyn Manzutto of the Jefferson County Coroner's Office in Colorado, Dr Ian Paul of the New Mexico Office of Medical Investigators, Dr Kenneth Gallagher and Dr Gary Zintek of the State of Alaska Medical Examiner's Office, Dr Victor Froloff of the Ramsey County Medical Examiner's Office in Minnesota, Dr John Worsing of Pierce County, Wisconsin, Ms Elizabeth Spratt and Dr Virginia Richards of the West Chester County Medical Examiner's Office in New York, Dr Mark Flomenbaum and Ms Alyssa Woodard of the Maine Office of the Medical Examiner, Mr Michael Burke of the Livingston County Coroner's Office in Illinois, Ms Reta Newman of the Pinellas County Forensic Laboratory in Florida, Dr Lynn Smith of the Lake County Coroner's Office in Ohio and Dr Ruth Winecker of the North Carolina Department of Health and Human Services: Division of Public Health, Office of the Chief Medical Examiner.

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